The change in the electronic character upon cisplatin binding to guanine nucleotide is transmitted to drive the conformation of the local sugar-phosphate backbone - A quantitative study

Article abstract of DOI:10.1039/a902330e

The microstructure alteration as a result of cisplatin binding to N7 of guanines in DNA has been herein assessed through the multinuclear temperature-, pH*- and concentration-dependent NMR study of the effect of Pt²⁺ complexation to 2′-deoxygua

Full text of DOI:10.1039/a902330e

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The change in the electronic character upon cisplatin binding to
guanine nucleotide is transmitted to drive the conformation of the
local sugar-phosphate backbone—a quantitative study
a
a
b
b
ˇ
Matjaz Polak, Janez Plavec,* Anna Trifonova, Andras Földesi and
Jyoti Chattopadhyaya*b
a National Institute of Chemistry, Hajdrihova 19, SI-1115 Ljubljana, Slovenia.
E-mail: janez.plavec@ki.si
b Department of Bioorganic Chemistry, Box 581, Biomedical Centre, University of Uppsala,
S–751 23 Uppsala, Sweden. E-mail: jyoti@bioorgchem.uu.se
Received (in Cambridge, UK) 23rd March 1999, Accepted 6th August 1999
The microstructure alteration as a result of cisplatin binding to N7 of guanines in DNA has been herein assessed
through the multinuclear temperature-, pH*- and concentration-dependent NMR study of the effect of Pt2ϩ
complexation to 2Ј-deoxyguanosine 3Ј,5Ј-bis(ethyl hydrogen phosphate) 1 and its ribo analogue 3 which mimic
the central nucleotide moiety in a trinucleoside diphosphate in the complete absence of intramolecular base–base
stacking interactions. The N S pseudorotational equilibrium shifts towards N-type conformers by 17 and 21
percentage points at 298 K, respectively, thereby showing that free energy of platination is transmitted to drive the
sugar conformation. The increase in the population of N-type conformers was rationalized with the strengthening
of the anomeric effect in both 2Ј-deoxy and ribo nucleotides upon the formation of the Pt–N7 bond which promotes
nO4Ј → σ*C1Ј-N9 orbital interactions due to the reduction of π-electron density in the imidazole part of guanine.
The additional stabilization of N-type conformers in Pt2ϩ complexes of ribonucleotides is due to the tuning of the
gauche effect of the [N9–C1Ј–C2Ј–O2Ј] fragment, which is absent in 2Ј–deoxy-ribo counterparts. The platination
of N7 favours N1 deprotonation in 2 and 4 by ∆pKa of 0.7 and 0.9 units in comparison with parent nucleotides 1
and 3, respectively. The N
S pseudorotational equilibrium in 1–4 showed classical sigmoidal dependence as a
function of pH with pKa-values at the inflection points. The population of S-type conformers has increased upon
N1 deprotonation in 1–4 because the anomeric effect weakened due to the increased π-electron density in the
imidazole part of the guanine moiety. The formation of the Pt–N7 bond in bifunctional complexes 2 and 4
simultaneously causes a shift of the syn
anti equilibrium towards anti by 43 and 63 percentage points, and the
increase in the population of εt,N conformers by 20 and 32 percentage points at 278 K, respectively. Only a minor
conformational redistribution along β, γ, βϩ1 and εϪ1 torsion angles has been observed, which suggests their weak
conformational cooperativity with the N S pseudorotational equilibrium as a result of platination to guanine. In
comparison with nucleotide phosphodiesters, apurinic 3Ј,5Ј-bis(ethyl hydrogen phosphate) sugars 5 and 6 showed no
interaction with Pt2ϩ and therefore no conformational changes.
and N-type conformers, respectively. The nucleobase in N-
nucleosides influences the drive of the N S equilibrium
Introduction
The anticancer drug cisplatin manifests its therapeutic activity
by reacting with DNA to form crosslinks as major lesions, thus
interfering with replication and transcription processes.1 Vari-
ous studies have shown that platinum is coordinated by the N7
of guanines and adenines which agrees, with its ‘soft’ metal ion
nature and thus has greater affinity for nitrogen donors than
‘hard’ oxygen ligands of phosphodiester functionalities.2–11 It
is well known from numerous X-ray studies12–14 that sugar
moieties in natural nucleosides adopt predominantly two
distinct North (N, roughly C3Ј-endo) and South (S, roughly
C2Ј-endo) conformational families (Fig. 1). NMR studies
in solution have shown that the sugar moieties of nucleosides
and nucleotides are involved in a two-state conformational
equilibrium between N and S puckered forms which are
dynamically interconverting on the NMR time-scale. We have
previously quantified the thermodynamics of N S pseudo-
rotational equilibrium and shown that it is driven by the inter-
play of stereoelectronic anomeric and gauche effects as well as
by the competing steric effects of the substituents.15,16 The
gauche effects of C3Ј–O3Ј and C2Ј–O2Ј bonds with C4Ј–O4Ј
and C1Ј–O4Ј bonds drive the N S equilibrium towards S-
through inherent steric effects and the O4Ј–C1Ј–N1/9 anomeric
effect. It has been shown that the anomeric effect of guanine
is strengthened by N7 protonation17 or by interaction with
‘soft’ M2ϩ ions.18,19 A conformational rearrangement has been
observed in DNA–cisplatin adducts20 which can clearly be
ascribed to the binding of Pt2ϩ to N7 of guanine. Little is,
however, known of how the Pt2ϩ binding to N7 of guanine
nucleobase changes its stereoelectronic properties and how
these are transmitted into the drive of the local sugar-
phosphate backbone conformation. In the present study we
have synthesized 2Ј-deoxyguanosine 3Ј,5Ј-bis(ethyl hydrogen
phosphate) 1 and its ribo analogue 3, which serve as model
systems mimicking the central nucleotide moiety in a tri-
nucleoside diphosphate in order to shed light on the nature and
strength of intramolecular stereoelectronic effects in the com-
plete absence of intramolecular base–base stacking interactions
(Chart 1). The temperature- and pH-dependent conformational
analysis on apurinic sugars 5 and 6 enabled the comparison
and dissection of the conformational changes caused by the
transmission of the strengthened anomeric effect in platinated
nucleotides 2 and 4 from the drive of the conformational equi-
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843
This journal is © The Royal Society of Chemistry 1999
2835

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Fig. 1 Pseudorotation cycle of the pentofuranose ring in nucleosides
and nucleotides. Two pseudorotational parameters P and Ψm, which
have been proposed by Altona and Sundaralingam,13 are used to
describe the geometry of the puckered five-membered ring; P is the
phase angle of pseudorotation and Ψm is the maximum puckering amp-
litude. In the pseudorotational cycle, the sugar geometries alternate
between envelope (E) and twist (T) conformations every P of 18Њ. Ψm
denotes the extent of puckering and increases with the radial displace-
ment. The conformational regions that are most commonly found by
X-ray crystallography and NMR studies in solution are shaded in the
North and South regions of conformational space.
libria along sugar-phosphate torsion angles due to the possible
interactions of Pt2ϩ with phosphodiester moieties in 5 and 6 to
be made.
Chart 1 Structural formulas of ligands 1, 3, 5 and 6 and schematical
formula of bifunctional platinum complexes 2 and 4.
Results and discussion
Formation and identification of reaction products between cis-
gave clear evidence for the lack of their interaction with Pt2ϩ in
the absence of the heterocyclic nucleobase (Table 1). The
∆δ(31P)-values of both 3Ј- and 5Ј-phosphodiesters in 5 and 6 are
below 0.06 ppm (Table 1),24 which indicates that electrostatic
interactions of positive platinum and negative phosphate
oxygen atoms are minimal.
[Pt(ND3)2(D2O)2]2؉ and 1, 3, 5 and 6
The reactions of cis-[Pt(ND3)2(D2O)2]2ϩ with 1, 3, 5 and 6 in
1
D2O were monitored by H NMR spectroscopy. Preliminary
experiments showed that the reaction of one equivalent of cis-
[Pt(ND3)2(D2O)2]2ϩ with 1 resulted in the fast formation21,22
(<10 min at 298 K) of monoplatinated complex (δH8 = 8.60),
which was further transformed into the bifunctional complex 2
(δH8 = 8.50) together with several other products. The complex
reaction mixture prevented the extraction of coupling constants
for the individual species. On the other hand, the complex-
ation of 1 and 3 with 0.5 equivalents of cis-[Pt(ND3)2(D2O)2]2ϩ
resulted in one predominant set of 1H NMR resonances corre-
sponding to bisnucleotide Pt2ϩ complexes 2 and 4, respectively
(Chart 1). The amount of complexes 2 and 4 in the NMR tubes
Self-association of nucleotides 1 and 3 and their platinated
complexes 2 and 4
The 1H NMR chemical shifts for 1–4 at eight concentrations in
the range from 227 mM to 1 mM have been collected to study
1
their self-association. The H NMR chemical-shift changes of
H8 and all sugar resonances of 1–4 showed only minute con-
centration dependence (∆δ < 0.045 ppm). Nevertheless, the
concentration-dependent chemical shifts were further analyzed
according to the isodesmic EK model.25 The analysis of small
∆δ-values of H8, H1Ј, H2Ј and H3Ј in 1–4 afforded small aver-
age individual association constants (Kav = 1 1 MϪ1) for 1–4
which indicated the presence of over 96% ( 3%) of monomeric
species at 20 mM concentration, which value was used in the
present multinuclear NMR conformational study.
1
was over 90%. The detailed H, 13C, 15N, 31P and 195Pt NMR
chemical-shift analysis given in Table 1 confirmed the presence
of two guanine moieties in the Pt2ϩ coordination plane and
therefore the structures of 2 and 4. 31P chemical-shift changes
upon coordination of 1 and 3 with Pt2ϩ are insignificant and
thus clearly exclude the formation of an N7, PO chelate23 in 2
and 4 (Table 1).
The effect of temperature on the H8 linewidths in bi-
functional complexes 2 and 4 was studied to assess the rate of
rotation about the Pt–N7 bonds. The H8 signals sharpen with
an increase in temperature from 278 to 358 K from 2.3 to 1.0 Hz
in 2 and from 3.7 to 1.1 Hz in 4, which indicates that nucleotide
moieties are in the fast Pt–N7 rotation-rate regime on the NMR
time-scale at all studied temperatures. For comparison, the H8
resonances in the parent nucleotides sharpened from 2.8 to 1.1
Hz in 1 and from 5.4 to 1.4 Hz in 3 as temperature was
increased from 278 to 358 K.
Acid–base properties of nucleotides 1 and 3 and their platinated
complexes 2 and 4
Nucleotides 1 and 3 and their Pt2ϩ complexes 2 and 4 may
release a proton from N1. Analysis of the H8 chemical shift in 1
and 3 as a function of pH* in the range from 5.3 to 12.5 showed
classical sigmoidal dependence (Fig. 2A) (For a definition
of pH* see Experimental section.). The least-squares fitting
procedure and Hill plots (not shown) gave pKa-values of 10.2
for both 1 and 3. The pH* dependence of H8 chemical shifts
in 2 and 4 which is shown in Fig. 2A yielded pKa-values of 9.5
and 9.1, respectively. The platination of N7 therefore favoured
N1 deprotonation in 2 and 4 by a ∆pKa of 0.7 and 0.9 units in
No chemical-shift changes were observed upon addition of
cis-[Pt(ND3)2(D2O)2]2ϩ to abasic compounds 5 and 6, which
2836
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843

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Table 1 1H, 13C, 31P, 15N, 195Pt NMR chemical shiftsa for 1–6 at 298 K at neutral pH*
Compound
1
2
3
4
5
5 ϩ Ptb
6
6 ϩ Ptb
H8
8.081
6.359
8.502
6.311
8.103
5.988
8.534
5.959
H1Ј
H1Љ
H2Ј
H2Љ
H3Ј
H4Ј
H5Ј
H5Љ
3Ј-CH2
5Ј-CH2
3Ј-CH3
5Ј-CH3
C6
C4
C2
C8
C5
C1Ј
C4Ј
C3Ј
C2Ј
3.985
4.085
2.230
2.148
4.690
4.186
3.920
3.871
3.947
3.947
1.278
1.274
3.986
4.086
2.232
2.149
4.691
4.188
3.921
3.872
3.947
3.947
1.278
1.274
3.840
4.104
4.438
3.842
4.106
4.440
2.930
2.723
5.014
4.435
4.063
4.063
3.996
3.783
1.304
1.122
162.24
157.10
154.82
140.87
119.52
88.14
86.93
78.98
40.91
68.39
65.73
65.46
18.89
18.63
1.00
2.844
2.756
5.002
4.423
4.093
4.043
3.989
3.828
1.298
1.103
159.81
157.74
153.59
143.25
117.36
88.43
88.04
78.19
41.26
68.24
65.81
65.52
18.94
18.68
4.945
HOD
4.830
4.836
4.537
4.109
4.109
4.024
3.846
1.303
1.162
162.21
157.17
155.26
140.86
119.59
89.66
86.45
77.39
75.93
68.09
65.86
65.54
18.90
18.69
1.00
4.522
4.165
4.089
3.930
3.983
3.950
1.288
1.274
4.523
4.168
4.091
3.932
3.984
3.951
1.288
1.275
4.515
4.176
4.129
4.014
3.901
1.294
1.159
159.82
157.80
153.75
143.39
117.32
91.26
86.33
76.80
76.33
67.84
65.91
65.58
18.90
18.71
0.98
66.99
83.48
76.55
32.82
64.82
61.93
61.89
15.20
15.16
1.29
67.00
83.49
76.57
32.84
64.85
61.95
61.91
15.22
15.18
1.30
74.88
83.16
78.18
73.45
68.23
65.55
65.36
18.66
18.59
1.26
74.90
83.17
78.39
73.46
68.25
65.57
65.38
18.68
18.61
1.27
C5Ј
3Ј-CH2
5Ј-CH2
3Ј-CH3
5Ј-CH3
P5Ј
0.94
P3Ј
N7
N9
Pt
0.36
Ϫ145.9
Ϫ208.2
0.36
0.57
Ϫ145.9
Ϫ213.5
0.58
Ϫ238.1
Ϫ209.2
Ϫ2442
0.53
0.54
0.72
0.72
Ϫ239.1
Ϫ203.4
Ϫ2441
Ϫ1615
Ϫ1615
a Chemical shifts are in ppm ( 0.001 ppm for 1H, 0.01 ppm for 13C and 31P, 0.05 ppm for 15N and 1 ppm for 195Pt). The methylene protons of
ethyl phosphodiester groups showed ∆δ < 16 Hz. HOD denotes that H2Ј was hidden under the residual water signal. b 5 ϩ Pt and 6 ϩ Pt represent
solutions of 5 and 6 in the presence of cis-[Pt(ND3)2(D2O2)]2ϩ, respectively.
comparison to parent nucleotides 1 and 3, respectively. The
increased acidity8,11 of N1 in 2 and 4 can be explained by the
reduction of electron density at N1 by the electron-withdrawing
effect of the cis-[Pt(ND3)2]2ϩ moiety. It is noteworthy that com-
pounds 1–4 are protonated at N1 by over 99.5% at a pH* of 6.8,
which was a pH* value used in our conformational studies.
ance of anomeric and gauche effects of [O4Ј–C4Ј–C3Ј–O3Ј]
fragments in 2Ј-deoxy analogues 1 and 2. In 2, the platination
of N7 modulates exclusively the anomeric effect which has been
observed by monitoring the increase in the relative ratio of
pseudoaxial (i.e. in N-type pseudorotamers) over pseudo-
equatorial (i.e. in S-type pseudorotamers) orientations of the
nucleobase. The comparison of ∆HЊ-values in 1 and 2 shows
that the anomeric effect of guanine in 2 is strengthened by a
value for ∆∆HЊ of 3.5 kJ molϪ1 which results in the drive of the
North South pseudorotational equilibrium
The conformational analysis of the pentofuranosyl moiety in
1–6 was based on the temperature-dependent JHH proton–
proton coupling constants (Table 2), which were interpreted
in terms of a two-state N S pseudorotational equilibrium
(Table 3). The experimental 3JHH coupling constants which were
acquired at five temperatures in the range from 278 to 358 K
were analysed with the computer program PSEUROT.26,27 The
N
S equilibrium towards N-type pseudorotamers. The N
S
3
pseudorotational equilibrium of ribo analogues 3 and 4 is in
contrast to that of 2Ј–deoxy counterparts 1 and 2 additionally
controlled by the gauche effects of [O4Ј–C1Ј–C2Ј–O2Ј] frag-
ments, which stabilize the N-type conformation, and by the
gauche effects of [N9–C1Ј–C2Ј–O2Ј] fragments, which stabilize
the S-type sugar conformation.15 The data in Table 3 show that
the overall drive of N S equilibrium towards N-type sugar
conformations upon platination of ribo nucleotide 3 is
strengthened by a value for ∆∆HЊ of 4.9 kJ molϪ1. It is therefore
apparent that a platinum atom bound to N7 of guanine in 4
modulates the anomeric effect as well as the gauche effect of the
[N9–C1Ј–C2Ј–O2Ј] fragment, which results in the additional
stabilization of N-type pseudorotamers in ribo analogue 4 in
comparison with its 2Ј-deoxy counterpart 2 (Table 3).
N
S pseudorotational equilibrium in both 1 and 3 is biased
towards S by 77% at 298 K. On the other hand, their platinated
complexes 2 and 4 exhibit smaller conformational purities of 60
and 56% of S-type sugar conformation at 298 K, respectively.
There is therefore a definite drive of the N S pseudorota-
tional equilibrium towards N-type sugar conformations by 17
percentage points in 2 compared with 1, and by 21 percentage
points in 4 compared with 3 at 298 K.
The mole fractions of N- and S-type conformers at five tem-
peratures between 278 and 358 K in 20 K steps were used in a
van’t Hoff-type analysis to obtain enthalpy (∆HЊ) and entropy
(∆SЊ) of the N S pseudorotational equilibrium in 1–6. Nucleo-
tides 1 and 3 exhibited negative ∆HЊ-values which predominate
The conformational analyses of 2Ј-deoxy sugar 5 showed a
high conformational bias of 85% towards S-type sugar pucker-
ing at 278 K (Table 3). The van’t Hoff analysis showed that
N
S equilibrium in 5 is driven towards S by larger ∆HЊvalues,
which is opposed by the weaker entropy term. The addition of
Pt2ϩ reagent caused no observable tuning of the N S equi-
librium in 5 (Table 3). The analysis of temperature-dependent
3JHH values in 6 before and after addition of Pt2ϩ (Table 2)
showed nearly identical conformational preferences of 73%
for N-type sugar at 278 K (Table 3). The pseudorotational
over the weaker ϪT∆SЊ contributions in the drive of their N
S
equilibrium towards S-type pseudorotamers (Table 3). Their
platinated complexes 2 and 4 were characterized by small ∆HЊ
values as compared with the relatively stronger ϪT∆SЊ contri-
butions (Table 3). The experimental ∆HЊ-values reflect the bal-
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843
2837

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caused by N1 deprotonation. Inspection of sigmoidal curves
in Fig. 2B shows that the increase in the population of
S-type conformers has been considerably more pronounced in
bifunctional platinated complexes 2 and 4 in comparison with
their parent nucleotides 1 and 3, respectively. The Pt2ϩ bound to
N7 obviously promotes the re-distribution of the electronic
density from anionic deprotonated N1 into the imidazole part
of the guanine moieties in 2 and 4. The increase of the electron-
rich character of imidazole weakens nO4Ј→σ*C1Ј–N9 interactions
which is manifested in the larger shift towards S-type con-
formers in platinum complexes 2 and 4 in comparison with their
parent nucleotides. The much higher preference for S-type con-
formers in ribo analogue 4 in comparison with its 2Ј-deoxy
counterpart 2 (Fig. 2B) can be explained by the additional
drive of the N S equilibrium by the gauche effects of [O2Ј–
C2Ј–C1Ј–N9] and [O4Ј–C1Ј–C2Ј–O2Ј] fragments. The combin-
ation of the weakened anomeric effect and the strengthened
gauche effect of the [O2Ј–C2Ј–C1Ј–N9] fragment in 4 upon N1
deprotonation results in the large conformational bias of the
N
S pseudorotational equilibrium towards S-type conformers
(Table 3, Fig. 2B).
Van’t Hoff-type analysis of temperature-dependent popu-
lations of the N- and S-type conformers in N1 deprotonated 1–
4 gave ∆HЊ- and ∆SЊ-values for the N S pseudorotational
equilibrium (Table 3). Deprotonation of 2Ј-deoxy nucleotides 1
and 2 unexpectedly resulted in an increase in the ∆HЊ-values of
the N S equilibrium, which indicates a drive towards N-type
conformations. However, the stronger opposing entropy term
has prevailed, which results in a decrease in ∆GЊ for 1 and 2
(Fig. 2C) and therefore the overall stabilization of the S-type
sugar conformation upon N1 deprotonation (Table 3). The
deprotonation of ribonucleotide 3 and its Pt2ϩ complex 4
results in a decrease in ∆HЊ by 1.4 and 7.0 kJ molϪ1, respectively
(Table 3). The weaker entropy terms in 3 and 4 drive N
S
equilibrium towards N, but the overall stabilization of S-type
sugar conformations has been observed upon N1 deprotona-
tion for both 3 and 4 (Table 3).
Fig. 2 pKa Determination of 1–4 on the basis of pH-dependent chem-
ical shifts of H8 (A), populations of S-type conformers (B) and ∆GЊ of
N
S pseudorotational equilibrium (C). Panel (A): Titration curves
Conformational equilibrium across the C1Ј–N9 (ꢀ) bond
constructed from H8 chemical shifts as a function of pH* at 298 K in 1
(filled circles), 2 (open circles), 3 (open triangles) and 4 (filled triangles).
The sigmoidal curves are the best iterative least-squares fit (r.m.s.
error < 0.003 ppm) of the experimental pH*-dependent chemical shifts
giving the pKa-values at the inflection points. The pKa-values are 10.2
for 1 and 3, 9.5 for 2 and 9.1 for 4. Panel (B): The population of S-type
conformers as a function of pH* for 1–4 at 298 K. The sigmoidal curves
which are the best iterative least-squares fit (r.m.s. error < 0.9 percent-
age points) of the pH*-dependent mole fractions of S-type conformers
in the two state N S pseudorotational equilibrium are characterized
by the following pKa-values: 10.2 for 1 and 3, 9.4 for 2 and 9.5 for 4. The
populations of S-type conformers on plateaux raised by 3.2 percentage
points (from 75.5 to 78.7%) for 1, by 9.6 percentage points (from 57.1 to
66.7%) for 2, by 3.7 percentage points (from 76.4 to 80.1%) for 3 and by
22.6 percentage points (from 50.5 to 73.1%) for 4. Panel (C): pH*-
dependent ∆GЊ for the N S equilibrium in 1–4 at 298 K. The best fit
through the experimental data points gave identical pKa-values to those
in panel (B). The changes in the experimental ∆GЊ-values upon N1-
deprotonation were: Ϫ0.45 kJ molϪ1 for 1, Ϫ1.0 kJ molϪ1 for 2, Ϫ0.5 kJ
molϪ1 for 3 and Ϫ2.55 kJ molϪ1 for 4.
Semiquantitative information about the conformational equi-
librium across glycosyl bonds in 1–4 has been obtained with
the use of 1D NOE spectroscopy.28 Analyses of H8→H1Ј NOE
enhancements have shown that the populations of anti con-
formers are 53 and 29% in 1 and 3, respectively. Their platinated
complexes 2 and 4 have shown a high conformational bias
towards anti of 96 and 92%, respectively. The strong preference
for anti conformers upon binding of Pt2ϩ to N7 in 2 and 4 can
be explained by the increased steric bulk which forces the
heterocyclic moiety to turn away from the sugar moiety. The
large increase in the population of anti conformers in 2 and 4
in comparison with 1 and 3, respectively, is correlated with the
increase in the population of N-type pseudorotamers. It is
noteworthy that the larger shift of N S equilibrium towards
N-type pseudorotamers upon platination of ribo analogue 3 in
comparison with its 2Ј-deoxy counterpart 1 (Table 3) correlates
with the larger shift of their syn anti equilibrium towards
anti.
equilibrium in 6 is driven towards N-type conformers by the
∆HЊ term, which is opposed by the twice weaker entropy term.
The N S pseudorotational equilibrium in 1–4 was also
assessed as a function of pH*, which showed classical sig-
moidal dependence (Fig. 2B). The population of S-type con-
formers at 298 K increased upon N1 deprotonation from 76
to 79% in 1, from 57 to 67% in 2, from 76 to 80% in 3 and from
51 to 73% in 4 (Fig. 2B). Our previous studies17 have shown that
the thermodynamics of N1 deprotonation in guanine nucleo-
sides is transmitted to drive the N S equilibrium towards
S-type pseudorotamers. The drive of N S equilibrium towards
S can be explained by the weakening of the anomeric effect due
to the increased electron density on the heterocyclic nucleobase
Conformational equilibrium across the C5Ј–O5Ј (β) bond
The conformational equilibrium across torsion angle β[C4Ј–
C5Ј–O5Ј–P] has been assessed with the use of temperature-
3
3
3
dependent JC4ЈP and the sum of JH5ЈP and JH5ЈP coupling
constants29–31 (Table 2). The analysis of both sets of coupling
constants gave comparable results with discrepancies below 3
percentage points, which is well within the accuracy of the
Karplus equations used. The average populations of βt rotam-
ers at the two limiting temperatures are reported in Table 4.
βt conformers are preferred by over 75% in 1–6 at 278 K. This
value is lowered by ≈4 percentage points when the temperature
2838
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843

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Table 2 JHH, JHP and JCP coupling constants for 1–6 at two limiting temperatures at neutral pH*a
JHH JHP
T(K) 1Ј2Ј 1Ј2Љ 1Љ2Ј 1Љ2Љ 2Ј3Ј 2Љ3Ј 3Ј4Ј 4Ј5Ј 4Ј5Љ H3ЈP3Ј H5ЈP5Ј H5ЉPЈ C2ЈP3Ј C4ЈP3Ј C4ЈP5Ј
JCP
Comp.
1
278
358
278
358b
278
358
278
358b
278
358
278
358b
278
358
278
358b
8.1 6.2
7.8 6.3
6.2 6.7
6.5 6.6
6.9
6.4
5.0
5.8
6.1
6.3
6.1
5.2
5.2
4.9
5.1
5.8
6.0
5.7
6.0
4.6
4.9
4.7
5.0
2.5
2.9
4.1
4.1
2.2
2.7
3.3
3.3
2.5
3.2
4.3
4.3
1.7
2.4
1.9
2.4
6.3
6.0
6.4
5.9
4.3
3.4
3.2
3.5
2.7
2.8
2.4
2.8
3.7
3.9
3.8
4.0
2.4
2.7
2.4
2.8
3.0
5.1
4.4
5.0
3.5
4.5
3.8
4.8
5.4
5.7
5.3
5.7
5.0
5.5
5.1
5.6
7.1a
7.5
7.2a
7.1
7.8
7.5
7.8
7.7
7.3
7.0
7.2
7.0
8.1
7.5
8.1
7.5
4.2
4.9
4.5
5.1
4.6
4.9
5.1
5.2
4.9
5.3
4.9
5.4
5.2
5.4
5.1
5.4
4.5
5.0
4.5
5.1
3.9
4.9
4.6
4.8
5.2
5.7
5.1
5.7
4.7
5.5
4.7
5.3
3.6
3.2
2.4
3.0
4.9
4.0
3.4
3.7
3.2
3.3
3.5
3.3
3.3
3.3
3.4
3.4
6.0
6.3
6.6
6.1
2.9
4.3
6.2
5.8
6.5
6.3
6.3
6.3
6.6
6.3
6.3
6.3
9.0
8.7
8.2
7.9
9.0
8.5
8.7
8.7
8.4
8.1
8.4
8.2
8.7
8.5
8.5
8.1
2
3
4
5.1
5
10.0 6.2
9.3 6.6
10.0 6.2
9.3 6.6
3.2
3.8
3.2
8.3
8.2
8.3
8.2
4.3
4.9
4.3
4.7
2.8
3.7
2.9
3.7
2.1
2.4
2.0
2.4
5 ϩ Pt
6
6 ϩ Pt
3.8
a Homo- and hetero-nuclear coupling constants are given in Hz ( 0.1 Hz for 3JHH and 3JHP
,
0.2 Hz for 3JCP). H3Ј signals were hidden under HOD
3
signal and JH3ЈP3Ј coupling constants at 278 K were extrapolated according to the trend with temperature. b The highest temperature for the
collection of 13C NMR data for complexes 2, 4 and for 5 ϩ Pt and 6 ϩ Pt was 338 K.
Table 3 Assessment of the N S pseudorotational equilibrium in 1–6
Compound
1
2
3
4
Protonation
state
5
5 ϩ Pt
6
6 ϩ Pt
neutral
neutral
deprot.
neutral
deprot.
neutral
deprot.
neutral
deprot.
neutral neutral
neutral
pH*
6.8
Ϫ4.8
33.2b
160.7
33.2
0.09
0.19
79.1
74.3
Ϫ2.8
0.7
12.5
Ϫ10.1
34.1b
158.7
34.1
0.04
0.10
79.2
76.9
Ϫ1.5
5.9
Ϫ1.8
Ϫ3.3
6.8
Ϫ10.0
32.3b
165.8
32.2b
0.06
0.14
59.7
61.3
0.7
12.1
Ϫ22.3b
32.8b
157.4
32.9
0.10
0.25
67.4
71.0
1.8
12.4
Ϫ3.7
Ϫ1.9
6.6
15.0
32.5b
158.1
32.5
0.04
0.06
79.8
71.8
Ϫ4.6
Ϫ5.1
1.5
12.1
17.0b
32.9b
158.6
32.9
0.06
0.13
82.8
73.2
Ϫ6.0
Ϫ8.4
2.5
6.9
16.2
32.5b
161.9
32.5b
0.08
0.23
54.8
55.5
0.3
12.2
20.1b
31.6b
156.6
31.6
0.04
0.08
81.3
68.8
Ϫ6.7
Ϫ12.3
3.7
6.8
Ϫ22.5
30.5b
153.2
30.5
0.28
0.52
85.3
77.9
6.8
Ϫ21.6
30.5b
151.7
30.5
0.30
0.58
85.0
77.8
6.7
Ϫ13.7
39.3
111.8
39.3b
0.07
0.17
26.8
35.5
4.2
6.8
Ϫ14.1
39.4
111.1
39.4b
0.07
0.15
26.8
35.3
4.1
PN
Ψm
N
PS
Ψm
S
r.m.s.
∆Jmax
%S (278 K)
%S (358 K)
∆HЊ
Ϫ5.0
Ϫ3.5
1.0
Ϫ4.9
Ϫ3.4
1.0
∆SЊ
5.8
Ϫ1.7
Ϫ1.0
2.6
Ϫ0.8
Ϫ0.5
6.6
Ϫ2.0
2.2
6.2
Ϫ1.9
2.2
ϪT∆SЊ (298 K) Ϫ0.2
∆GЊ (298 K) Ϫ3.0
Ϫ3.1
Ϫ3.5
Ϫ3.0
Ϫ4.0
Ϫ3.9
a Pseudorotational parameters are given in degrees, root-mean-square error and ∆Jmax are in Hz, ∆HЊ ( 0.2), ∆GЊ ( 0.2) and ϪT∆SЊ ( 0.5) are in
kJ molϪ1, ∆SЊ are in J molϪ1 KϪ1 b The pseudorotational parameter was kept fixed during the iterative optimization procedure.
.
is increased to 358 K. The platinum complexes 2 and 4 showed a
lower preference for βt rotamers by 2 and 5 percentage points at
278 K in comparison with their parent nucleotides 1 and 3,
respectively. In contrast, apurinic sugar analogues 5 and 6
showed only minor conformational changes below 2 percentage
points upon addition of Pt2ϩ reagent, suggesting that there are
no electrostatic interactions with their constituent phosphate
moieties, which is also in agreement with the negligible ∆δ(31P)-
values (Table 1).
Conformational equilibrium across the C3Ј–O3Ј (ꢂ) bond
3
3
The temperature-dependent JH3ЈP,
3JC4ЈP and JC2ЈP coupling
constants in 1–6 (Table 2) have been used for the evaluation of
conformational equilibria across C3Ј–O3Ј bonds (torsion angle
ε[C4Ј–C3Ј–O3Ј–P]). Preliminary interpretation of the experi-
mental coupling constants in terms of a two-state εt εϪ con-
formational equilibrium33 resulted in large errors of up to 1.0
3
Hz for individual JCP coupling constants. Therefore a further
3
3
attempt has been made to interpret experimental JH3ЈP, JC4ЈP
3
and JC2ЈP coupling constants in terms of a three-state εt,N
Conformational equilibrium across the C4Ј–C5Ј (ꢁ) bond
εt,S
ε
Ϫ,S conformational equilibrium (Fig. 3).34
3
3
Conformational analysis32 of the J4Ј5Ј and J4Ј5Љ coupling con-
stants (Table 2) has shown that γ+ rotamers are preferred by
over 59% at 278 K in 1–4 (Table 4). Comparison of the popu-
lations of γ rotamers in 1 and 3 with those in 2 and 4, respect-
ively, showed that N7 platination exerted only a minor, if any,
effect on the conformational equilibria across their C4Ј–C5Ј
bonds. Conformational analysis has also shown that γϩ and
γt rotamers are highly preferred over γϪ rotamers in apurinic
analogues 5 and 6 (Table 4). As no reaction occurred between
the Pt2ϩ reagent and apurinic sugars 5 and 6, no conformational
changes were observed, as expected.
The least-squares optimization procedure resulted in an
r.m.s. error below 0.6 Hz and a maximum discrepancy between
experimental and calculated coupling constants below 0.7 Hz
(Table 4). The population of εt,N conformers at 278 K was 28
and 17% in 1 and 3, respectively. Their Pt2ϩ complexes 2 and 4
showed considerably higher εt,N populations of 48 and 49% at
4
278 K, respectively. Similarly, the analysis of JH2ЈP coupling
constants35 in 1–4 showed that the population of εϪ rotamers
decreases (i.e., εt increases) upon platination of the nucleobase.
The higher preference for εt rotamers in Pt2ϩ complexes 2 and 4
is correlated with the higher preference for N-type pseudo-
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843
2839

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Table 4 Conformational equilibria across β, γ and ε torsion angles in 1–6 at two limiting temperatures
γ torsionb
ε torsionc
t,N (Њ)
β torsiona
%βt
Compd.
T(K)
%γϩ
%γt
%γϪ
ε
ε
t,S (Њ)
ε
Ϫ,S (Њ)
%εt,N
%εt,S
%εϪ,S
r.m.s. (Hz)
∆Jmax (Hz)
1
278
358
278
358
278
358
278
358
278
358
278
358
278
358
278
358
81
77
79
75
82
76
77
76
75
71
75
70
77
73
76
72
61
50
59
50
73
62
73
59
44
39
44
38
61
53
60
51
10
35
27
34
18
29
21
32
38
41
37
41
35
40
36
41
29
15
14
17
10
9
6
9
18
20
20
21
4
221
214
211
211
212
213
212
210
190
192
192
192
197
198
192
191
267
263
261
266
260
266
266
267
28
37
48
31
17
27
49
53
17
0
15
21
70
51
67
57
35
32
26
32
11
16
14
9
43
56
46
41
0
37
31
27
37
72
57
36
38
40
44
40
36
30
37
33
34
0.076
0.331
0.587
0.107
0.079
0.038
0.218
0.104
<0.1
0.4
2
3
0.7
4
0.1
5
0.1
5 ϩ Pt
6
<0.1
0.3
6
4
7
13
0
9
6 ϩ Pt
0.1
a The populations of βt conformers ( 3%) have been calculated independently from 3JCP and 3JHP acquired in the temperature range from 278 to 358
K in 20 K steps and their average values are reported. For 2, 4, 5 ϩ Pt and 6 ϩ Pt the populations of β t conformers at 358 K have been calculated
from 3JHP only. b The populations of γ conformers ( 3%) have been calculated from 3J4Ј5Ј and 3J4Ј5Ј c The highest temperature for the collection of
.
13C NMR data for complexes 2 and 4 and for 5 ϩ Pt and 6 ϩ Pt was 338 K, which is the highest temperature where the populations of εt,N, εt,S and
Ϫ,S conformers ( 5%) are reported.
ε
Fig. 4 Correlation plots between the populations of εt,N conformers
and the populations of S-type pseudorotamers in 1–6. The least-
squares-fitted straight lines were calculated from the mole fractions
of S-type pseudorotamers obtained by the PSEUROT analysis of
3
temperature-dependent JHH coupling constants, and the respective
mole fractions of εt,N conformers obtained from the analysis of 3JHP and
3JCP coupling constants. Their Pearson’s correlation coefficients are
Ϫ0.859 for 1, Ϫ0.965 for 2, 1.000 for 3 and 4, 0.951 for 5 and Ϫ0.966 for
6. Note that for 1, 2, 5 and 6 five experimental data points were available
from the temperature-dependent coupling constants in the range from
278 to 358 K in 20 K steps. In the case of platinum complexes 2 and 4
four sets of populations were determined in the temperature range from
278 to 338 K in 20 K steps. The populations of S-type pseudorotamers
in 4 change by <1 percentage point from 278 to 338 K in 20 K steps
which results in the overlap of some data points.
Fig. 3 A hypothetical three-state conformational equilibrium across
C3Ј–O3Ј bonds in 1–6.
rotamers.33 Correlations of populations of εt,N and S-type con-
formers in 1–6 are shown in Fig. 4. The least-squares fitting
procedure afforded straight lines which indicate that as the
population of S-type pseudorotamers decreases with increas-
ing temperature in 1 and 3, the population of εt,N conformers
increases.
between increased population of N-type pseudorotamers and
εt,N conformers has been observed (Fig. 4). It is interesting to
note that methylene protons of 3Ј-ethyl phosphodiester groups
in 2Ј-deoxynucleotide 1 and its platinum complex 2 are iso-
chronous at all temperatures from 278 to 358 K, which suggests
their unperturbed rotation. The methylene protons of 3Ј-ethyl
phosphodiester groups in 3 and 4 were non-isochronous
(∆δ = 10 and 7 Hz at 278 K and ∆δ = 5 and 4 Hz at 358 K,
respectively) which implies the specific role of 2Ј-OH interact-
ing with the vicinal ethyl phosphate. The conformational equi-
libria across βϩ1 torsion angles in 1–6 were evaluated on the
Conformational equilibria across CH2–O (ꢂ؊1) and O–CH2 (ꢃ؉1)
bonds
The structural make-up of 1–6 gives insight into the tunability
as well as the energetic transmission characteristics of the N
S
pseudorotational equilibrium to drive the rotamer distribution
along the phosphate backbone as a result of formation of the
new Pt–N7 bond in 2 and 4 compared with the parent nucleo-
tides 1 and 3 and abasic bis-phosphates 5 and 6. We argued that
this should in turn show how the dynamics of the conform-
ational cooperativity in the immediate micromilieu changes and
is transmitted as a result of ligand binding. There is, however,
no J-coupling that would give insight into α and ζ torsions
(Chart 1) which are the closest to ε, where a linear correlation
3
basis of both temperature-dependent JCH3P and the sum of
3JCH2P coupling constants.29–31 The population of βϩ1-trans
rotamers was ≈57% at 278 K and decreased by 4 to 5 percent-
age points upon an increase of temperature to 358 K in 1–4. On
2840
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843

View Article Online
the other hand, the platination of 1 and 3 caused negligible
conformational changes across their βϩ1 torsion angles. The
spectral overlap of methylene resonances of 3Ј-ethyl phos-
phodiester groups in 5 and 6 prevented determination of their
guanine complex with a cytosine moiety more strongly through
hydrogen bonding or binding to any other basic ligand, com-
pared with the free guanine nucleotide moieties. The N
S
pseudorotational equilibrium in 1–4 showed classical sigmoidal
dependence as a function of pH with pKa-values at the inflec-
tion points. The population of S-type conformers at 298 K
increased upon N1 deprotonation from 76 to 79% in 1, from 57
to 67% in 2, from 76 to 80% in 3 and from 51 to 73% in 4, which
showed that the thermodynamics of N1 deprotonation in guan-
ine nucleosides is transmitted through the tunable anomeric
effect to drive the N S equilibrium towards S-type pseudo-
rotamers. The Pt2ϩ-complex bound to N7 was found to pro-
mote the re-distribution of the π-electronic density from
anionic deprotonated N1, which results in the larger increase in
the population of S-type conformers by 10 percentage points in
2 and by 22 percentage points in 4 in comparison with parent
nucleotides 1 and 3 respectively.
The formation of the Pt–N7 bond simultaneously causes a
shift of the syn anti equilibrium towards anti by 43 and 63
percentage points in bifunctional complexes 2 and 4, and an
increase in the population of εt,N conformers by 20 and 32 per-
centage points at 278 K, respectively. Only minor conform-
ational re-distributions along β, γ, βϩ1 and εϪ1 torsion angles
have been observed which suggests weak conformational co-
operativity between the shift in the N S pseudorotational
equilibrium towards N-type conformers and the conform-
ational changes across phosphate torsion angles other than ε.
The apurinic 3Ј,5Ј-bis(ethyl hydrogen phosphate) sugars 5 and
6 showed no interaction with Pt2ϩ and therefore no conforma-
tional changes.
3
1H NMR chemical shifts and extraction of individual JCH2P
coupling constants. The analysis of 3JCH3P coupling constants in
3Ј-ethyl phosphodiester groups in 5 and 6 showed that the pref-
erence for βϩ1-trans rotamers drops by ≈4 percentage points
with the increase of temperature from 278 to 358 K.
The methylene protons of 5Ј-ethyl phosphodiester groups in
2 are non-isochronous (∆δ = 16 Hz at 278 K and 9 Hz at 358 K),
whereas they are isochronous in 1. In the case of ribo counter-
parts 3 and 4 we have observed the non-equivalence of the
methylene protons of 5Ј-ethyl phosphodiester groups up to 338
K in 3 (∆δ = 11 Hz at 278 K and ∆δ = 5 Hz at 318 K) and up to
358 K in 4 (∆δ = 10 Hz at 278 K and 5 Hz at 358 K). The
appearance of non-isochronous methylene protons of 5Ј-ethyl
phosphodiester groups in 2 and 4 in comparison to 1 and 3
suggests some electrostatic interactions between platinum and
phosphate oxygen atoms. Note that ∆δ-values for 31P upon
platination of 1 and 3 were below 0.06 ppm (Table 1) and did
not provide any conclusive evidence about such chelative inter-
actions. The conformational equilibria across εϪ1 torsion angles
in 1–6 were evaluated on the basis of both temperature-
3
3
dependent JCH3P and the sum of JCH2P coupling constants of
the 5Ј-ethyl hydrogen phosphate moiety. The population of εϪ1
trans rotamers was ≈57% at 278 K and decreased by 1 to 5
percentage points upon an increase of temperature to 358 K.
The platination of 1 and 3 caused negligible conformational
changes across εϪ1 torsion angle. In the case of abasic bisphos-
phate analogues 5 and 6, the heavy spectral overlap prevented
the extraction of ∆δ-values of their methylene protons and
3JCH2P coupling constants. The analysis of 3JCH3P coupling con-
stants in 5Ј-ethyl phosphodiester groups in 5 and 6 showed that
the preference for εϪ1-trans rotamers drops by ≈6 percentage
points with an increase of temperature from 278 to 358 K. The
addition of platination reagent to 5 and 6 caused negligible
change in the population of εϪ1 rotamers.
-
Experimental
General procedures in synthesis
Compounds 7 and 9 were synthesized using standard pro-
cedures37,38 starting from 2Ј-deoxyguanosine and guanosine,
respectively (Scheme 1). Protected nucleosides were converted
into 3Ј,5Ј-bis(phosphotriesters) 8 and 10 by treatment with (2-
cyanoethoxy)(N,N-diisopropylamino)(ethoxy)phosphine39 in
DMF–MeCN solution at r.t. under nitrogen atmosphere in the
presence of tetrazole followed by oxidation of the intermediary
phosphite triesters by aq. iodine solution with yields of 60 and
91%, respectively. Deprotections of these compounds were
carried out using aq. ammonia and 80% aq. acetic acid for
removing of MDMP (3-methoxy-1,5-dimethoxycarbonylpent-
3-yl) group of 11 to give fully deprotected 3Ј,5Ј-bis(phospho-
diesters) 1 (73%) and 3 (57%).
1-Deoxyribofuranose derivative 15 was obtained from 1,2,3,
5-tetra-O-acetyl-β--ribofuranose via conversion into the
1-phenylthio derivative using boron trifluoride–diethyl ether as
catalyst (70%) followed by reduction by a modified litera-
ture procedure (80%),15 deprotection using methanolic
ammonia (96%) and protection as the 3,5-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl) derivative (75%).40 Part of this
compound was converted to 15 according to the literature pro-
cedure (60%),41 whereas the other part was reduced (60%)42
and converted15 to the 1,2-dideoxy derivative 12 (60%). C5
of deoxyribose derivatives 12 and 15 was protected since
3-phosphotriesters 14 and 17 were to be used as intermediates
for the synthesis of 3,5-bis(phosphodiesters) having an O-
methyl hydrogen phosphate group at the C5 position. 3,5-
Bis(phosphotriesters) 18 (90%) and 19 (70%) were prepared by
phosphorylation of the 3-hydroxy group by (2-cyanoethoxy)-
(N,N-diisopropylamino)(ethoxy)phosphine in DMF–MeCN
solution at r.t. under nitrogen atmosphere in the presence of
tetrazole followed by oxidation by aq. iodine solution to give
compounds 13 (80%) and 16 (83%), followed by removal of
5-O-protection by 80% acetic acid (η 84% for compound 14) or
by 0.1 M trichloroacetic acid (η 83% for compound 17) and
Electron delocalization caused by Pt2؉ binding to N7 steers
sugar-phosphate conformation
It is known that the binding of cisplatin to DNA is followed by
structural distortions leading to duplex bending and unwinding
that are recognized by HMG proteins.36 Our data clearly show
that there are important electronic and thermodynamic con-
sequences of the formation of bifunctional complexes with cis-
platin. Two simple model mononucleotides, 2Ј-deoxyguanosine
3Ј,5Ј-bis(ethyl hydrogen phosphate) 1 and its ribo analogue 3,
were prepared in order to understand how the local micro-
structure changes as a consequence of Pt2ϩ binding to the
guanine moiety in a polynucleotide. Comparative analysis of
multinuclear NMR chemical-shift data of abasic bisphos-
phodiesters 5 and 6 clearly showed the absence of any
interactions of Pt2ϩ with anionic phosphates. The N
S
pseudorotational equilibrium in 2 and 4 has been shifted
towards N-type conformers by 17 and 21 percentage points at
298 K, respectively. The increase in the population of N-type
conformers caused by the formation of Pt–N7 bonds in 2 and
4 promotes nO4Ј→σ*C1Ј-N9 orbital interactions due to the re-
distribution of π-electron density and thus strengthens the
anomeric effect of guanine in both 2Ј-deoxy and ribo nucleo-
tides. The additional stabilization of N-type conformers in Pt2ϩ
complexes of the ribonucleotides is due to the tuning of the
gauche effect of [N9–C1Ј–C2Ј–O2Ј] fragment which is absent in
2Ј-deoxy-ribo counterparts.
The platination of N7 makes N1–H more acidic, and thus N1
deprotonations in 2 and 4 are favoured by ∆pKa of 0.7 and 0.9
units in comparison with parent nucleotides 1 and 3, respect-
ively, which might somewhat facilitate the base-pairing of Pt2ϩ–
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843
2841

View Article Online
Scheme 1 Synthesis of 1, 3, 5 and 6.
phosphorylation of 5-OH by the procedure described above.
reference for 15N NMR spectra, 85% aq. H3PO4 (δ = 0 ppm) as
3,5-Bis(phosphotriesters) of deoxyribofuranose, derivatives 18
and 19, were converted into appropriate bis(phosphodiesters) 5
(86%) and 20 by treatment with aq. ammonia. The tetrahydro-
pyranyl group of 20 was cleaved by 10% aq. acetic acid
(pH > 3)43 to afford compound 6 (57%). All 3,5-bis(phospho-
diesters) were converted into the dirodium salt by passage
through Dowex 50 WX8 resin (Naϩ-form).
external reference for 31P NMR spectra and Na2PtCl6 as
external reference for 195Pt spectra (δ = 0 ppm). The errors in
1
chemical-shift determinations were: 0.001 ppm for H, 0.01
ppm for 13C and 31P, 0.5 ppm for 15N, and 1 ppm for 195Pt.
The sample temperature was varied from 275 K to 358 K in 20
K steps and controlled to approximately 0.5 K. The error in
3JHH was 0.1 Hz as estimated from a comparison of the experi-
mental and simulated spectra.
Preparation of Pt2؉ complexes
Conformational analysis
A 10 mM solution of the activated form of cisplatin, cis-
[Pt(ND3)2(D2O)2]2ϩ, was prepared by suspending cis-[Pt(NH3)2-
Cl2] in D2O (99.9% D) containing two equivalents of AgNO3.
The suspension was stirred vigorously overnight. After removal
of AgCl, the solution was stored in a closed vial at 5 ЊC in the
dark (pH* ≈ 3). Fresh solutions of cis-[Pt(ND3)2(D2O)2]2ϩ
(δPt = Ϫ1613 ppm) were prepared no more than one day before
the experiment. Two molar equivalents of 1, 3, 5 and 6 were
dissolved in a 10 mM solution of cis-[Pt(ND3)2(D2O)2]2ϩ and
the reaction mixture was stirred at 25 ЊC for 20 h to form the
bifunctional complex.
Conformational analysis of the sugar moiety has been per-
formed by the computer program PSEUROT27 with the use of
λ electronegativities44 for the substituents along H–C–C–H
fragments. In the optimization procedure the geometries and
populations of both N- and S-type pseudorotamers were varied
to obtain the best fit between the experimental and the calcu-
lated coupling constants. Our optimization procedure started
with the following input values: PN = 18Њ, ΨmN = 36Њ, PS = 156Њ
and ΨmS = 36Њ. In the case of 1, 3, 5 and 6 the puckering ampli-
tude of the minor conformer was kept frozen during the
individual iterative least-squares optimization, whereas in the
case of 2 and 4 the puckering amplitudes of both conformers
NMR Measurements
N
(Ψm and ΨmS) were constrained while all other parameters
1H, 13C, 31P and 15N NMR spectra were acquired at 600.07,
150.91, 242.91 and 60.82 MHz, respectively, on a Varian Unity
Inova 600 NMR spectrometer at the National NMR Center of
Slovenia. 195Pt spectra were recorded at 64.325 MHz on a Var-
ian Unity Plus 300. Sample concentration was 20 mM in D2O
(99.9% D) at a neutral pH* of 6.8 0.3. The pH* values were
measured with a combined glass electrode calibrated with
standard buffers with pH of 4, 7 and 10 in H2O and were not
corrected for the deuterium isotope effect. 3-(Trimethylsilyl)
were freely optimized. The following ranges of P and Ψm were
found to fit with experimental JHH data within root-mean-
3
square error <0.3 Hz and ∆Jmax <0.5 Hz: Ϫ20Њ < PN < 20Њ,
33Њ < ΨmN < 34Њ, 157Њ < PS < 165Њ, 33Њ < ΨmS < 34Њ for 1,
Ϫ11Њ < PN < Ϫ7Њ, 29Њ < ΨmN < 35Њ, 163Њ < PS < 176Њ, 29Њ <
ΨmS < 35Њ for 2, Ϫ40Њ < PN < 50Њ, 33Њ < ΨmN < 40Њ, 140Њ < PS <
174Њ, 33Њ < ΨmS < 40Њ for 3, 16Њ < PN < 48Њ, 32Њ < ΨmN < 38Њ,
160Њ < PS < 198Њ, 32Њ < ψmS < 38Њ for 4, Ϫ25Њ < PN < Ϫ5Њ,
30Њ < ΨmN < 31Њ, 154Њ < PS < 156Њ, 31Њ < ΨmS < 32Њ for 5,
Ϫ20Њ < PN < Ϫ5Њ, 39Њ < ΨmN < 41Њ, 102Њ < PS < 123Њ, 39Њ <
ΨmS < 41Њ for 6.
1
propionic acid was used as internal standard for H and 13C
NMR (δ = 0 ppm) spectra, MeCN (δ = Ϫ135.8 ppm) as external
2842
J. Chem. Soc., Perkin Trans. 1, 1999, 2835–2843

View Article Online
The interpretation of the experimental 3JC4ЈP, 3JC2ЈP and 3JH3ЈP
coupling constants has been performed with the assumption of
10 B. Song, G. Oswald, J. Zhao, B. Lippert and H. Sigel, Inorg. Chem.,
1998, 37, 4857.
11 H. Sigel, B. Song, G. Oswald and B. Lippert, Chem. Eur. J., 1998, 4,
1053.
a three-state εt,N
εt,S
ε
Ϫ,S conformational equilibrium. The
three-parameter Karplus equations34 for 3JCP = 9.1 cos2Φ Ϫ 1.9
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cosΦ ϩ 0.8 and JHP = 15.3 cos2Φ Ϫ 6.2 cosΦ ϩ 1.5 have been
3
used in the calculation of 3JCP and 3JHP coupling constants from
the ε torsion angles in the hypothetical εt,S, εϪ,S and εt,N con-
formers and their respective populations, which were iterated to
find the best fit with the experimental temperature-dependent
3JC4ЈP, 3JC2ЈP and 3JH3ЈP coupling constants.
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27 C. Haasnoot, F. A. A. M. de Leeuw, D. Huckriede, J. van Wijk and
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Acid–base properties
The pKa-values of 1–4 (Fig. 2A) were calculated from the
sigmoidal curves which were the best iterative least-squares
fits (r.m.s. error < 0.003 ppm) of the pH*-dependent experi-
1
mental H NMR chemical shifts according to the Henderson–
Hesselbach equation (1):
[A]
1 Ϫ α
(1)
pH* = pKa ϩ log
= pKa ϩ log
[AH]
α
We have additionally constructed Hill plots of pH* as a
function of the logarithm of the ratio of the protonated and
deprotonated species. The Pearson’s correlation coefficients (R)
of the linear regression lines in Hill plots are above 0.994
for 1–4. The slopes are close to 1, which is the characteristic
indication for the protonation involving a single protonation
site. The populations of S-type pseudorotamers and ∆GЊ as a
function of pH* (Fig. 2B and 2C) were analyzed in a similar
way.
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Acknowledgements
We thank the Ministry of Science and Technology of Republic
of Slovenia (Grant No. Z1-8609-0104), and the Swedish
Natural Science Research Council (NFR), the Swedish Board
for Technical Development (NUTEK) and the Swedish
Engineering Research Council (TFR) for generous financial
support.
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