Int. J. PeptideProtein Rex 20,1982, 308-311

p -Endorphin : Synthesis and properties of analogs with replacement of lysine residues by arginine

JAMES BLAKE, CHOH HA0 LI and PIERRE NICOLAS

Hormone Research Laboratory, University o f California, San Francisco, CA, USA

Received 4 January, accepted for publication 30 March 1982

Human Pendorphin analogs, [Arg” 19i24*28*29]Q-endorphin (I) and [Arg24*28r29 1- /%endorphin (11), have been synthesized by the solid-phase method. Peptide I1 had 86% of the analgesic potency and 216% of the receptor binding activity of the parent molecule. Peptide I had only 18% analgesic potency but its binding activity was more than three time greater than that of human Pendorphin. Key words: analgesia; opiate receptor binding activity; rat brain membrane; solid-phase

peptide synthesis.

Studies on substitution, omission, or chain length analogs of P-endorphin (P-EP, Fig. 1) have shown that some of the lysine residues are important for biological activity. Thus, bioassay of peptides of varying chain length (1) has shown the importance of Lys-28,29, and sub- stitution of Ala for Lys-19 has demonstrated the significance of this residue for biological activity (2). In contrast, studies of omission analogs (3) have shown that most of the neutral amino acid residues at positions 6-31 are relatively less important for biological activity. To further delineate the significance of the lysine residues, we have synthesized two analogs of Ph-EP in which three or five of the lysine residues have been replaced by arginine. The opiate receptor binding activity and analgesic potency of these peptides is reported here.

RESULTS AND DISCUSSION

The peptide analogs [Arg9*’9*24p28329 IQ-EP (1)

Abbreviations: Ph-EP, human p-endorphin; Boc, terf.- butyloxycarbonyl.

and [Arg24,28*29] Q-EP (11) were synthesized by the solid-phase method (4) as previously des- cribed (2). Boc-Glu(Bz1) resin was alternately deblocked in 5 5% trifluoroacetic acid/methylene chloride, neutralized with 5% diisopropyl- ethylamine, and coupled to the symmetrical anhydrides of the Boc-amino acids. The final peptide resin was treated with liquid HF, and the desired products were obtained after chromatography on CM-cellulose and partition chromatography (5) on Sephadex G-50. The peptides were characterized by paper electro- phoresis, thin-layer chromatography and amino acid analysis of acid and enzyme hydrolysates (Table 1).

The synthetic analogs were assayed for anal- gesic potency and opiate receptor binding activity and the results are summarized in Table 2. It is seen that peptide I1 has almost the full analgesic potency of Ph-EP (see Fig. 2) and approximately twice the binding activity. Therefore, the lysine residues at positions 24,28,29 may be substituted with arginine without deleterious effect on the biological activity. The data for peptide I indicate that

308 0367-8377/82/090308-04 $02.00/0 0 1982 Munksgaard, Copenhagen

TABLE 1 Amino acid composition of the synthetic peptides

] Qh-EP I Arg ' '" 29 I-ph-EP

Acida Enzyme Acid Enzyme

[~,,~9,19.24,28,19

Asp 2.1(2)' 2.0(2) Thr 3.0(3) 7.3(8)d 6.9 (8)d Ser 1.9(2) 1.8 (2) Glu 3.1(3) 2.2(2) 3.0(3) 2.1 (2) Pro 1.0(1) 0 .2(1)~ I.O(I) 0 . 2 ~ ) ~ Gly 3.1(3) 3.0(3) 3.0(3) 3.0(3) Ala 2.0(2) 1.9(2) 2.0(2) 2.0(2) Val 1.0(1) 1.0(1) 1.0(1) 1.1(1) Met 1.0(1) 1.0(1) 1.0(1) 1.0(1) Ile 1.1(2) 1.8(2) 1.2 (2) 1.8 (2) Leu 2.1(2) 1.4(2) 2.0(2) 1.3(2) Tyr 2.0(2) 2.0(2) 2.0(2) 2.1(2) Phe 2.0(2) 2.0(2) 1.9 (2) 1.9 (2)

Arg 5.4(5) 4.7(5) 3.1(3) 2.9(3)

aHydrolysis in constant boiling HCI 22 h at 110". bTrypsin/chymotrypsin followed by leucineamino- peptidase. 'Numbers in parentheses are the theoretical values.

Represents the sum of Asn + Gln + Thr + Ser. eLow values are due to the resistance of prolyl peptides to enzymatic digestion.

LY s - - 2.0(2) 2.0(2)

1 5 10 H-Tyr-Gly-Gly-Phe-Met-Thr-Sec-Glu-Lys-Ser

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

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

FIGURE 1 Amino acid sequence of human pendorphin.

90

80

-

- 3 70-

u =o- Y

'$ 50- 5 40- 4 30-

20

10-

-

-u 0.05 0.1 0.2 0.5 1.0 2.0

5 L Dose ( f i g /rouse)

FIGURE 2 Log-probit dose response curves for antinociceptive effect produced by intracerebroventricular injection of ph-EP (+), [Arg'"'6''9 ]-ph-EP (0) and [Arg9'19' 2 4 ~ 2 8 . 2 9 ]-ph-EP (0). After control latency was obtained (before peptide injection) groups of eight mice were injected with various doses of synthetic peptides and the tail-flick response was determined at 15 min, 30 min, and 1 h after injection.

substitution of Lys-19 and Lys-9 with arginine greatly reduces analgesic potency (see Fig. 2) although binding activity is increased over that for peptide 11. In this respect, it should be noted that there has been a general qualitative correlation between analgesic potency and binding activity as seen in omission analogs (3) and analogs of varying chain lengths (1). Quanti- tative correlation has not always been observed. The ratio of binding/analgesic activities has usually been greater than one ( 6 , 7 ) . A high ratio of binding/analgesic activities as observed

TABLE 2 Biological activity of @endorphin analogs

Anaigesic potency

Synthetic peptide (A)

ADSO Relativea (Hg/mouse) potency

p-E ndorphin 0.094 100 ]-PEP (1) 0520 18

[ ~ ~ ~ z 4 . ' 8 , ~ 9 ]-PEP (11) 0.109 86 [ Arg 9,19,24,28,29

Opiate receptor binding activity

(B) BIA -~ ~

IC,, Relative (nM) potency

0.74 100 1 .o 0.22 336 18.7 0.34 217 2.5

molar basis.

309

J . Blake e f al.

for peptide I suggests a possible approach toward the design of an inhibitor to P-EP.

EXPERIMENTAL PROCEDURES

Protected peptide resins corresponding to the synthetic analogs BocGlu(OBz1)-resin (0.90 g, 0.42 mmol) was subjected to the following synthetic procedure: 1) washing with methylene chloride 4 times; 2) washing with 55% trifluoroacetic acid/ rnethylene chloride for 15 rnin; 3) washing with methylene chloride 2 times; 4) washing with 25% dioxane/methylene chloride 3 times; 5) repeat step 3 ; 6) reaction with 5% diiso- propylethylamine/methylene chloride for 2 rnin; 7) repeat step 3 ; 8) repeat step 6 ; 9) washing with methylene chloride 5 times; 10) reaction with 1.5 mmol of symmetrical anhydride of the Boc-amino acid in methylene chloride for 20min; 11) addition of 0.42mmol N-methyl- morpholine to the coupling mixture and continued reaction for 20min; 12) washing with methylene chloride 3 times; 13) washing with 33% ethanol/methylene chloride 3 times.

Nff-protection was by the Boc group. Side chain protection was as follows: Ser, 0-benzyl; Thr, 0-benzyl; Glu, y-benzyl ester; Lys, NE-o- bromobenzyloxycarbonyl; Arg, N gp-toluene- sulfonyl; Tyr, Oa-bromobenzyloxycarbonyl for residue 27, and 0-benzyloxycarbonyl for residue 1. The preformed symmetrical anhydrides were synthesized as previously described (8). Boc- Asn was coupled by the use of 1-hydroxybenzo- triazole (9) as previously reported (8). Solvent wash volumes were 15 ml. After the coupling of Asn-20 the peptide resin was divided into two parts for the synthesis of peptides I and 11. At this stage coupling was achieved with 1 .O mmol symmetrical anhydride. After the coupling of Tyr-1, the peptide resin was sub- jected to steps 1-5, washed with ethanol, and

acetic acid and chromatography on Sephadex G-10 gave 157 mg crude product. Ion-exchange chromatography on CM-cellulose gave 69 mg product. Partition chromatography on Sephadex G-50 in the system n-butanol: pyridine: water- (5 :2 : IO)gave40mgpeptide I at Rf0 .16 .

Paper electrophoresis at pH 3.7 and 6.7 gave single ninhydrin, Pauly, and chlorine positive spots at RfLYs 0.53 and 0.34, respectively. Thin- layer chromatography in the system n-butanol: pyridine:acetic acid:water (5 : 5 : 1 :4) showed a single spot at R f 0.56. Amino acid analysis of acid and enzyme hydrolysates of peptide I is shown in Table 1.

[Arg24*2s*29]Q-EP (11). A portion (380mg, 0.060mmol) of peptide resin was treated with HF/anisole as described for peptide I , The yield was 155mg crude peptide, and 64mg peptide after chromatography on CM-cellulose. Par- tition chromatography in the system n-butanol: pyridine:O.l% acetic acid(5:3:10)gave 44.2 mg peptide I1 at R f 0.44.

Paper electrophoresis at pH 3.7 and 6.7 showed single spots at RfLYs 0.52 and 0.34, respectively. Thin-layer chromatography showed a single spot at R f 0.50. Amino acid analysis of acid and enzyme hydrolysates is shown in Table 1.

Bioassay. Analgesic potency was estimated in male Swiss Webster mice weighing 20-25g (Simonson Laboratories, Gilroy, CA) by the tail-flick method (10) as described (11) using groups of eight mice per dose. The opiate receptor binding assay was performed with a rat brain membrane fraction using tritiated Ph-EP (12) as primary ligand and synthetic Ph-EP (13) as standard competing ligand as described (14, 15).

dried. ACKNOWLEDGMENTS

p r g 9 . 19,24,28,29 ]9-EP (I). A portion (390 mg, We thank W.F. Hain and J . Roeder for technical

was 0.8m1 and 'Om' '! National Institute of Mental Health (MH-30245), for 1 h at the National Institute of Health (GM-2907) and the and the peptide-resin mixture was washed with Hormone Research Foundation. P.N. is a European ethyl acetate. Peptide was dissolved in 6 ml 0.5 N Molecular Biology Organization Fellow.

0.0616mmol) Of the protected peptide resin assistance. This work was supported in by the

. The HF was evaporated Off at

310

Jlh-EP analogs

REFERENCES

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

2. Blake, J., Tseng, L.F. & Li, C.H. (1980) Int. J. Peptide Protein Res. 15,167-170

3. Li, C.H., Yamashiro, D., Tseng, L.F., Chang, W.C. & Ferrara, P. (1980) Proc. Natl. Acad. Sci.

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

5. Yamashiro, D. (1980) in Hormonal Proteins and Peptides (Li, C.H., ed.), vol. 10, pp. 25-107, Academic Press, New York

6. Li, C.H., Tseng, L.F., Ferrara, P. & Yamashiro, D. (1980) Proc. Natl. Acad. Sci. US 77,2303-2304

7. Li, C.H., Tseng, L.F., Jibson, M.D., Hammonds, R.G., Jr., Yamashiro, D. & Zaoral, M. (1980) Biochem. Biophys. Res. Cornmun. 97,932-938

8 . Blake, J. & Li, C H . (1975) Int. J. Peptide Protein Res. 7,495 -50 1.

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

US 77,3211-3214

2149 -2154

788-798

10. D’Amour, FE. & Smith, D.L. (1941) J. Pharma- COI. Exp. n e r . 72, 74-79

11. Loh, H.H., Tseng, L.F., Wei, E. & Li,C.H. (1976) Proc. Natl. Acad. Sci. US 73,2895-2898

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

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

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

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

Address: Dr. C.H. Li Hormone Research Laboratory 1088 HSW University of California San Francisco, CA 94143 USA

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