FR2617487A1 - Process for the chemical modification of protides and products thus modified - Google Patents

Abstract

Process for the modification of a protide by fixing an organic group to its molecule, reacting the protide with a dithioic ester, whose acid residue contains the organic group to be fixed, whereas the thiol residue has a leaving group structure, so that the dithio ester can react with the -NH2 or -NH of the protein molecule.

Description

 The present invention relates to a novel process for the chemical modification of proteins. It comprises a reaction for fixing one or more organic groups to the molecule of protide, capable of changing the physicochemical properties of this protein.

The invention aims, in particular, the modification of proteins and, in particular, that of enzymes. An important application of the invention is to change the solubility of peptides or proteins and, especially, to render enzymes soluble in organic solvents.

 The invention also includes protins bearing organic groups, modifying their physicochemical properties. It relates in particular to the enzymes made soluble in organic solvents, by fixing said -groups.

 It is known to change more or less the properties of a protein by the action of a chemical reagent. In the case of enzymes, this usually has the purpose of modifying their physical properties, such as solubility, mobility, ie molecular weight, stability, and so on. It is also sought, by this means, to make possible the use of these remarkable catalysts in less drastic conditions than those required by the native enzyme, including pH, temperature, ionic strength, resistance to inhibitors, etc. Other objectives lie in the modification of the catalytic properties themselves, that is to say in the widening of the range of substrates justiciable of the given enzyme, or - on the contrary - in the orientation of the catalytic activity in the sense of greater specificity; it may be, for example, to make a protease more specific for a certain peptide bond, if it hydrolyzes, in the native state, a too broad spectrum of peptides or the use of enzymes in a non-aqueous medium-or weakly concentrated in water which is likely to reverse the direction of the reactions in which the water intervenes.

 Most of the known methods of chemical protein modification involve placing specific compounds capable of reacting with functional groups, carried by the residues of the natural amino acids of the peptide ring or by the carbohydrate or nucleic portion of glycoproteins or of nucleoproteins. Thus the nucleophilic power of the -NH 2 'NH 2 group of lysine residues on activated electrophilic reagents is very commonly used. The reagents most often used in the prior art are acid anhydrides and trinitro benzene sulfonic acid. The enzymes, modified by these known methods, generally have a substantially lowered activity. The choice of reagents is very limited, because many possible reagents too much alter the activity of the enzyme, or require operating conditions that are incompatible with the preservation. catalytic activity, or react with other essential amino acids to maintain the active structure of the enzyme.

 As a result, given the interest of the appropriate modification of proteins, especially enzymes, the need for adequate reagents continues to be felt.

 The present invention provides in this field a significant progress: it relates to a class of compounds that have the property of easily reacting with amine residues of protides, to fix on them any desired organic group, lowering only relatively little catalytic activity When the protide is an enzyme, and making it soluble in an organic solvent.

 The process of the invention is characterized in that the given protide is reacted with a dithioi ester whose acid residue contains the organic group which it is desired to bind to the protein, while the thiol residue has a departing group structure.

où R désigne le groupe organique à fixer, par l'intermédiaire de C=S, sur le protide, alors que X est un groupement choisi de telle manière que X-S soit un bon groupe partant.The dithioic ester employed can be represented by the formula

where R denotes the organic group to be fixed, via C = S, on the protide, while X is a group chosen such that XS is a good leaving group.

By denoting by E the molecule of the latter, the reaction according to the invention can be schematized as follows:

Thus, HSX being eliminated, the protide molecule is modified by the attachment of one or more groups

This modifier group is chosen, of course,
according to the new properties which it is desired to acquire to the protide, for example solubility in hydrocarbons, reactivity with bones, resistance to heat, etc.

It can therefore comprise an R which is a hydrocarbon radical, aliphatic or aryl, therefore hydrophobic, in particular C 1 -C 20 alkyl or C 2 -C 20 alkenyl, phenyl, phenylenyl, naphthyl or naphthylenyl, or a corresponding alkylaryl or arylalkyl . R may also be a hydrophilic, polar, negative, positive or neutral organic group, it may, for example, contain a carboxyl, hydroxyl, halogen, sulphinyl, sulphonyl, phosphoryl, phosphonyl, sulphhydryl, amide, ammonium, phosphonium, etc. group.

 In a particular embodiment, R is a polymer or oligomeric cat, for example polyethylene, polypropylene, polyacrylic, polyethylene glycol or the like.

dans lequel R' désigne un groupe hydrocarboné divalent, notamment alkylène, cycloalkylène, arylène ou alkyl-arylène, le premier comportant de préférence 1 à 20 atomes de C, les autres 6 à 16.In addition, R can be constituted, itself, by a dithioester residue of the type

in which R 'denotes a divalent hydrocarbon group, in particular alkylene, cycloalkylene, arylene or alkylarylene, the former preferably containing 1 to 20 C atoms, the others 6 to 16.

Y is a group of the same type as X but not necessarily identical to this one. In this variant, the reagent is a bis-dithioester

The use of a bis-dithioester makes it possible to form a modified, special protein bearing a polymer chain, in particular polyacrylic chain, of polyvinyl alcohol, polyalkylene glycol or other oligomer or polymer capable of being aminated at least by end of chain.Naming Q-NH2 such an amino polymer, it can be combined with bis-dithioester by a reaction

Then, by reacting the compound of Q obtained in (4) [e p-5]
NOTE
In a variant X or / and Y, which are similar or different, are constituted by a hydrocarbon portion (-CH 2 -) n, where n is 1 to 6, bearing a halogen, carboxyl, sulfinic, sulphonic, phosphorous, phosphonous or phosphoric function, phosphonic, active H, SH, amide or hydroxy.

 A protide according to the invention, in particular protease or peroxidase, soluble in benzene, toluene, ether, ethanol, chloroform and / or dimethyl sulfoxide, comprises several lysine units which carry, via a -c = S- a group R consisting of a C1 to C20 aliphatic radical, a C6 to C16 aryl radical or a polymer chain, in particular polyethylene glycol.

c 'est-à-dire un bis-thioamide à la fois du polymère Q et du protide E.with a protide S NH2, leads to

that is, a bis-thioamide of both the Q polymer and the E-protide.

By way of nonlimiting illustration, the formulas of some of the dithioesters and bis-dithioesters used in the embodiment of the present invention are given below.

 Depending on the nature of the protein to be treated and that of the dithioester employed, the process according to the invention is carried out in aqueous solution, organic or hydro-organic, generally at a temperature between 0 and 700C.

In the case of enzymes, the preferred temperatures range from 100 to 500 ° C. and especially from 200 to 450 ° C.

The proportions of the reagents are calculated from the number of -NH- or -NH 2 groups of the protein that is to be combined with the dithioester. It is generally good to use an excess of dithioester functions by -NH- or -NH2 free group of the protein on which we want to fix the modifier group. The reaction medium can optionally be heterogeneous, which may for example be constituted by a dispersion of the protide in an organic solution of the dithioester; this is particularly the case where the reaction according to the invention solubilizes the protide in the organic solvent of the dithioester; thus, the protide, having undergone the reaction, goes into solution in this solvent.

 The preferred pH of the reaction medium is between the isoelectric point of the protein and 11.5, in the case of enzymes, the most favorable pH are between about 7.5 and 11.5, and especially between 8 and 11.

 The following examples provide, without limitation, details of the implementation of the invention.

EXAMPLE 1
The operations described here serve to show (I) that it is possible to prepare a dithioester from another, by simple change of nucleofuge, and - secondly (II) how the -NH2 of an amino acid reacts with a.dithioester.

I- Preparation of S-dithiobenzoyl N-Acetyl Cysteine (DTAC)
To 1.632 g of N-acetyl cysteine and 2.123 g of thiobenzoyl mercapto acetic acid (or carboxymethyl dithiobenzoate), dissolved in 50 ml of ethanol, 2 ml of aqueous NaOH solution are added. The mixture is stirred at 200 ° C., for 2 hours and then concentrated, by evaporation of ethanol, and acidified with 6N HCl.

By chromatography on silica, eluting with a hexane / chloroform mixture, 2.45 g of S-thiobenzoyl N-acetyl cysteine are isolated, which represents a yield of 86.5%, relative to the starting acetyl cysteine. , of the reaction

II- Preparation of N-thiobenzoyl lysine from
dithiobenzoyl acetyl-cysteine (DTAC) 0.425 g or 1.5 mmol of DTAC, prepared according to I, and 0.220 g of lysine, also 1.5 mmol, are dissolved in 10 ml of 0.25N aqueous NaOH solution. The solubilization of mixing is facilitated by the addition of a drop of ethanol.

After 5 hours of reaction at room temperature, the medium is acidified and liberates N-thiobenzoyl lysine

N-thiobenzoyl lysine
EXAMPLE 2
Modification of horseradish peroxidase with tridecyl thiomercaptoacetic acid C12H25CS-S-CH2COOH (ie carboxymethyl)
In 3 ml of 0.1 M phosphate buffer pH = 8.5 are dissolved 6 mg of commercial horseradish peroxidase (Sigma type II), ie 0.15 immolum (according to absorption at 402 nm with 3 = 102 mMl. Cm -1 ) and 1.6 mg of tridecyl thiomercaptoacetic acid is 5 immoles (previously dissolved in 0.1 ml of ethanol). The mixture is stirred for 18 hours at room temperature. The solution is then dialyzed for 18 hours at 40C against distilled water and then lyophilized. The enzyme obtained was analyzed. It has the following characteristics.

 Spectrum W: appearance of a band at 277 nm due to thioamide functions formed.

 Number of lysines affected; the free lysines are assayed by the conventional TNBS (trinitrobenzenesulphonic acid) method, thus 3 to 4 lysines are affected.

 Solubilities: While the native enzyme is only soluble in water, the new enzyme is soluble to more than 5 mg / ml in ethanol, chloroform, toluene, ether and water.

Specific activity: this is the activity measured on oxidation of orthodianisidine by oxy-genic water. A dimer is formed which absorbs at 444 nm (with -1
= 30 mu ~ 1. cl). The specific activity obtained is 850 U per mg of protein, ie 97% of the specific activity of the native enzyme.

Activity in organic solvent: Horseradish peroxidase is known to catalyze the oxidation reaction of 9-methoxyelipticin

This reaction is interesting because 9-oxoellipticin is readily reduced to 9-hydroxyellipticin by ascorbic acid. The only drawback lies in the low solubility of 9-methoxyellipticine in water. The low substrate concentration, which results for the enzyme, limits the rate of the oxidation reaction.

However, it was possible to carry out this reaction in ether with the modified peroxidase according to the invention. 0.5 ml of 1 mM 9-methylene-ellipticin in ether, 0.5 ml of modified peroxidase (1 mg / ml in ether), 10 to 50 μl of aqueous solution of water are mixed in a spectrophotometer tank. 200 mM oxygenated and supplemented with ether to a final volume of 2 ml. The adsorbance at 486 nm, the absorption wavelength of the 9-oxoellipticine formed (= 9, is then followed at 20 ° C. 2 mM 1.cm 1). Several tests, performed with different amounts of hydrogen peroxide, have shown that the oxidation kinetics is Michaelian with
KH2O2 = 2 mM
m
vm = 0.25 mol / min per mg lyophilisate of
modified peroxidase.

EXAMPLE 3
Fixation of thiobenzofc groups on papain making it soluble and active in dimethyl sulfoxide
In 40 ml of 0.1 M phosphate buffer pH = 8.5 are dissolved 0.32 g of commercial papain (Sigma Type IV) and 100 mg of thiobenzoyl-mercapto-acetic acid, previously dissolved in 3 ml of ethanol. The mixture is left for 2 hours at 400 ° C. and then dialyzed for 16 hours at 40 ° C. against water and then lyophilized. 0.26 g is recovered, ie 81% of chemical yield. The enzyme obtained has the following characteristics.

 Spectrum W: displacement of the 278 nm absorption band at 280 nm.

 Number of lysines affected: 2 of the 10 that includes papain.

 Solubility: Soluble in benzene, DMSO and water at 10 mg / ml, while the native enzyme is not soluble in benzene, only in water and DMSO.

Amidase activity: it is that of hydrolysis, at pH = 7.5 and 500C in tris buffer and in the presence of cysteine and EDTA, N-benzoyl D, L arginine para-nitroanilide (BAPA) which liberates the absorbent para-nitroaniline at 410 nm (with = 8.8 mu · cm -1). While the native enzyme has an activity of 28 mU per mg lyophilizate, the modified enzyme has 11 mU per mg lyophilizate, or 32% of the initial activity; despite this, the modified enzyme is very interesting because it has an organic solvent activity; it can therefore be used in DMSO, in the absence of water, for peptide synthesis.
DMSO is particularly suitable for solubilizing many peptides, but the native enzyme has no activity in this solvent: on the other hand, the modified enzyme has an activity of 1.2 mU per mg of lyophilizate.

sur la papaine
Pour cela, dans 4,5 ml de tampon phosphate 0,1 M pH = 8,5 on mélange 36 mg de papaïne commerciale (Sigma
Type IV) avec 23 mg de dithioester préalablement dissous dans 0,1 ml d'éthanol. Le mélange est porté à 400C pendant 2 heures, puis dialysé 16 heures à 40C contre du tampon phosphate 0,1 M pH = 7,2. La solution protéique obtenue est analysée et l'on trouve
Spectre UV : déplacement maximum d'absorption.EXAMPLE 4
Modification of the papain by the attachment of thiopropionate groups on its molecule
The dithioester carboxymethyl 3-sulphide ethyl dithiopropionate is reacted in an aqueous medium.

on the papain
For this, in 4.5 ml of 0.1 M phosphate buffer pH = 8.5, 36 mg of commercial papain (Sigma
Type IV) with 23 mg of dithioester previously dissolved in 0.1 ml of ethanol. The mixture is heated at 400C for 2 hours and then dialyzed 16 hours at 40C against 0.1M phosphate buffer pH = 7.2. The protein solution obtained is analyzed and found
UV spectrum: maximum absorption displacement.

from 278 nm to 270 nm
Number of lysines affected: 4.5 out of 10
Enzymatic activity in water, 50% loss.

The sites of the modified molecule can be represented, according to formula (2), by

This example shows that it is possible to replace a group -tH3 with a group -COO, which makes it possible to change the isoelectric point and the optimum pH range of the modified enzyme.

préalablement dissous dans 0,1 ml d'éthanol ; cela représente 250 moles de fonction dithioester Le pH se fixe à 10,8. On laisse agiter 48 heures à la température ambiante. La solution est ensuite dialysée 16 heures à température ambiante contre de l'eau distillée. On récupère alors 13 ml d'une solution de pH = 6,6 qui est analysée.EXAMPLE 5
Fixation of a polyethylene glycol bis-dithioester (PEGdithioester) on horseradish peroxidase
A functionalized polymer is prepared in the following manner. In 10 ml of distilled water, 0.25 g of
PEG 20000 bis-amino, commercial (Sigma), corresponding to 25 moles of free NH 2 functions; 0.4 ml of triethylamine and 44 mg of carboxymethyl tetrathiooctanedioate are added,

previously dissolved in 0.1 ml of ethanol; this represents 250 moles of dithioester function. The pH is 10.8. Stirring is carried out for 48 hours at room temperature. The solution is then dialyzed for 16 hours at room temperature against distilled water. 13 ml of a solution of pH = 6.6 are then recovered, which is analyzed.

le rapport des concentrations en fonction thioamide (1,31 mM) et dithioester (0,18 mu) permet de calculer le degré de polymérisation n ; ce rapport vaut
2n + 2
r = ~~~~~~ = n+ 1
2 expérimentalement : r = 1,31 = 7,3 soit n = 6,3 on obtient 0w18 6 mg de peroxydase de raifort commerciale (Sigma Type II) sont dissous dans la solution précédemment préparée (0,15 mole de peroxydase et 2,3 mole de dithioester) et le pH est porté à 8,5 par dissolution de Na2HPO4 dans le mélange.The UV spectrum makes it possible to assay at 308 nm the dithioester grafted functions (with = 12.2 mM 1 cm -1) and at 263 nm the thioamide functions formed (= 12.8 mM 1 cm -1). Having worked with an excess of dithioester functions, we can admit that there are no more functions -NH2 free. So we have a product of formula

the ratio of the concentrations of thioamide (1.31 mM) and dithioester (0.18 mu) makes it possible to calculate the degree of polymerization n; this report is worth
2n + 2
r = ~~~~~~ = n + 1
2 experimentally: r = 1.31 = 7.3, ie n = 6.3, 018 6 mg of commercial horseradish peroxidase (Sigma Type II) are dissolved in the previously prepared solution (0.15 mole of peroxidase and 2, 3 moles of dithioester) and the pH is brought to 8.5 by dissolving Na2HPO4 in the mixture.

It is stirred for 18 hours at room temperature, then the solution is dialyzed and finally lyophilized. The enzyme obtained has the following characteristics.

 Spectrum W: appearance of a band at 287 nm due to thioamide functions.

Number of lysines affected: 2
Solubility: the enzyme is soluble to more than 5 mg / ml in solvents: acetone, chloroform, dioxane, toluene, dimethylformamide, water
Specific activity: 780 U per mg of protein or 89% of the specific activity of the native enzyme.

Activity in chloroform: for the oxidation of 9-methoxyellipticine, the activity is satisfactory. In a spectrophotometer tank are mixed: 0.5 ml of 1 mM 9methoxyellipticin in chloroform, 0.3 ml of modified peroxidase at 1 mg / ml in chloroform, 10 to 40 ssl of hydrogen peroxide 200 mM (in solution aqueous) and the volume is brought to 1.5 ml with chloroform. For different amounts of hydrogen peroxide, there is a Michaelian kinetics with
K 2 2: 6.25 mM
m
Vm = 83 nmol / minute per mg of lyophilizate of
modified peroxidase.

1,9 mg de papaïne commerciale (Sigma type IV) et 89 mg de
Na2HPO4 sont dissous dans 23 ml de la solution susindiquée de polymère fonctionnalisé. Le volume est porté à 50 ml avec de l'eau distillée. Le pH obtenu est 8,5.La solution est portée à 400C pendant 2 heures puis dialysée 16 heures à 40C contre de l'eau distillée. La solution dialysée a un pH = 6,6 et un volume de 58 ml. Après lyophilisation on ré récupère 45 mg, soit un rendement chimique de 76%. L'analyse de l'enzyme ainsi modifiée donne les résultats que voici.EXAMPLE 6
Modification of papain using a polyethylene glycol bis-dithioester
The functionalized polymer was prepared as in Example 5, but under conditions of greater dilution. The same amounts of product were used in 100 ml of distilled water instead of 10 ml in Example 5. A lighter polymer was then obtained, since the assay W leads to n = 0. in the presence of

1.9 mg of commercial papain (Sigma type IV) and 89 mg of
Na2HPO4 are dissolved in 23 ml of the above-mentioned solution of functionalized polymer. The volume is brought to 50 ml with distilled water. The pH obtained is 8.5. The solution is heated at 400 ° C. for 2 hours and then dialyzed for 16 hours at 40 ° C. against distilled water. The dialyzed solution has a pH = 6.6 and a volume of 58 ml. After lyophilization, 45 mg is recovered, ie a chemical yield of 76%. The analysis of the modified enzyme gives the following results.

 Spectrum W: The 278 nm band is shifted to 269 nm with a shoulder at 308 nm showing that all dithioester functions have not reacted.

 Number of affected lysines: 5 out of 10 lysines.

 Solubility: The enzyme is soluble at 10 mg / ml in chloroform, DMSO and water.

Amidase activity: 1.5 mU per mg lyophilizate, ie a yield in activity:
45 x 1.5 = 125%
1N9 x 28
Activity in the -DMSO by the same test as in Example 3: 3.0 mU per mg lyophilisate. In chloroform, the substrate solution is prepared in a mixture of 80% chloroform with 208 DMSO to solubilize the ingredients necessary for the test. An activity of 4.2 mU per mg lyophilizate is found. On the other hand, the native enzyme, put in suspension (because insoluble in chloroform), in an identical test, gives no activity.

 Both Examples 5 and 6 show that the modification with PEG-dithioesters allows to have good activities in chloroform, which brings a significant technical progress.

Claims (13)

 1. A process for modifying a protide by fixing an organic group on its molecule, characterized in that the protide is reacted with a dithiol ester whose acid residue contains the organic group to be fixed, while the thiol residue has a leaving group structure so that the dithioester reacts with -NH2 or -NH- of the protein molecule.  2. Process according to claim 1, characterized in that the dithiolic ester corresponds to the formula

 where R denotes the organic group to be attached through C = S to the protide, while X is a group removable by the reaction of -S-X with an amine residue of the protein.  3. Process according to claim 2, characterized in that R is a hydrocarbon radical, aliphatic or aryl, especially C1-C20 alkyl, C2-alkenyl. C20, phenyl, phenylenyl, naphthyl, naphthylenyl, alkylaryl or arylalkyl.  4. Process according to claim 2, characterized in that R is an organic, hydrophilic, polar, negative, positive or neutral group, in particular carrying a carboxyl, hydroxyl, halogen, sulfinyl, sulphonyl, phosphoryl, phosphonyl, sulphydryl or amide.  5. Method according to claim 2, characterized in that R is a channe polymer or oligomer, in particular polyethylene, polypropylene, polyacrylic polyethylene glycol or polyvinyl alcohol.

 Process according to Claim 2, characterized in that R is of the type C16, while Y is a group that can be removed by the reaction of -S-Y with an amine residue of the protein. C20, or cycloalkylene, arylene or C6-alkyl arylene at R 'denoting a divalent hydrocarbon group, in particular a C1 to C4 alkylene  7. Process according to one of claims 2 to 6, characterized in that X or / and Y, which are similar or different, are constituted by a hydrocarbon portion (-CH 2 -) n, where n is 1 to 6, bearing a halogen function, carboxyl, sulfinic, sulfonic, phosphorous, phosphonous, phosphoric, phosphonic, H active, SH, amide or hydroxy.  8. Method according to one of claims 1 to 7, characterized in that it is carried out in an aqueous solvent, organic or hydro-organic, or in a heterogeneous medium, between 0 and 700C, preferably 200-450C, the pH of the medium being between the isoelectric point of the protide and 11.5, preferably between 8 and 11, in particular in the case of enzymes.  9. Chemically modified protide, characterized in that at least one of its nitrogen atoms carries a group

 where R is an organic radical, in particular a hydrocarbon radical comprising a polar part, positive, negative or neutral, or is constituted by a polymer chain.  10. Protide according to claim 9, consisting of an enzyme characterized in that it is soluble in organic solvents.  11. Protide according to claim 10, in particular protease or peroxidase, soluble in benzene, toluene, ether, ethanol, chloroform and / or dimethyl sulfoxide, several lysine units of which, via a

 a group R consisting of a C1 to C20 aliphatic radical, a C6 to C16 aryl radical or a polymer chain, in particular polyethylene glycol.  12. Protide according to claim 9, characterized in that it consists of an enzyme whose isoelectric point and the optimum pH range are modified.  13. Protide according to claim 10, characterized in that it consists of an enzyme, especially hydrolase, capable of catalyzing a reverse reaction, in particular synthesis of esters, amides, thioamides and peptides.