AU626543B2 - Aromatic acids - Google Patents

Abstract

The invention relates to aromatic acids, especially compounds of the formula <IMAGE> in which R1 is lower alkyl, R2 is lower alkyl, R3 is hydrogen, carboxyl or sulpho, R4 is carboxyl or sulpho, G is an optionally substituted 1,4-phenylene group or an optionally substituted 1,4-naphthylene group and in which either R5 and R6 together form an additional bond and L is an oxygen or sulphur atom, or in which R5 is hydrogen, R6 is halogenomethyl and L is an oxygen atom, and their salts, to the use of the compounds I and their salts, to a process for preparing the compounds I and their salts, to starting materials used in this preparation process and their salts, to a process for preparing these starting materials and their salts, to an apparatus in which the compounds I and their salts are used, and to a process in which this apparatus is used. The compounds of the formula I can be used as aids in the investigation of proteins and can be prepared in a manner known per se.

Description

I L Z :~FIC lliC~ICIILi-rl- Our Ref: 272816

AUSTRALIA

Patents Act 2 COMPLETE SPECIFICATION

(ORIGINAL)

Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Arts i Applicant(s): r a S e

S

Ciba-Geigy AG Klybeckstrasse 141 4002 BASLE

SWITZERLAND

ARTHUR S. CAVE CO.

Patent Trade Mark Attornerys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service; 0 S Complete specification for the invention entitled "Aromatic acids".

SThe following statement is a full description of this invention, including the best method of performing it known to me:- 5020 r 1A 4-17027/142.+ Aromatic acids The invention relates to novel aromatic acids, especially compounds of formula R3\ R4 wherein R 1 is lower alkyl, Ra is lower alkyl, R 3 is hydrogen, carboxy or sulfo, R4 is carboxy or sulfo, G is an unsubstituted or substituted 1,4-phenylene group or an unsubstituted or substituted 1,4-naphthylene group, and wherein either Rs and R6 together are an additional bond and L is an oxygen or sulfur atom or wherein Rs is hydrogen, Rs is halomethyl and L is an oxygen atom, and salts thereof, to the use of compounds I and their salts, to a process for the preparation of compounds I and their salts, to starting materials used in that preparation process, and salts S thereof, to a process for the preparation of those starting materials and their salts, to a device in which the compounds I and their salts are used, and to a process in which that device is used.

Suitable unsubstituted or substituted 1,4-phenylene groups are, for example, unsubstKiuted or carboxy- and/or sulfo-substituted 1,4-phenylene groups, the substituted 1,4-phenylene groups having from 1 up to and including 4, especially 1 or 2, of the mentioned substituents. If the substituted 1, 4 -phenylene groups contain more than one substituent, then some or all of those substituents may be identical. Examples that may be mentioned are the 2-sulfo-, 2,5- and 3,5-disulfo-, 2,3,5-trisulfoand 2 ,3,5,6-tetrasulfo-1,4-phenylene group, the 2-carboxy-, 2,3,5-tricarboxy- and 2,3,5,6-tetracarboxy-1,4-phenylene group, the 2-carboxy-3-sulfo-, 2-caCboxy-5-sulfo-, k.

-2- 2, 3 -dicarboxy-5-sulfo-, 3, 5-dicarboxy-2-sulfo-, 5-carboxy-2, 3-disulfoand 2 -carboxy-3,5--disulfo-1,4-phenylene group and especially the 1 4 -phenylene group.

Suitable unsubstituted or substituted 1,4-naphthylene groups are, for example, unsubstituted or carboxy- and/or sulfo-substituted 1,4-naphthylene groups, the substituted 1 ,4-naphthylene groups containing from 1 up to and including 6, especially from 1 up to and including 3, of the mentioned substituents. If the substituted 1,4-naphthylene groups contain more than one substituent, then some or all of those substituents may be identical. Examples that may be mentioned are the 5- and 6-sulfa-, 6,7- and 2,6-disulfo-, 2,3,5- and 2,3,6-trisulfo-and 2,3,5,7and 2,3,6,7-tetrasulfo-1,4-naphthylene group, the 5- and 6-carboxy-, 6,7- and 2,6-dicarboxy-, 2,3,5- and 2,3,6-tricarboxy- and 2,3,5,7- and 2,3,6,7-tetracarboxy-) ,4-naphthylene group, the 2-carboxy- 3-sulfo-, 2-carboxy-5-sulfo-, 3-carboxy-6-sulfo-, 5-carboxy-7-su,fo-, 2,3-dicarboxy-5--sulfo-, 3,5-dicarboxy-2-sulfo-, 6,7-dicarboxy-2-sulfo-, 3-carboxy-6, 7-disulfo-, 5-carboxy-2 ,3-disulfo-and 2-carboxy-3, 1,4-naphthylene group and especially the 1,4-naplithylene group.

I-nj invention relates, for example, to compounds I wherein R, is lower alkyl, R 2 is lower alkyl, R 3 is hydrogen, carboxy or sulfo, R4 is carboxy or sulfo, G is an unsubstituted or substituted 1,4-phenylene group or an upsubstituted or substituted 1,4-naphthylene group, Rs an~d '4 together are an additional bond and L is an oxygen or sulfur atom, and salts thereof.

C. S C S

S.

C.

C

S

S

C. SC Ge C C

C

C S 9

C.

C.

Some of the compounds I can be in the form of stereoisomers. For~ example, if the compounds I contain at least one chiral carbon atom (C atom) (for example a C atom of a corresponding tadical RI), they can be, for example, in the form of pure emantiomers or mixtures of enantiomers, such as racemates, and if there is at least one further chiral centre present (for example a C atom of a corresponding radical R2) they may also be in the form of diastereoisomers, mixtures of diastereoisomers or mixtures of racemates 1 3 Salts of compounds I are especially salts with bases, preferably pharmaceutically acceptable salts with bases, for example alkali metal salts or alkaline earth metal salts, for example sodium, potassium or magnesium salts, transition metal salts, such as zinc or copper salts, or salts with ammonia or organic amines, such as cyclic amines, such as mono-, dior tri-lower alkylamines, such as hydroxy-lower alkylamines, for example mono-, di- or tri-hydroxy-lower alkylamines, hydroxy-lower alkyl-lower alkylamines or polyhydroxy-lower alkylamines. Cyclic amines are, for example, morpholine, thiomorpholine, piperidine or pyrrolidine. Suitable mono-lower alkylamines are, for example, ethylamine or tert.-butylamine; suitable di-lower alkylamines are, for example, diethylamine or diisopropyl-amine; and suitable tri-lower alkylamines are, for example, trimethylamine or triethylamine. Suitable hydroxy-lower alkylamines are, for example, mono-, di- or tri-ethanolamine, and hydroxy-lower alkyllower alkylamines are, for example, N,N-dimethylamino- or N,N-diethylamino-ethanol, whilst a suitable polyhydroxy-lower alkylamine is, for Sexample, glucosamine. The compounds I can also form acid addition salts, preferably pharmaceutically acceptable acid addition salts, for example with strong inorganic acids, such as mineral acids, for example sulfuric Sacid, a phosphoric acid or a hydrohalic acid, with strong organic carboxylic acids, such as lower alkanecarboxylic acids, for example acetic acid, saturated or unsaturated dicarboxylic acidt, for example malonic, maleic or fumaric acid, or hydroxyarboxylic acids, for example tartaric or citric acid, or with sulfonic acids, such as lower alkanesulfonic acids or unsubstituted or substituted benzenesulfonic acids, for example methane- or p-toluene-sulfonic acid. The compounds I can also form inner salts.

Also included are salts of compounds I that are less suitable for pharmaceutical uses. These may be used, for example, for the isolation or purification of free compounds I according to the invention and their pharmaceutically acceptable salts.

Hereinbefore and hereinafter, unless defined otherwise, radicals or compounds designated "lower" are to be understood as being especially those radicals or compounds contaiing up to and including 7, especially up to and including 4, caron atoms.

I.

-4- Lower alkyl is, for example, Ci-C4alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl or tert.-butyl, and also includes Cs-C 7 alkyl radicals, that is to say pentyl, hexyl and heptyl radicals.

Halomethyl is fluoro-, chloro- or bromo-methyl, but especially iodomethyl.

The compounds I and their salts have valuable properties. For example, they can be used as adjuncts in the investigation of proteins, for example as reagents for the chemical modification of proteins. Hereinbefore and hereinafter the term "protein" is to be understood as including both peptides having a relative molecular weight of 10 000 or more atomic mass units, which are generally termed proteins, and peptides having a relative molecular weight of less than 10 000 atomic mass units, which are generally termed polypeptides, a lower limit of from approxia* mately 1000 to approximately 2000 atomic mass units applying to the *e* relative molecular weight of the polypeptides. Examples taken from the numerous applications in which the chemical modification of proteins can play a part are the structural analysis and the coloration of proteins.

It is known that the modification of a single amino acid building block or of a small number thereof can substantially alter the spatial structure of a protein and therefore also its function, for example its *9 biological activity. This opens up the possibility of using the specific :a chemical modification of amino acid building blocks as a widely applicable method of determining the contribution made by these building blocks to the spatial structure of a protein. The problem arises here of relating any alteration in the spatial protein structure occurring as a result of a certain chemical modification of amino acid building blocks to the nature and extent of that chemical modification. For this purpose the structure of a corresponding chemically modified protein must be determined and compared with the structure present before the chemical modification was carried out. One of the aims of this kind of structural analysis is to determine in what way the protein primary structure has changed during the chemical modification, that is to say which amino acid I; B 7 5 building blocks have been chemically modified. In primary structure analyses of this kind, the procedure is often as follows: a chemically modified protein M obtained after chemical modification of a a corresponding unmodified protein N and the unmodified protein N are, a a optionally after having carried out a denaturation step which may be necessary, each treated with the same protease, the two peptide mixtures

M

I and N I so obtained are each subjected to high-performance liquid a a chromatography (HPLC) and the peak patterns in the two resulting chromatograms M 2 and N 2 are compared with one another. The detection a a systems used for recording these chromatograms are usually detection systems which evaluate the light absorption behaviour of the peptides.

The peak pattern in chromatogram M 2 generally differs from the peak a pattern in chromatogram N 2 at those places at which peptides M 3 a a which contain at least one chemically modified amino acid building block, are detected, since the peptides M 3 generally exhibit a light a absorption behaviour different from that of the corresponding unmodified peptides N 3 The peptides MH are separated off and subjected to a a further primary structure analysis, for example to amino acid sequence analysis. Ideally, each chemically modified amino acid building block can so* be identified and chara-terised in this manner.

In many cases, however, the usefulness of the above-described procedure leaves something to be desired. For example, when the protein to be investigated has a relatively high relative molecular weight, there are S. normally so many different peptides present in the corresponding peptide mixture after the protease treatment that HPLC is unable to effect sufficient separation of the peptids mixture. Furthermore, the chemical modification itself is often too complex; it often proceeds in a nonspecific manner and therefore affects a large number of structurally different amino acid building blocks or even all the amino acid building blocks in the protein under investigation; advantageously, however, it should be possible to carry out the chemical modification as specifically as possible, that is to say specifically directed only at a class of amino acid building blocks that is defined as accurately as possible and advantageously that 4,s narrowly defined in terms of nature and number, Ic c i 6- It .is therefore desirable and of great practical interest to optimise the procedure described above by overcoming the disadvantages indicated. The compounds I disclosed within the framework of the present invention and their salts make such an optimisation possible. This optimisation is explained in detail below with reference to compounds I in which RI, R 2

R

3 R4 and G are as defined above, Rs and R6 together are an additional bond and L is an oxygen or sulfur atom, and salts thereof. These compounds are designated compounds IA below.

In the chemical modification of a protein, that is to say in the reaction with that protein, the compounds IA and their salts react in the first instance virtually exclusively with the amino groups in the e-position of lysine building blocks, with high reactivity being observed. Only in isolated cases is there additionally a reaction with the amino group of the N-terminal amino acid building block of the protein in question. The nature and number of the amino acid building blocks of a protein that are S affected by a chemical modification by a compound IA or a salt thereof are therefore clear and, advantageously, narrowly defined. Thus the number of chemically modified amino acid building blocks is generally not Sgreater than the number of lysine building blocks in the protein under investigation. Only in isolated cases, when there is an additional N-terminal modification, can it be greater by 1.

Furthermore, the use of a compound IA or a salt thereof for the chemical modification of a protein has the great advantage that after the protease treatment of the resulting chemically modified protein M b there is S obtained a peptide mixture in which by means of simple reversed phase HPLC it is possible to obtain separation, usually complete separation, of the peptides 3 which contain at least one chemically modified amino acid building block, from the remaining peptides N 3 In addition, the peptideAs 3 which contain at least one chemically modified amino acid building block, present in the peptide mixture on the one hand and the unmodified peptides N 3 on the other hand can each be detected at markedly different wavelengths, since the peptides

M

3 exhibit a light absorption behaviour that is markedly different from that of the peptides N This considerably facilitates tFe identificah' 61 I 7tion of the peptides Advantageously, the peptides M, as they have a colour, can be detected at wavelengths of visible light, that is to say Lt wavelengths between approximately 400 nm and approximately 800 nm for example in acidic solution at a wavelength of 535 nm or in alkaline solution at a wavelength of 465 nm, preferably at one of the wavelengths mentioned in Examples 10 to 15. The colourless peptides N 3 b' however, can be detected at wavelengths of ultraviolet light, for example at a wavelength of 200, 220 or 280 nm, and therefore do not interfere with the detection of the coloured peptides It is also of particular intetest that it is not only the peptides 3 that have a colour but also the corresponding chemically modified protein b from which the peptides M are obtained by protease treatment. Since the chemical modification of a protein by a compound IA or a salt thereof is therefore always associated with coloration oi the protein, the compounds IA and their salts can also be used as reagents for the coloration of proteins. The coloured proteins obtainable by reaction of the e-amino groups of lysine building blocks and, in isolated cases, additionally by reaction of the amino group of the N-terminal amino acid building block of corresponding colourless, unmodified proteins Nh with a compound IA or a salt thereof, can be used in a variety of ways for analytical and/or diagnostic purposes. The comments made above in connection with the separation and detection of peptides M that have a colour also apply in anaJloous manner to the separation and detection of the proteins Mb that have been coloured. The peptides H can also be used for a variety of analycical anr/or diagnostic purposes.

SO

S

Since the investigation of proteins generally takes place in aqueous or water-containing solution, the good water-solvbility of the compounds IA and their salts constitutes a further valuable property. Becwag'i the chemically modified proteins M and the peptides X which contiin at least one chemically modified a'-ino acid building block, are also distinguished by good water-slubility, both the reaction of proteins 'b with a compound IA or with a salt thereof and any other process steps customary in investigations of proteins which might be envisaged, for example of the kind mentioned below, can advantageously be carried out in aqueous solution, optionally with the addition of an organic solvent.

61--

U_

8 8 In the course of the reaction with a compound IA or with a salt thereof, the corresponding amino groups of a protein Nb of general formula H2N-R (Ia) wherein R in each case is the residue of the protein, that is to x say the E-amino groups of lysine building blocks and in isolated cases additionally the amino group of the N-terminal amino acid building block, are converted into carbamoyl or thiocarbamoyl groups, respectively, so that a chemically modified protein of formula NH--NH-R (Ib), R2 wherein R 1 Rz, R3, R4, R and G are as defined above and L is an oxygen or sulfur atom, is obtained. The speed of this conversion reaction increases the higher the temperature and the higher the pH value of the reaction medium, Examples of typical reaction conditions can be found in Examples 9 to 15. The stability of the carbamoyl or thiocarbamoyl bonds, respectively, in the proteins Ib is advantageously so great in normal cases that the proteins Ib can be stored for periods of several days Without any appreciable decomposition taking place and that they can also S* withstand unimpaired the further process steps customary in investigations of proteins, such as chromatographic separations, for example by Means of molecular exclusion chromatography, for example gel chromatography, or HPLC, enzymatic treatments, for example treatment with a protease, for example with trypsin or chymtrypsin, other standard S reactions, for example denaturation steps, such as the reduction of disulfide bridges and the subsequent carboxymethylation of the mercapto groups, or test reactions for the analysis of the biological activity.

Such process steps are known or can be carried out analogously to known process steps. Examples of details of such process steps can also be found in Examples 9 to 15. As regards the stability of the carbamoyl or thiocarbamoyl bonds in the peptides M 3 the comments made above for the proteins Ib apply in analogous manner, For the above-described optimisation of the procedure for the chemical modification of proteins and for the analysis of the primary structure of such chemically modified proteins, in addition to compounds IA and their 9 salts it is also possible to use compounds I wherein Ri, R 2 R3, R4 and G are as defined above, Rs is hydrogen, R6 is hlomethyl and L is an oxygen atom, and salts thereof. These compounds are ia..~l ed compounds IB below.

The comments made above for compounds IA and their salts generally apply in an analogous manner to compounds IB and their salts. However, in the chemical modification of a protein, that is to say in the reaction with that protein, the compounds IB and their salts react neither with the e-amino groups of lysine building blocks nor with the amino group of the N-terminal amino acid building block of that protein. On the contrary, in the course of the reaction with a compound IB or with a salt thereof specifically the mercapto groups of cysteine building blocks of a protein N of general formula HS-R wherein R in each case is the residue a y y of the protein, are converted into carbonylmethylthio groups, so that a chemically modified protein of formula R3 0 N=N- -NH 2-S-R (Ie), R4 herein RI, 2, R 3 RI, R and G are as defined above, is obtained, Thus when compounds IB and their salts are used, generally the nature and number of the amino acid building blocks affected by a chemical modification are likewise clear and, advantageously, narrowly defined, the number of chemically modified amino acid building blocks being not greater than the number of cysteine building blocks in the proo.- U4mder investigation, The invention therefore relates also to the use of compounds I and their salts as adjuncts in the investigation of proteins, for example as reagents for the chemical modification of proteins, especially as reagents for the chemical modification of proteins that is associated with c0loration, The commercial formulation of the adjuncts may also be included.

The present invention relates also, to a corresponding process for the chemical modification of proteins, especially a process for the chemical modification of proteins that is associated with coloration, which process comprises reacting the proteins with a compound I or with a salt thereof.

The invention relates especially to compounds of formula I wherein R 1 is lower alkyl, R 2 is low~er alkyl, R 3 is hydrogen, carboxy or sulfo, R4, is ca ue,~r sulfo, G i8 an unsubstituted or earboxy- and/or sulfo-substi.tuted 1,4-phenylene group or an unsubstituted or carboxcy-and/or sulfo-substituted 1,4-naphthylene group and wherein either R6i and R6 together are an additional bond, and L is an oxygen or sulfur atom, or wherein R5 is hydrogen, Rs is halomethyl and L is an oxygen atom, and salts thereof.

The invention relates especially to compounds of formula I wherein Ri is lower alkyl, R 2 iLs lower alkyl, R3 is hydrogen, carboxy or sulfo, Ri is carboxy or stqlfoiv G is an unsubstituted or carboxy- and/or sulfo-substituted 1,4-phenylene group or an unsubstituted or carboxy-and/oc' sul1fo-substituted 1,4-,-aphthylene group, Rs and R6~ together are an additional b'ond and L is an oxygen or sulfur atom, and salts thereof.

*0 9 S S 0* *9

S

S S

OOSS

SO**

S

9* 55 S S

S

The Invention relates more especially to compounds of formula I wherein Ri is CI-C441klyl, such as methyl, or ethyl, R2 is Ci-Ci~alkyl, such as methyl or ethyl, R 3 i; yrgn rslo R4, is sulfo, G i an unsukstituted or sulfo-substii!uted 1,4-phenylene group and wherein either Rs and RG together are an ,odditional bond and L is an oxygen or sulfur atom, or wh, roin R6 is hydrogen, RG is iodomethyl and L is an oxygen atom, and salts, thereof.

S. The inventi~.n relates especially to compounds of formula I wherein Ri is

C

1 -CiOAlYl, such as methyl or ethyl, R2 is Cl-Ci~a1~yl, such as 'nethyl or ethyl, R3 is hydro; en or sulfo, R4. is sulfa, G is an un:§ubstituted or sulfa-substituted 1,4-phenylene group, Rs and R6 together are an additional, bond and L is an oxygen or sulfur atom, and silts thereof.

11 The invention relates especially to compounds of formula I wherein R 1 is Ci-C4alkyl, such as methyl, R2 is C 1 -C4alkyl, such as methyl, R 3 is hydrogen, R4 is sulfo, G is an unsubstituted 1,4-phenylene group and wherein either Rs and R 6 together are an additional bond and L is a sulfur atom, or wherein Rs is hydrogen, Rs is iodomethyl and is an oxygen atom, and salts thereof.

The invention relates more especially to compounds of formula I wherein Ri is Ci-C4alkyl, such as methyl, R 2 is Ci-C4alkyl, such as methyl, R3 is hydrogen, R4 is sulfo, G is an unsubstituted 1,4-phenylene group, Rs and R.6 together are an additional bond and L is a sulfur atom, and salts thereof.

The invention relates specifically to the novel compounds of formula I mentioned in the Examples and their salts.

The present invention relates also to a process for the preparation of a S compound I or a salt thereof, which process comprises, for example: in a compound of formula

R\

SG-N=N- -N (Ii) and, if desired, separating a mixture of isomers obtainable in accordance with the process into the components and isolating the desired isomer I, tesolving a mixture of enantiomers or diastereoisomers obtainable in ml l accordance with there process rtinto the individual enantiomersoup of foiastr-mula and, if desired, separating a mixture of isomers obtainable n accordance with the process into the components and isolating the desired isomer I, resolving a mixture of enantiomers or diastereoisomers obtainable in accordance with the process into the individual enantiomers or diastereoisomers and isolating the desired enantiomer or diastereoisomer, and/or converting a free compound I obtainable in accordance with the process into a salt or converting a salt obtainable in accordance with the process into the free compound I or into a different salt.

12 The reactions described hereinbefore and hereinafter are carried out in a manner known per se, for example in the presence of a catalyst and/or in the absence or, usually, in the presence of a suitable inert solvent or diluent or a mixture thereof, the reactions being carried out, as necessary, with cooling, at room temperature or with heating, for example in a temperature range of from approximately -80°C £o the boiling temperature of the reaction medium, preferably from approximately -20 0

C

to approximately +150°C, and, if necessary, in a closed vessel, under pressure, in an inert gas atmosphere and/or under anhydrous conditions.

Some of the starting materials of formulae II, IIa, III, IV, IVa and V mentioned hereinbefore and hereinafter, which are used in the preparation of compounds I or their salts, are known or they can be prepared according to methods known per se.

S Starting materials having basic centres can, for example, be in the form of acid addition salts, for examp,- vith the acids mentioned above, whilst starting compounds having acidic groups can form salts with bases, for example of the kind mentioned above.

'The conversion of the NH2 group in a compound II, or in a salt thereof.

into a group Ic can be carried out, for example, by reacting the compound II or a salt thereof with a compound of formula gee1 C=L (IIa), r* wherein L is an oxygen or sulfur atom and ZI and Z2 either independently of one another each represent a .,Acleofugal leaving group GI or together represent free or functionally modified oxo G 2 or wherein L is an oxygen atom, Zi is a nucleofugal leaving group G and Z2 is halomethyl.

Nucleofugal leaving groups GI are, for example, free, etherified or esterified hydroxy or mercapto groups, also amino, ammonium or sulfonium groups. Etherified hydroxy is, for example, lower alkoxy, such as methoxy or ethoxy, or unsubstituted or substituted phenyl-lower alkoxy, such as unsubstituted or substituted benzyloxy. Esterified hydroxy is especially hydroxy esterified by a mineral acid or an organic sulfonic acid, 13 especially halogen, such as chlorine, bromine or iodine, sulfonyloxy, such as unsubstituted or halo-substituted lower alkanesulfonyloxy, for example methanesulfonyloxy or trifluoromethanesulfonyloxy, cycloalkanesulfonyloxy, for example cyclohexanesulfonyloxy, or unsubstituted or lower alkyl- or halo-substituted benzenesulfonyloxy, for example benzenesulfontyloxy, p-bromophenylsulfonyloxy or p-toluenesulfonyloxy, also lower alkanoyloxy, for example acetoxy or pivaloyloxy. Etherified mercapto is, for example, lower alkylthio, such as methylthio or ethylthio, or unsubstituted or substituted phenylthio, such an phenylthio or p-tolylthio. Esterified mercapto groups are, for exariple, lower alkanoylthio groups, such as acetylthio. Amino groups are, for example, amino, N-lower alkylamino, NN-di-lower alkylamino or N-lower alkanoylamino groups, also N,N-lower alkyleneamino or N,N-aza-, N,N-oxa- or N,N-thia-lower alkyleneamino groups, for example dimethylamino or diethylamino, also pyrrolidino, piperidino, morpholino or thiomorpholino, also anilino. Ammonium S groups are, for example, tertiary or quaternary ammonium groups corresponding to the amino groups mentioned above, such as tri-lower alkyl- S amionio or pyridinio. Sulfonium groups are, for example, di-lower alkylnulfonium groups, such as dimethylsulfonium.

ree or functionally modified oxo G 2 is, for example, oxo, thioxo or a group =N-Wi. Groups are, for example, those groups in which R' is hydrogen, lower !lkyl or an acyl radical, such as lower alkanoyl, untubstituted or substituted benzoyl, pyridoyl or lower alkanesulfonyl, for example imino, N-lower alkylimino, N-lower alkanoylimino, unsub- 4 stituted or substituted N-benzoylimino or N-lower alkanesulfonylimino groups, compounds I1a preferably used for the preparation of compounds I wherein R s and R 6 together are aii additional bond, or salts thereof, are, for example, thiophosgene (Z1 Z2 chlorine) and carbon disulfide (ZI and Z2 thioxo) which result in compounds I wheren L is a sulfur atom, or salts thereof, and phosgene (ZI Zz chlorine) which results in compounds I wherein L is an oxygen atom, or salts thereof.

14 Compounds IIa preferably used for the preparation of compounds I wherein Rs is hydrogen and R 6 is halomethyl, or salts thereof, are, for example, haloacetic acids (ZI hydroxy; Zz halomethyl; L oxygen atom) and corresponding reactive haloacetic acid derivatives (Zi for example halogen, lower alkoxy or sulfonyloxy).

The reaction of a compound II or a salt thereof with a compound IIa is carried out in customary manner, for example optionally in the presence of a condensation agent, such as a suitable base, and in the case of the reaction with compounds IIa wherein Zi and Zz together are thioxo, optionally in the presence of a sulfur-binding agent, and in the case of the reaction with compounds IIa wherein ZI is Gi Zz is halomethyl and L is an oxygen atom, optionally in the presence of a water-binding agent, in the absence or, usually, in the presence of a suitable inert solvent or diluent or a mixture thereof and, as necessary, with cooling, at room Stemperature or with heating, for example in a temperature range of from S approximately -80°C to approximately +200 0 C, preferably from approxi- S mately -20 0 C to approximately +1500C, and, if necessary, in a closed vessel, under pressure and/or under an inert gas, such as nitrogen.

685@ Suitable bases are, for example, alkali metal hydroxides, hydrides, O amides, alkanolates, carbonates, triphenylmethylides, di-lower alkylamiaes, amino-lower alkylamides and lower alkylsilylamides, or naphthaleneamines, lower alkylamines, basic heterocycles, ammonium hydroxides and carbocyclic amines. Examples that may be mentioned are sodium .8 hydroxide, hydride, amide, ethanolate or carbonate, potassium tert.- Sbutanolate or carbonate, lithium triphenylmethylide, lithium diisopropylamide, potassium 3-(aminopropyl)-amide or bis-(trimethylsilyl)amide, or dimethylaminonaphthalene, di- and tri-ethylamine, pyridine, benzyltrimethylammonium hydroxide, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and 1,5-diazabicyclo(5.4.0]undec-5-ene (DBU).

Suitable sulfur-binding agents for the reaction with compounds IIa wherein Zi and Zz together are thioxo are, for example, oxides of phosphorus, such as tetraphosphorus decaoxide, carbodiimides, such as

U

I I i __r 15 N,N'-dicyclohexyl carbodiimide, and derivatives of carbonic acid, such as carbonic acid esters, for example halocarbonic acid lower alkyl esters, such as chlorocarboiic acid ethyl esters.

Suitable water-binding agents for the reaction with compounds IIa wherein ZI is GI, Z 2 is halomethyl and L is an oxygen atom are, for example, oxides of phosphorus, such as tetraphosphorus decaoxide, and carbodiimides, such as N,N'-dicyclohexyl carbodiimide or N-[3-(N,N-dimethylamino)-propyl]-N'-ethyl carbodiimide and the acid addition salts thereof, for example the hydrochloride.

Inert solvents or diluents that may be mentioned are, for example, water and, also in the form of mixtures with water, cyclic ethers, unsubstituted or halogenated aromatic hydrocarbons, halogenated lower alkanes, N,N-di-lower alkyl-lower alkanoic acid amides, phosphoric acid-lower S alkylamides, di-lower alkylsulfoxides, basic heterocycles and lower alkanols, such as tetrahydrofuran, dioxane, benzene, toluene, xylene, chlorobenzene, 1,2-dichlorobenzene, trichloromethane, N,N-dimethylformamide, hexamethylphosphoric acid triamide, dimethyl sulfoxide, pyridine, N-methylmorpholine, methanol and ethanol.

The starting material II or a salt thereof can be prepared analogously to known methods, for example by hydrolysis, that is to say by reaction with water, of a compound of formula oRi *4 e R3 -NH-Y (III),

RR

wherein Y is an acyl radical, or a salt thereof.

Acyl radicals Y are, for example, acyl xadicals derived from an organic carboxylic or sulfonic acid.

Acyl derived from an organic carboxylic acid is, for example, the radical of an aliphatic or monocyclic-aromatic carboxylic acid, such as lower alkanoyl or unsubstituted or substituted benzoyl, also pyridoyl.

16 Acyl derived from an organic sulfonic acid is, for example, lower alkanesulfonyl.

Lower alkanoyl is, for example, Cz-Csalkanoyl, such as acetyl, propionyl, butyryl, isobutyryl or pivaloyl.

Unsubstituted or substituted benzoyl is, for example, benzoyl, p-chlorobenzoyl or p-nitrobenzoyl.

Lower alkanesulfonyl is, for example, Ci-C4alkanesulfonyl, such as methane- or ethane-sulfonyl.

The hydrolysis of a compound III or a salt thereof is carried out in customary manner, for example in the presence of a hydrolysing agent and optionally in the absence or, usually, in the presence of a suitable inert solvent or diluent or a mixture thereof, the operation being S* carried out, as necessary, with cooling, at room temperature or with heating, for example in a temperature range of from approximately -80 0

C

to approximately +200 0 C, preferably from approximately -20C to approximately +150C, and, if necessary, in a closed vessel, under pressure and/or under an inert gas, such as nitrogen.

Suitable hydrolysing agents are, for example, acids or bases. Suitable Sacids are, for example, inorganic or organic protonic acids, such as mineral acids, for example sulfuric acid or hydrohalic acids, for example a *a hydrochloric acid, sulfonic acids, for example lower alkanesulfonic acids a or unsubstituted or substituted benzenesulfonic acids, for qxample methane- or p-toluene-sulfonic acid, or carboxylic acids, for example lower alkanecarboxylic acids, for example acetic acid, whilst bases that can be used are, for example, those mentioned above, especially sodium or potassium hydroxides Suitable inert solvents or diluents are especially those mentioned above, more especially water and aqueous lower alkanols, such as aqueous methanol or ethanol.

I

17 The starting material III or a salt thereof can be prepared analogously to known methods, for example by reaction of a salt of formula R3\ A N2-' '-NH-Y (IV), R4 wherein A is the anion of a protonic acid, with a compound of formula Ri S-G-H (IVa) Rz or with a salt thereof.

Anions A of protonic acids in salts IV are, for example, anions of the acids mentioned above for the formation of acid addition salts of compounds I, especially anions of strong inorganic protonic acids, such as anions of m~ineral acids, for example sulfuric acidf a phosphoric acid or a hydrohalic acid, or of tetrafluoroboric acid, or anions of strong organic carboxylic acids, such as lower alkanecarboxylic acids, for example formic acid or acetic acid, for example the sulfate, phosphate, chloride, bromide, tetraflurorbrate or acetate ion.

o The reaction of a salt IV with a compound IVa or with a salt thereof is effected analogously to known procedures under the customary reaction conditions, for example in an inert solvent or diluent, for example of the kind mentioned above, preferably in water, optionally in the presencq of an acidic agent, for example in the presence of one of the acids mentioned above, and/or with cooling, at room temperature or with heating, for example in a temperature range of from approximately -20 to approximately +50 0 C, preferably from approximately C' to approximately +300C.

The starting material IV is known or Can be prepared analogously to known methods, for example by reaction of a compound of formula

R/

or a salt thereof, with nitrous acid, the reaction being carried out under the reaction conditions customarily used, for example in a solvent or diluent, preferably in water, and/or with cooling, at room temperature or with heating, for example in a temperature range of from approximately to approximately +50°C. The nitrous acid is preferably produced in situ, for example by reaction of an alkali metal nitrite, such as sodium nitrite, with a strong protonic acid, for example a hydrohalic acid, such as hydrochloric acid, or a lower alkanecarboxylic acid, such as formic acid or glacial acetic acid.

In an especially preferred form, a compound V or a salt thereof is reacted as described above with nitrous acid, which is, for example, formed in situ, and the salt IV initially formed is then further reacted, without being isolated and/or additionally purified, in _itu according to the invention with a compound IV'a or with a salt thereof to form the desired .ompound III, The compounds IVa and their salts are known or can be prepared analogously to known methods.

The starting material IIa is also known or can be prepared analogously to t known methods, Salts of compounds I can be produced in a manner known per se. Thus, for ea x. example, acid addition salts of compounds I are obtained by treatment with an acid or a suitable ion exchange reagent. Acidic compounds I can he -inverted into salts with bases, for example, by treatment with a base or with a suitable ion exchange reagent. Salts of compounds I can be converted into the free compounds I in cust 2mary manner; for example acid addition salts can be converted by treatment with a suitable bhAic agent, and salts with bases can be converted, for example, by treatment with a suitable acidic agent, 19 Depending upon the procedure and reaction conditions, the compounds I having salt-forming, especially acidic, properties may be obtained in free form or in the form of salts.

As a result of the close relationship between the novel compound I in free form and in the form of its salts, hereinbefore and hereinafter a free compound I or its salts should be understood as meaning also the corresponding salts or the free compound I, respectively, where appropriate and expedient.

The novel compounds I, including their salts, can also be obtained in the form of their hydrates or may include other solvents, for example those used (cr the crystallisation of compounds in solid form.

Depending upon The starting materials and procedures chosen, the novel i compounds I may be in the form of one of the possible isomers or in the form of a mixture thereof. Depending upon the molecular symmetry, for example depending upon the number and the absolute and relative confi- •*o guration of the chiral centres, such as asymmetric carbon atoms, as pure isomers there may be obtained, for example, pure enantiomers and/or pure S diastereoisomers, such as pure cis/trans isomers or meso-compounds.

Accordingly, as isomeric mixtures there may be obtained, for example, enantiomitic mixtures, such as racemates, diastereoisomeric mixtures or mixtures of racmates.

Resulting diastereoisomeric mixtures and mixtures of racemates can be separated into the pure diastereoisomers or racemates in known manner on the basis of the physico-chemical differences between the constituents, s..e for example by fractional crystallisation.

seeo Resulting enantiomeric mixtures, such as racemates, can be resolved into the enantiomers by known methods, for example by recrystallisation from an optically active solvent, by chromatography on chiral adsorbents, with the aid of suitable microorganisms, by cleaving with specific, immobilised enzymes, by means of the formation of inclusion compounds, for example using chiral Crown ethers, in which case only one enantiomer is complened, or by conversion into diastereoisomeric salts, for example by 20 reaction of a basic end product racemate with an optically active acid, such as a carboxylic acid, for example tartaric or malic acid, or a sulfonic acid, for example camphorsulfonic acid, and separation of the mixture of diastereoisomers obtained in this manner, for example on the basis of their different solubilities, into the diastereoisomers from which the desired enantiomer can be freed by the action of suitable agents. Advantageously, the more active enantiomer is isolated.

The inventi'i also relates to those forms of the preparation process according to which a compound obtainable as intermediate at any stage of the process is used as starting material and the remaining steps are carried out, or a starting material is used in the form of a derivative or salt and/or its racemates or enantiomers or, especially, is forped under the reaction conditions.

S* In the preparation process of the present invention it is preferable to r use those starting materials which result in the compounds I described at the beginning as being especially valuable. The invention relates also to Snovel starting materials which were developed specifically for the preparation of the compounds 1, to their use and to processes for their preparation, the variables Ri, Rz, RA, R4, Rs, R6, G and L having the meanings indicated for the groups of compounds of formula I that are preferred in each case.

S

In this connection, special mention should be made of compounds of formula Ri R3

>-NH

2

(II)

2* R and their salts, to which the comments made above for salts of compounds I apply in analogous manner. These can be used in an especially advantageous manner as starting materials for the preparation of compounds I or their salts, for example in accordance with the process described above, -21- The invention accordingly relates e'lso to compounds of formula II wherein R, is lowesr alkyl, R2 is lower alkyl, R3 is hydrogen. carboxy or sulfo, R4 is carboxcy or sulfo, and G is an unsubstituted or substituted 1,4phenylenc group or an unsubstituted or substituted 1,4-naphthylene group, and salts thereof, to the use of these compounds and their salts and to a process for the preparation o.7 theise compounds and their salts.

The variables in formula II have, for example, the preferred meanings given under formula I.

The invention relates in this respect especially to compounds of formula II wherein RI is lower alkyl, R2 is lower alkyl, R3 is hydrogen, carboxy or sulfp, R4 is carboxy or sulfo, and G is an unsubstituted or~ carboxyand/or sulfo-substituted 1,4-phenylene group or an unsubstituted or carboxy- and/or sulfo-stibstituted 1,4-naphthylene group, and salts thereof.

The invention relates in this respect more especially to compounds of *.*fornmula IT wherein Pj is C1-Cz4alkyl, such as methyl or ethyl., Rg is *eg CaI-C~alkyl, sticlh as methyl or ethyl, Pq is hydrogen or sulfo, 4~ is sulfa, and G I's an unsubstitutqd or stlfo-substituted 1,4-phenylene group, and salts thereof* The invention relates in this respect most especially to compounds of formula II Wherein R1 is CJ-C44akyl, such as methyl, R2, is C1-'C4allkyl, such as methyl, R) is hydrogen, R4 is sulfo, and 0 is an unsubstituted lf4-pbenylene group, and salts thereof, *og The invention. relates in, this respect specifically to the novel compounds of formula Ii mentioned In the Exmples and t~o their salts, The present inven~tion relates also to a process for the preparation of a compound 11 or a salt thereof, which pr'ocess comprisosj for example, hydrolysing a compound of formula -22- G-N=N NH-Y(III),

R

2

N--

wherein Y is an acyl radical, or a salt thereof, for example as described above, and, if desired, separating a mixture of isomers obtainable in accordance with the process into the components and isolating the desired isomer 11, itesollving a mixture of enantiomers or diastereoisomers obtainable in accordance with the process into the individual enantiomers or diastereoisomers and isolating the desired enantiomer or diastereoisomer, and/or converting a free compound II obtainable in accordance with the process into a salt or converting a salt obtainable in accordance with the process into the free compound II or into a daifferev't salt, The preparation of compouvrds III or salts thereof is described above.

ubsequent poptions which may, if desired, be carried out on compounds 11 or their salts obtainable in Accordance With, the process or by other means are especially separations of enantiomers or diastereoisomers so* and conversions into one another of salts And free compounds II analogous to those indicated for compotinda I and these operations are also carried out in analogous mannero invention relates also to the use of compoundts 11 or their salts as ".'starting materials for, the preparation of compounds I or their salts.

&it is possible, to carry otit the above-doscxribed optimiged procedure or the chemical modification of protqins and for the Analysis of the primary gos structure of such Chemically modified proteins, that is to say the sequence consisting of the chemical modification by moans of a compound I or a salt thereof, optional denaturation, protease treatment and MCPL, Also Automatically, using a device specifically designed for this Tfhe design of this device is schematically shown in figure 1. The device compises 4 syringe pump (24)0 which component (24) serves to diivor a sample of the protein, which is to be chemically modified, by means of a -23 compound I or a salt thereof, solvents, reagents and/or catalysts, that are necessary for the said steps of chemical modification by means of a compound I or a salt thereof, optional denaturation and piotease treatment, inert gas, and washing solution through the connection into the reactor which component (15) is equipped with a screw cap (16) and is installed within a heating-jacket a vacuum pump which component (25) enables by means of the connection the evacuation of the reactor (15) for the purpose of the concentration of the contents of the reactor a connection which component by means of a slight excess pressure of inert gas delivered from the syringe pump (24) through the connection serves to deliver the contents of the reactor when the chemical modification or the optional denaturation step has been carried out, through the valve the connect.in the valve (27) and the connection to the desalting unit or serves to deliver the contents of the reactor when the protease treatment step has been carried out, through the valve the connection the valve (27) and the connection to the HPLC component to which component (21) a plotter (22) and through the connection a fraction collector (20) are attached, or serves to deliver the washing solution contained in the reactor (15) through the I S valve (26) and the connection (11) to the waste reservoir a pump which component (23) delivers eluent through the connection the valve (27) and the connection to the desalting unit a connection which component (10) serves to deliver the eluate which is eluted from the desalting unit (19) to the detector (18), which component (18) serves to control the valve (28) in such a way that S the eluate which is eluted from the desalting unit when it contains the chemically modified protein prepared in the chemical modification j step or optionally the denat red chemically modified protein prepared in the optional denaturation step is delivered through the connection (4) into the reactor and when it contains no such chemically modified protein or denatured chemically modified protein is delivered through the connection (12) into the waste reservoir and a connection (13), which component (13) serves to deliver the eluate from the detector (18) to the valve The device according to the invention comprises the components to (28).

r i f 24 The reactor (15) is in its size and shape comparable to a 1.5 ml Zppendorf tube. The reactor (15) can be made of any inert material, for example of glass, virtually inert plastics, such as polytetrafluoro ethylene, or high-quality stainless steel, preferably of a transparent material, for example of glass or transparent, virtually inert plastics.

The steps of chemical modification, optional denaturation and protease treatment are carried out in the taper bottom part of the reactor with a total reaction volume oi from approximately 100 to approximately 300 il.

The reactor can be stoppered by means of the screw cap which preferably is made of the same material as the reactor. The connection should reach as closely as possible to the bottom of the reactor, in order to enable a virtually complete emptying of the reactor The syringe pump (24) can deliver the educt (the protein which is to be chemically midified), the necessary solvents, reagents, for example the compound I or a salt thereof used in the chemical modification step, Sand/or catalysts, as well as inert gas and washing solution into the reactor The syringe pump (24) is a commercially available apparatus, which i, onstructed in such a way that kind and quantity of the igent which is to be delivered into the reactor (15) can be chosen independently at a y moment. By means of a valve contained in the syringe pump the connection leading into the reactor (15) can be S stoppered at any time, for example, when the desired quantity of agent has been pumped into the reactor The quantities which can be pumped in a single step into the reactor (15) can vary within a wide volume range for example from approximately 1 pl1 to approximately 200 il.

As vacuum pump (25) any commercially available pump can be used, S preferably an oil pump. The aesalting unit (19) serves to remove excess reagents and by-products contained in the reaction mixture resulting aiter the chemical modification step and the optional denaturation step.

Customary chromatographic separation devices can be used as desalting unit for example devices used for column chromatography, such as molecular exclusion chromatography, for example gel chromatography. The detector (18) is preferably evaluating the light absorption behaviour of the molecules, for example the light absorption behaviour at wavelengths

P

79 25 of the ultraviolet or especially of the visible light. Typical wavelengths are those given hereinbefore for the detector of the HPLC system.

As heating jacket waste reservoir fraction collector HPLC component plotter (22) and pump (23) which delivers the eluent to the desalting unit (19) suitable commercially available components can be used.

The alve (26) can open on the one hand the connections and and at the same time stopper the connection On the other hand, valve (26) can open the connections and (11) and at the same time stopper the connection The valve (27) can either open and (9) and at the same time stopper and or open and and at the same time stopper and or open and and at the same time stopper an The valve (28) can either open and (13) and at the same time stopper or open (12) and (13) and at the same time stopper Commercially available valves of suitable type can be used.

The connections(1) and through (13) can be made of any inert material, for example of glass or virtually inert plastics, such as polytetrafluoroethylene, preferably of a transparent material, for example of glass or transparent, virtually inert plastics. The connection is made of vacuum resistant tubing.

a 0 B

S.

0ee0: 0 0000 0 In detail, the reaction sequence is carried out as follows: A solution of the protein which is to be chemically modified by a compound I or a salt thereof is delivered by the syringe pump (24) through connection into S* the reactor Subsequently, a solution of the reagent (a compound I or a salt thereof) is delivered in the same way into the reactor After the chemical modification is finished, a slight excess pressure of inert gas, for example argon, is built up in the reactor (15) (the inert gas is delivered from the syringe pump (24) via the connection The excess pressure causes rhe contents of the reactor (15) to be delivered via (27) and to the desalting unit The valves (26) and (27) are adjusted accordingly. The connection is stoppered by means of the valve When the whole contents uf the reactor (15) have been transferred to tle dct-ilting unit the excess pressure is abandoned. The sample is then palsed through the desalting 26 unit (19) in order to purify it, the pump (23) being delivering tne eluent via (27) and (the valve (27) is adjusted accordingly). By means of (28) and the purified chemically modified protein is delivered back into the reactor the valve (28) being adjusted accordingly. The detector (18) serves to adjust the valve (28) in such a way, that only the fraction of the eluate containing the desired product is delivered into the reactor whereas the other fractions are directly delivered via the connection (12) into the waste reservoir The contents of the reactor (15) (eluate with purified chemically modified protein) are concentrated by means of the application of vacuum via the connection using the vacuum pump Puring the evacuation the connection is stoppered by means of the valve contained in the syringe pump. Also, connections and respectively, (by means of the valve and (by means of the valve are stoppered.

C

C If a denaturation step is to be performed, then this is done in a manner analogous to that described for the chemical modification step, using the apropriate reagents &nd solvents, Thisdenaturation step is then followed v, again by a purificanion step using the desalting unit (19) analogously.

SThe resulting eluato is again delivered into the reactor (15) and concentrated.

e« Finally, the protease treatment *tep is carried out in the reactor in a manner analogous to that described for the chemical modification i step, again using the appropriate reagents and solvents. When the protease treatment is finished, the contents of the reactor (15) are delivered to the HPLC component (21) via (27) and (7) (the valves (26) and (27) are adjusted accordingly) in a manner analogous to that described before (by means of a slight excess pressure of inert gas). When the HPLC has been performed, the chromatogram is obtained on the plotter The fractions resulting from the HPLC can be collected by mean, of the fraction collector i i I 27 The reaction conditions (temperature, volume, reaction time, and so on) and the gradient of the HPLC are controlled by a pre-set program. Any catalysts or other adjuncts that are necessary in the described reactions can be delivered into the reactor (15) by means of the syringe pump (24) via The device is operated in such a fashion, that on the one hand the desalting unit (19) is continuously washed with eluent deli:.'ied from the pump while a reaction (chemical modification, optional denaturation or protease treatment) is going on in the reactor and that on the other hand the reactor (15) is cleaned with washing solution (delivered from the syringe pump (24) via while the sample (chemically modified protein or optionally denatured chemically modified protein) is being purified in the desalting unit The washing solution of the desalting unit (19) is delivered into the waste reservoir (17) via (28) and and the washing solution of the reactor S is delivered to the waste reservoir (17) via (26) and in each case in a manner analogous to that described before for the transfer of the products and eluates.

foee The invention also relates to a device of the type described hereinbefore specifically designed to carry out automatically the sequence consisting of the chemical modification of a protein by means of a compound I or a 1 salt thereof, the optional denaturation of the chemically modified protein, the protease treatment of the chemically modified and optionally denatured protein, and the HPLC of the peptide mixture resulting from the protease treatment step.

The invention also relates to a process for the manufacture of said device, characterised in that the components of the device described hereinbefore are combined in the way described hereinbefore to yield the desired device.

f r-i 28 The invention also relates to a process for the automatic chemical modification of a protein and for the automatic analysis of the primary structure of the chemically modified protein consisting of the sequence described hereinbefore, which process is characterised in that the protein is processed using the device described hereinbefore.

The invention also relates to the use of the device described hereinbefore for the automatic performance of the sequence described hereinbefore and to the use of a compound I or a salt thereof as reagent in the chemical modification step performed in said device.

The invention relates also to the following Examples which illustrate the invention described above but are not intended to limit the scope thereof in any way. Temperatures are given in degrees Celsius. HPLC represents high-performance liquid chromatography. Lys represents lysine. Val represents valine.

S.

S Example 1: 25 ml of 0.24M sodium carbonate solution and 0.91 g (7.9 mmol; 0.6 ml) of thiophosgene are added to 1 g (3.1 mmol) of 4-amino-4'-(N,Ndimethylamino)-azobenzene-2-sulfonic acid. The reaction mixture is stirred for 30 minutes at 70°. After cooling to room temperature, the resulting product is filtered off by means of a fritted glass filter and thoroughly washed in succession with 1N hydrochloric acid, toluene and again with IN hydrochloric acid. The black crystals are then dried in vacuo at room temperature, yielding 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. The product decomposes on heating and S has no definite melting point; in the infra-red spectrum there is a -1 strong band at 2110 cm produced by the asymmetric N=C=S stretch vibration.

4 -amino- 4 '-(N,N-dimethylamino)-azobenzene-2-sulfonic acid can be obtained, for example, as follows: 0.9 g (8.5 mmol) of anhydrous sodium carbonate and 38 ml if water are added to 3.5 g (15 mmol) of 5-(N-acetylamino)-2-aminobenriLl esulfonic acid. The reaction mixture is stirred until a clear solution hasbeen obtained This solution is then cooled to +100 by means of an ice bath.

II

I I :I i I I 1" ~a 29 After the addition of 1.22 g (17.7 mmol) of solid sodium nitrite the mixture is stirred for a further 2 minutes. The reaction mixture is then poured onto a mixture of 3.5 ml of concentrated hydrochloric acid and g of crushed ice. Stirring is continued for a further 30 minutes at 00 and then a solution of 2.01 g (16.6 mmol; 2.1 ml) of N,N-dimethylaniline in 3 ml of glacial acetic acid is added to the reaction mixture. The temperature of the reaction mixture is allowed to rise to room temperature over a period of 3 to 4 hours. The precipitated product is filtered off and dried overnight in vacuo at room temperature, yielding 4-(Nacetylamino)-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid in the form of black to purple crystals which decompose on heating and. have no definite melting point.

2 g (5.5 mmol) of 4-(N-acetylamino)-4'-(N,N-dimethylamino)-azobenzene-2sulfonic acid are dissolved in 8 ml of ethanol. After the addition of 4 ml of 11N sodium hydroxide solution, the mixture is stirred for 60 minutes at 90°. The mixture is then allowed to cool to room temperature and then 5 ml of 11.5N hydrochloric acid are added. The reaction mixture is then left overnight at +40. The precipitated product is filtered off ;nd vashed thoroughly with IN hydrochloric acid. After drying in vacuo at room temperature, 4-amino-4'-(N,N-dimethylamino)azobenzene-2-sulfonic acid is obtained in the form of black to purple crystals which decompose on hea",ng and have no definite melting point.

Example 2: 0.67 g (2.1 mmol) of 4-amino-4'-(N,N-dimethylamino)-azobenj* zene-2-sulfonic acid (Example 1) are dissolved in 5 ml of absolute pyridine. This solution is then added dropwise, with stirring and while cooling with ice/sodium chloride, to a mixture of 424 mg (2.1 mmol) of N,N'-dicyclohexyl carbodiimide, 5 ml of anhydrous pyridine and 1.26 g (16.6 mmol; 1 ml) of carbon disulfide. The reaction mixture is then stirred for 3 to 4 hours with the cooling being maintained. The mixture is then left to stand at room temperature for 17 hours. The pyridine and excess carbon disulfide are then removed as completely as possible under reduced pressure. Column chromatography of the residue on silica gel (150 to 200 mesh) with benzene as eluant yields 4'-(N,N-dimethylamino)- 4-isothiocyanato-azobenzene-2-sulfonic acid which crystallises out when the eluate is concentrated by evaporation.

Example 3: 0.67 g (2.1 mmol) of 4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1) are dissolved in 5 ml of absolute pyridine. With stirring and while cooling with ice, 1.26 g (16.6 mmol; 1 ml) of carbon disulfide and 0.22 g (2.2 mmol; 0.3 mi) of triethylamine are added to the solution. The reaction mixture is then stirred for 3 to 4 hours with the cooling being maintained. The mixture is then left to stand for 17 hours at room temperature and is then concentrated to dryness by evaporation. The triethylammonium dithiocarbamate remaining behind as residue is dried overnight in vacuo and is then dissolved in 12 ml of trichloromethane. While cooling with ice, 0.22 g (2.2 mmol; 0.3 mi) of triethylamine is added to the solution. To this mixture there is then added dropwise, within a period of 4 minutes, 0.34 g (3.1 mmol; 0.3 ml) of chlorocarbonic acid ethyl ester, with stirring And while cooling with ice. The reaction mixture is left to stand for 3 to 4 hours with the cooling being maintained and is then concentrated to diyness by evaporation under reduced pressure. The crude product remaining behind as residue is purified by column chromatography as described in Example 2, yielding 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic 'soo acid.

S Example 4: In a manner analogous to that described in Examples 1 to 3, starting from 4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and 4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-aminobenzene-2'sulfonic acid), it is also possible to obtain 4'-(N,N-dimethylamino)- 4-isothiocyanato-azobenzene-2,6-disulfonic acid and 4-(N,N-dimethylamino)-naphthalene-1-azo-(4'-isothiocyanatobenzene-2'-sulfonic acid), respectively.

The starting materials can each be obtained in a manner analogous to that described in Example 1, starting from 4-(N-acetylamino)-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and 4-(N,N-dimethylamino)naphthalene-1-azo-14'-(N-acetylamino)-benzene-2'-sulfonic acid), respectively.

31 :Eample 5: 20 ml of chlorobenzene are cooled to 0° by cooling with ice/sodium chloride. 1.1 g (11.1 mmol) of phosgene are introduced by condensation into the cooled chlorobenzene with the customary precautions being observed. With the cooling being maintained and with vigorous stirring, a solution of 1.79 g (5.6 mmol) of 4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1) in 30 ml of chlorobenzene is added dropwise at such a rate that the temperature of the reaction mixture does not exceed When the dropwi-e addition is complete, the cooling bath is replaced by a heating bath by means of which the reaction mixture is heated to 1300 within a period of 90 minutes while constant stirring is continued. In the course of this process, as soon as the reaction mixture has reached a temperature of 750, further phosgene is slowly introduced until a clear solution is obtained. The reaction mixture is then heated under reflux, with nitrogen being passed through the mixture, until phosgene can no longer be detected in the outgoing 1 stream of gas (about 2 hours). After cooling to room temperature, the w reaction mixture is concentrated as completely as possible under reduced pressure. Column chromatography of the residue on silica gel (150 to 200 mesh) witih benzene as eluant yields 4'-(N,N-dimethylamino)-4-isocyanato-azobenzene-2-sulfonic acid which crystallises out when the eluate is concentrated by evaporation.

Example 6: In a manner analogous to that described in Example 5, starting S from 4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-dislfonic acid and S 4-(N,N-dimethyl&,,ino)-naphthalene-l-azo-(4'-aminobenzene-2'-sulfonic acid) it is also possible to obtain 4'-(N,N-dimethylamino)-4-isocyanatoazobenzene-2,6-disulfonic acid and 4-(N,N-dimethylamino)-naphthalene-lazo-(4'-isocyanatobenzene-2'-sulfonic acid), respectively.

The starting materials can each be obtained in a manner analogous to that described in Example 4.

Example 7: 1.8 g (9.7 mmol) of iodoacetic acid and 1.2 g (6.3 mmol) of N-[3-(N,N-dimethylamino)-propyl]-N'-ethyl-carb>diimide hydrochloride are dissolved in 9 ml of water. This solution is immediately mixed, with stirring, with a solution of 0.5 g (1,6 mmol) of 4-amino-4'-(N,N-dimethylamino)-azobenzene-2-sulfonic acid (Example 1) in 7.6 ml of a buffer 32 solution which has a pH of 9 and consists of 50mM sodium hydrogen carbonate solution and 50mM sodium carbonate solution. 0.4 ml of 11N d sodium hydroxide solution is then added dropwise to the mixture which is then stirred for 10 minutes at room temperature. IN hydrochloric acid is then added dropwise until the mixture turns purple. The product that precipitates is filtered off, washed thoroughly with 100 ml of O.1N hydrochloric acid and dried in vacuo. The dried crude product is dissolved in a mixture of 16 ml of N,N-dimethylformamide and 0.4 ml of triethylamine. Further precipitation with 1N hydrochloric acid in the mannet described above, filtration and drying of the filter contents in vacuo yield the desired pure 4'-(N,N-dimethylamino)-4-(N-iodoacetyl)amino-azobenzene-2-sulfonic acid which decomposes on heating and has no definite melting point.

Example 8: In a manner analogous to that described in Example 7, starting I from 4-amino-4'-(N,N-dimethylamino)-azobenzene-2,6-disulfonic acid and v S 4-(N,N-diDsethylamino)-naphthalene-l-azo-(4'-aminobenzene-2'-sulfonic acid), it is also possible to obtain 4'-(N,N-dimethylamino)-4-(N-iodo,acetyl)-amino-azobenzene-2,6-disulfonic acid and 4-(N,N-dimethylamino)- *ca: naphthalene-l-azo-(4'-(N-iodoacetyl)-aminobenzene-2'-sulfonic acid), *a respectively.

The starting materials can each be obtained in a manner analogous to that described in Example 4.

B B *4 Example 9: Monoclonal antibodies that act specifically against eglin C are chemically modified at room temperature over a period of 18 hours in a 50mM sodium hydrogen carbonate solution by the action of a 2mM solution of 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid,, a Subsequent structural analysis shows that each antibody molecule has been chemically modified by 30 molecules of 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenene-2-sulfonic acid, With this degree of modification, the antibodies exhibit specificity against and affin,ty for recombinant eglin C that is unchanged in comparison with the unmodified antibodies.

These chemically modified antibodies which have also been coloured at the r -33same time can be used to demonstrate the presence of eglin C with the aid of the sandwich dot assay. In thac assay concentrations of 1 pg/ml and above can be detected with the naked eye.

Example 10: Hirudin, a specific inhibitor of thrombin, is chemically modified at 37° over a period of 7 hours in a 50mM sodium hydrogen carbonate solution by the action of a 2mM solution of 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. The hirudin so modified is subjected to gel chromatography on a Sephadex G 25 column. In the modification of the inhibitor, 1.96 mol of 4'-(N,N-dimethylamino)- 4-isothiocyanato-azobenzene-2-sulfonic acid are reacted per mol of hirudin. The structure of the modified inhibitor and its biological activity are analysed and the following findings are obtained: a) The chemically modified hirudin has a reduced inhibitor activity against thrombin, as can be seen from the fact that the corresponding dissociation constant Ki is 300 times greater.

l e b) Monoclonal antibodies against native hirudin act also against the so chemically modified hirudin, c) The chemically modified hirudin is carboxymethylated and treated with protease V8, The resulting peptides are subjected ta HPLC, At a wave- S length of 536 nm two peptides are detected. Structural analysis shows that these two coloured peptides correspond to amino acid building blocks 1 to 7 and 18 to 37 and that selectively the building blocks Val-1 and o* Lys-27 are modified by 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.

ls** Example 11: Human antithrombin is chemically modified at room temperature over a period of 15 minutes in a 50mM sodium hydrogen carbonate solution by the action of a ImM solution af 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid. The antithrombin so modified is subjected to gel chromatography on a Sephadex G 25 column. The structure of the modified antithrombin and its biological activity are analysed and t-he following findings are obtained: I II I all LI~Y~ 34 a) The chemically modified antithrombin retains only 25 of its .n cofactor activity, but its inhibitor activity against thrombin in the absence of heparin (progressive inhibitor activity) is virtually unchanged.

b) The chemically modified antithrombin is carboxymethylated and treated with trypsin. The resulting peptides are subjected to HPLC. At a wavelength of 436 nm three peptides are detected. Structural analysis shows that these three coloured peptides correspond to amino acid building blocks 91 to 111, 115 to 129 and 133 to 139 and that selectively the building blocks Lys-107, Lys-125 and Lys-136 are modified by 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid.

c) If antithrombin is incubated with heparin before being acted upon by 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid, the chemical modification of the building blocks Lys-107, Lys-125 and Lys-136 .is inhibited by 98 88 and 93 respectively, d) The building blocks Lys-107, Lys-125 and Lys-136 of human antithrombin are accordingly of particular importance to the bonding of heparin.

S*

Example 12: In a manner analogous to that described in Examples 9 to 11 it is also possible to use a different compound of formula I or a salt of a compound of formula I, for example according to Examples 1 to 8, for *5 the chemical modification of a protein, Example 13: The investigation of the heparin binding site of human antithrombin by means of a device of the type shown in figure 1 can be carried out as follows: a) Human antithrombin (432 amino acids) and the heparin-antithrombin complex are processed one after another, the folowing steps b) through g) being carried out in each case. The device used in these steps comprises the Lomponents shown shcematically in figure 1. The reactor (15) is made of glass. The detector (18) works on the basis of the absorption of light of a wavelength of 436 nm. The connections and through (13) are 35 made of glass. The connection is made of vacuum resistant tubing. An oil pump is used as vacuum pump The desalting unit (19) consists of a Sephadex G 25 column.

b) Chemical modification st~p Solvent: 50 mM sodium hydrogencarbonate solution (pH 8.3); Sample 1: antithrombin, 250 pg; Sample 2: heparin-antithrombin complex (2;1 parts by weight), 750 pg (250 pg of antithrombin and 500 pg of heparin); Reagent of the formula I for the chemical modification: 1mM solution of 4' -(',N-dimethylamino)-4-isothiocyanato-azobenzene-2-sulfonic acid in the solvent mentioned; Reaction volume: 200 pl; Reaction temperature; +250; Reaction time: 7,5 minutes.

Cc) Desalting step se.*Column: Seph~dex G 125 (length: 3.5 cm; internal diameterli 1 cm); E: luent: 50 mM ammonium, hydr~ogen carbonate solution (pH= d) Denaturation step Solvent; 0.5 8 a, U -tris (hydroxyme thyl) me thylamine hydro~hlo ride ("tr'is Ce. hydroch 1 orile") solution (pHit~ 8.4) and 5 X guanidinq hydroQ.hloridQ solution; Reactilon volume; 200 p1; *~Reduction reagent: threo-1,04 -dime rcapto-2, 3-bu tanediol (dithiothroitol, DTT), 1 mg; Reduction temperature; +37*; *moo Reduction timel 2 hours,- Carbo~ymothyla~ion reagent. iodoacetic acid, 2 mg; Caboxymetylkationtemperature: +3701; Carboxymethylation time: 15 minutes, e) De salting step Column., Sephadex G 25 (length: 3.5 cm; internal. diameter! I cm); tluent:' 50 ml' ammonium, hydrogen carbonate solution (pH -36 f) Protease treatment step Protease: trypsin, 10 pig; Solvent: 50 mM ammonium hydrogen carbonate solution (pH S'' R~eaction volume: 100 p1; Reaction temperature: +370; Reaction time: 2 hours.

g) HPLC step Column: Vydac C-18 for peptides and proteins; Column temperature: +250; Solvent A; 17,5 mM sodium acetate (pH =540); Solvont B; acetonitrile; Gradient: linear from 10 B to 70 B within 30 minutes; Flow rate: 1 ml per minute; Detector: absorption of light of a wavelength of 436 nm h) The comparison of the peak patterns in the two chromatograms resuilting from sample 1 and sample 2, respectively, and the sequence analysis of the coloured peptides which correspond to peak~s that appear only in the chromatogran of sample 1 rev.eal, that. the amino acid building blocks Lys-l07, Lys-125 and Lys-136 are situated within tho heparin binding site of human antithrombin, S. S S

S.

SO

0 0 55S*

S

*SOS

S See

C

505

S*

S

S

:04Example 141 In a manner ana~logous to that described in Example 13, the nvestigation of the hirudin binding site of humon thrombin can be carried out using the devi~e described in Example, 13. Human thrombin (sample 1; 250 pg) and tie hirudin-thronbin complex [sample 2; 750 pgt molar~ ratio (hirudin: thrombin) are processed one after another, steps b) through g) of Ex~ample 13 being carried out analogously in each case. The comparison of the peak patterns in~ the two chromato., grams resulting from. sample I and sample 2, respectively, and the sequence ano2.ysis of the coloured peptides which correspond to peaks that appear only in the thromatogrom of sample 1 reveal, that the amino acd building blooks Lys-.21, Lys-52, Lys-65, Lys-106, Lys-107 and Lys-154 are situated~ within the hiridi~n bending site of human thrombino 37 Example 15: In a manner analogous to that described in Examples 13 and 14 it is also possible to use a different compound of formula I or a salt of a compound of formula I, for example according tc~ Example~ 1 to 8, as reagent for the chemical modification in b) cE the processing sequence carried out in the devi'~ ci*~JScLd.' in Example 13.

*9 9 0 0090 S. Os S S

S

0**9 9 5* 9. 9. *0 L

S

9S 5* 5 9 See

S

Oeg

Claims (22)

1. 6. A compound of formula 1 accordiiig to claim 1 wherein R, 1 is C 1 -C 4 alkyl, R 2 is C 1 -G 4 alkyl, R 3 is hydrogen or sulfo, R 4 is sulfo, G is an unsubstituted or sulfo-substituted I,4-phenylene group, R 5 and R 6 together are an additional bond and L is an oxygen or sulfur toem, or a salt thereof, 7, A compound of formula I according to claim 1 wherein R, is C 1 -C 4 alkyl, R 2 is Cl-C 4 alkyl, R 3 is hydrogen, R 4 is suffo, G is an unsubstituted 1,4-phenylene group and wherein either and R 6 together are an additional bond and L is a sulfur atom, or wherein R 5 is hydrogen, R 6 is iodornethyl and L is an oxygen atom, or a salt thereof. 8, A compound of formiula I according to claim 1 wherein R, 1 is alkyl, R 2 is Cl-C 4 alkyl, R. 3 is hydrogen, R 4 is sulfo, G is an unsubstituted 1,4-phenyletne group, P. 5 and R6 together are an additionO2 bond and L is a sulfur atomn, or a sail thereof, .59, 4-(N,N-di'methylamino)-4-isothiocyanato-azobenzene-2-stilfD ic acid oi a salt thereof. 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene-2,6-disulfonic acid or a salt thereof. 11, 4-(N,N-dimethylaminio)-naphthalene- t-azo-(4'-isothiocya;4tobcnzene-2'-sulfonic acid) or asalt thereof, 12, 41 -(N,N-dimethylanmino)-4-Isocyanato-azobenzene-2slY inic acid or a salt thereof,
2. 13. 4' -(NN-dimethylaninilo)-4-isocyanato-a,,obenzene-2,6-dilsulfonic aci, or a salt thereof,
3. 14. 4-(N,N-.dinliethiylai nro)-naph thalenie- 1.-azo-(4' -isocyantbnee -sulfonic acid) or a salt thereof. 40 4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2-sulfonic acid or a salt thereof.
4. 16. 4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino-azobenzene-2,6-di-sulfonic acid or a salt thereof.
5. 17. 4-(N,N-dimethylamino)-naphthalene-1-azo-[4'-(N-iodoacetyl)-aminobenzene-2'- sulfonic acid] or a salt thereof.
6. 18. A process for the preparation of a compound of formula I or a salt thereof according to any one of claims 1 to 17, which process comprises: in a compound of formula Ra 3 R1 N-G-N=NN NH 2 (H) 2 3D- RR4 or in a salt thereof, converting the NH 2 group into a group of formula S: L (Ic) 1 5 R 6 j and, if desired, separating a mixture of isomers obtainable in accordance with the process S into the components and isolating the desired isomer I, resolving a mixture of enantiomers or diastereoisomers obtainable in accordance with the process into the individual enantiomers or diastereoisomers and isolating the desired enantiomer or diastereoisomer, and/or converting a free compound I obtainable in accordance with the process into a salt S* or converting a salt obtainable in accordance with the process into the free compound I or into a different salt.
7. 19. A compound of formula i -1 -41- R 3 N- G- N =N NH 2 (I wherein R, is lower alkyl, R 2 is lower alkyl, R 3 is hydrogen, carboxy or sulfo, R 4 is carboxy or sulfo, and G is an unsubstituted or substituted 1,4-phenylene group or an unsubstituted or substituted I ,4-naphthylene group, or a salt thereof. A compound of formula II according to claim 19 wherein R, is lower alkyl, R 2 'is lower alkyl, R 3 is hydrogen, carboxy or sulfo, R 4 is carboxy or sulfo, and G is an unsubstituted or carboxy- and/or sulfo-substituted 1 ,4..phenylene group or an unsubstituted or carboxy- and/or sulfo-substituted l,4-naphthylene group, or a salt thereof.
8. 21. A compound of formula II according to claim 19 wherein R, is C 1 -C 4 -alkyl, R 2 is C 1 -Calkl, R ishydrgenor slfo R 4 is sulfo, and G is an unsubstituted or sulfo substituted 1,4-phenylene group, or a salt thereof, 22, A opudo ormula 11 according to claim 19 wherein R, is C 1 -C 4 -alkyl, R 2 i C 1 -C 4 alkyl, R 3 is hydrogen, R 4 is sulfo, and G is an unsubstituted, 1,4-pheilylene group, or a salt thereof. 23 *-mn-'(,-iityiio-zbnee2sloi cdo atteef
9. 23. 4-amino-4'-(N,N-dimethylamino)-azobenzene-2,-sulf7nic acid or a salt thereof. ,imethiylamiino)-naphthalene- 1-azo-(4'-aminiobenzene-2'-sulfonic acid) or a salt thereof, 26,. A process for the preparation of a compound of formula 11 or a salt thereof according to any one of claims 19 to 25, which process comprises hydrolysing a compound of formula RA/41' -42- R 3 N-G-N=N NH-Y (HI), R2 R4 wherein Y is an acyl radical, or a salt thereof and, if desired, separating a mixture of isomers obtainable in accordance with the process into the components and isolating the desired isomer II, resolving a mixture of enantiomers or diastereoisomers obtainable in accordance with the process into the individual enantiomers or diastereoisomers and isolating the desired enantiomer or diastereoisomer, and/or converting a free compound II obtainable in accordance with the process into a salt or converting a salt obtainable in S accordance with the process into the free compound II or into a different salt.
10. 27. A process for the chemical modification of a protein, wherein the protein is reacted with a compound according to any one of claims 1 to 17 or with a salt thereof.
11. 28. A process for the chemical modification of a protein that is associated with coloration according to claim 27, wherein the procedure is as indicated in claim 27,
12. 29. A process for the chemical modification of lysine building blocks of a protein, wherein the protein is reacted with 4'-(N,N-dimethylamino)-4-isothiocyanato-azobenzene- S 2-sulfonic acid.
13. 30. A process for the chemical modification of lysine building blocks of a protein that is associated with coloration according to claim 29, wherein the procedure is as indicated in claim 29,
14. 31. A process for the chemical modification of cysteine building blocks of a protein, wherein the protein is reacted with 4'-(N,N-di-methylamino)-4-(N-iodoacetyl)-amino-ato- benzene-2-sulfonic acid, 32, A process for the chemical modification of cysteine building blocks of a protein that is associated with coloration according to claim 31, wherein the procedure is as indicated in claim 31. 43
15. 33. The end products substantially as herein described with reference to any one of Examples 1 to 8.
16. 34. The preparative process substantially as herein described with reference to any one of Examples 1 to 8. A method of investigating a protein comprising incorporating a compound according to any one of claims 1 to 17 or a salt thereof into the protein and comparing the protein so altered with an unaltered sample of the protein.
17. 36. The process for modifying a protein substantially as herein described with reference to any one of Examples 9 to 12.
18. 37. A device designed to carry out automatically the sequence consisting of the chemical modification of a protein by means of a compound of the formula I according to any one of claims 1 to 17 or a salt thereof, the optional denaturation of the chemically modified protein, the protease treatment of the chemically modified and optionally denatured protein, and the high-performance liquid chromatography (HPLC) of the peptide mixture resulting from the protease treatment step, said device comprising a syringe pump which serves to deliver a sample of the protein which is to be chemically modified by means of a compound I or a salt thereof, solvents, reagents and/. c catalysts, that are necessary for the said steps of chemical modification by means of a compound or a salt thereof, optional denaturatioan and protease treatment, inert gas, and washing solution through a connection into a reactor, which is equipped with 0434e/VMJ 44 a screw cap and is installed within a heating-jacket, a vacuum pump which enables by means of a connection the evacuation of the reactor for the purpose of the concentration of the contents of the reactor, a connection, which, by means of a slight excess pressure of inert gas delivered from the syringe pump through the connection, serves to deliver the contents of the reactor, when the chemical modification or the optional denaturation step has been carried out, through a valve, a connection, another valve and another connection to a desalting unit, or serves to deliver the contents of the reactor, when the protease treatment step has been carried out though a valve, a connection, a valve and a connection to a HPLC component, to which component a plotter and thrwugh a connection a fraction collector are attached, or serves to deliver the e washing solution contained in the reactor through a valve and a connection to a waste reservoir, a pump which delivers eluent through a connection, a valve and a connection to the desalting unit a connection which serves to deliver the eluate which is eluted from the desalting unit to a detector which serves to control the valve in such a way the eluate which is eluted from the desalting unit, when it contains the chemically modified protein prepared in the chemical i modification step or optionally the denatured chemically modified protein prepared in the optional denaturation step is delivered through a connection into the reactor and when it contains no such chemically modified protsin or denatured chemically modified protein is delivered throagh a connection into the waste reservoir, and a connection which serves to deliver the eluate from the detector to a valve.
19. 38. A process for carrying out automatically the sequence mentioned in claim 37, characterised in that the protein is processed by means of a device according to claim 37. 0434e/VMJ i i I 41( I r r 9 S S S S. 9 45
20. 39. A process according to claim 38, characterised in that in the chemical modification step the protein is reacted with 4'-(N,N-dimathylamino)-4-isothiocyanato-azobenzene- 2-sulfonic acid. A process according to claim 38, characterised in that in the chemical modification step the protein is reacted with 4'-(N,N-dimethylamino)-4-(N-iodoacetyl)-amino- azobenzene-2-sulfonic acid.
21. 41. Method of chemically modifying a protein comprising reacting the protein with a compound according to any one of claims 1 to 17 or with a salt thereof, said reaction being carried out in a device according to claim 37.
22. 42. The process for investigating a protein substantially as herein described with reference to any one of Examples 13 to DATED this 12th day of May, 1992. CIBA-GEIGY AG By Their Patent Attorneys DAVIES COLLIRON CAVE S 0434e/VMJ