De novo chemoenzymatic synthesis of sialic acid

Article abstract of DOI:10.1039/c2cc37305j

A chemoenzymatic synthesis of sialic acid from inexpensive N-acetyl-D-glucosamine is described. In a three-step Wittig-protection- ozonolysis strategy manno-configured aldehydes are obtained. Treatment with oxaloacetate in the presence of macrophomate syn

Full text of DOI:10.1039/c2cc37305j

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 11987–11989
www.rsc.org/chemcomm
COMMUNICATION
De novo chemoenzymatic synthesis of sialic acidw
Pierre Stallforth,zya Stefan Matthies,ya Alexander Adibekian,za Dennis G. Gillingham,8b
Donald Hilvertb and Peter H. Seeberger*a
Received 6th October 2012, Accepted 24th October 2012
DOI: 10.1039/c2cc37305j
A chemoenzymatic synthesis of sialic acid from inexpensive
N-acetyl-D-glucosamine is described. In a three-step Wittig–
protection–ozonolysis strategy manno-configured aldehydes are
obtained. Treatment with oxaloacetate in the presence of macro-
phomate synthase affords the signature a-keto-c-hydroxy acid
moiety with high diastereoselectivity.
Scheme 1 Sialic acid 1 and its open chain form 2 with the a-keto acid
Sialic acid 1 (Scheme 1) and its derivatives are ubiquitous in
both mammalian and bacterial glycomes.2 In mammals, sialic
acid is typically located at the distal end of glycan chains. As a
consequence, sialic acid is a key player in a variety of important
biological processes, including cell–cell and cell–pathogen
communication as well as tumor metastasis.3 Surface glyco-
proteins of the influenza virus, for instance, recognize sialic
acids on human cells in the upper respiratory tract and use
them for virus entry. In a later step, budding of virus progeny
from the infected cell is achieved by the action of viral
sialidases that detach the viruses from the host cell.4 As a
consequence, chemically homogeneous glycans containing
sialic acids and derivatives thereof have proven to be valuable
as drugs and as formidable tools in biomedical research.5
Structurally, sialic acids differ from other common mam-
malian carbohydrates in several important respects: their
backbones are nine carbon atoms long; they contain either a
free or acylated amine at C5; and they terminate in an a-keto
acid moiety (Scheme 1). These structural features render their
synthesis inherently difficult. Although many elegant approaches
for preparing sialic acid derivatives have been described in
recent years,6 short, flexible, and inexpensive routes to these
compounds are still needed.
motif highlighted in red. A retrosynthetic disconnection of sialic acid 1
reveals protected manno-configured aldehyde 3 and pyruvic acid 4 as
generic precursors.
Scheme 2 Retrosynthesis of sialic acid 1 starting from N-acetyl-D-
glucosamine 5.
De novo syntheses that transform inexpensive and commercially
available starting materials into carbohydrates or carbohydrate
building blocks via (dia)-stereoselective C–C-bond-forming
reactions have rendered many glycans chemically accessible.7
For sialic acids, such an approach would entail introduction of
the labile a-keto acid motif at a later stage by means of a
stereoselective pyruvate addition to a protected aldehyde like 3
(Scheme 2). However, to date, no single chemical method has been
developed that addresses this problem satisfactorily.8
We recently showed that the enzyme macrophomate
synthase1 (MPS) is an excellent catalyst for the stereoselective
installation of the a-keto acid motif9 and can be used for the
chemoenzymatic synthesis of 3-deoxysugars such as keto-
deoxy-nonulosonic acid (KDN).10 Here, we report that this
strategy also provides a convenient chemoenzymatic route to
sialic acid. While there are numerous examples of enzymatic
syntheses of sialic acids,6h the key strategic advantage of MPS
is its unusual tolerance to a wide range of protecting groups.
Protected building blocks are essential to allow the seamless
integration of sialic acid derivatives into synthetic strategies
towards large complex carbohydrates.
a Department of Biomolecular Systems, Max-Planck-Institute of
Colloids and Interfaces, Am Muhlenberg 1, 14476 Potsdam-Golm,
¨
Germany and Freie Universitat Berlin, Arnimallee 22, 14195 Berlin,
¨
Germany. E-mail: peter.seeberger@mpikg.mpg.de;
Fax: +49 331 567 9302; Tel: +49 331 567 9301
b Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich,
Switzerland
w Electronic supplementary information (ESI) available: Experimental
data and characterization data. See DOI: 10.1039/c2cc37305j
z Current address: Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, 240 Longwood Avenue,
Boston, MA 02115, USA.
y Both authors contributed equally.
z Current address: University of Geneva, Quai Enrest-Ansermet 30,
1211 Geneva 4, Switzerland.
8 Current address: University of Basel, St. Johannes-Ring 19, 4056
Basel, Switzerland.
According to the retrosynthetic analysis shown in Scheme 2,
protected aldehydes with D-manno-configuration are needed
to access sialic acids. Since N-acetyl-D-mannosamine itself
is prohibitively expensive (B$40 per g, Sigma-Aldrich),
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11987–11989 11987

View Article Online
Fig. 1 1H NMR spectra of synthetic sialic acid 1 (bottom, black) and
synthetic sialic acid spiked (1 : 1) with commercially available sialic
acid, Sigma-Aldrich (top, red). Note: sialic acid occurs in aq. solution
as a mixture of a/b-diastereomers.15
Scheme 3 Synthesis of manno-configured aldehyde 9 from D-GlcNAc
via a Wittig–protection–ozonolysis sequence.
N-acetyl-D-glucosamine 5 (B$1 per g, Sigma-Aldrich) was
utilized as an inexpensive alternative starting material.
accomplish this task. The MPS-catalysed addition reaction takes
place at near-neutral pH and thus provides an extremely mild
means to add the pyruvate moiety. The reaction was performed in
aqueous buffer with 5 mM MPS and magnesium(II) as a
cofactor. The pyruvate enolate nucleophile is generated in situ by
decarboxylation of oxaloacetate. Careful optimization of the
reaction conditions showed that the best results were obtained
at pH 7.5 and 25 1C to provide the desired 4S alcohol 10 as the
only diastereoisomer.
1H NMR analysis of the crude, deprotected cyclization
product 1 was used to assess the diastereoselectivity of the
enzymatic addition reaction and to provide unambiguous proof
of the C4 configuration (Fig. 1). To that end, bis-dioxolane 10
was treated with aqueous trifluoroacetic acid to give sialic acid 1.
Fig. 1 shows the 1H NMR spectrum of the crude product
resulting from the deprotection of 10 (bottom). Additionally,
the crude product was spiked (1 : 1 w/w) with commercially
available sialic acid (top). The data show that the stereoselectivity
of the MPS-catalysed pyruvate addition is excellent, with a
dr > 20 : 1 in favor of the desired 4S diastereomer. Thus,
manno-configured aldehydes are excellent substrates for MPS.
The outlined MPS strategy was also applicable for the
synthesis of sialic acid analogs. From the same common
precursor, olefin 7, a protected g-acetamido aldehyde, was
obtained (Scheme 5). Selective hydrolysis of the terminal
isopropylidene acetal of bis-acetal 7, and subsequent oxidative
cleavage of diol 11 gave aldehyde 12 in 93% yield over two
steps. The aldehyde was an excellent substrate for MPS in the
pyruvate addition reaction, with only a single C4 epimer of a
sialic acid analog 13 observed.
Conversion of D-GlcNAc into manno-configured aldehydes 3
requires an inversion step and subsequent transformation to the
open chain form. Subjecting 5 to the Wittig reagent benzylidene-
(triphenyl)phosphorane (BnPPh3Cl/K2CO3) accomplished both
tasks (Scheme 3).11 Due to the basic reaction conditions it is likely
that equilibration of the open chain epimers D-GlcNAc and
D-ManNAc occurs.11 Reaction of the ylid with open chain
D-ManNAc then leads to the formation of olefin 6. Treatment of
tetrol 6 with acetone and aqueous hydrochloric acid gave
bis-dioxolane 7, which was subsequently oxidized with ozone.
Because a-amino aldehydes are inherently unstable,12 they are not
typically isolated or purified.13 However, quantitative conversion
without racemization was mandatory to provide a pure substrate
for the subsequent aldol reaction. Unfortunately, reductive work-
up of the intermediate ozonide 8 to yield aldehyde 9 proved
difficult.14 None of the standard reduction protocols involving zinc
and either triphenyl phosphine or dimethylsulfide gave satisfactory
yields, and epimerization of the carbon bearing the acetamido
group was often observed. Nevertheless, careful optimization
revealed that a mixture of finely dispersed zinc and dimethylsulfide
reduced the ozonide quantitatively without any observable
epimerized by-product after 24 h. The reaction proceeded cleanly
and no chromatographic purification was required. Starting from
D-GlcNAc, aldehyde 9 could thus be easily obtained in only three
steps. This short and general route provides ready access to
protected manno-configured aldehydes.
With aldehyde 9 in hand, the enzymatic addition of a pyruvate
equivalent was investigated (Scheme 4). As a-acetamido aldehydes
are particularly prone to epimerization at the a-position, neither
strongly acidic nor strongly basic conditions could be used to
In summary, we describe a concise route to sialic acid and its
derivatives starting from inexpensive N-acetyl-D-glucosamine.
Scheme 4 Diastereoselective addition of pyruvate to aldehyde 9 using
Scheme 5 Synthesis of a protected sialic acid analog 13 from common
precursor 7.
macrophomate synthase (MPS).
c
11988 Chem. Commun., 2012, 48, 11987–11989
This journal is The Royal Society of Chemistry 2012

View Article Online
A three-step Wittig–protection–ozonolysis sequence provides
manno-configured, O-protected aldehydes, which are then
condensed with pyruvate enolate in a diastereoselective reaction
catalysed by the aldolase MPS. The desired S selectivity for the
newly formed stereogenic carbinol center proved to be excellent
with a dr > 20 : 1.
5, 163; (c) P. H. Seeberger and D. B. Werz, Nature, 2007,
446, 1046.
6 (a) B. Bressel and H.-U. Reissig, Org. Lett., 2009, 11, 527;
(b) I. Hemeon and A. Bennet, Synthesis, 2007, 1899;
(c) Z. Hong, L. Liu, C.-C. Hsu and C.-H. Wong, Angew. Chem.,
Int. Ed., 2006, 45, 7417; (d) M. J. Kiefel and M. von Itzstein, Chem.
Rev., 2002, 102, 471; (e) M. P. DeNinno, Synthesis, 1991, 583;
(f) R. Lorpitthaya, S. B. Suryawanshi, S. Wang, K. K. Pasunooti,
S. Cai, J. Ma and X.-W. Liu, Angew. Chem., Int. Ed., 2011,
50, 12054; (g) R. Csuk, M. Hugener and A. Vasella, Helv.
Chim. Acta, 1988, 71, 609; (h) H. J. M. Gijsen, L. Qiao, W. Fitz
and C.-H. Wong, Chem. Rev., 1996, 96, 443.
The high diastereoselectivity, the mild conditions of the
MPS-catalysed pyruvate addition, as well as its tolerance to
a variety of protecting groups open up possibilities to access
fully protected sialic acid for the assembly of sialic
acid-containing oligosaccharides, novel sialidase inhibitors,
and fluorinated sialic acids. The latter are the subject of
ongoing studies in our laboratories.
7 (a) A. Kirschning, M. Jesberger and K. Schoning, Synthesis, 2001,
¨
507; (b) M. Markert and R. Mahrwald, Chem.–Eur. J., 2008,
14, 40; (c) E. U. Sharif and G. A. O’Doherty, Eur. J. Org. Chem.,
2012, 2095; (d) R. S. Babu, M. Zhou and G. A. O’Doherty, J. Am.
Chem. Soc., 2004, 126, 3428; (e) P. Stallforth, A. Adibekian and
P. H. Seeberger, Org. Lett., 2008, 10, 1573; (f) T. Ohara,
A. Adibekian, D. Esposito, P. Stallforth and P. H. Seeberger,
Chem. Commun., 2010, 46, 4106; (g) D. C. Koester,
A. Holkenbrink and D. B. Werz, Synthesis, 2010, 3217.
8 A. Dondoni, A. Marra and P. Merino, J. Am. Chem. Soc., 1994,
116, 3324.
9 J. M. Serafimov, D. Gillingham, S. Kuster and D. Hilvert, J. Am.
Chem. Soc., 2008, 130, 7798.
10 D. G. Gillingham, P. Stallforth, A. Adibekian, P. H. Seeberger and
D. Hilvert, Nat. Chem., 2010, 2, 102.
11 A. Giannis and T. Henk, Liebigs Ann. Chem., 1991, 789.
12 (a) J. Jurczak, D. Gryko, E. Kobrzycka, H. Gruza and
P. Prokopowicz, Tetrahedron, 1998, 54, 6051; (b) J. Jurczak and
A. Go"e˛biowski, Chem. Rev., 1989, 89, 149; (c) D. Gryko,
J. Cha"ko and J. Jurczak, Chirality, 2003, 15, 514;
(d) M. T. Reetz, Chem. Rev., 1999, 99, 1121.
13 F. A. Davis, T. Ramachandar, J. Chai and E. Skucas, Tetrahedron
Lett., 2006, 47, 2743.
14 S. G. Van Ornum, R. M. Champeau and R. Pariza, Chem. Rev.,
2006, 106, 2990.
15 The chemical shifts of the spiked sample are slightly shifted from
those of the commercial material. Since this is a spike experiment
the identity of the compound is unequivocally demonstrated. We
believe the slight differences observed in the chemical shifts are a
result of the minor changes in concentration and/or pH that arise
from the spiking: (a) T. Klepach, I. Carmichael and A. S. Serianni,
J. Am. Chem. Soc., 2008, 130, 11892; (b) J. Haverkamp, H. van
Halbeek, L. Dorland, J. F. G. Vliegenthart, R. Pfeil and
R. Schauer, Eur. J. Biochem., 1982, 122, 305.
This work was supported by the Max-Planck-Society, ETH
Zurich, a Marie Curie International Incoming Fellowship
within the 7th European Community Framework Programme
to D. G. G. (IIF-AEOM) and a PhD fellowship from the
Studienstiftung des Deutschen Volkes to P.S. We would like to
thank Carl V. Christianson for the expression and purification
of MPS.
Notes and references
1 MPS crystal structure adapted from (a) T. Ose, K. Watanabe,
T. Mie, M. Honma, H. Watanabe, M. Yao, H. Oikawa and
I. Tanaka, Nature, 2003, 422, 185; (b) K. Arnold, L. Bordoli,
J. Kopp and T. Schwede, Bioinformatics, 2006, 22, 195;
(c) F. Kiefer, K. Arnold, M. Kunzli, L. Bordoli and T. Schwede,
¨
Nucleic Acids Res., 2009, 37, D387.
2 (a) A. Adibekian, P. Stallforth, M.-L. Hecht, D. B. Werz,
P. Gagneux and P. H. Seeberger, Chem. Sci., 2011, 2, 337;
(b) T. Angata and A. Varki, Chem. Rev., 2002, 102, 439;
(c) D. B. Werz, R. Ranzinger, S. Herget, A. Adibekian, C.-W. von
der Lieth and P. H. Seeberger, ACS Chem. Biol., 2007, 2, 685.
3 (a) R. Schauer, Curr. Opin. Struct. Biol., 2009, 19, 507;
(b) Y. N. Malykh, R. Schauer and L. Shaw, Biochimie, 2001,
83, 623; (c) P. Sillanaukee, M. Ponnio and I. P. Jaaskelainen, Eur.
J. Clin. Invest., 1999, 29, 413.
¨
¨
¨
¨
¨
4 J. J. Skehel and D. C. Wiley, Annu. Rev. Biochem., 2000, 69, 531.
5 (a) L. L. Kiessling and R. A. Splain, Annu. Rev. Biochem.,
2010, 79, 619; (b) X. Chen and A. Varki, ACS Chem. Biol., 2010,
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11987–11989 11989