AU611385B2 - Membrane anchor/active compound conjugate, its preparation and its use - Google Patents

Abstract

Conjugates are described in which the active ingredient is covalently bonded to the anchoring membrane, which is a compound of the formulae <IMAGE> in which A can be: sulphur, oxygen, disulphide (-S-S-), methylene (-CH2-) or -NH-; n = 0 to 5; m = 1 or 2; C* = an asymmetric carbon atom of R or S configuration, R, R' and R'' are identical or different and are an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms or are hydrogen which can optionally be substituted by hydroxyl, amino, oxo, acyl, alkyl or cycloalkyl groups and R1 and R2 are identical or different and are defined as R, R' or R'' or can be -OR, -OCOR, -COOR, -NHCOR or -CONHR and X is an active ingredient or a spacer active ingredient group. The anchoring membrane active ingredient conjugates increase antibody formation.

Description

.L I I I r- "rs 3 r J 113 3 For1o COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: 5 9 Ti Lodged: Complete Specification Lodged: Accepted: Published: Priority felated Art: Narh of Applicant: HOECHST AKTIENGESELLSCHAFT t.

ASdlress of Applicant 45 Bruningstrasse, D-6230 Frankfurt/Main Federal Republic of Germany Actual Inventor: 'AdGfess for Service: GUNTHER JUNG, KARL-HEINZ WIESMULLER, JORG METZGER, HANS-JORG BUHRING, GERHARD BECKER and WOLFGANG BESSLER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: MEMBRANE ANCHOR/ACTIVE COMPOUND CONJUGATE, ITS PREPARATION AND ITS USE The following statement is a full description of this invention, including the best method of performing it known to US 1.

-la-

P

HOECHST AKTIENGESELLSCHAFT HOE 85/F 301J Dr. MD/mk Membrane anchor/active compound conjugate, its preparation and its use The invention relates to membrane anchor/active compound conjugates having at Least one active compound covalently bonded to the membrane anchor compound(s), to a process for their preparation and to their use.

Membrane anchor compounds are compounds which can penetrate into biological and synthetic membranes.

For example, these membrane anchor compounds can be natural membrane lipoproteins as have already been isolated from the outer membrane of Escherichia coli and have now also been synthesized. The E. coli membrane anchor compound is composed in the N-terminal region of three fatty acids which are bonded to S-glyceryl-L-cysteine Jung et al. in "Peptides, Structure and Function", V.J. Hruby and D.H. Rich, pages 179 to 182, Pierce Chem.

Co. Rockford, Illinois, 1983).

Moreover, conformation-stabilized alpha-helical polypeptides have already been described for the investigation of biological membranes by means of models, see inter alia alamethicin, an alpha-helical amphiphilic eicosapep- Stide antibiotic which forms voltage-dependent ionically Sconducting systems in lipid membranes (Boheim, Hanke, Jung, Biophys. Struct. Mech. 9, pages 181 to 191 (1983); Schmitt, H. and Jung, Liebigs Ann. Chem.

pages 321 to 344 and 345 to 364 (1985)).

There is a description in European Patent A1-330 of the immunopotentiating action of Lipopeptides which are analogs of tne lipoprotein from E. coli which has been known since 1973. Another European patent application, A2-114787, deals with the ability of lipopeptides of this type to activate alveolar macrophages of rats and mice in vitro so that, I 2after incubation with the substance for 24 hours, the maciophages are able to eliminate tumor cells and, in particular, they significantly increase the production of antibodies, for example against porcine serum albumin.

It is proposed in European Patent A2-114787 to use these lipoprotein derivatives as adjuvants for immunization, that is to say to employ the lipoprotein derivatives of the E. coli membrane protein mixed with antigens to improve the immune response.

There is a great need for substances which stimulate and potentiate the immune response, in particular because purified antigens can often be obtained in only minuscule amounts; furthermore, when new batches of antigens are employed there is always the possibility of new contaminants or decomposition products.

It is furthermore desirable not to have to inoculate an experimental animal frequently but, where possible, to obtain the desired immune response by a single dose of the immunogenic material.

Hence it is an object of the present invention to increase the formation of antibodies against antigens or haptens and thus to obtain a specific immunopotentiating action.

The object is achieved according to the invention by the Snew membrane anchor/active compound conjugate having at least one membrane anchor compound and at Least one active compound covalently bonded to the membrane anchor compound(s).

According to the invention, a process for the preparation of membrane anchor compounds is also proposed, which process comprises synthesis of the peptide, which is protected with protective groups in a manner known per se on the functional groups at which no reaction is to take I 1 i- 3 place, by means of known coupling processes on a solid or soluble carrier, such as a polymer (for example Merrifield resin); covalent bonding of the carrier-bound peptides, which have been synthesized in this way, via N-termini or side-groups of the peptide to the membrane anchor compound; isolation of the poLymer/peptide conjugate, which has been prepared in this way, by cleavage of the protective groups and the peptide/carrier bond in a manner known per se, and thus the membrane anchor/peptide or the membrane anchor/active compound conjugate being obtained.

The invention also relates to the use of the compounds for the preparation of conventional and monoclonal antibodies in vivo and in vitro; however, it is also possible, in an advantageous manner, tc use the compounds according to 15 the invention in genetic engineering to facilitate cell fusion, for the preparation of synthetic vaccines, for the preparation of cell markers with fluorescence labels, spin labels, radioactive Labels or the like, for affinity chromatography, in particuLar for affinity coL.umns; for Liposome preparations; as additive to human foodstuffs or animal feeds, and as additive to culture media for microorganisms and, generally, for cell cultures. This may entail, where appropriate, the compounds according to the invention.being used, together with vehicles known per se, in solution, ointments, adsorbed onto solid carriers, in emulsions or sprays, for purposes in human or veterinary medicine.

The membrane anchor compound is preferably a compound of Sone of the following general formulae: R CO-O-CH 2 R O-CH R O-CO-CH CO-O-CH* O-CH*

O-CO-CH*

(CH2 n (CH2)n (CH 2 n A A A (CH2m (CH2 (CH2) R"-CO-NH-CH*-CO-X R"-CO-NH-CH*-CO-X

R"-CO-NH-CH*-CO-X

I.

II.

III.

R -NH-CO-CH R -NH-CO-CH* CC 2 n

A

(CH2 R"-O-H-C2mO- -4 R -CO-NH-CH 12 R -CO-NH-CH

(CH

2

A

(CH 2

~M

R -CO-NH..CH*-CO-X

B

S

(CH 2

R-NH-CO-CH'-CO-X

VI.

R

1

-CH

2 2H

A

(CH

;2 M R-O N -C *C

VII.

00 0 it being possibLe for A to be suLfur, oxygen, methyLene (-CH 2 or -NH-; d isu Lf idce orn-a-; t:m (a) carbon atom with R or S configuration; R, R' and "Rbeing identicaL or different and being an aLkyL, a enyL or aLkynyL group having 7 to 25 carbon ato or hydrogen, which can optionaLly be substitute y hydroxyL, amino, oxo, acyL, aLkyL or cycLoaLkyL>-4roups, and R, and R 2 being identical pr dlifferent an eing defined as R, R' and R" or possibLy being -C0 -OCOR, -C)OR, -NHCOR or -CONHR, and X being an acti compound or a spacer-active compound g ro up S 13 8OO-ey+- Fl d OR ftgzu au'.

membrane anchor/active-c-o-m-p-o-u-d conjugate according to the 4a n being 0 to 5; m being 1 or 2; C* being an asymmetric carbon atom with R or S configuration; R, R' and R" being identical or different and being an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms or hydrogen, which can optionally be substituted by hydroxyl, amino, oxo, acyl, alkyl or cycloalkyl groups, and R, and R 2 being identical or different and being defined as R, R' and R" or possibly being -OR, -OCOR, -COOR, -NHCOR or -CONHR, X being an active compound or a spacer-active compound group, and B can be any one of the substituted S-alkyl radicals defined by structural formulae I to V wherein A For example, B can denote each of the -S-(CH 2 )n- (substituted alkyl)-radicals as defined for structural formulae I to V.

It is also possible, in an advantageous manner, to use a mem rane anchor/active compound conjugate according to ,the invention of the following general formula: 0 Co 0 o 00 0 0 o S 0 I I I stJ o o 5

R

R

3 Nh CH CO X

VIII.

R

3 being an alpha-acyl-fatty acid residue having between 7 and 25 carbon atoms; preferably between 10 and 20 carbon atoms and very particularly preferably having between 14 and 18 carbon atoms; an alpha-alkyl-beta-hydroxy-fatty acid residue or its beta-hydroxy ester, the ester group being preferably straight-chain or branched chain and having more than 8 carbon atoms, preferably between about and 20 and very particularly preferably between 14 and 18 carbon atoms; it is possible and preferable for formula VIII to be an active compound conjugate with the S following membrane anchor compounds: N,N'-diacyllysine; N,N'-diacylornithine; di(monoalkyl)amide or ester of glutamic acid, di(monoalkyl)amide or ester of aspartic 15 acid, N,0-diacyl derivative of serine, homoserine or threonine and N,S-diacyl derivatives of cysteine or homocysteine; serine and homoserine; R 4 being a side chain of an amino acid or hydrogen; and X being hydrogen or a spacer-active compound group, it being possible when R 3 is S 20 a side chain of lysine, ornithine, glutamic acid, aspartic acid or their derivatives for the latter to be bonded, both in the manner of an ester and in the manner of an amide in the same molecule, in alpha or omega positions to R 4 2' 25 It is G"articularly preferred for the membrane anchor/ active compound conjugates to be prepared by synthesis of the peptide, which is protected with protective groups in a manner known per se on the functional groups at which no reaction is to take place, by means of known coupling processes on a solid or soluble carrier, such as a polymer (for example Merrifield resin); covalent bonding of the carrier-bound peptides, which have been synthesized in this way, via N-termini or side-groups of the peptide to the membrane anchor compound; isolation of the peptide conjugate, which has been prepared in this way, by 6 cleavage of the protective groups and the peptide/ carrier bond in a manner known per se, and thus the membrane anchor peptide or the membrane anchor/active compound conjugate being obtained.

The Linkage between the peptide and the membrane anchor compound can be produced by condensation, addition, substitution or oxidation (for example disulfide formation).

It is possible to use, in an advantageous manner, conformation-stabilizing alpha-alkylamino acid helices with an alternating amino acid sequence as the membrane anchor, it not being permissible for the alpha-helix to be destabilized by the other amino acids, such as of the type X-(Ala-Aib-ALa-Aib-ALa)n-Y, n being 2 or 4, and X and Y being protective groups which are known per se or -H, -OH or -NH 2 T, s c\ c po e \a o c. V .c.ed Qv- ow. rr-,0\So It may be advantageous for the active compound to be covalently Linked to two membrane anchor compounds which are, where appropriate, different.

In addition, it is also possible for the active compound to be covalently Linked to an adjuvant which is known per se for immunization purposes, such as, for example, muramyldipeptide and/or to a lipopolysaccharide.

Examples of active compounds which we propose are: an S antigen such as, for example, a Low moLecular weight par- 25 tiaL sequence of a protein or conjugated protein, for example of a glycoprotein, of a viral coat protein, of a bacterial cell wall protein or of a protein of protozoa (antigenic determinant, epitope), an intact protein, an antibiotic, constituents of bacterial membranes, such as muramyLdipeptide, Lipopolysaccharide, a natural or synthetic hapten, an antibiotic, hormones such as, for example, steroids, a nucleoside, a nucleotide, a nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, biotin, avidin, poLyethyLene glycol, a peptidic active compound 35 such as, for example, tuftsin, polylysine, a fLuorescence 1i \y7 7 marker (for example FITC, RITC, dansyL, luminol or coumarin), a bioluminescence marker, a spin Label, an alkaloid, a steroid, biogenic amine, vitamin or even a toxin such as, for example, digoxin, phalloidin, amanitin, tetrodoxin or the Like, a complex-forming agent or a drug.

The nature of the active compound determines the completely novel areas of use which emerge for the substances according to the invention.

It may also be beneficial for several membrane anchor/ active compound conjugate compounds to be crosslinked together in the Lipid part and/or active compound part.

The membrane anchor compounds and the active compound can also be connected together via a crosslinker, which results in the active compound becoming more remote from the membrane to which it is attached by the membrane anchor.

Examples of suita le crosslinkers are a dicarboxylic acid or a dicarboxylic acid derivative, diols, diaiines, polyethylene glycol, epoxides, maleic acid derivatives or the z like.

According to the invention, an unambiguously defined, Low molecular weight conjugate which is suitable, inter alia, Sfor immunization and which covalently links together the i' 25 carrier/antigen/adjuvant principles is prepared. The carrier and adjuvant can be not only a lipopeptide having mitogenic activity, such as, for example, tripaLmitoyl-S-glycerylcysteine (Pam 3 Cys) and its analogs, but also lipophilic conformation-stabilized alpha-helices and combinations thereof, such as Pam 3 Cys-antigen-helix, alpha-helixantigen-helix or even merely Pam3Cys-antigen or antigen- Pam 3 Cys or C-terminal Linkage), and antigen-helix or helix-antigen or C-terminal, or incorporated in the helix on side chains of Glu, Lys and the like). Thus, the I' i i ~bL

I

b 8 new compounds differ in essential aspects from all the high molecular weight conjugates of antigens with high molecular weight carrier substances which have hitherto been used, for example proteins, such as serum albumins, globulins or polylysine or, in general, high molecular weight linear or crosslinked polymers.

In particular, however, the new compounds also differ from all hitherto known adjuvants which are merely admixed and, accordingly, do not bring about specific presentation of the antigen on the cell surface. The adjuvants hitherto known have frequently rquired multiple immunizations and have also resulted in inflammatory reactions in animal experiments. A part -ular advantage according to the invention is the possibility of reproducible preparation of pyrogen-free, pure, unambiguously chemically defined compounds, and this in contrast to conventional compounds or mixtures of various substances also results in an improvement in the reproducibility of antibody formation. Hence, a particular area of use of the compounds according to the invention is viewed as being the area of antibody production, genetic engineering, the preparation of synthetic vaccines, diagnostic methods and therapy in veterinary and human medicine, since the new conjugates have for the first time an action which specifically stimulates the immune response, whereas the adjuvants hitherto used have merely stimulated the immune response non-specifically. Surprisingly, it is even possible with the compounds according to the invention to convert weakly immunogenic compounds into highly immunogenic compounds. Thus, a particular importance of the invention derives from the possibility of dispensing with animal experiments and costs for the preparatior of antibodies, since the new immunogens are also highly active in vitro. Moreover, because the immunization method is not inflammatory, an animal can be used several times for 0 0r fr- 0 0000o 000 Al 900 011 it1l.

8a obtaining different antibodies. The compounds can also be used in affinity chromatography, liposome preparation, all culture and microbiological culture media as well as in animal or human feedstuffs.

Finally, it might also be possible to use the new immunogens 0 a f f t B 6B s 4 formula: /3 9 to prepare polyvalent vaccines, i.e. for example a membrane anchor to whose side chains several antigens or haptens have been linked so that several different active antibodies can be prepared by means of one immunization.

One example of a water-soluble, mitogenic lipid anchor group is Pam 3 Cys-Ser(Lys)n-OH, which is particularly suitable for the preparation of the new immunogens as well as for the preparation of fluorescent, radioactive and biologically active cell markers. A particularly desirable property of the membrane anchor/active compound conjugates according to the invention is their amphiphilicity, i.e.

a partial water-solubility, since in this case it is considerably more straightforward to carry out biological tests on animals and investigations with living cells.

Moreover, the artificial lipid bilayer membranes, liposomes and vesicles which are required for some experiments can be prepared, and are stable, only in an aqueous medium.

An example of a suitable amphiphilic, biologically active membrane anchor is Pam 3 Cys-Ser(Lys)n-OH. The serine residue coupled to Pam3Cys favors immunogenic properties, whereas the polar, protonated epsilon-amino groups of the Lysine residues represent the hiydrophilic part of the molecule. Because of its multiple charges, this type of compound has further interesting properties. Owing to induction of interaction between cells, it can be used as Sa fusion activator in the preparation of hybridoma cells, °a especially when the lysine chain is relatively long, when coupling to polyethyLene glycol, or on covalent incorporation of the biotin/avidin system.

Furthermore, in an advantageous manner, it is possible to use the compounds according to the invention for the preparation of novel liposomes by crosslinking, it being possible for this to take place either in the fatty acid moiety or in the peptide moiety.

The membrane anchor (Pam 3 Cys and analogs, and the helices) i. formation.

10 are also suitable for potentiating the cell/cell interaction when, for example, they are covalently combined with the biotin/avidin system. Other advantageous properties of the compounds according to the invention are that they may facilitate cell fusion as is required, for example, for work in genetic engineering. Furthermore, the new immunogens can also be used in ELISA, RIA and bioluminescence assays.

Various Pam3Cys derivatives are lipid- and water-soluble and have potent mitogenic activity in vivo and in vitro.

They are also very suitable for labeling of cells with FITC and other markers such as RITC, dansyl and coumarin.

In particular, they can also be used for fluorescence microscopy and fluorescence activated cell sorting (FACS).

A reasonably priced membrane anchor having an analogous action to Pam3Cys is S-(1,2-dioctadecyloxycarbonyLethyl)cysteine, whose preparation is described in detail in the experimental.part.

Specific coupling of the mitogenically active lipid anchors to antigens can also be effected by crosslinkers, such as, for example, with dicarboxylic acid monohydrazide a derivatives of the general formula:

X-NH-NH-CO-A-CO-B-Y

or J 25 X-NH-NH-CO-A-COOH where A and B are amino acid or (CH2)n, and X and Y are protective groups known per se.

It is also possible to use every other suitable crosslinker or spacer for the preparation of the new substances, a particularly preferred embodiment of the invention always being represented by the principle (low molecular weight carrier and adjuvant)-(antigen) as long as it contains lipopeptide structures with lipid membrane anchor 11 functions and/or conformation-stabilized helices.

Particularly advantageous effects can be found by use of the compounds according to the invention in affinity chromatography, for which purpose Lipopeptide-antigen(hapten) conjugates are particularly suitable. The Latter are outstandingly suitable for loading conventional reversed phase HPLC columns (or preparation RP columns), this entailing, for example, anchoring of a tripalmitoyL compound, which has been applied in organic aqueous systems, in the apolar alkyL layer. The presentation of the antigen to the mobile aqueous phase remains the same as on cell surfaces, and thus it invites adsorption of the antibodies. Hence, it is possible to enrich or isolate antibodies, which specifically react with the relevant antigen, from dilute serum directly on an affinity column of this type. The elution of the antibodies is effected as with other affinity columns, for example by adjusting Sthe pH.

The intention now is to illustrate the invention in detail below by means of examples, but first the abbreviations used in them are Listed: Aib 2-methylalanine TFA trifluoroacetic acid EGF R epidermal growth factor receptor Pam palmitoyl radical St I DCC dicyclohexylcarbodiimide DMF dimethylformamide FITC fluorescein isothiocyanate Fmoc fluorenylmethoxycarbonyl But tert.-butyl radical PS DVB styrene/divinylbenzene copolymer with 4- (hydroxymethyl)phenoxymethyl anchor groups HOBt 1-hydroxybenzotriazoLe RITC rhodamine isothiocyanate Hu IFN-(Ly) 11-20 antigenic determinant of human interferon DCH Dicyclohexylurea EE Ethylacetate 12 The figures which are attached to illustrate the invention show: Fig. 1 the scheme for the preparation of Pam-Cys(C 8 g) 2 Ser-Ser-Asn-ALa-OH Fig. 2 the table of the 13C NMR spectra Fig. 3 the 1C NMR spectrum of Pam 3 Cys-Ser-(Lys) 4 -OH x 3TFA in CDCL3 Fig. 4 the 13C NMR spectrum of Pam-Cys(Pam)-OBut in CDCL3 Fig. 5 the 1C NMR spectrum of Pam-Cys(Pam)-OH in

CDCL

3 /CD30D 1:1 Fig. 6 the 13C NMR spectrum (J-moduLated spin-echo spectrum) of Pam(a-Pam)Cys-OBut Fig. 7 the 13C NMR (100 MHz) of the alpha-helix Fig. 8 the CD spectrum of the alpha-helix of HuIFN-(a- Ly)-11-20 Fig. 9 the obtaining of antibodies using Pam 3 Cys-Ser- EGF-R (516 to 529) Fig. 10 an in vivo immunization experiment Fig. 11 a comparison of the in vivo and in vitro immunization experiments and Fig. 12 the mitogenic activation of Balb/c mouse spleen cells using Pam3Cys-Ser-(Lys) 4

FITC.

First some preparation processes for substances according 25 to the invention and their precursors are now described below:

J

a o o~ rj a~ o &a r, i o o ii I. Preparation of Pam 3 Cys-EGF-R (516 529) After the customary stepwise synthesis (Merrifield synthesis protecting with N a-Fmoc/(But), with DCC/HOBt and symmetric anhydrides) of the EGF-R segment (526-529), the final amino acid attached was Fmoc-Ser(But)-OH. After elimination of the Fmoc group with piperidine/DMF (1:1, min), the resin-bound pentadecapeptide of EGF-R H-Ser- (But)-Asn-Leu-Leu-Glu-(OBut)-Gly-GLu(0But)-Pro-Arg(H+)-GLu- (OBut)-Phe-Val-Glu(OBut)-Asn-Ser(But)-0-p-alkoxybenzyldeaLs with the abiLity of Lipopeptides of this type to activate aLveoLar macrophages of rats and mice in vitro so that, 13 CopoLy(divinylbenzene/styrene) (1 g, Loading 0.5 mmol/g) was Linked with Pam-Cys(CH 2 -CH(OPam)CH 2 (OPam) (2 mmol, in

DMF/CH

2

CL

2 and DCC/HOBt (2 mmoL, preactivated at OOC for 20 min) (16h), folLowed by a second coupling (4 The Lipohexadecapeptide was cleaved off with trifluoroacetic acid (5 ml) with the addition of thioanisole (0.25 mi) within 2 h.

Yield: 960 mg Pam-Cys(CH 2 -CH(OPam)CH 2 (OPam))Ser-Asn-Leu- Leu-GLu-GLy-Glu-Pro-Arg-Glu-Phe-VaL-GLu-Asn-Ser-OH x

CF

3 COOH (correct amino acid analysis, no racemization).

II. Preparation of S-(1,2-dioctadecyoxycarbonylethy)-NpalmitoyL-L(or D)cysteine tert.-butyl ester Dioctadecy maleate can be obtained by the general procedure for esterifications of maleic acid Klostergaard, J. Org. Chem. 23 (1958), 108).

13 C NMR spectrum: see Fig. 2.

1.2 mmoL (500 mg) of N-palmitoyl-L-cysteine tert.-butyl ester and 1.2 mmol (745 mg) of dioctadecy maleate are dissolved in 20 mL of THF. After addition of 20 mmoL (3 ml) of N,N,N',N'-tetramethyethyenediamine, the mixture is stirred under nitrogen with a reflux condenser for 12 h. After addition of 100 mL of methanol and 5 mL of water, the coLorless precipitate is fiLtered off with suction, washed with water and methanol and dried in vacuo over P 2 0 5 YieLd: 1 g Melting point: 51 degrees Celsius L I IW 1 5 v o r a K 14- Thin-layer chromatography: RF 0.80; (mobile phase: CHCL 3 /ethyL acetate 14:1) 13 C NMR: see Fig. 2.

Molecular weight:

C

63

H

1 13 N0 7 S (1035.7) Elemental analysis: CalcuLated C 72.99 H 11.76 N 1.35 S 3.09 Found C 73.08 H 11.92 N 1.27 S 3.27 III. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)- N-palmitoylcysteine 0.48 mmoL (500 mg) of the t-butyl ester described under II is stirred in 65.3 mmoL (7.45 g, 5 mL) of trifluoroacetic acid in a closed vessel at room temperature for 1 h. The mixture is evaporated in a rotary evaporator under high vacuum, the residue is taken up in 1 mL of chloroform, 50 mL of petroleum ether is added to precipitate at -20 degrees C, and the product is dried in vacuo over P 2 0 5 Yield: 420 mg i Melting point: 64 degrees Celsius Thin-layer chromatography on silica gel plates: RF 0.73; (mobile phase: CHCI 3 /MeOH/H 2 0 65:25:4) 13C NMR: see Tab. 1.

Molecular weight:

C

5 9

H

113 N0 7 S (980.6)

III.

-B ,II "I 15 Elemental analysis: Calculated C 72.27 H 11.62 N 1.43 S 3.27 Found C 72.46 H 11.75 N 1.36 S 3.50 The new cysteine derivative and its t-butyl ester can be separated into the diastereomers on silica gel and RP chromatography. It is thus possible to prepare the two pairs of diastereomers of the L- and D-cysteine derivative.

IV. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)- N-palmitoyl-Cys-Ser(But)-Ser(But)-Asn-Ala-OBut 0.2 mmol (196 mg) of S-(1,2-dioctadecyloxycarbonylethyL)- N-paLmitoylcysteine is dissolved in 5 ml of dichloronethane, and preactivation is carried out with 0.2 mmol (27 mg) of HOBt in 0.5 mL of DMF and 0.2 mmol (41 mg) of DCC by stirring at 0 degrees C for 30 min.

After addition of 0.2 mmol (109 mg) of H-Ser(But)-Ser(But)- Asn-ALa-OBut in 3 ml of dichloromethane, the mixture is stirred at room temperature for 12 hours. Without further working up 40 mL of methanol are added to the reaction mixture. The colorless product can be filtered off with suction after 3 h. It is taken up in a Little dichloromethane and again precipitated with methanol. After washing with methanol, it is dried in vacuo over P 2 0 5 Yield: 260 mg (86%) Melting point: 194 degrees Celsius Thin-layer chromatography: RF 0.95; (mobile phase: CHC1 3 /MeOH/H 2 0 65:25:4) RF 0.70; (mobile phase: CHCL3/MeOH/glacia acetic acid 90:10:1) I- I j F''ll'Sj CL 0 V I OLw LI I U IneII LU LII !am .Of the f, L in -genera- forD u la.

116 13 SC

NMR:

see Fig. 2 Molecular weight:

C

84

H

1 58

N

6 0 14 S (1508.3) Elemental analysis: Calculated C 66.89 H 10.56 N 5.57 Found C. 67.10 H 10.41 N 5.52 V. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-NpalmitoyL-Cys-Ser-Ser-Asn-Ala 53 imoL (80 mg) of protected Lipopeptide (IV) are stirred o a e with 13 mmol (1.5 g; 1 ml) of trifluoroacetic acid in a Sclosed vessel at room temperature for 1 h. After evaporation under high vacuum, the residue is taken up twice with ml of dichloromethane each time and evaporated each time in a rotary evaporator. The residue is taken up in 3 ml of chloroform and precipitated with 5 ml of methanol at 4 degrees Celsius in 12 h. The product is filtered off with suction, washed with methanol and dried in a desiccator over P205.

Yield: ,o 63 mg (87%) Melting point: S"208 degrees Celsius (decomposition) Thin-layer chromatography: RF 0.63; (mobile phase: CHCl3/MeOH/glacial acetic acid/

H

2 0 64:25:3:4) RF 0.55; (mobile phase: CHCL3/MeOH/H 2 0 64:25:4) RF 0.06; (mobile phase: CHCl3/MeOH/glacial acetic acid 90:10:1) 17 Amino acid analys s: Cysteic acid 0.6; aspartic acid 0.93; serine 1.8; aLanine Molecular weight:

C

72

H

13 4

N

6 0 14 S (1340) VI. Preparation of Pam3Cys-Ser-(Lys) 4

-OH:

Pam3Cys-Ser(Lys) 4 -OH was synthesized by the solid-phase method (MERRIFIELD) on a p-aLkoxybenzyl aLcohoL/PS-DVB copolymer with N-Fmoc-amino acids and acid-labile side-chain protection (But for serine and Boc for lysine).

The symmetric anhydrides of the Fmoc-amino acids were used.

SThe coupling to Pam 3 Cys-OH was carried out by the DCC/HOBt method and repeated in order to achieve as near quantitative conversion as possible. In order to cleave the 15 Lipopeptide off the carrier resin and to remove the sidechain protection, the resin was treated twice with trifluoroacetic acid for 1.5 h and the acid was then removed in a rotary evaporator under high vacuum. The product was recrystallized from acetone.

13 The elemental analysis and the C spectrum indicate that the lipopeptide is in the form of the trifluoroacetate.

Assuming that Pam 3 Cys-Ser-(Lys) 4 -OH is in the form of a zwitterion, there are still three e-amino groups remaining which can be protonated by three trifluoroacetic acid molecules.

The C NMR spectrum of Pam 3 Cys-Ser-(Lys) 4 -OH x 3 TFA shows that the compound is in the form of the trifluoroacetate. (Quartet of the CF 3 group at 110-120 ppm, and carbonyl signals at 161-162 ppm). Owing to the aggregation of the polar part of the molecule, .the lines for the Lys and Ser carbon atoms are greatly broadened. The carbonyl signal at 206.9 ppm is produced by acetone which was used for the recrystallization and which is still adherent.

conjugate, which has been prepared in this way, by

I

18 Molecular weight: 1510.4 Elemental analysis: CalcuLated C 56.40 H 8.70 N 7.56 S 1.73 Found C 55.58 H 9.33 N 6.54 S 2.61 Amino acid analysis: The amino acid analysis showed that the ratio of serine to lysine is 1:4.2. The characteristic decomposition products of S-glyceryLcysteine produced during the hydrolysis (6 N HCL, 1100C, 18 h) were present (comparison with known standards). The peptide content was calculated to be 83%. 3 TFA molecules per Lipopeptide correspond to a peptide content of 80.2%, which agrees well with the analysis.

VII. Preparation of Pam3Cys-Ser-(Lys)4-OH x 3 TFA VII.1. Coupling of Fmoc-Lys(Boc)-OH to the carrier resin Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol) in 15 to 20 ml of DMF/

CH

2

CI

2 1:1 at 0 degrees C is mixed with DCC (0.99 g, 4.8 mmol). After 30 min, the precipitated urea is removed by filtration directly into a shaker vessel 2 which contains p-benzyloxybenzyl alcohol resin (2.5 g, 1.6 mmoL of OH groups). After addition of pyridine (0.39 ml, 4.8 mmol), the mixture is shaken at room tempera- I ture for 18 h. The solvent is removed by filtration with o. 25 suction, and the resin is washed 3 x each with 20 ml of

DMF/CH

2

CI

2 and DMF for each time. The resin is added to 20 ml of CH 2

CI

2 and then mixed first with pyridine (28.8 mmol, 6 equivalents) and then with benzoyl chloride (28.8 mmol, 6 equivalents). The mixture is shaken at room temperature for 1 h. The solvent is removed by filtration with suction, and the resin is washed 3 x each with 20 ml of CH 2

CL

2 DMF, isopropanol and PE 30/50.

I such as, for example, tuftsin, polylysine, a tluorescence 19 VII.2. Symmetric Fmoc-amino acid anhydride Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol, 3 equivalents) is dissolved in 15 mL of CH 2 CL2/DMF and, at 0 degrees CeLsius, DCC (4.8 mmol, 1.5 equivalents) is added. After 30 min at 0 degrees Celsius, the urea is removed by filtration directly into the reactor, and the process is continued as indicated in the table below.

The following procedure applies to 1/5 of the amount of resin used at the start (0.5 g, 0.32 mmoL of OH groups).

Fmoc-0-butyl-serine (0.74 g, 1.91 mmoL) is dissolved in 4 mL of CH 2

/CL

2 /DMF and, at 0 degrees CeLsius, DCC (0.96 mmoL) is added.

o pid Cs Table: Sequential synthesis of the peptide using symmetric Fmoc-amino acid anhydrides Operation Reagent Time Number of [min] times 1 CH 2

CL

2 2 2 2 DMF 2 2 oa 3 55% piperidine/DMF 5 1 4 55% piperidine/DMF 10 1 DMF 2 3 0 6 isopropanoL 5 2 7 DMF 2 3 8 CH 2

CL

2 2 3 9 DMF 2 2 0 10 Coupling with 3 eq. of symmetric Fmoc-amino acid anhydride in DMF/CH 2

CL

2 1:1 after 15 min addition of 3 eq. of NMM 11 DMF 2 3 12 CH 2

CL

2 2 3 helix on side chains of Glu, Lys and the like). Thus, the Operation Reagent Time Number of Smin times 13 Completeness of coupling checked by the Kaiser test; steps 10-12 repeated if necessary 14 Acetylation: 2 eq. of 15 1 and 0..5 eq. of NMM in ml of CH 2

CL

2 15 CH 2 C1 2 2 3 16 isopropanol 2 3 17 CH 2

CL

2 2 3 After 30 min, the urea is removed by filtration at 0 degrees C directly into the reactor, and the procedure S 15 is continued as usually.

VII.3. Coupling to Pam3Cys-OH Pam 3 Cys-OH (0.58 g, 0.64 mmol) is dissolved in 5 ml of

CH

2 CL2/DMF 1:1 and, at 0 0 C, is mixed with HOBt (93 mg, 0.64 mmol) and DCC (0.64 mmol). After 30 min at 0°C, the mixture is poured directly into the reactor.

After shaking for 16 h, a second coupling is carried out, with the same molar ratios as above, for 4 The solvent *is removed by filtration with suction, and the resin is Swashed 3 x each with 20 ml of DMF/CH 2 CL2 and DMF.

S 25 VII.4. Cleavage of the hexapeptide from the polymer The Boc-protected peptide/polymer resin compound (about 1 g) from VII.3 is thoroughly washed with CH 2

CI

2 and shaken 2 x 1.5 h with a mixture of 5 ml of TFA and 0.5 ml of anisole. The filtrate is evaporated in vacuo, and the residue is taken up in 5 ml of CHCL 3 Pam 3 Cys-Ser-(Lys)4- OH x 3TFA crystallizes out after addition of 50 ml of acetone at -20 degrees Celsius, is removed by centrifugation and is dried under high vacuum.

21 Yield: 0.41 g Melting point: 205 degrees Celsius (decomposition) Thin-layer chromatography on silica gel plates: RF 0.42; (mobile phase: n-BuOH/pyridine/H 2 0/gLacial acetic acid 4:1:1:2) RF 0.82 (mobile phase: n-BuOH/MeOH/H20/glacial acetic acid 10:4:10:6) Amino acid analysis: SSer 0.95 Lys 4 (4) SMolecular weight:

C

8 7

H

15 9

N

10 0 19

SF

9 (1852.6) Elemental analysis: Calculated C 56.40 H 8.70 N 7.56 S 1.73 i Found C 55.58 H 9.33 N 6.94 S 2.61 VIII. Preparation of Pam3Cys-Ser-(Lys)4-OH-FITC x 2 TFA Fluoresceine isothiocyanate (3.9 mg, 10 micromol) is dissolved in 2 ml of chloroform and added to a solution I of Pam 3 Cys-Ser-(Lys) 4 -OH x 3TFA (18.5 mg, 10 micromol) in 2 ml of chloroform. After addition of 4-methylmorpho- Sline (10 microliters, 10 micromol), the mixture is stirred for 1 h and the solvent is then removed in a rotary evaporator. The residue is dissolved in 10 ml of chloroform/acetone 1:1. The yellow product forms a voluminous precipitate at -20 degrees Celsius and is removed by centrifugation and dried under high vacuum.

i 22 Yield: 16 mg after purification oh Sephadex LH The product is in the form of the trifluoroacetate and fluoresces very strongly on excitation with UV Light of wavelength 766 nm. Compared with the starting material, a -amino group is covalently linked with FITC. This results in the molecular formula Pam 3 Cys-Ser-(Lys) 4

-OH-

FITC x 2TFA, assuming the zwitterionic structure is retained.

Molecular weight:

C

106

H

16 9

N

1 1 0 22

S

2

F

6 (2127.68) Thin-layer chromatography on silica gel plates: RF 0.72 (mobile phase: n-butanol/pyridine/water/glacial acetic acid 4:1:1:2) RF 0.73 (mobile phase: n-butanol/formic acid/water 7:4:2) Amino acid analysis: Ser 1.11 (1.00) Lys 4.00 (4.00) The hydrolysis products of glycerylcysteine are present.

IX. Preparation of Pam-Cys-Ser-(Lys) 4 -OH x 3HCI Pam 3 Cys-Ser-(Lys) 4 -OH Y 3 TFA (185.2 mg, 0.1 mmol) is just dissolved in a little chloroform, and approximately the same volume of ethereal HCL solution is added. The mixture is thoroughly shaken, whereupon there is some precipitation but the major part remains in solution. The mixture is evaporated to dryness in a rotary evaporator and ether/HCL is added once more. After this procedure has been repeated several times, the residue is dissolved in a little chloroform, and acetone is added until the solution becomes cLoudy. The product crystallizes as a colorless powder at -20 degrees C and is fiLtered off with suction and dried under high vacuum.

j t The membrane anchor (Pam 3 Cys and analogs, and the helices) -23 Yield: 153 mg Molecular weight:

C

8 1

H

15 9

N

10 0 13

SCL

3 (1619, 63) Elemental analysis: Calculated C 60.07 H 9.89 N 8.65 Found C 57.64 H 11.20 N 8.39 Excess HCL is still adherent to the product.

Field-desorption mass spectrometry: The M peak appears at m/e 1510, together with M and M The protonated fragments Pam 3 Cys-NH (908.5) at m/e 909, 910, 911 and 912 are characteristic.

X. N,S-Dipalmitoylcysteine tert.-butyl ester Palmitic acid (2.5 g, 9.6 mmol), dimethylaminopyridine (130 mg, 0.9 mmol) and dicyclohexylcarbodiimide (9.6 mmol) are dissolved in 100 ml of chloroform. The solution is stirred for half an hour and N-palmitoylcysteine tert.butyl ester (2 g, 4.8 mmol), which has previously been dissolved in 50 mL of chloroform, is added dropwise to the other solution. After 1 1/2 hours, the solvent is removed S.in a rotary evaporator, and the residue is taken up in 100 ml of chloroform/methanol 1:5. The product forms a voluminous precipitate at -20 degrees C. It is filtered S 'off with suction and dried under high vacuum.

Yield: 2.3 g (73%) Molecular weight: (mass spectrometer)

C

39

H

7 5 N0 4 S (655.20) Elemental analysis: Calculated: C 71.48 H 11.71 N 2.13 S 4.89 Found: C 71.72 H 12.14 N 2.12 S 4.77

LI

weight carrier and adjuvant)-(antigen) as long as it contains Lipopeptide structures with Lipid membrane anchor 24 Thin-layer chromatography on silica gel plates: RF 0.67 (mobile phase: chloroform/ethyl acetate 95:5)

R

F 0.73 (mobile phase: chLoroform/cycLohexane/MeOH 10:7:1) 13 C NMR: see Figure 4 XI. N-(a-TetradecyL-B-hydroxyoctadecanoy )cysteine tert.butyL ester N-(a-Palmitoylpalmitoyl)cysteine tert.-butyl ester (1.5 g, 2.3 mmol) is dissolved in 10 mL of i-propanol, and times the molar amount of sodium borohydride is added.

The mixture is stirred for two hours, and after completion of the reaction nitrogen-saturated 1 N hydrochloric acid is added until there is no further evolution of hydrogen.

This results in a voluminous precipitate of the product.

It is filtered off with suction, washed several times with nitrogen-saturated water and dried under high vacuum.

Yield: 1.4 g (93%) Molecular weight: (determined from the mass spectrum)

C

3 9

H

7 7 N0 4 S 656.11 Thin-layer chromatography on silica gel plates: SRF 0.84 (mobile phase: chloroform/ethyl acetate 95:5) Elemental analysis: Calculated: C 71.39 H 11.83 N 2.13 S 4.89 Found: C 71.32 H 12.39 N 2.04 S 5.33 XII. N-(a-TetradJecyl-B-hydroxyoctadecanoy )cysteine N-(a-Tetradecyl-B-hydroxyoctadecanoyL)cysteine tert.-butyl ester (1 g, 1.5 mmol) is treated with anhydrous trifluoroacetic acid for 1/2 hour, and the Latter is then removed i .j nu irN- Ly) i i- u antigenic determinant of human interferon DCH Dicyclohexylurea EE Ethylacetate in a rotary evaporator under high vacuum. The residue is dissolved in tert.-butanoL and is freeze-dried.

Yield: 0.7 g (78%) Molecular weight: (determined from the mass spectrum)

C

3 5

H

6 9 N0 4 S 600.0 Elemental analysis: Calculated: C 70.06 H 10.92 N 2.33 S 5.34 Found: C 70.36 H 10.44 N 2.45 S 5.01 Thin-layer chromatography on silica gel plates: RF 0.43 (mobile phase: chloroform/methanol/water 65:25:4) S" XIII. N,S-Dipalmitoylcysteine N,S-Dipalmitoylcysteine tert.-butyl ester (1 g, 1.5 mmol) is treated with anhydrous trifluoroacetic acid for 1 h.

The latter is then removed in a rotary evaporator under high vacuum, and the residue is taken up in tert.-butanol and freeze-dried.

Yield: 0.8 g (89%) Molecular weight: (determined from the mass spectrum) i o C 3 5

H

6 7 N0 4 S (598.00) Elemental analysis: Calculated: C 70.18 H 11.44 N 2.34 S 5.34 Found: C 69.97 H 11.31 N 2.50 S 5.17 Thin-layer chromatography: RF 0.30 (mobile phase: chloroform/methanol/glacial acetic acid 90:10:1) (But)-Asn-Leu-Leu-GLu-(OBut)-GLy-GLu(OBut)-Pro-Arg(H )-GLu- (OBut)-Phe-VaL-GLu(OBut)-Asn-Ser(But)-0-p-aLkoxybenzyl- 26 RF =0.75 (mobiLe phase: chloroform/methanol/water 65:25:4) RF 0.81 (mobiLe phase: chloroform/methanoL/ammonia 65:25:3:4) 13C

NMR:

see Fig. XIV. N-(a-PaLmitoyLpalmitoyl)-N'-paLmitoylcysteine ditert.-butyL ester PaLmitoyL chloride (8 g, 30 mmoL) is dissolved in 40 mL of nitrogen-saturated dimethyLformamide, and triethylamine (60 mL, 60 mmoL) is added. The mixture is stirred under refLux in a stream of nitrogen for three hours, during which the triethyLammonium chloride which is produced in the formation of the tetradecyLketene dimer precipitates out as a colorless salt. The reflux condenser is then replaced by a dropping funnel, and a solution of cysteine di-tert.-butyL ester (4.9 g, 15 mmoL) in 20 mL of dimethyLformamide is slowly added dropwise. After 6 hours, the solvent is removed in a rotary evaporator, and the residue is taken up in chloroform and washed twice with 100 mL of 5% strength potassium bisuLfate solution each time and once with 1,200 ml of water. The organic phase is dried over anhydrous sodium sulfate, and the solvent is removed once more. At -20 degrees Celsius a mixture of N-(a-palmitoylpaLmitoyl)-N'-palmitoyysteine tert.buty ester and N,N'-dipalmitoylcysteine di-tert.-butyL ester crystaLLizes out and the products are separated by gel f iLtration on Sephadex LH-20 in chLoroform/methanol 1:1.

Yield: 6.4 g Molecular weight: (mass spectrum)

C

6 2

H

1 1 8

N

2 0 7 S (1067.76) Melting point: 51 degrees Celsius 27 Thin-layer chromatography on silica gel plates: RF 0.69 (mobile phase: chloroform/ethyl acetate 91:5) XV. N-(a-Palmitoylpalmitoyl)cysteine tert.-butyl ester N-(a-PaLmitoyLpalmitoyl)-N'-palmitoyLcysteine di-tert.butyl ester (3.2 g, 3 mmoL) is dissolved in a Little methylene chloride, and 100 ml of 9.1 N methanolic hydrochloric acid are added. The solution is transferred into an electrolysis cell with a silver electrode as anode and mercury as cathode, and is reduced at a constant voltage of -1.1 V. The current falls from about 200 mA to almost zero at the end of the electrochemical reduction. The solvent is then removed in a rotary evaporator, and the mixture of products comprising N-(a-palmitoylpaLmitoyl)cysteine tert.-butyl ester and N-palmitoylcysteine tert.butyl ester is precipitated from methanoL at -20 degrees Celsius. These two compounds are separated by gel filtration on Sephadex LH-20 in chloroform/methanol 1:1.

Yield: g (76%) Molecular weight: (determined from the mass spectrum)

C

3 9

H

7 5 N0 4 S 654.09 Thin-layer chromatography on silica gel plates: RF 0.75 (mobile phase: chloroform/ethyl acetate 95:5) Elemental analysis: Calculated: C 71.48 H 11.71 N 2.13 S 4.89 Found: C 71.16 H 11.31 N 2.00 S 4.65 XVI. Preparation of an antigen conjugate with a conformation-stabilized a-helical membrane anchor Synthesis of HuIFN-a(Ly)(11-20)-(L-ALa-Aib-ALa-Aib-ALa) 2 OMe, a 20-peptide which has on the N-terminal end an antigenic determinant of human interferon

C

5 9

H

1 13 N0 7 S (980.6) 28 The synthesis of the lipophilic membrane anchor with a functional amino group at the end, H-(ALa-Aib-ALa-Aib- Ala) 2 -OMe, can be applied to other conjugates. The alphahelix can also be extended once or twice by the Ala-Aib- Ala-Aib-ALa unit. It is advantageous for this purpose to start from the pentapeptide Boc-Ala-Aib-L-Ala-Aib-L-Ala- OMe. Oekonomopulos, G. Jung, Liebigs Ann. Chem.

1979, 1151; H. Schmitt, W. Winter, R. Bosch, G. Jung, Liebigs Ann. Chem. 1982, 1304).

XVII. Preparation of Boc-Asn-Arg(N? )-Arg(NO?)-OH XVII.1. Boc-Arg(NO?)-OMe Boc-Arg(NO 2 )-OMe (15.97 g, 50 mmoL) and HOBt (6.67 g, mmol) in DMF (100 ml) were added at -10 degrees Celsius to HCL x H-Arg(NO 2 )-OMe (13.49 g, 50 mmol) and NMM (5.5 mL, 50 mmol) in CH 2 CL2 (12 mol) and the mixture was stirred at -10 degrees Celsius for 30 min, at 0 degrees Celsius for 1 hour and at room temperature for 3 hours. The reaction was then stopped with a few drops of glacial acetic acid. The precipitated DCU was removed by filtration, and the solvent was removed under high vacuum. The oily residue was dissolved in ethyl acetate with the addition of a little n-butanol. After the organic phase had been washed with 5% KHSO 4 solution, 5% KHCO 3 solution and saturated NaCL solution, it was dried over Na 2 S04, and petroleum ether (30-50) was added and the mixture was ,cooled to precipitate.

0 0 Y (i o o Yield: 20.30 g Melting point: 130 degrees Celsius (decomposition); Thin-layer chromatography: RF(I) 0.69, RF(II) 0.87, 29 RF(III) 0.81, RF(IV) 0.32, RF(V) 0.42.

Molecular weight determination

C

18

H

5 4

N

10 0 9 (534.5) ELemental analysis: Calculated C 40.45 H 6.41 N 26.20 Found C 40.39 H 6.55 N 26.11 XVII.2. HCL x H-Arg(N02)-Arg(NO2)-OMe Boc-Arg-(N0 2 )-Arg(NO 2 )-OMe (20.00 g, 37.42 mmol) was mixed with 1.2 N HCL/acetic acid (110 ml) and, after min, the mixture was poured into stirred ether (600 ml).

This resulted in precipitation of HCL x H-Arg(N0 2 )-Arg- (N0 2 )-OMe which was pure by thin-Layer chromatography.

YieLd: 17.3 g Thin-Layer chromatography on siLica geL pLates: RF(I) 0.37, RF(II) 0.29, RF(III) 0.44, RF(IV) 0.07, RF(V) 0.10.

XVII.3. Boc-Asn-Arg(N02)-Arg(NO 2 )-OMe Boc-Asn-OH (8.39 g, 36.10 mmoL) and HOBt (4.89 g, 36.10 mmoL) in DMF (75 ml) were added at -100C to HCL x H-Arg(N0 2 )-Arg(NO2)-OMe (17.00 g, 36.10 mmol) and NMM (3.98 mmoL) in DMF (75 mL). After addition of DCC (7.53 g, 36.50 mmol) in CH 2

CL

2 (10 ml), the mixture was stirred at -10 degrees Celsius for 30 min, at 0 degrees Celsius for 1 hour and at room temperature for 3 hours. After the reaction had been stopped with a few drops of glacial t 30 acetic acid, the solvent was removed by evaporation in vacuo, and the residue was taken up in a Little methanol.

This solution was added dropwise to stirred dry ether.

The residue was removed by filtration and taken up in methanoL. The pure product precipitated out in the cold.

Yield: 18.25 g (78%) Melting point: 170 degrees Celsius Thin-layer chromatography RF(I) 0.59, RF(II) 0.67, S RF(III) 0.66, RF(IV) 0.45, RF(V) 0.65.

Amino acid analysis: Asx 1.00 Arg 1.85 (2) SMolecular weight determination:

C

22

H

4 0

N

12 0 1 1 (648.6) S o 20 Elemental analysis: Calculated C 40.74 H 6.22 N 25.91 Found: C 40.70 H 6.40 N 25.79 S..o XVII.4. Boc-Asn-Arg(NO2)-Arg(N02)-OH Boc-Asn-Arg(N0 2 )-Arg(NO2)-OMe (18.00 g, 27.75 mmol) in methanol (180 mL) was hydroLyzed with 1 N NaOH (80 ml) at room temperature. After 2 h, the mixture was neutralized with dilute HCL, and the methanoL was removed by evaporatioh in vacuo. Exhaustive extraction with ethyl acetate was carried out at pH 3. The organic phases were washed with a little saturated NaCL solution, dried over Na 2

SO

4 and the product was crystallized from a methanolic solu- 31 tion at -20 degrees Celsius.

Yield: 15.84 g Melting point: 228 degrees Celsius (decomposition) Thin-layer chromatography RF(I) 0.49, RF(II) 0.21 RF(III) 0.26 RF(IV) 0.05, RF(V) 0.21.

XVIII. Boc-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib- Ala)?-OMe XVIII.1 Boc-Ala-Aib-Ala-Aib-Ala-OH Boc-Ala-Aib-Ala-Aib-Ala-OMe (10.03 g, 20 mmol) in MeOH (150 ml) was hydrolyzed with 1 N NaOH (40 ml, 40 mmol).

After 2.5 hours, the mixture was neutralized with 1 N HCL, evaporated in vacuo and partitioned between EA/5% KHCO 3 S(1:1; 1,000 ml). The aqueous phase was acidified to pH 4 with 5% KHSO 4 and was extracted five times with EA/1- Sbutanol The organic phase was dried over Na 2

SO

4 PE (30-50) was added, and the pentapeptide acid was precipitated in the cold.

,Yield: 6.54 g Melting point: 195 degrees Celsius (decomposition) Thin-layer chromatography RF(I) 0.72, RF(II) 0.80, 32 RF(III) 0.87, RF(IV) 0.95, RF(V) 0.80.

Amino acid analysis: Ala 3.08 Aib 1.98 (2) Molecular weight:

C

2 2

H

3 9

N

5 0 8 (501.6) Elemental analysis: Calculated C 52.68 H 7.84 N 13.96 Found C 52.70 H 7.90 N 13.89 XVIII.2. Boc-(ALa-Aib-Ala-Aib-Ala) 2 -OMe Boc-ALa-Aib-ALa-Aib-Ala-OH (1.75 g, 3.48 mmol) and HOBt (470 mg, 3.48 mmol) in DMF (10 mL) were added at degrees Celsius to HCL x H-ALa-Aib-Ala-Aib-Ala-OMe (1.57 g, 3.48 mmoL) and NMM (384 pl, 3.48 mmol) in DMF (8 ml).

After addition of DCC (825 mg, 4.00 mmol) in CH 2

CI

2 (3 ml) at -10 degrees Celsius, the mixture was stirred for 15 h allowing it slowly to warm up spontaneously. After the reaction had been stopped with a few drops of glacial acetic acid, the DCU which had precipitated out was removed by centrifugation, the residue was washed twice with cold DMF, and the solvent was removed by evaporation in vacuo. The residue was taken up in 10 ml of CHCL 3 /MeOH S 1:1 and chromatographed on Sephadex LH 20 in CHCL 3 /MeOH 1:1.

0 0 J 25 Yield: 2.246 g Melting point: 160 degrees Celsius, Thin-layer chromatography RF(I) 0.61, RF(II) 0.76, 33 RF(III) 0.83, RF(IV) 0.95, RF(V) 0.81.

Molecular weight determination:

C

40

H

70

N

10 0 13 (899.1) Elemental anaLysis: Calculated C 53.44 H 7.85 N 15.58 Found C 53.42 H 7.90 N 15.40 XVIII.3. HCL x H-(ALa-Aib-ALa-Aib-ALa) 2 -OMe Boc-(ALa-Aib-ALa-Aib-Aa) 2 -OMe (2.046 g, 2.276 mmoL) was mixed with 1.2 N HCL/AcOH (10 mL). After stirring for min, the hydrochloride was precipitated with ether, filtered off and dried over KOH under oil pump vacuum.

YieLd: 1.805 g Thin-Layer chromatography: RF(I) 0.50, RF(II) 0.38, RF(III) 0.71, RF(IV) 0.48, RF(V) 0.53.

XVIII.4. Boc-GLn-(ALa-Aib-ALa-Aib-ALa) 2 -OMe g 0 2 Boc-GLn-OH (997 mg, 4.05 mmoL) and HOBt (547 mg, 4.05 mmoL) in DMF (10 ml) were added at -10 degrees Celsius to HCL x H-(ALa-Aib-Ala-Aib-ALa) 2 -OMe (2.250 g, 2.70 mmoL) and NMM (298 p 1 2.70 mmoL) in DMF (13 mL). After addition of DCC (846 mg, 4.10 mmoL) in CH 2

CL

2 (2 mt) at degrees CeLsius, the mixture was stirred for 15 h aLLowing it sLowLy to warm up spontaneously. After the reaction had been stopped with a few drops of gLaciaL acetic acid, the precipitated DCU was removed by centri- 34 fugation, the residue was washed twice with a Little coLd DMF, and the solvent was removed under oil pump vacuum.

The residue was taken up in 10 mL of CHCI 3 /MeOH 1:1 and chromatographed on Sephadex LH 20 in CHCL 3 /MeOH Yield: 2.60 g (94%) Melting point 223 degrees Celsius (decomposition) Thin-layer chromatography: RF(I) 0.66, RF(II) 0.73, RFIII) 0.79, RF(IV) 0.94, RF(V) 0.80.

Molecular weight determination:

C

4 5

H

7 8

N

12 0 15 (1027.2) Elemental analysis: Calculated C 52.62 H 7.65 N 16.36 Found C 52.65 H 7.68 N 16.32 XVIII.5. HCL x H-Gln-(Ala-Aib-ALa-Aib-ALa) 2 -OMe Boc-GLn-(ALa-Aib-Ala-Aib-ALa) 2 -OMe (2.60 g, 2.701 mmoL) was mixed with 1.2 N HCL/AcOH (15 ml). After 40 min, the hydrochloride was precipitated with ether while stirring, removed by filtration and dried over KOH under oil pump vacuum.

Yield: 2.209 g Thin-layer chromatography: RF(I) 0.48, RF(II) 0.25, 35 RF(III) 0.54, RF(IV) 0.24, RF(V) 0.35.

XVIII.6. Boc-ALa-Leu-lle-Leu-ALa-Gln-(ALa-Aib-ALa-Aib- ALa)2-OMe Boc-Ala-Leu-ILe-Leu-Leu-Ala-OH (760 my, 1.07 mmol) and HOBt (145 mg, 1.07 mmol) in DMF (12 mL) were added at room temperature to HCL x H-GLn-(ALa-Aib-ALa-Aib-ALa) 2 -OMe (818 mg, 0.85 mmoL) and NMM (94 rl, U.85 mmoL) in DMF (10 mL). After addition of DCC (227 mg, 1.10 mmol) in

CH

2

CI

2 (1.5 mL), the mixture was stirred for 64 hours.

After the reaction had been stopped with a few drops of glacial acetic acid, the precipitated DCU was removed by centrifugation, the residue was washed twice with a little cold DMF, and the solvent was removed under oil pump vacuum. The residue was taken up in 8 mt of CHCL 3 /MeOH and chromatographed on Sephadex LH-20 in CHCL 3 /MeOH Yield: 774 mg Melting point: 260 degrees Celsius (decomposition); Thin-layer chromatography: RF(I) 0.80, RF(II) 0.86, RF(III) 0.91, RF(IV) 0.77 RF(V) 0.78.

Amino acid analysis: GLx 1.00 ILe 0.89 Leu 3.10 Aib 4.08 Ala 7.95 with suction and dried under high vacuum.

-36 MoLecular weight determination

C

75

H

132

N

18 0 2 1 (1622.0) Elemental analysis Calculated C 55.54. H 8.20 N 15.54 Found C 55.58 H 8.31 N 15.52 XIX. Preparation of Boc-Asn-Arg(N0 2 )-Arg(NO 2 )-AL a-Leu- ILe-Leu-ALa-GLn-(ALa-Aib-ALa-Aib-ALa) 2 -OMe XIX. 1. HCL x H-ALa-Leu-ILe-Leu-Leu-ALa-GLn-(ALa-Aib-ALa- A ib-Ala) 2 -OMe Boc-ALa-Leu-I Le-Leu-Leu-ALa-GLn-(AI a-Aib-ALa 1 ib-A a )2-OMe (754 mg, 0.465 mmoL) was mixed with 1.2 N HCI/AcOH (10 ml).

After 50 min, the mixture was partly evaporated under oil pump vacuum and, after addition of water (10 ml), freeze- Y ie Ld: 690 mg Thin-Layer chromatography: R F(I 1 0.71, RF(II) =0.52, RF(III) 0.78, R F (I V) 0.56,.

R F( V) 0.54.

XIX.2. Bos-Asn-Arg(N02 )-Arg(N02 )-ALa-Leu-I Le-Leu-Leu-AI a- G~n-CALa-Aib-ALa-Aib-ALa )-Me Boc-AMn-Arg(N0 2 )-Arg(NO 2 )-OH (634 mg, 0.995 mmoL) and HO~t (135 mg, 1.13 mmoL) in DMF (5 ml) were added at -5 degrees Celsius to HCI x H-ALa-Leu-ILe-Leu-Leu-ALa-GLn-(ALa-Aib- ALa-Aib-ALa) 2 -OMe (20 mg, 0.398 mmoL) and NMM (44 micro- Liters, 400 micromoLe) in DMF (7 ml). After addition of DCC (217 mg, 1.05 mmoL) in CH 2

CI

2 (1.5 ml) at -5 degrees Celsius, the mixture was stirred for 48 h allowing it to CaLculated: C 71.48 H 11.(1 N ?.13 S 4.89 Found: C 71.72 H 12.14 N 2.12 S 4.77 37 warm up spontaneously. After the reaction had been stopped with 3 drops of glacial acetic acid, the precipitated DCU was removed by centrifugation. The working up and purification by chromatography were carried out as described previously.

YieLd: 630 mg Melting point: 195 degrees Celsius (decomposition); Thin-layer chromatography: RF(I) 0.70, RF(II) 0.51, RF(III) 0.56, RF(IV) 0.45, RF(V) 0.68.

Amino acid analysis: Asx 0.94 GLx 1.00 Ite 0.89 Leu 3.16 Arg 1.95 MolecJLar weight determination: 120 C 9 1

H

160

N

30 0 29 (2135.5) Elemental analysis: Calculated C 51.11 H 7.54 N 19.65 Found C 51.14 H 7.60 N 19.66 1' a XX. Preparation of the free eicosapeptide XX.1. Boc-Asn-Arg-Arg-ALa-Leu-Ile-Leu-Leu-Ala-GLn-(Ala- Aib-ALa-Aib-Aa) 2 -OMe x 2HCI Boc-Asn-Arg(NO2)-Arg(NO2)-ALa-Leu-ILe-Leu-Leu-ALa-GLn- (Ala-Aib-ALa-Aib-Aa) 2 -OMe (350 mg, 0.164 mmoL) in 3 mL of anhydrous methanol was mixed with 35 mg of Pd/active charcoal and 12 vL (0.075 mmol) of 6 N HCL. Hydrogen was ester (1 g, 1.5 mmol) is treated with anhydrous trifluoroacetic acid fnr 1/2 hour, and the Latter is then removed -38 passed through the solution while stirring at room temperature. After 20 min 8 pi (49 micromoLe), and after 35 min 7 pl (42 micromoLe), of 6 N HCL were added. After a hydrogenation time of about 50 min the cleavage off, as checked by TLC, was quantitative. The catalyst was removed by filtration and washed several times with a little methanol. The solvent was rapidly removed by distillation in a rotary evaporator (oil pump vacuum, bath temperature 25 degrees Celsius), and the residue was taken up in a little water and freeze-dried.

Yield: 332 mg Thin-Layer chromatography: RF(I) 0.16, RF(II) 0.11, RF(III) 0.21, RF(IV) 0.10.

XX.2. H-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-Ala-GLn-(Ala- Aib-Ala-Aib-ALa) 2 -OMe x 3HCI Boc-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-ALa-GLn-(ALa-Aib-Ala- Aib-ALa) 2 -OMe x 2HCL (600 mg, 0.283 mmol) was mixed with 1,2 N HCI/AcOH (5 ml). After 30 min, the mixture was partly evaporated in a rotary evaporator, and the residue was mixed with water (10 ml) and freeze-dried.

j 25 Yield: 564 mg Melting point: 245 degrees Celsius (decomposition) Thin-layer chromatography: RF(I) 0.11 r, c 90:10:1) 39 Molecular weight determination:

C

86

H

157

N

2 8 0 2 3

CL

3 (2057.7) ELementaL anaLysis: Calculated Found C 50.20 C 50.31 H 7.69 H 7.78 N 19.06 N 18.95 CL 5.17 CL 5.28 Materials and methods for the experiments Chemicals Analytical grade solvents were obtained from Merck, while other solvents were dried and distilled by customary methods. N-MethyLmorpholine (Merck) was distilled over ninhydrin to remove sec. amines. 1-Hydroxybenzotriazole and dicycLohexylcarbodiimide likewise originated from Merck. ALL L-amino acid derivatives were obtained from Bachem. Boc-Aib-OH and H-Aib-OMe x HCL were synthesized by Literature methods.

Thin-layer chromatography Precoated silica gel 60 F 2 54 plates (supplied by Merck) and the following mobile phases were used: 1-Butanol/glaciaL acetic acid/water 3:1:1 (II) Chloroform/methanol/glacial acetic acid/water 65:25:3:4 (III) Chloroform/methanol/concentrated ammonia/water 65:35:3:4 (IV) Chloroform/methanol/water 65:25:4 Chloroform/methanol 1:1 The following spray reagents were used: ninhydrin reagent, chlorine/4,4'-bis(dimethyLamino)diphenyLmethane (TDM reagent) and Sakaguchi reagent. The reference used was dicycLohexylurea with the following values: RF(I) 0.91, RF(II) 0.82, RF(III) 0.92, RF(IV) 0.81, RF(V) 0.83.

Molecular weight: (mass spectrum)

C

6 2

H

11 8

N

2 0 7 S (1067.76) 40 Amino acid analyses To establish the identity of the intermediates approximateLy 200 microgram samples of each of the protected peptides were hydrolyzed in 6 N HCL at 110 degrees Celsius for 24 hours. The intermediates and the target sequence of the hexapeptide segment which contains the Leu-Leu bond were hydrolyzed for 72 hours under conditions which were otherwise identical. The amino acid analyses were carried out with a Biotronic LC 6000 E amino acid analyzer using the standard program.

Racemate determination The hydrolyzed amino acids were derivatized as the npropyl esters of the pentafluoropropionylamino acid and the enantiomers were separated by gas chromatography on glass capillary columns with Chirasil-Val. The reported percentages of D-amino acids have not been corrected for racemization caused by the hydrolysis.

Elemental analyses Single C, H and N-determinations were carried out using a model 1104 (Carlo Erba, Milan) elemental analyzer.

Melting points Melting points were determined according to Tottoli and are uncorrected.

Recording of the spectra 13 C NMR spectra: 30 mg of the protected eicosapeptide were dissolved in 400 microliters of 12

C

2

HCL

3 12

C

2

H

3 0 2

H

(supplied by Merck) and measured in a WM 400 Bruker NMR spectrometer at 30 0 C for 12 h. Circular dichroism spectra: solutions of the free eicosapeptide (c 1-1.7 mg/ ml) in ethanol, trifluoroethanol, methanol, 1,1,1,3,3,3hexafluoro-2-propanol, water and ethanol/water mixtures were measured in a Dichrograph II (Jouhan-Roussel).

Purification by chromatography The protected peptide intermediates were, after termina- L_ genic determinant of human interferon 41 tion of the coupling reaction and removal of the solvent under oil pump vacuum, dissoLved'by addition of the same volume of CHCI 3 /MeOH 1:1, the dicycLohexyLurea was removed by centrifugation, and the product was chromatographed on Sephadex LH 20: column 3 x 115 cm; eluting agent CHCL 3 /MeOH 1:1; amount applied 35 ml; flow rate 8.40 ml/10 min. The 3 ml fractions were examined by thinlayer chromatography in system II (TDM reagent). The peptides appeared in the elution volume 165-190 ml. The fractions were combined, the solvent was removed in vacuo, and the residue was dried over P 2 0 5 Amino acid analysis produced the expected values and a peptide content of 92-96%.

Immunization tests We have for the first time covalently linked a B-cell mitogen, which is simultaneously an outstanding carrier and a highly active adjuvant, to synthetic antigenic determinants. For this we have used, inter alia, the synthetic Lipopeptide S-(2,3-bis(palmitoyloxy)propyl)-N-paLmitoylcysteinylserine (Pam 3 Cys-Ser) which represents the N-terminal end of the lipoprotein from the outer membrane of Escherichia coli. The amphiphilic properties, which are particularly pronounced when covalently bonded to an antigen, ensure, on the one hand, stable anchoring of the S-gLyceryl compound, which carries three fatty acid residues, in the lipid layer of the cell membrane. On the other hand, this means that the antigen (or hapten), which is usually more polar, is presented in the outer hydrophilic layer of the membrane. Since the activating effect of the Lipoprotein is determined entirely by its Nterminal part, the immunostimulant effect of Pam 3 Cys-Ser, or analogs, is retained in aLL the conjugates which carry it.

As an example, we detail the use of the concept for the generation of specific antibodies against epidermal growth factor receptor (EGF-R) Fig. 9. For this purpose, a computer-assisted search for epitopes Led to selection of RF(I) U.6Y, RF(II) 0.87, 42 the extracytoplasmic region 516-529, which was constructed by Merrifield synthesis and finally Fmoc-Ser(But)-OH and then Pam 3 Cys'-OH were attached. The conjugate, which was found to be homogeneous by analysis, was cleaved off from the resin and then administered without further additives, in a single dose to mice. After only 2 weeks high titers of specific antibodies against the tetradecapeptide were found by ELISAs. An essential point is that no antibody titers were obtained with the tetradecapeptide, which is by itself obviously a weak immunogen, in control experiments.

Since Pam 3 Cys conjugates are Likewise highly immunogenic in cell cultures, it is possible in a rapid and elegant manner to obtain conventional and monoclonal antibodies, even against weakly immunogenic compounds, by in vitro immunization.

The advantages of our concept in association with cell cultures are: straightforward preparation of a chemicaLly unambiguously defined antigen-adjuvant conjugate in any desired amount, in contrast to other conjugates a single administration without multiple "boosters", and high efficiency in vivo and in vitro. The considerable saving in experimental animals, and frequently even dispensing completely with in vivo immunization and a drastic saving in Time, especially in genetic engineering procedures, are obvious. The experiments can also be carried out with Shuman cell culture systems.

Example of an in vivo immunization: 6- to 10-week old Balb/c mice were immunized by a single i.p. administration of 50 micrograms and 500 micrograms (0.2 ml of a 10 to 10 2 molar solution of adjuvant covalently coupled to antigen) of Pam 3 Cys-Ser-(EGF-R 515-529).

The controls used were antigen, adjuvant and a mixture of antigen and adjuvant, in each case in comparable molar amounts, and medium. Two weeks after the injection, blood was taken from the retroorbital venous plexus of the mice reaction had been stopped with a few drops of glacial 43 to obtain serum, and the antibody titer was determined by

ELISA.

Analogous immunizations can also be obtained by other administrations, for example oral, rectal, i.m.

and s.c.

The formation of specific antibodies without Freund's adjuvant against the tetradecapeptide EGF-R 516-529 after in vivo immunization was examined.

Balb/c mice were immunized once i.p. with 0.2 micromol of the conjugate I. Conjugate Pam 3 Cys-Ser (EGF-R 516-526) II. Free tetradecapeptide EGF-R 516-529 alone III. Pam 3 Cys-Ser alone IV. Free tetradecapeptide (EGF-R 516-529) mixed together with Pam 3 Cys-Ser as shown in Fig. 10. The antibody titer was determined by ELISA. (Ordinate OD at 405 nm) (Fig. 14 days after the immunization the mice were bled from the ophthalmic vein and the serum which was obtained was used in ELISA. The values emerge from the mean (3-5 mice) of the difference between the ELISA values of PEP 14 BSA conjugate and BSA (Fig. It is evident that only when the membrane anchor/active compound conjugate according to the invention is used are drastically elevated antibody concentrations, which exceed the activity of those with previous processes by a multiple, found.

Example of an in vitro immunization Samples of mouse spleen cells were cultivated for 5 days in the presence of the conjugate Pam 3 Cys-Ser-(EGF-R 515-529), of the adjuvant Pam 3 Cys-Ser, of the tetradecaand the product was crystaLLized from a methanoLic solu- 44 peptide EGF-R 516-529, of a mixture of the antigen and adjuvant, and of medium.

The lymphocytes were cultivated at a cell density of x 10 6 /ml in 0.2 mL aLiquots in RPMI-1640 medium enriched with 10% heat-inactivated FCS, gLutamine (2 mM), penicillin (100 U/ml), streptomycin (100 pg/ml) and 2- -5 mercaptoethanoL (5 x 10 for 48 h.

The supernatants were obtained for examination for specific antibodies by ELISA.

Mitogenic activation of mouse spleen cells The mitogenic activation of Balb/c spleen cells by Pam 3 Cys-Ser-(Lys) 4 -FITC (circles), Pam 3 Cys-Ser-(Lys) 4 -OH x 3HCL (triangles) and Pam3Cys-Ser-(Lys) 4 -OH x 2 TFA (squares) is shown in Fig. 12. The cell cultivation conditions have been described Immunforsch. 153, 1977, pp. 11 et seq.

and Eur. J. Biochem. 115, 1981). In the figure, the stimulation index for the incorporation of H-thymidine into the DNA (cpm for incorporation/cpm for the control without mitogen) is plotted as the ordinate against the concentration of active compound used.

In vivo/in vitro comparison In Fig. 11 the in vivo experiment detailed above is compared with an in vitro experiment: In vitro experiment in microtiter plates: cell density: S 25 2.5 x 106 cells/ml; substance concentration: 5 x 10 7 millimolar; incubation conditions: 370C, 5% C0 2 5 days.

Conj.: conjugate Pam 3 Cys-Ser-(EGF-R 515-529) Pep tetradecapeptide EGF-R 516-529 Adj. Pam 3 Cys-Ser Mix mixture of free tetradecapeptide EGF-R 516-529 and Pam 3 Cys-Ser.

The drastic rise in the antibody concentration also emerges in vitro, and this considerably extends the utilizability c RF(II) 0.80, 45 of ceLL cultures, in particuLar for the preparation of antibodies.

Claims (15)

1. A membrane anchor/active compound conjugate containing at least one membrane anchor compound and at least one active compound covalently bonded to the membrane anchor compound(s) wherein the membrane anchor compound is a compound of the following formulae: R -CO-O-CH R O-CH 2 R O-CO-CH 2 I 1 I R'-CO-O-CH* R'-O-CH Rf- O-CO-CH kCH 2 (OHi )n k(CH 2 ).n A AA 2 (GEL kCH 2) R"-CO-NH-CH -CO-X R"l-OO-NH-CH -OO-X R"-OO-NHI-CH -CO-X R-NH-CO-CH 2 I -CO-NH-CH 2 RI -NH-CO-OH RF-CO-NH-CH (CH 2 (CH 2 B A AS (CH 2 (OHki 2 ~m (CH 2 n R' '-OO-NH-CH -OO-X R' '-CO-NH-OH -CO-X R-NH-CO-CH -CO-X IV. V. VI. R 2 -OH (O 2 )n (O 2 ~m R-OO-NH-OH -CO-X VII. RF(II) 0.25, 47 it being possible for A to be sulfur, oxygen, disulfide methylene (-CH 2 or -NH-; n being 0 to 5; m being 1 or 2; C* being an asymmetric carbon atom with R or S configuration; R, R' and R" being identical or different and being an alkyl, alkenyl or alkynyl group having 7 to 25 carbon atoms or hydrogen, which can optionally be substituted by hydroxyl, amino, oxo, acyl, alkyl or cycloalkyl groups, and R I and R 2 being identical or different and being defined as R, R' and R" or possibly being -OR, -OCOR, -COOR, -NHCOR or -CONHR, X being an active compound or a spacer-active compound group, and B can be any one of the substituted S-alkyl radicals defined in relation to structural formulate I to V.

2. A membrane anchor/active compound conjugate as claimed in claim 1, which has the following formula: R 4 R 3 NH CH CO X VIII. R being an alpha-acyl -fatty acid residue having between 7 and 25 carbon atoms; preferably between 10 and 20 carbon atoms and very particularly preferably having between 14 and 18 carbon atoms; an alpha-alkyl-beta-hydroxy-fatty acid residue or its beta-hydroxy ester, the ester group being preferably straight-chain or branched chain and having more than 8 carbon atoms, preferably between about 10 and 20 and very particularly preferably between 14 and 18 carbon atoms; it is possible and peferable for formula VIII to be an Sactive compound conjugate with the following membrane anchor compounds: N,N'-diacyllysine; N,N'-diacylornithine; di(monoalkyl)amide or ester of aspartic acid, N,O-diacyl 48 derivative of serine, homoserine or threonine and N,S-diacyl derivatives of cysteine or homocysteine; serine and homoserine; R 4 being a side chain of an amino acid or hydrogen; and X being hydrogen or a spacer-active compound group, it being possible when R is a side chain of lysine, ornithine, glutamic acid, aspartic acid or their derivatives for the latter to be bonded, both in the manner of an ester and in the manner of an amide in the same molecule, in alpha or omega positions to R

3. A membrane anchor/active compound conjugate as claimed in claim 1, wherein the membrane anchor compound is Pam 3 -Cys or Pam 3 Cys-Ser or a Pam Cys-peptide having 1 to amino acids wherein Pam 3 Cys is a residue of the following formula: CO- S I CH 2 -CH-CH CH -CH-CH O 0 NH I I C=0 C=0 O=C

4. A membrane anchor/active compound conjugate as claimed in claim 1, wherein the membrane anchor compound is Pam-Cys(Pam)-OH or Pam(a-Pam)-Cys[Pam(a-Pam)]-OH. I. l DCC (217 mg, 1.05 mmol) in CH 2 CL 2 (1.5 mL) at -5 degrees Celsius, the mixture was stirred for 48 h allowing it to 49 A membrane anchor/active compound conjugate as claimed in claim 1, wherein the membrane anchor compound is an alpha-alkyl-B-hydroxyacyl-peptide (mycolylpeptide), the peptide chain having between one and 10 amino acids.

6. A membrane anchor/active compound conjugate as claimed in claim 1, wherein the membrane anchor compound is S-(1,2-dioctadecyloxycarbonylethyl)cysteine or a homolog of this compound.

7. A membrane anchor/active compound conjugate as claimed in claim 1, wherein the membrane anchor compound is a helix which is conformationally stabilised by alpha-alkyl-amino acids and has alternating amino acids, such as of the X-(Ala-Aib-Ala-Aib-Ala)n-Y type, n being 5 to 4; X and Y being protective groups which are known per se, S-H, -OH or -NH2, and it being possible for Ala also to be replaced by other amino acids.

8. A membrane anchor/active compound conjugate as claimed in one of the preceding claims, wherein the active compound is covalently linked to two, optionally different, membrane anchor compounds.

9. A membrane anchor/active compound conjugate as Su° 2 claimed in one of the preceding claims, wherein the active compound is additionally covalently linked to an adjuvant which is known per se for immunization purposes, for example muramyldipeptide and/or to a lipopolysaccharide. A membrane anchor/active compound conjugate as claimed in one of the preceding claims, wherein the active compound is an antigen such as, for example, a low molecular weight partial sequence of a protein or conjugated protein, for example of a glycoprotein, of a viral coat protein, of a OT annyouous metannoL was mixed with 35 rg of Pd/active charcoal and 12 vl (0.075 mmol) of 6 N HCL. Hydrogen was 50 bacterial cell wall protein or of a protein of protozoa tigenic determinant, epitope), constituents of bacterial membranes, such as muramyldipeptide, lipopolysaccharide, a natural or synthetic hapten, an antibiotic, a hormone, a nucleoside, a nucleotide, a nucleic acid, an enzyme, an enzyme substrate, an enzyme inhibitor, biotin, avidin, polyethylene glycol, a peptidic active compounds such as, for example, tuftsin, polylysine, a fluorescence marker FITC, RITC, dansyl, luminol or coumarin, bioluminescence markers, a spin label, an alkaloid, steroid, biogenic amine, vitamin or a toxin such as, for example, digoxin, phalloidin, amanitin, tetrodoxin or the like, a complex-forming agent or a drug. O Sc" 11. A membrane anchor/active compound conjugate as claimed in one of the preceding claims, wherein several membrane anchor/active compound conjugate compounds are crosslinked together in the lipid part and/or active S compound part. S12. A membrane anchor/active compound conjugate as claimed in one of the preceding claims, wherein the membrane anchor compound and the active compound are covalently bonded together via a crosslinker, for example via a dicarboxylic acid derivative, diols, diamines, polyethylene glycol, epoxides, maleic acid derivatives or the like. o So 13. A process for the preparation of membrane anchor compounds as claimed in one of the preceding claims, which o comprises synthesis of the peptide, which is protected with protective groups in a manner known per se on the functional S groups at which no reaction is to take place, by means of known coupling processes on a solid or soluble carrier, such as a polymer (for example Merrifield resin); covalent bonding of the carrier-bound peptides, which have been 51 synthesized in this way, via N-termini or side-groups of the peptide to the membrane anchor compound; isolation of the peptide conjugate, which has been prepared in this way, by cleavage of the protective groups and the peptide/carrier bond in a manner known per se, and thus the membrane anchor peptide or the membrane anchor/active compound conjugate being obtained.

14. The process as claimed in claim 13, wherein the peptide/membrane anchor linkage is set up by condensation, addition, substitution or oxidation, for example dirulfide formation. The process as claimed in one of claims 13 or 14, wherein the membrane anchor compounds are those of claims 1 to 7, in particular Pam 3 Cys, Pam 3 Cys-Ser, S-[1,2-dicar- boxyalkylethyl]cySteine, X-(Ala-Aib-Ala-Aib-Ala)n-Y, n being 2 to 4 and X and Y being peptide protective groups which are known per se or -OH or -NH 2 Pam(a-Pam)-peptide, a mycolylpeptide or its derivative, or Pam-Cys(Pam), Pam-Lys(Pam), Pam-Orn(Pam), Pam-Ser(Pam) or the di(hexadecyl-amide) of glutamic acid.

16. The use of the compounds as claimed in one of claims 1 to 12 for the preparation of conventional and monoclonal antibodies in vivo and in vitro. oo 17. The use of the compounds as claimed in one of claims 1 to 12 in genetic engineering to facilitate cell fusion.

18. The use of the compounds as claimed in one of claims 1 to 12 for the preparation of synthetic vaccines. RF(1) 0.91, RF(II) 0.82, RF(III) 0.92, RF(IV) 0.81, RF(V) 0.83. 52

19. The use of the compounds as claimed in one of claims 1 to 12 for the preparation of cell markers with fluorescence labels, spin labels, radioactive labels or the like. The use of the compounds as claimed in one of claims 1 to 12 for affinity chromatography, in particular for affinity columns.

21. The use of the compounds as claimed in one of claims 1 to 12 for liposome preparations.

22. The use of the compounds as claimed in one of claims 1 to 12 for purposes in human medicine and/or veterinary medicine, where appropriate together with vehicles which are know per se, in solution, in ointments, adsorbed on to solid carriers, in emulsions or sprays.

23. The use of the compounds as claimed in one of claims 1 to 12 for addition to human foodstuffs or animal feeds, and for addition to culture media for microorganisms and generally for cell cultures. DATED this 10th day of December 1990. HOECHST AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN, VICTORIA 3122 AUSTRALIA DBM/JMW/CH (2.26) '47 44 .7 L r C