Atropselective hydrolysis of chiral BiNoL-phosphate esters catalyzed by the phosphotriesterase from Sphingobium sp. TCM1

Article abstract of DOI:10.1021/acs.biochem.0c00831

The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1′-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb-PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, k_cat/K_m, of Sb-PTE toward the test phosphate triesters ranged from ～10 to 10⁵ M^?1 s^?1. The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.

Full text of DOI:10.1021/acs.biochem.0c00831

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Atropselective Hydrolysis of Chiral Binol-Phosphate Esters Catalyzed
by the Phosphotriesterase from Sphingobium sp. TCM1
Dao Feng Xiang, Tamari Narindoshvili, and Frank M. Raushel*
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ABSTRACT: The phosphotriesterase from Sphingobium sp.
TCM1 (Sb-PTE) is notable for its ability to hydrolyze a broad
spectrum of organophosphate triesters, including organophospho-
rus flame retardants and plasticizers such as triphenyl phosphate
and tris(2-chloroethyl) phosphate that are not substrates for other
enzymes. This enzyme is also capable of hydrolyzing any one of the
three ester groups attached to the central phosphorus core. The
enantiomeric isomers of 1,1′-bi-2-naphthol (BINOL) have become
among the most widely used chiral auxiliaries for the chemical
synthesis of chiral carbon centers. PTE was tested for its ability to
hydrolyze a series of biaryl phosphate esters, including mono- and
bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb-PTE was shown to be able to catalyze the hydrolysis of the
chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, kcat/Km, of Sb-PTE toward the test phosphate
triesters ranged from ∼10 to 105 M−1 s−1. The product ratios and stereoselectivities were determined for four pairs of
phosphorylated BINOL derivatives.
hosphotriesterases (PTEs) have been shown to be capable
Pof catalyzing the hydrolysis of organophosphates with an
unusual degree of regio- and stereoselectivity. For example, the
PTE first isolated from Pseudomonas dininuta (Pd-PTE)
catalyzes the hydrolysis of the SP enantiomer of compound 1
∼2 orders of magnitude faster than the hydrolysis of the RP
enantiomer with exclusive formation of p-acetylphenol as one
of the products.1 This observation has been interpreted to
mean that the active site of this enzyme binds (SP)-1 in a
manner that is more conducive to substrate turnover than it is
for the binding of (RP)-1. Consistent with this observation is
the fact that mutation of a single residue in the active site, Gly-
60, to an alanine enhances the stereoselective preference for
the hydrolysis of (SP)-1 by 5 orders of magnitude.2
Alternatively, mutation of His-257 in the active site to tyrosine
effectively eliminates any stereoselectivity, whereas construc-
tion of the I106G/F132G/H257Y mutant results in a mutant
enzyme that preferentially hydrolyzes the RP enantiomer of
compound 1 by 3 orders of magnitude. Thus, the identity of
only four residues (G60, I106, F132, and H257) in the active
site of this PTE can modulate the stereoselectivity by >8 orders
of magnitude.1
phenol.4 Remarkably, with the RP enantiomer, phenol is the
predominant reaction product (96%) with cyclohexanol at 4%
and no detectable formation of methanol. With Sb-PTE, the
product ratios are dictated by an earlier transition state,
effective protonation of the leaving group, and an active site
that accommodates any one of three possible substrate binding
orientations.4,5
Selective hydrolysis of structurally similar pairs of substrates
can also arise from compounds that are sterically restricted
from rotating about a carbon−carbon single bond.6 The most
common type of compound within this select group is 1,1′-bi-
2-naphthol (BINOL) as illustrated with (SP)-3 and (RP)-3 in
Scheme 1. These compounds are rotationally restricted with a
calculated t1/2 of 60 min at 220 °C in diphenyl ether solvent.7
Chemical syntheses of the R and S enantiomers of BINOL
have been reported, and individual enantiomers are available
commercially. Such compounds are widely used as chiral
auxiliaries in the chemical synthesis of chiral carbon centers.8
Enzymatic resolution of racemic BINOL esters has been
reported.9−14 Perhaps the most dramatic demonstration of this
achievement was obtained by Kazlauskas9 using cholesterol
The PTE from Sphingobium sp. TCM1 (Sb-PTE) also can
hydrolyze a broad spectrum of organophosphate triesters.
However, this enzyme is uniquely capable of hydrolyzing any
one of the three ester groups attached to the central
phosphorus core.3,4 Thus, with (SP)-2, 79% of the reaction
products occur via cleavage of methanol and 21% with
cyclohexanol. There is no detectable (<1%) formation of
Received: October 12, 2020
Revised: November 4, 2020
Published: November 9, 2020
https://dx.doi.org/10.1021/acs.biochem.0c00831
© 2020 American Chemical Society
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(S)-BINOL (0.28 g, 1.0 mmol, 1.0 equiv) in anhydrous
dichloromethane (8.0 mL) was added triethylamine (0.14 mL,
1.0 mmol, 1.0 equiv), and the mixture was stirred for 18 h at
room temperature (23 °C). The reaction mixture was filtered,
and after concentration, the residue was purified by silica gel
column chromatography (hexanes/ethyl acetate, 3:2) yielding
Scheme 1. Chiral Substrates for Phosphotriesterases
(compounds 1 and 2), Axially Restricted BINOL
(compound 3), and the Dipentanoyl Ester of Compound 3
(compound 4)
1
0.25 g (63%) of product after concentration: H NMR (400
MHz, CDCl3) δ 8.06 (d, J = 8.9 Hz, 1H), 7.96 (d, J = 7.9 Hz,
1H), 7.92 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.62
(d, J = 8.9 Hz, 1H), 7.54−7.48 (m, 1H), 7.41 (d, J = 8.6 Hz,
1H), 7.39−7.32 (m, 2H), 7.30−7.24 (m, 2H), 7.05 (d, J = 8.3
Hz, 1H), 6.08 (s, 1H), 3.60 (d, J = 11.4 Hz, 3H), 3.18 (d, J =
11.4 Hz, 3H); 31P NMR (160 MHz, CDCl3) δ −3.06 (s);
HRMS (ESI+) m/z [M + H]+ calcd for C22H20O5P 395.1048,
found 395.1038.
(R)-[1,1′-Binaphthalene]-2,2′-diyl Tetramethyl Bis-
(phosphate) (6). A stirred solution of (R)-BINOL (0.28 g,
1.0 mmol, 1.0 equiv) in anhydrous diethyl ether (7 mL) was
chilled to −78 °C, and 1.0 mL of LDA (2.0 M in THF, 2.0
mmol, 2.0 equiv) was added. The solution was stirred for 30
min and transferred via cannula to another flask at −78 °C that
contained dimethyl chlorophosphate (0.23 mL, 2.2 mmol,
96%, 2.2 equiv) in diethyl ether (20 mL). The cooling bath
was removed, and the reaction mixture was allowed to warm to
ambient temperature (23 °C) and stirred for 18 h. The
reaction mixture was filtered and concentrated, and the residue
purified by silica gel column chromatography (ethyl acetate)
yielding 0.25 g (50%) of product. Synthesis of the bi-
sphosphorylated binol product (6) required a stronger base,
relative to the synthesis of the monophosphorylated product
(5), presumably because of hindered access to the intermediate
esterase to selectively hydrolyze the S enantiomer of
compound 4 leaving the R enantiomer intact. From 200 g of
racemic 4, nearly 70 g each of (S)-4 and (R)-4 were isolated
with an enantiomeric excess of 99. In this report, we have
tested Sb-PTE for its ability to hydrolyze a series of biaryl
phosphate esters. These substrates include mono- and bis-
phosphorylated BINOL derivatives and cyclic phosphate
esters.
MATERIALS AND METHODS
■
General Materials. Tryptone was purchased from
Research Products International. General laboratory chemicals
and supplies were obtained from Sigma/Aldrich. The NMR
shift reagent Fmoc-Trp(Boc)-OH was obtained from Sigma/
Aldrich. Vivaspin columns (10 kDa) were purchased from GE
Healthcare.
1
after the first phosphorylation event: H NMR (400 MHz,
CDCl3) δ 7.90 (d, J = 9.2 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H),
7.67 (d, J = 9.2 Hz, 2H), 7.39−7.34 (m, 2H), 7.27−7.18 (m,
4H), 3.34 (d, J = 11.4 Hz, 6H), 3.09 (d, J = 11.4 Hz, 6H); 31
P
Expression and Purification of Sb-PTE. The wild-type
histidine-tagged Sb-PTE enzyme was expressed and purified
from Escherichia coli strain BL21(DE3) as previously
described.15 After purification, Sb-PTE was stored in 20 mM
HEPES buffer (pH 8.0), and 100 mM NaCl was added to
maintain protein stability. The protein purity was judged to be
>95% using Mini-PROTEIN TGX gel electrophoresis (Bio-
Rad). The yield of purified Sb-PTE was ∼10 mg from a 1.0 L
cell culture.
NMR (160 MHz, CDCl3) δ −5.31 (s); HRMS (ESI+) m/z [M
+ H]+ calcd for C24H25O8P2 503.1025, found 503.1010.
(S)-[1,1′-Binaphthalene]-2,2′-diyl Tetramethyl Bis-
(phosphate) (6). To a stirred solution of (S)-BINOL (0.28
g, 1.0 mmol, 1.0 equiv) in anhydrous diethyl ether (7 mL)
chilled to −78 °C was added 1.0 mL of LDA (2.0 M in THF,
2.0 mmol, 2.0 equiv). The solution was stirred for 30 min and
transferred via cannula to another flask at −78 °C that
contained dimethyl chlorophosphate (0.23 mL, 2.2 mmol,
96%, 2.2 equiv) in diethyl ether (20 mL). The cooling bath
was removed, and the reaction mixture was allowed to warm to
ambient temperature (23 °C) and stirred for 18 h. The
reaction mixture was filtered and concentrated, and the residue
purified by silica gel column chromatography (ethyl acetate)
yielding 0.25 g (50%) of product: 1H NMR (400 MHz,
CDCl3) δ 7.90 (d, J = 9.2 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H),
7.67 (d, J = 9.2 Hz, 2H), 7.39−7.34 (m, 2H), 7.27−7.18 (m,
Synthesis of (R)-2′-Hydroxy[1,1′-binaphthalen]-2-yl
Dimethyl Phosphate (5). To a stirred solution of dimethyl
chlorophosphate (0.11 mL, 1.0 mmol, 96%, 1.0 equiv) and
(R)-BINOL (0.28 g, 1.0 mmol, 1.0 equiv) in anhydrous
dichloromethane (8.0 mL) was added triethylamine (0.14 mL,
1.0 mmol, 1.0 equiv), and the mixture was stirred for 18 h at
room temperature (23 °C). The reaction mixture was filtered,
and after concentration, the residue was purified by silica gel
column chromatography (hexanes/ethyl acetate, 3:2) yielding
0.25 g (63%) of product: 1H NMR (400 MHz, CDCl3) δ 8.06
(d, J = 8.9 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 8.9
Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H),
7.54−7.48 (m, 1H), 7.41 (d, J = 8.6 Hz, 1H), 7.39−7.32 (m,
2H), 7.30−7.24 (m, 2H), 7.05 (d, J = 8.3 Hz, 1H), 6.08 (s,
4H), 3.34 (d, J = 11.4 Hz, 6H), 3.09 (d, J = 11.4 Hz, 6H); 31
P
NMR (160 MHz, CDCl3) δ −5.31 (s); HRMS (ESI+) m/z [M
+ H]+ calcd for C24H25O8P2 503.1025, found 503.1010.
(R)-Methyl 1,1′-Binaphthyl-2,2′-diylphosphate (7). To
a stirred solution of methyl dichlorophosphate (0.26 mL, 2.2
mmol, 85%, 1.1 equiv) and (R)-BINOL (0.57 g, 2.0 mmol, 1.0
equiv) in anhydrous dichloromethane (10 mL) was added
triethylamine (0.62 mL, 4.4 mmol, 2.2 equiv), and the mixture
was stirred for 18 h at room temperature (23 °C). The reaction
mixture was filtered, and after concentration, the residue was
purified by silica gel column chromatography (hexanes/ethyl
1H), 3.60 (d, J = 11.4 Hz, 3H), 3.18 (d, J = 11.4 Hz, 3H); 31
P
NMR (160 MHz, CDCl3) δ −3.06 (s); HRMS (ESI+) m/z [M
+ H]+ calcd for C22H20O5P 395.1048, found 395.1038.
(S)-2′-Hydroxy[1,1′-binaphthalen]-2-yl Dimethyl
Phosphate (5). To a stirred solution of dimethyl
chlorophosphate (0.11 mL, 1.0 mmol, 96%, 1.0 equiv) and
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acetate, 3:2) yielding 0.22 g (30%) of product after
concentration: H NMR (400 MHz, CDCl3) δ 8.06 (d, J =
−4.00 (s); HRMS (ESI+) m/z [M + H]+ calcd for C12H14O4P
253.0630, found 253.0632.
1
7.7 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.8 Hz, 1H),
7.54−7.48 (m, 3H), 7.43−7.30 (m, 4H), 4.01 (d, J = 11.4 Hz,
3H); 31P NMR (160 MHz, CDCl3) δ 3.83 (s); HRMS (ESI+)
m/z [M + H]+ calcd for C21H16O4P 363.0786, found
363.0780.
Activity Screening of Sb-PTE with Binol Esters. To
determine if Sb-PTE is capable of hydrolyzing the binol
phosphate esters, the hydrolysis products from compounds 5−
9 after incubation with Sb-PTE were analyzed by 31P NMR
spectroscopy. The enzymatic reaction for each substrate was
conducted in 50 mM HEPES buffer (pH 8.5) containing 100
μM MnCl2. DMSO [20% (v/v)] was included in the reactions
to increase the solubility of the binol esters. The reaction
mixtures were applied to a Vivaspin 500 column (GE
Healthcare) to remove the protein after the reaction mixture
was incubated (3−24 h) in glass vials at 30 °C. The 31P NMR
spectrum was obtained after the addition of 10 mM EDTA and
10% D2O. For each enzymatic reaction, a control without the
addition of Sb-PTE was conducted concurrently. The catalytic
activities of Sb-PTE toward the hydrolysis of compounds 5−9
and the ratios of multiple products from a single substrate were
determined.
Determination of the Enantioselective Activity of Sb-
PTE. To enable the identification of the specific enantiomers of
the phosphate esters hydrolyzed by Sb-PTE, 31P NMR analysis
was employed. The specific enantiomer being preferentially
hydrolyzed from a racemic mixture by Sb-PTE was identified
by isolating the remaining substrate from the reaction mixture
and then conducting 31P NMR analysis after the addition of
the chiral shift reagent Fmoc-Trp(Boc)-OH to the sample. For
compound 5, the reaction was conducted in a reaction volume
of 50 mL with 0.15 mM racemic 5, 100 μM MnCl2, 20%
DMSO, 50 mM HEPES (pH 8.0), and 0.6 μM Sb-PTE. The
progress of the reaction was monitored by removing small
aliquots and measuring the absorbance at 334 nm. At various
time points, a 10 mL sample was removed from the reaction
mixture and the unreactive substrate in the reaction mixture
was extracted using ethyl acetate (3 × 20 mL). The organic
phase was washed with water to remove DMSO and dried over
anhydrous Na2SO4, and the solvent removed by rotary
evaporation. The remaining substance was dissolved in 750
μL of CDCl3 with addition of 200 mg of Fmoc-Trp(Boc)-OH,
and the 31P NMR spectrum was then recorded. For compound
7, the enantiomer being preferentially hydrolyzed by Sb-PTE
was also determined by 31P NMR spectroscopy. Conditions
and purifications were as described above for compound 5
except 100 μM racemic compound 7 and 0.3 μM Sb-PTE were
used.
(S)-Methyl 1,1′-Binaphthyl-2,2′-diylphosphate (7). To
a stirred solution of methyl dichlorophosphate (0.26 mL, 2.2
mmol, 85%, 1.1 equiv) and (S)-BINOL (0.57 g, 2.0 mmol, 1.0
equiv) in anhydrous dichloromethane (10 mL) was added
triethylamine (0.62 mL, 4.4 mmol, 2.2 equiv), and the mixture
was stirred for 18 h at room temperature (23 °C). The reaction
mixture was filtered, and after concentration, the residue was
purified by silica gel column chromatography (hexanes/ethyl
acetate, 3:2) yielding 0.22 g (30%) of product after
1
concentration: H NMR (400 MHz, CDCl3) δ 8.06 (d, J =
7.7 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.8 Hz, 1H),
7.54−7.48 (m, 3H), 7.43−7.30 (m, 4H), 4.01 (d, J = 11.4 Hz,
3H); 31P NMR (160 MHz, CDCl3) δ 3.83 (s); HRMS (ESI+)
m/z [M + H]+ calcd for C21H16O4P 363.0786, found
363.0781.
(R)-Phenyl 1,1′-Binaphthyl-2,2′-diylphosphate (8). To
a stirred solution of phenyl dichlorophosphate (0.16 mL, 1.1
mmol, 95%, 1.1 equiv) and (R)-BINOL (0.28 g, 1.0 mmol, 1.0
equiv) in anhydrous dichloromethane (10 mL) was added
triethylamine (0.3 mL, 2.2 mmol, 2.2 equiv), and the mixture
was stirred for 18 h at room temperature (23 °C). The reaction
mixture was filtered and washed with 0.1 N HCl and a
saturated solution of NaHCO3 yielding 0.27 g (63%) of
1
product: H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.1 Hz,
1H), 8.04 (d, J = 9.6 Hz, 1H), 8.01−7.97 (m, 2H), 7.67(d, J =
8.7 Hz, 1H), 7.56−7.50 (m, 2H), 7.48 (d, J = 8.7 Hz, 1H),
7.46−7.29 (m, 8H), 7.27−7.21 (m, 1H); 31P NMR (160 MHz,
CDCl3) δ −3.44 (s); HRMS (ESI+) m/z [M + H]+ calcd for
C26H18O4P 425.0943, found 425.0931.
(S)-Phenyl 1,1′-Binaphthyl-2,2′-diylphosphate (8). To
a stirred solution of phenyl dichlorophosphate (0.16 mL, 1.1
mmol, 95%, 1.1 equiv) and (S)-BINOL (0.28 g, 1.0 mmol, 1.0
equiv) in anhydrous dichloromethane (10 mL) was added
triethylamine (0.3 mL, 2.2 mmol, 2.2 equiv), and the mixture
was stirred for 18 h at room temperature (23 °C). The reaction
mixture was filtered and washed with 0.1 N HCl and a
saturated solution of NaHCO3 yielding 0.27 g (63%) of
1
product: H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.1 Hz,
1H), 8.04 (d, J = 9.6 Hz, 1H), 8.01−7.97 (m, 2H), 7.67 (d, J =
8.7 Hz, 1H), 7.56−7.50 (m, 2H), 7.48 (d, J = 8.7 Hz, 1H),
7.46−7.29 (m, 8H), 7.27−7.21 (m, 1H); 31P NMR (160 MHz,
CDCl3) δ −3.44 (s); HRMS (ESI+) m/z [M + H]+ calcd for
C26H18O4P 425.0943, found 425.0931.
Determination of Extinction Coefficients. The ex-
tinction coefficients used to calculate the catalytic efficiency
of Sb-PTE toward the hydrolysis of substrates 5−8 were
determined in 50 mM HEPES at pH 8.5. Each substrate was
dissolved in DMSO and then diluted to 20−100 μM in 50 mM
HEPES (pH 8.5) containing 100 μM MnCl2 and 20% DMSO.
The ultraviolet−visible spectra of the solutions were recorded
between 240 and 400 nm. The same concentrations of the
substrate were completely hydrolyzed by Sb-PTE under the
same reaction conditions, and the spectrum was recorded. The
two spectra were subtracted from each other, identifying 334
Dimethyl Naphthalen-2-yl Phosphate (9). To a stirred
solution of dimethyl chlorophosphate (0.36 mL, 3.3 mmol,
96%, 1.1 equiv) and naphthalen-2-ol (0.43 g, 3.0 mmol, 1.0
equiv) in anhydrous dichloromethane (15 mL) was added
triethylamine (0.46 mL, 3.3 mmol, 1.1 equiv), and the mixture
was stirred for 18 h at room temperature (23 °C). The reaction
mixture was filtered and concentrated, and the residue purified
by silica gel column chromatography (hexanes/ethyl acetate,
nm as being optimal with a delta extinction coefficient, Δε334
,
of 3520 M−1 cm−1 for compound 5; 334 nm with a delta
extinction coefficient, Δε334, of 5240 M−1 cm−1 for compound
6; 310 nm with a delta extinction coefficient, Δε310, of 7400
M−1 cm−1 for compound 7; and 310 nm with a delta extinction
coefficient, Δε310, of 12450 M−1 cm−1 for compound 8.
1
2:1) yielding 0.41 g (54%) of a colorless oil: H NMR (400
MHz, CDCl3) δ 7.88−7.80 (m, 3H), 7.71−7.69 (m, 1H),
7.54−7.44 (m, 2H), 7.38 (dd, J1 = 2.1 Hz, J2 = 8.9 Hz, 1H),
3.91 (d, J = 11.4 Hz, 6H); 31P NMR (160 MHz, CDCl3) δ
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Scheme 2. Biaryl Phosphate Derivatives Tested as Substrates for Sb-PTE
Measurement of Kinetic Constants. Due to the limited
solubility of compounds 5−9, total hydrolysis with low
concentrations of the pure enantiomers of these substrates
was employed to determine the catalytic efficiency (kcat/Km) of
Sb-PTE for these substrates. In preliminary experiments, we
demonstrated that neither dimethyl phosphate (17) nor binol
(3) is inhibitory for the hydrolysis of an equivalent
concentration of diethyl p-nitrophenyl phosphate (paraoxon).
For compounds 5−8, total hydrolysis reactions were
conducted in a volume of 1.0 mL containing 50 mM HEPES
(pH 8.5), 100 μM MnCl2, and 20% DMSO. Stock solutions of
substrates were made in DMSO with final concentrations
between 5 and 100 μM in the actual assays. All reactions were
followed to completion in 1.0 cm cuvettes using a Molecular
Devices Spectramax 364 plus spectrophotometer. The fraction
of substrate hydrolyzed was plotted as a function of time. The
time courses were fitted to eq 1
RESULTS AND DISCUSSION
■
Catalytic Properties of Sb-PTE with Chiral Binol
Phosphate Esters. The Mn2+-dependent phosphotriesterase
from Sphingobium sp. strain TCM1 (Sb-PTE) was previously
reported to be capable of hydrolyzing a broad spectrum of
substrates, including organophosphate plasticizers, flame
retardants, and pesticides.16 Here we have measured the
catalytic properties of Sb-PTE for the hydrolysis of four pairs of
chiral binol phosphate esters (compounds 5−8) and achiral
building block 9 (Scheme 2). The enantiomeric preferences of
Sb-PTE for the hydrolysis of (R/S)-5 and (R/S)-7 were
determined by 31P NMR spectroscopy. The enantiomers of
racemic 5 were differentiated from one another using the chiral
shift reagent Fmoc-Trp(Boc)-OH, as illustrated in Figure 1A,
where (S)-5 resonates downfield of (R)-5 (Figure 1B). When
Sb-PTE is added to (R/S)-5, the S enantiomer is hydrolyzed
significantly faster than is the R enantiomer (Figure 1C−E).
The same approach was utilized for the hydrolysis of (R/S)-
7. The enantiomers of 7 were also differentiated from one
another by 31P NMR spectroscopy after the addition of the
chiral shift reagent Fmoc-Trp(Boc)-OH (Figure 2A). (S)-7
resonates at 3.665 ppm, whereas the (R)-7 enantiomer
resonates at 3.645 ppm (Figure 2B). The addition of Sb-
PTE demonstrated that this enzyme hydrolyzes the R
enantiomer significantly faster than the S enantiomer (Figure
1C,D). With racemic mixtures of compounds 6 and 8, there
were no significant differences in the overall rates of hydrolysis
of the enantiomers as measured by 31P NMR spectroscopy.
Determination of Product Ratios. Previously, it was
shown that Sb-PTE can hydrolyze any of the three ester groups
contained within a suitably activated organophosphate triester
substrate.3,4 Therefore, it was imperative to determine the
product ratios by 31P NMR spectroscopy for each of the
targeted substrates 5−9. The structures for the possible
products are depicted in Scheme 3. For substrate 5, the two
possible products are 3/17 and 10. For the hydrolysis of (R)-5,
dimethyl phosphate (17) is the major product (91%) and
product 10 (9%) is the minor product (Figure S1A,B). With
the (S)-5 enantiomer, the only product identified was dimethyl
phosphate (Figure S1C,D). For substrate 7, the two possible
products are 10 and 13. With the faster enantiomer, (R)-7, the
only observable product is 10, formed from the hydrolysis of
the naphthyl substituent to the phosphorus (Figure S2A,B). In
contrast, with the slower (S)-7 substrate, the two possible
products are formed in a 90:10 10:13 ratio (Figure S2C,D).
The enantiomers of substrate 8 are hydrolyzed at nearly the
same rate, and the two possible products are 14 and 13. With
F = a(1 − e−kt
)
(1)
where F is the fraction hydrolyzed, a is the magnitude of the
exponential phase, t is the time, and k is the rate constant. All
assays were performed in triplicate at a single fixed substrate
concentration (except where noted). The assays were
conducted at pH 8.5 because previous experiments have
shown that Sb-PTE exhibits optimal catalytic activity for the
hydrolysis of paraoxon above pH 8.0.5
For compound 9, the catalytic activities of Sb-PTE were
measured by anion exchange chromatography. The hydrolysis
of compound 9 was conducted in a reaction mixture
containing 100 μM compound 9 and 1.8 μM Sb-PTE in 50
mM HEPES (pH 8.5) containing 100 μM MnCl2 and 20%
DMSO. A 150 μL aliquot was injected onto a prepacked 1 mL
HiTrap Q HP anion exchange column (GE Healthcare) at
different time points to measure the change in product
formation at 255 nm. The elution buffer was 20 mM HEPES
(pH 8.5) containing 1.0 M NaCl. The reactions were followed
to completion, and the fraction of product formed was plotted
T
as a function of time. The value of (kcat/Km) was obtained
using eq 2
T(kcat/Km) = k/Et
(2)
When multiple products are observed, the specific value of kcat/
Y
Km for the formation of each product [X(kcat/Km) and (kcat/
Km)] was determined from the experimentally determined
product ratios.15
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initial products (5 and 11) can be hydrolyzed a second time,
potentially forming a mixture with 3/17, 10, and 12. With (S)-
6, the primary reaction product identified in the 31P NMR
spectrum of the reaction mixture is dimethyl phosphate (17) as
illustrated in panels C and D of Figure S4. There are, however,
two smaller resonances of equal intensity at −4.28 and −4.54
ppm (6.6% of the total phosphorus integration), which are due
to the separate resonances for the two phosphoryl groups of
product 11 (Figure 3C,D). These two resonances are also
found in the control sample incubated for the same amount of
time, but without the addition of enzyme (6.3% of total
phosphorus integration) as illustrated in Figure 3C and Figure
S4C. Therefore, the initial rate of formation of (S)-11 is <1%
of the rate of formation of (S)-5 from the hydrolysis of (S)-6.
For the enzymatic hydrolysis of (R)-6, the control spectrum
(Figure 3A and Figure S4A) also exhibits the non-enzymatic
formation of (R)-11 (6.1% of the phosphorus integration). In
the 31P NMR spectrum for the enzyme-catalyzed reaction
mixture, the relative integration for (R)-11 is 7.6% (Figure 3B
and Figure S4B). Because the subsequent enzymatic hydrolysis
of the initial product, (R)-5, is slower than the hydrolysis of
(R)-6, the accumulation of (R)-5 is observed at −4.39 ppm
(25.2%). A small amount of (R)-10 (2.4%) is observed at
−3.65 ppm. This product is formed from the enzymatic
hydrolysis of (R)-5. Therefore, for the enzymatic hydrolysis of
(R)-6 by Sb-PTE, the initial product is >98% (R)-5. There is
no evidence to suggest that either (R)-11 or (S)-11 is a
substrate for Sb-PTE.
Figure 1. 31P NMR spectra for the hydrolysis of (R/S)-5 by Sb-PTE.
(A) Racemic mixture of compound 5 in the presence of added Fmoc-
Trp(Boc)-OH. (B) Mixture of (S)-5 (−4.14 ppm) and (R)-5 (−4.27
ppm) in a molar ratio of 2:1. (C) One hour after the addition of Sb-
PTE to (R/S)-5. (D) Three hours after the addition of Sb-PTE to (R/
S)-5. (E) Eight hours after the addition of Sb-PTE to (R/S)-5. (F)
Spectrum E spiked with (R/S)-5.
For hydrolysis of naphthyl dimethyl phosphate (9), two
products are observed in the NMR spectrum of the reaction
mixture (Figure S5B). The primary product (16) results from
the hydrolysis of one of the two methyl groups from the
substrate, and the other product is dimethyl phosphate (17).
The two products are formed in an 81:19 ratio.
Measurement of Kinetic Constants. The kinetic
constants for the enzymatic hydrolysis of compounds 5−9
are listed in Table 1. Due to the limited solubility of
compounds 5−9 in water, the values of kcat/Km were
determined by measuring the first-order rate constant for the
complete hydrolysis of each substrate at low substrate
concentrations. Typical time courses for the hydrolysis of
compounds (S)-7 and (R)-7 are presented in Figure S6. The
value of kcat/Km was obtained by dividing the first-order rate
constant for hydrolysis by the enzyme concentration (eq 2).
The value of kcat/Km for hydrolysis of the S enantiomer of
compound 5 (5.3 × 102 M−1 s−1) is ∼72 times higher than that
for hydrolysis of the R enantiomer (7.4 M−1 s−1). In contrast,
the value of kcat/Km for hydrolysis of the R enantiomer of
compound 7 (1.1 × 104 M−1 s−1) is ∼58 times higher than that
for hydrolysis of the S enantiomer (1.9 × 102 M−1 s−1). The
values of kcat/Km for hydrolysis of (R)-8 and (S)-8 are 2.9 ×
104 and 6.4 × 104 M−1 s−1, respectively. The values of kcat/Km
for hydrolysis of (R)-6 and (S)-6 are 53 and 49 M−1 s−1,
respectively. Compound 9 has a kcat/Km of 1.1 × 103 M−1 s−1
when hydrolyzed by Sb-PTE.
Figure 2. 31P NMR spectra for the hydrolysis of (R/S)-7 by Sb-PTE.
(A) Racemic mixture of 7 in the presence of added Fmoc-Trp(Boc)-
OH. (B) Mixture of (S)-7 (at 3.665 ppm) and (R)-7 (at 3.645 ppm)
in a molar ratio of 2:1. (C) Ten minutes after the addition of Sb-PTE
to (R/S)-7. (D) One hundred ten minutes after the addition of Sb-
PTE to (R/S)-7. (E) Spectrum D spiked with racemic 7.
CONCLUSIONS
■
The eight chiral binol phosphate esters tested as potential
substrates for Sb-PTE were shown to be active. The values of
kcat/Km ranged from a low of ∼10 M−1 s−1 for (R)-5 to a high
that approached 105 M−1 s−1 for (S)-8. The best substrate
identified thus far for Sb-PTE is triphenyl phosphate with a
reported kcat/Km of 2 × 106 M−1 s−1.16 Stereoselective
(R)-8, the only observable product is 14 (Figure S3A,B).
However, with the (S)-8 enantiomer, the two products are
formed in a 93:7 (S)-14:(S)-13 ratio (Figure S3C,D).
The hydrolyses of the bisphosphorylated binol esters, (S)-6
and (R)-6, are a bit more complicated because the two possible
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Scheme 3. Structures of Products from the Hydrolysis of Substrates Depicted in Scheme 2
prefers the hydrolysis of the R enantiomer for hydrolysis of the
cyclic phosphate ester (compound 7). The three-dimensional
structure of Sb-PTE is that of a seven-bladed β-propeller with a
binuclear metal center embedded in the central core at the top
of the propeller.17 However, a structure of Sb-PTE has not
been determined in the presence of a bound ligand. The
experiments reported here demonstrate that Sb-PTE can be
used to kinetically resolve small quantities of racemic
phosphate esters from one another. No discrimination of the
stereoisomers was observed for the hydrolysis of compound 6
or 8.
Sb-PTE is known for its ability to hydrolyze any one of three
ester groups of an activated organophosphate triester
substrate.3,4 To the best of our knowledge, this is the only
known phosphotriesterase with this unique catalytic property.
For example, Sb-PTE hydrolyzes the RP enantiomer of
cyclohexyl, phenyl, p-nitrophenyl phosphate with the following
product distribution: phenol (55%), p-nitrophenol (38%), and
cyclohexanol (7%). With the SP enantiomer, the product
distribution is phenol (8%), p-nitrophenol (84%), and
cyclohexanol (7%). With the binol phosphate esters employed
here, the predominant product is always hydrolysis of the binol
ester. With (R)-5, (S)-7, and (S)-8, ≤10% of the alternate
product is observed. In contrast, with the naphthyl phosphate
ester (9), the predominant product is due to the hydrolysis of
either of the two methyl groups (81%). This observation must
reflect the differential binding of these compounds in the active
site of Sb-PTE. Given the broad substrate specificity of Sb-
PTE, it is likely that other axially chiral phenols will be
Figure 3. Selected region of the 31P NMR spectra for the product
from the Sb-PTE hydrolysis of (R)-6 and (S)-6. (A) Control sample
for (R)-6. (B) Product distribution after the addition of Sb-PTE. (C)
Control sample for (S)-6. (D) Product distribution after the addition
of Sb-PTE. The full 31P NMR spectra are provided in Figure S4.
Additional details are provided in the text.
hydrolysis was observed for hydrolysis of the enantiomers of
compounds 5 and 7. The S enantiomer of 5 was preferred by a
factor of 72, whereas with compound 7, the R enantiomer was
preferred by a factor of ∼58. Unfortunately, it is not possible at
this stage to rationalize why Sb-PTE prefers the S enantiomer
for hydrolysis of the acyclic phosphate ester (compound 5) but
a
Table 1. Kinetic Constants for the Hydrolysis of Compounds 5−9
substrate
product ratio X:Y
T(kcat/Ka) (M−1 s−1
)
X(kcat/Ka) (M−1 s−1
)
Y(kcat/Ka) (M−1 s−1
)
(R)-5 (50 μM)
(S)-5 (50 μM)
(R)-6 (50 μM)
(S)-6 (50 μM)
(R)-7 (25−100 μM)
(S)-7 (25−100 μM)
(R)-8 (5.0 μM)
(S)-8 (5.0 μM)
9 (100 μM)
(R)-3:(R)-10, 91:9
(S)-3:(S)-10, 100:0
(R)-5:(R)-11, 100:0
(S)-5:(S)-11, 100:0
(R)-10:(R)-13, 100:0
(S)-10:(S)-13, 90:10
(R)-14:(R)-13, 100:0
(S)-14:(S)-13, 93:7
15:16, 19:81
7.4 0.3
6.7 0.3
0.7 0.1
<10
<1
(5.3 0.2) × 102
(5.3 0.1) × 10
(4.9 0.1) × 10
(1.1 0.1) × 104
(1.9 0.1) × 102
(2.9 0.2) × 104
(6.4 0.2) × 104
(1.1 0.1) × 103
(5.3 0.2) × 102
(5.3 0.1) × 10
(4.9 0.1) × 10
(1.1 0.1) × 104
(1.7 0.2) × 102
(2.8 0.2) × 104
(6.0 0.2) × 104
(2.1 0.2) × 102
<1
<2 × 102
(1.9 0.2) × 10
<6 × 102
(4.5 0.5) × 103
(8.9 0.9) × 102
a
All enzymatic reactions were conducted in 50 mM HEPES (pH 8.5) containing 100 μM MnCl2 and 20% (v/v) DMSO at 30 °C. The
concentration of the substrate used for the kinetic measurements is provided in the first column.
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(9) Kazlauskas, R. J. (1989) Resolution of binaphthols and
spirobiindanols using cholesterol esterase. J. Am. Chem. Soc. 111,
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differentially recognized as substrates for this enzyme. To the
best of our knowledge, this is the only phosphatase that has
been demonstrated to differentially hydrolyze chiral binol
phosphate esters.
(10) Lin, G., Liu, S.-H., Chen, S.-J., Wu, F.-C., and Sun, H.-L. (1993)
"Triple enantioselection” by an enzyme-catalyzed transacylation
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.0c00831.
■
(11) Juarez-Hernandez, M., Johnson, D. V., Holland, H. L.,
McNulty, J., and Capretta, A. (2003) Lipase-catalyzed stereo-
selectivity resolution and desymmetrization of binaphthols. Tetrahe-
dron: Asymmetry 14, 289−291.
(12) Aoyagi, N., Ogawa, N., and Izumi, T. (2006) Effects of reaction
temperature and acyl group for lipase-catalyzed chiral binaphthol
synthesis. Tetrahedron Lett. 47, 4797−4801.
sı
NMR spectra of reaction products and time courses for
the hydrolysis of (S)-7 and (R)-7 (PDF)
(13) Takemoto, M., Suzuki, Y., and Tanaka, K. (2002)
Enantioselective oxidative coupling of 2-naphthol derivatives cata-
lyzed by Camellia sinensis cell culture. Tetrahedron Lett. 43, 8499−
8501.
(14) Sridhar, M., Vadivel, S. K., and Bhalerao, U. T. (1997) Novel
Horseradish peroxidase catalyzed enantioselective oxidation of 2-
naphthols to 1.1’-binaphthyl-2,2’-diols. Tetrahedron Lett. 38, 5695−
5696.
(15) Bigley, A. N., Narindoshvili, T., Xiang, D. F., and Raushel, F. M.
(2020) Stereoselective formation of multiple reaction products by the
phosphotriesterase from Sphingobium sp. TCM1. Biochemistry 59,
1273−1288.
Accession Codes
Sb-PTE, UniProt A0A077JBW9.
AUTHOR INFORMATION
Corresponding Author
■
Frank M. Raushel − Department of Chemistry, Texas A&M
University, College Station, Texas 77843, United States;
orcid.org/0000-0002-5918-3089; Email: raushel@
tamu.edu
Authors
(16) Xiang, D. F., Bigley, A. N., Ren, Z., Xue, H., Hull, K., Romo, D.,
and Raushel, F. M. (2015) Interrogation of the structure and catalytic
properties of the phosphotriesterase from Sphingobium sp. strain
TCM1: An Enzyme capable of hydrolyzing organophosphate flame
retardants and plasticizers. Biochemistry 54, 7539−7549.
(17) Mabanglo, M. F., Xiang, D. F., Bigley, A. N., and Raushel, F. M.
(2016) Structure of a novel phosphotriesterase from Sphingobium sp.
TM1: A familiar binuclear metal center embedded in a 7-bladed β-
propeller protein fold. Biochemistry 55, 3963−3974.
Dao Feng Xiang − Department of Chemistry, Texas A&M
University, College Station, Texas 77843, United States
Tamari Narindoshvili − Department of Chemistry, Texas
A&M University, College Station, Texas 77843, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.0c00831
Funding
This work was supported in part by grants from the National
Institutes of Health (GM 116894) and the Robert A. Welch
Foundation (A-840).
Notes
The authors declare no competing financial interest.
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