Studies on the two thymine residues in the catalytic core of 10-23 DNAzyme: The impact on the catalysis of their 5-substituted functional groups

Article abstract of DOI:10.3390/molecules22071011

In the 15-mer catalytic core of 10-23 DNAzyme, each residue contributes to the catalytic conformation differently. Here, the critically conserved T4 and the least conserved T8 were modified on their 5-position with hydroxyl, imidazolyl, and amino groups with a hydrogen-bonding ability. These external functional groups induced new interactions within the catalytic core, resulting in both negative and positive effects on the catalytic activity of 10-23 DNAzyme, and the different linkages could be used to modulate the effect of the functional groups. The conservation of T4 and T8 could be recognized at the level of the nucleobase, but at the level of the functional group, T4 is not completely conserved. Their 5-methyl groups could be modified for a better performance in terms of the DNAzyme.

Full text of DOI:10.3390/molecules22071011

molecules
Article
Studies on the Two Thymine Residues in the
Catalytic Core of 10-23 DNAzyme: The Impact on the
Catalysis of Their 5-Substituted Functional Groups
Pengyu Li 1, Shanshan Du 1, Yang Li 1 and Junlin He 2,
*
1
College of Life Sciences, Guizhou University, Guiyang 550025, China; lipengyu1@126.com (P.L.);
shanshandu1992@163.com (S.D.); liyang920312@163.com (Y.L.)
Laboratory of Bioorganic chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
Correspondence: hejunlin@bmi.ac.cn; Tel.: +86-010-6821-7656
2
*
Received: 26 May 2017; Accepted: 15 June 2017; Published: 22 June 2017
Abstract: In the 15-mer catalytic core of 10-23 DNAzyme, each residue contributes to the catalytic
conformation differently. Here, the critically conserved T4 and the least conserved T8 were modified
on their 5-position with hydroxyl, imidazolyl, and amino groups with a hydrogen-bonding ability.
These external functional groups induced new interactions within the catalytic core, resulting in
both negative and positive effects on the catalytic activity of 10-23 DNAzyme, and the different
linkages could be used to modulate the effect of the functional groups. The conservation of T4 and T8
could be recognized at the level of the nucleobase, but at the level of the functional group, T4 is not
completely conserved. Their 5-methyl groups could be modified for a better performance in terms of
the DNAzyme.
Keywords: 10-23 DNAzyme; chemical modifications; thymine; functional group; catalytic core;
observed catalytic constants
1. Introduction
10-23 DNAzyme is a small catalytic DNA molecule selected from artificial in vitro selection [
Its catalytic cleaving ability on complementary RNA has attracted much attention, from practical
therapeutics [ ] to catalytic mechanism studies [ ]. Furthermore, it is an ideal model for studying
1].
2
–4
5,6
the artificial functions and related tertiary structures of DNA molecules of non-genetic carriers,
especially when no counterpart of DNAzyme has yet been found in nature. The composition of
the secondary motifs (the duplex-loop-duplex) seems to be clear and simple, but no profile has
been generated for the tertiary structure and the critical catalytic conformation, in which the specific
catalytic groups conduct the cleavage reaction with the help of Mg2+. Screening approaches have been
used for the nucleobase [7–9], sugar-phosphate moiety [10], and non-bridging oxygen atom of the
phosphate group [11]. It could not be concluded that any residues or functional groups are directly
involved in the catalytic reaction, but the importance of each nucleotide has been recognized by these
screening approaches. Among the 15 residues in the catalytic core, T8 was suggested to be the least
conserved residue, which is in contrast to the other highly conserved T4 residue, indicating their
unique position-dependent contributions to the catalytic activity.
Considering the completely different conservation of T4 and T8, several chemical modifications
have been conducted to gain an insight into the structural basis. When phosphorothioate linkage and
2’-O-methyl were applied to T8, the two most frequently used chemical modifications for providing the
resistance of oligonucleotides against nucleases, no significant negative effect on the cleavage activity of
the DNAzyme was observed [12
,13]. In contrast, T4 is always negatively affected by these modifications.
But, it is noteworthy that both T4 and T8 are sensitive to its sugar conformation, as demonstrated by
Molecules 2017, 22, 1011; doi:10.3390/molecules22071011
www.mdpi.com/journal/molecules

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(R)- and (S)-2’-C-methyl-thymidine and LNA [14
,
15]. The nucleobase thymine was also demonstrated
to be sensitive to the modifications. A large caging group (6-nitropiperonyloxymethyl, NPOM) at N-1
of thymine at T4 could abrogate the catalytic activity completely, while partial activity remained with
the caged thymine of T8 [16]. The 5-substituted aromatic group of thymine could produce a favorable
or unfavorable effect at T4 and T8, depending on its geometric configuration [17]. The two thymine
residues of T4 and T8 were further modified with imidazolyl and ester groups, and the extra functional
groups at T8 were found to be helpful for the catalytic reaction, even in the absence of Mg2+
[18].
When T4 was conducted for these modifications, a negative effect was mostly observed, in agreement
with its critical conservation.
These studies demonstrated that the effect of T4 and T8 can be modulated by changing the
interactions around the two thymine residues. Here, we report new modifications on the two thymine
residues with the hydroxyl group (in compounds
1 and 2), imidazolyl group (in compound 3), and amino
group (in compounds ) at the 5-position by different linkages (Scheme 1). More hydrogen-bonding
4–7
interactions around T4 and T8 were expected to have an effect on the catalytic reaction of
10-23 DNAzyme (Scheme 1) against VEGFR 2 mRNA [19], and it was hoped that a chemical
modification approach for a positive effect was found.
5'- AGG TGC AGG A UGG AGA GCA- 3'
O
N
O
N
HN
O
N
3'- TCC ACG TCC
ACC TCT CGT- 5'
OH
N
HN
OH
HN
HN
A
G
G
C
G
O
O
O
13
C
HO
HO
HO
4
O
O
A
A
C
A
T
O
A
10-23 DNAzyme
G
OH
1
OH
2
OH
3
C
T
8
O
O
O
N
O
O
N
O
H
H
N
N
NH2
NH2
NH2
NH2
HN
HN
HN
N
H
HN
N
H
O
O
O
N
O
O
N
O
HO
HO
HO
HO
O
O
O
O
OH
4
OH
6
OH
5
OH
7
Scheme 1. 10-23 DNAzyme targeting against VEGFR2 mRNA and the 2’-deoxythymidine analogues
for the modifications on T4 and T8 at the catalytic core. The bold letter represents the RNA residues in
the substrate to be cleaved, as the arrow indicates.
2. Results and Discussion
2.1. Nucleoside Analogues
From chemical modifications on the thymine base and sugar-phosphate moieties of T4 and T8,
its functional groups and the aromatic plane are supposed to form hydrogen-bonding and stacking
interactions with other residues in the catalytic loop, and these interactions can be modulated
to exert more influence on the catalytic conformation. Here, chemical modifications were only
conducted at its 5-position, and the functional groups of its hydrogen-bonding ability were introduced.
The hydroxyl group was linked by an alkyl bridge in compounds
was linked by a vinylene group in compound , and the amino group was linked through an amido
group in compounds of different lengths. Compounds
synthesized according to the literature [20 21]. Compounds
1 and 2, the imidazolyl group
3
4
–
7
1
–
4
3
and their phosphoramidites were
to and their phosphoramidites
,
7
were synthesized as shown in Schemes 2 and 3. 5-methoxycarbonylmethyl-2’-deoxyuridine (4a
)
and 5-methoxycarbonylethyl-2’-deoxyuridine (5a) were prepared from the glycosylation of
5-methoxycarbonylmethyl-uracil [22]. With the ester-amide exchange reaction with 1,2-diaminoethane,
the 5-carbonylmethyl ester of compound 4a was converted to 5-[N-(2-aminoethyl)carbamoylmethyl]
of compound 4b and 5-[N-(3-aminoethyl)carbamoylethyl] of compound 5b; employing the same

Molecules 2017, 22, 1011
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reaction with 1,3-diaminopropane, 4a and 5a were converted to the compounds 6a and 7a
respectively. The 5-substituted terminal amino group was protected with the trifluoroacetyl group [23].
The tritylation of their 5’-OH offered the corresponding compounds 4c and 5c, as well as 6b and 7b
Further conversion to the phosphoramidites was conducted to obtain the phosphoroamidites 4d 5d
6c, and 7c, for the solid-phase synthesis of the DNAzymes. Compounds 4b 5a 6a, and 7a could
be deprotected to obtain the corresponding nucleosides , and . In these four compounds,
,
.
,
,
,
,
4
,
5,
6
7
the 5-positioned amino group was linked with a different linkage length and the amido group, in order
to produce different effects on the catalytic ability of the DNAzyme.
O
O
O
N
O
N
O
N
H
N
H
N
OCH3
CF3
CF3
N
H
N
H
HN
HN
HN
n
n
O
n
O
O
O
O
O
HO
HO
DMTrO
(i)
(iii)
O
O
O
HO
OH
OH
n = 1, 4a
n = 2, 5a
n = 1, 4b
n = 2, 5b
n = 1, 4c
n = 2, 5c
(ii)
(iv)
O
O
O
H
N
H
N
CF3
NH2
N
H
HN
HN
n
O
n
O
N
N
O
O
HO
DMTrO
O
O
OH
O
n = 1, 4
n = 2, 5
P
NC
N
O
n = 1, 4d
n = 2, 5d
(i) 1,2-diaminoethane, in methanol, 60 °C, then CF3COOC2H5, at r.t.; (ii) aq. ammonia, r.t.; (iii) DMTrCl, in pyridine r.t.;
(iv) diethylpropylammonium tetrazolide, (NCCH2CH2O)[(iPr)2N]2P, CH2Cl2.
.
Scheme 2. Synthesis of compounds 4 and 5 and the phosphoramidites 4d and 5d.
O
N
O
N
O
N
H
N
H
N
H
N
H
N
OCH3
CF3
CF3
HN
HN
HN
n
n
O
n
O
O
O
O
O
O
O
HO
DMTrO
HO
(i)
(iii)
O
O
O
HO
OH
OH
n = 1, 4a
n = 2, 5a
n = 1, 6a
n = 2, 7a
n = 1, 6b
n = 2, 7b
(ii)
(iv)
O
O
H
N
H
N
H
N
NH2
CF3
HN
HN
n
n
O
O
O
O
N
O
N
HO
DMTrO
O
O
OH
O
n = 1, 6
n = 2, 7
P
NC
N
O
n = 1, 6c
n = 2, 7c
(i) 1,3-diaminopropane, in methanol, 60 °C, then CF3COOC2H5, at r.t.; (ii) aq. ammonia, r.t.; (iii) DMTrCl, in pyridine r.t.;
(iv) diethylpropylammonium tetrazolide, (NCCH2CH2O)[(iPr)2N]2P, CH2Cl2.
.
Scheme 3. Synthesis of compounds 6 and 7 and the phosphoramidites 6c and 7c.
2.2. Oligonucleotides
The solid-phase synthesis was conducted on an ABI 392, with a 1 µmol scale. The coupling time of the
phosphoramidites of compounds 1–7 was extended to 3 min. The deprotection of the oligodeoxynucleotides

Molecules 2017, 22, 1011
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was conducted with conc. aq. ammonia at 55 ◦C for 16 h. For the oligonucleotides containing
compounds and , further cleavage of the tert-butyldiphenylsilyl group of the 5-substituted hydroxyl
1
2
group was conducted with 1 M TBAF/THF. All of the deprotected oligonucleotides were purified
with 20% denaturing PAGE (8 M urea). Desaltation was conducted with a Sep-Pak column and
washed with sterilized bidistilled water, and the DNAzyme was eluted with 70% methanol/water
(v/v) and lyophilized before being stored at
characterization results are listed in Table 1 (mass spectrum in Supplementary materials).
−
30 ◦C. The sequences of modified DNAzymes and the
Table 1. Characterization results of DNAzymes and substrates with molecular mass spectrum
measurements.
DNAzyme
Sequence
MS (Calc.)
MS (Found)
DZ01
DZ-T4-1
DZ-T8-1
DZ-T4-2
DZ-T8-2
DZ-T4-3
DZ-T8-3
DZ-T4-4
DZ-T8-4
DZ-T4-T8-4
DZ-C13-4
DZ-T4-5
DZ-T8-5
DZ-T4-T8-5
DZ-C13-5
DZ-T4-6
DZ-T8-6
DZ-T4-T8-6
DZ-C13-6
DZ-T4-7
DZ-T8-7
DZ-T4-T8-7
DZ-C13-7
D19
5’-d(tgc tct cca GGC TAG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 1AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C1A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 2AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C2A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 3AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C3A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 4AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C4A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 4AG C4A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG CTA 4AA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 5AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C5A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 5AG C5A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG CTA 5AA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 6AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C6A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 6AG C6A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG CTA 6AA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 7AG CTA CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG C7A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC 7AG C7A CAA CGA cct gca cct)-3’
5’-d(tgc tct cca GGC TAG CTA 7AA CGA cct gca cct)-3’
5’-d(AGG TGC AGG ATG GAG AGC A)-3’
9994.4
10010.5
10010.5
10038.5
10038.5
10072.5
10072.5
10080.53
10080.53
10166.62
10094.55
10094.6
10094.6
10194.7
10109.6
10094.55
10094.55
10194.67
10109.6
10108.7
10108.7
10222.8
10123.7
5966.9
9992.5
10015.1
10014.0
10038.5
10038.3
10072.8
10074.6
10079.3
10078.5
10164.8
10093.9
10093.4
10094.0
10196.1
10113.0
10092.9
10093.3
10193.4
10109.5
10110.6
10115.6
10223.1
10124.9
5966.4
D19AU
5‘-d(AGG TGC AGG)-r AU-d(TG GAG AGC A)-3’
5984.9
5983.0
2.3. The Effect of the Modified Catalytic Loop on the DNAzyme-Substrate Complexes
In the DNAzyme-substrate complex, the duplex-loop-duplex contributes to a stable and specific
tertiary structure for the catalytic reaction. Mg2+ was suggested to be delicately complexed for
stabilizing and catalyzing roles. Therefore, the influence of the modified loop on the complex formation
was studied with thermal stability (Tm) and circular dichroism (CD).
The DNAzyme-substrate complex was measured under the reaction conditions (50 mM Tris-HCl,
pH 7.5, 2 mM Mg2+), and the full-DNA substrate D19 was used instead of the chimeric substrate,
to avoid the cleavage of the substrate in the reaction buffer. From the measured Tm values (Table 2),
it seems that there was no significant difference between the stacking strength of the nucleobases in
the catalytic loop. The thermal stabilities ensured that the modified DNAzyme could bind with the
substrate, as the first step of the catalytic reaction. On the other hand, the changes of CD spectra shown
in Figure 1 could imply conformational differences related to the unmodified 10-23 DNAzyme (DZ01).
The 5-positioned external functional groups produced new interactions with other residues in the
catalytic core and exerted some influence on the stacking mode of the two thymine residues, leading
to conformational changes. The conformational changes are different from each other, depending on
the properties of different functional groups and their positions in the catalytic core.

Molecules 2017, 22, 1011
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Table 2. Thermal stabilities of the DNAzyme-substrate complexes under the catalytic reaction conditions.
◦
◦
◦
DNAzyme
Tm ( C)
DNAzyme
Tm ( C)
DNAzyme
Tm ( C)
DZ01
51.0
51.2
50.9
49.2
50.8
49.9
50.5
49.0
DZ-T4-1
DZ-T4-2
DZ-T4-3
DZ-T4-4
DZ-T4-5
DZ-T4-6
DZ-T4-7
DZ-T8-1
DZ-T8-2
DZ-T8-3
DZ-T8-4
DZ-T8-5
DZ-T8-6
DZ-T8-7
50.1
49.9
49.9
50.3
50.3
50.2
49.3
DZ-T4-T8-4
DZ-T4-T8-5
DZ-T4-T8-6
DZ-T4-T8-7
DZ-C13-4
DZ-C13-5
DZ-C13-6
50.3
49.7
50.1
50.5
50.6
51.3
51.3
49.7
DZ-C13-7
Three measurements were conducted for each Tm, with a standard error of 0.5 ◦C.
Figure 1. CD spectra of the DNAzyme-substrate complexes in the reaction conditions. Comparisons of
the CD spectra were made between the modified DNAzyme-substrate complexes with different
nucleoside analogues and at different positions: (
analogues ; ( ) Modified complexes at T8 with nucleoside analogues
at T4 and T8 with nucleoside analogues ; ( ) Modified complexes with compound 4
A
) Modified complexes at T4 with nucleoside
; ( ) Modified complexes
at different
1–7
B
1–7 C
4–
7
D
positions; (E) Modified complexes with compound 5 at different positions.
2.4. The Catalytic Activities of the Modified DNAzymes
The catalytic activities of the modified DNAzymes were evaluated under single-turnover
conditions against the chimeric substrate D19AU, with rAU as the cleavage site. No reaction was
observed without Mg2+, indicating that all of the extra functional groups could not behave like Mg2+
The cleavage profiles of the DNAzymes are shown in Figure 2.
.
With compound 1, the external hydroxyl group was introduced to the catalytic loop through
substitution at T4 (DZ-T4-1) or T8 (DZ-T8-1), and the observed rate constants indicated that the
5-substituted hydroxyl group at T8 could produce a slightly favorable effect on the cleavage reaction
(Table 3), while no influence was observed for T4 substitution, which is in contrast with the drastic
negative effect of most modifications at T4. The interaction related to this extra hydroxyl group was
then studied with compound 2. The decreased kobs of DZ-T4-2 indicated that the one-bond-longer
linkage permitted the hydroxyl group to induce different interactions, leading to an unfavorable effect
at T4. T8 was less affected by such a structural change (DZ-T8-2).

Molecules 2017, 22, 1011
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Figure 2. The cleavage reaction profiles of DNAzymes under single turnover conditions. (
A) The
DNAzymes modified at T4 were compared; ( ) The DNAzymes modified at T8 were compared.
B
Each DNAzyme was specified: DZ01 (solid square, black line), DZ-T4-1(solid circle, red line), DZ-T4-2
(solid triangle, blue line), DZ-T4-3 (solid downtriangle, dark cyan line), DZ-T4-4 (solid left triangle,
magenta line), DZ-T4-5 (solid right triangle, dark yellow line), DZ-T4-6 (solid diamond, navy line),
DZ-T4-7 (solid star, mine line), DZ-T8-1 (empty circle, red line), DZ-T8-2 (empty triangle, blue line),
DZ-T8-3 (empty down triangle, dark cyan line), DZ-T8-4 (empty left triangle, magenta line), DZ-T8-5
(empty right triangle, dark yellow line), DZ-T8-6 (empty diamond, navy line), DZ-T8-7 (empty star,
wine line).
Table 3. The observed rate constants of DNAzymes under single-turnover conditions.
−1
−1
)
DNAzyme
kobs (min
)
DNAzyme
kobs (min
DZ01
DZ-T4-1
DZ-T4-2
DZ-T4-3
DZ-T4-4
DZ-T4-5
DZ-T4-6
DZ-T4-7
DZ-T4-T8-4
DZ-T4-T8-5
DZ-T4-T8-6
DZ-T4-T8-7
0.0051 ± 0.0005
0.0060 ± 0.0005
0.0032 ± 0.0001
0.0009 ± 0.00006
0.0034 ± 0.0003
0.0035 ± 0.0004
0.0037 ± 0.0003
0.0026 ± 0.0004
0.0062 ± 0.0006
0.0064 ± 0.0006
0.0052 ± 0.0007
0.0059 ± 0.0005
DZ-T8-1
DZ-T8-2
DZ-T8-3
DZ-T8-4
DZ-T8-5
DZ-T8-6
DZ-T8-7
DZ-C13-4
DZ-C13-5
DZ-C13-6
DZ-C13-7
0.0076 ± 0.0005
0.0064 ± 0.0003
0.0022 ± 0.00006
0.0036 ± 0.0005
0.0088 ± 0.0005
0.0054 ± 0.0008
0.0072 ± 0.0008
-
-
-
-
It has been reported that the contribution of T8 could be modulated by changing its stacking
interaction with 5-substituted azobenzene groups, where the specific stacking of the aromatic group
with a different configuration could induce a favorable or unfavorable effect for the contribution of
T8 [17]. Compound 3 was used to introduce a rigidly conjugated imidazolyl group for the enlarged
base stacking and hydrogen-bonding interactions. The obvious decrease in the observed rate constants
of DZ-T4-3 and DZ-T8-3 indicated that these new interactions induced an unfavorable conformational
change of the catalytic loop. It seems that the original base stacking interaction of T4 and T8 is very
important, even for the least conserved T8. It was also convinced by the sugar modification of
a different configuration at T4 and T8, where the (R)- and (S)-2’-C-methyl-thymidine and the locked
conformation with LNA could lead to the different spatial occupation of thymine compared to the
2’-deoxyribose moiety, with an unfavorable impact on the catalytic activity [14,15].
With compounds 4–7, an amino group was introduced, together with the amido linkage at
the 5-position of thymine. As shown in Table 3, T4 is less compatible with these changes than T8.
However, the partial retention of the catalytic activity demonstrated that the conservation of T4 could
be recognized at the level of the thymine base, because any replacement of this thymine with other
nucleobases led to an almost complete loss of activity [8]. On the other hand, although it has been

Molecules 2017, 22, 1011
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demonstrated that several modifications led to a drastic loss of activity [15
,18], T4 is not critically
conserved at the 5-methyl group. This 5-methyl group could be modified for a better performance of
T4 with a delicately designed substituent.
At the least conserved T8, the influence of the new substituents from compounds 4–7 was
not always positive. The importance of the linkage was evident for the effect of the substituent.
The decreased kobs of DZ-T8-4 indicated that the new interactions related to the external amino and
amido groups induced an unfavorable effect on the catalytic conformation, but only one-carbon-longer
change in DZ-T8-5 led to a new positive effect. In addition, from the different activities between
DZ-T8-5 and DZ-T8-6 with the same length of the linkage, the amido group in the linkage seems to be
involved in the interactions of the new residues
5 and 6 at T8. Further extension of the terminal amino
group could not induce a more positive effect in DZ-T8-7. These results indicated that T8 is also very
sensitive to the chemical modifications at its 5-position.
Combinational modifications on both T4 and T8 with the same compound were conducted and
different combinatorial effects were observed for each of the four modified residues compared to
the corresponding single substitutions. The negative effect from the substitutions at T4 was always
compensated by the further substitution at T8, by which a favorable conformational change was
supposed to be driven. Especially in the case of compound 4, its negative effect at T4 or T8 was
deleted by the combined substitutions. These results further indicated the delicate influence of the
5-substituents of T4 and T8. On the other hand, these results could not imply that the modified residues
at T4 and T8 are directly related to each other, based on the completely different conservation of T4
and T8. In the catalytic core of 10-23 DNAzyme, each residue was suggested to have its own interaction
network with nearby residues, contributing to the catalytic conformation in its own way. Therefore,
the observed rate constants of the modified DNAzymes reflected the comprehensive result of complex
interactions between the external functional groups with the internal residues in the catalytic loop,
and the additive effect could not be expected, depending on the specific residues and the modifications.
At C13, its 4-amino group has been demonstrated to be very important for its contribution,
and partial activity could be retained by the replacement of adenine, while thymine could not be used
for this position [8]. Here, the amino-modified thymine analogues 4–7 at C13 led to the complete loss
of catalytic activity of the DNAzyme, which meant that the interactions derived from C13 could be
very specific for the right catalytic conformation, including its base-stacking mode and the original
functional groups for other interactions.
In previous studies on the conservation of residues, the uniqueness of each residue in the catalytic
core of 10-23 DNAzyme has been demonstrated by the inter-replacement of the canonical residues [7].
Therefore, the design of nucleoside analogues for the corresponding residues/nucleosides has been
the first consideration in our approach for the chemical modification of the catalytic core. With the
modification at the level of functional groups, the most conserved guanine residues [24] and T4
could be modified for a better performance. On the other hand, the selection of functional groups
and the linkage is very important for the catalytic activity, as reported for the modifications of the
6-amino group of adenine [18,25]. These results demonstrated that the catalytic conformation of 10-23
DNAzyme is very sensitive to any structural changes, but it could be optimized.
3. Materials and Methods
3.1. General
Commercially available chemicals were used without purification. Dichloromethane was
redistilled and dried on anhydr. K2CO3. 1,2-Diaminoethane and 1,3-diaminopropane were redistilled
before use. Silica gel for TLC and flash column chromatography was obtained from Qingdao Chemicals
1
Co. (Qingdao, China). H-NMR (400 MHz), 13C-NMR (100 MHz), and 31P-NMR (160 MHz) were
performed on JNM-ECA400 (JEOL, Tokyo, Japan), with TMS as an internal standard and 85% H3PO4
as the external standard, respectively. The high resolution mass spectrum for new compounds was

Molecules 2017, 22, 1011
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obtained with Agilent TOF G6230A (Agilent Technologies, Santa Clara, CA, USA). The mass spectrum
of oligonucleotides was obtained with a HTCS Oligo LC/MS system (Thermo Finnigan, Somerset,
NJ, USA).
5-[2-(Trifluoroacetylamino)ethylaminocarbonylmethyl]-2’-deoxyuridine (4b). To the solution of 1,2-diaminoethane
◦
(2.5 mL, 38 mmol) in methanol (2.5 mL) at 60 C, a solution of compound 4a (1.14 g, 3.8 mmol)
in methanol (5 mL) was slowly added. After stirring for 4 h, the solvent and extra reagents were
evaporated off in a vacuum. The residue was dissolved in methanol (10 mL), and triethylamine
(2.1 mL) and ethyl trifluoroacetate (5 mL) were added. The reaction mixture was stirred at r.t., until
the reaction was finished. By flash chromatography, the product was obtained as colorless foam
1
(1.1 g, 68.3%). Rf (DCM/methanol, 9:1) 0.32. H-NMR (400 MHz, DMSO-d6):
δ 2.08 (m, 2 H, C2’-H),
3.05 (s, 2 H, CH2), 3.18 (m, 4 H, CH2CH2), 3.56 (m, 2 H, C5’-H), 3.77 (m, 1 H, C4’-H), 4.22 (m, 1 H, C3’-H),
4.96 (t, J = 5.5, C5’-OH), 5.24 (d, J = 4.2, C3’-OH), 6.17 (t, J = 4.8, C1’-H), 7.72 (s, 1 H, C6-H), 7.96 (m, 1 H,
CONH), 9.37 (s, 1 H, CONHCOCF3), 11.34 (s, 1 H, 3-NH). 13C-NMR (400 MHz, DMSO-d6):
δ 35.3, 39.5,
63.4, 72,4, 85.9, 89.3, 110.5, 116.4, 119.3, 140.4, 152.2, 158.2, 158.6, 165.2, 171.7. HRMS (C15H19F3N4O7
+ H+, 425.1279): 425.1278; (C15H19F3N4O7 + Na+, 447.1098): 447.1097.
5-(2-aminoethylaminocarbonylmethyl)-2’-deoxyuridine (4). Compound 4a (400 mg, 0.94 mmol) was
dissolved in methanol (2 mL) and conc. aq. ammonia (40 mL), and after stirring at r.t. for 4 h,
the solution was concentrated for flash chromatography. Compound
(286 mg, 93%). Rf (DCM/methanol (with ammonia), 1:1) 0.47. H-NMR (400 MHz, DMSO-d6):
4
was obtained as white foam
2.09
1
δ
(m, 2 H, C2’-H), 2.85 (m, 2 H, CH2), 3.10 (s, 2 H, CH2), 3.27 (m, 2 H, CH2), 3.53 (m, 2 H, C5’-H),
3.78 (m, 1 H, C4’-H), 4.24 (m, 1 H, C3’-H), 5.31 (br, 1 H, C3’-OH), 6.18 (m, 1 H, C1’-H), 7.77 (s, 1 H,
C6-H), 8.09 (m, 1 H, NH), 8.55 (br, 1 H, NH). 13C-NMR (100 MHz, DMSO-d6):
δ 33.9, 37.1, 61.9, 70.9,
84.5, 87.9, 109.0, 139.0, 150.9, 163.8, 170.8. HRMS (C13H20N4O6 + H+, 329.1456): 329.1456; (C13H20N4O6
+ Na+, 351.1275): 351.1276.
5’-O-(4,4’-Dimethoxytrityl)-5-[2-(trifluoroacetylamino)ethylaminocarbonylmethyl]-2’-deoxyuridine (4c). After the
co-evaporation of compound 4b (1.20 g, 2.82 mmol) with dried pyridine (5 mL) twice, the residue was
dissolved in dried pyridine (2 mL) and stirred at r.t. To the solution, DMTrCl (1.16 g, 3.38 mmol) was
added in portions, with TLC monitoring the process. The reaction was stopped by adding methanol
(5 mL), and the solution was evaporated to a small volume for flash chromatography on a neutralized
silica gel column with triethylamine. The product was obtained as colorless foam (1.28 g, 62.5%).
1
Rf (DCM/methanol, 9:1) 0.56. H-NMR (400 MHz, DMSO-d6):
δ 2.18 (m, 2 H, C2’-H), 2.70 (s, 2 H, CH2),
3.17 (m, 6 H, CH2CH2, C5’-H), 3.73 (s, 6 H, 2 OCH3), 3.87 (m, 1 H, C4’-H), 411.25 (m, 1 H, C3’-H),
5.32 (d, J = 4.5, C3’-OH), 6.20 (t, J = 4.8, C1’-H), 6.86, 7.19-7.40 (2 m, 13 H, arom.H), 7.55 (s, 1 H, C6-H),
7.86 (m, 1 H, CONH), 9.33 (s, 1 H, CONHCOCF3), 11.38 (s, 1 H, 3-NH). 13C-NMR (400 MHz, DMSO-d6):
δ
33.1, 37.5, 55.0, 63.9, 70.5, 84.0, 85.4, 85.8, 108.8, 113.2, 114.5, 117.3, 123.9, 126.8, 127.7, 127.9, 129.7, 135.3,
135.5, 136.2, 138.3, 144.8, 149.6, 150.4, 158.1, 163.2, 169.4. HRMS (C36H37F3N4O9 + Na+, 749.2405): 749.2405.
3’-O-(2-Cyanoethyl-N,N-diisopropylaminophosphinoxy)-5’-O-(4,4’-dimethoxytrityl)-5-[2-(trifluoroacetylamino)
ethylaminocarbonylmethyl]-2’-deoxyuridine (4d). Compound 4c (0.5 g, 0.688 mmol) was dissolved
in redistilled dichloromethane (10 mL), and N,N-diisopropylammonium tetrazolide (0.14 g) and
2-cyanoethylbis(diisopropylamino)phosphoramidite (0.10 mL) were added to the solution in sequence.
After stirring at r.t. for 30 min, the reactin mixture was diluted with redistilled dichloromethane
(10 mL). The solution was washed once with ice-cold 2% aq. NaHCO3 and brine. The organic layer was
dried with MgSO4 and concentrated for flash chromatography on a silica gel column neutralized with
3% triethylamine. The product was obtained as colorless foam (0.42 g, 65.8%). Rf (DCM/methanol,
1
20:1) 0.42. H-NMR (400 MHz, CDCl3):
δ 1.16 (m, 12 H, 4 CH3), 2.36–2.76 (m, 6 H, C2’-H, CH2,
OCH2CH2CN), 3.24–3.90 (m, 16 H, NCH2CH2N, C5’-H, 2 OCH3, 2 CH, OCH2CH2CN), 4.14 (m, 1 H,
C4’-H), 4.70 (m, 1 H, C3’-H), 6.31 (m, C1’-H), 6.49 (1 H, NH), 6.83, 7.19-7.43 (2 m, 13 H, arom.H, C6-H),
7.81 (m, 1 H, CONH), 7.89 (s, 1 H, CONHCOCF3). 13C-NMR (100 MHz, CDCl3):
δ 20.2, 22.2, 23.2, 24.6,

Molecules 2017, 22, 1011
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34.7, 38.9, 40.5, 43.4, 55.3, 62.4, 62.6, 85.2, 87.0, 108.8, 112.7, 113.4, 113.6, 117.7, 127.5, 128.1, 128.3, 128.5,
130.2, 135.2, 138.8, 144.2, 150.0, 158.2, 158.8, 170.0, 171.3. 31P-NMR (160 MHz, CDCl3):
HRMS (C45H54F3N6O10P + Na+, 949.3483): 949.3483.
δ 149.46, 149.64.
5-[2-(Trifluoroacetylamino)ethylaminocarbonylethyl]-2’-deoxyuridine (5b). As described for 4b, 5b was
prepared from the reaction of 5a (1.14 g, 3.8 mmol) and 1,2-diaminoethane (2.5 mL, 38 mmol) and
subsequent protection with ethyl trifluoroacetate (5 mL) in methanol. The product was obtained
1
as colorless solid (1.1 g, 68.3%). Rf (DCM/methanol, 9:1) 0.34. H-NMR (400 MHz, DMSO-d6):
2.08 (m, 2 H, CH2), 2.26 (m, 2 H, C2’-H), 2.43 (m, 2 H, CH2), 3.21 (m, 4 H, CH2CH2), 3.58 (m, 2 H,
C5-H), 3.78 (m, 1 H, C4’-H), 4.25 (m, 1 H, C3’-H), 5.03 (t, 1 H, J = 5.1, C5’-OH), 5.26 (d, J = 4.2, C3’-OH),
6.18 (t, 1 H, J = 6.9, C1’-H), 7.66 (s, 1 H, C6-H), 7.98 (m, 1 H, NH), 9.43 (m, 1 H, NH), 11.32 (s, 1 H, NH).
13C-NMR (100 MHz, DMSO-d6): 23.8, 35.1, 38.3, 62.3, 71.4, 84.8, 88.3, 113.6, 137.5, 151.3, 164.3, 172.6.
HRMS (C16H21F3N4O7 + H+, 439.1435): 439.1434; (C16H21F3N4O7 + Na+, 461.1255): 461.1255.
5-(2-Aminoethylaminocarbonylethyl)-2’-deoxyuridine (5). As described for 4, compound 5 was prepared
from 5b (500 mg, 1.14 mmol) in conc. aq. ammonia (45 mL) as a colorless solid (368 mg, 94.6%),
1
Rf (DCM/methanol saturated with ammonia, 1:1) 0.50. H-NMR (400 MHz, DMSO-d6): 2.08 (m, 2 H,
CH2), 2.29 (m, 2 H, C2’-H), 2.45 (m, 2 H, CH2), 2.84 (m, 2 H, CH2), 3.26 (m, 2 H, CH2), 3.58 (m, 2 H,
C5-H), 3.78 (m, 1 H, C4’-H), 4.25 (m, 1 H, C3’-H), 5.30 (br, 1 H, C3’-OH), 6.18 (t, J = 6.9, 1 H, C1’-H),
7.67 (s, 1 H, C6-H), 8.04 (br, 2 H, NH, NH). 13C-NMR (100 MHz, DMSO-d6): 23.2, 34.6, 37.0, 61.9,
71.0, 84.4, 87.8, 113.1, 137.1, 150.8, 163.8, 172.7. HRMS (C14H22F3N4O6 + H+, 343.1612): 343.1611;
(C14H22F3N4O6 + Na+, 365.1432): 365.1433.
5’-O-(4,4’-Dimethoxytrityl)-5-[2-(trifluoroacetylamino)ethylaminocarbonylethyl]-2’-deoxyuridine (5c). As described
for 4c, compound 5c was prepared from 5b (1.20 g, 2.82 mmol) and DMTrCl (1.16 g, 3.38 mmol). The product
was purified with flash chromatography as a white foam (1.28 g, 62.5%). Rf (DCM/methanol, 9:1) 0.58.
1H-NMR (400 MHz, DMSO-d6): 2.09-2.26 (m, 6 H, C2’-H, CH2CH2), 3.13–3.20 (m, 6 H, C5’-H, CH2CH2),
3.73 (s, 6 H, 2 CH3O), 3.86 (m, 1 H, C4’-H), 4.22 (m, 1 H, C3’-H), 5.32 (d, 1 H, J = 4.8, C3’-OH), 6.16
(t, 1 H, J = 6.7, C1’-H), 6.89, 71.9–7.41 (m, 14 H, C6-H, arom.H), 7.89 (m, 1 H, NH), 9.40 (m, 1 H, NH),
11.38 (m, 1 H, NH). 13C-NMR (100 MHz, DMSO-d6): 23.8, 38.3, 55.9, 64.9, 71.4, 84.9, 86.3, 86.6, 113.8,
114.2, 127.7, 128.6, 128.8, 130.7, 136.4, 136.5, 137.5, 145.8, 151.2, 157.2, 157.5, 159.1, 164.2, 172.5. HRMS
(C37H39F3N4O9 + Na+, 763.2561): 763.2561.
3’-O-(2-Cyanoethyl-N,N-diisopropylaminophosphinoxy)-5’-O-(4,4’-dimethoxytrityl)-5-[2-(trifluoroacetylamino)
ethylaminocarbonylethyl]-2’-deoxyuridine (5d). As described for 4d, 5d was prepared from the reaction of
5c (0.51 g, 0.663 mmol) with 2-cyanoethylbis(diisopropylamino)phosphoramidite (0.10 mL) in the
presence of N,N-diisopropylammonium tetrazolide (0.14 g). The product was obtained as white
foam (0.42 g, 67.3%). Rf (DCM/methanol, 20:1) 0.48. 1H-NMR (400 MHz, CDCl3):
δ (ppm) 1.14
(m, 12 H, 4 CH3), 2.04–2.64 (m, 10 H, C2’-H, CH2CH2, 2 CH, CH2CN), 3.27–3.88 (m, 16 H, CH2CH2,
C5’-H, 2 CH3O, OCH2), 4.11 (m, 1 H, C4’-H), 4.63 (m, 1 H, C3’-H), 6.37 (m, 2 H, C1’-H, NH), 6.83,
7.21–7.41 (m, 14 H, C6-H, arom.H), 7.60 (s, 1 H, NH), 8.22 (br, 1 H, NH), 9.80 (br, 1 H, NH). 13C-NMR
(100 MHz, CDCl3):
δ (ppm) 20.7, 23.3, 24.7, 24.8, 24.9, 35.6, 38.8, 40.3, 41.5, 43.4, 43.5, 55.6, 58.3,
58.5, 63.3, 73.8, 73.9, 85.0, 85.2, 85.6, 87.0, 113.6, 113.9, 117.6, 118.1, 127.5, 128.3, 128.5, 130.4, 135.6,
137.5, 144.6, 150.6, 159.0, 164.5, 174.2. 31P-NMR (160 MHz, CDCl3):
(C46H56F3N6O10P + Na+, 963.3640): 963.3642.
δ (ppm) 149.26, 149.35. HRMS
5-[3-(Trifluoroacetylamino)propylaminocarbonylmethyl]-2’-deoxyuridine (6a). As described for the preparation
of compound 4b, 6a was prepared from the reaction of 4a (3.42 g, 11.4 mmol) with 1,3-diaminopropane
(7.5 mL, 114 mmol) in metanol (22.5 mL) at 60 ◦C, followed by the addition of triethylamine (6.3 mL)
and ethyl trifluoroacetate (15 mL). The product was purified with flash chromatography (3.30 g, 66.1%).
1
Rf (DCM/methanol, 9:1) 0.34. H-NMR (400 MHz, DMSO-d6):
δ (ppm) 1.60 (m, 2 H, CH2CH2CH2),
2.08 (m, 2 H, C2’-H), 2.98–3.10 (m, 6 H, CH2, CH2CH2CH2), 3.55 (m, 2 H, C5’-H), 3.77 (m, 1 H, C4’-H),
4.22 (m, 1 H, C3’-H), 5.00 (br, 1 H, C5’-OH), 5.27 (br, 1 H, C3’-OH), 6.16 (t, J = 5.7 Hz, 1 H, C1’-H), 7.74

Molecules 2017, 22, 1011
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(s, 1 H, C6-H), 7.88 (t, J = 5.3 Hz, NH), 9.40 (s, 1 H, NH), 11.34 (s, 1 H, 3-NH). 13C-NMR (100 MHz,
DMSO-d6): (ppm) 29.4, 34.4, 37.3, 38.0, 62.4, 71.4, 84.9, 88.3, 109.7, 139.3, 151.4, 157.0, 157.3, 164.2,
170.4. HRMS (C16H21F3N4O7 + H+, 439.1435): 439.1435; (C16H21F3N4O7 + Na+, 461.1255): 461.1252.
δ
5-[3-(Trifluoroacetylamino)propylaminocarbonylmethyl]-2’-deoxyuridine ( ). As described for compound
as a colorless solid (347 mg,
(ppm)
6
4,
compound 6a (500 mg, 1.14 mmol) was deprotected to obtain compound
88.9%). Rf (DCM/methanol saturated with ammonia, 1:1) 0.51. H-NMR (400 MHz, DMSO-d6):
6
1
δ
1.42 (m, 2 H, CH2), 2.05 (m, 2 H, C2’-H), 2.48 (m, 2 H, CH2), 3.05 (m, 4 H, 2 CH2), 3.53 (m, 2 H, C5’-H),
3.74 (m, 1 H, C4’-H), 4.20 (m, 1 H, C3’-H), 5.08 (br, 2 H, NH2), 6.15 (t, J = 6.9, 1 H, C1’-H), 7.70 (s, 1 H,
C6-H), 7.81 (m, 1 H, NH). 13C-NMR (100 MHz, DMSO-d6):
δ (ppm) 33.8, 34.3, 37.3, 62.3, 71.3, 84.9, 88.3,
109.7, 139.1, 151.4, 164.2, 170.1. HRMS (C14H22N4O6 + H+, 343.1612): 343.1611; (C13H20N4O6 + Na+,
365.1432): 365.1429.
5’-O-(4,4-Dimethoxytrityl)-5-[3-(trifluoroacetylamino)propylaminocarbonylmethyl]-2’-deoxyuridine
(6b).
As described for compound 4c 6b was preparaed from 6a (1 g, 2.28 mmol) and DMT-Cl (0.94 g,
,
2.74 mmol) in pyridine (2 mL). The product was obtained as colorless foam by flash chromatography
1
(1.04 g, 61.6%). Rf (DCM/methanol, 9:1) 0.56. H-NMR (400 MHz, DMSO-d6):
δ (ppm) 1.57 (m, 2 H,
CH2CH2CH2), 2.19 (m, 2 H, C2’-H), 2.65 (m, 2 H, C5’-H), 2.98-3.26 (m, 6 H, CH2, CH2CH2CH2),
3.73 (s, 6 H, 2 OCH3), 3.88 (m, 1 H, C4’-H), 4.29 (m, 1 H, C3’-H), 5.36 (d, J = 4.5 Hz, 1 H, C3’-OH), 6.22
(t, J = 6.7 Hz, 1 H, C1’-H), 6.87 (m, 4 H, arom.H), 7.19-7.38 (m, 9 H, arom.H), 7.58 (s, 1 H, C6-H), 7.77
(t, J = 5.6 Hz, NH), 9.39 (s, 1 H, NH), 11.41 (s, 1 H, 3-NH). 13C-NMR (100 MHz, DMSO-d6):
δ (ppm)
29.4, 34.2, 37.2, 38.0, 56.0, 64.8, 71.5, 84.9, 86.5, 86.8, 110.0, 114.2, 127.7, 128.7, 128.9, 130.7, 136.3, 136.4,
139.1, 145.7, 151.4, 159.1, 164.1, 170.1. HRMS (C37H39F3N4O9 + Na+, 763.2561): 763.2561.
3’-O-(2-Cyanoethyl-N,N-diisopropylaminophosphinoxy)-5’-O-(4,4-dimethoxytrityl)-5-[3-(trifluoroacetylamino)
propylaminocarbonylmethyl]-2’-deoxyuridine (6c). As described for compound 4d, compound 6c was
prepared from 6b (0.5 g, 0.675 mmol) and 2-cyanoethylbis(diisopropylamino)phosphoramidte
(0.28 mL) in dichloromethane (10 mL), in the presence of N,N-diisopropylammonium tetrazolide
(0.13 g). The product was obtained as a colorless solid (0.43 g, 67.7%). Rf (DCM/methanol, 20:1) 0.46.
1H-NMR (400 MHz, CDCl3):
δ (ppm) 1.14 (m, 12 H), 1.60 (m, 2 H, CH2CH2CH2), 2.34–2.68 (6 H, 2 CH,
CH2CN, C2’-H), 3.12–3.90 (m, 16 H, CH2CH2CH2, C5’-H, CH2, OCH2, 2 OCH3), 4.08 (m, 1 H, C4’-H),
4.69 (m, 1 H, C3’-H), 6.35 (m, 2 H, C1’-H, NH), 6.77–7.40 (m, 13 H, arom.H), 7.81 (m, 1 H, C6-H), 8.16
(m, 1 H, NH), 9.85 (br, 1 H, NH). 13C-NMR (100 MHz, CDCl3):
δ (ppm) 20.5, 20.6, 20.7, 23.2, 23.3, 24.7,
24.8, 24.9, 25.0, 29.2, 36.2, 36.3, 36.4, 43.4, 43.5, 43.6, 55.5, 55.6, 58.3, 58.5, 63.0, 63.1, 73.2, 73.4, 85.2, 85.3,
85.5, 86.0, 87.2, 109.4, 113.6, 117.8, 118.1, 127.5, 128.4, 128.6, 130.5, 135.5, 135.6, 139.2, 144.4, 150.5, 157.5,
159.0, 164.4, 164.5, 171.1, 171.2. 31P-NMR (160 MHz, CDCl3):
+ Na+, 963.3640): 963.3642.
δ (ppm) 149.41. HRMS (C46H56F3N6O10P
5-[3-(Trifluoroacetylamino)propylaminocarbonylethyl]-2’-deoxyuridine (7a). As described for 4b, the reaction
between 5a (2.28 g, 7.6 mmol) with 1,3-diaminopropane (5 mL, 76 mmol) followed by protection
with ethyl trifluoroacetate (10 mL) offered the compound 7a as a colorless solid (2.1 g, 61.1%).
1
Rf (DCM/methanol, 9:1) 0.36. H-NMR (400 MHz, DMSO-d6):
δ (ppm) 1.59 (m, 2 H, CH2CH2CH2),
2.06 (m, 2 H, C2’-H), 2.24, 2.42 (2 t, J = 7.42 Hz, 4 H, CH2CH2), 3.01-3.18 (m, 4 H, CH2CH2CH2),
3.56 (m, 2 H, C5’-H), 3.76 (m, 1 H, C4’-H), 4.22 (m, 1 H, C3’-H), 5.00 (t, J = 5.4, Hz, 1 H, C5’-OH),
5.22 (d, J = 4.2 Hz, 1 H, C3’-OH), 6.15 (t, J = 7.1 Hz, 1 H, C1’-H), 7.63 (s, 1 H, C6-H), 7.84 (m, 1 H,
NH), 9.36 (s, 1 H, NH), 11.29 (s, 1 H, 3-NH). 13C-NMR (100 MHz, DMSO-d6):
δ (ppm) 22.9, 28.5, 34.1,
36.1, 37.1, 61.4, 70.5, 83.9, 87.4, 112.6, 136.6, 150.4, 163.3, 171.4. HRMS (C17H23F3N4O7 + H+, 453.1592):
453.1592; (C17H23F3N4O7 + Na+, 475.1411): 475.1411.
5-[3-(Trifluoroacetylamino)propylaminocarbonylethyl]-2’-deoxyuridine (7). As described for compound 4,
compound 7a (300 mg, 0.66 mmol) was deprotected in conc. aq. ammonia (30 mL). The product was
obtained as a colorless solid (229 mg, 97%). Rf (DCM/methanol saturated with ammonia, 1:1) 0.53.
1H-NMR (400 MHz, DMSO-d6): 1.67 (m, 2 H, CH2), 2.08 (m, 2 H, C2’-H), 2.28 (m, 2 H, CH2), 2.42 (m, 2 H,

Molecules 2017, 22, 1011
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CH2), 2.78 (m, 2 H, CH2), 3.11 (m, 2 H, CH2), 3.59 (m, 2 H, C5’-H), 3.78 (m, 1 H, C4’-H), 4.25 (m, 1 H,
C3’-H), 5.34 (br, 1 H, C5’-OH), 6.17 (t, J = 6.9, 1 H, C1’-H), 7.67 (s, 1 H, C6-H), 8.04 (m, 1 H, NH, NH).
13C-NMR (100 MHz, DMSO-d6): 23.3, 28.0, 34.5, 36.0, 37.2, 61.9, 70.9, 84.4, 87.8, 113.1, 116.1, 119.1, 137.0,
150.8, 158.9, 159.2, 163.8, 172.3. HRMS (C15H24N4O6+ H+, 357.1769): 357.1769; C15H24N4O6 + Na+,
379.1588): 379.1582.
5’-O-(4,4-Dimethoxytrityl)-5-[3-(trifluoroacetylamino)propylaminocarbonylethyl]-2’-deoxyuridine (7b). As described
for 4c, compound 7a (1 g, 2.21 mmol) was reacted with DMTrCl (0.84 g, 2.65 mmol) in dried pyridine
1
(2 mL) to obtain the product as a colorless solid (1.18 g, 70.7%). Rf (DCM/methanol, 9:1) 0.55. H-NMR
(400 MHz, DMSO-d6):
δ (ppm) 1.57 (m, 2 H, CH2CH2CH2), 2.12-2.25 (m, 6 H, C2’-H, CH2CH2),
2.98-3.18 (m, 6 H, C5’-H, CH2CH2CH2), 3.73 (s, 6 H, 2 OCH3), 3.86 (m, 1 H, C4’-H), 4.22 (m, 1 H, C3’-H),
5.32 (d, J = 4.5 Hz, 1 H, C3’-OH), 6.16 (t, J = 6.8 Hz, 1 H, C1’-H), 6.88 (m, 4 H, arom.H), 7.19–7.41
(m, 10 H, C6-H, arom.H), 7.76 (m, 1 H, NH), 9.38 (s, 1 H, NH), 11.39 (s, 1 H, 3-NH). 13C-NMR (100 MHz,
DMSO-d6):
δ (ppm) 23.8, 29.4, 35.1, 36.9, 38.0, 55.9, 64.8, 71.3, 84.9, 86.3, 86.6, 113.8, 114.1, 127.6, 128.6,
128.8, 130.6, 136.3, 136.4, 137.3, 145.7, 151.2, 159.0, 164.1, 172.1. HRMS (C38H41F3N4O9 + H+, 754.2826):
754.2826; (C38H41F3N4O9 + Na+, 777.2713): 777.2713.
3’-O-(2-Cyanoethyl-N,N-diisopropylaminophosphinoxy)-5’-O-(4,4-dimethoxytrityl)-5-[3-(trifluoroacetylamino)
propylaminocarbonylmethyl]-2’-deoxyuridine (7c). As described for compound 4d, 7c was prepared
from 7b (0.5 g, 0.66 mmol) with 2-cyanoethylbis(diisopropylamino)phosphoramidite (0.17 mL)
in dichloromethane (10 mL), in the presence of N,N-diisopropylammonium tetrazolide (0.14 g).
The product was obtained as white foam (0.45 g, 71%). Rf (DCM/methanol, 20:1) 0.39. 1H-NMR
(400 MHz, DMSO-d6):
δ (ppm) 1.02–1.51 (m, 14 H, 2 NCH(CH3)2, CH2CH2CH2), 2.04–2.77
(m, 8 H, C2’-H, CH2CH2, OCH2CH2CN), 3.12–3.86 (m, 14 H, 2 NCH(CH3)2, CH2CH2CH2, 2 CH3O,
OCH2CH2CN), 4.18 (m, 2 H, C5’-H), 4.61 (m, 1 H, C4’-H), 5.76 (m, 1 H, C3’-H), 6.36 (m, 1 H, C1’-H),
6.84, 7.29 (2 m, 14 H, NH, arom.H), 8.16 (s, 1 H, C6-H), 9.12 (br, 1 H, NH), 11.15 (br, 1 H, NH). 13C-NMR
(100 MHz, CDCl3):
δ (ppm) 23.6, 24.1 24.9, 29.4, 36.0, 40.4, 43.8, 55.7, 58.4, 63.7, 85.2, 87.1, 113.7, 127.6,
128.5, 130.6, 136.1, 137.4, 145.0, 150.4, 159.2, 173.7. 31P-NMR (160 MHz, CDCl3):
HRMS (C47H58F3N6O10P + Na+, 977.3796): 977.3795.
δ (ppm) 149.26, 149.40.
3.2. Tm Measurement
The melting temperatures of the DNAzyme-substrate complexes were determined using a Varian
Bio 100 spectrometer (Varian, Palo Alto, CA, USA). An equal amount (2.0 M) of DNAzyme and
µ
the full-DNA substrate were mixed in a buffer containing 50 mM Tris-HCl pH 7.4 and 2 mM Mg2+.
The ◦solution was incubated at 85 ◦C for 10 min. Subsequently, it was cooled slowly to 10 ◦C, at a rate
of 1 C/min. The absorbance at 260 nm was measured over the temperature range. The first derivative
of the melting curve corresponds to the melting temperature (Tm) of the DNAzyme-substrate complex.
Tm was given as the mean value of the two measurements, with a variation of <0.5 ◦C.
3.3. Circular Dichroism
The samples (2.0 µ
M in a buffer of 50 mM Tris-HCl (pH 7.4) and 2 mM Mg2+) in 1 cm cuvettes
were used for CD spectra. Data were collected on a Jasco J810 spectropolarimeter (JASCO Corporation,
Tokyo, Japan) from 350 nm to 200 nm, with a scan speed of 100 nm/min, at 2 nm intervals. Each
sample was scanned three times and smoothed with the Jasco smoothing algorithm.
3.4. Cleavage Reactions under Single-Turnover Conditions
The substrate was radio-labeled with 50 µ◦Ci of [
γ
-32P]ATP using 10 U of T4 polynucleotide kinase
(Takara, Dalian, China). After incubation at 37 C for 1 h, the radioactive substrate was extracted with a
Sep-Pak column (Waters Corporation, Milford, MA, USA) and washed with sterilized bidistilled water.
The product was eluted from the column with methanol/water (70/30, v/v), lyophilized, and stored
at −30 ◦C.

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The DNAzyme cleavage reaction was conducted under single-turnover conditions, with 2 µM
of the DNAzyme and 20 nM of the radio-labeled substrate. Prior to the reaction, the DNAzyme and
◦
the substrate were mixed in the buffer of 50 mM Tris-HCl, and the solution was heated at 75 C
for 10 min. After cooling to 37 C, the buffer containing Mg2+ was added to initiate the reaction,
◦
with the final concentration of 2.18 mM Mg2+. Aliquots were taken at defined intervals and mixed
with stopping buffer (8 M urea and EDTA) immediately. These samples were analyzed with a 20%
denaturing polyacrylamide gel, the substrate and the product were separated, and their radioactivity
were recorded with a Phosphoimager (Cyclone Plus Phosphor Scanning System Molders C431200,
PerkinElmer, Downer Crove, IL, USA) as the percentage of product after different time intervals.
The cleavage reaction of each DNAzyme was repeated at least three times on different days, and
less than 15% variation was permitted. The results were reported as the average values of at least
three measurements.
4. Conclusions
In the complex tertiary structure of the catalytic core of 10-23 DNAzyme, the functional groups
around each nucleobase are supposed to play an active role in forming the catalytic conformation,
in addition to the base stacking interaction. With the 2’-deoxythymidine analogues 1–7 at T4 or T8,
the hydroxyl, imidazolyl, and amino groups were introduced, and modifications on the functional
group of the nucleobases were conducted. Both negative and positive effects on the catalytic activity of
DNAzyme were obtained, which indicated that more interactions were induced by these new external
functional groups. These results implied that the 5-methyl group of the most conserved T4 and the
least conserved T8 are supposed to be in contact with other residues in the catalytic conformation.
This kind of contact could be modulated by other functional groups. T8 is more compatible with
the 5-modifications than T4, but a positive effect could be expected at both residues by a delicate
5-substituent, although it is still far from a rational design. On the other hand, the conservation of T4
and T8 could be recognized at the level of the nucleobase, but might be more accurate at the level of
the functional group.
In the complex tertiary structure of 10-23 DNAzyme, chemical modification at the level of
functional groups from either the nucleobase or sugar-phosphate is the first step for the optimization
approach. Nucleoside analogues of the corresponding canonical residues could be used for this
purpose. Here, we learnt that the 5-position of T4 and T8 could be modified with the external hydroxyl
and amino groups. This kind of chemical modification presents a new start for a better catalytic ability,
and furthermore, the modified catalytic core could offer a stronger resistance to endonucleases, which
is helpful for the therapeutic applications of 10-23 DNAzyme. Further delicate design of 5-substitutents
of 2’-deoxythymine is in progress for more efficient DNAzymes.
Supplementary Materials: The mass spectrum of oligonucleotides is available online.
Acknowledgments: Financial support from National Natural Science Foundation of China (Grant No. 21572268)
is gratefully acknowledged.
Author Contributions: Pengyu Li performed the synthesis of nucleoside analogues and oligonucleotides, and
evaluation experiments; Shanshan Du performed the evaluation of some modified DNAzymes; Yang Li analyzed
the data; Junlin He conceived and designed the experiments, and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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