J. Org. Chem. 1986,51, 5153-5156 mg, 27%), and 4 (17 mg, 46%). 33: 'H NMR (CDC13)6 0.02 (s, 9 H), 1.27 (d, 2 H, J = 9 Hz), 1.60 (9, 3 H), 3.27 (s, 3 H), 3.30 (s, 3 H), 3.10-3.22 (m, 1H), 4.74-4.90 (m, 2 H), 6.80 (s, 1H); IR (neat) 1700,1660,1450cm-'; exact mass calcd for CI4HzaNzOzSi 280.1606, found 280.1599. 34: 'H NMR (CDC13)6 1.42 (s, 6 H), 3.30 (9, 3 H), 3.36 ( 8 , 3 H), 5.00 (d, 1 H, J = 17 Hz), 5.03 (d, 1 H, J = 9 Hz), 6.04 (dd, 1 H, J = 9, 17 Hz), 6.92 (s, 1 H); IR (neat) 1700, 1650,1350 cm-'; exact mass calcd for CllHI6N2O2208.1211, found 208.1201.
Acknowledgment. This work was supported by a Grant-in-Aid for Specially Promoted Research from the
5153
Ministry of Education of Japan (No. 61065003). We are grateful to Yamasa Shoyu Co., Ltd., for a generous gift of uridine derivatives. Registry No. 1, 40738-83-8; 2, 762-72-1; 3, 105183-69-5; 4, 874-14-6; 4 (5-flUOrO), 3013-92-1; 5, 1024-99-3; 6, 54-42-2; 7, 59240-49-2; 8,73-39-2; 9, 58-96-8; 10,951-78-0;11, 21428-19-3; 11 (R = I), 21418-68-8; 12, 105183-68-4; 13,99044-61-8;14,10518370-8; 15,18293-99-7;16,99044-62-9;17,105183-76-4;18,1192-37-6; 19, 105183-71-9; 20, 105183-73-1; 21, 625-27-4; 22, 105183-72-0; 23, 105183-74-2;29, 105183-75-3;33, 99044-64-1; 34,99044-63-0; PhCHzSiMe3,770-09-2;(Me(CH2)3)3SnCHzCH=CH2, 24850-33-7.
Preparation of 0,P-Dialkyl Analogues of Cysteine Suitable for Peptide Synthesis C. Freeman Stanfield, Wayne L. Cody,' and Victor J. Hruby* Department of Chemistry, University of Arizona, Tucson, Arizona 85721
Received April 7, 1986 A general method is described for the preparation of cysteine derivatives that are substituted with one or two alkyl groups at the p-carbon. The synthesis is based on the sulfenylation of N"-formyl-a,P-dehydroamino acid esters. The protected dehydro esters were synthesized by the condensation of ethyl isocyanoacetatewith a ketone. The sulfenylation of these compounds was accomplished by refluxing with phosphorus pentasulfide to form the intermediate thiazoline, which can be hydrolyzed to the hydrochloride salt of the free sulfhydryl amino acid by heating in acid. The free sulfhydryl amino acid salt was protected as the S-p-methylbenzylthioether, isolated as the zwitterion. The S-protectedamino acids were then protected as the N"-tert-butyloxycarbonylderivatives and are suitable for use in solution- or solid-phasepeptide synthesis.
Peptides comprise a large class of biologically active molecules, which, until recently, have been difficult to prepare synthetically. Within the past several years, advances in the chemistry of both solid- and solution-phase peptide synthesis have greatly facilitated the preparation of specific peptide ana1ogues.l Additionally, rational design of peptide hormones to possess specific biological properties (i.e., increased potency, prolonged activity, antagonism, and receptor specificity) is now f e a ~ i b l e . ~Since ,~ peptide hormones are generally highly flexible molecules, with a myriad of possible conformations-of which only one or a limited number may be responsible for the observed biological response-it is often necessary to reduce the number of possible conformations in order to induce a specific biological response or property. For the past several years, we have attempted to design "biologically specific" peptide hormones through conformational restriction of the mobility of the molecule (e.g., side-chain or backbone restriction). Specific examples of conformational restriction include the substitution of nonproteinogenic rigid or semirigid amino acids into a peptide hormone4 and cyclization of a peptide, either between individual side chains5 or between the N- and the C-terminus.6 In general, for an amino acid substitution to impart significant conformational constraint on a peptide, the amino acid itself must be conformationally restricted. This approach has been expanded upon and employed in our laboratory for the preparation of conformationally restricted disulfide-linked peptides by the substitution of &@-dimethylcysteine (penicillamine) for t Present address: Eastman Chemicals Division, Research Laboratories, Kingsport, T N 37662.
cysteine in e n k e ~ h a l i n ,somatostatin,8 ~ and o x y t ~ c i n . ~ Specifically in enkephalin, this substitution led to the preparation of a receptor-specific peptide hormone, [DPen2,~-Pen5]enkephalin(DPDPE), the most &selective enkephalin agonist known.7 In this molecule, conformational restriction is induced by disulfide bond formation between the penicillamine (@,@-dimethylcysteine) residues in positions 2 and 5 and by the geminal dimethyl groups. This method of conformational restriction has also been used in fragments of somatostatin to prepare analogues that exhibit a high degree of antagonistic activity a t the p opioid receptor.* As part of an investigation aimed a t preparing more highly constrained sulfur amino acids for incorporation into peptide hormones, we report a general and relatively simple synthesis of nonproteinogenic 0,Pdisubstituted sulfur amino acids. These amino acids have been subsequently orthogonally protected a t the a amine and the sulfur functionalities, such that they are suitable (1) Stewart, J. M.; Young, J. D. Solid Phase Peptide Synthesis; Pierce Chemical Co.: Rockford. IL. 1984. (2) Hruby, V. J.; Krstenansky, J. L.; Cody, W. L. Annu. Rep. Med. Chem. 1984,19,303. (3) Hruby, V. J. Trends Pharmacol. Scz. 1985, 6 , 259. (4) Roberts, D. C.; Vellaccio, F. In The Peptides: Analysis, Synthesis, Biology;Gross, E., Meienhoffer, J., Eds.; Academic: New York, 1983;Vol. 5, pp-341-449. ( 5 ) Sawyer, T. K.; Hruby, V. J.; Darman, P. S.; Hadley, M. E. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1751. (6) Mutulis, F. K.; Chipens, G. I.; Mutule, I. E.; Maccrops, G. H. Int. J. Pep. Protein Res. 1985, 26, 449. (7) Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burks, T. F. Proc. Natl. Acad. Sci. U.S.A. 1983,BO, 5871. (8) Pelton, J. T.; Gulya, K.; Hruby, V. J.; Duckles, S. P.; Yamamura, H. Proc. Natl. Acad. Sci. U.S.A. 1985,82, 236. (9) Schulz, H.; du Vigneaud, V. J. Med. Chem. 1966, 9, 647.
0022-3263/86/1951-5153$01.50/0 0 1986 American Chemical Society
5154 J . Org. Chem., Vol. 51, No. 26, 1986
Stanfield et al. Scheme I
CN - CH2- C02C2H5
+
,-.
NaH THF OOC'
H-CO-NH-C-CO,C$I,
It
H. p4s10
C$l6IA
R
~
&N\
c\s-c,
I R
H . ,cLC02C2Hs R
HCO-NH-CH-CO,C,H,
I
R-C-SH
I
R Q
Q
CI H,N-
CH-CO,H
I R-C-SH
I. Na/NH3 -78OC
0 0 H3N CH CO,
- -
+
I
2. CH3-Q14CH&I
R-C-S-CH,-C,H,-CH, I R
I
R ((CH3 S C -0CO-),O
-
(CH3 S C - OCO NH-CH - C W
dioxane I "p 211 pH 10.5 O°C
for solid- or solution-phase peptide synthesis. Previous efforts to prepare these amino acids, including (1) Michael addition of sulfur nucleophiles to suitably protected dehydro amino aciddo and (2) condensation of glycine equivalents with thioketones,'l met with only limited success. We report here the reaction of phosphorus pentasaide with Ne-formyl-protected dehydro amino acid esters (prepared by the methodology of Schollkopf et al.12J3) to prepare Ne-formyl-@,@-substitutedcysteine analogues (Scheme I). Following acid-catalyzed hydrolysis, the unprotected sulfur amino acids were suitably protected for either solution- or solid-phase peptide synthesis.
Results and Discussion The fully protected (suitable for peptide synthesis) 8,0-dialkylcysteine derivatives could be prepared in overall yields of 10% to 30% by starting from ethyl isocyanoacetate and utilizing the synthetic scheme outlined in Scheme I. The initial condensation of ethyl isocyanoacetate with a given ketone proceeded in only moderate (
