Melanotropin Receptors I. Synthesis and Biological Activity of N^α‐(5‐Bromovaleryl)‐N^α‐deacetyl‐α‐melanotropin

Abstract

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...

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

“…Pretreatment The present results are in complete agreement with our earlier results on fluorescence labeling of these cells using a water-soluble class of fluorescent melanotropic macromolecular reagents (6, 7) as well as with those of Schwyzer and coworkers, who developed conjugates of MSH with tobacco mosaic virus (8)(9)(10)(11)(12). Using the melanotropin fluorescein conjugate, we demonstrated that both melanotic as well as amelanotic human melanoma cells expressed membrane melanocortin receptors (6,7).…”

“…In this respect, these results are similar to electron microscopic visualization of internalized receptor-ligand complex for a glucagon-serum albumin preparation labeled with 17 nanometer gold particles (23). These results are consistent with those obtained earlier using fluorescent tobacco mosaic virus-MSH conjugates (8)(9)(10)(11)(12) and fluorescent polyvinyl alcohol-MSH conjugates (6, 7).…”

“…This binding undoubtedly resulted from the multiplicity of the peptide ligand on these solid-phase probes, such that a single assembly of the bead-peptide conjugate is most likely able to interact simultaneously with more than one receptor on the cell membrane. This behavior has earlier been reported on interaction of tobacco mosaic virus-MSH conjugates with mouse melanoma cells (8)(9)(10)(11)(12). Similarly, certain classes of polyvalent antibodies that also are capable of establishing multiple interactions exhibit much higher binding affinities (22).…”

“…These reagents complement our previously reported fluorescent polyvinyl alcohol-conjugated (6, 7) MSH (8)(9)(10)(11)(12) constructs for use in receptor identification. Most importantly, every cell of every melanoma cell line possessed melanotropin receptors as visualized by these reagents.…”

“…These methods provide simple alternatives to the use of fluorescence techniques previously described by Schwyzer and coworkers (8)(9)(10)(11)(12) and recently by us (6,7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established.…”