Investigation of the effect of amino acid chirality in the internucleoside linker on DNA:DNA and DNA:RNA duplex stability

Article abstract of DOI:10.1016/j.tet.2015.02.042

Abstract Enzymatically and chemically stable amide-linked di/oligonucleosides are highly desired synthetic targets in which the phosphodiester linkages in native DNA are replaced by amide linkers of appropriate length and stereochemistry. The five-atom am

Full text of DOI:10.1016/j.tet.2015.02.042

Tetrahedron 71 (2015) 2442e2449
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Investigation of the effect of amino acid chirality in the
internucleoside linker on DNA:DNA and DNA:RNA duplex stability
*
Seema Bagmare, Anita D. Gunjal, Vaijayanti A. Kumar
Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 January 2015
Received in revised form 10 February 2015
Accepted 11 February 2015
Available online 17 February 2015
Enzymatically and chemically stable amide-linked di/oligonucleosides are highly desired synthetic tar-
gets in which the phosphodiester linkages in native DNA are replaced by amide linkers of appropriate
length and stereochemistry. The five-atom amide-linked dimers, synthesized from 30-amino-30-deoxy
thymidine,
incorporated into the DNA backbone to achieve partial replacement of selected phosphodiester linkages.
The results stressed the importance of the chirality of linker amino acid. -Proline was found to be the
most compatible as an internucleoside linker in the DNA backbone to stabilize the complexes with DNA
or RNA as compared to -proline and glycine.
a-(L/D
) proline/prochiral glycine and thymidine/uridine-40carboxylic acid derivatives, were
D
Keywords:
DNA
Five-atom amide linkage
L
Ó 2015 Elsevier Ltd. All rights reserved.
a
-Amino acid
Chiral amide linkage
-Proline
L/D
1. Introduction
be useful for RNA recognition.8 Pallan et al. carried out a systematic
study for RNA binding affinity of five-atom amide-linked DNA
analogues.8c The amide linkers in 20-O-methyl substituted furanose
sugar also led to the stabilization of RNA duplexes.8c The re-
placement of 30-O- with a methylene group and addition of
a methyl group in either R or S configuration also led to the stabi-
lization of duplexes.8c Over the last decade, we and others have
been working towards the synthesis of four- and five-atom amide-
linked nucleic acid analogues for better RNA binding.8e12 Such
amide linkers when present in 20-OMe substituted nucleoside di-
mers could drive the sugar conformation to N-type and increase the
binding strength of the modified ON:RNA duplexes.8b,c Recently it
has been shown that such amide linkages are not only tolerated at
internal positions of both guide and passenger strands of siRNAs,
but may also increase the silencing activity when placed near the
50-end of the passenger strand.10e,f The five-atom amide-linked
oligonucleotides containing thymidine and thymidineecytosine
dimers were reported by our group where thioacetamido backbone
(TANA/isoTANA) exhibited discrimination in binding affinity to-
Enzymatically and chemically stable oligonucleotide (ON) ana-
logues that can form stable duplexes with target nucleic acids are
highly desired synthetic objectives because of the potential appli-
cation of such ONs in antisense,1 siRNA,1,2 miRNA3 and splice cor-
rection4 as better drug candidates. ONs with natural
phosphodiester linkages are susceptible for hydrolysis with various
enzymes like nucleases and this limits their use for various
biological applications. Phosphorothioate, the ‘First Generation’
antisense oligonucleotides are widely applicable, however, this
chemistry is associated with the disadvantage of getting
diastereomeric mixtures at each phosphorus and also affect DNA/
RNA recognition processes due to reduced binding strength and
nonspecific binding with nontargetted proteins.5 The most prom-
ising modification of the sugar-phosphodiester backbone was
achiral peptide nucleic acid (PNA) with neutral amide linkages.6
The removal of sugars in the backbone of PNA, however, lost the
sense of 3050 directionality of natural DNA. The partial replacement
of sugar-phosphodiester linkages by four-atom sugar-amide sub-
stitution led to the chimeric ONs with sugar-amide-sugar-
phosphodiester backbone. Such chimeric ONs bound to RNA with
a moderate RNA binding selectivity and with moderately di-
minished affinity in antiparallel orientation.7 Partial replacement of
phosphodiester linkages with five-atom amide linkers was found to
wards DNA/RNA.9a,b We also reported (thyminyl-
-amino acid)8 sequences where the five-atom-amide linkers
b-amino acidþ
a-
L
completely replaced the anionic four-atom-phosphodiester link-
ages, specifically recognized DNA and RNA with high binding af-
finity.12 Further, we reported that the change in chirality of the
amino acid proline in the TT-dimer blocks has profound effect on
the CD pattern and postulated that this effect could be because of
the differential orientation of base-stacking interactions.13 The
D-proline containing dimer in fact, showed maximum amplitude at
* Corresponding author. E-mail address: va.kumar@ncl.res.in (V.A. Kumar).
http://dx.doi.org/10.1016/j.tet.2015.02.042
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
2443
270 nm similar to the native DNA dimer whereas as the
L
-proline
gave the dimer blocks 6a/6b/6c/6d. Phosphitylation of the 30-OH
function of 6a, 6b, 6c and 6d was done using the reagent N,N-dii-
sopropylcyanoethyl chlorophosphine and diisopropylethyl amine
in DCM as the solvent to obtain the phosphoramidite derivatives 7a,
7b, 7c and 7d, respectively (Scheme 1). Formation of the product
was confirmed by 31P NMR spectroscopy. For NMR and CD studies,
50-O-DMT deprotection in 6a, 6b, 6c and 6d was effected with 3%
dichloroacetic acid to give Ia, Ib, Ic and Id, which were purified by
successive trituration of the crude product with diethyl ether
(Scheme 1). All the new compounds were appropriately charac-
terized using 1H, 13C and 31P NMR and confirmed by mass spectral
analysis. The structural integrity of the dimer blocks Ia, Ib, Ic and Id
after deprotection was confirmed by HRMS. All the four dimer units
were then extensively characterized by NMR. The 1H NMR spectra
of all four unprotected dinucleosides (Iaed) were recorded using
D2O as the solvent. All the peaks were found to be well resolved and
fully assigned with the aid of 2D COSY, 2D NOESY experiments
(Supplementary data) and chemical shift values are tabulated in
Table 1 (Table 1, Supplementary data). H50/H500 protons were
assigned according to the Remin and Shugar rule18 where the lower
field proton is assigned as H50 while the higher field protons are
assigned as the H500. The discrimination between 20 and 200 proton
was made using the rules devised by Altona et al.19 The 30-(Ring A)
and 50-(ring B) deoxyribose ring proton signals in the dimers were
assigned by COSY experiments and distinguished by absence of 50
and 500 protons in the 30 terminal sugar. This sugar also showed
noticeable downfield shift of H30 and H40 proton. In the case of
dimers Ia and Ib, the H40 proton signal merged with the D2O peak.
In the case of dimer Id, the H20-proton in uridine, which showed
containing dimer showed minima at 270 nm. The prochiral glycine
present as the linker did not show such distinct CD, either because
of the absence of base stacking or cancellation of opposing CD
signal due to prochiral glycine.13 The extended backbone has also
proven to be a good strategy in the case of PNA consisting of cyclic
and chiral monomers, as the binding with RNA/DNA was found to
be better in those studies.14e16
Here, we present our results of partial replacement of phos-
phodiester linkages with chiral amide linkage using prochiral gly-
cine cyclic and amino acids such as
D/L-proline. This strategy
precludes the tedious diastereomeric separation of the required
compounds due to the easy availability of chiral amino acids as both
enantiomers. The detailed synthesis of dimer blocks (TD-proT, TL-proT,
TglyT, and TD-proUOMe) from the starting nucleosides and their
characterization using NMR is presented. The presence of 20-OMe
group in TD-proUOMe is expected to drive the conformation of the
nucleoside towards N-type, suitable for RNA binding.8,12 We also
report the incorporation of these dimer blocks in DNA sequences
and evaluation of the effect of the chirally differentiated linker on
the ability of oligomers to form complexes with cDNA and RNA.
2. Results and discussion
2.1. Synthesis of dimer blocks
The synthesis of the dimer building blocks and their protected
phosphoramidite derivatives amenable for their incorporation into
oligonucleotides is depicted in Schemes 1. 50-Dimethoxytrityl-30-
deoxy-30-aminothymidine 19a was acylated using Fmoc-protected-
L
-proline (2a), -proline (2b) or glycine (2c) to get 3a, 3b and 3c,
D
respectively. The Fmoc protection was removed using relatively
volatile diethyl amine (50% DEA in DCM). The deprotection as
monitored by TLC was found to be complete in about 45 min. Iso-
lation of the intermediate products 4a, 4b and 4c with free amino
group was achieved by simple precipitation using petroleum ether
and the precipitated products were used in the next coupling step
without further purification. 30-O-tert-Butyldimethylsilylth-
ymidine-40-carboxylic acid 5a was obtained by TEMPOeBAIB oxi-
dation of the 30-O-tert-butyldimethylsilyl thymidine according to
reported procedure.12,17 30-O-tert-Butyldimethylsilyl-20-O-methyl-
uridine-40-carboxylic acid 5b was obtained from commercially
available 20-OMe-uridine using similar protection and oxidation
steps. The 30-protected thymidine or 20-OMe-uridine 40-carboxylic
acid (5a)/(5b) was then coupled with either 4a/4b/4c to yield 30-50
protected dinucleosides, which on deprotection with TBAF in THF
Table 1
N-Type and S-type preferencea for compounds 1, 5a/b and dimers Ia, Ib, Ic and Id
0
0
0
00
Compound
JH1 H2
JH1 H2
%S
%N
1 (Ring A)
5a(Ring B)
5b(Ring B)
Ia, R¼H
5.5
5.0
6.0
5.6
5.7
5.5
5.5
5.0
5.8
6.9
5.6
5.8
9.3
d
25
78
73
82
50
72
36
58
51
73
44
75
22
27
18
50
28
64
42
49
27
56
A
B
A
B
A
B
A
B
9.0
7.1
8.6
6.5
d
T-L-pro-T
Ib, R¼H
T-D-pro-T
Id, R¼OMe
T-D-pro-UOMe
Ic, R¼H
7
7.2
6.8
T-gly-T
a
20-Deoxyribonucleoside: %S¼(SJH1 ꢀ9.8)/5.9ꢁ100, where SH1 ¼J1 2 þJ1 2 , 2 -O-
0
0
0
0
0
0 00
0
methylribonucleoside S(%)¼100ꢁ(J1 )/6.9.
Scheme 1. Reagents and conditions: (i) HOBt, HBTU, DIPEA, DMF/CH3CN (1:1), (ii) Et2NH, CH2Cl2, (iii) TBAF, THF, (iv) DCA, CH2Cl2, (v) chloro-(cyanoethoxy-N,N-diisopropylamino)
phosphine, DIPEA, CH2Cl2.

2444
S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
a downfield shift helped to differentiate easily between the two
sugar rings. As the proline CH2 groups resonance mixed with both
50500 and 20200 protons resonance, it was not possible to derive all the
coupling constants due to spectral complexity and overlap.
2.3. Sugar conformations in dimer blocks
It is important to study the effect of chirality in the backbone on
conformational preferences of deoxyribose sugar in dimers. The
sugar ring conformations of nucleosides 1, 5a and 5b and of the
individual sugars in the dimers blocks Ia, Ib, Ic and Id were eluci-
dated with an empirical method of analysis using the vicinal cou-
2.2. Conformation about glycosidic bond
3
3
0019e21
0
pling constant J1 2 and J1 2 and is presented in Table 1. The
0
0
populations of 20/30-endo sugar conformations were calculated
from the equation used in the analysis. The predominant N-type
sugar conformation was observed for the 30-amino nucleoside (2, %
N¼75), however, 40-carboxylic acid nucleosides (5a, % N¼22) and
(5b, % N¼27) adopted predominantly S-type conformation. The 20-
OMe group had little effect in shifting the equilibrium to N-type
geometry of the monomer acid. In all the three dimers Ia, Ib and Ic,
the 30-sugar exhibited higher S-type geometry uniformly as com-
pared to the 50-thymidinyl units. The 20-OMe-uridine-40-carboxylic
acid 5b showed predominantly S-type sugar pucker but its N-type
character increased in dimer Id (ring A) from 27 % to 42%. The
percentage of N character was found to be higher where C30 of the
sugar ring was substituted by nitrogen in the ring B (50e64%) as
compared to the 30-OH sugar (18e28%) in ring A in the dimers
IaeIc, where the 30-end sugar was devoid of 20-OMe group (Table
1).
The conformation about the glycosidic bond in the dimers was
determined from 2D NOESY experiments (Supplementary data). An
anti conformation is preferred when a strong NOE between H6 of
thymidine (H8 of adenosine) and H20or H30of the sugar ring is
observed while a strong NOE between H6/H8 and H10 reveals
a preferred syn conformation.20 Schematic representation of some
of the long range NOEs seen in 2D NOESY spectrum of dimers Ia, Ib,
Ic and Id is depicted in Fig. 1. In -proline containing dimer Ia, there
L
are strong NOE cross peaks between H6 with H20 (1.6%) and H30
(1.3%) in the ring B and strong NOE between H6 with H20 (2.5%) in
the ring A, indicating anti glycosidic conformation (Fig. 1 A). In
D-proline containing dimer Ib, there are strong NOE cross peaks
between H6 with H20 (2.6%), H30 (1.7%) and H6 and H10 (1.5%) in 50
terminal sugar (ring B) indicates both glycosidic conformation with
more population of anti conformation (Fig. 1B). In the ring A strong
NOE between H6 with H20 (2.6%) indicates anti glycosidic confor-
mation of thymine (Fig. 1B). In 20-OMe substituted dimer Id, the
strong NOE between H6 and H20 (1.7%) in ring A and strong NOE
cross peaks between H6 and H20 (2.4%), H30 (2.3%) in ring B indicate
anti conformation of uracil and thymine, respectively (Fig. 1D) In
glycine containing dimer Ic, NOE cross peaks between H6eH30
(1.8%) and H6eH20 (3.2%) were observed only in the 50-end sugar
(ring B), which indicates the anti glycosidic conformation. In the 30
terminal sugar (ring A), however, strong NOE between
H6eH10(4.4%) and H6eH200 (4.3%) seems to indicate population of
syn conformation of thymine (Fig. 1C). The reason for this could be
the possibility of hydrogen bonding between the C2 carbonyl of
thymine and the hydrogen atom of the NH-amide bond of the 30
terminal A ring, facilitating the rotation of the glycosidic bond in
syn conformation (Fig. 1C). However absence of such possible hy-
drogen bonding in proline containing dimer Ia, Ib and Id probably
allows only the anti conformation of glycosidic bond, which allows
better stacking interactions as observed earlier by us using CD
spectroscopy.13 The CD band attributed to base-stacking in-
teractions was found to be insignificant in the case of Ic, probably
due to the anti to syn conformational change as observed by NMR in
these studies or due to prochiral glycine in the internucleoside
linker as predicted earlier.13
The north/south conformational equilibria of the sugar rings are
known to affect the base-stacking interactions.22 20-O-Methylation
has been shown to enhance base stacking of dipyrimidine ribodi-
nucleotides, which could be a consequence of the C30-endo sugar
conformational preference.22 Comparison of the CD for the T-D-pro-T
0
Ib and T-D-pro-U2 -OMe Id is shown in Fig. 2A. The strong positive
0
band at w270 nm in the T-D-pro-U2 -OMe Id, indicate that the base
0
stacking could be better in T-D-pro-U2 -OMe Id than in T-D-pro-T Ib
(Fig. 2A). The increased N-type propensity in 20-OMe substituted
sugar as studied by NMR (Table 1) could be the reason for the in-
crease in CD intensity band at w270 nm. The strong positive band
0
at w270 nm in the T-D-pro-U2 -OMe dimer Id displayed significant
temperature dependence from 10 to 80 ꢂC (Fig. 2B), also indicating
the presence of stacking interactions similar to that found in the
case of Ib.13
In our earlier studies, the dimer Ia, containing L-proline in the
internucleoside linker group was shown to have a CD minimum at
270 nm, which also disappeared with temperature.13 The obser-
vance of opposite CD signal in Ia compared to Ib was attributed to
differential base orientation of the stacked species in the two di-
mers containing enantiomeric proline amino acid. The positive CD
Fig. 1. Schematic representation of some of the long range NOEs seen in 2D NOESY spectrum of dimers Ia, Ib, Ic and Id.

S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
2445
Fig. 2. a) Comparison of CD spectra of D-proline containing dimers Ib and Id. (b) Temperature dependent CD of dimer Id.
band at w270 nm in
D
-proline containing amino acids (1b and 1d)
and control 19 mer (DNA4) is the antisense sequence for bcr/abl
b2a2 gene, which is held responsible for chronic myeloid leukaemia.24
The modified sequences (ON-1 to ON-12) were synthesized by the
incorporation of a single or two modified dimer blocks using
extended coupling time. After cleavage from the support, the
oligomers were purified by reverse phase HPLC. The purity of the
oligomers was again ascertained by analytical RP-HPLC and found
to be >95% and the oligomers were characterized by MALDI-TOF
mass spectrometric analysis. MALDI-TOF mass data are summa-
rized in Table 2.
is same as observed for the natural nucleic acid dimers containing
phoshodiester linkage.22 The glycine containing dimer Ic, on the
other hand, showed a very weak CD and showed no changes in CD
spectra with temperature. The increased amplitude of positive CD
band observed in the case of -proline containing dimer Id is very
D
similar to the N-type locked dimer blocks22 and hence could be
a compatible replacement of phosphodiester internucleoside link-
ages in DNA. The effect of the different base orientations and
stacking interactions in these dimers would affect the stability of
duplex structures and hence to study this effect we introduced
these dimer blocks (IaeId) into oligomers.
2.5. Hybridization properties of modified oligonucleotides
2.4. Synthesis of oligonucleotides
UV-Tm studies were carried out to investigate the effect of chiral
amino acid that replace the phosphodiester linker in the dimers, on
the binding efficiency of the modified oligomers to complementary
DNA and RNA sequences. The Tm experiments of duplexes were
carried out in 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl and 0.1 mM EDTA. Chimeric DNA oligonucleotides
were individually hybridized with the complementary DNA and
RNA strands, to obtain duplexes.
A series of chimeric oligonucleotides containing one or two di-
mer blocks incorporated at the predetermined positions were
synthesized on automated Bioautomation DNA Synthesizer using
the phosphoramidite approach. The two control sequences were
chosen of biological relevance: control 18 mer (DNA1) was specific
for the splice correction of an aberrant b
-globin intron (705 site)23
Table 2
Modified sequences and MALDI-TOF mass analysis and UV-Tm studies
Chimeric sequences containing phosphate and 5-atom amide
UV-Tm ꢂC ( Tm ꢂC)
D
linkages gly/D-pro and L-pro as a-amino acid (notation) mass calcd/observed
ONs and their expected/found MALDI mass
cDNA2a
52.8
mmDNA3b
45.5 (ꢀ7.2)
44.6 (ꢀ7.8)
39.9 (ꢀ7.9)
41.49 (ꢀ8.2)
43.6 (ꢀ7.9)
40.7 (ꢀ6.5)
32.0 (ꢀ8.3)
40.5 (ꢀ6.8)
cRNA1
56.6
mmRNA2b
1
2
3
4
5
6
7
8
50CCT CTT ACC TCA GTT ACA30 Control DNA1
50CCTCTTACC TCAGTD-proTACA30 (ON1) 5400.6/5403.4
50CCTCTTACCTCAGTL-proTACA30 (ON2) 5400.6/5403.6
50CCTCTTACCTCAGTglyTACA3 (ON3) 5360.6/5354.4
46.2 (ꢀ10.4)
44.8 (ꢀ10.7)
40.6 (ꢀ10.9)
42.3 (ꢀ12.1)
45.7 (ꢀ9.5)
42.3 (ꢀ10.9)
32.7 (ꢀ10.8)
36.4 (ꢀ12.4)
52.4 (ꢀ0.4)
47.8 (ꢀ5.0)
49.6 (ꢀ3.2)
51.5 (ꢀ1.3)
47.2 (ꢀ5.6)
40.3 (ꢀ12.5)
47.3 (ꢀ5.5)
55.5 (ꢀ1.1)
51.5 (ꢀ5.1)
54.4 (ꢀ2.2)
55.2 (ꢀ1.4)
53.2 (ꢀ3.4)
43.5 (ꢀ13.1)
48.8 (ꢀ7.8)
50CCTCTTACCTCAGTD-proU2 OMeACA3 (ON4) 5416.6/5417.3
0
50CCTCTD-proTACC TCAGTD-proTACA30 (ON5) 5431.7/5429.0
50CCT CTL-proT ACC TCA GTL-proT ACA30 (ON6) 5431.7/5433.6
50CCTCTglyTACCTCAGTglyTACA30 (ON7) 5351.6/5346
cDNA 5a
55.9
56.5 (þ0.6)
50.9 (ꢀ5.0)
53.5 (ꢀ2.4)
54.5 (ꢀ1.4)
50.5 (ꢀ5.4)
mmDNA6b
50.3 (ꢀ5.6)
50.4 (ꢀ6.1)
47.7 (ꢀ3.2)
47.0 (ꢀ6.5)
45.6 (ꢀ8.9)
nd
cRNA3a
56.5
56.0 (ꢀ0.5)
50.7 (ꢀ5.8)
53.1 (ꢀ3.4)
54.3 (ꢀ1.7)
50.2 (ꢀ6.3)
mmRNA4b
48.3 (ꢀ8.2)
48.7 (ꢀ7.3)
47.9 (ꢀ2.8)
46.1 (ꢀ7.0)
47.0 (ꢀ7.3)
nd
9
50GAA GGG CTT TTG AAC TCT T30 control DNA4
10
11
12
13
14
50GAA GGG CTT TD-proTG AAC TCT T30 (ON8) 5864.9/5866.6
50GAA GGG CTT TL-proTG AAC TCT T30 (ON9) 5864.9/5868.3
50GAA GGG CTT TglyTG AAC TCT T30 (ON10) 5824.9/5819.7
50GAA GGG CTT TD-proU2 OMe G AAC TCT T30(ON11) 5880.9/5880.5
0
50GAA GGG CTD-proU2 OMe TD-proU2 OMe G AAC TCT T30(ON12) 5928.0/5926.9
0
0
cDNA2¼50 TGT AAC TGA GGT AAG AGG 30; mmDNA3¼50 TGT AAC TGA GGT ATG AGG 30; c RNA1¼50UGU AAC UGA GGU AAG AGG 30; mmRNA2¼50UGU AAC UGC GGU AAG AGG
30; cDNA5¼50AAG AGT TCA AAA GCC CTT C 30; cRNA3¼50AAG AGU UCA AAA GCC CUU C 30; mmDNA6¼50AAG AGT TCT AAA GCC CTT C 30; RNA4¼50AAG AGU UCU AAA GCC CUU
C 30; Tm¼melting temperature (measured in the buffer 10 mM sodium phosphate, 100 mM NaCl, pH¼7.2), of T-(a-amino acid)-T/U2 -OMe modified ONs:DNA/RNA complexes. The
0
values reported here are the average of three independent experiments and are accurate to ꢃ1.0 ꢂC.
a
Values in parentheses denotes the difference between the Tm of modified sequences and Tm of control sequence.
Values in the parentheses are calculated relative to fully matched duplex.
b

2446
S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
0
2.6. UV-Tm of T-(a-amino acid)-T/U2 OMe modified 18mer oligo-
nucleotides(DNA1 and ON2eON7)
both single mismatch DNA6 and RNA4 with destabilization of
6e9 ꢂC (Table 2, entries 10, 12 and 13).
The strong positive CD bands observed for the dimers Ib and Id
The binding affinity of 18 mer chimeric ONs (ON2eON8) with
complementary DNA and RNA (cDNA/cRNA) was investigated by
measuring the melting temperatures (Tm) of the duplexes and the
results are summarized in Table 2. The unmodified 18 mer DNA1
sequence forms complexes with complementary DNA2 and RNA1
with higher melting temperature for DNA:RNA complex over
containing D-pro internucleoside linkage (Fig. 2) is thus clearly in-
dicative of the fact that they have the most compatible geometry
for duplex formation and are better candidates for the replacement
of phosphodiester linkage. The intermittent replacement of the
phosphodiester linkages probably causes discontinuity of the
phosphodiester backbone, causing some destabilizing the duplexes
when more than one dimer unit is replaced. Current studies
towards synthesis of fully amide-linked backbone modified se-
quences are in progress.
DNA:DNA (
D
Tm¼þ3.8 ꢂC, Table 2, entry 1). Oligomer ON1 bearing
single T-D-pro-T dimer unit towards 30-end was found to have affinity
with DNA and RNA similar to the unmodified oligomers
(
D
Tm¼ꢀ1.1 ꢂC, Table 2, entry 2). In comparison, T-L-pro-T dimer-
bearing oligomer ON2 destabilized the duplexes (
D
Tm¼ꢀ5 to
3. Conclusion
ꢀ6 ꢂC, Table 2, entry 3). In the case of T-gly-T dimer-bearing oligomer
ON3, the destabilization of the duplex (
D
Tm¼ꢀ2.2 to ꢀ3.2 ꢂC, Table
Chiral amide-linked dimers have been synthesized and in-
corporated into the DNA backbone by partial replacement of se-
lected phosphodiester linkages by phosphoramidite method using
an automated DNA synthesizer. Melting studies indicate that the
2, entry 4) was less as compared to T-L-Proline-T dimer modified oli-
gonucleotide. The ON4:DNA2 and ON4:RNA1 duplexes containing
T-D-pro-UOMe were also slightly destabilized (
D
Tm¼ꢀ1.3 and ꢀ1.4 ꢂC,
respectively, Table 2, entry 5) and this could be because of the in-
corporation of uridine in place of thymidine,25 in spite of better base
stacking in this case as seen by CD studies.. Incorporation of two
units of the modified dimers (ON5eON7, Table 2, entries 6e8) were
found to destabilize the duplexes, however, the degree of de-
incorporation of the single dimer block containing D-proline was
well accommodated in DNA:DNA and DNA:RNA duplexes as mar-
ginal (ꢃ) effect in the UV-Tm values was observed. Incorporation of
the second dimer block, however, caused destabilization of du-
plexes, in most cases.
stabilization was less with complementary RNA1 in the case of T-D
-
proline-T modified oligomers ON5 (
compared toT-L-pro-T modified ON6 (
and T-gly-T modified oligomer ON7 (
D
Tm¼ꢀ3.4 ꢂC, Table 2, entry 6) as
4. Experimental
D
D
Tm¼ꢀ13.1 ꢂC, Table 2, entry 7)
Tm¼ꢀ7.8 ꢂC, Table 2, entry 8).
4.1. General remarks
0
The thermal stabilities of T-(a
acid)-T/U2 -OMe oligonucleo-
-amino
tides ON1eON7 with DNA3 and RNA2 containing single mismatch
in the sequence were then studied. For oligonucleotides ON1eON4,
the decrease of binding affinity was 7.8 ꢂC, 7.9 ꢂC, 8.2 ꢂC and 7.9 ꢂC,
respectively, when they are hybridized mismatch DNA3 (Table 2,
entries 2e5). Increase in destabilization is more (9e12 ꢂC), when
we studied the binding with single base mismatch RNA2 (Table 2,
entries 2e5). For ON5, 6 and 7 having double modifications, there
was a decrease of UV-melting temperature 10.9 ꢂC, 10.8 ꢂC and
12.4 ꢂC, respectively, with the single base mismatch RNA 2 (Table 2,
entries 6e8). The destabilization is less when they hybridized with
single base mismatch DNA3 (6.5 to 8.5 ꢂC, Table 2, entries 6e8).
These results clearly indicate that these chimeric oligonucleotides
hybridize with their complementary DNA/RNA through Watsone
Crick H-bonding.
All the reagents used as purchased. All solvents used were dried
and distilled according to standard protocols. Analytical TLCs were
performed on Merck 5554 silica 60 aluminium sheets. Column
chromatography was performed for purification of compounds on
silica gel (60e120 mesh, Merck). For acid-sensitive compounds, the
column packed and equilibrated with 0.5% triethylamine. TLCs were
performed using dichloromethane/methanol or petroleum ether/
ethyl acetate solvent systems. Visualization was accomplished with
UV light and/or by spraying with perchloric acid reagent and heating.
1H, 13C NMR spectra were obtained using Bruker ACF 200 (200 MHz)
or 400 (400 MHz) spectrometers and chemical shifts are reported on
d
scale in parts per million (ppm) with the solvent indicated as the
internal reference.1H NMR data are reported in the order of chemical
shift, multiplicity (s, singlet; d, doublet; t, triplet; br, broad; br s, broad
singlet; m, multiplet and/or multiple resonance), number of protons.
0
2.7. UV-Tm of T-(a-amino acid)-T/U2 OMe modified 19mer oligo-
nucleotides(DNA4 and ON8eON12)
4.2. 30-Amino-50-O-(4,40-dimethoxytrityl)-30-deoxythymidine
(1)
The duplexes of ON8 with single modified dimer block having D-
proline (T-D-pro-T) showed excellent binding efficiency with com-
A mixture of 30-azido-50-O-(4, 40-dimethoxytrityl)-30-deoxy-
thymidine (1.8 g, 3.16 mmol) and 10% palladium on charcoal
(0.180 g) in 10 mL ethyl acetate was stirred under hydrogen at-
mosphere (40 psi) at room temperature for 4 h. The reaction mix-
ture was filtered through Celite and the solvent was evaporated in
vacuo to give 30-amino-50-O-(4, 40-dimethoxytrityl)-30-deoxy-
thymidine 1 (1.46 g, 85%) as yellow foam. 1H NMR (200 MHz, CDCl3)
plementary DNA5 and RNA3 (
spectively, Table 2, entry 10), which was as good as the control
D
Tm¼þ0.6 ꢂC and ꢀ0.5 ꢂC, re-
sequence. The ON9 modified with T-L-Proline-T dimer showed de-
stabilization of the duplexes both with DNA5 and RNA3 (
D
Tm¼ꢀ5
and ꢀ5.8 ꢂC, respectively, Table 2, entry 10). The T-gly-T modified
ON10 also showed little destabilization both with DNA5
(D
Tm¼ꢀ2.4 ꢂC, Table 2, entry 12) and RNA3 (
D
Tm¼ꢀ3.4 ꢂC, Table 2,
d 7.60 (s, 1H), 7.27e7.44 (m, 10H), 6.82e6.86 (d, 4H), 6.28 (t,
entry 12), but the degree of destabilization was less as compared to
ON9. Introduction of a single T-D-pro-U20-OMe dimer unit in ON11
showed a destabilization of 1.4 ꢂC and 1.7 ꢂC with complementary
DNA5 and RNA3, respectively (Table 2, entry 13). A cumulative ef-
fect was observed in destabilization in the ON12:DNA5/RNA3 when
two units of T-D-pro-U20-OMe dimer units were incorporated (Table
2, entry 14). Overall, in ON9eON12 showed more destabilization
J¼5.67 Hz, 1H), 3.78 (8H, singlet for 6H merged with other 2H),
3.33e3.59 (m, 2H), 2.23e2.36 (m, 2H), 1.50 (s, 3H).
4.3. 50-O-(4,40-Dimethoxytrityl)-30-[(N-Fmoc-
L-prolyl)-
amino]-30-deoxythymidine (3a)
To N-Fmoc- -proline 2a (1.00 g, 2.96 mmol) in dry acetonitrile/
L
with RNA as compared to DNA. Oligomers with either
glycine containing dimer, which stabilize duplexes with DNA and
RNA, showed discrimination in mismatch binding studies with
D
-proline or
dry DMF (4:1, 5 mL), HBTU (1.34 g, 3.55 mmol), HOBt (0.20 g,
1.48 mmol) and diisopropyl ethyl amine (1.52 mL, 8.89 mmol) were
added under nitrogen atmosphere. This reaction mixture was

S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
2447
allowed to stir for 15 min. 30-Amino-50-O-(4, 40-dimethoxytrityl)-
30-deoxythymidine 1 (1.44 g, 2.66 mmol) dissolved in dry aceto-
nitrile (5 mL) was added to the reaction mixture and further stirred
for 2 h. The reaction mixture was concentrated to dryness,
dissolved in ethyl acetate (100 mL) and washed with 5% NaHCO3
solution (2ꢁ30 mL). The organic layer was dried over Na2SO4 and
concentrated. Compound was purified by column chromatography
using 1.5e2.0% methanol in dichloromethane to afford 3a (1.78 g,
78%) as a pale yellow foam. 1H NMR (200 MHz, CDCl3): 9.95 (s, 1H),
8.33 (s, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 7.65 (m, 2H), 7.25e7.41 (m,
17H), 6.81 (d, J¼8.84 Hz, 4H), 6.63 (t, J¼8.4 Hz, 1H), 4.70 (s, 1H), 4.54
(m, 1H), 4.18e4.32 (m, 2H), 4.05 (s, 1H), 3.78 (s, 6H), 3.40e3.57 (m,
4H), 2.40e2.60 (m, 1H), 2,26e2.40 (m, 1H), 2.06e2.23 (m, 2H), 1.93
(m, 2H), 1.70 (s, 3H); MS (EI) m/z 862.3578, found 885.90 (MþNaþ).
(1 mmol) in dry THF (10 mL), 1 M solution of TBAF (1.5 mmol) was
added and the reaction mixture stirred at room temperature for 1 h.
THF was removed in vacuo and the residue redissolved in CH2Cl2
(50 mL). The solution was washed with water (2ꢁ20 mL). The or-
ganic layer was dried over anhydrous Na2SO4 and evaporated to
dryness. Silica gel column chromatography using 1e1.5% methanol
in dichloromethane as eluting system gave pure product 6a/6b/6c.
4.8. 50-O-(4,40-Dimethoxytrityl)-T-L-pro-T-30-OH (6a)
Yield 84%. 1H NMR (200 MHz, CDCl3): 10.47 (s, 1H), 9.91 (s, 1H),
8.05 (s, 1H), 7.64 (s,1H), 7.28 (m, 9H), 6.81 (d, J¼8.84 Hz, 4H), 6.47 (t,
J¼6.44, 6.07 Hz), 6.30 (t, J¼6.45, 5.81 Hz, 1H), 4.68 (m, 3H), 4.53 (m,
1H), 4.05 (s, 1H), 3.78 (s, 8H), 3.43 (m, 2H), 1.90e2.50 (m, 8H), 1.84
(s, 3H),1.33 (s, 3H); MS (EI) m/z 878.3487, found 901.7669 (MþNaþ).
4.4. 50-O-(4,40-Dimethoxytrityl)-30-[(N-Fmoc-
D-prolyl)-
amino]-30-deoxythymidine (3b)
4.9. 50-O-(4,40-Dimethoxytrityl)-T-L-pro-T-30-OH (6b)
This compound was obtained in 82% yield as pale yellow foam
by an analogous method described above in the synthesis of 3a,
Yield 72%.1H NMR (500 MHz, D2Oþdrop of DMSO-d6): 10.04 (br s,
2H), 8.41 (s,1H), 7.53 (s,1H), 7.38 (d, J¼8.37 Hz, 2H), 6.85 (m, 4H), 7.27
(m, 7H), 6.36 (dd, J¼9.08, 5.23 Hz, 1H), 6.23 (t, J¼6.53, 6.60 Hz, 1H),
5.84 (d, J¼3.54 Hz, 1H), 4.65 (s, 1H), 4.54 (m, 1H), 4.41 (s, 1H), 3.95 (s,
1H), 3.70 (s, 6H), 3.66 (m,1H), 3.56 (m,1H), 3.31 (m,1H), 3.16 (m,1H),
2.35 (m,1H), 2.05e2.23 (m, 3H),1.86 (m, 3H),1.77 (m,1H),1.56 (s, 3H),
1.37 (s, 3H); MS (EI) m/z 878.3487, found 902.3909 (MþHþþNaþ).
except N-Fmoc- -proline 2b was utilized as the starting material.
D
1H NMR (200 MHz, CDCl3): 9.88 (s, 1H), 8.34 (s, 1H), 7.78 (s, 1H),
7.75 (s, 1H), 7.65 (m, 2H), 7.25e7.44 (m, 16H), 6.79e6.84 (d,
J¼8.84 Hz, 4H), 6.63 (m, 1H), 4.69 (br s, 1H), 4.54 (m, 1H), 4.20e4.32
(m, 2H), 4.05 (s, 1H), 3.78 (s, 6H), 3.40e3.57 (m, 4H), 2.49e2.64 (m,
1H), 2.11e2.39 (m, 3H), 1.86e2.06 (m, 3H), 1.63 (s, 3H). MS (EI) m/z
862.3578, found 885.87 (MþNaþ).
4.10. 50-O-(4,40-Dimethoxytrityl)-T-gly-T-30-OH (6c)
4.5. 50-O-(4,40-Dimethoxytrityl)-30-[(N-Fmoc-glycyl)-amino]-
Yield 73%. 1H NMR (200 MHz, DMSO-d6): 8.07 (s, 1H), 7.57 (s,
1H), 7.39 (d, J¼7.53 Hz, 2H), 7.27 (m, 7H), 6.90 (m, 4H), 6.39 (dd,
J¼8.28, 6.52 Hz, 1H), 6.24 (t, J¼6.53 Hz, 1H), 5.67 (d, J¼4.02 Hz, 1H),
4.51 (m, 1H), 4.41 (s, 1H), 4.33 (s, 1H), 3.93 (s, 1H), 3.81 (dd, J¼16.57,
6.05 Hz, 1H), 3.73 (s, 6H and m, 1H overlapping), 3.30 (m, 1H), 3.31
(m, 1H), 2.39 (m, 1H), 2.21 (m, 1H), 2.09 (m, 2H), 1.72 (s, 3H), 1.46 (s,
3H); MS (EI) m/z 838.3174, found 862.9564 (MþHþþNaþ).
30-deoxythymidine (3c)
This compound was obtained in 76% yield as pale yellow foam
by an analogous method described above in the synthesis of 3a,
except N-Fmoc-glycine 2c was utilized. 1H NMR (200 MHz, CDCl3):
7.92 (s, 1H), 7.74 (s, 1H), 7.70 (s, 1H), 7.53e7.62 (m, 3H), 7.19e7.40
(m, 15H), 6.77e6.82 (m, 4H), 6.40 (dd, J¼7.96, 5.68 Hz, 1H), 5.80 (br
s, 1H), 4.71 (s, 1H), 4.37 (s, 1H), 4.34 (s, 1H), 4.01e4.19 (m, 2H), 3.87
(m, 2H), 3.74 (s, 6H), 3.41 (m, 2H), 2.38 (m, 2H), 1.35 (s, 3H). MS (EI)
m/z 822.3265, found 845.87 (MþNaþ).
4.11. 50-O-(4,40-Dimethoxytrityl)-T-D-pro-UOMe-30-OH (6d)
4.11.1. 30-O-(tert-Butyldimethylsilyl)-20-OMe-uridine. 20-OMe-Uri-
dine (1.0 g, 3.87 mmol) was dissolved in dry DMF (10 mL), then TBS-
Cl (1.39 g, 9.29 mmol) and imidazole (1.30 g, 19.36 mmol) were
added under nitrogen atmosphere. The reaction mixture was stir-
red at room temperature for 6 h. DMF was removed in vacuo. The
residue was dissolved into ethyl acetate (100 mL), washed with
several portions of water, dried over Na2SO4 and on concentration
gave 30,50-di-O-(tert-butyldimethylsilyl)-20-OMe-uridine (1.50 g,
3.08 mmol, 80%) as a white solid, which then dissolved into 30 mL
methanol and pyridinium-p-toluene sulfonate (3.10g, 12.34 mmol)
was added. The reaction mixture was stirred at room temperature
for 20 h. The solvent was removed in vacuo. The residue was dis-
solved into 50 mL of ethyl acetate, washed with water (3ꢁ25 mL)
and then with brine. The organic layer dried over Na2SO4 and
concentrated under reduced pressure. The resulting residue was
chromatographed on silica gel column. The product was eluted
with 30% ethyl acetate in petroleum ether to afford the required
intermediate compound 30-O-(tert-butyldimethylsilyl)-20-OMe-
uridine (0.74 g, 65%) as a white solid. 1H NMR (200 MHz, CDCl3):
8.92 (s, 1H, exchanges with D2O), 7.67 (d, J¼8.09 Hz, 1H), 5.72 (d,
J¼8.08 Hz, 1H), 5.62 (d, J¼4.04 Hz, 1H), 4.36 (m, 1H), 3.95e4.08 (m,
3H), 3.76 (m, 1H), 3.49 (s, 3H), 2.74 (br s, 1H, exchanges with D2O),
0.94 (s, 9H), 0.12 (s, 6H); 13C NMR (50 MHz, CDCl3): 163.7, 150.3,
142.0, 102.2, 90.7, 85.2, 82.6, 69.4, 60.8, 58.4, 25.6, 18.0, ꢀ4.7, ꢀ4.8;
HRMS (EI) m/z 395.1609, found 395.1601 (MþNaþ).
4.6. Deprotection of Fmoc group, general procedure for the
synthesis of (4a, 4b and 4c)
50-O-(4,40-Dimethoxytrityl)-30-[(N-Fmoc-aminoacyl)-amino]-
30-deoxythymidine 3a/3b/3c (1.00 g) was dissolved in dry
dichloromethane (10 mL). To it, diethyl amine (10 mL) was added
and the reaction mixture was allowed to stir for 45 min.
Dichloromethane and diethyl amine were removed in vacuo and
the residue co-distilled with dichloromethane. Isolation of the
product 4a/4b/4c with free amino group was obtained by simple
precipitation using petroleum ether and used in next coupling
reaction without further purification.
4.7. General procedure for synthesis of dinucleoside 6ae6c
To 30-O-(tert-Butyldimethylsilyl)-thymidin-40-carboxylic acid 5a
(0.43 g, 1.15 mmol) in dry acetonitrile (5 mL), HBTU (0.527 g,
1.39 mmol), HOBt (0.08 g, 0.57 mmol) and DIPEA (0.6 mL,
3.47 mmol) were added and stirred for 15 min. Appropriate com-
pound 4a/4b/4c (1.15 mmol) dissolved in dry acetonitrile (5 mL)
was then added to the reaction mixture and further stirred at room
temperature for 2 h. The reaction mixture was then concentrated to
dryness, dissolved in ethyl acetate (30 mL) and washed with 5%
NaHCO3 (10 mLꢁ2). The organic layer was dried over anhydrous
Na2SO4 and concentrated to get the dinucleosides as yellow foam in
90e92% crude yield. Then, to a stirred solution of dinucleoside
4.11.2. 30-O-(tert-Butyldimethylsilyl)-20-OMe-uridine-40-carboxylic
acid (5b). TEMPO (0.073 g, 0.47 mmol) and BAIB (1.33 g, 4.14 mmol)

2448
S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
were added to the solution of 30-O-(tert-butyldimethylsilyl)
20-OMe-uridine (0.7g, 1.88 mmol) in 1:1 acetonitrile/water (15 mL).
The reaction mixture was allowed to stir at room temperature for
2 h. Acetonitrile was removed in vacuo. The reaction mixture was
diluted with ethyl acetate (30 mL) and washed with several por-
tions of water, dried over Na2SO4 and concentrated. The resulting
residue was triturated sequentially with diethyl ether and acetone,
filtered and dried in vacuo. The product obtained as white solid
(0.68 g, 93%). 1H NMR (200 MHz, CDCl3): 8.22 (d, J¼8.21 Hz, 1H),
6.11 (d, J¼6.07 Hz,1H), 5.77 (d, J¼8.08 Hz,1H), 4.46 (m,1H), 4.38 (m,
1H), 3.72 (m, 1H), 3.40 (s, 3H), 0.91 (s, 9H), 0.12 (s, 6H); 13C NMR
(50 MHz, CDCl3): 172.8, 163.9, 150.6, 141.1, 102.8, 87.7, 87.6, 82.6,
73.1, 58.2, 25.4, 17.4, ꢀ5.05, ꢀ5.10; HRMS (EI) m/z 387.1586, found
387.1582 (Mþ), 405.1395(MþNa).
2.5 mmol) and chloro (2-cyanoethoxy) (N,N-diisopropylamino)-
phosphine (0.44 mL, 2 mmol) and the reaction mixture was stir-
red at room temperature for 2 h. The contents were then diluted
with dry DCM and washed with 5% NaHCO3 solution. The organic
phase was dried over anhydrous Na2SO4 and concentrated to
foam. The residue was dissolved in DCM and precipitated with
hexane to obtain 7a/7b/7c/7d. The phosphoramidite 7a/7b/7c/7d
was dried overnight over P2O5 and KOH in a desiccator before
applying on DNA synthesizer. TLC shows two close moving spots
for two diastereomers (Rf¼0.5, 5% methanol/DCM).
4.13.1. 50-O-(4,40-Dimethoxytrityl)-T-L-pro-T-30-O-(2-cyanoethyl-N,N0-
diisopropylphosphoramidate) dimer (7a). This compound was ob-
tained in 80% yield starting from 6a.
31P NMR (400 MHz, CDCl3)
d 150.28, 148.20.
Compound 6d obtained in 75% yield as white solid foam by an
analogous method described above in the synthesis of 6ae6c, using
30-O-(tert-butyldimethylsilyl)-20-OMe-uridine-40-carboxylic acid
5b and 4b as starting materials. 1H NMR (CDCl3, 200 MHz): 8.26 (br,
1H), 7.78 (br s, 1H), 7.69 (s, 1H), 7.40 (d, J¼7.83 Hz, 2H), 7.26e7.30
(m, 8H), 6.82e6.85 (m, 4H), 6.65 (m, 1H), 5.96 (s, 1H), 5.55 (d,
J¼8.07 Hz, 1H), 4.88 (m, 1H), 4.68 (m, 1H), 4.61 (m, 1H), 4.41 (t,
J¼6.36 Hz, 1H), 4.05 (m, 2H), 3.77 (s, 6H), 3.65e3.74 (m, 2H), 3.58 (s,
3H), 3.40e3.47 (m, 2H), 2.48 (m, 1H), 2.23e2.36 (m, 2H), 2.11e2.16
(m, 2H), 1.97 (m, 1H), 1.23 (s, 3H); MS (EI) m/z 894.3436, found
917.45 (MþNaþ).
4.13.2. 50-O-(4,40-Dimethoxytrityl)-T-D-pro-T-30-O-(2-cyanoethyl-
N,N0-diisopropylphosphoramidate) dimer (7b). This compound was
obtained in 82% yield starting from 6b. 31P NMR (400 MHz, CDCl3)
d
150.37, 148.44.
4.13.3. 50-O-(4,40-Dimethoxytrityl)-T-(Glycine)-T-30-O-(2-cyanoethyl-
N,N0-diisopropylphosphoramidate) dimer (7c). This compound was
obtained in 78% yield starting from 6c.
31P NMR (400 MHz, CDCl3)
d 148.80, 148.28.
4.13.4. 50-O-(4,40-Dimethoxytrityl)-T-D-pro-UOMe-30-O-(2-cyanoethyl-
N,N0-diisopropylphosphoramidate) dimer (7d). This compound was
obtained in 72% yield as starting from 6d. 31P NMR (400 MHz,
CDCl3): 151.27, 149.91.
4.12. General procedure for deprotection of trityl group:
synthesis of 30,50-OH free dinucleosides
The 50-O-DMT-protected dinucleosides 6a/6b/6c/6d (0.2 mmol)
were dissolved in 3% dichloroacetic acid (5 mL) and 20 mL triiso-
propyl silane was added as scavenger. Reaction mixture was stirred
for ½ h. The reaction mixture was concentrated and the product
was recovered by successive trituration with acetone and diethyl
ether to afford Ia/Ib/Ic/Id as a white solid in 63e65% yield. 1H NMR
recorded in D2O on 400 MHz for these dimers and values are
reported in Table 1.
4.14. Oligonucleotides synthesis and purification
The synthesis was carried out with a DNA synthesizer (Bio-
automation Mer-Made 4 synthesizer) by the standard
b-cya-
noethyl phosphoramidite method. For the modified dimers,
double coupling (300 sꢁ2) was performed. After synthesis, the
oligonucleotides were cleaved from solid support by ammonia
treatment. The crude oligonucleotides were initially purified by
NAP filtration followed by further purification by reverse phase
high performance liquid chromatography (RP-HPLC) on a Waters
system (Waters Delta 600e quaternary solvent delivery system and
2998 photodiode array detector and Empower 2 chromatography
Software) on C18 column using with increasing gradient of ace-
tonitrile in 0.1M triethylammonium acetate buffer (pH 7.0). The
oligonucleotides were analyzed for purity by HPLC and charac-
terized by MALDI-TOF mass spectroscopy. The MALDI-TOF spectra
were recorded on Voyager-De-STR (Applied Biosystems) in-
strument and the matrix used for analysis was THAP (20,40,60-
trihydroxyacetophenone).
4.12.1. 50,30-OH Free T-L-pro-T (Ia). 13C NMR (100 MHz, CDCl3): 173.9,
170.9, 169.8, 166.7, 151.9, 151.7, 137.8, 137.4, 111.3, 111.2, 85.9, 84.6,
84.0, 83.4, 73.4, 68.3, 60.7, 48.7, 47.6, 37.9, 36.1, 29.3, 24.6, 11.6, 11.5;
MS (EI) m/z 576.2180, found 577.56 (MþHþ), 599.55 (MþNaþ);
HRMS (ESI) calcd for C25H33N6O10: 577.2258 (MþHþ), found
577.2268.
4.12.2. 50,30-OH Free T-D-pro-T (Ib). MS (EI) m/z 576.2180, found
577.48 (MþHþ), 599.47 (MþNaþ); HRMS (ESI) calcd for
C
25H33N6O10: 577.2258 (MþHþ), found 577.2263.
4.12.3. 50,30-OH Free T-gly-T (Ic). MS (EI) m/z 536.1867, found 537.44
(MþHþ), 559.42 (MþNaþ); HRMS (ESI) calcd for C22H28N6O10
:
537.1945 (MþHþ), found 537.1969.
4.15. UV melting experiments
4.12.4. 50,30-OH Free T-D-pro-U2 -OMe (Id). 13C NMR (100 MHz, CDCl3):
173.9, 169.2, 167.3, 166.8, 152.5, 151.8, 141.6, 137.4, 111.3, 102.4, 87.3,
84.6, 83.9, 82.5, 81.1, 70.7, 68.4, 61.0, 60.7, 58.0, 48.6, 47.7, 36.1, 28.3,
24.4, 11.5 (extra peaks at 171.1 and 68.3 counts for dichloroacetic
acid). MS (EI) m/z 592.2129, found 615.17 (MþNaþ); HRMS (ESI)
calcd for C25H33N6O11: 593.2207 (MþHþ), found 593.2196.
0
UV melting experiments were carried out on a Varian Cary 300
UVevis spectrophotometer fitted with a Peltier-controlled tem-
perature programmer. The experiments were performed at 1 mM
concentrations. The complexes were prepared in 10 mM sodium
phosphate buffer, pH 7.4 containing NaCl (100 mM) and were
annealed by keeping the samples at 90 ꢂC for 2 min followed by
slow cooling to room temperature and refrigeration for at least 2 h
prior to running the experiments. The melting profiles were
recorded at 260 nm from 10 to 85 ꢂC at a scan rate of 0.5 ꢂC/min. Tm
was calculated as the temperature at which the duplexes were half
dissociated, determined by taking the first derivative of the
4.13. General procedure for synthesis of phosphoramidites:
synthesis of 7a/7b/7c/7d
Compound 6a/6b/6c/6d (1 mmol) was dissolved in dry DCM
(10 mL) followed by the addition of diisopropyl ethyl amine (0.42 mL,

S. Bagmare et al. / Tetrahedron 71 (2015) 2442e2449
2449
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2697e2700; (b) Gokhale, S. S.; Gogoi, K.; Kumar, V. A. J. Org. Chem. 2010, 75,
7431e7434.
melting curve. All values are an average of at least three experi-
ments and accurate to within ꢃ0.5 ꢂC.
10. (a) Tanui, P.; Kullberg, M.; Song, N.; Chivate, Y.; Rozners, E. Tetrahedron 2010, 66,
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Acknowledgements
S.B. thanks University Grants Commission, New Delhi for senior
research fellowship. V.A.K. thanks Council of Scientific and
Industrial Research, New Delhi for research grant (BSC0123)
Supplementary data
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