A new series of N-[2,4-dioxo-6-D-ribitylamino-1,2,3,4-tetrahydropyrimidin- 5-yl]oxalamic acid derivatives as inhibitors of lumazine synthase and riboflavin synthase: Design, synthesis, biochemical evaluation, crystallography, and mechanistic implications

Article abstract of DOI:10.1021/jo702631a

(Figure Presented) The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a K_i of 1.3 nM. The crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a model of the conformation of the intermediate occurring immediately after phosphate elimination, supporting a mechanism in which phosphate elimination occurs before a conformational change of the Schiff base intermediate toward a cyclic structure.

Full text of DOI:10.1021/jo702631a

A New Series of
N-[2,4-Dioxo-6-D-ribitylamino-1,2,3,4-tetrahydropyrimidin-5-yl]oxalamic
Acid Derivatives as Inhibitors of Lumazine Synthase and Riboflavin
Synthase: Design, Synthesis, Biochemical Evaluation,
Crystallography, and Mechanistic Implications
Yanlei Zhang,† Boris Illarionov,‡ Ekaterina Morgunova,§ Guangyi Jin,† Adelbert Bacher,‡
Markus Fischer,| Rudolf Ladenstein,§ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and
Pharmaceutical Sciences, and The Purdue Cancer Center, Purdue UniVersity, West Lafayette, Indiana
47907, Lehrstuhl fu¨r Biochemie, Technische UniVersita¨t Mu¨nchen, Lichtenbergstr 4, D-85747 Garching,
Germany, Institute of Biochemistry and Food Chemistry, Food Chemistry DiVision, UniVersity of
Hamburg, D-20146 Hamburg, Germany, and Karolinska Institute, NOVUM, Center of Bioscience,
S-14157 Huddinge, Sweden
cushman@pharmacy.purdue.edu
ReceiVed December 11, 2007
The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three
metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination
in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors.
All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus
subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved
to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.3 nM. The
crystal structure of M. tuberculosis lumazine synthase in complex with one of the inhibitors provides a
model of the conformation of the intermediate occurring immediately after phosphate elimination,
supporting a mechanism in which phosphate elimination occurs before a conformational change of the
Schiff base intermediate toward a cyclic structure.
Introduction
processes. While vertebrates obtain riboflavin from dietary
sources, pathogenic microorganisms acquire it through biosyn-
thesis. A variety of Gram-negative Enterobacteria as well as
Candida- and Saccharomyces-type yeasts lack an efficient
riboflavin uptake system and are therefore dependent on their
own endogenous biosynthesis mechanisms to secure the
vitamin.1-4 Since a clear rationale for selective toxicity to the
Riboflavin plays an indispensable role in the maintenance of
life through its participation in essential electron-transport
† Purdue University.
‡ Technische Universita¨t Mu¨nchen.
§ Karolinska Institute.
| University of Hamburg.
10.1021/jo702631a CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/11/2008
J. Org. Chem. 2008, 73, 2715-2724
2715

Zhang et al.
SCHEME 1. Last Two Steps in the Riboflavin Biosynthesis
Pathway
pathogen and not the host can be advanced, the enzymes in the
riboflavin biosynthesis pathway are attractive targets for the
design and synthesis of new antibiotics. In fact, expression of
the Salmonella riboflavin biosynthesis gene ribB has recently
been shown to play an essential role in enteritis induction and
systemic typhoid fever animal disease models.5,6
Increasing bacterial resistance to the known antibiotics has
seriously compromised their efficacy, and it is therefore prudent
for the medical scientific community to discover and develop
new types of antibiotics. The present communication focuses
on the last two enzymes in the riboflavin biosynthesis pathway,
lumazine synthase and riboflavin synthase. Lumazine synthase
catalyzes the condensation of 5-amino-6-D-ribitylamino-2,4-
(1H,3H)-pyrimidinedione (1) with 3,4-dihydroxybutanone 4-phos-
phate (2) to afford 6,7-dimethyl-8-D-ribityllumazine (3).7,8
Riboflavin synthase catalyzes a mechanistically unusual dis-
mutation of two molecules of 3 to form one molecule of
riboflavin (4) and one molecule of the lumazine synthase
substrate 1 (Scheme 1).9-13
A mechanism of the riboflavin synthase-catalyzed dismutation
reaction is outlined in Scheme 2. Intermediates 5 and 6 are
derived by deprotonation of the C-7 methyl group of one
substrate molecule 3 and addition of an undefined nucleophile
X- to the C-7 carbon of a second molecule of 3.14-16 Although
the identity of the nucleophile has not been rigorously estab-
lished, likely candidates include water or one of the ribityl
hydroxyl groups.17,18 Nucleophilic attack of the carbanion 5 on
the imine 6 would afford intermediate 7, which could tautomer-
ize to afford compounds 8 and then 9. Elimination of “X-”
would lead to the iminium ion 10, which could undergo
intramolecular nucleophilic attack by the enamine to afford the
intermediate 11. The pentacyclic compound 11 has been isolated
and shown to be a kinetically competent intermediate, and its
structure has been established by multinuclear NMR spectros-
copy.11 Two sequential elimination reactions would then produce
the final products 1 and 4. The reaction mechanism shown in
Scheme 2 is essentially the same as that proposed earlier,11
except it involves the iminium ion 10 instead of a direct
displacement of “X-” by the enamine in intermediate 9.
The details of the reaction catalyzed by lumazine synthase
have not been completely elucidated. The mechanism outlined
in Scheme 3 involves the condensation of the primary amino
group of the substituted pyrimidinedione 1 with the ketone 2
to give Schiff base 13, elimination of phosphate to yield the
enol 14, tautomerization of the enol 14 and isomerization of
the imine to produce the ketone 15, ring closure, and dehydration
of the covalent hydrate 16 to provide the product 3.19 It can be
assumed that the inorganic phosphate formed after elimination
from 13 would remain enzyme bound, at least for some time,
but that it would eventually have to be removed to make room
for another molecule of the substrate 2.
Although the mechanism outlined in Scheme 3 is certainly
very reasonable, the details of the pathway, such as the timing
of phosphate elimination relative to the conformational reor-
ganization of the side chain to allow cyclization, and the
isomerization of the trans Schiff base to a cis Schiff base, or
the possible initial formation of a cis Schiff base, remain
unknown.19 For example, the conformational change of the side
chain might occur with the phosphate still covalently bound to
form intermediate 17, which could then eliminate phosphate to
generate the enol in 18 close to the ribitylamino group (Scheme
4).20
(1) Wang, A. I. Chuan Hsueh Pao 1992, 19, 362-368.
(2) Oltmanns, O.; Lingens, F. Z. Naturforsch. 1967, 22 b, 751-754.
(3) Logvinenko, E. M.; Shavlovsky, G. M. Mikrobiologiya 1967, 41,
978-979.
(4) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1985,
127, 175-181.
(5) Rollenhagen, C.; Bumann, D. Infect. Immun. 2006, 74, 1649-1660.
(6) Becker, D.; Selbach, M.; Rollenhagen, C.; Ballmaier, M.; Meyer, T.
F.; Mann, M.; Bumann, D. Nature 2006, 440, 303-307.
(7) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1986,
139, 1111-1116.
(8) Kis, K.; Volk, R.; Bacher, A. Biochemistry 1995, 34, 2883-2892.
(9) Bacher, A.; Eberhardt, S.; Richter, G. In Escherichia coli and
Salmonella: Cellular and Molecular Biology, 2nd ed.; Neidhardt, F. C.,
Ed.; ASM Press: Washington, DC, 1996; pp 657-664.
(10) Gerhardt, S.; Schott, A.-K.; Kairies, N.; Cushman, M.; Illarionov,
B.; Eisenreich, W.; Bacher, A.; Huber, R.; Steinbacher, S.; Fischer, M.
Structure 2002, 10, 1371-1381.
(11) Illarionov, B.; Eisenreich, W.; Bacher, A. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 7224-7229.
(12) Illarionov, B.; Kemter, K.; Eberhardt, S.; Richter, G.; Cushman,
M.; Bacher, A. J. Biol. Chem. 2001, 276, 11524-11530.
(13) Plaut, G. W. E.; Harvey, R. A. Methods Enzymol. 1971, 18B, 515-
538.
(14) Beach, R. L.; Plaut, G. W. E. J. Am. Chem. Soc. 1970, 92, 2913-
2916.
(15) Beach, R. L.; Plaut, G. W. E. J. Am. Chem. Soc. 1971, 36, 3937-
3943.
One of the main goals of the present investigation has been
to differentiate between the two mechanisms represented in
(16) Paterson, T.; Wood, H. S. C. J. Chem. Soc., Perkin Trans. 1 1972,
1051-1056.
(17) Cushman, M.; Patrick, D. A.; Bacher, A.; Scheuring, J. J. Org. Chem.
1991, 56, 4603-4608.
(19) Volk, R.; Bacher, A. J. Am. Chem. Soc. 1988, 110, 3651-3653.
(20) Zhang, X.; Meining, W.; Cushman, M.; Haase, I.; Fischer, M.;
Bacher, A.; Ladenstein, R. J. Mol. Biol. 2003, 328, 167-182.
(18) Bown, D. H.; Keller, P. J.; Floss, H. G.; Sedlmaier, H.; Bacher, A.
J. Org. Chem. 1986, 51, 2461.
2716 J. Org. Chem., Vol. 73, No. 7, 2008

SCHEME 2. Hypothetical Mechanism of the Riboflavin
Synthase Catalyzed Reaction (X-, Undefined Nucleophile;
R, Ribityl Chain)
SCHEME 3. Hypothetical Reaction Mechanism of the
Lumazine Synthase-Catalyzed Reaction
SCHEME 4. Alternative Mechanism of the Lumazine
Synthase-Catalyzed Reaction
intermediate 14, crystallize a lumazine synthase complex with
a bound intermediate analogue and bound inorganic phosphate,
and determine the structure of the intermediate analogue by
crystallography. This would hopefully provide insight into the
question of whether a ternary complex consisting of the enzyme,
an analogue of 14, and inorganic phosphate can exist, and if
so, the structure of the complex.
Schemes 3 and 4. In the present case, an attempt was made to
synthesize metabolically stable analogues of the hypothetical
J. Org. Chem, Vol. 73, No. 7, 2008 2717

Zhang et al.
SCHEME 5. Synthesis of Fully Protected Ribitylamino-
pyrimidinedione 24a
Enzyme Kinetics. The hypothetical intermediate analogues
27, 30, and 33 were tested as inhibitors of recombinant lumazine
synthases from M. tuberculosis, B. subtilis, and S. pombe.
Compounds 27, 30, and 33 are product 1 analogues of the
riboflavin synthase-catalyzed reaction and might therefore be
expected to inhibit riboflavin synthase as well. The analogues
were therefore also tested for inhibition of recombinant E. coli
riboflavin synthase.
Representative Lineweaver-Burke plots for inhibition of M.
tuberculosis lumazine synthase by inhibitors 27, 30, and 33 are
presented in Figure S26 (Supporting Information). The inhibition
constants and inhibition mechanisms for these inhibitors are
listed in Table 1. The data reveal that the relative potencies of
compounds 27, 30, and 33 as inhibitors of lumazine synthase
depend entirely on the enzyme species. In the case of M.
tuberculosis lumazine synthase, the relative potencies increase
in the order 33 < 30 < 27, while in the case of B. subtilis
lumazine synthase, the order is reversed: 27 < 30 < 33. With
S. pombe lumazine synthase, the potencies do not change
significantly. In addition, the mechanism of inhibition can be
partial, competitive, or mixed depending on the inhibitor and
the species of enzyme (Table 1).
The ethyl oxalate derivative 27 proved to be an unusually
potent inhibitor of E. coli riboflavin synthase, with a Ki of 1.3
nM. The potency and the structure of this compound are
reminiscent of the potent 6,7-dihydro-6,7-dioxo-8-ribityllu-
mazine system 34, which was previously shown to be a potent
inhibitor of baker’s yeast riboflavin synthase (Ki 25 nM)21 and
Ashbya gossypii riboflavin synthase (Ki 9 nM).22 Evidently,
conformational restriction of the 1,2-diketone moiety of 27 in
a dioxolumazine system 34 is not required for potent riboflavin
synthase inhibitory activity. An antibiotic that could inhibit both
riboflavin synthase and lumazine synthase would make it more
difficult for a pathogenic microorganism to become resistant,
since in order to do so, it would have to mutate both enzymes
simultaneously.
a Reagents and conditions: (a) POCl3, diethylaniline, 0 °C (30 min),
23 °C (2 h); (b) BnONa/THF, -20 °C (2 h), 23 °C (4 h); (c) ribitylamine,
DMF, rt (2 h); (d) TBS triflate, triethylamine, 23 °C (11 h); (e) Na2S2O4,
MeOH, H2O, 90 °C (5.5 h).
Results and Discussion
Synthesis. Lumazine synthase and riboflavin synthase inhibi-
tors that are based on the structures of the hypothetical
intermediates in the enzyme-catalyzed reactions are generally
very polar molecules, making purification problematic. In the
present case, the pyrimidinedione ring was masked with benzyl
groups that were cleaved by hydrogenolysis in the final step.
By simply removing the catalyst and solvent, the final com-
pounds could be synthesized in good yields and the crude
products were fairly pure. In addition, TBS protection of the
hydroxyl groups of the ribityl side chain was employed to avoid
their reaction during acylation of the primary amino group
attached to the pyrimidine ring.
Reaction of 5-nitrobarbituric acid (19) with phosphorus
oxychloride provided the trichloride 20 (Scheme 5). Nucleo-
philic aromatic substitution of 20 with sodium benzyl oxide
yielded intermediate 21. A second nucleophilic aromatic
substitution with ribitylamine afforded compound 22 in good
yield. Due to its poor solubility in organic solvents, the four
hydroxyl groups of 22 were protected as their tert-butyldim-
ethylsilyl ethers to yield 23. The nitro group of 23 was then
reduced, resulting in the amine 24. The amine 24 was not stable,
and the crude product was used in the next reaction immediately
after the solvent was removed.
The key intermediate 24 was reacted with ethyl chlorooxoac-
etate, isobutyryl chloride, or propionyl chloride to afford the
corresponding amides 25, 28, and 31 (Scheme 6), which were
deprotected with HF-pyridine to yield 26, 29, and 32. Removal
of the benzyl protecting groups from 26, 29, and 32 by catalytic
hydrogenolysis using palladium on carbon provided the desired
products 27, 30, and 33 in high yield.
Crystallography. The complex formed from inhibitor 33 and
M. tuberculosis lumazine synthase was crystallized in sitting
2718 J. Org. Chem., Vol. 73, No. 7, 2008

SCHEME 6. Synthesis of Target Compounds 27, 30, and 33a
a Reagents and conditions: (a) ethyl chlorooxoacetate, triethylamine, THF, 0 °C (30 min), 23 °C (10 min); (b) HF-pyridine, THF, 23 °C (5 h); (c) Pd/C,
H2, MeOH-H2O, 23 °C (24 h); (d) isobutyryl chloride, triethylamine, THF, 0 °C (40 min), 23 °C (40 min); (e) HF-pyridine, THF, 23 °C (6 h); (f) Pd/C,
H2, MeOH-H2O, 23 °C (24 h); (g) propionyl chloride, triethylamine, THF, 0 °C (40 min) 23 °C (40 min); (h) HF-pyridine, THF, 23 °C (6 h); (i) Pd/C, H2,
MeOH-H2O, 23 °C (24 h).
TABLE 1. Inhibition Constants vs Lumazine Synthases from M. tuberculosis, B. subtilis, S. pombe, and Riboflavin Synthase from E. coli
M. tuberculosis
B. subtilis
S. pombe
E. coli riboflavin
compd
parameter
lumazine synthasee
lumazine synthasef
lumazine synthaseg
synthaseh
27
Ksa (ΜM)
kcatb (min-1
Kic (µM)
53 ( 11
0.11 ( 0.01
4.0 ( 1.7
35 ( 14
5.4 ( 0.5
2.5 ( 0.1
607 ( 177
1.0 ( 0.1
1.00 ( 0.03
1.1 ( 0.2
15 ( 3
2.61 ( 0.15
16.3 ( 0.34
0.0013 ( 0.0001
0.34 ( 0.08
mixed
5.9 ( 0.6
11.8 ( 0.5
)
Kisd (µM)
mechanism
partial
competitive
3.6 ( 0.3
0.40 ( 0.01
95 ( 36
64 ( 11
partial
3.2 ( 0.3
0.37 ( 0.01
20 ( 4
56 ( 12
partial
mixed
30
33
Ks (µM)
63 ( 8
1.1 ( 0.1
0.95 ( 0.02
7.9 ( 0.8
k
cat (min-1
)
0.11 ( 0.01
42 ( 16
Ki (µM)
K
is (µM)
77 ( 28
partial
>1000
mechanism
competitive
1.9 ( 0.3
1.3 ( 0.1
11 ( 1
uncompetitive
6.3 ( 0.7
12.3 ( 0.1
Ks (µM)
58 ( 8
0.095 ( 0.005
110 ( 14
k
cat (min-1
)
Ki (µM)
is (µM)
mechanism
K
>1000
uncompetitive
competitive
competitive
b
a Ks is the substrate dissociation constant for the equilibrium E + S a ES. Kcat is the rate constant for the process ES f E + P. c Ki is the inhibitor
d
dissociation constant for the process E + I a EI. Kis is the inhibitor dissociation constant for the process ES + I a ESI. e Recombinant lumazine synthase
from M. tuberculosis, assay performed in Tris hydrochloride buffer. f Recombinant lumazine synthase from B. subtilis, assay performed in potassium phosphate
buffer. g Recombinant lumazine synthase from S. pombe, assay performed in Tris hydrochloride buffer. h Recombinant riboflavin synthase from E. coli,
assay performed in phosphate buffer.
drops by the vapor diffusion technique with a macroseeding
procedure as previously reported for compounds 35 and 36, and
the crystal structure was determined (Figure 1).23 The diffraction
data were collected using beamline BM14 at the European
Synchrotron Light Source (ESRF, Grenoble, France), and the
structure was refined to a resolution of 2.3 Å. The active site
of M. tuberculosis lumazine synthase is known to be located at
the interface between two neighboring subunits in the pentameric
J. Org. Chem, Vol. 73, No. 7, 2008 2719

Zhang et al.
FIGURE 1. Stereodiagram of the 2|Fo|-|Fc| electron density map (σ )1.5) around the active site of M. tuberculosis lumazine synthase in complex
with inhibitor 33 and phosphate ion (magenta) (a) and the respective schematic drawings of the interactions between the enzyme and the ligand (b).
Red spheres indicate water molecules. The carbon atoms of the residues of different subunits are shown in green and in yellow, oxygen atoms are
in red, and nitrogen atoms are in blue.
assembly. It is formed by the residues from three â-loops
(residues 26-28, 58-61, 81-87) of one subunit as well as
residues 128-141 and residue Asn114 of the neighboring
subunit.23 The aromatic ring of the inhibitor 33 is packed in
the hydrophobic environment in the active site formed by Trp27,
Ile60, Val81 and Val82, Ile83, Phe90, and Val93 residues of
one subunit. The pyrimidine ring is in stacking interaction with
the indole ring of Trp27 at a distance of 4 Å. This interaction
was characterized by a slight deviation of the rings from their
parallel positions and was suggested to be weaker in comparison
to the previously reported purinetrione inhibitors 35, 36, and
related purinetriones because of the smaller size of the pyrimi-
dine ring of 33.23,24 The terminal methyl group of the inhibitor
side chain is directed toward the hydrophobic interior of the
pocket and is located at distances of 4.6 and 5.3 Å from the
side chains of Phe90 and Val93, respectively. The hydrophobic
interaction is supported by two hydrogen bonds formed between
the main chain O-Val81 and N3 from the pyrimidine ring and
the main chain N-Ala59 and the pyrimidine O2 (Figure 1).
The ribityl chain is embedded in a surface depression formed
by residues 56-62 from one subunit and residues 113-114 of
the adjacent subunit with seven H-bonds to both subunits in
total. A schematic diagram of protein-inhibitor interactions is
presented in Figure 1b.
The phosphate ion forms two hydrogen bonds with atoms N
and OG of Thr87 with a distance of 2.9 and 2.6 Å, respectively,
a hydrogen bond with the main chain nitrogen atom of Gln86
with a distance of 2.9 Å, and two ionic contacts with NE and
NH2 of Arg128 with a distance of 2.9 and 2.6 Å, respectively.
Two water molecules were observed in each of the 10 active
sites. One water molecule forms a hydrogen-bonding bridge
between the amide side chain carbonyl oxygen of the inhibitor
and the oxygen of the buffer-derived inorganic phosphate ion.
This water molecule has never been observed before in the
crystal structures of complexes formed between other inhibitors
and lumazine synthase, which may result from the fact that the
previously reported inhibitors do not bear a carbonyl oxygen at
the position of the amide carbonyl present in compound 33.
Also, none of the previous crystal structures display the enzyme
bound to both an inorganic phosphate and a C-5 pyrimidinedione
side chain resembling intermediates 14 and 15.10,23-29 Obvi-
(25) Ladenstein, R.; Meyer, B.; Huber, R.; Labischinski, H.; Bartels, K.;
Bartunik, H.-D.; Bachman, L.; Ludwig, H. C.; Bacher, A. J. Mol. Biol.
1986, 187, 87-100.
(26) Ladenstein, R.; Schneider, M.; Huber, R.; Bartunik, H. D.; Wilson,
K.; Schott, K.; Bacher, A. J. Mol. Biol. 1988, 203, 1045-1070.
(27) Schott, K.; Ladenstein, R.; Ko¨nig, A.; Bacher, A. J. Biol. Chem.
1990, 265, 12686-12689.
(28) Zhang, X.; Meining, W.; Fischer, M.; Bacher, A.; Ladenstein, R. J.
Mol. Biol. 2001, 306, 1099-1114.
(29) Persson, K.; Schneider, G.; Jordan, D. B.; Viitanen, P. V.; Sandalova,
T. Protein Sci. 1999, 8, 2355-2365.
(21) Al-Hassan, S. S.; Kulick, R. J.; Livingston, D. B.; Suckling, C. J.;
Wood, H. C. S.; Wrigglesworth, R.; Ferone, R. J. Chem. Soc., Perkin Trans.
1 1980, 2645-2656.
(22) Winestock, C. H.; Aogaichi, T.; Plaut, G. W. E. J. Biol. Chem. 1963,
238, 2866-2874.
(23) Morgunova, K.; Meining, W.; Illarionov, B.; Haase, I.; Jin, G.;
Bacher, A.; Cushman, M.; Fischer, M.; Ladenstein, R. Biochemistry 2005,
44, 2746-2758.
(24) Morgunova, E.; Illarionov, B.; Sambaiah, T.; Haase, I.; Bacher, A.;
Cushman, M.; Fischer, M.; Ladenstein, R. FEBS J. 2006, 273, 4790-4804.
2720 J. Org. Chem., Vol. 73, No. 7, 2008

FIGURE 2. Stereoview of the substrate binding site of LS from M. tuberculosis with bound purinetrione inhibitor 36. The inhibitor is shown in
blue and magenta (phosphate group), and amino acids interacting with compound are shown in yellow and green. The diagram is programmed for
walleyed (relaxed) viewing.
ously, the water molecule occupies an energetically favorable
position due to its presence in a hydrogen bond network between
the phosphate ion and the inhibitor molecule, which stabilizes
the position of the inhibitor. Its presence is significant because
it suggests that it may be present and participate in the enzyme-
catalyzed reaction. The following are possible roles of this water
molecule during catalysis: (1) it may be involved in hydrolysis
of the phosphate to form a 1,2-diol that then eliminates water
to generate the enol 14. This seems unattractive because the
phosphate is a better leaving group than a hydroxide or water.
(2) It could participate as a proton acceptor from carbon during
the phosphate elimination reaction. (3) It could stabilize the
inorganic phosphate formed after the elimination reaction by
hydrogen bonding. (4) It could act as a hydrogen donor or
acceptor during tautomerization of the enol 14 to the ketone
15. (5) Finally, it could stabilize the enol and keto structures
during the conformational reorganization of the side chain
toward a cyclic structure (14 f 15).
One of the goals of the present study was to gain insight
into the question of whether phosphate elimination occurs before
or after the conformational change toward a cyclic structure
(Schemes 3 vs 4). The crystal structure displayed in Figure 1
provides, for the first time, evidence for an extended conforma-
tion of the C-5 side chain with inorganic phosphate in the
phosphate-binding site of the enzyme. It provides a model of
the hypothetical intermediate 14 that would result from phos-
phate elimination while phosphate remains bound in the
phosphate-binding site. Our previously published crystal struc-
ture of the phosphonate 37 bound to S. cereVisiae lumazine syn-
thase provides a snapshot of the bound hypothetical intermediate
13 formed from the substrates 1 and 2 (Scheme 3).30 Crystal
structures are also available of an analogue of the substrate 1
bound to B. subtilis lumazine synthase in which the primary
amino group of the substrate 1 is replaced by a nitro group.26,27
The second water molecule found in the active site hydrogen
bonds to the Lys138 amino group of the enzyme as well as the
ribitylamino group of the inhibitor. The oxygen atom of this
water molecule mimics the carbonyl oxygen in the five-
membered ring of the purinetrione inhibitors, including com-
pounds 35 and 36 (Figure 2). This water molecule likely plays
a role that is similar to the carbonyl oxygen in the five-
membered ring of the purinetrione inhibitors.
binding site of the enzyme during catalysis. Presumably, the
binding free energy of the substrate 2 is larger than that of an
inorganic phosphate. Crystallization of lumazine synthase in
complex with bound organic phosphonate inhibitors (e.g., 37),
as well as isothermal titration calorimetry and crystallization
of lumazine synthase with bound organic phosphate inhibitors
(e.g., 35 and 36, Figure 2), have been carried out in phosphate
buffer. In these studies, the inhibitors were able to displace
inorganic phosphate from its binding site, so it is reasonable to
expect that the substrate 2 can as well.23,24,30,31
Experimental Section
2,4,6-Trichloro-5-nitropyrimidine (20). 5-Nitrobarbituric acid
(3.02 g, 17.48 mmol) was added to fresh POCl3 (18 mL) in a dry
flask. Diethylaniline (12.50 g, 83.9 mmol) was added dropwise
while the mixture was cooled with an ice bath. The speed of DEA
dropping was roughly one drop per second. After addition was
complete, the reaction mixture was stirred for 0.5 h on the ice bath.
The ice bath was removed, and the reaction mixture was stirred at
23 °C for 2 h and then poured slowly into ice-water to quench
the reaction. During this process, the temperature was kept below
5 °C. After all of the reaction mixture had been poured into ice-
water, the solution was stirred at 23 °C for a few min until it reached
23 °C. The solution was extracted with ethyl ether (4 × 35 mL),
and the ether layer was washed with water (2 × 10 mL) and dried
with Na2SO4. The ether solution was concentrated to yield a residue
that was extracted with boiling hexane (4 × 70 mL). The extracts
were combined and the solvent was removed to afford the product
(1.31 g, 33%) as a yellow powder: mp 56-58 °C (lit.32 mp 57-
58 °C).
2,4-Bis(benzyloxy)-6-chloro-5-nitropyrimidine (21). NaH (0.276
g, 11.5 mmol) was added to the solution of benzyl alcohol (1.24 g,
11.5 mmol) in THF (26 mL) at 23 °C, and the mixture was stirred
for 30 min. The solution was added dropwise into a solution of
compound 20 (1.31 g, 5.75 mmol) in THF (26 mL) at -20 °C.
The reaction mixture was stirred at -20 °C for 2 h and at 23 °C
for 4 h. A few drops of acetic acid were added to stop the reaction.
The reaction mixture was concentrated in vacuo, and the residue
was extracted with ether. The extracts were combined, and solvent
was removed. The residue was separated by silica gel TLC with
CH2Cl2-hexane 1:1 as the mobile phase to provide the product
1
2133 (667 mg, 31.3%) as yellow crystals: mp 105-107 °C; H
(30) Meining, W.; Mo¨rtl, S.; Fischer, M.; Cushman, M.; Bacher, A.;
Ladenstein, R. J. Mol. Biol. 2000, 299, 181-197.
Overall, the evidence provided by the crystal structure shown
in Figure 1 supports elimination of the phosphate before the
C-5 side chain rotates toward a cyclic structure (Scheme 3).
Additional studies will be required to clarify exactly how the
substrate 2 displaces inorganic phosphate from the phosphate-
(31) Morgunova, E.; Saller, S.; Haase, I.; Cushman, M.; Bacher, A.;
Fischer, M.; Ladenstein, R. J. Biol. Chem. 2007, 282, 17231-17241.
(32) Robins, R. K.; Dille, K. L.; Christensen, B. E. J. Org. Chem. 1954,
19, 930-933.
(33) Hashizume, K.; Inoue, S. Yakagaku Zasshi 1985, 105, 362-367.
J. Org. Chem, Vol. 73, No. 7, 2008 2721

Zhang et al.
NMR (300 MHz, CDCl3) δ 7.61-7.50 (m, 10 H), 5.65 (s, 2 H),
5.59 (s, 2 H); 13C NMR (75 MHz, CDCl3) δ 164.6, 163.3, 155.0,
136.0, 135.4, 130.0, 129.6, 129.2, 72.0, 71.7; ESIMS m/z 394
(MNa+).
(CDCl3, film) 2955, 2930, 2886, 1800, 1763, 1610, 1514, 1472,
1395, 1375, 1251, 1228 cm-1; MALDIMS m/z 1013 (MH+). Anal.
Calcd for C51H88N4O9Si4: C, 60.43; H, 8.75; N, 5.53. Found: C,
60.31; H, 8.56; N, 5.37.
N-[2,4-Bis(benzyloxy)-6-(ribitylamino)pyrimidin-5-yl]oxalam-
ic Acid Ethyl Ester (26). Compound 25 (35 mg, 0.034 mmol) was
added to THF (1 mL). HF‚Py (0.4 mL) was added to the THF
solution. The reaction mixture was stirred for 5 h at 23 °C. Saturated
NaHCO3 solution was added to the reaction mixture to neutralize
it to pH ) 8. The solution was extracted with chloroform, and the
extracts were combined and dried over Na2SO4. The chloroform
solution was evaporated and the residue was separated by TLC to
afford compound 26 (11 mg, 57%) as an oil: 1H NMR (300 MHz,
CDCl3) δ 8.73 (s, 1 H), 7.52-7.39 (m, 10 H), 6.35 (s, 1 H), 5.56
(s, 2 H), 5.43 (s, 2 H), 4.45-4.38 (q, J ) 7 Hz, 2 H), 3.92-3.81
(m, 5 H), 3.69-3.63 (m, 2 H), 1.46-1.42 (t, J ) 6 Hz, 3 H); 13C
NMR (75 MHz, CDCl3) δ 164.7, 162.4, 160.8, 160.4, 156.4, 136.8,
128.9, 128.4, 128.3, 128.1, 92.8, 74.0, 73.7, 71.7, 69.6, 68.9, 64.1,
63.8, 44.3, 14.2; MALDIMS m/z 557 (MH+). Anal. Calcd for
C27H32N4O9‚0.4 H2O: C, 57.52; H, 5.86; N, 9.94. Found: C, 57.78;
H, 5.79; N, 9.58.
N-[2,4-Dioxo-6-(ribitylamino)-1,2,3,4-tetrahydropyrimidin-5-
yl]oxalamic Acid Ethyl Ester (27). Starting material 26 (11 mg,
0.020 mmol) and Pd/C (10%, 2.5 mg) were added into a flask.
Methanol-water (2.5 mL-0.25 mL) was added, and the reaction
mixture was stirred under H2 for 24 h. The reaction mixture was
filtered, and the filtrate was evaporated to provide the product 27
(6.9 mg, 93%) as a white semisolid powder: 1H NMR (300 MHz,
D2O) δ 4.18-4.11 (q, J ) 7 Hz, 2 H), 3.67-3.24 (m, 7 H), 1.13-
1.08 (t, J ) 7 Hz, 3 H); 13C NMR (D2O) (75 MHz) δ 162.5, 160.1,
153.7, 151.6, 85.4, 72.6, 72.5, 70.8, 64.6, 62.7, 44.6, 13.4; ESI m/z
579 (MNa+). Anal. Calcd for C13H20N4O9‚0.4H2O: C, 39.85; H,
5.58; N, 14.29. Found: C, 39.84, H, 5.50, N, 14.72.
N-(2,4-Bis(benzyloxy)-6-[2,3,4,5-(tert-butyldimethylsilyl)ribi-
tylamino]pyrimidin-5-yl)isobutyramide (28). Compound 23 (600
mg, 0.636 mmol) was dissolved in methanol (12 mL) to make a
clear solution, and Na2S2O4 (841 mg, 4.83 mmol) was added. Water
(1.2 mL) was added, and the reaction mixture was stirred in a sealed
vial and heated at 115 °C for 4 h. The reaction mixture was then
cooled to 23 °C, and solvent was removed. The residue was
dissolved in dichloromethane (27 mL) and filtered to remove the
solid. Dichloromethane was evaporated to afford a crude product
24 (566 mg). Without purification, the crude product 24 was
dissolved in THF (15.8 mL). Triethylamine (0.70 mL, 5.02 mmol)
and isobutyryl chloride (118 µL, 1.12 mmol) were added dropwise
to the solution at 0 °C. The reaction mixture was stirred at 0 °C
for 40 min, and then it was stirred at 23 °C for 40 min. The solvent
was evaporated, and the residue was separated by silica gel flash
chromatography, eluting with hexane-ethyl acetate 5:1, to afford
product 28 as a clear oil (486 mg, total yield 78%): 1H NMR (300
MHz, CDCl3) δ 7.38-7.18 (m, 10 H), 6.33 (s, 1 H) 5.35 (s, 1 H),
5.31-5.19 (m, 4 H), 3.93-3.50 (m, 7 H), 2.49-2.38 (septet, J )
5.4 Hz, 1 H), 1.12 (s, 3 H), 1.10 (s, 3 H). 0.90-0.76 (m, 36 H),
0.05 - -0.10 (m, 24 H); 13C NMR (75 MHz, CDCl3) δ 176.5, 163.9,
161.7, 160.4, 137.2, 136.7, 128.4, 128.3, 128.2, 127.9, 94.8, 76.8,
74.7, 71.5, 68.8, 68.4, 64.6, 43.3, 35.6, 29.7, 26.1, 26.0, 19.7, 19.6,
18.4, 18.3, 18.2, 18.1, -4.0, -4.2, -4.4, -5.1, -5.3, -5.4; MS
(ESI) 983 m/z (MH+). Anal. Calcd for C51H90N4O7Si4: C, 62.27;
H, 9.22; N, 5.70. Found: C, 62.49; H, 9.06; N, 6.08.
2-Benzyloxy-5-nitro-4,6-bis(ribitylamino)pyrimidine (22). Com-
pound 21 (539 mg, 1.45 mmol) and ribitylamine (548 mg, 3.63
mmol) were added to DMF (8 mL). The reaction mixture was stirred
at 23 °C for 2 h. DMF was removed in vacuo. The residue was
separated by silica gel flash chromatography with CHCl3-methanol
3:1 as the mobile phase to afford the product as an amorphous
white powder (488 mg, 70%): 1H NMR (300 MHz, DMSO-d6) δ
9.08 (s, 1 H), 7.49-7.31 (m, 10 H), 5.50 (s, 2 H), 5.41 (s, 2 H),
5.03-5.02 (d, J ) 5.3 Hz, 1 H), 4.70-4.68 (d, J ) 4.9 Hz, 1 H),
4.43-4.40 (t, J ) 5.5 Hz, 1 H), 3.91-3.85 (m, 2 H), 3.61-3.30
(m, 5 H); 13C NMR (75 MHz, DMSO-d6) δ 165.9, 162.4, 158.3,
136.0, 135.9, 128.5, 128.3, 128.0, 127.5, 112.0, 73.5, 72.7, 69.3,
69.2, 69.0, 63.2, 44.0, 40.3, 40.1, 39.8, 39.2, 38.9, 38.7; ESIMS
m/z 487 (MH+). Anal. Calcd for C23H26N4O8: C, 56.79; H, 5.39;
N, 11.52. Found: C, 56.50; H, 5.42; N, 11.33.
2,4-Bis(benzyloxy)-5-nitro-6-[2,3,4,5-tetrakis-(O-tert-butyl-
dimethylsilanyl)ribitylamino]pyrimidine (23). Compound 22 (243
mg, 0.5 mmol) and TBS triflate (792 mg, 3 mmol) were added
into a flask. Triethylamine (7.74 mL) was added to the flask to
make a solution. The reaction mixture was stirred at 23 °C for 11
h. The TEA was removed in vacuo. The residue was added to ethyl
acetate (12.8 mL), and the mixture was stirred for 2 h at 23 °C.
The cloudy ethyl acetate solution was filtered through Celite to
afford a clear solution. The solvent was evaporated, and the residue
was separated by silica gel flash chromatography (hexane-ethyl
acetate 95:5) to provide the product 23 as a yellow oil (396 mg,
84%): 1H NMR (300 MHz, CDCl3) δ 8.96 (s, 1 H), 7.48-7.27
(m, 10 H), 5.51 (s, 2 H), 5.44-5.31 (q, J ) 12 Hz, 2 H), 4.14-
4.09 (m, 1 H), 3.99-3.89 (m, 2 H), 3.85-3.82 (m, 1 H), 3.73-
3.69 (m, 1 H), 3.57-3.52 (m, 2 H), 0.88-0.85 (m, 36 H), 0.11-
0.04 (m, 24 H); ESIMS m/z 944 (MH+). Anal. Calcd for
C47H82N4O8Si4: C, 59.83; H, 8.76; N, 5.94. Found: C, 59.78; H,
8.67; N, 5.76.
5-Amino-2,4-bis(benzyloxy)-6-[2,3,4,5-tetrakis-O-(tert-
butyldimethylsilanyl)ribityl]pyrimidine (24). Compound 23 (47
mg, 0.05 mmol) was added to a vial. Methanol (1 mL) and water
(0.1 mL) were added to the vial. Na2S2O4 (66 mg, 0.38 mmol) was
added to the reaction mixture. The reaction mixture was stirred for
5.5 h at 90 °C in a sealed vial and then cooled to 23 °C. The solution
was filtered through Celite, and the filtrate was dried to afford a
residue. The residue was separated by silica gel flash chromatog-
raphy with hexane-ethyl acetate 20:1 as mobile phase to afford
the product as a yellow oil (40 mg, 87.5%): 1H NMR (300 MHz,
CDCl3) δ 7.37-7.16 (m, 10 H), 5.26-5.23 (m, 4 H), 3.90-3.65
(m, 7 H), 0.83-0.81 (m, 36 H), 0.048 - -0.041 (m, 24 H); ESIMS
m/z 914 (MH+).
N-{2,4-Bis(benzyloxy)-6-[2,3,4,5-tetrakis-O-(tert-butyldimeth-
ylsilanyl)ribitylamino]-pyrimidin-5-yl}oxalamic Acid Ethyl Es-
ter (25). Compound 24 (40 mg, 0.044 mmol) was mixed with THF
(1 mL), and then TEA (40 mg, 0.4 mmol) was added to the THF
solution. The mixture was cooled to 0 °C, and ethyl chlorooxoac-
etate (7.4 µL, 9 mg, 0.066 mmol) was added dropwise. The reaction
mixture was stirred at 0 °C for 0.5 h. The reaction mixture was
warmed to 23 °C and stirred for 10 min. The mixture was
evaporated in vacuo to yield a residue that was purified by silica
gel flash chromatography (hexane-ethyl acetate 10:1) to provide
the product as a viscous yellow oil (25 mg, 56%): 1H NMR (300
MHz, CDCl3) δ 8.12 (s, 1 H), 7.38-7.20 (m, 10 H), 5.43-5.22
(m, 4 H), 4.34-4.27 (q, J ) 6 Hz, 2 H), 3.94-3.48 (m, 7 H),
1.36-1.31 (t, J ) 7 Hz, 3 H) 0.83-0.81 (m, 36 H), 0.06-0.06 (m,
24 H); 13C NMR (75 MHz, CDCl3) δ 163.8, 162.2, 160.0, 159.5,
154.7, 136.8, 136.4, 128.3, 128.2, 128.0, 127.8, 127.7, 127.6, 92.9,
76.6, 74.5, 71.4, 68.8, 68.2, 64.4, 63.2, 43.1, 26.0, 25.9, 25.8, 18.3,
18.2, 18.1, 17.9, 13.9, -4.1, -4.4, -4.7, -5.9, -5.7, -5.5; IR
N-2,4-Bis(benzyloxy)-6-(ribitylamino)pyrimidin-5-ylisobu-
tyramide (29). Compound 28 (164 mg, 0.167 mmol) was dissolved
in THF (5.1 mL). HF-Py solution (2.05 mL) was added to the
solution. The reaction mixture was stirred at 23 °C for 6 h. Then
NaHCO3 powder and its saturated aqueous solution were added to
the reaction mixture to neutralize it to pH 7-8. The solution was
extracted with CHCl3 (3 × 35 mL). The extracts were combined
and dried over Na2SO4 for 2 h. The mixture was filtered, and the
solvent was removed. The residue was separated by silica gel flash
chromatography, eluting with dichloromethane-ethanol 10:1, to
2722 J. Org. Chem., Vol. 73, No. 7, 2008

provide a semisolid product 29 (56 mg, 63.7%): 1H NMR (300
MHz, CD3OD) δ 7.33-7.16 (m, 10 H), 5.24 (s, 2 H) 5.19 (s, 2 H),
3.79-3.40 (m, 7 H), 2.56-2.47 (septet, J ) 6.9 Hz, 1 H), 1.05 (s,
3 H), 1.03 (s, 3 H); 13C NMR (75 MHz, CD3OD) δ 181.0, 166.4,
163.6, 163.3, 138.6, 138.3, 129.4, 129.0, 128.9, 128.7, 94.7, 74.4,
74.3, 72.7, 70.0, 69.3, 64.7, 44.7, 36.2, 19.9; MS (ESI) m/z 527
(MH+). Anal. Calcd for C27H34N4O7: C, 61.58; H, 6.51; N, 10.64.
Found: C, 61.22; H, 6.43; N, 10.28.
TABLE 2. Enzymes Used in Kinetic Assays
specific
concn of
enzyme in
activity
reaction mixture
enzyme
organism
(µM mg-1 h-1
)
(µg mL-1
)
lumazine synthases: M. tuberculosis
B. subtilis
1.1
5.1
30
2.0
0.9
1.0
S. pombe
4.2
riboflavin synthase: E. coli
11.6
N-6-(Ribitylamino)pyrimidine-2,4(1H,3H)-dione-5-ylisobu-
tyramide (30). Intermediate 29 (30 mg, 0.057 mmol) was added
to methanol-water (7 mL, 0.7 mL) to make a solution. Then Pd/C
(10%, 7.5 mg) was added to the solution. The reaction mixture
was stirred under 1 atm of H2 at 23 °C for 24 h. The reaction
mixture was filtered to remove the Pd/C. The solvent was removed
from the filtrate to provide a light yellow amorphous powder (18.7
methanol-water (9.5 mL, 0.95 mL) to make a solution. Then Pd/C
(10%, 10 mg) was added, and the reaction mixture was stirred under
1 atm H2 at 23 °C for 24 h and then filtered to remove the Pd/C.
The solvent was removed from the filtrate to provide a light yellow
solid powder (23 mg, 92%): mp 126-127 °C; 1H NMR (300 MHz,
D2O) δ 3.77-3.31 (m, 7 H), 2.31-2.23 (q, J ) 7.8 Hz, 2 H), 1.00-
0.95 (t, J ) 7.5 Hz, 3 H); 13C NMR (75 MHz, D2O) δ 180.1, 163.0,
154.1, 151.7, 86.8, 72.6, 70.7, 62.7, 44.5, 29.0, 9.3; MS (ESI) MH+
333. Anal. Calcd for C12H20N4O7: 43.37; H, 6.07; N, 16.86.
Found: C, 43.22; H, 5.71; N, 16.48.
1
mg, 95%): mp 184-185 °C; H NMR (300 MHz, D2O) δ 3.78-
3.27 (m, 7 H), 2.53-2.48 (p, J ) 7.0, 1 H), 1.01 (s, 3 H), 0.99 (s,
1 H); 13C NMR (75 MHz, D2O) δ 185.4, 165.0, 156.1, 153.8, 89.0,
74.7, 72.6, 64.9, 46.5, 37.3, 21.1; MS (ESI) m/z 347 (MH+). Anal.
Calcd for C13H22N4O7‚1.4 H2O: 42.02; H, 6.73; N, 15.08. Found:
C, 42.28; H, 6.55; N, 15.03.
Kinetic Assays. All assays were performed in 96-well microtiter
plates using computer-controlled microplate reader. Enzymes used
in kinetic assays are specified in Table 2.
N-(2,4-Bis(benzyloxy)-6-[2,3,4,5-(tert-butyldimethylsilyl)ribi-
tylamino]pyrimidin-5-ylpropionamide (31). Compound 23 (510
mg, 0.541 mmol) was dissolved in methanol (10 mL) to make a
clear solution, and Na2S2O4 (715 mg, 4.12 mmol) was added. Water
(1.0 mL) was added to the solution. The reaction mixture was stirred
in a sealed vial at 0 °C for 4 h. The reaction mixture was then
cooled to 23 °C, and the solvent was evaporated. The residue was
dissolved in dichloromethane (25 mL) and filtered to remove the
solid. Dichloromethane was evaporated to afford a crude product
24 (535 mg). Without purification, the crude product 24 was
dissolved in THF (15.0 mL). Triethylamine (0.66 mL, 4.74 mmol)
was added to this solution. Propionyl chloride (92.1 µL, 1.05 mmol)
was added dropwise to the solution at 0 °C. The reaction mixture
was stirred at 0 °C for 40 min, and then it was stirred at 23 °C for
40 min. The solvent was removed. The residue was separated by
silica gel flash chromatography, eluting with hexane-ethyl acetate
5:1, to afford product 31 as a clear oil (427 mg, total yield 80.3%):
1H NMR (300 MHz, CDCl3) δ 7.36-7.21 (m, 10 H), 6.40 (s, 1 H)
5.37 (s, 1 H), 5.32-5.26 (m, 4 H), 3.94-3.53 (m, 7 H), 2.31-
2.23 (q, J ) 7.8 Hz, 2 H), 1.14-1.09 (t, J ) 7.5 Hz, 3 H). 0.91-
0.82 (m, 36 H), 0.08- -0.05 (m, 24 H); 13C NMR (75 MHz, CDCl3)
δ 178.4, 173.6, 164.3, 163.0, 162.4, 162.1, 160.7, 137.5, 137.2,
136.9, 128.9, 128.7, 128.6, 128.4, 128.3, 95.1, 75.1, 72.0, 71.7,
69.3, 68.7, 68.5, 65.0, 43.7, 30.0, 26.5, 26.4, 25.3, 18.9, 18.7, 18.6,
18.5, 10.2, -3.6, -3.9, -4.0, -4.1, -4.6, -4.8, -5.0; MS (ESI)
m/z 969 (MH+). Anal. Calcd for C50H88N4O7Si4: C, 61.94; H, 9.15;
N, 5.78. Found: C, 61.67; H, 9.24; N, 6.15.
N-2,4-Bis(benzyloxy)-6-(ribitylamino)pyrimidin-5-ylpropiona-
mide (32). Compound 31 (176 mg, 0.179 mmol) was dissolved in
THF (5.5 mL). HF-Py solution (2.17 mL) was added to the
solution. The reaction mixture was stirred at 23 °C for 6 h. Then
NaHCO3 powder and its saturated aqueous solution were added to
the reaction mixture to neutralize to pH 7-8. The solution was
extracted with CHCl3 (3 × 37 mL). The extracts were combined
and dried over Na2SO4 for 2 h. The mixture was filtered, and the
solvent was removed. The residue was separated by silica gel flash
chromatography, eluting with dichloromethane-ethanol 10:1, to
provide a semisolid product 32 (60 mg, 65%): 1H NMR (300 MHz
CD3OD) δ 7.31-7.15 (m, 10 H), 5.22 (s, 2 H) 5.21 (s, 2 H), 3.78-
3.39 (m, 7 H), 2.31-2.23 (q, J ) 7.8 Hz, 2 H), 1.07-1.02 (t, J )
7.8 Hz, 3 H); 13C NMR (75 MHz, CD3OD) δ 178.3, 166.7, 164.0,
163.7, 139.0, 138.9, 129.8, 129.4, 129.0, 95.1, 74.8, 74.6, 73.3,
70.4, 69.6, 65.0, 45.2, 30.5, 10.73; MS (ESI) 513 (MH+). Anal.
Calcd for C26H32N4O7: 60.93; H, 6.29; N, 10.93. Found: C, 60.84;
H, 6.29; N, 10.58.
Lumazine Synthase Assay. Assay mixtures with variable
concentration of 1 contained 50 mM Tris hydrochloride, pH 7.0,
100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, 100 µM 2,
lumazine synthase, and 1 (3-150 µM) in a volume of 0.2 mL.
Assay mixtures were prepared as follows. A solution (175 µL)
containing 103 mM NaCl, 5.1 mM dithiothreitol, 114 µM 2, and
lumazine synthase in 51 mM Tris hydrochloride, pH 7.0 was added
to 4 µL of inhibitor in 100% (v/v) DMSO (inhibitor concentration
window, 0 - 300 µM) in a well of a 96-well microtiter plate. The
reaction was started by adding 21 µL of a solution containing 103
mM NaCl, 5.1 mM dithiothreitol, and substrate 1 (30-1500 µM)
in 51 mM Tris hydrochloride, pH 7.0. The formation of 6,7-
dimethyl-8-ribityllumazine (3) was measured online for the period
of 40 min at 27 °C at 408 nm (ꢀlumazine ) 10200 M-1 cm-1).
For kinetic assays in phosphate buffer, Tris hydrochloride and
NaCl in reaction mixtures were replaced by 100 mM K/Na-
phosphate pH 7.0. All other assay parameters were the same as
describe above.
Riboflavin Synthase Assay. Assay mixtures contained 100 mM
K/Na-phosphate pH 7.0, 1% (v/v) DMSO, 5 mM dithiothreitol,
enzyme, and variable concentrations of 3 (3-50 µM) in a vol-
ume of 0.2 mL. Assay mixtures were prepared as follows. A
solution (175 µL) containing 5.1 mM dithiothreitol and riboflavin
synthase in 103 mM K/Na-phosphate pH 7.0 was added to 5 µL of
inhibitor in 40% (v/v) DMSO (inhibitor concentration window,
0-400 µM) in a well of a 96-well microtiter plate. The reaction
was started by adding 21 µL of a solution containing 5.1 mM
dithiothreitol, and substrate 3 (30-500 µM) in 103 mM K/Na-
phosphate pH 7.0. The formation of riboflavin was measured online
for the period of 40 min at 27 °C at 470 nm (ꢀriboflavin ) 9600 M-1
cm-1).
Evaluation of Experimental Data. The velocity-substrate data
were fitted for all inhibitor concentrations with a nonlinear
regression method using the program DynaFit.34 Different inhibition
models were considered for the calculation. Ki and Kis values (
standard deviations were obtained from the fit under consideration
of the most likely inhibition model as described earlier.35
Acknowledgment. This research was made possible by NIH
Grant Nos. GM51469 and R21 NS053634, the Fonds der
Chemischen Industrie, the Hans Fischer Gesellschaft, and the
(34) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273.
(35) Cushman, M.; Jin, G.; Illarionov, B.; Fischer, M.; Ladenstein, R.;
Bacher, A. J. Org. Chem. 2005, 70, 8162-8170.
N-6-(Ribitylamino)pyrimidine-2,4(1H,3H)-dion-5-ylpropiona-
mide (33). Compound 32 (40 mg, 0.078 mmol) was added to
J. Org. Chem, Vol. 73, No. 7, 2008 2723

Zhang et al.
lumazine synthase by compounds 27, 30, and 33, the protein
crystallization procedure, diffraction data collection, data collection
and refinement statistics, and structure determination and refine-
ment. This material is available free of charge via the Internet at
http://pubs.acs.org.
Swedish Natural Science Research Council (Vetenskapsrådet).
This research was conducted in a facility constructed with
support from Research Facilities Improvement Program Grant
No. C06-14499 from the National Center for Research Re-
sources of the National Institutes of Health.
Supporting Information Available: 1H NMR and 13C NMR
spectra, Lineweaver-Burk plots for the inhibition of M. tuberculosis
JO702631A
2724 J. Org. Chem., Vol. 73, No. 7, 2008