Chemistry Reference
In-Depth Information
d(CGCG
TT
GTCC)
ยท
d(GGAC
TT
CGCG).
81
For this DNA duplex, the sequences
were designed specifi cally for the structure determination of the T-Hg
II
- T base pair.
On the addition of Hg
II
, two successive T - Hg
II
-T base pairs are formed in the middle
of the duplex. It should also be mentioned that the sequence of the DNA duplex
used in this experiment was made nonself-complementary, in order to avoid the loss
of structural information. This is because if duplexes with self-complementary
sequences are used, NOEs between symmetry-related protons around the dyad axis
of the duplexes will be lost due to the averaging of NMR signals. By contrast, with
the above nonself-complementary sequences, NOE cross peaks were not lost, and
unambiguous resonance assignments based on NOEs were able to be achieved.
82
During Hg
II
titration experiments on the duplex, imino proton signals from
Hg
II
- free duplex and Hg
II
- duplex (1 : 1) complexes (transient complexes) were inde-
pendently observed.
81
It was found that association/dissociation processes are slow
relative to the NMR timescale. Therefore, the disappearance of imino proton signals
upon the addition of Hg
II
supports the formation of the T-Hg
II
-T base pair, as was
the case in the studies by Kuklenyik and Marzilli.
80
For nonexchangeable protons,
such as the methyl protons of thymidine [CH
3
(T)], NMR signals from the Hg
II
- free
duplex, Hg
II
- duplex (1 : 1) complexes and the Hg
II
- duplex (2 : 1) complex were inde-
pendently observed.
82
More interestingly,
15
N NMR spectra of the Hg
II
complex of the same sequence
was reported in 2007 (Table 16.2).
83
In that study,
15
N -
15
N
J
- coupling across Hg
II
(
2
J
NN
) was observed within the T-Hg
II
-T base pairs in the duplex, providing direct
evidence of the formation of an
15
N - Hg
II
-
15
N covalent linkage (Scheme 16.4, Figure
16.4 and Table 16.3 ).
83
At this stage, the chemical structure of the T-Hg
II
- T base pair
was defi nitely determined. Interestingly, a theoretical value of
2
J
NN
for the T - Hg
II
- T
base pair was also reported by Bagno and Saielli in 2007; this showed a similar value
to the experimental data (Table 16.3).
84
For reference data, related metal-mediated
15
N -
15
N
J
- coupling values
85,86
and trans - hydrogen bond
15
N -
15
N
J
- coupling values
87 - 89
are also listed in Table 16.3 .
To evaluate chemical shift perturbations upon Hg
II
binding, chemical shifts of
Hg
II
-free thymidine residues were determined using
1
H -
15
N HSQC spectra (Figure
16.5 ).
83
From the spectra, N3 resonances in the T-T mismatches (Hg
II
- free form)
were assigned, and the chemical shift differences between Hg
II
- free and Hg
II
- bound
forms were determined (Table 16.2). It was found that approximately 30 ppm lower-
fi eld shifts were observed for the N3 resonances of thymidines upon Hg
II
binding
(Table 16.2). This large chemical shift change is discussed later.
O
O
Hg
II
15
N
15
N
N
N
O
O
R
R
2
J
NN
Scheme 16.4
