N-trifluoroacetyl sialyl phosphite donors for the synthesis of α(2 → 9) oligosialic acids

Article abstract of DOI:10.1021/ol0515210

(Chemical Equation Presented) A new method for the synthesis of α(2 → 9) oligosialic acids is developed using phosphite sialyl donors that are protected with a C-5 N-trifluoroacetyl (NHTFA) substituent. Compared with conventional donors, these donors gave a higher degree of α-anomeric selectivity during glycosidic bond formation and better yields during iterative sialylation in the synthesis of oligosialic acids.

Full text of DOI:10.1021/ol0515210

ORGANIC
LETTERS
2005
Vol. 7, No. 19
4169-4172
N-Trifluoroacetyl Sialyl Phosphite
Donors for the Synthesis of
Oligosialic Acids
r(2 f 9)
Chang-Ching Lin,†,‡ Kuo-Ting Huang,† and Chun-Cheng Lin*,†
Institute of Chemistry and Genomic Research Center, Academia Sinica,
Taipei, Taiwan, and Department of Chemistry, National Tsing Hua UniVersity,
Hsinchu, Taiwan
cclin@chem.sinica.edu.tw
Received June 29, 2005
ABSTRACT
A new method for the synthesis of
r(2 f 9) oligosialic acids is developed using phosphite sialyl donors that are protected with a C-5
N-trifluoroacetyl (NHTFA) substituent. Compared with conventional donors, these donors gave a higher degree of
r-anomeric selectivity
during glycosidic bond formation and better yields during iterative sialylation in the synthesis of oligosialic acids.
Sialic acids are nine-carbon carboxylated saccharides and
are often found at the nonreducing terminus of oligosaccha-
ride chains that are part of glycolipids or glycoproteins in
avian and mammalian tissues. Sialic acids are involved in a
number of biological processes, such as cell-cell interaction,
cell differentiation and proliferation, tumor metastasis,
malignant alteration, pathongen-host recognition, toxin-
receptor interaction, and neural network development.1
Among the more than 40 members of the sialic acid family,
the monomeric N-acetyl neuraminic acid (Neu5Ac) is a
prominent one because of its wide occurrence in bioconju-
gates.2 Linear polysialic acids consist of contiguous Neu5Ac’s
linked by R2-8, R2-9, and alternating R2-8 and R2-9
linkages; these chains are found on the surface of bacteria
(e.g., Escherichia coli K1 and Neisseria meningitides groups
B and C), where they function as virulence factors. Their
presence on the bacterial surface make them a good target
for bactericidal antibodies and potential antigens for anti-
bacterial vaccine development.3a
The polysaccharides used to prepare vaccines are generally
isolated from their natural sources. The natural isolates,
however, may be heterogeneous and/or contaminated with
other antigenic components. Thus, it is of great interest to
develop synthetic polysaccharide vaccines having greater
† Academia Sinica.
‡ Tsing Hua University.
(1) (a) Bomsel, M.; Alfsen, A. Nat. ReV. Mol. Cell Biol. 2003, 4, 57-
68. (b) Allende, M. L.; Proia, R. L. Curr. Opin. Struct. Biol. 2002, 12,
587-592. (c) Crocker, P. R. Curr. Opin. Struct. Biol. 2002, 12, 609-615.
(d) Muhlenhoff, M.; Eckhardt, M.; Gerardy-Schahn, R. Curr. Opin. Struct.
Biol. 1998, 8, 558-564. (e) Biology of the Sialic Acids; Rosenberg, A.,
Ed.; Plenum: New York, 1995.
(2) (a) Kiefel, M. J.; Itzstein von, M. Chem. ReV. 2002, 102, 471-490.
(b) Inoue, Y.; Inoue, S. Pure Appl. Chem. 1999, 71, 789-800.
10.1021/ol0515210 CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/17/2005

immunogenicity.3b However, the chemical synthesis of
polysialic acids presents a formidable challenge in that the
sialylation reaction often proceeds with low yield, low
R-stereoselectivity, and undesired 2,3-elimination due to an
electron-withdrawing group at the anomeric center, the lack
of a participating auxiliary substituent adjacent to the
anomeric center, and a sterically hindered tertiary anomeric
center.4 Fortunately, recent progress in the development of
sialic acid donors for better R-selectivity has been achieved
with moderate success by modifying the amino protecting
group at the C-5 position5-8 and inserting an auxiliary group
at the C-19 or C-310 position while phosphite,11 sulfide,12
xanthanate13 or hydroxyl group14 was used as a leaving group.
However, for oligosialic acids, the preparation of suitable
donors and acceptors with differentially protected hydroxyl
functions is a laborious task, although effective strategies
have been developed in recent years.4 In view of the
aforementioned points, we investigated whether a homo-
oligosialic acid could be synthesized by iterative glycosy-
lation using a minimum of reaction types; we also measured
the extent to which synthesis could be carried out with
acceptable R-stereocontrol. Here, we report our synthesis of
homooligosialic acids having R2-9 intersialyl linkages.
Before embarking on the synthesis of homooligosialic
acids, we took cognizance of literature precedence to design
appropriate sialyl donor-acceptor combinations for the
proposed synthesis. Over the past several years, it has been
well recognized that replacement of the N-acetyl functional
group at C-5 with N,N-diacetyl (NAc2),5 N-trifluoroacetyl
(NTFA),6 N-2,2,2-trichloroethoxycarbonyl (NTroc),7 or an
azido group8 in the sialyl donor results in higher yields and,
in some cases, better R-selectivity during sialylation. These
groups are also considered to inhibit hydrogen bond forma-
tion between NH at C-5 and OH at C-8/C-9. Thus, the
nucleophilicity of the C-9 OH group can be enhanced, which
would be advantageous if R2-9 sialylation is desired.4c,15
Although several leaving groups at the anomeric center can
enhance the R-selectivity of sialyl donors,11-14 the sulfide-
and phosphite-based donors are most commonly used,11,12
with the sulfide-based donors having the comparative
advantage of being very stable. Conversely, phosphite-based
donors can be activated by a catalytic amount of promoter
(usually TMSOTf) and usually lead to predominant formation
of the R-product during glycosylation.11c On the basis of the
above precedence, TFA and Troc were chosen as protecting
groups at the N-5 of sialic acid, phosphite was used as the
leaving group during sialylation, and thiocresol served as
the protecting group of the anomeric center.
As shown in Figure 1, three phosphite donors with NHAc,
NHTroc, and NHTFA at the C-5 position (1, 2, and 3,
respectively) were tested with regard to their R-selectivities
in sialylation with sulfide acceptors 416 and 5. The results
(3) (a) Jennings, H. J. In Carbohydrate-based Drug DiscoVery; Wong,
C.-H., Ed; Wiley-VCH: Weinheim, 2003; pp 357-380. (b) Verez-Bencome,
V.; Ferna´ndez-Santana, V.; Hardy, E.; Toledo, M. E.; Rodr´ıguez, M. C.;
Heynngnezz, L.; Rodriguez, A.; Baly, A.; Herrera, L.; Izquierdo, M.; Villar,
A.; Valde´s, Y.; Cosme, K.; Deler, M. L.; Montane, M.; Garcia, E.; Ramos,
A.; Aguilar, A.; Medina, E.; Yoran˜o, G.; Sosa, I.; Hernandez, I.; Mart´ınez,
R.; Muzachio, A.; Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.; Diaz,
M.; Roy, R. Science 2004, 305, 522-525.
(4) (a) Ress, D. K.; Linhardt, R. J. Curr. Org. Synth. 2004, 1, 31-46.
(b) Lin, C.-H.; Lin, C.-C. in The Molecular Immunology of Complex
Carbohydrates 2; Kluwer-Plenum: New York, 2001. (c) Boons, G.-J.;
Demchenko, A. V. Chem. ReV. 2000, 100, 4539-4565.
(5) Demchenko, A. V.; Boons, G.-J. Chem. Eur. J. 1999, 5, 1278-
1283.
(6) (a) Pan, Y.; Chefalo, P.; Nagy, N.; Harding, C.; Guo, Z. J. Med.
Chem. 2005, 48, 875-883. (b) Meijer, A.; Ellervik, U. J. Org. Chem. 2004,
69, 6249-6256. (c) De Meo, C.; Demchenko, A. V.; Boons, G.-J. J. Org.
Chem. 2001, 66, 5490-5498.
(7) (a) Tanaka, H.; Adachi, M.; Takahashi, T. Chem. Eur. J. 2005, 11,
849-862. (b) Ando, H.; Koike, Y.; Ishida, H.; Kiso, M. Tetrahedron Lett.
2003, 44, 6883-6886.
(8) (a) Lu, K.-C.; Tseng, S.-Y.; Lin, C.-C. Carbohydr. Res. 2002, 337,
755-760. (b) Yu, C.-S.; Niikura, K.; Lin, C.-C.; Wong, C.-H. Angew.
Chem., Int. Ed. 2001, 40, 2900-2903. (c) Scneider, R.; Freyhardt, C. C.;
Schmidt, R. R. Eur. J. Org. Chem. 2001, 9, 1655-1661.
(9) (a) Ishiwata, A.; Ito, Y. Synlett 2003, 9, 1339-1343. (b) Haberman,
J. M.; Gin, D. Y. Org. Lett. 2001, 3, 1665-1668. (c) Takahashi, T.;
Tsukamoto, H.; Yamada, H. Tetrahedron Lett. 1997, 38, 8223-8236.
(10) (a) Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schmidt, R. R. J. Am.
Chem. Soc. 1998, 120, 5434-5440. (b) Martichonok, V.; Whitesides, G.
M. J. Am. Chem. Soc. 1996, 118, 8187-8191. (c) Erce´govec, T.;
Magnusson, G. J. Org. Chem. 1996, 61, 179-184. (d) Ito, Y.; Numata,
M.; Sugimoto, M.; Ogawa, T. J. Am. Chem. Soc. 1989, 111, 8508-8510.
(e) Kondo, T.; Abe, H.; Goto, T. Chem. Lett. 1988, 1657-1660.
(11) (a) Martin, T. J.; Schmidt, R. R. Tetrahedron Lett. 1992, 33, 6123-
6126. (b) Kondo, H.; Ichikawa, Y.; Wong, C.-H. J. Am. Chem. Soc. 1992,
114, 8748-8750.
(12) (a) Zhang, Z.; Ollmann, I. R.; Ye, X.; Wischnat, R.; Baasov, T.;
Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 734-753. (b) Nicolaou, K. C.;
Ueno, H. In PreparatiVe Carbohydrate Chemistry; Hanessian, S., Ed.;
Marcel Dekker: New York, 1997; pp 313-338. (c) Hasegawa, A. In
Modern Methods in Carbohydrate Synthesis; Khan, S., O’Neill, R., Eds.;
Harwood Academic Publishers: New York, 1996; pp 277-300.
(13) Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35-42.
(14) Haberman, J. M.; Gin, D. Y. Org. Lett. 2003, 5, 2539-2541.
Figure 1. Acceptors, donors, and products of sialylation.
of sialylations were shown in Table 1 The reaction between
bulky acceptor 4 and 1 yielded the elimination product only
(15) Castro-Palomino, J. C.; Tsvetkov, Y. E.; Schneider, R.; Schmidt,
R. R. Tetrahedron Lett. 1997, 38, 6837-6840.
(16) Chen, M.-Y.; Patkar, L. N.; Lu, K.-C.; Lee, A. S.-Y.; Lin, C.-C.
Tetrahedron Lett. 2004, 60, 11465-11475.
4170
Org. Lett., Vol. 7, No. 19, 2005

3. Selective acetonation of 12 was followed by acetylation
and then acidic deprotection of acetonide to yield 5.
Table 1. Sialylation of C-5 N-Protected Sialic Acid
Derivativesa
For extending the sialic acid chain, we adopted a strategy
in which the chain was elongated from the nonreducing end
toward the reducing end. Thus, the free hydroxyl group at
C-8 of the reducing end sialyl unit of 9 was capped with an
acetyl group (95% yield), and subsequently the sulfide group
was converted to a hydroxyl group (84% yield) by treating
with N-bromosuccinimide (NBS)17 in aqueous acetone.
Treatment of the resulting intermediate with dibenzyl N,N-
diisopropylphosphoramidite produced the disialyl phosphite
donor (97% yield).11b The second generation of sialylation
was performed using the phosphite dimer with acceptor 5
to give the trimer, 14. Iterating the sialylation, acetylation,
thiocresol deprotection, and phosphite formation produced
the higher-order sialosides. The execution and results of this
strategy are summarized in Table 2. The R-selectivity of
entry
donor
acceptor
product
yield (%)
R/â
1
2
3
4
1
2
1
3
4
4
5
5
6
7
8
9
83
33
55
77
1.5/1
4.7/1
R
a Tol ) p-methyl phenyl.
(entry 1), whereas the reaction between 2 and 4 produced
the desired dimer with 33% yield. Although the R-selectivity
was not good (R/â ) 1.5), donor 2 had better reactivity for
the bulky acceptor (entry 2). When the less sterically hindered
acceptor 5 was sialylated with 1, as expected, only the
primary hydroxyl group reacted with the donor, and both
the reaction yield (55%) and the R-selectivity increased (entry
3). Thus, the donor with higher activity, 3, was reacted with
5 to produce the R2-9 sialic acid dimer 9 with 77% yield
(entry 4). These model studies demonstrated that the TFA
protecting group at the C-5 position of sialic acid is a good
choice for both sialyl donor and acceptor.
Table 2. Synthesis of R2-9 Oligosialic Acidsa
glycosylation
thiocresol
acetylation deprotection formation
phosphite
Synthesis of donor 3 and acceptor 5 are outlined in Scheme
1. The synthesis of the key intermediate 12 was achieved
entry
(R/â)
1
2
3
4
9
77% (R)
95%
94%
96%
95%
84%
88%
88%
82%
80%
85%
14 70% (4/1)
15 74% (1.6/1)
16 45% (1/1.3)
Scheme 1. Synthesis of Donor 3 and Acceptor 5
a Tol ) p-methyl phenyl.
sialylation decreased with increasing phosphite donor size.
Eventually, the â isomer became the major product in the
synthesis of pentasialic acid (â/R ) 1.3) using our newly
developed method. Notably, the R/â mixture of 14, 15, and
16 could not be separated without acetylation of the free
hydroxyl group. The new formed anomeric configuration of
9 was determined by the long-range JC-1,H-3ax coupling
constant.8a,18 By selective proton decoupled 13C NMR experi-
ments, the coupling pattern of C-1 of the R anomer gave a
doublet C-1 signal and the coupling constant was 5.6 Hz.
The configurations of nonreducing end sialic acids of 14,
15, and 16 were determined by empirical rules8a,b,19 and the
(17) Lin, C.-C.; Hsu, T.-S.; Lu, K.-C.; Huang, I.-T. J. Chin. Chem. Soc.
2000, 47, 921-928.
(18) Hori, H.; Nakajima, T.; Nishida, Y.; Ohrui, H.; Meguro, H.
Tetrahedron Lett. 1988, 29, 6317-6320.
(19) (a) Hasegawa, A.; Ohki; H.; Nagahama, T.; Ishida, H.; Kiso, M.
Carbohydr. Res. 1991, 212, 277-281. (b) Kanie, O.; Kiso, M.; Hasegawa,
A. J. Carbohydr. Chem. 1988, 7, 501-506. (c) Okamoto, K.; Kondo, T.;
Goto, T. Bull. Chem. Soc. Jpn. 1987, 60, 637-643. (d) Van der Vleugel,
D. J. M.; Van Heeswijk, W. A. R.; Vliegenthart, J. F. G. Carbohydr. Res.
1982, 102, 121-130. (e) Dabrowski, U.; Friebolin, H.; Brossmer, R.; Supp,
M. Tetrahedron Lett. 1979, 48, 4637-4640.
by acylation of 11,6c which was obtained from neuraminic
acid 10, as previously reported.5,8 Peracetylation of 12 was
followed by deprotection of thiocresol17 to give a hydroxyl
compound that was transformed to phosphite11b to produce
Org. Lett., Vol. 7, No. 19, 2005
4171

Several important features of this work should be empha-
sized. New phosphite-based sialyl donors have demonstrated
their utility for efficient construction of di-, tri-, tetra-, and
pentasialosides by iterative sialylation. The R-selectivity
displayed by the donors, especially during the formation of
di- and trisialosides, has very few parallels compared with
the reported sialyl donors.5,6c,8c The yields obtained in the
sialylation reactions are comparable or superior to those
previously reported for disialosides and trisialosides.8c Fi-
nally, to the best of our knowledge, this is the first chemical
synthesis of pentasialic acid through glycosidic bond forma-
tion.
In conclusion, we have demonstrated the utility of new
phosphite-based donors for efficient and highly R-selective
synthesis of oligosialic acids (up to pentasialoside) using
iterative sialylation. Although the R-selectivity decreased
with increasing size of the donor, pure pentasialic acid was
obtained on the 10 mg scale. We believe that these phosphite
donors will have applications in the development of polysialic
acid based vaccines.
Acknowledgment. This research was supported by Aca-
demia Sinica and the National Science Council in Taiwan
(NSC 92-2113-M-001-015).
Figure 2. Proton NMR spectrum of R2-9 pentasialic acid 16.
chemical shifts of H-3eq of nonreducing end sialic acids.
The chemical shifts of H-3eq of R anomers were more
downfield than those of â anomers. The proton NMR
spectrum of the acetylated R2-9 pentasialic acid derivative,
16, is shown in Figure 2. Note that the chemical shifts of
H-3eq protons of the nonreducing end saccharides are at the
same position and that of the reducing end sugar shows
downfield shift.
Supporting Information Available: Detailed synthetic
procedures and spectroscopic characterization of compounds
3, 5, 7-9, and 12-16. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL0515210
4172
Org. Lett., Vol. 7, No. 19, 2005