Peptides,Vol. 13, pp. 1145-1148, 1992
0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
Printed in the USA.
The Effect of N-Terminal Substitutions on the Biological Activity of MSH Fragments H. S L I L I - V A R G H A , 1 J. B O D I , H. M E D Z I H R A D S Z K Y - S C H W E I G E R
A N D K. M E D Z I H R A D S Z K Y
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest 112, P.O. Box 32, Hungary R e c e i v e d 7 F e b r u a r y 1992 SLILI-VARGHA, H., J. BODI, H. MEDZ1HRADSZKY-SCHWEIGER AND K. MEDZIHRADSZKY. The effect ofN-terminal substitutions on the biologicalactivity of MSH fragments. PEPTIDES 13(6) 1145-1148, 1992.--In order to study the role of N-terminal substitutions of peptide sequences related to the active site of a-melanotropin, [GIpS]a-MSH(5- 10), [GlpS,D-Phe7]aMSH(5-10), [Sar~,D-PheT]a-MSH(5-10),[NIe4,D-Phe7]a-MSH(4-10), [N-carbamoyl]a-MSH(5-10), and formyl and acetyl derivatives of a-MSH(5-10), [GIyS]a-MSH(5-10) and [GlyS,D-PheT]a-MSH(5-10),were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect. Melanotropin fragments
Synthesis
Biological activity
N-acyl peptides
N-alkyl peptide
C-18 column (4 × 150 mm, particle size 10 #), using the following solvents: HPLC-A, methanol/0.1 M phosphate-water at pH 3 (31:69, v/v); HPLC-B, methanol/0.1 M triethylamine (TEA)formate-water at pH 3 (49:41, v/v); HPLC-C, methanol/0.1 M TEA-formate-water at pH 3 (35:65, v/v); HPLC-D, methanol/ 0.1 M TEA-formate-water at pH 3 (28:72, v/v). Flow rate was 1.0 ml/min, detection at 220 nm. Ion exchange chromatography was carried out on QAE-Sephadex A-25 (acetate cycle) column. Amino acid analysis was after 24 h hydrolysis with 5.7 N HCI at 105°C. A general procedure for removing the tert-butyl ester group was used. The peptide tert-butyl ester was dissolved in trifluoroacetic acid (TFA) containing 10% anisole. The solution was left to stand for 45 min at room temperature, then poured into ether, and the precipitate was filtered off.
a-MELANOTROPIN (t~-MSH) represents one of the few polypeptides occurring in nature with an acetyl group on their Ntermini. In its acetylated form it is biologically more active than the one containing a free terminal amino group. More interestingly, this is valid not only for the whole sequence, but also for the smaller fragments of the hormone: in each case acetylation enhances the biological activity by two- to tenfold (!,4). Since it is not likely that the presence of an acetyl group on the Nterminus of a-MSH is a local structural requirement of the melanotropin receptor, other aspects of this substitution have come into consideration. For example the N-terminal acetyl group may be important in connection with enzymatic degradation, with transport processes influenced by electric charge distribution on the whole hormone molecule, or with general hydrophiliclipophilic features required by the melanotropin receptor (1). In the present study we further explored the role of N-terminal acyl and alkyl functionalization on MSH active site fragments in terms of their structure-activity relationships using an in vitro bioassay.
H-Glu-His-Phe-Arg-Trp-Gly-OMe 1 H-Glu(OBut)-His-Phe-Arg-Trp-Gly-OMe• 3HC1 (7) was deprotected by the general procedure, then purified by HPLC and ion exchange chromatography to give I with 45% yield. Rf 0.16 (TLC-C), 0.28 (TLC-D); k' 6.15 (HPLC-D). Amino acid analysis: Glu 1.02, His 1.04, Phe 1.00, Arg 0.99, Gly 0.98.
METHOD Thin layer chromatography was performed on precoated silica gel plates (Merck) in the following solvent systems: TLC-A, ethyl acetate/pyridine/acetic acid/water (60:20:6:11, v/v); TLC-B, chloroform/methanol (8:2, v/v); TLC-C, ethyl acetate/pyridine/ acetic acid/water (60:40:12:22, v/v); TLC-D, butanol/pyridine/ acetic acid/water (4:1:1:1, v/v); TLC-E, ethyl acetate/pyridine/ acetic acid/water (60:10:3:5.5, v/v). HPLC was performed on BST (Bioseparation Technology Company, Budapest) SI-100S
For-Glu-His-Phe-Arg-Trp-Gly-OMe H H-Glu(OBu%His-Phe-Arg-Trp-Giy-OMe • 3HC1 110 mg (0.1 mmol) was dissolved in 0.5 ml dimethylformamide (DMF) 0.045 ml (0.3 mmol) TEA and 24 mg (0.11 retool) trichloro. phenyl formate were added with stirring. After 24 h the DMF
Requests for reprints should be addressed to Dr. Helga Siili-Vargha.
1145
1146 was evaporated, the residue was triturated with ether, and filtered to give the crude For-Glu(OBut)-His-Phe-Arg-Trp_Gly_OMe. Deprotection was carried out by the general procedure and purification was carried out by HPLC and ion exchange chromatography, producing 47 mg (49%) II. Rf0.26 (TLC-C), 0.38 (TLCD); k' 3.65 (HPLC-B). Amino acid analysis: Glu 1.07, Phe 1.00, His 0.97, Arg 0.95, Gly 1.0 I.
Ac-Glu-His-Phe-Arg- Trp-Gly-OMe Ill To the solution of 110 mg (0.1 mmol) H-Glu(OBut)-HisPhe-Arg-Trp-Gly-OMe. 3HC1 0.045 ml (0.3 mmol) TEA and 21 mg (0.11 mmol) p-nitrophenyl acetate were added. After 24 h the DMF was distilled in vacuo, the residue triturated with ether and filtered. Deprotection and purification were done with the same procedure as in the case of I. Yield 51 mg (52%); Rf 0.27 (TLC-C), 0.40 (TLC-D); k' 4.33 (HPLC-C). Amino acid analysis: Giu 1.04, Phe 0.99, His 1.03, Arg 0.98, Gly 0.97.
H2N-CO-Glu-His-Phe-Arg- Trp-Gly-OMe IV H-Glu(OBut)-His-Phe-Arg-Trp-Gly-OMe• 3HCI 110 mg (0.1 mmol) and 12 mg (0.15 mmol) KOCN were let to stand for 1 day in 1.0 ml phosphate buffer (pH = 8). The solvent was evaporated, the residue was triturated with absolute ethanol, and the inorganic salts were filtered off. The filtrate was evaporated, triturated with ether, and filtered. The crude HzN-CO-Glu(OBut)His-Phe-Arg-Trp-Gly-OMe was deprotected and purified as compound I to give 38 mg (39%) IV. Rf 0.25 (TLC-C), 0.38 (TLC-D); k' 2.10 (HPLC-C). Amino acid analysis: Phe 0.99, His 1.03, Arg 1.00, Gly 0.98, Glu 0.70 (the low value obtained for the glutamic acid may reflect the relative stability of the carbamoyl group).
Glp-His-Phe-OMe. HCI Va The solution of 660 mg (5.1 retool) Glp-OH, 700 mg (5.4 mmol) l-hydroxybenzotriazole (HOBO, and 1.12 g (5.4 retool) dicyclohexylcarbodiimide (DCC) in 5 ml D M F was stirred at 0°C. After 15 min the filtrate of 2 g (5.1 mmol) H-His-PheOMe. HC1 and 0.75 ml (5.4 mmol) TEA was added. Stirring was continued for 2 h at 0°C, then the reaction mixture was left overnight in the refrigerator. Dicyclohexylurea (DCU) was filtered off and the solvent was evaporated in vacuo. The residue was triturated with ethylacetate and crystallized from methanolether to give 1.6 g (66%) Va. Rf 0.29 (TLC-A), 0.50 (TLC-D). [a]~ -24.1 ° (c = 1, 1 M HC1). Analysis calculated for CzIHz6NsCI (463.91): C 54.37, H 5.65, N 15.09, C! 7.64. Found: C 54.31, H 5.87, N 15.18, C! 8.10%.
Glp-His-D-Phe-OMe. HCI Xa The title compound was prepared by the same procedure as Va. Rf 0.30 (TLC-A), 0.50 (TLC-D). [a]~5 +2.4 ° (c = 1, 1 M HCI). Analysis found: C 53.90, H 5.77, N 14.75, C1 8.15%.
GIp-His-Phe-N2H~ Vb To the suspension of 1.6 g (3.45 mmol) Glp-His-PheOMe.HC1 (Va) in 15 ml D M F 3 ml (60 mmol) hydrazine hydrate was added. Several drops of pyridine were added until clear solution was formed. The reaction mixture stood in a refrigerator overnight, then ether was added, the precipitate was filtered off and crystallized from acetic acid-ether to give 1.4 g (87%) Vb. Rf 0.34 (TLC-C), 0.42 (TLC-D). [a]~5 -17.5 ° (c = 1, DMF). Analysis calculated for C20H25N704 (427.46): C 56.20, H 5.90, N 22.93. Found: C 55.78, H 5.90, N 22.76%.
SISLI-VARGHA ET AL.
Glp-His-D-Phe-N2H3 Xb The title compound was prepared by the same method as Vb. Rf0.34 (TLC-C), 0.42 (TLC-D). [a]~5 +25.5 ° (c = 1, DMF). Analysis found: C 55.61, H 6.00, N 22.34%.
Glp-His-Phe-Arg- Trp-Gly-OMe V V was prepared from Vb and Arg-Trp-Gly-OMe by the same procedure as VI. The crude peptide was purified by HPLC and ion exchange chromatography to give the pure product with 38% yield. Rf 0.35 (TLC-C), 0.40 (TLC-D); k' 3.59 (HPLC-C). [a]2o5 -15.6 ° (c = 0.64, methanol). Amino acid analysis: Glu 1.05, Phe 0.99, His 1.00, Arg 0.98, Gly 0.98.
Glp-His-D-Phe-Arg- Trp-Gly-OMe X The title compound was prepared by the same way as V with 35% yield. Rf 0.36 (TLC-C), 0.43 (TLC-D); k' 1.60 (HPLC-C). Amino acid analysis: Glu 1.05, Phe 0.99, His 1.00, Arg 0.97, Gly 0.98.
Boc-Gly-His-Phe-OMe Via To the solution of 9 g (25.5 mmol) H-His-Phe-OMe. HC1 in 50 ml D M F 3.6 ml TEA and 12.9 g (30 mmol) Boc-GIy-OPCP (Boc, tert-butyloxycarbonyl; OPCP, pentachlorophenyl) were added at 0°C with stirring. Then the reaction mixture stood overnight at room temperature, the TEA- HCI was filtered off, and the DMF was distilled in vacuo. The residue was dissolved in ethyl acetate, the solution was washed with NaHCO3 solution and water. The ethyl acetate phase was dried over MgSO4 and evaporated. The residue was triturated with ether, filtered, and crystallized from ethanol-ether to give 8.2 g (68%) Via. Rf0.44 (TLC-A), 0.49 (TLC-B). m.p. 156-158°C; [a]~5 -12.2 ° (c = 1, methanol). Analysis calculated for C23H3IN506 (473.54): C 58.34, H 6.61, N 14.80. Found: C 57.89, H 6.58, N 14.70%.
Boc-Gly-His-Phe-N2H3 Vlb To the solution of 7.1 g (15 mmol) Via in 70 ml methanol 3 ml (60 mmol) hydrazine hydrate was added. The next day the reaction mixture was evaporated and the residue crystallized from methanol-ether to give 6.3 g (89%) VIb. Rf 0.29 (TLC-A), 0.19 (TLC-B). m.p. 192-193°C (d). [a]~5 = -22.05 ° (c = 1, methanol). Analysis calculated for C22Ha~NTO5 (473.58): C 55.80, H 6.61, N 20.7. Found: C 55.44, H 6.57, N 20.50%.
H-Gly-His-Phe-Arg- Trp-Gly-OMe VI VIb 1.0 g (2.1 mmol) was suspended in 10 ml D M F and at - 2 5 ° C the solution o f equivalent amount of NOC1 in CH2C12 was added with stirring. Stirring was continued for 30 min and then 0.3 ml (2.1 mmol) TEA was added. The solution was cooled down to - 5 0 ° C and was combined with the cold solution of 1.0 g (1.7 mmol) H-Arg-Trp-Gly-OMe.2HOAc and 0.5 ml (3.4 mmol) TEA in 10 ml DMF. Stirring was continued at - 1 0 ° C for 1 h and then the reaction mixture was left for one night in the refrigerator. TEA. HCI was filtered off, the filtrate was evaporated in vacuo, and the residue was triturated with ether and filtered to give 1.8 g (97%) crude Boe-Gly-His-Phe-Arg-Trp-GlyOMe VIc. Rf0.6 (TLC-C), 0.61 (TLC-D). To 1.2 g (1.3 mmol) VIc 1.2 ml anisole and 12 ml 2 N HCIformic acid were added. After 10 rain ether was added and the precipitate was filtered off and purified on silica gel column in solvent system C. The pure product was dissolved in ethanol containing equivalent amounts of HC1 and precipitated with ether to give 0.75 g (62%) VI. Rr0.34 (TLC-D), 0.31 (TLC-C).
N-TERMINAL SUBSTITUTION OF MSH FRAGMENTS
[c~]~5 -18.9 ° (c = 1, methanol), k' 1.81 (HPLC-D). Amino acid analysis: Phe 1.00, His 1.04, Arg 1.01, Gly 1.94.
For-Gly-His-Phe-Arg-Trp-Gly-OMe VII The title compound was prepared from VI with the aid of trichlorophenyl formate, and purified by HPLC and ion exchange chromatography with 61% yield. Rr 0.31 (TLC-C), 0.43 (TLC-D); k' 1.91 (HPLC-C). Amino acid analysis: Gly 2.07, Phe 0.98, His 1.00, Arg 0.95.
Ac-Gly-His-Phe-Arg-Trp-Gly-OMe VIII VIII was prepared from VI with p-nitrophenyl acetate, and purified by HPLC and ion exchange chromatography with 51% yield. Rf0.29 (TLC-C), 0.43 (TLC-D); k' 3.80 (HPLC-C). Amino acid analysis: Gly 1.95, Phe 1.01, His 1.03, Arg 1.01.
H-Glu-His-D-Phe-Arg-Trp-Gly-OMe IX Produced by the same way from H-Glu(OBut)-His-D-PheArg-Trp-Giy-OMe (7) as compound I, with 39% yield. Rf 0.22 (TLC-C), 0.33 (TLC-D); k' 3.70 (HPLC-A). Amino acid analysis: Glu 0.99, His 1.02, Phe 1.03, Arg 1.02, Gly 0.98.
For-Gly-His-D-Phe-Arg-Trp-Gly-OMe XIII XIII was prepared from Gly-His-D-Phe-Arg-Trp-Gly-OMe (8) similar to VII with 56% yield. Rf 0.32 (TLC-C), 0.45 (TLCD); k' 1.43 (HPLC-C). Amino acid analysis: Gly 2.01, Phe 1.00, His 1.00, Arg 1.00.
Boc-Sar-His-D-Phe-OMe XVa The solution of 1.76 g (5 mmol) H-His-D-Phe-OMe.HCI (8), 0.7 ml (5 mmol) TEA, and 2.4 g (5 mmol) Boc-Sar-OPCP in 10 ml D M F stood overnight at room temperature. After concentration in vacuo the residue was applied on a silica gel column in solvent system E. The purified tripeptide ester was crystallized from ethylacetate petroleum ether to give 890 mg (37%) XVa. Re0.36 (TLC-E), 0.76 (TLC-B). m.p. 146°C. Analysis calculated for C24H33NsO6 (487.56): C 59.12, H 6.82, N 14.36. Found: C 58.55, H 6.73, N 13.98%.
1147 protected with the aid of 4 N HCI/EtOAc under N2 atmosphere in the presence ofanisole. After 15 min the precipitate was filtered off and on the filter washed with ether to give XV with 90% yield. Rf 0.22 (TLC-C), 0.34 (TLC-D). A sample for biological measurements was further purified by HPLC. k' 1.68 (HPLCD). Amino acid analysis: Gly 1.05, Phe 0.98, His 0.98, Arg 0.99.
Biological Measurement For the estimation of the melanocyte-stimulating activity, the frog skin reflectometric method of Shizume et al. (9) was used with slight modification. The pH of the Ringer solution was maintained between 7.0-7.2. Synthetic a-melanotropin served as reference substance. Before the experiments animals (Rana ridibunda) were kept at constant illumination for 24 h at + 15°C. Light reflectance was measured with a Zeiss SPEKOL spectrophotometer equipped with reflectance accessories. Typically, measurements consisted of 60-rain preincubation of the skin in the Ringer, 60 min with the standard a-MSH, 60-rain rinse, and the actual measurement with the substance in question. For each peptide 10-12 skins were used and the mean values of the activities are given. RESULTS It is widely accepted that one of the shortest sequences required for melanotropic activity is the His-Phe-Arg-Trp tetrapeptide, the so-called active site of the hormone (6). Since this fragment possesses a very moderate activity, for our experiments we chose the more active (106 U/mmol) hexapeptide I, Glu-HisPhe-Arg-Trp-Gly, commonly found in all naturally occurring melanotropins derived from the pro-opiomelanocortin. Earlier experiments proved that the negative charge of the glutamic acid 3,-carboxylate group is not essential for the biological activity of this fragment, since substitution of glutamic acid by glycine (8) or glutamine (3) does not influence the melanocyte-stimulating activity of this hexapeotide.
TABLE 1 THE MELANIN-DISPERSINGACTIVITYOF a-MSH FRAGMENT ANALOGSON FROG (Ranaridibunda)SKIN Potencies Relative to a-MSH
Boc-Sar-His-D-Phe-NeH3 XVb The methanolic solution of XVa with 10 equivalent hydrazine hydrate stood at room temperature for 2 days. Methanol was evaporated, the residue was dissolved in methanol and evaporated again. Crystallization was performed from ethanol:water 1: l to give the hydrazide with 90% yield. Rf 0.13 (TLC-E), 0.43 (TLC-B). m.p. 191°C (dec). Analysis calculated for C23Ha3N705 (487.56): C 56.65, H 6.82, N 20.11, O 16.40. Found: C 57.01, H 6.54, N 19.77%.
Sar-His-D-Phe-Arg-Trp-Gly-OMe XV To the solution of 245 mg (0.5 mmol) Boc-Sar-His-D-PheNzH3 (XVb) in 2 ml DMF 55 mg (1.5 mmol) HC1 in 0.75 ml T H F and 0.073 ml (0.6 mmoi) isoamylnitrite were added at - 2 0 ° C under stirring. Stirring was continued for 20 rain, then the solution was poured into the cold solution of 275 mg (0.5 mmol) H-Arg-Trp-Gly-OMe.2CH3COOH and 0.22 ml (2 mmol) NMM in 2 ml DMF. The reaction mixture stood overnight in a refrigerator, then the solvent was evaporated in vacuo, and the residue triturated first with NaHCO3 solution, then with ether and filtered to give 290 mg (63%) Boc-Sar-His-D-Phe-Arg-TrpGly-OMe (XVc). Rf 0.91 (TLC-C), 0.79 (TLC-D). XVc was de-
Peptides a-MSH H-Glu-His-Phe-Arg-Trp-Gly-OMe H-CO-Glu-His-Phe-Arg-Trp-Gly-OMe CH3-CO-Glu-His-Phe-Arg-Trp-GIy-OMe NH2-CO-Glu-His-Phe-Arg-Trp-GIy-OMe Glp-His-Phe-Arg-Trp-Gly-OMe
I II III IV V
1.0 2.5 × 1.2 X 2.5 × 1.0 X 5.0 ×
H-Gly-His-Phe-Arg-Trp-Gly-OMe H-CO-Gly-His-Phe-Arg-Trp-Gly-OMe CH3-CO-Gly-His-Phe-Arg-Trp-GIy-OMe
VI VII VIII
2.5 × 10-5 1.5 x 10-4 1.0 × 10-4
H-Glu-His-D-Phe-Arg-Trp-Gly-OMe Glp-His-D-Phe-Arg-Trp-Gly-OMe Nle-Glu-His-D-Phe-Arg-Trp-Gly-OMe
IX X XI*
1.2 × 10-2 5.0 × 10-2 5.0 × 10-2
XII'~ XIII XIV t XV
1.7 × 5.0 × 5.0 × 1.5 ×
H-Gly-His-D-Phe-Arg-Trp-Gly-OMe H-CO-Gly-His-D-Phe-Arg-Trp-Gly-OMe CH3-CO-GIy-His-D-Phe-Arg-Trp-GIy-OMe CH3-Gly-His-D-Phe-Arg-Trp-Gly-OMe
*t For preparation of* see (10) and o f t see (5).
10-5 10-4 10-4 10-4 10-5
10-2 10-2 10-2 10-2
1148
SIJLI-VARGHA ET AL.
In order to study the effect of the structure of the N-terminal acyl group on biological activity, a series derived from the natural hexapeptide sequence (I), containing formyl (II), acetyl (III), carbamoyl (IV), and pyroglutamyl (V) residues was synthesized. All compounds used in this work had a methyl ester group on the C-terminal glycine residue, in order to simplify the charge interaction relationships. As can be seen from Table l, the blocking of the free terminal amino group enhanced the biological activity (melanin dispersion) in all cases, with the highest effect observed in the case of the acetyl derivative, and the lowest when the glutamic acid was self-acylated in the form of pyroglutamic acid. In a second series of model peptides the effect of acylation was investigated on a similar hexapeptide containing glycine (VI) in place of glutamic acid. Again, acylation by the formyl (VII) or acetyl group (VIII) led to an increase of the biological potency. In this case, however, the formyl derivative was somewhat more potent than the acetyl-substituted compound (Table l). Furthermore, this series confirmed again the lack of requirement for the glutamic acid side chain. It is well known that melanotropin peptides containing Dphenylalanine are much more active than the ones with the naturally occurring L form (7). It was interesting to learn whether acylation of the N-terminal amino group leads to enhanced biological activity in this case, too. And it was found to be the case: converting glutamic acid (IX) to pyroglutamic
acid (X) or acylation of the corresponding glycine analog (XI1) either with formyl (XIII) or with acetyl (XIV) group led to increased potency. DISCUSSION It is not easy to draw unequivocal conclusions for the structure-activity relationships from the data presented in Table 1. Certainly, the structural features of the acyl groups, at least when they are small and comparable in size, do not play a significant role in eliciting the biological response. One would assume that a positively charged N-terminus is disadvantageous for the biological activity; this seems to be confirmed by the investigation of the sarcosyl derivative (XV) possessing an alkylated N-terminus, whose activity is approximately the same as that of the glycine or glutamic acid derivative but less than that of the Nacylated peptides. On the other hand, simple elongation of the peptide chain, thus preserving the terminal positive charge as in the case of compound XI, leads to the enhancement of the biological activity, a well-known fact in the melanotropin field. It is also known that the [desamino-Tyr2]a-MSH(2-13) is less potent than ~-MSH(2-13) (2). Therefore, the most likely explanation of the observed changes in the biological activity seems to be the introduction o f a carbonyl group into the N-terminus, capable of forming new hydrogen bonds either with the receptor, or rather intramolecularly, leading to conformational changes required by the melanotropin receptor.
REFERENCES
1. Eberle, A. N. The melanotropins. Basel: Karger; 1988. 2. Eberle, A. N.; Hfibscher, W. a-Melanotropin labelled at its tyrosine 2 residue: synthesis and biological activities of Y-iodotyrosine2-y~25iodotyrosine2-,Y,5'-diiodo-tyrosine2- and (Y,5'-3H2)-tyrosine2-amelanotropin, and of related peptides. Helv. Chim. Acta 62:24602483; 1979. 3. Kappeler, H. Synthese von L-giutaminyl-L-histidyl-L-phenylalanylL-arginyl-L-tryptophyl-glycin. Helv. Chim. Acta 44:476-491; 1961. 4. Medzihradszky, K. The bio-organic chemistry of a-melanotropin. Med. Res. Rev. 2:247-270; 1982. 5. Medzihradszky-Schweiger, H.; Siili-Vargha, H.; B6di, J.; Medzihradszky, K. The melanocyte stimulating activity of N-nitroso-2chloroethyl-carbamoyl derivatives of a-melanotropin fragments. Coll. Czech. Chem. Commun. 53:2574-2582; 1988. 6. Otsuka, H.; Inouye, K. Synthesisofpeptides related to the N-terminal structure ofcorticotropin, lII. Synthesis of L-histidyl-L-phenylalanyl-
7.
8. 9. 10.
L-arginil-L-tryptophan, the smallest peptide exhibiting the melanocyte stimulating and the lipolytic activities. Bull. Chem. Soc. Jpn. 37:1465-1471; 1964. Sawyer, T. K.; Hruby, V. J.; Wilkes, B. C.; Draelos, M. T.; Hadley, M. E.; Bergsneider, M. Comparative biological activities of highly potent active-site analogues of a-melanotropin. J. Med. Chem. 25: 1022-1027; 1982. Schnabel, E.; Li, C. H. Synthesis of glycyI-L-histidyl-L-phenylalanylL-arginil-L-tryptophyl-glycine and its melanocyte-sfimulating activity. J. Biol. Chem. 235:2010-2012; 1960. Shizume, K.; Lemer, A. B.; Fitzpatrick, T. B. In vitro bioassay for the mdanocyte-stimulating hormone. Endocrinology 54:553-560; 1964. Siili-Vargha, H.; Botyfinszki, J.; Medzihradszky-Schweiger, H.; Medzihradszky, K. Synthesis of a-MSH fragments containing phenylalanine mustard for receptor studies. Int. J. Pept. Protein Res. 36:308-315; 1990.
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Printed in the USA.
The Effect of N-Terminal Substitutions on the Biological Activity of MSH Fragments H. S L I L I - V A R G H A , 1 J. B O D I , H. M E D Z I H R A D S Z K Y - S C H W E I G E R
A N D K. M E D Z I H R A D S Z K Y
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, H-1518 Budapest 112, P.O. Box 32, Hungary R e c e i v e d 7 F e b r u a r y 1992 SLILI-VARGHA, H., J. BODI, H. MEDZ1HRADSZKY-SCHWEIGER AND K. MEDZIHRADSZKY. The effect ofN-terminal substitutions on the biologicalactivity of MSH fragments. PEPTIDES 13(6) 1145-1148, 1992.--In order to study the role of N-terminal substitutions of peptide sequences related to the active site of a-melanotropin, [GIpS]a-MSH(5- 10), [GlpS,D-Phe7]aMSH(5-10), [Sar~,D-PheT]a-MSH(5-10),[NIe4,D-Phe7]a-MSH(4-10), [N-carbamoyl]a-MSH(5-10), and formyl and acetyl derivatives of a-MSH(5-10), [GIyS]a-MSH(5-10) and [GlyS,D-PheT]a-MSH(5-10),were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect. Melanotropin fragments
Synthesis
Biological activity
N-acyl peptides
N-alkyl peptide
C-18 column (4 × 150 mm, particle size 10 #), using the following solvents: HPLC-A, methanol/0.1 M phosphate-water at pH 3 (31:69, v/v); HPLC-B, methanol/0.1 M triethylamine (TEA)formate-water at pH 3 (49:41, v/v); HPLC-C, methanol/0.1 M TEA-formate-water at pH 3 (35:65, v/v); HPLC-D, methanol/ 0.1 M TEA-formate-water at pH 3 (28:72, v/v). Flow rate was 1.0 ml/min, detection at 220 nm. Ion exchange chromatography was carried out on QAE-Sephadex A-25 (acetate cycle) column. Amino acid analysis was after 24 h hydrolysis with 5.7 N HCI at 105°C. A general procedure for removing the tert-butyl ester group was used. The peptide tert-butyl ester was dissolved in trifluoroacetic acid (TFA) containing 10% anisole. The solution was left to stand for 45 min at room temperature, then poured into ether, and the precipitate was filtered off.
a-MELANOTROPIN (t~-MSH) represents one of the few polypeptides occurring in nature with an acetyl group on their Ntermini. In its acetylated form it is biologically more active than the one containing a free terminal amino group. More interestingly, this is valid not only for the whole sequence, but also for the smaller fragments of the hormone: in each case acetylation enhances the biological activity by two- to tenfold (!,4). Since it is not likely that the presence of an acetyl group on the Nterminus of a-MSH is a local structural requirement of the melanotropin receptor, other aspects of this substitution have come into consideration. For example the N-terminal acetyl group may be important in connection with enzymatic degradation, with transport processes influenced by electric charge distribution on the whole hormone molecule, or with general hydrophiliclipophilic features required by the melanotropin receptor (1). In the present study we further explored the role of N-terminal acyl and alkyl functionalization on MSH active site fragments in terms of their structure-activity relationships using an in vitro bioassay.
H-Glu-His-Phe-Arg-Trp-Gly-OMe 1 H-Glu(OBut)-His-Phe-Arg-Trp-Gly-OMe• 3HC1 (7) was deprotected by the general procedure, then purified by HPLC and ion exchange chromatography to give I with 45% yield. Rf 0.16 (TLC-C), 0.28 (TLC-D); k' 6.15 (HPLC-D). Amino acid analysis: Glu 1.02, His 1.04, Phe 1.00, Arg 0.99, Gly 0.98.
METHOD Thin layer chromatography was performed on precoated silica gel plates (Merck) in the following solvent systems: TLC-A, ethyl acetate/pyridine/acetic acid/water (60:20:6:11, v/v); TLC-B, chloroform/methanol (8:2, v/v); TLC-C, ethyl acetate/pyridine/ acetic acid/water (60:40:12:22, v/v); TLC-D, butanol/pyridine/ acetic acid/water (4:1:1:1, v/v); TLC-E, ethyl acetate/pyridine/ acetic acid/water (60:10:3:5.5, v/v). HPLC was performed on BST (Bioseparation Technology Company, Budapest) SI-100S
For-Glu-His-Phe-Arg-Trp-Gly-OMe H H-Glu(OBu%His-Phe-Arg-Trp-Giy-OMe • 3HC1 110 mg (0.1 mmol) was dissolved in 0.5 ml dimethylformamide (DMF) 0.045 ml (0.3 mmol) TEA and 24 mg (0.11 retool) trichloro. phenyl formate were added with stirring. After 24 h the DMF
Requests for reprints should be addressed to Dr. Helga Siili-Vargha.
1145
1146 was evaporated, the residue was triturated with ether, and filtered to give the crude For-Glu(OBut)-His-Phe-Arg-Trp_Gly_OMe. Deprotection was carried out by the general procedure and purification was carried out by HPLC and ion exchange chromatography, producing 47 mg (49%) II. Rf0.26 (TLC-C), 0.38 (TLCD); k' 3.65 (HPLC-B). Amino acid analysis: Glu 1.07, Phe 1.00, His 0.97, Arg 0.95, Gly 1.0 I.
Ac-Glu-His-Phe-Arg- Trp-Gly-OMe Ill To the solution of 110 mg (0.1 mmol) H-Glu(OBut)-HisPhe-Arg-Trp-Gly-OMe. 3HC1 0.045 ml (0.3 mmol) TEA and 21 mg (0.11 mmol) p-nitrophenyl acetate were added. After 24 h the DMF was distilled in vacuo, the residue triturated with ether and filtered. Deprotection and purification were done with the same procedure as in the case of I. Yield 51 mg (52%); Rf 0.27 (TLC-C), 0.40 (TLC-D); k' 4.33 (HPLC-C). Amino acid analysis: Giu 1.04, Phe 0.99, His 1.03, Arg 0.98, Gly 0.97.
H2N-CO-Glu-His-Phe-Arg- Trp-Gly-OMe IV H-Glu(OBut)-His-Phe-Arg-Trp-Gly-OMe• 3HCI 110 mg (0.1 mmol) and 12 mg (0.15 mmol) KOCN were let to stand for 1 day in 1.0 ml phosphate buffer (pH = 8). The solvent was evaporated, the residue was triturated with absolute ethanol, and the inorganic salts were filtered off. The filtrate was evaporated, triturated with ether, and filtered. The crude HzN-CO-Glu(OBut)His-Phe-Arg-Trp-Gly-OMe was deprotected and purified as compound I to give 38 mg (39%) IV. Rf 0.25 (TLC-C), 0.38 (TLC-D); k' 2.10 (HPLC-C). Amino acid analysis: Phe 0.99, His 1.03, Arg 1.00, Gly 0.98, Glu 0.70 (the low value obtained for the glutamic acid may reflect the relative stability of the carbamoyl group).
Glp-His-Phe-OMe. HCI Va The solution of 660 mg (5.1 retool) Glp-OH, 700 mg (5.4 mmol) l-hydroxybenzotriazole (HOBO, and 1.12 g (5.4 retool) dicyclohexylcarbodiimide (DCC) in 5 ml D M F was stirred at 0°C. After 15 min the filtrate of 2 g (5.1 mmol) H-His-PheOMe. HC1 and 0.75 ml (5.4 mmol) TEA was added. Stirring was continued for 2 h at 0°C, then the reaction mixture was left overnight in the refrigerator. Dicyclohexylurea (DCU) was filtered off and the solvent was evaporated in vacuo. The residue was triturated with ethylacetate and crystallized from methanolether to give 1.6 g (66%) Va. Rf 0.29 (TLC-A), 0.50 (TLC-D). [a]~ -24.1 ° (c = 1, 1 M HC1). Analysis calculated for CzIHz6NsCI (463.91): C 54.37, H 5.65, N 15.09, C! 7.64. Found: C 54.31, H 5.87, N 15.18, C! 8.10%.
Glp-His-D-Phe-OMe. HCI Xa The title compound was prepared by the same procedure as Va. Rf 0.30 (TLC-A), 0.50 (TLC-D). [a]~5 +2.4 ° (c = 1, 1 M HCI). Analysis found: C 53.90, H 5.77, N 14.75, C1 8.15%.
GIp-His-Phe-N2H~ Vb To the suspension of 1.6 g (3.45 mmol) Glp-His-PheOMe.HC1 (Va) in 15 ml D M F 3 ml (60 mmol) hydrazine hydrate was added. Several drops of pyridine were added until clear solution was formed. The reaction mixture stood in a refrigerator overnight, then ether was added, the precipitate was filtered off and crystallized from acetic acid-ether to give 1.4 g (87%) Vb. Rf 0.34 (TLC-C), 0.42 (TLC-D). [a]~5 -17.5 ° (c = 1, DMF). Analysis calculated for C20H25N704 (427.46): C 56.20, H 5.90, N 22.93. Found: C 55.78, H 5.90, N 22.76%.
SISLI-VARGHA ET AL.
Glp-His-D-Phe-N2H3 Xb The title compound was prepared by the same method as Vb. Rf0.34 (TLC-C), 0.42 (TLC-D). [a]~5 +25.5 ° (c = 1, DMF). Analysis found: C 55.61, H 6.00, N 22.34%.
Glp-His-Phe-Arg- Trp-Gly-OMe V V was prepared from Vb and Arg-Trp-Gly-OMe by the same procedure as VI. The crude peptide was purified by HPLC and ion exchange chromatography to give the pure product with 38% yield. Rf 0.35 (TLC-C), 0.40 (TLC-D); k' 3.59 (HPLC-C). [a]2o5 -15.6 ° (c = 0.64, methanol). Amino acid analysis: Glu 1.05, Phe 0.99, His 1.00, Arg 0.98, Gly 0.98.
Glp-His-D-Phe-Arg- Trp-Gly-OMe X The title compound was prepared by the same way as V with 35% yield. Rf 0.36 (TLC-C), 0.43 (TLC-D); k' 1.60 (HPLC-C). Amino acid analysis: Glu 1.05, Phe 0.99, His 1.00, Arg 0.97, Gly 0.98.
Boc-Gly-His-Phe-OMe Via To the solution of 9 g (25.5 mmol) H-His-Phe-OMe. HC1 in 50 ml D M F 3.6 ml TEA and 12.9 g (30 mmol) Boc-GIy-OPCP (Boc, tert-butyloxycarbonyl; OPCP, pentachlorophenyl) were added at 0°C with stirring. Then the reaction mixture stood overnight at room temperature, the TEA- HCI was filtered off, and the DMF was distilled in vacuo. The residue was dissolved in ethyl acetate, the solution was washed with NaHCO3 solution and water. The ethyl acetate phase was dried over MgSO4 and evaporated. The residue was triturated with ether, filtered, and crystallized from ethanol-ether to give 8.2 g (68%) Via. Rf0.44 (TLC-A), 0.49 (TLC-B). m.p. 156-158°C; [a]~5 -12.2 ° (c = 1, methanol). Analysis calculated for C23H3IN506 (473.54): C 58.34, H 6.61, N 14.80. Found: C 57.89, H 6.58, N 14.70%.
Boc-Gly-His-Phe-N2H3 Vlb To the solution of 7.1 g (15 mmol) Via in 70 ml methanol 3 ml (60 mmol) hydrazine hydrate was added. The next day the reaction mixture was evaporated and the residue crystallized from methanol-ether to give 6.3 g (89%) VIb. Rf 0.29 (TLC-A), 0.19 (TLC-B). m.p. 192-193°C (d). [a]~5 = -22.05 ° (c = 1, methanol). Analysis calculated for C22Ha~NTO5 (473.58): C 55.80, H 6.61, N 20.7. Found: C 55.44, H 6.57, N 20.50%.
H-Gly-His-Phe-Arg- Trp-Gly-OMe VI VIb 1.0 g (2.1 mmol) was suspended in 10 ml D M F and at - 2 5 ° C the solution o f equivalent amount of NOC1 in CH2C12 was added with stirring. Stirring was continued for 30 min and then 0.3 ml (2.1 mmol) TEA was added. The solution was cooled down to - 5 0 ° C and was combined with the cold solution of 1.0 g (1.7 mmol) H-Arg-Trp-Gly-OMe.2HOAc and 0.5 ml (3.4 mmol) TEA in 10 ml DMF. Stirring was continued at - 1 0 ° C for 1 h and then the reaction mixture was left for one night in the refrigerator. TEA. HCI was filtered off, the filtrate was evaporated in vacuo, and the residue was triturated with ether and filtered to give 1.8 g (97%) crude Boe-Gly-His-Phe-Arg-Trp-GlyOMe VIc. Rf0.6 (TLC-C), 0.61 (TLC-D). To 1.2 g (1.3 mmol) VIc 1.2 ml anisole and 12 ml 2 N HCIformic acid were added. After 10 rain ether was added and the precipitate was filtered off and purified on silica gel column in solvent system C. The pure product was dissolved in ethanol containing equivalent amounts of HC1 and precipitated with ether to give 0.75 g (62%) VI. Rr0.34 (TLC-D), 0.31 (TLC-C).
N-TERMINAL SUBSTITUTION OF MSH FRAGMENTS
[c~]~5 -18.9 ° (c = 1, methanol), k' 1.81 (HPLC-D). Amino acid analysis: Phe 1.00, His 1.04, Arg 1.01, Gly 1.94.
For-Gly-His-Phe-Arg-Trp-Gly-OMe VII The title compound was prepared from VI with the aid of trichlorophenyl formate, and purified by HPLC and ion exchange chromatography with 61% yield. Rr 0.31 (TLC-C), 0.43 (TLC-D); k' 1.91 (HPLC-C). Amino acid analysis: Gly 2.07, Phe 0.98, His 1.00, Arg 0.95.
Ac-Gly-His-Phe-Arg-Trp-Gly-OMe VIII VIII was prepared from VI with p-nitrophenyl acetate, and purified by HPLC and ion exchange chromatography with 51% yield. Rf0.29 (TLC-C), 0.43 (TLC-D); k' 3.80 (HPLC-C). Amino acid analysis: Gly 1.95, Phe 1.01, His 1.03, Arg 1.01.
H-Glu-His-D-Phe-Arg-Trp-Gly-OMe IX Produced by the same way from H-Glu(OBut)-His-D-PheArg-Trp-Giy-OMe (7) as compound I, with 39% yield. Rf 0.22 (TLC-C), 0.33 (TLC-D); k' 3.70 (HPLC-A). Amino acid analysis: Glu 0.99, His 1.02, Phe 1.03, Arg 1.02, Gly 0.98.
For-Gly-His-D-Phe-Arg-Trp-Gly-OMe XIII XIII was prepared from Gly-His-D-Phe-Arg-Trp-Gly-OMe (8) similar to VII with 56% yield. Rf 0.32 (TLC-C), 0.45 (TLCD); k' 1.43 (HPLC-C). Amino acid analysis: Gly 2.01, Phe 1.00, His 1.00, Arg 1.00.
Boc-Sar-His-D-Phe-OMe XVa The solution of 1.76 g (5 mmol) H-His-D-Phe-OMe.HCI (8), 0.7 ml (5 mmol) TEA, and 2.4 g (5 mmol) Boc-Sar-OPCP in 10 ml D M F stood overnight at room temperature. After concentration in vacuo the residue was applied on a silica gel column in solvent system E. The purified tripeptide ester was crystallized from ethylacetate petroleum ether to give 890 mg (37%) XVa. Re0.36 (TLC-E), 0.76 (TLC-B). m.p. 146°C. Analysis calculated for C24H33NsO6 (487.56): C 59.12, H 6.82, N 14.36. Found: C 58.55, H 6.73, N 13.98%.
1147 protected with the aid of 4 N HCI/EtOAc under N2 atmosphere in the presence ofanisole. After 15 min the precipitate was filtered off and on the filter washed with ether to give XV with 90% yield. Rf 0.22 (TLC-C), 0.34 (TLC-D). A sample for biological measurements was further purified by HPLC. k' 1.68 (HPLCD). Amino acid analysis: Gly 1.05, Phe 0.98, His 0.98, Arg 0.99.
Biological Measurement For the estimation of the melanocyte-stimulating activity, the frog skin reflectometric method of Shizume et al. (9) was used with slight modification. The pH of the Ringer solution was maintained between 7.0-7.2. Synthetic a-melanotropin served as reference substance. Before the experiments animals (Rana ridibunda) were kept at constant illumination for 24 h at + 15°C. Light reflectance was measured with a Zeiss SPEKOL spectrophotometer equipped with reflectance accessories. Typically, measurements consisted of 60-rain preincubation of the skin in the Ringer, 60 min with the standard a-MSH, 60-rain rinse, and the actual measurement with the substance in question. For each peptide 10-12 skins were used and the mean values of the activities are given. RESULTS It is widely accepted that one of the shortest sequences required for melanotropic activity is the His-Phe-Arg-Trp tetrapeptide, the so-called active site of the hormone (6). Since this fragment possesses a very moderate activity, for our experiments we chose the more active (106 U/mmol) hexapeptide I, Glu-HisPhe-Arg-Trp-Gly, commonly found in all naturally occurring melanotropins derived from the pro-opiomelanocortin. Earlier experiments proved that the negative charge of the glutamic acid 3,-carboxylate group is not essential for the biological activity of this fragment, since substitution of glutamic acid by glycine (8) or glutamine (3) does not influence the melanocyte-stimulating activity of this hexapeotide.
TABLE 1 THE MELANIN-DISPERSINGACTIVITYOF a-MSH FRAGMENT ANALOGSON FROG (Ranaridibunda)SKIN Potencies Relative to a-MSH
Boc-Sar-His-D-Phe-NeH3 XVb The methanolic solution of XVa with 10 equivalent hydrazine hydrate stood at room temperature for 2 days. Methanol was evaporated, the residue was dissolved in methanol and evaporated again. Crystallization was performed from ethanol:water 1: l to give the hydrazide with 90% yield. Rf 0.13 (TLC-E), 0.43 (TLC-B). m.p. 191°C (dec). Analysis calculated for C23Ha3N705 (487.56): C 56.65, H 6.82, N 20.11, O 16.40. Found: C 57.01, H 6.54, N 19.77%.
Sar-His-D-Phe-Arg-Trp-Gly-OMe XV To the solution of 245 mg (0.5 mmol) Boc-Sar-His-D-PheNzH3 (XVb) in 2 ml DMF 55 mg (1.5 mmol) HC1 in 0.75 ml T H F and 0.073 ml (0.6 mmoi) isoamylnitrite were added at - 2 0 ° C under stirring. Stirring was continued for 20 rain, then the solution was poured into the cold solution of 275 mg (0.5 mmol) H-Arg-Trp-Gly-OMe.2CH3COOH and 0.22 ml (2 mmol) NMM in 2 ml DMF. The reaction mixture stood overnight in a refrigerator, then the solvent was evaporated in vacuo, and the residue triturated first with NaHCO3 solution, then with ether and filtered to give 290 mg (63%) Boc-Sar-His-D-Phe-Arg-TrpGly-OMe (XVc). Rf 0.91 (TLC-C), 0.79 (TLC-D). XVc was de-
Peptides a-MSH H-Glu-His-Phe-Arg-Trp-Gly-OMe H-CO-Glu-His-Phe-Arg-Trp-Gly-OMe CH3-CO-Glu-His-Phe-Arg-Trp-GIy-OMe NH2-CO-Glu-His-Phe-Arg-Trp-GIy-OMe Glp-His-Phe-Arg-Trp-Gly-OMe
I II III IV V
1.0 2.5 × 1.2 X 2.5 × 1.0 X 5.0 ×
H-Gly-His-Phe-Arg-Trp-Gly-OMe H-CO-Gly-His-Phe-Arg-Trp-Gly-OMe CH3-CO-Gly-His-Phe-Arg-Trp-GIy-OMe
VI VII VIII
2.5 × 10-5 1.5 x 10-4 1.0 × 10-4
H-Glu-His-D-Phe-Arg-Trp-Gly-OMe Glp-His-D-Phe-Arg-Trp-Gly-OMe Nle-Glu-His-D-Phe-Arg-Trp-Gly-OMe
IX X XI*
1.2 × 10-2 5.0 × 10-2 5.0 × 10-2
XII'~ XIII XIV t XV
1.7 × 5.0 × 5.0 × 1.5 ×
H-Gly-His-D-Phe-Arg-Trp-Gly-OMe H-CO-Gly-His-D-Phe-Arg-Trp-Gly-OMe CH3-CO-GIy-His-D-Phe-Arg-Trp-GIy-OMe CH3-Gly-His-D-Phe-Arg-Trp-Gly-OMe
*t For preparation of* see (10) and o f t see (5).
10-5 10-4 10-4 10-4 10-5
10-2 10-2 10-2 10-2
1148
SIJLI-VARGHA ET AL.
In order to study the effect of the structure of the N-terminal acyl group on biological activity, a series derived from the natural hexapeptide sequence (I), containing formyl (II), acetyl (III), carbamoyl (IV), and pyroglutamyl (V) residues was synthesized. All compounds used in this work had a methyl ester group on the C-terminal glycine residue, in order to simplify the charge interaction relationships. As can be seen from Table l, the blocking of the free terminal amino group enhanced the biological activity (melanin dispersion) in all cases, with the highest effect observed in the case of the acetyl derivative, and the lowest when the glutamic acid was self-acylated in the form of pyroglutamic acid. In a second series of model peptides the effect of acylation was investigated on a similar hexapeptide containing glycine (VI) in place of glutamic acid. Again, acylation by the formyl (VII) or acetyl group (VIII) led to an increase of the biological potency. In this case, however, the formyl derivative was somewhat more potent than the acetyl-substituted compound (Table l). Furthermore, this series confirmed again the lack of requirement for the glutamic acid side chain. It is well known that melanotropin peptides containing Dphenylalanine are much more active than the ones with the naturally occurring L form (7). It was interesting to learn whether acylation of the N-terminal amino group leads to enhanced biological activity in this case, too. And it was found to be the case: converting glutamic acid (IX) to pyroglutamic
acid (X) or acylation of the corresponding glycine analog (XI1) either with formyl (XIII) or with acetyl (XIV) group led to increased potency. DISCUSSION It is not easy to draw unequivocal conclusions for the structure-activity relationships from the data presented in Table 1. Certainly, the structural features of the acyl groups, at least when they are small and comparable in size, do not play a significant role in eliciting the biological response. One would assume that a positively charged N-terminus is disadvantageous for the biological activity; this seems to be confirmed by the investigation of the sarcosyl derivative (XV) possessing an alkylated N-terminus, whose activity is approximately the same as that of the glycine or glutamic acid derivative but less than that of the Nacylated peptides. On the other hand, simple elongation of the peptide chain, thus preserving the terminal positive charge as in the case of compound XI, leads to the enhancement of the biological activity, a well-known fact in the melanotropin field. It is also known that the [desamino-Tyr2]a-MSH(2-13) is less potent than ~-MSH(2-13) (2). Therefore, the most likely explanation of the observed changes in the biological activity seems to be the introduction o f a carbonyl group into the N-terminus, capable of forming new hydrogen bonds either with the receptor, or rather intramolecularly, leading to conformational changes required by the melanotropin receptor.
REFERENCES
1. Eberle, A. N. The melanotropins. Basel: Karger; 1988. 2. Eberle, A. N.; Hfibscher, W. a-Melanotropin labelled at its tyrosine 2 residue: synthesis and biological activities of Y-iodotyrosine2-y~25iodotyrosine2-,Y,5'-diiodo-tyrosine2- and (Y,5'-3H2)-tyrosine2-amelanotropin, and of related peptides. Helv. Chim. Acta 62:24602483; 1979. 3. Kappeler, H. Synthese von L-giutaminyl-L-histidyl-L-phenylalanylL-arginyl-L-tryptophyl-glycin. Helv. Chim. Acta 44:476-491; 1961. 4. Medzihradszky, K. The bio-organic chemistry of a-melanotropin. Med. Res. Rev. 2:247-270; 1982. 5. Medzihradszky-Schweiger, H.; Siili-Vargha, H.; B6di, J.; Medzihradszky, K. The melanocyte stimulating activity of N-nitroso-2chloroethyl-carbamoyl derivatives of a-melanotropin fragments. Coll. Czech. Chem. Commun. 53:2574-2582; 1988. 6. Otsuka, H.; Inouye, K. Synthesisofpeptides related to the N-terminal structure ofcorticotropin, lII. Synthesis of L-histidyl-L-phenylalanyl-
7.
8. 9. 10.
L-arginil-L-tryptophan, the smallest peptide exhibiting the melanocyte stimulating and the lipolytic activities. Bull. Chem. Soc. Jpn. 37:1465-1471; 1964. Sawyer, T. K.; Hruby, V. J.; Wilkes, B. C.; Draelos, M. T.; Hadley, M. E.; Bergsneider, M. Comparative biological activities of highly potent active-site analogues of a-melanotropin. J. Med. Chem. 25: 1022-1027; 1982. Schnabel, E.; Li, C. H. Synthesis of glycyI-L-histidyl-L-phenylalanylL-arginil-L-tryptophyl-glycine and its melanocyte-sfimulating activity. J. Biol. Chem. 235:2010-2012; 1960. Shizume, K.; Lemer, A. B.; Fitzpatrick, T. B. In vitro bioassay for the mdanocyte-stimulating hormone. Endocrinology 54:553-560; 1964. Siili-Vargha, H.; Botyfinszki, J.; Medzihradszky-Schweiger, H.; Medzihradszky, K. Synthesis of a-MSH fragments containing phenylalanine mustard for receptor studies. Int. J. Pept. Protein Res. 36:308-315; 1990.
