Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.


Introduction
The selective scission of the amide bonds of proteins and peptides is of crucial importance for numerous biochemical and biotechnological applications 1 and plays a decisive role in physiological processes. 2,3 Considering the inertness of the peptide bond (t 1/2 nonenz ∼ 500 years), 4,5 proficient proteolytic enzymes with high catalytic efficiencies (k cat /K M ) need to be used in order to degrade proteins or peptides into smaller fragments or for promoting site-specific cleavage. However, in terms of practical applications, proteolytic enzymes are not always applicable since they often lead to the generation of rather short fragmentation products, lack sequence-specificity, and cleave at multiple sites. Thus, artificial proteases based on synthetic reagents that could favor the selective cleavage of proteins under mild conditions are highly sought after. In this context, transition metal complexes were suggested as prime candidates to serve as biomimetic chemical proteases since they are often more effective than organic residues at promoting the scission of inert bonds. 1,6,7 Moreover, metal centers are often combined with reagents such as β-cyclodextrins and porphyrins that facilitate the recognition and the binding to the target site and thus reach appreciable catalytic efficiencies for the scission of amide linkages in peptides and even proteins. [8][9][10][11] However, even though some of these artificial, transition metal-based proteases achieve cleavage with multiple turnover 12 and with some site selectivity, 8,13 they suffer from severe drawbacks such as side-reactions, low k cat values, and in general perform rather poorly when compared to protein enzymes.
DNA enzymes or DNAzymes have emerged as a new and prominent class of biomolecular catalysts, and have been selected to accelerate an increasing number of chemical processes. [14][15][16][17][18] Indeed, application of SELEX and related combinatorial methods of in vitro selection 19,20 resulted in the identification of nucleic acid enzymes catalyzing an abundance of reactions ranging from the formation of C-C, [21][22][23][24] C-N, 25 C-S, 26 and P-O, 27,28 bonds to the scission of ribophosphodiester linkages. 29,30 Surprisingly, the scission of amide linkages is a reaction that has eluded catalytic nucleic acids so far. 31 Indeed, in an early effort towards this goal, Joyce et al. initially reported on the selection of a ribozyme capable of cleaving a substrate containing an embedded 3′-NH-C(O)-CH 2 -5′ linkage 32 but later realized that the reaction proceeded through a different mechanism leading to scission of a phosphodiester bond adjacent to the target site rather than the intended amide bond. 33 Similarly, Silverman et al. set out to select a DNAzyme capable of the selective hydrolysis of amide bonds of a tripeptide sequence, 34 which culminated in the serendipitous discovery of a class of very potent DNA-cleaving DNAzymes. 35,36 Recently, blending of the chemical functionalities found in the active sites of enzymes together with tools of in vitro selection resulted in the identification of potent DNAzymes. 37,38 In particular, this approach allowed for the development of highly functionalized DNA-based RNase A mimics that operate either in the presence of cofactors [39][40][41] or in an M 2+ -independent regime. [42][43][44][45][46][47] Moreover, all the functional groups encompassed in these modified DNAzymes seem to act in synergy and in a cooperative manner, one of the fundaments in the design of de novo enzyme mimics and catalysts in general. 48 Finally, this methodology is applicable to other reactions than the cleavage of ribophosphodiester bonds 25,49 provided that the functional groups can easily be introduced into DNA, i.e. that the modified nucleoside triphosphates (dN*TPs) are compatible with methods of in vitro selection.
In this context, the enzymatic polymerization of dN*TPs has advanced as a versatile platform for the inclusion of functional groups into nucleic acids. 50 Indeed, dN*TPs have been used to introduce a myriad of functionalities including amino acids, 51-57 boronic acids, 58,59 ligands for transition metals, 60-65 thiols, 66-68 diamondoid-like residues, 69 bile acids, 70 side chains capable of organocatalysis, 71 and even oligonucleotides. 72 Herein, I report on the synthesis and biochemical characterization of modified dN*TPs bearing the side chains found in the active site of serine proteases. 31,73,74 Indeed, dA Hs TP (7-propargylamido-histamine-dA) 1, dU POH TP (5-pentynol-dU) 2, and dC Val TP (5-valeric acid-dC) 3 ( Fig. 1) are equipped with imidazole, hydroxyl, and carboxylic acid residues, respectively, that are reminiscent of the side chains of the amino acids His, Ser, and Asp that form the catalytic triad of serine proteases. These modified dN*TPs are good substrates for a variety of DNA polymerases in the context of primer extension reactions. In addition, when used in conjunction with the terminal deoxynucleotidyl transferase (TdT), efficient tailing reactions that rival those of the natural counterparts could be observed. Finally, these modified dN*TPs were readily incorporated into DNA by the polymerase chain reaction (PCR), further underscoring their compatibility with in vitro selection techniques.

Synthesis of the modified nucleoside triphosphates
The nucleoside analogues considered herein all bear modifications either at the C5 position of the pyrimidine nucleobase or at the N7 of 7-deaza-2′-deoxyadenosine. Indeed, these anchoring sites were chosen for synthetic purposes (the modifications can easily be introduced via palladiumcatalyzed coupling reactions) and because of their minimal disturbance and impact on substrate acceptance and duplex stability. 53,71,[75][76][77] The adenosine analogue 1 adorned with a histamine residue was obtained in a 6-step sequence that started with the DMTr-protection of the known propargylamino modified 7-deaza-2′-deoxyadenosine 4 (Scheme 1). 53,75,78 After removal of the TFA masking group, nucleoside 6 was converted into derivative 7 in 64% yield under standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated amide bond-formation conditions using the suitably protected carboxylic acid S2 (ESI †). The resulting intermediate 7 was then 3′-O-acetylated under standard conditions and in good yield (84%). Following a global acid-mediated deprotection step (quantitative yield), nucleoside analogue 9 could be converted into the corresponding triphosphate dA Hs TP 1 in acceptable yields (17%) by application of the one-pot four-step triphosphorylation method developed by Ludwig and Eckstein. 79 The synthetic path leading to dU POH TP 2 commenced with the Sonogashira cross-coupling reaction of the DMTr-protected deoxyuridine analogue 11 (which was easily obtained by tritylation of the commercially available 5-iodo-2′-deoxyuridine 10, ESI †) and free 4-pentyn-1-ol as outlined in Scheme 2. After an uneventful acetylation-detritylation sequence, precursor 14 could be converted to the corresponding triphosphate 2 by application of the Ludwig and Eckstein conditions and was obtained in 14% yield after a thorough RP-HPLC purification step. 79 The synthesis of the modified dC*TP analogue 3 proceeded in an analogous manner as highlighted in Scheme 3. Indeed, commercially available 5-iodo-2′-deoxycytidine 15 was converted into the ester functionalized nucleoside 16 in good yield (90%) using a Sonogashira cross-coupling reaction with 4-pentynoic acid methyl ester. 80 The free 5′-hydroxyl residue of

Primer extension reactions
An important prerequisite for modified dN*TPs to be applicable in selection experiments is that they serve as substrates for DNA polymerases in primer extension reactions. 71 Furthermore, not only do primer extension reactions constitute the first step of an in vitro selection experiment, but they represent the main tool for gauging the enzymatic recognition of modified dN*TPs in general. Consequently, I explored the substrate acceptance of the modified dN*TPs 1-3 with different DNA polymerases under primer extension reaction conditions ( Fig. 2 and ESI †). Therefore, a variety of DNA polymerases (Vent (exo − ), Pwo, 9°N m , and the Klenow fragment of E. coli DNA polymerase I) were used in conjunction with the 98-mer long template T1 and the 25-mer primer P1 (see ESI † for the sequence compositions). 70,71 The modified dN*TPs were then investigated as to whether they could act as surrogates for their natural counterparts, either as lone modifications (lanes 1-3, Fig. 2), as combinations of two modified and two natural dNTPs (lanes 4-6, Fig. 2), or in conjunction with the lone natural dGTP (lane 7, Fig. 2). Vent (exo − ) was revealed to be highly proficient at extending primer P1 to full length since no faster running bands were apparent in all the combinations that were investigated. Moreover, 16 dA Hs 1 and 21 dU POH 2 that were incorporated in the nascent chain led to a significant and expected gel retardation as compared to the natural control sequence (compare lanes 1 and 2 with lane 8, Fig. 2). Surprisingly, the inclusion of 18 dC Val 3 had little impact on the gel mobility of the resulting modified DNA (lane 3 vs. lane 8, Fig. 2). Furthermore, besides Vent (exo − ), all the other DNA polymerases that were tested in primer extension reactions showed very high substrate tolerance for all the dN*TPs and led to fully extended products ( Fig. S1-S3, ESI †). Interestingly, the Klenow fragment, a prominent member of the family A polymerases, was as efficient at incorporating the modified dN*TPs as the more tolerant family B polymerases (Fig. S2, ESI †). 53

TdT-mediated polymerization
The terminal deoxynucleotidyl transferase (TdT) is a Co 2+dependent family X DNA polymerase that catalyzes the random and template-independent polymerization of nucleoside triphosphates at the 3′-OH termini of single-stranded oligonucleotides. 83,84 Recently, TdT-mediated reactions using modified dN*TPs have been employed for the generation of 3′-fluorescently labelled oligonucleotides, 85,86 to confer exonuclease stability to specific sequences, 87 and to synthesize ssDNAs containing stretches of nucleotides bearing redox active tags. 88 However, the tailing reaction was never used for the incorporation of nucleotides bearing side chains that potentially could confer catalytic activity to the ensuing modified ssDNA. Consequently, the modified dN*TPs 1-3 were assayed for their capacity to act as substrates in TdT-mediated tailing reactions. Thus, the 5′-32 P-labelled 15 nt long primer P3 (ESI †) was incubated at 37°C for 60 min in the presence of TdT and varying concentrations of the modified dN*TPs (Fig. 3).
The 7-substituted 7-deaza-deoxyadenosine derivative dA Hs TP 1 was revealed to be the best substrate for TdT, since the tailing efficiencies are better than those of its natural counterpart (compare lanes 1 and 4, Fig. 3). Indeed, at a concentration as low as 10 μM, mainly 7-15 additional dA Hs nucleotides were incorporated along with some longer tailed sequences, while the natural dATP led to the appendage of a rather narrow 4-17 nucleotide distribution with little polydisperse longer-sized oligonucleotides. Furthermore, at a concentration of 50 μM several tens of both the dA Hs and dA nucleotides were appended. Moreover, dU POH TP 2 (lanes 2 in Fig. 3) led to the best tailing efficiencies amongst the pyrimidine triphosphates, a tendency that is opposite to that observed with natural dNTPs (vide infra). Indeed, the TdTmediated homopolymerization was rather ineffective at 10 μM since only 2-5 additional dU POH units were appended, but at higher concentrations (>50 μM) only highly modified slowrunning products could be observed. Unlike its natural counterpart, dC Val TP 3 stifles the TdT and only a few additional residues (∼3 to 15 dC Val MPs) are added, even at 200 μM (lanes 3 in Fig. 3).

Polymerase chain reactions (PCR)
An additional and important part of the biochemical characterization of modified dN*TPs is the assessment of their substrate acceptance by DNA polymerases under PCR conditions. Consequently, the dN*TPs 1-3 were evaluated for their capacity to amplify the 98-mer template T1

Organic & Biomolecular Chemistry Paper
In terms of in vitro selections, it is of primordial importance that single-stranded modified DNAs can be transformed into wild-type oligonucleotides that can serve as templates for subsequent rounds of selection. 30,44 Thus, in a different PCR experiment a 5′-phosphorylated 79-mer long template T2 (ESI †) was amplified using the 19-and 20-mer primers P4 and P5 in the presence of Vent (exo − ) and 500 μM dNTPs. The resulting modified dsDNA was then λ-exonuclease digested, and used as a template for a subsequent PCR using natural dNTPs, the same set of primers, and Vent (exo − ) (Fig. S5, ESI †). 71 This experiment clearly underscores the compatibility of the modified dN*TPs 1-3 with methods of in vitro selection since a modified oligonucleotide could serve as a template to regenerate natural DNA (Fig. S5, ESI †).
Finally, in order to assess the fidelity of the PCR amplification with the modified dN*TPs 1-3, a sequencing experiment based on the dideoxynucleoside triphosphate (ddNTP) method developed by Sanger et al. was carried out ( Fig. S6 and S7, respectively, ESI †). 52,89 Briefly, the 5′-phosphorylated template T2 and primer P4 were used in a primer extension reaction with Vent (exo − ). The unmodified template was then λ-exonuclease digested, and the resulting modified ssDNA was used as a template for PCR with each one of the 5′-biotinylated primers P4B and P5B. Following immobilization of the duplexes on streptavidin coated magnetic beads, the unbiotinylated oligonucleotides were eluted with hydroxide washes and then sequenced. Comparison of the sequencing patterns of both the forward and reverse directions ( Fig. S6 and S7, ESI †) clearly reveals that no loss of sequence information occurs upon converting a heavily modified ssDNA into its complementary natural sequence, and that the fidelity of PCR with the modified dN*TPs is high.

Conclusions
Three modified dN*TPs adorned with side-chains reminiscent of the Ser, His, and Asp residues found in the active site of serine proteases could easily be accessed in 5 or 6 steps starting from known and/or commercially available precursors. The functionalities were anchored on the C5 of pyrimidines and the N-7 of 7-deaza-adenosine in order to minimize the destabilization of duplexes and disruption of Watson-Crick base pairing, and concomitantly, to maximize the substrate acceptance. All of the modified dN*TPs were found to be excellent substrates for all four DNA polymerases, including the Klenow fragment of DNA polymerase I, in primer extension reactions. It is noteworthy that the polymerases readily accept and tolerate the presence of the negative charge of the carboxylic acid of dC Val TP 3 and allow for the incorporation of up to 3 consecutive dC Val residues.
Moreover, the substrate acceptance for TdT is heavily dependent on the nature and position of the substituent and followed the order dA Hs TP > dU POH TP > dC Val TP. This trend is opposite to what is observed for natural dNTPs, since TdT utilizes dGTP and dCTP much more efficiently than dTTP and more markedly, dATP. 90 The additional negative charge on dC Val TP might account for its limited substrate acceptance by TdT.
Finally, these modified dN*TPs were shown to be good substrates for the Vent (exo − ) DNA polymerase under PCR conditions. However, unlike what was observed for primer extension reactions, only optimized conditions and the sole Vent (exo − ) DNA polymerase led to clean and robust amplifications. The resulting modified oligonucleotides could serve as a template to regenerate natural DNA, which is an important prerequisite for the use of these dN*TPs in selection experiments. Moreover, PCR amplification using the modified dN*TPs and the subsequent conversion into natural DNA was shown to proceed with high fidelity.
Thus, these three nucleoside triphosphate analogues, equipped with functionalities that are reminiscent of the residues found in the active site of serine proteases, could be shown to be fully compatible with methods of in vitro selection. Consequently, their simultaneous polymerization will considerably increase the chemical landscape that can be covered in the course of a selection experiment for the generation of DNAzymes that can promote the catalytic scission of amide bond linkages, which is a long-standing goal in the field of nucleic acid enzymes. Such an artificial peptidase would be an invaluable synthetic and biochemical tool, and selection for such an enzyme mimic is currently under way.

Experimental part
General method for the Sonogashira coupling reactions The nucleoside was dissolved in dry DMF (5 mL mmol −1 ). CuI (0.1 eq.) and NEt 3 (2 eq.) were then added and the reaction mixture was stirred for 10 min at room temperature. The alkyne (4 eq.) and Pd(PPh 3 ) 4 (0.05 eq.) were then added in turn. The reaction mixture was cooled down to −78°C and thoroughly degassed. After stirring at room temperature overnight, the DMF was removed under reduced pressure. The blackened crude product was then dissolved in CH 2 Cl 2 (50 mL) and washed with brine (50 mL). Following drying (MgSO 4 ) and removal of the solvent in vacuo, the residue was purified by flash column chromatography.

General method for detritylation
The nucleoside analogue was dissolved in dry CH 2 Cl 2 (10 mL) and dichloroacetic acid (0.1%, 0.1 mL) was added. The orangecoloured reaction mixture was stirred at room temperature for 40 min. MeOH (3 mL) was added and the solution was stirred for another 5 min at room temperature. Following removal of the solvent under reduced pressure, the residue was purified by flash column chromatography.

Triphosphorylation of nucleosides
General method for the triphosphorylation of nucleosides. The nucleoside was co-evaporated twice with dry pyridine (1 mL) and then dried under vacuum overnight. After dissolving the nucleoside in dry pyridine (0.2 mL), dry dioxane (0.4 mL) was added, followed by the addition of 2-chloro-1,3,2benzodioxaphosphorin-4-one (1.1 eq.). The reaction mixture was stirred at room temperature for 45 min. A solution of tributylammonium pyrophosphate (1.3 eq.) in dry DMF (170 μL) and tributylamine (58 μL) was then added and the resulting solution was stirred at room temperature for 45 min. Iodine (1.6 eq.) in a mixture of pyridine (980 μL) and water (20 μL) was then added and the resulting dark solution was stirred at room temperature for 30 min. Excess iodine was then quenched by addition of a solution of sodium thiosulfate (10%) and the solvents were removed under reduced pressure (water bath ≤30°C). Water (5 mL) was then added and the mixture was allowed to stand at room temperature for 30 min. Following removal of the various protecting groups (see below) and evaporation of the solvent, the crude triphosphates were precipitated with NaClO 4 in acetone (2%) and then purified by RP-HPLC. The appropriate fractions were freeze-dried and coevaporated several times with water.

HPLC of triphosphates
The purification of the various triphosphates was carried out on a Phenomenex Jupiter semi-preparative RP-HPLC column (see ESI †) at a flow rate of 3.5 mL min −1 , using a triethylammonium bicarbonate (TEAB) buffer system (eluent I: 50 mM TEAB, pH 7.7; eluent II: 50 mM TEAB in CH 3 CN-H 2 O 1 : 1), and one of the programs highlighted in Table 1.
Modified dATP 1 (dA Hs TP). This modified triphosphate was obtained by application of the general triphosphorylation procedure starting from 9 (35 mg, 0.065 mmol). Deprotection was carried out by incubation in aqueous NH 3 (10 mL) at rt for 1.5 h. RP-HPLC purification ( program A, R t = 26.9 min) yielded 1 as its triethylammonium salt ( pale yellow solid, 12 mg, 17%). 1  Modified dCTP 3 (dC Val TP). Analogue 3 was obtained by application of the general triphosphorylation procedure starting from 19 (45 mg, 0.119 mmol). Deprotection: the crude reaction mixture was dissolved in H 2 O (1.69 mL). After cooling to 0°C, NaOH (380 μL, 2.5 M; 0.46 M final concentration) was added and the reaction stirred at 0°C for 5 min. The mixture was then neutralized by adding NaH 2 PO 4 buffer (925 μL; 1 M, pH 1.9) and further diluted by addition of H 2 O (3 mL). The solvent was then removed in vacuo and the residue purified by RP-HPLC using program B (R t = 21.8 min). dC Val TP 3 was obtained as its triethylammonium salt (white solid, 10 mg, 9%). 1   Primer extension experiments. The 5′-32 P-labelled primer P1 (ca. 1 pmol) and 10 pmol of primer P1 were annealed to appropriate template T1 (10 pmol) in the presence of 10× enzyme buffer ( provided by the supplier of the DNA polymerase) by heating to 95°C and then gradually cooling to room temperature (over 30 min). The appropriate DNA polymerase (1 U) was then added to the annealed oligonucleotides mixture at 4°C. Finally, dNTPs (final concentration of 100 μM) were added for a total reaction volume of 20 μL. Following incubation at the optimal temperature for the enzyme, the reactions were quenched by adding stop solution (20 μL; formamide (70%), ethylenediaminetetraacetic acid (EDTA; 50 mM), bromophenol (0.1%), xylene cyanol (0.1%)). The reaction mixtures were subjected to gel electrophoresis in denaturing polyacrylamide gel (15%) containing trisborate-EDTA (TBE) 1× buffer ( pH 8) and urea (7 M). Visualization was performed by phosphorimaging.
Standard conditions for TdT-mediated insertion of modified triphosphates into a DNA primer. A solution containing 7 pmol of primer P3 (ESI †), ∼2 pmol of 5′-32 P-radio-labelled P3, and 4 U of TdT was added to a mixture composed of the appropriate concentration of dNTPs (10, 50, 100 or 200 μM final), reaction buffer, and H 2 O (for a total reaction volume of 10 μL). The reaction mixtures were then incubated at 37°C for 60 min and quenched by addition of 10 μL of loading buffer. The reaction products were then resolved by electrophoresis (PAGE 15%) and visualized by phosphorimager analysis.
Polymerase chain reactions. The PCR reaction mixtures (20 μL total) contained both primers (400 nM each; P1 and P2, see the ESI † for the sequences), template T1 (25 nM), dNTPs (200 μM; both natural and modified), and Vent (exo − ) (1 U) in the reaction buffer (Thermopol buffer) provided by the manufacturer. The PCRs were carried out in a Gene Q Thermal Cycler from Bioconcept and 30 cycles were performed. Each cycle included denaturation at 94°C for 1 min, annealing for 1 min at 55°C, and extension for 1.5 min at 72°C. A final extension step of 5 min at 72°C was included. After the completion of the reactions, 20 μL of 2× sucrose loading buffer was added. All PCR products were analysed by agarose gels (2%) in 1× TBE buffer, containing ethidium bromide. The gels were visualized by phosphorimager analysis.