Synthesis of Nonproteinogenic Amino Acids to Probe Lantibiotic Biosynthesis

Abstract

The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid phase peptide synthesis is described. These amino acids are (2S,3R)-vinylthreonine, (2S)-(E)-2-amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-β²-homoserine 3, (S) and (R)-β³-homocysteine, and (2R,3R)-methylcysteine. Once incorporated into peptides, these compounds were envisioned to serve as alternative substrates for the lantibiotic synthases that dehydrate serine and threonine residues in their peptide substrates and catalyze the subsequent intramolecular Michael-type addition of cysteines to the dehydroamino acids.

Introduction

Nonproteinogenic amino acids with highly functionalized side chains are frequently found as constituents of biologically important peptides. As a result, there has been significant interest in the synthesis, biosynthesis, and biological activities of these compounds.¹ In addition, β-amino acids have gained recent attention both for their natural occurrence and as building blocks for oligomers with well-defined folding behavior.² The so-called lantibiotics are a group of ribosomally synthesized and post-translationally modified peptide antibiotics.³ These modifications involve dehydrations of Ser and Thr residues followed by intramolecular Michael-type additions of Cys residues to the dehydroamino acids in a regio- and stereoselective fashion (Scheme 1). The enzymatic system that carries out these modifications during the biosynthesis of lacticin 481 has been recently reconstituted in vitro.⁴ The enzyme was shown to display a high degree of substrate promiscuity opening the possibility of introducing nonproteinogenic amino acids into its peptide substrate. Since the ribosomal origin of the lantibiotics limits in vivo engineering of their structures to the 20–22 proteinogenic amino acids, a synthetic approach would greatly expand the functional and structural diversity that can be incorporated into lantibiotics via the biosynthetic enzymes. We report here the preparation of a series of nonproteinogenic amino acids designed to investigate this premise. These compounds are the vinylthreonine derivative 1, fluoroallylglycine 2, the β-amino acids 3–5, and cysteine derivative 6 (Chart 1). All compounds were appropriately protected for use in Fmoc-based solid phase peptide synthesis (SPPS).

Scheme 1.

Chart 1.

Once incorporated into the peptide substrate for the dehydratase, amino acids 1–3 were envisioned as possible Ser or Thr analogs that would serve to further investigate the substrate specificity of the dehydration reaction (Scheme 2). Similarly, the β³-homocysteines 4 and 5 and 3-methylcysteine 6 were anticipated as potential alternative nucleophiles for the cyclase catalyzed Michael-type addition. In the case of 6 this would provide (2R,3R,6S)-3-methyllanthionine instead of (2S,3S,6R)-3-methyllanthionine that has been found in all lantibiotics characterized to date.³

Scheme 2.

Results and Discussion

Synthesis of vinylthreonine 1, (E)-2-amino-5-fluoro-pent-3-enoic acid 2, and β²-homoserine 3

A number of elegant approaches have been described for the asymmetric synthesis of various β-hydroxy α-amino acids, but the previous methods for the asymmetric synthesis of derivatives of vinylthreonine produced moderate yields and/or stereoselectivity and utilized protecting groups that are not directly amenable for SPPS.⁵ Hence, we explored alternative routes towards the synthesis of 1. Protected amino acids 1 and 2 were both accessed from the D-Garner aldehyde⁶ 7. Vinyllithium was combined with anhydrous zinc bromide in diethyl ether followed by addition of aldehyde 7 to produce alcohol 8 as a solid with 5:1 syn-diastereoselectivity in 90% yield (Scheme 3).^7c Recrystallization of the crude reaction mixture produced syn-8 in 24:1 d.r. The recrystallized allylic alcohol was converted to the corresponding tert-butyl ether 9 in 78% yield by treatment with tert-butyl trichloroacetimidate and a catalytic amount of boron trifluoride etherate in cyclohexane at room temperature. Initial attempts to deprotect the isopropylidene and Boc groups of compound 9 with 10% HCl, followed by protection of the resulting free amino group with FmocCl afforded compound 13 in low yield (32%). By-product 10 resulting from cleavage of the tert-butyl ether was also formed in the deprotection step. A survey of a series of reaction conditions, showed that treatment with 1 N HCl-dioxane at room temperature for 24 h maximized the desired product although significant amounts of 10 were still formed. The crude mixture of compounds 10 and 11 was treated with FmocCl and NaHCO₃ to provide compound 13 in 45% yield (2 steps), along with 29% (2 steps) of compound 12. Compounds 12 and 13 were separated by silica gel chromatography. To increase the overall yield, compound 12 was transformed into 13 in three steps (Scheme 3). Subsequent oxidization of alcohol 13 with Jones reagent afforded the protected target amino acid 1 in 76% yield.

Scheme 3.

For the preparation of fluorinated amino acid 2, the (E)-α,β-unsaturated aldehyde 15 was prepared by Wittig reaction of aldehyde 7 with (triphenyl-phosphoranylidene)acetaldehyde in 86% yield (Scheme 4).⁸ DIBAL-H reduction of aldehyde 15 provided allylic alcohol 16 in 69% yield. With this key intermediate in hand we turned our attention to the introduction of fluorine. A wide variety of fluorinating reagents are available.⁹ We opted for the use of diethylaminosulfur trifluoride (DAST), as it is a highly effective reagent for the direct, one-step, and high yielding conversion of alcohols into fluorides under mild conditions. Treatment of allylic alcohol 16 with DAST afforded fluorinated compound 17 in 56% yield. The moderate yield is probably due to competitive S_N2′ reaction¹⁰ although this product was not isolated. Deprotection of the isopropylidene and Boc groups of compound 17 with 4 N HCl-dioxane, followed by protection of the free amino group with FmocCl and NaHCO₃ afforded compound 18 in 80% yield (2 steps). Oxidization of 18 with Jones reagent afforded the target amino acid 2 in low yield 32%. Attempts to improve the yield using either PDC or the Dess Martin reagent failed to give the desired target compound 2. Under the latter conditions, the conjugated product 19 was formed instead of protected amino acid 2.

Scheme 4.

For the preparation of Fmoc-β²-homoserine(But)-OH 3, the common approach of conjugate addition of nitrogen nucleophiles to α,β-unsaturated carboxylic acid derivatives is not practical. Several years ago, Seebach and coworkers developed an asymmetric synthesis method to access β²-homoserine derivatives.¹¹ In this method, a modified Evans auxiliary was used and excellent stereoselectivity was obtained. We applied this methodology to prepare the β²-homoserine derivative 20. The hydroxyl group of compound 20 was protected as the tert-butyl ether typically used for Fmoc-based solid phase peptide synthesis in the presence of isobutylene and catalytic H₂SO₄ in CH₂Cl₂. Removal of the chiral auxiliary from compound 21 with LiOH and H₂O₂ proceeded smoothly, but as previously observed,¹² the phthaloyl group is not stable under the reaction conditions and ring opening took place to give compound 22 in 95% yield. Rather than opting for other deprotection conditions that leave the phtaloyl group intact but that lead to an erosion in e.r.,^11c we chose to reinstall the phtaloyl group with CDI in THF¹² to afford compound 23 in 95% yield. Subsequent hydrazinolysis of the phthaloyl group proceeded smoothly, and the product mixture was carried on without further purification. Protection of the resulting amino group with FmocOSu and NaHCO₃ in THF and H₂O afforded amino acid 3 in 60% yield (2 steps).

Synthesis of β³-homocysteines 4 and 5, and (2R,3R)-methylcysteine 6

Since the stereochemical requirement for acceptance of β-amino acids by the lantibiotic synthases is unknown, both enantiomers of β³-homocysteine were prepared. The preparation of the (R)-isomer followed the Arndt-Eistert homologation protocol developed by Seebach and coworkers^13a using the S-tert-butyl disulfide protecting group for the thiol functionality of L-Fmoc-cysteine (Scheme 6). After formation of diazoketone 24, silver-mediated Wolf rearrangement¹⁴ resulted in the desired product but in disappointing yield (48%). However, when the rearrangement was induced by photolysis the protected β³-homocysteine derivative 4 was obtained in excellent yield (82%). This protected β³-amino acid can be incorporated into the N-terminal position of peptides by Fmoc-based SPPS and provides, after global deprotection, a disulfide masked N-terminal thiol that can be used immediately for expressed protein ligation^4,15 to provide the desired 51-mer substrate analogs for LctM.

Scheme 6.

The same protocol can in principle be used for the preparation of (S)-β ³-homocysteine but this route suffers from the high cost of Fmoc-D-Cys(StBu) (1 g $ 225, BACHEM 2005 catalog). Hence an alternative route was developed starting from L-aspartic acid. The advanced intermediate 25 was obtained in three steps¹⁶ from commercially available N-α-Boc-L-aspartic acid β-tert-butyl ester N-hydroxysuccinimide ester (Boc-Asp(OtBu)-Osu, Scheme 7). Treatment of 25 with sodium methoxide in methanol quantitatively yielded the corresponding thiol,¹⁷ which was converted to intermediate 26 (Scheme 7). Without further purification, treatment of activated compound 26 with tert-butylthiol in the presence of triethylamine gave the tert-butyldisulfide 27 in 51% yield from 25. Subsequent removal of the Boc group with TFA in methylene chloride followed by protection of the primary amine using Fmoc-OSu yielded the target compound 5 in 79% yield.

Scheme 7.

For the preparation of disulfide 6, benzyl protected methylcysteine derivative 28 was prepared from L-threonine using previously developed aziridine methodology.¹⁸ Acidic removal of both the carboxybenzyl group and hydrolysis of the methyl ester, followed by protection of the primary amine with Fmoc provided compound 29 in 76% yield (Scheme 7). The benzyl group was subsequently removed using silicon tetrachloride in the presence of diphenylsulfoxide and trifluoro acetic acid¹⁹ to provide the desired (2R,3R)- methylcystine for immediate use in solid phase peptide synthesis¹⁹ and subsequent expressed protein ligation.

In summary, nonproteinogenic amino acids 1–6 were prepared using stereoselective syntheses. The incorporation of these amino acids into the 51-mer peptide substrate for lacticin 481 synthase is currently in progress.

Experimental Section

(4R, 1′R)-4-(1-Hydroxy-allyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (syn-8)

MeLi (1.6 M in Et₂O, 42.36 mL, 67.77 mmol) was added to a solution of tetravinyltin (3.05 mL, 16.94 mmol) in Et₂O (127 mL) at 0 °C and the mixture was stirred for 15 min at the same temperature. Then ZnBr₂ (15.24 g, 67.77 mmol) was added to the mixture and stirred for 1 h at room temperature. The resulting solution of vinyl zinc bromide in Et₂O was slowly added to a suspension of 7 (3.88 g, 16.94 mmol) and ZnBr₂ (3.82 g, 16.94 mmol) in Et₂O (43 mL) at −78°C. Then the reaction mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was cooled to 0°C, saturated aqueous NH₄Cl was added. The organic layer was separated and the aqueous layer was extracted with ether (40 mL ×3). The combined organic phases were washed with brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel (Hexane/EtOAc = 3:1, R_f = 0.3) to give a mixture of syn and anti 8 (syn:anti = 5:1) (3.90 g, 90%) as a white solid. Recrystallization of the solid from a solution of hexane/EtOAc (5:1) gave syn-8 (2.89 g, 74 %) as a white solid. mp 80 −81 °C (lit. 80 –80.5 °C); [α]_D20 = 47.07 (c 1.25, CHCl₃) (lit. 48.9 (c 1.25, CHCl₃)); ¹H NMR (400 MHz, CDCl₃) δ5.80 (m, 1H), 5.32 (d, J = 17.2 Hz, 1H), 5.21 (d, J = 10.0 Hz, 1H), 4.34 (br, 1H), 4.20 (br, 1H), 3.96 (m, 1H), 3.91 – 3.87 ( m, 2H), 1.56 (s, 3H), 1.48 (s, 12H); ¹³C NMR (100 MHz, CDCl₃) δ155.1, 137.7, 117.9, 94.3, 81.4, 76.1, 64.5, 61.8, 28.3, 27.1, 24.2; MS m/z 258 (M⁺ + H⁺, 0.24), 202 ( 5.9 ), 144 ( 9.1 ), 100 ( 27.2 ), 57 (100 ).

(4R, 1′R)-4-(1-tert-Butoxy-allyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (9)

To a solution of syn-8 (2.60 g, 10.12 mmol) in cyclohexane (20 mL) were added tert-butyl-2,2,2-trichloroacetimide (4.09 mL, 23.16 mmol), followed by BF₃•Et₂O (202 μL) at room temperature. After stirring for 2.5 days, the reaction was quenched with NaHCO₃, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 10:1, R_f = 0.4) to give product 9 (1.341 g) as a clear oil. Elution of the column with hexane/EtOAc = 2:1 recovered reactant syn-8 (1.993 g). The recovered compound syn-8 was dissolved in cyclohexane (15.5 mL) again. tert-Butyl-2,2,2-trichloroacetimide (2.74 mL) and BF₃•Et₂O (155 uL) were added to the reaction mixture. After stirring for 2.5 days at room temperature, the reaction was quenched with NaHCO₃, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 10:1) to give product 9 (523 mg) as a clear oil, and recovered reactant syn-8 (1.257 g) (hexane/EtOAc = 2:1). The recovered compound syn-8 was dissolved in cyclohexane (8 mL) once more. tert-Butyl-2,2,2-trichloroacetimide (2.2 mL) and BF₃•Et₂O (83 uL) were added to the reaction mixture. After stirring for 2.5 days at room temperature, the reaction was quenched with NaHCO₃, filtered, concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 12:1, R_f = 0.3) to give product 9 (605 mg) as a clear oil. The overall product 9 from this procedure was 3.121 g (yield 78%). [α]_D20 = 43.86 (c 0.93, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ5.85 (m, 1H), 5.23 – 5.13 (m, 2H), 4.51 and 4.38 (t, J = 5.6 Hz, 1H), 4.06 (ddd, J = 17.2, 9.6, 1.6 Hz, 1H), 3.93 and 3.81 (m, 1H), 3.88 (dd, J = 8.8, 6.4 Hz, 1H), 1.53 – 1.39 (m, 15 H), 1.17 and 1.14 (s, 9H). Note: this compound exists as different rotamers at 25 °C; ¹³C NMR (100 MHz, CDCl₃) δ152.6 and 152.1, 137.3 and 137.1, 116.7 and 116.4, 94.6 and 93.9, 79.9 and 79.8, 74.5 and 74.3, 71.4 and 70.6, 63.6 and 63.3, 61.1 and 60.5, 28.7, 28.4, 26.4 and 25.5, 24.4 and 23.0; IR (thin film, cm⁻¹) 3076, 1704, 1694, 1642; Ms m/z (ESI) 336.4 (M⁺ + Na⁺), 314.5 (M⁺ + H⁺). Anal. Calcd for C₁₇H₃₁NO₄: C, 65.14; H, 9.97; N, 4.47. Found: C, 65.07; H, 10.12; N, 4.63

(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-tert-butyl-pent-4-enoic acid (13)

Method A

A solution of compound 9 (107 mg, 0.342 mmol) in MeOH (6 mL) and aq. 10% HCl (3 mL) was heated to 40 °C. After stirring for 3 h, the reaction mixture was diluted with CH₂Cl₂. The organic layers were separated and the aqueous layer was extracted with EtOAc (20 mL ×3). The combined organic layers were washed with satd. aqueous NaHCO₃, dried over Na₂SO₄, filtered, and concentrated to give crude amino alcohol 11 (38 mg) as a clear oil, which was used without further purification. The crude amino alcohol was dissolved in THF/H₂O (2:1, 3 mL). FmocCl (72 mg, 0.257 mmol) was added, followed by NaHCO₃ (42 mg, 0.5 mmol) at room temperature. After stirring for 24 h, the reaction mixture was diluted with ether. The organic layers were separated and the aqueous layer was extracted with ether (20 mL ×3). The combined organic layers were dried over Na₂SO₄, filtered, concentration. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 3:1, R_f = 0.24) to give compound 13 (43 mg, 32%, two steps) as a foam.

Method B

Compound 9 (1.785 g, 5.702 mmol) was dissolved in 1 N HCl-dioxane (60 mL) at room temperature. The reaction mixture was stirred overnight. Volatiles were removed, the residue was diluted with THF and concentrated again. The residue was dissolved in THF/H₂O (2:1, 75 mL). FmocCl (2.246 g, 8.017 mmol) was added, followed by NaHCO₃ (1.106 g, 13.167 mmol) at room temperature. After stirring for 24 h, the reaction mixture was diluted with EtOAc. The organic layers were separated and the aqueous layer was extracted with ether (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 3:1) to give compound 13 (1.020 g, 45%, two steps, R_f = 0.24) as a foam and compound 12 (551 mg, 28.5%, two steps, R_f = 0.1) as a solid. Compound 13: [α]_D20 = −3.72 (c 0.835, CHCl₃); 1H NMR (400 MHz, CDCl₃) δ7.76 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 5.89 – 5.85 (m, 1H), 5.29 (d, J = 17.2 Hz, 1H), 5.26 (m, 1H), 5.18 (d, J = 10.8 Hz, 1H), 4.44 (dd, J = 10.4, 6.8 Hz, 1H), 4.36 (dd, J = 10.4, 6.8 Hz, 1H), 4.28 – 4.22 (m, 2H), 3.71 ( br, 3H), 2.71 (br, 1H), 1.22 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ156.9, 144.1, 141.5, 139.0, 127.9, 127.3, 125.3, 120.2, 116.6, 75.4, 72.6, 67.0, 63.3, 56.6, 47.5, 28.8; IR (thin film) 3437, 3377, 1713, 1605, 1505 cm⁻¹; MS m/z (ESI) 418 (M⁺ + Na⁺), 396 (M⁺ + H⁺). HRMS Calcd for C₂₄H₂₉NO₄ (M+ + Na⁺): 418.1994. Found: 418.2010. Compound 12: mp 79 – 82 °C; [α]_D20 = 3.06 (c 0.82, CHCl₃); 1H NMR (400 MHz, CDCl₃)δ 7.76 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 5.91 – 5.84 (m, 1H), 5.46 (d, J = 8.4 Hz, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.4 Hz, 1H), 4.46 – 4.42 (m, 2H), 4.36 (m, 1H), 4.20 (t, J = 6.8 Hz, 1H), 3.81 (br, 2H), 3.74 – 3.73 (m, 1H), 3.14 (br, 2H); ¹³C NMR (100 MHz, CDCl₃) δ157.2, 144.0, 141.5, 137.6, 128.0, 127.3, 125.3, 120.2, 116.8, 72.8, 67.1, 63.9, 55.7, 47.4; IR (thin film) 3400, 3067, 1694 cm⁻¹; MS m/z (ESI) 340.1 (M⁺ + H⁺). HRMS Calcd for C₂₀H₂₁NO₄ (M+ + Na⁺): 362.1368. Found: 362.1348.

Carbonic acid allyl ester (2S,3R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-pent-4-enyl ester (12a)

To a solution of compound 12 (100 mg, 0.295 mmol) in CH₂Cl₂ (4 mL) and dry pyridine (72 μL, 3.0 eq) was added allyl chloroformate (57 μL) at 0 °C. Then the reaction was warmed to room temperature and stirred for 24 h. Water was added, the organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 2:1, R_f = 0.4) to give compound 12a (102 mg, 82%) as a white solid. mp 102 – 105 °C; [α]_D20 = 5.61 (c 0.425, CHCl₃); 1H NMR (400 MHz, CDCl₃) δ7.76 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.31 (td, J = 7.6, 0.8 Hz, 2H), 5.96 – 5.86 (m, 2H), 5.36 (m, 2H), 5.29 – 5.24 (m, 3H), 4.63 (d, J = 6.0 Hz, 2H), 4.45 – 4.34 (m, 3H), 4.30 – 4.20 (m, 3H), 3.98 (m, 1H), 2.51 (d, J = 3.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ156.7, 155.3, 144.0, 141.5, 137.0, 131.5, 128.0, 127.3, 125.3, 120.2, 119.5, 117.3, 71.0, 69.1, 67.2, 66.8, 53.8, 47.4; IR (thin film) 3400, 3067, 1747, 1723, 1701, 1522 cm⁻¹; MS m/z (ESI) 446.2 (M⁺ + Na⁺), 430.2 (M⁺ + Li⁺), 424.2 (M⁺ + H⁺). HRMS Calcd for C₂₄H₂₆NO₆ (M+ + H⁺): 424.1760. Found: 424.1761.

Carbonic acid allyl ester (2S,3R)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pent-4-enyl ester (14)

To a solution of compound 12a (54 mg, 0.128 mmol) in CH₂Cl₂ (2 mL) was added a catalytic amount H₂SO₄ (5 μL) at 0 °C. Then isobutylene was bubbled through the solution for 15 min. The reaction was sealed and stirred overnight. The reaction mixture was diluted with ethyl acetate and washed with water and brine, and dried over Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 4:1, R_f = 0.6) to give compound 14 (40 mg, 66%). [α]_D20 = −4.20 (c 0.58, CHCl₃); 1H NMR (400 MHz, CDCl₃)δ 7.76 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.31 (td, J = 7.6, 3.2 Hz, 2H), 5.91 – 5.80 (m, 2H), 5.35 (dd, J = 16.8, 1.2 Hz, 1H), 5.28 (dt, J = 12.9 Hz, 1.2 Hz, 1H), 5.26 (dd, J = 10.4, 0.8 Hz, 1H), 5.17 (dt, J = 10.4, 1.2 Hz, 1H), 5.12 (d, J = 8.8 Hz, 1H), 4.63 (dt, J = 6.0 Hz, 1.2 Hz, 2H), 4.44 – 4.34 (m, 2H), 4.24 – 4.17 (m, 4H), 3.94 – 3.93 (m, 1H), 1.19 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ156.1, 154.7, 143.9, 141.3, 138.4, 131.4, 127.7, 127.0, 125.1, 120.0, 119.1, 116.6, 74.9, 70.4, 68.6, 66.8, 66.3, 54.1, 47.2, 28.5; IR (thin film) 3424, 3339, 3068, 1748, 1731, 1715, 1651 cm⁻¹; MS m/z (ESI) 502 (M⁺ + Na⁺), 480 (M⁺ + H⁺). HRMS Calcd for C₂₈H₃₄NO₆ (M+ + H⁺): 480.2386. Found: 480.2362.

(2R,3R)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-tert-butyl-pent-4-enoic acid (13)

To a solution of compound 14 (15 mg, 0.031 mmol) and (Ph₃P)₄Pd (2 mg) in THF (0.5 mL) was added morpholine (3 μL, 0.037 mmol) at room temperature under N₂. The reaction mixture was stirred for 30 min. Ethyl acetate was added, the resulting mixture was washed with 1 N HCl, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with satd. NaHCO₃, dried over Na₂SO₄, concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 3:1, R_f = 0.2) to give compound 12 (8 mg, 70%) as a foam.

(2S,3R)-3-tert-Butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pent-4-enoic acid (1)

To a solution of compound 13 (0.79 g, 2.01 mmol) in acetone (56 mL) was added Jones reagent (1.0 M in H₂O, 6.02 mL) at 0 °C. After stirring for 1 h, the reaction mixture was warmed to room temperature and stirred for an additional 5 h. Then the reaction was quenched with 2-propanol (1.2 mL). After the resulting mixture was stirred for 2 h, water was added. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by preparative TLC (MeOH/MeOH = 20:1, R_f = 0.1) to give amino acid 1 (624 mg, 76%) as a foam. mp 64 °C (foam); [α]_D20 = 9.12 (c 1.05, MeOH); ¹H NMR (400 MHz, MeOH-d₄) δ7.78 (d, J = 7.6 Hz, 2H), 7.65 (m, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.29 (td, J = 7.2, 0.8 Hz, 2H), 6.62 (d, J = 9.6 Hz, 1H), 5.91 – 5.85 (m, 1H), 5.34 (dd, J = 17.2, 1.2 Hz, 1H), 5.16 (dd, J = 10.8, 1.2 Hz, 1H), 4.62 (m, 1H), 4.36 – 4.28 (m, 2H), 4.24 – 4.20 (m, 2H), 1.18 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ174.0, 158.6, 145.3, 145.1, 142.6, 140.2, 128.8, 128.2, 126.3, 126.0, 120.9, 116.8, 76.0, 74.2, 68.2, 60.7, 28.8; IR (thin film) 3429, 3294, 3073, 1724, 1715, 1607 cm⁻¹; MS m/z (ESI) 410.2 (M⁺ + H⁺). HRMS Calcd for C₂₄H₂₇NO₅ (M+ + Na⁺): 432.1787. Found: 432.1796.

(S)-2,2-Dimethyl-4-((1E)-3-oxo-propenyl)-oxazolidine-3-carboxylic acid tert-butyl ester (15)

Triphenylphosphoranylidene-acetaldehyde (320 mg, 1 mmol) was added to a solution of D-Garner aldehyde 7 (229 mg, 1 mmol) in dry toluene (12 mL). The reaction mixture was heated to 70 °C. After stirring for 24 h, the solution was concentrated and the residue was purified by column chromatography on nutralized silica gel (hexane/EtOAc/Et₃N = 70/20/1, R_f = 0.3) to giveα,β-unsaturated aldehyde 15 (220 mg, 86%) as an ample oil. ¹H NMR (400 MHz, CDCl₃) δ9.55 (d, J = 17.2 Hz, 1H), 6.71 (td, J = 15.6, 5.7 Hz, 1H), 6.16 (m, 1H), 4.66 − 4.51 (m, 1H), 4.15 – 4.05 (m, 1H), 3.85 – 3.82 (m, 1H), 1.63 – 1.40 (m, 15H).

(S)-((1E)-3-Hydroxy-propenyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (16)

To a solution of aldehyde 15 (220 mg, 0.863 mmol) in dry THF (4 mL) was added DIBAl-H (1.2 mL, 1.2 mmol, 1.3 eq, 1.0 M in hexanes) within 10 min at −75 °C. After the reaction mixture was stirred at the same temperature for 2 h, the reaction mixture was slowly warmed to −40 °C and stirred for 1 h. Then the resulting reaction mixture was cooled to −75 °C, and 0.2 mL of MeOH was slowly added so as to keep the internal temperature below −70 °C. The resulting mixture was poured into 0.1 M HCl, the organic layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 2:1, R_f = 0.3) to give alcohol 16 (154 mg, 69%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ5.78 – 5.64 (m, 2H), 4.40 – 4.28 (m, 1H), 4.13 (t, J = 4.8 Hz, 2H), 4.02 (dd, J = 8.8, 6.0 Hz, 1H), 3.72 (dd, J = 8.8, 2.0 Hz, 1H), 1.59 – 1.41 (m, 15H); IR (thin film) 3435, 1696 cm⁻¹.

(S)-((1E)-3-Fluoro-propenyl)-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester (17)

To a cooled solution (−78 °C) of DAST (0.8 mL, 6 mmol) in dry CH₂Cl₂ (20 mL) was added dropwise a solution of alcohol 16 (514 mg, 2 mmol) in dry CH₂Cl₂ (20 mL) over a period of 1 h. After the reaction mixture was stirred at −78 °C for 3 h, water was added. The organic layer was separated and the aqueous layer was extracted with CH₂Cl₂. The combined organic layers were washed with brine, dried over dried Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 7:1, R_f = 0.3) to give compound 17 (292 mg, 56%) as a yellow oil. [α]_D20 = 4.53 (c 0.99, CHCl₃); 1H NMR (400 MHz, CDCl₃) δ5.78 (m, 2H), 4.85 (dd, J = 48.6, 4.4 Hz, 2H), 4.44 – 4.30 (m, 1H), 4.04 (dd, J = 8.4, 6.8 Hz, 1H), 3.75 (d, J = 8.4 Hz, 1H), 1.61 – 1.38 (m, 15H); ¹³C NMR (100 MHz, acetone-d₆) δ152.3, 135.0 (d, J = 11.4 Hz), 127.2 (d, J = 16.7 Hz), 94.3, 83.2 (d, J = 160.0 Hz), 79.7, 68.5, 59.2, 28.5, 26.8, 23.7; ¹⁹F NMR (376 MHz, CDCl₃) δ − 212.9 and –214.1 (t, J = 48.6 Hz), Note: this compound exists as different rotamers at 25 °C; IR (thin film) 2981, 1694, 1386, 1367, 1100 cm⁻¹; MS m/z (ESI) 282.3 (M⁺ + Na⁺), 260.2 (M⁺ + H⁺); HRMS calcd for C₁₃H₂₂NO₃F (M+ + Na⁺): 282.1481. Found: 282.1490.

(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-fluoro-pent-3-enoic acid (18)

Compound 17 (287 mg, 1.108 mmol) was dissolved in 4 N HCl-dioxane (2.5 mL) at room temperature. After stirring for 45 min, the reaction mixture was concentrated. The residue was dissolved in THF and evaporated again to give crude amino alcohol, which was used without further purification. The crude amino alcohol was dissolved in dioxane/H₂O (1:1, 16 mL). FmocCl (434 mg, 1.551 mmol) was added, followed by NaHCO₃ (215 mg, 2.659 mmol). The reaction mixture was stirred overnight. Ethyl acetate was added, the organic layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 1:1, R_f = 0.3) to give compound 18 (302 mg, 80%) as a white solid. mp 137 – 139 °C; [α ]_D20 = −14.94 (c 0.29, MeOH); ¹H NMR (400 MHz, MeOH-d₄) δ7.77 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 6.8 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.29 (td, J = 7.6, 1.2 Hz, 2H), 5.81 (br, 2H), 4.82 (dd, J = 47.6, 3.2 Hz, 2H), 4.42 – 4.35 (m, 2H), 4.20 – 4.17 (m, 2H), 3.54 – 3.52 (m, 2H); ¹³C NMR (100 MHz, MeOH-d₄) δ157.2, 144.1 (d, J = 10.9 Hz), 141.4, 131.9 (d, J = 10.9 Hz), 127.6, 126.9, 126.6 (d, J = 17.1 Hz), 125.0, 119.7, 82.4 (d, J = 162.6 Hz), 66.5, 63.7 (d, J = 2.3 Hz), 54.5, 47.3; ¹⁹F NMR (376 MHz, MeOH-d₄) δminus; 215.5 (t, J = 47.6 Hz); IR (thin film) 3315, 1684, 1589, 1542 cm⁻¹; MS m/z (ESI) 364.1 (M⁺ + Na⁺), 348.2 (M⁺ + Li⁺), 342.1 (M⁺ + H⁺); HRMS calcd for C₂₀H₂₀NO₃F (M+ + Na⁺): 364.1325. Found: 364.1328. Anal. Calcd for C₂₀H₂₀NO₃F: C, 70.37; H, 5.91; N, 4.10; Found: C, 70.06; H, 5.96; N, 4.18.

(S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-5-fluoro-pent-3-enoic acid (2). (Jones oxidation)

To a solution of compound 18 (58 mg, 0.170 mmol) in acetone (4.7 mL) was added Jones reagent (1.0 M, 0.51 mL, 0.510 mmol) at 0 °C. Then the reaction was warmed to room temperature and stirred overnight. The reaction was quenched with 0.5 mL of 2-propanol. After the resulting mixture was stirred for 2 h, water was added. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentration. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 4:1, R_f = 0.4) to give compound 2 (19 mg, 32%) as a white solid. mp 129 – 132 °C; [α]_D20 = −5.67 (c 0.35, CHCl₃); 1H NMR (400 MHz, MeOH-d₄) δ7.76 (d, J = 7.6 Hz, 2H), 7.64 (t, J = 6.4 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 6.01 (dt, J = 15.6 Hz, 4.0 Hz, 1H), 5.86 (m, 1H), 4.82 (dd, J = 47.2, 5.6 Hz, 2H), 4.67 (br, 1H), 4.40 – 4.31 (m, 2H), 4.19 ( t, J = 6.8 Hz, 1H); ¹³C NMR (100 MHz, MeOH-d₄) δ176.4, 156.8, 144.1 (d, J = 23.5 Hz), 141.4, 131.6 (d, J = 11.3 Hz), 127.6, 127.0, 125.6 (d, J = 15.9 Hz), 125.0, 119.7, 82.4 (d, J = 161.6 Hz), 66.6, 57.6, 47.2; ¹⁹F NMR (376 MHz, MeOH-d₄) δ −215.9 (td, J = 47.2, 12.0 Hz); IR (thin film) 3392, 3073, 3035, 1694, 1682, 1586 cm⁻¹; MS m/z (ESI) 378.1 (M⁺ + Na⁺), 362.2 (M⁺ + Li⁺); HRMS calcd for C₂₀H₁₈NO₄F (M+ + Na⁺): 378.1118. Found: 378.1124.

2-[(2S)-2-tert-Butoxymethyl-3-((4R)-4-isopropyl-2-oxo-5,5-diphenyl-oxazolidin-3-yl)-3-oxo-propyl]-isoindole-1,3-dione (21)

To a solution of compound 20 (631 mg, 1.23 mmol) in CH₂Cl₂ (20 mL) was added a catalytic amount of H₂SO₄ (41 μL). The resulting mixture was cooled to 0 °C, isobutylene was bubbled into the solution for 15 min, and then the reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was diluted with CH₂Cl₂ and was washed with water and brine successively, dried over Na₂SO₄, filtered, concentration. The residue was purified by silica gel column chromatography (Hexane/EtOAc = 2:1, R_f = 0.5) to give product 21 (532 mg, 76 %) as a white solid. mp 151 – 153 °C; [α]_D20 = 128.8 (c 0.945, CHCl₃); 1H NMR (400 MHz, CDCl₃) δ7.75 – 7.73 (m, 2H), 7.69 – 7.66 (m, 2H), 7.42 – 7.25 (m, 7H), 7.21 – 7.12 (m, 3H), 5.49 (d, J = 2.8 Hz, 1H), 4.38 – 4.33 (m, 1H), 3.96 – 3.85 (m, 2H), 3.78 (dd, J = 8.8 Hz, 4.4 Hz, 1H), 3.67 (dd, J = 8.8 Hz, 5.6 Hz, 1H), 1.96 – 1.89 (m, 1H), 1.12 (s, 9H), 0.93 (d, J = 7.2 Hz, 3H), 0.74 (d, J = 6.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ172.0, 167.8, 152.5, 142.1, 138.4, 133.7, 131.9, 128.7, 128.33, 128.26, 127.7, 125.8, 125.6, 123.2, 89.0, 73.3, 64.0, 61.4, 43.5, 36.5, 30.2, 27.3, 21.7, 15.8; IR (thin film, cm⁻¹) 3057, 1779, 1716, 1610; MS m/z (ESI) 591.2 (M⁺ + Na⁺), 569.6 (M⁺ + H⁺); Anal. calcd for C₃₄H₃₆N₂O₆: C, 71.81; H, 6.38; N,4.93. Found: C, 71.75; H, 6.43; N, 5.06.

(S)-N-(3-tert-Butoxy-2-carboxy-propyl)-phthalamic acid (22)

To a solution of compound 21 (1.32 g, 2.32 mmol) in THF/H₂O (125 mL, 2.4:1) were added H₂O₂ (30% aq. 955 μL, 0.4 mmol) at 0 °C. After the mixture was stirred for 5 min, LiOH•H₂O (198 mg, 4.65 mmol) was added. The mixture was stirred at the same temperature for 3 h. Na₂SO₃ (1.16 g) was added at 0 °C, and the reaction mixture was stirred for 30 min. THF was evaporated and 16 mL of Et₂O was added. The suspension was stirred for about 15 min and the solid was filtered, which was washed with 1 mL of 1 N NaOH (16 mL), H₂O (16 mL), Et₂O (10 mL) and pentane (10 mL) successively to give auxiliary 600 mg (recovery 92%). The filtrate was diluted with EtOAc and the aqueous layer was acidified to pH 2 with 1 M HCl, which was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated to give compound 22 (713 mg, 95%) as a sticky oil. ¹H NMR (400 MHz, CD₃OD) δ7.95 (dt, J = 8.0 Hz, 1H), 7.59 (td, J = 7.6 Hz, 1.2 Hz, 1H), 7.52 (td, J = 7.6 Hz, 1.6 Hz, 1H), 7.42 (dd, J = 7.6 Hz, 1.2 Hz, 1H), 3.72 – 3.67 (m, 2H), 3.61 – 3.58 (m, 2H), 3.61 – 3.58 (m, 2H), 2.95 – 2.94 (m, 1H), 1.19 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ176.4, 173.0, 169.2, 139.9, 133.0, 131.3, 130.7, 130.5, 128.8, 74.2, 62.1, 46.9, 39.9, 27.7; IR (thin film) 3569 – 2620 (br), 1772, 1716, 1557; MS m/z (ESI) 346.1 (M⁺ + Na⁺), 324.1 (M⁺ + H⁺); HRMS calcd for C₁₆H₂₂NO₆ + H⁺: 324.1447; Found: 324.1446.

(S)-2-tert-Butoxymethyl-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-propionic acid (23)

To a solution of compound 22 (375 mg, 1.162 mmol) in dry THF (23 mL) was added 1,1-carbonyl diimidazole (CDI, 570 mg, 3.486 mmol) at room temperature. The reaction mixture was stirred for 47 h and then was concentrated. The residue was redissolved in 10% NaHCO₃ (16 mL). Then the aqueous solution was acidified to pH 1 with 1 N HCl. The resulting precipitate was extracted with EtOAc and the combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH = 12:1, R_f = 0.4) to give product 23 (337mg, 95%) as a white solid. mp 121 – 124 °C; [α]_D20 = −2.04 (c 0.595, CHCl₃); 1H NMR (400 MHz, CDCl₃) δ7.86 – 7.24 (m, 2H), 7.72 – 7.70 (m, 2H), 4.09 (dd, J = 14.4 Hz, 6.8 Hz, 1H), 3.95 (dd, J = 14.4 Hz, 8.0 Hz, 1H), 3.62 (d, J = 5.6 Hz, 2H), 3.20 – 3.16 (m, 1H), 1.12 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ175.9, 168.4, 134.3, 132.2, 123.6, 74.3, 60.6, 44.5, 37.0, 27.4; IR (thin film) 3574, 3472, 3138, 1774, 1715, 1610; MS m/z (ESI) 328.1 (M⁺ + Na⁺), 306.1 (M⁺ + H⁺); HRMS calcd for C₁₆H₂₀NO₅ + H⁺: 306.1341; Found: 306.1338. Anal. calcd for C₃₄H₃₆N₂O₆: C, 62.94; H, 6.27; N,4.59. Found: C, 62.44; H, 6.15; N, 4.58.

(S)-2-tert-Butoxymethyl-3-(9H-fluoren-9-ylmethoxycarbonylamino)-propionic acid (3)

Compound 23 (335 mg, 1.1 mmol) was dissolved in EtOH (13 mL). Hydrazine (140 μL) was added and the reaction mixture was heated to reflux. After being stirred for 30 min under reflux, the reaction was cooled to room temperature and the solvent was evaporated to give a white solid. Then the solid was suspended in CH₂Cl₂, and the solvent was filtered. The organic layer was discarded, and the solid (370 mg) was used to do the next step without purification. The solid was dissolved in THF/H₂O (1/1, 40 mL), the reaction mixture was cooled to 0 °C, and FmocOSu (450 mg, 1.32 mmol) was added, followed by NaHCO₃ (914 mg, 11 mmol). After the reaction mixture was stirred overnight, the solvent was removed, the residue was dissolved in 3 mL of 10% NaHCO₃, and the resulting solution was washed with Et₂O. The aqueous layer was acidified to pH = 1 with 1 N HCl and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO_4, filtered, and concentrated. The residue was purified with silica gel column chromatography (Hexane/EtOAc = 2:1 then CH₂Cl₂/MeOH = 16:1, R_f = 0.1) to give product 3 (262 mg, 60%, two steps) as a foam. [α]_D20 = 7.32 (c 0.68, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ7.79 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.38 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.10 (t, J = 5.6 Hz, 1H), 4.33 (d, J = 7.0 Hz, 2H), 4.20 (t, J = 7.0 Hz, 1H), 3.58 (d, J = 5.6 Hz, 2H), 3.41 –3.32 (m, 2H), 2.72 (m, 1H), 1.16 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ176.8, 158.7, 145.3, 142.6, 128.8, 128.1, 162.2, 120.9, 74.2, 67.7, 61.9, 48.4, 48.0, 41.0, 27.7; IR (thin film, cm⁻¹) 339, 3073, 1718, 1708, 1525; MS m/z (ESI) 420.2 (M⁺ + Na⁺), 398.2 (M⁺ + H⁺); HRMS calcd for C₂₃H₂₈NO₅ + H⁺: 398.1967; Found: 398.1985.

(R)-Diazomethyl ketone compound 24

To a solution of FmocCys(StBu)OH (86 mg, 0.2 mmol) in THF (−15 °C) was added NMM (15.6 μL, 0.2 mmol), followed by the addition of methyl chloroformate (22 μL, 0.2 mmol). The mixture was stirred under a N₂ atmosphere at −15 °C for 2 h. Filtration gave the crude anhydride, which was slowly treated with diazo methane in ether solution at −5 °C. The reaction mixture was allowed to warm up to room temperature and left overnight. A couple of drops of HOAc were added to quench the reaction. The mixture was washed with sat. aqueous NaHCO₃ (15 mL), NH₄Cl (15 mL), dried over MgSO₄, and concentrated under reduced pressure. Purification over silica gel gave the desired product 24 (EtOAc/Hexane = 1:1, R_f = 0.65) as a white solid (74 mg, 76%). [α]_D20 = (−48.1 (c 0.86, CHCl₃). 1H NMR (500 MHz, CDCl₃): δppm 1.30 (s, 9 H), 3.06 (m, 2H), 4.21 (t, J=6.5 Hz), 4.44–4.52 (m, 3 H), 5.45 (br, 1 H), 5.76 (d, 1 H, J=8.0), 7.29–7.40 (m, 4H), 7.60 (m, 2H), 7.75 (d, 2 H, J=7.5 Hz). ¹³C NMR (CDCl₃): δppm 29.7, 41.9, 47.7, 48.3, 54.8, 57.1, 60.3, 66.8, 119.9, 119.9, 125.0, 127.0, 127.6, 141.2, 141.3, 143.5, 143.6, 155.7, 191.9. IR (thin film, cm(⁻¹) 3306, 2959, 2108, 1362; MS m/z (ESI) 427.9 (M⁺-N₂+H), 478.0 (M++Na); HRMS (ESI+): calculated (M⁺-N₂+H) C₂₃H₂₅N₃NaS₂ 478.1235, found 478.1219.

(R)-4-tert-Butyldisulfanyl-3-(9H-fluoren-9-ylmethoxycarbonylamino)-butyric acid (4)

Method A

A mixture of AgNO₃ (3.3 mg, 0.02 mmol) and compound 24 (81.1 mg, 0.18 mmol) was dissolved in 9.6 mL of THF/H₂O (5:1) with the exclusion of light at −15 °C, followed by the addition of TEA (78 μL, 0.52 mmol). The mixture was stirred overnight at −15 °C, and the reaction mixture was acidified by addition of 10% aqueous citric acid (2 mL). The reaction mixture was diluted with EtOAc (30 mL), washed with brine, and concentrated. Purification over silica gel gave the desired product 4 (CH₃OH/DCM = 1:10, R_f = 0.3) as a foam-like white solid.

Method B

A solution of diazoketone 24 (74 mg, 0.17 mmol) in 9.6 mL of degassed THF/H₂O (5:1) was irradiated with long wavelength UV light (Hg lamp) under nitrogen for 3 h. The reaction mixture was diluted with EtOAc (30 mL), washed with brine and concentrated. Purification over silica gel gave the desired product 4 (CH₃OH/DCM = 1:10, R_f = 0.3) as a foam-like white solid (56 mg, 82%). [α]_D20 = (−8.80 (c 0.8, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δppm 1.32 (s, 9 H), 2.77–3.2.99 (m, 4 H), 4.22–4.40 (m, 4 H), 5.45(br, 1 H), 7.26–7.41 (m, 4H), 7.41 (d, 2H, J=7.6 Hz), 7.75 (d, 2 H, J=7.6 Hz). ¹³C NMR (CDCl₃): δppm 29.7, 36.8, 43.5, 47.1, 47.7, 66.9, 119.9, 125.1, 127.0, 127.6, 141.2, 143.8, 155.6, 176.2. IR (thin film, cm(⁻¹) 3324, 3066, 2961, 1713, 1517, 1451, 1248; MS m/z (ESI) 446.1 (M⁺+H); HRMS (ESI⁺): calculated (M⁺+H) C₂₃H₂₈NO₄S₂ 446.1460, found 446.1445.

(S)-3-tert-Butoxycarbonylamino-4-mercapto-butyric acid tert-butyl ester (25a)

Compound 25 (261 mg, 0.783 mmol) was dissolved in methanol solution (0.1 M NaOCH₃ in 54 mL CH₃OH) and the solution was stirred at room temperature under nitrogen for 15 min. The mixture was acidified with acetic acid and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over MgSO₄, and concentrated and the product was dried under reduced pressure to give the crude thiol product 25a, which was used immediately in the next step reaction without further purification. ¹H NMR (500 MHz, CDCl₃): δppm 1.43 and 1.42 (2 s, 18 H), 2.48–2.68 (m, 4 H), 4.02 (m, 1H), 5.28 (br, 1 H).

(S)-(Methoxycarbonylsulfenyl)-3-tert-butoxycarbonylamino-4-mercapto-butyric acid tert-butyl ester (26)

To a stirred ice-cold solution of methoxycarbonylsulfenylchloride (0.14 mL, 1.56 mmol) in methanol (5 mL), was added dropwise a solution of the thiol 25a (0.783 mmol) in methanol (4 mL) that was prepared as described above. The clear solution was stirred for 2 h at 0 °C. Then the methanol was evaporated under reduced pressure and a white crystalline residue 26 was obtained. ¹H NMR (500 MHz, CDCl₃): δppm 1.43 and 1.49 (2 s, 18 H), 2.61 (m, 1 H), 2.70 (m, 1 H), 2.96–3.08 (m, 2 H), 3.88 (s, 3 H), 4.05 (m, 1H), 5.26 (m, 1 H). Compound 26 was used immediately in the next step without further purification.

(1-tert-Butyldisulfanylmethyl-5,5-dimethyl-3-oxo-hexyl)-carbamic acid tert-butyl ester (27)

To a solution of tert-butyl mercaptan (0.17 mL, 0.16 mmol) and TEA (0.1 mL, 0.72 mmol) in methanol (13 mL), was added a solution of compound 26 in methanol (10 mL). The mixture was stirred at room temperature for 20 min and concentrated. The residue was dissolved in EtOAc and washed with water, brine, dried over MgSO₄ and concentrated. Purification over silica gel gave desired product 27 (EtOAc/Hexane = 1:2, R_f = 0.6) as colorless oil in 51% yield over three steps (from compound 25 to compound 27). ¹H NMR (500 MHz, CDCl₃): δppm 1.31 (s, 9 H), 1.39 and 1.40 (2 s, 18 H), 2.55–2.62 (m, 2 H), 2.85–2.98 (m, 2 H), 4.10 (m, 1 H), 5.09–5.11 (m, 1 H). ¹³C NMR (CDCl₃): δppm 27.8, 27.9, 28.1, 28.2, 29.3, 29.5, 29.7, 29.8, 38.1, 44.3, 47.5, 47.8, 79.2, 80.9, 154.8, 170.6. IR (thin film, cm(⁻¹) 3366, 2972, 2932, 1717, 1500, 1366, 1248, 1161; HRMS (ESI⁺): calculated (M⁺ +H) C₁₇H₃₄NO₄S₂ 380.1929, found 380.1927.

4-tert-Butyldisulfanyl-3-(9H-fluoren-9-ylmethoxycarbonylamino)-butyric acid tert-butyl ester (5)

Compound 27 (0.105 g, 0.28 mmol) was dissolved in a solution of TFA/CH₂Cl₂ (2 mL/2 mL) and stirred at room temperature for 4 h. After removal of solvent, the residue was dissolved in a solution of THF/H₂O (2 mL/2 mL), followed by the addition of FmocOSu (0.104 g, 0.28 mmol) and NaHCO₃ (237 mg, 2.8 mmol). The resulting mixture was stirred overnight and was adjusted to ~ pH 3 with 6 N HCl. The mixture was diluted with EtOAc and the layers were separated. The aqueous phase was extracted further with EtOAc. The combined EtOAc extracts were concentrated. Purification over silica gel gave desired product 5 (CH₃OH/CH₂Cl₂ = 1:10, R_f = 0.3) as a white solid (60 mg, 79%). [α]_D20 = +7.17 (c 0.8, CHCl₃). 1H NMR (400 MHz, CDCl₃): δppm 1.33 (s, 9 H), 2.77–3.02 (m, 4 H), 4.20–4.87 (m, 4 H), 5.47–5.49 (m, 1 H), 7.29–7.41 (m, 4H), 7.41 (d, 2H, J=7.6 Hz), 7.75 (d, 2 H, J=7.6 Hz). ¹³C NMR (CDCl₃): δppm 29.8, 36.8, 43.4, 47.1, 47.7, 48.2, 66.9, 1199, 125.1, 127.0, 127.6, 141.1, 143.8, 155.6, 176.4. IR (thin film, cm(⁻¹) 3323, 3066, 2961, 1713, 1518, 1450, 1248, 1044; MS m/z (ESI) 446.1 (M⁺+H); HRMS (ESI⁺): calculated (M⁺ +H) C₂₃H₂₈NO₄S₂ 446.1460, found 446.1460.

(2R,3R)-3-Benzylsulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyric acid (29)

Compound 28 (0.965 g, 2.69 mmol) was dissolved in HOAc (9.5 mL) and HCl (6 M, 9.5 mL). The reaction mixture was refluxed at 100°C for 9 h and concentrated. The residue was lyophilized to give a white solid. The solid was washed twice with EtOAc. The resulting salt was dissolved in a mixture of THF (9.5 mL) and H₂O (9.5 mL), and to this solution was added FmocOSu (1.0 g, 2.96 mmol), followed by the addition of NaHCO₃ (2.26 g, 16.9 mmol). The reaction mixture was stirred at room temperature overnight and extracted with Et₂O. The basic aqueous layer was acidified to pH around 2 with 3 N HCl, and extracted times with EtOAc (3 ×100 mL). The combined EtOAc layers were washed with brine, dried over MgSO₄, and concentrated. Purification by silica gel chromatography (CH₃OH: CH₂Cl₂ = 1:10, R_f = 0.3) afforded the desired product 29 (0.9015 g, 76%) as a white solid. [α]_D20 = +41.2, (c 2.0, CHCl₃); m.p. 64 °C–66 °C. ¹H NMR (500 MHz, CD₃OD + CDCl₃): δppm 1.26 (d, 3 H, J = 7.0 Hz), 2.82 (m, 1 H), 3.18 (d, 1 H, J = 6.4 Hz), 3.76 (Abq, 2H), 4.23 (t, 1H, J = 7.0 Hz), 4.29–4.44 (m, 3H), 7.20–7.39 (m, 9H), 7.64–7.78 (m, 4H). IR (thin film, cm(⁻¹) 3307, 3064, 3030, 2967, 2927, 1718, 1513, 1449, 1233; ¹³C NMR (CDCl₃): δppm 18.5, 34.8, 41.6, 46.3, 58.1, 66.3, 119.1, 124.3, 126.3, 126.9, 127.6, 128.1, 137.4, 140.5, 143.0, 143.2, 156.4, 172.8. HRMS (ESI⁺): calculated (M⁺ + Na) C₂₆H₂₅NO₄NaS 470.1402, found 410.1387.

(2R,3R)-3-[2-Carboxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-1-methyl-ethyl disulfanyl]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-butyric acid (6)

SiCl₄ (1.6 mL, 13.5 mmol) and Ph₂SO (0.683 g, 3.38 mmol) in 3 mL of TFA were added to compound 29 (0.318 g, 0.677 mmol) at 0 °C. The resulting mixture was stirred for 1 hour and diluted with Et₂O (30 mL). The solution was treated with saturated aq. NaHCO₃ (50 mL). The aqueous layer was then acidified to pH 1, and extracted with EtOAc (3 × 100 mL). The combined EtOAc layers was dried over MgSO₄ and concentrated. Purification by silica gel chromatography (CH₃OH: CH₂Cl₂ = 1:2, R_f = 0.3) gave title compound (97 mg, 40%). [α]_D20 = (−27.3, (c 1.0, CHCl₃); as a white solid. m.p. 180 °C–182 °C. ¹H NMR (400 MHz, CDCl₃): δppm 1.31 (d, 3 H, J = 7.2 Hz), 3.62 (m, 1 H), 4.23 (t, J = 7.0 Hz), 4.32–4.65 (m, 3H), 7.20–7.39 (m, 4H), 7.50–7.78 (m, 4H). ¹³C NMR (DMSO): δppm 17.5, 46.6, 47.6, 58.0, 66.0, 120.2, 124.3, 125.4, 125.5, 126.1, 127.2, 129.6, 129.8, 130.8, 131.2, 131.4, 131.9, 132.6, 140.8, 143.8, 156.1, 171.7. IR (thin film, cm(⁻¹) 3306, 3066, 2965, 2926, 1693, 1517, 1449, 1210; LRMS (ESI⁺): calculated M⁺ for C₃₈H₃₆N₂O₈S₂ 712.2, found 712.9. HRMS (ESI⁺): calculated (M⁺ + H) for C₃₈H₃₇N₂O₈S₂ 713.1991, found 713.1975.

Supplementary Material

si20050627_104. Supporting Information Available.

Copies of ¹H NMR and ¹³C NMR spectra of all unknown compounds. This information is available free of charge via the internet at http://pubs.acs.org.

Scheme 5.