Design, synthesis and properties of artificial nucleic acids from (R)-4-amino-butane-1,3-diol

Article abstract of DOI:10.1039/c3ob42291g

A new artificial nucleic acid analogue, (R)-Am-BuNA, was developed with a simplified acyclic (R)-4-amino-butane-1,3-diol phosphodiester backbone. Phosphoramidite monomers of (R)-Am-BuNA were incorporated into DNA oligonucleotides (ODNs) and G-quadruplexes. Their thermal stability, conformation change and biological stability were further investigated using UV-melting, circular dichroism (CD) and gel electrophoresis. The results suggested that thermal stability of the duplexes of (R)-Am-BuNA modified ODNs and their complementary ODN is highly dependent on the substitution position. Substitution of thymidine at the 7th position in a thrombin-binding DNA aptamer (TBA) results in a slight increase in T_m with no effect on quadruplex conformation on the CD spectrum in comparison to that of the natural G-quadruplex. Further enzymatic experiments with fetal bovine serum (FBS) and snake venom phosphodiesterase (SVPDE) indicated that only single replacement of a (R)-Am-BuNA modified nucleobase greatly inhibited oligonucleotide degradation, which shows their promising applications as capping nucleotides in nucleic acid drugs. This journal is

Full text of DOI:10.1039/c3ob42291g

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Biomolecular Chemistry
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PAPER
Design, synthesis and properties of artificial
nucleic acids from (R)-4-amino-butane-1,3-diol†
Pengfei Li,‡a Jingjing Sun,‡a Meng Su,a Xiaogai Yangb and Xinjing Tang*a
Cite this: Org. Biomol. Chem., 2014,
12, 2263
A new artificial nucleic acid analogue, (R)-Am-BuNA, was developed with a simplified acyclic (R)-4-
amino-butane-1,3-diol phosphodiester backbone. Phosphoramidite monomers of (R)-Am-BuNA were
incorporated into DNA oligonucleotides (ODNs) and G-quadruplexes. Their thermal stability, confor-
mation change and biological stability were further investigated using UV-melting, circular dichroism (CD)
and gel electrophoresis. The results suggested that thermal stability of the duplexes of (R)-Am-BuNA
modified ODNs and their complementary ODN is highly dependent on the substitution position. Substi-
tution of thymidine at the 7th position in a thrombin-binding DNA aptamer (TBA) results in a slight
increase in Tm with no effect on quadruplex conformation on the CD spectrum in comparison to that of
the natural G-quadruplex. Further enzymatic experiments with fetal bovine serum (FBS) and snake venom
phosphodiesterase (SVPDE) indicated that only single replacement of a (R)-Am-BuNA modified nucleo-
base greatly inhibited oligonucleotide degradation, which shows their promising applications as capping
nucleotides in nucleic acid drugs.
Received 17th November 2013,
Accepted 30th January 2014
DOI: 10.1039/c3ob42291g
www.rsc.org/obc
with PNAs, unlocked nucleic acids (UNAs), as the most studied
acyclic analogues of nucleotides, were developed and opti-
Introduction
Nucleic acids are important biological molecules for storing mized for the applications of oligonucleotide-based technol-
genetic information as well as regulating biological functions. ogy.6 UNAs have a highly flexible scaffold with removing the
Artificial nucleic acids with modifications of nucleosides and/ C2′–C3′ bond in contrast with natural ribonucleotides. Nucleic
or backbone have been developed to mimic natural nucleic acids with UNA modification lead to the decrease of duplex
acids and have shown broad applications such as diagnostics, thermal stability in both DNA and RNA.7 The destabilization
therapeutic agents and building blocks for constructing nano- of the UNA modified-duplex limits its use in antisense oligo-
structures.1 Among many artificial nucleic acids, modifications nucleotides in some way, but UNA modified siRNA has shown
of oligonucleotide sugar rings have also been extensively broad potential applications due to its low toxicity and
investigated to improve the binding ability for target oligonu- reduction of off-target effects.8 In addition, Benner et al.
cleotides and the resistance to nucleases.2
reported an acyclic nucleotide analogue from glycerol derived
One of the directions was focused on artificial nucleotide building blocks.9 Oligonucleotides modified with this nucleo-
mimics without sugar rings. With pseudopeptide linkage tide analogue formed less stable duplexes with their comp-
instead of a phosphodiester backbone, peptide nucleic acids lementary sequences.10 To search for more artificial nucleic
(PNA)3 show remarkably strong hybridization with its comp- acids, many new acyclic nucleotides have been developed. The
lementary DNA or RNA due to a non-charged backbone Meggers group disclosed a new nucleic acid analogue–glycol
and resistant to various proteases and nucleases.4 However, nucleic acid (GNA) with structural simplicity and atom
low solubility and poor cellular uptake of PNAs limit their economy.11 GNA maintains the canonical Watson–Crick base
applications if no further modifications are applied.5 Together pairing combined with an acyclic three-carbon propylene
glycol phosphodiester backbone. Propylene glycol nucleosides
constitute the most simplified possible building blocks for a
phosphodiester-bond-containing nucleic acid. In addition, the
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China, and State Key Laboratory of Drug
thermal stability of the GNA duplex significantly surpasses
DNA and RNA duplexes with the same sequence. Thermo-
dynamic parameters of duplex formation indicate that the
enthalpy of GNA duplex formation is less favorable than that
of DNA duplex formation, but its entropy is significantly favor-
able.12 Further crystal structure of GNA showed the existence
Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China. E-mail: xinjingt@bjmu.edu.cn; Tel: +86-010-82805635
bDepartment of Chemical Biology, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
†Electronic supplementary information (ESI) available: NMR, ESI-Mass and
additional figures and tables are provided. See DOI: 10.1039/c3ob42291g
‡These authors have equal contributions.
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of the large average slide (around 3.4 Å) between neigh-
boring base pairs in (S)-GNA duplex itself due to a large
backbone-base inclination compared with B-DNA.13 Even
though GNA can form a more stable duplex itself, its duplex
with DNA or RNA oligonucleotides is still less stable than the
duplex of DNA or RNA oligonucleotide themselves. Recently
Asanuma et al. reported two artificial nucleic acid analogues
(TNA and SNA) based on acyclic threoninol and serinol
scaffolds. TNA and SNA contained three carbons between two
phosphates instead of two carbons for GNA. In addition, SNA
could form a relative stable duplex with its target DNA or RNA
irrespective of the sequence.14
In TNA and SNA analogues, an amide bond is formed as a
linkage between a nucleobase and the backbone instead of
direct attachment of a nucleobase to the backbone. When we
first started to develop artificial nucleic acids, (S)-3-amino-1,2-
propanediol and (R)-4-amino-butane-1,3-diol were chosen as
scaffolds to demonstrate the effect of the length of the back-
bone unit on thermal stability of their duplexes. In the design
of each scaffold monomer, the amine group was attached with
Scheme 1 (a) Structures of DNA (RNA) and (R)-Am-BuNA. (b) Phosphor-
amidite of (R)-Am-BuNA.
sequences were then chosen for the incorporation of (R)-Am-
BuNA. One sequence (CACACCTTGCCATCG) is an antisense
oligonucleotide for matrix metalloproteinase 2 (mmp-2),15 and
the other sequence (GGTTGGTGTGGTTGG) is an anti-throm-
bin-binding aptamer (TBA), a G-rich oligonucleotide that can
form a G-quadruplex aptamer.16
Synthesis of (R)-Am-BuNA phosphoramidite building blocks
a nucleobase through an amide bond formation, while the For constructing the (R)-4-amino-butane-1,3-diol backbone,
primary hydroxyl group was coupled with trityl group and the ethyl-(R)-4-chloro-3-hydroxybutanoate was used as the starting
secondary hydroxyl group was used to form a phosphor- material as shown in Scheme 2. This compound was converted
amidite. These artificial nucleotides were efficiently incorpor- to the diol compound B1 through reduction with sodium boro-
ated into oligonucleotides by a DNA solid phase synthesizer. hydride complex in THF in the quantitative yield. With the
Oligonucleotides with the incorporation of these monomers treatment of sodium azide in DMF, azide compound B1-1 was
contained the extended distance of nucleobases/backbone and obtained with less than 20% yield and its purification was
two or three carbons between two backbone phosphates. difficult. Then the diol compound B1 reacted with 1.1 equi-
Further studies of the formation of duplexes and quadru- valence of DMT chloride to afford the compound B2. B2-1 was
plexes, and their enzymatic stability were investigated.
prepared through an azide SN2 reaction, while the yield was
still not satisfactory. Finally, another route was chosen. With
the treatment of 5 M sodium hydroxide solution at room tem-
perature, epoxy compound B3 was obtained from B2 in the
quantitative yield. DMTr-epoxy compound B3 was subject to
ring opening with ammonia-saturated methanol solution to
afford the scaffold, DMTr-(R)-4-aminobutane-1,3-diol (B4) with
Results and discussion
Rational design of artificial nucleic acids from (R)-4-amino-
butane-1,3-diol ((R)-Am-BuNA)
According to the reported nucleic acid analogues, we are 70% yield over four steps. In comparison, we also synthesized
devoted to the design and evaluation of novel artificial nucleic another set of artificial nucleic acid analogues based on (S)-3-
acids, which could have potential applications in pharma- amino-1,2-propanediol as a scaffold (see ESI S2† for detail).
ceutical sciences. Since the phosphodiester bonds in nucleic
In order to attach nucleobases to the acyclic backbone via
acids play an important role in biological functions of nucleic amide linkage, an acetic modification of nucleobases was
acids, which guarantees the solubility in water and allows carried out as shown in Scheme 2 and Scheme S1.† Ethyl-3-(3-
organisms to adapt biophysical conditions, we maintained dimethylaminopropyl) carbodiimide hydrochloride (EDC-Cl)
the phosphodiester bond for our artificial nucleic acids. In and N-hydroxybenzotriazole (HOBt) were used as coupling
addition, the acyclic backbone was based on six-bond-per- reagents for the amide bond formation between four acetic-
backbone units of vicinal phosphodiester groups similar to nucleobases and acyclic scaffold B4. After purification, the
DNA and RNA with the purpose of maintaining the distance designed products A1/G1/C1/T1 were obtained with 60–84%
between conventional Watson–Crick or Hoogsteen base-pair- yields. Under anhydrous and anaerobic conditions, four phos-
ings. Based on the structures of DNA or RNA, the scaffold of phoramidite monomers A2/G2/C2/T2 for solid phase synthesis
1,3-butanediol with three carbon atoms and six-bond-per-back- were obtained and were further characterized with 1H-NMR
bone units was implemented in (R)-Am-BuNA (Scheme 1). In and 31P-NMR spectra (Scheme 3).
addition, a chiral carbon is introduced in the acyclic monomer
Phosphoramidite monomers A2/G2/C2/T2 were then used
with the same conformation as 3′-C in ribose. And nucleobases to synthesize oligonucleotides (see Table 1) by an automated
were coupled to the amine group on a phosphate backbone solid-phase DNA synthesizer under the same conditions
through an amide bond linkage based on the feasibility as natural nucleoside phosphoramidite monomers with
of synthesis and the stability of anti-nucleases. Two model similar coupling efficiencies. Cleavage and deprotection of the
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Scheme 2 Synthesis of (R)-4-aminobutane-1,3-diol. (a) NaBH4, dry THF, 8 h; (b) DMT-Cl, dry DCM, overnight; (c) 5 M NaOH: 1,4-dioxane, over-
night; (d) NH3, MeOH. Yield 70% for four steps (a,b,c,d). (e) and (f) NaN3, DMF, 90 °C.
Table 1 Sequences of oligonucleotides used in our studies
Entry
Oligonucleotidea
Calculated Mw
Measured Mw
mmp-0
mmp-1
mmp-2
mmp-3
mmp-4
mmp-5
mmp-6
mmp-7
mmp-8
mmpsn-0
mmpsn-2
mmpsn-4
mmpsn-5
TBA-0
CACACCTTGCCATCG
cACACcTTgCCaTCg
4′-cacaccttgccatcg-2′
cACACCTTGCCATCG
CACaCCTTGCCATCG
CACACCTtGCCATCG
CACACCTTGCCaTCG
CACACCTTGCCATCg
CACACCTcGCCATCG
CGATGGCAAGGTGTG
4′-cgatggcaaggtgtg-2′
CGATGGCAAGGtGTG
CGATGGCaAGGTGTG
CGTTGGTGTGGTTGG
GGTTGGtGTGGTTGG
GGTTggTGTGGTTGG
GGtTGGtGTGGtTGG
GGttGGTGTGGttGG
4472.9
4618.1
4908.4
4501.9
4501.9
4501.9
4501.9
4501.9
4486.9
4673.1
5108.5
4702.1
4702.1
4726.1
4755.1
4784.1
4813.1
4842.1
4474.0
4619.9
4909.4
4503.1
4502.8
4503.3
4503.2
4503.1
4487.8
4675.0
5110.2
4702.6
4702.6
4725.4
4755.3
4785.1
4813.6
4843.1
TBA-1
TBA-2
TBA-3
TBA-4
a Lower case letters represent (R)-Am-BuNA nucleotides and upper case
letters represent DNA nucleotides.
Scheme 3 Synthesis of phosphoramidite building blocks of (R)-Am-
BuNA.
oligonucleotide resulted in only a slight decrease in ΔTm of
−1.5 °C and −1.9 °C/modification in comparison to natural
mmp-0/mmpsn-0 duplex (see Table 2, entries 3 and 7). This
suggested that (R)-Am-BuNA modified oligonucleotides were
better tolerated for the duplex formation with its complemen-
tary oligonucleotide when Am-BuNA was incorporated close to
two ends of oligonucleotides due to the end-fraying phenom-
enon. If an acyclic cytosine instead of thymine was incorpor-
ated into the middle of mmp-0 sequence, the obtained
oligonucleotide (mmp-8) can still form a duplex with the
complementary sequence (mmpsn-0), but its melting tempera-
ture was 12.2 °C lower. In comparison to the duplex of mmp-5/
mmpsn-0, the duplex of mmp-8/mmpsn-0 with one mismatch
was less stable with lower Tm. This indicated that acyclic
thymidine in mmp-5 may still have some degree of hydrogen
bonding with its complementary adenine in mmpsn-0 even
though the interaction of acyclic thymine/adenosine is much
less stable than thymidine/adenosine of mmp-0/mmpsn-0
duplex. If all nucleotides of mmp-0 were replaced with
oligonucleotides are performed according to the standard pro-
tocols. After subsequent purification by RP-HPLC, the
obtained oligonucleotides were characterized by ESI-MS (see
Table 1).
Hybridization properties of (R)-Am-BuNA substituted DNA
strands
In order to investigate the effect of (R)-Am-BuNA nucleotide
substitution on ODN duplexes, we measured duplex thermal
stability by UV-melting at 260 nm (see Table 2 and Fig. 1).
Incorporation of one acyclic thymine or one acyclic adenine
nucleotide into the middle of the sequence (see Table 2,
entries 5 and 4, 6) decreased the duplex stability with ΔTm/
modification = −10 °C, −9.1 °C and −9.0 °C, respectively, com-
pared to unmodified mmp-0/mmpsn-0 duplex. Replacement
with acyclic nucleotide in the 4th- or 2nd- end of the
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Table 2 UV-melting studies and thermodynamic parameters (T = 310 K) of (R)-Am-BuNA modified duplexes and (S)-3-amino-1,2-propanediol
nuclei acid (AmpNA) modified duplexesc
Entry
Duplexa
Tm (°C)
ΔTm b (°C)
−ΔH (kJ mol−1
)
−ΔS (J (mol K)−1
)
−TΔS (kJ (mol K)−1
)
ΔG310 K (kJ mol−1
)
1
2
mmp-0/mmpsn-0
mmp-2/mmpsn-0
60.0
nt
—
—
293.1
—
878.7
—
272.4
—
−20.7
—
3
4
5
6
7
8
9
10
11
12
13
14
mmp-3/mmpsn-0
mmp-4/mmpsn-0
mmp-5/mmpsn-0
mmp-6/mmpsn-0
mmp-7/mmpsn-0
mmp-8/mmpsn-0
AmpNA-mmp-2/mmpsn-0
AmpNA-mmp-3/mmpsn-0
AmpNA-mmp-4/mmpsn-0
AmpNA-mmp-5/mmpsn-0
AmPNA-mmp-6/mmpsn-0
AmpNA-mmp-7/mmpsn-0
58.5
50.9
50.0
51.0
58.1
47.8
nt
57.3
52.1
52.0
52.0
56.5
−1.5
−9.1
−10.0
−9.0
−1.9
−12.2
—
−2.7
−7.9
−8.0
−8.0
−3.5
239.3
221.4
219.3
185.4
317.8
185.3
—
214.8
208.9
200.4
214.2
284.4
719.0
687.7
678.5
568.1
993.4
574.6
—
653.7
642.9
613.1
654.2
892.8
222.9
213.2
210.3
176.1
308.0
178.1
—
202.7
199.3
190.1
202.8
276.8
−16.5
−8.2
−9.0
−9.3
−9.8
−7.2
—
−12.1
−9.6
−10.3
−11.4
−7.6
a See Table 1 and Table S1 for oligonucleotides sequences. b Decrease in melting temperature ΔTm was calculated with respect to unmodified
DNA duplex (entry 1). nt = no melting temperature was observed. Experiment conditions: 10 mM phosphate buffer, 100 mM NaCl, pH 7.2, 1 μM
of each oligo strand. c Part of the data of AmpNA came from the published thesis of a master student from our laboratory (for comparison):
Meng Su, “Functionalization and Reconstrunction of Oligonucleotides”, Master thesis, Peking University, 2012.
acyclic nucleotides, no Tm was observed for the duplex of
the obtained oligonucleotide (mmp-2) and mmpsn-0, which is
normal for most of the nucleic acids modified with acyclic
nucleotide analogues, such as (S)-BuNA,17 UNA7 or TNA14a
under similar salt conditions. By shortening the distance of
three carbons to two carbons between two backbone phos-
phates, we obtained another artificial nucleic acid analogues,
(S)-AmpNA) with (S)-3-amino-1,2-propanediol as scaffold
which was previously synthesized in our lab. These artificial
nucleic acid analogues did not show much better improvement
on the thermodynamic stability on duplex formation (see
Table 2 and ESI S2†). Incorporation of one AmpNA nucleotide
into the end of the oligonucleotide (Table 2, entries 10 and 14)
leads to decrease in Tm of about −3 °C/modification. When we
incorporated AmpNA nucleotides in the middle of the
sequences (Table 2, entries 11–13), a sharp decrease of ΔTm
values was observed. These unstable duplexes between acyclic
nucleotide modified oligonucleotide and its complementary
natural DNA was thought to be the flexible backbone. For oli-
gonucleotide analogues containing all (R)-Am-BuNA and
(S)-AmpNA monomers, no better hybridization property was
found in comparison to natural DNA, probably due to the fact
that there is a more flexible linkage between the nucleobase
and backbone, in addition to the flexible backbone as others.
The flexibility of the acyclic nucleotide might cause an un-
favorably large loss in entropy upon duplex formation.
Thermodynamic parameters in the process of duplex for-
mation (see Table 2) were then evaluated using the van’t Hoff
plot. Under a two-state transition model, Ka, the equilibrium
constant, refers to the folding and unfolding of the duplex.
Assuming that enthalpy and entropy are independent of temp-
Fig. 1 (a) UV-melting profile at 260 nm of (R)-Am-BuNA modified DNA
duplex. Experiment conditions: 1 μM for each strand of oligonucleotides
in 10 mM phosphate buffer, 100 mM NaCl, pH 7.2. (b) UV-melting profile
at 260 nm of mmp-5/mmpsn-0 duplex at different sodium concen-
trations. Experiment conditions: 1 μM for each strand of oligonucleotides
in 10 mM phosphate buffer, pH 7.2 with different concentrations of
NaCl.
erature and Gibbs equation ΔG = −RTln(Ka) = ΔH − TΔS, the
plot of ln(Ka) against 1/T (K−1) gave a straight line with r2
>
0.99 (ESI S4†). The slope and intercept of the straight line cor-
responded to (−ΔH/R) and (ΔS/R), respectively, as shown in
Table 2. As expected, the lower thermal stabilities of the acyclic
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nucleotide modification duplex correlated with lower thermo-
dynamic parameters (310 K) except for the 2′-end modified duplex
mmp-7/mmpsn-0. The −TΔS value of mmp-7/mmpsn-0 exceeded
that of mmp-0/mmpsn-0, indicating that a 2′-end guanine modi-
fied duplex showed higher enthalpy and entropy than natural
DNA, which is quite different from other acyclic nucleotide modi-
fied duplexes in Table 2. This observation is similar to a previous
study with GNA, in which acyclic nucleotide modification
duplex formation is less exothermic than DNA duplex for-
mation, but at the same time entropically significantly less
unfavorable due to a stacking interaction of acyclic modifi-
cation at the 2′-end.9 The free energy (ΔG) of the duplexes at
310 K was also calculated as listed in Table 2, which suggested
that all (R)-Am-BuNA modified duplexes could be formed
under physiological pH and salt concentrations. The values of
ΔG correlated with the Tm of the corresponding duplexes.
Furthermore, considering the effect of salt bridge on the
nucleic acid duplex, we investigated the effect of Na+ concen-
tration on the Tm value. The duplex of mmp-5/mmpsn-0 was
significantly stabilized with increasing NaCl concentration from
0.05, 0.10, and 0.20 to 0.40 M, respectively, as shown in Fig. 1b.
Fig. 2 shows the melting curves of the duplexes of (R)-Am-
BuNA, double-stranded ODNs with two complementary acyclic
nucleotide modifications and double-stranded natural ODNs.
The thermal stability of the (R)-Am-BuNA duplex mmp-2/
mmpsn-2 (Tm = 41.2 °C) was significantly lower than one
acyclic nucleotide modification duplex (Tm = 51.2 °C) and DNA
duplex (Tm = 60.0 °C) with the decrease of Tm by 8.8 °C and
18.8 °C, respectively. This observation of lower stability of pure
duplex of (R)-Am-BuNA is different from previous reports of
GNA, TNA or SNA, but similar to (S)-BuNA. We further investi-
gated the effect of salt concentration on the stability of
acyclic nucleotide modified oligonucleotide duplex, however,
no apparent differences were observed with the increase in salt
concentrations from 0.05 M to 0.20 M. Slight increase of Tm
(∼1.0 °C) under a strong electrolyte condition (salt concentration,
0.40 M) was observed. The results showed a weak connection
between salt concentrations and Tm values in comparison to that
of one acyclic nucleotide modified oligonucleotide duplex.
A possible reason for the inefficient reorganization of oligonu-
cleotide duplexes with acyclic nucleotide modification may be
ascribed to the longer and more flexible linkage between the
base and backbone than that of natural DNA, leading to an
unstable duplex and a lower contribution of ionic bond between
negative charge in the backbone and positive charge ions to the
stabilization of duplex in physiological environments.
Fig. 2 (a) UV-melting profile at 260 nm of (R)-AM-BuNA duplex. Experi-
ment conditions: 1 μM for each strand of oligonucleotides in 10 mM
phosphate buffer, 100 mM NaCl, pH 7.2. (b) UV-melting profile at
260 nm of mmp-2/mmpsn-2 duplex at different sodium concentrations.
Experiment conditions: 1 μM for each strand of oligonucleotides in
10 mM phosphate buffer, pH 7.2 with different concentrations of NaCl.
CD experiments were carried out to elucidate the duplex
conformation of mmp-2/mmpsn-2 as well as natural ODNs. As
shown in Fig. 3, the CD spectrum of mmpsn-2 alone was
associated with weak cotton effect and showed weak broad
positive peak at 230–260 nm, which indicated that no preorga-
nized structure was formed. Further investigation of the mmp-
2/mmpsn-2 duplex showed that its CD spectrum contained
strong positive peaks at 280 nm and 240 nm and a negative
peak at 260 nm with a crossover at 257 nm. This observation
Fig. 3 CD profiles of natural and acyclic nucleotide-modified oligo-
nucleotide duplexes. Experiment conditions: 5 μM for each strand of oligo-
clearly indicated duplex formation between these two strands. nucleotides in 10 mM phosphate buffer, 100 mM NaCl, pH 7.2, at 25 °C.
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Table 3 UV-melting studies and thermodynamic parameters of (R)-Am-BuNA modified TBA (T = 310 K)
Entry
Duplexa
Tm (°C)
ΔTm b (°C)
−ΔH (kJ mol−1
)
−ΔS (J (mol K)−1
)
−TΔS (kJ (mol K)−1
)
ΔG310 K (kJ mol−1
)
1
2
3
4
5
TBA-0
TBA-1
TBA-2
TBA-3
TBA-4
49.8
50.8
36.0
nt
—
178.3
162.2
111.4
—
554.2
501.1
353.5
—
171.8
155.3
109.6
—
−6.6
−6.8
−1.9
—
1.0
−13.8
nt
−10.2
39.6
149.7
480.6
149.0
−0.7
a See Table 1 for oligonucleotides sequences. b Decrease in melting temperature ΔTm was calculated with respect to unmodified TBA
oligonucleotide (entry 1). nt = no melting temperature was observed. Experiment conditions: 5 μM oligonucleotides in 10 mM Tris-HCl buffer,
100 mM KCl, pH 7.4.
In addition, the positive peak at 280 nm and a crossover at
257 nm of the acyclic nucleotide modified nucleic acid duplex
were in accordance with the characteristics of right-handed
nucleic acids.
Quadruplex formation of (R)-Am-BuNA modified TBA
aptamers
According to previous NMR and X-ray structural studies, TBA
forms an intramolecular antiparallel G-quadruplex with two
G-tetrads linked by three loops: a central TGT and two TT
loops.18 Further studies on the properties of modified TBA
shows that modifications with acyclic nucleotide monomers at
3rd, 7th and 12th positions of this sequence resulted in an
increased thermal stability, especially at the 7th position.19
Thus, we synthesized two TBA oligonucleotides by incorpor-
ation of one acyclic monomer at the 7th position (TBA-1) and
three acyclic monomers at 3rd, 7th and 12th positions (TBA-3).
Modifications with acyclic (R)-Am-BuNA monomers at
G-tetrads (TBA-2) and two TT loops (3rd and 4th, 12th and
13th positions, TBA-4) were also achieved to investigate their
thermal stability. The results of UV melting studies at 295 nm
are shown in Table 3 and Fig. 4a. It is clearly seen that modifi-
cation at the 7th position slightly increased the thermal stabi-
lity with the ΔTm value of 1.0 °C compared to that of natural
TBA-0. Unexpectedly, for TBA-3 with three acyclic nucleotide
modifications at the 3rd, 7th and 12th positions, no Tm was
observed. Acyclic nucleotide modification in the region of
Fig. 4 (a) UV-melting profile at 295 nm of (R)-Am-BuNA modified TBA.
Experiment conditions: 10 mM Tris-HCl buffer, 100 mM KCl, pH 7.4,
5 μM oligonucleotide strand concentration. (b) UV-melting profile at
295 nm of TBA-2 at different potassium concentrations. Experiment
G-tetrads and two TT loops also lowered the thermal stability of
modified aptamers with ΔTm values of −13.8 °C and −10.2 °C,
respectively. These results suggested that incorporation of
(R)-Am-BuNA monomer only at the 7th position in the TBA
conditions: 10 mM Tris-HCl buffer, pH 7.4, 5 μM oligo strand concen-
sequence was favorable for quadruplex formation. Next, the
effect of potassium concentration on quadruplex formation
was also investigated. The 7th modified quadruplex TBA-1 was
treated with different concentrations of potassium ion (0.01 M,
0.05 M, 0.10 M, 0.20 M and 0.40 M). As we expected, gradual
increase of Tm values was observed with the increase of potass-
ium concentrations, as shown in Fig. 4b.
Melting curves of each modified TBA quadruplex provided
the thermodynamic parameters as shown in Table 3. Modifi-
cation at G-tetrads (TBA-2) resulted in the destabilization of
the quadruplex structure due to a sharp decrease of ΔH. This
suggested that acyclic modification in the G-tetrads region
was unfavorable for quadruplex formation. Thermodynamics
tration with different concentrations of KCl.
parameters of the 7th modified G-quadruplex (TBA-1) shows a
favorable ΔG compared to that of natural TBA-0. However,
incorporation of multiple acyclic thymines (TBA-3) caused a
disadvantageous effect, which indicated that more flexible
structures might restrict the spatial folding process of the
G-quadruplex. CD spectra were further recorded for natural
TBA and (R)-Am-BuNA modified TBAs (Fig. 5). It is clearly seen
that positive peaks at 250 nm and 290 nm and a negative peak
at 270 nm were observed in the CD spectrum of the 7th posi-
tion modified quadruplex (TBA-1) which is in accordance with
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Fig. 6 (a) Stability of unmodified and acyclic nucleotide-modified
single oligonucleotide strand in FBS. Experiment conditions: 10 μM
mmp-0 or mmp-7 in 20% FBS at 37 °C. (b) Stability of unmodified
(TBA-0) and acyclic nucleotide-modified anti-thrombin-binding
aptamer (TBA-1) in FBS. Experiment conditions: 10 μM TBA-0 or TBA-1
in 40% FBS at 37 °C. (c) Stability of unmodified and acyclic nucleotide-
modified single oligonucleotide strand in SVPDE. Experiment conditions:
5 μM mmp-0 or mmp-7 with 220 mU SVPDE in 100 mM Tris-HCl buffer,
10 mM MgCl2, 100 mM NaCl, pH 7.4, at 37 °C. (d) Stability of unmodified
and acyclic nucleotide-modified thrombin-binding DNA aptamer in
SVPDE. Experiment conditions: 5 μM TBA-0 or TBA-1 with 330 mU
SVPDE in 100 mM Tris-HCl buffer, 10 mM MgCl2, 100 mM KCl, pH 7.4, at
37 °C.
Fig. 5 CD profile of natural TBA and (R)-Am-BuNA modified TBAs.
Experiment conditions: 1 μM for each strand of oligonucleotides in
10 mM Tris-HCl buffer, 100 mM KCl, pH 7.4, at 25 °C.
that of natural TBA-0. This suggested that acyclic modification
at the 7th position did not cause any disadvantageous effect
on the formation of antiparallel G-quadruplex. TBA-2 with
acyclic monomer incorporation into G-tetrads showed a broad
positive CD spectrum between 230 and 310 nm that is comple-
tely different from that of the antiparallel G-quadruplex of the
natural TBA. The complicated CD spectra may be due to
multiple conformations of TBA-2, which was consistent with
the observation of unclear transition in the Tm melting
curve. Incorporation of multiple acyclic nucleobases (TBA-4)
further caused the loss of CD signals due to non-organized
conformations.
Conclusions
Artificial nucleic acid analogues, (R)-Am-BuNA with a (R)-4-
aminobutane-1,3-diol backbone, were synthesized and their
thermal stability of duplexes and G-quadruplexes was demon-
strated. Similar to other acyclic nucleotide analogues, in-
corporation of acyclic (R)-4-aminobutane-1,3-diol scaffold at
different positions in DNA strands induced a decreased
Enzymatic stability of (R)-Am-BuNA modified ODNs
Together with the natural sequences of mmp-0 and TBA-0, thermal stability. A significantly less effect on the decrease of
(R)-Am-BuNA modified ODNs (mmp-7 and TBA-1) were treated Tm was experienced with acyclic nucleotide modifications
with fetal bovine serum (FBS) and snake venom phosphodi- close to the two terminals, and modifications in the middle of
esterase (SVPDE), respectively. All oligonucleotides species the sequences resulted in a marked decrease in the Tm value.
were individually incubated in FBS and SVPDE, and aliquots Increasing the backbone length by one carbon atom resulted
were then sampled at regular intervals, quickly frozen and in no significant difference of duplex thermal stability of
stored for gel-shift analysis. (Fig. 6). When incubated in 20% nucleic acid analogues with (R)-4-amino-butane-1,3-diol in
FBS at 37 °C, mmp-0 was almost completely degraded in comparison to (S)-3-amino-1,2-propanediol as scaffolds. It
480 min, while mmp-7 with single terminal (R)-Am-BuNA seems that the long linkage of an amide bond in the side
modification showed little degradation even after 960 min chain shows no favorable effect on the formation of duplex in
incubation, as shown in Fig. 6a. If SVPDE was used, mmp-0 comparison to that of (S)-BuNA due to the flexible backbone
degraded very fast and almost disappeared in 20 min. Instead, and flexible linkage of the backbone and nucleobases. The
only a small amount of degradation of mmp-7 was observed in data of acyclic (R)-Am-BuNA modified TBAs indicated that
60 min incubation at the same concentration of SVPDE modification at the 7′ position of the TBA sequence resulted in
(Fig. 6c). Similar to mmp-7, we observed the increased enzy- comparable thermal stability and maintained the same confor-
matic stability of (R)-Am-BuNA modified TBA-1 than that of mation of anti-parallel G-quadruplex as that of the natural
TBA-0, as shown in Fig. 6b and 6d. These results indicated TBA. Multiple modifications of TBA sequences at the loop and
that single modification of oligonucleotides with (R)-Am-BuNA G-tetrads are unfavorable. Further enzymatic experiments
could greatly enhance their resistance to enzymatic degra- revealed that oligonucleotides with even single acyclic (R)-Am-
dation. This is because the artificial nucleotide is not recog- BuNA modification greatly enhanced their enzymatic stability.
nized by some exo-nucleases, which is similar to a previously These studies indicated that the extended distances between
reported glycerol derived building block as terminal the backbone units or the backbone and nucleobase caused an
modification.9
unfavorable effect on the duplex thermodynamic stability,
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providing additional information on DNA base pairing. The (c = 1.16, methanol); 1H-NMR (400 MHz, CDCl3) δ = 7.44 (d, J =
enzymatic stability of oligonucleotides with single modifi- 7.6 Hz, 2 H), 7.16–7.34 (m, 7 H), 6.83 (d, J = 8.8 Hz, 4 H), 3.75
cation of (R)-Am-BuNA analogues shows their possible appli- (s, 6 H), 3.20–3.26 (m, 2 H), 3.09–3.12 (m, 1 H), 2.80 (dd, J =
cations as capping nucleobases for high stable nucleic acids 8.2, 5.0 Hz, 1 H), 2.51 (dd, J = 5.0, 2.7 Hz, 1 H), 1.79–1.83 (m,
drugs.
2 H); 13C-NMR (100 MHz, CDCl3) δ = 158.2, 144.9, 136.2, 129.9,
129.0, 127.6, 126.6, 113.0, 85.9, 60.3, 55.1, 50.3, 47.1, 33.2; m/z
MS (ESI-TOF+) measured [M + Na]+ 413.32, C25H26O4, calcu-
lated [M + Na]+ 413.18.
(3)-4-Amino-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-
butanol (B4). The product B3 (0.32 g, 0.82 mmol) was dis-
Experimental section
Synthesis of acyclic (R)-Am-BuNA monomers
(3R)-4-Chloro-1,3-butanediol (B1). Sodium borohydride solved in a saturated solution of ammonium in methanol. The
(0.68 g, 18.0 mmol) was dissolved in THF (10 mL). The mixture mixture was stirred for 24 h and then concentrated in vacuo.
was warmed at 40 °C for 30 min. To this mixture was added The crude residue was purified by flash chromatography
dropwise ethyl-(3R)-4-chloro-3-hydroxyl butyrate (98% ee, (DCM–MeOH–TEA, 10/1/1%) to afford B4 (245 mg, 0.60 mmol)
2.15 g, 12.9 mmol, Beijing Ouhechem Technology Co. Ltd). as a colorless viscous oil with a yield of 75%; [α]2D5 = 2.3 (c =
The reaction continued for another 5 hours and was then 1.06, methanol); 1H-NMR (400 MHz, CDCl3) δ = 7.43(d, J =
quenched with hydrochloric acid (2 mL concentrated HCl in 7.4 Hz, 2 H), 7.18–7.32 (m, 7 H), 6.83 (d, J = 8.8 Hz, 4 H), 3.78
5 mL water) at 0 °C. The mixture was stirred for one more hour (s, 6 H), 3.72 (m, 1 H), 3.30 (m, 1 H), 3.22 (m, 1 H), 2.75 (dd,
and was then adjusted to pH = 7 with aqueous sodium hydroxide. J = 12.6, 3.5 Hz, 1 H), 2.58 (dd, J = 12.7, 7.8 Hz, 1 H), 1.67–1.77
The solution was extracted with ethyl acetate (50 mL) and the (m, 2 H); 13C-NMR (100 MHz, CDCl3) δ = 158.5, 145.0, 136.2,
organic layer was combined and dried with anhydrous Na2SO4. 136.1, 130.0, 128.1, 127.9, 126.8, 113.2, 86.6, 71.6, 61.8, 55.3,
After filtration, the organic solvent was removed in vacuo and 47.7, 34.5; m/z: MS (ESI-TOF+) measured [2M + H]+ 815.4250,
the crude residue was purified by flash column chromato- C25H29NO4, calculated [2M + H]+ 815.4266.
graphy (PE–EA, 1/10) to afford the compound B1 (1.55 g,
General method for coupling of acetic-bases and acyclic
backbone (B4)
12.4 mmol) as a pale-yellow oil with 96% yield. [α]2D5 = 22.3 (c =
1
1.09, methanol); H-NMR (400 MHz, CDCl3) δ = 3.99–4.05 (m,
1 H), 3.82 (m, J = 10.4, 9.0, 3.9 Hz, 2 H), 3.59 (dd, J = 11.1, Acyclic backbone B4 (1.0 eq.), acetic-base (A, G, C, T, 1.1 eq.),
4.3 Hz, 1H), 3.50 (dd, J = 11.1, 6.6 Hz, 2 H), 1.72–1.78 (m, 1 H). EDC-HCl (1.5 eq.), HOBt (1.5 eq.), and TEA (3.0 eq.) were sus-
13C-NMR (100 MHz, CDCl3) δ = 70.8, 60.4, 49.7, 35.7.
pended in anhydrous DMF (0.5 mmol mL−1). After stirring for
(3R)-4-Chloro-1-(bis(4-methoxyphenyl)(phenyl)methoxy)- 1 hour at room temperature, chloromethane was added to
3-butanol (B2). B1 (400 mg, 3.2 mmol) and DMT-Cl (1.08 g, dilute the mixture. The solution was washed with brine three
3.2 mmol) were dissolved in dichloromethane (15 mL). Under times and the organic layer was combined and concentrated.
N2, 1 mL of triethylamine was added dropwise carefully at Cold ether was added to the residue and the mixture was left
0 °C. The mixture was stirred overnight, and then diluted with at −4 °C overnight. The desired building blocks (A1, G1, C1
dichloromethane (50 mL). The obtained solution was washed and T1) were obtained by filtration and used for the following
with saturated sodium bicarbonate solution (20 mL × 3). The steps without further purification.
organic layer was combined and dried with anhydrous Na2SO4.
A1 for adenine (A1). White solid (67% yield), [α]2D5 = −5.6
After filtration, the organic solvent was removed in vacuo to (c = 1.03, methanol) 1H-NMR: (400 MHz, CDCl3) δ = 8.65 (s,
afford the compound B2 (1.23 g, 2.88 mmol) as a pale-yellow 1 H), 8.09 (s, 1 H), 7.98 (d, J = 7.6 Hz, 2 H), 7.58 (d, J = 7.1 Hz,
oil with a yield of 90%. Rf value was given 0.5 in PE–EA 3/1. 1 H), 6.80–7.55 (m, 11 H), 6.80 (d, J = 8.4 Hz, 4 H), 4.90 (s,
1H-NMR (400 MHz, CDCl3) δ = 7.38 (d, J = 7.3 Hz, 2H), 2 H), 3.94 (s, 1 H), 3.76 (2, 1 H) 3.52–3.76 (m, 1 H), 3.24 (m,
7.24–7.29 (m, 7 H), 6.80 (d, J = 8.8 Hz, 4 H), 3.97–4.00 (m, 1 H), 1 H), 3.13–3.18 (m, 2 H), 1.71–1.76 (m, 2H); 13C-NMR
3.75 (s, 6 H), 3.43–3.52 (m, 2 H), 3.34 (dd, J = 5.4, 4.3 Hz, 1H), (100 MHz, CDCl3) δ = 166.0, 165.0, 158.4, 152.5, 152.0, 149.3,
3.25 (dd, J = 5.5, 4.0 Hz, 1 H), 1.79–1.82 (m, 2 H); 13C-NMR 144.7, 143.8, 135.9, 135.8, 133.5, 132.7, 129.9, 128.7, 128.0,
(100 MHz, CDCl3) δ = 158.6, 144.8, 136.0, 135.9, 128.1, 128.0, 127.9, 126.8, 122.4, 113.2, 86.7, 69.4, 61.4, 55.2, 46.3, 45.8, 34.4;
127.0, 113.2, 86.8, 70.8, 61.3, 55.3, 49.5, 34.0.
(3R)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3,4-epoxy- culated [M + Na]+ 709.28.
m/z MS (ESI-TOF+) measured [M + Na]+ 709.58, C39H38N6O6 cal-
butane (B3). To a solution of B2 (450 mg, 1.1 mmol) in 1,4-
G1 for guanine (G1). White solid (59% yield), [α]2D5 = −2.3
dioxane (5 mL), 5 M sodium hydroxide solution was added (c = 0.97, methanol) 1H-NMR (400 MHz, DMSO-d6): 8.21 (s,
dropwise. The solution was stirred overnight at 40 °C and was 1 H), 7.90 (s, 1 H), 7.36 (d, J = 7.8 Hz, 2 H), 7.30 (d, J = 7.6 Hz,
then diluted with ethyl acetate (50 mL). The mixture was 2 H), 7.22–7.35 (m, 5 H), 6.87 (d, J = 8.6 Hz, 4 H), 5.75 (s, 1 H),
washed with saturated sodium chloride solution (10 mL × 3). 4.79 (s, 2 H), 4.65–4.66 (d, J = 5.4 Hz, 2 H), 3.73 (s, 6 H), 3.67
The organic layer was combined and dried with anhydrous (br, 1 H), 3.20–3.32 (m, 1 H), 3.03 (m, 3 H), 2.50–2.78 (m, 2 H),
Na2SO4. After filtration, the organic solvent was removed 1.51–1.72 (m, 2 H), 1.09 (d, J = 6.7 Hz, 6 H); 13C-NMR
in vacuo to afford pale-yellow oil B3 (420 mg, 1.1 mmol) with a (100 MHz, DMSO-d6) δ = 180.1, 166.1, 157.9, 154.9, 149.0,
yield of 98%. Rf value was given 0.3 in PE–EA (10/3). [α]2D5 = 6.7 147.9, 145.2, 140.8, 129.6, 127.7, 127.6, 126.5, 119.7, 113.1,
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85.3, 66.4, 60.2, 55.0, 45.6, 45.3, 34.9, 34.7, 18.9. m/z MS 4H), 4.50 (m, 2 H), 4.12 (m, 1 H), 3.78 (s, 6 H), 3.12–3.78 (m,
(ESI-TOF+) measured [M + Na]+ 691.58, C36H40N6O7 calculated 8 H), 2.23 (s, 3 H), 1.82–1.99 (m, 4 H), 1.01–1.16 (m, 12 H).
[M + Na]+ 691.73.
31P-NMR (162 MHz, CDCl3) δ = 146.8, 148.1. m/z MS (ESI-TOF+)
C1 for cytosine (C1). White solid (84.4% yield), [α]2D5 = −8.7 measured [M + Na]+ 824.29, C42H53N6O8P calculated [M + Na]+
1
(c = 0.98, methanol); H-NMR (400 MHz, CDCl3) δ = 9.38 (s, 1 823.88.
H), 7.56–7.63 (m, 1 H), 7.53–7.55 (m, 1 H), 7.40–7.52 (m, 3 H),
T2. Eluent solution: dichloromethane–TEA, 100/2, 51%
1
7.28–7.50 (m, 10 H), 6.81 (d, J = 8.9 Hz, 4 H), 4.46–4.60 (m, yield; H-NMR (400 MHz, CDCl3) δ = 7.15–7.32 (m, 9 H), 7.10
2 H), 3.96 (m, 1H), 3.78 (s, 7 H), 3.44–3.55 (m, 1 H), 3.24–3.37 (s, 1 H), 7.05 (s, 1 H), 6.85 (t, J = 4.8, 8.8 Hz, 4 H), 4.36 (m,
(m, 1 H), 3.20 (m, 1 H), 3.07 (m, J = 13.3, 8.1, 5.4 Hz, 1 H), 2.15 2 H), 3.89 (m, 1 H), 3.80 (s, 6 H), 3.16–3.78 (m, 8 H), 2.51 (m,
(s, 3 H), 1.71 (m, 2 H); 13C-NMR (100 MHz, CDCl3) δ = 170.8, 1 H), 2.17 (m, 1 H), 1.84–1.92 (m, 5 H), 1.03–1.17 (m, 12 H);
166.7, 162.6, 157.9, 155.2, 151.4, 145.2, 136.0, 129.6, 127.7, 127.6, 31P-NMR (162 MHz, CDCl3) δ = 146.6, 148.0. m/z MS (ESI-TOF+)
126.5, 113.1, 94.6, 85.2, 66.4, 60.1, 55.0, 51.4, 45.7, 45.5 (triethyl- measured [M + Na]+ 797.14, C41H52N5O8P calculated [M + Na]+
amine), 34.8, 24.3, 11.6 (triethylamine); m/z MS (ESI-TOF+) 796.85.
measured [M + Na]+ 623.13, calculated C33H36N4O7 [M + Na]+
623.66.
T1 for thymine (T1). White solid (75% yield), [α]2D5 = −4.4
(c = 1.00, methanol), H-NMR (400 MHz, CDCl3), δ = 7.40–7.41
Acknowledgements
1
(m, 1 H), 7.38 (s, 1 H), 7.27–7.30 (m, 6 H), 7.21 (m, J = 7.2 Hz, This work was supported by the National Basic Research
1H), 7.04–7.18 (m, 1H), 6.82 (m, J = 8.9 Hz, 2 H), 4.27 (s, 1 H), Program of China (973 Program; grant no. 2012CB720600), the
3.92 (m, 1H), 3.79 (s, 6 H), 3.44–3.49 (m, 1 H), 3.32–3.40 (m, National Natural Science Foundation of China (grant no.
1 H), 3.20–3.27 (m, 1 H), 3.04–3.17 (m, 1 H), 1.88 (s, 3 H), 21072015), Program for New Century Excellent Talents in Uni-
1.69–1.77 (m, 2 H); 13C-NMR (100 MHz, CDCl3) δ = 167.00, 164.5, versity (grant no. NCET-10-0203) and the State Key Laboratory
158.6, 151.4, 144.8, 140.9, 136.0, 130.0, 128.1, 128.0, 127.0, 113.3, of Drug Research (grant no. SIMM1106KF-15).
111.1, 86.9, 69.9, 61.8, 55.3, 50.55, 46.2, 45.6 (triethylamine), 36.6
(DMF), 34.4, 12.4. m/z MS (ESI-TOF+) measured [M + Na]+ 596.32,
C32H35N3O7 calculated [M + Na]+ 596.24.
Notes and references
General method for phosphoramidition
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tetraisopropylphosphorodiamidite (4.0 eq.) was dissolved in
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tion system. The mixture was stirred at room temperature for
2 h, the corresponding (R)-Am-BuNA monomers (A2, G2, C2,
T2) were then purified through flash silica gel column
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A2. Eluent solution: dichloromethane–TEA, 100/2. 54%
yield, 1H-NMR (400 MHz, CDCl3) δ = 8.79 (s, 1 H), 8.20 (s, 1 H),
8.15 (s, 2 H), 7.29–7.61 (m, 12 H), 6.82 (d, J = 8.5 Hz, 4 H),
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148.1. m/z MS (ESI-TOF+) measured [M
+
Na]+ 910.29,
C48H55N8O7P calculated [M + Na]+ 909.97.
G2. Eluent solution: dichloromethane–acetone–TEA, 100/
10/2. 54% yield, 1H-NMR (400 MHz, CDCl3) δ = 7.70 (d, 1 H, J =
12.6 Hz), 7.37–7.39 (m, 2 H), 7.19–7.22 (m, 8 H), 6.81 (m, 4 H),
4.70 (m, 2 H), 4.12 (m, 1 H), 3.78 (s, 6 H), 3.12–3.78 (m, 8 H),
2.63 (s, 2 H), 1.78–1.88 (m, 3 H), 1.01–1.19 (m, 12 H). 31P-NMR
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891.96.
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C2. Eluent solution: dichloromethane–TEA, 100/3. 33%
1
yield, H-NMR (400 MHz, CDCl3) δ = 7.71 (d, J = 7.3 Hz, 1 H),
7.28–7.43 (m, 9 H), 7.20 (m, 1 H), 6.81–6.84 (m, J = 4.2, 8.4 Hz,
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