Conformational rigidity introduced by 2′,5′-phosphodiester links in DNA

Article abstract of DOI:10.1081/NCN-100107190

Conformational properties of 2′,5′-linked 3′-deoxyribonucleotides have been compared with their natural isomer using CD spectroscopy. It is inferred from the salt induced titration curves that the 2′,5′-linked3′deoxyribonucleotides have rigid phosphodiest

Full text of DOI:10.1081/NCN-100107190

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CONFORMATIONAL RIGIDITY INTRODUCED BY 2′,5′-
PHOSPHODIESTER LINKS IN DNA
Anil Kumar a , Debasis Dass b , M. Atreyi b , M. V.R. Rao b & S. B. Katti c
a Division of Biopolymers , Central Drug Research Institute , P.O. Box-173, Lucknow, 226 001,
India
b Department of Chemistry , University of Delhi , Delhi, India
c Division of Biopolymers , Central Drug Research Institute , P.O. Box-173, Lucknow, 226 001,
India
Published online: 19 Aug 2006.
To cite this article: Anil Kumar , Debasis Dass , M. Atreyi , M. V.R. Rao & S. B. Katti (2001) CONFORMATIONAL RIGIDITY
INTRODUCED BY 2′,5′-PHOSPHODIESTER LINKS IN DNA, Nucleosides, Nucleotides and Nucleic Acids, 20:10-11, 1783-1796
To link to this article: http://dx.doi.org/10.1081/NCN-100107190
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(10&11), 1783–1796 (2001)
CONFORMATIONAL RIGIDITY INTRODUCED
BY 20,50-PHOSPHODIESTER LINKS IN DNA
Anil Kumar,1 Debasis Dass,2 M. Atreyi,2 M. V. R. Rao,2
and S. B. Katti1,*
1Division of Biopolymers, Central Drug Research Institute,
P.O. Box-173, 226 001 Lucknow, India
2Department of Chemistry, University of Delhi, Delhi, India
ABSTRACT
Conformational properties of 20,50-linked 30-deoxyribonucleotides have
been compared with their natural isomer using CD spectroscopy. It is
inferred from the salt induced titration curves that the 20,50-linked-
30deoxyribonucleotides have rigid phosphodiester backbone.
INTRODUCTION
The transcription and the replication of nucleic acids are the central
reactions responsible for the transmission of hereditary information in all
living organisms. Biological systems use RNA- and DNA polymerases to
condense the mononucleotide units to form the polynucleotides having ex-
clusively 30,50-phosphodiester linkages1. In contrast to this a non-enzymatic
polymerization of activated ribonucleotides, either in the presence=absence of
a template2,3 or in the presence of divalent cations4, is known to result in the
production of oligonucleotides containing predominantly the unnatural 20,50-
phosphodiester linkages. The preponderance of 20,50- over 30,50- linked iso-
mers in these reactions was thought to be due to the greater nucleophilic
*Corresponding author.
1783
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1784
KUMAR ET AL.
reactivity of the 20-OH group. If 20,50- phosphodiester linkages are easier to
form then a basic question arises ‘Why nature has selected 30,50 phosphodie-
ster linkages in nucleic acids and not 20,50-phosphodiester linkage?’ Earlier
studies done by Usher and McHale5 had shown that a dodecanucleotide
containing one 20,50-phosphodiester linkage and its natural isomer hydrolyzed
at a comparable rate in the absence of any template. However, the presence of
a template caused a 7fold increase in the rate of hydrolysis of the oligomer
containing 20,50-bond and a 5 fold decrease in the rate of hydrolysis of the
natural oligomer. Dhingra and Sarma6 with the help of NMR studies have
shown that in 20,50-linked dimers, the intrinsic molecular stereodynamics are
such that in order to attain base-base interactions, the magnitude of the
glycosyl torsion angle changes so much that they can’t support helical
structures. Whereas, the geometries of 30,50-linkages allow the formation of
helical configurations for RNA. Their findings were further supported by
X-ray studies done by Parthasarathy et al7. Subsequently other experimental8
and theoretical9,10 studies led to contradictory predictions about the possi-
bility of forming helices with 20,50-linked ribonucleotides. If the slower rate of
hydrolysis for 30,50-linkages and=or the inability of 20,50-linked nucleotides to
form helix are the reasons for the natural preference for 30,50-linkages in RNA,
then what are the reasons underlying the preponderance of 30,50-phospho-
diester linkages in DNA? In order to know the details of the intrinsic prop-
erties of 30,50-linkages, which the 20,50-phosphodiester linkages lack, a few
reports have been published in the recent past11–24. Major efforts have gone
into the comparison of their melting curves, their thermodynamic stability
and base stacking properties. However, a limited attention25 has been paid to
unravel their conformational properties. In this article we describe a com-
parative study of the isomeric linkages of oligodeoxyribonucleotides by Cir-
cular Dichroism (CD) spectroscopy to assess their conformational flexibility.
Towards this purpose we have synthesized 30,50-linked DNA decamers 5 and 6
and their corresponding 20,50-linked DNA decamers 7and 8 respectively. The
present data enabled us to compare conformational properties of 30,50 versus
20,50-linked DNAs. We have also calculated the thermodynamic parameters of
these decamers by Tm studies. Significance of the conformational and ther-
modynamic properties of 30,50-viz.-a-viz. 20,50 DNA that led to the selection of
the former linkage in the biological system is also described.
RESULTS AND DISCUSSIONS
The phosphoramidite synthons were obtained from the corresponding
30-deoxynucleosides26 as shown in Scheme1. Subsequently oligomer synthesis
was carried out (0.2 mmol) on DNA synthesizer using the appropriate
phosphoramidite monomers and the DMT-off protocol. At the end of
synthesis oligomers were deblocked from the solid support and the remaining

CONFORMATIONAL RIGIDITY
1785
Scheme 1.
protecting groups were removed by the concentrated ammonia treatment.
Completely deblocked decamers were purified to homogeneity by the
reversed phase (C-18) HPLC before using for Tm and CD studies.
1a, B ¼ C
1b, B ¼ G
Decamer 5:
Decamer 6:
Decamer 7:
Decamer 8:
2a, B0 ¼ CBz
3a, B0 ¼ CBz
2b, B0 ¼ GIbu
3b, B0 ¼ GIbu
CGCGCGCGCG
GCGCGCGCGC
CGCGCGCGCG
GCGCGCGCGC
30,50-linked
30,50-linked
20,50-linked
20,50-linked
Tm Studies
UV absorbance versus temperature profiles of self-complementary 20,50-
linked decamers 7 and 8 exhibited sigmoidal curves, similar to their corre-
sponding isomeric 30,50-linked decamers 5 and 6 at 0.15 M NaCl
concentration, indicative of the association between 20,50-linked deoxy-
ribonucleotides. It was also evident that the helix to coil transition in 20,50-
linked decamers 7 and 8 was not that sharp as observed in the case of 30,50
linked decamers 5 and 6. The thermodynamic parameter viz., enthalpy
(DHꢀ), entropy (DSꢀ) and free energy (DGꢀ) for a transition were derived
from the absorbance melting curves. This was possible by assuming a two
state (all or none) model27, which assumes that single strands are in equili-
brium with only one base paired native structure and there are no partially
base paired intermediates in the melting process. This approximation is most
appropriate for short (< 12 base pairs) complexes28. Thermodynamic prop-
erties of the four decamers are summarized in Table 1. It can be easily in-
ferred from the data that values of each decamer vary with the strand
concentration. Although, the change is very less, due to a small difference in
strand concentrations, it supports a bimolecular association in all decamers.
Further, the Tm values of 20,50-linked decamers 7 and 8 is 20–30 ꢀC lower as
compared to their 30,50 linked isomers 5 and 6. The average values of DHꢀ

1786
KUMAR ET AL.
Table 1. Thermodynamic Parameters of Decamers 5–8
Strand Conc.
(mM)
À DHꢀ
À DSꢀ
DGꢀ
Decamers
30,50 [CG]5
Tm (ꢀC)
(Kcal MÀ 1
)
(Cal MÀ 1 KÀ 1
)
(Kcal MÀ 1
)
11.39
8.98
1.296.477 110.40
72.61
71.79
98.51
90.92
163.14
145.84
134.86
61.7
55.1
50.7
8.20
6.34
8.74
70.40
69.81
69.32
87.83
96.97143.54
90.66
130.53
48.9
50.5
30,50 [GC]5
20,50 [CG]5
20,50 [GC]5
54.2
134.69
8.33
6.71
4.71
49.84
48.70
46.78
34.1755.68 .57
35.68
35.15
17
57.42
56.24
18.56
18.39
11.99
9.24
6.89
60.35
59.92
58.54
37.23
45.03
39.93
59.26
70.23
62.45
19.56
24.09
21.32
and DGꢀ for decamers 5 and 6 reported in the Table 1 were found to be in
good agreement with the theoretical values, calculated on the basis of near-
est-neighbour thermodynamic data reported in the literature29. It is also clear
that the DHꢀ and DGꢀ values obtained for 20,50-linked decamers are
approximately half that of the corresponding natural decamers 5 and 6.
Our results support the previous observations for 20,50-DNA by Jung
and Switzer16 that the 20,50-linked oligonucleotides do associate in a bimo-
lecular fashion but with a less thermodynamic stability as compared to the
natural DNA. On the other hand, since this bimolecular association is ob-
served in 150 mM salt concentration, our findings are contradictory to the
results reported recently by Breslauer et al.15 who have shown that 20,50-
deoxyribonucleotide strands do not associate at low to intermediate sodium
ion concentration, instead they form a triplex, that too only at high salt
concentration. This difference could be due to the difference in length and
composition of the oligomer sequences. The oligomers employed in the
present study are palindromic having alternating dC and dG sequences with
inherently high affinity for duplex formation. Whereas, the sequence reported
by Breslauer15 are homopolymeric (dA16=dT16) with a higher propensity for
triplex formation, because of the favourable Hoogsteen-base pairs, suggest-
ing thereby that 20,50-linked oligomers like their 30,50-isomers exhibit sequence
dependent structural variation.
CD Studies
As expected, the CD spectrum of decamer 5 in 1 M SSc buffer (Fig.
1A) showed a positive band at 275 nm and a negative band at 248 nm,

CONFORMATIONAL RIGIDITY
1787
Figure 1. A). CD profiles of 30,50(C-G)5 in 1 M NaCl (—), 4 M NaCl (--6---6--) and
20,50(C-G)5 in water (--ꢀ---ꢀ--), 4 M NaCl (ꢁꢁ—ꢁꢁ—ꢁꢁ—). B). CD profiles of 30,50(G-C)5 in 1 M
NaCl (—ꢁꢁ—ꢁꢁ—), 4M NaCl (--ꢀ---ꢀ--) and 20,50(G-C)5 in water (—), 4 M NaCl (–u–u–).
characteristic of B form of DNA. On titrating with NaCl solution, the CD
spectra showed marked changes; the intensity of the 275 nm band progres-
sively decreased and ultimately it was replaced by an intense negative band
centered at 290 nm accompanied by a new positive band at 264 nm at 4 M
salt concentration. The crossover point was shifted from 264 to 277 nm.
Thus, the final spectra had features of the left-handed Z-DNA30. As already
known31 that in the process of B to Z transition, the sugar puckering of dG
residue changes from C20-endo to C30-endo and the glycosyl bond changes
to syn position whereas the dC residue remains unchanged. As a con-
sequence, the distance between the phosphate group attached to the C50 and
C30 ribose positions is shortened, leading to an increased charge-charge
repulsion between the adjacent phosphate residues under normal conditions,
this repulsion acts as a barrier in favour of B-DNA. However, in the pre-
sence of excess NaCl, the Naþ ions shield the negative charges and thus
stabilize the Z-form.
The CD spectra of decamer 6 in 1 M SSc buffer (Fig. 1B) also showed a
standard B DNA like pattern a positive band at 282 nm and a negative band

1788
KUMAR ET AL.
at 250 nm. By increasing the salt concentration, the intensity of the positive
and negative bands was decreased with concomitant shift in the crossover
point towards longer wavelength.
However, the pattern of the CD spectra remained unchanged even at
4.0 M salt concentration. These observations are consistent with earlier
findings that the synthetic d[GC]n sequences (where n ¼ 3,4,5) do not assume
the left handed conformation even at high ionic strength. A systematic study
carried out by Quadrifoglio et al.32 have shown that at least a hexadecamer
or octadecamer d[GC] sequence is required to observe salt induced left-
handed conformation. On the contrary, even a short d[CG]n sequence (where
n ¼ 3) can undergo B-Z conformation change.
The 20,50-linked decamers 7 and 8, however, showed the CD pattern
which resemble with that of A form DNA. In 1 M SSc buffer, the CD pattern
of decamer 7showed a negative band at 295 nm and a broad positive band
centered at 264 nm (with a shoulder at 232 nm). The crossover point was
observed at 286 nm (data not shown). Assuming that this could be a tran-
sition stage between B-A (or B-Z) conformational change, the CD pattern of
decamer 7was checked in H 2O and change in the CD profile was monitored
by titration with increasing salt concentration. In H2O the CD pattern
showed two negative bands at 301 and 242 nm, and two positive bands at 271
and 223 nm. The crossover points were observed at 291, 251 and 232 nm. On
increasing the salt concentration, the intensities of negative bands were de-
creased. A positive band at 310 nm in 4 M salt concentration ultimately re-
placed the 301 nm negative band, whereas the 242 nm negative band
disappeared. A shoulder accompanied the main positive band at 270 nm.
However, the overall CD pattern remains the same (A-DNA like) all
throughout the titration, from H2O to 4.0 M salt concentration (Fig. 1A).
Similar behaviour in the CD pattern of decamer 8 was observed under
identical conditions used above in the case of decamer 7(Fig. 1B). The above
results indicate that the 30,50-linked oligodeoxyribonucleotides are con-
formationally flexible and exhibit polymorphism depending on changes in the
environment. Whereas the 20,50-linked nucleotides exist in A-DNA like
structure which is conformationally rigid.
CONCLUSIONS
The results of Tm and CD studies, in aggregate, indicate significant
differences in the properties of complexes formed by the association of
strands with identical base sequences but different phosphodiester linkages. It
is apparent from the Tm studies that 20,50-linked oligo-30-deoxyribonucleo-
tides do form duplex structures likewise their natural 30,50-isomers. However,
relative to its component single strands, the 20,50-duplexes are thermo-
dynamically much less stable than the corresponding 30,50-duplexes.

CONFORMATIONAL RIGIDITY
1789
Inspection of the thermodynamic data reveal that the enthalpy required to
disrupt a 20,50-duplex to its component strands is considerably less than that
required to disrupt the corresponding 30,50-duplex. With the assumption that
the final single-stranded states are energetically equivalent, these data in-
dicate that the forces that stabilize the 20,50-duplex are 50–75 Kcal=mole less
enthalpic than the forces that stabilize the corresponding 30,50-duplex. This is
also clear from the free energy change values that relative to their component
single strands, the 20,50-duplex is stabilized by only 17–24 Kcal=mole of du-
plex compared with 48–61 Kcal=mole for the 30,50-duplex. This dramatic
differential thermodynamic stability (DGꢀ ¼ 31–37Kcal =mole at 25 ꢀC)
probably reflects better base stacking and pairing interaction when the nu-
cleotide are connected via 30,50-phosphodiester linkages rather than via 20,50-
linkages. Furthermore, the CD studies reveal that the two isomeric duplexes
are conformationally nonequivalent. The duplexes containing 30,50-phos-
phodiester linkages exist in B-DNA like structure which is flexible (as it
shows B-Z transition on increasing salt concentration), whereas, the duplex
formed by 20,50-linked nucleotides exist in A-DNA-like structure which is
conformationally rigid. Since the context dependent conformational flex-
ibility of oligonucleotides is necessary for various biochemical functions,
rather rigid or unstacked conformation of the 20,50-DNA could be a negative
selective pressure in addition to the weak duplex forming ability. The
structural information deduced here for the formation of 20,50-duplexes
suggest that synthetic oligodeoxyribonucleotides containing all 20,50-linkages
as well as chimeric linkages (20,50=30,50) could serve as ideal probes for DNA-
protein interaction studies. Such chimeric oligonucleotides have already been
used for the targeted destruction of RNA in the antisense oligonucleotide
technology33.
EXPERIMENTAL
All reactions were performed in freshly prepared solutions and dry
solvents. Protected 20-deoxyribonucleosidephosphoramidites and Controlled
Pore Glass (CPG) loaded 20-deoxyribonucleosides were purchased from
Cruachem, Scotland. Succinic anhydride, 2,20-Dithiobis-(5-nitropyridine), 2-
Cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodi-amidite and Long chain
alkylamine-CPG (mesh 80–120, pore size 500 Aꢀ) were obtained from Sigma
Chemical Company, USA. 4-Dimethylaminopyridine, 1H-Tetrazole, 4,40-
Dimethoxytritylchloride (DMT-Cl), Isobutyric anhydride and Triphenyl-
phosphine were supplied by Fluka, Switzerland.
30-Deoxyribonucleotides were synthesized by using published proce-
dure26. Protected 30-deoxyriboncleoside-phosphoramidites and CPG loaded
30-deoxyribonucleosides were prepared by following procedure. Phosphity-
lation reactions were carried out in dry argon atmosphere. The Thin layer

1790
KUMAR ET AL.
chromatography (TLC) was performed on readymade plates (MERCK DC-
Alufolien Kieselgel 60 F254, 0.2 mm). Synthesis of decamers were carried out
on Pharmacia Gene Assembler Plus using the manufacturers’ recommenda-
tions for phosphoramidite chemistry. Deblocked decamers were purified on
Waters HPLC system using reversed phase column (MERCK 50983-
Lichrosopher 100 RP-18, 5 mm, 25064). All UV measurements were carried
out on Perkin Elmer (Lambda 15) and Gilford (ResponseTM) UV=VIS
spectrophotometer. NMR spectra were recorded on Bruker WM-400 MHz
spectrometer. The FAB mass spectra were recorded on Jeol SX-102=DA-
6000 double focusing spectrometer.
For enzyme studies, TDW and the buffers were freshly prepared and
sterilized before use. Phosphodiesterase (Crotallus durissus) was purchased
from Boehringer Mannheim, Germany and alkaline phosphatase from
Sigma, USA.
General Method of N-Acylation
To a stirred suspension of predried 30-deoxynucleoside (1a=b, 10 mmol)
in dry pyridine (50 ml) was added freshly distilled TMS-Cl (6.4 ml, 50 mmol)
dropwise at 5 ꢀC and the mixture was stirred at room temperature. After
20 min, the acylating reagent* (30 mmol) was added dropwise, under cool
condition. The stirring was continued for another 2 h at room temperature.
After completion of reaction, the excess of acylating reagent was quenched
with chilled MeOH (5 ml). The aqueous NH3 solution (10 ml) was added
dropwise followed by stirring at room temperature for 30 min. The reaction
mixture was concentrated under reduced pressure and the residue was dis-
solved in a solution of 1:9 MeOH:CHCl3 (25 ml). The salt thus precipitated
was filtered and washed with 1:9 MeOH:CHCl3 (10 ml). Combined organic
layer was concentrated. The product was purified by silica gel column
chromatography using a linear gradient of 0–10% MeOH in CHCl3. Ap-
propriate fractions were pooled and concentrated to give the product as a
white solid (TLC in 10% MeOH in CHCl3).
N4-Benzoyl-30-deoxyribocytidine. Yield 82%; Rf 0.58 (), PMR
(CDCl3 þ DMSOd6)d: 9.9 (brs, 1H, N-H); 8.7(d, 1H, J
¼ 8 Hz, H-6); 8.0
5–6
(d, 2H, J ¼ 7.8 Hz, Ar-H); 7.56–7.49 (m, 3H, Ar-H); 7.48 (d, J5–6 ¼ 8 Hz, H-
5); 5.83 (s, 1H, H-10); 5.31 (d, 1H, J2 OH-2 H ¼ 4.5 Hz, 20-OH); 4.85 (t, 1H,
0
0
J5 OH-5 H ¼ 6 Hz, 50-OH); 4.6 (m, 1H, H-40); 4.4 (s, 1H, H-20); 4.06 and 3.7
(2 m, 2H, Ha-50 and Hb-50); 2.08 and 1.88 (2 m, 2H, Hb-30 and Ha-30), FABMS
(m=z): 332 [M þ H]þ .
0
0
*Benzoyl chloride was used for the protection of 30-Deoxyribocytidine, and Isobutyric anhy-
dride for the protection of 30-Deoxyriboguanosine.

CONFORMATIONAL RIGIDITY
1791
N2-Isobutyryl-30-deoxyriboguanosine. Yield 76%, Rf 0.23, PMR
(CDCl3 þ DMSOd6)d: 11.5 (brs, 1H, N-H); 8.55 (s, 1H, H-8); 7.8 (s, 1H, N-
H); 6.18 (s, 1H, H-10); 5.52 (d, 1H, J2 OH-2 H ¼ 4.5 Hz, 20-OH); 4.94 (t, 1H,
0
0
J5 OH-5 H ¼ 5.8 Hz, 50-OH); 4.58 (m, 1H, H-20); 4.48 (m, 1H, H-40); 3.97and
3.65 (2 m, 2H, Ha-50 and Hb-50), 2.2 and 1.9 (2 m, 2H, Hb-30 and Ha-30), 1.2
(d, 6H, J ¼ 7.6 Hz, CH3), FABMS (m=z): 338 [M þ H]þ .
0
0
General Method of Dimethoxytritylation
A mixture of N-Acyl-30-deoxyribonucleoside (10 mmol) was co-evapo-
rated with dry pyridine (2615 ml). The dried residue was dissolved in dry
pyridine (70 ml). To this was added DMT-Cl (3.7 g, 11 mmol) at 5 ꢀC and the
mixture was stirred at room temperature for 2–4 h. After completion of re-
action, the mixture was concentrated and the residue was dissolved in CHCl3
(100 ml). The organic layer was washed successively with aqueous NaHCO3,
H2O and brine, dried over anhydrous Na2SO4 and concentrated. The pro-
duct was purified by silica gel column chromatography using a linear gradient
of 0–10% MeOH in CHCl3 containing 1% Triethylamine (TEA). Appro-
priate fractions were pooled and concentrated. The residue was re-dissolved
in fresh CHCl3 (10 ml) and precipitated in dry hexane (250 ml). The pre-
cipitate was filtered and dried to give the desired products in excellent yields
(TLC in 4% MeOH in CHCl3).
N4-Benzoyl-50-O-dimethoxytrityl-30-deoxyribocytidine (2a). Yield 85%,
Rf 0.51 (), PMR (CDCl3)d: 7.9 (d, 1H, J6,5 ¼ 7.8 Hz, H-6); 7.43–7.24 (m, 14H,
Ar-H); 6.88 (m, 5H, Ar-H and H-5); 5.78 (s, 1H, H-10); 4.72 (brs, 1H, H-40);
4.53 (brs, 1H, H-20); 3.8 (s, 6H, OCH3); 3.53 (dd, 1H, J5 –4 ¼ 4 Hz;
0
0
0
0
0
0
0
0
0
0
J5 –5 ¼ 11 Hz, Ha-5 ); 3.3 (dd, 1H, J5 –4 ¼ 3.9 Hz, J5 –5 ¼ 11.2 Hz, Hb-5 ), 2.25
and 2.1 (2 m, 2H, Hb-30 and Ha-30); FABMS(m=z): 632 [M-H]À .
N2-Isobutyryl-50-O-dimethoxytrityl-30-deoxyriboguanosine (2b). Yield
82%, Rf 0.62, PMR (CDCl3)d: 9.05 (brs, 1H, N-H); 8.15 (s, 1H, H-8); 7.2–
7.45 (m, 9H, Ar-H); 6.82 (m, 4H, Ar-H); 6.15 (s, 1H, H-10); 4.72 (brs, 1H, 20-
0
OH); 4.57(s, 1H, H-2 ); 4.45 (s, 1H, H-40); 3.8 (s, 6H, O-CH3); 3.4 and 3.3
(2 m, 2H, Ha-50 and Hb-50); 2.13 (m, 2H, Hb-30 and Ha-30); 1.28 (d, 6H,
J ¼ 7.4 Hz, CH3), FABMS (m=z):638 [M-H]À .
Synthesis of N-Acyl-50-O-dimethoxytrityl-30-deoxyribonucleoside-20-(2-
cyanoethyl)-N,N-diisopropyl-phosphoramidites (3a=b). Predried N-Acyl-50-
O-dimethoxytrityl-30-deoxyribonucloside (3a=b, 0.5 mmol) was dissolved in
dry Acetonitrile (5.0 ml). To this was added 2-cyanoethyl-N,N,N0,N0-tetra-
isopropyl-phosphorodiamidite (0.32 ml, 1 mmol) followed by 0.5 M solution
of 1-H Tetrazole in dry Acetonitrile (1.0 ml), dropwise with stirring.

1792
KUMAR ET AL.
The mixture was stirred at room temperature for additional 2 h. It was di-
luted with a solution of 2% TEA in CHCl3 (50 ml) and washed successively
with aqueous NaHCO3, H2O and brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated. The residue was dissolved in CHCl3
and precipitated in hexane. The precipitate was centrifuged and dried in a
vacuum desiccator, TLC [TEA=EtOAc=DCM (1:2:7)]. 3a: Yield 80%, Rf
0.67, 31P NMR (CDCl3)d: 148.8, 150.5 and 3b: Yield 79%, Rf, 0.65, 31P
NMR (CDCl3)d: 150, 151.3.
Synthesis of N-Acyl-50-O-dimethoxytrityl-30-deoxyribonucleoside-20-O-
succinates. A mixture of N-Acyl-50-O-dimethoxytrityl-30-deoxyribonu-
cleo-side (0.3 mmol) and DMAP (0.15 mmol) was co-evaporated with dry
pyridine (262 ml) and dissolved in dry DCM (10 ml). To this was added dry
TEA (0.5 ml, 3.6 mmol) and succinic anhydride (0.15 g, 1.5 mmol) at 5 ꢀC.
The reaction mixture was stirred at room temperature for 6 h. After com-
pletion of reaction, the mixture was diluted with 1% TEA=CHCl3 (50 ml)
and washed successively with aqueous citric acid, aqueous NaHCO3, H2O
and brine. The organic layer was dried over anhydrous Na2SO4 and con-
centrated. The residue was dissolved in CHCl3 and precipitated in hexane.
The precipitate was centrifuged and dried to give the desired product as a
white solid (TLC in 5% MeOH in CHCl3).
N4-Benzoyl-50-O-dimethoxytrityl-30-deoxyribocytidine-20-O-succi-
nate. Yield 93%, Rf 0.33 PMR (CDCl3)d: 8.0 (d, 1H, J6,5 ¼ 8 Hz, H-6);
7.44–7.2 (m, 13H, Ar-H); 6.82 (m, 5H, Ar-H and H-5), 5.92 (s, 1H, H-10), 5.4
(s, 1H, H-40), 4.38 (brs, 1H, H-20), 3.75 (s, 6H, O-CH3); 3.56 and 3.28 (2 m,
2H, Ha-50 and Hb-50), 2.46–2.3 (m, 6H, -CH2CH2-, Hb-30 and Ha-30), FABMS
(m=z): 732 [M-H]À .
N2-Isobutyryl-50-O-dimethoxytrityl-30-deoxyriboguanosine-20-O-succi-
nate. Yield 95%, Rf 0.38, PMR (CDCl3)d: 8.23 (s, 1H, H-8); 7.5–7.12 (m,
9H, Ar-H); 6.75 (m, 4H, Ar-H); 5.8 (s, 1H, H-10); 4.6 (s, 1H, H-20); 3.88 (m,
1H, H-40); 3.78 (s, 6H, O-CH3); 3.56 and 3.4 (2 m, 2H, Ha-50 and Hb-50); 2.8–
2.4 (m, 6H, -CH2CH2-, Hb-30 and Ha-30); 1.06 (s, 6H, CH3), FABMS (m=z):
738 [M-H]À .
Loading of protected-30-deoxyribonucleoside on lcaa-CPG. To a stirred
solution of N-Acyl-50-O-dimethoxytrityl-20-O-succinate (0.2 mmol), DMAP
(49 mg, 0.4 mmol) and 2,20-Dithiobis-(5-nitropyridine) (62 mg, 0.2 mmol) in
acetonitrile:1,2-dichloroethane (3:1, 4 ml), was added Triphenylphosphene
(52.5 mg, 0.2 mmol) and the mixture was stirred for 5 min to get a clear so-
lution. To this was added lcaa-CPG (1 g, 0.1 mmol of free amino groups) and
the suspension was agitated for 45 min at room temperature. It was filtered,
washed successively with 1,2-Dichloroethane, acetonitrile and 1%

CONFORMATIONAL RIGIDITY
1793
TEA=ether (10 ml of each) and dried in a vacuum desiccator. The residual
amino groups on the support were capped by treating with a mixture of
Ac2O: TEA: N-Methylimidazole: Dichloromethane (DCM) (1:1:0.3:6, 10 ml)
at room temperature for 30 min. The derivatized CPG was filtered again and
washed with DCM and ether (10 ml of each) followed by drying in a vacuum
desiccator. N2-Isobutyryl-50-O-dimethoxytrityl-30-deoxyribo-guanosine loa-
ded-CPG and N4-Benzoyl-50-O-dimethoxytrityl-30-deoxyribo-cytidine loa-
ded-CPG were obtained with a loading capacity of 36.0 and 39.57 mmol=g of
CPG respectively.
Synthesis and Purification of Decamers 5–8
The decamers 5^8 were synthesized on DNA synthesizer employing
the suitably protected appropriate phosphoramidite monomers and DMT-
off protocol recommended by the manufacture (0.2 mmol scale). Completely
deblocked oligomers were purified to homogeneity by the reversed phase
HPLC using a linear gradient of 10–30% B in A (30 min), flow ¼ 1 ml=min
[Where A ¼ 0.1 M triethylammoniumacetate, pH ¼ 7.2 and B ¼ acetonitrile].
Appropriate fractions containing the major peak were pooled and con-
centrated on a Speedvac by co-evaporation with MeOH to give 6–8 O. D.
units of each of the purified decamers. The purified decamers were
subjected to analytical HPLC wherein, only one peak was observed in all
the cases suggesting the homogeneity of the decamers. The retention time
of the decamers 5^8 was found to be 12.16, 12.5, 5.63 and 9.5 min
respectively.
Confirmation of Composition
Each of the purified decamer (0.3 A260) was dissolved separately in a
mixture of 1 M Tris (pH 8.5, 25 mL), 1 M MgCl2 (25 mL) and H2O (175 mL) in
an eppendorf tube. It was mixed vortexed, heated at 75 ꢀC and then cooled
suddenly to 0 ꢀC. The SVPD (0.015units dissolved in 25 mL 0.1 M Tris HCl)
was then added and the mixture was incubated at 37 ꢀC. After 2 h, alkaline
phosphatase (0.6 units dissolved in 1 mL 0.1 M Tris HCl) was added and the
mixture was again incubated at 37 ꢀC for 1.5 h. HPLC profile of the crude
reaction mixture was checked with a linear gradient of 0–15% B in A
(20 min), flow ¼ 1 ml=min. Disappearance of the peaks corresponding to their
respective decamers confirmed the complete hydrolysis. Also, the peaks of
equal intensities, corresponding to 20-dC and 20-dG were obtained in the
HPLC pattern of decamers 5 and 6. Similarly, the HPLC profiles of dec-
amers 7 and 8 showed the peaks of equal intensities corresponding to 30-dC
and 30-dG. Retention time (in min) for 20-dC, 20-dG, 30-dC and 30-dG was

1794
KUMAR ET AL.
found to be 7.42, 11.68, 9.27 and 13.61 respectively, in the above gradient
system.
Tm Studies
Solutions of decamers 5^8 were prepared in 165 mM SSc buffer
(150 mM NaCl and 15 mM sodium citrate) adjusted to pH 7.1. Strand
concentrations (Ct) were determined from the high temperature absorbance
at 260 nm. The UV melting curves at three different concentrations of each
decamer were recorded at 260 nm with an automated reference compensator
that allowed melting curves to be obtained on three separate samples
simultaneously. Four cuvettes (1 cm pathlength, 400 mL capacity, sealed with
teflon stoppers, three cuvettes containing samples and the forth one for the
reference solvent) were placed in a thermistor-controlled thermoelectric cell
holder, circulated by water at a flow rate of 100 ml=min for the control of
heating rate. Absorbance data were collected over the temperature range
from 20ꢀ–90ꢀ C at a heating rate of 1 ꢀC=min. After each melting experiment,
the samples were cooled to the starting temperature (in 15 min) and the final
absorbance values were compared with the initial absorbance before starting
the experiment. Less than1% error was found between the absorbance values
indicating that no evaporation of the solvent or hydrolysis of the sample
occurred during the experiment. The absorbance data thus obtained were
used to calculate the Tm and the thermodynamic parameters (DH0, DS0and
DG0) using a software programme developed in our laborꢀatory. Using this
programme the absorbance values at each temperature (in K) were entered
to draw A Vs T graph. Upon selecting the lower and upper base lines, the ‘‘f ’’
vs ‘‘1=T’’ plot and the Tm value (in ꢀ K) were obtained. This was followed by
entering the values of n (molecularity, in this case 2) and the strand con-
centration (Ct) to get Vant Hoff’s plot and the values of DH0, DS0, and DG0
were calculated.
CD Studies
All CD spectra were recorded on JASCO J-500 spectropolarimeter
equipped with a DP-500N data processor. The base line of the instrument
was properly adjusted. An average of two scans was recorded at a sensitivity
of 1.0 m0cmÀ 1 and a scan speed of 50 nm=min. Quartz cells of 1.0 mm path
length was used. Solutions of decamers ( ꢂ 25 mM) were prepared in 1.1 M
SSc buffer (1 M NaCl þ 100 mM sodium citrate) adjusted at pH 7.2. Titra-
tions were carried out at 20 ꢀC by adding aliquots of 5 M aqueous NaCl
solution. The spectra recorded were corrected by multiplying with an ap-
propriate factor so that the resulting spectra correspond to that of same
concentration as the original one.

CONFORMATIONAL RIGIDITY
1795
ACKNOWLEDGMENTS
The authors acknowledge RSIC for providing the spectral data of the
compounds reported here. One of us (AK) thanks CSIR for financial assistance in
the form of SRF.
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Received December 5, 2000
Accepted June 12, 2001