Synthesis of urea-tethered neoglycoconjugates and pseudooligosaccharides in water

Article abstract of DOI:10.1021/ja056253f

A novel approach to the synthesis of urea glycosides in aqueous media has been explored. Steyermark's glucopyranosyl oxazolidinone was found to be a good synthon for anchoring glucosyl moieties onto amines and thiols. The present method was successfully a

Full text of DOI:10.1021/ja056253f

Published on Web 03/04/2006
Synthesis of Urea-Tethered Neoglycoconjugates and
Pseudooligosaccharides in Water
Yoshiyasu Ichikawa,*,† Yohei Matsukawa,‡ and Minoru Isobe‡
Contribution from the Faculty of Science, Kochi UniVersity, Akebono-cho, Kochi 780-8520,
Japan, and Laboratory of Organic Chemistry, School of Bioagricultural Sciences,
Nagoya UniVersity, Chikusa, Nagoya 464-8601, Japan
Received September 11, 2005; E-mail: ichikawa@cc.kochi-u.ac.jp
Abstract: A novel approach to the synthesis of urea glycosides in aqueous media has been explored.
Steyermark’s glucopyranosyl oxazolidinone was found to be a good synthon for anchoring glucosyl moieties
onto amines and thiols. The present method was successfully applied to establish a new route for the
synthesis of urea-tethered neoglycoconjugates and pseudooligosaccharides in water.
Scheme 1
Introduction
Development of a new method for anchoring carbohydrate
moieties onto biomolecules via nonnative glycosidic linkages,
which is an alternative to glycosylation, has been an active area
of numerous synthetic endeavors.1 This trend motivated us to
explore the synthesis of urea-tethered neoglycoconjugates and
pseudooligosaccharides during our synthetic effort for the natural
product, glycocinnasperimicin D (1).2
the hydrolysis of isocyanate 3. In the course of this research
work, we were fascinated with an approach to the urea glycoside
synthesis using unprotected sugars, because such a protocol
would lead to a new bioconjugate method in water.
This aminosugar antibiotic possesses a unique structural
feature, the urea-glycosyl linkage, which connects two highly
functionalized aminosugars. The most challenging problem
inherent in the total synthesis of 1 is the construction of this
urea-glycosyl bond. To solve this problem, we developed a new
method for the synthesis of urea glycoside, which involves the
reaction of glycopyranosyl isocyanate with amine (Scheme 1).3
Oxidation of isonitrile 2 with pyridine N-oxide in the presence
of a catalytic amount of iodine generated highly reactive
isocyanate 3, which was immediately treated with aminosugar
4 to afford the urea-linked disaccharide 5 in good yield. It should
be noted that the reaction was carried out in organic solvent
(CH3CN) in the presence of water scavenger (MS3A) to avoid
In 1961, Steyermark reported the reaction of â-D-gluco-
pyranosylamine 6 with phosgene, expecting to produce the
known N,N′-di-â,â-D-glucopyranosyl urea 7; however, the
isolated product was actually the cyclic carbamate 8 in poor
yield ranging from 6 to 30%.4 The improved synthesis of
oxazolidinone 8 was reported by Pinter and co-workers, who
employed the reaction of glucopyranosyl phosphinimine with
carbon dioxide and proposed the correct stereochemistry at the
anomeric position in 8.5 The large coupling constant between
H-1 and H-2 for 8 (J1,2 ) 9.2 Hz) unambiguously established
the structure of diequatorial trans-annulation of the oxazolidi-
none to the pyranose ring. Since this Steyermark’s glucopyra-
nosyl oxazolidinone 8 has twisted structure and delocalization
of the lone pair electrons on nitrogen into the carbonyl group
was prevented,6 the carbonyl group in oxazolidinone has
increased kinetic reactivity toward nucleophilic attack with a
decrease of the ring strain. In fact, we have disclosed that this
unprotected glucopyranosyl derivative 8 behaves chemically as
† Kochi University.
‡ Nagoya University.
(1) (a) Taylor, C. M. Tetrahedron 1998, 11317. (b) Davis, B. G. Chem. ReV.
2002, 102, 579.
(2) (a) Dobashi, K.; Nagaoka, K.; Watanabe, Y.; Nishida, M.; Hamada, M.;
Takeuchi, T.; Umezawa, H. J. Antibiot. 1985, 1166. For the total synthesis
of 1, see: (b) Nishiyama, T.; Isobe, M.; Ichikawa, Y. Angew. Chem., Int.
Ed. 2005, 44, 4372.
(3) (a) Ichikawa, Y.; Nishiyama, T.; Isobe, M. Synlett 2000, 1253. (b) Ichikawa,
Y.; Nishiyama, T.; Isobe, M. J. Org. Chem. 2001, 66, 4200.
(4) Steyermark, P. R. J. Org. Chem. 1962, 27, 1058.
(5) Kovacs, J.; Pinter, I.; Messmer, A. Carbohydr. Res. 1985, 141, 57.
9
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J. AM. CHEM. SOC. 2006, 128, 3934-3938
10.1021/ja056253f CCC: $33.50 © 2006 American Chemical Society

Synthesis of Neoglycoconjugate and Pseudooligosaccharide
A R T I C L E S
Scheme 2
Scheme 3
Scheme 4
an acylating agent in water: oxazolidinone 8 undergoes smooth
ring-opening reaction with amines to give rise to the corre-
sponding urea glucosides 9 (Scheme 2).7
Herein, we wish to report the full details and further
development of our approach for the synthesis of urea-tethered
neoglycoconjugates and pseudooligosaccharides in aqueous
media utilizing Steyermark’s glucopyranosyl oxazolidinone.
methanol to furnish the urea glucoside 12 in 87% yield. The
structure of 12 was confirmed by transforming into the corre-
sponding acetate 13, authentic sample of which was prepared
by the reaction of Fischer’s glucopyranosyl isocyanate 39 with
2-phenylethylamine in organic solvent (anhydrous toluene).
Further studies focused on the reaction of 8 with a variety of
amines to evaluate the generality and scope of this ring-opening
reaction and to identify the steric factors of the alkyl groups in
amine. The results are summarized in Table 1. Primary amines
having no branches at the R-carbon, such as n-butylamine and
benzylamine (entries 1 and 2), reacted with 1.2 equiv of 8 to
give the urea glucosides 14a and 14b in good yields (95 and
88%). Alkyl substituents at the R-position of primary amine
had considerable steric effects; in the case of (S)-(-)-R-
methylbenzylamine and cyclohexylamine (entries 3 and 4), a
slight excess of 8 (1.5 equiv), longer reaction time (2.0 h), and
keeping the reaction mixture at 40 °C (entry 4) was necessary
to obtain the urea glucosides 14c and 14d with satisfactory yields
(80 and 84%). Secondary amines, diethylamine and pyrrolidine
(entries 5 and 6), were less reactive than primary amines;
however, use of 2 equiv of 8, heating the reaction mixtures at
50-60 °C, and longer reaction times (3-6 h) resulted in
sufficient yields (80 and 71% yield). The steric effect at the
â-position of secondary amine considerably interfered with the
reaction. For example, when diisobutylamine was employed
(entry 7), hydrolysis of 8 predominated and only 8% of the
product 14g was isolated. In the case of more sterically
congested secondary amine, diisopropylamine (entry 8), only
hydrolysis of 8 was observed even after extended reaction times.
Finally, aniline (entry 9) underwent urea glycosylation in water
at 70 °C for 12 h to afford 14i in 75% yield. It should be noted
that heating a reaction mixture did not accelerate the hydrolysis
of 8 due to the weaker basicity of aniline.
Results and Discussions
Stability of Oxazolidinone 8 in Water. Our initial effort
focused on the hydrolysis of Steyermark’s glucopyranosyl
oxazolidinone 8, which was prepared by the modified procedure
of Pinter (Scheme 3). Treatment of glucopyranosyl azide 10
with triphenylphosphine in acetone gave phosphinimine 11,
which was successively treated with carbon dioxide in a one-
pot process. The resulting white precipitate was collected and
washed with acetone to furnish 8 in excellent yield. Surprisingly,
hydrolysis of oxazolidinone in 8 occurred even in neutral media.
1
In fact, a solution of 8 in D2O at 40 °C was monitored by H
NMR, which showed that about 60% of 8 was hydrolyzed after
24 h.8
Reaction of 8 with Amines in Water. Satisfied with a high
reactivity of 8, we next turned our attention to examine the
chemical behavior of 8 as an acylating reagent with amines in
water. Gratifyingly, Steyermark’s glucopyranosyl oxazolidinone
8 underwent smooth ring-opening reaction with 2-phenylethyl-
amine in water; the reaction was completed within 1 h (Scheme
4). The resultant aqueous reaction mixture was directly loaded
on reversed-phase column chromatography, and the product was
eluted with stepwise gradient from water to 30% aqueous
(6) The twisted structure of 8 was recognized in the highly reactive nature of
nitrogen in oxazolidinone. Acetylation of 8 occurred on both the hydroxy
groups and nitrogen in oxazolidinone under mild conditions (Ac2O, pyridine,
room temperature, 20 h) to furnish the tetraacetate i. See ref 4.
(7) (a) Ichikawa, Y.; Matsukawa, Y.; Isobe, M. Synlett 2004, 1019. For the
only reported example, to our knowledge, of the reaction of 8 with
N-methylpiperazine in water, see: (b) Pinter, I.; Kovacs, J.; Toth, G.
Carbohydr. Res. 1995, 273, 99. Analogous O-unprotected Zemplen’s
glucopyranosyl thiocarbamate and its reaction with amines was reported
during our reserarch work. See: (c) Maya, I.; Lopez, O.; Fernandez-Bolanos,
J. G.; Robina, I.; Fuentes, J. Tetrahedron Lett. 2001, 42, 5413. (d) Lopez,
O.; Maya, I.; Fernandez-Bolanos, J. G. Tetrahedron 2004, 60, 61.
(8) 1H NMR and TLC analysis indicated that the major hydrolysis products
were R-, â-D-glucopyranose and N,N′-di-â,â-D-glucopyranosyl urea 7, which
was further confirmed by acetylation of the resultant hydrolysate and
comparison with authentic samples. We thank Fumiyo Ohara (Kochi
University) for these experiments.
Reaction with Thiol. The importance of thiol-reactive
reagents for the synthesis of glycoconjugates was well recog-
nized in the field of neoglycoproteins and neoglycopeptides.
(9) (a) Fischer, E. Ber. 1914, 47, 1377. (b) Johnson, T. B.; Bergmann, W. J.
Am. Chem. Soc. 1932, 54, 3360. (c) Ichikawa, Y.; Matsukawa, Y.;
Nishiyama, T.; Isobe, M. Eur. J. Org. Chem. 2004, 586.
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J. AM. CHEM. SOC. VOL. 128, NO. 12, 2006 3935

A R T I C L E S
Table 1
Ichikawa et al.
Scheme 6
formed N-glucosyl thiolcarbamate 15 was unstable and appeared
to go back to the starting material, which was confirmed by
the following experiments: treatment of 8 with ethanethiol in
water afforded an aqueous solution of 15 (checked by TLC
analysis), which was subsequently subjected with benzylamine.
Monitoring the reaction mixture showed that 15 gradually
disappeared with concomitant formation of urea glucoside 14b.
After workup followed by acetylation provided the urea
glucoside 17 in 65% yield. These experimental results confirmed
that initial product 15 gradually regenerated 8 by intramolecular
attack of C2 hydroxy group to the ethyl thiolcarbamate moiety,12
and the resultant 8 was subsequently trapped with benzylamine
to afford 14b.
Synthesis of Glycosyl Amino Acid Conjugates. The reactiv-
ity of 8 toward amine and thiol appears to promise that
Steyermark’s glucopyranosyl oxazolidinoned 8 is a good
synthon for anchoring carbohydrate moieties onto biomolecules
in aqueous media. In this regard, we examined the reaction of
8 with amino acids in water (Scheme 6). Synthesis of urea-
tethered glucosyl amino acid conjugate was carried out by the
reaction of 8 with lysine ꢀ-amino group in 18 in the presence
of triethylamine. The reaction was completed at room temper-
ature within 1 h, and the urea-tethered glucosyl lysine conjugate
19 was obtained in 85% yield. The reaction of 8 with L-cysteine
methyl ester hydrochloride 20 in the presence of triethylamine
was completed at room temperature for 10 min. The resulting
reaction mixture was immediately treated with acetic anhydride
to protect the remaining amino group in the cysteine moiety.13
Concentration followed by acetylation furnished glucopyranosyl
cysteine conjugate 21 in 71% yield.
a Unless otherwise noted, yields were calculated after reversed-phase
column purification. b The product was isolated after acetylation, because
purification by reversed-phase column was difficult. c Since the product is
hygroscopic, the yield was determined after acetylation.
Scheme 5
Several successful approaches to site-specific ligation of
carbohydrate moieties at cysteine residues in proteins and/or
peptides have been reported.10 In this context, we were interested
in the reactivity of 8 with sulfur nucleophiles, and we performed
exploratory experiments testing the reaction of 8 with simple
ethanethiol in water (Scheme 5). Although reaction of 8 with
ethanethiol in water did not occur, addition of 1 equiv of
triethylamine resulted in the smooth ring-opening reaction within
1 h, and the product 16 was isolated in 81% yield after
acetylation.11 During this investigation, we found that initially
Synthesis of Urea-Tethered Pseudooligosaccharide in
Aqueous Media. The chemistry of Steyermark’s glucopyranosyl
(11) Authentic sample of 16 was prepared by the reaction of Fischer’s
glucopyranosyl isocyanate 3 with ethanethiol and triethylamine in toluene.
(12) The existence of such an intramolecular attack of the C2 hydroxy group
was also supported by the following experiments: treatment of 16 with
benzylamine in toluene resulted in the near recovery of starting material.
(10) (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1991, 32, 6793. (b)
Macindoe, W. M.; Oijen, A. H.; Boons, G.-J. Chem. Commun. 1998, 847.
(c) Davis, B. G.; Lloyd, R. C.; Jones, J. B. J. Org. Chem. 1998, 63, 9614.
(d) Shin, I.; Jung, H.-J.; Lee, M.-R. Tetrahedron Lett. 2001, 42, 1325. (e)
Cohen, S. B.; Halcomb, R. L. J. Am. Chem. Soc. 2002, 124, 2534. (f) Zhu,
X.; Pachamuthu, K.; Schmidt, R. R. J. Org. Chem. 2003, 68, 5641.
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Synthesis of Neoglycoconjugate and Pseudooligosaccharide
A R T I C L E S
Scheme 7
Scheme 8
oxazolidinone 8 is much more precious than we initially
expected, which finally led us to the synthesis of urea-linked
pseudooligosaccharides in aqueous media. The synthetic plan
is outlined in Scheme 7. Starting with commercially available
D-glucosamine hydrochloride A, we set up to prepare phenyl
2,6-diaminoglycoside B as a core segment, which has two
reactive sites for the urea glycosylation with Steyermark-type
glycosyl oxazolidinones for the construction of â-(1 f 6) and
â-(1 f 4) urea glycosyl bond. In addition, the phenyl group in
B was envisioned to facilitate the final purification step by
reversed-phase chromatography.
Scheme 9
Synthesis of the phenyl 2,6-diaminoglycoside began with
tin(IV) chloride-catalyzed glycosylation of the Troc-oxazolidi-
none 22 with phenol (Scheme 8). In this phenyl glycosylation,
prolonged reaction time (2 days) resulted in a gradual decrease
of the â-isomer proportion and concomitant increase in the
formation of R-anomer to furnish R-phenyl glycoside 23
predominantly in 89% yield.14 Treatment of 23 with zinc in a
mixture of acetic acid and THF cleaved Troc-carbamate, and
the resultant amine was immediately reprotected as its Cbz-
derivative 24 in a 78% yield for two steps. Hydrolysis of acetyl
groups in 24 (Et3N, MeOH) gave triol, whose primary alcohol
was selectively tosylated (TsCl, pyridine), and the remaining
two hydroxy groups were acetylated (subsequent addition of
Ac2O) to afford 25 in an 88% yield. Displacement of the tosylate
25 with sodium azide in DMF and hydrolysis of the acetyl
groups (Et3N, MeOH) furnished the core segment 26 in an 88%
yield.
2-phenylethylamine were carried out in water to result in the
smooth formation of the corresponding urea glycosides 31 and
32 in 92 and 87% yield, respectively (Scheme 9).
The synthesis of urea-tethered pseudopentasaccharide began
with the Staudinger reaction of azide 26 with triphenylphosphine
in a mixture of water and tert-butyl alcohol (Scheme 10). Under
this reaction condition, initially formed phosphinimine was
hydrolyzed to afford the amine 33, which was dissolved in
aqueous media (H2O/MeOH, 1:2) and then treated with 1.5
equiv of lactosyl oxazolidinone 30. The formation of â-(1 f
6) urea intersaccharide bond was completed at room temperature
within 24 h. The resultant mixture was treated with triethylamine
to quench the excess 30 and then concentrated under reduced
pressure to afford the residue, which was repeatedly washed
with ethyl acetate to remove triphenylphosphine and triphen-
ylphosphine oxide.15 Hydrogenolysis of the Cbz group in 34
(H2, 5% palladium on carbon, H2O) gave the amine 35, which
was subsequently subjected to the second urea glycosylation
with cellobiosyl oxazolidinone 29 (1.5 equiv) in water. The
reaction mixture was stirred at room temperature for 24 h and
then loaded on the reversed-phase column chromatography
(eluted with H2O followed by 9:1 H2O/MeOH) to furnish urea-
linked pseudopentasaccharide 36 in 73% overall yield starting
from 26. It should be noted that the whole procedure in this
Disaccharides 29 and 30, containing the structural motif of
Steyermark’s glucopyranosyl oxazolidinone, were prepared from
cellobiosyl and lactosyl azides 27 and 28 by using procedures
similar to those described in Scheme 3. To check the reactivity
of these disaccharide oxazolidinones 29 and 30, reactions with
(13) Without protection of the amino group with acetic anhydride, the product
gradually decomposed during isolation. These optimized conditions were
established by the following experiments: competitive reaction of 8 with
ethanethiol and benzylamine showed that initially formed major product
was 15. However, prolonged reaction time resulted in the disappearance
of 15 and almost exclusive formation of 14b. Upon concentration, the
resultant product was treated with acetic anhydride and pyridine to afford
the urea glucoside 17 in 80% yield.
(15) This operation was necessary for smooth hydrogenolysis of the Cbz group
(14) Nishiyama, T.; Ichikawa, Y.; Isobe, M. Synlett 2004, 89.
in 34.
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J. AM. CHEM. SOC. VOL. 128, NO. 12, 2006 3937

A R T I C L E S
Scheme 10
Ichikawa et al.
Synthesis of Pseudopentasaccharide 36. To a solution of 26 (50
mg, 0.12 mmol) dissolved in a mixture of water (0.50 mL) and tert-
butyl alcohol (2.0 mL) was added triphenylphosphine (35 mg, 0.13
mmol) in a single portion. After being stirred at 60 °C for 4 h, the
reaction mixture was concentrated under reduced pressure to give 33,
which was dissolved in a mixture of water (1.0 mL) and MeOH (2.0
mL). Lactosyl oxazolidinone 30 (66 mg, 0.18 mmol) in methanol (1.0
mL) was added, and the resulting reaction mixture was stirred at room
temperature for 24 h. Addition of triethylamine (0.10 mL) followed
by stirring for about 6 h resulted in the disappearance of lactosyl
oxazolidinone 30 (checked by TLC). The resultant reaction mixture
was concentrated under reduced pressure to afford the residue, which
was repeatedly washed with ethyl acetate to remove triphenylphosphine
and triphenylphosphine oxide. The resulting 34 was dissolved in H2O
(5.0 mL), and palladium on carbon (5%, 20 mg) was added. The
suspension was vigorously stirred under atmosphere of hydrogen at
room temperature for 24 h. Palladium catalyst was removed by filtration,
and the filtrate was concentrated under reduced pressure. Finally, amine
35 was dissolved in H2O (5.0 mL) and then treated with cellobiosyl
oxazolidinone 29 (66 mg, 0.18 mmol). After being stirred at room
temperature for 24 h, the resultant reaction mixture was loaded on the
reversed-phase column chromatography (eluted with H2O and 9:1 H2O/
MeOH) to furnish pseudopentasaccharide 36 (87 mg, 73% overall yield)
reaction sequence (26 f 33 f 34 f 35 f 36) was carried out
in a one-pot operation.
While most 1H NMR signals of 36 are overlapped, 13C NMR
and FAB mass spectra confirmed the structure of this novel
pseudopentasaccharide.16 The 13C NMR spectrum of 36 showed
four aromatic carbons (δ 118.2, 124.0, 130.7, and 156.7), two
urea carbonyl carbons (δ 159.8 and 160.2), and 30 carbons
corresponding to the five hexopyranoses. Among 13C NMR
signals associated with the carbohydrate portions, the prominent
chemical shifts assigned to two anomeric carbons linked to
ureido nitrogen atom are δ 81.5 and 81.6, which are consistent
with the â-stereochemistry.17 The 103.2 and 103.6 ppm signals
are assigned to the anomeric carbons of lactose and cellobiose
moieties, and the upfield signal at 97.6 ppm to that of phenyl
glycoside.
Conclusion
In this work exploring the chemitry of Steyermark’s glu-
copyranosyl oxazolidinone 8, we uncovered its utility for the
synthesis of neoglycoconjugates in water. In fact, this strained
cyclic carbamate 8 onto unprotected carbohydrate reacted with
amine and thiol, which provides access to a variety of neogly-
coconjugate and pseudooligosaccharide. Steyermark’s gluco-
pyranosyl oxazolidinone 8, an old but renewed synthon for urea
glycosylation, can be considered to be a water-soluble synthetic
equivalent of Fischer’s glucopyranosyl isocyanate 3.
as a white solid: mp 222-223 °C; [R]24 +43.9 (c 0.90, H2O); IR
D
(KBr) νmax ) 1654, 1560, 1279 cm-1 13C NMR (D2O, 100 MHz): δ
;
41.0, 54.8, 60.6, 60.8, 61.3, 61.7, 69.2, 70.1, 71.6, 71.9, 72.1, 72.2,
72.4, 72.5, 73.2, 73.8, 75.7, 75.9, 76.0, 76.1, 76.52, 76.56, 76.63, 78.8,
79.0, 81.5, 81.6, 97.6, 103.2, 103.6, 118.2, 124.0, 130.7, 156.7, 159.8,
160.2; HRMS (FAB) calcd for C38H61N4O26 [M + H]+ 989.3574, found
989.3561.
Experimental Section18
General Method for the Preparation of Urea Glucoside in Water.
To a solution of n-butylamine (21 mg, 0.29 mmol) in water (4.0 mL)
was added Steyermark’s glucopyranosyl oxazolidinone 8 (71 mg, 0.35
mmol) in a single portion. After being stirred at room temperature for
1.0 h, the reaction mixture was directly passed through a reversed-
phase column (H2O followed by 10:1 H2O/MeOH as eluent) to afford
the urea glucoside 14a as a viscous gum (76 mg, 95%).
Acknowledgment. We are grateful for the financial support
from MEXT Grant-in-Aid for Specially Promoted Research
(16002007). Y.I. thanks Grant-in-Aid for Scientific Research
on Priority Areas (17035061) from MEXT and The Naito
Foundation for financial support.
(16) For a recent example of related glycooligomers with thiourea linkage, see:
Jimenez Blanco, J. L.; Bootello, P.; Ortiz Mellet, C.; Gutierrez Gallego,
R.; Garcia Fernandez, J. M. Chem. Commun. 2004, 92.
Supporting Information Available: Experimental procedures
and spectral data for all relevant compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(17) The 13C NMR of â-glucopyranosyl urea shows anomeric carbons in the
region of 80-81 ppm, and the 13C NMR chemical shift of R-ureido
glycosidic carbon appears around 75-78 ppm. See ref 3b.
(18) For materials and methods, see the Supporting Information.
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