Stereochemical sensitivity of the human UDP-glucuronosyltransferases 2B7 and 2B17

Article abstract of DOI:10.1021/jm051142c

A set of 28 enantiomers comprising rigid and flexible secondary alcohols was synthesized by the asymmetric Corey-Bakshi-Shibata reduction. The enantiomerically pure alcohols were subjected to enzymatic glucuronidation assays employing the human UDP-glucuronosyltransferases (UGTs) 2B7 and 2B17. Both UGTs displayed high levels of stereoselectivity, favoring the conjugation of the (R)-enantiomers over their respective (S)-stereoisomers at eudismic ratios up to 256. The spatial arrangement of the hydroxy group determined the diastereoselectivity of the UGT2B17-catalyzed reaction in agreement with Pfeiffer's rule (eudismic activity quotient = 0.83 ± 0.14). Inhibition studies revealed that the enantiomers had similar affinities toward the enzymes. The diastereoselectivity of the UGT-catalyzed conjugation stemmed, therefore, from the arrangement of the substrates in the catalytic site, rather than from distinct affinities toward the enzymes. Taken together, this study showed that metabolic enzymes that are generally conceived to be rather "flexible" in nature are capable of displaying high levels of chiral distinction.

Full text of DOI:10.1021/jm051142c

1818
J. Med. Chem. 2006, 49, 1818-1827
Stereochemical Sensitivity of the Human UDP-glucuronosyltransferases 2B7 and 2B17
Ingo Bichlmaier,† Antti Siiskonen,† Moshe Finel,‡ and Jari Yli-Kauhaluoma*,†
Faculty of Pharmacy, DiVision of Pharmaceutical Chemistry, UniVersity of Helsinki, FI-00014 Helsinki, Finland, and
Faculty of Pharmacy, Drug DiscoVery and DeVelopment Technology Center, UniVersity of Helsinki, FI-00014 Helsinki, Finland
ReceiVed NoVember 14, 2005
A set of 28 enantiomers comprising rigid and flexible secondary alcohols was synthesized by the asymmetric
Corey-Bakshi-Shibata reduction. The enantiomerically pure alcohols were subjected to enzymatic
glucuronidation assays employing the human UDP-glucuronosyltransferases (UGTs) 2B7 and 2B17. Both
UGTs displayed high levels of stereoselectivity, favoring the conjugation of the (R)-enantiomers over their
respective (S)-stereoisomers at eudismic ratios up to 256. The spatial arrangement of the hydroxy group
determined the diastereoselectivity of the UGT2B17-catalyzed reaction in agreement with Pfeiffer’s rule
(eudismic activity quotient ) 0.83 ( 0.14). Inhibition studies revealed that the enantiomers had similar
affinities toward the enzymes. The diastereoselectivity of the UGT-catalyzed conjugation stemmed, therefore,
from the arrangement of the substrates in the catalytic site, rather than from distinct affinities toward the
enzymes. Taken together, this study showed that metabolic enzymes that are generally conceived to be
rather “flexible” in nature are capable of displaying high levels of chiral distinction.
Introduction
transfer of the hydrophilic R-D-glucuronic acid (GlcA) from
UDP-glucuronic acid (UDPGlcA) to lipophilic substrates bearing
hydroxy, thiol, amino, carboxy groups, and even enolate
moieties.12 This conjugation reaction increases the water solubil-
ity of the aglycones and aids in their excretion to prevent the
accumulation of endobiotics and xenobiotics to undesirable, or
even toxic, levels. Prominent chiral endobiotics and xenobiotics
that are glucuronidated by UGTs include the opioid analgesic
morphine, the nonsteroidal anti-inflammatory drugs ibuprofen
and ketoprofen, the steroids testosterone, estriol, and ethi-
nylestradiol, and the anxiolytic oxazepam as well as the
terpenoid alcohols menthol and thymol.13
It has long been known that the stereochemistry of chiral
xenobiotics determines their pharmacodynamic, pharmacoki-
netic, and toxicological profiles.1 These differences in pharma-
cological and toxicological action are based on the distinct
interactions between chiral entities and the homochiral functional
constituents of the human body such as receptors, enzymes, and
transport proteins.2,3 In recent years, the advent of efficient
asymmetric synthesis enabling the production of single-enan-
tiomer entities at high optical purities, together with progresses
in developing efficient purification and analytical methods for
these chiral compounds, has paved the way for investigations
on the stereoselective outcomes of chiral interactions in the
organism.4 In drug design and development, these advances
resulted in the development of optically pure drugs by neglecting
the nonactive, less active, or even toxic stereoisomeric “ballast”.5
Detailed insights into stereoselective pharmacological events
have led to the understanding that single-enantiomer drugs are
often superior over the administration of isomeric mixtures and,
therefore, new chiral drugs are approved today virtually
exclusively as enantiopure entities.6-8 It is therefore important
to identify biochemical pathways in the human organism
susceptible to stereoselective events such as the metabolism by
UDP-glucuronosyltransferases (UGTs,a EC 2.4.1.17), involved
in the deactivation, activation, and toxication of achiral and
chiral compounds.9-11
The mechanism of the UGT-catalyzed glucuronidation is
essentially unknown, and the crystal structure of these membrane-
bound proteins has not yet been resolved. The transition from
reactants to products proceeds with inversion of configuration
at the anomeric carbon atom of GlcA, and it is therefore assumed
that the transfer step is concerted and resembles the bimolecular
nucleophilic substitution reaction (SN2). In addition, it is
presumed that the cosubstrate binding site resides in the carboxy-
terminal half of the protein, which represents a highly conserved
region among different UGT isoforms.14-16 The substrate
binding site is presumably located in the amino terminus of the
enzymes. Recently, it was shown that UGT1A isoforms display
a compulsory ordered mechanism in which UDPGlcA is the
first-binding substrate.17
UGTs are important enzymes of the human phase-II metabolic
system. These enzymes are functionally characterized by the
UGTs, like other metabolic enzymes, are multifunctional
and accept substrates with a wide range of different chem-
ical structures. In the case of UGTs, this is also referred to as
promiscuity, or loose fit, and these enzymes are, therefore,
commonly described to possess a high degree of “flexibility”
to depict their complex and partly overlapping substrate
specificities.18 This could be the reason metabolic enzymes
are generally not subjected to eudismic analysis using whole
series of chiral compounds.2 However, this study showed that
also these “flexible” UGTs can display great stereochemical
selectivity similar to highly specialized receptor proteins. It
should be noted here that the complex substrate selectivity of
the UGT-catalyzed glucuronidation has been often addressed
in the literature, and studies employing computational chem-
* Corresponding author. Address: Faculty of Pharmacy, Division of
Pharmaceutical Chemistry, P.O. Box 56, University of Helsinki, FI-00014
Helsinki, Finland. Phone: +358 9 19159170. Fax: +358 9 19159556.
E-mail: jari.yli-kauhaluoma@helsinki.fi.
† Division of Pharmaceutical Chemistry.
‡ Drug Discovery and Development Technology Center.
a Abbreviations: AICC, corrected Akaike’s information criterion; CI95%
,
95% confidence interval; EA, eudismic analysis; EAQ, eudismic activity
quotient; EI, eudismic index; ER, eudismic ratio; GlcA, glucuronic acid;
IC50, concentration of inhibitor which causes 50% inhibition; Kic, competitive
inhibition constant; Kiu, uncompetitive inhibition constant, R2, goodness of
fit; Km, Michaelis-Menten constant; SAR, structure-activity relationship;
SD, standard deviation; UDPGlcA, UDP-glucuronic acid; UGT, UDP-
glucuronosyltransferase; Vmax, maximum velocity; VR, rate of (R)-enantiomer
conjugation; VS, rate of (S)-enantiomer conjugation.
10.1021/jm051142c CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

Stereochemical SensitiVity of UGT2B7 and 2B17
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1819
Table 1. The Structures of the Enantiomerically Pure Secondary
entities such as steroids, oxazepam, and profen as well as opioid
analgesics.13,21-23 In addition, we showed previously by con-
ducting screening assays that both enzymes glucuronidated the
enantiomers of 1-tetralol, 1-indalol, and 1-benzosuberol.24
By employing the enantiomerically pure enantiomers to
measure their affinities to the UGTs 2B7 and 2B17 and their
rates of UGT-catalyzed conjugation, the influence of the spatial
orientation of the hydroxy group and of the substitution pattern
on these processes could be directly studied. Eudismic analysis
(EA), in general, provides a valuable approach to elucidate the
interaction between chiral compounds and their target pro-
teins.25,26 In addition, this analytical method is used in drug
design and development to increase the selectivity and potency
of chiral lead compounds toward their receptors.27 In this study,
the tools of EA were employed to identify the factors determin-
ing affinity and GlcA-transfer to edge closer to the mechanism
of the UGT-catalyzed glucuronidation reaction at the molecular
level.
Alcohols Employed in This Studya
Results and Discussion
The enantiomers were synthesized by the asymmetric reduc-
tion developed by Corey et al.28,29 The Corey-Bakshi-Shibata
(CBS) catalyst was prepared in situ by the addition of trimethyl
borate to a solution of (R)- or (S)-1,1-diphenylprolinol in
toluene.30,31 The addition of a borane N,N-diethylaniline complex
serving as the reducing agent, followed by the prochiral ketone,
afforded the enantiomeric alcohols in excellent yields and high
optical purities (cf. Supporting Information).32 Subsequently,
the synthesized substrates were recrystallized several times until
an enantiomeric excess of >99.9% for each enantiomer was
reached because it was previously shown that eudismic analysis
depends strongly on the optical purity of the stereoisomers.33
In the case of liquid products, the enantiomers were resolved
by high-performance liquid chromatography (HPLC) with the
use of a chiral (R,R)-ULMO column.
a The rigid enantiomers include the entries 1-10, and the flexible
stereoisomers comprise the entries 11-14.
The first part of this study is concerned with the differences
between the synthesized enantiomerically pure alcohols with
respect to the rate of the UGT-catalyzed GlcA-transfer reaction.
Therefore, the diastereoselectivity, or substrate-product ste-
reoselectivity,34 of the enzymatic glucuronidation was studied
by comparing the rates of (R)- and (S)-enantiomer conjugation.
In this respect, the velocity of the conjugation reaction catalyzed
by UGT2B7 and UGT2B17 for each enantiomer was measured.
The concentrations of formed glucuronide for the bicyclic
alcohols 1-10 were, however, not sufficient for UV detection,
and therefore, the enzyme assays were conducted employing
radiolabeled [14C]UDPGlcA in combination with “cold” UD-
PGlcA at a final concentration of 120 µM.35 This procedure
had the advantages that the formed [14C]â-D-glucuronides were
readily detected and unambiguously identified and that the
amount of product could be directly quantified.36
The reaction rates were determined by recording progression
curves (Figure 1). This analytical method ensured the compa-
rability and consistency of the data because nonlinear kinetic
behavior within the time frame of the enzyme assays was readily
detected and subsequently omitted from regression analysis. It
should be noted here that as a rule of thumb the formed [14C]-
glucuronides of (R)-enantiomers were readily detected at
incubation times between 10 and 60 min and the [14C]GlcA-
conjugates of (S)-enantiomers between 70 and 120 min of
incubation (Figure 1). The bicyclic alcohols 1-10 were assayed
at a saturating concentration of 2.0 mM, yielding a good estimate
of the Vmax under the assay conditions (Figure 1). The flexible
alcohols 11-14, however, were employed at unsaturating
istry tools led to predictive models for some UGT1A iso-
forms.19,20
In this study we investigated the stereoselective events during
the formation of the enzyme-substrate complex and the GlcA-
transfer reaction, employing a large set of enantiopure com-
pounds comprising both rigid cyclic and flexible aliphatic
alcohols (Table 1). The enantiomers possess only one element
of chirality, i.e., the asymmetric carbon atom bearing the
nucleophilic hydroxy group, the very site of the UGT-catalyzed
glucuronidation. Therefore, the only difference between the
enantiomers of each compound is in the spatial arrangement of
this nucleophilic group, whereas the physicochemical properties
remain per definitionem identical in an achiral environment.
By including both flexible and rigid secondary alcohols, we
could address the question whether the freely rotating hydroxy
group of the flexible alcohols was able to overcome stereose-
lective events that were observed for rigid alcohols during UGT-
catalyzed glucuronidation. The rigid stereoisomers comprise the
bicyclic alcohols 1-10 (Table 1), in which the spatial flexibility
of the hydroxy group is impaired and, therefore, its position in
space is rather well-defined. This is due to the partially
unsaturated bicyclic structure hindering the rotation along the
σ-bonds in the vicinity of the hydroxy group. In the case of the
flexible alcohols, however, the hydroxy group rotates freely
because the aliphatic nature of the secondary alcohols does not
impair the rotation along the σ-bonds (Table 1, entries 11-
14). The UGTs 2B7 and 2B17 were chosen because these
enzymes play an important role in the glucuronidation of chiral

1820 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Bichlmaier et al.
Figure 1. Progression curves for the UGT2B17-catalyzed glucuronida-
tion of (R)-2 (A) and (S)-2 (B). Both enantiomers were assayed at a
saturating concentration of 2.0 mM as can be seen from the insets
(ordinate, velocity in pmol min-1 mg-1; abscissa, substrate concentration
in mM). The enzyme was employed at 1.0 µg/µL, and the UDPGlcA
concentration was 120 µM. The slope (velocity) was determined by
linear regression (the intercept was constrained to zero). The rate for
the (R)-2 conjugation was determined to be 84 pmol min-1 mg-1, the
velocity for the (S)-2 glucuronidation was 1.4 pmol min-1 mg-1. The
detection limit for the (S)-2 glucuronide was ∼125 nM, corresponding
to ∼10 pmol glucuronide (80 µL injection). The signal-to-noise ratio
for the smallest [14C]glucuronide peak was 5.7. The ordinate displays
the amount of product formed per milligram of protein. The mean values
and the standard deviation (n ) 2) are shown.
Figure 2. The velocities of the UGT-catalyzed glucuronidation for
the enantiomeric alcohols 1-14 as determined from the slope of the
respective progression curves (the error bars represent the respective
standard deviations, n ) 12). Panel A displays the conjugation rates
for the glucuronidation catalyzed by UGT2B7, and panel B shows the
glucuronidation velocities for the UGT2B17-catalyzed conjugation. The
white and black bars represent the velocities for (R)- and (S)-
enantiomers, respectively. Both enzymes displayed high levels of
stereoselectivity with respect to the GlcA-transfer reaction, strongly
favoring the glucuronidation of the (R)-enantiomers over their respective
(S)-enantiomers (with the only exception of compound 11 in panel A).
The enantiomers of compound 14 were not found to be glucuronidated
either by UGT2B7 or by UGT2B17.
concentrations of 0.10 mM because of their low solubility, and
therefore, their measured rates did not resemble their Vmax
values.
diastereoselective GlcA-transfer is the spatial orientation of this
group. However, when the ERs of the compounds were
compared, it became clear that the substituents exerted a
significant influence on the stereoselectivity of this process. This
can be readily seen by comparing the ERs of the 4-chromanol
derivatives 6 and 7 glucuronidated by UGT2B17 (Table 2). The
ER for compound 6 was 50.2, whereas its methylated derivative
7 displayed a 2.6-fold increased stereoselectivity (ER ) 131),
and the reason for this shift toward higher stereoselectivities
could be ascribed to the introduction of the methyl group to
the 4-chromanol scaffold of compound 6 as subsequently
described. The enantiomer (S)-6 was glucuronidated at a rate
of 1.2 pmol min-1 mg-1, and its methylated derivative (S)-7
was glucuronidated at a rate of 2.4 pmol min-1 mg-1 (Table
2). Therefore, the methylation of (S)-6 affording (S)-7 induced
a 2-fold increase in the glucuronidation rate. In contrast, the
enantiomer (R)-6 was conjugated by UGT2B17 at 60.8 pmol
min-1 mg-1, and its derivative (R)-7 was transformed at 314
pmol min-1 mg-1. Hence, the introduction of the methyl group
into (R)-6 yielding (R)-7 corresponded to a ∼5-fold enhanced
glucuronidation rate, whereas for the respective (S)-enantiomers
the conjugation rate was only doubled (Table 2). The increase
in the ER induced by the methylation of (R)-6 and (S)-6 yielding
(R)-7 and (S)-7, respectively, was equal to the ratio of the
resulting changes in glucuronidation rates, in this example 5
divided by 2. In general, shifts to higher stereoselectivities were
observed when the introduction or the exchange of functional
groups induced only a small increase (high decrease) in the
glucuronidation rate for (S)-enantiomers (VS) accompanied by
a higher increase (smaller decrease) in the conjugation rate for
the respective (R)-enantiomers (VR). Therefore, structure-
activity relationships (SARs) with respect to the diastereose-
lectivity of the GlcA-transfer reaction were derived by relating
The results of the rate measurements are displayed in Figure
2, and both enzymes were found to display high degrees of
diastereoselectivity during the UGT-catalyzed GlcA-transfer
reaction toward the employed enantiomeric alcohols. In this
respect, the UGTs 2B7 and 2B17 strongly favored the glucu-
ronidation of the (R)-enantiomers over their respective (S)-
stereoisomers. The sole exception was the flexible alcohol 11
because its enantiomers (R)-11 and (S)-11 were glucuronidated
by UGT2B7 at comparable rates of approximately 30 pmol
min-1 mg-1 (Figure 2). It can also be seen that UGT2B17
displayed higher levels of stereoselectivity than UGT2B7. This
finding was quantified by calculating the eudismic ratios (ER)
for each pair of enantiomers, and the results of this analysis are
shown in Table 2. The ER is a useful indicator for the
stereoselectivity of biochemical processes. It is calculated by
dividing the velocity of the more active enantiomer (eutomer)
by the rate of the corresponding less active stereoisomer
(distomer). The higher this ratio, the more pronounced the
stereoselectivity, or chiral distinction,37 during the process under
investigation. The ERs for UGT2B7 ranged between 1.10 and
58.1, and for 10 out of the 13 compounds the ERs were below
10.0. In contrast, UGT2B17 favored the (R)-enantiomers over
their corresponding (S)-stereoisomer by factors ranging from
8.22 to 256, and only 1 out of the 13 values was found to be
below 10.0 (Table 2). Notably, the highest ERs were measured
for the UGT2B17-catalyzed conjugation of compounds 5, 7,
and 10 (ER > 100). Alcohol 5 displayed the highest degree of
stereoselectivity, as indicated by its ER of 256.
The only difference between the enantiomers of each
compound was in the spatial arrangement of the nucleophilic
hydroxy group. Therefore, the underlying principle for the

Stereochemical SensitiVity of UGT2B7 and 2B17
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1821
Table 2. Velocities and Eudismic Ratios for the Enantiomerically Pure Alcohols
UGT2B7
UGT2B17
a
b
a
b
VR
VS
VR
VS
compd
pmol min-1 mg-1
pmol min-1 mg-1
ERc
pmol min-1 mg-1
pmol min-1 mg-1
ERc
1
2
3
4
5
6
7
8
29.5
139
64.5
269
253
28.4
114
5.71
29.1
41.2
30.5
23.0
17.8
NDd
3.56
9.60
1.11
32.1
38.5
4.45
2.93
2.10
5.03
6.01
27.8
3.05
3.90
NDd
8.29
14.5
58.1
8.38
6.57
6.38
38.9
2.72
5.79
6.86
1.10
7.54
4.56
d
56.8
83.6
54.1
92.2
460
60.8
314
12.5
28.4
219
1.30
1.40
1.20
5.28
1.80
1.21
2.40
1.10
1.99
2.01
1.34
3.90
1.29
NDd
43.7
59.7
45.1
17.5
256
50.2
131
11.4
14.3
109
14.0
13.9
8.22
d
9
10
11
12
13
14
18.8
54.3
10.6
NDd
a Glucuronidation rate for the (R)-enantiomer. SD, CI95%, and R2 are included in the Supporting Information. b Glucuronidation rate for the (S)-enantiomer.
SD, CI95%, and R2 are included in the Supporting Information. c The eudismic ratio (ER) was calculated by dividing VR by VS. d The [14C]glucuronide was
not detected (ND) and, therefore, the respective ER was not calculated.
highest VR values. For UGT2B7, however, the increase in
stereoselectivity resulted either from the decrease in VR ac-
companied by a higher decrease in VS or from an increase in VR
with an approximately constant VS. Therefore, the highest ERs
were associated with compounds displaying low VS and moderate
VR values (Figure 3).
The SARs for the UGT2B17-catalyzed glucuronidation were
derived by relating the substitution pattern of the bicyclic
alcohols 1-10 to their stereoselectivities and conjugation rates,
on the basis of the principles outlined in detail above (Figure
3). The methylated alcohols 5, 7, and 10 displayed the highest
ERs, ranging from 109 to 256 (Table 2). The common
characteristic of these alcohols was the methyl group attached
to carbon atom 7 in the case of compound 5 and to carbon atom
6 in the case of the indalol derivative 5 and chromanol derivative
7 (Figure 4). The methyl group in this particular position of
the bicyclic scaffold played an important role in shifting the
stereoselectivities to higher levels. This finding was derived by
comparing the ERs of the methylated compounds 5, 7, and 10
to those of their unsubstituted derivatives, namely, alcohols 1,
6, and 8. The latter displayed significantly lower ERs of 43.7,
50.2, and 11.4, respectively (Table 2). Furthermore, the methyl
group was superior in shifting the stereoselectivity toward higher
levels compared to a methoxy group attached to the same carbon
atom, because the ER of alcohol 3 was significantly smaller
(45.1) compared to that of the methylated compounds 5, 7, and
10. However, the shift of the methoxy group from carbon atom
7 (compound 3) to position 5 (compound 2) had only a minor
effect on both ER and VR. The exchange of the methyl group
from carbon atom 5 (compound 9) to position 7 (alcohol 10)
induced a 7.6-fold increase in the respective ERs. Furthermore,
the shift from the indalol derivatives 8-10 to the respective
tetralol or 4-chromanol derivatives accomplished by the intro-
duction of one additional methylene group or oxygen atom into
the indalol scaffold increased the stereoselectivity of the
UGT2B17-catalyzed GlcA-transfer reaction (Figure 4). The
change of the tetralol scaffold to the heterocyclic 4-chromanol
had only a minute effect on the stereoselectivity (compounds 1
and 6). In contrast, the attachment of the nitro group into the
bicyclic system had a significant effect on the VR/VS ratio. In
this respect, compound 4 was the only observed “outlier” within
this series of bicyclic compounds because its VS value was 3.8-
fold higher than that of compound 2, and both were glucu-
ronidated at comparable VR (Table 2 and Figure 4). This might
indicate that the nitro group exerted a significant disturbance
Figure 3. Correlation between the ER, VR, and VS for the alcohols
1-10: (A) UGT2B7; (B) UGT2B17. For UGT2B17 it was observed
that the stereoselectivity increased with increasing VR values ac-
companied by minor changes, or even no variations, in the respective
VS values (B). In the case of UGT2B7, however, the highest ERs were
obtained for compounds displaying moderate VR values and small VS
values (A).
the changes in glucuronidation rates for a pair of structurally
related (R)- and (S)-enantiomers to changes in their substitution
pattern. The three-dimensional plot in Figure 3 relates the
measured velocities, VR and VS, to the respective ERs. For
UGT2B17, a clear trend was observed, insofar as the increase
in the ER was caused by a sharp increase in VR accompanied
by an approximately constant VS (Figure 3). Therefore, in the
case of UGT2B17, the highest ERs were associated with the

1822 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Bichlmaier et al.
their respective (S)-enantiomers (Table 2 and Figure 2). There-
fore, the high rotational “freedom” of the hydroxy group had
no significant effect on the substrate-product stereoselectivity
of the GlcA-transfer step. We therefore concluded that UGT2B17
displayed high levels of chiral distinction during the glucu-
ronidation reaction, independently of the flexibility of the chiral
substrate.
In the case of UGT2B17, clear trends were observed that led
to the derivation of SARs with respect to higher levels of chiral
distinction. For UGT2B7, however, no comparable pattern was
found (Figure 4), but some valid conclusions could nevertheless
be made. In contrast to UGT2B17, the compounds 2, 4, and 5,
displaying the highest VR values, did not exhibit the highest
stereoselectivities during the UGT2B7-catalyzed conjugation
reaction (Table 2, Figures 3 and 4). Instead, the highest ERs
were associated with compounds 3 and 7, displaying moderate
VR and small VS values (Table 2 and Figure 3). In this respect,
within the set of the tetralol derivatives 1-5, the introduction
of the methoxy group in position 7 (compound 3) caused a clear
shift to higher ERs, primarily induced by the decrease in VS
(Table 2 and Figure 3). However, the increase in VR was
accompanied by an analogous change to higher VS values, as
indicated by similar ERs for 6 out of 8 bicyclic alcohols, ranging
from 5.79 to 14.5, corresponding to an increase in the stereo-
selectivity of at most 2.5-fold (Table 2, Figures 3 and 4).
The relationship between VR and ER is presented in Figure
4. It can be seen from the inset that in the case of UGT2B17
the data obeyed Pfeiffer’s rule, stating that the higher the activity
is of the eutomer the higher the separation is in activities
between eutomer and distomer.38 Correlation analysis afforded
a clear relationship between these two parameters (Pearson r
) 0.904, two-tailed P ) 3.0 × 10-4, R ) 0.05), showing that
the eudismic index (EI), defined as log(VR/VS), increased linearly
toward higher velocities for the eutomer, log(VR) (Figure 4).
The determination of the slope of this straight line by linear
regression yielded the eudismic activity quotient (EAQ) of 0.83
( 0.14 (Figure 4). This value quantifies the rate of change in
the EI with velocity. This high EAQ strongly indicated a
significant participation of the element of chirality during the
glucuronidation reaction catalyzed by UGT2B17. We therefore
concluded that the two enantiomers of each compound displayed
high similarities in their orientation within the active site of
UGT2B17. With both enantiomers taking the same arrangement,
the hydroxy group of the (S)-enantiomer was unfavorably placed
(“out of reach”) for efficient GlcA-transfer, resulting in con-
stantly low VS values. On the other hand, the (R)-enantiomers
were conjugated rapidly; hence, the respective hydroxy group
was favorably placed within the catalytic site, resulting in a
fast GlcA-transfer (VR . VS). In the case of UGT2B7, however,
the obtained data violated Pfeiffer’s rule, insofar as there was
no correlation between the EI and log(VR). Instead, the data
points seemed to be randomly scattered within the limits of the
two parameters (Figure 4). This lack of correlation showed that
factors other than the spatial arrangement of the hydroxy group
became dominant, suggesting that the element of chirality did
not significantly participate in the GlcA-transfer reaction
catalyzed by UGT2B7. The two enantiomers of each compound
seemed, therefore, to display distinct orientations within the
active site, meaning that the catalytic site of UGT2B7 allowed
for the different arrangement of the (R)- and its respective (S)-
enantiomer during the actual transfer reaction. This conclusion
was supported by the result that the two enantiomers of the
flexible compound 11 were glucuronidated at similar rates, and
therefore, the respective ER was close to unity (Table 2 and
Figure 4. The glucuronidation rates (white bars) for the bicyclic (R)-
enantiomers and the ERs (black bars) for the enantiomeric pairs 1-10
as determined for UGT2B7 (A) and UGT2B17 (B). The compounds
were sorted by increasing velocity. In panel B, the ER increased with
increasing VR. Therefore, the (R)-enantiomers bearing the methyl group
in m-position to the benzylic hydroxy function, namely (R)-5, (R)-7,
and (R)-10, displayed the highest velocities and simultaneously the
highest stereoselectivities. The inset shows that the EI increased with
increasing log(VR) consistent with Pfeiffer’s rule. The curve in the inset
was fitted by linear regression (R2 ) 0.82). The EAQ was 0.83 ( 0.14,
indicating a strong participation of the element of chirality. Compound
4 displayed a significant deviation from the regression curve as
discussed in the text. In panel A, in the case of UGT2B7, there was no
clear trend for the relationship of the glucuronidation velocity and the
stereoselectivity. The nitro compound 4 was the best substrate in this
series accompanied by a low ER. The highest stereoselectivities were
associated with compounds 3 and 7, which were glucuronidated at
moderate rates. The inset of panel A shows no correlation between the
EI and log(VR) (violation of Pfeiffer’s rule). This indicated no
participation of the element of chirality in the GlcA-transfer reaction
catalyzed by UGT2B7.
to the system, possibly because this residue is bulkier and
possesses a higher electron density in comparison to the methyl
and methoxy groups. This finding might indicate a different
orientation of compound 4 within the active site of the enzyme
rendering also the (S)-enantiomer a better substrate for UGT2B17
leading to its higher VS value and, hence, inducing a lower level
of chiral distinction during the glucuronidation reaction (Figure
4). In conclusion, even the flexible, aliphatic alcohols 11-13
were glucuronidated by UGT2B17 at pronounced diastereose-
lectivities, favoring the conjugation of the (R)-enantiomers over

Stereochemical SensitiVity of UGT2B7 and 2B17
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1823
Table 3. IC50 Values for the Enantiomerically Pure Enantiomers
UGT2B7a
UGT2B17b
compd IC50 (R) (µM)c IC50 (S) (µM)c IC50 (R) (µM)c IC50 (S) (µM)c
1
2
3
4
5
6
7
8
337 ( 8
366 ( 10
284 ( 9
599 ( 26
158 ( 4
507 ( 23
225 ( 10
275 ( 8
317 ( 8
394 ( 11
130 ( 7
79 ( 3
489 ( 14
301 ( 10
351 ( 5
651 ( 10
166 ( 8
518 ( 7
182 ( 6
513 ( 14
179 ( 7
540 ( 13
214 ( 15
105 ( 4
532 ( 27
242 ( 7
112 ( 3
162 ( 5
217 ( 4
144 ( 6
448 ( 10
365 ( 8
517 ( 10
682 ( 15
489 ( 5
389 ( 14
578 ( 23
703 ( 46
374 ( 10
132 ( 3
216 ( 13
305 ( 14
98 ( 4
319 ( 8
325 ( 11
545 ( 21
608 ( 20
728 ( 15
537 ( 40
657 ( 27
606 ( 21
9
10
11d
12d
13d
344 ( 10
a Assayed with 500 µM scopoletin (Km ) 531 µM). b Assayed with 300
µM scopoletin (Km ) 309 µM). c The mean value ( standard deviation is
displayed. The CI95%, R2, and the Hill coefficient are included in Supporting
Information. d The enantiomers were only employed at concentrations
ranging from 5 to 100 µM, because of their low solubilities. The
concentration of the substrate scopoletin was lowered to 50 µM, because
of the high IC50 values and the low maximum concentrations of these
compounds. This renders the IC50 value incomparable to the values obtained
for the bicyclic enantiomers 1-10.
Figure 5. Semilogarithmic concentration-response plot for the
inhibition assays using (R)-2 (A) and (S)-2 (B). Both were assayed
with UGT2B7. Scopoletin was used as the substrate at a concentration
similar to its Km for that enzyme (500 µM). The reaction assay contained
5.0 mM cosubstrate (UDPGlcA), and the protein concentration was
0.10 µg/µL (incubation time of 15 min). The IC50 for (R)-2 was
366 ( 10 µM (CI95% ) 345-388) and 301 ( 10 µM for (S)-2 (CI95%
) 280-322).
of each compound toward the UGTs 2B7 and 2B17 were of
the same order of magnitude and even nearly identical.40 Plots
of the IC50 values of the (S)-enantiomers versus the IC50 values
of the (R)-enantiomers are presented in Figure 6. The affinities
of a pair of enantiomers toward both enzymes were rather similar
because the data points were distributed near to the bisecting
line (Figure 6). The ERs of the IC50 values for UGT2B7 ranged
between 1.1 and 1.9, and those for UGT2B17 were between
1.0 and 1.6 (Figure 6). Furthermore, we observed no general
bias toward (R)- or (S)-enantiomers because the data points were
scattered randomly below and above the bisecting line. These
results were confirmed by measuring the IC50 values of each
racemic mixture of the compounds 5, 7, and 10, and the results
showed that the potency of each racemate was intermediary
between the two respective enantiomers (results not shown).
The inset in Figure 6 revealed the lack of correlation between
the EI and the affinity of the eutomer toward both UGT2B7
and UGT2B17, a violation of Pfeiffer’s rule. These findings
indicated that the element of chirality did not significantly
participate during the formation of the enzyme-substrate
complex. However, these findings could also have been a result
of unspecific, noncompetitive, and uncompetitive binding to the
enzymes, blurring the stereoselectivities of the active-site
directed, competitive interaction. Therefore, we determined the
mechanism of inhibition for the racemates of compounds 5, 7,
and 10 by the direct plot method and simultaneous nonlinear
regression analysis (velocity versus scopoletin concentration).
The data were fitted to the competitive, noncompetitive, mixed-
type, and uncompetitive inhibition models. The different models
were ranked according to the corrected Akaike’s information
criterion (AICC). On the basis of these results, the active-site
directed, competitive inhibition model was chosen and the
resulting Kic values were in good agreement with those
calculated from the respective IC50 values by the Cheng-Prusoff
equation (Figure 7 and Supporting Information). Therefore, we
assumed that the spatial arrangement of the hydroxy group had
indeed no effect on the affinity toward the enzymes. This might
indicate that the nucleophilic hydroxy group was negligible
during the formation of the enzyme-substrate complex, mean-
ing that the enzyme merely recognized the lipophilic core of
the structure. The (R)- and its respective (S)-enantiomer
displayed very similar affinities toward both UGTs 2B7 and
Figure 2). Nevertheless, the (R)-enantiomers were arranged more
favorably during the GlcA-transfer step than their corresponding
stereoisomers because they were conjugated at higher rates than
their respective (S)-enantiomers (VR g VS).
In the preceding sections we have discussed the influences
of the element of chirality on the actual GlcA-transfer reaction,
and we will now turn to its effect on the formation of the
enzyme-substrate complex. It was asked whether the preferred
glucuronidation of (R)-enantiomers over their corresponding (S)-
enantiomers by both UGT2B7 and UGT2B17 resulted from vast
differences in their affinities toward these enzymes. To answer
this question, we employed the IC50 value as a measure for the
affinity toward the enzymes. The IC50 is correlated to its Ki by
the equations derived by Cheng and Prusoff, and it therefore
relates to the dissociation (formation) of the enzyme-inhibitor
complex.39 This complex is called here the enzyme-substrate
complex because the inhibitors, i.e., the enantiomers of com-
pounds 1-13, are substrates of the enzymes (the so-called
“second substrates” in the inhibition assay). Scopoletin was
chosen as the “first-substrate” because its glucuronide was
conveniently identified by fluorescence detection and the Km
values of this substrate toward UGT2B7 and UGT2B17 (531
and 309 µM, respectively) were optimal for the IC50 determi-
nation for the bicyclic enantiomeric alcohols 1-10 at a
scopoletin concentration identical to its Km (Figure 5). However,
the measurement at a scopoletin concentration resembling its
Km was not possible in the case of the aliphatic alcohols 11-
13 because of their low solubilities. Therefore, the scopoletin
concentration was lowered to 50 µM to obtain meaningful IC50
values for these lipophilic alcohols. It should be mentioned here
that the IC50 values for the enantiomers of compounds 11-13
were not comparable to the values for compounds 1-10,
because the IC50 depends on the scopoletin concentration.
However, the values within each set of compoundssalcohols
1-10 and 11-13scould be compared to each other. Compound
14 was excluded from this study because it was not a substrate
of both enzymes.
The results of the inhibition assays are presented in Table 3.
As can be seen from the data, the IC50 values for the enantiomers

1824 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Bichlmaier et al.
Figure 6. IC50 values of the (S)-enantiomers versus the IC50 values of
the respective (R)-enantiomers of compounds 1-13. Panel A displays
the data for UGT2B7, and panel B shows the results for UGT2B17.
The bisecting solid line corresponds to a IC50 ratio of unity. Data points
on this line represent identical IC50 values for the (R)- and (S)-
enantiomer. Points above the bisecting line indicate a higher affinity
for the (S)-enantiomer toward the enzyme, and points below the
bisecting line indicate bias toward (R)-enantiomers. The slopes of the
dashed lines represent the maximal and minimal IC50 ratios corre-
sponding to the maximal bias toward the (S)-enantiomer and the
maximal bias toward the (R)-enantiomer, respectively. The error bars
represent the standard deviations as presented in Table 3. The insets
of panels A and B display the lack of correlation between the EI and
the pIC50 for both enzymes (violation of Pfeiffer’s rule) indicating no
significant participation of the element of chirality during the formation
of the enzyme-enantiomer complex.
Figure 7. Inhibition of the UGT2B7-catalyzed scopoletin glucuronida-
tion by rac-5. The data were fitted by simultaneous nonlinear regression
(global fitting) to the competitive (A), uncompetitive (B), mixed-type
(C), and noncompetitive inhibition models. The noncompetitive model
did not converge. Model comparison was carried out by employing
the corrected Akaike’s information criterion (AICC). The AICC values
were -313, -262, and -311 for the competitive, uncompetitive, and
mixed-type model, respectively. The evidence ratios showed that the
competitive inhibition model was ∼12 × 1010 times more likely than
uncompetitive inhibition and ∼2.7 times more likely than mixed-type
inhibition. In addition, the mixed-type inhibition model yielded a very
high and therefore negligible uncompetitive inhibition constant (Kiu)
of ∼2.1 × 103 µM, whereas its competitive inhibition constant (Kic)
was similar to that obtained for competitive inhibition (∼100 µM). On
the basis of these results, competitive inhibition was chosen to be most
likely to explain the data correctly (cf. Supporting Information).
2B17, and therefore, the enzymes displayed no significant chiral
distinction during the formation of the enzyme-substrate
complex. Hence, the binding site of UGT2B7 and UGT2B17
seemed to be quite “flexible” in accepting the enantiomers of
each compound at similar affinities, and it may be concluded
that the preferential glucuronidation of the (R)-enantiomers by
the UGTs 2B7 and 2B17 did not result from differences in the
affinities toward the enzymes.
Interestingly, the results of the affinity assays were in sharp
contrast to the findings of the glucuronidation assays. Both
enzymes displayed diastereoselective conjugation favoring the
(R)- over its respective (S)-enantiomer at eudismic ratios up to
256. In contrast, the formation of the enzyme-substrate complex
was not influenced by the spatial arrangement of the hydroxy
group, because the enzymes accepted the stereoisomers at very
similar affinities. This study showed that the observed stereo-
selectivities resulted from the preferential conjugation of the
(R)- over its (S)-enantiomer during the catalytic transfer reaction
per se and not from distinct affinities between the stereoisomers
to the enzymes.
not display high levels of chiral distinction. In this study,
however, we showed that in particular UGT2B17 was highly
stereoselective in its glucuronidation reaction and the “flex-
ibility” was merely limited to the formation of the enzyme-
substrate complex. Therefore, also enzymes of the human
metabolic system are capable of displaying high levels of
substrate-product stereoselectivity. In addition, the findings of
this study suggest that eudismic analysis may be used to edge
closer to understanding the mechanism of the UGT-catalyzed
glucuronidation reaction at the molecular level.
Experimental Section
Materials. UDPGlcA (trisodium salt, CAS 63700-19-6), sco-
poletin (CAS 92-61-5), and saccharic acid-1,4-lactone (CAS 61278-
30-6) were obtained from Sigma (St. Louis, MO). [14C]UDPGlcA
was acquired from PerkinElmer Life and Analytical Sciences
(Boston, MA). HPLC grade solvents were used throughout the
study. The recombinant human UGT2B7 was expressed in bacu-
lovirus-infected insect cells as previously described.41 The cDNA
for the human UGT2B17 was a generous gift from Prof. Peter
Mackenzie (Flinders University, Flinders Medical Centre, Bedford
Park, South Australia, Australia), and it was also expressed as a
His-tagged protein in insect cells. 5-Methoxy-1-tetralone (CAS
33892-75-0), 7-methoxy-1-tetralone (CAS 6836-19-7), 5,7-dimeth-
yl-1-tetralone (CAS 13621-25-5), 4-chromanone (CAS 491-37-2),
Conclusion
To date, studies on metabolic enzymes such as UGTs,
sulfotransferases, and acyltransferases employing whole sets of
chiral compounds have not been conducted.2 This lack of
eudismic data is probably due to the general conception that
these enzymes are quite “flexible” in nature and, therefore, do

Stereochemical SensitiVity of UGT2B7 and 2B17
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1825
(R)-2: (-)-(R)-5-Methoxy-1-tetralol. Rf ) 0.28 (15% EtOAc
6-methyl-4-chromanone (CAS 39513-75-2), 4-methyl-1-indanone
(CAS 24644-78-8), 6-methyl-1-indanone (CAS 24623-20-9), 1′-
acetonaphthone (CAS 941-98-0), 2′-acetonaphthone (CAS 93-08-
3), 9-acetylanthracene (CAS 784-04-3), 9-acetylphenanthrene (CAS
2039-77-2), borane N,N-diethylaniline complex (CAS 13289-97-
9), and anhydrous toluene were acquired from Aldrich (Schnelldorf,
Germany). 7-Nitro-1-tetralone (CAS 40353-34-2) was purchased
from Alfa Aesar/Lancaster (Lancashire, U.K.).
in n-hexane); mp 97.5-97.9 °C (toluene/n-hexane); [R]23 -17.3
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C11H14O2) C, H.
(S)-2: (+)-(S)-5-Methoxy-1-tetralol. Rf ) 0.28 (15% EtOAc
in n-hexane); mp 97.8-98.2 °C (toluene/n-hexane); [R]23 +16.9
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C11H14O2) C, H.
(R)-3: (-)-(R)-7-Methoxy-1-tetralol. Rf ) 0.24 (20% EtOAc
in n-hexane); mp 45.7-46.4 °C (toluene/n-hexane); [R]23 -45.4
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C11H14O2) C, H.
(S)-3: (+)-(S)-7-Methoxy-1-tetralol. Rf ) 0.24 (20% EtOAc
Analytics. The progress of chemical reactions was monitored
by thin-layer chromatography on silica gel 60-F254 plates acquired
from Merck (Darmstadt, Germany). Optical purities were deter-
mined applying HPLC by use of a chiral (R,R)-ULMO column (25
cm × 4.6 mm; Regis Technologies, Morton Grove, IL). The eluent
consisted of 2-propanol in n-hexane, and signal detection was
conducted at 254 nm. Melting points were measured using an
IA9100 digital melting point apparatus (Electrothermal Engineering,
Essex, U.K.). The synthesized compounds were analyzed by
NMR on a Varian Mercury 300 MHz spectrometer (Varian, Palo
Alto, CA) and by GC-MS. The GC-MS system consisted of a
5890A gas chromatograph and a 5970 mass selective detector
(Hewlett-Packard, Palo Alto, CA). NMR spectra were processed
using the Varian VNMR software (Varian, Palo Alto, CA) on a
UNIX Solaris workstation. GC-MS spectra were analyzed with
the HP ChemStation program on a Windows 3.1 workstation.
Optical rotatory powers were determined with a Polartronic E
polarimeter by use of a microtube for small sample volumes (100
mm, 1.10 mL; Schmidt + Haensch, Berlin, Germany). The
elemental analyses were conducted by Robertson-Microlit Labo-
ratories (Madison, NJ).
The HPLC system consisted of the Agilent 1100 series degasser,
binary pump, autosampler, thermostated column compartment
(sustained at 40 °C), multiple wavelength detector, fluorescence
detector, and fraction collector (Agilent Technologies, Palo Alto,
CA). Radiolabeled [14C]glucuronides were detected by use of a 9701
HPLC radioactivity monitor (Reeve Analytical, Glasgow, Scotland)
at 16 kcounts/s after separation on a Hypersil BDS-C18 column
(4.6 mm × 150 mm; Agilent Technologies, Palo Alto, CA) with
the use of a mixture of methanol and phosphate buffer (50 mM,
pH 3) as an eluent. Signal separation for the scopoletin assays was
carried out using a reversed-phase C18 column (Chromolith
SpeedROD RP18e 50 mm × 4.6 mm; Merck, Darmstadt, Germany).
The resulting spectra were analyzed with Agilent ChemStation
software (revision B.01.01) on a Windows 2000 workstation.
Regression and correlation analyses were conducted using Prism4
software (version 4.02; GraphPad Software, San Diego, CA).
General Procedure for the CBS Reduction. All reactions were
performed in oven-dried glassware under an atmosphere of dry
argon. (R)- and (S)-2-(diphenylhydroxymethyl)pyrrolidine were
synthesized as previously described.24
To a solution of (R)- or (S)-2-(diphenylhydroxymethyl)pyrroli-
dine (253 mg, 1.00 mmol) in dry toluene (10.0 mL) was added
trimethyl borate (125 mg, 1.20 mmol), and the mixture was stirred
under argon atmosphere at room temperature for 2 h (23 °C). Borane
N,N-diethylaniline complex (1.78 mL, 10.0 mmol) was added via
cannula, followed by dropwise addition of a solution of ketone (10.0
mmol) in dry toluene (10.0 mL) over 2.5 h with a syringe pump.
The mixture was stirred overnight at room temperature. The
resulting solution was quenched with methanol (5.0 mL) at 0 °C
and concentrated under reduced pressure. The residue was puri-
fied by flash chromatography on a silica gel Flash 25+M cartridge
(25 × 150 mm) with a SP4 purification system (Biotage AB,
Uppsala, Sweden; 50 mL/min flow rate, detection at 254 nm). The
eluent consisted of a mixture of ethyl acetate and n-hexane (cf.
Supporting Information). Subsequently, the eluent was evaporated
in vacuo and the resulting solid was recrystallized several times to
increase the enantiomeric excess. The obtained crystals were
filtered, washed with n-hexane, and dried in vacuo. In the case of
liquid compounds, the enantiomers were resolved by HPLC
purification on an (R,R)-ULMO column. The enantiomers of
compounds 1 and 8 were prepared according to the procedure as
previously described.24
in n-hexane); mp 46.0-46.5 °C (toluene/n-hexane); [R]23 +47.3
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C11H14O2) C, H.
(R)-4: (-)-(R)-7-Nitro-1-tetralol. Rf ) 0.31 (25% EtOAc in
n-hexane); mp 79.9-80.6 °C (CH2Cl2/n-hexane); [R]23 -63.7
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H11NO3) C, H, N.
(S)-4: (+)-(S)-7-Nitro-1-tetralol. Rf ) 0.31 (25% EtOAc in
n-hexane); mp 80.2-80.8 °C (CH2Cl2/n-hexane); [R]23 +62.3
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H11NO3) C, H, N.
(R)-5: (-)-(R)-5,7-Dimethyl-1-tetralol. Rf ) 0.26 (10% EtOAc
in n-hexane); mp 87.5-88.0 °C (10% EtOAc in n-hexane); [R]23
D
-48.5 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C12H16O) C, H.
(S)-5: (+)-(S)-5,7-Dimethyl-1-tetralol. Rf ) 0.28 (10% EtOAc
in n-hexane); mp 87.4-88.0 °C (CH2Cl2/n-hexane); [R]23 +49.4
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C12H16O) C, H.
(R)-6: (+)-(R)-4-Chromanol. Rf ) 0.26 (15% EtOAc in
n-hexane); mp 76.2-76.7 °C (toluene/n-hexane); [R]23 +46.0
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C9H10O2) C, H.
(S)-6: (-)-(S)-4-Chromanol. Rf ) 0.25 (15% EtOAc in
n-hexane); mp 76.2-76.6 °C (toluene/n-hexane); [R]23 -43.9
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C9H10O2) H, C: calcd,
71.98; found, 71.22.
(R)-7: (+)-(R)-6-Methyl-4-chromanol. Rf ) 0.27 (20% EtOAc
in n-hexane); mp 84.3-84.9 °C (CH2Cl2/n-hexane); [R]23 +37.5
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O2) C, H.
(S)-7: (-)-(S)-6-Methyl-4-chromanol. Rf ) 0.27 (20% EtOAc
in n-hexane); mp 83.9-84.4 °C (CH2Cl2/n-hexane); [R]23 -38.6
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O2) C, H.
(R)-9: (-)-(R)-4-Methyl-1-indalol. Rf ) 0.29 (20% EtOAc in
n-hexane); mp 69.3-69.8 °C (20% EtOAc in n-hexane); [R]23
D
-36.3 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O) C, H.
(S)-9: (+)-(S)-4-Methyl-1-indalol. Rf ) 0.31 (20% EtOAc in
n-hexane); mp 69.0-69.6 °C (CH2Cl2/n-hexane); [R]23 +36.6
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O) C, H.
(R)-10: (-)-(R)-6-Methyl-1-indalol. Rf ) 0.23 (15% EtOAc
in n-hexane); mp 94.6-95.7 °C (CH2Cl2/n-hexane); [R]23 -32.3
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O) C, H.
(S)-10: (+)-(S)-6-Methyl-1-indalol. Rf ) 0.25 (15% EtOAc in
n-hexane); mp 94.0-95.0 °C (CH2Cl2/n-hexane); [R]23 +31.3
D
deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C10H12O) C, H.
(R)-11: (+)-(R)-1-Naphthalen-1-ylethanol. Rf ) 0.21 (10%
EtOAc in n-hexane); [R]23 +62.7 deg‚mL‚dm-1‚g-1 (c 1.5,
D
CHCl3). Anal. (C12H12O) C, H.
(S)-11: (-)-(S)-1-Naphthalen-1-ylethanol. Rf ) 0.21 (10%
EtOAc in n-hexane); [R]23 -61.2 deg‚mL‚dm-1‚g-1 (c 1.5,
D
CHCl3). Anal. (C12H12O) C, H.
(R)-12: (+)-(R)-1-Naphthalen-2-ylethanol. Rf ) 0.23 (15%
EtOAc in n-hexane); mp 69.7-70.4 °C (CH2Cl2/n-hexane); [R]23
D
+51.1 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C12H12O) C, H.
(S)-12: (-)-(S)-1-Naphthalen-2-ylethanol. Rf ) 0.22 (15%
EtOAc in n-hexane); mp 70.1-70.7 °C (CH2Cl2/n-hexane); [R]23
D
-49.9 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C12H12O) C, H.
(R)-13: (+)-(R)-1-Anthracen-9-ylethanol. Rf ) 0.29 (20%
EtOAc in n-hexane); mp 120.6-121.5 °C (10% EtOAc in n-
hexane); [R]23 +13.0 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal.
D
(C16H14O) C, H.
(S)-13: (-)-(S)-1-Anthracen-9-ylethanol. Rf ) 0.29 (20%
EtOAc in n-hexane); mp 120.6-121.5 °C (10% EtOAc in n-
hexane); [R]23 -13.7 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal.
D
(C16H14O) C, H.

1826 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
(R)-14: (+)-(R)-1-Phenanthren-9-ylethanol. Rf ) 0.32 (30%
Bichlmaier et al.
addition of perchloric acid (4.0 M), and the mixtures were
transferred to ice. The mixtures were centrifuged (16000g, 10 min),
and aliquots of the supernatants were subjected to HPLC analysis.
The eluent consisted of methanol in phosphate buffer (50 mM, pH
3). Fluorescence excitation and emission wavelengths of 335 and
455 nm, respectively, were optimal for the detection of scopoletin
glucuronide. The quantification was carried out by recording
standard curves with dilutions of scopoletin glucuronide, which was
previously synthesized in our laboratory. The data were analyzed
by nonlinear regression applying the two-parameter Hill equation
(concentration-response curve). The Kic values and the mechanism
of inhibition for the racemates of compounds 5, 7, and 10 were
determined by measuring the initial velocities for scopoletin
glucuronidation. Scopoletin was used at 100, 250, 500, 1000, and
2000 µM for both UGT isoforms. The enantiomers were applied
at three concentrations bracketing their estimated Kic values, which
were calculated by the Cheng-Prusoff equation for competitive
inhibition from their respective IC50 values.39 The assays were
conducted similarly to the determination of IC50 values. The results
reflect a minimum of three replicate determinations.
EtOAc in n-hexane); mp 126.7-127.7 °C (toluene/n-hexane); [R]23
D
+79.5 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C16H14O) C, H.
(S)-14: (-)-(S)-1-Phenanthren-9-ylethanol. Rf ) 0.34 (30%
EtOAc in n-hexane); mp 126.4-127.2 °C (toluene/n-hexane); [R]23
D
-76.8 deg‚mL‚dm-1‚g-1 (c 1.5, CHCl3). Anal. (C16H14O) C, H.
Racemization Study. It was determined that the enantiomerically
pure substrates did not undergo racemization under the reaction
conditions of the enzyme assays. To determine the magnitude of
racemization, each (R)-enantiomer was dissolved in methanol and
diluted to a final concentration of 1 mg/mL with 50% methanol/
water (v/v). To this solution, phosphate buffer (20 mM, pH 7.4)
was added to a final concentration of 10% (v/v), and the mixture
was sustained at 40 °C overnight. The methanol was removed under
reduced pressure, and the solution was then extracted with diethyl
ether. The organic phase was washed with water and brine and
was evaporated in vacuo, and the residue was dissolved in a small
amount of dichloromethane. The resulting solution was subjected
to HPLC analysis to determine the optical purity.
Activity Assays. The progression curves were recorded using
20 µM radiolabeled [14C]UDPGlcA (20.0 µCi/mL, 196 mCi/mmol)
in combination with 100 µM cold UDPGlcA yielding a total
concentration of 120 µM of this cosubstrate.36 The assay volume
was 700 µL and consisted of 50 mM phosphate buffer (pH 7.4),
10 mM MgCl2, and 5.0 mM saccharic acid-1,4-lactone. The
enzymes UGT2B7 and UGT2B17 were employed at 1.0 µg/µL.
The enzyme reactions were initiated after 5 min of preincubation
(37 °C) by the addition of a solution of the bicyclic enantiomer in
50% water/DMSO (v/v) to a final, saturating concentration of 2.0
mM. The flexible compounds 11-14 were employed at a final
concentration of 0.1 mM. The DMSO concentration in the assay
mixtures was 2.5% (v/v). The enzyme reactions, incubated at 37
°C, were terminated after a set amount of time by pipetting 100
µL of the reaction mixture to a vial containing 10 µL of perchloric
acid (4.0 M). The acidified mixtures were vortexed, transferred to
ice, centrifuged (16000g, 10 min), and aliquots of the supernatants
were subjected to HPLC analysis. The eluent consisted of methanol
in phosphate buffer (50 mM, pH 3). The signal detection was carried
out as described above. The results reflect a minimum of two
replicate determinations. It was demonstrated that a concentration
of 2.0 mM for the bicyclic alcohols was saturating by determining
the velocity of the glucuronidation reaction at concentrations of
0.5, 1.0, and 2.0 mM showing that the plateau in the Michaelis-
Menten plot (velocity versus substrate concentration) was reached
between approximately 0.5 and 1.5 mM depending on the substrate
used. Substrate inhibition was not detected. Therefore, the velocities
measured at a concentration of 2.0 mM yielded good estimates of
the Vmax values under the conditions of the assay (unsaturating
cosubstrate concentration). Nonlinear kinetic behavior due to
enzyme denaturation occurred for UGT2B7 after an incubation time
of 140 min and for UGT2B17 after 160 min, as demonstrated by
progression curves using scopoletin as well as UDPGlcA at
saturating concentrations.
Inhibition Assays. The IC50 values of the substrates 1-10 were
determined at concentrations of 10, 25, 50, 100, 250, 500, and 1000
µM, and the IC50 values for the enantiomers of compounds 11-14
were determined at 5, 10, 25, 50, and 100 µM. Scopoletin was
employed at 500 µM for UGT2B7 and 300 µM for UGT2B17,
and it resembled its Km for the respective enzyme (531 and 309
µM, respectively). The Km values of scopoletin for UGT2B7 and
UGT2B17 were determined frequently in our laboratory, and the
values represent the average values of experiments conducted during
the last year using the same enzyme batches. The reaction mixture
consisted of 5.0 mM phosphate buffer (pH 7.4), 10 mM MgCl2,
and 5.0 mM saccharic acid-1,4-lactone. The enzymes were em-
ployed at 0.1 µg/µL. One control assay in the absence of inhibitor
and one blank run in the absence of cosubstrate were included in
each inhibition assay. The enzyme reaction was initiated after a 5
min preincubation time (37 °C) by the addition of a solution of
UDPGlcA to a final concentration of 5.0 mM. The enzyme reactions
were terminated after an incubation time of 15 min at 37 °C by the
Acknowledgment. This work was supported by a fellowship
from the Finnish Cultural Foundation (I.B.) and the Academy
of Finland (Project 207535).
Supporting Information Available: Degree of purity (elemen-
tal analysis), spectroscopic data, statistical data for Tables 2 and 3,
results of the inhibition-model selection, and equations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM051142C