Light-Activated Chemical Probing of Nucleobase Solvent Accessibility Inside

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1 Light-Activated Chemical Probing of Nucleobase Solvent Accessibility Inside Cells Chao Feng 1,$, Dalen Chan 1,$, Jojo Joseph 2, Mikko Muuronen 3, William H. Coldren 2, Nan Dai 4, Ivan R. Corrêa Jr. 4, Filipp Furche 3, Christopher M. Hadad 2, and Robert C. Spitale 1,3,5 1 Department of Pharmaceutical Sciences, University of California, Irvine. Irvine, California Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18 th Avenue, Columbus Ohio Department of Chemistry, University of California, Irvine. Irvine, California New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA. 5 Correspondence to: R.C.S. (rspitale@uci.edu) $ These authors contributed equally to this project.

2 Supplementary Notes on Nitrenium Reactivity We considered two mechanistic scenarios for the reaction between electrophilic nitrenium ion and guanosine to form the observed C 8 -N product 14 (Supplementary Figure 1). In scenario (a), the N 7 -N adduct 13a is formed via a nucleophilic addition from guanosine N 7 to nitrenium N after which the C 8 -N adduct 13b is formed via a N 7 -C 8 isomerization. In scenario (b), we consider direct formation of C 8 -N adduct 13b via an electrophilic aromatic substitution reaction according to previous studies 1. In both scenarios, the final C 8 -N adduct 14 is formed after deprotonation of the intermediate 13b and is assumed to be the rate-determining step of the reaction based on analogues reaction with 2-fluorenylnitrenium ion and 2 -deoxyguanosine 2. The reaction may proceed in either the singlet and triplet potential energy surfaces (PES): The ground state of the nitrenium ion is likely a triplet, but depending on the rates of the ISC and triplet pathways, both spin states may be reactive. Thus, the formation of 14 may involve multi-state reactivity 3.

3 Supplementary Note 2 Fig. 1. Computationally studied mechanistic scenarios. The computed pathways (a) and (b) are depicted in detail in Supplementary Figure 2 for both spin states. Guanosine and the nitrenium ion can either form hydrogen-bonded complexes (12a) yielding N 7 -N adducts, or stacked complexes (12b) yielding C 8 -N adducts. The formation of these complexes from isolated triplet nitrenium and guanosine species is strongly exergonic (- 35 kcal/mol at TPSS-D3/def2-TZVP level) and is not considered to limit or direct the reactivity. All intermediates are also accessible within a 2 kcal/mol energy window. Large differences between the reactivity of the singlet and triplet nitreniums appear in the transition state (TS) structures: The singlet TSs are significantly more stable than the corresponding triplet TSs. For pathway (a), the barrier for N 7 -N bond formation is 4.4 kcal/mol in the singlet potential energy surface (PES), while the corresponding barrier is 22.0 kcal/mol on the triplet PES. For pathway (b), the C 8 -N bond forms with a low barrier of only 1.6 kcal/mol on the singlet PES, but the barrier is 10.1 kcal/mol on the triplet PESs. In addition, the triplet reactions are predicted to be slightly endergonic and thus reversible, while the singlet reactions are strongly exergonic and thus irreversible. Both singlet transition states have open-shell singlet diradical character, and spin pairing occurs soon after the TSs. The reaction energies are rather insensitive to the computational methodology (Table S3). While barriers increase slightly with inclusion of EXX (TPSS vs. TPSSh), the free energy differences between the reactive transition states change little, and the def2-tzvp and def2-qzvp results are nearly identical. Solvation effects, however, change the relative barriers slightly: Solvation makes the N 7 less nucleophilic thus increasing the barriers for N7-N additions, and also stabilizes the C 8 -N transition state at singlet PES thus lowering its barrier.

4 In summary, these results support the mechanistic proposal (b), whereby the C 8 -N adduct is formed on the singlet PES via an almost barrierless (1.6 kcal/mol) electrophilic aromatic substitution. This mechanism requires the triplet nitrenium species to undergo ISC from the triplet ground state to the reactive singlet state. The formation of N 7 -N adduct requires slightly higher activation energy (4.4 kcal/mol). Moreover, the N 7 -C 8 isomerization is associated with a high activation energy barrier 4, which further supports the direct formation of the C 8 -N adduct 13b. If ISC is slow, the C 8 -N could form at room temperature reversibly in the triplet PES with a reasonably low barrier (10 kcal/mol) and undergo ISC later to form stable singlet species. Supplementary Notes and Figures on TRIR Experiments. TRIR experimental set-up: Ultrafast IR measurements were performed using home-built spectrometers at The Ohio State University. The time resolution is about 300 fs for the TRIR instruments. The absorbance of the sample solutions was about 1.0 in a 1 mm cell at the excitation wavelength (273 nm). Sample solutions were excited in a stainless-steel flow cell equipped with 2 mm thick BaF 2 front and 2 mm thick CaF 2 back windows for the TRIR instrument. After passing through the sample, the reference and probe beam were spectrally dispersed with a polychromator and independently imaged on a liquid-nitrogen cooled HgCdTe detector (2 x 32 pixels. The pump pulse energy was 4 μj at the sample position, and the pump beam diameter (fwhm) was equal to about 250 μm. The entire set of pump-probe delay positions (cycle) is repeated at least three times, to observe data reproducibility from cycle to cycle. To avoid rotational diffusion effects, the angle between polarization of the pump beam

5 and the probe beam was set to the magic angle (54.7 ). Kinetic traces were analyzed by fitting to a sum of exponential terms. All experiments were performed at room temperature. Time-resolved IR Experiments The photochemistry of NAz was studied with femtosecond (fs) time-resolved infrared (TRIR) spectroscopy in chloroform (CHCl 3 ), carbon tetrachloride (CCl 4 ), and acetonitrile (MeCN) at ambient temperature. The TRIR spectra obtained after 273 nm excitation of NAz in chloroform reveal the presence of many positive and negative bands as shown in Figure S2. TRIR spectra in the range of cm -1 (Supplementary Figure 3-6) showed two ground state azide bleaches at 2180 and 2137 cm -1 corresponding to the two conformational isomers. These ground state bleaches did not recover on a 3 ns timescale. A new band around 2105 cm -1 was born immediately (< 1 ps) after the laser pulse and is attributed to the first singlet excited state (S 1 ) of the azide. The S 1 state decay is biphasic with an ultrafast growth component of ~19 ps and a longer-lived component of 247 ps. The fast component is due to vibrational cooling of the initially formed, hot S 1 state of azide, and the longer component is assigned to the S 1 state lifetime. Another positive band was observed in the range of cm -1 (Supplementary Figure 3-6) with a lifetime of 37 ps, and this band is readily assigned to the isocyanate since it was observed as a product in steady-state photolysis in chloroform at 2264 cm -1 (Supplementary Figure 3-6). The isocyanate is formed as a vibrationally hot species and its significantly different rate of formation from the decay rate of S 1 state confirms the generation of isocyanate mainly through a competing Curtius rearrangement pathway from hot S 1 or S n states as previously observed by others 5,6. The C=O vibrations of the S 1 state is observed as two broad bands between cm -1 (Supplementary Figure 3-6). Calculations predicted singlet nitrene to be around 1752 cm -1 ; however, we did not observe the singlet nitrene formation in chloroform on the ns timescale (Supplementary Figure 3-6). Peaks characteristic of

6 triplet nitrene were also not observed. The absence of nitrene in chloroform is not surprising since solvents like chloroform are known to favor the isocyanate formation over the nitrene formation. TRIR experiments were repeated in carbon tetrachloride (Supplementary Figure 3-6) since singlet nitrene formation from other aroyl nitrene precursors was previously observed in carbon tetrachloride 6. The isocyanate formation in carbon tetrachloride was biphasic with a rise time of 30ps (64%) and 149 ps (36%) (Supplementary Figure 3-6). A weak, broad, band centered around 1734 cm -1 was observed in carbon tetrachloride (Supplementary Figure 3-6) and is assigned to singlet nitrene (calculations predicted singlet nitrene to be around 1752 cm -1 ). The singlet nitrene (1734 cm -1 ) was immediately formed after the laser pulse indicating initial formation of singlet nitrene from higher singlet excited state (S n, where n 2) of NAz. It is important to note that the 273 nm light promotes NAz to the higher excited state S 3 according to calculations. It was previously reported that benzoyl nitrenes are formed and have stabilities that are on the order of microseconds 7,8 and is not surprising that we did not observe the nitrene formation from the azide S 1 state on the ns timescale. Interestingly, we do not see the band centered at ~1734 cm -1 in TRIR experiments in acetonitrile, which strongly indicates that the nitrene is indeed reacting with acetonitrile (Supplementary Figure 3-6). In order to verify this, we irradiated NAz solution in acetonitrile-d 3 with 270 nm light and a positive absorption band was detected around 1628 cm -1 in the postphotolysis steady-state FTIR absorption spectrum and it matched well with the FTIR spectrum of oxadiazole (Supplementary Figure 3-6). The product isolation of the steady-state photolysis mixture revealed the formation of isocyanate (Supplementary Figure 3-6) and oxadiazole products; further confirming the singlet nitrene formation upon photoexcitation 9.

7 References for Supplementary Note 2 1 McClelland, R. A., Ahmad, A., Dicks, A. P. & Licence, V. E. Spectroscopic characterization of the initial C8 intermediate in the reaction of the 2-fluorenylnitrenium ion with 2 -deoxyguanosine. J. Am. Chem. Soc. 121, (1999). 2 Staroverov, V. N., Scuseria, G. E., Tao, J. & Perdew, J. P. Comparative assessment of a new nonempirical density functional: Molecules and hydrogen-bonded complexes. J. Chem. Phys. 119, (2003). 3 Schröder, D., Shaik, S. & Schwarz, H. Two-State Reactivity as a New Concept in Organometallic Chemistry. Acc. Chem. Res. 33, (2000). 4 Yang, Z.-Z. & Qi, S.-F. Mechanism of direct conversion between C8 adducts and N7 adducts in carcinogenic reactions of arylnitrenium ions with purine nucleosides: A theoretical study. J. Phys. Chem. B 111, (2007). 5 Kubicki, J. et al. Direct Observation of Acyl Azide Excited States and Their Decay Processes by Ultrafast Time Resolved Infrared Spectroscopy. J. Am. Chem. Soc. 131, (2009). 6 Kubicki, J. et al. Photochemistry of 2-Naphthoyl Azide. An Ultrafast Time-Resolved UV Vis and IR Spectroscopic and Computational Study. J. Am. Chem. Soc. 133, (2011). 7 Pritchina, E. A. et al. Matrix isolation, time-resolved IR, and computational study of the photochemistry of benzoyl azide. Phys. Chem. Chem. Phys. 5, (2003). 8 Desikan, V., Liu, Y., Toscano, J. P. & Jenks, W. S. Photochemistry of sulfilimine-based nitrene precursors: Generation of both singlet and triplet benzoylnitrene. J. Org. Chem. 72, (2007). 9 Liu, J., Mandel, S., Hadad, C. M. & Platz, M. S. A Comparison of acetyl-and methoxycarbonylnitrenes by computational methods and a laser flash photolysis study of benzoylnitrene. J. Org. Chem. 69, (2004).