Int. J. Peptide Protein Res. 15, 1980, 167-170

S Y N T H E S I S A N D A N A L G E S I C A C T I V I T Y OF H U M A N

17, 1 8 , O R 19 0 - E N D O R P H I N A N A L O G S S U B S T I T U T E D A T P O S I T I O N S

JAMES BLAKE, LIANG-FU TSENG* and CHOH H A 0 LI

Hormone Research Laboratory, University of California, San Francisco, California, and *Department o f Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Received 10 May, accepted for publication 13 June 1979

Three peptide analogs of 0-endorphin which are substituted in positions 17, 18 or 19 have been synthesized and their analgesic potencies have been measured by the tail-flick method in mice. The results showed that the replacement of Phe-18 or Lys-19 by alanine reduced the potency to 15% whereas the replacement of Leu-1 7 by alanine reduced the analgesic potency to 68%.

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

A continuing investigation in this laboratory has been directed toward delineating the struc- tural requirements for opiate and analgesic activities of Oendorphin (O-EP*, Fig. 1 ; see Li (1977) for a review on 0-endorphin). Structure- activity studies within the enkephalin region of 0-EP indicate the importance of the Tyr-1, Phe- 4, and Met-5 residues for analgesic activity (Yamashiro et d , 1977, 1978; Blake et QZ., 1978). The contribution of the amino acid residues outside of the enkephalin region is less clearly defined, although the following obser- vations have been made: ( 1 ) Large segments cannot be deleted from the middle of 0-EP without severe reduction in analgesic activity (Li et d , 1 9 7 8 ~ ) . (2) Substitutions of Tyr-27 and Glu-31 by Phe and Gly, respectively, d o not decrease analgesic potency (Blake et a[., 1978). (3) Bioassay of a series of analogs of camel @endorphin (Yamashiro, 1979) which

~ -

*Abbreviations: ph-EP, human pendorphin; Boc, t-butyoxycarbonyl. All asymmetric amino acids mentioned in this paper are of the L-configuration unless otherwise indicated.

2 5 i i A l a - I l e - ~ le-~~~-~sn-~la-lvr-') s - L y s - ~ l ~ : - C l u - O H

FIGURE 1 Amino acid sequence of ohendorphin. The amino terminal pentapeptide corresponds to the sequence of Metenkephalin.

differ from the parent molecule by the omission of single uncharged amino acid residues indi- cates that most of those residues outside of the enkephalin region make n o vital contribution to analgesic activity (Li et ~ l . , 1979). (4) The requirement of the entire 31 amino acid chain for full analgesic activity and particularly the great difference in activity between &-EP- (1 -28) and 0h-EP-(-30) suggest that Lys- 28,29 may also make important contributions to analgesic potency (Li et QI., 1978b).

We now report the synthesis and analgesic potency of three analogs of Ph-EP which are substituted in positions 17, 18, or 19.

0367-8377/80/020167-04 $02.00/0 0 1980 Munksgaard, Copenhagen 167

J . BLAKE ET AL.

RESULTS A N D DISCUSSION

There were two reasons for the investigation o f residues 17. 18. and 19 in the b-EP molecule. First. these residues are outside of the region of 0-EP that has previously been studied by sub- stitution or omission analogs. This study would clarify their smtr ibut ion to the analgesic activity of (3-EP. The second reason was suggested by the recent report (Grif er 41.. 1977) that the incubation of porcine 0-lipotropin with porcine pituitary honiogenate resulted in enzymatic cleavage between the Leu7'-P1ie 78

and LysW-AsnSO bonds in P-lipotropin. These correspond to the L e ~ ' ~ - P h e ' ~ and L y ~ " - A s n ~ ~ bonds in 0-EP. Thus, aminb acid substitutions which stabiliLed the 18-19 and 19-20 peptide bonds t o enzymatic digestion could increase the potency of the O-EP analog. Although the usual nieans for stabilizing a peptide bond t o enzy- matic digestion is by substitution with D-amino acids or other umatural amino acids, we decided that our purpose here would be best served by substituting each of residues 17, 18 and 19 with Ala. This would remove the side chain of each

residue that might serve as a site for speafic cleavage by proteolytic enzymes without Severe disruption of the peptide chain conformation. In addition, we retained the substitution o f Phe for Tyr-27 and Gly for Glu-31 that has beer1 previously shown t o have no deleterious effect 011 analgesic potency (Blake et aZ., 1978).

The three peptides [AlaI7, Phe27, Gly3']- Oh-EP (I) , [Ala18. PheZ7, Gly31 ] &-EP (11). and [Ala". Phe27. G1y3l ] &,-EP (111) were syn- thesized by the solid-phase method (Merrifield, 1963) as previously described for [Phe27. Gly31]-flh-EP (Blake e l a/., 1978). The pro- tected resin was treated with liquid HF and the crude peptide product was purified by ion- exchange chromatography on carboxymethyl- cellulose (Peterson & Sober, 1956) as previously described (Li et al., 1976), and partition chromatography on Sephadex G-SO (Yama- shiro & Li, 1973: Li et al., 1976). The peptide products were characterized by paper electro- phoresis, thin-layer chromatography, and amino acid analysis of acid and enzyme hydrolysates (Table 1 ) .

TABLE 1 Amino acid composition of the svtzt/?etic peptides

Peptide I Peptide I1 Peptide 111 -

Amino acid Acid Enzymeb Acid Enzyme Acid Enzyme

LYS

ASP Asn Gln T h r Ser Clu Pro GlY Ah Val Met Ile Leu TYI Phe

5.0 wa 2.1 (2)

-

3.0 (3) 1.9 (2) 2.0 (2) 1.0 (1) 4.0 (4) 3.1 (3) 1 . 1 (1) 1.0 (?)

1.0 (1) 1.1 (1) 3.1 (3)

1 .4d (2)

4.9 (5) -

7.2 (8)'

1.1 (1) 1.0 (1) 3.6 (4) 2.9 (3) 1.0 (1) 1 .o (1) 2.1 (2) 1.1 (1) 1.1 (1) 3.0 (3)

4.9 (5) 2.1 (2)

- 3.0 (3) 1.9 (2) 2.1 (2) 1.0 (1) 4.0 (4) 3.1 ( 3 ) 1.0 (1) 1.0 (1)

2.0 (2) 1 .1 (1) 2.0 (2)

1.2d ( I )

4.8 (5) -

1.3 (8)'

1.0 (1) 1.1 (1) 3.7 (4) 2.8 (3) 1.1 (1) 0.9 ( 1 ) 1.8 (2) 1.9 (2) 1.0 (1) 1.9 (2)

3.8 (4) 2.0 (2)

-

2.8 (3) 1.8 (2) 2.0 (2) 0.9 (1) 3.8 (4) 3.0 (3) 1.2 (1) 1.0 (1)

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

l . ld (2)

3 .9 (4) -

7.3 (8)'

1.0 (1) 1.0 (1.1 3.7 (4) 3.0 (3) 1.0 (1) 1.0 (1 ) 2.1 (2) 2.1 (2) 1.1 (1) 3.0 (3)

aNumbers in parentheses are the expected values. bDigestion with trypsin/chymotrypsin followed by leucine aminopeptidase (see Blake et oL, 1978). 'Corresponds to sum of Asn + Gln + Ser + Thr. dLow value is due to resistance of Ile-Ile bond to acid hydrolysis.

168

SYNTHESIS AND ANALGESIC ACTIVITY OF P-ENDORPHIN ANALOGS

Analgesic potency of the synthetic peptides was determined in mice by the tail-flick method (DAmour & Smith, 1941; Loh et al., 1976; Tseng ef al., 1976). The results are summarized in Table 2. The data indicate that Phe-18 and Lys-19 are necessary for the full analgesic activity of 0-EP. Although the substitution of Ala for Leu-17 decreases the analgesic potency of peptide I , the difference is not as great as that existing for the Ala-18 and Ala-19 analogs and indicates that the Leu side chain is not as important for the biological activity of P-EP as are the side chains of Phe and Lys.

EXPERIMENTAL PROCEDURES

Protected pept ide resins corresponding to the synthetic analogs Boc-glycyl resin (1.49 g, 0.71 mmol) was sub- jected to the following synthetic procedure: (1) washing with rnethylene chloride, 4 times; ( 2 ) washing with 55% trifluoracetic acid/ methylene chloride; (3) reaction with 55% tri- fluoracetic acid/rnethylene chloride for 15 min; (4) washing with methylene chloride, 2 times; ( 5 ) washing with 25% dioxane/methylene chloride, 3 times; (6) repeat step 4 ; (7) reaction with 5% diisopropylethylaminelrnethylene chloride for 2min; (8) repeat step 4 ; (9) repeat step 7 ; (10) washing with methylene chloride, 5 times; (1 1) reaction with 2 mmol of the sym- inetrical anhydride of the Boc-amino acid in nethylene chloride for 20 min; (1 2) addition of 0.35 mrnol of N-methylrnorpholine to the coupling mixture and continued reaction for 20min; (13) washing with methylene chloride, 3 times; (14) washing with 33% ethanol/methyl- ene chloride, 3 times.

Na-Protection was by the Boc group for all amino acids. Side chain protection was as follows: Ser, 0-benzyl; Thr, 0-benzyl; Tyr, 0- benzyloxycarbonyl; Glu, y-benzyl ester; Lys, NE-O-bromobenzyloxycarbonyl. The preformed symmetrical anhydrides were synthesized as previously described (Blake & Li, 1975) and Boc-Asn was coupled by the use of l-hydroxy- benzotriazole (Konig & Geiger, 1970) as pre- viously reported (Blake & Li, 1975). For the first 10 cycles of synthesis solvent wash volumes were 20ml. After the coupling of Asn-20, the peptide resin was washed with ethanol and dried to yield 2.70 g peptide resin.

Portions of the undecapeptide resin (0.65 g, 0.17rnmol) were used for the synthesis of the desired analogs. At the reduced scale of syn- thesis, solvent wash volumes were 12ml and coupling was achieved with 0.6mmol sym- metrical anhydride. After the coupling of Tyr-1, the peptide resin was subjected to steps 1-5, washed with ethanol, and dried.

[AIa", Phe2', Gly31]-Ph-EP (1) A portion (400mg, 0.065mmol) of the final protected peptide resin corresponding to peptide I was treated with 1.3 ml anisole and 8ml liquid HF at 0" for 1 h. HF was then evaporated at 0" and the peptide-resin mixture was stirred with 30ml ethyl acetate. The mixture was filtered, and the precipitate was washed with ethyl acetate and air dried. Peptide was dissolved in 5 m l of 0 . 5 N acetic acid. Filtration gave a filtrate that was chromato- graphed on Sephadex G-10 to give 158mg crude peptide. Chromatography on carboxy- methyl-cellulose as previously described (Li et aL, 1976) gave 88 mg partially purified peptide

TABLE 2 Analgesic potency of synthetic ph-endorphin analogs

Relative potency

Synthetic peptides AD 50 a

0.047 (0.033-0.065) 100 [Phe" , Gly3' ] Qh-EP

[Ah", Phe*', Glf' ] Qh-EP (11) [ A h i 9 , Phe" , Gly3' ] Qh-EP (111)

0.069 (0.027-0.17) 68 0.32 (0.22-0.47) 15 0.31 (0.24-0.52) 15

[Ah'', Phe" , Gly" ] Qh-EP (1)

aAD5, in nmol/mouse (95% confidence limit).

169

J. BLAKt bl AL.

I. Partition chromatography on Sephadex G-50 in the system n-butanol : pyridine : 0.6 M am- monium acetate (S:3.10) showed a major peak at Rf 0.54. The corresponding fractions were isolated, 30 nil water was added,and the mixture was evaporated to a volume of cu. 20ml. Then 40ml water was added and the solution was lyophilized. Two more lyophilizations gave 69mg peptide I (32% yield based on starting Boc-glycyl resin).

Paper electrophoresis (400 V, 2.5 h) at pH 3.7 and 6.7 showed ninhydrin, chlorine positive spots at RfLYs 0.58 and 0.51, respect- ively. Thin-layer chromatography on silica gel in the systems n-butanol : pyridine : acetic acid : water (5:5:1:4) (BPAW) an+ n-butano1:acetic acid : water (4:3:3) (BAW) gave single ninhydrin, chlorine positive spots at Rf 0.43 and 0.18, respectively. Amino acid analysis of acid and enzyme hydrolysates of peptide I is indicated in Table 1. [A1aI8, Phe27, GI . v~~] -&-EP (II) A portion of the peptide resin (383 mg, 0,065 mmol) was treated as described for peptide I to give 88 mg peptide after carboxymethylcellulose chromatography. Partition chromatography on Sephadex G-50 in the system n-butanol : pyridine:O.l M anunonium acetate (5:3:10) gave 63 nig peptide I1 (29%) at Rf 0.41. Paper electrophoresis at pH3.7 and 6.7 gave ninhydrin, chlorine positive spots at RfLYs 0.58 and 0.52, respectively. Thin-layer chromatography in the BPAW and BAW systems gave single ninhydrin, chlorine positive spots at Rf 0.43 and 0.18, respectively. Amino acid analysis of acid and enzyme hydrolysates of peptide I1 is indicated in Table 1. [A la l9 , PheZ7, Glv3’ ]-&,-EP (III) A portion of the peptide resin (360mg, 0.064 mmol) was treated as described for peptide I to give 42 mg peptide after carboxyrnethylcellulose chromatography. Partition chromatography on Sephadex G-50 in the system n-butanol: pyridine:O.l% acetic acid (5:1.5:10) gave 26.4 mg peptide 111 (12% yield) a t Rf 0.31. Paper electrophoresis of peptide 111 at pH 3.7 and 6.7 gave ninhydrin, chlorine positive spots at RfLyS 0.48 and 0.44, respectively. Thin-layer chro- matography in the BPAW and BAW systems gave single ninhydrin, chlorine positive spots at Rf 0.46 and 0.23, respectively. Amino acid

170

analysis of acid and enzyme hydrolysates is indicated in Table 1.

ACKNOWLEDGMENTS

We thank W.F. Hain and K. Hoey for technical assist- ance. This work was supported in part by the US National Institute of Health (MH-30245 to CHL) and NIDA (DA41314 to LFT).

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Address: Dr. James Blake Hormone Research Laboratory 1088 HSW University of California San Francisco, California 94143 U.S.A.