This paper introduces a unique amino acid that can readily be incorporated into peptides to make them fold into beta-sheetlike structures that dimerize through beta-sheet interactions. This new amino acid, Orn(i-PrCO-Hao), consists of an ornithine residue with the beta-strand-mimicking amino acid Hao [J. Am. Chem. Soc. 2000, 122, 7654-7661] attached to its side chain. When Orn(i-PrCO-Hao) is incorporated into a peptide, or appended to its N-terminus, the Hao group hydrogen bonds to the three subsequent residues to form a beta-sheetlike structure. The amino acid Orn(i-PrCO-Hao) is readily used in peptide synthesis as its Fmoc derivative, Fmoc-Orn(i-PrCO-Hao)-OH (3). Fmoc-Orn(i-PrCO-Hao)-OH behaves like a regular amino acid in peptide synthesis and was uneventfully incorporated into the peptide o-anisoyl-Val-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe (4) through standard automated Fmoc solid-phase peptide synthesis, with DIC and HOAt as the coupling agent for Fmoc-Orn(i-PrCO-Hao)-OH and o-anisic acid and HATU as the coupling agent for all other couplings. A second synthetic strategy was developed to facilitate the preparation of peptides with N-terminal Orn(i-PrCO-Hao) residues, which avoids the need for the preparation of Fmoc-Orn(i-PrCO-Hao)-OH. In this strategy, Boc-Orn(Fmoc)-OH is used as the penultimate amino acid in the peptide synthesis, and i-PrCO-Hao-OH (2) is used as the final amino acid. N-Terminal Orn(i-PrCO-Hao) peptide H-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe.TFA (5) was prepared in a fashion similar to that for 4, using DIC and HOAt as the coupling agent for i-PrCO-Hao-OH and HATU as the coupling agent for all other couplings. 1H NMR transverse-ROESY, coupling constant, and chemical shift studies establish that peptide 4 forms a dimeric beta-sheetlike structure in CDCl3 solution. The 1H NMR studies also suggest that the ornithine unit adopts a well-defined turn conformation. Analogous 1H NMR studies of peptide 5 indicate that this TFA salt folds but does not dimerize in CD3OD solution. Collectively, these synthetic and spectroscopic studies establish that the amino acid Orn(i-PrCO-Hao) induces beta-sheet structure and interactions in peptides in suitable organic solvents. Unlike the Hao amino acid, which acts as a prosthetic to replace three residues of the peptide strand, the Orn(i-PrCO-Hao) amino acid acts as a splint that helps enforce a beta-sheetlike structure without replacing the residues and their side chains. This feature of Orn(i-PrCO-Hao) is important, because it allows the creation of beta-sheet structure with minimal perturbation of the peptide sequence.

This paper introduces polar and hydrophobic variants of the unnatural amino acid Hao, which mimics the hydrogen-bonding functionality of one edge of a β-strand. In these variants, the methyl side chain of Hao is replaced with acidic, basic, and hydrophobic groups. These modifications can impart improved solubility and additional side-chain interactions to peptides containing Hao.Peptidomimetic templates that mimic or induce helix, turn, or β-sheet structures are useful for studying and controlling the conformations and interactions of peptides and proteins. 1 Our research group previously introduced the unnatural amino acid Hao as a tripeptide β-strand mimic that forms hydrogen bonds from only one edge (Figure 1). 2 We have developed Haocontaining peptides that fold to form β-sheet structure, dimerize through edge-to-edge β-sheet interaction, and antagonize β-sheet aggregation. 3 Other research groups have investigated Hao and related structures in peptidomimetic compounds and hydrogen-bonded assemblies. 4,5The original unnatural amino acid Hao provides the hydrogen-bonding functionality of the peptide main chain but lacks side-chain functionality. In this paper, we introduce variants of Hao with acidic, basic, and hydrophobic side chains: Hao K , Hao D , Hao F , and Hao L (Figure 1). We have developed these variants to address specific problems with solubility and folding of Hao-containing peptides that we have encountered in our own research, and we anticipate that these variants and types of side chains will be useful to others. 6,7 jsnowick@uci.edu. (Figure 2). 8 NIH Public AccessThe syntheses of Hao analogues 1a-1d are similar to the synthesis of Fmoc*-Hao-OH that we reported previously but require an alkylation reaction to introduce the different side chains and tactical changes to tolerate the functional groups and protecting groups of the side chains. The syntheses of 1a-1d begin with ethyl or allyl 5-nitrosalicylate and involve alkylation of the phenol group to introduce the side chains, conversion of the ester group to the hydrazide, and conversion of the nitro group to the oxamic acid. 9Alkylation of ethyl 5-nitrosalicylate with Boc-protected 3-amino-1-bromopropane, 10 t-butyl bromoacetate, benzyl bromide, or isopentyl bromide gives ethers 2a-2d (Scheme 1). Alkylation to form 2a and 2c proceed smoothly at 70-100 °C. For 2b, the temperature must be kept below 50 °C to minimize undesired reactions. For 2d, sodium iodide is added to increase the rate of alkylation. Saponification of the ethyl ester groups of 2a-2d is sluggish at room temperature but occurs in 2-5 h upon heating at reflux in aqueous THF (Scheme 1). Carboxylic acids 3a, 3c, and 3d are readily isolated by neutralization with strongly acidic ion exchange resin (Amberlite IR-120) and removal of THF. Competing reactions during the hydrolysis of 2b are a problem. To circumvent this problem, we selected the orthogonal allyl protecting group and have used allyl 5-nitrosalicylate to prepare acid 3b. Alkylation of allyl 5-nitrosalicylate w...

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