Divergent synthesis of sialylated glycan chains: Combined use of polymer support, resin capture-release, and chemoenzymatic strategies

Article abstract of DOI:10.1002/anie.200500777

(Chemical Equation Presented) Carbo loading! The synthesis of α(2,3)- or α(2,6)-sialylated biantennary glycans is possible with a new approach. The common precursor 1 was synthesized with a soluble polymer support strategy (a) in combination with a resin capture-release protocol. Hexasaccharide 1 can then be diverged to various polysaccharides by enzymatic glycosylation (b). Bn = benzyl, TBS = tert-butyldimethylsilyl.

Full text of DOI:10.1002/anie.200500777

Communications
Carbohydrate Chemistry
Divergent Synthesis of Sialylated Glycan Chains:
Combined Use of Polymer Support, Resin
Capture–Release, and Chemoenzymatic
Strategies**
Shinya Hanashima, Shino Manabe, and Yukishige Ito*
As key constituents of cell-surface glycoproteins and glyco-
sphingolipids, sialic acid containing glycan chains are involved
in a variety of important physiological events such as cell–cell
recognition, adhesion, and signal transduction.[1] It has been
reported that the increased levels of sialic acid and sialyl-
transferase expression in tumor cells are closely related to
their metastatic potential and malignancy progression.[2] Sialic
acid residues of cell-surface glycoconjugates are also recog-
nized by virulent proteins such as influenza virus hemagglu-
tinins, which initiate the viral invasion into the host cell
cytoplasm.[3] In cell-mediated immunity, the rolling of leuko-
cytes is dependent on the levels of endothelial sialic acid.[4]
Modulation of the recognition events exerted by sialylated
glycans, therefore, seems promising for therapeutic pur-
poses.[5]
Among the types of glycoprotein structures, asparagine-
linked complex-type oligosaccharides (N-glycans) are most
prominent[6] in terms of diversity as well as complexity. These
N-glycans are often terminated with a sialic acid (Neu5Ac)
residue attached to a penultimate galactose (Gal) moiety
through an a(2,3) or a(2,6) linkage (Scheme 1a). The syn-
thesis of sialylated N-glycans has been reported by several
groups.[7] Most notably, Unverzagt and co-workers developed
chemoenzymatic routes to afford a variety of biantennary
glycans.[7d,e,8] More recently, Kajihara and co-workers
reported the systematic preparation of biantennary com-
plex-type glycans and glycopeptides, starting from glycopep-
tides that are isolated from egg yolks as the common
[*] Dr. Y. Ito
RIKEN (The Institute of Physical and Chemical Research)
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
and
CREST, Japan Science and Technology Agency (JST)
Kawaguchi, Saitama 332-1102 (Japan)
Fax: (+81)48-482-4680
E-mail: yukito@riken.jp
Dr. S. Hanashima
RIKEN (The Institute of Physical and Chemical Research)
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
Dr. S. Manabe
RIKEN and PRESTO, JST (Japan)
[**] This work was partly supported by the Special Postdoctoral
Researchers Program at RIKEN and by a Grant-in-Aid for Young
Scientists (B) (16710165) from the Ministry of Education, Science,
Culture, Sports, and Technology in Japan (to S.H.). We thank Dr.
Teiji Chihara and his staff for elemental analyses, and Ms. Tamiko
Chijimatsu for NMR measurements. We thank Ms. Akemi Takahashi
for her technical assistance.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 1. a) Structures of sialylated complex-type N-glycans; b) retrosynthetic analysis for polysaccharides 1–6. Bn=benzyl,
Bz=benzoyl, Gal=galactose, GlcNAc=N-acetylglucosamine, Man=mannose, Neu5Ac=sialic acid, PEG=poly(ethylene glycol),
Phth=phthalyl, TBDPS=tert-butyldiphenylsilyl.
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precursor.[9] In terms of chemical synthesis, the preparation of
(2,6)-[7a] and (2,3)-linked disialylated N-glycans[7b] have been
reported.
Polymer-supported synthesis is considered to be a prom-
ising technology that should facilitate, and ultimately auto-
mate, the synthesis of oligosaccharides. To date, however,
there are only a few reported syntheses of branched complex
structures such as sialylated N-glycans.[10] As part of our
efforts to develop methodologies for the efficient construc-
tion of glycan chains based on soluble polymer-support
technology,[11] we set out to synthesize disialylated com-
pounds 1–4 as well as monosialylated saccharides 5 and 6 in a
divergent manner. Low molecular weight (average MW ꢀ 750)
monomethyl polyethylene glycol (PEG) was chosen as the
polymer support. For purification, the PEG-supported prod-
uct can be adsorbed on silica gel, washed to remove the non-
PEG supported materials (e.g. excess donor), and then
retrieved by elution with polar solvents.[11e] With a unique
resin capture–release purification, which uses a chloroacetyl
group as the purification handle, fully assembled oligomers
can be distinguished from shorter products. A nitro-modified
Wang-resin-type linker, which has been shown to endure most
of the typical glycosylation conditions,[12] was used.
Scheme 2. Reagents and conditions: a) TFA, Et3SiH, CH2Cl2, ꢁ408C,
96%; b) (ClAc)2O, pyridine, CH2Cl2, 96%; c) NBS, DAST, CH2Cl2, 98%;
d) AgOTf, [Cp2HfCl2], molecular sieves (4 ꢀ), toluene, 458C, 85%;
e) NBS, DAST, CH2Cl2, 84%. TFA=trifluoroacetic acid,
Cp=cyclopentadienyl.
The synthetic plan for the preparation of target glycans 1–
6 is depicted in Scheme 1b. With hexasaccharide 7 as the
common precursor, the scheme involves the use of glycosyl-
transferases to introduce the terminal Neu5Ac residues and
penultimate Gal of the (1,6) branch. An initial glycosylation
with either (2,6)- or (2,3)-sialyltransferase should provide
monosialylated heptasaccharide 5 or 6, which can then serve
as substrates of sequential galactosylation–sialylation to
afford 1–4. Thus, simply by changing the type of glycosyl-
transferase, all positional isomers of Neu5Ac2Gal2GlcNAc2-
Man3, 1 (2,6/2,6), 2 (2,3/2,6), 3 (2,6/2,3), and 4 (2,3/2,3), as well
as monosialylated 5 and 6, can be prepared from the single
precursor 7. Compound 8 was designed as a protected
hexasaccharide that would be assembled from Man3 9,
GlcNAc 10, and LacNAc 11 derivatives.
As shown in Scheme 2, lactosamine fluoride 11 was
prepared from galactosyl donor 15[13] and glucosamine
component 19.[14] Removal of the trityl group of 15 afforded
16, which was reprotected with a chloroacetyl (ClAc) group to
give 17. Conversion of the phenylthio group to fluoride was
carried out by using N-bromosuccinimide (NBS) and diethyl-
aminosulfur trifluoride (DAST)[15] to afford 18 in nearly
quantitative yield (a/b = 46:54). Coupling with 19 was then
conducted through activation with [Cp2HfCl2] and AgOTf[16]
in toluene to afford 20, which was then converted into fluoride
11.
with 2-O-Ac- and 2-O-ClAc-protected donors 13 and 14. The
initial glycosylation with chloride 13[21] proceeded smoothly
under standard conditions to afford disaccharide 27 with
excellent purity in 98% yield,[22] then desilylation produced
28. Subsequent glycosylation with methylthiomannoside
14,[11d] when conducted through activation with NIS and
TMSOTf (0.2–0.4 equiv) in CH2Cl2 (ꢁ208C), gave a mixture
of the desired product 9 [dH = 5.18 (d, J = 1.7 Hz), 4.99 ppm
(d, J = 1.7 Hz)] and the corresponding orthoester 30 [dH =
5.25 (d, J = 2.4 Hz), 5.19 ppm (s)] in variable ratios (1:5–
1:0.7). In contrast, the use of a stoichiometric amount of
TfOH with NIS at low temperatures drastically suppressed
formation of the orthoester and provided 9 as the major
product. Capture–release purification was then carried out by
using Merrifield resin loaded with N-protected (Boc or Fmoc)
Cys (29a,b)[11d] in the presence of excess iPr2NEt to specif-
ically capture 9. We found that Boc-protected 29a was more
expedient than the Fmoc-protected 29b for the chemo-
selective reaction. Notably, orthoester 30 did not react under
these conditions and remained in the solution phase, along
with unreacted 9. Removal of the Boc group was carried out
with 10% TFA in CH2Cl2. Treatment with 10% piperidine in
THF initiated the cyclo-release to afford product 31 (60%
overall yield from 12).
The synthesis of the trimannose core is depicted in
Scheme 3. Phenylthiomannoside 21[17] was silylated and
chloroacetylated to give 22 in 93% yield. The nitro-modified
Wang-resin-type linker was introduced with 23[18] under
activation with N-iodosuccinimide (NIS) and triflic acid
(TfOH),[19] to afford 24 in 92% yield. Removal of the allyl
group afforded phenol 25, to which PEG was introduced
under standard Mitsunobu conditions to provide 12. Follow-
ing the selective deprotection with hydrazinedithiocarbonate
(HDTC),[20] the resultant 26 was glycosylated sequentially
The construction of hexasaccharide 8 is shown in
Scheme 4. For the elongation of the a(1,6) branch, thioglyco-
side 10[23] was used as the donor to afford 32,[22] which was
deacetylated to give 33. Reaction with LacNAc donor 11 with
[Cp2HfCl2] and AgOTf gave hexasaccharide 8 in 92% yield,
which was subjected to the same capture–release purification
used for 31 to afford 34 in 81% yield (from 31).
Cleavage from the linker and global deprotection are
depicted in Scheme 5. After acetylation of 34, the nitro group
was reduced to an amine function by using Mo(CO)6.[24]
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Scheme 3. Reagents and conditions: a) 1) TBDPSCl, DMAP, Et3N, DMF; 2) (ClAc)2O, pyridine, CH2Cl2, 93%; b) NIS, TfOH, molecular sieves
(4 ꢀ), CH2Cl2, 48C, 92%; c) [Pd(PPh3)4], Et3SiH, AcOH, toluene, 98%; d) HO-PEG-OMe, DEAD, Ph3P, THF, 94%; e) HDTC (0.42m), CH3CN,
98%; f) 13, AgOTf, CH2Cl2, molecular sieves (4 ꢀ), ꢁ20!08C, 98%; g) HF-pyridine, THF, 98%; h) 14, NIS, TfOH, molecular sieves (4 ꢀ), ꢁ408C,
94%; i) iPr2NEt, CH3CN/CH2Cl2; j) 1) 10% TFA in CH2Cl2, 2) 10% piperidine, THF, 60% from 12. All=allyl, DEAD=diethyl azodicarboxylate,
DMAP=4-(N,N-dimethylamino)pyridine, DMF=dimethylformamide.
Scheme 4. Reagents and conditions: a) 10, NIS, TfOH, molecular sieves (4 ꢀ), CH2Cl2, ꢁ10!08C, 92%; b) NaOMe (0.05m) in MeOH/THF,
96%; c) 11, AgOTf, [Cp2HfCl2], toluene, molecular sieves (4 ꢀ), ꢁ20!08C, 92%; d) 1) 29, iPr2NEt, DMF, 808C, 2) TFA (10%) in CH2Cl2,
3) piperidine (10%) in THF, 88% from 31.
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Scheme 5. Reagents and conditions: a) 1) Ac2O, pyridine, 2) Mo(CO)6, EtOH, dichloroethane; b) EtOCOCl, iPr2NEt, CH2Cl2; c) TFA (15%) in
CH2Cl2, 88%; d) BTBSA, TBAF (0.05 equiv) in THF-NMP; e) 1) ethylenediamine, nBuOH, 958C; 2) Ac2O, pyridine, 56%; f) 1) NaOMe (0.05m) in
MeOH, 93%; 2) Pd(OH)2/C (20%), MeOH, H2O, 92%. Ts=tosyl.
Amine 35 was then converted into ethyl carbamate 36a, then
treated with 15% TFA in CH2Cl2 to give 37 in 88% yield
(from 34). Interestingly, the corresponding acetyl 36b and
tosyl 36c derivatives were more resistant to cleavage under
these conditions. The liberated hydroxy group was protected
as a tert-butyldimethylsilyl (TBDMS) ether with N,O-bis(tert-
late buffer; upon completion of the galactose transfer, a(2,6)-
or a(2,3)-sialyltransferase and CMP-Neu5Ac were added to
the reaction mixture to give nonasaccharides 1 and 2 in
quantitative yields (CMP = cytidine-5’-monophosphoryl).
Similarly, heptasaccharide 6 was used to afford nonasacchar-
ides 3 and 4 in high yields. During the enzymatic trans-
formations, the TBDMS group served as a hydrophobic tag
that allowed product isolation by using reversed-phase solid-
phase extraction cartridges.[28]
In summary, the a(2,3)- or a(2,6)-sialylated biantennary
glycans 1–6, which correspond to the branched portions of
typical complex-type N-glycans, were systematically synthe-
sized with a polymer–resin hybrid strategy and enzymatic
glycosylation. By using low molecular weight (average MW
ꢀ 750) monomethyl polyethylene glycol as the polymer
support, the purification procedures were simple and involved
only loading on silica gel, washing to remove the non-PEG-
supported materials (e.g. excess donor), then eluting with
polar solvents. Introduction of sialic acid on the nonreducing
ends, and of the penultimate galactose moiety on the (1,6)
branch was successful with commercially available glycosyl-
transferases and the appropriate sugar nucleotides. The
common precursor 7 was synthesized by using a soluble
polymer support strategy, in which Merrifield resin loaded
butyldimethylsilyl)acetamide (BTBSA) and
a catalytic
amount of tetrabutylammonium fluoride (TBAF)[25] in N-
methylpyrrolidinone (NMP) to yield 38. Following conver-
sion of the phthalimide 38 to the acetamide 39, the acetyl and
benzyl groups were removed to afford 7.
As several types of sialyltransferases are commercially
available, and chemical sialylation is technically demanding,
enzymatic glycosylation is an attractive strategy for the
preparation of oligosaccharides that contain sialic acids.[26]
Hexasaccharide 7 possesses a terminal LacNAc residue,
which is an excellent substrate for sialyltransferases. In fact,
7 was smoothly converted into 5 and 6 with a(2,6)-N-
sialyltransferase (EC 2.4.99.1, Toyobo) and a(2,3)-N-sialyl-
transferase (EC 2.4.99.5, Calbiochem), respectively, then
subjected to sequential galactosylation/sialylation[27] to
afford 1–4 (Scheme 6). As an example, heptasaccharide 5
was treated with b(1,4)-galactosyltransferase (EC 2.4.1.22,
Toyobo) and UDP-galactose in MnCl2 containing a cacody-
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Scheme 6. Enzymatic transformations of 7 to 1–6: a) a2,6- or a2,3-sialyltransferase, CMP-Neu5Ac, alkaline phosphatase, BSA, cacodylate buffer
(50 mm); b) 1) b1,4-galactosyltransferase, UDP-galactose, BSA, alkaline phosphatase, MnCl2, cacodylate buffer (50 mm); 2) a2,6- or a2,3-sialyl-
transferase, CMP-Neu5Ac. UDP=uridine diphosphate.
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Keywords: carbohydrates · glycoproteins · glycosylation ·
oligosaccharides · sialic acids
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