Pyrrolidin-1-yl Acetic Acid Derivatives of Thymine
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Table 1. PNA2:DNA sequences and UVTmꢀC (DTmꢀC, % Hyperchromicity).
DNA 50-G C A A A A A A A A C G-30
Stereochemistry
(2R,4R) (2S,4R)
43.5
PNA
(2S,4S)
(2R,4S)
1. H-T T T T T T T T-NH-b-alanine-OH
2. H-T T T T T T T t-NH-b-alanine-OH
3. H-T T T T T T T t-NH-b-alanine-OH
Control PNA
37( ꢁ 6.5, 10.6) 44( þ 0.5, 23.5) 46.5( þ 3, 19.3) ND(ꢁ, 7.6) 3.
ND(ꢁ, 4.7) ND(ꢁ, 7.6) ND(ꢁ, 12.4) ND(ꢁ, 5.8)
t indicates incorporation of modified PNA unit in aegPNA, ND not detected, Buffer conditions-10 mM sodium
phosphate. 100 mM Nacl. 0.01 mM EDTA pH 7.0.
Mitsunobu conditions. Hydrolysis of N-3-benzoyl group in compound 5, followed
by hydrogenation using Pd-C to remove the ring nitrogen protection and alkylation
with ethyl bromoacetate gave the target protected 2S,4R thymine monomer unit. 6.
It was hydrolyzed using aqueous methanolic sodium hydroxide to obtain the
thymine monomer 7 that could be used for solid phase synthesis of PNA-Pyr-
rolidinePNA oligomer=mixmers. All the new compounds were characterized using
suitable spectroscopic analysis.
A similar set of reactions starting from trans 4-hydroxy-d-proline (2R,4S) gave
the other isomer. PNA oligomers containing the unmodified aegPNA and
modified pyrrolidine-PNA backbone units were synthesized by SPPS using the
BOC-protection strategy. DNA oligomers were synthesized on Pharmacia GA plus
synthesizer gave employing phosphoramidite chemistry.
The preliminary DNA binding studies (Table 1) indicate that the 2S,4R stereo-
chemistry of pyrrolidine PNA I induced the PNA conformation to be in agreement
for binding with complementary DNA as positive changes are observed in both Tm
and % Hyperchromicity when a single unit is present at the C-terminus. The effect
was not found to be synergistic as an additional unit in the center of the sequence
completely destabilizes the triplex. It is possible that the complementarity to the
internucleobase distances in DNA could be disturbed due to the constrained pyrro-
lidine-amide linker as compared to the control PNA.
CONCLUSIONS
We report here the synthesis of all four diastereomers of the pyrrolidine PNA
monomer I and their incorporation at predetermined positions in PNA oligomers.
DNA complementation studies indicate that the 2S,4R stereochemistry could be well
suited for better DNA binding. Extended amide backbone may be better for inter
nucleobase distance complemetarity to target DNA as compared to the shorter amide
linkage. Further studies to exploit this understanding are in progress in our laboratory.
ACKNOWLEDGMENTS
VAK thanks Department of Science and Technology, New Delhi, and National
Chemical Laboratory, Pune, for financial support. Meena thanks CSIR, New Delhi,
for research fellowship.