Recognition of platinum(II) amine complexes by nucleotides: Role of phosphate and carbonyl groups in ([15N3]diethylenetriamine)-(guanosine 5′-monophosphate)platinum(II)

Article abstract of DOI:10.1039/a606275j

Two-dimensional [¹H,¹⁵N] NMR studies show that in the model monofunctional DNA adduct [Pt([¹⁵N₃]dien)(5′-GMP-N⁷)]²⁺ [5′-GMP = guanosine 5′-monophosphate, dien = 1,5-diamino-3-azapentane (pr

Full text of DOI:10.1039/a606275j

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Recognition of platinum(ii) amine complexes by nucleotides: role of
phosphate and carbonyl groups in ([15N3]diethylenetriamine)-
(guanosine 5A-monophosphate)platinum(ii)
Zijian Guo, Peter J. Sadler*† and Erle Zang
Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London, UK WC1H 0PP
Two-dimensional [1H,15N] NMR studies show that in the
duced to [15N2]monotritylethylenediamine by LiAlH4.
1-([15N]Tritylglycyl)-2-([15N2]trityl)ethylenediamine was ob-
tained as a condensation product from [15N2]monotritylethyl-
enediamine and [15N]tritylglycine. After reduction and cleav-
age of the protecting group, the hydrochloride salt of [15N3]dien
was obtained in 20% overall yield based on [15N]glycine. Full
details of the synthesis will be published elsewhere.
model monofunctional DNA adduct [Pt([15N3]dien)(5A-
GMP-N7)]2+ [5A-GMP
= guanosine 5A-monophosphate,
dien = 1,5-diamino-3-azapentane (prepared by a novel
synthesis from glycine)], stereospecific hydrogen-bonding
interactions involving Pt–NH protons are promoted by
deprotonation of both the 5A-phosphate group and N1H of
coordinated 5A-GMP.
The platinum complex [Pt([15N3]dien)Cl]Cl was prepared
according to the literature method for the unlabelled complex.7
The two-dimensional [1H,15N] HSQC NMR spectrum of a fresh
solution (i.e. prior to hydrolysis) of [Pt([15N3]dien)Cl]+ 1 in
H2O–D2O (9:1) showed three sets of cross-peaks at d
5.23/233.9, 5.02/233.9 and 6.65/8.13 (see Fig. 1). On the basis
of their shifts,8 the former two peaks can be assigned to non-
equivalent protons on the two NH2 groups, and the latter to the
NH group.
A surprising recent finding is that monofunctional adducts of
[PtCl2(NH3)2] (cisplatin) and [Pt(NH3)2(H2O)2]2+ with one of
the guanine bases in GG oligonucleotides can be very long
lived.1,2 It is therefore important to understand the factors which
lead to stabilization of particular adducts between platinum
am(m)ine complexes and DNA. Complexes of [Pt(dien)]2+ with
nucleotides have been widely studied since they represent stable
monofunctional adducts, and the ability of [Pt(dien)]2+ to
introduce local denaturation of DNA has been demonstrated, for
example by Brabec et al. using chemical probes.3
A′
The NH groups on platinum am(m)ine ligands are thought to
play a major role in determining the nature of the adducts via
hydrogen-bonding interactions with DNA,4 and we have shown
previously that these can be investigated in solution using two-
dimensional [1H,15N] NMR and 15N-labelled ligands.5 Use of
this method with dien complexes is complicated by the lack of
an efficient synthesis of dien which can be adapted to 15N
labelling. We report here briefly a novel efficient synthesis of
[15N3]dien, and show that use of this allows an NMR study of
hydrogen-bonding interactions in nucleotide complexes such as
[Pt([15N3]dien)(5A-GMP-N7)]2+. The NMR data implicate both
the 5A-phosphate and C6 carbonyl groups in pH-dependent
stereospecific interactions with the Pt–NH2 groups.
A
c
d
a
b
e
B
–30
–10
10
15N-Labelled dien cannot readily be prepared by the Gabriel
method6 since [15N]diethanolamine is required as a starting
material. The preparation of this from ethylene oxide and excess
15NH3 is uneconomic since the yield is low, the product cannot
be readily separated from ethanolamine and triethanolamine,
and the intermediate 2,2A-dichlorodiethylamine is unstable and
difficult to handle. Therefore we have developed the new
method shown in Scheme 1, which uses [15N]glycine as starting
material. The trityl group was used to protect the amino group
throughout the synthesis. Amidation of [15N]tritylglycine with
15NH3 afforded [15N2]tritylglycinamide, which was then re-
He
Hb
Ha
N
N
N
Pt
N7
Hc
Hd
7
5
6
O
O
1
δ( H)
i–iii
iv
TrN*H
v, vi
Fig.
1
Reaction of [Pt([15N3]dien)Cl]+
1 with 5A-GMP. This two-
OH
N*H2
N*H
N*HTr
N*H2
TrN*H
dimensional [1H,15N] HSQC NMR spectrum was recorded 1.2 h after
mixing and shows NH2 and NH peaks for complex 1 labelled A, AA and B,
respectively, and for the GMP adduct 2 labelled a–e (all five NH protons are
non-equivalent). After 12 h the reaction was complete and only peaks for 2
were observable. The large downfield shift of peak a is notable. 195Pt
satellites in both the 1H and 15N dimensions are evident for complex 1 but
not for 2 (satellites broaden with increases in molecular size and chemical
shift anisotropy). Conditions: mol ratio Pt:GMP 0.8:1 (5 mm), H2O–D2O
(9:1), pH 6.83, 298 K.
• 3HCl
N*H
N*H2
N*H2
Scheme 1 Reagents and conditions: i, TrCl, Tr = Trityl, PriOH–H2O, room
temp.; ii, ethyl chloroformate, 15NH3, CHCl3, room temp.; iii, LiAlH4, thf,
room temp.; iv, ethyl chloroformate, 15N-tritylglycine, CHCl3, room temp.;
v, LiAlH4, thf, 75 °C; vi, 4.8 m HCl, reflux
Chem. Commun., 1997
27

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As an initial model for DNA binding, we studied the adduct
formed from the reaction of 5A-GMP with 1 (5 mm, 1:1
mol ratio) at pH 6.8, 298 K. The two-dimensional spectrum after
1.2 h of reaction is shown in Fig. 1, where peaks assignable to
the starting material 1 and to the product [Pt([15N3]dien)(GMP-
N7)]2+ 2 are well resolved. Most notable is the presence of five
cross-peaks for 2 showing that all the NH protons are
magnetically non-equivalent. This might be expected in view of
the chirality of the bound nucleotide. In order to understand the
origin of the low-field shifts of the peaks for 2, their pH
dependence was studied over the range pH 3–11. There was
little change in the 1H,15N shift of peak e assigned to the central
NH group over the range pH 3–9.5, but above this pH it
broadened and disappeared, probably due to an increased
exchange rate. The behaviour of the NH2 peaks a, b, c and d is
shown in Fig. 2. All four peaks show two-step titrations with
pKa values of 5.58 and 8.54 (averages for peaks a and c). The
shift changes for peaks a and c are much larger than those for
peaks b and d.
The two pKa values can be associated with deprotonation of
the 5A-phosphate (5.58) and the N1H group (8.54) of coordinated
5A-GMP in 2. For comparison, the phosphate pKa value for
[Pt(en)(5A-GMP)2]2+ is 0.38 units lower than the value for free
5A-GMP (6.20) indicative of the presence of Pt–NH···5A-
phosphate hydrogen bonding.5 There are previous reports that
the pKa of N1H of guanine (ca. 9.5) is lowered by ca. 0.9–1.3
when guanine derivatives are coordinated to PtII via N7.9 The
large low-field PtNH2 1H NMR shifts observed for complex 2
(peaks a and c) on deprotonation of the N1H group of bound
GMP are intriguing since these PtNH2 protons appear to be far
away from N1. This can be explained by an increase in the
strength of hydrogen bonding between PtNH2 and the C6
carbonyl group due to an increase in electron density at C6O on
deprotonation of N1. It is assumed that there is free rotation
about Pt–N7 and therefore both NH2 groups are involved in
hydrogen bonding. Keto–enol tautomerism could increase the
negative charge on C6O, as illustrated in the structure presented
for complex 2. Such hydrogen bonding involving C6O has been
found to play a role in determining the structure of Pt am(m)ine
nucleotide complexes in the crystalline state, occurring for
example in [Pt(dien){AGA-N7(2)}],10 but evidence for its
presence in solution has previously proved difficult to obtain.
The present data appear to provide evidence for such inter-
actions at pH values close to those of biological relevance.
Related NH···carbonyl interactions have been reported to play a
major role in the stabilization of ternary complexes of zinc(ii)
cyclen complexes with thymine derivatives.11
The methods described here should also readily allow studies
of chelate ring-opening reactions of [Pt([15N3]dien)]2+ com-
plexes. These have been the subject of much recent interest.12 It
is notable from our preliminary work that complex 2 showed no
evidence for ring opening at pH 3.
We thank the Association for International Cancer Research,
Biomolecular Sciences programme of the EPSRC and BBSRC,
and EC HCM (ERBCHRXCT920016) and COST (D1-
92/0002) programmes for their support for this work, and the
MRC and ULIRS for the provision of NMR facilities.
H
H
N
H
N
O–
Pt
Footnote
H
N
H
N
† Present address: Department of Chemistry, The University of Edinburgh,
King’s Buildings, West Mains Road, Edinburgh, UK EH9 3JJ.
N
6
7
H
2–O3PO
N
H
5′
O
N
NH2
1′
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5.6
5.2
2
4
6
8
10
12
pH
Fig. 2 Dependence of the NH 1H NMR chemical shifts of complex 2
[Pt([15N3]dien)(5A-GMP-N7)]2+ on pH; peak a (5), b (-) c (.), d (3). The
large downfield shifts of two of the resonances between pH 5 and 7 can be
associated with deprotonation of the 5A-phosphate group, and between 7 and
9 with deprotonation of N1H of GMP. Little shift of peak e (Fig. 1) was
observed over this pH range.
12 See for example, M. Mikola, J. Vihanto and J. Arpalahti, J. Chem. Soc.,
Chem. Commun., 1995, 1759.
Received, 11th September 1996; Com. 6/06275J
28
Chem. Commun., 1997