Chimeric (aeg-pyrrolidine)PNAs: Synthesis and stereo-discriminative duplex binding with DNA/RNA

Article abstract of DOI:10.1039/b407292h

The design and facile conversion of naturally occurring 4-hydroxyproline to all four diastereomers of thymine pyrrolidine PNA monomer, (2R,4S)-adenine, -guanine and -cytosine monomers and their incorporation into duplex forming PNA oligomers is reported,

Full text of DOI:10.1039/b407292h

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A R T I C L E
Chimeric (aeg-pyrrolidine)PNAs: synthesis and stereo-
discriminative duplex binding with DNA/RNA†
Pallavi S. Lonkar, Krishna N. Ganesh and Vaijayanti A. Kumar*
Division of Organic Chemistry, Synthesis, National Chemical Laboratory, Pune 411008, India.
E-mail: vakumar@dalton.ncl.res.in; Fax: +9120 2589 3135; Tel: +9120 2589 3135
Received 14th May 2004, Accepted 19th July 2004
First published as an Advance Article on the web 17th August 2004
The design and facile conversion of naturally occurring 4-hydroxyproline to all four diastereomers of thymine pyrrolidine
PNA monomer, (2R,4S)-adenine, -guanine and -cytosine monomers and their incorporation into duplex forming PNA
oligomers is reported. The interesting results of the hybridization studies with complementary DNA/RNA sequences in
either parallel or antiparallel orientation reveal the stereochemistry-dependent DNA vs. RNA discriminations and parallel/
antiparallel orientation selectivity.
five-membered ring conformation and stability of the resulting
Introduction
complexes. It was thought that the design of the (pyrrolidine)PNA
III where the nucleobase attachment to the backbone is through a
flexible methylene linker might prove to be more appropriate for
uniform results. The introduction of nucleobases besides thymine
will allow access to PNAs with mixed purine–pyrimidine sequences
for examining the consequence of the chiral constraints and the
nucleobase effects on PNA:DNA duplex stability.
We herein report the synthesis of (2R,4S)-pyrrolidine monomers
of the natural nucleobases cytosine (5a), adenine (5b) and guanine
(5d). The synthetic strategies to obtain the other two diastereo-
meric pyrrolidine thymine monomers 14 (2S,4R) and 15 (2R,4R)
(Fig. 2) requires an additional Mitsunobu reaction in the scheme
to invert the stereochemistry at C4. The synthesis of chimeric
(aeg-pyrrolidine)PNAs and their binding to complementary
DNA/RNA sequences is also presented in this paper. The results
presented indicate that the preferred stereochemistry of the
pyrrolidine ring varied with the different structural requirements
for the PNA2 :DNA triplexes, PNA:DNA duplexes and PNA:RNA
duplexes.4 The DNA:PNA duplexes were found to be uniformly
more stable in antiparallel orientation.
Peptide nucleic acids, in which the sugar phosphate backbone of
natural nucleic acids is replaced by the pseudopeptide backbone
derived from N-(2-aminoethyl)glycyl units (aegPNA II), have
emerged as promising DNA/RNA (I) mimics (Fig. 1).1 Several
analogues and derivatives of aegPNA have been synthesized to
overcome the limitations of PNA, such as poor water solubility,
cellular permeability and orientational ambiguity in complementary
DNA recognition2 by making them cationic and chiral. As part of
our continuing studies of conformationally constrained chiral
analogues of PNA based on the pyrrolidine core structure,3 we have
recently reported3f a pyrrolidine PNA III derived by introducing
a methylene bridge between the  carbon atom of the nucleobase
acetamide linker and the ′ carbon atom of the aminoethyl segment
of aegPNA backbone. The introduction of a single modification
of (2R,4S)-pyrrolidine-T unit III in aegPNA-T8 largely stabilized
the PNA2 :DNA complex (Tm = +16 °C) while the (2S,4S)-III
diastereomer destabilized the complex (Tm = −16 °C) compared
to unmodified aegPNA. The binding efficiency of chimeric
(aeg-pyrrolidine)PNA oligomers with DNA, in triplex mode,
seems to be influenced by the stereochemistry of the backbone
chiral unit.
Fig. 2 (Pyrrolidine)PNA diastereomeric structures.
Results and discussion
Fig. 1 DNA/RNA/PNA structures.
Synthesis of (pyrrolidine)PNA monomers 5a,b,d and 14, 15
In view of the above encouraging results on the (2R,4S)-
pyrrolidine thymine isomer III, we considered the synthesis
of the corresponding monomers of the other nucleobases and
other diastereomeric pyrrolidine PNA thymine monomers. In the
aegPNA backbone, substitution with a pyrrolidine modification
such as aepPNA IV was reported to exhibit nucleobase
dependent selectivity in DNA recognition properties.3c The direct
attachment of the nucleobases with different electronegativities
to the pyrrolidine ring as in aepPNA IV, perhaps affected the
The previously reported3f intermediate (2R,4S)-1 was reacted with
individual protected nucleobases, N4-benzyloxycarbonylcytosine,
N6-benzoyladenine and 2-amino-6-chloropurine in the presence of
K2CO3 and 18-crown-6 in DMF to give the desired pure nucleobase
derivatives 2a,b,c in 30, 40 and 45% isolated yields respectively
(Scheme 1).3d,5 The ring nitrogen was deprotected and the free
amine was immediately alkylated with ethyl bromoacetate in the
presence of diisopropylethyl amine to obtain 3a–c. The C-4 azide
in 3 was subjected to catalytic hydrogenation over Pd–C to obtain
the primary amine that was immediately protected as a tert-butyl
carbamate to give 4a–c. The ethyl ester in 4a–c was hydrolyzed
using sodium hydroxide in aqueous methanol to give free acids
of the pyrrolidine–cytosine, –adenine and –guanine monomers
(5a,b,d).
† Electronic supplementary information (ESI) available: HPLC profiles
and MALDI-TOF mass spectra of the chimeric (aeg-pyrrolidine)PNAs
17–23. UV-mixing Job’s plot of PNA:DNA 19:24 indicating 1:1 complex
formation. First derivative plots of (aeg-pyrrolidine)PNA (17–23):DNA
(24) complexes. See http://www.rsc.org/suppdata/ob/b4/b407292h/
2 6 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Scheme 1 Reagents and conditions: (i) K2CO3, 18-crown-6, BH, DMF;
(ii) TFA, DCM; (iii) BrCH2COOEt, DIPEA, THF (92%); (iv) H2, Pd/C or
Raney-Ni, (Boc)2O (90%); (v) NaOH/water–MeOH.
The synthesis of (2R,4R)-14 and (2S,4R)-15 pyrrolidine thymine
diastereomers was accomplished by a set of reactions shown in
Scheme 2. The (2R,4R)-N-benzyloxycarbonyl-4-hydroxyproline
methyl ester 6 was converted to (4S)-tosylate 7 with inversion
at C4 under Mitsunobu conditions using DIAD/PPh3 and methyl
tosylate. The (4S)-tosylate 7 was treated with sodium azide to
give the (4R)-azide 8, the reaction proceeding by inversion of
configuration at C-4. The azide 8 was selectively reduced by
catalytic hydrogenation in presence of Raney-Ni to give free
(4R)-amine, which was in situ protected as a Boc derivative 9.
The sodium borohydride reduction of the ester function in 9 to
alcohol 10 was followed by its conversion to O-mesyl derivative
11 using mesyl chloride in the presence of triethylamine. N-1
alkylation of thymine was effected by its reaction with the mesyl-
ate 11 in the presence of K2CO3 and 18-crown-6 to give (2R,4R)-
N-1 thyminylmethyl derivative 12 in 30% yield. The pyrrolidine
ring nitrogen in 12 was deprotected by hydrogenation and im-
mediately alkylated with ethyl bromoacetate to obtain 13. Sub-
sequent hydrolysis of the ethyl ester 13 with sodium hydroxide
in aqueous methanol gave (4S)-(N-Boc-amino)-(2R)-(thymin-
1-ylmethyl)pyrrolidin-N-1-acetic acid 14. Starting from 1-(N-
benzyloxycarbonylamino)-(4R)-hydroxy-(2S)-proline methyl ester
gave (4R)-(N-Boc-amino)-(2S)-(thymin-1-ylmethyl)pyrrolidin-N-
1-acetic acid 15. During the alkylation of nucleobases, minor by-
products are formed that could be the regiomers due to alkylations
at N3/N7 ring nitrogens. These were carefully separated during
column chromatography and the yields correspond to the isolated
pure products. Fig. 3 represents the mirror image relationship in
CD spectroscopy of the enantiomeric thymine pyrrolidine-PNA
monomer pairs (2S,4S3f and 2R,4R) and (2S,4R and 2R,4S3f). The
monomers 5a,b,d and 14, 15 were used for solid phase synthesis of
(aeg-pyrrolidine)PNAs 17–23. All new compounds were character-
ized using 1H, 13C NMR and mass spectrometry analysis. The pKa
of the pyrrolidine ring nitrogen atom in the monomeric unit was
determined by acid–base titration to be 6.8, indicating that in the
modified oligomers the pyrrolidine ring nitrogen could be at least
partially protonated at physiological pH.
Scheme 2 Reagents and conditions: (i) methyl tosylate, PPh3, DIAD,
THF (70%); (ii) NaN3, DMF (85%); (iii) Raney-Ni, H2, (Boc)2O
(85%); (iv) NaBH4, LiCl, EtOH, THF (80%); (v) MsCl, DCM, Et3N
(95%); (vi) thymine, K2CO3, 18-crown-6, DMF (35%); (vii) H2,Pd/C;
(viii) BrCH2COOEt, DIPEA, THF (80%); (ix) NaOH/water–MeOH (90%).
Solid phase synthesis of chimeric (aeg-pyrrolidine)PNA
oligomers
The modified (2S,4S), (2S,4R), (2R,4R), (2R,4S) thymine
monomers and (2R,4S) cytosine, adenine and guanine monomers,
were individually introduced at pre-defined positions into
duplex forming aegPNA sequence 16. The (aeg-pyrrolidine)PNA
oligomers 17–20 were synthesized to study the effect of stereo-
chemistry of the thymine monomer at the same position, while
PNAs 20–23 were required for examining the effect of nucleobases
with the same stereochemistry. The aegPNA sequence 16 was used
as control. The synthesis of all PNAoligomers was carried out using
standard solid-phase synthesis protocols.6 Appropriate modified
PNAmonomers were used for coupling at the desired positions. The
oligomers were cleaved from the support using trifluoroacetic acid–
trifluoromethanesulfonic acid7 to yield the PNA oligomers carrying
-alanine at their carboxy termini. The oligomer 22 required an
additional deprotection of N6-benzoyl group.3d The oligomers
were purified by FPLC on a C8-PepRPC column. The oligomers
were rechecked for purity by HPLC on an RP-C18 column and
were characterized by MALDI-TOF mass spectrometry (Table 1)
(ESI†). No precipitation was observed in samples of chimeric (aeg-
pyrrolidine)PNAeven after prolonged storage, in comparison to the
control aegPNA 16, indicative of their water solubility possibly as
a consequence of ring N-protonation. The complementary oligo-
nucleotides DNA 24, 25, RNA 26 and DNA 27 with a mismatched
base were synthesized on automated DNA synthesizer and were
used for complementation studies. All synthesized PNA, DNA and
RNA sequences are listed in Table 1.
UV-Tm studies
UV-Tm data of duplexes of PNAs 16–23 in both antiparallel (ap)
and parallel (p) modes with DNA (24 and 25, respectively) and (ap)
RNA (26) was obtained as a measure of duplex stability (Figs. 4
and 5). The 1:1 stoichiometry of PNA:DNA complexation was
confirmed by UV-mixing data (Job’s plot,8 ESI†). The Tm data is
summarized in Table 2. The Tm values were obtained by the first
derivative curves from individual melting curves (ESI†).
(aeg-Pyrrolidine)PNA:DNA duplexes. The control aeg-
PNA:DNA antiparallel duplex (16:24) was only marginally more
stable (Tm = +1 °C) than the parallel duplex (16:25).
All (aeg-pyrrolidine)PNA oligomers 17–23 formed stable
duplexes preferentially in the ap mode with complementary
DNA. The PNAs 18 (t 2S,4R) and 19 (t 2R,4R) were found to
stabilize the complexes with ap DNA 24 (Tm = +11 and +5 °C,
Fig. 3 CD spectra of diastereomeric thymine pyrrolidine-PNA
monomers.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1
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Table 1 PNA, DNA and RNA oligomer sequences
No.
PNA Sequence
M (Calc.)
M (Found)a
16
17
18
19
20
21
22
23
H–GTAGATCACT–NH(CH2)2COOH
H–GTAGA t (2S,4S) CACT–NH(CH2)2COOH
H–GTAGA t (2S,4R) CACT–NH(CH2)2COOH
H–GTAGA t (2R,4R) CACTNH(CH2)2COOH
H–GTAGA t (2R,4S) CACT–NH(CH2)2COOH
H–GTA g (2R,4S)ATCACT–NH(CH2)2COOH
H–GT a (2R,4S) GATCACT–NH(CH2)2COOH
H–GTAGAT c (2R, 4S) ACT–NH(CH2)2COOH
2796
2796
2796
2796
2796
2796
2796
2796.0
2798.0
2796.0
2796.75
2797.0
2797.7
2796.0
T, C, G, A represent aegPNA unit and t, c, g, a represent pyrrolidine PNA unit. 24 5′-AGT GAT CTA C-3′ DNA (ap), 25 5′-CAT CTA GTG A-3′ DNA (p),
26 5′-AGU GAU CUA C-3′ RNA (ap), 27 5′-AGT GTT CTA C-3′ DNA (ap) with a T mismatch. a Molecular weights as obtained by MALDI-TOF mass
spectrometry.
Table 2 UV Tm (in °C) studies of PNA:DNA/RNA duplexes
PNA
DNA 24 (ap)
DNA 25 (p)
RNA 26 (ap)
H–GTAGATCACT–NH(CH2)2COOH 16
47
34
58 (44)
52 (48)
39
29
36
34
46
30
48
47
38
27
34
31
41
76
44
78
43
33
38
36
H–GTAGA t (2S,4S) CACT–NH(CH2)2COOH 17
H–GTAGA t (2S,4R) CACT–NH(CH2)2COOH 18
H–GTAGA t (2R,4R) CACT–NH(CH2)2COOH 19
H–GTAGA t (2R,4S) CACT–NH(CH2)2COOH 20
H–GTA g (2R,4S)ATCACT–NH(CH2)2COOH 21
H–GT a (2R,4S) GATCACT–NH(CH2)2COOH 22
H–GTAGATc (2R, 4S) ACT–NH(CH2)2COOH 23
Buffer: 10 mM sodium cacodylate, 100 mM NaCl, 0.01 mM EDTA. pH 7.3. T, C, G, A represent aegPNA units and t, c, g, a represent pyrrolidine PNA unit.
Melting experiments were performed with a PNA:DNA strand concentration of 1 M each. The concentrations were calculated on the basis of the absorbance
at 260 nm using the molar extinction coefficients of A, T, G and C for DNA. Values in parenthesis indicate melting temperature with DNA sequence 27 having
one base mismatch.
19 also stabilized the ap duplex with DNA 24 (Tm = +4 °C) but to
a lesser extent. PNA oligomers 18 and 19 with DNA 27 having a
single mismatch at the site complementary to the pyrrolidine unit
were destabilized by 14 and 4 °C, respectively. This indicates that
the recognition process could be primarily through WC base pairing
aided by steric fit and not overruled by steric fitting or electrostatic
interactions. The oligomers 21–23 comprising other bases g, a and
c with (2R,4S) stereochemistry of the pyrrolidine ring, destabilized
the corresponding DNA duplexes indicating that this stereo-
chemistry indeed is not suitable for duplex formation independent
of the nucleobase.
Fig. 4 UV melting profiles of PNA:DNA complexes: (A) (a) 16:24,
(b) 17:24, (c) 18:24, (d) 19:24, (e) 20:24; (B) (a) 16:24, (b) 21:24,
(c) 22:24, (d) 23:24.
(aeg-Pyrrolidine)PNA:RNA duplexes. Considering the
differential structural requirements4 for PNA:RNA duplexes
compared to PNA:DNA duplexes, we studied the duplex binding
properties of the (aeg-pyrrolidine)PNAs 17–23 with ap RNA(Fig. 5,
Table 2). The control aegPNA:RNA antiparallel duplex (16:26)
was found to be less stable compared to control PNA:DNA duplex
with a lower Tm of 41 °C. Interestingly, all four (aeg-pyrrolidine)
PNAthymine oligomers comprising different diastereomers (17–20)
formed more stable duplexes than control aegPNA. PNAoligomers
with cis diastereomers as in (t 2S,4S) 17 and (t 2R,4R) 19 which
destabilized the corresponding duplexes with DNA, conferred very
large stabilization (Tm = +35, +37 °C, respectively) of the result-
ing RNAduplexes. This is an interesting observation and the reason
could be either effective better steric fitting of PNAstereochemistry
with RNA or other complex intrinsic structural differences in DNA
and RNA. The oligomers 20–23 comprising other bases a, g and c
with (2R,4S) stereochemistry of the pyrrolidine ring, destabilized
the corresponding RNA duplexes. Apart from the stereochemical
Fig. 5 The first derivative plots of % hyperchromicity vs. temperature
for PNA:RNA complexes: (a) 16:26, (b) 17:26, (c) 18:26, (d) 19:26,
requirements, the reason for the destabilization could also be the
position of the modification in the sequence as observed in case
of fluorinated olefinic PNAs.9 The (2R,4S) stereochemistry that
stabilized the PNA2 :DNA triplex structures was thus found to be
unsuitable to form duplexes with RNA in ap orientation. The PNAs
with an L-lysine unit at the C-terminus is known to bind better to
RNA over DNA.10 In our studies with the unmodified aegPNA 16,
the presence of -alanine at C-terminus allowed better binding to
DNA over RNA.
(e) 20:26.
respectively). Complexes formed by PNA 17 (t 2S,4S) and PNA
20 (t 2R,4S) with DNA 24 were quite destabilized (Tm = −13
and −8 °C, respectively) compared to control aegPNA 16:DNA
hybrids. The stereochemistry of the pyrrolidine ring in 18 (t 2S,4R)
could thus exercise preferential structural pre-organization of (aeg-
pyrrolidine)PNA that stabilized PNA:DNA 18:24 duplex in ap
orientation. The diastereomeric (t 2R,4R) pyrrolidine monomer in
2 6 0 6
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Parallel/antiparallel (p/ap) orientation selectivity. The largest
ap/p differentiation (Tm = +12 °C) was observed for PNA 18
having a (2S,4R) pyrrolidine–thymine unit. This stereochemistry
was thus found to be better suiting for ap binding than binding
in p orientation. The diastereomer in 19 (t 2R,4R), also stabilized
ap duplex with DNA 24 with comparatively less discrimination
of ap/p orientation selectivity. The results reported here show the
differential p/ap DNA and RNA binding properties conferred by
a single pyrrolidine unit in an aegPNA sequence as a consequence
of the stereochemistry of the pyrrolidine ring. Such an effect was
earlier observed when a chiral cyclic monomer was present in the
middle of the sequence along with an L-lysine unit at the C-terminus
or N-terminus of the sequence.11 We have used -alanine at the
C-terminus so that the C-terminus remains achiral and the results
obtained are solely because of the presence of the chiral pyrrolidine
unit in the middle of the PNA sequence and not due to the positive
charge or chirality of the terminal lysine unit.
Thus, the different structural features of the PNA2 :DNA,
PNA:DNA vs. PNA:RNA complexes seem to have an impact on
the selection of the stereochemistry of the pyrrolidine unit while
binding to either RNA or DNA complementary sequence, in duplex
binding mode. It is interesting to note that the pyrrolidine backbone
with (2R,4S) stereochemistry in the homopyrimidine sequence
stabilized the triplex with complementary DNA,3f but was found
to destabilize the derived DNA/RNA duplexes. Further work that
will reveal the suitable stereochemistry in sequence context and
with homo-pyrrolidine backbone is currently undergoing in our
laboratory.
(iii) cis-(2S,4S) and -(2R,4R) remarkably enhance PNA:RNA
duplex stability, (iv) all pyrrolidine modifications show prefer-
ence for antiparallel DNA binding. The interesting stability
differences seen in the complexes of different pyrrolidinyl PNAs
with DNA/RNA and stereochemical preferences will be useful to
design and arrive at PNA analogues having selective DNA/RNA
recognition properties.12 Such studies are currently under progress
in our laboratory.
Experimental
General
All the reagents were purchased from Sigma-Aldrich and used
without purification. DMF and pyridine were dried over KOH and
4 Å molecular sieves. THF was passed over basic alumina and dried
by distillation over sodium. Ethanol was dried over Mg/iodine.TLCs
were run on Merck 5554 silica 60 aluminium sheets. All reactions
were monitored by TLC and usual work-up implies sequential
washing of the organic extract with water and brine followed
by drying over anhydrous sodium sulfate and evaporation under
vacuum. Column chromatography was performed for purification
of compounds on silica gel (100–200 mesh, LOBA Chemie). TLCs
were performed using dichloromethane–methanol or petroleum
ether–ethyl acetate solvent systems for most compounds. Purity of
the free acids was checked by TLC using isopropanol–acetic acid–
water (9:1:1, v/v) solvent system. Compounds were visualized
with UV light and/or by spraying with ninhydrin reagent subsequent
1
to Boc-deprotection (exposing to HCl vapourS) and heating. H
(200 MHz) and 13C (50 MHz) NMR spectra were recorded on a
Bruker ACF 200 spectrometer fitted with an Aspect 3000 computer
and all the chemical shifts (/ppm) are referred to internal TMS for
1H and chloroform-d for 13C NMR. Optical rotations were measured
on a JASCO DIP-181 polarimeter. Mass spectra were recorded on
a Finnigan-Matt mass spectrometer, while MALDI-TOF spectra
were obtained from a KRATOS PCKompact instrument. UV
experiments were performed on a Perkin Elmer 35 UV-VIS
spectrophotometer fitted with a Peltier temperature programmer
and a Julabo water circulator. The DNA oligomers were
synthesized on CPG solid support using a Pharmacia GA plus DNA
synthesizer by -cyanoethyl phosphoramidite chemistry followed
by ammonia treatment13 and their purities checked by HPLC prior
to the use. aegPNA monomers were synthesized according to
literature procedures.6
Gel-shift assay
The formation of PNA:DNA ap duplexes was confirmed by
the non-denaturing gel-shift assay (Fig. 6) which is useful to
establish the binding of (aeg-pyrrolidine)PNAs to complementary
oligonucleotides by observed mobility retardation of the complexes
on the gel. The PNAs modified with one unit of (pyrrolidine)PNA
and the control PNA were individually treated with complementary
DNA and complexation were monitored by non-denaturing gel
electrophoresis at 10 °C. The spots were visualized on a fluorescent
TLC background. The PNA:DNA complexes derived from
(pyrrolidine)PNA with single modified unit were significantly
retarded in the gel (lanes 3, 4; PNA:DNA 18:24, 19:24).
Synthesis, cleavage and purification of the PNA oligomers
from the solid support
The PNA oligomers were synthesized manually by solid-
phase peptide synthesis using the Boc-protection strategy and
employing HBTU and 1-hydroxybenzotriazole (HOBT) as the
coupling agents. The solid support used was Merrifield resin
that was derivatized with -alanine (0.19 meq./g resin) as the
spacer amino acid. Synthesis involved repetitive cycles, each
comprising (i) deprotection of the N-protecting Boc-group using
50% trifluoroacetic acid (TFA) in CH2Cl2, (ii) neutralization of the
TFA salt formed with diisopropylamine (5% solution in CH2Cl2,
v/v) and (iii) coupling of the free amine with the free carboxylic
acid group of the incoming monomer (3–4 equivalentS) in the
presence of HBTU and HOBt, in DMF or NMP as the solvent.
The deprotection of the N-Boc protecting group and the coupling
reaction were monitored by Kaiser’s test.14 Since the coupling
efficiencies were found to be >98%, capping of the unreacted amino
groups was not found necessary.
Fig. 6 Non-denaturing gel: lane 1: PNA 18 ss; lane 2: DNA 24, lane 3:
PNA 18:DNA 24, lane 4: PNA 19:DNA 24.
These results were in accordance with the data obtained from
the UV-thermal melting studies with these oligomers, which also
underline the specificity of the hydrogen bonding between the
complementary base pairs.
In summary, this paper presents detailed studies on the synthesis
and comparative complexation of PNA oligomers incorporating
diastereomeric pyrrolidine monomers having nucleobases
T, A, G and C with complementary DNA and RNA sequences. It
is found that (i) (2R,4S)-PNA oligomers that stabilize PNA2 :DNA
homopyrimidine:homopurine triplexes destabilize the mixed
pyrimidine:homopurine duplexes, (ii) the presence of (2S,4R)
and (2R,4R) stereoisomers affects enhanced DNA duplex stability,
The PNA oligomers were cleaved from the solid support using
the TFA–trifluoromethanesulfonic acid (TFMSA) method to yield
oligomers with free carboxylic acids at their carboxy termini.7
The resin-bound PNA oligomer (10 mg) was stirred in an ice-bath
with thioanisole (20 l) and 1,2-ethanedithiol (8 l) for 10 min.
TFA (120 l) was then added and the stirring was continued for
another 10 min. TFMSA (16 l) was added while cooling the
reaction in an ice-bath and stirring was continued for 2 h. The
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reaction mixture was filtered through a sintered funnel, the residue
washed with TFA(3 × 2 ml) and the combined filtrate and washings
were evaporated under vacuum. The residual pellet was re-dissolved
in methanol (~0.1 ml) and re-precipitated by adding ether to obtain
the crude PNA oligomer. (In the case of oligomer 22, the exocyclic
benzoyl protection of adenine was removed by treating the resin
with ethylene diamine–ethanol3d,15 in 1:1 ratio overnight at room
temperature prior to TFA–TFMSA cleavage.) This was purified by
gel filtration over Sephadex G25. The purity of the PNA oligomer
was checked by analytical RP HPLC on a C18 column and found
to be >90% (ESI†).
128.2, 128.0, 127.7, 95.0, 80.3, 67.2, 58.5, 55.7, 51.3, 36.0, 34.3,
27.8. MS: calc. for C22H28N7O5 (M+) m/z 470.5, found 470.0.
(4S)-Azido-(2R)-(N4-benzyloxycarbonylcytosin-1-ylmethyl)-
pyrrolidin-N-1-acetic acid ethyl ester (3a)
To
a
solution of 1-(N-Boc)-(4S)-azido-(2R)-(N4-benzyloxy-
carbonylcytosin-1-ylmethyl)pyrrolidine (0.27 g, 0.57 mmol) in
dry CH2Cl2 (1 ml) was added TFA (1 ml) and the reaction was
stirred at room temperature for 10 min, when TLC indicated a
complete absence of starting material. The resulting free amine
was immediately subjected to alkylation using ethyl bromo-
acetate (0.07 ml, 0.63 mmol) in dry THF (2 ml) in the presence
of DIPEA (0.060 ml, 0.35 mmol). The amine was completely
consumed within 3 h, upon which, the solvents were removed
under vacuum and the crude product purified by silica gel column
chromatography to obtain the pure product 3a in the form of a gum
(0.24 g, 93%).
1H NMR (CDCl3) : 7.92 (d, 1H, J = 7.32 Hz), 7.35 (s, 5H), 7.16
(d, 1H, J = 7.33 Hz), 5.19 (s, 2H), 4.14 (m, 4H), 3.89 (m, 1H), 3.70
(m, 1H), 3.40 (m, 2H), 3.29 (m, 1H), 2.58 (m, 1H), 2.04 (m, 1H),
1.85 (m, 1H), 1.24 (t, 3H, J = 7.32 Hz). 13C NMR (CDCl3) : 170.4,
162.4, 155.5, 152.7, 149.8, 135.0, 128.2, 128.0, 127.7, 94.4, 67.2,
60.5, 59.2, 58.3, 55.4, 51.6, 40.3, 34.3, 13.7. ESIMS: calc. for
C21H25N7O4 (M+) m/z 439.4, found 440 (M + 1).
UV-melting studies
The concentration of the PNAoligomers was calculated on the basis
of the absorption at 260 nm, at 80 °C6 assuming the molar extinction
coefficients of the nucleobases to be as in DNA, i.e., T, 8.8 cm2
mol−1; C, 7.3 cm2 mol−1; G, 11.7 cm2 mol−1 and A, 15.4 cm2
mol−1. Molar extinction coefficients for the (pyrrolidine)PNA
monomers were calculated and found to be almost the same as
that of the nucleobases in DNA (molar extinction coefficients
for T with (2R,4S), (2R,4R), (2S,4S) and (2S,4R): 8.7, 8.6, 9.0
and 9.1 cm2 mol−1; (2R,4S) G, 11.9 cm2 mol−1; C, 7.8 cm2
mol−1; A, 16.0 cm2 mol−1) The PNA oligomers (16–23) and the
relevant complementary DNA/RNA oligonucleotide (24–26) were
mixed together in a 1:1 molar ratio in 10 mM sodium cacodylate,
100 mM NaCl, 0.01 mM EDTA, (pH 7.3) to obtain a final strand
concentration of 1 M. The samples were annealed by heating at
85 °C for 1–2 min, followed by slow cooling to room temperature,
kept at room temperature for ~30 min and then, refrigerated
overnight. The samples were heated at a rate of 0.5 °C min−1 and
the absorbance at 260 nm was recorded at every 1 min. The percent
hyperchromicity at 260 nm was plotted as a function of temperature
and the melting temperature was deduced from the peak in the first
derivative plots.
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(N4-benzyloxy-
carbonylcytosin-1-ylmethyl)pyrrolidin-N-1-acetic acid ethyl
ester (4a)
(4S)-Azido-(2R)-(N4-benzyloxycarbonylcytosin-1-ylmethyl)-
pyrrolidin-N-1-ethyl acetate (0.24 g, 0.55 mmol) was reduced to the
(4S)-amine by hydrogenation using Raney–Ni as a catalyst (0.16 g)
in ethyl acetate at room temperature for 2 h, which was converted
in situ to the corresponding tert-butyl carbamate by Boc-anhydride
(0.14 g, 0.66 mmol). The product was purified by silica gel column
chromatography to obtain pure 4a in the form of a gum (0.25 g,
90% yield).
1-(N-Butyloxyoxycarbonyl)-(4S)-azido-(2R)-(O-mesylmethyl)-
pyrrolidine (1)
1
[]24D −33.1 (c 0.024, MeOH); H NMR (CDCl3) : 7.72 (br s,
Toastirred, cooledmixtureof1-(N-Boc)-(4S)-azidopyrrolidin-(2S)-
methanol3f (1.0 g, 4.1 mmol) and triethylamine (1.44 ml, 10.3 mmol)
in dry dichloromethane (10 ml), methanesulfonyl chloride (0.8 ml,
10.3 mmol) was added dropwise. Stirring was continued at room
temperature overnight. The solvent was completely removed under
vacuum. The residue was taken up in water and the crude product
obtained by extraction in dichloromethane, followed by drying of
the organic layer sodium sulfate and evaporation under vacuum.
Silica gel column chromatography yielded the pure product in the
form of a gum (0.6 g, 90% yield).
1H NMR (CDCl3) : 4.45 (dd, 1H), 4.15 (br m, 3H), 3.65 (m,
1H), 3.35 (m, 1H), 3.03 (s, 3H), 2.30 (m, 1H), 2.13 (m, 1H), 1.45 (s,
9H). 13C NMR (CDCl3) : 164.9, 154.2, 152.2, 152.1, 149.4, 143.2,
133.3, 132.2, 128.2, 127.7, 122.4, 80.5, 58.3, 55.8, 51.4, 44.5, 34.4,
27.9. ESIMS: calc. for C22H25N9O3 (M+) m/z 463.6, found: 464
(M + 1).
1H), 7.37 (s, 5H), 7.17 (br s, 1H), 5.20 (s, 2H), 5.08 (m, 1H), 4.13
(m, 4H), 3.79 (m, 2H), 3.69 (m, 1H), 3.42 (m, 2H), 3.31 (m, 1H),
2.55 (m, 1H), 2.03 (m, 1H), 1.41 (s, 9H), 1.24 (br s, 3H). 13C NMR
(CDCl3) : 171.1, 162.8, 156.0, 155.6, 152.9, 150.0, 135.3, 128.7,
128.5, 128.3, 94.8, 79.6, 67.8, 60.8, 59.1, 58.7, 54.1, 53.5, 49.8,
36.1, 28.4, 14.2. ESIMS: calc. for C26H36N5O7 m/z 530.6, found
530.0.
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(N4-benzyloxy-
carbonylcytosin-1-ylmethyl)pyrrolidin-N-1-acetic acid (5a)
To
a
solution of (4S)-(N-Boc-amino)-(2R)-(N4-benzyloxy-
carbonylcytosin-1-ylmethyl)pyrrolidin-N-1-ethyl acetate 4a (0.1 g,
0.44 mmol) in methanol–water, 1:1 (3 ml) was added 1 M NaOH
(1 ml) and stirred for 20 min at room temperature. The excess alkali
was neutralized using Dowex H+ resin, which was then filtered off
to obtain the product as a white foam (90% yield).
1
[]24D −5.0 (c 0.002 MeOH); H NMR (D2O) : 7.41–7.31 (m,
1-(N-Butyloxyoxycarbonyl)-(4S)-azido-(2R)-(N4-benzyloxy-
2H), 6.99–6.91 (m, 1H), 5.18 (m, 1H), 2.55 (s 1H), 2.31–1.68 (m,
2H), 1.46 (s, 2H), 1.45–1.32, 2.3 (s, 2H). 13C NMR (D2O) : 172.13,
170.97, 158.79, 155.63, 155.28, 153.95, 150.84, 142.66, 138.64,
137.54, 128.63, 128.48, 128.29, 128.17, 128.06, 127.97, 127.85,
127.80, 126.77, 126.58, 85.06, 63,07, 62.97, 59.61, 58.59, 57.72,
53.85, 52.56, 51.66, 40.38, 34.33, 28.37, 27.97. ESIMS: calc. for
C25H35N5O6 m/z 501.5, found 502 (M+); max = 270 nm,  = 7.8 cm2
mol−1.
carbonylcytosin-1-ylmethyl)pyrrolidine (2a)
A
mixture of 1-(N-Boc)-(4S)-azido-(2R)-(O-mesylmethyl)-
pyrrolidine 1 (0.80 g, 2.50 mmol), anhydrous potassium carbonate
(0.86 g, 6.25 mmol), N4-benzyloxycarbonylcytosine (0.60 g,
3.00 mmol) and 18-crown-6 (0.13 g, 0.75 mmol) in dry DMF was
stirred at 60 °C under argon atmosphere overnight. The solvent
was completely removed under vacuum and the crude residue was
purified by column chromatography subsequent to filtering and
evaporation of the filtrate to obtain the pure product in the form of
liquid gum 2a (0.27 g, 30% yield).
[]24D −25.0 (c 0.04, MeOH); 1H NMR (CDCl3) : 7.65 (d, 1H),
7.40 (s, 5H), 7.15 (d, 1H), 5.20 (s, 2H), 4.20 (m, 2H), 4.05 (m, 1H),
3.80 (m, 1H), 3.55 (m, 1H), 3.35 (m, 1H), 2.15 (m, 2H), 1.45 (s,
9H). 13C NMR (CDCl3) : 162.3, 155.8, 154.3, 152.6, 148.7, 134.9,
1-(N-Butyloxyoxycarbonyl)-(4S)-azido-(2R)-(N6-benzoyl-
adenin-9-ylmethyl)pyrrolidine (2b)
A mixture of N6-benzoyladenine (0.80 g, 3.15 mmol) and K2CO3
(0.86 g, 6.25 mmol) in dry DMF was stirred at 60 °C for 1 h.
A
solution of 1-(N-Boc)-(4S)-azido-(2R)-(methyl-O-mesyl)-
2 6 0 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1

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pyrrolidine (0.84 g, 2.63 mmol) in DMF was added dropwise to
this mixture and stirring was continued at 60 °C for 5 h, when TLC
inspection indicated absence of the starting material. The solvent
was completely removed under vacuum and the crude residue
purified by column chromatography to obtain the pure product 2b
in the form of a gum (0.26 g, 40% yield).
[]24D +35.7 (c 0.014, MeOH); 1H NMR (CDCl3) : 8.77 (s, 1H),
8.02 (m, 3H), 7.54 (m, 3H), 4.60 (m, 2H), 4.31 (m, 1H), 3.79 (m,
1H), 3.50 (m, 1H), 2.06 (m, 2H), 1.49 (s, 9H). 13C NMR (CDCl3)
: 164.9, 154.2, 152.2, 152.1, 149.4, 143.2, 133.3, 132.2, 128.2,
127.7, 122.4, 80.5, 58.3, 55.8, 51.4, 44.5, 34.4, 27.9. ESIMS: calc.
for C22H25N9O3 (M+) m/z 463.5, found 464.0 (M + 1).
(0.22 g, 1.56 mmol), 2-amino-6-chloropurine (0.13 g, 0.78 mmol)
and 18-crown-6 (0.03 g, 0.09 mmol) in dry DMF were stirred
at 65 °C under argon atmosphere overnight. The solvent was
completely removed under vacuum and the crude residue, purified
by column chromatography subsequent to filtering and evaporation
of the filtrate to obtain the pure product 2c in the form of a gum
(45% yield).
[]24D +5.83 (c 0.012, MeOH); 1H NMR (CDCl3) : 7.60 (s, 1H),
5.15 (br, 2H), 4.50 (m, 1H), 4.20 (m, 2H), 3.73 (m, 1H), 3.40 (m,
1H), 3.10 (m, 1H), 1.95 (m, 2H), 1.40 (s, 9H). 13C NMR (CDCl3)
: 159.2, 153.9, 150.6, 142.4, 123.9, 80.3, 58.2, 55.5, 51.4, 44.1,
34.1, 27.6. ESIMS: calc. for C15H20N9O2Cl m/z 393.3, found 394
(M + 1).
(4S)-Azido-(2R)-(N6-benzoyladenin-9-ylmethyl)pyrrolidin-N-
(4S)-Azido-(2R)-(2-amino-6-chloropurin-9-ylmethyl)-
pyrrolidin-N-1-ethyl acetate (3c)
1-ethyl acetate (3b)
To a solution of 1-(N-Boc)-(4S)-azido-(2R)-(N6-benzoyladenin-
9-ylmethyl)pyrrolidine 2b (0.39 g, 0.84 mmol) in dry CH2Cl2
(1 ml) was added TFA (1 ml) and the reaction was stirred at
room temperature for 10 min, when TLC indicated a complete
absence of starting material. The resulting free amine was
immediately subjected to alkylation using ethyl bromoacetate
(0.1 ml, 0.93 mmol) in dry THF (4 ml) in the presence of DIPEA
(0.22 ml, 1.26 mmol). The amine was completely consumed within
2.5 h, upon which, the solvents were removed under vacuum and
the crude product purified by silica gel column chromatography to
obtain the pure product 3b in the form of a gum (95% yield).
[]24D +35.7 (c 0.004, MeOH); 1H NMR (CDCl3) : 8.70, (s, 2H),
8 (m, 3H), 7.6 (m, 4H), 4.2, (m, 2H), 3.5 (m, 2H), 3.4 (m, 2H), 2.7,
(m, 1H), 2, (m, 1H), 1.3 (t, 3H). 13C NMR (CDCl3) : 176.5, 170.2,
146.7, 146.3, 145.5, 140.9, 135.2, 131.6, 129.4, 127.7, 120.7, 60.8,
60.5, 58.6, 58.1, 54.1, 45.4, 34.3, 13.8. ESIMS: calc. for C22H25N9O3
m/z 463.6, found 464.0 (M+).
To
a
solution of 1-(N-Boc)-(4S)-azido-(2R)-(2-amino-6-
chloropurin-9-ylmethyl)pyrolidine 2c (0.08 g, 0.2 mmol) in dry
CH2Cl2 (1 ml) was added TFA (1 ml) and the reaction was stirred
at room temperature for 10 min, when TLC indicated a complete
absence of starting material. The TFA salt was neutralized with
DIPEA and the free amine was immediately subjected to alkylation
using ethyl bromoacetate (0.025 ml, 0.20 mmol) in dry THF (1 ml)
in the presence of DIPEA (0.053 ml, 0.31 mmol). The amine was
completely consumed within 2 h, upon which, the solvents were
removed under vacuum and the crude product purified by silica gel
column chromatography to obtain the pure product 3c in the form
of a gum (89% yield).
[]24D +36.66 (c 0.012, MeOH), 1H NMR (CDCl3) : 8.05 (s, 1H),
5.25 (s, 2H), 4.20 (m, 2H), 4.05 (m, 2H), 3.70 (m, 1H), 3.40 (m, 2H),
2.65 (m, 1H), 2.00 (m, 1H), 1.75 (m, 1H), 1.30 (t, 3H). 13C NMR
(CDCl3) : 170.3, 159.4, 150.7, 143.4, 124.2, 60.9, 60.5, 58.8, 58.3,
54.7, 45.4, 34.4, 31.1, 13.8. ESIMS: calc. for C15H20O2N9Cl m/z
393.8, found 394 (M+).
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(N6-benzoyladenin-
9-ylmethyl)pyrrolidin-N-1-ethyl acetate (4b)
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(2-amino-6-
chloropurin-9-ylmethyl)pyrrolidine-N-1-ethyl acetate (4c)
(4S)-Azido-(2R)-(N6-benzoyladenin-9-ylmethyl)pyrrolidin-N-1-
ethyl acetate 3b (0.34 g, 0.76 mmol) was reduced to the (4S)-amine
by hydrogenation using 10% Pd–C as catalyst at room temperature
for 2 h. The resulting amine (0.30 g, 0.71 mmol) was converted in
situ to the corresponding tert-butyl carbamate by Boc-anhydride
(0.19 g, 0.85 mmol). The crude product was purified by silica gel
column chromatography to obtain pure product 4b in the form of a
gum (0.37 g, 98% yield).
1H NMR (CDCl3) : 8.10 (m, 2H), 7.90 (s, 1H), 7.45 (m, 3H),
7.25 (s, 1H), 5.10 (m, 1H), 4.20 (m, 4H), 4.05 (m, 1H), 3.75 (m,
1H), 3.40 (m, 4H), 2.60 (m, 1H), 1.45 (s, 9H), 1.28 (t, 3H). 13C
NMR (CDCl3) : 171.2, 162.7, 155.5, 152.4, 152.0, 149.7, 144.6,
133.7, 132.4, 128.4, 128.2, 12.8.0, 122.5, 78.9, 60.7, 60.1, 58.8,
53.1, 49.0, 45.9, 35.7, 28.2, 14.1. ESIMS: calc. for C26H25N7O5 m/z
515.5, found 515.0.
(4S)-Azido-(2R)-(2-amino-6-chloropurin-9-ylmethyl)pyrrolidine-
N-1-ethyl acetate 3c (0.07 g, 0.18 mmol) was reduced to the
(4S)-amine by hydrogenation using 10% Pd–C as catalyst (0.05 g)
in methanol, which was converted in situ to the corresponding
tert-butyl carbamate by Boc-anhydride (0.08 g, 0.46 mmol) to
obtain the crude product that was purified by silica gel column
chromatography to obtain pure 4c in the form of a gum (85%
yield).
1H NMR (CDCl3) : 7.95 (s, 1H), 5.28 (s, 2H), 5.13 (m, 1H), 4.14
(m, 5H), 3.48 (m, 1H), 3.28 (m, 2H), 2.93 (m, 2H), 2.51 (m, 1H),
2.28 (m, 1H), 1.37 (s, 9H), 1.26 (t, 3H). 13C NMR (CDCl3) : 171.2,
159.3, 155.4, 154.3, 151.2, 143.5, 124.7, 77.9, 61.0, 59.8, 59.2,
58.8, 55.1, 46.0, 36.5, 28.4, 14.2. ESIMS: calc. for C19H27O4N2Cl
m/z 439.8, found 456 (M + 17, M + NH3).
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(N6-benzoyladenin-
9-ylmethyl)pyrrolidin-N-1-acetic acid (5b)
(4S)-(N-Butyloxyoxycarbonylamino)-(2R)-(guanin-9-
ylmethyl)pyrrolidin-N-1-acetic acid (5d)
To a solution of (4S)-(N-Boc-amino)-(2R)-(N6-benzoyladenin-9-
ylmethyl)pyrrolidin-N-1-ethyl acetate 4b (0.34 g, 0.76 mmol) in
methanol–water, 1:1 (3 ml) was added 1 M NaOH (1 ml) and the
reaction mixture was stirred for 20 min at room temperature when
TLC indicated complete absence of starting material. The excess
alkali was neutralized using Dowex H+ resin which was then filtered
off. The filtrate was then evaporated to obtain the product as a white
foam (90% yield).
To a solution of (4S)-(N-Boc-amino)-(2R)-(2-amino-6-chloropurin-
9-ylmethyl)pyrrolidin-N-1-ethyl acetate (0.2 g, 0.5 mmol) in
methanol–water, 1:1 (5 ml) was added 1 M NaOH (2 ml) and stirred
overnight at room temperature. The excess alkali was neutralized
using Dowex H+ resin which was then filtered off. The filtrate was
then concentrated to obtain the pure product as a white foam (88%
yield).
[]24D −5.0 (c 0.002, MeOH); 1H NMR (D2O) : 8.37–8.16
(m, 1H), 4.47–4.19 (m, 2H), 4.26–3.96 (m, 5H), 3.71–3.66 (m,
1H), 3.38–3.21 (m, 1H), 2.36–2.21 (m, 1H), 2.15–1.89 (m, 1H),
1.57–1.30 (m, 9H). 13C NMR (D2O) : 170.6, 169.9, 159.5, 156.9,
154.2, 140.5, 139.7, 69.5, 65.4, 65.0, 63.0, 62.4, 59.6, 59.2, 58.6,
57.3, 56.6, 55.2, 54.8, 47.1, 42.9, 30.3, 29.5, 27.3. ESIMS: calc.
for C16H24O5N7 m/z 406.2, found 408 (M + 1); max = 270 nm,
 = 11.9 cm2 mol−1.
[]24D +20 (c 0.002, MeOH); ESIMS: calc. for C24H29N7O5 m/z
495.5, found 497 (M + 1); max = 270 nm,  = 16.0 cm2 mol−1.
1-(N-Butyloxyoxycarbonyl)-4-(S)-azido-2(R)-(2-amino-6-
chloropurin-9-ylmethyl)pyrrolidine (2c)
A
mixture of 1-(N-Boc)-(4S)-azido-(2R)-(methyl-O-mesyl)-
pyrrolidine 1 (0.2 g, 0.6 mmol), anhydrous potassium carbonate
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1
2 6 0 9

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(2R/S,4S)-N-1-(Benzyloxycarbonyl)-4-(p-toluenesulfonyloxy)-
proline methyl ester (7)
2H), 3.58 (s, 1H), 3.45–3.34 (m, 1H), 2.28–2.15 (m, 2H), 1.45 (s,
9H). 13C NMR (CDCl3) : 172.2, 172.0, 154.8, 154.5, 153.8, 135.9,
128.1, 127.7, 127.6, 77.0, 76.7, 67.0, 57.5, 52.1, 51.9, 51.5, 36.6,
35.5, 28.0. ESIMS: calc. for C19H26N2O6 m/z 378.2, found 379.0
(M + 1).
(2R/S,4R)-N-1-(benzyloxycarbonyl)-4-hydroxyproline methyl ester
6 (5 g, 7.85 mmol), PPh3 (3.97 g, 19.6 mmol) and methyl-p-toluene
sulfonate (3.65 g, 19.4 mmol) were taken in dry THF (100 ml)
and the mixture was cooled to −10 °C in an ice-salt bath. To this,
a solution of diisopropylazodicarboxylate (5.14 ml, 25 mmol)
in dry THF (10 ml) was added using a syringe over a period of
1 h. The reaction mixture was further stirred for 1 h at −10 °C
followed by 12 h at room temperature, when the TLC indicated
the complete conversion of starting material. The reaction mixture
was concentrated and was subjected to silica gel column chromato-
graphy. The separation proved to be difficult as the diisopropyl-
hydrazodicarboxylate moves very closely with the product.
7a: []24D +15.31 (c 0.322, MeOH); 1H NMR (CDCl3) : 7.9–7.5
(m, 2H) 7.3–7.1 (m, 2H) 5.1–4.8 (m, 3H) 4.5–4 (m, 2H) 3.7–3.5 (s,
3H), 3.7 3.2–3.0 (s, 2H), 2.5–2.3 (s, 3H), 2.2–2.0 (m, 3H). 13C NMR
(CDCl3):  172.5, 156.0, 143.0, 136.2, 132.6, 132.1, 130.4, 129.9,
126.3, 125.6, 65.36, 62.0, 51.2, 22.5, 20.1, 13.4. ESIMS: calc. for
C20H23O8NS m/z 433.4, found 434.0 (M + 1).
(2R/S,4R)-N-(Benzyloxycarbonyl)-4-(tert-butyloxycarbonylamino)-
pyrrolidine-2-methanol (10)
To a stirred mixture of dry ethanol (20 ml) and dry THF (30 ml), was
added lithium chloride (2.16 g, 50 mmol) and sodium borohydride
(1.88 g, 50 mmol) and stirring continued for ~10 min under argon
atmosphere. A solution of (2R,4R)-N-(benzyloxycarbonyl)-4-(tert-
butyloxycarbonylamino)proline methyl ester 9 (5.49 g, 14 mmol)
in THF (10 ml) was added dropwise and stirring was continued at
room temperature under an argon atmosphere for 6 h. When TLC
indicated complete disappearance of starting material, the pH of
the reaction mixture was adjusted to 4 with aqueous ammonium
chloride. The reaction mixture was stirred for 10 min and the
solvents were removed on a rotary evaporator. The residue was
taken up in water and extracted with ethyl acetate (4 × 20 ml). The
organic layer was then dried over sodium sulfate and evaporated to
obtain the crude product. Silica gel column chromatography yielded
the pure product in the form of a gum (80% yield).
7b: []24D −1.1 (c 0.012, MeOH); 1H NMR (CDCl3) : 7.9–7.10
(m, 2H), 7.4–7.1 (m, 2H), 5.4–4.8 (m, 2H), 4.6–3.9 (m, 2H), 3.8–
3.49 (m, 4H), 2.5 (s, 3H), 2.2–1.9 (m, 2H). 13C NMR (CDCl3) :
173.5, 156.7, 145.0, 136.2, 133.6, 132.1, 131.5, 129.8, 127.3, 127.6,
67.36, 62.0, 52.2, 21.5, 20.6, 14.3. ESIMS: calc. for C20H23O8NS
m/z 433.4, found 434.0 (M + 1).
10a: []24D +5.0 (c 0.024 MeOH); 1H NMR (CDCl3) : 7.42–7.30
(s, 5H), 5.18–5.12 (m, 2H), 4.86 (br s, 1H), 4.27–3.96 (m, 4H),
3.87–3,79 (m, 1H), 3.76–3.52 (m, 3H), 3.49–3.39 (m, 1H), 2.46–2.0
(m, 2H), 1.44 (s, 9H). 13C NMR (CDCl3) : 154.0, 128.2, 127.8,
127.5, 79.5, 67.0, 65.4, 55.7, 52.7, 51.9, 48.8, 34.2, 33.6 28.0.
ESIMS: calc. for C18H26O5N2 m/z 350.4, found 350.0.
(2R/S,4R)-N-1-(Benzyloxycarbonyl)-4-azidoproline methyl
ester (8)
1
10b: []24D −27.5 (c 0.004, MeOH); H NMR (CDCl3)  7.40–
The mixture from the above step was taken in dry DMF (50 ml)
and to this was added NaN3 (7.64 g, 117 mmol). The mixture was
stirred at 60 °C for 12 h when TLC indicated complete conversion
of starting material, DMF was removed under vacuum on a rotary
evaporator, 100 ml of water was added to the mixture and extracted
with ethyl acetate (30 ml × 3). The organic layers were pooled,
washed with water (20 ml) and concentrated to give the crude
product that was further purified by column chromatography as a
thick liquid (85% yield).
7.32 (m, 4H), 5.16 (br s, 2H), 4.72–4.64 (m, 1H), 4.27–4.03 (m,
2H), 3.79–3.6 (m, 3H), 3.5–3.4 (s, 2H), 2.01–1.75 (m, 2H), 1.44 (s,
9H). 13C NMR (CDCl3) : 156.0, 154.9, 136.0, 128.2, 127.9, 127.6,
79.5, 66.0, 58.8, 52.8, 51.0, 34.4, 28.0. ESIMS: calc. for C18H26O5N2
m/z 350.4, found 350.0.
N-1-(Benzyloxycarbonyl)-(4R)-(tert-butyloxycarbonylamino)-
(2R/S)-(O-mesylmethyl)pyrrolidine (11)
8a: []24D +20.45 (c 0.022, MeOH); 1H NMR (CDCl3) : 7.8–7.4
(m, 2H), 7.3–7.2 (m, 2H), 5.2–4.7 (m, 3H), 4.45 (m, 2H), 3.66 (s,
3H), 3.42 (m, 2H), 2.13–1.48 (m, 2H). 13C NMR (CDCl3) : 172.5,
154.2, 153.8, 128.3, 128.2, 127.8, 127.8, 127.6, 69.5, 58.9, 52.2,
51.1, 50.8, 35.8, 21.7. ESIMS: calc. for C14H16O4N4 m/z 304.3,
found 305.0 (M + 1).
To a stirred, cooled mixture of alcohol 10 (1.64 g, 4.6 mmol)
and triethylamine (1.95 ml, 19.30 mmol) in dry dichloromethane
(10 ml) was added dropwise methanesulfonyl chloride (0.64 ml,
5.5 mmol). Stirring was continued for further 30 min when TLC
indicated absence of starting material. The solvent was removed
under vacuum, the residue was taken up in water and extracted
with ethyl acetate (2 × 10 ml). The organic layer was dried over
sodium sulfate, and then evaporated to dryness. Silica gel column
chromatography yielded the pure product in the form of a gum (95%
yield).
1
8b: []24D −50.9 (c 0.022, MeOH); H NMR (CDCl3) : 7.4 (s,
5H), 5.35–5 (m, 2H), 4.5–4.32 (m, 1H), 4.25–4.15 (m, 1H), 3.75 (s,
3H), 3.6–3.4 (m, 2H), 2.4–2.3 (m, 2H). 13C NMR (CDCl3) : 171.5,
171.2, 154.2, 153.8, 136.0, 128.1, 127.6, 67.1, 67.0, 58.9, 57.5,
57.2, 52.2, 51.1, 50.8, 35.8, 34.8, 21.6. ESIMS: calc. for C14H16O4N4
m/z 304.2, found 321 (M + 17, M + NH3).
1
11a: []24D +25.5 (c 0.02, MeOH); H NMR (CDCl3) : 7.4 (s,
5H), 5.0 (s, 2H), 4.9–4.8 (m, 1H), 4.35–3.7 (m, 4H), 2.8 (s, 3H),
2.49–2.3 (m, 1H), 1.8–1.7 (m, 1H), 1.4 (s, 9H). 13C NMR (CDCl3)
: 154.0, 155.0, 136.0, 128.2, 127.7, 80.3, 68.0, 67.3, 66.0, 55.1,
52.2, 48.5, 36.7, 33.2, 28.0, 21.6. ESIMS: calc. for C19H28O8N2S m/z
446.3, found 448.0 (M + 1).
11b: 1H NMR (CDCl3) : 7.4 (s, 5H), 5.0 (s, 2H), 4.9–4.8 (m, 1H),
4.35–3.7 (m, 4H), 2.8 (s, 3H), 2.49–2.3 (m, 1H), 1.8–1.7 (m, 1H),
1.4 (s, 9H). 13C NMR (CDCl3) : 154.8, 155.6, 154.1, 136.0, 128.6,
79.7, 68.9, 66.8, 60.1, 54.8, 54.3, 52.2, 36.9, 36.7, 33.7, 31.2, 28.0.
ESIMS: calc. for C19H28O8N2S m/z 446.3, found 448.0 (M + 1).
(2R/S,4R)-N-1-(Benzyloxycarbonyl)-4-(tert-butyloxycarbonyl-
amino)proline methyl ester (9)
Compound 8 (4.74 g, 15 mmol) was dissolved in a dry ethyl acetate
and to this was added Raney-Ni (4 ml) and Boc-anhydride (4 ml,
18 mmol). The mixture was subjected to hydrogenation for 2 h at
a pressure of 40 psi in a Parr-hydrogenation apparatus when TLC
indicated complete conversion of starting material. The crude
product was purified by column chromatography to obtain 9 as a
gum (95% yield).
N-1-(Benzyloxycarbonyl)-(4R)-(tert-butyloxycarbonylamino)-
(2R/S)-(thymin-1-ylmethyl)pyrrolidine (12)
1
9a: []24D +9.0 (c 0.02 MeOH); H NMR (CDCl3) : 7.5 (s,
5H), 5.20–5.1 (m, 2H), 4.27 (m, 1H), 3.87 (m, 1H), 3.6–3.4 (m,
3H), 3.39–3.32 (m, 1H), 2.46–2 (m, 2H), 1.44 (s, 9H). 13C NMR
(CDCl3) : 173.5, 154.8, 154.4, 153.7, 136.0, 128.1, 127.7, 127.5,
79.36, 67.0, 57.7, 57.3, 53.0, 52.8, 51.9, 49.7, 48.7, 36.6, 35.5, 28.0.
ESIMS: calc. for C19H26N2O6 m/z 378.2, found 378.0.
9b: []24D −17.20 (c 0.022 MeOH); 1H NMR (CDCl3) : 7.4–7.29
(m, 5H), 5.22–5.11 (m, 2H), 5.08–5.04 (m, 1H), 4.66 (br s, 1H),
4.88–4.40 (m, 1H), 4.37–4.26 (m, 1H), 3.89–3.84 (m, 1H), 3.76 (s,
To compound 11 (1 g, 2.7 mmol) in dry DMF was added anhydrous
potassium carbonate (0.6 g, 5 mmol), thymine (0.51 g, 4 mmol) and
18-crown-6 (0.26 g, 0.98 mmol). The reaction mixture was allowed
to stir at 60 °C for 4 h. When TLC indicated complete conversion
of starting material, solvent was removed under vacuum. Silica gel
column chromatography yielded the pure product in the form of a
gum (0.4 g, 35%).
2 6 1 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1

View Article Online
12a: []24D +41.25 (c 0.008, MeOH); 1H NMR (CDCl3) : 7.3 (s,
5H), 5.1 (s, 2H), 4.2–3.6 (m, 5H), 3.49–3.3 (m, 1H), 2.3–2.1 (m,
1H), 1.9–1.7 (m, 5H), 1.45 (s, 9H). 13C NMR (CDCl3) : 163.8,
161.0, 155.4, 154.9, 151.7, 140.1, 135.9, 128.2, 127.9, 127.6, 127.5,
66.9, 56.6, 52.7, 49.5, 48.5, 36.1, 32.8, 28.0, 11.8. ESIMS: calc. for
C23H30O6N4 m/z 458.7, found 458.0.
(2S,4R)-4-(tert-Butoxycarbonylamine)-2-(thymin-1-ylmethyl)-
pyrrolidin-N-1-acetic acid (15)
1H NMR (D2O) : 7.31 (s, 1H), 4.45 (s, 1H), 4.12–3.9 (m, 5H),
3.04 (m, 1H), 2.78 (s, 1H), 2.06 (m, 2H), 1.70 (s, 3H), 1.20 (s,
9H). 13C NMR (D2O) : 172.18, 169.52, 164.79, 156.97, 152.76,
140.83, 110.88, 81.88, 65.20, 62.98, 60.10, 57.47, 57.18, 55.20,
48.53, 43.06, 36.72, 32.87, 29.50, 27.41, 11.75. ESIMS: calc.
for C17H26N4O6 m/z 382.4, found 383.0 (M + 1); max 270 nm,
 = 9.0 cm2 mol−1.
1
12b: []24D −10.0 (c 0.01, MeOH); H NMR (CDCl3)  7.3 (s,
5H), 5.1 (s, 2H), 4.2–3.6 (m, 5H), 3.49–3.3 (m, 1H), 2.3–2.1 (m,
1H), 1.9–1.7 (m, 5H), 1.45 (s, 9H). 13C NMR (CDCl3) : 163.83,
155.4, 154.9, 151.7, 140.1, 135.9, 128.2, 127.9, 127.6, 127.5, 79.0,
66.9, 56.6, 52.7, 49.5, 48.5, 36.1, 32.8, 28.0, 11.8. ESIMS: calc. for
C23H30O6N4 m/z 458.7, found 458.0.
Acknowledgements
P. S. L. thanks CSIR, New Delhi for a research fellowship. V. A.
K. thanks Department of Science and Technology, New Delhi for
financial support.
(2S,4S)-4-(tert-Butyloxycarbonylamino)-2-(thymin-1-ylmethyl)-
pyrrolidin-N-1-ethyl acetate (13)
Compound 12 (0.35 g, 0.8 mmol) was taken in methanol and to
this 10% Pd–C (0.3 g) was added. The mixture was subjected
to hydrogenation at a pressure of 50 psi for 6 h when TLC
indicated complete absence of starting material. The free amine was
immediately used for alkylation using ethyl bromoacetate (0.2 g,
1.6 mmol) in the presence 2.5 equivalents of DIPEA (0.08 ml,
0.35 mmol). The reaction was complete after 2 h as evident from
TLC. The solvent was completely removed under vacuum and the
crude product was purified by silica gel column chromatography to
obtain the pure product in the form of a gum (90% yield).
13a: []24D +5.0 (c 0.002, MeOH); 1H NMR (CDCl3) : 9.39 (s,
1H), 7.29 (s, 1H), 4.96 (s, 1H), 4.18 (q, 2H), 4.02–3.94 (m, 1H),
3.6–3.37 (m, 2H), 3.26–3.23 (s, 1H), 3.14–3.04 (m, 1H), 3.00–2.91
(m, 1H), 2.80–2.69 (m, 1H), 2.34–2.20 (m, 1H), 1.89 (s, 3H), 1.63–
1.52 (m, 1H), 1.39 (s, 9H), 1.28 (t, 3H). 13C NMR (CDCl3) : 154.9,
128.3, 127.9, 127.6, 66.9, 55.8, 51.9, 48.9, 34.4, 29.4, 28.0, 13.8,
11.8. ESIMS: calc. for C19H30N4O6 m/z 410.4, found 411.
13b: []24D −12.01 (c 0.004, MeOH); 1H NMR (CDCl3) : 9.41 (s,
1H), 7.29 (s, 1H), 4.77–4.58 (q, 2H), 4.33–3.99 (m, 3H), 3.90–3.28
(m, 3H), 2.97–2.83 (m, 2H), 2.64–2.48 (br s, 1H), 1.95 (s, 3H) 1.76–
1.59 (br s, 2H), 1.45 (s, 9H) 1.31–1.26 (t, 3H). 13C NMR (CDCl3) :
167.5, 162.8, 154.9, 151.1, 139.7, 63.4, 61.2, 60.9, 59.4, 58.6, 52.8,
52.1, 49.1, 41.8, 36.4, 28.1, 13.8, 12.6. ESIMS: calc. for C19H30N4O6
m/z 410.4, found 410.0.
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(2R,4R)-4-(tert-Butyloxycarbonylamino)-2-(thymin-1-
ylmethyl)pyrrolidin-N-1-acetic acid (14)
1H NMR (D2O) : 7.28 (s, 1H), 4.47–4.39 (m, 1H), 4.12–4.09 (m,
1H), 4–3.91 (m, 2H), 3.77–3.64 (m, 3H), 3.49–3.39 (m, 1H), 3.22
(br s, 1H), 2.54–2.48 (m, 1H), 1.64 (s, 4H), 1.14 (s, 9H). 13C NMR
(D2O) : 170.04, 166.51, 157.43, 153.63, 143.07, 111.26, 81.65,
65.70, 60.37, 55.92, 47.75, 47.28, 46.55, 34.44, 32.72, 31.83, 29.53,
27.34, 11.10. ESIMS: calc. for C17H26N4O6 m/z 382.4, found 383.0
(M + 1); max = 270 nm,  = 8.6 cm2 mol−1.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 0 4 – 2 6 1 1
2 6 1 1