HETEROCYCLES

Paper | Regular issue | Vol. 81, No. 1, 2010, pp. 79-89
Received, 18th September, 2009, Accepted, 22nd October, 2009, Published online, 22nd October, 2009.
DOI: 10.3987/COM-09-11834

■ Synthetic Studies on Callipeltins: Stereoselective Syntheses of (3S,4R)-3,4-Dimethyl-L-pyroglutamic Acid and Fmoc-D-allothreonine from Serine Derivatives

Hiroyuki Konno,* Yoko Takebayashi, Kazuto Nosaka, and Kenichi Akaji

Yamagata University, 3-16 Jonan 4-Chome, Yonezawa 992-8510, Japan

Abstract

The non-proteinogenic amino acids contained in callipeltins, (3S, 4R)-3, 4-dimethyl-L-pyroglutamic acid and D-allothreonine, were synthesized from D- or L-serine. The stereoselective synthesis of two methyl groups of (3S, 4R)-3, 4-dimethyl-L-pyroglutamic acid was accomplished by diastereoselective hydrogenation and alkylation. Kinetic epimerization of the C-4 methyl substituent followed by Boc-deprotection with 10% TFA gave the desired (3S, 4R)-3, 4-dimethyl-L-pyroglutamic acid as a single isomer.

INTRODUCTION
Callipeltin A (1)1 and B (2)2 are cyclic depsipeptides isolated from a shallow water sponge of the genus Callipelta by Zampella et al. Callipeltins have antiviral and antifungal properties, and callipeltin A (1) is the first natural peptide found to act against HIV. Callipeltin A (1) was also found to be a selective and powerful inhibitor of cardiac interest as a regulator of myocardial contractility.3 Similar biological activities were reported for cyclic depsipeptides isolated from other marine sponges.4 Because of the unique structure containing unusual amino acid residues and biological properties of these natural products, intensive efforts have been directed toward their synthesis5 including the solid phase synthesis of callipeltin B (2).6 Although the total synthesis of callipeltin A (1) has not yet been achieved, several groups7 have generated the unusual amino acids contained in callipeltins. We also reported the synthesis and identification of the absolute configuration of β-methoxytyrosine in callipeltin E to be 2R, 3R.8
As part of our efforts toward the total synthesis and elucidation of the structure-activity relationship of callipeltin B (2), we report herein the synthesis of two unusual amino acids contained in callipeltin B (2), (3S,4R)-3,4-dimethyl-L-pyroglutamic acid (3) and Fmoc-D-allothreonine (4), from a serine derivative. Two other groups, Lipton’s group9 and Hamada’s group,10 have achieved the synthesis of 3 using an intermediate derived from L-pyroglutamic acid, independently. The key step, the diastereoselective induction of methyl groups, was achieved by Micheal-type addition and subsequent enolate alkylation. In the present study, serine derivatives, a versatile synthon used in the synthesis of a variety of unusual amino acids, was employed as the starting compound for the synthesis of both 3 and 4. For the total synthesis of callipeltin A (1), 3,4-dimethyl-L-glutamine has been briefly transformed from 3.

RESULTS AND DISCUSSION
Our method of generating 3 and 4 is outlined in Scheme 1. As a common starting compound, protected serine derivatives (5) prepared from D- or L-serine were selected. For the diastereoselective introduction of methyl groups, Hanessian’s 1, 2-asymmetric induction reaction11 was employed to give anti-product 6.

Scheme 2 shows the synthetic route for 3. The protected serine derivative (5) derived from D-serine was first transformed through hydrolysis of methyl ester and subsequent Weinreb amidation into an amide (7) in 75% yield. Subsequent alkylation with methyllithium in THF afforded ketone (8) in 53% yield, which was then treated with triethyl phosphonoacetate to give the α,β−unsaturated ester (9) in 65% yield as E/Z mixture (3:1). Diastereoselective hydrogenation of 9 as an E/Z mixture in the presence of 5% Pd/C gave the saturated ester (6) in a quantitative yield with stereoselection of 95:5. The desired anti-product was easily separated from the minor syn-product. The result indicates that the stereo-controlled reaction in the acyclic system exploiting internal 1, 2-asymmetric induction proceeded effectively.11 Treatment of the potassium enolate of 6 with methyl iodide gave the anti-syn product (10) as a single product isolated in 77% yield, which suggests that diasteroselection in the alkylation of α-alkyl substituted enolate esters12 is applicable to the syn-alkylation to yield α, β-dimethyl groups in 10. Deprotection of acetonide (10) with a catalytic amount of p-TsOH gave hydroxyl amine, which cyclized to afford the corresponding δ-lactone (11) as a single diastereoisomer in 67% yield. The stereostructure of the two methyl groups of 11 was further confirmed by conversion to known hydroxyl γ-lactam (4-epi-13)10 by Boc-deprotection and subsequent DBU treatment. The trans-product (11) was converted to the desired cis-lactone (4-epi-11) by kinetic epimerization of a α-methyl group of δ-lactone (11). Treatment with sodium hexamethyldisilazide in THF at -78 oC and stereoselective protonation gave a single diastereoisomer (4-epi-11) in 67% yield, as expected: NOE experiments showed the stereoinversion of the α-position.11 The 4-epi-11 was then efficiently converted to 13 without epimerization by treatment with 10% TFA/CH2Cl2 at 0oC over 10 h. Treatment of 4-epi-11 with neat TFA gave undesired hydroxyl γ-lactam (4-epi-13) having trans-methyl groups on the quantitative acid-catalyzed epimerization of the α-methyl group. The same isomerization was observed by Lipton et al. in the synthesis of Fmoc-protected dimethylglutamine derivative by the indirect conversion of Boc to Fmoc group.9a Finally, the hydroxyl γ-lactam (13) was converted to 3, 4-dimethyl-pyroglutamic acid (3) by TEMPO-mediated oxidation13 in 80% yield. The spectroscopic data for 3 are well consistent with the reported data.9

Transformation of the alcohol group in 4-epi-13 to the desired 3, 4-dimethyl glutamine has already been completed by Hamada’s group.10,14

Then, D-allothreonine was prepared from the protected L-serine derivative (ent-5) by the route shown in Scheme 3. First, the alcohol derivative (14) was prepared from L-serine in 24% overall yield (6 steps) according to a published procedure.15 Protection of a hydroxyl group of 14 with MEMCl/iPr2NEt followed by deprotection of acetonide with a catalytic amount of p-TsOH afforded the corresponding alcohol (15) in 59% yield. Oxidation of the alcohol with Jones reagent gave D-allothreonine derivative (16) quantitatively. Deprotection of the Boc group followed by Fmoc protection afforded the desired Fmoc-D-allothreonine (4). The overall yield of 4 was 57% in 5 steps from 14.

In conclusion, two unusual amino acid derivatives, (3S, 4R)-3, 4-dimethyl-L-pyroglutamic acid (3) and Fmoc-D-allothreonine (4), were synthesized stereoselectively starting from a serine derivative. In the synthesis of 3, the stepwise diastereoselective introduction of methyl groups proceeded to give the desired compound as a single isomer. In addition, deprotection of the Boc group by 10% TFA/CH2Cl2 at 0 oC effectively suppressed the acid-catalyzed epimerization of the α-methyl group. The synthesis of other components of callipeltins and total synthesis on a solid support are now actively underway.

EXPERIMENTAL
General. All manipulations were conducted under an inert atmosphere (N2). All solvents were of reagent grade. THF was distilled from sodium and benzophenone ketyl. CH2Cl2 was distilled from CaH2. All commercial reagents were of the highest purity available. Analytical TLC was performed on silica gel (60F-254, plates 0.25 mm). Column chromatography was carried out on Wakogel 60 (particle size, 0.063-0.200 mm). 1H (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Bruker AM-300. Chemical shifts are expressed in ppm relative to TMS (0 ppm) or CHCl3 (7.28 ppm for 1H and 77.0 ppm for 13C). IR spectra were obtained on a HORIBA FREEXACT-II FT-710 spectrometer. Optical rotations were recorded on a HORIBA SEPA-300 polarimeter at the sodium D line. Low-resolution mass spectra (LRMS) and high-resolution mass spectra (HRMS) were obtained on either a JEOL JMS-HX-211A or a JMS-HX-110A (EI or FAB), or a JMS-T100LC (ESI).

(R)-N,O-Isopropylidenyl-2-(N-tert-butyloxycarbonylamino)-3-oxo-propanamide-N,O-dimethyl-
hydroxyamine (7). To a solution of 5 (10.0 g, 38.6 mmol) in MeOH (100 mL) was added LiOH·H2O (455 mg, 77.2 mmol) and the mixture was stirred for 4 h at room temperature. H2O and Et2O were then added, and the organic layer was dried over MgSO4 and concentrated in vacuo. To a solution of crude product in CH2Cl2 (100 mL) were added Et3N (7.81 mL, 77.2 mmol), BOP (20.0 g, 45.1 mmol), and NH(OMe)Me·HCl (4.52 g, 46.3 mmol) and the mixture was stirred for 3 h at room temperature. 1N HCl (pH=4) and Et2O were added and the organic layer was washed with sat. aq. NaHCO3, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=2:1) to the give amide 7 (8.29 g, 28.8 mmol, 75%) as a colorless oil. [α]D23 +31.5o (c 1.0, CHCl3). IR (film) ν max cm-1: 2978, 1707, 1685, 1390, 1101, 999, 849, 769. 1H NMR (CDCl3) δ: 1.42 (4.5H, s), 1.50 (4.5H, s), 1.53 (1.5H, s), 1.58 (1.5H, s), 1.69 (1.5H, s), 1.71 (1.5H, s), 3.22 (3H, s), 3.71 (1.5H, s), 3.75 (1.5H, s), 3.93 (0.5H, dd, J=9.0, 3.6 Hz), 3.98 (0.5H, dd, J=9.3, 3.0 Hz), 4.19 (0.5H, dd, J=9.0, 7.2 Hz), 4.20 (0.5H, dd, J=9.0, 7.5 Hz), 4.73 (0.5H, dd, J=7.5, 3.6 Hz), 4.81 (0.5H, dd, J=7.2, 3.0 Hz). 13C NMR (CDCl3) δ: 24.6, 24.7, 25.5, 25.7, 28.35, 28.42, 32.6, 36.7, 57.7, 57.9, 61.2, 65.9, 66.1, 80.0, 80.5, 94.4, 95.0, 151.3, 152.2, 170.6, 171.4. HRFABMS (M+H)+ calcd for C13H25N2O5: 289.1763, found: 289.1747.

(R)-N,O-Isopropylidenyl-4-(N-tert-butyloxycarbonylamino)-3-oxo-2-butanone (8). To a solution of 7 (4.15 g, 14.4 mmol) in THF (100 mL) was added MeLi (1.0 M in THF solution; 25.0 mL, 25.0 mmol) at -78 ℃ and the mixture was stirred for 1 h. H2O and Et2O were then added and the organic layer was washed with sat. aq. NaHCO3, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=2:1) to give the ketone 8 (1.80 g, 7.69 mmol, 53%) as a colorless oil. [α]D23 +48.0o (c 1.0, CHCl3). IR (film) ν max cm-1: 2979, 1732, 1705, 1392, 1250, 1176, 1092, 850, 769. 1H NMR (CDCl3) δ: 1.21 (3H, s), 1.30 (9H, s), 1.46 (3H, s), 1.98 (3H, s), 3.73 (1H, brs), 3.93 (1H, brs), 4.16 (1H, brs). 13C NMR (CDCl3) δ: 23.3, 24.3, 25.0, 25.5, 25.9, 27.8, 64.7, 65.0, 79.9, 80.0, 93.9, 94.6, 150.9, 151.9, 205.7. HRFABMS (M+H)+ calcd for C12H22NO4: 244.1549, found: 244.1570.

(S) Ethyl N,O-isopropylidenyl-4-(N-tert-butyloxycarbonylamino)-3-methyl-5-oxo-2-pentenoate (9). To a solution of triethyl phosphonoacetate (2.49 mL, 11.1 mmol) in THF (20 mL) was added NaH (444 mg, 11.1 mmol) at 0 ℃. The mixture was stirred for 1 h and then the ketone 8 (1.80 g, 7.41 mmol) was added dropwise. After 24 h of stirring, sat. aq. NH4Cl and AcOEt were added, and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=4:1) to give the ethyl ester 9 (1.50 g, 4.79 mmol, 65%) as a colorless oil. [α]D23 +10.4o (c 1.0, CHCl3). IR (film) ν max cm-1: 2979, 1700, 1376, 1243, 1213, 1161, 1092, 1058, 852. 1H NMR (CDCl3) δ: 1.27 (3H, m), 1.39 (3H, brs), 1.52 (9H, brs), 1.68 (3H, brs), 2.14 (3H, s), 3.75 (1H, dd, J=9.3, 3.3 Hz), 4.13 (1H, dd, J=9.3, 7.2 Hz), 4.15 (2H, m), 4.32 (1H, dd, J=7.2, 3.3 Hz), 5.77 (1H, s). 13C NMR (CDCl3) δ: 14.0, 14.7, 23.1, 25.5, 28.0, 29.4, 63.8, 67.1, 68.9, 79.8, 94.6, 115.8, 151.6, 156.6. HRFABMS (M+H)+ calcd for C16H28NO5: 314.1967, found: 314.1951.

(3S,4S) Ethyl N,O-isopropylidenyl-4-(N-tert-butyloxycarbonylamino)-3-methyl-5-oxo-pentanoate (6). To a solution of ethyl ester 9 (1.50 g, 4.81 mmol) in MeOH/AcOEt (1:1; 20 mL) was added Pd-C (50 mg) at room temperature. The mixture was stirred for 12 h in a hydrogen atmosphere. It was then filtered on a Celite pad and concentrated in vacuo. The crude product was purified with silica gel column chromatography (hexane/AcOEt=4:1) to give the ester 6 (1.52 g, 4.81 mmol, 100%) as a colorless oil. [α]D23 +6.3o (c 1.0, CHCl3). IR (film) ν max cm-1: 2979, 1736, 1697, 1458, 1375, 1257, 1176, 1084, 854. 1H NMR (CDCl3) δ: 0.87 (3H, d, J=5.4 Hz), 1.18 (3H, td, J=6.9, 1.8 Hz), 1.40 (9H, m), 1.41 (3H, brs), 1.53 (3H, brs), 2.05 (1H, m), 2.43 (2H, d, J=13.2 Hz), 3.73 (2H, m), 3.83 (1H, m), 4.06 (2H, qd, J=6.9, 1.5 Hz). 13C NMR (CDCl3) δ: 14.1, 16.4, 26.1, 28.2, 32.5, 32.8, 36.4, 60.0, 61.0, 64.1, 79.8, 94.0, 152.4, 172.9. HRFABMS (M+H)+ calcd for C16H30NO5: 316.2124, found: 316.2126.

(2S,3S,4S) Ethyl N,O-isopropylidenyl-4-(N-tert-butyloxycarbonylamino)-2,3-dimethyl-5-oxo-pentanoate (10). To a solution of the ester 6 (1.52 g, 4.82 mmol) in THF (20 mL) was added NaHMDS (1.0 M in THF solution; 7.82 mL, 7.82 mmol) at -78 ℃ and the mixture was stirred for 30 min. Methyl iodide (0.675 ml, 10.8 mmol) was added, the solution was stirred for 2 h, sat. aq. NH4Cl and AcOEt were added, and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=4:1) to give the ethyl ester 10 (1.22 g, 3.70 mmol, 77%) as a colorless oil. [α]D29 +3.0o (c 1.0, CHCl3). IR (film) ν max cm-1: 2979, 1734, 1697, 1456, 1383, 1255, 1176, 1086, 860. 1H NMR (CDCl3) δ: 0.93 (3H, d, J=7.2 Hz), 1.13 (3H, d, J=7.2 Hz), 1.26 (3H, t, J=6.7 Hz), 1.50 (9H, s), 1.52 (3H, s), 1.60 (3H, s), 2.34 (1H, brs), 2.52 (1H, m), 3.87 (3H, m), 4.15 (2H, q, J=6.9 Hz). 13C NMR (CDCl3) δ: 10.4, 11.5, 14.2, 14.3, 16.1, 22.9, 24.5, 26.7, 27.2, 28.3, 28.4, 32.6, 38.0, 40.5, 40.9, 59.9, 60.1, 60.2, 64.2, 66.6, 79.9, 93.8, 94.2, 152.4, 153.3, 175.7. HRFABMS (M+H)+ calcd for C17H32NO5: 330.2280, found: 330.2289.

(3S,4S,5S)-N-(tert-Butyloxycarbonyl)-5-amino-3,4-dimethyl-pyran-2-one (11). To a solution of the ethyl ester 10 (300 mg, 0.911 mmol) in MeOH (5 mL) was added p-TsOH (10 mg) at room temperature. After the solution had been stirred for 4 h, H2O and AcOEt were added and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=4:1) to give the lactone 11 (149 mg, 0.612 mmol, 67%) as a colorless oil. [α]D27 -13.2o (c 1.1, CHCl3). IR (film) ν max cm-1: 3356, 2978, 1740, 1712, 1522, 1367, 1248, 1171. 1H NMR (CDCl3) δ: 1.22 (3H, d, J=6.9 Hz), 1.27 (3H, d, J=6.9 Hz), 1.45 (9H, s), 1.55 (1H, m), 2.21 (1H, dq, J=11.1, 6.9 Hz), 3.68 (1H, brs), 4.15 (1H, dd, J=12.0, 4.2 Hz), 4.33 (1H, dd, J=12.0, 3.6 Hz), 4.94 (1H, m). 13C NMR (CDCl3) δ: 13.9, 18.2, 28.1, 36.7, 40.0, 51.4, 51.8, 68.8, 79.6, 155.1, 174.5. HRFABMS (M+H)+ calcd for C12H22NO4: 244.1549, found: 244.1530.

(3S,4S,5S)-5-Hydroxymethyl-3,4-dimethyl-pyrrolidin-2-one (4-epi-13). To a solution of the lactone 11 (20 mg, 0.0821 mmol) in CH2Cl2 (1.0 mL) was added TFA (0.10 mL) at room temperature. After 1 h of stirring, the solvent was removed in vacuo. To a solution of crude product in THF (2 mL) was added DBU (5 µL, 0.0328 mmol) and then the mixture was stirred for 3 h at room temperature. To the mixture were added H2O and AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (CHCl3/MeOH=19:1) to give the lactam 4-epi-13 (12 mg, 0.0821 mmol, 100%) as a colorless solid. Mp=81-84 oC. [α]D23 -2.0o (c 1.0, CHCl3). IR (film) ν max cm-1: 3384, 2992, 2981, 1685, 1460, 1063. 1H NMR (CDCl3) δ: 1.12 (6H, brs), 2.16 (2H, brs), 3.58 (2H, m), 3.72 (1H, m), 4.71 (1H, brs), 7.51 (1H, brs). 13C NMR (CDCl3) δ: 13.1, 13.9, 39.8, 42.6, 57.3, 62.0, 181.6. HRFABMS (M+H)+ calcd for C7H14NO2: 144.1025, found: 144.1031.

(3R,4S,5S)-N-(tert-Butyloxycarbonyl)-5-amino-3,4-dimethyl-pyran-2-one (4-epi-11). To a solution of 11 (462 mg, 1.90 mmol) in THF (10 mL) was added NaHMDS (1.0 M in THF solution; 5.70 mL, 5.70 mmol) at -78 ℃ and the mixture stirred for 30 min. Sat. aq. NH4Cl and AcOEt were then added and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=2:1) to give the lactone 4-epi-11 (311 mg, 1.28 mmol, 67%) as a colorless oil. [α]D28 +13.8o (c 1.1, CHCl3). IR (film) ν max cm-1: 3238, 2976, 1743, 1701, 1281, 1255, 1163, 1101, 855, 792. 1H NMR (CDCl3) δ: 1.12 (3H, d, J=6.9 Hz), 1.17 (3H, d, J=6.9 Hz), 1.48 (9H, s), 2.08 (1H, ddq, J=10.2, 8.1, 6.9 Hz), 2.23 (1H, dq, J=10.2, 6.9 Hz), 3.72 (1H, ddt, J=8.1, 3.6, 1.2 Hz), 3.93 (1H, dd, J=10.8, 8.1 Hz), 4.22 (1H, dd, J=10.8, 3.6 Hz), 5.92 (1H, brs). 13C NMR (CDCl3) δ: 13.4, 13.8, 27.7, 39.9, 41.7, 53.8, 66.7, 82.8, 153.3, 179.6. HRFABMS (M+H)+ calcd for C12H22NO4: 244.1549, found: 244.1544.

(3R,4S,5S)-5-Hydroxymethyl-3,4-dimethyl-pyrrolidin-2-one (13). To a solution of the lactone 4-epi-11 (311 mg, 1.28 mmol) in CH2Cl2 (10 mL) was added TFA (1 mL) at room temperature. After 1 h of stirring, the solvent was removed in vacuo. The product was purified with silica gel column chromatography (CHCl3/MeOH=19:1) to give the lactam 13 (105 mg, 0.733 mmol, 57%) as a colorless solid. Mp=76-79 oC. [α]D23 +81o (c 0.2, CHCl3). IR (film) ν max cm-1: 3325, 2962, 1682, 1458, 1267, 1065. 1H NMR (CDCl3) δ: 1.02 (3H, d, J=7.3Hz), 1.09 (3H, d, J=7.6 Hz), 2.25 (1H, m), 2.54 (1H, m), 3.33 (1H, m), 3.50 (1H, dd, J=11.2, 7.3 Hz), 3.74 (1H, dd, J=11.2, 2.9 Hz), 4.32 (1H, brs), 6.05 (1H, brs). 13C NMR (CDCl3) δ: 10.7, 13.8, 34.6, 39.8, 62.1, 64.6, 181.4. HREIMS (M)+ calcd for C7H13NO2: 143.0943, found: 143.0944.

(2S,3S,4R)-3,4-Dimethylpyroglutamic acid (3). To a solution of 13 (105 mg, 0.733 mmol) in MeCN-pH 6.6 NaH2PO4 buffer (1:1; 10 mL) were added a 10~15% NaOCl solution (12 mL), NaClO2 (150 mg, 1.65 mmol) and TEMPO (15 mg, 0.1 mmol). The mixture was stirred for 48 h at room temperature. To the mixture were added 1N HCl and AcOEt. The organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (CHCl3/MeOH=9:1) to give the dimethyl pyroglutamic acid 3 (92 mg, 0.587 mmol, 80%) as a yellow solid. [α]D23 +41o (c 0.1, CHCl3). IR (film) ν max cm-1: 3262, 2979, 1719, 1654, 1243. 1H NMR (D2O) δ: 0.89 (3H, d, J=6.6 Hz), 0.97 (3H, d, J=6.2 Hz), 2.53 (2H, m), 3.80 (1H, d, J=3.9 Hz). 13C NMR (D2O) δ: 9.4, 13.7, 38.0, 39.2, 61.4, 176.2, 183.8. HRFABMS (M+H)+ calcd for C7H12NO3: 158.0817, found: 158.0815.

(2S,3R)-2-N-(tert-Butyloxycarbonyl)-amino-3-methoxyethoxymethoxybutan-1-ol (15). To a solution of 14 (3.04 g, 12.4 mmol) in CH2Cl2 (50 mL) were added iPr2NEt (3.75 g, 37.0 mmol) and MEMCl (2.31 g, 28.7 mmol) at 0 oC and the mixture stirred for 10 h at room temperature. 1N HCl and CH2Cl2 were then added and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=4:1) to give the MEM ether (3.98 g, 11.9 mmol, 96%) as a colorless oil. 1H NMR (CDCl3) δ: 1.17 (3H, d, J=6.6 Hz), 1.49 (9H, s), 1.52 (3H, s), 1.58 (3H, s), 3.41 (3H, s), 3.56 (2H, t, J=4.2 Hz), 3.70 (2H, m), 3.76 (1H, m), 3.93 (1H, m), 4.08 (1H, dd, J=8.4, 1.8 Hz), 4.11 (1H, m), 4.77 (1H, d, J=6.9 Hz), 4.83 (1H, d, J=6.9 Hz). To a solution of the MEM ether (3.23 g, 9.67 mmol) in MeOH (50 mL) was added p-TsOH (10 mg) at room temperature and the mixture stirred for 10 h. Sat. aq. NaHCO3 and AcOEt were then added and the organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The product was purified with silica gel column chromatography (hexane/AcOEt=2:1) to give the alcohol 15 (1.78 g, 6.07 mmol, 63%) as a colorless oil. [α]D25 -1.3o (c 0.6, CHCl3). IR (film) ν max cm-1: 3448, 3363, 2978, 2931, 1712, 1511, 1365, 1172, 1041, 856. 1H NMR (CDCl3) δ: 1.19 (3H, d, J=6.3 Hz), 1.45 (9H, s), 3.07 (1H, brs), 3.40 (3H, s), 3.54-3.63 (6H, m), 3.79 (1H, m), 4.11 (1H, q, J=5.7 Hz), 4.64 (1H, d, J=7.2 Hz), 4.77 (1H, d, J=7.2 Hz), 4.91 (1H, brs). 13C NMR (CDCl3) δ: 16.4, 28.3, 56.0, 59.0, 62.6, 67.0, 70.6, 71.6, 79.3, 93.0, 156.3. HRESIMS (M+Na)+ calcd for C13H27NO6Na: 316.1736, found: 316.1748.

(2R,3R)-2-N-(tert-Butyloxycarbonyl)-3-methoxyethoxymethyl-D-allothreonine (16). To a solution of 15 (1.36 g, 4.64 mmol) in acetone (20 mL) was added Jones reagent (3 mL; 2.67 M solution) at 0 oC and the mixture was stirred for 30 min. 2-Propanol (1 mL) was then added and the mixture was filtered on a Celite pad and concentated in vacuo. The crude product was treated with 1N HCl and extracted with AcOEt. The organic layer was dried over MgSO4, and concentrated in vacuo. The product was purified with silica gel column chromatography (CHCl3/MeOH=9:1) to give 16 (1.30 g, 4.63 mmol, 91%) as a colorless oil. [α]D23 +1.4o (c 1.3, CHCl3). IR (film) ν max cm-1: 3459, 3330, 2978, 2934, 1716, 1506, 1367, 1167, 1039, 858. 1H NMR (CDCl3) δ: 1.21 (3H, d, J=6.3 Hz), 1.42 (9H, s), 3.28 (3H, s), 3.51-3.70 (5H, m), 4.31 (2H, m), 4.61-4.74 (2H, m), 5.35 (1H, m). 13C NMR (CDCl3) δ: 16.8, 28.2, 58.0, 58.8, 67.0,
71.6, 72.9, 79.9, 93.6, 156.1, 173.9. ESIMS (M)+ calcd for C13H25NO7: 307.16, found: 307.12.

(2R,3R)-2-N-(tert-9-Fluorenylmethyloxycarbonyl)-D-allothreonine (4). A solution of 16 (67 mg, 2.19 mmol) in 90% TFA/H2O (2 mL) was stirred for 10 h at room temperature and the solvent was removed in vacuo. To a solution of the crude product in H2O (2 mL) were added 1N NaOH (0.4 mL) and Fmoc-OSu (147 mg, 0.436 mmol) in dioxane (1 mL) and then the mixture was stirred for 15 min at room temperature. 1N HCl was added and the resulting precipitate was filtered to give 4 (73 mg, 0.214 mmol, 98%) as a white powder. All spectra for the synthetic 4 were well consistent with those reported. 1H NMR (CDCl3) δ: 1.26 (3H, m), 2.65 (1H, brs), 4.16 (1H, brs), 4.35 (3H, brs), 5.39 (1H, brs), 6.05 (1H, brs), 7.30 (2H, m), 7.37 (2H, t, J=7.2 Hz), 7.58 (2H, brs), 7.73 (2H, d, J=7.2 Hz). ESIMS (M+Na)+ calcd for C19H19NO5Na7: 364.12, found: 364.05.

ACKNOWLEDGEMENTS
We thank Dr. Nobutaka Fujii and Dr. Shinya Oishi (Kyoto University) for the measurement of mass spectra. This work was supported in part by Chisso, Co. Ltd., and a Grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (Grant 21689004 to H.K.).

References

1. A. Zampella, M. V. D’Auria, L. Gomez-Paloma, A. Casapulla, C. Debitus, and Y. Henin, J. Am. Chem. Soc., 1996, 118, 6202. CrossRef
2. M. V. D’Auria, A. Zampella, L. Gomez-Paloma, and L. Minare, Tetrahedron, 1996, 52, 9589; CrossRef A. Zampella, A. Randazzo, N. Borbone, S. Luciani, L. Trevisi, C. Debitus, and M. V. D’Auria, Tetrahedron Lett., 2002, 43, 6163; CrossRef M. V. D’Auria, V. Sepe, R. D’Orsi, F. Rellota, C. Debitus, and A. Zampella, Tetrahedron, 2007, 63, 131. CrossRef
3. L. Trevisi, S. Bova, G. Cargnelli, D. Danieli-Betto, M. Floreani, M. V. D’Auria, and S. Liciani, Biochem. Biophys. Res. Commun., 2000, 279, 219; CrossRef L. Trevisi, G. Cargnelli, G. Ceolotte, I. Papparella, A. Semplicini, A. Zampella, M. V. D’Auria, and S. Luciani, Biochem. Pharmacol., 2004, 68, 1331. CrossRef
4. P. W. Ford, K. R. Gustafson, T. C. McKee, N. Shigematsu, L. K. Maurizi, Pannell, D. E. Williams, E. D. de Silva, P. Lassota, T. M. Allen, R. Van Soest, R. J. Andersen, and M. R. Boyd, J. Am. Chem. Soc., 1999, 121, 5899; CrossRef M. A. Rashid, K. R. Gustafson, L. K. Cartner, N. Shigematsu, L. K. Pannell, and M. R. Boyd, J. Nat. Prod., 2001, 64, 117; CrossRef N. Oku, K. R. Gustafson, L. K. Cartner, J. A. Wilson, N. Shigematsu, S. Hess, L. K. Pannell, M. R. Boyd, and J. B. McMahon, J. Nat. Prod., 2004, 67, 1407; CrossRef A. Zampella, V. Sepe, P. Luciano, F. Bellota, M. C. Monti, M. V. D’Auria, T. Jepson, S. Petek, M.-T. Adeline, O. Laprevote, A.-M. Aubertin, C. Debitus, C. Poipat, and A. Ahond, J. Org. Chem., 2008, 73, 5319. CrossRef
5. W. Xie, D. Ding, W. Zi, G. Li, and D. Ma, Angew. Chem. Int. Ed., 2008, 47, 2844. CrossRef
6. R. Krishnamoorthy, L. D. Vazquez-Serrano, J. A. Tunk, J. A. Kowalski, A. G. Benson, N. T. Breaux, and M. A. Lipton, J. Am. Chem. Soc., 2006, 128, 15392; CrossRef R. Krishnamoorthy, B. L. Richardson, and M. A. Lipton, Bioorg. Med. Chem. Lett., 2007, 17, 5136. CrossRef
7. K. A. Parker and Q. Xie, Org. Lett., 2008, 10, 1349; CrossRef J. Joen, S. K. Hong, J. S. Oh, and Y. G. Kim, J. Org. Chem., 2006, 71, 3310; CrossRef D. C. Cranfill, A. I. Morales-Ramos, and M. A. Lipton, Org. Lett., 2005, 7, 5881; CrossRef and references cited therein.
8. H. Konno, S. Aoyama, K. Nosaka, and K. Akaji, Synthesis, 2007, 3666. CrossRef
9. C. Selcuk and M. A. Lipton, J. Org. Chem., 2005, 70, 6218; CrossRef C. M. Acevedo, E. F. Kogut, and M. A. Lipton, Tetrahedron, 2001, 57, 6353. CrossRef
10. N. Okamoto, O. Hara, K. Makino, and Y. Hamada, Tetrahedron: Asymmetry, 2001, 12, 1353. CrossRef
11. S. Hanessian, W. Wang, and Y. Gai, Tetrahedron Lett., 1996, 37, 7477; CrossRef S. Hanessian, Y. Gai, and W. Wang, Tetrahedron Lett., 1996, 37, 7473. CrossRef
12. Y. Morizawa, A. Tasuda, and K. Uchida, Tetrahedron Lett., 1986, 27, 1833. CrossRef
13. M. M. Zhao, Z. Li, E. Mano, Z. J. Song, and D. M. Tschaen, Organic Syntheses, 2005, 81, 195; J. Z. Song, M. Zhao, R. Desmond, P. Devine, D. M. Tschaen, R. Tiller, L. Frey, R. Heid, F. Xu, B. Foster, J. Li, R. Reamer, R. Volante, E. J. Grabowski, U. H. Dolling, P. J. Reider, S. Okada, Y. Kato, and E. Mano, J. Org. Chem., 1999, 64, 9658. CrossRef
14. B. Liang, P. J. Carroll, and M. M. Joullié, Org. Lett., 2000, 2, 4157. CrossRef
15. L. Williams, Z. Zhang, F. Shao, P. J. Carroll, and M. M. Joullié, Tetrahedron, 1996, 36, 11673; CrossRef L. A. Paquette, T. M. Mitzel, M. B. Issac, C. F. Crasto, and W. W. Schomer, J. Org. Chem., 1997, 62, 4293; CrossRef X. E. Hu, N. K. Kim, and B. Ledoussai, Org. Lett., 2002, 4, 4499. CrossRef