Synthesis, Photophysical Properties, and Enzymatic Incorporation of ...

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Nov 30, 2016 - The authors express sincere thanks for a Grant-in-Aid Priority. Research from Japan ... G. N. Pandian and H. Sugiyama, Bull. Chem. Soc.
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Synthesis, Photophysical Properties, and Enzymatic Incorporation of an Emissive Thymidine Analogue

Izumi Okamura, Soyoung Park,* Ryota Hiraga, Seigi Yamamoto, and Hiroshi Sugiyama*

Advance Publication on the web November 30, 2016 doi:10.1246/cl.161024

© 2016 The Chemical Society of Japan Advance Publication is a service for online publication of manuscripts prior to releasing fully edited, printed versions. Entire manuscripts and a portion of the graphical abstract can be released on the web as soon as the submission is accepted. Note that the Chemical Society of Japan bears no responsibility for issues resulting from the use of information taken from unedited, Advance Publication manuscripts.

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Synthesis, Photophysical Properties, and Enzymatic Incorporation of an Emissive Thymidine Analogue Izumi Okamura,1 Soyoung Park,*1 Ryota Hiraga,1 Seigi Yamamoto1 and Hiroshi Sugiyama*1,2 1 2

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-Ushinomiyacho, Sakyo-ku, Kyoto 606-8501 E-mail: [email protected]

Herein, we describe the synthesis, photophysical characterization and DNA incorporation of a fluorescent nucleobase analogue, thieno[3,4-d]-pyrimidine T-mimic deoxynucleoside, thdT. thdT indicates the comparable thermal stability and selectivity of base pairing to that of natural T and the noteworthy photophysical behaviors depending on its microenvironments in DNA. A thio-analogue of thymidine triphosphate, thdTTP, has been synthesized and enzymatically incorporated into DNA by naturally occurring polymerases and replication systems with assistance from dTTP. The present results show the utility of thdT as a fluorescent surrogate of thymidine for further applications. The development of fluorescent nucleoside analogues has engaged the interest of many investigators because of the nonemissive nature of natural nucleic acid bases.1–3 For a greater understanding of biological phenomena, fluorescent nucleosides have provided powerful and essential tools, and their applications have enabled a variety of investigations, such as detecting single nucleotide polymorphisms (SNPs),4,5 monitoring the structural dynamics of nucleic acids,6,7 and elucidating enzyme activities.8,9 Fluorescent nucleobases are also important in the context of the expansion of the artificial genetic alphabet with useful functionality.10–13 Furthermore, the application of fluorescent nucleobases have contributed to advance in various research areas including DNA nanotechnology and biotechnology.14–21 Among the fluorescent nucleobases, challenging tasks include the design and synthesis of isomorphic nucleobases, which have strong structural resemblance to natural nucleobases and native Watson–Crick base pairing. Recently, Tor and coworkers developed isomorphic fluorescent RNA nucleosides derived from thieno[3,4-d]-pyrimidine and reported that the nucleosides have important photophysical properties including visible light emission and a high quantum yield.22 In addition, they have showed the usefulness of the nucleoside analogues in various applications.23–29 We have taken note of the potential of isomorphic nucleoside analogues based on a thieno[3,4-d]-pyrimidine core and exploited the emissive DNA nucleoside analogues. Previously, we synthesized the 2aminothieno[3,4-d]-pyrimidine G-mimic deoxyribonucleoside, th dG, and demonstrated that thdG enabled the visual detection of Z-DNA.30 This can be applied in solid-phase oligonucleotide synthesis; moreover, a guanosine triphosphate analogue, thdGTP, was also synthesized and enzymatically incorporated into DNA through primer extension and PCR amplification.31 Very recently, we have focused the nucleotide modifications to enhance the utility of thdG and synthesized a highly emissive 2-O-methylated guanosine analogue, 2OMe-thG.32 The bright blue light of 2-OMe-thG combined

with the distinctive B–Z transition of DNA has the potential for application as a visible nanothermometer. Herein, we describe the synthesis, photophysical properties, and DNA incorporation of a fluorescent base analogue, thieno[3,4-d]-pyrimidine T-mimic deoxynucleoside, thdT. The fluorescence intensity of oligonucleotides including thdT was investigated depending on their microenvironments. In addition, a thio-analogue of thymidine triphosphate, thdTTP, was synthesized and enzymatically incorporated into DNA by naturally occurring enzymes and replication systems.33–36 The synthesis of thdT and thdTTP was achieved by following reported procedures for the synthesis of RNA nucleosides and nucleotides (Scheme 1).22,37,38

Scheme 1 Synthesis of thdT and thdTTP. Reagents and conditions: (a) KOCN, AcOH (aq.), RT, 83%; (b) NaOMe, MeOH, RT, 96%; (c) D-deoxyribofuranose 1-acetate 4,5dibenzoate, BSTFA, TMSOTf, RT, 96%; (d) NH4OH, dioxane, 60 oC, 77%; (e) DMTrCl, Py, 27%; (f) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, i-Pr2NEt, DCM, RT; (g) POCl3, (MeO)3PO, 0–4 oC, then tributylammonium pyrophosphate, Bu3N, 0 oC, 9% The fluorescent base moiety was synthesized from the commercially available methyl 4-aminothiophene-3carboxylate hydrochloride 1 by reaction with KOCN, followed by cyclization. The thienothymine 3 was coupled with benzoyl-protected 1-O-acetyl-2-deoxy-,-dribofuranose in the presence of BSTFA and TMSOTf. This glycosylation reaction yielded a mixture of - and -anomer

2

Table 1 Photophysical data of thdT solvent water dioxane MeOH

abs /nm (/103M -1cm -1)

em /nm ()

303 (3.10) 308 (3.59) 304 (2.98)

420 (0.64) 383 (0.13) 414 (0.42)



/ns

1984

16.0

9194

467

1.2

6358

1252

9.6

8740

Stokes shift/cm-1

The fundamental photophysical properties of thdT were investigated and are shown in Table 1. The thdT deoxyribonucleoside shows absorption at 303 nm and visible fluorescence at 420 nm in water. The absorption and emission spectra of thdT change depending on the solvent polarity. In solvents with lower polarity, the emission intensity of thdT decreases and the emission maxima are shifted toward a shorter wavelength (see SI).22 To evaluate oligonucleotides containing the isomorphic dT analogue, the phosphoramidite of thdT was incorporated into the center of 18-mer DNA oligonucleotides of 5-(CGTCCGTCXTACGCACGC)-3 by automated solid-phase synthesis, where X = thdT. The complementary strands of ODN1 containing matched or mismatched bases with thdT, ODN3–7, and corresponding natural DNA duplexes with thymine, ODN2, were also prepared. In thermal denaturation experiments, matched DNA duplexes including the thdT-A base pair indicated a thermal stability comparable to that of a native duplex with a T–A base pair (Fig. 1a and 1b). Mismatched (G, C, T) or abasic site-containing duplexes, ODN1•4, 1•5, 1•6, and 1•7, showed decreased melting temperatures similar to those of Tcontaining mismatched duplexes. The thermal stability and the base pairing selectivity suggest that thdT can replace native T bases without significant structural perturbation. Therefore, the fluorescence properties of DNA duplexes containing thdT by hybridization of ODN1 with ODN3–7 were examined (Fig. 1c). thdT did not afford distinct fluorescence differences between the matched base pair (thdTA) and the mismatched base pairs (thdT-G, thdT-C, thdT-T). However, significant enhancement of the fluorescence intensity was observed when ODN1 was hybridized with a complementary strand having an abasic site opposite thdT. This fluorescence intensity change shows the potential of thdT

as a fluorescent probe for detecting abasic sites in DNA duplexes considering with base-excision repair system to correct DNA damage. ODN1-2: 5’-CGTCCGTCXTACGCACGC-3’ X= thdT or T ODN3-7: 3’-GCAGGCAGYATGCGTGCG-5’ Y= A,G,T,C or dSpacer (c) thdT:A thdT:G thdT:T thdT:C thdT:dS

1 0.8 0.6 0.4

3.5

0

71 69 67 65 63 61 59 57 55

80

3 2.5

1.5

2 1

1.5 1

0.5

0.5 0

0 220

thdT:A dT:A thdT:G dT:G thdT:T dT:T thdT:C dT:C thdT:dS dT:dS

(b)

40 60 Temperature (C)

4

2

0.2 20

2.5

Fluorescence intensity

Normalized Abs. at 260 nm

1.2

Absorbance

(a)

Temperature (C)

at a : ratio of 3:2. After the benzoyl protecting groups of the deoxyribonucleoside 4 were removed in aqueous ammonia, the 5-hydroxy group of the nucleoside analogue 5 was protected with dimethoxytrityl ether (DMTr), and the desired -anomer was isolated in 27% yield by column chromatography. The configuration at the C-1 carbon of each anomer was confirmed by 1D and 2D (NOESY) 1H NMR spectroscopy (see SI). The 3-hydroxy group of the nucleoside 6 was converted into the phosphoramidite group, and compound 7 was used for the automated solid-phase synthesis of oligonucleotides. The fluorescent deoxythymidine triphosphate thdTTP 8 was also synthesized for the enzymatic incorporation. The synthesized thymidine analogue 5 was treated with POCl3 in trimethylphosphate, and then tributylammonium pyrophosphate was added to the reaction mixture. Subsequently, thdTTP was obtained through HPLC purification and its structure confirmed with 1H- and 31PNMR and ESI-TOF MS.

320 420 Wavelength /nm

520

ss

thdT:A

thdT:G

thdT:T

thdT:C

thdT:dS

ss

thdT:A

thdT:G

thdT:T

thdT:C

thdT:dS

Dashed lines: absorbance / Solid lines: fluorescence

Figure 1 (a) and (b) Thermal stability and selectivity of base pairing. (a) Thermal melts of thdT containing DNA. ODN1: 5’-CGTCCGTCthdTTACGCACGC-3’ paired with ODN3-7: 5’-GCGTGCGTAYGACGGACG-3’ Y = A, T, C, G, or dSpacer. (b) Comparison of Tm values. (c) Absorbance (dashed lines) and fluorescence properties (solid lines) of ODN1 hybridized with complementary strands containing matched or mismatched bases. ODN1-2: 5’CGTCCGTCXTACGCACGC-3’ X = thdT or T paired with ODN3-7. All samples contained 5 M of each oligonucleotide strand, 20 mM Na cacodylate (pH 7.0) and 100 mM NaCl. 5’O-Dimethoxytrityl-1’,2’-dideoxyribose-3’-[(2-cyanoethyl)(N,N-diisopropyl)]-phosphoramidite was used as dSpacer. Excitation at 325 nm. Based on the biophysical analysis of thdT in the character of an isomorphic thymidine analogue, we investigated the photophysical behavior of thdT-containing oligonucleotides to verify the utility of thdT as an environmentally responsive probe (Fig. 2). ODN8–10, in which one or multiple positions were replaced with thdT, were synthesized by solid-phase automated synthesis. Complementary strands ODN11–12 were also prepared. The fluorescence intensity of modified single strands was observed in the order of ODN10 > ODN9 > ODN8. It is generally known that the fluorescence intensity of a single strand including a fluorescent nucleobase analogue (e.g. 2-aminopurine) decreases with duplex formation. When ODN10, with the adjoining incorporation of thdT at the center position, was hybridized with its complementary strand ODN12, a decrease in the fluorescence intensity was observed. We observed a similar change in fluorescence intensity with the duplex ODN1•3, as shown in Fig. 1. By contrast, when ODN8, including a single incorporation of thdT at the center position, was hybridized with its complementary strand ODN11, the fluorescence intensity of the duplex significantly increased compared with single-strand ODN8. ODN9, including three alternating thdTs, also showed increased fluorescence intensity upon the formation of a duplex with ODN11. This interesting result indicates that thdT could produce various emissive outputs depending on its microenvironment through its DNA sequence configuration, because of the stacking of thdT with flanking bases and

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ODN8 : ODN9 : ODN10: ODN11: ODN12:

5’-GCGCGATAXATAGGAGC-3’ 5’-GCGCGAXAXAXAGGAGC-3’ 5’-GCGCGAAXXXAAGGAGC-3’ 5’-GCTCCTATATATCGCGC-3’ 5’-GCTCCTTAAATTCGCGC-3’ X = thdT

9 8 7 6 5 4 3 2 1 0

8 8·11 9 9·11 10 10·12 325

425 Wavelength /nm

525

Figure 2 Fluorescence spectra of DNA containing multiple dT. (a) DNA sequences. (b) Fluorescence spectra of duplexes (solid lines) 8·11 (blue), 9·11 (orange) and 10·12 (green) and single strands (dashed lines) 8, 9 and 10. All samples contained 5 M of each oligonucleotide strand, 20 mM Na cacodylate (pH 7.0) and 100 mM NaCl. Excitation at 303 nm. th

To explore the utility of thdT, we turned to enzymatic incorporation using naturally occurring enzymes and replication systems. To investigate whether thdTTP can be taken up in DNA polymerase, a primer extension reaction was performed by using the 35 exonuclease-proficient Klenow fragment, a FAM-labeled 10-mer primer, and 17- or 21-mer templates 1–3. The primer extension assays were analyzed by denaturing gel electrophoresis to determine the length of the extended chain, as shown in Fig. 3b. When template 1, which contains a single adenine residue in the extension area, was used in primer extension using thdTTP, full-length products were obtained. In the absence of both thdTTP and dTTP, the elongation was not observed (see SI). Therefore, these results suggest that thdTTP can be incorporated on the opposite site of an adenine residue by KF polymerase as a dTTP analogue. Templates 2 and 3, which each involve two adenine residues at the 11th and 14th, and the 11th and 17th, positions respectively, were also investigated. Unfortunately, primer extension using template 2 and thdTTP was halted. However, when we performed the primer extension with template 3, the fully extended product was obtained. The results indicate that the consecutive incorporation of thdT depends on the distance between two adenine residues in the extension area of the template. (a)

(b) 3’

3’

A

Template dTTP

KF polymerase thdTTP

5’ FAM 3’

1

5’

thdT

3’

A

5’

control

5’ FAM

2

3

+

-

+

-

+

-

-

+

-

+

-

+

Template 1: 3’-AGTGCTC-5’ Template 2: 3’-AGTACTC-5’ Template 3: 3’-AGTGCTACGTC-5’

Figure 3 (a) Primer extension experiments with a FAMlabeled primer and template 1 to 3, in the presence of the natural dTTP or thdTTP, and three dNTPs (dATP, dGTP and

Regarding PCR amplification, the primer extension assays imply that it is difficult to completely replace dTTP with th dTTP (Fig. 3b). We therefore designed PCR amplification using a mixture of dTTP and thdTTP to compensate for the insufficient incorporation of thdT in the proximal adenine residue regions. A 298-mer PCR amplification was conducted by using pUC18 F413-R710 as a template, KOD Plus polymerase, and a mixture of dTTP and thdTTP. Although PCR amplification using thdTTP only instead of dTTP did not give the expected PCR product, the desired PCR product was gradually observed by adding dTTP in fractions from 1% to 50%. The amplified PCR products using 0%, 50%, 75%, 90%, 95%, and 97% thdTTP were fractionated and analyzed by agarose gel electrophoresis, as shown in Fig. 4a. After gel extraction followed by purification using a Wizard® SV Gel and PCR Clean-Up System (Promega) to remove the unreacted dNTPs and the primers, fluorescence spectra of each purified DNA solution were measured to determine the fluorescent labeling of the long DNA construct (Fig. 4b). In addition, to determine the ratio of incorporation of thdTTP into amplified DNA, the purified PCR product was degraded to the corresponding nucleosides by using P1 Nuclease and Antarctic Phosphatase. The nucleosides obtained were analyzed by HPLC and the ratios of thdT to dT in the amplified products were calculated based on the decrease of dT to dA after the incorporation of thdTTP. As shown in Fig. 4c, the incorporation ratio of thdT reached 18% when 97% th dTTP was used for PCR amplification. The results from the enzyme digestion and HPLC analysis indicate that thdTTP could be incorporated into DNA by PCR amplification with KOD Plus polymerase, albeit at low efficiency. dTTP / thdTTP 100/0 50/50 25/75 10/90 5/95 3/97 1/99 0/100

(a)

(b)

(c) 100 Incorporation ratio (%)

(b) Fluorescent intensity

(a)

dCTP). (b) Analyses of primer-extended products by denaturing gel electrophoresis.

Fluorescencent intensity

hydrogen bond interaction with neighboring bases.40 In addition, the observed results indicate that thdT, by fusing its thiophene ring with pyrimidine, has different photophysical behavior from 5-(thiene-2-yl)-2-deoxyuridine, a fluorescent T analogue with a thiophene residue conjugated to the 5position of the pyrimidines, as developed by the Tor group.39

35 30 25 20 15 10 5 0

dTTP/thdTTP left: 100/0 right: 3/97 350

400 450 500 wavelength /nm thdTTP ratio (%) 0 50 75 90 95

550

97

80 60 thdT

40

dT

20 0 100/0 50/50 25/75 10/90 5/95 dTTP / thdTTP

3/97

dTTP/thdTTP

100/0

50/50

25/75

10/90

5/95

3/97

Incorporation ratio dT/thdT

100/0

97/3

94/6

91/9

90/10

82/18

Figure 4 (a) Analyses by agarose gel electrophoresis of amplified 298 bp products. PCR amplification was performed with dATP, dCTP, and dGTP (200 M each) and a combined total of 200 M dTTP and thdTTP mixture (100/0, 50/50, 25/75, 10/90, 5/95, 3/97, 1/99, and 0/100). (b) Fluorescence spectra of purified DNA solutions obtained as described in (a) from dTTP/thdTTP ratios. All samples contained 30 ng/L DNA. The excitation wavelength was 303 nm. (c) Relationship between the dTTP/thdTTP ratio and the extent of th dT incorporation. Averages of more than two runs. We have synthesized thdT as a visible light-emissive thymidine analogue and successfully incorporated the nucleotide into oligonucleotides using phosphoramidite chemistry. In addition to having thermal stability and base pairing selectivity comparable to those of an isomorphic dT

4

analogue, thdT has shown a significant fluorescence intensity change for abasic sites in DNA duplexes. In addition, singleor multiple-thdT-containing oligonucleotides have shown environmentally responsive photophysical behaviors according to DNA sequence and duplex formation. We have also synthesized a fluorescent thio-analogue of thymidine triphosphate, thdTTP, and achieved the enzymatic incorporation of thdTTP using natural polymerases and replication systems with assistance from dTTP. We continue to explore the synthesis and application of thieno[3,4-d]pyrimidine deoxyribonucleosides to expand the repertoire of fluorescent base analogues. Supporting Information is http://dx.doi.org/10.1246/cl.******.

available

on

Acknowledgements The authors express sincere thanks for a Grant-in-Aid Priority Research from Japan Society for the Promotion of Science (JSPS) and the grant from the WPI program (iCeMS, Kyoto University). We also thank KAKENHI program (Grant-in-Aid for Young Scientists B) for support to S. P. We like to thank Karin Nishimura (Graduate School of Engineering, Kyoto University) for technical assistance to measure mass spectra of synthetic compounds. We also thank Fumitaka Hashiya for his help and useful discussions. Conflict of interest statement. None declared.

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References and Notes

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1 2 3

40

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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