J. Am. Chem. SOC. 1994,116, 6431-6432
Synthesis and Nuclear Magnetic Resonance Structure Determination of an a-Helical, Bicyclic, Lactam-Bridged Hexapeptide Clay Bracken, J6zsef GulyBs, John W. Taylor,' and Jean Baume Rutgers University Department of Chemistry Piscataway, New Jersey 08855
( I)
Attempts to generate short a-helical peptides in solution have so far met with limited success, due to the unfavorable nature of helix initiation.14 This has led to the use of non-native amino acids, and side-chain or backbone bridges to serve as elements for stabilizing helices in short peptide sequence^.^^ Rigid structures that maintain multiple hydrogen-bond donors and/or acceptorsin the appropriategeometry should overcome the highly unfavorable entropic barrier for helix initiation.5 Such structures would facilitate the more favorable propagation of helix into neighboring amino acid sequences in the appropriate direction. This communication outlines the synthesis and NMR structure determination of a conformationally constrained bicyclic hexapeptide containing two overlapping i, i + 4 side-chain lactam bridges and selectively cleavable protecting groups on the N-terminal and C-terminal ends (I, Figure 1). NMR characterization indicates that this hexapeptideadopts a rigid, a-helical conformationin solution. This extremely short helical structure should serve as a useful a-helical peptidomimetic scaffold or a-helix initiator. Compound I was designed to include two Lys', side chain to side chain lactam bridges, chosen for their helix-stabilizing properties839 and arranged to constrain overlapping peptide structures in the shortest possible amino acid sequence. The synthesisof I followed oxime resin-based assembly and cyclizationcleavage methods reported earlier? but modified to allow a second cyclization in solution by using temporary fluorenylmethyl-based protecting groups for Lys1 and Asps. This strategy, along with the use of Nu-Boc and 0-phenacyl ester (OPac) protecting groups, allows peptide coupling to I in either direction, as well as future substitution of Ala3 and Ala4 by any other residues. Conformationalanalysis of peptide I by NMR was performed in TFE-dJH20 solutions, because the peptide showed limited solubility in water.10 However, circular dichroism spectra of peptide I in water at lower concentrations matched very closely those measured in TFE/H20 solution, indicating that solvent-
* To whom correspondence should be addressed (1) Zimm, B. H.; Bragg, J. K. J. Chem. Phys. 1959,31, 526-535. (2) Lifson, S.;Roig, A. J. Chem. Phys. 1961, 34, 1963-1974. (3) Scheraga. H. A. Pure Appl. Chem. 1978, 50, 315-324. (4) Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1989,86, 5286-5290. (5) (a) Kemp, D. S.;Boyd, J. G.; Muendel, C. C. Nature 1991,352,451454. (b) Chiang, L.; Cabezas, E.; Noar, B.; Arrhenius, T.; Lerner, R. A.; Satterthwaite, A. C. In Peptides 1990: Proceedings of the 21st European Peptide Symposium; Giraldt, E., Andreu, D., Eds.; ESCOM: Leiden, The Netherlands, 1991; pp465-467. (c) Jackson, D. Y.;King, D. S.;Chmielewski, J.; Singh, S.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 9391-9392. (d) Pease, J. H. B.;Storrs, R. W.; Wemmer, D. E. Proc.Natl. Acad. Sci. U.S.A. 1990,87, 5643-5647. (6) Rizo, J.; Gierasch, L. M. Annu. Rev. Biochem. 1992, 61, 387-418. (7) Burgess, A. W.; Leach, S.J. Biopolymers 1973, 12, 2599-2605. (8) Felix, A. M.; Heimer, E. P.; Wang, C.-T.; Lambros, T. J.; Fournier, A.; Mowles, T. F.; Maines, S.;Campbell, R. M.; Wegrzynski, B. B.; Toome, V.; Fry D.;Madison, V. S.Int. J. Pept. Protein Res. 1988, 32, 441454. (9) bsapay, G.; Taylor, J. W:J. Amer. Chem. Soc. 1992,114,6966-6973. (10) Spectra were collected in 40% (w/w) TFE-d,/HZO at 0 "C and in 50% (w/w) TFE-d3/HzO a t 0 "C and-10 "C;all NMR solutionswere buffered with 10 mM sodium acetate-d3 adjusted to pH 4.0.
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Received January 24, 1994
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6431
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Figure 1. N M R data for compound I. Coupling constants and NOES were measured at -10 "C in 50% (w/w) TFE-d3/H20, p H 4.0. The thickness of the lines indicates relative intensities of the N O E cross peaks. Amide exchange rates (k,) were measured by 1D NMR at p H 3.7 at 22 "Cin 50%(w/w) TFE-d,/DzO bymeasuringthedecayofpeakheight.
induced perturbationsof the peptide structure were slight." Amino acid assignments of the NMR spectra of peptide I were made using standard 1D and 2D lH NMR methods.12 Significant structural informationwas obtained from NOESY spectral data. A summary of the observed interresidueNO& is shown in Figure 1. A string of d"(i,i+l) NOEs are observed from Lys' to Asp6 in addition to NO& from Ha(i)-HN(i+3) and Ha(i)-H@(i+3). This pattern of NOEs is consistent with that generally seen in helical regions of proteins.l3 Side-chain cross-linkage through the two lactam bridges was also confirmed by NO& between the HNE(i) to Hj3(i+4) protons for Lysl to Asp5 and Lys2 to Asp6. No NOEs were observed from the Boc and OPac end groups to the amino acids. The conformation of peptide I, shown in Figure 2, was determined by distance-restrained annealing and restrained molecular dynamics"I8 using 85 NOE constraints measured at -10 OC. An average of fewer than one NOEviolation (>0.5A) per structure generated was observed, excluding the Lys side chains (discussed below). Vander Waalsoverlaps (>0.1 A) were alsorarely observed. All of the structures were helical throughout the peptide backbone (Figure 2), and the pattern of calculated hydrogen bonds corresponded predominantly to the two Lys-toAsp backbone hydrogen bonds expected for an a-helical conformation. H bonds between the Nu-Boccarbonyl and Ala3 (3!0 helix) or Ala4 (a-helix) were observed in only a few of the structures, and other H bonds were also observed only rarely. The average 4 angles in the models grow increasingly negative from the N terminus to the C terminus, starting at around -50° for Lysl and Lys2 and increasing to - 7 O O to - 8 O O for residues 3-6. These 4 angles agree well with the small experimentalvalues (1 1) CD spectra (180-250 nm) were compared using 0% and 50% (w/w) TFE/HzO buffered with lOmM sodiumphosphateat pH 6.0, at temperatures ranging from -10 "C to 70 "C. (12) Wuthrich,K.NMRofProteinrandNucleicAcids; Wiley: New York, 1986. (13) Wuthrich, K.; Billeter, M.; Werner, B. J. Mol. Biol. 1984,180,715740. (14) Randomized coordinates were used to generate 500 starting structures for distance-restrained annealing using DSPACE." The 10 lowest energy structures (backbone root mean squaredeviation0.88 A) wererefined further." ( 15) Hare Research Inc., Woodinville, WA, 1990. (16) Clore, G. M.; Gronenborn, A. M. Crit. Reo. Biochem. Mol. Biol. 1989, 24,479-564. (17) Each of the 10 distance restrained annealing models were refined using five separate runsof restrained molecular dynamics using DISCOVER.l8 Each run employed a different distance-dependent dielectric function. The backbone root mean square deviation for the 50 resultant structures was 0.44 A. The structures dis layed in Figure 2 resulted from molecular dynamics runs employing a 10-fcutoff, with the two highest energy structuresomitted. (18) Biosym Technologies Inc., 10065 Barnes Canyon Rd., San Diego, CA, 1991.
OOO2-7863/94/1516-6431$04.50/0 0 1994 American Chemical Society
Communications to the Editor
6432 J. Am. Chem. Sa.,Vol. 116, No. 14, 1994
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Figure 2. Structures from restrained molecular dynamics.Structures, excludingnon-amidehydrogens, were obtained by distance-restrainedannealing1' and molecular dynamicsrefinement.17Theyare shownascross-eyedstereoviews with superpositionof all atoms except those of the Boc and OPac groups.
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