Synthesis of a deoxysugar dinucleotide containing an exo-difluoromethylene moiety as a mechanistic probe for studying enzymes involved in unusual sugar biosynthesis

Full text of DOI:10.1021/jo0103672

6810
J . Org. Chem. 2001, 66, 6810-6815
terized an important enzyme involved in the biosynthesis
Syn th esis of a Deoxysu ga r Din u cleotid e
of yersiniose A,11 which is an immunodominant branched-
chain sugar found in the O-antigen of Yersinia pseudo-
tuberculosis VI. This enzyme, YerE, is a thiamine pyro-
phosphate (TPP) dependent flavoenzyme that catalyzes
the attachment of the branched-chain to a 3,6-dideoxy-
4-hexulose precursor 3 to give 6 using pyruvate 2 as the
side chain donor. As illustrated in Scheme 1, the reaction
is initiated by the addition of TPP to pyruvate 2, followed
by the decarboxylation of the initial adduct to generate
a nucleophilic carbanionic species 4. This two-carbon acyl
anion equivalent will then be used to attack the 4-keto
group of 3 to give 5. The subsequent breakdown of the
coupling adduct 5 produces 6 and regenerates the TPP
coenzyme. The last step of yersiniose A biosynthesis is
the reduction of 6 by an NADPH-dependent reductase,
YerF, to yield CDP-yersiniose A (1). Since the activity of
YerE was not affected by the presence of the reducing
agent dithionite, the enzyme bound FAD is unlikely to
play a redox role in this transformation.11
In an attempt to develop methods of controlling this
enzymatic transformation, which is essential to the
biosynthesis of yersiniose, we prepared substrate ana-
logues that, upon incubation with YerE, would lead to
either inhibition or turnover, depending on the mode of
catalysis. In view of the unique chemical, as well as steric,
properties of the difluoromethylene group and the mecha-
nistic insights gained from our early studies on the
catalysis of YerE, we chose to pursue the synthesis of an
exo-difluoromethylene containing analogue 7 as a poten-
tial inhibitor. If this compound is recognized by YerE,
its difluoromethylene moiety may react with the nucleo-
philic acyl anion equivalent 4 at the active site to
generate 8. As illustrated in Scheme 2, the breakdown
of this adduct may occur via two distinct routes. Pathway
A is the same as that observed in normal catalysis.
Carbon-carbon bond scission via this route resulted in
a doubly branched-chain sugar 9 as the new product,
together with the regeneration of TPP coenzyme after
each catalytic cycle. In contrast, cleavage by route B gives
a new product 10, accompanied by the release of a
fluoride anion. Since the TPP coenzyme is acetylated
after each turnover, the enzyme is expected to be
inactivated. This modified TPP coenzyme 11 may also
function as an acylating agent to label an active-site
nucleophile. Therefore, even the TPP coenzyme may be
regenerated at the end, the covalently modified enzyme
12 may still be impaired.
Con ta in in g a n exo-Diflu or om eth ylen e
Moiety As a Mech a n istic P r obe for
Stu d yin g En zym es In volved in Un u su a l
Su ga r Biosyn th esis
Zongbao Zhao and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and
Department of Chemistry and Biochemistry,
University of Texas, Austin, Texas 78712
h.w.liu@mail.utexas.edu
Received April 9, 2001
In tr od u ction
Fluorine-containing compounds, while few are natu-
rally occurring,1 have attracted much attention due to
their potential as enzyme inhibitors/chemotherapeutic
agents.2 It has been well documented that when fluorine-
(s) are introduced at appropriate loci, the chemical
properties as well as the biological activities of the
resulting compounds could be significantly altered.3 In
view of their potential applications, a diverse array of
fluorine-containing molecules have been prepared and
their activities evaluated. Particularly notable are com-
pounds carrying a difluoromethylene moiety which can
act as a bioisostere for aldehydes and ketones.4 This
functional group is chemically reactive under both ionic5
and radical reaction conditions6 and has been incorpo-
rated in the structure of a few mechanism-based inhibi-
tors7 of enzymes including squalene epoxidase,7a oxido-
squalene cyclase,7b and monoamine oxidase.7c In addition,
a number of difluoromethylene-containing compounds
have been shown to possess antiviral8 and/or antifungal
activities.9
In an effort to explore nature’s strategy for making
unusual sugars,10 we have recently isolated and charac-
* Corresponding author. Fax: 1-512-471-2746.
(1) O’Hagan, D.; Harper, D. B. J . Fluorine Chem. 1999, 100, 127.
(2) (a) Filler, R.; Kirk, K. In Chemistry of Organic Fluorine Com-
pounds II; Hudlicky, M., Pavlath, E., Eds.; American Chemical
Society: Washington, DC, 1995; p 1011. (b) Ternansky, R. J .; Hertel,
L. W. Organofluorine Compounds in Medicinal Chemistry and Bio-
medical Applications; Filler, R., Kobayashi, Y., Yagupolski, L. M., Eds.;
Elsevier: Amsterdam, 1993; p 23. (c) Smart, B. E. In Chemistry of
Organic Fluorine Compounds II; Hudlicky, M., Pavlath, A. E., Eds.;
American Chemical Society: Washington, DC, 1995; pp 979-1010.
(3) Welch, J . T.; Eswarakrishnan, S. Fluorine in Bioorganic Chem-
istry; J ohn Wiley & Sons: New York, 1990.
(4) Motherwell, W. B.; Tozer, M. J .; Ross, B. C. J . Chem. Soc., Chem.
Commun. 1989, 1487.
(5) (a) Huang, X.-h.; He, P.-y.; Shi, G.-q. J . Org. Chem. 2000, 65,
627. (b) Hayashi, S.; Nakai, T.; Ishikawa, N.; Burton, D. J .; Naae, D.
G.; Kesling, H. S. Chem. Lett. 1979, 983.
It should be noted that nucleophilic addition to 1,1-
difluoroolefins usually occurs at the terminal difluoro-
methylene carbon.12 Therefore, if the acceptor 7 binds in
the active-site in a manner that it is accessible to the
(6) (a) Piettre, S. R. Tetrahedron Lett. 1996, 37, 2233. (b) Piettre,
S. R. Tetrahedron Lett. 1996, 37, 4707. (c) Herpin, T. F.; Motherwell,
W. B.; Roberts, B. P.; Roland, S.; Weibel, J .-M. Tetrahedron 1997, 53,
15085.
(7) (a) Bey, P.; McCarthy, J . R.; McDonald, I. A. ACS Symp. Ser.
1991, 456, 105 and references cited therein. (b) Moore, W. R.;
Schatzman, G. L.; J arvi, E. T.; Gross, R. S.; McCarthy, J . R. J . Am.
Chem. Soc. 1992, 114, 360. (c) Madden, B. A.; Prestiwich, G. D. Bioorg.
Med. Chem. Lett. 1997, 7, 309. (d) McDonald, I. A.; Lacoste, J . M.; Bey,
P.; Palfreyman, M. G.; Zreika, M. J . Med. Chem. 1985, 28, 186.
(8) Chung, S.-K.; Ryoo, C. H.; Yang, H. W.; Shim, J .-Y.; Kang, M.
G.; Lee, K. W.; Kang, H. I. Tetrahedron 1998, 54, 15899.
(9) Serafinowski, P. J .; Barnes, C. L. Synthesis 1997, 225.
(10) (a) Liu, H.-w.; Thorson, J . S. Annu. Rev. Microbiol. 1994, 48,
223. (b) Kirschning, A.; Bechthold, A. F.-W.; Rohr, J . In Bioorganic
Chemistry Deoxysugars, Polyketides & Related Classes: Synthesis,
Biosynthesis, Enzymes; Rohr, J ., Ed.; Springer: Berlin, 1997; p 1. (c)
J ohnson, D. A.; Liu, H.-w. In Comprehesive Chemistry of Natural
Products; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon:
New York, 1999; Vol. 3, p 311. (d) Hallis, T. M.; Liu, H.-w. Acc. Chem.
Res. 1999, 32, 579. (e) He, X.; Agnihotri, G.; Liu, H.-w. Chem. Rev.
2000, 100, 4615.
(11) Chen, H.; Guo, Z.; Liu, H.-w. J . Am. Chem. Soc. 1998, 120,
11796.
(12) Chambers, R. D.; Mobbs, R. H. Adv. Fluorine Chem. 1965, 4,
50.
10.1021/jo0103672 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/31/2001

Notes
J . Org. Chem., Vol. 66, No. 20, 2001 6811
Sch em e 1
Sch em e 2
Sch em e 3
nucleophile 4, the chemical reactivity of the difluoro-
methylene moiety may direct the acyl anion equivalent
4 to attack its terminal carbon as shown in Scheme 3.
The anion intermediate 13 may be protonated to give 14,
or it may undergo elimination to yield 15. In either case,
the enzyme is expected to be inactivated. Even if 15 can
be further converted to 16 to generate TPP coenzyme,
the resulting product 16 is a potential Michael acceptor
that may trap an active site nucleophile to give 17,
leading to enzyme inactivation. Clearly, compound 7

6812 J . Org. Chem., Vol. 66, No. 20, 2001
Notes
Sch em e 4
holds promise as a mechanism-based inhibitor. In this
paper, we report an efficient synthesis of this nucleotide
sugar and the incubation results with the target enzyme
YerE.
Methyl pyranosides have been frequently used as
starting material for carbohydrate synthesis; however,
difficulties are often encountered when trying to remove
the anomeric methoxy group after chemically sensitive
functionalities are introduced in the target molecules.
Indeed, complication was observed in our attempt to
eliminate the C-1 methoxy moiety of 23 while keeping
both the ester at C-2 and the difluoroolefin group at C-4
intact. Due to these complications, a three-step sequence
was developed to convert 23 to its C-1 acetate derivative.
As shown in Scheme 4, the pivaloyl ester of 23 was
hydrolyzed first and the resulting product 24 was treated
with an acidic resin in a mixture of acetonitrile and
water. Then, acylation under basic condition produced
25 as a mixture of two anomeric isomers. Direct acetolysis
occurred by treating 24 with acetic anhydride in the
presence of catalytic amount of either sulfuric acid or
triflic acid; however, this led to a defluorinated mixture
as shown by 19F NMR. It is worth mentioning that when
a benzyl ether was used instead of pivaloyl ester to
protect the C-2 hydroxy group in 20, it could not be
removed without destroying the difluoromethylene func-
tionality.18a
Resu lts a n d Discu ssion
To test our idea, we have developed a convenient
strategy to synthesize the desired molecule. The first
phase of our synthesis called for the construction of
4-keto-3,6-dideoxyhexose 22. As depicted in Scheme 4, a
two-step procedure that was originally reported by Clas-
son et al.13 was used to make the 3,6-dideoxysugar
precursor 20 from methyl R-D-glucopyranoside 18. Fresh
2,3,5-tribromoimidozole14 is essential to obtain the di-
bromide intermediate 19 in good yield. The C-2 hydroxyl
group of 20 was then selectively acylated with pivaloyl
chloride.15 The C-4 hydroxy group of 21 was oxidized by
PCC to give the 4-keto-sugar 22. The incorporation of an
exo-difluoromethylene moiety into 22 was accomplished
using a Wittig-type reagent generated in situ with
hexamethylphosphorus triamide (HMPT) and dibromo-
difluoromethane.16 In this experiment, HMPT was added
dropwise to the solution of 22 and CF2Br2 in dry THF at
0 °C, followed by warming the reaction mixture to room
temperature. This modified procedure not only gave a
very clean reaction with high yield but also alleviated
the requirement of zinc metal in the reaction.17
Problems were also encountered during the deprotec-
tion of C-1 hydroxyl in 25. Commonly used reagents such
as hydrazine, hydrazine acetate, and benzylamine pro-
duced complicated results. Although the products were
not well characterized, in most cases, the double bond
seemed to have migrated into the pyranose ring upon
treatment with these organic bases. Fortunately, this
problem could be overcome by heating 25 with tributyltin
methoxide in methylene dichloride.19 The subsequent
(13) Classon, B.; Garegg, P. J .; Samuelsson, B. Can. J . Chem. 1981,
59, 339.
(14) Balaban, P. J . Chem. Soc. 1922, 121, 949.
(15) J iang, L.; Chan, T.-H. J . Org. Chem. 1998, 63, 6035.
(16) Vinson, W. A.; Prickett, K. S.; Spahic, B.; de Montellano, P. R.
O. J . Org. Chem. 1983, 48, 4661.
(17) (a) Serafinowski, P. J .; Barnes, C. L. Tetrahedron 1996, 52,
7929. (b) Houlton, J . S.; Motherwell, W. B.; Ross, B. C.; Tozer, M. J .;
Williams, D. J .; Slawin, A. M. Z. Tetrahedron 1993, 49, 8087.
(18) (a) Hallis, T. M.; Zhao, Z.; Liu, H.-w. J . Am. Chem. Soc. 2000,
122, 10493. (b) Chen, H.; Zhao, Z.; Hallis, T. M.; Guo, Z.; Liu, H.-w.
Angew. Chem. Int. Ed. 2001, 40, 607.
(19) Gurjar, M. K.; Mainkar, A. S. Tetrahedron 1992, 48, 6729.

Notes
J . Org. Chem., Vol. 66, No. 20, 2001 6813
solution of phosphomolybdic acid (3% in EtOH). Dry THF was
distilled over sodium and benzophenone.
conversion of 26 to 27 was initially attempted by treat-
ment of 26 with dibenzyl N,N-diethylphosphoramidite,
followed by an oxidation step.20 However, this two-step
procedure via a phosphite intermediate gave very poor
yield along with troubles in column purification. The
transformation, however, could be readily achieved using
dibenzyl phosphochloridate as the phosphatylation re-
agent.21 This procedure has proven to be very dependable
in our hands for the synthesis of diphosphonucleotidyl
sugars in this laboratory.18b J udging from the coupling
P r ep a r a tion of 2,3,5-Tr ibr om oim id a zole. To a stirred
solution of imidazole (68 g, 1.0 mol) in chloroform (500 mL) was
added dropwise Br2 (51.3 mL, 1.0 mmol) at room temperature.
The resulting mixture was stirred for 1 h. The yellowish solid
was collected by filtration, washed thoroughly with water, and
then recrystallized from 95% ethanol. The desired tribromide14
was isolated as a yellow solid (56 g, 55%).
Meth yl 3,6-Dibr om o-3,6-d id eoxy-r-D-a llo-h exop yr a n o-
sid e (19). A mixture of methyl R-D-glucoside 18 (22 g, 0.11 mol),
PPh3 (130 g, 0.49 mol), and tribromoimidazole (75 g, 0.24 mol)
in toluene (1 L) was heated at 85 °C for 1 h and at 120 °C for 5
h. The mixture was cooled to room temperature and mixed with
saturated sodium bicarbonate solution (600 mL). After the
mixture was stirred for 10 min, I2 was introduced in small
increments with vigorous stirring until the solution turned
purple. Excess I2 was quenched with aqueous sodium thiosulfate
solution. The organic layer was separated, and the aqueous layer
was extracted with ether. The combined organic extracts were
washed with brine, dried over sodium sulfate, and concentrated
in vacuo. The solid residue was redissolved in a minimum
amount of hot ethyl acetate. After cooling of the mixture to room
temperature, the precipitate was filtered off and the filtrate was
concentrated. The crude product was purified by silica gel
chromatography (hexanes:ethyl acetate, 3:1) to give the 3,6-
dibromosugar 1913 (20.5 g, 56%) as a white solid.
Meth yl 3,6-Did eoxy-r-D-r ibo-h exop yr a n osid e (20). A sus-
pension of the 3,6-dibromosugar 19 (20.4 g, 63 mmol), tributyltin
hydride (42 g, 144 mmol), and AIBN (0.5 g, 3 mmol) in benzene
(700 mL) was refluxed for 9 h under a nitrogen atmosphere. The
solvent was removed in vacuo, and the residue was chromato-
graphed on silica gel (hexanes:ethyl acetate, 2:1 to 1:2) to afford
the 3,6-dideoxysugar 2013 (10.0 g, 96%) as a colorless oil. 1H NMR
(CDCl3): δ 4.58 (1H, d, J ) 3.6 Hz), 3.70 (1H, m), 3.46 (1H, m),
3.43 (3H, s), 3.26 (1H, m), 2.18 (1H, m), 2.10 (1H, br s), 1.80
(1H, br s), 1.63 (1H, m), 1.25 (3H, d, J ) 6.9 Hz). 13C NMR
(CDCl3): δ 98.3, 70.8, 68.7, 67.6, 55.1, 37.0, 17.4.
Meth yl 3,6-Did eoxy-2-O-p iva loyl-r-D-r ibo-h exop yr a n o-
sid e (21). To a solution of 20 (4.8 g, 30 mmol) in pyridine (50
mL) was added portionwise an excess of pivaloyl chloride (12
mL) at 0 °C. The mixture was stirred for 2 h at the same
temperature and then diluted with ethyl acetate. The resulting
solution was washed successively with 5% HCl, brine, saturated
sodium bicarbonate solution, and brine and dried over sodium
sulfate. The solvents were removed in vacuo, and the residue
was purified by silica gel chromatography (hexanes:ethyl acetate,
5:1) to give the pivalate 21 (6.7 g, 91%) as a colorless oil. 1H
NMR (CDCl3): δ 4.73 (1H, m), 4.66 (1H, m), 3.52 (1H, m), 3.34
(3H, s), 3.30 (1H, m), 2.50 (1H, br s), 2.05 (1H, m), 1.82 (1H, m),
1.21 (3H, d, J ) 6.0 Hz), 1.14 (9H, s). 13C NMR (CDCl3): δ 178.1,
96.1, 70.7, 68.9, 68.5, 55.1, 38.7, 32.5, 27.0, 17.4. HRMS (CI):
calcd for C12H23O5 [M + H]+, 247.1545; found, 247.1538.
Meth yl 3,6-Did eoxy-2-O-p iva loyl-r-D-er yth r o-h exop yr a -
n osid -4-u lose (22). A mixture of pivalate 21 (0.58 g, 2.3 mmol),
sodium acetate (0.39 g, 4.7 mmol), and PCC (2.06 g, 9.6 mmol)
in dry CH2Cl2 (25 mL) was vigorously stirred at room temper-
ature for 12 h. The resulting dark-brown mixture was filtered
through a short silica gel column and washed with ether. The
filtrate was collected and concentrated in vacuo. The residue was
chromatographed on silica gel (hexanes:ethyl acetate, 5:1) to
afford the keto sugar 22 (0.47 g, 81%) as a colorless oil. 1H NMR
(CDCl3): δ 5.11 (1H, ddd, J ) 9.3, 6.9, 3.6 Hz), 4.93 (1H, d, J )
3.6 Hz), 4.16 (1H, q, J ) 6.6 Hz), 3.46 (3H, s), 2.72 (1H, d, J )
6.9 Hz), 2.71 (1H, d, J ) 9.3 Hz), 1.28 (3H, d, J ) 6.6 Hz), 1.18
(9H, s). 13C NMR (CDCl3): δ 205.8, 177.5, 96.3, 70.4, 68.3, 55.9,
39.8, 38.7, 27.0, 14.8. HRMS (CI): calcd for C12H21O5 [M + H]+,
245.1389; found, 245.1347.
3
3
constants of 3.3 Hz for J H-H and 6.3 Hz for J H-P
associated with the 1-H signal, product 27 clearly has
the desired a configuration at C-1.
Compound 27 was then hydrogenolyzed and treated
with 2 N lithium hydroxide to give 28. The reaction
sequence and conditions for the deprotection of 27 were
very critical to produce 28 in good yield with high purity.
Hydrogenolysis of the O-benzyl group of 27 should be
performed for less than 10 min to prevent over reduction
of the difluoromethylene double bond. Furthermore,
alkaline hydrolysis to remove the acetyl group should be
carried out after hydrogenation; this avoided deletion of
the dibenzyl phosphate functionality. The final step of
this synthesis was the 1H-tetrazole-promoted coupling
of 28 with cytidine 5′-monophosphomorpholidate.22 The
desired product 7 was isolated by Bio-Gel P2 column
eluted with 50 mM ammonium bicarbonate and charac-
terized by NMR and MS.
Unfortunately, incubation of YerE with compound 7
showed no apparent effect on the enzyme activity. 19F
NMR analysis also failed to detect the release of fluoride
anion during incubation. In addition, no new product was
discernible in the incubation mixture by HPLC analysis.
It is thus clear that 7 is neither an inhibitor nor a
substrate for YerE. This result is disappointing; however,
it only indicates that the active site of YerE is less flexible
to accommodate this specific compound. Since many
enzymes are known to be promiscuous with respect to
substrate recognition, the strategy discussed in this paper
may have general applicability to the design of probes to
further the study of the mechanisms of related enzymatic
catalyses. The chemistry described here should also be
useful for the synthesis of compounds, especially modified
sugar dinucleotides, containing an exo-difluoromethylene
functionality.
Exp er im en ta l Section
All chemicals were products of Aldrich Co. (Milwaukee, WI),
unless otherwise specified. NMR spectra (1H at 300 MHz, 13C
at 75 MHz, 19F at 282 MHz, and 31P at 121 MHz) were recorded
with a Varian U-300 Spectrometer in CDCl3, unless otherwise
specified in the text. Chemical shifts (δ in ppm) are given relative
to those for Me4Si (for 1H), CDCl3 (for 13C), external CFCl3 (for
19F), and external aqueous 85% H3PO4 (for 31P). High-resolution
mass spectroscopy was performed on a Finnigan Mat 95 (for HR-
CI and HR-EI) or a VG Analytical 7070E-HF (for FAB) instru-
ment. Flash chromatography was performed on Lagand Chemi-
cal silica gel (230-400 mesh) by elution with the specified
solvents. Analytical thin-layer chromatography (TLC) was car-
ried out on Polygram Sil G/UV254 plates (0.25 mm). Bio-Gel P2
resin (extra fine) for size-exclusion chromatography was pur-
chased from Bio-Rad Laboratories (Hercules, CA). TLC spots
were visualized by heating the plate previously stained with a
Meth yl 3,4,6-Tr id eoxy-4-C-d iflu or om eth ylen e-2-O-p iv-
a loyl-r-D-er yth r o-h exop yr a n osid e (23). To a solution of keto
sugar 22 (2.56 g, 10.4 mmol) and dibromodifluoromethane (2.9
mL, 31 mmol) in dry THF (70 mL) was added dropwise HMPT
(7.8 mL, 41 mmol) at 0 °C. After being vigorously stirred at room
temperature for 2 h, the mixture was diluted with water and
extracted with ethyl acetate. The organic extracts were dried
over sodium sulfate and concentrated in vacuo. The residual oil
was chromatographed on silica gel with hexanes to afford the
(20) Sim, M. M.; Kondo, H.; Wong, C.-H. J . Am. Chem. Soc. 1993,
115, 2260.
(21) Macher, I. Carbohydr. Res. 1987, 162, 79.
(22) Wittmann, V.; Wong, C.-H. J . Org. Chem. 1997, 62, 2144.

6814 J . Org. Chem., Vol. 66, No. 20, 2001
Notes
difluoro sugar 23 (2.51 g, 86%) as a colorless oil. 1H NMR
(CDCl3): δ 4.78 (1H, m), 4.75 (1H, m), 4.41 (1H, m), 3.40 (3H,
s), 2.51 (1H, m), 2.38 (1H, m), 1.40 (3H, dd, J ) 6.6, 5.4 Hz),
1.19 (9H, s). 13C NMR (CDCl3): δ 177.7, 151.6 (dd, J ) 289.2,
284.4 Hz), 96.8, 86.5 (dd, J ) 18.5, 15.4 Hz), 68.7, 62.1 (t, J )
4.2 Hz), 55.4, 38.6, 26.9, 23.3 (d, J ) 2.6 Hz), 18.2 (d, J ) 7.4
Hz). 19F NMR (CDCl3): δ -99.1 (1F, d, J ) 54.2 Hz), -101.7
(1F, dd, J ) 54.2, 2.8 Hz). HRMS (CI): calcd for C13H21F2O5
[M + H]+, 279.1408; found, 279.1412.
(d, J ) 5.3 Hz), 85.4 (dd, J ) 19.5, 14.2 Hz), 69.3 (d, J ) 4.8
Hz), 69.2 (d, J ) 4.2 Hz), 68.2 (m), 64.5 (t, J ) 4.8 Hz), 22.8,
20.5, 18.0 (d, J ) 7.9 Hz). 19F NMR (CDCl3): δ -97.6 (1F, d, J
) 49.7 Hz), -99.9 (1F, dd, J ) 49.7, 5.4 Hz). 31P NMR (CDCl3):
δ -1.7. HRMS (FAB) calcd for C23H26F2PO7 [M + H]+, 483.1378;
found, 483.1381.
3,4,6-Tr id eoxy-4-C-d iflu or om eth ylen e-r-D-er yth r o-h exo-
p yr a n osyl P h osp h a te (28). A suspension of compound 27 (131
mg, 0.27 mmol) and 10% Pd/C catalyst (45 mg) in a mixture of
methanol and ethyl acetate solution (1:1, 10 mL) was hydroge-
nated with a hydrogen balloon at room temperature for 8 min.
The mixture was filtered through Celite. The filtrate was mixed
with 3 drops of triethylamine and concentrated under reduced
pressure to give a colorless oil. The oil was dissolved in 2 N LiOH
solution (2 mL) and stirred for 11 h at room temperature. The
mixture was then diluted with water (4 mL) and stirred with
Dowex 50WX-200, a strongly acidic cation resin (H+ type, ∼ .5
g), for 30 min. The resin was filtered off, and the aqueous
solution was neutralized with triethylamine. Lyophilization of
the solution gave the coupling precursor 28 (64 mg, 62%) as a
white powder. 19F NMR (D2O): δ -100.4 (1F, d, J ) 55.0 Hz),
-104.5 (1F, d, J ) 55.0 Hz). 31P NMR (D2O): δ 0.8. HRMS
(FAB): calcd for C7H10F2PO6 [M - H]-, 259.0182; found,
259.0175.
Meth yl 4-C-Diflu or om eth ylen e-3,4,6-tr ideoxy-r-D-er yth r o-
h exop yr a n osid e (24). A solution of compound 23 (2.48 g, 8.9
mmol) in methanol (60 mL) containing potassium hydroxide
(1.75 g, 31 mmol) was stirred at room temperature for 2 h, then
filtered through a short silica gel column, and washed with THF.
The filtrates were concentrated, and the residue was chromato-
graphed on silica gel (hexanes:ethyl acetate, 5:1 to 2:1) to afford
1
24 (1.8 g, 93%) as a colorless oil. H NMR (CDCl3): δ 4.69 (1H,
d, J ) 3.3 Hz), 4.39 (1H, m), 3.74 (1H, m), 3.47 (3H, s), 2.57
(1H, dd, J ) 13.5, 5.4 Hz), 2.21 (1H, m), 2.06 (1H, br s), 1.41
(3H, dd, J ) 6.9, 5.1 Hz). 13C NMR (CDCl3): δ 151.5 (dd, J )
289.7, 284.4 Hz), 98.7, 86.4 (dd, J ) 19.1, 14.2 Hz), 67.3, 62.6
(dd, J ) 4.7, 3.7 Hz), 55.4, 27.2, 18.0 (d, J ) 7.4 Hz). 19F NMR
(CDCl3): δ -89.7 (1F, d, J ) 54.1 Hz), -92.4 (1F, dd, J ) 54.1,
6.2 Hz). HRMS (CI): calcd for C8H16NF2O3 [M + NH4]+,
212.1098; found, 212.1090.
Cytid in e 5′-(3,4,6-Tr id eoxy-4-C-d iflu or om eth ylen e-r-D-
er yth r o-h exop yr a n osyl d ip h osp h a t e) (7). A suspension of
precursor 28 (64 mg, ∼0.17 mmol) and cytidine 5′-monophos-
phomorpholidate (200 mg, 0.29 mmol) in dry pyridine (1.5 mL)
was evaporated over an oil pump to form a white powder. To
ensure the removal of any residual moisture, this procedure was
repeated three times. Dry pyridine (2.0 mL) and 1H-tetrazole
(33 mg, 0.47 mmol) were then added to the above solid. After
the mixture was stirred at room temperature for 60 h, the
solvents were removed under vacuum. The residue was purified
by Bio-Gel P2 column (2 cm × 1.1 m), eluted with 50 mM
ammonium bicarbonate. The desired fractions, as indicated by
their UV absorption at 267 nm, were collected and lyophilized
to give the desired product 7 (45 mg, 45%) as a white powder.
1H NMR (D2O): δ 7.81 (1H, d, J ) 7.2 Hz), 5.95 (1H, d, J ) 7.2
Hz), 5.79 (1H, d, J ) 3.6 Hz), 5.35 (1H, dd, J ) 7.5, 2.7 Hz),
4.54 (1H, m), 4.0-4.2 (5H, m), 3.66 (1H, m), 2.39 (1H, m), 2.19
(1H, m), 1.22 (3H, dd, J ) 6.9, 3.9 Hz). 13C NMR (D2O): δ 164.2,
155.2, 151.5, 142.0, 96.1, 94.2, 94.1, 89.4, 85.1 (dd, J ) 19.8,
15.8 Hz), 82.6 (d, J ) 9.0 Hz), 74.3, 68.9, 66.5, 65.3, 64.3 (d, J )
5.3 Hz), 25.0, 17.0 (d, J ) 6.6 Hz). 19F NMR (D2O): δ -99.7 (1F,
d, J ) 54.2 Hz), -103.3 (1F, d, J ) 52.8 Hz). 31P NMR (D2O): δ
-10.7 (1P, d, J ) 21.4 Hz), -12.4 (1P, d, J ) 20.8 Hz). HRMS
(FAB): calcd for C16H22F2N3O13P2 [M - H]-, 564.0604; found,
564.0585.
P u r ifica tion of Yer E.23 The yerE gene was amplified from
Yersinia pseudotuberculosis VI by polymerase chain reaction
(PCR) and cloned into a pET24(+) vector. After transformation
into Escherichia coli DH5R, the positive clone was isolated and
the corresponding plasmid DNA, pHC32, was transformed into
E. coli BL21(DE-3). The BL21(DE-3)-pHC32 cells were grown
in Terrific Broth medium supplemented with kanamycin (30 µg/
mL) at 37 °C until the OD600 reached 0.6. The culture was then
induced with isopropyl-thio-â-galactoside (IPTG, 0.1 mM), and
grew at 24 °C for an additional 15 h. The cells were harvested
by centrifugation at 6500g for 5 min at 4 °C, resuspended in
lysis buffer (50 mM sodium phosphate, 150 mM NaCl, 10 mM
imidazole, pH 8.0), and purified by Ni-NTA affinity chroma-
tography. The YerE protein was further purified by FPLC-
MonoQ chromatography with a linear gradient of 100-500 mM
NaCl in 20 mM Tris-HCl buffer, pH 7.5. The collected fractions
were concentrated by an Amicon concentrator using a YM-10
membrane.
2-O-Acet yl-3,4,6-t r id eoxy-4-C-d iflu or om et h ylen e-r/â-D-
er yth r o-h exop yr a n osyl Aceta te (25). A suspension of com-
pound 24 (0.45 g, 2.3 mmol) and Dowex 50WX4-200 strongly
acidic cation resin (H+ type, ∼1 g) in a mixture of acetonitrile
(4 mL) and water (16 mL) was stirred at 50 °C for 12 h. The
resin was filtered off, and the filtrate was extracted with ethyl
acetate. The combined organic extracts were dried over sodium
sulfate and concentrated in vacuo. The residue was treated with
a mixture of pyridine (3.0 mL) and acetyl anhydride (3.5 mL)
for 2.5 h at 0 °C. The mixture was diluted with ethyl acetate
and washed with 5% HCl, saturated sodium bicarbonate, and
brine successively. After being dried over sodium sulfate, the
solvents were evaporated under reduced pressure. The residue
was chromatographed on silica gel (hexanes:ethyl acetate, 5:1
to 3:1) to give diacetyl sugar 25 (0.37 g, 60%) as a colorless oil,
which was
a
mixture of two anomeric isomers. 1H NMR
(CDCl3): δ 6.17 (0.4H, d, J ) 3.3 Hz), 5.87 (0.6H, d, J ) 3.3
Hz), 4.95 (0.4H, ddd, J ) 8.4, 5.1, 3.3 Hz), 4.67 (0.6H, m), 4.63
(0.6H, m), 4.54 (0.4H, m), 2.66 (1H, m), 2.42 (1H, m), 2.11 (1.2H,
s), 2.09 (1.8H, s), 2.07 (1.8H, s), 2.03 (1.2H, s), 1.41 (3H, m). 13C
NMR (CDCl3): δ 169.9, 169.3, 169.0, 155.7, 155.3, 151.9, 151.5,
147.7, 91.4, 89.9, 89.3, 67.2, 65.2, 23.4, 21.2, 21.0, 20.9, 20.8,
18.2, 18.1. 19F NMR (CDCl3): δ -98.5 (0.43F, dd, J ) 50.2, 2.8
Hz), -99.5 (0.57F, d, J ) 51.4 Hz), -100.9 (0.43F, dd, J ) 50.2,
2.8 Hz), -105.3 (0.57F, d, J ) 51.4 Hz). HRMS (CI): calcd for
C11H15F2O5 [M + H]+, 265.0888; found, 265.0900.
Diben zyl 2-O-Acetyl-3,4,6-tr id eoxy-4-C-d iflu or om eth yl-
en e-r-D-er yth r o-h exop yr a n osyl P h osp h a te (27). A solution
of peracetylated sugar 25 (0.36 g, 1.36 mmol) and tributyltin
methoxide (0.5 mL) in dry CH2Cl2 (10 mL) was refluxed for 37
h under a nitrogen atmosphere. The mixture was concentrated,
and the residue was chromatographed on silica gel (hexanes:
ethyl acetate, 2:1) to yield the C1-deprotected intermediate 26
(0.17 g, 63%) as a colorless oil.
The intermediate 26 (0.168 g, 0.75 mmol) was dissolved in
dry THF (10 mL) and cooled to -78 °C. n-BuLi solution (1.6 M
in hexanes, 0.5 mL) was then added dropwise to this solution.
After the mixture was stirred for 10 min at the same temper-
ature, a solution of dibenzyl phosphorochloridate (prepared from
0.27 g of dibenzyl phosphite and 0.13 g of NCS in 4 mL of
benzene)21 in dry THF (4 mL) was added. The resulting solution
was stirred at -78 °C for 1 h and then at 0 °C for 1 h. The
mixture was concentrated at room temperature, diluted with
ether, washed with water, and dried over sodium sulfate. The
solvents were removed in vacuo, and the residue was purified
by flash chromatography on silica gel (hexanes:ethyl acetate,
5:1 to 1:1) to give the desired product 27 (0.27 g, 74%) as a
In cu ba tion Resu lts. Compound 7 (2.3 mg, 4.0 µmol) was
incubated with thiamine pyrophosphate (2 µmol), pyruvate (83
µmol), MgCl2 (1 µmol), and YerE (0.25 nmol) in 100 mM
potassium phosphate buffer prepared with D2O (pD 8.0, 450 µL)
at 24 °C. Aliquots were taken at appropriate time intervals to
check enzyme activity under standard assay conditions.11,23 It
1
colorless oil. H NMR (CDCl3): δ 7.34 (10H, m), 5.79 (1H, dd, J
) 6.3, 3.3 Hz), 5.08 (4H, m), 4.84 (1H, m), 4.50 (1H, m), 2.60
(1H, dd, J ) 13.5, 5.4 Hz), 2.35 (1H, m), 1.91 (3H, s), 1.32 (3H,
dd, J ) 6.3, 6.0 Hz). 13C NMR (CDCl3): δ 189.8, 151.8 (dd, J )
291.3, 285.0 Hz), 135.6, 135.5, 128.6, 128.4, 127.8, 127.7, 94.5
(23) Chen, H. Ph.D. Thesis, University of Minnesota, Minneapolis,
MN, 1999.

Notes
J . Org. Chem., Vol. 66, No. 20, 2001 6815
was found that preincubation of YerE with 7 led to no loss of
activity as compared to the control. The incubation was also
monitored by 19F NMR to detect the possible release of fluoride
ion. Again, no new signal was discernible after incubation for
15 h. The reaction mixture was finally analyzed by HPLC
equipped with a SAX anion exchange column. No new peaks
were found.
National Institutes of Health (GM35906 and GM54346).
H.-w.L. also thanks the National Institute of General
Medical Sciences for a MERIT Award.
Su p p or tin g In for m a tion Ava ila ble: Reproductions of 1H
NMR spectra of compounds 7 and 19-28. This material is
available free of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. We thank Dr. Huawei Chen for
his help on YerE purification and incubation studies.
This work was supported in part by grants from the
J O0103672