Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA †

Nucleosides and 2’-deoxyribonucleoside triphosphates (dNTPs) bearing phenothiazine (PT) attached to a nucleobase (cytosine or 7-deazaadenine) either directly or through an acetylene linker were prepared through Suzuki or Sonogashira cross-coupling and triphosphorylation, and were studied as building blocks for polymerase construction of modified DNA. The directly PT-substituted dNTPs were better substrates for polymerases than the alkyne-linked dNTPs but all of them were used in enzymatic synthesis of DNA using primer extension, nicking enzyme amplification, PCR or 3’-tail labelling by terminal deoxynucleotidyl transferase. The phenothiazine served as an oxidizable redox label (giving two analytically useful signals of oxidation on electrode) for nucleosides and DNA and was also used in orthogonal combination with previously developed benzofurazane or nitrophenyl labels for redox coding of DNA bases. Therefore, the title PT-linked dNTPs are useful additions to the portfolio of nucleotides for enzymatic synthesis of redox-labelled DNA for electrochemical analysis.


Introduction
Electrochemical DNA sensors 1 are widely applied in diagnostics and DNA labelling by redox-active groups is of great importance. 2 We and others have previously reported labelling of DNA by diverse redox labels, including ferrocene, 3,4 aminoand nitrophenyl, 5 Os(bpy) 3 -, 6 anthraquinone, 3,7,8 benzofurazane, 9 azidophenyl, 10 methylene-blue, 11,12 polyoxometalates 13 or methoxyphenol 14 through polymerase incorporation of redox-labelled 2′-deoxyribonucleoside triphosphate (dNTP). Combination of several orthogonal redox labels could be used for multipotential redox-coding of DNA nucleobases for development of electrochemical minisequencing techniques. The orthogonality requires labelling of each nucleobase by a different redox-active group (having a different redox potential), and each label should be "readable" in the presence of all the other labels and give a ratiometric signal intensity. Our first generations of redox coding 5,7 suffered from weak and overlapping signals, limited stability and difficult incorpor-ation of some labels into DNA. More recently, we reported the first orthogonal and ratiometric set of two reducible labels (nitrophenyl and benzofurazane) useful for electrochemical minisequencing of short DNA stretches. 9 However, we need a set of at least four fully orthogonal labels for diagnostic applications and, given the rather narrow window of potentials available for electrochemical analysis of DNA, we should be able to combine some reducible and some oxidizable labels. While previously developed reducible labels, i.e. nitrophenyl, 5 benzofurazane 9 or azidophenyl, 10 give strong signals due to multi-electron reductions, most of the oxidizable labels 3,14 typically gave weak signals due to one-electron oxidation. Therefore, there is a great need for other oxidizable labels for DNA and we turned our attention to phenothiazine (PT).
PT derivatives are known redox-active molecules giving either two waves of single electron oxidations 15 or one strong two-electron oxidation. 16,17 They have been extensively used as redox and fluorescent label for polymers and other compounds. 18,19 Some PT derivatives have also been used for labelling of nucleic acids, mostly for photosensitizing 20 or chargetransfer study. [21][22][23][24] One example of a PT-linked dNTP (through a flexible non-conjugate amide linker) was shown in a recent work 12 for polymerase labelling of DNA and for redox coding in a single-nucleotide polymorphism study. However, the PTlinked dNTP was not fully characterized and the biochemistry of the enzymatic incorporation was not reported either. 12 Therefore, we decided to prepare a set of PTZ-modified nucleosides and dNTPs either directly linked or tethered through a conjugate acetylene linker and characterize them chemically, † Electronic supplementary information (ESI) available: Additional gels and copies of spectra. See DOI: 10.1039/c7ob01439b a Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, CZ-16610 Prague 6, Czech Republic b electrochemically and biochemically in order to develop them as useful redox labels for DNA.

Synthesis
From previous studies it is well known that dNTPs bearing modifications at position 5 of pyrimidines or at position 7 of 7 deazapurines are good substrates for DNA polymerases 25,26 and some dNTPs bearing less bulky aryl or alkynyl groups can even be better substrates than natural dNTPs. [27][28][29] Therefore, we selected 2′-deoxycytidine linked through position 5 and 2′-deoxy-7-deazaadenine linked through position 7 for attachment of PT either directly via s single bond or through a conjugate ethynyl tether. The syntheses started from commercial 2′-deoxy-5-iodocytidine (dC I ) or from well-known 2′-deoxy-7iodo-7-deazaadenosine (dA I ), 30 which were triphosphorylated to the corresponding dNTPs (dC I TP and dA I TP). 5 The Suzuki-Miyaura cross-coupling of iodinated nucleosides (dC I or dA I ) with PTZ-linked pinacolborane 1 under aqueous conditions 31 in the presence of Pd(OAc) 2 and tris(3-sulfonatophenyl)phosphine (TPPTS) gave the arylated nucleosides (dC PT and dA PT ) in good yields of 75% or 96%, respectively (Scheme 1, Table 1). The Sonogashira cross-coupling reactions of dC I or dA I with ethynylphenothiazine 2 were performed in the presence of Pd (PPh 3 ) 2 Cl 2 and CuI in DMF to give labelled nucleosides dC EPT and dA EPT in similarly good yields (76% and 93%). The Suzuki-Miyaura reactions of iodinated dN I TPs with PT-Bpin (1) under aqueous conditions gave the desired PT-linked dN PT TPs (dC PT TP in 53% and dA PT TP in 68%), whereas the aqueous Sonogashira reactions of dN I TPs with EPT (2) afforded the PT-acetylene-linked dN EPT TPs (dC EPT TP in 48% and dA EPT TP in 49%). Taking into account partial hydrolysis of the dNTPs during the reaction and isolation, these yields are very good. Alternatively, the dN EPT TPs were prepared by triphosphorylation of the corresponding nucleosides dN EPT in moderate yields (43 and 45%). In all cases the PT-labelled dN PT TPs or dN EPT TPs were isolated in ca. 30-60 mg amounts by HPLC and fully characterized.

Biochemistry
The four new dNTPs (dA PT TP, dC PT TP, dA EPT TP and dC EPT TP) were then tested as substrates for DNA polymerases in primer extension experiments (PEX) using KOD XL, Vento(exo-) and Pwo polymerases (for sequences of primers, templates and products, see Table 2). The first experiment was a single nucleotide incorporation of each of the dN X TPs into a 15-nt primer followed by three natural dG using temp A or temp C templates (Fig. 1). In all cases, we obtained fully extended products as shown by the analysis by polyacrylamide gel electrophoresis (PAGE). All the products were also characterized by MALDI-TOF (see Table 3).
The kinetic experiments (see Fig. S1-S4 in the ESI †) in the presence of KOD XL polymerase showed that the modified dN X TPs were incorporated at a slightly slower rate compared to dATP or dCTP but all of them were fully incorporated within max. 5-10 minutes. The slowest extension proceeded with dA EPT TP.
Then, each of the modified dN X TPs was incorporated into a longer 31-mer oligonucleotide (ON) using temp rnd16 designed to encode for four incorporations at each of the four nucleotides and four modifications if one of the dNTPs is modified. Furthermore, the PEX experiments gave full-length products in a For magnetoseparation of the extended primer strands, the templates were 5′-end biotinylated. b Primer sequences in the template are underlined. c 6-Carboxyfluorescein-(6-FAM-) labeled primer rnd was used for visualization in PEX experiments and TDT elongation. the presence of all tested polymerases (Fig. 2). The PEX products have slightly different electrophoretic mobility but their correct length and sequence was verified by MALDI (Table 3). In order to test the possibility of use of the PT-labelled nucleotides for redox coding of DNA bases, we synthesized ON containing redox-labelled A and in combination with redoxlabelled C or T (A PT + C EBF or A MOP + U NO2 ). The PEX experiments combining the use of dA PT TP with previously reported dC EBF TP 9 or dU NO2 TP 5 were also successful to give full-length ONs bearing four A PT labels in combination with either four C EBF or four U NO2 modifications ( Fig. 3 and 4). These ON products were also characterized by MALDI (Table 3).
We also tried the use of the dN XPT TPs in PCR amplifications, but the PCR reactions using KOD XL did not give a significant product (see Fig. S6 in ESI †). Since the dN XPT TPs are reasonably good substrates for these polymerases in the PEX experiments, the problem is probably in the limited ability of the enzymes to read through the PT-modified templates. We also tested PCR reactions with mixtures of natural dATP and    modified dA PT TP or dA EPT TP, and only the experiment with 50% of dA PT TP in the presence of natural dATP gave a significant product of amplification (see Fig. S7 and S8 †). Next, we tested whether the new dN XPT TPs could be used in the nicking enzyme amplification reaction (NEAR), which is used for enzymatic synthesis of short single-stranded ONs. 32 The principle of the method is that a PEX reaction is performed in the presence of Vent (exo-) polymerase and a nicking endonuclease (in our case Nt·BstNBI), which cleaves the extended strand to release the modified ssON. 33 We tested the NEAR amplification using three different templates designed for the synthesis of ON containing either one, two or four modifications (10-mer ON containing either one or two modifications or 16-mer ON containing four modifications).  Fig. S5 in the ESI †) shows the outcome of these experiments. The ssONs containing one modification were obtained efficiently in case of each of the four dN XPT TPs, whereas the products containing two or four labels were still obtained (though in lower yield).
The last enzymatic method tested in this work was the nontemplated 3′-tail labelling by terminal deoxynucleotidyl transferase (TdT). 34,35 Fig. 6 shows that the TdT-catalyzed elongation of the primer using dA PT TP gave almost the perfect product of single nucleotide extension, whereas the other dN XPT TPs were less efficient. The single nucleotide extension can advantageously be used for specific incorporation of one PT-redox label at the 3′-end of ON analytes.

Fluorescence
In order to verify the possible applications in fluorescence labelling of DNA, we measured the absorption and emission spectra of PT-modified nucleosides and triphosphates. Table 4 shows that the nucleosides in ethanol showed significant fluorescence with emission maxima at 462-476 nm, whereas the fluorescence of dN XPT TPs in water was negligible. The fluorescence of the PEX product ON rnd16 A EPT containing four A EPT modifications was moderate (see Fig. S13 in ESI †).

Electrochemistry
Electrochemical properties of PT-and EPT-deoxynucleoside conjugates (dN PT or dN EPT , respectively) and of PT-modified oligonucleotides were studied by means of cyclic (CV) and square-wave (SWV) voltammetry at the basal-plane pyrolytic graphite electrode (PGE). First, we compared electrochemical oxidation of free PT with individual modified nucleosides ( Fig. 7, 8, S36 and S37 †). PT alone yielded two oxidation signals in the potential range between +0.5 and +1.0 V. According to the literature, 15 the first signal (denominated here as peak PT ox1 , around +0.6 V, Fig. 7a) corresponds to oneelectron reversible oxidation to form a radical cation at the PT nitrogen atom. The second one-electron oxidation step (reflected in peak PT ox2 around +0.9 V) is irreversible and results in the formation of the corresponding sulfoxide. Cyclic voltammograms of PT (Fig. 7a) accorded with the above mechanism: when the anodic CV scan was turned back at +0.8 V, i.e., without applying potentials sufficiently positive for the sulfoxide formation, a cathodic counter peak to the peak PT ox1 was observed, giving evidence about the reversibility of the first oxidation step. On the other hand, when the CV scan was turned "after" the peak PT ox2 , the cathodic signal disappeared in agreement with the overall irreversibility of the two-step PT oxidation. Another irreversible anodic signal was observed Fig. 5 Incorporation of modified dNTPs in NEAR using Nick_1A, Nick_1C and Nick_4A(4C) templates. L: DNA ladder; A PT : product of NEAR with dA PT TP, dCTP, dGTP, dTTP; A EPT : product of NEAR with dA EPT TP, dCTP, dGTP, dTTP, C PT : product of NEAR with dC PT TP, dATP, dGTP, dTTP, C EPT : product of NEAR with dC EPT TP, dATP, dGTP, dTTP. Fig. 6 TdT-catalyzed DNA chain elongation. Pr: primer rnd ; S: standard (PEX product of temp termA with dATP or temp termC with dCTP); A+, A PT and A EPT : products of primer rnd elongation using terminal transferase and either dATP, dA PT TP or dA EPT TP respectively; C+, C PT and C EPT : products of primer rnd elongation using terminal transferase and either dCTP, dC PT TP or dC EPT TP respectively (for the full gel image, see Fig. S9 and S10 †). around +1.3 V, suggesting further oxidation of the PT sulfoxide (Fig. 7a). Results of SWV measurements (Fig. 8), including inspection of the forward and backward components of the SWV current to evidence the (ir)reversibility of the processes (see Fig. S37 †), accorded with CV data. Basically, CVs and SWVs of dA PT and dC PT displayed signals characteristic for the PT moiety ( Fig. 7b and c, S36 †); in the case of the conjugates only the peak PT ox1 was apparently split into two distinct signals. The reversibility of PT oxidation was retained when the CV scan was turned after the more positive one of them (at +0.80 V), but lost when the scan was turned at +1.0 V. The signals around +0.85-+0.90 V were thus identified as the peak PT ox2 corresponding to the second irreversible oxidation step. In addition to the PT-specific signals, the dC PT conjugate yielded a well-developed, irreversible peak close to +1.2 V. An analogous peak was produced by dC EPT but not by either of the dA PT or dA EPT conjugates (see CVs in Fig. S36 † and SWVs in Fig. 8), suggesting involvement of the cytosine moiety in the corresponding electrode process. The absence of a base-specific signal on the voltammograms of dA PT or dA EPT may be rather surprising as 7-deazaadenine was reported to undergo irreversible electrochemical oxidation at the PGE both in its underivatized form (around +1.1 V) 36 and in conjugates with, e.g., trisbipyridine complexes of Os or Ru (between +0.9 and +1.0 V). 6 An explanation of the absence of a distinct 7-deazaA peak on voltammograms of its PT conjugates may lie in its overlap with a PT signal, possibly peak PT ox2 . A more extensive electrochemical study, which is out of the scope of this report, will be required to understand the electrooxidation processes of (E)PT and nucleobase moieties in the dN PT conjugates in more detail.
In the next experiments, we used SWV to measure the electrochemical responses of ONs with incorporated PT-or EPT-labelled nucleotides. In the first series, we analyzed short 10-or 16-mer ON-products of NEAR bearing 1, 2 or 4 dA PT conjugates (for sequences of templates see Table 2). Control unmodified NEAR products (blue curves in Fig. 9a and b, shown for ON Nick_1C ) yielded well-developed signals of the oxidation of natural purine bases ( peaks G ox and A ox ), the intensities of which reflected relative contents of G and A in the given ON (see sequences in Table 2). For the dA PT -modified ONs, peaks corresponding to individual oxidation steps of PT were observed; even for the ON nick_1A A PT bearing a single PT moiety, small but distinct peaks PT ox1 , PT ox2 and PT ox3 were detected. Their potentials were in general shifted to less positive values, as compared to the corresponding peak potentials measured with the nucleosides (see Table S1 †). The first two peaks occurred at potentials sufficiently less positive than the potentials of the purine oxidation signals, making it possible to measure them independently, while peak PT ox3 was overlapping with peak A ox and its observation was possible only owing to the fact that the dA PT -modified NEAR products did not contain any natural adenine residues (unlike the PEX products that always contained adenines, see Fig. 10). Following the  intensities of the measured signals in dependence on the number of PT moieties incorporated, one can see a significantly non-linear behavior, with a large difference between NEAR products containing 1 and 2 PT labels (black and green curve in Fig. 9a) and very similar peak heights obtained for 2 and 4 PT moieties (green and red curve in Fig. 9a). Nevertheless, it should be noted that the latter ON nick_4A A PT was 16-mer (while ON nick_1A A PT and ON nick_2A A PT were 10-mers), and thus the relative content of PT in the 16-mer was higher only by a factor of 1.25 (instead of 2), compared to ON nick_2A A PT . Thus, the small difference between 4 and 2 PT moieties can be ascribed primarily to this fact. On the other hand, the difference between the decanucleotides may be due to a strong effect of the PT moieties on interactions of the ON with the PGE surface, such as preferential adsorption of the PT tags.
A different behavior was observed with analogous NEAR products labelled with dA EPT (Fig. 9b). While the peak PT ox1 produced by these modified ONs was similar to that in the case of ONs bearing dA PT , well separated from other signals and exhibiting similar changes in peak heights depending on the number of EPT conjugates incorporated, peak PT ox2 was shifted to a more positive potential, making it overlap with peak G ox . As a consequence, it was not possible to differentiate between these two signals and measure their intensities. Again, involvement of the ethynyl linker in the electrooxidation processes of EPT-modified DNA is a matter of specialized study and will be published elsewhere. Inspection of the SWV current components (Fig. 9c) confirmed the reversibility of the electrode process giving rise to the peak PT ox1 for the (E)PT moiety incorporated into DNA, and the irreversibility of the more negative signals produced by either the PT or the nucleobases. Based on the above observations, for the next experiments we chose the dA PT conjugates producing distinct PTspecific and natural base-specific signals without mutual overlaps.
To test applicability of PT as a label in a "multicolor" redox coding system, we prepared PEX products modified with dA PT combined with either of the following reducible labels, ethynyl benzofurazane (as dC EBF ) and nitrophenyl (as dU NO2 ). Again, the PT labels yielded characteristic oxidation peaks PT ox1 and PT ox2 on SWVs measured in the anodic direction, where the purine-specific peaks were also detected (Fig. 10). EBF and PhNO 2 yielded, in agreement with previous literature, 5,9 specific reduction signals around −0.95 V and −0.55 V, respectively. When the measurements were performed with ONs freshly adsorbed at the electrode and the initial potential (E i ) was set at 0.0 V, no interfering anodic signals of ONs modified with BF or PhNO 2 were detected in the anodic scan. Similarly, no interfering reduction peak originating from PT labels was observed in the cathodic scans starting from 0.0 V (Fig. 10). Therefore, all three labels could easily be differentiated and determined independently by their peak potentials and/or by the direction of electron flow.

Conclusions
We developed the synthesis of 2′-deoxycytidine and 2′-deoxy-7deazaadenosine nucleosides and dNTPs bearing phenothiazine linked directly or through an acetylene tether at position 5 of cytosine or at position 7 of 7-deazaadenine. The dN XPT TPs  were good substrates for DNA polymerases and served as building blocks for enzymatic synthesis of modified ON and DNA through PEX and NEAR (but did not work well in PCR amplification). Interestingly, dA PT TP and dC PT TP gave efficient and selective non-templated single-nucleotide extension catalyzed by TdT. The nucleosides showed interesting fluorescence in EtOH but the nucleotides and EPT-modified DNA showed low fluorescence in water. The phenothiazine moiety is a useful redox label for nucleosides and DNA, giving two anodic peaks of PT oxidation, which are analytically useful. The directly linked A PT is more useful than the ethynyl-linked A EPT base where the second oxidation peak overlaps with oxidation of guanine. The PT-redox label is orthogonal to previously reported benzofurazane 9 and nitrophenyl labels 5 and can be used for multipotential redox coding of DNA bases. 12 The characteristic reversibility of the first PT signal and the irreversibility of the two-step oxidation process can be exploited for unambiguous identification of the PT label among other oxidizable moieties. Thus, although the presence of two oxidation peaks apparently limits the potential space for a combination of more oxidizable DNA labels, using proper measuring parameters can help one to attain satisfactory selectivity even in the case of overlapping primary oxidation signals. On the other hand, further studies of other alternative oxidizable labels are still needed to introduce the last (fourth) redox label in prospective four-label coding.

Experimental
Chemicals, synthetic oligodeoxyribonucleotides, and enzymes were purchased from commercial suppliers and were used without further purification. Purification of nucleoside triphosphates was performed using HPLC on a column packed with 10 μm C18 reversed phase. NMR spectra were measured on a 500 MHz ( 1 H at 500.0 MHz, 13 C at 125.7 MHz and 31 P at 202.3 MHz) or a 600 MHz ( 1 H at 600.1 MHz and 13 C at 150.9 MHz) NMR spectrometer in DMSO-d 6 or D 2 O solutions at 25°C. Chemical shifts (in ppm, δ scale) were referenced to the residual solvent signal in 1 H spectra (δ((CHD 2 )SO(CD 3 )) = 2.5 ppm) or to the solvent signal in 13 C spectra (δ((CD 3 ) 2 SO) = 39.7 ppm). 1,4-Dioxane was used as an internal standard for D 2 O solutions (3.75 ppm for 1 H and 69.3 ppm for 13 C). Coupling constants ( J) are given in Hz. The complete assignment of 1 H and 13 C signals was performed by an analysis of the correlated homonuclear H,H-COSY, and heteronuclear H, C-HSQC and H,C-HMBC spectra. Known starting compounds dA I TP, dC I TP, dU NO2 TP, 5 dC EBF TP, 9 EPT, 37 and PT-Bpin 38 were prepared by published procedures.
Synthesis of modified nucleosides -Suzuki-Miyaura cross-coupling (dC PT , dA PT ). Method A: A 1 : 1 mixture of H 2 O-CH 3 CN (2 mL) was added through a septum to an argon-purged flask containing a halogenated nucleoside dN I (1 equiv.), PT-Bpin (2 equiv.) and Cs 2 CO 3 (3 equiv.). In a separate flask, Pd(OAc) 2 (10 mol%), and TPPTS (2.5 equiv. with respect to Pd) were combined, the flask was evacuated and purged with argon, and then a 1 : 1 mixture of H 2 O-CH 3 CN (1 mL) was added. This catalyst solution was injected into the reaction mixture, which was then stirred at 50°C for 40 min until complete consumption of the starting material, and then evaporated in vacuo. The products were purified by silica gel column chromatography using chloroform/methanol (100 : 0 to 90 : 10) as the eluent.
Synthesis of modified nucleotides triphosphates -Suzuki-Miyaura cross-coupling (dC PT TP or dA PT TP). Method C: A 1 : 1 mixture of H 2 O-CH 3 CN (1 mL) was added through a septum to an argonpurged flask containing a halogenated nucleotide dN I TP (1 equiv.), PT-Bpin (2 equiv.) and Cs 2 CO 3 (3 equiv.). In a separate flask, Pd(OAc) 2 (10 mol%), and TPPTS (2.5 equiv. with respect to Pd) were combined, the flask was evacuated and purged with argon, and then a 1 : 1 mixture of H 2 O-CH 3 CN (0.5 mL) was added. This catalyst solution was injected into the reaction mixture, which was then stirred at 50°C for 40 min until complete consumption of the starting material, and then evaporated in vacuo. The product was isolated from the crude reaction mixture by HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H 2 O to 0.1 M TEAB in H 2 O-MeOH (1 : 1) as the eluent. Several co-distillations with water and conversion to sodium salt form (Dowex 50WX8 in Na + cycle) followed by freeze-drying from water gave the solid product.
Synthesis of modified nucleotide triphosphates -Sonogashira cross-coupling (dC EPT TP or dA EPT TP). Method D: A 1 : 1 mixture of H 2 O-CH 3 CN (2 mL) was added through a septum to an argonpurged flask containing a halogenated nucleotide dN I TP (1 equiv.), PTE (1.5 equiv.), CuI (10 mol%), and (iPr) 2 EtN (10 equiv.). In a separate flask, Pd(OAc) 2 (5 mol%), and TPPTS (2.5 equiv. with respect to Pd) were combined, the flask was evacuated and purged with argon, and then a 1 : 1 mixture of H 2 O-CH 3 CN (0.5 mL) was added. This catalyst solution was injected into the reaction mixture, which was then stirred at 75°C for 1 h until complete consumption of the starting material, and then evaporated in vacuo. The product was isolated from the crude reaction mixture by HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H 2 O to 0.1 M TEAB in H 2 O-MeOH (1 : 1) as the eluent. Several co-distillations with water and conversion to sodium salt form (Dowex 50WX8 in Na + cycle) followed by freeze-drying from water gave solid product.
Synthesis of modified nucleosides triphosphatestriphosphorylation (dC EPT TP or dA EPT TP). Method E: POCl 3 (1.2 equiv.) in PO(OMe) 3 (1 ml) was added through a septum to an argonpurged flask containing modified nucleosides dN EPT (1 equiv.). The reaction mixture was then stirred at 0°C for 3 h until complete consumption of the starting material. Then an ice-cooled solution of (NHBu 3 ) 2 H 2 P 2 O 7 (5 equiv.) and Bu 3 N (4.2 equiv.) in dry DMF (2 ml) was added and the mixture was stirred at 0°C for another 1.5 h. The reaction was quenched by addition of 2 M aqueous TEAB (2 ml), the solvents were evaporated in vacuo and the residue was co-distilled with water three times. The product was isolated by HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H 2 O to 0.1 M TEAB in H 2 O-MeOH (1 : 1) as the eluent. Several co-distillations with water and conversion to the sodium salt form (Dowex 50WX8 in Na + cycle) followed by freeze-drying from water gave the solid product.
dC PT : Compound dC PT was prepared from dC I according to the general procedure (Method A). The product was isolated as a white solid (59 mg, 75%); m.p. 145°C; 1  In the case of Pwo polymerase, 1.25 U of the enzyme, 0.25 μl of dGTP (4 mM) and 2 μl of either dATP or dA XTP TP (4 mM) were used. The reaction mixture was incubated for 40 minutes at 60°C. The PEX reaction was stopped by the addition of PAGE stop solution [20 μL, formamide (80%, v/v), EDTA (20 mM), bromophenol blue (0.025%, w/v), xylene cyanol (0.025%, w/v) and Milli-Q water] and heated for 2 minutes at 95°C. Samples were analyzed by use of 12.5% denaturing polyacrylamide gel electrophoresis (1 h, 50°C) and visualized by fluorescence imaging. Method B: PEX reactions with temp tempC were performed in the same way as described for temp tempA except either dCTP or dC XTP TP (4 mM, 1 μl for KOD XL and VENT (exo-) polymerases and 2 μl for Pwo polymerase) were used.