Oligosaccharide synthesis in microreactors

Article abstract of DOI:10.1021/ol0705503

Described is the combination of microreactors and fluorous phase chemistry to assemble oligosaccharides. The synthesis of a β-(1→6) linked D-glucopyranoside homotetramer serves to illustrate this approach. Glycosylations employing a Fmoc-protected glucosy

Full text of DOI:10.1021/ol0705503

ORGANIC
LETTERS
2007
Vol. 9, No. 12
2285-2288
Oligosaccharide Synthesis in
Microreactors
Fre´de´ric R. Carrel, Karolin Geyer, Jeroen D. C. Code´e, and Peter H. Seeberger*
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology (ETH) Zu¨rich,
Wolfgang-Pauli-Strasse 10, 8093 Zu¨rich, Switzerland
seeberger@org.chem.ethz.ch
Received March 5, 2007
ABSTRACT
Described is the combination of microreactors and fluorous phase chemistry to assemble oligosaccharides. The synthesis of a
â-(1f6) linked
D-glucopyranoside homotetramer serves to illustrate this approach. Glycosylations employing a Fmoc-protected glucosyl phosphate building
block were performed in a silicon-based micro-structured device to optimize reaction conditions and for reaction scale-up. A perfluorinated
linker at the reducing end of the oligosaccharides allowed for purification by fluorous solid-phase extraction (FSPE) and further functionalization.
Microreactors are receiving increasing interest for conducting
chemical transfomations.1 The small dimensions of micro-
structured reactors allow for high heat- and mass-transfer
rates, efficient mixing by laminar diffusion, and exact control
of reaction parameters. Microreactors lend themselves par-
ticularly well for the use of unconventional reaction condi-
tions such as high coupling temperatures.2 Less consumption
of material compared to traditional processes, rapid screening
of reaction conditions and numbering-up, as well as scale-
out processes circumvent traditional challenges for synthetic
chemists.1 The silicon-glass microreactor2a used here was
chosen for its excellent thermal conductivity and stability to
a broad range of organic solvents and reagents. The internal
volume of 78.3 µL renders it suitable for reaction screening
and larger scale production; several successful applications
have already been reported.2 In the field of oligosaccharide
synthesis, microreactors had only been used to prepare
disaccharides.2a,c,3
Here, we demonstrate the synthesis of a protected â-(1f6)
linked D-glucopyranoside homotetramer in a microfluidic
device by iterative glycosylations using the Fmoc-protected
glycosyl phosphate 14 and perfluorinated linker 25 (Scheme
(1) For reviews, see: (a) Ehrfeld, W.; Hessel, V.; Lo¨we, H. Micro-
reactors: New Technology for Modern Chemistry; Wiley-VCH: Weinheim,
Germany, 2000. (b) Jensen, K. F. Chem. Eng. Sci. 2001, 56, 293-303. (c)
Haswell, S. J.; Middleton, R. J.; O’Sullivan, B.; Skelton, V.; Watts, P.;
Styring, P. Chem. Commun. 2001, 5, 391-398. (d) Ja¨hnisch, K.; Hessel,
V.; Lo¨we, H.; Baerns, M. Angew. Chem., Int. Ed. 2004, 43, 406-446. (e)
Geyer, K.; Code´e, J. D. C.; Seeberger, P. H. Chem. Eur. J. 2006, 12, 8434-
8442. (f) Brivio, M.; Verboom, W.; Reinhoudt, D. N. Lab. Chip 2006, 6,
329-344. (g) Mason, B. P.; Price, K. E.; Steinbacher, J. L.; Bogdan, A.
R.; McQuade, D. T. Chem. ReV. published online Mar. 21, http://dx.doi.org/
10.1021/cr050944c, and the references cited therein. See also the following
articles: (h) Kawaguchi, T.; Miyata, H.; Ataka, K.; Mae, K.; Yoshida, J.
Angew. Chem., Int. Ed. 2005, 44, 2413-2416. (i) Iwasaki, T.; Kawano,
N.; Yoshida, J. Org. Process Res. DeV. 2006, 10, 1126-1131. (j) Iwasaki,
T.; Nagaki, A.; Yoshida, J. Chem. Commun. In press; and references cited
therein.
(2) (a) Ratner, D. M.; Murphy, E. R.; Jhunjhunwala, M. D.; Snyder, A.;
Jensen, K. F.; Seeberger, P. H. Chem. Commun. 2005, 5, 578-580. (b)
Flo¨gel, O.; Code´e, J. D. C.; Seebach, D.; Seeberger, P. H. Angew. Chem.,
Int. Ed. 2006, 45, 7000-7003. (c) Geyer, K.; Seeberger, P. H. HelV. Chim.
Acta 2007, 90, 395-403.
(3) Fukase, K.; Takashina, M.; Hori, Y.; Tanaka, D.; Tanaka, K.;
Kusumoto, S. Synlett 2005, 15, 2342-2346.
(4) Carrel, F.; Seeberger, P. H. J. Carbohydr. Chem. 2007, in press.
(5) The design of linker 2 was inspired by the octenediol linker reported
in: Andrade, R. B.; Plante, O. J.; Melean, L. G.; Seeberger, P. H. Org.
Lett. 1999, 1, 1811-1814.
10.1021/ol0705503 CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/10/2007

coupling reactions involving the fluorinated acceptor (2, 8,
9, or 10), glycosyl phosphate 1, and the activator (TMSOTf).
A mixture of DMF and piperidine, containing methyl 2,3,4,6-
tetra-O-benzyl-R-D-glucopyranoside as internal reference for
LC/MS analysis, was injected to quench the reaction and to
cleave the Fmoc group (Scheme 3).
Scheme 1. Synthetic Strategy and Linker Functionalization
(n ) 1, 2, 3, 4)
Scheme 3. Optimization of Glycosylation Reactions with Use
of a Silicon-Based Microreactor
1). The new linker system allowed for the use of fluorous
solid-phase extraction (FSPE) as an efficient purification
method.6 After completion of the synthesis, the fluorous tag
can be transformed into aldehydes, glycosyl trichloroacet-
imidates, or n-pentenyl glycosides (Scheme 1).7 Alternatively,
noncovalent attachment to perfluorinated glass-slides is
possible.8
The synthesis of fluorinated linker 2 commenced with
monotritylation of 1,4-butanediol to afford 3. Hydroxyl-
halogen exchange furnished 4.9 Subsequent treatment of alkyl
iodide 4 with PPh3 in refluxing benzene yielded the desired
phosphonium salt 5. Oxidation of the commercially available
3-(perfluorooctyl)propan-1-ol with Dess-Martin periodinane
afforded aldehyde 6. Wittig olefination of 5 and 6 delivered
7, exclusively as the Z-isomer. Final detritylation under
aqueous acidic conditions yielded the fluorinated acceptor
2 (Scheme 2).
Each glycosylation reaction was screened at seven different
reaction times, as summarized in Table 1. Since the
Table 1. Screening of Glycosylation Conditionsa
nucleophile
1
TMSOTf
temp
reaction
times (s)
entry
1
(µmol)
(equiv) (equiv) solvent (°C)
Scheme 2. Synthesis of the Fluorous Linker
2 (1.25)
2 (1.25)
8 (1.25)
9 (1.25)
9 (0.83)
10 (0.83)
2
2
2
2
3
3
2
2
2
2
3
3
TFT
TFT
0
10, 20, 30, 60,
120, 300
2
3
4
5
6
20 10, 20, 30, 60,
120, 300
CH2Cl2 20 10, 20, 30, 60,
120, 300, 600
CH2Cl2 20 10, 20, 30, 60,
120, 300, 600
CH2Cl2 20 10, 20, 30, 60,
120, 300, 600
CH2Cl2 20 10, 20, 30, 60,
120, 300, 600
a Experiments were performed in triplicate, except for entry 6.
To rapidly investigate the iterative glycosylations, the
silicon-glass microreactor was employed to examine the
fluorinated acceptor 2 was poorly soluble in CH2Cl2 at
0 °C, the first glycosylation was carried out in trifluoro-
toluene (TFT) (entries 1 and 2).10 For the first glycosylation,
(6) For reviews on F-tag chemistry, see: (a) Zhang, W. Tetrahedron
2003, 59, 4475-4489. (b) Miura, T. Trends Glycosci. Glycotechnol. 2003,
15, 351-358 and references cited therein. For F-tag oligosaccharide
synthesis, see: (c) Miura, T.; Satoh, A.; Goto, K.; Murakami, Y.; Imai, N.;
Inazu, T. Tetrahedron: Asymmetry 2005, 16, 3-6. (d) Manzoni, L.; Castelli,
R. Org. Lett. 2006, 8, 955-957. (e) Mizuno, M.; Goto, K.; Miura, T.; Inazu,
T. QSAR Comb. Sci. 2006, 25, 742-752 and references cited therein.
(7) Buskas, T.; So¨derberg, E.; Konradsson, P.; Fraser-Reid, B. J. Org.
Chem. 2000, 65, 958-963.
(8) (a) Ko, K. S.; Jaipuri, F. A.; Pohl, N. L. J. Am. Chem. Soc. 2005,
127, 13162-13163. (b) Mamidyala, S. K.; Ko, K. S.; Jaipuri, F. A.; Park,
G.; Pohl, N. L. J. Fluorine Chem. 2006, 127, 571-579.
2286
Org. Lett., Vol. 9, No. 12, 2007

a clear conversion optimum was found at 20 °C and 30 s
reaction time (Figure 1). For batch syntheses, reaction times
are typically longer and reaction temperatures are lower.11
Following oligosaccharide assembly, the perfluorinated
linker of tetrasaccharides 11 and 12 was transformed into
different functional groups (Scheme 5). Olefin cross-
Scheme 5. Functionalization of the Linker
Figure 1. Optimization of the first glycosylation.
After FSPE,12 the monoglycoside 8 was contaminated with
the C(6)-O-TMS derivative that was desilylated by treatment
with silica gel.13 Complete conversion to disaccharide 9
required a reaction time of 20 s at 20 °C (entry 3).13 The
same reaction temperature was applied to the third glyco-
sylation to form trisaccharide 10 (entry 4).13 In all cases,
unreacted starting material 9 was observed. Increasing
amounts of glycosyl phosphate 1 (3 equiv) drove the
glycosylation to completion at an optimal reaction time of
60 s (entry 5). For the final glycosylation step, formation of
the desired tetrasaccharide 11 was investigated by employing
again 3 equiv of glycosyl phosphate 1 (entry 6).13 Complete
conversion to tetrasaccharide 11 required 60 s of reaction
time.
metathesis of 11 and ethylene with use of Grubbs’ second
generation catalyst afforded the desired n-pentenyl gly-
coside 13 (56%), while starting material 11 remained (44%).
The acetylated derivative 12 was hydrolyzed under Fraser-
Reid’s conditions14 to yield the lactol that was further
transformed into glycosyl trichloroacetimidate 14 (ratio R:â
) 6:1, 98% yield for 2 steps). Ozonolytic cleavage of the
double bond of 12 afforded the desired aldehyde 15 in 62%
yield.
In summary, we report the synthesis of a â-(1f6) linked
D-glucopyranoside homotetramer in a silicon-glass micro-
reactor using iterative glycosylations. Each glycosylation was
optimized and scaled-out in the microfluidic device to obtain
the desired oligosaccharides in excellent yield. Notably, the
continuous-flow microreactor allowed for glycosylations by
using â-glycosyl phosphates at ambient temperature. A
perfluorinated linker was incorporated and allowed for the
successful purification of the oligosaccharides by FSPE and
additionally served as an n-pentenyl-type linker for further
functionalization.
The optimized reaction conditions for each glycosylation
were scaled-out to furnish larger quantities of the desired
oligosaccharides (Scheme 4). The different oligosaccharides
Scheme 4. Oligosaccharide Synthesis with Use of the
Optimized Reaction Conditions
(10) At 0 °C in TFT, clogging of the microchannels occurred at 10 s
and 20 s reaction time at the quench inlet due to increased back-pressure.
This problem was not encountered at lower flow rates. Therefore, no data
points are given in Graph 1 for these two reaction times.
(11) Generally, â-glycosylphosphates are activated at temperatures
in a range of -78 to -40 °C and typical reaction times range from 5 to 30
min.
(12) A “Siliabond Tridecafluoro, Silicycle” self-packed column was used
for FSPE.
(13) Only for the first two glycosylations were the observed C(6)-O-
TMS derivatives transformed into the desired glycosides 8 and 9 respectively
by treatment with silica gel. For large-scale syntheses, formation of the
undesired side-products was circumvented by addition of TBAF to the
quenching solution.
were obtained after FSPE in excellent yield and purity (8,
99%; 9, 97%; 10, 90%; and 11, 95%) and were directly used
for the next step. The first glycosylation produces 11.3 mmol
of product per day.
(9) Nicolaou, K. C.; Nikovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.;
Vallberg, H.; Finlay, M. R. V.; Yang, Z. J. Am. Chem. Soc. 1997, 119,
7974-7991.
(14) Motoo, D. R.; Date, V.; Fraser-Reid, B. J. Am. Chem. Soc. 1988,
110, 2662-2663.
Org. Lett., Vol. 9, No. 12, 2007
2287

Acknowledgment. We thank the ETH, the Swiss Na-
tional Science Foundation (SNF grant 200020-109555), and
The Netherlands Organization for Scientific Research (N.W.O.)
(for J.D.C.C.) for financial support. We gratefully acknowl-
edge Prof. K. F. Jensen and Dr. E. R. Murphy (MIT, USA)
for providing the silicon-glass microreactor and Silicycle
(Quebec, Canada) for the fluorinated silica gel used for FSPE.
Supporting Information Available: Detailed experi-
mental procedures and compound characterization data,
including NMR spectra for all described compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0705503
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Org. Lett., Vol. 9, No. 12, 2007