NO174207B - Analogous Process for the Preparation of Therapeutically Active Conjugate of Diaphragm Anchor - 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

The present invention relates to an analogous method for the production of a therapeutically active conjugate of membrane-anchoring active substance, where an active substance is covalently bound to a membrane-anchoring compound.

Membrane anchor compounds are compounds that can be introduced into biological and artificial membranes.

For example, such membrane anchor compounds can be natural membrane lipoproteins, as they have already been isolated from the outer membrane of Escherichia coli and in the meantime also synthesized. The E. coli membrane anchor compound consists in the N-terminal region of three fatty acids bound to S-glyceryl-L-cysteine (G. Jung et al. in "Peptides, Structure and Function", V.J. Hruby and D.H. Rich, p 179-182, Pierce Chem. Co. Rockford, Illinois, 1983).

In model-based investigations of biological membranes, conformationally stabilized alpha-helix polypeptides have also already been described, see e.g. alameticin, an alpha-helical amphiphilic eicosapeptide antibiotic, which forms voltage-dependent ion conducting systems in lipid membranes (Boheim, G., Hanke W., Jung G., Biphys. Struct. Mech. 9, pp. 181-191 (1983); Schmitt, E. and Jung, G., Liebig's Ann. Chem., pp. 321 to 344 and 345 to 364 (1985).

In EP-A1-330, it is described that lipopeptides, which are the analogues of the 1-lipoproteins from E. coli known since 1973, have an immunopotentiating effect. A further European patent publication, EP-A2-114787, relates to the ability of such lipopeptides to activate rat and mouse alveolar macrophages in vitro, so that after 24 hours of incubation with the substance these can eliminate the tumor cells and, in particular, significantly increase antibody production, e.g. e.g. against porcine serum albumin.

In EP-A2-114787 it is proposed to use these lipoprotein descendants as adjuvants in the immunization, i.e. to use the lipoprotein descendants of the E. coli membrane protein in mixture with antigens to improve the immune response.

There is a great need for substances that stimulate and enhance the immune response, especially as purified genes are often only present in very small quantities; furthermore, when new portions of antigens are used, there is always the possibility of new contaminants, respectively decomposition products.

Furthermore, there is a desire not to have to vaccinate an experimental animal often, but, on the other hand, preferably by a single supply of the immunogenic material to be able to achieve the desired immune response.

The present invention is therefore based on the task of increasing antibody formation against antigens or haptens in order to achieve a specific immunopotentiating effect.

According to the present invention, a therapeutically active conjugate of membrane-anchor-active substance is produced, where the membrane-anchor compound is a compound of the following general formula:

wherein A can be sulfur, oxygen, disulfide (-S-S-), methylene (-CH2-) or -NH-;

n = 0-5; m = 1 or 2; C<*> = asymmetric carbon atom with R— or S configuration; R, R', R" are the same or different and represent an alkyl, alkenyl or alkynyl group with 7-25

carbon atoms or hydrogen, which may optionally be substituted with hydroxy groups and X is an active substance or a spacing active substance group.

According to the invention, the therapeutically active conjugate of membrane anchor-active substance is produced by a method which includes that the protected peptide, which is protected with protective groups in a manner known per se at the functions where no reaction is to take place, is synthesized using known methods coupling methods to a solid or soluble support, such as a polymer, (e.g. Merrifield resin), in such a way that the synthesized, support-bound peptide is covalently bound to the membrane-anchor compound via the N-termini or side functions of the peptide ; so that the prepared peptide conjugate can be isolated by cleaving the protective groups and the bond peptide/carrier in a manner known per se, so that the membrane anchor peptide or the conjugate of membrane anchor active substance is recovered.

The compounds can be used for the production of conventional and monoclonal antibodies in vitro; preferably, the compounds produced according to the invention can also be used within genetic technology to facilitate cell fusion, for the production of synthetic vaccines, for the production of cell markers with fluorescent labels, "spin" labels, radioactive labels, etc., for affinity chromatography, especially for affinity columns; for liposome preparations; as an additive to foodstuffs or precursors in human and animal nutrition, as well as as an additive to culture media for microorganisms and in general for cell cultures. Hereby, the compounds produced according to the invention can optionally be used together with per se known carriers in solution, such as ointments, absorbed into solid carriers, in emulsions or sprays for human or veterinary medicinal purposes.

The connection peptide/membrane anchor connection can be produced by condensation, addition, substitution, oxidation (for example disulphide formation). Preferably, conformation-stabilizing alpha-alkyl amino acid helices with an alternating amino acid sequence can be used as membrane anchors, where the alpha helix must not be destabilized by other amino acids, such as of the type X-(Ala-Aib-Ala-Aib-Ala)n-Y, where n = 2 respectively 4, and X, Y are per se known protecting groups or -H, -OH or -NH2.

It can be advantageous for the active substance to be covalently bound to two possibly different membrane anchor compounds.

In addition, the active substance can also be covalently bound to an adjuvant known per se for immunization purposes, such as e.g. muramyl peptide and/or a lipopolysaccharide.

Proposed as active substances are, for example: an antigen, such as a low molecular weight partial sequence of a protein or proteid, for example a glycoprotein, a viral envelope protein, a bacterial cell wall protein or proteins of protozoa (antigenic determinants, epitope), an intact protein, an antibiotic , bacterial membrane constituents, such as muramyl dipeptide, lipopolysaccharide, a natural or synthetic hapten, an antibiotic, hormones, such as steroids, a nucleoside, a nucleotide, a nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, blotin, avidin, polyethylene glycol, a peptide active substance, such as tuftsin, polylysine, a fluorescence marker (e.g. FITC, RITC, dancyl, luminol or coumarin), a bioluminescence marker, a "spin" label, an alkanoid, a steroid, biogenic amine, vitamin or also a toxin such as e.g. digoxin, phalloidin, amanthine, tetrodoxin etc., a complexing agent or a pharmacon.

Depending on the type of active substance, completely new areas of application arise for the substances produced according to the invention.

It can also be useful when several membrane anchor-active substance conjugate compounds are cross-linked with each other in the lipid and/or active substance part.

The membrane anchor connections and the active substance can also be bound to each other via a cross-linker, this has the effect that the active substance is removed more from the membrane to which it is attached with the membrane anchor. Suitable crosslinkers are, for example, a dicarboxylic acid or a dicarboxylic acid derivative, diols, diamines, polyethylene glycol, epoxides, maleic acid derivatives, etc.

According to the invention, an unambiguously defined low molecular weight conjugate is produced which, among other things, suitable for immunization, which covalently connects the carrier-antigen-adjuvant principles with each other. Carrier and adjuvant can both be a mitogenically active lipopeptide, such as e.g. tripalmitoyl-S-glycerylcysteine (Pam3Cys) and analogues thereof, but also lipophilic, conformation-stabilizing alpha-helices as well as combinations of such, such as Pam3Cys-antigen-helix, alpha-helix-antigen-helix, or also just Pam3Cys-antigen or antigen-Pam3Cys (N - or C-terminal connection), as well as antigen-helix or helix-antigen (N- or C-terminal), or by side chains of Glu, Lys, etc. built into the helix). Thus, the new compounds differ in important respects from all hitherto used high-molecular conjugates of antigens with high-molecular carriers, for example proteins such as serum albumins, globulins or polylysine or generally high-molecular, linear or cross-linked polymers.

In particular, however, the new compounds also differ from all hitherto known adjuvants, which are optionally added and consequently do not enable any targeted presentation of the antigen on the cell surface. The hitherto known adjuvants often require several immunizations and also lead to inflammatory reactions in animal experiments. A particular advantage of the invention is the reproducibility of pyrogen-free, pure, chemically unambiguously defined compounds, which - in contrast to conventional compounds or mixtures of different substances - also leads to improved reproducibility for antibody formation. Thus, a special area of application for the compounds produced according to the invention is the area linked to antibody extraction, genetic engineering, the production of synthetic vaccines, diagnostics and therapy within veterinary and human medicine, as the new conjugates primarily have a specific stimulating effect on the immune response, while they the adjuvants used so far have often had a non-specific stimulating effect on the immune response. Surprisingly, with the help of the compounds produced according to the invention, weakly immunogenic compounds can also be converted into highly immunogenic compounds. Thus, a special significance of the invention lies in the possibility of saving on animal experiments as well as costs for the production of the antibodies, as the new immunogens are also highly active in vitro. Due to the non-inflammatory immunization procedure, an animal can also be used several times for different antibody extractions.

Finally, with the new immunogens, multivalent vaccination substances can also be produced, i.e. for example a membrane anchor to whose side chain several antigens or haptens are attached, so that with the help of an immunization, several different active antibodies can be produced.

A water-soluble, mitogenic lipid anchor group is, for example, PamsCys-Ser(Lys)n-OH, which is particularly suitable for the production of the new immunogens, but also for the production of fluorescent, radioactive and biologically active cell markers. A particularly desired property of the membrane anchor-active substance conjugates produced according to the invention is their amphiphilicity, i.e. a partial water solubility, as biological experiments on animals and investigations with living cells can thereby be carried out significantly more easily. Also the artificial lipid bilayer membranes, liposomes and vesicles that are required for many experiments are also only ready-made and stable in an aqueous medium.

A suitable amphiphilic, biologically active membrane anchor is e.g. Pam3Cys-Ser(Lys)n-OH. The serine residue linked to Pan^Cys promotes immunogenic properties, while the polar, protonated epsilon amino groups of the lysine residue constitute the hydrophilic part of the molecule. Because of the many charges, this type of compound exhibits other interesting properties. By inducing cell interaction, it can be used as a fusion activator in the production of hybridoma cells, especially with longer lysine chains, by coupling to polyethylene glycol or by covalent incorporation of the biotin/avidin system.

Furthermore, the compounds produced according to the invention can advantageously be used for the production of new liposomes by cross-linking, which can either take place in the fatty acid or in the peptide part.

The membrane anchor (Pam3Cys and analogues, as well as the helices) is also suitable for strengthening the cell/cell interaction, when, for example, they are covalently combined with the biotin/avidin system. Further advantageous properties of the compounds produced according to the invention are that they can facilitate cell fusion, this is, for example, required for genetic engineering work. In this way, the new Immunogens can also be used in ELISA, RIA and Bioluminescence analyses.

Various Pam3Cys derivatives are lipid- and water-soluble, and are highly mitogenically active in vivo and in vitro. They are also very suitable for labeling cells with FITC and other markers such as RITC, dansyl and cumarln. In particular, they can also be used in fluorescence microscopy and "Fluorescence Activated Cell Sorting" (FACS).

A cost-effective membrane anchor with an analogous effect to Pam3Cys is S-(1,2-dioctadecyloxycarbonylmethyl)-cysteine, the preparation of which is described in detail in the experimental section.

Specific links of the mitogenically active lipid anchor to antigens can also take place via crosslinkers, such as for example with the dicarboxylic acid monohydrazine derivatives of the general formula: or also

in which A, B = amino acid or (CH2)n and X, Y are per se known protecting groups.

However, any other crosslinker or spacer can also be used for the production of the new substances, whereby the principle (low molecular weight carrier and adjuvant) (antigen) constitutes a particularly preferred embodiment of the invention, as long as they contain lipopeptide structures with a lipid membrane anchor function and/or conformationally stabilized helices.

Special beneficial effects can be found by using the compounds produced according to the invention within affinity chromatography, for this purpose lipopeptide-antigen (hapten) conjugates are particularly suitable. These are excellently suited for coating ordinary reversed-phase HPLC columns (or also preparative RP columns), whereby, for example, a tripalmitoyl compound applied in organic aqueous systems is fixed in the apolar alkyl layer. The antigen is presented e.g. on the cell surfaces of the mobile aqueous phase and thus invites the antibodies to adsorb. Thus, antibodies that react specifically with the relevant antigen can be enriched or isolated directly on such an affinity column. The elution of the antibodies takes place as with other affinity columns, e.g. when setting the pH value.

In the following, the invention will be described in more detail by means of examples, whereby first the abbreviations used will be reviewed:

Aib = 2-methyl-alanine

TFE = trifluoroacetic acid

EGF R = receptor for epidermal growth factor

Pam = palmitoyl residue

DCC = dicyclohexylcarbodiimide

DMF = dimethylformamide

FITC = fluorescein isothiocyanate

Fmoc = fluorenylmethoxycarbonyl

But = tert.butyl residue

PS-DVB = styrene - divinylbenzene copolymers with 4-(hydroxymethyl)phenoxymethyl anchor groups

HOBt = 1-hydroxybenzotriazole

RITC = rhodamin isothiocyanate

Hu IFN-(Ly) 11-20 = antigenic determinants of human

interferon

DCH = N,N'-dicyclohexylurea

EE = ethyl acetate

The attached figures, which will serve to illustrate the invention, show: Fig. 1 - production scheme for Pam-Cys(C^g)2-Ser-Ser-Asn-Ala-OH

Fig. 2 - the tables for the <13>C-NMR spectra

Fig. 3 - <13>C-NMR spectrum of Pam3Cys-Ser-(Lys)4-OH x 3

TFA in CDC13

Fig. 4 - <13>NMR spectrum for Pam-CysCPanO-OBu<t> in CDCl3 Fig. 5 - <13>C-NMR spectrum for Pam-Cys(Pam)-OH in

CDCl3/CD3OD 1:1

Fig. 6 - <13>C-NMR spectrum (J-modulated spin-echo spectrum) for Pam(alpha-Pam)Cys-OBu<t>

Fig. 7 - <13>C-NMR (100 MHz) for alpha helix

Fig. 8 - the CD spectrum of α-helix of HuIFN-(alpha-Ly)-ll-20 Fig. 9 - the antibody extraction with Pam3Cys-Ser-EGF-R (516

to 529)

Fig. 10 - an in vivo immunization experiment

Fig. 11 - a comparison of in vivo/in vitro immunization ring experiment, and Fig. 12 - the mitogenic activation of Balb/c mouse spleen cells with Pam3Cys-Ser-(Lys^FITC.

In what follows, some manufacturing methods for substances as mentioned above, as well as their precursors, will first be described.

I. Preparation of Pam3Cys-EGF-R(516-529)

After usual stepwise construction (Merrifield synthesis under N alpha-Fmoc/(Bu<t>) protection, DCC/EOBt and symmetrical anhydrides of the EGF-R segment (516-529) the amino acid Fmoc-Ser(Bu< t>)-OH was added. After cleavage of the Fmoc group with piperidine/DMF (1:1, 15 min.), the resin-bound pentadecapeptide of EGF-RE:Ser(Bu<*>)-Asn-Leu-Leu- Glu- ( OBu* ) -Gly-Glu( OBu* )-Pro-Arg(H+)-Glu(OBu*)-Phe-Val-Glu(0But )-Asn-Ser-(Bu* )-0-p- Alkoxy-benzyl-copoly(dlvinyl-benzene-styrene) (1 g, coating 0.5 mmol/g) coupled with Pam-Cys (CH2-CH(0Pam(CE2(0Pam) (2 mmol, in DMF/CE2Cl2 ( 1:1)) and DCC/EOBt (2 mmol, preactivated at 0"C, 20 minutes) (16 hours), then a back-coupling (4 hours) was carried out. The lipohexadecapeptide was cleaved with trifluoroacetic acid (5 ml) with the addition of thioanisole (0.25 ml) over 2 hours.

Dividend:

960 mg = (76*) Pam.Cys (CH2-CH(0PAM)CH2(0Pam))Ser-Asn-Leu-Leu-Glu-Gly-Glu-Pro-Arg-Glu-Phe-Val-Glu-Asn- Ser-OH X CF3C00H (correct amino acid analysis, no racemization).

II. Preparation of S-(12,2-dioctadecyloxycarbonylethyl-N-palmitoyl-L(or D)cysteine tert-butyl ester

Maleic acid di-octadecyl ester is available according to the general procedure for the esterification of maleic acid (H. 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 maleic acid di-octadecyl ester are dissolved in 20 ml of THF. After adding 20 mmol (3 ml) of N,N,N',N'-tetramethylethylenediamine, it is stirred under nitrogen at a reflux condenser for 12 hours. After adding 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 vacuum over P2n5.

Yield: 1 g (83*).

Melting point: 51°C.

Thin layer chromatography:

Rf = 0.80 (eluent; CHCl 3 /acetic ester = 14.1).

13C-NMR: See fig. 2.

Molecular weight: C63H121N07S (1035.7).

III. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-N-palmitoyl-cysteine

0.48 mmol (500 mg) of the t-butyl ester mentioned under II is stirred in 65.3 mmol (7.45 g, 5 ml) of trifluoroacetic acid for 1 hour at room temperature in a closed container. It is evaporated on a rotary evaporator in high vacuum, the residue is taken up in 1 ml of chloroform, precipitated with 50 ml of petroleum ether at -20°C and dried in vacuum over P2°5•

Yield: 420 mg (89*).

Melting point: 64°C.

Thin layer chromatography on silica gel plates:

R f = 0.73; eluent; CHCl3/MeOH/H2O 65:25:4)

<13>C-NMR: See Table 1.

Molecular weight: C5gH113N07S (980.6)

Elemental analysis:

The new cysteine derivative and its t-butyl ester can be separated into the diastereoisomers on silica gel and by RP-phase chromatography. Both pairs of diastereomers of the L- and D-cysteine derivative can be prepared.

IV. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-N-palmitoyl-Cys-SertBu*)-Ser(But J-Asn-Ala-OBu*

0.2 mmol (196 mg) S-(1,2-dioctadecyloxycarbonylethyl-N-palmitoyl-cysteine) is dissolved in 5 ml dichloromethane and preactivated with 0.2 mmol (27 mg) HOBt in 0.5 ml DMF and 0.2 mmol (41 mg) DCC for 30 minutes at 0° C. with stirring After addition of 0.2 mmol (109 mg) H-Ser(But )-Ser (Bu* J-Asn-Ala-OBu* In 3 ml of dichloromethane, stir it for 12 hours at room temperature.Without further work-up, 40 ml of methanol are added

the reaction mixture. After 3 hours, a colorless product can be sucked off. It is taken up in a little dichloromethane and reprecipitated with methanol. After washing with methanol, dry in vacuum over <P>2°5-

Yield: 260 mg (86*).

Melting point: 194°C.

Thin layer chromatography:

Rf - 0.95; (eluent: CHCl3/MeOH/H20 = 65:25:4)

R f = 0.70; (eluent: CHCl3/MeOH/glacial vinegar = 90:10:1)

13C-NMR: See fig. 2

Molecular weight: Cq^ E^58N6°14S (1508.3)

Elemental analysis:

V. Preparation of S-(1,2-dioctadecyloxycarbonylethyl)-N-palmitoyl-Cys-Ser-Ser-Asn-Ala

53 pmol (80 mg) of protected lipopeptide (IV) is stirred with 13 mmol (1.5 g; 1 ml) of trifluoroacetic acid for 1 hour at room temperature in a closed container. After evaporation in high vacuum, the mixture is taken up twice with 10 ml of dichloromethane and it is evaporated on a rotary evaporator each time. The residue is taken up in 3 ml of chloroform and precipitated with 5 ml of methanol at 4°C over the course of 12 hours. After extraction, wash with methanol and dry in a desiccator at P2°5'

Yield: 64 mg (87*)

Melting point: 208°C (decomposition)

Thin layer chromatography:

R f = 0.63; (eluent: CHCl3/MeOH/glacial acetic acid/H20 64:25:3:4)

R f = 0.55; (eluent: CHCl3/MeOH/H20 = 64:25:4)

R f = 0.06; (eluent: CHCl3/MeOH/glacial acetic acid = 90:10:1).

Amino acid analysis:

Cysteine 0.6; aspartic acid 0.93; serine 1.8; alanine 1.0.

Molecular weight: C72<H>134<N>6014S (1340)

IV. Preparation of Pam3Cys-Ser-(Lys)4~OH Pam3Cys-Ser(Lys)4-OH was built up according to the solid-phase method (Merrifield) on a p-Alkoxybenzyl alcohol-PS-DVB(15) copolymer with N-Fmoc- amino acids and acid labile side chain protection (tBu for serine and Boe for lysine). The symmetrical anhydrides of the Fmoc amino acids were used. The coupling to Pam3Cys-OH was carried out according to the DCC/HOBt procedure and was repeated to achieve the most quantitative conversion possible. To cleave the lipopeptide from the carrier resin and to remove the side chain protection, the resin was treated twice for 1.5 hours with trifluoroacetic acid, which was then removed on a rotary evaporator 1 high vacuum. The product was recrystallized from acetone.

The elemental analysis indicates, in the same way as the <13>C spectrum, that the lipopeptide is present as trifluoroacetate. Under the assumption that Pam3Cys-Ser-(Lys)4-OH exists as a double ion, there are still 3 epsilon-amino groups left over, these can be protonated by three trifluoroacetic acid molecules.

The <13>C-NMR spectrum for Pam3-Cys-Ser-(Lys)4-OH x 3 TFE shows that the compound exists as trifluoroacetate. (Quartet for the CF3~ group at 110-120 ppm, as well as carbonyl signals at 161-162 ppm). When the polar molecular part is aggregated, the lines for the Lys and Ser-C atoms are strongly propagated. The carbonyl signal at 206.9 originates from acetone still present which was used for the recrystallization.

Molecular weight: 1510.4

Elemental analysis:

Amino acid analysis: The amino acid analysis gave a ratio between serine and lysine of 1:4.2. The characteristic decomposition products of S-glyceryl-cysteine resulting from the hydrolysis (6N HCl, 110°C, 18 hours) were present (comparison with known standards). The peptide proportion was calculated to be 83*. 3 TFE molecules per lipopeptide corresponds to a peptide proportion of 80.2*, in good agreement with the analysis.

VII. Preparation of Pam3Cys-Ser-(Lys>4-0H x 3 TFE).

VII. l. Coupling of Fmoc-Lvs(Boc)-OH to the support resin Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol) is mixed in 15-20 ml of DMF/CH2Cl2 1:1 (volume/volume) at 0'C with DCC (0.99 g, 4.8 mmol). After 30 minutes, the precipitated urea is filtered directly into a shaking funnel in which there is p-benzyloxy-benzyl alcohol resin (2.5 g, 1.6 mmol OH groups). After addition of pyridine (0.39 ml, 4.8 mmol), it is shaken for 18 hours at room temperature. The solvent is sucked off and the resin is washed 3 times, each time with 20 ml of DMF/CH 2 Cl 2 and DMF. The resin is mixed in 20 ml of CH 2 Cl 2 first with pyridine (28.8 mmol, 6 equivalents) and then with benzoyl chloride (28.8 mmol, 6 equivalents). It is shaken for 1 hour at room temperature. The solvent is sucked off and the resin is washed 3 times, each time with 20 ml of CH 2 Cl 2 , DMF, isopropanol and PE-30/50.

VII. 2. Symmetrical Fmoc amino acid anhydride

Fmoc-Lys(Boc)-OH (4.5 g, 9.6 mmol, 3 equivalents) is dissolved in 15 mL of CH 2 Cl 2 /DMF and mixed at CC with DCC (4.8 mmol, 1.5 equivalents). After 30 minutes at 0°C, the urea is filtered directly into the reactor and it is processed further as indicated in the following table.

The following procedure applies to 1/5 of the initially used amount of resin (0.5 g, 0.3 mmol OH groups).

Fmoc-O-butyl-serine (0.74 g, 1.92 mmol) is dissolved in 4 mL of CH 2 Cl 2 /D]VIF and mixed at 0°C with DCC (0.96 mmol).

After 30 minutes, the urea is filtered off directly in the reactor at 0°C, and processed in the usual way.

VII.3. Link to Pam3Cys-0H

Pam3Cys-OH (0.58 g, 0.64 mmol) is dissolved in 5 mL of CH2Cl2/DMF 1:1 (v/v) and mixed at 0°C with HOBt (0.93 mg, 0.64 mmol) and DCC (0.64 mmol). After 30 minutes at 0°C, the mixture is charged directly into the reactor. After 16 hours of shaking, the reaction is followed for 4 hours with the molar ratio indicated above. The solvent is sucked off and the resin is washed 3 times, each time with 20 ml of DMF/CH2C12 and DMF.

VI1. 4. Cleavage of the hexapeptide from the polymer

The Boc-protected peptide-polymer resin compound (ca. 1 g) from VII.3 is washed well with CH 2 Cl 2 and shaken for 2 x 1.5 hours with a mixture of 5 ml TFE and 0.5 ml anisole. The filtrate is evaporated in vacuo and the residue taken up in 5 ml of CHCl3. Pam3Cys-Ser-(Lys)4-0H x 3 TFE is crystallized after the addition of 50 ml of acetone at -20°C, it is centrifuged off and dried in a high vacuum.

Yield: 0.41 g (85*)

Melting point: 205"C (decomposition)

Thin layer chromatography on silica gel plates:

R f = 0.42; (eluent: n-BuOH/pyridine/H 2 O/glacial acetic acid = 4:1:1:2).

R f = 0.82; (eluent: n-BuOH/MeOH/H2O/acetic acid = 10:4:10:6).

Amino acid analysis:

Looks 0.95 (1); Light 4 (4).

Molecular Weight: CgyHjLs<gN>jgOigSFg (1852.6).

Elementary analysis: VIII. Preparation of Pam3Cys-Ser-(Lys)4-OH-FITC x 2 TFE Fluorescein isothiocyanate (3.9 mg, 10 jjmol) is dissolved in 2 ml of chloroform and added to a solution of Pam3Cys-Ser-(Lys)4-OH x 3 TFE (18.5 mg, 10 µmol) in 2 mL chloroform. After addition of 4-methylmorpholine (10 µl, 10 pmol) stir for 1 hour 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 precipitates voluminously at -20°C and is centrifuged off and dried in high vacuum.

Dividend:

16 mg after purification on "Sephadex LH20".

The product is available as trifluoroacetate and fluoresces very strongly when activated with UV light of wavelength 366 nm. Compared to the starting product, an epsilon amino group is covalently bound to FITC. This results in the sum formula Pam3Cys-Ser-(Lys)4-OH-FITC x 2 TFE, provided that the doubly ionic structure is preserved.

Molecular weight: C106<H>16g<N>11<0>22S2F6 (2127.68)

Chromatography on silica gel plates:

Rf = 0.72 (eluent: n-butanol/pyridine/water/glacial vinegar = 4:1:1:2)

Rf = 0.73 (eluent: n-butanol/formic acid/water = 7:4:2).

Amino acid analysis:

Looks 1.11 (1.00), Lights 4.00 (4.00)

The hydrolysis products of glycerylcysteine are present.

IX. Preparation of Pam3Cys-Ser-(Lys )4-OH x 3 HC1 Pani3Cys-Ser-(Lys )4-OH x 3 TFE (185.2 mg, 0.1 mmol) is just dissolved in a little chloroform, and approximately the same volume of ethereal HCl solution is added. It is shaken well, whereby a part is precipitated, the largest part, however, remains in solution. It is evaporated to dryness on a rotary evaporator and ether/HCl is added again. After repeating this procedure several times, the residue is dissolved in a little chloroform and acetone is added to the solution until cloudiness. The product crystallizes out as a colorless powder at -20"C and is sucked off and dried in a high vacuum.

Yield: 153 mg.

Molecular weight: C18H15g<N>1o<O>i3SCl3 (1619.63)

Elemental analysis:

There is still excess HC1 in the product.

Field desorption mass spectrometry:

The M<+->peak appears at m/e 1510, as well as M<+>+l and M<+>+2. Characteristic are the protonated fragments Pam3Cys-NH (908.5) at m/e 909, 910, 911 and 912.

X. N,S-dipalmitoyl-cysteine tert-butyl ester

Palmitic acid (2.5 g, 9.6 mmol), dimethylaminopyridine (130 ml, 0.9 mmol) and dicyclohexylcarbodiimide (9.6 mmol) are dissolved in 100 ml of chloroform. The solution is stirred for 1/2 hour and N-palmitoyl-cysteine tert-butyl ester (2 g, 4.8 mmol), which has previously been dissolved in 50 ml of chloroform, is added dropwise to the second solution. After 1.5 hours, the solvent is removed on a rotary evaporator, and the residue is taken up in 100 ml of chloroform/methanol 1:5. The product precipitates voluminously at -20°C. It is suctioned off and dried in a high vacuum.

Yield: 2.3 g (73*).

Molecular weight (mass spectrometry): C3gH75N04S (655.20)

Elemental analysis:

Thin layer chromatography on silica gel plates:

Rf = 0.67 (eluent: chloroform/acetic ester 95:5).

Rf = 0.73 (eluent: chloroform/cyclohexane/MeOH 10:7:1).

13C-NMR: See fig. 4.

XI. N-(α-tetradecyl-p<->hydroxy-octadecanoyl)-cysteine tert-butyl ester

N-(α-palmitoyl-palmitoyl)-cysteine tert-butyl ester (1.5 g, 2.3 mmol) is dissolved in 10 ml of i-propanol and 1.5 times the molar amount of sodium borohydride is added. It is stirred for 2 hours, and after the reaction has finished, nitrogen-saturated N hydrochloric acid is added until no more hydrogen evolution takes place. Thereby, the product is voluminously precipitated. It is suctioned off, washed several times with nitrogen-saturated water and dried in a high vacuum.

Yield: 1.4 g (93*).

Molecular weight: (determined from mass spectrum): C39H77NO4S = 656.11

Thin layer chromatography on silica gel plates:

Rf = 0.84 (eluent: chloroform/acetic ester 95:5).

Elemental analysis:

XII. N-(α-tetradecyl-p<->nydroxy-octadecanoyl)-cysteine

N-(cx-tet radecyl-3-hydroxy-octadecanoyl)-cysteine tert-butyl ester (1 g, 1.5 mmol) is treated for 1/2 hour with anhydrous trifluoroacetic acid and this is then removed in high vacuum on a rotary evaporator. The residue is dissolved in 1 tert.butanol and lyophilized.

Yield: 0.7 g (78*).

Molecular weight: (determined from mass spectrum) C35H69NO4S (600.0)

Elemental analysis:

Thin layer chromatography on silica gel plates:

Rf = 0.43 (eluent: chloroform/methanol/water 65:25:4)

XIII. N,S-dipalmitoyl-cysteine

N,S-dipalmitoyl-cysteine tert-butyl ester (1 g, 1.5 mmol) is treated for 1 hour with anhydrous trifluoroacetic acid. This is then removed on a rotary evaporator in high vacuum, the remainder is taken up in tert.butanol and lyophilized.

Yield: 0.8 g (89*).

Molecular weight: (determined from mass spectrum) C35H57NO4S (598.00)

Elemental analysis:

Thin layer chromatography:

Rf = 0.30 (eluent: chloroform/methanol/glacial acetic acid 90:10:4 )

Rf = 0.75 (eluent: chloroform/methanol/water 65:24:4) Rf = 0.81 (eluent: chloroform/methanol/ammonia (25*)/water 65:25:3:4)

13C-NMR: See fig. 5.

XIV. N-(α-palmitoyl-palmitoyl)-N'-palmitoyl-cysteine-di-tert-butyl ester

Palmitoyl chloride (8 g, 30 mmol) is dissolved in 140 ml of nitrogen-saturated dimethylformamide and triethylamine (60 ml, 60 mmol) is added. The mixture is boiled for 3 hours in a stream of nitrogen under reflux, whereby the triethylammonium chloride which occurs during the formation of the tetradecylketene dimer is precipitated 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 added slowly and dropwise. After 6 hours, the solvent is removed on a rotary evaporator, the residue is taken up in chloroform and washed twice with 100 ml of 5* potassium hydrogen sulphate solution and once with 1200 ml of water. The organic phase is dried over anhydrous sodium sulfate and the solvent is removed again. At -20°C, a mixture of N-(α-palmitoyl-palmitoyl)-N'-palmitoyl-cysteine-tert-butyl ester and N,N'-dipalmitoyl-cysteine-di-tert-butyl ester crystallizes out, which is separated by gel filtration on "Sephadex LH-20" in chloroform/methanol 1:1.

Yield: 6.4 g (40*).

Molecular weight: (mass spectrum)

Thin layer chromatography on silica gel plates:

Rf = 0.69 (eluent: chloroform/acetic ester (91:5)).

XV. N-(α-palmitoyl-palmitoyl)-cysteine tert-butyl ester N-(α-palmitoyl-palmitoyl)-N'-palmitoyl-cysteine di-tert-butyl ether (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 in an electrolysis cell with a silver electrode as anode and mercury as cathode, and is reduced with a constant voltage of -1.1V. The current decreases at the end of the electrochemical reduction from approx. 200 mA to approximately 0. The solvent is then removed on a rotary evaporator and the product mixture of N-(α-palmitoyl-palmitoyl)-cysteine tert-butyl ester and N-palmitoyl-cysteine tert-butyl ester is precipitated from methanol at -20°C. The compounds are separated by gel filtration on "Sephadex LH-20" in chloroform/methanol 1:1.

Yield: 1.5 g (76*).

Molecular weight: (determined by mass spectrum) €39117511048 (654 .09).

Thin layer chromatography on silica gel plates:

Rf = 0.75 (eluent: chloroform/acetic ester 95:5).

Elemental analysis:

XVI. Preparation of an antigen conjugate with a conformationally stabilized α-helical membrane anchor

Synthesis of HuIFN-α(Ly)(ll-20)-(L-Ala-Aib-Ala-Aib-Ala)2-OMe, a 20-peptide bearing N-terminally an antigenic determinant from human interferon (ot(LY )).

The synthesis of the lipophilic membrane anchor with functional amino head group H-(Ala-Aib-Ala-Aib-Ala)2-0Me can be transferred to other conjugates. The alpha helix can also be extended once or twice with the unit Ala-Aib-Ala-Aib-Ala. The starting point here is preferably from the pentapeptide Boc-Ala-Aib-L-Ala-Aib-L-Ala-OMe. (R. 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(NO2)-Arg(NO2)-OH

XVII.l. Boc-Arg(NO2)-OHMe

Boc-Arg(NO 2 )-OHMe (15.97 g, 50 mmol) and EOBt (6.67 g, 50 mmol) in DMF (100 mL) were stirred at -10 °C in HCl x H.Arg(NO 2 ) -0Me (13.49 g, 50 mmol) and NMM (5.5 mL, 50 mmol) in CH 2 Cl 2 (12 mL) for 30 min at -10 °C, 1 h at 0 °C, and 3 h at room temperature. The precipitated DCH was filtered off and the solvent was evaporated under high vacuum. The oily residue was dissolved in glacial acetic acid while adding some n-butanol. After washing with 5* KHSO4, 5* KECO3 and saturated NaCl solution, the organic phase was dried over Na2SO4, mixed with petroleum ether (30-50) and precipitated while cooling.

Yield: 20.30 g (76*).

Melting point: 130°C (decomposition).

Thin layer chromatography:

Rf (I) = 0.69

R f (II) = 0.87

R f (III) = 0.81

R f (IV) = 0.32

Rf (V) = 0.42.

Molecular weight: C18H54<N>10°9 (534.5)

Elemental analysis:

XVII.2. EC1 x H-Arg(NO 2 )-Arg(NO 2 )-OMe

Boc-Arg-(NO 2 )-Arg(NO 2 )-OMe (20.00 g, 37.42 mmol) was mixed with 1.2 N HCl/acetic acid (110 mL) and after 30 min the whole in stirred ether (600 mL ). Thin-layer chromatographically pure HC1 x H-Arg(NO2)-Arg(NO2)-0Me was thereby precipitated.

Yield: 17.3 g (98*).

Thin layer chromatography on silica gel plates:

Rf (I) = 0.37

R f (II) = 0.29

R f (III) = 0.44

Rf (IV) = 0.07

Rf (V) = 0.10.

XVII.3. Boc-Asn-Arg(NO2)-Arg(NO2)-OMe

Boc-Asn-OE (8.39 g, 36.10 mmol) and EOBt (4.89 g, 36.10 mmol) in DMF (75 mL) were added at -10°C to EC1 x E-Arg(NO2 )-Arg(NO 2 )-OMe (1.700 g, 36.10 mmol) and NMM (3.98 mmol) in DMF (75 mL). After addition of DCC (7.53 g, 36.50 mmol) in CE 2 Cl 2 (10 ml) it was stirred for 30 minutes at -10°C, 1 hour at CC and 3 hours at room temperature. After interrupting the reaction with a few drops of glacial acetic acid, the solvent is evaporated in vacuo and the residue is taken up in a little methanol. This solution is added dropwise with stirring to dry ether. The residue is filtered off and taken up in methanol. Pure product precipitates during cooling.

Yield: 18.25 g (78*)

Melting point: 170°C

Thin layer chromatography:

Rf (I) = 0.59

R f (II) = 0.67

R f (III) = 0.66

R f (IV) = 0.45

Rf (V) = 0.65

Amino acid analysis:

Asx 1.00 (1), Arg 1.85 (2).

Molecular weight determination: C22E4o<N>i2On (648.6)

Elemental analysis:

XVII.4. Boc-Asn-Arg(NO2)-Arg(NO2)-OE

Boc-Asn-Arg(NO 2 )-Arg(NO 2 )-OMe (18.00 g, 27.75 mmol) was saponified in methanol (180 mL) with 1N NaOE (80 mL). After 2 hours it was neutralized with dilute EC1 and the methanol was evaporated in vacuo. At pH 3, it was extracted thoroughly with acetic acid. The organic phase was washed with slightly saturated NaCl solution, dried over Na 2 SO 4 and crystallized from a methanolic solution at -20°C.

Yield: 15.84 g (90*)

Melting point: 228"C (decomposition)

Thin layer chromatography:

Rf (I) = 0.49

R f (II) = 0.21

Rf (III) - 0.26

Rf (IV) = 0.05

R f (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) was saponified in MeOH (150 mL) with 1N NaOH (40 mL, 40 mmol). After 2.5 hours it was neutralized with 1N HCl, evaporated in vacuo and partitioned between EE/5* KHCO 3 (1:1; 1000 mL). The aqueous phase was acidified with 5* KHSO 4 to pH 4 and extracted 5 times with EE/1-butanol (5:1). The organic phase was dried over Na 2 SC 4 , mixed with PE (30-50) and the pentapeptide acid was precipitated under cooling.

Yield: 6.54 g (65*)

Melting point: 195<0>C (decomposition)

Thin layer chromatography:

R f (I) = 0.72

R f (II) = 0.80

R f (III) = 0.87

Rf (IV) = 0.95

Rf (V) = 0.80

Amino acid analysis:

Ala 3.08 (3), Aib 1.98 (2).

Molecular Weight: 022<2>39^<0>3 (501.6)

Elemental analysis:

XVIII.2. Boc-(Ala-Aib-Ala-Aib-Ala)2-OMe

Boc-Ala-Alb-Ala-Aib-Ala-OH (1.75 g, 3.48 mmol) and HOBt (470 mg, 3.48 mmol) in DMF (10 mL) at -10 °C was added to HCl x H-Ala-Aib-ala-Aib-Ala-OMe (1.57 g, 3.48 mmol) and NMM (384 µl, 3.48 mmol) in DMF (8 mL). After addition of DCC (825 mg, 4.00 mmol) in CH 2 Cl 2 (3 mL) at -ICC, it was slowly stirred under independent heating for 15 h. After the reaction was stopped with a few drops of glacial acetic acid, the precipitated DCH was centrifuged off, the residue was washed 2 times with cold DMF and the solvent was evaporated in vacuo. The residue was taken up in 10 ml of CHCl3/MeOH 1:1 and chromatographed on "Sephadex LH 20" in CHCl3/MeOH 1:1.

Yield: 2.246 g (72*)

Melting point: 160°C

Thin layer chromatography:

Rf (I) = 0.61

R f (II) = 0.76

R f (III ) = 0.83

Rf (IV) = 0.95

R f (V) = 0.81

Molecular weight determination: C40H70N10<O>13 (899.1)

Elemental analysis:

XVIII .3. HCl x H-(Ala-Alb-Ala-Ail)-Ala)2-0Me

Boc-Ala-Aib-ala-Aib-Ala2-OHMe (2.046 g, 2.276 mmol) was mixed with 1.2 N ECl/AcOH (10 mL). After stirring for 30 minutes, the hydrochloride was precipitated with ether, filtered off and dried over KOH in an oil pump vacuum.

Yield: 1.805 g (95*)

Thin layer chromatography:

Rf (I) = 0.50

R f (II) = 0.38

R f (III) = 0.71

R f (IV) = 0.48

Rf (V) = 0.53

XVIII.4. Boc-Gln-(Ala-Aib-Ala-Aib-Ala)2-OMe

Boc-Gln-OH (997 mg, 4.05 mmol) and HOBt (547 mg, 4.05 mmol) in DMF (10 mL) were added at -10°C to HCl x H-(Ala-Aib-Ala- Aib-Ala)2-OMe (2.250 g, 2.70 mmol) and NMM (298 µl, 2.70 mmol) in DMF (13 mL). After addition of DCC (846 mg, 4.10 mmol) in CH 2 Cl 2 (2 ml) at -10°C, it was stirred under slow independent heating for 15 hours. After interrupting the reaction with a few drops of glacial acetic acid, the precipitated DCH was centrifuged off, the residue was washed 2 times with a little cold DMF and the solvent was removed in an oil pump vacuum. The residue was taken up in 10 ml CHCl3/MeOH 1:1 and chromatographed on "Sephadex LH 20" in CHCl3/MeOE (1:1).

Yield: 2.60 (94*)

Melting point: 223°C (decomposition)

Thin layer chromatography:

Rf (I) = 0.66

R f (II) = 0.73

R f (III) = 0.79

Rf (IV) - 0.94

Rf (V) = 0.80.

Molecular weight determination: C45H7g<N>i2°i5 (1027.2)

Elemental analysis:

XVIII.5. HCl x H-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me

Boc-Gln-(Ala-Aib-Ala-Aib-Ala)2-OMe (2.60 g, 2.701 mmol) was dried with 1.2 N HCl/AcOH (15 mL). After 40 min, the hydrochloride was precipitated with ether with stirring, filtered off and dried in oil pump vacuum over K0H.

Yield: 2.209 g (85*)

Thin layer chromatograph in:

R f (I) = 0.48

R f (II) = 0.25

R f (III) = 0.54

R f (IV) = 0.24

Rf (V) = 0.35

XVIII.6. Boc-Ala-Leu-Ile-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-OMe

Boc-Ala-Leu-Ile-Leu-Leu-Ala-OH (760 mg, 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 µl, 0.85 mmol) in DMF (10 mL). After addition of DCC (227 mg, 1.10 mmol) in CH 2 Cl 2 (1.5 mL) it was stirred for 64 h. After interrupting the reaction with a few drops of ice water, the precipitated DCH was centrifuged off, the residue was washed 2 times with a little cold DMF and the solvent was removed in an oil pump vacuum. The residue was taken up in 8 ml of CHCl3/MeOH (1:1) and chromatographed on "Sephadex LH 20" CHCl3/MeOH (1:1).

Yield: 774 mg (57*)

Melting point: 260°C (decomposition)

Thin layer chromatography:

Rf (I) = 0.80

R f (II) = 0.86

R f (III) = 0.91

R f (IV) = 0.77

Rf (V) = 0.78

Amino acid analysis:

Glx 1.00 (1), Ile 0.89 (1), Leu 3.10 (3), Aib 4.08 (4), Ala 7.95 (8).

Molecular weight determination: C75Hi32<N>i8°21 (1622.0)

Elemental analysis:

XIX. Preparation of Boc-Asn-Arg(NO2 )-Arg(NO2 )-Ala-Leu-Ile-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me

XIX.l. HC1 x H-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me

Boc-Ala-Leu-Ile-Leu-Leu-Ala-Gln - (Ala-Aib-Ala-Aib-Ala)2-0Me (754 mg, 0.465 mmol) was mixed with 1.2 N HCl/AcOE (10 mL ). After 50 minutes, some was evaporated in oil pump vacuum, and after addition of water (10 ml) it was lyophilized.

Yield: 690 mg (95*)

Thin layer chromatography:

R f (I) = 0.71

R f (II) = 0.52

R f (III) = 0.78

R f (IV) = 0.56

Rf (V) = 0.54

XIX.2. Boc-Asn-Arg(NO2)-Arg(NO2)-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me

Boc-Asn-Arg(NO 2 )-Arg(NO 2 )-OH (634 mg, 0.995 mmol) and HOBt (135 mg, 1.13 mmol) in DMF (5 mL) at -5 °C was added to HCl x A -Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me (20 mg, 0.398 mmol) and NMM (44 µl, 400 pmol) in DMF (7 mL ). After addition of DCC (217 mg, 1.05 mmol) 1 CH 2 Cl 2 (1.5 ml) at -5°C, it was stirred under independent heating for 48 hours. After stopping the reaction with 3 drops of glacial acetic acid, the precipitated DCH was centrifuged off. Processing and purification by chromatography takes place as described previously.

Yield: 630 mg (74*)

Melting point: 195°C (decomposition)

Thin layer chromatography:

Rf (I) = 0.70

R f (II) = 0.51

R f (III) = 0.56

R f (IV) = 0.45

Rf (V) - 0.68

Amino acid analysis:

Asx 0.94 (1), Glx 1.00 (1), Ile 0.89 (1), Leu 3.16 (3), Arg 1.95 (2).

Molecular weight determination: Cgi<H>i6ø<N>3o<0>2g (2138.5) Elemental analysis:

XX. Preparation of the free eicosapeptide

XX.1. Boc-Asn-Arg-Årg-Ala-Leu-Ile-Leu-Åla-Gln-(Ala-Aib-Ala-Aib-Ala )2-0Me x 2 HCl

Boc-Asn-Arg(NO2 )-Arg(NO2 )-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me (350 mg, 0.164 mmol) was in 3 ml anhydrous methanol mixed with 35 mg Pd/activated carbon and 12 µl (0.075 mmol) 6 N HCl. During stirring, hydrogen is passed through the solution at room temperature. After 20 minutes, 8 µl (49 pmol) and after 35 minutes 7 µl (42 pmol) of 6 N BX1 were added. After a hydration for approx. 50 minutes, the cleavage was quantitative according to DC control. The catalyst was filtered off and washed several times with a little methanol. The solvent was quickly distilled off on a rotary evaporator (oil pump vacuum, bath temperature 25°C), the residue was taken up in a little water and lyophilized.

Yield: 332 mg (95*)

Thin layer chromatography:

Rf (I) = 0.16

R f (II) = 0.11

R f (III) = 0.21

R f (IV) = 0.10

XX.2. H-Asn-Arg-Arg-Ala-Leu-Ile-1le-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala )2-0Me x 3 HCl

Boe-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-Ala-Gln-(Ala-Aib-Ala-Aib-Ala)2-0Me x 2 HCl (600 mg, 0.283 mmol) was mixed with 1 .2 N HCl/AcOH (5 mL). After 30 minutes, some was evaporated on a rotary evaporator, mixed with water (10 ml) and lyophilized.

Yield: 564 mg (97*)

Melting point: 45°C (decomposition)

Thin layer chromatography:

Rf (I) - 0.11

Molecular weight determination: Cg6Hi57N2gC>23Cl3 (2057.7)

Elemental analysis:

Materials and procedures for the experiments.

Chemicals

Solvent p.a. was provided from Fa. Merck, otherwise dried according to usual methods and distilled. N-methylmorpholine (Merck) was distilled over ninhydrin to remove secondary amines. 1-Hydroxybenzotriazole and dicyclohexylcarbodiimide were also from Merck. All L-amino acid derivatives came from Fa. Bachem. Boc-Aib-OH and H-Aib-OMe x HC1 were synthesized by methods reported in the literature.

Tvnns. 1 optical chromatograph i

Ready plates "Kieselgel 60 F254" (Fa. Merck) and the following eluents were used:

(I) 1-butanol/glacial vinegar/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

(V) chloroform/methanol 1:1

As flushing reagents, the following were used: ninhydrin reagent, chlorine/4,4'-bis(dimethylamino)diphenylmethane (TDM reagent) and Sakaguchi reagent. Dicyclohexyl urea with the following values served as a reference: Rf (I) 0.91, Rf (II) 0.82, Rf (III) 0.92, Rf (IV) 0.81, Rf (V) 0.83.

Amlnosvreanlvser

To determine the identity of the intermediates, 20 µg samples of the protected peptides were hydrolyzed in 6 N HCl for 24 hours at 110°C. The intermediate step and the target sequence of the heptapeptide segment, which contains the Leu-Leu bond, were hydrolyzed under otherwise identical conditions for 72 hours. The amino acid analyzes were carried out using a Biotronik amino acid analyzer "LC 6000 E" using standard programming.

Racemate determination

The hydrolyzed amino acids were derived as pentafluoropropionyl amino acid n-propyl ester and the enantiomers were separated gas chromatographically on glass capillary columns with "Chirasil-Val". The indicated percentages of D-amino acids are not corrected for racemization due to hydrolysis.

Elementary anal<y>se

Single C,E,N determinations were carried out on an "Elemental Analyzer Model 1104" (Carlo Erba, Milan).

Melting points

The melting points were determined according to Tottoli and are uncorrected.

Recording of the spectra

<13>C-NMR spectrum: 30 mg of the protected eicosapeptide was dissolved in 400 µl 12C2HC13/12C2H0<2>H (1:1) (Fa. Merck) and measured in the NMR spectrometer "WM 400" (Fa. Bruker ) for 12 hours at 30°C. "Circulardichroismus" spectrum: solutions of the free eicosapeptide (c = 1-1.7 mg/ml) in ethanol, trifluoroethanol, methanol, 1,1,1,3,3,3-hexafluoropropanol-(2 ), water and ethanol/water mixtures were measured with "Dichrograph II" (Fa. Jouhan-Roussel).

Chromatographic purification

After completion of the coupling reaction and removal of the solvent in an oil pump vacuum, the protected peptide intermediates were dissolved by adding an equal volume of CHCl3/MeOH 1:1, centrifuged from dicyclohexylurea and chromatographed on "Sephadex LH 20": column 3 x 115 cm ; eluent CHCls/MeOH 1:1; application amount 35 ml; flow rate 8.40 ml/10 min. The 3 ml fractions were examined thin-layer chromatographically in system II (TDM reagent). The peptides appear in the elution volumes 165-190 ml. The fractions were collected, the solvent removed in vacuo, and the residue dried over ?2®5' The amino acid analysis gives the expected values and a peptide content of 92-96*.

Immunization trials

First, a B-cell mitogen, which is both an excellent carrier and a highly effective adjuvant, was covalently linked to synthetic antigenic determinants. For this purpose, it was a. used the synthetic lipopeptide S-(2,3-bis(pal-mitoyloxy)propyl)-N-palmitoylcystelnyl-serine (Pam3Cys-Ser), which forms the N-terminus of the lipoprotein from the outer membrane of Escherichia coli. The particularly pronounced amphiphilic properties in a covalent connection with an antigen ensure, on the one hand, a stable anchoring of the S-glyceryl compound, which carries the 3 fatty acid residues, in the lipid layer of the cell membrane. On the other side, the most polar antigen (respectively the hapten) is also presented in the outermost hydrophilic layer of the membrane. As the activating effect of the lipoprotein alone is determined by this N-terminal part, this immunostimulating effect is found in all conjugates bearing Pan^Cys-Ser or analogues thereof.

As an example, the application of the concept to obtain specific antibodies against the epidermal growth factor receptor (EGF-R) is cited fig. 9. For this purpose, by means of computer-assisted epitope searching, the extra cytoplasmic region 516-529 was selected, built up by Merrifield synthesis and finally Fmoc-Ser(Bu<*>)-OH and then Pan^ Cys-OH added. After cleavage of the resin, the analytically uniform conjugate without further additives was administered in a single dose to mice i.p. Using ELISA analyses, high titers of specific antibodies against the tetradecapeptide were determined already after 2 weeks. It is significant that no antibody titers were obtained in control experiments with the obviously weakly immunogenic tetradecapeptide alone.

As Pam3Cys conjugates are also highly immunogenic in cell cultures, conventional and monoclonal antibodies can also be obtained against weakly immunogenic compounds by in vitro immunization in a fast and elegant way.

The advantages of the present concept in connection with cell cultures are: simple production of a chemically uniquely defined antigen-adjuvant conjugate in any amount, in contrast to other conjugates by single administration or multiple "boosts", with high efficiency in vivo and in vitro. A significant saving of experimental animals, often in vivo immunizations can also be completely dispensed with, and a drastic time saving, especially in genetic engineering work, is also achieved. Experiments can also be carried out with human cell culture systems.

Example of an ln vivo immunization

6- to 10-week-old Balb/c mice are immunized by single i.p. supply of 50 µg and 500 µg (0.2 ml of a 10"<1> to 10"<2 >molar solution of adjuvant covalently linked to antigen). Pam3Cys-Ser-(EGF-R 515-529). Antigen, adjuvant and a mixture of antigen and adjuvant in comparable molar amounts, as well as medium, served as controls. Two weeks after the injection, blood was taken from the mice at the retroorbital venous plexus to extract serum, and the antibody titer was determined by ELISA.

Analogous immunizations can also be achieved by other delivery methods, e.g. i.v., oral, rectal, i.m., s.c.

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

Balb/c mice were, as shown in fig. 10, immunized once i.p. with

I. Conjugate Pam3Cys-Ser (EGF-R 516-526)

II. Free tetradecapeptide EGF-R 516-529, alone

III. Pam3Cys-Ser alone

IV. Free tetradecapeptide (EGF-R 516-529) in mixture with

Pam3Cys-Ser,

with 0.2 pmol of the conjugate. The antibody titer was determined using ELISA analysis. (Ordinate OD at 405 nm) (Fig. 10). 14 days after the immunization, blood samples were taken from the mice at the eye vein and the recovered serum was used 1 ELISA. The values are given from the mean value (3-5 mice) of the difference between the ELISA values of PEP 14 - BSA conjugate and BSA (Fig. 10).

It turned out that only by using the membrane anchor-active substance conjugate prepared according to the invention were drastically elevated antibody concentrations found, which far exceed the activities of previously known methods.

Example of an ln vitro Immunization

Spleen cells from mice were cultured in the presence of conjugate Pam3Cys-Ser-(EGF-R 515-529), of adjuvant Pan^Cys-Ser, of tetradecapeptide EGF-R 516-529, a mixture of antigen and adjuvant and medium for 5 days.

At a cell density of 2.5 x 10^/ml, the lymphocytes were cultured for 48 hours in 0.2 ml portions in TPMI-1640 medium, enriched with 10* heat-inactivated FCS, glutamine (2 mM), penicillin (100 U/ml) , streptomycin (100 µg/ml) and 2-mercaptoethanol (5 x 10-<5> M).

The supernatants were recovered for examination of specific antibodies by ELISA analysis.

Mitogenic activation of mouse spleen cells

The mitogenic activation of Balb/c spleen cells by Pam3Cys-Ser-(Lys)4-FITC (circles), Pam3Cys-Ser-(Lys)4-OH x 3 HC1 (triangles) and Pam3Cys-Ser-(Lys)4- 0H x 2 TFE (squares) appears from fig. 12. The conditions for cell cultivation are described. (Z. Immunforsch. 153, 1977, p. 11 and Eur. J. Biochem. 115, 1981). In the drawing, the stimulation index for incorporation of <3>H-thymidine into DNA (cpm values for incorporation/cpm values for the control without mitogen) is plotted on the ordinate as a function of the concentration of the active substance used.

Comparison of in vivo/ln vitro

In fig. 11, the above experiment is compared to an in vitro experiment: In vitro experiment in microtiter plates: cell density: 2.5 x 10^ cells/ml; substance concentration: 5 x 10"<7> mmolar; incubation conditions: 37°C, 5* CO2, 5 days.

Conj.: Conjugate Pam3Cys-Ser-(EGF-R 515-529)

Pep.: Tetradecapeptide EGF-R 516-529)

Adj.: Pam3Cys-Ser

Mixture: Mixture of free tetradecapeptide EGF-R 516-529 and

Pam3Cys-Ser.

Also in vitro there is a drastic increase in the antibody concentration, whereby the applicability of the cell cultures, especially for the production of antibodies, is significantly expanded.

Claims (1)

Analogy method for the preparation of therapeutically active conjugate of membrane anchor-active substance where the membrane anchor compound is a compound of the following general formula: wherein A can be sulfur, oxygen, disulfide"(—S—S—), methylene (-CH2-) or -NE-; n = 0-5; m = 1 or 2; C<*> = asymmetric carbon atom with R— or S— configuration; R, R', R" are the same or different and stand for an alkyl, alkenyl or alkynyl group with 7-25 carbon atoms or hydrogen, which may optionally be substituted with hydroxy groups and X is an active substance or a spacer active substance group , characterized in that it includes that the protected peptide, which is protected with protective groups in a manner known per se at the functions where no reaction is to take place, is synthesized by means of known coupling methods to a solid or soluble support, such as a polymer, (e.g. Merrifield resin), in such a way that the synthesized, carrier-bound peptide is covalently bound to the membrane anchor compound via the N-termini or side functions of the peptide; so that the prepared peptide conjugate can be isolated by cleaving the protective groups and the bond peptide/carrier in a manner known per se, so that the membrane anchor peptide or the conjugate of membrane anchor active substance is recovered.