Design, synthesis, biophysical and primer extension studies of novel acyclic butyl nucleic acid (BuNA)

Article abstract of DOI:10.1039/c3ob41244j

A novel nucleic acid analogue called acyclic (S)-butyl nucleic acid (BuNA) composed of an acyclic backbone containing a phosphodiester linkage and bearing natural nucleobases was synthesized. Next, (S)-BuNA nucleotides were incorporated in DNA strands and their effect on duplex stability and changes in structural conformation were investigated. Circular dichroism (CD), UV-melting and non-denatured gel electrophoresis (native PAGE) studies revealed that (S)-BuNA is capable of making duplexes with its complementary strands and integration of (S)-BuNA nucleotides into DNA duplex does not alter the B-type-helical structure of the duplex. Furthermore, (S)-BuNA oligonucleotides and (S)-BuNA substituted DNA strands were studied as primer extensions by DNA polymerases. This study revealed that the acyclic scaffold is tolerated by enzymes and is therefore to some extent biocompatible.

Full text of DOI:10.1039/c3ob41244j

Organic &
Biomolecular Chemistry
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PAPER
Design, synthesis, biophysical and primer extension
studies of novel acyclic butyl nucleic acid (BuNA)†
Vipin Kumar,a Kiran R. Gore,b P. I. Pradeepkumarb and Venkitasamy Kesavan*a
Cite this: Org. Biomol. Chem., 2013, 11,
5853
A novel nucleic acid analogue called acyclic (S)-butyl nucleic acid (BuNA) composed of an acyclic back-
bone containing a phosphodiester linkage and bearing natural nucleobases was synthesized. Next,
(S)-BuNA nucleotides were incorporated in DNA strands and their effect on duplex stability and changes
in structural conformation were investigated. Circular dichroism (CD), UV-melting and non-denatured gel
electrophoresis (native PAGE) studies revealed that (S)-BuNA is capable of making duplexes with its
complementary strands and integration of (S)-BuNA nucleotides into DNA duplex does not alter the
B-type-helical structure of the duplex. Furthermore, (S)-BuNA oligonucleotides and (S)-BuNA substituted
DNA strands were studied as primer extensions by DNA polymerases. This study revealed that the acyclic
scaffold is tolerated by enzymes and is therefore to some extent biocompatible.
Received 17th June 2013,
Accepted 9th July 2013
DOI: 10.1039/c3ob41244j
www.rsc.org/obc
novel acyclic nucleotides, which are tolerated well in DNA or
RNA after incorporation offers new possibilities to explore
Introduction
Nucleic acid is the genetic material that stores the hereditary their structure and function. In 1990 Steven A. Benner and co-
information passed from one generation to the next. The workers constructed the first acyclic nucleic acid analogue
growing field of DNA biotechnology requires new chemically from glycerol derived building blocks.4a Extensive studies of
modified nucleic acid analogues that can perform advanced these oligonucleotides indicated very low duplex stability (Tm
=
and superior functions. Artificial nucleotide analogues have 16 °C) of alternating adenine and thymine monomers within
been widely explored to modulate the properties of nucleic 19 mer oligonucleotides.4c Similarly, 2′-deoxy-1′,2′-seco-D-
acids, making them promising therapeutic agents1 and impor- ribosyl oligonucleotides were not able to form duplexes with
tant building blocks for constructing molecular devices.2 their complementary strands.4e In 2005, a breakthrough was
Researchers have investigated various artificial nucleic acid achieved by Eric Meggers and co-workers who synthesized
systems containing non-ribose sugars3 as well as acyclic structurally simplified glycol nucleic acid (GNA) and demon-
scaffolds. Based on the number of carbon atoms between the strated its capability to form a highly stable duplex with its
two phosphate groups acyclic nucleic acids can be constructed complementary strands.4f (S)-GNA adopts a right-handed helix
as C-2 or C-3 building blocks.4 The straightforward chemical and notably substituting a single thymine (S)-GNA nucleotide
synthesis of acyclic phosphoramidite building blocks, facili- in normal DNA duplex reduces the stability of the duplex by
tates the construction of fully modified artificial nucleic acids 13 °C.5j (S)-GNA oligonucleotide does not bind with DNA but it
or incorporation of these nucleotides in DNA or RNA exhibits some capability to make duplexes with complemen-
strands.5,6 Another interesting aspect of acyclic nucleotides is tary RNA strands.5j Similarly threoninol nucleic acid (aTNA)4h
the effect of different stereoisomers on the nucleic acid struc- and serinol nucleic acid (SNA)4i reported as C-3 scaffold based
ture.5i,j It is evident from the literature that the handedness of acyclic nucleic acids, adopt A-form and B-form-like helical
the helical structure of acyclic nucleic acids depends upon the structures respectively. In contrast to GNA and SNA, the aTNA
chirality of the nucleotide building blocks.7 Hence, designing building block contains two chiral centres but surprisingly no
preorganized structure was observed for the single strands.
These literature findings suggest that introduction of a second
chiral centre induces an unexpected structural conformation.
Collectively, these reports suggest the construction of a novel
acyclic oligonucleotide which is tolerated in natural nucleic
acids and is capable of hybridizing with DNA/RNA. Moreover,
a new structurally simplified nucleic acid may help us to
understand, how self-replicating RNA could have usurped the
role of genetic material over nucleic acid-like molecules.8
aChemical Biology Laboratory, Department of Biotechnology, Indian Institute of
Technology Madras (IITM), Chennai 600036, India. E-mail: vkesavan@iitm.ac.in;
Tel: +91-44-22574124
bDepartment of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai
400076, India
†Electronic supplementary information (ESI) available: Characterization, NMR
spectra, MALDI-TOF and additional figures are provided. See DOI: 10.1039/
c3ob41244j
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Fig. 1 (a) Chemical structures of DNA, (S)-GNA and (S)-BuNA. Numbers were
assigned in order to show six-bonds-per-backbone for DNA, BuNA and five-
bonds-per-backbone for (S)-GNA. (b) (S)-BuNA nucleosides.
Scheme 1 (a) Me2S–BH3, B(OCH3)3 dry THF, 90%. (b) Benzaldehyde dimethyl
acetal, camphorsulfonic acid, dry DCM, 60%. (c) Benzoyl protected nucleobases,
DEAD, PPh3, dry THF, 40 to 60%. (d) NaOH–EtOH solution, acetic acid, 90%. (e)
10% Pd/C, H2, MeOH, 90%. (f) DMT-Cl, dry pyridine, 80%. (g) 2-Cyanoethyl
N,N-diisopropylchlorophosphoramidite, DIPEA, dry DCM, 70%.
When these C-2 and C-3 acyclic scaffold based nucleotides
were incorporated into DNA and RNA strands, they have been
found to be stable against nucleases.4b,6 Moreover, acyclic
scaffolds have been utilized to tether nucleobase surrogates
(universal artificial nucleobases)9a or functional moieties.9b
aTNA and SNA have been reported as effective C-3 scaffolds for
the wedge type insertion of functional moieties working as
photo switches9c and for tethering nucleobases.4d,5g,h
There are only very few acyclic scaffolds which are tolerated
by enzymes in DNA strands.10 Hence, it is important to syn-
thesize new chiral acyclic building blocks which can be incor-
porated in DNA or RNA strands and can be used to construct
new artificial nucleic acids. Herein, we report the synthesis of
acyclic butyl nucleic acid (BuNA) with six-bonds-per-backbone
of vicinal phosphodiester groups analogous to DNA and RNA
(Fig. 1). Furthermore, these BuNA nucleotides were tolerated
among DNA strands (primers) (Fig. 1) which was demon-
strated by primer extension studies on a DNA template by Bsu
and Bst DNA polymerases.
Synthesis of (S)-BuNA phosphoramidite building blocks
In our first attempt, (S)-malic acid was used as the starting
material for constructing the phosphoramidite monomers as
shown in Scheme 1. (S)-Malic acid 1 was converted to the triol
by reduction with borane dimethyl sulphide complex in THF
in 90% yield. Butane triol 2 was protected with benzaldehyde
dimethyl acetal yielding the corresponding 1,3 protected
alcohol 3 in 60% yield.11b The protected nucleobases and the
alcohol were condensed by a Mitsunobu reaction. By this syn-
thetic route we could condense uracil 4a, thymine 4b and
adenine 4c with 40–60% yield. The benzoyl group was cleaved
under basic conditions to obtain 5a and 5b in 90% yield.
Deprotection of the benzylidene protecting group was carried
out by treatment with H2 in the presence of 10% palladium/
carbon. Using these conditions it was possible to deprotect
only the uracil substituted compound, resulting in 6a with
90% yield. Hydrogenation with 10% palladium/carbon of the
thymine and adenine analogues to achieve nucleobase diols
6b and 6c was unsuccessful. Additionally, deprotection using
BCl3 led to decomposition of the substrate. The primary
alcohol of the uracil nucleobase diol 6a was converted to
dimethoxy trityl chloride (DMT) alcohol 7 in 80% yield and
subsequently the uracil phosphoramidite building block 8 in
70% yield. It is important to mention that we have not utilized
compound 8 for oligonucleotide synthesis. In future, it might
be useful for incorporation in modified siRNA.
Results and discussion
Rational design of acyclic butyl nucleic acid (BuNA)
Due to the structurally simplified structure and stable duplex
formation by (S)-GNA, we intended to increase the back-bone
length by one carbon atom, which we hoped would enable
hybridization to DNA and RNA strands. The rational design of
the acyclic scaffold was based on the hypothesis that keeping
the number of atoms between two repeating units of mono-
mers (Fig. 1) similar to DNA/RNA should enable the conven-
tional Watson–Crick base-pairing to occur as in DNA/RNA. It
was reported that cleavage of the C2′–C3′ bond of deoxynucleo-
side resulted in the (S)-isomer of acyclic nucleosides.4b In
order to acquire the same configuration in BuNA as that of
DNA, we synthesized the required 1,3-(S)-diol nucleosides11a
from (R)-aspartic acid.
In order to synthesis the A, T, G and C phosphoramidite
building blocks, we followed an established synthetic methodo-
logy of epoxide ring opening by the nucleobase with a catalytic
amount of sodium hydride in DMF as a solvent at 110 °C
(Scheme 2).4f,11d This was achieved using (R)-aspartic acid 9 as
the starting material. The aspartic acid was subjected to a di-
azotization reaction to give the corresponding bromosuccinic
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Scheme 2 Synthesis of pyrimidine phosphoramidite building blocks of (S)-
BuNA.
acid 10 in 60% yield. The resulting bromosuccinic acid was
converted to the bromodiol 11 by treatment with borane
dimethyl sulphide complex in THF at 0 °C in 87% yield. The
bromodiol was treated with cesium carbonate in dry dichloro-
methane and converted to epoxide 12 with 65% yield.11c Sub-
sequently, this epoxide was treated with DMT chloride to
afford the corresponding DMT-epoxide 13 with 60% yield. The
DMT-epoxide was subjected to ring opening with nucleobase
(14, 15 and 16) in the presence of 0.3 equivalent of NaH in dry
DMF to afford DMT-alcohols 17, 18 (Scheme 2) and 19
(Scheme 3). Next, the exocyclic amine of compound 19
was protected by a benzoyl group to afford 20 in 60% yield.
Phosphitylation of DMT-alcohols in the presence of 2-cya-
noethyl N,N,N′,N′-tetraisopropylphosphordiamidite and diiso-
propyl ammonium tetrazolide afforded phosphoramidite
building blocks 21, 22 and 23. In order to synthesize the
guanine phosphoramidite monomer, 2-amino-6-chloropurine
24 was treated with 1,4-diazabicyclo[2.2.2]octane (DABCO)
yielding the corresponding DABCO purine 25 (Scheme 3).
Compound 25 was converted to O6-benzylguanine 26 in the
presence of benzyl alcohol and sodium hydride.11e
In order to obtain DMT-alcohol 27, compound 26 was
treated with 13 under similar conditions of epoxide ring
opening. The exocyclic amine of 27 was protected by an iso-
butyryl group to obtain title compound 28 in 67% yield. Finally,
treatment of 28 with phosphoramidite reagent and tetrazolide
salt provides compound 30 in 65% yield. The regioselectivities
of N-alkylation of nucleobases were confirmed by 2-D NMR
(HMBC) (Fig. S4†).
Phosphoramidite monomers 21, 22, 23 and 30 were used to
synthesize oligonucleotides (Table 1) by automated solid-
phase oligonucleotide synthesis. It is noteworthy that the pres-
ence of a vicinal diol functionality in the C-2 scaffold based
acyclic nucleic acid could give transesterification as a side reac-
tion,4b,10c but we did not observe any side product during
BuNA synthesis. Therefore, deprotection and cleavage of the
Scheme 3 Synthesis of purine phosphoramidite building blocks of (S)-BuNA.
Table 1 Sequences of oligonucleotides used in this study
Entry
Oligonucleotidesa
ON-1
ON-2
ON-3
ON-4
ON-5
ON-6
ON-7
ON-8
ON-9
ON-10
ON-11
ON-12
ON-13
ON-14
ON-15
ON-16
ON-17
ON-18
ON-19
ON-20
ON-21
ON-22
T1
4′-aaaaaaaaaaaaaaA-3′ poly(a14)A
4′-ttttttttttttttT-3′ poly(t14)T
4′-atatatatatatataT-3′
4′-aaaaaaaattttttT-3′
4′-aaaatttatattattA-3′
4′-taataatataaatttT-3′
4′-gtgtaataacaacaT-3′
4′-gtgttataacaacat-2′
4′-atgttgttattacaC-3′
4′-atgttgttattacac-2′
5′-AAAAAAAAAAAAAAA-3′
5′-TTTTTTTTTTTTTTT-3′
5′-GTGTAATAACAACAT-3
5′-ATGTTGTTATTACAC-3′
5′-AAAAAAAaAAAAAAA-3′
5′-AAAaAAAAAAAaAAA-3′
5′-TTTTTTTtTTTTTTT-3′
5′-TTTtTTTTTTTtTTT-3′
5′-ATGTTGTtATTACAC-3′
5′-GTGTAATaACAACAT-3′
5′-ATGTTGttaTTACAC-3′
5′-GTGTAAtaaCAACAT-3′
5′-CATTCACTTCCCTACGCCATGTTGTTATTACAC-3′
5′-CATTCACTTCCCTACGCCAAAAAAAAAAAAAAA-3′
5′-CATTCACTTCCCTACGCCTTTTTTTTTTTTTTT-3′
T2
T3
a Lower case letters represent (S)-BuNA nucleotides and upper case
letters represent DNA nucleotides. 3′ notation for BuNA shows that
oligonucleotides were synthesized on DNA nucleotide containing CPG
solid support.
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oligonucleotides from the solid support and subsequent
purification can be performed by standard oligonucleotide
synthesis protocols. The characterization of the resulting
acyclic oligonucleotides was carried out by MALDI-TOF or
LC-MS (Table S1†).
Table 2 UV-melting studies of modified (S)-BuNA strands and DNA
Duplex (oligo pairs)
Tma (°C)
Tmb (°C)
Tmc (°C)
1 (ON-1/ON-2)
2 (ON-3/ON-3)
3 (ON-4/ON-4)
4 (ON-5/ON-6)
5 (ON-8/ON-10)
nt
40
39
37
22
nd
nd
42
31
43
45
43
40
50
21, 28
Pairing properties of (S)-BuNA
a UV-melting at 260 nm was carried out in 10 mM phosphate buffer
CD and UV-melting experiments were carried out to demon-
strate the interaction between various sequences of (S)-BuNA
with their complementary strands as well as DNA or RNA.
Initially, conformational studies of poly(a14)A (ON-1) and poly-
(t14)T (ON-2) were carried out individually. At 20 °C the CD
spectrum of ON-1 in 10 mM phosphate buffer containing
150 mM sodium chloride at pH 7.0 revealed the preorganized
structure of ON-1 with a positive cotton effect at 269 nm and a
negative cotton effect at 217 nm with crossover at 247 nm
(Fig. 2). Under similar experimental conditions, the CD of
ON-2 was associated with the cotton effect to a much smaller
extent (random coil), and showed positive peaks at 273 nm,
219 nm and negative peaks at 242 nm (Fig. 2). The confor-
mational rigidity of ON-1 was confirmed by performing a
containing 150 mM sodium chloride at pH 7. b Melting temperature in
1
M
sodium chloride. c Melting temperature of corresponding
unmodified DNA duplexes in 150 mM NaCl. nt = no melting transition
was observed. nd = not determined.
no sharp melting transition was observed by UV at 260 nm
with 150 mM sodium chloride. However, by increasing the salt
concentration to 1 M NaCl, a sharp melting transition was
observed with Tm of 22 °C (Fig. S1c†). Eschenmoser and co-
workers also reported the weak pairing between poly(A12) and
poly(T12) of α-threose nucleic acid (TNA).12 The reported CD
spectrum of a stretch of adenines of GNA was found to be com-
pletely different from BuNA.4g These observations clearly
suggest that by introducing one additional carbon atom in the
C-2 scaffold of GNA, the resulting C-3 scaffold of BuNA
organizes poly adenines in a different fashion.
In order to minimize the loss of entropy in the single
strands, ON-1 and ON-2, we investigated other (S)-BuNA oligo-
nucleotide strands (Table 1, ON-3 to ON-10). Self-complemen-
tary (S)-BuNA strands ON-3 and ON-4 which contain both
“a and t” acyclic nucleotides were subjected to conformational
studies using CD and UV-melting under similar experimental
temperature-dependent CD experiment, which showed
a
decrease in cotton effect at 269 nm and 217 nm (Fig. S1a†).
Next, we investigated the duplex formation between ON-1 and
ON-2 by mixing equimolar quantities of each strand in the
same buffer. It is not unusual to obtain a different CD spec-
trum, when compared with their isolated components, if they
are forming a duplex. Indeed, we observed similar results.
Positive and negative cotton effects of single strands were
transformed to give positive peaks at 273 nm, 217 nm and
negative peaks at 245 nm, 203 nm (Fig. 2). This observation
clearly indicates duplex formation between the two strands.
Temperature-dependent CD studies demonstrated that
ON-1 has a highly organized structure unlike ON-2 and there-
fore duplex formation resulted in loss of entropy when both
single strands adopt a new conformation (Fig. S1b†). Hence,
conditions.
A stable duplex formation was observed by
UV-melting at 260 nm with a Tm of 40 °C and 39 °C of ON-3
and ON-4 respectively. Here we cannot rule out the formation
of a hairpin-like secondary structure of self-complementary
strands of BuNA similar to DNA.13
Next, we investigated the duplex stability of mixed
sequences of “a and t” (Table 2, duplex 4) and “a, t, g and c”
(Table 2, duplex 5). Duplex 4 showed a sigmoidal melting
curve with Tm = 37 °C (Fig. 3a). To our surprise, introduction
of “g and t” nucleotides in (S)-BuNA strands (duplex 5) dis-
played two melting transitions (Fig. 3b). In order to under-
stand the two melting transitions, we investigated UV-melting
of both single strands, which revealed the internal folding of
ON-8 compared to ON-10 (Fig. S1f†). Importantly, duplex 5 dis-
played a single transition in the presence of 1 M NaCl. Duplex
4 showed an increase in melting temperature at 1 M sodium
chloride in 10 mM phosphate buffer (Fig. 3a).
Table 2 displays the duplex stability of (S)-BuNA in compari-
son to DNA duplexes. These data reveal that BuNA duplexes
show less stability compared to DNA duplexes, although they
exhibit a less cooperative melting transition than DNA.
CD dependent conformational studies revealed that (S)-
BuNA duplexes of ON-1/ON-2, self-complementary strands
(ON-3 and ON-4) and mixed sequences (ON-5/ON-6 and ON-8/
ON-10) were associated with A-form-like structure with positive
Fig. 2 CD profile of ON-1, ON-2 and their duplex. Single strand of ON-1
(20 µM) and ON-2 (20 µM) in 10 mM phosphate buffer containing 150 mM
sodium chloride at pH 7. Duplex of ON-1 and ON-2 in equimolar concentration
(10 µM) adopted a different conformation than single strands.
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duplexes therefore, (S)-BuNA duplexes were also investigated in
the presence of magnesium chloride, which indicated that
10 mM and 100 mM magnesium ions provide more folded
structures than sodium ions alone (Fig. 4). This effect of mag-
nesium was more pronounced for duplex ON-1/ON-2 than
ON-5/ON-6 (Fig. 4a and b). In the case of mixed sequences of
nucleotides (a, t, g and c) ON-8/N-10 was found to be more
folded than ON-5/ON-6 (containing “a and t” only). This study
indicates that like natural nucleic acids BuNA also folded more
effectively in the presence of divalent cations.
The preorganized structure adopted by a single strand is a
very important characteristic of oligonucleotides. Therefore,
we investigated temperature-dependent structural confor-
mation studies of the (S)-BuNA single strand (Fig. 5a). In order
to compare the helicity of the corresponding double strand
(ON-8/ON-10), the CD was recorded at 10 °C for the duplex,
where it showed a strong cotton effect with positive ellipticity
at 275 nm, 221 nm and negative ellipticity at 243 nm, 205 nm
(Fig. 5a). This observation clearly indicates that double
stranded (S)-BuNA has a more folded and rigid structure than
single strand (S)-BuNA. It is important to note that the CD
signals disappear gradually upon increasing temperature for
single and double stranded BuNA alike. These observations
demonstrate that the structural conformation of mixed-
sequence (a, t or a, t, g and c) of individual complimentary
strands of BuNA, gain similar helical structure unlike ON-1
and ON-2 as discussed earlier (Fig. S1d and S1e†). To further
demonstrate the duplex formation, the ON-8/ON-10 duplex was
analysed by non-denatured polyacrylamide gel electrophoresis
(PAGE) analysis. Fig. 5b clearly demonstrates the duplex for-
mation by (S)-BuNA (Fig. 5, lane 4).
Next, we investigated the cross-hybridization between
(S)-BuNA and DNA or RNA. CD studies have revealed that
(S)-BuNA adopts a left-handed helix. Therefore, it was expected
that the (S)-isomer of BuNA would not form a duplex with DNA
or RNA. To investigate this, we carried out heteroduplex
studies of BuNA with DNA and RNA by CD. The complemen-
tary parallel sequence of DNA/BuNA duplex was also studied
but no hybridization was observed by these heteroduplexes
(data not shown). We also investigated these heteroduplex and
homoduplex pairings by non-denatured gel electrophoresis
Fig. 3 UV-melting profile at 260 nm. (a) (S)-BuNA duplex 4 (ON-5/ON-6). (b)
Duplex 5 (ON-8/ON-10). Experimental conditions: 10 mM phosphate buffer,
150 mM NaCl or 1 M NaCl, pH 7.0, 2 µM of each oligo strand.
bands at 270 nm, 223 nm and negative bands at 247 nm,
207 nm (Fig. 4). Based on previous literature reports,7 our
experimental data suggests that (S)-BuNA forms a left-handed
helical structure.
It is evident from the literature that magnesium ions
provide more stability to the folded structure of DNA and RNA
Fig. 4 Effect of magnesium ions on folding of (S)-BuNA duplexes. (a) ON-1/ON-2. (b) ON-5/ON-6. (c) ON-8/ON-10. Experimental conditions: [oligonucleotide] =
5 µM, 10 mM phosphate buffer containing 150 mM NaCl at pH 7.0; 10 mM tris buffer containing 100 mM MgCl2 or 10 mM MgCl2, 150 mM NaCl at pH 7.
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Fig. 5 Temperature-dependent CD profile and non-denatured PAGE (UV
shadow) of single strands and duplexes. (a) 20 µM of ON-10 in 10 mM
phosphate buffer containing 100 mM sodium chloride at pH 7.0. Under the
same experimental conditions CD was measured for the duplex (ON-8 and
ON-10) containing 10 µM of each strand. (b) 20% non-denatured PAGE of
single and duplex of BuNA and DNA. Lanes: 1 = ON-14, 2 = ON-10, 3 = duplex
(ON-13/ON-14), 4 = duplex (ON-8/ON-10), 5 = ON-8, 6 = ON-13, hetero-
duplexes 7 = (ON-10/ON-13), 8 = (ON-8/ON-14), 9 = bromophenol blue. The
gel was run using 1 × 89 mM tris-borate-magnesium (TBM) buffer at 10 °C with
40 to 60 V.
Fig. 6 UV-melting profile at 260 nm of (S)-BuNA modified DNA duplex. Experi-
ment condition: 10 mM phosphate buffer, 150 mM NaCl, pH 7.0, 2 µM each
oligo strand concentration. Tm values in Table 3.
Table 3 UV-melting studies at 260 nm of modified DNA strands
Duplex (oligo pairs)a
Tm (°C)
ΔTmb (°C)
1 (ON-12/ON-15)
37
33
41
44
31
25
28
31
37
38
31
31
−6
−9
(Fig. 5b) which showed the absence of oligonucleotide bands 2 (ON-11/ON-17)
3 (ON-13/ON-19)
4 (ON-14/ON-20)
5 (ON-12/ON-16)
−9
corresponding to heteroduplexes (Fig. 5b, lanes 7 and 8).
Moreover, the flexible backbone of BuNA might be another
−6
−12
−18
−22
−19
−13
−12
−19
−19
reason for failing to form the heteroduplex in addition to the 6 (ON-11/ON-18)
7 (ON-13/ON-21)
8 (ON-14/ON-22)
9 (ON-19/ON-20)
incompatibility of handedness. Our attempt at visualizing the
gel by ethidium bromide staining did not succeed in detecting
single and double stranded BuNA. Similar observations have 10 (ON-21/ON-22)
been reported for GNA.14 This proves the importance of the
11 (ON-20/ON-21)
12 (ON-19/ON-22)
sugar moieties on the intercalation properties of DNA and
a See Table 1 for oligonucleotide sequences. b Decrease in melting
temperature. ΔTm was calculated with respect to unmodified DNA
duplexes Tm = 43 °C (ON-11/ON-12), Tm = 50 °C (ON-13/ON-14).
RNA. These observations revealed that chirality is an important
parameter in determining the handedness of the helical struc-
ture. In the literature, it has been illustrated that a peptide
nucleic acid (PNA) containing all the chiral monomers can be
cross-hybridized with right-handed DNA; in addition no inter-
action was observed with left-handed PNA.7e
melting at 260 nm (Fig. 6 and Table 3). Table 3 shows the
effect of (S)-BuNA nucleotides in DNA oligomers on the
melting transition. Experiments were carried out in 10 mM
phosphate buffer, containing 150 mM sodium chloride at
pH 7.0 with 2 µM of each oligonucleotide. Introduction of one
Pairing properties of (S)-BuNA substituted DNA strands
In order to investigate the effect of (S)-BuNA nucleotide substi-
tution in DNA strands, we measured duplex stability by UV
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extension on a DNA template by DNA polymerases. As dis-
cussed earlier, we demonstrated that the B-form helical struc-
ture remained intact for modified DNA strands and, these
results encouraged us to use these modified DNA strands as
primers. For this study, we investigated nine modified primers
of 15 nucleotides on 33 nucleotides of DNA templates (Fig. 8).
Bst, Bsu, Taq and Therminator polymerases were employed to
perform primer extension. Previously, Bst DNA polymerase has
been used to demonstrate DNA polymerization on a (S)-GNA
template,10c and therefore we started our studies by employing
Bst as thermophilic enzyme. Moreover, structural studies of
the Bst enzyme have been investigated by X-ray crystallography
together with a primer/template complex.15 Modified primer
sequences containing one (S)-BuNA nucleotide at position 7,
two nucleotides at positions 4, 12 and three consecutive
nucleotides at position 7, 8, 9 were investigated. Our hypo-
thesis was that the positions of the acyclic nucleotides exist in
the DNA duplex binding region of the enzyme. When (S)-BuNA
substituted DNA strands were employed for primer extension,
it resulted in full extension of the modified primers (Fig. 8a,
lanes 1, 2, 6, 7) similar to unmodified DNA primers (Fig. 8a,
lanes 4, 9) by Bst polymerase. No extension was observed for
fully modified primers (Fig. 8a, lanes 3, 8). It is important to
Fig. 7 Temperature-dependent CD profile of (S)-BuNA substituted DNA duplex
(ON-13/ON-21) in phosphate buffer at pH 7 containing 150 mM NaCl.
(S)-BuNA thymine or one adenine nucleotide decreases the
DNA duplex stability by ΔTm/mod = −9.0 °C and ΔTm/mod =
−6.0 °C respectively (Table 3, duplex 1, 2, 3 and 4). These
results suggest that (S)-BuNA adenine nucleotide was better
tolerated than thymine nucleotide in DNA duplexes. Moreover,
the destabilization effect was found to be additive for incorpor-
ation of more than one (S)-BuNA nucleotide (Table 3, duplex 5
and 6).
note that ON-18 was found to form an unstable duplex (Tm
=
25 °C) with its complementary strand, and consequently
resulted in a lower yield (Fig. 8a, lane 6). Similar results were
obtained when extension was carried out with Bsu polymerase
(mesophilic enzyme) at 25 °C. Gel results show that primers
ON-19 and ON-22 (Fig. 8b, lanes 5, 6) extended with efficien-
cies comparable to that of unmodified primer ON-13 (Fig. 8b,
lanes 4). As in the case of Bst, fully modified BuNA primers
did not result in any extension by Bsu DNA polymerase
(Fig. 8b, lanes 7, 8). Even incubation at 5 °C for 12 h with Bsu
polymerase did not show any extension. This study clearly
suggests an incompatibility of fully modified BuNA strands
with DNA polymerases. Based on the fully modified (S)-BuNA
strands inability to engage in hybridization with normal DNA
we anticipated their failure in the primer extension. Similar
results were obtained for Taq and Therminator polymerase
(data not shown). It is reasonable to assume that a BuNA sub-
stituted primer/template duplex is compatible with DNA poly-
merases for their elongation. Hence, DNA was successfully
modulated by incorporation of BuNA nucleotides and still
recognized by the enzymes.
Next, we investigated the effect of three consecutive (S)-
BuNA nucleotides in the middle of the DNA strand. This
demonstrates that “tta” (ON-21) and “aat” (ON-22) incorpor-
ation destabilize the duplex by ΔTm = −22 °C and ΔTm
−19 °C respectively (Table 3, duplex 7 and 8).
=
On the other hand hybridization of complementary substi-
tuted (S)-BuNA nucleotides within a double stranded DNA
sequence revealed a more stable duplex (Tm = 38 °C) than
(S)-BuNA nucleotides substituted in one DNA strand (Table 3,
duplex 10). To further test this hypothesis, we analysed the
duplex stability of modified duplexes with one strand contain-
ing one substitution and the complementary strand containing
three modifications. These duplexes were better stabilized
(Table 3, duplex 11, 12) than duplex 7 and equally stabilized as
duplex 8, suggesting that (S)-BuNA modifications were better
accommodated when equal number of modifications were
present in both strands and opposite to each other. These
studies clearly indicate that (S)-BuNA nucleotides are tolerated
in DNA duplexes when they are incorporated as base pairs.
Furthermore, the temperature dependent CD profile showed
that incorporation of three consecutive BuNA nucleotides
(ON-22) resulted in a considerable decrease of the cotton
effects of the DNA duplex, and still maintained a B-form
helical structure (Fig. 7).
Conclusions
In summary, we report the synthesis of a novel acyclic scaffold
of (S)-butyl nucleic acid (BuNA) and demonstrate its ability to
form a duplex with its complementary strands. To the best of
our knowledge this is the first reported synthesis of BuNA,
Primer extension studies of modified DNA strands
In literature acyclic universal base analogues have been incor- where monomers are conjugated through phosphodiester
porated in primers for PCR and sequencing.10b We wanted to linkages and tethering natural nucleobases. Furthermore, we
investigate whether integrating (S)-BuNA nucleotides in DNA demonstrated the incorporation of (S)-BuNA nucleotides in
primers would disrupt their ability to participate in primer DNA strands, which resulted in a destabilized duplex, known
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Fig. 8 Primer extension studies of (S)-BuNA incorporated DNA primers and fully modified BuNA primers. (a) Thermophilic enzyme (Bst polymerase) was used for
primer extension studies at 55 °C. (b) Mesophilic enzyme (Bsu polymerase) was used for primer extension studies at 25 °C. Primer, enzyme, DNA template and dNTPs
were incubated for 2 h at 25 °C for Bsu enzyme and at 55 °C for Bst DNA polymerase. After completion of enzymatic reaction, the mixture was loaded in non-
denatured gel electrophoresis of 14% PAGE and the system was run in a cold room (10 °C) at 50 V in 1 × 89 mM tris-borate-magnesium (TBM) buffer. The gel was
stained by ethidium bromide.
as an intrinsic property of acyclic scaffolds. These studies temperature is not mentioned. CD data were collected at a
revealed that increasing the backbone length by one carbon scan rate 100 nm min−1 at 1 nm data intervals and are pres-
atom of GNA resulted in a decrease in melting temperature of ented as an average of three successive scans. CD spectra
(S)-BuNA duplexes in comparison to GNA duplexes. Although were baseline subtracted and smoothed via a five point adja-
(S)-BuNA adopts left-handed structure, it might possible that cent averaging algorithm. UV melting studies were carried out
(R)-BuNA will adopt a right-handed structure resulting in by increasing the temperature at a rate of 1 °C min−1. Melting
duplex formation with natural nucleic acids. We also investi- temperature (Tm) was determined by first derivatives of
gated primer extension studies of fully modified (S)-BuNA melting curves. Experiments were repeated three times and
strands and (S)-BuNA substituted DNA primers by DNA poly- average values were taken for calculating reported parameters.
merases. This study revealed that the (S)-BuNA scaffold is bio- Unmodified oligonucleotides and DNA templates were pur-
compatible with DNA polymerase and this provides a platform chased from Sigma-Aldrich. DNA polymerases were purchased
where other organic moieties or fluorescent functionalities from New England Biolabs.
can be tethered to this scaffold and can be introduced in
DNA oligomers to modulate its function. In order to obtain
a right handed helix of BNA the synthesis of (R)-BuNA is in
Primer extension studies
progress.
Unmodified, modified and fully modified primers (0.5 µM)
and the corresponding templates (0.5 µM) were annealed in a
reaction mixture of 1 × reaction buffer in 200 µl PCR tube.
After brief cooling at room temperature, DNA polymerase (2 to
10 units) was added followed by the addition of dNTPs
(200 µM). Reaction tubes were incubated at the required temp-
Experimental section
General methods
CD experiments were performed in Milli-Q water using a Jasco erature for the optimal time period. Elongation of primers was
J-815 instrument and UV experiments were carried out by a analyzed by non-denatured gel electrophoresis (14% PAGE)
Varian Cary 300 BIO instrument. CD experiments were carried using 1 × 89 mM tris-borate-magnesium (TBM) buffer at 10 °C
out with 1 mm path length quartz cuvettes at 20 °C, if with 40 to 60 V.
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(R)-2-Bromosuccinic acid (10)
dichloromethane. The organic layer was collected and washed
with brine, dried over sodium sulphate and concentrated
under vacuum to afford a crude yellow colored paste. The com-
pound was purified by silica gel chromatography (2% ethyl
acetate–hexane with 1.0% triethylamine) giving a pasty mass
(7.5 g, 60% yield). [α]2D5 = −0.5 (c = 1 in dichloroethane).
1H NMR (500 MHz, CDCl3) δ 7.51–7.49 (m, 2 H), 7.40–7.37 (m,
4 H), 7.35–7.32 (m, 2 H), 7.28–7.20 (m, 1 H), 6.90–6.87 (m,
4 H), 3.82 (s, 6 H), 3.34–3.25 (m, 2 H), 3.17–3.13 (m, 1 H),
2.84–2.82 (m, 1 H), 2.56 (dd, J = 2.5, 5.0 Hz, 1 H), 1.89–1.85 (m,
2 H); 13C NMR (125 MHz CDCl3) δ 158.34, 145.05, 136.25,
129.91, 128.05, 127.69, 126.60, 113.00, 85.96, 60.36, 55.15,
55.05, 50.26, 47.13, 33.32; HRMS calcd for C25H26O4Na
[M + Na]+ 413.1729, found [M + Na]+ 413.1728.
To a solution of sodium bromide (60 g, 588 mmol) in 6 N sul-
furic acid (25 ml), (R)-aspartic acid 9 (20 g, 150 mmol) was
added and cooled at 0 °C. Sodium nitrite (12.4 g, 180 mmol)
was added slowly over a time period of 30 min under nitrogen
pressure and temperature was maintained 0 °C. The reaction
mixture was stirred for the next 3 h. Urea (4.6 g) was added
and the reaction mixture stirred for 20 min. The compound
was extracted with diethyl ether (3 × 100 ml). The organic layer
was dried over sodium sulphate and concentrated to give
a pale yellow solid in 60% yield. [α]2D5 = +5.9 (c = 1 in metha-
nol). 1H NMR (500 MHz, DMSO) δ 4.51 (dd, J = 6.3, 8.8 Hz,
1 H), 3.07 (dd, J = 8.5, 17.3 Hz, 1 H), 2.91–2.84 (m, 1 H); 13C
NMR (125 MHz, DMSO) δ 171.46, 170.59, 41.01, 40.03.
(S)-1-(4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxybutyl)-5-methylpyrimidine-2,4(1H,3H)-dione (17)
(R)-2-Bromobutane-1,4-diol (11)
Dry THF was added to (R)-2-bromosuccinic acid 10 (16.0 g,
81.6 mmol). A 2 M solution of borane dimethyl sulfide
complex in THF (2.7 equiv.) was added drop by drop at 0 °C.
The reaction was allowed to stir for 5 h at room temperature.
After completion, the reaction was quenched with methanol
drop by drop and the solvent was removed under vacuum.
Three times co evaporation was carried out with methanol to
give a pale yellow oil as crude product. Purification was carried
out by silica gel chromatography with (5 to 10% methanol–
dichloromethane) to afford a colorless oil (12.1 g, 87%)
yield. [α]2D5 = +2.9 (c = 1 in methanol). 1H NMR (500 MHz,
CDCl3) δ 5.88 (br s, 1 H), 4.34–4.29 (m, 1 H), 3.90–3.86 (m,
1 H), 3.85–3.84 (m, 2 H), 3.82–3.77 (m, 1 H), 2.18–2.05 (m,
2 H); 13C NMR (125 MHz, CDCl3) δ 67.22, 60.17, 55.03, 37.93.
To a stirred solution of thymine 14 (500 mg, 3.96 mmol) in dry
DMF. 0.3 equiv. NaH was added and the reaction mixture was
stirred for 1 h at room temperature. DMT epoxide (1.3 g,
3.3 mmol) dissolved in dry DMF was added and the reaction
mixture was stirred for 12 h at 110 °C. The solvent was
removed under vacuum and ethyl acetate was added and
washed with saturated bicarbonate solution. The organic
phase was washed with brine and dried over sodium sulphate.
The organic layer was concentrated and loaded over a silica gel
column. The compound was eluted with 5 to 50% ethyl
acetate–hexane with 1% triethylamine to afford a white foam
product (800 mg, 45%). [α]2D5 = +22.9 (c = 1 in dichloroethane).
1H NMR (500 MHz, CDCl3) δ 7.33–7.30 (m, 2 H), 7.24–7.19 (m,
6 H), 7.16–7.12 (m, 1 H), 7.08 (s, 1 H), 6.78–6.74 (m, 4 H),
4.01–3.94 (m, 1 H), 3.85 (dd, J = 2.5, 13.9 Hz, 1 H), 3.72 (s,
6 H), 3.40 (dd, J = 8.2, 13.9 Hz, 1 H), 3.37–3.34 (m, 1 H),
3.25–3.19 (m, 1 H), 1.83 (s, 3 H), 1.70–1.65 (m, 2 H); 13C NMR
(125 MHz, CDCl3) δ 164.11, 158.60, 151.20, 144.40, 142.12,
135.52, 129.90, 127.99, 127.93, 126.95, 113.27, 109.75, 87.11,
70.33, 62.14, 55.22, 53.67, 33.77, 12.26; HRMS calcd
for C30H32N2O6Na [M + Na]+ 539.2158, found [M + Na]+
539.2156.
(S)-2-(Oxiran-2-yl)ethanol (12)
Dry dichloromethane was added to (R)-bromodiol 11 (7.3 g,
4.43 mmol) and stirred for 5 min. 1.8 equiv. cesium carbonate
(25.2 g, 77.3 mmol) was added under argon. The reaction was
stirred for 72 hours at room temp. After completion, the reac-
tion mixture was filtered and washed with dichloromethane.
The filtrate was dried over magnesium sulphate and organic
solvent was removed by vacuum to give a slightly pale yellow
oily liquid (S)-epoxide (2.4 g, 60%). The next step was carried
out without further purification. [α]2D5 = −2.6 (c = 1 in metha-
nol). 1H NMR (500 MHz, CDCl3) δ 3.85–3.78 (m, 2 H),
3.12–3.08 (m, 1 H), 2.81–2.80 (m, 1 H), 2.59 (d, J = 1.9 Hz, 1 H),
2.02–1.96 (m, 1 H), 1.77–1.70 (m, 1 H); 13C NMR (125 MHz
CDCl3) δ 60.09, 50.64, 46.67, 34.75.
(S)-N-(1-(4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxybutyl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (18)
N4-Benzoyl cytosine 15 (1.0 g, 4.6 mmol) was dried under
vacuum and dry DMF was added under nitrogen. 0.3 equiv. of
NaH was added under nitrogen and stirred for 1 h. DMT
epoxide (2.153 g, 5.5 mmol) dissolved in dry DMF was added
and the reaction mixture was allowed to stir for 20 h at 110 °C.
DMF was removed under vacuum and ethyl acetate was added
to residue and washed with saturated sodium bicarbonate
(S)-2-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)oxirane
(13)
(S)-Epoxide 12 (3.0 g, 34 mmol) was dissolved in dry dichloro- solution. The organic layer was washed with brine solution
methane followed by the addition of triethylamine (8.6 g, and dried over sodium sulphate. The ethyl acetate was
85 mmol) and dimethoxy trityl chloride (13.8 g, 40 mmol) at removed by vacuum and the residue was loaded over silica gel
room temp under a nitrogen atmosphere. The reaction was column. The compound was eluted by (20 to 100)% ethyl
allowed to stir overnight. After completion of the reaction, the acetate–hexane) with 1% triethylamine to afford a white
product was filtered and the filtrate poured into a saturated foam solid (1.44 g, 50%). [α]2D5 = +40.6 (c = 1 in dichloroethane).
aqueous sodium bicarbonate solution and extracted with 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 7 Hz, 2 H), 7.67
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(d, J = 7.5 Hz, 1 H), 7.54–7.50 (m, 1 H), 7.44–7.40 (m, 2 H), dichloroethane). H NMR (500 MHz, CDCl3) δ 9.17 (br s, 1 H),
7.32–7.29 (m, 2 H), 7.22–7.18 (m, 7 H), 7.14–7.11 (m, 1 H), 8.65 (s, 1 H), 7.99 (s, 1 H), 7.94 (d, J = 7 Hz, 2 H), 7.52–7.47 (m,
6.76–6.72 (m, 4 H), 4.15–4.07 (m, 2 H), 3.70 (s, 6 H), 3.52 (dd, J 1 H), 7.44–7.38 (m, 2 H), 7.33–7.28 (m, 2 H), 7.23–7.16 (m,
= 8.0, 13.2 Hz, 1 H), 3.35–3.31 (m, 1 H), 3.25–3.21 (m, 1 H), 6 H), 7.15–7.09 (m, 1 H), 6.76–6.70 (m, 4 H), 4.29 (dd, J = 2.5,
1.78–1.65 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ 162.36, 13.9 Hz, 1 H), 4.18–4.03 (m, 3 H), 3.69 (s, 6 H), 3.36–3.30 (m,
158.59, 150.57, 144.51, 135.81, 135.67, 133.12, 129.93, 129.90, 1 H), 3.25–3.21 (m, 1 H), 1.76–1.67 (m, 1 H), 1.66–1.56 (m,
129.02, 128.00, 127.62, 126.94, 113.29, 96.16, 87.00, 69.42, 1 H); 13C NMR (125 MHz, CDCl3) δ 164.94, 158.79, 152.59,
61.90, 56.08, 55.24, 34.03; HRMS calcd for C36H35N3O6Na [M + 152.39, 149.56, 144.72, 144.56, 135.98, 135.85, 133.96, 132.90,
Na]+ 628.2424, found [M + Na]+ 628.2424.
130.11, 129.01, 128.18, 128.12, 127.14, 122.76, 113.47, 87.16,
69.85, 61.76, 55.43, 50.00, 34.20; HRMS calcd for C37H36N5O6
[M + H]+ 630.2716, found [M + H]+ 630.2725.
(S)-1-(6-Amino-9H-purin-9-yl)-4-(bis(4-methoxyphenyl)-
(phenyl)methoxy)butan-2-ol (19)
Thymine phosphoramidite (21)
To a stirred suspension of adenine 16 (2.1 g, 15.5 mmol) in dry
DMF, 0.3 equiv. NaH was added and reaction mixture was Compound 17 (703 mg, 1.4 mmol) was dried under high
stirred for one hour at room temperature. DMT epoxide (6.0 g, vacuum and anhydrous dichloromethane was added followed
15.4 mmol) dissolved in dry DMF was added and the reaction by the addition of diisopropyl ammonium tetrazolide (120 mg,
mixture was stirred for 12 h at 110 °C. DMF was removed by 0.5 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphor-
vacuum and ethyl acetate was added, which was washed with diamidite (633 mg, 1.5 mmol) and the reaction was allowed to
saturated bicarbonate solution. The organic layer was washed stir at room temperature. After 3 h the reaction was diluted
with brine and dried over sodium sulphate. The solvent was with dichloromethane and poured into saturated aqueous
concentrated and the compound was purified by silica gel sodium bicarbonate. The product was extracted in dichloro-
column chromatography. The compound was eluted with 20 to methane and dried over magnesium sulphate. The product
80% ethyl acetate–hexane with 1% triethylamine to afford was purified by silica gel column with dichloromethane–
product (4.0 g, yield 50%). [α]2D5 = +2.7 (c = 1 in dichloro- hexanes (1 to 80%) with 1% triethylamine to afford the
ethane). 1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1 H), 7.83 (s, product (766 mg, yield 73%) as a white foam solid. 31P NMR
1 H), 7.42–7.39 (m, 2 H), 7.33–7.28 (m, 6 H), 7.25–7.21 (m, (121 MHz, CDCl3) δ 149.119, 148.406; HRMS calcd for
1 H), 6.86–6.82 (m, 4 H), 5.88 (br, 2 H), 4.32 (dd, J = 2.5, C39H49N4O7PNa [M + Na]+ 739.3237, found [M + Na]+ 739.3234.
14.2 Hz, 1 H), 4.26–4.19 (m, 1 H), 4.16–4.10 (m, 1 H), 3.80 (s,
Cytosine phosphoramidite (22)
6 H), 3.44–3.40 (m, 1 H), 3.32–3.28 (m, 1 H), 1.84–1.69 (m,
2 H); 13C NMR (125 MHz, CDCl3) δ 158.69, 155.59, 152.86, Compound 18 (605 mg, 1 mmol) was dried under high
144.70, 141.87, 135.99, 135.88, 130.04, 128.15, 127.05, 119.48, vacuum and anhydrous dichloromethane was added and
113.37, 86.98, 69.78, 61.60, 60.53, 55.36, 50.25, 34.19; HRMS subsequently diisopropyl ammonium tetrazolide (85 mg,
calcd for C30H31N5O4 [M]+ 526.2454, found [M]+ 526.2452.
0.5 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphor-
diamidite (452 mg, 1.5 mmol) was added. The reaction was
allowed to stir at room temperature. After 4 h, the reaction
mixture was diluted with dichloromethane and poured in satu-
(S)-N-(9-(4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxybutyl)-9H-purin-6-yl)benzamide (20)
Compound 19 (500 mg, 0.95 mmol) was dried well and dis- rated aqueous sodium bicarbonate. Product was extracted
solved in anhydrous pyridine under nitrogen. TMS-Cl in dichloromethane and dried over magnesium sulphate.
(412.8 mg, 3.8 mmol) was added at 0 °C followed by stirring at The product was purified by silica gel column by dichloro-
0 °C for 30 min and at room temperature for the next 4 h. methane–hexanes (10 to 60%) with 1% triethylamine to afford
Benzoyl chloride (200 mg, 1.42 mmol) was added drop by drop product as white foam (593 mg, yield 73%). 31P NMR
at 0 °C and the reaction was stirred for 4 h at room tempera- (121 MHz, CDCl3) δ 149.094, 148.922; HRMS calcd for
ture. After completion of the reaction, water was added to stop C45H52N5O7PNa [M + Na]+ 828.3502, found [M + Na]+ 828.3505.
the reaction and aqueous ammonia was added at 0 °C and
Adenine phosphoramidite (23)
stirred for 1 h. The compound was extracted with dichloro-
methane. The organic layer was dried over sodium sulphate Compound 20 (629 mg, 1 mmol) was dried under high
and concentrated to give an yellow oily paste. The resulting vacuum and anhydrous dichloromethane was added followed
oily paste was dissolved in THF and 1 M solution tetrabutyl- by the addition of diisopropyl ammonium tetrazolide (85 mg,
ammonium fluoride in THF was added and stirred for 30 min 0.5 mmol) under nitrogen. After 1 min 2-cyanoethyl N,N,N′,N′-
at room temperature. Dichloromethane was added to the reac- tetraisopropylphosphordiamidite (376 mg, 1.25 mmol) was
tion mixture and the product was washed with water and the added and the reaction was allowed to stir at room tempera-
organic layer was washed with brine and dried over sodium ture. After 3 h the reaction mixture was diluted with dichloro-
sulphate. The organic layer was concentrated and loaded over methane and poured into saturated aqueous sodium
a silica gel column. The compound was eluted with 20 to 90% bicarbonate. The product was extracted in dichloromethane
ethyl acetate–hexane with 1% triethylamine to afford a white and dried over magnesium sulphate. The product was purified
colored foam (400 mg, 60%). [α]D25
=
+12.2 (c = 1 in by silica gel column using dichloromethane–hexanes (20 to
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30%) with 1% triethylamine to afford the product as a HRMS calcd for C37H37N5O5Na [M + Na]+ 654.2692; found
white foam (560 mg, yield 67%). 31P NMR (121 MHz, CDCl3) [M + Na]+ 654.2692.
δ 149.119, 148.406; HRMS calcd for C46H52N7O6P [M + H]+
(S)-N-(6-(Benzyloxy)-9-(4-(bis(4-methoxyphenyl)(phenyl)-
methoxy)-2-hydroxybutyl)-9H-purin-2-yl)isobutyramide (28)
830.3795, found [M + H]+ 830.3796.
1-(2-Amino-9H-purin-6-yl)-4-aza-1-azoniabicyclo[2.2.2]octyl
chloride (25)
Compound 27 (740 mg, 1.2 mmol) was dried well under high
vacuum and dissolved in anhydrous pyridine under nitrogen.
TMS-Cl (956 mg, 8.8 mmol) was added at 0 °C and stirred at
room temperature for 2 h. Isobutyryl chloride (639 mg,
5.9 mmol) was added drop by drop at 0 °C and the reaction
mixture was stirred for an additional 4 h at room temperature.
After completion of the reaction water was added to stop the
reaction and the aqueous ammonia was added at 0 °C and
stirred for 1 h. The compound was extracted with dichloro-
methane. The organic layer was dried over sodium sulphate
and concentrated to give a yellow oily paste. The resulting oily
paste was dissolved in THF and 1 M solution tetrabutyl-
ammonium fluoride in THF was added and stirred for 30 min
at room temperature. The compound was washed with water
in dichloromethane and further the organic layer was washed
with brine and dried over sodium sulphate. The organic layer
was concentrated and loaded over a silica gel column. Column
chromatography was carried out with 20 to 90% ethyl acetate–
To a stirred solution of 2-amino-6-chloropurine 24 (1.0 g,
5.9 mmol) in dry DMSO, DABCO (3.64 g, 32.45 mmol) was
added and the reaction mixture was stirred overnight. After
completion of the reaction, the product was filtered and
washed with dichloromethane. The product was dried under
1
high vacuum to afford 87% yield as a white powder. H NMR
(500 MHz, D2O) δ 8.25 (s, 1 H), 4.18 (t, J = 10 Hz, 6 H), 3.43
(t, J = 10 Hz, 6 H); 13C NMR (125 MHz, D2O) δ 159.08,
158.11, 151.33, 143.66, 117.03, 53.51, 44.33.
6-(Benzyloxy)-9H-purin-2-amine (26)
Benzyl alcohol (2.5 g, 23.1 mmol) was added to stirred solution
of 25 and NaH (342 mg, 8.4 mmol) in dry DMSO. After 1 h of
stirring, DABCO purine 19 (1.2 g, 4.2 mmol) was added and
the reaction mixture was stirred overnight. DMSO was removed
under vacuum and the compound was dissolved in ethyl
acetate. The organic layer was washed with water and dried
over sodium sulphate. The solvent was removed by vacuum
and the solid was purified by silica gel chromatography (20 to
80% ethyl acetate–hexane) to give gave the product (600 mg,
60% yield) as a light brown powder. 1H NMR (500 MHz,
DMSO) δ 12.45 (br s, 1 H), 7.84 (s, 1 H), 7.51 (d, J = 7.6 Hz,
2 H), 7.40 (t, J = 7.5 Hz, 2 H), 7.34 (d, J = 7 Hz, 1 H); 13C NMR
(125 MHz, DMSO) δ 159.65, 155.23, 137.81, 136.77, 128.42,
128.37, 127.98, 113.51, 66.69.
hexane with 1% triethylamine to afford
a white foam
(842.17 mg, 67%). 1H NMR (500 MHz, CDCl3) δ 7.78 (br s,
1 H), 7.67 (s, 1 H), 7.44–7.41 (m, 2 H), 7.32–7.26 (m, 4 H),
7.23–7.19 (m, 8 H), 7.15–7.11 (m, 1 H), 6.76–6.73 (m, 4 H), 5.53
(s, 2 H), 4.23 (dd, J = 2.2, 14.2 Hz, 1 H), 4.13–4.07 (m, 1 H),
4.05–4.01 (m, 1 H), 3.70 (s, 6 H), 3.3–3.27 (m, 1 H), 3.21–3.17
(m, 1 H), 2.90–2.81 (m, 1 H), 1.75–1.64 (m, 2 H), 1.19 (d, J =
6.9 Hz, 6 H); 13C NMR (125 MHz, CDCl3) δ 175.74, 160.75,
158.63, 153.23, 151.62, 144.82, 142.97, 136.11, 136.02, 135.98,
130.04, 128.60, 128.32, 128.16, 128.02, 126.97, 118.08, 113.31,
86.77, 69.57, 68.88, 61.30, 55.33, 51.04, 36.08, 34.43, 19.41;
HRMS calcd for C41H43N5O6Na [M + Na]+ 724.3111, found
[M + Na]+ 724.3112.
(S)-1-(2-Amino-6-(benzyloxy)-9H-purin-9-yl)-4-(bis-
(4-methoxyphenyl)(phenyl)methoxy)butan-2-ol (27)
To a suspension of O-benzyl purine 26 (2.4 g, 9.9 mmol) in dry
DMF, 0.3 equiv. NaH was added and the reaction mixture was
stirred for one hour at room temperature. DMT epoxide (4.2 g,
10.2 mmol) dissolved in dry DMF was added and the reaction
(S)-N-(9-(4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxybutyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide
(29)
mixture was stirred for 12 hours at 110 °C. The solvent was Compound 28 (1.7 g, 2.42 mmol) was dissolved in ethyl acetate
removed by vacuum and workup was done with ethyl acetate (20 ml). 10% Pd/C (300 mg) was added under argon. The reac-
and saturated sodium bicarbonate solution. The organic layer tion was allowed to stir under hydrogen atmosphere for 5 h.
was washed with brine and dried over sodium sulphate. The The compound was filtered and washed with methanol. The
solvent was removed by rotavapour and loaded over a silica gel organic layer was concentrated to afford the product (1.45 g,
column. The compound was eluted with 20 to 80% ethyl 97% yield) as a white foam solid. [α]2D5 = +5.8 (c = 1 in dichloro-
acetate–hexane with 1% triethylamine to afford the product as ethane). 1H NMR (500 MHz, CDCl3) δ 11.77 (br s, 1 H), 8.64
1
a yellow foam (2.2 g, 35% yield). H NMR (500 MHz, CDCl3) δ (br s, 1 H), 7.52 (s, 1 H), 7.38 (d, J = 8.20 Hz, 2 H), 7.27 (d, J =
7.47 (s, 1 H), 7.42–7.41 (m, 2 H), 7.33–7.31 (m, 2 H), 7.28–7.23 8.20 Hz, 4 H), 7.21–7.18 (m, 2 H), 7.13–7.10 (m, 1 H), 6.76–6.73
(m, 2 H), 7.23–7.18 (m, 7 H), 7.14–7.11 (m, 1 H), 6.76–6.73 (m, (m, 4 H), 5.07 (br s, 1 H), 4.32–4.29 (m, 1 H), 4.09 (dd, J = 2.84,
4 H), 5.46 (s, 2 H), 4.75 (br s, 2 H), 4.12–4.06 (m, 2 H), 14.19 Hz, 1 H), 3.76–3.71 (m, 1 H), 3.70 (s, 6 H), 3.34–3.30 (m,
3.91–3.84 (m, 1 H), 3.70 (s, 6 H), 3.28–3.24 (m, 1 H), 3.19–3.14 1 H), 3.25–3.21 (m, 1 H), 2.51 (sept, J = 7 Hz, 1 H), 1.76–1.65
(m, 1 H), 1.66–1.62 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ (m, 2 H), 1.13 (dd, J = 6.9, 18.3 Hz, 6 H); 13C NMR (125 MHz,
161.17, 159.00, 158.67, 154.22, 144.85, 140.76, 136.57, 136.15, CDCl3) δ 178.76, 158.61, 155.09, 148.33, 147.25, 145.14, 140.57,
136.05, 130.09, 128.51, 128.38, 128.19, 128.12 128.05, 126.99, 136.34, 136.30, 130.22, 128.28, 128.01, 126.91, 120.33, 113.31,
115.58, 113.34, 86.81, 69.53, 68.25, 61.27, 55.36, 50.26, 34.30; 86.68, 68.15, 60.91, 55.36, 50.67, 36.50, 34.72, 19.11, 18.88;
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HRMS calcd for C34H37N5O6Na [M + Na]+ 634.262, found
[M + Na]+ 634.262.
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Guanine phosphoramidite (30)
Compound 29 (617 mg, 1 mmol) was dried under high
vacuum and anhydrous dichloromethane was added and
subsequently diisopropyl ammonium tetrazolide (85 mg,
0.5 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphor-
diamidite (452 mg, 1.5 mmol) were added under nitrogen. The
reaction was allowed to stir at room temperature. After 4 h, the
reaction was diluted with dichloromethane and poured into
saturated aqueous sodium bicarbonate. The product was
extracted with dichloromethane and dried over magnesium
sulfate. The product was purified by silica gel chromatography
dichloromethane–hexanes (2 to 80%) with 1% triethyl-
amine to afford the product as a white foam (532 mg, yield
65%). 31P NMR (121 MHz, CDCl3) δ 148.824, 147.865; HRMS
calcd for C43H54N7O6PNa [M + Na]+ 834.3720, found [M + Na]+
834.3724.
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Acknowledgements
The authors acknowledge financial support from the DBT and
ICMR, Govt. of India (BT/PR/10064/AGR/36/30/07). The
authors gratefully thank Dr Yamuna Krishnan from National
Center for Biological Sciences (NCBS) India for their kind
support for UV-melting studies and Department of Chemistry
IIT Madras, SAIF-IIT Madras for analytical data. Vipin thanks
IIT Madras for fellowship. Vipin thanks Dr Thomas Torring
from Center for DNA Nanotechnology (CDNA), Department of
Chemistry and iNANO, Aarhus University for proof reading the
article.
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