reduced peptide bond cyclic somatostatin based opioid octapeptides synthesis, conformational...

Int. J. Peptide Protein Res. 39, 1992, 401-414

Reduced peptide bond cyclic somatostatin based opioid octapeptides Synthesis, conformational properties and pharmacological characterization

WIESLAW M. KAZMIERSKI', RON D. FERGUSON ', RICHARD J. KNAPP', GEORGE K. LU12, HENRY I. YAMAMURA' and VICTOR J. HRUBY '

Department of Chernistiy, University of Arizona, Department of Phnrmcicology, Arizona Health Sciences Center, Tucson, Arizona, USA

Received 6 June, accepted for publication 20 November 1991

The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the p opioid receptor selective antag-

onist ~-Phe~-Cys~-Tyr~-~-Trp~-Orn~-Thr"-Pen~-Thr~-NH2 (IV), have been investigated. Peptide analogues that contain [CH,NH] and [CHzN] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp" with D- 1,2,3,4-tetrahydro-~-carboIine(~-Tca~), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for p opioid receptors. Con- formational analysis based on extensive NMR investigations demonstrated that the backbone conformations

of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr- NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the p opioid receptor. Peptide bond modifications were made including (Tyr3-$[ C H ~ N H I - D - T ~ ~ " ) , 3; (Tyr3-$[ CH~NI-D-TC~') , 2; and (Cys2-$[ CH2N]-Tic3)), 6. These analogues showed decreases in their p opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 'H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between p and 6 opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.

Key words: CH2NR>(R# H ) amide bond surrogate; conformation; p opioid receptors; NMR; opioid peptides; reduced

I I

I 1

peptide bonds; tetrahydrocarboline

In earlier work (1-3) we have developed an approach (referred t o a s "Topographical design on a stable template") t o control peptide topography by utilizing

Abbreviations of the common amino acids and their derivatives are in accordance with the recomniendations of IUPAC-IUB (J. Biol. Chmi. 264, 668-673, 1986). Additional abbreviations include: Tic, 1,2,3,4-tetrahydroisoquinoline carboxylate; Tca, 1,2,3,4-tetrahydro- P-carboline; TLC, thin-layer chromatography; H PLC, high performance liquid chromatography; FAB-MS, fast atom bombardment mass spectrometry; pMBHA, p-methylbenzhydrylamine; TFA. tri- fluoroacetic acid; BOP, bcnzotnazol-I-yloxy-tris-(dimethyl-amino)- phosphonium hexafluorophosphate; and DCM, dichloromethane; LAH, lithium aluminum hydride.

unusual, side chain-conformationally biased amino acids in the peptide sequence, but which still retain the backbone conformation of the peptide. For this pur- pose we have designed several somatostatin-derived octapeptide opioid antagonists with specific topographical features (e.g. I, 11, 111, and IV, Table l), and ex- amined their ability to recognize (bind to) either p or 6 opioid receptors (2,3). F o r example, replacement of D-Phe' in I by D- 1,2,3,4-tetrahydroisoquinoline carboxylate (D-Tic) resulted in a substantial increase in the selectivity and affinity of I1 for p opioid receptors (2).

'H NMR conformational analysis showed that the N-terminal D-Tic' residue has exclusively a g( + ) side chain conformation (3-5), resulting in an arrangement

40 1

W.M. Kazmierski et ul.

of aromatic side chains of D-Tic' and Tyr3 on the same face of the molecule (1, 3). This topography could be altered, resulting in an opposite arrangement (mis- match) of these aromatic moieties (3). by acylation of the N-terminal D-Tic with Gly (111, Table l), leading to the gciuche( - ) side chain conformation of D-Tic'. 111 showed a large decrease in affinity for p opioid receptors, and a modest increase of affinity for 6 receptors (2). Thus, we concluded that the N-terminal D-Tic prefers a gauche( + ) side chain conformation, whereas when a D-Tic residue is in the peptide chain, it prefers a gauche( - ) side chain Conformation. In our efforts to further understand the conformational properties of pipecolic acid systems such Tic, and their possible ap- plication in topographical design, we have turned our attention towards replacement of the peptide bond, centered on the pipecolic acid amino group, by an ami- nomethylene group.

We reasoned that the strong 1,2 diequatorial inter- actions of the N- and C-substituents present in the gauche( + ) conformation (Fig. 1. II), that cause the gauche( - ) conformation to be more stable for an acylated amino group Tic (3, 6), could be relieved if the N-substituent (CH2-N-C,) were forced to attain an out of plane conformation. To achieve this, the properties that cause its planar arrangement (resonance of the carbonyl group and the free electron pair of the amino group) needed to be modified. In principle, this can be done by replacing the carbonyl group preceding the pipecolic acid amino with a methylene unit. If, indeed, the resulting sp3 hybridization allows for nonpla- narity of both the C- and N-substituents, then it is possible that a 1,2-diequatorial relation may be energetically tolerated by the molecule in a gccuche( + ) side chain conformation. The required alkylamine may be obtained synthetically by reductive alkylation of a pipecolic acid derivative with an amino aldehyde in the presence of sodium cyanoborohydride. This procedure

T A B L E I Chemicul sfrucrure.T of .sytherir opioid peprides -

I D-Phe-Cys-Tqr-D-Trp-Lq s-Pen-Thr-,VHH- (CTP) - I1 D-Tic-Cys-Tyr-D-Trp-Lys-Pen-Thr-,~H- (TCTP)

111 Gly-D-Tic-C)s-Tyr-D-Trp-Om-Thr-Pen-Thr-hH_.

IV D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-,~H- (CTOP)

I I

I I

I 1 D-Phc-Cys-Tyr-o-Tca-Orn-Thr-P~n-Thr-,,r~'H-

I I 2 D-Phe-Cys-Tyr-Q [ CH2N ]-~-Tca-Orn-Thr-Pen-Thr-"H.

3 D-Phc-C;s-Tyr-Q [CH,NH]-~-Trp-Orn-Thr-Pkn-Thr-h'H.

5 ~-Pgl-C;,s-+ [ CH2N 1-Tic-o-Trp-Om-Thr-Pkn-Thr-IZIH.

has been successfully used to obtain CHzNH units both in a solution (7) and on solid supports (8), with a goal to increase peptide resistance to proteases (9). To our knowledge, CH2NR2 (where R # H) units have not yet been constructed in peptide syntheses, particularly on solid supports.

In this paper we present a series of six peptides (1-6, Table l), including four with modified peptide bonds, that address the above design questions. We have synthesized analogues of peptide I (with Orn5 replacing Lys5) and withTic' or D-Tca4 (Tca = 1,2,3,4-tetrahydro- P-carboline) (peptides 4 and 1, respectively, Table 1). Thus, the peptide bonds preceding the pipecolic acid- derived amino acids in the peptide chain were replaced by either CH2N (peptides 2, 5, 6 ) or CHzNH (in a control peptide 3). We also present the pharmacological properties of these new peptides and their conformational properties.

MATERIALS AND METHODS

Geiieral methods Syntheses of peptides 1 through 6 were accomplished by methods reported previously (1, 2) utilizing standard solid phase synthetic techniques (10, 11) on a Vega (Tucson, AZ) Model 250 or 1000 peptide synthesizer. N"-Boc protected amino acids were either purchased from Bachem (Torrance, CA) or were prepared by literature methods (1 1). Carboxamide terminal peptides were synthesized using a p-rnethylbenzhydrylamine (pMBHA) resin that was prepared by literature methods (12); resin substitution was 1.0 mM/g. A 1.5 M excess of preformed symmetrical anhydrides or a 3 M excess of N-hydroxybenzotriazole active esters was used for coupling reactions, which were monitored by ninhydrin (13) or chloranil tests (14). Purity of the final peptides was assessed by TLC in four different solvents, RP-HPLC, FAB-MS (Table 2), and 'H NMR. Capillary melting points were determined on a Thomas- Hoover apparatus and are uncorrected. Purity for each amino acid was established by the ninhydrin test, 'H NMR, optical rotation (sodium D line, Rudolph Re- search Auto-Pol 111 polarimeter), and TLC. Purifica- tion of peptides was accomplished by a combination of gel filtration, partition chromatography and reversed phase high performance liquid chromatography (RP- HPLC). For most cases gel filtration (G-15) followed by RP-HPLC was sufficient to obtain a peptide of high purity (> 95%). Gel filtrations were performed on a Sephadex G-15 (Pharmacia Fine Chemicals, Piscat- away, NJ ) column (2.65 x 75 cm), applying a 5% solution of acetic acid isocratically, with a Buchler Mono- static Pump (20-30 mL/h, over 20 h), a Buchler Fracto- Scan (254nm), and a Buchler Automatic Fraction Collector. Preparative RP-HPLC was performed on a Perkin-Elmer Series 3B Liquid Chromatograph equipped with an LC-75 Spectrophotometric Detector and an LCI-100 Laboratory Integrator, or on a Spectra-

402

Opioid peptides

" \ I

gauche (-)

FIGURE 1

gauche (-) gauche (+)

Conformational transformations of D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (D-Tic). I. D-Tic with a free amino group prefers gauche( + ) side chain conformation. 11. Peptide chain attachment on N-tcrmini of D-Tic 1,2 pseudoequatorial strains (bend arrows) dcsta- bilizing the gauche( + ) conformation. 111. The heterocyclic ring flips to a gauche( - ) side chain conformation, in which both the N- and C- substituents bear a 1.2 pseudoaxial relationship devoid of strains. IV. Sp3 hybridized N allows an out of plane relationship of both C- and N-termini, possibly allowing for gauche( + ) conformation. Note that guuche( + ) for a D-alnino acid corresponds to the same topography as gauche( - ) for an L-amino acid.

TABLE 2 Analvtical data for peptides prepared in this work

Peptide Thin-layer chromatograph)" HPLCb FAB-MS R, valucs k.'

I I1 111 IV [M + HI<,h7 [ M + H l c a ~ L

1 0.32 0.79 0.77 0.76 11.45 1074 1074 2 0.29 0.78 0.76 0.73 7.25 1060 1060 3 0.29 0.77 0.75 0.73 6.47 1048 1048 4 0.3 1 0.78 0.75 0.72 8.36 1058 1058 5 0.26 0.76 0.76 0.73 7.66 1037* 1037 6 0.25 0.80 0.78 0.74 6.12 1050* 1050

Silica gel G F 250 microns (Analtech) glass plates were used. The following solvent systems were used: ( I ) n-BuOH/AcOH/H20, 4:1:5: (v/v); (11) iPr-OH/NH3/H20.3:1:1: (byvol.); (111) n-BuOH/AcOH/HzO/Pyr, 6:1:5:6 (by vol.); (IV) n-BuOH/AcOH/H20/Pyr 15:3:10: 12 (by vol.).

* [ M t Li] recorded Vydac 218 TP 104 C18 column (25 cm x 4.6 mm), 0.1 ?; TFA/CH3CN 80:20, flow rate 1.5 mL/min, monitored at 2 = 214 mm.

Physics Liquid Chromatograph equipped with a Spectra-Physics SP 8800 ternary pump, SP 8500 dynamic mixer, Spectroflow 757 Absorbance Detector and SP4270 Integrator. Fractions were monitored at 280 nm or 254 nm (if no Tyr residue was present). A Vydac semi-preparative column (10 mm x 25 cm) was used with either isocratic or linear gradient elution in a mobile phase of varying concentrations of acetonitrile in aqueous TFA (0.10%). Cleavage of all side protecting groups as well as peptide from the resin was achieved with liquid H F (approximately 15 mL/g resin) with addition of 1 mL anisole, at 0'. The product was

washed with ethyl ether (3 x 20 mL), extracted with 100, aqueous HOAc (3 x 20 mL) followed by glacial HOAc (2 x 20 mL); both fractions were lyophilized separately. Next the linear peptide was cyclized by dis- solving the crude linear peptide in 1.5 L of water (pH adjusted with aqueous ammonia to 8.5), and oxidizing with 0.01 N K3Fe(CN)6 until the yellow color persisted for 1 h. After the reaction was terminated, the pH was adjusted to 4.5 with AcOH, ferrocyanide and ferricy- anide were removed by treatment with 15 mL of Am- berlite IRA-45 (mesh 15-60, C1- form), the mixture was stirred for 1 h, filtered, and the solution concen-

403

W.M. Kazmierski et 01.

trated in vucuo and lyophilized. Gel filtration on a 1 0 0 ~ 2.5 cm Sephadex G-15 column with 5 " , (v,/v) aqueous HOAc was generally applied. Final purification was achieved by RP-HPLC using a gradient of acetonitrile and O.lo,, TFA. Mass spectra were recorded on a Hewlett-Packard 5988A (University of Arizona Microanalysis Center) spectrometer using either FAB or EI techniques. Elemental analyses were performed by Desert Analytics (Tucson, AZ).

~-1,2,3.4-Tetruhydro-~carboline, D-TCO (Fig. 2). The compound was synthesized by methods described in the literature (15). D-Trp (45 g, 242 mM; Sigma) was suspended in 89 mL of distilled water. The 9.77 g (245 mM) NaOH was added, the temperature brought to 37". and the mixture stirred until the D-Trp was dissolved. Then 22 g of 37"" formaldehyde was added, and the reaction proceeded for 3 h under nitrogen. The reaction was terminated and the product precipitated with a stoichiometric amount of 6 N HC1 (final pH 3). The yellowish precipitate was left overnight in the re- frigerator, filtered, suspended in 3,1'2 (v;v) EtOH,'water (400 mL), and refluxed for 30 min. The suspension was filtered off, washed and dried it7 wcuo; Yield 88.5",. Anal. calc. for C12H12N202: C 66.660,, H 5.59";, N 12.960,;. Found: C 66.829,, H 5.480;, N 12.95",.

M S ( M + H+)216(216 Calc.);13CNMR(250 MHz. DzO + HCI): 173.80 (CO), 139.2 (Ar), 128.2 (Ar), 127.7 (Ar), 122.47 (Ar). 120.5 (Ar), 119.52 (Ar), 114.13 (Ar), 106.80 (Ar), 57.07 (Cx), 43.8 (CH?N), 23.93 (C,j).

~-Phe-C~s-Tyr-~-Tca-Om-Thr-P~n-Thr-NH~. I . The title peptide was synthesized by stepwise chain elongation, yielding Boc-~-Phe-Cys(S-4' -MeBzl)- Tyr(O-2,6-C1~-Bzl)-~-Tca-Orn(Cbz)-Thr(O-Bzl)-Pen- (S-4-MeBzl)-Thr(O-Bzl)-MBHA resin. Workup as outlined above with RP-HPLC as the final purification using a gradient of 10-307, acetonitrile in 0.1 TFA over a period of 20 min at a flow rate of 3.5 mL/min gave the title compound 1 in a yield of 23 yo. The structure of peptide 1 was confirmed by IH NMR analysis; the analytical data is given in Table 2.

H

H-

H

gauche (-) gauche (+)

FIGURE 2 GoirLhe( ~ ) and g~ruc/re( + ) sidc chain conformations of D-TU

404

N '-t-Bict~'lo.~~'carhotz~~l-~-l,2,3,4-tetruhydro-~-carboline IN1-Boc-D-Tca). The title compound was synthesised by standard methods (1 1) in a yield of 93.4%, 'H NMR (250 MHz, db-DMSO, ppni): 10.86 (indole, J = 11.5), 7.41(d,J=7.6),7.27(d,J=7.29),7.04(t ,J=7.0),6.95 (t, J =7.2), 5.14+ 5.04 (d, J = 5.8+d, J = 5.7), 4.73+4.44 (d, J = 1 6 . 3 + d , J=16.3), 4.69+4.33 (d, 16.6+d, J = 16.7), 3.28 (m), 2.96 (m); [ a ] g -83.53 (c 0.5; HOAc); TLC, Rf= 0.87 (n-butonal/acetic acid/ water, 4/ 1 / 1. by vol.).

SJnthesis of Nx-Boc- T ~ V ~ O - ~ , ~ - C I ~ - B Z ~ ) - C H O First, Nz-Boc-Tyr(O-2,6-Cl~-Bzl)CON(OCH~)CH~ was synthesized by the racemization free method of Fehrentz & Castro (16), as modified by others (17, 18). A sample of 4.75 g (10.8 mM) Nx-Boc-Tyr(O-2,6-C12- Bzl) was dissolved in 7 mL DCM, and 1.05 g ( 10.4 mM) triethylamine (TEA) was added. After cooling to O " , 4.60 g benzotriazol- 1-yloxy-tris-(dimethy1amino)phosphonium hexafluorophosphate (BOP) (in 7 mL DCM) was added. After 5 min, 1.47 g (14.5 mM) of O-N- dimethylhydroxylamineand 1.50 g( 14.9 mM)oftriethyl- amine were added, transferred to the reaction vessel and the resulting mixture was warmed to room temperature. After 60min the volume was diluted with DCM (150 mL). and the mixture successively extracted with 3 N HCI (3 x 15 mL), saturated sodium hydrogen carbonate solution (3 x 10 mL), and saturated sodium chloride solution (3 x 20 mL). The organic phase was dried with magnesium sulfate and the solvent evaporated. The crude product was purified on silica gel (230-400 mesh, Aldrich), yicld, 83%. TLC: Rr= 0.65 (95151 1, by vol., chloroform/methanol/acetic acid, An- altech silica gel plates), R f = 0.73 (ethyl acetate, Anal- tech silica gel plates); 'H NMR (250 MHz, dG-DMSO): 7.50 (m, 2,6-C1.-Bzl, 3H), 7.07 (d + d, J = 8.5 Hz, 4H), 7.15 (d, NH, lH), 5.18 ( s , CH?Bzl, 2H), 4.50 (m, aH,

(dd + dd, 4.0 Hz, 9.9 Hz, PH, 2H), 1.30 (s, Boc, 9H). MS (M + 1): 482 (Calc., 482). Anal. calc.: C 57.26y0, H 5.81, N 5.80:;. Found: C 57.32",, H 6.02:&, N 5.87%.

Second, a sample of 3.7 g (7.6 mM) of Boc-Tyr(0- ~ , ~ - C ~ ~ - B Z ~ ) C O N ( O C H ~ ) C H ~ was dissolved in a mixture of 100 mL anhydrous ethyl ether and 60 mL of freshly distilled THF, followed by cooling by 0". LAH (0.87 g, 22.8 mM) was added portionwise while stirring over 15 min. The reaction was allowed to proceed for an additional 20 min at 0 ', and then the excess LAH was quenched with about 10 mL ethyl acetate, and the lithium-amide complex hydrolyzed with 5 7; citric acid solution (added dropwisc). The mixture was vigorously stirred for 30 more min, ethyl ether added (100 mL), and the aqueous phase separated and extracted repeat- edly with ether (3 x 40 mL). The organic phases were combined, washed with 3 N hydrochloric acid (3 x 20 mL), saturated sodium hydrogen carbonate (3 x 20 mL). and saturated sodium chloride solution

lH), 3.72 ( s , OCH3, 3H), 3.10 ( s , N-CH3, 3H), 2.76

Opioid peptides

NMR methods (vide infra). Yield 98.35, of the final solid product as a hydrochloride salt. TLC, Rf- 0.56 (4/1/1, butanol/water/acetic acid, by vol., Analtech silica gel plates); 'H NMR (250 MHz, d6-DMSO): 10.85 (s, indole NH, IH), 10.34 (broad, HCl), 7.47 (d, 7.7 Hz, Ar, lH), 7.36 (d, 8.0 Hz, Ar, lH), 7.10 (t, 7.1 Hz, Ar. lH), 7.01 (t, 7.5 Hz,Ar, 1H),4.63 (dd, 5.3 Hz, 10.0 Hz, a-H, lH), 4.39 (s, N-CH2, 2H), 3.81 ( s , OCH2, 3H), 3.29 (5.3 Hz, 16 Hz, dd, P-CH-, lH), 3.06 (10.1 Hz, 16.0 Hz, dd,P-CH2, 1H). MS (M + H), 230 (Calc. 230).

0-Phe-Cys- Tjw- $[CH~N]-D- Tca-Orti-Thr-Pen- Thr-NH? (21 The title peptide was synthesized in a manner similar to 3 except that N-'-Boc-D-Tca was used instead of N'-Boc-D-Trp in the coupling scheme. Chloranil was used to monitor the extent of reductive alkylation. To perform it in a reliable way, the resin sample was care- fully neutralized with DIEA, followed by three DCM washes. The yield of the pure peptide was 22.3 O O . The composition of 2 was corroborated by FAB-MS (Table 2) and IH NMR experiments.

o-Phe- Cys- Tyr- $[CH~NH]-D- Trp-Orn- Thr-Ph- Thr- NH2 (3) A 1 mM solution of TFA*D-Trp-Orn(Z)-Thr(0-Bz1)- Pen(S-4-MeBzl)-Thr(O-Bzl)-resin fragment was synthesised as in 1. Following TFA-mediated cleavage of the N"-t-butoxycarbonyl protecting group of D-Trp, the resin was washed 4 times with DCM without neutral- ization. Then, 1.66 g of Boc-Tyr(O-2.6-Cl2-BzI)CHO was dissolved in 30 mL of fresh DMF containing 1 O 0

acetic acid and added to the reaction vessel containing the peptide fragment on the resin. Finally, 0.19g of sodium cyanoborohydride was added portionwise and reaction allowed to proceed for 1 h (8). Due to the positive result of the ninhydrin test, reductive alkylation reaction was repeated with the same quantities of reagents and quenched after 3 h (negative ninhydrin test). The resulting N"-Boc-T~~(O-~,~-C~~-BZI)+[CH~NH]- D-Trp-Orn(Z)-Thr(0-Bz1)-Pen( S-4-MeBzl)-Thr(O- Bz1)-resin was deprotected and then N"-Boc-Cys( S-4- MeBzl) coupled and deprotected, followed by a coupling and deprotection of Nm-Boc-Phe. The resulting peptide resin was cleaved with HF and worked up as for 1 yielding 14.60, of the title peptide. The composition of 3 was independently confirmed by FAB-MS and 'H NMR investigation (Table 2). The analytical data are given in Table 2.

D-Phe-Cvs- Tic-0- Trp-Om- Thr-Pen- Thr-NH2 (4) The synthesis of the title peptide 4 was accomplished as for 1, except that N"-Boc-Tic (1) was used instead of N"-Boc-Tyr(O-2,6-C12-Bzl) in the coupling scheme. Coupling of the next amino acid, N"-Boc-Cys( S-4- MeBzl) was monitored by a chloranil test. The resulting N"-Boc-~-Phe-Cys( S-4-MeBzl)-Tic-~-Trp- Orn(Z)-Thr(0-Bz1)-Pen( S-4-MeBzl)-Thr(O-Bzl)-resin

405

I I

I

r I

(3 x 20 mL), and dried over magnesium sulfate. The solvents were evaporated leaving an oil (TLC pure) in 947, yield; 'H NMR (250 MHz, dh-DMSO); 9.51 (s, CHO, lH), 7.50 (m, 2,6-C12-Bzl, 3H), 7.28 (d, NH, 7.6Hz, lH), 7.06 ( d + d , 8.5Hz, ar., 4H), 5.17 (s, CHzBzl, 2H), 4.03 (m, aH, lH), 3.03 (dd, 4.5 Hz, 14 Hz, PH, lH), 2.65 (dd, 10.0 Hz. 13.9 Hz, PH, lH), 1.34 (s. Boc, 9H). TLC: Rr= 0.54 (95/5/1, chloroform/ methanol/acetic acid, by vol., Kieselgel), MS, (M + H) 423 (Calc. 423).

Synthesis of NZ-Boc-Cys(S-4-MeBzl)CHO N'-Boc-Cys(S-4'-MeBzl)-CON(OCH3)CH3 was synthesized in a manner similar to that outlined above for Boc-Tyr-(2,6-C12-Bzl)CON(OCH3)CH3 (vide supra). After silica gel purification (hexane/ethyl acetate, v/v, l / l ) the yield was 78%; TLC: Rf=0.62 (95/5/1, v/v/v, chloroform/methanol/acetic acid, Analtech silica gel plates); [a12 -18.7 (c 1.80, MeOH); 'H NMR (250 MHz, d6-DMSO): 7.15 (d + d, J = 8.0 Hz, Ar, 4H), 7.14 (d, NH, lH), 4.60 (m, a-H, lH), 3.69

2.26 (MeBzl, 3H), 1.38 (t-Bu, 9H). MS (M+H): 368 (Calc. 368). Anal. calc.: C 58.705,. H 7.66%, N 7.60%. Found: C 58.637,, H 7.61%, N 7.64%.

The synthesis of N"-Boc-Cys(S-4-MeBzl)CHO was carried out as described for NX-Boc-Tyr(O-2,6-C12- Bz1)CHO (vide supra), except that 5 % aqueous citric acid and not 3 N hydrochloric acid was used for the acidic extraction. A clear oil (yield 91.5%) was obtained; TLC: Rr= 0.50 (95/5/1, by vol., chloroform/ methanol/acetic acid, Analtech silica gel plates); 'H

(d,7.9Hz,NH, 1H),7.13 ( d + d , 8.0Hz, ar,4H),4.05 (m, a-H, lH), 3.70 (s, CH2Bz1, 2H), 2.79 (dd, 5.0 Hz,

(CH~BZI, 2H), 2.56 (dd + dd, 5.5 Hz, 8.9 Hz, fl-H, 2H),

NMR (250 MHz, dh-DMSO): 9.43 ( s , CHO, lH), 7.43

13.8Hz, P-H, lH), 2.51 (dd, 9.0Hz, 13.8 Hz, 8-H, lH), 2.26 (s, CH3Bzl,3H), 1.32 (s, t-Bu, 9H). MS: 309 (Calc.), 309 (Found). MS (M + H), 309 (Calc. 309).

Nx-Boc- TvY(O-~,~-CI~-BZ~)- $[CH~N]-D- Tca-OMe Na-Boc-Tyr(O-2,6-Cl~-Bzl)CH0 (0.5 g, 1.13 mM) and 0.96 g (2.5 mM) of D-Tca-OMe (19) were dissolved in 2 m L D M F containing 1 % acetic acid. Then 0.20g (3 mM) sodium cyanoborohydride (Aldrich) was added portionwise (over 20 min) and the mixture stirred for 2 h. The reaction was monitored by TLC (4/1/1, by vol., butanol/acetic acid/water, Analtech silica gel plates). D M F was evaporated in vacuo, then 10 mL of saturated NaHCO3, 4 mL of saturated sodium chloride, and 20 mL ethyl acetate were added, and the organic phase extracted (repeated twice, organic phase collected). The organic phase was dried over magnesium sulfate, and the solvent evaporated to yield a clear oil, yield 0.70 g, (75 01~). Purifica!ion was carried out on a silica gel column (Aldrich, 60 A, 230-400 mesh) using 1:l ethyl acetate/hexane, Rf= 0.82 (ethyl acetate, An- altech silica gel plates). The structure was confirmed by

W.M. Kazmierski et ol.

was cleaved with H F and subjected to the work-up procedure described for peptide 1; Yield, 18.9"". The structure of peptide 4 was confirmed by IH NMR and by FAB-MS experiments (Table 2). The analytical data are given in Table 2.

0-Pgl- Cbs- $1 C€€?N]- Tic-0- Trp-Orti- Thr- P h - Thy- N H? (5) and

D-Phe-C,vs-$/CH2N/- Tic-D-Trp-Oni- Thr-Pen- Thr-NH? (6) The synthesis of the title peptide 5 was analogous to that of 4, except that after incorporation and deprotection of Nz-Boc-Tic, resulting in a peptide fragment Nz- Tic-D-Trp-Orn(Z)-Thr(0-Bzl)-Pen( S-4-MeBzl)-Thr- (0-Bz1)-resin, reductive alkylation was carried out, analogous to that described for peptide 2. Nx-Boc- Cys(S-4-MeBzl)CHO, (1.04 g, 3.25 mM) was dissolved in 30mL of fresh D M F containing l o o acetic acid, followed by a stepwise addition of 0.6 g of sodium cyanoborohydride. After 80 min the reaction was terminated and repeated with the same amounts of reagents to assure its completeness. Deprotection of the fragment Nz-Boc-Cys(S-4-MeBzl)-$[CH~N]-Tic-~-Trp- Orn(Z)-Thr(0-Bzl)-Pen( S-4-MeBzI)-Thr(O-Bzl)-resin was followed by coupling and deprotection of Nx-Boc- D-Pgl. The resulting peptide-resin underwent similar work-up procedure to that of 2, yielding 17.6"; of the final peptide 5. Peptide 6 was synthesized as 5, except that N"-Boc-D-Phe was used instead of N"-Boc-D-Pgl, with a yield of 19.9":. The structures of 5 and 6 were confirmed by IH NMR and FAB-MS analysis (Ta- ble 2). The analytical data are given in Table 2.

I I

Experimental conditiotis for the NMR experitnetits Each sample ( 5 mg) was dried in vc~cuo, dissolved in 0.3 mL of [2H6]-DMS0 (100"~ D atom, Aldrich), run through several thaw and freeze cycles and sealed. All spectra (recorded at 303 K. except for variable temperature experiments) were acquired with a Bruker AM-250 or WM-250 spectrometer equipped with an Aspect 3000 or 2000 computer, respectively.

Signals in the 1 D spectrum (digital rcsolution 0.1 Hz,' pt) were assigned using a combination of 2D NMR techniques. Phase sensitive COSY (20, 21) using the time-proportional phase increments (TPPI) method, allowed the assignments of the intraresidual resonances. Pulse sequence: D1-90-DO-90-D3-90-FID, D 1 = 1.05, DO = 0.000003 S.

Zero and first order phase corrections from the 1D spectrum were applied, along with zero filling in the F 1 dimension; shifted sine-bell multiplication was applied in both dimensions prior to FT. Digital resolution in F1 was 5.2 Hz/pt, and in F2 was 2.6 Hz,'pt, in all cases.

Homonuclear dipolar correlated 2D NMR (NOESY) was used to trace interresidual connectivities NH' + I

and CH:, as well as to provide important information regarding the secondarj structure of the peptide. 406

The pulse sequence was: D 1-90-DO-90-D3-90-FID, D1 = 1.5 s, D9 = 0.3 s was used. Data manipulation: zero filling in F 1, square sine-bell multiplication was applied in both directions prior to FT. Digital resolution in F1 and F2 = 2.6 Hz, in all cases.

Confirmation of the assignments in congested spectral regions ( p protons) was accomplished with homonuclear shift-correlated 2D NMR with delay (22), em- phasizing JJ coupling constants between o-hydrogens of aromatic acids and the 8-protons. The pulse sequence was: D1-90-DO-D2-90-D2-FID, D 1 = 1.5 s, D2 = 0.08 s. Data manipulation: zero filling in F1, square sine-bell multiplication in both directions was applied prior to FT. Digital resolution in both dimensions was 2.6 Hz/pt. in all cases.

Tcniperature studies (303-328 "K range, 5 O inter- vals) were carried on to identify amide protons possibly involved in H-bonding, as well as to investigate the conformational rigidity of these peptides.

Radioreceptor binding assays were carried as described previously (2).

RESULTS AND DISCUSSION

Srnrcture activitj, studies Table 3 lists the binding properties of peptides under investigation in the present work. Compared with the parent compound I(2). all the peptides synthesized in this series exhibit lower affinity for p opioid receptors. The binding affinity of 1 was decreased 36 fold from that of I as did its affinity towards the 6 opioid rcccp- tor (2). Thus, opioid receptor selectivity (p vs. 6) does not seem to suffer much as a result of D-Tca4 replacing D-Trp'. In light of our NMR investigations (vide it!fra), D-Tca" in 1 has a similar side chain population distribution to D-Trp in I (mostly gauche( + )). Consid- ering their identical backbones (vide irfra), there is an overall spatial similarity of both peptides resulting in a roughly similar receptor selectivity though reduced po- tencj .

TABLE 3 Binding ofiniries of S U ~ I I U I U S I ~ I ~ ~ I ~ mlologue F in conlpetition with ['HICTOP nnd [-'H[DPDPE rn recepor binding to rnr hroin rnerribrarres

Peptide ICSO, n M

Binding \is. Binding vs. [ 'H ICTOP [ 'HIDPDPE

133 5 f 2.5 > 10000 17 800 f 4400

5534 f 2014 881 0 5 6.0

91.2 5 11.9

1439 f 215 6419 f 104.7

891.9 f 72.7 3.7 f 0.8

9 10000 9 10000 s 10000

11532 116

Opioid peptides

chain conformers. This result encouragingly showed that alkylated D- 1,2,3,4-tetrahydro-p-carboline can exhibit bias towards the g( + ) side chain conformation unlike the exclusive g( - ) observed for acjilated tetrahydroisoquinoline carboxylate (3).

The proton spectral assignments and coupling con-

Much more dramatic effects of amide bond reduc- tions on the p opioid receptor affinity and selectivity can be seen with peptides 2 (with p vs. 6 selectivity of 20.2), 5 and 6 (Table 3). An interesting observation comes from comparing the binding potencies of the amide reduced peptides 2, 3, 6 with their corresponding peptides 1, 111, 4, respectively (2). Though they are less potent, there is only a modest decrease in their selec- tivities for p vs. 6-opioid receptors. This suggests that while amide bond reduction decreased affinity for the p-opioid receptor, it does not contribute much to the discrimination between p and 6 opioid receptor types. The low affinity of 4 has been explained by a mis- matched topography ofthat peptide, due to agauche( + ) side chain conformation of Tic', leading to an opposite arrangement of D-Phe' and Tic3 aromatic pharmaco- phores, this geometry not being recognized well by a p opioid receptor (3,23).

Conformational studies In order to establish the side chain preference of alkylated 1,2,3,4-tetrahydro-/3-carboline, conformational studies were performed on the simple model compound, Boc-Tyr(O-2,6-Cl~-Bz1)-~[CH~N]-~-Tca-OMe, the synthesis of which additionally allowed us to establish the best conditions for the desired series of reductive alkylation reactions on the resin. Spectral assignments were obtained on the basis of a phase sensitive COSY experiment, and are tabulated in Table4. Because of considerable spectral overlap of CH,/CH2 signals of D-Tca, detailed spectral decoupling experiments were needed. The nonequivalence of both vicinal coupling constants and the relatively low value of J,p suggest a dynamic equilibrium between the g( + ) and g( - ) side

TABLE 4 Chemicnl shjr und coupling constant ussignnients for model cornpound

N"-Boc-T~r~0-2,6-C12-Bzl)-~[C~~NJ-D-TcnOMe, FH6J-DMSO, 303 K

Residue Chem. shift [ppm] J [Hzl

=Y r But 1.29 (s)

r 3.80 (m) 8' 2.84 (dd) alp' = 4.4

NH 6.67 a/NH = 8.7

B'/8" = 13.9 B" 2.55 ( d 4 x/B" = 8.9 CH, 2.75 (d)

D-Tca i( (dd) ./B' = 3.3

a/P" = 5.9 8 2.99 (m)

3.93 (d) OMe 3.53 (S)

N-CH2 4.07 (d 1 NCH/NCH = 14.8

stants for D-Phe-Cis-Tyr-D-Tca-Orn-Thr-Pkn-Thr- NH2 (1) are shown in Table 5 ; calculated side chain conformer populations and accessible $(NH-CH,) angles are given in Table 6. Several features of these data are noteworthy. First, quite unexpectedly, the acylated D-Tca4 residue has a side chain conformation biased towards a g( + ) conformation. Due to the intermediate values of the coupling constants (J,D) -2.5; 6.8 Hz) in peptide 1 it is postulated that a dynamic exchange between two conformational states, g( - ) and g( + ), takes place. At present the different behavior of D-Tic in peptides I11 and 3, and of D-Tca in 1 is not clear, since both amino acids are pipecolic acid analogues. A comparison of $J angles with those of I (21) and I1 (1) suggests that they are still compatible with a type 11' p conformation (Table 6) . Similarly to I, the side chain conformation of peptide 1 is biased towards g( - ) for D-Phel, but there is a high population of a trans rotamer of Cys' (t and g( - ) for 1). Also, the side chains of the amino acid preceding and following D-Tca (Tyr3 and Om5, respectively) exhibit some conformational disor- der in comparison with I (21) . While the side chain of Orn' in 1 seems to be almost equally divided among all three staggered states (mostly g( - ) for Lys5 in I), Tyr' has a preference for a trans (g( - ) in I) side chain Conformation.

Comparing 1 with I there is an upfield shift of the Cys: ( -0.32 ppm) and amide protons ( -0.13), a downfield shift of the Tyri (0.44 ppm), as well as a dramatic downfield shift of Orn: (0.80 ppm with respect to Lys5 of I) protons. As has been observed for other peptides with constrained amino acids, the ring current anisotropy of the D-Tca4 residue seems to be responsible for these effects. Nuclear Overhauser effects are in good agreement with the above analysis. There is a very strong cross-relaxation signal for alpha protons of Cys' and Pen', attributable to a negative disulfide helicity (24). Additionally, the characteristic CH;/NH' + cross- relaxation signals are observed for the following pairs of amino acids: 112, 415, 611, 718. Thus these parts of amino acids are linked via trans peptide bonds (25). No Cys:/Tyr3NH (2/3) or Orn5CH,/Thr6NH (5/6) cross- relaxations have been observed, but a significant cross- relaxation effect was detected for Orn5NH/Thr6NH. In light of these results, and even with the presence of a conformationally constrained D-Tca residue in the core of the turn, a type 11' p turn is the best description of the backbone conformation of peptide 1. This is supported by the very low temperature coefficient of Thr6NH, suggesting that the amide is either hydrogen bound (to the Tyr3 carbonyl group) or solvent shielded.

407

W.M. Kazmierski e ta / .

TABLE 5

Chemical shifrs (~HJ, coupling wisfanr a~iigrinierirs, arid aniide reniperariire coeficients for peptide.7 u-Phe-C~:s-T~r-D-Tca-Om-~hv-P~n-Thr-

NH,, 1 . arid o-Phe-C;,s-Tic-o-Trp-Orn-Thr-Ph-Thr-NH,. 4, /-'HI,-DMSO, 303'K

Residue Chcmical shift [ppm] Coupling constants [Hz] Tciiip. factor [ 10 ~ 'ppm:deg]

1 4 1 4

D-Phe' NH; A z

P' B"

Cys' N H A 2

B' P"

Tyr' 1 Tic' 4

N H A x B' B" N-CHz

D-Tca' 1 D-Trp4 4

N H A 2

B' P" N-CHZ

8.07 (rn) - 1.04

3.20 (rn) 3.29 3.00

8.06 (m) - 0.44

4.02 3.23 1.96

zip' = 5.2 x l p " = 9.9

P ' ) B ' ' = 14.0

4.8 9.5

13.8

9 '71

5.32 3.05 2.80

-5.1 9.25

- 4 08 5.47 3.20 2.92

r / N H = 10.2 8.7

~ p ' = 5.9 2 D" = 10.1

8'1"' = 15.5

4.6 10.0 13.6

8.59

5 03 >.99 2.50

- 4.0

-

r / N H = 7.9

5.22 2.95 2 84

4.89 (m)

218' = 7.0 z/b" = 8.5

B'iB' ' = 13.2 -

4.1 5.8

15.8

6.2 8.09 4.7 4.27 3.04 2.96

- 5 25 3 07 2 93

4.70 J 50 (dd)

ZIP ' = 2.5 2,'P'' = 6.8

P ' /D ' ' = 16.5 14.8

6.6 8.8

14.2

om5 N H A 2

8' 8" 7 6 6-NH;

Thr6 NH A 2

B i

OH

8.4

4.00 1.92 1.54 1 4 9 2.65

- 7 7

8.29

4.07 1.70 1.25 1.18 2.58 7.63

- 3.1 z:NH = 7.5 8.0

218' = 4.8 218" = 6.4

4.7 9.1

7 40 - 0 10

4 43 4 03 I .03

-1 8

7.28 - 0.84

4.39 3.88 0.91

-4 .9

r / N H = 7.4 7.8

zip= 7.4 lr'i6= 6.3

4.5 6.3

408

Opioid peptides

TABLE 5

(continued) ~

Residue Chemical shift [ppm] Coupling constants [Hz] Temp. factor [ 10-3ppm/deg]

1 4 1 4

Pen’ NH A 2

i Thrn

NH A 2

B Y OH

8.27

4.88 1.241 1.28

0.041

- 6.3

8.31

4.30 3.98 1.06

x5 .01

- 4.1

7.92

4.80 1.27/1.40

0.13 1

- 2.0

8.32

4.26 4.01 1.05 4.90

- 4.4

2,”H = 9.4

r/NH = 9.7

@= 3.6 fill.= 6.3

9.0

8.3

3.9 6.4

In summary, substitution of D-Trp4 by D-Tcal does not alter the peptide backbone conformation. The constrained nature of this cyclic amino acid, and its ap- parent ability to flip between both gauche( - ) and gauche( + ) conformational states, perturbs the side chain conformation of the neighbouring residues: Cys2, Tyr3, and O r d . Another interesting observation from the NOESY experiments is a strong cross-correlation between the Tyr’CH, and both N-CH2 protons of D- Tca4.

The chemical shifts, coupling constants, and temper-

ature coefficients of ~-Phe-Cys-Tyr-flCHzN]-~-Tca-

Om-Thr-Pen-Thr-NH2, 2, are listed in Table 7. Due to the low resolution of some signals in the 1D spectrum (its possible origin will be discussed later) at 303 O K , we decided to examine the molecule at 365 K. However, the 2D experiments (low spectral resolution is accept- able) were carried out at 303 ’ K. Interestingly, peptide 2 consists of two domains, a flexible one (amino acids 2-4) and a rigid one (amino acids 6-8). Coupling constants could be measured for the semi-rigid domain amino acids, but not for the flexible ones. Differential decoupling experiments failed to extract the vicinal coupling constants for amino acids directly connected with the flexible peptide bond isostere (Cys’, Tyr3, D-Tca4, Om5). Visible broadening of amide and alpha proton signals for these residues may be attributed to conformational averaging. Signals of residues not in the “flexibility domain” (Thr6, Pen7, Thrs) had sharp resonances.

Comparison of the 1D spectra of 2 at 303°K and 365 OK besides improving spectral resolution (due to faster molecular tumbling) revealed some interesting dynamic processes. The diastereotopic protons of the niethylene group adjacent to the amino group in the

I

pseudopeptide bond are in intermediate exchange at 303 OK, and as such are observed as two broad humps. This observation may be related to a two-site exchange between the gauche( - ) and gauche( + ) conformations of the D-Tca4 side chain. The 4 dihedral angles between

TABLE 6

Side chain conforiner population and accessible @ angles derived,froru

‘ H NMR artalpis of D-Phe-C?k7)r-o-Tca-Orn-Thr-Ph-Tlrr-NH2, 1, f-’Hri]-DMSO. 303 K

Residue I$ angles Rotarner population ( ” ” )

- 120

- 156, - 87, 60

-

- 160. - 82, 38, 82

- 162, - 80, 36, 84

- 144, - 96. 60

- 150, - 100

0 28.6 71.3

0 83.5 16.5

0 64.1 35.3

*

39.5 39.6 20.9

-

-

-

409

W.M. Kazmierski et a/.

TABLE 7

Chemical shifrs, coupling constunrs and crnride rerirperariire coeficienrs .for o - P h e - C ~ , . s - T , . r - ~ [ C H ~ ~ ~ H l - ~ - Trp-Om- Tlir-Ph-Thr-NH2 (3).

o-Plie-C~s-T~r-~[CH~N~-o-Tca-Orn-Tl ir-P~ri- Thr-NH 2 (21. u-Pgl-Ci'~s-~/CH~~V/-Tic-n-Trp-Onr-Thr-P~,~-Tlir-NH~ (5). in 12Hn]DMS0

Residue Chernictil shifi [ppin J Coiipl. c'onsiani [Hi] A . Tenrp. jictor 10 ~ '[pph:deg/

3 2 5 333 K 365 K 333 K

3 2 5

D-Pgl' 5 D-Phe' 3

NH x

8' 8"

Cys' NH A x 8' B" CH? 5

Tyr3 Tic' 5

NH A x B' B" CH: 3,2

D-Tca4 2 D-T$ 3 3

NH r

P' P"

Om5 NH A x B' 8"

8 8-N;

Thr6 NH A r

B 7 OH

Pen' NH A x .,' " , , I

i

8.04 4.13 3.23 2.97

9.02

4.86 3.23 2.97

- 4.6

-

8.15

4.17 2.87 2.61 ND

- 5.5

ND 4.0 I 3.23 3.34

8.56

3.92 1.32d 1.02'' 0.98* 2.44d 7.70

7.28

4,31 3.93 0.98

z 5.3"

- 1.7

- 1.8

8.15

4.69 1.31 1.37

- 2.4

8.21 4.19 3.23 2.95

8.80

4.80 3.15 2.95

- 3.5

-

7.87

4.16 2.72 2.66 2.8 1

- 5.7

-

4.18 ND 2.96

8.20

4.25 1.85 1.61 1.61 2.81 7.77d

7.57

4.35 4.00 I .06'

- 1.4

- 3.8

8.04

4.64 1.32 1.38

- 4.5

8.63 5.26 - -

7.67" ND 3.60

-3.12 s 3.08

2.77

- - 3.88" 2.82 2.75 -

8.44 4.65 3.16 3.04

8.44 ND 4.12 1.75 1.38 1.32 2.67 7.70

7.32

4.3 1 3.94 0.98

7.78

4.51 1.19 1.22

zip' = 4.5 rip'' = 9.4

B' iB" = 14.3

ziNH = 8.9

x,B' = 3.5 r;B" = 9.4 8'8" = 14.3

x/NH = niL

zip' = 5.7 x / 8 " = 9.7

B','"' = 14.3

r/NH = ND rip' = ND zip" = 8.7

B','B" = 13.5

r /NH = 7.8

z/B'-ND x ' /8" -N D

r/NH = 7 . 6

Z I P = 4.5 8:;l = 6.3

xjNH = 9.1

5.1 9.1

14.1

8.3

ND ND ND

7.6

ND ND ND

- ND 9.1 s

8.0

ND ND

7.2

5.3 5.9

NDb

ND ND ND

ND ND ND

7.8 6.2 8.2

16.6

7.8

ND ND

3.9

6.1 6.3

9.0 6.9

ND ND

4 10

Opioid peptides

TABLE 7 (continued)

Residue Chemical shift [ppm] Coupl. constant [Hz] A, Temp. factor 10-3[ppb/deg]

3 2 5 333 K 365 K 333 K

3 2 5

Thr* NH A

B

OH

r

I

7.90

4.26 4.00 1.05 4.98d

- 1.6 - 7.80 7.96 r /NH = 9.1 8.5 8.6 3.4 4.24 4.20 a/B= 3.9 3.7 3.8 4.00 4.01 6.3 6.3 6.3 1.08" 1.06

a Probable assignment. ND could not be determined, see text. Multiplet. 303'K. Assignments could be reversed (Thr6/ThrM).

NHi-CH1cr are compatible with either a type 11' P turn or a reverse y turn conformation.

Analysis of the NOESY spectra reveals that two important cross-peaks between D-Phe'a/Cys2NH and Cys2a/Tyr CHiNH were not present. These amino acids are part of the flexible domain in 2. It is well known that internal flexibility in molecules greatly in- fluences the magnitude of NOE cross-peaks. Nonethe- less, the presence of a 4/5 and the absence of a 5/6 CH:/NHi+ NOE can be interpreted as evidence for a type 11' P-turn conformation involving residues 3-6. However, protons of Om5 and Thr6 was missing. More- over, analysis of amide temperatures factors did not indicate H-bond formation by the Thr6 NH. On the other hand, the low temperature factor for the Om5 amide NH may be indicative of a y- or inverse y-turn involving residues 3-5 (26).

Analysis of the amide chemical shifts for compounds 1,2 and 3 (Table 5 and Table 7, respectively) reveals no essential differences. The major discrepancy, an upfield shift of the Tyr2 CH2 amide proton of 2 (-0.28 pprn relative to 3) is expected as a result of the peptide bond replacement. The same, to a lesser extent, is true for the Cys' amide proton ( -0.22 ppm upfield shift relative to 3) . Otherwise, the chemical shifts for other residues are very uniform (with a small exception for the Thr8 amide).

I I D-Phe-Cys- Tyr- IC/ICH~NH]-D- Trp-Om- Thr-Pen- Thr- NH2, 3, is a model compound with a single peptide bond isostere modification, designed to test whether there is any significant influence of CH2/C=O substitution on peptide conformation before considering more complex cases involving cyclic amino acids such as Tic

or D-Tca. Since chemical shift anisotropy is an important tool in conformational analysis, it was of considerable interest to establish the magnitude of influence of this structural modification on the chemical shifts of the neighboring nuclei.

There are several features that can be noticed imme- diately in the IH NMR spectrum of 3. First, it was possible to obtain only limited information about vicinal coupling constants for ~ - T r p ~ , and the signal for the ~ - T r p ~ NH was not found. Additionally, the Tyr3 amide appeared as a broad singlet instead of the expected doublet. These irregularities in the 'H NMR spectrum of 3 were limited to the 3rd and 4th residues only. The aromatic moiety in the Tyr3 residue showed particularly interesting properties. Instead of the commonly seen pair of complex doublets of an AA'XX' system of Tyr (2', 6' and 3',5' going upfield, respectively), a split signal (two doublets of uneven intensity) for the 2', 6' protons were found at 303 OK (Fig. 3). With an increase in temperature both signals started to fuse and at 333 "K they coalesced into a doublet. An estimation of the activation enthalpy for the two site jump of the tyrosyl ring gives about 77.3 kJ/mol. This finding represents a rare experimental demonstration of restricted rotation of an aromatic side chain in a small peptide, which is slow enough to be observed on the NMR time scale (less than lo3 s - I ) (27).

Chemical shifts for 3 in Table 7 are strikingly different than these for I(24). The expected upfield shifts (as a result of isosteric peptide bond replacement) of the Cys2NH ( -0.32 ppm) and the Tyr3 N H ( -0.46 ppm) are easily identified. Other significant upfield shifts include the ThrhNH ( -0.41 ppm), Pen' NH ( -0.34 ppm) and Thrs NH ( -0.49 pprn), whereas a downfield shift is observed for the Oms amide proton (0.32 ppm). Sim-

411

W.M. Kazmierski et al.

r I 1 i 1 ~ 1 1 1 v ~ i

7.20 7.10 7.00 6.90 6.80 6.70 6.60 FIGURE 3

Temperature dependence of aromatic resonances in D-Phe-Cj s-T!r- I

+[ CH2NH]-o-Trp-Om-Thr-P&-Thr-NH-. 3. ['H,]-DMSO.

ilarly, for alpha protons, there are dramatic upfield shifts of Cys' (-0.81 ppm), Pen7 (-0.32ppm). and Tyr' (-0.42 ppm), all a consequence of the peptide bond replacement. Some of the unexpected shifts (e.g. for Pen7) may be related to the oscillation of the tyrosine aromatic ring between two sites and a transannular anisotropic effect.

Analysis of the NH-CH, dihedral angles (obtained from Table 7) supports the possibility of a type 11' 8- turn of the backbone, though the temperature coefficient of Thr6 NH is not as small as usually observed in H-bonded amides. On the other hand, the observed

412

NOE cross-relaxation signals for the D-Trp:/ Orn'NH and OrnsNH/Thr6NH pairs of protons are diagnostic for type 11' p-turns. There also is cross- relaxation between the alpha protons of Cys2 and Pen7, and unexpectedly, cross-relaxation between Cysi and Pen: protons. Other NOE cross-peaks, expected if the peptide bond is trcms, also are found: CH:/NH'+ I , for i and i + 1 in residue pairs 1/2, 2/3, 6/7, and 7/8, respectively. A type 11' turn precludes the spatial proximity of Orn5CH, and Thr6 NH, and indeed, cross relaxation from these residues is not detected.

Side chain population analysis is incomplete because it is not possible to extract the appropriate vicinal coupling constants, presumably due to the flexibility. How- ever, some important observations can be made. For example, there is a large tram side chain population of Cys' (90"". compared with the almost equal distribution among irons and gauche( - ) states for I). Also, the side chain of the Tyr' CH. is mostly trans populated (gcruche( - ) for Tyr in I). This effect may cause the observed chemical shift variations bctween equivalents residues of I and peptide 3. The low value of the Thr6 vicinal coupling constant J,o (4.5 Hz) vs. 6.8 Hz for CTP is another discrepancy between these two peptides.

In summary, peptide 3 still may be best characterized by a type 11' 8-turn conformation of its backbone involving residues 3 to 6, though there is substantial flexibility of its backbone, best manifested by a slow two- site jump of the Tyr3 ring.

The chemical shifts, coupling constants, and temper-

ature data for D-Phe-CGs-Tic-D-Trp-Om-Thr-Pkn-Thr- NH? (4), coefficients are listed in Table 5. Karplus- Bystrov (28) analysis of accessible @ angles does not indicate any major difference with those of I (24). Anal- ysis of the coupling constants for Tic3 indicates that both vicinal coupling constants are almost equal as a result of the somewhat skewed gauche( + ) conformation of the Tic3 side chain group (Table 5). Similar to peptide 3, one observes an increased trans side chain population of Cys' (probably the result of repulsions from the Tic3 aromatic ring). This allows only moder- ate cross-relaxation between the alpha protons of Cys2 and Pen7. On the other hand, NOESY experiments reveal two important cross peaks: Cys2 CH,/Tic3 NCH2 and Tic3 CH,/Tic3 NCH?. The first effect can be observed only if the corresponding peptide bond is in a trans configuration (25). The 1D spectrum of 4 suggests that only one conformer is observed. Thus, in contrast to the proline ring where cis and trans peptide bond isomers are often in equilibrium, the tetrahydroisoquinoline ring seems to exclusively prefer a trans conformation of the peptide bond. NOESY experiments reveal that the following pairs of protons CH',/NH'+ ' are in close proximity: 1/2, 3/5,4/5, 6/7, 7/8. These peptide bonds are tram. Lack of a 5 i6 cross-relaxation peak in the presence of a strong NH5/NH6 signal again is in-

Opioid peptides

Psi ($) angles calculated from the vicinal coupling constants for ~ - T r p ~ and Thr6 exhibit a significant de- viation compared with the corresponding values in I (24). NOESY experiments did not add any additional information. Only intraresidue NOE signals were detected. Thus, the conformational data attained was in- conclusive for this peptide.

The above analysis clearly shows that reduction of peptide bonds in cyclic constrained peptides with an otherwise rather semi rigid backbone (23,29), can result in a greater degree of conformational flexibility. In some specific cases, a slow interconversion between two states (as in peptide 3) may be observed. These observations are supported by the complementary results of Marraud etal. (29) who found different conformations for protonated vs. non-protonated forms of reduced amide bond peptides.

terpreted as evidence for a type 11' p-turn backbone conform ation.

The chemical shift difference between the diastereotopic methyl groups of Pen7 is much larger in 4 (0.132 ppm) than in I (0.06 ppm). Table 5 reveals that there is a substantial upfield shift of the D - T ~ ~ ~ N H (-0.74 ppm in comparison to I), which possibly is a result of ring anisotropy from the gauche( + ) populated side chain of Tic, in contrast to the mostly gauche( - ) populated side chain of Tyr in CTP, I. Upfield shifts of the Thr6, Pen7, and Thr8 amide resonances, and the absence of this phenomenon for Orn5NH, indicates the presence of transannular ring current anisotropy effects on these residues.

Strong indications of conformational averaging were

identified for ~-Pgl-C~!s-flCH2N]-Tic-~-Trp-Orn- I Thr-Pen-Thr-NH2 (S), in the form of broad lines for some signals at 303 O K . Increased resolution was achieved by doing NMR experiments at 333 OK. Chem- ical shifts and coupling constants obtained are listed in Table 7. However, even at elevated temperatures, the signals of the 2nd, 3rd, and 7th residues still remained sufficiently broadened that no accurate coupling constant information could be measured. To obtain qual- itative information about the conformation and dynam- ics of this molecule, chemical shift analysis (in a similar manner as done before) was utilized. Considering the alpha protons first, there was a significant upfield shift of the Cys2CH, protons in 5 (-0.87 ppm compared to 4). This is much greater than one would expect solely as a result of isosteric peptide bond replacement with an alkyl amino group (about -0.4 pprn). In addition, there was a tremendous upfield shift of the Cys2NH resonance in 5 relative to 4 ( -1.58 pprn). These results strongly implicate dramatic ring current anisotropic effects, and point to very different orientations of the Tic aromatic side chains in 5 and 4. Both the alpha and amide protons of ~ - T r p ~ in 5 experience a downfield shift relative to 4 (0.38 and 0.35 ppm, respectively). A closer look at other alpha and amide protons of peptides 4 and 5 (Tables 5 and 7) reveals that with the exception of the alpha protons of Pen7, most of the corresponding resonances are not very different. Fi- nally, the alpha proton of Tic in 5 experiences an upfield shift of - 1.34 ppm, which is much larger than expected from an alkyl amine peptide bond modification. Again, anisotropic effects of the aromatic ring of Tic3 must be involved.

Analysis of a 1D spectrum of 5 revealed that while D-Trp4, Om5 and Thr6 gave reasonably sharp signals, the next amino acid Pen7 again gave broad (slow exchange) alpha and amine proton resonances. Most convincing, however, is a comparison of the chemical shift difference between the diastereotopic methyl groups of Pen7. This value is only 0.033 pprn for 5, compared to 0.13 1 ppm for the more rigid 4 (Tables 5 and 7).

CONCLUSIONS

We have demonstrated that peptides with secondary reduced peptide bonds can be easily constructed in a reductive alkylation reaction on a solid support as has been previously done for peptides with primary peptide bonds (8). There is an increasing interest in this class of peptide amide bond modified peptides, since several of them possess antagonistic (30) or inhibitory (31) properties.

In agreement with our earlier observations (3, 28), acylated Tic residues prefer a gauche( + ) side chain conformation, as was found for peptide 4 in this series. In contrast, in the closely related cyclic D-tryptophan - i.e. D-Tca-containing peptide 1, there seems to bc a bias towards a gauche( - ) side chain conformer, with possible dynamic equilibrium between the two states (gauche( - ) and gauche( + )). Both Tic and D-Tca fa- vored trans peptide bonds unlike some other amino acids (Pro, etc.). We attempted to stabilize the gauche( - ) conformation of Tic and the gauche ( + ) conformation of D-Tca by synthesizing peptides with flCHzN]Tic (5, 6 ) and flCH2N] D-Tca (2) reduced peptide bonds. This would decrease 1,2 diequatorial repulsions (Fig. 1. 11) as a result of thc presence of tetrahedral nitrogens (Fig. 1,lV). Interestingly, peptides 2, 3, 5 and 6 exhibited a significant degree of conformational mobility (on the NMR time scale) in the vi- cinity of the reduced peptide bonds, thus resulting in broadening of resonances for amino acids adjacent to the reduced peptide bonds. As a result, it was not al- ways possible to obtain sufficient NOE and J-coupling data for comprehensive conformational analysis of these peptides. However, amino acid residues away from the peptide bond modification showed character- istics of a relatively stable backbone conformation (sharp resonances, significant NOES, etc.). The receptor binding data (Table 3), demonstrate fairly compa- rable binding potencies to the p opioid receptor for analogues 1 and 3 as well as for 4 and 6. Thus, despite

413

W.M. Kazmierski et al.

the findings from NMR studies that the reduced peptide bond analogues are substantially more flexible than their closely related analogues with standard peptide bonds, they apparently are still able to attain receptor bound conformations, similar to those of their respec- tive parent compounds.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service Grant NS 19972, DA 04248 and DA 06284. The mash spectral determinations were performed by the Midwest Center for Mass Spectrometr) at the University of Nebraska. a National Science Foundation Regional Instrumentation Facility (Grant No. CHE 811 1164).

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22, 399-484

33. 1954- 1958

SOC. 79. 5697-5703

cOl7t lJlUtl . 113, 967-974

425-428

Address:

Dr. L'rctor J . Hruhj Regents Professor Department of Chemistry University of Arizona Tucson, AZ 85721 USA

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