5′-Modified G-Quadruplex Forming Oligonucleotides Endowed with Anti-HIVActivity: Synthesis and Biophysical Properties

Jennifer D’Onofrio,† Luigi Petraccone,‡ Eva Erra,‡ Luigi Martino,‡ Giovanni Di Fabio,† Lorenzo De Napoli,†

Concetta Giancola,*,‡ and Daniela Montesarchio*,†

Dipartimento di Chimica Organica e Biochimica, Universita` degli Studi di Napoli “Federico II”, and Dipartimentodi Chimica, Universita` degli Studi di Napoli “Federico II”, Complesso Universitario di Monte S. Angelo, via Cintia, 4,I-80126 Napoli, Italy. Received February 23, 2007; Revised Manuscript Received April 19, 2007

Oligodeoxyribonucleotides of sequence d(5′TGGGAG3′) carrying bulky aromatic groups at the 5′ end were foundto exhibit potent anti-HIV activity [Hotoda, H., et al. (1998)J. Med. Chem. 41, 3655-3663 and references therein].Structure-activity relationship investigations indicated that G-quadruplex formation, as well as the presence oflarge aromatic substituents at the 5′-end, were both essential for their antiviral activity. In this work, we synthesizedsome representative examples of the anti-HIV active Hotoda’s 6-mers and analyzed the resulting G-quadruplexesby CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case ofthe sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drugoptimization, we developed an alternative protocol to synthesize the 5′-DBB-thymidine phosphoramidite buildingblock in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes ofthe 5′-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of themolecular modeling studies. The aromatic groups at the 5′ position of d(5′TGGGAG3′) dramatically enhance boththe equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigatedquadruplexes was found to correlate with the reported IC50 values, thus furnishing quantitative evidence for thehypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biologicalactivity.

INTRODUCTION

In the past decade, many synthetic oligonucleotides (ODNs)have been found to be potent antiviral agents, and a number ofmodified ODNs have been selected as promising drug candidatesagainst HIV (1-3). The mechanisms of action of syntheticODNs as antivirals are based on the inhibition and/or interfer-ence with the viral cycle at different levels, including HIVbinding to target cells. Recently, Hotoda and co-workersdemonstrated that various G-rich oligonucleotides, bearingaromatic moieties as the 4,4′-dimethoxytriphenylmethyl (DMT)group at their 5′-end, are non-antisense anti-HIV active com-pounds (4-7). Particularly, they identified the lead sequence5′d(TGGGAG)3′, active against HIV-1 only when conjugatedat the 5′ position with large aromatic groups. The 6-mer bearingthe 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end andthe 2-hydroxyethylphosphate group at the 3′-end (R-95288)showed the most potent activity and the least cytotoxicity amongthe synthesized analogues. This specific anti-HIV activity wascorrelated with the ability of G-rich sequences to form stablequadruplex structures (8, 9), postulated to be the ultimate activespecies, directly interacting with thein ViVo targets. Evidencefor quadruplex formation in 5′-substituted d(5′TGGGAG3′)sequences was obtained by CD spectra, showing a large positiveband at 263 nm, diagnostic of parallel, tetramolecular G-quadruplex structures (4). The hypothesized mechanism ofaction of these 5′-conjugated G-rich ODNs is based on theinterference/inhibition of both cell-to-cell transmission and virus-to-cell transmission of HIV-1 by interaction with both the V3loop and the CD4 binding site on viral gp120, in analogy with

the behavior proposed for the phosphorothioate oligonucleotidesd(5′TTGGGGTT3′) (10) and d(5′GGGTTTTGGG3′) (11), form-ing, respectively, tetramolecular and bimolecular G-quadruplexcomplexes.

Other examples of G-rich oligonucleotides folding into highlystable quadruplexes, found to be potent inhibitors of HIVreplication in cell culture, are also known from the literature(12, 13). In particular, a recent study (14) has identified the16-mer guanine-rich sequence d(GGGGTGGGAGGAGGGT),designated93del, which inhibits both the processing and strandtransfer functions of HIV-1 integrase at nanomolar concentra-tions; and the NMR-based solution structure of this aptamer,adopting an unusually stable dimeric quadruplex architecture,has been described in detail (15).

Interestingly, Hotoda’s unmodified d(5′TGGGAG3′) showedno antiviral effects; no specific antiviral activity was also foundwhen a relatively small aromatic residue, as the benzyl group,was inserted at the 5′ position of the same sequence. Theantiviral activity of 5′-modified ODNs was therefore attributedto the presence of large aromatic groups at the 5′-end of thenative ODN backbone, which could produce hydrophobic effectsstabilizing quadruplex structures. CD spectra of the investigatedODNs were in agreement with the hypothesized relationshipbetween G-quadruplex formation and antiviral activity, but nosuccessive data appeared in the literature to analyze theseG-quadruplex forming systems in a quantitative manner or toshed light on their conformational differences.

The final conclusion of the authors is thatboth the G-quadruplex forming ODN backboneand the cluster of 5′-endsubstituents contribute to the interaction of the 6-mers with viralgp120. These results can also be correlated to a previous studyon the 20-mer phosphorothioate d(5′TGGGGCTTACCTTG-CGAACA3′) carrying covalently attached cholesteryl tails (16).

* E-mail: montesar@unina.it.† Dipartimento di Chimica Organica e Biochimica.‡ Dipartimento di Chimica.

1194 Bioconjugate Chem. 2007, 18, 1194−1204

10.1021/bc070062f CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 06/15/2007

In this work, the 3′-conjugated oligomer was showed to form astable quadruplex aggregate; conversely, the corresponding 5′-conjugate was essentially present as a single strand, which wasexplained as a consequence of negative steric effects impedingefficient G-quartet formation.

Aiming at the design of novel, more efficient oligonucleotide-based antivirals, we focused our attention on Hotoda’s 5′-conjugated d(5′TGGGAG3′) sequences, chosen as model com-pounds, to determine the main physicochemical parameters ofthe corresponding quadruplex structures, presumably presentalso in physiological conditions. In this paper, we intend toexplore possible direct relationships between the specific effectsinduced by the terminal groups and the thermodynamic andkinetic aspects of quadruplex formation. To this purpose, wehave selected a set of 5′-substituted Hotoda’s ODNs, amongthe ones showing the highest antiviral activity (4), i.e., 5′-DMT,5′-tert-butyldiphenylsilyl (TBDPS), and 5′-DBB-ether modified6-mers (1, 2, and3, Figure 1 and Table 1), and compared themto the natural sequence (4, Figure 1). In the case of ODN3, analternative and more efficient procedure has been developed(17) for the synthesis of 5′-DBB-thymidine-phosphoramiditebuilding block13 (Scheme 2), used in the last coupling step ofthe automated ODN chain assembly.

In all the studied cases, a detailed CD investigation for theanalysis of the conformational behavior, as well as a DSCanalysis, have been carried out for the complete description ofthe thermodynamic and kinetic parameters governing thetetraplex formation processes in solution. The obtained experi-mental data have been examined in light of the molecularmodels, generated to directly investigate the main conforma-tional differences of the quadruplex structures formed by 5′-conjugated ODNs1-3 (here described, respectively, as Q-DMT,Q-TBDPS, and Q-DBB), in comparison to the quadruplexobtained by unmodified ODN4 (Q-TGGGAG).

EXPERIMENTAL PROCEDURES

Synthesis and Characterization of Oligonucleotides 1-4.Phosphoramidite building blocks6 and 13 were synthesized

following the reactions described respectively in Schemes 1 and2. In the case of 5′-O-tert-butyldiphenylsilylthymidine-3′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite6, known pro-cedures were essentially followed (18). An alternative, moreefficient procedure was developed for the synthesis of 5′-DBBoligomer3, based on the usage of novel building block13 (17).A full description of the synthetic procedures adopted for thepreparation of 5′-modified thymidine phosphoramidites6 and13, of the NMR and ESI-MS characterization for all thenucleoside intermediates and final compounds, and of thegeneral experimental procedures are reported in the SupportingInformation.

Oligonucleotides1-4 were synthesized using standard solid-phaseâ-cyanoethyl phosphoramidite chemistry on a 15µmolscale. In the case of oligomers1 and4, starting from 100 mgof functionalized CPG1-dG support with 0.10 mequiv/g initialloading, the sequence5′d(TG3AG)3′ was assembled by exploitingstandard commercially available 3′-phosphoramidite 2′-deoxy-nucleoside monomers, respectively adopting the DMT-on, foroligomer 1, and the DMT-off protocol, for oligomer4.Analogously, the solid-phase assembly of oligomers2 and 3was carried out starting from 100 mg of functionalized CPG-dG support with 0.10 mequiv/g initial loading, using, respec-tively, 5′-TBDPS- and 5′-DBB-thymidine phosphoramidite (6,Scheme 1; and13, Scheme 2) in the last coupling step of thechain assembly. Oligomers1, 2, and4 were detached from thesolid support and deprotected by standard treatment with concaq ammonia at 55°C for 12 h. In the case of oligomer3,detachment from the solid support and full oligonucleotidedeprotection, including complete thymidine unmasking, wassuccessfully achieved by an overnight treatment at 50°C witha 0.1 M NaOH solution.

The combined filtrates and washings were concentrated underreduced pressure, redissolved in H2O, and analyzed and purifiedby HPLC on a Nucleosil RP18 column (Supelco, 100-5, C18,4.6 × 250 mm, 7µm), using a linear gradient from 20% to100% of CH3CN in 0.1 M TEAB in 20 min, flow rate 0.8 mL/min, and detection atλ ) 260 nm. The isolated oligomers hadthe following retention times:1 ) 17.5 min;2 ) 14.0 min;3) 11.5 min. After HPLC purification, the oligonucleotidesamples were desalted on a Sephadex G25 column eluted withH2O/EtOH (4:1, v/v). The isolated oligomers proved to be morethan 98% pure by1H NMR and RP-HPLC analysis and werecharacterized by ESI-MS and/or MALDI-TOF mass spectrom-etry.

Purification of the crude natural oligomer4 was carried outon an analytical anion exchange Nucleogel SAX column(Macherey-Nagel, 1000-8/46, 4.6× 50 mm, 5µm), adoptingthe following eluents: buffer A, 20 mM KH2PO4/K2HPO4 aqsolution (pH 7.0), containing 20% (v/v) CH3CN; buffer B, 1 MKCl, 20 mM KH2PO4/K2HPO4 aq solution (pH 7.0), containing20% (v/v) CH3CN. Using a linear gradient from 0% to 100%B in 40 min, flow rate 0.8 mL/min, and detection atλ ) 260nm, two main peaks were observed, with retention times 15.5min and 26.7 min. As also reported by Hotoda (4), in all thepurification attempts on an anionic exchange HPLC column,the fastest eluting peak, attributed to the single-strand 6-mer,was always accompanied by a major peak, having a higherretention time. This additional peak was attributed to a G-quadruplex structure generated under HPLC elution conditions.In fact, by reinjecting on HPLC the isolated and desalted fastest

1 Abbreviations: ADDP) 1,1′-(azodicarbonyl)dipiperidine; mCPBA) 3-chloroperbenzoic acid; CPG) controlled pore glass; DMAP)4-dimethylaminopyridine; DMF) N,N-dimethylformamide; TBAF)tetrabutylammonium fluoride; TEA) triethylamine; TEAB) triethy-lammonium bicarbonate; TFA) trifluoroacetic acid; THF) tetrahy-drofuran.

Figure 1. Oligonucleotides used.

Oligonucleotides with Anti-HIV Activity Bioconjugate Chem., Vol. 18, No. 4, 2007 1195

eluting peak, one could always observe the slowest eluting onealso; in addition, the two isolated peaks, analyzed in the sameconditions by1H NMR and ESI-MS spectra, always showedsuperimposable spectra.

In a typical experiment, starting from 100 mg of function-alized CPG-dG support, 110-150 OD units of purified1-4could be obtained.

Oligomer 1: Calcd mass for C81H92N27O36P5: 2173.489; ESI-MS, foundm/z 2174.57.

Oligomer 2: Calcd mass for C76H92N27O34P5Si: 2109.476;ESI-MS, foundm/z 2110.96.

Oligomer 3: Calcd mass for C81H92N27O36P5: 2173.489;MALDI-TOF, found [M - H]-: m/z 2172.76.

Oligomer 4: Calcd mass for C60H74N27O34P5: 1871.358; ESI-MS, foundm/z 1872.01.

Quadruplex Preparation. The quadruplex complexes wereformed by dissolving the lyophilized compound in the appropri-ate buffer and annealed by heating at 95°C for 5 min and slowly

Table 1. Physical Data of ODNs 1-4

MS datab

entry sequenceHPLC,a

tR (min) calcd foundIC50 valuesc

(µM)

1 DMT-5′TGGGAG3′ 17.5 2173.49 2174.57 1.0( 0.212 TBDPS-5′TGGGAG3′ 14.0 2109.48 2110.96 0.88( 0.0703 DBB-5′TGGGAG3′ 11.5 2173.49 2172.76 0.37( 0.0604 5′TGGGAG3′ 15.5 and 26.7 1871.36 1872.01 not active up to 40µM

a HPLC conditions are described in Experimental Procedures.b See Supporting Information.c See ref 4.

Scheme 1a

a Reagents and conditions:a, TBDPSCl, imidazole, DMF, 5 h, rt (95%);b, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, TEA, CH2Cl2,30 min, rt (92%).

Scheme 2a

a Reagents and conditions:a, TBDMSCl, imidazole, DMF, 5 h, rt (quant.);b, DMTCl, DMAP, pyridine, 12 h, rt (95%);c, PhSCH2CH2OH,PBu3-ADDP in benzene, 18 h, 0°C to rt (90%);d, 1 M TBAF in THF, 30 min, rt (98%);e, NaH, DBBCl, NaI, DMF, 18 h, rt (70%);f, mCPBA,CH2Cl2, 1 h, rt (98%);g, 3% TFA in CH2Cl2, 30 min, rt (94%);h, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, TEA, CH2Cl2, 30 min, rt(85%).

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cooling to room temperature. The buffer used was 10 mMpotassium phosphate, 200 mM KCl, 0.1 mM EDTA at pH)7.0. The concentrations of the dissolved oligonucleotides1-3were determined by UV measurements at 260 nm and at 95°C,using, as the molar extinction coefficients, the values calculatedby the nearest-neighbor model (19) for the sequence5′GGGAG3′,to which theε coefficient determined for the corresponding 5′-conjugated thymidylic acid residue (9100, 8900, and 9000 cm-1

M-1, respectively, for 5′-DMT, 5′-TBDPS, and 5′-DBB-thymidine-3′-monophosphate derivatives; for the synthesis ofthese nucleotides, see Supporting Information) was added.

Thermal Dissociation and Reassociation.Thermal dissocia-tion and reassociation of the different quadruplexes weremonitored by recording the CD spectra as a function of thetemperature. The molar ellipticity was calculated from theequation [ϑ] ) 100ϑ/cl, whereϑ is the ellipticity value,c isthe concentration of the single strand (2.0× 10-4 M), and l isthe path length of the cell in centimeters. The temperature wasincreased or decreased at a rate of 1.0°C/min. The melting andannealing profiles were not superimposable, indicating that thedissociation/reassociation reactions were not in thermodynamicequilibrium. Since the heating curves are not equilibrium curves,the melting temperatures obtained from these experiments wereindicated asT1/2 to distinguish them from the thermodynamicmelting temperatures (indicated asTm).

Dissociation Kinetics.The quadruplex dissociation kineticswere measured by rapidly increasing the temperature andfollowing the change with time of the CD signal at 263 nm.All the experiments were performed at 2.0× 10-4 M single-strand concentration and in 60-80 °C temperature range,conditions under which the reassociation reaction can beneglected. The kinetic constants were obtained by fitting therecorded time-dependent decrease in CD signal with a single-exponential function.

Isothermal Kinetic Analysis for Association.The associa-tion of single strands into a well-defined quadruplex structurewas monitored for each sequence in the buffer 10 mM potassiumphosphate, 200 mM KCl, 0.1 mM EDTA at pH) 7.0. Theexperiments were carried out at various strand concentrations(between 5× 10-6 and 2× 10-4 M single-strand concentration),depending on the kinetics. The samples were incubated at 90°Cfor 5 min to allow for dissociation. The structural transitionfrom the single strands to the tetramolecular quadruplex wasmonitored in the temperature range 10-30 °C by recording theCD spectra as a function of time. Assuming that the dissociationis negligible, the single-strand concentration at any time is givenby

where [S]t is the single-strand concentration at timet, [S]0 isthe initial single-strand concentration,n is the order of thereaction with respect to the single-strand concentration, andkon

is the rate constant for the single-strand disappearance. Aspreviously described (20), the changes in CD spectra at a giventime t can be related with the single-strand concentrationaccording to the equation

whereΘ∞ andΘ0 correspond to the ellipticity of the final andinitial state, respectively.Θt is the ellipticity at timet, andk′ )4(Θ∞ - Θ0/[S]0)1-nkon.

The association rate constants and the reaction orders wereobtained by fitting the ellipticity at 263 nm as a function oftime by means of eq 2 usingOrigin 7.5 software.

DSC Experiments.Differential scanning calorimetry mea-surements were carried out on a third-generation Setaram Micro-DSC. The excess heat capacity function⟨∆CP⟩ was obtainedafter baseline subtraction, assuming that the baseline is givenby the linear temperature dependence of the native-state heatcapacity (21). No baseline difference was observed before andafter the transition, indicating a negligible heat capacity differ-ence between the initial and final states. The transition enthal-pies, ∆H°cal, were obtained by integrating the area under theheat capacity vs temperature curves. The enthalpy change valuesare the averages of at least three different heating experiments.The reported errors are the standard deviations of the mean fromthe multiple determinations.

Molecular Modeling. The conformational features of thequadruplexes generated by modified ODNs1-3 have beenexplored by means of molecular modeling studies. All thecalculations were performed on a personal computer runningthe HyperChem 7.5suite of programs. To build the [d(TG3-AG)]4 quadruplex, the T and dG nucleotide residues weregenerated by using the coordinates of the corresponding residuesin a previously reported quadruplex structure (22), whereas theA-tetrad was generated by using the coordinates of the dAresidues in the reported structure of the [d(TTAG3T)]4 (23).

The DMT, DBB, and TBDPS groups were generated usingthe HyperChem 7.5building tool. Partial charges for all theatoms in the DMT, DBB, and TBDPS moieties were assignedusing the Gasteiger-Marsili algorithm (24) implemented in theQSAR module of the HyperChem suite. The Q-DMT, Q-DBB,and Q-TBDPS quadruplexes were then built by linking theappropriate groups to the 5′-ends of the unmodified quadruplex.The resulting coordinates of the modified quadruplexes wereenergy-minimized in vacuum for 500 steps of the steepest-descent method, keeping the DNA residues in fixed position,allowing only the DMT, DBB, and TBDPS atoms to relax.Potassium ions were positioned in the central channel betweenthe G-tetrads to allow for coordination, for each potassium ion,with the eight carbonyl oxygen atoms of the two adjacentG-quartets completing the octahedral coordination sphere. Theremaining potassium counterions were added to give electricalneutrality, and then the neutralized systems were solvated in apreequilibrated box of TIP3P water (25). The water moleculeswithin a 2.6 Å radius from any solute atoms were removed.The Amber force field was used (26). A simulated annealingwas performed on the modified T residues before the energystructure minimization. During the simulated annealing, theDNA coordinates were kept fixed, and only the modified Tresidues and the solvent (water molecules and ions) wereallowed to move. The initial conformation was first heated to500 K over 10 ps, simulated at 500 K for 70 ps, and finallycooled to 10 K for 20 ps. Then, the whole system was minimizedusing 1000 cycles of the steepest-descent method followed bythe conjugate gradient method until convergence to an rmsgradient of 0.1 kcal mol-1 Å-1.

The contributions of the DBB, TBDPS, and DMT groups tothe total solvent-accessible surface area (SASA) were calculatedusing the grid method (27) implemented in the QSAR moduleof the HyperChem suite. The SASA was obtained for eacharomatic group both in the quadruplex (SASAquadruplex) and inthe dissociated single strand (SASAsingle strand). The SASAquadruplex

values were calculated using the minimized quadruplex struc-tures, whereas the SASAsingle strandvalues were obtained underthe assumption that the aromatic groups in the single strandsare completely exposed to the solvent. The difference∆(SASA)) SASAquadruplex- (4 × SASAsingle strand) represents the relativereduction of the solvent-accessible surface area of the 5′-aromatic substituents that occurs upon quadruplex formation.

[S]t1-n - [S]0

1-n ) 4(n - 1)kont (1)

Θt ) Θ∞ - [(Θ∞ - Θ0)1-n + (n - 1)k′t]1/1-n (2)

Oligonucleotides with Anti-HIV Activity Bioconjugate Chem., Vol. 18, No. 4, 2007 1197

RESULTS

For a detailed investigation of the structural and biophysicalproperties of 5′-modified d(5′TGGGAG3′) sequences, the prepa-ration of the model oligonucleotides in the 3-5 mg scale wasa prerequisite. The 5′-modified oligomer1 could be easilyachieved by standard ODN chain assembly procedures, simplyinvolving a DMT-on synthetic routine to preserve the 4,4′-dimethoxytriphenylmethyl group at the 5′-end of the oligo-nucleotide. On the other hand, for the preparation of ODNs2and3 special protocols were required. In their papers, Hotodaand co-workers synthesized the 5′-modified oligonucleotides bymeans of standard phosphoramidite chemistry (28) using tailored5′-substituted thymidine derivatives as the 5′-terminal residues.5′-O-TBDPS-thymidine derivative6 was synthesized in twosteps, starting from thymidine, with 46% overall yields. 5′-O-(3,4-dibenzyloxy)benzylthymidine-3′-O-(2-cyanoethyl)-N,N-di-isopropylphosphoramidite was obtained in three steps and 20%overall yields starting from 3′-O-tert-butyldimethylsilylthymi-dine (3′-O-TBDMS-thymidine), which in turn can be typicallyprepared from thymidine in three straightforward, high-yieldingreactions (18).

In our hands, the preparation of phosphoramidite6 wassuccessfully achieved with 88% overall yields. Conversely, wealways failed in the synthesis of 6-mer3 following Hotoda’sprocedure, particularly at the crucial stage of the insertion ofthe 3,4-DBB group at the 5′-OH of the thymidine monomer. InHotoda’s protocol, the 5′-DBB-ether was synthesized by react-ing, in the presence of NaH, DBB bromide (29) with 3′-O-TBDMS-thymidine in THF. The 5′-alkylated compound, re-covered by the authors in very modest yields (23%), afterTBDMS removal, was then phosphitylated in 87% yields to givethe corresponding 3′-phosphoramidite derivative, used as thelast addition monomer in the oligonucleotide chain growth.Following the same alkylation procedure, with the sole differ-ence of using the commercially available DBB chloride in lieuof the bromide, we could exclusively isolate from the reactionmixtures the undesired N3-alkylation product, even whenvarying several experimental conditions. When further forcingthe reaction, two compounds, identified after column chroma-tography as the N3, O-bis-alkylated derivatives, were observed.In fact, as also reported by Kobe et al. (30), starting from 3′-O-TBDMS-thymidine the concomitant formation, in almost 1:1ratio, of both the N3, O-5′- and the N3, O-3′-bis-alkylatedderivative occurred, as a result of the hydride-mediated migra-tion of the TBDMS group from the 3′- to the 5′-position. Theseresults led us to develop an alternative protection strategy forthe thymidine moieties to more efficiently obtain the 5′-alkylatednucleoside, based on (i) protection of the 3′-OH function witha group completely stable under basic conditions and reluctantto migrate to the 5′-OH and (ii) protection of the thymine base.

Having discarded the silyl ethers as the 3′-OH protectinggroups, we moved to the DMT group, completely stable evento harsh basic media, and searched for a suitable heterocycleprotection. A novel, two-stage protecting group, namely, the2-(phenylthio)ethyl group, proved to be useful to our purposes,preventing otherwise predominant N-alkylation of thymine (17).The phenylthioethyl residue proved to be completely stable ontothe heterocycle as such, but after oxidation of the tioether tosulfone, the resulting group could be easily removedVia aâ-elimination reaction. Treatment with a 0.1 M NaOH solutionat 50°C allowed complete thymine deblocking after 12 h,i.e.,under conditions usually adopted also for oligonucleotidedeprotection and detachment from the solid support.

We therefore synthesized 5′-O-TBDMS-3′-O-DMT-thymidine7 as the starting material and reacted it with 2-(phenylthio)-ethanol under standard Mitsunobu conditions (Scheme 2).Following this protocol, the N3-alkylated nucleoside8 was

obtained in 85% yields for the three steps. Subsequent standardtriethylammonium fluoride treatment yielded the 5′-OH deriva-tive 9 in almost quantitative yields, which was next reacted withDBBCl and NaH with cat. NaI, to give the desired 5′-DBBadduct10 in 70% yields. After oxidation with mCPBA of thethioether function to sulfone11, addition of 3% TFA in CH2-Cl2 led to12, which was finally phosphitylated to give desired13. Starting from 5′-O-TBDMS-3′-O-DMT-thymidine, 3′-phos-phoramidite building block13 was obtained in six straightfor-ward steps and 48% overall yields (46% overall yields startingfrom thymidine).

The assembly of oligomers1-4 was then achieved on anautomated DNA synthesizer by exploiting classical phosphora-midite chemistry, using a standard DMT-on protocol for 5′-conjugated1-3. In the case of the synthesis of2 and 3, thelast coupling was carried out, respectively, using the buildingblock 5′-TBDPS-thymidine phosphoramidite6 or 5′-DBB-thymidine phosphoramidite13. Once the assembly of the desiredsequence was completed, detachment from the solid supportand full deprotection of the 5′-conjugated oligomer, includingthymine protecting group removal for oligomer3, were achievedby an overnight treatment with 0.1 M NaOH.

Purification of the detached oligomers was carried out byHPLC on an analytical anionic exchange column in the case of4 and on an analytical RP18 column for1-3 (see ExperimentalProcedures). The isolated products, desalted on a Sephadex G25column, were checked for purity by HPLC and characterizedby mass analysis (Table 1).

The dissociation and reassociation of the tetramolecularquadruplexes were followed by circular dichroism. The CDspectra at 10°C for all the studied sequences showed a positiveband at 263 nm and a negative band at 243 nm (Figure 2),diagnostic of parallel-stranded tetramolecular quadruplex struc-tures (4, 31). In Figure 3, the melting and annealing CD profilesare shown for all the samples at the heating and cooling rate of1.0 °C/min. All the quadruplexes show hysteresis between themelting and annealing profiles, which indicates that the as-sociation/dissociation reaction is not at thermodynamic equi-librium during the heating and cooling experiments (32, 33).The melting temperatures obtained at 1.0°C/min scan rate arevery similar for the modified quadruplexes. Particularly, theT1/2

values are 89°C for the Q-TBDPS, 86°C for the Q-DMT, and83 °C for the Q-DBB, whereas theT1/2 for the unmodifiedquadruplex is 60°C. Q-DBB and Q-TBDPS show lesspronounced hysteresis between melting and annealing curvesthan Q-DMT. In addition, almost no reassociation of theunmodified strands (Figure 3) was observed during the coolingcurve. These findings suggest that Q-TBDPS and Q-DBBcomplexes are formed faster than Q-DMT and that all themodified strands associate faster than the unmodified strands.In the case of Q-DMT, the melting and annealing profiles were

Figure 2. CD spectra of the Q-TGGGAG (squares), Q-DBB (opentriangles), Q-TBDPS (open circles), and Q-DMT (diamonds). Thespectra were recorded at 10°C in a 10 mM potassium phosphate buffer(pH 7.0) supplemented with 200 mM KCl.

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always found to be not fully reproducible (data not shown).This behavior was not completely unexpected, since theprolonged exposition to high temperatures is well-known tocause partial 5′-deprotection of 5′-DMT-oligonucleotides. Toconfirm this hypothesis, the Q-DMT samples were analyzedfor integrity by RP HPLC after each heating experiment,resulting in a mixture of1 and4. For this reason, the Q-DMTsystem could not be investigated in detail, and our efforts werethen concentrated on more chemically stable ODNs2-4.

The kinetics of Q-DBB, Q-TBDPS, and Q-TGGGAG qua-druplex association was directly measured. The rate of theassociation reaction was studied by monitoring the ellipticityat 263 nm as a function of time after heating the sample at 95°Cfor 5 min and cooling rapidly. Representative molar ellipticity

vs time profiles at 15°C are shown in Figure 4. Similar profileswere obtained at the other investigated temperatures (see FiguresS1-S3 in the Supporting Information).

The experimental curves were fitted by means of eq 2. Allthe data for the studied sequences were fitted well by reactionorder of 4.0. The kinetic constants (kon) for the studied sequencesare compared in Table 2. The rate of quadruplex associationfollows the order Q-TBDPS> Q-DBB > Q-TGGGAG. Theassociation rate constants at 25°C were 8.6× 1010 M-3 s-1

and 2.2× 109 M-3 s-1 for Q-TBDPS and Q-DBB, respectively.The corresponding value found for the unmodified sequence atthe same temperature is 4.1× 105 M-3 s-1.

The kinetic experiments were performed over the temperaturerange 10-30 °C. For all the sequences, the association rate

Figure 3. CD melting and annealing profiles for the studied quadruplexes. The solid lines are relative to melting profiles and the dot lines toannealing profiles. All the experiments were performed in a 10 mM potassium phosphate buffer (pH 7.0) supplemented with 200 mM KCl.

Figure 4. Kinetics of the quadruplex formation. Association profiles for4 (A), 2 (B), and3 (C) at 15°C. Single-strand concentrations used: 2×10-5 M for 2, 4.4× 10-5 M for 3, and 4.4× 10-4 M for 4. Quadruplex formation leads to an increase of molar ellipticity at 263 nm. Mathematicalfits are shown in solid line. (D) Arrhenius plots for the association of4 (squares),2 (circles), and3 (triangles). All the experiments were performedin a 10 mM potassium phosphate buffer (pH 7.0) supplemented with 200 mM KCl.

Oligonucleotides with Anti-HIV Activity Bioconjugate Chem., Vol. 18, No. 4, 2007 1199

decreases upon increasing the temperature, and this effect ismore pronounced for the unmodified sequence. From the slopeof the Arrhenius plots (Figure 4D), the activation energies (Eon)could be derived (Table 2). We found negative activationenergies for all the sequences, as already reported in the literaturefor other quadruplexes and attributed to the existence of a rapidpreequilibrium step involving intermediate species (20, 32, 34,35).

The kinetics of quadruplex dissociation was also investigatedby performing temperature-jump experiments. In each experi-ment, the temperature of the sample was rapidly increased, andthe rate of quadruplex dissociation was monitored by followingthe change of CD signal with time. Representative kineticprofiles obtained at 65°C are shown in Figure 5. Similar profileswere obtained at the other investigated temperatures (see FiguresS4-S6 in the Supporting Information). The dissociation rateconstants were obtained by fitting these kinetic profiles with asingle-exponential curve. The kinetic constants were then usedto construct the Arrhenius plots (Figure 5D), so allowing thedetermination of the corresponding activation energies. The

kinetic parameters for quadruplexes dissociation are summarizedin Table 3. Both Q-TBDPS and Q-DBB havekoff values 10-100 times smaller than the corresponding value of Q-TGGGAGat the same temperature. Analogously, the modified quadru-plexes have higher activation energies in comparison with theunmodified one. The values found for Q-TBDPS and Q-DBBare about 100 kJ mol-1 higher than the one found forQ-TGGGAG, proving that the presence of the aromatic groupssignificantly stabilizes the quadruplex structure. The kineticallymost stable complex is Q-TBDPS, with a half-life of 591 daysat 37°C, whereas at the same temperature, the half-life for theunmodified quadruplex is 69 min.

Differential scanning calorimetry (DSC) experiments wereperformed on each quadruplex, and the corresponding calori-metric profiles at scan rate of 1.0°C/min are shown in Figure6. The calorimetric profiles and melting temperatures wereinfluenced by the scan rate (Figure 7), indicating nonequilibriumconditions, analogous to the results already found in the CD-monitored melting experiments. Although the∆S°, ∆G°, andTm values cannot be derived from these measurements as theyare not at equilibrium, the enthalpy changes measured can becorrectly attributed to the quadruplex-single-strand transition,provided that the oligonucleotides are 100% structured in thequadruplex form at the starting temperature of the DSC scan.To ensure complete quadruplex formation, before DSC experi-ments, each sequence was annealed by heating at 95°C for 5min and slowly cooling to room temperature, and then incubatedat low temperature for an appropriate time, depending on theknown association kinetics. The enthalpy changes for all thequadruplexes are reported in Table 4. Note, however, that thevalue obtained here for Q-DMT is assumed to be less precise;in fact, it cannot be excluded that partial detritylation occurredwhen the sample was exposed to high temperatures. This eventwas indeed directly analyzed in the case of the samples preparedfor the CD-monitored melting investigations.

The thermodynamic parameters for the dissociation of thequadruplexes were calculated from the rate constants and theactivation energies (35, 36). These kinetically derived thermo-dynamic parameters are reported in Table 4. There is a goodagreement between the calorimetric∆H°cal∆ values and thekinetically derived values (∆H° ) Eon - Eoff). It is noteworthythat all the modified quadruplexes show an equilibrium meltingtemperature (Tm) much higher than 37°C, whereas the unmodi-fied quadruplex show a lower equilibriumTm (26 °C). The∆S°and∆G° values were calculated using the more accurate∆H°calvalues.

The ∆H°cal value for the unmodified quadruplex is 310 kJmol-1, which accounts for the formation of four G-tetrads (31,37, 38) and suggests that both the dA and dG nucleotide at the3′-ends of the ODN strand participate in the overall quadruplexstabilization. All the modified quadruplexes have higher en-thalpic terms in comparison with the unmodified quadruplex.Particularly, the∆H°cal values of the modified quadruplexesfollow the order Q-TBDPS> Q-DMT > Q-DBB. The sametrend was observed for the∆S° values of Q-TBDPS andQ-DBB, thus counterbalancing the contribution given by the∆H°cal and leading to comparable data for theTm and freeenergy values for Q-TBDPS and Q-DBB.

We used molecular modeling to explore the main conforma-tional differences induced by the presence of the DBB, DMT,and TBDPS groups at the 5′-end of the d(5′TGGGAG3′)quadruplex structures. In Figure 8, the minimized structures ofthe three modified quadruplexes are shown. In the top view,the central channel of the quadruplex structure is clearly visiblefor Q-TBDPS and Q-DMT, whereas in Q-DBB, it is hidden bythe DBB groups, suggesting that only this moiety is able tospan the distance between the 5′-ends of nonadjacent strands.

Table 2. Kinetic Parameters for the Quadruplex Formationa

quadruplextemp

(°C) (1 n kon (M-3 s-1)b Eon(kJ mol-1)

Q-TBDPS 15 4 (2.9( 0.5)× 1011

20 4 (1.5( 0.6)× 1011

25 4 (8.6( 0.5)× 1010 -125( 1930 4 (2.3( 0.8)× 1010

Q-DBB 15 4 (6.9( 0.4)× 109

20 4 (4.9( 0.4)× 109

25 4 (2.2( 0.7)× 109 -90 ( 930 4 (1.5( 0.3)× 109

Q-TGGGAG 10 4 (3.0( 0.5)× 107

15 4 (4.5( 0.8)× 106

20 4 (3.2( 0.2)× 106 -190( 2025 4 (4.1( 0.3)× 105

a All the values have been determined in a 200 mM KCl, 10 mMpotassium phosphate, pH 7.0 buffer.b kon is defined as d[S]/dt ) -4d[S4]/dt ) -kon[S]4.

Table 3. Kinetic Parameters for the Quadruplex Dissociationa

quadruplextemp

(°C) (1koff × 104 b

(s-1) t1/2c

Eoff

(kJ mol-1)

Q-TBDPS 70 0.50( 0.0373 0.83( 0.0275 1.4( 0.4 591 days77 2.3( 0.1 220( 2080 3.5( 0.3

Q-DBB 65 1.8( 0.567 2.2( 0.570 5.0( 0.4 41 days73 9.1( 0.3 210( 975 15( 3

Q-TGGGAG 63 40( 165 46( 167 67( 1 69 min70 84( 5 105( 1473 120( 9

a All the values have been determined in a 200 mM KCl, 10 mMpotassium phosphate, pH 7.0 buffer.b koff is defined as d[S]/dt ) -d[S4]/dt ) koff[S4]. c Half-dissociation times of tetramolecular quadruplex at 37°C.

Table 4. Thermodynamic Parameters for the QuadruplexDissociation

quadruplexTm

(°C) (1∆H°cal

(kJ mol-1)∆H°kin

(kJ mol-1)∆S°

(kJ mol-1 K-1)∆G°298

(kJ mol-1)

Q-TGGGAG 26 310( 12 295( 34 0.82( 0.03 66( 21Q-DBB 72 320( 15 300( 17 0.71( 0.03 108( 15Q-TBDPS 75 355( 15 345( 39 0.80( 0.03 117( 15Q-DMT 330( 13

1200 Bioconjugate Chem., Vol. 18, No. 4, 2007 D’Onofrio et al.

Close inspection of the models reveals that the DBB aromaticrings form a network of noncovalent face-to-face and face-to-edge (T-shaped) aromatic interactions, so that the planar fourthymines at the 5′-end of the strands are not exposed to thesolvent.

Still being isomers, the DBB and the DMT groups conferdifferent properties to the resulting quadruplex complexes. Inthe DBB group, the three phenyl rings are spaced by the flexible

O-CH2- linkers and can adopt different relative orientations,whereas in the DMT group, the three phenyl residues, beingattached to the same carbon atom, show a significantly reducedflexibility and, to avoid steric crowding, are obliged to beapproximately perpendicular to each other. In Q-DMT, the DMTgroup does not stack over the surface formed by the fourthymine bases, with the three phenyl rings largely projectedaway from the helix axis, to minimize mutual steric hindranceinteractions. However, in Q-DMT, a certain number of van derWaals contacts were observed between the aromatic groups ofadjacent strands.

In Q-TBDPS, the TBDPS groups are not large enough todirectly interact with each other over the quadruplex surface atthe 5′-end and only show stacking interactions with the adjacentthymine base.

To compare the relative contributions of the modified groupsto the hydrophobic interactions involved in the quadruplex

Figure 5. Kinetics of the quadruplex dissociation. Dissociation profiles for Q-TGGGAG (A) and Q-DBB (C), measured at 65°C, and for Q-TBDPS(B) measured at 70°C. Quadruplex dissociation leads to a decrease of the molar ellipticity at 263 nm. Mathematical fits are shown in solid line.(D) Arrhenius plots for the dissociation of the Q-TGGGAG (squares), Q-TBDPS (circles), and Q-DBB (triangles). All the experiments were performedin 10 mM potassium phosphate buffer (pH 7.0) supplemented with 200 mM KCl.

Figure 6. Calorimetric heat capacity vs temperature profiles at1 °C/min for the studied quadruplexes. Excess capacity values havebeen shifted along they-axis for ease of presentation. All theexperiments were performed in a 10 mM potassium phosphate buffer(pH 7.0) supplemented with 200 mM KCl.

Figure 7. Calorimetric heat capacity vs temperature profiles at differentscan rate 0.3°C/min (open triangles), 0.5°C/min (solid line), and1 °C/min (open circles) for the Q-TGGGAG. All the experiments wereperformed in a 10 mM potassium phosphate buffer (pH 7.0) supple-mented with 200 mM KCl.

Oligonucleotides with Anti-HIV Activity Bioconjugate Chem., Vol. 18, No. 4, 2007 1201

formation, we calculated the relative decrease in the solvent-accessible surface area (SASA) for the DBB, TBDPS, and DMTgroups in the quadruplex structure (SASAquadruplex) with respectto the dissociated single strands (SASAsingle strand). Inspectionof the ∆(SASA) values (Table 5) reveals that the highestreduction in the solvent-accessible surface area upon quadruplexformation is observed in the case of the DBB group. This resultsuggests that the hydrophobic interactions play a major role inthe Q-DBB quadruplex formation with respect to the other twomodified quadruplexes.

DISCUSSION

In this work, we synthesized and characterized a set of 5′-substituted Hotoda’s ODNs, among the ones showing the highestantiviral activity (4), i.e., 5′-DMT, 5′-TBDPS, and 5′-DBB-ethermodified 6-mers (1, 2, and 3, Figure 1 and Table 1). TheseODNs were compared to the unmodified sequence, whichshowed no antiviral effects, in an attempt to put on a quantitativebasis the hypothesized correlation between biological activityand physicochemical properties of these G-quadruplex-formingODNs. Our results point out remarkable physicochemicaldifferences between the Hotoda’s 5′-substituted ODNs and thenatural sequence. The insertion of large aromatic groups, asDMT, DBB, or TBDPS, at the 5′-end of the d(5′TGGGAG3′)sequence has dramatic effects on the kinetics and thermodynam-ics of quadruplex formation. All the 5′-modified quadruplexes,showing comparable thermal stability, are far more stable than

the natural quadruplex. Inspection of Table 4 reveals that thehigher thermodynamic stability of Q-DBB and Q-TBDPS withrespect to the natural quadruplex complex has different ther-modynamic origins. Indeed, Q-DBB is entropically stabilized,whereas Q-TBDPS is mainly enthalpically stabilized in com-parison with Q-TGGGAG. The low entropic term observed forthe dissociation of the Q-DBB quadruplex can be due to adecrease of the solvation entropy of the DBB groups after thequadruplex dissociation. This hypothesis is supported by ourmolecular modeling results, revealing a significant increase ofthe solvent-accessible surface area (∆SASA) of the DBB groupswhen they are not involved in the quadruplex structure. On theother hand, the formation of structured DBB groups in theQ-DBB quadruplex very weakly affects the overall enthalpicchange involved in quadruplex formation, with this being theresult of two opposite contributions: a positive contribution,due to hydrophobic factors, and a negative contribution, due toincreased stacking and van der Waals interactions between theDBB groups. As a consequence, the enthalpic change forQ-DBB formation is very similar to the one measured forQ-TGGGAG.

Conversely, the higher thermodynamic stability of Q-TBDPSin comparison with the natural quadruplex is essentially due tothe increased enthalpic term, suggesting that the hydrophobiceffects play a minor role in this case, when compared to Q-DBB.This result is in agreement with the smaller∆(SASA) valueestimated from the molecular models (Table 5). Further, thestacking interactions between the TBDPS groups and theadjacent thymine bases, observed in the molecular models, canaccount for the higher enthalpic term measured in comparisonwith the natural quadruplex. In addition, in both Q-DBB andQ-TBDPS complexes, high activation energies were measuredfor the dissociation processes, consistent with the additionalstacking and van der Waals interactions induced by the aromaticgroups in these modified quadruplexes.

Another striking result of our study is that the introductionof the aromatic groups deeply affects the rate of quadruplex

Figure 8. Top and lateral views of the molecular models of Q-TBDPS (left), Q-DMT (middle), and Q-DBB (right). The 5′-aromatic groups areshown with a space-filling representation.

Table 5. Contributions of the 5′-Aromatic Groups to the TotalSolvent-Accessible Surface Area (SASA)

5′-aromaticgroups

SASAquadruplex

(Å2)SASAsingle strand

(Å2)∆(SASA)a

(Å2)

DBB 1245 520 -835TBDPS 1251 442 -517DMT 1826 541 -338

a ∆(SASA) ) SASAquadruplex- (4 × SASAsingle strand).

1202 Bioconjugate Chem., Vol. 18, No. 4, 2007 D’Onofrio et al.

formation. Under the same conditions, Q-TBDPS is assembled105 times faster than Q-TGGGAG. The faster associationkinetics of the modified single strand is probably due tohydrophobic interactions, which is consistent with the higheractivation energies (hydrophobic interactions give a positivecontribution to the totalEon value) measured for the modifiedstrands with respect to the unmodified sequence.

Taken together, our data show that all the modified d(5′-TGGGAG3′) sequences are able to adopt stable G-quadruplexstructures. On the contrary, the unmodified sequence forms aweak complex, with aTm of 26 °C under the investigatedconditions. Large aromatic groups at the 5′-end of the oligo-nucleotide chain dramatically stabilize the resulting G-quadru-plex complexes on both a kinetic and a thermodynamic basis,with hydrophobic contributions on one side and additionalstacking and van der Waals interactions on the other side playingmajor roles in the quadruplex formation processes.Tm valueshigher than 70°C were found for Q-DBB and Q-TBDPS; similarindications were obtained also for Q-DMT from∆H°cal asobtained by DSC experiments, even if the latter case could notbe further investigated in detail due to the intrinsic instabilityto high temperatures of the DMT ether linkage. High thermalstability, associated with a very fast association and slowdissociation, account for the 5′-modified ODNs fully structuredin the form of quadruplex complexes; on the contrary, only avery small percentage of the unmodified oligomer is present inthe aggregated form under physiological conditions. Since thephysicochemical parameters for quadruplex stability reflect wellthe trend observed for the anti-HIV activity (see IC50 datareported in Table 1), our data can furnish indirect evidence that5′-conjugated d(5′TGGGAG3′) oligomers interact with the HIV-1gp120 target only if structured in the quadruplex form.

In conclusion, a systematic physicochemical characterizationby DSC and CD analyses has been carried out on a representa-tive set of 5′-modified d(5′TGGGAG3′) sequences, able to adoptstable G-quadruplex structures, to better understand the subtlefeatures involved in the formation and stability of thesecomplexes. The present results demonstrate that the largearomatic groups at the 5′-position of d(5′TGGGAG3′) play adecisive role in favoring the quadruplex formation processes.The unmodified sequence is indeed able to adopt a G-quadruplexstructure, though it is not thermodynamically stable at physi-ological temperature and its formation is, in addition, very slow.Conversely, the 5′-modified quadruplexes are formed faster and,once formed, are very thermodynamically stable, withTm valueshigher than 70°C. These findings suggest a correlation betweenstability and rate of formation of the G-quadruplex structureson one side and anti-HIV activity of 5′-modified ODNs on theother side.

These results are useful not only to gain a critical insightinto the biological behavior of Hotoda’s 6-mers, but also todevelop a general frame to better understand and, ultimately,roughly predict the effective potential as drugs of quadruplex-forming oligonucleotides. The very slow kinetics of quadruplexformation is one of the main drawbacks limiting efficientpharmacological applications of aptamers based on tetramo-lecular quadruplex structures. For this reason, it is highlydesirable that, in a drug optimization process, most researchefforts are concentrated on the rational search for well-tailoredmodifications to be inserted within the oligonucleotide backbone.Large aromatic groups may indeed be regarded as suitableappendages to enhance the biological activity of G-rich,quadruplex-forming ODNs, positively affecting both stabilityand rate of formation of quadruplex complexes.

This study, developed in the more general frame of the searchfor novel ODN analogues suitable for therapeutic applications,adds a further contribution to the research of biologically active

quadruplex forming oligonucleotides, showing kinetic aspectsas relevant as thermodynamic parameters in influencing theoverall quadruplex formation process and therefore in affectingtheir biological role within the cell.

ACKNOWLEDGMENT

We acknowledge MIUR (PRIN) and Regione Campania (L5)for grants in support of this investigation. We also thankC.I.M.C.F., Universita` degli Studi di Napoli “Federico II”, forthe NMR, MS, and CD facilities.

Supporting Information Available: Synthetic procedures andcharacterization of phosphoramidite building blocks6 and13and general experimental procedures and synthetic methods forthe preparation of the 5′-DMT, 5′-TBDPS, and 5′-DBB nucleo-side-3′-monophosphates for theε calculation are reported.Kinetic profiles for the association process and for the dissocia-tion process of Q-DBB, Q-TBDPS, and Q-TGGGAG at differenttemperatures are also available. This material is available freeof charge via the Internet at htpp://pubs.acs.org/BC.

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