Automated solid-phase synthesis of oligosaccharides containing sialic acids

Article abstract of DOI:10.3762/bjoc.11.69

A sialic acid glycosyl phosphate building block was designed and synthesized. This building block was used to prepare α-sialylated oligosaccharides by automated solid-phase synthesis selectively.

Full text of DOI:10.3762/bjoc.11.69

Automated solid-phase synthesis of oligosaccharides
containing sialic acids
Chian-Hui Lai1, Heung Sik Hahm1,2, Chien-Fu Liang1 and Peter H. Seeberger*1,2,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 617–621.
1Department of Biomolecular Systems, Max-Planck-Institute of
doi:10.3762/bjoc.11.69
Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany
and 2Freie Universität Berlin, Institute of Chemistry and Biochemistry,
Arnimallee 22, 14195 Berlin, Germany
Received: 11 February 2015
Accepted: 20 April 2015
Published: 04 May 2015
Email:
Peter H. Seeberger* - peter.seeberger@mpikg.mpg.de
This article is part of the Thematic Series "Multivalency as a chemical
organization and action principle".
*
Corresponding author
§
Fax: +49 30 838-59302; Tel: +49 30 838-59301
Associate Editor: S. Flitsch
Keywords:
© 2015 Lai et al; licensee Beilstein-Institut.
License and terms: see end of document.
α-sialylation; automated synthesis; glycosylation; sialic acid;
solid-phase synthesis
Abstract
A sialic acid glycosyl phosphate building block was designed and synthesized. This building block was used to prepare α-sialylated
oligosaccharides by automated solid-phase synthesis selectively.
Introduction
N-Acetylneuraminic acid (sialic acid, Neu5Ac) is an important monosaccharide building blocks to construct particular link-
component of mammalian glycans and key to many recognition ages. To date, α-(2,3)- and α-(2,6)-sialylated glycans have been
events of biomedical relevance including cell–cell recognition, accessible by automation only via incorporation of sialic
signaling, and the immune response [1]. Sialic acids are present acid–galactose disaccharide building blocks [5,11]. Here, we
in tumor-associated carbohydrate antigens (TACAs) such as the describe a sialic acid building block that can be utilized for
sialyl-Tn antigen (sTn) [2]. Neu5Ac is often the terminal automated glycan assembly.
residue and is usually linked via an α-(2,3) or α-(2,6) linkage to
galactose (Gal) (Figure 1) [3].
Results and Discussion
Sialylating oligosaccharides in high yield and α-selectivity was
Automated glycan assembly enables rapid access to structurally challenging since the presence of a C-1 carboxyl electron-with-
defined oligosaccharides [4,5] including glycopeptides [6], drawing group at the quaternary anomeric center decreases the
glycosaminoglycans [7-9], and chains as long as 30-mers [10]. reactivity. In addition, no participating group on C-3 can be
Key to automated assembly is the identification of reliable used to direct the stereochemistry at the anomeric carbon (C-2)
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Beilstein J. Org. Chem. 2015, 11, 617–621.
Figure 1: Terminal sialic acids are typically α-(2,3) or α-(2,6) linked to galactose (Gal) such as in the tumour-associated antigen sialyl Tn (sTn).
[
2]. Efficient chemical sialylation reactions utilize the cyclic “Approved building blocks” for automated glycan assembly
4
O,5N-oxazolidinone protecting group [12-15], where the have to be accessible in sufficient quantities, stable for storage
trans-fused cyclic protecting group in the glycosylation tran- and activated at a specific temperature to provide the desired
sition state likely stabilizes the positive charge on the intermedi- linkage in high yield. The optimal glycosylation temperature
ate acetonitrile adduct and decreases the generation of a posi- was determined to ensure fast and efficient reactions at the
tive charge at the anomeric center by their strong dipole highest possible temperature [19,20]. Rather than slowly
moment [2,16,17].
warming a reaction mixture as is done in solution phase, on the
automated synthesizer, the building block will be delivered at
Based on these considerations sialyl phosphate building blocks the optimal temperature and reacted for a predetermined time.
and 5 [14] were selected for automated glycan assembly using For sialic acid building block 4, the activation temperature was
4
monosaccharides (Scheme 1). The synthesis of building block 4 determined to be −20 °C (Table S1 in Supporting Information
commenced with the placement of a C-9 Fmoc protecting group File 1). The synthesis of trisaccharides 14 illustrates how opti-
on thioglycoside 1 [14] to produce 2. Installation of O-chloro- mization of the activation temperature resulted in increased
acetyl groups on C-7 and C-8 for better α-stereoselectivity [12] yields (Table S4 in Supporting Information File 1).
produced 3. An α-anomeric phosphate leaving group was
chosen since it had previously shown high reactivity [14,18] Six di- and trisaccharides (12–17, Scheme 2) served as targets
and selectivity [15]. Building block 4 was obtained in 54% to develop an automated method for chemical sialylation.
yield over three steps from 1.
Monosaccharide building blocks 4, 5 [14], 6, 7 [21], 8, 9 [21],
Scheme 1: (a) FmocCl, py, CH2Cl2, rt, 4 h, 77%, (b) 2-chloroacetyl chloride, py, CH2Cl2, 0 °C to rt, 3 h, 88%, (c) HOPO(OBu)2, NIS, TfOH, 4 Å MS,
CH3CN/CH2Cl2, −78 °C to 0 °C, 2 h, 80%.
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Beilstein J. Org. Chem. 2015, 11, 617–621.
Scheme 2: Automated synthesis of oligosaccharides with α(2,3)-, α(2,6)-sialic acid linkages. Glycosylations: a) 2 × 5 equiv TMSOTf, ACN/DCM (1:1),
−50 °C (5 min), −30 °C (10 min), −20 °C (80 min), −10 °C (10 min), 0 °C (10 min) for 4 and 5. b) 2 × 5 equiv TfOH, NIS, DCM, −40 °C (5 min), −20 °C
(
(
30 min) for 6 and 7. c) 2 × 5 equiv TMSOTf, DCM/dioxane (3:2), 20 °C (90 min), for 8. d) 2 × 5 equiv TfOH, NIS, DCM, −30 °C (5 min), −10 °C
25 min) for 9 and 10. Fmoc Deprotection: e) 3 × 20% NEt3 in DMF, 5 min. Photocleavage: f) UV irradiation using a continuous flow reactor, DCM, rt.
Synthesis of 12 or 13: (1) 6, b, (2) e, (3) 4 or 5, a (4) f, 30% for four steps to yield 12, 40% for four steps to yield 13; synthesis of 14: (1) 9, d, (2) e, (3)
, b, (4) e, (5) 4, a, (6) f, 22% for six steps; synthesis of 15: (1) 10, d, (2) e, (3) 6, b, (4) e, (5) 4, a, (6) f, 7% for six steps; synthesis of 16: (1) 7, b, (2)
6
e, (3) 4, a, (4) f, 19% for four steps; synthesis of 17: (1) 8, c, (2) e, (3) 3 × Ac2O, py, 25 °C for 60 min, (4) 4, a, (5) f, 10% for five steps.
and 10 [5] were employed for these syntheses. Merrifield poly- glycosylations. These building blocks resulted in complete
styrene resin equipped with a photocleavable linker, 11, was conversion as determined by Fmoc quantification [5] and HPLC
placed in the reaction chamber of the automated synthesizer and analysis.
the coupling cycles were initiated following programmed
maneuvers. Each cycle starts with a TMSOTf acidic wash at Sialyl phosphate building blocks 4 and 5 resulted in good
−
20 °C to ensure that no base from previous deprotection reac- α-selectivity for the installation of α-(2,6)-linkages in disaccha-
tions remains and quenches the subsequent coupling. This rides 12 and 13, both sialyl phosphate building blocks 4 and 5
problem had been observed earlier (data not shown) and can be showed exclusive α-selectivity. However, building block 4 was
overcome by this extra washing step. In addition, TMSOTf more reactive than 5 as the synthesis of disaccharide 13 resulted
eliminates any moisture that may have resided on the resin or in almost in full α-sialylation as observed by HPLC analysis of the
the reaction vessel.
crude product following photocleavage from the resin that
showed only one peak while 12 was not the only product.
Glycosylations were carried out using the optimized tempera- Disaccharide 12 was obtained in 30% and 13 in 40% overall
tures for each building block using twice five equivalents of yield for four steps based on resin loading. The absolute
building block and activator. Removal of the Fmoc protecting anomeric configurations of glycans that contain sialylic acid
group with triethylamine uncovered the hydroxy group to serve were determined by recording the long-range coupling
as the nucleophile in the next coupling. Participating protecting constants of C1 with axial H3 (3J C-1,H-3ax) using 1D coupled
groups at the C2 position of building blocks 6, 7, 9 and 10 HMQC experiments. Coupling constant higher than 5 Hz
ensured selective formation of β-glycosidic linkages during the correspond to α-configurations [12].
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Two trisaccharides (14 and 15) that are α-(2,6)-sialylated were [11] or by enzymatic sialylation [27] following the cleavage and
obtained in 22% and 7% yield after HPLC purification based on deprotection of an oligosaccharide.
resin loading for six steps. The sialylation proceeded with
α-stereoselectivity in both cases. The synthesis of 14 was higher
Supporting Information
yielding than 15. The major structural difference of 14 and 15 is
the first sugar attached on the resin. The N-protecting TCA
Supporting Information File 1
group of glucosaminoside has more electron-withdrawing char-
acter in the synthesis of 15 than the benzoate ester groups of the
glucoside in the synthesis of 14 which resulted in a less favor-
able sialylation for 15.
Experimental part.
http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-11-69-S1.pdf]
[
To demonstrate that α-(2,3)-sialylations are possible, model Acknowledgements
disaccharide 16 was synthesized in 19% yield. The secondary We thank the Max Planck Society and the ERC (Advanced
C3 hydroxy group in galactose is less reactive and conse- Grant AUTOHEPARIN) for very generous financial support.
quently, even after optimization, the chemical sialylation of the We thank Dr. Fabian Pfrengle, Dr. Mattan Hurevich and Mr.
C3 position of galactose did not result in a satisfactory yield and Frank Schuhmacher for assistance in operating the synthesizer,
demonstrates a current limitation of the automated glycan HPLC and fruitful discussions.
assembly approach. Recently, placement of an isothiocyanate
moiety on the C5 position was reported to be an effective References
method to construct alpha linkages [22] and may prove useful
1. Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.;
Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of glycobiology,
for solid-phase synthesis in the future as well.
2
nd ed.; Cold Spring Harbor Laboratory Press: New York, 2009.
Adak, A. K.; Yu, C.-C.; Liang, C.-F.; Lin, C.-C. Curr. Opin. Chem. Biol.
013, 17, 1030–1038. doi:10.1016/j.cbpa.2013.10.013
Varki, A. Nature 2007, 446, 1023–1029. doi:10.1038/nature05816
2
.
.
The tumor associated sTn carbohydrate antigen (Neu5Ac-
α(2,6)GalNAc-α(1,1)linker) disaccharide 17, that resembles the
sTn antigen glycan framework (Neu5Ac-α(2,6)GalNAc-
α(1,1)Ser/Thr) was synthesized. In order to install the cis-glyco-
side formed by the union of the galactosamine and the linker,
galactosamine building block 8 relies on remote participating
protecting group effects of esters at C3 and C4 [23,24]. The
selectivity of the cis-glycosylation improved with higher reac-
tion temperatures due the strongly deactivating effect of three
electron withdrawing ester and carbonate protecting groups
2
3
4. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001, 291,
523–1527. doi:10.1126/science.1057324
1
5.
Kröck, L.; Esposito, D.; Castagner, B.; Wang, C.-C.; Bindschädler, P.;
Seeberger, P. H. Chem. Sci. 2012, 3, 1617–1622.
doi:10.1039/c2sc00940d
6.
Hurevich, M.; Seeberger, P. H. Chem. Commun. 2014, 50, 1851–1853.
doi:10.1039/c3cc48761j
7. Eller, S.; Collot, M.; Yin, J.; Hahm, H. S.; Seeberger, P. H.
Angew. Chem., Int. Ed. 2013, 52, 5858–5861.
doi:10.1002/anie.201210132
[
23,25]. The addition of dioxane to CH2Cl2 resulted in preferred
8.
Kandasamy, J.; Schuhmacher, F.; Hahm, H. S.; Klein, J. C.;
Seeberger, P. H. Chem. Commun. 2014, 50, 1875–1877.
doi:10.1039/c3cc48860h
formation of the α-anomer, an effect that is well known from
solution phase syntheses [26] (Table S6, Figure S1 in
Supporting Information File 1). When five equivalents of 9. Walvoort, M. T. C.; Volbeda, A. G.; Reintjens, N. R. M.;
van den Elst, H.; Plante, O. J.; Overkleeft, H. S.; van der Marel, G. A.;
building block 8 were used at 20 °C for 90 min with a solvent
Codée, J. D. C. Org. Lett. 2012, 14, 3776–3779.
ratio of CH2Cl2 and dioxane of 3:2, mainly the desired
doi:10.1021/ol301666n
α-anomer was obtained (2:1). A double coupling of building
1
0.Calin, O.; Eller, S.; Seeberger, P. H. Angew. Chem., Int. Ed. 2013, 52,
block 8 to install the α-galactosamine linker was followed by a
capping step. Incorporation of building block 4, cleavage from
the resin and purification by HPLC yielded disaccharide 17 in
5
862–5865. doi:10.1002/anie.201210176
11.Esposito, D.; Hurevich, M.; Castagner, B.; Wang, C.-C.;
Seeberger, P. H. Beilstein J. Org. Chem. 2012, 8, 1601–1609.
doi:10.3762/bjoc.8.183
1
0% yield.
1
2.Lin, C.-C.; Lin, N.-P.; Sahabuddin, L. S.; Reddy, V. R.; Huang, L.-D.;
Hwang, K. C.; Lin, C.-C. J. Org. Chem. 2010, 75, 4921–4928.
doi:10.1021/jo100824s
Conclusion
In summary, we demonstrated that a 5N,4O-carbonyl-7,8-di-O-
chloroacetyl-9-O-Fmoc-protected sialic acid phosphate building
block 4 can be used to install α(2,6)-sialic acid linkages effi-
ciently, while it did not give satisfactory results for α(2,3)-
sialylations. The latter linkage has to be incorporated either by
using a preformed sialic acid–Gal disaccharide building block
1
3.Tanaka, H.; Nishiura, Y.; Takahashi, T. J. Am. Chem. Soc. 2006, 128,
7124–7125. doi:10.1021/ja0613613
1
4.Chu, K.-C.; Ren, C.-T.; Lu, C.-P.; Hsu, C.-H.; Sun, T.-H.; Han, J.-L.;
Pal, B.; Chao, T.-A.; Lin, Y.-F.; Wu, S.-H.; Wong, C.-H.; Wu, C.-Y.
Angew. Chem., Int. Ed. 2011, 50, 9391–9395.
doi:10.1002/anie.201101794
620

Beilstein J. Org. Chem. 2015, 11, 617–621.
1
5.Hsu, C.-H.; Chu, K.-C.; Lin, Y.-S.; Han, J.-L.; Peng, Y.-S.; Ren, C.-T.;
Wu, C.-Y.; Wong, C.-H. Chem. – Eur. J. 2010, 16, 1754–1760.
doi:10.1002/chem.200903035
1
1
1
1
2
6.Kancharla, P. K.; Navuluri, C.; Crich, D. Angew. Chem., Int. Ed. 2012,
5
1, 11105–11109. doi:10.1002/anie.201204400
7.Kancharla, P. K.; Kato, T.; Crich, D. J. Am. Chem. Soc. 2014, 136,
472–5480. doi:10.1021/ja501276r
8.Weishaupt, M. W.; Matthies, S.; Seeberger, P. H. Chem. – Eur. J.
013, 19, 12497–12503. doi:10.1002/chem.201204518
5
2
9.Seeberger, P. H. Chem. Soc. Rev. 2008, 37, 19–28.
doi:10.1039/B511197H
0.Ratner, D. M.; Murphy, E. R.; Jhunjhunwala, M.; Snyder, D. A.;
Jensen, K. F.; Seeberger, P. H. Chem. Commun. 2005, 578–580.
doi:10.1039/B414503H
2
1.Hofmann, J.; Hahm, H. S.; Seeberger, P. H.; Pagel, K. submitted for
publication.
2
2.Mandhapati, A. R.; Rajender, S.; Shaw, J.; Crich, D.
Angew. Chem., Int. Ed. 2015, 54, 1275–1278.
doi:10.1002/anie.201409797
2
2
2
2
2
3.Kalikanda, J.; Li, Z. J. Org. Chem. 2011, 76, 5207–5218.
doi:10.1021/jo1025157
4.Ngoje, G.; Addae, J.; Kaur, H.; Li, Z. Org. Biomol. Chem. 2011, 9,
6
825–6831. doi:10.1039/c1ob05893b
5.Park, J.; Kawatkar, S.; Kim, J.-H.; Boons, G.-J. Org. Lett. 2007, 9,
959–1962. doi:10.1021/ol070513b
1
6.Demchenko, A.; Stauch, T.; Boons, G.-J. Synlett 1997, 818–820.
doi:10.1055/s-1997-5762
7.Fair, R. J.; Hahm, H. S.; Seeberger, P. H. Chem. Commun. 2015, 51,
6183–6185. doi:10.1039/C5CC01368B
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