Synthesis of γ-lactone-type sialic acid, an isomer of 2,3-dehydrosialic acid

Article abstract of DOI:10.1271/bbb.90912

An efficient synthesis of γ-lactone-type sialic acid, which is an isomer of 2,3-dehydrosialic acid, was achieved from the corresponding sialic ester. The sialic ester was reconstructed from the γ-lactone in a 95% yield. The γ-lactone structure was determined by methylating to its corresponding methyl ether.

Full text of DOI:10.1271/bbb.90912

Biosci. Biotechnol. Biochem., 74 (5), 1106–1107, 2010
Note
Synthesis of ꢀ-Lactone-Type Sialic Acid, an Isomer of 2,3-Dehydrosialic Acid
y
Yoshiharu SAWADA and Shin-ichi NAKATSUKA
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Received December 9, 2009; Accepted January 12, 2010; Online Publication, May 7, 2010
[doi:10.1271/bbb.90912]
An efficient synthesis of ꢀ-lactone-type sialic acid,
which is an isomer of 2,3-dehydrosialic acid, was
achieved from the corresponding sialic ester. The sialic
ester was reconstructed from the ꢀ-lactone in a 95%
yield. The ꢀ-lactone structure was determined by
methylating to its corresponding methyl ether.
Two mechanisms can be proposed for the synthesis of
ꢀ-lactone 3 from 2 via bicyclo-intermediate I or ring-
opened intermediate II in Scheme 1. The instability of
ꢀ-lactone 3 suggested that the original pyranose-type
backbone found in sialic acid may be reconstructed from
linear conjugated ꢀ-lactone 3.
We attempted to recover sialic acid by treating
ꢀ-lactone 3 with an aqueous alkali solution like NaOH,
KOH or NaHCO3, but the yield was low. The ring-
construction reaction was performed in various meth-
anolic alkali solutions containing NaOH, NaHCO3 or
NEt3, achieving a spot identical with that of methyl ester
2. The product was purified by silica gel column
chromatography to produce pure sialic ester 2 in a
Key words: ꢀ-lactone-type sialic acid; 2,3-dehydrosialic
acid; isomerization
Sialic acid (N-acetylneuraminic acid, 1) is widely
distributed in animal tissues as glycoproteins and
gangliosides and plays an important role in human
influenza infections. The structure of sialic acid is a
unique C9-carbohydrate that can be biologically synthe-
sized from N-acetylmannosamine and pyruvic acid, and
that exists as a mixture of two anomers.1) However,
sialic acid (1) exists primarily as the ꢁ-form in a
solution. 2,3-Dehydrosialic acid2,3) is one of the sialic
acid derivatives and has interesting bioactivities. The
reactivity of sialic acid (1) was evaluated in this study,
since 1 and its derivatives are medicinally interesting
compounds.
We first examined the reactivity of sialic acid (1) and
its sialic ester (2). Sialic ester (2) was easily prepared
from sialic acid in methanol containing either HCl4) or
an acidic resin.5) Compound 1 or 2 was treated with
many reagents under various reaction conditions, and a
new clean spot was found on a silica gel TLC plate upon
treating 2 with 0.5 M sodium methoxide in methanol for
30 min. However, the major product decomposed during
silica gel p-TLC or column chromatography separation
and could not be isolated from the reaction mixture.
Changing to crystallization allowed us to isolate the
unstable crystalline product upon neutralization with
CK08P acidic resin. The UV absorption of this crystal
was observed at 229 nm (ꢂmax, " ¼ 6;800), while the
proton signal for the methyl ester disappeared and a
vinyl proton signal appeared at 6.53 ppm (doublet,
J ¼ 2:1 Hz) in the 1H-NMR spectrum, suggesting the
presence of a conjugated lactone.
1
95% yield, its structure being confirmed by H-NMR.
Methyl ether 4a was isomerised into an equilibrium
mixture (4a/4b ¼ 3/1) in 1% triethylamine-methanol at
110 ꢀC for 4 h in a sealed tube (Scheme 1). Treating
lactone 4a with n-butylamine enabled a ring-opening
reaction to proceed, affording linear conjugated amide 5
in an 81% yield (Scheme 1). 2,3-Dehydrosialic acid
analogues 3, 4a, 4b, and 5 may be more activated
structures than the original sialic acid itself, because
their conjugated ꢀ-lactone or amide function is a potent
Michael accepter. Further chemical studies on the
application of these dehydrosialic acid derivatives are
currently in progress.
Experimental
All reactions were carried out in an N2 atmosphere. Silica gel
column chromatography was performed with silica gel M-300H
(Nagara Science Co.), and optical rotation values were recorded by a
JASCO P-2300 digital polarimeter. 1H- and 13C-NMR spectra were
measured by a JEOL ECA 600 spectrometer, with tetramethylsilane
used as an internal standard in CD3OD or t-BuOH in D2O. Mass
spectra were recorded with a JMS-700 (ESI) spectrometer, and UV
spectra were recorded with a Hitachi 4000 U spectrophotometer.
Melting point (mp) data were measured with Yanaco MP-J3 micro-
melting point apparatus.
ꢀ-Lactone-type sialic acid (3). Five ml of 1 M CH3ONa in CH3OH
was added to a solution of 2 (1.0 g, 3.1 mmol) in anhydrous CH3OH
(5.0 ml), and the mixture was stirred at 25 ꢀC for 30 min. The base was
trapped with an CK08P acidic resin (500 mg) column and washed 3
times with anhydrous CH3OH (30 ml). After evaporating the solvent,
the crude product was recrystalized from CH3OH/CH2Cl2 (1/4) to
give 0.56 g (62%) of 3 as colorless prisms. 1H-NMR (600 MHz, D2O)
ꢃ: 6.53 (1H, d, J ¼ 2:1 Hz, H-3), 5.81 (1H, t, J ¼ 2:1 Hz, H-4), 4.60
(1H, dd, J ¼ 10:3, 2.1 Hz, H-5), 4.11 (1H, dd, J ¼ 12:4, 2.8 Hz, H-9),
4.03 (1H, m, H-8), 3.89 (1H, dd, J ¼ 11:7, 6.2 Hz, H-9), 3.77 (1H, d,
J ¼ 8:3 Hz, H-7), 2.20 (3H, s, N-Ac); 13C-NMR (150 MHz, D2O)
ꢃ: 170.98 (C-1), 170.34 (COCH3), 143.63 (C-2), 118.75 (C-3), 78.31
(C-4), 72.27 (C-8), 70.69 (C-7), 68.92 (C-6), 64.77 (C-9), 51.76 (C-5),
23.43 (COCH3). HR-ESI-MS (positive): calcd. for C11H17NNaO8
Ogura et al.6,7) reported in 1987 the synthesis of
compound 4a. We therefore prepared the methyl ether
by methylating that crystalline compound to determine
the structure. Treating the crystalline compound with
diazomethane in methanol afforded corresponding
1
methyl ether 4a in a 96% yield. The H-NMR spectrum
of purified methyl ether 4a was identical to the data
reported by Ogura et al. The result proved to be
ꢀ-lactone 3, and the yield from 2 was 62%.
y
To whom correspondence should be addressed. Tel/Fax: +81-58-293-2919; E-mail: nakatsu@gifu-u.ac.jp

Synthesis of ꢀ-Lactone-Type Sialic Acid
1107
AcNH
O
OH
OH
OH
HO
H
OH
OH
OH
OH
AcNH
H3CO
HO
1) CH3ONa / CH3OH
2) resin
HO
OH
OR
O
or
O
O
OH
AcNH
O
O
HO
OH
O
I
II
1 R= H: Sialic acid
2 R=CH3: Sialic ester
1) NEt3 / CH3OH
2) resin
95%
62%
NHAc
H
NHAc
H
OH
OH
O
O
CH2N2
O
O
OCH3
HO
HO
OH OH
OH OH
96%
OH
3
4a
Et3N /CH3OH
110°C (sealed tube), 4h
4a / 4b = 3 / 1
n-BuNH2
81%
NHAc
HO
NHAc
HO
H
OCH3
O
O
HO
HO
OH
O
OH OH
OH OH
N
H
OCH3
5
4b
Scheme 1. Synthesis of ꢀ-Lactone-Type Sialic Acids (3 and 4ab) and Derivative 5.
(M þ Na), 314.0852; found, m=z 314.0859. Mp 192 ꢀC (decomp.); UV
Open-chain conjugated amide of sialic acid (5). n-Butylamine
(18 ml) was added to a solution of 4a (50 mg, 0.16 mmol) in CH3OH
(0.5 ml), and the mixture stirred for 2 h at 25 ꢀC while being monitored
by HPLC. After evaporating the solvent, the product was purified by
HPLC (35% CH3OH, 1% AcOH/H2O) to give amide 5 (50.3 mg, 81%)
as a colorless syrup. 1H-NMR (CD3OD) ꢃ: 5.40 (1H, dd, J ¼ 7:6,
2.1 Hz, H-4), 5.14 (1H, d, J ¼ 7:6 Hz, H-3), 4.02 (1H, dd, J ¼ 9:6,
2.1 Hz, H-5), 3.96 (1H, d, J ¼ 10:3 Hz, H-6), 3.80 (1H, dd, J ¼ 11:2,
3.4 Hz, H-9a), 3.72 (1H, m, H-8), 3.62 (1H, dd, J ¼ 11:2, 6.2 Hz,
H-9b), 3.59 (3H, s, OCH3), 3.43 (1H, d, J ¼ 8:9 Hz, H-7), 3.24 (1H, dt,
J ¼ 6:9, 2.1 Hz, N-CH2-C3H7), 2.02 (3H, s, H-Ac), 1.52 (2H, m,
N-CH2-CH2-C2H5), 1.36 (2H, m, N-C2H4-CH2-CH3), 0.94 (3H, t,
20
(CH3OH) ꢂmax 229 nm (" 6,800); ½ꢄꢁD ꢂ137ꢀ (c 1.0, CH3OH).
N-Acetylneuraminic acid methyl ester (2). Triethylamine (25 ml)
was added dropwise to 3 (100 mg, 0.34 mmol) in CH3OH (5 ml), and
the mixture stirred for 30 min at 25 ꢀC. After removing the solvent, the
residue was purified by silica gel column chromatography with 5%
CH3OH/CH2Cl2 to afford pure sialic ester 2 (105 mg, 95%), whose
structure was confirmed by 1H-NMR. The respective yields of 2 using
NaOH or NaHCO3 were 92% and 93%.
2-O-Methyl ether (4a). Five ml of an ethereal solution of diazo-
methane was added to 3 (50 mg, 0.17 mmol) in CH3OH (5 ml). After
stirring the mixture for 30 min at 25 ꢀC, 1 drop of acetic acid was added
to terminate the reaction. After evaporation, the residue was purified by
silica gel open column chromatography with 5% CH3OH in CH2Cl2 to
give 2-O-methyl derivative 4a (50.3 mg, 96%) as a colorless syrup.
The 1H-NMR spectrum of 4a was identical with that of 4a reported by
Ogura et al.6)
J ¼ 7:6 Hz, N-C3H6-CH3). HR-ESI-MS (positive): calcd. for
20
C
16H30N2NaO8 (M þ Na), 401.1900; found, m=z 401.1896. ½ꢄꢁD
ꢂ30ꢀ (c 1.0, CH3OH).
Acknowledgments
Isomerization of 2-O-methyl ether (4a). Triethylamine (10 ml) was
added to a solution of 4a (50 mg, 0.16 mmol) in CH3OH (1 ml), and the
mixture was heated at 110 ꢀC in a sealed tube. The reaction was
monitored by 1H-NMR for 4 h until the mixture had been converted to
a 3/1 equilibrium mixture (4a/4b). Neither 4a nor 4b could be
isolated, so the mixture was acetylated overnight with Ac2O/pyridine
at 20 ꢀC. The peracetates were isolated to the natural R-isomer (4a
peracetate) and unnatural S-isomer (4b peracetate).
4a peracetate. 1H-NMR (600 MHz, CDCl3) ꢃ: 5.96 (1H, d,
J ¼ 2:1 Hz, H-3), 5.59 (1H, d, J ¼ 10:3 Hz, NH), 5.50 (1H, dd,
J ¼ 9:6, 2.1 Hz, H-6), 5.41 (1H, dd, J ¼ 8:9, 2.1 Hz, H-7), 5.08 (1H,
m, H-8), 4.91 (1H, t, J ¼ 2:1 Hz, H-4), 4.53 (1H, t, J ¼ 10:3 Hz, H-5),
4.24 (1H, dd, J ¼ 12:4, 2.8 Hz, H-9), 4.01 (1H, dd, 12.4, 5.5 Hz, H-9),
2.16, 2.11, 2.08, 2.06 [4 ꢃ (3H, s), 4 ꢃ -OAc], 1.88 (3H, s, N-Ac).
4b peracetate. 1H-NMR (600 MHz, CDCl3) ꢃ: 6.10 (1H, d,
J ¼ 2:1 Hz, H-3), 5.81 (1H, brd, J ¼ 9:6 Hz, NH), 5.41 (1H, dd,
J ¼ 8:3, 2.8 Hz, H-6), 5.39 (1H, dd, J ¼ 7:6, 2.8 Hz, H-7), 5.06 (1H,
m, H-8), 4.97 (1H, dd, J ¼ 2:1, 4.1 Hz, H-4), 4.55 (1H, dt, J ¼ 9:6,
4.1 Hz, H-5), 4.25 (1H, dd, J ¼ 12:4, 3.4 Hz, H-9), 4.02 (1H, dd,
J ¼ 12:4, 5.5 Hz, H-9), 2.14, 2.11, 2.06, 2.05 [4 ꢃ (3H, s), 4 ꢃ -OAc],
1.96 (3H, s, N-Ac).
We are indebted to Nagara Science Co. for supplying
sialic acid and for financial support.
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