influence of pna containing 8-aza-7-deazaadenine on structure stability and binding affinity of...

1958 Mol. BioSyst., 2013, 9, 1958--1971 This journal is c The Royal Society of Chemistry 2013

Cite this: Mol. BioSyst.,2013,9, 1958

Influence of PNA containing 8-aza-7-deazaadenine onstructure stability and binding affinity of PNA�DNAduplex: insights from thermodynamics, counter ion,hydration and molecular dynamics analysis†

Sharad K. Gupta,za Souvik Sur,zb Rajendra Prasad Ojhac and Vibha Tandon*b

This paper describes the synthesis of a novel 8-aza-7-deazapurin-2,6-diamine (DPP)-containing peptide nucleic

acid (PNA) monomer and Boc protecting group-based oligomerization of PNA, replacing adenine (A) with DPP

monomers in the PNA strand. The PNA oligomers were synthesized against the biologically relevant SV40

promoter region (2494-AATTTTTTTTATTTA-2508) of pEGFP-N3 plasmid. The DPP-PNA�DNA duplex showed

enhanced stability as compared to normal duplex (A-PNA�DNA). The electronic distribution of DPP monomer

suggested that DPP had better electron donor properties over 2,6-diamino purine. UV melting and

thermodynamic analysis revealed that the PNA oligomer containing a diaminopyrazolo(3,4-d)pyrimidine moiety

(DPP) stabilized the PNA�DNA hybrids compared to A-PNA�DNA. DPP-PNA�DNA duplex showed higher water

activity (Dnw = 38.5) in comparison to A-PNA�DNA duplex (Dnw = 14.5). The 50 ns molecular dynamics

simulations of PNA�DNA duplex containing DPP or unmodified nucleobase-A showed average H-bond distances

in the DPP–dT base pair of 2.90 Å (O� � �H–N bond) and 2.91 Å (N� � �H–N bond), which were comparably shorter

than in the A–dT base pair, in which the average distances were 3.18 Å (O� � �H–N bond) and 2.97 Å (N� � �H–N

bond), and there was one additional H-bond in the DPP–dT base pair of around 2.98 Å (O2� � �H–N2 bond),

supporting the higher stability of DPP-PNA�DNA. The analysis of molecular dynamics simulation data showed

that the system binding free energy increased at a rate of approximately �4.5 kcal mol�1 per DPP base of the

PNA�DNA duplex. In summary, increased thermal stability, stronger hydrogen bonding and more stable

conformation in the DPP-PNA�DNA duplex make it a better candidate as antisense/antigene therapeutic agents.

1. Introduction

Peptide nucleic acids (PNA) are synthetic nucleic acid analoguesin which the sugar phosphate backbone has been replaced by2-aminoethylglycine units with nucleobases attached through amethylene carbonyl linker, resulting in an achiral and unchargednucleic acid mimic which is not susceptible to enzymatic cleavage.1,2

PNA is capable of sequence-specific binding to DNA as well asRNA to form PNA�DNA and PNA�RNA duplexes, obeying Watson–Crick hydrogen-bonding rules,3,4 but unlike DNA or RNA, whichare susceptible to enzymatic degradation, PNA is not easilydegraded by proteases or nucleases.5 These properties makePNA an attractive reagent for biotechnology applications.

Although several PNA-based applications have been success-fully developed, the potential of PNAs as gene therapeutic drugshas been hampered because of their low water solubility andcellular uptake.6,7 However considerable efforts were carriedout recently to improve the water-solubility and cellular uptake.For example, several modifications in the nucleobases have already

a Dr B. R. Ambedkar Center for Biomedical Research, Delhi, Indiab Department of Chemistry, University of Delhi, Delhi, India.

E-mail: [email protected] Biophysics Unit, Department of Physics, DDU Gorakhpur University, Uttar Pradesh,

India

† Electronic supplementary information (ESI) available: Fig. S1–S5 – NMR spectralcharacterization of compounds 2–6; Table S1 – HPLC method for PNA oligomerpurification; Fig. S6 and S7 – HPLC chromatograms; Fig. S8–S11 – ESI-MS-TOFspectra of different PNA oligomers; Fig. S12 and S13 – Tm dependence on saltconcentration for different PNA�DNA duplexes; Table S2 and S3 – Tm data for meltingof DPP-PNA2�DNA duplex; Fig. S14 and S15 – Tm dependence on water of hydrationfor different PNA�DNA duplexes; Fig. S16 and S17 – uncertainty estimation duringcalculation of water of hydration; Fig. S18 – molecular dynamics protocol forequilibration and data collection; Fig. S19 – total energy graph for DPP-PNA2�DNAduplex and A-PNA�DNA; Fig. S20 – H-bonding distance between adenine thymine(A� � �T) in A-PNA�DNA; Table S4 – RMSD values of PNA and DNA strands inDPP-PNA2�DNA duplex and A-PNA�DNA; Table S5 – detailed H-bond informationin A-PNA�DNA duplex of averaged PDB structure after 50 ns simulation; Table S6 –detailed H-bond information in DPP-PNA2�DNA duplex of averaged PDB structureafter 50 ns simulation; Table S7 – average H-bonding distance in A/DPP–T base pairsin DPP-PNA2�DNA duplex and A-PNA�DNA. See DOI: 10.1039/c3mb25561a‡ First and second authors have equal contribution to the work.

Received 7th December 2012,Accepted 5th March 2013

DOI: 10.1039/c3mb25561a

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This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst., 2013, 9, 1958--1971 1959

been reported,8–11 and several other strategies have been developedto overcome these drawbacks including electroporation,12

microinjection,13 cationic liposome carriers,14,15 and conjugationto cell-penetrating peptides (CPPs),16,17 but none of these werefound to be up to the mark.

The stability of natural DNA duplexes depends on base paircomposition, sequence and length; similar trends were observedfor PNA�DNA duplex stability when traditional adenine/thyminebases were used. Efforts were undertaken to increase the PNA�DNAduplex stability by chemical modification of the nucleobase aswell as backbone modification.18–24 Nucleobase modificationswere previously reported in DNA duplexes.25–27 All approachesfocused either on strengthening of the H-bonds between thebases and/or increasing base-stacking interactions amongthem. Besides, it was also reported that, in the case of theDNA-duplexes, the modified base with extra amino group inadenine did not increase the stability of the bi-dentate dA–dTpair to the level of a tri-dentate dG–dC pair.25 The GC base pair ismore stable than the AT base pair, which was attributed to the thirdH-bond (Scheme 1). PNA�DNA duplexes containing other tridentatebase-pairs were also expected to show higher stability. Nielsenet al.28,29 and Hudson et al.30 had shown that substitution ofadenine by di-aminopurine in PNA oligomer increased the Tm ofPNA�DNA duplexes by 2.5–6.5 1C per di-aminopurine base. Seelaet al. showed that 4,6-diaminopyrazolo(3,4-d)pyrimidine provide

higher stability to DNA and RNA duplexes in comparison to2,6-di-aminopurine and/or adenine.25

We have observed that incorporation of DPP monomer(s) in PNA(DPP-PNA1, DPP-PNA2, DPP-PNA3) (Table 1) increases the meltingtemperature of DPP-PNA�DNA duplexes. We noticed that thenumber of water molecules per base released upon melting ofDPP-PNA3�DNA was more compared to A-PNA�DNA, suggesting notonly tridentate base pairing as reported earlier23 but also that thenumber of water molecules contributes to higher thermal meltingof the DPP-PNA�DNA duplex. In order to investigate whether thestructural perturbations produced by incorporation of the DPPmonomer in PNA�DNA duplexes could explain their vast stabilitydifference from normal PNA�DNA duplexes, CD spectra of PNA�DNAduplexes were recorded. In order to further explicate the experi-mental data, 50 ns MD simulation studies were done with PNA�DNAduplexes using DPP-PNA2 and A-PNA as the PNA template using theAMBER10.0 suite of programs, and H-bond distances betweenDPP–dT and A–dT pair were calculated. MD simulation studiesconfirmed that DPP-PNA�DNA duplexes have more a stable con-formation as compared to A-PNA�DNA duplexes. Our studies onmore stable base pairs led us to the DPP-PNA monomer, which canbe considered as an ideal shape mimic of the parent adenine.

2. Results and discussions2.1. Synthesis

The synthesis of normal PNA monomer (without any modification)was done according to the literature method,31,32 however thesynthesis of modified PNA monomer 6 (DPP) was carried out strictlyfrom compound 1 as shown in Scheme 2. Compound 1 was alkyl-ated at N-(1) by methyl bromoacetate in DMF using K2CO3 as a baseand then benzoylation of exocyclic 4,6-diamino group of compound2 was done using benzoyl chloride in pyridine. Finally 4,6-dibenzoyl-aminopyrazolo(3,4-d)pyrimidinylacetic acid 4 was prepared byhydrolysis of the ester product (3) under aqueous alkaline condi-tions at room temperature. The compound 4 was coupled to ethylN-(2-t-butyloxycarbonylaminoethyl)glycinate (backbone) utilizingHBTU as a coupling agent. The (4,6-dibenzoylaminopyrazolo-[3,4-d]pyrimidin-1-ylacetyl)-(2-t-butyloxycarbonylaminoethyl)amino-acetic acid 6 was finally obtained after alkaline hydrolysis with1 M lithium hydroxide.

2.2. Stability of PNA�DNA duplexes

On the addition of an extra amino group at the 6-position,tridentate base pairing increases the stability of the base pairs.

Scheme 1 Base-pairing between A–T and DPP–T, canonical structures ofadenine (A) and 4,6-diaminopyrazolo(3,4-d)pyrimidine(DPP).

Table 1 PNA sequences and DNA sequence

Name Sequencea,b

A-PNA H-Lys-TAA ATA AAA AAA ATT-NH2

DPP-PNA1 H-Lys-TAA ATA AAA (�D�P�P)AA ATT-NH2

DPP-PNA2 H-Lys-TA(�D�P�P) ATA (�D�P�P)AA (�D�P�P)AA (�D�P�P)TT-NH2DPP-PNA3 H-Lys-T(�D�P�P)(�D�P�P)(�D�P�P)T(�D�P�P) (�D�P�P)(�D�P�P)(�D�P�P)(�D�P�P)(�D�P�P)(�D�P�P)(�D�P�P)TT-NH2DNA AAT TTT TTT TAT TTA

a All the sequences start from N to C terminal or 50 to 30 and all the duplexes resulting from these sequences are anti-parallel. b The N-terminal ofthe peptide backbone of PNA is shown by ‘H’ and the C-terminal (amidated carboxyl terminal) of the peptide backbone of PNA is shown by ‘NH2’.

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The increased thermal stability using 4,6-diaminopyrazolo-(3,4-d)pyrimidine in place of adenine can be explained on thebasis of: (i) Electron density at N1 in DPP being higher due topresence of adjacent N2 with a negative charge in the mesomericstructure (Scheme 1, structure II) compared to adenine-N9. Thetwo adjacent nitrogen atoms in pyrazolo(3,4-d)pyrimidine increasethe proton donor property (through formation H-bond withthymine) of the DPP moiety as compared to adenine (A) andeven to purin-2,6-diamine; for that reason, we preferred tochoose 4,6-diaminopyrazolo(3,4-d)pyrimidine over A and purin-2,6-diamine. In case of A, the negative charge developed at C-8does not move to N9 as compared to N1 (in DPP), where N2increases the charge density on N1. (ii) The presence of nitrogen atthe 2-position (in DPP) allows the formation of mesomeric struc-tures (Scheme 1) increasing the amide character of the two aminogroups. As a result, they are now both in plane with the heterocycleand in more favorable situations for base pairing and/or stacking,and thus increase the stability of the PNA�DNA duplexes.

It was observed (Fig. 1) that substituting single DPP foradenine A increased the thermal stability (DTm) of the complexesby Z3 1C per substitution in DPP-PNA1�DNA, DPP-PNA2�DNAand in DPP-PNA3�DNA (Table 2). It was reported that PNA�DNAduplex containing an additional 2-amino group on adenine (i.e.2,6-diaminopurine instead of adenine) showed an increase inthermal melting.10,30 In addition, the DNA duplex containingpyrazolo(3,4-d)pyrimidine–thymine pairs showed an increase inthermal stability as compared to normal A–dT pairs.25 Theincrease in the thermal stability (DTm) was attributed to theformation of a third hydrogen bond. Besides, the reason of thisexpected increase may be sought in the difference in counter ionbinding (and/or release), hydration, and/or a combination ofthese. In view of the above, it became imperative to determinethe thermal melting temperature, water activity, and salt effect

on the stability of PNA�DNA duplexes. The enthalpy change(�DH) was also calculated from differentiated melting curves(Table 2). Enthalpy calculations from the upper half-width athalf-height of the differentiated melting curve are relativelyinsensitive to the choice of the lower base line,33,34 and thereforewill provide better estimates for DH. Thus we decided to use theenthalpies obtained from the upper half-width at half-height ofthe differentiated melting curve as the most reliable values forfurther calculations. We found a good correlation between thethermal stability (Tm) and the change in standard free energy(DG), as well as the change in enthalpy (DH), which was observedfor all of the complexes (Table 2). The Tm increased from 53.07 1Cfor A-PNA�DNA duplex to 56.17, 64.02 and 82.46 1C forDPP-PNA1�DNA, DPP-PNA2�DNA, DPP-PNA3�DNA, respectively(Fig. 1), which was in agreement to the corresponding values of�DG, i.e.�11.5, �12.6, �14.3, �17.4 kcal mol�1 respectively forthe above four duplexes. These results were further supportedby enthalpy (DH), which showed the same trend, i.e. �68.01,

Scheme 2 Synthesis of (4,6-dibenzoylaminopyrazolo[3,4-d]pyrimidin-1-ylacetyl)-(2-t-butoxycarbonylaminoethyl)aminoacetic acid (6): (i) methyl bromo acetate,K2CO3, DMF, rt, 5 h, 60% (2); (ii) benzoyl chloride, pyridine, rt, 20 h, 40% (3); (iii) NaOH, MeOH, HCl, rt, 0.5 h, 37% (4); (iv) ethyl N-(2-t-butoxycarbonylaminoethyl)-glycinate, HBTU, triethylamine, DMF; (v) LiOH, THF, HCl, rt, 0.75 h, 70%.

Fig. 1 Relative absorbance vs. temperature profile of the UV melting curve of allPNA�DNA duplexes in 10 mM phosphate buffer containing 150 mM NaCl and0.1 mM EDTA, pH 7.2 � 0.01.

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�80.45, �89.34, �108.13 kcal mol�1 for these duplexes, respec-tively. This enthalpy/entropy compensation to DG value wasexhibited by all DPP-modified PNA�DNA duplexes. Our resultssuggest that DPP substitution leads to a larger increase in thefavorable enthalpy term that overrides the effect of unfavorableentropy, thereby making all DPP-PNA�DNA duplexes enthalpicallymore favorable than normal A-PNA�DNA duplexes, suggesting majorstructural rearrangements occurred in these duplexes during theduplex to coil transition in comparison to the A-PNA�DNA duplex.

2.3. Counter ion uptake upon duplex formation

The PNA molecule is a unique system that represents a combi-nation of aspects of proteins as well as nucleic acids. Therefore, interms of electrostatic effects PNA is expected to respond differentlyto variations in ionic environments compared to DNA and protein.Cations destabilize PNA�DNA duplexes whereas they stabilizeDNA�DNA duplexes at a range of 0.01–1.0 M. It was found thatincreasing ionic strength had a destabilizing effect on the PNA�DNAduplexes.3 In order to address any differences in counter ionbinding and/or release that account for the differential stabilityof DPP-modified PNA�DNA duplexes from normal A-PNA�DNA

duplexes, UV melting curves of all four duplexes (5 mM) at fourdifferent salt concentrations (10–1000 mM Na+) were studied(Fig. S12 and S13, ESI†). The thermodynamics of the uptake of thecounter ion for the modified and unmodified PNA�DNA duplexeswere obtained from the slopes of these curves dTm/d(ln[Na+])(Table 3). We compared the Tm of normal A-PNA�DNA duplex withDPP-modified duplexes. At increasing salt concentration (0.01 to1.0 M), the Tm of A-PNA�DNA decreased from 56.93 to 49.09 1C, whilethe decrease for DPP-PNA1�DNA was from 59.11 to 52.07 1C, forDPP-PNA2�DNA was from 70.80 to 63.54 1C and for DPP-PNA3�DNAwas from 82.46 to 64.58 1C. These Tm values in turn give the values ofthe changes in the number of moles of bound sodium ions, i.e.DCNa+ (Table 3). The obtained DCNa+ values suggest that there werealmost equal changes in the number of moles of bound Na+ uponmelting of these duplexes (B0.04). Thus, these results showed nosignificant differential effect of the ionic strength, indicating similarcation-binding and release for all the duplexes.

2.4. Hydration effect

It is well established that water molecules bound at specificand/or nonspecific sites of a biopolymer can influence the

Table 2 Melting temperatures (Tm) and thermodynamic parameters (DH and DS) for PNA�DNA duplexes using 10 mM sodium phosphate buffer containing 150 mMNaCl and 0.5 mM Na2EDTA, at pH 7.0

Duplex name Tma (derivative)/1C Tm

a (van’t Hoff)/1CDG a (van’t Hoff)at 37 kcal mol�1

DS a (van’t Hoff)/cal mol�1 K�1

DH a (van’t Hoff)/kcal mol�1

DH a [da/d(1/Tm) vs. Tm]/kcal mol�1

A-PNA�DNA 53.07 � 0.28 52.14 � 0.15 �11.5 � 0.11 �180.4 � 7.89 �67.41 � 3.91 �68.01 � 4.12DPP-PNA1�DNA 56.17 � 0.21 54.74 � 0.25 �12.6 � 0.27 �187.9 � 6.13 �70.85 � 4.13 �80.45 � 2.08DPP-PNA2�DNA 64.02 � 0.30 64.89 � 0.29 �14.3 � 0.33 �199.3 � 8.06 �76.14 � 4.77 �89.34 � 3.69DPP-PNA3�DNA 82.46 � 0.12 79.13 � 0.43 �17.4 � 0.58 �241.8 � 4.12 �92.12 � 3.85 �108.13 � 2.56

a Standard deviations are given.

Table 3 Counter ion release/binding

Duplex name [Na+] (M) ln[Na+] Tm (van’t Hoff)/1C Tm (van’t Hoff)/KDH [da/d(1/Tm) vs. Tm]/

kcal mol�1 dTm/d(ln[Na+]) DCNa+a

A-PNA�DNA 0.01 �4.600 56.93 330.08 �74.24 1.7157 �0.04140.10 �2.300 52.25 325.40 �69.330.50 �0.693 49.90 323.05 �65.581.00 �0.000 49.09 322.24 �64.68

DPP-PNA1�DNA 0.01 �4.600 59.11 332.26 �84.33 1.5112 �0.04150.10 �2.300 56.04 330.19 �81.560.50 �0.693 53.48 326.73 �77.761.00 �0.000 52.07 326.22 �74.39

DPP-PNA2�DNA 0.01 �4.600 70.80 343.95 �95.25 1.5815 �0.04470.10 �2.300 66.63 339.78 �89.600.50 �0.693 64.42 337.57 �84.581.00 �0.000 63.54 336.69 �80.14

DPP-PNA3�DNA 0.01 �4.600 82.46 355.46 �112.14 1.7732 �0.05010.10 �2.300 73.18 346.48 �98.250.50 �0.693 67.15 340.15 �92.471.00 �0.000 64.58 337.58 �87.53

a The change in the number of moles of bound Na+ upon melting of the duplexes (DCNa+) has been calculated using the slope of the plot betweendTm and d(ln[Na+]) (Fig. S12 and S13, ESI) in the equation:

DCNaþ ¼1

anu

dTm

dð ln½Naþ�Þ

� �DHRTm

2

where a is approximately 0.9 in this range of salt concentration, nu is the number of phosphates per cooperative melting unit.


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stability of that biopolymer including nucleic acids.35,36 Withinthe crystal structures of PNA�DNA and PNA�PNA duplexesnumerous ordered water molecules have been identified,37

many of which specifically contact nucleobases. Thereforespecific hydration of all four PNA�DNA duplexes could be quitedifferent and could differently affect the duplex stability.

To address this issue, we performed osmotic stressingexperiments and studied the effect of organic solvent (20%DMF) on duplex stability, as DMF would diminish water activityand thereby enhance any differential hydration effects. We choseDMF as organic solvent as this is aprotic and still polar enough toretain sufficient solubility of the PNA�DNA complexes even up to50% concentration. Interestingly, the results suggest that 20%DMF significantly destabilized PNA�DNA duplexes. In the case ofnormal A-PNA�DNA duplex, destabilization was about 10 1C, i.e.without DMF Tm was 52.14 1C, whereas with 20% DMF the Tm

decreased to 42.07 1C (Table 4). In the case of DPP-containingduplexes, the melting temperatures were decreased by approxi-mately 12, 17 and 22 1C for DPP-PNA1�DNA, DPP-PNA2�DNA andDPP-PNA3�DNA, respectively (Table 4).

It was also seen that DPP-PNA�DNA and A-PNA�DNA duplexbehave differently when considering the enthalpic and entropiccontributions to the free energy. The DPP-containing duplexes wereenthalpically destabilized and relatively entropically stabilizedby 20% DMF, resulting in considerable enthalpy–entropy com-pensation (Fig. 2). For example, when comparing the enthalpic

and entropic contributions to free energy for DPP-containingduplexes in 20% DMF and without DMF, it was found that theenthalpic contribution to destabilization was more than theentropic gain that results in overall enthalpic destabilizationand enthalpy–entropy compensation. Thus the influence ofDMF (organic solvent) on the thermodynamic properties ofthese PNA�DNA duplexes is quite remarkable. The obviousdifferential effect of aqueous versus organic solvent on thebehavior of the DPP-containing duplexes proves that theseduplexes are hydrated differently. In this manuscript, we arediscussing the enthalpy, entropy and free energy in terms of thestability of the PNA�DNA duplexes rather the hydration effect onthe increase of entropic stability.

Any equilibrium that involves changes in the water mole-cules associated with a biopolymer is sensitive to changes inthe water activity.38–40 Water activity can be manipulated by theaddition of low molecular weight co-solutes, which themselvesdo not interact with the biopolymer but are assumed to changethe water activity. To detect the differences in the hydration ofduplexes induced by the incorporation of DPP monomers inplace of A monomer, glycerol was used as co-solute. The valuesof water activity (Dnw) per base in the case of DPP-containingduplexes were observed to be 1.57, 2.10 and 2.54 for DPP-PNA1�DNA,DPP-PNA2�DNA and DPP-PNA3�DNA, respectively, and theuncertainty of these measurements was in between 0.20 to0.30 (Table 5), which suggests that increasing the amount ofDPP modification in PNA led to a higher uptake of watermolecules by the modified duplex compared to unmodifiedA-PNA�DNA duplex (Dnw = 0.97). Our results suggest that thehigher stability of DPP-PNA�DNA duplexes compared to theunmodified counterpart is not only because of the tridentatebase pairing with thymine as reported earlier23 but also becauseof higher hydration of DPP-modified duplexes than of theircorresponding unmodified counterparts.

To further explore the contribution of the extent of hydra-tion of the PNA�DNA duplexes, the change in the number ofbound water molecules in the thermal melting process wasdetermined using a method described by Rozners et al.41 Astraight-forward equation was then applied, which gives thenumber of water molecules uniquely bound to the double helixthat were released upon melting or unfolding of the duplex.42

The UV melting data of each duplex (2.5 mM) at increasingconcentrations of glycerol (0–20%) in phosphate buffer (10 mM,pH 7.0, 150 mM NaCl) was measured (Fig. S14 and S15, ESI†).We observed that at increasing concentration of glycerol Tm

shifts much lower in the case of DPP-PNA�DNA compared to

Table 4 Melting temperatures (Tm) PNA�DNA duplexes using 10 mM sodiumphosphate buffer containing 150 mM NaCl and 0.5 mM Na2EDTA, at pH 7.0 withand without DMF

Duplex nameTm

a (van’t Hoff)without DMF/1C

Tma (van’t Hoff)

with 20% DMF/1C

A-PNA�DNA 52.14 � 0.15 42.07 � 0.12DPP-PNA1�DNA 54.74 � 0.25 42.56 � 0.10DPP-PNA2�DNA 64.89 � 0.29 47.59 � 0.29DPP-PNA3�DNA 79.13 � 0.43 57.58 � 0.42

a Standard deviations are given.

Fig. 2 Effect of polar aprotic solvent (DMF) on the duplex properties of all fourPNA�DNA duplexes. A schematic comparison of the values of thermodynamicsparameters, free energy change (DG), enthalpy change (DH) and entropy change(DS) for all PNA�DNA duplexes with and without DMF.

Table 5 Calculation of Dnw and error estimatesa

Duplex name DH s(DH) d(1/Tm)/d(ln aw) Dnw s(Dnw)

A-PNA�DNA �68.01 �4.12 0.00045356 0.97 �0.16DPP-PNA1�DNA �80.45 �2.08 0.00053054 1.57 �0.20DPP-PNA2�DNA �89.34 �3.69 0.00062055 2.10 �0.27DPP-PNA3�DNA �108.13 �2.56 0.00074716 2.54 �0.30

a 10 mM sodium phosphate buffer containing 150 mM NaCl and0.5 mM Na2EDTA, (pH 7.0) was used for this study.


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A-PNA�DNA. The results showed dependence of 1/Tm on ln aw

for each PNA�DNA duplex (Fig. S16 and S17, ESI†) at differentco-solute concentrations.

2.5. Circular dichroism analysis

It is known that a DNA double helix with B-type DNA structurehas positive bands around 280 nm and 220 nm and an intensenegative band in the 250 nm region; on the other hand, aspectrum of A-type right-handed duplex has positive bandsaround 270 and 220 nm and a relatively weak and negativepeak around 235 nm.43

The introduction of a DPP-monomer unit in the PNA oligo-mers lead to a change in the CD spectrum, as compared tonormal A-PNA�DNA duplex (Fig. 3). The CD signature of anormal A-PNA�DNA duplex showed a positive Cotton effect at222 nm and 258 nm and a weak negative Cotton effect around245 nm, suggestive of the A-form of duplex. While comparingthe positive ellipticity at 258 nm of the A-PNA�DNA duplex withDPP-modified duplexes, this region showed a gradual changein shape. In DPP-PNA3�DNA, this long wavelength regionstarted from a negative ellipticity at around 290 nm and apositive band around 260 nm suggesting an entirely differentconformation from the right handed A-form of the A-PNA�DNAduplex. The intense negative band in DPP-PNA3�DNA duplexindicated that its conformation is similar to DNA. It wasobserved that as the number of DPP monomers increased fromDPP-PNA1�DNA to DPP-PNA3�DNA, the 222 nm band showednot only a positive Cotton effect but also showed a bathochromicshift from B220 nm to 232 nm, indicating an enhancedinter-strand interaction between the DPP-PNA oligomer andthe DNA strand. This might also account for the higher stabilityof DPP-PNA�DNA over A-PNA�DNA.

2.6. Molecular dynamics analysis

Molecular dynamics simulation offers the prospect of a detaileddescription of the dynamic structure of ions and water at themolecular level. We calculated the energy for different combi-nations of strands as the ligand and receptor for all structures.

Since the force-field parameters for the modified nucleotides(PNA) were not available in the literature, their parameters weregenerated using literature values44 using the GAUSSIAN 03 andRESP program of AMBER 10.

The macroscopic properties of the systems such as tempera-ture, pressure, volume, density etc. (data not shown) stayedfairly constant during whole 50 ns simulation, indicatingsuccessful simulation. The analysis of the trajectories showedthat the simulation process led to stable structures (Fig. 4aand b). No major helical unfolding or transitions to doublehelical structures were observed during the simulations. Thegraph of total energy vs. time clearly showed the stability ofmodified DPP-PNA2�DNA duplex over normal A-PNA�DNAduplex (Fig. S19, ESI†).

As far as the conformational features of PNA�DNA duplexand (PNA)2�DNA triplex in aqueous solution concerned, theywere known to exist as a P-type helix,44 which is different fromcanonical A- and B-DNA structures. Finally, it was confirmed bythe solid-state structure of a PNA duplex21,45–47 that the P-typehelix is the preferred conformation of duplexes and triplexesinvolving PNA. In our case, the comparison of the DPP-PNA2�DNAor A-PNA�DNA duplex to those described in the literaturerevealed that all PNA torsion angles were very similar to thoseof the PNA duplex and (PNA)2�DNA triplex (Table 6).

The RMSD (root mean square deviation) fluctuation duringwhole 50 ns simulation time was reported (Fig. 4c) and theRMSD values of each duplex, including individual strands, werecalculated (Table S4, ESI†). 500 frames were selected every100 ps from a 50 ns simulation to calculate RMSD values. Boththe duplexes were seen to be helical with well-defined basepairing and base stacking. The molecular structure shows allbase pairs coupled in the Watson–Crick manner, all sugarspuckered, and glycosidic torsion angles were like the anticonformation in DNA strand. The puckering involved in themodified PNA was well maintained throughout the simulation.The simulated structure of DPP-PNA�DNA showed an ensembleconformation with 3–4 Å RMSDs, compared to A-PNA�DNAshowing 2–3 Å RMSD. Although modified PNA�DNA duplexshowed a little higher RMSD, no breakage of molecules wasobserved.

The helicoidal parameters like twist, roll, tilt, and inclina-tion were calculated for the average structures of the duplexesusing 3DNA,48 version 2.0 Beta (Table 6). The inter-base pairparameters ‘‘twist’’, ‘‘roll’’ and ‘‘tilt’’ describe the linear shear-ing displacements of stacked bases. The A-PNA�DNA duplex hasan average twist value of 35.121 (�6.391) while DPP-PNA2�DNAduplex has a twist value 29.081 (�5.121), which indicates thatthe A-PNA�DNA duplex is more or less like B-DNA (twist value361), whereas DPP-PNA2�DNA duplex is of A-DNA type (i.e.30.31).49,50 The values in parentheses indicate the fluctuationof twist over all of the base pairs. Besides, the lower value of thetwist suggests a higher number of bases per turn. The positivevalue of ‘‘roll’’(+2.601) in the case of the DPP-PNA2�DNA duplexcorresponds to a local expansion on the major groove sidecompared to the A-PNA�DNA duplex, which had a value �3.681(�5.01). DPP-PNA2�DNA duplex showed a larger fluctuation

Fig. 3 Relative circular dichroism vs. wavelength spectra of all four PNA�DNAduplexes.


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value of �10.041, indicating strong irregularity in sequencescompared to the A-PNA�DNA duplex.

Similarly, the ‘‘tilt’’ values showed a noticeable variation from0.891 compared to 1.921 in the case of normal PNA�DNA duplex;

here also a large fluctuation value of �5.561 suggests a strongsequence dependability on changing the normal adenine toone modified with DPP. The inclination values here also supportthe variation from modified to normal PNA�DNA duplex.The completely reversed values �6.961 and 3.261 suggest aremarkable change in the conformation, with large fluctuationvalue �16.311 in the case of the DPP-PNA2�DNA duplex. Theintra-base pair parameters ‘‘shear’’ and ‘‘stretch’’ indicated thelinear in-plane displacement of one base pair with respect toanother; the values were found to have a more or less similartrend in normal as well as modified duplex, which suggests thatthere was not much conformational change in the modifiedduplexes compared to A-PNA�DNA duplex (data not shown).Twist, roll, tilt, and inclination suggest a mixed helical natureof both duplexes and indicate an overall conformational fluctua-tion of DPP-PNA2�DNA duplex from A-PNA�DNA duplex.

The backbone torsion angles of individual strands of PNAand DNA were obtained (Table 6). The seven torsion angles ofthe DNA backbone, named a, b, g, d, e, z and X, are valuable forconformational studies, whereas in the case of PNA they are a,b, g, d, e, X1, X2 and X3. In our study, torsion angles of DNA fallbetween those of canonical structures (A- and B-DNA). If we comparethe values of A-PNA and DPP-PNA2, the torsion angles are compar-able to each other and are quite different from the parameters ofDNA. The average torsion angles of PNA and DNA were earlierreported in for PNA�DNA duplex by Carlo et al., who suggestedthat PNA�DNA duplex and even individual strands exist as P-typehelical structures47 different from A- and B-DNA conformations.

Fig. 4 The structure of (a) A-PNA�DNA duplex and (b) DPP-PNA2�DNA duplex after 50 ns showing base pairing between PNA�DNA strands [DPP-T base pairs in DPP-PNA2�DNA duplex, the adenines replaced by modified base are shown in yellow in the four positions in the PNA strand]; (c) RMSD plot for A-PNA�DNA duplex (red) andDPP-PNA2�DNA (black).

Table 6 Comparison of backbone torsion angles and helicoidal parameters forthe average structures of the duplexes after 50 ns simulation

Helicoidal parameters

Duplex name Twist Roll Tilt Inclination Bases per turn

A-PNA�DNA 35.2 �3.7 1.92 �6.96 14(6.4) (7.0) (4.2) (12.1)

DPP-PNA2�DNA 29.1 2.6 0.89 3.26 14(5.2) (10.1) (5.6) (16.3)

A-DNAa 30.3 12.4 2.8 13.0 10B-DNAa 36.0 1.7 2.5 2.4 11

Torsion angles (DNA backbone)

Duplex name (a) (b) (g) (d) (e) (z) (X)

A-PNA �68.3 172.4 59.9 120.8 179 �91 �121DPP-PNA2 �72.8 171.9 59.6 134.9 �174 �94 �118A-DNAa �50 172 41 79 �146 �78 �154B-DNAa �61 180 57 122 173 �91 �119

Torsion angles (PNA backbone)

Duplex name (a) (b) (g) (d) (e) (X1) (X2) (X3)

A-PNA �120 B80 B85 �80 �180 10 �160 B90DPP-PNA2 �105 B70 B85 100 �180 �10 �160 B75

a Values are given for standard A- and B-DNA; all the angles and helicalparameters are denoted in degrees.


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The values of the torsion angles in our measurements alsoindicate the same phenomenon, although uniform torsionangles throughout the PNA�DNA duplex suggest that PNA incor-poration does not perturb the duplex conformation.

The MM_PBSA module in AMBER was used to study thermo-chemical properties of both PNA�DNA duplexes and to provide asemi-quantitative estimate of their stability. The duplex withlower binding energy is expected to be more stable than thehigher value (Table 7). The total energy of the solute (EGAS)includes the electrostatic energy, van der Waals energy derivedfrom a Lennard-Jones potential and the internal energy.51,52

Finite difference Poisson–Boltzmann equations were used tocalculate the electrostatic potential field. The binding energycalculation suggested a favorable contribution for van derWaals energy (EVDW) for both duplexes. The total energy (ETOT)of normal and DPP-PNA2�DNA duplex were found to be�4269.91 and�5739.29 kcal mol�1 respectively (Table 8). Thesevalues showed that DPP-PNA2�DNA duplex was much morestable compared to A-PNA�DNA duplex, having the most favor-able binding energy (�176.45 kcal mol�1) compared to A-PNA�DNA (�158.12 kcal mol�1) duplex (Table 7). Since both the

duplexes had sufficient negative binding energy, they wereconsidered as stable structures as a whole.

The free energy components for both duplexes (where theDNA-receptor strand is bound with the PNA-ligand strand) aresummarized in Table 8. Here, electrostatic energy (DEELE) makesunfavorable contributions while van der Waals energy (DEVDW)provides favorable contributions. Moreover, the nonpolar solvationenergy (DESNP) also has favorable contributions to the total bindingfree energy. This implies that van der Waals and nonpolar solvationenergy play important roles in the binding of PNA and DNA strand.The solvent electrostatic energy (DESP) also makes a favorablecontribution. Moreover, we observed unfavorable entropy contribu-tions (TDS) for both duplexes. The solute entropy terms of bindingfor both duplexes are found to be �47.67 and �48.99 kcal mol�1

for normal and modified duplexes, respectively, showing an unfavor-able contribution towards free energy. The binding free energy(DGTOT) of the two duplexes were found to be �158.12 and�176.45 kcal mol�1 for normal and modified duplexes, respectively.These values show that DPP-PNA2�DNA duplex, which containeddi-modified bases in the PNA strand, is stable compared to theproposed duplexes because it has the highest binding free energy(�176.45 kcal mol�1).

From the careful structural analysis of DPP–dT and A–dT, weobserved that the higher stability of DPP-PNA2�DNA compared toA-PNA�DNA is shown by the average H-bond distances betweenDPP–dT base pair being 2.90 Å (for O� � �H–N bond) and 2.91 Å (forN� � �H–N bond), which are shorter than for the A–dT base pair, inwhich the average distances are 3.18 Å (for O� � �H–N bond) and2.97 Å (for N� � �H–N bond) (Fig. 5 and Fig. S20, ESI†). The fluctua-tion in H-bond distances in A–dT and DPP–dT base pairs through-out the simulation was observed and one extra H-bond was foundin the DPP–dT base pairs (Fig. 5a, b; in Fig. 5b the distancefluctuation for the extra H-bond is denoted in magenta). The extraH-bond was found between DPP–dT base pairs (for O2� � �H–N2;2.98 Å) supporting the higher stability of the DPP-PNA2�DNA duplex(Fig. 5c and Table S5–S7, ESI† over the A-PNA�DNA duplex.

3. Conclusions

This study was undertaken to synthesize and evaluate the effect ofincorporation of 4,6-diaminopyrazolopyrimidine in PNA oligomerto replace adenine leading to the assembly of PNA�DNA duplex. Weobtained a detailed insight into the hybridization thermodynamicsof the modified PNA oligomer with DNA. In addition, we have usedstate-of-the-art MD simulations to understand in detail the struc-ture and dynamics of nucleic acid duplexes formed by a modifiedPNA strand. We also performed simulation with regular unmodi-fied duplex. Furthermore, tracking the effects of PNA modificationsin different sequence contexts will allow us to formulate guidelinesfor the optimum changes of PNA sequences so that maximumfunctional efficiency could be drawn out of minimum modifica-tions. This study deepened our understanding of PNA-basednucleic acids in following aspects:

1. The thermal denaturation experiments with differentiallymodified duplexes suggested a higher thermal stability for theDPP-containing PNA�DNA over A-PNA�DNA.

Table 7 Comparison of binding free energies of both duplexes

EnergyA-PNA�DNA

duplex (kcal mol�1)DPP-PNA2�DNA

duplex (kcal mol�1)

DEELE �616.57 � 56.43 �572.15 � 51.20DEVDW �111.12 � 12.79 �87.76 � 10.05DEGAS �727.69 � 56.07 �659.91 � 52.08DESOLV 519.88 � 55.44 435.79 � 50.11DETOT-ELE �6.75 � 16.99 �68.62 � 12.94DEGAS+SOLV �207.81 � 8.77 �224.12 � 9.95TDS �48.99 � 9.51 �47.67 � 11.24DGTOT �158.12 � 9.51 �176.45 � 11.24

DEELE = coulombic energy. DEVDW= van der Waal’s energy. DEINT =internal energy. DEGAS = DEELE + DEVDW + DEINT. DESOLV = DESNP +DESP = total solvation energy. DETOT-ELE = DEELE + DESP = total electro-static energy. DEGAS+SOLV = DEGAS + DESOLV = enthalpy. TDS = soluteentropy. DGTOT = DEGAS-SOLV � TDS = absolute free energy.

Table 8 Total energy calculation froma MM_PBSA.pl module of A-PNA�DNA andDPP-PNA2�DNA duplex

EnergyPNA�DNA duplex

(kcal mol�1)DNA strand(kcal mol�1)

PNA strand(kcal mol�1)

A-PNA�DNA duplexEELE �3272.98 � 71.73 189.84 � 20.54 �2846.25 � 25.08EVDW �229.17 � 19.68 �87.85 � 7.02 �30.21 � 8.32EGAS �1689.05 � 65.50 968.23 � 26.50 �1929.59 � 25.57ESOLV �2580.86 � 62.91 �2787.63 � 18.68 �313.11 � 14.09ETOT-ELE �6092.32 � 26.68 �2760.07 � 8.18 �3325.50 � 17.13EGAS+SOLV �4269.91 � 28.16 �1819.40 � 19.04 �2242.70 � 17.85

DPP-PNA2�DNA duplexEELE �4691.41 � 56.83 170.88 � 23.31 �4290.14 � 28.35EVDW �186.50 � 5.38 �72.80 � 7.60 �25.93 � 9.53EGAS �3075.28 � 62.11 907.66 � 24.35 �3323.02 � 26.24ESOLV �2664.01 � 54.15 �2777.43 � 22.08 �322.37 � 13.18ETOT-ELE �7616.91 � 20.35 �2769.60 � 8.56 �4778.68 � 18.29EGAS+SOLV �5739.29 � 28.77 �1869.78 � 16.00 �3645.39 � 19.87

a MM_PBSA = molecular mechanics Poisson–Boltzmann surface area.


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2. Our results proved the higher affinity of DPP-containingPNA analogs to DNA was not only because of tridentate basepairing as reported earlier, but also the average hydrogen bonddistance in the DPP–dT base pair was less compared to thedA–dT base pair, suggesting a stronger affinity between DPP-PNAand DNA strands.

3. DPP-containing PNA�DNA duplexes were more hydratedcompared to A-PNA�DNA.

4. CD experiments suggest a large structural perturbationleading to a thermodynamically stable conformation.

5. An integrated computational approach by combining MDsimulations and MM_PBSA binding free-energy calculationswas used to characterize the stability of modified PNA�DNAduplex over normal PNA�DNA duplex. The comparison of thetotal potential energy curves from trajectory analysis of bothduplexes throughout the time scale of 50 ns suggests the higherstability of the modified one.

6. On the basis of the helicoidal and torsional parameteranalysis we conclude that DPP-PNA�DNA duplex has higherconformational rigidity and stability than A-PNA�DNA duplex.

The investigation of hybridization thermodynamics ofmodified PNA aided prediction of the stability of modifiedPNA, which in turn correlated with functional efficiency.

4. Experimental sections4.1. Materials and sample preparation

Chemicals and reagents used in the present study were analyticalgrade or equivalent and were procured from Sigma Chemicals(St. Louis, MO, USA), E Merck (Germany), Fine Chemicals (India).

The completion of all reactions was monitored by thin layerchromatography (TLC), silica 60 F254 (Merck) using the followingsolvent systems: (A) n-butyl alcohol/acetic acid/water (3 : 1 : 1 v/v), (B)dichloromethane (DCM)/ethyl acetate (1 : 1 v/v), (C) DCM/methanol(9 : 1 v/v). The products were visualized by spraying with ninhydrin(1% w/v in ethanol) with heating for 5 min or by UV irradiation.Flash chromatography was carried out for the purification of theproducts, using silica gel (60–120 mesh size, Merck). Compoundswere analyzed by QSTAR XL quadrupole time-of-flight (QqTOF)hybrid mass spectrometer (Applied Biosystems, Applera Inter-national Inc., Rotkreuz, Switzerland) equipped with a standardheated capillary Turbolon Spray Source. 1H NMR spectra wererecorded on a Bruker 300 MHz NMR Instrument and Varian 400MHz NMR spectrophotometer (Fig. S1–S5 ESI†).

pEGFP-N3 SV40 promoter region (2494-AATTTTTTTTATTTA-2508) was taken as the sequence of choice for all studies. Thispromoter region not only lies within the origin of replication ofthis plasmid but also the neomycin resistance gene is underdirect control of this promoter region. Thus any cell-linecontaining this plasmid vector can easily be controlled by theantisense inhibition of this plasmid sequence. We have synthe-sized four penta-decameric PNA oligomers complementary tothe above sequence with one lysine at the N-terminal.

4.2. Synthesis

The synthesis of 4,6-diaminopyrazolo(3,4-d)pyrimidine wasdone according to a known procedure (Scheme 2).

Synthesis of diaminopyrazolo(3,4-d)pyrimidin-yl-acetic acidmethyl ester (2). To the solution of 1 [4,6-diaminopyrazolo-(3,4-d)pyrimidine] [2.25 g, 15 mmol] in DMF (50 ml), K2CO3

Fig. 5 Hydrogen bond distance between PNA and DNA base pairs during whole production dynamics (1–50 ns). Here, time is represented by frames in the X-axis.(a) Between adenine and thymine (A–T) in A-PNA�DNA duplex, and (b) between 4,6-diaminopyrazolo(3,4-d)pyrimidine and thymine (DPP–T) in DPP-PNA2�DNA duplex.(c) Figure showing triple H-bonding between 4,6-diaminopyrazolo(3,4-d) pyrimidine and thymine (DPP = T) in DPP-PNA2�DNA duplex.


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(12.4 g, 90 mmol) was added slowly and the reaction mixturewas stirred at rt for 15 min. To this reaction mixture methylbromoacetate (2.5 mL, 27 mmol,) was added over 3 h withcontinual stirring. The reaction mixture was stirred at rt for afurther 2 h, then the solution was concentrated to a semisolidmass. Water (20 ml) was added and the precipitate wascollected and recrystallized from 60% ethanol to give the titleester. The product was obtained as a white solid, yield 60%,Rf = 0.31 (DCM/ethyl acetate (1 : 1 v/v). Mp 192–194 1C. 1H NMR(400 MHz, DMSO-d6): d(ppm) = 3.66 (s, 3H), 5.18 (s, 2H), 7.68(s, 2H), 7.88 (s, 2H), 8.12 (s, 1H); 13C NMR (400 MHz, DMSO-d6)d 166.3, 162.4, 156.9, 154.5, 134.4, 101.2, 55.2, 51.6; ESI-MS(m/z): 223.0829 (M+1), calculated 222.0865. Anal. calcd forC8H10N6O2: C, 43.28; H, 4.54; N, 37.82; O, 14.40%. Found: C,43.21; H, 4.49; N, 37.92; O, 14.38%.

Synthesis of dibenzoylaminopyrazolo(3,4-d)pyrimidin-yl-acetic acid methyl ester (3). The ester 2 [1.6 g, 7.2 mmol] wascoevaporated with pyridine (3 � 3 ml) then redissolved inpyridine (15 ml). Benzoyl chloride (0.916 ml, 7.92 mmol) inpyridine (15 ml) was added dropwise over 3 h and stirring wascontinued for 16 h at rt. Water (30 ml) was added and thereaction mixture was extracted with DCM (3 � 30 ml). Thecombined extracts were washed with NaHCO3 (0.5 N, 3 �100 ml), saturated NaCl (30 ml), dried over anhyd. Na2SO4,then evaporated to an oily residue. The oil was purified by silicagel column chromatography with DCM/methanol (0–6%) as aneluent. The product was obtained as white solid, (yield 40%).Mp 211–215 1C. Rf 0.56 (DCM/methanol (9 : 1 v/v): 1H NMR(400 MHz, DMSO-d6): d(ppm) = 3.66 (s, 3H), 4.94 (s, 1.25H), 4.98(s, 0.75H), 7.32–7.57 (m, unresolved, 10H), 8.79 (s, 1H), 10.53(br, 1H); 13C NMR (400 MHz, DMSO-d6) d 166.3, 164.8, 157.1,155.6, 154.5, 134.4, 134.2, 132.2, 128.9, 127.5, 101.2, 55.2, 51.6;ESI-MS (m/z): 431.1391 (M+1), calculated 430.139. Anal. calcd forC8H9N5O2: C, 61.39; H, 4.22; N, 19.53; O, 14.87%. Found: C,61.29; H, 4.55; N, 19.92; O, 14.24%.

Synthesis of dibenzoylaminopyrazolo(3,4-d)pyrimidin-yl-acetic acid (4). The ester 3 [1.5 g, 3.5 mmol] was suspendedin methanol (20.3 ml), NaOH (20.3 ml, 2 N) was added to theabove solution at 0 1C, reaction mixture stirred at rt for 30 min.The solution was washed with DCM (2 � 20.3 ml) and the pHof the solution was adjusted to 1 by the addition of conc. HCl(1.27 ml). The title acid was dried over P2O5 under vacuum. Theproduct was obtained as white solid, (yield 37%). Rf = 0.34(n-butyl alcohol/acetic acid/water). Mp 218–221 1C. 1H NMR(400 MHz, DMF); d(ppm) = 5.02 (s, 1.25H), 5.16 (s, 0.75H),7.47–7.67 (m, unresolved, 10H), 8.17 (s, 1H), 11.14 (br, 1H),11.95 (s, 1H), 13C NMR (400 MHz, DMSO-d6) d 175.0, 164.8,157.1, 155.6, 154.5, 134.4, 134.2, 132.2, 128.9, 127.5, 101.2, 57.7;ESI-MS (m/z): 415.1203 (M�1), calculated 416.1233. Anal. calcdfor C21H16N6O4: C, 60.57; H, 3.87; N, 20.18; O, 15.37%. Found:C, 61.09; H, 3.71; N, 19.70; O, 15.50%.

Synthesis of dibenzoylamino-pyrazolo[3,4-d]pyrimidin-1-yl)-acetyl-(2-t-butoxycarbonyl amino-ethyl)amino-acetic acid-ethylester (5). Ethyl-n-[2-(t-butoxycarbonylamino)ethyl]glycinate(0.492 g, 2.0 mmol) and 4 [0.830 g, 2.0 mmol] were dissolvedin DMF (4.5 ml) and triethylamine (6.0 mmol, 1.1 ml) was

added to the above mixture. HBTU (2.0 mmol, 0.758 g) wasadded and the reaction mixture was stirred at ambient tem-perature for 16 h. The solvent was removed by evaporation andDCM (45 ml) was added to the residue. The resultant solutionwas washed with NaHCO3 (0.5 N, � 18 ml), citric acid (10% w/v,2 � 18 ml) and saturated NaCl (18 ml), dried over anhyd.Na2SO4 and then concentrated to an oil which was purified bysilica gel column chromatography with DCM/methanol (0–3%)as an eluent. The product was obtained as a foamy solid, (yield45%). Rf = 0.94 (DCM/MeOH (9 : 1 v/v). Mp 103–105 1C. 1H NMR(300 MHz, DMSO-d6): d(ppm) = d 1.24 (t, 3H), 1.35–1.39 (s, 9H),3.86 (s, 1.6H) and 3.98 (s, 0.4H), 4.71–4.82 (q, 2H), 5.20 (s, 0.4H)and 5.33 (s, 1.6H), 6.91 (t, 2H, J = 5.5), 7.08 (t, 2H, J = 5.5),7.55–7.58 (m, unresolved, 6H), 8.02 (d, 2H), 8.14 (d, 2H), 8.34(s, 0.8H) and 8.39 (s, 0.2H); 13C NMR (400 MHz, DMSO-d6)d 169.6, 169.4, 164.8, 157.1, 155.6, 154.5, 134.4, 134.2, 132.2,128.9, 127.5, 101.2, 61.0, 59.4, 50.1, 48.0, 38.1, 14.1; ESI-MS(m/z): 645.2702 (M+1), calculated 644.2707. Anal. calcd forC32H36N8O6: C, 61.13; H, 5.77; N, 17.82; O, 15.27%. Found: C,60.95; H, 5.74; N, 17.67; O, 15.64%.

Synthesis of dibenzoylamino-pyrazolo(3,4-d)pyrimidin-1-yl)-acetyl-(2-t-butoxycarbonyl amino-ethyl)amino-acetic acid (6).The ester 5 [0.644 g, 1 mmol] was suspended in THF(2.42 ml) and LiOH�H2O (1 M, 1.45 ml) was added. The reactionwas stirred for 45 min at room temperature, and then filtered;water (0.5 ml) was added and washed with DCM (2 � 3.75 ml).The aqueous solution was then cooled to 0 1C and the pH wasadjusted to 2 with HCl (1 M). The crude compound wasextracted with ethyl acetate (9 � 2.5 ml) and the combinedextracts were dried using anhyd. Na2SO4 and then evaporated todryness in vacuo. The residue was evaporated with methanol toget a colorless white solid after drying overnight, yield 70%,Rf = 0.20 (DCM/MeOH (9 : 1 v/v). Mp 265 1C. 1H NMR (400 MHz,DMSO-d6): d(ppm) = 1.33–1.37 (s, 9H), 3.86 (s, 1.6H) and 3.95(s, 0.4H), 5.19 (s, 0.4H) and 5.32 (s, 1.6H), 6.96 (t, 2H, J = 5.6Hz),7.07 (t, 2H, J = 5.6Hz), 7.50–7.65 (m, unresolved, 6H), 8.00 (m,2H), 8.13 (m, 2H), 8.36 (s), 11.11 (br, 1H,), 11.60 (br, 1H);13C NMR (400 MHz, DMSO-d6) d 173.2, 169.4, 164.8, 157.1,155.6, 154.5, 134.4, 134.2, 132.2, 128.9, 127.5, 101.2, 59.4, 50.2,50.1, 38.1; ESI-MS (m/z): 615.2363 (M�1), calculated 616.2394.Anal. calcd for C30H32N8O7: C, 58.43; H, 5.23; N, 18.17; O,18.16%. Found: C, 58.00; H, 5.45; N, 18.20; O, 18.35%.

4.3. Oligomerization on resin support

PNA oligomers were synthesized by Boc-strategy on a solid support(MBHA resin, PE Biosystems) using a published protocol.53,54

Polystyrene beads carrying 4-methyl benzhydrylamine groups aremost frequently used as a solid support in PNA synthesis.

The loading value of MBHA resin was estimated by thepicrate assay and was found to be 0.6 mmol g�1, which waslowered to 0.15 mmol g�1 by partial coupling of first monomerto resin and the rest of the free amino groups were capped byacetic anhydride as a capping reagent. Similar to solid phasepeptide synthesis, PNA oligomerization also takes place fromthe ‘‘C’’ terminus to the ‘‘N’’ terminus on a solid support. Thefree carboxylic functional group of PNA monomers was activated


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using the HBTU and HOBt was used as an additive to suppressthe level of racemization and enhance the coupling efficiency ofthe growing PNA oligomer chain. Deprotection of the N-terminalBoc-group was carried out with 50% TFA in dichloromethaneand after coupling any unreacted amino functionalities werecapped with acetic anhydride in pyridine/dichloromethane. Thebase-labile acyl groups (benzoyl and isobutyryl groups) wereremoved by treating with 40% methylamine.

Cleavage of PNA from resin, purification and characterizationof PNAs. Final cleavage was done with m-cresol/thioanisole/trifluromethanesulphonic acid) (TFMSA)/trifluoroacetic acid(TFA) (1/1/1/7 v/v), in 40% methylamine for 16 h. The PNAoligomers (Table 1) were desalted over Sephadex G25 column.The desalted PNA were purified on a C18 reverse-phase HPLC(>95%) with the mobile phase consisted of solution A (0.1% TFAin water) and solution B (0.1% TFA in acetonitrile). It hasbeen found that PNA containing DPP modifications (DPP�PNA1,DPP�PNA2, DPP�PNA3) eluted out earlier on the C18 reverse phasecolumn than normal A-PNA oligomer. This trend of retention timewere perhaps because DPP-modification is more hydrophilic andthus contributes to the weak column binding compared to A-PNAoligomer, thus retaining more on the column. After HPLCpurification (Table S1 and Fig. S6 and S7, ESI†), these PNAsequences were characterized by positive ion electrospray ioniza-tion (ESI) mass spectroscopy (QqTOF-MS) (Fig. S8–S11, ESI†).

4.4. Temperature-dependent UV Spectroscopy(UV melting profile)

The PNA concentrations were determined spectrophotometricallyat 65 1C using molar extinction coefficients for correspondingdeoxyribonucleotides: e260 of adenine = 15 400 M�1 cm�1, e260 ofguanine = 11 700 M�1 cm�1, e260 of thymine = 8800 M�1 cm�1 ande260 of cytosine = 7400 M�1 cm�1. The PNA strand and itscomplementary DNA strand were mixed at a concentration ratioof 1 : 1 to obtain PNA�DNA duplexes. Absorbance versus tempera-ture profiles (melting curves) for each duplex were measured at260 nm with a thermoelectrically controlled multi-chamber Cary300 (Varian) spectrophotometer. To prevent evaporation, a drop ofpure mineral oil was placed on top of the solution. The temperaturewas scanned at a heating rate of 0.2 1C min�1. To prepare samples,all PNA and DNA sequences were dissolved in 10 mM sodiumphosphate buffer pH 7.0 containing 150 mM NaCl and 0.5 mMNa2EDTA. They were heated to 97 1C for 5 min in a dry bath, thenslowly cooled to room temperature, and then incubated for 2 h at4 1C prior to the melting experiment.

4.5. Determination of thermodynamic parameters

The thermodynamic parameters, namely enthalpy change (DH),entropy change (DS), and Gibbs’ free energy change (DG), wereevaluated using the hyperchromicity method and differentiatedmelting curve method.

The hyperchromicity method. The hyperchromicity methodutilizes alpha curves and van’t Hoff plots (ln KT vs. T�1).55 Thefraction (aT) of single strands that remained hybridized in theduplex at a particular temperature T in kelvin is represented as:(aT = As� A/As� Ad), where Ad is the absorbance of the duplex in

a fully hybridized condition, As is the absorbance of the singlestrands in fully denatured condition, and A is absorbance at aparticular point on the thermal melting curve at temperature T.For a bimolecular transition of non-self complementary strands,the equilibrium constant at particular temperature T is:

KT = aT/[(Cts/n)n�1(1 � a)n]

where Cts represents the total concentration of strands and n is themolecularity of the complex. Assuming a two-state model, theequilibrium constant is represented as KT = 2aT/[(Cts (1 � aT)2].The van’t Hoff plot (ln K versus 1/Tm in K) is linear with �DH/R asthe slope and DS/R as the intercept (R is the universal gas constant1.986 cal mol�1 K�1) and is represented as ln KT = [(�DH/R)1/T +(DS/R). The value of DG at a particular temperature T can becalculated from DG = �RT ln KT = DH � TDS. All fittingand calculation operations were done using Cary Varian software(version 3.00) using settings for a bimolecular transition of non-self-complementary strands. The final �DH and �DS is theaverage of at least 5–8 measurements.

Differentiated melting curve method. In this method, typicalabsorbance vs. temperature curves are first converted intodifferentiated melting curves [da/d(1/T)], which were thenplotted against T, i.e. [da/d(1/T)] vs. T. The upper half-width of thedifferentiated melting curve at the half-height is inversely propor-tional to the van’t Hoff transition enthalpy; for a bimoleculartransition DH = 4.38/(Tmax

�1� T2�1), where Tmax is the temperature

at the maximum and T2 is the upper temperature at one-half of[da/d(1/Tm)]. The final DH is the average of 5–8 measurements.

4.6. Thermodynamic uptake of counter ions

The thermodynamic uptake of counter ions, DnNa+, associatedwith the process of duplex formation can be obtained accordingto the equation:

DnNa+ = 1.11(DH/RTm2)dTm/d(ln [Na+])

where 1.11 is a proportionality constant for converting ionicactivity into concentrations and dTm/d(ln [Na+]) represents theslope of a plot of Tm versus the logarithm of different concen-trations (10–1000 mM Na+) of sodium ions (ln [Na+]). R repre-sents the universal gas constant (1.986 cal mol�1 K�1) (Table 3and Fig. S12 and S13, ESI†).

4.7. Evaluation of water activity (Dnw)

We can calculate number of water molecules released perbase pair using osmotic stressing method. The change in thenumber of water molecules released is directly correlated withthe increment of thermal melting of the duplex. Osmotic stressmonitors the depression of the melting temperature upondecreasing the water activity and calculates the changes inthe number of water molecules, Dnw.42

Dnw = (�DH/nR)[d(Tm � 1)/d(ln aw)]

where �DH1 is the enthalpy determined from the upper half-width at the half-height of differentiated melting curve in purebuffer, n is the number of base pairs in the duplex, and R is theuniversal gas constant (1.986 cal mol�1 K�1). The experimentally


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determined values of water activity (ln aw) at given co-soluteconcentrations have been taken from Rozners and Moulder.41

The slope of the plot of reciprocal temperature (ln KT) of meltingversus the logarithm of water activity (ln aw) at different concen-trations (0, 5, 10, 15 and 20%) of small co-solutes gave the valueof d(Tm � 1)/d(ln aw) (Table S2 and S3, Fig. S14–S17, ESI†). Weused that methodology to determine the stability of PNA–DNAduplexes in the presence of different concentrations separatelyof glycerol and DMF (Table 4).We used this technique andcalculated the water activity (Dnw) of DPP-PNA�DNA duplexes incomparison to A-PNA�DNA duplex.

4.8. Error analysis

The uncertainties in the final Dnw were estimated from standarddeviations of experimental melting temperatures and DH accordingto standard procedures.41 Uncertainties in the slopes of the 1/Tm

versus ln aw plots [sd(Tm � 1)/d(ln aw)] were estimated by con-structing alternative linear fits using error bars (standard deviationsof 1/Tm) as the data points. Two alternative plots were obtained bylinear fits through 1/Tm plus or minus standard deviation at 0 and20% co-solute, e.g. 1/Tm plus standard deviation at 0% co-soluteand 1/Tm minus standard deviation at 20% co-solute gave onealternative, whereas the 1/Tm minus standard deviation at 0%co-solute and the 1/Tm plus standard deviation at 20% co-solutegave the other alternative. The deviations of both alternative plotsfrom the original plot were averaged. If necessary, to maintain thealternative plots inside the limits of error bars of the original plot,error bars at 5 or 15% co-solute were also included to construct thealternative plots. The final uncertainty in Dnw was calculated as:

sDnw = Dnw[(sDH/DH)2 + (s slope/slope)2]0.5

where sDH is the standard deviation, and slope = d(Tm � 1)/d(ln aw).

4.9. Circular dichroism experiments

CD spectra were scanned in the wavelength range of 195–320 nm,with a response time of 1.0 s, scan speed of 100 nm min�1,resolution of 1.0 nm, and a bandwidth of 1.0 nm on a JascoJ-810 Spectropolarimeter (Tokyo, Japan). Each CD spectrum wasaveraged from 3 accumulations and was corrected for baseline andnoise. 5 mM duplex concentrations were used for CD.

4.10. MD simulations

Model generation and parametrization. Since PNA is not astandard bio-molecule, its parameters are not included in thelibrary of the Amber MD package. We used standard B-DNA as atemplate56 for the model generation of PNA as double helix,stabilized mainly by the base pairing between the complemen-tary bases and the stacking of the base, while the electrostaticrepulsion between charged backbones tends to destabilize thestructure in the case of DNA.57 Since the force field parametersfor the modified nucleotides (PNA) are not available in theliterature, their parameters were generated using the GAUSSIAN 03and RESP58 program of AMBER10. All ab initio calculationswere done at the HF level of theory with 6-31G(d) basis set usingGAUSSIAN suite. In order to get RESP charges, the PNA residues

were separated into peptidic and basic fragments whereN-acylated DPP base was used as a basic fragment precededwith geometry optimization using the HF/6-31G(d) level oftheory. After the proper optimization and RESP charge fitting,parameters and forcefields were generated from antechamberand Leap module of Amber MD package using parmbsc0 forcefield.42

Equilibration and production dynamics. After the systempreparation and prior to the MD simulation, the structureswere minimized to remove the steric clashes. The completesystem was neutralized with twelve Na+ ions and immersed intothe truncated octahedral shell of TIP-3P59 water of dimension10.0 Å. The systems were then gradually heated from 10 to300 K over a period of 200 ps and then maintained in theisothermal–isobaric ensemble (NPT) at a target temperature of300 K and target pressure of 1 bar using Langevin thermostat60

and Barendsen barostat61 with collision frequency of 2 ps andpressure relaxation time of 1 ps, respectively. The hydrogenbonds were constrained using SHAKE.62 We have used thevelocity-Verlet algorithm (default algorithm for the Amber MDpackage) for MD simulations. Particle Mesh Ewald (PME) wasused to treat long-range electrostatic interactions using defaultparameters.63 After getting the systems at our target tempera-ture and pressure of 300 K and 1 bar, respectively, the dynamicscontinued up to 1 ns to equilibrate the system, with slowlydecreasing force constant from 500 to 5 kcal mol�1 Å2 in 11steps. For the analysis of the systems, the molecular mechanicalproduction dynamics phase was initiated and continued foranother 50 ns and maintained in the isothermal–isobaric ensembleat the target temperature of 300 K and target pressure of 1 bar usingthe same Langevin thermostat and Barendsen barostat as in theequilibration process, for each duplex separately. The structures inthe trajectories were collected at 10 ps intervals. All analyses oftrajectories were performed with the ptraj module of Amber 10.

MM_PBSA calculation. We performed standard MM_PBSA(molecular mechanics Poisson–Boltzman surface area)64

method for free energy calculation. Here, the total free energyof binding is expressed as the sum of the contribution from thegas phase and solvation energy and an additional term of soluteentropy. This can be expressed by following equation:

DGTOT = DGgas + DGsol + TDS

where DGgas is the total gas phase energy given by

DGgas = DEint + DEvdw + DGelec

Here DEint corresponds to bond, angle and torsion terms in themolecular mechanical force field, DGsol is the total solvationenergy (polar and non-polar), and TDS corresponds to soluteentropy effect. The analysis is done for the constant portionfrom RMSD plots for each simulation. The snapshots for theseduplexes are extracted at intervals of 20 ps. Prior to the analysis,all water molecules and sodium ions were stripped from thetrajectory. Solvation free energy is computed as the sum ofpolar and nonpolar contributions using a continuum solventrepresentation. The polar contribution is calculated by Molsurf,implemented in AMBER10. The non-polar solvent contribution


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is estimated from a SASA (solvent-accessible surface area)dependent term

DEsnp = gSASA + b

Here g is set to 0.0072 kcal Å�2 and b is set to 0. The calculationfor solute entropy contribution is performed with the NMODEmodule in AMBER10. The snapshots were minimized in the gasphase using the conjugate gradient method for 1000 steps,using a distance-dependent dielectric of 4r (r is inter-atomicdistance) and with a convergence criterion of 0.1 kcal mol�1 Åfor the energy gradient.

The trajectories were imaged using the ptraj module ofAMBER. The MOIL-View package was used to animate andvisualize the trajectories. The ptraj module of AMBER was usedto calculate the RMS deviations of all of the duplexes.

Acknowledgements

SS is thankful to UGC for funding. SKG is thankful to IndianCouncil of Medical Research (ICMR), Delhi, India, for providing afellowship as Senior Research Fellow. We want to acknowledgeProf. B. Jayaram (Department of Chemistry, IIT-Delhi, India) forproviding AMBER facility at the Supercomputing facility forBioinformatics & Computational Biology, IIT-Delhi, and Prof. S.Jain (Department of Physics & Astrophysics, University of Delhi)for providing Cluster facility. We also thankful to Dr K. D. Dubey,DDU Gorakhpur University, for his assistance for the computa-tional study and Dr Akhilesh K. Verma (Department of Chemistry,University of Delhi) for processing the NMR data of thecompounds.

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