Inhibition of mandelate racemase by the substrate-intermediate-product analogue 1,1-diphenyl-1-hydroxymethylphosphonate

Article abstract of DOI:10.1016/j.bmcl.2005.06.060

Mandelate racemase has been studied as a paradigm for enzyme-catalyzed abstraction of a proton from carbon acids with relatively high pK_a values. 1,1-Diphenyl-1-hydroxymethylphosphonate is a substrate-intermediate- product analogue and is a mod

Full text of DOI:10.1016/j.bmcl.2005.06.060

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4342–4344
Inhibition of mandelate racemase
by the substrate–intermediate–product analogue
1,1-diphenyl-1-hydroxymethylphosphonate
*
Rodney K. M. Burley and Stephen L. Bearne
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5
Received 18 May 2005; revised 9 June 2005; accepted 13 June 2005
Available online 20 July 2005
Abstract—Mandelate racemase has been studied as a paradigm for enzyme-catalyzed abstraction of a proton from carbon acids with
relatively high pKa values. 1,1-Diphenyl-1-hydroxymethylphosphonate is a substrate–intermediate–product analogue and is a
modest competitive inhibitor of the enzyme (Ki = 1.41 0.09 mM), suggesting that simultaneous binding of the two phenyl groups
obviates mimicry of the aci-carboxylate function of the intermediate by the phosphonate group.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Mandelate racemase (MR; EC 5.1.2.2) from Pseudomo-
nas putida catalyzes the Mg2+-dependent 1,1-proton
transfer that interconverts the enantiomers of mandelate
via a two-base mechanism as shown in Scheme 1.1–5 MR
has been studied as a paradigm for enzymes that cata-
lyze rapid carbon–hydrogen bond cleavage of carbon
acids with relatively high pKa values.6
Our interest in understanding how protein–ligand inter-
actions within the active site of MR stabilize the transi-
tion state for a-proton abstraction led us to survey
a series of intermediate analogues as potential transi-
tion state or intermediate analogue inhibitors.7,8 We
identified a-hydroxybenzylphosphonate (a-HBP) as
a potent, competitive intermediate analogue inhibitor
(Ki = 4.7 lM) of MR.7 Recently, we demonstrated that
MR binds benzilate as
a
competitive inhibitor
(Ki = 0.67 mM) with an affinity similar to that exhibited
for substrate.9 Benzilate may be regarded as a substrate–
product analogue because it bears two phenyl groups
that mimic the phenyl groups of (R)- and (S)-mandelate
as if they were bound simultaneously to MR (i.e., in an
R-pocket and an S-pocket; see Scheme 2).
Scheme 1.
To further explore the topology of the active site, we
examined the interaction of 1,1-diphenyl-1-hydroxy-
methylphosphonate (DPHMP) with MR. This sub-
Keywords: Mandelate racemase; Inhibition; Phosphonate.
*
Corresponding author. Tel.: +1 9024941974; fax: +1
9024941355; e-mail: sbearne@dal.ca
Scheme 2.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.060

Rodney K. M. Burley, S. L. Bearne / Bioorg. Med. Chem. Lett. 15 (2005) 4342–4344
4343
strate–intermediate–product analogue combines the
structural features of a-HBP and benzilate.
(150 ng/mL). Apparent values of Vmax and Km were
determined from nonlinear regression analysis of
Michaelis–Menten plots, and the competitive inhibition
constants (Ki) were determined from plots of the appar-
ent Km/Vmax values versus inhibitor concentrations.13
All reagents were purchased from Sigma–Aldrich
Canada Ltd. (Oakville, ON, Canada). Dimethyl
DPHMP10 and DPHMP11 were prepared using modi-
1
fied literature procedures. Chemical shifts (d) for H,
MR binds benzilate with an affinity that is similar to
that which it exhibits for substrate, indicating that MR
can accommodate the simultaneous binding of two
phenyl groups within its active site.9 We therefore de-
signed DPHMP as an analogue of benzilate that incor-
porates the substrate and product characteristics
present in benzilate (i.e., two phenyl rings), and includes
the phosphonate group that, as is the case for a-HBP,
mimics the aci-carboxylate group of the putative
intermediate. Hence, DPHMP may be regarded as a
substrate–intermediate–product analogue (Scheme 2).
Surprisingly, MR bound DPHMP with an affinity that
was ꢀ3-fold less than that exhibited for substrate (Table
1) and 300-fold less than that exhibited for a-HBP. In
previous studies, we examined the pH dependence of
inhibition by a-HBP and showed that MR preferentially
binds the monoanionic form of a-HBP.7 This does not
appear to be true for DPHMP. At pH values 7.5 and
8.7, the relative affinities of MR for substrate and
DPHMP are not significantly different; however, the rel-
ative affinity for DPHMP is reduced 2-fold at pH 6.3,
suggesting that binding of the phosphonate monoanion
is not preferred relative to the dianion.
13C, and 31P NMR spectra are reported in ppm relative
to the calibrated deuterium lock signal. Circular dichro-
ism (CD) assays were conducted using a JASCO J-810
spectropolarimeter. Elemental analyses were conducted
by the Canadian Microanalytical Service Ltd. (Delta,
BC, Canada). MR was overexpressed, purified, and
assayed as described previously.12 Inhibition assays con-
taining DPHMP (1.5–6.0 mM) were conducted at 25 ꢁC
in Na+–Hepes buffer (0.1 M, pH 7.5) containing MgCl2
(3.3 mM), (R)-mandelate (0.25–10.0 mM), and MR
Table 1. Inhibition of MR by DPHMP
Inhibitor
Ki (mM)a
1,1-Diphenyl-1-
hydroxymethylphosphonate
1.41 ( 0.09)
(DPHMP)
pH 6.3
5.91 ( 0.04)
6.93 ( 0.04)
0.0047 ( 0.0007)b
pH 8.7
(R,S)-a-Hydroxybenzylphosphonate
(a-HBP)
Substrate
Km (mM)a
Ki/Km
A stereoelectronic feature of the ligands that must be
considered is their angular charge distribution.7,15 The
negative charges on the carboxylate of (R)-mandelate
and on the aci-carboxylate of the intermediate (Scheme
3A and B) are rotationally symmetrical about the line
that bisects the angle made by the carboxyl carbon and
the two anionic oxygens. This line also makes an angle
(R)-Mandelate
pH 6.3
pH 8.7
0.44 ( 0.01)
0.95 ( 0.03)
2.02 ( 0.06)
3.2 ( 0.2)
6.2 ( 0.2)
3.4 ( 0.1)
a Values are the means of three experiments; standard deviation is
given in parentheses. All values determined at pH 7.5 unless indicated
otherwise.
b Value from Ref. 14.
Scheme 3.

4344
Rodney K. M. Burley, S. L. Bearne / Bioorg. Med. Chem. Lett. 15 (2005) 4342–4344
6. Gerlt, J. A.; Gassman, P. G. Biochemistry 1993, 32, 11943.
7. St. Maurice, M.; Bearne, S. L. Biochemistry 2000, 39,
13324.
8. St. Maurice, M.; Bearne, S. L.; Lu, W.; Taylor, S. D.
Bioorg. Med. Chem. Lett. 2003, 13, 2041.
9. Siddiqi, F.; Bourque, J. R.; Jiang, H.; Gardner, M.; St.
Maurice, M.; Blouin, C.; Bearne, S. L. Biochemistry 2005,
44, 9013.
of 180 ꢁ relative to the bond between the carboxyl carbon
and the a-carbon. The negative charge of the a-HBP
monoanion is not rotationally symmetrical with respect
to such a line. Consequently, it has been suggested that
MR binds the a-HBP monoanion in a skewed orienta-
tion so that the orientation of the vector of the negative
charge of the monoanionic phosphonate function aligns
with that of the carboxylate group of the substrate or aci-
carboxylate intermediate (Scheme 3C).7 This require-
ment for phosphonate binding may account for why
a-HBP is an inhibitor and not a substrate.7 It may also
permit the phenyl ring of a-HBP to closely approximate
the binding orientation assumed by the phenyl ring of the
planar intermediate. The observed weak binding of
DPHMP, relative to a-HBP, suggests that binding of
DPHMP in a skewed orientation similar to a-HBP and
the simultaneous binding of the two phenyl groups with-
in the active site of MR is not favored.
10. Dimethyl
1,1-diphenyl-1-hydroxymethylphosphonate
(dimethyl DPHMP) was prepared following a procedure
similar to that described by Maeda et al. for the
corresponding diethyl phosphonate diester.16 Dimethyl
benzoylphosphonate17 (3.21 g, 15 mmol) in dry THF
(30 mL) was cooled to À78 ꢁC for 10 min. Phenylmagne-
sium bromide (16.5 mL of a 1.0 M solution in THF) was
added dropwise and after the slow addition was complete,
the reaction was stirred for 30 min at À78 ꢁC. The reaction
mixture was then allowed to warm to room temperature
and quenched by addition of satd aq NH4Cl (25 mL) and
stirred for 10 min. The entire reaction mixture was then
added to satd aq NH4Cl (400 mL) and stirred for an
additional 10 min. The mixture was extracted with CH2Cl2
(3 · 50 mL) and the combined extracts were washed with
satd NaCl (50 mL) and dried over anhydrous Na2SO4.
The CH2Cl2 was removed in vacuo giving a yellow-white
solid. This solid was transferred to a sintered glass funnel
and washed with cold THF to give a white solid (1.05 g,
24%): mp 167–169 ꢁC (lit.18 171 ꢁC); 1H NMR (CDCl3,
250 MHz, d) 7.28–7.37 (m, 6H), 7.68–7.71 (m, 4H), and
3.65 (d, J = 10.5 Hz, 6H); 13C NMR (1H-decoupled,
CDCl3, 500 MHz, d) 54.24 (d, J = 7.5 Hz), 78.75 (d,
J = 160.2 Hz), 127.09 (d, J = 5.3 Hz), 127.86, 128.19, and
141.19 (d, J = 2.6 Hz). 31P NMR (1H-decoupled, CDCl3,
500 MHz, d) 22.81. Anal. Calcd for C, 61.63; H, 5.87; P,
10.60. Found: C, 61.60; H, 6.14; P, 10.97.
For example, either the added steric bulk of the second
phenyl group or the simultaneous binding of the two
phenyl groups in their respective ground state R- and
S-pockets, may cause the phosphonate to bind with an
orientation similar to the substrate carboxylate (i.e., with
rotationally symmetrical negative charge as in Scheme
3D). Consequently, the ability of DPHMP to mimic
the intermediate/transition state would be lost, although
the additional negative charge on dianionic DPHMP
may enhance binding to some degree. It is the presence
of only the single phenyl group on a-HBP that permits
this inhibitor to adopt the appropriate conformation
upon binding such that it can better mimic the interme-
diate or transition state. Hence, substrate–product fea-
tures of an inhibitor may enforce a binding mode that
obviates the ability of another functional group to mimic
transition state/intermediate characteristics.
11. Disodium
1,1-diphenyl-1-hydroxymethylphosphonate
(DPHMP) was prepared by slowly adding trimethylsilyl-
bromide (3.0 mL, 22.8 mmol) to a solution of dimethyl
DPHMP (1.0 g, 3.42 mmol) in dry CH3CN (30 mL). After
refluxing the solution for 30 min under argon, the solvent
was removed in vacuo. Methanol (10 mL) and water
(10 mL) were added and then removed in vacuo leaving a
light brown oil. CH2Cl2 (20 mL) was added to this oil
which produced the phosphonic acid as a white solid after
10–20 min (0.85 g, 94%). The phosphonic acid (0.5 g,
1.89 mmol) was dissolved in ethanol/water (1:1, 10 mL
each) and passed through an AG 50-X8 column (3 · 15 cm,
Na+-form). Fractions containing the phosphonate were
combined and the water was removed using lyophilization
Acknowledgments
We thank the Natural Sciences and Engineering Re-
search Council (NSERC) of Canada for support
through a discovery grant (S.L.B.) and an undergradu-
ate summer research award (R.K.M.B.). We thank
Bob Berno of the Atlantic Regional Magnetic Reso-
nance Centre for assistance in obtaining NMR spectra.
1
to yield a white solid (0.50 g, 86%): mp 157 ꢁC (dec); H
NMR (D2O, 250 MHz, d) 7.35–7.38 (m, 6H), 7.58–7.61 (m,
4H); 13C NMR (1H-decoupled, D2O, 500 MHz, d) 79.06 (d,
J = 154 Hz), 127.43 (d, J = 5 Hz), 127.52, 128.14, and
142.88. 31P NMR (1H-decoupled, D2O, 500 MHz, d) 18.35.
Anal. Calcd for C, 50.66; H, 3.60; P, 10.05. Found: C,
51.07; H, 4.54; P, 11.37.
References and notes
1. Landro, J. A.; Gerlt, J. A.; Kozarich, J. W.; Koo, C. W.;
Shah, V. J.; Kenyon, G. L.; Neidhart, D. J.; Fujita, S.;
Petsko, G. A. Biochemistry 1994, 33, 635.
12. Bearne, S. L.; St. Maurice, M.; Vaughan, M. D. Anal.
Biochem. 1999, 269, 332.
2. Landro, J. A.; Kallarakal, A. T.; Ransom, S. C.; Gerlt, J.
A.; Kozarich, J. W.; Neidhart, D. J.; Kenyon, G. L.
Biochemistry 1991, 30, 9274.
3. Neidhart, D. J.; Howell, P. L.; Petsko, G. A.; Powers, V.
M.; Li, R. S.; Kenyon, G. L.; Gerlt, J. A. Biochemistry
1991, 30, 9264.
4. Powers, V. M.; Koo, C. W.; Kenyon, G. L.; Gerlt, J. A.;
Kozarich, J. W. Biochemistry 1991, 30, 9255.
5. Gerlt, J. A. In Bioorganic Chemistry: Peptides and
Proteins; Hecht, S. M., Ed.; Oxford University Press:
New York, 1998, pp 279–311.
13. Segel, I. H. Enzyme Kinetics; John Wiley and Sons: New
York, 1975, pp 100–111.
14. St. Maurice, M.; Bearne, S. L. Biochemistry 2004, 43,
2524.
15. Bearne, S. L.; Kluger, R. Bioorg. Chem. 1992, 20, 135.
16. Maeda, H.; Takahashi, K.; Ohmori, H. Tetrahedron 1998,
54, 12233.
17. Berlin, K. D.; Taylor, H. A. J. Am. Chem. Soc. 1964, 86,
3862.
18. Texier-Boullet, F.; Lequitte, M. Tetrahedron Lett. 1986,
27, 3515.