CHEMICAL/BIOCHEMICAL RESEARCH to reveal general structural characteristics in biologi- Innovative Foldamers: Engineering cal molecules. Our initial studies began in methanol, Heterochiral Peptides an alcohol solvent often employed as a surrogate for water. In methanol, the CD spectrum is character- T.D. Clark and J.L. Kulp III istic of a β-helical structure. As we added increasing Chemistry Division amounts of water, the peptide partially unfolded into a bracelet-like structure. Innovative Foldamers: The field of foldamer We further probed the peptide’s aggregation state design promises new routes to important compounds and thermal stability by CD. The CD spectra of peptide for use in sensors, smart materials, and catalysts. The 1 in methanol and water are independent of concentra- term “foldamer” refers to a molecule that folds into tion, suggesting the peptide is monomeric under the a structurally stable state in solution.1 Proteins and conditions studied. The CD signals in methanol and peptides are an important class of natural foldamers water do not change appreciably from 5 to 65 °C, indi- that carry out a host of essential functions in biology, cating both structures are surprisingly thermostable; including molecular recognition, information storage, for comparison, most proteins unfold at 45 °C. catalysis, and controlled crystallization of inorganic Another technique that allows several different materials. The desire to mimic such functions with means for establishing molecular structure is nuclear synthetic molecules inspires the field of foldamer magnetic resonance (NMR). NMR perturbs and moni- design. tors the nuclear spins of atoms, which are sensitive Of the foldamers under development, β helices — to local chemical environments, thus making NMR a peptide helices containing amino acids with alternat- powerful technique for investigating the three-dimen- ing chirality — represent an intriguing and relatively sional structure of molecules. Computer modeling unexplored subclass of peptide-based foldamers. Very using NMR data with simulated annealing molecular few β-helical peptides exist in nature, and all of these dynamics (SAMD) generates structures consistent compounds adopt their active β-helical structures in with the experimental measurements — effectively hydrophobic membrane environments. However, for allowing us to determine the coordinates of the atoms many potential biomimetic or bioinspired applications, in the molecule. Using NMR and SAMD, we were water or other polar solvents will likely be the medium able to generate images of the molecular structures of of choice. In our research, therefore, we pose the ques- peptide 1 in methanol (Fig. 5(b)) and water (Fig. 5(c)). tion: Can engineered β helices discretely fold in polar In water, the molecule consists of three subgroups media such as methanol, and ultimately water? all having topography with antiparallel strands and two turn regions; in methanol, the peptide forms the Peptide Engineering: In designing β helices, intended structure — a well-defined β helix (Fig. 5(b)). we must overcome several challenges, including the tendency of β helices to adopt multiple structures in Second-Generation Peptide: Having achieved solution (structural polymorphism), and the tendency a β-helical structure in methanol, we next sought to of water to disrupt the hydrogen bonds that stabilize design a new peptide that would adopt a similar struc- folded peptide structures. Structural polymorphism ture in water. For this second-generation peptide, we and aggregation are undesirable for functional foldam- increased the length of the peptide, which we expected ers as these physical phenomena effectively decrease the would increase the number of helical hydrogen bonds, concentration of the desired structure, leading to lower thus increasing the overall stability of the helical activity. To limit structural polymorphism, we devel- fold. Furthermore, we designed peptide 2 (Fig. 6) to oped a method to trap a singular β-helical structure by be stabilized by six electrostatic interactions in the linking the two strands and forming a cyclic peptide expected helical structure. The CD spectrum of peptide (Fig. 4).2 The cyclic construction also helps to stabilize 2 in buffered water confirmed the formation of a β the folded structure against the disrupting effect of helix and validated our approach to the problem. This water. To further stabilize the peptide in polar media, demonstration of a β helix in water furthers our goal of we designed hydrophilic sites and two stabilizing elec- developing β helices as a versatile class of foldamers. trostatic interactions into the structure (peptide 1, Fig. 5(a)). After synthesis and purification, we found that Conclusions and Outlook: We have presented the resulting peptide folded stably in the highly polar the design and biophysical characterization of the first solvent methanol — an unprecedented achievement in β-helical peptide foldamers that adopt stable structures the field of β-helical peptide foldamers.3 in polar solvents. By joining the strands with turns, we trapped the peptides into a discrete structural state — a Structural Characterization: Circular dichroism prerequisite for a fully functional foldamer. Our studies (CD) uses differential adsorption of polarized light prove the β helices can fold stably in polar solvents; 2009 NRL REVIEW 149 CHEMICAL/BIOCHEMICAL RESEARCH the fundamental principles established in this work will enable the future design of predictable β-helical structures. These innovative heterochiral peptides promise numerous potential applications as sensors, smart materials, and catalysts, thus enhancing NRL’s multidisciplinary technology platform. [Sponsored by ONR and AFOSR] References 1 S. Hecht and I. Huc, eds., Foldamers: Structure, Properties, and Applications (Wiley-VCH, 2007). 2 M. Sastry, C. Brown, G. Wagner, and T.D. Clark, “Cyclic Peptide Helices: A Hybrid β-Hairpin, β-Helical Supersecondary Structure,” J. Am. Chem. Soc. 128, 10650–10651 (2006). 3 J.L. Kulp III and T.D. Clark, “Engineering a β-helical D,L Peptide for Folding in Polar Media,” Chem. Eur. J., 2009, 15(44), 11867-11877. FIGURE 4 Peptide backbone structure of a β-helical supersecondary structure developed, synthesized, and characterized at NRL. 150 2009 NRL REVIEW CHEMICAL/BIOCHEMICAL RESEARCH 22 G L - P 21 1 D - L D - A 20 2 L - E L - T 19 3 D - V D - L 18 4 L - R L - T 17 5 D - L D - V 16 6 L - T L - K 15 7 D - A D - L 14 8 L - T L - E 13 9 D - V D - A 12 10 L - P G 11 1 (a) (b) (c) FIGURE 5 (a) Sequence and numbering scheme for peptide 1 using one-letter codes for amino acid residues; D,L-convention for denoting chirality describes amino acids that are non-superimposable on its mirror image — human hands being the most common example of chiral- ity; D-amino acids and glycine are boxed in black, L-amino acids are boxed in red. (b) and (c) Nuclear magnetic resonance derived structures, computed by simulated annealing molecular dynamics, of peptide 1 in methanol (b) and buffered water (c). FIGURE 6 (a) Sequence and numbering scheme for second-generation peptide 2 using three-letter codes for amino acid residues; D-amino acids and glycine are boxed in black, L-amino acids are boxed in red. (b) Energy minimized model based on CD data. 2 (a) (b) 2009 NRL REVIEW 151