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Solid-Phase Synthesis of Peptides via a,ß-Unsaturated Amino Acids. Incorporation of the Amide Gronp in encZo-Positions

Kosaku Nöda* and Erhard Gross** Section on Molecular Structure, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205, U.S.A. Dedicated to Professor Helmut Holzer on the occasion of his 60th birthday Z. Naturforsch. 36b, 1345-1347 (1981); received July 15, 1981 Amide Group, Dehydroalanine, Peptide, Solid-Phase Synthesis

Dehydroalanine is introduced as pseudo-protecting group for the to-amide function of Asn and Gin in solid-phase peptide synthesis. Using Boc-X(Dha-NHMe)-OH (X = Asp or Glu), the model peptides, L-Leu-L-Asn-Gly-NH2 and L-Leu-Lr-Gln-Gly-NH2, were synthesized.

In previous papers [1, 2], we reported a new method of peptide amide synthesis using Dha [3] as protecting group for the amide function and vehicle for the attachment of the growing peptide chain to the support in solid-phase peptide synthesis [4]: Pept idyl-Dha assembled on the solid support was easily cleaved at the N- a C bond of Dha under mildly acidic conditions to give the peptide amide (Fig. 1).

CH2

PEPTIDE-CO-NH-<Ü-CO-ro— 1 eq. H20 w HCl/AcOH CH3

PEPTIDE -CO-NH2 + C-CO-resin A

Fig. 1. Solid-phase synthesis of peptide amides via dehydroalanine.

Now, this novel approach has been employed successfully for the incorporation of the amide group of Asn and Gin in emZo-positions using Boc-Y(Dha-NHMe)-OH (1) ( la , X-Asp; lb , X-Glu). This paper describes the preparation and

* Visiting Scientist from Fukuoka Women's Uni-versity, Fukuoka, Japan.

Boc-X-OBzl I

Boc-MCys-OH I HOSu, DCC I NH2-Me

Y Boc-MCys-NHMe (2)

j DCC J-MCys-NHMe

Boc-X-OBzl I oxidation

(3 a) (X = Asp) (3 b) (X = Glu)

-NHMe Boc

J~MCys(02) -X-OBzl

I NaOH

TDha-NHMe Boc-X-OH

(la) (X = Asp) ( l b ) (X = Glu)

Fig. 2. Synthesis of Boc-Asp(Dha-NHMe)-OH and Boc-Glu(Dha-NHMe )-OH.

properties of 1 and the solid-phase synthesis of model peptides with Asn or Gin using 1.

The route to the synthesis of 1 is outlined in Fig. 2. Boc-L-MCys-NHMe (2) (m.p. 85-86 °C; R/A 0.74 [5]; CHN) was prepared by coupling Boc-L-MCys-OH (m.p. 87-89 °C) [6] and methyl-amine using the N-hydroxysuccinimide ester (yield, 88%). Removal of the Boc-group of 2 with 2 N HCl in ethyl acetate and coupling with Boc-L-Asp-OBzl [7] or Boc-L-Glu-OBzl [8] afforded Boc-Ir-Asp(L-MCys-NHMe)-OBzl (3a) (71 % ; m.p. 92-94 °C; R/A 0.71; CHN). Compounds 3 were treated with hydrogen peroxide in glacial acetic acid to give the sulfones, which, without further purification, were converted to 1 by treatment with NaOH. The products contaminated by a few minor components were purified by partition chromato-graphy on a silica gel column using a solvent system consisting of chloroform-methanol-acetic acid (10:1:1, v/v) l a (41%; m.p. 132-135 °C; Rf A0.34; CHN); l b (51%; m.p. 117-120 °C: iü/AO.38; CHN).

leg. H20 HCl/AcOH

CH CO-NH-(Ü-CO-NHMe

(CH2)„ Boc-NH-iH-COOH

la, n = 1 l b , n = 2

CO-NH2

(CH2)n CH3

NHa-^H-COOH + 0 = dj-C0-NHMe Asn, n = 1 Gin, n = 2

Fig. 3. Acid-treatment of Boc-Asp(Dha-NHMe)-OH and Boc-Glu (Dha-NHMe )-OH.

BOC-L-ASP- /i- DH A-CON HCHg + 1 N HCI/AcOH; 55'C

Boc-L-GLU- K-DHA-CONHCH3 + 1 N HCI/AcOH; 55'C

120 0 TIME (min)

Fig. 4. Conversion of Boc-Asp(Dha-NHMe)-OH to Asn (left) and Boc-Glu(Dha-NHMe)-OH to Gin (right): conditions: 1 N HCl in glacial acetic acid, 1 equivalent of water, 55 °C.

Treatment of 1 a or 1 b with IN HCl in glacial acetic acid in the presence of one equivalent of water at 55 °C [9] cleaved the peptides at the N~aC bond of Dha and removed the Boc-group to give Asn and/or Gin (Fig. 3). Quantitative studies using the amino acid analyzer showed that the conversion of 1 a to Asn and I b to Gin was complete within 30 and 50 min, respectively (Fig. 4).

Using the Dha-derivatives, l a and lb , the model peptides, H-L-Leu-L-Asn-Gly-NH2 (4 a) and H-L-Leu-L-Gln-Gly-NH2 (4b), were synthesized via the solid-phase method. Following the attach-ment of Boc-Gly-Dha-OH [1] to the chloro-methylated styrene-divinylbenzene copolymer, two successive cycles of solid-phase synthesis with l a or l b and Boc-L-Leu-OH were carried out under the following reaction conditions: (1) CH2Cl2 wash (1.5 min X 4); (2) deprotection with 25% trifluoro-acetic acid in CH2CI2 (1.5 min x l , 3 0 m i n x l ) ; (3) CH2CI2 wash (1.5 m i n x 5); (4) neutralization with 10% triethylamine in CH2CI2 (1.5 min x l , 10 min x 1); (5) addition of Boc-amino acid (4-fold excess) in CH2CI2; (6) coupling with N,N'-dicyclo-hexylcarbodiimide (4-fold excess, 2.5 h); (7) CH2Cl2 wash (1.5 m i n x 4); (8) EtOH wash (1.5 minx 3). Treatment of the resulting Boc-L-Leu-L-Asp(Dha-NHMe)-Gly-Dha-resin (5 a) or Boc-L-Leu-L-Glu(Dha-NHMe)-Gly-resin (5 b) with 1 N HCl in glacial acetic acid in the presence of two equivalents of water (55 °C; 90 min) afforded the desired tripeptides with the simul-taneous incorporation of the amide functions at the carboxyl-terminal residues and the w-carboxyl groups of Asp and Glu and the removal of the Boc-group (Fig. 5). After purification by partition chromatography on silica gel using the solvent system n-butanol-acetic acid-water (4:1:1, v/v), the products were shown to be homogeneous on thin-layer chromatography: 4a (68%; i?/B 0.31: RfC

CH2

CO-NH-C-CO-NHMe

(^H2)n CH2

Boc-Leu-NH-CH-CO-Gly-NH-C-CO-O-resin 2eqs. H 2 0 | HCI/AcOH

CO-NH2

(CH2)„ I

H-Leu-NHCH-CO-Gly-NHa 4 a , n = 1; 4b , n = 2 Fig. 5. Solid-phase synthesis of Leu-Asn-Gly-NH2 and Leu-Gln-Gly-NH2 via dehydroalanine.

Table I. Amino acid analysis of L-Leu-L-Asn-Gly-NH2 and L-Leu-L-Gln-Gly-NH2 after acid hydrolysis and aminopeptidase M (AP-M) digestion.

Leu-Asn-Gly-NH2 Leu-Gln-Gly-NH2 Amino acid Acid AP-M Acid AP-M

hydrolysis hydrolysis

Asp 0.94 _ _ _ Asn - 0.96 - -

Glu — - 0.96 -

Gin - — — 0.95 Gly 0.98 1.01 0.93 Leu 1.00 1.00 1.00 1.00 ammonia 2.25 1.07 2.10 1.05

0.63); 4b (49%; Rf B 0.33; Rf C 0.68). The identities of the products were established by amino acid analysis of the acid hydrolysates and the amino peptidase M digests [10] as shown in Table I.

[1] E. Gross, K. Nöda, and B. Nisula, Angew. Chem. Int. Ed. 12, 664 (1973).

[2] E. Gross, K. Nöda, and S. Matsuura, in Proc. 13th Eur. Peptide Symp., Kiryat Anavim, Israel, April 28-May 3, 1974 (Y. Wolman (ed.): p. 403, John Wiley and Sons, New York 1975.

[3] Abbreviations: Dha, dehydroalanine; MCys, S-methylcysteine; HOSu, N-hydroxysuccinimide; DCC, N,N'-dicyclohexylcarbodiimide.

[4] R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963).

[5] Thin-layer chromatography was carried out on silica gel G (Merck) with the following solvent systems: A, chloroform-MeOH-AcOH (85:10:5);

B, n-butanol-AcOH-water (4:1:1); C, n-butanol-AcOH-pyridine-water (4:1:1:2).

[6] Broadbent et al. reported this compound to be an oil: W. Broadbent, J. S. Morley, and B. E. Stone, J. Chem. Soc. (C) 1967, 2632.

[7] J. Halstrom, O. Schou, K. Kovacs, and K. Brun-feldt, Hoppe-Seyler's Z. Physiol. Chem. 351, 1576 (1970).

[8] E. Schroder and E. Klieger, Ann. Chem. 673, 196 (1964).

[9] E. Gross and J. L. Morell, J. Am. Chem. Soc. 93, 4634 (1971).

[10] R. J. Delange and E. L. Smith, "The Enzymes", (P. D. Boyer, ed.): p. 102, Academic Press, New York, NY 1971.