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. Author manuscript; available in PMC: 2015 Oct 7.
Published in final edited form as: Chemistry. 2009 Aug 3;15(31):7569–7577. doi: 10.1002/chem.200900481

Synthesis and ESR studies of 2'-deoxyuridines tethered with alkynyl, rod-like linkages#

Adam Sniady [a], Michael D Sevilla [a], Srinivasarao Meneni [a], Tadeusz Lis [b], Slawomir Szafert [b], Deepthi Khanduri [a], John M Finke [a], Roman Dembinski [a],*
PMCID: PMC4596529  NIHMSID: NIHMS726705  PMID: 19609983

Abstract

Sonogashira coupling of diacetyl 5-ethynyl-2'-deoxyuridine with diacetyl 5-iodo-2'-deoxyuridine gave the acylated ethynediyl-linked 2'-deoxyuridine dimer (3b) (63%) that was deprotected with ammonia/methanol to ethynediyl-linked 2'-deoxyuridines (3a) (79%). Reaction of 5-ethynyl-2'-deoxyuridine (1a) with 5-iodo-2'-deoxyuridine gave the furopyrimidine linked to 2'-deoxyuridine (78%). Catalytic oxidative coupling of 1a (O2, CuI, Pd/C, DMF) gave the butadiynediyl-linked 2'-deoxyuridines (4) (84%). Double Sonogashira coupling of 5-iodo-2'-deoxyuridine with 1,4-bis(ethynyl)benzene gave 1,4-phenylenediethyne-bridged 5-ethynyl-2'-deoxyuridines (5, 83%). Cu-catalyzed cycloisomerization of dimers 4 and 5 gave their furopyrimidine derivatives. One electron addition to 1a, 3a and 4 gave the anion radical whose ESR spectra showed the unpaired electron largely localized at C6 of one uracil ring (17 G doublet) at 77 K. For the ethynediyl- and butadiynyl-linked uridines 3a and 4 the ESR spectra of their one electron oxidized species at 77 K showed that the unpaired electron is delocalized over both rings. Thus structures 3a and 4 provide an efficient electronic link for hole conduction between the uracil rings. However, for the excess electron, an activation barrier prevents coupling to both rings. These dimeric structures could provide a gate that could separate hole transfer from electron transport between strands in DNA systems. In the crystal structure of acylated dimer 3b the bases were found in the anti position to each other across the ethynyl link. Similar anti conformation was preserved in the derived furopyrimidine–deoxyuridine dinucleoside.

Keywords: Alkynes, Electron delocalization, ESR, Deoxyuridine, Furopyrimidine, Nucleosides, Synthesis

Introduction

Covalently linked nucleosides have been synthesized for a variety of applications. For example, DNA duplexes with interstrand cross-linkers,[i],[ii],[iii],[iv],[v],[vi] hydrogen (Watson-Crick) base pair bonding models (Figure 1),[vii],[viii] inhibitors of ribonucleotide reductase[ix] or HIV reverse transcriptase,[x] a model of the mechanism for the repair of DNA photolesion,[xi] supramolecular self-assembly,[xii] as well as protein binding[8] have all been studied with their aid. Some of the interstrand cross-linked oligonucleotides exhibit interesting biological properties such as thrombin inhibition.[5],[xiii]

Figure 1.

Figure 1

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An example of covalently linked uridines.[7a]

Acetylenes and their higher conjugated homologues have been found to promote strong electronic communication between terminal subunits and to favor rigid, rod-like structures, which have found application in the design of molecular wires.[xiv] The linear, sp carbon chain facilitates exact positioning of the alkynyl substituents for oligonucleotides arrays, and provides an opportunity for substituent-nucleobase communication. Interest in the utilization of the ethynyl (acetylenic) fragment for modification of nucleoside bases, in particular uridines, has resulted in a great number of contributions in recent years.[xv],[xvi] By comparison, butadiynyl or 1,4-phenylenediethynediyl fragments have been used for nucleoside modifications to a much lesser extent.[xvii],[xviii]

Internucleoside alkynyl modifications involving sugar groups are also known. For example, for backbone modification purposes, the phosphodiester bridge has been replaced by acetylenic links.[xix],[xx] Homo– and heterodimers of uridine and adenosine linked at C-3' by a butadiynyl group have also been reported.[xxi]

Purine-purine,[xxii] purine-pyrimidine,[xxiii] and pyrimidine-pyrimidine[xxiv] base conjugates linked with ethynyl or butadiynyl fragments have also been synthesized.[xxv] Initial screening has shown cytostatic activity of bis(purin-6-yl)ethyne and –butadiyne.[22]

We have approached alkynyl-nucleoside chemistry of the μ-ethynediyl,[xxvi] μ-butadiynediyl,[xxvii] and μ-1,4-phenylenediethynediyl[xxviii] linked uridines to study electronic communication or electron delocalization between the linked pyrimidines for hole and excess electron states that are of interest to DNA electronic chips.[xxix],[xxx] Since the alkynyl conformationally well-defined rigid linker may also serve as an interstrand linkage,[6] detailed investigation of dimers’ properties has been warranted. In addition, the tethered nucleosides offer a starting point for further synthetic transformations, such as cyclization to furopyrimidine nucleosides. Such structures, containing a bicyclic base, are known for their highly potent and selective antiviral properties.[xxxi] Furthermore, synthesis of halofuropyrimidines[xxxii] or metallation to dicobalt hexacarbonyl complexes,[xxxiii] with a resulting potential for biological activity, can be envisioned.

Results and Discussion

Synthesis

Preparation of the 2'-deoxyuridine dinucleosides, which contain rod-like tethers of different lengths anchored to position C5 of their pyrimidine base, have been approached in a systematic way. A series of compounds with conformationally well-defined units, namely ethynediyl, butadiyndiyl, and 1,4-phenylenediethynediyl (lengths[xxxiv] ca. 4.0,[xxxv] 6.5, and 10.8 Å respectively), were synthesized. Two key substrates, 5-ethynyl-2'-deoxyuridine (1a), and 5-iodo-2'-deoxyuridine (2a), were used (Scheme 1). Both unprotected nucleosides 1a and 2a are commercially available, although it has been more economically feasible to synthesize 1a from 2a.[16],[xxxvi]

Scheme 1.

Scheme 1

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Synthesis of ethynediyl-, butadiynediyl-, phenylenediethynediyl-bridged 2'-deoxyuridine dimers 3a, 4, 5, and their furopyrimidine derivatives 6a, 7, and 8.

The ethynediyl-linked 2'-deoxyuridines (3a) were prepared first by Sonogashira coupling at 37 °C,[xxxvii] as we also observed that elevated temperature leads to a cyclization product.[36],[xxxviii] Although the ethynediyl-linked dimer 3a was obtained in 64% yield, due to its poor solubility synthesis of ribose-acetylated derivative 3b was explored. A DMF solution of diacetyl 5-ethynyl-2'-deoxyuridine (1b, 2.5 equiv) was used in an excess when reacted with diacetyl 5-iodo-2'-deoxyuridine (2b, 1 equiv) in the presence of Pd(PPh3)4 (0.1 equiv), CuI (0.1 equiv), and Et3N (37 °C). After workup tetraacetyl ethynyl-bridged dimer of 2'-deoxyuridine 3b was isolated in 63% yield (Scheme 1). Deprotection with NH3/MeOH gave 3a in 79% yield.

A homocoupling reaction was subsequently investigated. The dimer of 5-ethynyl-2'-deoxyuridine was obtained by Eglinton-Glaser oxidative coupling.[xxxix] The unprotected ethynyl deoxyuridine 1a (1.0 equiv) and Cu(OAc)2 (1.5 equiv) were combined in pyridine below 55°C, to avoid formation of the cyclized product(s). Column chromatography gave the μ-butadiynediyl 2'-deoxyuridine (4).[27] However, the leaching of CuII salts during column chromatography was occasionally observed. Thus, an alternative catalytic procedure, utilizing less copper, was sought. Oxidative coupling of ethynyl nucleoside 1a (1.0 equiv) in the presence of oxygen and catalytic amounts of Pd/C (0.01 equiv) and CuI (0.03 equiv, DMF, rt)[xl] gave, after workup, an identical dimer 4 in 84% yield, as shown in Scheme 1. In this case ribose acylation was not applied since purification was accomplished effectively.

Subsequently, the internucleoside linkage was elongated by a phenylene group. Unprotected iododeoxyuridine 2a (2 equiv) was combined with 1,4-bis(ethynyl)benzene (1 equiv) under Sonogashira conditions at 40°C to yield dimer 5 in 83% yield.

The temperature of the reactions needs to be precisely controlled to find an optimum balance between the rate of coupling and of cyclization to furopyrimidines. Since furopyrimidines, as mentioned earlier, constitute an antiviral-active class of structures, we were stimulated to investigate the cyclization process of dimers that will lead to novel furopyrimidine-pyrimidine or furopyrimidine-furopyrimidine dinucleosides. When the above-mentioned reaction of 1a and 2a was carried out in a higher temperature (45 °C), dinucleoside 6a with a bicyclic base component were isolated and characterized. To increase the potential crystallinity, dimer 6a was acylated to yield 6b. When dimers 4 and 5 were treated with CuI in the presence of Et3N in DMF at an elevated temperature, furopyrimidine dimers 7 and 8 were obtained with 46% and 60% yields, respectively.

The 1H and 13C NMR spectra confirmed the structural integrity of compounds 3–8. The 1H NMR spectra for 3-5 exhibited resonances at 11.68–11.76 and 8.01–8.48 ppm corresponding to the NH and H6 protons, respectively ([D6]DMSO). The characteristic signals were observed for furopyrimidines. In the spectrum of 6-8 peaks at 8.58–8.91 and 7.13–7.41 ppm, consistent with a H4 and H5 resonances, respectively, were present.[xli] The isomer 6a exhibited both NH, H6 (11.81, 8.41 ppm) and H4, H5 proton signals, as expected for pyrimidine and furopyrimidine rings, respectively. Monitoring of these 1H NMR signals is convenient for observation of reaction progress. The C≡C 13C signals for 3-5 showed chemical shifts characteristic for alkynyl uridines.[xlii] Assignment of carbons, where possible, was established for 4 by a gated decoupling experiment based upon C-H coupling constants J(C,H). Strong parent ions and adequate fragmentation were commonly observed in the MS spectra. Elemental analyses usually indicated hydrates that were also observed by X-ray crystallography for 3b.

Fluorescence of furopyrimidines has been noted in previous studies.[32a],[xliii],[xliv] The properties of 3a and 6a were investigated as fluorescent nucleosides may find practical applications as nucleic acids probes.[44],[xlv],[xlvi] Due to the limited solubility of 3a/6a in water, fluorescence measurements were carried out in water/DMSO 1:1. The excitation maxima were found at λex = 344/356 nm and the emissions maxima were found at λem = 425/441 nm for 3a/6a (Figure 2). Both fluorescence emission spectra were slightly red-shifted relative to the alkynyl mononucleosides.[xlvii] Measurements of the fluorescence lifetime of 3a indicated a very fast (<0.1 ns) decay, which could not be accurately determined with the available instrumentation. However, the fluorescence decay of 6a (for λex = 295 nm) produced experimentally accessible lifetime(s) in the ns regime. Based on the χr2 parameters and inspection of the fit residuals, a two-exponential model gave the optimal fit with a 0.8 ns component (41% amplitude) and a 3.7 ns component.[xlviii]

Figure 2.

Figure 2

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Comparison of excitation (---) and emission (—, λex 320 nm) spectra for 3a (top) and 6a (bottom) (H2O/DMSO 1:1, 22°C).

Crystallography

Molecular structures of 3b, 6b (and 1b) were confirmed by X-ray analysis. Crystallization of acylated dinucleoside from dichloromethane/hexanes gave single crystals of a hydrate 3b•(H2O)1.25 suitable for X-ray analysis. Figure 3 illustrates the selected molecular structure of the expected ethynediyl dimer. Views of all ORTEPS for 3b as well as an X-ray structure data for 1b are available in the Supporting Information. Compound 3b crystallized in space group P1 with both uracil rings positioned almost in the same plane similarly for crystallographically independent molecules (Z = 4). The dihedral angle between the two calculated planes of six atoms of pyrimidine rings ranges from 1.8(5) to 15.2(5)°, with all conformations being anti. However, molecules of 3b differ more significantly in ribose configuration. Similar anti conformation of heterocyclic bases was also observed for 6b (P21; Figure 3). The angles/distances for 1b, 6b, and all four crystallographically independent molecules of 3b were provided in the Supporting Information.

Figure 3.

Figure 3

Figure 3

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An ORTEP view of the representative molecule of 3b (top), and 6b (bottom) illustrating atom labeling scheme and thermal ellipsoids (50% probability level, asymmetric unit). Selected interatomic distances: 3b C5–C7 1.411(12), C7–C7A 1.219(12), C5A–C7A 1.436(12), N1–C1' 1.472(10), N1A–C1'A 1.482(10); 6b C4A–C5 1.447(2), C5–C6 1.363(2), C6–C5A 1.440(2), N3–C1' 1.497(2), N1A–C1'A 1.465(2). Key angles: 3b C5–C7–C7A 177.1(11), C7–C7A–C5A 175.9(9); 6b C4A–C5–C6 105.74(14), C5–C6–C5A 133.74(14).

ESR Experiments

Electronic coupling between DNA components is becoming of increasing interest for DNA electronic technologies such as DNA chips,[xlix] and electronic DNA sequence recognition.[l] In this work we have produced one electron reduced radical species of 1a, 3a, and 4 and one electron oxidized radical species of 3a and 4.[li] The radicals were produced in aqueous glassy solutions at low temperature after gamma irradiation. A critical measure of the electronic coupling between the two uracil rings in the anion radicals of 3a, and 4 is the ESR hyperfine structure. The ESR spectrum of the anion radical of 1a at 77 K in 7 M LiBr (D2O) is shown in Figure 4, where we find a 17 G doublet. This results from a single proton hyperfine coupling from the hydrogen at C6 of the uracil ring. This spectrum shows identical coupling to that found for the uridine anion radical and other substituted uracil anion radicals (such as thymidine anion radical).[lii],[liii],[liv] Thus in structure 1a at 77 K no significant delocalization into the side chain at C5 occurs. The 17 G hyperfine coupling clearly indicates the spin density distribution is largely localized to the C6 position (ca. 70%).[lv] The ESR spectra of the anion radical for both of the dimeric structures 3a and 4 are nearly identical to that of mononucleoside 1a (Figure 4). No significant delocalization to the second ring on an ESR time scale is found for 3a or 4. The electron transfer rate between rings is therefore slow at 77 K as compared to the ESR time scale.[lvi] However, at elevated temperature the small activation barrier to exchange in the anion radicals of 3a and 4 would be overcome and these dimers would also be expected to show fast electron exchange between rings.[56]

Figure 4.

Figure 4

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First derivative X-band Electron Spin Resonance spectra of the one electron reduced radicals of 1a, 3a, and 4, in a aqueous glassy matrix (7 M LiBr, D2O) at 77 K. The three vertical dashed lines in the figure are field calibration (13.09 G separations) and g value (2.0056 central line) markers.

The spectra of one electron oxidized dimeric structures 3a and 4 are shown in Figure 5.[lvii] Both show unresolved singlets spectra, which are consistent with delocalization over both rings of the structures at 77 K. If the radical were localized to one of the structures, a resolved nitrogen hyperfine coupling from N1 and a proton hyperfine coupling from the beta proton at C1' would be observed.[lviii] Thus the sharp singlets are strong evidence for delocalization between rings. A second ESR signal seen at low intensity in the spectrum is observed for 4 that can be attributed to a side reaction during the production of oxidized form.

Figure 5.

Figure 5

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First derivative X-band Electron Spin Resonance spectra of the one electron oxidized radicals of 3a and 4 (7 M LiCl, D2O) at 77 K. The three vertical dashed lines in the figure are field calibration (13.09 G separations) and g value (2.0056 central line) markers.

The exchange between the two rings in these dimeric structures is influenced by several factors. First, in the anion radical of uracil and its analogues its major spin distribution occurs at C6 with little at C5, thus the coupling between the rings is weak (Figure 6).[55] This is not so for the cation radical, which has a maximum in its spin density at the C5 position (Figure 6)[58] and thus p-type conduction is expected to be favored over n-type conduction through this coupling site. Other factors that influence the transfer between rings is nuclear reorganization,[lix] counter ion placement, and possible reversible protonation of the anion radical at a carbonyl group.[lx],[lxi] These events create an activation energy toward electron exchange between the rings.

Figure 6.

Figure 6

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Calculated spin densities for analogue of 3 (ribose rings replaced by methyl; B3LYP/6-31G*). Protonated anion at O4 (left) and cation radical (right).

These results were in line with the theoretical calculations. Using density functional theory (DFT) the structure of an analogue of 3a (ribose moieties were replaced by methyl groups), in its anionic, cationic, and O4-protonated (anion protonated at O4) radical states, was fully optimized in anti conformation as observed in an X-ray structure of 3b. Figure 6 illustrates the B3LYP/6-31G* calculated spin densities for an analogue of 3a. In cationic state (top/left) the spin densities are delocalized on both of the uracil ring. The calculated spin density is spread over both rings in the anion radical as well. However, the corresponding anion protonated at O4 (bottom/right), that corresponds to experimentally investigated anion (pKa ca. 7),[60] shows localization of the spin densities within a single uracil ring (for color figures see Supporting Information; nodal negative spin densities are visualized in green).

In conclusion, the synthesis and characterization of alkynyl modified nucleoside dimers with conformationally rigid linkages that may serve as interstrand cross-linkers, as well as their furopyrimidine derivatives, have been presented. In the crystal structure of acylated ethynediyl-linked 2'-deoxyuridine dimer and its furopyrimidine analogue the bases have been found in the anti position to each other across the link. It was experimentally established by ESR spectroscopy that the unpaired electron was mainly localized at C6 of the one uracil ring in one electron reduced 3a and 4, but for the one electron oxidized species delocalization to both rings has been found. These results show that these dimeric structures may provide a gate that could separate hole transfer from electron transport in DNA electronic systems.

Experimental

General

Commercial chemicals were treated as follows: DMF, distilled from CaH2 and degassed (freeze and thaw) three times prior to use; Et3N and pyridine distilled from P2O5. 5-Iodo-2'-deoxyuridine (Yamasa Corporation), p-diethynylbenzene (GFS Chemicals), Pd(PPh3)4 (Pressure Chemicals), copper(I) iodide 99.999%, and copper(II) acetate (Aldrich) were used as received. Chromatography media: silica gel 40-63 μm and TLC plates (Dynamic Adsorbents). Other materials not listed were used as received. Sonogashira coupling reactions were carried out under an N2 atmosphere. IR and UV-visible spectra were recorded on a Varian 3100 Excalibur or Bio Rad FTS-175C and Cary 50 or 100 spectrometers. NMR spectra were obtained on a Bruker Avance III 400 and Avance 200 spectrometers. Mass spectra were recorded on a Bruker MicrOTOF-Q (ESI) instrument. Microanalyses were conducted by Atlantic Microlab. Fluorescence including time-resolved was observed on a Photon Technologies Quantum Master/Easy Life instrument.

5,5'-ethyne-1,2-diylbis(3',5'-di-O-acetyl-2'-deoxyuridine) (3b)

A Schlenk flask was charged with 3',5'-di-O-acetyl-5-iodo-2'-deoxyuridine 2b[38] (0.280 g, 0.639 mmol), Pd(PPh3)4 (0.074 g, 0.064 mmol), CuI (0.012 g, 0.064 mmol), DMF (6 mL), Et3N (0.18 mL, 1.3 mmol), and 3',5'-di-O-acetyl-5-ethynyl-2'-deoxyuridine 1b[16] (0.537 g, 1.60 mmol). The yellow mixture was stirred at 37°C for 20 h (1H NMR showed complete conversion of the substrate). The solvent was removed by oil pump vacuum. Silica gel column chromatography (25 × 2.5 cm; hexanes/EtOAc 100:0 → 0:100) gave a pale-yellow fraction. Solvent was removed by rotary evaporation and the residue was dried by oil pump vacuum. MeOH (ca. 10 mL) was added and the solid residue after column chromatography was extracted/sonicated (ultrasonic bath) for 0.5 h. The precipitate was filtered off and the product was dried by oil pump vacuum for 3 h to give 3b as a white powder (0.260 g, 0.402 mmol, 63%).

1H NMR (200 MHz, CDCl3, 22°C, TMS): δ = 11.76 (s, 2 H; N3), 8.01 (s, 2 H; H6), 6.15 (t, 3J(H,H) = 6.5 Hz, 2 H; H1'), 5.26-5.11 (m, 2 H; H3'), 4.39-4.09 (m, 6 H; H4', H5'), 2.63-2.21 (m, 4 H; H2'), 2.07 (s, 6 H; 2COCH3), 2.06 ppm (s, 6 H; 2COCH3); 13C NMR (50 MHz, CDCl3, 22°C, TMS): δ = 170.1 (s; COCH3), 170.0 (s; COCH3), 161.2 (d, 2J(C,H) = 9.2 Hz; C4), 149.3 (d, 2J(C,H) = 7.5 Hz; C2), 143.5 (d, 1J(C,H) = 184.5 Hz; C6), 98.7 (d, 2J(C,H) = 4.2 Hz; C5), 84.9 (d, 1J(C,H) = 178.4 Hz; C1'),[lxii] 84.5 (d, 3J(C,H) = 5.1 Hz; C≡C), 81.5 (d, 1J(C,H) = 153.2 Hz; C4'),[62] 73.7 (d, 1J(C,H) = 158.5 Hz; C3'), 63.5 (t, 1J(C,H) = 148.9 Hz; C5'), 36.3 (d, 1J(C,H) = 135.4 Hz, C2'), 20.8 (q, 1J(C,H) = 129.7 Hz; COCH3), 20.6 ppm (q, 1J(C,H) = 129.7 Hz; COCH3); IR (KBr): ν bar = 3194 br, 3076 br, 1701 vs, 1459 s, 1367 m, 1297 s, 1236 vs, 1105 m, 1063 m cm−1; UV/Vis (CH3OH, 2.0 × 10−5 M): λmax (ε) = 321 (19000), 252 sh (11000), 238 nm (13000 mol−1 dm3 cm−1); MS (ESI): m/z (%): 669 ((M + Na)+, 100%), 469 ((M – ribose + Na)+, 23%); elemental analysis calcd (%) for C28H30N4O14•0.5H2O: C 51.30, H 4.77; found: C 51.17, H,4.66.

5,5'-ethyne-1,2-diylbis(2'-deoxyuridine) (3a)

A round bottom flask was charged with 3b (0.111 g, 0.170 mmol), MeOH (5 mL), and ammonia (7.0 M in MeOH; 0.80 mL, 5.6 mmol). The solution was stirred at rt for 20 h. 1H NMR showed complete conversion of the substrate. Solvent was removed by oil pump vacuum and the residue was extracted (sonicated) with CHCl3/MeOH (80:20, 15 mL) for 1 h. The precipitate was filtered off and washed with CHCl3/MeOH (80:20, 3 × 5 mL). The solid was dried by oil pump vacuum for 3 h to give 3a (0.054 g, 0.11 mmol, 66%). The solvent was removed from the filtrate by rotary evaporation and the residue was suspended extracted (sonicated) with CHCl3/MeOH (80:20, 5 mL) for 0.5 h. Filtration and drying by oil pump vacuum gave an additional amount of 3a as a white solid (0.010 g, 0.021 mmol, 12%; total 0.064 g, 1.3 mmol, 79%).

1H NMR (200 MHz, [D6]DMSO, 22°C, TMS): δ = 11.68 (s, 2 H; N3), 8.24 (s, 2 H; H6), 6.12 (t, 3J(H,H) = 6.5 Hz, 2 H; H1'), 5.25 (d, 3J(H,H) = 4.2 Hz, 2 H; OH3'), 5.11 (t, 3J(H,H) = 4.8 Hz, 2 H; OH5'), 4.23 (p, 3J(H,H) = 3.5 Hz, 2 H; H3'), 3.89-3.72 (m, 2 H; H4'), 3.71-3.47 (m, 4 H; H5'), 2.13 ppm (t, 3J(H,H) = 6.0 Hz, 4 H; H2'); 13C NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 161.5 (d, 2J(C,H) = 9.2 Hz; C4), 149.4 (d, 2J(C,H) = 8.5 Hz; C2), 144.0 (d, 1J(C,H) = 183.5 Hz; C6), 98.3 (s; C5), 87.6 (d, 1J(C,H) = 146.3 Hz; C4'),[61] 84.8 (d, 1J(C,H) = 168.1 Hz; C1'),[61] 84.5 (d, 3J(C,H) = 5.4 Hz; C≡C), 70.2 (d, 1J(C,H) = 150.6 Hz; C3'), 61.0 (t, 1J(C,H) = 140.3 Hz; C5'), 40.3 ppm (d, 1J(C,H) = 133.0 Hz; C2'); IR (KBr): ν bar = 3404 br, 3066 br, 1701 vs, 1466 m, 1297 s, 1293 m, 1101 m cm−1; UV/Vis (H2O/DMSO 1:1, 1.0 × 10−5 M): λmax (ε) = 238 nm (13000 mol−1 dm3 cm−1); MS (ESI): m/z (%): ((2M + Na)+, 55%), 737 (unassigned, 38%), 501 ((M + H + Na)+, 100%), 479 ((M + H)+, 48%), 385 ((M – ribose + Na)+, 33%); elemental analysis calcd (%) for C20H22N4O10•H2O: C 48.39, H, 4.87; found: C 48.01, H, 4.58.

5,5'-buta-1,3-diyne-1,4-diylbis(2'-deoxyuridine) (4)[6b]

A Schlenk flask was charged with 1a (0.253 g, 1.00 mmol), Pd/C (10%; 0.011 g, 0.01 mmol), CuI (0.0057 g, 0.03 mmol), and DMF (1mL). The flask was sealed with septum, the air inside the flask was replaced with oxygen by five vacuum-oxygen (balloon) cycles. The mixture was stirred for 12 h at rt. 1H NMR showed complete conversion of the substrate. The reaction mixture was passed through a 1 cm silica gel pad, followed by an additional amount of DMF (10 mL). Solvent was removed by oil pump vacuum and the residue was extracted (sonicated) with CHCl3/MeOH (60:40, 25 mL) for 2 h. The precipitate was filtered off, washed with CHCl3/MeOH (60:40, 3 × 10 mL), and dried by oil pump vacuum for 3 h to give 4 as a white solid (0.210 g, 0.418 mmol, 84%).

1H NMR and MS spectra matched those reported earlier;[6b] 13C NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 161.6 (d, 2J(C,H) = 9.0 Hz; C4), 149.2 (d, 2J(C,H) = 8.1 Hz; C2), 146.1 (d, 1J(C,H) = 184.3 Hz; C6), 96.7 (s; C5), 87.7 (d, 1J(C,H) = 147.8 Hz; C4'),[61] 85.2 (d, 1J(C,H) = 170.2 Hz; C1'),[61] 76.5 (s; C≡CC≡C), 75.4 (d, 3J(C,H) = 5.6 Hz; C≡CC≡C), 69.7 (d, 1J(C,H) = 149.2 Hz; C3'), 60.7 (t, 1J(C,H) = 141.2 Hz; C5'), 40.9 ppm (d, 1J(C,H) = 134.8 Hz; C2'); IR (KBr): ν bar = 3426 br, 3181 br, 3061 br, 1702 vs, 1459 m, 1281 m, 1087 m cm−1; UV/Vis (CH3OH, 2.6 × 10−5 M): λmax (ε) = 359 (19000), 336 (28000), 316 (25000), 295 sh (20000), 274 (16000), 255 sh (23000), 240 nm (26000 mol−1 dm3 cm−1); elemental analysis calcd (%) for C22H22N4O10•H2O: C 50.77, H, 4.65; found: C 50.98, H 4.34.

5,5'-(1,4-phenylenediethyne-2,1-diyl)bis(2'-deoxyuridine) (5)

A Schlenk flask was charged with 5-iodo-2'-deoxyuridine 2a (1.70 g, 4.80 mmol), Pd(PPh3)4 (0.555 g, 0.480 mmol), CuI (0.092 g, 0.48 mmol), DMF (5 mL), Et3N (1.4 mL, 9.6 mmol), and 1,4-bis(ethynyl)benzene (0.303 g, 2.40 mmol). The yellow mixture was stirred at 40°C for 18 h (1H NMR showed complete conversion of the substrate). Solvent was removed by oil pump vacuum and the residue was extracted (sonicated) with CHCl3/MeOH (50:50, 25 mL) for 20 h. The precipitate was filtered off and extracted in a Soxhlet apparatus (CHCl3/MeOH (50:50, 150 mL) for 20 h. The solid was dried by oil pump vacuum for 3 h to give 5 as a pale-yellow solid (1.15 g, 1.99 mmol, 83%).

1H NMR (200 MHz, [D6]DMSO, 22°C, TMS): δ = 11.72 (br s, 2 H; NH), 8.43 (s, 2 H; H6), 7.48 (s, 4 H; C6H4), 6.13 (t, 3J(H,H) = 6.3 Hz, 2 H; H1'), 5.27 (d, 3J(H,H) = 4.3 Hz, 2 H; OH3'), 5.19 (t, 3J(H,H) = 4.7 Hz, 2 H; OH5'), 4.26 (p, 3J(H,H) = 3.9 Hz, 2 H; H3'), 3.88-3.77 (m, 2 H; H4'), 3.75-3.52 (m, 4 H; H5'), 2.17 ppm (t, 3J(H,H) = 5.3 Hz, 4 H; H2'); 13C{1H} NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 161.4 (C4), 149.4 (C2), 144.3 (C6), 131.4 (m,o-C6H4), 122.4 (i,p-C6H4), 97.9 (C5), 91.4 (C≡CC6H4), 87.6 and 84.9 (C4' and C1'), 84.7 (C≡CC6H4), 69.9 (C3'), 60.8 ppm (C5');[lxiii] IR (KBr): ν bar = 3413 br, 3055 br, 2690 vs, 1461 s, 1301 s, 1274 s, 1091 s cm−1; UV/Vis (DMSO, 1.9 × 10−5 M): λmax (ε) = 343 (38000), 365 sh nm (28000 mol−1 dm3 cm− 1); MS (ESI): m/z (%): 1179 ((2M + Na)+, 9%), 601 ((M + Na)+, 44%), 413 (unassigned, 100%); elemental analysis calcd (%) for C28H26N4O10•H2O: C 56.37, H 4.73; found: C 56.63, H 4.73.

1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-[3-(2-deoxy-β-D-erythro-pentofuranosyl)-2-oxo-2,3-dihydrofuro[2,3-d]pyrimidin-6-yl]uracil (6a)

A Schlenk flask was charged with 5-iodo-2'-deoxyuridine 2a (1.00 g, 2.90 mmol), Pd(PPh3)4 (0.335 g, 0.290 mmol), CuI (0.055 g, 0.29 mmol), DMF (8 mL), Et3N (0.84 mL, 5.8 mmol), and 5-ethynyl-2'-deoxyuridine (1.10 g, 4.35 mmol). The yellow mixture was stirred at 45°C for 55 h (1H NMR showed complete conversion of the substrate). Solvent was removed by oil pump vacuum and the residue was extracted (sonicated) with MeOH (30 mL). The precipitate was filtered off, washed with MeOH (3 × 5 mL), and extracted (sonicated) in CHCl3/MeOH (95:5, 30 mL). The precipitate was filtered off and washed with cold CHCl3 (3 × 5 mL). The solid was dried by oil pump vacuum for 3 h to give 6a (0.760 g, 1.59 mmol, 55%). The solvent was removed from filtrate by rotary evaporation and remaining product was suspended in CHCl3/MeOH (95:5, 5 mL) and sonicated. Filtration and drying by oil pump vacuum gave an additional amount of 6a as a white solid (0.320 g, 0.670 mmol, 23%; total 1.08 g, 2.26 mmol, 78%).

1H NMR (200 MHz, [D6]DMSO, 22°C, TMS):[lxiv] δ = 11.82 (s, 1 H; NH), 8.77 (s, 1 H), 8.41 (s, 1 H), 7.13 (s, 1 H; H5), 6.18 (q, 3J(H,H) = 6.3 Hz, 2 H; 2H1'), 5.30 (d, 3J(H,H) = 4.2 Hz, 2 H; 2OH3'), 5.12 (q, 3J(H,H) = 4.6 Hz, 2 H; 2OH5'), 4.35-4.18 (m, 2 H; 2H3'), 3.98-3.82 (m, 2 H; 2H4'), 3.76-3.58 (m, 4 H; 2H5'), 2.54-2.32 (m, 1 H), 2.22 (t, 3J(H,H) = 5.4 Hz, 2 H), 2.16-1.98 ppm (m, 1 H); 13C NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 170.3 (t, 2J(C,H) = 9.2 Hz; C7A), 160.1 (d, 2J(C,H) = 9.2 Hz), 153.9 (d, 2J(C,H) = 5.6 Hz), 149.2 (d, 2J(C,H) = 8.1 Hz), 147.8 (m; C6), 137.6 (d, 1J(C,H) = 187.6) Hz, 137.2 (d, 1J(C,H) = 182.9 Hz), 106.8 (s; C4A), 103.5 (s; C5), 101.2 (d, 1J(C,H) = 187.2 Hz; C5A), 88.3/87.9 (d, 1J(C,H) = 148.6 Hz/d, 1J(C,H) = 147.3 Hz; C4' and C4'A),[62] 87.7/85.3 (d, 1J(C,H) = 174.5 Hz/d, 1J(C,H) = 172.0 Hz; C1' and C1'A),[62] 70.4/69.8 (d, 1J(C,H) = 148.6 Hz/d, 1J(C,H) = 148.5; C3' and C3'A), 61.1/60.9 ppm (t, 2J(C,H) = 140.2 Hz/t, 2J(C,H) = 141.6 Hz; C5' and C5'A);[63] IR (KBr): ν bar = 3415 br, 1697 vs, 1093 m, 1057 m cm−1; UV/Vis (CH3OH, 2.5 × 10−5 M): λmax (ε) = 355 (22000), 316 sh (13000), 274 sh (13000), 260 (18000), 243 sh nm (14000 mol−1 dm3 cm−1); MS (ESI): m/z (%): 1457 ((3M + Na)+, 10%), 979 ((2M + Na)+, 48%), 479 ((M + H)+, 100%), 363 ((M – ribose + H)+, 32%); elemental analysis calcd (%) for C20H22N4O10•0.5H2O: C 49.28, H 4.76; found: C 49.30, H 4.54.

1-(3,5-di-O-acetyl-2-deoxy-β-D-erythro-pentofuranosyl)-5-[3-(3,5-di-O-acetyl-2-deoxy-β-D-erythro-pentofuranosyl)-2-oxo-2,3-dihydrofuro[2,3-d]pyrimidin-6-yl]uracil (6b)

A flask was charged with 6a (0.205 g, 0.429 mmol), pyridine (2 mL), and acetic anhydride (1.0 mL, 1.1 mmol). The mixture was stirred at rt for 16 h (time not optimized), then poured into HCl (1 N; ca. 20 mL) and extracted with chloroform (3 × 15 mL). The organic layer was washed with water (2 × 100 mL), brine (100 mL), and dried over anhydrous MgSO4. The solvent was removed by rotary evaporation. Ethyl acetate (10 mL) was added to the oily residue, and the solid was collected on a fritted funnel. The solid was dried by oil pump vacuum for 2 h to give 6b (0.194 g, 0.300 mmol, 70%) as a white powder.

1H NMR (400 MHz, [D6]DMSO, 22°C, TMS):[64] δ = 11.96 (s, 1 H; NH), 8.58 (s, 1 H; H4), 8.11 (s, 1 H; H6A), 7.24 (s, 1 H; H5), 6.37-6.23 (m, 2 H; 2H1'), 5.33-5.12 (m, 2 H; 2H3'), 4.49-4.10 (m, 6 H; 2H4' and 2H5'), 2.73-2.25 (m, 4 H; 2H2'), 2.20 (s, 3 H; CH3), 2.09 (s, 6 H; 2CH3), 2.01 ppm (s, 3 H; CH3); 13C NMR (100 MHz, [D6]DMSO, 22°C, TMS): δ = 170.5, 170.3, 170.1, 170.02, 170.00, 159.9 (d, 2J(C,H) = 9.3 Hz; C4), 153.7 (d, 2J(C,H) = 6.1), 149.0 (dd, 2J(C,H) = = 8.1, 2.0 Hz), 147.5 (dd, 2J(C,H) = 8.6, 4.9 Hz), 137.6 (dd, 1J(C,H) = 189.0 Hz, 2J(C,H) = 2.8 Hz), 136.2 (dd, 1J(C,H) = 182.3 Hz, 2J(C,H) = 3.0 Hz), 107.0 (dd, 2J(C,H) = 4.5, 2.9 Hz; C4A), 103.8 (s; C5), 101.6 (d, 1J(C,H) = 188.9 Hz; C5A), 88.1/85.6 (dm, 1J(C,H) = 173.1 Hz/dm, 1J(C,H) = 169.8 Hz; C1' and C1'A),[62] 82.5/82.0 (d, 1J(C,H) = 152.4 Hz/d, 1J(C,H) = 152.9 Hz; C4' and C4'A),[62] 74.2/74.1 (br d, 1J(C,H) = 158.6 Hz/br d, 1J(C,H) = 157.7 Hz; C3' and C3'A), 63.6 (t, 1J(C,H) = 149.2 Hz, C5' and C5'A), 37.9/37.0 (dd, 1J(C,H) = 137.7, 134.6 Hz/dd, 1J(C,H) = 137.1, 134.4 Hz; C2' and C2'A), 20.73 (q, 1J(C,H) = 129.7 Hz; 2CH3), 20.71 (q, 1J(C,H) = 129.8 Hz; CH3), 20.5 ppm (q, 1J(C,H) = 129.6 Hz; CH3); IR (KBr): ν bar = 3075 br, 1747 vs, 1716 vs, 1671 vs, 1233 vs cm−1; UV/Vis (CH3OH, 1.9 × 10−5 M): λmax (ε) = 355 (24000), 260 nm (18000 mol−1 dm3 cm−1); MS (ESI): m/z (%): 1315 ((2M + Na)+, 15%), 669 ((M + Na)+, 100%), 447 ((M – ribose + H)+, 54%); elemental analysis calcd (%) for C28H30N4O14: C 52.01, H 4.68; found: C 51.72, H 4.61.

3,3'-bis(2-deoxy-β-D-erythro-pentofuranosyl)-6,6'-bifuro[2,3-d]pyrimidine-2,2'(3H,3'H)-dione (7)

A flask was charged with 4 (0.196 g, 0.390 mmol), CuI (0.015 g, 0.075 mmol), DMF (7 mL), and Et3N (3 mL). The brown mixture was stirred at 120°C for 60 h. 1H NMR showed complete conversion of the substrate. The precipitate was filtered off and dried by oil pump vacuum for 3 h and extracted (sonicated) with CHCl3/MeOH (70:30, 20 mL) for 1 h. The solid was filtered off, washed with CHCl3/MeOH (70:30, 3 × 10 mL), and dried by oil pump vacuum for 3 h to give 7 as a grey-brown solid (0.090 g, 0.18 mmol, 46%).

1H NMR (200 MHz, [D6]DMSO, 22°C, TMS): δ = 8.91 (s, 2 H; H4), 7.20 (s, 2 H; H5), 6.16 (t, 3J(H,H) = 6.0 Hz, 2 H; C1'), 5.31 (d, 3J(H,H) = 4.3 Hz, 2 H; OH3'), 5.16 (t, 3J(H,H) = 5.1 Hz, 2 H; OH5'), 4.35-4.15 (m, 2 H; H3'), 4.03-3.87 (m, 2 H; H4'), 3.80-3.55 (m, 4 H; H5'), 2.55-2.35 (m, 2 H; H2'), 2.20-2.00 ppm (m, 2 H; H2'); 13C NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 171.0 (apparent t, 3J(C,H) = 7.5 Hz; C7a), 153.6 (d, 2J(C,H) = 5.7 Hz; C2), 143.5 (d, 2J(C,H) = 9.6 Hz; C6), 139.6 (d, 1J(C,H) = 188.0 Hz; C4), 105.7 (s; C4a), 102.8 (d, 1J(C,H) = 186.5 Hz; C5), 88.4 (d, 1J(C,H) = 145.1 Hz; C4'),[62] 88.0 (d, 1J(C,H) = 176.0 Hz; C1'),[62] 69.6 (d, 1J(C,H) = 147.9 Hz; C3'), 60.7 ppm (t, 1J(C,H) = 139.2 Hz; C5');[63] IR (KBr): ν bar = 3410 br, 1660 vs, 1572 m, 1175 w cm−1; UV/Vis (CH3OH, 3.2 × 10−5 M): λmax (ε) = 416 sh (18000), 387 sh (26000), 374 (27000), 291 nm (27000 mol−1 dm3 cm−1); MS (ESI): m/z (%): 503 ((M + H)+, 100%); HRMS (C22H22N4O10 + Na 525.1228) 525.1254.

6,6'-(1,4-phenylene)bis[3-(2-deoxy-β-D-erythro-pentofuranosyl)furo[2,3-d]pyrimidin-2(3H)-one] (8)

A flask was charged with 5 (0.500 g, 0.864 mmol), CuI (0.050 g, 0.255 mmol), DMF (10 mL), and Et3N (3 mL). The brown mixture was stirred at 70°C for 15 h (1H NMR showed complete conversion of the substrate). The precipitate during was filtered off and dried by oil pump vacuum for 3 h. The precipitate was extracted (sonicated) with CHCl3/MeOH (70:30, 20 mL) for 1 h. The solid was filtered off, washed with CHCl3/MeOH (70:30, 3 × 10 mL), and dried by oil pump vacuum for 3 h to give 8 as a dark yellow solid (0.300 g, 0.518 mmol, 60%). 1H NMR (200 MHz, [D6]DMSO, 22°C, TMS): δ = 8.91 (s, 2 H; H4), 7.95 (s, 4 H; C6H4), 7.41 (s, 2 H; H5), 6.18 (t, 3J(H,H) = 5.8 Hz, 2 H; H1'), 5.31 (d, 3J(H,H) = 4.1 Hz, 2 H; OH3'), 5.20 (t, 3J(H,H) = 5.0 Hz, 2 H; OH5'), 4.33-4.19 (m, 2 H; H3'), 4.00-3.88 (m, 2 H; H4'), 3.81-3.57 (m, 4 H; H5'), 2.50-2.35 (m, 2 H; H2'), 2.21-2.03 ppm (m, 2 H; H2'); 13C{1H} NMR (50 MHz, [D6]DMSO, 22°C, TMS): δ = 170.8 (C7a), 153.3/152.6 (C6 and C2), 138.1 (i-C6H4), 128.6 (C4), 128.6 (4C, C6H4), 106.4 (C4a), 100.2 (C5), 88.0/87.4 (C4' and C1'), 69.5 (C3'), 60.6 ppm (C5');[63] IR (KBr): ν bar = 3387 br, 1664 vs, 1571 vs, 1382 s, 1342 s, 1179 s, 1099 s, 1059 s, 1026 s, 1000 s, 832 m, 779 s, 695 w cm−1; UV/Vis (DMSO, 1.3 × 10−5 M): λmax (ε) = 413 (52000), 390 (50000), 321 (13000), 258 nm (10000 mol−1 dm3 cm−1); MS (ESI): m/z (%): 601 ((M + Na)+, 100%); elemental analysis calcd (%) for C28H26N4O10•2H2O: C 54.72, H 4.92; found: C 55.10, H 4.54.

Crystallography

X-ray quality crystals of 1b, 3b, and 6b (all colorless plates) were grown by slow evaporation from the chloroform (1b and 6b), and dichloromethane/hexanes (3a) solutions. Selected crystallographical tables are provided in Supporting Information. Crystallographic data for the structures of 1b, 3b, and 6b were also deposited with Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-711025, CCDC-711026, and CCDC-715301, respectively. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

ESR

Samples for ESR investigation were prepared by dissolving approximately 1 mg of 1a, 3a, or 4 in 7 M LiBr (D2O) or 7M LiCl (D2O). These solutions were drawn into 4 mm suprasil quartz tubes and cooled to 77 K. On cooling these solutions form glasses and are essentially supercooled liquid states. These samples were γ-irradiated (500 Gy, 60Co) at 77 K, which forms excess electrons and Br2−· (Cl2−·) from the irradiation of the 7 M LiBr (LiCl) solution. The solute which makes up only 0.1% of the sample mass is not directly irradiated to any observable extent. The electrons formed by the irradiation add to the solute and the Br2−· (Cl2−·) formed by the ionization of the matrix produces a very broad background ESR signal that does not interfere in the g = 2 region significantly.[54]

The ESR spectra observed of the solute anions are a result of addition to the π electron system (LUMO) of the structures. One electron oxidation is performed by attack of Cl2−· on the solutes (3a, 4) on annealing of the 7 M LiCl glasses to 155K where the glass softens and Cl2−· becomes mobile. For these oxidative studies 5mg/mL K2S2O8 was added to scavenge the electrons. This results in the sulfate radical (SO4-·) which simply forms additional Cl2−·.lxv Thus the system has only one electron oxidative processes. All ESR spectra were recorded at 77 K after γ-irradiation and were taken with a Varian Century Series EPR spectrometer operating at X-band with a dual cavity and a 200-mW klystron, with Frémy’s salt (g = 2.0056, AN = 13.09 G) as a reference.

Computations

The geometries of dimer 3a methyl analogue in their cationic, O4-protonated (and anionic, Supporting Information) radical states were fully optimized using DFT as implemented in Gaussian 03 suite of programs.[lxvi] The B3LYP functional with 6-31G* basis set was used in the calculation. The B3LYP functional is a combination of Becke's three-parameter hybrid exchange functional[lxvii],[lxviii] and the Lee-Yang-Parr correlation functional.[lxix] The use of B3LYP functional, for the study of radicals, is well documented in the literature.[lxx],[lxxi] GaussView molecular modeling software[lxxii] was used to plot the spin density distributions in the molecules.

Supplementary Material

supporting information

Acknowledgment

We thank the National Institute of Health (NIH, CA111329, CA045424), Oakland University, and its Research Excellence Program in Biotechnology for support of this research. National Science Foundation instrumentation award (NSF, CHE-0821487) is also acknowledged. T.L. and S.S. thank the Polish State Committee for Scientific Research (Grant N204 140 31/3236) for support. A.S. is grateful for the Provost's Graduate Student Research Award. Dr. Agnieszka Mikus and Dr. Anil Kumar are acknowledged for assistance with fluorescence experiments and DFT calculations, respectively. We are also thankful to Dr. Hiroyuki Hayakawa (Yamasa Corporation, Biochemicals Division) for generous supply of iodonucleosides.

Footnotes

#

Presented in part: R. Dembinski, M. S. Rao, 225th National Meeting of the American Chemical Society, New Orleans, LA, Mar 23–27, 2003; Abstract of Papers, Vol. 225, American Chemical Society: Washington, DC, 2003; 54-CARB (p U260).

Supporting Information. NMR spectra for compounds 3-8; X-ray tables for compounds 1b, 3b, and 6b, ORTEPs for 1b and 3b; fluorescence figures; computational figure for anion of analogue of 3 and Figure 6 in color. This material is available free of charge via the Internet at http://onlinelibrary.wiley.com/doi/10.1002/chem.200900481/abstract.

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