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The correlation between β2-, β3-, and β2,3-amino acid-residue configuration and stability of helix and hairpin-turn secondary structures of peptides consisting of homologated proteinogenic amino acids is analyzed (Figs. 4;1–3). To test the power of Zn2+ ions in fortifying and/or enforcing secondary structures of β-peptides, a β-decapeptide, 1, four β-octapeptides, 2–5, and a β-hexadecapeptide, 10, have been devised and synthesized. The design was such that the peptides would a) fold to a 14-helix (1 and 3) or a hairpin turn (2 and 4), or form neither of these two secondary structures (i.e., 5), and b) carry the side chains of cysteine and histidine in positions, which will allow Zn2+ ions to use their extraordinary affinity for RS− and the imidazole N-atoms for stabilizing or destabilizing the intrinsic secondary structures of the peptides. The β-hexadecapeptide 10 was designed to a) fold to a turn, to which a 14-helical structure is attached through a β-dipeptide spacer, and b) contain two cysteine and two histidine side chains for Zn complexation, in order to possibly mimic a Zn-finger motif. While CD spectra (Figs. 4;6–8 and 17) and ESI mass spectra (Figs. 4;9 and 18) are compatible with the expected effects of Zn2+ ions in all cases, it was shown by detailed NMR analyses of three of the peptides, i.e., 2, 3, 5, in the absence and presence of ZnCl2, that i) β-peptide 2 forms a hairpin turn in H2O, even without Zn complexation to the terminal β3hHis and β3hCys side chains (Fig. 4;11), ii) β-peptide 3, which is present as a 14-helix in MeOH, is forced to a hairpin-turn structure by Zn complexation in H2O (Fig. 4;12), and iii) β-peptide 5 is poorly ordered in CD3OH (Fig. 4;13) and in H2O (Fig. 4;14), with far-remote β3hCys and β3hHis residues, and has a distorted turn structure in the presence of Zn2+ ions in H2O, with proximate terminal Cys and His side chains (Fig. 4;15).

The Ser, Cys, and His side chains play decisive roles in the syntheses, structures, and functions of proteins and enzymes. For our structural and biomedical investigations of β-peptides consisting of amino acids with proteinogenic side chains, we needed to have reliable preparative access to the title compounds. The two β3-homoamino acid derivatives were obtained by Arndt–Eistert methodology from Boc-His(Ts)-OH and Fmoc-Cys(PMB)-OH (Schemes 4;2–4), with the side-chain functional groups' reactivities requiring special precautions. The β2-homoamino acids were prepared with the help of the chiral oxazolidinone auxiliary DIOZ by diastereoselective aldol additions of suitable Ti-enolates to formaldehyde (generated in situ from trioxane) and subsequent functional-group manipulations. These include OH→OtBu etherification (for β2hSer; Schemes 4;5 and 6), OH→STrt replacement (for β2hCys; Scheme 4;7), and CH2OH→CH2N3→CH2NH2 transformations (for β2hHis; Schemes 4;9–11). Including protection/deprotection/re-protection reactions, it takes up to ten steps to obtain the enantiomerically pure target compounds from commercial precursors. Unsuccessful approaches, pitfalls, and optimization procedures are also discussed. The final products and the intermediate compounds are fully characterized by retention times (tR), melting points, optical rotations, HPLC on chiral columns, IR, 1H- and 13C-NMR spectroscopy, mass spectrometry, elemental analyses, and (in some cases) by X-ray crystal-structure analysis.

Although they are less abundant than their α-analogues, β-amino acids occur in nature both in free form and bound to peptides. Oligomers composed exclusively of β-amino acids (so-called β-peptides) might be the most thoroughly investigated peptidomimetics. Beside the facts that they are stable to metabolism, exhibit slow microbial degradation, and are inherently stable to proteases and peptidases, they fold into well-ordered secondary structures consisting of helices, turns, and sheets. In this respect, the most intriguing effects have been observed when β²-amino acids are present in the β-peptide backbone. This review gives an overview of the occurrence and importance of β²-amino acids in nature, placing emphasis on the metabolic pathways of β-aminoisobutyric acid (β-Aib) and the appearance of β²-amino acids as secondary metabolites or as components of more complex natural products, such as peptides, depsipeptides, lactones, and alkaloids. In addition, a compilation of the syntheses of both achiral and chiral β²-amino acids is presented. While there are numerous routes to achiral β²-amino acids, their EPC synthesis is currently the subject of many investigations. These include the diastereoselective alkylation and Mannich-type reactions of cyclic- or acyclic β-homoglycine derivatives containing chiral auxiliaries, the Curtius degradation, the employment of transition-metal catalyzed reactions such as enantioselective hydrogenations, reductions, C–H insertions, and Michael-type additions, and the resolution of rac. β²-amino acids, as well as several miscellaneous methods. In the last part of the review, the importance of β²-amino acids in the formation of β-peptide secondary structures is discussed. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 2004

β-Peptides offer the unique possibility to incorporate additional heteroatoms into the peptidic backbone (Figs. 1 and 2). We report here the synthesis and spectroscopic investigations of β²-peptide analogs consisting of (S)-3-aza-β-amino acids carrying the side chains of Val, Ala, and Leu. The hydrazino carboxylic acids were prepared by a known method: Boc amidation of the corresponding N-benzyl-L-α-amino acids with an oxaziridine (Scheme 1). Couplings and fragment coupling of the 3-benzylaza-β²-amino acids and a corresponding tripeptide (N-Boc/C-OMe strategy) with common peptide-coupling reagents in solution led to β²-di, β²-tri-, and β²-hexaazapeptide derivatives, which could be N-debenzylated (4–9; Schemes 2–4). The new compounds were identified by optical rotation, and IR, ¹H- and ¹³C-NMR, and CD spectroscopy (Figs. 4 and 5) and high-resolution mass spectrometry, and, in one case, by X-ray crystallography (Fig. 3). In spite of extensive measurements under various conditions (temperatures, solvents), it was not possible to determine the secondary structure of the β²-azapeptides by NMR spectroscopy (overlapping and broad signals, fast exchange between the two types of NH protons!). The CD spectra of the N-Boc and C-OMe terminally protected hexapeptide analog 9 in MeOH and in H2O (at different pH) might arise from a (P)-314-helical structure. The N-Boc-β²-tri and N-Boc-β²-hexaazapeptide esters, 7 and 9, were shown to be stable for 48 h against the following peptidases: pronase, proteinase K, chymotrypsin, trypsin, carboxypeptidase A, and 20S proteasome.

Two new β³-homohistidine- and β³-homocysteine-containing β-peptides have been prepared by solid-phase synthesis. A β-octapeptide (2) contains seven β³-amino acids and one β²-amino acid. The β²/β³ segment has been placed in the middle of this peptide, which contains β³-amino acids of alternating configuration, to induce the formation of a hairpin secondary structure. A β-decapeptide (3) has been designed to fold to a 314-helical secondary structure with neighboring His side chains in 6- and 9-positions. Circular-dichroism (CD) measurements show the capability of both peptides to bind Zn²⁺ ions in aqueous solution. In the case of the β-octapeptide, binding of Zn²⁺ causes a dramatic change of the CD spectrum, indicating a change or a stabilization of its secondary structure. Zn²⁺ Ions clearly stabilize the 314-helix of the β-decapeptide, in neutral and basic solution. For the construction of the two new β-peptides, we needed to have a supply of the β-amino acid derivatives Fmoc-β³hCys(Trt)-OH and Fmoc-β³hHis(Trt)-OH, the preparation of which is described herein.

Multigram amounts of suitably protected β²-amino acids with 17 of the 20 proteinogenic side chains are prepared by diastereoselective reactions of Li, B, or Ti enolates of the corresponding 3-acyl-4-isopropyl-5,5-diphenyloxazolidin-2-ones (acyl-DIOZ; 1) with appropriate electrophiles (amidomethylation, hydroxyalkylation, (benzyloxycarbonyl)methylation) in yields of 55–90% and with diastereoselectivities of 80 to >97% (Scheme). The primary products 2–8 thus obtained are converted to protected β²-amino acids by standard procedures (Table 1). Many of the DIOZ derivatives are highly crystalline compounds (31 X-ray crystal structures in Table 2). The chiral auxiliary DIOZ, readily prepared in either enantiomeric form, is recovered with high yield.

The structural properties of four mixed β-peptides with alternating β²/β³- or β³/β²-sequences have been analyzed by two-dimensional homonuclear ¹H-NMR- and CD spectroscopic measurements. All four β-peptides fold into (P)-helices with twelve- and ten-membered H-bonded rings (Figs. 3–6). CD Spectra (Fig. 2) of the mixed β³/β²-hexapeptide 4a and β³/β²-nonapeptide 5a, indicating that peptides of this type also adopt the 12/10-helical conformation, were confirmed by NMR structural analysis. For the deprotected β³/β²-nonapeptide 5d, NOEs not consistent with the 10/12 helix have been observed, showing that the stability of the helix decreases upon N-terminal deprotection. From the NMR structures obtained, an idealized helical-wheel representation was generated (Fig. 7), which will be used for the design of further 12/10 or 10/12 helices.