CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201402018

8-Cyclopropyl-2’-Deoxyguanosine: A Hole Trap for DNAMediated Charge Transport Jiun Ru Wong and Fangwei Shao*[a] DNA duplexes containing 8-cyclopropyl-2’-deoxyguanosine (8CPG) were synthesized to investigate the effect of the C8modified deoxyguanosine as a kinetic trap for transient hole occupancy on guanines during DNA-mediated hole transport (HT). Thermal denaturation and CD spectra show that DNA duplexes containing 8CPG are able to form stable B-form duplexes. Photoirradiation of terminal tethered anthraquinone can

induce oxidative decomposition of 8CPG through DNA HT along adenine tracts with lengths of up to 4.8 nm. Shallow and periodic distance dependence was observed in a long adenine tract with intervening guanines. The efficient charge transport indicates that 8CPG can electronically couple well with a DNA bridge and form HT-active conformational domains to facilitate transient hole delocalization over an adenine tract.

Introduction The double helix structure adopted by DNA duplexes is an efficient medium for conducting electrons.[1] Various experiments have confirmed that oxidative electron transfer, (also called hole transport, HT) can span over 200  along duplex DNA and that it is sensitive to sequence, base pair integrity, and secondary structure of the nucleic acid.[2] Guanine—the naturally occurring DNA base most vulnerable to oxidation—has been used predominantly as a hole trap in probing DNA HT and studies of the HT mechanism.[3] The resulting guanine radical cations (G· + ) of HT further react with water and/or oxygen to form a mixture of permanent oxidative products.[4] However, because of the long lifetime of G· + (millisecond), mechanistic studies on HT have been complicated by processes such as hole trapping and back electron transfer.[5] Remarkably, recent studies have shown that DNA HT might play vital roles in DNA repair, mitochondria DNA maintenance, and oncogenic protein signaling.[6] Hole transport via nucleobase pair stacking occurs within the lifetime of the guanine radical and induces redox reactions between the metal clusters and amino acid residues of distantly bound proteins. The biological functions of DNA HT have inspired studies of the DNA mechanism on a faster timescale than that of the trapping reaction of guanine radicals.[7] The development of a kinetic radical trap, N2-cyclopropyl-deoxyguanosine (CPG), circumvents the drawback of slow trapping in the case of guanine doublets and has been applied to study the dynamics of HT through DNA bridges.[8] Rapid and irreversible ring opening of the cyclopropyl ring on the exocyclic amine upon one-electron oxidation allows pre-equilibrium [a] J. R. Wong, Dr. F. Shao Division of Chemistry and Biological Chemistry School of Physical and Mathematical Sciences Nanyang Technological University 21 Nanyang Link, Singapore 637371 (Singapore) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402018.

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hole occupation to be examined on a pico- to nanosecond timescale. Such chemical modification has been extended to include other nucleobases (CPA and CPC).[9] Although the current kinetically fast traps are effective in probing HT, there are certain limitations in terms of preparation and application. Studies have shown that 7,8-dihydro-8oxo-2’-deoxyguanosine (8-oxoG), one of the dominant lesions in DNA oxidation, is generated from hydroxylation of C8 of G· + after a photosensitized reaction.[10] Further oxidation of the resulting C8-OH adduct radical might then degrade to 8-oxoG. While CPG has been helpful in gaining insights into the dynamic mechanism of HT, formation of a radical cation at the exocyclic amine might not bear close resemblance to the fate of guanine radical cations generated in vivo, as previous studies have strongly indicated that C8 of G· + is the preferred site for a further irreversible reaction.[10] Postsynthetic strategies involving nucleophilic substitution of commercially available 2fluoro-deoxyinosine as the precursor by aqueous cyclopropylamine have been the general approach for the synthesis of cyclopropylamine-modified oligonucleotides. Such treatments are straightforward and less time consuming, as chemical modification, deprotection, and cleavage of the oligonucleotides from the resin proceed concurrently. However, the synthesis is restricted to DNA–conjugate systems that contain no other functionalities that are labile or unstable under alkylamine treatment, such as certain fluorescent dyes and unnatural nucleobases commonly used in DNA bioconjugation. For example, the presence of a dye such as TAMRA or a base-labile monomer such as 5,6-dihydro-dT would require a milder deprotection method to prevent possible undesired side reactions.[11] Such chemical incompatibility with the postsynthesis conditions might make the experimental designs difficult to accomplish if incorporation of CPG and a labile nucleobase analogue into the same DNA strand is desired. In addition (and unlike direct modification on the phosphoramidite that introduces the nucleobase surrogates as a whole entity during ChemBioChem 2014, 15, 1171 – 1175

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CHEMBIOCHEM FULL PAPERS solid-phase synthesis), postsynthetic methods unavoidably often result in partial completion and/or formation of multiple by-products, not only on the intended precursor, but also likely on natural nucleobases. The complication of synthetic crudes and low yields can significantly increase difficulties in the purification. The limitations of current cyclopropylamine-substituted hole traps motivated us to develop an alternative cyclopropyl-substituted guanine phosphoramidite to circumvent postsynthetic alkylamine treatment. In order to reflect the process of oxidative degradation of guanine in vivo more accurately, trapping C8-G· + would be attractive. We envisioned that a cyclopropyl ring on the C8 position of G would be ideal for this purpose. Here, we describe the synthesis of 8-cyclopropyl-2’-deoxyguanosine (8CPG) and the incorporation of 8CPG into DNA oligonucleotides by the corresponding phosphoramidite. Synthesis followed conventional phosphoramidite chemistry and deprotection conditions, so that postsynthetic adaptation is not required. In addition, we demonstrated the application of 8CPG as a kinetics trap for radical cations in mechanistic studies of DNA-mediated hole transport.

Results and Discussion Synthesis of

8CP

G-modifed DNA

The synthetic route of 8CPG phosphoramidite as the building block for solid phase oligonucleotides synthesis is shown in Scheme 1. The attachment of cyclopropane ring to the C8 carbon of G was accomplished by aqueous-phase Suzuki– Miyaura cross-coupling of a halogenated nucleoside with cyclopropylboronic acid in the presence of Pd(OAc)2/TPPTS catalyst and K2CO3 to generate the desired nucleoside, 8-cyclopropyl-2’-deoxyguanosine (8CPG, 1), with good yield (82 %). The exocyclic amine and the 5’-hydroxyl group were protected by a dimethylforamidine group and 4,4’-dimethoxyltrityl chloride, respectively, to yield 2. 8CPG phosphoramidite (3) was then prepared from 2 with 89 % yield after chromotographic purification. After verification by 31P NMR, 3 was dissolved in anhydrous CH3CN (with good solubility) and incorporated into oligonucleotides by standard automated solid-phase synthesis on a DNA synthesizer. The coupling time for 3 was extended to 3 min to ensure high coupling yield (see Figure S1 in the Supporting Information). Standard deprotection with ammonium hydroxide/aqueous methylamine (1:1, v/v) at 37 8C for one

www.chembiochem.org hour was applied to remove the protection groups on all nucleobases and simultaneously cleave the oligonucleotides from the resin. The 8CPG-containing oligonucleotides were purified by HPLC before and after removal of the 4,4’-dimethoxytrityl (DMT) group. A series of 8CPG-modified DNA strands (8CPG-DNA) were obtained in high yields and were confirmed by ESI-MS (Table S1). 8CP

G-DNA forms stable B-form duplex

To determine the effects of 8CPG on the structure and thermal stability of duplex DNA, thermal denaturation and circular dichroism spectroscopy were performed. The melting curve of 8CP G-containing duplex (Figure S2) exhibited a typical sigmoidal transition (as for canonical duplex DNA); the melting temperature decreased by only 1.8 8C with incorporation of one 8CPG into the duplex (Table S2). Substituting guanine with 8CPG in duplex had a negligible effect on the absorption spectrum around 260 nm upon duplex annealing (Figure S3). The CD spectrum of 8CPG-DNA also revealed the characteristic B-form duplex, with a positive peak at around 280 nm and a negative peak at around 250 nm (Figure S4).[12] End-capping anthraquinone (ecAQ) can increase the thermal stability of duplexes. Consistent with previous reports, an enhancement of approximately 3 8C was observed for AQ-containing duplexes, with or without 8CPG (Table S2).[2b, 13] This implies that 8CPG readily base-pairs with cytosine and can be accommodated in B-form DNA without disrupting either p-stacking of base pairs or the secondary structure.

One-electron oxidation of

8CP

G

Cyclic voltammetry was used to determine the oxidation potential of 8CPG. A peak for an irreversible oxidation was observed at 0.85 V (vs. Ag/AgCl) for 8CPG; G is observed to be oxidized irreversibly at 1.00 V (vs. Ag/AgCl) in 0.1 m sodium phosphate buffer, pH 7.0 (Figure S5). The difference (150 mV) in oxidation potential is comparable to that between CPG and G.[8a] Similar to cyclopropyl modification on an exocyclic amino group, the cyclopropyl modification at C8 only slightly lowered the oxidation potential of G, and hence is unlikely to function as a thermodynamic sink but rather as a kinetic trap, analogously to the previous reported CPG. We first examined the direct one-electron oxidation of 8CPG by photoexcited anthraquinone in trinucleotides. AQ was se-

Scheme 1. Synthesis of 8CPG phosphoramidite (3). a) Cyclopropylboronic acid, K2CO3, Pd(OAc)2, TPPTS, H2O, 100 8C; b) (OMe)2CHNMe2, pyridine, 40 8C; c) DMTCl, pyridine, RT; d) iPr2NPClO(CH2)2CN, DIPEA, CH2Cl2, RT.

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CHEMBIOCHEM FULL PAPERS lected to initiate the oxidation chemistry, as the reduction potential of the excited state in AQ is sufficient to oxidize nucleobases.[14] To avoid both poor aqueous solubility of deoxynucleosides and limitations of the diffusion process on oxidation rate, an AQ derivative (AQdU) was placed 5’ to the hole trap (8CPG or G) in a trinucleotide to allow direct contact and thereby accelerate redox reactions between AQ and the hole traps (Figure 1 A). Oxidation of 8CPG and G by photoirradiating AQ at

www.chembiochem.org Table 1. DNA assemblies used to investigate the distance dependence of hole transfer between ecAQ and 8CPG. Sequence[a,b]

DNA 8CP

GG1/ecAQCC1

8CP

GG2/ecAQCC2

8CP

GG3/ecAQCC3

8CP

GG4/ecAQCC4

5’-ecAQTTTT TCCTTT TTTTTA GAGATA G-3’ 3’-AAAAAG XAAAAA AAATCT CTATC-5’ 5’-ecAQTTTT TTTCCT TTTTTA GAGATA G-3’ 3’-AAAAAA AGXAAA AAATCT CTATC-5’ 5’-ecAQTTTT TTTTTC CTTTTA GAGATA G-3’ 3’-AAAAAA AAAGXA AAATCT CTATC-5’ 5’-ecAQTTTT TTTTTT TCCTTA GAGATA G-3’ 3’-AAAAAA AAAAAG XAATCT CTATC-5’ 5’-TTTTTC CTTTTT TTTAGA GATAG-3’

TCC1 [a] X:

Figure 1. Direct oxidation of 8CPG and G by AQ in trinucleotides. A) Trinucleotides containing photo-oxidant AQ and hole traps (G or 8CPG). Bottom: structure of AQdU derivative. B) Percentage decomposition of hole traps G of AQ dUGT () and 8CPG of AQdU8CPGT (*) as a function of irradiation time: trinucleotide (10 mm) in sodium phosphate buffer (20 mm, pH 7.0) was irradiated at 350 nm for the indicated time. Mean with error bars are given for irradiation of 1 min.

350 nm was detected by HPLC analysis of the nucleoside decomposition after enzymatic digestion. Rapid decomposition of 8CPG was observed with increasing irradiation time (Figure 1 B). In contrast, consumption of guanine occurred at a much slower rate under the same reaction condition. The rapid decomposition of 8CPG clearly suggests that after the generation of a radical cation (8CPG· + or G· + ) through one-electron oxidation, 8CPG is able to trap the radical cation more readily and rapidly than guanine, through irreversible cyclopropyl ring opening. Hole trapping by

8CP

G during hole transfer through DNA

Having established that 8CPG can undergo one-electron oxidation as a kinetic trap, we then probed the ability of 8CPG to trap transient hole occupancy during hole transport over a series of duplex DNA bridges with 13 consecutives adenines (Table 1). End-capped AQ covalently attached 5’ to the thymine strand stacked with the terminal AT pair and served as the photo-oxi 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

8CP

G. [b] Structure of ecAQ:

dant to inject an electron hole into DNA bridges upon photoexcitation. A guanine doublet with 8CPG on 5’ side was initially positioned five adenines away from the photo-oxidant in the purine strand of duplex 8CPGG1/ecAQCC1, and was sequentially moved away by two adenines in each step in duplex 8CPGGn/ ec AQCCn (n = 2–4). Figure 2 A shows HT-induced oxidative damage of 8CPG in 8CP GG1/ecAQCC1. After irradiation at 350 nm and subsequent digestion with phosphodiesterase I and alkaline phosphate, the resulting deoxynucleosides were analyzed by reversedphase HPLC (RP-HPLC). The 8CPG nucleoside peak decreased with longer irradiation time (from 0 to 15 min), whereas the other the nucleobases in the duplex remained essentially unchanged. Percentage 8CPG decomposition was quantified by normalizing the peak areas of irradiated samples to a non-irradiated sample. The significant attenuation of the 8CPG peak indicates that electron holes injected by photoexcited AQ can propagate five AT base pairs to induce 8CPG oxidation and be trapped by undergoing rapid ring opening reactions. Next we used 8CPG-modified DNA to examine the distance dependence of DNA-mediated HT along the adenine tract with fixed length of 4.8 nm. When using 8CPG as a kinetic trap, an exponential distance dependence was obtained from a linear fitting of the plot of ln(decomposition of 8CPG) against distance. The shallow distance dependence (slope 0.002 1) is strikingly similar to that observed previously (0.001 1) in a long adenine tract (without interruptions by either poorly stacked or electronically-coupled traps).[4, 15] Absence of HT efficiency attenuation up to 4.8 nm indicates that 8CPG electronically couples well with the adenine bridges. More remarkably, decomposition of 8CPG showed a periodic oscillation when more intervening AT base pairs were present between the photo-oxidant and hole trap. Maximum oxidation of 8CPG was observed at 5 and 9 bridging base pairs from photo-oxidant, and minima were at 7 and 11 adenines (Figure 2 B). As the periodic distance ChemBioChem 2014, 15, 1171 – 1175

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www.chembiochem.org sient hole delocalization within electronic-coupled domains, with sizes of four base pairs. Intervening guanine doublets can form transient base-pair domains by good electron coupling with adenine tracts, and no attenuation on pre-equilibrium hole delocalization over 4.8 nm was observed. Future work in our lab will focus on the implementation of 8CPG to investigate HT through various high-order DNA structures.

Experimental Section

Figure 2. A) Overlaid HPLC profiles (260 nm) for digested nucleosides from 8CP GG1/ecAQCC1 after irradiated at 350 nm for 0, 5, 10, and 15 min. dU was added as external reference. Inset: decomposition of 8CPG peak with increasing irradiation time. B) Decomposition of 8CPG (Y) in A-tract as a function of r (distance in  between ecAQ and 8CPG) after irradiation for 30 s.

dependence was observed only when the hole trapping rate was faster than the back electron transfer rate,[16] 8CPG was able to reveal the pre-equilibrium hole occupancy along the bridge bases. The oscillatory period of four base pairs is consistent with a delocalized domain hopping mechanism, in which the optimum size of the bridge domain has been observed to be four to five base pairs for uninterrupted adenine tracts.[17] The shallow and periodic distance dependence indicates that long adenine tracts can tolerate mild energetic disturbance from guanines during transient hole delocalization. With one intervening guanine doublet, adenine bridges can accommodate a electronic coupled domain structure similar to that for the uninterrupted A-tracts, regardless of the positions of the internal guanine doublet, and hence achieve effective DNA mediated charge transport up to 4.8 nm by hopping or dissolving over adjacent adenine domains.

Conclusions In summary, an efficient synthesis method was developed to prepare 8CPG phosphoramidite and synthesize 8CPG-containing DNA strands by conventional phosphoramidite chemistry. Electrochemical characterization and AQ-induced photo-oxidation in trinucleotides revealed that 8CPG can undergo one-electron oxidation more readily than guanine, energetically and kinetically. Upon incorporating into AQ-capped DNA duplexes, 8CPG was able to trap radical cations induced effectively by photo-irradiation of AQ and delivered by DNA HT. Shallow distance dependence with periodic oscillation shows that 8CPG shows tran 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

8-cyclopropyl-2’-deoxyguanosine (8CPG, 1): A mixture of 8-bromo2’-deoxyguanosine (8-BrG; 100 mg, 0.29 mmol), palladium(II) acetate (2.6 mg, 0.0116 mmol), cyclopropylboronic acid (57 mg, 0.667 mmol), TPPTS (16.5 mg, 0.029 mmol) and potassium carbonate (232 mg, 1.682 mmol) were placed in a nitrogen-purged sealed tube. Degassed water (5 mL) was added and the reaction was stirred in an oil bath (100 8C, ~ 18 h) or until RP-HPLC showed completed consumption of the starting material (8-BrG lmax 261 nm; 8CP G lmax 257 nm). The mixture was concentrated under reduced pressure and purified by dry column flash chromatography. The eluent for the silica column was prepared by mixing n-butanol/ water/acetic acid (10:10:1, v/v/v) and extracting the organic layer. Desired fractions were collected and concentrated in vacuo to yield 8CPG as white solid (73 mg, 82 %). 1H NMR (400 MHz, D2O with MeOH as internal standard): d = 6.49 (t, J = 7.21 Hz, 1 H), 4.64–4.62 (m, 1 H), 4.12–4.10 (m, 1 H), 3.84 (qd, J = 4.01, 12.53 Hz, 2 H), 2.88 (quin, J = 7.34 Hz, 1 H), 2.33–2.27 (m, 1 H), 2.01 (sep, J = 4.94 Hz, 1 H), 1.07–1.04 (m, 2 H), 0.97–0.95 (m, 1 H), 0.93–0.90 ppm (m, 1 H); 13 C NMR (400 MHz, D2O with MeOH as internal standard): d = 158.4, 153.2, 152.8, 152.1, 115.4, 87.8, 85.3, 72.4, 62.7, 38.4, 8.0, 7.2, 6.7 ppm; HRMS (ESI): m/z calcd for C13H18O4N5 + : 308.1359 [M+H] + ; found: 308.1361. N2-Dimethylaminomethylene-8-cyclopropyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyguanosine (2): 8CPG (1; 61.4 mg, 0.2 mmol) was coevaporated with anhydrous pyridine (1 mL) twice and redissolved in anhydrous pyridine (2 mL). Dimethylformamide dimethylacetal (290 mg, 2.4 mmol) was then added and stirred under nitrogen at 40 8C. After 1 h, TLC showed that the reaction was complete, and the solvents were removed under reduced pressure. The residue was coevaporated with anhydrous pyridine (1 mL) twice before being redissolved in anhydrous pyridine (2 mL). Dimethoxytrityl chloride (86 mg, 0.24 mmol) was added portion-wise to the mixture and stirred for another 1 h. The reaction was then quenched with methanol (4 mL) and the solvents were removed under reduced pressure. The residue was purified by dry column flash chromatography (pre-equilibrated with 1 % NEt3) and eluted with CH2Cl2/MeOH (0–10 %) to give the product as white solid (120 mg, 90 %). TLC: Rf = 0.46 (CH2Cl2/MeOH, 10:1); 1H NMR (400 MHz, CDCl3): d = 9.03 (s, 1 H), 8.45 (s, 1 H), 7.37 (d, J = 7.81 Hz, 2 H), 7.27– 7.23 (m, 5 H), 7.21–7.18 (m, 1 H), 6.77 (d, J = 8.39 Hz, 4 H), 6.52 (t, J = 7.22 Hz, 1 H), 4.79–4.75 (m, 1 H), 4.05–4.02 (m, 1 H), 3.76 (s, 6 H), 3.40 (qd, J = 4.65, 11.16 Hz, 2 H), 3.14–3.07 (m, 1 H), 3.00 (d, J = 8.93 Hz, 6 H), 2.38–2.32 (m, 1 H), 2.13 (sep, J = 5.06 Hz, 1 H), 1.94 (br s, 2 H), 1.33–1.28 (m, 1 H), 1.01–0.97 (m, 1 H), 0.83–0.78 (m, 1 H), 0.71–0.68 ppm (m, 1 H); 13C NMR (400 MHz, CDCl3): d = 158.6, 157.8, 157.5, 155.6, 152.1, 150.9, 144.6, 135.7, 135.6, 130.0, 128.1, 127.8, 126.9, 118.4, 113.1, 86.4, 84.9, 82.4, 72.0, 63.5, 41.3, 38.9, 35.1, 9.3, 8.9, 7.8 ppm; HRMS (ESI): m/z calcd for C37H41O6N6 + : 665.3088 [M+H] + ; found: 665.3090. N2-Dimethylaminomethylene-8-cyclopropyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxyguanosine 3’-(2-cyanoethyl N,N-diisopropylphosChemBioChem 2014, 15, 1171 – 1175

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phoramidite) (3): Compound 2 (100 mg, 0.15 mmol) was coevaporated with anhydrous CH2Cl2 (1 mL) thrice then redissolved in anhydrous CH2Cl2 (2 mL). Diisopropylethylamine (77 mg, 0.6 mmol) was added with stirring under argon. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (53 mg, 0.225 mmol) was added dropwise, and the mixture was stirred for 1 h at room temperature or until TLC showed complete consumption of the starting material. The solvents were removed under reduced pressure and purified by dry column flash chromatography (pre-equilibrated with 1 % NEt3), and eluted with CH2Cl2/MeOH (0–10 %) to give 3 as a white foam (115 mg, 89 %). TLC: Rf = 0.48 (CH2Cl2/MeOH, 10:1); 31P NMR (400 MHz, CDCl3): d = 149.2, 148.8 ppm. Oligonucleotides synthesis: Oligonucleotides (1 mmol) were synthesized by standard phosphoramidite protocols, except with 3 min coupling time for 8CPG phosphoramidite. 8CPG-containing oligonucleotides were then deprotected and cleaved from the resin by treatment with concentrated NH4OH/NHMe2 (1:1, v/v) at 37 8C for 1 h. The crude products were concentrated and purified by RPHPLC (CH3CN (5–35 %) in triethylammonium acetate (TEAA) buffer (0.1 m, pH 7.0) over 30 min.). The oligonucleotides were detritylated with acetic acid (80 %) for 15 min and repurified by HPLC (5–18 % CH3CN in TEAA buffer (0.1 m, pH 7.0) over 30 min). Anthraquinonetethered DNA strands were prepared according to previous procedures.[13] Thermal analysis, circular dichroism spectroscopy, and cyclic voltammetry: See the Supporting Information. Photo-oxidation: DNA aliquots (10 mm, 30 mL) for irradiation were prepared by annealing equimolar amounts of the desired DNA complementary strands. Aliquots were then irradiated (350 nm, 30 s) from a 450 W Xenon lamp equipped with a monochromater and a 320 nm long-pass filter. After irradiation, duplex samples were digested by enzymes (phosphodiesterase I and alkaline phosphatase) by incubating at 37 8C for 24 h to yield the free nucleosides. Then the samples were analyzed by RP-HPLC with a