Chemical N-Glycosylation by
Asparagine under Integrated Microfluidic/Batch Conditions

Katsunori Tanaka; Takuya Miyagawa; Koichi Fukase

doi:10.1055/s-0029-1217343

LETTER

Chemical N-Glycosylation by Asparagine under Integrated Microfluidic/Batch Conditions

Katsunori Tanaka, Takuya Miyagawa, Koichi Fukase*
Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan
Fax: +81(6)68505419; e-Mail: koichi@chem.sci.osaka-u.ac.jp;

Abstract

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Abstract

An integrated microfluidic/batch system was applied to the chemical N-glycosylation by the asparagine amide group, a key glycosyl bond-formation reaction in the synthesis of N-glycopeptides. By applying the advantageous features of microfluidic conditions, that is, efficient mixing and rapid heat transfer, the GlcNTrocβAsn and the Fucα(1-6)GlcNTrocβAsn fragments were efficiently prepared.

Key words

N-glycosylation - asparagine - microreactor - oligosaccharide - N-glycopeptide

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Referenzen

References and Notes

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IMM micromixer: http://www.imm-main2.de/

Comet X-01 micromixer: http://homepage3.nifty.com/techno-applications/ or e-mail: yukio-matsubara@nifty.com.

Procedure of N-Glycosylation Using an Integrated Microfluidic/Batch System
A solution of TMSOTf (33 µL, 180 µmol, 43 mM) in CH₂Cl₂ (4.2 mL) was injected, in advance, into the micromixer by a syringe pump at a flow rate of 1.0 mL/min. Then a solution of donor 1a (1.0 g, 1.1 mmol, 260 mM) and acceptor 2 (110 mg, 360 µmol, 86 mM) dissolved in CH₂Cl₂ (4.2 mL) was injected into the IMM micromixer by another syringe pump at a flow rate of 1.0 mL/min. The reaction was mixed at r.t. After the reaction mixture was allowed to flow at r.t. for an additional 94 s through a Teflon tube reactor (Φ = 1.0 mm, l = 1.0 m), the mixture was introduced into a flask, and stirred for 12 h at this temperature. Then the mixture was quenched by an aq NaHCO₃ solution. The resulting mixture was extracted with EtOAc, washed with brine, dried over Na₂SO₄, filtered, and concentrated in vacuo to give the
crude product. The residue was purified by column chromatography on silica gel (from 25-33% EtOAc in hexane) to give N-glycoside 3a as a white solid (376 mg, 85%). ESI-MS: m/z calcd for C₅₃H₅₃Cl₃N₃O₁₃ [M + H]⁺: 1044.3; found: 1044.2. ^¹H NMR (500 MHz, CDCl₃): δ = 7.76 (d, J = 7.6 Hz, 1 H), 7.73 (d, J = 7.5 Hz, 1 H), 7.59 (d, J = 7.6 Hz, 1 H), 7.55 (d, J = 7.4 Hz, 1 H), 7.41-7.17 (m, 19 H), 6.80 (d, J = 8.5 Hz, 1 H), 5.91 (d, J = 9.2 Hz, 1 H), 5.88-5.80 (m, 1 H), 5.27 (dd, J = 17.2, 1.3 Hz, 1 H), 5.19 (dd, J = 10.5, 1.3 Hz, 1 H), 5.14-5.04 (m, 4 H), 4.89 (dd, J = 9.2, 9.2 Hz, 1 H), 4.75 (d, J = 12.1 Hz, 1 H), 4.69 (d, J = 12.0 Hz, 1 H), 4.68-4.58 (m, 3 H), 4.52-4.35 (m, 6 H), 4.15 (dd, J = 6.9, 6.9 Hz, 1 H), 3.65-3.61 (m, 3 H), 3.56-3.49 (m, 2 H), 2.86 (dd, J = 16.7, 3.8 Hz, 1 H), 2,68 (dd, J = 16.4, 4.2 Hz, 1 H).
Data for 3b
ESI-MS: m/z calcd for C₆₃H₆₇Cl₃N₃O₁₉ [M + H]⁺: 1274.3; found: 1274.2. ^¹H NMR (500 MHz, CDCl₃): δ = 7.76 (d, J = 7.6 Hz, 1 H), 7.73 (d, J = 7.5 Hz, 1 H), 7.61 (d, J = 7.6 Hz, 1 H), 7.60 (d, J = 7.3 Hz, 1 H), 7.41-7.19 (m, 19 H), 6.89 (d, J = 8.2 Hz, 1 H), 5.94 (d, J = 8.6 Hz, 1 H), 5.86-5.79 (m, 1 H), 5.31-5.25 (m, 3 H), 5.18 (d, J = 11.6 Hz, 1 H), 5.13 (d, J = 12.2 Hz, 1 H), 5.06 (d, J = 12.4 Hz, 1 H), 4.99 (d, J = 3.5 Hz, 1 H), 4.90-4.87 (m, 2 H), 4.76 (d, J = 12.2 Hz, 1 H), 4.68 (dd, J = 12.1, 4.0 Hz, 2 H), 4.62-4.52 (m, 10 H), 4.37 (d, J = 11.8 Hz, 1 H), 4.23 (dd, J = 6.7, 6.7 Hz, 1 H), 3.80 (dd, J = 10.2, 3.5 Hz,1 H), 3.71 (dd, J = 11.8, 2.2 Hz, 1 H), 3.67-3.52 (m, 3 H), 2.85 (dd, J = 16.5, 3.6 Hz, 1 H), 2.69 (dd, J = 16.5, 3.9 Hz, 1 H), 2.09 (s, 3 H), 1.96 (s, 3 H), 1.06 (d, J = 6.5 Hz, 3 H).