Conversion of the carboxy group of sialic acid donors to a protected hydroxymethyl group yields an efficient reagent for the synthesis of the unnatural beta-linkage

Article abstract of DOI:10.1039/b102166b

New sialyl donors with a protected hydroxymethyl group at the anomeric center are over 1000 times more reactive than the normal ester containing sialylation reagent and give excellent yield (> 90%) with unusually high β-stereoselectivity in sialylation.

Full text of DOI:10.1039/b102166b

Conversion of the carboxy group of sialic acid donors to a protected
hydroxymethyl group yields an efficient reagent for the synthesis of
the unnatural beta-linkage
Xin-Shan Ye, Xuefei Huang and Chi-Huey Wong*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: wong@scripps.edu
Received (in Corvallis, OR, USA) 5th March 2001, Accepted 4th April 2001
First published as an Advance Article on the web 10th May 2001
New sialyl donors with a protected hydroxymethyl group at
the anomeric center are over 1000 times more reactive than
the normal ester containing sialylation reagent and give
excellent yield ( > 90%) with unusually high b-stereoselectiv-
ity in sialylation.
N-Acetylneuraminic acid (sialic acid) residues are often located
at the non-reducing end of glycoconjugates, and play important
roles in many biological recognition events such as cancer
metastasis and bacterial or viral infection.1,2 The synthesis of
sialyl glycoconjugates, however, remains a challenge.3
Scheme 1 Reagents and conditions: (i) p-Thiocresol, BF3·Et2O, CH2Cl2,
20% for 4a, 75% for 4b; (ii) NaOMe, MeOH, 100%; (iii) NaH, BnBr, DMF,
55%; (iv) LiBH4, THF, MeOH, 90%; (v) Ac2O, pyridine, 96%; (vi)
BOMCl, Pri2NEt, CH2Cl2, 86%; (vii) TBDPSCl, imidazole, DMF, 96%.
comparable to or even higher than perbenzylated -fucose 6
L
which was the most reactive thioglycoside measured pre-
viously.12
With the RRV values in hand, sialylation of donors 1a–1d
was performed. The TBDPS protected sialyl donor 1c failed to
undergo glycosylation with galactose acceptor 7, presumably
due to the large TBDPS group. With the smaller acetyl
protective group, donor 1a underwent smooth sialylation with
galactose acceptor 7 to give disaccharides 8 and 9 in 95% yield
(8+9 = 15+1) in acetonitrile using dimethyl(methylthio)sulfon-
ium triflate (DMTST) as the promoter13,14 (Scheme 2a). No
elimination product was isolated. The hydroxymethyl moiety of
the products can be subsequently unmasked after sialylation and
selectively oxidized to the carboxy group in three high yielding
steps as demonstrated by transformation of compound 8 to 10
(Scheme 2b) and 9 to 12 (Scheme 2c). However, quite
unexpectedly the predominant product 8 formed in the sialyla-
tion contains a b linkage between the two monosaccharides. The
near zero 3JC1,H3a value in the EXCIDE5,15 spectra of 8 and 11‡
In a typical sialylation reaction, the electron withdrawing
carboxy group at the anomeric center of sialic acids sig-
nificantly destabilizes the oxonium ion forming transition state
thus lowering the reactivity of sialyl donors towards nucleo-
philes. Furthermore, the steric hinderance at the anomeric center
leads to elimination and low yields of sialylation. Various
methods have been developed to overcome these problems,
including, for example, introduction of an additional N-acetyl
moiety,4,5 installation of an anchimeric assisting group at the C3
position5–8 or a carboxy equivalent at the anomeric center.9,10
The latter approach includes utilizing a less electron with-
drawing furyl substituent as the carboxy surrogate.9 This
method, however, failed to yield any disaccharides with
secondary sugar alcohols. Increased reactivities towards N-
iodosuccinimide (NIS) have also been observed with the
reduced 2,3-didehydro sialic acid derivatives, but these reac-
tions gave very low yields ( ~ 20%).10
Table 1 Relative reactivity values (RRV) of various thio-glycosidesa
To tackle the aforementioned problems, we converted the
carboxy group of sialic acid to the hydroxymethyl group and
prepared derivatives 1a–1d for investigation (Scheme 1). The
peracetylated sialic acid 311 was treated with p-thiocresol to
give thio-sialic acid 4b in 75% yield together with 20% of the a
isomer 4a. The protective groups of 4b were exchanged for
benzyl groups to give compound 5 in two steps in 55% yield.
Reduction of the ester moiety was accomplished with LiBH4 in
90% yield to give the hydroxymethyl sialic acid derivative 2,
the hydroxy group of which was protected with the acetyl,
benzyloxymethyl (BOM) or tert-butyldiphenylsilyl (TBDPS)
group to give sialyl donors 1a–1c. The a sialyl donor 1d was
prepared from 4a in a similar manner as the b sialyl donor
1a.
Compound
RRV Compound
RRV
The relative reactivity values (RRV) of known and new sialic
acid donors 1a–1d, 2, 4b and 5 were measured as previously
described12 and shown in Table 1. Compared to the benzyl
protected sialic acid 5, over three orders of magnitude increase
in RRV was observed with all the reduced sialic acid derivatives
1a†–1d and 2 (RRV for 1a, 1b, 1c, 1d and 2 are 4.0 3 104, 2.3
3 105, 7.8 3 104, 8.0 3 104 and 3.3 3 105 respectively). The
reactivities of all these reduced sialic acids (1a–1d and 2) are
a The RRV is based on the reactivity of 1-thiotolyl-2,3,4,6-tetraacetyl-b-
D-
mannopyranoside.
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Chem. Commun., 2001, 974–975
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b102166b

Scheme 2 Reagent and conditions: (i) DMTST, molecular sieves, CH3CN, 240 °C to rt, overnight; (ii) NaOMe, MeOH, rt, 1 h; (iii) PhI(OAc)2, TEMPO,
rt, 12 h; (iv) NaClO2, 2-methylbut-2-ene, rt, 4 h; (v) NaOH, H2O, rt, 1 h.
3
indicated the b-configuration, while the JC1,H3a value of 13§
was determined to be 6.1 Hz indicating its a-configuration.
Comparison of the chemical shifts of H3eq of compound 11
(2.64 ppm) and 13 (2.72 ppm)16 further confirmed the
assignment following the empirical rules of chemical shift.4 The
stereoselectivity does not vary much with different acceptors.
Sialylation of various acceptors such as isopropyl alcohol 14,
lactose derivative 15 as well as primary alcohol 16, glucosamine
17 and galactose 18, with donor 1a using DMTST as the
promoter, gave predominantly the b-linked disaccharide17 (b+a
> 10+1) in high yields ( > 90%). The exception was the sialic
This work was supported by the National Institutes of Health
(GM-44154). We thank Dr Zhiyuan Zhang for helpful discus-
sions.
Notes and references
† Selected data for 1a: 1H NMR (400 MHz, CDCl3) d 7.03 (d, J = 7.9 Hz,
2H), 5.06 (d, J = 8.5 Hz, 1H), 3.69 (dd, J = 3.8, 10.4 Hz, 1H), 2.26 (s, 3H),
2.20 (dd, J = 3.7, 13.5 Hz, 1H), 1.92 (dd, J = 11.0, 13.4 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 170.44, 170.15, 88.86, 69.17, 68.18, 51.77, 35.62,
23.69, 21.07, 20.66; HRMS (M + Cs) calcd for C48H53O8NSCs 936.2546,
found 936.2577.
‡ Selected data for 11: 1H NMR (400 MHz, CD3OD) d 5.32 (s, 1H), 2.66
(dd, J = 4.4, 13.2 Hz, 1H), 2.16 (t, J = 7.6 Hz, 2H), 1.77 (s, 3H), 1.72 (dd,
J = 11.3, 13.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 174.36, 170.04,
102.54, 101.90, 100.26, 69.93, 69.19, 68.84, 52.04, 51.60, 35.92, 33.69,
28.65, 25.36, 24.25, 23.22; 3JC1,H3a ~ 0 Hz; HRMS (M 2 H + 2 Na+) calcd
for C58H65O16NNa2 1054.4196, found 1054.4202.
§ Selected data for 13: 1H NMR (600 MHz, CD3OD) d 5.32 (s, 1H), 2.72
(dd, J = 3.7, 12.6 Hz, 1H), 2.16 (t, J = 7.2 Hz, 2H), 1.94 (s, 3H), 1.88 (t,
J = 12.6 Hz, 1H); 13C NMR (150 MHz, CDCl3) d 182.25, 174.40, 172.78,
103.45, 101.61, 101.28, 69.84, 68.80, 50.95, 38.15, 35.95, 29.43, 26.48;
3JC1,H3a = 6.1 Hz; HRMS (M 2 H + 2 Na+) calcd for C58H65O16NNa2
1054.4196, found 1054.4175.
acid derivative 19 which gave a ratio of 3+1 favoring b-
disaccharide in 90% total yield. Sialylations in solvents such as
ether, toluene and dichloromethane gave even more b anomer
than those performed in acetonitrile. The acetonitrile effect18
could not significantly alter the anomeric selectivity. Sialylation
of galactose 7 with the BOM protected donor 1b or a sialyl
donor 1d gave the product with similar yield and ster-
eoselectivity to those with donor 1a.
Sialylations with promoters other than DMTST were also
tested. MeOTf19 failed to activate sialyl donor 1a while with
PhSOTf20 only the b isomer was isolated when galactose 18 was
sialylated with 1a. The use of NIS and triflic acid (TfOH)
improved the a-selectivity (a+b = 1+2.5) when galactose
acceptor 7 was sialylated with 1a in 90% total yield.
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In conclusion, it has been demonstrated that the reactivity of
sialic donors can be dramatically increased by reducing the
carboxy group at the anomeric center to the hydroxymethyl
moiety. Subsequent sialylation with these novel sialyl donors
proceeded in excellent yield ( > 90%) but with unusually high b
stereoselectivity, probably due to a significant anomeric effect.
The hydroxymethyl moiety can be easily oxidized to the
carboxy group in high yield. The high reactivity of sialyl donors
could find uses in the preparation of enzymatically stable
unnatural oligosaccharides containing b-sialic acid. Oligo-
saccharides with unnatural glycosidic linkage could have
important biological implications, as illustrated in the study of
CD-1 mediated T-cell activation.21 The new glycosylation
reagents can also be utilized in programmable one-pot synthe-
sis, where the sialylation reaction often has to be the first and
most reactive as sialic acid is often located at the non-reducing
end of bio-active oligosaccharides.12 Introduction of a C-3
auxiliary may give the a-linkage.
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11 (2.64 ppm) and 13 (2.72 ppm). The formation of lactone 12 caused
an upfield shift of H3eq to 2.60 ppm while the chemical shift of H3eq of
10 remained 2.64 ppm.
17 The stereochemistry of the disaccharides was determined by measuring
3JC1,H3a from the EXCIDE spectra as described for compounds 8, 11
and 13.
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