First gram-scale synthesis of a heparin-related dodecasaccharide

Article abstract of DOI:10.1021/ol303112y

The first example of a gram-scale synthesis of a structurally defined, heparin-related dodecasaccharide is reported. An iterative 14-step process using an iduronate donor disaccharide delivers >1g quantities of the dodecasaccharide sequence [GlcNS-IdoA2S]₆-OMe in 15% overall yield from the reducing terminal disaccharide, a 2 orders of magnitude increase in scale for access to synthetic heparanoid dodecasaccharide mimetics. The synthesis also delivers multigram amounts of the protected oligosaccharides from tetra- through to dodecasaccharide.

Full text of DOI:10.1021/ol303112y

ORGANIC
LETTERS
2013
Vol. 15, No. 1
88–91
First Gram-Scale Synthesis
of a Heparin-Related Dodecasaccharide
Steen U. Hansen,† Gavin J. Miller,† Gordon C. Jayson,‡ and John M. Gardiner*,†
Manchester Institute of Biotechnology, School of Chemistry, Faculty of EPS,
The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.,
and School of Cancer and Enabling Sciences, The University of Manchester,
Wilmslow Road, Manchester M20 4BX, U.K.
gardiner@manchester.ac.uk
Received November 12, 2012
ABSTRACT
The first example of a gram-scale synthesis of a structurally defined, heparin-related dodecasaccharide is reported. An iterative 14-step process
using an iduronate donor disaccharide delivers >1g quantities of the dodecasaccharide sequence [GlcNS-IdoA2S]6-OMe in 15% overall yield from
the reducing terminal disaccharide, a 2 orders of magnitude increase in scale for access to synthetic heparanoid dodecasaccharide mimetics. The
synthesis also delivers multigram amounts of the protected oligosaccharides from tetra- through to dodecasaccharide.
Heparin and heparan sulfate (H/HS) are highly charged,
ubiquitous, naturally occurring glycosaminoglycans (GAGs)
which are involved in regulating a wide range of biologically
important cellular signaling events that control a variety of
biological functions.1
Capabilities for the provision of structurally defined
GAG sequences from the H/HS sulfate family are at the
forefront of current oligosaccharide synthetic challenges.
Considerations in this area relate to the significance of
sequence length and the relevance of both the level and
location of final sulfations. Despite advances in separation
techniques,2 access to structurally specific heparin-related
oligosaccharides sufficient for biological investigations is
incumbent on chemical synthesis. A range of synthetic ap-
proaches have been described to generate heparin-related
fragments both in solution and also on solid support,3
including recent chemoenzymatic approaches.4 Such syn-
theses have previous requirements for lengthy routes and,
combinedwiththe inclusion of iduronate asthe uronicacid
common in many biologically relevant target types, have
thus limited accessibility to small scale, particularly for the
longest sequences. The longest heparin-related synthetic
fragments reported to date have thus been high profile
examples of small-scale syntheses of dodecasaccharides
yielding tens of milligrams.1e,h The challenges facing syn-
thesis of H/HS oligosaccharides of this type on scales of 1ꢀ2
orders of magnitude above prior reports (i.e. hundreds of
milligrams to gram scale) include the availability of eco-
nomic syntheses of iduronate building blocks on scale and
enabling multiple sequential glycosylations which retain
† Manchester Institute of Biotechnology.
‡ School of Cancer and Enabling Sciences.
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r
10.1021/ol303112y
Published on Web 12/14/2012
2012 American Chemical Society

both high chemical yields and anomeric selectivities at
each glycosylation step.
Strategies employed previously which have succeeded in
delivering deca- and dodecasaccharide level targets have
included the use of glucosamine-based donor disaccha-
rides and monosaccharides (thereby introducing both
GlcN-IdoA and IdoA-GlcN linkages during homologation)
and the introduction of iduronate using iditol with later-stage
oxidation of iditol to iduronate.
Access to scalable syntheses of such long IdoA-containing
oligosaccharides must be underpinned by access to suffi-
cient quantities of IdoA-containing precursors. We re-
cently described a new route for the synthesis of iduronic
building blocks suitable for oligosaccharide homologa-
tion, specifically for multigram access to key disaccharide
iduronate thioglycoside donors.5
This significantly facilitates the viability of up-scaling
the synthesis of long sequence heparin-related oligosac-
charides, both through significant improvement in economic
scalability of the iduronate reagents upstream and, most
importantly, by directing homologation through the use of
shelf-stable thioglycoside-based disaccharides. The use of
uronic thioglycosides in both glucuronic6 and iduronic
systems3e has been the subject of a number of evaluations.
The utility of monosaccharide iduronate thioglycosides has
been demonstrated previously and for the use of disaccha-
rides this is also an important factor for scalable syntheses as
it circumvents using reactive trichloracetimidates as donors,
which are not as readily amenable to storage.
Figure 1. Iteration strategy for scalable HS oligomer synthesis.
Our strategy was to use an acceptor reducing end methyl
glycoside capped disaccharide of type A and a disaccharide
thioglycosidedonor moduleof type B asthetwo basicunits
for assembling oligosaccharides (see Figure 1). Adding on
the disaccharide unit B in an iterative two-step per cycle
process (glycosylation/deprotection at O-4) would give
access to a sequence of defined length oligosaccharides.
The fully protected oligomer could then be deprotected
and sulfated to yield the target dodecasaccharide.
Scheme 1. Preparation of Reducing End-Cap Disaccharide 3
and Iterative Disaccharide Donor 5
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M.; Fugedi, P. Carbohydr. Res. 2008, 343, 596. (g) De Paz, J. L.; Angulo, J.;
Lassaletta, J. M.; Nieto, P. M.; Horcajo, M.-R.; Lozano, R. M.; Gallego,
The capability to approach such a dodecasaccharide
synthesis on scale was underpinned by our route to multi-
gram batches of both the disaccharide donor type B, i.e. 5
(Scheme 1), from iduronate 4, and initiator disaccharide
type A, i.e. 3.5 We chose the TCA (trichloroacetyl) ester
group asthe O-4 protecting group (replacing benzylic ether
protection) as this can be removed under mild basic
conditions to which the other esters present are stable.
Multigram synthesis of the iduronate methylglycoside 2
allowed us to prepare the novel initiator disaccharide 3 in
good yield using standard glycosylation conditions. The
reaction gave a 6:1 (R/β) mixture which could be separated
by column chromatography. With multigram access to the
two requisite disaccharide modules, the iterative assembly
of longer oligosaccharides was thus pursued.
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G. G.; Martın-Lomas, M. ChemBioChem 2001, 2, 673. (h) Bonnaffe, D. C.
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T.-C.; Zulueta, M. M. L.; Zhong, Y.-Q. Org. Biomol. Chem. 2012, 10,
760. (m) Lee, J.-C.; Lu, X.-A.; Kulkarni, S. S.; Wen, Y.-S.; Hung, S.-C.
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A facile deprotection of the glucosamine O-4 of dis-
accharide 3 afforded the reducing-end-capping moiety
which was then glycosylated with pure R-anomer donor
disaccharide 5 to afford tetrasaccharide 7 in very good
yield on multigram scale (4.5 g, 88%). Facile deprotection
of 7 was completed to afford tetrasaccharide acceptor 8 in
excellent yield (Scheme 2).
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Org. Lett., Vol. 15, No. 1, 2013
89

Scheme 2. Synthesis of Acceptor Tetrasaccharide 8
The selection of trichloroacetyl as the iterative growing-
terminus O-4 protecting group proved vital in ensuring
reproducible efficiency for chain elongation. Thus, with tetra-
saccharide acceptor 8 in hand, a deprotection/coupling two-
step cycle of iterations using thioglycoside donor 5 was com-
pleted (Scheme 3). For these remaining iterations a mixture of
R/β thioglycoside donor 5 was employed which proved to be
equally efficient and selective in the glycosylation reactions.
Figure 2. TLC analysis of oligosaccharide polarity versus chain
length and eluent. (A) Oligosaccharides 3, 7, 9, 11, 13, and 15
using EtOAc/hexane 1:2 as eluent. (B) Oligosaccharides 3, 7, 9, 11,
13, and 15 cospotted with relevant acceptor starting materials 6, 8,
10, 12, and 14 using EtOAc/hexane 1:2 as eluent. (C) Oligosaccha-
rides 3, 7, 9, 11, 13, and 15 cospotted with relevant acceptor starting
materials 6, 8, 10, 12, and 14 using toluene/acetone 10:1 as eluent.
More importantly the difference in polarity between
acceptor and product oligosaccharides was significantly
increased (relative to EtOAc/hexanes) and only changed
slightly with oligosaccharide length. This ensured that
high purity at each cycle could be achieved with a single
standard silica flash chromatography column to obtain
multiple grams of pure oligosaccharide products as well as
very effective recovery of unreacted (and thus recyclable)
acceptor oligosaccharide. Such efficient recovery of accep-
tors is particularly important on scale.
Scheme 3. Iterative Scale Route to Dodecasaccharide 15
Protected dodecasaccharide 15 was then elaborated via
deesterification, O-sulfation, azide reduction/debenzyla-
tion, and final N-sulfation (Scheme 4). The debenzylation
required a relatively large amount of Pd(OH)2/C catalyst
and heating for 2 days to go to completion (atmospheric
pressue of hydrogen).
This iteration approach proved efficient on multiple
grams at each step and remained extremely reliable with
yields for each glycosylation step not falling below 80%
and each deprotection step averaging 88% per cycle. Each
iteration was efficiently performed using only a slight
excessofdonor(1.04ꢀ1.23equiv). Thisprovidedprotected
decasaccharides 13 and 14 on 4.5ꢀ5.0 g scales.
Scheme 4. Deprotection and Sulfation to Dodecasaccharide 17
With such large scale access to decasaccharide 14 avail-
able for the first time, a further iterative cycle using 5
provided the uniformly protected dodecasaccharide back-
bone 15 (4.8 g scale).
One major issue necessary to resolve during the synthesis
of such extended oligosaccharides on large scale was to
ensure high purity at each iteration to avoid accumulation
of side products. TLC analysis showed that the polarity of
the oligosaccharides increased as a function of chain length
(Figure 2A). More importantly the difference in polarity
between the starting material acceptor and the product
decreased significantly in longer oligomers using EtOAc/
hexaneeluent mixtures(Figure2B). Itwas found thatupon
switching to toluene/acetone eluent mixtures the polarity
of different oligosaccharides was much less dependent on
chain length (Figure 2C).
This provided the dodecasaccharide 17 on 1.1 g scale
in a total of 14 steps from the initiator disaccharide
methylglycoside 3in an overall yield of 15%. This equates to
approximately a gram-to-gram throughput from disaccharide
3 to final dodecasaccharide 17 and is the first reported exam-
ple of the synthesis of any heparin-related dodecasaccharide
on such a scale. It is also a significant increase (2 orders of
magnitude) in scale provision over prior H/HS-related
dodecasaccharide syntheses.1e,h
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Org. Lett., Vol. 15, No. 1, 2013

Figure 3. (a) MALDI-TOF mass spectrum of 15; (b) 1H NMR (400 MHz, D2O) assignment of dodecasaccharide 16.
Characterization and confirmation of high purity for
these long sequences are important, particularly when
establishing a large scale route. The final protected dode-
casaccharide was analytically pure, and in addition to
NMR data, MALDI-TOF mass spectroscopy provided a
definitive [M þ Na]þ peak at m/z 4710 (Figure 3a).
disaccharides are effective intermediates for large scale
heparanoid syntheses. This, combined with a multigram
access to the two disaccharide components and an efficient
iterative process, has enabled us to report here a very sig-
nificant increase in scale of synthetic access to a biologically
active/relevant heparin-related oligosaccharide for the first
time. We envision this modular and iterative approach can
now be further extended to provide a general strategy to a
variety of heparin-like oligosaccharides with programmed
sulfation patterns and of different sequence lengths. This
approach also demonstrates the accessibility of such materi-
als for the first time on a scale suitable for in vivo evaluations
of candidates emerging from in vitro investigations.
The dodecasaccharides16and 17thence derived from15
consist of repeating disaccharide units, so it was expected,
after deprotections/sulfation, and particularly with a methyl
1
glycoside terminus, that H NMR spectra would show a
significant overlap of similar protons. However 16 showed
distinctive peaks for the reducing and nonreducing end
monosaccharide units (A and L rings) well-separated from
A
the larger clusters (Figure 3b). In particular H4L, H1
,
Acknowledgment. The MRC [G0601746and G902173],
CRUK [C2075/A9106] are thanked for funding, and the
EPSRC National Mass Spectrometry Service, Swansea are
also thanked for mass spectroscopic analyses.
and H5A could be identified and compared to the integra-
tion of the clearly defined Ido2A and GlcN anomeric
signal groupings. The spectrum is a good indication of
high purity (>95%).
Proof of purity at this stage was important, as the
purification of deprotected and sulfated saccharides on
such scales is difficult, and was achieved here using size-
exclusion chromatography.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
The reliable selectivity and yields of this route on multigram
scale throughout demonstrate that iduronate thioglycoside
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 1, 2013
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