CDP-6-deoxy-6,6-difluoro-D-glucose: A mechanism-based inhibitor for CDP- D-glucose 4,6-dehydratase [10]

Full text of DOI:10.1021/ja982198h

9698
J. Am. Chem. Soc. 1998, 120, 9698-9699
Scheme 1
CDP-6-deoxy-6,6-difluoro-D-glucose: A
Mechanism-Based Inhibitor for CDP-D-glucose
4,6-Dehydratase
Cheng-Wei T. Chang, Xuemei H. Chen, and Hung-wen Liu*
Department of Chemistry
UniVersity of Minnesota
Minneapolis, Minnesota 55455
ReceiVed June 24, 1998
CDP-D-glucose 4,6-dehydratase (Eod),1 isolated from Yersinia
pseudotuberculosis, is a homo-dimeric enzyme that catalyzes the
transformation of CDP-D-glucose (1) to CDP-6-deoxy-L-threo-
D-glycero-4-hexulose (4).2 The catalysis, shown in Scheme 1,
has been established to proceed via three discrete steps, oxidation
of CDP-D-glucose to 4-keto-glucose 2, C-5/C-6 dehydration to a
4-keto-∆5,6-glucoseen intermediate 3, and reduction at C-6 of 3
to give the final product 4.3 This intramolecular oxidation-
reduction is characteristic for an internal hydrogen transfer from
C-4 of the substrate 1 to C-6 of the resulting 4-keto-6-deoxyhexose
product 4, and the hydride carrier is an enzyme-bound NAD+.
Since NAD+ is regenerated at the end of each catalytic cycle, it
is in essence a prosthetic group, contrary to most of the other
nicotinamide dinucleotide-dependent enzymes in which NAD(P)+
functions merely as a cosubstrate.4 To develop methods to control
and/or regulate this intriguing enzymatic conversion, we decided
to prepare substrate analogues that, upon incubation with Eod,
would lead to either inhibition or turnover, depending on the mode
of catalysis. Reported herein are the synthesis and characteriza-
tion of a CDP-difluoroglucose derivative 5, which has been shown
to be the first mechanism-based inhibitor for Eod.
The designed inhibitor 5 was synthesized from methyl R-D-
glucoside (6) according to the reactions delineated in Scheme 2.
Selective tritylation of the 6-OH, followed by perbenzylation and
removal of the 6-trityl group, afforded 7 in 37% combined yield.
The exposed C-6 hydroxyl group was oxidized under Swern
conditions, and the crude product was fluorinated with diethyl-
aminosulfur trifluoride (DAST)5 to give 6-deoxy-6,6-difluoro-
glucoside 8 in nearly quantitative yield. Conversion of 8 to 9
involved acid treatment and peracetylation (74% yield). Subse-
quent treatment with hydrazine in DMF6 selectively removed the
1-O-acetyl group. Phosphorylation was then carried out in the
presence of N,N-diisopropyl dibenzylphosphamidite and 1H-
tetrazole.7 After oxidation of the resulting phosphite with
m-chloroperbenzoic acid (m-CPBA), the phosphate product 10
was purified by triethylamine-treated silica gel chromatography
Scheme 2
(ether/hexane gradient) to separate the R and â anomers (R:â )
3:1). The desired R-anomer, isolated in 31% yield (from 9), was
subjected to hydrogenation and basic hydrolysis to remove the
acetyl groups to form 11 (90% combined yield). The final step
involved coupling of 11 with cytidine 5′-monophospho-morpholi-
date in pyridine to give 5,6 which, after purification by Dowex
1X-8 (formate form) ion-exchange chromatography (NH4HCO3
gradient), was isolated in 25% yield.8
9
When compound 5 was incubated with Eod at 25 °C in 20
mM Tris‚HCl buffer (pH 7.5), time-dependent inactivation
occurred.10 The kinact of 2.4 × 10-2 min-1 and KI of 0.94 mM of
this inactivation were deduced from a plot of t1/2 versus [I]-1
.
Since extensive dialysis failed to regenerate the enzyme activity,
the observed inactivation is clearly irreversible. Considering the
fact that Eod was fully protected from inhibition by 5 (0.8 mM)
in the presence of substrate (0.8 mM), we deduced that the effect
of 5 on Eod must be active-site directed. Similar to the catalytic
situation, we could detect no apparent accumulation of NADH
spectrofluorometrically during the course of inactivation.
However, a fluorine peak at δ -119.6, which is due to the
(1) Abbreviations: CDP, cytidine 5′-diphosphate; NAD+, â-nicotinamide
adenine dinucleotide; NADH, â-nicotinamide adenine dinucleotide, reduced
form; Tris, tris(hydroxymethyl)amino-methane; GC, gas chromatography; MS,
mass spectroscopy.
(2) (a) Gonzalez-Porque´, P.; Strominger, J. L. J. Biol. Chem. 1972, 247,
6748-6756. (b) Russell, R. N.; Liu, H.-w. J. Am. Chem. Soc. 1991, 113,
7777-7778. (c) Yu, Y.; Russell, R. N.; Thorson, J. S.; Liu, L.-d.; Liu, H.-w.
J. Biol. Chem. 1992, 267, 5868-5875. (d) He, X.; Thorson, J. S.; Liu, H.-w.
Biochemistry 1996, 35, 4721-4731.
(8) Spectral data of 5: 1H NMR (D2O) δ 7.71 (1 H, d, J ) 7.5 Hz, cytidine
H-6), 5.97 (1H, t, JHF ) 52.6, 6-H), 5.93 (1 H, d, J ) 7.5, cytidine H-5), 5.79
(1 H, d, J ) 3.0, cytidine H- 1′), 5.44 (1H, dd, JHP ) 6.9, JHH ) 3.5, H-1),
3.8-3.4 (6 H, m, cytidine H-2′, 3′, 4′, 5′), 3.60 (1 H, t, J ) 9.4, H-3), 3.45
(1 H, t, J ) 9.4, H-4), 3.40 (1 H, ddd, J ) 9.4, 3.5, 2.0, H-2). 13C NMR
(3) (a) Glaser, L.; Zarkowsky, R. In The Enzymes; Boyer, P., Ed.; Academic
Press: New York, 1971; Vol. 5, p 465-480. (b) Gabriel, O. In Carbohydrates
in Solution; Gould, R., Ed.; Advances in Chemistry Series, No. 117, American
Chemical Society: Washington, D. C., 1973; p 387-410. (c) Snipes, C. E.;
Brillinger, G.-U.; Sellers, L.; Mascaro, L.; Floss, H. G. J. Biol. Chem. 1977,
252, 8113-8117. (d) Liu, H.-w.; Thorson, J. S. Annu. ReV. Microbiol. 1994,
48, 223-256.
(D2O) δ 159.6, 149.1, 144.0, 114.3 (t, JCF ) 242.2 Hz, 6-C), 95.4 (d, JCP
)
6.1, 1-C), 89.7, 83.3 (d, JCP ) 9.0, 4′-C), 74.5, 72.5, 71.2 (d, JCP ) 8.2, 2-C),
70.7 (t, JCF ) 10.2, C-5), 69.1, 68.6, 64.4 (d, JCP ) 5.7, 5′-C). 31P NMR
(D2O) δ -11.1 (d, J ) 19.2 Hz), -13.0 (d, J ) 19.2). 19F NMR (D2O) δ
-133.0 (dd, J ) 53.6, 10.9 Hz), -133.3 (dd, J ) 53.6, 16.4). High-resolution
MALDI-MS calcd for C15H21F2N3O15P2 (M + H)+ 583.0416, found 583.0488.
(9) Eod was prepared from Escherichia coli strain HB101-pJT8 according
to the published procedure.2c.
(10) Activity of Eod was determined spectrophotometrically by measuring
the formation of CDP-4-keto-6-deoxy-D-glucose (4) as previously described.2c
The blank was prepared by boiling the enzyme for 5 min prior to the addition
of other reagents. One unit of enzyme activity corresponds to the formation
of 1 µmol of product per hour under the assay conditions.
(4) Frey, P. A. In Pyridine Nucleotide Coenzymes (B); Dolphin, D., Poulson,
R., Avramovic, O., Ed.; Wiley: New York, 1987; Part B, pp 461-511 and
references therein.
(5) (a) Middleton., W. J. J. Org. Chem. 1975, 40, 574-578. (b) May, J.
A.; Sartorelli, A. C. J. Med. Chem. 1979, 22, 971-976.
(6) Excoffier, G.; Utille, D. G. Carbohydr. Res. 1975, 39, 368-373.
(7) Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144-2147.
S0002-7863(98)02198-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/02/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9699
Scheme 3
developed GC-MS assay lent further credence to this model.2d,14
As summarized in Scheme 3, the inactivated Eod was removed
by a Centricon-10, and the filtrate, after lyophilization, was
subjected to sodium borodeuteride reduction, acid hydrolysis,
further reduction, and acetylation. The mass data (m/z of M +
NH4+ ) 453) of the resulting glycidol peracetate 14 is consistent
with the data of a turnover product with a 4-keto-glucose skeleton
(2), the formation of which may be rationalized by the hydration
of 3.15
It is worth mentioning that the important features making the
incorporation of fluorine(s) into biological molecules particularly
attractive include the favorable steric interactions toward the
biological targets, the unique effects of fluoridation on the bond
strength, and the susceptibility of fluoride ion as a leaving group.16
Clearly, compound 5, as a mechanism-based inhibitor for Eod, is
a new addition to a great variety of organofluorine compounds
that have been developed and established to exhibit potent
biological activities.17 It should be noted that the Eod reaction
has been distinguished as the common entry in the formation of
all 6-deoxyhexoses,2,3 many of which play key roles as cellular
components or are an indispensable part of secondary metabo-
lites.18 With the development of effective inhibitors for Eod and
perhaps other enzymes of this class, we may be able to better
control and regulate the biosynthesis of many unusual sugars.
Further characterization of the modified protein will certainly
provide valuable information on the active site of this important
class of enzymes, for which a crystal structure is still lacking.
released fluoride ion,11 was discernible by 19F NMR after
incubation at 37 °C.
A sample of inactivated Eod, from which the unreacted inhibitor
1 had been removed by thorough dialysis, was subjected to
electron spray ionization MS analysis.12 In addition to the intact
Eod, which has a molecular mass of 40 280 ( 5.4 Da per
monomer, a new species with a molecular mass of 40 825 ( 1.7
Da per monomer was found. The mass increase of 545 Da
indicates that the covalently modified Eod is likely an adduct
(Scheme 3, 13) with a CDP-4-keto-6-deoxyglucosyl moiety which
has a calculated molecular weight of 545. On the basis of these
data and the above findings, a plausible mechanism can now be
proposed. As depicted in Scheme 3, the inactivation may be
initiated by C-4 oxidation followed by C-5/C-6 elimination to
give 12, with the subsequent reductive elimination leading to the
regeneration of NAD+ and the formation of 3 as the nascent
product. In normal catalysis, the key intermediate 3 will be
reduced by NADH (see Scheme 1). However, in the absence of
a hydride source, this conjugated 4-keto-∆5,6-glucoseen may serve
as a Michael acceptor and trap an active site nucleophile to give
13 and subsequently impair the enzyme.13 The detection of a
turnover product (2) in the incubation mixture by a previously
Acknowledgment. This work was supported by a National Institutes
of Health grant GM 35906.
JA982198H
(14) (a) Rubenstein, P. A.; Strominger, J. L. J. Biol. Chem. 1974, 249,
3776-3781. (b) Weigel, T. M.; Liu, L.-d.; Liu, H.-w. Biochemistry 1992, 31,
2129-2139.
(15) The assignment was based on the observation that the MS fragments
of 14 containing the C-4 oxygen linkage (m/z 187, 259, 289, and 361) are all
uniformly shifted by one mass unit.2d The increment of one mass unit of these
fragments bearing the C-4 ester linkage is indicative of a deuterium
incorporation through the reduction of a 4-keto-glucose derivative 2.
(16) (a) Organofluorine Chemistry, Principles and Commercial Applica-
tions, Banks, R. E., Smart, B. E., Tatlow, J. C., Eds.; Plenum Press: New
York, 1994. (b) Hudlicky, M.; Pavlath, A. E. Chemistry of Organic Fluorine
Compounds II, American Chemical Society: Washington, D. C., 1995. (c)
Chambers, R. D. Fluorine in Organic Chemistry, John Wiley: New York,
1973.
(17) For sugar examples, see Fluorinated Carbohydrates, Chemical and
Biochemical Aspects, ACS Symposium Series 374, Taylor, N. F. Ed.; American
Chemical Society: Washington, D. C., 1988; for sugar examples.
(18) (a) Williams, N. R.; Wander, J. D. In The Carbohydrates: Chemistry
and Biochemistry; Pigman, W., Horton, D., Eds.; Academic Press: New York,
1980; Vol. 1B, pp 761-798. (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, 1-84.
(c) Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99-110. (d) Johnson,
D. A.; Liu, H.-w. In ComprehesiVe Chemistry of Natural Products Chemistry,
Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon: New York; 1998,
in press.
(11) McCarthy, J. R.; Jarvi, E. T.; Matthews, D. P.; Edwards, M. L.;
Prakash, N. J.; Bowlin, T. L.; Mehdi, S.; Sunkara, P. S.; Bey, P. J. Am. Chem.
Soc. 1989, 111, 1127-1128.
(12) The MS measurements were performed at the MS facility of the Cancer
Research Institute, University of Minnesota.
(13) Alternatively, intermediate 12 could also act as a Michael acceptor to
trap the active-site nucleophile and the resulting adduct after reduction would
lead to an identical final product 13. While both routes are feasible, the one
shown in Scheme 3 is more likely, in view of its close resemblance to the
normal catalytic mechanism.