Synthesis and Characterization of Phenothiazine Labeled

A facile procedure for the incorporation of phenothiazine at the terminus of oligodeoxynucleotides is reported. Phenothiazine is covalently linked to ...
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Bioconjugate Chem. 2002, 13, 83−89

83

Synthesis and Characterization of Phenothiazine Labeled Oligodeoxynucleotides: Novel 2′-Deoxyadenosine and Thymidine Probes for Labeling DNA Xi Hu, Mark T. Tierney, and Mark W. Grinstaff* Department of Chemistry, Paul M. Gross Chemical Laboratory, Duke University, Durham, North Carolina 27708. Received April 15, 2001; Revised Manuscript Received August 15, 2001

A facile procedure for the incorporation of phenothiazine at the terminus of oligodeoxynucleotides is reported. Phenothiazine is covalently linked to the 5′-position of 2′-deoxyadenosine and thymidine. Next, the corresponding phosphoramidites are prepared, and then the labeled nucleosides are incorporated in DNA using an automated DNA solid-phase synthesizer. Phenothiazine labeled oligodeoxynucleotides form stable B-form duplexes with similar melting temperatures, CD spectra, and DSC traces compared to unlabeled DNA duplexes. The favorable photophysical properties of phenothiazine are also retained after covalent attachment to the oligodeoxynucleotide.

Scheme 1. Synthesis of PTZ-Acida

INTRODUCTION

Organic and inorganic chromophores possessing welldefined spectroscopic and redox properties are of interest for mechanistic studies of DNA mediated charge transfer (1-15) and for optimizing DNA diagnostic devices (1621). Both of these interests require synthetic control over the site-specific location of the probe on the duplex. Current automated DNA synthesis technology offers a convenient and high-yielding procedure for synthesizing such duplexes. By selecting the appropriate derivatized phosphoramidite probe, this approach enables labeling at specific sites (phosphate, ribose, nucleobase) and sequence locations on the oligodeoxynucleotide. Intercalating probes such as anthraquinone and Ru(phen)2(dppz)2+ when covalently attached to DNA are typically tethered to the 5′- or 3′-terminal phosphate of the oligodeoxynucleotide (1, 2). Yet intercalating probes alter the DNA duplex structure as evident by an increase in melting temperature and by crystallographic examination of the DNA π-stack (22). Nonintercalating tethered probes are less explored and provide an opportunity to assess the role of location site and the mode of attachment on duplex hybridization and on charge transfer chemistry. To expand the current repertoire of probes available for study (23), we are synthesizing oligodeoxynucleotides labeled at the 5-position of uridine (24-27), C8 position of 2′-deoxyadenosine (28), 5′-terminal phosphate (29, 30), and the 5′-position of thymidine (31) with spectroscopic and/or redox-active chromophores. Herein, we describe the synthesis of two novel phenothiazine-2′deoxyadenosine and -thymidine nucleosides and the incorporation of these labeled pyrimidine and purine nucleosides in oligodeoxynucleotides. EXPERIMENTAL PROCEDURES

Reagents were purchased from Aldrich or Acros as highest purity grades and used without further purification. All solvents were freshly distilled under inert atmosphere prior to use unless otherwise noted. Dichlo* Corresponding author: [email protected]; http://www.chem.duke.edu/∼mwg/labgroup.

a Key: (a) acrylonitrile, NBu +OH-, 87% yield; (b) NaOH, 4 aqueous MeOH, 56% yield.

romethane (CH2Cl2) and acetonitrile (CH3CN) were distilled from CaH2. Ethyl ether and THF were distilled from sodium/benzophenone. Anhydrous DMF was obtained from Aldrich. All reactions were performed under inert atmosphere unless otherwise noted. Absorption spectra and melting curves were measured on a diode array spectrometer. CD spectra were recorded on a spectropolarimeter. Calorimetric data were obtained using a VP-DSC from MicroCal Inc. MALDI-TOF mass spectra of oligodeoxynucleotides were obtained using a PerSeptive Biosystems Voyager-DE Biospectrometry Workstation operating in the negative ion mode using a hydroxypicolinic acid matrix. NMR spectra were recorded on a spectrometer operating at 400 MHz. Fast atom bombardment mass spectra (FABMS) were obtained on a JEOL JMS-SX102A spectrometer using a 3-nitrobenzyl alcohol matrix. Syntheses. 3-Phenothiazin-10-ylpropionitrile 2. In a modification of a previous report by Godefroi and Wittle (32), phenothiazine (2.0 g; 1.0 mmol) was suspended in acrylonitrile (15 mL) and cooled to 0 °C. Tetrabutylammonium hydroxide (50% in water; 0.1 mL) was added and the mixture allowed to slowly warm to room temperature (Scheme 1). An exothermic reaction occurred immediately. Once the reaction subsided, dioxane (25 mL) was added and the mixture was heated to reflux for 1 h. The mixture was poured in water with vigorous stirring, and the resulting tan solid was filtered. Recrystallization from

10.1021/bc0100509 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/27/2001

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Scheme 2. Synthesis of PTZ-Phosphoramiditesa

a Key: (a) TMSCl, BzCl/pyridine, 25 °C, 2.5 h, H O, NH OH, 0 °C, 10 min; (b) MsCl/pyridine, 0 °C, 12 h; (c) LiN /DMF, 90 °C, 3 2 4 3 h; (d) H2, Pd/C, 50 psi, 25 °C 5 h; (e) 3, CDI/DMF, LH-20/THF, 25 °C, 18 h; (f) 2-cyanoethyl N,N′-diisopropylchlorophosphoramidite, DIPEA, CH3CN, 25 °C, 2 h.

acetone gave 2 as a white solid (2.2 g; 87% yield). This material was used directly in the next step. 3-Phenothiazin-10-ylpropionic Acid 3. This compound was prepared from 2 using the hydrolysis method described by Godefroi and Wittle (56% yield) (32). FABHRMS calcd (found) for C15H13NO2S (M+) 271.0667 (271.0666). 6-N-Benzoyl-2′-deoxyadenosine 4c. The reaction was performed following the literature procedure by Jones (33) to afford 4c in 93% yield. This procedure also required transient protection of the 5′- and 3′-hydroxyls by TMS-Cl in pyridine, followed by addition of benzoyl chloride to protect the free amine and subsequent deprotection of the hydroxyls by ammonium hydroxide (33). 1 H NMR (DMSO-d6): δ 2.32-2.40 (m, 1H, C2′), 2.772.83 (m, 1H, C2′), 3.51-3.67 (m, 2H, C5′), 3.90 (q, J ) 2.8 Hz, 1H, C4′), 4.45-4.46 (m, 1H, C3′), 5.05 (s, 1H, 5′OH), 5.39 (s, 1H, 3′-OH), 6.48 (t, J ) 6.8 Hz, 1H, C1′), 7.55-8.06 (m, 5H, benzoyl), 8.69 (s, 1H, C2), 8.75 (s, 1H, C8), 11.20 (s, 1H, amide). FAB-MS calcd (found) for M+ + H: 356.13 (356.09). 5′-O-Methanesulfonylthymidine 5a. The reaction was based on a literature procedure (34). Thymidine 4a (2.90 g, 12.0 mmol) was dissolved in 10 mL of dry pyridine and cooled to -10 °C. Next, mesyl chloride (14 mmol) was added dropwise over a period of 20 min (Scheme 2). The reaction mixture was then held at 0 °C for 12 h. The next day, 10 mL of methanol was added to quench the reaction, and the solvents were evaporated via high vacuum. The resulting crude product was checked by TLC and purified by column chromatography (silica gel, CH3OH:CHCl3 ) 1:9). A white solid 5a was obtained (2.95 g, 77% yield). 1H NMR (DMSO-d6): δ 1.78 (s, 3H, 5-methyl), 2.08-2.22 (m, 2H, C2′), 3.22 (s, 3H, mesyl), 3.98 (q, 1H, C4′), 4.23-4.28 (m, 1H, C3′), 4.36-4.39 (m, 2H, C5′), 5.49 (d, J ) 4.4 Hz, 1H, 3′-OH), 6.22 (t, J ) 7.2

Hz, 1H, C1′), 7.48 (s, 1H, C6), 11.25 (s, 1H, N3). FABMS calcd (found) for M+ + H: 321.07 (321.06). 5′-O-Methanesulfonyl-6-N-benzoyl-2′-deoxyadenosine 5b. Synthesized from 4c in a similar manner as 5a, 75% yield. 1H NMR (DMSO-d6): δ 2.40-2.47 (m, 1H, C2′), 2.88-2.96 (m, 1H, C2′), 3.16 (s, 1H, mesyl), 4.11-4.15 (m, 1H, C4′), 4.38-4.49 (m, 2H, C5′), 4.54-4.56 (m, 1H, C3′), 5.64 (d, J ) 4.0 Hz, 1H, 3′-OH), 6.54 (t, J ) 6.8 Hz, 1H, C1′), 7.55-8.06 (m, 5H, benzoyl), 8.65 (s, 1H, C2), 8.76 (s, 1H, C8), 11.20 (s, 1H, amide). FAB-MS calcd (found) for M+ + H: 434.11 (434.10). 5′-Azido-5′-deoxythymidine 6a. A solution of 5a (1.84 g, 5.75 mmol) in 15 mL of DMF containing lithium azide (1.60 g, 32.6 mmol) was refluxed at 90 °C under nitrogen. After 3 h, the reaction was stopped and the solvent was removed under vacuum. The resulting crude product was purified by column chromatography (silica gel, CH3OH: CHCl3 ) 1:9) to afford 6a (1.12 g, 73% yield). 1H NMR (DMSO-d6): δ 1.78 (s, 3H, 5-methyl), 2.16-2.34 (m, 2H, C2′), 3.38-3.56 (m, 2H, C5′), 3.92 (q, J ) 4.8 Hz, 1H, C4′), 4.31-4.33 (m, 1H, C3′), 5.50 (s, 1H, 3′-OH), 6.13 (t, J ) 7.2 Hz, 1H, C1′), 7.48 (s, 1H, C6), 11.25 (s, 1H, N3). FAB-MS calcd (found) for M+ + H: 268.10 (268.12). 5′-Azido-6-N-benzoyl-2′,5′-dideoxyadenosine 6b. Synthesized from 5b in a similar manner as 6a, 76% yield. 1H NMR (DMSO-d ): δ 2.45 (m, 1H, C2′), 2.97 (m, 1H, 6 C2′), 3.51-3.67 (m, 2H, C5′), 3.99-4.03 (m, 1H, C4′), 4.47-4.49 (m, 1H, C3′), 5.52 (d, J ) 4.0 Hz, 1H, 3′-OH), 6.52 (t, J ) 6.8 Hz, 1H, C1′), 7.52-8.04 (m, 5H, benzoyl), 8.68 (s, 1H, C2), 8.76 (s, 1H, C8), 11.20 (s, 1H, amide). FAB-MS calcd (found) for M+ + H: 381.13 (381.14). 5′-Amino-5′-deoxythymidine 7a. A solution of 5′-azido2′-deoxythymidine 6a (1.12 g, 4.2 mmol) in 30 mL of methanol containing 10% Pd/C was shaken under 50 psi of hydrogen for 5 h. The catalyst was removed by filtration, and the filtrate was then concentrated to

Phenothiazine Labeled Oligodeoxynucleotides

dryness via evaporation. The resulting crude product was purified by column chromatography (silica gel, CH3OH: CHCl3 ) 1:7) to yield 7a (0.91 g, 90% yield). 1H NMR DMSO, δ 1.78 (s, 3H, 5-methyl), 2.08-2.18 (m, 2H, C2′), 2.75 (s, 2H, amine), 3.50-3.60 (m, 2H, C5′), 3.64 (q, J ) 3.2 Hz, 1H, C4′), 4.18-4.20 (m, 1H, C3′), 5.20 (s, 1H, 3′OH), 6.14 (t, J ) 7.2 Hz, 1H, C1′), 7.68 (s, 1H, C6). FABHRMS calcd (found) for C10H16N3O4 (MH+): 242.1143 (242.1141). 5′-Amino-6-N-benzoyl-2′,5′-dideoxyadenosine 7b. Synthesized from 6b in a similar manner as 7a, 62% yield.1H NMR (DMSO-d6): δ 2.31-2.36 (m, 1H, C2′), 2.71-2.89 (m, 3H, C5′+C2′), 3.16 (s, 2H, amine), 3.81 (q, J ) 4.4 Hz, 1H, C4′), 4.43-4.45 (m, 1H, C3′), 5.31 (s, 1H, OH), 6.45 (t, J ) 6.8 Hz, 1H, C1′), 7.52-8.05 (m, 5H, benzoyl), 8.70 (s, 1H, C2), 8.74 (s, 1H, C8). FAB-HRMS calcd (found) for C17H19N6O3 (MH+): 355.1520 (355.1518). N-(5′-Amino-5′-deoxythymidine)-3-phenothiazin-10-ylpropionamide 8a. A solution of 3-phenothiazin-10-ylpropionic acid 3 (0.28 g, 1.0 mmol) and carbonyldiimidazole (CDI1) (0.24 g, 1.5 mmol) in 8 mL of dry DMF was stirred under nitrogen for 1 h at 25 oC. The mixture was then diluted with 17 mL of dry THF, followed by addition of the LH-20 resin (0.43 g, excess) to quench the excess CDI. An hour later, LH-20 was removed, and 7a (0.24 g, 1.0 mmol) was added to the reaction mixture. After stirring for 12 h, the reaction was stopped, and the solvents were removed. Column chromatagraphy (silica gel, CH3OH: CHCl3 ) 1:9) yielded a white solid (0.40 g, 81% yield). 1 H NMR (DMSO-d6): δ 1.72 (s, 3H, 5-methyl), 1.98-2.08 (m, 2H, C2′), 2.55 (t, J ) 7.2 Hz, 2H, PTZ N-CH2), 3.203.38 (m, 2H, C5′), 3.68-3.69 (m, 1H, C4′), 4.07-4.12 (m, 3H, carbonyl-CH2 and C3′), 5.24 (d, J ) 4.4 Hz, 1H, 3′OH), 6.08 (t, J ) 7.2 Hz, 2H, C1′), 6.87-7.16 (m, 8H, aromatic H of PTZ), 7.42 (s, 1H, C6), 8.08 (t, J ) 6.0 Hz, 1H, amide NH), 11.25 (s, 1H, C3). 13C NMR: δ 13.0, 34.1, 39.4, 41.9, 43.9, 72.1, 84.6, 85.8, 110.6, 116.3, 123.5, 124.1, 128.0, 128.6, 137.1, 145.3, 151.4, 164.6, 171.3. FAB-HRMS calcd (found) for C25H26N4O5S (M+): 494.1618 (494.1621). N-(5′-Amino-6-N-benzoyl-2′,5′-dideoxyadenosine)-3-phenothiazin-10-ylpropionamide 8b. Synthesized from 7b in a similar manner as 8a, 82% yield. 1H NMR (DMSO-d6): δ 2.25-2.31 (m, 1H, C2′), 2.55-2.60 (m, 2H, PTZ-N-CH2), 2.75-2.78 (m, 1H, C2′), 3.27-3.43 (m, 2H, C5′), 3.863.90 (m, 1H, C4′), 4.03-4.08 (m, 2H, carbonyl-CH2), 4.37 (m, 1H, C3′), 5.37 (d, J ) 4.0 Hz, 1H, 3′-OH), 6.41 (t, J ) 6.8 Hz, 1H, C1′), 6.84-7.14 (m, 8H, PTZ), 7.48-8.01 (m, 5H, benzoyl), 8.12 (t, J ) 6.0 Hz, 1H, PTZ amide), 8.62 (s, 1H, C2), 8.68 (s, 1H, C8), 11.14 (s, 1H, benzoyl amide). 13C NMR: δ 34.1, 39.5, 42.1, 43.9, 72.5, 84.8, 86.8, 116.4, 123.4, 124.1, 127.1, 128.0, 128.6, 129.4, 133.4, 134.3, 144.4, 145.3, 151.4, 152.4, 152.8, 166.6, 171.2. FABHRMS calcd (found) for C32H29N7O4S (M+): 607.2002 (607.1996). N-(5′Amino-3′-cyanoethoxydiisopropylaminophosphine5′-deoxythymidine)-3-phenothiazin-10-ylpropionamide 9a. 2-Cyanoethyl N,N′-diisopropylcholophosphoramidite (140 µL, 0.62 mmol) was added to a solution of 8a (0.191 g, 0.39 mmol) in 20 mL of dry CH3CN containing diisopropylethylamine (0.31 mL). The reaction mixture was stirred under nitrogen for 2 h. The solvent was then removed and the product checked by 31P NMR(CDCl3): δ 149.5 and 149.9 observed. TLC: >95% yield. 1 1Abbreviations: DIPEA ) N,N-diisopropylethylamine; CDI ) carbonyldiimidazole; DMT ) dimethoxytrityl; PTZ ) phenothiazine.

Bioconjugate Chem., Vol. 13, No. 1, 2002 85

N-(5′-Amino-3′-cyanoethoxydiisopropylaminophosphine6-N-benzoyl-2′,5′-dideoxyadenosine)-3-phenothiazin-10-ylpropionamine 9b. Synthesized in a similar manner as 9a. 31P NMR(CDCl3): δ 149.6 and 150.0 observed. TLC: >95% yield. Oligodeoxynucleotide Syntheses. Oligodeoxynucleotide syntheses were performed on an ABI 395 DNA synthesizer from the 3′ to 5′ end using standard automated DNA synthesis protocols as shown in Scheme 3 (1.0 µmol scale). A 0.1 M solution of 9a (or 9b) in dry acetonitrile was prepared and installed on the DNA synthesizer in a standard reagent bottle. Normal solidphase oligodeoxynucleotide synthesis was performed. In the last step, the PTZ-modified phosphoramidite was introduced and allowed to react with the oligodeoxynucleotide for 15 min. The PTZ-labeled oligodeoxynucleotides were cleaved from the column and deprotected in 30% NH4OH at 55 °C for 16 h. MALDI mass spectrometry of the PTZ-oligodeoxynucleotides confirmed their formation.

calcd found

10 5436.8 5437.8

11 5129.9 5128.5

12 4205.7 4207.1

13 5445.6 5448.4

14 5141.4 5144.7

HPLC Purification of the Oligodeoxynucleotides. HPLC purification of the PTZ-labeled oligodeoxynucleotides was accomplished on a Rainin HPLC instrument. Reverse phase chromatography was performed on a C18 column (25 cm × 4.6 mm) with acetonitrile (ACN) and 0.1 M triethylamine acetate (TEAA) as eluting solvents. A flow rate of 3 mL/min was used, and the concentration of ACN was increased from 5% to 50% over 40 min. The retention times of the labeled oligodeoxynucleotides were well separated from those of the unlabeled oligodeoxynucleotide products (12-13 min). Melting Curves. The stability of the duplex formed between two complementary oligodeoxynucleotides was determined from the melting curve profiles as a function of temperature. The Tm value was determined from the first derivative. The concentrations of stock solutions of oligodeoxynucleotide single strands were determined from the UV-vis absorbance spectra. Enzyme digestion of the oligdeoxynucleotides to yield the individual nucleotides with Nuclease P1 Penicillium citrinum was performed by the addition of 1 µL of a 1 mg/mL solution of enzyme to 5.0 µL of oligodeoxynucleotide single strand solution and 44.0 µL of 20 mM sodium acetate buffer at pH 5.5. The solutions were incubated at 55 °C for 30 min. A sample of 20.0 µL of digest solution was diluted to 2.00 mL with water, and the absorption spectrum was measured. The total molar absorptivity was determined from the sum of all the individual contributions of each nucleoside and PTZ. The concentrations of the stock solutions were then calculated. Equimolar amounts of each single strand were measured out of the stock solutions, combined, and diluted with phosphate buffer (5 mM NaH2PO4, 50 mM NaCl, pH ) 7) to the appropriate concentration for UV-vis measurements. The solution was heated to 90 °C for 3 min and allowed to slowly cool to room temperature. After cooling, the thermal denaturation experiment was performed using the following parameters on a HP UV-vis: (a) monitoring wavelength, 260 nm, (b) temperature range, 25-70 °C, (c) temperature step, 1.0 °C, (d) averaging time constant, 15 s, (e) 0.5 °C rate of change. Calorimetry. A VP-DSC from MicroCal Inc. with a cell volume of 0.5136 mL was used to determine the calorimetric profiles of labeled and unlabeled DNA

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Scheme 3. Synthesis of PTZ-Labeled Oligodeoxynucleotidesa

a

Key: (a) normal synthesis; (b) 30% NH3, 55 °C, 16 h. B ) A, C, G, or T.

duplexes. Samples were scanned from 10 °C to 80 °C using a constant rate of 30 °C/h. Prior to each experiment, the buffer was scanned to produce a stable baseline, and then the DNA sample (37.5 µM) was scanned repeatedly until a reproducible calorimetric transition curve was obtained. Subtraction of buffer baseline and normalization of sample concentration yielded the desired heat capacity versus temperature curve. The enthalpy of transition during the melting process was determined from the area under this heat capacity peak. RESULTS AND DISCUSSION

To capitalize on solid-phase DNA synthesis technology and the flexibility that this automated and modular approach brings to the synthesis of site-specifically labeled oligodeoxynucleotides (35), we require redox probes that are (1) sufficiently soluble as the labeled nucleoside phosphoramidite in acetonitrile for solid-phase reactions, (2) stable to the standard oligodeoxynucleotide syntheses and deprotection reactions so as to prevent undesired side reactions leading to loss of Watson-Crick base pairing selectivity (36) or decomposition, and (3) amenable to covalent attachment to both purine and pyrimidine nucleosides. The chromophore, phenothiazine (PTZ), satisfies these requirements and is known to be a low potential reductant (PTZ+•/PTZ; 0.59 V vs SCE) (37). The one-electron oxidized product, PTZ+•, is also spectroscopically characterized (λmax ) 510 nm) (38, 39). Moreover, previous reports have successfully described

the use of PTZ with photoexcited inorganic chromophores such as *Ru(bpy)32+ to study reductive electron-transfer reactions in peptide (37, 39) and DNA (40) assemblies. The PTZ-derivatized 5′-deoxythymidine and N-benzoyl2′,5′-dideoxyadenosine phosphoramidites, 9a and 9b, respectively, for automated DNA synthesis were synthesized as shown in Schemes 1-3. The 3-phenothiazin-10ylpropionic acid, 3, was prepared by first reacting PTZ with acrylonitrile in the presence of tetrabutylammonium hydroxide (87% yield), followed by hydrolysis with sodium hydroxide in aqueous methanol to afford the product in 56% yield (32). As shown in Scheme 2, the 5′-position of thymidine was converted from a hydroxyl to an amine in three consecutive steps. First, thymidine was treated with an equal amount of methanesulfonyl chloride in dry pyridine to afford 5a (34). Next, the 5′-mesyl group was substituted with lithium azide to yield 6a. Finally, 6a was reduced with H2 and Pd/C to give the 5′-amino-5′deoxythymidine, 7a in 90% yield. This reduction method was preferred to that of using triphenylphosphine and ammonium hydroxide (72% yield). The primary hydroxyl of 2′-deoxyadenosine was converted to an amine in a similar manner, except that the primary amine was first protected with a benzoyl group (Scheme 2). This step required transient protection of the 5′- and 3′-hydroxyls by TMS-Cl in pyridine, followed by addition of benzoyl chloride and subsequent deprotection of the hydroxyls by ammonium hydroxide (33). Unlike the thymidine synthesis, the reduction of 6b to 7b was best achieved using

Phenothiazine Labeled Oligodeoxynucleotides

Bioconjugate Chem., Vol. 13, No. 1, 2002 87

Figure 1. Melting curve for PTZ-dT labeled (11‚21; dashed line) and unlabeled (16‚21; solid line) oligodeoxynucleotide duplexes.

Figure 2. A heat capacity vs temperature transition curve for PTZ-dT labeled (11‚21; dashed line) and unlabeled (16‚21; solid line) oligodeoxynucleotide duplexes.

Table 1. PTZ-Labeled Oligodeoxynucleotides

sides (>98%) and 9a and 9b coupled in ≈80%. Once synthesized, the PTZ-labeled oligodeoxynucleotides were cleaved from the column, and the nitrogenous bases and phosphate groups were deprotected in 30% ammonium at 55 °C. The oligodeoxynucleotides (see Table 1) were subsequently purified by RP-HPLC and analyzed by MALDI mass spectrometry. A temperature change of approximately 1 °C is observed in the melting temperature between the unlabeled and PTZ-dA or PTZ-dT labeled oligodeoxynucleotides as determined by temperature-dependent optical measurements (see Figure 1 and Table 2). This difference in melting temperature is small indicating that labeling the 5′-terminal nucleotide of the oligodeoxynucleotide does not dramatically alter the DNA duplex structure. Differential scanning calorimetry (DSC) data provide additional insight into the thermodynamic consequences of PTZ labeling at the ribose (42, 43). As shown in Table 2, the transition enthalpies (∆H°) of the PTZ-T and PTZ-A labeled and unlabeled 16-mer duplexes are 71.0, 70.6, 66.8, and 68.0 kcal/mol, respectively. The differences in transition enthalpy change (∆∆H°) for the unlabeled and labeled dT and dA duplexes are 0.4 and 1.2 kcal/mol, respectively. These ∆Ho values are deduced from the heat capacity/temperature relationship, such as the one shown in Figure 2 for the PTZ-dT (11•21) labeled and unlabeled (16•21) duplex. The free energies (∆G°) at 25 °C for the PTZ-dT and -dA labeled and unlabeled 16-mer duplexes are 5.4, 5.6, 4.9, and 5.0 kcal/mol, respectively. There is only a modest decrease in free energy (∆∆G°) of 0.2 kcal/ mol or less for the labeled duplex relative to the unlabeled duplex. The heat capacity traces shown in Figure 2 for the PTZ-T-labeled duplex can also be fit to a two-state model, and the calculated van’t Hoff enthalpy (∆Hv) is 70.4 kcal/mol. This value is similar to the measured enthalpy. These data suggest that a two-state (all-ornone) helix to coil transition occurs in the labeled and

5′-PTZTTGCTACAAACTGTTG A-3′ 5′-PTZTGCTACAAACTGTTG A-3′ 5′-PTZTTCAACAGTTTGT-3′ 5′-PTZATGCTACAAACTGTTGA-3′ 5′-PTZAGCTACAAACTGTTGA-3′ 5′-TTGCTACAAACTGTTG A-3′ 5′-TGCTACAAACTGTTG A-3′ 5′-TTCAACAGTTTGT-3′ 5′-ATGCTACAAACTGTTG A-3′ 5′-AGCTACAAACTGTTG A-3′ 5′-TCAACAGTTTGTAGCAA-3′ 5′-TCAACAGTTTGTAGCA-3′ 5′-ACAAACTGTTGAA-3′ 5′-TCAACAGTTTGTAGCAT-3′ 5′-TCAACAGTTTGTAGCT-3′

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Pd/C hydrogenation. The carboxylic acid derivatized PTZ, 3, was then coupled to 5′-amino-5′-deoxythymidine (7a) or N-benzoyl 5′-amino 2′,5′-dideoxyadenosine (7b) in DMF using CDI (Scheme 2). Other amide forming strategies using DCC or the NHS activated ester were explored, but yielded less product. Finally, 8a and 8b were reacted with 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite in dry CH3CN to afford the PTZ-phosphoramidite, 9a and 9b. The PTZ-thymidine and PTZ-2′-deoxyadenosine phosphoramidites were now ready for use in an oligodeoxynucleotide synthesizer (ABI 392). Standard solid-phase oligodeoxynucleotide syntheses were performed (35, 41), except that in the last coupling step the PTZ-modified thymidine or 2′-deoxyadenosine phosphoramidite was introduced as shown in Scheme 3. All syntheses were performed at the 1.0 µmol scale using the standard coupling protocol except that the final step proceeded for 15 min to ensure sufficient reaction time for 9a and 9b. Collection and analysis of the DMTfractions during automated synthesis showed efficient phosphoramidite couplings throughout the procedure, with both the standard pyrimidine and purine nucleo-

Table 2. Optical and DSC Thermodynamic Data for the PTZ-dT Labeled and Unlabeled Duplexes optical data

DSC data

duplex

Tm °C ( 0.5

Tm °C ( 0.5

∆H (kcal/mol) ( 1.0

∆Hv (kcal/mol) ( 1.0

∆S (cal/mol) ( 3.0

∆G at 25 °C (kcal/mol) ( 0.1

11‚21 16‚21 14‚24 19‚24

49.7 49.6 51.0 49.7

49.6 50.7 48.4 48.6

71.0 70.6 66.8 68.0

70.4 74.6 69.5 70.9

220.1 218.1 207.8 211.4

5.4 5.6 4.9 5.0

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Hu and Grinstaff CONCLUSION

Figure 3. CD spectra of PTZ-dT labeled (11‚21; solid line) and unlabeled (16‚21; dashed line) oligodeoxynucleotide duplexes.

The site-specific labeling of oligodeoxynucleotides at a 5′-terminal thymidine or 2′-deoxyadenosine with phenothiazine is reported. The PTZ-labeled oligodeoxynucleotides form stable B-form duplexes with melting temperatures comparable to the unlabeled analogues. Similar results are also observed with oligodeoxynucleotides labeled with PTZ at the nucleobase (28) and 5′-terminal phosphate (30) indicating that PTZ is amenable to covalent attachment to DNA at different locations. Calorimetric data on the unlabeled and PTZ labeled duplexes reveal only a slight decrease in stability upon labeling with PTZ. The spectroscopic properties of PTZ are retained when the complex is covalently attached to DNA at the 5′-terminal thymidine or 2′-deoxyadenosine position. In summary, these two novel PTZ-modified thymidine and 2′-deoxyadenosine increase the number of well-characterized probes available for study, and the corresponding PTZ-labeled oligodeoxynucleotides are of use for DNA-mediated charge-transfer studies. Photophysical studies are currently underway to monitor the electron-transfer intermediate (PTZ+•) in a site-specifically labeled duplex containing PTZ and the inorganic chromophore, Ru(bpy)32+. ACKNOWLEDGMENT

This work was supported in part by the Army Office of Research. We thank Dr. T. Christensen for help with the calorimetry experiments. M.W.G. also thanks the Pew Foundation for a Pew Scholar in the Biomedical Sciences, the Alfred P. Sloan Foundation for a Research Fellowship, and the Dreyfus Foundation for a Camille Dreyfus Teacher Scholar. Figure 4. UV-vis spectra of PTZ-acid (3, dashed line), thymidine (4a, dotted line), and PTZ-thymidine (8, solid line).

unlabeled duplexes, and that introduction of PTZ does not generate intermediate states. The formation of a welldefined DNA duplex structure is also supported by circular dichroism (CD) spectroscopy. Figure 3 shows the CD spectra for the unlabeled (16•21) and PTZ-thymidine labeled (11•21) duplexes. Circular dichroism spectra (44) of the unlabeled, PTZ-thymidine labeled, and PTZadenosine labeled are similar, and the spectral features for B-DNA are present. Covalent attachment of PTZ to the 2′-deoxynucleoside or oligodeoxynucleotide does not alter its spectroscopic properties. The UV-vis spectra of PTZ-labeled thymidine (8a), phenothiazine (3), and thymidine (4a) are shown in Figure 4 (1:1 CH3CN:H2O). The electronic spectrum of 8a is a summation of PTZ and thymidine. Specifically the PTZ π-to-π and weak π-to-n absorption are observed at 250 and 306 nm, respectively. The thymidine π-to-n absorption is at 267 nm (Figure 4) (45). The PTZ-labeled oligodeoxynucleotide possess an absorption at 260 nm, but the absorption of PTZ at lower energy is masked by the DNA. Excitation of 8a or 8b at 315 nm produces an emission centered at 450 nm in 1:1 CH3CN:H2O in agreement with the reported literature value (45). Likewise, the excitation spectra of ss 14, and ds 14•24 in phosphate buffer (50 mM NaCl, 5 mM NaH2PO4, pH ) 7) possess a maximum at 315 nm, and the corresponding emission spectra possess a broad emission centered at 450 nm.

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