(5′R)-5′,8-Cyclo-2′-deoxyadenosine: NMR and DFT study of its influence on TPOcdA structure

Article abstract of DOI:10.1016/j.tetasy.2008.10.025

Oxidatively generated damage to DNA frequently appears in the human genome as an effect of aerobic metabolism or as the result of exposure to exogenous oxidizing agents, such as ionizing radiation and solar light, a product of radiation. 5′,8-Purine cyclo

Full text of DOI:10.1016/j.tetasy.2008.10.025

Tetrahedron: Asymmetry 19 (2008) 2390–2395
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
(50R)-50,8-Cyclo-20-deoxyadenosine: NMR and DFT study of its influence
on TPOcdA structure
*
Bolesław T. Karwowski
´
´
Department of Biopharmacy, Medical University of Lodz, Muszynskiego Street 1, 90-151, Łodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxidatively generated damage to DNA frequently appears in the human genome as an effect of aerobic
metabolism or as the result of exposure to exogenous oxidizing agents, such as ionizing radiation and
solar light, a product of radiation. 50,8-Purine cyclonucleosides constitute an important class of oxida-
tively generated tandem lesions. The present study deals with the synthesis of the (50R)-diastereomer
of 50,8-cyclo-20-deoxyadenosine (cdA) containing TPOcdA, in an attempt to delineate the conformational
changes induced in the DNA fragments by the presence of this lesion. For this purpose, extensive 1D and
2D NMR measurements were performed and completed by theoretical calculations. It was found that the
covalent bond between C(50) and C(8) in the (50R)-50,8-cyclo-20-deoxyadenosine induces an unusual West
(0T1) conformation of the furanose ring. It is similar to the opposite (50S)-diastereoisomer; however, the
three-dimensional structure of the dinucleoside investigated has shown an analogy to natural d(TPOA).
Thus, it can be postulated that the rigid structure of (50R)-50,8-cyclo-20-deoxyadenosine would perturb
the global geometry of oligonucleotides at the site of the modification less strongly than (50S) and there-
fore be less toxic for cells. In this article, the influence of the (50R)-diastereomer of 50,8-cyclo-20-deoxya-
denosine on the dinucleotide structure will be presented for the first time.
Received 1 September 2008
Accepted 23 October 2008
Available online 19 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
taneous damage of the sugar residue and of the base (tandem le-
sions)—for example, 50,8-cyclo-20-deoxyadenosine (cdA)—are not
Nucleotides and nucleosides in the natural environment are
constantly exposed to different kinds of radicals.1 Among them
the major portions are hydroxyl radicals formed in cells as a result
eliminated by the BER (base excision repair) system, but by the
complex NER (nucleotide excision repair) system.4 Experimental
data show that (R)- and (S)-diastereomers of cdA are formed in a
2:1 ratio for free nucleoside and 1:1 in the case of oligonucleo-
tides.5 Moreover, depending on the C50 atom configuration in cdA
as (R) or (S) (Fig. 1), they are characterized with a different biolog-
ical impact.6 For example, (50S)-50,8-cyclo-20-deoxyadenosine com-
pletely blocks the progression of polymerases involved in
of water c
-radiolysis, or during anaerobic cellular processes.2 The
reactions of a radical with oligonucleotides and nucleosides may
lead to nucleic base modifications or other DNA lesions, including
strand breaks. The majority of them can be removed during the
BER repair process.3 However, modifications which involved simul-
NH2
NH2
NH2
N
N
N
N
N
N
N
N
HO
H
*
*
N
N
N
φ4
φ0
φ1
N
O
O
O
H
HO
HO
φ3
HO
φ2
HO
HO
(R)-isomer
(S)-isomer
dA
Figure 1. The (50R)- and (50S)-diastereomers of 50,8-cyclo-20-deoxyadenosine with indication of torsion angles in the furanose ring by symbols /0, /1, /2, /3, /4.
* Tel.: +48 42 677 91 21; fax: +48 42 677 91 20.
E-mail address: Bolek.Karwowski@wp.pl
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.10.025

B. T. Karwowski / Tetrahedron: Asymmetry 19 (2008) 2390–2395
2391
CH3
O
O
NH2
HN
HO
O
N
NH
N
N
N
N
N
N
NH2
HO
H
HO
H
O
O
O
O
N
O
N
N
N
O
N
P
-O
HO
O
N
O
H
Si
HO
2
3
1
Figure 2. Structure of (50R)cdA 1, (50R)cdANTBBDzMSi 2, TPO(50R)cdA 3.
oligonucleotide chain extension, whereas the (50R)-diastereomer
has a less pronounced effect.7 Moreover, the (50R)-form was found
to be more efficiently removed from cdA containing oligonucleo-
tides than the (50S)-diastereomer via an NER mechanism.
The present study is aimed at delineating the conformational
features of the (50R)-diastereomer of either free cdA 1, the O-30
and N-6 cdA-substituted derivative N6-benzoyl-30-O-(tert-butyldi-
methylsilyl)-50,8-cyclo-50-oxo-20-eoxyadenosine ðcdATNBBDzMSiÞ 2, or
when inserted into a dinucleotide, namely TPOcdA 3 (Fig. 2). This
was achieved by both extensive NMR studies in aqueous solutions
and theoretical DFT (density functional theory) calculations.
Table 1
Value of torsion angles and pseudorotation parameters in the sugar moiety (DFT
**
method B3LYP/6-31G
Angle
)
Torsion angle value (°)
(50R) cdATNBBDzMSi
cdA
(50R)
TPO(50R)cdA
(50S)a
T
cdA
0
0
O
C
O
C
4 –C1 –N1–C2
v
ꢀ123.9
ꢀ20.7
35.4
0
0
0
0
0
4 –O4 –C1 –C2
/
48.1
48.9
48.7
ꢀ36.5
11.0
48.0
ꢀ31.6
4.8
0
0
0
0
4 –C1 –C2 –C3
0
/
/
/
/
s
ꢀ36.0
ꢀ36.5
1
2
3
4
0
0
0
1 –C2 –C3 –C4
10.8
11.0
ꢀ35.9
0
0
0
0
0
C2 –C3 –C4 –O4
16.9
17.1
16.9
25.0
22.9
0
0
0
C
C
O
3 –C4 –O4 –C1
ꢀ40.7
ꢀ41.5
ꢀ41.0
ꢀ2.8
ꢀ44.5
0
0
0
3 –C4 –C5 –C8
68.3
67.4
67.4
70.5
0
0
4 –C1 –N9–C4
v
ꢀ149.6
ꢀ153.3
ꢀ153.0
ꢀ150
2. Results and discussion
Pseudorotation parameters
NBz
TBDMSi
2.1. Synthesis of cdA
, cdA and cdA containing dinucleotide
Phase
P
283.2
47.4
0T1
283.2
47.8
0T1
283.3
47.8
0T1
165.6
37.0
2T3
275.6
49.2
0T1
Amplitude
Puckering
/
max
NBz
(50R)-50,8-Cyclo-20-deoxyadenosine 1, cdA
2 and their
TBDMSi
Data taken directly from the literature.13
a
derivatives for short oligonucleotides synthesis were obtained by
the method described by Cadet.8 The TPOcdA 3 was synthesized
via solid-phase synthesis on the 1 lmol scale using a classical
phosphoamidite strategy9 with a slight modification as reported
by Brooks.10 The synthetic products were purified by a one-step
angel is almost the same for 50S and 50R forms. Another point to be
noted from the DFT calculations is that the 50-furanose ring from
thymidine adopts the (2T3) S form in 3. This observation is in oppo-
sition to the data obtained for TPO(50S)cdA, in which sugar moiety
of thymidine adopts (3T4) N conformation13 (Table 1). It should
be mentioned that this change originates from the fact that the cal-
culations were carried out for the gas phase.
purification method using RP-HPLC DMTOFF level
.
NBz
TBDMSi
2.2. Molecular structure of cdA, cdA
studies
, TPOcdA from DFT
It should be noted that X-ray data are only available for both the
(50R)- and (50S)-diastereoisomers of the ribofuranose-derivatives of
cA (50,8-cyclo adenosine).11 Therefore, quantum mechanics was
chosen for the DFT calculations12 with the goal of cross-checking
the experimental data obtained from NMR. It has been found that
the conformation of the fixed sugar ring is independent of the pres-
ence of substituents. The torsion angles and the distances in cdA
are similar to those formed in cA. Moreover, I have not observed
any changes in the (50R)-50,8-cyclo-20-deoxyadenosine structure,
NBz
TBDMSi
2.3. cdA, dA
and TPOcdA structure determination by NMR
spectroscopy
2.3.1. Conformational features of 2-deoxyribose moiety
In the following step, the quantum calculations have been com-
pleted using the data obtained from NMR investigations of the
molecular structures of 1, 2 and 3. In particular, for the determina-
tion of the influence of the (50R)cdA on the global geometry and
flexibility of the modified oligonucleotide, 1H NMR investigations
have been performed. NMR signals collected at 29 °C were used
for the determination of the chemical shifts via straightforward
2D COSY14 NMR experiments. The set of data, which was obtained,
constituted as the starting point for further investigations (Table
2). Following a previous study,13 it was expected that changes
should be observed in the sugar moiety of the nucleoside adjacent
to the (50R)cdA. The conformation of the furanose ring of thymidine
should not correspond to the (S)-form. Moreover, the introduction
of the substituents at the O30 and N6 positions of the cdA moiety
should facilitate the formation of other more or less probable con-
independent of whether it has been modelled as the cdA monomer,
NBz
TBDMSi
cdA
, or as conjugates such as TPOcdA. Surprisingly, the crystal-
lographic structural data11 of cA were consistent with data inferred
from the quantum chemical calculations for all structures studied
that exhibit the 0T1 conformation for the sugar moiety. The homol-
ogy of the conformation of the furanose ring forced the position of
adenine in relation to the sugar unit. Therefore, the values of the
glycosidic dihedral angle (O40–C10–N9–C4) between both 50R and
50S diasrereoizomers differ only by 4.1°. Additionally, this differ-
ence has been determined by the position of the 50OH group and
moreover, the value of the newly created C30–C40–C50–C8 dihedral

2392
B. T. Karwowski / Tetrahedron: Asymmetry 19 (2008) 2390–2395
Table 2
that in 3 the population of g+, t and g– conformers was 55%, 28%
and 17%, respectively. This means that in the investigated struc-
tures, the S-type of the sugar pucker is most abundant for the nor-
mal nucleosides on the 50 end. Moreover, these results are in
agreement with the estimation of the conformers. This was
Chemical shifts [ppm] of sugar and base protons from 300 MHz 1H NMR spectra at
29 °C in D2O
Proton
(50R)cdAa
(50R)cdATNBBDzMSi
TPO(50R)cdA
TPO
cdA
3
achieved regarding the values of JH–H measured by 1H NMR and
10
20
200
30
40
50
500
2
6.54
2.56
2.21
4.45
4.75
4.89
6.62
2.53
2.31
4.34
4.62
4.97
6.24
2.31
2.60
6.64
2.60
2.25
4.52
4.98
5.37
using the following equation:
XS = 100(J1 2 /9.3); Keq = S/N = J1 2 /J3 4 (for (50R)cdA XS = 67.7 and
0
0
0
0
0
0
4.79b
4.16
3.76
3.71
Keq = 1.9).
Finally, 1H–31P coupling constants were assigned with the aim
of determining the dihedral angles in C40–C30–O30–P and P–O50–
C50–C40 using the suitable Karplus equation16 parameterized by
Mooren.17,18 Unfortunately, due to the overlap of the signal from
8.12
8.76
8.23
5
6
1.85
7.61
HDO and 30H from the thymidine unit, only the constant coupling
3
JH5 –P for the PꢀO50–C50–C40 dihedral angle was measured, and
Proton chemical shifts of (50R)cdATNBBDzMSi obtained in CDCl3.
Obscured by signal from HDO.
a
0
b
this corresponded to 160°.
2.3.3. Determination of the sugar moiety conformation by 2D
NOESY experiments
formations. In fact, no significant changes in the vicinal proton cou-
pling constants were observed for the cdA units of 1, 2 and 3, the
largest variation being 0.9 Hz for H30–H20 (Table 3), which corre-
sponds to a change in the dihedral angle by ca. 3.7°. Moreover,
the chemical shifts of sugar moiety protons of cdA exhibit almost
the same value independent of the NMR solvent. The largest differ-
ences of the chemical shifts, for protons in cdA, were observed for
N2 and C50 protons and amount to 0.64 and 0.48 ppm, respectively.
This difference is the result of changes in the electron-withdrawing
effect of the substituents in the nearest environment. Emphasis
should be placed on the fact that 50 adjacent to the nucleoside
adopts a 1T2 conformation, which is characteristic of B-DNA. This
is contrary to the data (DFT study) previously obtained for the
(50S)-diastereomer of cdA, and was additionally confirmed by the
presented theoretical study.
In order to elucidate these results further and to verify the pro-
posed working hypothesis, attempts were made to determine the
distances between the sugar protons by 2D NOESY19 experiments,
at 29 °C (Table 4). From these data and with the help of the molec-
ular mechanistic calculations,20 a three-dimensional structure of
compound 3 can be obtained by providing the previously deter-
mined distances.
Table 4
Selected distances of sugar protons calculated by DFT B3LYP 6-31G** level and from
2D NOESY NMR experiment
System
Inter-molecular distances (Å) between inter-ring protons
(50R) cdANTBBDzMSi (50R)cdA TPO(50R)cdA
T
cdA
DFT
NMR
DFT
NMR
DFT
NMR
DFT
NMR
10–20
10–200
20–30
200–30
30–40
30–50
40–50
2.8
2.5
2.4
3.0
2.9
2.4
2.6
2.7
2.4
2.3
2.9
2.7
2.4
2.5
2.8
2.5
2.4
3.0
2.8
2.6
2.6
2.9
2.5
2.5
2.8
2.9
2.4
2.7
3.1
2.4
2.4
2.7
2.8
3.0
2.4
2.3
2.8
3.0
2.8
2.5
2.4
3.0
2.8
2.5
2.6
2.7
2.4
2.3
3.0
2.8
2.5
2.6
Table 3
3
Vicinal coupling constants JH–H (Hz) obtained at 29 °C for the sugar moieties of
investigated compounds
NBz
TBDMSi
(50R)cdA
(50 R)cdA
TPO(50R)cdA
cdA
<0.2
TPO
6.3
7.0
2.4
2.4
10–20
10–200
20–200
30–20
30–200
30–40
40–50
40–500
50–500
<0.2
4.9
<0.2
4.6
4.7
ꢀ13.7
7.9
The conformation of the five-membered ring is described using
the pseudorotation concept developed by Kilpatrick. Thus, the rela-
tionship between the five torsion angles (Fig. 1) determined the
phase (P) and the amplitude of the sugar unit21 (Table 5). It has
been shown that the sugar in (50R)cdA adopts an unusual W-west
conformation, 0T1. Moreover, it can be inferred when considering
ꢀ13.9
ꢀ13.6
ꢀ15.24
7.5
7.0
6.7
4.5
<0.2
<0.2
4.6
<0.2
<0.2
3.2
4.4
3.4
3.6
<0.2
<0.2
4.5
ꢀ12.6
3
the JH–H data obtained for the investigated derivatives that the
conformation of the rigid (50R)cdA unit is not affected by substitu-
ents in different positions. Contrary to (50S)cdA in the NMR spectra
of (50R)cdA and investigated derivatives, the characteristic H50;H30
interaction was present (Table 4). In addition, the results obtained
suggest that the 50-end sugar of the nucleosides attached to cdA
predominantly adopts an (S)-conformation. Moreover, this obser-
vation has been confirmed by its existence in the 2D NOESY spectra
of a cross-peak corresponding to 2.5 Å distance between H6 and
H20 of the thymidine unit with the absence of signals from H6–
H200 and H6–H10 interactions.
2.3.2. The orientation of the hydroxyl group with respect to the
-bond
The spatial orientation around the C40–C50 bond should have the
c
most pronounced influence on the geometry of the 50 internucleo-
tide phosphodiester linkage. The frequency of the distribution of
three possible staggered conformers, g+ (gauche +), t (trans) and
gꢀ (gauche ꢀ), is not equivalent. Their populations depend on
the sugar puckering, which is strongly connected to the substitu-
ents presented in different positions of the nucleoside. This situa-
tion does not concern 50,8-cyclo-20-deoxyadenosine due to the
presence of a new covalent bond between C50 and C8, which locks
the rotation of 50-OH around the C40–C50 bond, therefore determin-
ing a gauche—conformer for (50S)cdA and a trans-conformer for
(50R)cdA. For a natural nucleoside, the conformer populations have
been estimated from the coupling constants between H40 and H50,
H500 using the method described by Westhof et al.15 It was found
Based on the data derived from the aforementioned measure-
ments and calculations, it was possible to build three-dimensional
models of 1, 2 and 3. Figure 3B presents the deduced structure of
the most significant compound 3, TPO(50R)cdA.
From data currently available in literature,22 it is known that
TPO(50S)cdA is not fully depredated to free nucleosides by enzymes.
These results suggest that the (S)-diastereoisomer blocks the

B. T. Karwowski / Tetrahedron: Asymmetry 19 (2008) 2390–2395
2393
could bypass (50R)cdA, which is present
Table 5
hand, polymerase Pol
g
in the matrix chain of DNA.8 As mentioned in the introduction,
508-cyclo-20-deoxynucleosides are substrates for the nucleotide re-
pair system, but the (R)-diastereomer is being more efficiently re-
moved from genome than the (S)-diastereomer.23 These data
suggest that the presence of different isomeric forms of cdA in
the genome leads to various results. From a chemical-physical
point of view, the influence of the stereogenic centre at C50 of
50,8-cyclo-20-deoxyadenosine can be well demonstrated by a sim-
ple RP-HPLC analysis. The (50R)cdA and TPO(50R)cdA exhibit a short-
er retention time than the relative derivatives of the opposite
diastereoisomer; surprisingly, TPO(50R)cdA is eluted faster than
natural 20-deoxyadenosine (Fig. 3A).
Value of inter-sugar dihedral angles, sugar conformation phase P and amplitude /max
of sugar calculated from 2D NOESY NMR experiments
Angle
Torsion angle value (°)
(50R) cdATNBBDzMSi
cdA
(50R)
TPO(50R)cdA
T cdA
(50S)a
0
0
O
C
O
C
C
C
C
O
4 –C1 –N9–C2
vb
60.4
0
0
0
0
4 –O4 ––C1 –C2
/
45.2
ꢀ27.3
1.3
25.1
ꢀ44.8
48.3
43.6
ꢀ26.3
1.6
ꢀ10.0
45.9
ꢀ30.5
4.0
0
0
0
0
0
4 –C1 –C2 –C3
0
/
ꢀ37.2
18.2
1
0
0
0
1 –C2 –C3 –C4
/
4.2
ꢀ18.2
13.1
2
0
0
0
0
0
2 –C3 –C4 –O4
/
23.9
ꢀ44.9
71.2
23.3
23.0
3
0
0
0
3 –C4 –O4 –C1
/
ꢀ42.0
ꢀ2.1
–42.6
67.3
4
0
0
0
3 –C4 –C5 –C8
sb
74.5
40
10
–C –N9–C4
vb
ꢀ148.1
ꢀ149.5
ꢀ159
This observation can be used to explain the different biological
consequences described above. To explain these phenomena,
molecular mechanics calculations of the investigated dinucleotides
were performed at a periodic box of water.24 As a starting point for
this study, the d[TPOA] was extracted from ds-DNA.25 The modified
dinucleosides TPO(50S)cdA and TPO(50R)cdA were obtained by a suit-
able rearrangement of d[TPOA]. As presented in Figure 4, TPO(50R)c-
dA has shown a related structure to d[TPOA], and due to this feature
it probably has been better recognized by enzymes. Moreover, the
diagnostic distance between H2 of adenine and H3 of thymine
from d[TPOA] was 3.06 Å; the same distances for TPOcdA containing
the (50R)- and (50S)-diastereomer of cdA were 5.58 Å and 6.45 Å,
Pseudorotation parameters
Phase (P)
272
37
275
48
272
46
166
19
275
46
Amplitude (/max
)
Puckering
0T1
0T1
0T1
2T3
0T1
Data taken directly from the literature.13
Value obtained after molecular mechanics calculations with distances inferred
a
b
from 2D NOESY experiments.
hydrolytic activity of endo- or egzo-nucleases. Moreover, from a
study reported by Cadet et al., it is known that (50S)cdA completely
blocks the polymerization process of DNA chain.8 On the other
Figure 3. (A) Reverse-phase HPLC analysis of the mixture of natural 20-deoxynucleosides (1—dC, 3—dG, 4—T, 7—dA), 50,8-cyclo-2-deoxyadenosine (2—50R, 5—50S),
dinucleotides (6—TPO(50R)cdA, 8—TPO(50S)cdA); (B) three-dimensional structure of TPO(50R)cdA inferred from NMR data.
Figure 4. Graphical visualization of the difference in 3D structure between TPO(50S)cdA, d[TPOA] and TPO(50R)cdA obtained by molecular mechanics calculation in a periodic
box of water.

2394
B. T. Karwowski / Tetrahedron: Asymmetry 19 (2008) 2390–2395
respectively. Additionally, the dihedral angel determined by N9–C8
from adenine and N1–C2 from thymine, which described the
arrangement between neighbouring bases, was ꢀ170.1° for
d[TPOA], and ꢀ169.4° and 158.2°, respectively, for TPO(50R)cdA
and TPO(50S)cdA.
T), 12.1 (5-(50S)cdA), 12.4 (6-TPO(50R)cdA), 13.31 (7-dA), 14.8
(TPO(50S)cdA).
(50R)cdATNBBDzMSi
1 yield of inversion of configuration step
((50S)cdATNBBDzMSi?(50R)cdANTBBDzMSi) described by the J. Cadet group26
20%; (50R)cdA 2 RP-HPLC Rt = 15,0 min; UV (H2O) kmax = 267 nm;
TPO(50R)cdA 3; yield 80% (under HPLC integration), RP-HPLC Rt =
15.5 min; UV (H2O) kmax = 267 nm; m/z (MALDI ToF) 552.1
([MꢀH]ꢀ, requires 552.42); 31P NMR (D2O) d: ꢀ1.2 ppm.
3. Conclusion
In conclusion, (50R)cdA as a model of the tandem base modifica-
tion occurring in DNA as the result of an ionization radiation or an
oxidative stress has been synthesized and studied by NMR and
DFT. It has been demonstrated that the covalent bond between
C50 and C8 in the nucleoside induces an unusual West conforma-
tion (0T1) of the furanose ring. Moreover, the postulated geometry
is independent of the substituents on the furanose ring and in the
adenine moiety. It should be pointed out that despite the changes
in the configuration on C50 atom (S?R), the sugar conformation re-
mains the same—0T1. The excellent agreement between the avail-
able crystallographic, DFT and NMR data should also be noted. As
a result, they converge to essentially the same puckering, ampli-
tude of sugar moiety and distances between protons in investi-
gated molecules.
4.2. Computation methodology
4.2.1. Quantum mechanics study
The molecular structure of the (50R)cdA 1, (50R) cdANTBBDzMSi 2 and
TPOcdA 3 was calculated using a DFT (Density Functional Theory)
approach with B3LYP (Becke0s three-parameter exchange func-
tional and the gradient-corrected functional of Lee, Yang and Parr).
For 1, 2 and 3, the 6-31G** base was used. The calculation of all
structures was achieved with GAUSSIAN 03 Revision D.01.27
4.2.2. Molecular mechanics study
The structures of 1, 2 and 3 were calculated using HyperChem
8.0 software, evaluation version, (HyperCube, Inc.), using MM+
molecular force files, RMS gradient 0.001 Kcal/mol, the Polak-Ribi-
ere Conjugate Gradient Algorithm; also the periodic box 20/20/
20 Å (w/h/d) was used. The minimum distance between the solvent
and solute atom was 2.3 Å.
The results strongly indicate that the sugar’s 5-membered ring
adopts the same conformations for 20-deoxynucleosides with a ri-
gid geometry. Moreover, from previous13 and present data, it was
found that the structure of the (S)- and (R)-diastereomer of 50,8-cy-
clo-20-deoxyadenosine is probably both temperature and derivati-
zation independent. In the 1H NMR experiments at 29 °C, no
4.3. NMR parameters
3
significant changes in the values of JH–H have been observed for
(50R)cdA, (50R)cdATNBBDzMSi and dTPO(50R)cdA, as in the case of previ-
ously studied derivatives of ((50S)cdA).13 Finally, by combining
2D NOESY experiments with simple molecular mechanics calcula-
tions and using fixed inter-proton distances, it has been demon-
All NMR spectra were recorded on a Varian ‘Mercury 300’
(300 MHz) spectrometer. 1H spectra were recorded at 29 °C and
collected in 65.3 K data points including 500 transients; the spectra
width was 3108 Hz at an operation frequency of 300.1 MHz.
31P spectra were acquired at 29 °C in 20.5 K, over 128 transients
with a spectral width of 10000 Hz and an operating frequency of
121.5 MHz.
strated that the 50-end sugar residue of nucleosides
3
predominately adopts an (S)-conformation, namely 2T3. In the
same way, the conformation of the furanose five-membered ring
of (50R)cdA derivatives adopts a 0T1 conformation. It can therefore
be postulated that the rigid and fixed structure of cdA can strongly
influence the global geometry of oligonucleosides. Moreover, by
comparison of the obtained three-dimensional structure of d[TPOA]
with TPO(50R)cdA and TPO(50S)cdA by molecular mechanics study, it
has been found that (50R)-derivatives adopt a structure more re-
lated to natural dinucleotide TPOdA than the opposite (50S)-
diastereomer.
1H spectra were referenced to the residual HOD of D2O fixed at
4.80 ppm.
2D COSY spectra and 2D NOESY spectra were obtained by a
standard puls-programme implemented in the standard VARIAN
software.
The distances between suitable protons were obtained from 2D
NOESY experiments. In NOESY spectra, the intensity of cross-peaks
is related to the distances between spins via the relaxation ma-
trix.14 For the distances assignment, the following equation was
used rij = rref (Iref/Iij)1/6, where rref is the reference distance. This is
usually done with the help of a reference NOE corresponding to a
known distance. For compounds 1, 2 and 3, as a reference (rref), dis-
tance between H-20 and H-200 from CH2 group was used
(rref = 1.78 Å). For compound 3, as an additional reference, the dis-
tance between the H-6 and CH3 proton of thymine was used
(rref = 2.70 Å).28 As an Iref, the integration of the cross-peak corre-
sponding to the known distance (rij) was assigned as 1.
All spectra were processed on a personal computer after accu-
mulation, using MESTRENOVA 5.2.3 (Mestreclab Research) software.
4. Experimental
NBz
TBDMSi
4.1. Synthesis of cdA, cdA
and TPOcdA
The (50R)-50,8-cyclo-20-deoxyadenosine 1, cdATNBBDzMSi 2 and deriv-
atives for short oligonucleotides synthesis were obtained by a
method described by Cadet.8 Dinucleotides TPOcdA 3 were synthe-
sized according to the solid-phase synthesis approach on a 1 lmol
scale using the classical phosphoramidite strategy9 with the slight
modification reported by Brooks et al.10 The synthesized products
were purified in one-step RP-HPLC in the DMTOFF mode. For this
purpose, a Supelco column, DiscoverÒ RP-C18 (25 cm ꢁ 4.6 mm,
Acknowledgement
5 lm), was used. The elution was by achieved using 0.1 M ammo-
I would like to thank the Medical University of Lodz (502-13-
704) for support.
nium acetate (AA) as the buffer A with a gradient for compound 1
from 0% to 10% of acetonitrile in 25 min, and for compounds 3 from
0% to 14% of acetonitrile in 25 min; detection k = 260 nm; the chro-
matogram presented in Figure 3A was achieved using buffer A with
a gradient from 0% to 100% of buffer B (40% acetonitrile in buffer A)
in 30 min, retention times, given in minutes, observed in the elu-
tion profile: 8.7(1-dC), 10.27 (2-(50R)cdA), 10.92 (3-dG), 11.5 (4-
References
1. (a) Burrows, C. J.; Muller, G. J. Chem. Rev. 1998, 98, 1109–1152; (b) Knapp, W.;
Pogozelski, B.; Tullius, D. T. Chem. Rev. 1998, 98, 1089–1108.
2. Cadet, J.; Douki, T.; Gasparutto, D.; Ravanat, J.-L. Mutat. Res. 2003, 531, 5–23.

B. T. Karwowski / Tetrahedron: Asymmetry 19 (2008) 2390–2395
2395
3. Sancar, A.; Lindsey-Boltz, L.; Unsal-Kacmaz, K.; Linn, S. Annu. Rev. Biochem.
19. Gochin, M.; James, T. Biochemistry 1990, 29, 11172–11180.
20. Wang, J.; Kollman, A. P. J. Comput. Chem. 2001, 22, 1219–1228.
2004, 73, 39–85.
4. Scancar, A. Chem. Rev. 2003, 103, 2203–2238.
5. Dizdaroglu, M.; Jurga, P.; Rodrigez, H. Free Radic. Biol. Med. 2001, 30, 774–784.
6. Brooks, P. J. DNA Repair 2008, 7, 1168–11799.
21. Houseknecht, B. J.; Altona, C.; Hadad, Ch. M.; Lowary, T. L. J. Org. Chem. 2002, 67,
4647–4651.
22. Jaruga, P.; Theruvathu, J.; Dizdaroglu, M.; Brooks, P. J. Nucleic Acids Res. 2004,
32, e87. doi:10.1093/nar/gnh087.
23. Jaruga, P.; Dizdaroglu, M. DNA Repair 2008. doi: 10.1016/j.dnarep.2008. 06.005.
24. Zacharias, M. In Computational Study of DNA and RNA; Sponer, J., Lankas, F., Eds.;
Springer: Berlin, 2006; pp 95–120.
25. Goodsell, D. S.; Kaczor-Grzeskowiak, M.; Dickerson, R. E. J. Mol. Biol. 1994, 240,
79–96.
7. Kuraoka, I.; Robins, P.; Masutani, C. H.; Hanaoka, F.; Gasparutto, D.; Cadet, J.;
Wood, R. D.; Lindahl, T. J. Biol. Chem. 2001, 276, 49283–49288.
8. Romieu, A.; Gasparutto, D.; Molko, D.; Cadet, J. J. Org. Chem. 1998, 63, 5245–5249.
9. Romieu, A.; Gasparutto, D.; Cadet, J. Chem. Res. Toxicol. 1999, 12, 412–421.
10. Brooks, P. J.; Wise, D. S.; Berry, D. A.; Kosmoski, V. J.; Smerdon, M. J.; Somer, R.
L.; Mackie, H.; Spoonde, A. Y.; Ackerman, E. J.; Coleman, K.; Taronc, R. E.;
Robbins, J. H. J. Biol. Chem. 2002, 275, 22355–22362.
11. Birnbaum, G. I.; Cygler, M.; Dudycz, L.; Stolarski, R.; Shugar, D. Biochemistry
1981, 20, 3294–3301.
12. Shishkin, O. V.; Pelmenschikov, A.; Hovorun, M. D.; Leszczynski, J. J. Mol. Struct.
2000, 526, 326–341.
13. Karwowski, B. T.; Gaillard, J.; Grand, A.; Cadet, J. Org. Biomol. Chem. 2008. DOI:
101039/b807046f.
14. Wijmenga, S. S.; van Buuren, B. N. M. Prog. Nucl. Magn. Reson. Spectrosc. 1998,
32, 287–387.
15. Westhof, E.; Roder, O.; Croneiss, L. I.; Ludeman, H.-D. Z. Naturforsch. Teil C. 1975,
30, 131–140.
16. Davis, D. B.; MacCoss, M.; Danyluk, S. S. J. Chem. Soc., Chem. Commun. 1984, 8,
536–538.
17. Mooren, M. M.; Wijmenga, S. S.; van der Marel, G. A.; van Boom, J. H.; Hilbers, C.
W. Nucleic Acids Res. 1994, 22, 2658–2666.
26. Romieu, A.; Gasparutto, D.; Cadet, J. Chem. Res. Toxicol. 1999, 12, 412–
421.
27. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, J. A.; Vreven, T. Jr.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;. Voth, G.
A; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill,, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.;
Gaussian, Inc., Wallingford CT, 2004.
18. Wijmenga, S. S.; Mooren, M. M.; Hilbers, C. W. In NMR of Macromolecules, A
Practical Approach ed.; Roberts, G. C. K., Ed.; Oxford University Press: New York,
1993.
28. Gronenborn, A. M.; Clore, G. M. Prog. NMR Spectrosc. 1985, 17, 1–32.