Synthesis, Biophysical Characterization, and Anti-HIV Activity of Glyco

Bioconjugate Chem. 2008, 19, 607–616

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Synthesis, Biophysical Characterization, and Anti-HIV Activity of Glyco-Conjugated G-Quadruplex-Forming Oligonucleotides Jennifer D’Onofrio,† Luigi Petraccone,‡ Luigi Martino,‡ Giovanni Di Fabio,† Alfonso Iadonisi,† Jan Balzarini,§ Concetta Giancola,*,‡ and Daniela Montesarchio*,† Dipartimento di Chimica Organica e Biochimica and Dipartimento di Chimica “Paolo Corradini”, Università degli Studi di Napoli “Federico II”, Via Cintia 4, I-80126 Napoli, Italy, and Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroederstraat, B-3000 Leuven, Belgium. Received September 4, 2007; Revised Manuscript Received December 11, 2007

Novel hybrid oligonucleotides carrying the G-quadruplex-forming d(5′TGGGAG3′) sequence, conjugated with mono- or disaccharides at the 3′ or 5′-end through phosphodiester bonds, have been synthesized as potential anti-HIV agents, Via a fully automated, online phosphoramidite-based solid-phase strategy. CD-monitored thermal denaturation studies on the resulting quadruplexes indicated the insertion of a single monosaccharide at the 3′end as the optimal modification, conferring improved stability to the quadruplex complex. In addition, the 3′conjugation with glucose or mannose converted the anti-HIV inactive unmodified oligomer into active compounds. On the contrary, the 5′-tethering with these monosaccharides, as well as the conjugation, either at the 5′ or 3′end, with sucrose, were in all cases detrimental to quadruplex stability and did not improve the biological activity. On the basis of the assumption that the kinetically and thermodynamically favored formation of the quadruplex complex is a prerequisite for efficient antiviral activity, a novel bis-conjugated oligonucleotide was designed. This combined a mannose residue at the 3′-phosphate end with bulky aromatic tert-butyldiphenylsilyl (TBDPS) group at the 5′-end, previously shown to markedly favor the formation of quadruplex complexes. The 5′,3′-bisconjugated 6-mer, for which a detailed biophysical characterization has been carried out, resulted in 3-fold greater antiviral activity against HIV-1 than the sole 3′-glyco-conjugated oligonucleotide.

INTRODUCTION Glyco-conjugates participate in a variety of biological processes of paramount importance, including molecular recognition, signal trafficking, and recognition of cell surface through carbohydrate-binding proteins, as well as intracellular lectins in a sugar-dependent manner (1–3). Synthetic glyco-conjugates are therefore useful diagnostic tools to identify and characterize endogenous sugar-binding proteins, but they can also show high potential in several therapeutic applications for specific drug delivery (4, 5). Methods to obtain glyco-conjugatesssuch as neoglycoproteins, glycosylated polymers, or glycopeptidesshave been largely developed; on the contrary, synthetic strategies to prepare glyco-oligonucleotides are still far from being fully optimized (6). Indeed, for several in ViVo applications of oligonucleotides (ODNs), end derivatization with carbohydrates can result in a very convenient strategy to improve their otherwise poor internalization, eliciting specific sugar-binding processes to cell membranes and thus finally enhancing their cellular uptake by endocytosis. Through glyco-conjugation, further desirable properties can be conveyed to oligonucleotide-based drugs, as increased bioavailability, due to protection toward nucleases, and reduction of unwanted aggregation. DNA-carbohydrate conjugates have been prepared by direct, online solid-phase * Corresponding author. Prof. Daniela Montesarchio, PhD, Dipartimento di Chimica Organica e Biochimica, Complesso Universitario di Monte S.Angelo, Via Cynthia, 4, I-80126 Napoli, Italy, FAX number: +39-081-674393, e-mail address: [email protected]. † Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli “Federico II”. ‡ Dipartimento di Chimica “Paolo Corradini”, Università degli Studi di Napoli “Federico II”. § Katholieke Universiteit Leuven.

glycosidation of the 5′-end of oligonucleotides with suitable trichloroacetimidate sugar scaffolds (7, 8). A convenient procedure involved the use of unprotected carbohydrates, conjugated to 5′-amino-modified oligonucleotides by exploiting a reductive amination procedure (9). Recently, site-specifically galactosylated oligonucleotides, obtained from a 2′-deoxyuridine phosphoramidite carrying one galactose unit on the base, were synthesized, and periodic glycoclusters using a half-sliding oligonucleotide strategy could be obtained (10, 11). An alternative strategy to efficiently conjugate carbohydrates to ODNs was first explored by Akhtar and co-workers, who synthesized a suitable phosphoramidite derivative of mannose, incorporated onto the 5′-OH terminus of the solid-supported ODN by standard phosphoramidite chemistry (12). A similar approach has been recently proposed also by Joyce et al., who designed a new class of DNA-carbohydrate conjugates, called nucleo-glycoconjugates, based on the synthesis of three new phosphoramidite carbohydrate derivatives, inserted at the extremities or at defined, internal positions of the growing oligonucleotide chain (13, 14). In this frame, we recently synthesized suitably protected phosphoramidite derivatives of model mono- or disaccharides. When used in conjunction with DMT1-protected sugars immobilized on solid supports, they allowed us to prepare, through an online automated oligonucleotide synthesis protocol, a variety of 5′- and 3′-glycoconjugates of ODNs with stable phosphodiester linkages connecting the saccharide residues to one or both OH termini of the oligonucleotide chain (15, 16). In all the studied cases, the presence of the saccharide dangling ends was 1 Abbreviations: Ac ) acetyl; CE ) 2-cyanoethyl; CPG ) controlled pore glass; DBB ) 3,4-dibenzyloxybenzyl; DCC ) N,N-dicyclohexylcarbodiimide; DIPEA ) N,N-diisopropylethylamine; DMAP ) 4-dimethylaminopyridine; DMT ) 4,4′-dimethoxytriphenylmethyl; LCAA ) long chain alkylamine; TBDPS ) tert-butyldiphenylsilyl; TEA ) triethylamine; TEAB ) triethylammonium bicarbonate.

10.1021/bc7003395 CCC: $40.75  2008 American Chemical Society Published on Web 02/07/2008

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Scheme 1. Synthetic Scheme for the Preparation of Phosphoramidite Building Blocks 16, 17, and 18, and of Functionalized CPG Solid Supports 19, 20, and 21

shown to protect the ODN chains from nuclease digestion, while not hampering their specific recognition properties. In the search for ODNs endowed with significant antiviral properties, Hotoda and his group recently investigated a series of G-quadruplex-forming ODNs, finally focusing their research interest on modified oligonucleotides of sequence d(5′TGGGAG3′). These were found to exhibit potent anti-HIV activity associated with low cytotoxicity when carrying at the 5′-end bulky aromatic residues, as the 3,4-dibenzyloxybenzyl (DBB) or tert-butyldiphenylsilyl (TBDPS) groups (17, 18). Structure–activity relationship analysis indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5′-end, were both essential for their antiviral activity. In this frame, we studied a selected set of Hotoda’s 5′-modified ODNs: our data, combining CD, DSC, and molecular modeling studies on the modified G-quadruplexes in comparison with the unmodified one, showed that the overall stability of the investigated complexes correlated well with the

reported IC50values, thus furnishing quantitative evidence, even if still indirect, to the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the desired antiviral effects (19). Aiming at the preparation of novel oligonucleotide-based antivirals, we adopted the d(5′TGGGAG3′) sequence as a suitable platform to investigate the effect of glyco-conjugation on both quadruplex formation and biological properties of G-rich ODNs. Here, we here describe the synthesis, biophysical characterization, and biological properties of d(5′TGGGAG3′) oligomers, carrying a monosaccharide or disaccharide tail (with glucose, mannose, and sucrose chosen here as model carbohydrates) at either the 5′ or 3′-end. The best modification, in terms of both quadruplex formation and antiviral activity enhancement, i.e., the 3′-conjugation with a mannose residue, was then combined with the insertion of TBDPS at the 5′-end. The resulting 5′,3′-bis-conjugated TBDPS-5′TGGGAG3′-p-mannose was found to be 3-fold more active than the sole 3′-conjugated

Glyco-Conjugated G-Quadruplex-Forming Oligonucleotides.

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Scheme 2. Synthetic Scheme for the Preparation of Glyco-Conjugated Oligonucleotides II-VII

′TGGGAG3′-p-mannose oligonucleotide. To gain a deeper insight into the thermodynamic and kinetic parameters governing the quadruplex association/dissociation processes in solution, detailed CD analysis and CD-monitored thermal denaturation studies for the 5′,3′-bis-conjugated d(5′TGGGAG3′) have been carried out and discussed. 5

EXPERIMENTAL PROCEDURES Synthesis and Characterization of Oligonucleotides I-IX. DMT-protected succinylated sugars 13, 14, and 15; phosphoramidite building blocks 16, 17, and 18; and functionalized solid supports 19, 20, and 21 were synthesized following the reactions described in Scheme 1, according to previously reported protocols (15, 16). Oligonucleotides I-IX were synthesized using standard solid-phase β-cyanoethyl phosphoramidite chemistry on a 15 µmol scale, in all cases adopting DMT-off protocols. In the case of oligomer I, starting from 100 mg of commercially available 2′-deoxyguanosine-functionalized controlled pore glass (CPG-dG) support with 0.10 mequiv/g initial loading, the sequence 5′d(TGGGAG)3′was assembled by exploiting standard commercially available 3′-phosphoramidite 2′deoxynucleoside monomers. As depicted in Scheme 2, the synthesis of oligomers III, V, and VII was realized starting from 100 mg of functionalized CPG supports 19, 20, and 21, respectively, with 0.06–0.08 mequiv/g initial loading, on which the sequence 5′d(TGGGAG)3′ was assembled in a standard manner. In the case of oligomers II, IV, and VI, starting from 100 mg of commercially available CPG-dG support with 0.10 mequiv/g initial loading, after the assembly of the sequence 5 ′d(TGGGAG)3′, an additional coupling with phosphoramidite building blocks 16, 17, or 18, respectively, was then realized. The synthesis of oligomer IX was achieved starting from functionalized support 21, as described in Scheme 3, which was first used in the solid-phase assembly of the sequence 5′d(GGGAG)3′, then coupled with 5′-TBDPS-thymidine-3′-phosphoramidite building block 29, synthesized as previously described (19). Once the chain elongation was complete, target oligomers I-IX were detached from the solid support and deprotected by standard treatment with conc aq ammonia at 55 °C for 12 h. The combined filtrates and washings were concentrated under reduced pressure, redissolved in H2O, and analyzed and purified by HPLC. Purification of the crude natural oligomer I and of conjugated oligonucleotides II-VII was carried out on an

Scheme 3. Synthetic Scheme for the Preparation of 5′,3′-Bis-Conjugated Oligonucleotide IX


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Table 1. Oligonucleotide Characterization sequence (5′-3′)

HPLCa retention time, min

MS data calcd

ESI-MS data found

′TGGGAG3′ (I) Glucose-p-5′TGGGAG3′ (II) 5 ′TGGGAG3′-p-glucose (III) sucrose-p-5′TGGGAG3′(IV) 5 ′TGGGAG3′-p-sucrose (V) mannose-p-5′TGGGAG3′(VI) 5 ′TGGGAG3′-p-mannose (VII) TBDPS-5′TGGGAG3′(VIII) TBDPS-5′TGGGAG3′p-mannose (IX)

15.5; 26.7 16.9; 24.8 17.5; 22.3 16.6; 27.2 16.0; 21.8 15.9; 26.4 16.2; 23.8 14.0 9.2; 11.5

C60H74N27O34P5: 1872.26 C67H86N27O42P6: 2127.40 C67H86N27O42P6: 2127.40 C72H95N27O47P6: 2276.52 C72H95N27O47P6: 2276.52 C67H86N27O42P6:2127.40 C67H86N27O42P6:2127.40 C76H92SiN27O34P5: 2109.48 C83H105SiN27O42P6:2366.81

1872.01 2128.08 2128.27 2277.10 2276.88 2127.88 2127.79 2110.96 2366.28

5

a

For HPLC conditions, see Experimental Procedures.

Figure 1. CD spectra of the natural ODN 5′TGGGAG3′ (I) and of glyco-conjugated oligonucleotides II-VII. The spectra were recorded at 20 °C in a 10 mM potassium phosphate buffer (pH 7.0), supplemented with 200 mM KCl, at 5 × 10-5 M quadruplex concentration.

analytical anion exchange Nucleogel SAX column (MachereyNagel, 1000–8/46, 4.6 × 50 mm, 5 µm), adopting the following eluents: buffer A, 20 mM KH2PO4/K2HPO4 aq solution (pH 7.0), containing 20% (v/v) CH3CN; buffer B, 1 M KCl, 20 mM KH2PO4/K2HPO4 aq solution (pH 7.0), containing 20% (v/v) CH3CN. In all cases, using a linear gradient from 0% to 100% B in 40 min, flow rate 0.8 mL/min, and detection at λ ) 260 nm, two main peaks were observed. As previously found (18, 19), in all the purification 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 higher retention time, due to G-quadruplex aggregates generated under HPLC elution conditions. Oligonucleotide VIII, synthesized as previously described (19), was purified by HPLC on a NUCLEOSIL RP18 column (Supelco, 100–5, C18, 4.6 × 250 mm, 7 µm), using a linear gradient of CH3CN in 0.1 M TEAB in H2O, pH 7.0, from 20% to 100% in 20 min, flow rate 0.8 mL/min, with detection at λ ) 260 nm. Oligonucleotide IX was purified by HPLC on a RP18 Eclipse column (Agilent, XD8-C18); buffer A, 0.1 M TEAB in H2O, pH 7.0; buffer B, CH3CN; a linear gradient from 20%

to 100% B in 20 min, flow rate 0.8 mL/min, with detection at λ ) 260 nm, was used. The isolated oligomers had the retention times reported in Table 1. After HPLC purification, the oligonucleotide samples were desalted on a Sephadex G25 column eluted with H2O/EtOH (4:1, v/v). Successively, HPLC analysis of the desalted oligomers I-IX on a Partisphere Whatman RP18 analytical column (125 × 4.0 mm, 5 µm), eluted with a linear gradient from 0% to 100% in 60 min of CH3CN in TEAB buffer (0.1 M, pH 7.0), flow ) 0.8 mL/min, confirmed the expected purity for all the samples (higher than 98%). The modified oligonucleotides were characterized by ESI-MS mass spectrometry in the negative mode, in all cases giving masses in accordance with the expected values (Table 1). In a typical experiment, starting from 100 mg of commercially available CPG-dG supports, or of sugar-functionalized supports 19, 20, or 21, 80–120 OD units of purified I-IX could be obtained. Quadruplex Preparation. The quadruplex complexes were obtained by dissolving the lyophilized oligonucleotide in the appropriate buffer, annealed by heating at 95 °C for 5 min and slow cooling to room temperature. The buffer used was 10 mM



Table 2. Melting Temperatures of the Quadruplexes Formed by ODNs I-IXa oligonucleotide sequence (5′-3′)

Tm (°C)

′TGGGAG ′ (I) Glucose-p-5′TGGGAG3′ (II) ′TGGGAG3′-p-glucose (III) sucrose-p-5′TGGGAG3′(IV) 5 ′TGGGAG3′-p-sucrose (V) mannose-p-5′TGGGAG3′(VI) 5 ′TGGGAG3′-p-mannose (VII) TBDPS-5′TGGGAG3′ (VIII) TBDPS-5′TGGGAG3′-p-mannose (IX)

41 ( 1 26 ( 1 44 ( 1 35 ( 1 36 ( 1 31 ( 1 45 ( 1 75 ( 1b 81 ( 1

5

3

5

a As determined by CD-monitored thermal denaturation studies (see also Figure 1 in Supporting Information). b Data from ref (19).

Table 3. Anti-HIV-1 and Anti-HIV-2 Activity and Cytostatic Properties of Oligonucleotides II-VII in Human T-Lymphocyte (CEM) Cells oligonucleotide glucose-p-5′TGGGAG3′ (II) 5 ′TGGGAG3′-p-glucose (III) sucrose-p-5′TGGGAG3′ (IV) 5 ′TGGGAG3′-p-sucrose (V) mannose-p-5′TGGGAG3′ (VI) 5 ′TGGGAG3′-p-mannose (VII)

EC50a (µM) EC50a (µM) HIV-1(IIIB) HIV-2(ROD)

CC50b (µM)

S.I.c

87 ( 4.9

99 ( 70

110

21 ( 9.8

140

>140

>6.7

>30

>30

57 ( 13

30

74 ( 34

e2.5

>30

>30

41 ( 34

140 >140 g140 64.9 ( 9.5 >140 >140 g140

5

3

a IC50 ) 50% inhibitory concentration or compound concentration required to inhibit the HIV-1 RT-catalyzed reaction by 50%.

potassium phosphate, 200 mM KCl, 0.1 mM EDTA at pH ) 7.0. The concentration of the dissolved oligonucleotides I-VII was determined by UV measurements at 260 nm and at 95 °C, using, as the molar extinction coefficients, the values calculated by the nearest neighbor model (20) for the sequence 5′d(TGGGAG)3′, assuming the contribution of the terminal sugar residues to be negligible at this wavelength. For oligonucleotide IX, the contribution of the TBDPS group at the 5′-end was taken into account, as previously described in the case of VIII (19). CD Experiments. All circular dichroism (CD) experiments were registered on a JASCO J-715 spectrophotometer equipped with a thermostatically controlled cuvette holder (JASCO PTC348), in a 0.2 cm or 0.5 path length cuvette. CD spectra were collected from 220 to 340 nm, at 20 nm/min, with a response time of 4 s and at 1 nm bandwidth. The molar ellipticity was calculated from the equation [θ] ) 100 · θ /cl where θ is the ellipticity value, c is the quadruplex concentration (5.0 × 10-5 M), and l is the path length of the cell in cm. Thermal

dissociation of the studied quadruplexes was monitored by following the CD signal at 263 nm upon increasing the temperature. The quadruplex dissociation kinetics was measured by rapidly increasing the temperature and following the change of the CD signal at 263 nm with time. All the experiments were performed in a temperature and concentration range where the reassociation reaction can be neglected. The kinetic constants were obtained by fitting the recorded time-dependent decrease in CD signal with a single exponential function. For each sequence, the kinetic constant was determined at five different temperatures. The structural transition from the single strands to the tetramolecular quadruplex was monitored in the temperature range 10–30 °C by recording the CD spectra as a function of time. The samples were incubated at 90 °C for 5 min and then rapidly cooled. The kinetic constant and reaction order were obtained by analyzing the CD vs time profiles as previously described (21). Biological Assays. For the anti-HIV assays, CEM cells (4.5 × 105 cells/mL) were suspended in fresh culture medium and infected with HIV-1 at 100 CCID50 per mL of cell suspension in the presence of appropriate dilutions of the test compounds. After 4 to 5 days incubation at 37 °C, giant cell formation was recorded microscopically in the CEM cell cultures. The 50% effective concentration (EC50) corresponds to the compound concentrations required to prevent syncytium formation by 50% in the virus-infected CEM cell cultures. To determine the cytostatic activity, all assays were performed in 96-well microtiter plates. To each well were added (5–7.5) × 104 CEM cells and a given amount of the test compound. The cells were allowed to proliferate for 72 h at 37 °C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The CC50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited CEM cell proliferation by 50%. In the cocultivation assays, 5 × 104 persistently HIV-1infected HUT-78 cells (designated HUT-78/HIV-1) were mixed with 5 × 104 Sup-T1 cells, along with appropriate concentrations of the test compounds. After 16 to 20 h, marked syncytium formation was noted in the control cell cultures, and the number of syncytia was determined under a microscope. For determination of the 50% inhibitory concentration (IC50) of the test compounds against HIV-1 RT, the RNA-dependent DNA polymerase assay was performed as follows: the reaction mixture (50 µL) contained 50 mM Tris · HCl (pH 7.8), 5 mM DTT, 300 µM glutathione, 500 µM EDTA, 150 mM KCl, 5 mM MgCl2, 1.25 µg of bovine serum albumin, a fixed concentration of the labeled substrate [3H]dGTP (1.6 µM, 1 µCi; specific activity, 12.6 Ci/mmol; Amersham Pharmacia Biotech), a fixed concentration of the template/primer poly(rC) · oligo(dG) 12–18 (0.1 mM; Amersham Pharmacia Biotech), 0.06% Triton X-100, 5 µL of the inhibitor solution [containing various concentrations (5-fold dilutions) of the compounds], and 5 µL of the RT preparations. The reaction mixtures were incubated at 37 °C for 30 min, at which time 200 µL of yeast RNA (2 mg/mL) and 1 mL of trichloroacetic acid (5%, v/v) in water were added. The solutions were kept on ice for at least 30 min, after which the acid-insoluble material was filtered over Whatman GF/C glass-fiber filters and washed 10 times with 2 mL of 5% trichloroacetic acid in water and once with 70% ethanol. The filters were then analyzed for radioactivity in a liquid scintillation counter (Canberra Packard, Zellik, Belgium). The


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Table 5. Anti-HIV-1 and Anti-HIV-2 Activity and Cytostatic Properties of Oligonucleotide IX in Human T-Lymphocyte (CEM) and MT-4 Cellsa EC50b (µM)

CC50c (µM)

MT-4 oligonucleotide TBDPS-5′TGGGAG3′-p-mannose (IX) TBDPS-5′TGGGAG3′ (VIII)

CEM

HIV-1

HIV-2

HIV-1 (IIIB)

HIV-2 (ROD)

6.7 ( 0.1 0.88 ( 0.070e

>70

4.2 ( 2.0 1.8 ( 0.5

>70 >14

MT-4

CEM

S.I.d

>70 >40e

>70 >14

>17 on CEM; >10 on MT-4 >8 on CEM; >45 on MT-4

a For comparison, data from ref (18) for oligonucleotide VIII, determined using MT-4 cells, are also reported. b EC50 ) effective concentration or concentration required to protect CEM cells against the cytopathicity of HIV by 50%. c CC50 ) cytotoxic concentration or concentration required to reduce CEM or MT4 cell viability by 50%. d S.I. ) selectivity index for HIV-1, calculated as the ratio of CC50/EC50. e Data from ref (18).

Table 6. Inhibitory Activity of the Glyco-Conjugates against Syncytia Formation between Persistently HIV-1-Infected HUT-78/ HIV-1 Cells and Uninfected SupT1 Cells oligonucleotide

EC50a (µM)

II III IV V VI VII VIII IX

>140 >140 >30 >140 >140 >140 >14 1.3 ( 0.49

a EC50 ) 50% effective concentration or compound concentration required to prevent giant (syncytium) cell formation between HUT-78/ HIV-1 and SupT1 cells by 50%.

IC50 for each test compound was determined as the compound concentration that inhibited HIV-1 RT activity by 50%.

RESULTS Several modified oligonucleotides have been shown to inhibit HIV-1 in cell culture. If, in most cases, a non-sequence-specific inhibition is hypothesized, sequence-specific effects are also invoked for aptamers (22). Particularly, for G-rich ODNs the putative mechanism of action is based on the interference/ inhibition of both cell-to-cell and virus-to-cell transmission of HIV-1 by interaction with the V3 loop and the CD4 binding site on viral gp120 (23–25). As it is accepted that G-rich ODNs exert their anti-HIV activity through formation of G-quadruplex complexes, the correlation between thermodynamic stability and rate of formation of the quadruplexes, on one end, and the antiviral properties, on the other, was assumed here as the basis for the rational design of novel oligonucleotide-based antivirals. Starting from the model oligonucleotide d(5′TGGGAG3′), indicated here as I, a small library of 5′- and 3′-glyco-conjugates carrying the same sequence was synthesized and their ability to generate four-stranded quadruplexes analyzed by CDmonitored thermal denaturation experiments. 5′- and 3′conjugated 6-mers II-VII, as well as unmodified 6-mer I (Scheme 2 and Table 1), were synthesized using standard solidphase β-cyanoethyl phosphoramidite chemistry protocols on a 15 µmol scale (26). Glucose and sucrose-containing-phosphoramidites 16 and 17, as well as sugar-functionalized supports 19 and 20, were prepared essentially as previously reported (15, 16). Novel mannose building block 18 and mannose-functionalized support 21 were synthesized in analogy with the corresponding glucose and sucrose derivatives and exploited for the synthesis of mannose-containing oligomers VI and VII, respectively, here chosen as examples of glyco-conjugates which can find a direct application for lectin recognition. In all cases, starting from 100 mg of commercially available DMT-protected guanosinefunctionalized CPG support (0.10 mequiv/g, used in connection with phosphoramidites 16–18 for the synthesis of 5′-conjugated oligomers) or from LCAA-CPG functionalized with the appropriate DMT-protected sugar residue (19–21, 0.06–0.08 mequiv/g, used for the 3′-conjugation), on average, 80–120 OD

units of pure I-VII could be isolated, after HPLC purification, and characterized by ESI-MS data (Table 1). CD spectra at 20 °C for all the studied sequences were diagnostic of parallel-stranded quadruplex structures showing a positive band at 263 nm and a negative band at 243 nm (Figure 1) (18, 19, 27). The melting temperatures and melting profiles for each system were largely influenced by the scan rate, revealing kinetic control of the quadruplex dissociation process. From the CD-monitored melting profiles for all the samples, registered at 263 nm (see Supporting Information) at the slowest heating rate investigated (0.05 °C/min), the corresponding melting temperature values were determined (see Table 2). Inspection of Table 2 reveals that 6-mers III and VII, carrying a monosaccharide residue at their 3′-end, formed quadruplex complexes with moderately increased stability with respect to unmodified sequence I (∆Tm ) +3/+4 °C). Remarkably, the same modifications at the 5′-end of the ODN chain caused a dramatic destabilization, with ∆Tm of -15 and -10 °C for II and VI, respectively. When inserted at either the 5′ or 3′-end of the sequence, the disaccharide tail produced quadruplex complexes with comparable thermal stability; however, both IV and V showed lower melting temperature values than unmodified oligomer I (∆Tm ) -6/-5 °C). The activity of the synthesized glyco-conjugated oligonucleotides against-HIV-1 and HIV-2 was evaluated in human T-lymphocyte (CEM) cell cultures, as well as their cytotoxic properties. Interestingly, the EC50 values of the glyco-conjugated oligonucleotides were found to correlate well with the thermal stabilities of the corresponding quadruplex complexes, and activity against HIV-1 in the 10–20 µM range for 3′-glycoconjugated ODNs III and VII was observed (Table 3). No obvious correlation was found between the lack of anti HIV RT activity, reported in Table 4, and the anti-HIV activity. These data further confirmed that specific antiviral activity of Gquadruplex-forming ODNs, with well-defined three-dimensional structures, is not due to HIV-1 reverse transcriptase inhibition, but can be attributed to efficient interaction with the viral entry process. From the data in our hands, we concluded that, among the investigated conjugations, the insertion of a monosaccharide, and more specifically, of a mannose residue at the 3′-phosphate end of the studied ODN sequence, was the most promising modification in terms of both quadruplex formation and biological activity. Assuming VII as the best candidate for further drug optimization, we thought of combining the 3′-glyco-conjugation present in this oligomer with an additional 5′-tethering, chosen among the ones previously demonstrated by Hotoda et al. to convert the anti-HIV inactive 5′TGGGAG3′ into an active drug, effective at micromolar concentration (18). In our previous work, the tert-butyldiphenylsilyl (TBDPS) group at the 5′-end of the 5 ′TGGGAG3′ sequence (VIII) was shown to markedly enhance both the equilibrium and the rate of formation of the quadruplex complexes, in comparison with the unmodified sequence (19). In an effort to improve the biophysical and pharmacological profile of this G-rich oligonucleotide, we synthesized the oligonucleotide containing both the TBDPS residue at the 5′-



Figure 2. CD melting profile (on the left) and CD spectrum at 20 °C (on the right) of the 5′,3′-bis-conjugated oligonucleotide IX, at 5 × 10-5 M quadruplex concentration.

end and the mannose moiety at the 3′-phosphate end, thus obtaining the oligomer TBDPS-5′TGGGAG3′-p-mannose IX. The synthetic procedure to obtain IX, described in Scheme 3, is based on the usage of functionalized solid support 21 in conjunction with 5′-TBDPS-thymidine-3′-phosphoramidite building block 29 (18, 19), exploited in the last coupling of the solidphase ODN assembly. Antiviral evaluation on IX showed that the insertion of the TBDPS group at the 5′-end of 5′TGGGAG3′p-mannose notably affected its biological activity, with a 3-fold increase in the anti-HIV potency, with respect to ODN VII, carrying the sole mannose as the tethering group (Table 5). However, bis-conjugate IX was found to be 2.3-fold less active than TBDPS-5′TGGGAG3′ (VIII) on HIV-infected CEM cells. When the series of compounds were examined for their inhibitory activity against syncytium formation between persistently HIV-1-infected HUT-78/HIV-1 cells and uninfected SupT1 cells, only IX was measurably inhibitory (Table 6). In order to gain a critical insight into the peculiar structural features of bis-conjugated ODN IX, a detailed physicochemical characterization of the corresponding quadruplex complex was therefore carried out and comparison with previously studied TBDPS-5′TGGGAG3′ (VIII), to better investigate the role of the mannose-phosphate appendage at the 3′-end on the quadruplex structure, is reported here. The CD spectrum of TBDPS-5′-TGGGAG-3′-p-mannose IX is consistent with the formation of a parallel quadruplex structure (Figure 2). From the CD melting profile obtained at 0.05 °C/ min, shown in Figure 2, the melting temperature of the quadruplex complex was found to be 81 °C, with a ∆Tm of +40 °C with respect to unmodified I, and of +36 °C with respect to the quadruplex formed by VII, bearing mannosephosphate tails at the 3′-ends (Table 2). In order to especially focus on the kinetic aspects, recognized to primarily affect the in ViVo existence of four-stranded quadruplex complexes, a complete biophysical characterization of the quadruplex formed by ODN IX was carried out. The obtained data were compared with those previously determined for TBDPS-5′TGGGAG3′ VIII (19). The kinetics of TBDPS-5′TGGGAG3′-p-mannose quadruplex association was investigated in detail. The rate of the association reaction was studied by monitoring the ellipticity at 263 nm as a function of time after heating the sample at 95 °C for 5 min followed by fast cooling. The molar ellipticity vs time profiles are shown in Figure 3. The experimental curves were fitted following a previously described procedure (21). All the data were consistent with a reaction order of 4.0. The kinetic experiments were performed over the temperature range 15–30 °C. The activation energy (Eon) was derived from the slope of

the Arrhenius plot (see Supporting Information). The kinetic parameters are summarized in Table 7. We found that the association rate decreased upon increasing the temperature, due to a negative value for the activation energy, in accordance with previously obtained data for the TBDPS-5′TGGGAG3′ quadruplex. The activation energy for the formation of the quadruplex complex of IX is about half the activation energy determined in the case of VIII. In the 15–30 °C temperature range, the kinetic constants for the TBDPS-5′TGGGAG3′ and TBDPS5 ′TGGGAG3′-p-mannose quadruplexes are comparable. However, when the values are extrapolated at higher temperatures, the association reaction for quadruplex formation in IX is much faster in comparison to VIII, due to the difference in the absolute value of their activation energies. The kinetics of quadruplex dissociation for TBDPS-5′TGGGAG3′-p-mannose was also investigated by performing temperature-jump experiments. In each experiment, the temperature of the sample was rapidly increased and the rate of quadruplex dissociation was monitored by following the change of CD signal with time. The kinetic profiles at different temperatures are shown in Figure 4. The dissociation rate constants were then obtained by fitting these kinetic profiles with a single exponential curve. The activation energy (Eoff) was derived from the corresponding Arrhenius plot (see Supporting Information). The kinetic parameters for quadruplex dissociation are summarized in Table 8. The quadruplex formed by IX has koff values comparable with those previously determined for VIII. The Eoff value for the quadruplex formed by IX is about 130 kJ mol-1 higher than the value found for VIII. Taken together, these data showed that bis-conjugated TBDPS-5′TGGGAG3′-p-mannose IX formed more kinetically and thermodynamically stable four-stranded quadruplex structures than TBDPS-5′TGGGAG3′(VIII). Therefore, we can conclude that the presence of mannose residues at the 3′-phosphate end of the G-rich oligonucleotide chain, in addition to providing several desirable properties for the in ViVo activity, as increased resistance toward nucleases and improved cellular uptake, also significantly contributes to the overall stabilization of the quadruplex complex. However, contrarily to what we have found for monoconjugated 6-mers II-VII when referred to unmodified oligonucleotide I, in the comparison of IX vs VIII, the higher thermal stability for the quadruplex complex did not result in improved biological activity. Further experiments, including a detailed NMR-based conformational analysis study, are currently underway to further


D’Onofrio et al.

Figure 3. Kinetics of quadruplex formation for 5′,3′-bis-conjugated IX at different temperatures. Quadruplex formation leads to an increase of molar ellipticity at 263 nm. Mathematical fits are showed in dotted lines. All the experiments were performed in 10 mM potassium phosphate buffer (pH 7.0), supplemented with 200 mM KCl, at 5 × 10-6 M quadruplex concentration. Table 7. Kinetic Parameters for the Quadruplex Formation of IX with Respect to VIIIa quadruplex

temp (°C) ( 1 n

kon (M3 s-1)b

TBDPS-5′TGGGAG3′p-mannose

15

4 (9.8 ( 0.5) × 1010

TBDPS-5′TGGGAG3′c

20 25 30 15 20 25 30

4 4 4 4 4 4 4

(5.1 (3.6 (2.3 (2.9 (1.5 (8.6 (2.3

( ( ( ( ( ( (

0.6) 0.5) 0.8) 0.5) 0.6) 0.5) 0.8)

× × × × × × ×

Eon (kJ mol-1)

1010 1010 -70 ( 19 1010 1011 1011 -125 ( 19 1010 1010

a All the values have been determined in a 200 mM KCl, 10 mM potassium phosphate, pH 7.0 buffer. b kon is defined as d[S]/dt ) -4d[S4]/dt ) -kon[S]4. c Data from ref (19).

clarify the structure–activity relationships of these short, fourstranded G-quadruplex-forming oligonucleotides.

DISCUSSION After more than two decades, the search for new chemically modified ODNs is a very active field of research, and novel structures are continuously being proposed. Aiming at ODNs endowed with potential anti-HIV activity, end-conjugation with sugars was investigated here as a strategy to improve the pharmacological profile of the oligonucleotide 5′TGGGAG3′, chosen as a model compound. G-rich oligonucleotides are thought to be active against HIV-1 through interactions of the corresponding G-quadruplex complexes with viral gp120 protein. Our results, furnishing an in-depth analysis of the stability and biological activity of the quadruplex structures formed by modified 5′TGGGAG3′ oligonucleotides, further corroborated

this hypothesis, showing a good correlation between thermal stability of the corresponding quadruplex complexes and antiviral activity. In detail, the presence of a monosaccharide (glucose or mannose) at the 3′-phosphate end of the ODN chain was identified as the best modification, stabilizing the quadruplex structures and also converting the anti-HIV inactive natural oligomer into moderately active compounds. On the contrary, the same modification, when present at the 5′-end, led to a dramatic destabilization of the quadruplex structure. Still unclear is the reason behind this behavior, but a plausible explanation could be related to the fact that, in the case of 3′-conjugation, the sugar directly interacts with the G-quartet at the 3′-end of the sequence, which in the natural oligonucleotide would be exposed to the solvent. Therefore, in this case, stabilization of the quadruplex complex might arise from removal of the hydration spheres covering the hydrophobic surface of the G quartet due to the presence of the sugar. In the case of 5′-conjugation, this effect is not operative, since the sugar is attached to a dangling thymidine, not directly involved in the four-stranded quadruplex complex, and in turn other steric effects may be effective. Starting from the most promising modified oligonucleotide investigated here, i.e., oligomer VII, further optimization of the target was obtained by conjugating the bulky aromatic group tert-butyldiphenylsilyl (TBDPS) group at its 5′-end, previously shown to markedly enhance the formation of quadruplex complexes. The 5′,3′-bis-conjugated TBDPS-5′TGGGAG3′-pmannose IX, onto which a detailed biophysical characterization has been carried out, has been shown to form a four-stranded quadruplex complex with remarkably improved stability, and was 3-fold more active than the sole 3′-glyco-conjugated oligonucleotide VII. The mechanism of antiviral action of TBDPS-5′TGGGAG3′-p-mannose is most likely inhibition of



Figure 4. Kinetics of quadruplex dissociation for 5′,3′-bis-conjugated IX at different temperatures. Quadruplex dissociation leads to a decrease of molar ellipticity at 263 nm. Mathematical fits are showed in dotted lines. All the experiments were performed at 5 × 10-5 M quadruplex concentration. Table 8. Kinetic Parameters for the Quadruplex Dissociation of IX with Respect to VIIIa temp (°C) ( 1 koff × 104 (s-1)b Eoff (kJ mol-1)

quadruplex TBDPS- ′TGGGAG ′p-mannose

70

0.55 ( 0.03

TBDPS-5′TGGGAG3′c

73 75 77 80 70 73 75 77 80

1.5 ( 0.02 2.6 ( 0.4 4.0 ( 0.1 6.5 ( 0.3 0.50 ( 0.03 0.83 ( 0.02 1.4 ( 0.4 2.3 ( 0.1 3.5 ( 0.3

5

3

250 ( 20

220 ( 20

a

All the values have been determined in a 200 mM KCl, 10 mM potassium phosphate, pH 7.0 buffer. b koff is defined as d[S]/dt ) d[S4]/ dt + koff[S4]. c Data from ref (19).

viral entry (fusion), as is evident from coculture experiments. When compared to the sole 5′-conjugated TBDPS5 ′TGGGAG3′VIII, we determined that the 5′,3′-bis-conjugation sensibly favoredsboth kinetically and thermodynamicallysthe quadruplex formation, still IX exhibited lower anti-HIV activity. This last result indicates that the antiviral properties of short G-quadruplex forming oligonucleotides are indeed subordinate to the formation of tetrameric complexes, but the in ViVo picture is obviously much more complicated than simply expected on the basis of a single point of view. In all cases, the quadruplex formation can be considered as a fundamental prerequisite for the in ViVo biological activity of G-rich oligomers; however, their activity does not exclusively rely on a favored equilibrium between single strands and quadruplex structures, with other mechanisms also successively involved. Therefore, in the search for antiviral properties in the libraries of modified G-rich oligonucleotides, the correlation between thermal stability of the corresponding quadruplex complexes and their anti-HIV activity can be helpful, but this has to be strictly intended only for a first level of prediction, since a variety of different, complex processes may eventually affect their in ViVo behavior.

Taken together, these findings further expand the repertoire of known anti-HIV active oligonucleotides and can be useful for the future design of novel modified G-rich ODNs with improved antiviral activity.

ACKNOWLEDGMENT We acknowledge MUR (PRIN) for grants in support of this investigation. We also thank C.I.M.C.F., Università degli Studi di Napoli “Federico II”, for the NMR, MS, and CD facilities, and the Rega Centers of Excellence of the K.U.Leuven. This work is dedicated to Prof. Guido Barone and Matteo Adinolfi on the occasion of their 70th birthdays. Supporting Information Available: General experimental methods; CD melting profiles for ODNs II-VII; Arrhenius plots for the association and for the dissociation of the 5′,3′-bisconjugated quadruplex formed by ODN IX. This material is available free of charge via the Internet at http://pubs.acs.org.

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Synthesis, Biophysical Characterization, and Anti-HIV Activity of Glyco

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