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Synlett 2002(1): 0057-0060
DOI: 10.1055/s-2002-19355
LETTER
© Georg Thieme Verlag Stuttgart · New York

An Expeditious Route to GlcNAc-Cbz-Asn by Chemo-enzymatic Synthesis

Yoshihide Tokudaa, Yoichi Takahashia, Kaori Matoishia, Yukishige Itob, Takeshi Sugai*a
a Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, Japan
e-Mail: sugai@chem.keio.ac.jp;
b RIKEN (The Institute of Physical and Chemical Research), 2-1, Hirosawa, Wako-shi, 351-0198, Japan
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Publication History

Received 10 October 2001
Publication Date:
01 February 2007 (online)

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Abstract

A short-step route to GlcNAc-Cbz-Asn was developed. Treatment of GlcNAc in sat. aq. NH4HCO3 solution and subsequent electorodialytic desalting provided ammonia-free glycosylamine in large quantity. The product was coupled with Cbz-Asn α-isobutyl ester β-fluoride, and finally, the isobutyl ester was deprotected by enzyme-catalyzed hydrolysis under mild conditions.

Key words

amino acids - coupling - enzymes - glycosides - hydrolyses

10

GlcNAc (2, 40.4 g, 182.9 mmol) was dissolved in H2O (220 mL) and saturated with NH4HCO3. The conversion of GlcNAc into glucosylamine 3 was determined by 1H NMR spectroscopy by comparing anomeric protons. After stirring for 4 days at 35 °C, the conversion reached 86%, and the solution was diluted with H2O (150 mL). The mixture was desalted by AC-220-550 on Asahi Chemical Micro Acylyzer S3. At the initial stage, the conductivity was 87.8 mS and after the desaltation at the 15 V (1.07 A), it reached 0.6 mS. NH4HCO3 was estimated to be 5.6 mmol/L based on the calibration linear as described in Figure [2] . The yields of 3 and 2 were quantitatively estimated to be 54% and 9%, respectively, by 1H NMR using methyl β-d-glucopyranoside as internal standard. The mixture was frozen and lyophilized to give a highly hygroscopic white powder (30.1 g). Again at this stage, the amounts of 3 and 2 in the solid were estimated to be 22.3 g (101 mmol) and 3.0 g (14 mmol), respectively. No decomposition of 3 was observed into 2 and ammonia through the lyophilization. 1H NMR (400 MHz, D2O) δ 4.16 (d, 1 H, J = 9.3 Hz), 3.85 (dd, 1 H, J = 2.1, 12.4 Hz), 3.69 (ddd, 1 H, J = 2.1, 4.6, 12.4 Hz), 3.62 (t, 1 H, J = 9.3 Hz), 3.52 (m, 1 H), 3.55-3.40 (m, 2 H), 2.07 (s, 3 H); 13C NMR (100 MHz, D2O) δ 175.26, 85.01, 77.63, 75.36, 70.90, 61.71, 57.21, 23.21; IR (KBr) 3352, 1652, 1558, 1375, 1047 cm-1.

11

Ester 5a was obtained from oxazolidinone 6a [12] with sodium isobutoxide. The alkoxide solution prepared from isobutyl alcohol (37.8 mL) and sodium (0.83 g, 36.2 mmol) was added dropwise into the solution of 6a (10.1 g, 36.2 mmol) in isobutyl alcohol (55 mL) at 65 °C with stirring. After the stirring was continued for 3 h, AcOH (10 g) was added to the reaction mixture. Conventional work up procedure and subsequent silica gel column chromatography of the residue [CHCl3-MeOH-AcOH (90:3:2)] gave 5a (5.5 g, 47% yield). Recrystallization from n-hexane-Et2O afforded fine colorless needles. Mp 65.0-66.0 °C [lit. [12] mp 68-69 °C]; [α]D 22 -4.4 (c 1.3, AcOH); [α]435 22 -10.3 (c 1.3, AcOH) [lit. [12] [α]427 22 -11.8 (c 1.3, AcOH)]; 1H NMR (400 MHz, CDCl3) δ 7.28 (m, 5 H), 5.71 (d, 1 H, J = 8.3 Hz), 5.06 (m, 2 H), 4.58 (ddd, 1 H, J = 3.9, 4.4, 8.3 Hz), 3.92 (d, 2 H, J = 6.8 Hz), 3.04 (dd, 1 H, J = 3.9, 17.1 Hz), 2.85 (dd, 1 H, J = 4.4, 17.1 Hz), 1.86 (m, 1 H), 0.83 (d, 6 H, J = 6.3 Hz); 13C NMR (100 MHz, CDCl3) δ 175.83, 170.36, 155.89, 135.84, 128.40, 128.01, 127.96, 71.98, 67.23, 50.24, 36.46, 27.63, 19.06; IR (KBr) 3350, 3127, 2954, 1761, 1727, 1692, 1280, 1224, 1063 cm-1. Its NMR spectrum was in good accordance with that reported previously. [12] No β-ester was detected. In contrast, the ring-opening reaction [20] of a cyclic anhydride of N-Cbz-aspartate with isobutyl alcohol gave a mixture of α-ester and β-ester (ca. 10:1, δ 3.92 for α-ester and 3.86 for β-ester). Attempts for enzyme-catalyzed selective hydrolysis [21] of diethyl N-Cbz-aspartate or ethyl N-Cbz-asparagine gave no fruitful results, such as the formation of a diacid, or entirely no reaction.

15

Mp 37.5-40.0 °C (colorless fine needles from CH2Cl2-hexane); 1H NMR (400 MHz, CDCl3) δ 7.37 (m, 5 H), 5.72 (d, 1 H, J = 7.3 Hz), 5.14 (d, 1 H, J = 12.2 Hz), 5.10 (d, 1 H, J = 12.2 Hz), 4.67 (ddd, 1 H, J = 2.4, 4.8, 7.3 Hz), 3.98 (d, 2 H, J = 6.8 Hz), 3.23 (dd, 1 H, J = 2.4, 18.1 Hz), 3.13 (dd, 1 H, J = 4.8, 18.1 Hz), 1.95 (m, 1 H), 0.92 (d, 6 H, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 160.90 (d, J = 357 Hz), 169.21, 155.65, 135.67, 128.44, 128.20, 128.01, 72.43, 67.38, 49.98, 35.14 (d, J = 52 Hz), 27.64, 18.93; IR (KBr) 3341, 2961, 1840, 1727, 1691, 1533, 1294, 1269, 1096 cm-1.

16

To a solution of 3 (782.9 mg, 2.42 mmol) in anhydrous DMF (15 mL) was added NaHCO3 (383 mg, 4.56 mmol) and fluoride (5b, 872.8 mg, 2.94 mmol) at room temperature. After stirring for 2 h, the reaction mixture was filtered through a Celite pad and washed with 1,4-dioxane. The filtrate was evaporated and purified by silica gel column chromatography. Elution with CHCl3-MeOH (5:1) afforded 1c (1.07 g, 84%). Mp 195.0-196.5 °C; [α]D 25 +6.9 (c 0.81, MeOH); 1H NMR (400 MHz, CD3OD) δ 7.28 (m, 5 H), 5.09 (d, 1 H, J = 12.2 Hz), 5.05 (d, 1 H, J = 12.2 Hz), 4.93 (d, 1 H, J = 9.8 Hz), 4.58 (dd, 1 H, J = 4.9, 7.3 Hz), 3.88 (d, 2 H, J = 6.8 Hz), 3.81 (dd, 1 H, J = 1.5, 12.2 Hz), 3.73 (t, 1 H, J = 9.8 Hz), 3.64 (ddd, 1 H, J = 0.9, 3.4, 12.2 Hz), 3.44 (m, 1 H), 3.30 (m, 2 H), 2.79 (dd, 1 H, J = 4.9, 16.0 Hz), 2.68 (dd, 1 H, J = 7.3, 16.0 Hz), 1.90 (s, 3 H), 1.89 (m, 1 H), 0.90 (d, 6 H, J = 6.8 Hz); 13C NMR (100 MHz, CD3OD) δ 175.45, 172.73, 172.26, 158.96, 137.83, 129.31, 128.92, 128.82, 80.24, 79.67, 76.24, 72.50, 71.76, 67.74, 62.62, 56.06, 52.14, 38.49, 28.97, 22.92, 19.40; IR (KBr) 3295, 3091, 2957, 1749, 1703, 1654, 1546, 1296, 1271 cm-1. Anal. Calcd. For C24H35N3O10: C, 54.85; H, 6.71; N, 8.00. Found: C, 54.59; H, 6.84; N, 7.56.

19

The coupling product (1c, 200.9 mg, 0.38 mmol) was dissolved in a mixture of DMF (2.0 mL) and 0.2 M phosphate buffer (pH 7.0, 2.0 mL). The pH was adjusted to 7.0 with 2 mol/L HCl, and papain (Sigma, P3375, 56.4 mg, 107 units) and l-cysteine (11.0 mg, 0.09 mmol) were added. Then the mixture was stirred at 35 °C with a controlled addition of 0.5 mol/L NaOH to maintain the pH at 7.0. After stirring for 7 h, the reaction mixture was brought to pH 8.0 with 0.5 mol/L NaOH and then filtered through a Celite pad and washed with H2O. The filtrate was adjusted to be pH 4.7 by the addition of 2 mol/L HCl, concentrated in vacuo, and the residue was purified by silica gel column chromatography. Elution with EtOAc-MeOH-H2O (7:2:1) afforded 1b (119.2 mg, 67%). Analytical sample (colorless fine needles from acetone-H2O); mp 174.0-176.0 °C(dec) [lit. [4] mp 172-174 °C]; [α]D 20 +8.1 (c 1.00, H2O) [lit. [4] [α]D 25.5 +5.2 (c 1, H2O)]; 1H NMR (400 MHz, D2O) δ 7.45 (m, 5 H), 5.17 (m, 2 H), 5.09 (d, 1 H, J = 9.8 Hz), 4.57 (dd, 1 H, J = 4.3, 7.8 Hz), 3.89 (dd, 1 H, J = 2.0, 12.2 Hz), 3.82 (dd, 1 H, J = 9.8, 10.3 Hz), 3.76 (dd, 1 H, J = 4.9, 12.2 Hz), 3.63 (dd, 1 H, J = 8.3, 10.3 Hz), 3.52 (m, 2 H), 2.89 (dd, 1 H, J = 4.4, 16.1 Hz), 2.79 (dd, 1 H, J = 7.8, 16.1 Hz), 1.96 (s, 3 H); 13C NMR (100 MHz, D2O) δ 175.39, 173.42, 172.19, 158.47, 136.89, 129.48, 129.13, 128.49, 79.09, 78.41, 74.96, 70.31, 68.02, 61.35, 55.17, 51.82, 38.31, 22.91; IR (KBr) 3292, 3073, 1716, 1689, 1655, 1542, 1374, 1256, 1065 cm-1. Anal. Calcd. For C20H27N3O10: C, 51.17; H, 5.80; N, 8.95. Found: C, 51.00; H, 5.90; N, 8.75. The NMR spectrum of fully deprotected form(1a) was identical with that reported previously. [22]