Absolute stereochemistry and preferred conformations of urate degradation intermediates from computed and experimental circular dichroism spectra

Article abstract of DOI:10.1039/c1ob05433c

The enzymatic oxidation of urate leads to the sequential formation of optically active intermediates with unknown stereochemistry: (-)-5-hydroxyisourate (HIU) and (-)-2-oxo-4-hydroxy-4-carboxy-5- ureidoimidazoline (OHCU). In accordance with the observation that a defect in HIU hydrolase causes hepatocarcinoma in mouse, a detoxification role has been proposed for the enzymes accelerating the conversion of HIU and OHCU into optically active (+)-allantoin. The enzymatic products of urate oxidation are normally not present in humans, but are formed in patients treated with urate oxidase. We used time-dependent density functional theory (TDDFT) to compute the electronic circular dichroism (ECD) spectra of the chiral compounds of urate degradation (HIU, OHCU, allantoin) and we compared the results with experimentally measured ECD spectra. The calculated ECD spectra for (S)-HIU and (S)-OHCU reproduced well the experimental spectra obtained through the enzymatic degradation of urate. Less conclusive results were obtained with allantoin, although the computed optical rotations in the transparent region supported the original assignment of the (+)-S configuration. These absolute configuration assignments can facilitate the study of the enzymes involved in urate metabolism and help us to understand the mechanism leading to the toxicity of urate oxidation products. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob05433c

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PAPER
Absolute stereochemistry and preferred conformations of urate degradation
intermediates from computed and experimental circular dichroism spectra†
Silvio Pipolo,‡a Riccardo Percudanib and Roberto Cammi*a
Received 18th March 2011, Accepted 19th April 2011
DOI: 10.1039/c1ob05433c
The enzymatic oxidation of urate leads to the sequential formation of optically active intermediates
with unknown stereochemistry: (-)-5-hydroxyisourate (HIU) and (-)-2-oxo-4-hydroxy-4-carboxy-5-
ureidoimidazoline (OHCU). In accordance with the observation that a defect in HIU hydrolase causes
hepatocarcinoma in mouse, a detoxification role has been proposed for the enzymes accelerating the
conversion of HIU and OHCU into optically active (+)-allantoin. The enzymatic products of urate
oxidation are normally not present in humans, but are formed in patients treated with urate oxidase. We
used time-dependent density functional theory (TDDFT) to compute the electronic circular dichroism
(ECD) spectra of the chiral compounds of urate degradation (HIU, OHCU, allantoin) and we
compared the results with experimentally measured ECD spectra. The calculated ECD spectra for
(S)-HIU and (S)-OHCU reproduced well the experimental spectra obtained through the enzymatic
degradation of urate. Less conclusive results were obtained with allantoin, although the computed
optical rotations in the transparent region supported the original assignment of the (+)-S
configuration. These absolute configuration assignments can facilitate the study of the enzymes
involved in urate metabolism and help us to understand the mechanism leading to the toxicity
of urate oxidation products.
The enzymatic pathway for the oxidative degradation of urate to
1
Introduction
allantoin occurs in bacteria, fungi, plants, and animals, including
most mammals. Among mammals, notable exceptions are humans
and apes. In these organisms the pathway is truncated due
to inactivation of the three genes involved in urate catabolism
(urate oxidase, HIU hydrolase, OHCU decarboxylase).2 As a
consequence, the levels of urate in human serum are tenfold higher
than those in other primates. Uric acid is only marginally soluble,
and its salt can precipitate in joints to cause gout and in the
kidney to cause renal stones. An elective treatment for abnormally
high levels of uric acid (hyperuricemia)—particularly for the
surge of uric acid that characterizes the tumor lysis syndrome—
is the intravenous administration of urate oxidase.3 In normal
conditions, small amounts of the urate oxidation products can
be formed in humans by chemical oxidation. Following the urate
oxidase treatment, however, the unstable intermediates of uric acid
oxidation are formed in significant amounts in the patient’s blood
serum (the blood serum urate of patients subjected to the urate
oxidase treatment can be as high as 14 mg dL-1). In humans,
the physiological consequences of the presence of such chemicals
species are presently unknown, but recent studies in mouse indicate
that HIU has strong toxic effects leading to hepatocarcinoma in
the absence of a functioning HIU hydrolase.4
The assignment of the absolute configuration of chiral species
involved in enzymatic reactions is essential for the understanding
of biochemical processes. In this paper we present an experi-
mental and computational study of the absolute configurations
of intermediates involved in the enzymatic degradation of uric
acid. Recent studies have been made on the purine degradation
pathway (Scheme 1), supporting the presence of additional steps
in the enzymatic conversion of uric acid (a purine derivative)
to allantoin: oxidation of urate by the enzyme urate oxidase
yields optically active 5-hydroxyisourate (HIU)*; the enzymatic
or spontaneous hydrolysis of HIU yields optically active 2-oxo-
4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU), an unstable
intermediate that is converted into optically active allantoin by
OHCU decarboxylase, or into racemic allantoin by spontaneous
decay.1
aDipartimento di Chimica “G.I.A.F”, Universita´ di Parma, 43100, Parma,
Italy. E-mail: roberto.cammi@unipr.it; Fax: +39 0521905557; Tel: +39
0521905442
bDipartimento di Biochimica e Biologia Molecolare, Universita´ di Parma,
43100, Parma, Italy
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05433c
‡ Present address: Dipartimento di Fisica, Universita´ di Modena, 41125
Modena, Italy; S3 Center CNR-NANO, 41125 Modena, Italy
Complete information on the absolute configuration of the
intermediates involved in the oxidation of the urate pathway is
presently lacking. The absolute configuration for the allantoin
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Scheme 1 The mammalian urate degradation pathway. HIU and OHCU isomers are drawn according to the results presented in this work.
produced during purine catabolism has already been proposed,
with the assignment of the S configuration to the (+) enantiomer
based on CD spectra comparison.5 Although this assignment
has never been called into question in the literature,1,6,7 no
direct crystallographic evidence is available. Recently, specific
13C-labeling of allantoin, and NMR monitoring of the reaction
catalyzed by allantoate amidohydrolase, an enzyme with known
stereospecificity, have provided support to this assignment.8 The
intermediate species (HIU, OHCU) of the urate degradation
pathway are unstable and establishing their absolute configuration
is a challenge.
Recent developments in computational methods based on the
time-dependent density functional theory (TDDFT)9 for the
calculation of chiroptical properties such as optical rotatory
dispersion (ORD) and electronic circular dichroism (ECD) have
increased their reliability to determine the absolute configuration
of chiral molecules in vitro.10–13 The absolute configuration is then
assigned on the basis of the agreement between both computed
and experimentally measured ORD and ECD spectra. In principle,
results from only ORD or only ECD may be used for the
assignment of the absolute configuration, however the assignment
of absolute configuration is more robust when consistent results
are obtained with both methods. Usually, one looks for a
semi-quantitative agreement, as quantitative differences may be
introduced by the limits of TDDFT inherent to the choice of
the density functional/basis set, and from the non-consideration
of the solvent effects on both conformational flexibility and
on the molecular electronic responsive properties (i.e. rotational
strengths and excitation energies14,15) determining the ORD and
ECD spectra.
Solvent effects may be introduced by using the Polarizable
Continuum Model (PCM),16 in which the molecular solute treated
at Quantum Mechanical (QM) level is placed in a cavity within
the solvent represented as a dielectric medium characterized by its
dielectric permittivity. Within the PCM framework, the ORD and
ECD spectra of the chiral solutes may be computed considering
both the solvent effect on the conformational equilibrium and
both the effects on rotational strengths and excitation energies by
exploiting the specific formulation of the TDDFT developed for
PCM methods.17,18 The TDDFT-PCM scheme provides a good
picture of the ORD and ECD phenomena in solution,19–23 and
we exploit this methodology in order to assign absolute configu-
rations of HIU, OHCU and allantoin within the urate degrada-
tion.
2
Experimental and computational procedures
Approximate ECD spectra of HIU, OHCU, and allantoin in
the 200–350 nm range have been reported previously.2 For the
purpose of this work, accurate ECD spectra of the enzymatic
products of urate degradation were collected in the 190–550
nm range. ECD measurements were carried out in a 10 mm
pathlength cuvette with a Jasco J715 spectropolarimeter; data were
collected at 0.5 nm intervals in 1 ml solution of 20 mM potassium
phosphate, pH 7.6. HIU, OHCU, and allantoin were generated
enzymatically from urate (0.1 mM) using 0.1 g of urate oxidase
from Candida utilis (Sigma), plus 0.022 g of recombinant HIU
hydrolase from zebrafish (for OHCU, allantoin), plus 0.17 g of
recombinant OHCU decarboxylase from zebrafish (for allantoin).
The experimental ORD spectra were obtained as Kramers-Kronig
(KK) transform of the measured ECD spectra using the utility
implemented in the Jasco Spectra Manager Software.
Theoretical simulations of chiro-optical properties (ECD and
ORD) were carried out as the weighted sum of the computed
chiro-optical responses Pi(n) of the various isomers present in
solution:
n
(1)
P(n) =
xiP (n)
i
∑
i=1
where xi is the fractional population of the ith isomer. The chiro-
optical responses Pi(n) (i.e. the specific rotation [ai(n)] for ORD
and the differential dichroic absorption Dei(n) for ECD) were
computed using the TDDFT theory, and the effects of the solvent
are treated using PCM formalism.
All the studied systems present in solution amino–imino
tautomerism and/or conformational mobility (OHCU and allan-
toin). The corresponding molecular structures were determined
by a systematic computational procedure at the DFT/B3LYP24,25
level using the 6-31G** basis set26 with the Polarizable Continuum
Model (PCM) for solvation. In this procedure, the tautomeric
species were selected from the most stable tautomers, within a
window of 10 kcal/mol, of the various amino–imino forms in the
gas phase. The equilibrium geometries of the selected tautomers
were optimized in solution at the PCM/B3LYP/6-31G** level.
For the selected tautomers of OHCU and allantoin a conforma-
tional analysis was performed, at the same level of theory, using
both scan-relaxed and simple-scan procedures27 in order to locate
stable conformers as minima of the corresponding molecular po-
tential energy surface. All the resulting minimum structures were
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re-optimized without constraints, and confirmed as stable struc-
tures by the analysis of the Hessian matrix. The fractional
populations of all the molecular species in solution (tau-
tomers/conformers) were calculated with a Boltzmann approach:
where (sl) is the half-height width of the gaussian function (set to
0.25 eV), the rotatory strengths, R0i, are expressed in 10-40 esu2 cm2
and the differential absorption molar coefficient, De, is expressed
in L mol-1 cm-1.
All the calculations were carried out with a development
version of the Gaussian package.31 The atomic coordinates of the
equilibrium geometries and the computed rotatory strengths are
reported in the ESI.†
/ RT
e−DG
1,i
xi =
(2)
−DG / RT
1,i
n
e
i=1
∑
where DG1,i is the standard free energy of the i-th conformer,
relative to the most stable conformer (denoted by 1), and it is
evaluated as
3
Results and discussions
3.1 Tautomerism and conformational analysis
(3)
DG1,i = Gi–G1
A critical issue for exploring the chiro-optical properties of
enantiomers in solution is to obtain a careful description of the
different tautomers and conformers that may be present in solution
under the experimental conditions. The Boltzmann population of
the different chemical species in solution has been computed using
the density functional theory with a polarizable continuum model
for solvation (See eqn (2) of Computational Procedures).
where Gi is the standard free energy of the i-th conformer in
solution, relative to a reference state given by non-interacting
electrons and nuclei and by the unperturbed solvent. The standard
free energy G1,i for each species was computed as the sum of the
standard free energy in solution at 0 K, the unscaled zero point
energy and the change of the free energy from 0 to 298 K. For
each conformer, the standard free energy in solution at 0 K has
been evaluated using the PCM/B3LYP theory and the AUG-
cc-pVDZ28 basis set. The zero-point energies and the thermal
contributions were computed using the PCM/B3LYP theory with
the 6-31G** basis set. In all the optimization and free-energy
calculations in solution phase we employed the PCM model using
the integral equation formulation (IEF-PCM),29 with a solvent
excluding surface cavity model16 and UFF atomic radii30 with
scaling factor a = 1.31
The single point calculations of the chiro-optical properties
of the various species in solution were computed using the
PCM-TDDFT theory in a non-equilibrium framework16 with the
AUG-cc-pVDZ basis set using two different exchange-correlation
functionals, B3LYP and CAM-B3LYP.32 Only species with relative
population values higher than 5% were considered. The choice
of the two different functionals is justified by the fact that the
CAM-B3LYP functional is found to be particularly promising
for the description of Rydberg-type electronic states that are
frequently involved in the electronic transitions of the studied
chromophores.33 In all the single point PCM-TDDFT calculations
we employed the scaling factors a of the UFF atomic radii
optimized in a previous work on a set of molecular systems
similar34 to that considered in this work. The optimized scaling
factors a are 0.91 for neutral species and 0.83 for anions. In the
present work the solvent effect on the rotational strengths and
on the optical rotations due to the local field correction were not
considered.35
3.1.1 HIU isomers. Under the experimental conditions
(pH = 7.6) HIU is present as a monoanionic species (see Schemes
S1, S2 and Table S1 in the ESI† for details of the pKa calculations)
which may present tautomerism due to proton transfer.37,38 In
order to obtain a systematic description of the tautomeric struc-
tures of HIU, a tri-anionic species (see Scheme S3 in the ESI†) was
chosen as the starting point, and from the analysis of its molecular
electrostatic potential function computed at the B3LYP/6-31G**
level 19 tautomers were selected. Their equilibrium geometries
were computed in the gas phase, and finally, the five most stable
structures were optimized in solution and considered for study of
the fractional tautomeric populations in solution.
The values of free energy differences show a full predominance
of the HIU7,1 isomer (Fig. 1). On the basis of these fractional
populations (Table 1) only the HIU7,1 isomer was considered in
the calculations of the ECD and ORD spectra of HIU.
Optical rotations were calculated using a gauge-invariant for-
malism (GIAO).36 Rotational strengths were computed by means
of both length and velocity formalisms, to check a posteriori the
degree of gauge-invariance. In all cases the rotational strengths
obtained with the two formalisms do not differ significantly, so we
only report the results for the length formalism.
The simulated ECD spectra were than obtained by super-
position of Gaussian functions centered on each absorption
wavelength (l0i):
2
2
l
1
1
)
/ 2s
]
l0iR0ie[−(l−l
Fig. 1 HIU7,1, OHCU3,6, OHCU1,3, ALL-T1, ALL-T2, S isomers. Atomic
coordinates are available in the ESI.†
0i
De(l) =
(4)
2.294×10−39
∑
sl 2p
i
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Fig. 2 Experimental and computed HIU spectra. Computed spectra (red) for the HIU7,1 isomer were calculated at the PCM/TDDFT/B3LYP level.
ORD spectra are shown in the inset. Red solid and red dotted lines refer to the S and R configurations of HIU7,1, respectively.
Table 1 Gibbs free energy differences in kcal mol-1 and relative popula-
tions of HIU, OHCU and allantoin isomers in solution
liminary B3LYP/6-31G** calculations in the gas phase only a
chemically relevant tautomer was obtained. This tautomeric form
is characterized by a high conformational flexibility, as also noted
HIU7,1
OHCU1,3
OHCU3,6
ALL-T1
ALL-T2
in a previous ab initio HF study on allantoin,6 due to the torsion
angles involving atoms C5N6C7N8 and N1C5N6C7, respectively
(Fig. 1).
DG
xi
0.0
1.00
0.0
0.57
0.2
0.43
0.0
0.93
7.1
0.05
By exploiting a scan-relaxed procedure six low energy conform-
ers are located in the gas phase and all of them were considered for
the study in solution. The fractional populations of the dominant
conformers are reported in Table 1. The ALL-T1 conformer
is found to be the predominant species in aqueous solutions,
but ALL-T2 shows a significant, although numerically small,
population. Therefore, the chiro-optical properties of allantoin
were computed considering the presence of these two conformers.
3.1.2 OHCU isomers. The OHCU monoanions exhibits
both proton tautomerism and conformational flexibility.37,38 The
tautomeric forms were selected by a procedure similar to that used
for HIU. Starting from a tri-anionic species (see Scheme S3 in the
ESI†), six protonation sites were located and 13 tautomers were
initially selected. Their equilibrium geometries were first computed
in the gas phase, then optimized in solution and finally the two
most stable structures, denoted as OHCU1,3 and OHCU3,6, were
considered for the study of optical properties in solution. The
two tautomers present conformational flexibility with respect to
the torsion angles involving atoms N1C5N6C7 and C5N6C7N8,
respectively (Fig. 1).
Eight stable conformers in solution were first determined as
minima of the potential energy surface scanned along these two
conformational degrees of freedom.§ All the eight conformers were
confirmed as true minima of the PES by complete optimization
of their geometries. The values of free energy difference and the
corresponding fractional populations are reported in Table 1 only
for the two conformers that have significant fractional populations.
The data show a slight prevalence of the most stable conformer
of the tautomeric form OHCU1,3 over the most stable conformer of
taumeric form OHCU6,3. These two conformers were considered
in the calculations of the ECD and ORD spectra of OHCU in
solution.
3.2 ECD and ORD spectra and absolute configuration
assignments
In the following sections we present the computed ORD and
ECD spectra of HIU, OHCU, and allantoin in order to obtain an
assignment of the absolute configuration of the species generated
by the enzymatic degradation of urate (see Fig. 1) by comparison
with the experimental spectra.
3.2.1 HIU spectra. In Fig. 2, the B3LYP theoretical ECD
and ORD spectra of the S stereoisomer of the HIU7,1 tautomer
are compared with the experimental data.
The experimental ECD spectrum of HIU shows a negative peak
(I) centered around 308 nm, a positive peak (II) centered around
275 nm and a negative peak (III) around 235 nm. The experimental
ORD curve presents negative rotation in the spectral range
considered (350–465 nm). The computed ECD spectrum gives
a good reproduction of the absorption wavelengths and intensities
related to the three experimental peaks. The experimental negative
rotation is also well reproduced by the theoretical ORD curve.
The CAM-B3LYP results show a similar behaviour (see ESI,†
Figure S1), with a systematic shift toward shorter wavelengths
3.1.3. Allantoin isomers. Allantoin (pKa = 8.96) is a neutral
species in aqueous solutions under the experimental conditions,
and presents a complex conformational flexibility. From pre-
§ A step size of 18◦ was chosen for both the torsion angles, and a total of
400 PES points have been collected.
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Fig. 3 Experimental and computed OHCU spectra. Computed spectra (red) for the Boltzmann population of OHCU1,3 and OHCU6,3 isomers, were
calculated at the PCM/TDDFT/B3LYP level. ORD spectra are shown in the inset. Red solid and red dotted lines refer to the S and R configurations of
the OHCU isomers, respectively.
in the ECD spectrum, and a systematic underestimation of the
optical rotation signal in the ORD spectrum.
ORD curve presents a positive rotation in all the spectral range
considered (250–400 nm).
On the basis of the good agreement between the computed
spectra and the experimental data we assign the S configuration to
the (-)-HIU intermediate generated by the enzymatic degradation
of urate.
The B3LYP computed ECD spectrum of (S)-allantoin is
consistent with the sign of the band II, but not with the sign
of the weak band I. The corresponding CAM-B3LYP spectrum
(see ESI,† Figure S5) shows a similar behaviour. However, both
the B3LYP and CAM-B3LYP calculated ORD spectra of (S)-
allantoin (see Fig. 3 and Figure S5) are in agreement with the
experimentally observed positive rotation in the spectral region
from 250 nm to 400 nm.
Although the TDDFT-ECD results cannot provide conclusive
evidence on the absolute configuration of allantoin stereoisomers,
the agreement between the computed and experimental ORD
spectra supports the previous assignment of the S configuration
to (+)-allantoin.5,40
3.2.2 OHCU spectra. In Fig. 3, the B3LYP computed ECD
and ORD spectra of the Boltzmann distribution of OHCU1,3 and
OHCU6,3 tautomers in the S configuration, are compared with
the experimental data. The corresponding spectra for the single
tautomers are reported in Figure S4 of the ESI.†
The experimental ECD spectrum of OHCU presents as major
signatures a negative peak (I) centered around 257 nm, and a
positive peak (II) centered around 230 nm. The experimental
ORD curve presents negative rotation in the spectral range
considered (300–400 nm). The computed ECD spectrum gives
a good reproduction of the absorption wavelengths and intensities
of the two experimental peaks. The experimental negative rotation
is also well reproduced by the theoretical ORD curve.
The CAM-B3LYP results show a similar behaviour (see ESI,†
Figure S3), with a systematic shift toward shorter wavelengths
in the ECD spectrum, and a systematic underestimation of the
optical rotation signal in the ORD spectrum.
On the basis of the good agreement between the computed spec-
tra and the experimental data, we assign the S configuration to the
(-)-OHCU intermediate generated by the enzymatic degradation
of urate.
4
Concluding remarks
The TDDFT-PCM computed chiro-optical ECD and ORD
spectra for HIU and OHCU species are in good agreement with the
corresponding experimentally measured spectra. This agreement
leads to a non-ambiguous assignment for both intermediates
HIU and OHCU to the (-)-S configuration. Given that the
formation of OHCU through the hydrolysis of HIU does not
change the configuration at the chiral center (see Scheme 1), the
independent assignment of the same configuration to HIU and
OHCU gives further support to the proposed stereochemistry (the
same reasoning does not apply to allantoin, in which a different
chiral center is involved).
The TDDFT-PCM computed ORD and ECD spectra for allan-
toin reproduce the main features of the experimentally measured
spectra, confirming the (+)-S configuration in agreement with the
previous assignments based on CD experimental measurements
and biochemical evidence. Some minor features of the computed
ECD spectra in the long-wavelength region do not agree in
sign with the experimental ECD spectra, due to the limitations
3.2.3 Allantoin spectra. In Fig. 4, the computed B3LYP ECD
and ORD spectra of the Boltzmann distribution of ALL-T1
and ALL-T2 conformers of allantoin in the S configuration are
compared with the experimental data. The corresponding spectra
for the single tautomers are reported in Figure S4 of the ESI.†
The experimental ECD spectrum of allantoin presents as major
signatures a weak broad negative peak (I) around 240 nm, and
strong positive peak (II) centered at 208 nm. The experimental
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Fig. 4 Experimental and computed allantoin spectra. Computed spectra (red) for the Boltzmann distribution of ALL-T1 and ALL-T2 isomers were
calculated at the PCM/TDDFT/B3LYP level. ORD spectra are shown in the inset. Red solid and red dotted lines refer to the S and R configurations of
the allantoin isomers, respectively; the black dashed line shows experimental data of (+)-allantoin from Ref. 5.
of the TDDFT-PCM method. However, it is well known that
given the actual TDDFT technology one should not expect for
the computation of ECD spectra the same reliability of the
computations of ORD spectra in the assignment of absolute
configurations.39
TDDFT
GIAO
time-dependent density functional theory
gauge invariant atomic orbitals
Acknowledgements
Although the stereochemistry of urate degradation has not been
directly investigated in crystallographic studies, previous evidence
obtained by the structural analysis of the enzymes involved in
the metabolic pathway is consistent with our results. Detailed
investigations of the urate oxidase structure in complex with
inhibitors have localized a water molecule candidate for the
hydroxylation step of the reaction near to the si-face of urate,
thus suggesting formation of (S)-HIU.41,42 Docking of the unstable
substrate at the active site of OHCU decarboxylase has suggested
binding of the S enantiomer of OHCU and has localized a
histidine residue candidate for catalysis in a position consistent
with the formation of (S)-allantoin.43–45
We thank Vincenzo Puggioni for help with CD measurements.
This work has been supported by the University of Parma.
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Abbreviations
HIU
OHCU
QM
5-hydroxyisourate
2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
quantum mechanical
PCM
IEF
ECD
ORD
DFT
polarizable continuum model
integral equation formalism
electronic circular dichroism
optical rotatory dispersion
density functional theory
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