Reversal of Selectivity for Riboflavin Synthase
water and 10% HCOOH. After concentration of the pooled
fractions, the residue was treated with methanol to afford a
white precipitate 13 (0.3 g, 85%) as an amorphous solid: 1H
NMR (300 Hz, D2O) δ 3.82 (m, 1 H), 3.70 (m, 2 H), 3.59-3.45
(m, 3 H), 3.39 (m, 1 H), 2.40 (t, J ) 7.3 Hz, 2 H), 1.7-1.9 (m,
2 H), 1.65-1.5 (m, 2 H). PDMS m/z 427 (MH+). Anal. Calcd
for C13H23N8O10P(2.0H2O + 0.9HCO2H): C, 34.54; H, 5.64; N,
10.89. Found C, 34.53; H, 5.47; N, 11.07.
(9) (0.5 g, 1.6 mmol) and triethylamine (2 mL) in water (20
mL). The mixture was stirred at room temperature for 12 h
and then evaporated to dryness. The residue was then dis-
solved in a mixture of 1:1 MeOH-water (20 mL) and hydro-
genated under an H2 atmosphere in the presence of Pd(OH)2
on charcoal (0.1 g, 20%) for 12 h. The catalyst was removed
by filtration, and the filtrate was concentrated to dryness. The
residue was basified with a few drops of triethylamine and
then applied on an anion-exchange resin column (Dowex 1 ×
2-400), eluted with distilled water and 10% HCOOH. After
concentration of pooled fractions, the residue was treated with
ethanol to precipitate 26 (0.29 g, 40%) as an amorphous solid:
1H NMR (300 Hz, D2O) δ 3.86 (m, 3 H), 3.68 (m, 2 H), 3.58-
3.45 (m, 3 H), 3.39 (m, 1 H), 2.38 (m, 2 H), 1.62 (m, 4 H); ESMS
m/z 457 (MH+). Anal. Calcd for C14H25N4O11P‚5.0H2O‚
0.7HCOOH: C, 35.48; H, 5.55; N, 11.26. Found C, 35.59; H,
5.78; N, 11.03.
Molecu la r Mod elin g. Using Sybyl (version 6.5; Tripos,
Inc., 1998), we downloaded the X-ray crystal structure of E.
coli riboflavin synthase (1I8D), and two molecules of the ligand
13 were docked and oriented as suggested by the published
model of the binding of two molecules of the substrate 3 in
the active site.20 The C- and N-terminal groups were changed
to neutral carboxylic acid and amino groups, and hydrogens
were added to the protein structure and to the oxygens of the
water molecules. MMFF94 charges were loaded, and a 6 Å
spherical subset including and surrounding the two ligand
molecules was then energy minimized using the Powell method
to a termination gradient of 0.05 kcal/mol while employing the
MMFF94 force field. The remaining portion of the protein was
held rigid using the aggregate function during energy mini-
mization. Figure 3 was constructed by displaying the amino
acid residues in the C- and N-barrels surrounding the two
ligand molecules. The maximum distance between donor and
acceptor atoms contributing to the hydrogen bonds shown in
Figure 3 was set to 2.8 Å.
5-(5-P h osp h on ova ler yl)a m in o-6-D-r ibityla m in ou r a cil
(23). 5-(Dibenzylphosphone)valeric acid (20) (1.5 g, 4.1 mmol)
was dissolved in dichloromethane (20 mL) and a solution of
oxalyl chloride in dichloromethane (2M, 2 mL), and two drops
of DMF were added. After being stirred at room temperature
for an additional 2 h, the reaction mixture was evaporated and
then coevaporated twice with dichloromethane (20 mL) to
remove traces of oxalyl chloride. The residue was dissolved in
dry acetonitrile (10 mL) and added to a solution of 5-amino-
6-D-ribityluracil hydrochloride17,23,24 (0.5 g, 1.5 mmol) and
triethylamine (2 mL) in water (20 mL). The mixture was
stirred at room temperature for 12 h and then evaporated to
dryness. The residue was washed with acetonitrile to give 22
(0.7 g, 75%) as a hygroscopic solid that was used without
further purification. A mixture of compound 22 (0.5 g, 0.8
mmol) and Pd(OH)2 on charcoal (0.1 g, 20%) in 1:1 MeOH-
water (20 mL) was hydrogenated under an H2 atmosphere for
12 h. The catalyst was removed by filtration, and the filtrate
was concentrated to dryness. The residue was treated with
methanol to afford an amorphous white precipitate 23 (0.3 g,
85%): 1H NMR (300 Hz, D2O) δ 3.81 (m, 1 H), 3.68 (m, 2 H),
3.59-3.45 (m, 3 H), 3.39 (m, 1 H), 2.35 (t, J ) 7.2 Hz, 2 H),
1.63-1.48 (m, 6 H). Anal. Calcd for C14H25N8O10P‚2.1 H2O: C,
35.17; H, 6.16; N, 11.72. Found C, 35.08; H, 5.90; N, 11.48.
Eth yl 5-(Diben zylp h osp h on oxy)va ler a te (24). A mix-
ture of ethyl 5-bromovalerate (16) (0.87 g, 4.15 mmol) and
silver dibenzyl phosphate (1.6 g, 4.15 mmol) in toluene (50 mL)
was heated at reflux for 5 h under N2. After the mixture was
cooled, the precipitate was removed by filtration and the
filtrate was concentrated to give 24 (1.6 g, 95%) as a colorless
oil: 1H NMR (300 Hz, CDCl3) δ 7.35 (m, 10 H), 5.05-5.00 (m,
4 H), 4.14 (q, J ) 7.1 Hz, 2 H), 4.02 (m, 2 H), 2.29 (m, 2 H),
1.64 (m, 4 H), 1.26 (t, J ) 7.1 Hz, 3 H); 13C NMR (CDCl3) δ
173.1, 135.8, 128.5, 127.8, 69.1, 67.2, 60.2, 33.5, 29.4, 20.8, 14.1;
EIMS m/z 429 (100, MNa+). Anal. Calcd for C21H27O6P: C,
62.06; H, 6.70. Found C, 62.00; H, 6.79.
5-(Diben zylph osph on oxy)valer ic Acid (25).Ethyl5-(diben-
zylphosphono)valerate 24 (1.5 g, 3.6 mmol) and lithium
hydroxide (0.2 g, 8 mmol) were mixed in 1:1 THF-water (25
mL) for 5 h. THF was then removed under reduced pressure,
and the remaining aqueous solution was acidified with con-
centrated HCl and extracted with diethyl ether (3 × 50 mL).
The ether layer was washed with brine, dried over sodium
sulfate, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (1:2:0.1 hexane-AcOEt-
AcOH) to afford 25 (1.1 g, 81%) as a colorless oil: 1H NMR
(300 Hz, CDCl3) δ 7.37 (m, 10 H), 5.10-4.98 (m, 4 H), 4.02
(m, 2 H), 2.34 (m, 2 H), 1.67 (m, 4 H); 13C NMR (CDCl3) δ
177.9, 135.7, 128.5, 127.9, 69.3, 67.3, 33.2, 29.4, 20.6; EIMS
m/z 379 (100, MH+). Anal. Calcd for C19H23O6P: C, 60.32; H,
6.13. Found C, 60.45; H, 6.22.
5-(5-P h osp h on oxyva le r yl)a m in o-6-D -r ib it yla m in o-
u r a cil (26). 5-(Dibenzylphosphonoxy)valeric acid (25) (1.0 g,
2.6 mmol) was dissolved in dichloromethane (20 mL) at 0 °C
and a solution of oxalyl chloride in dichloromethane (2M, 2
mL), and two drops of DMF were added. After being removed
from the cooling bath and stirred at room temperature for an
additional 2 h, the reaction mixture was evaporated and then
coevaporated twice with dichloromethane (20 mL) to provide
the acid chloride as a colorless oil: 1H NMR (300 Hz, CDCl3)
δ 7.37 (m, 10 H), 5.10-4.98 (m, 4 H), 3.98 (q, J ) 6 Hz, 2 H),
2.82 (t, J ) 6 Hz, 2 H), 1.65 (m, 4 H). The acid chloride was
dissolved in dry acetonitrile (10 mL) and added dropwise to a
solution of 5-amino-6-D-ribitylaminouracil,17,23,24 freshly pre-
pared by catalytic reduction of 5-nitro-6-D-ribitylaminouracil
Lu m a zin e Syn th a se Assa y.25 Reaction mixtures contained
100 mM potassium phosphate, pH 7.0, 5 mM EDTA, 5 mM
dithiothreitol, inhibitor (0-86 µM), 170 µM 5-amino-6-ribity-
lamino-2,4(1H,3H)-pyrimidinedione (1), and lumazine syn-
thase (30 µg, specific activity 12.5 µmol mg-1 h-1) in a total
volume of 560 µL. Inhibitor concentrations ranged between 0
and 862 µM. The solution was incubated at 37 °C, and the
reaction was started by the addition of a small volume (20 µL)
of L-3,4-dihydroxy-2-butanone 4-phosphate to a final concen-
tration of 50-310 µM. The velocity/substrate data were fitted
for all inhibitor concentrations with a nonlinear regression
method using the program DynaFit.26 Different inhibition
models were considered for the calculation. Ki values (
standard deviations were obtained from the fit under consid-
eration of the most likely inhibition model.
Ribofla vin Syn th a se Assa y.27 Reaction mixtures con-
tained buffer (100 mM potassium phosphate, 10 mM EDTA,
10 mM sodium sulfite), inhibitor (0-87 µM), and riboflavin
synthase (10 µg, specific activity 50 µmol mg-1h-1). Inhibitor
concentrations ranged between 0 and 1.6 mM. After preincu-
bation, the reactions were started by the addition of various
amounts of 6,7-dimethyl-8-ribityllumazine (3) (20-200 µM) to
a total volume of 570 µL. The formation of riboflavin (4) was
measured online with a computer-controlled photometer at 470
nm (ꢀriboflavin ) 9100 M-1cm-1). The Ki evaluation was per-
formed in the manner described above.
Ack n ow led gm en t. This research was made possible
by NIH Grant GM51469 as well as by support from the
Deutsche Forschungsgemeinschaft and Fonds der Che-
mischen Industrie.
J O020144R
(25) Kis, K.; Bacher, A. J . Biol. Chem. 1995, 270, 16788-16795.
(26) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273.
(27) Eberhardt, S.; Richter, G.; Gimbel, W.; Werner, T.; Bacher, A.
Eur. J . Biochem. 1996, 242, 712-718.
J . Org. Chem, Vol. 67, No. 20, 2002 6877