Microreactor-based reaction optimization in organic chemistry - Glycosylation as a challenge

Article abstract of DOI:10.1039/b414503h

Glycosylation reactions are performed rapidly over a wide range of conditions as an example of microreactor-based method optimization and process development in organic chemistry. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b414503h

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Microreactor-based reaction optimization in organic chemistry—
glycosylation as a challenge{
Daniel M. Ratner,{a Edward R. Murphy,{b Manish Jhunjhunwala,b Daniel A. Snyder,a Klavs F. Jensen*b and
Peter H. Seeberger*ac
Received (in Cambridge, UK) 22nd September 2004, Accepted 10th November 2004
First published as an Advance Article on the web 17th December 2004
DOI: 10.1039/b414503h
products. The five-port silicon microreactor (Fig. 1a) was designed
with three primary inlets to mix and react glycosylating agent,
nucleophile (acceptor), and activator. In order to ensure adequate
mixing and long residence times, the reactor is split into a mixing
and a reaction zone. Once mixed, the reactants enter a reaction
zone which is terminated by a secondary inlet used to quench the
reaction. The quenched reaction stream then exits the reactor for
collection and analysis (Fig. 1b).
Glycosylation reactions are performed rapidly over a wide
range of conditions as an example of microreactor-based
method optimization and process development in organic
chemistry.
Although glycosylations have been carried out for more than a
century, the union of glycosylating agent and nucleophile to form
a glycosidic linkage is a notoriously difficult undertaking.1
Glycoside formation depends on the conformation, sterics, and
electronics of both reaction partners. The challenge in accurately
predicting the reactivity of the coupling partners makes it difficult
to foresee the outcome of the reaction. In addition, reaction
variables such as concentration, stoichiometry, temperature, reac-
tion time, and activator play an indisputable role in the outcome of
a given glycosylation.2 This complexity is shared by many other
organic transformations in which multiple factors determine the
outcome of the reaction. In both academic and industrial settings,
much of the effort spent by synthetic organic chemists is consumed
searching for optimal reaction conditions to achieve a particular
transformation. Method optimization frequently requires the
commitment of time and large quantities of valuable starting
materials.3 The ability to find ideal reaction conditions quickly and
efficiently therefore has a major impact on the practice and pace of
research and development in organic chemistry.
Microfluidic channels were etched into a silicon wafer and
capped by a Pyrex wafer via an anodic bond. This construction
was chosen for its compatibility with a wide range of chemical
reagents, as well as the high thermal conductivity of silicon—
facilitating rapid thermal equilibration and temperature control.6
Moreover, the silicon can be oxidized to create a glass surface
throughout the resulting microchannels. Deep reactive ion etching
techniques (DRIE)7 make it easier to realize deep aspect ratio
structures in silicon than glass. Thus, the use of DRIE and
subsequent oxidation and anodic bonding to pyrex facilitates
making microreactors with glass surface properties. All ports were
directly connected by soldering joint to stainless steel tubing to
allow the use of solvents, such as CH2Cl2.
An UV-active compound (methyl 2,3,4,6-tetra-O-benzyl-a-D-
mannopyranoside)8 was used as an HPLC standard and added to
Microfluidic-based devices are capable of performing a wide
range of single and multiphase organic reactions.4 In addition to
requiring small quantities of reagent, submillimeter reaction
channels allow for the precise control of reaction variables, such
as reagent mixing, flow rates, reaction time, and heat and mass
transfer. Microfluidic devices are also amenable to integrated
reaction monitoring, using UV/VIS, IR, NMR, mass spectrometry
(MS), and LC/MS.5 Unlike conventional bench-top batch
reactions, microreactors are easily scalable, rendering a device
capable of both analytical and semi-preparative scales of
production. Finally, the microreactor format is amenable to
automation of reaction optimization.
Here, we use continuous flow microreactors to systematically
study the glycosylation reaction as an example of a challenging
organic transformation. Optimization of yield and the selection of
optimal reaction time and temperature is the goal, in addition to
gaining an understanding of the formation of different side
{ Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/cc/b4/b414503h/
{ These authors contributed equally to this work.
*kfjensen@mit.edu (Klavs F. Jensen)
Fig. 1 a) Silicon microfluidic microreactor. b) Schematic of microreactor
system, comprised of three primary inlets, a mixing and reaction zone, a
secondary inlet for quench, and an outlet for analysis/collection. c)
Soldered joints of microreactor, also perspective of device from side.
seeberger@org.chem.ethz.ch (Peter H. Seeberger)
578 | Chem. Commun., 2005, 578–580
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the quench syringe which enters through the secondary inlet (after
the reaction zone). The standard was selected for high UV/Vis
absorbance, and compatibility with the reacting species. The
HPLC standard normalizes the output stream for HPLC analysis
by compensating for solvent evaporation and variability in the
volume of collected sample.
Initially, the microchemical system was used to carry out the
mannosylation of diisopropylidene galactose 1 with mannosyl
trichloroacetimidate 2 upon activation with 0.2 equivalents
TMSOTf in anhydrous dichloromethane. The reaction was
quenched with a solution of triethylamine that also contained
the HPLC standard (Scheme 1a). Since the concentration of the
reagents in the reactor is determined not only by the concentration
inside the syringe but also the flow rate of each stream, all flow
rates were maintained in proportion to that of the donor inlet
stream. The reaction temperature was varied from 278 to 20 uC,
with glycosyl donor stream flow rates of 10, 20, 40 and
80 ml min21, that resulted in reactor residence times (reaction
time) of 26.7, 53.4, 106.8, and 213.5 seconds. Glycosylating agent 2
(1.2 equivalents) and nucleophile 1 (1.0 equivalents) were flowed
through the microreactor with reaction zone concentrations of
0.0136 M and 0.0114 M respectively. The triethylamine quench
contained the HPLC standard (1.0 equivalent) and dichloro-
methane to increase solubility.
HPLC analysis of the crude samples, normalized with internal
standard, illustrates a clear relationship between reaction tempera-
ture, reaction time and formation of product (Fig. 2a). For a given
reaction time, the yield of product increases with temperature until
maximum conversion is achieved. Correspondingly, at tempera-
tures lower than the optimum, yield increases with increasing
reaction time (i.e. decreasing flow rate). Importantly, we were able
to observe the formation of orthoester 4 as a major side product at
lower temperatures. Frequently encountered in glycosylations
involving a C2-O-acetate, the orthoester is observed at low
Fig. 2 (a) Normalized HPLC results for glycosylation of nucleophile 1
with mannosyl donor 2. (b) Normalized HPLC results for glycosylation of
nucleophile 5 with mannosyl donor 1. Legend, reaction times: e 213.5 s,
6 106.8 s, n 53.4 s, # 26.7 s.
temperatures. The rapid formation of orthoester, was most
pronounced around 270 uC. Over time, rearrangement of the
orthoester to the desired product is also evident.
Scheme 1 (a) Sample glycosylation of glycosyl donor 2 and nucleophile (acceptor) 1 to fashion disaccharide 3. Formation of orthoester 4 is also often
observed. (b) Glycosylation reaction involving glycosylating agent 2 (mannosyl donor) and nucleophile 5 (acceptor) to fashion disaccharide 6. Formation
of orthoester 7 is also often observed.
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Daniel M. Ratner,{a Edward R. Murphy,{b Manish Jhunjhunwala,b
Following the success with the initial glycosylation, 2,3,4-
tri-O-benzyl-methyl mannoside 5 was mannosylated with 2
(Scheme 1b). The more sterically hindered nucleophile is more
difficult to glycosylate and contains a benzyl group that facilitates
monitoring during HPLC analysis.
Daniel A. Snyder,a Klavs F. Jensen*b and Peter H. Seeberger*ac
aDepartment of Chemistry, Massachusetts Institute of Technology,
Cambridge, MA, USA
bDepartment of Chemical Engineering, MIT, 77 Massachusetts, Avenue,
Cambridge, MA 02139, USA. E-mail: kfjensen@mit.edu;
Fax: 617-258-8224; Tel: 617-253-4589
In contrast to the results obtained for the coupling of 1 and 2,
microreactor-HPLC analysis of the union of 2 and 5 shows a
unique reaction profile (Fig. 2b). Optimal product yields are
obtained from 260 to 240 uC, the same temperature range that
fosters orthoester formation. The reaction outcome is optimal at
260 uC with a reaction time of just over 213 seconds. However,
this analysis also demonstrates that nearly the same yield is
achievable by running the reaction at 235 uC for 25.7 seconds.
With very little change in overall yield, it would be possible to
increase production by nearly an order of magnitude over the
slower reactions run at lower temperatures. The microreactor-
HPLC study reveals important information regarding process
development for scale-up, in addition to reaction optimization.
From the perspective of developing a method for semi-preparative
or preparative scale, significant advantage can be found from the
results of this continuous-flow study, over a much more
cumbersome and costly batchwise optimization.
cLaboratorium fu¨r Organische Chemie, ETH Ho¨nggerberg, HCI F315,
Wolfgang-Pauli-Str. 10, CH-8093, Zurich, Switzerland.
E-mail: seeberger@org.chem.ethz.ch; Fax: 41 1 633 12 35;
Tel: 41 1 633 21 03
Notes and references
1 J. Lehmann, in Carbohydrates: Structure and Biology, Thieme, Stuttgart,
1998, p. 1–45.
2 For review, see: K. Toshima and K. Tatsuta, Chem. Rev., 1993, 120,
1503; T. Nukada, A. Berces, M. Z. Zgierski and D. M. Whitfield, J. Am.
Chem. Soc., 1998, 120, 13291.
3 R. Carlson, Design and Optimization in Organic Synthesis, Elsevier
Science: Amsterdam, New York, 1992.
4 K. Ja¨hnisch, V. Hessel, H. Lo¨we and M. Baerns, Angew. Chem. Int. Ed.,
2004, 43, 406; H. Pennemann, P. Watts, S. J. Haswell, V. Hessel and
H. Lo¨we, Org. Process Res. Dev., 2004, 8, 422; P. D. I. Fletcher,
S. J. Haswell, E. Pombo-Villar, B. H. Warrington, P. Watts, S. Y.
F. Wong and X. Zhang, Tetrahedron, 2002, 58, 4735.
5 M. Schilling, W. Nigge, A. Rudzinski, A. Neyer and R. Hergenro¨der,
Lab. Chip, 2004, 4, 220; R. J. Jackman, T. M. Floyd, R. Ghodssi,
M. A. Schmidt and K. F. Jensen, J. Micromech. Microeng., 2001, 11,
263; H. Lu, M. A. Schmidt and K. F. Jensen, Lab. Chip., 2001, 1, 22.
6 K. F. Jensen, Chem. Eng. Sci., 2001, 56, 293.
7 M. W. Losey, R. J. Jackman, S. L. Firebaugh, M. A. Schmidt and
K. F. Jensen, J. Microelectromech. Sys., 2002, 11, 709.
8 P. Fu¨gedi, A. Lipta´k and P. N. Neszme´lyi, Carbohydr. Res., 1982,
107, C5; A. Vasella, C. Witzig, C. Waldraff, P. Uhlmann,
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76, 2847.
Unlike batch methods, which are challenged by the difficulty of
handling microliter quantities of volatile solvents and the
possibility of external contamination, the enclosed microreactor
system serves to rapidly obtain comprehensive information about
a given transformation. With a single preparation of reagents, 44
reactions were completed at varying temperatures and reaction
times requiring just over 2 mg of glycosylating agent for each
reaction.
580 | Chem. Commun., 2005, 578–580
This journal is ß The Royal Society of Chemistry 2005