Recognition of 8-Oxo-2′-deoxyguanosine in DNA Using the Triphosphate of 2′-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2′-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2′-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.


Introduction
DNA and nucleotides in living cells are constantly at risk of damage.This can be caused by external factors, such as ultraviolet radiation, or internal factors, such as reactive oxygen species (ROS), both of which are unavoidable in the process of life.In particular, ROS are very familiar to aerobic organisms because they are generated during immune responses and normal metabolic activities.Therefore, reactive oxygen species remove enzymes, such as superoxide dismutase (SOD), and damage repair mechanisms have been developed.However, a decline in their functions due to illness or mental stress leads to the accumulation of damage, which affects cells, tissues, genes, and other organs at various levels.In general, "the state in which the oxidative capacity of ROS in vivo exceeds its antioxidant capacity" is called oxidative stress [1], and the definition and quantification methods of indicators have been actively studied in relation to diseases and stress.Oxidative stress damages all biomolecules, and DNA damage, in particular, is an extremely important issue because DNA itself has genetic information.DNA oxidative metabolites have also attracted attention as good biomarkers for assessing the burden of oxidative stress on living cells [2].It has been reported that nucleobases containing purine bases have a low oxidation potential and can easily induce oxidative damage [3][4][5][6][7][8][9].Among them, 8-oxo-2 ′ -deoxyguanosine (oxodG), in which the 8-position of 2 ′ -deoxyguanosine (dG) is oxidized, has been actively studied because several repair enzymes have been found in vivo, and it is most commonly used as an indicator of nucleobase oxidation.Although oxodG occurs randomly in DNA [10,11], its presence at specific positions is thought to play an important role in biochemistry, which makes its analysis by DNA sequencing increasingly important [12].However, no simple method has been developed to identify the location of 8-oxo-dG occurrence in DNA using conventional detection methods [13][14][15][16][17][18][19][20][21][22][23].
Our group has been investigating the use of artificial nucleoside analogs to directly recognize oxodG in DNA through specific hydrogen bonding formation and to apply them to damaged DNA sequencing methods.Recently, we have succeeded in developing novel nucleoside derivatives, Adenosine-1,3-diazaphenoxazine (Adap) and Purine-1,3diazaphenoxazine (Pdap) (Figure 1A), consisting of a phenoxazine unit attached to a purine skeleton, which can recognize 8-oxodG in DNA (Figure 1B) [24][25][26].However, these artificial trinucleotide derivatives have a drawback in that not only the oxodG but also the T of the template strand is incorporated into the primer strand in the single nucleotide insertion reaction using DNA polymerase.We are currently investigating various approaches to overcome this issue, and as one of them, we focused on new derivatives with a pyrimidine skeleton.Therefore, in this paper, based on the previous concept of recognition mode of artificial nucleoside analogs, we investigate the length of the linker that connects the phenoxazine unit from the pyrimidine skeleton to form a hydrogen bonding with oxodG in the syn-conformation (Figure 1C).
Our group has been investigating the use of artificial nucleoside analogs to directly recognize oxodG in DNA through specific hydrogen bonding formation and to apply them to damaged DNA sequencing methods.Recently, we have succeeded in developing novel nucleoside derivatives, Adenosine-1,3-diazaphenoxazine (Adap) and Purine-1,3diazaphenoxazine (Pdap) (Figure 1A), consisting of a phenoxazine unit attached to a purine skeleton, which can recognize 8-oxodG in DNA (Figure 1B) [24][25][26].However, these artificial trinucleotide derivatives have a drawback in that not only the oxodG but also the T of the template strand is incorporated into the primer strand in the single nucleotide insertion reaction using DNA polymerase.We are currently investigating various approaches to overcome this issue, and as one of them, we focused on new derivatives with a pyrimidine skeleton.Therefore, in this paper, based on the previous concept of recognition mode of artificial nucleoside analogs, we investigate the length of the linker that connects the phenoxazine unit from the pyrimidine skeleton to form a hydrogen bonding with oxodG in the syn-conformation (Figure 1C).

Results and Discussion
2.1.Synthesis of 3 ′ -,5 ′ -Diol Compounds First, we synthesized 1,3-diazaphenoxazine units with a C2-linker or C3-linker (Scheme 1).The C2-linker compound 5 was synthesized according to a previous report [24].The C3-linker derivative, on the other hand, was synthesized using the Fmoc-protected propanolamine, 3-(Fmoc-amino)-1-propanol, and compound 6 in the Mitsunobu reaction to obtain compound 7. Immediately after a quick purification process, target compound 8 was successfully obtained in good yield by the cycloaddition reaction and deprotection reaction of the Fmoc group using ammonia methanol.Then, using thymidine or 2 ′ -deoxyuridine as starting materials, TBS protection of the hydroxyl groups (9 or 10) and TPS of the carbonyl group at 4-position of the corresponding nucleobase were performed to obtain compounds 11 or 12 [27,28].Phenoxazine units with different linker lengths were connected to these compounds by the S N Ar reaction to obtain compounds 13-16.The TBS groups of the hydroxyl groups of these compounds were deprotected to obtain the corresponding 3 ′ -,5 ′ -diol compounds of Cdap derivatives (1)(2)(3)(4).
Molecules 2024, 29, x FOR PEER REVIEW 3 of 11 target compound 8 was successfully obtained in good yield by the cycloaddition reaction and deprotection reaction of the Fmoc group using ammonia methanol.Then, using thymidine or 2′-deoxyuridine as starting materials, TBS protection of the hydroxyl groups (9 or 10) and TPS of the carbonyl group at 4-position of the corresponding nucleobase were performed to obtain compounds 11 or 12 [27,28].Phenoxazine units with different linker lengths were connected to these compounds by the SNAr reaction to obtain compounds 13-16.The TBS groups of the hydroxyl groups of these compounds were deprotected to obtain the corresponding 3′-,5′-diol compounds of Cdap derivatives (1)(2)(3)(4).

Synthesis of Triphosphate Compounds
In this reaction, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one and tributylammonium pyrophosphate were first mixed to produce a cyclic triphosphate reagent [29].Then, the hydroxyl group at the 5′-position of the sugar part was modified by mixing the adjusted reagent with the corresponding 3′-,5′-diol compounds.The cyclic triphosphate was oxidized and hydrolyzed to synthesize the desired triphosphate compounds 17-20.Purification was performed by HPLC, and each structure was determined by 31 P-NMR and high-resolution mass measurements (Scheme 2).For compounds with poor isolated yields, the corresponding 3′-,5′-diol compound was confirmed by HPLC.This may be due to a problem with the reagents or the 3′-,5′-diol compound's physical properties that prevented the first reaction from completing.However, we did not study the reaction conditions further because we obtained a sufficient amount of trisphosphate compounds to evaluate their functions.Scheme 2. Synthesis of triphosphate compounds of dCdap derivatives.

Evaluation of oxodG Recognition Ability by Single Nucleotide Elongation Reaction
We tested the recognition ability between oxodG and dG in oligonucleotides of new triphosphate derivatives using Klenow Fragment (KF) and Bst 3.0 DNA polymerase.The gel results of the single nucleotide elongation reaction are shown in Figure 2. When using KF, the 5Me dCdapTP with a C2-linker was not incorporated into the primer chain.On the Scheme 1. Synthesis of 3 ′ -,5 ′ -diol compounds of Cdap derivatives.

Synthesis of Triphosphate Compounds
In this reaction, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one and tributylammonium pyrophosphate were first mixed to produce a cyclic triphosphate reagent [29].Then, the hydroxyl group at the 5 ′ -position of the sugar part was modified by mixing the adjusted reagent with the corresponding 3 ′ -,5 ′ -diol compounds.The cyclic triphosphate was oxidized and hydrolyzed to synthesize the desired triphosphate compounds 17-20.Purification was performed by HPLC, and each structure was determined by 31 P-NMR and high-resolution mass measurements (Scheme 2).For compounds with poor isolated yields, the corresponding 3 ′ -,5 ′ -diol compound was confirmed by HPLC.This may be due to a problem with the reagents or the 3 ′ -,5 ′ -diol compound's physical properties that prevented the first reaction from completing.However, we did not study the reaction conditions further because we obtained a sufficient amount of trisphosphate compounds to evaluate their functions.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 11 target compound 8 was successfully obtained in good yield by the cycloaddition reaction and deprotection reaction of the Fmoc group using ammonia methanol.Then, using thymidine or 2′-deoxyuridine as starting materials, TBS protection of the hydroxyl groups (9 or 10) and TPS of the carbonyl group at 4-position of the corresponding nucleobase were performed to obtain compounds 11 or 12 [27,28].Phenoxazine units with different linker lengths were connected to these compounds by the SNAr reaction to obtain compounds 13-16.The TBS groups of the hydroxyl groups of these compounds were deprotected to obtain the corresponding 3′-,5′-diol compounds of Cdap derivatives (1)(2)(3)(4).

Synthesis of Triphosphate Compounds
In this reaction, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one and tributylammonium pyrophosphate were first mixed to produce a cyclic triphosphate reagent [29].Then, the hydroxyl group at the 5′-position of the sugar part was modified by mixing the adjusted reagent with the corresponding 3′-,5′-diol compounds.The cyclic triphosphate was oxidized and hydrolyzed to synthesize the desired triphosphate compounds 17-20.Purification was performed by HPLC, and each structure was determined by 31 P-NMR and high-resolution mass measurements (Scheme 2).For compounds with poor isolated yields, the corresponding 3′-,5′-diol compound was confirmed by HPLC.This may be due to a problem with the reagents or the 3′-,5′-diol compound's physical properties that prevented the first reaction from completing.However, we did not study the reaction conditions further because we obtained a sufficient amount of trisphosphate compounds to evaluate their functions.

Evaluation of oxodG Recognition Ability by Single Nucleotide Elongation Reaction
We tested the recognition ability between oxodG and dG in oligonucleotides of new triphosphate derivatives using Klenow Fragment (KF) and Bst 3.0 DNA polymerase.The gel results of the single nucleotide elongation reaction are shown in Figure 2. When using KF, the 5Me dCdapTP with a C2-linker was not incorporated into the primer chain.On the Scheme 2. Synthesis of triphosphate compounds of dCdap derivatives.

Evaluation of oxodG Recognition Ability by Single Nucleotide Elongation Reaction
We tested the recognition ability between oxodG and dG in oligonucleotides of new triphosphate derivatives using Klenow Fragment (KF) and Bst 3.0 DNA polymerase.The gel results of the single nucleotide elongation reaction are shown in Figure 2. When using KF, the 5Me dCdapTP with a C2-linker was not incorporated into the primer chain.On the other hand, for the 5Me dCdapTP with a C3-linker, the elongation reaction proceeded only when the template DNA contained dG (Figure 2A, left).Interestingly, primer elongation reactions were observed for template DNA containing oxodG or dG using dCdapTPs, especially when dCdapTP with a C3-linker was well incorporated into the primer strand opposite the oxo-dG template DNA (Figure 2A, right).Therefore, the uptake percentages were calculated from the fluorescence intensity of these bands (Figure 2B).The value of dCdapTP with a C2-linker was 11% for oxodG template DNA and 66% for dG template DNA.On the other hand, the value of dCdapTP with a C3-linker was 85% for oxodG template DNA and 71% for dG template DNA (Figure 2A, right).These results indicated that the longer linker length may have enabled the artificial nucleotide analog to successfully recognize the syn-oriented oxodG (Figure 3).In contrast, the linker length does not seem to affect the anti-oriented dG since there is no significant change in the uptake percentage (Figure 3).When using Bst 3.0 DNA polymerase, 5Me dCdapTP with a C2-or C3-linker was almost never incorporated into the primer strand (Figure 2C).In the case of dCdapTPs, the single nucleotide elongation reaction was confirmed; however, the results showed that it was well incorporated at the dG template DNA.
Molecules 2024, 29, x FOR PEER REVIEW 4 of 11 other hand, for the 5Me dCdapTP with a C3-linker, the elongation reaction proceeded only when the template DNA contained dG (Figure 2A, left).Interestingly, primer elongation reactions were observed for template DNA containing oxodG or dG using dCdapTPs, especially when dCdapTP with a C3-linker was well incorporated into the primer strand opposite the oxo-dG template DNA (Figure 2A, right).Therefore, the uptake percentages were calculated from the fluorescence intensity of these bands (Figure 2B).The value of dCdapTP with a C2-linker was 11% for oxodG template DNA and 66% for dG template DNA.On the other hand, the value of dCdapTP with a C3-linker was 85% for oxodG template DNA and 71% for dG template DNA (Figure 2A, right).These results indicated that the longer linker length may have enabled the artificial nucleotide analog to successfully recognize the syn-oriented oxodG (Figure 3).In contrast, the linker length does not seem to affect the anti-oriented dG since there is no significant change in the uptake percentage (Figure 3).When using Bst 3.0 DNA polymerase, 5Me dCdapTP with a C2-or C3-linker was almost never incorporated into the primer strand (Figure 2C).In the case of dCdapTPs, the single nucleotide elongation reaction was confirmed; however, the results showed that it was well incorporated at the dG template DNA.other hand, for the 5Me dCdapTP with a C3-linker, the elongation reaction proceeded only when the template DNA contained dG (Figure 2A, left).Interestingly, primer elongation reactions were observed for template DNA containing oxodG or dG using dCdapTPs, especially when dCdapTP with a C3-linker was well incorporated into the primer strand opposite the oxo-dG template DNA (Figure 2A, right).Therefore, the uptake percentages were calculated from the fluorescence intensity of these bands (Figure 2B).The value of dCdapTP with a C2-linker was 11% for oxodG template DNA and 66% for dG template DNA.On the other hand, the value of dCdapTP with a C3-linker was 85% for oxodG template DNA and 71% for dG template DNA (Figure 2A, right).These results indicated that the longer linker length may have enabled the artificial nucleotide analog to successfully recognize the syn-oriented oxodG (Figure 3).In contrast, the linker length does not seem to affect the anti-oriented dG since there is no significant change in the uptake percentage (Figure 3).When using Bst 3.0 DNA polymerase, 5Me dCdapTP with a C2-or C3-linker was almost never incorporated into the primer strand (Figure 2C).In the case of dCdapTPs, the single nucleotide elongation reaction was confirmed; however, the results showed that it was well incorporated at the dG template DNA.

Steady-State Kinetic Study of dCapTP Using Klenow Fragment
In order to investigate the uptake efficiency for dCdapTP using KF in more detail, a steady-state kinetic analysis was carried out (Table 1).In comparing the uptake efficiencies, dCdapTP with a C2-linker is clearly incorporated at the complementary position of dG template DNA (7.22 × 10 6 %min −1 M −1 ), followed by the oxo-dG template DNA (2.91 × 10 6 %min −1 M −1 ) (Table 1, entries 2 and 1).It is then taken up against dC, T, and dA template DNA (1.13 × 10 6 , 0.88 × 10 6 , and 0.69 × 10 6 %min −1 M −1 , respectively), in that order, but with lower efficiency.On the other hand, in the case of dCdapTP with a C3linker, it is well incorporated against the complementary position of oxodG template DNA (8.00× 10 6 %min −1 M −1 ) (Table 1, entry 6).The higher V max value compared to a C2-linker suggests that an effective elongation reaction is occurring (V max values: 0.85 %min −1 and 2.86 %min −1 for the C2-linker and C3-linker, respectively) (Table 1, entries 1 vs. 6).This is expected to result in the formation of an artificial base pair between dCdap with a C3-linker and oxodG.For dG template DNA, almost no differences in parameters are shown at dCdapTP with a C2-linker or C3-linker, which supports the results of the interaction with anti-oriented dG with no influence of the linker (for the C2-linker vs. C3-linker, V max : 1.95 vs. 2.20 %min −1 , K m : 0.27 vs. 0.29 µM, respectively) (Table 1, entries 2 vs. 7).Moreover, it is then taken up against T, dC, and dA template DNA (0.64 × 10 6 , 0.50 × 10 6 and 0.28 × 10 6 %min −1 M −1 , respectively), in that order, but with much lower efficiency.

General Procedure of the Deprotection Reaction in TBS Groups
To a solution corresponding to a TBS-protected compound (0.4 mmol) in pyridine (5.0 mL), triethylamine trihydrofluoride (0.32 mL, 1.96 mmol) was added.After stirring for 22 h at room temperature, the solution was removed under reduced pressure.

Figure 2 .
Figure 2. (A) Gel results of single nucleotide primer elongation reactions using Klenow Fragment; (B) bar graph of the uptake percentage of dCdapTP using KF; and (C) gel results of single nucleotide primer elongation reactions using Bst 3.0 DNA polymerase.Conditions: 1.0 µM 15 mer primer and 25 mer template (X) duplex DNA, 0.2 or 0.1 unit/µL of KF or 0.2 unit/µL of Bst 3.0 polymerase in the corresponding reaction buffer, 5 µM of dCdapTP derivatives, and incubation for 10-30 min in a reaction volume of 10 µL.

Figure 2 .
Figure 2. (A) Gel results of single nucleotide primer elongation reactions using Klenow Fragment; (B) bar graph of the uptake percentage of dCdapTP using KF; and (C) gel results of single nucleotide primer elongation reactions using Bst 3.0 DNA polymerase.Conditions: 1.0 µM 15 mer primer and 25 mer template (X) duplex DNA, 0.2 or 0.1 unit/µL of KF or 0.2 unit/µL of Bst 3.0 polymerase in the corresponding reaction buffer, 5 µM of dCdapTP derivatives, and incubation for 10-30 min in a reaction volume of 10 µL.

Figure 2 .
Figure 2. (A) Gel results of single nucleotide primer elongation reactions using Klenow Fragment; (B) bar graph of the uptake percentage of dCdapTP using KF; and (C) gel results of single nucleotide primer elongation reactions using Bst 3.0 DNA polymerase.Conditions: 1.0 µM 15 mer primer and 25 mer template (X) duplex DNA, 0.2 or 0.1 unit/µL of KF or 0.2 unit/µL of Bst 3.0 polymerase in the corresponding reaction buffer, 5 µM of dCdapTP derivatives, and incubation for 10-30 min in a reaction volume of 10 µL.