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Synlett 2009(10): 1571-1574  
DOI: 10.1055/s-0029-1217343
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

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;
Further Information

Publication History

Received 16 February 2009
Publication Date:
02 June 2009 (online)

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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

    References and Notes

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9

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

10

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

11

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).