Glycosylation with in situ separation: Carbohydrate chemistry on a TLC plate

Article abstract of DOI:10.1039/b509417h

Iodine vapour promotes thioglycoside-based glycosylation chemistry on TLC plates, which in turn permits in situ separation by conventional elution with solvent. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b509417h

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C O M M U N I C A T I O N
Glycosylation with in situ separation: carbohydrate chemistry on a
TLC plate†
Balaram Mukhopadhyay,a Peter Cura,b K. P. Ravindranathan Kartha,‡a Catherine H. Bottingb
and Robert A. Field*a
a Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of
East Anglia, Norwich, UK NR4 7TJ. E-mail: r.a.field@uea.ac.uk
b Centre for Biomolecular Sciences, University of St Andrews, St Andrews, UK KY16 9ST
Received 5th July 2005, Accepted 9th August 2005
First published as an Advance Article on the web 23rd August 2005
Iodine vapour promotes thioglycoside-based glycosylation
chemistry on TLC plates, which in turn permits in situ
separation by conventional elution with solvent.
no glycoside formation was apparent, as judged by comparison
with authentic a/b-disaccharide, 4, produced by conventional
solution phase coupling. It was evident that the moisture present
in the silica caused the hydrolysis of the donor. When a similar
reaction was carried out on pre-dried silica gel plates, traces of
disaccharide products, 4, became apparent, but we were unable
to produce useful quantities of material for characterisation. The
principle product of the reaction still proved to be hemiacetal
3,20 along with smaller amounts of a compound that co-ran with
authentic per-O-benzylated 1,1ꢀ-linked Gal–Gal disaccharide
5.21 Repetition of the experiment (fifteen 1.5 × 10 cm plates)
and extraction of relevant material from the silica (with CHCl3)
produced sufficient material for accurate mass characterisation,
confirming formation of the 1,1ꢀ-linked sugar.22 NMR and mass
spectral data for all disaccharides isolated are included in the
electronic supplementary information.
Thorough drying of silica TLC plates is not straightforward;
when heated at more than 100 ◦C for a couple of hours, the silica
gel on the plate detached from the glass surface. Considering
this point, and taking into account that hydrogen iodide
generated in situ might lead to acid-catalysed product hydrolysis,
we were minded to investigate alumina as an alternative to
silica. Glass-backed alumina TLC plates are robust and remain
intact after drying at 200 ◦C for 3 h. The result changed
dramatically when pre-dried alumina plates were employed in
on-plate glycosylation reactions; reaction between thioglycoside
1 and galactoside primary alcohol 2 led to approximately 40%
conversion to the desired disaccharides 4 (Fig. 2). In an attempt
to increase the conversion, reaction times were varied. However,
for donor 1 the most productive results were obtained from
approx. 30–60 min reactions. In the reaction of thioglycoside
1 and alcohol 2, repetition of the on-plate experiment (fifteen
1.5 × 10 cm plates) and extraction of relevant material from
the TLC plates19 produced sufficient material for accurate mass
measurement as well as for 1H and 13C NMR characterisation.
The disaccharide obtained proved to be an approx. 2 : 1 a :
b-mixture, as judged by integration of 1H NMR signals for
the methyl groups of the reducing terminal sugars (assignment
assisted by the relative intensity of anomeric carbon signals in
the 13C NMR data).
Over the past twenty years, the largely neglected biological
function of carbohydrates and glycoconjugates has received
much attention. However, the precise molecular detail of the role
of such biomolecules remains to be established in many, perhaps
most, cases.1 There is, therefore, a need for methodologies
to provide rapid access to structurally diverse carbohydrate
structures. Synthetic procedures have developed substantially
of late,2 with orthogonal glycosylation strategies offering scope
for minimising manipulations at the anomeric centre during
multi-step synthesis,3 as does the application of reactivity tuning4
based on the concept of ‘armed’ and ‘disarmed’ glycosylation
reagents.5 Further extension of this strategy has led to ‘pro-
grammable’ oligosaccharide synthesis.6 Perhaps the ultimate in
oligosaccharide target synthesis has been achieved by Seeberger
and co-workers, who adapted technologies from peptide syn-
thesis to develop instrument-based, automated oligosaccharide
synthesis.7 Further work from the same group has explored
microreactors for optimisation of glycosylation chemistry.8
On a more general front, “random” strategies for preparing
carbohydrate libraries have been explored.9
This laboratory has a long-standing interest in the use of
iodine in carbohydrate chemistry,10 particularly in relation to
glycosyl donor activation.11 Since iodine is readily vapourised
and is often used to detect organic compounds on TLC
plates, we reasoned that thioglycosides co-spotted with sugar
alcohols onto TLC plates might give rise to glycosides on
exposure to iodine vapour. We were encouraged to follow this
line by literature reports of low-tech chemistries for accessing
compound libraries12 and by studies showing silica-supported,
solvent-free synthesis of nucleosides,13 the impact of silica on
the AgNO3/NCS-mediated cyclisation of an alcohol onto a
dithioketal14 and on NBS-promoted glycosylation reactions,15
and the established use of silver silicate as a heterogeneous
promoter of SN2-like glycosylation processes.16 Here we report a
proof-of-concept for iodine vapour-promoted glycosylation on
TLC plates employing armed thioglycosides as donors.17,18
In initial experiments, armed thiogalactoside donor 1
and primary alcohol acceptor methyl 2,3,4-tri-O-benzyl-b-D-
galactopyranoside 2 were co-spotted on a standard analytical
silica TLC plate and exposed to iodine vapour.19 After 30 min,
the plate was removed from the reaction vessel and developed
with organic solvent. Subsequent charring of the plate with
ethanolic sulfuric acid showed only the conversion of the thio-
glycoside donor to the corresponding hemiacetal, 3 (Fig. 1);20
In light of the above success, a range of armed donor
thioglycosides and variously protected acceptor sugar alcohols
were also investigated. A combination of 3 donors (1, 6, 7)
and 4 acceptors (2, 8, 9, 10) was assessed (Table 1); in 6 of
the 12 reactions investigated, conversion was sufficient (30–
60%) to enable characterisation of the disaccharide products
formed. In some of the other reactions investigated, disaccharide
formation was evident but tlc separation was poor.23 The
relatively mild nature of these on-plate reactions is evident from
their compatibility with potentially acid labile protecting groups
in all of the acceptor alcohols investigated. Reactions with the
‘armed’ thioglycosides of deoxysugar L-fucose, 7, gave maximum
conversion within 15–30 minutes. These observations can be ra-
tionalized considering the higher reactivity of the thioglycosides
† Electronic supplementary information (ESI) available: experimental
procedures, NMR spectra. See http://dx.doi.org/10.1039/b509417h
‡ Present address: Department of Medicinal Chemistry, National Insti-
tute of Pharmaceutical Education and Research, Sector 67, SAS Nagar,
Punjab, 160062, India.
3 4 6 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 4 6 8 – 3 4 7 0
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Fig. 1 Glycosylation on TLC plates. A–C on a silica plate, D–F on an alumina plate. A – Hemiacetal 3; B – reaction of donor 1 and acceptor 2; C –
donor 1 and acceptor 2; D – donor 1 and acceptor 2; E – reaction of donor 1 and acceptor 2 on an alumina plate; F – authentic a/b-disaccharides 4.
Table 1 Results of on-plate glycosylation reactions with armed thioglycoside donors and assorted sugar alcohol acceptors. In all cases conversion
to disaccharide was in the 30–60% range. Anomeric ratios, as judged by NMR spectroscopy, are given below
Acceptors
Donors
(4) 2 : 1
N. i.
(11) 1:1
N. i.
(12) 2 : 1
N. i.
(13) 3 : 1
N. i.
(14) 1 : 1
N. i.
N. i.
(15) 1 : 1
N.i. - no disaccharide isolable from tlc plate.
of NMR assignment after acetylating the disaccharide obtained
(downfield shift of H-2 of galactose from 3.65 to 5.03 ppm upon
acetylation), these were identified as the anomeric 1,6-linked
sugars, as it to be expected given the greater reactivity of Gal
6-OH over 2-OH.24
In conclusion, armed thioglycoside donor substrates spotted
onto TLC plates can be activated in situ by iodine vapour. Whilst
poor results were obtained on silica, alumina plates gave rise to
coupling efficiencies of up to ca. 50–60%, enabling the synthesis
of a range of disaccharides and their subsequent separation
and characterisation on the tens of micrograms scale. This
microscale, iodine-mediated glycosylation strategy on a TLC
plate minimizes the time and labour of the glycosylation reaction
and subsequent purification process. With further optimisation,
this approach offers scope for multiple parallel synthesis of
glycoside libraries; alternative formats for scale-up are currently
being investigated.
Fig. 2 Model glycosylation reaction conducted on TLC plates and
promoted by iodine vapour.
Acknowledgements
This work was supported by the EPSRC, the Weston Founda-
tion, the Wellcome Trust and the University of St Andrews. We
thank Dr Alan Haines for useful discussions during the course
of this work and Colin Macdonald for assistance with acquiring
NMR spectra. We gratefully acknowledge the EPSRC Mass
Spectrometry Service Centre, Swansea for invaluable support.
of deoxy-sugars. Of a number of secondary alcohol acceptors
investigated (not shown), only the 6-deoxy acceptor 9 gave
identifiable disaccharide products, consistent with the greater
reactivity of 6-deoxysugar acceptors. In experiments with diol
11, only disaccharide products were identifiable. On the basis
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 4 6 8 – 3 4 7 0
3 4 6 9

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17 We have previously shown that armed, but not disarmed, thio-
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18 Other armed glycosyl donors (glycosyl bromides and glycosyl-
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from quenched donor were obtained.
19 Typical procedure for glycosylation on a TLC plate: a solution
of donor (0.12 lmol) and acceptor (0.10 lmol) in DCM (10 lL)
was spotted (1 lL) on a pre-dried TLC plate using a micro-pipette
(alumina plates were heated at 200 ◦C for 2 h and allowed to cool
over P2O5 in a desiccator prior to use). The plate was placed in the
main chamber of the apparatus (see the electronic supplementary
information) and dried over P2O5 under reduced pressure for 1 h.
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in the adjacent flask. The TLC plate was kept exposed to iodine
vapour for 30–60 min (Table 1). After the reaction, the TLC plate
was kept under vacuum for 30 min to remove iodine vapour and it was
developed by using a suitable solvent system (e.g. n-hexane–EtOAc
2 : 1). Compounds were identified under UV-light, a relevant band of
silica was scratched from the plate, triturated with CH2Cl2–MeOH
(1 : 1) and filtered through Celite. The filtrate was evaporated and
material obtained was used directly for NMR and mass spectrometry
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22 It did not immediately prove possible for this particular experiment
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23 A change to use of more lipophilic protecting groups offers scope
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3 4 7 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 4 6 8 – 3 4 7 0