Int. J. Peptide Protein Ref . 25, 1985,515-519

Psndorphin Synthesis and biological activity of analogs with disulfide bridges

JAMES BLAKE, DAIGA M. HELMESTE and CHOH H A 0 LI

Laboratory of Molecular Endocrinology. University of California, Son Francisco, CA, USA

Received 1 1 October, accepted for publication 29 October 1984

Two analogs of human &endorphin (0-EP) which contain cystine bridges, [Cys"- C y ~ ~ ~ , P h e ~ ' ,Gly31 ]+EP (I) and [ Cys'6Cy~26,Phe27,GIy31 ] 4-EP (11). were syn- thesized by the solid-phase method. Peptides I and I1 were shown to contain 2-2.5 times the opiate receptor binding activity of &endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these

(Cam)'2*26, Phe2', Gly31 I , were shown to have approximately the same opiate C~s(Carn)*'.~~'Phe~~,Gly~~ ] and [Arg9r'9,w*28.29 , CYS- peptides, [kg9*19,24,28,29

receptor activity as &endorphin.

Key words: opioid-receptor binding assay; synthesis

r:.t brain membranes; solid-phase peptide

Previous structure-activity studies in this labora- tory have centered on the inclusion of cystine bridges in otherwise linear peptides such as corticotropin (1) and @endorphin (2,3) (Fig. 1) to determine the effect of the resulting confor- mational restrictions on biological activity. We observed that for the case of P-EP, th; cyclic analogs retained significant and sometimes enhanced biological activity (2-4). We now report the synthesis of two analogs of 8-EP which contian cystine bridges and two analogs which contain reduced alkylated cysteine residues. The opiate receptor binding activity of the four synthetic peptides is presented here.

RESULTS

The five cyclic analogs which we have pre- viously synthesized (2, 3) contained cystine

bridges between positions 7-26, 11-26, 14- 26, 17-26 and 21-26. For their combination of high receptor binding affinity and high analgesic potency, the peptides which contained cystine bridges between positions 14-26 and 17-26 were of special interest (4). Accordingly we decided to synthesize analogs with cystine bridges between positions 15-26 and 16-26 to determine the effect of a small structural change on biological activity. In addition we retained the substitutions of Phe2' and Gly" which have been shown to increase biological activity slightly (5 ) .

5 10 H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-

15 20 Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-

25 31 Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-OH

Abbreviations: ph-EP, human pendorphin; Cys(Cm), S-carboxymethylcysteine; Cys(Cam), S-carbamoyl- me th ylcysteine.

FIGURE 1 Amino acid sequence of human pendorphin.

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J. Blake et ul.

The peptides [ C y ~ ' ~ - C y s ~ ~ ,Phe2',Gly3' 1 $h-

EP (I) and [ C ~ s ' ~ - C y s ~ ,Phe2',Gly3'] gh-EP (11) were synthesized by the solid-phase method (6). Boc-Gly resin was subjected to conventional procedures ( 5 ) and the final protected peptide resin was treated with HF/anisole. The crude deprotected peptide was oxidized with K3Fe- (CNlk and the product was purified by chro- matography on CM-cellulose and partition chromatography on Sephadex G-50. The highly purified peptides I and I1 were characterized by paper electrophoresis HPLC (Fig. 2) and amino acid analysis (Table 1).

The synthetic peptides were assayed for their opioid-receptor binding activity in rat brain membranes and the results are sum- marized in Table 2. Each of the peptides (I and 11) possessed 2-2.5 times the binding affinity of Ph-EP. This is comparable to the results previously obtained for the 14-26 and 17-26 bridges peptides (4) and indicates that slight changes in the size of the peptide ring in the central part of PBP have only modest effects on opioid-receptor binding activity.

r I

I

0.3

E h c 0

1 - I I I I I

10 20 30 10 20 30 TIME (min)

FIGURE 2 HPLC of 20pg samples of peptides I-IV on a Vydac- 201TP column; 4.6 X 250mm. Elution was by a linear gradient of 10-50% isopropyl alcohol in 0.1% tri- fluoroacetic acid over a period of 30 min

576

Arg containing analogs. The second half of our study was suggested by the results from two previous studies. In the first of these we observed (2, 4) that a peptide analog which contained a cystine bridge between positions 11 and 26 had full opioid-receptor binding activity but only 5% analgesic potency. A similar dif- ferential effect on the two biological acitivities was observed for an analog in which all five lysine residues of Ph-EP were replaced by arginine (7). It occurred to us that if both of the above structural changes were incorporated into the same molecule the effect would be to give an analog with a high binding/analgesic ratio, and that such a peptide could be useful as an inhibitor of Ph-EP.

The method of solid-phase peptide synthesis was used to assemble peptide chains that con- tained cysteine residues in positions 11 and 26, or 12 and 26. However, when the peptides were cleaved and deprotected by reaction with HF/ anisole, followed by oxidation with K&(CN)6, none of the desired cystine-bridged analogs could be isolated. Apparently the presence of arginyl residues in the peptide chain prevented the folding necessary for the formation of intra- molecular disulfide bonds, and thus only inter- molecular disulfide bonds could be formed leading to the production of dimers and/or polymers. The extensive precipitation observed during oxidation supports this explanation.

Since the desired cystine-bridged analogs could not be obtained, we decided to isolate the reduced alkylated peptides. Accordingly when the peptide resins were treated with HF/ anisole the crude product was immediately alkylated with iodoacetamide and the highly purified products were isolated after chromato- graphy on CM-cellulose and partition chro- matography on Sephadex (3-50. The products

EP (111) and ' [Cy~(Cam)'~*~~,Arg~*'~*~~*~~*~~, Phe27,Gly31] -Ph-EP (IV) were characterized by paper electrophoresis, HPLC (Fig. 2) and amino acid analysis (Table 1).

Peptides 111 and IV were assayed for their opioid-receptor binding activity and the results are shown in Table 2. Each of the peptides has approximately the same activity as oh-EP. However, the previous report (7) that [Arg9>l9* 24*28*29] -0h-EP has three times the activity of

[Cys(Cam)' 9 l6 Arg9. 19*24*28*29$ Phe2' ,Gly3' ]-oh-

Synthetic Pendorphin analogs

TABLE 1 Amino acid analysis of the synthetic peptides

Peptide I Peptide I1 Peptide 111 Peptide IV Acid' Enzymeb Acid' Enzymeb Acid' Enzymeb Acid' Enzymeb

ASP 2.0 (2) 2.0 (2) 3.8 (4)' 4.0 (4)' Thr 2.8 (3) 1.9 (2) 2.9 (3) 2.1 (2)

Ser 1.7 (2) 1.8 (2) 1.7 (2) 1.9 (2) 1.5 (8)d 6.8 (7)d 1.2 (9)e 8.0 (9)e

Glu 1.9 (2) 1.0 (1) 2.0 (2) 1.0 (1) 1.0(1) 1.0(1) 2.2 (2) 1.2 (1) Pro 1.0 (1) 1.0 (1) 1.1 (1) 1.1 (1) 0.9 (1) 0.9 (1) 1.0 (1) 1.2 (1) t c y s 1.9 (2) 1.3 (2) 1.9 (2) 1.3 (2) - - - -

GlY 3.7 (4) 4.0 (4) 4.0 (4) 4.0 (4) 4.0 (4) 3.8 (4) 3.9 (4) 4.0 (4)

Val - 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 0.9 (1) Met 0.9 (1) 0.9 (1) 0.9 (1) 0.8 (1) 1.1 (1) 0.9 (1) 1.0 (1) 0.9 (1)

Ala 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) l .O(l) l .O(l) 1.0 (1) 1.0 (1)

I le 1.4 (2) 1.9 (2) 1.2 (2) 1.8 (2) 1.4 (2) 1.9 (2) 1.2 (2) 2.0 (2) Leu 2.0 (2) 2.1 (2) 2.0 (2) 2.0 (2) 2.0 (2) 2.0 (2) 2.1 (1) 2.0 (2) TYr 0.9 (1) 1.0 (1) 0.9 (1) 1.0 (1) 1.0 (1) 1.1 (1) 0.9 (1) 1.0 (1) Plie 2.9 (3) 2.8 (3) 3.0 (3) 2.8 (3) 2.9 (3) 2.8 (3) 3.0 (3) 2.6 (3)

5.2 (5) 5.1 ( 5 ) 5.3 (5) 5.1 (5) - - - - LYS - - - - 5.1 (5) 4.8 (5) 5.1 (5) 5.2 ( 5 ) Arg

*Hydrolysis in constant-boiling HCl; 22 h at 110". bTrypsin/chymotrypsin followed by amino peptidase M. 'Corresponds to sum of Asp + Cys(Cm). dCorresponds to sum of Asn + Gln + Thr + Ser. eCorresponds to sum of Asn + Gln + Ser + Thr + Cys(Cam). Cys(Cam) is partially destroyed during enzyme digestion; see Yamashiro et al. (13).

&-EP indicates that the incorporation of the Cys(Cam) residues in peptides I11 and IV reduces opioid-receptor binding potency.

EXPERIMENTAL PROCEDURES

Protected peptide resins corresponding to pep- tides I-IK Boc-Gly resin (1.2g, 0.60mmol) was treated by the following procedure: 1) four washings with methylene chloride; 2) one wash with 55% trifluoroacetic acid/methylene chlo- ride; 3) 15-min reaction with 55% trifluoroacetic acidlmethylene chloride; 4) two washings with methylene chloride; 5) three washings with 25% dioxane/methylene chloride; 6) repeat step 4; 7) 2-min reaction with 5% diisopropylethyl- aminelmethylene chloride; 8) repeat step 4; 9) repeat step 7; 10) five washings with methylene chloride; 1 1) 20-min coupling with 1.8 mmol of symmetrical anhydride of a Boc amino acid; 12) addition of 0.6 mmolN-methylmorpholine and

continued coupling for 20min; 13) three washings with 33% ethanol/methylene chloride.

Solvent wash volumes were 18 ml. Peptides I and I1 were synthesized from the same starting batch of Boc-Gly resin. After the coupling of Leu", the resin was divided into two equal parts which were used for the syntheses of peptides I and 11. Peptides I11 and IV were synthesized by a similar protocol. Side chain protecting groups were as follows: Ser, benzyl; Thr, benzyl; Glu, benzyl; Lys, 2chlorobenzyl- oxycarbonyl; Tyr, benzyloxycarbonyl; Arg, p - toluenesulfonyl; Cys, 3,4-dimethylbenzyl. The same cysteine protecting group was used in the synthesis of all the synthetic peptides (14%.

After the coupling of Tyr' , the peptide resin was subjected to steps 1-5, washed with ethanol and dried.

/Cys' 5-Cys26,Phe27Gly3'] -&-endorphin (I). Peptide resin (371 mg) was treated with 0.74 ml

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J. Blake et al.

TABLE 2 Opioid-receptor binding activity of synthetic &,-endorphin analogs

Synthetic IC,, * Relative peptides ( nM) potency

Clh-EP [Cys"€ys" ,Phe7' ,Gly3' ] -PwEP** [Cys"-Cysz6 ,Phe",Gly3' 1 -&,-EP (I) [Cys" Cys" ,Phe" ,Gly3' ] %-EP (11) [Cys'' Cy% ,Phe" ,Gly3' 1 -&-EP**

0.65 f 0.080

0.26 f 0.003 0.29 f 0.030

100 360 250 224 200

100 100 I9

*Man f SEM. **Taken from ref. 4.

anisole plus 8 ml HF at 0' for 1 h. The HF was evaporated at 0" and the peptide-resin mixture was washed with ethyl acetate. Peptide was dissolved in 6 m10.5 N acetic acid, 1 14 ml water was added and 1 N ammonium hydroxide was added to give pH 8.2. An aliquot (100fi) was analyzed with the Ellman reagent (10) and indicated the presence of 0.069 mmol of thiol

added (ca. 6.5ml) until a slight yellow color persisted in the peptide solution and an aliquot showed the absence of detectable thiol S-H. There was extensive precipitation during the oxidation. Acetic acid was added to give pH 4.5 and the mixture was centrifuged. The super- natant was lyophilized to a residue that was chromatographed on Sephadex G-10 in 0.5N acetic acid, The crude peptide that was isolated was chromatographed on CM-cellulose as pre- viously described (1 1) to give 17.3 mg peptide. The peptide was subjected to partition chro- matography (12) on Sephadex G-50 in the system n-butanol:pyridine:0.07 M ammonium acetate (5:3:10) to give 9.0mg peptide I at

Paper electrophoresis of peptide I at pH 3.7 and 6.7 gave single ninhydrin, chlorine spots at R P 0.65 and 0.55, respectively. Analysis by HPLC is shown in Fig. 2 and amino acid analysis of acid and enzyme hydrolysate is shown in Table 1.

S-H. A 0.01 M solution Of K,Fe(CN6 Was

Rf 0.47.

/ C y ~ ' ~ - C y s ~ ~ , P h e ~ ~ , -&-endophin (II). A sample (369 mg) of peptide resin was treated as described in the preparation of peptide I. A yield of 9.5 mg peptide could be isolated at Rf 0.43 after partition chromatography on Sephadex G-50 in the system n-butanol: pyridine:0.03 M ammonium acetate (5:3:10). Paper electrophoresis at pH 3.7 and 6.7 gave single ninhydrin, chlorine positive spots at R P 0.58 and 0.53, respectively. Analysis by HPLC is shown in Fig. 2 and amino acid analysis of acid and enzyme hydrolysates is shown in Table 1.

/Os( Cam)' 1 s 16,Argg* 19 * 249 28*29,phe27, Gly31/- P,, - endorphin (III). A sample (400mg) of peptide resin was treated with 0.8 ml anisole plus 8 ml HF at 0" for 1 h. HF was evaporated at Oo, and the peptide-resin mixture was washed with ethyl acetate. Peptide was disolved in 3 ml 6 M guanidine hydrochloride/Od M K2P04. The pH was adjusted to 8.3 and a lop1 aliquot was analyzed for thiol S-H by the Ellman reagent. Then 37 mg iodoacetamide in 0.3 ml water was added to the stirring peptide solution. Sodium hydroxide was added to maintain pH 8.3. After 6min there was no detectable thiol S-H. The solution was chromatographed on Sephadex G-10 in 0.5 N acetic acid and the crude peptide that was isolated was chromatographed on CM- cellulose to give 40 mg peptide. Partition

578

chromatography of 83 mg peptide (isolated from two experiments) on Sephadex G-50 in the system n-butano1:O.l M ammonium acetate (1:2) gave 10.8mg peptide I11 at Rf 0.26.

Paper electrophoresis at pH 3.7 and 6.7 gave single ninhydrin, chlorine positive spots at Rfys 0.50 and 0.48, respectively. Analysis by HPLC is shown in Fig. 2 and amino acid analysis of acid and enzyme hydrolysates is shown in Table 1.

/QJ,~( Cam 12.16 *A rgQ, 19924.28 ~ ~ ~ , P h e 2 ’ , cly 3 ’1 -oh- endorphin (ZV). A sample (393 mg) of peptide resin was treated as described in the preparation of peptide 111. A yield of 9.1 mg of peptide IV was isolated at Rf 0.32 from partition chroma- tography on Sephadex G-50 in the system n-butano1:O.l M ammonium acetate (1 :2). Paper electrophoresis at pH 3.7 and 6.7 gave single ninhydrin, chlorine positive s ots at

HbLC is shown in Fig. 2 and amino acid analysis of acid and enzyme hydrolysates is shown in Table 1.

RLYS 0.53 and 0.48, respectively. An s ysis by

Bioassay. The opiate-receptor binding assay was performed with a rat brain membrane fraction using tritiated &-EP (14) as a primary ligand (0.4 nM final concentration) and synthetic &-EP (15) as standard competing ligand as described (16,17).

ACKNOWLEDGMENTS

We thank W.F. Hain for technical assistance. This work was supported in part by the National Institutes of Health (GM-30072, and GM-2907) and the National Institute of Drug Abuse (DA-03434). D.M.H. is a recipient of a Fellowship from the Medical Research Council of Canada, and is on leave from the Clarke Institute of Psychiatry. Toronto, Canada.

Synthetic Pendorphin analogs

REFERENCES

1. Blake, J., Rao, A.J. & Li, C.H. (1979) Int. J. Peptide Protein Res. 13, 346-352

2. Blake, J., Chang, W . C . & Li, C.H. (1979) Int. J. PeptideProtein Res. 14, 275-280

3. Blake, J., Ferrara, P. & Li, C.H. (1981) Int. J. Peptide Protein Res. 17,239-242

4. Li, C.H. (1981) in Hormonal Proteins and Peptide (Li. C.H., ed.), vol. X, pp. 2-34, Academid Press, New York

5. Blake, J., Tseng, L.-F., Chang. W . C . & Li, C.H. (1978) Int. J. Peptide Protein Rex 11, 323- 328

6. Merrifield, R.B. (1963) J. Am. Chem. SOC. 85,

7. Blake, J., Li, C.H. & Nicolas, P. (1982) Int. J. Peptide Protein Res. 20, 308 - 3 1 1

8. Blake, J. & Li, C.H. (1975) Inr. J. Pepride Protein Res. 7,495-501

9. Konig, W. & Greiger, R. (1970) Chem. Ber. 103,

10. Ellman, G.L. (1959) Arch. Biochem. Biophys.

11. Li, C.H., Lemaire, S., Yamashiro, D. & Doneen, B.A. (1976) Biochem. Biophys. Res. Commun.

12. Yamashiro, D. (1 980) in Hormonal Proteins and Peptides (Li, C.H., ed.), vol. I X , pp. 25-107, Academic Press, New York

13. Yamashiro, D., Noble, R.L. & Li, C.H. (1973) J. Org. Chem. 38,3561-3565

14. Houghten, R.A. & Li, C.H. (1978) Int. J. Peptide Protein Res. 12, 325-326

15. Li, C.H., Yamashiro, D., Tseng, L.F. & Loh, H.H. (1977)J. Med. Chem. 20,325-328

16. Ferrara, P. & Li, C.H. (1980) Int. J. Peptide Pro- tein Res. 16, 66-69

17. Hammonds, R.G., Nicolas, P. & Li, C.H. (1982) Int. J. Peptide Protein Res. 19,556-564

Address:

Dr. James Blake Laboratory of Molecular Endocrinology 1018 HSE University of California San Francisco, CA 94143 USA

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