Hydroperoxylation Mechanism
A R T I C L E S
for HEPD that is followed by a Criegee-like rearrangement. As
such, the enzyme has similarities with flavin dependent
bombardment (FAB) mass spectrometry for characterization of
synthetic compounds was performed by the University of Illinois
Mass Spectrometry Center using a Waters 70-SE-4F mass
2
1-24
Bayer-Villigerases,
except that the substrate for HEPD is
spectrometer.
at a lower oxidation level than the carbonyl-containing substrates
of these enzymes. The corollary of the hydroperoxylation
mechanism of HEPD is that the O-O bond of molecular oxygen
is not broken prior to substrate activation, as has been proposed
for the aforementioned nonheme iron enzymes MIOX and IPNS,
4
2
-HEP and HMP were synthesized as previously reported. The
following substrate analogues were synthesized according to
18
14
12
literature procedures: 2- OH-HEP, (2R)- and (2S)-HPP, 3-hy-
32
33
droxypropylphosphonate, 2-hydroxybutylphosphonate, 3-fluoro-
34
35
2
-hydroxypropylphosphonate, 2-fluoroethylphosphonate, phospho-
2
5
36 37
for a diiron(II/III) superoxide model complex, and for the
copper-dependent enzymes dopamine ꢀ monooxygenase, pep-
tidyl glycine R-hydroxylating monooxygenase, and galactose
noacetaldehyde, and1-hydroxyethylphosphonate. Ethylphosphonate
and aminoethylphosphonate were purchased from Sigma-Aldrich.
2-Nitrophenylhydrazine (NPH) was purchased from Acros Organics.
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) was pur-
chased from Chem-Impex (Wood Dale, IL). Dichloromethane was
2
6-30
oxidase.
We note that Seto and co-workers in pioneering
studies had proposed that phosphonoacetaldehyde was converted
2
distilled over CaH . All other chemicals were purchased from Sigma
to HMP via a Baeyer-Villiger like oxidation to OFHMP
31
Aldrich at the highest purity and used without further purification.
Synthesis of O-Formyl-HMP. A round-bottom flask equipped
with a magnetic stir bar and reflux condenser was charged with
followed by nonenzymatic hydrolysis. Although the molecular
details turn out to be different, the overall biosynthetic logic is
remarkably similar.
38
dibenzyl HMP (500 mg, 1.7 mmol), which was dissolved in
formic acid (3 mL) and heated under reflux while stirring overnight.
The solution was allowed to cool to 25 °C, and Et O (20 mL) was
2
Experimental Procedures
added. A solution of aqueous NaOH (1 M) was added dropwise
until a second layer formed. The layers were separated and water
was removed from the aqueous (bottom) layer under reduced
pressure to afford product as a white solid. This material was a 1:1
mixture of the desired product and HMP. OFHMP was relatively
General. HEPD was overexpressed in E. coli as an N-terminal
His -fusion protein and purified via affinity chromatography as
6
4
previously described. Protein concentrations were determined by
the method of Bradford using bovine serum albumin as the standard.
In addition, a theoretical extinction coefficient of 79 995 M- cm
1
-1
1
stable in buffered HEPES solution (25 mM, pH ) 7.5). H NMR
was calculated using Expert Protein Analysis System (ExPASy,
http://www.expasy.org) and concentrations determined using this
value were within ∼10% of the concentrations determined by the
Bradford assay. Thus, while small deviations from the reaction
stoichiometries reported herein are possible, they would not change
the interpretation of these studies. All activity assays were
performed in HEPES buffer (25 mM, pH ) 7.5) unless otherwise
noted. LC-MS was performed on an Agilent 1200 series quad pump
system equipped with a diode array detector and a G1956B mass
spectrometer with a multimode-electrospray/atmospheric pressure
chemical ionization (MM-ES+APCI) source. The column used for
separation was a Synergi 4 µ Fusion-RP 80A column (150 × 4.6
mm, 4 µm, Phenomenex Torrance, CA) using a flow rate of 0.5
mL/min. UV-visible absorption spectra were recorded on a Varian
Cary 4000 UV-vis spectrophotometer. GC-MS was performed on
an Agilent 6890N gas chromatograph equipped with an electron
impact (EI) ionization source.
(
500 MHz, D
2
2
O) δ 4.09 (d, J ) 8.5 Hz, 2H, CH ), 7.99 (s, 1H
13
CHO); C NMR (125 MHz, D
2
O) δ 59.4 (d, J ) 156.9 Hz), 163.5;
O) δ 13.7.
Synthesis of 1-Hydroxy-2,2,2-trifluoroethylphosphonate. Un-
3
1
P NMR (202 MHz, D
2
der a nitrogen atmosphere a round-bottom flask equipped with a
magnetic stir bar and reflux condenser was charged with com-
mercially available diethyl 1-hydroxy-2,2,2-trifluoroethylphospho-
nate (1 g, 4.2 mmol, 1 equiv), which was dissolved in dry DCM
(
10 mL). To the solution was added bromotrimethylsilane (2 mL,
5.1 mmol, 3.6 equiv) and the orange solution was heated under
reflux while stirring for 2 h. The solution was allowed to cool to
5 °C and solvent was removed under reduced pressure. The
resulting residue was taken up in 1:1 H O:EtOH (10 mL) and
solvent was removed under reduced pressure to afford product (663
1
2
2
1
mg, 3.7 mmol, 88%) as a tan oil. H NMR (500 MHz, D
2
O) δ
1
3
4
(
D
.09 (m); C NMR (125 MHz, D O) δ 27.76 (d, J ) 8.5), 66.01
2
31
19
m); P NMR (202 MHz, D
O) δ 17.34 (at); HRMS (FAB ) calcd for (C
80.9878 m/z found 180.9880 m/z.
Identification of Assay Products via LC-MS. Enzymatic assays
were quenched by adding H SO (30 mM) and the protein was
2
O) δ 11.13 (bs); F NMR (215 MHz,
NMR spectra were recorded on a Varian Unity 500 or Varian
Unity Inova 600 spectrometer. Proton and carbon chemical shifts
are reported in δ values relative to an external standard of 0.1%
+
+
2
2 4 3 4
H F O P+H )
1
3
tetramethylsilane in CDCl (0.00 ppm). Phosphorus shifts are
2
4
reported in δ values relative to an external standard of 85%
pelleted via microcentrifugation (1 min at 16.1g). The supernatant
was analyzed via LC-MS (positive mode) using aqueous 0.1%
formic acid as an isocratic mobile phase and scanning masses of
phosphoric acid (0.00 ppm). Fluorine shifts are reported in δ values
3
relative to an external standard of CFCl (0.00 ppm). Fast atom
5
0-500 m/z.
(
21) Ryerson, C. C.; Ballou, D. P.; Walsh, C. Biochemistry 1982, 21, 2644–
Identification of Assay Products via 31P NMR Spectro-
2
655.
scopy. HEPD was separated from the assay via centrifugal filtration
(
22) Kamerbeek, N. M.; Moonen, M. J.; Van Der Ven, J. G.; Van Berkel,
W. J.; Fraaije, M. W.; Janssen, D. B. Eur. J. Biochem. 2001, 268,
(Millipore Micron YM-30, 30 kDa nominal molecular weight limit).
D
2
O (20% v/v) was added to the flow-through and the 31P NMR
2
547–2557.
(
(
(
(
(
23) Sheng, D.; Ballou, D. P.; Massey, V. Biochemistry 2001, 40, 11156–
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(
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