Hydroxyl-functionalized DNA: An efficient orthogonal protecting strategy and duplex stability

Article abstract of DOI:10.1080/15257770903307276

Tert-butyldiphenylsilyl (TBDPS) was found to be an effective orthogonal protecting strategy for the 5-substituted hydroxyl groups of de novo synthesized deoxyuridine analogues 1-3 and 7-(3-hydroxypropynyl)- of 8-aza-7- deazadeoxyadenosine 4 for their inco

Full text of DOI:10.1080/15257770903307276

Nucleosides, Nucleotides and Nucleic Acids, 28:924–942, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903307276
HYDROXYL-FUNCTIONALIZED DNA: AN EFFICIENT ORTHOGONAL
PROTECTING STRATEGY AND DUPLEX STABILITY
Di Zhang,1,2 Liang Xu,2 Xia Wei,2 Yaming Li,2 Junlin He,2 and Keliang Liu2
1School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang, China
2Beijing Institute of Pharmacology and Toxicology, Beijing, China
Tert-butyldiphenylsilyl (TBDPS) was found to be an effective orthogonal protecting strategy
2
for the 5-substituted hydroxyl groups of de novo synthesized deoxyuridine analogues 1–3 and
7-(3-hydroxypropynyl)- of 8-aza-7-deazadeoxyadenosine 4 for their incorporation into oligodeoxynu-
cleotides by phosphoramidite chemistry. It could be completely cleaved under normal and ultra-mild
deprotection conditions applied to DNA synthesis, without extra cleaving operation. The new
phosphoramidites were coupled as usual with high yields. The new modified oligodeoxynucleotides
were characterized by MALDI-TOF and enzymatic cleavage analysis. The thermal stability and
conformation of these hydroxyl-functionalized DNA duplexes were evaluated.
Keywords Hydroxyl; TBDPS; nucleosides; DNA; functionalization
INTRODUCTION
Modification of nucleic acid residues with various functional groups
provided an alternative choice for in vitro selection of new functional
RNA or DNA, such as artificial ribozymes,[1] deoxyribozymes,[2] and
aptamers.[3]When the basic base pairing of the modified residues was
maintained, more interactions and functions could expect for functional-
ized DNA assemblies used in DNA nanotechnology and materials.[4] The
functional groups selected for these purposes were mostly those actively
involved in proton-transfer or hydrogen-bonding. For example, amino
and imidazolyl groups were introduced to the nucleic acid libraries for
Received 16 March 2009; accepted 20 August 2009.
Financial support by Beijing Institute of Pharmacology and Toxicology is gratefully acknowledged.
Address correspondence to Junlin He, Beijing Institute of Pharmacology and Toxicology, 27 Taiping
Road, 100850, Beijing, China. E-mail: hejunlin@yahoo.com
924

Hydroxyl-Functionalized DNA
925
artificial ribozymes.[5,6] Several kinds of functional group-bearing residues
have been tested as the substrates of polymerases for building function-
alized DNA assemblies and libraries.[4,7] The hydroxyl group is known
for hydrogen bonding, and it plays a critical role in the catalysis and
interference of ribozymes and siRNA, respectively,[8,9] but it has been
seldom used for the modification of oligonucleotides, partly because of
the orthogonal protection for the extra hydroxyl group. 5-Hydroxymethyl-
deoxyuridine (HMdU, 1)[10,11] has been converted to its phosphoramidite
for DNA solid-phase synthesis, with acetyl as the orthogonal protecting
group for the 5-substituted hydroxyl group. However, this acetyl could not
be applied for hydroxyl groups linked with other alkyl groups, such as in
nucleoside analogues 2–3.[12] In compound 1, the 5-positioned hydroxyl
group could be selectively protected by acetyl because it is more acidic
than the 5ꢁ-hydroxyl group in the sugar moiety.[11] In compounds 2 and
3, however, the two hydroxyl groups at 5- and 5ꢁ-positions hold almost
the same reactivity toward acylation. Therefore, this orthogonal protec-
tion method was only specific for the 5-hydroxymethyl of compound 1.
The bulky tert-butyldimethylsilyl (TBDMS), which is often used for the
protection of the 2ꢁ-hydroxyl of RNA residues as well as the 5-positioned
hydroxyl group of 5-hydroxypropynyl-deoxyuridine,[13] was also used for
compound 1, producing a selective silylation with a big ratio between
its 5-positioned hydroxyl and 5ꢁ-hydroxyl groups, but HPLC has to be
used for the difficult separation.[10] It seems that these two protection
groups were not feasible as a general orthogonal protection strategy for
the hydroxyalkyl groups in 1–3. Here we report an efficient orthogonal
protection strategy for the incorporation of hydroxyl-bearing nucleoside
analogues 1–3 into oligodeoxynucleotides with phosphoramidite chemistry.
De novo synthesis of these nucleoside analogues was utilized to avoid
the simultaneous presence of the 5-positioned hydroxyl and 5ꢁ-hydroxyl
groups.
For the hydroxyl-containing purine nucleosides, we synthesized a de-
oxyadenosine analogue 4 to study the orthogonal protection method for
its hydroxyl groups. To our best knowledge, 8-aza-7-deaza-deoxyadenosine
(5)[14] possesses the same base-pairing ability as dA in the context of
DNA duplex, its 7-substituent protrudes into the major groove, without
much influence on the duplex conformation. Therefore, its 7-position is
an ideal position for the introduction of various functional groups. Here,
3-hydroxypropynyl was introduced to its 7-position to obtain compound 4
by a cross-coupling reaction. This post-synthesis modification could allow
the orthogonal protection between its two primary hydroxyl groups. The
effect of these four hydroxyalkyl-containing nucleoside analogues on DNA
duplex thermal stability was also evaluated.

926
D. Zhang et al.
RESULTS AND DISCUSSION
Synthesis of the Phosphoramidites
Our strategy toward the phosphoramidites 1f–3f was based on de novo
synthesis of nucleoside analogues 1–3.[12,15,16] as described in Scheme
1. The sugar moiety of compounds 2b and 3b was protected with
toluoyl group after the glycosylation reaction of the hydroxyl-bearing
nucleobases 2a and 3a with 1-chloro-3,5-ditoluoyl-2-deoxypentofuranose
(chlorosugar).[17]The toluoyl group would be cleaved by basic treatment
(0.1 M CH3ONa/CH3OH) in the next step. Therefore, the 5-positioned
hydroxyl group must be protected before this deprotection step and its
protecting group could go through such basic treatment. It is apparent
that acetyl and toluoyl groups were inappropriate for the protection of the
5-positioned hydroxyl group. Although a selective silylation with TBDMS
for 2b–3b could be realized in this synthetic scheme, it was observed
that TBDMS could be cleaved simultaneously with the 5ꢁ-toluoyl group by
the basic treatment. Another silyl group, however, tert-butyldiphenylsilyl
SCHEME 1 Synthesis of phosphoramidites 1f–3f. Reagents: (i) HMDS, CuI; (ii) TBDPSCl, imidazole;
(iii) 0.1 M MeONa/MeOH; (iv) DMTCl, in pyridine; (v) NC(CH2)2O(iPr)2NPCl, DIEA, in CH2Cl2.

Hydroxyl-Functionalized DNA
927
SCHEME 2 Synthesis of phosphoramidite 4d. Reagents: (i) 3-tert-butylediphenylsilyloxy-1-propyne or
3-hydroxy-1-propyne, Pd(PPh3)4, CuI, Et3N, in DMF; (ii) (n-Butyl)2NCH(OMe)2, in MeOH, room
temperature; (iii) DMTCl, in pyridine; (iv) NC(CH2)2O(iPr)2NPCl, DIEA, in CH2Cl2.
(TBDPS) in 2c–3c was stable enough to go through this basic treatment
to the key intermediates 2d–3d. More importantly, this protecting group
could be cleaved efficiently by incubation in conc. aq. ammonia or 1 M
TBAF/THF used in DNA/RNA synthesis.
After
the
determination
of
this
orthogonal
protecting
group, 5-hydroxymethyluracil was silylated or protected with
tert-butyldiphenylsilylchloride (TBDPS-Cl) in advance because of its
poor solubility in the glycosylation reaction (Scheme 1) to obtain the
silylated nucleobase 1a. 1c was obtained from the glycosylation reaction of
1a, and then it was deprotected with 0.1 M CH3ONa/CH3OH to obtain 1d.
Compounds 1d–3d were tritylated with DMT-Cl to offer 1e–3e, followed by
subsequent conversion to the phosphoramidites 1f–3f.[18]
In compound 4, the 3-hydroxypropynyl group was introduced by cross-
coupling reaction to the 7-position of 8-aza-7-deaza- deoxyadenosine 5[14]
(Scheme 2). For the synthesis of phosphoramidite 4d, the 7-positioned
hydroxyl group was protected before it was introduced to the purine base.
Tert-Butyldiphenylsilyl, tert-butyldimethylsilyl, and acetyl could be used in
this route. The presence of the triple bond required a mild-fast depro-
tection condition for the 7-positioned hydroxyl and the 6-amino groups,
for example, both concentrated aqueous ammonia at room temerpature
for 2–4 hours and 1 M TBAF/THF were tolerable for the triple bond.
TBDPS was used as the protecting group of the 7-positioned hydroxyl group
(4a), because it could be cleaved completely by incubation with conc. aq.
ammonia for 4 hours at room temperature, or 1 M TBAF/THF. The di-
n-butylaminomethylidene group was applied to protect the 6-amino group
(4b). 4b was further protected with DMT (4c) and then converted to the
phosphoramdite 4d. Nucleoside 5 and its phosphoramidite were synthesized
according to literature.[19]All the compounds were fully characterized by el-
emental analysis, 1H NMR, 13C NMR (Table 1), and/or UV data.

928
D. Zhang et al.
TABLE 1 13C NMR Chemical shifts of nucleoside derivatives thereofa
C(2)b,c C(4)b,c C(5)b C(6)b,c
,
C(6)c,d C(7a)c,d C(3a)d C(4)c,d C(3)d
C(1ꢁ) C(2ꢁ) C(3ꢁ) C(4ꢁ) C(5ꢁ)
1a
1c
1d
1e
2c
2d
2e
3c
3d
3e
4
151.2
150.0
150.2
150.2
150.3
150.4
150.2
150.2
150.3
150.2
157.7
157.6
157.1
157.4
163.4
162.2
162.3
162.2
163.0
163.3
163.1
163.2
163.3
163.2
156.8
156.8
155.4
156.0
111.2
112.9
112.8
113.2
110.8
110.2
110.4
113.9
113.1
113.7
100.8
100.9
106.1
108.0
138.2
136.4
136.5
135.1
137.2
137.6
137.1
135.6
136.0
135.6
153.7
153.7
155.3
154.4
81.4
84.0
83.9
81.1
83.8
84.0
81.2
83.9
83.8
84.0
84.1
83.8
83.9
36.7
74.6
70.6
70.6
74.5
70.7
70.5
74.7
70.5
70.6
71.0
71.0
71.3
70.6
85.2
87.4
85.6
84.3
87.4
85.7
84.5
87.3
85.5
87.8
87.8
87.6
85.3
64.3
61.7
64.2
64.3
61.8
63.8
64.2
62.9
63.7
62.4
62.4
63.0
64.2
e
e
36.1
e
e
36.2
e
126.7
126.3
127.8
126.4
37.9
38.0
37.9
38.1
4a
4b
4c
alkyl, alkynyl, phenyl, and carboxyl groups
1a
1c
1d
1e
2c
18.8, 26.6, 58.9, 128.0, 130.0, 132.7, 135.0
18.7, 21.2, 26.5, 58.8, 127.8, 129.2, 129.3, 129.4, 135.0, 143.8, 144.1, 165.2, 165.4
18.8, 26.6, 59.0, 128.0, 130.0, 132.6, 132.7, 135.1
18.7, 26.5, 59.0, 85.0, 126.7, 127.8, 127.9, 129..7, 129.9, 135.0, 144.8, 158.1
18.6, 21.2, 26.5, 29.9, 61.7, 126.5, 127.7, 129.3, 129.4, 129.7, 133.1, 134.9, 143.9, 144.0,
165.2, 165.5
2d
2e
3c
18.8, 26.7, 30.2, 61.4, 127.8, 129.8, 133.2, 135.0
18.7, 26.6, 30.0, 61.8, 85.3, 113.2, 127.6, 127.7, 127.8, 129.7, 133.1, 134.9, 144.8, 158.1
18.7, 21.1, 21.2, 23.0, 26.6, 31.0, 63.0, 126.5, 127.9, 129.3, 129.4, 129.5, 129.8, 133.2,
135.0, 143.9, 144.1, 165.3, 165.5
3d
3e
18.7, 22.9, 26.6, 30.6, 61.3, 127.8, 129.7, 133.2, 135.0
18.7, 23.0, 26.6, 31.1, 63.1, 85.8, 113.2, 127.7, 127.8, 129.7, 129.8, 133.2, 135.0, 135.2,
135.3, 144.6
4
49.8, 75.3, 95.2
4a
4b
18.8, 26.8, 53.2, 76.2, 93.2, 128.1, 130.1, 132.4, 135.1
13.6, 13.7, 18.7, 19.1, 19.8, 24.8, 26.6, 28.6, 30.4, 31.0, 45.3, 51.4, 62.6, 127.8, 129.7,
133.2, 134.9, 146.9, 162.5
4c
13.6, 18.8, 19.1, 19.6, 26.5, 28.6, 30.4, 44.9, 51.1, 52.8, 54.9, 77.9, 85.2, 90.3, 113.0, 127.6,
128.0, 129.6, 129.7, 130.1, 132.2, 135.1, 145.1, 157.9, 162.2
aMeasured in (D6)DMSO at 303 K.
bPyrimidine base numbering.
cTentative.
dSystematic numbering for purine base.
eSuperimposed by (D6)DMSO.
Oligonucleotides
The complementary duplex, 5ꢁ-d (GCG CGA TAA GGC CG)-3ꢁ (6),
5ꢁ-d (CGG CCT TAT CGC GC)-3ꢁ (7), was used in this study. In the
solid-phase synthesis of modified DNA sequences 8-19, the coupling yields
of the phosphoramidites 1f–3f and 4d (0.15– 0.2 M in acetonitrile)
were all above 92%, with 4 minutes of coupling time, in the context

Hydroxyl-Functionalized DNA
929
FIGURE 1 (A) HPLC purification profile of the DMT-on intermediate of sequence 8 after incubation
in concentrated aqueous ammonia at 60◦C for 18 hours. (B) HPLC purification profile of the DMT-on
intermediate of sequence 10 after incubation in concentrated aqueous ammonia at 55◦C for 19 hours
of the sequences. For the cleavage of TBDPS of compounds 1–4 in the
oligodeoxynucleotides 8–16, normal deprotection or mild deprotection
methods were used. The normal deprotection procedure was conducted
with the oligodeoxynucleotides containing 1–3. For the sequences 8–9
containing nucleoside 1, the optimum cleavage of TBDPS was performed
at 60◦C for 18 hours (8, (Figure 1A). For the sequences 10–13 containing
2 or 3, 55◦C and 19 hours in concentrated aqueous ammonia was enough
for a complete cleavage (10, Figure 1B). 1 M TBAF/THF was also efficient
for the cleavage of TBDPS in DMT-on or DMT-off sequences (such as 12),
which is compatible with ultra-mild DNA synthesis.[20] TBDPS has also been
used for 5-hydroxymethyl pyrimidine in PNA synthesis, but its cleavage
was conducted in a cold solution of trifluoroacetic acid and triethylsilane
which is not suitable for DNA deprotection.[21] The cleavage of TBDPS in
sequences 14–16 was also conducted in conc. aq. ammonia for 4 hours at
room temperature, a complete deprotection was obtained (Figure 2). The
sequences 6–19 were characterized with MALDI-TOF (Table 2) and some
representative sequences were analyzed by enzymatic hydrolysis to be the
expected hydroxyl-bearing oligodeoxynucleotides.
Thermal Stability of Hydroxyl-Functionalized DNA Duplexes
As presented in Table 3, the base pair 4-dT in the duplex was more
stable compared to the corresponding dA-dT and the unsubstituted 5-
dT,[22] and the increase of Tm was position dependent. More 4-dT base
pairs led to higher thermal stability of the modified duplexes. It is well
known that the planar propynyl linker contributes to the helix stability. The
functional group at the end of the linker also contributed to the duplex

930

Hydroxyl-Functionalized DNA
931
FIGURE 2 (A) HPLC purification profile of the DMT-on intermediate of sequence 14 after cleavage
with concentrated aqueous ammonia at room temperature for 4 hours and (B) the target sequence 14
after detritylation.
stability. Compared to the amino group (of 3-aminopropynyl),[23,24] the
hydroxyl group is a neutral group, which makes its contribution less than
aminopropynyl to the duplex and triplex stability.[13]
When the hydroxyl group was linked to the nucleobase by alkyl chains,
as shown in Table 3, the incorporation of the base pairs 1-dA, 2-dA, or 3-dA
in place of dT-dA, from one to three incorporations, led to the decrease
of the Tm values, and more incorporations resulted in lower Tm. The
effect of these hydroxyalkyl groups was contrary to that of the rigid planar
3-hydroxypropynyl, probably because these alkyl linkers were flexible and
not contributable to base stacking, and their hydrophobic property was not
compatible with the hydrophilic environment of the major groove. The
duplexes containing 3-dA were the least stable, because nucleoside 3 has
the longest alkyl chain with the most hydrophobic and flexible properties.
All the melting curves of the duplexes exhibited a typical sigmoidal
profile. The CD spectra of the control duplex and all the modified duplexes
showed a positive band at around 280 nm and a negative band at around
250 nm that were attributable to B-like DNA conformation. It indicated that
these hydroxyl-bearing base pairs did not result in significant deformation
on the overall duplex conformation.
CONCLUSIONS
The orthogonal protective group TBDPS was successfully applied for
the incorporation of 5-hydroxyalkyl deoxyuridine analogues and 7-(3-
hydroxypropynyl)-8-aza-7-deaza-deoxyadenosine to oligodeoxynucleotides.
It was compatible with DNA solid-phase synthesis with either normal or
ultra-mild deprotection method. Modification of DNA duplex with these

932
D. Zhang et al.
TABLE 3 The Tm values of duplexes containing compounds 1–4a
Duplex
Tm (◦C)
Duplex
Tm (◦C)
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·7)
68.1
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CG4 TAA GGC CG)-3ꢁ
(14·8)
69.3
70.0
69.6
71.8
72.4
67.6
66.7
65.2
5ꢁ- d(GCG CG5 TAA GGC CG)-3ꢁ
(17·7)
68.7
68.2
68.1
68.7
68.5
65.8
65.8
63.5
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·16)
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·19)
3ꢁ- d(CGC GCT 4TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T44 GGC CG)-3ꢁ
(15·7)
3ꢁ- d(CGC GCT 5TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T55 GGC CG)-3ꢁ
(18·7)
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CG4 TAA GGC CG)-3ꢁ
(14·16)
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CG5 TAA GGC CG)-3ꢁ
(17·19)
3ꢁ- d(CGC GCT 4TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T44 GGC CG)-3ꢁ
(15·16)
3ꢁ- d(CGC GCT 5TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA T55 GGC CG)-3ꢁ
(18·19)
3ꢁ- d(CGC GCT 4TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA 1AA GGC CG)-3ꢁ
(9·7)
3ꢁ- d(CGC GCT 5TT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·10)
3ꢁ- d(CGC GCT ATT CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·8)
3ꢁ- d(CGC GCT AT2 CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·11)
3ꢁ- d(CGC GCT AT1 CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·12)
3ꢁ- d(CGC GCT A22 CCG GC)-5ꢁ
5ꢁ- d(GCG CGA TAA GGC CG)-3ꢁ
(6·13)
3ꢁ- d(CGC GC3 ATT CCG GC)-5ꢁ
3ꢁ- d(CGC GCT A33 CCG GC)-5ꢁ
aMeasured in 100 mM NaCl, 10 mM MgCl2, and 10 mM Na-cacodylate (pH 7.0) with 5 µM
oligonucleotides.
hydroxyl-bearing nucleoside analogues resulted in thermal stability changes
which were dependent on the linker used for the hydroxyl group and
the position of the modified nucleosides in the duplex, but had no
significant influence on the whole duplex conformation. This kind of
modification affords extra hydrogen bonding and reactivity for the modified
oligodeoxynucleotides in the various applications of DNA in gene research
and biotechnologies.
EXPERIMENTAL
General
¨
Chemicals bought from ACROS were reagent grade or better and used
without further purification. Chloroform was refluxed and distilled from
P2O5, and pyridine was dried with calcium hydride. Silica gel TLC was
carried out on HS GF254 (Yantai Institute of Chemical Industry, China)

Hydroxyl-Functionalized DNA
933
and spotted by UV shadowing. Silica gel (200–300 mesh, Qingdaohaiyang
Chemicals. Co., China) was used for flash column chromatography. The
1H and 31P NMR were recorded on a JNM-ECA-400 spectrometer (JEOL,
Japan). Chemical shifts (δ) were in p.p.m. downfield from internal TMS
(1H, 13C) or external 85% H3PO4 (31P). The J values were given in Hz,
λmax in nm, and ε in dm3 mol-1 cm-1. Microanalyses were performed by
National Center of Biomedical Analysis (Beijing, China) on a Fisons-1108
(Fisons, Italy). Reversed phase (RP)-HPLC was performed on an SPD-10AT
(Shimadzu, Japan) equipped with XTerraꢀR MS column (C-18, 4.6 × 250
mm, 5 µm, Waters, USA). Solid-phase extraction was conducted on SEP-PAK
Cartridge (Oasis MAX, C18, Waters, USA). The solvent gradient system
for HPLC is A: 0.07 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN.
MALDI-TOF was performed on Kratos Axima-CFR-plus (Shimatzu, Japan)
with THAP (2ꢁ,4ꢁ,6ꢁ-trihydroxyacetophenone) as the matrix.
Silylation of Nucleobase-bearing Hydroxyl Groups
5-Tert-butyldiphenylsiloxymethyl-1H,3H-pyrimidin-2,4-dione (1a)
To a suspension of 5-hydroxymethyluracil (1 g, 7.0 mmol) in dried
THF (10 mL), TBDPS-Cl (2.3 g, 8.4 mmol ) and imidazole (0.58 g, 8.4
mmol ) were added. The mixture was stirred at room temperature until the
reaction was finished. Methanol (6 mL) was added to the mixture. After
stirring for 5 minutes, the solution was concentrated for purification by
flash chromatography (CH2Cl2/CH3OH from 80:1 to 40:1 as the eluent)
to obtain 1a as colorless solid (2.2 g, 84%). Rf (CH2Cl2/CH3OH 40:1): 0.2.
1H NMR (400 MHz, [D6]DMSO): δ = 1.01 (s, 9 H, tert-Bu), 4.36 (s, 2 H,
CH2), 7.27 (s, 1 H, 6-H), 7.41–7.66 (m, 10 H, arom. H), 10.74, 11.12 (2 s, 2
H, 2 NH). C21H24N2O3Si (M 380.51): C, 66.29; H, 6.36; N, 7.36. Found: C,
66.71; H, 6.57; N, 7.02.
1-(2-Deoxy-3,5-di-O-p-toluoyl-β-D-erythro-pentofuranosyl)-5-(2-tert-
butyldiphenylsilyloxyethyl)-1H,3H-pyrimidin-2,4-dione (2c)
To the solution of 3ꢁ,5ꢁ-di-O-p-toluoyl-5-(2-hydroxyethyl)-2ꢁ-deoxyuridine
(1.0 g, 1.97 mmol) in CH2Cl2 (40 mL), TBDPS-Cl (0.32 g, 27.5 mmol), and
imidazole (0.45 g, 6.62 mmol) were added. The solution was stirred at room
temperature for 4 hr. Methanol (4 mL) was added to the solution. After
stirring for 5 minutes, the solution was concentrated for flash chromatogra-
phy (CH2Cl2/CH3OH from 80:1 to 40:1 as the eluent). 2c was obtained as
colorless solid (1.4 g, 95.2%). Rf (CH2Cl2/CH3OH 20:1): 0.63. UV (MeOH):
λmax 206 (46 000), 242 (33 000). 1H NMR (400 MHz, [D6]DMSO): δ = 0.92
(s, 9 H, tert-Bu), 2.17–3.67 (m, 10 H, 2ꢁ-H, CH2CH2, 2 CH3), 3.65 (m, 2 H,
CH2CH2), 4.48–4.56 (m, 3 H, 4ꢁ-H, 5ꢁ-H), 5.60 (m, 1 H, 3ꢁ-H), 6.32 (t, J =
7.3, 1 H, 1ꢁ-H), 7.29–7.54, 7.87–7.94 (m, 19 H, 6-H, arom. H), 11.40 (s, 1 H,

934
D. Zhang et al.
NH). C43H46N2O8Si (M 746.92): C, 69.15; H, 6.21; N, 3.75. Found: C, 68.92;
H, 6.36; N, 3.96.
1-(2-Deoxy-3,5-di-O-p-toluoyl-β- D-erythro-pentofuranosyl)-5-(3-tert-
butyldiphenylsilyloxyprop-1-yl)-1H,3H-pyrimidin-2,4-dione (3c)
As described for 2c, from 3ꢁ, 5ꢁ-di-O-p-toluoyl-5-(3-hydroxypropyl)-2ꢁ-
deoxyuridine (9.7 g, 18.56 mmol), TBDPS-Cl (16.5 ml, 63.02 mmol) and
imidazole (4.27 g, 62.79 mmol) in CH2Cl2 (130 ml), 3c was obtained as
colorless foam (13.4 g, 95%). Rf (CH2Cl2/CH3OH 20:1): 0.59. UV (MeOH):
1
λmax 207 (75 000), 270 (14 600). H NMR (400 MHz, [D6]DMSO): δ =
0.96 (s, 9 H, tert-Bu), 1.58 (m, 2 H, CH2CH2CH2), 2.11 (t, J = 7.4, 2 H,
CH2CH2CH2), 2.29, 2.40 (2 s, 6 H, 2 CH3), 2.57 (m, 2 H, 2ꢁ-H), 3.50 (t, J =
6.3, 2 H, CH2CH2CH2), 4.50–4.63 (m, 3 H, 4ꢁ-H, 5ꢁ-H), 5.62 (m, 1 H, 3ꢁ-H),
6.30 (t, J = 7.10, 1 H, 1ꢁ-H), 7.24–7.94 (m, 19 H, 6-H, arom. H), 11.40 (s,
1 H, NH). C44H48N2O8Si (M 760.95): C, 69.45; H, 6.13; N, 3.68. Found: C,
69.18; H, 6.11; N, 3.88.
1-(2-Deoxy-3,5-di-O-p-toluoyl-β-D-erythro-pentofuranosyl)-5-tert-
butyldiphenylsilyloxymethyl-1H,3H-pyrimidin-2,4-dione (1c)
The suspension of 1a (1.5 g, 3.96 mmol) in hexamethyldisilazane
(HMDS) (10 mL, 48.0 mmol) was refluxed for 3 hours to a clear solu-
tion, with ammonium sulfate (0.052 g, 0.39 mmol) as the catalyst. Extra
HMDS was evaporated off under vacuum, and the residue was dissolved
in dried chloroform (100 mL). To the solution 1-chloro-2-deoxy-3,5-di-O-
α-D-toluoylpentose (1.5 g, 3.87 mmol) and CuI (0.42 g, 2.19 mmol) were
added. The mixture was stirred at room temperature overnight, and then
poured to sat. aq. NaHCO3. The organic layer was washed with water, dried
with anhydr. Na2SO4, and concentrated for flash chromatography (ethyl
acetate/petroleum ether from 15:1 to 8:1 as the eluent). The product was
obtained as colorless solid (1.13 g, 41 %). Rf (ethyl acetate/petroleum ether
1:2): 0.6. UV (MeOH): λmax 206 (44 600), 242 (14 600).1H NMR (400 MHz,
[D6]DMSO): δ = 0.94 (s, 9 H, tert-Bu), 2.33, 2.40 (2 s, 6 H, CH3), 2.55
(m, 2 H, 2ꢁ-H), 4.30 (m, 2 H, 5-CH2), 4.54 (m, 3 H, 4ꢁ-H, 5ꢁ-H), 5.55 (m,
1 H, 3ꢁ-H), 6.27 (t, J = 7.0, 1 H, 1ꢁ-H), 7.25–7.94 (m, 19 H, 6-H, arom. H).
C42H44N2O8Si (M 732.89): C, 68.83; H, 6.05; N, 3.82. Found: C, 68.72; H,
5.95; N, 3.87.
Deprotection Procedure for Compounds 1c–3c
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-tert-butyldiphenylsilyloxymethyl-
1H,3H-pyrimidin-2,4-dione (1d)
The solution of 1c (1.8 g, 2.46 mmol) in 0.1 M CH3ONa/ CH3OH
(50 mL) was stirred at room temerpature for 2 hours. Then it was neu-
tralized with glacial acetic acid. The solution was concentrated for flash

Hydroxyl-Functionalized DNA
935
chromatography (CH2Cl2/CH3OH from 40:1 to 15:1 as the eluent) to
obtain the product as colorless foam (0.94 g, 75.8%). Rf (CH2Cl2/CH3OH
1
9:1): 0.50. UV (MeOH): λmax 209 (49 000), 229 (50 000). H NMR (400
MHz, [D6]DMSO): δ = 1.02 (s, 9 H, tert-Bu), 2.03 (m, 2 H, 2ꢁ-H), 3.51 (m, 2
H, 5ꢁ-H), 3.79 (m, 1 H, 4ꢁ-H), 4.22 (m, 1 H, 3ꢁ-H), 4.39 (s, 2 H, 5-CH2), 4.92
(t, J = 5.3, 1 H, C5ꢁ-OH), 5.28 (d, J = 4.2, 1 H, 3ꢁ-OH), 6.19 (t, J = 6.7, 1 H,
1ꢁ-H), 7.42–7.66 (m, 10 H, arom. H), 7.74 (s, 1 H, 6-H), 11.39 (s, 1 H, NH).
C26H32N2O6Si (M 496.63): C, 62.88; H, 6.49; N, 5.64. Found: C, 62.48; H,
6.57; N, 5.39.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-(2-tert-butyldiphenylsilyloxyethyl)-
1H,3H-pyrimidin-2,4-dione (2d)
As described for 1d, 2c (1.2 g, 1.6 mmol) was dissolved in 0.1 M
CH3ONa/ CH3OH (50 mL), and the solution was stirred for 2 hours. After
neutralization with glacial acetic acid, the solution was concentrated for
flash chromatography (CH2Cl2/CH3OH from 40:1 to 15:1 as the eluent)
to obtain the product as colorless foam (0.78 g, 95%), Rf (CH2Cl2/CH3OH
1
9:1): 0.54. UV (MeOH): λmax 250 (12 900), 287 (14 200). H NMR (400
MHz, [D6]DMSO): δ = 0.97 (s, 9 H, tert-Bu), 2.02 (m, 2 H, 2ꢁ-H), 2.50 (m,
2 H, CH2CH2), 3.56 (m, 2 H, CH2CH2), 3.72–3.79 (m, 3 H, 4ꢁ-H, 5ꢁ-H), 4.23
(m, 1 H, 3ꢁ-H), 5.01 (t, J = 4.9, 1 H, 5ꢁ-OH), 5.22 (d, J = 3.9, 1 H, 3ꢁ-OH),
6.18 (t, J = 6.9, 1 H, 1ꢁ-H), 7.75 (s, 1 H, 6-H), 7.38–7.58 (m, 10 H, arom. H),
11.28 (s, 1 H, NH). C27H34N2O6Si (M 510.65): C, 63.50; H, 6.71; N, 5.49.
Found: C, 63.06; H, 6.56; N, 5.40.
1-2-Deoxy-β- D-erythro-pentofuranosyl)-5-(3-tert-butyldiphenylsilyloxyprop-1-
yl)-1H,3H-pyrimidin-2,4-dione (3d)
As described for 1d, the solution of 3c (14.6 g, 19.2 mmol) in 0.1 M
CH3ONa/ CH3OH (500 mL) was stirred at room temperature for 2 hours.
3d was obtained as colorless foam (10.5 g, 98%), Rf (CH2Cl2/CH3OH 9:1):
1
0.68. UV (MeOH): λmax 207 (47 200), 250 (12 900), 287 (14 200). H
NMR (400 MHz, [D6]DMSO): δ = 0.99 (s, 9 H, tert-Bu), 1.72 (m, 2 H,
CH2CH2CH2), 2.04 (m, 2 H, 2ꢁ-H), 2.30 (m, 2 H, CH2CH2CH2), 3.54 (m,
2 H, CH2CH2CH2), 3.64 (m, 2 H, 5ꢁ-H2), 3.77 (m, 1 H, 4ꢁ-H), 4.22 (m, 1
H, 3ꢁ-H), 4.98 (t, J = 5.2, 1 H, 5ꢁ-OH), 5.22 (d, J = 4.2, 1 H, 3ꢁ-OH), 6.16
(t, J = 6.9, 1 H, 1ꢁ-H), 7.39–7.65 (m, 11 H, arom. H, 6-H), 11.26 (s, 1 H,
NH). C28H36N2O6Si.0.5H2O (M 533.68): C, 62.96; H, 6.93; N, 5.25. Found:
C, 62.70; H, 6.90; N, 5.09.
Tritylation of 5ꢁ-Hydroxyl Group of Compounds 1d–3d
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-tert-
butyldiphenylsilyloxymethyl-1H,3H-pyrimidin-2,4-dione (1e)
Compound 1d (1.4 g, 2.8 mmol) was firstly dried by repeated co-
evaporation with anh. pyridine (3 × 5 mL), then it was dissolved in dried

936
D. Zhang et al.
pyridine (4 mL). To the stirred solution DMT-Cl (1.15 g, 3.36 mmol) was
added in portions. After stirring at room temperature for 4 hours, methanol
(4 mL) was added to the mixture, and stirring was continued for 15 minutes.
The mixture was concentrated and then co-evaporated with methanol (3 ×
5 mL), the residue was dissolved in CH2Cl2 (3 mL) for flash chromatography
(CH2Cl2/CH3OH from 80:1 to 40:1 as the eluent) to obtain the product as
colorless foam (1.8 g, 80%). Rf (CH2Cl2/CH3OH 40:1): 0.3. UV (MeOH):
λmax 208 (57 500), 266 (11 500). 1H NMR (400 MHz, [D6]DMSO): δ = 0.88
(s, 9 H, tert-Bu), 2.08, 2.21 (2 m, 2 H, 2ꢁ-H), 3.16 (m, 2 H, 5ꢁ-H), 3.68 (s, 6 H,
2 CH3O), 3.89 (m, 1 H, 4ꢁ-H), 4.07 (m, 1 H, 3ꢁ-H), 4.27 (m, 2 H, CH2), 5.42
(d, J = 4.5, 1 H, 3ꢁ-OH), 6.20 (t, J = 6.3, 1 H, 1ꢁ-H), 6.82–7.58 (m, 24 H,
6-H, arom. H). C47H50N2O8Si (M 798.99): C, 70.65; H, 6.31; N, 3.51. Found:
C, 70.31; H, 6.18; N, 3.57.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-(2-tert-
butyldiphenylsilyloxyethyl)-1H,3H-pyrimidin-2,4-dione (2e)
As described for 1e, with 2d (5.6 g, 11 mmol) and DMT-Cl (4.87 g, 14.37
mmol) in dried pyridine (15 mL), 2e was obtained as colorless foam (8.86
g, 98 %) after purification with flash chromatography (CH2Cl2/CH3OH
from 80:1 to 40:1 as the eluent). Rf (CH2Cl2/CH3OH 20:1): 0.44. UV
1
(MeOH): λmax 205 (52 000), 249 (37 800), 326 (49 500). H NMR (400
MHz, [D6]DMSO): δ = 0.91 (s, 9 H, tert-Bu), 2.09 (m, 2 H, 2ꢁ-H), 2.27 (t, J
= 1.6, 2 H, CH2CH2), 3.18 (m, 2 H, 5-CH2CH2), 3.65 (m, 2 H, 5ꢁ-H), 3.70
(s, 3 H, OCH3), 3.88 (m, 1 H, 4ꢁ-H), 4.21 (m, 1 H, 3ꢁ-H), 5.34 (d, J = 4.5, 1
H, 3ꢁ-OH), 6.17 (t, J = 6.7, 1 H, 1ꢁ-H), 6.85–7.53 (m, 24 H, 6-H; arom. H),
11.34 (s, 1 H, NH). C48H52N2O8Si (M 813.02): C, 70.91; H, 6.45; N, 3.45.
Found: C, 70.83; H, 6.40; N, 3.58.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-(3-tert-
butyldiphenylsilyloxyprop-1-yl)-1H,3H-pyrimidin-2,4-dione (3e)
As described for 1e, with 3d (1 g, 1.91 mmol) and DMT-Cl (0.77 g, 2.3
mmol) in dried pyridine (2 mL), 3e was obtained as colorless foam (1.3
g, 82.5%) after purification with flash chromatography (CH2Cl2/CH3OH
from 80:1 to 40:1 as the eluent). Rf (CH2Cl2/CH3OH 20:1): 0.49. UV
(MeOH): λmax 208 (82 500), 284 (30 500), 329 (30 300). 1H NMR (400 MHz,
[D6]DMSO): δ = 0.94 (s, 9 H, tert-Bu), 1.51 (m, 2 H, CH2CH2CH2), 1.92 (m,
2 H, CH2CH2CH2), 2.16 (m, 2 H, 2ꢁ-H), 3.17 (m, 2 H, CH2CH2CH2), 3.40
(m, 2 H, 5ꢁ-H), 3.67 (2 s, 6 H, 2 OCH3), 3.88 (m, 1 H, 4ꢁ-H), 4.28 (m, 1 H,
3ꢁ-H), 5.32 (d, J = 4.5, 1 H, 3ꢁ-OH), 6.19 (t, J = 6.9, 1 H, 1ꢁ-H), 6.82–7.57
(m, 24 H, 6-H, arom.H), 11.32 (s,1 H, NH). C49H54N2O8Si (M 827.05): C,
71.16; H, 6.58; N 3.39. Found: C, 71.30; H, 6.65; N, 3.22.

Hydroxyl-Functionalized DNA
937
Phosphitylation Procedure for Compounds 1e–3e
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-tert-
butyldiphenylsilyloxymethyl-1H,3H-pyrimidin-2,4-dione 3ꢁ-[(2-cyanoethyl)
N,N-diisopropylphosphoramidite] (1f)
To a solution of 1e (0.8 g, 1.00 mmol) and diisopropylethylamine (1
mL, 6.05 mmol) in CH2Cl2 (10 mL) was added 2-cyanoethyl diisopropy-
lphosphoramido chloridite (0.45 mL, 2.0 mmol) until complete conver-
sion. The solution was diluted with CH2Cl2 (20 mL), washed with 5%
aqueous NaHCO3 (30 mL) and sat. brine (30 mL), dried with anhydr.
Na2SO4, and concentrated to an oily residue for flash chromatography
(CH2Cl2/CH3COCH3 from 60:1 to 40:1 as the eluent). The product was
obtained as a mixture of two isomers (0.8 g, 80%). Rf (CH2Cl2/CH3COCH3
20:1) 0.47, 0.64. 1H NMR (400 MHz, CDCl3): δ = 0.99 (s, 9 H, tert-Bu), 1.17
[m, 12 H, 2 N (CH(CH3)2)], 2.63, 2.76 (2 t, J = 5.9, 2 H, NCCH2CH2O),
3.20 (m, 2 H, 5ꢁ-H), 3.45–3.75 [m, 10 H, NCCH2CH2O, 2 CH3O, 2 N
(CH(CH3)2)], 4.02 (m, 1 H, 4ꢁ-H), 4.27 (m, 3 H, CH2, 3ꢁ-H), 6.17 (m, 1
H, 1ꢁ-H), 6.81–7.59 (m, 24 H, 6-H; arom. H). 31P NMR (400 MHz, CDCl3):
148.18, 148.72.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-(2-tert-
butyldiphenylsilyloxyethyl)-1H,3H-pyrimidin-2,4-dione 3ꢁ-[(2-cyanoethyl)
N,N-diisopropylphosphoramidite] (2f)
As described for 1f, with 2e (1.8 g, 2.21 mmol), diisopropylethylamine
(2.5 mL, 15.13 mmol), and 2-cyanoethyl diisopropylphosphoramido chlo-
ridite (0.9 mL, 4.06 mmol) in CH2Cl2 (30 mL), 2f was obtained as
colorless foam (1.33 g, 59%). Rf (CH2Cl2/CH3COCH3 20:1): 0.65, 0.75.
1H NMR (400 MHz, CDCl3): δ = 0.99 (s, 9 H, tert-Bu), 1.08, 1.12 [m, 12
H, 2N (CH(CH3)2)], 2.27 (m, 2 H, 2ꢁ-H), 2.11–2.64 (m, 6 H, CH2CH2,
NCCH2CH2O), 3.30–3.78 (m, 6 H, CH2CH2, NCCH2CH2O, 5ꢁ-H), 3.74 (2
s, 6 H, OCH3), 4.17 (m, 1 H, 4ꢁ-H), 4.52 (m, 1 H, 3ꢁ-H), 6.28 (t, J = 6.9, 1
H, 1ꢁ-H), 6.79–7.56 (m, 24 H, 6-H; arom. H). 31P NMR (400 MHz, CDCl3):
149.06, 149.65.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-β-D-erythro-pentofuranosyl]-5-(3-tert-
butyldiphenylsilyloxyprop-1-yl)-1H,3H-pyrimidin-2,4-dione 3ꢁ-[(2-cyanoethyl)
N,N-diisopropylphosphoramidite] (3f)
As described for 1f, with 3e (1 g, 1.21 mmol), diisopropylethylamine (0.6
mL, 2.7 mmol), and 2-cyanoethyl diisopropylphosphoramido chloridite (1.2
mL, 5.4 mmol) in CH2Cl2 (20 mL), 3f was obtained as colorless foam (0.65
1
g, 52.4%). TLC (CH2Cl2/CH3COCH3 20:1) Rf 0.60, 0.63. H NMR (400
MHz, CDCl3): δ = 0.98 (s, 9 H, tert-Bu), 1.21, 1.29 [m, 6 H, N(CH(CH3)2)],
1.46–2.73 [m, 14 H, 2 N(CH(CH3)2), 5-CH2CH2CH2, 2ꢁ-H], 2.41 (t, J = 6.5,
2 H, CH2CH2CH2), 3.30–3.68 (m, 6 H, CH2CH2CH2, NCCH2CH2O, 5ꢁ-H),

938
D. Zhang et al.
3.71 (2 s, 6 H, 2 OCH3), 4.17 (m, 1 H, C4ꢁ-H), 4.60 (m, 1 H, 3ꢁ-H), 6.35 (m,
1 H, 1ꢁ-H), 6.77–7.61 (m, 24 H, 6-H, arom. H). 31P NMR (400 MHz, CDCl3):
149.06, 149.53.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-(prop-1-ynyl)-1H-pyrazolo[3,
4-d]pyrimidin-4-amine (4)
To the solution of compound 7-iodo-8-aza-7-deaza-2ꢁ-deoxyadenosine
(1.95 g, 5.17 mmol) in DMF (10 ml) was added Pd [P(C6H5)3]4 (300
mg, 0.25 mmol) and CuI (200 mg, 1.04 mmol), the mixture was stirred
under N2 at room temperature 3-hydroxypropyne (0.28 ml, 5 mmol) and
triethylamine (2.0 ml, 11.48 mmol) were added to the mixture subsequently.
The stirring was continued overnight. The solvent was evaporated off under
vacuum and the residue was subjected to flash chromatography, yielding
the product as colorless solid (1.45 g, 92%). TLC (CH2Cl2/CH3OH 5:1) Rf
0.58. UV (MeOH): λmax 207 (24 500), 249 (11 400), 286 (13 000). 1H NMR
(400 MHz, [D6]DMSO): δ = 2.22 (m, 1 H, C2ꢁ-Hα), 2.72 (m, 1 H, C2ꢁ-Hβ),
3.25–3.48 (m, 2 H, C5ꢁ-H), 3.81 (m, 1 H, C4ꢁ-H), 4.38–4.43 (d, J = 6.16, 3
H, 5-C≡CCH2OH, C3ꢁ-H), 4.76 (t, J = 5.9, 1 H, C5ꢁ-OH), 5.27 (d, J = 4.48,
1 H, C3ꢁ-OH), 5.47 (t, J = 6.02, 2 H, 5-C≡CCH2OH), 6.50 (t, J = 6.44, 1 H,
C1ꢁ-OH), 6.66, -8.09 (br, 2 H, NH2). C13H15N5O4 (M 305.29): C, 51.14; H,
4.95; N, 22.94. Found: C, 51.18; H, 4.86; N, 22.92.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-3-(3-tert-butyldiphenylsilyloxyprop-1-
ynyl)-1H-pyrazolo[3, 4-d]pyrimidin-4-amine (4a)
The coupling reaction of 7-iodo-8-aza-7-deaza-2ꢁ-deoxyadenosine (0.38
g, 1.0 mmol) with 3-tert-butyldiphenylsilyloxypropyne (0.57 g, 2.0 mmol)
was conducted with the procedure described for compound 4, with
Pd[P(C6H5)3]4 (100 mg, 0.09 mmol) and CuI (60 mg, 0.33 mmol) as
the catalysts and triethylamine (0.5 ml, 2.87mmol) as the base in DMF (5
ml). After purification by flash chromatography, the product was yielded
as colorless solid (0.45 g, 84%). TLC (CH2Cl2/CH3OH 9:1) Rf 0.58. UV
1
(MeOH): λmax 206 (44 100), 250 (12 800), 287 (13 700). H NMR (400
MHz, [D6]DMSO): δ = 1.0 (s, 9 H, tert-Bu), 2.29 (m, 1 H, C2ꢁ-Hα), 2.80 (m,
1 H, C2ꢁ-Hβ), 3.56, 3.41 (2 m, 2 H, C5ꢁ-H), 3.85 (m, 1 H, C4ꢁ-H), 4.47 (m,
1 H, C3ꢁ-H), 4.73 (s, 2 H, 5-C≡CCH2), 4.81 (t, J = 5.8, 1 H, C5ꢁ-OH), 5.32
(d, J = 4.4, 1 H, C3ꢁ-OH), 6.58 (t, J = 6.4, 1H, C1ꢁ-H), 7.72–7.44 (2m, 10 H,
arom. H), 8.26 (s, 1 H, C2-H). C29H33N5O4Si.(M 543.69): C, 64.06; H, 6.12;
N, 12.88. Found: C, 63.56; H, 6.06; N 12.76.
1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(di-n-
butylamino)methylidene]amino}-3-(3-tert-butyldiphenylsilyloxyprop-1-ynyl)-
1H-pyrazolo[3, 4-d]pyrimidine (4b)
To the solution of compound 4a (1.16 g, 2.19 mmol) in methanol
(10 ml) N, N -di-n-butylformamide dimethyl acetal (0.45 g, 2.2 mmol)

Hydroxyl-Functionalized DNA
939
was added. After being stirred at 40◦C for 4 h, the reaction mixture
was evaporated under vacuum, and the residue was subjected to flash
chromatography. The product was yielded as colorless foam (1.3 g, 91%).
TLC (CH2Cl2/CH3OH 15:1) Rf 0.57. UV (MeOH): λmax 218 (47 000), 329
1
(27 700). H NMR (400 MHz, [D6]DMSO): δ = 0.76, 0.90 [2 t, 6 H, N =
CHN(CH2CH2CH2CH3)2]. 1.03 (s, 9 H, tert-Bu), 1.16, 1.25, [2 m, 4 H, N =
CHN(CH2CH2CH2CH3)2], 1.51 [m, 4 H, N = CHN(CH2CH2CH2CH3)2],
2.29 (m, 1 H, C2ꢁ-Hα), 2.82 (m, 1 H, C2ꢁ-Hβ), 3.50 [m, 6 H, N =
CHN(CH2CH2CH2CH3)2, C5ꢁ-H], 3.84 (m, 1 H, C4ꢁ-H), 4.46 (m, 1 H,
C3ꢁ-H), 4.64 (s, 2 H, 5-C≡CCH2), 4.79 (t, J = 5.7, 1 H, C5ꢁ-OH), 5.32 (d,
J = 4.5,1 H, C3ꢁ-OH), 6.61 (t, J = 6.3, 1 H, C1ꢁ-H), 7.70∼7.42 (m, 10 H,
arom. H), 8.47 (s, 1 H, C2-H), 8.95 [s, 1 H, N = CHN(CH2CH2CH2CH3)2].
C38H50N5O4Si (M 682.93): C, 66.83; H, 7.38; N, 12.31. Found: C, 66.57; H,
7.53; N, 11.98.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-4-
˝[(di-n-butylamino)methylidene]amino˝-3-(3-tert-butyldiphenylsilyloxyprop-
1-ynyl)-1H-pyrazolo[3, 4-d]pyrimidin-4-amine (4c)
As described for 1e, with compound 4b (1.6 g, 2.39 mmol) and 4, 4ꢁ-
dimethyltriphenylchloride (DMT-Cl) (0.97 g, 2.87 mmol) in dried pyridine
(5ml), the product 4c was obtained as colorless foam (1.46 g, 63%), TLC
(CH2Cl2/CH3OH 20:1) Rf 0.39. UV (MeOH): λmax 206 (82 300), 328 (29
1
800). H NMR (400 MHz, [D6]DMSO): δ = 0.73, 0.90 [2 t, 6 H, N =
CHN(CH2CH2CH2CH3)2], 1.01 (s, 9 H, tert-Bu), 1.14, 1.25 [2 m, 4 H, N =
CHN(CH2CH2CH2CH3)2], 1.50 [m, 4 H, N = CHN(CH2CH2CH2CH3)2],
2.33 (m, 1 H, C2ꢁ-Hα), 2.82 (m, 1 H, C2ꢁ-Hβ), 3.06 (m, 2 H, C5ꢁ-H), 3.47 [m,
4 H, N = CHN(CH2CH2CH2CH3)2], 3.66, 3.65 (2 s, 6 H, 2 OCH3), 3.94
(m, 1 H, C4ꢁ-H), 4.55 (m, 1 H, C3ꢁ-H), 4.63 (s, 2 H, 5-C≡CCH2), 5.35 (d, J
= 4.8, 1 H, C3ꢁ-OH), 6.63 (m, 1 H, C1ꢁ-H), 7.70∼6.70 (m, 23 H, arom. H),
8.49 (s, 1 H, C2-H), 8.95 [s, 1 H, N = CHN(CH2CH2CH2CH3)2]. 13C NMR
(400 MHz, [D6]DMSO): 13.6, 18.8, 19.1, 19.6, 26.5, 28.6, 30.4, 38.1, 44.9,
51.1, 52.8, 54.9, 64.2, 70.6, 77.9, 83.9, 85.2, 85.3, 90.3, 108.0, 113.0, 126.4,
127.6, 128.0, 129.6, 129.7, 130.1, 132.2, 135.1, 145.1, 154.4, 156.0, 157.4,
157.9, 162.2. C59H68N6O6Si (M 985.29): C, 71.92; H, 6.96; N, 8.53. Found:
C, 71.91; H, 7.01; N, 8.40.
1-[2-Deoxy-5-O-(4,4ꢁ-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-4-
{[(di-n-butylamino)methylidene]amino}-3-(3-tert-butyldiphenylsilyloxyprop-1-
ynyl)-1H-pyrazolo[3, 4-d]pyrimidine 3ꢁ-[(2-cyanoethyl)
N,N-diisopropylphosphoramidite] (4d)
As described for 1f, this phosphoramidite was prepared from the
reaction of compound 4c (1.0 g, 1.03 mmol) with 2-cyanoethyl N ,
N ꢁ-(diisopropyl)-phosphoramidochloridite (0.5 g, 2.1 mmol) in dried

940
D. Zhang et al.
dichloromethane (10 ml) in the presence of diisopropylethylamine (1 ml,
6.05 mmol). The product 4d was obtained as colorless foam (0.57 g, 47.3%),
TLC (CH2Cl2/CH3OH 30:1) Rf 0.66, 0.75. 1H NMR (400 MHz, CDCl3): δ =
0.82, 0.95 [2 t, 6 H, N = CHN(CH2CH2CH2CH3)2], 1.01 (s, 9 H, tert-Bu),
1.10–1. 57 [m, 14 H, NCH(CH3)2, N = CHN(CH2CH2CH2CH3)2], 2.63,
2.45 (2 m, 2 H, C2ꢁ-H), 3.15–3.74 [m, 10 H, N = CHN(CH2CH2CH2CH3)2,
C5ꢁ-H, OCH2CH2CN], 3.72, 3.71 (2 s, 6 H, 2 OCH3), 4.23 (m, 1 H, C4ꢁ-H),
4.59 (s, 2 H, 5-C≡CCH2), 4.80 (t, 1 H, C3ꢁ-H), 7.75∼6.71 (m, 24 H , arom.
H, C1ꢁ-H), 8.50 (s, 1 H, C2-H), 8.84 (s, 1 H, N = CHN(CH2CH2CH2CH3)2).
31P NMR (400 MHz, CDCl3): 149.06, 148.90.
Synthesis and Purification of Oligodeoxynucleotides
The oligodeoxynucleotide synthesis was carried out on an ABI 392
DNA/RNA synthesizer (Applied Biosystems, USA) with 1 µmol scale, ac-
cording to the User Protocol for phosphoramidite chemistry with DMT-on
or DMT-off mode. For the sequences containing compounds 1-3, normally
protected phosphoramidites were used. The sequences containing nucleo-
side 1 were deprotected in conc. aq. ammonia at 60◦C for 18 hours; and
19 hours at 55◦C for the sequences containing nucleoside 2 or 3. The
DMT-on sequences 14–16 containing compound 4 were synthesized with
tac(tert-butylphenoxyacetyl)-protected phosphoramidites. TBDPS could be
cleaved by incubation in conc. aq. ammonia at room temperature for 4
hours.
The TBDPS of the DMT-on or DMT-off sequences containing 1–4 was
also cleaved with 1 M TBAF/THF efficiently. After the DMT-on or DMT-
off sequence was cleaved from CPG, the ammonic soln. was concentrated
and dried; the residue was then dissolved in 1 M TBAF/THF and incubated
at room temperature overnight. The soln. was desalted with SEP-PAK for
HPLC purification.
The DMT-on oligodeoxynucleotide was purified by HPLC with the
Gradient One (0–20 minutes, 10–50% B in A with a flow rate of 1.0 ml/min)
and pooled together; the solution was concentrated and extracted with SEP-
PAK column. After washing the column with water, 2.5% trifluoroacetic acid
was eluted through the column to remove the DMT group. The DMT-off
oligodeoxynucleotide was then eluted with methanol/water (70:30, V/V)
for further purification by HPLC with gradient two (0–20 minutes, 0–16 %
B in A, with a flow rate of 1 ml/min).
The purified oligodeoxynucleotides were desalted on SEP-PAK columns,
lyophilized and stored at −18◦C. The homogeneous product was character-
ized by MALDI-TOF and enzymatic hydrolysis[17] with snake venom phos-
phodiesterase (EC 3.1.15.1, Crotallus adamanteus) and alkaline phosphatase
(EC 3.1.3.1, E. coli) (Sigma, USA). The following extinction coefficients ε260
were used: dA 15400, dG 11700, dT 8800, and dC 7300, the extinction

Hydroxyl-Functionalized DNA
941
coefficients of compounds 1–4 were assumed to be the same as that of
canonical nucleosides.
Tm Measurement
The Tm measurement of the complementary oligodeoxynucleotides
was performed with a Cary-100 Bio UV-Visible spectrophotometer (Varian,
USA) equipped with a Cary temperature controller. The buffer used for
the measurement consisted of 100 mM NaCl, 10 mM MgCl2 and 10 mM
sodium cacodylate (pH 7.0)[17] with 5 µM of each oligodeoxynucleotide.
The two complementary oligodeoxynucleotides were mixed in the buffer
(1 ml), and the solution was heated to 85◦C. After halting for 10 minutes, it
was cooled to 35◦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.
Circular Dichroism Measurement
The CD experiments were conducted on a Jasco J-810 spectropolarime-
ter equipped with a Jasco PTC-423S temperature controller. The duplex so-
lution was the same as that for Tm measurement. Before the measurement,
the sample was cooled to 5◦C in a cuvette with a path length of 10 mm. The
ellipticities of duplexes were recorded from 220 to 350 nm. The spectra
were the averages of three accumulations and smoothed using a 25-point
adaptive smoothing algorithm.
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