Synthesis and conformational studies of amide-linked cyclic homooligomers of a thymidine-based nucleoside amino acid

Article abstract of DOI:10.1016/j.tet.2005.07.089

Cyclic homooligomers of a thymidine-based nucleoside amino acid were synthesized from the linear dimer using BOP reagent in the presence of DIPEA under dilute conditions. Conformational analysis by NMR and constrained MD studies revealed that all the cyclic products had symmetrical structures. The NH and CO groups in these molecules point in opposite directions with near perpendicular orientation with respect to the plane of the macrocyclic ring having CO on the same side as the base.

Full text of DOI:10.1016/j.tet.2005.07.089

Tetrahedron 61 (2005) 9506–9512
Synthesis and conformational studies of amide-linked cyclic
homooligomers of a thymidine-based nucleoside amino acid*
*
Tushar Kanti Chakraborty, Dipankar Koley, Sripadi Prabhakar, Rapolu Ravi
*
and Ajit Chand Kunwar
Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 30 May 2005; revised 11 July 2005; accepted 28 July 2005
Available online 18 August 2005
Abstract—Cyclic homooligomers of a thymidine-based nucleoside amino acid were synthesized from the linear dimer using BOP reagent in
the presence of DIPEA under dilute conditions. Conformational analysis by NMR and constrained MD studies revealed that all the cyclic
products had symmetrical structures. The NH and CO groups in these molecules point in opposite directions with near perpendicular
orientation with respect to the plane of the macrocyclic ring having CO on the same side as the base.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Cyclisation of linear biopolymers is widely used to
constrain their conformational degrees of freedom and
induce desirable structural biases permitting enhanced
receptor selectivity and binding affinity with additional
properties like decreased susceptibility to degradation in
biological systems.1 Cyclic DNAs and RNAs, for example,
have been studied extensively for their unusual chemical
and biological activities.2 However, synthesis of such cyclic
DNAs remains a challenging task,3 thereby limiting
exploratory studies, especially in discovering potential
leads for drug discovery. It was envisaged that the
replacement of the phosphodiester linkages with amide
bonds would not only facilitate the assembly of such
substrates using standard solid- or solution-phase peptide
synthesis methods, but would also help to enhance their
stability towards nucleases. Amide-linked oligonucleotides
have been studied extensively for potential therapeutic
applications involving antisense strategy.4 However, their
cyclic versions have remained largely unexplored. Herein,
we report the synthesis and conformational studies of
amide-linked cyclic homooligonucleotides 1 and 2, which
were prepared, as shown in Scheme 1, by cyclisation of the
Scheme 1. Reagents and conditions: (i) BOP reagent, DIPEA, DMF, 0 8C to
rt, 10 h; (ii) H2, Pd-C (10%), THF–MeOH (1:1), rt, 0.5 h.
1.1. Synthesis of the cyclic homooligomers
The starting material for our synthesis was the fully
protected monomer Taa 5a.5,6 While the tert-butoxycarbo-
nyl (Boc) group was deprotected using TFA–CH2Cl2 (1:3),
saponification of the ethyl ester was carried out with LiOH
in dioxane–water (1:1). Reaction of Boc-Taa(BOM)-OH
with H2N-Taa(BOM)-OEt using the conventional solution
phase method using N,N,N0,N0-tetramethyl-O-(benzo-
triazol-1-yl)uronium tetrafluoroborate (TBTU) and
1-hydroxybenzotriazole (HOBt) as coupling agents in
linear dimer 3 of the monomeric building block 4,4h
a
thymidine-based nucleoside amino acid (Taa).
* IICT Communication no. 050409.
Keywords: Nucleoside amino acids; Cyclic DNAs; Cyclic RNAs; Cyclic
peptides; Conformation; NMR.
*
Corresponding authors. Tel.: C91 40 27193154; fax: C91 40 27193108/
27160387; e-mail: chakraborty@iict.res.in
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.089

T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
9507
presence of N-methylmorpholine (NMM) in CH3CN gave
the protected dimer, Boc-[Taa(BOM)]2-OMe in 75% yield.
Saponification of the protected dimer was followed by
Boc-deprotection under the conditions mentioned above to
furnish the intermediate 3, which was directly subjected to
cyclisation using benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP reagent) in the
presence of N,N-diisopropylethylamine (DIPEA) in amine-
free DMF as solvent to furnish a mixture of products, 1a and
2a, in 16 and 28% yields, respectively. They were separated
by standard silica gel column chromatography and
hydrogenated using 10% Pd-C in THF–MeOH (1:1) to
furnish the BOM-deprotected products 1b and 2b in
quantitative yields. The purified products were fully
characterized by spectroscopic methods before using them
in the conformational studies.
value of [MC2Na]2C ion matches with that of [MCNa]C
ion of lower homologue, these ions were identified by
isotopic distribution patterns and also by the mass
differences between the 12C and 13C isotopic peaks, that
is, a difference of 1 Da in the case of [MCNa]C ion and
0.5 Da in the case of [MC2Na]2C ion. Further the [2MC
Na]C and [MC2Na]2C ions were confirmed by MS/MS,
which resulted in the corresponding [MCNa]C ion in
addition to the other characteristic fragment ions.8
1.2. Conformational analysis. NMR studies
The conformational analysis of 1a and 2a were carried out
by NMR spectroscopy at 500 MHz. The studies were
undertaken in 5–10 mM solution at 30 8C in CDCl3 and
DMSO-d6 for 1a, whereas structures of 2a, due to its
inadequate solubility in CDCl3, were investigated in
DMSO-d6. The presence of only one set of peaks in 1a
and 2a is consistent with a two and four fold symmetry,
respectively, in the NMR time frame.
All the products were characterized by positive ion
electrospray ionization (ESI) mass spectra.7 The spectra
showed the expected [MCNa]C ion to confirm the
molecular weight of the product, and sometimes [2MC
Na]C ion was also found. Interestingly, the spectra of some
of the products showed [MC2Na]2C. Although, the m/z
While extensive decoupling experiments and simulations
of the spectra were used to obtain the couplings, the
1
Table 1. H NMR chemical shifts (d, ppm) and coupling constants (J, Hz) of 1a (CDCl3, 500 MHz, 30 8C)
Ring protons
Base protonsa
H100
6.15 (t, JZ6.9 Hz)
C5-Me
H6
H7
2.01 (s)
7.14 (s)
2.27 (ddd, JZ6.2, 10.2, 14.3 Hz)
1.71 (ddd, JZ6.6, 7.6, 14.3 Hz)
4.32 (dddd, JZ6.6, 8.2, 8.3, 10.2 Hz)
3.55 (ddd, JZ3.2, 8.2, 11.0 Hz)
2.37 (dddd, JZ1.8, 11.0, 12.2, 13.5 Hz)
2.07 (dddd, JZ2.0, 3.2, 7.1, 13.5 Hz)
2.33 (ddd, JZ1.8, 7.1, 14.3 Hz)
2.03 (ddd, JZ2.0, 12.2, 14.3 Hz)
H2
H200
H30
H40
H50
H5000
H6
5.68 (d, JZ10.2 Hz)
5.47 (d, JZ10.2 Hz)
4.72 (d, JZ11.6 Hz)
4.70 (d, JZ11.6 Hz)
6.55 (d, JZ8.3 Hz)
7.29–7.40 (m)
0
H7
H9
0
H9
NH
Ph
H600
a H7,70; H9,90 and Ph protons are from BOM groups.
1
Table 2. H NMR Chemical shifts (d, ppm) and coupling constants (J, Hz) of 1a (DMSO-d6, 500 MHz, 30 8C)
Ring protons
Base protonsa
H10
H20
H200
H30
H400
H5
H500
H60
H600
6.04 (t, JZ6.4 Hz)
2.30 (ddd, JZ5.7, 9.5, 13.7 Hz)
1.99 (m)
C5-Me
H6
H7
H70
H9
H90
NH
Ph
1.86 (s)
7.52 (s)
5.34 (d, JZ9.8 Hz)
5.32 (d, JZ9.8 Hz)
4.58 (br s)
4.58 (br s)
7.80 (d, JZ7.8 Hz)
7.25–7.35 (m)
4.10 (m)
3.71 (ddd, JZ3.5, 7.9, 10.0 Hz)
2.12 (m)
1.95 (m)
2.24 (m)
2.12 (m)
a H7,70; H9,90 and Ph protons are from BOM groups.
1
Table 3. H NMR chemical shifts (d, ppm) and coupling constants (J, Hz) of 2a (DMSO-d6, 500 MHz, 30 8C)
Ring protons
Base protonsa
H100
6.15 (t, JZ6.7 Hz)
2.30 (ddd, JZ14.2, 9.1, 5.7 Hz)
2.15 (m)
C5-Me
H6
H7
H70
H9
H90
NH
Ph
1.86 (s)
7.57 (s)
5.33 (d, JZ9.8 Hz)
5.31 (d, JZ9.8 Hz)
4.58 (S)
4.58 (S)
8.12 (d, JZ8.0 Hz)
7.24–7.34 (m)
H2
H200
H30
H40
H500
H500
H6
4.25 (m)
3.60 (ddd, JZ8.6, 7.0, 4.2 Hz)
1.95 (m)
1.86 (m)
2.26 (ddd, JZ5.8, 9.1, 14.9 Hz)
2.16 (m)
H600
a H7,70; H9,90 and Ph protons are from BOM groups.

9508
T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
assignments were carried out with the help of DQFCOSY
experiments,9 and ROESY experiments10 provided the
information on the proximity of protons. The spectral
parameters are given in Tables 1–3. Some of the important
long-range NOEs seen in the ROESY spectra of 1a and 2a
are shown in Figures 1 and 3, respectively.
Figure 3. Schematic representation of some of the diagnostic long-range
NOEs seen in the ROESY spectrum of 2a in DMSO-d6.
Figure 1. Schematic representation of some of the diagnostic long-range
NOEs seen in the ROESY spectrum of 1a in CDCl3.
The cross-peak intensities in the ROESY spectra were used
for obtaining the restraints in the molecular dynamics (MD)
calculations13 on 1a. Molecular dynamics calculations were
carried out using Sybyl 6.8 program on a Silicon Graphics
O2 workstation. The Tripos force field, with default
parameters, was used throughout the simulations. The
detailed protocol of the MD calculations is provided in the
Section 4. Figure 2 depicts the ensemble of the backbone-
superimposed structures of the 20 samples, collected during
600 ps simulated annealing protocol, which clearly shows
the proposed structure of the molecule. The average pair
The conformational analysis of 1b and 2b could not be
carried out due to line-broadening and overlapping signals
both in CDCl3 and DMSO-d6, making it very difficult to
derive the spectral parameters.
1.3. Conformational analysis of 1a
The spectral data of 1a suggested that its 12-membered
macrocyclic ring was very rigid. The vicinal couplings,
14
˚
3
3
3
wise backbone RMSD for the structures is 0.24C0.15 A.
0
0
0
00
00
00
JH4 –H5 Z11.0 Hz, JH5 –H6 Z12.2 Hz, JH5 –H6 Z2.0 Hz,
3
3
0
0
0
00
JH5 –H6 Z1.8 Hz, JH4 –H5 Z3.2 Hz are consistent with
1.4. Conformational analysis of 2a
values of about 608and K608 for C4–C5–C6–CO and
C3–C4–C5–C6, respectively. This is further supported by
the NOE correlations H404H600 and H304H50 shown in
Figure 1. The resulting structure had the NH and CO
pointing approximately perpendicular to the plane of the
macrocyclic ring with CO on the same side as the base. The
information on the sugar pucker was derived with the help
of PSUEROT programme,11 which indicates that the sugar
ring takes a single 4OT conformation12 with PZ69.2 and
nmaxZ39.68. In nucleosides O4 T pucker is in between the C20
endo and C30 endo sugar puckerings, which are the lowest
energy conformations. The structure is consistent with the
NOEs between H204H50, H104H40 and H304H50
(Fig. 1). The information on the orientation of the base
was obtained from the distinct NOEs between the base
proton, H6 and sugar protons. The presence of NOEs
between H204H6, H304H6, and H504H6 very clearly
supported the presence of anti conformation of the base. Yet
the NOE correlation H104H6 implies a significant
population of molecule with syn conformation. Such a
situation is often encountered in nucleosides and nucleo-
tides where both syn and anti conformations12 are observed
in solution.
For 2a, though the chemical shift values were obtained from
the DQFCOSY spectra, it was not possible to derive all the
couplings due to spectral complexity and overlap. However,
ROESY data showed NOEs (Fig. 3), which were similar to
those for 1a, suggesting a very similar structure for the
tetramer.
Few of the couplings, which could be obtained, as well as
weaker NOEs, point towards averaging of the spectral
parameters, which may arise due to several other
conformations contributing to the structure, due to the
larger macrocyclic ring. The predominant structure,
however, resembles that of 1a. The NOEs between the
base proton, H6 and the protons in the sugar ring, are
consistent with the presence of both syn and anti
conformation, with the latter being dominant.
Cyclic homooligomers of nucleoside amino acids constitute
a new class of novel molecular entities that display
interesting 3-D structures, reminiscent of the structures of
peptide nanotubes. The NH and CO groups in these
molecules point in opposite directions with near
Figure 2. Stereo view of the 20 superimposed energy-minimized structures of 1a sampled during 100 cycles of the 600 ps constrained MD simulations
following the simulated annealing protocol. For clarity the protons (except amide protons) and the BOM groups are not shown.

T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
9509
perpendicular orientation with respect to the plane of the
macrocyclic ring having CO on the same side as the base.
This study will be useful in creating various de novo amide-
linked cyclic homo- as well as heterooligomers using other
nucleoside amino acids as well. The well-defined structures
of these macrocyclic peptides will be useful to carry out
investigations into many interesting molecular recognition
processes, especially those involving base-pairing and may
find many applications similar to those exhibited by cyclic
DNAs and RNAs.
workstation. The Tripos force field, with default parameters,
was used throughout the simulations.
A dielectric constant of 47 Debye for DMSO solvent was
used in all minimizations as well as in MD runs.
Minimizations were done first with steepest decent,
followed by conjugate gradient methods for a maximum
of 2000 iterations each or RMS deviation of 0.005 kcal/mol,
whichever was earlier. The energy-minimized structures
were then subjected to MD studies. A number of inter
atomic distance constraints (more than three bond away)
were used in the MD studies that were derived from the rOe
cross-peaks from the ROESY spectrum. Distance con-
straints have been obtained from 0.3 s mixing time ROESY
experiments by using the volume integral and two-spin
2. Experimental
2.1. General procedures
˚
approximation. Force constant of 15 kcal/A were applied in
All reactions were carried out in oven or flame-dried
glassware with magnetic stirring under nitrogen atmosphere
using dry, freshly distilled solvents, unless otherwise noted.
Reactions were monitored by thin-layer chromatography
(TLC) carried out on 0.25 mm silica gel plates with UV
light, I2, 7% ethanolic phosphomolybdic acid-heat and 2.5%
ethanolic anisaldehyde (with 1% AcOH and 3.3% concd
H2SO4)-heat as developing agents. Silica gel finer than 200
mesh was used for flash column chromatography. Yields
refer to chromatographically and spectroscopically homo-
geneous materials unless otherwise stated. Melting points
are uncorrected. IR spectra were recorded as KBr pellets on
FT-IR. Mass spectra were obtained under liquid secondary
ion mass spectrometric (LSIMS) and electrospray ionisation
(ESI) techniques. Optical rotations were measured with a
digital polarimeter.
the form of flat bottom potential well with the lower and
upper bounds obtained by subtracting and adding 10% to the
distances obtained above. These constraints are given in
Table 4.13
Table 4. NOE constraints used in MD simulation study of compound 1a
S.no.
From
To
Upper
bound
Lower
bound
H20
H30
H50
H30
H10
NH
1
2
3
4
5
6
H6
H6
H6
H50
H40
H40
2.713
2.868
3.425
2.860
2.496
3.439
2.220
2.347
2.802
2.340
2.042
2.810
No H-bonding constraint was used. The energy-minimized
structures were subjected to constrained MD simulations for
duration of 600 ps using 100 cycles, each of 6 ps period, of
the simulated annealing protocol. The atomic velocities
were applied following Boltzmann distribution about the
center of mass, to obtain a starting temperature of 700 K.16
2.2. Details of NMR studies
NMR spectra were recorded using a 500 MHz spectrometer
at 30 8C with 5–10 mM solutions in appropriate solvents
using TMS as internal standard and the solvent signals as
secondary standards and the chemical shifts (d) are shown in
ppm. Multiplicities of NMR signals are designated as s
(singlet), d (doublet), t (triplet), q (quartet), br (broad), m
(multiplet, for unresolved lines), etc. 13C NMR spectra were
recorded at 75 and 125 MHz with complete proton
decoupling. The proton chemical shift assignments were
carried out with the help of two-dimensional double
quantum filtered correlation spectroscopy (DQFCOSY)9
and nuclear Overhauser effect spectroscopy (NOESY)/
rotating frame nuclear Overhauser effect spectroscopy
(ROESY) experiments,10 the later also provided the
information on the proximity of protons. All the experi-
ments were carried out in the phase sensitive mode.15 The
spectra were acquired with 2!256 or 2!192 free induction
decays (FID) containing 8–16 transients with relaxation
delays of 1.0–1.5 s. The ROESY experiments were
performed with mixing time of 0.3 s and a spin-locking
field of about 2.5 kHz was used. The two-dimensional data
were processed with Gaussian apodization in both the
dimensions.
After simulating for 1 ps at high temperature, the system
temperature was reduced exponentially over a 5 ps period to
reach a final temperature of 300 K. Structures were sampled
after every five cycles, leading to an ensemble of total 20
structures. The sampled structures were energy-minimized
without constraints, by using the above-mentioned protocol
and the superimposed structures obtained by backbone
alignment are shown in the paper, in Figure 2. To determine
the backbone and the average pair-wise heavy atom RMSD,
the structures were analyzed using the MOLMOL
program.14
2.3.1. Synthesis of 5a. To a solution of Boc-Taa-OEt (5b,
1.21 g, 2.94 mmol) in CH3CN (10 mL) at room temperature
was added DBU (1.32 mL, 8.83 mmol) with stirring. After
5 min at the same temperature, BOM-Cl (0.61 mL,
4.41 mmol) was added to it and stirring continued for 1 h.
The reaction was quenched with saturated aqueous NH4Cl
solution, extracted with EtOAc. The combined organic
extracts were washed with brine, dried (Na2SO4) and
concentrated in vacuo. Purification by column chromato-
graphy (SiO2, 28–30% EtOAc in petroleum ether eluant)
gave the desired compound 5a (1.32 g, 85%) as a white
semisolid. Data for 5a: RfZ0.4 (silica gel, 30% EtOAc in
2.3. Details of molecular dynamics studies
Molecular mechanics/dynamics calculations were carried
out using Sybyl 6.8 program on a Silicon Graphics O2

9510
T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
petroleum ether); [a]D26 C42.1 (cZ5.5 in CHCl3); IR (neat)
nmax 3373, 2927, 1710, 1660, 1523 cmK1 1H NMR
(20) [MCH]C; HRMS (LSIMS) calcd for C47H60N6O13Na
939.4116, found 939.4089.
;
(200 MHz, CDCl3) d 7.32–7.28 (m, 5H, aromatic), 7.13 (s,
1H, H6), 6.16 (t, JZ6.0 Hz, 1H, H10), 5.48 (s, 2H, N–CH2),
4.70 (m, 3H, BocNH and Ph–CH2), 4.14 (q, JZ7.15 Hz, 2H,
ester CH2), 3.98 (m, 1H), 3.72 (m, 1H), 2.50 (m, 2H), 2.25
(m, 2H), 2.15 (m, 2H), 1.95 (s, 3H, C5–CH3) 1.45 (s, 9H,
Boc), 1.26 (t, JZ7.15 Hz, 3H, ester-CH3); 13C NMR
(75 MHz, CDCl3) d 172.8, 163.1, 155.2, 150.7, 137.8,
133.6, 128.1, 127.4, 110.4, 84.3, 83.2, 79.9, 77.2, 72.1, 70.4,
60.4, 53.7, 38.1, 30.5, 28.5, 28.2, 14.0, 13.1; MS (LSIMS)
m/z (%) 532 (16) [MCH]C; HRMS (LSIMS) calcd for
C27H37N3O8Na 554.2478, found 554.2498.
To a solution of Boc-[Taa(BOM)]2-OEt (295 mg,
0.321 mmol) in dioxane–water (4 mL, 1:1) at 0 8C was
added LiOH–H2O (40.5 mg, 0.965 mmol) with stirring.
Stirring was continued for 3 h as the temperature was
allowed to rise slowly from 0 8C to room temperature. Then
the reaction mixture was neutralized by DOWEX 50!80–
100 ion exchange acidic resin and filtered. The filtrate was
concentrated in vacuo. The mixture was then diluted with
CH2Cl2, washed with brine, dried (Na2SO4), filtered and
concentrated in vacuo to obtain the acid Boc-[Taa(BOM)]2-
OH. The acid was dissolved in CH2Cl2 (3 mL) To this
solution was added trifluoroacetic acid (0.75 mL) with
stirring at 0 8C. Stirring was continued for 3 h as the
temperature was allowed to rise slowly from 0 8C to room
temperature. The reaction mixture was then concentrated in
vacuum to give the TFA salt of the crude acid, 3, which was
used directly in the next step.
2.3.2. Synthesis of 3. To a solution of 5a (334 mg,
0.629 mmol) in dioxane–water (6 mL, 1:1) at 0 8C was
added LiOH–H2O (79.2 mg, 1.88 mmol) with stirring and
the temperature was allowed to rise from 0 8C to room
temperature over 3 h. Then the reaction mixture was
neutralized by DOWEX 50!80–100 ion exchange acidic
resin and filtered. The filtrate was concentrated in vacuo.
The mixture was then diluted with CH2Cl2, washed with
brine, dried (Na2SO4), filtered and concentrated in vacuo to
obtain the acid Boc-[Taa(BOM)]-OH.
2.3.3. Synthesis of 1a and 2a. To the TFA salt of the crude
acid 3 in amine free dry DMF (32.1 mL, 10K2 M) was
added BOP reagent (156.5 mg, 0.353 mmol) at 0 8C and the
reaction mixture was stirred for 15 min. This was followed
by the slow addition of DIPEA (0.27 mL, 1.6 mmol) to the
reaction mixture and stirring was continued for 10 h at room
temperature. Evaporation of the DMF under reduced
pressure gave a residue that was dissolved in CH2Cl2,
washed with saturated aqueous NH4Cl solution, saturated
aqueous NaHCO3 solution, brine, dried (Na2SO4) and
concentrated in vacuum. The crude was purified by column
chromatography (SiO2, 3.8–4.0% MeOH in CHCl3 as
eluant) to afford the cyclic dimer 1a (39 mg, 16%) and the
cyclic tetramer 2a (139 mg, 28%) as solids. Data for 1a:
RfZ0.24 (silica gel, 7% MeOH in CHCl3); [a]2D6 C17.5
(cZ0.95 in CHCl3); IR (KBr) nmax 3291, 2924, 2854, 1713,
1650, 1547 cmK1; 1H NMR (CDCl3, 500 MHz) see Table 1;
1H NMR (DMSO-d6, 500 MHz) see Table 2; 13C NMR
(75 MHz, CDCl3) d 170.7, 163.1, 151.3, 137.1, 133.7,
128.6, 128.2, 111.7, 83.0, 82.4, 73.0, 71.0, 51.6, 39.0, 31.3,
29.6, 27.9, 13.2; MS (ESI) m/z (%) 793 (100) [MCNa]C,
810 (20) [MCK]C; HRMS (ESI) calcd for C40H46N6O10Na
793.3173, found 793.3211. Data for 2a: RfZ0.32 (silica gel,
7% MeOH in CHCl3), [a]2D6 C30.6 (cZ0.76 in CHCl3), IR
In another round bottom flask, a solution of 5a (341 mg,
0.692 mmol) in CH2Cl2 (4 mL) was taken. To this solution
was added trifluoroacetic acid (1 mL) with stirring at 0 8C.
Stirring was continued for 2 h as the temperature was
allowed to rise slowly from 0 8C to room temperature. The
reaction mixture was then concentrated in vacuo to give
TFA [Taa(BOM)]-OEt.
The crude acid Boc-[Taa(BOM)]-OH was dissolved in
CH3CN (4 mL) and NMM (0.07 mL, 0.619 mmol) was
added. Then it was sequentially treated with HOBT$H2O
(42.5 mg, 0.314 mmol) and TBTU (222 mg, 0.619 mmol) at
room temperature. After 30 min, compound TFA
[Taa(BOM)]-OEt dissolved in CH3CN (4 mL) containing
NMM (0.1 mL, 0.963 mmol) was added to the reaction
mixture at room temperature. After being stirred for 12 h at
room temperature, the reaction mixture was quenched with
saturated aqueous NaH2PO4 solution and the solvent was
evaporated under reduced pressure. The water phase was
extracted with CH2Cl2, washed with water, brine, dried
(Na2SO4), concentrated in vacuo. Purification by column
chromatography (SiO2, 1.8% MeOH in CHCl3 as eluant)
afforded the protected linear dimer Boc-[Taa(BOM)]2-OEt
(432 mg, 75%). Data for Boc-[Taa(BOM)]2-OEt: RfZ0.45
(silica gel, 3% MeOH in CHCl3); [a]2D6 C36.2 (cZ1.9 in
1
(KBr) nmax 3296, 2924, 2854, 1713, 1650, 1547 cmK1; H
NMR (DMSO-d6, 500 MHz) see Table 3; 13C NMR
(125 MHz, DMSO-d6) d 170.9, 162.1, 150, 137.5, 135.1,
127.6, 126.8, 126.7, 108.6, 83.4, 81.9, 70.4, 69.7, 51.3, 35.6,
31.5, 28.4, 12.0; MS (ESI) m/z (%) 1563 (100) [MCNa]C;
HRMS (ESI) calcd for C80H92N12O20Na 1562.6370, found
1562.6345.
CHCl3); IR (KBr) nmax 3348, 2975, 1711, 1665, 1531 cmK1
;
1H NMR (400 MHz, CDCl3) d 7.3–7.17 (m, 10H, aromatic),
7.08 (s,02H, H6), 6.93 (d, JZ7.15 Hz, 1H, N–H), 6.08 (m,
2H, H1 ), 5.51 (d, JZ7.15 Hz, 1H, BocNH), 5.41 (s, 4H,
N–CH2), 4.64 (s, 4H, Ph–CH2), 4.19 (m, 1H), 4.1 (m, 2H),
3.95 (m, 1H), 3.72 (m, 2H), 2.5–2.4 (m, 2H), 2.38–2.08 (m,
10H), 1.97 (s, 3H, one of the C5-methyls), 1.95 (s, 3H, other
C5–CH3) 1.42 (s, 9H, Boc), 1.26 (t, JZ7.15 Hz, 3H, ester-
CH3); 13C NMR (75 MHz, CDCl3) d 173.1, 172.3, 163.1,
155.4, 150.7, 137.8, 133.8, 133.6, 128.1, 127.5, 110.5, 84.7,
84.4, 83, 80.1, 77.1, 72.1, 70.5, 60.5, 53.8, 52.4, 37.7, 32.4,
30.3, 29.1, 28.5, 28.2, 14.0, 13.1; MS (LSIMS) m/z (%) 918
2.3.4. Synthesis of 1b. To a solution of 1a (30 mg,
0.038 mmol) in THF–MeOH (2 mL, 1:1) was added 10%
Pd on C (10 mg). The mixture was hydrogenated under
atmospheric pressure using of a H2-filled balloon for
30 min. The reaction mixture was filtered through a short
pad of Celite, and the filter cake was washed with MeOH.
The filtrate and the washings were combined and
concentrated in vacuum to get a quantitative yield of 1b
(20 mg). Data for 1b: RfZ0.18 (silica gel, 20% MeOH in
CHCl3), [a]2D6 K21.9 (cZ0.47 in DMSO), IR (KBr) nMax

T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
9511
3421, 1664, 1562 cmK1; 1H NMR (500 MHz, DMSO-d6) d
11.06 (s, 1H, NH of thymine), 8.24 (br s, 1H, CONH), 7.46
(s, 1H, H6), 6.06 (d, JZ6.6 Hz, H10), 3.91 (m, 1H, H30),
3.77 (m, 1H, H40), 2.35–1.92 (m, 6H), 1.79 (s, 3H, 5-Me);
13C NMR (75 MHz, DMSO-d6) d 170.9, 163.6, 150.2,
136.0, 109.6, 82.4, 79.9, 52.2, 37.2, 30.5, 28.5, 11.9; MS
(ESI) m/z (%) 553 (100) [MCNa]C; HRMS (ESI) calcd for
C24H30N6O8Na 553.2022, found 553.2043.
Neamati, N.; Sunder, S.; Jaworska-Maslanka, M.; Wickstrom,
E.; Zeng, F.; Jones, R. A.; Mandes, R. F.; Chenault, H. K.;
Pommier, Y. Mol. Pharmacol. 1997, 51, 567–575. (l) Kool,
E. T. Acc. Chem. Res. 1998, 31, 502–510. (m) Escaja, N.;
Pedroso, E.; Rico, M.; Gonzalez, C. J. Am. Chem. Soc. 2000,
122, 12732–12742. (n) Li, T.; Liu, D.; Chen, J.; Lee, A. H. F.;
Qi, J.; Chan, A. S. C. J. Am. Chem. Soc. 2001, 123,
12901–12902.
3. (a) Hsu, C.-Y. J.; Dennis, D.; Jones, R. A. Nucleosides
Nucleotides 1985, 4, 377–389. (b) Barbato, S.; De Napoli, L.;
Mayol, L. Tetrahedron Lett. 1987, 28, 5727–5728. (c)
Capobianco, M. L.; Carcuro, A.; Tondelli, L.; Garbesi, A.;
Bonara, G. M. Nucleic Acids Res. 1990, 18, 2661–2669.
(d) Ross, P.; Mayer, R.; Weinhouse, H.; Amikam, D.;
Huggirat, Y.; Benziman, M.; de Vroom, E.; Fidder, A.; de
Paus, P.; Sliedregt, L. A. J.; van der Marel, G. A.; van Boom,
J. H. J. Biol. Chem. 1990, 265, 18933–18943. (e) De Napoli,
L.; Montesarchio, D.; Picciall, G.; Santacroce, C.; Mayol, L.;
Galeone, A.; Messere, A. Gazz. Chim. Ital. 1991, 121,
505–508. (f) Battistini, C.; Fustinoni, S. Tetrahedron 1993,
49, 1115–1132. (g) Zeng, F.; Jones, R. A. Nucleosides
Nucleotides 1996, 15, 1679–1686. (h) Alazzouzi, E.; Escaja,
N.; Grandas, A.; Pedroso, E. Angew. Chem., Int. Ed. 1997, 36,
1506–1508. (i) Frieden, M.; Grandas, A.; Pedroso, E. Chem.
Commun. 1999, 1593–1594. (j) Smietana, M.; Kool, E. T.
Angew. Chem., Int. Ed. 2002, 41, 3704–3707. (k) Smietana,
M.; Johnson, R. B.; Wang, Q. M.; Kool, E. T. Chem. Eur. J.
2004, 10, 173–181.
2.3.5. Synthesis of 2b. Compound 2b was synthesized from
2a in quantitative yield following the same procedure
described above for the synthesis of 1b. Data for 2b: RfZ
0.17 (silica gel, 20% MeOH in CHCl3), [a]2D6 C57.1 (cZ
0.24 in DMSO); IR (KBr) nMax 3434, 2927, 1700,
1
1551 cmK1; H NMR (500 MHz, DMSO-d6) d 11.28 (s,
1H, NH of thymine), 8.19 (d, JZ9.1 Hz, 1H, CONH), 7.50
(s, 1H, H6), 6.10 0(d, JZ6.9 Hz, 1H, H10), 4.23 (m, 1H, H30),
3.55 (m, 1H, H4 ), 2.33–1.86 (m, 6H), 1.81 (s, 3H, 5-Me);
13C NMR (75 MHz, DMSO-d6) d 171.4, 163.6, 150.3,
136.1, 109.8, 82.9, 82.3, 51.8, 36.1, 32.0, 29.0, 11.9; MS
(ESI) m/z (%) 1083 (20) [MCNa]C; HRMS (ESI) calcd for
C48H60N12O16Na 1083.4147, found 1083.4190.
Acknowledgements
We thank CSIR, New Delhi for research fellowships (D.K.
and R.R.) and DST, New Delhi for financial support.
4. (a) Lebreton, J.; Mesmaeker, A. D.; Waldner, A.; Fritsch, V.;
Wolf, R. M.; Freiser, S. M. Tetrahedron Lett. 1993, 34,
6383–6386. (b) Lebreton, J.; Waldner, A.; Fritsch, V.; Wolf,
R. M.; Mesmaeker, A. D. Tetrahedron Lett. 1994, 35,
5225–5228. (c) Mesmaeker, A. D.; Lebreton, J.; Waldner,
A.; Fritsch, V.; Wolf, R. M. Bioorg. Med. Chem. Lett. 1994, 4,
873–878. (d) Mesmaeker, A. D.; Waldner, A.; Lebreton, J.;
Hoffmann, P.; Fritsch, V.; Wolf, R. M.; Freier, S. M. Angew.
Chem., Int. Ed. Engl. 1994, 33, 226–229. (e) Bergmann, F.;
Bannwarth, W.; Tam, S. Tetrahedron Lett. 1995, 36,
6823–6826. (f) Waldner, A.; Mesmaeker, A. D.; Wendenborn,
S. Bioorg. Med. Chem. Lett. 1996, 6, 2363–2366.
(g) Mesmaeker, A. D.; Lesueur, C.; Bevierre, M.-O.; Fritsch,
V.; Wolf, R. M. Angew. Chem., Int. Ed. Engl. 1996, 35,
2790–2794. (h) Fujii, M.; Yamamoto, K.; Hidaka, J.
Tetrahedron Lett. 1997, 38, 417–420. (i) Mesmaeker, A. D.;
Leberton, J.; Jouanno, C.; Fritsch, V.; Wolf, R. M.;
Wendeborn, S. Synlett 1997, 1287–1290. (j) Goodnow,
R. A., Jr.; Richou, A.-R.; Tam, S. Tetrahedron Lett. 1997,
38, 3195–3198. (k) Goodnow, R. A., Jr.; Tam, S.; Pruess,
D. L.; McComas, W. W. Tetrahedron Lett. 1997, 38,
3199–3202. (l) Robins, M.; Doboszewski, B.; Nilsson, B. L.;
Peterson, M. A. Nucleosides Nucleotides Nucleic Acids 2000,
19, 69–86. (m) Wilds, C. J.; Minasov, G.; Natt, F.; von Matt,
P.; Altmann, K.-H.; Egli, M. Nucleosides, Nucleotides Nucleic
Acids 2001, 20, 991–994. (n) Li, X.; Zhan, Z.-Y.J.; Knipe, R.;
Lynn, D. G. J. Am. Chem. Soc. 2002, 124, 746–747. (o) Li, X.;
Lynn, D. G. Angew. Chem., Int. Ed. 2002, 41, 4567–4569.
5. Compound 5a was synthesized from 5b, which was prepared
from 30-Azido-30-deoxythymidine (AZT) by a slight modifi-
cation of the reported procedure [Ref. 4h], in which the
oxidation of AZT and olefination of the resulting aldehyde was
accomplished in an one-pot process using iodoxybenzoic acid
(IBX) in the presence of stabilized ylide, Ph3PZCHCO2Et
(Ref. 6). The resulting product A was then transformed into 5b
References and notes
1. (a) Deber, C. M.; Madison, V.; Blout, E. R. Acc. Chem. Res.
1976, 9, 106–113. (b) Milner-White, E. J. Trends Pharmacol.
Sci. 1989, 10, 70–74. (c) Fairlie, D. P.; Abbenante, G.; March,
D. R. Curr. Med. Chem. 1995, 2, 654–686. (d) Cuniasse, P.;
Raynal, I.; Yiotakis, A.; Dive, V. J. Am. Chem. Soc. 1997, 119,
5239–5248. (e) Cavallaro, V.; Thompson, P.; Hearn, M.
J. Pept. Sci. 1998, 4, 335–343. (f) Benito, J. M.; Blanco,
´
J. L. J.; Mellet, C. O.; Fernandez, J. M. G. Angew. Chem., Int.
Ed. 2002, 41, 3674–3676. (g) Fan, L.; Hindsgaul, O. Org. Lett.
2002, 4, 4503–4506. (h) Jaunzems, J.; Oelze, B.; Kirschning,
A. Org. Biomol. Chem. 2004, 2, 3448–3456. (i) Kazmaier, U.;
Hebach, C.; Watzke, A.; Maier, S.; Mues, H.; Volker Hu, V.
Org. Biomol. Chem. 2005, 3, 136–145.
2. (a) Hsu, C.-Y.L.; Dennis, D. Nucleic Acids Res. 1982, 10,
5637–5647. (b) Ulanovsky, L.; Bodner, M.; Trifonov, E. N.;
Choder, M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 862–866.
(c) Ross, P.; Weinhouse, H.; Aloni, Y.; Michaeli, D.;
Weinberg-Ohana, P.; Mayer, R.; Braun, S.; de Vroom, E.;
van der Marel, G. A.; van Boom, J. H.; Benziman, M. Nature
1987, 325, 279–290. (d) Rao, M. V.; Reese, C. B. Nucleic
Acids Res. 1989, 17, 8221–8239. (e) Rumney, S., IV; Kool,
E. T. Angew. Chem., Int. Ed. Engl. 1992, 31, 1617–1619.
(f) Prakash, G.; Kool, E. T. J. Am. Chem. Soc. 1992, 114,
3523–3527. (g) Gamper, H. B.; Reed, M. W.; Cox, T.; Virosco,
J. S.; Adams, A. D. Nucleic Acids Res. 1993, 21, 145–150.
(h) Diener, T. O. Trends Microbiol. 1993, 1, 289–294.
(i) Rubin, E.; Rumney, S.; Kool, E. T. Nucleic Acids Res. 1995,
23, 3547–3553. (j) Kool, E. T. Annu. Rev. Biophys. Biomol.
Struct. 1996, 25, 1–28. (k) Mazumder, A.; Uchida, H.;

9512
T. K. Chakraborty et al. / Tetrahedron 61 (2005) 9506–9512
following the earlier reported steps (Ref. 4h). Treatment of 5b
with BOM-Cl in the presence of DBU gave 5a in 85% yield
9. (a) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G., III;
Skelton, N. J. Protein NMR Spectroscopy; Academic: San
Diego, 1996. (b) Wu¨thrich, K. NMR of Proteins and Nucleic
Acids; Wiley: New York, 1986.
10. Hwang, T. L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114,
3157–3159.
11. de Leeuw, F. A. A. M.; Altona, C. Quant. Chem. Progr. Exch.,
No. 463, University of Indiana at Bloomington, 1983.
12. Saenger, W. Principles of Nucleic Acid Structure; Springer:
New York, 1984.
13. Kessler, H.; Griesinger, C.; Lautz, J.; Mu¨eller, A.; van
Gunsteren, W. F.; Berendsen, H. J. C. J. Am. Chem. Soc.
1988, 110, 3393–3396.
14. Etezady-Esfarjani, T.; Hilty, C.; Wu¨thrich, K.; Rueping, M.;
Schreiber, J.; Seebach, D. Helv. Chim. Acta 2002, 85,
1197–1209.
6. Lerner, C.; Siegrist, R.; Schweizer, E.; Diederich, F.;
Gramlich, V.; Jakob-Roetne, R.; Zurcher, G.; Borroni, E.
Helv. Chim. Acta 2003, 86, 1045–1062.
15. States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson.
1982, 48, 286–292.
16. For details see: (a) Sybyl Force Field Manual, version 6.6,
1999, pp 217—229 (http://www.tripos.com/custResources/
applicationNotes/triad/index.html). (b) Evans, J. N. S. Bio-
molecular NMR Spectroscopy; Oxford University Press:
Oxford, 1995.
7. (a) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem.
1987, 59, 2642–2646. (b) Iribane, J. V.; Thomson, B. A.
J. Chem. Phys. 1976, 64, 2287–2294.
8. Cole, R. B. Electrospray Ionization Mass Spectrometry;
Wiley: New York, 1997.