Synthesis and thermal stabilities of oligonucleotides containing 2′-: O,4′- C -methylene bridged nucleic acid with a phenoxazine base

Article abstract of DOI:10.1039/c7ob01874f

We designed and synthesized a novel artificial 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward compleme

Full text of DOI:10.1039/c7ob01874f

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Takeharu Haino et al.
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DOI: 10.1039/C7OB01874F
PAPER
Synthesis and thermal stabilities of oligonucleotides containing 2′-
O,4′-C-methylene bridged nucleic acid with a phenoxazine base
Received 00th January 20xx,
Accepted 00th January 20xx
Yuki Kishimoto,a,b Akane Fujii,a,b Osamu Nakagawa,*a,b Tetsuya Nagata,b,c Takanori Yokota,b,c
Yoshiyuki Hari,d and Satoshi Obika*a,b
DOI: 10.1039/x0xx00000x
We designed and synthesized
a novel artificial 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) with a
www.rsc.org/
phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher
binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2′,4′-BNA/LNA with 5-
methylcytosine or 2′-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits
properties associated with the phenoxazine moiety in DNA/DNA duplex es and characteristics associated with the 2′,4′-
BNA/LNA moiety in DNA/RNA duplexes.
place of the four natural bases (A, G, C and T) and these analogues
exhibit novel functionalities and work effectively in oligonucleotide
technology applications.8) For example, we have developed 2′,4′-
BNA/LNAs with several artificial nucleobases for application in
antigene strategies using triplex-forming oligonucleotides (TFOs).9)
TFOs containing 2′,4′-BNA/LNA analogues exhibited improved
recognition ability toward CG or TA base pairs that could not be
recognized by natural nucleobases.
Introduction
Various artificial nucleic acids have been developed for
application in oligonucleotide technologies such as
antisense/antigene, small interfering RNA, aptamers, and single
nucleotide polymorphism (SNP) analysis.1) These modified nucleic
acids exhibit useful properties, including improved duplex-forming
ability, stability against enzymatic degradation, and accurate base
discrimination. One of the most promising modified nucleic acids is
2′-O,4′-C-methylene bridged nucleic acid (abbreviated as either
2′,4′-BNA or LNA) independently developed by our laboratory2) and
Wengel’s group3). 2′,4′-BNA/LNA is strictly locked into the N-type
sugar conformation (C3′-endo) and shows high duplex-forming
ability and gene knockdown efficiency in vivo (Figure 1, a). The
design concept behind 2′,4′-BNA/LNA is to prevent entropy loss by
pre-organizing the structure into the N-type sugar conformation
preferred in A-form duplexes. Based on this concept, we have
developed a series of 2′,4′-bridged nucleic acids such as AmNA4),
GuNA5), scpBNA6), and SeLNA7). In addition, various artificial
nucleobases can be introduced into the original 2′,4′-BNA/LNA in
Here, we focused on the nucleobase modification of 2′,4′-
BNA/LNA. The artificial phenoxazine base is a tricyclic cytosine
analogue that shows high binding affinity towards complementary
guanine (Tm: approximately +6°C compared with cytosine) (Figure
1, b)10) due to effective π-π stacking interaction of the tricyclic
aromatic ring with the flanking nucleobase in an
oligonucleotide.10a,b) Pyrimidine bases in the 5′-flanking site interact
effectively with phenoxazine. The phenoxazine base and its 5-sulfur
analogue (phenothiazine base) have promising properties such as
fluorescence11), enzymatic stability12) and polymerase recognition13)
.
Moreover, a phenoxazine derivative bearing an aminoethoxy unit at
9-position (named G-clamp) is well-studied.14)
a. Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka 565-0871, Japan.
b. Core Research for Evolutional Science and Technology (CREST), Japan Sciences
and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.
c. Department of Neurology and Neurological Science, Graduate School of Medical
and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8519, Japan.
d. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Nishihama,
Yamashiro-cho, Tokushima 770-8514, Japan
E-mail: obika@phs.osaka-u.ac.jp (S.Obika),
osamu_nakagawa@phs.osaka-u.ac.jp (O. Nakagawa)
Figure 1. Design of 2′,4′-bridged nucleic acid with a phenoxazine
base (BNAP).
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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of 4 was chlorinated using the Appel reaction,14a) then reacted with
Both 2′,4′-BNA/LNA and the phenoxazine base improve the
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2-aminophenol and 1,8-diazabicyclo[5.4.0]DunOdI:e1c0-7.1-0e3n9e/C7(DOBB0U18)74toF
afford 5. After deprotection of the 3′,5′-acetyl groups, formation of
the phenoxazine skeleton with 6 was achieved in 59% yield using
basic conditions with DBU in ethanol at reflux. Next, the 5′-hydroxy
group of 7 was protected with a 4,4′-dimethoxytrityl (DMTr) group
to give 8. The reaction under typical conditions (DMTrCl in
pyridine) did not proceed effectively whereas DMTrOTf
successfully gave the desired product. Finally, the 3′-hydroxy group
of 8 was phosphitylated to afford phosphoramidite 9.
duplex-forming ability of oligonucleotides but through different
molecular mechanisms. Therefore, combining these two moieties
could be a good strategy for the design of new oligonucleotides with
high binding affinity. We designed 2′,4′-Bridged Nucleic Acid with
Phenoxazine base (abbreviated BNAP) and here describe its
synthesis and properties (Figure 1, c).
Subsequently, the phosphoramidite
9
was satisfactorily
incorporated into oligodeoxynucleotides (ODNs) using an automated
DNA synthesizer by following the standard phosphoramidite
protocol with
a
prolonged coupling time, using 5-[3,5-
bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42®) as the
activator. The synthesized ODNs were cleaved from the controlled
pore glass (CPG)-supported columns using a solution of aqueous
methylamine and aqueous ammonia (v/v, 1/1) at 65°C for 10 min,
purified by reverse-phase HPLC, and their composition was
confirmed by MALDI-TOF-MS spectrometry (Table 1).
The chemical stabilities of ODNs containing BNAP (B) (termed
B-ODNs) were similar to that of the corresponding ODNs containing
2′-deoxyribonucleotide with phenoxazine (P) (P-ODNs). The B-
ODNs were unstable in the aqueous ammonia solution conditions
(10 h at 55°C) used in typical deprotection/cleavage steps, and
therefore deprotection was conducted with the above aqueous
ammonia/methylamine (v/v, 1/1) solution at 65°C for 10 min. It is
known that phenoxazine base exhibits fluorescence (Em:
approximately 450 nm, Ex: 365 nm).11f,h) The B-ODNs showed
fluorescence similar to P-ODNs (see Supporting Information, Figure
S3).
Table 1. MALDI-TOF mass data of ODNs containing BNAP.
MALDI-TOF Mass
Sequences
(B: BNAP)
Calcd. (M-H)-
Found
Scheme 1. Synthesis of BNAP phosphoramidite.
5′-d(GCGTTBTTTGCT)-3′
5′-d(GCGTCBATTGCT)-3′
5′-d(GCGTCBCTTGCT)-3′
5′-d(GCGTABATTGCT)-3′
5′-d(GCGTGBGTTGCT)-3′
5′-d(GCGTBCBTTGCT)-3′
5′-d(GCGTCBBTTGCT)-3′
5′-d(GCGTBBBTTGCT)-3′
3735.5
3729.5
3705.4
3753.5
3785.5
3823.5
3823.5
3941.6
3735.2
3728.2
3705.1
3754.5
3786.5
3822.0
3823.4
3941.1
Reagents and conditions: (a) Ac2O, pyridine, rt, 7 h (99%); (b) NBS,
DMF, 0°Crt, 2.5 h (81%); (c) PPh3, CCl4, CH2Cl2, reflux, 2 h, then
2-aminophenol, DBU, rt, 12 h; (d) 7 M NH3 in MeOH, rt, 2 h (81%
in 2 steps); (e) DBU, EtOH, reflux, 30 h (59%); (f) DMTrCl, AgOTf,
2,6-lutidine, CH2Cl2, 0°C, 3 h (58%); (g) i-Pr2NP(Cl)OCH2CH2CN,
DIPEA, CH2Cl2, rt, 3 h, (74%).
Results
Evaluation of the duplex-forming abilities of BNAP
Synthesis of BNAP nucleoside and incorporation into
oligodeoxynucleotides
The hybridization properties of B-ODNs with various flanking
sequences were evaluated by UV melting (Tm) experiments and the
results are shown in Table 2. ODNs bearing 2′-deoxycytidine (C),
and 2′,4′-BNA/LNA with 5-methylcytosine (L) and P, were also
investigated as controls.
As shown in Scheme 1, the synthesis of BNAP nucleoside started
with known 2′,4′-BNA/LNA-uridine (2).15) After protection of the
3′,5′-hydroxy groups of 2 with acetyl groups, 3 was brominated at C-
5 with N-bromosuccinimide (NBS) to give 4. The 4-carbonyl group
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C7OB01874F
Table 2. Tm values of duplexes formed by ODNs with complementary (c) DNA and cRNA.
Tm (Tm: (Tm (L, P or B) -Tm (C))) (°C)
Sequences
Target DNA
(5′-NNN-3′)
Target RNA
X:
C
40
44
45
41
46
45
45
45
L
P
B
C
L
P
B
TXT
CXA
CXC
AXA
GXG
XCX
CXX
XXX
44 (+4)
48 (+4)
51 (+6)
45 (+4)
48 (+2)
55 (+10)
53 (+8)
58 (+13)
44 (+4)
49 (+5)
51 (+6)
38 (−3)
46 (±0)
54 (+9)
56 (+11)
61 (+16)
46 (+6)
50 (+6)
54 (+9)
40 (−1)
48 (+2)
59 (+14)
57 (+12)
60 (+15)
41
46
54
38
46
54
54
54
48 (+7)
53 (+7)
61 (+7)
44 (+6)
51 (+5)
67 (+13)
65 (+11)
71 (+17)
45 (+4)
49 (+3)
56 (+2)
36 (−2)
45 (−1)
59 (+5)
64 (+10)
70 (+16)
50 (+9)
54 (+8)
62 (+8)
40 (+2)
49 (+3)
69 (+15)
67 (+13)
73 (+19)
UV melting profiles were measured in 2 mM sodium phosphate buffer (pH 7.2) containing 20 mM NaCl at a scan rate of 0.5 °C/min at 260 nm. The
concentration of oligonucleotide used was 2 μM for each strand. The error in Tm values was ±0.5°C.
C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, P: 2′-deoxyribonucleoside with phenoxazine, B: BNAP.
Sequence: 5′-d(GCGTNNNTTGCT)-3′/3′-CGCQGQAACGA-5′ (X: C, L, P or B) (Q: Corresponding matching base).
First, for single modifications of B-ODN with complementary In contrast, all B-ODN/cRNAs showed excellent affinities compared
DNA (cDNA), the B-ODN/cDNA duplexes showed Tm values to P-ODN/cRNAs (Tm: +2°C to +6°C). Overall, the Tm results of
ranging from −1°C to +9°C; unless otherwise specified, Tm B-ODN/cRNAs were +1°C to +3°C higher than that of B-
indicates the difference in melting temperature compared to C- ODN/cDNAs. Conversely, the Tm values of P-ODN/cRNAs were
ODN/cDNA (or cRNA) with the same sequence. Note that ODNs ±0°C to −4°C, which were equal or lower than those of P-
with specific adjacent bases are denoted as, for example, B- ODN/cDNAs. Furthermore, the - stacking interactions of the
ODN(CBC). The highest increase in Tm value was observed for the phenoxazine base in A-form duplexes such as DNA/RNA may be
B-ODN(CBC)/cDNA (Tm: +9°C). Moreover, this Tm value was insufficient. In contrast, the Tm values of the L-ODN/cRNAs were
+3°C higher than those of L-ODN(CLC)/cDNA and P- +1°C to +3°C higher than those of L-ODN/cDNAs. Adequate pre-
ODN(CPC)/cDNA. However, B-ODN(ABA)/cDNA and B- organization of 2′,4′-BNA/LNA to the N-type sugar conformation
ODN(GBG)/cDNA with 5′-flanking purine bases did not show favours DNA/RNA A-form duplex (N-type) rather than DNA/DNA
comparable stabilization effects [Tm: −1°C and +2°C, respectively], B-form duplex (S-type). Thus, in the case of B-ODN/cRNA with 5′-
and rather exhibited Tm values lower than or equal to L- flanking pyrimidines, the influence of B may be mainly due to the
ODN(ALA)/cDNA and L-ODN(GLG)/cDNA but +2°C higher than 2′,4′-BNA/LNA sugar rather than the phenoxazine base. On the
those of P-ODN(APA)/cDNA and P-ODN(GPG)/cDNA. Overall, contrary, in the case of B-ODN/cRNA with 5′-adjacent purines, the
the Tm values of all P-ODN/cDNAs were 1°C to 3°C lower than that influence of the phenoxazine base in B increased, destabilizing the
of the corresponding B-ODN/cDNA. On the other hand, both L- duplex.
ODN/cDNA and P-ODN/cDNA with 5′-adjacent pyrimidine bases
Finally, B-ODN/cRNAs containing multiple modifications of B
showed almost equal Tm values [Tm: +4°C to +6°C], but in the case were evaluated. B-ODN/cRNAs containing two or three B residues
of 5′-flanking purines, P-ODN/cDNAs were less stable than L- showed higher thermal stability (Tm/modification: +6.3°C to
ODN/cDNAs. These results suggest that the sequence dependency of +7.5°C). Furthermore, as with cDNA, interval modification (BCB)
B-ODN/cDNA may be mainly due to the phenoxazine moiety.
Next, B-ODNs containing multiple
rather than continuous modifications (CBB or BBB) showed higher
modifications were duplex-forming abilities per modification.
B
evaluated. B-ODN/cDNAs containing two or three B residues
showed higher thermal stability (Tm/modification: +5.0°C to
+7.0°C). Moreover, interval modifications (BCB) rather than
continuous modifications (CBB or BBB) resulted in larger increases
in thermal stability per modification.
Evaluation of the base selectivity of BNAP
The base discrimination ability of B was investigated and compared
with that of C, L, and P (Table 3).
ODNs with the flanking sequence 5′-CXC-3′ (X: C, L, P or B),
The duplex-forming ability of B-ODNs was also evaluated with
complementary RNA (cRNA). With single modifications, B-
ODN/cRNAs showed Tm values ranging from +2°C to +9°C. Tm
values of the B-ODN/cRNAs bearing 5′-flanking pyrimidines were
+1°C to +2°C higher than those of the corresponding L-ODN/cRNA.
However, 5′-flanking purines resulted in decreased affinities to B-
ODN/cRNAs compared with L-ODN/cRNAs (Tm: −2°C to −4°C).
which showed effective duplex-forming abilities, were used. B-ODN
showed excellent discrimination toward C compared with C-ODN,
L-ODN and P-ODN [Tm(G-C): −28°C (B-ODN)], and the
discrimination of B-ODN toward A and T was similar to those of C-
ODN and L-ODN [Tm(G-A): −15°C (B-ODN), Tm(G-T): −15°C
(B-ODN)]. On the other hand, P-ODN showed lower base
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discrimination towards T compared with C-ODN, L-ODN and P- ODN/cRNA duplexes produced typical spectra of B- and A-type
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ODN [Tm (G-T): −12°C (P-ODN)], whereas P-ODN exhibited helices, respectively. The incorporation of eDacOhI: m10o.1d0i3fi9c/aCt7ioOnB0(1L8,74PF,
similar discrimination towards A and C [Tm(G-A): −12°C (P-ODN), and B) into DNA/DNA and DNA/RNA duplexes resulted in no
Tm(G-C): −23°C (P-ODN)] as did C-ODN and L-ODN. These significant change in the CD spectra, which suggests that these
findings strongly support the hypothesis that the 2′,4′-BNA/LNA modifications do not induce significant change in duplex structure.
moiety in B compensates for the low selectivity of the phenoxazine Some small changes were observed: for example, the P-ODN/cDNA
base toward the targeted T, and the superior base discrimination of B and B-ODN/cDNA duplexes exhibited a unique small negative
toward the targeted C is due to new properties acquired via the Cotton band at 230–240 nm. Conversely, the CD patterns of both P-
fusion of 2′,4′-BNA/LNA and phenoxazine.
ODN/cRNA and B-ODN/cRNA duplexes were similar in the 220–
The base discrimination of B towards cRNA was also investigated 240 nm region, and different from those of duplexes containing C
(see Supporting Information, Table S2) and was found to exhibit and L. Thus, the CD spectrum around 220–240 nm may exhibit
efficient base selectivity.
features derived from the phenoxazine base.
Thermodynamic parameters of ODNs containing BNAP
A detailed thermodynamic analysis was conducted to determine
whether the 2′,4′-BNA/LNA sugar or the phenoxazine base in BNAP
predominantly contributes to the function of BNAP. The analysis of
duplexes with 5′-CXC-3′ was conducted by constructing van’t Hoff
plots based on Tm results obtained at various concentrations (Table 3,
see also Supporting Information, Figure S6).
For duplexes with cDNA, B-ODN/cDNA showed a small
favourable entropy change (∆∆H = +2.2 kcal/mol, ∆∆S: +14
cal/mol·K). [H, S or G indicate the difference with the
corresponding H, S or G values obtained for C-ODN/cDNA (or
cRNA).] In contrast, L-ODN/cDNA showed a favourable large
entropy change though significant enthalpy loss was observed (∆∆H:
+15.1 kcal/mol, ∆∆S: +52 cal/mol·K), and P-ODN/cDNA showed
small enthalpy and entropy changes (∆∆H: −0.9 kcal/mol, ∆∆S: +2
cal/mol·K). Based on these results, the S and H changes of B-
ODN/cDNA are similar to those of P-ODN/cDNA rather than those
of L-ODN/cDNA. The ΔG (Gibbs' free energy change) of L-
ODN/cDNA was small (G: −1.1 kcal/mol), whereas P-
ODN/cDNA and B-ODN/cDNA showed high stability (G: −1.6
Table 3. Comparison of matched versus mismatched hybridization
5′-d(GCGTCXCTTGCT)-3′
3′-d(CGCAGYGAACGA)-5′
Tm (Tm: Tm[mismatch]-Tm[match]) (°C)
X =
Y =
G
A
T
C
C
L
P
45
51
51
32 (−13)
37 (−14)
39 (−12)
29 (−16)
33 (−18)
39 (−12)
24 (−21)
27 (−24)
28 (−23)
B
54
39 (−15)
39 (−15)
26 (−28)
Values in parentheses are the difference between the Tm when the ODN was
bound with the guanine-containing target and the Tm when the ODN was
bound with a mismatched base. The buffer conditions were the same as given
in Table 2. C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine,
P: 2′-deoxyribonucleoside with phenoxazine, B: BNAP.
CD measurements of duplexes containing BNAP
The duplex structures of B were analysed by circular dichroism
(CD) measurements (Figure 2). The C-ODN/cDNA and C-
(a)
(b)
70
60
40
10
30
0
-20
-50
-30
220
240
260
280
300
320
220
240
260
280
300
320
Wavelength (nm)
Wavelength (nm)
Figure 2. CD spectra of DNA/DNA (panel a) and DNA/RNA (panel b) duplexes.
Conditions: 2 mM sodium phosphate buffer (pH 7.2), 20 mM NaCl, 2 μM each strand. C: 2′-deoxycytidine (blue), L: 2′,4′-BNA/LNA with 5-
methylcytosine (green), P: 2′-deoxyribonucleoside with phenoxazine (orange), B: BNAP (red).
Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-CGCGCGAACGA-5′ (X: C, L, P or B).
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Table 3. Thermodynamic parameters of duplexes with cDNA and cRNA.
Target DNA
Target RNA
X =
H
S
G(37°C)
H
S
G(37°C)
H
S
G(37°C)
H
S
G(37°C)
C
L
P
B
−93.3
−78.2
−94.2
−91.1
−265
−213
−263
−251
−11.0
−12.1
−12.6
−13.4
-
-
-
−97.4
−104.6
−96.0
−271
−286
−264
−300
−13.4
−16.0
−14.1
-
-
-
+15.1
−0.9
+2.2
+52
+2
−1.1
−1.6
−2.4
−7.2
+1.4
−15
+7
−2.6
−0.7
−3.3
+14
−109.8
−16.7 −12.4
−29
C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, P: 2′-deoxyribonucleoside with phenoxazine, B: BNAP.
Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-CGCGCGAACGA-5′ (X: C, L, P or B).
The units of H, S and G are kcal/mol, cal/mol・K and kcal/mol, respectively. H, S and G indicate the difference of the corresponding H, S
and G value from that of C-ODN/cDNA (or cRNA) duplex.
DNA/DNA10b) but poorly in A-type duplex such as DNA/RNA.10a)
Conformational change of the duplex significantly affects π-π
overlap between phenoxazine and the 5′-flanking nucleobase.
Therefore, the thermal stability of B is largely dependent on the
duplex-structure and flanking sequence. During the design of BNAP,
we anticipated a powerful additive effect, without loss of affinity,
resulting from the fusion of 2′,4′-BNA/LNA and phenoxazine.
BNAP exhibits greatly enhanced duplex-forming ability compared to
2′,4′-BNA/LNA, but this ability is not equivalent to the summed
ability exhibited by 2′,4′-BNA/LNA and phenoxazine. This lack of
additivity suggests that (a) the spatial position of phenoxazine base
fixed by 2′,4′-BNA/LNA sugar modification in B is not completely
fit for duplex structures, and/or (b) the stabilization mechanisms of
both modifications partially overlap. It has been reported that the
duplex-forming ability of 2′,4′-BNA/LNA might be due to π-π
stacking interactions,17) similar to that of phenoxazine base.
Thermodynamic analysis (∆H, S and ∆G) clearly revealed that in
the sequence 5′-CXC-3′, BNAP exhibits properties associated with
the phenoxazine moiety in DNA/DNA duplex and characteristics
associated with the 2′,4′-BNA/LNA moiety in DNA/RNA duplex. It
is interesting that the predominance of each characteristic changed
depending on whether the duplex-structure was DNA/DNA or
DNA/RNA.
and −2.4 kcal/mol, respectively). Thus, the thermodynamic
parameters of B-ODN/cDNA were similar to those of P-ODN/cDNA
rather than those of L-ODN/cDNA, indicating that the phenoxazine
base in BNAP predominantly works within the DNA/DNA duplex
(ODN/cDNA).
Next, thermodynamic analysis was conducted with cRNA. B-
ODN/cRNA showed the most favourable enthalpy change (∆∆H:
−12.4 kcal/mol, ∆∆S: −29 cal/mol·K). L-ODN/cRNA and P-
ODN/cRNA showed a favourable enthalpy change and a small
favourable entropy change, respectively. Furthermore, the ΔΔG of
B-ODN/cRNA (−3.3 kcal/mol) was similar to that of L-ODN/cRNA
(−2.6 kcal/mol) but significantly different from the G of P-
ODN/cRNA (−0.7 kcal/mol). In DNA/RNA (ODN/cRNA), unlike
DNA/DNA, the thermodynamic parameters of B-ODN/cDNA were
similar to that of L-ODN/cRNA rather than that of P-ODN/cRNA.
Thus, in the above sequences with 5′-CXC-3′, the phenoxazine
moiety in BNAP mainly affects the duplex-forming ability of
DNA/DNA, and conversely, the 2′,4′-BNA/LNA moiety
predominantly influences the duplex forming ability of DNA/RNA.
Thermodynamic analysis of duplexes with 5′-ABA-3′ (see
Supporting Information, Table S3) showed that the thermodynamic
parameters of duplex formed with B-ODN(ABA) and duplex formed
with B-ODN(CBC) were completely different. These results
revealed that the thermodynamic parameters of B-ODN depend
significantly on the flanking sequence.
Conclusions
In conclusion, we demonstrated the synthesis and evaluation
Discussion
of 2′,4′-BNA/LNA bearing a phenoxazine base.
showed superior duplex-forming abilities compared to
B
C
-ODN
-ODN,
-ODN, and P-ODN. Thermodynamic analysis of B revealed
Modifications of the phenoxazine base in oligonucleotide
significantly improve its duplex-forming ability due to effective π-π
stacking between adjacent nucleobases.10a,b) This π-π stacking
interaction has been confirmed by molecular modeling10a,b) and X-
ray analysis16) using phenoxazine and its analogues. B-ODN with 5′-
flanking pyrimidine bases showed much better binding affinity than
did the corresponding L-ODN and P-ODN due to the π-π stacking
interaction between the phenoxazine base and the pyrimidine,
whereas this interaction did not stabilize duplexes if the B-ODN
contained 5′-flanking purine sequences. The π-π stacking interaction
of the phenoxazine base functions well in B-type duplex such as
L
that the phenoxazine moiety is dominant in DNA/DNA duplex
and that the 2′,4′-BNA/LNA moiety is dominant in DNA/RNA
duplex.
Thus, BNAP is
a
promising candidate for
oligonucleotide therapeutics and DNA nanotechnologies,
especially for those requiring high duplex-forming ability. The
synthesis and evaluation of 2′,4′-BNA/LNA with
a 9-
(aminoethoxy)phenoxazine (G-clamp) base are now underway
for further improvement of the duplex-forming ability.
This journal is © The Royal Society of Chemistry 20xx
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5-Bromo-N4-(2-hydroxyphenyl)- 2′-O,4′-C-mDetOhIy: 1le0n.1e0c3y9t/iCd7inOeB0(618)74F
Experimental
To a solution of compound 4 (850 mg, 2.00 mmol) in CH2Cl2 (90
ml) was added triphenylphosphine (Ph3P), then CCl4 (20 ml) was
added. The resultant mixture was refluxed for 2 h under an Ar
atmosphere, then cooled to room temperature. 2-Aminophenol (533
mg, 4.88 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
(600 μl, 4.01 mmol) were added, and the mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated under
reduced pressure and the residue was dissolved in CH3Cl. This
organic layer was washed with 5% citric acid aqueous solution, dried
over Na2SO4, and concentrated under reduced pressure. The residue
General
Dry dichloromethane, N,N-dimethylformamide, tetrahydrofuran,
acetonitrile and pyridine were used as purchased. H-, 13C- and 31P
1
NMR spectra were recorded on JEOL JNM-ECS300, JNM-ECS400
and JNM-ECA500 spectrometers. Chemical shift values are
expressed in δ values (ppm) relative to internal tetramethylsilane
(0.00 ppm), CHD2OD (3.30 ppm) or CHD2S(=O)CD3 (2.50 ppm) for
1H NMR, chloroform-d1 (77.0 ppm) or DMSO-d6 (39.5 ppm) for 13C
NMR, and 1% H3PO4 (0.00 ppm) for 31P-NMR. IR spectra were
recorded on a JASCO FT/IR-4200 spectrometer. Optical rotations
were recorded on a JASCO DIP-370 instrument. MALDI-TOF mass
spectra of all new compounds were measured on a JEOL SpiralTOF
JMS-S3000. MALDI-TOF mass spectra of ODNs were measured on
a Bruker Daltonics Autoflex II TOF/TOF mass spectrometer. Fuji
Silysia PSQ-60B or PSQ-100B silica gel was used for column
chromatography.
was quickly passed through
a short silica gel column (n-
hexane/AcOEt = 1/1 to 1/2). Without further purification, compound
5 contaminated with Ph3P=O (1.93 g) was dissolved in 7 M NH3 in
MeOH (12 ml) and the reaction mixture was stirred at room
temperature for 2 h. The mixture was then concentrated to remove
MeOH under reduced pressure and the residue was purified by silica
gel column chromatography (n-hexane/AcOEt = 1/3 to 0/1) to give
compound 6 (693 mg, 81% in 2 steps) as a white solid. [훂]ퟐ퐃ퟓ +84.6
(c 0.1, 1,4-dioxane); IR νmax (KBr): 3355, 2956, n 1716, 1646, 1618,
1558, 1494, 1460, 1290, 1232, 1117, 1083, 1051 cm-1; 1H NMR
(500 MHz, CD3OD): δ 3.76, 3.96 (2H, AB, J = 8 Hz), 3.89, 3.92 (2H,
AB, J = 13 Hz), 4.05 (1H, s), 4.36 (1H, s), 5.58 (1H, s), 6.85 (1H, t,
J = 8 Hz), 6.86 (1H, d, J = 8 Hz), 6.99 (1H, t, J = 8 Hz), 8.28 (1H, s),
8.40 (1H, d, J = 8 Hz); 13C NMR (101 MHz, DMSO-d6): δ 55.6,
68.2, 71.0, 78.6, 87.1, 87.8, 89.0, 115.0, 119.1, 122.3, 125.2, 125.9,
140.7, 148.2, 152.9, 157.4; HRMS (MALDI) calcd. for
C16H1679BrN3NaO6 [M+Na]+: 448.0115, found: 448.0113.
3′,5′-di-O-Acetyl-2′-O,4′-C-methyleneuridine (3)
To a solution of compound 2 (139 mg, 0.54 mmol) in pyridine (2 ml)
was added acetic anhydride (110 μL, 1.16 mmol) at 0°C, and the
resultant mixture was stirred at room temperature for 7 h. After
addition of saturated aqueous NaHCO3 solution, the solution was
extracted with AcOEt. The organic extracts were washed with
saturated aqueous NaHCO3 solution, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (n-hexane/AcOEt = 1/2 to 1/3) to
give compound 3 (183 mg, 99%) as a white foam. [훂]ퟐ퐃ퟖ +68.7 (c 0.3,
CHCl3); IR νmax (KBr): 3524, 3167, 3055, 1748, 1687, 1455, 1374,
1,3-Diaza-3-(2′-O,4′-C-methylene-β-D-ribofuranosyl)-2-
oxophenoxazine (7)
1234, 1110, 1083, 1049 cm-1; H NMR (500 MHz, CDCl3): δ 2.14
1
To a solution of compound 6 (1.20 g, 2.80 mmol) in EtOH (300 ml)
was added DBU (460 μl, 3.08 mmol) and the resultant mixture was
refluxed for 30 h. The reaction mixture was concentrated under
reduced pressure. After precipitation with MeOH, the residue was
purified by silica gel column chromatography (AcOEt/MeOH = 1/0
to 9/1) to give compound 7 (569 mg, 59%) as an orange solid. [훂]퐃ퟐퟔ
−76.7 (c 0.1, EtOH); IR νmax (KBr): 3392, 3299, 2961, 2362, 2338,
2328, 1677, 1636, 1577, 1508, 1472, 1421, 1337, 1260, 1231, 1085,
1049 cm-1; 1H NMR (500 MHz, DMSO-d6): δ 3.60, 3.80 (2H, AB, J
= 8 Hz), 3.73 (2H, s), 3.92 (1H, s), 4.11 (1H, s), 5.27 (1H, brs), 5.34
(1H, s), 5.68 (1H, brs), 6.78-6.89 (4H, m), 7.45 (1H, s), 10.6 (1H,
brs); 13C NMR (101 MHz, DMSO-d6): δ 55.7, 68.3, 70.9, 78.8, 86.6,
88.8, 115.2, 116.6, 121.3, 123.7, 123.8, 126.9, 127.2, 142.2, 152.6,
154.0; HRMS (MALDI) calcd. for C16H16N3O6 [M+H]+: 346.1034,
found: 346.1027.
(3H, s), 2.15 (3H, s), 3.99, 4.04 (2H, AB, J = 8 Hz), 4.40, 4.50 (2H,
AB, J = 13 Hz), 4.69 (1H, s), 4.86 (1H, s), 5.71 (1H, s), 5.79 (1H, d,
J = 8 Hz), 7.60 (1H, d, J = 8 Hz); 13C NMR (126 MHz, CDCl3): δ
20.6, 20.7, 58.8, 71.1, 71.8, 77.9, 85.6, 87.2, 102.4, 137.9, 149.7,
163.0, 169.7, 170.1; HRMS (MALDI) calcd. for C14H16N2NaO8
[M+Na]+: 363.0799, found: 363.0791.
5-Bromo-3′,5′-di-O-acetyl-2′-O,4′-C-methyleneuridine (4)
To a solution of compound 3 (6.69 g, 19.7 mmol) in anhydrous N,N-
dimethylformamide (DMF) (190 ml) was added
N-
bromosuccinimide (NBS) (5.24 g, 29.0 mmol) at 0°C, and the
resultant mixture was stirred at 0oC for 1.5 h under an Ar atmosphere,
then stirred at room temperature for 1 h. After removal of DMF in
vacuo with heat, the residue was purified by silica gel column
chromatography (CH3Cl/MeOH = 97/3) to give compound 4 (6.45 g,
81%) as a white solid. [훂]ퟐ퐃ퟕ +3.5 (c 0.4, CHCl3); IR νmax (KBr):
3525, 3190, 3084, 2823, 1749, 1697, 1619, 1438, 1370, 1232, 1133,
1,3-Diaza-3-[5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylene-β-D-
ribofuranosyl]-2-oxophenoxazine (8)
1
1094, 1056 cm-1; H NMR (500 MHz, CDCl3): δ 2.15 (3H, s), 2.24
To a solution of silver trifluoromethanesulfonate (AgOTf) (364 mg,
1.42 mmol) in CH2Cl2 (5 ml) was added 4,4′-dimethoxy chloride
(DMTrCl) (515 mg, 1.52 mmol) in CH2Cl2 (5 ml), and the resultant
mixture was stirred for 1 h at room temperature under an Ar
atmosphere to give DMTrOTf solution. Then, to a solution of
compound 7 (99 mg, 0.28 mmol) in anhydrous 2,6-lutidine (8 ml)
was added DMTrOTf solution (4.8 ml) at 0oC, and the mixture was
(3H, s), 3.96, 4.03 (2H, AB, J = 8 Hz), 4.41, 4.46 (2H, AB, J = 13
Hz), 4.68 (1H, s), 4.86 (1H, s), 5.68 (1H, s), 7.99 (1H, s), 8.56 (1H,
brs); 13C NMR (126 MHz, DMSO-d6): δ 20.5, 20.6, 58.8, 70.8, 71.2,
77.4, 85.4, 86.7, 95.8, 138.2, 149.4, 159.3, 169.6, 169.8; HRMS
(MALDI) calcd. for C14H1579BrN2NaO8 [M+Na]+: 440.9904, found:
440.9903.
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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ARTICLE
stirred for 3 h. After addition of NaHCO3 aq. at 0°C, the mixture was detritylated with 0.5% aqueous trifluoroacetic acid VsieowluAtrtiioclne,Onalninde
extracted with AcOEt. The organic layer was washed with saturated eluted with 35% aqueous MeOH solutiDonO.I: 1T0h.1e039p/rCo7dOuBc0t 18w74aFs
aqueous NaHCO3 solution and copper (II) sulphate aqueous solution subjected to reverse-phase HPLC using a Waters XBridgeTM OST
(3.00 g in 20 ml H2O), dried over Na2SO4, and then concentrated C18 2.5 μm (10 × 50 mm) column using 0.1 M triethylammonium
under reduced pressure. The residue was purified by silica gel acetate (TEAA) buffer (pH 7.0) as buffer A and 50% MeCN in 0.1
column chromatography (0.5% triethylamine in n-hexane/AcOEt = M TEAA buffer (pH 7.0) as buffer B. The purity of the ODNs was
1/9 to 0/1) to give compound 8 (105 mg, 58%) as a yellow foam. confirmed by reverse-phase HPLC using a Waters XBridgeTM OST
[훂]ퟐ퐃ퟕ −82.0 (c 0.3, CHCl3); IR νmax (KBr): 3413, 3086, 2952, 2838, C18 2.5 μm (4.6 × 50 mm) column and by MALDI-TOF mass
2357, 2334, 1677, 1640, 1607, 1576, 1499, 1472, 1419, 1331, 1282, spectrometry.
1254, 1178, 1082, 1048 cm-1; H NMR (500 MHz, CDCl3): δ 3.44
(2H, s), 3.73, 3.88 (2H, AB, J = 7 Hz), 3.78 (3H, s), 3.79 (3H, s),
UV melting experiments were carried out on SHIMADZU UV-
4.08 (1H, brs), 4.40 (1H, s), 4.78 (1H, s), 5.63 (1H, s), 6.60-6.68 (3H,
1
UV melting experiments (Tm measurements)
1650PC and UV-1800 spectrometers equipped with Tm analysis
accessory quartz cuvettes with 1 cm optical path lengths. The UV
m), 6.87 (4H, dd, J = 3, 9 Hz), 7.05 (1H, s), 7.20 (1H, t, J = 7 Hz),
7.33 (2H, t, J = 8 Hz), 7.37 (4H, dd, J = 2, 9 Hz), 7.48 (2H, d, J = 8
melting profiles were recorded in 2 mM sodium phosphate buffer
Hz), 7.73 (1H, s), 11.2 (1H, brs); 13C NMR (101 MHz, CDCl3): δ
(pH 7.2) containing 20 mM NaCl to give a final strand concentration
55.2, 58.6, 70.3, 72.1, 79.9, 86.5, 87.2, 87.8, 113.3, 114.7, 118.0,
of 2 μM. The samples were annealed by heating at 100°C, followed
120.2, 123.3, 124.2, 126.5, 126.8, 127.5, 127.9, 128.0, 130.1, 130.2,
by slow cooling to room temperature. The melting profile was
135.2, 135.4, 142.5, 144.9, 153.3, 155.3, 158.5, 158.5; HRMS
recorded at 260 nm from 5 to 90°C at a scan rate of 0.5°C /min. A
(MALDI) calcd. for C37H33N3NaO8 [M+Na]+: 670.2160, found:
two-point average method was used to obtain the Tm values and the
final values were determined by averaging three independent
measurements which were accurate to within 1°C.
670.2162.
1,3-Diaza-3-[3′-O-(N,N-diisopropylamino-2-
cyanoethoxyphosphino)-5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-
methylene-β-D-ribofuranosyl]-2-oxophenoxazine (9)
CD measurements
CD spectra were measured using
a
JASCO J-720W
To a solution of compound 8 (107 mg, 0.160 mmol) in CH2Cl2 (1
ml) was added N,N-diisopropylethylamine (DIPEA) (85 l, 0.48
mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (80
μl, 0.36 mmol) at 0°C and the mixture was stirred for 3 h at room
temperature under an Ar atmosphere. After addition of saturated
aqueous NaHCO3 solution at 0°C, the mixture was extracted with
AcOEt. The organic layer was washed with saturated aqueous
NaHCO3 solution, water, and brine, dried over Na2SO4, and then
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (0.5% triethylamine in n-
hexane/AcOEt = 1/1 to 3/7) to give compound 9 (100 mg, 74%) as a
spectrophotometer. The spectra were recorded at 10°C in a quartz
cuvette with a 1 cm optical path length. The samples were prepared
in the same manner as described for the UV melting experiments.
The molar ellipticity was calculated from the equation [θ] = θ/cl,
where θ is the relative intensity, c is the sample concentration, and l
is the cell path length in centimetres.
Conflicts of interest
There are no conflicts of interest to declare.
1
yellow foam. H NMR (300 MHz, CDCl3): δ 0.99-1.29 (13H, m),
2.38 (13/20H, t, J = 7 Hz), 2.54 (7/20H, t, J = 6 Hz), 3.35-3.85 (14H,
m), 4.28 (7/20H, d, J = 12 Hz), 4.39 (13/20H, d, J = 15 Hz), 4.65
(13/20H, s), 4.72 (7/20H, s), 5.72 (1H, s), 6.55-6.59 (1H, m), 6.80-
6.92 (6H, m), 7.25-7.41 (7H, m), 7.47-7.64 (4H, m); 31P NMR (203
MHz, CDCl3): δ 149.1, 149.2; HRMS (MALDI) calcd. for
C46H50N5NaO9P [M+Na]+: 870.3238, found: 870.3240.
Acknowledgements
This work was supported by the Core Research for Evolutional
Science and Technology (CREST) program of the Japan
Science and Technology Agency (JST), and JSPS KAKENHI
Grant Number 15K08024. This research was also partially
supported by the Platform Project for Supporting Drug
Discovery and Life Science Research from the Japan Agency
for Medical Research and Development (AMED).
Synthesis of the ODNs containing BNAP
The ODNs containing BNAP were synthesized using an automated
DNA/RNA (Gene Design, nS-8) Nucleic Acid Synthesis System at
0.2 or 1.0 mmol scale. 5-[3,5-Bis(trifluoromethyl)phenyl]-1H-
tetrazole (Activator 42®) was used for all coupling steps as the
activator. The standard phosphoramidite protocol coupling time of
70 s was increased to 15 min. The BNAP phosphoramidite 13 was
prepared in 0.1 M acetonitrile solution. The ODN syntheses were
performed using DMTr-on mode. Solid (CPG)-supported ODNs
were cleaved and the protecting groups on each nucleobase were
removed with a mixture of aqueous methylamine (40%) and aqueous
ammonia (28%) solution (v/v, 1/1) at 65°C for 10 min. The crude
ODNs bearing a DMTr group were detritylated and purified using a
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01874F
Table of contents
Synthesis and thermal stabilities of oligonucleotides containing 2′-O,4′-C-methylene bridged
nucleic acid with a phenoxazine base
Yuki Kishimoto, Akane Fujii, Osamu Nakagawa,* Tetsuya Nagata, Takanori Yokota, Yoshiyuki Hari
and Satoshi Obika*
BNAP-modified ODNs showed higher binding affinities toward complementary DNA and RNA as compared to ODNs
bearing 2′,4′-BNA/LNA with 5-methylcytosine or 2′-deoxyribonucleoside with phenoxazine.