Hydroxyl-functionalized DNAs with different linkers and their complmentary duplex stability

Article abstract of DOI:10.1080/15257770.2010.510124

Tert-butyldiphenylsilyl (TBDPS) was testified to be an appropriate orthogonal protecting group for novel 7-hydroxyl-functionalized 8-aza-7-deaza-2′-deoxyadenosine analogues. It was stable in partial and complete hydrogenation reactions used for the differ

Full text of DOI:10.1080/15257770.2010.510124

Nucleosides, Nucleotides and Nucleic Acids, 29:734–747, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.510124
HYDROXYL-FUNCTIONALIZED DNAS WITH DIFFERENT LINKERS
AND THEIR COMPLMENTARY DUPLEX STABILITY
Di Zhang,1,2 Yami n Li , 2,3 Liang Xu,2 Maosheng Cheng,1 Ying Zhou,3
Junlin He,2 and Keliang Liu1,2
1School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang, P. R. China
2Beijing Institute of Pharmacology and Toxicology, Beijing, P. R. China
3College of Life Science, Guizhou University, Guizhou, P. R. China
Tert-butyldiphenylsilyl (TBDPS) was testified to be an appropriate orthogonal protecting group
2
for novel 7-hydroxyl-functionalized 8-aza-7-deaza-2ꢁ-deoxyadenosine analogues. It was stable in
partial and complete hydrogenation reactions used for the different linker preparation. The cor-
responding phosphoramidites and hydroxyl-functionalized oligodeoxynucleotides were synthesized
and identified. The thermal effect of the hydroxyl group with different linkers on DNA duplexes was
evaluated. It provided a feasible strategy for the preparation of hydroxyl-functionalized DNAs for the
nucleic acid research.
Keywords Tert -butyldiphenylsilyl; 8-aza-7-deazaza-2ꢁ-deoxyadenosine; hydroxypropar-
gyl; hydroxypropenyl; oligodeoxynucleotides; thermal stability
INTRODUCTION
As more and more attention has been focused on exploring new func-
tions of nucleic acids, DNA and RNA modified with active functional
groups—which are involved in protein functions such as amino, hydroxyl,
and imidazolyl residues—have been used as an important approach for
new functional nucleic acids.[1–3] In our previous article,[4] we reported
an orthogonal protecting strategy for the synthesis of hydroxyl-containing
oligodeoxynucleotides (ODNs) in which tert-butyldiphenylsilyl (TBDPS)
group was used as the orthogonal protecting group of the extra hydroxyl
Received 7 March 2010; accepted 19 July 2010.
Financial support by the National Key Technologies R&D Program for New Drugs 2009ZX09301-002
is gratefully acknowledged.
Address correspondence to Keliang Liu, Beijing Institute of Pharmacology and Toxicology,
27 Taiping Road, 100850, Beijing, P. R. China. E-mail: keliangliu@yahoo.com; or Junlin He, E-mail: hejun-
lin@
yahoo.com
734

Hydroxyl-Functionalized DNAs
735
OH
OH
NH2
NH2
NH2
NH2
6
4
OH
3
7
N8
1
N
N
N
N
N
N
N
N2
1
N
N
N
N
O
N
O
N
N
HO
HO
HO
HO
O
O
1'
1'
OH
OH
OH
OH
1
2
3
4
systematicnumbering
purinenumbering
group linked to the position 5 of 2ꢁ-deoxyuridine or position 7 of 8-aza-7-
deaza-2ꢁ-deoxyadenosine. Here, we continued our efforts for the synthesis of
hydroxyl-functionalized ODNs with new 2ꢁ-deoxyadenosine analogues 1 and
2, and their influence on DNA duplex stability was evaluated.
Nucleoside analogue 3 was synthesized from Pd (0)-catalyzed cross-
coupling reaction between 7-iodo-8-aza-7-deaza-2ꢁ-deoxyadenosine and 3-
hydroxypropyne.[4–6] Complete hydrogenation of the triple bond in nucle-
oside 3 with Pd/C (10%) was used to obtain the hydroxypropyl-containing
nucleoside 1. The partial hydrogenation of the triple bond in nucleoside
3 with NiCl2/NaBH4 as the catalyst[7] led to the Z-double bond linker in
compound 2 (Scheme 1). The coupling constant of the hydrogen atoms
attached to the double bond of compound 2 is around 12 Hz, which is con-
sistent with other Z- alkenes.[8–10] However, the value for the hydrogen atoms
of E-double bond is normally in the range of 15 to 18 Hz.[8–10] The chemical
shifts of the alkenyl carbon atoms were 116.7 and 139.2 ppm, respectively.
Based on such synthetic routes for compounds 1 and 2, the preparation
of their corresponding phosphoramidites used in hydroxyl-functionalized
ODNs synthesis required that the 7-positioned hydroxyl group must be pro-
tected before it was introduced to the nucleosides. Therefore, the hydrox-
ypropargyl group of compound 3 needs a proper protecting group, and the
protecting group should be stable in the subsequent hydrogenation con-
ditions. Tert -butyldiphenylsilyl was testified to be stable to go through the
hydrogenation conditions. Compound 3a was used as the key precursor,
which was successfully converted to the two key intermediates 1a and 2a
by partial and full hydrogenation, respectively (Scheme 1). Compound 2a
exhibited the same characteristic for the Z-alkenyl linker, the coupling con-
stant of the hydrogen atoms attached to the Z double bond was about 12 Hz
and the chemical shifts of the alkenyl carbon atoms were 117.5 and 136.6
ppm, respectively.[8–10] Therefore, orthogonal protection was smoothly real-
ized with TBDPS between the two primary hydroxyl groups, the 7-positioned
hydroxyl group and the 5ꢁ-hydroxyl of the sugar moiety in nucleoside ana-
logues 1 and 2. In addition, compound 1 and 2 could be prepared from

736
D. Zhang et al.
OH
OH
NH2
NH2
NH2
OH
N
N
N
N
N
N
N
N
N
N
O
N
O
(ii)
(i)
N
O
HO
HO
HO
20%
90.7%
OH
OH
OH
2
1
3
(iii) 88%
NH2
OTBDPS
OTBDPS
NH2
NH2
OTBDPS
N
N
N
N
N
N
N
N
N
N
O
(i)
N
O
(ii)
N
HO
HO
HO
O
90.3%
40%
OH
3a
OH
2a
OH
1a
SCHEME 1 Synthesis of nucleosides 1–2 and their intermediates. Conditions: (i) H2, Pd/C, room
temperature at 3 hours; (ii) NiCl2/NaBH4, MeOH, −78◦C; (iii) 1 M TBAF/THF, room temperature.
the deprotection of 1a and 2a with TBAF/THF, respectively. Especially, this
route is a preferred choice for compound 2, because yield of the partial
hydrogenation of compound 3 was low (20%).
Further protection of the amino groups at position 4 in compounds
1a and 2a with N , N -di-n-butylformamide dimethyl acetal (1b and 2b) and
5ꢁ-hydroxyl with DMT (1c and 2c), and subsequent phosphitylation yielded
the corresponding phosphoramidites 1d (Scheme 2) and 2d (Scheme 3),
respectively.
For the synthesis of ODNs 7–9 containing 1, the protected monomer
1d with canonical residues was used. The normal deprotection condition,
concentrated ammonia at 55–60◦C for 18 hours, for DNA synthesis was
capable of cleaving the tert-butyldiphenylsilyl group. For the synthesis of
ODNs 10 and 11 containing 2, mild deprotection was required because
of the existence of the double bond linker. Tert -butyldiphenylsilyl could be
cleaved by incubation in 1 M TBAF/THF overnight at room temperature.[4,8]
Purification was carried out with denaturing PAGE. After desalting with a
Sep-Pak Column (C18, Waters, Milford, MA, USA). MALDI-TOF was used
for characterization (Table 1).
With TBDPS as an orthogonal protecting group, the synthesis of
hydroxyl-functionalized ODNs with extra hydroxyl group in purine or pyrim-
idine bases was realized. TBDPS could be cleaved either with 1 M TBAF/THF
meeting ultra-mild deprotection, or under the normal deprotection

Hydroxyl-Functionalized DNAs
737
CH2OTBDPS
CH2OTBDPS
(n-Bu)2NCH
NH2
N
N
N
N
N
N
N
N
N
O
(i)
HO
HO
O
90%
OH
1a
OH
1b
CH2OTBDPS
CH2OTBDPS
(n-Bu)2NCH
N
(n-Bu)2NCH
N
N
N
N
N
N
N
N
N
(ii)
(iii)
35.4%
DMTO
DMTO
O
O
61%
OH
1c
O
P
N(iPr)2
1d
NCCH2CH2O
SCHEME 2 Synthesis of phosphoramidite 1d. Conditions: (i) (C4H9)2NCH(OCH3)2, MeOH, 2 hours;
(ii) DMTCl, in pyridine, at room temperature for 1 hour; (iii) (NCCH2CH2O)(iPr)2NPCl, (iPr)2EtN, in
CH2Cl2, at room temperature for 30 minutes.
(n-Bu)2NCH
NH2
N
CH2OTBDPS
CH2OTBDPS
N
N
N
N
N
N
N
O
N
(i)
HO
HO
O
90%
OH
2a
OH
2b
(n-Bu)2NCH
N
(n-Bu)2NCH
N
N
N
N
N
CH2OTBDPS
CH2OTBDPS
N
N
N
N
(ii)
60%
(iii)
88%
DMTO
DMTO
O
O
OH
2c
O
P
N(iPr)2
2d
NCCH2CH2O
SCHEME 3 Synthesis of phosphoramidite 2d. Conditions: (i) (C4H9)2NCH(OCH3)2, MeOH, 2 hours;
(ii) DMTCl, in pyridine, at room temperature for 1 hour; (iii) (NCCH2CH2O)(iPr)2NPCl, (iPr)2EtN, in
CH2Cl2, at room temperature for 30 minutes.

738
D. Zhang et al.
TABLE 1 Molecular weights of oligodeoxynucleotides 5–11 determined by MALDI-TOF mass
spectrometry
Molecular
Molecular
No.
Sequences
weight (calc.)
weight (found)
5
6
7
8
9
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
5ꢁ- d(CGG CCT TAT CGC GC)-3ꢁ
5ꢁ- d(GCG CG1 TA1 GGC CG)-3ꢁ
5ꢁ- d(GCG CGA T11 GGC CG)-3ꢁ
5ꢁ- d(CGG CCT T1T CGC GC)-3ꢁ
5ꢁ- d(GCG CG2 TAA GGC CG)-3ꢁ
5ꢁ- d(CGG CCT T2T CGC GC)-3ꢁ
4313.8
4214.7
4430.0
4430.0
4272.8
4369.9
4270.9
4314.2
4216.5
4429.4
4429.1
4271.3
4369.5
4270.4
10
11
condition (55◦C conc. aq. ammonia). Therefore, TBDPS is a good choice
for the synthesis of hydroxyl-functionalized DNAs.
Thermal Stability of Hydroxyl-Functionalized DNA Duplexes
It has been demonstrated that the propargyl linker at 5-position of
2ꢁ-deoxyuridine or the 7-position of 8-aza-7-deaza-2ꢁ-deoxyadenosine is
favorable for the stability of corresponding base pairs and increases the
thermal stability of duplexes and triplexes.[4,5,11–13] Hydroxylpropynyl of 8-
aza-7-deaza-2ꢁ-deoxyadenosine (3) behaves similarly, as we have reported
previously, the incorporation of compound 3 into DNA duplexes always
leads to an increase of thermal stability.[4] When the linker was changed to
propyl in compound 1, a negative effect on duplex stability was observed (see
Table 2) and more incorporations resulted in a lower Tm. The decrease of Tm
was dependent on the number and positions of compound 1 in ODNs. For
example, three incorporations of compound 1 in duplex 7·9 (see Table 2)
gave a Tm of 67.3◦C, while a lower Tm of 63.7◦C was observed for the other
duplex 8·9 with differently located three incorporations. The hydrophobic-
ity and flexibility of the alkyl linker was supposed to be responsible for the
negative effect. With the incorporation of compound 2 with a Z-propenyl
linker, a similar position-dependent negative effect was observed. However,
this negative effect could not exclude the positive contribution of the pla-
nar double bond linker to the base-stacking, as observed with the planar
triple bond linker in compound 3. It is probably that the geometry of the Z-
propenyl linker buried the group deeply within the major groove, the regular
duplex structure was disturbed and a negative effect on duplex stability was
generated. This negative contribution was possibly greater than the positive
base-stacking of the double bond. On the contrary, the triple bond extruded
outward linearly; there was less agitation in the major groove and a posi-
tive effect was always obtained. The similar phenomenon was also observed
with triplexes modified with 5-aminopropargyl and 5-(Z)-aminopropenyl-2ꢁ-
deoxyuridines.[8] A Tm increase was observed for the triplexes containing the

Hydroxyl-Functionalized DNAs
739
TABLE 2 The Tm values of duplexes containing deoxyadenosine analogues 1–4a
No.
Duplex
Tm (◦C)
68.1b
67.3
5·6
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CG1 TA1 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 1TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 1TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T11 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T11 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 1TT CCG GC)-5ꢁ
5ꢁ- d(GCG CG2 TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 2TT CCG GC)-5ꢁ
5ꢁ- d(GCG CG2 TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 2TT CCG GC)-5ꢁ
5ꢁ- d(GCG CG3 TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CG3 TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 3TT CCG GC)-5ꢁ
5ꢁ-d(GCG CGA TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 3TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T33 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T33 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 3TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T44 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T44 GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 4TT CCG GC)-5ꢁ
5ꢁ- d(GCG CG4 TAA GGC CG)-3ꢁ
3ꢁ- d(CGC GCT 4TT CCG GC)-5ꢁ
7·9
5·9
66.9
8·6
65.6
8·9
63.7
11·6
5·12
11·12
15·6
15·13
5·13
14·6
14·13
16·6
16·17
18·17
66.8
66.1
66.0
69.3b
71.8b
70.0b
69.6b
72.4b
68.1b
68.5b
68.7b
aMeasured in 100 mM NaCl, 10 mM MgCl2, and 10 mM Na-cacodylate (pH 7.0) with 5 µM
oligodeoxynucleotides.
bData from reference 4.
triple bond linker, while no Tm increase could be observed for the triplexes
appended with the Z-double bond linker. The distinct influence of the link-
ers on thermal stability of DNA helix was supposed to be closely related
to their inherent properties.[11,14] In our study, the stabilities of base pairs
3-dT > 4-dT > 1-dT ∼ 2-dT, reflected the contribution of the 7-positioned
linkers of 8-aza-7-deaza-2ꢁ-deoxyadenosine in the order of propargyl > H >
alkyl ∼ (Z)-alkenyl.
Computer aided calculation on the duplexes 5·9, 5·12, 5·13 was con-
ducted with AMBER 8.0 (University of California, San Francisco, CA, USA),
employing the Amber 2003 force field (15–17) on a SGI Altix 350 worksta-
tion (SGI, Freemont, CA, USA). The calculation results showed that total
energy of these three duplex systems was very similar to each other. The

740
D. Zhang et al.
FIGURE 1 Calculated averaged conformations of duplex 5·9 (left), 5·12 (middle), 5·13 (right), with
arrows pointing to the 7-positioned substituents of nucleoside analogues 1, 2, and 3, respectively.
three 7-substitutents located in the major groove (Figure 1), but with a
different extending mode for each of them, depending on the geometry of
the linkers. The propyl link is flexible, the propargyl extends straight out-
sides fully, and the propenyl linker bends a little towards the duplex. Thus,
the local environment was affected differently, and different Tm values were
observed for them, respectively.
In summary, our research provided the approach for the synthesis of
hydroxyl-functionalized ODNs, with TBDPS as the orthogonal protecting
group. These primary results demonstrated that the influence of hydroxyl
group on the thermal stability of duplexes is closely related to the properties
of the linker. The oligodeoxynucleotides with hydroxyl group might be ap-
plied in the finding of new functional nucleic acids or other biotechnologies.
MATERIALS AND METHODS
General
Commercially available reagents were used without further purification.
Pyridine was dried and distilled over calcium hydride. Thin layer chromatog-
raphy (TLC) and flash column chromatography were run on HS GF254
(Qingdao, China) and silica gel (200–300 mesh, Qingdao, China), respec-
tively. All 1H, 13C, and 31P-NMR were recorded on a JNM-ECA-400 spectrom-
eter (JEOL, Tokyo, Japan), chemical shifts (δ) are in ppm downfileld from
internal TMS (1H, 13C) or external 85% H3PO4 (31P), and the J values were

Hydroxyl-Functionalized DNAs
741
given in Hz. Melting points were obtained using a RY-1 apparatus and are
uncorrected (Tianjin, China). Ultraviolet (UV) spectra were recorded on a
Cary-100 Bio UV-visible spectrophotometer, λmax in nm, ε in dm3mol−1cm−1.
Microanalyses were performed on a Fisons-1108 (Fisons, Milan, Italy).
4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-(3-hydroxyprop-1-yl)-
1H-pyrazolo[3,4-d]pyrimidine (1). 10% Pd/C (0.6 g) was added to the
methanolic solution (50 ml) of compound 3 (0.5 g, 1.64 mmol). The
mixture was sealed in a stainless oven under hydrogen (5 Kg pressure) and
stirred at 30◦C. After complete hydrogenation, the catalyst was filtered off
and the product was crystallized from the concentrated filtrate as a colorless
solid (0.46 g, 90.7%), m.p. 150–152◦C. Rf (CH2Cl2/CH3OH 5:1) 0.52. UV
1
(MeOH): λmax 209 (30600), 259 (8100), 278 (8700). H-NMR (DMSO-d6):
δ 1.79 (dt, J = 7.00, 2 H, 5-CH2CH2CH2OH), 2.20 (m, 1 H, C2ꢁ-Hα), 2.78
(m, 1 H, C2ꢁ-Hβ), 2.94 (m, 2 H, 5-CH2CH2CH2OH), 3.28–3.56 (m, 4 H,
C5ꢁ-H, 5-CH2CH2CH2OH), 3.78 (m, 1 H, C4ꢁ-H), 4.42 (m, 1 H, C3ꢁ-H),
4.66 (t, J = 5.0, 1 H, 5-CH2CH2CH2OH), 4.80 (t, J = 5.9, 1 H, C5ꢁ-OH),
5.22 (d, J = 4.5, 1 H, C3ꢁ-OH), 6.50 (t, J = 6.6, 1 H, C1ꢁ-H), 7.32, 8.15 (br,
2 H, NH2), 8.16 (s, 1 H, C2-H). 13C-NMR (DMSO-d6): δ 24.2, 31.6, 37.9,
59.7, 62.6, 71.2, 83.6, 87.5, 98.9, 145.8, 154.8, 155.9, 158.2. Anal. Calcd for
C13H19N5O4 (M 309.32): C, 50.48; H, 6.19; N, 22.64. Found: C, 50.54; H,
6.10; N 22.55.
4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-(3-tert-butyldiphenyl
silyloxyprop-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (1a). With the same proce-
dure described for compound 1, compound 1a was prepared from 3a (2.9 g,
3.07 mmol) and 10% Pd/C (0.52 g) in methanol (50 ml) as a colorless
solid (2.7 g, 90.3%), Rf (CH2Cl2/CH3OH 9:1) 0.53. UV (MeOH): λmax 207
(24450), 259 (7900), 278 (8000). 1HNMR (DMSO-d6): δ 0.98 (s, 9 H, tBu),
1.94 (m, 2 H, 5-CH2CH2CH2OH), 2.16 (m, 1 H, C2ꢁ-Hα), 2.73(m, 1 H,
C2ꢁ-Hβ), 3.02 (m, 2 H, 5-CH2CH2CH2OH), 3.36, 3.51 (2 m, 2 H, C5ꢁ-H),
3.76 (m, 3 H, C4ꢁ-H, 5-CH2CH2CH2OH), 4.41 (m, 1 H, C3ꢁ-H), 4.81 (t,
J = 6.4, 1 H, C5ꢁ-OH), 5.22 (d, J = 4.5, 1 H, C3ꢁ-OH), 6.49 (t, J = 6.3,
1 H, C1ꢁ-H), 7.10–7.70 (2 m, 12 H, arom. H, NH2), 8.16 (s, 1 H, C2-H).
13C-NMR (DMSO-d6): δ 18.8, 24.0, 26.7, 31.1, 37.9, 62.6, 71.3, 83.7, 87.6,
98.9, 127.8, 127.9, 129.8, 133.3, 135.0, 145.4, 154.8, 155.8, 158.2. Anal. Calcd
for C29H37N5O4Si. 0.5 H2O (M 556.73), C, 62.56; H, 6.88; N, 12.58. Found:
C, 62.72; H, 6.77; N, 12.28.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(di-n-butylamino)methylide
ne]amino}-3-(3-tert-butyldiphenylsilyloxyprop-1-yl)-1H-pyrazolo[3,4-d]pyrimi
dine (1b). N , N -di-n-butylformamide dimethyl acetal (0.58 g, 2.82 mmol)
was added to the methanolic solution (50 ml) of compound 1a (1.50 g,
2.81 mmol). The solution was stirred at 40◦C for 2 hours, and then
concentrated for flash chromatography (CH2Cl2/CH3OH 40:1 as the
eluent). The product was obtained as a colorless syrup (1.70 g, 90%), Rf
(CH2Cl2/CH3OH 15:1) 0.57. UV (MeOH): λmax 214 (22500), 319 (21500).

742
D. Zhang et al.
1HNMR (DMSO-d6): δ 0.83, 0.90 [2 t, 6 H, N CHN(CH2CH2CH2CH3)2],
0.96 (s, 9 H, tBu), 1.26 [m, 4 H, N CHN(CH2CH2CH2CH3)2], 1.56 [m, 4
H, N CHN(CH2CH2CH2CH3)2], 2.06 (m, 2 H, 5-CH2CH2CH2OH), 2.19
(m, 1 H, C2ꢁ-Hα), 2.77 (m, 1 H, C2ꢁ-Hβ), 3.07 (m, 2 H, 5-CH2CH2CH2OH),
3.37, 3.49 [2 m, 6 H, N CHN(CH2CH2CH2CH3)2, C5ꢁ-H], 3.72 (t, J = 6.4,
2 H, 5-CH2CH2CH2OH), 3.81 (m, 1 H, C4ꢁ-H), 4.43 (m, 1 H, C3ꢁ-H), 4.80
(t, J = 5.9, 1 H, C5ꢁ-OH), 5.25 (d, J = 4.5, 1 H, C3ꢁ-OH), 6.53 (t, J = 6.6,
1 H, C1ꢁ-H), 7.34–7.58 (m, 10 H, arom. H), 8.42 (s, 1 H, C2-H), 8.97 [s, 1
H, N CHN(CH2CH2CH2CH3)2]. 13C-NMR (DMSO-d6): δ 13.5, 13.6, 18.7,
19.1, 19.7, 24.7, 26.7, 28.6, 30.4, 31.0, 37.9, 45.3, 51.3, 62.6, 63.0, 71.2, 83.8,
87.5, 106.1, 127.7, 129.7, 133.2, 134.9, 146.9, 155.3, 155.4, 157.1, 162.4. Anal.
Calcd for C38H54N6O4Si (M 686.96): C, 66.44; H, 7.92; N, 12.23. Found: C,
66.68; H, 8.06; N, 11.92.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofura
nosyl]-4-{[(di-n-butylamino)methylidene]amino}-3-(3-tert-butyldiphenylsily
loxyprop-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (1c). Compound 1b (1.20 g,
1.78 mmol) was coevaporated with dried pyridine for three times,
dissolved in dried pyridine (2 ml). DMT-Cl (0.76 g, 2.23 mmol) was
added to the solution in portions with stirring at room temerpa-
ture. Then methanol was added to the solution, and the solution was
concentrated for flash chromatography (CH2Cl2/CH3OH from 80:1 to 40:1
as the eluent). The product was obtained as a colorless solid (1.06 g, 61%). Rf
(CH2Cl2/CH3OH 20:1) 0.46. UV (MeOH): λmax 205 (77000), 318 (30400).
1HNMR (DMSO-d6): δ 0.80, 0.91 [t, 6 H, N CHN(CH2CH2CH2CH3)2],
0.95 (m, 9 H, tBu), 1.20 [m, 4 H, N CHN(CH2CH2CH2CH3)2], 1.55 [m,
4 H, N CHN(CH2CH2CH2CH3)2], 1.83 (m, 2 H, 5-CH2CH2CH2OH),
2.27 (m, 1 H, C2ꢁ-Hα), 2.71 (m, 1 H, C2ꢁ-Hβ), 3.00 (m, 4 H, C5ꢁ-H,
5-CH2CH2CH2OH), 3.47 [m, 4 H, N CHN(CH2CH2CH2CH3)2], 3.64 (m,
8 H, 2 OCH3, 5-CH2CH2CH2OH), 3.95 (m, 1 H, C4ꢁ-H), 4.50 (m, 1 H,
C3ꢁ-H), 5.28 (d, J = 5.0, 1 H, C3ꢁ-OH), 6.58–7.58 (m, 24 H, arom. H, C1ꢁ-H),
8.44 (s, 1 H, C2-H), 8.98 [s, 1 H, N CHN(CH2CH2CH2CH3)2]. 13C-NMR
(DMSO-d6): δ 13.6, 18.7, 19.1, 19.7, 24.8, 26.6, 28.6, 30.4, 31.0, 38.3, 45.2,
51.3, 54.8, 63.2, 64.9, 71.4, 83.4, 85.1, 85.5, 106.1, 112.8, 112.9, 126.3, 127.5,
127.6, 127.7, 127.8, 129.5, 129.6, 129.7, 133.2, 134.9, 135.6, 145.1, 147.0,
155.3, 155.4, 157.0, 157.8, 157.9, 162.4. Anal. Calcd for C59H72N6O6Si (M
989.33): C, 71.63; H, 7.34; N, 8.49. Found: C, 71.31; H, 7.50; N, 8.30.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofura
nosyl]-4-{[(di-n-butylamino)methylidene]amino}-3-(3-tert-butyldiphenylsily
loxyprop-1-yl)-1H-pyrazolo[3,4-d]pyrimidine 3ꢁ-[(2-Cyanoethyl) N,N-diiso
propylphosphoramidite] (1d). Compound 1c (0.85 g, 0.87 mmol) was
dissolved in dried dichloromethane (10 ml), DIEA (1 ml, 5.75 mmol)
and diisopropylphosphoramido chloridite (0.25 g, 1.0 mmol) were
added to the solution. After stirring at room temperature for 2 hours,
the solution was diluted with dichloromethane (20 ml), washed with

Hydroxyl-Functionalized DNAs
743
cold aq. 5% NaHCO3 and brine, dried with anhydr. Na2SO4, and con-
centrated for flash chromatography (CH2Cl2/CH3COCH3 20:1 as the
eluent). The product was obtained as colorless foam (0.35 g, 35.4%).
Rf (CH2Cl2/CH3OH 30:1) 0.61, 0.71. 1H NMR(CDCl3): δ 0.86, 0.98
[t, 6 H, N CHN(CH2CH2CH2CH3)2], 1.01 (m, 9 H, tBu), 1.14–1.40
[m, 10 H, NCH(CH3)2, N CHN(CH2CH2CH2CH3)2], 1.60 [m, 4 H,
N CHN(CH2CH2CH2CH3)2], 1.94–2.17 (m, 3 H, 5-CH2CH2CH2OH,
C2ꢁ-Hα), 2.36–2.63 (m, 3 H, 5-CH2CH2CH2OH, C2ꢁ-Hβ), 2.92–3.91
[m, 18 H, C5ꢁ-H, 5-CH2CH2CH2OH, N CHN(CH2CH2CH2CH3)2, 2
OCH3, OCH2CH2CN], 4.23 (m, 1 H, C4ꢁ-H), 4.80 (m, 1 H, C3ꢁ-H),
6.66–7.64 (m, 24 H, arom. H, C1ꢁ-H), 8.48 (s, 1 H, C2-H), 8.86 [s, 1 H,
N CHN(CH2CH2CH2CH3)2]. 31P NMR (CDCl3): 148.75, 148.49.
4-Amino-1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[3-tert-butyldipheny
lsilyloxy-(Z)-prop-1-enyl]-1H-pyrazolo[3,4-d]pyrimidine (2a). The methano-
lic solution of compound 3a (2 g, 3.68 mmol) and NiCl2.6H2O (0.9 g, 3.78
mmol) was cooled to −78◦C. NaBH4 (0.21 g, 5.56 mmol) was added in
portions to the solution. After stirring for 30 minutes, the reaction mixture
was warmed naturally until it turned black. The mixture was concentrated
for flash chromatography (CH2Cl2/CH3OH from 40:1 to 20:1 as the
eluent). The product was obtained as colorless needles (0.8 g, 40%), m.p.
150–153◦C (recrystallized from methanol). Rf (CH2Cl2/CH3OH 10:1) 0.45.
1
UV (MeOH): λmax 208 (37800), 251 (13800). H NMR (DMSO-d6): δ 1.02
(s, tBu), 2.04 (m, 1 H, C2ꢁ-Hα), 2.44 (m, 1 H, C2ꢁ-Hβ), 3.18, 3.28 (2 m, 2 H,
C5ꢁ-H), 3.75 (m, 1 H, C4ꢁ-H), 4.22 (m, 1 H, C3ꢁ-H), 4.64 (t, J = 5.6, 1 H,
C5ꢁ-OH), 4.67–4.86 (ddd, J = 2.2, 4.5, 16.2, 2 H, CH = CHCH2), 5.18 (d,
J = 4.5, 1 H, C3ꢁ-OH), 6.11 (dt, J = 5.0, 11.5, 1 H, CH CHCH2), 6.48 (t,
J = 6.7, 1 H, C1ꢁ-H), 6.93 (dt, J = 11.8, 1 H, CH CHCH2), 7.35–7.63 (m,
arom., NH2), 8.17 (s, 1 H, C2-H). 13C NMR (DMSO-d6): δ 18.8, 26.7, 37.4,
62.6, 63.1, 71.2, 83.3, 87.7, 98.6, 117.5, 127.8, 129.8, 134.9, 136.6, 140.9,
154.4, 156.0, 158.0. Anal. Calcd for C29H35N5O4Si.(M 545.7): C, 63.83; H,
6.46; N, 12.83. Found: C, 63.62; H, 6.35; N 12.60.
4-Amino-1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-[3-hydroxy-(Z)-prop-
1-enyl]-1H-pyrazolo[3,4-d]pyrimidine (2). To the anhydrous THF solution
(20 ml) of compound 2a (0.6 g, 1.1 mmol), TBAF (0.48 g, 1.5 mmol)
was added, the solution was stirred at room temperature for 10 minutes.
TLC indicated a complete deprotection reaction. The reaction mixture
was concentrated for flash chromatography (CH2Cl2/CH3OH 10:1 as the
eluent), and the product was obtained as colorless solid (0.3 g, 88%), m.p.
170–173◦C. Rf (CH2CH2:CH3OH 5:1) 0.4. UV (MeOH): λmax 209 (22600),
1
250 (14000), 282 (10200). H NMR (DMSO-d6): δ 2.27 (m, 1 H, C2ꢁ-Hα),
2.76 (m, 1 H, C2ꢁ-Hβ), 3.38, 3.54 (2 m, 2 H, C5ꢁ-H), 3.81 (m, 1 H, C4ꢁ-H),
4.47 (m, 3 H, C3ꢁ-H, CH CHCH2OH), 4.77 (br, C5ꢁ-OH), 4.95 (br, 1
H, CH CHCH2OH), 5.29 (d, J = 4.5, C3ꢁ-OH), 6.02 (2 t, J = 5.1, J =
12.1, 1 H, CH CHCH2OH), 6.60 (t, J = 6.2, 1 H, C1ꢁ-H), 6.87, 6.90 (2 t,

744
D. Zhang et al.
J = 2.2, 11.8, 1 H, CH CHCH2OH), 7.50 (br, 2 H, NH2), 8.18 (s, 1 H,
C2-H). 13C NMR (DMSO-d6): δ 37.9, 60.2, 62.6, 71.1, 83.4, 87.6, 98.6, 116.7,
139.2, 141.3, 154.3, 155.9, 158.0. Anal. Calcd for C13H17N5O4.(M 307.31):
C, 50.81; H, 5.58; N, 22.79. Found: C, 51.20; H, 5.53; N 22.33.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(di-n-butylamino)methylide
ne]amino}-3-[3-tert-butyldiphenylsilyloxy-(Z)-prop-1-enyl]-1H-pyrazolo[3,4-
d]pyrimidine (2b). Compound 2a (1.50 g, 2.81 mmol) was dissolved in
methanol (50 ml), followed by the addition of N , N -di-n-butylformamide
dimethyl acetal (0.58 g, 2.82 mmol). The solution was stirred at 40◦C
for 2 hours, and then was concentrated for flash chromatography
(CH2Cl2/CH3OH 40:1 as the eluent). The product was obtained
as a colorless syrup (1.70 g, 90%), Rf (CH2Cl2/CH3OH 15:1) 0.57.
UV (MeOH): λmax 205 (20500), 316 (28500). 1HNMR (DMSO-d6):
δ 0.96, 1.00 [2 t, 6 H, N CHN(CH2CH2CH2CH3)2], 1.07 (s, 9 H,
tBu), 1.39 [2 m, 4 H, N CHN(CH2CH2CH2CH3)2], 1.67 [2 m, 4 H,
N CHN(CH2CH2CH2CH3)2], 2.13 (m, 1 H, C2ꢁ-Hα), 2.58 (m, 1 H, C2ꢁ-Hβ),
3.24, 3.34 (2 m, 2 H, C5ꢁ-H), 3.61 [m, 4 H, N CHN(CH2CH2CH2CH3)2],
3.82 (m, 1 H, C4ꢁ-H), 4.31 (m, 1 H, C3ꢁ-H), 4.71 (t, J = 5.8, 1 H, C5ꢁ-OH),
4.81, 4.95 (2 m, 2 H, CH CHCH2), 5.27 (d, J = 4.5, 1 H, 3ꢁ-OH), 6.14
(m, 1 H, CH CHCH2), 6.58 (t, J = 6.4, 1 H, C1ꢁ-H), 7.23 (m, 1 H,
CH CHCH2), 7.41–7.69 (2 m, 10 H, arom. H), 8.48 (s, 1 H, C2-H), 9.02
[s, 1 H, N CHN(CH2CH2CH2CH3)2]. 13C-NMR (DMSO-d6): δ 13.5, 13.6,
18.8, 19.1, 19.8, 26.7, 28.5, 30.4, 37.5, 45.6, 51.5, 62.6, 63.1, 71.2, 83.5, 87.7,
105.9, 117.8, 127.7, 127.8, 129.7, 133.1, 134.9, 142.5, 154.8, 155.6, 157.0,
162.4. Anal. Calcd for C38H52N6O4Si.0.25H2O (M 689.45): C, 66.20; H,
7.68; N, 12.19. Found: C, 66.39; H, 7.88; N, 11.70.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofura
nosyl]-4-{[(di-n-butylamino)methylidene]amino}-3-[3-tert-butyldiphenylsily
loxy-(Z)-prop-1-enyl]-1H-pyrazolo[3,4-d]pyrimidine (2c). Compound 2b
(0.35 g, 0.51 mmol) was coevaporated with dried pyridine for three times
firstly, then it was dissolved in dried pyridine (2 ml), DMT-Cl (0.2 g,
0.59 mmol) was added in portions to the solution. After stirring at room
temperature for 2 hours, methanol (5 ml) was added, and the solution was
concentrated for flash chromatography (CH2Cl2/CH3OH from 60:1 to 40:1
as the eluent). The product was obtained as a colorless solid (0.3 g, 60%). Rf
(CH2Cl2/CH3OH 20:1) 0.45. UV (MeOH): λmax 206 (61700), 322 (29400).
1HNMR (DMSO-d6): δ 0.92, 0.97 [2 t, 6 H, N CHN(CH2CH2CH2CH3)2],
0.99 (s, 9 H, tBu), 1.34 [2 m, 4 H, N CHN(CH2CH2CH2CH3)2],
1.63 [2 m, 4 H, N CHN(CH2CH2CH2CH3)2], 2.16 (m, 1 H, C2ꢁ-Hα),
2.47 (m, 1 H, C2ꢁ-Hβ), 2.95 (2 m, 2 H, C5ꢁ-H), 3.50, 3.59 [2 m, 4 H,
N CHN(CH2CH2CH2CH3)2], 3.66, 3.67 (2 s, 2 OCH3), 3.93 (m, 1 H,
C4ꢁ-H), 4.30 (m, 1 H, C3ꢁ-H), 4.38, 4.87 (2 m, CH CHCH2), 5.21 (d, J =
5.0, 1 H, C3ꢁ-OH), 6.05 (m, 1 H, CH CHCH2), 6.57 (m, 1 H, C1ꢁ-H),
6.63–7.58 (4 m, 24 H, CH CHCH2, arom. H), 8.46 (s, 1 H, C2-H), 8.98

Hydroxyl-Functionalized DNAs
745
[s, 1 H, N CHN(CH2CH2CH2CH3)2]. 13C-NMR (DMSO-d6): δ 13.6, 18.8,
19.1, 19.8, 26.7, 28.6, 30.5, 37.8, 45.6, 51.5, 54.8, 54.9, 63.2, 64.9, 71.3, 83.6,
85.1, 85.9, 106.0, 112.9, 126.4, 127.5, 127.7, 127.8, 129.7, 134.9, 142.4, 145.0,
156.9, 157.8, 157.9, 162.4. Anal. Calcd for C59H70N6O6Si (M 987.31): C,
71.77; H, 7.15; N, 8.51. Found: C, 71.88; H, 7.20; N, 8.43.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofura
nosyl]-4-{[(di-n-butylamino)methylidene]amino}-3-[3-tert-butyldiphenylsily
loxy-(Z)-prop-1-enyl]-1H-pyrazolo[3,4-d]pyrimidine 3ꢁ-[(2-Cyanoethyl) N,N-
diisopropylphosphoramidite] (2d). To the solution of compound 2c (0.21
g, 0.2 mmol) in dichloromethane (6 ml), DIEA (0.6 ml, 2.8 mmol) and
diisopropylphosphoramido chloridite (0.1 g, 0.4 mmol) were added. After
stirring at room temperature for 1 hour, the solution was diluted with
dichloromethane (10 ml), and it was washed with cold aq. 5% NaHCO3 and
brine. The solution was then dried with anhydr. Na2SO4, and concentrated
for flash chromatography (CH2Cl2/CH3COCH3 from 30:1 to 15:1 as the
eluent). The product 2d was obtained as colorless foam (0.22 g, 88%).
1
Rf (CH2Cl2/CH3OH 30:1) 0.61, 0.71. H NMR(CDCl3): δ 0.89–1.68 [m,
29 H, N CHN(CH2CH2CH2CH3)2, tBu, N(CH(CH3)2], 2.23–3.07 [m,
8 H, C2ꢁ-H, C5ꢁ-H,, N CHN(CH2CH2CH2CH3)2], 3.50–4.15 (m, 11 H, 2
OCH3, OCH2CH2CN, C4ꢁ-H), 4.53 (m, 2 H, CH CHCH2), 4.89 (m, 1 H,
C3ꢁ-H), 6.03 (1 H, CH CHCH2), 6.57 (m, 1 H, C1ꢁ-H), 6.65, 7.08–7.60
(4 m, 24 H, CH CHCH2, arom. H), 8.46 (s, 1 H, C2-H), 8.98 [s, 1 H,
N CHN(CH2CH2CH2CH3)2]. 31P NMR (CDCl3): 148.30, 148.57.
Synthesis and Purification of Oligonucleotides
Deoxyoligonucleotides were synthesized on an ABI 392 DNA/RNA syn-
thesizer (Applied Biosystems, USA) on a 1 µmol scale by standard proto-
col for phosphoramidite chemistry with DMT-off mode, the coupling yields
of unnatural phosphoramidites were all above 95% with extending cou-
pling time to 2 minutes. The deoxyoligonucleotides containing monomer 1
were synthesized with normal phosphoramidites and deprotected in conc.
aq. ammonia at 55◦C for 18 hours. The ammoniac solution was concen-
trated for further purification. Tac-protected naturally phosphoramidites
were used for the synthesis of deoxyoligonucleotides containing monomer
2, and the deprotection was conducted in conc. aq. ammonia overnight at
room temperature. The solution was dried and the residue was immersed
in 1 M TBAF/THF overnight at room temperature. 1 M (Et3NH)OAc was
added to the solution, and the mixture was desalted with Sep-Pak column.
After washing thoroughly with water, the DMT-off oligonucleotide was eluted
with methanol/water (70:30, V/V), and concentrated for further purifica-
tion. The concentrated residues were purified with 20% polyacrylamide /7
M urea denaturing gel, extracted with 0.3 M sodium acetate. The solution
was desalted on Sep-Pak. The homogeneous product was characterized by

746
D. Zhang et al.
MALDI-TOF (Table 1) performed on Kratos Axima-CFR-plus (Shimatzu,
Kyoto, Japan) with BHB as matrix.
Tm Measurements
Tm of the DNA duplexes was performed with a Cary-100 Bio UV-Visible
spectrophotometer (Varian, Palo Alto, CA, USA) equipped with a Cary tem-
perature controller. A buffer consisting of 100 mM NaCl, 10 mM MgCl2 and
10 mM Na-cacodylate (pH 7.0)[4,13] with 5 µM of each oligonucleotide was
used. The solution was heated to 85◦C, after halting for 10 minutes, it was
cooled to 25◦C at a rate of 0.5◦C/min. The UV absorbance was recorded
at 260 nm during the cooling process. The Tm values were obtained from
the melting curves. Each melting curve was fit to a non-self-complementary
two-state model.
REFERENCES
1. Ja¨ger, S.; Famulok, M. Generation and enzymatic amplification of high-density functionalized DNA
double strands. Angew. Chem. Int. Ed. 2004, 43, 3337–3340.
2. Borsenberger, V.; Howorka, S. Diene-modified nucleotides for the Diels–Alder-mediated functional
tagging of DNA. Nucleic Acids Res. 2009, 37, 1477–1485.
3. Thomas, J.M.; Yoon, J.-K.; Perrin, D.M. Investigation of the catalytic mechanism of a synthetic
DNAzyme with protein-like functionality: an RNaseA mimic? J. Am. Chem. Soc. 2009, 131, 5648–5658.
4. Zhang, D.; Xu, L.; Wei, X.; Li, Y.; He, J.L.; Liu, K.L. Hydroxyl-functionalized DNA: an efficient
orthogonal protecting strategy and duplex stability. Nucleosides, Nucleotides and Nucleic Acids, 2009,
28, 924–942.
5. Seela, F.; Zulauf, M. Synthesis of 7-alkynylated 8-aza-7-deaza-2ꢁ-deoxyadenosines via the Pd-catalysed
cross-coupling reaction. J. Chem. Soc., Perkin Trans 1 1998, 3233–3240.
6. Hobbs, F.W. Palladium-catalyzed synthesis of alkynylamino nucleosides. A universal linker for nucleic
acids. J. Org. Chem. 1989, 54, 3420–3422.
7. Lee, S.E.; Vyle, J.S.; Williams, D.M.; Grasby, J.A. Novel syntheses of (Z)-alkene and alkane base-
modified nucleosides. Tetrahedron Lett. 2000, 41, 267–270.
8. Brazier, J.A.; Shibata, T.; Townsley, J.; Taylor, B.F.; Frary, E.; Williams, N.H.; Williams, D.M. Amino-
functionalized DNA: the properties of C5-amino-alkyl substituted 2ꢁ-deoxyuridines and their appli-
cation in DNA triplex formation. Nucleic Acids Res. 2005, 33, 1362–1371.
9. Robins, M.J.; Barr, P.J. Nucleic acid related compounds. 39. Efficient conversion of 5-iodo to 5-alkynyl
and derived 5-substituted uracil bases and nucleosides. J. Org. Chem. 1983, 48, 1854–1862.
10. Ruth, J.L.; Bergstrom, D.E. C-5 substituted pyrimidine nucleosides. 1. Synthesis of C-5 allyl, propyl,
and propenyl uracil and cytosine nucleosides via organopalladium intermediates. J. Org. Chem. 1978,
43, 2870–2876.
11. Froehler, B.C.; Wadwani, S.; Terhorst, T.J.; Gerrard, S.R. Oligodeoxynucleotides containing C-5
propyne analogs of 2ꢁ- deoxyuridine and 2ꢁ-deoxycytidine. Tetrahedron Lett. 1992, 33, 5307–5310.
12. Ahmadian, M.; Zhang, P.M.; Bergstrom, D.E. A comparative study of the thermal-stability of
oligodeoxyribonucleotides containing 5-substituted 2ꢁ-deoxyuridines. Nucleic Acids Res. 1998, 26,
3127–3135.
13. He, J.L.; Seela, F. Propynyl groups in duplex DNA: stability of base pairs incorporating 7-substituted
8-aza-7-deazapurines or 5-substituted pyrimidines. Nucleic Acids Res. 2002, 30, 5485–5496.
´
´
¨
14. Sa´gi, J.; Szemzo¨, A.; Ebinger, K.; Szabolcs, A.; Sa´gi, G.; Ruff, E.; Otvo¨s, L. Base-modified oligodeoxynu-
cleotides. I. Effect of 5-alkyl, 5-(1-alkenyl) and 5-(1-alkynyl) substitution of the pyrimidines on duplex
stability and hydrophobicity. Tetrahedron Lett. 1993, 34, 2191–2194.
15. Case, D.A.; Darden, T.A.; Cheatham, III T.E.; Simmerling, C.I.; Wang, J.; Duke, R.E.; Luo, R.; Merz,
K.M.; Wang, B.; Peralman, D.A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak,

Hydroxyl-Functionalized DNAs
747
V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J.W.; Ross, W.S.; Kollma, P.A. Amber 8, University
of California, San Francisco, USA. 2004.
ˇ
16. Lankaˇs, F.; Sponer, J.; Langowski, J.; Cheatham, III T.E. DNA basepair step deformability inferred
from molecular dynamics simulations. Biophysical J. 2003, 85, 2872–2883.
17. Castrignano, T.; Chillemi, G.; Desideri, A. Structure and hydration of BamHI DNA recognition site:
a molecular dynamics investigation. Biophysical J. 2000, 79, 1263–1272.

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