Automated synthesis of sialylated oligosaccharides

Article abstract of DOI:10.3762/bjoc.8.183

Sialic acid-containing glycans play a major role in cell-surface interactions with external partners such as cells and viruses. Straightforward access to sialosides is required in order to study their biological functions on a molecular level. Here, automated oligosaccharide synthesis was used to facilitate the preparation of this class of biomolecules. Our strategy relies on novel sialyl α-(2→3) and α-(2→6) galactosyl imidates, which, used in combination with the automated platform, provided rapid access to a small library of conjugation-ready sialosides of biological relevance.

Full text of DOI:10.3762/bjoc.8.183

Automated synthesis of sialylated oligosaccharides
Davide Esposito1,2, Mattan Hurevich1,2, Bastien Castagner3,
Cheng-Chung Wang4 and Peter H. Seeberger*1,2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1601–1609.
1Max-Planck-Institute of Colloids and Interfaces, Department of
Biomolecular Systems, Am Mühlenberg 1, 14476 Potsdam, Germany,
2Freie Universität Berlin, Institute of Chemistry and Biochemistry,
Arnimallee 22, 14195 Berlin, Germany, 3Institute of Pharmaceutical
Sciences, Swiss Federal Institute of Technology (ETH) Zurich, 8093
Zurich, Switzerland and 4Institute of Chemistry, Academia Sinica,
Taipei, 11529, Taiwan
doi:10.3762/bjoc.8.183
Received: 25 June 2012
Accepted: 16 August 2012
Published: 21 September 2012
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Email:
Peter H. Seeberger* - Peter.Seeberger@mpikg.mpg.de
Guest Editor: T. K. Lindhorst
* Corresponding author
© 2012 Esposito et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
automated synthesis; disaccharide building blocks; solid-phase
synthesis; sialic acid; sialosides
Abstract
Sialic acid-containing glycans play a major role in cell-surface interactions with external partners such as cells and viruses.
Straightforward access to sialosides is required in order to study their biological functions on a molecular level. Here, automated
oligosaccharide synthesis was used to facilitate the preparation of this class of biomolecules. Our strategy relies on novel sialyl
α-(2→3) and α-(2→6) galactosyl imidates, which, used in combination with the automated platform, provided rapid access to a
small library of conjugation-ready sialosides of biological relevance.
Introduction
Sialic acid (Sia) belongs to a family of nonulosonic acids, i.e., mated synthesis of oligosaccharides has been significantly im-
monosaccharides equipped with a carboxylic moiety and a nine- proved since the first report in 2001 [4]. Currently, the platform
carbon backbone, which play a unique role in glycobiology. enables the rapid assembly of complex oligosaccharides and
Sia-containing glycans mediate pathogen invasion [1] and are accommodates the most commonly employed glycosylation
involved in signalling cascades, which have been extensively reactions [5-7]. However, accessing sialosides by automation
studied [2]. The distinctive structure of Sia confers special prop- has been hampered by several factors. Chemical silalylation
erties to membrane oligosaccharides [3] resulting in sialosides represents a significant challenge, and is usually plagued by low
having exceptional biological significance. Rapid access to syn- yields and anomeric mixtures [8]. To avoid synthetic complica-
thetic sialylated glycans would contribute greatly to the bio- tions, Sia has often been introduced by enzymatic methods [9].
logical studies on this important class of molecules. The auto- In order to allow for access to synthetic sialosides, an intense
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Beilstein J. Org. Chem. 2012, 8, 1601–1609.
effort has been devoted to identifying sialic acid building blocks goal, we developed a method to provide sialic acid containing
with superior sialylation properties [10]. In turn, only limited disaccharide glycosylating agents with minimal synthetic effort.
attention has been given to the design of more efficient nucleo- Simple N-acetyl building blocks such as 1 (Figure 1) were used
philes for sialylations. In naturally occurring N- and O-glycans due to their facile syntheses, in contrast to other commonly
the terminal sialic acid residue is most often connected to the employed N-5 modified building blocks [14-17]. Most of the
C3 or C6 hydroxy group of galactose. Therefore, differently N-acetyl sialic acid glycosylating agents reported in the litera-
protected galactose precursors have been exploited for sialyla- ture can be accessed from the common intermediate 6 [18],
tion reactions. The obtained disaccharides have been used to which is prepared in two steps from commercially available Sia
prepare synthetic sialosides [11-13]. A disaccharide building (Figure 1b).
block approach is an attractive possibility for solid-phase syn-
thesis, since it avoids performing a low yielding and unse- Compound 6 was converted in a single step into various
lective sialylation on a solid support.
sialylating agents, such as the N-phenyl trifluoroacetimidoyl
glycoside 10, and glycosyl phosphites 8 and 1 as previously
Here, we describe a method for the rapid preparation of described (Table 1) [19-21]. Galactal 2 was identified recently
different sialosides relying on a new automated solid-phase syn- as an efficient acceptor for sialylation [11,12]. Its efficiency can
thesis platform [5]. Central to the success of this approach is be attributed to a combination of reduced steric hindrance and
the use of galactals as nucleophiles for chemical sialylation, good nucleophilicity of the C3 hydroxy group. Thus, galactal 2
which allows for efficient access to the novel sialyl α-(2-3) and was glycosylated with different N-acetyl sialic acid building
α-(2-6) galactosyl imidate disaccharide building blocks. blocks (Table 1).
The combination of the automated platform and sialylated
building blocks proved successful for the synthesis of represen- We started our screening by comparing the glycosylation of
tative Sia-containing oligosaccharides ready for biological eval- building blocks 1, 8 and 10 with galactal 2 under similar condi-
uation.
tions (Table 1, entries 1–3). Sialylation with building block 8
failed to yield any disaccharide 9 (Table 1, entry 1), while gly-
cosylating agents 10 and 1 gave moderate yields and good
selectivity (Table 1, entries 2 and 3). The glycosylation of
Results and Discussion
Building-blocks preparation
Many sialylation strategies utilize building blocks that require galactal 2 with phosphite 1, which we described in the
multistep syntheses [10]. In contrast, solid-phase automated supporting information of [5], was further optimized. In particu-
synthesis requires readily accessible building blocks that can be lar, elevating the reaction temperature proved beneficial and
used in excess to drive reactions to completion. As our initial disaccharide 9 was isolated in higher overall yield, albeit with a
Figure 1: (a) Synthesis sequence for the preparation of building blocks 4 and 5; (b) Retrosynthetic analysis for the preparation of 1.
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Table 1: Synthesis of building blocks 9 and 11.
entry
glycosylating agent
nucleophile
conditionsa
product
yield α/β ratio
1
a
–
–
2 (1.5 equiv)
2 (1.5 equiv)
8 (1.0 equiv)
9
9
2
a
30%
9/1
10 (1.0 equiv)
3
4
2 (1.5 equiv)
2 (1.7 equiv)
a
b
9
9
27%
80%
9/1
4/1
1 (1.0 equiv)
1 (1.0 equiv)
5
1 (1.0 equiv)
b
75%
2.5/1
3 (2.0 equiv)
11
aReagents and Conditions: (a) TMSOTf (0.2 equiv), EtCN, AW-4Å MS, −78 °C; (b) TMSOTf (0.2 equiv), CH3CN, AW-4Å MS, −42 °C.
slight decrease in selectivity. The best results were obtained by Solution-phase studies
using 1.7 equivalents of 2 at −42 °C, using acetonitrile as the In order to evaluate the utility of building block 4 for the solid-
solvent instead of the more expensive propionitrile (Table 1, phase synthesis of sialosides we undertook a model solution-
entry 4). The conditions established for the synthesis of com- phase synthesis of the glycan portion of GM3 ganglioside 16
pound 8 were applied to synthesize sialyl α-(2→6) galactal 11 (Scheme 2). GM3 serves as an important receptor for viral
(Table 1, entry 5) in good yield upon glycosylation of galactal 3 infection [24,25] and contains the common sialyl α-(2→3)
[22]. In all cases, the desired anomer was readily purified by galactose motif. The key step en route to compound 16 was the
column chromatography.
glycosylation of compound 14 with building block 4
(Scheme 2), which proceeded efficiently in the presence of
In order to convert the disaccharide products into the corres- trimethylsilyl triflate (TMSOTf) as promoter at −10 °C to afford
ponding glycosyl imidates, the double bond in compounds 9 trisaccharide 15 with a yield of 80%. It is worth mentioning that
and 11 was oxidized by treatment with PhI(OAc)2 and catalytic glycosylation of an analogue of glucose 14 equipped with a
amounts of BF3·Et2O [23] and gave disaccharides 12 and 13, benzoyl group at the C3 hydroxy position resulted in a lower
respectively after acetylation (Scheme 1). Removal of the glycosylation yield (36%), suggesting that an ester can lower
anomeric acetate mediated by hydrazine acetate provided the the nucleophilicity of the vicinal C4-hydroxy. The synthesis
hemiacetals, which was followed by introduction of the was completed by deacetylation of compound 15 under
anomeric N-phenyl trifluoroacetimidate to furnish disaccharide Zemplén's conditions, followed by saponification and hydro-
building blocks 4 and 5. It should be noted that building block 4 genolysis affording good yields of the trisaccharide 16,
can be prepared with higher overall yield than the recently equipped with an amino spacer for conjugation. The synthesis
disclosed N-Troc protected disaccharide building block [11] of GM3 trisaccharide 16 proved that compound 4 is efficient for
obtained with a similar method.
installing the capping sialyl α-(2→3) galactose unit into syn-
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Beilstein J. Org. Chem. 2012, 8, 1601–1609.
Scheme 1: Reagents and conditions: (i) PhI(OAc)2, BF3·Et2O, CH2Cl2, −40 °C; then Ac2O, pyridine; (ii) N2H4·AcOH, DMF; (iii) CF3C(NPh)Cl,
Cs2CO3, CH2Cl2, DCM, 66% over three steps for 4 (for a detailed description of the synthesis of compound 4 see the supporting information of [5]);
62% over three steps for 5.
Scheme 2: Reagents and conditions. (i) TMSOTf, DCM, −10 °C, 80%; (ii) NaOMe, MeOH; then KOH, MeOH, 60 °C; then Pd/C, H2, AcOH, MeOH,
THF, H2O, rt, 76% over three steps.
thetic oligosaccharides. Furthermore, conditions applied to the (TCA) reduction, ester removal and cleavage from the solid
preparation of 16 can be easily adapted for solid-phase syn- support by automation. In many cases, only hydrogenolytic
thesis making 4 a valuable candidate for automation.
cleavage of the remaining benzyl ethers and carbamates has to
be performed manually at the end of an automated sequence.
These features make the automated platform very attractive for
sialoside synthesis. Based on the encouraging results obtained
Automated synthesis of sialosides
Automated synthesis of linear α-(2→3) sialosides
The new integrated platform for automated synthesis of oligo- for the solution-phase synthesis of 16, building blocks 4 and 5
saccharides [5] offers the possibility to construct a diverse set of were used for the automated solid-phase synthesis of various
glycans rapidly and efficiently. The automated synthesizer sialosides. Some of the results presented herein have been
proved capable of performing iterative glycosylation–deprotec- communicated in preliminary form [5]. Sialyl lactosamine 20
tion cycles under conditions commonly employed for solution- (Scheme 3) [25], which serves as a site of attachment for
phase oligosaccharide synthesis. The synthetic strategy relies on viruses during infections, and sialyl lactose (GM3) 16
the solid support-bound linker 17 (Scheme 3), which contains a (Scheme 4) were chosen to confirm the viability of building
latent amino spacer useful for conjugation. In addition, manual block 4 for the automated solid-phase synthesis of linear sialo-
operations are minimized by performing the trichloroacetyl sides.
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Beilstein J. Org. Chem. 2012, 8, 1601–1609.
Scheme 3: Automated synthesis of 20. Reagents and conditions: (i) (a) NIS, TfOH, dioxane, DCM, −40 to −20 °C, 40 min; (b) piperidine, DMF. (ii) (a)
TMSOTf, DCM, 0 °C, 2 h; (b) piperidine, DMF. (iii) AIBN (cat.), Bu3SnH (10 equiv), xylene, 90 °C. (iv) NaOMe, MeOH, DCM, 1.5 h, 33%. (v) Pd/C, H2,
MeOH/H2O, cat. AcOH, 78% (for experimental details see the supporting information of [5]).
Scheme 4: Automated synthesis of 16. Reagents and conditions: (i) (a) NIS, TfOH, dioxane, DCM, −40 to −20 °C, 40 min; (b) piperidine, DMF. (ii) (a)
TMSOTf, DCM, 0 °C, 2 h; (b) piperidine, DMF. (iii) NaOMe, MeOH, DCM, 1.5 h. (iv) KOH, MeOH, H2O, THF, 60 °C, 40%. (v) Pd/C, H2, MeOH/H2O,
cat. AcOH, 91% (for experimental details see the supporting information of [5]).
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Beilstein J. Org. Chem. 2012, 8, 1601–1609.
For all sialosides, a similar synthetic route was followed, onment could, in principle, additionally reduce the accessibility
consisting of automated glycosylation and deprotection cycles, of a sterically hindered nucleophile. Thus, the possibility of
followed by TCA reduction (when glucosamines are present) accessing branched structures was explored on the solid support
and final ester removal/cleavage to afford the semi-protected [5].
oligosaccharide. The automated synthesis of 20 (Scheme 3)
started with the glycosylation of resin-bound linker 17 with Sialyl LewisX tetrasaccharide 27 (Scheme 5), has been impli-
glucosamine building block 18 (2 × 5 equiv) [5] in the presence cated in inflammation and cancer metastasis [26], and was
of N-iodosuccinimide and triflic acid. Fluorenylmethoxycar- chosen as a model glycan for the construction of branched com-
bonyl (Fmoc) removal was followed by glycosylation with pounds. Glucosamine building block 23, containing
building block 4 (2 × 5 equiv) for 1 h at −10 °C with TMSOTf C3-levulinoyl (Lev) and C4-Fmoc protecting groups [27], was
used for activation. Radical reduction using tributyltinhydride first reacted with the linker. The glycosylation was followed by
and azobisisobutyronitrile (AIBN) was performed to convert the Fmoc removal from the C4 hydroxy group and a second
trichloroacetamide into an N-acetyl moiety, followed by meth- glycosylation was performed with building block 4 under the
oxide-mediated cleavage to provide compound 19. Analysis of conditions optimized in the context of the synthesis of tri-
the crude mixture by LC–MS showed incomplete glycosylation saccharide 20. Removal of the levulinoyl ester from C3 by
of the resin bound glucosamine by building block 4. The reac- treatment with hydrazine hydrate and acetic acid exposed the
tion was optimized to identify the best glycosylation conditions second hydroxy nucleophile on the central glucosamine. Our
for building block 4 under the solid-phase paradigm. first attempt to glycosylate using fucose thioglycoside building
Performing the glycosylation at a higher temperature (0 °C) and block 25 afforded the product in low yield and as a mixture of
longer time (2 h) proved sufficient to drive the reaction to anomers as confirmed by LC–MS analysis. The use of N-phe-
higher conversion and trisaccharide 19 was isolated in 33% nyl trifluoroacetimidate building block 24 proved more effi-
overall yield with respect to resin loading. Hydrogenolysis cient. Nevertheless, when fucosylation with building block 24
under standard conditions afforded the fully deprotected tri- was performed in dichloromethane, a mixture of anomers of
saccharide 20 in 78% yield. These conditions were applied to compounds 26 was detected by NMR analysis. Only running
the synthesis of GM3 trisaccharide 16 previously prepared in the reaction in ether, which is a strong α-directing solvent [28],
solution phase (see above). Glucose thioglycoside building ensured stereoselective introduction of the fucose residue.
block 21 and disaccharide building block 4 served for the Under these optimized conditions, the branched tetrasaccharide
assembly of 16 (Scheme 4). Final saponification afforded the 26 was isolated in 51% overall yield after TCA reduction,
partially protected glycan 22 in 40% overall yield before hydro- cleavage, ester saponification and HPLC purification. Finally,
genolysis gave the final trisaccharide 16 in 91% yield. The effi- solution-phase hydrogenolysis gave tetrasaccharide 27.
ciency of the solid-phase and the solution-phase syntheses was
compared. The solution-phase synthesis of trisaccharide 16 was Automated synthesis of linear α–(2→6) sialosides
completed with an overall yield of 42% (taking into account the α-(2→6) Sialylated oligosaccharides have been identified in
preparation of compound 14, 69%) in about one week. The humans as a recurring constituent of the upper respiratory
same number of steps was executed in the solid-phase synthesis epithelial glycocalix [29]. For instance, tetrasaccharide 30 has
with little operator interference, to yield the desired compound been reported to bind to haemagglutinins isolated from different
in a comparable overall yield (36%) in shorter time. An average H1N1 human viral strains, and was chosen as a target to show-
time of 3 h per glycosylation cycle (coupling and deprotection) case the solid-phase automated synthesis of α-(2→6) sialosides
or cleavage from the support, allows the assembly of a tri- (Scheme 6).
saccharide in roughly 10 h. In general, although an excess of
building block is used in the automated solid-phase synthesis, The synthesis of tetrasaccharide 30 started with the glycosyl-
the method provides the final assembled oligosaccharide with ation of linker 17 by using galactose building block 28 under
much greater efficiency than in the solution-phase synthesis, standard conditions for the activation of thioglycosides, fol-
and avoids the loss of material encountered when performing lowed by Fmoc removal and a glycosylation with building
purifications in between steps.
block 18. The solid-phase bound disaccharide was further
elongated, following removal of the temporary protecting
group, by reaction with building block 5. For this reaction, we
Automated synthesis of sialyl LewisX
Branching is often observed in naturally occurring sialosides. applied the reaction conditions optimized for building block 4
Assembly of branched oligosaccharides is particularly challen- without further optimization. Thus, standard TCA reduction,
ging due to the steric hindrance of the branching sites, which cleavage from the support, saponification and isolation afforded
can affect glycosylation yields. Working in a solid-phase envir- semiprotected tetrasaccharide 29 in 16% overall yield. As
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Scheme 5: Automated synthesis of 27. Reagents and conditions: (i) (a) NIS, TfOH, dioxane, DCM, −40 to −20 °C, 40 min; (b) piperidine, DMF. (ii) (a)
TMSOTf, DCM, 0 °C, 2 h; (b) NH2NH2·H2O, AcOH, pyridine, DCM. (iii with 24) TMSOTf, Et2O, −10 °C, 1 h. (iii with 25) NIS, TfOH, dioxane, DCM,
−40 °C to −20 °C, 40 min. (iv) AIBN (cat.), Bu3SnH (10 equiv), xylene, 90 °C. (v) NaOMe, MeOH, DCM, 1.5 h. (vi) KOH, MeOH, H2O, THF, 60 °C,
51%. (vii) Pd/C, H2, MeOH/H2O, cat. AcOH, 30% (for experimental details see the supporting information of [5]).
Scheme 6: Automated synthesis of 30. Reagents and conditions: (i) (a) NIS, TfOH, dioxane, DCM, −40 to −20 °C, 40 min; (b) piperidine, DMF. (ii)
NIS, TfOH, dioxane, DCM, −40 to −20 °C, 40 min. (iii) TMSOTf, Et2O, 0 °C, 2 h. (iv) AIBN (cat.), Bu3SnH (10 equiv), xylene, 90 °C. (v) NaOMe,
MeOH, DCM, 1.5 h. (vi) KOH, MeOH, H2O, THF, 60 °C, 16%. (vii) Pd/C, MeOH/H2O/EtOAc, cat. AcOH, 51%.
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Beilstein J. Org. Chem. 2012, 8, 1601–1609.
Scheme 7: Reagents and conditions: (i) 10% DMF in PBS buffer pH 7.5, overnight, 80%.
shown by LC–MS analysis (see Supporting Information File 1) overall yields from readily accessible starting materials. In
the crude mixture contains the desired tetrasaccharide, but also combination with a fully automated synthesizer, the disac-
some deletion sequences that can be attributed to the non-opti- charide building blocks have been exploited for the solid-phase
mized conditions used for the synthesis. Moreover, building synthesis of several oligosaccharides ready for biological evalu-
blocks 18 and 28 have been observed to be a non-ideal ation. This work represents the first full account of an auto-
donor–acceptor pair (unpublished results). Nevertheless, simple mated solid-phase synthesis of sialosides.
reverse-phase HPLC was sufficient to isolate semiprotected
tetrasaccharide 29 in milligram quantities. To complete the syn-
Supporting Information
thesis, hydrogenolysis was performed to give final tetrasac-
charide 30 in 51% yield. The above example shows that the
Supporting Information File 1
platform can provide access to target oligosaccharides by using
generalized coupling protocols even when conditions are not
optimized.
Experimental procedure and characterization data for new
compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-183-S1.pdf]
Formation of biotinylated probes
Supporting Information File 2
The syntheses of glycans 27 and 30 showed the efficiency of
sialyl building blocks 4 and 5 in combination with the auto-
mated solid-phase platform for the rapid and reliable access to
complex sialosides. Our synthetic strategy makes use of linker
17, which incorporates an amino spacer for conjugation into the
final oligosaccharide. In this way, the synthetic sialosides can
1H and 13C NMR spectra for new compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-183-S2.pdf]
be easily conjugated to probes for biological evaluation or Acknowledgments
labelled for instance with UV-active tags. Biotinylation is a We acknowledge the Max-Planck Society and the Swiss
typical example of a commonly employed labelling technique National Science Foundation (200020-117889) for generous
[30] and has been extensively used for instance as a functional- financial support. We thank the National Science Council
ization technique for antigens in antibody selection by phage- Taiwan for a fellowship to C.-C. Wang and the Minerva Stif-
display methods [31]. Thus, trisaccharide 16 (Scheme 7) was tung for a fellowship to M. Hurevich.
reacted with biotin derivative 31 in PBS buffer to afford com-
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