J. Am. Chem. SOC.1991, 113, 2275-2283 and extends the findings of Saito et aLs and suggests that this is a general property of all G T steps. Recent observations have demonstrated that the cleavage resulting from 4’-hydrogen abstraction at GT steps occurs most frequently as part of a staggered double-strand break.23 Additional studies using oligomers to (23) Dedon, P. C.; Goldberg, 1. H. J . Biol. Chem. 1990,265,14713-14716.
2275
explore the effects of thiols on the partitioning between 4’- and S’-hydrogen abstraction and on the reduction of peroxide intermediates are in progress. Acknowledgment. This research was supported by the National Institutes of Health G M 34454 (J.W.K. and J.S.) and National Institutes of Health CA 44257 and G M 12573 (I.H.G.).
Topographic Design of Peptide Neurotransmitters and Hormones on Stable Backbone Templates: Relation of Conformation and Dynamics to Bioactivity’ Wieslaw M. Kazmierski,” Henry I. Yamamura,2band Victor J. Hruby*.2P Contribution from the Departments of Chemistry and Pharmacology, University of Arizona, Tucson, Arizona 85721. Received June 4, I990
Abstract: We have proposed that development of methods for controlling the side-chain topography of amino acid residues
in peptides and proteins provides a new approach to the topographical design of biologically active peptides. An example of this approach is the use of the 1,2,3,4-tetrahydroisoquinolinecarboxylicacid (Tic) residue, which favors a gauche (-) side-chain conformation when in the N-terminal position, whereas in its acylated form (internal position), the most stable side-chain conformation is gauche (+). This approach has been tested by incorporating D-Tic or Tic at different positions of 1.1 opioid I 1 receptor specific octapeptides such as D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH, (CTP, l), examination of the biological consequences of these modifications, and detailed ’HNMR based conformational analysis. The compounds prepared and r their biological activities were as follows: ~-Tic-Cys-Tyr-~-Trp-Lys-Thr-Pen-Thr-NH~ (2; gauche (-), 6/1.1 = 7800, ICso 1.1 , = 1.2 nM); Gly-~-Tic-Cys-Tyr-~-Trp-Orn-Thr-Pen-Thr-NH~ (3; gauche (+), 6/1.1 = 19, ICso p = 278.7 nM); and D-Phe, Cys-Tic-~-Trp-Orn-Thr-Pen-Thr-NH~ (4; gauche (+), 6 / p = - 7 , ICso p = 1439.0 nM). In the absence of a geminal pair of protons suitable for distance calibration, a new technique (Davis, D. G. J . Am. Chem. SOC.1987, 109, 3471-3472) of transverse and longitudinal cross-relaxation rate measurements has been utilized in conjunction with other 2D NMR methods in order to determine the three-dimensionalsolution conformations for the peptides 1-4, with subsequent application of restrained molecular dynamics (GROMOS). The average backbone conformations in peptides 1-4 were very similar, but the side-chain conformational preferences in the analogues differed, suggesting that the different affinities and selectivitiesfor 1.1opioid receptors were primarily due to differences in the side-chain conformationsof Tic (D-Tic), and thus due to differences in the topographies of these peptides, and not the backbone conformations. A detailed analysis of these relationships is presented.
.
Introduction A dogma in molecular biology is that the biological function (“function” code) of biologically active peptides is determined by the conformations coded in their primary structure (“structure” code). For peptide neurotransmitters and hormones, transfer of their biological messages to the target cells via specific receptors requires at least two consecutive events: (1) binding of the hormone or neurotransmitter to its receptor; (2) transduction of the information from the hormone-receptor complex into the cell, leading to a biological r e s p ~ n s e . ~Since the structural, confor-
mational, and dynamic properties of the peptide hormone and its receptor play a key role in both steps, their recognition and control are essential prerequisites to understanding the molecular basis of information transfer in these systems. In principle, the most direcl approach would be the use of transferred nuclear Overhauser effect (TRNOE) studies of the neurotransmitter bound to its receptor. This method has provided insights into the conformations of small ligands bound to Currently there are no isolated opioid receptors available. However, in principle this goal also may be pursued by examination of the conformational and dynamic properties of constrained synthetic hormone analogues carefully selected for their complementary biological properties. Opioid peptides, owing to the multiplicity of opioid receptors, display a variety of biological actions. At least four major classes of opioid receptors have been postulated so far; p , 6, K , and e. Recognition of the conformational and dynamic features of ligands
( I ) Preliminary communication of this work was presented in part during the 10th American Peptide Symposium, May 23-28, 1987,St. Louis, MO, and the 13th International Conference on Magnetic Resonance in Biological Systems, Madison, WI, August 14-19, 1988. Symbols and abbreviations are in accord with the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. 1972, 247, 977). All optically active amino acids are of the L variety unless otherwise stated. Other ab(3) Hruby, V. J. In Perspectives in Peptide Chemistry: Structure, Conbreviations include the following: Tic, I , 2, 3, 4-tetrahydroisoquinolineformation and Aclioity; Eberle, A,, Geiger, R., Wieland, T., Eds.; Karger: carboxylic acid; Pen, penicillamine or P,B-dimethylcysteine; Orn, ornithine; Basel, Switzerland, 1981,pp 207-220. TRNOE, transferred nuclear Overhauser effect; NOE, nuclear Overhauser (4) Clore, G.M.;Gronenborn, A. M. J . Mugn. Reson. 1982,48,402-417. effect; DIEA, diisopropylethylamine; p-MBHA, p-methylbenhydrylamine; ( 5 ) Andersen, N.H.; Eaton, H. L.; Nguyen, - . K. T. Magn. Reson. Chem. TPPI, time-proportional phase increments; CTP, o-Phe-Cys-Tyr-o-Trp1987,25, 1025-1034. Lys-Thr-Pen-Thr-NH2; CTOP, D-Phe-Cys-Tyr-o-Trp-Orn-Thr-Pen-Thr- (6) Hallenga, K.;Nirmala, N. R.; Smith, D. D.; Hruby, V. J. In Peptides: NH2-Na-ter,-butyloxycarbonyl;Cbz, benzyloxycarbonyl; GPI, guinea pig Chemistry and Biology, Proceedings of the Tenth American Peptide Symileum; MVD, mouse vas deference. posium; Marshall, G. R., Ed., ESCOM: Leiden, The Netherlands, 1988;pp (2) (a) Department of Chemistry. (b) Department of Pharmacology. 39-41.
-
0002-786319 1115 13-2275$02.50/0 0 . 1991 American Chemical Societv
Kazmierski et al.
2276 J . Am. Chem. SOC.,Vol. 113, No. 6, 1991
Table 1. Binding Affinites of Peptides 1-4 in Competition with ['HICTOP" and ['H]DPDPEb and Their Inhibitory Activity in the in Vivo Hot Plate TestC peptide nM vs [-'H]CTOP ICSO$nM vs [-'H]DPDPE in vivo PA*
,
1
~-Phe-Cys-Tyr-o-Trp-Lys-Thr-Pen-Thr-NH~ (1) I o-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (2) Gly-D-Tic-Cys-Tyr-o-Trp-Orn-Thr-Pen-Thr-NHz (3) D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH2 (4) I
b
3.7 f 0.8 1.2 f 0.03 278.1 i 0.5 1439.0 f 215
1153 f 116 9324 f 546 5352 i 503
> 10000
11.17 1 1.29
ND
ND
4
6 opioid selective agonist,62DPDPE, Tyr-D-Pen-Gly-Phe" w opioid selective antagonis,' CTOP, ~-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH~. 1
D-Pen. cPercent in vito measurement of antagonist potency.E Rat brain binding assays. ND, not determined due to low antagonist potency. dIC50, inhibitory concentration at which 50% of a receptor-bound radioligand is displaced by a ligand (agonist or antagonist) under investigation. e PA2 refers to the -log of the concentration of inhibitor requiring twice the concentration of agonist (e&, morphine) to induce its original biological response.
selective for only one of them may provide important insights into the basis of molecular recognition and information transfer in the opioid receptor system. We have synthesized and biologically evaluated a number of peptide p receptor antagonists from which four have been selected for further conformational studies due to their interesting structures and p opioid receptor potency and selectivity relationships (Table I). I Replacement of the D-Phel in D-Phe-Cys-Tyr-D-Trp-LysI Thr-Pen-Thr-NH2 (1, CTP) by a D-tetrahydroisoquinohecarboxylic acid (D-Tic) residue resulted in an analogue (2) with a substantial increase in selectivity and affinity for the p opioid receptor.'^^ On the other hand, attachment of Gly to the N terminal of peptide 2 (analogue 3), resulted*in a dramatic decrease of potency and selectivity for the p opioid receptor. Similarly, substitution of Tyr3 by Tic3, as in peptide 4, decreases its potency at p opioid receptors. These results became even more intriguing, in light of our preliminary findings9 that all of those peptides exhibited very similar backbone conformations. Thus, we have investigated the conformational and topographical properties of these peptides in greater detail. A useful tool in this approach is the determination of the interproton distances in peptides and proteins as measured by the nuclear Overhauser effect,I0 providing that the molecule contains a pair of geminal protons that can serve as a calibration standard of the interproton distance and magnitude of the Overhauser effect. Generally, a-protons of Gly, or 6-CH2 protons of Pro are used for this purpose. Since these are not present (except in 3) in peptides 1-4, the P-CH2 protons of other amino acids (e&, D-Trp, Tyr, Om, etc.) might be considered. However, it has been shown in several lab~ratories'l-~~ that there is a nonnegligible effect of internal molecular motions on cross-relaxation for these protons both in laboratory and rotating frames. In practical terms, the presence of fast internal motions can alter (usually increase) the calculated interproton distances on the basis of NOE effects up to 20%. While this may not constitute a problem for proteins, which usually have hundreds of observable NOEs that can be used in conjunction with distance geometry routines to provide averaged and self-consistent conformations, it is of considerable concern in the case of small peptides, which usually possess much fewer observable NOEs. (7) Pelton, J. T.; Kazmierski, W.; Gulya, K.; Yamamura, H. 1.; Hruby, V. J. J . Med. Chem. 1986, 29, 2370-2375. (8) Kazmierski, W.; Wire, W. S.;Lui, G. K.; Knapp, R. J.; Shook, J . E.: Burks, T. F.; Yamamura, H. 1.; Hruby, V. J. J . Med. Chem. 1988, 31,
2170-21 - . 77. (9) Kazmierski, W.; Yamamura, H. I.: Burks, T. F.; Hruby, V. J. In Peptides 1988; Bayer, E., Young,G., Eds.; Walter de Gruyter & Co.: Berlin, 1989; pp 643-645. (IO) Noggle, J . H.; Schirmer, R. E. In The Nuclear Overhauser Effect; Academic Press: New York and London, 1971. ( I 1) (a) Lipari, G.; Szabo, A. J . Am. Chem. Sac. 1982, 104,4546-4559. (b) Lipari. G.; Szabo. A. J . Am. Chem. SOC.1982. 104, 4459-4570. ( I i ) Olejniczak, E. T.; Dobson, C. M.: Karplus, M.; Levy, R. M. J . Am. Chem. Soc. 1984, 106, 1923-1930. (13) Farmer, B. T., 11; Macura, S.;Brown, L. R. J . Magn. Reson. 1988, -.
80, 1-22.
Recently, Davis14 and Mirau and Bovey15 have suggested NMR experiments capable of determining internuclear distances as well as correlation times ( T ~ )for pairs of protons without any a priori assumptions about their internal motions. The method of Davis involves measurements of longitudinal (q)and transverse (ul) cross-relaxation rates and is based on the observation that u,, and u1 have different dependencies on T~ (eqs 1 and 2). The ratio R = q / u L is r A B independent and
gives an entry into T~ correlations times between pairs of protons AB, whereas r A B can be calculated from either eq 1 or eq 2, providing that 7,is calculated for a given pair of protons (eq 3). -(1 - 22R) - [(l - 22R)* 80(1 + 2R)(1 - R)]'I2 7: = (3) -8(1 2R)u20 In this article, we will examine the utility of this novel approach for studies of the conformation and dynamics of the 20-membered rings of the cyclic octapeptides 1-4. We will then correlate the stereochemical ( r A B ) and dynamic ( T ~ )features determined with the observed activities and selectivities for p opioid receptors for these peptides and postulate those structural elements that are responsible for the high degree of molecular recognition for ligand-p opioid receptor interactions. Finally, the applicability of tetrahydroisoquinoline-carboxylicacid (Tic) to protein/peptide design will be discussed.
+
+
Experimental Section Materials and Methods. The synthesis and biological properties of Compound 4 was synthesized peptides 1-3 have already been according to published using solid-phase synthesis techn i q u e ~ ' ~ with ~ ' ' a Vega (Tucson, AZ) Model 250 peptide synthesizer. Amino aicds either were purchased from Bachem (Torrance, CA) or were prepared by literature methcds.I6 The synthesis was accomplished on a p-methylbenzhydrylamine @MBHA) resinIE (substitution 1.O mM/g of resin). A 1.5 M excess of preformed symmetrical anhydrides or 3 M excess of hydroxybenzotriazole active esters was used for coupling reactions, which were monitored by ninhydrinI9or chlorani120tests. The protected peptide resin ( I mM) of 4 was synthesized by sequential coupling, deprotection with trifluoroacetic acid (TFA), and neutralization with diisopropylethylamine (DIEA) of N'-Boc-Thr(O-Bzl), N"-BocPen(S-4-MeBzl) (Pen, P,P-dimethylcysteine), N"-Boc-Thr(O-Bzl), NuBoc-Orn(Nb-Cbz), NO-Boc-Tic, Nu-Boc-Cys(S-4-MeBzl), and N*-Boco-Phe. After N"-Boc-D-Trp was coupled, the deprotecting TFA solution (V/V/V:48% TFA, 2% anisole, and 50% dichloromethane (DCM)), was modified to contain 48% TFA, 2% anisole, 10% dithioethane, 20% di(14) Davis, D. G. J . Am. Chem. Sac. 1987, 109, 3471-3472. (15) Mirau, P. A.; Bovey, F. A. J . Am. Chem.Soc. 1986,108,5130-5134. (16) Stewart, J. M.; Young, J. D. I n Solid Phase Peptide Synthesis, 2nd ed.; Pierce Chemical Co.: Rockford, IL, 1984. (17) Upson, D. A.; Hruby, V . J. J . Org. Chem. 1976, 41, 1353-1358. (18) Orlowski, R. C.; Walter, R.; Winkler, D. J . Orn. Chem. 1976, 41, 3702-3705. (19) Keiser. E.: Colescott. R. L.: Bossinaer, - C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595-598. (20) Christensen, T. In Peptides, Structure and Biological Functions; Gross, E., Meienhofer, J., Eds.; Pierce Chemical Co.: Rockford, IL,1979; pp 385-388.
Peptide Neurotransmitter and Hormone Design
J. Am. Chem. SOC.,Vol. 113, No. 6, 1991 2277
Table 11. ' H N M R Spectral Assignments (ppm) for the Aliphatic and Amide Resonances of Gly-~-Tic'-Cys-Tyr~-~-Trp-Orn-Thr-Pen-Thr-NH~ , (3) and ~-Phe'-Cvs-Tic~-~-Tr~-Orn-Thr-Pen-Thr-NH, (4) in 12H,1DMS0. 303 KO residue 3 4 (3) CIyo N H 8.02 (3j H - C Y - C H ~ 4.85/4.54 (J9 = 15.4) (3) residue 1 N H 8.06 (m) 5.05 (Jap = 6.3, 6.3) (4) CY-CH 4.02 (Jus = 4.9, 9.5) 3.34 (J9 = 15.4) 3.23 (J9 = 13.8) 0-CH 2.96 3.13 4.57 (m) N-CH2 Cys2 N H 9.25 ( J = 8.7, AdlAT = -4.1) 8.63 ( J = 9.8, A6lAT = -4.7) 5.37 (Jap = 7.3, 3.7) 5.47 (Jap = 4.6 10.0) CH, 3.20 ( J 9 13.6) 2.78 (J9 = 15.4) CHI5 2.68 2.92 8.56 ( J = 7.8, A6fAT = -4.1) (3) residue 3 N H 4.55 (Jap = 8.7, 6.1) (4) residue 3 a - C H 5.22 2.68 2.95 (Jus = 4.1, 5.8) 0-CH 2.84 ( J g 15.8) 4.89 (m) N-CH2 o-Trp4 N H 8.87 ( J = 5.4, A 6 / A T = -4.0) 8.09 ( J = 6.2, A 6 / A T = -4.7) CY-CH 4.15 (Jap = 8.4, 7.3) 4.27 (Jus = 6.6, 8.8) 0-CH 3.04 (J9 = 14.2) 3.00 2.36 2.73 orn5N H 8.29 ( J = 8.0, A 6 / A T = -3.1) 8.33 ( J = 9.3, A6/AT = -2.7) CY-CH 4.09 (Jab = 10.5, 3.2) 4.07 (Jup = 4.7, 9.1) 0-CH 1.70 1.85 1.25 1.14 Y-CH 1.18 0.92 6-CH 2.58 2.58 6-NH3" 7.58 Thr6 N H 7.28 ( J = 7.8, AblAT = -0.8) 7.65 ( J = 9.1, A s / A T = -0.2) CY-CH 4.58 (JOB = 7.0) 4.39 (Jus = 4.5) 3.88 (Jp, = 6.3) P-CH 3.92 (Jp = 6.5) 1.05 0.9 1 7-CH Pen' N H 8.41 ( J = 9.8, A s l A T -6.0) 7.92 ( J = 9.0, A6/AT =: -2.0) 4.78 CY-CH 4.80 Y-CH 1. I 7/0.90 1.27/ 1.40 Thrs N H 7.95 ( J = 3.0, A6/AT = -2.1) 8.32 ( J = 8.3, A 6 / A T = -4.4) CY-CH 4.26 (Jep = 3.9) 4.23 (Jab = 3.9) P-CH 4.00 (Jp, = 6.3 4.01 (Jp, = 6.4) y-CH 0.99 1.05 a Coupling constants in hertz. All samples (ca. 4-5 mg each) were dried overnight in vacuo, dissolved methyl su!fide and 20% dichloromethane. After the last amino acid was in [2H6]DMS0,degassed by repeated freeze-thaw cycles, and sealed. All coupled, the Nu-Boc protecting group was removed, the amino acid the spectra were recorded at 20 OC, unless mentioned otherwise. neutralized with DIEA, and the peptide resin dried in vacuo. The reThe phase-sensitive corrleation experiments with double-quantum sulting (H)-o-Phe-Cys(S-4-MeBzl)-Tic-o-Trp-Orn(Z)-Thr(O-Bzl)-Penfilter, using time-proportional phase increments (TPPI) were performed (S-4-MeBzl)-Thr(O-Bzl)-resin was cleaved with 15 mL of liquid H F and by methods described by Marion and WiithrichZ3and Rance et aLZ4with of I mL of anisole at 0 "C. The product was washed with ethyl ether 256 t l experiments and 64 scans of 1K data points. Multiplication by (3 X 20 mL) and extracted with 10%aqueous acetic acid (HOAc; 3 X a shifted sine bell was applied in both dimensions. Zero filling in the ti 20 mL) followed by glacial HOAc (2 X 20 mL), and both fractions were dimension followed by Fourier transformation and phasing in both dilyophilized separately. The linear peptide was cyclized by dissolving the mensions resulted in a final matrix of 1 K X 1 K points. Pulse sequence lyophilized powder in 1.5 L of water (pH adjusted with aqueous ammonia D 1 -9O-DO-90-D3-90-FID, to 8.5) followed by oxidation with 0.01 M aqueous K,Fe(CN), until the Homonuclear shift-correlated 2D N M R experiments with a delay yellow color persisted for 20 min. After the reaction was terminated, the period to emphasize long-range or small couplings were run by the mepH was adjusted to 4.5 with AcOH, and excess ferro- and ferricyanides thod of Bax and Freeman.25 Pulse sequence D1-90-DO-D2-90-FID; were removed by 15 mL of Amberlite IRA-45 (mesh 15-60, Cl- form). D2 = 0.08 s; 256 t , experiments with 128 scans of I K data points. The mixture was stirred for 1 h and filtered, and the solution was conMultiplication by a sine bell in both dimensions and zero filling in the centrated in vacuo and lyophilized. Gel filtration on 100 X 2.5 cm t l dimension followed by Fourier transformation and phasing in both Sephadex (3-15 with 5% (v/v) aqueous HOAc was followed by reversedimensions resulted in a final matrix of 1 K X I K. phase HPLC with a gradient of IO-30% acetonitrile and 0.1% aqueous Homonuclear dipolar correlated 2D N M R experiments in phase-senTFA on a Vydac C I Scolumn: total yield 18.9%; FAB-MS [M + H]c,Ic sitive mode using TPPIZ6were performed by acquisition of 256 ti ex1058, [M + HIobs1058. The structure was confirmed additionally with periments with 128 scans of 2K data points. Pulse sequence D1-90'H NMR spectroscopy (Table 11). Purity was assessed by thin-layer DO-90-D9-90-F1D. Zero-quantum scalar coupling correlations were chromatography in three solvent systems and RP-HPLC. suppressed by random variation (*20%) of the mixing time D9. Boc-Tic was obtained analogously to Boc-o-Tic*in 71.3% yield for the first step (Pictet-Spengler reaction), and a 94% yield for the Na-Boc At least five experiments, with different mixing times ranging from IO to 450 ms (represented on cross-relaxation buildup rate graphs) were protection procedure. Binding Studies. Binding studies were performed as described in r u n for each pcptidc investigated (1-4). previous After acquisition, all data were transferred to a microVax 3200 for processing with the FTNMR program, version 5.1 (Hare Research, Inc.). NMR Studies. The spectral assignments of peptides 1 and 2 in [2H,]DMS0 were determined previously.2's22 The 'H N M R spectra of compounds 3 and 4 were acquired with a Bruker AM250 spectrometer (23) Marion, D.; Wiithrich, K. Biochem. Biophys. Res. Commun. 1983, equipped with an Aspect 3000 computer. 113, 967-914. (24) Rance, M.; Sarensen, 0. W.; Bcdenhausen, G.;Wagner, R. R.; Ernst, R. R.; Whthrich, K. Biochni. Biophys. Res. Commun. 1983, 117,471-478. (25) Bax, A,; Freeman, R. J . Magn. Reson. 1981, 44, 542-561. (21) Sugg, E. E.; Tourwe, D.; Kazmierski, W.; Hruby, V. J.; van Binst, (26) Bcdenhausen, G.; Kogler, H.;Ernst, R. R. J . Magn. Reson. 1984, 58, G . Int. J . Pept. Protein Res. 1988, 31, 192-200. 370-388. (22) Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 41, 697-710. ~~
Kazmierski et al.
2278 J. Am. Chem. Soc., Vol. 113, No. 6,1991
Table 111. Selected Longitudinal (uII), Measured Transverse (uI (exp ROE)), Corrected Transverse ( u ~ ) Cross-Relaxation ’ ~ ~ Rates, Correlation Times ( T ~ ) , ‘Internuclear Distances (r”),“ and Calculated Internuclear Distances (#ja,c)e for ~-Phe-C;s-Tyr-~-Trp-Lys-Thr-Pkn-Thr-NH~, (CTP, l ) , [D-T~c’ICTP(2), [Glyo,~-Tic’]CTOP (3), and [Tic3]CTOP (4), in [2H6]DMS0, 303 Kf ull (exp uI (exp uI (corr 6 1 1 (exp uI (exp ui (corr atom pair NOE) ROE) ROE) T ~ Jns , r’j, A rai,’j, A atom pair NOE) ROE) ROE) ns #i, A ral;l, A Compound 1 2.1 a5/p511 0.0 0.26 0.27 0.71 2.8 4.10 4.37 1.01 1.8 2.8 a1/NH2 -0.69 2.2 a7/y7,1 0.0 0.8 1 0.08 0.84 1.9 0.71 3.4 3.30 3.30 a2/NH3 -0.26 4.16 2.5 NHS/NH6-0.33 1.03 1.8 4.28 0.84 1.8 4.31 4.62 3.2 a3/NH4 -0.78 1.23 2.3 @/NH4 -0.11 1.32 0.93 1.7 0.84 2.2 -0.99 7.46 7.82 3.0 a4/NHS 1.84 @ll/NH4 -0.28 3.2 2.0 1.02 2.2 0.96 2.1 1.62 1.64 -0.27 aS/NH6 0.33 2.4 0.34 1.04 1.9 flSI1/NIf 0.0 0.71 2.7 2.1 3.13 3.31 -0.57 a6/NH7 0.24 0.24 1.22 1.8 2.3 $/NH7 -0.09 0.76 2.9 3.4 4.96 4.99 a7/NH8 -1.17 0.73 1.28 2.0 2.2 $/NH 0.0 0.71 2.4 0.77 4.3 2.67 a2/a7 -0.67 2.67 0.81 0.71 2.5 2.5 $/NH8 0.0 0.82 0.61 0.62 0.71 2.3 3.7 0.0 a’lP‘, 0.02 0.71 2.4 2.7 /3ll1/NH2 0.0 0.02 0.64 0.0 0.71 4.2 0.61 a3i~3/ 0.69 0.71 2.7 2.8 @‘,t/NH2 0.0 0.75 0.34 0.71 2.4 0.0 0.33 a4/@ll 0.71 2.4 2.7 0.74 0.0 0.73 a5IP511 Compound 2 2.9 a5/~51, 0.0 -0.24 2.32 2.55 0.86 2.0 0.51 0.52 0.71 2.5 2.6 a1/NH2 1.39 0.86 1.9 2.0 NHs/NH6 -0.40 1.36 2.3 3.24 3.63 1.40 4.2 -0.33 a2/NH3 0.96 1.8 p l 1 / N H 4 0.0 2.0 5.23 0.56 0.60 0.71 2.5 -0.72 4.88 3.2 a3/NH4 0.78 1.6 1.7 p l 1 / N H 4 0.0 9.14 1.25 1.34 -0.41 8.93 1.9 a4/NHS 0.71 2.2 3.6 PSII/NHS0.0 0.78 1.9 aS/NH6 0.71 2.5 -0.16 3.47 3.54 0.53 0.56 2.5 2.5 PsIl/NHs not 0.85 1.8 a6/NH7 -0.39 4.43 4.68 observed 3.4 2.91 2.93 1.05 2.0 a7/NH8 -0.52 1.8 p6/NH7 3.71 0.91 1.9 0.0 0.82 0.87 -0.43 3.71 0.71 2.3 a2/a7 4.3 0.0 0.71 2.7 2.7 b3l/NH3 0.0 0.35 0.63 0.67 0.35 0.71 2.4 3.5 a5/~51 Compound 3 -0.20 3.27 3.47 0.80 1.9 0.95 1.1 0.79 2.3 ari/NH2 2.2 2.2 a4/p11 -0.05 a2/NH3 0.92 2.0 2.0 a2/P211 -0.05 -0.29 2.30 2.43 0.68 0.68 0.84 2.5 2.4 0.96 1.8 2.4 a’/$ 0.0 -0.61 4.02 4.31 0.50 a3/NH4 0.71 2.5 0.50 4.9 1.07 1.9 1.7 a 7 / ~ 8 0.0 -0.63 3.25 3.42 1.07 a4/NHS 1.08 0.71 2.2 5.0 0.73 2.1 3.7 NHS/NH6 -0.28 1.70 1.76 1.79 1.61 aS/NH6 1.03 2.2 -0.02 3.0 2.4 PSI/NHS 0.0 3.78 0.09 0.92 1.8 a6/NH7 0.10 0.71 3.3 -0.48 3.98 2.9 0.82 1.8 a7/NH8 2.1 @,t/NH4 0.0 0.59 0.71 2.5 -0.26 3.94 2.1 3.80 0.55 0.71 3.2 p 1 , / N H 4 0.0 0.71 2.1 1.58 0.13 0.12 1.48 0.0 ao//NH3 3.3 2.1 p 6 1 ~ ~ 6-0.08 0.97 1.9 3.78 3.78 a2/a7 -0.54 0.74 1.8 3.75 3.2 3.65 @/NH7 0.0 0.70 1.26 0.76 2.4 0.71 2.2 -0.02 1.32 4.2 0.67 0.71 2.7 0.0 $)NH~ 0.0 0.71 2.1 0.36 1.80 0.36 1.77 Compound 4 -0.54 2.96 3.24 1.03 1.9 2.6 PZl/a7 0.0 0.56 0.71 2.5 a‘/NH2 0.56 4.6 0.92 2.0 2.4 .4/p 0.0 -0.32 2.67 2.68 0.1 1 a’/NH4 0.1 1 0.71 3.3 2.7 0.77 1.9 1.34 -0.13 3.08 3.12 a4/NHs 2.4 NHS/NH6 -0.05 1.40 0.76 2.2 4.2 -0.20 1.81 1.88 a6/NH7 0.89 2.1 2.1 y71,/NH7 0.0 1.42 1.37 0.71 2.1 0.85 2.2 3.4 @,/NH4 0.0 0.71 2.0 2.1 a7/NH8 -0.12 1.34 1.35 2.06 1.98 a2Ia7 0.87 2.4 1.9 @$/NH4 0.0 -0.08 0.82 0.82 0.56 0.53 0.71 2.5 3.1 0.0 0.71 2.3 pSl/NyS 0.0 0.0 0.94 0.94 0.0 0.71 2.5 2.5 @/NH 0.0 0.31 0.32 0.71 2.7 4.4 0.50 0.50 0.71 2.9 0.0 1.6 B2,,/NH2 0.0 0.65 0.71 2.4 0.7 1 0.23 0.23 3.1 0.71 2.2 4.3 b2;)NH2 0.0 0.52 0.51 0.71 2.5 1.30 1.30 3.3 ‘In recriprocal seconds. bCalculated from eq 6. ‘Calculated from eq 3. “Calculated from eq I . eCalculated by using GROMOS with r” distance constrains. /The rf carrier was positioned at 5.67 ppm, r B p l = 3030 Hz. Offsets can be calculated by using data in Table II for 3 and 4 (see ref 21 for 1 and ref 22 for 2 ) . PI, y l iefer to downfield h:lastereGipic protons pIl,yIl refer to upfield diasiereotopic protons.
y/>
The IK complex point FIDs (2K by Bruker nomenclature) were multiplied by a shifted (n/2) sine-bell function extending to I024 points in F2 and to 256 points in FI dimensions, followed by zero filling in the FI dimension. Fourier transformation and phase correction in both dimensions resulted in a IK X I K matrix.
D1 = 1.5 s
DO = 3 X
The homonuclear dipolar correlated spectroscopy experiments in a rotating frame (CAMELSPIN, ROESY) were performed utilizing principles described by B 0 t h n e r - B ~and ~ ~ Bax and Davis,28 in a phasesensitive mode using TPPI; pulse sequence DI-90-DO-SL-FID. The spin-lock field, which used yBsL = 3030 Hz, was generated by a lowpower transmitter. At least five experiments for each peptide 1-4 with different spin-lock times ranging from 0.01 to 0.15s (presented on cross relaxation in the rotating-frame buildup rate graphs-see text) were made. After acquisition of 256 t l experiments with 32 scans of 2K data points, the data were transferred and processed analogously to the procedure outlined for 2D NOE spectra.
I n each of the 2D NOE and 2D ROE experiments the measurements of cross-peak and corresponding diagonal-peak volumes were carried out by defining an oval around each peak and volume integration routine provided by FTNMR. For each given experiment the cross-peak volumes were scaled according to a procedure suggested by Macura et eq 4, where 7, is the mixing (NOE) or spin-lock time (ROE), uAB is the %(Tm)
=
aAB(7m) (%)nBaAA(Tm)
+ (%)nAaBB(Tm)
(4)
cross-peak intensity, uAA is the diagonal-peak intensity, a B B is the diagonal-peak intensity, nA is the number of equivalent atoms A, and nB is the number of equivalent atoms B. This methodology allows for corrections of any variations between experiments (number of scans, signal/noise ratio, etc.), and most of all, a2(~,,,)is a monotonically increasing function that is linear over a wider range with respect to either spin-lock or mixing times than the absolute cross-peak volumes are. I f both diagonal peaks (AA and BB) are not resolved well enough for integration, the cross-diagonal peak volumes are scaled with regard to
(27) Bothner-By,A. A.: Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz,
R. W.J . Am. Chem. SOC.1984, 106, 811-813. (28) Bax, A.; Davis, D.G.J . Magn. Reson. 1985,63, 565-569.
(29) Macura, S.; Farmer, B. T., 11; Brown, L. R. J . Magn. Reson. 1986, 70, 493-499.
Peptide Neurotransmitter and Hormone Design
J . Am. Chem. SOC.,Vol. 113, No. 6, 1991 2219
only one of them29(eq 5 ) . In this work, eq 5 was used, even if resolution a1(7m)
= aAB(7m)/nAaBB(7m)
(5)
of both diagonal peaks was good enough for integration purposes, and only the statistically better of the two values (with regard to A and B) was selected for further computations. The linear regression and statistical data analysis were performed (Lotus Development Corp., release 2.01) for all the 2D-NOE and 2DROE experiments and selected values (see supplementary material for complete sets) are presented in Table 111 for peptides 1-4, respectively. Transverse cross-relaxation is observed in the rotating frame with the spins oriented along an effective spin-locking field we = yBerf= y ( A 2 + BsL2)'/z makes an angle @ = sin-l (BsL/Ben)with the longitudinal component A in the rotating frame.14 Therefore a 2-fold correction of these offset effects has to be made. Since the intensity of the ROE cross-peak between spins A and B is proportional to sin PA X sin Be, while the intensity of the diagonal peaks is proportional to sin2 PA and sin2 for spins A and B, respectively, the cross-relaxation rate ~ ~ (or 7a,(rsL) ~ ~must ) be corrected by a factor of sin BA/sin PB if the cross-peak was normalized relative to B diagonal peak, or by sin &/sin PA if the cross-peak was referenced (eq 5) relative to an A diagonal peak.30 Another correction is necessary based on the observation that the measured transverse cross-relaxation rate contains a component of longitudinal cross-relaxation,14 eq 6, where uRF is the experimental ( ~ R F ) A B = COS
PA COS P B ( ~ ~ I )+A sin B PA sin B B ( ~ ~ ) A+ B (y4h2/10PAB) sin2 P A sin2PB[(1+ 4ue7,2)-' - I]r, (6)
transverse cross-relaxation rate, ull is the longitudinal transverse crossrelaxation rate, rCis the correlation time, and TAB is the internuclear distance between protons A and B. Since in solution W ~ T ,
