Chemoenzymatic synthesis of the 9-deoxy-9-fluoro-[3-13c]-NeuAc-α- (2→6)-[U-13c]-Gal-β-sequence on an intact glycoprotein [17]

Full text of DOI:10.1021/ja982950e

J. Am. Chem. Soc. 1999, 121, 1411-1412
1411
Scheme 1a
Chemoenzymatic Synthesis of the 9-Deoxy-
9-fluoro-[3-13C]-NeuAc-r-(2f6)-[U-13C]-Gal-â-
Sequence on an Intact Glycoprotein
Tatsuo Miyazaki,† Tohru Sakakibara,† Hajime Sato,‡ and
Yasuhiro Kajihara*,†
Graduate School of Integrated Science, and Faculty of Science
Yokohama City UniVersity, 22-2, Seto, Kanazawa-ku
Yokohama 236-0027, Japan
Bruker Japan Co., Ltd.
21-5, Ninomiya, 3-chome, Tsukuba
Ibaraki 305-0051, Japan
ReceiVed August 17, 1998
The synthesis of sialyloligosaccharides and their analogues has
enabled researchers to study the conformation and the structure-
function relationship of these important cellular components.1
However, the synthesis of sialyloligosaccharide analogues on
intact glycoprotein backbones has proven far more difficult than
the chemical synthesis of gangliosides and their analogues.2 If
sialyloligosaccharide analogues synthesized on glycoprotein
backbones could be easily analyzed by NMR, we would be able
to study the dynamics and conformation of sialyloligosaccharide
analogues on proteins. Recent research using glycosyltransferases
has centered on the transfer of a sugar analogue to the nonreducing
terminal of the oligosaccharide of a glycoprotein.3 However, in
most cases, analysis by NMR of the resulting sugar analogue is
difficult due to overlap with the proton resonances of the amino
acids in the protein.4,5 We report here a concise 13C-labeling6
method of a NeuAc analogue, synthesis of the 9-deoxy-9-fluoro-
[3-13C]-NeuAc-R-(2f6)-[U-13C]-Gal-â- sequence on an intact
glycoprotein by sialyl (STase) and galactosyltransferase (GTase),
and its structure analysis by NMR.
a Reagents: (a) aldolase, lactate dehydrogenase, â-NADH, yields are
summarized in Table 1; (b) aldolase, [3-13C]-sodium pyruvate, yields are
summarized in Table 1; (c) (1) Dowex 50W-X8 (H+), MeOH, (2) HClO4,
Ac2O, y ) 82%; (d) (1) 1H-tetrazole, MeCN, (2) t-BuOOH, MeCN, (3)
DBU, THF, (4) NaOMe, MeOH:H2O ) 1:2, y ) 27%; (e) UDP-[U-
13C]-glucose, UDP-glucose-4-epimerase, bovine â-(1f4)-galactosyltrans-
ferase; (f) (1) reaction of bacterium R-(2f6)-sialyltransferase (three
times), 11, (2) Diplococcus pneumoniae â-galactosidase.
Table 1. Stable Isotope Labeling of NeuAc Analogues by Use of
NeuAc Aldolasea
ManNAc analogue
[3-13C]-Neu5Ac analogue
Since a large number of NeuAc analogues have been synthe-
aldolase aldolase +
sized from NeuAc,7 their synthetic route including selective
substrate R1 R2
(%)c LDHb (%)d
product
(%)e
† Yokohama City University.
1
2
3
4
OH OH 69%
90%
5
6
7
8
87%
91%
93%
90%
‡ Bruker Japan Co., Ltd.
F
OH 48%
94%
98%
45%
(1) (a) Sabesan, S.; Bock, K.; Paulson, J. C. Carbohydr. Res. 1991, 218,
27-54. (b) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.; Garcia-
Junceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.; Paulson,
J. C.; Wong, C.-H. J. Am. Chem. Soc. 1992, 114, 9283-9298. (c) Sabesan,
S.; Neira, S.; Davison, F.; Duus, J. O.; Bock, K. J. Am. Chem. Soc. 1994,
116, 1616-1634. (d) Salvatore, B. A.; Ghose, R.; Prestegard, J. H. J. Am.
Chem. Soc. 1996, 118, 4001-4008.
(2) (a) Hasegawa, A.; Kiso, M.; Ishida, H. In Methods in Enzymology; Lee,
Y. C.; Lee, R. T., Eds.; 1994; Vol. 242, pp 158-197. (b) Ogawa, T. Chem.
Soc. ReV. 1994, 23, 397-407.
N3 OH 60%
OH H
26%
a Conversion yields were determined by NMR spectra. b LDH:lactate
dehydrogenase. c Reaction used NeuAc analogue (32 mM) and aldolase
(3.8 U). d Reaction used NeuAc analogue (32 mM), aldolase (3.8 U),
and LDH (20 U). e Reaction used ManNAc analogue (63 mM) and
aldolase (10 U).
(3) (a) Higa, H. H.; Paulson, J. C. J. Biol. Chem. 1985, 260, 8838-8849.
(b) Gross, H. J.; Brossmer, R. Eur. J. Biochem. 1988, 177, 583-589. (c)
Kajihara, Y.; Endo, T.; Ogasawara, H.; Kodama, H.; Hashimoto, H. Carbohydr.
Res. 1995, 269, 273-294. (d) Witte, K.; Sears, P.; Martin, R.; Wong, C.-H.
J. Am. Chem. Soc. 1997, 119, 2114-2118.
protection of hydroxyl groups has become fairly common.
Therefore, if [3-13C]-NeuAc analogues can be obtained from
NeuAc analogues, the published route for the NeuAc analogues
will be practically utilized. As is shown in Scheme 1, we
performed the aldolase reaction twice: first, degradation8 of the
NeuAc analogue to a ManNAc analogue and, second, condensa-
tion of the ManNAc analogue with [3-13C]-pyruvic acid. However,
degradation of analogues 2-4 did not afford ManNAc analogues
in sufficient yields (26-69%) due to the competing reverse
reaction. Therefore, the reactions were run in the presence of
lactate dehydrogenase in order to push the equilibrium toward
the ManNAc analogues. When 1 equiv of â-NADH was used,
the degradation yields increased to the range 45-98% (Table 1).
After purification, ManNAc analogues were condensed with 3
equiv of [3-13C]-pyruvic acid. Among these analogues, the fluoro
analogue 6 in which the fluorine atom can be utilized for
(4) Two research groups have performed transfer of 13C-labeled galactose
by galactosyltransferase in order to analyze galactoside on protein, see: (a)
Goux, W. J.; Perry, C.; James, T. L. J. Biol. Chem. 1982, 257, 1829-1835.
(b) Gilhespy-Muskett, A. M.; Partridge, J.; Homans, S. W. Glycobiology 1994,
4, 485-489.
(5) Analysis of a glycan chain on small proteins (less than 15 KDa) could
be performed, see: (a) Berman, E.; Walters, D. E.; Allerhand, A. J. Biol.
Chem. 1981, 256, 3853-3857. (b) de Beer, T.; van Zuylen, C. W. E. M.;
Hård, K.; Boelens, R.; Kaptein, R.; Kamerling, J. P.; Vliegenthart, J. F. G.
FEBS Lett. 1994, 348, 1-6.
(6) 13C-labeling of carbohydrates is convenient for conformational analysis:
(a) Low, D. G.; Probert, M. A.; Embleton, G.; Sehadri, K.; Field, R. A.;
1b,d
Homans, S. W.; Windust, J.; Davis, P. J. Glycobiology 1997, 7, 373-381.
(b) Probert, M. A.; Milton, M. J.; Harris, R.; Schenkman, S.; Brown, J. M.;
Homans, S. W.; Field, R. A. Tetrahedron Lett. 1997, 38, 5861-5864. (c)
Bose, B.; Zhao, S.; Stenutz, R.; Cloran, F.; Bondo, P. B.; Bondo, G.; Hertz,
B.; Carmichael, I.; Serianni, A. S. J. Am. Chem. Soc. 1998, 120, 11158-
11173.
(7) Ogura, H.; Hasegawa, A.; Suami, T. Carbohydrates Synthetic Methods
and Applications in Medicinal Chemistry; KODANSHA, Tokyo, 1992; pp
243-339.
(8) Synthesis of 14C-labeled N-glycoylneuraminic acid in analytical scale
see: Terada, T.; Kitazume, S.; Kitajima, K.; Inoue, S.; Ito, F.; Troy, F. A.;
Inoue, T. J. Biol. Chem. 1993, 268, 2640-2648.
10.1021/ja982950e CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/29/1999

1412 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Communications to the Editor
Table 2. 1H and 13C Chemical Shifts and Coupling Constants of Sialoside 14a
1H
coupling constants (Hz)
13C
1JC,H
NOE
NeuAc
3ax
3eq
4
5
6
8
9
1
2
3
4
5
6R
6S
4
1.70
2.66
3.64
3.82
3.72
4.10
4.80-4.66
4.46
3.53
3.66
3.92
3.82
3.98
3.54
3.79
3JH3ax,H4 (ca. 14.7)
2Jgem (ca. 14.7)
3JH4,H5 (8.0)
40.84
129.8
132.1
NeuAc-H 4, 5, 6
3JH5,H6 (10.0)
3JH6,H7 (ca. 0.0)b
3JF,H8 (21.5)
2JF,H9 (46.6)
Gal
103.64
71.49
72.99
69.13
74.35
64.10
162.9
140.9
136.5
146.7
143.8
152.6
152.6
Gal-H 3, GlcNAc-H4
Gal-H 3
Gal-H 3, 5, 6R, 6S
Gal-H 3, 4, 6R, 6S
GlcNAc
3JH4,H5 (8.0)
3JH3,H4 (8.0)
a 1H chemical shifts were measured at 298 K (HOD ) 4.81 ppm), 13C chemical shifts were measured at 298 K (1,4-dioxane ) 67.48 ppm), 19F
chemical shift is -234.64 ppm at 298 K (trifluorotoluene ) 0.00 ppm); b Because HMQC-TOCSY did not show the H-7 resonance from H-6 of
NeuAc (spin locking time, 45, 60, 100 ms)
1H-19F HMQC was selected to synthesize CMP-9′′-deoxy-9′′-
fluoro-[3′′-13C]-NeuAc (11).9 As shown in Scheme 1, CMP-
NeuAc analogue 11 was prepared by a previously reported
method.10
the 6 position of the galactoside should result in the gt-rotamer
being the predominant orientation. These data are in good
agreement with those of the sialyloligosaccharide.15 Furthermore,
there appears to be an NOE between H-1 of the galactoside and
1
H-416 of GlcNAc, and the JC1,H1 value of the galactoside was
For synthesis of sialoside 14, hen ovalbumin 12 was used,
because this glycoprotein has only one glycan chain on its
backbone.11 Transfer of the [U-13C]-Gal residue proceeded
smoothly toward this glycoprotein. However, when the NeuAc
analogue was transferred by repetition of the R-(2f6)-STase
reaction,12 only about 60% of galactoside was estimated to be
sialylated according to the HMQC spectrum. Therefore, the
[U-13C]-Gal which was not sialylated was subject to digestion
by galactosidase. Consequently, the sialoside analogue was
obtained in an analytically pure state. To assign chemical shifts
and to analyze the conformation, 1D/2D HMQC-TOCSY, 1D/
2D 1H-19FHMQC, and 2D HMQC-NOESY analyses were
performed, and then the difference in the 13C chemical shifts
values between galactoside 13 and sialoside 14 was measured in
order to determine the sialylated position.12 The C-6 and C-5
chemical shifts of galactoside were shifted downfield and upfield,
respectively, and the H-6 (pro-R) and H-6 (pro-S)13 peaks were
also shifted downfield and upfield, respectively. Therefore, the
sialylated position was determined to be the 6 position of the
galactoside 13. The changes in the 1JC,H values at the 2, 5, and 6
positions of the galactoside residues in compounds 13 and 14,
suggest that the galactose-ring is strained14 by sialylation (Table
2 and Table 3 in Supporting Information). Although an NOE
between the NeuAc and the galactoside was not observed,1a,15
based on the NOE data obtained (Table 2) free rotation around
found to be 162.9 Hz. These data suggest that the galactose is
bound to the 4 position of the GlcNAc by a â-bond. The 1H vicinal
and geminal coupling constant values observed indicate the ring
3
of NeuAc residue adopts a C1 chair form, and the JF,H8 value
4
(21.5 Hz) suggests that the dihedral angle (9F-9C-8C-8H)
adopts an antiperiplanar orientation.17 Comparison of the hydrogen
and carbon chemical shift data between sialoside 14 and sialyl-
N-acetyl-â-D-lactosaminide,1a,18 reveals that the chemical shifts
are almost identical. These results suggest that conformational
properties of sialoside on the protein are similar to those of sialyl-
N-acetyl-â-D-lactosaminide.18 Research is in progress to synthesize
other 13C-labeled sialyloligosaccharide analogues.
Acknowledgment. This work was partially supported by a Grant-
in-Aid for Scientific Research from the Ministry of Education, Science,
Sports and Culture, Japan (No. 09780531) and the Takamura Foundation.
The authors thank Dr. Markus Wa¨lchli (Bruker Japan Co., Ltd.) for helpful
discussions and Mr. Masayoshi Kusama for mass spectroscopic analysis.
Supporting Information Available: Synthesis of NeuAc analogues
2-9, 11, 13, 14 and its NMR spectra (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA982950E
(9) Enzymatic synthesis of CMP-9′′-deoxy-9′′-fluoro-[2′′-14C]-NeuAc has
been performed in analytical scale, see: Conradt, H. S.; Bu¨nsch, A.; Brossmer,
R. FEBS Lett. 1984, 170, 295-300.
(14) (a) Podlasek, C. A.; Wu, J.; Stripe, W. A.; Bondo, P. B.; Serianni, A.
S. J. Am. Chem. Soc. 1995, 117, 11158-11173. (b) Bandyopadhyay, T.; Wu,
J.; Stripe, W. A.; Carmichael, I.; Serianni, A. S. J. Am. Chem. Soc. 1997,
119, 1737-1744.
(10) Kajihara, Y.; Ebata, T.; Koseki, K.; Kodama, H.; Matsushita, H.;
Hashimoto, H. J. Org. Chem. 1995, 60, 5732-5735.
(15) Breg, J.; Kroon-Batenburg, L. M. J.; Strecker, G.; Montreuil, J.;
Vligenthart, J. F. G. Eur. J. Biochem. 1989, 178, 727-739.
(16) The chemical shift is not consistent with those of any other hydrogens
of sialylgalactoside 14. H-7 of NeuAc appears as doublet at ca. 3.7 ppm.
(17) Csuk, R.; Gla¨nzer, B. I. AdV. Carbohydr. Chem. Biochem. 1988, 46,
73-177.
(11) The glycan chain is either a hybrid type containing one or two GlcNAc
residues at the nonreducing end or a high-mannose type, see: Tomiya, N.;
Awaya, J.; Kurono, M.; Endo, S.; Arata, Y.; Takahashi, N. Anal. Biochem.
1988, 171, 73-90.
(12) Kajihara, Y.; Yamamoto, T.; Nagae, H.; Nakashizuka, M.; Sakakibara,
T.; Terada, I. J. Org. Chem. 1996, 61, 8632-8635.
(18) Structure of sialyl-N-acetyl-â-D-lactosaminide here is NeuAc-R-(2f6)-
Gal-â-(1f4)-GlcNAc-â-O(CH2)8CO2Me,1a and its chemical shift data com-
pared to 14 is summarized in Supporting Information.
(13) Pro R and Pro S were tentatively determined based on a report: Ohrui,
H.; Nishida, Y.; Itoh, H.; Meguro, H. J. Org. Chem. 1991, 56, 1726-1731.