Determination of the absolute configuration of sialic acids in gangliosides from the sea cucumber Cucumaria echinata

Article abstract of DOI:10.1248/cpb.55.1051

Enantiomeric pairs of sialic acid, D- and L-NeuAc (N-acetylneuraminic acid), were converted to D- and L-arabinose, respectively, by chemical degradation. Using this method, the absolute configuration of the sialic acid residues, NeuAc and NeuGc (N-glycolylneuraminic acid), in the gangliosides from the sea cucumber Cucumaria echinata was determined to be the D-form. Although naturally occurring sialic acids have been believed to be the D-form on the basis of biosynthetic evidence, this is the first report of the determination of the absolute configuration of the sialic acid residues in gangliosides using chemical methods.

Full text of DOI:10.1248/cpb.55.1051

July 2007
Chem. Pharm. Bull. 55(7) 1051—1052 (2007)
1051
Determination of the Absolute Configuration of Sialic Acids in
Gangliosides from the Sea Cucumber Cucumaria echinata
*
Fumiaki KISA, Koji YAMADA, Tomofumi MIYAMOTO, Masanori INAGAKI, and Ryuichi HIGUCHI
Faculty of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan.
Received March 15, 2007; accepted April 6, 2007
Enantiomeric pairs of sialic acid, D- and L-NeuAc (N-acetylneuraminic acid), were converted to D- and L-
arabinose, respectively, by chemical degradation. Using this method, the absolute configuration of the sialic acid
residues, NeuAc and NeuGc (N-glycolylneuraminic acid), in the gangliosides from the sea cucumber Cucumaria
echinata was determined to be the D-form. Although naturally occurring sialic acids have been believed to be the
D-form on the basis of biosynthetic evidence, this is the first report of the determination of the absolute configu-
ration of the sialic acid residues in gangliosides using chemical methods.
Key words sialic acid; absolute configuration; ganglioside; sea cucumber; Cucumaria echinata
Gangliosides, an important group of glycosphingolipids 2-O-methylneuraminate (3). Protection of the diol in the side
due to their biological functions,1) are characterized by the chain of 3 with acetone afforded an isopropyridene derivative
presence of one or more moles of sialic acid, N-acetylneu- (4). Oxidation of 4 with NaIO4 gave a dial derivative (5). Fi-
raminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), nally, compound 5 was hydrolyzed to separate arabinose (6).
in their carbohydrate parts. In general, naturally occurring The absolute configuration of 6 was verified as being the D-
sialic acids have been believed to be the D-form on the basis form using the Hara method. On the other hand, L-NeuAc
of biosynthetic evidence, i.e., NeuAc is synthesized from (7), which was synthesized from L-glucose using the authen-
pyruvic acid and N-acetyl-D-mannosamine enzymatically.2) tic method,6) gave L-arabinose in the same manner as 1. Thus
However, there has been no report on the determination of the fact that D(L)-sialic acid could be converted to D(L)-arabi-
the absolute configuration of the sialic acid residues in gan- nose was confirmed, as shown in Fig. 1.
gliosides. We attempted to determine the absolute configura-
tion (D or L) of the sialic acid residues in the gangliosides3,4) CG-1, CEG-3, CEG-4, CEG-5, CEG-6, HLG-3, CEG-8, and
from the sea cucumber Cucumaria echinata.
CEG-93,4) from the sea cucumber C. echinata. The nine gan-
Next, this method was applied to the gangliosides SJG-1,
For the determination of the absolute configuration of al- gliosides were each methanolyzed and the sialic acid deriva-
doses in gangliosides, we have used the Hara method.5) How- tives (3) produced were converted to ketals (4). Periodic acid
ever, this method cannot be applied to sialic acids directly. oxidation of 4 followed by acidic hydrolysis of the oxidation
Therefore we prepared aldose from sialic acid, preserving the products 5 gave arabinoses (6). Compound 6, obtained from
absolute configuration of the parent sialic acid, and then ap- each ganglioside, was determined to be the D-form using the
plied the Hara method to the aldose. As shown in Fig. 1, Hara method, as shown in Fig. 2. Accordingly, the sialic acid
D(L)-arabinose should be obtained from D(L)-sialic acid in the residues (NeuAc and NeuGc) in the gangliosides from C.
process. First, this assumption was proved.
echinata must be the D-form.
D-NeuAc (1) was acetylated to give pentaacetate (2),
To the best of our knowledge, this is the first report of the
which was methanolyzed with HCl–MeOH to yield methyl determination of the absolute configuration of the sialic acid
Fig. 1. Preparation of D(L)-Arabinose from D(L)-NeuAc
Reagents and conditions: (a) Ac2O, pyridine; (b) 10% HCl–MeOH, 80 °C, 18 h; (c) DMF, acetone, 10% HCl–MeOH; (d) NaIO4, H2O; (e) 90% HCOOH–10% CF3COOH (1/1),
100 °C, 12 h.
∗ To whom correspondence should be addressed. e-mail: rhiguchi@phar.kyushu-u.ac.jp
© 2007 Pharmaceutical Society of Japan

1052
Vol. 55, No. 7
was concentrated in vacuo. The residue was chromatographed on silica
gel (solvent CHCl3–MeOH, 8 : 2) to give 4 (10.5 mg, 90%). [a]D ꢁ17.1°
1
(cꢂ1.0, H2O). Positive-ion FAB-MS m/z: 336 [MꢃH]ꢃ. H-NMR (D2O) d:
4.44 (1H, m, 4-H), 4.34 (1H, m, 7-H), 4.14 (1H, m, 8-H), 4.12 (1H, m, 9-H),
3.90 (3H, s, COOCH3), 3.77 (1H, m, 9-H), 3.56 (1H, m, 6-H), 3.30 (3H, s,
OCH3), 2.80 (1H, m, 5-H), 2.47 (1H, dd, Jꢂ12.9, 11.5 Hz, 3-H), 1.85 (1H,
dd, Jꢂ12.9, 5.1 Hz, 3-H), 1.45, 1.40 [each 3H, s, C(CH3)2].
Preparation of D-Arabinose (6) from 4 To the solution of 4 (10.5 mg)
in H2O (0.5 ml), NaIO4 (1.0 mg) was added and stirred at room temperature
for 1 h. The reaction mixture was diluted with H2O (5 ml) and a small
amount of ethylene glycol, and extracted with n-BuOH (3 mlꢀ3). The
organic layer was washed with H2O and concentrated in vacuo to give 5
as a crude product. Compound 5 was heated with 90% HCOOH–10%
CF3COOH (1 : 1) (0.5 ml) at 100 °C for 12 h, and the reaction mixture was
concentrated in vacuo. The residue was purified on silica gel TLC (solvent
CHCl3–MeOH–H2O, 7 : 3 : 0.5) to give 6 (2.3 mg, 50% from 4).
Preparation of L-Arabinose (8) from L-NeuAc (7) In the same manner
as for 1, compound 7 (8.4 mg) afforded 8 (1.8 mg, 44.1%).
Determination of the Absolute Configuration of 6 and 8 (Hara
Method) Compounds 6 (1 mg) and 8 (1 mg) were each heated with L-cys-
teine methyl ester hydrochloride (1.5 mg) and pyridine (0.2 ml) at 70 °C for
1 h. Then, 0.1 ml of 1-(trimethylsilyl) imidazole was added and the mixture
was heated at 70 °C for a further 15 min. The reaction mixture was diluted
with 50% MeOH (0.8 ml), extracted with n-hexane (0.5 mlꢀ3), and the n-
hexane layer was concentrated in vacuo to yield trimethylsilyl ether of the
methyl (4R)-thiazolidine-4-carboxylate derivative. Each derivative was ana-
lyzed using gas chromatography [column temperature: 180—300 °C (rate of
temperature increase 2.5 °C/min)]; tR [min]ꢂ23.5 (derivative of 6), 22.1 (de-
rivative of 8) (derivative of D-arabinose, 23.5, L-arabinose, 22.1).
Determination of the Absolute Configuration of Sialic Acid Residues
of Gangliosides from C. echinata Each ganglioside (2 mg) shown in Fig.
2 was heated with 10% HCl in MeOH (0.5 ml) at 80 °C for 18 h and concen-
trated in vacuo. The residue was dissolved in DMF (0.3 ml), and acetone
(0.3 ml) and 10% HCl in MeOH (10 ml) were added. The mixture was heated
at 60 °C for 4 h, neutralized with Ag2CO3, centrifuged, and the clear super-
natant solution was concentrated in vacuo. To the solution of the product in
H2O (0.5 ml), NaIO4 (1.0 mg) was added and stirred at room temperature for
1 h. The reaction mixture was diluted with H2O (5 ml) and a small amount of
ethylene glycol, and extracted with n-BuOH (3 mlꢀ3). The organic layer
was concentrated in vacuo, the reaction product was heated with 90%
HCOOH–10% CF3COOH (1 : 1) (0.5 ml) at 100 °C for 12 h, and the reaction
mixture was concentrated in vacuo. The arabinose from sialic acid, con-
tained in the reaction product, was induced to the thiazolidine derivative, and
the derivative was analyzed using gas chromatography in the same manner
as in 6; tR [min] (original ganglioside)ꢂ23.5 (SJG-1), 23.5 (CG-1), 23.5
(CEG-3), 23.4 (CEG-4), 23.4 (CEG-5), 23.5 (CEG-6), 23.5 (HLG-3), 23.5
(CEG-8), 23.4 (CEG-9) (derivative of D-arabinose, 23.5, L-arabinose, 22.1).
Fig. 2. Determination of the Absolute Configuration of Sialic Acid
Residues of Gangliosides from Cucumaria echinata by Conversion to Arabi-
nose
SJG-1: NeuGca2→6Glcb1→1 ceramide
CG-1: 8-O-sulfo-NeuGca2→6Glcb1→1 ceramide
CEG-3: 4-O-acetyl-Fuca1→11NeuGca2→6Glcb1→1 ceramide
CEG-4: Fuca1→11NeuGca2→6Glcb1→1 ceramide
CEG-5: Fuca1→11NeuGca2→6Glcb1→1 ceramide
CEG-6: Fuca1→11NeuGca2→4NeuAca2→6Glcb1→1 ceramide
HLG-3: Fuca1→11NeuGca2→4NeuAca2→6Glcb1→1 ceramide
CEG-8: NeuGc2→11NeuGc2→4NeuAc2→6Glc1→1 ceramide
CEG-9: NeuGc2→11NeuGc2→4NeuAc2→6Glc1→1 ceramide
Bold: sialic acid
residues in gangliosides using chemical methods and thus is
noteworthy.
Experimental
Optical rotations were measured with a Jasco Dip-370 digital polarimeter
1
at 23 °C. H-NMR spectra were recorded on a Jeol GX-270 spectrometer
(270 MHz) or a Varian Unity-500 spectrometer (500 MHz). Positive- and
negative-ion FAB-MS spectra were acquired with a Jeol JMS-SX-102 mass
spectrometer [xenon atom beam; matrix, m-nitrobenzylalcohol (positive-ion
mode) and triethanolamine (negative-ion mode)]. Gas chromatographs were
recorded with a Shimadzu QP-5050A [EI mode; ionizing potential, 120 eV;
column, Neutra Bond-5 (0.25 mmꢀ30 m, GL Science Inc.); carrier gas, He].
N-Acetyl-2,4,7,8,9-penta-O-acetyl-D-neuraminic Acid (2) D-NeuAc
(1) (20 mg) was heated with Ac2O (0.5 ml) and pyridine (0.5 ml) at 60 °C for
2 h. The reaction mixture was concentrated in vacuo to give 2 (33.4 mg).
Acknowledgments We thank Mr. Y. Tanaka and Ms. Y. Soeda of the
Faculty of Pharmaceutical Sciences, Kyushu University, for the NMR meas-
urements. This work was supported in part by a Grant-in-Aid for Scientific
Research (No. 13024260, Priority Area A) from the Ministry of Education,
Culture, Science, Sports and Technology, Japan, and a grant (No. 16510163,
18510187) from the Japan Society for the Promotion of Science, which are
gratefully acknowledged.
1
[a]D ꢁ10.4° (cꢂ1.0, H2O). Negative-ion FAB-MS m/z: 518 [MꢁH]ꢁ. H-
NMR (D2O) d: 5.43 (1H, dd, Jꢂ7.7, 1.8 Hz, 7-H), 5.29 (1H, ddd, Jꢂ11.5,
10.3, 5.1 Hz, 4-H), 5.17 (1H, ddd, Jꢂ7.7, 4.1, 3.0 Hz, 8-H), 4.45 (2H, dd,
Jꢂ12.8, 3.0 Hz, 9-H2), 4.10 (1H, dd, Jꢂ10.6, 1.8 Hz, 6-H), 3.94 (1H, t,
Jꢂ10.4 Hz, 5-H), 2.45 (1H, dd, Jꢂ13.6, 11.5 Hz, 3-H), 1.87 (1H, dd,
Jꢂ13.6, 5.1 Hz, 3-H), 1.99, 2.02, 2.03, 2.03, 2.12, 2.14 (each 3H, s,
COCH3ꢀ6).
References and Notes
1) Hakomori S., Igarashi Y., J. Biochem. (Tokyo), 118, 1091—1103
(1995).
2) Comb D. G., Roseman S., J. Biol. Chem., 235, 2529—2537 (1960).
3) Kisa F., Yamada K., Miyamoto T., Inagaki M., Higuchi R., Chem.
Pharm. Bull., 54, 982—987 (2006).
4) Kisa F., Yamada K., Miyamoto T., Inagaki M., Higuchi R., Chem.
Pharm. Bull., 54, 1293—1298 (2006).
5) The Hara method is a well-known method using determination of the
absolute configuration of aldoses in which trimethylsilyl ethers of the
methyl (4R)-thiazolidine-4-carboxylate derivative of aldose enan-
tiomers can be separated by GLC; Hara S., Okabe H., Mihashi K.,
Chem. Pharm. Bull., 35, 501—506 (1987).
Methyl 2-O-Methyl-D-neuraminate (3) Compound 2 (33.4 mg) was
heated with 10% HCl in MeOH (0.5 ml) at 80 °C for 18 h. The reaction mix-
ture was concentrated in vacuo and the residue was chromatographed on sil-
ica gel (solvent CHCl3–MeOH–H2O, 6 : 4 : 1) to give 3 (10.3 mg, 54% from
1). [a]D ꢁ23.2° (cꢂ1.0, H2O). Positive-ion FAB-MS m/z: 296 [MꢃH]ꢃ. 1H-
NMR (D2O) d: 4.42 (1H, m, 4-H), 4.19 (1H, m, 7-H), 4.05 (1H, m, 8-H),
3.95 (2H, m, 9-H2), 3.92 (3H, s, COOCH3), 3.53 (1H, m, 6-H), 3.30 (3H, s,
OCH3), 2.84 (1H, m, 5-H), 2.68 (1H, dd, Jꢂ13.2, 12.0 Hz, 3-H), 1.78 (1H,
dd, Jꢂ13.2, 4.5 Hz, 3-H).
Methyl 8,9-O-Isopropylidene-2-O-methyl-D-neuraminate (4) Com-
pound 3 (10.3 mg) was dissolved in DMF (0.5 ml), and acetone (0.5 ml) and
10% HCl in MeOH (20 ml) were added. The mixture was heated at 60 °C for
4 h, neutralized with Ag2CO3, centrifuged, and the clear supernatant solution
6) Kozlov I. A., Mao S., Xu Y., Huang X., Lee L., Sears P. S., Gao C.,
Coyle A. R., Janda K. D., Wong C.-H., ChemBioChem, 2, 741—746
(2001).