Int. J. Peptide Protein Res. 19, 1982,54-62

Dehy droenkephalins 111. Synthesis and biological activity of [AAla’ , Leu5 ] enkephalin

YASUYUKI SHIMOHIGASHI** and CHARLES H. STAMMER

Department of Chemistry, University of Georgia, Athens, Georgia, USA

Received 11 May, accepted for publication I4 July 198 1

[AAla’ , Leu5 1 enkephalin has been prepared and shown to be more active than the parent saturated enkephalin in a binding assay using rat brain membranes and [3H]dihydromorphine as a tracer. In a comparison of potencies against [ HI dihydromorphine and [ 3 H ] -[ D-Ala’ , D-Leu’ ] enkephalin as tracers, [ AAla2, Leu5 1 enkephalin showed preference for 1.1 opiate receptors, possibly due to the hydrophobicity of the AAla’ residue. A synthetic tetrapeptide enkephalin [ AAla’ 1 -desLeu’ enkephalin had weak activity and high selectivity for the p receptors. 0-Acylation of a serine residue in the peptide was achieved by coupling between the peptide and a carboxylic acid using DCC and a catalytic amount of 4-dimethylaminopyridine.

Key words: enkephalin analog; dehydroenkephalin; dehydroalanine; receptor binding; serine 0-acylation.

For a preliminary communication on this subject see ref. 1. Abbreviations according to IUPAC-IUB Commission (1972), Biochemistry 11, 1726-1732, are used throughout. Additional abbreviations: A, dehydro (a, 0-unsaturated); Boc, tert. -bu tyloxy- carbonyl; Dabco, 1,4diazabicyclo[ 2.2.23 octane; DBU, 1 ,5diazabicyclo[ 5.4.0]undec-5ene; DCC, N,N’- dicyclohexylcarbodiimide; DCHA, dicyclohexylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N- dimethylformamide; DMSO, dimethyl sulfoxide; EDC, lethyl-3-(3-dimethylaminopropyl)carbodiimide; H- DADLE, [’HI -[ D-Ala’ , D-LeU’ ] enkephalin; H- DHM, [’ H] dihydromorphine; HOBt, l-hydroxy- benzotriazole; TFA, trifluoroacetic acid; THF, tetra- hydrofuran; TosC1, p-tohenesulfonyl chloride; Z, benzyloxy carbonyl. *Present address: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205, USA.

With numerous synthesis of analogs, the struc- ture-activity relationships of endogenous opioid enkephalins have been clarified (2,3). Our current interest in the incorporation of dehydroamino acid residues into peptide hormones led to the synthesis of the highly potent [D-Na’ , APhe4, Met’] enkephalin amide (4), which is five times as active as the corresponding saturated enkephalins in the stimulated guinea pig ileum assay and the mouse tail-flick test (5). Simple a, P-dehydro- genation of an amino acid residue results in the formation of more rigid, hydrophobic and reactive moiety. In general, such unsaturated sites in peptide hormones may act to enhance activity by virtue of an increased receptor binding affinity or by their ability to react irreversibly with a nucleophile on the receptor

54 0367--8377/82/010054-09 $02.00 0 1982 Munksgaard. Copenhagen

Synthesis of AAlaenkephalin

sometimes encountered, however, cause poor yields and difficult purifications. Schmidt et al. (18) have reviewed these reactions: oxazoline formation, aziridine formation, hydantoin formation and nucleophilic substi- tution of the 0-tosyl group. In our preliminary syntheses of di- or tripeptides containing AAla, some of these side reactions were observed and resulted in inseparable mixture formation.

Recently, Vedejs & Fuchs(l9) reported a Pelimination reaction from 3-acetoxy-4,4- diethoxybutanal catalysed by DBU yielding the a,P-unsaturated aldehyde (C2H50)2CHCH= CHCHO in a high yield. It seemed reasonable that an 0-acylated serine might also be induced to undergo a 8-elimination by such strong bases as DBU or Dabco. Since the facile syn- thesis of depsipeptides using N,N’-dicyclohexyl carbodiimide (DCC) and a 4-dimethylamino- pyridine (DMAP) catalyst has been reported (20), we applied this procedure to the acylation of serine peptides. Z-Ser-Gly-OMe (I) was acylated with acetic, chloroacetic, benzoic and p-nitrobenzoic acids using DCC and a catalytic amount of DMAP within a rather short time (1 h) at 0’ in excellent yields. However, these acylated peptides were quite stable to treat- ment with the appropriate bases (DBU, Dabco and Et2NH) and no AAla residues were pro- duced. Z-Ser(Ochloroacety1)-Gly-OMe (111) reacted with Et, NH to yield Z-Ser(0-dimethyl- aminoacety1)Gly-OMe (VI) apparently by an SN2 reaction. Thus, in the present study, AAla was derived from serine in the final penta- or tetrapeptides by the standard tosy- lation Pelimination sequences using Et, NH.

Syntheses of AAh’ enkephalins We selected [D-Ala’, Leu’ ] enkephalin, H-Tyr- D -Ala-Gly-Phe-Leu-OH as the saturated enkephalin on which to model our systematic syntheses of analogs containing AAla’ Or 3 ,

APhe4 and ALeu’. The choice of the Leu5 compounds avoids complications due to sulfur and amide functions present in Met’ amide analogs and ALeu’ compounds can be pre- pared by known methods (21). The phenolic hydroxyl group of Tyr’ must be protected during the formation of AAla’ from Ser’ and in the final deprotection step, we planned to use anhydrous liquid HF because the AAla

55

surface. Although the syntheses of unsaturated peptide hormone analogs such as oxytocin (6) , angiotensin (7) and bradykinin (8) have been reported as highly potent agonists or antag- onists, the effect of a dehydroamino acid moiety on biological activity has not been clearly elucidated. Through the synthesis of systematically unsaturated analogs of enkephalin, dehydroenkephalins, we are attempting to clarify this point and, simul- taneously, to develop further structure-activity relationships.

The enzymatic degradation of enkephalins occurs most rapidly at Tyr’-Gly2 peptide linkage in rat brain homogenates(9) and in plasma (10). Substitution of D-alanine in position 2 for glycine gives a peptide with good agonist properties having a longer duration of action (1 1-13). @Dehydrogenation of the D - alanine residue in position 2 could also result in complete resistance to enzymatic hydrolysis as demonstrated for APhe peptides (14). Thus we designed the synthesis of [AAla2, Leu5 ] - enkephalin (XXV) with the expectation of increased affinity for opiate receptors and resistance to peptidases. The finding that tetrapeptide enkephalin analogs lacking the C-terminal amino acids are often more potent than the pentapeptide (1 5,16) encouraged us to synthesize also the [AAla’ ] -desLeu5 - enkephalin. We describe here the syntheses and biological activity of (AAla’ , Leu’ ] enkephalin and [AAla* ] -desLeu’ enkephalin methyl ester.

OAcylation of the serine residue in a peptide in an attempt to incoporate dehydroalanine A dehydroalanine residue can be derived from a serine-0-sulfonate such as 0-tosylserine by a 0-elimination reaction (1 7). The side reactions

CH2 I1

H-Tyr-NH-C-CO-Gly-Phe-Leu-OH (XXW

CH3 I

H-Tyr-NH-CH-CO-Gly-Phe-Leu-OH (STi 1

FIGURE 1 Structure of unsaturated [ AAla* , LeuS ] enkephah (XXV) and saturated [D-Ala’, Leu’ j-enkephalin (ST, )

Y. Shimohigashi and C.H. Stammer

lyr’

BOC--OH

BOC CI,Bzl

Z-‘OH H - IXV) IEDC - z ’

z -‘ z -- A A I ~

TABLE 1 Binding activity of dehydro-enkephalins

Ser’ GIP Phe‘ Leu‘

BOC--OH H-- OBzl*TosOH

BOC - 0821 IHCI- EtOAcl

IDCCI

BOC--OH H - OBzI*HCI

BOC - OBzl IDCCI

IHCI- EtOAcl H- ‘- OBzl*HCI

IEDC - HOBtl I OBrl

OBzI-HCI IHCl- EtOAcl

HOBtl -0Bzl

lTosCI1 Tos 1 ’ I -0621 lEt2NHI

1 0621

Enkephalin ED;^ vs H-DADLE ED,, vs H-DHM

H

H-Tyr-AAlaCly-Phe-Leu-OH (XXV) H-Tyr-AAlaCly-Phe-OMe (XII) H-Tyr-D-Ala-Gly-Phe-Leu-OH (ST, ) H-Tyr-GlyGly-Phe-Leu-OH (ST, ) Pendorphin

IHFI - OH

2.8 (86)b

1.9 (130) 2.4 (100) 2.3 (100)

180 (1) 6.1 (250)

76 (20) 7.6 (200)

15 (100) 2.8 (540)

a b n.M. Relative potency (%) against leucineenkephalin (ST, ),

moiety is very sensitive to aqueous acidic con- ditions yielding readily the peptide amide and pyruvic acid derivatives (22). Hydrogenolytic deblocking can not be used in dehydropeptide synthesis.

Z-Tyr(Bz1)-Ser-Gly-Phe-OMe (XI) and Z- Tyr(C1, Bz1)-Ser-Gly-Phe-Leu-OBzl (XXII) were prepared by stepwise elongation from the C-terminal using the carbodiimide/ 1 -hydroxy- benzotriazole coupling method. The synthetic scheme for XXII is illustrated in Fig. 2. Com- pounds XI and XXII were tosylated with p-toluenesulfonyl chloride in pyridine, and tosylated peptides XI1 and XXIII were treated with diethylamine in DMF to yield the AAla residue. Purification was carried out by silica gel column chromatography (35-45% yield), and pure peptides containing AAla’ were treated with anhydrous liquid HF for 60 min at 0”. Purifications of free AAla’ -enkephalins were carried out by gel filtration followed by partition column chromatography using n-BuOH-AcOH-H, 0 (4 : 1 : 5) as the solvent system.

The AAla moiety was confirmed by ’ H-n.m.r. showing two distinct singlet peaks (5 .5-5.6 and 6.1-6.5p.p.m.) for the vinyl protons and a sharp singlet peak (9.2-9.5 p.p.m.) for the amide proton, and also by difference spectro- scopy (~4000, A240 nm) in U.V. measurements (23).

Biological uctivity of AAla’-enkephalins As shown in Table 1, [AAla, Leu’ ] -enkephalin (XXV) was 2.5 times as active as Leu’- enkephalin (ST,) and 1.3 times as active as the saturated stereoisomer [D-Ala’, D-L~u’ ] - enkephalin as measured against [3 HI dihydro-

morphine (3H-DHM) as a tracer, and almost as active as both standards against [3H] -[D-Ma’, D-Leu’ ] enkephalin (3 H-DADLE). The dose response curves were parallel to the saturated analogs as shown in Figs. 3 and 4. In these binding assays using rat brain membranes, enzymatic hydrolysis was minimized by the addition of bacitracin in order to examine only the binding ability of the dehydro- enkephalins. The results obtained for [AAla2, Leu’ ] enkephalin indicate that the change in molecular shape caused by the rigid unsaturated site sustains the interaction with the opiate receptors, but the incorporation of A d a Z

IXVll

(XVII-HCI)

IXVIII)

IXIX*HCI)

(XXJ

lXXI*HCIJ

(XXIIJ

IXXlllJ

(XXIVJ

(XXVJ

FIGURE 2 Syntheses of [ AAla’ , Leu’ ] enkephalin (XXV) via pentapeptide (XXII) containing a Ser residue.

56

Synthesis of AAla-enkephalin

a preference for one type of receptor (Table 2). 0-Endorphin has almost the same affinity for both receptors, while all enkephalin analogs have more affinity for 6-receptors. Surprisingly, our AAla'-tetrapeptide analog (XW) is an exception to this, since analog XIV shows selectivity for the p receptors. This result may be mainly due to the removal of the C-terminal residue as demonstrated by Ronai e l al. (25). In a comparison of the influence of changes in the amino acid residue at position 2, replacement of Gly' by D-Ala* and then by AAla' facilitates the selectivity towards the p sites as shown in Fig. 5. This indicates that increased hydro- phobicity of position 2 increases selectivity for p receptors. In order to elucidate further effects of the unsaturated moiety on bioactivity, such as possible irreversible receptor cite reactions and resistance to peptidase, other biological assays are in progress.

g m K I 4- x s m -

l m K m "t

\\ IWU Wl

W.ml-'

FIGURE 3 Displacement curves of AAla' enkephalins using [ ' H] -[ D-Ala', D-Leu' ] enkephalin as indicator ligand.

0

does not afford a strongly enhanced binding ability.

On the other hand, the tetrapeptide analog [AAla' ] -deskus enkephalin ester (XIV) showed very weak activities (1 -200/0) against both tracers. McGregor et al. (15) reported [D-Ala' ] -desLeuS enkephalin amide to be equipotent with the pentapeptide analog, and Fujino et al. (16) synthesized some highly potent tetrapeptide acylhydrazide analogs. The presence of amide type bonding of the C-terminus of R e 4 may be necessary for activity. In this synthesis, however, C-terminal Phe4-NH2 would be easily dehydrated to yield a nitrile function during tosylation of the Ser' residue by p-toluenesulfonyl chloride, so that i t could not be prepared.

The influence of the dehydroalanine moiety on the peptide affinity for two types of opiate receptors (24) has been examined by using 3H-DADLE for peptide receptors (6) and 3H-DHM for opiate receptors (p) as tracers. The ratio of the potencies against these two tracers, EDSO vs H-DHM/EDSo vs H-DADLE, suggests

EXPERIMENTAL PROCEDURES

All the melting points were measured with 6427-HI 0 Thomas-Hoover Melting Point

Displacement curves of AAla'-enkephalins using [ H] dihydromorphine as indicator ligand.

TABLE 2 Ratio of potency against ['H]dihydromorphhe and [ 'HJ -[D-Ah-D-LeuS 1-enkephalin

Enkephalin ED,, vs H-DHM

ED,, vs H-DADLE

H-Tyr-AAlaGly-Phe-LeuUH (XXV) 2.2 H-Tyr-AAlaGlyPhe-OMe (XII) 0.4 H-Tyr-D-AlaCly-Phe-Leu-OH (ST, ) 4.0 H-TyrGlyGly-Phe-Leu-OH (ST,) 6.2 Pendorphin 1.2

57

Y. Shimohigashi and C.H. Stammer

7 r

f > a

0 % "P

GIy2 D-Ala* AAla2 MlaZ-desLeu5

FIGURE 5 Potency ratio (ED,, vs H-DHM/ED,, vs H-DADLE) for different substitution in position 2 of enkephalin. ratio = 1 , equipotent; ratio >1, more potent for 6 receptors; ratio < 1 , more potent for ct receptors.

Apparatus, and are uncorrected. T.1.c. was carried out on Silica Gel K6GF (Whatmail) with following solvent systems: (by volume) Ri , CHC13-MeOH (5:l); R:, CHCI,-MeOH (9:l); R:, CHC13-MeOH-AcOH (95:5:1); R:, CHC13- EtOAc (1:l); R:, CHC1,-EtOAc (3:l); RP, n-Bu0I-I-AcOH-pyridine-H, 0 (4: 1 : 1 :2); R: , n-BuOH-AcOH-H,O (4: 1 : 5 ) , organic phase; R:, 0.1% Ac0H-n-BuOH-pyridine ( 1 1:5:3), organic phase. Silica gel columns were packed with Silica Gel 60 (230-400 mesh, Merck). Electrophoreses were carried out on a Whatman 3MM chromatography paper at pH 1.9 in a solvent mixture of HCOOH-MeOH-AcOH-H, 0 (1 :3:6: 10). Migration values are reported with respect to lysine as RLys. Optical rotations were measured with a Perkin-Elmer Model 141 Polarimeter. The ' H-n.m.r. spectra were recorded on a Varian EM-390 90MHz n.m.r. Spectrometer with tetramethylsilane as internal standard. Amino acid analyses were performed on a Beckman Model 119CL Amino Acid Analyzer. The U.V. spectra were measured on a Varian Cary 219 Spectrophotometer. Elemental analyses were carried out by Atlantic Microlab, Atlanta, Georgia. 0-Acylation of Z-Ser-GIy-OMe(I) by DCC- DMAP method General procedure. To a solution of I (621 mg, 2mmol) and carboxylic acid (3mmol) in

CH2Clz (5ml) were added DMAP (24mg, 0.2 mmol) and DCC (619 mg, 3 mmol) at 0". The reaction mixture was stirred for 1 h at 0". After filtration the solvent was evaporated in vacuo and the residual oil was dissolved in EtOAc. The solution was washed successively with 4% NaHCO,, 2% HCl and H,O, dried (anhyd. Na, SO4), and evaporated. Purifications were carried out by silica gel chromatography using a column (1.9 x 40cm) and eluants of CHCl, -EtOAc ( 1 -3: 1) . The fractions containing product were pooled, evaporated and the residual solid was recrystallized from EtOAc- ether-petroleum ether.

Z-Ser(O-acety1)-Gly-OMe( I I ) . Yield, 5 33 mg (75%); m.p. 89-91'; [a]&' + 11.4" (c 1.0, CHCI,); Ri 0.92, R: 0.62, R; 0.48, RI 0.28; n.m.r. (DMSOd6) 6: 2.00 (s, 3H, OCOCH,) 3.68 (s, 3H, COOCH,), 3.90 (d, J = 6&, 2H, GIyC,H,), 4.1-4.6 (m, 3H, SerC,!,

ArH), 7.68 (d, J = 9 c p s , i H , Ser NH), 8.56 ( t , l H , GlyNH). Anal. calc. fz C16H2007NZ (352.3): C 54.54, H 5.72, N 7.95. Found: C, 54.31; H, 6.01; N, 8.09.

CflHZ), 5.12 (s, 2H, PhCHZO), 7.47 (s, 5H, -

Z - Ser(0 - chloroacety1)- Gly- OMe (W). Yield, 647mg (84%); m.p. 118-120"; [a]: + 12.6' (c 1.0, CHC13), R: 0.74, R;' 0.52; n.m.r.

(d, J = 6cps, 2H, GlyC,H,), 4.114.6 (m, 3H, SerC,H, CpH,), 4.33 (s, 2H, COCHzC1), 5.1 1 (s, 2H, PhCHzO), 7.42 (s, 5IJ,-ArH), 7.72 (d, J = 8 c p s , l H , SerNH), - 8.58 (t, l H , GlyNH). AnaLcalc. for C16H1907N2C1(386.8): C 49.68, H 4.95, N 7.24. Found: C 49.68, H 4.99, N 7.26.

(DMSO-d,) 6 : 3.65 (s, 3H, COOCHj), 3.90

Z-Ser(~~enzoyl)-GIy-OMe (ZV). Yield, 624 mg (75%); m.p. 118-120"; [a12 -4.3' (c 1.0, DMF); R: 0.92, R: 0.73, R: 0.64, Ri 0.37; n.m.r. (DMSOd6) 6: 3.75 (s, 3H, COOCH3), 3.93 (d, J = 6 cps, 2H, Gly C,H,), 4.3-4.8<m, 3H, SerC,H, CpH,), 5.12 (s, 2H, PhCFJ,O), 7.43 (s, 5H, Arg, 7.5-8.2 (m, SH, Arg, 7.78 (d, J = 8 cps. 1 H, SerNH), - 8.68 (t, 1 H, GlyNH). -

58

Synthesis of AAla-enkephalin

Anal. calc. for Cz1Hzz07N, (414.4): C 60.86, H 5.35, N 6.76. Found: C 61.00, H 5.39, N 6.75.

Z-Ser(0-pJzitrobenzoy1)GlyOMe ( V ) . Yield 790mg (86%); m.p. 175-176'; [ a l g - 3.5" (c 1.0, DMF);R: 0.58,Rt 0.69;RI 0.44;n.m.r. (DMSOdI,) 6: 3.67 (s, 3H, COOCH,), 3.95 (d, J = 6 cps, 2H, GlyC&), 4.4-4.8 (m, 3H, SerC,H, CpH,), 5.13 (s, 2H, PhCHzO), 7.43 (s, 5HrArH), 7.90 (d, J = 7 cps, 1H, SerNH), 8.35 (m, 4H, ArH), 8.70 (t, lH, GlyNH). Anal. calc. for G1 Hzl 0 9 N 3 (459.4)TC 54.90, H 4.61, N 9.15. Found: C 54.82, H 4.66, N 9.13.

Z-Ser (O-die thy laminoace ty l )GZy-OMe ( VZ) . To a solution of 111 (77 mg, 0.2 mmol) in DMF was added 2mM EtzNH-DMF (0.2ml) at room temperature. The reaction mixture was allowed to stand for 3 days at room temperature, and evaporated. The residual oil was dissolved in EtOAc. The solution was washed with Hz 0, and evaporated. The residual oil was crystallized from ether-petroleum ether; yield, 46 mg (54%); m.p. 60-61'; [ a ) g i-13.7" (c 1.0, CHCl,); R: 0.09, R': 0.13; n.m.r. (DMSOd6)

(9, J = 7cps, 2H, NCHzCH3), 3.43 (s, 2H, OCOCHzN), 3.70 (s, 3 H , COOCg,), 3.90 (d, J =-6cps, 2H, GlyC,H2), 4.0-4.8 (m, 3H, SerC& C p H 2 ) , 5.1 1 (s, 2H, PhC_H20), 7.45 (s, 5H, ArH), 7.63 (d, J = 8 cps, lH, SerNH), - 8.57 (t, lH, GlyNH). Anal. calc. for Cz&g07N3 .1/2 HzO(432.5): C 55.54, H 6.99, N 9.72. Found: C 55.52, H 7.01, N 9.70.

6 : 0.98 (t, J = ~ C P S , 3H, NCHZCH,), 2.61

Synthesis of tetradehydroenkephalin analog

Boc-Gly-Phe-OMe (VII). To a chilled solution of Boc-Gly-OH (3.85g, 22mmol) and Et,N (3.08 ml, 22 mmol) in THF (40 ml) was added isobutyl chloroformate (2.88 ml, 22 mmol) at 0". After 10min a chilled solution of H-Phe- OMe.HCl(4.74g, 22mmol) and Et,N (3,08ml, 22mmol) in CHCI, (40ml) was added. The reaction mixture was stirred for 1 h at 0" and left overnight at room temperature. The solvent was evaporated and the residual oil was dissolved in EtOAc. The solution was washed successively

with 4% NaHC03, 1% citric acid and HzO, dried, and evaporated. The residual oil was purified on a column (3.1 x 48cm) of silica gel by elution with CHC1,-EtOAc (3:l). The fractions (300-450 ml) were pooled and evaporated to yield 4.96g of an oily product (67%); Ri 0.87, R: 0.44, R: 0.44.

H-Gly-Phe-OMe . HCI (VIZI. HCI). Compound VII (4.96 g, 14.8 mmol) was dissolved in 2.9 M HC1-EtOAc (50ml). After 2 h at room tem- perature, the resulting precipitate was collected with the aid of petroleum ether, washed with petroleum ether, and dried over KOH and P 2 0 5 ; yield 3.73g (92%); m.p. 158-159"; [a] g + 9.5" (c 1 .O, MeOH); R: 0.08. Anal. calc. for C1zH1703NZC1 (272.7): C 52.84, H 6.28, N 10.27. Found: C 52.88, H 6.30, N 10.28.

Boc-Ser-Gly-Phe-OMe ( I X ) . Boc-Ser-OH (3.6 1 g, 17.6 mmol), VIII .HCI (4.80 g, 17.6 mmol) and Et,N (2.5m1, 17.6rnmol) were dissolved in CHCl, (70ml). To the solution were added HOBt (3.23 g, 21.1 mmol) and EDC.HC1 (3.72g, 19.4mmol) at - 10". The reaction mixture was stirred for 2 h at 0", left overnight at room temperature, and evaporated. The residual oil was dissolved in EtOAc, and the solution was washed successively with 4% NaHCO,, 1oo/o citric acid and HzO, dried, and evaporated. The residual solid was recrystallized from MeOHether-petroleum ether; yield 6.13 g (28%); m.p. 166-168"; [a12 + 45.6" (c 1.0, CHCl,); Rj 0.59, R: 0.39. Anal. calc. for CzoH290,N3 (423.5): C 56.72, H 6.90, N 9.93. Found: C 56.72, H 6.94, N 9.94.

Z-~r(Bzl)Ser-Gly-Phe-OMe (XI) . Compound IX (2.12g, 5 mmol) was dissolved in TFA ( 1 7 ml) at 0'. After 30 min at 0" the solution was evaporated to leave an oil, which was solidi- fied by the addition of ether; yield of H-Ser- Gly-Phe-OMe-TFA (X-TFA), 2.13 g (10%). Z-Tyr(Bz1)-OH (2.03 g, 5 mmol), X*TFA (2.13g, 5mmol) and Et,N (0.7m1, 5mmol) were dissolved in DMF (20 ml). To this solution were added HOBt (0.92g, 6mmol) and EDC.HCI (1.05 g, 5.5 mmol) at - 10". The

59

Y . Shimohigashi and C.H. Stammer

reaction mixture was treated as described for IX; yield J.13g (88%); m.p. 144-146 ; [ a ] g

Anal. calc. for C39H4209N4 (710.8): C 66.90, H 5.96, N 7.88. Found: C 66.66, H 5.98, N 7.82.

- 0.8" (C 0.5, DMF); Ri 0.71, R; 0.32.

2-Tyr-(Bzl) -AAla-Cly-Phe-OMe (XIII). To a chilled solution of XI (3.10g, 4.4mmol) in dry pyridine (1 1 ml) was added TosCl (1.68 g, 8.8 mmol) at -- 5'. After 9 h at 0" the solution was evaporated, and cold water was added to yield a solid. The collected solid was washed successively with H,O, 10% citric acid, H,O and ether-petroleum ether (1 :2, 90 ml); yield of crude Z-Tyr(Bz1)-Ser(Tos)-Gly-Phe-OMe (XII), 3.77g (100%). To a solution of XI1 (3.77g, 4.4mmol) in DMF (10ml) was added Et,NH (0.9 ml, 8.8 mmo1)at room temperature. After 6 h, the solution was evaporated to leave a solid, which was collected with the aid of cold water. The purification was carried out on a silica gel column (2.8 x 10cm) eluted with CHCI, -EtOAc (1 : 1). The fractions (1 600- 2050ml) were pooled and evaporated. The resulting solid was recrystallized from dioxane- ether-petroleum ether; yield, 1.37 g (45%); m.p. 129-131"; [ a ] g - 19.4" (c 0.5, DMF); R: 0.44, R$ 0.38, R: 0.12; n.m.r. (DMSOd6) 6 : 5.64 (s, IH, C=CH.H), 6.26 (s, 1H, C=CH . H), 9.40 (s, 1 H, AaaNH); - U.V. (MeOH), ~4100 (x241 nm). Anal. calc. for C39H400sN4 (692.8): C 67.61, H 5.82, N 8.09. Found: C 67.44, H 5.84, N 8.04.

H-Tyr-AAla-Gly-Phe-OMe AcOH (XIV . AcOH). Compound XI11 (416 mg,0.6 mmol) was treated with anhydrous liq. HF (5ml) and anisole (0.4 ml) for I h at 0". The solution was evapor- ated to leave an oil, which was dissolved in 30% AcOH. The aqueous solution was washed with ether, evaporated to a small volume, and subjected to gel filtration on a column (1.9 x 90 cm) of Bio Gel P-2 (200-400 mesh) in 30% AcOH. The fractions (1 30-1 70 ml) were pooled and lyophilized; yield, 286 mg (90%). Further purification was carried out on partition chromatography column (1.9 x 82 cm) of

Sephadex G-10 with the solvent system of n-BuOH-AcOH-H,O (4: 1 :5). The fractions (140- 190 ml) were pooled and lyophilized repetitively with 30% AcOH; yield of pure XIV. AcOH, 178 mg (56%); m.p. 57-59"; [ a ] g + 37.4" (c 0.5, 95% AcOH); RP 0.83, R: 0.71, R! 0.86; RLys 0.53; U.V. (MeOH), €4000 (h241 nm). Amino acid ratios in acid hydrolyzate : Tyr 0.92, Gly 1.04, Phe 1 .OO,

Anal. calc. for CZ6H3,O8N4.1/2 H,O (537.6): C 58.09, H 6.19, N 10.42. Found: C 57.94, H 6.22, N 10.40.

NH3 1.07.

Synthesis of H-Tyr-AA la-Gly-Phe-Leu-OH ( [AAla2, Leu' ] -enkephalin)

Z-i')r(Cl,Bzl).OH ( X V ) . This compound was prepared from 0 -2,6-dichlorobenzyl-~-tyrosine (26) (3.0 g, 8.8 mmol) and carbobenzoxy chloride (1.41 ml, 8.8 mmol) in 1 M NaOH (1 7.6 ml) and acetone (1 ml) by the procedure of Bergmann & Zervas (27). The crude product was converted into its DCHA salt (XV . DCHA), which was recrystallized from MeOH-EtOAc- ether; yield, 3.94g (68%); m.p. 164-165"; [a] g + 20.6" (c 1 .O, MeOH); R: 0.45.

C 65.95, H 6.76, N 4.27. Found: C 66.02, H 6.79, N 4.25. Salt XVeDCHA (3.8g, 5.8 mmol) was converted into free acid XV by washing with 10% citric acid in EtOAc, which was recrystallized from MeOH-ether-petroleum ether; yield, 2.3g (83%); m.p. 149-150"; [a15 - 2.5" (c 1 .O, MeOH); R: 0.45. Anal. calc. for C24HZi 05NC12 (474.3): C 60.77, H 4.46, N 2.95. Found: C 60.84, H 4.50, N 2.91.

Anal. calc. for C ~ ~ H M O ~ N ~ C ~ Z (655.6):

Boc-Phe-Leu-OBzl (XVI) . To a solution of Boc- Phe-OH (2.65 g, 10 mmol) H-Leu-OBzl . TosOH (3.93g, 1Ommol) and Et3N (1.40m1, 1Ommol) in CH, C1, (30 ml) was added EDC * HCl(2.11 g, 11 mmol) at 0'. The reaction mixture was treated as described for IX, and finally the residual oil was crystallized from ether-pet- roleum ether; yield, 3.86 g (82%); m.p. 85-86"; [ c x ] ~ - 14.5" (C l.O,CHCI,);Ri 0.94,R: 0.83. Anal. calc. for C2,H3605NZ (468.6): C 69.20, H 7.74, N 5.99. Found: C 69.58, H 7.92, N 6.03.

60

Synthesis of AAla-enkephalin

EDC . HCI (0.42 g, 2.2 mmol) as described for IX. The crude product was purified by recrys- tallization twice from DMF-EtOAcether; yield, 1.75g (90%); m.p. 187-189"; [ a ] g -- 29.2" (c 0.5, DMF); R: 0.90, R: 0.38. Anal. calc. for C51H55010NSC12 .1/2 H 2 0 (977.9): C 62.63, H 5.83, N 7.16. Found: C 62.63, H 5.91, N 7.21.

H-Phe-Leu-OBzl- HCI (XVII . H a ) . Compound XVI (23.4g, 50mmol) was dissolved in 3.6M HCI-EtOAc (140ml). After 2h at room tem- perature the solvent was evaporated, and the resulting solid was collected with the aid of ether; yield, 20.1 g (99%); m.p. 160-162"; R: 0.28. Reported values; m.p. 159-160" (28).

Boc-Gly-Phe-Leu-OBzl (X VIIZ). This compound was prepared from Boc-Gly-OH (8.76 g, SOmmol), XVII*HCI (20.1 g, 50mmol), Et,N (714 50mmol) and DCC (10.32g, 50mmol) as described for IX; yield of an oil, 24.6 g (94%); R: 0.91, R: 0.58.

Boc-Ser- Gly -Phe- Leu - OBzl ( X X ) . Compound XVIII (6.3 1 g, 12 mmol) was dissolved in 3.0 M HCI-EtOAc (40 ml). After 4 h at room tempera- ture the solution was evaporated; yield of oily H-Gly-Phe-Leu-OBzl . HCI (XIX . HCl), 5 S O g (99%). Compound XX was prepared from Boc- Ser-OH (2.46 g, 12 mmol), XIX . HCl (5 S O g, 12 mmol), Et, N (1.68 ml, 12 mmol), HOBt (2.2 1 g, 14.4 mmol) and EDC . HCI (2.53 g, 13.2 mmol) as described for IX; yield of an oil, 7.66 g. The oil was purified by silica gel column chromatography using a column (3.0 x 45 cm) and elution with CHC1,-EtOAc (3-0:l) and a second silica gel column (3.0 x 45 cm) eluted with CHCI,-acetone (3: 1). The fractions con- taining a pure product were pooled and evapor- ated and the residual oil was crystallized from ether-petroleum ether; yield, 4.80 g (66%); m.p. 75-76"; [ a ] g - 10.5" (c 1.0, CHCl,); R: 0.33, R': 0.14, R: 0.06. Anal. calc. for C32H4408N4 (612.7): C 62.72, H 7.24, N 9.15. Found: C 62.66, H 7.27, N 9.14.

Z-Tyr(C12 Bzl )Ser-Gly-Phe-Leu-OBzI (XXZI). Compound XX (1.23 g, 2.0 mmol) was dissolved in 2.8M HC1-EtOAc (7.2ml). After 1.5 h at room temperature, the solution was evaporated; yield of oily H-Ser-Gly-Phe-Leu-OBzl . HCI (XXI . HCI), 1. I 0 g (1 00%). Compound XXII was prepared from XV (0.95 g, 2mmol), XXI.HCI ( ] . log , 2mmol), Et,N (0.28m1, 2 mmol), HOBt (0.37 g, 2.4 mmol) and

Z- 5r (CI2 Bz l ) AA la-Gly-Phe-Leu-OBzl (XXZV). Compound XXII (484 mg, 0.5 mmol) was treated with TosCl (191 mg, 1 mmol) as described for XII; yield of Z-Tyr(C12 Bz1)- Ser(Tos)-Gly-Phe-Leu-OBzl (XXIII), 545 mg (97%). Compound XXIII (545 mg, 0.49 mmol) was treated with Et2NH (0.1 ml, 0.98 mmol) in DMF as described for XIII. In order to remove any remaining XXII and XXIII, the solid obtained was stirred in EtOAc (30mI) overnight. The filtrate containing XXIV was evaporated to a small volume and the solution was chromatographed on a silica gel column (1.9 x 30 cm) by elution with CHCl, -EtOAc (3-1 : 1). The fractions (120-2 10 ml) were pooled and evaporated, and the residual oil was crystallized from EtOAc-ether-petroleum ether; yield, 159mg (34%); m.p. 76-77"; [a]:: -26.2" (C 0.5, DMF); R: 0.52, R: 0.53, R: 0.34, RI 0.06; n.m.r. (DMSOd6) 6 : 5.60 (s, IH, C=CH :H), 6.20 (s, IH, C=CH H), 9.1 I (s. lH, AAlaNFJ); U.V. (MeOH) €4100 (A241 nm). Anal. calc. for CS1 HS3 O9 NS C12 . H2 0 (968.9): C 63.22, H 5.72, N 7.23. Found: C 63.1 1, H 5.84, N 7.17.

H-Tyr-AALz-Gly-Phe-Leu-OH ([AAla', Leus ] - enkephalin) ( X X V ) . Compound XXIV (1 24 mg, 0.13 mmol) was treated with HF (2 ml) and anisole (0.1 ml) as described for XIV .AcOH. Purifications were carried out on a column (1.9 x 89 cm) of Bio-Gel P-2 (200-400 mesh) in 30% AcOH, followed by a partition column (1.5 x 82 cm) of Sephadex G- 10 using a solvent system of n-BuOH-AcOH-H20 (4: 1 5). The fractions containing pure XXV were pooled and lyophilized repetitively with 30% AcOH; yield, 25 mg (34%); m.p. 138" (decom.); [a] 2 + 8.4" (c 0.25, 95% AcOH). R$' 0.83, Rf7 0.87, RP 0.90; RLys 0.47;u.v. (MeOH) €4200 (A240 nm). Amino acid ratios in acid hydro1yzate;Tyr 0.95, Gly 1.06, Phe 1 .OO, Leu 1.03, NH3 1.06.

61

Y. Shimohigashi and C.H. Stammer

Binding assays 10. Hambrook, J.M., Morgan, B.A., Rance, M.J. & Receptor binding assays [3H] -[D-!4ki2, D- Smith, C.F.C. (1976)Naiure 262,782-783 b u s ] enkephalin (40 Ci mmol) and [3 H] di- 11. Pert, C.B., Pert, A., Chang, J. & Fong, B. (1976) hydromorphine (47.5 Ci mmol) were performed Science 1949 330-332

12. Coy, D.H., Kastin, A.J., Schally,A.V., Martin, O., as described by Pert ’ Snyder (29)’ Each Caron, N.G., Labrie, F., Walker, J.M., Fertel, R., peptide was assayed at least three times with G-.G. & Sandman, C.A. (1976) each t m e r and bacitracin (Sigma, fina1 Biochem,, Biophys. Res, Commun. 73,632-638 1 00 pglml). 13. Walker, J.M., Berntson, G.G., Sandman, C.A.,

Potencies expressed in EDso (i.e. dose which Coy, D.H., Schally, A.V. & Kastin, A.J. (1977) produces a 50% inhibition of binding) have Science 196,85-87 been estimated using an R.I.A. computer 14. English, M.L. & Stammer, C.H. (1978) Biochem. program which provides a weighted least square fitting of binding data after “logit” transform- ation (30). Nonspecific binding, obtained for each tracer in the presence of lpM of the corresponding unlabeled ligand, was subtracted from all the data. It was 10% of the binding for

H-DADLE and 30-3576 for H-DHM.

15.

16.

17. 18.

Biophys. Res. Commun. 83,1464-1467 McGregor, W.H., Stein, L. & Belluzzi, J.D. (1978) Life Sci. 23, 1371-1378 Fujino, M., Shinagawa, S., Kawai, K. 4 Ishii, H. (1979) Natunvissenschaften 66,625-626 Photaki, I. (1963) J. Am. Chem. SOC. 85,1123 Schmidt, U., Hausler, J., Ohler, E. & Poisel, H. (1979) Progress in the Chemistry of Natural Products, 37,251-327

19. Vedejs, E. & Fuchs, P.L. (1971) J. Org. Chem. ACKNOWLEDGMENTS 36.366-367

20. Gilon, C., Klausner, Y. & Hassner, A. (1979) Tetrahedron Lett., 381 1-3814

21. Poisel, H. & Schmidt, U. (1976) Angew. Chem. Inf, Ed, Engl. 294-295

Chem. Int. Ed. Engl. 12,664-665

We thank Dr. T. Costa, National Institutes of Health, for his biological assays and helpful discussions during this work. We gratefully acknowledge the financial

No. DA 02091. support Of the Institutes Of Grant 22. Gross, E., Noda, K. & Nisula, B. (1973) Angew,

1.

2.

3.

4.

5.

6.

7.

8.

9.

62

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Shimohigashi, Y., Costa, T. & Stammer, C.H. (1981) Biochem. Biophys. Res. Commun., in press Morley, J.S. (1980) Ann. Rev. Pharmacol. Toxicol. 20,8 1 - 1 10 Coy, D.H. & Kastin, A.J. (1980) Pharmac. Ther.

English, M.L. & Stammer, C.H. (1978) Biochem. Biophys. Res. Commun. 85,780-782 Chipkin, R.E., Stewart, J.M. & Stammer, C.H. (1979) Biochem. Biophys. Res. Commun. 87,

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Address: Prof. Charles H. Stammer Department of Chemistry University of Georgia Athens, Georgia 30602 USA