Photochemical Reactivity of dTPT3: A Crucial Nucleobase

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Photochemical Reactivity of dTPT3: A Crucial Nucleobase Derivative in the Development of Semi-Synthetic Organisms Brennan Ashwood, Steffen Jockusch, and Carlos E. Crespo-Hernández J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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The Journal of Physical Chemistry Letters

Photochemical Reactivity of dTPT3: A Crucial Nucleobase Derivative in the Development of Semi-Synthetic Organisms Brennan Ashwood,1 Steffen Jockusch,2 Carlos E. Crespo-Hernández1* 1

Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, Cleveland, Ohio 44106, United States 2

Department of Chemistry, Columbia University, New York, New York 10027, United States

ACS Paragon Plus Environment

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Abstract In 2017, the Romesberg’s group successfully developed the dTPT3 dNaM unnatural base pair to ●

create a semi-synthetic living organism with enhanced genetic fidelity and the ability to store additional genetic information indefinitely. It is also desirable that the newly developed genetic material remains stable upon exposure to radiation. However, the photochemical properties of dTPT3 are presently unknown. In this contribution, excitation of dTPT3 with near-visible radiation is shown to efficiently populate a reactive triplet state in a sub-1 ps time scalea state that is able to survive for up to a few microseconds in aqueous solution. The triplet state can also generate singlet oxygen in ca. 30% yield, suggesting that dTPT3 can act as a pervasive photosensitizer to accelerate oxidatively-generated damage within DNA and to other biological molecules within cells.

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Expansion of the genetic alphabet has long been desired as a means to enhance the genetic storage capacity of an organism.1-6 More recently, it has been used to drive biotechnological applications like site-specific tagging of DNA/RNA, Systematic Evolution of Ligands by EXponential enrichment (SELEX), and production of novel amino acids.6-8 In 2014, the genetic alphabet was expanded in vivo for the first time using the hydrophobic bases d5SICS [2-(2-deoxyβ-D-erythro-pentofuranosyl)-5-methyl-isoquinolinethione] and dNaM [2-methoxy-3-(2-deoxy-βD-erythro-pentofuranosyl)-napthalene], effectively creating the first semi-synthetic living organism.9 Unfortunately, this unnatural base-pair exhibits poor retention in many sequence contexts due to chemical instability and dephosphorylation of the triphosphate groups, requiring further work to develop a stable semi-synthetic organism. In 2017, the Romesberg group demonstrated that an unnatural base-pair consisting of dNaM and dTPT3 [(2-deoxy-β-D-erythropentofuranosyl)-thieno[3,4]pyridine-2-thione]

is

retained

in

much

higher

yield

than

dNaMd5SICS in vitro and in vivo within most sequence contexts.10-11 In addition, it was shown that a β,γ-CF2 modification of the dNaM and dTPT3 triphosphates significantly reduces their degradation by desphosphorylation, leading to greater retention of the unnatural base pair.12 Even though the β,γ-CF2 modification reduces DNA polymerase recognition of dNaMTP (i.e., the triphosphate nucleotide of dNaM), this approach is a promising step in the optimization of cellular conditions for developing a stable semi-synthetic living organism. Current advances in the development of stable semi-synthetic organisms have overlooked the stability of the genetic code necessary upon exposure to radiation―either emanating from the sun or from standard room lighting. It is currently thought that the canonical DNA bases were not only selected for their ability to replicate and encode our genetic information,13-14 but also due to their photochemical integrity upon exposure to sunlight.15-20 Upon exposure to radiation, the


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canonical DNA bases release ca. 99% of the excess electronic energy efficiently and on an ultrafast time scale,15, 21-23 which minimizes the likelihood of photochemical reactions and damage to the genetic code. Similarly, the integrity of the expanded genetic code should be preserved upon exposure to radiation for the development of semi-synthetic living organisms. Therefore, it is important to investigate the photochemical properties of the unnatural dTPT3 nucleoside as a necessary first step to evaluate the potential unintended consequences of expanding the genetic code.24-25

dTPT3

Normalized Intensity

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1.0 0.8 0.6 0.4 0.2 0.0 260 280 300 320 340 360 380 400 420

Wavelength (nm)

Figure 1. Molecular structure and absorption spectrum of dTPT3 (blue) and absorption spectrum of random double-stranded DNA (black) in phosphate buffer saline (PBS) solution at pH 7.4. The solar irradiance spectrum at the Earth’s surface (orange) is also shown.

Figure 1 presents the molecular structure and absorption spectrum of dTPT3 in aqueous solution at physiological pH overlaid with the solar irradiance spectrum at the Earth’s surface. Also shown in this figure is the absorption spectrum of random double-stranded DNA under similar experimental conditions for comparison. Contrary to the canonical DNA nucleosides, dTPT3


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absorbs near-visible radiation efficiently, with an absorption maximum at 354 nm. Atmospheric ozone absorbs UV wavelengths below 310 nm, effectively protecting the canonical DNA bases from excitation by sunlight. However, UVA wavelengths (315 – 400 nm) account for 95% of the UV solar radiation reaching the Earth’s surface (Figure 1),26-27 indicating that dTPT3 readily absorbs UVA to near-visible sunlight radiation. dTPT3 exhibits a broad steady-state emission spectrum following excitation at 383 nm (see Figure S1). The emission band centered at 530 nm is quenched in the presence of O2. The large Stokes shift of this emission band relative to the absorption maximum along with its quenching by O2 are indicative of phosphorescence.

2

4

A (10-3)

a)

Time (ps) -0.10 0.07

1

-0.01 0.15

3

Time (ps)

b)

0.15 0.32 0.61 1.8 15

2 1

0.24 0.42 0.81 5.2

0 0 -2 -2

-3

450

500

550

600

650

450

500

550

600

650

Wavelength (nm) 3

A (10-3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


c)

3

d)

S1 T1 (Hot) + Tn

500 nm 625 nm 695 nm

2

1

0 0.1

T1

2

1

0 1

10

100

1000

450

500

Time (ps)

550

600

650

Wavelength (nm)

Figure 2. Broadband transient absorption spectra of dTPT3 in phosphate buffer saline solution at pH 7.4 from femtosecond (a) to picosecond (b) time delays upon 383 nm excitation. The negativeamplitude absorption band near 440 nm at early time delays originates from stimulated Raman emission from the solvent. (c) Representative kinetic traces and fit curves over the first 3 ns time window. The broadband data was globally fit to a two-lifetime sequential model containing a constant offset to account for the excited-state population that does not decay within the 3 ns time window that was probed. (d) Decay-associated spectra generated from the target, global analysis.


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The observation of steady-state phosphorescence at room temperature indicates that a large fraction of the excited-state population intersystem crosses to the triplet manifold. However, in order to determine the photoreactivity of dTPT3, it is necessary to elucidate the electronic relaxation pathways following excitation with UVA radiation. Hence, the excited-state relaxation dynamics of dTPT3 were investigated using broadband transient absorption spectroscopy with 383 nm excitation and femtosecond time resolution. As shown in Figure 2a, excitation of dTPT3 results in an initial broad spectrum with absorption maximum around 650 nm, which grows and blueshifts to ca. 535 nm at a time delay of 0.15 ps. This absorption band further blueshifts to 510 nm during the initial 12 ps, with a simultaneous formation of an absorption band above 650 nm that has an absorption maximum around 690 nm. After a time delay of 12 ps, the transient absorption spectra remain constant for the time window of 3 ns (Figure 2c). The time-domain data was globally fit to a two-component sequential kinetic model plus a constant offset (Figure 2c), resulting in lifetimes of 248 ± 15 fs and 4.3 ± 0.2 ps. Decay-associated spectra were generated from the target and global analysis (Figure 2d), and the spectra match the broadband transient absorption data.


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1.2

Normalized A

a) 1.0

N2

0.8

Air

0.6 0.4 0.2 0.0 0

2

4

6

8

10

Time (s) 1.0

Intensity @ 1270 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b)

dTPT3 Phenalenone

0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

Time (s)

Figure 3. (a) Normalized kinetic traces of dTPT3 at 500 nm prepared in air- and N2-saturated pH 7.4 phosphate buffered solution with an absorbance of 0.2 at 383 nm. (b) Singlet oxygen phosphorescence decay traces monitored at 1270 nm and generated by excitation at 335 nm (7 ns pulse length) of dTPT3 (blue) and standard phenalenone28 (black) in O2-saturated Tris-buffered D2O solution.

An electronically-triggered white light source, which has an instrument response function of 400 ps, was used to monitor the full decay of the long-lived transient absorption signal. The broadband spectrum of the long-lived transient species decays monotonically (Figure S2). Figure 3a shows a representative decay trace at the probe wavelength of 500 nm under air- and N2-saturated conditions. The broadband data were globally fit to a single exponential model generating lifetimes of 0.95 ± 0.05 and 2.4 ± 0.1 μs in air- and N2-saturated solutions, respectively. When the concentration of dTPT3 is doubled (i.e., by doubling the absorbance to 0.4 at 383 nm), the decay lifetimes decrease to 0.88 ± 0.03 and 1.1 μs in air- and N2-saturated solutions (Figure S3),


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respectively, indicating that the decay lifetime of this species depends on the concentration of the nucleoside used. Extrapolation to infinity dilution yields a 3.7 s lifetime under N2-saturared conditions, assuming a linear relationship with concentration. The long-lived nature of this transient species, the quenching of its excited-state decay lifetime by O2, and the observed steady-state phosphorescence unequivocally show that it should be assigned to the lowest-energy triplet state of dTPT3. Because the excited-state absorption spectrum at microsecond time delays closely resembles that observed within 1 ps (Figure 2b), we can confidently propose that dTPT3 undergoes intersystem crossing from the singlet to triplet manifold on a sub-1 ps timescale. Ultrafast intersystem crossing has been observed for many thiocarbonylcontaining aromatic hydrocarbons in solution,23-24, 29-38 which has recently been explained as due to the presence of three-state near-degeneracy regions between the singlet and triplet potential energy surfaces that have large spin-orbit coupling matrix elements upon thionation.39 Those compounds exhibit nearly-unity triplet yields in solution, suggesting that dTPT3 should also has a close to unity triplet yield in aqueous solution.


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3.6

Energy (eV)

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1

*

3.3

1

*

3

*

3

*

*

3

1

n*

3

1

n*

n*

3.0

*

3

3

n*

2.7

*

3

*

3

2.4

Vacuum

Water

Figure 4. Vertical excitation energies of dTPT3 in vacuum and water environments calculated at the TD-PBE0/IEFPCM/6-311++G(d,p) level of theory. Vertical excitation energies were computed using the ground-state optimized structure at the PBE0/6-311++G(d,p) level of theory (Figure S4) to assist in the elucidation of an excited-state relaxation mechanism of dTPT3 following UVA excitation. As depicted in Figure 4, these calculations were performed both in vacuum and in water environments at the TD-PBE0/6311++G(d,p) level of theory. The integral equation formalism of the polarizable continuum model (IEFPCM) was used to describe the dielectric solvent reaction field. The singlet vertical excitation energies in water are in good agreement with the ground-state absorption spectrum (Figure S5), and indicate that excitation with 383 nm radiation initially populates the bright S2(ππ*) state. From this state, the excited-state population can intersystem cross to the triplet manifold or internally convert to the dark S1(nπ*) state according to this level of theory. As discussed above, the 248 fs lifetime is assigned to intersystem crossing from the singlet to triplet manifold. According to the El-Sayed propensity rules,40-41 it is expected that the following intersystem crossing pathways may compete in the eventual population of the T1(ππ*) state:


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S2(ππ*)  T2(nπ*), S1(nπ*)  T3(ππ*), and S1(nπ*)  T1(ππ*). The calculated energy gaps between these states are 0.49, -0.03, and 0.67 eV, respectively. At this level of theory, intersystem crossing from the S1(nπ*) state to T3(ππ*) state is expected to be the most competitive pathway due to the near degeneracy of these excited states. Previous theoretical and experimental studies indicate that thiocarbonyl-containing nucleobases generally undergo ultrafast internal conversion from the S2(ππ*) state to the S1(nπ*) state, followed by intersystem crossing to the T1(ππ*) state,23, 35, 39, 42-44

as may be expected by Kasha’s rule.45 Hence, assuming that dTPT3 exhibits similar

dynamics as the thiobases do, the first lifetime can be assigned to a combination of ultrafast internal conversion from the S2(ππ*) state to the S1(nπ*) state, followed by intersystem crossing from the S1(nπ*) state to the T3(ππ*) state (Scheme 1), as the main relaxation pathways. The observation of a ca. 20 to 25 nm blueshifts in the absorption maximum of the short-wavelength absorption band with a lifetime of 4.3 ± 0.2 ps (Figure 2b) can be assigned to a combination of absorption from a high-energy triplet state (i.e., T3 and/or T2 in Scheme 1) and vibrational relaxation in the T1 state. The ultrafast nature of the intersystem crossing pathway and the large energy gap of 0.7 eV between the T3 and T1 states lend support to the assumption that the T1 state is initially populated with a large excess of vibrational energy. As mentioned above, other pathways may also compete in the intersystem crossing process, and static and dynamics calculations performed at a higher level of theory are necessary to evaluate the likelihood of their participation.


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S2(*)

1 UVA Excitation

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T3(*) S1(n*)

T2(n*)

2 VR

3

T1(*)

S0 Scheme 1. Proposed excited-state relaxation mechanism for dTPT3 in aqueous solution upon UVA excitation. The lifetimes correspond to the experimental time constants. VR stands for vibrational energy redistribution to the solvent, intramolecularly, or both.

Importantly, the significant quenching of the T1(ππ*) state population by O2 shown in Figure 3a suggests that singlet oxygen and possibly other reactive oxygen species (ROS) can be generated upon excitation of dTPT3 with near-visible radiation. Thus, time-resolved emission experiments were recorded at 1270 nm to monitor the possible generation of singlet oxygen upon UVA excitation of dTPT3 in aqueous buffer solution. Figure 3b demonstrates that dTPT3 can generate singlet oxygen efficiently, with quantum yields of 0.31 ± 0.02 and 0.33 ± 0.02 under air- and O2saturated conditions, respectively. Furthermore, calculations reported in the SI provide thermodynamic evidence for the exergonic generation of superoxide and possibly other ROS by competitive electron transfer from the triplet state of dTPT3 to molecular oxygen. The favorable electron transfer processes can explain the ca. 30% singlet oxygen yield quantified herein despite


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the highly efficient intersystem crossing to the triplet manifold. Similar observations have been reported recently for other thionated nucleobase derivatives.25, 30, 35 Table 1. Photophysical constants determined for dTPT3 and compared with d5SICS and Thymidine in pH 7.4 aqueous solution. Compound

λmax (nm)a

τISCb (fs)

τTc (Air, μs) τTd (N2, μs)

ΦΔ (Air)e

ΦΔ (O2)f

dTPT3

354

248 ± 15

0.95 ± 0.05

0.31 ± 0.02

0.33 ± 0.02

d5SICS

365

200 ± 2024

0.67 ± 0.0425 1.4 ± 0.125

0.21 ± 0.02

0.36 ± 0.0225

Thymidine

26746

76047

n.d.g

Photochemical Reactivity of dTPT3: A Crucial Nucleobase

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