A purine-like nickel(II) base pair for DNA

Article abstract of DOI:10.1002/anie.200462047

Metal in the middle: A metallo base pair incorporating 6-pyridylpurine (Pur^p) was synthesized and characterized. Pur^p binds nickel (II) preferentially over other divalent ions and yields a base pair that is more stable than natural W

Full text of DOI:10.1002/anie.200462047

Angewandte
Chemie
Nucleobases
A Purine-like Nickel(ii) Base Pair for DNA**
Christopher Switzer,* Surajit Sinha, Paul H. Kim, and
Benjamin D. Heuberger
Nucleic acids rely on complementary functionalized purine
and pyrimidine heterocycles to encode genetic information as
G·C and A·T(U) base pairs. Several approaches have been
developed to expand the number of available base pairs
beyond the two natural pairs, including the use of non-
standard hydrogen-bond donor/acceptor patterns,[1] van der
Waals and hydrophobic interactions,[2] and metal coordina-
tion.[3] Herein we report the realization of a naturally inspired
metallo base pair with a purine core whose design derives
from minimal modification of adenine. The resulting base pair
(Purp·Ni·Purp, Figure 1) is found to have the following novel
features and properties: 1) greater stability than a G·C base
pair, 2) a surprising dimensional resemblance to natural
purine–pyrimidine base pairs, and 3) the potential to serve
in an ion-activated switch.
Figure 1. Left: The 6-(2’-pyridyl)-purine (Purp) metallo base pair.
Right: A representation of a hypothetical helix composed of purely
PurP·Ni2+·PurP base pairs.
Formally, PurP is derived from adenine by replacing the 6-
amino group with a pyridyl group. This functional-group
interchange places two Lewis basic donor atoms (the purine
N1 and pyridine N1’ atoms) in an optimal 1,4-relationship for
coordinating metal ions. Scheme 1 summarizes the synthesis
of PurP. The key transformation involved a modified Negishi
coupling of pyridyl zinc bromide with chloropurine deoxy-
riboside 1[4] to provide pyridylpurine deoxyriboside 2. We
[*] Prof. C. Switzer, S. Sinha, P. H. Kim, B. D. Heuberger
Department of Chemistry
University of California
Riverside, CA 92521 (USA)
Fax: (+1)951-787-2435
E-mail: christopher.switzer@ucr.edu
[**] This work was supported by DOD/DARPA/DMEA under award
no. DMEA90-02-2-0216 and the NASA exobiology program under
award no. NAG5-9812.
Angew. Chem. Int. Ed. 2005, 44, 1529 –1532
DOI: 10.1002/anie.200462047
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Table 1: DNA-duplex melting temperatures, Tm, in the presence and
absence of divalent ions.[a]
5’-d-CTTTCTXTCCCT
3’-d-GAAAGAYAGGGA
Entry
X·Y
Duplex
Metal
Tm
D[b]
+6.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
PurP·PurP
T·PurP
4/5
4/5
4/5
4/5
4/5
4/5
4/5
4/5
4/5
4/5
4/5
6/5
7/5
8/5
9/5
NiCl2
Ni(NO3)2
CoCl2
46.1
46.6
38.8
31.4
30.8
30.5
28.8
29.2
29.1
27.3
28.5
27.1
26.6
27.0
29.1
36.8
37.4
36.7
40.2
40.1
39.9
+6.5
À1.3
CuCl2
À8.7
ZnSO4
AgNO3
FeSO4
À9.3
À9.6
À11.3
À10.9
À11.0
À12.8
À11.6
À13.0
À13.5
À13.1
À11.0
À3.3
MnCl2
Eu(NO3)3
Pd(NO3)2
[c]
–
NiCl2
NiCl2
NiCl2
NiCl2
C·PurP
A·PurP
G·PurP
T·A
[c]
6/10
6/10
6/10
7/11
7/11
7/11
–
[d]
T·A
T·A
C·G
C·G
NiCl2
À2.7
[d]
CoCl2
À3.4
Scheme 1. Synthesis of 2’-deoxyribosyl-N9-[6-(2’-pyridyl)-purine]
[c]
–
+0.1
0.0
phosphoramidite 3. Tol=4-toluoyl, THF=tetrahydrofuran, DMT=4,4’-
dimethoxytriphenylmethyl, Pyr=pyridine, OCE=cyanoethyl,
DIPEA=N,N-diisopropylethylamine.
[d]
NiCl2
[d]
C·G
CoCl2
À0.2
[a] Samples contained 2.5 mm of each DNA strand, 5 mm divalent ion
where indicated, 50 mm NaCl, and 10 mm NaH2PO4 (pH 7.0). All
measurements were performed at least in triplicate. [b] Difference in Tm
value relative to that of duplex 7/11 (X·Y=C·G) in the presence of NiCl2
(entry 20). [c] No divalent metal ions were added. [d] Divalent ion was
added in these cases as a control.
initially used the conditions reported for the preparation of
ribosyl and acyclic analogues of 2[5] but found that an
improved yield was possible by switching to
a
was
[PdCl2(PPh3)2]catalyst.[6] Pyridylpurine nucleoside
2
transformed in three steps into phosphoramidite 3. DNA
containing PurP was prepared by using 3 and standard
phosphite triester methodology on an ABI394 synthesizer.
Complementary dodecamer DNA strands were prepared,
each bearing a single PurP residue: 5’-d-CTTTCTPurpTCCCT
(4) and 5’-d-AGGGAPurPAGAAAG (5). These oligomers
were purified by PAGE, and their identities were confirmed
by MALDI mass spectrometry.
To assess the viability of PurP as the organic component of
a metallo base pair, UV-monitored thermal denaturation of
complementary dodecamers 4 and 5 bearing single PurP
residues was performed in the presence of the divalent ions
noted in Table 1. Representative denaturation profiles are
shown in Figure 2. In all, nine divalent metal ions were
screened for their abilities to coordinate to the PurP·PurP site
contained in the 4/5 duplex by measuring the Tm value; the Tm
value of a metal-free control with the same duplex (Table 1,
entry 11) was also recorded. As is apparent from the table,
only four of the seven divalent ions gave Tm values for 4/5 that
differed significantly from the metal-free control: Ni2+, Co2+,
Cu2+, Zn2+, and Ag+. Of these five metals, Ni2+ is the most
stabilizing. PurP·Co2+·PurP leads to a duplex Tm value roughly
in between those of the 6/10 and 7/11 duplexes bearing T·A
and C·G pairs, respectively. More significantly, the PurP·Ni2+·
PurP base pair is more stabilizing to a double helix than a C·G
base pair by a margin of 68C under the conditions reported in
Table 1, with 5 mm NiCl2. Importantly, the PurP·PurP 4/5
duplex is highly destabilized in the absence of Ni2+ or other
Figure 2. Absorbance versus temperature denaturation profiles.
Conditions are as reported in Table 1.
divalent ions (Table 1, entry 11): there is a À17.68C difference
in Tm value between the PurP·Ni2+·PurP (Tm = 46.18C) and
PurP·PurP (Tm = 28.58C) base pairs (Table 1, entry 1 versus
entry 11). As a control, both the T·A 6/10 and the C·G 7/11
duplexes were denatured in the presence and absence of Ni2+
or Co2+, and no significant changes were observed in the Tm
values (Table 1, entries 16–21).
The stability of mismatches between the four natural
bases and PurP was investigated in the presence of Ni2+
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(Table 1, entries 12–15). Clearly, none of the natural bases
make stable pairs with PurP·Ni2+. Indeed, the DTm values of
the mismatched pairs relative to the PurP·Ni2+·PurP base pair
(not the D values listed in Table 1 which are relative to the
C·G pair) range from À17 to À19.58C. By contrast, T·G and
C·A mismatches of the parent natural duplex under the same
conditions show DTm values of À7.4 and À18.58C under the
same conditions.[7] Thus, all four PurP·Ni2+ mismatches with
natural bases are much less stable than the PurP·Ni2+·PurP
match. A further point to note is that the instabilities seen for
all four PurP·Ni2+ mismatches with natural bases rival the
most severe natural nucleobase mismatches such as C·A.
Within the double helix, three possible geometries could
be envisioned for divalent metal ion complexation by PurP2 :
square planar, tetrahedral, and Dd2 (a geometry intermediate
between square planar and tetrahedral). In the absence of a
geometric preference by the metal ion, the most productive
geometry for a metallo base pair in forming the double helix is
expected to be square planar because this will maximize
favorable nearest-neighbor stacking interactions. Low-energy
square-planar geometries should be accessible for Ni2+, Co2+,
Cu2+, Ag+, and Pd2+ ions. The first two of these five ions
appreciably stabilize the PurP2-bearing helix, whereas the
latter three do not. As a result, it may be concluded that
geometry alone is an insufficient predictor of metal-ion
affinity for PurP. A circular dichroism spectrum of duplex 4/5
in the presence of Ni2+ is consistent with a B-DNA structure.
To assess the viability of a square-planar geometry for
PurP·Ni2+·PurP, an ab initio geometry optimization was per-
formed on the complex by using Gaussian98[8] at the B3LYP/
6-31G*(CHN)/SDD(Ni) level of theory. Figure 3 (left panel)
shows the square-planar geometry found to be a (local)
minimum on the energy surface. Interestingly, this structure
bears an N9–N9’ (purine numbering) PurP–PurP, distance of
9.54 ꢀ, which nearly replicates the N9–N1 purine–pyrimidine
distance of 9.05 ꢀ that occurs in natural B-DNA helices for
both G·C and A·T base pairs. This suggests PurP·Ni2+·PurP is a
good dimensional mimic of natural base pairs despite the fact
that the metallo base pair incorporates two purine-like
bases towards the major groove, with a corresponding short-
ening of the distance between interstrand N9 atoms.
The PurP·Ni2+·PurP structure in Figure 3 results from
head-to-head dimerization (N1,N1’-PurP·Ni2+·N1,N1’-PurP).
An alternative head-to-tail dimerization mode of PurP
(N1,N1’-PurP·Ni2+·N7,N1’-PurP) was also investigated compu-
tationally. The geometry of this latter complex was found to
be highly nonplanar due to ligand encroachment resulting
from the 1,5-relationship of the nitrogen atoms (N7,N1’)
presented by the “tail”-oriented PurP. (In contrast, the
opposing “head”-oriented PurP bears the optimal 1,4-rela-
tionship of nitrogen atoms, as found in bipyridine.) Therefore,
the head-to-tail dimer is predicted to be less compatible with a
helix than the head-to-head dimer.
Metallo base pairs could become functional elements of
oligonucleotides that activate or suppress enzymatic activity
(for example, transcription or translation) in the presence or
absence of a metal ion. For a proof of principle, we have
incorporated three consecutive PurP residues into a helix to
attain “on” and “off” states (in the presence and absence of
Ni2+, respectively) that are sufficiently insulated from one
another to be effectively binary (0 or 1). The following
tetradecamer DNA strands were prepared: 5’-d-
CTTTCTPurpPurpPurpTCCCT (12) and 5’-d-AGGGAPurp-
PurpPurPAGAAAG (13). Gratifyingly, the Tm values for the
12/13 duplex under the conditions reported in Table 1 were
64.38C in the presence of 10.0 mm NiC12 (1.3 equiv per Purp
residue) and 20.68C in the absence of Ni2+. Thus, the Tm
values between the “on” (Ni2+ present) and “off” (Ni2+
absent) states of this system are separated by 43.78C, a
difference sufficient to produce binary behavior at 378C (that
is, at this temperature, in the presence of Ni2+ the helix is
present and in the absence of Ni2+ the helix is absent).
In summary, PurP leads to a metallo base pair with Ni2+
selectivity that is more stable than natural G·C and A·T base
pairs. Additionally, PurP·Ni2+·PurP is orthogonal in its pairing
properties relative to the four genomic nucleobases: all
mismatches are highly destabilizing to the helix in comparison
to the parent metallo base pair. Finally, PurP·Ni2+·PurP
appears to resemble natural base pairs dimensionally and
has the potential to serve as the functional component of an
ion-activated switch. Given the resemblance between natural
purines and PurP it is possible that enzymes, including those
components.
Superposition
of
ab initio
optimized
PurP·Ni2+·PurP and A·T base-pair structures (Figure 3, right
panel) further supports this idea and shows that PurP
coordination of Ni2+ is attended by rotation of the PurP
Figure 3. Left: Stereoview of the structure of PurP·Ni2+·PurP with optimized geometry obtained with Gaussian98.[8] Right: Superposition of opti-
mized PurP·Ni2+·PurP and A·T structures. The superposition was guided by PurP N9/A N9, PurP N9/T N1, and the hydrogen atoms attached to
these nitrogen atoms.
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involved in DNA replication, might recognize the PurP·Ni2+·
PurP base pair. We are actively pursuing this possibility.
Received: September 21, 2004
Published online: January 28, 2005
Keywords: DNA · metallo base pairs · molecular switches ·
.
nickel · nucleobases
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