Synthesis and evaluation of prolyl carbamate nucleic acids (PrCNA)

Article abstract of DOI:10.1081/NCN-100002517

Carbamate linked prolyl nucleic acids are obtained in high yield and purity under mild conditions in solution and solid phase. p-Nitrophenylchloroformate is used as the activating reagent for alcohol. Homooligomers of PrCNA do not bind to DNA. The introduction of this modification in PNA sequences destabilizes the triplxes, inspite of enhancement in the base stacking.

Full text of DOI:10.1081/NCN-100002517

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SYNTHESIS AND EVALUATION OF PROLYL CARBAMATE
NUCLEIC ACIDS (PrCNA)
Meena a , V. A. Kumar a & K. N. Ganesh b
a Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune, 411008,
India
b Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune, 411008,
India
Published online: 07 Feb 2007.
To cite this article: Meena , V. A. Kumar & K. N. Ganesh (2001) SYNTHESIS AND EVALUATION OF PROLYL CARBAMATE
NUCLEIC ACIDS (PrCNA), Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1193-1196
To link to this article: http://dx.doi.org/10.1081/NCN-100002517
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1193–1196 (2001)
SYNTHESIS AND EVALUATION OF PROLYL
CARBAMATE NUCLEIC ACIDS (PrCNA)
Meena, V. A. Kumar, and K. N. Ganesh∗
Division of Organic Chemistry (Synthesis), National Chemical
Laboratory, Pune 411008, India
ABSTRACT
Carbamatelinkedprolylnucleicacidsareobtainedinhighyieldandpurityunder
mildconditionsinsolutionandsolidphase. p-Nitrophenylchloroformateisused
as the activating reagent for alcohol. Homooligomers of PrCNA do not bind to
DNA. The introduction of this modification in PNA sequences destabilizes the
triplxes, inspite of enhancement in the base stacking.
Carbamate linked nucleic acids are known to bind with complementary DNA
strands with higher thermal stability (1,2). Carbamate linkage being uncharged
these are expected to be capable of penetrating the cell membrane and are more
stable towards enzymes (3). Earlier, prolyl nucleic acids having peptide backbone
were prepared in our laboratory and it was found that the homooligomers of prolyl
PNA did not bind to complementary DNA strand (4). This was probably because of
the constraint in the flexible PNA backbone (5). We replace the peptide linkages in
prolyl nucleic acids with carbamate linkages thus increasing the number of atoms
in the backbone while keeping the stereogenic centers intact. Here, we report the
synthesis and the binding studies of the carbamate linked prolyl nucleic acids.
The activated monomer building blocks 4a and 4b were synthesized from
trans-L-hydroxy proline 1 (Scheme 1). After stepwise protection of ring nitrogen as
benzylcarbamate and acid function as methyl ester the hydroxy group was converted
tothecorrespondingazideintwosteps. Theazidewasthenselectivelyhydrogenated
using Raney Ni and the resultant amine was protected as t-butoxycarbamate. The
∗Corresponding author.
1193
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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1194
MEENA, KUMAR, AND GANESH
Scheme 1. (a) CBzCl, NaHCO3 Water/Dioxane (b) SOCl2, MeOH (c) MsCl/py (d) NaN3, DMF,
70◦C (e) H2, Raney Ni, MeOH (f) BocN3, TEA, DMSO, 50◦C (g) LIBH4, THF (h) H2, Pd-C, MeOH
(i)Thyminylacetic acid, DCC, HOBt, DMF (j) 4-N-CBz-cytosinylacetic acid, DCC, HOBt, DMF (k)
p-nitrophenylchloroformate, Dioxane, Py (l) p-nitrophenylchloroformate, Dioxane, TEA (m) 50%
TFA./DCM, DIPEA (n) DMF/DIPEA.
resulting protected ester was reduced using LiBH4/THF to get 2. The Ring nitrogen
was then deprotected followed by coupling with nucleobaseacetic acid in presence
of DCC and HOBt to obtain 3a (B = Thymine, T) and 3b (B = Cytosine, CCBz). The
Boc protected amino alcohols 3a and 3b were activated to give the corresponding
p-nitrophenylcarbonates (6) 4a and 4b respectively. All the new compounds were
characterized by 1H, 13C NMR and mass spectrometry (7).
The activated monomer synthons 4a and 4b along with standard PNA
monomers (8) were used to synthesize the oligomer sequences 6 (in solution phase)
(1,2) and 7–13 (on solid support) using standard protocols. The sequences 7–13
were cleaved from the support using standard conditions used for the cleavage of
peptides (8). Stability of the carbamate linkage under these conditions was estab-
lished by independent treatment of 6 with TFMSA/TFA and methanolic ammonia
and HPLC and mass spectral analysis of the samples. The carbamate linkage was
found to be stable in acidic condition but degradation was observed under basic
condition. The oligomers 7–13 were purified by FPLC and purity was checked by
HPLC. The homogeneity of each of the oligomer was established by MALDI-TOF
DNA sequences
PrCNA/PNA sequences
14 5ꢁ-G C A A A A A A A A C G-3ꢁ
15 5ꢁ-A A A A A G A G A-3ꢁ
6
7
8
9
Boc-T∗T–OH∗
H-T∗T∗T∗-(β-ala)-OH
H-T T T T T T T T-(β-ala)-OH
H-T T T T T T T∗T-(β-ala)-OH
10 H-T T T T∗ T T T T-(β-ala)-OH
11 H-T∗ T T T T T T T-(β-ala)-OH
12 H-T∗ T∗ T∗ T∗ T∗ T∗ T∗ T∗-(β-ala)-OH
13 H-T∗ C∗ T∗ C∗ T∗ T∗ T∗ T∗-(β-ala)-OH
Scheme 2.

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PROLYL CARBAMATE NUCLEIC ACIDS
1195
Figure1. Meltingcurvesfor(PrCNA/PNA)2:DNAcomplexesinSodiumphosphatebufferatpH7.3.
Concentration of DNA strand was taken as 1.5 µM.
mass spectrometry (7). The effect of the carbamate linked prolyl units on the struc-
tural features of the backbone of the oligomers was studied using CD spectroscopy.
Compounds 5, 6 and 7 showed sequential enhancement in the CD signal at 260 nm
with increasing number of three prolyl thymine units and plateaued in the prolyl
octamer.
The UV melting studies of the complexes of oligomers sequences 8–13 with
complementary DNA strands 14–15 were performed to evaluate the thermal stabil-
ity (Fig. 1). The homooligomers 12 and 13 did not bind to DNA. PNA sequence, 10,
with T∗ at the center of the sequence did not bind to DNA. When the modification is
at the N-terminus as in PNA 11, marginal destabilization compared to PNA2:DNA
Figure 2. CD curves for triplexes in Sodium phosphate buffer at pH 7.3.

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1196
MEENA, KUMAR, AND GANESH
Table 1. UV Melting Temperatures of Triplexes
Complexes
(8)2:14
44.5
(9)2:14
31.5
(10)2:14
—
(11)2:14
44
(12)2:14
—
(13)2:15
—
Tm ◦C
% Hyperchromicity
18.2
18.4
6.8
22.2
7.1
9.3
complex was observed whereas the C-terminal modification in 9 destabilized the
triplex by 13◦C. The reassociation process of DNA hybridization was monitored
by decrease in absorbance with lowering temperature. Absorbance Vs temperature
profiles for PrCNA:DNA complexes showd that the rate of reassociation to form
triplexes is slower than the rate of dissociation and is similar to DNA:PNA com-
plexes. CD curves obtained for complexes (9)2:14 and (11)2:14 were similar in
pattern to (8)2:14 (Fig. 2).
Inconclusion, wehavesuccessfullydevelopedsolidphasesynthesischemistry
for carbamate linked oligomers and the insertion of prolyl carbamate linkage into
PNA. The results from DNA binding studies show that carbamate linked prolyl
homooligomers do not bind to DNA possibly due to incompatible internucleobase
distances in the PrCNA. Placement of carbamate linkage either at C-terminal or
N-terminal in PNA causes destabilization of triplexes.
REFERENCES
1. Stirchak, E. P.; Summerton, J. E.; Weller, D. D. J. Org. Chem. 1987, 52, 4202.
2. Stirchak, E. P.; Summerton, J. E.; Weller, D. D. Nucl. Acids Res. 1989, 15, 6129.
3. Habus, I.; Tesamani, J.; Agarwal, S. BioMed. Chem. Lett. 1994, 4, 1065.
4. Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetrahedron. 1999, 55, 177.
5. Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 9, 916.
6. Letsinger, R. L.; Ogilvie, K. K. J. Org. Chem. 1967, 32, 296.
1
7. Compound 4a H NMR in CDCl3 δ ppm 8.25 (d 2H phenyl J = 0.2), 7.4 (d 2H
phenyl J = 0.2), 7 (s 1H thy 6H), 4.5 (m 5H OMe & Pro C5), 1.9 (3H Thy Me),
1
1.4 (d 9H Boc). Compound 4b H NMR in CDCl3 δ ppm 8.25 (d 2H nitroPhe), 7.6
(d 1H Cyt C6 J = 0.15), 7.25 (m 7H cbz, nitroPhe, Cyt C5), 5.2 (s 2H benzyl CH2),
4.5 (m 5H benzyl CH2, Proline C2-mehyl, C5), 1.4 (s 9H t-Boc). Mass (FAB) m/z =
667(M+1), 689(M+Na) calculated mass 666. Compound 6 Mass(FAB) m/z = 691
(M+1), 713(M+Na) Calculated mass 690. Compound 9 Mass (MALDI TOF) m/z =
2268.8 calculated mass 2259 and 10 m/z = 2266.5 calculated mass 2259.
8. Nielsen, P. E.; Egholm, M. Eds. In Peptide Nucleic Acids: Protocols and Applications,
1999, Horizon Scientific Press, Norfolk, U.K.

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