Preparation of a fluorous protecting group and its application to the chemoenzymatic synthesis of sialidase inhibitor

Article abstract of DOI:10.1039/b605519b

2-(Perfluorohexyl)ethoxymethyl chloride was prepared as a novel fluorous protecting reagent. Neu5Ac aldolase-catalyzed chemoenzymatic transformation of N-acetyl-d-mannosamine to Neu5Ac derivatives was achieved successfully by using the fluorous reagent not only for hydroxy group protection but also for fluorous tagging. This chemoenzymatic method was applied to the synthesis of 2-deoxy-2,3-didehydrosialic acid 1 known as a potent sialidase inhibitor. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b605519b

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Preparation of a fluorous protecting group and its application to the
chemoenzymatic synthesis of sialidase inhibitor
Kiyoshi Ikeda,* Hitomi Mori and Masayuki Sato*
Received (in Cambridge, UK) 18th April 2006, Accepted 19th May 2006
First published as an Advance Article on the web 13th June 2006
DOI: 10.1039/b605519b
Our concept of chemoenzymatic synthesis of 1 using a fluorous tag
is shown in Scheme 1.
2-(Perfluorohexyl)ethoxymethyl chloride was prepared as a
novel fluorous protecting reagent. Neu5Ac aldolase-catalyzed
chemoenzymatic transformation of N-acetyl-D-mannosamine
to Neu5Ac derivatives was achieved successfully by using the
fluorous reagent not only for hydroxy group protection but
also for fluorous tagging. This chemoenzymatic method was
applied to the synthesis of 2-deoxy-2,3-didehydrosialic acid 1
known as a potent sialidase inhibitor.
The first step of the synthesis of 7 began with the per-
O-acetylation of D-ManNAc, followed by the glycosylation and
subsequent deprotection of O-acetyl group to give a-glycoside 2 in
68% yield in three steps. Compound 2 was protected by
isopropylidene group and then acetylated to give 3 in 84% yield
in two steps. Next, the treatment of 3 with 80% AcOH afforded
diol 4 in quantitative yield. We synthesized 2-(perfluorohexyl)-
ethoxymethyl chloride 58 as a fluorous acetal protecting reagent by
the reaction of 2-(perfluorohexyl)ethanol and paraformaldehyde in
the presence of dry hydrogen chloride. The 2-(perfluorohexyl)-
ethoxymethyl protecting group would have minimal effect on the
reactivity of the attached molecules for enzyme reaction, and might
be useful for separation by the FRPS method. Selective installation
of a fluorous protecting group to the primary hydroxyl function of
4 was successfully carried out with 5 and diisopropylethylamine in
DMF to give 6 in 81% yield. Finally, protective acetyl groups and
the glycosidic benzyl group of 6 were removed to afford substrate 7
in 94% yield in two steps. Compound 7 was obtained in 52% yield
in nine steps from D-ManNAc (Scheme 2).
Sialic acids displayed on the surface of mammalian cells are
involved in many cell-surface interactions including cell–cell
recognition processes, cell adhesion, and viral receptor recogni-
tion.1 Current interests in sialic acid and related molecules stem
from their growing importance in biological systems that
eventually trigger a variety of biological responses.2 Among the
diverse array of compounds related to sialic acid family, 2,3-
unsaturated sialic acid derivatives based on 5-acetamido-2,6-
anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (1)3
are promising candidates for designing chemotherapeutics agents
against influenza virus.4
The search for rapid and efficient protocols for the purification
of organic compounds is a major concern of modern chemistry.
Fluorous techniques for organic synthesis are very attractive for
strategic separation of reaction mixtures. Early fluorous synthesis
technologies relied on heavy fluorous tags and liquid–liquid
extraction to separate tagged-fluorous molecules from untagged
organics.5 In the recently introduced light fluorous synthesis, fewer
fluorines are used in the tag and the fluorous solid-phase extraction
(FSPE) over fluorous reverse-phase silica gel (FRPS) is proving far
superior to binary liquid–liquid extractions for compounds with
fewer fluorine atoms.6 The fluorous tagging method has the
advantage of allowing the use of silica gel TLC to monitor the
reaction process and allowing us ready purification by a single
chromatographic method, greatly reducing time for isolation.
To our knowledge, there is no application of fluorous tagging
method to chemoenzymatic synthesis of biologically active
carbohydrates. As part of a program aimed at the new sialidase
inhibitors, we described the Neu5Ac aldolase-catalyzed chemoen-
zymatic synthesis of sialic acid derivatives and their inhibitory
activities against enzyme.7 Herein we wish to report the first
chemoenzymatic synthesis of sialic acid derivatives from D-
ManNAc derivative bearing a fluorous protecting group at the
C-6 position of D-ManNAc and its application to synthesis of 1.
The crucial incubation of Neu5Ac aldolase9 with 7 and sodium
pyruvate in potassium phosphate buffer (KpB) (pH 7.5) in the
presence of DTT and MgCl2 at 37 uC for 7 days afforded 8 in 62%
yield (Table 1, entry 1), which was desalted by Bio-Gel P-2 gel
filtration chromatography using water and purified by FRPS
chromatography eluting first with 80% MeOH–H2O and then with
MeOH, without tedious and time consuming ion-exchange
chromatography. It should be noted that the addition of HFE-
720010 for improving the solubility of 7 in KpB resulted in
increasing yield of 8 (71%) (Table 1, entry 2).11
As depicted in Scheme 3, after esterification of 8 with
TMSCHN2, further transformation of 9 into 1012 was accom-
plished with the treatment of AcCl, followed by elimination of HCl
with pyridine to give crude 2,3-deoxy sialic acid 10, which was
Department of Organic Chemistry, School of Pharmaceutical Sciences,
University of Shizuoka, 52-1 Yada Suruga-Ku, Shizuoka, 422-8526,
Japan. E-mail: ikeda@ys2.u-shizuoka-ken.ac.jp; Fax: +81-54-264-5108;
Tel: +81-54-264-5108
Scheme 1 Rf = fluorous protecting group.
This journal is ß The Royal Society of Chemistry 2006
Chem. Commun., 2006, 3093–3094 | 3093

View Article Online
its application to the synthesis of sialidase inhibitor 1. Further
application to the synthesis of the bioactive carbohydrates is now
in progress.
The authors thank MARUKIN BIO, INC. (Kyoto, Japan) for
the generous gift of D-ManNAc. This work was performed
through the Noguchi Fluorous Project.
Notes and references
1 Biology of Sialic Acids, ed. A. Rosenberg, Plenum Press, New York,
London, 1995.
2 M. J. Kiefel and M. von Itzstein, Chem. Rev., 2002, 102, 471.
3 C. T. Holzer, M. von Itzstein, B. Jin, M. S. Pegg, W. P. Stewart and
W.-Y. Wu, Glycoconjugate J., 1993, 10, 40.
Scheme 2 Reagents and conditions: (a) (i) Ac2O, pyridine, CH2Cl2, rt,
24 h, (ii) BnOH, BF3?OEt2, CH3NO2, 80 uC, 7 h, (iii) NaOMe, MeOH,
0 uC, 3 h, 68% from D-ManNAc; (b) (i) 2,2-dimethoxypropane, PTSA,
DMF, rt, 1 h, (ii) Ac2O, pyridine, rt, 24 h, 84% from 2; (c) 80% AcOH,
80 C, quant.; (d) RfCl 5, iPr2NEt, DMF, 45 uC, 54 h, 81%; (e) (i) NaOMe,
MeOH, 0 uC, 2 h, (ii) 20% Pd(OH)2/C, H2, MeOH, rt, 24 h, 94% from 6.
4 (a) K. Ikeda, M. Sato and Y. Torisawa, Curr. Med. Chem., 2004, 3, 339;
(b) J. C. Wilson, R. J. Thomson, J. C. Dyason, P. Florio, K. J. Quelch,
S. Abo and M. von Itzstein, Tetrahedron: Asymmetry, 2000, 11, 53.
5 (a) I. T. Horvath and J. Rabai, Science, 1994, 266, 72; (b) D. P. Cuuran,
Angew. Chem., Int. Ed., 1998, 37, 1174; (c) K. Goto, T. Miura,
M. Mizuno, H. Takaki, N. Imai, Y. Murakami and T. Inazu, Synlett,
2004, 2221; (d) D. P. Curran, Pure Appl. Chem., 2000, 72, 1649.
6 (a) D. P. Curran and Z. Y. Luo, J. Am. Chem. Soc., 1999, 121, 9069; (b)
D. P. Curran, Synlett, 2001, 1488; (c) Z. Y. Luo, Q. S. Zhang,
Y. Oderaotoshi and D. P. Curran, Science, 2001, 291, 1766.
Table 1 Neu5Ac aldolase-catalyzed reaction using fluorous protect-
ing group
7 (a) M. Murakami, K. Ikeda and K. Achiwa, Carbohydr. Res., 1996, 280,
101; (b) K. Ikeda, F. Kimura, K. Sano, Y. Suzuki and K. Achiwa,
Carbohydr. Res., 1998, 312, 183.
8 Experimental data for 5: Anhydrous hydrogen chloride was passed
through a mixture of 2-(perfluorohexyl)ethanol (10.9 g, 30 mmol) and
paraformaldehyde powder (0.99 g, 33 mmol) at 20–25 uC for 2 h. The
reaction mixture was transferred into a separating funnel and the upper
layer was diluted with pentane. The organic layer was dried over
anhydrous MgSO4 and concentrated to give 10.9 g (86%) of crude 5 as a
colorless oil. Distillation of this oil gave pure 5 (45 mmHg, bp 83–85 uC).
1H NMR (CDCl3, 500 MHz): d 2.43–2.49 (m, 2H, ClCH2OCH2CH2),
3.96–4.00 (m, 2H, ClCH2OCH2CH2), 5.50 (m, 2H, ClCH2OCH2CH2).
13C NMR (CDCl3, 125 MHz): d 29.1 (t, JCF = 22.2 Hz), 60.4, 80.4,
106.7–116.5 (m, C6F13). 19F NMR (CDCl3, 470 MHz): d 2126.7,
2124.2, 2123.4, 2122.4, 2114.0, 281.4. Negative FABMS (TEA) m/z:
377 (M-Cl)+.
Entry
Condition
Yield (%)
1
2
Neu5Ac aldolase KpB (pH 7.5) MgCl2,
DTT, 37 uC, 7 d
Neu5Ac aldolase HFE-7200-KpB (pH 7.5)
62
71
(1 : 5) MgCl2, DTT, 37 uC, 7 d
9 J. L.-C. Lin, G. -J. Shen, Y. Ichikawa, J. F. Rutant, G. Zapata,
W. F. Vann and C. -H. Wong, J. Am. Chem. Soc., 1992, 114, 3901.
10 EtOC4F9 is a commercially available fluorocarbon solvent (3 M,
Tokyo), which is called Novec2 HFE-7200.
11 Experimental data of Neu5Ac aldolase catalyzed reaction: Neu5Ac
aldolase [10 unit] was added to a solution of 7 (60 mg, 0.10 mmol) and
sodium pyruvate (110 mg, 1.0 mmol) in 0.05 M KpB (pH 7.5) (2 ml)
and HFE-7200 (0.4 ml) in the presence of DTT (1.4 mg) and 0.1 M
MgCl2 (0.10 ml) at 37 uC. After 2 days, an additional amount of
aldolase (10 unit) and sodium pyruvate (110 mg, 1.20 mmol) was added.
The mixture was incubated for a further 5 days at 37 uC. The course of
the reaction was monitored by TLC. The whole solution was passed
through a column of Bio-Gel P-2 gel filtration chromatography using
water as eluant. Fractions containing the product were purified on a
column of FRPS chromatogaraphy eluted first with 80% MeOH2H2O
and then with MeOH to give 8 (50 mg, 71%), as amorphous after freeze
drying. Positive FABMS (NBA) m/z: 708 (M + H)+, 730 (M + Na)+.
12 Selected data for 10: 1H NMR (CDCl3, 500 MHz): d 1.90 (s, 3H,
NHAc), 2.04, 2.06, 2.09 (s, each 3H, OAc), 2.3422.48 (m, 2H,
OCH2OCH2CH2), 3.63 (dd, 1H, J9a,9b = 11.5, J9a,8 = 6.9 Hz H-9a), 3.77
Scheme 3 Reagents and conditions: (a) TMSCHN2, CH2Cl2, rt, 24 h,
quant. from 8; (b) (i) AcCl, rt, 24 h; (ii) pyridine, rt, 1 h, 87% from 9; (c) (i)
NaOMe, MeOH, 0 uC, 2 h; (ii) TMSBr, CH2Cl2, 0 uC, 12 h, 67% from 10.
(s, 3H, OMe), 3.80–3.84 (m, 2H, OCH2OCH2CH2), 4.07 (dd, 1H, J9b,8
=
3.5 Hz H-9b), 4.68, 4.69 (d, each 1H, Jgem = 16.6 Hz, OCH2OCH2CH2),
5.98 (d, 1H, J3,4 = 3.5 Hz, H-3). 13C NMR (CDCl3, 125 MHz): d 20.7,
20.8, 20.9, 23.2, 31.4 (t, JCF = 21.6 Hz), 46.5, 52.6, 60.0, 65.8, 67.8, 68.1,
71.9, 95.7, 107.9, 116.1–118.4 (m, C6F13), 145.0, 161.7, 170.1, 170.3,
170.5, 170.8. 19F NMR (CDCl3, 470 MHz): d 2126.7, 2124.2, 2123.4,
2122.4, 2114.1, 281.3. Positive FABMS (NBA) m/z: 830 (M + Na)+.
13 S. Hanessian, D. Delorme and Y. Dufresne, Tetrahedron Lett., 1984, 25,
2515.
separated over FRPS chromatography. Finally, after the stepwise
removal of acetyl groups and methyl ester group, deprotection of
the fluorous acetal group with trimethylsilyl bromide (TMSBr)13
under mildly acidic conditions gave 1.14
In conclusion, we have demonstrated the first chemoenzymatic
synthesis of sialic acid derivatives by using the FSPE technique and
14 The spectral data of 1 were consistent with an authentic sample.
3094 | Chem. Commun., 2006, 3093–3094
This journal is ß The Royal Society of Chemistry 2006