HPLC-assisted automated oligosaccharide synthesis

Article abstract of DOI:10.1021/ol301105y

A standard HPLC was adapted to polymer supported oligosaccharide synthesis. Solution-based reagents are delivered using a software-controlled solvent delivery system. The reaction progress and completion can be monitored in real time using a standard UV detector. All steps of oligosaccharide assembly including loading, glycosylation, deprotection, and cleavage can be performed using this setup.

Full text of DOI:10.1021/ol301105y

ORGANIC
LETTERS
2012
Vol. 14, No. 12
3036–3039
HPLC-Assisted Automated
Oligosaccharide Synthesis
N. Vijaya Ganesh, Kohki Fujikawa, Yih Horng Tan, Keith J. Stine,* and
Alexei V. Demchenko*
Department of Chemistry and Biochemistry and the Center for Nanoscience,
University of Missouri;St. Louis, One University Boulevard, St. Louis,
Missouri 63121, United States
kstine@umsl.edu; demchenkoa@umsl.edu
Received April 25, 2012
ABSTRACT
A standard HPLC was adapted to polymer supported oligosaccharide synthesis. Solution-based reagents are delivered using a software-
controlled solvent delivery system. The reaction progress and completion can be monitored in real time using a standard UV detector. All steps of
oligosaccharide assembly including loading, glycosylation, deprotection, and cleavage can be performed using this setup.
Solid-phase synthesis1,2 has been widely utilized in the
routine preparation of oligopeptides3 and oligonucle-
otides.4 The use of polymer supports in oligosaccharide
synthesis has also been reported.5À7 The use of these tech-
niques helps to expedite the synthesis of oligosaccharides
and glycoconjugates8À16 by minimizing the necessity for
purifying reaction intermediates and simplifying the re-
moval of excess reagents that is usually achieved by filtra-
tion. To expedite solid-phase oligosaccharide synthesis,
Seeberger et al. developed an automated approach, which
was first accomplished by using a modified peptide
synthesizer17À19 and very recently extended to “the first
fully automated solid-phase oligosaccharide synthesizer”.20
Despite being a relatively new technique, it has already been
applied to the synthesis of a variety of oligosaccharide
sequences.20À23 Other recent enhancements of the sup-
ported synthesis of oligosaccharides include, but are not
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P.; Seeberger, P. H. Chem. Sci. 2012, 3, 1617–1622.
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r
10.1021/ol301105y
Published on Web 05/30/2012
2012 American Chemical Society

limited to, approaches employing fluorous tag,24,25 ionic
liquid,26À28 nanoparticle,29 and nanoporous gold30 supports.
As a part of an ongoing research effort, presented herein
is the development of a new HPLC-based automated syn-
thesis. To obtain clear evidence of the advantages of the new
technology in comparison to the state-of-the-art polymer-
supported synthesis, we chose the most common approaches
for all aspects of our synthesis:5,7 a solution-based trichloro-
acetimidate donor, a Tentagel resin-bound glycosyl acceptor
attached via the anomeric center, TMSOTf as the activator/
promoter, CH2Cl2 as the reaction solvent, and Fmoc as the
temporary hydroxyl protecting group.11,16,31
In accordance with the traditional manual synthesis, a
glycosyl acceptor bound to the polymer beads is shaken
with an excess of the solution phase glycosyl donor and
promoter (Figure 1A). Periodic monitoring of the solution
phase by TLC can provide information on whether the
glycosyl donor still remains. The completeness of the
coupling can be determined experimentally by performing
test reactions, such as the Kaiser test,32 or by cleaving the
product off the polymer support followed by characteriza-
tion. Routinely, the coupling step is repeated two or three
times using fresh reagents. Alternatively (or as necessary),
the remaining hydroxyls can be capped to ensure that they
will not interfere with subsequent steps.
The new setup is based on an unmodified HPLC instru-
ment, which is readily available in practically any synthetic
or analytical laboratory. In brief, a chromatography col-
umn was packed with the preswelled polymer resin. The
column was then connected to the HPLC system contain-
ing a pump (a three-head pump was used), a detec-
tor (a variable UV range detector), and a computer with
standard HPLC-operating software installed (Figure 1B).
The column was loaded with the glycosyl acceptor and
purged with solvent, and then two separate solutions
containing glycosyl donor and promoter were delivered
concomitantly. After a relatively short reaction time,
typically 30À60 min, the system was purged (washed) with
solvent. At this time, the resin is loaded with the disacchar-
ide derivative that can be either cleaved off of the polymer
support or the oligosaccharide elongation can be contin-
ued via alternating deprotection/glycosylation steps. All
steps can be monitored using a standard HPLC detection
system set to record changes in the UV absorbance of the
Figure 1. Comparison of the manual polymer-supported synthe-
sis (A) with the new automated setup described here (B).
solution eluting off the column. A solution of reagents can
be recirculated to reduce the amount required for each
transformation.
During our initial experimentation, the attachment of
glycosyl acceptor precursor 1a (3.0 equiv based on the
theoretical loading capacity of the resin) to TentaGel MB-
NH2 resin was accomplished using a conventional setup in
the presence of EDC (3.0 equiv) and DMAP (1.0 equiv) in
CH2Cl2. The Kaiser test32 was conducted on the resin to
ensure complete loading (48 h). The resin was then treated
with 10% trifluoroacetic acid in wet CH2Cl2 to afford
polymer bound acceptor 2a (30 min, loading 0.29 mol/g).
Next, resin 2a (190 mg, 55 μmol of the glycosyl acceptor)
was swelled in CH2Cl2 for 4À16 h, loosely packed in the
Omnifit SolventPlus chromatography column equipped
with an adjustable end-piece (http://www.omnifit.com)
and the column was integrated into the HPLC.
The relative inefficiency of this protocol motivated us to
use the HPLC experimental setup that has allowed us to
expedite the initial loading. For instance, loading using
a recirculating solution containing 1a (5 equiv), EDC
(5 equiv), and DMAP (1 equiv) in CH2Cl2 (Pump B,
1.0 mL/min) was much more effective and the desired loading
was achieved in 8 h. After that, the system was purged with
CH2Cl2 for 10 min (Pump A, 2.0 mL/min flow rate)
followed by detritylation that was accomplished using
recirculating TFA/CH2Cl2/H2O (10/88/2, v/v/v, Pump C,
1.0 mL/min, 5 min) to afford 2a. The system was purged
with CH2Cl2 for 10 min (Pump A, 2.0 mL/min flow rate).
Reagent bottles, one containing a 39 mM solution of
glycosyl donor 3a33 in CH2Cl2 and another one containing
a 0.28 M solution of TMSOTf in CH2Cl2, were connected
to inlets for pumps B and C, respectively. Pumps B/C were
programmed to deliver the mixed solution of donor/pro-
moter concomitantly in the ratio 4/1 (v/v) at the total flow
rate 0.3 mL/min. After 60 min (18 mL total), pumps B and C
were stopped and by this time ∼10 mol equiv of donor 3a
ꢀ
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Org. Lett., Vol. 14, No. 12, 2012
3037

(562 μmol) has passed through the column. It should be
noted that the amount of reagents used for this initial study
were chosen to mimic that used in the conventional poly-
mer-supported synthesis. Apparently, the flow rate and the
donor/promoter ratio can be easily adjusted by simple
reprogramming of the pump operation. Since fresh re-
agents are delivered constantly, this eliminates the need for
multiple reiterations of glycosylation, common in manual
and other automated techniques. The column was purged
with CH2Cl2 (pump A, 2.0 mL/min) for 10 min. The
formation of disaccharide 4a was determined by cleaving
off the sugar molecule from the resin using a 0.1 M solution
of NaOCH3 in CH3OH/CH2Cl2 (5 mL, 1/1, v/v, pump C,
1.0 mL/min, 60 min) carried out by continuous recircula-
tion followed by washing with CH3OH (2.0 mL/min) for
10 min. Standard (see the Supporting Information (SI))
neutralization and acetylation (Ac2O/pyridine) afforded
product 4a in 98% yield (Table 1, entry 1).
Table 1. Exploratory Comparative Study of the Coupling
Efficiency of Glycosyl Donors 3aÀf with the Polymer-Bound
Acceptor 2a
Encouraged by this result, we explored other donors
using essentially the same experimental setup and reaction
time. Thus, glycosidation of galactosyl donor 3b34 was
equally effective and disaccharide 4b was isolated in 96%
yield(Table 1, entry 2). Glycosidations using mannosyl and
lactosyl donors 3c35 and 3d36 were somewhat less efficient,
and the resulting oligosaccharides 4c and 4d were isolated
in 78% and 67% yield, respectively (entries 3 and 4).
Finally, glycosidations of glucosyl donors 3e and 3f
equipped with an easily removable Fmoc protecting group
at the C-6 and C-4 positions, afforded 4a in 95% and 92%
yield, respectively (entries 5 and 6). Overall, these results
were very indicative of the high efficiency of the new
experimental setup based on HPLC, but that generalized
reaction conditions (60 min) may not be universally
applicable across all sugar series. This result implies
that an extended reaction time would be beneficial for
driving the glycosidation the less reactive37À39 of man-
nosyl donor 3c to completion, as well as for the reaction
with lactose imidate 3d.
This called for further study, and we noted that the
progress of the reaction could be determined from changes
in the UV absorbance of the mixture eluting off the column
(measured at 254 nm) in comparison to that measured for
the initial 0.39 mmol solution of the donor that is entering
the column and the expected total absorbance of the donor
and TMSOTf (Figure 2A). Once no change is detected (the
detector trace reaches a plateau), this can serve as an
indication that the reaction had ended. Similar monitoring
of the washing stage of the process can provide the desired
information about its completion, the trace reaches the
baseline corresponding to the standard absorbance of neat
CH2Cl2.
Real-time monitoring has helped us to optimize the
reaction time and reduce the amount of reagents required.
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Figure 2. Idealized sketches for the UV detector monitoring: (A)
glycosylationÀwashing steps; (B) Fmoc deprotectionÀwashing.
3038
Org. Lett., Vol. 14, No. 12, 2012

deprotection of the Fmoc group in 5a was carried out as
follows: purge with DMF for 1 min (2.0 mL/min) and then
pass a 20% solution of piperidine in DMF (0.5 mL/min).
The release of the dibenzofulveneÀpiperidine adduct was
monitored by the UV detector at 312 nm. Based on the real-
time Fmoc deprotection curve (Figure 2B), we were able to
reduce the reaction time to 5 min (step 3).
Table 2. Experimental Data for the HPLC-Assisted Synthesis of
Pentasaccharide 8
This transition was followed by a 10-min wash with
CH2Cl2 (step 2) to afford disaccharide acceptor 5b. The
synthesis of trisaccharide 6 was continued using polymer-
bound disaccharide acceptor 5b and donor 3e via sequential
execution of steps 1À2À3À2 (glycosylateÀwashÀdeprotectÀ
wash). The same sequence was repeated to obtain tetrasac-
charide 7. Finally, pentasaccharide was obtained by steps 1
and 2 followed by the cleavage step 4 using a recirculating
0.1 M solution of NaOCH3 in CH3OH/CH2Cl2 followed
by acetylation to afford compound 8 in 62% yield. A
comparative yield of pentasaccharide 8 was obtained
using the manual setup, but the experimental time was
significantly longer.
In conclusion, we developed a new technology for
automated oligosaccharide synthesis that can now be based
on practically any standard HPLC or LC instrumentation.
The new technology offers the following advantages in
comparison to that of manual oligosaccharide synthesis on
polymer supports: faster reaction times, real-time reaction
monitoring using an HPLC detection system, and that all
steps and sequences can be automated using standard HPLC-
managing computer software. This work was greatly
inspired by and is complementary to other automated
approaches19,25,40,41 developed for the synthesis of oligo-
saccharides and related compounds including on-
column,42 flow-through,24,43 microfluidic,44 and other
related processes.45,46 Further optimization of the HPLC-
based technology and its application to other platforms and
targets is currently underway.
Acknowledgment. This work was supported by awards
from the NIGMS (GM090254 and GM077170). Dr. Winter
and Mr. Kramer (UM;St. Louis) are thanked for HRMS
determinations.
Thus, the optimized reaction times for glycosylation of
acceptor 2a using a 0.3 mL/min combined flow rate for the
donor and promoter solutions (4/1, v/v) are as follows: 3a,
e,f (30 min), 3b (25 min), 3c (70 min), and 3d (85 min).
Typical washing times for all reactions at 1.0 mL/min of
CH2Cl2 are 5À10 min. We also found that slight lowering
the concentration of glycosyl donors or performing partial
recirculation of the “used” solution by connecting the
column outlet with the pump intake (recirculation) is
nearly as efficient as using the standard “fresh” 39 mmol
solution. However, UV monitoring of experiments where-
in reagents are continuously recirculated is cumbersome.
Having optimized model glycosylations we decided to
undertake the synthesis of an oligosaccharide chain using
the UV detection system. This synthesis began with glyco-
syl acceptor 2b and donor 3e equipped with the temporary
Fmoc protecting group. The standard glycosylation pro-
tocol (60 min, step 1, Table 2) followed by a 10-min wash
(step2) led totheformationofdisaccharide 5a. Subsequent
Supporting Information Available. Experimental pro-
1
cedures, extended experimental data, H and 13C NMR
spectra for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 12, 2012
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