Synthesis of trans-L/D-2-(tert-butoxycarbonylaminomethyl)-4-(thymin-1-yl) pyrrolidin-1-yl acetic acid

Article abstract of DOI:10.1081/NCN-120022947

To delineate the binding preferences of stereochemically divergent pyrrolidine PNAs, synthesis of all four diastreomeric monomers of I and the systematic complexation studies of the resultant PNAs with complementary DNA/ RNA is essential. We herein report the synthesis of trans-L/D-2-(tert-butoxycarbonylaminomethyl)-4-(thymin-1-yl) pyrrolidin-1-yl acetic acids I, their incorporation in PNA oligomers and DNA binding studies will be presented.

Full text of DOI:10.1081/NCN-120022947

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Synthesis of trans-L/D-2-(Tert-butoxycarbonyl-
aminomethyl)-4-(thymin-1-yl) Pyrrolidin-1-yl Acetic
Acid
Vaijayanti A. Kumar a b & Meena a
a Division of Organic Chemistry , National Chemical Laboratory , Pune, India
b Division of Organic Chemistry , Synthesis , National Chemical Laboratory , Dr. Homi Bhabha
Road, Pune, 411008, India
Published online: 31 Aug 2006.
To cite this article: Vaijayanti A. Kumar & Meena (2003) Synthesis of trans-L/D-2-(Tert-butoxycarbonyl- aminomethyl)-4-
(thymin-1-yl) Pyrrolidin-1-yl Acetic Acid, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1285-1288, DOI: 10.1081/
NCN-120022947
To link to this article: http://dx.doi.org/10.1081/NCN-120022947
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1285–1288, 2003
Synthesis of trans-L=D-2-(Tert-butoxycarbonyl-
aminomethyl)-4-(thymin-1-yl) Pyrrolidin-1-yl
Acetic Acid
*
Vaijayanti A. Kumar and Meena
Division of Organic Chemistry, National Chemical Laboratory,
Pune, India
ABSTRACT
To delineate the binding preferences of stereochemically divergent pyrrolidine
PNAs, synthesis of all four diastreomeric monomers of I and the systematic
complexation studies of the resultant PNAs with complementary DNA=RNA is
essential. We herein report the synthesis of trans-L=D-2-(tert-butoxycarbonyl-
aminomethyl)-4-(thymin-1-yl) pyrrolidin-1-yl acetic acids I, their incorporation
in PNA oligomers and DNA binding studies will be presented.
Key Words: Pyrrolidine-PNA; PNA-DNA=RNA complexes.
INTRODUCTION
In the recent years cationic analogues of peptide nucleic acids are gaining atten-
tion because of their favorable properties for therapeutic applications.[1] From
the interesting results of others[2,3] and our own earlier work,[4] PNAs based on
*Correspondence: Vaijayanti A. Kumar, Division of Organic Chemistry, Synthesis,
National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India; Fax: þ91 20
5893153; E-mail: vkumar@dna.ncl.res.in.
1285
DOI: 10.1081/NCN-120022947
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1286
Kumar and Meena
pyrrolidine acetic acid seem to hold some potential for the development as important
antisense molecules. The pyrrolidine based PNAs are chiral, conformationally con-
strained molecules with a methylene bridge inserted between a carbon atom of ethy-
lenediamine and b0 carbon atom of linker to nucleobase and simultaneously
removing the rigid carbonyl group. Their DNA=RNA binding preferences may be
dictated by the geometry of the backbone as well as the orientation of the nucleo-
base. To delineate the recognition preferences of stereochemically divergent pyrroli-
dine PNAs, synthesis of all four diastereomers of I and systematic studies of binding
with complementary DNA=RNA are essential. Here, we report the synthesis of
trans-2-(Boc-aminomethyl)-4-(thymin-1-yl)-pyrrolidine-N-acetic acid.
RESULTS AND DISCUSSION
The suitably protected trans-4-hydroxy proline 2 was converted to the trans-2-
Bocaminomethyl-4-hydroxy pyrrolidine derivative 4 by employing a number of steps
(Sch. 1). These sequentially consist of reduction of ester function, tosylation of the
resulting primary hydroxy group and reaction with sodium azide to get 3 which
was then selectively hydrogenated using Ra-Ni and Boc-protected to get the 2-ami-
nomethyl pyrrolidine 4. Trans-4-N3-benzoyl-Thymin-1-yl-2-Bocaminomethyl pyrro-
lidine derivative 5 was synthesized from 4 by double inversion steps under
Scheme 1.

Pyrrolidin-1-yl Acetic Acid Derivatives of Thymine
1287
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.

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Kumar and Meena
REFERENCES
1. Kumar, V.A. Structural pre-organization of peptide nucleic acids: Chiral catio-
nic analogs with five- or six-membered ring structures. Eur. J. Org. Chem. 2002,
2021 pp.
2. Puschl, A.; Tedeschi, T.; Nielsen, P.E. Pyrrolidine PNA: A novel conformation-
ally restricted PNA analogue. Org. Lett. 2000, 2, 4161–4163.
3. Hickman, D.T.; King, P.M.; Cooper, M.A.; Slater, J.M.; Mickelfield, J. Unusual
RNA and DNA binding properties of a novel pyrrolidine-amide oligonucleotide
mimic (POM). Chem. Commun. 2000, 2251–2252.
4. Kumar, V.A.; Pallan, P.S.; Meena; Ganesh, K.N. Pyrrolidine nucleic acids:
DNA=PNA oligomers with 2-hydroxy=aminomethyl-4-(thymin-1-yl)pyrrolidine-
N-acetic acid. Org. Lett. 2001, 3, 1269–1272.