Fig. 5.6 Molecular conformations observed in the crystals of peptides: (a) Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-DAla-DLeu-Leu-Val-Phe-Val-Aib-DVal-Leu-Phe-Val-Val-OMe [24]; (b) Zervamicin analog [18b]; (c) Antiamoebin [18d].

the group of Hanessian [26], is based on an early observation that the diproline-containing sequences can form incipient 310-helical structures in organic solvents, where consecutive type III b-turn formation is driven by successive hydrogen bond formation [27]. Figure 5.7a shows the consecutive type III b-turn structure proposed for the model peptide Pivaloyl-Pro-Pro-LAla-NHMe. Here, the Pro2 residue occupies the i + 2 position of the first b-turn and i + 1 position of the second b-turn. This motif, stabilized by two successive C10 hydrogen bonds, constitutes a single turn of a 310 helical structure in which the torsion angles of all three residue lies in the aR (-60°, -30°) [27]. Bridging the Cg atom of Pro(1) and the Cd atom of Pro(2) by a thiomethylene group constrains diproline conformations to local helical structures at both proline residues, thus providing a rigid template for helix nucleation in attached peptide segments. Extensions of this approach to

Fig. 5.7 Templates, used to nucleate helical structures (a) NMR model of Piv-LPro-LPro-LAla NHMe [27]; (b) Structure of Kemp's template (2S,5S,8S,-17S)-1-acetyl-1,4-diaza-3-keto-5-carboxy-10-thiatricyclo []-tridecane(Ac-Hel1-OH) in crystals [25b]; (c) Ac-L-TcaP-L-Pro-OH (TcaP = tricyclic constrained proline)[26a]; (d) (3S,6S,8S,9S)-6-acetylamino-8-methoxy-6-methyl-5-oxooctahydroindolizine-3-carboxylic acid (LBcaP) [26b]; (e) Crystal structure of the alamethicin segment Ac-Aib-Pro-Aib-Ala [61]; (f) Crystal structure of L-BcaP-L Ala-L Ala-OtBu [26a, 62].

other constrained proline derived structures have been reported [28]. Figure 5.7 summarizes the structures of parent Pro-Pro sequences and related structures.

Application of the diproline mimetic organic templates to synthetic protein design is limited by the complexity of the synthetic protocols used in preparing them. A more readily accessible approach would be to examine the use of unconstrained diproline segments in generating helical structures. A recent analysis focuses on the model hexapeptide Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe [29]. Solu-

tion NMR studies demonstrate a significant population of helical conformations encompassing the entire length of the peptide, including the N-terminus dipro-line segment. However, populations of the cis Pro-Pro conformer and an apparently unfolded structure, with Prol adopting the Pn conformation are also described by NMR in solution. In single crystals, a helical fold is observed over the segment, residues 2-5, while Prol adopts a PII conformation. This study illustrates the conformational heterogeneity that may be anticipated for diproline segments. The observation of aR, aR conformations at Prol and Pro2 is encouraging, suggesting that an attempt to bias conformational choices by using local sequence effects, may be worthwhile. An analysis of X-Pro-Pro segments in 1741 protein structures reveals about 25 examples of diproline segments occurring in the aR, aR conformations at the N terminus of a helix [29]. The predominant conformation, that is favored for a Pro-Pro unit, is the PII-PII structure, with 256 examples being found in the data set. Directing an unconstrained diproline segment into a helical fold will require a detailed understanding of near neighbor effects on conformational choice.

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