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4-Norleucine, 7-D-Phenylalanine-A-Melanocyte

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4-Norleucine, 7-D-Phenylalanine-A-Melanocyte Proc. Natl. Acad. Sci. USA Vol. 77, No. 10, pp. 5754-5758, October 1980 Biochemistry 4-Norleucine, 7-D-phenylalanine-a-melanocyte-stimulating hormone: A highly potent a-melanotropin with ultralong biological activity (amino acid racemization/peptide degradation/adenylate cyclase/tyrosinase/melanoma) ToMI K. SAWYER*, PAULINE J. SANFILIPPO*, VICTOR J. HRUBY*t, MICHAEL H. ENGELt, CHRISTOPHER B. HEWARD§, JEAN B. BURNETT§T, AND MAC E. HADLEY§ *Department of Chemistry and Biochemistry; tLaboratory of Organic Geochemistry, Department of Geosciences; and §Department of General Biology, University of Arizona, Tucson, Arizona 85721 Communicated by C. S. Marvel, July 7,1980 ABSTRACT a-Melanocyte-stimulating hormone (a-MSH) cemization within the primary sequences of peptides, we ob- reversibly darkens frog skins by stimulating melanosome tained additional evidence which suggested that stereochemical movement (dispersion) within melanophores. Heat-alkali substitution at position 7 (replacement of L-phenylalanine by treatment of a-MSH results in prolonged biological activity of a-MSH or would provide the hormone. Quantitative gas chromatographic analysis of the D-phenylalanine) of [Nle4]-a-MSH hydrolyzed heat-alkali-treated peptide revealed partial race- an analogue with the desired biological properties. Previous mization particularly at the 4 (methionine) and 7(phenylalanine) investigations have shown that [Nle4]-a-MSH is more potent positions. [Nle4J-caMSH, a synthetic analogue of a-MSH, re- than a-MSH on both amphibian melanophores (2, 6) and on versibly darkens frog skins and also exhibits prolonged activity stimulating melanoma adenylate cyclase (6, 13), and it is also after heat-alkali treatment. Synthesis of [Nle4, DPhe7-a-MSH resistant to inactivation by chloramine-T (14, 15), an oxidant provided an analogue with prolonged biological activity, used in peptide iodination. Because heat-alkali treatment of identical to that observed with heat-alkali-treated a-MSH or [Nle4ja-MSH. [Nle4, D-Phe7J-a-MSH was resistant to enzymatic this analogue also resulted in "potentiation," "prolongation," degradation by serum enzymes. In addition, this peptide ex- and "retardation," it was clear that alteration of the methionine hibited dramatically increased biological activity as determined residue was not a requirement for the expression of these by frog skin bioassay, activation of mouse melanoma adenylate properties. Thus, it was decided to retain the benefits of the cyclase, and stimulation of mouse melanoma cell tyrosinase norleucine substitution in position 4 in the synthesis of the activity. This Nle4, D-Phe7 synthetic analogue ofa-MSH is a very "definitive" peptide. potent melanotropin, 26 times as potent as a-MSH in the ade- We report here the synthesis of [Nle4, D-Phe7]-a-MSH and nylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged bio- present data demonstrating its unique biological properties. logical activity should make this analogue of a-MSH an im- These include prolonged biological activity, enhanced potency portant molecular probe for studying the melanotropin receptors relative to a-MSH in a number of biological systems, and re- of both normal and abnormal (melanoma) melanocytes. sistance to degradation by serum enzymes. The biological properties of this analogue provide insights into the struc- a-Melanotropin (a-MSH, a-melanocyte-stimulating hormone) ture-activity relationships of the melanotropins. is a tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg- Trp-Gly-Lys-Pro-Val-NH2) that is synthesized and secreted by MATERIALS AND METHODS the pars intermedia of the vertebrate pituitary (1). The amino The amino acids used were of the L configuration unless oth- acid residues that are important in the expression of melano- erwise stated. NO-tert-butyloxycarbonyl (Na-Boc) amino acids tropic activity have been elucidated through systematic and amino acid derivatives were purchased from Vega Bio- structure-function investigations of a-MSH and a-MSH chemical (Tucson, AZ) or from Biosynthetica (Oberdorf, fragments on amphibian melanophores (2, 3) and, to a lesser Switzerland), or were prepared by published procedures. The extent, on mammalian melanoma cells (4-6). Very little in- [Nle4]-a-MSH was purchased from Penninsula Laboratories formation is available, however, regarding the stereochemical (San Carlos, CA), or was prepared in our laboratories (15; un- and conformational correlates of biological activity in either published). of these two biological systems. Solid-Phase Peptide Synthesis. The a-MSH was prepared Earlier reports have shown that heat-alkali treatment of as described (16). [Nle4, D-Phe7]-a-MSH was synthesized by crude or purified preparations of naturally occurring a-MSH using a p-methylbenzhydrylamine resin (17). The method for produces a partially racemized product with altered activity the preparation of this resin from beaded polystyrene/1% di- on amphibian melanophores both in vivo and in vitro. Such vinylbenzene was identical to published procedures for the changes in biological effects have been discussed in terms of preparation of benzhydrylamine resin (18), except for the "potentiation," "prolongation," and "retardation" (7-12). Al- substitution of p-toluoyl chloride for benzoyl chloride in the though the precise biochemical mechanism by which these intermediate step of ketone resin formation. Amino acids were unusual biological properties were produced is unknown, it coupled successively as their Na-Boc derivatives. Reactive appeared possible that synthetic stereostructural tailoring of amino acid side-chains were protected as follows: serine, 0- a-MSH might produce an analogue that would also possess benzyl; tyrosine, 0-2,6-dichlorobenzyl; glutamic acid, y-benzyl these properties. Utilizing a high-resolution gas chromato- graphic method to localize and quantitate specific sites of ra- Abbreviations: a-MSH, a-melanotropin, a-melanocyte-stimulating hormone; NMe, norleucine; Na-Boc, No-tert-butyloxycarbonyl; 1- The publication costs of this article were defrayed in part by page BuOH, 1-butanol; Pyr, pyridine. charge payment. This article must therefore be hereby marked "ad- t To whom reprint requests should be addressed. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate I Present address: Department of Biomechanics, Michigan State this fact. University, East Lansing, MI 48824. 5754 Downloaded by guest on September 25, 2021 Biochemistry: Sawyer et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5755 Table 1. Analytical properties of a-MSH analogues Residue Residue Physicochemical properties in in Thin-layer chromatography [a]25 in posi- posi- RF in various systems 10% AcOH, tion 4 tion 7 A B C D degrees Met L-Phe 0.24 0.58 0.72 0.78 -58.0 (c = 0.50) Nle L-Phe 0.20 0.70 0.69 ND -45.5 (c = 0.44) Nle D-Phe 0.24 0.79 0.74 0.72 -59.7 (c = 0.49) Amino acid analyses, mol/mol of analogue Trp Lys His Arg Ser Glu Pro Gly Val Met Nle Tyr Phe Met L-Phe 0.90 1.04 0.90 0.98 1.72 1.00 1.08 1.08 1.07 0.97 0.92 1.06 Nle L-Phe 0.85 1.04 0.99 1.04 1.58 0.95 0.97 0.92 0.87 0.93 1.05 1.10 Nle D-Phe 0.93 1.07 1.00 1.01 1.75 1.00 1.06 1.07 1.00 0.91 0.87 0.96 c, Concentration, g/100 ml; ND, not determined. ester; lysine, N1-2,4-dichlorobenzyloxycarbonyl; arginine, HOAc/H20, 6:6:1.2:4.8; D, 2-propanol/25% aqueous NH3/ Ng-p-toluenesulfonyl; histidine, Nim-p-toluenesulfonyl; tryp- H20, 3:1:1. Optical rotation values were measured at the tophan, Ni-formyl. The coupling reactions in the solid phase mercury green line (546 nm) in a Perkin-Elmer 241 MC po- syntheses were achieved with a 3-fold excess of Na-Boc amino larimeter. Amino acid analyses were performed on a Beckman acid and a 2.4-fold excess of dicyclohexylcarbodiimide. Re- 120C amino acid analyzer after hydrolysis (100°C, 22 hr) in 4 moval of the Na-Boc protection at each step was effected by M methanesulfonic acid containing 0.2% 3-(2-aminoethyl)- treatment (2 and 20 min) with 45% (vol/vol) trifluoroacetic acid indole. No corrections were made for destruction of amino acids in CH2Cl2 containing 2% (vol/vol) anisole. Other solid-phase during hydrolysis. Analytical results for all peptides are shown procedures were similar to those used in the synthesis of a-MSH in Table 1. (16). After all of the amino acid residues had been coupled to Quantitation of Heat-Alkali-Catalyzed Racemlization. Gas the resin, the amino terminus of the peptide-resin was acety- chromatographic determination of the extent of racemization lated with N-acetylimidazole. The protected peptide was of heat-alkali-treated a-MSH and [Nle4]-a-MSH was adopted cleaved from the resin and all protecting groups were removed from procedures previously described (20-22) with some (except the Ni-formyl group of tryptophan) by treatment (30 modifications. min, 0°C) with anhydrous HF containing 16% anisole and 0.5% Heat-alkali treatment of a-MSH and [Nle4]-a-MSH was 1,2-ethanedithiol. The formylated (Ni-formyl-Trp) peptide was accomplished by heating the peptides (100°C, 10 min) in 0.1 deformylated (19) by adding 4 M NaOH to pH 11.5 (3 min), M NaOH in sealed tubes. The samples were then hydrolyzed and the reaction was terminated by addition of glacial acetic in 6 M HCI (100°C, 24 hr) and desalted by cation-exchange acid to a final pH of 4.5. chromatography (Bio-Rad AG 50-X8). The purified amino acid Purification and Homogeneity. The crude [Nle4, D- residues were esterified with 2-4 M (+)-2-butanol/HCI and Phe7]-a-MSH was purified by ion-exchange chromatography acylated with pentafluoropropionic anhydride. The resultant on a carboxymethyl-cellulose column (2.0 X 18.0 cm) using a amino acid diastereomeric derivatives were separated on a discontinuous gradient of 250 ml of 0.01 M ammonium acetate Carbowax-20 M nickel (wall coated open tubular) capillary (pH 4.5) and then 250 ml of each of 0.1 M, 0.2 M, and 0.4 M column (60 m X 0.5 mm).
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