Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y1-receptor

Article abstract of DOI:10.1021/jm049771u

In the companion paper, part 1, we described the construction of an improved molecular model for the h-P2Y₁ receptor (h-P2Y ₁-R) and proposed a rational for the stereoelectronic selectivity of the receptor. Here, we extend our studies on the molecular recognition of the h-P2Y₁-R to the exploration of the diastereoselectivity of this receptor. For this purpose, we implemented an integrative approach combining synthesis, spectral analysis, biochemical assays, and computational analysis. Specifically, we selected and synthesized novel ATP analogues bearing a chiral center on the phosphate chain. We analyzed the conformation of the chiral ATP analogues in solution by ¹H/¹³C NMR and assigned the absolute configuration of the diastereoisomers. The coordination mode of these analogues with a Mg²⁺ ion was evaluated by ³¹P NMR. These chiral analogues were biochemically evaluated and found to be potent h-P2Y ₁-R ligands. An EC₅₀ difference of ca. 20-fold was observed between the diastereoisomers. Their spectral absolute configuration assignment was confirmed by comparison of the biochemical results to those of ATP-α-S diastereoisomers whose chirality is known. Finally, a computational analysis was performed for the elucidation of molecular recognition employing molecular mechanics (docking) studies on the receptor:ligands complexes. On the basis of the current results, we hypothesize that h-P2Y₁-R's chiral discrimination originates from the requirement that the nucleotide analogue interacts with a Mg²⁺ ion within the receptor binding site. This Mg2+ ion is possibly coordinated with both Asp204 and the ATP's α, β, γ-phosphates in a Λ configuration.

Full text of DOI:10.1021/jm049771u

J . Med. Chem. 2004, 47, 4405-4416
4405
Molecu la r Recogn ition in P u r in er gic Recep tor s. 2. Dia ster eoselectivity of th e
h -P 2Y1-Recep tor †
Dan T. Major,‡ Victoria Nahum,‡ Yingfei Wang,§ Georg Reiser,§ and Bilha Fischer*,‡
Department of Chemistry, Gonda-Goldschmied Medical Research Center,
Bar-Ilan University, Ramat-Gan 52900, Israel, and Institute for Neurobiochemistry,
Faculty of Medicine, Otto von Guericke University, Leipziger Str. 44 D-39120 Magdeburg, Germany
Received March 24, 2004
In the companion paper, part 1, we described the construction of an improved molecular model
for the h-P2Y1 receptor (h-P2Y1-R) and proposed a rational for the stereoelectronic selectivity
of the receptor. Here, we extend our studies on the molecular recognition of the h-P2Y1-R to
the exploration of the diastereoselectivity of this receptor. For this purpose, we implemented
an integrative approach combining synthesis, spectral analysis, biochemical assays, and
computational analysis. Specifically, we selected and synthesized novel ATP analogues bearing
a chiral center on the phosphate chain. We analyzed the conformation of the chiral ATP
analogues in solution by 1H/13C NMR and assigned the absolute configuration of the
diastereoisomers. The coordination mode of these analogues with a Mg2+ ion was evaluated by
31P NMR. These chiral analogues were biochemically evaluated and found to be potent h-P2Y1-R
ligands. An EC50 difference of ca. 20-fold was observed between the diastereoisomers. Their
spectral absolute configuration assignment was confirmed by comparison of the biochemical
results to those of ATP-R-S diastereoisomers whose chirality is known. Finally, a computational
analysis was performed for the elucidation of molecular recognition employing molecular
mechanics (docking) studies on the receptor:ligands complexes. On the basis of the current
results, we hypothesize that h-P2Y1-R’s chiral discrimination originates from the requirement
that the nucleotide analogue interacts with a Mg2+ ion within the receptor binding site. This
Mg2+ ion is possibly coordinated with both Asp204 and the ATP’s R, â, γ-phosphates in a Λ
configuration.
Sch em e 1. ATP Analogues Used as Stereo- and
Diastereoprobes of the P2Y1-R
In tr od u ction
Studies of the molecular recognition of ATP receptors
(P2-Rs), and especially of the P2Y1-R subtype, have
focused over the past decade on the stereoelectronic
requirements of the receptors. The question of the
molecular recognition of the P2Y1-R was addressed by
SAR studies of novel ATP analogues,1 site-directed
mutagenesis, and molecular modeling.2
However, to the best of our knowledge, the question
of the receptor’s diastereoselectivity has not been ad-
dressed. The need for addressing this question arose as
we discovered that the P2Y1-R demonstrates a clear
preference for one of the diastereoisomers of our chiral
PR-phosphate ATP ligands (Scheme 1). These ligands
include ATP-R-B analogues (3, 4, 7, 8, 11, 12)3 and ATP-
R-S analogues (5, 6, 9, 10) (described below). Studies of
this chiral selectivity are expected both to provide the
origin of the receptor’s diastereoselectivity and to help
design novel potent and selective agonists.
For elucidating the origin of the receptor’s chiral
discrimination, we implemented the following strat-
egy: (1) synthesis and separation of the PR-chiral-ATP
analogues, (2) biochemical analysis of the pure diaste-
reoisomers of analogues 3-12 as P2Y1-R ligands, (3)
conformational analysis of one of the diastereoisomeric
pairs in solution and elucidation of the absolute config-
uration around the PR in analogues 3-12, and (4)
docking studies of the diastereoisomeric pairs of ana-
logues 3-12 at the P2Y1-R binding-site, as obtained by
molecular modeling (preceding companion paper),4 in
order to reveal the origin of chiral selectivity of the
P2Y1-R.
Here, we report on the identification of the preferred
P2Y1-R ligands’ diastereoisomers and their absolute
configuration and propose explanations for the recep-
tor’s diastereoselectivity.
† This work has been supported by Innodia Inc, Montreal, Canada.
* Corresponding author. Fax: 972-3-5351250. Tel: 972-3-
5318303. E-mail: bfischer@mail.biu.ac.il.
Resu lts
Selection a n d P r ep a r a tion of ATP An a logu es for
P r obin g Ch ir a l Discr im in a tion of th e h -P 2Y1-R.
‡ Bar-Ilan University.
§ Otto von Guericke University.
10.1021/jm049771u CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/03/2004

4406 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
Sch em e 2. Substitution of Nonbonding Oxygen on PR
by BH3 Group or Sulfur Atom Induces the Formation of
Sp and Rp Distereoisomers
Phosphorothioate analogues of ATP have been widely
used for the determination of the stereoselectivity of
enzymes and the configuration of the metal-ATP
substrate complex in enzymatic reactions by measuring
kinetic parameters of the enzymatic reaction with the
diastereoisomers of ATP-R-S and ATP-â-S in the pres-
ence of various metal ions.5
For probing the chiral discrimination of the h-P2Y1-
R, we chose ATP analogues 3-12 possessing a chiral
PR (Scheme 2).
F igu r e 1. NMR spectra of 2-MeS-ATP-R-S (mixture of two
diastereoisomers): (A) 31P NMR spectrum. Signals of PR of Rp-
and Sp-isomers measured at 81 MHz. (B) H NMR spectrum.
1
In previous studies, we synthesized various ATP-R-S
analogues and established their P2Y1-R affinity.6 There-
fore, we applied ATP-R-S analogues 5/6 and 9/10, estab-
lished in this study to possess P2Y1-R affinity, as probes
for exploring the chiral discrimination of P2Y1-R. For this
purpose, we also applied ATP-R-B probes 3/4, 7/8, and
11/12, which we recently synthesized and characterized
as potent P2Y1-R ligands.3 Furthermore, these ATP-R-B
analogues show no potency at the P2Y2-receptor.7
The diastereoisomeric pair of 2-MeS-ATP-R-S ana-
logues 9/10 was prepared here in two steps from 2-SH-
adenosine. Highly facile and selective S-alkylation
afforded 2-MeS-adenosine in quantitative yield. This
derivative was then triphosphorylated in a one-pot
reaction6 to afford the product as a diastereomeric pair
9/10 (Scheme 3). The products were analyzed by 1H and
31P NMR spectra (Figure 1) and the diastereoisomers
were separated by HPLC (Figure 2).
Signals of H8 of Rp- and Sp-isomers measured at 200 MHz.
F igu r e 2. HPLC chromatogram of a mixture of 2-MeS-ATP-
R-S diastereoisomers 9,10. Separation was performed on a
LichroCART LiChrospher 60 RP-select B column (250 × 10
mm), flow rate 5 mL/min. A linear gradient of methanol:0.1
M triethylammonium acetate, 10:90 to 30:70, in 25 min was
applied.
Finally, for the comparison of P2Y1-R affinity of
analogues 9/10 to the parent compound and for estab-
lishing the absolute configuration of analogues 9/10, we
also biochemically assayed commercial ATP-R-S Rp and
Sp diastereoisomers 5/6.
tion of th e Ch ir a l P r obes. Adenine nucleotides are
expected to possess conformational flexibility, due to
possible rotations around the glycosidic bond and pseu-
dorotation of the ribose ring, as well as possible rota-
tions around the C4′-C5′, C5′-O5′, and O5′-PR bonds.
Con for m a tion a l An a lysis of 2-MeS-ATP -r-B in
Solu tion a n d Assign m en t of Absolu te Con figu r a -
Sch em e 3. Preparation of Chiral Probes 9/10

Molecular Recognition in Purinergic Receptors. 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4407
1
An a lysis of Sp ectr a . Here, we employed H and 13C
NMR spectra to study the solution conformation of both
diastereoisomers of 2-MeS-ATP-R-B, 7/8, and to eluci-
date the absolute configuration around PR for both
diastereoisomers. This absolute configuration assign-
ment enabled also the assignment of chirality of the
other probes under investigation. The chemical shifts
and splitting pattern of adenine and sugar protons of
0.03 M D2O solutions of both diastereoisomers of 2-MeS-
ATP-R-B were assigned from 1H NMR spectra at 300
and 600 MHz (Tables 1 and 2). Diastereoisomers 7 and
8 were denoted as A and B, respectively, where the
A-isomer is the first eluting isomer from HPLC.
Con for m a tion Ar ou n d th e Glycosid ic Bon d . Pu-
rine nucleotides can adopt two main conformations, syn
or anti, in which the aromatic proton H8 points above
or away from the sugar ring. The quantitative deter-
mination of the conformation around the glycosidic bond
can be obtained by monitoring the vicinal coupling
F igu r e 3. Concentration-response curves for 9,10 isomers
and ATP by measuring intracellular calcium ([Ca2+]i) mobiliza-
tion in HEK cells expressing the P2Y1 receptor. Black squares,
triangles, and circles represent the concentration-response
curves for 9, 10, and ATP, respectively. The data points
represent the mean ( SEM of 57-220 cells from at least three
different experiments and three different cells preparations.
3
constants J C8-H1′ and 3J C4-H1′, which are extracted from
13C NMR spectra for each diastereomer of 7 and 8. A
practical rule for the orientation of the base relative to
the ribose was formulated: a value of 3J C4-H1′ < 3J C8-H1′
indicates that ø is in the anti conformation, whereas the
reverse indicates that ø is in the syn conformation.11
The J -coupling constants in Table 2 provide strong
support for the preferred anti-conformer in solution for
both diastereoisomers of 2-MeS-ATP-R-B.
The magnitude of a three-bond scalar coupling J
depends on the value of the corresponding torsion angle
θ by the following expression, which is known as the
Karplus equation:
3J (θ) ) A cos2 θ + B cos θ + C
Ippel et al. reparametrized and generalized the Karplus
equations for the glycosidic bond conformation of purine
and pyrimidine nucleosides and nucleotides.11
The following equations11 were used in this study to
calculate the glycosidic bond angle ø for A- and B-
isomers of 2-MeS-ATP-R-B (7 and 8):
3J C6/8-H1′ ) 4.5 cos2(ø - 60°) - 0.6 cos(ø - 60°) + 0.1
3J C2/4-H1′ ) 4.7 cos2(ø - 60°) + 2.3 cos(ø - 60°) + 0.1
F igu r e 4. Concentration-response curves for Rp-ATP-R-S (6),
Sp-ATP-R-S (5), and ATP by measuring intracellular calcium
([Ca2+]i) mobilization in HEK cells expressing the P2Y1 recep-
tor. Black squares, triangles, and circles represent the con-
centration-response curve for Rp-ATP-R-S, Sp-ATP-R-S, and
ATP, respectively. The data points represent the mean ( SEM
of 64-220 cells from at least three different experiments and
three different cells preparations.
The first equation provides values of ø ) 228.2° for the
A-isomer, and ø ) 210.1° for B-isomer in øanti range. The
second equation yields ø ) 217.8°, since the J values for
both diastereoisomers are identical (3J C2/4-H1′ ) 2 Hz).
Su ga r P u ck er in g.
However, it is well-established that most 5′-nucleotides
adopt a predominant preferred conformation in solution
(which is nevertheless in equilibrium with other con-
formations).8
The conformation of the D-ribose ring of 2-MeS-ATP-
R-B was analyzed in terms of a dynamic equilibrium
between two favored puckered conformations: a type
N conformer and a type S conformer.12,13 N and S
equilibrium populations were calculated from observed
Thus, it has been shown that a majority of purine and
pyrimidine 5-nucleotides favor an anti conformation of
base ring with respect to the sugar ring.9 Likewise, the
ribose ring exhibits a puckered conformation in which
either the C2′ or C3′ atom is furthest from the plane of
the other atoms of the ribose ring, named as South (S)
and North (N) conformations, respectively.10 Finally, it
has been shown that the ribose exocyclic group exists
predominantly in a gauche-gauche (gg) conformation
about the C4′-C5′ bond, with the O5′ atom projecting
over the furanose ring.
J
1′2′ and J 3′4′ couplings as reported previously.14 Accord-
ing to this method, the observed vicinal couplings are
related to the relative proportion of conformers by eqs
1-3:
J 1′2′ ) 9.3(1 - XN) ) 9.3XS
J 2′3′ ) 4.6XN + 5.3(1 - XN)
J 3′4′ ) 9.3XN
(1)
(2)
(3)

4408 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
Ta ble 1. Chemical Shifts and Splitting Pattern of 2-MeS-ATP-R-B in D2O
isomer A
H8
H1′
H2′
H3′
H4′
H5′
H5′′
chemical shift (δ)
coupling constants (Hz)
8.46
s
6.14
d 5.3
4.82
dd 5.3, 5.0
4.69
dd 5.0, 4.0
4.38
ddd 2.4, 2.8, 4.0
4.35
4.14
ddd 2.4, 4.2, 11.8
ddd 2.8, 4.5, 11.8
isomer B
H8
H1′
H2′
H3′
H4′
H5′
H5′′
chemical shift (δ)
coupling constants (Hz)
8.42
s
6.13
d 5.7
4.86
dd 5.7, 5.1
4.61
dd 5.1, 3.7
4.39
ddd 2.9, 3.2, 3.7
4.29
4.19
ddd 3.2, 7.7, 11.8
ddd 2.9, 5.8, 11.8
Ta ble 2. 3J Coupling Constants (Hz) of 2-MeS-ATP-R-B in D2O
1′, 2′
2′, 3′
3′, 4′
4′, 5′
4′, ′′5
5′, PR
5′′, PR
C8, H1′
C4, H1′
isomer A
isomer B
5.3
5.7
5.0
5.1
4.0
3.7
2.4
3.2
2.8
2.9
4.2
7.7
4.5
5.8
5
4
2
2
Ta ble 3. Conformational Analysis of 2-MeS-ATP-R-B in Solution
sugar pucker C4′-C5′ bond
C5′-O5′ bond
isomer A %
isomer B %
isomer A%
isomer B%
isomer A%
isomer B%
XS ) 57.0
XN ) 43.0
Keq )1.32
XS ) 60.6
XN ) 39.4
Keq )1.54
gg ) 88.4
tg ) 3.7
gt ) 7.9
gg ) 79.1
tg ) 12
gt ) 8.9
gg ) 73.3
tg ) 12.6
gt ) 14.1
gg ) 48.2
tg ) 30.9
gt ) 20.9
Sch em e 4. Possible Nucleotide Rotamers: (A)
Rotamers around the C4′-C5′ Bond and (B) Rotamers
around the C5′-O5′ Bond
The coupling constants for pure rotamers were estimat-
ed as J g ) 2.04 Hz and J t ) 11.72 Hz from the appro-
priate Karplus relation.15,12 The observed proton signals
which are labeled H5′ and H5′′ refer to downfield and
upfield signals, respectively. From the results presented
in Table 3, there is a marked preference for a gg rotamer
about C4′-C5′ bond in both diastereoisomers.
The computations of conformational properties for
exocyclic carbinol groups and furanose ring conforma-
tions suggest a destabilization of the gg conformer in a
C2′-endo (S) pucker relative to a C3′-endo (N) pucker
of the furanose ring.16 Table 3 shows that as the S con-
former population increases (57% for A-isomer and
60.6% for B-isomer), the gg conformer population de-
creases (88.4% and 79.1%, respectively).
2. C5′-O5′ Bon d . Rotamer populations about C5′-
O5′ are calculated from 31P-H5′ and 31P-H5′′ coupling
constants by procedures analogous to those used for
C4′-C5′, using eqs 8-10:
Fgg ) [(J t + J g) - (J P5′ + J P5′′)]/(J t - J g)
Ftg ) (J P5′ - J g)/(J t - J g)
(8)
(9)
The equilibrium constant Keq can be calculated directly
from the observed values of J 1′2′ and J 3′4′
:
Keq ) XS/XN ) J 1′2′/J 3′4′
(4)
Fgt ) (J P5′′ - J g)/(J t - J g)
(10)
Using the assigned J -coupling constants (Table 2) and
the above equation, the mole fraction of conformers S
and N and Keq for both diastereoisomers were calculated
and the results are summarized in Table 3. For both A-
and B-isomers the predominant ribose pucker is S (57%
and 61% for A- and B-isomers, respectively).
Con for m a tion s of Exocyclic CH2OR Gr ou p (R )
P h osp h a te). 1. C4′-C5′ Bon d . The coupling constants
J 4′5′ and J 4′5′′ can be interpreted in terms of three
classical staggered rotamers shown in Scheme 4A with
a preferred gauche-gauche (gg) conformation.12 The
mole fractions of each staggered rotamer of C4′-C5′
were calculated from the following expressions (5-7):
Where J t ) 20.9 Hz and J g ) 1.8 Hz, as reported
previously.12 Relative rotamer populations were then
calculated from observed J (HCOP) magnitudes (Table
3). Clearly, the two diastereoisomers have different
conformational behavior around the C5′-O5′ bond,
because of the asymmetric center present in PR.
Assign m en t of th e Absolu te Con figu r a tion of th e
Ch ir a l P r obes. A difference in the chemical shift of H8
is observed between the two diastereoisomers of 2-MeS-
1
ATP-R-B. The signal of H8 of B-isomer in the H NMR
spectra is more shielded than the signal of H8 of
A-isomer due to the influence of the vicinal BH3 group
on PR (8.46 vs 8.42 ppm). Because of the difference in
torsion angle of the C5′-O5′ bond between isomer A and
B, as shown above, PR is much further away from H8
in A-isomer than in B-isomer (B-isomer is shown in
Scheme 5). This small, although indicative, ∆δ between
H8 of isomers A and B may be explained in terms of
Fgg ) [(J t + J g) - (J 4′5′ + J 4′5′′)]/(J t - J g)
Ftg ) (J 4′5′ - J g)/(J t - J g)
(5)
(6)
(7)
Fgt ) (J 4′5′′ - J g)/(J t - J g)

Molecular Recognition in Purinergic Receptors. 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4409
Ta ble 4. Differences of Chemical Shifts (∆δ in ppm) between
Adenine Nucleotides Mg2+ Complexes and Parent Compounds
ATP analogue
analogue no.
PR
Pâ
Pγ
ATP
2-MeSATP-R-B
2-MeSATP-R-S
1
0.33
1.96
2.14
2.44
2.72
2.25
0.51
1.06
0.78
7, 8
9, 10
Sch em e 5. Sp Configuration Is Attributed to
Diastereoisomer 8 (B-isomer)
F igu r e 5. Dihedral angles investigated during force field
development for borano- or thiodiphosphate.
the greater proximity of the negative charge on BH3 to
H8 as compared to O. The greater proximity is due to
the longer P-B bond (1.89 Å) as compared to P-O (1.60
Å), in addition to the contribution of the B-H bond (1.25
Å).17 Furthermore, the upfield shift of H8 of the B-
isomer may be due to an unconventional H-bond (known
to exist between borane-amines and acidic protons)18
between the BH3 group and the relatively acidic H8.8,19
Such a preferential intramolecular H-bond results in the
shielding of H8.
F igu r e 6. Water-Me-boranodiphosphate or -Me-thiodiphos-
phate interaction geometries.
Ta ble 5. Comparison of EC50 Values of Several ATP Analogues
as P2Y1-R Agonists
Only in the gt rotamer is the BH3 group near the H8
(population of this rotamer in B-isomer is higher than
in A-isomer, 21% vs 14%). In the gt rotamer, the H8 is
closer to the BH3 group when PR is in the Sp configu-
ration with an expected H-bond length between H8 and
BH3 of ca. 1.9 Å18 (Scheme 5).
Thus, the Sp configuration can be attributed to
B-isomer, 8, and Rp to A-isomer, 7, of 2-MeS-ATP-R-B.
Since the group priorities around PR for ATP-R-S
analogues are opposite to those of ATP-R-B, and assum-
ing the same order of elution in HPLC, the absolute
configuration assignments for 2-MeS-ATP-R-S ana-
logues are that the Sp configuration is attributed to
A-isomer, 9, and Rp to B-isomer, 10.
analogue absolute
ATP analogue
no.
config
EC50 (nM)
ATP
1
2
590
1.0
200
>1000
9.4
75
2.6
53
1.0
20
2-MeS-ATP
ATP-R-B (A-isomer)
ATP-R-B (B-isomer)
ATP-R-S
3
4
5
6
7
8
9
10
11
12
Rp
Sp
Sp
Rp
Rp
Sp
Sp
Rp
Rp
Sp
ATP-R-S
2-MeS-ATP-R-B (A-isomer)
2-MeS-ATP-R-B (B-isomer)
2-MeS-ATP-R-S (A-isomer)
2-MeS-ATP-R-S (B-isomer)
2-Cl-ATP-R-B (A-isomer)
2-Cl-ATP-R-B (B-isomer)
4.5
36
performed in HEK cells that were stably transfected
with P2Y1 receptor. The density of the overexpressed
P2Y1-R is several orders of magnitude higher than that
of the endogenous receptors. Therefore, the interaction
of other P2Y-R subtypes with the tested ligands is
negligible and does not contribute to the observed EC50
values.
Indeed, this absolute configuration assignment was
confirmed later by the biochemical assays and compu-
tational studies described below.
Coor d in a tion An a lysis of Mg2+ Com p lexes of
ATP a n d An a logu es 7,8 a n d 9,10 in Solu tion . To
learn the possible modes of coordination of Mg2+ with
chiral probes 7,8 and 9,10 in solution, we explored the
31P NMR spectra of these complexes at pH 7.4 and 0.04
M concentration of each nucleotide and MgCl2 and
compared the spectra to those of the ATP:Mg2+ complex
(Table 4).
Concentration-response curves of 9 and 10 show that
both substances are more potent in inducing the [Ca2+
]
i
mobilization than ATP (Figure 3). The EC50 value of
ATP is about 590 nM. However, the agonist effect of
isomer 9 (EC50 1 nM) is about 20-fold higher than that
of its isomer 10 (EC50 20 nM). 2-MeS-ATP is as potent
as isomer 9 with a comparable EC50 value of 1 nM, Table
5 (curve not shown). The amplitude caused by 100 nM
ATP was very small compared to the Ca2+ response
caused by 1 µM ATP, which was close to the maximal
response. The slopes for the concentration-response
curves appear to differ between ATP and isomers 9/10.
The concentration-response curves of the parent Rp-
ATP-R-S and Sp-ATP-R-S isomers demonstrate that
these two chiral analogues of ATP are more potent than
ATP (Figure 4). In addition, isomer Sp-ATP-R-S (5) with
an EC50 value of 9.4 nM is about 8-fold more potent than
its isomer Rp-ATP-R-S (6) (EC50 75 nM). Taken together,
Apparently, all three phosphates in both chiral dias-
tereoisomeric pair probes are involved in metal ion
coordination, with Pâ, being mostly affected. Yet, mixed
chelate populations, i.e., PR,â-Mg2+ and Pâ,γ-Mg2+ coor-
dinated species, may exist at equilibrium. For ATP the
effect of Mg2+ coordination is significantly lower at PR
and Pγ as compared to those values for analogues 7,8
and 9,10.
Eva lu a tion of Isom er s of ATP -r-S (5,6) a n d
2-MeS-ATP -r-S (9,10) a s P 2Y1-R Agon ists. To study
the efficacy of 2-MeS-ATP-R-S isomers 9,10, we exam-
ined the [Ca2+]i mobilization induced by these analogues
and compared it to that of commercial Sp-ATP-R-S (5)
and Rp-ATP-R-S (6) and ATP. These studies were

4410 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
Ta ble 6. Comparison of Ab Initio and Empirical Interaction
Energies (kcal/mol) and Geometries (Å) between Water and
Me-Diphosphate (MePP),a Me-R-Thiodiphosphate (MeSPP), and
Me-R-Boranodiphosphate (MeBPP)
ab initio (HF/6-31G*)
CHARMM
interaction
Rmin
Emin
Rmin
Emin
MePP P1-OW
-20.8
-23.2
-23.2
-17.7
-12.8
-22.5
-16.7
-12.8
-23.7
3.64
1.73
1.73
4.13
2.43
1.76
4.03
1.82
1.75
-19.9
-18.5
-18.5
-16.8
-10.6
-22.7
-17.9
-9.3
3.51
1.64
1.64
3.38
2.14
1.61
3.43
1.68
1.61
MePP O13-HW
MePP O14-HW
MeSPP P1-OW
MeSPP SA-HW
MeSPP O14-HW
MeBPP P1-OW
MeBPP BA-HW
MeBPP O14-HW
-22.4
a
From ref 35.
isomer 9 is the most powerful analogue of ATP com-
pared with the other three indicated substances; isomer
5 is second in potency between the studied phospho-
rothioate analogues of ATP with increased metabolic
stability, indicating the role of the C2 methylthio ether
moiety in enhancing affinity toward the P2Y1-R. Isomers
10 and 6 are also good agonists of P2Y1-R, but with
lower potency in comparison with isomers 9 and 5. Thus,
the finding that Sp-ATP-R-S is more potent than its Rp
counterpart supports our spectral assignment of the
absolute configuration of 2-MeS-ATP-R-S isomers.
Molecu la r Mod elin g of th e h -P 2Y1-R Agon ists
1-12 a n d t h eir Com p lexes w it h t h e R ecep t or .
Over view . For the computational analysis of the chiral
discrimination by the h-P2Y1-R, ATP analogues 1-12
were employed as probe ligands.
Initially, the CHARMM 27 force field was augmented
with new parameters for these novel ATP analogues,
to enable reliable docking experiments. These param-
eters account for changes to the adenine ring due to C2-
substituents4 and changes to the phosphate chain due
to PR phosphorothioate and boranophosphate modifica-
tions.
F igu r e 7. ATP docked into the binding pocket of the h-P2Y1-
R. A putative Mg2+ ion is coordinated to the phosphate moiety
of ATP and to Asp204. Extracellular loops are deleted for
clarity.
energies of the parent compounds. Sulfur forms rela-
tively weak H-bonds,20 with a longer interaction dis-
-
tance than that of oxygen, and BH3 may participate
in weak H-bonds as H-bond donors via the hydrogen
atoms.18 These results emphasize the lower potential
-
of sulfur and BH3 compared to that of oxygen to
participate in electrostatic interactions.
The CHARMM interaction distances and energies
agree well with the ab initio results. The correlation
coefficients, R2, from a least-squares fit between ab initio
QM and empirical data for MeSPP yield a perfect fit of
1 for both interaction distances and energies. For
MeBPP, the interaction energy correlation coefficient
drops to 0.89, while R2 for the interaction distances is
1.
Dock in g Stu d ies. The stereoelectronic recognition
by the h-P2Y1-R was investigated by docking ATP
analogues 1-12. These docking studies used the
h-P2Y1-R model described in the companion paper.4 In
this model, ATP is bound approximately 5 Å below the
membrane plane and is involved in a multitude of
interactions with the receptor. The phosphate chain is
tightly bound by several positively charged amino acid
residues, including Arg128, Lys280, and Arg310. The
adenine moiety is more loosely bound, interacting with
Arg310, Ser314, and possibly Tyr58. In this model, a
Mg2+ ion was introduced into the binding site, to account
for the diastereoselectivity observed for this receptor
(see Discussion). A typical view of ATP docked into the
receptor binding pocket with the Mg2+ coordinated by
the phosphate moiety and Asp204 is presented in Figure
7. The ATP possibly coordinates with the Mg2+ ion via
the R, â, and γ phosphates, supported by the above NMR
analysis of Mg2+-nucleotide complexes, with the free
octahedral position of the Mg2+ ion probably occupied
by a water molecule. Metal ion coordination with the
ATP phosphates induces new chiral centers at the P
atoms. The conformation around the R-phosphate of
bound ATP may be defined as either pro-Rp or con-
versely as the Λ configuration (Scheme 6). Thus, ac-
cording to the current h-P2Y1-R model, the phosphate-
Mg2+ configuration required for recognition by the
receptor is Λ.
Subsequently, the ATP analogues were docked into
the receptor model to obtain information regarding the
molecular recognition determinants at the h-P2Y1-R
binding site. Thereafter, we performed a quantitative
estimation of the importance of specific H-bonding
interactions employing quantum mechanical calcula-
tions on model compounds.
P h osp h a te Mod ifica tion s. To generate parameters
for the chiral PR-ATP derivatives used in this study,
methyl diphosphate (MePP) was used as a model
compound.35 Thus, to represent the PR-phosphorothioate
and PR-boranophosphate functions, Me-PR-thiodiphos-
phate (MeSPP) and Me-PR-boranodiphosphate (MeBPP)
were employed (Figure 5, see Methods). The interaction
energies between MeSPP and MeBPP and individual
water molecules were computed at the ab initio QM
level and the CHARMM partial atomic charges were
fitted to reproduce the interaction distances and ener-
gies (Figure 6). The interaction energies obtained using
HF/6-31G* and the new CHARMM parameters are
presented in Table 6. The values for MePP35 are
presented for comparison. The main difference is the
considerably lower interaction energies in the diphos-
phate derivatives, where sulfur or BH3- participates in
the hydrogen bonding. The interaction energies of these
diphosphate derivatives are approximately half the

Molecular Recognition in Purinergic Receptors. 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4411
phate oxygen may participate in Mg2+ coordination and
-
not the BH3 group or the sulfur atom, respectively.
Thus, the docking experiments clearly demonstrate the
chiral discrimination of the h-P2Y1-R for one diastere-
omer. For instance, in the case of the Rp-isomer of
2-MeS-R-B-ATP (7) the coordination with Mg2+ is pre-
dicted to be with R, â, γ-phosphates (Figure 8). Thus,
for the Rp diastereomer, interaction with the Mg2+ ion
is maximized. However, in the case of the Sp-isomer (8),
the ligand loses the coordination with the R-phosphate
-
due to the lack of coordination of BH3 with Mg2+. In
this case, the coordination of the phosphate moiety with
the Mg2+ ion is via â,γ-phosphates only. This loss of
R-coordination with the Mg2+ ion considerably lowers
the interaction energy of the Sp ligand with the receptor-
Mg2+, relative to the Rp-isomer. The same argument
holds for the diastereomeric pairs of phosphorothioate
analogues 5/6 and 9/10. Here, the clear preference of
Mg2+ for the “hard”, namely, small and not easily
polarizable, phosphate oxygen as opposed to the “soft”
sulfur atom, large and fairly polarizable, accounts for
the receptor’s selectivity for the Rp-isomer.
F igu r e 8. 2-MeS-R-B-ATP docked into the h-P2Y1-R model:
gold, Rp-isomer; silver, Sp-isomer. pink, Mg2+ ion.
Sch em e 6. Screw Sense of Nucleotide-Mg2+ Complexes
Used as Chiral Probes of P2Y1-R
Discu ssion
Con for m a tion a l Ch a n ges of th e ATP An a logu e
Ar e In d u ced u p on Diffu sion fr om th e Solu tion to
th e P 2Y1-R Bin d in g P ock et. Upon diffusion of 2-MeS-
ATP-R-B (7, Rp-isomer) from the aqueous solution to its
binding site within the h-P2Y1-R, several conformational
changes occur. Thus, this isomer, which in solution has
a slight preference to the Southern ribose pucker (57%),
transforms exclusively into the Northern conformation
with a pseudorotation angle of 43.5° and ring puckering
of 29.3°.4 The predominant gg conformation around
C4′-C5′ for 7 in solution (88%) is exclusively occupied
in the P2Y1-R with a value of -64.4° ( 9°.4 Likewise,
the ø angle around the glycosidic bond of 7 in solution
remains practically the same as that of 7 in the receptor,
228° vs 239° ( 11°.
Ta ble 7. Interaction Energiesa between the h-P2Y1-R and
Various ATP Derivatives
analogue
ATP and derivatives
ATP
no.
∆E (kcal/mol)
1
2
3
4
5
6
7
8
-1214.5 ( 12.5
-1218.7 ( 12.7
-1220.0 ( 10.6
-1180.3 ( 9.4
-1237.1 ( 11.8
-1188.7 ( 9.3
-1221.2 ( 9.3
-1182.6 ( 8.2
-1236.4 ( 10.5
-1193.2 ( 8.8
-1222.3 ( 11.4
-1177.0 ( 8.6
2-MeS-ATP
ATP-R-B (Rp-isomer)
ATP-R-B (Sp-isomer)
ATP-R-S (Sp-isomer)
In this way, the P2Y1-R ligand 7 acquires a more
extended, elongated conformation upon binding to the
receptor.
ATP-R-S (Rp-isomer)
2-MeS-ATP-R-B (Rp-isomer)
2-MeS-ATP-R-B (Sp-isomer)
2-MeS-ATP-R-S (Sp-isomer)
2-MeS-ATP-R-S (Rp-isomer)
2-Cl-ATP-R-B (Rp-isomer)
2-Cl-ATP-R-B (Sp-isomer)
9
A Meta l Ion P ossibly P la ys a P ivota l Role in th e
Ch ir a l Discr im in a tion of th e h -P 2Y1-R. In this study
we used chiral probes 3-12 to explore the diastereose-
lectivity of the h-P2Y1-R. Indeed, these probes indicated
a chiral discrimination by the receptor (Table 5). EC50
values for these analogues presented a 8-20-fold po-
tency enhancement for one of the diastereoisomers as
compared to their diastereoisomeric counterparts.
The absolute configuration assignment of our chiral
probes necessitated X-ray crystal structure data for
these analogues. Yet, due to the inherent difficulties in
the crystallization of nucleotides, all our attempts to
crystallize R-boranophosphate analogues 7 and 8 proved
unsuccessful.
Alternatively, we applied an integrated approach
combining 1H/13C NMR spectroscopy, biochemical as-
says, and molecular modeling for assigning the absolute
configuration of analogues 3-4 and 7-12.
The conformational analysis of 2-MeS-ATP-R-B iso-
mers (7, 8) in solution by NMR enabled the assignment
of the Rp and Sp absolute configuration to isomer A and
B (7 and 8), respectively (Scheme 5). Subsequently, the
10
11
12
a
The interaction energy was defined as ∆Einter ) ∆ER‚L - (ER
+ EL).
ATP derivatives 1-12 were docked into the h-P2Y1-R
model, using MC simulations. According to this scheme,
some flexibility is added to binding-site side chains in
addition to ligand flexibility. The interaction energy,
which accounts for the nonbonded interactions between
the receptor and the ligand, was employed as a crude
estimate of the binding free energy. The average inter-
action energies obtained for ATP derivatives 1-12 over
100 000 steps of MC docking are presented in Table 7.
For the R-boranophosphate derivatives (3/4, 7/8, 11/
12), the Rp-isomer displays the greatest interaction
energy. On the other hand, for the R-phosphorothioate
derivatives (5/6, 9/10), the greatest interaction energies
were obtained for the Sp-isomer. The common denomi-
nator for the R-boranophosphate Rp-isomers and the
R-phosphorothioate Sp-isomers is that only the R-phos-

4412 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
absolute configuration of 2-MeS-ATP-R-S was obtained
with A- and B-isomers (9, 10) possessing the Sp and Rp
configuration.
sphere coordinationsthe distance of metal-phosphorus
is 3 Å) or via bridging water molecules (outer sphere
coordinationsthe distance of metal-phosphorus is 6 Å).
In the next phase, 2-MeS-ATP-R-S isomers with the
above putative absolute configuration were evaluated
as P2Y1-R ligands in a functional assay. In addition,
their EC50 values were compared to those of commercial
ATP-R-S isomers, whose absolute configuration is known.
The significantly higher potency of the Sp-isomer 9 as
compared to Rp-isomer 10 (20-fold difference) was in
accordance with that obtained for ATP-R-S Sp-isomer 5
vs Rp-isomer 6 (8-fold difference) (Table 5). This result
supports our NMR analysis of the absolute configuration
of ATP-R-B and ATP-R-S analogues.
Furthermore, computational docking studies indi-
cated clearly for chiral probes 7,8 and 9,10 that only
the Rp- and Sp-isomers of each, respectively, will form
strong binding interactions, thus supporting the abso-
lute configuration assignment mentioned above.
Finally, recent X-ray crystallography data for the
NDPK-thymidine-R-borano-diphosphate complex21 fully
support our absolute configuration assignment.
For the elucidation of the origin of diastereoselectivity
in the h-P2Y1-R, we compared our biochemical and
computational results with reports on the diastereose-
lectivity in nucleotide-binding enzymes.
The pivotal role of metal ions in determining the
stereospecificity of enzymes was demonstrated in the
classic report of J affe and Cohn.22 In this work, dias-
tereoisomers of ATP-R-S and ATP-â-S were used in
combination with hard and soft metal ions. The pref-
erential coordination of Mg2+ to oxygen and of Cd2+ to
sulfur in the phosphorothioate analogues23 resulted in
the inversion of yeast hexokinase and rabbit muscle
pyruvate kinase diastereomeric selectivity upon substi-
tuting Cd2+ for Mg2+ in the phosphoryl transfer reac-
tions using a chiral ATP substrate (ATP-â-S).
The most common pattern, especially for kinases,
seems to be a reversal of stereoselectivity with the ATP-
â-S diastereoisomers coupled, with no reversal at the
PR. The results are usually interpreted as meaning that
the â,γ-bidentate metal-nucleotide chelate is the active
one in these enzyme-catalyzed reactions. Indeed, ATP-
Mg2+ in kinases forms the â,γ bidentate chelate in the
Λ screw sense.24
Therefore, the ATP analogue bound to the P2Y1-R is
most likely metal ion bound, and the metal ion is most
likely to be Mg2+, although, to the best of our knowledge,
no experimental evidence has been provided so far for
the presence of a metal ion in the P2Y1-R. Furthermore,
in enzymes, metal ions, usually Mg2+, play an important
role in partially shielding the phosphate negative
charges and aligning the substrate in proper orientation
to produce the catalytic reaction.26 In the P2Y1-R, the
putative Mg2+ ion can stabilize the ligand in a position
so as to induce conformational changes of the receptor,
in addition to neutralization of the ligand negative
charges.
In ATP, various metal chelates of the R, â, and γ
phosphate groups have been identified by X-ray crystal-
lography and by 31P NMR spectroscopy. Seven mono-
dentate, bidentate, and tridentate metal-ATP isomers
may form, including all of the permutations of R; â; γ;
R,â; γ,â; R,γ; and R,â,γ coordination. Our 31P NMR
results for the Mg2+ complexes of chiral probes 7/8 and
9/10 indicate that in solution coordination involves all
phosphates (Table 4). This could be either a simulta-
neous R,â,γ metal coordination or R,â and â,γ coordi-
nated species existing in equilibrium. This finding is in
agreement with earlier reports.28
The mode of ATP coordination with Mg2+ in the
protein may be different than the mode in solution. For
some proteins, the tridentate R,â,γ metal coordination
complex is the active species, and for others, it is the
bidentate â,γ complex.
Metal binding to the R- or â-phosphate groups of
nucleotides imposes chirality on those phosphorus at-
oms. The bidentate â,γ Mg2+ complex has a chiral center
on the Pâ and the tridentate R,â,γ complex has chiral
centers at both the PR and the Pâ. For the â,γ-bidentate
chelate, the resulting two isomers have been designated
Λ and ∆.
In our docking experiments, ATP putatively coordi-
nates the Mg2+ ion via the R, â, and γ phosphates.
Moreover, the computational results emphasize the
receptor’s preference for the Λ configuration. Therefore,
the h-P2Y1-R preferentially binds one diastereomer of
ATP-R-S and ATP-R-B derivatives. In the case of the
PR-thiophosphate analogues, the Sp-isomer is predicted
to be the more potent ligand, while for the PR-borano-
phosphate derivatives, the Rp-isomer should be more
potent, as was experimentally verified.
Therefore, the chiral discrimination we have observed
by the P2Y1-R for the diastereoisomers of ATP-R-B
analogues3 and for ATP-R-S analogues (Table 5) strongly
supports the involvement of a metal ion as the origin of
the receptor diastereoselectivity.
It is estimated that more than 90% of the ATP in cells
is bound to Mg2+ ion.25 In fact, because the intracellular
Mg2+ concentration is generally an order of magnitude
higher than the dissociation constant for MgATP, almost
all intracellular ATP is complexed with Mg2+ (log
stability constant for ATP4- complex with Mg2+ is
Here we found a difference of ca. 20-fold between the
EC50 values of the diastereoisomer pairs of 7,8 and 9,10
(Table 5). This difference is apparently small as com-
pared to up to several orders of magnitude in catalytic
efficiency of various enzymes with pairs of substrate
diastereoisomers.22
4.29).26 Since the concentration of extracellular Mg2+
,
Indeed, small discriminatory preferences between di-
astereoisomers of ATP-R-S in enzymes are usually in-
dicative of the involvement of PR only in binding and
not in catalysis. The changes in catalysis may be up to
several orders of magnitude, whereas changes in kinetic
parameters that are due to binding are around 1 order
of magnitude.5b In our case, only binding plays a role
ca. 0.5-1 mM, is close to the intracellular concentration,
ca. 0.25-1 mM,27 we assume that extracellular ATP
exists also mostly as the Mg2+ complex. The Mg2+ ion
coordination occurs exclusively at the ATP’s oxygen
atoms, and not with N7, with water occupying the
remaining octahedral positions about the metal.25 Mg2+
can bind directly to the phosphate oxygen atoms (inner

Molecular Recognition in Purinergic Receptors. 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4413
in the chiral discrimination, and this may explain a dis-
crimination of ca. 20-fold between the diastereoisomers.
Therefore, we hypothesize that ATP-R-B and ATP-
R-S analogues are recognized by the P2Y1-R as follows:
The Mg2+ ion that is held in the vicinity of the
phosphate binding pocket by mono- or bidentate coor-
dination with Asp204 coordinates also with the phos-
phate chain via PR, Pâ, and Pγ, with the remaining
octahedral positions possibly occupied by water mol-
ecules (Figure 7). As the docking studies revealed, the
required Mg2+-phosphate configuration at the h-P2Y1-R
is the Λ configuration. Namely, for ATP, the pro-R
oxygen atom will be coordinated preferentially to the
pro-S oxygen atom.
The hard Mg2+ ion binds preferentially the hard
oxygen atom rather than the soft sulfur atom in ATP-
R-S analogues. Likewise, only the PR oxygen atom in
ATP-R-B analogues can coordinate with the metal ion.
Thus, in the ATP-R-S (Rp) and ATP-R-B (Sp) isomers,
the PR oxygen is not in a position to coordinate the Mg2+
ion, and in this case the coordination is probably â,γ
(Figure 8). Hence, these isomers lose a tight interaction
with Mg2+ ion that has energetic implications and
possibly also consequences for the orientation of the
ligand, resulting eventually in their different EC50
values.
Our hypothesis regarding the P2Y1-R origin of dias-
tereoselectivity is supported by a recent related study
on a complex of R-boranothymidine diphosphate with
nucleoside diphosphate kinase (NDPK).21 The 1.92 Å
resolution crystal structure of the NDPK complex
demonstrates that a Mg2+ ion ligates the R- and
â-phosphates of the R-boranophosphate analogue, as
observed for natural nucleotides, in addition to four
water molecules. For the active R-borano thymidine
diphosphate Rp-isomer, it was found that the R-borano
group does not interact with the protein, and the metal
ion coordination with the oxygen atoms of the phos-
phates is likely to determine the stereochemistry of
recognition. Furthermore, the structures of our R,â,γ-
Mg2+-coordinated ligand-receptor complexes are sup-
ported by the crystal structure of 2′,3′-didehydro 2′,3′-
dideoxythymidine triphosphate in NDPK. Here it was
found that the Mg2+ ion coordinates with all three
phosphates.
Meth od s
Syn th esis. Gen er a l. All air- and moisture-sensitive reac-
tions were conducted in flame-dried, nitrogen-flushed, two-
neck flasks sealed with rubber septa, and the reagents were
introduced with a syringe. The progress of the reactions was
monitored by TLC on precoated Merck silica gel plates (60F-
254). Compounds were characterized by nuclear magnetic
resonance using Bruker DMX-600, DPX-300, or AC-200 spec-
trometers. 1H NMR spectra were measured in D2O, and the
chemical shifts are reported in ppm relative to HOD (4.78 ppm)
as an internal standard. Nucleotides were characterized also
by 31P NMR in D2O, using 85% H3PO4 as an external reference.
Final products were characterized on an AutoSpec-E FISION
VG high-resolution mass spectrometer desorbed from a glyc-
erol matrix under FAB (fast atom bombardment) conditions.
Primary purification of the nucleotides was achieved on an
LC (Isco UA-6) system using a Sephadex DEAE-A25 column,
which was swelled in 1 M NaHCO3 in the cold for 1 day. Final
purification of the nucleotides and separation of the diastere-
oisomer pair was achieved on a HPLC (Merck-Hitachi) system
using a semipreparative reverse-phase column. Conditions for
LC and HPLC separation are described below. The purity of
the new nucleotides was determined on a Lichrospher 60 RP-
select B column (250 × 4 mm) in the following solvent
systems: system I, MeOH:0.1 M TEAA (triethylammonium
acetate), 10:90 to 30:70 linear gradient in 25 min; system II,
5 mM TBAP (tetrabutylammonium dihydrogen phosphate) in
MeOH:60 mM ammonium phosphate and 5 mM TBAP in 90%
H2O/10% MeOH, 10:90 to 40:60, linear gradient in 25 min.
2-Methylthioadenosine was synthesized from 2-SH-adenosine
as described before.29 2-SH-adenosine was obtained from
adenosine in three steps according to the procedure previously
reported.30
P r ep a r a tion of 2-Meth ylth ioa d en osin e-5′-O-(r-P -th io)-
tr ip h osp h a te 9/10. 2-Methylthioadenosine-5′-O-(R-P-thio)-
triphosphate was prepared according to our previously re-
ported method.6 The resulting residue was applied on an
activated Sephadex DEAE-A25 column using a 0-0.6 M
NH4HCO3 linear gradient (2000 mL). The relevant fractions
were collected and freeze-dried, and excess NH4HCO3 was
removed by repeated freeze-drying with deionized water to
yield a mixture of 9 and 10 as a triammonium salt. Final
purification and separation of diastereoisomers of 2-MeS-ATP-
R-S was achieved on a semipreparative HPLC column. Sub-
sequently, the triethylammonium counterions in each of the
pure diastereoisomers were exchanged for Na+ by passing the
nucleotide through a Sephadex-CM C-25 column. Both dias-
tereoisomers were obtained from 2-methylthioadenosine in
34% yield.
Rever se-P h a se HP LC Sep a r a tion of Dia ster eoisom er s
9/10. The separation of diastereoisomers 9/10 was accom-
plished using a semipreparative reverse-phase Lichro CART
250-10 column and linear gradient [100 mM triethylammo-
nium acetate (TEAA), pH 7 (A):MeOH (B), 90:10 to 70:30] for
25 min with flow rate of 5 mL/min. Fractions containing the
same isomer (similar retention time) were freeze-dried. The
excess buffer was removed by repeated freeze-drying with
deionized water. The isomer with the shorter retention time
(tR) is designated isomer A and the other, isomer B.
Con clu sion s
In this study, we observed a significant 20-fold
difference in the potency of the diastereoisomers of both
ATP-R-S and ATP-R-B analogues regarding the h-P2Y1-
R. We identified the absolute configuration of the potent
h-P2Y1-R ligands. Thus, it is the Rp ATP-R-B analogues
3, 7, and 11 and the Sp ATP-R-S analogues 5 and 9, that
induce higher affinity to the h-P2Y1-R. Furthermore, we
hypothesize that the P2Y1-R’s chiral discrimination
originates from the requirement that the nucleotide
analogue interacts with a Mg2+ ion within the receptor
binding site. We identified the mode of coordination of
the ATP analogues’ triphosphate chain with a Mg2+ ion
both in solution (experimentally) and in the receptor
(computationally) and determined the active conforma-
tion as Λ. Furthermore, the conformational changes of
the ligand occurring upon diffusion from the solution
to the receptor’s binding site were explained.
2-SMe-ATP -r-S, 9 (A-Isom er ). tR: 15.26 min. 1H NMR
(D2O, 300 MHz, Na+ form): δ 8.50 (s, H-8, 1H), 6.12 (d, J )
5.8 Hz, H-1′, 1H), 4.88 (dd, J ) 5.8, 5 Hz, H-2′, 1H), 4.66 (dd,
J ) 3.8, 5 Hz, H-3′, 1H), 4.40 (ddd, J ) 3.8, 3.0, 3.1 Hz, H-4′,
1H), 4.34 (ddd, J ) 3.1, 7.6, 11.7 Hz, H-5′, 1H), 4.26 (ddd, J )
3.0, 5.8, 11.7 Hz, H-5′, 1H), 2.58 (s, S-CH3, 3H) ppm. 31P NMR
(D2O, 81 MHz, Et3N+H form): δ 44.12 (d, J ) 30.7 Hz, PR),
-10.26 (d, J ) 21.2 Hz, Pγ), -23.64 (t, J ) 21.4 Hz, Pâ) ppm.
UV: λmax 275.7 nm. MS FAB (negative) m/z: 611.959 (M -
3H + 2Na)-, 590.012 (M - 2H+ + Na+)-, 568.023 (M - H)-.
tR: 17.77 min (97.4% purity) using solvent system I. tR: 17.75
min (95.9% purity) using solvent system II.
2-SMe-ATP -r-S, 10 (B-Isom er ). tR: 16.83 min. 1H NMR
(D2O, 300 MHz, Na+ form): δ 8.42 (s, H-8, 1H), 6.12 (d, J )
5.8 Hz, H-1′, 1H), 4.86 (dd, J ) 5.8, 5.1 Hz, H-2′, 1H), 4.65

4414 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
(dd, J ) 3.6, 5.1 Hz, H-3′, 1H), 4.40 (ddd, J ) 2.9, 3.1, 3.6 Hz,
H-4′, 1H), 4.33 (ddd, J ) 2.9, 7.6, 11.7 Hz, H-5′, 1H), 4.26 (ddd,
J ) 3.1, 6.3, 11.7 Hz, H-5′, 1H), 2.56 (s, S-CH3, 3H) ppm. 31P
NMR (D2O, 81 MHz, Et3N+H form): δ 43.87 (d, J ) 25.3 Hz,
PR), -9.63 (d, J ) 16.3 Hz, Pγ), -23.43 (t, J ) 23.2 Hz, Pâ)
ppm. UV: λmax 275.7 nm. MS FAB (negative) m/z: 611.978
(M -3H+ + 2Na+)-, 590.028 (M - 2H+ + Na+)-, 568.034 (M
- H)-. tR: 19.76 min (95.8% purity) using solvent system I.
tR: 17.68 min (91.2% purity) using solvent system II.
Bioch em istr y. Ma ter ia ls. Adenosine-5′-O-(1-thiotriphos-
phate), Rp-isomer (Rp-ATP-R-S), and adenosine-5′-O-(1-thiot-
riphosphate), Sp-isomer (Sp-ATP-R-S), were purchased from
BIOLOG Life Science Institute (Bremen, Germany).
equilibrium values. The torsional term is based on a cosine
function, where Kφ is the force constant, n is the periodicity,
and δ is the phase. The nonbonded terms are described by a
Lennard-J ones term plus the Coulombic term. ꢀ is the Len-
nard-J ones well depth, Rmin is the distance at the Lennard-
J ones minimum, and qi is the partial atomic charge. A
dielectric constant of unity was employed throughout.
The vdW parameters of sulfur were taken directly from the
existing CHARMM parameters for sulfur, while for borane the
recently developed parameters of Otkidach and Pletnev were
used.36 However, the vdW parameters for the borane hydro-
gens were optimized to fit ab initio interaction geometries and
energies, as described below.
[Ca 2+]i Mea su r em en ts. Transfection of HEK 293 cells with
the rP2Y1 receptor was carried out as described.31 These HEK
293 cells were cultured on coverslips and used for pharmaco-
logical analysis.32 For Ca2+ measurements, these HEK cells
were loaded for 30 min with 2 µM Fura 2/AM in HEPES-
buffered saline (HBS) containing 145 mM NaCl, 5.4 mM KCl,
1.8 mM MgCl2, 25 mM glucose, and 20 mM HEPES, pH 7.4.
Cells were assayed under continuous superfusion of 37 °C
prewarmed HBS (2 mL/min) in the presence of varying
concentrations of different nucleotides, as indicated.
The relative enzymatic stability of the analogues tested, in
addition to the fast superfusion system used to apply the
different ATP analogues, precludes any appreciable enzymatic
conversion. Thus, problems related to responses that may
result from degraded or released nucleotides are circumvented.
Constant superfusion of the prewarmed buffer excluded un-
specific Ca2+ responses caused by mechanical stress, temper-
ature variation, or different components of the buffer.
Fluorescence changes of single cells were detected with an
imaging system (TILL Photonics GmbH) attached to a Zeiss
axioscope, using alternative excitation at 340/380 nm and
emission at 510 nm. EC50 values were calculated from con-
centration-effect curves, as represented in Figures 3 and 4
and fitted with the Sigmaplot program. The data points in
Figures 5 and 6 represent the mean ( SEM of 60-200 cells
from at least three different experiments and two different
preparations. Concentration-response data were analyzed
with the Excel program applying ∆(F340nm/F380nm) before and
after the addition of the agonist. EC50 values represent the
agonist concentration at which 50% of the maximal effect is
achieved.
Initially, the MeSPP and MeBPP geometries were optimized
at the quantum mechanical (QM) ab initio MP2/6-31+G*
level.37 For water, the TIP3 geometry was used.38 Thereafter,
the interaction energies between MeSPP or MeBPP and the
water molecules were calculated at the HF/6-31G* level39 and
the CHARMM partial atomic charges were varied to obtain
an optimal fit between the ab initio QM and empirical energies
and interaction distances. In this case, no scaling of the ab
initio energies was used.35 Moreover, no diffuse functions were
added to the 6-31G* basis set, to be consistent with previous
work on MePP.35
Subsequently, we investigated the influence of substitution
-
of the R-phosphate oxygen with either sulfur or BH3 on the
rotation around the P-O bonds of the phosphate backbone (see
Supporting Information). The PES map at the HF/6-31+G*
level was compared with the one obtained using the existing
dihedral parameters for MePP and the newly developed partial
atomic charges for MeSPP and MeBPP. The force field PES
map reproduced the main features of the ab initio surface and
therefore the phosphate backbone dihedral angle parameters
of MeSPP and MeBPP were set to those of MePP.
Dock in g Stu d ies. The ATP derivatives addressed here
were docked into the h-P2Y1-R model developed in the
companion paper.4 In this model, the receptor is embedded in
a fully hydrated lipid bilayer. However, for the current docking
studies, the lipid bilayer and bulk water were not accounted
for, since no changes to the receptor outside the binding site
are performed.
Initially, the ligands were rigidly docked into the binding
site using the location of ATP in our previous studies as a
template.4 Specifically, modifications to the ATP scaffold in
its binding configuration were performed to obtain each of the
derivatives 2-12. Subsequently, the ligands were subjected
to 100 000 steps of Monte Carlo (MC) simulations at 310 K.
The docking studies employed MC simulations, where flex-
ibility is added to the side chains that are in close proximity
to the ligand.4 The MC moves defined for the receptor were
dihedral rotations around side chain bonds. Rigid rotations of
the entire ligand of up to 30° and rigid translations of up to
0.5 Å were allowed per MC step, respectively. In addition,
flexibility was added to all rotatable bonds within the ligand
in addition to the rigid ligand rotations and translations. The
complexation free energy was approximated crudely as the
nonbonded interaction energy between ligand and receptor.
Thus ligand desolvation, changes in internal energy, and
entropy were ignored. However, here the main purpose of the
interaction energy is to differentiate between different dia-
stereomers, and a simple nonbonded interaction expression
was deemed sufficient for this purpose.
Com p u t a t ion a l Met h od s. F or ce F ield Develop m en t .
The CHARMM 27 force field33-35 includes parameters for ATP.
However, to describe the various ATP derivatives introduced
in the current study, new parameters must be developed.
Therefore, the CHARMM 27 force field was extended to include
our ATP derivatives 2-12 that were investigated in the
current docking studies. The CHARMM energy function is
described by the following expression of internal and non-
bonded terms:
VT )
Kb(b - b0)2 +
Kθ(θ - θ0)2 +
Kφ[1 +
∑
∑
∑
bonds
angles
dihedrals
cos(nφ - δ)]
+
KUB(S - S0)2 +
Kimp(æ - æ0)2
∑
∑
1,3pairs
improper
12
6
Rmin,ij
Rmin,ij
qiqj
A cutoff scheme of 8-12-13 Å was used for both electro-
statics and vdW interactions in the simulations. Force and
energy switching were used for electrostatic and vdW interac-
tions, respectively.40 This cutoff scheme entails turning on the
switching functions at 8 Å, ignoring interactions beyond 12
Å, and keeping a list of all pair interactions within 13 Å.
All molecular mechanics calculations employed the CHARMM
software package,33 while all quantum mechanics computa-
tions used the Gaussian 98 program.41
+
ꢀij
- 2
+
∑
( (
[
)
(
)
)
]
rij
rij
4πDrij
nonbonded
In the above equation the first five terms relate to the internal
energy, while the last two terms relate to the nonbonded
energy. In the current study, only parameters for the dihedral
and nonbonded terms were developed, since these are the only
terms that are variable in the docking procedure. Kb, Kθ, KUB
,
and Kimp are the bond, angle, Urey-Bradley, and improper
dihedral force constants, respectively. b, θ, S, and æ are the
bond length, bond angle, Urey-Bradley 1,3 distance, and
improper dihedral angle, respectively. The subscript denotes
Ack n ow led gm en t. This work was supported in part
by BMBF-MOS, Grant No. 1812, and the Marcus Center

Molecular Recognition in Purinergic Receptors. 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4415
(13) (a) Altona, C.; and Sundaralingam, M. Conformational Analysis
of the Sugar Ring in Nucleosides and Nucleotides. New Descrip-
tion Using the Concept of Pseudorotation. J . Am. Chem. Soc.
1972, 94, 8205-8212. (b) Altona, C.; Sundaralingam, M. Con-
formational Analysis of the Sugar Ring in Nucleosides and
Nucleotides. Improved Method for the Interpretation of Proton
Magnetic Resonance Coupling Constants. J . Am. Chem. Soc.
1973, 95, 2333-2344.
for Medicinal Chemistry. D.T.M. acknowledges the
financial support provided by the Israeli Ministry of
Science.
Su p p or tin g In for m a tion Ava ila ble: Figures related to
the CHARMM force field development and the partial atomic
charges developed for derivatives 2-12 are provided. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Davies, D. B.; Danyluk, S. S. International Symposiuym on NMR
Spectroscopy, University of Surrey, 1972.
(15) Blackburn, B. J .; Grey, A. A.; Smith, I. C. P.; Hruska, F. E.
Determination of the Molecular Conformation of Uridine in
Aqueous Solution by Proton Magnetic Resonance Spectroscopy.
Comparison with â-Pseudouridine. Can. J . Chem. 1970, 48,
2866-2870.
Refer en ces
(1) (a) Fischer, B. Therapeutic Applications of ATP-(P2)-Receptors
Agonists and Antagonists. Exp. Opin. Therap. Pat. 1999, 9, 385-
399. (b) Guile, S. D.; Ince, F.; Ingall, A. H.; Kindon, N. D.;
Meghani, P.; Mortimore, M. P. The Medicinal Chemistry of the
P2 Receptor Family. Prog. Med. Chem. 2001, 38, 115-187. (c)
Halbfinger, E.; Major, D. T.; Ritzmann, M.; Ubl, J .; Reiser, G.;
Boyer, J . L.; Harden, K. T.; Fischer, B. Molecular Recognition
of Modified Adenine Nucleotides by the P2Y1-Receptor. 1. A
Synthetic, Biochemical, and NMR Approach. J . Med. Chem.
1999, 42, 5325-5337. (d) Major, D. T.; Halbfinger, E.; Fischer,
B. Molecular Recognition of Modified Adenine Nucleotides by
the P2Y1-Receptor. 2. A Computational Approach. J . Med. Chem.
1999, 42, 5338-5347. (e) Major, D. T.; Laxer, A.; Fischer, B.
Protonation Studies of Modified Adenine and Adenine Nucle-
otides by Theoretical Calculations and 15N NMR. J . Org. Chem.
2002, 67, 790-802.
(2) (a) J acobson, K. A.; J arvis, M. F.; Williams, M.Purine and
Pyrimidine (P2) Receptors as Drug Targets J . Med. Chem. 2002,
45, 4057-4093. (b) van Rhee, A. M.; Fischer, B.; van Galen, P.
J . M.; J acobson, K. A. Modelling the P2Y Purinoceptor Using
Rhodopsin As Template. Drug Des. Discovery 1995, 13, 133-
154. (c) Moro, S.; Guo, D.; Camaioni, E.; Boyer, J . L.; Harden,
T. K.; J acobson, K. A Human P2Y(1) Receptor: Molecular
Modeling and Site-Directed Mutagenesis as Tools to Identify
Agonist and Antagonist Rrecognition Sites. J . Med. Chem. 1998,
41, 1456-1466. (d) Moro, S.; Hoffmann, C.; J acobson, K. A. Role
of the Extracellular Loops of G Protein-Coupled Receptors in
Ligand Recognition: A Molecular Modeling Study of the Human
P2Y(1) Receptor. Biochemistry 1999, 38, 3498-3509. (e) Kim,
H. O.; Barak, D.; Harden, T. K.; Boyer, J . L.; J acobson, K. A.
Acyclic and Cyclopropyl Analogues of Adenosine Bisphosphate
Antagonists of the P2Y(1) Receptor: Structure-Activity Rela-
tionships and Receptor Docking. J . Med. Chem. 2001, 44, 3092-
3108.
(3) Nahum, V.; Zu¨ndorf, G.; Le´vesque, S. A.; Beaudoin, A. R.; Reiser,
G.; Fischer B. Adenosine 5′-O-(1-Boranotriphosphate) Deriva-
tives as Novel P2Y1 Receptor Agonists. J . Med. Chem. 2002, 45,
5384-5396.
(4) Major, D. T.; Fischer, B. Molecular Recognition in Purinergic
Receptors. 1. A Comprehensive Computational Study of the
h-P2Y1-Receptor. J . Med. Chem. 2004, 47, 4391-4404.
(5) (a) Eckstein, F. Nucleoside Phosphorothioates. Annu. Rev.
Biochem. 1985, 54, 367-402. (b) Garcia, G. A.; Leatherbarrow,
R. J .; Eckstein, F.; Fersht, A. R. Metal-Ion Dependence of
Phosphorothioate ATP Analogues in the Bacillus-Stearother-
mophilus Tyrosyl-Transfer RNA-Synthetase Reaction. Biochem-
istry 1990, 29, 1643-1648.
(6) Fischer, B.; Chulkin, A.; Boyer, J . L.; Harden, T. K.; Gendron,
F.-P.; Beaudoin, A. R.; Chapal, J .; Hillaire-Buys, D.; Petit, P.
2-Thioether-5′-O-(1-thiotriphosphate) Adenosine Derivatives as
New Insulin Secretagogues Acting Through P2Y-receptors. J .
Med. Chem. 1999, 42, 3636-3646.
(7) Tulapurkar, M. E.; Laubinger, W.; Nahum, V.; Fischer, B.;
Reiser, G. Subtype Specific Internalization of P2Y1 and P2Y2
Receptors Induced by Novel Adenosine 5′-O-(1-boranotriphos-
phate) Derivatives. Br. J . Pharmacol. 2004, 142, 869-878.
(8) Saenger W. In Principles of nucleic acid structure; Springer-
Verlag: New York, 1984; pp 81-82.
(16) Hruska, F. E. In Conformations of Biological Molecules and
Polymers (Proceedings of J erusalem Symposium); Bergmann, E.
D., Pullman, B., Eds.; Israel Academy of Science and Humani-
ties: J erusalem, 1973; p 345.
(17) Nahum, V. and Fischer, B. Eur J . Inorg. Chem. 2004, in press.
(18) Fujii, A.; Patwari, G. N.; Ebata, T.; Mikami, N. Vibrational
Spectroscopic Evidence of Unconventional Hydrogen Bonds. Int.
J . Mass Spect. 2002, 220, 289-312.
(19) Srivastava, P. C.; Robins, R. K.; Meyer, R. B., J r. In Chemistry
of nucleosides and nucleotides; Townsend, L. B., Ed.; Plenum
Press: New York, 1988; Vol. 1, pp 197-198.
(20) Platts, J . A.; Howard, S. T.; Bracke, B. R. F. Directionality of
Hydrogen Bonds to Sulfur and Oxygen. J . Am. Chem. Soc. 1996,
118, 2726-2733.
(21) Meyer, P.; Schneider, B.; Sarfati, S.; Deville-Bonne, D.; Guer-
reiro, C.; Boretto, J .; Canard, B. Structural Basis for Activation
of R-Boranophosphate Nucleotide Analogues Targeting Drug-
resistant Reverse Transcriptase. EMBO J . 2000, 19, 3520-3529.
(22) J affe, E. K.; Cohn M. Divalent Cation-Dependent Stereospecifity
of Adenosine 5′-O-(2-thiotriphosphate) in the Hexokinase and
Pyrivate Kinase Reactions. The Absolute Stereochemistry of the
Diastereoisomers of Adenosine 5′-O-(2-thiotriphosphate). J . Biol.
Chem. 1978, 253, 4823-4825.
(23) J affe, E. K.; Cohn, M. 31P Nuclear Magnetic Resonance Spectra
of the Thiophosphate Analogues of Adenine Nucleotides; Effects
of pH and Mg2+ Binding. J . Biol. Chem. 1978, 17, 652-657.
(24) Romaniuk, P. J .; Eckstein, F. Structure of the Metal-Nucleotide
Complex in the Acetate Kinase Reaction. A Study with gamma-
32P-labled Phosphorothioate analogues of ATP. J . Biol. Chem.
1981, 256, 7322-7328.
(25) Lippard, S. J .; Berg, J . M. In Principles of Bioinorganic Chem-
istry, University Science Books: Mill Valley, CA, 1994; pp 64-
66, 132-133, 276-277.
(26) Klevickis & Grisham In Metal ion in biological systems; Sigel,
A., Sigel, H., Eds.; Marcel Dekker: New York, 1996; Vol. 32.
(27) (a) Lane, M.; Boatman, D. E.; Albrecht, R. M.; Bavister, B. D.
Intracellular Divalent Cation Homeostasis and Developmental
Competence in the Hamster Preimplantation Embryo. Mol.
Reprod. Dev. 1998, 50, 443-50. (b) Touyz, R. M.; Yao, G.
Modulation of Vascular Smooth Muscle Cell Growth by Magne-
sium-role of Mitogen-Activated Protein Kinases. J . Cell Physiol.
2003, 197, 326-335. (c) Grubbs, R. D. Intracellular Magnesium
and Magnesium Buffering. BioMetals 2002, 15, 251-259.
(28) Cohn, M. In Biological actions of extracellular ATP; Dubyak, G.
R., Fedan, J . S., Eds.; Annals of the New York Academy of
Sciences, Vol. 603; The New York Academy of Sciences: New
York, 1990.
(29) Macfarlane, D. E. 2-Methylthioadenosine [â-32P]diphosphate:
Synthesis and Use as Probe of Platelet ADP Receptors. Methods
Enzymol. 1992, 215, 137-142.
(30) (a) Kikugawa, K.; Suehiro, H.; Inchino, M. Platelet Aggregation
Inhibitors. 6. 2-Thioadenosine Derivatives. J . Med. Chem. 1973,
16, 1381-1388. (b) Kikugawa, K.; Suehiro, H.; Yanase, R.; Aoki,
A. Platelet Aggregation Inhibitors. IX. Chemical Transformation
of Adenosine into 2-Thioadenosine Derivatives. Chem. Pharm.
Bull. 1977, 25, 1959-1969.
(31) Vo¨hringer C, Scha¨fer R, Reiser G. A Chimeric Rat brain P2Y1
Receptor Tagged with Green-Fluorescent Protein: High-Affinity
Ligand Recognition of Adenosine Diphosphates and Triphos-
phates and Selectivity Identical to that of the Wild-Type Recep-
tor. Biochem. Pharmacol. 2000, 59, 791-800.
(32) Zu¨ndorf, G.; Scha¨fer, R.; Vo¨hringer, C.; Halbfinger, E.; Fischer,
B.; Reiser, G. Novel Modified Adenosine 5′-Triphosphate Ana-
logues Pharmacologically Characterized in Human Embryonic
Kidney 293 Cells Highly Expressing Rat Brain P2Y1 Receptor:
Biotinylated Analogue Potentially Suitable for Specific P2Y1
Receptor Isolation. Biochem. Pharmacol. 2001, 61, 1259-1269.
(33) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J .;
Swaminathan, S.; Karplus, M. CHARMM. A program for mac-
romolecular energy, minimization, and dynamics calculations.
J . Comput. Chem. 1983, 4, 187-217.
(9) Donohue, J .; Trueblood, K. N. Base Pairing in DNA. J . Mol. Biol.
1960, 2, 363-371.
(10) Sundaralingam, M. Stereochemistry of Nucleic Acids and their
Constituents. IV. Allowed and Preferred Conformations of
Nucleosides, Nucleoside Mono-, Di-, Tri-, Tetraphosphates,
Nucleic Acids, and Polynucleotides. Biopolymers 1969, 7, 821-
860.
(11) Ippel, J . H.; Wijmenga, S. S.; de J ong, R.; Heus, H. A.; Hilbers,
C. W. Heteronuclear Scalar Coupling in the Bases and Sugar
Rings of Nucleic Acids: Their Determination and Application
in Assignment and Conformational Analysis. Magn. Reson.
Chem. 1996, 34, S156-S176.
(12) Davies, D. B.; Danyluk, S. S. Nuclear Magnetic Resonance
Studies of 5′-Ribo- and Deoxyribonucleotide Structures in Solu-
tion. Biochemistry 1974, 13, 4417-4434.

4416 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Major et al.
(34) Foloppe, N.; MacKerell, A. D., J r. All-atom empirical force field
for nucleic acids: I. Parameter optimization based on small
molecule and condensed phase macromolecular target data. J .
Comput. Chem. 2000, 21, 86-104.
(35) Pavelites, J . J .; Gao, J .; Bash, P. A.; Mackerell, A. D., J r. A
molecular mechanics force field for NAD(+), NADH, and the
pyrophosphate groups of nucleotides. J . Comput. Chem. 1997,
18, 221-239.
(36) Otkidach, D. S.; Pletnev, I. V. Conformational analysis of boron-
containing compounds using Gillespie-Kepert version of molec-
ular mechanics. J . Mol. Struct. (THEOCHEM) 2001, 536, 65-72.
(37) Møller, C.; Plesset, M. S. Note on an approximate treatment for
many-electron systems. Phys. Rev. 1934, 46, 618-622.
(38) J orgensen, W. L.; Chandrasekhar, J .; Madura, J . D.; Impey, R.
W.; Klein, M. L. Comparison of simple potential functions for
simulating liquid water. J . Chem. Phys. 1983, 79, 926-935.
(39) (a) Hall, G. G. The molecular orbital theory of chemical valency
VIII. A method for calculating ionisation potentials. Proc. R. Soc,
London Ser. A 1951, 205, 541-552. (b) Roothan C. C. J . New
developments in molecular orbital theory. Rev. Mod. Phys. 1951,
23, 69-89.
(40) Steinbach, P. J .; Brooks, B. R. New spherical-cutoff methods for
long-range forces in macromolecular simulation. J . Comput.
Chem. 1994, 15, 667-683.
(41) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J . R.; Zakrzewski, V. G.; Montgomery,
J . A.; Stratmann, R. E.; Burant, J . C.; Dapprich, S.; Millam, J .
M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.;
Tomasi, J .; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.;
Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J .; Petersson, G.
A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck,
A. D.; Raghavachari, K.; Foresman, J . B.; Cioslowski, J .; Ortiz,
J . V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J .; Keith, T.;
Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.;
Challacombe, M.; Gill, P. M. W.; J ohnson, B. G.; Chen, W.; Wong,
M. W.; Andres, J . L.; Head-Gordon, M.; Replogle, E. S.; Pople,
J . A. Gaussian 98 (Revision A.7); Gaussian, Inc., Pittsburgh, PA,
1998.
J M049771U