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Dedicated to Prof. Dr. Hum Neu~urh on the occasion of his 75th birthday (26.X.1984) Asymmetric disulfide conjugates of mercaptosuccinyl tobacco mosaic virus (TMV -SH) with Na-desacetyl-N1-5-(mercaptovaleryl)-a -melanotropin were prepared via the S-sulfoderivative of the peptide. The conjugates, TMV -S-S -a-MSH(n), contained up to n = 330 disulfide-linked peptide molecules/virion. Similarly, fluorescent conjugates, Rh(m) -TMV -S-S -a-MSH(n) were prepared, containing m Y 200 rhodamine molecules linked to the virions by thiourea bridges. Such conjugates were designed to study a-MSH receptor localization and dynamics (mainly internalization), because the carrier virions which served to enhance specific receptor binding and as fluorescent or radioactive markers may be detached from the ncuropeptides at will by reduction. Reduction occurred in solution and on the cell surface, but not in the cytoplasm, thus allowing detection of internalized agonist-receptor complexes. The conjugates were superpotent agonists for tyrosinase stimulation in Cloudmun S-Y1 melanoma cell cultures, but were inactive for c y c k AMP accumulation. Their rather rapid internalization and the influence of reducing agents and other agonists on their biologic activity suggest a close connection between receptor location and biologic response as well as the presence of essential receptor HS-groups.Introduction. ~ The recent discovery of regulatory peptides common to the nervous and endocrine systems has, because of its fundamental and practical implications [3], revived interest in peptides tremendously. Our laboratory is dealing with two aspects of neuropeptides and hormones: structure-activity relationships [4] and molecular mechanisms of peptide-receptor interactions, including the influence of lipid membranes [5]. This report is concerned with receptors, particularly with development of new tools for studying receptor localization and dynamics in tissue s h e s and target cells.Virions of tobacco mosaic virus (TMV) carrying peptides covalently attached to a considerable number (50 to 500) of the identical capsomers (Fig. I) react almost irreversibly with peptide receptors on a target cell surface to produce the biological effects typical of the attached peptides. Thus, they display properties expected of receptor-specific 'artificial antibodies ' [6]. Such TMV-peptide conjugates, additionally labeled with fluorescent molecules, have been used for the study of cell-surface receptors with fluorescence microscopy [7]. In order to analyze the observed effects of receptor local-

Chemical synthesis and biological activities of a new a-melanotropin derivative are described. Nu-(5-Bro-movalery1)-Na-deacetyl-a-melanotropin contains the 5-bromopentanoyl group as a chemical 'handle' in place of the acetyl group of the natural hormone. The synthesis involved a new protected intermediate which allowed the selective deprotection of either the N" or N' amino group. The title compound reacted with sodium thiosulfate to give N"-deacetyl-Na-(5-(sulfothio)valeryl)-a-melanotropin, a key intermediate for the preparation of tobaccomosaic virus/a-melanotropin disulfide conjugates. As a basis for the study of the conjugates, biological activities of the title compound on Cloudman S-YI mouse melanoma cell cultures (tyrosinase stimulation, binding, and cyclic AMP accumulation) were determined. They proved to be quite similar to the corresponding a-melanotropin activities. Differences in bindings may be explained by stronger hydrophobic interaction of the new derivative with the lipid phase of the target cell membranes. ') Parts of this report have appeared as a thesis [I]. Nomenclature and abbreviations [2]. Additional abbreviations: MSH = rnelanophore stimulating hormone (melanotropin), DCC = N,N-dicyclohexylcarbodiimide, DCU = N,N'-dicyclohexylurea, DMF = dimethylformamide, HOBt = 1-hydroxybenzotriazole, Boc = tert-butoxycarbonyl, OBu' = tert-butoxy, Msoc = 2-(methylsulfonyl)ethoxycarbonyl, Np = 4-nitrophenyl, Z = benzyloxycarbonyl, TFA = trifluoroacetic acid. Chiral amino acids are in their L-configuration. Culture media see E.xperimentu1. Synthesis of N"-(5-Bromovaleryl)-N"-deacetyl-a-melanotropin (10).-Two approaches are illustrated in the Scheme. One (steps 11+12-+10) was based on an earlier synthesis of a-MSH [7] wherein the Lys (Msoc)-11 protection is removed by alcaline /I-elimination in the last step. This worked here for the relatively stable 5-bromovaleryl derivative. However, for more reactive handles, such as bromoacetyl or maleimido groups [5] [8], deblocking in the final stage must be effected by mild acid treatment. We, therefore, reversed the type of protection, using Msoc for the IT-atom and protecting groups derived from t-butyl alcohol for the side chains [9]. The key intermediates 7 and 8 can serve a wider range of handles than 11.Suitable educts for the second approach (steps l+lO) were the Boc-tetrapeptide methyl ester 1 [lo] and the Z-protected nonapeptide 5 [ll]. The former was saponified to the free acid 2 which was treated with TFA to remove the N-terminal protection. The crystalline tetrapeptide salt 3. TFA was reacted with 4-nitrophenyl 2-(methyl-sulfony1)ethyl carbonate [7] [ 121 to yield crystalline, analytically pure Msoc-tetrapeptide 4. Its isolation was rather difficult because of a pronounced solubility in H,O and organic solvents.Compound 4 was condensed with the mixed HOBt.HC1 salt 6 of partially protected nonapeptide amide (produced from 5 by catalytic hydrogenation in the presence of HOBt), using the DCC/HOBt method without base. This procedure is known to minimize sid...

Durch Kondensation der Fragmente (I) und (II) und Umsetzung des Produkts (IIIa) nach Deblockierung [→(IIIb)] mit dem Aktivester (IV) wird das geschützte Tridecapeptid (Va) erhalten, dessen Deblockierung (HCl/HCOOH) die Titelverbindung (Vb), die am N‐terminalen Molekülende die 5‐Brompentanoyl‐Gruppe anstelle der Acetylgruppe im natürlichen α‐Melanotropin aufweist, liefert.

Asymm. Disulfid‐Konjugate von Mercaptosuccinyl‐Tabakmosaikvirus (TMV∼SH) mit dem α‐Melanotropin (Ia) (α‐MSH‐Analogen (Id) werden über das S‐Sulfoderivat (Ic) [zugänglich aus (Ib)] dargestellt.