Synthesis of 5-deoxy-5-phospho-D-ribonohydroxamic acid: A new competitive and selective inhibitor of type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tetlet.2005.03.151

5-Deoxy-5-phospho-d-ribonohydroxamic acid, a mimic of the 1,2-cis-enediolate high-energy intermediate species of the allose-6-phosphate isomerase reaction, was obtained by a six-step synthesis from d-erythronolactone. In contrast to the known competitive

Full text of DOI:10.1016/j.tetlet.2005.03.151

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 3691–3694
Synthesis of 5-deoxy-5-phospho-D-ribonohydroxamic acid:
a new competitive and selective inhibitor of type B
ribose-5-phosphate isomerase from Mycobacterium tuberculosis
Emmanuel Burgos,a Annette K. Roos,b Sherry L. Mowbrayc and Laurent Salmona,*
a
´
´
Laboratoire de Chimie Bioorganique et Bioinorganique, CNRS-UMR 8124, Institut de Chimie Moleculaire et des Materiaux
ˆ
´
d’Orsay (ICMMO), Batiment 420, Universite Paris-Sud XI, F-91405 Orsay, France
bDepartment of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden
cDepartment of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Box 590,
SE-751 24 Uppsala, Sweden
Received 31 January 2005; revised 21 March 2005; accepted 22 March 2005
Available online 6 April 2005
Abstract—5-Deoxy-5-phospho-D-ribonohydroxamic acid, a mimic of the 1,2-cis-enediolate high-energy intermediate species of
the allose-6-phosphate isomerase reaction, was obtained by a six-step synthesis from D-erythronolactone. In contrast to the
known competitive ribose-5-phosphate isomerase (Rpi) inhibitors 4-deoxy-4-phospho-D-erythronohydroxamic acid, 4-deoxy-4-phos-
pho-D-erythronate, and 4-deoxy-4-phosphonomethyl-D-erythronate, the new hydroxamic acid selectively inhibits Mycobacterium
tuberculosis RpiB (Ki = 0.40 mM, Km/Ki = 4.5) versus Spinacia oleracea RpiA, and hence appears as a promising lead for the design
of potent species-specific inhibitors of the bacterial enzyme.
Ó 2005 Elsevier Ltd. All rights reserved.
Ribose-5-phosphate isomerase (Rpi, E.C. 5.3.1.6), an
CHO
OH
OH
OH
CH2OPO3
O-
OH
OH
CH2OH
O
OH
OH
CH2OPO3
1
2
aldose–ketose isomerase involved in the pentose phos-
phate pathway, catalyzes the reversible isomerization
of D-ribose 5-phosphate (R5P) and D-ribulose 5-phos-
phate (Ru5P) (Scheme 1).1 The reaction is thought to
proceed via a proton-transfer mechanism that involves
a 1,2-cis-enediolate high-energy intermediate. Two com-
pletely distinct types of Rpi (A and B) have been
differentiated.2 Recently, the hypothesis was put forth
that the RpiB type of enzyme might also catalyze the
reversible isomerization of the six-carbon sugars D-
allose 6-phosphate (All6P) and D-allulose 6-phosphate
(Allu6P) (Scheme 1). Studies of D-allose catabolism in
Escherichia coli3,4 identified the product of alsR as the
repressor for the rpiB gene, as well as for a neighboring
operon of genes linked with allose transport and metab-
olism. Allose was a stronger inducer of these genesÕ
expression than ribose. The rpiB gene was thus sug-
gested to encode the isomerase in D-allose catabolism,
Rpi
Rpi
OH
2-
2-
2-
CH2OPO3
R5P
Ru5P
1,2-cis-enediolate
high-energy intermediates
CHO
OH
O-
OH
OH
OH
OH
CH2OH
O
1
2
AlsI
AlsI
OH
OH
OH
OH
OH
OH
2-
2-
2-
CH2OPO3
CH2OPO3
CH2OPO3
All6P
Allu6P
Scheme 1. Isomerization reactions catalyzed by D-ribose-5-phosphate
isomerases (Rpi) and D-allose-6-phosphate isomerase (AlsI).
Keywords: Competitive inhibitor; Phosphate sugar; 5-Phosphate
D-ribose isomerase; 6-Phosphate D-allose isomerase; RpiB.
and given the additional designation of alsI. Further-
more, the similarity of All6P and R5P gives substance
to this hypothesis.
*
Corresponding author. Tel.: +33 169 156311; fax: +33 169 157281;
e-mail: lasalmon@icmo.u-psud.fr
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.03.151

3692
E. Burgos et al. / Tetrahedron Letters 46 (2005) 3691–3694
to Ru5P isomerization reaction catalyzed by SoRpiA
and MtRpiB is also presented.
OH
NH
O
OH
NH
O
-O
O
-O
O
OH
OH
OH
Our synthetic strategy to obtain compound 4 started
from the commercially available D-ribono-1,4-lactone
(5, Scheme 3). The primary alcohol position was trityl-
ated according to reported procedures to give com-
pound 6 in 81% yield.8,9 Protection of the secondary
hydroxyl groups of 6 was achieved with PhCOCl in pyr-
idine to yield 710 in 80% yield. Deprotection of the
primary hydroxyl group through hydrogenolysis over
10% Pd/C gave compound 811 in 96% yield, which was
next phosphorylated with diphenylphosphochloridate
in pyridine to yield 912 in 95%. The product was finally
converted quantitatively into 1013 by both hydrogen-
ation and hydrogenolysis over PtO2. As reported in
our previous related synthesis,5 the nucleophilic substi-
tution at C-1 of the precursor 10 and deprotection of
the hydroxyl groups on C-2 and C-3 were achieved in
one step by reaction with hydroxylamine and gave 5-
deoxy-5-phospho-D-ribonohydroxamic acid (5PRH, 4)
as the bis(hydroxylammonium) salt14 in 91% yield.
OH
OH
CH2OPO3
OH
OH
CH2OPO3
OH
OH
CH2CH2PO3
2-
2-
2-
2-
CH2OPO3
1
2
3
4
Scheme 2. Synthesized models of the 1,2-cis-enediolate high-energy
intermediate species of the Rpi- and AlsI-catalyzed isomerization
reactions.
The first three compounds depicted in Scheme 2, 4-
deoxy-4-phospho-D-erythronohydroxamic acid (4PEH,
1), 4-deoxy-4-phospho-D-erythronate (4PEA, 2), and
4-deoxy-4-phosphonomethyl-D-erythronate (4PMEA,
3), have been described as the best competitive inhibitors
with regard to Spinacia oleracea (So) RpiA5,6 and
M. tuberculosis (Mt) RpiB7 (Table 1). Compound 1
(4PEH), the strongest MtRpiB competitive inhibitor
ever reported, and compound 2 (4PEA), led recently
to new information about the reaction mechanism
through structural studies of the enzyme-inhibitor com-
plexes.7 All three inhibitors appear selective for the SoR-
piA catalyzed R5P isomerization, with selectivity factors
for the type B enzyme (R, Table 1) ranging from 0.004 to
0.12. To date, no selective inhibitor of any type B Rpi
has been reported. Such RpiB inhibitors would be valu-
able tools for structural and mechanistic studies of this
type of Rpi, and for comparative studies of the A and
B types. Furthermore, in some pathogenic organisms,
such as Trypanosoma cruzi (the causative agent of Cha-
gas disease) and Mt (which causes tuberculosis), the
only representative of Rpi activity is the B-type enzyme.
Therefore, a strong and selective competitive inhibitor
of the RpiB versus the human RpiA enzyme might lead
to molecules of therapeutic interest, and help determine
whether the enzymes are viable targets for drug treat-
ment. We report in this paper the synthesis of 5-
deoxy-5-phospho-D-ribonohydroxamic acid (5PRH, 4,
Scheme 2), a reaction-intermediate analogue of the
All6P to Allu6P isomerization reaction catalyzed by
AlsI. A comparison of its inhibitory properties with
those of 4PEH (1), 4PEA (2), and 4PMEA (3) for R5P
The new hydroxamic acid 4 was evaluated as a potential
inhibitor of the R5P to Ru5P isomerization reaction of
both SoRpiA (Aldrich) and MtRpiB.7 Kinetic constants
(Km, Ki) and IC50 values were obtained as previously de-
scribed for SoRpiA5 and MtRpiB.7 Hydroxamic acid 4
behaves as a competitive inhibitor of both enzymes.
With a Ki value similar to the Km value of the substrate
R5P, 5PRH (4) can be considered as a poor competitive
inhibitor of the SoRpiA-catalyzed reaction compared to
the values previously obtained for compounds 1, 2, and
3 (Table 1).5,6 For MtRpiB, compound 4 appears as a
good inhibitor with a Km/Ki ratio of 4.5, a value between
those obtained for 1 (Km/Ki = 32) and 2 (Km/Ki = 1).
Such a feature makes compound 4 viable for biochemi-
cal and crystallographic studies, valuable in itself, con-
sidering that RpiB inhibitors are not widespread. The
most interesting point for this compound, however, is
its selectivity factor for the type B enzyme (Table 1):
5PRH (4) appears as the most selective inhibitor for
MtRpiB versus SoRpiA ever reported, with an R value
of 4.5, approximately 37-fold higher than the corre-
sponding value for 4PEH (1) (R = 0.12). Our results thus
lend strong support for the proposal that the rpiB gene
Table 1. Inhibitory effect of 4-deoxy-4-phospho-D-erythronohydroxamic acid 1 (4PEH), 4-deoxy-4-phospho-D-erythronate 2 (4PEA), 4-deoxy-4-
phosphonomethyl-D-erythronate 3 (4PMEA), and 5-deoxy-5-phospho-D-ribonohydroxamic acid 4 (5PRH) on Spinacia oleracea and Mycobacterium
tuberculosis ribose-5-phosphate isomerases (respectively SoRpiA and MtRpiB)
Inhibitor
SoRpiA
Ki (mM)
MtRpiB
Ki (mM)
Rc
IC50 (mM)
Km/Kia
IC50 (mM)
Km/Kib
1
2
3
4
0.018 0.003d
0.010 0.002d
0.15 0.02
5.0 0.5
0.029 0.003d
0.028 0.005d
0.074 0.009f
6.2 0.5
260
270
100
1
0.040 0.005e
6.0 0.6e
ꢀ12.0e
0.057 0.006e
1.7 0.2e
2.0 0.2e
0.40 0.04
32
1
0.12
0.004
0.01
4.5
1
4.5
—
a Km (R5P) = 7.5 0.8 mM.
b Km (R5P) = 1.8 0.2 mM.
c R = (Km/Ki)Mt/(Km/Ki)So
d See Ref. 5.
.
e See Ref. 7.
f See Ref. 6.

E. Burgos et al. / Tetrahedron Letters 46 (2005) 3691–3694
OTr
3693
OH
OH
OTr
O
a
b
c
O
O
O
O
O
O
O
HO
PhOCO
PhOCO
HO
OH
OCOPh
OCOPh
OH
5
6
7
8
O
P
O
P
O
P
OPh
OPh
O
OH
OH
O
ONH3OH
ONH3OH
OH
OH
O
O
O
d
e
f
O
O
PhOCO
C6H11OCO
HO
OCOPh
OCOC6H11
NHOH
O
9
10
4
Scheme 3. Reagents and conditions: (a) i. TrCl (1.1 equiv), pyridine, 4-DMAP (0.03 equiv), 45 °C, 12 h; ii. silica gel chromatography (AcOEt/
pentane, 2:1, Rf = 0.33); iii. Crystallization (CHCl3/n-hexane/acetone, 100/100/1), 81%; (b) i. PhCOCl (2.1 equiv), pyridine, 24 h, 25 °C; ii. Silica gel
chromatography (AcOEt/pentane, 2:1, Rf = 0.66); iii. Crystallization upon MeOH addition (F = 182 °C), 80%; (c) H2, 15 bar, Pd/C, CH2Cl2, 6 days,
96%; (d) (PhO)2POCl (1 equiv), pyridine, 12 h, 25 °C, 95%; (e) H2, 25 bar, PtO2, CH2Cl2, 6 days, 100%; (f) i. Solid NH2OH, MeOH, 12 h, 4 °C;
ii. Precipitation upon Et2O addition; iii. Water dissolution, freeze-drying, 91%.
encodes both RpiB and AlsI activities.3,4 The longer
8. Ireland, R. E.; Anderson, R. C.; Badoud, R.; Fitzsim-
mons, B. J.; McGarvey, G. J.; Thaisrivongs, S.; Wilcox, C.
S. J. Am. Chem. Soc. 1983, 105, 1988–2006.
9. Taylor, C. M.; Barker, W. D.; Weir, C. A.; Park, J. H.
sugar in the All6P case could easily be accommodated
in the active site, via small movements of the long flexi-
ble basic residues that interact with the phosphate
J. Org. Chem. 2002, 67, 4466–4474.
group; the other end of the sugar, that which is isomer-
ized, is expected to dock in a very similar manner in the
10. 2,3-Di-O-benzoyl-5-O-triphenylmethyl-D-ribono-1,4-lactone
(7). A mixture of 6 (7.67 g, 19.6 mmol) and benzoyl
two cases.7 In conclusion, the synthesis of 5-deoxy-5-
chloride (4.9 mL, 42.5 mmol) in dry pyridine (35 mL) was
phospho-D-ribonohydroxamic acid 4 (5PRH), a new
stirred under argon for 19 h at 25 °C. After concentration
competitive and selective inhibitor of the R5P to Ru5P
isomerization reaction catalyzed by MtRpiB (vs SoR-
piA) appears to be very promising for the further
kinetic, mechanistic, and structural investigations on
the presumed second enzymatic activity of MtRpiB, that
is, AlsI activity. Such investigations will hopefully lead
to the development of more selective and efficient inhib-
itors of MtRpiB and other RpiBs of therapeutic interest.
of the residue and addition of water (30 mL), the product
was extracted with chloroform (30 mL). The organic layer
was dried over anhydrous sodium sulfate, filtered, and
concentrated (10 mL). Upon addition of methanol
(40 mL), 9.42 g (80% yield) of 7 were obtained as white
crystals: mp = 182 °C. Rf = 0.66 (AcOEt/pentane 2/1). 13
C
NMR (90 MHz, CDCl3) d (ppm) 171.9 (C-1), 166.0, 165.5
(C-11, C-110), 143.4 (C-7), 134.4, 134.3 (C-12, C-120),
130.7, 130.4 (C-14, C-140), 129.3 (C-9), 129.2, 129.1 (C-15,
C-150), 129.1–128.9 (C-8, C-13, C-130), 128.2 (C-10), 88,9
(C-6), 83.1 (C-4), 72.2 (C-2), 68.5 (C-3), 63.5 (C-5). Anal.
Calcd for C38H30O7 (598.6): C, 76.24; H, 5.05. Found C,
76.25; H, 5.03.
Acknowledgements
11. 2,3-Di-O-benzoyl-D-ribono-1,4-lactone (8). A mixture of 7
(6.65 g, 11.1 mmol) and 10% Pd/C (2.50 g) in anhydrous
CH2Cl2 (130 mL) was hydrogenolyzed (15 bar) at 25 °C
for 6 days. After filtration of the mixture and evaporation
of the solvent, the residue was purified by silica gel
chromatography (eluting triphenylmethane with CHCl3,
then the desired product with CHCl3/AcOEt 1/1) to afford
3.82 g of 8 (96% yield) as a colorless oil: Rf = 0.54 (CHCl3/
AcOEt 1/1). 13C NMR (62 MHz, CDCl3) d (ppm)
172.4 (C-1), 165.8, 165.3 (C-6, C-60), 134.1 (C-7, C-70),
130.2, 130.0 (C-9, C-90), 128.8, 128.4 (C-10, C-100), 128.7
(C-8, C-80), 84.5 (C-4), 71.8 (C-2), 68.3 (C-3), 61.7
(C-5).
12. 2,3-Di-O-benzoyl-5-deoxy-5-diphenylphosphate-D-ribono-
1,4-lactone (9). To a solution of compound 8 (3.82 g,
10.7 mmol) in anhydrous pyridine (15 mL) at 0 °C under
argon was added diphenylphosphochloridate (2.22 mL,
10.7 mmol). Thereafter, the solution was stirred at 25 °C
for 15 h. After evaporation of the solvent and addition of
AcOEt (15 mL), the precipitated pyridinium hydrochlo-
ride was filtered off and the solvent evaporated. The
residue was purified by silica gel chromatography (CHCl3)
to afford 6.00 g of 9 (95% yield) as a colorless oil: Rf = 0.23
(CHCl3). 13C NMR (90 MHz, CDCl3) d (ppm) 170.4
`
Financial support from The Ministere de lÕEducation
Nationale et de la Recherche (E. B., 2001-2004), the
Swedish Research Council (VR) and the Swedish Foun-
dation for Strategic Research (SSF) are gratefully
acknowledged.
References and notes
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E. Burgos et al. / Tetrahedron Letters 46 (2005) 3691–3694
(C-1), 165.4, 165.0 (C-6, C-60), 150.5 (C-11, C-110), 134.2,
134.0 (C-7, C-70), 130.4, 130.3 (C-9, C-90), 130.0, 129.8
(C-10, C-100), 129.7 (C-14, C-140), 128.7, 128.4 (C-8, C-80),
126.1 (C-13, C-130), 120.3 (d, C-12, C-120, J = 4.8 Hz),
81.3 (d, C-4, J = 7.9 Hz), 70.2 (C-2), 67.5 (d, C-5,
J = 5.6 Hz), 67.4 (C-3).
handled in a fume hood, wearing gloves; because
hydroxamic acids are known to readily form stable metal
complexes, a glass spatula was used to handle solid
NH2OH, and thereafter 5PRH). Upon addition of diethyl
ether, compound 4 precipitated from the reaction mixture
as the bis(hydroxylammonium) salt. Following water
dissolution and freeze-drying, 3.04 g of 4 was recovered
(91% yield) as a white solid (hygroscopic): 1H NMR
(250 MHz, D2O) d (ppm) 3.82–4.04 (m, 4 H, H-3, H-4,
CH2), 4.36 (d, J = 3.8 Hz, H-2). 13C NMR (90 MHz, D2O)
d (ppm) 171.2 (C-1), 72.8 (C-2), 72.3 (C-3), 71.0 (d,
J = 6.8 Hz, C-4), 66.1 (C-5). 31P NMR (90 MHz, D2O) d
(ppm) 4.98. FT-IR m (cmꢁ1) 3380, 3215 (NH, OH), 1653
(C@O), 1056, 966 (C–O, PO23ꢁ). MS (negative ion
electrospray) m/z (%) 260.1 [MH]ꢁ (78, MW = 260.0),
245.1 [MHꢁNH]ꢁ (21), 227.1 [MHꢁNH2OH] (100). Anal.
Calcd for C5H18N3O11P (327.2): C, 18.35; H, 5.55. Found:
C, 18.41; H, 6.01.
13. 2,3-Di-O-cyclohexanoyl-5-deoxy-5-dihydrogenophosphate-
D-ribono-1,4-lactone (10). A mixture of compound 9
(6.00 g, 10.2 mmol) and PtO2 (250 mg) in anhydrous
CH2Cl2 (100 mL) was placed under H2 (25 bar) for 6 days.
Removal of the catalyst by filtration and evaporation of the
solvent afforded 4.57 g of 10 (100% yield) as a colorless oil:
13C NMR (50 MHz, CDCl3) d (ppm) 175.3, 174.9 (C-6, C-
60), 171.7 (C-1), 82.1 (d, C-4, J = 8.5 Hz), 70.2 (C-2), 67.3
(C-3), 66.1 (C-5).
14. 5-Deoxy-5-phospho-D-ribonohydroxamic
acid,
bis-
(hydroxylammonium) salt (5PRH, 4). A solution of 10
(4.57 g, 10.2 mmol) in anhydrous methanol was stirred at
4 °C for 24 h with an excess of solid NH2OH15 (CAU-
TION, this very volatile and toxic product must be
15. Brauer, G. In Handbook of Preparative Inorganic Chem-
istry, 2nd ed.; Academic, 1963; Vol. I, p 504.