Design, synthesis and DNA/RNA binding studies of nucleic acids comprising stereoregular and acyclic polycarbamate backbone: Polycarbamate nucleic acids (PCNA)

Article abstract of DOI:10.1039/c003405n

The designed, chiral, acyclic polycarbamate nucleic acids (PCNA) exhibited sequence and orientation specific binding to nucleic acids. Complexes of PCNA with DNA were as stable as PNA:DNA complexes and those with RNA were as stable as natural DNA:RNA complexes.

Full text of DOI:10.1039/c003405n

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design, synthesis and DNA/RNA binding studies of nucleic acids comprising
stereoregular and acyclic polycarbamate backbone: polycarbamate nucleic
acids (PCNA)†
Vangala Madhuri and Vaijayanti A. Kumar*
Received 18th February 2010, Accepted 7th June 2010
First published as an Advance Article on the web 24th June 2010
DOI: 10.1039/c003405n
The designed, chiral, acyclic polycarbamate nucleic acids (PCNA) exhibited sequence and orientation
specific binding to nucleic acids. Complexes of PCNA with DNA were as stable as PNA:DNA
complexes and those with RNA were as stable as natural DNA:RNA complexes.
a secondary amide linker to 2,3-diamino-1-propanol. The linking
Introduction
of individual monomers is via a carbamate linkage to form
Complete replacement of sugar-phosphate backbone by achiral,
polycarbamate nucleic acids (PCNA) (Fig. 1).
acyclic and uncharged scaffold in aminoethylglycyl peptide nucleic
acids (aegPNA) has been extensively studied in the last two
decades since its discovery in 1991.1 The strong and sequence
specific binding to both DNA and RNA along with its unique
strand invasion properties has proved to be extremely beneficial
to both chemists and biologists working towards the development
of ultimate antisense oligonucleotides.2 Extensive work has been
done to improve the properties of PNA and a large number of
structurally interesting PNA analogues have been synthesized over
the last decade. The balance between rigidity and flexibility has
been achieved by having an extended backbone with constrained
ring structures3 but so far no PNA analogue has been found to be
Fig. 1 Proposed PCNA compared with DNA and PNA structures.
better than PNA itself from an applications perspective.
The carbamate linkages were earlier utilized to form DNA
mimics by a few select groups as early as in 19744 but were
not further exploited in the development of antisense oligomers.
function to attach the nucleobase may circumvent the likelihood
The carbamate linker being shorter than the phosphate linker
The simple variation in the structure of PNA leading to PCNA
allows the presence of a chiral center in the monomer unit
and conserves six atom repeating backbone. A secondary amide
of cis/trans isomers present in tertiary amide linker groups in
probably destabilized the complexes with DNA or RNA. To
PNA. This may render the present backbone conformationally
ease the constraint of the linker group, acyclic carbamate linked
more flexible than PNA. The additional hydrogen bonding sites
dimer blocks were synthesized from sugar precursors and were
in the PCNA backbone might render it more water soluble as
compared with PNA. The hydroxy group is activated as a p-
incorporated into oligomers replacing a dimer sugar-phosphate
block. This led to large destabilization of the duplexes probably
nitrophenyl carbonate and the oligomerization may be effected
because of conformational freedom of the modified backbone
by the formation of carbamate linker groups.
in an otherwise unmodified DNA.5 The cytosine-carbamate
The synthesis of thymine, cytosine, guanine and adenine con-
hexamer joining morpholino-nucleosides formed highly stable
taining activated monomer blocks is accomplished from naturally
complexes with DNA but failed to recognize complementary
occurring chiral amino acid, L-serine, that would yield stereo-
RNA sequences.6 We, in our laboratory, synthesized pyrrolidinyl
regular polypyrimidine and mixed purine/pyrimidine PCNA
carbamate oligonucleotides, but too much flexibility in the linker
sequences using solid phase synthesis methodology. We also
group led to destabilization of the complexes.7 In this paper we
present our results of their binding studies to complementary
introduce a fresh approach to make the DNA mimics with an
DNA/RNA sequences.
uncharged polycarbamate backbone which is structurally closely
related to PNA. The chiral centre corresponds to C-4¢ in natural
nucleic acids and the individual nucleobases are attached through
Results and Discussion
Synthesis of monomer units
Division of Organic Chemistry, National Chemical Laboratory, Pune 411008,
India. E-mail: va.kumar@ncl.res.in; Fax: +91 20 25602623; Tel: +91 20
25602623
The simple organic transformations to get the desired monomers
are outlined in Scheme 1. The free hydroxy group in protected L-
serine derivative 1 was converted to TBDMS ether. The reduction
of the ester group using NaBH4 in MeOH gave alcohol 2. The
alcohol 2 was then converted into azide via mesylate followed
† Electronic supplementary information (ESI) available: 1H, 13C NMR
and mass spectra data for all new compounds, HPLC and MALDI-TOF
of PCNA sequences 1–4, UV Job’s Plot and UV-Tm studies of all the
complexes. See DOI: 10.1039/c003405n
3734 | Org. Biomol. Chem., 2010, 8, 3734–3741
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Scheme 1 Synthesis of monomer units to form PCNA oligomers.
by reduction over Ra–Ni/H2 and in situ Boc protection of the
primary amino group to get 3c. Compound 3c was subjected
to hydrogenation over Pd–C to remove the CBz protection
and the free amino group was acetylated using chloroacetyl
chloride/NaHCO3 in dioxane–water to get 4b. The individual
nucleobases thymine, N6-CBz-adenine, N4-CBz-cytosine and 2-
amino-6-chloropurine were reacted with 4b in the presence of
K2CO3 to get individual protected monomer units 5a–d. The
exocyclic amino group of cytosine and adenine was protected as
CBz but in the case of 2-amino-6-chloropurine and thymine, the
base protection was not necessary. The 2-amino-6-chloropurine
derivative 5d was converted to 5e with 0.75 M NaOH in 1 : 1
methanol–water. The enol ether in 6-OMe guanine derivative
is hydrolysed in acidic medium and gets deprotected to give
guanine derivative at the end of solid phase synthesis during TFA–
TFMSA treatment.8 5e was therefore carried forward in further
steps as protected guanine derivative. Individual compounds
5a–c and 5e were deprotected using TBAF in dry THF or
I2/MeOH to get intermediate protected monomer units 6a–c and
6e. Individual monomers 6a–c and 6e were then activated using p-
nitrophenyloxycarbonyl chloride to get 7a–c and 7e in good overall
yield. All the new compounds were adequately characterized by
1H, 13C NMR and LC-MS mass spectroscopic analysis.
attached to the solid support was deprotected using 50% TFA in
DCM. The resin was washed with DCM and was treated with
5% DIPEA in DCM. The activated monomers 7a–c and 7e (3–
5 equivalents) were dissolved in 200 mL of dry DMF and the
solution was added to the resin under dry conditions. After 2 h the
completion of the coupling reaction was confirmed by negative
Kaiser test.9 The cycles including these steps (Scheme 2, a, b and
c) were then continued until completion of the oligomer synthesis.
After each coupling step (Scheme 2, c), the excess monomer could
be recovered as no other reagent is used for coupling. The resin
bound oligomers were subjected to treatment with TFA/TFMSA4
to yield completely deprotected carbamate oligomers with a
C-terminal amide group. The oligomers were precipitated using
ether and were further purified by RP-HPLC. The purity of the
oligomers was rechecked by analytical RP-HPLC. The complete
Synthesis of PCNA oligomers
The synthesis of PCNA oligomers was carried out in a sequential
manner using MBHA resin to which L-lysine is attached as a
first amino acid. The sequences were planned so that we could
evaluate the feasibility of synthesis with t8 oligomer (PCNA1),
polypyrimidine sequences containing mixed t and c monomers,
to evaluate parallel/antiparallel triplex binding (PCNA2 and
PCNA3), and a mixed purine–pyrimidine sequence (PCNA4) to
evaluate the duplex binding as well as parallel/antiparallel binding
selectivity. The Boc-protection of a-amino group of the lysine
Scheme 2 Solid phase synthesis of PCNA, small letters a, t, g and c denote
PCNA monomer units.
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Table 1 PCNA sequences
Seq. code
PCNA1
PCNA2
PCNA3
PCNA4
PCNA Sequencea
Ret. time/min
Calculated/Found MW (mol. formula)
H-ttt ttt tt- Lys NH2
H-tct ctt tct t- Lys NH2
H-ctt ctt cct t- Lys NH2
H-gta gat cac t- Lys NH2
10.0
9.8
9.2
8.9
2401.43/2402.74
(C94H127O41N35+H+)
2922.56/2921.79
(C113H155O48N46)
2905.18/2906.03
(C112H151O47N47+H+)
3012.46/3021.6
(C114H147O41N60+Li+)
a t, c, g, a are PCNA monomers.
Table 2 Comparative UV-Tm (◦C) data for PCNA, PNA and DNA complexes with cDNA and RNA sequencesa
Sequences
UV-Tm/◦C
PCNA1 H-tttttttt-Lys
DNA 5¢TTTTTTTT3¢
PNA1 H-TTTTTTTT-Lys
DNA1: 5¢GCAAAAAAAACG3¢
RNA1: 5¢GCAAAAAAAACG3¢
50.1
nd11,b
45.012,c
30.1, (32.9)d, (37.7)e
PCNA2 H-tctctttctt-Lys
54.3
2011
DNA14 5¢TCTCTTTCTT3¢
26.0
27.6
DNA15 5¢CTTCTTCCTT3¢
29.1
30.9
DNA16 5¢GTAGATCACT3¢
31.6
32.0
DNA2: 5¢AAGAAAGAGA3¢
RNA2: 5¢AAGAAAGAGA3¢
31.6
PCNA3 H-cttcttcctt-Lys
PNA3 H-CTTCTTCCTT-Lys
DNA3: 5¢AAGGAAGAAG3¢
RNA3: 5¢AAGGAAGAAG3¢
51.4
52.013,c
31.9, (34.6)d, (39.4)e
PCNA4 H-gtagatcact-Lys
53.4
30.4
PNA4 H-GTAGATCACT-Lys
DNA4: 5¢AGTGATCTAC3¢
RNA4: 5¢AGUGAUCUAC3¢
52.014
a 10 mM Sodium phosphate buffer and 10 mM NaCl, 1 mM PCNA and hyperchromicity 8–12%. All values are an average of at least 3 experiments and
accurate within 0.5 ◦C. b nd not detected, c please refer to ESI page 42, d 10 mM sodium phosphate buffer and 10 mM NaCl, 1.66 mM PCNA, e 10 mM
sodium phosphate buffer and 10 mM NaCl, 2.5 mM PCNA.
deprotection of adenine, guanine and cytosine residues and
structural integrity of the oligomers was established by MALDI-
TOF mass spectral analysis (Table 1).
the literature12–14 (Table 2, column 4, also see ESI†). We confirm
this strong DNA:PCNA binding by gel shift assay where the triplex
mobility was found to be similar to aegPNA:DNA triplex (please
refer to ESI†). The mixed purine–pyrimidine sequence PCNA4
was also found to be highly stable duplex (DNA4:PCNA4) and the
Tm was found to be 53.4 ◦C.
UV-Tm studies
The most interesting results are those for RNA binding. The
observed UV-Tm for PCNA:RNA triplex were much less at 1 mM
concentration, compared to the PCNA:DNA triplexes. To confirm
the binding of PCNA with complementary RNA, we studied the
UV-Tm for RNA1:PCNA1 and RNA3:PCNA3 at higher strand
concentrations (1.66 mM and 2.5 mM, Table 2). As expected, we
observed concentration dependent increase in the triplex melting.
The mixed purine–pyrimidine duplex PCNA4:RNA4 was also
less stable than the PCNA4:DNA4 duplex (Table 2) but the Tm
was comparable with natural DNA16:DNA4 or DNA16:RNA4
duplexes at high salt concentrations.
The effect of salt concentration on the stability of these duplexes
was then studied◦. The change in Tm was found to be comparable
(DTm +0.1–3.7 C) to that of PNA:DNA/RNA complexes15
(please refer to ESI†). In parallel cDNA orientation (p), the bind-
ing of PCNA (PCNA2:DNA9, PCNA3:DNA13, PCNA4:DNA12)
is reduced considerably (Table 3) in contrast to achiral PNA
sequences.14 The mismatch studies were carried out to study the
sequence specific binding of PCNA to DNA. The complexes with
mismatched base pair show reduced Tm with broad transitions
(Table 3), and DTm was found to be the least for a G:T mismatch
as found in the case of the mismatched DNA:DNA duplexes.16
The polypyrimidine sequences PCNA1, PCNA2 and
PCNA3 were subjected to UV-Job’s plot10 with DNA1
(5¢GCAAAAAAAACG 3¢), DNA2 (5¢AAGAAAGAGA 3¢) and
RNA3 (5¢AAGGAAGAAG 3¢) respectively. The stoichiometry of
binding was found to be 2 : 1 in each case. To find the strength
of binding, UV-Tm experiments were carried out using the
annealed samples of DNA:PCNA and RNA:PCNA in 1 : 2
stoichiometry. The parallel DNA sequences used were the same
as DNA2 and DNA3, but in the reverse direction. The single
mismatch discrimination was studied using corresponding DNA
sequences with a single mismatch in the central position. The
mixed purine–pyrimidine sequence with either PCNA or DNA
backbone PCNA4 or DNA16 (5¢GTAGATCACT 3¢) was used in
1 : 1 stoichiometry with DNA4/RNA4 (5¢AGTGATCTAC 3¢/5¢
AGUGAUCUAC 3¢) for the melting studies. The results are
presented in Table 2.
The results indicate that sequences with PCNA backbone (N-
terminus of PCNA corresponding to 5¢-terminus of DNA) bind
strongly with cDNA in antiparallel orientation. The binding of
PCNA sequences PCNA1, PCNA3 and PCNA4 with antiparallel
DNA sequences at 1 mM concentration was found to be compara-
ble with the binding of known PNA sequences earlier reported in
3736 | Org. Biomol. Chem., 2010, 8, 3734–3741
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Table 3 UV-Tm data for PCNA complexes with mismatched DNA and
singlet; d, doublet; t, triplet; br, broad; br s, broad singlet; m,
multiplet and/or multiple resonance), number of protons. 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 l35 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 b-cyanoethyl
phosphoramidite chemistry followed by ammonia treatment and
their purities checked by HPLC prior to the use. The RNA
oligomers were procured from Sigma–Aldrich Bangalore.
parallel cDNA sequencesa
Sequences
UV-Tm/◦C
PCNA1
43.6c
DNA5: 5¢ GCAAAGAAAACG 3¢ (G:T) mismatch
¯
¯
DNA6: 5¢GCAAATAAAACG 3¢ (T:T) mismatch
42.4c
¯
¯
PCNA2
DNA7: 5¢ AAGAGAGAGA 3¢ (G:T) mismatch
49.7c
DNA8: 5¢ AAGACAGAGA 3¢ (C:T) mismatch
45.4c
¯
¯
b
DNA9: 5¢ AGAGAAAGAA 3¢(p)
24.2c
¯
¯
PCNA3
DNA10: 5¢ AAGGTAGAA G 3¢ (T:T) mismatch
43.4c
DNA13: 5¢ GAAGAAGGA A 3¢(p)b
22.0c
¯
¯
PCNA4
DNA11: 5¢ AGTGTTCTA C 3¢ (T:T) mismatch
48.3c
b
DNA12: 5¢ CATCTAGTG A 3¢(p)
ndd
¯
¯
R-1-O-tert-butyldimethylsilyl-2-benzyloxycarbonylamino-1,3-
propanediol (2). A solution of O-tert-butyldimethylsilyl-2-N-
benzyloxycarbonylamino-L-serine methyl ester 1 (20 g, 54.4 mmol)
in methanol (200 mL) was cooled to 0 ◦C in an ice bath. Solid
NaBH4 (12.4 g, 32.64 mol) was added in portions for a period
of 30 min. The reaction mixture was stirred for a period of 3 h
and finally quenched using NH4Cl solution till pH is neutral.
Methanol was removed under reduced pressure and the residue
was extracted into EtOAc (4 ¥ 100 mL), washed with water and
brine. The organic layer was dried over anhydrous Na2SO4 and
solvent was removed under reduced pressure. The residue was
purified by column chromatography (20% EtOAc in petroleum
ether) affording compound 2 (15.6 g, 85%) as colourless oil.
[a]D20 + 8.0◦ (c 1.0, CHCl3); IR (CHCl3) nmax cm-1: 3436, 3106,
a 10 mM Sodium phosphate buffer and 10 mM NaCl, 1 mM PCNA, b p
parallel orientation, c Broad transitions with 3–6% hyperchromicity, d nd
no detectable transition.
The RNA targeting with uncharged or positively charged
oligomers with very high binding affinities sometimes leads to
unwanted non-targeted affinities and hence toxicity in cellular
assays.17 The modest stability of such sequences comprising novel
PCNA backbone may find application in antisense therapeutics.
Conclusions
In summary, a novel, uncharged, chiral backbone of PCNA
presented in this paper could be quite attractive for its applications
in therapeutics. The possibility of recovery of the excess monomer
used in the synthesis allows the use of large excess of the same
during the coupling step to improve yield at each coupling step
in addition to reducing the cost of synthesis for therapeutic
applications.
1
2954,1712, 1510; H NMR (200 MHz, CDCl3) d (ppm) 0.04 (s,
6H, Si–(CH3)2), 0.87 (s, 9H, Si–C(CH3)3), 3.61–3.87 (m, 5H 1-
¯
¯
¯ ¯
¯ ¯
CH2, 2-CH, 3-CH2), 5.10 (s, 2H, NHCOOCH2Ph), 5.41 (br, 1H,
¯
¯ ¯
carbamate NH), 7.35 (s, 5H, Ph); 13C NMR (50 MHz, CDCl3) d
(ppm) -5.6 (Si–(CH3)2),18.6 (Si–C–(CH3)3), 25.9 (Si–C–(CH3)3),
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
53.3 (2-CH), 62.9 (3-CH2), 63.6 (1-CH2), 67.2 (NHCOCH2Ph),
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
127.1 (Ph), 127.6 (Ph), 128.9 (Ph), 136.1 (Ph), 155.6 (NHCOO);
EI-MS calcd for C17H29NO4Si (M+): 339.19; found 340.47 (M++1),
Experimental
362.35 (M++Na).
R-1-O-tert-butyldimethylsilyl-2-benzyloxycarbonylamino-3-O-
mesyl-1,3-propane-diol (3a). To a solution of alcohol 2 (4 g,
11.7 mmol) in dry DCM (40 mL) triethylamine (4.8 mL,
35.1 mmol) was added dropwise at 0 ◦C under nitrogen followed
by dropwise addition of methanesulfonyl chloride (1.82 mL,
23.4 mmol) over a period of 15 min. The mixture was stirred for
a period of 2 h. The reaction mixture was checked for completion
of the reaction by TLC and was extracted in DCM (2 ¥ 75 mL),
washed with 5% KHSO4 sol. and dried over anhydrous Na2SO4.
The crude mixture was used for the next reaction without any
further purification.
General
All the reagents were purchased from Sigma–Aldrich and used
without purification. DMF and pyridine were dried over KOH
˚
and 4 A 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 per-
formed for purification of compounds on silica gel (100–200 mesh,
LOBA Chemie). TLCs were performed using dichloromethane–
methanol or petroleum ether-EtOAc solvent systems for most
compounds. Compounds were visualized with UV light and/or by
spraying with ninhydrin reagent subsequent to Boc-deprotection
(exposing to HCl vapours) and heating. 1H (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 (d (ppm)) are referred to internal TMS
R-1-O-tert-butyldimethylsilyl-2-benzyloxycarbonylamino-3-
azido-1-propanol (3b). A stirred mixture of mesylate 3a (6 g,
13.8 mmol) and NaN3 (10.78 g, 165.8 mmol) was heated to 65 ◦C
for a period of 5 h. After cooling, the solvent was removed under
reduced pressure at low temperature and the residue was extracted
into EtOAc (4 ¥ 75 mL). The combined organic layer was washed
with water and brine and dried over anhydrous Na2SO4. The
residue was further purified by column chromatography (10%
EtOAc in petroleum ether) to afford a colorless oil of azide 3b
(3.82 g, 76%). [a]D20 - 4.0◦ (c 1.0, CHCl3); IR (CHCl3) nmax cm-1:
1
1
for H and chloroform-d, DMSO-d6 for 13C NMR. H NMR
data are reported in the order of chemical shift, multiplicity (s,
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3334, 2954, 2929, 2102, 1724, 1255; 1H NMR (200 MHz, CDCl3)
(3-CH2), 42.5 (NHCOOCH2Ph), 51.4 (2-CH), 62.9 (1-CH2), 79.6
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
d (ppm) 0.05 (s, 6H, Si–(CH3)2), 0.88 (s, 9H, Si–C(CH3)3),
(NHCOOC(CH3)3), 156.6 (NHCOO-), 166.2 (NHCOCH2); EI-
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
3.48–3.87 (m, 5H, 1-CH2, 2-CH, 3-CH2), 5.0–5.10 (br s, 3H,
NHCOOCH2Ph, carbamate NH), 7.35 (s, 5H, Ph); 13C NMR
(50 MHz, CDCl3) d (ppm) -5.6 (Si–(CH3)2), 18.1 (Si–C–(CH3)3),
25.7 (Si–C–(CH3)3), 50.9 (3-CH2), 51.5 (2-CH), 61.7 (1-CH2), 66.8
MS calcd for C16H33ClN2O4Si (M+): 380.19; found 381.31 (M++1),
403.16 (M++Na).
¯
¯ ¯
¯
¯ ¯
R-1-O-tert-butyldimethylsilyl-2-(N1-thyminylacetylamino)-3-
tert-butyloxycarbonylamino-1-propanol (5a). A mixture of com-
pound 4b (3 g, 7.89 mmol), thymine (0.99 g, 7.89 mmol) and
anhydrous K2CO3 (1.19 g, 8.67 mmol) in dry DMF (40 mL)
was stirred at room temperature for 12 h under nitrogen. The
solvent was removed under reduced pressure and the residue was
extracted into EtOAc (2 ¥ 100 mL), washed with water, brine
and dried over anhydrous Na2SO4. The solvent was evaporated
and crude compound was purified by column chromatography
(70% EtOAc in petroleum ether) to afford protected thymine
monomer as white foam 5a (2.96 g, 80%). [a]2D0 +18. 0◦ (c 1.0,
CHCl3); IR (CHCl3) nmax cm-1: 3018, 1693, 1215, 757; 1H NMR
(200 MHz, CDCl3) d (ppm) 0.04 (s, 6H, Si–(CH3)2), 0.87 (s, 9H,
¯
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
(NHCOCH2Ph), 128.0 (Ph), 128.4 (Ph), 128.7 (Ph), 136.1, 155.0
¯
¯ ¯
(NHCOO-); EI-MS calcd for C17H28N4O3Si (M+): 364.19; found
¯ ¯
365.36 (M++1), 387.26 (M++Na).
R-1-O-tert-butyldimethylsilyl-2-benzyloxycarbonylamino-3-
tert-butyloxycarbonyl-amino-1-propanol (3c). To a solution of
azide 3b (6 g, 16.4 mmol) in dry EtOAc (25 mL) placed in
hydrogenation flask was added di-tert-butyl dicarbonate (4.9 mL,
R
21.4 mmol) and RANEYꢀ-nickel (2 mL). The mixture was
hydrogenated in Parr apparatus at room temperature (35–40 psi)
for 3 h. The slurry was filtered through celite and the filtrate
was evaporated under reduced pressure. The residue was purified
by column chromatography (15% EtOAc in petroleum ether) to
afford compound 3c (6.26 g, 87%) as colorless oil. [a]2D0 +22.0
(c 1.0, CHCl3);IR (CHCl3) nmax cm-1: 3348, 3012, 2954, 1704,
1514, 837; 1H NMR (200 MHz, CDCl3) d (ppm) 0.03 (s, 6H, Si–
(CH3)2), 0.88 (s, 9H, Si–C(CH3)3), 1.41 (s, 9H, tBoc), 3.27–3.43
¯
¯ ¯
Si–C(CH3)3), 1.42 (s, 9H, tBoc), 1.91 (s, 3H, thy-CH3), 3.22–3.33
¯
¯
¯ ¯
¯ ¯
(m, 2H, 3-CH2), 3.5–3.76 (m, 2H, 1-CH2), 3.91–4.03 (m, 1H,
2-CH), 4.27–4.30 (m, 2H, NHCOCH2-thy), 5.0 (br, 1H, NH),
¯
¯ ¯
7.02 (s, 1H, thy-CH C–CH3), 8.0 (br, 1H, thy-NH); 13C NMR
=
¯ ¯
(50 MHz, CDCl3) d (ppm) -5.4 (Si–(CH3)2), 12.3 (thy-CH3), 18.1
¯
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
(m, 2H, 1-CH2), 3.56–3.83 (m, 3H, 3-CH2, 2-CH), 4.98 (br, 1H,
NH), 5.09 (s, 2H, NHCOOCH2Ph), 5.32 (br, 1H, NH), 7.35 (s, 5H,
Ph); 13C NMR (50 MHz, CDCl3) d (ppm) -5.5 (Si–(CH3)2), 18.2
(Si–C–(CH3)3), 25.0 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3),
42.5 (3-CH2), 52.5 (1-CH), 63.6 (2-CH2), 66.7 (NHCOOCH2Ph),
(Si–C–(CH3)3), 25.8 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3),
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
41.9 (3-CH2), 50.3 (NHCOCH2-thy), 52.0 (2-CH), 62.6 (1-CH2),
79.7 (NHCOOC(CH3)3), 110.9 (thy-CH C–CH3), 140.9 (thy-
CH C–CH3), 151.1 (thy-2-CO), 157.1 (NHCOO-), 164.3 (thy-
4-CO), 166.6 (NHCOCH2-thy); EI-MS calcd for C21H38N4O6Si
(M+): 470.26; found 470.89 (M+), 492.71 (M++Na).
¯
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
=
¯
¯ ¯
=
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
79.5 (NHCOOC(CH3)3), 128.0 (Ph), 128.5 (Ph), 136.5 (Ph), 156.6
(NHCOO-); EI-MS calcd for C22H38N2O5Si (M+): 438.25; found
R-1-O-tert-butyldimethylsilyl-2-[N1-(N4 -benzyloxycarbonyl-
cytosinyl)-acetylamino]-3-tert-butyloxycarbonylamino-1-propanol
(5b). A mixture of compound 4b (2.5 g, 6.57 mmol), N4-
benzyloxycarbonyl-cytosine (1.611 g, 6.57 mmol) and anhydrous
K2CO3 (0.99 g, 7.23 mmol) in dry DMF (30 mL) was stirred
at room temperature for 12 h under nitrogen. The solvent was
removed under reduced pressure and the residue was extracted
to EtOAc (2 ¥ 100 mL) and dried over anhydrous Na2SO4. The
solvent was evaporated and the crude compound was purified by
column chromatography (75% EtOAc in petroleum ether) to afford
a white foam of cytosine monomer 5b (3.05 g, 79%). [a]2D0 +28. 0◦
(c 1.0, CHCl3); IR (CHCl3) nmax cm-1: 3341, 3018, 1689, 1215,
757; 1H NMR (200 MHz, CDCl3) d (ppm) 0.03 (s, 6H, Si–(CH3)2),
¯ ¯
439.01 (M++1), 460.86 (M++Na).
R-1-O-tert-butyldimethylsilyl-2-amino-3-tert-butyloxycarbonyl
amino-1-propanol (4a). To a solution of compound 3c (5 g,
11.4 mmol) in methanol (15 mL) placed in hydrogenation flask was
added Pd–C (0.5 g, 10 mol%). The mixture was hydrogenated in
Parr apparatus (70 psi) at room temperature for 5 h. The slurry was
filtered through celite and filtrate was evaporated under reduced
pressure. This crude compound was used for the next reaction
without further purification.
R-1-O-tert-butyldimethylsilyl-2-chloroacetylamino-3-N -tert-
butyloxycarbonylamino-1-propanol (4b). To a stirred solution of
the amine 4a (3.47 g, 11.4 mmol) in 1,4-dioxane (10 mL) was
added 10% Na2CO3 (8.45 g, 79.8 mmol) in water (85 mL) and 1,4-
dioxane (85 mL). The mixture was cooled to 0 ◦C, and chloroacetyl
chloride (4.53 mL, 57.0 mmol) was added in two portions. After
30 min dioxane was removed under reduced pressure and the
residue was extracted in EtOAc (2 x100 mL). The organic layer
was dried over anhydrous Na2SO4 and the solvent was evaporated
under reduced pressure. The residue was purified by column
chromatography (30% EtOAc in petroleum ether) affording the
chloro compound 4b (2.4 g, 80%) as colourless oil. [a]2D0 +18.0
(c 1.0, CHCl3); IR (CHCl3) nmax cm-1: 3411, 3016, 2954, 1704,
¯
¯ ¯
0.87 (s, 9H, Si–C(CH3)3), 1.39–1.45 (d, 9H, rotamers tBoc), 3.1–
¯
¯ ¯
4.25 (m, 6H, 1-CH2, 3-CH2, NHCOCH2-cyt), 4.49 (br, 1H, 2-CH),
¯
¯ ¯
5.20 (s, 2H, NHCOCH2Ph), 7.28–7.34 (br s, 6H, Ph, cyt-5-CH),
¯
¯ ¯
7.61–7.65 (d, 1H, cyt-6-CH, DJ = 7.43 Hz), 8.65 (br,1H, NH);
13C NMR (50 MHz, CDCl3) d (ppm) -5.5 (Si–(CH3)2), 18.4 (Si–
¯
¯ ¯
C–(CH3)3), 25.8 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3), 41.7
¯
¯
¯ ¯
¯ ¯
¯ ¯
(3-CH2), 50.4 (2-CH), 62.8 (NHCOCH2-cyt), 67.3 (1-CH2), 80.1
(NHCOOC(CH3)3), 95.8 (cyt-5-CH), 127.6 (Ph), 128.1 (Ph), 128.5
(Ph), 135.3 (Ph), 149.6 (cyt-6-CH), 153.1 (NHCOOCH2Ph), 156.3
(NHCOOC(CH3)3), 157.8 (NHCOOCH2Ph), 166.7 (NHCOCH2-
cyt); EI-MS calcd for C28H45N5O7Si (M+): 591.31; found 590.6543
(M+), 612.6613 (M++Na).
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
1
1670, 1525, 1255; H NMR (200 MHz, CDCl3) d (ppm) 0.06 (s,
6H, Si–(CH3)2), 0.89 (s, 9H, Si–C(CH3)3), 1.42 (s, 9H, tBoc), 3.29–
¯
¯
¯ ¯
¯ ¯
3.45 (m, 2H, 3-CH2), 3.56–3.83 (m, 2H, 1-CH2), 4.01 (m, 1H,
2-CH), 4.02 (s, 2H, NHCOCH2Cl), 5.02 (br, 1H, carbamate NH);
13C NMR (50 MHz, CDCl3) d (ppm) -5.5 (Si–(CH3)2), 18.2 (Si–
R-1-O-tert-butyldimethylsilyl-2-[N9-(N6 -benzyloxycarbonyl-
adeninyl)-acetylamino]-3-tert-butyloxycarbonylamino-1-propanol
¯
¯ ¯
(5c).
A mixture of compound 4b (1.8 g, 4.73 mmol),
¯
¯ ¯
C–(CH3)3), 25.7 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3), 42.1
N6- benzyloxycarbonyladenine (1.274 g, 4.73 mmol) and
¯
¯
¯ ¯
¯ ¯
¯ ¯
3738 | Org. Biomol. Chem., 2010, 8, 3734–3741
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
anhydrous K2CO3 (0.719 g, 5.2 mmol) in dry DMF (25 mL)
under nitrogen was stirred at room temperature for 12 h. The
solvent was removed under reduced pressure and the residue was
extracted to EtOAc (2 ¥ 100 mL), washed with water, brine and
dried over anhydrous Na2SO4. The solvent was evaporated and the
crude compound was purified by column chromatography (80%
EtOAc in petroleum ether) to afford a white solid of protected
monomer 5c (1.8 g, 65%). [a]2D0 +8.0 (c 1.0, CHCl3); IR (CHCl3)
(s, 6H, Si–(CH3)2), 0.76 (s, 9H, Si–C(CH3)3), 1.41 (s, 9H, tBoc),
3.28–3.67 (m, 5H, 1-CH2, 2-CH, 3-CH2), 4.04 (s, 3H, OMe), 4.66–
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
4.68 (br, 2H, NHCOCH2-pu), 5.14 (br, 2H, pu-2-C–NH2), 7.61
¯
¯
¯ ¯
(s, 1H, pu-8-CH); 13C NMR (50 MHz, CDCl3) d (ppm) -5.6 (Si–
(CH3)2), 18.0 (Si–C–(CH3)3), 25.6 (Si–C–(CH3)3), 28.3 (NHCOO–
¯
¯ ¯
¯ ¯
¯ ¯
¯
C–(CH3)3), 41.8 (3-CH2), 46.8 (NHCOCH2-pu), 51.6 (2-CH), 54.0
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
(pu-OMe), 62.7 (1-CH2), 79.7 (NHCOOC(CH3)3), 114.9 (pu-5-C),
¯
¯ ¯
139.5 (pu-8-C), 153.5 (pu-4-C), 156.7 (NHCOOC(CH3)3), 159.7
(pu-2-C), 161.6 (pu-6-C), 166.6 (NHCOCH2-pu), 174.61; EI-MS
calcd for C22H39N7O5Si (M+): 509.28; found 510.5521 (M++1),
¯ ¯
1
nmax cm-1: 3339, 3018, 1690, 1215, 757; H NMR (200 MHz,
¯ ¯
CDCl3) d (ppm) 0.01 (s, 6H, Si–(CH3)2), 0.83 (s, 9H, Si–C(CH3)3),
¯
¯
¯ ¯
¯ ¯
1.37 (s, 9H, tBoc), 3.28–3.75 (m, 4H, 1-CH2, 3-CH2), 3.96 (m,
532.5439 (M++Na).
1H, 2-CH), 4.86 (s, 2H, NHCOCH2-ade), 7.35–7.5 (m, 5H, Ph),
¯
¯ ¯
R-2-(N1-thyminylacetylamino)-3-tert-butyloxycarbonyl amino-
1-propanol (6a). A solution of 5a (1.5 g, 3.189 mmol) in dry
THF (10 mL) was cooled to 0 ◦C and TBAF (2.1 mL of 1M
TBAF, 2.12 mmol) was added slowly. The reaction was allowed
to stir for 2.5 h. THF was removed and the residue was extracted
into EtOAc (2 ¥ 100 mL), dried over anhydrous Na2SO4. The
solvent was evaporated and the crude compound was purified
by column chromatography (90% EtOAc in petroleum ether) to
afford a white solid 6a (0.84 g, 75%). [a]2D0 -6.0 (c 1.0, MeOH);
IR (CHCl3) nmax cm-1: 3427, 3106, 2954, 1712, 1510, 1215, 757;
1H-NMR (200 MHz, CDCl3) d (ppm) 1.43 (s, 9H, tBoc), 1.89
(s, 3H, thy-CH3), 3.86–4.26 (m, 6H, 1-CH2, 3-CH2, NHCOCH2-
8.05 (s, 1H, ade-8-CH), 8.19 (s, 1H, NH), 8.75 (s, 1H, ade-2-
CH); 13C NMR (50 MHz, CDCl3) d (ppm) -5.5 (Si–(CH3)2), 18.4
¯
¯ ¯
(Si–C–(CH3)3), 25.8 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3),
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
41.7 (3-CH2), 50.4 (NHCOCH2-ade), 52.5 (2-CH), 62.8 (1-CH2),
67.3 (NHCOOCH2Ph), 80.1 (NHCOOC(CH3)3), 128.2 (Ph), 128.5
(Ph), 128.3 (Ph), 139.4 (Ph), 140.1 (ade-8-CH), 149.7 (ade-4-
CH), 153.0 (ade-2-C), 156.3 (NHCOO(CH3)3), 157.8 (ade-6-C),
163.6 (NHCOOCH2Ph), 166.7 (NHCOCH2-ade); EI-MS calcd
for C29H43N7O6Si (M+): 613.30; found 612.4265 (M+), 634.2770
(M++Na).
¯
¯
¯ ¯
¯ ¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
R-1-O-tert-butyldimethylsilyl-2-[N9-(2-amino-6-chloropurinyl)-
¯
¯ ¯
acetylamino]-3-tert-butyloxycarbonylamino-1-propanol (5d).
A
thy), 5.30 (br, 1H, 2-CH), 7.05 (s, 1H, thy-6-CH), 8.14 (br,1H,
thy-NH); 13C-NMR (50 MHz, CDCl3) d (ppm) 12.4 (thy-CH3),
mixture of compound 4b (3 g, 7.89 mmol), 2-amino-6-
chloropurine (1.33 g, 7.89 mmol) and anhydrous K2CO3 (1.19 g,
8.67 mmol) in dry DMF (40 mL) was stirred at room temperature
for 12 h under nitrogen. The solvent was removed under reduced
pressure and the residue was extracted to EtOAc (2 ¥ 100 mL),
washed with water, brine and dried over anhydrous Na2SO4. The
solvent was evaporated and the crude compound was purified
by column chromatography (75% EtOAc in petroleum ether)
to afford a white solid of monomer 5d (3.3 g, 82%). [a]2D0 -8.
0◦ (c 1.0, CHCl3); IR (CHCl3) nmax cm-1: 3411, 3016, 2950,
¯
¯ ¯
28.9 (NHCOO–C– (CH3)3), 40.9 (3-CH2), 49.9 (2-CH), 52.1
¯
¯
¯
¯ ¯
¯ ¯
(NHCOCH2-thy), 61.5 (1-CH2), 78.5 (NHCOOC(CH3)3), 107.9
(thy-HC C-CH3), 142.3 (thy-HC C-CH3), 151.7 (thy-2-CO),
155.9 (NHCOOC(CH3)3), 165.1 (thy-4-CO), 166.7 (NHCOCH2-
thy); EI-MS calcd for C15H24N4O6 (M+): 356.17; found 379.20
(M++Na).
¯
¯
¯ ¯
¯ ¯
=
=
¯ ¯
¯ ¯
¯ ¯
R-2-[N1-(N4-benzyloxycarbonyl-cytosinyl)-acetylamino]-3-tert-
butyloxycarbonylamino-1-propanol (6b). Protected cytosine
derivative 5b (1 g, 1.69 mmol) was taken in dry methanol (12 mL)
and solid I2 (179 mg, 3.39 mmol) was added. After stirring the
reaction mixture for 6–8 h, it was quenched by adding Na2S2O3
till the brown solution becomes colorless. Methanol was removed
and the residue extracted into EtOAc (2 ¥ 100 mL), dried over
anhydrous Na2SO4. The solvent was evaporated and the crude
compound was purified by column chromatography (EtOAc to
5% EtOAc in methanol) to afford a colourless solid of compound
6b (0.6 g, 75%). [a]2D0 -6.0 (c 1.0, MeOH); IR (CHCl3) nmax cm-1:
1
1704, 1525, 1255, 757; H NMR (200 MHz, CDCl3) d (ppm)
0.02 (s, 6H, Si–(CH3)2), 0.79 (s, 9H, Si–C(CH3)3), 1.40 (s, 9H,
¯
¯
¯ ¯
¯ ¯
tBoc), 3.26–3.72 (m, 4H, 1-CH2, 3-CH2), 3.97–4.08 (br, 1H,
2-CH), 4.7 (m, 2H, NHCOCH2-am), 4.97 (br, 1H, carbamate
¯
¯ ¯
NH), 5.34 (br, 2H, am-2-C-NH2), 7.81 (s, 1H, am-8-C); 13C
¯
¯ ¯
NMR (50 MHz, CDCl3) d (ppm) -5.6 (Si–(CH3)2), 18.0 (Si–
¯
¯ ¯
C–(CH3)3), 25.6 (Si–C–(CH3)3), 28.3 (NHCOO–C–(CH3)3), 41.7
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
(3-CH2), 46.3 (NHCOCH2-am), 51.9 (2-CH), 62.6 (1-CH2), 79.7
(NHCOOC(CH3)3), 124.3 (am-5-C), 142.9 (am-8-C), 151.1 (am-
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
1
3432, 3110, 2958, 1710, 1510, 1215, 757; H NMR (200 MHz,
4-C), 153.8 (am-6-C), 159.5 (NHCOOC(CH3)3), 162.6 (am-2-
¯ ¯
CDCl3) d (ppm) 1.38 (s, 9H, tBoc), 3.4–3.9 (m, 6H, 1-CH2, 2-CH,
C), 166.1 (NHCOCH2-am); EI-MS calcd for C21H36ClN7O4Si
¯ ¯
(M+): 513.23; found 514.5155 (M++1), 516.5043 (M++2), 536.5173
3-CH2), 4.55 (s, 2H, NHCOCH2-cyt), 5.2 (s, 2H, cyt-NHCOCH2-
¯
¯
¯ ¯
¯ ¯
(M++Na), 538.5051 (M++2+Na).
Ph), 7.17 (br, 1H, NH), 7.3 (br s, 5H, Ph), 7.9 (m, 2H, cyt-5-CH,
6-CH); 13C NMR (50 MHz, DMSO-d6) d (ppm) 28.1 (NHCOO–
C– (CH3)3), 40.5 (3-CH2), 51.2 (NHCOCH2-cyt), 51.4 (2-CH),
R-1-O-tert-butyldimethylsilyl-2-[N9-(2-amino-6-methoxy-
purinyl)-acetylamino]-3-tert-butyloxycarbonylamino-1-propanol
(5e). Compound 5d (2 g) was taken in methanol (10 mL) and 0.75
N NaOH (10 mL) was added. The reaction mixture was allowed to
stir for 3.5 h. Reaction mixture was directly extracted into EtOAc
(2 ¥ 100 mL), washed with water, brine and dried over anhydrous
Na2SO4. The solvent was evaporated and the crude compound was
purified by column chromatography (80% EtOAc in petroleum
ether) to af◦ford colourless flakes of compound 5e (1.58 g, 80%).
[a]2D0 -12. 0 (c 1.0, CHCl3); IR (CHCl3) nmax cm-1: 3408, 3016,
2955, 1704, 1525, 757; 1H NMR (200 MHz, CDCl3) d (ppm) 0.05
¯
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
¯ ¯
60.7 (1-CH2), 66.4 (NHCOOCH2Ph), 77.8 (NHCOOC(CH3)3),
¯
¯
¯ ¯
¯ ¯
93.7 (cyt-5-C), 127.9 (Ph), 128.3 (Ph), 128.4 (Ph), 135.9 (Ph),
150.8 (cyt-6-C), 153.0 (NHCOOC(CH3)3), 154.9 (cyt-2-C), 155.8
¯ ¯
(NHCOOCH2Ph), 162.9 (cyt-4-C); EI-MS calcd for C22H31N5O7
(M+): 477.22; found 476.24 (M+), 498.21 (M++Na).
¯ ¯
R-2-[N1-(N6 -benzyloxycarbonyladeninyl)-acetylamino]-3-tert-
butyloxycarbonylamino-1-propanol (6c). A solution of protected
adenine compound 5c (1 g, 1.63 mmol) in dry THF (10 mL) was
cooled to 0 ◦C and TBAF (1.6 mL of 1M TBAF, 1.63 mmol) was
This journal is
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Org. Biomol. Chem., 2010, 8, 3734–3741 | 3739
©

View Article Online
added slowly. The reaction was allowed to stir for 2.5 h. THF was
removed and the residue was extracted into EtOAc (2 ¥ 100 mL),
dried over anhydrous Na2SO4. The solvent was evaporated and
the crude compound was used for the next step without further
purification.
crude compound was purified by column chromatography (85%
EtOAc in petroleum ether) to afford pale yellow foam of monomer
7b (0.93 g, 70%). IR (CHCl3) nmax: 3261, 3018, 1760, 1693, 1504,
1
1215, 757 cm-1; H NMR (200 MHz, CDCl3) d (ppm) 1.46 (s,
9H, tBoc), 3.25–3.49 (m, 2H, 3-CH2), 4.20–4.67 (m, 5H, 1-CH2,
2-CH, NHCOCH2-cyt), 5.19 (s, 2H, NHCOOCH2Ph), 7.33–7.37
¯
¯
¯ ¯
¯ ¯
R-2-[N9-(2-amino-6-methoxypurinyl)-acetylamino]-3-tert-
butyloxycarbonylamino-1-propanol (6e). A solution of silyl com-
pound 5e (1 g, 1.96 mmol) in dry THF (10 mL) was cooled to
(br s,7H, Ph, Ph-ortho to CO), 8.20–8.25 (d, 2H, Ph-meta to CO,
DJ = 9.09 Hz), 7.64–7.84 (m, 2H), 9.0 (br,1H, NH); EI-MS calcd
for C30H32N8O10 (M+): 642.23; found 663.23 (M++Na).
◦
0 C and TBAF (1.9 mL of 1 M TBAF, 1.96 mmol) was added
R-1-O-p-nitrophenoxycarbonyl-2-[N1-(N6 -benzyloxycarbonyl-
adeninyl)-acetylamino]-3-tert-butyloxycarbonyl amino-1-propanol
(7c). Compound 6c (0.25 g, 0.5 mmol) was taken in dry DCM
(5 mL) and cooled to 0 ◦C in an ice bath. Dry pyridine
(0.12 mL, 1.50 mmol) was added and allowed to stir for 10 min.
p-Nitrophenyl chloroformate (0.25 g, 1.25 mmol) dissolved in
DCM (2 mL) was added and the reaction stirred at room
temperature for 2 h. After completion, the reaction mixture was
extracted into DCM (2 ¥ 50 mL), washed with water, brine
and dried over anhydrous Na2SO4. The solvent was evaporated
and the crude mixture was purified by column chromatography
(80% EtOAc in petroleum ether) to afford pale yellow foam of
activated monomer 7c (0.19 g, 60%). IR (CHCl3) nmax cm-1:
slowly. The reaction was allowed to stir for 2.5 h. THF was
removed and the residue was extracted into EtOAc (2 ¥ 100 mL),
dried over anhydrous Na2SO4. The solvent was evaporated and
the crude compound was purified by column chromatography
(EtOAc to 5% EtOAc in methanol) to afford a white solid of
monomer 6e (0.54 g, 70%). [a]2D0 -8.0 (c 1.0, MeOH); IR (CHCl3)
1
nmax cm-1: 3434, 3116, 2964, 1712, 1504, 1215, 757; H NMR
(200 MHz, CDCl3 + DMSO-d6) d (ppm) 0.88 (s, 9H, tBoc), 2.45–
2.70 (m, 4H, 1-CH2, 3-CH2), 3.26 (m, 1H, 2-CH), 3.46 (s, 3H,
OMe), 4.19 (s, 2H, NHCOCH2-pu), 5.87 (s, 2H, pu-2-C–NH2),
¯
¯
¯ ¯
¯ ¯
¯ ¯
6.1 (br, 1H, NH), 7.57 (s, 1H, pu-8-CH); 13C NMR (50 MHz,
DMSO-d6) d (ppm) 28.1 (NHCOO–C– (CH3)3), 40.5 (3-CH2),
¯
¯
¯
¯ ¯
¯ ¯
¯ ¯
44.7 (NHCOCH2-pu), 51.4 ((2-CH), 53.1 (OMe), 60.7 (1-CH2),
¯
¯
1
¯ ¯
¯ ¯
3257,3018,1760,1658,1214,757; H NMR (200 MHz, CDCl3) d
(ppm) 1.40 (s, 9H, tBoc), 3.30–3.41 (m, 2H, 3-CH2), 4.21–4.30
77.8 (NHCOOC(CH3)3), 113.27 (pu-6-C), 140.6 (pu-8-C), 154.3
(pu-1-C), 155.8 (NHCOOC(CH3)3), 159.7 (pu-3-C), 160.5 (pu-5-
¯ ¯
C), 166.3 (NHCOCH3-pu); EI-MS calcd for C16H25N7O5 (M+):
(m, 3H, 2-CH, NHCOCH2-ade), 4.9 (m, 2H, 1-CH2), 5.29 (s, 2H,
¯
¯ ¯
¯ ¯
395.19; found 396.2658 (M++1), 418.2670 (M++Na).
NHCOOCH2-Ph), 7.35–7.41 (br s, 7H, Ph, Ph-ortho to CO), 8.03
¯
¯ ¯
(s, 1H, ade-8-CH), 8.12 (s, 1H, ade-2-CH), 8.23–8.28 (d, 2H, Ph-
meta to CO, DJ = 9.21 Hz), 8.71 (br, 1H, ade-6-C–NH); EI-MS
calcd for C30H32N8O10 (M+): 664.22; found 664.9990 (M++1).
R-1-O-p-nitrophenoxycarbonyl-2-(N1-thyminylacetylamino)-3-
tert-butyloxycarbonylamino-1-propanol (7a). Compound 6a (1 g,
2.807 mmol) was taken in dry DCM (12 mL) and cooled to 0 ◦C
in an ice bath. Dry pyridine (0.68 mL, 8.42 mmol) was added and
allowed to stir for 10 min. p-Nitrophenyl chloroformate (1.41 g,
7.019 mmol) dissolved in DCM (3 mL) was added and the reaction
allowed to stir at room temperature for 2 h. After completion,
the reaction mixture was extracted into DCM (2 ¥ 50 mL),
washed with water, brine and dried over anhydrous Na2SO4. The
solvent was evaporated and the crude compound was purified
by column chromatography (80% EtOAc in petroleum ether) to
afford pale yellow foam of activated monomer 7a (1.02 g, 70%). IR
(CHCl3) nmax cm-1: 3305, 3018, 1770, 1681, 1525, 1215; 1H NMR
(200 MHz, CDCl3) d (ppm) 1.41 (s, 9H, tBoc), 1.87 (s, 3H, thy-
CH3), 3.37 (m, 2H, 3-CH2), 4.32 (m, 4H, 1-CH2, NHCOCH2-thy),
R-1-O- p-nitrophenoxycarbonyl-2-[N9-(2-amino-6-methoxypurinyl)-
acetylamino]-3-tert-butyloxycarbonylamino-1-propanol
(7e).
Compound 6e (0.5 g, 1.26 mmol) was dissolved in dry DCM
(6 mL) and cooled to 0 ◦C in an ice bath. Dry pyridine
(0.307 mL, 3.79 mmol) was added and allowed to stir for 10 min.
p-Nitrophenyl chloroformate (0.635 g, 3.163 mmol) dissolved in
DCM (3 mL) was added and the reaction allowed to stir at room
temperature for 2 h. After completion, the reaction mixture was
extracted into DCM (2 ¥ 50 mL), washed with water, brine and
dried over anhydrous Na2SO4. The solvent was evaporated and
the crude compound was purified by column chromatography
(90% EtOAc in petroleum ether) to afford pale yellow foam of
activated monomer 7e (0.45 g, 65%). IR (CHCl3) nmax cm-1:
¯
¯ ¯
=
5.23 (br,1H, 2-CH), 7.06 (s, 1H, thy-CH C–CH3), 7.35–7.4 (d, 2H,
1
¯ ¯
3347, 3257, 3018,1760, 1658, 1214, 757; H NMR (200 MHz,
Ph-ortho to CO, DJ = 9.13 Hz), 8.24–8.28 (d, 2H, Ph-meta to CO,
CDCl3) d (ppm) 1.42 (s, 9H, tBoc), 3.28–3.46 (m, 2H, 3-CH2), 4.03
DJ = 9.08 Hz); 13C NMR (50 MHz, CDCl3) d (ppm) 12.17, 28.26,
36.56, 50.59, 53.18, 74.93, 79.95, 110.91, 122.0, 125.20, 141.21,
145.32, 151.40, 151.85, 155.36, 156.46, 164.62, 167.47; EI-MS
calcd for C22H27N5O10 (M+): 521.18; found 544.2196 (M++Na).
(s, 3H, OMe), 4.31 (br s, 3H, 2-CH, NHCOCH2-pu), 4.89 (br, 2H,
¯
¯ ¯
¯ ¯
¯ ¯
ade-2-C-NH2), 5.03 (br, 1H, 2-CH), 7.33–7.38 (d, 2H, Ph-ortho
¯
to CO, DJ = 9.14 Hz), 7.63 (br,1H, NH), 7.85 (br,1H, pu-8-CH),
8.26–8.3 (d, 2H, Ph-meta to CO, DJ = 9.16 Hz); EI-MS calcd
for C23H28N8O9 (M+): 560.20; found 561.0808 (M++1), 583.0296
(M++Na).
R-1-O-p-nitrophenoxycarbonyl-2-[N1-(N4 -benzyloxycarbonyl-
cytosinyl)-acetylamino]-3-tert-butyloxycarbonyl amino-1-propanol
(7b). Compound 6b (1 g, 2.104 mmol) was taken in dry DCM
(10 mL) and cooled to 0 ◦C in an ice bath. Dry pyridine (0.51 mL,
6.312 mmol) was added and allowed to stir for 10–15 min. p-
Nitrophenyl chloroformate (1.05 g, 5.26 mmol) dissolved in DCM
(3 mL) was added and the reaction was allowed to stir at room
temperature for 2 h. After completion, the reaction mixture was
extracted into DCM (2 ¥ 50 mL), washed with water, brine and
dried over anhydrous Na2SO4. The solvent was evaporated and the
Synthesis, cleavage from the solid support and purification of the
PCNA oligomers
The PCNA oligomers were synthesized manually by solid phase
peptide synthesis using the Boc-protection strategy. The solid
support used was Merrifield resin that was derivatized with
L-lysine (0.27 mmol g-1 resin) as the spacer amino acid. Synthesis
3740 | Org. Biomol. Chem., 2010, 8, 3734–3741
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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 diisopropy-
lamine (5% solution in CH2Cl2, v/v) and (iii) coupling of the free
amine with p-nitrophenyl carbonate activated monomer units 7a–
c,e (3 to 4 equivalents) 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.8 Since the coupling
efficiencies were found to be >98%, capping of the unreacted
amino groups was not found necessary.
The PCNA oligomers were cleaved from the solid support
using the TFA–TFMSA method to yield completely deprotected
oligomers with free amide at their carboxy termini.18 The resin-
bound PCNA oligomer (10 mg) was stirred in an ice-bath with
thioanisole (20 mL) and 1,2-ethanedithiol (8 mL) for 10 min.
TFA (120 mL) was then added and the stirring was continued
for another 10 min. TFMSA (16 mL) was added while cooling the
reaction in an ice-bath and stirring was continued for 1 h 15 min.
The 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 precipitated by adding cold ether. The precipitate was then re-
dissolved in 200 mL of deionized water to obtain the crude PCNA
oligomer. (In the case of oligomer PCNA4, the exocyclic methyl
protection of guanine was found to be cleaved with TFMSA–
TFA treatment). This was purified by HPLC-(RP-C18 analytical
column 25 ¥ 0.2 cm. 5 mm) with gradient elution: A to 100% B in
20 min. A = 0.1% TFA in H2O:ACN (95 : 5), B = 0.1% TFA in
H2O:ACN (50 : 50) with flow rate 1.5 mL min-1 (Linear gradient
from A to B in 20 min). The purity of the PCNA oligomer was
checked by analytical RP-HPLC on a C18 column and found to
be >90% (Supplementary Information†).
Acknowledgements
VM thanks CSIR, New Delhi for senior research fellowship. VAK
thanks CSIR (NWP0036), New Delhi, for research grants.
Notes and references
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The concentration of the PCNA oligomers was calculated on the
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complementary DNA/RNA oligonucleotide were mixed together
in a 1 : 1 molar ratio for mixed purine/pyrimidine sequence and
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buffer and 10 mM NaCl, 0.01 mM EDTA, (pH 7.3) to get a
final strand conc◦entration of 1 mM. The samples were annealed
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temperature, kept at room temperature for ~30 min and then,
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