GB2029825A - Benzothiazolyl- and pyridyl-N- oxide-disulphide derivatives - Google Patents

Abstract

Disulphide compounds of the general formula:- R1-S-S-R2-@(CO-R3-)n-R4 wherein R1 is a 2-benzothiazolyl or 2- pyridyl-N-oxide radical, R2 is an alkylene radical optionally containing a free or protected functional group, R3 is a carboxyl residual group of an amino acid or lower peptide, R4 is a carboxyl group, a reactive derivative thereof or a protected carboxyl or imidate group and n is 0 or 1, are obtained by reacting disulphides of the formula R1-S-S-R6(R6 being a group capable of forming a mercaptan R6SH from a mercapto group) with mercaptans H-S-R2-(CO-R3-)n-R5 R5 being R4 or a group capable of being changed into an imidate group. The compounds have -S-S- exchange reactivity, e.g. with proteins.

Description

SPECIFICATION Disulphide compounds and the preparation thereof The present invention is concerned with disulphide derivitives having S-S exchange reactivity and with the preparation thereof.

The disulphide derivatives according to the present invention are compounds of the general formula

wherein R1 is a 2-benzothiazolyl or 2-pyridyl-N-oxide radical, R2 is an alkylene radical optionally containing a free or protected functional group, R3 is a carboxyl residual group of an amino acid pr lower polypeptide, Ra is a carboxyl group, a reactive derivative thereof or a protected carboxyl or imidate group and n is O or 1.

The disulphide derivatives (i) are useful reagents having an S-S exchange reactivity and the reactive derivatives of the carboxyl group thereof can be used as a thiol group-introducing reagent.

Disulphide derivatives having an S-S exchange reactivity for compounds having a thiol group are known and are used in covalent chromatography (see Biochem. J., 133, 573-584/1 973: "Affinity Chromatography: Practice and Application", pp. 64-65/1976); and Farmacia, 14(1)47-52/1978).

Some of the disulphide derivatives are used, based on their S-S exchange reactivity, as cross-linking reagents for the reaction between a protein having a thiol group and a protein having an amino group (see Biochem., 17(8), 1499--1506/1978).

However, the S--S exchange reaction rate of the known disulphide derivatives is quite slow and thus requires a long time, so that the use of the compounds is not very advantageous.

We have found that in the case of the novel disulphide derivatives of general formula (I), in which R, is a 2-benzothiazolyl or 2-pyridyl-N-oxide radical and R2 is an alkylene radical, the S-S exchange reaction proceeds 2-10 times more rapidly than in the case of the known compounds.

In the disulphide compounds of general formula (I) hereinafter designated as disulphide derivatives (i), R1 is a straight or branched chain alkylene radical optionally containing a free or protected functional group, the functional group being, for example, an amino group or a carboxyl group, and R3 is a spacer group which does not have a detrimental effect on the S-S exchange reactivity, for example a carboxyl residual group of an amino acid or lower polypeptide. Examples of amino acids include the known aamino acids and amino acids. Examples of lower polypeptides include peptides comprising 2 to 5 amino acid residues. R4 is a carboxyl group ora functional derivative thereof such as an active ester or acid halide, or a protected carboxyl or imidate group.

Examples of disulphide derivatives of the present invention are illustrated in the following Table 1.

Since the disulphide derivatives (I) contain, as R1, a benzo-thiazolyl or pyridyl-N-oxide radical and, as R2, an alkylene radical, the S-S exchange reactivity is extremely advantageous.

TABLE 1

R1 R2 2 R3 n R4 -CH2- -NHCH2(cH2)3. CH2- O -COOH N//CN2CN2 -NH.CH2- 0 -CH- 1 -COO 0 CH3 -NHCH2COMCH2- O CH-CH -NHCH2CONHCH- -COONO2 CH C'N 3 1. 3 CH3 -COOCH -C-CH CH 3NH2 -COC1 -C" CH2COOH -COBr -CH-CH2 XNH2 COON -C . OCH3 -CHZ-CH NHCOCH 2CH 2-C0H-NH2 COON The disulphide derivatives (I) can be prepared, for example, by the following synthesis:

R1 -S-s- Rg (1X) l 02 4 Co-R3t Rs (iso) R- S-S- Co from R3+ Rg St-S-S- 82*Co- from wherein R6 is the same as R4 or is a group which can be changed into an imidate group, R6 is a group capable of reacting with a mercapto group with the elimination of R6-SH and R1, R2, R3, R4 and n have the same meanings as above.

Examples of compounds R,-SS-S-R6 (II) include 2,2'-dithio-bis-(benzothiazole) and 2,2'-dithio bis-(pyridine-N-oxide).

Examples of compounds HS-F2CORR5 (III) include thiolcarboxylic acid or the carboxylic acid derivatives thereof which react with the compound (II) for example, a functional derivative or protective derivative of an active ester or acid halide, and a compound containing a nitrile group which can be changed into an imidate group, such as a thiolnitrile. Examples thereof include thioglycolic acid, ,B-mercaptopropionic acid, thiolactic acid (a-mercaptopropionic acid), thiomalic acid, cysteine, peniciilamine, glutathione, a condensation product of p-mercaptopropionic acid and E-aminocaproic acid and p-mercaptopropionitrile or the carboxylic acid derivatives thereof.In the above compounds, the functional group in the molecule, such as an amino or carboxyl group, can, if desired, be protected by acid formation using an organic or inorganic acid or base, or can be protected by a known protective group. These protective groups are known in the field of peptide synthesis and can easily be removed by known methods, such as hydrolysis, acid decomposition, reduction, aminolysis or hydrozinolysis.

Examples of protective groups for the amino group include acyl radicals, such as the formyl, trifluoroacetyl, phthaloyl, benzenesulphonyl, p-toluenesulphonyl, o-nitrophenylsulphenyl or 2,4dinitrophenylsulphenyl radical; aralkyl radicals, such as the benzyl, diphenylmethyl or triphenylmethy! radical (these radicals can be optionally substituted by a lower alkoxy radical, such as an o-methoxy or p-methoxy radical; benzyloxycarbonyl radicals, such as the benzyloxycarbonyl, o- bromobenzyloxyca rbonyl, o- or p-ch lorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, pmethoxybenzyloxyca rbonyl, p-phenylazobenzyloxycarbonyl or p-(p'-methoxyphenylazo)benzyloxycarbonyl radical; aliphatic oxycarbonyl radicals, such as the cyciopentyloxycarbonyl, trichloroethyloxycarbonyl, t-a myloxycarbonyl, t-butoxycarbonyl or diisopropylmethoxycarbonyl radical; and aralkyloxyca rbonyl radicals, such as the 2-phenylisop ropoxycarbonyl, 2-tolylisopropoxycarbonyl or 2-p-diphenylisopropoxycarbonyl radical. Amino groups can also be protected by enamine formation by reaction with a 1,3-diketone, such as benzoylacetone, acetylacetone or dimedone. Carboxyl groups can be protected by amide formation, hydrazide formation of esterification. An amide group can be substituted by a 3,4-dimethoxybenzyl, bis-(p-methoxyphenyl)-methyl or the like radical.The hydrazide group can be substituted by the benzyloxycarbonyl, trichloroethyloxycarbonyl, trifluoroacetyl, tbutoxycarbonyl, trityl or 2-p-diphenylisopropoxycarbonyl radical. The ester group can be derived from an alkanol, such as methanol, ethanol, t-butanol or cyanomethanol, an aralkanol, such as benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, p-methoxybenzyl alcohol, p-nitrobenzyl alcohol, 2,4,6trimethylbenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol orpchlorobenzolymethyi alcohol; a phenoi, such as 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol 2,4-dinitrophenol, p-cyanophenol or p-methane-sulphonylphenol, or a thiophenol, such as thiophenyl, thiocresol or p-nitrothiophenol.

The hydroxyl group can be protected by esterification or etherification. Examples of esterification groups include lower alkanoyl radicals, such as the acetyl radical; aroyl radicals, such as the benzoyl radical; or a radical derived from the benzyloxycarbonyl or ethyloxy-carbonyl radical. Examples of etherification groups include the benzyl, tetrahydropyranyl and t-butyl radicals. Further preferable examples of protective groups for the hydroxyl group include the 2,2,2-trifluoro-1 -tbutyloxycarbonylaminoethyl and 2,2,2-trifluoro- 1 -benzyloxycarbonylami noethyl radicals. However, the hydroxyl group need not always be protected.As a protective group for the imino group, there can be mentioned the benzyl, trityl, benzyloxycarbonyl, tosyl, adamantyloxycarbonyl, 2,2,2-trifluoro-l-t- butyloxycarbonylamino ethyl and 2,2,2-trifluoro-1-benzyloxycarbonylamino ethyl radicals. Here again, the imino group need not aiways be protected.

The thiolcarboxylic acids comprising residues of amino acids or of lower peptides containing 2 to 4 amino acids can be prepared by reacting an amino acid or peptide having a protected a-amino group and an activated terminal carboxyl group with amino acid or peptide having a free a-amino group and a protected terminal carboxyl group, or by reacting an amino acid or peptide having an activated a-amino group and a protected terminal carboxyl group with an amino acid or peptide having a free terminal carboxyl group and a protected a-amino group.

The compounds of general formula (II) are usually reacted with compounds of general fdrmula (III) in a solvent, such as methanol, ethanol, acetone, benzene, chloroform or carbon tetrachloride. The reaction components (II) and (III) are added to the solvent in equimolar ratio and reacted at 10 to 700 C.

and preferably at about 700C. for 10 minutes to 5 hours and preferably for 2 to 3 hours.

After the reaction, the product formed of general formula (IV) can be obtained in the usual way, for example by cooling and extracting.

In this product, the carboxyl group remains as it is or a carboxyl or nitrile group can be changed in known manner into a reactive derivative of a carboxyl group or protected carboxyl group. Furthermore, a nitrile group can be changed into an imidate group to give the product of general formula (I).

Examples of the reactive derivatives of the carboxyl group include conventional derivatives, such as acid azides, acid anhydrides, acid imidazolides, active esters and acid halides, for example, cyanomethyl esters, thiophenyl esters, p-nitrothiophenyl esters, p-methoane-sulphenyl esters, thiodyl esters, p-nitrophenyl esters, 2,4-dinitrophenyl esters, 2,4,5-trich lorophenyl esters,2,4,6-trichlorophenyl esters, pentachlorophenyl esters, N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, -8hydroxyquinoline esters and N-hydroxypiperidine esters. Other reactive derivatives can be obtained by using carbodiimide, N,N'-carbonyldiimidazole or isoxazolium such as Woodward reagents.

The following Table 2 gives some examples of disulphide compounds of the present invention compared with 3-(pyridine-2'-yl-dithio)-propionic acid.

The S-S exchange reaction rates were measured by reaction with a solution of dithiothreitol containing 1 mM ethylene-diamine-tetraacetic acid (EDTA) in 0.2 M Tris-HC1 buffer (pH 7.5) as a compound having a thiol group. Subsequently, the increased absorbancy was measured at its maximurr absorbancy wavelength to determine the molar ratio of S-S exchange reaction per minute.

As illustrated in Table 2, the disulphide derivatives of the present invention have a higher S-S exchange reaction rate.

T A B L E 2

RISsR2 ( CO-R3 ej R4 S-S exchange rartion rate 7substance wavelength reaction rate RI ( CO- fl R4 - measured measured mole/m n.

-2-CH2- O -COOH SH 310 nn 39.0 C 'OCH o 4011 = 37.4 CH3 42.CH2 O ( > N02 " 0 35.7 CR2 CR2- -CONHCH2(CH2)3CH2 1 'COOT 34.3 -CR CR- O -COOH r 35.6 RH CtH.3 CH3NH2 O -COOH 35.0 -CR- O -COOH r cH2coa8, COB 2 C -COOR 36.2 -CH2.CR2- 0 -COOH OSH 1 333 nm 93.6 t"ro } -CH2 CH2 O OOO2 . 93.5 O 2 , 2 O -COCH 90.3 NH2 CHO 1 -C-CH- O -COOH r 91.6 CR RH 3 2 g} c! CH2 CH2 | | O | -COOH Ns 343 nm 8.7 0 N S The disulphide derivatives (I) are useful as thiol group-introducing reagents and as cross-linking reagents.For example, a disulphide derivative (I) can be reacted with a compound having a reactive hydrogen atom, such as an amine compound, in a solvent, such as benzene, toluene, chloroform, acetone, tetrahydrofuran or dimethylformamide, optionally in the presence of a condensation reagent, to form an ester, amidino or amide linkage by reacting the reactive group R4 of the disulphide derivative with a hydroxyl or amino group in a compound having a hydroxyl group or with an amine. Subsequently, the disulphide bond of the compound is hydrolysed to introduce the thiol group into the compound having a hydroxyl group or the amine.The above compound with an ester or amide linkage can be reacted with a compound having a thiol group in the presence of an aqueous medium at pH 7 to 8 to achieve an S-S exchange reaction, thereby cross-linking the compound having a thiol group and the compound having a hydroxyl group or the amine.

Examples of the compounds having a hydroxyl group and of amines include insulin, albumin, growth hormone, calcitonin, prolactin, ACTH, PTH, glucagon, gastricsin, secretin and y-globulin, and immune components, such as IgG, lgM, lgA, secondary antibody, oestrogen, ATP, catecholamine and triiodothyronine, antigers of antibiotics and hypnotics, antibodies, secondary antibodies of haptens, and oxide-reductases, such as peroxidase, catalase, cholesterol oxidase, glyceroldehydrogenase and choline oxidase; hydrolases, such as alkaline phosphatase, gl ucoamylase, phospholipase,- D, p-galactosidase and lysozyme; and other transferases, lyases and isomerases; polysaccharides, such as cellulose, a minated cellulose, aga rose, dextrin, dextran or their water-insoluble derivatives; and water-insoluble materials hainv hydroxyl or amino groups, such as aminated polyamides, polyacrylonitriles and silanes.

There are also included known compounds and novel compounds which can be used as carriers in the present invention. Examples of novel compounds having amino groups include y-aminopropylated polyamides, amino derivatives of polyacrylonitrile polymers and polyacrylonitrile group polymers. A yaminopropylated polyamide can be prepared by heating a polyamide carrier, such as 6,6-nylon and 6nylon, in y-aminopropyltriethoxysilane at 1 000C. for 3 hours in order partly to introduce y-aminopropyl radicals into the amide groups of the polyamide compound.The amino derivatives of the polyacrylonitrile polymers or polyacrylonitrile group polymers can be prepared by heating under reflux a polyacrylonitrile polymer or polyacrylonitrile group polymer in the presence of lithium aluminium hydride in a medium, such as diethyl ether, dioxan or tetrahydrofuran, for 1 to 48 hours to give amino groups with partly reduced nitrile groups.

Examples of compounds having a thiol group include enzymes, such as peroxidase, catalase, galactosidase and alkaline phosphatase, and haptens, as well as compounds into which a thiol group has been introduced by means of S-acetylmercaptosuccinic anhydride (see Arch. Biochem. Biophys., 96.605-612/1962) our a thiol group-containing compound having a hydroxyl group prepared by hydrolysis of a disulphide derivative (I) or an amine.

The S-S exchange reaction is performed, as explained above, by optionally combining the above compounds conventionaily in an aqueous medium, for example in a buffer at pH 7 to 8, at ambient temperature. The reaction product is recovered by conventional separation and purification methods, such as salting out, phase separation, extraction, dialysis or adsorption chromatography.The compounds thus produced are advantageous and useful compounds for immobilising enzymes, crosslinking enzymes and water-insoluble carriers, for immobilising antigens and antibodies, cross-linking antigens or antibodies and water-insoluble carriers, for antigenic haptens, cross-linking proteins and haptens, or as enzyme immunoassay components, and cross-linking enzymes and immune components, The following Examples are given for the purpose of illustrating the present invention: EXAMPLE 1 To 13.2 g. 2,2'-dithio-bis-(benzothiazole) are added 400 ml. benzene and 6 g. 3mercaptopropionate and the mixture stirred at-700C. for 3 hours. The reaction mixture is then cooled in an ice-bath to precipitate the crude product (13.8 g.), which is recrystallised from benzene to give 12 g.

of crystalline 3-(henzothiazol-2'-yl-dithio)-propionate.

m.p. 162-1640C.

max:272 nm (methanol) Rf: 0.33 (TLC, silica gel; benzene:ethyl acetate=1 :2 v/v).

EXAMPLE 2 4.3 g. 2,2'-Dithio-bis-(pyridine-N-oxide) are added to 200 ml. chloroform and 3 g. 3mercaptopropionate and the mixture reacted at 700C. for3 hours. The reaction mixture is then cooled.

The crude crystalline product precipitates out and is recrystallised from chloroform to give 4.1 g. of crystalline 3-(pyridine-N-oxide-2'-yl-dithio)-propionate.

: 126-2180C.

max: 270 nm (methanol) Rf: 0.66 (TLC, silica gel; n-butanol:acetic acid:water=4:1 :1 v/v/v).

EXAMPLE 3 To 3 g. 3-(benzothiazole-2'-yl-dithio)-propionat in 20 ml. ethyl acetate are added 1 g. N- hydroxysuccinimide and 1.7 g. dicyclohexylcarbodiimide, whereafter the reaction mixture is stirred at ambient temperature for 3 hours. Precipitated dicyclohexylurea is filtered off and the filtrate is washed with phosphate buffer (pH 7.5) to remove unreacted free acid. The ethyl acetate layer is dried with an hydros sodium sulphate, evaporated to dryness and the residue recrystaliised from hot petroleum ether to give 2.4 g. of crystalline 3-(benzothiazole-2 '-yl-d ithio)-propionatesuccinimide ester.

:m.p. 114-1150C; max: 270 nm (pH 7.5, 10% aqueous dimethylformamide) Rf: 0.53 (TLC, silica gel; benzene:ethyl acetate=3:1 v/v).

EXAMPLE 4 A mixture of 3 g. 3-(benzothiazole-2'-yl-dithio)-propionate (obtained by the process as described in Example 1), 1.2 g. p-nitrophenol and 2.1 g. dicyclehexylcarbodiimide is dissolved in 20 ml. ethyl acetate and stirred at ambient temperature for 3 hours. The remaining procedure is carried out as described in Example 3 to give 1.85 g. of crystalline 3-(benzothiazole-2'-yl-dithio)-propionate-p- nitrophenyl ester.

max: 279 nm (pH 7.5,10 /0 aqueous dimethylformamide) Rf: 0.84 (TLC, silica gel; benzene:ethyl acetate=5:1 v/v).

EXAMPLE 5 3 g. 3-(Benzothiazole-2'-yl-dithio)-propionate are dissolved in 10 ml. thionyl chloride and reacted at 250C. for 2 hours, whereafter excess thionyl chloride is removed in vacuo to give 3-(benzothiazole-21- yl-dithio)-propionyl chloride as an oily substance.

max=272 nm (methanol) Rf=0.25 (TLC, benzene).

EXAMPLES 6 AND 7 1.3 g. g.3-(Benzothiazole-2'-yl-dithio)-propionate-succinimide ester, prepared by the same procedure as described in Example 3, and 0.6 g. E-aminocaproic acid are added io 50 ml.

tetrahydrofuran and the mixture is reacted at ambient temperature overnight. The tetrahydrofuran is then removed in vacuo and the residue is dissolved in hot isopropanol and cooled to give 0.8 g.

crystalline 6-N-[3-(benzothiazole-2'-yl-dithio)-propionyl]-aminohexanoic acid.

max=272 nm (in methanol) Rf=0.07 (TLC, silica gel; benzene:ethyl acetate=1 :2 v/v).

500 mg. of the above-obtained compound, 200 mg. N-hydroxysuccinimide and 340 mg.

dicyclohexylcarbodiimide are dissolved in 10 ml. tetrahydrofuran and reacted at ambient temperature for 3 hours. Precipitated dicyclohexylurea is filtered off and the tetrahydrofuran is distilled off. The residue is dissolved in hot petroleum ether and cooled to give 430 mg. crystalline 6-N-[3 (benzothiazole-2'-yl-dithio)-propionyl]aminohexanate su ccinimide ester.

max: 271 nm Rf: 0.42 (TLC, silica gel; benzene:ethyl acetate=3:1 v/v).

EXAMPLES 8,9 AND 10 3-(Pyridine-N-oxide-2'-yl-dithio)-propionate, obtained by the process described in Example 2, is used to prepare the succinimide ester, p-nitrophenyl ester and acid chloride therof, according to the methods of Examples 3, 4 and 5, respectively: Succinimide ester:

max: 260 nm (methanol) Rf: 0.25 (TLC, silica gel; benzene:ethyl acetate=3:1 v/v).

p-nitrophenyl ester:

max: 391 nm (methanol) Rf: 0.82 (TLC, silica gel; benzene:ethyl acetate=3:1 v/v).

Acid chloride:

max: 270 nm (methanol) Rf: 0.15 (TLC,silca gel; benzene:ethyl acetate=3:1 v/v).

EXAMPLE 11 1.1 g. 2,2'-Dithio-bis-(benzothiazole) and 3-mercaptopropionitrile are dissolved in 50 ml. benzene and reacted at 700 C. for 3 hours, while stirring. The reaction mixture is cooled in an ice-water bath to precipitate crude crystals which are crystallised from benzene to give 750 mg. 3-(benzothiazole-2'-yl- dithio)-propionitrile. 700 mg. thereof are added to 50 ml. methanol containing 19 g. hydrogen chloride and reacted at 50C. overnight. The solvent is then distilled off in vacuo to give a crude powder which is washed with benzene to give 720 mg. methyl 3-(benzoth iazole-2 methyl 3-(benzothiazole-2'-yl-dithio)-propionimidate hydrochloride.

max: =272 nm (methanol) Rf=0.05 (TLC, silica gel; benzene:ethyl acetate=1 :2 v/v).

EXAMPLES 12-35 2,2'-Dithio-bis (benzothiazole) and 2,2'-dithio-bis-(pyridine-N-oxide), prepared as described above, are reacted with thioglycolic acid, thiolactic acid, cysteine, thiomalic acid, penicillamine, N-(2 mercaptopropionyl)-glycine and glutathione, respectively, under the same reaction conditions as described above to give the following compounds, the Rf values being obtained by silica gel thin layer chromatography:

max=272 nm (methanol) Rf=0.36 (benzene:methanol=1 :2 v/v) S-S exchange reaction rate: 25.6,umoles/min.

max: 272 nm Rf: 0.38 (benzene:methanol=1 :2 v/v)

max: 271 nm (methanol) Rf: 0.08 (benzene:methanol=1 :2 v/v) 0.60 (upper layer; n-butanol:acetic acid:water=4:1 :5 v/v/v).

max: 271 nm (methanol) Rf: 0.15 ibenzene:ethyl acetate=1 :2 v/v).

max: 271 nm (pH 7.5, phosphate buffer) Rf: 0.60 (n-butanol:acetic acid:water=4:1 :5 v/v/v, upper layer).

max: 271 nm (methanol) Rf: 0.30 (upper layer: n-butanol:acetic acid:water=4:1 :5 v/v/v) S-S exchange reaction rate: 35.5,umoles/min.

max: 271 nm, 418 nm (methanol) Rf: 0.23 (upper layer; n-butanol:acetic acid:water=4:1 :5 v/v/v).

max: 271 nm (methanol) max: 0.48 (benzene:ethyl acetate=3:1 v/v).

max: 271 nm (methanol) Rf: 0.12 (benzene:ethyl acetate=3:1 vfiv) S-S exchange reaction rate: 35.0 ymoles/min.

max: 278 nm Rf: 0.80 (benzene:ethyl acetate=3:1 v/v).

max: 271 nm (methanol) Rf: 0.15 (benzene:ethyl acetate=3:1 v/v) S-S exchange reaction rate: 36.6 umoles/min.

max: 271 nm (methanol) Rf: 0.80 (benzene:ethyl acetate=3:1 v/v).

max: 271 nm (methanol) Rf: 0.10 (benzene:ethyl acetate=3:1 v/v) S-S exchange reaction rate: 35.7 moles/min.

max: 271 nm (methanol) Rf: 0.05 (benzene:ethyl acetate=3:1 v/v).

max: 272 nm (methanol) 'Rf: 0.31 (upper layer; n-butanol:acetic acid:water=4:1 :5 v/v/v) S-S exchange reaction rate: 35.2 Mmoles/min.

max: 270 nm (methanol) Rf: 0.7 (upper layer; n-butanol:acetic a.cid:water=4:1 :1 v/v/v) S-S exchange reaction rate: 92.8,umoles/min)

max: 270 nm (methanol) Rf: 0.66 (n-butanol:acetic acid:water=4:1 :1 v/v/v), upper layer) S-S exchange reaction rate: 93.2 umolesSmin.

max: 310 nm (methanol) Rf: 0.82 (benzene:ethyl acetate=3:1 v/v)

max: 271 nm (methanol) Rf: 0.41 (n-butanol:acetic acid:water=4:1 :1 v/v/v) S-S exchange reaction rate: 92.6 moles/min.

max: 271 nm (methanol) Rf: 0.25 (n-butanol:acetic acid:water=4:1 :1 v/v/v).

max: 270 nm (methanol) Rf: 0.32 (n-butanol:acetic acid:water=4:1 :1 v/v/v) S-S exchange reaction rate: 90.1,umoles/min.

max: 270 nm Rf: 0.20 (N-butanol:acetic acid:water=4:1 :1 v/v/v) S-S exchange reaction rate: 91.5 moles/min.

max: 270 nm Rf: 0.41 (n-butanol:acetic acid:water=4:1 :1 v/v/v)

max: 270 nm Rf: 0.25 (n-butanol:acetic acid:water=4:1 :1 v/v/v) EXAMPLE 36 To 0.6 mg. bovine insulin, dissolved in 10 ml. 0.1 M veronal buffer (pH 7.5) is added 1 ml. of a dimethyl formamide solution of 3.1 mg. 3-(benzothiazole-2-yl-dithio)-propionate-succinimide ester, followed by stirring for 4 hours at ambient temperature.The reaction mixture is then adjusted at pH 5.0 to precipitate the product, which was centrifuged at 3000 r.p.m. for 10 minutes to give the insulin derivative with an introduced 3-(benzothiazole-2'-yl-dithio)-propionitrile radical. This insulin derivative is washed with citrate buffer (pH 5) and dissolved in 10 ml. of 0.1 M phosphate buffer (pH 7.5).

To 0.1 ml. of this solution are added 5 mg. /3-glactosidase dissolved in 5 ml. phosphate buffer (pH 7.5) and reacted at ambient temperature for one hour. Thereafter, the reaction mixture is applied to a column of "Sephadex" G-100 (1 x 80 cm.), eluted with 0.01 M phosphate buffer (pH 7.5) containing 0.15 M sodium chloride and 4 ml. fractions collected. Fractions Nos. 7-8 contain the cross-linked compound of insulin and p-galactosidase. ("Sephadex" is a Registered Trade Mark).

To 0.02 ng-1 0 ng of bovine insulin in test tubes is added the above cross-linked compound of insulin and ss-galactosidase and anti-bovine insulin serum, incubated at 50C. overnight and further incubated at 50C. overnight with the addition of rabbit anti-guinea pig lgG serum as secondary antibody. The incubation mixture is centrifuged at 3000 r.p.m. for 10 minutes and the precipitate thus obtained is incubated at 440 C. for 20 minutes by p-galactosidase colorimetry with the addition of 200 ,ul. o-nitrophenylgalactoside (5 mg./ml., 0.1% bovine serum albumin, 10 mM mercaptoethanol in 0.1 M phosphate buffer, pH 6.7).The reaction is subsequently stopped by adding 25 ml. 0.1 M glycine buffer (pH 10.5) and colorimetrically assayed at 420 nm to measure the p-galactosidase activity. This constitutes an enzyme immunoassay of insulin, using the insulin-ss-galactosidase cross-linkage compound, anti-insulin serum and secondary antibody. The results obtained are shown in Fig. 1 of the accompanying drawings, a good linear relationship being obtained.

EXAMPLE 37 The bovine insulin used in Example 36 is replaced by guinea pig anti-bovine serum which is reacted with 3-(benzothiazo le-2'-yl-dithio)-p ropionate-succina mide ester to give guinea pig anti-bovine insulin-ss-galactosidase cross-linked compound.

EXAMPLE 38 To 5 mg. ss-galactosidase in 5 ml. M veronal buffer (pH 7.5) is added 3.1 mg. 3-(benzothiazole-2'yl-dithio)-propionate-succinimide ester in 1 ml. dimethylformamide. After stirring at ambient temperature for 4 hours, there is obtained 3-(be nzothi azole-2'-yl-dith io) -propionated-P-ga lactosidase which is subsequently added to an aqueous solution (pH 9.0) adjusted by the addition of 0.1 N aqueous sodium carbonate solution and left to stand for 2 hours. The S-S bond thereof is hydrolysed to give ss- thiopropionated ss-galactosidase (20 molecules of ss-thiopropionyl group is introduced in p ss- galactosidase molecule).

Claims (5)

1. Disulphide compounds of the general formula.

R1SS#C2OR3)fl4 wherein R, is a 2-benzothiazolyl or 2-pyridyl-N-oxide radical, R2 is an alkylene radical optionally containing a free or protected functional group, R3 is a carboxyl residual group of an amino acid or lower peptide, R4 is a carboxyl group, a reactive derivative thereof or a protected carboxyl or imidate group and n is O.or 1.

2. Disulphide compounds according to claim 1, which are hereinbefore specifically exemplified.

3. Process for the preparation of disulphide compounds of the general formula given in claim 1, wherein a compound of the general formula R1-S-S-R6, in which R, has the same meaning as in claim 1 and R6 is a group capable of reacting with a mercapto group with the elimination of RRSH, is reacted with a compound of the general formula HSRCOR3)R5, in which R2, R3 andn have the same meanings as in claim 1 and R5 is the same as R4 or is a group which can be changed into an imidate group, whereafter, if necessary, R5 is converted into R4.

4. Process for the preparation of disulphide compounds according to claim 1, substantiaily as hereinbefore described and exemplified.

5. Disulphide compounds according to claim 1 , whenever prepared by the process according to claim 3 or 4.