J. Am. Chem. SOC.1994,116,3201-3219
3201
Structural Studies of a Family of High Affinity Ligands for GPIIb/III a Alvin C. Bach, 11,**1Charles J. Eyermaql John D. Gross,t*lMichael J. Bower,*v* Richard L. Harlow,$ Patrica C. Weber,l and William F. DeCradol Contributionfrom the Dupont Merck Pharmaceutical Company, Experimental Station, Box 80336, Wilmington, Delaware 19880-0336, and Central Research & Development Department, E. I. du Pont de Nemours and Company, P.O. Box 80228, Wilmington, Delaware 19880-0228 Received September 20, 1993"
Abstract: A class of potent orally active cyclic peptide antagonists of the glycoprotein IIb/IIIa adhesion molecule have been prepared by linking a tetrapeptide, RGD-containing sequence between the two ends of a semirigid linker, m-(aminomethy1)benzoic acid (Mamb). Todetermine how this aminoacid constrainstheconformationof theintervening peptide sequence and to shed some light on the receptor-bound conformation of the peptide, we examined the solution and solid-state structures of nine cyclic analogues whose receptor binding constants span approximately 4 orders of magnitude. The general structure of these analogues is cyclo(Xxx-Arg-Gly-Asp-Mamb). The backbone conformations of each compound trace out a rectangular shape with a @-turncentered at the Xxx-Arg bond. In the most potent compounds in this series Xxx is a small, aliphatic D-amino acid, and Na of the Arg residue is methylated: peptides containing these features are highly rigid and contain a type 11' &turn centered at the D-Abu-N-MeArg dipeptide, a highly extended Gly residue, and a C7 turn centered at the Asp. Peptides lacking the N-methyl group and/or with reversed chirality at Xxx are more flexible. The Na-methyl group also restricts the conformationof the Arg side chain. In addition the aliphatic side chain of the D-aminO acid, the "-methyl of N-MeArg, and the phenyl group of the Mamb linker form a continuous hydrophobic surface, which presumably interacts favorably with the receptor. Finally, the effects of conformationalconstraints introduced at the Mamb and the Asp residues were investigated to determine their effects on receptor binding.
Introduction The discovery and optimization of peptides capable of binding to biological receptor molecules is an activity of fundamental interest to the field of molecularrecognition and practical interest to the pharmaceuticalindustry. In recent years, several biological and chemical approaches have been developed to prepare and sort through vast libraries of peptides in search of compounds with high affinity for a receptor of interest.14 One potential drawback of this approach is that peptides are highly flexible molecules, and hence it is difficult to define their biologically active conformation and lock them into this state. One way to partially overcome this limitationis to prepare libraries of disulfide cross-linked peptides5 or to embed the random sequences within the tertiary structure of a protein6 of known structure, although the resulting peptides still retain a large degree of flexibility. Therefore, we have begun to develop new approaches to the solidphase synthesis of highly conformationallyconstrained peptides for use in compound libraries. In a previous communication, we described template constrained cyclic peptides, in which one introduces a semirigid template into a cyclic ~ e p t i d e .The ~ template is intended to limit ~~~~
+ Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA. t Present address: Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA. E. I. DuPont de Nemours, Wilmington, DE. Dupont Merck Pharmaceutical Co. 0 Abstract published in Advance ACS Absrracrs, February 15, 1994. (1) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354.82-84. (2) Cwirla, S. E.; Peters, E.A.; Barrett, R. W.; Dower, W. J. Proc. Narl.
Acad. Sci. U.S.A. 1990, 87, 6378-6382. (3) Devlin, J. J.; Ranganiban, L. C.; Devlin, P. E. Science 1990,249,406 406. (4) Houghton, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84-86. ( 5 ) ONeil, K. T.; Hoess, R. H.; Jackson, S. A.; Ramachandran, N. S.; Mousa, S. A.; DeGrado, W. F. Proteins 1992, 14, 509-515. (6) ONeil, K. T.; DeGrado, W. F.; Hocss, R. H. in Techniques in Protein Chemistry V; Academic Press: Orlando, FL, in press.
the conformationalflexibility of the intervening peptide sequence. This strategy was evaluated with impressive results using the Arg-Gly-Asp (RGD) sequence as a test case. Linear peptides containing this tripeptide bind to the receptor, IIb/IIIa, with about 10 pM dissociation constants, whereas cyclic RGDcontaining peptides that incorporated the template, m-(aminomethy1)benzoic acid, bound with considerably higher affinity (up to 100 pM). A systematic set of analogues were prepared todetermine the features that gave rise to this high affinity (Figure 1). The most tight binding compound were cyclo(D-Abu-NMeArg-Gly-Asp-Mamb) (1) and cyclo(D-Val-N-MeArg-GlyAspMamb) (2) where D-Abu is a-aminobutyric acid and N-MeArg is N"-methylarginine. The D-chirality of the amino acid at position 1 of the sequence and the N-methylation of the Arg were found to be particularly important for obtaining high binding affinity. Additional conformation1constraints that were compatible with high activity (but did not necessarily lead to tighter binding) included the introduction of a phenyl ring onto the methylene group of the Mamb or the addition of a methyl group onto the j3-methylene of the Asp residue. In these cases, two isomers were prepared, and one was shown to be considerably more active than the other. In the present paper, we examine a number of different peptides (Figure 1) to determine the extent to which each of these features influence the conformation of the peptide. These peptides include compounds 1-3, all of which are highly active and have a D-amino acid (D-Abu, D-Val, or D-Ala, respectively) at position 1 and N-MeArg at position 2. To assess the role of the N-methyl group, cyclo(D-Abu-Arg-Gly-Asp-Mamb)(4) has also been examined. Further, comparison of this compound with cyclo(A1a-Arg-GlyAsp-Mamb) (5), which has L-Ala at position 1, helps to define the effect of the first chiral center. Finally, comparison of the two isomers of the Ca-methyl-Asp-containingpeptides (6and 7), and the two isomers of the phenyl-Mamb derivatives (8 and 9) (7) Jackson, S. A.; Harlow, R. L.; Dwivedi, A.; Parthasarathy, A.; Higley, A. C.; Krywko, J.; Rockwell, A.; Markwalder, J. A.; Wells, G. J.; Mousa, S. A.; DeGrado, W. F. J . Am. Chem. Soc., following paper in this issue.
OOO2-7863/94/1516-3207$04.50/00 1994 American Chemical Society
3208 J . Am. Chem. SOC.,Vol. 116, No. 8, 1994
R
Bach et al.
Two-dimensional data sets were processed using the FELIX program (Biosym Inc.). Matrix files were 1024 X 1024 points in size. All t2 time domain transforms were weighted with a skewed sine bell, shifted 80 degrees, with a skew factor of 1.1. The t l interferograms were also weighted with the same apodization function of the appropriate length. In most cases a high-order polynomial was then applied to flatten the baseline in both dimensions. Most ROESY data sets were collected with the t l delay set to the calculated value of the second point in the interferogram.lS The first point was back-calculated before transformation using the linear predication routine built into FELIX. In the other cases, the first point of each interferogram was multiplied by 0.5 before transformation to eliminate t 1 artifacts.19 Proton chemical shifts assignments were made using standard techniques.20 1cycZo(D-Abu-N-MeArg-Gly- Asp-Mamb) ROE (rotating frame nuclear Overhauser effect) crosspeak volumes were measured using FELIX software. These were converted to 2 cycZo(D-Val-N-MeArg-Gly-Asp-Mamb) interproton distances using the ROE between the two HB protons of N-MeArg or Arg as a standard distance which was set to 1.8 A. The 3 cycZo(D-Ala-N-MeArg-Gly- Asp-Mamb) calculated distances were independent of spin locking time between 50 4 cycZo(D-Abu-Arg-Gly-Asp-Mamb) and 300 ms. The spin locking times used for each calculation are listed below. ROE data beyond CB was not used in structure calculation. 5 cycZo(Ala-Arg-Gly-Asp-Mamb) Computational Methods. Conformations consistent with the NMR data were generated based on well-established procedures.2l-23 For 6 cycZo(D-Val-N-MeArg-Gly-B-Me-Asp-Mamb) Isomer 1 compounds 1, 4, and 5, 500 ROE-constrained conformations were 7 cycZo(D-Val-N-MeArg-Gly-P-Me-Asp-Mamb) Isomer 2 generated using the distance geometry program DGEOM (available from QCPE, Department of Chemistry, Indiana University, Bloomington, IN 8 cycZo(D-Val-N-MeArg-Gly-Asp-a-phenyl-Mamb) Isomer 1 47405). Cis-amide bonds were allowed, and no distance correlation was used in the distance sampling. Lower-bound distance constraints were 9 cycZo(D-Val-N-MeArg-Gly-Asp-a-phenyl-Mamb) Isomer 2 Figure 1. Chemical formula of cyclo(D-Abu-N-MeArg-Gly-Asp-Mamb) set to 20% less than the derived interproton distance, and upper bounds were set to 30% greater than this value. Antidistance constraints (ADCs) (1) and the sequences of the other cyclic peptides in this study. The corresponding to interproton distances which did not produce an observed proton at the two position of the phenyl ring is indicated. ROE were assigned a lower bound of 3 A.24Distance constraints were applied using a squarewell potential with a 50 kcal mol-’ A-2forceconstant have allowed us to better define the receptor-bound conformation applied outside the lower and upper bounds. Chiral constraints were of these peptides. applied to all chiral and prochiral centers. ROESinvolvingmethyl protons were represented as distances to the methyl carbon. The Gly Ha protons were stereochemically assigned using Pacoupling Methods constants and a transannular ROE between the Mamb H2 ring proton and one of the Gly Ha resonances (see Figure 2 ) . The conformational NMR Methods. NMR experiments were performed at 499.8 MHz calculations described below were run in parallel, with the assignment on a Varian VXR-SOOS spectrometer. NMR samples were prepared of the transannular ROE switched between the Glypro-R and pro-S Ha under dry nitrogen gas in 0.8 mL of 99.996% DMSO-& (Merck) at the protons. The chirality of the Gly Ca was fixed in all distance geometry concentrations listed in the table and figure captions. TMS (1 pL) was calculations. used as an internal chemical shift reference. Spectra in D20 were Constrained minimization of the initial distance geometry structures referenced to the HOD peak at 4.79 ppm at 25 OC. A 1 5 s relaxation was done using the TRIPOS force fieldZSwithout including electrostatic delay was used in all experiments. terms. The N-MeArg(Arg) and Asp side chains were kept neutral. One-dimensional FT experiments were collected with 16 384 complex Conformations with a distance constraint energy of 2 kcal/mol or less points. For variable-temperature experiments, the sample was allowed were then minimized with ROE constraints and electrostatic terms to equilibrate in the probe for 30 min before data collection. calculated using partial atomic charges based on the Gasteiger-Marsili DQF-COSY,Q’ TOCSY,lOJ1 and ROESYIZJ3two-dimensional spectra Those conformations with constraint energies of 2 kcal/ were recorded. TOCSY data sets were recorded using the clean-TOCSY mol or less and a total energy within 15 kcalof the lowest energy conformer pulse sequence with 35-50 ms spin locking times and 1-ms trim pulses. were finally checked for consistency with all the NMR data, including ROESY spectra were recorded with a 3.0 KHz spin locking field using pacoupling constants using the equation of Bystr0v.2~ Conformations the method of Kessler.14 RFcompensationwas achieved using the method which did not have ROE errors greater than 0.5 A and which had of Dezheng.Is For each sample, data at several spin locking times in the angles within 30 degrees of the allowedvaluescalculated fromPUcoupling range 50-300 ms were recorded. constants were taken as viable NMR solution conformations. Clustering Two-dimensional experiments were obtained with spectral widths of either 5800 or 7000 Hz in both dimensions. Complex points (2048) were (18) Marion, D.; Bax, A. J . Magn. Reson. 1989,83, 205-211. (19) Otting, G.; Widmer, H.; Wagner, G.; Wuethrich, K. J. Magn. Reson. collected in t2, and 256 complex points were collected in t l . Transients 1986, 66, 187-193. (32 or 64) were coadded for each t l value. All spectra were acquired (20) Wiithrich, K.; Billeter, M.; Braun, W. J . Mol. Biol. 1984, 180, 715in the phase-sensitive absorption mode with quadrature detection in both 740. dimensions.l6J7 (21) Peishoff, C. E.; Bean, J. W.; Kopple, K. D. J . Am. Chem. SOC.1991, 113,4416-4421. (8) Piantini, U.; Sorensen, 0. W.; Ernst, R. R. J . Am. Chem. SOC.1982, (22) Peishoff, C. E.; Ali, F. E.; Bean, J. W.; Calvo, R.; DAmbrosio, C. 104,6800-6801. A.; Eggleston, D. S.; Hwang, S. M.; Kline, T. P.; Koster, P. F.; Nichols, A.; (9) Shaka, A. J.; Freeman, R. J. Mugn. Reson. 1983, 51, 169-173. Powers, D.; Romoff, T.; Samanen, J. M.; Stadel, J.; Vasko, J. A.; Kopple, K. (10) Davis, D. G.: Bax, A. J . Am. Chem. SOC.1985, 107, 2820-2821. D. J. Med. Chem. 1992, 35, 3962-3969. (1 1) Griesinger, 6.;Otting, G.; Wuethrich, K.; Ernst,-R.R.. J . Am. Chem. (23)Kopple,K.D.;Baures,P.W.;Beans,J.W.;DAmbrosio,C.A.;Hughes, SOC.1988,110, 7870-7872. J. L.; Peishoff, C. E.; Eggleston, D. S.J . Am. Chem. SOC.1992, 114,96159623. (12) Bothner-By, A. A.; Stephens, R. L.;Lee,J.; Warren, C. D.; Jeanloz, (24) Briischweiler, R.; Blackledge, M.; Ernst, R. R. J . Biomol. NMR 1991, R. W. J. Am. Chem.Soc. 1984, 106,811-813. (13) Bax, A,; Davis, D. G. J. Mugn. Reson. 1985, 63, 207-213. I, 3-11. (14) Kessler, H.; Griesinger, C.; Kerssebaum, R.; Wagner, K.; Ernst, R. (25) Clark, M.; Cramer, R. D., 111; Opdenbosch, N. V. J. J . Comput. R. J . Am. Chem. SOC.1987, 109, 607-609. Chem. 1989, 10, 892-1012. (15) Dezheng, 2.;Fujiwara, T.; Nagayama, K. J. Magn. Reson. 1989,81, (26) Gasteiger, J.; Marsili, M. Tetrahedron 1980, 36, 3219-3228. 628-630. (27) Marsili, M.;Gasteiger, J. Croat. Chem. Acta 1980, 53, 601-614. (16) Miiller, L.;Ernst, R. R. Mol. Phys. 1979, 38, 963-992. (28) Gasteiger, J.; Marsili, M. Org. Mugn. Reson. 1981, 15, 353-360. (17) States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson. 1982, (29) Bystrov, V. F.; Portnova, S. L.;Bahashova, T. A.; Koz’min, S. A.; 48, 286-292. Gavrilov, Y.D.; Afanas’ev, V. A. Pure Appl. Chem. 1973, 36, 19-34. HN
Structural Studies of High Affinity Ligands for GPIIbjIIIa
J. Am. Chem. SOC..Vol. Il6, No. 8, 1994 3209 Table 1. NOE-Derived Distance Constrants for cyclo(D-Abu-N-MeArg-Gly-Asp-Mamb) 1 in DMSO-& at 25 'C (150 ms Mix) from D-Abu D-Abu Gly Gly Gly Asp Asp Asp Asp Mamb Mamb Mamb Mamb Mamb Mamb Mamb Mamb Mamb
Ib v1
r-
8
I
to NH NH NH NH NH NH NH NH NH NH NH H2 H2 H2 H2 H2 H4 H4
Mamb Mamb Mamb Nmra Nmr Gly Gly Asp Asp Asp Mamb Asp Nmr Gly Mamb Mamb Mamb Mamb
H2 H6 H2 Me Ha Hal Ha2 Hfll Hf12 Ha H2 Ha Me Hal Ha2b Halb Halb Ha2b
calcd distance
lower bound
upper bound
2.9 2.3 2.7 2.5 2.5 2.2 2.5 2.4 2.6 2.1 2.6 3.2 3.6 3.1 2.7 2.8 2.5 2.5
2.3 1.8 2.2 2.0 2.0 1.8 2.0 1.9 2.1 1.7 2.1 2.5 2.9 2.5 2.2 2.2 2.0 2.0
3.7 3.0 3.5 3.2 3.2 2.9 3.3 3.1 3.4 2.8 3.4 4.1 4.7 4.0 3.5 3.6 3.3 3.3
a Nmr-N-MeArg. Numerals indicate nondegenerate resonances but do not imply prochirality.
0 '
-m
2
4 " 8.$ 8 5.5
5.0
4.5
3.5
4.0
DI
3.0
(ppn)
ic
0
z
between 10.0 and 10.5 Hz for thepro-R proton and between 0.6 and 1.0 Hz for the p r e s proton. When the Gly H a pro-R assignment is used in calculating conformations of 4, three families of conformers result. The first has Gly @values between 65' and 7S0, corresponding to pavalues between 6.5 and 6.8 Hz for the pro-R and between 5.0 and 5.9 Hz for thepro-S protons. The second conformational family has Gly Q values between 49' and 56', corresponding to JNavalues between 6.7 and 6.8 Hz for the pro-R and between 3.0 and 3.8 Hz for the p r o 3 protons. The third family has Gly Q values between 150° and 160', corresponding to Pavalues between 0.4 and 1.O Hz for the pro-R proton and between 6.8 and 8.4 Hz for the pro-S proton. The experimentally observed values of 8.3 and 3.5 for 4 agree best with the ROE assigned to p r o 8 Ha proton. For compound 5, the two Gly JNa coupling constant values are too close in value, making this type of analysis invalid. They were therefore assigned based on chemicalshift, assumingthe more upfield shifted proton was S, as in 1 and 4. The accurate determination of the solution conformation of 1,4, and 5 is predicated on the correct stereochemical assignment of the Gly Ha protons. In the recent work on small RGD peptides the sterochemical assignment of the Gly Ha protons has always been reported as being a m b i g u o ~ s . ~In~our ~ ~study, ' ~ ~thepresenceofa ~~~~ consistentlyobervable transannular ROE (see Figure 2) and extremeJNavalues for the Gly N H enables the assignment of the pro-S proton as closest to the H2 proton of the Mamb residue across the backbone ring (see Figure 1). For 1, only one conformational family was obtained with the reversed stereochemicalassignment, which has Gly Q values inconsistent with the observed coupling constants. For 4, three families are obtained under the same reversed assignment; two are completely inconsistent with the observed values. The range of JNa'sfound for the third family places the experimentally observed JNa's at the edge of the range and is unlikely to be correct. StructureGenerationof 1,4,and5. Todetermine thesolution structure of 1, a total of 18distance constraints were derived from a 150-ms ROESY 2D spectrum of 1 (Table 1). An additional 85 ADCs (antidistance constraints) with a 3.0 A lower bound were also included in the calculations to generate the initial 500 conformations. A total of 172 conformations passed the 2 Kcal fixed distance range energy test of the first constrained minimization; 121of these conformerspassed another 2 Kcal fied distance range energy test of the second constrained minimization, and 119 were within the lowest 15 Kcals of energy. These 119 conformers were tested against consistent Q values (130O) calculated from the JNQcoupling constants (see Table 2). The D-Abu 6 was constrained to be near 70°, 170°, -20°, or -looo. The Gly Q was constrainedtobenear*135'. TheAspQwasconstrained tobenear 38', 8 l 0 , -8S0, or -155O. A total of 38 conformers satisfied all four paconstraints, containing no distanceconstraint violationsgreater than 0.1 A. These 38 conformers were within 0.48 & . RMS difference of each other and clustered into one conformational family for 1, based on the X-R-G-D backbone.
Buch et al.
3210 J . Am. Chem. SOC.,Vol. 116, No. 8, 1994 Table 2. Proton Chemical Shifts of cyclo(D-Abu-N-MeArg-Gly-Asp-Mamb) 1 (6.9 mM) in DMSO-& at 25 residue
0
D-Abu N-MeArg
HNQ 8.88 2.95 (Me)
GlY ASP Mamb
7.64 8.55 8.46
Aa/ A p
Ha
-6.6
4.0 -3.9 -5.5
He"
H'"
4.60 5.15
1.77 1.58/2.02
0.95 1.35
3.66s/4. 1ER 4.5 1 4.0414.62
2.5112.71
O C
othef
P ae
5.1 H6: 3.11 eNH: 7.53 H2: H4: H5: H6:
8.1,3.0 7.5 7.5, 5.0
7.48 7.37 7.37 7.71
PPM. b ppbfdeg. Hz.
Table 3. NOE-Derived Distance Constrants for cyclo(D-Abu-Arg-Gly-Asp-Mamb) 4 in DMSO-d6 at 25 OC (200 ms Mix) from D-Abu D-Abu D-Abu Arg Arg Arg Arg Gly Gly Gly Asp Asp Asp Asp Asp Mamb Mamb Mamb Mamb Mamb Mamb
to NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH H2 H2 H2 H4 H4
Mamb Mamb D-Abu Mamb Mamb D-Abu Arg Mamb D-Abu Arg Mamb Gly Gly Asp Asp Mam Gly Mamb Mamb Mamb Mamb
H2 H4 Ha H2 H2 H" HB1U H2 H" H" H2 Ha2 HaL HB1 HmQ H2 Hal Halu HU2O Ha2" Hal'
calcd distance
lower bound
upper bound
2.6 2.2 2.6 3.1 2.5 2.0 2.3 3.0 3.4 2.1 3.2 2.7 2.2 2.5 2.7 2.4 2.8 3.2 2.8 2.6 2.4
2.0 1.8 2.1 2.9 2.0 1.6 1.9 2.4 2.7 2.2 2.6 2.1 1.8 2.0 2.2 1.9 2.2 2.5 2.3 2.1 2.0
3.3 2.9 3.4 4.8 3.2 2.6 3.0 3.9 4.5 3.5 4.2 3.5 2.9 3.2 3.5 3.2 3.6 4.1 3.7 3.4 3.2
Numerals indicate nondegenerate resonances but do not imply prochirality
.
Table 4. Proton Chemical Shifts of cyclo(D-Abu-Arg-Gly-Asp-Mamb) (23.3 mM) 4 in DMSO-& at 25 OC residue HNa A a / A P Ha0 D-Abu 8.83 -6.4 4.17 Arg 8.58 -5.6 4.33 Gly 8.19 Asp 8.41 Mamb 8.48
-3.5 -3.8 -5.8
3.69'14.10R 4.60 4.0614.62
Hfla HTO otheP Pc 1.77 0.92 6.4 1.4812.03 1.48 HJ: 3.11 9.2 cNH: 7.71 8.3, 3.5 2.4812.71 8.0 H2: 7.60 7.7,4.8 H4: 7.36 H5: 7.36 H6: 7.66
Table 5. NOE-Derived Distance Constrants for cyclo(L-Ala-Arg-Gly-Asp-Mamb) 5 in DMSO-& at 25 OC (200 ms Mix) from Ala Ala Ala Arg Arg Gly Gly Gly Asp Asp Asp Asp Asp Asp Mamb Mamb Mamb Mamb Mamb Mamb Mamb Mamb
NH NH HH NH NH NH NH NH NH NH NH NH NH NH NH NH NH H2 H2 H2 H4 H4
Arg Mamb Ala Ala Arg Arg Mamb Ala Mamb Gly Gly Asp Asp Mamb Asp Asp Mamb Gly Mamb Mamb Mamb Mamb
calcd distance
lower bound
uppcr bound
2.7 2.1 2.4 2.8 4.1 2.5
2.1 1.7 2.0 2.2 3.3 2.0 2.4 2.8 2.6 2.0 2.5 2.3 2.2 2.2 2.7 1.7 1.9 2.5 2.4 2.2 1.8 2.1
3.5 2.8 3.2 3.6 5.4 3.2 3.9 4.6 4.2 3.2 4.1 3.7 3.6 3.5 4.3 2.8 3.1 4.1 3.9 3.7 2.9 3.4
NH H6 Hfl H" HBl a H" H2 H" H2 Hul Hu2 H@lU HB20 H2 NH Ha H2 Hal Hala HaZu Hala Hula
3.0 3.5 3.2 2.4 3.1 2.8 2.8 2.1 3.3 2.1 2.3 3.1 3.0 2.8 2.3 2.6
Numerals indicate nondegenerate resonances but do not imply prochiralit y.
Table 6. Proton Chemical Shifts of cyclo(L-Ala-Arg-Gly-Asp-Mamb) (17.8 mM) 5 in DMSO46 at 25 OC residue HN" A 6 / A P L-Ala Arg
8.87 7.86
-7.4 -2.0
Gly 7.90 Asp 8.01 Mamb 8.51
-1.2 -0.9 -6.4
PPM. ppb/deg. Hz. a
For determining the structure of 4, a total of 21 distance constraints were derived from a 200-ms ROESY 2D spectrum (Table 3). An additional 86 ADCs with a 3.0 A lower bound were also included in the calculations to generate the initial 500 conformations. A total of 190 conformers passed the 2 Kcal fixed distance range energy test of the first constrained minimization; 128 of these conformations passed the 2 Kcal fixed distance range energy test of the second constrained minimization all of which were within 15 Kcals of each other. The 128 conformers were tested against consistent @ values (f30°) calculated from the pucoupling constants (see Table 4).29 The D-Abu @ was constrained to be near 78O, 162O, -30°, or -9OO. The Arg @was constrained to be near -96O or -144'. The Gly @ was constrained to be near f130°. The Asp @ was constrained to be near 45O, 76O, -87O, or -153'. A total of 46 conformers satisfied the five Paconstraints, which were all within 0.20 A R M S difference of each other. They clustered into two conformational families, based on the X-R-G-D backbone.
to
Ha"
Hea
Hya
othep
JNac
4.11 4.34
1.42 4.8 1.4511.98 1.37 H6: 3.09 9.2 tNH: 7.59 3.53SI3.99R 6.8,4.9 4.79 2.2712.70 8.7 4.0614.52 H2: 7.61 7.0, 5.4 H4: 7.39 HS: 7.38 H6: 7.66
PPM. ppbfdeg. Hz.
The solution structures of 5 determined from 22 distance constraints were derived from a 200-ms ROESY 2D spectrum (Table 5). An additional 201 ADCs with a 3.0 A lower bound were also included in the calculations to generate the initial 500 conformations. A total of 348 conformers passed the 2 Kcal fixed distance range energy test of the first constrained minimization; 128 of these conformers passed the 2 Kcal fixed distance range energy test of the second constrained minimization, and 322 were within the lowest 15 Kcals of energy. The 328 conformers were tested against consistent @ values (&30°) calculated from the p acoupling constants (see Table 6).29 The L-Ala @ was constrained to be near 18O, l o l o , 4 8 O , or -171O. The Arg @ was constrained to be near -96O or -144'. The Gly @ was constrained to be near *131° and f58O. Further tightening of the Gly @ constraint was not possible because the two Gly P''s are too similar to each other. The Asp @ was constrained to be near 60°, -93O, or -148'. Seventy conformers satisfied all five Paconstraints and clustered into four conformational families which were within 1.38 A R M S difference
Structural Studies of High Affinity Ligands for GPIIblIIIa
J. Am. Chem. SOC.,Vol. 116, No. 8, 1994 3211
a
?Ili
I
8
7
6
8
7
6
5
3
4
b
C
I
9
5
.
,
.
.
,
4
.
.
.
~
,
'
3
'
,
.
2
,
.
.
'
.
,
~
~
ZPpm
Figure 3. 500-MHz 'HNMR spectra of I (a), 4 (b), and 5 (c) all in DMSO-& at 25 OC. Sample concentrations: 1, 6.9 mM; 4, 23.3 mM; 5, 17.8 mM. All three spectra show dispersed amide and Ha protons. of each other, based on the X-R-G-D backbone. Two are extended and two are bent at the Gly residue. The bent conformations cannot be ruled out because the two observed Gly F avalues are not as different from each other as they are in 1 and 4 (see Tables 2, 4, and 6).
Results and Discussion General NMR. Compounds 1, 4, and 5 all give singlecomponent spectra with no indication of slow conformational
Bach et al.
3212 J. Am. Chem. Soc., Vol. 116, No.8, 1994 Table 7. Proton Chemical Shifts of cvclo(D-Val-N-MeAr~-GIy-B-Me-Asp-Mamb) 6 (8.5 mM) in DMSO-& at 25 O C residue
HN'
As/ A p
D-Val N-MeArg
9.00 3.00 (Me)
-7.3
GIY 0-Me-Asp Mamb
7.68 8.52 8.38
-4.5 -5.1 -6.1
a
HB'
H7"
4.35 5.22
H"'
2.13 1.5812.06
0.9511.14 1.38
3.6414.36 4.40 4.02/4.71
2.83
othee
Jd=
P a
5.5 Ha: 3.15 cNH: 7.56 &Me: 1.14 H2: 7.43 H4: 7.38 H5: 7.38 H6: 7.67
9.5,2.7 8.5 7.9,4.6
9.7
PPM. b ppb/deg. Hz.
Table 8. Proton Chemical Shifts of cyclo(D-Val-N-MeArg-Gly-B-Me-Asp-Mamb) 7 (8.0 mM) in DMSO-& at 25 OC residue HN' A6/AP H"' HS' HT' othee D-Val N-MeArg
9.00 3.02 (Me)
-7.4
GIY b-Me-Asp Mamb
7.62 8.50 8.70
-3.6 -5.2 -6.2
4.30 5.20
2.13 1.5912.07
3.6014.25 4.41 4.0114.70
2.71
0.94f1.11 1.38
JQB
P ae
5.6 Ha: 3.13 cNH: 7.56 @-Me: 1.09 H2: 7.41 H4: 7.38 H5: 7.38 H6: 7.67
8.5,2.3 8.2 7.5,4.9
10.7
PPM. ppbfdeg. Hz. Table 9. Proton Chemical Shifts of cyclo(D-Val-N-MeArg-Gly-Asp-cr-Ph-Mamb) 8 (16.4 mM) in DMSO-& at 25 OC
a
residue D-Val N-MeArg
HN' 8.99 3.01 (Me)
H"' 4.39 5.21
HB' 2.16 1.60/2.08
GlY ASP a-Ph-Mamb
7.67 8.65 8.64
3.6314.23 4.68 6.10
2.5 112.7 1
H7' 0.95/1.12 1.38
other'
P"b 6.2
H6: 3.14 tNH: 7.61 H2: 7.71 H4: 6.96 H5: 7.30 H6: 7.63 Ph-0: 7.30 Ph-m: 7.37 Ph-p: 7.37
8.3, 3.2 7.5 8.6
PPM. Hz.
averaging near room temperature (Figure 3). Each was easily assigned using standard N M R techniques. There is no evidence for cis-peptide bonds in any of these compounds, or the other six compounds examined, as judged by the lack of Ha to H a ROES. Several N M R parameters point to the presence of a predominant, stable solution conformation or a family of rapidly interconverting, highly similar conformations of all seven compounds in DMSO-&. First, the amide proton resonances are well dispersed with maximum chemical shift differences (As) of 0.64 ppm in 1 and 4 to 1.56ppm in 9 (see Tables 2,4,and 6-10). Second, the P a coupling constants show a range of values which are stable between 25 OC and 65 O C . Particularly striking are the extreme .Pa coupling constant values for the Gly and Mamb N H resonances. Suchvalues are strong indicators for a preferred solution c ~ n f o r m a t i o n .Third, ~ ~ there are sizable variations in the amide A6/AT values, indicating that all the amide protons are not in the same averaged environment. Finally, the two diasterotopic Gly Ha proton resonances have A6's ranging from 0.41 (in 4) to 0.72 ppm (in 6, see Tables 2,4,and 6-10), again indicating that the methylene protons are not in an averaged environment in solution.32 The two Mamb methylene proton resonances are similarly dispersed with values of A6 from 0.46 ppm in 5 to 0.69 ppm in 6. (30) Bogusky, M. J.; Naylor, A. M.; Pitzenberger, S . M.; Nutt, R. F.; Brady, S . F.; Colton, C. D.; Sisko, J. T.; Anderson, P. S.; Veber, D. F. In?. J. Peptide Protein Res. 1992, 39, 63-76. (31) McDowell, R. S.; Gadek, T. R. J. Am. Chem. SOC.1992,114,92459253. (32) Kessler, H. Angew. Chem., Inr. Ed. Engl. 1982, 21, 512-523.
The preference toward a single conformation is also seen in H2O. Two related cyclic peptides, 2, cyclo(D-Val-N-MeArgGly-AspMamb), and 3, cyclo(sAla-N-MeArg-Gly-AspMamb), exhibit nearly identical Pacoupling constants in both DMSO-& and HzO (Table 11). The data in Table 1 1 are nearly identical to the P a coupling contants for 1,4,5,6,7,8, and 9 in DMSO-& (see Tables 2,4,and 6-10). Conformational analyses of 2 and 3 in DMSO-& (data not shown) clearly show that they adopt solution conformations similar to those observed for 1,4, and 5 (see below). These strong indicators for a preferred conformation are also consistentwith several X-ray crystal structures of 1wheredifferent crystal forms all give rise to essentially the same peptide backbone conformation in the solid state (see below). Solution Conformation of cyclo(D-Abu-N-MeArg-Gly-AspMamb), 1. The solution conformation of 1 was determined as described in the Experimental Section. A striking feature of 1 is that it appears to adopt a single conformation in solution. Examination of 4, plots (Figure 4) for 38 distance geometry generated structures of 1 show that these angles fall within very narrow regions of the map, suggesting the presence of narrow and well-defined energy wells. Furthermore, the orientation of the Arg side chain appears to be reasonably well-defined up to the C* atom as assessed from the fact that x1 and x2 values lie almost exclusively within one of the two regions of the plot, centered a t -180°, -180°, and -50°, -180O. The predominant solution structure (Figure 5 and Table 12) is similar to classical cyclic hexapeptides which have an overall
Structural Studies of High Affinity Ligands for GPIIblIIIa
J . Am. Chem. SOC.,Vol. 116, No. 8, 1994
3213
Table 10. Proton Chemical Shifts of cyclo(D-Val-N-MeArg-Gly-Asp-a-Ph-Mamb) 9 (16.4 mM) in DMSO-ds at 25 OC
residue D-Val N-MeArg
HNa 9.11 3.11 (Me)
Ha0 4.37 5.20
HBa 2.22 1.6012.08
GlY
7.55 8.16 8.65
3.83 f 4.26 4.31 6.20
2.5412.63
ASP
a-Ph-Mamb
0
otheP
Hra 0.9411.12 1.36
P ab 6.1
H6: 3.13 cNH: 7.62 H2: H4: H5: H6:
7.82 7.37 7.37 7.85 Ph-0: 7.16 Ph-m: 7.31 Ph-p: 7.24
8.6,2.8 6.8 9.1
PPM. Hz.
Table 11. Comparison of p ain H20 and DMSO-d6 for 2, cyclo(D-Val-N-MeArg-Gly-Asp-Mamb), and 3, cyclo(D-Ala-N-MeArg-Gly- Asp-Mamb) 3
2
residue 2 = D-Val 3 = D-Ala GlY ASP Mamb
DMSO-&
HzO 5.0
DMSO-&
5.9
5.5
H2O 4.6
E-11.2 7.5 = 12.7
E-11.5 6.4 1 = 12.8
c=11.1 7.6 = 12.4
c=ll.5 6.8 = 12.8
rectangular shape with two connected 8 - t ~ r n s . 3In ~ compound 1, the first is an almost ideal type II'@-turn centered at the D-AbuN-MeArg residues (Table 12). Only the N-MeArg $ is significantly different from the ideal angles for this type of turn.34 The presence of this turn is supported by the relatively small A6/AT value for the Gly NH resonance (see Table 2) and an ROE between the Gly NH and the Mamb H2 protons. The methyl group of the N-MeArg residue points up when viewed as in the top panel of Figure 5 . Because the Mamb residue lacks appropriate H-bonding functionality, a second@-turnis not found. However, a y-turn is found a t the Asp residue. The Gly residue is generally extended, with the molecule presenting an elongated face between the N-MeArg and Asp residues. The possibility of conformational averaging in 1 could be assessed by comparing the calculated interproton distances (in the distance geometry-generated structures) with those expected from the intensities of the ROES. The only distance violations were very small (
