CN118401538A - Oligosaccharide compounds and complexes - Google Patents

Abstract

The present disclosure provides compounds of formula I, as well as complexes and compositions comprising compounds of formula I, wherein the compositions and complexes are useful for delivering certain agents, including, for example, nucleic acids.

Description

Oligosaccharide compounds and complexes

manual

Background technique

Due to the instability of nucleic acids and their inability to penetrate cell membranes, there are certain challenges in the targeted delivery and expression of therapeutic agents and/or preventive agents (such as nucleic acids). Certain methods (including the use of lipid- or polymer-based systems) show good prospects, but there are also shortcomings associated with manufacturing difficulties, poor structural definition and high polydispersity. In addition, many such compositions lack satisfactory safety and efficacy when used to deliver therapeutic agents.

Summary of the invention

The present disclosure provides, inter alia, cationic and/or ionizable oligosaccharide complexes that overcome certain deficiencies associated with previous compositions and are useful for the targeted delivery of biological agents, including small molecules and nucleic acids.

In some embodiments, the present disclosure provides oligosaccharides that are compounds of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

A is ^A1 , ^A2 or ^A3 :

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , and wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and

p is an integer selected from 1, 2, 3, 4 or 5;

The premise is that when A is A ¹ , then:

(i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ - and Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(ii) each of R ¹ and R ² is not –(CH ₂ ) ₅ -CH ₃ or –(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and

(iv) Cy is not a triazole group.

In some embodiments, the present disclosure provides a complex comprising an oligosaccharide described herein and a nucleic acid.

In some embodiments, the present disclosure provides a method of increasing or causing an increase in RNA expression in a target in a subject, the method comprising administering to the subject a complex as described herein.

In some embodiments, the present disclosure provides a method of treating and/or preventing a disease, disorder, or condition in a subject, comprising administering to the subject a complex as described herein.

In some embodiments, the present disclosure provides a method for preparing an oligosaccharide of formula II:

It comprises reacting a compound of formula III:

Contacting with a compound of formula IV:

in

Each ^Ra is an optionally substituted _C1 - _C20 aliphatic;

Z ¹ is a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^Za - ^PG1 is ^Z1 , wherein one ^Rz has been replaced by a ^PG1 group; and

PG ¹ is a suitable nitrogen protecting group.

BRIEF DESCRIPTION OF THE DRAWINGS

1A is a graph illustrating the hydrodynamic diameters of example compositions comprising modRNA formulated with certain oligosaccharides at different N/P ratios.

FIG. 1B is a graph illustrating the zeta potential of example oligosaccharides of varying N/P ratios.

1C is a bar graph illustrating quantitative ex vivo luminescence 6 hours after injection of example compositions comprising modRNA in different organs and at different N/P ratios.

FIG. 1D is a bar graph illustrating the hydrodynamic diameters of example compositions comprising modRNA.

IE is a bar graph illustrating the zeta potential of example compositions.

FIG. 1F is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of example compositions comprising modRNA.

FIG. 1G is a bar graph illustrating the hydrodynamic diameters of example compositions comprising modRNA.

FIG. 1H is a bar graph illustrating the zeta potential of example oligosaccharide compositions.

FIG. 1I is a bar graph illustrating quantitative ex vivo luminescence in the spleen 6 hours after injection of a composition comprising modRNA.

FIG. 2A is a bar graph illustrating the hydrodynamic diameters of example compositions comprising modRNA formulated at different N/P ratios. FIG.

2B is a bar graph illustrating the zeta potential of example compositions at different N/P ratios.

2C is a bar graph illustrating quantitative ex vivo luminescence 6 hours after injection of example compositions comprising modRNA in different organs and at different N/P ratios.

FIG2D is a bar graph illustrating the hydrodynamic diameters of example compositions comprising modRNA.

FIG. 2E is a bar graph illustrating the zeta potential of example compositions.

FIG2F is a bar graph illustrating quantitative ex vivo luminescence in the spleen 6 hours after injection of a composition comprising modRNA.

FIG. 2G is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of a composition comprising modRNA.

3A is a bar graph illustrating the percentage of free RNA quantified by gel electrophoresis of example compositions comprising modRNAs with different N/P ratios between 0 and 3 for JRL13 or JRL45.

FIG. 3B is a graph illustrating the zeta potential after formulating example compositions comprising modRNA at different N/P ratios and JRL13 or JRL45.

FIG. 3C is a graph illustrating the hydrodynamic diameters after formulating example compositions comprising modRNA at different N/P ratios and JRL13 or JRL45.

3D is a bar graph illustrating expression of luciferase encoding modRNA following transfection of C2C12 cells, where example compositions were prepared at N/P 6 and comprised 25 ng RNA/well of modRNA with either JRL13 or JRL45.

3E is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of 10 μg of an example composition comprising modRNA formulated at N/P 3 with JRL13 or JRL45.

FIG. 3F is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection and evaluating the ratio of total flux [p/s] of the JRL13 to JRL45 groups.

FIG3G is a bar graph illustrating quantitative ex vivo luminescence in different organs after injection of a composition comprising pDNA complexed with JRL13 (Compound 8) or JRL45 (Compound 10).

FIG. 4A is a graph illustrating the hydrodynamic diameters of example complexes formulated at different N/P ratios and comprising RNA.

4B is a bar graph illustrating the zeta potential of example compositions comprising RNA at different N/P ratios.

4C is a bar graph illustrating the quantitative bioluminescence of example compositions comprising modRNA and JRL13 having specified N/P ratios over the duration of the experiment.

FIG4D is a bar graph illustrating the quantitative bioluminescence of example compositions comprising saRNA and JRL13 having a specified N/P ratio over the duration of the experiment.

FIG. 5A is a bar graph illustrating the hydrodynamic diameters of complexes formulated at N/P 6 and comprising saRNA and different oligosaccharides.

FIG. 5B is a bar graph illustrating the quantitative bioluminescence over the duration of the experiment and normalized to the JRL13-saRNA group.

FIG. 5C is a graph illustrating the quantitative bioluminescence of an example composition at each measurement time point.

FIG. 6A is a bar graph illustrating the hydrodynamic diameters of example complexes formulated with modRNA and different oligosaccharides at different N/P ratios.

FIG. 6B is a bar graph illustrating quantitative bioluminescence over the duration of the experiment.

FIG. 7A is a bar graph illustrating the hydrodynamic diameters of complexes formulated with N/P 6 modRNA and various oligosaccharides.

FIG. 7B is a bar graph illustrating quantified bioluminescence over the duration of the experiment and normalized to the JLF41-modRNA group.

FIG. 8A is a bar graph illustrating the hydrodynamic diameters of complexes formulated at different N/P ratios.

FIG8B is a bar graph illustrating the zeta potential of compositions comprising modRNA at different N/P ratios.

FIG. 8C is a bar graph illustrating the quantitative bioluminescence of various example compositions over the duration of the experiment.

9A is a bar graph illustrating the hydrodynamic diameters of example compositions.

FIG. 9B is a bar graph illustrating the quantitative bioluminescence of example oligosaccharide preparations over the duration of the experiment.

FIG. 10A is a bar graph illustrating the hydrodynamic diameters of certain complexes.

FIG. 10B is a bar graph illustrating the quantitative bioluminescence of example oligosaccharide preparations over the duration of the experiment.

FIG. 10C is a bar graph illustrating the quantitative response of CD8-positive T cells in spleen samples of treated mice 20 days after subcutaneous application.

FIG. 11A is a bar graph illustrating the hydrodynamic diameters of complexes formulated at different N/P ratios and comprising RNA.

11B is a bar graph illustrating the zeta potential of example compositions comprising RNA at different N/P ratios.

FIG. 11C is a bar graph illustrating the quantitative bioluminescence of the modRNA groups over the duration of the experiment.

FIG. 11D is a bar graph illustrating the quantitative bioluminescence of the saRNA group over the duration of the experiment.

FIG. 12A is a bar graph illustrating the hydrodynamic diameters of complexes formulated at different N/P ratios and comprising RNA.

FIG. 12B is a graph illustrating serological levels of specific IgG against HA at a 1:300 serum dilution during the course of the experiment.

FIG. 12C is a graph illustrating endpoint titration of serological levels of specific IgG against HA.

FIG. 12D is a bar graph illustrating CD8/CD4 responses to HA peptide pools.

13A is a graph illustrating the hydrodynamic diameters of complexes formulated with N/P 6 but with different surfactants and having surfactant to JLF99 ratios ranging from 0:1 to 0.5:1 (wherein C14-pMeOx45 is _C14 alkyl-poly(2-methyl-2-oxazoline) ₄₅ , C14pSar23 is C14 alkyl-poly(sarcosine) ₂₃ , and Tween20 is polysorbate 20).

13B is a graph illustrating the hydrodynamic diameters of complexes formulated at N/P 6 with either Tween 20 or Tween 40 and within a surfactant to JLF99 ratio of 0 to 1.5.

Figure 13C is a graph illustrating the expression of luciferase encoding modRNA 24h after transfection of C2C12 cells, the example composition has N/P 6 and is formulated with modRNA and Tween20 or Tween40 at different surfactant to JLF99 ratios at 100ng RNA/well. This example represents 3 independent experiments (n=3), technically triplicate.

Figure 13D is a graph illustrating the expression of luciferase encoding modRNA 24 hours after transfection of HepG2 cells, the example composition has N/P 6 and is formulated with modRNA and different surfactant to JLF99 ratios of Tween20 or Tween40 at 100ng RNA/well. This example represents 3 independent experiments (n=3), technically triplicate.

FIG. 13E is a bar graph illustrating the hydrodynamic diameters of complexes formulated with N/P 6 at different oligosaccharide to surfactant ratios (where T20 refers to Tween20).

FIG. 13F is a bar graph illustrating quantified bioluminescence over the duration of the experiment, expressed as area under the curve (where T20 refers to Tween20).

FIG. 13G is a graph illustrating quantitative bioluminescence over the duration of the experiment (where T20 refers to Tween20).

FIG. 13H is an image showing bioluminescence of the ventral axis 6 hours after injection of the composition comprising modRNA into each group (wherein T20 refers to Tween20).

FIG. 14A is a graph illustrating the hydrodynamic diameters of compositions comprising saRNA complexed with different oligosaccharides.

FIG. 14B is a graph illustrating the changes in serological levels of specific IgG for the indicated preparations over time.

FIG. 14C is a graph illustrating endpoint titration of serological levels of specific IgG against HA of provided formulations.

FIG. 14D is a bar graph illustrating the CD8/CD4 responses of provided formulations to a pool of HA peptides.

15A is a bar graph illustrating the hydrodynamic diameters of example compositions comprising modRNA formulated at N/P 6 with JLF99 and different polysorbates.

FIG. 15B shows the quantitative bioluminescence at the injection site at different time points during the experiment.

FIG. 15C shows quantitative bioluminescence in the liver 6 hours and 24 hours after injection of modRNA.

FIG. 16A is a graph illustrating serological levels of specific IgG against HA for the provided formulations at the indicated times (Day 0, Day 14, Day 28, and Day 49).

16B is a graph illustrating the in-process specific neutralization titers of provided formulations over the indicated time.

FIG. 16C is a graph illustrating endpoint titration of serological levels of specific IgG against HA for provided formulations.

FIG. 16D is a graph illustrating the CD8/CD4 responses of provided formulations to a pool of HA peptides.

FIG. 17A is a graph illustrating serological levels of specific IgG against HA with different RNA complexes formulated with JLF99 at different time points during the experiment.

FIG. 17B is a graph illustrating endpoint titration of serological levels of specific IgG against HA with different RNA platforms formulated with JLF99.

FIG. 17C is a graph illustrating CD8/CD4 responses to HA peptide pools from different RNA platforms formulated with JLF99.

FIG. 18A is a graph illustrating mean liver irradiance for example compositions comprising JFL99 or JLF218.

FIG. 18B is an image showing bioluminescence of the abdominal axis 6 hours after injection of the example formulations into each group.

FIG. 19A is a graph illustrating IL-6 secretion by hPBMCs following exposure to different doses of test formulations.

FIG. 19B is a graph illustrating TNF-α secretion by hPBMCs after exposure to different doses of the test formulations.

FIG. 19C is a graph illustrating IL-1β secretion by hPBMCs after exposure to different doses of the test formulations.

FIG. 19D is a graph illustrating IFN-γ secretion by hPBMCs after exposure to different doses of test formulations.

FIG. 19E is a graph illustrating serological levels of specific IgG against HA in the tested formulations.

FIG. 19F is a graph illustrating endpoint titration of serological levels of specific IgG against HA.

Figure 19G is a bar graph illustrating CD8/CD4 responses to HA peptide pools.

FIG. 19H is a bar graph illustrating the hydrodynamic diameters of different formulations tested.

FIG. 20A illustrates serological levels of specific IgG against HA during the Example process.

FIG. 20B illustrates endpoint titration of serological levels of specific IgG against HA.

FIG. 20C exemplifies CD8/CD4 responses to HA peptide pools.

FIG. 21A illustrates serological levels of specific IgG against HA during the Example process.

FIG. 21B illustrates endpoint titration of serological levels of specific IgG against HA.

Figure 21C illustrates the CD8/CD4 response to the HA peptide pool.

FIG. 22A illustrates serological levels of specific IgG against HA during the course of the experiment.

FIG. 22B illustrates endpoint titration of serological levels of specific IgG against HA.

FIG. 22C exemplifies CD8/CD4 responses to HA peptide pools.

FIG. 23A illustrates serological levels of specific IgG against Gc during the Example process.

FIG. 23B illustrates endpoint titration of serological levels of specific IgG against Gc.

FIG. 23C exemplifies T cell responses to Gc peptide.

Detailed ways

definition

Compounds of the present disclosure include compounds generally described above, and are further described by the categories, subclasses and species disclosed herein. As used herein, unless otherwise indicated, the following definitions should be applied. For the purposes of the present disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. In addition, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th edition, editors: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

Unless otherwise stated, the structure depicted herein is intended to include all stereoisomers (e.g., enantiomers or diastereomers) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are considered to be part of the disclosure. Therefore, the single stereochemical isomers of the provided compound and enantiomers, diastereomers, and geometric (or conformational) mixtures are within the scope of the disclosure. For example, in some cases, Table 1 shows one or more stereoisomers of the compound, and unless otherwise stated, represents each stereoisomer in a separate and/or mixture form. Unless otherwise stated, all tautomeric forms of the provided compound are within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having structures of the present invention including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by ¹³ C or ¹⁴ C enriched carbon are within the scope of the present disclosure.

About or approximately: As used herein, the term "about" or "approximately" as applied to one or more values of interest refers to a value similar to the stated reference value. Generally speaking, those skilled in the art who are familiar with the context will understand the relevant degree of difference covered by "about" or "approximately" in the context. For example, in some embodiments, the term "about" or "approximately" may cover (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or a range of values within a smaller range of the value referred to.

Administration: As used herein, the term "administering" or "administration" generally refers to the application of a composition to a subject to achieve delivery of an agent as a composition or contained in a composition to a target site or site to be treated. One of ordinary skill in the art will appreciate the various routes that can be used for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or include, for example, one or more of dermal topical, intradermal, interdermal, transdermal, and the like), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous administration, and the like. In some embodiments, administration may be parenteral administration. In some embodiments, administration can be oral administration. In some specific embodiments, administration can be intravenous administration. In some specific embodiments, administration can be subcutaneous administration. In some embodiments, administration can only involve a single dose. In some embodiments, administration can involve the application of a fixed number of doses. In some embodiments, administration can involve intermittent (e.g., multiple doses separated in time) and/or periodic (e.g., separate doses separated by the same time period) administration. In some embodiments, administration can involve continuous administration (e.g., perfusion) for at least a selected time period. In some embodiments, administration can include a primary immunization scheme and a booster scheme. The primary immunization scheme and the booster scheme can include the administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine), followed by the administration of a second dose or a subsequent dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) after a period of time. In the case of an immunogenic composition, the primary immunization scheme and the booster scheme can lead to an enhanced immune response in the patient.

Aliphatic: The term "aliphatic" refers to a straight chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon (also referred to herein as "alicyclic") that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single point of attachment or more than one point of attachment to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-12 aliphatic carbon atoms. As used herein, it is understood that an aliphatic group may also be divalent (e.g., encompassing a divalent hydrocarbon chain that is saturated or contains one or more unsaturated units, such as, for example, _-CH2- , _-CH2 - _CH2- , _-CH2 - _CH2 - _CH2- , etc.). In some embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms (e.g., _C1-6 ). In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms (e.g., _C1-5 ). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C _1-4 ). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C _1-3 ), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C _1-2 ). In some embodiments, "alicyclic" refers to a monocyclic C _3-8 hydrocarbon or a bicyclic C 7-10 hydrocarbon that is completely saturated or contains one or more unsaturated units but is not aromatic, and has a single connection point or more than one connection point with the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight or branched, substituted or unsubstituted alkyl, alkenyl or alkynyl _{, and their hybrids. Preferred aliphatic groups are C 1-6 alkyl} .

Alkyl: The term "alkyl," used alone or as part of a larger moiety, refers to a saturated, optionally substituted, straight or branched chain hydrocarbon radical having (unless otherwise specified) 1-12, 1-10, 1-8, _1-6 , 1-4, 1-3, or 1-2 carbon atoms (e.g., C _1-12 , C _1-10 , C _1-8 , C _1-6 , C _1-4 , C 1-3 , or C _1-2 ). Exemplary alkyl radicals include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

Alkenyl: The term "alkenyl," used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon radical having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, _2-6 , 2-4, or 2-3 carbon atoms (e.g., _C2-12 , _C2-10 , _C2-8 , C2-6, _C2-4 , or _C2-3 ). Exemplary alkenyls include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

Alkynyl: The term "alkynyl," used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon radical having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., _C2-12 , _C2-10 , _C2-8 , _C2-6 , _C2-4 , or _C2-3 ). Exemplary alkynyl radicals include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or portions with a reference substance. Typically, an "analog" exhibits significant structural similarity to a reference substance (e.g., a shared core or common structure) but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be produced from a reference substance, such as by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by performing a synthetic method that is substantially similar to (e.g., shares multiple steps with) a synthetic method that produces a reference substance. In some embodiments, an analog is generated by or can be generated by performing a synthetic method that is different from the synthetic method used to produce the reference substance.

Aryl: The term "aryl" refers to monocyclic and bicyclic ring systems having a total of five to fourteen ring members (e.g., C ₅ -C ₁₄ ), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, "aryl" contains a total of six to twelve ring members (e.g., C ₆ -C ₁₂ ). The term "aryl" can be used interchangeably with the term "aryl ring". In certain embodiments of the present invention, "aryl" refers to an aromatic ring system, which includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may bear one or more substituents. Unless otherwise specified, "aryl" is a hydrocarbon. In some embodiments, an "aryl" ring system is an aromatic ring (e.g., phenyl) fused to a non-aromatic ring (e.g., cycloalkyl). Examples of fused aryl rings include

Correlation: Two events or entities are "associated" with each other, as the term is used herein, if the presence, level, and/or form of one event or entity is associated with the other. For example, if the presence, level, and/or form of a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is associated with the incidence and/or susceptibility of a disease, disorder, or condition (e.g., in a related population), the particular entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly so that they are physically close to and/or remain close to each other. In some embodiments, two or more entities that are physically associated with each other are covalently attached to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently attached to each other, but are non-covalently associated, such as by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

Biological sample: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a biological source of interest as described herein (e.g., a tissue or an organism or a cell culture). In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or comprises a biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph fluid; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washing fluid or lavage fluid, such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirate; scraping; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids (e.g., semen, sweat, tears), secretions and/or excretions; and/or cells therefrom, etc. In some embodiments, biological sample is or includes the cell obtained from individual.In some embodiments, the cell obtained is or includes the cell from the individual who obtains the sample.In some embodiments, sample is " primary sample " obtained directly from the source of interest by any appropriate means.For example, in some embodiments, primary biological sample is obtained by the method selected from the group consisting of: biopsy (for example, fine needle aspiration or tissue biopsy), surgery, body fluid (for example, blood, lymph, feces, etc.) collection, etc.In some embodiments, as will be clear from the context, the term " sample " refers to the preparation obtained by processing primary sample (for example, by removing one or more components of primary sample and/or by adding one or more agents to primary sample).For example, using semipermeable membrane filtration.This " processed sample " may include nucleic acid or protein extracted from sample, for example, or nucleic acid or protein obtained by subjecting primary sample to technology (such as amplification or reverse transcription of mRNA, separation and/or purification of some components, etc.).

Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, the carrier may include sterile liquids such as, for example, water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents or modes) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, the regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, the agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administering one or more agents or modes to a subject who is receiving other agents or modes in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily administered simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition sets, etc., which may not be identical to each other, but are similar enough to allow comparison between them, so that a person skilled in the art will understand that a conclusion can be reasonably drawn based on the observed differences or similarities. In some embodiments, a comparable condition set, situation, individual or group is characterized by a plurality of substantially identical features and one or a small number of different features. A person of ordinary skill in the art will understand that, in the context, in any given case, what degree of consistency is required for two or more such agents, entities, situations, condition groups, etc. to be considered comparable. For example, a person of ordinary skill in the art will understand that when characterized by a sufficient number and type of substantially identical features, a situation set, individual or group is comparable to each other to ensure a reasonable conclusion that the differences in the results or observed phenomena obtained under or using different situation sets, individuals or groups are caused by changes in those changed features or indicate changes in those changed features.

Composition: Those skilled in the art will appreciate that the term "composition" can be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition can be in any form - e.g., gas, gel, liquid, solid, etc.

Cycloaliphatic: As used herein, the term "cycloaliphatic" refers to a monocyclic C3-8 hydrocarbon or a bicyclic C7-10 hydrocarbon that is fully saturated or contains one _or more unsaturated units but is not aromatic, having a single point of attachment or more than one point of attachment to the _rest of the molecule.

Cycloalkyl: As used herein, the term "cycloalkyl" refers to an optionally substituted saturated cyclic monocyclic or multicyclic ring system having about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Dosage form or unit dosage form: It will be understood by those skilled in the art that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic agent or a diagnostic agent) for administration to a subject. Typically, each of the units contains a predetermined amount of an active agent. In some embodiments, such an amount is a unit dose (or an entire portion thereof) suitable for administration according to a dosing regimen that has been determined to be associated with a desired or beneficial outcome when administered to a relevant population (i.e., using a therapeutic dosing regimen). It will be understood by those of ordinary skill in the art that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

Dosing regimen or treatment regimen: It will be understood by those skilled in the art that the terms "dosing regimen" and "treatment regimen" can be used to refer to a collection of (usually more than one) unit doses that are administered separately to a subject, usually separated by time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each dose separated from the other doses in time. In some embodiments, each dose is separated from each other by a time period of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separating each dose. In some embodiments, all doses within the dosing regimen are the same unit dose. In some embodiments, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first administration of a first dose, followed by one or more additional administrations of a second dose that is the same as the first dose. In some embodiments, the dosing regimen includes a first administration of a first dose amount, followed by one or more additional administrations of a second dose amount that is the same as the first dose amount. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered in a relevant population (i.e., it is a therapeutic dosing regimen).

Excipients: As used herein, the term "excipient" refers to non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like.

Heteroaliphatic: As used herein, the term "heteroaliphatic" or "heteroaliphatic group" means an optionally substituted hydrocarbon moiety having one to five heteroatoms in addition to carbon atoms, which may be straight chain (i.e., unbranched), branched, or cyclic ("heterocycle") and may be fully saturated or may contain one or more unsaturated units, but which is not aromatic. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, as well as any quaternized form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms, of which 1-3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, of which 1-2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, of which 1 carbon atom is optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, straight or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, heteroaliphatic groups of 1 to 10 atoms include the following exemplary groups: -O- _CH3 , _-CH2 -O- _CH3 , -O- _CH2 - _CH2 -O-CH2 _{- CH2} -O- _CH3 , and the like.

Heteroaryl: The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety (e.g., "heteroaralkyl" or "heteroaralkoxy"), refer to monocyclic or bicyclic groups having 5 to 12 ring atoms (e.g., 5 to 6 membered monocyclic heteroaryl or 9 to 12 membered bicyclic heteroaryl); having 6, 10 or 14 pi electrons shared in the ring array; and having one to five heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyrimidinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrrolopyridinyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., bicyclic heteroaryl rings having from 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyrido [2,3-b] -1, 4-oxazine -3 (4H) -one, 4H-thieno [3,2-b] pyrrole and benzisoxazolyl. Heteroaryl can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the term "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

Heteroatom: As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of the nitrogen or sulfur, and any quaternized form of a basic nitrogen.

Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic group" and "heterocyclic ring" are used interchangeably and refer to a stable 3-8-membered monocyclic, 7-12-membered bicyclic or 10-16-membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (such as one to four) heteroatoms as defined above in addition to carbon atoms. When used to refer to the ring atoms of the heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR ⁺ (as in N-substituted pyrrolidinyl). The heterocycle may be attached to its side group at any heteroatom or carbon atom that produces a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepine, oxazepine, thiazepine, morpholinyl, and thiamorpholinyl. The heterocyclic group can be monocyclic, bicyclic, tricyclic or polycyclic, preferably monocyclic, bicyclic or tricyclic, more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl substituted with a heterocyclic group, wherein the alkyl and heterocyclic moieties are independently optionally substituted. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, tetrahydroquinolinyl, The bicyclic heterocycle can also be a spiro ring system (e.g., a 7- to 11-membered spiro fused heterocycle having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms). The bicyclic heterocycle can also be a bridged ring system (e.g., a 7- to 11-membered bridged heterocycle having one, two or three bridging atoms).

Nucleic acid/polynucleotide: As used herein, the term "nucleic acid" refers to a polymer having at least 10 or more nucleotides. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a peptide nucleic acid (PNA). In some embodiments, the nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, the nucleic acid comprises a single-stranded and double-stranded portion. In some embodiments, the nucleic acid comprises a main chain containing one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a main chain containing phosphodiester linkages and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a main chain containing one or more thiophosphates or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, for example, as in "peptide nucleic acids". In some embodiments, the nucleic acid comprises one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all non-natural residues. In some embodiments, the non-natural residues comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine-oxoadenosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugar in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by separation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, such as in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the length of the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 350 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides.

Nucleic acid particles: "Nucleic acid particles" can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer (such as protamine) or a mixture thereof and nucleic acids. Nucleic acid particles include lipid nanoparticle (LNP)-based formulations and lipid complex (LPX)-based formulations.

Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used as an indication of size (e.g., the size of a polynucleotide), a certain number of nucleotides refers to the number of nucleotides on a single strand (e.g., a single strand of a polynucleotide).

Oral: As used herein, the phrases "oral administration" and "administered orally" have their art-understood meanings and refer to administration of a compound or composition by mouth.

Parenteral: As used herein, the phrases "parenteral administration" and "administered parenterally" have their art-understood meanings and refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety containing at least one double or triple bond between ring atoms. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties as defined herein.

Patient or subject: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition is or can be applied, e.g., for experimental, diagnostic, preventive, cosmetic, and/or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. In some embodiments, the patient or subject suffers from or is susceptible to one or more conditions or disorders. In some embodiments, the patient or subject exhibits one or more symptoms of a condition or disorder. In some embodiments, the patient or subject has been diagnosed with one or more conditions or disorders. In some embodiments, the patient or subject is receiving or has received certain therapies to diagnose and/or treat a disease, condition, or disorder.

Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a treatment or dosing regimen that shows a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), pills, powders, granules, pastes for application to the tongue; parenteral administration, e.g., in the form of sterile solutions or suspensions or sustained release formulations, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical application, e.g., in the form of creams, ointments, or controlled-release patches or sprays, to the skin, lungs, or oral cavity; intravaginally or intrarectally, e.g., in the form of vaginal suppositories, creams, or foams; sublingually; ophthalmically; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates, and/or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations.

Pharmaceutically acceptable salts: The term "pharmaceutically acceptable salts" as used herein refers to salts of such compounds suitable for use in a pharmaceutical context, i.e., salts suitable for use in contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and commensurate with a reasonable benefit/risk ratio, within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, S.M.Berge et al. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts of amino groups formed using inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or using organic acids (such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods used in the art (such as ion exchange). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates and arylsulfonates, as appropriate.

Polycyclic: As used herein, the term "polycyclic" refers to a saturated or unsaturated ring system having two or more rings (e.g., a heterocyclyl ring, a heteroaryl ring, a cycloalkyl ring, or an aryl ring) having 7 to 20 atoms, wherein one or more carbon atoms are shared by two adjacent rings. For example, in some embodiments, a polycyclic ring system refers to a saturated or unsaturated ring system having three or more rings (e.g., a heterocyclyl ring, a heteroaryl ring, a cycloalkyl ring, or an aryl ring) having 14 to 20 atoms, wherein one or more carbon atoms are shared by two adjacent rings. The rings in a polycyclic system may be fused (i.e., bicyclic or tricyclic), spirocyclic, or a combination thereof. An example of a polycyclic ring is a steroid.

Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three or more amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently general so as to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional, biologically active or characteristic fragments, portions or domains of such complete polypeptides (e.g., fragments, portions or domains that retain at least one activity). In some embodiments, the polypeptide may contain L-amino acids, D-amino acids, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise a peptide mimetic).

Prevent or prevention: As used herein, the term "prevent" or "prevention" when used in conjunction with the occurrence of a disease, disorder, and/or condition refers to reducing the risk of developing a disease, disorder, and/or condition and/or delaying the onset of one or more characteristics or symptoms of a disease, disorder, and/or condition. Prevention is considered complete when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

Reference: As used herein, describes a standard or control relative to which comparison is made. For example, in some embodiments, an agent, animal, individual, colony, sample, sequence or value of interest is compared to a reference or control agent, animal, individual, colony, sample, sequence or value. In some embodiments, the reference or control is tested and/or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to the assessment conditions or circumstances. Those skilled in the art will understand when there is sufficient similarity to justify reliance on a particular possible reference or control and/or comparison with a particular possible reference or control is reasonable.

Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications, including but not limited to, for example (a) terminal modifications, such as 5' terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), 3' terminal modifications (e.g., conjugation, reverse linkage, etc.), (b) base modifications, such as replacement with modified bases, stable bases, destabilizing bases, or bases that are base-paired with all components of the amplified partner or conjugated bases, (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars, and (d) internucleoside linkage modifications, including modification or replacement of phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, RNA is single-stranded. In some embodiments, RNA is double-stranded. In some embodiments, RNA comprises both single-stranded and double-stranded portions. In some embodiments, RNA may comprise a main chain structure as described above in the definition of "nucleic acid/polynucleotide". RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, wherein RNA is mRNA. In some embodiments where RNA is mRNA, RNA typically comprises a poly (A) region at its 3' end. In some embodiments where RNA is mRNA, RNA typically comprises a cap structure recognized in the art at its 5' end, for example, for identifying mRNA and connecting it to a ribosome to initiate translation. In some embodiments, RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by an enzymatic synthesis method and/or by a chemical synthesis method).

Sample As used herein, the term "sample" generally refers to an aliquot of a material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell, tissue, or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest may be or include a biological tissue or fluid. In some embodiments, the source of interest may be or include a prepared product produced in a production run. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from the context, the term "sample" refers to a prepared product obtained by processing a primary sample (e.g., by removing one or more components of a primary sample and/or by adding one or more agents to a primary sample).

Substituted or optionally substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens (e.g., At least and means at least ). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. The combination of substituents contemplated by the present invention is preferably those that result in the formation of stable or chemically feasible compounds. The term "stable" as used herein refers to a compound that does not substantially change when subjected to conditions that allow its production, detection, and in certain embodiments, its recovery, purification, and use for one or more purposes provided herein. A group described as "substituted" preferably has 1 to 4 substituents, more preferably 1 or 2 substituents. A group described as "optionally substituted" may be unsubstituted or "substituted" as described above.

Suitable monovalent substituents on the substitutable carbon atoms of the "optionally substituted" group are independently halogen; -(CH ₂ ) _0-4 R ^o ; -(CH ₂ ) _0-4 OR ^o ; -O(CH ₂ ) _0-4 R ^o , -O-(CH ₂ ) _0-4 C(O)OR ^o ; -(CH ₂ ) _0-4 CH(OR ^o ) ₂ ; -(CH ₂ ) _0-4 SR ^o ; -(CH ₂ ) _0-4 Ph, which may be substituted by R ^o ; - ₍ CH ₂ ) _0-4 O(CH ₂ ) _0-1 Ph, which may be substituted by R ^o ; -CH═CHPh, which may be substituted by R ^o ; -(CH ₂ ) _0-4 O(CH ₂ ) _0-1 -pyridyl, which may be substituted by R ^o ; -NO ₂ ; -CN; -N ₃ ; (CH ₂ ) _0–4 N(R ^o ) ₂ ;–(CH ₂ ) _0–4 N(R ^o )C(O)R ^o ;–N(R ^o )C(S) ^{R o} ;–( _{CH 2} ) _0–4 N(R ^o )C(O)NR ^o ₂ ;–(CH ₂ ) _0–4 N(R ^o )C(O)OR ^o ;–N(R ^o )N(R ^o )C(O)R ^o ;N(R ^o )N(R ^o )C(O)NR ^o ₂ ;N(R ^o )N(R ^o )C( ^O )OR ^o ;–(CH ₂ ) _0–4 C(O)R ^o ;C(S)R ^o ;–(CH ₂ ) _0–4 C(O)OR ^o ;–(CH ₂ ) _0–4 C(O)SR ^o ; (CH ₂ ) _0–4 C(O)OSiR ^o ₃ ;–(CH ₂ ) _0–4 OC(O)R ^o ;–OC(O)(CH ₂ ) _{0– 4} SR ^o ;–(CH ₂ ) _0–4 SC(O)R ^o ;–(CH ₂ ) _0–4 C(O)NR ^o ₂ ;–C(S)NR ^o ₂ ;–C(S)SR ^o ;–SC(S)SR ^o , (CH ₂ ) _0–4 OC(O)NR ^o ₂ ;C(O)N(OR ^o )R ^o ;–C(O)C(O)R ^o ;–C(O)CH ₂ C(O)R ^o ;–C(NOR ^o )R ^o ;(CH ₂ ) _0–4 SSR ^o ;-(CH ₂ ) _0–4 S(O) ₂ R ^o ; –(CH ₂ ) _0–4 S(O) ₂ OR ^{o ;} –(CH ₂ ) _0–4 OS(O) ₂ R ^o ; –S(O) ₂ NR ^o ₂ ;(CH ₂ ) _0–4 S(O)R ^o ; N(R ^o )S(O) ₂ NR ^o ₂ ; –N(R ^o )S(O) ₂ R ^o ; –N(OR ^o )R ^o ; –C(NH)NR ^o ₂ ; –P(O) ₂ R ^o ; P(O)R ^o ₂ ; OP(O)R ^o ₂ ; –OP(O)(OR ^o ) ₂ ; SiR ^o ₃ ; –(C _1–4 straight or branched alkylene)O–N(R ^o ) ₂ ; or –(C _1–4 straight or branched alkylene)C(O)O–N(R ^o ) ₂ , wherein each R ^o may be substituted as defined below and is independently hydrogen, C _1-6 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, -CH ₂ -(5- to 6-membered heteroaryl ring) or a 3- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent occurrences of R ^o together with their intervening atoms form a 3- to 12-membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R ^o (or the ring formed by two independent occurrences of R ^o together with their middle atom) are independently halogen, –(CH ₂ ) _0–2 R ^· , –(haloR ^· ), –(CH ₂ ) _0–2 OH, –(CH ₂ ) _0–2 OR ^· , –(CH ₂ ) _0–2 CH(OR ^· ) ₂ , O(haloR ^· ), –CN, –N ₃ , –(CH ₂ ) _0–2 C(O)R ^· , –(CH ₂ ) _0–2 C(O)OH, –(CH ₂ ) _0–2 C(O)OR ^· , –(CH ₂ ) _0–2 SR ^· , –(CH ₂ ) _{0– 2} SH, –(CH ₂ ) _0–2 NH ₂ , –(CH ₂ ) _0–2 NHR ^·. , –(CH ₂ ) _0-2 NR ^· ₂ , –NO ₂ , –SiR ^· ₃ , –OSiR ^· ₃ , C(O)SR ^· , –(C _1-4 straight or branched alkylene)C(O)OR ^· or –SSR ^· , wherein each R ^{· is} unsubstituted or, when preceded by “halo”, substituted only by one or more halogens, and is independently selected from C _1-4 aliphatic, –CH ₂ Ph, –O(CH ₂ ) _0-1 Ph or a 3- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on the saturated carbon atom of R ^o include =O and =S.

Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0 (“oxo”), =S, =NNR ^* ₂ , =NNHC(O)R ^* , =NNHC(O)OR ^* , =NNHS(O) ₂ R ^* , =NR ^* , =NOR ^* , —O(C(R ^* ₂ )) _2-3 O—, or —S(C(R ^* ₂ )) _2-3 S—, wherein each independent occurrence of R ^* is selected from hydrogen, a C _1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bound to an ortho-substitutable carbon of an "optionally substituted" group include: -O(CR ^* ₂ ) _2-3 O-, wherein each independent occurrence of R ^* is selected from hydrogen, a C _1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the aliphatic group of R ^* include halogen, —R ^· , —(haloR ^· ), OH, —OR ^· , —O(haloR ^· ), —CN, —C(O)OH, —C(O)OR ^· , —NH ₂ , —NHR ^· , —NR ^· ₂ or —NO ₂ , wherein each R ^· is unsubstituted or, when preceded by “halo”, substituted only by one or more halogens, and is independently C _1-4 aliphatic, —CH ₂ Ph, —O(CH ₂ ) _0-1 Ph or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include or Each of these is independently hydrogen, a C _1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent occurrences of Together with its middle atoms, it forms an unsubstituted 3- to 12-membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the aliphatic group are independently halogen, —R ^· , (haloR ^· ), —OH, —OR ^· , —O(haloR ^· ), —CN, —C(O)OH, —C(O)OR ^· , —NH ₂ , —NHR ^· , —NR ^· ₂ or NO ₂ , wherein each R ^{· is} unsubstituted or, when preceded by “halo”, substituted only by one or more halogens, and is independently C _1-4 aliphatic, —CH ₂ Ph, —O(CH ₂ ) _0-1 Ph or a 3- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. Typically, a "small molecule" is a molecule of a size less than about 5 kilodaltons (kD). In some embodiments, a small molecule is less than about 4kD, 3kD, about 2kD, or about 1kD. In some embodiments, a small molecule is less than about 800 daltons (D), about 600D, about 500D, about 400D, about 300D, about 200D, or about 100D. In some embodiments, a small molecule is less than about 2000g/mol, less than about 1500g/mol, less than about 1000g/mol, less than about 800g/mol, or less than about 500g/mol. In some embodiments, a small molecule is not a polymer.

A person of ordinary skill in the art will understand upon reading this disclosure that certain small molecule compounds described herein, including, for example, the oligosaccharide compounds described herein, can be provided and/or utilized in any of a variety of forms, such as, for example, crystalline forms (e.g., polymorphs, solvates, etc.), salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and/or structural isomers), isotopic forms, etc.

Those of ordinary skill in the art will appreciate that certain small molecule compounds (e.g., oligosaccharide compounds described herein) have structures that can exist in one or more stereoisomeric forms. In some embodiments, such small molecules can be utilized in the form of individual enantiomers, diastereomers, or geometric isomers according to the present disclosure, or can be in the form of a mixture of stereoisomers; in some embodiments, such small molecules can be utilized in the form of a racemic mixture according to the present disclosure.

Those skilled in the art will appreciate that certain small molecule compounds (e.g., oligosaccharide compounds described herein) have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules can be utilized according to the present disclosure in the form of individual tautomers or in a form that is mutually converted between tautomeric forms.

Those skilled in the art will appreciate that certain small molecule compounds (e.g., the oligosaccharide compounds described herein) have structures that allow isotopic substitution (e.g., ² H or ³ H for H; ¹¹ C, ¹³ C or ¹⁴ C for ¹² C; ¹³ N or ¹⁵ N for ¹⁴ N; ¹⁷ O or ¹⁸ O for ¹⁶ O; ³⁶ Cl for ³⁵ Cl or ³⁷ Cl; ¹⁸ F for ¹⁹ F; ¹³¹ I for ¹²⁷ I; etc.). In some embodiments, such small molecules may be utilized in accordance with the present disclosure in one or more isotopically modified forms or mixtures thereof.

In some embodiments, reference to a particular small molecule compound (e.g., an oligosaccharide compound described herein) may refer to a specific form of the compound. In some embodiments, a particular small molecule compound may be provided and/or utilized in a salt form (e.g., an acid addition salt or a base addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

In some embodiments, when a small molecule compound is a compound that exists or is found in nature, the compound may be provided and/or utilized in accordance with the present disclosure in a form different from that in which it exists or is found in nature. One of ordinary skill in the art will appreciate that in some embodiments, a preparation of a particular small molecule compound (e.g., an oligosaccharide compound described herein) is different from the compound as it exists in a reference preparation or source when it contains an absolute or relative amount of the compound or a particular form thereof that is different from the absolute or relative (relative to another component of the preparation, including, for example, another form of the compound) amount present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source). Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound from a racemic mixture of the compound; a specific salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation of a compound form containing only one conformer ((Z) or (E)) containing a double bond may be considered to be a different form of the compound from another conformer ((E) or (Z)) containing a double bond; a preparation in which one or more atoms are isotopes different from the isotopes present in a reference preparation may be considered to be a different form; etc.

Those skilled in the art will also understand that in small molecule structures, as used herein, the symbol It refers to the connection point between two atoms.

Therapeutic agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that causes a desired pharmacological effect when applied to an organism. In some embodiments, an agent is considered a therapeutic agent if it shows a statistically significant effect throughout an appropriate population. In some embodiments, an appropriate population may be a model organism population. In some embodiments, an appropriate population may be defined according to various criteria, such as a certain age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent one or more symptoms or features of a disease, disorder, and/or illness, delay its onset, reduce its severity, and/or reduce its incidence. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a government agency before it can be marketed for application to a person. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription to be applied to a person.

Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to subjects who do not show signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to subjects who show only early signs of a disease, disorder, and/or condition, for example, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and/or condition.

Polycationic oligosaccharide compounds

The present disclosure provides, inter alia, compositions comprising a compound that is an oligosaccharide of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

A is ^A1 , ^A2 or ^A3 :

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , and wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and

p is an integer selected from 1, 2, 3, 4 or 5;

The premise is that when A is A ¹ , then:

(i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(ii) each of R ¹ and R ² is not –(CH ₂ ) ₅ -CH ₃ or –(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and

(iv) Cy is not a triazole group.

As used herein, A is A ¹ , A ² or A ³ :

In some embodiments, A is ^A1 or ^A2 . In some embodiments, A is ^A1 . In some embodiments, A is ^A2 . In some embodiments, A is ^A3 . In some embodiments, A is ^A1 and the oligosaccharide is a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, the compound is an oligosaccharide of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , and wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

the Prerequisite is:

(i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(ii) each of R ¹ and R ² is not –(CH ₂ ) ₅ -CH ₃ or –(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and

(iv) Cy is not a triazole group.

In some embodiments, A is A ² and the oligosaccharide has Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, the compound is an oligosaccharide having Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , and wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S.

In some embodiments, A is A ³ and the oligosaccharide has Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein p, R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, the oligosaccharide is a compound of Formula Ic:

or a pharmaceutically acceptable salt thereof, wherein

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , and wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and

p is 1, 2, 3, 4, or 5.

In some embodiments, p is 1, and the oligosaccharide has formula Ic-i:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, p is 2, and the oligosaccharide has formula Ic-ii:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, p is 3, and the oligosaccharide has formula Ic-iii:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, p is 4, and the oligosaccharide has formula Ic-iv:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

In some embodiments, p is 5, and the oligosaccharide has Formula Ic-v:

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , X ¹ , X ² , Y ¹ , Y ² , Z ¹ , and Z ² are as described in classes and subclasses herein, individually or in combination.

As generally described herein, R ¹ and R ² are each independently selected at each occurrence from H, ^Ra , and -C(O) ^-Ra , and wherein at least one occurrence of R ¹ or R ² is not H. In some embodiments, R ¹ and R ² are each independently selected at each occurrence from ^Ra and -C(O) ^-Ra . In some embodiments, R ¹ and R ² are each ^Ra . In some embodiments, R ¹ and R ² are each ^Ra , and each ^{Ra is} independently selected from C ₁ -C ₂₀ aliphatic, C ₃ -C ₂₀ alicyclic, C ₅ -C ₆ aryl, 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S, wherein each ^Ra is optionally substituted with one or more ^R ;

In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₁ -C ₂₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₁ -C ₁₄ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₁ -C ₁₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₅ -C ₁₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₅ -C ₁₀ alkyl optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently C ₅ -C ₁₀ linear alkyl. In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl. In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently selected from

In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₃ -C ₂₀ alicyclic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently a C ₃ -C ₆ alicyclic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is independently C ₅ -C ₆ aryl optionally substituted with one or more R ^b . In some embodiments, R ¹ and R ² are each ^Ra , and each ^Ra is phenyl.

In some embodiments, ^R and ^R are each ^Ra , and each ^Ra is independently 3 to 12 membered heterocyclyl containing 1 to 3 ^heteroatoms selected from N, O and S, optionally substituted with one or more R. In some embodiments, ^R and R are each ^Ra , and each ^Ra is independently 3 to ⁶ membered heterocyclyl containing 1 to 3 ^heteroatoms selected from N, O and S, optionally substituted with one or more R.

In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^{a is} independently selected from C ₁ -C ₂₀ aliphatic, C ₃ -C ₂₀ alicyclic, C ₅ -C ₆ aryl, 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each R ^a is optionally substituted by one or more R ^b ;

In some embodiments, R ¹ and R ² are each -C (O) -R ^a . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₁ -C ₂₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₁ -C ₁₄ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₁ -C ₁₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₅ -C ₁₀ aliphatic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently a C ₅ -C ₁₀ alkyl group optionally substituted with one or more R ^b . In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently a C ₅ -C ₁₀ linear alkyl group. In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl or n-nonyl. In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently selected from

In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₃ -C ₂₀ alicyclic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a C ₃ -C ₆ alicyclic optionally substituted by one or more R ^b . In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, R ¹ and R ² are each -C (O) -R ^a , and each R ^a is independently a polycyclic C ₁₀ -C ₂₀ alicyclic optionally substituted by one or more R ^b . In some embodiments, one or more of R ¹ or R ² are:

In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently C ₅ -C ₆ aryl optionally substituted with one or more R ^b . In some embodiments, R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently phenyl.

In some embodiments, ^R and R are each -C(O) ^-R , and each R ^is independently 3 to ¹² membered heterocyclyl containing 1 to 3 ^heteroatoms selected from N, O and S, optionally substituted with one or more R. In some embodiments, R and R are each -C(O) ^-R , and each R ^is independently ³ to ⁶ membered heterocyclyl containing 1 to 3 ^heteroatoms selected from N, O and S, optionally substituted with one or more R.

In some embodiments, ^R1 and ^R2 are each independently selected from:

As described herein, each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c , and -NHC(O)NHR ^c . In some embodiments, R ^b is hydrogen. In some embodiments, R ^b is -N ₃ . In some embodiments, R ^b is R ^c . In some embodiments, R ^b is -OR ^c . In some embodiments, R ^b is -SR ^c . In some embodiments, R ^b is -NHR ^c . In some embodiments, R ^b is -C(O)-R ^c . In some embodiments, R ^b is -OC(O)R ^c . In some embodiments, R ^b is -NHC(O)R ^c . In some embodiments, R ^b is -C(O)NHR ^c . In some embodiments, R ^b is -NHC(O)NHR ^c .

As described herein, each R ^c is independently selected from C ₁ -C ₂₀ aliphatic, C ₃ -C ₂₀ alicyclic, C ₅ -C ₆ aryl, 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S. In some embodiments, R ^c is an optionally substituted C ₁ -C ₂₀ aliphatic. In some embodiments, R ^c is an optionally substituted C ₃ -C ₂₀ alicyclic. In some embodiments, R ^c is an optionally substituted C ₅ -C ₆ aryl. In some embodiments, R ^c is a 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S. In some embodiments, R ^c is a 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S.

As described herein, each of ^Xi and ^X2 is independently selected from -S- and -NH-. In some embodiments, ^Xi is -S-. In some embodiments, Xi is -NH-. In some embodiments, ^X2 is -S-. In some embodiments, ^X2 is -NH-. In some embodiments, each of ^Xi and ^X2 is -S-. In some embodiments, each of Xi and ^X2 is -NH-. In some embodiments, ^Xi is ^-S- and ^X2 is -NH-. In some embodiments ^{, Xi} is -NH- and ^X2 is -S-.

As described herein, Y ¹ and Y ² are each independently an optionally substituted C _1-30 aliphatic group, wherein one or more carbons of the Y ¹ and/or Y ² groups are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO ₂ -, -SO ₂ NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -SO- or -SO ₂ -. As described herein, it is understood that Y ¹ and Y ² are divalent moieties, one end of which is bonded to the X ¹ or X ² group, and the other end is bonded to the Z ¹ or Z ² group.

In some embodiments, ^Y1 and ^Y2 are each independently selected from _C1 - _C10 aliphatic, _C0 - _C4 -aliphatic-NHC(O)NH- _C0 - _C4 aliphatic, C0- _C4 -aliphatic-NHC(S)NH- _C0 -C4 aliphatic, C0-C4-aliphatic-C(O)NH- _C0 - _C4 aliphatic, _C0 - _C4 -aliphatic-C(S)NH- _C0 - _C4 aliphatic, _C0 - _C4 -aliphatic-NHC(O)NH- _{C0-C4 aliphatic, C0-C4-aliphatic-C(S)NH-C0 - C4 aliphatic ,} C0-C4-aliphatic-NHC(O)-C0 _-C4 aliphatic, _C0 - _C4 -aliphatic- _NHSO2 - _C0 - _C4 aliphatic, and _C0 - _C4 -aliphatic-C(O) _-C0 - _C4 aliphatic.

In some embodiments, Y ¹ and Y ² are each C ₁ -C ₁₀ aliphatic. In some embodiments, Y ¹ and Y ² are each methylene, ethylene, propylene, or butylene. In some embodiments, Y ¹ and Y ² are each -CH ₂ -CH ₂ -.

In some embodiments, Y ¹ and Y ² are each C ₀ -C ₄ -aliphatic -NHC(O)NH-C ₀ -C ₄ -aliphatic. In some embodiments, Y ¹ and Y ² are each C ₁ -C ₄ -aliphatic -NHC(O)NH-C ₁ -C ₄ -aliphatic. In some embodiments, Y ¹ and Y ² are each -CH ₂ -CH ₂ -NHC(O)NH-CH ₂ -CH ₂ -.

In some embodiments, Y ¹ and Y ² are each C ₀ -C ₄ -aliphatic-NHC(S)NH-C ₀ -C ₄ -aliphatic. In some embodiments, Y ¹ and Y ² are each C ₁ -C ₄ -aliphatic-NHC(S)NH-C ₁ -C ₄ -aliphatic. In some embodiments, Y ¹ and Y ² are each -CH ₂ -CH ₂ -NHC(S)NH-CH ₂ -CH ₂ -.

In some embodiments, ^Y1 and ^Y2 are each C ₀ -C ₄ -aliphatic -C(O)NH-C ₀ -C ₄ -aliphatic. In some embodiments, ^Y1 and ^Y2 are each C ₁ -C ₄ -aliphatic -C(O)NH-C ₁ -C ₄ -aliphatic. In some embodiments, ^Y1 and ^Y2 are each -CH ₂ -CH ₂ -C(O)NH-CH ₂ -CH ₂ -.

In some embodiments, ^Y1 and ^Y2 are each C ₀ -C ₄ -aliphatic-NHC(O)-C ₀ -C ₄ -aliphatic. In some embodiments, ^Y1 and ^Y2 are each C ₁ -C ₄ -aliphatic-NHC(O)-C ₁ -C ₄ -aliphatic. In some embodiments, ^Y1 and ^Y2 are each -CH ₂ -CH ₂ -NHC(O)-CH ₂ -CH ₂ -.

In some embodiments, ^Y1 and ^Y2 are each C ₀ -C ₄ -aliphatic-NHSO ₂ -C ₀ -C ₄ aliphatic. In some embodiments, ^Y1 and ^Y2 are each C ₁ -C ₄ -aliphatic-NHSO ₂ -C ₁ -C ₄ aliphatic. In some embodiments, ^Y1 and ^Y2 are each -CH ₂ -CH ₂ -NHSO ₂ -CH ₂ -CH ₂ -.

In some embodiments, ^Y is selected from C ₁ -C ₁₀ aliphatic, C ₀ -C ₄ -aliphatic-NHC(O)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-NHC(S)NH-C ₀ -C ₄ aliphatic, C 0 -C 4 -aliphatic-C(O)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-C(S)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-C(O)NH-C ₀ -C ₄ aliphatic, C 0 -C 4 -aliphatic-C(O)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-NHSO ₂ -C ₀ -C ₄ aliphatic, and C ₀ -C ₄ -aliphatic-C(O)-C ₀ -C ₄ aliphatic.

In some embodiments, Y ¹ is C ₁ -C ₁₀ aliphatic. In some embodiments, Y ¹ is methylene, ethylene, propylene, or butylene. In some embodiments, Y ¹ is -CH ₂ -CH ₂ -.

In some embodiments, Y ¹ is C ₀ -C ₄ -aliphatic-NHC(O)NH-C ₀ -C ₄ aliphatic. In some embodiments, Y ¹ is C ₁ -C ₄ -aliphatic-NHC(O)NH-C ₁ -C ₄ aliphatic. In some embodiments, Y ¹ is —CH ₂ —CH ₂ —NHC(O)NH—CH ₂ —CH ₂ —.

In some embodiments, Y ¹ is C ₀ -C ₄ -aliphatic-NHC(S)NH-C ₀ -C ₄ aliphatic. In some embodiments, Y ¹ is C ₁ -C ₄ -aliphatic-NHC(S)NH-C ₁ -C ₄ aliphatic. In some embodiments, Y ¹ is —CH ₂ —CH ₂ —NHC(S)NH—CH ₂ —CH ₂ —.

In some embodiments, Y ¹ is C ₀ -C ₄ -aliphatic-C(O)NH-C ₀ -C ₄ -aliphatic. In some embodiments, Y ¹ is C ₁ -C ₄ -aliphatic-C(O)NH-C ₁ -C ₄ -aliphatic. In some embodiments, Y ¹ is —CH ₂ —CH ₂ —C(O)NH—CH ₂ —CH ₂ —.

In some embodiments, Y ¹ is C ₀ -C ₄ -aliphatic-NHC(O)-C ₀ -C ₄ aliphatic. In some embodiments, Y ¹ is C ₁ -C ₄ -aliphatic-NHC(O)-C ₁ -C ₄ aliphatic. In some embodiments, Y ¹ is —CH ₂ —CH ₂ —NHC(O)-CH ₂ —CH ₂ —.

In some embodiments, Y ¹ is C ₀ -C ₄ -aliphatic-NHSO ₂ -C ₀ -C ₄ aliphatic. In some embodiments, Y ¹ is C ₁ -C ₄ -aliphatic-NHSO ₂ -C ₁ -C ₄ aliphatic. In some embodiments, Y ¹ is —CH ₂ —CH ₂ —NHSO ₂ —CH ₂ —CH ₂ —.

In some embodiments, ^Y2 is selected from _C1 - _C10 aliphatic, _C0 - _C4 -aliphatic-NHC(O)NH- _C0 - _C4 aliphatic, C0- _C4 -aliphatic-NHC(S)NH- _C0 -C4 aliphatic, C0 _{-C4 -} aliphatic _- C(O)NH- _C0 - _C4 aliphatic, C0- _{C4-aliphatic-C(S)NH-C0-C4 aliphatic ,} C0-C4-aliphatic-C(O)NH- _C0 -C4 aliphatic, _C0 - _C4 -aliphatic-C(O)NH- _C0 - _C4 aliphatic, _{C0 - C4} -aliphatic- _NHSO2 - _C0 - _C4 aliphatic, and _C0 - _C4 -aliphatic-C(O) _-C0 - _C4 aliphatic.

In some embodiments, Y ² is C ₁ -C ₁₀ aliphatic. In some embodiments, Y ² is methylene, ethylene, propylene, or butylene. In some embodiments, Y ² is -CH ₂ -CH ₂ -.

In some embodiments, Y ² is C ₀ -C ₄ -aliphatic-NHC(O)NH-C ₀ -C ₄ aliphatic. In some embodiments, Y ² is C ₁ -C ₄ -aliphatic-NHC(O)NH-C ₁ -C ₄ aliphatic. In some embodiments, Y ² is -CH ₂ -CH ₂ -NHC(O)NH-CH ₂ -CH ₂ -.

In some embodiments, Y ² is C ₀ -C ₄ -aliphatic-NHC(S)NH-C ₀ -C ₄ aliphatic. In some embodiments, Y ² is C ₁ -C ₄ -aliphatic-NHC(S)NH-C ₁ -C ₄ aliphatic. In some embodiments, Y ² is —CH ₂ —CH ₂ —NHC(S)NH-CH ₂ —CH ₂ —.

In some embodiments, Y ² is C ₀ -C ₄ -aliphatic-C(O)NH-C ₀ -C ₄ aliphatic. In some embodiments, Y ² is C ₁ -C ₄ -aliphatic-C(O)NH-C ₁ -C ₄ aliphatic. In some embodiments, Y ² is —CH ₂ —CH ₂ —C(O)NH—CH ₂ —CH ₂ —.

In some embodiments, Y ² is C ₀ -C ₄ -aliphatic-NHC(O)-C ₀ -C ₄ aliphatic. In some embodiments, Y ² is C ₁ -C ₄ -aliphatic-NHC(O)-C ₁ -C ₄ aliphatic. In some embodiments, Y ² is —CH ₂ —CH ₂ —NHC(O)-CH ₂ —CH ₂ —.

In some embodiments, Y ² is C ₀ -C ₄ -aliphatic-NHSO ₂ -C ₀ -C ₄ aliphatic. In some embodiments, Y ² is C ₁ -C ₄ -aliphatic-NHSO ₂ -C ₁ -C ₄ aliphatic. In some embodiments, Y ² is —CH ₂ —CH ₂ —NHSO ₂ —CH ₂ —CH ₂ —.

As generally described herein, each Cy is independently an optionally substituted C ₃ -C ₁₄ cycloaliphatic, a 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and a 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

In some embodiments, the moiety X ¹ -Y ¹ -Z ¹ is:

In some embodiments, the moiety X ² -Y ² -Z ² is:

As generally described herein, ^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

In some embodiments, Z ¹ and Z ² are each independently selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

In some embodiments, Z ¹ and Z ² are each an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ and Z ² are each an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ and Z ² are each an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ and Z ² are each selected from optionally substituted piperidinyl, piperazinyl, and morpholinyl.

In some embodiments, Z ¹ and Z ² are each an optionally substituted 5- to 14-membered heteroaryl ring having 1-3 heteroatoms selected from N, O, and S.

In some embodiments, Z ¹ and Z ² are each -N ⁺ (M) ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ (C ₀ -C ₆ aliphatic -R ^z ) ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ (R ^z ) ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ H ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ H(R ^z ) ₂ . In some embodiments, Z ¹ and Z ² are each -N ⁺ H(C ₁ -C ₆ aliphatic) ₂ . In some embodiments, Z ¹ and Z ² are each -N ⁺ (H) ₂ CH ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ H(CH ₃ ) ₂ . In some embodiments, Z ¹ and Z ² are each -N ⁺ (CH ₃ ) ₃ .

In some embodiments, Z ¹ and Z ² are each -N ⁺ (R ^z ) ₂ -C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, Z ¹ and Z ² are each -N ⁺ (R ^z )(C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ) ₂ . In some embodiments, Z ¹ and Z ² are each:

In some embodiments, ^Z1 and ^Z2 are each:

In some embodiments, ^Z1 and ^Z2 are each:

In some embodiments, ^Z1 and ^Z2 are each:

In some embodiments, ^Z1 and ^Z2 are each:

In some embodiments, ^Z1 and ^Z2 are each:

In some embodiments, Z ¹ is an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ is an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ is an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, Z ¹ is selected from optionally substituted piperidinyl, piperazinyl, and morpholinyl.

In some embodiments, Z ¹ is -N ⁺ (M) ₃ . In some embodiments, Z ¹ is -N ⁺ (C ₀ -C ₆ aliphatic -R ^z ) ₃ . In some embodiments, Z ¹ is -N ⁺ (R ^z ) ₃ . In some embodiments, Z ¹ is -N ⁺ H ₃ . In some embodiments, Z ¹ is -N ⁺ H(R ^z ) ₂ . In some embodiments, Z ¹ is -N ⁺ H(C ₁ -C ₆ aliphatic) ₂ . In some embodiments, Z ¹ is -N ⁺ (H) ₂ CH ₃ . In some embodiments, Z ¹ is -N ⁺ H(CH ₃ ) ₂ . In some embodiments, Z ¹ is -N ⁺ (CH ₃ ) ₃ .

In some embodiments, Z ¹ is —N ⁺ (R ^z ) ₂ —C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, Z ¹ is —N ⁺ (R ^z )(C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ) ₂ . In some embodiments, Z ¹ is:

In some embodiments, Z ¹ is:

In some embodiments, Z ¹ is:

In some embodiments, Z ¹ is:

In some embodiments, ^Z is

In some embodiments, ^Z is

In some embodiments, ^Z is an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, ^Z is an optionally substituted 5- to 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, ^Z is an optionally substituted 6-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O, and S. In some embodiments, ^Z is selected from optionally substituted piperidinyl, piperazinyl, and morpholinyl.

In some embodiments, Z ² is -N ⁺ (M) ₃ . In some embodiments, Z ² is -N ⁺ (C ₀ -C ₆ aliphatic -R ^z ) ₃ . In some embodiments, Z ² is -N ⁺ (R ^z ) ₃ . In some embodiments, Z ² is -N ⁺ H ₃ . In some embodiments, Z ² is -N ⁺ H(R ^z ) ₂ . In some embodiments, Z ² is -N ⁺ H(C ₁ -C ₆ aliphatic) ₂ . In some embodiments, Z ² is -N ⁺ (H) ₂ CH ₃ . In some embodiments, Z ² is -N ⁺ H(CH ₃ ) ₂ . In some embodiments, Z ² is -N ⁺ (CH ₃ ) ₃ .

In some embodiments, Z ² is -N ⁺ (R ^z ) ₂ -C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, Z ² is -N ⁺ (R ^z )(C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ) ₂ . In some embodiments, Z ² is:

In some embodiments, ^Z2 is:

In some embodiments, ^Z2 is:

In some embodiments, ^Z2 is:

In some embodiments, ^Z2 is

In some embodiments, ^Z2 is

As generally described herein, each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, each M is C ₀ -C ₆ aliphatic-R ^z . In some embodiments, each M is -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, each M is N ⁺ (R ^z ) ₃ . In some embodiments, each M is -C ₁ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ . In some embodiments, each M is N ⁺ (R ^z ) ₃ and each R ^z is H or C ₁ -C ₆ aliphatic. In some embodiments, each M is N ⁺ (R ^z ) ₃ and two or more R ^z are taken together with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl comprising 1 to 3 heteroatoms selected from N, O and S.

As generally described herein, each R ^is independently selected from H, an optionally substituted _C1 - _C6 aliphatic, an optionally substituted _C3 - _C20 alicyclic, an optionally substituted _C5 - _C6 aryl, an optionally substituted 3- to 12-membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S, and an optionally substituted 4- to 12-membered heteroaryl containing 1 to 3 heteroatoms selected from N, O, and S; or two or more ^R may be taken together with the atoms to which they are attached to form an optionally substituted 3- to 12-membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S or an optionally substituted 4- to 12-membered heteroaryl containing 1 to 3 heteroatoms selected from N, O, and S.

In some embodiments, each R ^z is H. In some embodiments, each R ^z is an optionally substituted C ₁ -C ₆ aliphatic. In some embodiments, each R ^z is an optionally substituted C ₃ -C ₂₀ alicyclic. In some embodiments, each R ^z is an optionally substituted C ₅ -C ₆ aryl. In some embodiments, each R ^z is a 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S. In some embodiments, each R ^z is a 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S.

In some embodiments, two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O, and S.

In some embodiments, R ^z on the Z ¹ moiety and R ^z on the Z ² moiety can be taken together to form a 4- to 6-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, and S.

As used herein, a "cationic" moiety is a group with a net positive charge. As used herein, an "ionizable" moiety is a group that may have a neutral charge at certain pH but may be charged (e.g., a cation) at different pH. For example, in some embodiments, the ionizable moiety becomes a cation (i.e., positively charged) at physiological pH (e.g., about pH 7.4). In addition, in some embodiments, Z ¹ and Z ² moieties (which are cations or ionizable moieties described herein) also include one or more suitable counterions (e.g., anions of each cation or ionizable moiety). For example, in some embodiments, Z ¹ and Z ² moieties include counterions, which are halogen ions, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In some embodiments, Z ¹ and Z ² moieties include counterions as acetate (CH 3 COO-), chloride (Cl-), iodide (I-), bromide (Br-) or fluoride (F-). Reference to specific counterions (eg, as shown in Table 1) is intended to encompass isolated charged moieties, as well as all chemically feasible counterions.

In some embodiments, ^Z1 and ^Z2 are each independently selected from:

In some embodiments, ^Z1 and ^Z2 are each

As described herein, with respect to Formula I, when A is ^A1 , then (i) each of ^R1 and ^R2 is not –CO-( _CH2 ) ₄ - _CH3 , ^X1 and ^X2 are not both –S-, ^Y1 and ^Y2 are not both –( _CH2 ) _2- , and ^Z1 and ^Z2 are not both –N ⁺ (H) ₃ ; (ii) each of ^R1 and ^R2 is not –( _CH2 ) ₅ - _CH3 or –( _CH2 ) _{13-CH3, X1 and X2 are not both –S-, Y1 and Y2 are not both –(CH2)2-, and Z1 and Z2 are not both –N+(H)3; (iii) each of R1 and R2 is not –CO-(CH2)4} ^{- CH3 , X1} _{and X2} ^{are not} _both ^{–S- ,} _{Y1 and} ^{Y2 are} not _both –(CH2)2-, and ^Z1 and Z2 are not both –N ⁺ (H) ₃ . ₍ ^iv ₎ ^{Cy is not} _{triazolyl . ​}

In some embodiments, the compound of Formula I is a compound of Formula Id:

or a pharmaceutically acceptable salt thereof, wherein n and m are each independently selected from 0, 1, 2, 3, 4, 5 or 6, and ^Ra , ^Y1 , ^Y2 , ^Z1 and ^Z2, individually or in combination, are as described in classes and subclasses herein.

In some embodiments, the oligosaccharide is a compound of Formula Id:

or a pharmaceutically acceptable salt thereof, wherein

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and

n and m are each independently selected from 0, 1, 2, 3, 4, 5 or 6;

the Prerequisite is:

(i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(ii) each of R ¹ and R ² is not –(CH ₂ ) ₅ -CH ₃ or –(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and

(iv) Cy is not a triazole group.

In some embodiments, the compound of Formula Id is a compound of Formula Id-i:

or a pharmaceutically acceptable salt thereof, wherein ^Ra , ^Z1 , and ^Z2, individually or in combination, are as described in classes and subclasses herein.

In some embodiments, the present disclosure provides compounds of Table 1.

Table 1

In some embodiments, provided compounds are provided and/or utilized in salt form (e.g., pharmaceutically acceptable salt form). Unless otherwise indicated, reference to a compound provided herein should be understood to include reference to a salt thereof. In addition, reference to a particular salt herein (e.g., a salt in Table 1) is also intended to include reference to the free base form of the compound, as long as such compound is chemically feasible.

It should be understood that while the exemplary compounds in Table 1 are represented as specific salts (e.g., chloride salts, iodide salts, etc.) containing counterions (e.g., ^Cl- , ^I-, etc.), those skilled in the art will appreciate that any suitable counterion can be used in conjunction with the cationic nitrogen groups in Table 1. Thus, Table 1 is intended to encompass any positively charged nitrogen group coupled to any chemically feasible counterion. Thus, the specific counterions provided above are provided by way of example only and are not intended to be limiting.

In some embodiments, the present disclosure provides a composition comprising a compound selected from the group consisting of:

In some embodiments, the compositions described herein do not comprise JRL13 or JRL45.

Compounds and compositions

The compounds described herein exhibit unexpectedly improved characteristics when incorporated into complexes comprising RNA, particularly relative to previously described complexes. In addition, the present disclosure encompasses the insight that cationic and/or ionizable oligosaccharides are surprisingly useful for the delivery and targeted expression of specific therapeutic agents (e.g., nucleic acids, RNA).

Previous studies related to cationic oligosaccharides have focused on their application to DNA. In particular, Carbajo-Gordillo et al. reported the use of certain oligosaccharides for DNA nanocomplexes and delivery. See Carbajo-Gordillo et al., Chem. Comm., 55: 8227-8230 (2019). However, it is worth noting that the oligosaccharide-DNA complex reported by Carbajo-Gordillo et al. shows surprising different properties from the complex reported in this application, especially when compounded with RNA. For example, in some embodiments, the compositions reported herein show different behaviors in vivo, such as targeting (i.e., causing an increase in RNA expression relative to other parts of the body) different systems in the body, and can be prepared with a significantly reduced oligosaccharide to nucleic acid ratio. At least for these reasons, the complex reported at present shows unexpected benefits compared to those previously reported.

For example, and by comparison only, and as described in detail in Example 2, pDNA complexed with JRL13 provided a complex that was primarily targeted to the liver. See Figure 3G. In contrast, JRL13 complexed with mRNA provided a complex that was primarily targeted to the lungs. See Figure 3E. Similarly, pDNA complexed with JRL45 provided a complex that was primarily expressed in the lungs. See Figure 3G. In contrast, JRL45 complexed with mRNA targeted the spleen, with very little complex expressed in the lungs. See Figure 3E. These results were surprising. There was no indication in previous studies that a particular oligosaccharide complexed with DNA would provide a composition that targeted one system, while the same oligosaccharide complexed with RNA would provide a complex that targeted a different system.

In addition, the composition reported at present shows an improved ratio of a cationic part or a group (on oligosaccharide) that can be ionized as a cationic group to an anionic part (on nucleic acid). For example, as described herein, the complex comprises a molar ratio of a cationic group to an anionic group, which is referred to as an "N/P ratio". As described herein, the N/P ratio refers to a molar ratio of a cationic group (or an ionizable group that can become a cation, referred to as "N" of N/P) in a composition comprising a cationic oligosaccharide compound relative to an anionic group (referred to as "P" of N/P) in a composition comprising mRNA. Relative to previously reported DNA complexes, the composition reported at present shows a favorable ratio, thereby providing a concentrated complex for RNA delivery. For example, the complex of Carbajo-Gordillo requires an N/P of about 20:1 to effectively target and deliver pDNA. In contrast, the complex of the present invention comprises an N/P ratio of less than 20:1. The N/P ratio can also be expressed as a single digit, such as 5, where it is understood that the indicated number is referred to as a ratio of X:1. For example, N/P is 5 and is intended to refer to N/P as 5:1. Similarly, N/P of 10 is intended to refer to N/P of 10:1, etc.

For example, in some embodiments, the N/P ratio of the complexes reported herein is less than 20:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 15:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 12:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 10:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 5:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 2:1. In some embodiments, the N/P ratio of the complexes reported herein is less than or equal to 1:1. In some embodiments, the N/P ratio of the complexes reported herein is about 1:1 to about 15:1. In some embodiments, the N/P ratio of the complexes reported herein is about 1:1 to about 12:1. In some embodiments, the N/P ratio of the complexes reported herein is about 1:1 to about 10:1. In some embodiments, the N/P ratio of the complex reported herein is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, or about 19:1.

In some embodiments, the complexes described herein have a diameter of about 30nm to about 300nm. In some embodiments, the complexes described herein have a diameter of about 50nm to about 200nm. In some embodiments, the complexes described herein have a diameter of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200nm. In some embodiments, the complexes described herein have a diameter of about 30nm to about 150nm. In some embodiments, the complexes described herein have a diameter of about 30nm to about 100nm. In some embodiments, the complexes described herein have a diameter of less than 100nm. In some embodiments, the complexes described herein have a diameter of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100nm.

In some embodiments, the compositions or complexes described herein further comprise a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is selected from polysorbates (e.g., polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80)), poloxamer, and amphiphilic groups comprising a portion selected from the following items: polyalkylene glycols (e.g., polyethylene glycol), poly (2-oxazoline), poly (2-methyl-2-oxazoline), polysarcosine, polyvinyl pyrrolidone, and poly [N- (2-hydroxypropyl) methacrylamide, wherein the portion is combined with one or more C ₁₂ -C ₂₀ aliphatic groups.

In some embodiments, the molar ratio of oligosaccharide to surfactant is about 1:0.0075 to about 1:3. In some embodiments, the molar ratio of oligosaccharide to surfactant is about 1:0.0075 to about 1:1.5. In some embodiments, the molar ratio of oligosaccharide to surfactant is about 1:1. In some embodiments, the molar ratio of oligosaccharide to surfactant is about 1:0.0075, about 1:0.01, about 1:0.1, about 1:0.5, about 1:1, about 1:1.5, about 1:2, about 1:2.5, or about 1:3.

In some embodiments, the surfactant is a polysorbate. In some embodiments, the molar ratio of oligosaccharides to polysorbates is 1:0.0075 to 1:3. In some embodiments, the surfactant is a polysorbate. In some embodiments, the molar ratio of oligosaccharides to polysorbates is 1:0.0075 to 1:1.5. In some embodiments, the surfactant is a polysorbate. In some embodiments, the molar ratio of oligosaccharides to polysorbates is 1:0.0075 to 1:1. In some embodiments, the molar ratio of oligosaccharides to polysorbates is about 1:0.0075, about 1:0.01, about 1:0.1, about 1:0.5, about 1:1, about 1:1.5, about 1:2, about 1:2.5 or about 1:3.

In some embodiments, the complex comprising an oligosaccharide and a pharmaceutically acceptable surfactant has a diameter of about 30 nm to about 150 nm. In some embodiments, the complex described herein has a diameter of about 30 nm to about 100 nm. In some embodiments, the complex comprising an oligosaccharide and a pharmaceutically acceptable surfactant has a diameter of less than 100 nm. In some embodiments, the complex comprising an oligosaccharide and a pharmaceutically acceptable surfactant has a diameter of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm.

RNA

In some embodiments, the complexes described herein comprise one or more oligosaccharide compositions and a nucleic acid. In some embodiments, the nucleic acid is RNA.

In some embodiments, RNA suitable for the technology described herein is single-stranded RNA. In some embodiments, RNA as disclosed herein is linear RNA. In some embodiments, single-stranded RNA is non-coding RNA because its nucleotide sequence does not include an open reading frame (or its complementary sequence). In some embodiments, single-stranded RNA has a nucleotide sequence encoding one or more polypeptides (e.g., epitopes) of the present disclosure (or a complementary sequence of a sequence encoding the polypeptide).

In some embodiments, the RNA is or includes siRNA, miRNA, or other non-coding RNA.

In many embodiments, the related RNA includes at least one open reading frame (ORF) (eg, is an mRNA); in some embodiments, the related RNA includes a single ORF; in some embodiments, the related RNA includes more than one ORF.

In some embodiments, RNA comprises ORF, for example, ORF encoding one or more polypeptides of interest. In some embodiments, RNA produced according to the technology provided herein comprises multiple ORFs (for example, ORFs encoding multiple polypeptides). In some embodiments, RNA produced according to the technology herein comprises a single ORF encoding multiple polypeptides. In some such embodiments, polypeptide is or comprises an antigen or its epitope (for example, a related antigen).

In some embodiments, the ORF used in accordance with the present disclosure encodes a polypeptide that includes a signal sequence (e.g., a signal sequence that is functional in mammalian cells, such as an intrinsic signal sequence or a heterologous signal sequence). In some embodiments, the signal sequence directs secretion of the encoded polypeptide, and in some embodiments, the signal sequence directs trafficking of the encoded polypeptide to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosomal-lysosomal compartment.

In some embodiments, the ORF encodes a polypeptide comprising a multimerization element (e.g., an intrinsic multimerization element or a heterologous multimerization element). In some embodiments, an ORF encoding a surface polypeptide (e.g., comprising a signal sequence directing surface localization) comprises a multimerization element.

In some embodiments, the ORF encodes a polypeptide comprising a transmembrane element or domain.

In some embodiments, the ORF is codon-optimized for expression in cells of a particular host (eg, a mammalian host such as a human).

In some embodiments, RNA includes unmodified uridine residues; RNA that includes only unmodified uridine residues may be referred to as "uRNA". In some embodiments, RNA includes one or more modified uridine residues; in some embodiments, such RNA (e.g., RNA including fully modified uridine residues) is referred to as "modRNA". In some embodiments, RNA may be self-amplifying RNA (saRNA). In some embodiments, RNA may be trans-amplifying RNA (taRNA) (see, e.g., WO2017/162461).

In some embodiments, the related RNA includes one or more polypeptide encoding portions. In some specific embodiments, such one or more portions may encode one or more polypeptides, which are or include biologically active polypeptides or portions thereof (e.g., enzymes or cytokines or therapeutic proteins, such as replacement proteins or antibodies or portions thereof). In some specific embodiments, such one or more portions may encode one or more polypeptides, which are or include antigens (or their epitopes), cytokines, enzymes, etc. In some embodiments, one or more encoded polypeptides may be or include one or more neoantigens or neoepitopes associated with tumors. In some embodiments, one or more encoded polypeptides may be or include one or more antigens (or their epitopes) of infectious agents (e.g., bacteria, fungi, viruses, etc.). In certain embodiments, the encoded polypeptide may be a variant of a wild-type polypeptide.

In some embodiments, the single-stranded RNA (e.g., mRNA) may include a secretion signal coding region (e.g., a secretion signal coding region that allows one or more encoded target entities to be secreted after being translated by the cell). In some embodiments, such a secretion signal coding region may be or include a non-human secretion signal. In some embodiments, such a secretion signal coding region may be or include a human secretion signal.

In some embodiments, a single-stranded RNA (e.g., mRNA) may include at least one non-coding element (e.g., to improve RNA stability and/or translation efficiency). Examples of non-coding elements include, but are not limited to, a 3' untranslated region (UTR), a 5'UTR, a cap structure (e.g., in some embodiments, an enzymatically added cap; in some embodiments, a co-transcriptional cap), a polyadenine (polyA) tail (e.g., in some embodiments, may be or include 100A or more residues, and/or in some embodiments may include one or more "interrupting" [i.e., non-A] sequence elements) and any combination thereof. Exemplary embodiments of such non-coding elements can be found in, for example, WO2011015347, WO2017053297, US10519189, US10494399, WO2007024708, WO2007036366, WO2017060314, WO2016005324, WO2005038030, WO2017036889, WO2017162266, and WO2017162461, each of which is incorporated herein by reference in its entirety.

form

At least four formats have been developed that can be used in RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines), namely, unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), self-amplifying mRNA (saRNA), and trans-amplifying RNA.

Characteristics of the unmodified uridine platform may include, for example, one or more of an intrinsic adjuvant effect, good tolerability and safety, and strong antibody and T cell responses.

Features of the modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, attenuated immune innate immune sensor activation ability and thus enhanced antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As noted herein, the present disclosure provides insights that such strong antibody and CD4 T cell responses may be particularly useful for vaccination.

Characteristics of a self-amplifying platform may include, for example, long duration of polypeptide (eg, protein) expression, good tolerability and safety, and a higher likelihood of achieving efficacy at very low vaccine doses.

In some embodiments, the self-amplification platform (e.g., RNA) comprises two nucleic acid molecules, wherein one nucleic acid molecule encodes a replicase (e.g., a viral replicase), and the other nucleic acid molecule is capable of trans-replication (trans-replication system) by the replicase (e.g., a replicon). In some embodiments, the self-amplification platform (e.g., RNA) comprises a plurality of nucleic acid molecules, wherein the nucleic acid encodes a plurality of replicase and/or replicons.

In some embodiments, the replication system in trans comprises the presence of two nucleic acid molecules in a single host cell.

In some such embodiments, the nucleic acid encoding the replicase (e.g., viral replicase) is unable to replicate itself in the target cell and/or target organism. In some such embodiments, the nucleic acid encoding the replicase (e.g., viral replicase) lacks at least one conserved sequence element important for (-) strand synthesis based on a (+) strand template and/or for (+) strand synthesis based on a (-) strand template.

In some embodiments, the self-amplifying RNA comprises a 5'-cap; in some trans-replication systems, at least one RNA encoding a replicase is capped. Without wishing to be bound by any one theory, it has been found that the 5'-cap may be important for high-level expression of a gene of interest in trans.

In some embodiments, the self-amplification platform does not require propagation of viral particles (eg, is not associated with undesired viral particle formation). In some embodiments, the self-amplification platform is incapable of forming viral particles.

In some embodiments, the RNA may comprise an internal ribosome entry site (IRES) element. In some embodiments, the RNA does not comprise an IRES site; specifically, in some embodiments, the saRNA does not comprise an IRES site. In some such embodiments, the translation of the gene of interest and/or replicase is not driven by the IRES element. In some embodiments, the IRES element is replaced by a 5'-cap. In some such embodiments, the replacement of the 5'-cap does not affect the sequence of the polypeptide encoded by the RNA.

In some embodiments, the complex described herein includes modRNA, saRNA, taRNA or uRNA. In some embodiments, the complex includes modRNA. In some embodiments, the complex includes saRNA. In some embodiments, the complex includes taRNA. In some embodiments, the complex includes uRNA.

Instructions

The complexes described herein can be used to treat and prevent the diseases, disorders and conditions described herein in a subject. In some embodiments, the present disclosure provides a method of treating a disease, disorder or condition comprising administering a complex described herein to a patient. In some embodiments, the present disclosure provides the use of the complexes described herein for treating a disease, disorder or condition. In some embodiments, the disease, disorder or condition is an infectious disease, cancer, autoimmune disease or a rare disease.

In some embodiments, the infectious disease is caused by or is associated with a viral pathogen. In some embodiments, the viral pathogen belongs to a family selected from the group consisting of poxviridae, rhabdoviridae, filoviridae, paramyxoviridae, hepadnaviridae, coronaviridae, caliciviridae, picornaviridae, reoviridae, retroviridae, and orthomyxoviridae. In some embodiments, the infectious disease is caused by or is associated with a virus selected from the group consisting of SARS-CoV-2, influenza, Crimean-Congo Hemorhhagic Fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

In some embodiments, the infectious disease is caused by or associated with a bacterial pathogen. In some embodiments, the bacterial pathogen is of a species selected from the group consisting of Actinomyces israelii, Bacillus antracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium idphteriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae pneumoniae, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, Yersinia pestis.

In some embodiments, the infectious disease is caused by or is associated with a parasite. In some embodiments, the parasite belongs to a family selected from the group consisting of Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomes, Ascaris, Echinococcus, and Taeniidae.

In some embodiments, the disease, disorder or condition is cancer. In some embodiments, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral/oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer and uterine cancer.

In some embodiments, the disease, disorder or condition is a genetic disorder. In some embodiments, the genetic disorder is associated with a gain-of-function mutation or a loss-of-function mutation.

In some embodiments, the disease, disorder or condition is an autoimmune disease. In some embodiments, the autoimmune disease is selected from Addison disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, psoriasis, pernicious anemia, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and vasculitis Sjögren's syndrome. syndrome).

In some embodiments, the disease, disorder or condition is a rare disease. As used herein, a rare disease refers to a life-threatening or chronically debilitating disease that has a very low prevalence (e.g., less than 1 in 2000 people) and requires special concerted efforts to address.

In some embodiments, the present disclosure provides a complex that can selectively target a specific system in vivo. As used herein, reference to "targeting" a specific system refers to causing an increase in RNA expression of a cargo derived from the complex in the desired system. For example, in some embodiments, the complex described herein can selectively target the lungs, liver, spleen, heart, brain, lymph nodes, bladder, kidneys, and pancreas. As described herein, after administration, when the amount of mRNA expressed by a single target is 65% or more than the amount expressed in other organs, the complex "selectively targets" the organ (e.g., 65% or more of the mRNA in the whole body is expressed by a single organ, and the remaining 35% is distributed between one or more different organs). In some embodiments, the complex described herein selectively targets the lungs. In some embodiments, the complex described herein selectively targets the liver. In some embodiments, the complex described herein selectively targets the spleen. In some embodiments, the complex described herein selectively targets the heart.

Delivery Method

The present disclosure particularly provides a complex to be administered to a subject (e.g., a pharmaceutical composition or pharmaceutical formulation as mentioned herein). For example, in some embodiments, the complex is administered as a monotherapy. In some embodiments, the complex is administered as part of a combination therapy. In some embodiments, the total RNA concentration (e.g., the total concentration of all one or more RNA molecules) in the pharmaceutical composition described herein is about 0.01 mg/mL to about 0.5 mg/mL or about 0.05 mg/mL to about 0.1 mg/mL.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which as used herein includes any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. suitable for the desired particular dosage form. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients for formulating pharmaceutical compositions and known preparation techniques thereof. Unless any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or interacting in a harmful manner with any other component or components of the pharmaceutical composition, its use is considered within the scope of the present disclosure.

In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients for making pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifiers, disintegrants, adhesives, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical preparations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or aromatics may be present in the composition at the discretion of the formulator.

General considerations in the formulation and/or manufacture of medicaments can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, the pharmaceutical compositions provided herein can be formulated according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy 21st Edition, Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety), with one or more pharmaceutically acceptable carriers or excipients, and any other known adjuvants and excipients.

The pharmaceutical complexes and compositions described herein can be administered by appropriate methods known in the art. It will be appreciated by those skilled in the art that the route and/or mode of administration may depend on a variety of factors, including, for example but not limited to, the stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

In some specific embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous (s.c) administration. In some specific embodiments, the pharmaceutical compositions described herein are formulated for intramuscular (i.m) administration.

The therapeutic composition must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticles or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyols (such as mannitol, sorbitol) or sodium chloride. In some embodiments, the extended absorption of the injectable composition can be achieved by including an agent (e.g., monostearate and gelatin) that delays absorption in the composition.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

In some embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be ensured by aseptic procedures and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents (such as sugars, sodium chloride, etc.) in the pharmaceutical compositions described herein. In addition, extended absorption of injectable drug forms may be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed thereafter. Generally speaking, such preparation methods include the steps of combining the active ingredient with a diluent or another excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, shaping and/or packaging the product into a desired single or multiple dose unit.

Pharmaceutical compositions according to the present disclosure can be prepared, packaged and/or sold in batches as a single unit dose and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and/or methods described herein.

In some embodiments, the active agent that may be included in the pharmaceutical composition described herein is or is included in a therapeutic agent administered in the combination therapy described herein. The pharmaceutical composition described herein may be administered in combination therapy, i.e., in combination with other agents. In some embodiments, such therapeutic agents may include agents that cause the depletion or functional inactivation of regulatory T cells. For example, in some embodiments, the combination therapy may include a provided pharmaceutical composition having at least one immune checkpoint inhibitor.

In some embodiments, the pharmaceutical compositions described herein may be administered in conjunction with radiation therapy and/or autologous peripheral stem cell or bone marrow transplantation.

In some embodiments, the pharmaceutical compositions described herein can be frozen to allow for long-term storage.

Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals are well known, and a veterinary pharmacologist of ordinary skill can design and/or perform such modifications using only ordinary, if any, experimentation.

Methods of making the compositions described herein

As described herein, the present disclosure provides, inter alia, a method for preparing an oligosaccharide of formula II:

It comprises reacting a compound of formula III:

Contacting with a compound of formula IV:

in

Each ^Ra is an optionally substituted _C1 - _C20 aliphatic;

Z ¹ is a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1-3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

and

each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^Za - ^PG1 is ^Z1 , wherein one ^Rz has been replaced by a ^PG1 group; and

PG ¹ is a suitable nitrogen protecting group.

In some embodiments, the method of preparing a compound of formula III comprises treating a compound of formula V

Contact with an acid, and then contacting the resulting product with _CSC12 to give a compound of formula III; wherein ^PG2 is a suitable acid-sensitive nitrogen protecting group.

In some embodiments, the method of preparing a compound of formula V comprises treating a compound of formula VI

Contacting with a compound of formula VII:

Ra ^- COCl

VII

To obtain the compound of formula V.

In some embodiments, the method of preparing a compound of Formula VI comprises making a compound of Formula VIII:

Contacting with a compound of formula IX:

HS(CH ₂ ) ₂ NHPG ²

IX

To obtain a compound of formula VI, wherein LG is a suitable leaving group.

In some embodiments, PG ¹ and PG ² are each independently selected from Boc (tert-butoxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Cbz (benzyl chloroformate), Troc (2,2,2-trichloroethoxycarbonyl), trityl (triphenylmethyl), phthalimide, tetrachlorophthalimide and trifluoroacetamide.

In some embodiments, LG is selected from iodine, bromine, chlorine, methanesulfonate, trichloromethanesulfonate, and p-toluenesulfonate.

Exemplary embodiments

The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the present disclosure:

Embodiment 1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

A is ^A1 , ^A2 or ^A3 :

R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , wherein at least one instance of R ¹ or R ² is not H;

Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ;

each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ;

each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^X1 and ^X2 are each independently selected from -S- and -NH-;

^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;}

each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, a 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and

p is an integer selected from 1, 2, 3, 4 or 5;

The premise is that when A is A ¹ , then:

(i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ - and Z ¹ and Z ² are not both –N ⁺ (H) ₃ ,

(ii) each of R ¹ and R ² is not -(CH ₂ ) ₅ -CH ₃ or -(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both -S-, Y ¹ and Y ² are not both -(CH ₂ ) ₂ -, Z ¹ and Z ² are not both -N ⁺ (H) ₃ ,

(iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and

(iv) Cy is not a triazole group.

Embodiment 2. The compound of embodiment 1, wherein the compound has formula Ia:

or a pharmaceutically acceptable salt thereof.

Embodiment 3. The compound of Embodiment 1, wherein the compound has Formula Ib:

or a pharmaceutically acceptable salt thereof.

Embodiment 4. The compound of Embodiment 1, wherein the compound has Formula Ic:

or a pharmaceutically acceptable salt thereof.

Embodiment 5. The compound of any one of embodiments 1-4, wherein R ¹ and R ² are each independently selected from ^Ra and -C(O) ^-Ra , and ^Ra is C ₁ -C ₂₀ aliphatic.

Embodiment 6. The compound of any one of embodiments 1-5, wherein R ¹ and R ² are each —C(O)—R ^a , and R ^a is C ₁ -C ₁₄ aliphatic.

Embodiment 7. The compound of any one of embodiments 1-6, wherein each ^Ra is a _C5 - _C10 straight chain alkyl.

Embodiment 8. The compound of embodiment 1, wherein R ¹ and R ² are each -C(O)-R ^a , and each R ^{a is} independently selected from

Embodiment 9. The compound of any one of embodiments 1-8, wherein X ¹ and X ² are each -S-.

Embodiment 10. A compound as described in any of embodiments 1-9, wherein ^Y1 and ^Y2 are each selected from _C1 - _C10 aliphatic, _C0 - _C4 -aliphatic-NHC(O)NH- _C0 - _C4 aliphatic, C0-C4-aliphatic-NHC(S)NH- _C0 - _C4 aliphatic, C0-C4-aliphatic-C(O)NH- _C0 -C4 aliphatic, _C0 - _C4 -aliphatic-C(S)NH- _C0 - _C4 aliphatic, _C0 - _C4 -aliphatic-NHC(O)NH- _C0 - _C4 aliphatic, C0- _C4 -aliphatic-C(S)NH- _C0 -C4 aliphatic, _C0 -C4-aliphatic-NHC(O)-C0- _C4 aliphatic, _{C0 -C4 -} aliphatic- _NHSO2 - _C0 - _C4 aliphatic and _C0 - _C4 -aliphatic-C(O) _-C0 - _C4 aliphatic.

Embodiment 11. A compound as described in any of embodiments 1-10, wherein Y ¹ and Y ² are each C ₁ -C ₄ aliphatic -NHC(S)NH-C ₁ -C ₄ aliphatic.

Embodiment 12. The compound of any one of embodiments 1-11, wherein Y ¹ and Y ² are each —CH ₂ —CH ₂ —NHC(S)NH—CH ₂ —CH ₂ —.

Embodiment 13. A compound as described in any one of embodiments 1-12, wherein the moiety X ¹ -Y ¹ -Z ¹ is

Embodiment 14. A compound as described in any of embodiments 1-13, wherein the moiety X ² -Y ² -Z ² is

Embodiment 15. A compound as described in any one of embodiments 1-14, wherein Z ¹ and Z ² are each independently selected from: N ⁺ (M) ₃ ,

Embodiment 16. A compound as described in any one of embodiments 1-14, wherein Z ¹ and Z ² are each independently selected from:

Embodiment 17. The compound of embodiment 1, wherein the oligosaccharide is a compound of formula Id:

or a pharmaceutically acceptable salt thereof, wherein n and m are each independently selected from 0, 1, 2, 3, 4, 5 or 6.

Embodiment 18. The compound of embodiment 17, wherein the oligosaccharide is a compound of formula Id-i:

or a pharmaceutically acceptable salt thereof.

Embodiment 19. The compound of any one of embodiments 1-18, further comprising one or more suitable counterions.

Embodiment 20. The compound of Embodiment 1, wherein the compound is selected from Table 1.

Embodiment 21. A complex comprising the compound of any one of embodiments 1-20 and a nucleic acid.

Embodiment 22. The complex of Embodiment 21, wherein the nucleic acid is RNA.

Embodiment 23. A complex as described in Embodiment 22, wherein the RNA is modRNA, saRNA, taRNA or uRNA.

Embodiment 24. A complex as described in embodiment 22 or 23, wherein the N/P ratio is less than 20:1.

Embodiment 25. A complex as described in Embodiment 24, wherein the N/P ratio is less than or equal to 12:1.

Embodiment 26. A complex as described in any of Embodiments 21-25, wherein the complex has a diameter of about 30 nm to about 300 nm.

Embodiment 27. A complex as described in any of Embodiments 21-26, wherein the complex has a diameter of about 50 nm to about 200 nm.

Embodiment 28. The complex of any one of embodiments 21-27, further comprising a pharmaceutically acceptable surfactant.

Embodiment 29. The complex of embodiment 28, wherein the pharmaceutically acceptable surfactant is polysorbate.

Embodiment 30. The complex of embodiment 28 or 29, wherein the molar ratio of the compound to the pharmaceutically acceptable surfactant is from about 1:0.0075 to about 1:3.

Embodiment 31. A method of increasing or causing an increase in RNA expression in a target in a subject, comprising administering to the subject a complex as described in any one of embodiments 21-30.

Embodiment 32. The method of embodiment 31, wherein the target is selected from the group consisting of lung, liver, spleen, heart, brain, lymph node, bladder, kidney and pancreas.

Embodiment 33. A method of treating a disease, disorder or condition in a subject, comprising administering to the subject the complex of any one of embodiments 21-30.

Embodiment 34. The method of embodiment 33, wherein the disease, disorder or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease or a rare disease.

Embodiment 35. The method of any one of embodiments 31-34, wherein the complex is administered intramuscularly.

Embodiment 36. A method as described in Embodiment 35, wherein the complex has a diameter between 50 nm and 100 nm.

Embodiment 37. The method of any one of embodiments 31-34, wherein the complex is administered subcutaneously.

Embodiment 38. Use of a compound as described in any one of Embodiments 1-20 in medicine.

Embodiment 39. Use of the complex according to embodiment 21 in medicine.

Embodiment 40. Use of the complex as described in Embodiment 21 for increasing RNA expression in a target or causing an increase in the RNA expression.

Embodiment 41. Use of the complex of embodiment 21 for treating a disease, disorder or condition.

Embodiment 42. The use according to embodiment 41, wherein the disease, disorder or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease or a rare disease. Embodiment 43. A method for preparing an oligosaccharide of formula II:

It comprises reacting a compound of formula III:

Contacting with a compound of formula IV:

in

Each ^Ra is an optionally substituted _C1 - _C20 aliphatic;

^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ ,

and

each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ;

each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or

Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S;

^Za - ^PG1 is ^Z1 , wherein one ^Rz has been replaced by a ^PG1 group; and

PG ¹ is a suitable nitrogen protecting group,

The prerequisite is that Z ¹ and Z ² are not both –N ⁺ (H) ₃ .

Embodiment 44. The method according to embodiment 43, comprising firstly treating a compound of formula V

contacting with an acid, and then contacting the resulting product with _CSCl2 to obtain a compound of formula III;

Where ^PG2 is a suitable acid-sensitive nitrogen protecting group.

Embodiment 45. The method of embodiment 44, comprising making a compound of formula VI

Contacting with a compound of formula VII:

Ra ^- COCl

VII

To obtain the compound of formula V.

Embodiment 46. The method of embodiment 45, comprising making a compound of formula VIII:

Contacting with a compound of formula IX:

HS(CH ₂ ) ₂ NHPG ²

IX

To obtain a compound of formula VI,

Among them, LG is a suitable leaving group.

The foregoing clauses have described certain non-limiting embodiments of the subject matter described herein. It should therefore be understood that the embodiments described in this specification are merely for the purpose of illustrating the subject matter reported therein. Reference to the details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features that are considered essential.

It is contemplated that the systems and methods of the claimed subject matter encompass variations and adaptations developed using information from the embodiments described therein. Adaptations, modifications, or both of the systems and methods described herein may be performed by one of ordinary skill in the relevant art.

Throughout the specification, where a system is described as having, including, or comprising particular components, or where a method is described as having, including, or comprising particular steps, it is contemplated that there are additionally systems encompassed by the subject matter that consist essentially of, or consist of, the recited components, and there are methods encompassed by the subject matter that consist essentially of, or consist of, the recited process steps.

It should be understood that the order of steps or the order in which certain actions are performed is not important, as long as the embodiments of the subject matter described herein remain operable. In addition, two or more steps or actions may be performed simultaneously.

Acknowledgements

The applicants would like to thank the Consejo Superior de Investigaciones Científicas and FIUS/UNIVERSITY OF SEVILLE for their support and contributions to the development of this work.

Example

The examples provided herein record and support certain aspects of the present disclosure, but are not intended to limit the scope of any claim. The following non-limiting examples are provided to further illustrate certain teachings provided by the present disclosure. According to the application, it will be understood by those skilled in the art that various changes may be made to the specific embodiments described in the present example without departing from the spirit and scope of this teaching.

The following abbreviations may be used in the following examples: aq. (aqueous solution); ACN (acetonitrile); CSA (camphorsulfonic acid); d (day); Da/kDa (Dalton/kilodalton); DCM (dichloromethane); d# (day, e.g., d0 = day 0, d20 = day 20); ddH ₂ O (redistilled H ₂ O); DEA (diethylamine); DHP (dihydropyran); DMF (N,N-dimethylformamide); DIPEA (N,N-diisopropylethylamine); DMAP (4-dimethylaminopyridine); DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane); DODMA (1,2-dioleyloxy-3-dimethylaminopropane); DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); DMSO (dimethyl sulfoxide); EA (ethyl acetate); ee (enantiomeric excess); equiv. (equivalent); Ethanol (EtOH); h or hr (hour); Hex (hexane); HPLC (high performance liquid chromatography); IPA (isopropanol); im (intramuscular); iv (intravenous); KHMDS (potassium bis(trimethylsilyl)amide); LAH (lithium aluminum hydride); LCMS (liquid chromatography-mass spectrometry); LDA (lithium diisopropylamide); LiHMDS (lithium bis(trimethylsilyl)amide); MeOH (methanol); min (minute); NMR (nuclear magnetic resonance); Pd/C (palladium on carbon); PEI (polyethyleneimine); PPh ₃ O (triphenylphosphine oxide); Pt/C (platinum on carbon); rb (round bottom); Rf (retention factor); rt or RT (room temperature); sc (subcutaneous); SM (starting material); TEA (triethylamine); THF (tetrahydrofuran); THP (tetrahydropyran); TLC (thin layer chromatography); TsOH (p-toluenesulfonic acid or toluenesulfonic acid); and UV (ultraviolet light).

Example 1 - Composites and methods of making composites

The following composites were prepared using various embodiments described herein:

i.m. = intramuscular; s.c. = subcutaneous; and i.v. = intravenous

This example describes certain complexes comprising an oligosaccharide and a nucleic acid (eg, RNA). In the examples provided, paOs refers to "polycationic amphiphilic oligosaccharides".

As described herein, the calculation of the N/P ratio is determined by finding the molar ratio of the positive charge in the cationic oligosaccharide (e.g., the protonated or protonatable or permanent charge (N) of the nitrogen atom of the amine/guanidine) and the anionic charge (phosphate) (P) in the RNA. Therefore, the amount of RNA and oligosaccharide in a particular complex is determined by the N/P ratio calculation and the desired final RNA concentration of the formulation. The complex itself is generated when asymmetric volumes of RNA-containing solution and oligosaccharide-containing solution are mixed. For the RNA solution, the desired concentration of RNA is prepared by diluting the appropriate volume of ddH ₂ O, RNA stock solution and buffer (6x). In a separate test tube, an oligosaccharide-containing solution is prepared by diluting the oligosaccharide to the desired concentration in DMSO. Complexation of RNA occurs by mixing the RNA-oligosaccharide phases at a volume ratio of 9:1 (RNA: oligosaccharide). The formulated complex is incubated at room temperature for 5-10 minutes to stabilize. The buffer used for formulation in all experiments is MGB (MES buffered glucose (10mM MES, pH6.1, 5% D-glucose)).

The size of the complex described in this example was analyzed by a DynaPro plate reader II, and the hydrodynamic diameter of the complex was calculated using dynamic light scattering (DLS). The preparation was measured by diluting the sample to 0.0125 mg/mL RNA in 120 μl MGB (10 mM MES, pH 6.1, 5% D-glucose) 5% in a well of a 96-well plate. Ten acquisitions were recorded for each well, each lasting 5 seconds.

Example 2 - Intravenous Injection Example

Example 2a - Targeting lung expression

Nine female BALB/c mice were divided into three research groups. All groups received the same amount of prepared luciferase encoding modRNA (5 μg), applied intravenously (tail vein). After 6 hours, the in vitro luciferase expression of each extracted organ was quantified. All groups received modRNA prepared with JRL13, and modRNA was prepared with N/P1, N/P3, N/P6 or N/P12. All N/P ratios were calculated based on oligosaccharide-RNA charge ratios. For all cases, in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA solution and oligosaccharide solution was 9:1 (volume: volume). The preparation was characterized before injection to confirm the hydrodynamic diameter and zeta potential. Figure 1A is a diagram illustrating the hydrodynamic diameter of an example composition comprising modRNA prepared with different N/P ratios and certain oligosaccharides. Figure IB is a graph illustrating the zeta potential of example oligosaccharides at different N/P ratios.Figure 1C is a bar graph illustrating quantitative ex vivo luminescence 6 hours after injection of example compositions comprising modRNA in different organs and at different N/P ratios.

It was found that the organ-selective expression of modRNA was strongly dependent on the N/P ratio of the JRL13 preparation. Although signals were observed in the spleen at N/P1, the ratio of biodistribution shifted completely to the lungs as N/P increased. At N/P3, there was mixed expression in the lungs and spleen, while at N/P 6, >90% of the signal came from the lungs. In Figures 1A and 1B, no differences in surface charge or hydrodynamic diameter were observed. In addition, increasing N/P to greater than 6 did not change the biodistribution.

The effects of altering the fatty chains in JRL13 were also studied to elucidate the relevance of these elements in the targeting properties of JRL13. Six female BALB/c mice were divided into two study groups. All groups received the same amount of formulated luciferase-encoding modRNA (5 μg), applied intravenously (tail vein). After 6 hours, the in vitro luciferase expression of each extracted organ was quantified. One group received RNA formulated with JRL13, and the other group received RNA formulated with another JRL13 derivative in which the fatty chains had been replaced by longer chains (e.g., C10), as observed in JLJB617. All groups received a composition comprising modRNA formulated with N/P6. The N/P ratio was calculated based on the oligosaccharide-RNA charge ratio. For all cases, the co-formulations were in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the RNA final concentration was 0.05 mg/ml and the mixing ratio of the RNA-containing solution and the oligosaccharide-containing solution was 9:1 (volume:volume). FIG. 1D is a bar graph illustrating the hydrodynamic diameter of an example composition comprising modRNA. FIG. 1E is a bar graph illustrating the zeta potential of an example composition. FIG. 1F is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of an example composition comprising modRNA.

Compared to JRL13, the introduction of longer aliphatic chains (e.g., C10, as observed in JLJB617) did not affect the overall surface charge or hydrodynamic diameter of the complexes formed by JLJB617. On the other hand, the introduction of longer aliphatic chains affected biodistribution (see Figure 1F). While JLR13 showed >90% modRNA expression from the lungs, in JLJB617, the distribution ratio was reduced to only 21% from the lungs, and at the same time spleen expression increased from 5% in JRL13 to 70% in JLJB617.

The effect of disaccharide scaffold on the targeting properties of JRL13 was studied. Six female BALB/c mice were divided into two research groups. All groups received the same amount of formulated luciferase encoding modRNA (5 μg), applied intravenously (tail vein). After 6 hours, the in vitro luciferase expression of each extracted organ was quantified. One group received RNA formulated with JRL13, and the other group received RNA formulated with JRL13 analogs, in which trehalose was replaced by sucrose-JLJB626-. All groups received modRNA formulated with N/P3. For all cases, the compound formulation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume: volume). Figure 1G is a bar graph illustrating the hydrodynamic diameter of an example composition comprising modRNA. Figure 1H is a bar graph illustrating the zeta potential of example oligosaccharide compositions. Figure 1I is a bar graph illustrating quantitative ex vivo luminescence in spleen 6 hours after injection of a composition comprising modRNA.

Example 2b - Targeting the liver or spleen

In this example, JLJB604 was formulated with modRNA at two different N/Ps: 3 and 6. Biodistribution was analyzed based on organ expression of modRNA encoding luciferase. Six female BALB/c mice were divided into two research groups. All groups received the same amount of formulated luciferase encoding modRNA (5 μg), applied intravenously (tail vein). After 6 hours, the in vitro luciferase expression of each extracted organ was quantified. All groups received modRNA formulated with JLJB604, wherein the modRNA was formulated with N/P3 or N/P6. For all cases, the compound formulation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume: volume). The formulation was characterized before injection to confirm the hydrodynamic diameter and ζ potential. Figure 2A is a bar graph illustrating the hydrodynamic diameter of example compositions comprising modRNA formulated at different N/P ratios. Figure 2B is a bar graph illustrating the zeta potential of example compositions at different N/P ratios. Figure 2C is a bar graph illustrating quantitative ex vivo luminescence 6 hours after injection of example compositions comprising modRNA in different organs and at different N/P ratios.

Example 2c - Targeting spleen expression

In the present embodiment, modRNA is directly prepared with N/P 6 using a combination of JLJB619 and JLJB604, compared with a preparation (F12, comprising a 3:1 molar ratio of DOTMA:DOPE and a 0.65 N/P ratio) currently in clinical development and widely disclosed for mRNA expression in the spleen. Biodistribution is analyzed based on the organ expression of modRNA encoding luciferase. Six female BALB/c mice were divided into two research groups. All groups received the same amount of prepared luciferase encoding modRNA (5 μg), applied intravenously (tail vein). After 6h, the in vitro luciferase expression of each extracted organ was quantified. The first group received modRNA prepared together with an equimolar mixture of JLJB619 and JLJB604, and modRNA was formulated into final N/P6. The formulations were prepared in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1) with a final RNA concentration of 0.05 mg/ml and a mixing ratio of 9:1 (volume:volume) of RNA-containing solution and oligosaccharide solution. The second group received modRNA formulated with F12, where the N/P ratio was calculated based on the cation:anion charge between DOTMA:RNA. F12 was prepared in _ddH2O with a final NaCl concentration of 0.112 mM and a final RNA concentration of 0.05 mg/ml. All formulations were characterized prior to injection to confirm the hydrodynamic diameter and zeta potential. Figure 2D is a bar graph illustrating the hydrodynamic diameter of example compositions comprising modRNA.

Figure 2E is a bar graph illustrating the zeta potential of example compositions. Figure 2F is a bar graph illustrating quantitative ex vivo luminescence in spleen 6 hours after injection of a composition comprising modRNA. Figure 2G is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of a composition comprising modRNA.

Example 2d—modRNA delivery versus DNA delivery

Six female BALB/c mice were divided into two research groups. All groups received the same amount of prepared luciferase encoding modRNA (10 μg), applied intravenously (tail vein). After 6 hours, the in vitro luciferase expression of each extracted organ was quantified. All groups received modRNA prepared with N/P 3 together with JRL13 or JRL45. In addition, different N/P ratios of modRNA were prepared together with JRL13 or JRL45 to more extensively study the binding properties with modRNA and the zeta potential of different preparations with modRNA. For all cases, the compound preparation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume: volume). The preparation was characterized before injection to confirm the hydrodynamic diameter and zeta potential. FIG. 3A is a bar graph illustrating the percentage of free RNA quantified by gel electrophoresis of an example composition comprising modRNAs at different N/P ratios between 0 and 3 for JRL13 or JRL45. FIG. 3B is a graph illustrating the zeta potential after preparing an example composition comprising modRNAs at different N/P ratios with JRL13 or JRL45. FIG. 3C is a graph illustrating the hydrodynamic diameter after preparing an example composition comprising modRNAs at different N/P ratios with JRL13 or JRL45. FIG. 3D is a bar graph illustrating the expression of luciferase encoding modRNAs after transfection of C2C12 cells, wherein the example composition is prepared at N/P 6 and comprises modRNAs at 25 ng RNA/well with JRL13 or JRL45. The data here represent 3 independent experiments (n=3), technically in triplicate. Figure 3E is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection of 10 μg of an example composition comprising modRNA formulated with JRL13 or JRL45 at N/P 3. Figure 3F is a bar graph illustrating quantitative ex vivo luminescence in different organs 6 hours after injection and evaluating the total flux [p/s] ratio of the JRL13 group to the JRL45 group.

JRL45 and JRL13 oligosaccharides are two molecules in the library described in the literature for delivering pDNA after intravenous injection (García Fernández et al. 2019, DOI 10.1039/c9cc04489b). In the publication of García Fernández, JRL45 is referred to as compound 10 and JRL13 is referred to as compound 8. The complex reported by García Fernández contains oligosaccharides and pDNA with an N/P ratio of 20. For example, Figure 3G is a bar graph illustrating quantitative in vitro luminescence in different organs after injection of a composition comprising pDNA complexed with JRL13 (compound 8) or JRL45 (compound 10).

García Fernández's formulation was prepared in Hepes buffer (20 mM) at pH 7.4, including diluting the pDNA in the buffer to the desired concentration and diluting the oligosaccharides by simple dispersion and mixing. The pDNA expression of JRL13 (Compound 8) was mainly in the liver, while the pDNA expression of JRL45 (Compound 10) was mainly in the lungs. See Figure 3G. When the complex of the present invention contains RNA, as shown in Figure 3E, JRL13 is mainly expressed in the spleen and lungs, while the expression of JRL45 is significantly reduced (Figure 3F) and is only observed in the spleen. The results obtained in this example not only contradict the published results, but also emphasize the influence of appropriate formulation parameters in providing the desired vehicle for RNA relative to DNA.

Example 3 - Intramuscular Delivery

Example 3a - modRNA and saRNA delivery and N/P ratio

Eighteen female BALB/c mice were divided into eleven research groups. All groups received the same amount of prepared luciferase encoding modRNA or saRNA (1 μg), which was applied intramuscularly to each leg. At seven time points (6 hours, 24 hours, 72 hours, the 6th day, the 8th day, the 20th day), the in vivo luciferase expression in the applied tissue was measured. Three modRNA groups were prepared with JRL13 at three different N/P ratios (1, 6 or 12), and three saRNA groups were prepared with JRL13 at three different N/P ratios (1, 6 or 12). N/P ratios were calculated based on JRL13-RNA charge ratios, as described herein. For all cases, in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume: volume). The formulations were characterized prior to injection to confirm the hydrodynamic diameter and zeta potential. FIG. 4A is a graph illustrating the hydrodynamic diameter of example complexes formulated at different N/P ratios and comprising RNA. FIG. 4B is a bar graph illustrating the zeta potential of example compositions comprising RNA at different N/P ratios. FIG. 4C is a bar graph illustrating the quantitative bioluminescence of an example composition comprising modRNA and JRL13 with a specified N/P ratio over the duration of the experiment. FIG. 4D is a bar graph illustrating the quantitative bioluminescence of an example composition comprising saRNA and JRL13 with a specified N/P ratio over the duration of the experiment.

After intramuscular application of modRNA, expression of negatively charged N/P ratio (N/P1) can be observed, while increasing the overall N/P ratio in the formulation leads to a complete loss of modRNA expression. The JRL13 formulation containing modRNA seems to have almost no difference in complex size with the increase of N/P (Fig. 4A), but the surface charge changes from negative charge to positive charge (Fig. 4B). In contrast, saRNA formulations show that size is affected by the N/P ratio. For example, at a lower N/P ratio (e.g., N/P1), the complex is about 170nm, and at a higher N/P ratio (e.g., N/P is 6-12), the size is reduced to 90nm. At a given dose, saRNA formulations have the highest expression at N/P12, while modRNA achieves the highest modRNA expression at N/P1 (Fig. 4C-Fig. 4D).

Example 3b - Cationic Oligosaccharide Screening

The present embodiment examines the effects of different cationic groups on the intramuscular delivery of saRNA. Forty-eight female BALB/c mice were divided into sixteen research groups. All groups received the same amount of prepared luciferase encoding saRNA (2 μg), which was applied intramuscularly to each leg. At six time points (6 hours, 24 hours, 72 hours, the 6th day, the 9th day, the 20th day), the in vivo luciferase expression in the applied tissue was measured. All groups received saRNA prepared with N/P 6. Each group received saRNA prepared with one of the following oligosaccharides: JRL13, JLJB619, JLF58, JLF41, JLF42, JLF102, JLF120, JLF94, JLF96, JLF97, JLF135, JLF90, JLF130, JLF82, JLF93, JLF99. For all cases, the co-formulations were in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.1 mg/ml, and the mixing ratio of the RNA-containing solution and the oligosaccharide-containing solution was 9:1 (volume:volume). The formulations were characterized before injection to confirm the hydrodynamic diameter. FIG5A is a bar graph illustrating the hydrodynamic diameter of complexes formulated at N/P 6 and comprising saRNA and different oligosaccharides. FIG5B is a bar graph illustrating the quantitative bioluminescence during the duration of the experiment and normalized to the JRL13-saRNA group. FIG5C is a graph illustrating the quantitative bioluminescence of the example composition at each measurement time point.

While most of the tested formulations of saRNA with N/P 6 had hydrodynamic diameters ranging from 50 to 100 nm ( FIG. 5A ), the diameter of the formulation containing JLF99 was approximately 150 nm. Of the different oligosaccharides tested, at least 4 were identified as comparable to JRL13: JLF102, JLF94, JLF97, and JLF93 ( FIG. 5B ). In addition, certain combinations of primary and secondary amines (e.g., JLF102) exhibited a positive effect, as reflected by a two-fold signal increase compared to JRL13.

Example 3c - Screening of selected oligosaccharides for N/P ratio

Based on previous observations (e.g., as shown in FIG. 4C ), additional cationic derivatives were tested with different N/P ratios to identify amine types suitable for delivery of modRNA. Oligosaccharides with different types of amines were tested, such as primary amines (JRL13), secondary amines (JLJB619 and JLF58), tertiary amines (JLF41), and quaternary ammoniums (JLF42).

Thirty female BALB/c mice are divided into ten research groups.All groups all accept the luciferase encoding modRNA (2 μ g) of the preparation of the same amount, and intramuscular application is to each leg at five time points (6 hours, the 1st day, the 2nd day, the 3rd day, the 6th day), and the body luciferase expression in the tissue of measurement application is measured.Half of the group accepts the modRNA prepared with N/P 1, and the other half accepts the modRNA prepared with N/P6.Two groups accept the modRNA prepared with JRL13, two groups accept the modRNA prepared with JLJB619, two groups accept the modRNA prepared with JLF58, two groups accept the modRNA prepared with JLF41, and two groups accept the modRNA prepared with JLF42. For all cases, the complex formulations were prepared in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the RNA final concentration was 0.1 mg/ml, and the mixing ratio of the RNA-containing solution and the oligosaccharide-containing solution was 9:1 (volume:volume). The formulations were characterized before injection to confirm the hydrodynamic diameter. FIG6A is a bar graph illustrating the hydrodynamic diameter of example complexes formulated with modRNA and different oligosaccharides at different N/P ratios. FIG6B is a bar graph illustrating the quantitative bioluminescence during the duration of the experiment.

In all tested preparations, the lowest N/P ratio (N/P 1) tested in this embodiment leads to the formation of significantly larger nanoparticles (Fig. 6 A). For JLJB619, JLF58 and JLF41, the particle size difference between N/P 1 and 6 is larger, while in JRL13 and JLF42 preparations, the particle size difference between N/P 1 and N/P 6 is smaller. Not bound by theory, it is believed that this is because JRL13 has a primary amine that is almost charged at any physiological pH, and JLF42 has a quaternary ammonium as a permanent cation. These two groups interact with RNA and have suitable condensation ability. JLJB619, JLF58 and JLF41 have amines with pKa in the physiological range. JLF58 and JLF41 include sterically hindered amines. Under the two N/Ps tested, the bioluminescence in JLF141 is the highest (Fig. 6 B). N/P 6 groups are 1.5 times higher than N/P 1 groups. JRL13 and JLF58 showed no significant differences in modRNA expression between the two N/Ps tested.

Example 3d - Screening for additional oligosaccharides for modRNA delivery

Forty-eight female BALB/c mice were divided into sixteen research groups. All groups received the same amount of prepared luciferase encoding saRNA (2 μg), applied intramuscularly to each leg at six time points (6 hours, day 1, day 2, day 3, day 6), and the in vivo luciferase expression in the applied tissue was measured. All groups received modRNA prepared with N/P 6. Each group received modRNA prepared with one of the following oligosaccharides: JRL13, JLJB619, JLF58, JLF42, JLF41, JLF128, JLF142, JLF131, JLF96, JLF97, JLF135, JLF94, JLF90, JLF99, JLF82 and JLF138. For all cases, the co-formulations were in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.1 mg/ml, and the mixing ratio of the RNA-containing solution to the oligosaccharide-containing solution was 9:1 (volume:volume). The formulations were characterized before injection to confirm the hydrodynamic diameter. FIG. 7A is a bar graph illustrating the hydrodynamic diameter of the complexes formulated with N/P 6 modRNA and different oligosaccharides. FIG. 7B is a bar graph illustrating the quantitative bioluminescence during the experiment and normalized to the JLF41-modRNA group.

JLF99 showed promising modRNA expression ( FIG. 7B ). Certain oligosaccharides (JLF142, JLF131, JLF99) formed complexes with modRNAs exceeding 100 nm.

Example 3e - Screening of cationic headgroups with different N/P ratios for modRNA delivery

Twenty-four female BALB/c mice were divided into eight research groups. All groups received the same amount of luciferase encoding modRNA (5 μg) prepared, which was applied subcutaneously to the right flank. At five time points (6 hours, 24 hours, 48 hours, 72 hours, the 6th day), the in vivo luciferase expression in the tissue of application was measured. Two groups received modRNA prepared with N/P 1 and N/P 6 together with JRL13. Two groups received modRNA prepared with N/P 1 and N/P 6 together with JLJB619. Two groups received modRNA prepared with N/P 1 and N/P 6 together with JLJB604. The last two groups received modRNA prepared with N/P 1 and N/P 6 together with JLJB605. For all cases, the co-formulations were prepared in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.05 mg/ml, and the mixing ratio of the RNA-containing solution to the oligosaccharide-containing solution was 9:1 (volume:volume). The formulations were characterized prior to injection to confirm the hydrodynamic diameter and zeta potential. FIG. 8A is a bar graph illustrating the hydrodynamic diameter of complexes formulated at different N/P ratios. FIG. 8B is a bar graph illustrating the zeta potential of compositions comprising modRNA at different N/P ratios. FIG. 8C is a bar graph illustrating the quantitative bioluminescence of various example compositions over the duration of the experiment.

Example 3f - Screening of aliphatic chain lengths for modRNA delivery

Thirty-six female BALB/c mice were divided into twelve research groups. All groups received the same amount of luciferase encoding modRNA (5 μg) prepared, which was applied subcutaneously to the right flank. The in vivo luciferase expression in the tissue of application was measured at seven time points (6 hours, 24 hours, 72 hours, the 6th day, the 9th day, the 20th day). Three groups received oligosaccharides of primary amines carrying different aliphatic chain lengths, i.e., C ₆ (JRL13), C ₁₀ (JLJB617), C ₁₄ (JLJB618). Three groups received oligosaccharides of secondary amines carrying different aliphatic chain lengths, such as C ₆ (JLJB619), C ₁₀ (JLF43), C ₁₄ (JLF31). Three groups received oligosaccharides of tertiary amines carrying different aliphatic chain lengths, such as C ₆ (JLJB604), C ₁₀ (JLF41), C ₁₄ (JFL25). Three groups received oligosaccharides carrying quaternary amines of different aliphatic chain lengths, such as C ₆ (JLJB605), C ₁₀ (JLF42), C ₁₄ (JLF26). All formulations were tested with an N/P ratio of 6. For all cases, the formulations were compounded in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final RNA concentration was 0.05mg/ml, and the mixing ratio of the RNA-containing solution and the oligosaccharide-containing solution was 9:1 (volume:volume). The formulations were characterized before injection to confirm the hydrodynamic diameter and zeta potential. FIG. 9A is a bar graph illustrating the hydrodynamic diameter of the example composition. FIG. 9B is a bar graph illustrating the quantitative bioluminescence of the example oligosaccharide formulations during the duration of the experiment.

Example 3g - Screening of aliphatic chain length for saRNA delivery

Thirty-six female BALB/c mice were divided into twelve research groups. All groups received the same amount of prepared luciferase encoding saRNA (5 μg), which was applied subcutaneously to the right flank. The in vivo luciferase expression in the tissues applied was measured at seven time points (6 hours, 24 hours, 72 hours, the 6th day, the 8th day, the 20th day). Three groups received oligosaccharides of primary amines carrying different aliphatic chain lengths, such as C ₆ (JRL13), C ₁₀ (JLJB617), C ₁₄ (JLJB618). Three groups received oligosaccharides of secondary amines carrying different aliphatic chain lengths, such as C ₆ (JLJB619), C ₁₀ (JLF43), C ₁₄ (JLF31). Three groups received oligosaccharides of tertiary amines carrying different aliphatic chain lengths, such as C ₆ (JLJB604), C ₁₀ (JLF41), C ₁₄ (JFL25). Three groups received oligosaccharides carrying quaternary amines of different aliphatic chain lengths, such as C ₆ (JLJB605), C ₁₀ (JLF42), C ₁₄ (JLF26). All preparations were tested with an N/P ratio of 6. For all cases, the preparations were compounded in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume:volume). The preparations were characterized before injection to confirm the hydrodynamic diameter and zeta potential. Figure 10A is a bar graph illustrating the hydrodynamic diameter of certain complexes. Figure 10B is a bar graph illustrating the quantitative bioluminescence of the example oligosaccharide preparations during the experimental duration. Figure 10C is a bar graph illustrating the quantitative response of CD8 positive T cells in spleen samples of treated mice 20 days after subcutaneous application. The CD8 response to the luciferase peptide presented by murine MHC was quantified via IFN release in ELISPOT. An irrelevant peptide was used as a control, showing that the CD8 response to the luciferase peptide was specific.

Example 3h - Effect of N/P ratio on modRNA and saRNA delivery

Eighteen female BALB/c mice were divided into six research groups. All groups received the same amount of luciferase encoding saRNA (5 μg) or modRNA (5 μg) prepared, subcutaneously applied to the right flank. The in vivo luciferase expression in the tissue of application was measured at seven time points (6 hours, 24 hours, 72 hours, the 6th day, the 8th day, the 20th day). Three groups received modRNA prepared with JLJB619 using three different N/Ps (3, 6 and 12), and the other three groups received saRNA prepared with JLJB619 using three different N/Ps (3, 6 and 12). Three groups received oligosaccharides carrying secondary amines of different aliphatic chain lengths, such as C6 (JLJB619), C10 (JLF43), C14 (JLF31). Three groups received oligosaccharides carrying tertiary amines of different aliphatic chain lengths, such as C6 (JLJB604), C10 (JLF41), C14 (JFL25). All N/P ratios were calculated based on oligosaccharide-RNA charge ratios. For all cases, the final concentration of RNA was 0.05 mg/ml in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), and the mixing ratio of RNA-containing solution and JLJB619 solution was 9:1 (volume: volume). The formulation was characterized before injection to confirm the hydrodynamic diameter and zeta potential. Figure 11A is a bar graph illustrating the hydrodynamic diameter of a complex prepared with different N/P ratios and comprising RNA. Figure 11B is a bar graph illustrating the zeta potential of an example composition of different N/P ratios and comprising RNA. Figure 11C is a bar graph illustrating the quantitative bioluminescence of the modRNA group during the duration of the experiment. FIG. 11D is a bar graph illustrating the quantitative bioluminescence of the saRNA group over the duration of the experiment.

Example 3i - Immunization against H1N1/Cal07/HA after saRNA delivery

Fifteen female BALB/c mice were divided into 3 groups. One group received only buffer as a negative control, and the other groups received 2 μg of saRNA compounded with JLJB619, and the saRNA encoded the hemagglutinin (HA) protein of influenza strain California/7/2009. The third group received 2 μg of saRNA compounded with LNP (DODMA, cholesterol, DOPE and PEG2000-ceramide C16, mol ratio of 40:48:10:2), and the saRNA encoded the hemagglutinin (HA) protein of influenza strain California/7/2009. All saRNA preparations were applied subcutaneously. The saRNA compounded with JLJB619 was prepared with N/P6. The saRNA prepared with LNP was prepared with N/P4. The N/P ratio of LNP is determined by the DODMA/RNA ratio.

The JLJB619 formulation was compounded in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final RNA concentration was 0.05mg/ml, and the mixing ratio of the RNA-containing solution and the paOs-containing solution was 9:1 (volume:volume); the LNP formulation was produced in PBS according to the standard LNP production method widely described in the literature. See Nogueira et al., ACS Appl. Nano. Mater., 3(11): 10634-10645 (2020). LNPs were produced by mixing an ethanol solution of the lipid mixture with a citrate buffer solution (pH 4.5) of saRNA at a ratio of 3:1, and the mixing speed was 12mL/min. The LNPs were then dialyzed in PBS for 3 hours. Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to influenza HA peptides. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of splenocytes. FIG. 12A is a bar graph illustrating the hydrodynamic diameter of complexes prepared at different N/P ratios and containing RNA. FIG. 12B is a diagram illustrating the serological levels of specific IgG for HA at a 1:300 serum dilution during the experimental process. FIG. 12C is a diagram illustrating the endpoint titration of the serological levels of specific IgG for HA. FIG. 12D is a bar graph illustrating the CD8/CD4 response to the HA peptide pool.

Example 3j - Cationic oligosaccharide combination complex

The present embodiment contemplates the JLF99 and modRNA comprising N/P6 and the compound of polysorbate (for example, Tween20 and Tween40) combination.Nine female BALB/c mice are divided into 3 research groups.All groups receive the luciferase encoding modRNA (2 μ g) of the same amount of preparation, and intramuscular application is to each leg at five time points (6 hours, the 1st day, the 2nd day, the 3rd day, the 6th day), and the body luciferase expression in the tissue of application is measured.The first group receives the modRNA prepared only with JLF99, and the second group receives the modRNA prepared with the mixture of JLF99+Tween20 (1:0.5 mol ratio), and the third group receives the modRNA prepared with the mixture of JLF99+Tween20 (1:1 mol ratio).All groups receive the modRNA prepared with N/P6. For all cases, co-formulations were made in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), RNA final concentration was 0.1 mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide/Tween20-containing solution was 9:1 (volume:volume). The formulations were characterized before injection to confirm the hydrodynamic diameter. FIG. 13A illustrates the hydrodynamic diameter of the complexes formulated with N/P6 and different surfactant combinations and having a surfactant to JLF99 ratio of 0:1 to 0.5:1 (where C14-pMeOx45 is _C14 alkyl-poly(2-methyl-2-oxazoline) ₄₅ , C14pSar23 is C14 alkyl-poly(sarcosine) ₂₃ , and Tween20 is polysorbate 20). Figure 13B illustrates the hydrodynamic diameter of the complex prepared with N/P6 and Tween20 or Tween40 and within the ratio of surfactant to JLF99 of 0 to 1.5. Figure 13C illustrates the expression of luciferase encoding modRNA at 24h after transfection of C2C12 cells, and the example composition has N/P6 and is prepared with Tween20 or Tween40 of 100ng RNA/well modRNA and different surfactants and JLF99 ratios. This example represents 3 independent experiments (n=3), technically in triplicate. Figure 13D illustrates the expression of luciferase encoding modRNA at 24 hours after transfection of HepG2 cells, and the example composition has N/P 6 and is prepared with Tween20 or Tween40 of 100ng RNA/well modRNA and different surfactants and JLF99 ratios. This example represents 3 independent experiments (n=3), technically in triplicate. Figure 13E is a bar graph illustrating the hydrodynamic diameter of complexes formulated with different oligosaccharide to surfactant ratios at N/P 6 (where T20 refers to Tween20). Figure 13F illustrates the quantitative bioluminescence over the entire duration of the experiment, expressed as the area under the curve (where T20 refers to Tween20). Figure 13G illustrates the quantitative bioluminescence over the entire duration of the experiment (where T20 refers to Tween20). Figure 13H is an image showing the bioluminescence of the ventral axis 6 hours after injection of a composition comprising modRNA into each group (where T20 refers to Tween20).

Example 3k - Intramuscular vaccination using cationic oligosaccharides and saRNA

Forty female BALB/c mice were divided into six groups. One group received only buffer as a negative control, and the other five groups received 1 μg of saRNA, which encodes the hemagglutinin (HA) protein of influenza strain California/7/2009. All saRNA preparations were applied intramuscularly to one of the legs. All saRNA groups were prepared with N/P12, but prepared with different oligosaccharides (JRL13, JLF102, JLF97, JLF94), or prepared with linear polyethyleneimine 22.5kDa (AVROXA, Ghent, Belgium) PEI500 as a benchmark. The N/P ratio of saRNA prepared with PEI500 is determined by secondary amine: RNA charge ratio. For all cases, the co-formulations were in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.05 mg/ml, and the mixing ratio of the RNA-containing solution and the cationic oligosaccharide-containing solution was 9:1 (volume:volume). The animals will be non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days, and the spleen will be extracted to quantify the CD4/CD8 T cell response to influenza HA peptides. Anti-influenza HA antibodies in serum will be quantified by enzyme-linked immunosorbent assay. Virus neutralization titers will be quantified by VNT assay. CD4/CD8 T cell responses are quantified via IFN–ELISPOT of splenocytes.

Example 4 - Synthesis Example

Unless otherwise stated, all reagents and solvents used in the preparations disclosed in the following examples were purchased from commercial sources and used without further purification. Thin layer chromatography (TLC) was performed on aluminum plates coated with silica gel 60F ₂₅₄ Merck, visualized by UV light (λ254 nm), and charred with 10% ethanolic H ₂ SO ₄ , 0.1% ethanolic ninhydrin and heated at 100° C. Column chromatography was performed on Silice 60A.CC Chromagel (SDS 70-200 and 35-70 μm). The structures of all compounds described herein, including synthetic intermediates and precursors, were confirmed by ¹ H and ¹³ C nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR experiments were performed at 300 (75.5) and 500 (125.7, 202) MHz using Bruker 300ADVANCE and 500DRX. 1D TOCSY, 2D COSY, HMQC and HSQC experiments are used to assist in NMR distribution.Mass spectrum (high resolution mass spectrum) is carried out on Bruker Daltonics Esquire6000 ^TM (LTQ-Orbitrap XL ETD).Introduce sample by the solid probe heated from 30 ℃ to 280 ℃.Use ESI as ionization source (electrospray ionization), use methanol as solvent.Use Cole-Parmer syringe to introduce sample via direct injection with the flow velocity of 2 μ l/min.Use the resonance jet of the multipole resonance of one-third radio frequency (Ω＝781.25kHz), scan ion between 300 to 3000Da with the scanning speed of 13000Da/s with unit resolution.

method

Scheme 1 depicts the synthesis of compounds of Formula I, wherein A is A ¹ , R ¹ and R ² are both -C(O)-R ^a , X ¹ and X ² are both -S-, Y ¹ and Y ² are both -(CH ₂ ) ₂ -, and Z ¹ and Z ² are NH ₃ ⁺ (compound 4T in Scheme 1 ). Scheme 1 also shows the general routes (Routes A1, A2, B1, and B2) carried out to synthesize compounds of Formula I, wherein A is A ¹ , R ¹ and R ² are both -C(O)-R ^a , X ¹ and X ² are both -S-, Y ¹ and Y ² are both -(CH ₂ ) ₂ -NHC(S)NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are the same (compound 6T in Scheme 1 ):

Scheme 2 depicts the synthesis of compounds of Formula I, wherein A is A ² , R ¹ and R ² are both —C(O)--R ^a , X ¹ and X ² are both —S—, Y ¹ and Y ² are both —(CH ₂ ) ₂ -, and Z ¹ and Z ² are NH ₃ ⁺ (compound 4S in Scheme 2 ). Scheme 2 also shows the general routes (Routes A1 and A2) carried out to synthesize compounds of Formula I, wherein A is A ² , R ¹ and R ² are both —(C═O)R ^a , X ¹ and X ² are both —S—, and Y ¹ and Y ² are both —(CH ₂ ) ₂ -NHC(S)NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are the same:

Scheme 3 depicts the synthesis of compounds of Formula I, wherein A is A ¹ , R ¹ and R ² are both —C(O)R ^a , X ¹ and X ² are both —S—, Y ¹ and Y ² are both —(CH ₂ ) ₂ —, and Z ¹ and Z ² are —N(CH ₃ )H ₂ ⁺ (compound 8T in Scheme 3) or —N(CH ₃ ) ₂ H ⁺ (compound 9T in Scheme 3):

Scheme 4 depicts the synthesis of compounds of Formula I, wherein A is A ³ , R ¹ is —C(O)-R ^a , R ² is —C(O)-R ^a or methyl, X ¹ and X ² are both —S—, Y ¹ and Y ² are both —(CH ₂ ) ₂ -, Z ¹ and Z ² are NH ₃ ⁺ , and p is 1 (compound 4M in Scheme 4). Scheme 4 also shows the general routes (Routes A1, A2, B1, and B2) carried out to synthesize compounds of Formula I, wherein A is A ³ , R ¹ is —C(O)-R ^a , R ² is —(C═O)R ^a or methyl, X ¹ and X ² are both —S—, Y ¹ and Y ² are both —(CH ₂ ) ₂ -NHC(S)NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are the same:

In some embodiments, the synthesis of compounds of Formula I ^wherein R ¹ and R ² are both -C(O)-Ra comprises the following lipid reagents:

In an exemplary embodiment, the synthesis of compounds of Formula I according to Scheme 1 or 2 involves the following isothiocyanate or amine reagents in the reaction steps leading to the installation of cationic heads ^Z1 and ^Z2 :

Exemplary cationic moieties

Material

In some exemplary embodiments, the following precursors used to synthesize the specified compounds described herein are prepared according to known methods.

6,6'-Diiodo-6,6'-dideoxy-α,α'-trehalose (1T) was prepared from commercially available reagents following procedures described in the literature. J.M. García Fernández, C. Ortiz Mellet, J.L. Jiménez Blanco, J. Fuentes Mota, A. Gadelle, A. Coste-Sarguet, J. Defaye, Carbohydr. Res. 1995, 268, 57-71.

6,6'-Diiodo-6,6'-dideoxy-sucrose (1S) was prepared from commercially available reagents according to procedures described in the literature. See J. M. García Fernández, A. Gadelle, J. Defaye, Carbohydr. Res. 1994, 265, 249-269.

Methyl 6,6'-diiodo-6,6'-dideoxy-2,3,2',3',4'-penta-O-acetyl-β-maltoside (1M) was prepared from commercially available reagents following procedures described in the literature. G.O. Aspinall, R.C. Carpenter, L. Khondo, Carbohydr. Res. 1987, 165, 281-298.

As used herein, an arrow followed by a code in parentheses, such as "(→ code)", indicates that the preceding reagent provides a lipophilic tail ( ^R1 and ^R2 substituents in compounds of Formula I) or a cationic head ( ^Z1 and ^Z2 substituents in compounds of Formula I) during synthesis. For example, "hexanoic anhydride (→L1)" is intended to refer to the moiety L1 as described in the above table being bonded to the compound by using hexanoic anhydride.

Hexanoic anhydride (Cat. No. 2051-49-2, →L1) and octanoyl (Cat. No. 111-64-8, →L2), decanoyl (Cat. No. 112-13-0, →L3), 2-butylhexanoyl (Cat. No. 39053-77-5, →L3.1), 4-ethyloctanoyl (Cat. No. 16493-81-5, →L3.2), lauroyl (Cat. No. 112-16-3, →L4), myristoyl (Cat. No. 112-64-1, →L5), stearoyl (Cat. No. 112-76-5, →L7), and oleoyl (Cat. No. 112-77-6, →L7.1) chlorides were purchased from commercial sources.

2-(N-tert-Butyloxycarbonylamino)ethanethiol (Catalog No. 67385-09-5, C1SH), 2-(N,N-dimethylamino)ethyl isothiocyanate (Catalog No. 7097-89-4, →C3), 2-(4-methylpiperazin-1-yl)ethylamine (Catalog No. 934-98-5, →C7), 2-(4-isopropylpiperazin-1-yl)ethylamine (Catalog No. 132740-59-1, →C7.1), tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (Catalog No. 192130-34-0, →C 8), 2-morpholinoethylamine (Catalog No. 2038-03-1, →C9), 2-morpholinoethyl isothiocyanate (Catalog No. 2038-03-1, →C9), 1-(2-aminoethyl)piperidine (Catalog No. 63224-35-1, →C9.1), 2-(1H-imidazol-1-yl)ethylamine (Catalog No. 5739-10-6, →C10), 2-(1H-imidazol-2-yl)ethylamine (Catalog No. 19225-96-8, →C10.1), 2-(1H-imidazol-4-yl)ethyl-1-amine (Catalog No. 5 1-45-6, →C10.2), 2,2'-(piperazine-1,4-diyl)diethylamine (Catalog No. 6531-38-0, →C14), tert-butyl (2-aminoethyl)(ethyl)carbamate (Catalog No. 105628-63-5, →C15), N,N-diethylethylenediamine (Catalog No. 100-36-7, →C16), 1,4-bis-Boc-1,4,7-triazaheptane (Catalog No. 120131-72-8, →C18), 1-(2-aminoethyl)pyrrolidine (Catalog No. 7154- 73-6, →C47.1), N-(2-aminoethyl)-1,4-oxazepan (catalog number 878155-50-1, →C48), 1-(2-aminoethyl) homopiperidine (catalog number 51388-00-2, →C48.1), N-(2-aminoethyl)-N-(2-hydroxyethyl) carbamic acid tert-butyl ester (catalog number 364056-56-4, →OH1), N,N-bis(2-hydroxyethyl)ethylenediamine (catalog number 3197-06-6, →OH5), 3, 3'-((2-aminoethyl)azanediyl)bis(propan-1-ol) (Catalog No. 50331-68-5, →OH6), 4,4'-((2-aminoethyl)azanediyl)bis(butan-1-ol) (Catalog No. 197966-97-5, →OH7), 2-((2-aminoethyl)amino)-2-(hydroxymethyl)propane-1,3-diol (Catalog No. 1211477-35-8, →OH10), 2-(4-(2-aminoethyl)piperazin-1-yl )ethanol (Catalog No. 20542-08-9, → OH12), 1-(2-aminoethyl)piperidin-4-one (Catalog No. 1196887-97-4, → C54), N-(2-aminoethyl)-4-piperidinol (Catalog No. 129999-60-6, → C59), [1-(2-amino-ethyl)-piperidin-3-yl]-methanol (Catalog No. 857637-03-7, → C59.1), 2-(4-methoxy-piperidin-1-yl)-ethylamine (Catalog No. 911300-69-1, → C59.2 ), 2-(4,4-difluoropiperidin-1-yl)ethylamine (Catalog No. 605659-03-8, →C60), 4-(2-aminoethyl)-thiomorpholine (Catalog No. 53515-36-9, →C61), 4-(2-aminoethyl)thiomorpholine-1,1-dioxide (Catalog No. 89937-52-0, →C63), 1-[4-(2-aminoethyl)piperazin-1-yl]ethylene ketone (Catalog No. 148716-35-2, →C66), 4-(2-aminoethyl)piperazin-2-one (Catalog No. 145 625-71-4, →C68), 1-(2-aminoethyl)piperidine-4-carboxamide (Catalog No. 443897-95-8, →C69), [1-(2-aminoethyl)piperidin-4-yl]methanol dihydrochloride (Catalog No. 1311315-83-9, →C70), 2-[N-methyl-N-(tert-butoxycarbonyl)amino]ethanethiol (Catalog No. 134464-53-2, C2SH) and 2-(N,N-dimethylamino)ethanethiol (Catalog No. 108-02-1, C3SH) were purchased from commercial sources.

2-(N-tert-Butoxycarbonylamino)ethyl isothiocyanate (→C1), D.M.Kneeland, K.Ariga, V.M.Lynch, C.Y.Huang, E.V.Anslyn, J.Am.Chem.Soc.1993,115,10042-10055. 2-(N-Methyl-N-tert-butoxycarbonylamino)ethyl isothiocyanate (→C2), T.Kim, Y.-J.Kim, I.-H.Han, D.Lee, J.Ham , K.S. Kan, J.W. Lee, Bioorg. Med. Chem. Lett. 2015, 25, 62-66. 2-(N,N,N-Trimethylamino)ethyl isothiocyanate (→C4), Y.-J. Ghang, J.J. Lloyd, M.P. Moehlig, J.K. Arguelles, M. Mettry, X. Zhang, R.R. Julian, Q. Cheng, R.J. Hooley, Langmuir 2014, 30, 10160-10166. 2-(2-Aminoethyl)-1,3-di-(tert-butoxycarbonyl)guanidine (→C11), S.M. Hickey, T.D. Ashton, J.M. White, J. Li, R.L. Nation, H.Y. Yu, A.G. Elliott, M.S. Butler, J.X. Huang, M.A. Cooper, F.M. Pfeffer, RSC Adv. 2015, 5, 28582-28596. N,N-bis[2-(tert-butoxycarbonylamino)ethyl]ethylenediamine (→C43), Y. Wu, A. Kaur, E. Fowler, M. M. Wiedmann, R. Young, W. R. J. D. Galloway, L. Olsen, H. F. Sore, A. Chattopadhyay, T. T. -L. Kwan, W. Xu, S. J. Walsh, P. de Andrade, M. Janecek, S. Arumugam, L. S. Itzhaki, Y. Heng Lau, D. R. Spring, ACS Chem. Biol. 2019, 14, 526-533. And N,N-bis(2-acetamidoethyl)ethylenediamine (→C45) was prepared according to the procedure. L. Liu, M. Rozenman, R. Breslow, J. Am. Chem. Soc. 2002, 124, 12660-12661.

^N1 ,N1-dimethyl-N1-tert-butoxycarbonyl-diethylenetriamine (→C12), N-( ₃ -hydroxypropyl)-N-(tert-butoxycarbonyl)ethylenediamine (→OH2), and N-(4-hydroxybutyl) ^-N- (tert-butoxycarbonyl)ethylenediamine (→OH3) were synthesized from commercially available ^N1 , ^N1 -dimethyl-diethylenetriamine, N-(3-hydroxypropyl)ethylenediamine, and N-(4-hydroxybutyl)ethylenediamine, respectively, by sequential primary amine trifluoroacetylation with ethyl trifluoroacetate ^, carbamoylation of the remaining amino groups using Boc2O, and base-promoted trifluoroacetamide hydrolysis.

N-(2-Aminoethyl)-1,3-oxazolidine (Cat. No. 67626-78-2, → C47) was synthesized from commercially available N-( ₂ -hydroxyethyl)-1,3-oxazolidine (Cat. No. 20073-50-1) by sequential hydroxymesylation with methanesulfonyl chloride, substitution by sodium azide, and finally catalytic hydrogenation using H2-Pd/C.

4-(2-Aminoethyl)-thiomorpholine-1-oxide (→C62) was synthesized by sequential primary amine carbamylation with _Boc2O , thioether oxidation with m-chloroperbenzoic acid, and final acid-promoted carbamate cleavage.

^N1- (tert-butoxycarbonylmethyl) ^-N1- (2-isothiocyanatoethyl)-ethylenediamine (→C5) was synthesized from commercially available diethylenetriamine according to the following scheme (Scheme 5).

The synthetic intermediates 2T, 2S, 2M, 3T, 3S and 3M in the reaction sequences depicted in Schemes 1, 2 and 4 were prepared as follows:

6,6'-bis[2-(tert-butoxycarbonylamino)ethylthio)-α,α'-trehalose (2T). Under _N2 atmosphere, to a mixture of diiodotrehalose 1T (14.3 g, 25.44 mmol) and _Cs2CO3 ( _23.45 g, 71.23 mmol) in anhydrous DMF (150 mL) was added a solution of 2-(N-tert-butoxycarbonylamino)ethanethiol (C1SH, 13.6 mL, 76.32 mmol) in anhydrous DMF (100 mL). The reaction mixture was stirred at room temperature overnight, and the remaining solid in the suspension was decanted and filtered out. The solvent was then removed under reduced pressure, and the resulting residue was ground with _Et2O (500 mL), which now produced a sticky solid. The residue was filtered and ground with EtOH (250 mL), and the remaining solid was filtered out again. Finally, the solvent was evaporated to dryness to obtain analytically pure compound 2T (16.8 g) in almost quantitative yield. Chemical formula: C ₂₆ H ₄₈ N ₂ O ₁₃ S ₂ . Molecular weight: 660.26. ESI-MS (m/z) 705.51 ([M+HCO ₂ ] ⁻ ).

6,6'-bis[2-(tert-butoxycarbonylamino)ethylthio]sucrose (2S). To a mixture of diiodo-sucrose 1S (1.07 g, 1.9 mmol) and Cs ₂ CO ₃ (1.75 g, 5.33 mmol) in anhydrous DMF (30 mL) under N2 atmosphere was added 2-(tert-butoxycarbonylamino)ethanethiol (1.0 mL, 5.7 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was then removed under reduced pressure, and the resulting residue was ground with Et2O (100 mL), at which time a sticky solid was produced. The residue was filtered and ground with EtOH (50 mL), and the remaining solid was filtered out again. The solvent was finally evaporated to dryness to obtain an analytically pure compound 2S (1.25 g) in almost quantitative yield. Chemical formula: C26H48N2O13S2. Molecular weight: 660.26. ESI-MS(m/z)705.56([M+HCO2]-).

Methyl 6,6'-bis[2-(tert-butoxycarbonylamino)ethylthio]-β-maltoside (2M). To a mixture of methyl 6,6'-dideoxy-6,6'-diiodo-β-maltoside 1M (1.20 g, 1.5 mmol) and Cs ₂ CO ₃ (1.38 g, 4.20 mmol) in anhydrous DMF (40 mL) under N ₂ atmosphere was added 2-(tert-butoxycarbonylamino)ethanethiol (C1SH, 0.80 mL, 4.50 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with 1:1 Et ₂ O-toluene mixture (200 mL) and extracted with water (2×100 mL). The organic phase was dried over Na ₂ SO ₄ , filtered, and evaporated to dryness. The residue was chromatographed using a 1:1 EtOAc-petroleum ether mixture as eluent to give the peroxyacetylated intermediate in 90% yield (1.20 g). Acetate removal was quantitatively performed by treatment with NaOMe (0.67 mmol, 36 mg) in MeOH (25 mL) for 1 h, followed by neutralization with amberlite IR120 (H ⁺ ) ion exchange resin, filtration and evaporation to dryness. Chemical formula: C ₂₇ H ₂₀ N ₂ O ₁₃ S ₂ . Molecular weight: 674.82. ESI-MS (m/z) 719.50 ([M+HCO ₂ ] ⁻ ).

General procedure for exhaustive hydroxyl acylation of 2T, 2S and 2M to give reaction intermediates 3T, 3S and 3M. To a solution of the corresponding acylating agent (→L1 to L7.1, as listed in the above lipid reagent list, ₄₆ mmol for 2T and 2S; 38 mmol for 2M) in anhydrous DMF (20 mL) was added dropwise over 10 min at 0 °C under N2 inert atmosphere. The reaction mixture was allowed to warm to room temperature over 2 h and further stirred overnight. The reactants were then diluted in a 1:1 water-DCM mixture (400 mL). The organic layer was decanted and further washed with water (100 _mL ), _{2N H2SO4} (2 x 100 mL), saturated _NaHCO3 (100 mL) and water (100 mL), dried over _MgSO4 or _Na2SO4 , filtered and evaporated under reduced pressure. The resulting syrup residue was purified by column chromatography using the eluent indicated in each case to give the corresponding 3T, 3S or 3M reaction intermediates, wherein the ^Ra substituent is as defined by codes L1 to L7.1 in the above list of lipid reagents.

Compound 3T-L1 Eluent 1:4 EtOAc-petroleum ether Yield 82%

Compound 3T-L2 Eluent 1:4→2:3 EtOAc-petroleum ether Yield 37%

Compound 3T-L3 Eluent 1:10→1:3 EtOAc-petroleum ether Yield 54%

Compound 3T-L3.1 Eluent 1:10→1:3 EtOAc-petroleum ether Yield 51%

Compound 3T-L3.2 Eluent 1:10→1:3 EtOAc-petroleum ether Yield 47%

Compound 3T-L4 Eluent 1:10→1:5 EtOAc-petroleum ether Yield 27%

Compound 3T-L5 Eluent 1:10→1:5 EtOAc-petroleum ether Yield 63%

Compound 3T-L7 Eluent 1:10→1:5 EtOAc-petroleum ether Yield 34%

Compound 3T-L7.1 Eluent 1:10→1:5 EtOAc-petroleum ether Yield 39%

Compound 3S-L1 Eluent 1:4 EtOAc-petroleum ether Yield 72%

Compound 3S-L3 Eluent 1:10→1:3 EtOAc-petroleum ether Yield 17%

Compound 3S-L3 Eluent 1:10→1:3 EtOAc-petroleum ether Yield 62%

General procedure for hydrolysis of the tert-butoxycarbonyl group in the reaction intermediates 3T (→ 4T), 3S (→ 4S) and 3M (→ 4M). Trifluoroacetic acid (TFA, 10 mL) was added to a solution of 3T, 3S or 3M (1 mmol) in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 h, then the solvent was evaporated under reduced pressure, and trace acid was eliminated by repeated coevaporation with water. The resulting residue was lyophilized from 10 mM HCl to obtain the corresponding diamines 4T, 4S and 4M in quantitative yield as hydrochlorides. In some preferred embodiments, according to the above procedure, compounds of the following formula I characterized by structure 4T are synthesized according to Scheme 2: JLF19, JLF150, JLF20, JLF155, JLF23, JLF46.

In some preferred embodiments, following the above procedure, the following compounds of Formula I characterized by Structure 4S are synthesized according to Scheme 1: JLF50, JLF62.

General procedure for the synthesis of diisocyanate reaction intermediates 5T, 5S and 5M in Schemes 1, 2 and 4. To a solution of 4T, 4S or 4M (1 mmol) in a heterogeneous mixture of water and DCM (1: 1, mL) was added CaCO ₃ (0.48 g, 4.8 mmol) and Cl ₂ CS (0.22 ml, 2.4 mmol) in sequence. The mixture was vigorously stirred at room temperature for 1 h, and then transferred to a decanting funnel and diluted with water and DCM (1: 1, 100 mL). The organic layer was decanted and washed with water (50 mL) and saturated NaHCO ₃ aqueous solution (50 mL), dried over MgSO ₄ or Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give the target diisocyanate reaction intermediates 5T, 5S or 5M, wherein the ^Ra substituent is defined by the code in the lipid reagent list:

Compound 5T-L1: Yield 99%. Chemical formula: C ₅₄ H ₈₈ N ₂ O ₁₅ S ₄ . Molecular weight: 1133.54. ESI-MS (m/z) 1155.58 ([M+Na] ⁺ ).

Compound 5T-L3): Yield 90%. Chemical formula: C ₇₈ H ₁₃₆ N ₂ O ₁₅ S ₄ . Molecular weight: 1470.19. ESI-MS (m/z) 1514.87 ([M+HCO ₂ ] ^- ).

Compound 5T-L3.1: Yield 78%. Chemical formula: C ₇₈ H ₁₃₆ N ₂ O ₁₅ S ₄ . Molecular weight: 1470.19. ESI-MS (m/z) 1513.88 ([M+HCO ₂ ] ^- ).

Compound 5T-L3.2: Yield 94%. Chemical formula: C ₇₈ H ₁₃₆ N ₂ O ₁₅ S ₄ . Molecular weight: 1470.19. ESI-MS (m/z) 1487.22 ([M+NH ₄ ] ⁺ )

Compound 5S-L1: Yield 99%. Chemical formula: C ₅₄ H ₈₈ N ₂ O ₁₅ S ₄ . Molecular weight: 1133.54. ESI-MS (m/z) 1155.58 ([M+Na] ⁺ ).

Compound 5S-L3: Yield 90%. Chemical formula: C ₇₈ H ₁₃₆ N ₂ O ₁₅ S ₄ . Molecular weight: 1470.19. ESI-MS (m/z) 1514.87 ([M+HCO ₂ ] ^- ).

Compound 5M-L3: Yield 93%. Chemical formula: C ₆₉ H ₁₂₀ N ₂ O ₁₅ S ₄ . Molecular weight: 1329.96. ESI-MS (m/z) 1375.09 ([M+HCO ₂ ] ^- ).

General procedures for the synthesis of compounds of Formula I according to Schemes 1, 2 and 4, Routes A1, A2, B1 and B2.

Route A1 (Scheme 1, 2 or 4): To a solution of the corresponding diamine dihydrochloride (4T, 4S or 4M, 0.1 mmol) in DCM (10 mL) and triethylamine ( _Et3N , 0.3 mmol, 41 μL) is added sequentially a solution of the corresponding isothiocyanate reagent SCN-( _CH2 ) ₂ -Za ^{- PG1} (0.3 mmol) in DCM (5 mL). The reaction mixture is stirred at room temperature for 16 h while monitoring the pH to ensure that it remains alkaline (if the pH is found to be below neutral, it should be readjusted to slightly alkaline (pH about 8) by adding an aliquot of _Et3N ). After completion of the reaction (TLC reveals complete disappearance of the starting material 4T, 4S or 4M and the formation of a new spot at a higher _Rf ), the reaction mixture is concentrated to dryness and the crude material is purified by column chromatography using the eluent indicated in each case. The resulting intermediate with a Boc-protected amino group was then treated with a 1:1 DCM-TFA mixture (10 mL) at room temperature for 1 h, followed by evaporation of the solvent under reduced pressure. Traces of TFA were removed by coevaporation with toluene (3×10 mL). Lyophilization from 10 mM HCl gave the desired products 6T, 6S or 6M, with the yields indicated in each case.

In some embodiments, the following compounds of Formula I characterized by structure 6T are synthesized according to Scheme 1, Route A1: JLF31, JLF32, JLF33, JLF35, JLF43, JLF58, JLF78, JLJB617, JLJB618, JLJB619.

In some embodiments, the following compounds of Formula I characterized by structure 6S are synthesized according to Scheme 2, Route A1: JLF59, JLF60, JLF68, JLF69, JLF76, JLF77.

Route A2 (Scheme 1, 2 or 4): Similar to Route A1, Route A2 involves the coupling reaction of compounds of structure 4T, 4S or 4M with isothiocyanate reagent SCN-(CH ₂ ) ₂ -Z ¹ , using a similar protocol. After completion of the reaction, the reaction mixture is transferred to a decanting funnel and partitioned between DCM and 0.1N aqueous HCl (1:1, 20 mL). The organic layer is decanted, dried over MgSO ₄ or Na ₂ SO ₄ , filtered, and evaporated under reduced pressure. Column chromatography purification is performed in these cases. The resulting product is lyophilized from 10 mM HCl to give the desired product 6T, 6S or 6M, the yields are indicated in each case.

In some embodiments, the following compounds of Formula I characterized by structure 6T are synthesized according to Scheme 1, Route A2: JLF25, JLF26, JLF29, JLF30, JLF41, JLF42, JLF154, JLF158, JLF170, JLJB604, JLJB605.

In some embodiments, the following compounds of Formula I characterized by Structure 6S are synthesized according to Scheme 2, Route A2: JLF54, JLF55, JLF66, JLF67.

Route B1 (Scheme 1, 2 or 4): To a solution of the corresponding diisothiocyanate reaction intermediate 5T, 5S or 5M (1 mmol) in DCM (10 mL) was added a solution of the corresponding amine reagent H ₂ N-(CH ₂ ) ₂ -Z ^a -PG ¹ (0.3 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 16 h while monitoring the pH to ensure that it remained basic. After completion of the reaction (TLC revealed complete disappearance of the starting material 5T, 5S or 5M and the formation of a new spot at a lower R _f ), the reaction mixture was concentrated to dryness and the crude material was purified by column chromatography using the indicated eluent. The intermediate obtained was further treated with a 1:1 DCM-TFA mixture (10 mL) at room temperature for 1 h, followed by evaporation of the solvent under reduced pressure, co-evaporation of acidic traces with toluene (3×10 mL), and lyophilization from 10 mM HCl to give the desired product 6T, 6S or 6M in the indicated yields in each case.

In some embodiments, compounds of the following formula I characterized by structure 6T are synthesized according to Scheme 1, Route B1: JLF93, JLF94, JLF96, JLF97, JLF98, JLF102, JLF103, JLF111, JLF115, JLF120, JLF121, JLF139, JLF141, JLF200, JLF201, JLF220, PER20.

Route B2: Similar to Route B1, Route B2 involves the coupling reaction of compounds of structure 5T, 5S or 5M with the amine reagent _H2N- ( _CH2 ) ₂ - ^Z1 , using a similar protocol. After completion of the reaction, the reaction mixture is transferred to a decanting funnel and partitioned between DCM and 0.1N aqueous HCl (1:1, 20 mL). The organic layer is decanted, dried over _MgSO4 or _{Na2SO4 ,} filtered, and evaporated under reduced pressure. In these cases, column chromatography purification is not necessary. The resulting product is lyophilized from 10 mM HCl to give the desired product 6T, 6S or 6M in the indicated yield.

In some embodiments, compounds of Formula I characterized by Structure 6T are synthesized according to Scheme 1, Route B2: JLF81, JLF82, JLF87, JLF90, JLF95, JLF99, JLF106, JLF108, JLF110, JLF111, JLF125, JLF128, JLF130, JLF131, JLF134, JLF135, JLF138, JLF142, JLF163, JLF164, JLF165, JLF190, JLF197, JLF223, JLF234, JLF237, JLF238, JLF244, JLF245, JLF248, JLF301, JLF601, JLF602, JLF604, JLF605, JLF606, JLF607, JLF608, PRX 051, NC117, NC118.

In some embodiments, the following compounds of Formula I characterized by Structure 6S are synthesized according to Scheme 2, Route B2: JLF218, PRX052, PRX093, PRX094, PRX096.

In some embodiments, the following compounds of Formula I characterized by Structure 6M are synthesized according to Scheme 4, Route B2: JMB500, JMB501, JMB503.

General procedures for the synthesis of compounds of Formula I according to Scheme 3, Routes C1 and C2.

Following a procedure similar to the general procedure for the exhaustive hydroxyl acylation of 2T and 2S, the diiodotrehalose derivative 1T was peroxidoacylated to afford the above-mentioned reaction intermediates 3T and 3S. The resulting crude product was purified by column chromatography (1:10 EtOAc-petroleum ether) to afford the reaction intermediate 7T in 67% yield.

In a preferred embodiment, the diiodide reaction intermediate 7T is prepared according to this procedure, wherein the ^Ra substituent is defined by code L1 (7T-L1) as shown in the list of lipid reagents.

Route C1: To a mixture of 7T (0.1 mmol) and Cs ₂ CO ₃ (91 mg, 0.28 mmol) in anhydrous DMF (3 mL) under N ₂ atmosphere was added 2-[N-methyl-N-(tert-butoxycarbonyl)amino]ethanethiol (C 2 SH, 52 μL, 0.3 mmol), and the reaction mixture was stirred at room temperature overnight. The solvent was then removed under reduced pressure, and the resulting residue was purified by column chromatography (1:6 EtOAc-petroleum ether). The resulting intermediate was treated with a 1:1 DCM-TFA mixture (4 mL) at room temperature for 1 h. The solvent was evaporated under reduced pressure, trace acid was repeatedly co-evaporated with water, and the residue was lyophilized from 10-mM HCl to give 8T in 60% yield.

In a preferred embodiment, the compound of Formula I characterized by Structure 8T is synthesized according to Scheme 3, Route C1: JLF40.

Route C2: Similar to Route C1, Route C2 involves a coupling reaction between 7T and 2-(N,N-dimethylamino)ethanethiol (C3SH), following a similar protocol. After completion of the reaction, the reaction mixture was transferred to a decanting funnel and partitioned between DCM and 0.1N aqueous HCl (1:1, 20 mL). The organic layer was decanted, dried over MgSO ₄ or Na ₂ SO ₄ , filtered, and evaporated under reduced pressure. In this case, column chromatography purification was unnecessary. The resulting product was lyophilized from 10 mM HCl to afford compound 8T in 82% yield.

In a preferred embodiment, the compound of Formula I characterized by Structure 8T is synthesized according to Scheme 3, Route C2: JLF48.

Example 4a - Compound JLF19

Synthesized in quantitative yield from compound 3T-L7 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₁₂₄ H ₂₃₈ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 2132.29 ESI-MS (m/z) 1031.08 ([M-2Cl] ²⁺ ).

Example 4b - Compound JLF20

Synthesized in quantitative yield from compound 3T-L5 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₁₀₀ H ₁₉₀ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1795.64 ESI-MS (m/z) 1723.23 ([M-2HCl+H] ⁺ ), 862.20 ([M-2Cl] ²⁺ ).

Example 4c - Compound JLF23

Synthesized in quantitative yield from compound 3T-L3 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₇₆ H ₁₄₂ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1458.99 ESI-MS (m/z) 1385.93 ([M-2HCl+H] ⁺ ), 693.98 ([M-2Cl] ²⁺ ).

Example 4d - Compound JLF25

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L5 and the corresponding isothiocyanate reagent (Code C3) were synthesized. Yield: 72%. Chemical formula: C ₁₁₀ H ₂₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2056.06. ESI-MS (m/z) 992.56 ([M-2Cl] ²⁺ ).

Example 4e - Compound JLF26

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L5 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 76%. Chemical formula: C ₁₁₂ H ₂₁₄ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2267.02 ESI-MS (m/z) 1006.49 ([M-2I] ²⁺ ).

Example 4f - Compound JLF29

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L7 and the corresponding isothiocyanate reagent (Code C3) were synthesized. Yield 77%. Chemical formula: C ₁₃₂ H ₂₅₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2264.65 ESI-MS (m/z) 1160.74 ([M-2Cl] ²⁺ ).

Example 4g - Compound JLF30

According to Scheme 1, Route A2 (4T to 6T steps), compound 4T-L7 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 87%. Chemical formula: C ₁₃₄ H ₂₅₈ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2575.61 ESI-MS (m/z) 1174.68 ([M-2I] ²⁺ ).

Example 4h - Compound JLF31

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L5 and the corresponding isothiocyanate reagent (Code C2) were synthesized. Yield 91%. Chemical formula: C ₁₀₈ H ₂₀₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2028.00 ESI-MS (m/z) 978.06 ([M-2Cl] ²⁺ .

Example 4i - Compound JLF32

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L7 and the corresponding isothiocyanate reagent (Code C1) were synthesized. Yield 66%. Chemical formula: C ₁₂₈ H ₂₄₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2308.54 ESI-MS (m/z) 1132.20 ([M-2Cl] ²⁺ ).

Example 4j - Compound JLF33

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L7 and the corresponding isothiocyanate reagent (Code C2) were synthesized. Yield 86%. Chemical formula: C ₁₃₀ H ₂₅₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 2336.60 ESI-MS (m/z) 1146.25 ([M-2Cl] ²⁺ ).

Example 4k - Compound JLF35

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L1 and the corresponding isothiocyanate reagent (Code C5) were used for synthesis. Yield 89%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₄ N ₈ O ₁₉ S ₄ . Molecular weight: 1601.78 ESI-MS (m/z) 728.30 ([M-2HCl-2Cl] ²⁺ ).

Example 41 - Compound JLF40

According to Scheme 3, Route C2 (7T to 8T steps), it was synthesized from precursor 7T-L1 and HS-(CH ₂ ) ₂ NMe ₂ (C3SH). Yield 82%. Chemical formula: C ₅₆ H ₁₀₂ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1178.45 ESI-MS (m/z) 1105.68 ([M-HCl-Cl] ⁺ ), 553.47 ([M-2Cl] ²⁺ ).

Example 4m - Compound JLF41

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L3 and the corresponding isothiocyanate reagent (Code C3) were synthesized. Yield 55%. Chemical formula: C ₈₆ H ₁₆₂ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1719.41 ESI-MS (m/z) 1645.95 ([M-HCl-Cl] ⁺ ), 824.06 ([M-2Cl] ²⁺ ).

Example 4n - Compound JLF42

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L3 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 57%. Chemical formula: C ₈₈ H ₁₆₆ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1930.37 ESI-MS (m/z) 838.06 ([M-2I] ²⁺ ).

Example 4o - Compound JLF43

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L3 and the corresponding isothiocyanate reagent (Code C2) were used for synthesis. Yield: 52%. Chemical formula: C ₈₄ H ₁₅₈ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1691.36. ESI-MS (m/z) 810.09 ([M-2Cl] ²⁺ ).

Example 4p - Compound JLF46

Synthesized in quantitative yield from compound 3T-L7.1 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₁₂₄ H ₂₂₆ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 2021.19 ESI-MS (m/z) 1024.38 ([M-2Cl] ²⁺ ).

Example 4q - Compound JLF48

According to Scheme 3, Route C1 (7T to 8T steps), it was synthesized from precursor 7T-L1 and HS-(CH ₂ ) ₂ N(Boc)Me(C2SH). Yield 60%. Chemical formula: C ₅₄ H ₉₈ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1150.40 ESI-MS (m/z) 1077.63 ([M-HCl-Cl] ⁺ ), 536.44 ([M-2Cl] ²⁺ ).

Example 4r - Compound JLF50

Synthesized from compound 3S-L1 in quantitative yield according to Scheme 2 (3S to 4S steps). Chemical formula: C ₅₂ H ₉₄ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1122.34 ESI-MS (m/z) 1049.63 ([M-HCl-Cl] ⁺ ), 525.44 ([M-2Cl] ²⁺ ).

Example 4s - Compound JLF54

According to Scheme 2, Route A2 (4S to 6S steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code C3) were used to synthesize the product. Yield: 59%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1382.76. ESI-MS (m/z) 1309.71 ([M-HCl-Cl] ⁺ ), 655.57 ([M-2Cl] ²⁺ ).

Example 4t - Compound JLF55

According to Scheme 2, Route A2 (4S to 6S steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 59%. Chemical formula: C ₆₄ H ₁₁₈ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1593.72 ESI-MS (m/z) 1338.63 ([M-HI-I] ⁺ ), 1338.63 ([M-HI-Me ₃ NI] ⁺ ), 669.80 ([M-2I] ²⁺ ), 639.99 ([MI-Me ₃ NI] ²⁺ ).

Example 4u - Compound JLF58

According to Scheme 1, Route A1 (4T to 6T steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code OH1) were synthesized. Yield 69%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1414.76 ESI-MS (m/z) 1341.70 ([M-HCl-Cl] ⁺ ), 671.61 ([M-2Cl] ²⁺ ).

Example 4v - Compound JLF59

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code C1) were synthesized. Yield: 62%. Chemical formula: C ₅₈ H ₁₀₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1326.65 ESI-MS (m/z) 1253.69 ([M-HCl-Cl] ⁺ ), 627.59 ([M-2Cl] ²⁺ ).

Example 4w - Compound JLF60

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code C2) were used for synthesis. Yield: 49%. Chemical formula: C ₆₀ H ₁₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1354.71 ESI-MS (m/z) 1284.69 ([M-HCl-Cl] ⁺ ), 641.59 ([M-2Cl] ²⁺ ).

Example 4x - Compound JLF62

Synthesized from compound 3S-L3 in quantitative yield according to Scheme 2 (3S to 4S steps). Chemical formula: C ₇₆ H ₁₄₂ Cl ₂ N ₂ O ₁₅ S ₂ . Molecular weight: 1458.99 ESI-MS (m/z) 1385.98 ([M-2HCl+H] ⁺ ), 693.95 ([M-2Cl] ²⁺ ).

Example 4y - Compound JLF66

According to Scheme 2, Route A2 (4S to 6S steps), compound 4S-L3 and the corresponding isothiocyanate reagent (code C3) were synthesized. Yield 81%. Chemical formula: C ₈₆ H ₁₆₂ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1719.41 ESI-MS (m/z) 1646.99 ([M-HCl-Cl] ⁺ ), 824.14 ([M-2Cl] ²⁺ ).

Example 4z - Compound JLF67

According to Scheme 2, Route A2 (4S to 6S steps), compound 4S-L3 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 85%. Chemical formula: C ₈₈ H ₁₆₆ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1930.37 ESI-MS (m/z) 1615.91 ([M-HI-I] ⁺ ), 838.21 ([M-2I] ²⁺ ).

Example 4aa - Compound JLF68

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L3 and the corresponding isothiocyanate reagent (code C1) were synthesized. Yield 57%. Chemical formula: C ₈₂ H ₁₅₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1663.30 ESI-MS (m/z) 1590.90 ([M-HCl-Cl] ⁺ ), 796.06 ([M-2Cl] ²⁺ ).

Example 4ab - Compound JLF69

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L3 and the corresponding isothiocyanate reagent (code C2) were synthesized. Yield 73%. It was synthesized by coupling of compound 4S-L3 and C2 from Route A1. The overall yield of 4S-L3 (2 steps) was 73%. Chemical formula: C ₈₄ H ₁₅₈ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1691.36 ESI-MS (m/z) 1618.92 ([M-HCl-Cl] ⁺ ), 810.09 ([M-2Cl] ²⁺ ).

Example 4ac - Compound JLF76

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L1 and the corresponding isothiocyanate reagent (code C5) were synthesized. Yield 66%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₄ N ₈ O ₁₉ S ₄ . Molecular weight: 1601.78 ESI-MS (m/z) 1454.66 ([M-4HCl-H] ^- ).

Example 4ad - Compound JLF77

According to Scheme 2, Route A1 (4S to 6S steps), compound 4S-L3 and the corresponding isothiocyanate reagent (code C5) were synthesized. Yield: 62%. Chemical formula: C ₉₀ H ₁₇₀ Cl ₄ N ₈ O ₁₉ S ₄ . Molecular weight: 1938.43. ESI-MS (m/z) 897.20 ([M-2HCl-2Cl] ²⁺ ).

Example 4ae - Compound JLF78

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L3 and the corresponding isothiocyanate reagent (Code C5) were synthesized. Yield 68%. Chemical formula: C ₉₀ H ₁₇₀ Cl ₄ N ₈ O ₁₉ S ₄ . Molecular weight: 1938.43 ESI-MS (m/z) 897.20 ([M-2HCl-2Cl] ²⁺ ).

Example 4af - Compound JLF81

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C9). Yield 86%. Chemical formula: C ₆₆ H ₁₁₈ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1466.84 ESI-MS (m/z) 1393.77 ([M-HCl-Cl] ⁺ ), 697.87 ([M-2Cl] ²⁺ ).

Example 4ag - Compound JLF82

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C7). Yield 66%. Chemical formula: C ₆₈ H ₁₂₆ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1565.84 ESI-MS (m/z) 1419.81 ([M-3HCl-Cl] ⁺ ), 710.90 ([M-2HCl-2Cl] ²⁺ ), 1418.96 ([M-4HCl-H] ^- ).

Example 4ah - Compound JLF87

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C10). Yield 89%. Chemical formula: C ₆₄ H ₁₀₈ Cl ₂ N ₈ O ₁₅ S ₄ . Molecular weight: 1428.75 ESI-MS (m/z) 1355.67 ([M-HCl-Cl] ⁺ ), 678.77 ([M-2Cl] ²⁺ ).

Example 4ai - Compound JLF90

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C10.2). Yield 73%. Chemical formula: C ₆₄ H ₁₀₈ Cl ₂ N ₈ O ₁₅ S ₄ . Molecular weight: 1428.75 ESI-MS (m/z) 1355.68 ([M-HCl-Cl] ⁺ ), 678.77 ([M-2Cl] ²⁺ ), 1354.61 ([M-2HCl-H] ^- ).

Example 4aj - Compound JLF93

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C8). Yield 81%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1537.78 ESI-MS (m/z) 1391.74 ([M-3HCl-Cl] ⁺ ), 696.90 ([M-2Cl] ²⁺ ), 647.65 ([M-hexanoyl-2Cl] ²⁺ ), 1390.84 ([M-4HCl-H] ^- ).

Example 4ak - Compound JLF94

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C11). Yield 95%. Chemical formula: C ₆₀ H ₁₁₀ Cl ₂ N ₁₀ O ₁₅ S ₄ . Molecular weight: 1410.74 ESI-MS (m/z) 1337.68 ([M-HCl-Cl] ⁺ ), 669.73 ([M-2Cl] ²⁺ ), 1336.62 ([M-2HCl-H] ^- ).

Example 4al - Compound JLF95

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C10.1). Yield 42%. Chemical formula: C ₆₄ H ₁₀₈ Cl ₂ N ₈ O ₁₅ S ₄ . Molecular weight: 1428.75 ESI-MS (m/z) 1355.65 ([M-HCl-Cl] ⁺ ), 678.79 ([M-2Cl] ²⁺ ), 1354.62 ([M-2HCl-H] ^- ).

Example 4am - Compound JLF96

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH2). Yield 97%. Chemical formula: C ₆₄ H ₁₁₈ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1442.81 ESI-MS (m/z) 1369.76 ([M-HCl-Cl] ⁺ ), 685.79 ([M-2Cl] ²⁺ ).

Example 4an - Compound JLF97

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH3). Yield 34%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1470.87 ESI-MS (m/z) 1397.78 ([M-HCl-Cl] ⁺ ), 699.81 ([M-2Cl] ²⁺ ).

Example 4ao - Compound JLF98

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C11). Yield 99%. Chemical formula: C ₈₄ H ₁₅₈ Cl ₂ N ₁₀ O ₁₅ S ₄ . Molecular weight: 1747.38 ESI-MS (m/z) 838.07 ([M-2Cl] ²⁺ ).

Example 4ap - Compound JLF99

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C9). Yield 45%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1803.48 ESI-MS (m/z) 1731.03 ([M-HCl-Cl] ⁺ ), 866.19 ([M-2Cl] ²⁺ ).

Example 4aq - Compound JLF102

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C18). Yield 94%. Chemical formula: C ₆₂ H ₁₁₈ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1485.71 ESI-MS (m/z) 1339.69 ([M-HCl-Cl] ⁺ ), 670.54 ([M-2Cl] ²⁺ ), 1338.93 ([M-2HCl-H] ^- ).

Example 4ar - Compound JLF103

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C18). Yield 62%. Chemical formula: C ₈₆ H ₁₆₆ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1822.36 ESI-MS (m/z) 1677.15 ([M-3HCl-Cl] ⁺ ), 839.10 ([M-2HCl-2Cl] ²⁺ ).

Example 4as - Compound JLF106

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C14). Yield 94%. Chemical formula: C ₆₂ H ₁₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1378.73 ESI-MS (m/z) 1305.94 ([M-HCl-Cl] ⁺ ), 653.86 ([M-2Cl] ²⁺ ), 1350.66 ([M-2HCl+HCO ₂ ] ^- ).

Example 4at - Compound JLF108

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C7). Yield 78%. Chemical formula: C ₉₂ H ₁₇₄ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1902.49 ESI-MS (m/z) 1757.06 ([M-3HCl-Cl] ⁺ ), 879.22 ([M-2HCl-2Cl] ²⁺ ), 1755.25 ([M-4HCl-H] ^- ).

Example 4au - Compound JLF110

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C10). Yield 72%. Chemical formula: C ₈₈ H ₁₅₆ Cl ₂ N ₈ O ₁₅ S ₄ . Molecular weight: 1765.40 ESI-MS (m/z) 1691.98 ([M-HCl-Cl] ⁺ ), 847.16 ([M-2Cl] ²⁺ ).

Example 4av - Compound JLF111

According to Scheme 1, Route B2 (5T to 6T steps) was synthesized from diisothiocyanate precursor 5T-L3 and the corresponding amine reagent (code C10.2). Yield 49%. Synthesized from Route B1 by coupling of compound 5T-L3 and C10.2. The overall yield of 5T-L3 (2 steps) was 49%. Chemical formula: C ₈₈ H ₁₅₆ Cl ₂ N ₈ O ₁₅ S ₄ . Molecular weight: 1765.70ESI-MS (m/z) 1692.93 ([M-HCl-Cl] ⁺ ), 847.13 ([M-2Cl] ²⁺ ), 1691.14 ([M-2HCl-H] ^- ).

Example 4aw - Compound JLF115

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C8). Yield 68%. Chemical formula: C ₉₀ H ₁₇₀ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1874.43 ESI-MS (m/z) 1571.05 ([M-3HCl-Cl] ⁺ ), 865.23 ([M-2HCl-2Cl] ²⁺ ).

Example 4ax - Compound JLF120

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C43). Yield 88%. Chemical formula: C ₆₆ H ₁₃₀ Cl ₆ N ₁₀ O ₁₅ S ₁₀ . Molecular weight: 1644.76 ESI-MS (m/z) 1426.74 ([M-5HCl-Cl] ⁺ ), 713.56 ([M-4HCl-2Cl] ²⁺ ), 1424.65 ([M-6HCl-H] ^- ).

Example 4ay - Compound JLF121.

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisothiocyanate precursor 5T-L3 and the corresponding amine reagent (code C43). Yield 55%. It was synthesized from Route B1 by coupling of compound 5T-L3 and C43. The overall yield of 5T-L3 (2 steps) was 55%. Chemical formula: C ₉₀ H ₁₇₈ Cl ₆ N ₁₀ O ₁₅ S ₁₀ . Molecular weight: 1981.41ESI-MS (m/z) 1763.11 ([M-5HCl-Cl] ⁺ ), 882.20 ([M-4HCl-2Cl] ²⁺ ), 1761.32 ([M-6HCl-H] ^- ).

Example 4az - Compound JLF125

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C16). Yield 60%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1438.87 ESI-MS (m/z) 1365.77 ([M-HCl-Cl] ⁺ ), 683.99 ([M-2Cl] ²⁺ ), 1364.81 ([M-2HCl-H] ^- ).

Example 4ba - Compound JLF128

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisothiocyanate precursor 5T-L3 and the corresponding amine reagent (code C18). Yield 76%. It was synthesized from Route B2 by coupling compound 5T-L3 and C18. The yield of 5T-L3 was 76%. Chemical formula: C ₉₀ H ₁₇₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1775.52ESI-MS (m/z) 1703.07 ([M-HCl-Cl] ⁺ ), 852.21 ([M-2Cl] ²⁺ ), 1701.28 ([M-2HCl-H] ^- ).

Example 4bb - Compound JLF130

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH12). Yield 71%. Chemical formula: C ₇₀ H ₁₃₀ Cl ₄ N ₈ O ₁₇ S ₄ . Molecular weight: 1625.89 ESI-MS (m/z) 1479.81 ([M-3HCl-Cl] ⁺ ), 740.95 ([M-2HCl-2Cl] ²⁺ ), 1478.81 ([M-4HCl-H] ^- ).

Example 4bc - Compound JLF131

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C46). Yield 40%. Chemical formula: C ₉₈ H ₁₈₂ Cl ₂ N ₁₀ O ₁₉ S _4. Molecular weight: 2003.73ESI-MS (m/z) 1931.17 ([M-HCl-Cl] ⁺ ), 966.35 ([M-2Cl] ²⁺ ), 1929.40 ([M-2HCl-H] ^- ), 1965.40 ([M-HCl-H] ^- ).

Example 4bd - Compound JLF134

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH5). Yield 74%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₂ N ₆ O ₁₉ S _4. Molecular weight: 1502.87ESI-MS (m/z) 1429.97 ([M-HCl-Cl] ⁺ ), 715.89 ([M-2Cl] ²⁺ ), 1428.72 ([M-2HCl-H] ^- ).

Example 4be - Compound JLF135

According to Scheme 1, Route B2 (steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH10). Yield 61%. Chemical formula: C ₆₆ H ₁₂₂ Cl ₂ N ₆ O ₂₁ S ₄ . Molecular weight: 1534.86 ESI-MS (m/z) 1461.79 ([M-HCl-Cl] ⁺ ), 731.90 ([M-2Cl] ²⁺ ), 1460.71 ([M-2HCl-H] ^- ).

Example 4bf - Compound JLF138

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C7.1). Yield 90%. Chemical formula: C ₇₂ H ₁₃₄ Cl ₄ N ₆ O ₁₅ S ₄ . Molecular weight: 1621.95 ESI-MS (m/z) 1475.94 ([M-3HCl-Cl] ⁺ ), 739.04 ([M-2HCl-2Cl] ²⁺ ), 1475.01 ([M-4HCl-H] ^- ).

Example 4bg - Compound JLF139

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C15). Yield 99%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1382.76 ESI-MS (m/z) 1309.84 ([M-HCl-Cl] ⁺ ), 655.88 ([M-2Cl] ²⁺ ), 1308.96 ([M-2HCl-H] ^- ).

Example 4bh - Compound JLF141

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C12). Yield 78%. Chemical formula: C ₆₆ H ₁₂₆ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1541.82 ESI-MS (m/z) 1395.90 ([M-3HCl-Cl] ⁺ ), 698.68 ([M-2HCl-2Cl] ²⁺ ), 1394.92 ([M-4HCl-H] ^- ).

Example 4bi - Compound JLF142

According to Scheme 1, Route B2 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code OH5). Yield 39%. Chemical formula: C ₆₆ H ₁₂₆ Cl ₄ N ₈ O ₁₅ S ₄ . Molecular weight: 1541.82 ESI-MS (m/z) 1767.15 ([M-3HCl-Cl] ⁺ ), 884.24 ([M-2HCl-2Cl] ²⁺ ), 1765.37 ([M-4HCl-H] ^- ).

Example 4bj - Compound JLF150

Synthesized in quantitative yield from precursor 3T-L2 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₆₄ H ₁₁₈ Cl ₂ N ₂ O ₁₅ S ₄ . Molecular weight: 1290.67. ESI-MS (m/z) 1217.91 ([M-HCl-Cl] ⁺ ), 609.45 ([M-2Cl] ²⁺ ).

Example 4bk - Compound JLF154

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L2 and the corresponding isothiocyanate reagent (code C9) were synthesized. Yield 72%. Chemical formula: C ₇₈ H ₁₄₂ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1635.16. ESI-MS (m/z) 1561.98 ([M-HCl-Cl] ⁺ ), 781.99 ([M-2Cl] ²⁺ ).

Example 4b1 - Compound JLF155

Synthesized in quantitative yield from diamine precursor 3T-L4 according to Scheme 1 (3T to 4T steps). Chemical formula: C ₈₈ H ₁₆₆ Cl ₂ N ₂ O ₁₅ S ₄ . Molecular weight: 1627.32. ESI-MS (m/z) 1555.21 ([M-HCl-Cl] ⁺ ), 778.15 ([M-2Cl] ²⁺ ).

Example 4bm - Compound JLF158

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L4 and the corresponding isothiocyanate reagent (code C9) were synthesized. Yield 61%. Chemical formula: C ₁₀₂ H ₁₉₀ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1971.81. ESI-MS (m/z) 950.19 ([M-2Cl] ²⁺ ).

Example 4bn - Compound JLF163

According to Scheme 1, Route B2 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C30). Yield 87%. Chemical formula: C ₆₈ H ₁₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1450.80. ESI-MS (m/z) 1377.69 ([M-HCl-Cl] ⁺ ), 689.41 ([M-2Cl] ²⁺ ).

Example 4bo - Compound JLF164

According to Scheme 1, Route B2 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (Code C9.1). Yield 99%. Chemical formula: C ₆₈ H ₁₂₂ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1462.89. ESI-MS (m/z) 1389.81 ([M-HCl-Cl] ⁺ ), 695.45 ([M-2Cl] ²⁺ ).

Example 4bp - Compound JLF165

According to Scheme 1, Route B2 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH6). Yield 72%. Chemical formula: C ₇₀ H ₁₃₂ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1558.79. ESI-MS (m/z) 1485.80 ([M-HCl-Cl] ⁺ ), 743.90 ([M-2Cl] ²⁺ ).

Example 4bq - Compound JLF170

According to Scheme 1, Route B2 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C9). Yield 86%. Chemical formula: C ₁₁₄ H ₂₁₄ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 2140.13. ESI-MS (m/z) 2106.80 ([M-HCl-Cl] ⁺ ), 1034.29 ([M-2Cl] ²⁺ ).

Example 4br - Compound JLF200

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C52). Yield 68%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1382.76. ESI-MS (m/z) 1309.80 ([M-HCl-Cl] ⁺ ), 655.44 ([M-2Cl] ²⁺ ).

Example 4bs—Compound JLF201

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (Code C53). Yield 70%. Chemical formula: C ₆₀ H ₁₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1354.71. ESI-MS (m/z) 1281.7 ([M-HCl-Cl] ⁺ ), 641.42 ([M-2Cl] ²⁺ ).

Example 4bt - Compound JLF218

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4S-L3 and the corresponding isothiocyanate reagent (code C9) were synthesized. Yield 56%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1803.48. ESI-MS (m/z) 1731.08 ([M-HCl-Cl] ⁺ ), 866.11 ([M-2Cl] ²⁺ ).

Example 4bu - Compound JLF220

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code C65). Yield 79%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₁₀ O ₁₅ S ₄ . Molecular weight: 1438.79. ESI-MS (m/z) 1365.77 ([M-HCl-Cl] ⁺ ), 683.45 ([M-2Cl] ²⁺ ).

Example 4bv - Compound JLF223

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L1 and the corresponding amine reagent (code OH7). Yield 75%. Chemical formula: C ₇₄ H ₁₃₈ Cl ₂ N ₆ O ₁₉ S ₄ . Molecular weight: 1615.08. ESI-MS (m/z) 1541.90 ([M-HCl-Cl] ⁺ ), 771.97 ([M-2Cl] ²⁺ ).

Example 4bw - Compound JLF234

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C70). Yield 51%. Chemical formula: C ₉₄ H ₁₇₄ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1859.59. ESI-MS (m/z) 1787.35 ([M-HCl-Cl] ⁺ ), 894.28 ([M-2Cl] ²⁺ ).

Example 4bx - Compound JLF237

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C26). Yield 23%. Chemical formula: C ₈₈ H ₁₆₀ Cl ₂ N ₁₆ O ₁₅ S ₄ . Molecular weight: 1881.49. ESI-MS (m/z) 905.17 ([M-2Cl] ²⁺ ).

Example 4bx - Compound JLF238

According to Scheme 1, Route B1 (4T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C59.2). Yield 66%. Chemical formula: C ₉₄ H ₁₇₄ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1859.59. ESI-MS (m/z) 894.19 ([M-2Cl] ²⁺ ).

Example 4by—Compound JLF244

According to Scheme 1, Route B1 (4T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C63). Yield 66%. Chemical formula: C ₉₄ H ₁₇₂ Cl ₂ N ₈ O ₁₇ S ₄ . Molecular weight: 1885.59. ESI-MS (m/z) 1813.12 ([M-HCl-Cl] ⁺ ), 907.18 ([M-2Cl] ²⁺ ).

Example 4bz - Compound JLF245

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C68). Yield 60%. Chemical formula: C ₉₀ H ₁₆₄ Cl ₂ N ₈ O ₁₇ S ₄ . Molecular weight: 1829.48. ESI-MS (m/z) 1757.08 ([M-HCl-Cl] ⁺ ), 879.15 ([M-2Cl] ²⁺ ).

Example 4ca - Compound JLF248

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (Code C59.1). Yield 58%. Chemical formula: C ₉₄ H ₁₇₄ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1859.59. ESI-MS (m/z) 1786.13 ([M-HCl-Cl] ⁺ ), 894.17 ([M-2Cl] ²⁺ ).

Example 4cb - Compound JLF301

According to Scheme 1, Route B1 (Steps 4T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C66). Yield 77%. Chemical formula: C ₉₄ H ₁₇₂ Cl ₂ N ₈ O ₁₇ S ₄ . Molecular weight: 1885.59. ESI-MS (m/z) 1812.25 ([M-HCl-Cl] ⁺ ), 907.19 ([M-2Cl] ²⁺ ).

Example 4cc - Compound JLJB604

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L1 and the corresponding isothiocyanate reagent (Code C3) were used for synthesis. Yield 69%. Chemical formula: C ₆₂ H ₁₁₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1382.76 ESI-MS (m/z) 1309.62 ([M-HCl-Cl] ⁺ ), 655.53 ([M-2Cl] ²⁺ ).

Example 4cd - Compound JLJB605

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L1 and the corresponding isothiocyanate reagent (code C4) were synthesized. Yield 94%. Chemical formula: C ₆₄ H ₁₁₈ I ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1593.72 ESI-MS (m/z) 1278.58 ([MI-Me ₃ NI] ⁺ ), 669.80 ([M-2I] ²⁺ ), 1382.20 ([M-2HI+Cl] ^- ), 1323.49 ([M-HI-Me ₃ NI+HCO ₂ ] ⁺ ).

Example 4ce - Compound JLJB617

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L3 and the corresponding isothiocyanate reagent (Code C1) were synthesized. Yield: 21%. Chemical formula: C ₈₂ H ₁₅₄ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1663.30. ESI-MS (m/z) 795.97 ([M-2Cl] ²⁺ ).

Example 4cf - Compound JLJB618

According to Scheme 1, Route A1 (Steps 4T to 6T), compound 4T-L5 and the corresponding isothiocyanate reagent (Code C1) were synthesized. Yield 50%. Chemical formula: C ₁₀₆ H ₂₀₂ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1999.95 ESI-MS (m/z) 1927.39 ([M-HCl-Cl] ⁺ ), 964.29 ([M-2Cl] ²⁺ ).

Example 4cg - Compound JLJB619

According to Scheme 1, Route A2 (Steps 4T to 6T), compound 4T-L1 and the corresponding isothiocyanate reagent (Code C2) were used for synthesis. Yield: 52%. Chemical formula: C ₆₀ H ₁₁₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1354.71 ESI-MS (m/z) 1281.40 ([M-HCl-Cl] ⁺ ), 641.22 ([M-2Cl] ²⁺ ).

Example 4ch - Compound PER20

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code OH1). Yield 59%. Chemical formula: C ₈₆ H ₁₆₂ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1751.41 ESI-MS (m/z) 840.14 ([M-2Cl] ²⁺ ).

Example 4ci - Compound JLF190

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C9.1). Yield 87%. Chemical formula: C ₉₂ H ₁₇₀ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1799.54. ESI-MS (m/z) 1727.11 ([M-HCl-Cl] ⁺ ), 864.11 ([M-2Cl] ²⁺ ).

Example 4cj - Compound JLF601

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C47). Yield 10%. Chemical formula: C ₈₈ H ₁₆₀ N ₆ O ₁₇ S ₄ . Molecular weight: 1702.51. ESI-MS (m/z) 1701.11 ([MH] ^- ).

Example 4ck - Compound JLF602

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C48). Yield 84%. Chemical formula: C ₉₂ H ₁₇₀ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1831.54. ESI-MS (m/z) 880.17 ([M-2Cl] ²⁺ ).

Example 4cl - Compound JLF604

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C54). Yield 24%. Chemical formula: C ₉₂ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1827.51. ESI-MS (m/z) 1773.11 ([M-HCl-Cl+H ₂ O] ⁺ ), 896.21 ([M-2Cl+2H ₂ O] ²⁺ ).

Example 4cm - Compound JLF605

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C59). Yield 58%. Chemical formula: C ₉₂ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1831.54. ESI-MS (m/z) 1759.10 ([M-HCl-Cl] ⁺ ), 880.15 ([M-2Cl] ²⁺ ).

Example 4cn - Compound JLF606

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C60). Yield 90%. Chemical formula: C ₉₂ H ₁₆₆ Cl ₂ F ₄ N ₆ O ₁₅ S ₄ . Molecular weight: 1871.50. ESI-MS (m/z) 1799.06 ([M-HCl-Cl] ⁺ ), 900.11 ([M-2Cl] ²⁺ ).

Example 4co - Compound JLF607

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C61). Yield 85%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₆ . Molecular weight: 1835.51. ESI-MS (m/z) 1762.30 ([M-HCl-Cl] ⁺ ), 881.65 ([M-2Cl] ²⁺ ).

Example 4cp - Compound JLF608

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C62). Yield 51%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₆ . Molecular weight: 1867.60. ESI-MS (m/z) 1795.05 ([M-HCl-Cl] ⁺ ), 898.12 ([M-2Cl] ²⁺ ).

Example 4cq - Compound JLF197

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisothiocyanate precursor 5T-L3 and the corresponding amine reagent (code C63). Yield 95%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₉ S ₆ . Molecular weight: 1899.60. ESI-MS (m/z) 1827.02 ([M-2Cl] ²⁺ ).

Example 4cr - Compound PRX051

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (Code C48.1). Yield 86%. Chemical formula: C ₉₂ H ₁₇₀ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1827.59. ESI-MS (m/z) 1755.18 ([M-HCl-Cl] ⁺ ), 878.18 ([M-2Cl] ²⁺ ).

Example 4cs - Compound PRX052

According to Scheme 1, Route B1 (5T to 6T steps), it was synthesized from diisocyanate precursor 5T-L3 and the corresponding amine reagent (code C47.1). Yield 90%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1771.49. ESI-MS (m/z) 1699.11 ([M-HCl-Cl] ⁺ ), 850.12 ([M-2Cl] ²⁺ ).

Example 4ct - Compound PRX093

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5S-L3 and the corresponding amine reagent (code C10). Yield 88%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1765.40. ESI-MS (m/z) 1692.15 ([M-HCl-Cl] ⁺ ), 847.10 ([M-2Cl] ²⁺ ).

Example 4cu - Compound PRX094

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5S-L3 and the corresponding amine reagent (code C59). Yield 76%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1831.54. ESI-MS (m/z) 1759.23 ([M-HCl-Cl] ⁺ ), 880.18 ([M-2Cl] ²⁺ ).

Example 4cv - Compound PRX096

According to Scheme 1, Route B1 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5S-L3 and the corresponding amine reagent (code C66). Yield 63%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₅ S ₄ . Molecular weight: 1885.59. ESI-MS (m/z) 1812.23 ([M-HCl-Cl] ⁺ ), 907.22 ([M-2Cl] ²⁺ ).

Example 4cw - Compound NC117

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5S-L3.1 and the corresponding amine reagent (code C9). Yield 76%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1803.48. ESI-MS (m/z) 1731.20 ([M-HCl-Cl] ⁺ ), 866.17 ([M-2Cl] ²⁺ ).

Example 4cx - Compound NC118

According to Scheme 1, Route B2 (Steps 5T to 6T), it was synthesized from diisocyanate precursor 5S-L3.2 and the corresponding amine reagent (code C9). Yield 65%. Chemical formula: C ₉₀ H ₁₆₆ Cl ₂ N ₆ O ₁₇ S ₄ . Molecular weight: 1803.48. ESI-MS (m/z) 1731.21 ([M-HCl-Cl] ⁺ ), 866.18 ([M-2Cl] ²⁺ ).

Example 4cy - Compound JMB500

According to Scheme 4, Route B2 (5M to 6M steps), it was synthesized from diisocyanate precursor 5M-L3 and the corresponding amine reagent (code C9). Yield 91%. Chemical formula: C ₈₁ H ₁₅₀ Cl ₂ N ₆ O ₁₆ S ₄ . Molecular weight: 1663.26. ESI-MS (m/z) 1591.13 ([M-HCl-Cl] ⁺ ), 796.10 ([M-2Cl] ²⁺ ).

Example 4cz - Compound JMB501

According to Scheme 4, Route B2 (5M to 6M steps), it was synthesized from diisocyanate precursor 5M-L3 and the corresponding amine reagent (code C10.1). Yield 89%. Chemical formula: C ₇₉ H ₁₄₀ Cl ₂ N ₆ O ₁₆ S ₄ . Molecular weight: 1625.17. ESI-MS (m/z) 1552.16 ([M-HCl-Cl] ⁺ ), 777.06 ([M-2Cl] ²⁺ ).

Example 4da - Compound JMB503

According to Scheme 4, Route B2 (5M to 6M steps), it was synthesized from diisocyanate precursor 5M-L3 and the corresponding amine reagent (code C59). Yield 95%. Chemical formula: C ₈₃ H ₁₅₄ Cl ₂ N ₆ O ₁₆ S ₄ . Molecular weight: 1691.31. ESI-MS (m/z) 1619.17 ([M-HCl-Cl] ⁺ ), 810.13 ([M-2Cl] ²⁺ ).

Example 5 - Intramuscular vaccination

Forty female BALB/c mice were divided into six groups. One group received only buffer as a negative control, and the other five groups received 1 μg of saRNA, which encoded the hemagglutinin (HA) protein of influenza strain California/7/2009, formulated with different oligosaccharides (JRL13, JLF102, JLF97, JLF94). All saRNA preparations were applied intramuscularly to one of the legs. All saRNA groups were formulated with N/P 6 for different oligosaccharides, or with N/P 12 for linear polyethyleneimine 22.5kDa (AVROXA, Ghent, Belgium) PEI500 as a benchmark. The N/P ratio was calculated based on the oligosaccharide-RNA or secondary amine-RNA charge ratio of the saRNA formulated with PEI. For all cases, the compound formulation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final RNA concentration was 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide-containing solution was 9:1 (volume:volume). Polyethyleneimine was prepared by mixing RNA-containing solution and polymer solution in an equal volume ratio. Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days, and spleens were extracted at the end of the experiment. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified by IFN–ELISPOT of splenocytes. Figure 14A is a graph illustrating the hydrodynamic diameter of a composition comprising saRNA complexed with different oligosaccharides. Figure 14B illustrates the serological levels of specific IgG for HA during the experiment. Figure 14C illustrates the endpoint titration of the serological levels of specific IgG for HA. FIG. 14D exemplifies CD8/CD4 responses to HA peptide pools.

Among all tested groups, JLF94 carrying a guanidine group showed the best overall performance at both the humoral and cellular levels (Figure 14B to Figure 14D). JLF97 elicited a strong humoral response, but the overall T-cell response was lower compared to JLF94 (Figure 14D).

Example 6 - Surfactant Effect

Fifteen female BALB/c mice are divided into three research groups. All groups receive the same amount of luciferase encoding modRNA (2 μg) prepared, and intramuscular application is applied to each leg. Each group receives the combination of 1:0.5 molar ratio (oligosaccharide: Tween (20/40/60)) of JLF99 and Tween20, Tween40 or Tween60. All groups receive modRNA prepared with N/P 6. N/P ratio is calculated based on oligosaccharide-RNA charge ratio. For all cases, in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), composite formulation, RNA final concentration is 0.1mg/ml, and containing RNA solution and containing JLF99+Tween (20/40/60) solution The mixing ratio is 9:1 (volume: volume). Preparation is characterized before injection to confirm hydrodynamic diameter. In vivo luciferase expression in applied tissues was measured at five time points (6 hours, day 1, day 2, day 3, day 6). FIG. 15A is a bar graph illustrating the hydrodynamic diameter of a composition comprising modRNA formulated with JLF99 and different polysorbates at N/P 6. FIG. 15B illustrates quantitative bioluminescence at different injection sites at different time points during the experiment. FIG. 15C illustrates quantitative bioluminescence in the liver at 6 hours and 24 hours after injection of modRNA.

Example 7 - Intramuscular vaccination using JLF99 and modRNA

Fifteen female BALB/c mice were divided into three research groups. All groups received the same amount (1 μg) of the preparation modRNA of the hemagglutinin (HA) protein encoding influenza strain California/7/2009. All preparations were applied intramuscularly in one leg according to the initial immunity/boosting schedule (day 0/day 28). All modRNA groups were prepared with N/P 6. The N/P ratio was calculated based on the ratio of amine to phosphate charge. One group received the modRNA prepared with the combination of JLF99 and Tween20, and the mol ratio of the combination of the JLF99 and Tween20 was 1:0.5 (oligosaccharide: Tween20). One group received the modRNA prepared with benchmark LNP, and the benchmark LNP included ionizable lipids, cholesterol, DSPC and C14-pSar23 with a mol ratio of 47.5:38.5:10:4. One group only received buffer injection as a negative control. The composition of JLF99 was complexed with RNA in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.05 mg/ml, and the mixing ratio of RNA-containing solution and JLF99+Tween20-containing solution was 9:1 (volume:volume).

LNP preparations are produced in HEPES buffered sucrose (HBS, HEPES 10mM, 10% weight/volume sucrose) according to the standard LNP production method widely described in the literature. See Nogueira et al., ACS Appl.Nano.Mater., 3(11): 10634-10645(2020). In short, LNPs are produced in a molar ratio of 3: 1 mixing an ethanol solution of a lipid mixture with a citrate buffer solution (pH 4.5) of modRNA, and a mixing speed of 12mL/min. The LNPs are then dialyzed for 3 hours and finally exchanged to HBS.

Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to influenza HA peptides. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. Quantitative virus neutralization titers were determined by VNT. CD4/CD8 T cell responses were quantified by IFN–ELISPOT of spleen cells. Figure 16A illustrates the serological levels of specific IgG for HA during the experiment. Figure 16B illustrates influenza-specific neutralization titers during the experiment. Figure 16C illustrates the endpoint titration of serological levels of specific IgG for HA. Figure 16D illustrates the CD8/CD4 response to the HA peptide pool.

The results presented (Figure 16A to Figure 16D) show that the compositions provided provide powerful immunological results, which are at least comparable to the current LNP standards in the art. In Figure 16A, during the experiment, after the evolution of IgG for HA antigen, no significant difference between the JLF99 preparation and the LNP benchmark was detected. The same findings were obtained using the virus neutralization titer shown in Figure 16B. Finally, the antibody concentration at the experimental endpoint (49th day) as illustrated in Figure 16C also shows that the serological concentration of IgG is comparable to the LNP group. The CD4-CD8 reaction of the LNP benchmark and JLF99 does not show significant differences (Figure 16D).

Example 8 - Intramuscular vaccination using JLF99 and different RNA platforms

Twenty female BALB/c mice were divided into four research groups. All groups received the same amount (1 μg) of different RNA platforms, which all encoded the hemagglutinin (HA) protein of influenza strain California/7/2009, and were prepared with JLF99 and Tween20 (1:0.5 molar ratio). In the first group, mice received the RNA platform consisting of trans-amplification RNA (taRNA), which was composed of two kinds of RNA: one encoding viral replicase, and another RNA encoding HA antigen. In the second group, mice received RNA comprising self-amplification RNA (saRNA). In the third group, RNA was composed of m1Y modified mRNA (modRNA). In the third group, mice only received buffer as a negative control. All preparations were applied intramuscularly in one of the legs according to the initial exemption/boosting schedule (i.e., the dosage applied on the 0th and 28th days). All preparations were prepared with N/P 6. The JLF99-containing formulation was complexed with RNA in MBG buffer (final concentration 5% weight/volume glucose, 10 mM MES, pH 6.1), the final RNA concentration was 0.05 mg/ml, and the mixing ratio of the RNA-containing solution and the JLF99+Tween20-containing solution was 9:1 (volume:volume).

Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days. Spleens were extracted at the end of the experiment, and the response of CD4/CD8 T cells to influenza HA peptides was quantified. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified by IFN–ELISPOT of splenocytes. Figure 17A is a diagram illustrating the serological levels of specific IgG for HA with different RNA complexes at different time points during the experiment, and the RNA complexes were formulated with JLF99. Figure 17B illustrates the endpoint titration of the serological levels of specific IgG for HA with an RNA platform formulated with JLF99. Figure 17C shows the CD8/CD4 response to the HA peptide pool, which comes from different RNA platforms formulated with JLF99.

This embodiment illustrates that oligosaccharides described herein provide the delivery of a variety of different mRNAs. In this experiment, the length of RNA varies greatly: saRNA is about 9200 nucleotides in length, taRNA is about 7200 nucleotides in length (replicase RNA) and about 2100 nucleotides (HA mRNA), and modRNA is 2100 nucleotides in length. Regardless of the length of RNA and the replication or non-replication properties of RNA, JLF99 functionally delivers all RNAs after intramuscular injection and induces a strong immune response.

Example 9 - Effects of intravenously delivered heterocyclic amines on the liver

This example compares two preparations of modRNA (luciferase encoding) complexed with either JLF99 or JLF218.

Six female BALB/c mice were divided into two groups. One group received modRNA formulated with JLF218 and Tween40. The other group received modRNA formulated with JLF99 and Tween40. A total of 2 μg of prepared modRNA was injected into the orbit. After 6 hours, the in vivo biodistribution was quantified by measuring the luciferase expression in different organs. All RNA groups were prepared with N/P 6. The N/P ratio was calculated based on the ratio of amine to phosphate charge. In MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.02 mg/ml, and the mixing ratio of RNA solution and oligosaccharide+Tween40 solution was 9: 1 (volume: volume). Figure 18 A is a diagram illustrating the average liver radiance of an example composition comprising JFL99 or JLF218.

FIG. 18B is an image showing bioluminescence of the abdominal axis 6 hours after injection of the example formulations into each group.

Example 10 - Adjuvant properties of vaccines

This example reports the adjuvant properties of six different formulations of the present disclosure:

preparation Oligosaccharides additive Molar ratio (oligosaccharide: additive) 1 JLF218 Tween40 1:0.5 2 JLF99 Tween40 1:0.5 3 JLF110 Tween40 1:0.5 4 JLF244 Tween40 1:0.5 5 JLF605 Tween40 1:0.5 6 JLF238 Tween40 1:0.5

All groups receive modRNA prepared with N/P 6. For all cases, in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), preparation is compounded with RNA, RNA final concentration is 0.15mg/ml, and the mixing ratio containing RNA solution and containing oligosaccharide+Tween40 solution is 9:1 (volume: volume). MSD test kit standard protocol was used to assess the secretion of IL-6, TNF-α, IL-1β and IFN-γ in the supernatant after transfection of 10 ⁶ hPMBCs for 24 hours.

Figure 19A is a graph illustrating IL-6 secretion by hPBMC after exposure to different doses of test formulations. Figure 19B is a graph illustrating TNF-α secretion by hPBMC after exposure to different doses of test formulations. Figure 19C is a graph illustrating IL-1β secretion by hPBMC after exposure to different doses of test formulations. Figure 19D is a graph illustrating IFN-γ secretion by hPBMC after exposure to different doses of test formulations. In the second part of the experiment, the same test formulation was used in the vaccination study.

Thirty-five female BALB/c mice were divided into seven groups. Each group received 0.5 μg of modRNA encoding the hemagglutinin (HA) protein of influenza strain California/7/2009 intramuscularly in one leg according to the prime/boost schedule (day 0/day 28). A total of 6 different formulations with HA encoding modRNA were tested:

preparation Oligosaccharides additive Molar ratio (oligosaccharide: additive) 1 JLF218 Tween40 1:0.5 2 JLF99 Tween40 1:0.5 3 JLF110 Tween40 1:0.5 4 JLF244 Tween40 1:0.5 5 JLF605 Tween40 1:0.5 6 JLF238 Tween40 1:0.5

One group received buffer as a negative control.

All groups received modRNA formulated with N/P6. The N/P ratio was calculated based on the oligosaccharide-RNA charge ratio. For all cases, the compound formulation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final RNA concentration was 0.025mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide+Tween40 solution was 9:1 (volume: volume). Animals were non-invasively serologically monitored at different time points during the experiment (Day 0, Day 14, Day 21, Day 28, Day 49), and their spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to influenza HA peptides. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of splenocytes. Figure 19E is a diagram illustrating the serological levels of specific IgG for HA formulated with different oligosaccharides. Figure 19F is a graph illustrating an endpoint titration of serological levels of specific IgG against HA. Figure 19G is a bar graph illustrating CD8/CD4 responses against a pool of HA peptides. Figure 19H is a bar graph illustrating the hydrodynamic diameters of different test formulations.

The results presented illustrate that JLF99 and other tested oligosaccharides exhibit strong adjuvant properties. As shown in Figures 19A to 19D, JLF244 triggered the highest levels of proinflammatory cytokines IL-6, TNF-α, IL-1β and IFN-γ secretion, while JLF110 caused a moderate level of secretion, followed by JLF99 and JLF218. The secretion level of proinflammatory cytokines is related to the immune response to HA antigens triggered. As shown in Figure 19E, the highest IgG concentration was detected in JLF244, followed by JLF110, and then JLF99 and JLF218.

Example 11 - Mycobacterium tuberculosis-Ag85a antigen

This example illustrates the use of antigens associated with tuberculosis. The Ag85a antigen of tuberculosis is used here. A total of fifteen female BALB/c are divided into three groups. Each group receives 1 μg of modRNA in one leg intramuscularly according to the primary immunity/boosting schedule (day 0/day 21), and the modRNA encodes the secretable form of Mycobacterium tuberculosis Ag85a protein. A total of 2 different preparations with AG85a encoding modRNA were tested: a preparation comprising JLF99 and Tween40 (1:0.5 molar ratio) and LNP benchmark. The last group of receiving buffer is used as a negative control. All groups receive modRNA prepared with N/P6. In MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the JLF99 preparation is compounded, and the final concentration of RNA is 0.05mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide+Tween40 solution is 9:1 (volume:volume). Benchmark LNP comprises ionizable lipid, cholesterol, DSPC and C14-pSar23 in a molar ratio of 47.5:38.5:10:4. LNP preparations are produced in hepes buffered sucrose (HBS, hepes 10mM, 10% weight/volume sucrose) according to the standard LNP production method widely described in the literature. In brief, LNP is produced in a mixing ratio of 3:1 mixing of an ethanol solution of a lipid mixture with a citrate buffer solution (pH 4.5) of modRNA, and a mixing speed of 12mL/min. Then LNP is dialyzed for 3 hours and finally exchanged to HBS.

Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 49) over 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to the AG85a peptide. Anti-AG85a antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of splenocytes. Figure 20A illustrates the serological levels of specific IgG for HA during the present embodiment. Figure 20B illustrates the endpoint titration of the serological levels of specific IgG for HA. Figure 20C illustrates the CD8/CD4 response to the HA peptide pool.

Example 12 - Structural changes in oligosaccharides for vaccination

This example examines the effects of structural changes in the oligosaccharides described herein when incorporated into vaccines. Thirty female BALB/c were divided into six groups. Each group received 0.5 μg modRNA intramuscularly in one leg according to the primary immunization/boost schedule (day 0/day 28), the modRNA encoding the hemagglutinin (HA) protein of the influenza strain California/7/2009. A total of 5 different formulations with HA encoding modRNA were tested:

preparation Oligosaccharides additive Molar ratio (oligosaccharide: additive) 1 JLF99 Tween40 1:0.5 2 JLF110 Tween40 1:0.5 3 JLF301 Tween40 1:0.5 4 JLF608 Tween40 1:0.5 5 JLF197 Tween40 1:0.5

The last group received buffer as a negative control.

All groups received modRNA formulated with N/P6. For all cases, the compound formulation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.025mg/ml, and the mixing ratio of RNA-containing solution and paOs+Tween40 solution was 9:1 (volume: volume). Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) within 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to influenza HA peptides. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of spleen cells. Figure 21A illustrates the serological levels of specific IgG for HA during the embodiment. Figure 21B illustrates the endpoint titration of the serological levels of specific IgG for HA. Figure 21C exemplifies the CD8/CD4 response to the HA peptide pool.

In the data set, JLF197 has the lowest calculated theoretical pKa, followed by JLF608<JLF301<JLF110<JLF110.

Example 13 - Further study of oligosaccharide structural changes

This embodiment examines the effects of structural changes in oligosaccharides described herein when incorporated into vaccines comprising taRNA. Twenty female BALB/c mice were divided into five groups. Each group received 0.5 μg of taRNA intramuscularly in one leg according to the primary immunization/boosting schedule (day 0/day 28), and the taRNA encoded hemagglutinin (HA) protein of influenza strain California/7/2009. A total of 5 different preparations with HA encoding taRNA were tested:

preparation Oligosaccharides additive Molar ratio (oligosaccharide: additive) 1 JLF99 Tween40 1:0.5 2 JLF218 Tween40 1:0.5 3 JLF110 Tween40 1:0.5 4 JLF244 Tween40 1:0.5 5 JLF99 Tween40 1:0.5

The last group is replication-defective taRNA, so HA protein cannot be expressed, and the taRNA is prepared as a negative control together with JLF99 and Tween40.

All groups received taRNA formulated with N/P6. For all preparations, the compound preparation was prepared in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final concentration of RNA was 0.025mg/ml, and the mixing ratio of RNA-containing solution and oligosaccharide+Tween40 solution was 9:1 (volume: volume). Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) within 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to influenza HA peptides. Anti-influenza HA antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of spleen cells. Figure 22A illustrates the serological levels of specific IgG for HA during the experiment. Figure 22B illustrates the endpoint titration of the serological levels of specific IgG for HA. FIG. 22C exemplifies CD8/CD4 responses to HA peptide pools.

Example 14 - Antigenic changes

JLF99 was further tested with different viral antigens, and its immunogenicity was much lower than that of influenza HA. In this embodiment, twelve female C57BL/6IFNAR-/- mice were divided into two research groups. All groups received the same amount (1.3 μg) of prepared saRNA, which encodes the glycoprotein (Gc) of Crimean-Congo hemorrhagic fever virus (CCHFV). All preparations were applied intramuscularly in one of the legs according to the initial immunization/boosting schedule (Day 0/Day 28). All saRNA groups were prepared with N/P6.

The first group received saRNA formulated in a combination of JLF99 and Tween40 (1:0.5 molar ratio), and the second group received saRNA formulated in a benchmark LNP. The JLF99 formulation was compounded in MBG buffer (final concentration 5% weight/volume glucose, 10mM MES, pH 6.1), the final RNA concentration was 0.065mg/ml, and the mixing ratio of the RNA-containing solution and the JLF99+Tween40 solution was 9:1 (volume:volume). The benchmark LNP contained ionizable lipids, cholesterol, DSPC, and C14pSar23 in a molar ratio of 47.5:38.5:10:4. The LNP formulation was produced in hepes buffered sucrose (HBS, hepes 10mM, 10% weight/volume sucrose) according to the standard LNP production method widely described in the literature. Briefly, LNPs were produced by mixing an ethanol solution of a lipid mixture with a citrate buffer solution of modRNA (pH 4.5) at a ratio of 3:1, and the mixing speed was 12 mL/min. The LNPs were then dialyzed for 3 hours and finally exchanged to HBS.

Animals were non-invasively serologically monitored at different time points (day 0, day 14, day 21, day 28, day 49) over 49 days, and spleens were extracted at the end of the experiment to quantify the response of CD4/CD8 T cells to CCHFV-Gc peptides. Anti-CCHFV-Gc antibodies in serum were quantified by enzyme-linked immunosorbent assay. CD4/CD8 T cell responses were quantified via IFN–ELISPOT of splenocytes. Figure 23A illustrates the serological levels of specific IgG for Gc during the experiment. Figure 23B illustrates the endpoint titration of the serological levels of specific IgG for Gc. Figure 23C illustrates the T cell response to the Gc peptide.

JLF99 was able to elicit a strong immune response, even against low immunogenic antigens, such as CCHFV-Gc. Antibodies at the time point (day 14) were lower than those of LNP, but after day 28 (before boost), antibody levels were the same as those achieved with the LNP benchmark. T cell responses were equally strong under both test conditions.

Claims (46)

1. A compound of formula I: or a pharmaceutically acceptable salt thereof, wherein: A is ^A1 , ^A2 or ^A3 : R ¹ and R ² are each independently selected from H, ^Ra and -C(O) ^-Ra , wherein at least one instance of R ¹ or R ² is not H; Each ^{Ra is} independently selected from _C1 - _C20 aliphatic, _C3 - _C20 alicyclic, _C5 - _C6 aryl, 3-12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, wherein each ^Ra is optionally substituted by one or more ^Rb ; each R ^b is independently selected from halogen, -N ₃ , -R ^c , -OR ^c , -SR ^c , -NHR ^c , -C(O)-R ^c , -OC(O)R ^c , -NHC(O)R ^c , -C(O)NHR ^c and -NHC(O)NHR ^c ; each R ^c is independently selected from optionally substituted C ₁ -C ₂₀ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; ^X1 and ^X2 are each independently selected from -S- and -NH-; ^Y1 and ^Y2 are each independently an optionally substituted _C1-30 aliphatic group, wherein one or more carbons are optionally and independently replaced by -Cy-, -NH-, -NHC(O)-, -C(O)NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)NH-, -NHC(S)NH-, -C(S)NH-, -C(O)NHSO2- _, -SO2NHC(O)-, -OC(O)O-, -O-, -C(O)-, -OC(O)-, -C(O)O-, _{-SO-, or -SO2- ;} each Cy is independently an optionally substituted C ₃ -C ₁₄ alicyclic, a 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, and an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S; ^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ , Each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ; each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; and p is an integer selected from 1, 2, 3, 4 or 5; The premise is that when A is A ¹ , then: (i) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ - and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , (ii) each of R ¹ and R ² is not –(CH ₂ ) ₅ -CH ₃ or –(CH ₂ ) ₁₃ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , (iii) each of R ¹ and R ² is not –CO-(CH ₂ ) ₄ -CH ₃ , X ¹ and X ² are not both –S-, Y ¹ and Y ² are not both –(CH ₂ ) ₂ -NH-C(S)-NH-(CH ₂ ) ₂ -, and Z ¹ and Z ² are not both –N ⁺ (H) ₃ , and (iv) Cy is not a triazole group. 2. The compound of claim 1, wherein the compound has formula Ia: or a pharmaceutically acceptable salt thereof. 3. The compound of claim 1, wherein the compound has Formula Ib: or a pharmaceutically acceptable salt thereof. 4. The compound of claim 1, wherein the compound has Formula Ic: or a pharmaceutically acceptable salt thereof. 5. The compound of any one of claims 1 to 4, wherein ^R1 and ^R2 are each independently selected from ^Ra and -C(O) ^-Ra , and ^Ra is _C1 - _C20 aliphatic. 6. The compound of any one of claims 1-5, wherein ^R1 and ^R2 are each -C(O)-R ^a , and R ^a is C ₁ -C ₁₄ aliphatic. 7. The compound of any one of claims 1-6, wherein each ^Ra is a _C5 - _C10 straight chain alkyl group. 8. The compound of claim 1, wherein R ¹ and R ² are each -C(O)-R ^a , and each R ^a is independently selected from 9. The compound of any one of claims 1-8, wherein ^X1 and ^X2 are each -S-. 10. The compound of any one of claims 1 to 9, wherein Y ¹ and Y ² are each selected from C ₁ -C ₁₀ aliphatic, C ₀ -C ₄ -aliphatic-NHC(O)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-NHC(S)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-C(O)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-C(S)NH-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-NHC(O)-C ₀ -C ₄ aliphatic, C ₀ -C ₄ -aliphatic-NHSO ₂ -C ₀ -C ₄ aliphatic, and C ₀ -C ₄ -aliphatic-C(O)-C ₀ -C ₄ aliphatic. 11. The compound of any one of claims 1-10, wherein ^Y1 and ^Y2 are each _C1 - _C4 aliphatic -NHC(S)NH- _C1 - _C4 aliphatic. 12 . The compound of claim 1 , wherein Y ¹ and Y ² are each —CH ₂ —CH ₂ —NHC(S)NH—CH ₂ —CH ₂ —. 13. The compound of any one of claims 1 to 12, wherein the moiety ^X1 - ^Y1 - ^Z1 is 14. The compound of any one of claims 1 to 13, wherein the moiety ^X2 - ^Y2 - ^Z2 is 15. The compound of any one of claims 1 to 14, wherein Z ¹ and Z ² are each independently selected from: N ⁺ (M) ₃ , 16. The compound of any one of claims 1 to 14, wherein Z ¹ and Z ² are each independently selected from: 17. The compound of claim 1, wherein the oligosaccharide is a compound of formula Id: or a pharmaceutically acceptable salt thereof, wherein n and m are each independently selected from 0, 1, 2, 3, 4, 5 or 6. 18. The compound of claim 17, wherein the oligosaccharide is a compound of formula Id-i: or a pharmaceutically acceptable salt thereof. 19. The compound of any one of claims 1-18, further comprising one or more suitable counterions. 20. The compound of claim 1, wherein the compound is selected from Table 1. 21. A complex comprising the compound according to any one of claims 1 to 20 and a nucleic acid. 22. The complex of claim 21, wherein the nucleic acid is RNA. 23. The complex of claim 22, wherein the RNA is modRNA, saRNA, taRNA or uRNA. 24. The complex of claim 22 or 23, wherein the N/P ratio is less than 20:1. 25. The composite of claim 24, wherein the N/P ratio is less than or equal to 12:1. 26. The complex of any one of claims 21-25, wherein the complex has a diameter of about 30 nm to about 300 nm. 27. The complex of any one of claims 21-26, wherein the complex has a diameter of about 50 nm to about 200 nm. 28. The complex of any one of claims 21-27, further comprising a pharmaceutically acceptable surfactant. 29. The complex of claim 28, wherein the pharmaceutically acceptable surfactant is a polysorbate. 30. The complex of claim 28 or 29, wherein the molar ratio of the compound to the pharmaceutically acceptable surfactant is from about 1:0.0075 to about 1:3. 31. A method of increasing or causing an increase in RNA expression in a target in a subject, comprising administering to the subject a complex as described in any one of claims 21-30. 32. The method of claim 31, wherein the target is selected from the group consisting of lung, liver, spleen, heart, brain, lymph node, bladder, kidney, and pancreas. 33. A method of treating a disease, disorder or condition in a subject comprising administering to the subject the complex of any one of claims 21-30. 34. The method of claim 33, wherein the disease, disorder or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease, or a rare disease. 35. The method of any one of claims 31-34, wherein the complex is administered intramuscularly. 36. The method of claim 35, wherein the complex has a diameter between 50 nm and 100 nm. 37. The method of any one of claims 31-34, wherein the complex is administered subcutaneously. 38. A compound as claimed in any one of claims 1 to 20 for use in medicine. 39. Use of the complex according to claim 21 in medicine. 40. Use of the complex of claim 21 for increasing RNA expression in a target or causing an increase in RNA expression. 41. Use of the complex of claim 21 for treating a disease, disorder or condition. 42. The use of claim 41, wherein the disease, disorder or condition is an infectious disease, cancer, a genetic disorder, an autoimmune disease or a rare disease. 43. A method for preparing an oligosaccharide of formula II: It comprises reacting a compound of formula III: Contacting with a compound of formula IV: in Each ^Ra is an optionally substituted _C1 - _C20 aliphatic; ^Z1 and ^Z2 are each independently a cationic or ionizable group selected from an optionally substituted 5- to 14-membered heterocyclyl ring having 1 to 3 heteroatoms selected from N, O and S, an optionally substituted 5- to 14-membered heteroaryl ring having 1 to 3 heteroatoms selected from N, O and S, -N ⁺ (M) ₃ , and each M is independently -C ₀ -C ₆ aliphatic-R ^z or -C ₀ -C ₆ aliphatic-N ⁺ (R ^z ) ₃ ; each R ^z is independently selected from H, optionally substituted C ₁ -C ₆ aliphatic, optionally substituted C ₃ -C ₂₀ alicyclic, optionally substituted C ₅ -C ₆ aryl, optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, and optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; or Two or more R ^z can be combined with the atoms to which they are attached to form an optionally substituted 3 to 12 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O and S, or an optionally substituted 4 to 12 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; ^Za - ^PG1 is ^Z1 , wherein one ^Rz has been replaced by a ^PG1 group; and PG ¹ is a suitable nitrogen protecting group, The prerequisite is that Z ¹ and Z ² are not both –N ⁺ (H) ₃ . 44. The method of claim 43, comprising first treating a compound of formula V contacting with an acid, and then contacting the resulting product with _CSCl2 to obtain a compound of formula III; Where ^PG2 is a suitable acid-sensitive nitrogen protecting group. 45. The method of claim 44, comprising subjecting a compound of formula VI to Contacting with a compound of formula VII: Ra ^- COCl VII To obtain the compound of formula V. 46. The method of claim 45, comprising treating a compound of formula VIII: Contacting with a compound of formula IX: HS(CH ₂ ) ₂ NHPG ² IX To obtain a compound of formula VI, Among them, LG is a suitable leaving group.