Int. J. P e p t i d e h t e i n Res. 11,1978, 323-328 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

THE SYNTHESIS A N D OPIATE ACTIVITY O F HUMAN P-ENDORPHIN ANALOGS

Substituted at Residue Positions 27 and 3 1

JAMES BLAKE, LIANGFU TSENG*, WENCHANG CHANG and CHOH HA0 LI

Hormone Research Laboratory and *Department o f Psychiatry and Pharmacology, University of California, San Francisco, California, U.S.A.

Received 3 1 October, accepted for publication 2 1 November 1977

Four analogs o f human @endorphin have been synthesized and their opiate activities have been determined. One of the analogs, [27-phenylalanine, 31 - g1ycine]-&,-endorphin, was shown t o possess an analgesic potency greater than that o f the natural peptide.

Key words: p-endorphin; opiate activity; peptide synthesis.

The isolation of met enkephalin and pendor- phin (0-EP, Fig. 1) from brain and pituitary extracts (Hughes et al., 1975; Li & Chung, 1976a; Li et al., 1976a; Bradbury et al., 1976a, 19766; Guillemin et al., 1976; Lazarus et al., 1976; Graf et al., 1976a, 19766; Ling & Guillemin, 1976; Chretien et al., 1976), and the ability of these peptide fragments of 0-lipo-

5 10 H-TyrGly Gly-Phe-Met-Thr-SerGlu-LysSer

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

25 31 Ala-Ile-Ile-Lys- Asn-Ala-T yr-Lys-Lys-Gly Glu-OH

FIGURE 1 The amino acid sequence of phendorphin. The amino acid sequence of met-enkephalin corresponds to the amino-terminal pentapeptide.

tropin @-LPH) to affect the central nervous system, has prompted extensive studies into their structure-activity relationships. Results from these studies indicate that for peptides containing the native sequence, virtually the entire length of the P-EP chain is required for full analgesic activity (Li e t al., 1978; W . Yeung, D. Yamashiro, and C. H. Li, manu- script in preparation). Although the analgesic activity of met-enkephalin is almost nil, several of its analogs have been reported to have an analgesic activity comparable to that of 0-EP (Pert et al.. 1976; SzCkely et al., 1977; Roemer et al., 1977; Wei et al., 1977; Yamashiro et al., 1977a). Similar studies with amino acid substi- tution within the met-enkephalin portion of 0-EPaimed at increasing the activity of the parent molecule have given discouraging results (Yamashiro et al., 1977b). In only one case, [D-Ala2] -ph-EP, has full analgesic activity been retained, and in no case has there been an increase in activity.

Abbreviations: p-EP, p-endorphin (subscripts ‘h’ and ‘c’ indicate pendorphin from human and camel pituitaries); iv, intravenous; icv, intracerebroventricular; Boc, t-butyloxycarbonyl. All asymmetric amino acids mentioned in this paper are of the Lconfiguration unless otherwise indicated.

3 23

J . BLAKE ET AL.

TABLE 1 Amino acid composition of the synthetic peptides

Amino Peptide I Peptide I1 Peptide 111 Peptide 1V acid Acida Enzymeb Acid Enzyme Acid Enzyme Acid Enzyme

Lys 4.9 (5F Asp 2.1 (2) Asn -

Gin - Thr 3.0 (3) Ser 1.9 (2) Giu 2.1 (2) Pro 0.9 (1) Gly 4.3 (4) Ala 2.0 (2) Val 0.9 (1) Met l . O ( l ) Ile 1.3 (2) Leu 2.0 (2) Nle -

TY r 1.0 (1) Phe 2.9 (3)

4.6 (5) -

1.1 (8)d

1.1 (1) 1.0 (1) 3.1 (4) 1.9 (2) 1.1 (1) 1.1 (1) 1.9 (2) 2.1 (2)

1.0 (1) 3.0 (3)

-

4.1 (5) 2.1 (2) - -

1.9 (2) 0.9 (1) 2.1 (2) 1.0 (1) 4.2 (4) 3.9 (4) 1.0 (1) 1.1 (1) 1.3 (2) 2.1 (2)

1.0 (1) 3.0 (3)

-

4.1 (5) -

5.6 (6)d

1.1 (1) 0.9 (1) 3.9 (4) 3.9 (4) 1.2 (1) 1.0 (1) 2.0 (2) 2.2 (2)

1.1 (1) 3.0 (3)

-

5.1 (5) 2.0 (2)

-

- 2.8 (3) 1.9 (2) 2.0 (2) 1.0 (1) 3.8 (4) 2.0 (2) 1.0 (1)

1.2 (2) 1.9 (2) 1.0 (1) 1.0 (1) 3.0 (3)

-

5.1 ( 5 ) -

1.9 (8)d

1.0 (1) 1.0 (1) 3.6 (4) 1.9 (2) 0.9 (1)

1.8 (2) 2.0 (2) 1.0 (1) 1.0 (1) 2.8 (3)

-

5.1 (5) 2.1 (2)

-

- 3.1 (4) 1.9 (2) 2.1 (2) 0.9 (1) 3.0 (3) 2.0 (2) 1 .o (1) 1.0 (1) 1.1 (2) 2.0 (2)

1 .o (1) 3.1 (3)

-

5.1 (5) -

1.1 (9)*

1.1 (1) 0.9 (1) 1.9 (3) 2.0 (2) 1.1 (1) 1 .o (1) 1.8 (2) 2.3 (2)

1.0 (1) 2.2 (3)

-

a Hydrolysis with constant boiling HCI; 22 h at 110". See Experimental section. Numbers in parentheses are the expected values. Corresponds to sum of Asn + Gln + Thr + Ser.

In an effort to further delineate the struc- tural requirements for full or even increased opiate activity of human 0-EP, we have synthesized four analogs with amino acid substitutions within the metenkephalin and COOH-terminal portions of the peptide. The opiate activities of these analogs are also reported here.

Synthesis. The four peptides [Phez7, Gly31 ] - [Nle', Phez7, Gly3'] -Ph-EP (111), and (D-Thr', PheZ7, Gly31] -flh-EP (IV) were synthesized by improved procedures of the solid-phase method (Merrifield, 1963) as previously described (Yamashiro & Li, 1974; Li et ~ l . , 1977). Boc-glycyl resin was alternately subjected to deblocking in 55% trifluoroacetic acid/ methylene chloride, neutralization with diisopropylethylamine, and coupling with the preformed symmetrical anhydride of the Boc amino acid (Hagenmaier & Frank, 1972). Treatment of the final peptide resin with liquid HF (Sakakibara el ul., 1967; Lenard &

3 24

&-EP (I), [Ala6* 7 , Phez7, G1y3l ] -&-EP (II),

Robinson, 1967) followed by purification of the crude product by chromatography on carboxymethylcellulose and partition chromatography (Yamashiro, 1964) on Sephadex G-50 gave the highly purified peptides I-IV. The synthetic peptides were characterized by amino acid analysis (Spackman et ul., 1958) of acid hydrolysates and enzyme digests (Table l), paper electro- phoresis, and thin-layer chromatography.

Opiate activity. The opiate activities of pep- tides I-IV as assayed by the in vitro and in vivo procedures are summarized in Tables 2 and 3. It may be noted that there is little correlation between the in vitro assay by the guinea pig ileum method and the in vivo assay in mice by either intravenous (iv) or intracerebroventricu- lar (icv) administration. All of the synthetic analogs are slightly more potent than Oh-EP by the guinea pig ileum assay but, with the exception of peptide I, are significantly less potent in the mouse. The analgesic activity following iv injection of peptide I is shown in Fig. 2. The duration of analgesic activity after iv injection appeared to be shorter than after icv injection. To our knowledge, peptide I is the first synthetic analog of &-EP to possess a greater in vivo analgesic activity than the

SYNTHESIS AND OPIATE ACTIVITY OF HUMAN 0-EP ANALOGS

TABLE 2 Opiate activity of synthetic &endorphin analogs by guinea pig ileum assay

Preparations IC 50 Relative potencya mol/l

4.5 x lo-’ 100 3.5 x lo-* 128 3.8 X lo-’ 118 3.8 X lo-’ 118 3.3 x lo-’ 136

a Relative potencies are calculated using the IC,, of Ph-endorphin as 100%.

TABLE 3 Analgesic potency of synthetic &,-endorphin analogs in mice

Preparation Intracerebroventricular Intravenous

pg/mouse potency mdkg potency ADSO Relativea ADSO Relativea

~~

ph-cndorphin 0.11 (0.07-0.17) 100 11.4 (6.4-19.6) 100

[Alab*’, Phe”, Gly” ] -&-EP (11) 0.46 (0.28-0.77) 23 22.1 (14.3-34.3) 49 [Phe” , Gly” 1 -&-EP (I) 0.09 (0.08-0.11 119 7.5 (3.4-16.5) 148

[Nle’, Phe”, Gly” ] -&-EP (111) 0.31 (0.14-0.72) 35 ca. 17.0 65 [D-Thr’, Phea’, Gly” ] -@h-EP (IV) 0.53 (0.24-1.16) 21 ca. 32.0 32

Numbers in parentheses are the 95% confidence limits. a Relative potencies are calculated on a molar basis using the AD,, of &,endorphin as 100%.

c c 0)

a : 40

20

0

5 10 20 30 45 60

Time After Injection ( m i n ) FIGURE 2 The analgesic effect following iv injection of [Phe”, GlY” ] -&-endorphin in mice. The analgesic response was measured by the tail-flick test. The peptide was injected via the tail vein. The numbers in parentheses denote the number of mice used.

natural peptide. It should be noted that the positions (27 and 3 1) of substitution in peptide I correspond to the positions where the amino acid sequences of human (Li et al., 1976a) and camel (Li & Chung, 197Q) 0-EP differ. The comparable analgesic potencies of @,- and ph-EP (Li et d., 1977) led us to conclude that the Tyr or His residue at position 27 could be substituted by Phe, and the Glu or Gln residue at position 31 could be substituted by Gly without deleterious effect on biological potency. The results support t h i s belief. In fact the analgesic potency of peptide I by iv admin- istration is somewhat greater than that of the natural peptide.

The low analgesic potency of peptide I1 is an indication that the Thr4 and/or the Ser-7 residues of P-EP are necessary for the full bio- logical response of this molecule. Of particular interest is the result of substitution of the Met-5 residue by Me in peptide 111. Previous

325

J . BLAKE ET AL.

results have indicated that Met-5 in met- enkephalin could be replaced by other amino acids with relative impunity. Thus the substi- tution of Met-5 by Leu (Pert er al., 1976), Pro (Szdkely et al., 1977), met(0)-ol (Roemer et al., 1977), or L-thiazolidine-4-carboxylic acid (Yamashiro et al., 1977a), when combined with substitutions in other positions, gave highly potent analogs of met-enkephalin. The anal. ogous substitutions of Leu and Pro in the 5-position of &-EP gave peptides with greatly reduced analgesic activity (Yamashiro er al, 1978). In the present work it is also seen that the replacement of Met-5 by its non-sulfur- steric equivalent, Nle, reduces analgesic potency. Thus it is evident that the analgesic activity of 0-EP is highly sensitive to even conservative amino acid changes in sequence.

While this study was in progress, it was observed that the substitution of D-Thr for Gly-2 in an enkephalin analog augmented the analgesic potency (Yamashiro et al., 1977~) . The analogous substitution was made in P-EP and the resulting peptide (IV) was shown to have a lower analgesic activity than &EP or peptide I . This demonstrates again the diffi- culty in extrapolating structure-activity results in enkephalin analogs to that for P-EP.

EXPERIMENTAL PROCEDURES

Protected pep t ide resins corresponding t o pept ides I - I V . Boc-glycyl resin (1.51 g, 0.60mmol glycine) was subjected to the following reaction procedure: (1) washing with methylene chloride, 4 x 20 ml; (2) washing with 55% trifluoroacetic acid/methylene chloride, 1 x 20ml; (3) reaction with 55% trifluoroacetic acid/methylene chloride, 20 ml for 15min; (4) washing with methylene chloride, 2 x 20 ml; (5) washing with dioxane- methylene chloride (1 :2), 3 x 20 ml; (6) repeat step 4; (7) reaction with 5% diisopropylethyl- amine/methylene chloride, 20 ml for 2 min; (8) repeat step 4; (9) repeat step 7 ; (10) washing with methylene chloride, 5 x 20 ml; (1 1) reaction with 1.8mmol of the preformed symmetrical anhydride of the Boc-amino acid in 13 ml of methylene chloride for 20 min; (1 2) addition of 0.3 mmol of N-methylmorpholine

in 2ml of methylene chloride to the above reaction mixture and continued shaking for 20 min; (13) washing with methylene chloride, 3 x 20ml; (14) washing with ethanol-methylene chloride (1 : 2), 3 x 20 ml.

Na-protection for all amino acids was by the Boc group and side chain protection was as follows: Ser, 0-benzyl; Thr, 0-benzyl; Glu, y-benzyl ester; Lys, N'-o-bromobenzyloxy- carbonyl; Tyr, 0-benzyloxycarbonyl. The pre- formed symmetrical anhydrides of the Boc- amino acids were prepared as previously described (Blake & Li, 1975) and Boc- asparagine was coupled to the peptide resin in the presence of 1-hydroxybenzotriazole (Konig & Geiger, 1970) as previously reported (Blake & Li, 1975).

After coupling of the y-benzyl glutamic acid residue corresponding to position 8 in the P-EP sequence, the peptide resin was dried to yield 3.29 g of protected peptide resin. A one-fifth portion (ca. 600 mg, 0.1 2 mmol peptide) of the peptide resin was submitted to the above reaction procedure and the final seven amino acid residues were coupled. At the reduced scale, solvent wash volumes were 10 ml and coupling was effected by reaction of the peptide resin with 0.5 mmol of symmetrical anhydride in 6.5ml of methylene chloride. After coupling of the final amino acid residue the peptide resin was subjected to steps 1-8, washed with ethanol, and dried.

Isolation of pept ides I - IV . The same procedure was used to isolate each peptide from the corre- sponding peptide resin and the details for the isolation of peptide I are typical. The peptide resin (302 mg, 0.060 mmol) was treated with 1 ml of anisole and 8 ml of liquid HF for 1 h at 0". The HF was evaporated at O", 40 ml of cold ethyl acetate was added to the peptide-resin residue, and the resultant mixture was stirred for 10min at room temperature. The mixture was filtered, and the precipitate was washed with ethyl acetate and air dried. Peptide was extracted from the precipitate by stirring with 6 m l of 0.5N acetic acid. Filtration gave a filtrate that was chromatographed on Sephadex G-10; material corresponding to the major peak was isolated by lyophilization to give 97 mg of crude peptide I . Chromatography on carboxy-

3 26

SYNTHESIS AND OPIATE ACTIVITY OF HUMAN 0-EP ANALOGS

methylcellulose as previously described (Li et the Republic of China. This work was supported in af., 19763) gave 41.2 mg of peptide I. Further part by grants from the Hormone Research

chromatography on Sephadex C-50 (column: NIDA No- Da41314 (to L-F-T.).

REFERENCES 1.9 x 44 cm) in the system n-butanol: pyridine: 0.1% acetic acid (5:3: 11). Material correspond- ing to the major peak at Rf 0.23 was combined, Blake, J. & Li, C.H. (1975) Int. J. Pept. Prof. Res. 7 , diluted with an equal volume of water, and evaporated in vacuo to a volume of ca. 10ml. Bradbury, A.F., Smyth, D.G. & Snell, C.R. ( 1 9 7 6 ~ ) The concentrated solutjon was lyopmzed and Biochem. BioPhYs. Res. Commun. 72,1542-1547 the residue was redissolved in dilute acetic acid Bradbury, A.F., Smyth, D.G., S n e b C.R. & Hulme, and to give 29*2 mg Of peptide I Chrttien, M., Benjannet, S., Dragon, N., Seidah, N.G.

purification was achieved by partition Foundation and NIMH NO. MH30245 ( to C.H.L.) and

495-501

E.C. (19766) Nature (Lond.) 260,793-795

(14'4% yield based On starting Boc%lycyl & Lis, M. (1976) Biochem. Biophys, Res, resin). Peptides 11, 111, and IV were isolated in yields of 10 ,28 , and 21%, respectively. D'Amour, F.E. & Smith, D.L. (1941) J. Pharmacol.

Paper electrophoresis of peptide I at pH 3.7 and 6.7 (400V, 3.5 h) gave a single ninhydrin Grdf, L., Rondi, A.Z., Bajusz, S., Cseh, G . & Szdkely, positive, chlorine positive spot at R P 0.56 and 0.38, respectively. Thin-layer chromatography Grdf, L., Sztkely, J . I . , Ron& A.Z., Dunai-K6vacs, Z. on silica gel in the system n-butanol:pyridine: & Bajusz, S. (19766) Nafure (Lond.) 263, acetic acid:water ( 5 : 5 : 1 :4) gave a single 240-241 nihydr in , chlorine positive spot at R~ 0.5 1. Guillemin, R., Ling, N. c% Burgus, R. (1976) ComPt.

Thin-1ayer chromatography in the system Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill,

single ninhydrin, chlorine positive spot at Rf 0.82. For enzymatic digestion, a solution of K O , , ~ , W, & ~ ~ b ~ ~ , R. (1970) (-hem. B ~ ~ . 103, 0.7mg of peptide plus 14pg of chymotrypsin and 14pg of trypsin in 0.15ml of Tris buffer Kosterlitz, H.W., Lydon, R.J. & Watt, A.J. (1970) (PH 8.5,O.Ol M Mg2+) was incubated at 37" for 24 h. Then the solution was heated in boiling Lazarus, L.H., Ling, N. & Guillemin, R. (1976) Proc. water for 15 min, cooled to room temperature,

leucineaminopeptidase for 44 h. A portion of Li, C.H., Chung, D. & Doneen, B.A. (1967a)Eiochem. the enzyme digest was submitted to the amino Eioph,,s, Res. Commun. 72, 1542-1547

acid and the are shown in Li, C.H., Lemaire, S., Yamashiro, D. & Doneen, B.A. Table 1 together with the amino acid analyses (19766) Biochem. Biophys. Res. Commun. 71 ,

Li, C.H., Yamashiro, D., Tseng, L-F. & Loh, H.H. Bioassay. I n vitro assay of opiate activity was by the guinea pig ileum method (Kosterlitz et Li, C.H., Yamashiro. D., Tseng, L-F. 8~ Lob, H.H. af., 1970) and in vivo assay in mice was by the

The calculation has been outlined elsewhere (Loh et al., 1976; Tseng et al., 1976).

Commun. 72 , 472-478

EXP. Therap. 72 ,74-79

J.I. (19760) FEESLeffers64, 181-184

Rend. Ser. D, 283,783-785

n-butanol: acetic acid:water (4:3:3) gave a L.A., Morgan, B.A. & Morris, H.R. (1975) Nature (Land,) 258, 577-579

788-798

Brit. J. Pharmacol. 39, 398-413

Natz.Acad. SCi. U.S.A. 739 2156-2159 and further incubated at 37" with 2gPg of Lenard, J . & Robinson, A.B. (1967) J. Am. Chem.

SOC. 89,181-182

of acid hydrolysates of peptides I-IV. 19-25

(1978) fnt . J. Pept. Prof. Res. 11,154-158

(1977)J . Med. Chem. 20* 325-328

U.S.A. 73,1145-1148 procedure and method Of Li, C.H. & Chung, D. (19766) Nafure (Lond.) 260,

622-624 Li, C.H., Barnafi, L., Chrdtien, M. & Chung, D.

(1965) Nature (Lond.) 208, 1093-1094; (1966) Excerpta Med. Inf. Congr. Ser, 112,349-364

Ling, N. & Guillemin, R. (1976)Proc. Nafl. Acad. Sci.

tdl-flick method ( D V A ~ ~ ~ ~ & Smith, 1941). Li, C.H. & Chung, D. (19760) PrOC. Natl. Acad. sci.

ACKNOWLEDGMENTS U.S.A. 73,3308-3310 We thank W. F. Hain, K. Hoey, M. O'Rourke and D.

Cheng for technical assistance. W4.C. is a recipient of a Fellowship from the National Science Council of

Loh, H.H., Tseng, L-F., Wei, E. & Li, C.H. (1976) Proc. Natl. Acad. Sci U.S.A. 7 3 , 2895-2898

321

J. BLAKE ET AL.

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

Pert, C.B., Pert, A., Chang, J.K. & Fong, B.T.W. (1976) Science 194,330-332

Roemer, D., Buescher, H.H., Hill, R.C., Pless, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D. & Huguenin, R. (1977) Nature (Lond.] 268, 547-549

Sakakibara, S., Shimonishi, Y., Kishida, Y., Okada, M. & Sugihara, H. (1967) Bull. Chem. SOC. Japan

Spackman, D.H., Stein, W.H. & Moore, S. (1958) Anal. Chem. 30,1190-1206

Szdkely, J.I., Rdnai, A.Z., Dunai-Kdvacs, Z., Miglecz, E., Barzetri, I., Bajusz, S. & Grrlf, L. (1977) Eur. J. Pharm. 43,392-394

Tseng, L-F., Loh, H.H. & Li, C.H. (1976) Nature (Lond.] 263,239-240

Wei, E.T., Tseng, L-F., Loh, H.H. & Li, C.H. (1977) Life Sci. 21,321-328

2149-2154

40,2164-2167

Yamashiro, D. (1964) Nature (Lond.] 201, 76-77 Yamashiro, D. & Li, C.H. (1974) Proc. Natl. Acad.

Sci. V.S.A. 71,4945-4949 Yamashiro, D., Tseng, L-F. & Li, C.H. (19770)

Biochem. Biophys. Res. Commun. 78,1124- 1 129 Yamashiro, D., Tseng, L-F., Doneen, B.A., Loh,

H.H. & Li, C.H. (19776) Int. J. Pept. Prot. Res. 10,

Yamashiro, D., Li, C.H., Tseng, L-F. & Loh, H.H. 159-166

(1978)Int. J. Pept. Prot. Rex 11,251-257

Address:

Dr. C.H. Li Hormone Research Laboratory University of California San Francisco, California 94 143

328