A practical method for the stereoselective generation of β-2-deoxy glycosyl phosphates

Article abstract of DOI:10.1021/ol049187f

(Equation Presented) β-2-Deoxy sugar nucleotides are substrates used by a variety of glycosyltransferases (Gtfs). We have developed a chemical route to synthesize β-2-deoxy sugar phosphates that starts from α-glycosyl chlorides. Our approach reliably prov

Full text of DOI:10.1021/ol049187f

ORGANIC
LETTERS
2004
Vol. 6, No. 17
2873-2876
A Practical Method for the
Stereoselective Generation of
â-2-Deoxy Glycosyl Phosphates
Markus Oberthu1r, Catherine Leimkuhler, and Daniel Kahne*
Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544
dkahne@princeton.edu
Received May 4, 2004
ABSTRACT
â-2-Deoxy sugar nucleotides are substrates used by a variety of glycosyltransferases (Gtfs). We have developed a chemical route to synthesize
â-2-deoxy sugar phosphates that starts from r-glycosyl chlorides. Our approach reliably provides access to a range of NDP â-2-deoxy sugars
essential for studying glycosyltransferases involved in the synthesis of biologically active natural products.
Deoxy sugars are essential constituents of a variety of
biologically active natural products that include vancomycin,
erythromycin, and daunomycin (Figure 1).1 As a conse-
quence, considerable effort has been directed toward devel-
oping methods that will attach deoxy sugars to various natural
product aglycones. Enzymes are proving to be efficient tools
for glycosylating these aglycones, particularly since the
relaxed substrate specificity of many glycosyltransferases
(Gtfs) in terms of both glycosyl donors and glycosyl
acceptors makes it possible to prepare analogues.2-4 One
limitation of enzymatic glycosylation methods is the dif-
ficulty sometimes encountered in obtaining the required NDP
deoxy sugar5 donors. Although enzymatic methods for
making NDP sugars have been investigated,6 chemical
methods still represent the most flexible approach. However,
the â-linked 2-deoxy L-sugar donors, which are used by many
natural product Gtfs, are especially difficult to synthesize
chemically because intermediates can be unstable and the
desired â-anomers are thermodynamically disfavored.1a,7,8
Here, we report the first stereoselective method for preparing
2-deoxy â-L-glycosyl phosphates from R-glycosyl chlorides.
Such phosphates can easily be transformed to the corre-
(4) Examples for the chemoenzymatic synthesis of analogues using
purified glycosyltransferases: (a) Losey, H. C.; Peczuh, M. W.; Chen, Z.;
Eggert, U. S.; Dong, S. D.; Pelczer, I.; Kahne, D.; Walsh, C. T. Biochemistry
2001, 40, 4745-4755. (b) Losey, H. C.; Jiang, J.; Biggins, J. B.; Oberthu¨r,
M.; Ye, X.-Y.; Dong, S. D.; Kahne, D.; Thorson, J. S.; Walsh, C. T. Chem.
Biol. 2002, 9, 1305-1314. (c) Fu, X.; Albermann, C.; Jiang, J.; Liao, J.;
Zhang, C.; Thorson, J. S. Nature Biotechnol. 2003, 21, 1467-1469. (d)
Albermann, C.; Soriano, A.; Jiang, J.; Vollmer, H.; Biggins, J. B.; Barton,
W. A.; Lesniak, J.; Nikolov, D. B.; Thorson, J. S. Org. Lett. 2003, 5, 933-
936.
(5) NDP ) nucleotide diphosphoryl; TDP ) thymidine diphosphoryl;
TMP ) thymidine monophosphoryl; UDP ) uridine diphosphoryl.
(6) Recently, two TDP â-2-deoxy-L-sugars were synthesized enzymati-
cally: (a) TDP epi-vancosamine: Chen, H.; Thomas, M. G.; Hubbard, B.
K.; Losey, H. C.; Walsh, C. T.; Burkart, M. D. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 11942-11947. (b) TDP L-olivose: Amann, S.; Dra¨ger, G.;
Rupprath, C.; Kirschning, A.; Elling, L. Carbohydr. Res. 2001, 335, 23-
32.
(7) (a) Niggemann, J.; Lindhorst, T. K.; Walfort, M.; Laupichler, L.;
Sajus, H.; Thiem, J. Carbohydr. Res. 1993, 246, 173-183. (b) Uchiyama,
T.; Hindsgaul, O. J. Carbohydr. Chem. 1998, 17, 1181-1190. (c) Mu¨ller,
T.; Schmidt, R. R.; Tetrahedron Lett. 1997, 38, 5473-5476. (d) Komatzu,
H.; Awano, H. J. Org. Chem. 2002, 67, 5419-5421.
(8) This problem is also encountered in the construction of â-2-deoxy
O-glycosides. For approaches to overcome this problem, see for example:
Marzabadi, C. H.; Franck, R. W. Tetrahedron 2000, 56, 8385-8417.
(1) (a) Kirschning, A.; Rohr, J.; Bechthold, A. Top. Curr. Chem. 1997,
188, 1-84. (b) Thorson, J. S.; Hosted, T. J.; Jiang, J.; Biggins, J. B.; Ahlert,
J. Curr. Org. Chem. 2001, 5, 139-167.
(2) For recent reviews, see: (a) Walsh, C. T. ChemBioChem 2002, 3,
124-134. (b) Mendez, C.; Salas, J. A. Trends Biotech. 2001, 19, 449-
456.
(3) For recent examples of syntheses of analogues using an in vivo
approach, see: (a) Hoffmeister, D.; Dra¨ger, G.; Ichinose, K.; Rohr, J.;
Bechthold, A. J. Am. Chem. Soc. 2003, 125, 4678-4679. (b) Remsing, L.
L.; Gonzalez, A. M.; Nur-e-Alam, M.; Fernandez-Lozano, M. J.; Brana,
A. F.; Rix, U.; Oliveira, M. A.; Mendez, C.; Salas, J. A.; Rohr, J. J. Am.
Chem. Soc. 2003, 125, 5745-5753.
10.1021/ol049187f CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/20/2004

UDP epi-vancosamine derivative 2 in only a 1:2 â/R-ratio,
we obtained after deprotection sufficient material for the
initial characterization of Gtfs involved in the biosynthesis
of vancomycin and chloroeremomycin.4a
Unfortunately, this method proved to be unreliable, giving
significantly lower â/R-ratios in closely related systems. For
example, when vancosamine lactol 3, which differs from 1
only in the stereochemistry at C-4, was subjected to these
reactions, only the R-isomer 4 was obtained (Scheme 1).
Therefore, we began to explore alternative methods to make
â-2-deoxy sugar nucleotides.
We speculated that the low â-selectivities obtained using
the in situ-generated R-glycosyl iodides resulted from a
substitution reaction that proceeded predominantly via an SN1
pathway.9 More stable anomeric leaving groups, while still
being reactive enough to be replaced by suitable nucleophiles,
might therefore shift the substitution toward an SN2 pathway,
leading to higher â-selectivities starting from an R-configured
donor. To address this possibility, we prepared the L-
acosamine (4-epi-daunosamine) derivative 5 (Scheme 2),
Scheme 2. Model System for â-Phosphate Formation
Figure 1. Deoxy sugar-containing natural products.
sponding TDP 2-deoxy sugars,5 which are substrates for Gtfs
in the biosynthesis of various glycosylated natural products.
As part of our ongoing investigations into the substrate
selectivity of various glycosyltransferases, we desired an
efficient method for the preparation of â-2-deoxy sugar
nucleotides. We were able to obtain one substrate, UDP
L-epi-vancosamine,5 as an R/â-mixture4a using an approach
similar to that reported by Hindsgaul.7b This procedure
involved coupling of the protected epi-vancosamine lactol
1, via the anomeric iodide, with the tetrabutylammonium salt
of UDP (Scheme 1). Although this method generated the
which is easily obtained from 3,4-di-O-acetyl-L-rhamnal.10
L-Acosamine is a model system for a 2,6-dideoxy sugar
found in many glycosylated natural products. Efforts to
generate the anomeric phosphate from the corresponding
anomeric trichloroacetimidate or phosphite failed due to the
instability of these glycosyl donors. However, R-chloride 6,
which was obtained from lactol 5 (oxalyl chloride, DMF),11
and R-bromide 7, generated via the anomeric acetate using
TMS-Br,12 proved to be stable, enabling us to investigate a
variety of reaction conditions for the generation of â-phos-
phates.
Scheme 1. Synthesis of Protected UDP epi-Vancosamine 2 and
UDP Vancosamine 4
(9) Coupling is conducted at pH > 7; therefore, no acid-catalyzed product
equilibration should occur.
(10) Abbaci, B.; Florent, J.-C.; Monneret, C. Bull. Soc. Chim. France
1989, 669-672.
(11) Ko¨pper, S.; Lundt, I.; Pedersen, C.; Thiem, J. Liebigs Ann. Chem.
1987, 531-535.
(12) Gillard, J. W.; Israel, M. Tetrahedron Lett. 1981, 22, 513-516.
2874
Org. Lett., Vol. 6, No. 17, 2004

Scheme 3. 2-Deoxy Phosphates from Glycosyl Chloridesa
Table 1. Effect of Solvent and Nucleophile on Glycosyl
Phosphate Formation
entry donor product solvent
nucleophilea
â/Rb (yield)
1
2
3
4
5
6
7
6c
7d
6c
6c
6c
7d
6c
8
8
8
8
8
8
9
CH2Cl2
CH2Cl2
toluene
TBAP
TBAP
TBAP
2:1 (56%)
1:2 (51%)
2:1 (48%)
2:1 (54%)
5:1 (58%)
4:1 (52%)
2:1 (54%)
CH3NO2 TBAP
CH3CN
CH3CN
CH3CN
TBAP
TBAP
TDP Bu4N-salt
a TBAP ) Bu4NH2PO4. b â/R-ratios were determined by 1H NMR.
c DIPEA (pH > 7), 3 Å molecular sieves, 0 °C f rt, 3 h. d DIPEA (pH >
7), 3 Å molecular sieves, -30 °C f rt, 3 h.
a Reaction conditions: Bu4NH2PO4, CH3CN, 0 °C to rt.
Table 1 shows the stereochemical outcome of glycosyla-
tion of tetrabutylammonium dihydrogen phosphate (Bu4NH2-
PO4, TBAP) with chloride 6 and bromide 7. A correlation
between increased â-selectivity (2:1 vs 1:2) in the formation
of phosphate 8 and increasing stability of the donor (Cl >
Br) was observed in dichloromethane (entries 1 and 2).
Variation of the solvent polarity did not have a significant
effect on the stereochemical outcome, as evidenced by the
fact that nitromethane (entry 4) gave comparable results to
dichloromethane and toluene (entry 3). However, when
acetonitrile, which is similar in polarity to nitromethane, was
used as a solvent, the â/R-ratios improved to 5:1 (chloride
6) and 4:1 (bromide 7) (entries 5 and 6). It has previously
been observed that nitriles favor the â-anomer in many
glycosylation reactions, perhaps because nitrile-containing
solvents participate in the reaction, forming an R-nitrilium
ion so that attack from the â-face is favored.13 On the basis
of these model studies, we selected anomeric chlorides as
our donors of choice for further work both because they gave
better â-selectivities and were easier to generate and handle
than the corresponding anomeric bromides.
Having established favorable donor and solvent conditions,
we next examined the use of different phosphate nucleo-
philes.14 Obviously, the most straightforward approach to
sugar nucleotides would be to couple the anomeric chloride
directly to TDP. However, when chloride 6 was coupled with
the tetrabutylammonium salt of TDP, the corresponding
protected TDP sugar 9 could be isolated, but a decrease in
â-selectivity from 5:1 to 2:1 was observed (Table 1, entry
7). Therefore, we decided that better stereochemical control
could be achieved by coupling the glycosyl phosphate to an
activated nucleotide monosphosphate rather than attempting
to install the NDP group in a single step. Under optimized
conditions, coupling of chloride 6 with Bu4NH2PO4 in
acetonitrile gave phosphate 8 (â/R ) 5:1) in 58% yield.15
To explore the utility of the conditions established above,
we prepared the R-chloro derivatives of the five â-2-deoxy
sugars shown in Scheme 3 (see Supporting Information).
Compounds 10 and 12 are precursors of sugar moieties found
in the anthracycline group of antibiotics, e.g., daunomycin
and aclacinomycin A, while 14 is part of tetrocarcin A.
Chlorides 16 and 18 are precursors of epi-vancosamine and
vancosamine, respectively, which are constituents of the
glycopeptide antibiotics chloroeremomycin and vancomycin,
respectively. Each of these 2-deoxy glycosyl chlorides were
subjected to phosphorylation, and the â-phosphate was the
predominant product in all cases, with â/R-ratios ranging
from 5:1 to 9:1.16 Hence, the method reliably produces the
desired â-anomer for a range of 2-deoxy sugars with high
selectivity.
Phosphates 8 (L-acosamine), 11 (L-daunosamine), 13 (2-
deoxy L-fucose), 17 (L-vancosamine), and 19 (L-epi-van-
cosamine) were prepared on a larger scale and converted to
the corresponding TDP derivatives by coupling with TMP
morpholidate5 using the conditions developed by Wong
(Scheme 4).17 The undesired R-isomers could usually be
removed at this stage by careful separation using preparative
reversed-phase HPLC. Deprotection and, for azido-containing
(15) Synthesis of L-Acosaminyl Phosphate 8. To a solution of lactol 5
(216 mg, 1.0 mmol) in CH2Cl2 (10 mL) was added freshly activated
molecular sieves (3 Å, 1.0 g), and the mixture was stirred at room
temperature for 30 min under argon. Then, a catalytic amount of DMF and
oxalyl chloride (2 M in CH2Cl2, 0.6 mL, 1.2 mmol) was added, and stirring
was continued for 1 h. The mixture was filtered, evaporated without heating,
and coevaporated with toluene (three times). The crude glycosyl chloride
was dissolved in CH3CN (10 mL) under argon, and the pH was adjusted to
9 by the dropwise addition of DIPEA. After the addition of freshly activated
molecular sieves (3 Å, 1.0 g), the mixture was cooled (ice bath), and Bu4-
NH2PO4 (0.4 M in CH3CN, 5.0 mL, 2 mmol) was added. The mixture was
stirred for 3 h while warming to room temperature and then filtered and
evaporated. The residue was purified by reversed-phase HPLC (5 min with
H2O/0.1% NH4HCO3 and then a linear gradient to 100% MeOH/0.1% NH4-
HCO3 over 50 min; flow rate ) 45 mL/min). Evaporation of the product-
containing fractions (Rf ) 0.30, CH2Cl2/MeOH/H2O/Et3N 8:4:0.2:0.3) gave
the ammonium salt of 8 as a white powder upon lyophilization (180 mg,
58%, â/R 5:1, based on 1H NMR). 1H NMR (500 MHz, CD3OD): δ 5.55
(m, 1 H, 1-HR), 5.21 (ddd, J1,P ) 8.0, J1,2a ) 2.2, J1,2b ) 9.6 Hz, 1 H,
1-Hâ). For complete NMR data, see Supporting Information.
(13) Schmidt, R. R.; Behrendt, M.; Toepfer, A. Synlett 1990, 694-696.
(14) When protected phosphates, e.g., dibenzyl phosphate, were used in
the coupling reaction, the products proved to very labile and difficult to
purify. Consistent with this observation, phosphoramidite coupling of lactol
6 ((1) iPr2NP(OBn)2, tetrazole; (2) mCPBA) did not generate the dibenzyl
phosphate.
(16) When a daunosaminyl chloride carrying a 3-NHCbz group instead
of the 3-N3 function was used in the coupling reaction, a decrease in
â-selectivity was observed. Therefore, azido derivatives were used for all
nitrogen-containing 2-deoxy sugars.
(17) Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144-2147.
Org. Lett., Vol. 6, No. 17, 2004
2875

In summary, we have developed a practical chemical
method for obtaining the â-anomers of TDP 2-deoxy sugar
donors stereoselectively. This method reliably provides
access to substrates essential for studying a variety of
glycosyltransferases, including Gtfs involved in anthra-
cycline18 and glycopeptide biosynthesis.19 Access to epimers
and other derivatives of these glycosyl donor substrates will
permit further characterization of the specificity of these Gtfs.
More importantly, perhaps, our work will allow chemoen-
zymatic methods to be more thoroughly explored for
generating novel sugar-containing natural products.
Scheme 4. Synthesis of TDP 2-Deoxy Sugars 20-24a
Acknowledgment. This work was supported by National
Institutes of Health Grants 66174 and 69721 (to D.K.)
Supporting Information Available: Experimental pro-
cedures and spectral characterization of phosphates 11, 13,
15, 17, and 19 and TDP sugars 20-24. This material is
available free of charge via the Internet at http://pubs.acs.org.
a Reaction conditions for 21-24: (1) TMP morpholidate, tet-
razole, pyridine. (2) HPLC separation. (3) H2/Pd-C, MeOH. (4)
MeOH/H2O/Et3N or NaOMe/MeOH.
OL049187F
(18) Lu, W.; Leimkuhler, C.; Oberthu¨r, M.; Kahne, D.; Walsh, C. T.
Biochemistry 2004, 43, 4548-4558.
(19) Lu, W.; Oberthu¨r, M.; Leimkuhler, C.; Tao, J.; Kahne, D.; Walsh,
C. T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4390-4395.
compounds, reduction thereafter produced the 2-deoxy
glycosyl TDP donors 20-24 (see Supporting Information
and Scheme 4).
2876
Org. Lett., Vol. 6, No. 17, 2004