Towards genetic code expansion

4.4. Application of the pyrazole ligandoside

79 Scheme 4-9 Synthesis of triphosphate 22: a) Condition: 1) bis-tetrabutylammonium pyrophosphate, tributylamine, 2-chloro-1,3,2-benzodioxaphosphorin-4-one 23, DMF; 2) I2, pyridine, H2O, RP-HPLC purification, 11%. b) Mechanism of the triphosphate addition.

Figure 4-22 Characterization of triphosphate 22: a) ESI+, C20H35N3O13P3+ [M+H⁺+TEA], calc.

618.1383, found 618.1380, C26H50N4O13P3+ [M+H⁺+2TEA], calc. 719.2587, found 719.2585; and b) ESI- of 22, C14H18N2O13P3- [M-H⁺], calc. 515.0022, found 515.0017; c) ³¹P NMR of 22.

After HPLC purification, the triphosphate product was characterized by ³¹P-NMR and ESI-Mass spectrum. ESI-Mass in the positive charge mode showed two high peaks at 618.1380 and 719.2585 (Figure 4-22a), which equal to the mass of the triphosphate plus one and two molecules of triethylamine (calc. 618.1383 and 719.2587), while ESI-Mass in the negative detection mode gave the exact triphosphate molecular weight of 515.0017 (calc. 515.0022, Figure 4-22b). ³¹P-NMR showed two set of peaks, one set of peaks at around -11.3 ppm, and a set of the triplet at -23.6 ppm (Figure 4-22c). α- and γ- phosphorus atoms were crowded into the low field, leaving the β-phosphorus atom at

high field. Thus, we affirmed that the pyrazole triphosphate was obtained.

As a template, a 30 mer oligonucleotide (ODN 11a) with Pz at the 24^th position from 3’

terminus was synthesized using solid-phase synthesis (Table 4-1). A 5’-fluorescein- labeled 23 mer oligonucleotide was designed as a primer. Certainly, the primer can be labeled with radioactive ³²P using polynucleotide kinase for direct detection.

4.4.1.2. Replication with pyrazole triphosphate

With all the three components in hand, primer extension experiments were performed.

First, a collection of polymerases were screened to find out which allowed the incorporation of dPzTP (22) in the 24^th position of the primer. Taq, Q5, Pwo, Phusion, T4, T7, Φ29, KF (exo^-), Bst Pol large fragment, Deep Vent and Therminator were tried. In the reaction system, an excessive amount of template (2 μM) and primer (1.5 μM) as well as 100-fold excess of 22 (200 μM) was employed. Experiment with and without Cu²⁺ (200 μM) at both 37°C and 50°C were performed.

Deep vent (exo-) polymerase and Therminator polymerase, which belong to B family polymerase and which are isolated from archaea, are able to incorporate dPzTP with the primer opposite a Pz base on the template. Deep Vent achieved the goal at 50°C in 24 h, while Therminator DNA Polymerase succeeded in 2 h at 37°C or in 5 min at 75°C. It found out to be the best polymerase able to incorporate the ligandoside 22.

Considering the incorporation efficiency and temperature consistency, 75°C was fixed as the reaction temperature for all further investigations.

Therminator polymerase is a mutant variant of the 9^oN exo^- polymerase (Thermococcus species 9^oN-7), in which the alanine 485 has been replaced by a leucine residue. This provides the polymerase with an enhanced ability to incorporate modified substrates, e.g. dideoxynucleotides, ribonucleotides, and acyclic- nucleotides.^310-313

Next, we studied the dPzTP incorporation efficiency under different conditions. At 75°C within 5 min, the incorporation was achieved to a full extend, independent of

81

present of copper or magnesium ions (Figure 4-23).

Figure 4-23 PAGE assay shows dPzTP incorporation and extension. Condition: 2 μM template 11a, 1.5 μM primer 11b, 200 μM dPzTP 22, 200 μM each dNTP, 1 U Therminator, 1× Thermopol buffer, 10 mM MgSO4, the final volume of 20 μL.

The extension towards full length seemed more challenging than the dPzTP incorporation. For polymerase Therminator, at 50°C, the yield of full length was less than 50%. When the reaction temperature was raised to 60°C with the additive Cu²⁺

(200 μM) or Mg²⁺ (10 μM), the yield reached a maximum value of 65% in 9 h and then further decayed. At 75°C, the reaction could be finished in 2 h with additional Cu²⁺ and Mg²⁺ (Figure 4-23). The co-enzyme Mg²⁺ is a requisite for the extension, especially at the high temperature. Extension experiments with variable Cu²⁺ concentration showed that 200 μM of copper ions give the best condition.

Further studies showed that the yield reached 80% in 90 min (Figure 4-24). Due to the dissatisfied efficiency, PCR amplification is not practical because the failed synthesis will be accumulated exponentially in PCR cycles.

After PAGE analysis, the corresponding gel band was cut out and the DNA contatining material was dissolved and desalted. MALDI-TOF analysisi gave a clear signal (Figure 4-27), corresponding to the 31 mer without the fluorescein tag but with a Pz base at the 3’-overhang. We speculated this is due to the lack of a 3’-5’ exonuclease activity of the polymerase. We think hat the loss of 5’-fluorescein is happening during work up.

Figure 4-24 Time dependent study of dPzTP extension: a) PAGE assay shows the primer extends to full length in 2 h at 75°C after dPzTP full incorporated in 5 min at 75°C; b) Plot of the percentage of the full-length product by the integration from PAGE assay. Data are mean ± SD of three replicates.

Condition: 2 μM template 11a, 1.5 μM primer 11b extended with 22, 200 μM each dNTP, 1 U Therminator, 1× Thermopol buffer, 10 mM MgSO4, the final volume of 20 μL.

To study if the Pz base was orthogonal to the natural bases, we added four natural triphosphates to Pz-containing template ODN 11a, and reversely, dPzTP to four different templates with A/C/G/T at the 24^th position (ODN 11c/d/e/f).

At 75°C for 5 min, all the four natural bases were incorporated into the primer complementary to the Pz. However, no 25 mer product was visible showing again that elongation was blocked. dPzTP could also be complementarily incorporated opposite the natural bases on the template. Extension afterward was not discernible either (Figure 4-25), which could be good news for the plasmid replication experiments.

83 Figure 4-25 PAGE assay shows incorporation of dPzTP on 11c/d/e/f with primer 11b and incorporation of dATP/dCTP/dGTP/dTTP with template 11a on primer 11b. Condition: 2 μM template, 1.5 μM primer 11b, 200 μM dPzTP or dNTP, 1 U Therminator, 1× Thermopol buffer, 10 mM MgSO4, the final volume of 20 μL.

Figure 4-26 PAGE assay shows incorporation of dPzTP mixed with dNTP extend with template 11a on primer 11b. Condition: 2 μM template, 1.5 μM primer 11b, 200 μM dPzTP and dNTP, 1 U Therminator, 1× Thermopol buffer, 10 mM MgSO4, the final volume of 20 μL.

In all furture studies we fixed the reaction conditions (75°C, 2 h, 1U Therminator DNA polymerase, 200 μM Cu²⁺ and additive Mg²⁺) constant and investigate the effect of the different ratio of dPzTP and dNTP was investigated (Figure 4-26). The best result were obtained of a ratio of 1/1, i.e. 200 μM dPzTP, and 200 μM dNTP. Although the primary product was a 31 mer oligonucleotide, the yield was lower than when it was done in two separate steps. MALDI-TOF failed to give a signal after analysis of band cut out of the the PAGE gel. To solve the problem, we cut the gel and extracted the DNA with phenol and chloroform, desalted, and measured again. The spectrum, shown in Figure 4-27, indicated formation of the same product formed in two steps.

Figure 4-27 MALDI-TOF spectrum for a) extended primer in two steps, found 9567.3; b) extended primer in one pot, found 9569.5; calc. 9566.7, corresponding to the complementary template strand with Pz at 3’-overhang without 5’-fluorescein, listed in c).

In summary, we showed the possibility to incorporate the dPzTP into a primer opposite a templating dPz base using the Therminator polymerase. Even for such a powerful polymerase, neither the efficiency nor the fidelity was sufficient to continue with PCR experiments.