Int. J . Peptide Profein Res. 7 , 1975, 495-501 PVLlished by Munksgaard, Copenhagen. Denmark No'part may be reproduced by any process without written permission from the author(s)

T H E SYNTHESIS AND BIOLOGICAL ACTIVITY O F [165, 182, 189-S-CARBAMIDOMETHYLCYSTEINEI-HUMAN GROWTH HORMONE-(140-191)* JAMES BLAKEand CHOHHAOLI

Hormone Research Laboratory, University of California, San Francisco, California, U.S.A.

Received 14 April 1975 The peptide [ 165, 182, I89-S-carbamidomethylcysteine]-human growth hormone-(140191) has been synthesized by the solid-phase method. The complement fixation, lactogenic and growth-promoting activities of the synthetic peptide were comparable to that of [165, 182, 189-S-carbamidomethyIcysteine]-humanzrowth hormone-(141-191)that was derived from the native hormone.

characterization of [Cys(Cam)16svl E 2lE9]-HGH(140-191) (I). The amino acid sequence of peptide I is shown in Fig. 1. An additional amino acid was added beyond the sequence of the peptide derived from HGH to avoid the difficulties inherent in working with any peptide containing an amino terminal glutamine, i.e., the tendency of the amino terminal glutamine residue to cyclize to a pyroglutamyl residue and the uncertainty this confers on the structural identity of the peptide product. Peptide I was synthesized by the solidphase method ( 1 l), using the modified carbodiimide coupling procedure (12, 13). Instead of adding equimolar amounts of Boc amino acid and DCC to the peptide resin and allowing these two reagents to react in situ (1l), three equivalents (with respect to the peptide resin) of symmetrical anhydride (12) were added to the peptide resin. The advantage of this procedure is that it reduced the extent of chain termination during peptide synthesis (Yamashiro & Noble, unpubl. results). Moreover, it has been shown that this procedure * Paper 43 in the Human Pituitary Growth Hor- can be used to couple Boc-Gln without danger of mone series. Abbreviations: HGH, human growth dehydrating the amide side-chain (14). Dimethylhormone; Boc, t-butyloxycarbonyl ; Boc-AA, N"-Boc amino acid; DMF, dimethylformamide; DCC, formamide (15 ) and diisopropylethylamine (Yamdicyclohexylcarbodiimide;TFA, trifluoroacetic acid; ishiro & Blake, unpubl. results, 16) were added to DIA, diisopropylethylamine; Cys(Cm), S-carboxy- the peptide resin during coupling (Table 4, steps methylcysteine; Cys(Cam), S-carbamidomethyl- 1l b and 1lc) because they were found to aid the coupling reaction. cysteine; Tos, p-toluenesulfonyl. Continuing investigations in this and other laboratories have been directed towards the elucidation of the structural requirements for biological activity of HGH and other mammalian growth hormones (1, 2). This problem has been investigated either by modification of the sidechain residues of the intact hormone (l), fragmentation of the hormone by enzymatic means (3-9, or chemical synthesis of peptide fragments that have small, but measurable biological effects (6-8). It was recently observed (4) that plasmin digestion of HGH followed by reduction and alkylation gives two peptides which exhibit measurable response in the tibia and crop sac and [Cys assays: [Cy~(Cam)~~]-HGH-(1-134) (Cam) 5 * 8 2 * 89]-HGH-(141-19 1). Both peptides possess immunological activity as determined by radioimmunoassay and complement fixation, also gives a and [Cys(Cam)s3]-HGH-(1-134) precipitin line with antiserum to HGH (9) and possesses metabolic activities of the natural hormone (10). This paper reports the synthesis and

495

JAMES BLAKE AND CHOH H A 0 LI

H2N-Lys-Gln-Thr-Tyr-Ser-Lys-Phe-Asp-Thr-Asn 140

Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-L ys-Asn150 Tyr-Gly-Leu-Leu-Tyr-Cys(Cam)-Phe- Arg-Lys-Asp-

FIGURE I

160

The amino acid sequence of [Cys(Cam)16s* 1 8 z , ls9]-HGH-

Met-Asp-Lys-Val-GIu-Thr-Phe-Leu-Arg-Ile170

(I 40- 191).

Val-Gln-Cys(Cam)-Arg-Ser-Val-G lu-Gly-Ser-Cys(Cam)180

Gly-Phe-OH 190

Treating the protected peptide resin correspondR f 0.39 ing to the sequence of peptide I with liquid HF (17,18) gave the crude peptide, which was then 0.3 1 purified by chromatography on Sephadex G-10 and G-50, isoelectric precipitation, and partition chromatography (Fig. 2a) on Sephadex G-50(19, 20). In purification by isoelectric precipitation we took advantage of the low solubility of peptide I at neutral pH and the relatively higher solubility of some of the smaller peptide impurities. Amino 8 h O*'t acid analysis of side products'obtained from the last two separation methods (see Experimental J I I I I I Procedures) showed that the amino acids concenR f 0.32 trated in the amino terminal part of peptide I (Ser, Thr, Asp, Ala, Leu, Tyr) were lower n than expected. This indicated that the side products were a mixture of truncated peptides corres0.3 ponding to the carboxyl region of peptide I. Determination of the amino terminal in these side products by the dansyl technique (22) showed the expected amino terminal amino acid for peptide I, lysine, and about 10% tyrosine Thus, most of the peptide side products were blocked at their amino terminuses and probably were the result of trifluoroacetylation occurring during the coupling reaction (Yamashiro & Noble, unpubl. results, 23). L L I 1 I I I 10 20 30 40 50 60 The final product, peptide I, was shown to be highly purified by amino acid analysis (Table l), TUBE NUMBER partition chromatography (Fig. 2b), and thin FIGURE 2 layer chromatography (Fig. 3). Fig. 4 indicates that the synthetic peptide (a) Lower: Partition chromatography of 40 mg of possesses complement fixing activity comparable crude peptide I on Sephadex G-50 in the system 0.52% trichloroacetic acid in sec-butanol :0.029 N HCI : to that of [Cys(Cam)16s-1 8 2 . '89]-HGH-(141-191) as revealed by the microtechnique of Wasserman acetic acid (145:205:5); column, 2.56 x 36 cm. The eluant volume per tube was 4.7 ml and the peptide & Levine (24). Prolactin activity of peptide I in concentration was measured by the method of Lowry the pigeon crop sac test (25) was also comparable et al. (21). (b) Upper: Partition chromatography of 1 to thenatural fragment as shown inTable 2. When mg of peptide I on Sephadex (3-50 in the same system peptide I was assayed for its growth-promoting described above; column, 1 . 0 6 ~ 17 cm. The eluant activity by the tibia test in hypophysectomized volume per tube was 0.55 ml.

I-

6

5-

t

IJ

496

d

SYNTHESIS AND BIOLOGICAL ACTIVITY OF

HGH-(140-191)

TABLE 1 Amino acid analysis" of synthetic [Cys(Cam) 165.

182*

1-HGH-(140-19 I )

Amino acid

Found

Theory

LYS

5.0

5

His Arl3 Cys(Cm) ASP Thr Ser

1 .o

I

3.2 3.1 8.2 3.1

3 3 8 3 4 4 3 1 3 1 1 5 3 4

Glu

GlY Ala Val Met Ile Leu TYr Phe a

3.7

4.0 3.1 1 .o 2.9 1 .o 0.7b 5.0 3.0

4.0

22 h hydrolysate. Amino acid analysis of a 69 h hydrolysate gave Ile 1.o.

=

rats (26), it was found that it had measurable activity but was lower than that exhibited by the natural fragment (Table 3).

FIGURE 3

T.1.c. of synthetic [Cys(Cam)165* 1 8 2 * 189]-HGH-(140191) (right) and a derivative of the native hormone, [Cys(Cam)16sv 1 8 2 , 189]-HGH-(141-191) (left), on silica gel in the solvent system n-butanol: pyridine: acetic acid: water ( 6 : 6 :1.2:4.8). Peptide spots were detected by ninhydrin spray.

EXPERIMENTAL PROCEDURES

NWoc-Gly-Phe Resin by limited coupling. Bocphenylalanine resin (3.88 g, 0.50 mmol/g) (20) was deblocked and neutralized by the standard procedure described below, and was then treated for 15 min with 1.6 mmol of Boc-glycine and 1.6 mmol of DCC in 32 ml of methylene chloride. The resin was washed with methylene chloride and the amine content was 0.1 1 mmollg, as determined by the Gisin test (27). Treating the resin with 10% acetic anhydride in methylene chloride for 15 min gave a resin with an amine content of O.OOO1 mmol/g. A small sample of dipeptide resin was deblocked with trifluoroacetic acid and neutralized, and the amine content was 0.34 mmol/g. Another sample of dipeptide resin was treated with propionic acid-conc. HCI (1 :1) for 19 h at 1 lo", and amino acid analysis of the hydrolysate by the procedure of Spackman et al. (28) gave 0.32

mmol of glycine and 0.52 mmol of phenylalanine per gram of resin. Protected peptide resin of [Cys(Cam)165s' "'* 189]-HGH-(140-191).Boc-Gly-Phe resin (1.47 g, 0.48 mmol of dipeptide) was subjected to 50 cycles of synthesis on the Beckman Model 990 peptide synthesizer according to the procedure outlined in Table 4.The Boc group gave N"-protection for all amino acids, and side-chain protection was as follows: Ser, 0-benzyl; Thr, 0-benzyl; Tyr, O-obromobenzyloxycarbonyl ; Glu, y-benzyl ester; Asp, 8-benzyl ester; Arg, NG-p-toluenesulfonyl; Lys, N"-o-bromobenzyloxycarbonyl; and His, N'"-Boc. The following procedure was used to prepare the Boc amino acid symmetrical anhydrides. Three mmol of Boc amino acid were dissolved in 9 ml of methylene chloride (Table 4) and the solution was cooled to 0".Then, 1.5 mmol of DCC 497

JAMES BLAKE AND CHOH H A 0 1-1

100

80 Z

0 c a

60

c

zw

FIGURE 4

5

M icrocomplement fixaticn curves obtained with [Cys (Cam)165~ 182. '89]-HGH(141-191) (0-0) or synthetic [ C y ~ ( C a m ) ' ~ ~ .I8']-HGH(140-191) (0-0) and guinea pig antiserum to [Cys(Cam) 165. 1 * 2 * 1*9]-HGH-(141-191) diluted 1/900.

2

Q

z

0

40

U

x 20

I

I

I

100

300

500

A NTlGEN,n g TABLE3

TABLE 2 Prolactin activity of HGH derivatives in the pigeon crop sac assay

Growth-promothg activity of HCH derivatives as assa-vedby the rat tibia test

Preparation

Preparation

Total dose Response" P-value (Pg)

Saline 0 [Cys(Cam) 5 * 8 2 * 189]-HGH-(141-191) 120 Synthetic [Cys(Cam) 165. 182, 189 1-HGH(140-190) I20 HGH

6

10.750.2

Total dose Response" Fvalue (Pep)

Saline 0 [Cys(Cam) 6 5 8 2 . 15.351.5