FR2676732A1 - Monosubstituted tetrahalogenated and disubstituted trihalogenated pyridines photochemically grafted by their 4-carbon atom and processes for their preparations - Google Patents

Abstract

The present invention relates to monosubstituted tetrahalogenated and disubstituted trihalogenated pyridines photochemically grafted by their 4-carbon atom to a wide variety of small or large, inert or functionalised molecules which are soluble or insoluble in their medium of use, to their method of preparation and to some of their fields of application in biochemistry, in biotechnologies and in macromolecular chemistry.

Description

difluoro-2,6 pyridine (B) (PAC) possèdent deux propriétés essentielles.Perhalogenated pyridyl azides such as azido-4 tetrafluoro 2,3,5,6 pyridine (PAF) (A) or azido-4 dichloro-3,5

2,6-difluoro pyridine (B) (PAC) has two essential properties.

 In position 2, there is a nucleofuge fluorine atom which can be replaced by a nucleophilic function (nitrogen-sulfur, for example).

They have on the carbon-4 an azide function which, under ultraviolet irradiation, for example, leads to an intermediate nitrene which is inserted in a CN, CO, CH or CC bond or any other bond being in its vicinity. These two properties led one of us to use them a) as crosslinking agents to study the topology of
components of the bacterial ribosome (R. Millon et al, Biochim.

Biophys. Res. Comm. 1981, 101, 784-791) b) as agents capable of reacting in position 2 with the
amino function of chitosan, a glucosamine homopolymer
in order to produce a photoactivable resin (S. Sicsic et al,
French Patent No 80-190-31) c) as agents capable of reacting with the amino function of
glycine thus leading to an N-substituted glycine
photoactivatable (C. Bouthier de la Tour et al, Eur. J. Med.

Chem. 1983, 18, 315-318) d) as agents capable of reacting with an amino function
grafted beforehand on a purine basis, which leads to a
immobilized cofactor (ATP or NAD +) (S. Sicsic et al, Eur. J.

 Biochem. 1989, 179, 435-440).

 If one of these substances A or B is reacted with an inert or functionalized molecule, small or large, soluble or not in the medium used, these two molecules subjected to appropriate irradiation, in general from 260 nm to 320 nm, lead to very short-lived nitrites which are immediately inserted into a CC, CH, CN, CO, CS bond, located in their vicinity.

représente une molécule petite ou grosse sur laquelle la pyridine tétrahalogénée A ou B est maintenant greffée par l'atome d'azote se trouvant en position 4 de la pyridine halogénée.Such a reaction, presented in Figure I (page 1/7), leads to the two structures C and D in which

represents a small or large molecule on which the tetrahalogenated pyridine A or B is now grafted by the nitrogen atom located in position 4 of the halogenated pyridine.

représente une macromolécule naturelle ou synthétique telle que - du polyéthylène - du polypropylène - des latex diversement fonctionnalisés - du polyéthylène glycol - des polysaccharides (cellulose ou dérivé de cellulose,
dextranes), chitosane, chitine - du PVDF - des polyamides ou des polyesters - des protéines tcollagène, enzymes, anticorps) - des acides nucléiques - des lipides - ou une combinaison des systèmes précédents - des cellules procaryotes ou eucaryotes.In general, and without being limiting

represents a natural or synthetic macromolecule such as - polyethylene - polypropylene - variously functionalized latexes - polyethylene glycol - polysaccharides (cellulose or cellulose derivative,
dextrans), chitosan, chitin - PVDF - polyamides or polyesters - tcollagen proteins, enzymes, antibodies) - nucleic acids - lipids - or a combination of the above systems - prokaryotic or eukaryotic cells.

These new structures thus acquire a new functionality since, as indicated in Figure II (page 1/7), they can react with molecules having nucleophilic functions represented by HNuX to lead to the trihalogenated E and F systems disubstituted in 2 and 4 of pyridine in which Nu is a primary or secondary nitrogen atom, a residue
S- and X can be
- a hydrocarbon chain
- a hydrocarbon chain with various functions
monovalent, divalent or trivalent
- a hydrocarbon chain having in its structure
heteroatoms (O, S, N) and possibly having
monovalent, divalent or trivalent functions
- a dye, a fluorescent residue
- a cofactor such as biotin or imminobiotin,
mono, di or triphosphate adenosine, NAD + or NADH
- a monosaccharide, an oligosaccharide, a polysaccharide
- an α-amino acid, a peptide, a protein
- a fatty acid, a lipid
- a nucleoside, a mononucleotide
- a purine or pyrimidine base, an oligonucleotide, a
nucleic acid (ribo or deoxyribonucleic)
- a combination of the above structures.

 Of course, the preceding systems which do not have a nucleophilic function will have to be structurally modified beforehand.

molécules A ou 3 présentes dans le milieu et que le nombre de résidus -NuX fixés dépendra bien sûr du nombre de pyridines tétrahalogénées accessibles. Obviously, systems C and D can thus be used to prepare a wide variety of structures having very significant advantages compared to the photoactivable resin described in French patent 80-190-31. Indeed - systems C and D are not photoactivable and can by
therefore be stored and handled in the light without altering
their chemical reactivity therefore their potential for reaction with
molecules with nucleophilic functions - macromolecules supporting halogenated pyridines can
have very diverse structures and therefore respond
to a great number of needs and to satisfy many
requirements in the fields of chemistry and biotechnology - they have a very large capacity since the quantity of
fixed pyridine will essentially depend on the molecule concentration ratios

molecules A or 3 present in the medium and that the number of -NuX residues fixed will of course depend on the number of accessible tetrahalogenated pyridines.

The molecules E and F will be used, for example, and without this being limiting to - the manufacture of supports, soluble or insoluble, basic
(NH2) or acids (COOH, S03H, PO3H2, B (-OH) 2 usable in
purification processes by ion exchangers or by
complexation, to carry out solid phase synthesis
(peptides, oligonucleotides), to fix macromolecules - the fixing of amino or acid functions on proteins,
lipids, nucleic acids or polysaccharides,
way to modify their physicochemical properties and
possibly biological as well as improving their properties
of fixation on supports: covalent fixation of an enzyme
on a support (beads or membranes) of activated cellulose
beforehand with cyanogen bromide, for example - passivation to hydrophilic chromatographic supports or
hydrophobic - the manufacture of usable biotinylated supports, for example,
to purify avidin or streptavidin - the production of biotinylated proteins (enzymes-antibodies)
usable in immunology and, more specifically, in
immunodiagnosis - the production of oligonucleotides or nucleic acids
biotinylates in which biotin acts as a cold probe
in immunoassay the immobilization of oligo or polysaccharides and, more precisely, of cyclodextrins (a, ss - the manufacture of usable colored or fluorescent supports
in several fields of biology and immunology
(latex, for example) the particularly effective immobilization of enzymes, such
that, for example, papain, proteinase K,
ribonucleases, deoxyribonucleases, enzymes
restriction, phosphodiesterases, DNA ligases,
lipid A degradation enzymes. These different enzymes
find, indeed, quite relevant applications in
biology and biotechnology - immobilization of proteins with no properties
catalytic: bovine and human albumin serum, antibodies,
various receivers.

 By way of example, and without this being limiting, we describe below some of the products thus obtained, as well as their method of preparation.

EXAMPLE I PAF fixed on Bovine Serum Albumin (BISA) 1. Preparation of the BSA solution (Bovine Serum Albumin)
The BSA is dissolved in a phosphate buffer pH 7.5. A starting solution of 7.6 mg / ml is prepared.

2. Preparation of PAF solutions
A range of concentrations of PAF in solution in methanol is prepared.

 Concentrations used in pmoles / liter: 12.5 - 2t - 37.5 - 50 - 100 - 150 - 200 - 300 - 400 - 700 - 900.

3. Fixing the PAF on the BSA
Into 30 ml test tubes, 10 ml of the BSA solution (76 mg) and 10 ml of the different concentrations of the
PAF. Each mixture is stirred to obtain a homogeneous solution then the tubes are irradiated under UV for 15 minutes. The contents of each tube are then dialyzed against water. Then the volume of each tube is adjusted to 10 ml by lyophilization. To each of the tubes is added 10 ml of a 200 pmolar solution of aminoethyl dabsyl sulfonamide and then stirred at room temperature for 24 h.

The solutions obtained are lyophilized and then the residue is washed with methylene chloride (3 x 10 ml). The residue is then dissolved in 30 ml of physiological saline then the optical density of each of the preparations is measured at 434 nm. This measurement makes it possible to determine the amount of PAF fixed on the BSA as a function of increasing concentrations of PAF used (FIG. III, page 2/7).

EXAMPLE II PAF fixed on dextran 60 1. Preparation of the dextran 60 solution
Dextran 60, which is a glucose polymer, is dissolved in water at a concentration of 14 mg / ml.

2. Preparation of PAF solutions
For PAF, a range of four concentrations was used by dissolving it in methanol:
25 pM - 37.5 uM - 50 pM - 100 uM.

3. Attachment of PAF to dextran 60
In test tubes, 10 ml of the starting solution of dextran 60 (140 mg) and 10 ml of the 4 concentrations are mixed.
PAF. After shaking, the tubes are irradiated under UV for approximately 15 minutes. The ethanol contained in the tubes is then eliminated in a rotary evaporator. Then each fraction is washed several times with ethyl acetate (40 ml each wash) until the excess PAF disappears.

 Each solution of PAF dextran is then treated as in Example I, which makes it possible to measure the quantity of PAF fixed as a function of increasing concentrations of PAF used.

The results of these experiments are shown in Figure IV, page 2/7).

EXAMPLE III: PAC fixed on a PVDF membrane (Difluoride of
polyvinylidene) 1. Preparation of PAC solutions
A range of concentrations of PAC in solution in methanol is prepared: 0.1; 0.25; 0.5; 0.75 and 1 M.

2. Fixing of PAC on PVDF membranes
Each PVDF filter (5 cm 2) is soaked in the methanolic PAC solution and dried in the dark. As soon as the filter is dry, it is irradiated at 254 nm for 15 minutes. It is then washed with methanol until absorption at 254 nT.

of the washing solution is zero. The filters are then dried and then stored at room temperature without taking any special precautions.

EXAMPLE IV PAC fixed on cellulose membrane 1. Preparation of PAC solutions
A range of concentrations of PAC in solution in methanol is prepared: 0.1; 0.25; 015; 0.75 and 1 M.

2. Fixing of PAC on cellulose membrane
Each cellulose filter (5 cm 2) is soaked in the methanolic PAC solution and then dried in the dark. As soon as the filter is dry, it is irradiated for 15 minutes. It is then washed with methanol until the absorption at 254 nm of the washing solution is zero. The filters are then dried and then stored at room temperature without taking any special precautions.

EXAMPLE V: PAF fixed on cellulose membrane 1. Preparation of PAF solutions
A range of concentrations of PAF in solution in methanol is prepared: 0.1; 0.25; 0.5; 0.75 and 1 M.

2. Fixation of PAF on cellulose membrane
Each cellulose filter (5 cm 2) is soaked in methanolic PAF solution and then dried in the dark. As soon as the filter is dry, it is irradiated for 15 minutes. It is then washed with methanol until the absorption at 25 nm of the washing solution is zero. The filters are then dried and then stored at room temperature without taking any special precautions.

EXAMPLE VI. Deoxyribonuclease I EC3.1.2.1.1. (DNase-I)
immobilized on PVDF-PAC membrane 1. Radioactive labeling of DNase-I
10 .g of DNase-I are dissolved in 3 ml of 0.2 M borate buffer pH 9.5 to which 250 μCi of 14C formaldehyde and 10 mg of CNBHNa are added. This mixture is stirred at room temperature for 2 hours, then the 14C-labeled enzyme is dialyzed extensively against 3 l of a 0.8% ClNa solution. The volume of the enzyme solution is then adjusted to 10 ml by addition of 0.8% CiNa 2. DNase-I immobilized on a PVDF-PAC membrane
The filters activated by PAC of Example III are incubated for 12 h at 250C in the presence of a DNase-I solution at 2 mg / ml to which a small amount of DNase 14C has been added as a tracer. The filters are then washed with a 0.8% NaCl solution. The washing is stopped when the radioactivity of the washing solutions reaches an almost zero value. The radioactivity fixed on the filters is then measured by liquid scintillation. The results of fixing DNase-I on the filters are shown in Figure V, page 3/7.

3. Catalytic activity of the PVDF-PAC-DNase-I membrane
The catalytic power of DNase-I is determined by measuring the optical density of a DNA solution, according to J.

Kunitz, J. Gen. Physiol. 1950, 33, 349, in the presence of the PVDF-PAC-DNase-I membrane at 260 nm as a function of time.

 Figure VI, page 3/7, shows the result thus obtained.

EXAMPLE VII DNase-I immobilized on cellulose-PAC membrane
The cellulose filters activated by PAC of Example IV are incubated for 12 h at 250C in the presence of a solution of
DNase-I at 2 mg / ml to which a small amount of
DNase 14C as a radioactive tracer. The filters are then washed with a 0.8% NaCl solution. The washing is stopped when the radioactivity of the washing solutions reaches a quasi-zero value. The radioactivity fixed on the filters is then measured by liquid scintillation. The results of fixing the
DNase-I on the filters are shown in Figure VII.

Catalytic activity of the PAC-DNaseI cellulose membrane
The catalytic power of DNase-I is determined by measuring the optical density of a DNA solution in the presence of
the PAC-DNase-I cellulose membrane at 260 nm depending on the
time, according to J. Kunitz, J Gen. Physiol. 1950, 33, 349.

Figure VIII, page 4/7) reports the results
obtained. Table 1 compares this result with that
obtained by condensation of DNase I on a membrane of
cellulose activated by cyanogen bromide.

 DNase4 activation method g) AImn.

 PAC 34 5.66.

CNBr 290 6,28.10-3
Table I
Comparison of immobilization of DNasé-I by PAC or CNBr
EXAMPLE VIII RNase-A immobilized on PAC 1 cellulose membrane. Radioactive mark of RNase-A
10 mg of RNase-A (beef pancreas) are dissolved in 3 ml of 0.2 M borate buffer, pH 9.5. Then added 250 uCi of formaldehyde 14C and 10 mg of CNBH3Na. The mixture is stirred at room temperature for 2 hours.

 RNase-A 14C is dialyzed extensively against 3 liters of a 0.8% tIaCl solution.

2. Immobilization of RNase-A
The filters activated by the PAC of Example IV are incubated for 12 hours at 250 ° C. in the presence of a RNase-A solution at 2 mg / ml to which RNase-A 14C has been added as a radioactive tracer. The filters are then washed with a 0.8 3 NaCl solution. The washing is stopped when the radioactivity of the washing solutions reaches a virtually zero value. The radioactivity on the filters is then measured by liquid scintillation. The results of the attachment of RITase-A to the filters are shown in Figure IX, page 5/7.

3. Catalytic activity on the PAC RNase-A cellulose membrane
The catalytic activity of immobilized RNase-A is determined by measuring the absorbance of a yeast RNA solution in the presence of the PAC-RNase-A cellulose membrane at 260 nm as a function of time (Kalnitsky and al J. Biol. Chem. 1959, 234, 1512).

 The PAC-R> Jase-A cellulose membrane is soaked in 25 ml of 0.01% RNA in 0.1 M acetate buffer, pH 5.0 in a reactor thermostatically controlled at 15 C. The absorbance is measured at 260 nm every 5 minutes. The results obtained are shown in Figure X, page 5/7.

EXAMPLE IX DNase-I immobilized on cellulose-PAF membrane
The cellulose filters activated by PAF, from the example
IV, are incubated for 12 h at 250C in the presence of a solution of
DNase-I at 2 mg / ml to which a small amount of
DNase 14C as a radioactive tracer. The filters are then washed with a 0.8% NaCl solution. The washing is stopped when the radioactivity of the washing solutions reaches a quasi-zero value. The radioactivity fixed on the filters is then measured by liquid scintillation.

EXAMPLE X RNase-A immobilized on PAF cellulose membrane
The filters activated by the PAF of Example IV are incubated for 12 hours at 250C in the presence of a RNase-A solution at 2 mg / ml to which RNase-A 14C has been added as a radioactive tracer. The filters are then washed with a 0.8% NaCl solution. The washing is stopped when the radioactivity of the washing solutions reaches an almost zero value. The radioactivity on the filters is then measured by liquid scintillation.

 The results of fixing Examples IX and X are shown in Figure XI, page 6/7.

EXAMPLE XI: Attachment of Biotin Hexamethylene Diamine to
PVDF-PAF filters 1. Synthesis of biotin-hexamethylene diamine
In a 500 ml run-in flask, 5.86 mmol (2 g) of biotin-hydroxysuccinimide are dissolved in 150 ml of DMF.

5.86 mmol (1.36 mg) of hexamethylene diamine dissolved in 50 ml of DMF are added. The mixture is stirred at room temperature for 24 hours. At the end of the reaction, the solvent is evaporated and the residue is washed with dichloromethane. The residue is then deposited on an appropriate column of silica equilibrated with methanol. The elution is made with an increasing gradient in ammonia 28%. Biotin hexamethylene diamine is eluted at 25% NH3. The fractions are collected and the solvent is evaporated.

The product is obtained in the form of a white powder. The reaction yield is 76%.

Characteristics - TLC: methanol: NH3 28% 4: 1 Rf = 0.52; revealing
ninhydrin; p.DACA (p.dimethylamino cinnamaldehyde).

 - Mass (DCI / NHs) m / e: MH MH + = 343.

2. Fixation of PAF on PVDF membranes
Each PVDF filter (5 cm 2) is soaked in a methalonic solution of PAF at different concentrations (0.1; 0.2 0.3; 0.4 and 0.5 M) and then dried in the dark. As soon as the filter is dry7 it is irradiated for 15 minutes. It is then washed with methanol until the absorption at 254 nm of the washing solution is zero. The filters are then dried and stored at room temperature.

3. Attachment of biotin-hexamethylene diamine to filters
PVDF-PAF
Each of the PVDF-PAF filters is incubated for 12 hours at 250C in the presence of 2 ml of a 0.1 M solution of biotin hexamethylene diamine in 0.1 M DJaHCO3 buffer pH 9.5 The filters are then washed with the same buffer and the amount of biotin hexamethylene diamine is measured in the washing solutions by the method described by McCormick et al, Anal. Biochem. 1970, 34, 226-236
The fixation results are shown in Figure XII, page 7/7.

EXAMPLE XII Fixation and Release of Streptavidin on
PVDF PAF-Biotin membrane
(Affinity chromatography
A solution of streptavidin (20 ml) is prepared in
0.2 M NaHCO3; pH 8.7 at a concentration of 0.5 mg / ml. The membrane is then soaked in this solution and stirred.

Teenager. at 282 nm is taken every 5 minutes. After 1.5 hours, the d.o. reaches a maximum and the membrane is soaked in the washing buffer: guanidine-HCl 3 M; pH 5.5, then the streptavidin is eluted in 20 ml of 6 M guanidine HCl buffer; pH 1.5.

The results are shown in Figures XIII and XIV, page 7/7.

 This membrane has been shown to have a capacity of approximately 3 mg.

Claims (8)

1. Molecules of tetrahalogenated pyridines fixed by a nitrogen atom  found in position 4 of tetrahalogenated pyridine on  inert or functionalized molecules, small or large, soluble  or not.  thus an extremely reactive nitrene.  halogenated having in position 4 an azide function which generates  suitable, usually 260 to 320 nm of tetra pyridines  claim 1. obtained by irradiation at one wavelength  2. Tetrahalogenated pyridine molecules substituted in 4 according to the 3. Molecules of tetrahalogenated pyridines fixed by a nitrogen atom  being in position 4 according to claims 1. and 2. in  which the molecule attached to the pyridine can be  - polyethylene  - polypropylene  - variously functionalized latexes  - polyethylene glycol  - polysaccharides (cellulose or cellulose derivative, dextran,  chitosan, chitin)  - PVDF  - polyamides or polyesters  - proteins  - nucleic acids  - lipids  - combinations of previous systems  - prokaryotic and eukaryotic cells obtained by  insertion of the nitrene according to claim 2. into a  C-C, C-H, C-N, C-O, C-S link located in their vicinity.  being in position 4 according to claims 1 to 3.  4. Molecules of tetrahalogenated pyridines fixed by a nitrogen atom  (PAC) pyridine on a wide variety of molecules.  azido-4 pyridine (PAF) and difluoro-2,6 dichloro-3,5 azido-4  obtained by photochemical condensation of 2,3,5,6-tetrafluoro 5. Trihalogenated pyridine molecules attached by a nitrogen atom  found in position 4 of trihalogenated pyridine on  inert or functionalized molecules, small or large,  soluble or not in which we find in position 2 a  NuX function. 6. Process for the preparation of fixed trihalogenated pyridine molecules  by a nitrogen atom located in position 4 of pyridine  trihalogenated on inert or functionalized molecules, small  or large, soluble or not, and obtained by reacting  tetrahalogenated pyridines of claims 1. to 4. with  molecules of general formula HNuX in a solvent such as water,  phosphate, borate or acetate buffer, DMF or methanol. 7. Trihalogenated pyridine molecules obtained according to the process of  preparation of claim 6. where X may be  - a hydrocarbon chain  - a hydrocarbon chain with various functions  monovalent, divalent or trivalent  - a hydrocarbon chain having in its structure  heteroatoms (O, S, N) and possibly having  monovalent, divalent or trivalent functions  - a dye, a fluorescent residue  - a cofactor such as biotin or imminobiotin,  mono, di or triphosphate adenosine, NAD + or NADH  - a monosaccharide, an oligosaccharide, a polysaccharide  - an amino acid, a peptide, a protein  - a fatty acid, a lipid  - a nucleoside, a mononucleotide  - a purine or pyrimidine base, an oligonucleotide, a  nucleic acid (ribo or deoxyribonucleic)  - a combination of the above structures 8. Trihalogenated pyridine molecules according to claims 5.  various receivers.  catalytic: bovine and human albumin serum, antibodies,  biology and biotechnology immobilization of proteins with no properties  find, indeed, quite relevant applications in  lipid A degradation enzymes. These different enzymes  restriction, phosphodiesterases, DNA ligases,  ribonucleases, deoxyribonucleases, enzymes  that, for example, papain, proteinase K,  (latex, for example) the particularly effective immobilization of enzymes, such  in several fields of biology and immunology  specifically, cyclodextrins (a, ss, Y - the manufacture of usable colored or fluorescent supports  in immunoassay immobilization of oligo or polysaccharides and, more  biotinylates in which biotin acts as a cold probe  immunodiagnosis - the production of oligonucleotides or nucleic acids  usable in immunology and, more specifically, in  to purify avidin or streptavidin - the production of biotinylated proteins (enzymes-antibodies)  hydrophobic - the manufacture of usable biotinylated supports, for example,  beforehand with cyanogen bromide, for example - the passivation of hydrophilic chromatographic supports or  on a support (beads or membranes) of activated cellulose  of fixation on supports: covalent fixation of an enzyme  possibly biological as well as improving their properties  way to modify their physicochemical properties and  lipids, nucleic acids or polysaccharides,  (peptides, oligonucleotides), to fix macromolecules - the fixing of amino or acid functions on proteins,  complexation to carry out solid phase synthesis  purification processes by ion exchangers or by  (NH2) or acids (COOH, SO3H, PO3H2, B (-OH) 2 usable in  and 7. usable in: - the manufacture of supports, soluble or insoluble, basic