2184
`
`J. Am. Chem. Soc. 1998, 120, 2184-2185
`
`Unexpected Enzymatic Fucosylation of the Hindered
`Tertiary Alcohol of 3-C-Methyl-N-Acetyllactosamine
`Produces a Novel Analogue of the LeX-Trisaccharide
`
`Xiangping Qian, Ole Hindsgaul,* Hong Li, and
`Monica M. Palcic*
`Department of Chemistry, UniVersity of Alberta
`Edmonton, Alberta T6G 2G2, Canada
`ReceiVed September 25, 1997
`
`Mammalian oligosaccharides are biosynthesized through the
`action of glycosyltransferases that sequentially transfer single
`pyranosyl residues from nucleotide mono- or diphosphate sugars
`to growing carbohydrate chains.1 The increasing availability of
`recombinant glycosyltransferases has resulted in a corresponding
`increase in the use of these enzymes to achieve efficient combined
`chemical-enzymatic syntheses of such oligosaccharides.2 Nu-
`merous examples have also appeared where glycosyltransferases
`have been shown to either transfer an unnatural sugar to the
`natural substrate oligosaccharide or transfer a natural sugar to an
`unnatural oligosaccharide substrate, thus increasing the scope of
`the enzymatic step to include the preparation of oligosaccharide
`analogues.3
`Most oligosaccharide analogues that have been synthesized to
`probe the molecular specificity of carbohydrate-protein recognition
`have the hydroxyl groups on the pyranose rings either derivatized
`or replaced with other functional groups.4 Very few carbon-
`branched sugar residues have been reported5 and even fewer where
`the branching occurs at a carbon bearing a glycosylated OH-
`group.6,7 This is undoubtedly because the chemical glycosylation
`of complex hindered tertiary alcohols is notoriously difficult.8 The
`scope of analogues that have been prepared using glycosyltrans-
`ferases also does not yet include sugar residues that are branched
`at the carbon bearing the hydroxyl group undergoing glycosyla-
`tion. Since such carbon-branched sugar units do not exist in
`mammalian oligosaccharides,
`it could be expected that
`the
`available glycosyltransferases might not be able to catalyze their
`formation. Oligosaccharides containing such branched sugar units
`
`(1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Schachter, H. In Molecular
`Glycobiology; Fukuda, M., Hindsgaul, O., Eds.; IRL Press: Oxford, U.K.,
`1994; pp 88-97. (c) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto,
`T. Angew. Chem., Int. Ed. Engl. 1995, 34, 412. (d) Wong, C.-H.; Halcomb,
`R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34,
`521. (e) Dwek, R. A. Chem. ReV. 1996, 96, 683.
`(2) For reviews on chemoenzymatic synthesis, see: (a) Crawley, S. C.;
`Palcic, M. M. In Modern Methods in Carbohydrate Synthesis; Khan, S. H.,
`O’Neil, R. A., Eds.; Harwood Academic: Amsterdam, 1995; pp 492-517.
`(b) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. ReV. 1996, 96,
`443. (c) McGarvey, G. J.; Wong, C.-H. Liebigs Ann. 1997, 1059.
`(3) Palcic, M. M.; Hindsgaul, O. Trends Glycosci. Glycotechnol. 1996, 8,
`37 and references therein.
`(4) (a) Lemieux, R. U. Chem. Soc. ReV. 1989, 18, 347. (b) Kihlberg, J.;
`Hultgren, S. J.; Normark, S.; Magnusson, G. J. Am. Chem. Soc. 1989, 111,
`6364. (c) Glaudemans, C. P. J. Chem. ReV. 1991, 91, 25. (d) Sierks, M. R.;
`Bock, K.; Refn, S.; Svensson, B. Biochemistry 1992, 31, 8972. (e) Kanie, O.;
`Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Bioorg. Med. Chem. 1994, 2,
`1231. (f) Reck, F.; Meinjohanns, E.; Springer, M.; Wilkens, R.; van Dorst, J.
`A. L. M.; Paulsen, H.; Mo¨ller, G.; Brockhausen, I.; Schachter, H. Glyco-
`conjugate J. 1994, 11, 210.
`(a)
`(5) For reviews on the synthesis of branched-chain sugars, see:
`Yoshimura, J. AdV. Carbohydr. Chem. Biochem. 1984, 42, 69. (b) Chapleur,
`Y.; Chre´tien, F. In PreparatiVe Carbohydrate Chemistry; Hanessian, S., Ed.;
`Marcel Dekker: New York, 1997; pp 207-262.
`(6) Beau, J.-M.; Jaurand, G.; Esnault, J.; Sinay¨, P. Tetrahedron Lett. 1987,
`28, 1105.
`(7) For some examples on such naturally occurring sugars, see: (a) Kimura,
`Y.; Kobayashi, Y.; Takeda, T.; Ogihara, Y. J. Chem. Soc., Perkin Trans. 1
`1981, 1923. (b) Chatterjee, D.; Bozic, C.; Aspinall, G. O.; Brennan, P. J. J.
`Biol. Chem. 1988, 263, 4092. (c) Shimomura, H.; Sashida, Y.; Mimaki, Y.;
`Adachi, T.; Yoshinari, K. Chem. Pharm. Bull. 1989, 37, 829.
`(8) Kahne, D.; Walker, S.; Cheng, Y.; van Engen D. J. Am. Chem. Soc.
`1989, 111, 6881
`
`would be of interest for probing which face of a given sugar is
`making contact with a protein binding site. The resulting
`glycosidic linkage to a tertiary alcohol would additionally be
`expected to be conformationally much less flexible than that to
`the natural secondary OH group.
`We report here the chemical synthesis of the N-acetyllactos-
`amine analogue 3 bearing a methyl branch at C-3 of the GlcNAc
`residue. This compound was found to be a kinetically competent
`acceptor for a fucosyltransferase that transfers an R-Fuc residue
`to the branched alcohol, yielding the trisaccharide analogue 4 of
`the well-known blood group LeX trisaccharide 2. Remarkably,
`4 was also found to be an excellent substrate for a fucosidase
`known to act on the natural LeX structure 2.
`The glycosyl acceptor 6 was glycosylated by reaction of the
`glycosyl donor 5,9 promoted by AgOTf, yielding the expected
`(cid:226)-linked disaccharide 7 (81%) (Scheme 2). The O-allyl group
`was then removed using PdCl2, providing 8 in quantitative yield.
`Oxidation of 8 was achieved using DMSO-Ac2O. Treatment
`of the resulting ketone with methyllithium gave compound 9, with
`an axial C-methyl group.11 O-Deacetylation with NaOMe in
`MeOH followed by hydrogenolysis using Pd(OH)2 provided 3.
`The configuration of C-3 was established through NMR TROESY
`studies which show the significant NOE between 3-C-methyl
`protons and H-1 of the GlcNAc residue.
`Disaccharide 1 is a known acceptor for the R(1f3/4)-
`fucosyltransferase12 that can be readily isolated from human
`milk.13 The Km value for 1 was found to be 0.4 mM using an
`established radioactive “Sep-Pak assay”,14 and the relative velocity
`of fucosyl transfer was arbitrarily set to 100. The C-methyl-
`branched acceptor 3 was also found to be an acceptor in the same
`radioactive assay, with 20-fold elevated Km (to 8.0 mM) but a
`70% increase in Vrel (synthesis) (Scheme 1).
`It was very surprising that the enzyme tolerated the introduction
`of a C-methyl group directly at the site of transfer, implying that
`there was substantial flexibility in the active site at the transition
`state of the reaction. To be certain that the reaction proceeded
`normally, i.e. that OH-3 was indeed the alcohol that became
`fucosylated, a preparative reaction was performed15 to yield the
`C-methyl-branched trisaccharide 4, whose structure was confirmed
`by 1H NMR and MS analyses.16 In particular, H-5 of the newly
`
`(9) Spohr, U.; Lemieux, R. U. Carbohydr. Res. 1988, 174, 211.
`(10) The ketone product was not isolated but reacted directly in the next
`step.
`(11) Selected 1H NMR data for 3 (500 MHz, D2O): (cid:228) 4.55 (d, 1H, J )
`8.8 Hz, H-1), 4.47 (d, 1H, J ) 7.8 Hz, H-1¢), 3.92 (d, 1H, J ) 3.2 Hz, H-4¢),
`1.22-1.34 (m, 13H, (CH2)5CH3 and C-3-CH3). HR-ESMS m/e calcd for (M
`+ H+) C23H44NO11: 510.2914. Found: 510.2915.
`(12) (a) Johnson, P. H.; Donald, A. S. R.; Feeney, J.; Watkins, W. M.
`Glycoconjugate J. 1992, 9, 251. (b) de Vries, T.; Srnka, C. A.; Palcic, M. M.;
`Swiedler, S. J.; van den Eijnden, D. H.; Macher, B. A. J. Biol. Chem. 1995,
`270, 8712. (c) Murray, B. W.; Takayama, S.; Schultz, J.; Wong, C.-H.
`Biochemistry 1996, 35, 11183. (d) Gosselin, S.; Palcic, M. M. Bioorg. Med.
`Chem. 1996, 4, 2023.
`(13) Palcic, M. M. Methods Enzymol. 1994, 230, 300
`(14) Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycoconjugate
`J. 1988, 5, 49.
`(15) The reaction mixture contained 3 (1.0 mg, 2.0 (cid:237)mol), GDP-Fuc (1.7
`mg, 2.9 (cid:237)mol), 30 mU of milk FucT (in 300 (cid:237)L of 25 mM sodium cacodylate
`buffer, pH 6.5, containing 5 mM MnCl2 and 25% glycerol), 30 (cid:237)L of
`concentrated buffer (200 mM Hepes, pH 7.0, containing 200 mM MnCl2,
`and 2% BSA), and 3 (cid:237)L of calf intestine alkaline phosphatase (1 U/(cid:237)L). The
`reaction was incubated at 37 (cid:176)C with rotation for 2 days, then at room
`temperature for 4 days. Additional GDP-Fuc was added daily (total 1.6 mg).
`The reaction was stopped by filtering through a 0.22-(cid:237)m Millex GV filter
`unit, and product 4 was isolated by loading the mixture onto two sequential
`Sep-Pak C18 reverse phase cartridges. The cartridges were washed with water
`to remove enzyme and unreacted nucleotide donor, with 30% aqueous
`methanol to remove guanosine, and then with 50% aqueous MeOH to elute
`4 (1.0 mg, 75% yield).
`(16) Selected 1H NMR data for 4 (600 MHz, D2O): (cid:228) 5.29 (d, 1H, J )
`4.0 Hz, H-1¢¢), 4.68 (bq, 1H, H-5 ¢¢, J ) 6.8 Hz), 4.49 (d, 1H, J ) 8.4 Hz,
`H-1), 4.48 (d, 1H, J ) 7.7 Hz, H-1¢). HR-ESMS m/e calcd for (M + H+)
`C29H54NO15: 656.3493. Found: 656.3498.
`
`S0002-7863(97)03361-1 CCC: $15.00 © 1998 American Chemical Society
`Published on Web 02/20/1998
`
`Illumina Ex. 1077
`IPR Petition - USP 10,435,742
`
`

`

`Communications to the Editor
`
`Scheme 1
`
`J. Am. Chem. Soc., Vol. 120, No. 9, 1998 2185
`
`Scheme 2a
`
`The surprising finding in this work is that both the fucosyl-
`transferase and the fucosidase enzymes tolerate the introduction
`of a large methyl substituent on the same carbon that bears the
`oxygen which must be activated in both reactions.
`It will be
`interesting to see if this will be the case also for other glycosyl-
`transferases.
`
`a Reagents and Conditions: (a) 3 (1.5 equiv), AgOTf (2 equiv), 4 Å molecular sieves, CH2Cl2, -30 to 0 (cid:176) C, 3 h, 81%; (b) PdCl2 (0.5 equiv),
`MeOH, room temperature (rt), 2 h, quantitative; (c) DMSO, Ac2O, rt, 4 h; (d) MeLi (1.5 equiv), THF, -78 (cid:176) C, 2 h, 20% (two steps); (e) NaOMe,
`MeOH, rt, 27 h, 92%; (f) H2, 20% Pd(OH)2/C, MeOH, 20 h, 85%.
`introduced R-Fuc residue was strongly downfield-shifted (to 4.68
`ppm), which is diagnostic of 3-O-fucosyl-N-acetyllactosamine
`sequences such as 2, where it is found near 4.8 ppm.17
`Having access to the C-methyl-branched trisaccharide 4, we
`also examined if the fucosidase from almond meal that is known18
`to cleave the Fuc residue in the LeX structure 2 would tolerate
`the introduction of a methyl group so close to the glycosidic
`oxygen that must be protonated by the enzyme as glycoside
`hydrolysis is initiated. Remarkably, the introduction of the methyl
`group was found to have very little effect on the kinetics of the
`hydrolysis of 4, resulting in improved recognition of the substrate
`(with a 40% decrease in Km) and only a modest reduction (30%)
`in the reaction velocity (Scheme 1).19
`
`Acknowledgment. This work was supported by grants from the
`Natural Sciences and Engineering Research Council of Canada (to O.H.)
`and the Medical Research Council of Canada (to M.M.P.).
`
`Supporting Information Available: Spectral data for compounds
`3, 4, and 7-9 (3 pages). See any current masthead page for ordering
`and Web access instructions.
`
`(17) For NMR data of Lewis X, see: (a) Hindsgaul, O.; Norberg, T.; Pendu,
`J. L.; Lemieux, R. U. Carbohydr. Res. 1982, 109, 109. (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. (c) Ball, G. E.; O’Neil, R. A.; Schultz, J. E.; Lowe, J.
`B.; Weston, B. W.; Nagy, J. O.; Brown, E. G.; Hobbs, C. J.; Bednarski, M.
`D. J. Am. Chem. Soc. 1992, 114, 5449. (d) Yan, L.; Kahne, D. J. Am. Chem.
`Soc. 1996, 118, 9239.
`(18) Zhao, J. Y.; Dovichi, N. J.; Hindsgaul, O.; Gosselin, S.; Palcic, M.
`M. Glycobiology 1994, 4, 239.
`
`JA973361W
`(19) Compounds 2 or 4 (0.1-4 mM) were incubated at 37 (cid:176) C with 6.3 (cid:237)U
`almond meal fucosidase (Sigma, E.C. 3.2.1.111) in 25 (cid:237)L of 50 mM sodium
`citrate buffer, pH 5.0, for 24 h. Fucose released was quantitated by removing
`10-(cid:237)L aliquots, transferring them to microtiter wells containing 13 mU of
`Pseudomonas sp. fucose dehydrogenase (Sigma) and 0.98 mM NADP+ in
`130 (cid:237)L of 0.6 M diethanolamine buffer, pH 9.0, and monitoring the increase
`in absorbance at 340 nm.
`
`