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. CuCN (0.030 g) was added to a dry three-neck flask fitted with a reflux condenser under argon. Then 25 ml of 1 M t-butyl MgCl in THF was added via syringe, followed by the slow addition (drop-wise; 12 min) of trimethyl chlorosilane (3.10 ml). Reaction was refluxed for 2 h. After cooling to room temperature, 5 ml of water was added carefully to the reaction, and the resulting mixture was filtered through a Buchner funnel. The resulting filtrate was dried with sodium sulfate, and the solvent was removed in vacuo. The resulting oil was chromatographed on silica gel (1:1 hexanes:EtOAc), giving 3.37 g of a light yellow oil (16.7 mmol; 67% yield). ESI-MS: calculated, 202.18; actual, 202.2.

Product 1 was dissolved in methanolic citric acid (1 g of citric acid in 20 ml of methanol) and allowed to stir for 30 min at room temperature. The solvent was removed in vacuo, and the resulting oil was taken up into EtOAc and washed with water (2 ´ 25 ml). The organic layer then was dried with sodium sulfate, and the solvent was removed in vacuo. The resulting oil then was taken up in 25 ml of 48% HBr and allowed to reflux for 3 h. The resulting solution was cooled to room temperature and extracted with 1:1 CH₂Cl₂:Et₂O (2 ´ 25 ml). The organic layer was combined and washed with NaHCO₃ (2 ´25 ml) and brine (1 ´ 25 ml) and dried with sodium sulfate, and the solvent was removed in vacuo. The resulting oil then was purified via silica gel flash chromatography (4:1 EtOAc:hexanes), giving 2.98 g of a slightly yellow oil (15.4 mmol, 92% yield). APCI-MS: calculated, 192.1; actual, 192.2. NMR (400 MHz, CDCl₃): 3.565 (t, 2H), 1.974 (m, 2H), 1.337 (m, 2H), 1.024 (s, 9H).

A 250-ml round-bottom flask was charged with (R,R)-(-)pseudoephedrine (10 g; 60.5 mmol) and glycine methyl ester (9.88 g; 78.7 mmol) in 50 ml of THF, followed by 15 min of stirring at room temperature under argon. Lithium tert-butoxide powder (6.78 g; 84.7 mmol) was added in one portion. The reaction then was stirred for 2 h, followed by the addition of water (75 ml), and the resulting mixture was concentrated in vacuo. The resulting oil was taken up in hot THF (50 ml), and water (2 ml) was added to the hot solution. Crystallization occurred upon cooling to room temperature. The solution was allowed to sit at -20°C overnight. Crystals were collected by filtration and dried after washing with diethyl ether. After leaving under vacuum overnight, the white crystalline powder weighed 9.66 g (40.4 mmol; 67% yield). ESI-MS: calculated, 239; actual, 239.1. Spectroscopic data matched published data. NMR (400 MHz,CDCl3): 7.27-7.38 (m, 5H), 4.50-4.60 (m, 1.5H), 3.81 (m, 0.5H), 3.681 (d, 0.5H), 3.388 (d, 1H), 3.313 (d, 0.5H), 2.934 (s, 1.5H), 2.774 (s, 1.5H), 2.420 (s(br), 3H), 1.029 (d, 1.5H), 0.951 (d, 1.5H).

An oven-dried three-neck round-bottom flask was charged with dry LiCl (0.606 g; 14.4 mmol) and allowed to cool to room temperature under vacuum. The flask then was flushed with argon, and THF was added (20 ml). The resulting suspension was allowed to stir for 20 min at room temperature. Solid PSE glycinamide 3 (0.862 g; 3.6 mmol) then was added in one portion, and the solution was immersed in an isopropanol bath maintained at 0°C with a Neslab Cryotrol cold finger and allowed to stir for 30 min. Next, LiHMDS was added via dry syringe (drop-wise; 14 ml of a 1 M solution in THF), followed by 20 min of stirring. Finally, product 2 (0.802 g; 4.2 mmol) was added slowly over 10 min via syringe to the enolate solution. The reaction mixture then was allowed to stir for 23 h at 0°C. The reaction was quenched with the addition of 10 ml of H₂O and acidified to near pH 0 with 6 M HCl. The resulting acidic solution was extracted with ethyl acetate (3 ´ 50 ml), dried with sodium sulfate, and rotovapped to dryness. The resulting oil then was purified via silica gel flash chromatography via gradient elution (column-washed with 100 ml of CH₂Cl₂, followed by washes of 20:1 CH₂Cl₂:MeOH (400 ml), 10:1 CH₂Cl₂:MeOH (220 ml), and 5:1 CH₂Cl₂:MeOH (200 ml). (Yield: 0.770 g; 2.3 mmol; 64% yield). NMR (600 MHz, CDCl3): ESI-MS: calculated, 334.26; actual, 334.3.

Product 5 was taken up in 4.6 ml of 1 M NaOH and 20 ml of H₂O. The resulting mixture was refluxed for 5.5 h, allowed to cool to room temperature, and filtered through a Buchner funnel. The filtered solution was washed with ethyl acetate (1 ´ 25 ml), frozen, and lyophilized to dryness. The resulting oil was dissolved in 25 ml of H₂O with stirring. Then 0.387 g of sodium bicarbonate was added, followed by 25 ml of dioxane. The resulting solution was cooled in an ice bath for 30 min. Fmoc-OSu (0.935 g; 2.8 mmol) was dissolved in 10 ml of dioxane and added to the chilled solution drop-wise via pipette. The reaction was stirred for 1 h at ice-cold temperature and 3 h at room temperature. The reaction was followed by TLC (10:1 CH₂Cl₂:MeOH). Water then was added (30 ml), and the resulting solution was extracted with 75 ml of 1:1 EtOAc/ether. The organic layer was separated and back-extracted with 2% sodium bicarbonate (2 ´ 25 ml). The aqueous layers were combined and acidified to pH 1, followed by extraction with EtOAc (2 ´ 50 ml). The organic layer was dried with sodium sulfate, and the solvent was removed in vacuo, giving product 6 as a clear oil (0.454 g; 48% overall yield for two steps). NMR (600 MHz, CDCl3): 7.7465 (d, 2H), 7.582 (d, 2H), 7.383 (t, 2H), 7.297 (t, 2H), 5.2355 (d, 1H), 4.397 (m, 2H), 4.215 (t, 1H), 1.898 (m, 1H), 1.708 (m, 1H), 1.4-1.5 (m, 6H), 0.842 (s, 9H). ESI-MS: calculated, 409.23; actual, 409.3.

The hairpin WtbutylNle exhibits aggregation behavior in the 1 mM and above concentration range. To determine the most suitable concentrations for NMR analysis, a concentration study was performed by CD (10 mM sodium phosphate buffer, pH 6.4, 298 K). The peptide was found to be monomeric near and below »0.5 mM concentration. As a result, the NMR studies of the WtbutylNle hairpin were carried out at concentrations of 0.5 mM or less. The thermal denaturation, for example, was executed at 0.316 mM.

Methods used to indicate the formation of b-hairpin structure include the analysis of Ha shifting relative to random coil, backbone amide shifts relative to random coil, and the identification of cross-strand NOEs. The b-hairpin should have backbone hydrogen bonded amides between cross-strand residue pairs Arg-Gln, Val-Ile, and Val-Orn in all hairpin peptides. The presence of these hydrogen bonds readily is demonstrated by downfield shifting of the amide hydrogens in these positions relative to random coil. As seen below, both peptides exhibit significant downfield shifting at key positions along the strand. As expected for b-hairpins, the termini are frayed and show little or no amide shifting. The Asn amide shows significant downfield shifting as expected for a type I' turn.

Peptides were analyzed assuming a two-state system. The equilibrium constant was determined from the fraction folded (f) by K = f/(1 - f). The free energy then was calculated from DG° = -RT lnK. To determine the thermodynamic parameters, DH°, DS°, and DCp°, the temperature dependence of the Gly chemical shift difference was fit to the following equation (3):

Fraction folded = [exp(x/RT)]/[1 + exp(x/RT)],

where x = T(DS°₂₉₈ + a ln(T/298) + b (T - 298) - (c/2) (1/T² - 1/298²)) - (DH°₂₉₈ + a (T - 298) + (b/2) (T² - 298²) - c (1/T - 1/298)).

1. Shirahata A (1989) Tetrahedon Lett 30:6393-6394.

2. Rybczynski PJ, Zeck RE, Dudash J, Jr, Combs DW, Burris TP, Yang M, Osborne MC, Chen X, Demarest KT (2004) J Med Chem 47:196-209.

3. Maynard AJ, Sharman GJ, Searle MS (1998) J Am Chem Soc 120:1996-2007.