CN114349800B - Ferrocene derivative and synthetic method and application thereof - Google Patents
Ferrocene derivative and synthetic method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of organic chemical synthesis, and discloses a ferrocene derivative, a synthesis method and application thereof. The ferrocene derivative of the present invention comprises at least one of a divalent ferrocene derivative, a tetravalent ferrocene derivative, an octavalent ferrocene derivative or a fourteen-valent ferrocene derivative. In the oligonucleotide molecule, the succinimidyl ester of the ferrocene derivative can be modified on amino groups of nucleotides at different positions to multiply label the oligonucleotide molecule, and the oligonucleotide molecule is provided with ferrocene groups. Ferrocene has good electrochemical activity, so that the target nucleotide molecule can be used as an electrochemical sensing indicator in electrochemical detection, the detection sensitivity and the signal intensity can be effectively improved, and the method has wide application prospect in the field of gene detection.
Description
Technical Field
The invention belongs to the field of organic chemical synthesis, and particularly relates to a ferrocene derivative, and a synthesis method and application thereof.
Background
Ferrocene, also known as dicyclopentadiene, is a "sandwich" like compound formed from two cyclopentadiene anions and a ferrous ion. The special sandwich structure of ferrocene has outstanding aromaticity, is not easy to generate addition reaction and reduction reaction, is especially easy to generate electrophilic substitution reaction, synthesizes the derivative containing ferrocenyl functional group, and has much higher reactivity than benzene.
Based on the low toxicity, unique electrochemical activity and photochemical property, special redox and excellent stability of ferrocene and the derivative thereof, the ferrocene derivative can be used for modifying electrodes, functional materials, biological materials and some small molecules.
Nucleic acids are synthesized in basic units of nucleotides as important genetic materials for organisms. The nucleotide is often modified to form modified nucleotide, the synthesis of the modified nucleotide and the incorporation of the modified nucleotide into a nucleic acid sequence provide a plurality of possibilities for changing the chemical properties of the nucleotide, so that the practical application fields of the nucleotide, such as the biotechnology fields of gene detection, polymerase Chain Reaction (PCR), microarray and the like, are greatly widened; the modification group can be used as a labeling molecule for fluorescence and electrochemical detection. However, the signal generated by a single labeled modified nucleotide is weak, and needs to be recognized by the detection element at a higher concentration, which limits the improvement of the detection sensitivity.
Ferrocene derivatives can be used to modify nucleotides, for labelling of nucleotides, due to their excellent chemical properties. The ferrocene derivative disclosed in the related art has good prospect in the application of labeled nucleotide, but only the 5' end of the nucleotide can be modified, which has certain limit on improving detection signals and sensitivity. Therefore, it is of great importance to find ferrocene derivatives that can label nucleotides and increase the intensity and sensitivity of the detection signal.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. For this purpose, the invention provides a ferrocene derivative, a plurality of which can be simultaneously combined on an oligonucleotide molecule to label the oligonucleotide molecule.
The invention also provides a synthesis method of the ferrocene derivative.
The invention also provides application of the ferrocene derivative in modifying oligonucleotide molecules.
The invention also provides an oligonucleotide molecule modified with the ferrocene derivative.
The invention also provides a method for modifying the oligonucleotide molecules by the ferrocene derivatives.
The invention also provides application of the oligonucleotide molecule modified with the ferrocene derivative.
According to one aspect of the present invention, a ferrocene derivative is provided, having the structural formula:
in the method, in the process of the invention,
at least one of (a) and (b);
wherein,,
according to a preferred embodiment of the invention, there is at least the following advantageous effect:
1. the ferrocene derivative can be used as a nucleotide modification monomer, because the ferrocene derivative contains succinimidyl ester, the group can be connected to various oligonucleotide molecules with aliphatic amino sites, and the modification of the oligonucleotide molecules is realized.
2. Due to the existence of ferrocenyl, the ferrocene derivative is endowed with good electrochemical activity, and the intensity and sensitivity of detection signals can be effectively improved when the ferrocene derivative is used in electrochemical detection.
In some embodiments of the invention, the ferrocene derivative comprises at least one of a divalent ferrocene derivative, a tetravalent ferrocene derivative, an octavalent ferrocene derivative, or a fourteen valent ferrocene derivative.
According to a second aspect of the present invention, there is provided a method for synthesizing the ferrocene derivative, comprising the steps of:
s1: taking amino donor to react with ferrocenyl aldehyde substance to form ferrocene intermediate;
s2: reacting the ferrocene intermediate in the step S1 with N-succinimidyl carbonate to form a ferrocene derivative;
in the synthesis method of the divalent ferrocene derivative, the amino donor is amino alcohol, the ferrocene intermediate is double ferrocene amino alcohol, and the ferrocene derivative is double ferrocene amino alcohol succinimidyl ester;
in the synthesis method of the tetravalent ferrocene derivative, the amino donor is diamino-acid, the ferrocene intermediate is di-double ferrocene amino acid, and the ferrocene derivative is di-double ferrocene amino acid succinimidyl ester;
in the synthesis method of the octavalent ferrocene derivative, the amino donor is tetra-O-amino-beta-D-glucopyranoside, and the structural formula is as follows
The ferrocene intermediate is tetra-O-bisferrocenyl amino-beta-D-glucopyranoside, and the ferrocene derivative is tetra-O-bisferrocenyl amino-beta-D-glucopyranoside succinimidyl ester;
in the synthesis method of the fourteen-valent ferrocene derivative, the amino donor is tetra-O-amino-beta-D-galactopyranosyl-O-tri-O-amino-beta-D-glucopyranoside, and the structural formula is as follows
The ferrocene intermediate is tetra-O-dicyclopentadienyl amino-beta-D-galactopyranosyl-O-tri-O-dicyclopentadienyl amino-beta-D-glucopyranoside, and the ferrocene derivative is tetra-O-dicyclopentadienyl amino-beta-D-galactopyranosyl-O-tri-O-dicyclopentadienyl amino-beta-D-glucopyranoside succinimidyl ester.
According to a preferred embodiment of the invention, there is at least the following advantageous effect:
the synthesis method of the invention can prepare the divalent ferrocene derivative through two-step chemical reaction, and can skillfully use lysine-like compounds as frameworks to construct and synthesize the tetravalent ferrocene derivative, or use glucose as frameworks to construct and synthesize the octavalent ferrocene derivative, or use lactose as frameworks to construct and synthesize the decatetravalent ferrocene derivative. The succinimidyl ester in the ferrocene derivative can be modified on amino groups of oligonucleotide molecules, the modification can be carried out at different positions of nucleotide, the reaction condition is mild, and the post-treatment and the purification are simple.
In some embodiments of the invention, the amino alcohol comprises at least one of amino methanol, amino ethanol, amino propanol, amino butanol, amino pentanol, amino hexanol, or polyethylene glycol-like chain amino alcohols.
Preferably, the polyethylene glycol chain amino alcohol has a general formula of NH 2 -(CH 2 -CH 2 -O) k -H, wherein k = 2-6.
In some preferred embodiments of the invention, the amino alcohol comprises 6-amino-1-hexanol.
In some preferred embodiments of the invention, the diamino-acid comprises at least one of diaminopropionic acid, diaminobutyric acid, diaminovaleric acid, or diaminocaproic acid.
In some embodiments of the invention, the ferrocenyl aldehydes include at least one of ferrocenecarboxaldehyde or ferrocenedicarboxyaldehyde.
In some preferred embodiments of the invention, the ferrocenyl aldehydes comprise ferrocenecarboxaldehyde.
In some embodiments of the invention, the amino donor of step S1 is dissolved in an organic solvent.
In some embodiments of the invention, the organic solvent comprises at least one of anhydrous tetrahydrofuran or methyltetrahydrofuran.
In some preferred embodiments of the invention, the organic solvent comprises anhydrous tetrahydrofuran.
In some embodiments of the present invention, step S1 further comprises adding a catalyst comprising a borohydride reagent.
In some embodiments of the invention, the catalyst comprises at least one of sodium borohydride, sodium triacetoxyborohydride, sodium cyanoborohydride, or sodium borohydride acetate.
In some preferred embodiments of the invention, the catalyst comprises sodium triacetoxyborohydride.
In some embodiments of the invention, the reaction of step S1 is performed at room temperature, with an aldehyde amine condensation reaction occurring.
In some embodiments of the invention, the N-succinimidyl carbonate comprises at least one of N, N' -disuccinimidyl carbonate, cyclopentyl-N-succinimidyl carbonate, or benzyl-N-succinimidyl carbonate.
In some preferred embodiments of the invention, the N-succinimidyl carbonate comprises an N, N' -disuccinimidyl carbonate.
In some embodiments of the invention, the ferrocene intermediate of step S2 is dissolved in a chlorinated alkane solvent comprising at least one of anhydrous dichloromethane or chloroform.
In some preferred embodiments of the present invention, the chlorinated alkane solvent comprises anhydrous methylene chloride.
In some preferred embodiments of the present invention, a catalyst is further added in step S2, wherein the catalyst comprises triethylamine.
In some embodiments of the invention, the reaction of step S2 is performed at room temperature, and the esterification reaction occurs.
According to a third aspect of the present invention there is provided the use of the ferrocene derivative in the modification of an oligonucleotide molecule.
According to a preferred embodiment of the invention, there is at least the following advantageous effect:
the succinimidyl ester of the ferrocene derivative realizes the multiple marking of oligonucleotide molecules by modifying amino groups of nucleotides at different positions.
According to a fourth aspect of the present invention there is provided an oligonucleotide molecule having said ferrocene derivative modified thereon.
In some embodiments of the invention, the 5' end of the oligonucleotide molecule is modified with at least 1 of the ferrocene derivatives.
In some preferred embodiments of the invention, the 5' end of the oligonucleotide molecule is modified with at least 2 of the ferrocene derivatives.
In some more preferred embodiments of the invention, the 5' end of the oligonucleotide molecule is modified with a plurality of the ferrocene derivatives.
In some embodiments of the invention, the 3' end of the oligonucleotide molecule is modified with at least 1 of the ferrocene derivatives.
In some preferred embodiments of the invention, the 3' end of the oligonucleotide molecule is modified with at least 2 of the ferrocene derivatives.
In some more preferred embodiments of the invention, the 3' end of the oligonucleotide molecule is modified with a plurality of the ferrocene derivatives.
In some embodiments of the invention, the middle portion of the oligonucleotide molecule is modified with at least 1 of the ferrocene derivatives.
In some preferred embodiments of the invention, the middle portion of the oligonucleotide molecule is modified with at least 2 of the ferrocene derivatives.
In some more preferred embodiments of the invention, the intermediate portion of the oligonucleotide molecule is modified with a plurality of the ferrocene derivatives.
In some more preferred embodiments of the invention, the oligonucleotide molecule is modified with a plurality of the ferrocene derivatives.
According to a fifth aspect of the present invention, there is provided a method of modifying an oligonucleotide molecule comprising the steps of:
providing an oligonucleotide molecule, at least one nucleotide of the oligonucleotide molecule being modified with an amino group;
mixing the oligonucleotide molecule with the ferrocene derivative for reaction, so that the ferrocene derivative is modified on the oligonucleotide molecule.
In some embodiments of the invention, the method for modifying the 5' end of the oligonucleotide molecule comprises the following specific reaction formula:
wherein, the oligonucleotides are different nucleotide sequences;
at least one of (a) and (b);
wherein,,
in some embodiments of the invention, the method for modifying the 3' end of the oligonucleotide molecule comprises the following specific reaction formula:
wherein, the oligonucleotides are different nucleotide sequences;
at least one of (a) and (b);
wherein,,
in some embodiments of the invention, the method for modifying the middle portion of the oligonucleotide molecule is specifically represented by the following formula:
in the method, in the process of the invention,
m1, M2 and M3 are different nucleotide sequences; a is more than or equal to 0; b is more than or equal to 1;
at least one of (a) and (b);
wherein,,
in some preferred embodiments of the invention, the oligonucleotide molecule is dissolved in a buffer at ph=9 before mixing with the ferrocene derivative.
In some preferred embodiments of the invention, the ferrocene derivative is dissolved in acetonitrile and then mixed with the oligonucleotide molecule for reaction to complete the modification of the oligonucleotide molecule.
According to a sixth aspect of the invention, the use of said oligonucleotide molecules in electrochemical detection, genetic detection is proposed.
After the oligonucleotide molecule is modified by the ferrocene derivative, the oligonucleotide molecule is provided with a ferrocene group, and the ferrocene group has good electrochemical activity, so that the oligonucleotide molecule is used for the electrochemical detection and the gene detection, and the detection sensitivity and the signal intensity can be effectively improved.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a chemical structure diagram of ferrocene derivatives prepared according to the present invention;
FIG. 2 is a graph showing the results of monitoring the high performance liquid phase of the reaction system for preparing oligonucleotide 1 at 1h according to example 5 of the present invention;
FIG. 3 is an electrospray ionization Mass (ESI Mass) chart of oligonucleotide 1 prepared in example 5 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, unless explicitly defined otherwise, terms such as dissolution, modification, labeling and the like should be construed broadly, and those skilled in the art can reasonably determine the specific meaning of the terms in the present invention in combination with the specific contents of the technical scheme.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," and the like mean that the particular agent, method, or material described in connection with the embodiment is included in at least one embodiment of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment. Moreover, the particular reagents, methods, or materials described may be combined in any suitable manner in one or more embodiments.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1
The bivalent ferrocene derivative is prepared by the specific process:
(1) 0.5g (4.27 mmol) of 6-amino-1-hexanol was dissolved in 25mL of anhydrous tetrahydrofuran, 2.1g (9.81 mmol,2.31 eq) of ferrocene formaldehyde was added at room temperature and stirred, and 2.3g (10.90 mmol,2.5 eq) of sodium triacetoxyborohydride was added to the system in portions to react overnight. After completion of the reaction by Thin Layer Chromatography (TLC), it was quenched with 30mL of a saturated sodium carbonate solution, extracted with 50mL of ethyl acetate, then sequentially washed with 20mL of a saturated brine and high-purity water, and the organic phase was dried over anhydrous sodium sulfate, concentrated and column-stirred to give 1.8g of 6-N, N-bisferrocenylmethylamino-1-hexanol (dark brown solid) in 85% yield.
The parameters of the structure of the 6-N, N-bisferrocenyl methylamino-1-hexanol verified by nuclear magnetic resonance spectroscopy are as follows: 1 H NMR(400MHz，CDCl 3 )：δ4.22(s，4H，Cp)，4.15(s，4H，Cp)，4.10(s，10H，Cp)，3.61(t，J＝6.5Hz，2H，CH 2 )，3.50(s，4H，CH 2 )，2.82(dd，J＝14.2，7.0Hz，2H)，2.38-2.26(m，2H)，1.52(dd，J＝14.2，6.9Hz，4H)，1.35-1.19(m，6H)。
(2) Taking 0.5g of 6-N, N-bisferrocenyl methylamino-1-hexanol obtained in the step (1), dissolving in 10mL of anhydrous dichloromethane, stirring at room temperature, then sequentially adding 300mg (1.2 eq) of N, N' -disuccinimidyl carbonate (DSC), 295mg (3 eq) of Triethylamine (TEA), reacting at room temperature for 2h, directly adding a proper amount of silica gel into a reaction system for stirring a sample after TLC detection reaction is completed, evaporating under reduced pressure, and performing column chromatography (the silica gel needs to be alkalified by triethylamine) to obtain 0.5g of 6-N, N-bisferrocenyl methylamino-1-hexanol-O-succinimido ester, wherein the yield is 78%.
The synthetic route for the above bivalent ferrocene derivatives can be expressed as:
example 2
The tetravalent ferrocene derivative is prepared by the embodiment, and the specific process is as follows:
(1) 0.5g (3.42 mmol) of L-lysine was dissolved in 25mL of anhydrous tetrahydrofuran, 3.37g (15.73 mmol,4.6 eq) of ferrocene formaldehyde was added at room temperature and stirred, and 3.62g (17.10 mmol,5 eq) of sodium triacetoxyborohydride was added to the system in portions and reacted overnight. After completion of the reaction by Thin Layer Chromatography (TLC), it was quenched with 30mL of a saturated sodium carbonate solution, extracted with 50mL of ethyl acetate, then sequentially washed with 20mL of a saturated sodium chloride solution and high-purity water, and the organic phase was dried over anhydrous sodium sulfate, concentrated and column-chromatography with stirring to give 2.7g of 2,6-N, N-di-ferrocene methylamino caproic acid (dark brown solid) in 84% yield.
(2) Taking 0.5g of 2,6-N, N-di-ferrocene methylamino caproic acid obtained in the step (1), dissolving in 10mL of anhydrous dichloromethane, stirring at room temperature, then sequentially adding 164mg (1.2 eq) of N, N' -disuccinimidyl carbonate (DSC), 162mg (3 eq) of Triethylamine (TEA), reacting at room temperature for 2h, directly adding a proper amount of silica gel into a reaction system for stirring a sample after TLC detection reaction is completed, evaporating under reduced pressure, and performing column chromatography (the silica gel needs to be alkalified by triethylamine) to obtain 0.5g of 2,6-N, N-di-ferrocene methylamino-1-caproic acid-O-succinimido ester with the yield of 90%.
The synthetic route for the above tetravalent ferrocene derivatives can be represented as:
example 3
The octavalent ferrocene derivative is prepared by the following specific processes:
(1) 0.5g (983 mu mol) of 6-hydroxyhexyl-2, 3,4, 6-tetra-O-propylamino-beta-D-glucopyranoside is dissolved in 25mL of anhydrous tetrahydrofuran, 1.94g (9.4 mmol,9.2 eq) of ferrocenecarboxaldehyde is added at room temperature and stirred, 2.1g (9.83 mmol,10 eq) of sodium triacetoxyborohydride is added to the system in portions, and the reaction is carried out overnight. After completion of the reaction by Thin Layer Chromatography (TLC), it was quenched with 30mL of a saturated sodium carbonate solution, extracted with 50mL of ethyl acetate, then sequentially washed with 20mL of a saturated sodium chloride solution and high purity water, the organic phase was dried over anhydrous sodium sulfate, concentrated, and column chromatography with stirring to give 1.5g of 6-hydroxyhexyl-2, 3,4,6-N, N, N-tetra-ferrocene-O-propylamino-beta-D-glucopyranoside (dark brown solid) in 73% yield.
(2) Taking 0.5g of 6-hydroxyhexyl-2, 3,4,6-N, N, N, N-tetra-double ferrocene-O-propylamino-beta-D-glucopyranoside obtained in the step (1), dissolving in 10mL of anhydrous dichloromethane, stirring at room temperature, then sequentially adding 74mg (1.2 eq) of N, N' -disuccinimidyl carbonate (DSC) and 73mg (3 eq) of Triethylamine (TEA), reacting at room temperature for 2h, directly adding a proper amount of silica gel into a reaction system for stirring after the reaction is detected to be complete by TLC, evaporating under reduced pressure, and carrying out column chromatography (the silica gel needs to be alkalified by triethylamine) to obtain 0.45g of 6-hexanol-O-succinimidyl ester-1-hexyl-2, 3,4,6-N, N, N-tetra-double ferrocene-O-propylamino-beta-D-glucopyranoside (light yellow oily substance) with the yield of 84%.
The synthetic route for the octavalent ferrocene derivatives described above can be represented as:
example 4
The fourteen-valent ferrocene derivative is prepared by the following specific processes:
(1) 0.5g (594. Mu. Mol) of 6-hydroxyhexyl-2, 3, 6-tri-O-propylamino-4-O- (2, 3,4, 6-tetra-O-propylamino-beta-D-galactopyranosyl) -beta-D-glucopyranoside is dissolved in 25mL of anhydrous tetrahydrofuran, 2.03g (9.5 mmol,16 eq) of ferrocenecarboxaldehyde is added at room temperature and stirred, and 2.3g (10.7 mmol,18 eq) of sodium triacetoxyborohydride is added to the system in portions for reaction overnight. After completion of the reaction by Thin Layer Chromatography (TLC), it was quenched with 30mL of saturated sodium carbonate solution, extracted with 50mL of ethyl acetate, then sequentially washed with 20mL of saturated brine and high purity water, the organic phase was dried over anhydrous sodium sulfate, concentrated, and column chromatography with stirring to give 1.5g of 6-hydroxyhexyl-2, 3,6-N, N, N-tris-ferrocene-O-propylamino-4-O- (2, 3,4,6-N, N, N, N-tetra-ferrocene-O-propylamino-beta-D-galactopyranosyl) -beta-D-glucopyranoside (dark brown solid) in a yield of 70%.
(2) Taking 0.5g of 6-hydroxyhexyl-2, 3,6-N, N, N-tri-bisferrocene-O-propylamino-4-O- (2, 3,4,6-N, N, N-tetra-bisferrocene-O-propylamino-beta-D-galactopyranosyl) -beta-D-glucopyranoside obtained in the step (1), dissolving in 10mL of anhydrous dichloromethane, stirring at room temperature, sequentially adding 43mg (1.2 eq) of N, N' -disuccinimidyl carbonate (DSC), 42mg (3 eq) of Triethylamine (TEA), reacting at room temperature for 2h, directly adding a proper amount of silica gel into a reaction system for stirring after TLC detection reaction is completed, evaporating to dryness under reduced pressure, and performing column chromatography (silica gel needs to be alkalified by triethylamine) to obtain 6-hexanol-O-succinimidyl-ester-1-hexyl-2, 3,6-N, N-tri-bisferrocene-O-propylamino-4-O- (2, 3,4,6-N, N-tetra-bisferrocene-O-beta-galactopyranoside with a light yellow oil yield of 42.80%.
The synthetic route for the fourteen-valent ferrocene derivatives described above can be represented as:
wherein,,
example 5
The present example prepared an oligonucleotide 1, in which the amino group at the 5' end of the oligonucleotide molecule was modified, by the following steps:
30mg of amino-modified oligonucleotide 5' -NH was taken 2 Oligo (Oligo fragment 5' -NH) 2 -CAAGCGGATACACCAGGATTG-3 ') was dissolved in 1mL of sodium carbonate-sodium bicarbonate buffer at ph=9, 10mg (large excess) of 6-N, N-bis-ferrocenylmethylamino-1-hexanol-O-succinimido ester was taken and dissolved in 1mL of acetonitrile and added to the buffer system, stirred at room temperature for 1h, and the starting oligonucleotide 5' -NH was detected by High Performance Liquid Chromatography (HPLC) 2 Liquid phase off-peak position of Oligo (FIG. 2), indicating that the starting oligonucleotide was not reacted completely;
after the reaction is continued for 1h, the liquid phase detects the peak elimination of the peak positionLoss of oligonucleotide 5' -NH 2 The oligoo reaction is complete, the reaction is stopped, and the high-pressure preparation liquid chromatographic separation is continued: the chromatographic column is Durashell C18 (L) of Agela Technologies, 5 μm,30x250mm, eluent organic phase is chromatographic acetonitrile, gradient is 20% acetonitrile-90% acetonitrile, aqueous phase is 0.1M triethylamine-carbonic acid (TEAB) buffer solution, time is 30min, and product peak position is 12.2min.
Finally, 15mg of oligonucleotide 1 was obtained in 45% yield; electrospray ionization mass was measured using a linear ion trap mass spectrometer as 7182.1 (fig. 3) and molecular weight was 7182.69.
Example 6
The present example prepared an oligonucleotide 2, in which the amino group at the 3' end of the oligonucleotide molecule was modified, by the following steps:
30mg of amino-modified oligonucleotide was taken
Wherein the nucleotide fragment of Oligo is 5'-AGGTCTAACGAAACGTCG-NH2-3';
the oligonucleotide was dissolved in 1mL of sodium carbonate-sodium bicarbonate buffer at ph=9, and 10mg (large excess) of 6-N, N-bis-ferrocenylmethylamino-1-hexanol-O-succinimidyl ester was dissolved in 1mL of acetonitrile and added to the buffer system, stirred at room temperature until the reaction was detected to be complete by High Performance Liquid Chromatography (HPLC), the reaction was stopped, and high pressure preparative liquid chromatography was continued to separate: the chromatographic column is Durashell C18 (L) of Agela Technologies, 5 μm,30X250mm, eluent organic phase was chromatographic acetonitrile, gradient 20% acetonitrile-90% acetonitrile, aqueous phase 0.1M triethylamine-carbonic acid (TEAB) buffer for 30min.
Example 7
The present example prepared an oligonucleotide 3, in which the amino group in the middle part of the oligonucleotide molecule was modified, by the following steps:
30mg of amino-modified oligonucleotide R, R having a nucleotide fragment of 5'-ATAGACGCC-NH2-CCGACTAAA-NH2-CTCCGCAGAGCT-3' was taken, the oligonucleotide R was dissolved in 1mL of sodium carbonate-sodium bicarbonate buffer at pH=9, 10mg (large excess) of 6-N, N-bisferrocenylmethylamino-1-hexanol-O-succinimido ester was taken and dissolved in 1mL of acetonitrile and added to the buffer system, and stirred at room temperature until the reaction was complete as detected by High Performance Liquid Chromatography (HPLC), the reaction was stopped, and high pressure preparative liquid chromatography was continued to separate: the chromatographic column is Durashell C18 (L) of Agela Technologies, 5 μm,30X250mm, eluent organic phase was chromatographic acetonitrile, gradient 20% acetonitrile-90% acetonitrile, aqueous phase 0.1M triethylamine-carbonic acid (TEAB) buffer for 30min.
Example 8
The present example prepared an oligonucleotide 4, in which the amino group at the 5' end of the oligonucleotide molecule was modified, by the following steps:
30mg of amino-modified oligonucleotide 5' -NH was taken 2 Oligo (Oligo fragment 5' -NH) 2 CAAGCGGATACACCAGGATTG-3 '), oligonucleotide 5' -NH 2 -Oligo was dissolved in 1mL sodium carbonate-sodium bicarbonate buffer at ph=9, 10mg (large excess) of 2,6-N, N-di-ferrocenylmethylamino-1-hexanoic acid-O-succinimidyl ester was taken and dissolved in 1mL acetonitrile and added to the buffer system, stirred at room temperature until the reaction was detected to be complete by High Performance Liquid Chromatography (HPLC), the reaction was stopped, and high pressure preparative liquid chromatography separation was continued: the chromatographic column is Durashell C18 (L) of Agela Technologies, 5 μm,30X250mm, eluent organic phase was chromatographic acetonitrile, gradient 20% acetonitrile-90% acetonitrile, aqueous phase 0.1M triethylamine-carbonic acid (TEAB) buffer for 30min.
Example 9
The present example prepared an oligonucleotide 5, in which the amino group at the 3' end of the oligonucleotide molecule was modified, by the following steps:
30mg of amino-modified oligonucleotide was taken
Wherein the nucleotide fragment of Oligo is 5'-AGGTCTAACGAAACGTCG-NH2-3';
the oligonucleotide was dissolved in 1mL of sodium carbonate-sodium bicarbonate buffer at ph=9, 10mg (large excess) of 6-hexanol-O-succinimidyl ester-1-hexyl-2, 3,4,6-N, N-tetra-ferrocene-O-propylamino- β -D-glucopyranoside was taken out in 1mL of acetonitrile and added to the buffer system, stirred at room temperature until the reaction was detected to be complete by High Performance Liquid Chromatography (HPLC), the reaction was stopped, and high pressure preparative liquid chromatography separation was continued: the chromatographic column is Durashell C18 (L) of Agela Technologies, 5 μm,30X250mm, eluent organic phase was chromatographic acetonitrile, gradient 20% acetonitrile-90% acetonitrile, aqueous phase 0.1M triethylamine-carbonic acid (TEAB) buffer for 30min. />
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (6)
1. A ferrocene derivative, characterized by the following structural formula:
octavalent ferrocene derivativeOr tetradecyl ferrocene derivative，。
2. The method for synthesizing ferrocene derivatives according to claim 1, comprising the steps of:
s1: taking amino donor to react with ferrocene formaldehyde to form ferrocene intermediate;
s2: reacting the ferrocene intermediate in the step S1 with N, N' -disuccinimidyl carbonate to form a ferrocene derivative;
in the synthesis method of the octavalent ferrocene derivative, the amino donor is tetra-O-amino-beta-D-glucopyranoside, and the structural formula is as follows
，
The ferrocene intermediate is tetra-O-bisferrocenyl amino-beta-D-glucopyranoside, and the ferrocene derivative is tetra-O-bisferrocenyl amino-beta-D-glucopyranoside succinimidyl ester;
in the synthesis method of the fourteen-valent ferrocene derivative, the amino donor is tetra-O-amino-beta-D-galactopyranosyl-O-tri-O-amino-beta-D-glucopyranoside, and the structural formula is as follows
，
The ferrocene intermediate is tetra-O-dicyclopentadienyl amino-beta-D galactopyranosyl-O-tri-O-dicyclopentadienyl amino-beta-D-glucopyranoside, and the ferrocene derivative is tetra-O-dicyclopentadienyl amino-beta-D-galactopyranosyl-O-tri-O-dicyclopentadienyl amino-beta-D-glucopyranoside succinimidyl ester.
3. Use of a ferrocene derivative of claim 1 for the preparation of a product of a modified oligonucleotide molecule.
4. An oligonucleotide molecule having a ferrocene derivative of claim 1 modified thereon.
5. A method for modifying an oligonucleotide molecule, comprising the steps of:
providing an oligonucleotide molecule, at least one nucleotide of the oligonucleotide molecule being modified with an amino group;
mixing the oligonucleotide molecule with the ferrocene derivative of claim 1 for reaction, so that the ferrocene derivative is modified on the oligonucleotide molecule.
6. Use of an oligonucleotide molecule according to claim 4 or obtained according to the modification method of claim 5 for the preparation of an electrochemical detection product, a genetic detection product.
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| CN103459407A (en) * | 2010-12-22 | 2013-12-18 | 阿特莱斯遗传学有限公司 | Novel ferrocene labels for electrochemical assay and their use in analytical methods |
| CN103601761A (en) * | 2013-09-24 | 2014-02-26 | 中山大学达安基因股份有限公司 | Synthetic method of ferrocene derivatives |
| CN104781269A (en) * | 2012-06-22 | 2015-07-15 | 阿特莱斯遗传学有限公司 | 1,1'-[[(substituted alkyl)imino]bis(alkylene)]bis-ferrocenes and their use in electrochemical assays by labelling substrates of interest |
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| CN104781269A (en) * | 2012-06-22 | 2015-07-15 | 阿特莱斯遗传学有限公司 | 1,1'-[[(substituted alkyl)imino]bis(alkylene)]bis-ferrocenes and their use in electrochemical assays by labelling substrates of interest |
| CN103601761A (en) * | 2013-09-24 | 2014-02-26 | 中山大学达安基因股份有限公司 | Synthetic method of ferrocene derivatives |
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