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

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.

amino acids - coupling - enzymes - glycosides - hydrolyses

References

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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.

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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.

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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]

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