J. Ory. Chem., Vol. 38, No. 3, 1W3
TYROSINE IN PEPTIDE SYXTHESIS
591
Protection of Tyrosine in Solid-Phase Peptide Synthesis' DONALD YAMASHIRO AND CHOHHAOLI" The Hormone Research Laboratory, University of California, S a n Francisco, California 94121 Received August 24, 1972 iv"-tert-Butyloxycarbonyl-O-(o-bromobenzyloxycarbonyl)tyrosine has been synthesized and employed in the solid-phase peptide synthesis of the octapeptide Phe-Lys-Gln-Thr-Tyr-Ser-Lys-Phe, which occurs in the human growth hormone sequence. The new protecting group for tyrosine was found to be stable and gave no significant side product upon its removal in hydrogen fluoride. R f z0.23 In solid-phase peptide synthesis2 benzyl protection of the phenolic hydroxyl group in tyrosine has generally been employed. This protection is known to be unsatisfactory since it is not only unstable under the acidic conditions required for the removal of N"-Boc protection3-: but also yields a side p r o d u ~ t , 3-benzyl~,~ tyrosineJ4 when it is removed in hydrogen fluoride.'j l\Iodification of the tyrosine residue in HF has also been observed in solid-phase synthesis of tyrosinecontaining peptides.' We recently proposed use of the very stable Z(o-Br)8 group for the protection of the side chain of l y ~ i n e . ~ R e have now successfully applied it for similar protection of tyrosine in the solid-phase synthesis of the octapeptide Phe-Lye-Gln-Thr-Tyr-Ser-Lys-Phe (1) corresponding to amino acid residues 135-145 in the human growth hormone r n o l e c ~ l e . ~ Evidence ~~~ is presented that this protection is st'able and gives no signifiFRACTION NUMBER cant side product upon its removal in HF. Quantitative data on the stabilities of protecting Figure 1.-Partition chromatography of octapeptide 1 on Sephadex G-25; absorbance by Folin-Lowry analysis. groups commonly used in solid-phase synthesis have been obtained on acetylated amides of amino acids virtually quantitative yield (9Syo) of tyrosine. Octawith protected side-chain function^.^ A comparable peptide l , which has alrcady bccn syntliesizcd by solidtest was carried out n-ith Ne-acetyl-0-Z(o-Br)tyrophase procedurcs,: was resynthesized with the followsinamide by treatment with 50% TFA in CH2C12for 24 ing side-chain protecting groups: Z(o-Br) for Lys3 hr. As judged by tlc only 1% of the protection was and Tyr; Bzl for Scr and Tlir. lost as compared to standard benzyl protection where The finished peptidc was cleaved from tlic polymer at least 50% is lost. The new prot'ecting group was and dcprotected in HFGl 1 and purified as described completely removed in HF in 10 min at 0" and only previously.5 The partition chromatography on Sephaa single product was detected. Since the new prodex G-2,; is shown in Figure 1. Tlic Rfvalur of 0.23 tecting group forms a phenolic ester with tyrosine, the is in close agreement with thc valuc of 0.22 previously possibility of instability under basic conditions exists. reportedj for chromatography of 1 under these condiTherefore, the acetylated amide was treated with 10% tions. In tlic previous synthesis of 1, cithrr with Bzl diisopropylethylamine in D N F for 24 hr; only about protection of tyrosine or Bzl(nz-Br) protection, sig5% of the protection was lost,. nificant amounts of side products were observcd travelFor use of the new protecting group in peptide synling faster than 1 in the partition chromatography.12 thesis N"-Boc-0-Z (0-Br)Tyr was synthesized and isoIn the present synthesis only a minute trace of peptide lated as its dicyclohexylamine salt. The compound side product (Rf0.75) could be detected. Whcn pepwas treated in HF and amino acid analysis showed a tide 1 from the partition chromatography was then sub(1) This work was supported in part by the American Cancer Society, jected to chromatography on C~I-cellulose13only onc the Allen Foundation, and the Geffen Foundation. peak was obtained (I:igure 2 ) . Thr overall yield of (2) R . B. Merrifield, Biochemistry, 3, 1385 (1964). (3) D. Yamashiro, R. L. Noble, and C. 13. Li, presented a t the 3rd Amerihighly purified octapeptide 1 waq about SG% based on can Peptide Symposium, Boston, Mass., J u n e 1972. the starting Boc-l'he polymer, liighcr than the yields (4) 13. W.Erickson and R . B. Merrifield, presented a t the 3rd American prcvioudy attaincd. Peptide Symposium, Boston, Mass., .June 1972.
1
( 5 ) D . Yamashiro and C. H. Li.Int. J . P e p t i d e Protein Res., 4, 181 (1972). (6) S. Sakakibara, . ' 1 Shimonishi. Y . liishida, BI. Okada, and 13. Sugihara, Bull. Chem. Soc. J a p . , 40, 2164 (1967).
(7) R. Shapira, F. C-H. Chou, S. McKneally, E. Urban, and 12. F. Kibler. Science, 173, 736 (1971). ( 8 ) Symbols and abbreviations are in accordance with the recommenda-
tions of the IUP.1C-IUB Commission on Uiochemical Xomenclature, J . B i d . Chem., 247, 977 (1971). Other abbreviations: TFA, trifluoroacetic acid; D1.L diisopropylethylamine; CM-cellulose, carboxymethylcellulose; tlc, thin layer chromatography. (9) C. H. Li, J. 8. Dixon, a n d W.-I
