Developing Photoactive Coumarin-Caged N-Hydroxysulfonamides for Generation of Nitroxyl (HNO)

Photoactive N-hydroxysulfonamides photocaged with the (6-bromo-7-hydroxycoumarin-4-yl)methyl chromophore have been successfully synthesized, and the mechanisms of photodecomposition investigated for two of the compounds. Upon irradiation up to 97% of a diagnostic marker for (H)NO release, sulfinate was observed for the trifluoromethanesulfonamide system. In the absence of a species that reacts rapidly with (H)NO, (H)NO instead reacts with the carbocation intermediate to ultimately generate (E)-BHC-oxime and (Z)-BHC-oxime. Alternatively, the carbocation intermediate reacts with solvent water to give a diol. Deprotonation of the N(H) proton is required for HNO generation via concerted C-O/N-S bond cleavage, whereas the protonation state of the O(H) does not affect the observed photoproducts. If the N(H) is protonated, C-O bond cleavage to generate the parent N-hydroxysulfonamide will occur, and/or O-N bond cleavage to generate a sulfonamide. The undesired competing O-N bond cleavage pathway increases when the volume percentage of water in acetonitrile/water solvent mixtures is increased.


Introduction
Nitroxyl (HNO) is now recognized to be an important biological molecule, due to its unique chemical reactivity and significant promise in medicine [1][2][3][4][5][6].HNO shows distinctly different physiological and pharmacological properties from the well-studied nitric oxide [6][7][8].HNO is a vasodilator and improves myocardial contractility, two vital features that render HNO a promising therapeutic agent for the treatment of acute congestive heart failure [4,5,[9][10][11].Furthermore, the HNO-generating compounds CXL-1020 and CXL-1427 (Cimlanod) have shown improved cardiovascular performance in failing hearts [4,5,9,[11][12][13].HNO is likely to be generated endogenously via a range of reactions.However, rapid dimerization of HNO in solution complicates studies of HNO's fundamental chemical and biological reactivity.Molecules that release HNO in situ (HNO donors) are therefore an integral part of investigations on the reactivity of this emerging biological signaling molecule.
A range of HNO-releasing compounds have been developed, where the release of HNO at physiological pH conditions is typically on the minutes to hours timescale, although the half-life is less than a minute in rare cases [14,15].Since HNO reacts rapidly with biomolecules, including reacting with thiols [16], metalloproteins [5] and oxidants [17], this has inspired us and others to investigate the potential of light for rapid and spatiotemporally controlled release of HNO from photoactive molecules.Photoactive HNO donor molecules are valuable chemical tools for in situ investigations of the physiological and pharmacological roles of the unstable and highly reactive HNO in biological systems.These molecules could also be used for in vitro studies to directly obtain kinetic information on the rapid rates of HNO's reactions with biomolecules.Nakagawa et al. investigated the photochemical generation of HNO from hetero-Diels-Alder cycloadducts [18][19][20].This group also recently reported the synthesis of (7-diethylaminocoumarin-4-yl)methyl photocaged derivatives of the Piloty's acid derivatives (2-Br)PhSO 2 NHOH and (2-NO 2 )PhSO 2 NHOH [21].It was suggested that the HNO-generating pathway proceeds via C-O bond cleavage although detailed studies on the mechanism of photodecomposition were not carried out.Our group has also investigated the potential of photocaged N-hydroxysulfonamides to generate HNO, using O-(3-hydroxynaphthalen-2-yl)methyl (3,2-HNM) [22,23], O-(6-hydroxynaphthalen-2-yl)methyl (6,2-HNM) [24], O-2-nitrobenzyl (2-NB) [25], and 2-(2-nitrophenyl)ethyl phototriggers [26].Interestingly, whether or not the HNO-generating pathway dominates is highly dependent on the chromophore, sulfohydroxamate and solvent.For 6,2-HNM-caged N-hydroxysulfonamides, up to 98% HNO was observed in acetonitrile/aqueous phosphate buffer (pH 7.0) solvent mixtures.
The thermal stabilities of 8a and 8b were assessed in anaerobic CD 3 CN in the absence of light, by 19 F and/or 1 H NMR spectroscopy.No detectable decomposition was observed after ~10 min for both 8a and 8b.After 8 days, 8a had undergone 10% decomposition to give CF 3 SO 2 NH 2 ( 19 F NMR spectroscopy), whereas 8b was stable over this time period.
The UV-vis spectra of 8a and 8b are shown in Section S1 in the Supplementary Information.The wavelength maxima of 8a and 8b (370 and 366 nm, respectively) can be assigned to a π-π* transition [34].These transitions are lower in energy compared to 7-hydroxycoumarin (λ max = 350 nm [35]) due to the presence of the 6-bromo substituent [36].The molar extinction coefficients for 8a and 8b are 2.50 × 10 4 (370 nm) and 1.42 × 10 4 (366 nm) M −1 cm −1 , respectively.8a and 8b absorb significantly in the visible region.The photostability of 8a in aerobic CD 3 CN under typical lab light conditions was therefore investigated.After 12 h, 58% 8a had decomposed to give CF 3 SO 2 NH 2 (33%), CF 3 SO 2 − (17%) and CF 3 SO 3 − (8%) ( 19 F NMR), Section S2, SI. 8a was found to be stable in the solid form under the same light conditions.The photostability of 8b in CD 3 CN was also examined under typical lab light conditions, with no detectable photodecomposition after 12 h ( 1 H NMR spectroscopy).Both 8a (see Section S3, SI) and 8b were found to be stable under red light conditions for at least 4 h in CD 3 CN.Therefore, all the photolysis experiments were carried out under red light, including sample preparation.8a and 8b absorb significantly in the visible region.The photostability of 8a in aerobic CD3CN under typical lab light conditions was therefore investigated.After 12 h, 58% 8a had decomposed to give CF3SO2NH2 (33%), CF3SO2 − (17%) and CF3SO3 − (8%) ( 19 F NMR), Section S2, SI. 8a was found to be stable in the solid form under the same light conditions.The photostability of 8b in CD3CN was also examined under typical lab light conditions, with no detectable photodecomposition after 12 h ( 1 H NMR spectroscopy).Both 8a (see Section S3, SI) and 8b were found to be stable under red light conditions for at least 4 h in CD3CN.Therefore, all the photolysis experiments were carried out under red light, including sample preparation.
The effect of the solvent composition (CD 3 CN and phosphate buffer (0.10 M, pH 7.0)) on the photoproducts obtained upon irradiation of 8a was investigated under anaerobic conditions.The samples were irradiated using a Rayonet photoreactor (350 nm bulbs).The percentage of the fluorinated aliphatic photoproducts was established by integrating the peaks in the 19 F NMR spectrum of the photoproducts upon complete photodecomposition of 8a.The release of one equivalent of RSO 2 − (R = CF 3 for 8a) for each (H)NO generated, Pathway 1 Scheme 2, is a convenient marker for determining the percentage of the HNOgenerating pathway for photocaged N-hydroxysulfonamides by NMR spectroscopy [23].The aromatic photoproducts were characterized using 1 H NMR spectroscopy.The effect of the solvent composition (CD3CN and phosphate buffer (0.10 M, pH 7.0)) on the photoproducts obtained upon irradiation of 8a was investigated under anaerobic conditions.The samples were irradiated using a Rayonet photoreactor (350 nm bulbs).The percentage of the fluorinated aliphatic photoproducts was established by integrating the peaks in the 19 F NMR spectrum of the photoproducts upon complete photodecomposition of 8a.The release of one equivalent of RSO2 − (R = CF3 for 8a) for each (H)NO generated, Pathway 1 Scheme 2, is a convenient marker for determining the percentage of the HNO-generating pathway for photocaged N-hydroxysulfonamides by NMR spectroscopy [23].The aromatic photoproducts were characterized using 1 H NMR spectroscopy.
Figure 1a shows the 19 F NMR spectrum obtained upon completely photolyzing 8a in anaerobic 80:20 v/v CD3CN:phosphate buffer (pH 7.0, 0.1 M).Upon photolysis, 8a released CF3SO2 − (94%, -88.1 ppm); that is, almost stoichiometric release of (H)NO.In addition, a small amount of CF3SO2NH2 (6%, -80.5 ppm) was generated via competing photoinduced O-N cleavage of 8a (Scheme 2, Pathway 3).The chemical shifts of both fluorinated photoproducts were confirmed using authentic samples of these compounds.No formation and subsequent decomposition of CF3SO2N(H)O(H) was observed, in line with results found on related systems [22][23][24].If CF3SO2N(H)O(H) was an intermediate, we would have expected to observe it in the 19 F NMR spectra during data collection since the half-life (t1/2) for decomposition of this compound is ~13 min in pH 7.4 buffer solution [41].Hence, CF3SO2 − can be presumed to form exclusively via Pathway 1, Scheme 2.
The effect of the solvent composition on the photoproducts obtained from 8a is shown in The photoproducts obtained upon the irradiation of 8b were also determined as a function of solvent composition in phosphate buffer (30 mM, pH 7.0)/CD 3 CN solvent mixtures.8b solutions were irradiated using the Rayonet photoreactor (350 nm).The photoproducts were characterized by recording the 1 H NMR spectra of fully and partially photodecomposed samples.Products were assigned by recording the 1 H NMR spectra of authentic samples in the same solvent mixture.3) are observed at 7.65 and 6.72, with the remaining two peaks (6.22, 4.66 ppm) overlapping with the peaks of other compounds.The minor peaks at 6.88 ppm and 8.43 ppm could not be assigned.The peak at 5.27 ppm is an impurity in the CD3CN solvent.
The effect of the solvent composition on the photoproducts obtained from 8a is shown in Figure 2. The percentage of the desired pathway (Pathway 1, Scheme 2) increases as the volume percentage of CD3CN in the CD3CN/phosphate buffer (0.10 M, pH 7.0) increases, with 97% CF3SO2 − (and (H)NO) generated in 95:5 v/v CD3CN:phosphate buffer.However, in pure CD3CN only ~36% CF3SO2 − is produced.A dramatic decrease in the percentage of the (H)NO-generating pathway in pure CD3CN compared to solvent mixtures of CD3CN and phosphate buffer (pH 7.0) was also observed for the (6-hydroxylnapthylen-2-yl)methyl-ON(H)SO2CF3 system, with only O-N bond cleavage observed in pure CD3CN [23,24].The photoproducts obtained upon the irradiation of 8b were also determined as a function of solvent composition in phosphate buffer (30 mM, pH 7.0)/CD3CN solvent mixtures.8b solutions were irradiated using the Rayonet photoreactor (350 nm).The photoproducts were characterized by recording the 1 H NMR spectra of fully and partially pho- Figure 3a shows the 1 H NMR spectrum of a completely photodecomposed sample of 8b in the 2-5.5 ppm region in 60:40 v/v, CD 3 CN to 30 mM phosphate buffer, pH 7.0.CH 3 SO 2 NH 2 was observed, consistent with O-N bond cleavage occurring.A very small amount of an unknown species was also observed at 3.21 ppm, and a trace of CH 3 SO 2 NHOH. Figure 3b shows the 1 H NMR spectrum of a partially irradiated (16 s) sample of 8b.The peaks at 9.93, 7.90, 6.51 and 6.38 ppm can be assigned to BHC-CHO (10) by comparison with an authentic sample of 12. BHC-CHO was found to be photostable under the irradiation conditions.There was no evidence for (E)-BHC-oxime (13a; 8.30, 8.27, 6.56 and 6.18 ppm), (Z)-BHC-oxime (13b; 7.57, 7.67, 6.54 and 6.The small peak at 5.37 ppm is an impurity in the CD3CN solvent.The peaks at 8.41, 6.46, 6.14 and 5.98 ppm could not be assigned.

Effect of O 2
The aerobic photolysis of 8a was performed, to determine the effect of O 2 on photodecomposition.Since photodecomposition of 8a and release of HNO is predicted to occur via the singlet excited state, this would suggest that the impact of O 2 (a triplet species in the ground state) on the observed photoproducts should be minimal.Like anaerobic photolysis, aerobic photolysis of 8a predominantly occurred via C-O/N-S bond cleavage, generating 94% CF 3 SO 2 − .Surprisingly, however, aerobic photolysis of 8a resulted in the generation of larger amounts of diol 3 (a secondary diagnostic marker of HNO release) (30% vs. 4% observed under anaerobic conditions), implying enhanced release of (H)NO from the solvent cage under aerobic conditions.Direct quantification of HNO released in the presence of air was not possible using HNO trapping agents like aquacobalamin (H 2 OCbl(III)) [23] or a phosphine, due to instability of the diagnostic products and interference from secondary reactions of these traps with O 2 .The release of larger amounts of diol 3 under aerobic conditions would be consistent with competing oxidation of released (H)NO to NO by air, prior to any trapping with the incipient carbocation to afford an oxime byproduct.NO is unlikely to react rapidly with this carbocation intermediate, leading to increased trapping of the carbocation by solvent to form diol 3 [51].

Determination of the pKa Values for 8a and 8b
To understand the role of the protonation state of the ground state molecule on the mechanism of photodecomposition, the ground state pKa values for 8a and 8b were determined.There are two sites where deprotonation may occur-the 7-hydroxy substituent and the N(H) of the sulfonamide.Control experiments showed that 8a is stable under the experimental conditions used for determining the pKa, Section S6, SI.
The pKa values of 8a were initially investigated using NMR spectroscopy.The 19 F and 1 H NMR spectra of 8a were recorded in D 2 O with the pD of the aqueous component of the solvent mixture varying from pD 2.03-11.23, to obtain an estimate of pKa values of both sites.A small amount of CH 3 CN (8% v/v CH 3 CN) was required to ensure that the compound was fully dissolved.The 19 F NMR spectra are shown in Figure S10, SI, and a plot of chemical shift of the CF 3 signal versus pD is shown in Figure 4.The chemical shift of the CF 3 signal of 8a moves upfield upon deprotonation of the N(H) proton, due to shielding by the electron-rich N − proximate to the CF 3 group.and the N(H) of the sulfonamide.Control experiments showed that 8a is stable under the experimental conditions used for determining the pKa, Section S6, SI.
The pKa values of 8a were initially investigated using NMR spectroscopy.The 19 F and 1 H NMR spectra of 8a were recorded in D2O with the pD of the aqueous component of the solvent mixture varying from pD 2.03-11.23, to obtain an estimate of pKa values of both sites.A small amount of CH3CN (8% v/v CH3CN) was required to ensure that the compound was fully dissolved.The 19 F NMR spectra are shown in Figure S10, SI, and a plot of chemical shift of the CF3 signal versus pD is shown in Figure 4.The chemical shift of the CF3 signal of 8a moves upfield upon deprotonation of the N(H) proton, due to shielding by the electron-rich N − proximate to the CF3 group.Fitting the data in Figure 4 to the equation ( 10) 1 ( 10) where δobs = observed chemical shift (ppm), δHA = the chemical shift (ppm) of the acid form of 8a and δA− = the chemical shift (ppm) of the conjugate base of 8a, gives a pKa value of 3.42 ± 0.02.This pKa value is assigned to the N(H) proton of the N-hydroxysulfonamide, based on its value and the large changes in the chemical shift of the CF3 peak under varying pD conditions. 1 H NMR spectroscopy was not useful for determining this pKa value, Fitting the data in Figure 4 to the equation where δ obs = observed chemical shift (ppm), δ HA = the chemical shift (ppm) of the acid form of 8a and δ A− = the chemical shift (ppm) of the conjugate base of 8a, gives a pKa value of 3.42 ± 0.02.This pKa value is assigned to the N(H) proton of the N-hydroxysulfonamide, based on its value and the large changes in the chemical shift of the CF 3 peak under varying pD conditions. 1 H NMR spectroscopy was not useful for determining this pKa value, since the BHC-CH 2 -ON(H)-SO 2 CF 3 peak overlapped with the HDO peak.The pKa value of 3.42 ± 0.02 is similar to that observed for 6,2-HNM-ON(H)-SO 2 CF 3 (4.4 ± 0.1 in aqueous solution) [24] and 2-NPE-ON(H)-SO 2 CF 3 (3.77± 0.03 (in D 2 O with 5% v/v CH 3 CN, I = 1.0 M, NaCF 3 SO 3 )) [26].Essentially no change in the CF 3 shift was observed from pD 5.05 to 11.22 (where the coumarinyl OH proton is lost) (≤0.2 ppm), because of the distal location of the CF 3 moiety relative to the 7-hydroxy substituent.
The pKa for deprotonation of the second ionizable site, the 7-hydroxy substituent of 8a, was determined by recording 1 H NMR spectra from pD 4.90 to 9.43.The assignments of the aromatic protons are shown in Figure 5.The chemical shifts were unchanged (≤0.02 ppm) from pD 2.02 to 4.92.Deprotonation of the O(H) moiety resulted in an upfield shift of chemical shifts a, b, and c, due to shielding effects by the more electron-rich aryloxy anion with increasing pD.As expected, greater shielding effects at high pD were seen for protons b and c due to resonance delocalization of the oxyanion to the carbons directly bearing these two protons.Selected 1 H NMR spectra are shown in Figure S11, SI.The chemical shift of protons d overlapped with the HDO peak of the aqueous component.The chemical shifts of protons a, b and c were plotted as a function of pD, Figure 6, and data were fitted to Equation (1), giving pKa = 6.35, (proton a), pKa = 6.28 (proton b) and pKa = 6.32 (proton c), with an average pKa value of 6.32 ± 0.03.The pKa of 6-bromo-7-hydroxy-4-methylcoumarin was separately determined by UV-vis spectroscopy (pKa 6.91 ± 0.03 in 8:92 v/v CH 3 CN:H 2 O; Section S7, SI).pKa values of 6.2 (aqueous solution) and 5.88 (10:90 v/v DMSO:H 2 O) have been reported by others for the 7-hydroxy substituent of BHCM-OH and (6-bromo-7hydroxycoumarin-4-yl)methyl acetate (BHCM-OAc), respectively [52].
data were fitted to Equation (1), giving pKa = 6.35, (proton a), pKa = 6.28 (proton b) and pKa = 6.32 (proton c), with an average pKa value of 6.32 ± 0.03.The pKa of 6-bromo-7-hydroxy-4-methylcoumarin was separately determined by UV-vis spectroscopy (pKa 6.91 ± 0.03 in 8:92 v/v CH3CN:H2O; Section S7, SI).pKa values of 6.2 (aqueous solution) and 5.88 (10:90 v/v DMSO: H2O) have been reported by others for the 7-hydroxy substituent of BHCM-OH and (6-bromo-7-hydroxycoumarin-4-yl)methyl acetate (BHCM-OAc), respectively [52].The ground state pKa for the 7-hydroxy substituent of 8a was also determined using UV-vis spectroscopy.A control experiment showed that 8a does not decompose under the conditions used for these experiments, Section S8, SI.Significant changes in the UVvis spectra were only observed upon deprotonation of the O(H) and not the N(H), since the O(H) is an aromatic substituent while the N lone pair is not conjugated into the arene  The ground state pKa for the 7-hydroxy substituent of 8a was also determined using UV-vis spectroscopy.A control experiment showed that 8a does not decompose under the conditions used for these experiments, Section S8, SI.Significant changes in the UV-vis spectra were only observed upon deprotonation of the O(H) and not the N(H), since the O(H) is an aromatic substituent while the N lone pair is not conjugated into the arene chromophore.As the O(H) site deprotonates, the wavelength maximum shifts from 330 nm to 365 nm, Figure 7a.The observed absorbance, A obs , at 367 nm was plotted as a function of pH, Figure 7b and the data fitted to Equation (2).
where A HA = the absorbance of the conjugate acid and A A− = the absorbance of the conjugate base.Fitting the data to Equation ( 2) gives pKa = 6.35 ± 0.03 (Figure 7b).
Molecules 2024, 29, x FOR PEER REVIEW 10 of 28 chromophore.As the O(H) site deprotonates, the wavelength maximum shifts from 330 nm to 365 nm, Figure 7a.The observed absorbance, Aobs, at 367 nm was plotted as a function of pH, Figure 7b and the data fitted to Equation (2).
(10 1 10 ) where AHA = the absorbance of the conjugate acid and AA− = the absorbance of the conjugate base.Fitting the data to Equation ( 2) gives pKa = 6.35 ± 0.03 (Figure 7b).The pKa of the O(H) of 8a determined by 1 H NMR spectroscopy was 6.31 ± 0.03.This study was conducted in D2O with 8% v/v CH3CN, I = 1.0 M (CF3SO3Na).The value obtained by UV-vis spectroscopy was 6.35 ± 0.03, in 8:92 v/v CH3CN:H2O.The pKa values in H2O are typically 0.05-0.6lower than in D2O [53][54][55]; that is, acids are stronger in H2O versus D2O.The differences in ionic strength will also have an effect on the pKa value.The pKa of the O(H) of 8a determined by 1 H NMR spectroscopy was 6.31 ± 0.03.This study was conducted in D 2 O with 8% v/v CH 3 CN, I = 1.0 M (CF 3 SO 3 Na).The value obtained by UV-vis spectroscopy was 6.35 ± 0.03, in 8:92 v/v CH 3 CN:H 2 O.The pKa values in H 2 O are typically 0.05-0.6lower than in D 2 O [53][54][55]; that is, acids are stronger in H 2 O versus D 2 O.The differences in ionic strength will also have an effect on the pKa value.

Determination of the pKa Values for 8b
The pKa values for 8b were initially investigated using NMR spectroscopy. 1   The chemical shift of the CH 3 (e) proton signal moved upfield from 3.22 ppm to 2.88 ppm as pD of the solution was increased from 9.62 to 10.50, Figure S14, SI ( 1 H NMR spectra).Since proton e is adjacent to the N(H) moiety of the N-hydroxysulfonamide of 8b, the change in the chemical shift is attributed to deprotonation of this site.The chemical shift of the protons e was plotted as a function of pD (Figure 9).Fitting the data to Equation (1) gives pKa = 10.11 ± 0.03.Like 8a, the BHC-CH 2 -ON(H)-SO 2 CH 3 peak overlapped with the HDO peak from the solvent, so protons d could not be used to determine the pKa of the N(H) proton of 8b.This pKa value is very similar to the pKa for the N(H) in 2-NPE-ON(H)-SO 2 CH 3 (10.06, in D 2 O with 5% v/v CH 3 CN, I = 1.0 M, CF 3 SO 3 Na [26]).
The pKa value of 8b was also investigated by UV-vis spectroscopy.A UV-vis spectroscopic titration experiment for 8b was carried out to determine the pKa value of the O(H) substituent.A UV-vis spectrophotometric titration of 8b (3.1 × 10 −5 M, in 8:92 v/v CH 3 CN:H 2 O, 30 mL) was conducted using the same flow set up used for 8a (Figure 10a).The absorbance at 370 nm was plotted as a function of pH.Data were fitted to Equation (2), giving pKa = 6.47 ± 0.03 (8:92 v/v CH 3 CN:H 2 O, Figure 10b).The pKa for the O(H) proton from the NMR spectroscopic studies reported above was 6.40 ± 0.03 (in D 2 O with 8% v/v CH 3 CN, I = 1.0 M, CF 3 SO 3 Na).This is once again consistent with the pKa values in H 2 O being typically 0.05-0.6lower than in D 2 O [53][54][55].
The chemical shift of the CH3 (e) proton signal moved upfield from 3.22 ppm to 2.88 ppm as pD of the solution was increased from 9.62 to 10.50, Figure S14, SI ( 1 H NMR spectra).Since proton e is adjacent to the N(H) moiety of the N-hydroxysulfonamide of 8b, the change in the chemical shift is attributed to deprotonation of this site.The chemical shift of the protons e was plotted as a function of pD (Figure 9).Fitting the data to Equation (1) gives pKa = 10.11 ± 0.03.Like 8a, the BHC-CH2-ON(H)-SO2CH3 peak overlapped with the HDO peak from the solvent, so protons d could not be used to determine the pKa of the N(H) proton of 8b.This pKa value is very similar to the pKa for the N(H) in 2-NPE-ON(H)-SO2CH3 (10.06, in D2O with 5% v/v CH3CN, I = 1.0 M, CF3SO3Na [26]).The pKa value of 8b was also investigated by UV-vis spectroscopy.A UV-vis spectroscopic titration experiment for 8b was carried out to determine the pKa value of the O(H) substituent.A UV-vis spectrophotometric titration of 8b (3.1 × 10 −5 M, in 8:92 v/v CH3CN: H2O, 30 mL) was conducted using the same flow set up used for 8a (Figure 10a).The absorbance at 370 nm was plotted as a function of pH.Data were fitted to Equation (2), giving pKa = 6.47 ± 0.03 (8:92 v/v CH3CN:H2O, Figure 10b).The pKa for the O(H) proton from the NMR spectroscopic studies reported above was 6.40 ± 0.03 (in D2O with 8% v/v CH3CN, I = 1.0 M, CF3SO3Na).This is once again consistent with the pKa values in H2O being typically 0.05-0.6lower than in D2O [53][54][55].To summarize, two pKa values were determined for 8a and 8b using both NMR and UV-vis spectroscopy.For 8a, the N(H) site deprotonates first followed by the O(H) site.For 8b, the O(H) site instead deprotonates first followed by the N(H) site.Deprotonation sites for 8a and 8b are shown in Scheme 3.

Effect of pH on the Photoproducts
The effect of the pH of the aqueous component of a solvent mixture on the photoproducts obtained after irradiation of 8a was investigated in 10% v/v CD3CN in aqueous solution.Prior to doing these experiments, the stability of 8a under the ambient red-light conditions used for these experiments was checked at pH 2.1, 5.0, 7.0 and 10.0 using 1 H and 19 F NMR spectroscopy.8a (1.0 mM) was found to be photostable for at least 210 min under these conditions.
Figure S15a in Section S10, SI, shows 19 F NMR spectra of 8a recorded before and after 1.To summarize, two pKa values were determined for 8a and 8b using both NMR and UV-vis spectroscopy.For 8a, the N(H) site deprotonates first followed by the O(H) site.For 8b, the O(H) site instead deprotonates first followed by the N(H) site.Deprotonation sites for 8a and 8b are shown in Scheme 3. To summarize, two pKa values were determined for 8a and 8b using both NMR and UV-vis spectroscopy.For 8a, the N(H) site deprotonates first followed by the O(H) site.For 8b, the O(H) site instead deprotonates first followed by the N(H) site.Deprotonation sites for 8a and 8b are shown in Scheme 3.

Effect of pH on the Photoproducts
The effect of the pH of the aqueous component of a solvent mixture on the photoproducts obtained after irradiation of 8a was investigated in 10% v/v CD3CN in aqueous solution.Prior to doing these experiments, the stability of 8a under the ambient red-light conditions used for these experiments was checked at pH 2.1, 5.0, 7.0 and 10.0 using 1 H and 19 F NMR spectroscopy.8a (1.0 mM) was found to be photostable for at least 210 min under these conditions.
Figure S15a in Section S10, SI, shows 19 F NMR spectra of 8a recorded before and after 1.0 and 3.0 min total irradiation, in a solution of 10:90 v/v CD3CN: phosphate buffer, pH 2.1.At pH 2.1, only CF3SO2NH2 (O-N bond cleavage) was observed.To characterize the aromatic photoproducts at this pH condition, the 1 H NMR spectrum of a partially decomposed sample (1.0 min irradiation) was recorded, Figure S15b.The chemical shifts at 10.01, 8.66, 8.11 and 7.00 ppm were assigned to BHC-CHO using authentic BHC-CHO.

Effect of pH on the Photoproducts
The effect of the pH of the aqueous component of a solvent mixture on the photoproducts obtained after irradiation of 8a was investigated in 10% v/v CD 3 CN in aqueous solution.Prior to doing these experiments, the stability of 8a under the ambient red-light conditions used for these experiments was checked at pH 2.1, 5.0, 7.0 and 10.0 using 1 H and 19 F NMR spectroscopy.8a (1.0 mM) was found to be photostable for at least 210 min under these conditions.
Figure S15a in Section S10, SI, shows 19 F NMR spectra of 8a recorded before and after 1.0 and 3.0 min total irradiation, in a solution of 10:90 v/v CD 3 CN:phosphate buffer, pH 2.1.At pH 2.1, only CF 3 SO 2 NH 2 (O-N bond cleavage) was observed.To characterize the aromatic photoproducts at this pH condition, the 1 H NMR spectrum of a partially decomposed sample (1.0 min irradiation) was recorded, Figure S15b.The chemical shifts at 10.01, 8.66, 8.11 and 7.00 ppm were assigned to BHC-CHO using authentic BHC-CHO.
Table 2. Effect of the pH of the aqueous component of the solvent on the photoproducts derived from the N-hydroxysulfonamide moiety for photodecomposition of 8a (1.0 mM) in 10:90 v/v CD 3 CN:aqueous buffer (30 mM).Note that small peaks (≤2%) from secondary products were also observed at pH 2.1 and 10.0.The effect of the pH on the photoproducts obtained upon irradiating 8b was determined under anaerobic conditions in 10% v/v CD 3 CN in aqueous buffered solution, Section S11, SI.The photoproducts generated at pH 2.5, 5.0, 7.0, 10.0 and 10.7 were characterized using 1 H NMR spectroscopy, using both fully and partially photolyzed samples.Spectra are shown in Figures S19-S22.Table 3 summarizes the percentages of the aliphatic photoproducts upon complete photodecomposition.The percentage of each photoproduct is unchanged from pH 2.1 to pH 7.0 within experimental error, with O-N bond cleavage (~80%) dominating to give CF 3 SO 2 NH 2 and BHC-CHO (12).At pH 10.0 and 10.7 both O-N bond cleavage (40% CH 3 SO 2 NH 2 ) and concomitant C-O/N-S bond cleavage (48-54% CF 3 SO 2 − ) bond cleavage pathways are important.The amount of C-O bond cleavage is small and is almost pH independent within experimental error.Peaks were observed in partially irradiated samples that could be attributed to BHCM-OH (3) and BHC-CHO (12).

Effect of the Excitation Wavelength on the Photoproducts
The photoproducts obtained upon irradiation of 8a and 8b (1.0 mM) at two different excitation wavelengths (355 and 270 nm) were investigated in a mixture of phosphate buffer (30 mM, pH 7.0) and CD 3 CN (40:60, v/v), using a xenon lamp in conjunction with a monochromator.In each system, the excitation wavelength did not have any effect on the observed photoproducts (Table S3, SI).This is consistent with a π-π* transition occurring for this system regardless of the excitation wavelength.

Discussion
Three (6-bromo-7-hydroxycoumarin-4-yl)methyl-caged N-hydroxysulfonamides were synthesized.Detailed mechanistic studies were carried out for two of these compounds (8a and 8b).Studies by others of molecules photocaged with the (6-bromo-7-hydroxycoumarin-4-yl)methyl moiety have shown that for these systems, upon π-π* excitation, internal conversion to the lowest-energy singlet excited state occurs, followed by rapid heterolytic C-O bond cleavage, to release the molecule of interest and generate a carbocation intermediate [27,29,57,58,60].The carbocation rapidly reacts with nucleophiles including solvent For both of our systems, the photoproduct percentages were not affected by the excitation wavelength selected using a xenon lamp in conjunction with a monochromator, consistent with π-π* excitation occurring regardless of the excitation wavelength.The triplet state quenchers oxygen (aerobic versus anaerobic conditions), p-terphenyl and cyclohexadiene had no significant effect on the observed percentages of the photoproducts, which indicates no involvement of triplet excited state species in the bond cleavage events leading to the primary photoproducts.Hence, both 8a and 8b undergo bond cleavage from singlet excited state species.The photoproduct quantum yields of both systems were also determined.The photoproduct quantum yield values for 8a and 8b were 0.038 and 0.079, respectively, at 313 nm in CH 3 OH.The quantum yield of 6-bromo-7-hydroxy-4methylcoumarin was reported to be 0.13 (350 nm, aqueous solution) [36].
The pKa of the O(H) of the coumarin photocage and N(H) of the N-hydroxysulfonamide moiety played a crucial role in determining the mechanism of photodecomposition for the analogous (6-hydroxynapthalen-2-yl)methyl (6,2-HNM) photocaged N-hydroxytrifluoromethanesulfonamide system (6,2-HNM-ON(H)SO 2 CF 3 ) [62].This latter molecule also generates a carbocation, (H)NO and CF 3 SO 2 − upon rapid concomitant C-O/N-S heterolytic bond cleavage in the lowest singlet excited state of this system [62].The ground state pKa values for the N(H) and O(H) protons of 8a and 8b were therefore determined using a combination of 19 F NMR spectroscopy, 1  The pKa values were also determined for 8b.The pKa of the N(H) of 8b is 10.11 ± 0.03 (8:92 v/v CH 3 CN:D 2 O, I = 1.0 M, NaCF 3 SO 3 , 1 H NMR spectroscopy).The pKa of the O(H) was found to be 6.47 ± 0.03 for 8b (8:92 v/v CH 3 CN:H 2 O) using UVvis spectroscopy.The pKa of the O(H) in these systems therefore follows the order 8a (6.35 ± 0.03) < 8b (6.47 ± 0.03) < 6-bromo-7-hydroxy-4-methylcoumarin (6.91 ± 0.03).Interestingly, this order indicates that the electron-withdrawing ability of the deprotonated ON − SO 2 CF 3 moiety in 8a is slightly better than the neutral N(H)SO 2 CH 3 group in 8b.
Studying the effect of varying the volume percentages of phosphate buffer (pH 7.0) and CD 3 CN gave valuable information on the mechanisms of photodecomposition of 8a.Analysis of the photoproducts showed that two pathways for decomposition occur-concomitant C-O/N-S bond cleavage and O-N bond cleavage, Pathways 1 and 3, Scheme 2. The aromatic photoproducts observed in partially photolyzed solutions were BHCM-OH and (E)-and (Z)-BHC-oximes from C-O/N-S bond cleavage, Pathway 1, and BHC-CHO from O-N bond cleavage, Pathway 3. The generation of HNO was also demonstrated, using an established phosphine trapping agent, which reacts with HNO to produce the characteristic phosphine ylide; hence not all the released NO − generated upon C-O/N-S bond cleavage is trapped by the carbocation intermediate to give an oxime.The amount of C-O/N-S bond cleavage progressively decreases as the percentage of phosphate buffer in the solvent mixture, Figure 2. Hence, increasing the volume percentage of H 2 O in the solvent mixture results in a decrease in the desired HNO-releasing pathway.This result parallels what was observed for the 6,2-HNM-ON(H)SO 2 CF 3 system [24].However, only O-N bond cleavage was observed for 6,2-HNM-ON(H)SO 2 CF 3 in pure CD 3 CN, whereas 36% C-O/N-S bond cleavage occurs for 8a in this solvent.For 6,2-HNM-ON(H)SO 2 CF 3 it was proposed that the addition of a trace amount of water or another weak base was required for C-O/N-S bond cleavage since the N(H) of the N-hydroxysulfonamide of the reactant must be deprotonated [23].DFT calculations are currently underway, with preliminary results suggesting that deprotonation of the N(H) of the reactant is required for HNO generation.One important difference between the two systems is the excited state pKa* for the O(H), with the value anticipated to be lower for (6-bromo-7-hydroxycoumarin-4-yl)methyl-caged molecules (pKa* for O(H) = 0.9 for 7-hydroxy-4-methylcoumarin compared with 6,2-HNM-ON(H)SO 2 CF 3 (pKa* for O(H)~3.4 ± 0.4)) [62,63].Unfortunately, a fluorescence titration experiment could not be conducted to obtain an estimate of pKa* for the O(H) substituent of the singlet excited state of 8a, since the compound was not stable at pH values less than 0.9.
The effect of pH on the photoproducts and hence the mechanism of photodecomposition was also studied for 8a, in 10:90 v/v mixtures of CD 3 CN:aqueous buffer.When the pH of the aqueous component was pH 2.1, essentially only O-N bond cleavage was observed.However, at pH 5.0, 7.0 and 10.0, 20% CF 3 SO 2 − and 80% CF 3 SO 2 NH 2 was observed at all pH conditions.This is consistent with deprotonation of the N(H) of 8a being essential for concomitant C-O/N-S bond cleavage (pKa 3.42 ± 0.02 in 8:92 v/v CH 3 CN:D 2 O, I = 1.0 M, NaCF 3 SO 3 ).Interestingly, deprotonation of the 6-hydroxy substituent of 8a (pKa 6.35 ± 0.03 in 8:92 v/v CH 3 CN:H 2 O) had no effect on the percentages of the observed photoproducts in 92:8 v/v mixtures of aqueous buffer:CD 3 CN.Given that the N(H) is some distance from the chromophore and that bond conjugation does not extend to this site, pKa(NH)* is probably not that different from the ground state pKa(NH).However, this will not be the case for the 6-hydroxy substituent of the coumarin, as it is well-known that the pKa of aromatic OH groups can drop several orders of magnitude upon excitation of the molecule [63], consistent with a pKa* value of 0.9 for the 7-hydroxy substituent of 7-hydroxy-4-methylcoumarin [63].Hence, it is likely that this site is deprotonated in the excited state at even the lowest pH condition (pH 2.1) of this study.Unpublished data from our labs show that substituting the OH substituent of both Examination of the results for the related 8b system revealed some key differences.Firstly, unlike the 8a system, increasing the volume percentage of aqueous phosphate buffer (pH 7.0) in CD 3 CN/phosphate buffer mixtures does not have much effect on the photoproduct percentages, Table 1.For this system, 79-93% CH 3 SO 2 NH 2 was observed.Others have reported that CH 3 SO 3 − can be formed from the reaction of CH 3 SO 2 − with the hydroxyl radical [64].Trace amounts of the hydroxyl radical are generated upon photolysis of aqueous solutions.Importantly, the pKa of the N(H) of 8b was 10.11 ± 0.03 (in D 2 O with 8% v/v CH 3 CN, I = 1.0 M, CF 3 SO 3 Na); hence generation of HNO via C-O/N-S bond cleavage is not favorable since this site is protonated in all but the most basic solvent systems studied.This conclusion is supported by the results obtained upon varying the pH of the aqueous component for 92:8 v/v aqueous solution:CD 3 CN mixtures, Table 3. Specifically, while the amount of HNO generation remained low when the pH of the aqueous component was pH 5.0 and 7.0 (~3% CH 3  The latter molecule would then decompose further to generate HNO and the sulfinate product.Alternatively, subsequent nucleophilic attack of the N lone pair of the anion of the N-hydroxysulfonic acid was proposed, to give the oxime product via a nitroso compound which subsequently tautomerizes.The possible role of the protonation state of the N(H) was not investigated in this study and there was no experimental evidence for C-O bond cleavage to give the anion of the parent N-hydroxysulfonic acid in addition to the carbocation.Based on our results, we think that deprotonation of the N(H) will also be required for HNO generation for these two systems, and that the HNO-generating pathway instead likely involves concomitant C-O/N-S bond cleavage.It is likely that NO − is released and is then trapped by the resulting carbocation before it can escape the solvent cage.

Conclusions
Three photoactive N-hydroxysulfonamides photocaged with the (6-bromo-7-hydroxycoumarin-4-yl)methyl chromophore have been successfully synthesized, and the mechanisms of photodecomposition investigated for 8a and 8b.The pKa values for the N(H) and O(H) sites were determined for the ground state molecules by UV-vis and/or NMR spectroscopy titration experiments.The release of HNO from these molecules depends on the pH of the aqueous component of the system, with deprotonation of the N(H) proton required for efficient concerted C-O/N-S bond cleavage to give a carbocation, NO − and a sulfinate.The protonation state of the O(H) does not have any effect on the observed photoproducts.In the absence of a species that reacts rapidly with (H)NO, this species reacts with the carbocation intermediate to ultimately generate (E)-BHC-oxime and (Z)-BHC-oxime.However, O-N bond cleavage competes with the desired HNO-generating pathway and becomes more favorable when the volume percentage of water in acetonitrile/water solvent mixtures is increased.We hypothesize that one of the roles of water is to deprotonate the methylene carbon in the excited state molecule, which leads to O-N bond cleavage.Additionally, water can solvate the developing anionic leaving group, stabilizing the transition state required for O-N bond cleavage.Water is also expected to stabilize the carbocation intermediate generated upon C-O and concomitant C-O/N-S bond cleavage.
With photolysis under neutral or basic conditions, only C-O/N-S and O-N bond cleavage are observed for 8a, whereas a small amount of C-O bond cleavage is also observed for 8b.From a comparison of the photoproducts at pH conditions where the N(H) is deprotonated for both systems, it is clear that the presence of the electron-withdrawing CF 3 SO 2 moiety of 8a promotes undesired O-N bond cleavage compared with the 8b system, presumably by weakening the C-H bond and lowering the pKa of the methylene proton, resulting in O-N bond cleavage.It is likely that bond cleavage occurs in the singlet excited state as observed for a closely related (6-hydroxynapthalen-2-yl)methyl-photocaged system [62], since triplet quenchers had no effect on the observed photoproducts.To summarize, detailed mechanistic studies have allowed us to obtain fundamental information of the factors that ultimately determine the pathway(s) of photodecomposition for photoactive Piloty's acid-based systems.

General Information
All precursors, reagents and solvents were purchased from commercially available sources and used without purification, unless otherwise stated.Reagent-grade dichloromethane (CH 2 Cl 2 ) was distilled from CaH 2 before use.Anhydrous THF and anhydrous pyridine were purchased from commercial sources.Flash column chromatography was performed using silica gel (60 Å, 40-63 µm or 230-400 mesh), typically with elution using ACS-grade ethyl acetate and petroleum ether (boiling point range: 30-60 • C).Commercial anhydrous ethanol was used for trituration.All solid products were ground and dried in high vacuo before characterizing by 1 H, 13 C and 19 F NMR spectroscopy.NMR spectra were recorded using a Bruker 400 MHz NMR spectrometer with a 5 mm probe at 25 ± 1 • C and analyzed by MestReNova (MNOVA, version 11.3) or TOPSPIN NMR software (version 4.3.0).TSP (3-(trimethylsilyl)propionic-2,2,3,3-d 4 acid, sodium salt) was used as an internal reference standard for 1 H NMR spectroscopy, and an α,α,α-trifluorotoluene solution in CD 3 CN (−62.9 ppm) was used as an external reference standard for 19 F NMR spectroscopy.Anaerobic NMR solutions were prepared and transferred to NMR tubes in a glovebox.
Preparation of the anaerobic sample solutions was carried out inside an MBraun Labmaster 130 glove box equipped with O 2 and H 2 O sensors.Solutions were prepared in dim or red light conditions.Buffer solutions (0.10 M and 30 mM; phosphate, acetate or carbonate buffers) were adjusted to the desired pH (pD) with the addition of a solution of either HCl or NaOH solutions and subsequently degassed with argon or nitrogen.
pH measurements were carried using a Thermo Scientific Orion Star A211 pH meter connected to Mettler-Toledo pH combination micro electrode or an Orion Model 710A pH meter equipped with Mettler-Toledo Inlab 423 or 421 electrodes.Measurements were carried out at room temperature.The pH was adjusted using either NaOH or HCl solutions in water or D 2 O.For solvent mixtures the reported pH (pD) value is for the aqueous component.High-Resolution mass spectra were performed using an Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany).Mass spectra were recorded in the positive ionization mode with a scan range of 50-700 m/z, a mass resolving power setting of 140,000.The reported compounds were measured as pure solids ionized by electronically excited Helium gas using an ID-cube DART-source (Ionsense, Saugus, MA, USA).Isotopic patterns were simulated using the software Thermo Xcalibur 3.0.63(Thermo Scientific, Bremen, Germany).

Photolysis Experiments
A Rayonet mini-photoreactor (RMR-600, 8 bulbs, 4 W) equipped with a fan was used to irradiate the sample solutions using 350 nm light bulbs (8 bulbs, 4 W).The samples (~1.0 mM) were prepared under anaerobic conditions inside a glovebox by dissolving in a mixture of buffer and CD 3 CN and transferred in air-tight J-Young quartz NMR tubes.After each irradiation, the sample was analyzed by 1 H and/or 19 F NMR spectroscopy.

Determination of pKa by UV-Vis Spectroscopy
8a or 8b were dissolved in a mixture of aerobic CH 3 CN and H 2 O (8:92 v/v CH 3 CN:H 2 O).The solution was circulated using a peristaltic pump through a 1 cm path length quartz flow-through cell at 25.0 • C. A small volume of acid (~1 M HCl; negligible change in total volume of the solution) was added to the solution (3.5 × 10 −5 M, 30 mL).Absorbance spectra were recorded after the pH had stabilized in the reservoir flask.

Determination of pKa by NMR Spectroscopy
The following aqueous solutions were prepared in D 2 O: 0.10 M HCl (pD 1.4), phosphate buffer (30 mM, pD 2.4-3.9 and 6.2-8.2),acetate buffer (30 mM, pD 4.0-6.0),borate buffer (30 mM, pD 8.9-9.9),carbonate buffer (30 mM, pD 9.6-11.0),and 0.010 M NaOH (pD 12.4).The pD of the deuterated buffered solutions were adjusted to the desired value using either HCl or NaOH (pD = pH + 0.4) [65].A small volume of 8a or 8b dissolved in CH 3 CN was then added to an aerobic aqueous buffered solution.The final solution was ~1.0 mM, with 8% v/v CH 3 CN in deuterated aqueous buffer.A constant ionic strength of 1.0 M was maintained, by the addition of NaCF 3 SO 3 .

Determination of the Photoproduct Quantum Yields (Φ)
The photoproduct quantum yields for 8a and 8b at 313 nm were determined by actinometry in aerobic CH 3 OH, using the isomerization of trans-azobenzene to its cis isomer as a reference compound (Φ = 0.14 at 313 nm [57]), following our earlier published procedure [25].The percentage of trans-azobenzene converted to cis-azobenzene upon irradiation was followed by UV-vis spectroscopy, whereas the photodecomposition of 8a (1.00 mM) and 8b (1.00 mM) was followed by 19 F or 1 H NMR spectroscopy.was added all at once to a stirred solution of 4-bromoresorcinol (1, 5.26 g, 27.8 mmol) in methanesulfonic acid (40 mL) under nitrogen at room temperature.The reaction mixture was stirred for 2 h at room temperature before the resulting reddish-brown solution was slowly poured into ice-water (200 mL) and stirred for 30 min to give an off-white precipitate.The precipitate was collected by vacuum filtration, washed with cold water (3 × 50 mL) and dried in vacuo.The off-white solid was triturated with a 20:80 mixture of ethyl acetate/petroleum ether (100 mL), filtered, washed with petroleum ether, and dried in vacuo to afford the title compound as a pale pink solid (6.66 g, 83%). 1 H NMR (400 MHz, DMSO-d6) δ 11.57(s, 1H), 8.00 (s, 1H), 6.92 (s, 1H), 6.48 (t, 1H, J = 0.8 Hz), 5.00 (d, 2H, J = 0.8 Hz) [30].

6-Bromo-7-O-(methoxymethyl)-4-hydroxymethylcoumarin (4)
Anhydrous N,N-diisopropylethylamine (DIPEA, 3.21 mL, d = 0.742 g/mL at 25 °C, 18.4 mmol) was added all at once to a stirred suspension of 3 (5.12g, 18.9 mmol) in anhydrous CH2Cl2 (210 mL) at room temperature under Ar.The resulting brown suspension was cooled to 0 °C before adding chloromethyl methyl ether (MOMCl, 1.36 mL, d = 1.06 g/mL at 25 °C, 18.0 mmol) dropwise over 5 min at 0 °C.The resulting brown suspension was stirred at 0-16 °C for 2 h before being allowed to warm to room temperature and stirred overnight.The resulting light brown suspension was quenched by mixing with aqueous citric acid (0.5 M, 160 mL).A light brown precipitate was observed which we attempted to dissolve by adding CHCl3 (150 mL) but the precipitate remained undissolved.The precipitate was filtered, washed with CHCl3 (50 mL) and dried in vacuo to afford an off-white solid (3.77 g).The light brown organic layer from the filtrate was separated, and the aqueous layer was extracted with CHCl3 (2 × 100 mL).The combined organic extracts were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and the solvent was removed in vacuo to obtain an off-white solid.The solid was purified by trituration using Methyl 4-chloroacetoacetate (4.82 mL, d = 1.305 g/mL at 25 • C, 41.8 mmol) was added all at once to a stirred solution of 4-bromoresorcinol (1, 5.26 g, 27.8 mmol) in methanesulfonic acid (40 mL) under nitrogen at room temperature.The reaction mixture was stirred for 2 h at room temperature before the resulting reddish-brown solution was slowly poured into ice-water (200 mL) and stirred for 30 min to give an off-white precipitate.The precipitate was collected by vacuum filtration, washed with cold water (3 × 50 mL) and dried in vacuo.The off-white solid was triturated with a 20:80 mixture of ethyl acetate/petroleum ether (100 mL), filtered, washed with petroleum ether, and dried in vacuo to afford the title compound as a pale pink solid (6.66 g, 83%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.57(s, 1H), 8.00 (s, 1H), 6.92 (s, 1H), 6.48 (t, 1H, J = 0.8 Hz), 5.00 (d, 2H, J = 0.8 Hz) [30].was added all at once to a stirred solution of 4-bromoresorcinol (1, 5.26 g, 27.8 mmol) in methanesulfonic acid (40 mL) under nitrogen at room temperature.The reaction mixture was stirred for 2 h at room temperature before the resulting reddish-brown solution was slowly poured into ice-water (200 mL) and stirred for 30 min to give an off-white precipitate.The precipitate was collected by vacuum filtration, washed with cold water (3 × 50 mL) and dried in vacuo.The off-white solid was triturated with a 20:80 mixture of ethyl acetate/petroleum ether (100 mL), filtered, washed with petroleum ether, and dried in vacuo to afford the title compound as a pale pink solid (6.66 g, 83%). 1 H NMR (400 MHz, DMSO-d6) δ 11.57(s, 1H), 8.00 (s, 1H), 6.92 (s, 1H), 6.48 (t, 1H, J = 0.8 Hz), 5.00 (d, 2H, J = 0.8 Hz) [30].

6-Bromo-7-O-(methoxymethyl)-4-hydroxymethylcoumarin (4)
Anhydrous N,N-diisopropylethylamine (DIPEA, 3.21 mL, d = 0.742 g/mL at 25 °C, 18.4 mmol) was added all at once to a stirred suspension of 3 (5.12g, 18.9 mmol) in anhydrous CH2Cl2 (210 mL) at room temperature under Ar.The resulting brown suspension was cooled to 0 °C before adding chloromethyl methyl ether (MOMCl, 1.36 mL, d = 1.06 g/mL at 25 °C, 18.0 mmol) dropwise over 5 min at 0 °C.The resulting brown suspension was stirred at 0-16 °C for 2 h before being allowed to warm to room temperature and stirred overnight.The resulting light brown suspension was quenched by mixing with aqueous citric acid (0.5 M, 160 mL).A light brown precipitate was observed which we attempted to dissolve by adding CHCl3 (150 mL) but the precipitate remained undissolved.The precipitate was filtered, washed with CHCl3 (50 mL) and dried in vacuo to afford an off-white solid (3.77 g).The light brown organic layer from the filtrate was separated, and the aqueous layer was extracted with CHCl3 (2 × 100 mL).The combined organic extracts were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and the solvent was removed in vacuo to obtain an off-white solid.The solid was purified by trituration using A stirred suspension of compound 2 (1.86 g, 6.42 mmol) in water (500 mL) was heated under reflux for 5 days before the resulting suspension was allowed to cool to room temperature.The precipitate settled down at the bottom of the flask.Most of the water from the top was carefully decanted and the remaining water was removed in vacuo.The resulting off-white residue was triturated with diethyl ether/petroleum ether (1:2, 240 mL) and filtered off to afford the title compound as an off-white solid (1.51 g, 87%). 1  was added all at once to a stirred solution of 4-bromoresorcinol (1, 5.26 g, 27.8 mmol) in methanesulfonic acid (40 mL) under nitrogen at room temperature.The reaction mixture was stirred for 2 h at room temperature before the resulting reddish-brown solution was slowly poured into ice-water (200 mL) and stirred for 30 min to give an off-white precipitate.The precipitate was collected by vacuum filtration, washed with cold water (3 × 50 mL) and dried in vacuo.The off-white solid was triturated with a 20:80 mixture of ethyl acetate/petroleum ether (100 mL), filtered, washed with petroleum ether, and dried in vacuo to afford the title compound as a pale pink solid (6.66 g, 83%). 1 H NMR (400 MHz, DMSO-d6) δ 11.57(s, 1H), 8.00 (s, 1H), 6.92 (s, 1H), 6.48 (t, 1H, J = 0.8 Hz), 5.00 (d, 2H, J = 0.8 Hz) [30].

6-Bromo-7-O-(methoxymethyl)-4-hydroxymethylcoumarin (4)
Anhydrous N,N-diisopropylethylamine (DIPEA, 3.21 mL, d = 0.742 g/mL at 25 °C, 18.4 mmol) was added all at once to a stirred suspension of 3 (5.12g, 18.9 mmol) in anhydrous CH2Cl2 (210 mL) at room temperature under Ar.The resulting brown suspension was cooled to 0 °C before adding chloromethyl methyl ether (MOMCl, 1.36 mL, d = 1.06 g/mL at 25 °C, 18.0 mmol) dropwise over 5 min at 0 °C.The resulting brown suspension was stirred at 0-16 °C for 2 h before being allowed to warm to room temperature and stirred overnight.The resulting light brown suspension was quenched by mixing with aqueous citric acid (0.5 M, 160 mL).A light brown precipitate was observed which we attempted to dissolve by adding CHCl3 (150 mL) but the precipitate remained undissolved.The precipitate was filtered, washed with CHCl3 (50 mL) and dried in vacuo to afford an off-white solid (3.77 g).The light brown organic layer from the filtrate was separated, and the aqueous layer was extracted with CHCl3 (2 × 100 mL).The combined organic extracts were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and the solvent was removed in vacuo to obtain an off-white solid.The solid was purified by trituration using Anhydrous N,N-diisopropylethylamine (DIPEA, 3.21 mL, d = 0.742 g/mL at 25 • C, 18.4 mmol) was added all at once to a stirred suspension of 3 (5.12g, 18.9 mmol) in anhydrous CH 2 Cl 2 (210 mL) at room temperature under Ar.The resulting brown suspension was cooled to 0 • C before adding chloromethyl methyl ether (MOMCl, 1.36 mL, d = 1.06 g/mL at 25 • C, 18.0 mmol) dropwise over 5 min at 0 • C. The resulting brown suspension was stirred at 0-16 • C for 2 h before being allowed to warm to room temperature and stirred overnight.The resulting light brown suspension was quenched by mixing with aqueous citric acid (0.5 M, 160 mL).A light brown precipitate was observed which we attempted to dissolve by adding CHCl 3 (150 mL) but the precipitate remained undissolved.The precipitate was filtered, washed with CHCl 3 (50 mL) and dried in vacuo to afford an off-white solid (3.77 g).The light brown organic layer from the filtrate was separated, and the aqueous layer was extracted with CHCl 3 (2 × 100 mL).The combined organic extracts were washed with brine (500 mL), dried over anhydrous Na 2 SO 4 , filtered, and the solvent was removed in vacuo to obtain an off-white solid.The solid was purified by trituration using a mixture of petroleum ether and CH 2 Cl 2 (5:1, 120 mL) to afford an off-white solid (1.66 g).Total isolated yield of the title compound was 5.43 g (96%). 1  N-Hydroxyphthalimide (4.32 g, 26.5 mmol) and triphenylphosphine (6.93 g, 26.4 mmol) were added all at once to a stirred solution of 4 (5.56 g, 17.6 mmol) in anhydrous THF (350 mL) at 2 °C under Ar.Diisopropyl azodicarboxylate (DIAD, 5.20 mL, d = 1.027 g/mL at 25 °C, 26.4 mmol) was added dropwise to the reddish solution over 10 min at 0 °C.At this point, the reaction mixture turned to a pale yellow emulsion, which was stirred at 0-10 °C for 1 h before being allowed to warm to room temperature and stirred overnight.The resulting suspension was filtered to afford a white solid, which was washed with EtOH (50 mL) followed by petroleum ether (20 mL), before being dried in vacuo to give an off-white crude solid.The reddish filtrate was concentrated in vacuo, and the resulting solid was triturated with EtOH (100 mL), filtered and the resulting solid washed with EtOH (20 mL) followed by petroleum ether (20 mL) to obtain an off-white crude solid.The combined crude solid product was ground and washed sequentially with H2O (30 mL), EtOH (30 mL) and petroleum ether (20 mL) and dried in vacuo to afford the title compound as an off-white solid (6.21 g, 76%).Mp: 226-227 °C. 1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.91-7.85(m, 2H), 7.83-7.76(m, 2H), 7.18 (s, 1H), 6.51 (t, 1H, J = 1.2 Hz), 5.34 (s, 2H), 5.29 (d, 2H, J = 1.2 Hz), 3.54 (s, 3H). 13

O-[[6-Bromo-7-O-(methoxymethyl)coumarin-4-yl]methyl]hydroxylamine (6)
Hydrazine monohydrate (2.2 mL, d = 1.032 g/mL at 25 °C, 45 mmol) was added all at once to a stirred white emulsion of 5 (6.19 g, 13.5 mmol) in CH2Cl2 (350 mL) at 0 °C.The reaction mixture was stirred 0-4 °C for 30 min before being allowed to warm to room temperature and stirred overnight.The resulting suspension was filtered, and the resulting white solid was washed with CH2Cl2 (100 mL).The white solid was stirred in CH2Cl2 (100 mL) for 30 min and filtered off.The combined organic filtrates were washed with H2O (2 × 450 mL) and brine (500 mL), and dried over anhydrous Na2SO4 for 30 min.The drying agent was filtered off, and the solvent was removed in vacuo to afford the title compound as a pure white solid (4.02 g, 91%).Mp: 155-158 °C. 1   C for 1 h before being allowed to warm to room temperature and stirred overnight.The resulting suspension was filtered to afford a white solid, which was washed with EtOH (50 mL) followed by petroleum ether (20 mL), before being dried in vacuo to give an off-white crude solid.The reddish filtrate was concentrated in vacuo, and the resulting solid was triturated with EtOH (100 mL), filtered and the resulting solid washed with EtOH (20 mL) followed by petroleum ether (20 mL) to obtain an off-white crude solid.The combined crude solid product was ground and washed sequentially with H 2 O (30 mL), EtOH (30 mL) and petroleum ether (20 mL) and dried in vacuo to afford the title compound as an off-white solid (6.21 g, 76%).Mp: 226-227 • C. 1 H NMR (400 MHz, CDCl 3 ) δ 8.28 (s, 1H), 7.91-7.85(m, 2H), 7.83-7.76(m, 2H), 7.18 (s, 1H), 6.51 (t, 1H, J = 1.2 Hz), 5.34 (s, 2H), 5.29 (d, 2H, J = 1.2 Hz), 3.54 (s, 3H). 13  N-Hydroxyphthalimide (4.32 g, 26.5 mmol) and triphenylphosphine (6.93 g, 26.4 mmol) were added all at once to a stirred solution of 4 (5.56 g, 17.6 mmol) in anhydrous THF (350 mL) at 2 °C under Ar.Diisopropyl azodicarboxylate (DIAD, 5.20 mL, d = 1.027 g/mL at 25 °C, 26.4 mmol) was added dropwise to the reddish solution over 10 min at 0 °C.At this point, the reaction mixture turned to a pale yellow emulsion, which was stirred at 0-10 °C for 1 h before being allowed to warm to room temperature and stirred overnight.The resulting suspension was filtered to afford a white solid, which was washed with EtOH (50 mL) followed by petroleum ether (20 mL), before being dried in vacuo to give an off-white crude solid.The reddish filtrate was concentrated in vacuo, and the resulting solid was triturated with EtOH (100 mL), filtered and the resulting solid washed with EtOH (20 mL) followed by petroleum ether (20 mL) to obtain an off-white crude solid.The combined crude solid product was ground and washed sequentially with H2O (30 mL), EtOH (30 mL) and petroleum ether (20 mL) and dried in vacuo to afford the title compound as an off-white solid (6.21 g, 76%).Mp: 226-227 °C. 1

N-[(6-Bromo-7-O-(methoxymethyl)coumarin-4-yl)methoxy]methanesulfonamide (7b)
DMAP (0.093 g, 0.76 mmol) and anhydrous pyridine (0.060 mL, d = 0.978 g/mL at 25 °C, 0.74 mmol) were added all at once to a stirred colorless solution of 6 (0.252 g, 0.763 mmol) in anhydrous CH2Cl2 (30 mL) at room temperature under Ar.A solution of CH3SO2Cl (0.065 mL, d = 1.48 g/mL at 25 °C, 0.84 mmol,) in anhydrous CH2Cl2 (3 mL) was added dropwise to the solution over 5 min at room temperature.The reaction mixture was stirred at room temperature for 2 h under Ar and completion of the reaction was confirmed by 1 H NMR spectroscopy.The solution was concentrated in vacuo and the resulting white sticky solid was purified by flash column chromatography (silica gel, 40:60 ethyl acetate:petroleum ether) to afford the title compound as a white solid (0.105 g, 34%).Mp: 190-193 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.03 (s, 1H), 7.28 (s, 1H), 6.48 (br s, 1H), 5.43 (s, 2H), 5.16 (br s, 2H), 3.43 (s, 3H), 3.07 (s, 3H). 13  The pale yellow reaction mixture was stirred at −65 • C for 2 h before being allowed to warm to room temperature and stirred overnight.An aliquot was removed and analyzed by 1 H NMR spectroscopy, which showed some unreacted starting material.Additional CF 3 SO 2 Cl (0.24 mL, d = 1.583 g/mL at 25 • C, 2.3 mmol) in CH 2 Cl 2 (2 mL) was added in one portion to the pale yellow solution at room temperature and the mixture was stirred overnight.Complete consumption of the starting material was observed by 1 H NMR spectroscopy.The reaction mixture was passed through a short silica plug, eluting with CH 2 Cl 2 (300 mL) followed by EtOAc (100 mL), and the solution was concentrated in vacuo to obtain a pale yellow sticky solid.The crude product was purified by flash column chromatography (silica gel, 20:80 ethyl acetate:petroleum ether) to afford the title compound as an off-white solid (0.383 g, 27%).Mp: 152-154 • C. 19  Sulfonylation of 6 with CF 3 SO 2 Cl under these reaction conditions also generate a small amount of bis-sulfonylation product 9 (0.114 g, 6%), which was isolated from the flash silica chromatography column using a mixture of 20:80 v/v ethyl acetate:petroleum ether. 1 H NMR (400 MHz, CD 3 Cl) δ 7.84 (s, 1H), 7.18 (s, 1H), 6.46 (d, 1H, J = 0.4 Hz), 5.33 (s, 2H), 4.61 (d, 2H, J = 0.9 Hz), 3.53 (s, 3H), and 19 F NMR (470 MHz, CD 3 Cl) δ −77.45.The pale yellow reaction mixture was stirred at -65 °C for 2 h before being allowed to warm to room temperature and stirred overnight.An aliquot was removed and analyzed by 1 H NMR spectroscopy, which showed some unreacted starting material.Additional CF3SO2Cl (0.24 mL, d = 1.583 g/mL at 25 °C, 2.3 mmol) in CH2Cl2 (2 mL) was added in one portion to the pale yellow solution at room temperature and the mixture was stirred overnight.Complete consumption of the starting material was observed by 1 H NMR spectroscopy.The reaction mixture was passed through a short silica plug, eluting with CH2Cl2 (300 mL) followed by EtOAc (100 mL), and the solution was concentrated in vacuo to obtain a pale yellow sticky solid.The crude product was purified by flash column chromatography (silica gel, 20:80 ethyl acetate:petroleum ether) to afford the title compound as an off-white solid (0.383 g, 27%).Mp: 152-154 °C. 19F NMR (376 MHz, DMSO-d6) δ -73.1 (s). 1 H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.28 (s, 1H), 6.49 (br s, 1H), 5.44 (s, 2H), 5.21 (br s, 2H), 3.42 (s, 3H, overlap with H2O peak), NH peak was not observed. 13 Sulfonylation of 6 with CF3SO2Cl under these reaction conditions also generate a small amount of bis-sulfonylation product 9 (0.114 g, 6%), which was isolated from the flash silica chromatography column using a mixture of 20:80 v/v ethyl acetate:petroleum ether.Sulfonylation of 6 with CH 3 SO 2 Cl under these reaction conditions also generated a small amount of bis-sulfonylation product 10 (0.24 g, 8%), which was isolated from the flash silica column chromatography using a mixture of 40:60 v/v ethyl acetate: petroleum ether. 1  were added all at once to a stirred solution of 6 (0.354 g, 1.07 mmol) in anhydrous CH2Cl2 (50 mL) at −45 °C under Ar.A solution of (2-methanesulfonyl)benzenesulfonyl chloride (0.414 g, 1.63 mmol) in anhydrous CH2Cl2 (20 mL) was added to the resulting clear solution in one portion at −45 °C and the reaction mixture was allowed to stir at −40 °C for 30 min before being allowed to warm to room temperature and stirred for 16 h (completion of the reaction was confirmed by 1 H NMR spectroscopy).The resulting pale yellow mixture was passed through a short silica plug using an eluent mixture of CH2Cl2 and EtOAc (4:1, 300 mL).The solvent was removed in vacuo to afford a yellow solid (0.624 g).The crude product was purified by flash column chromatography (silica gel, 2:98 MeOH: CH2Cl2) to afford the title compound as a white solid (0.353 g, 60%).Mp: 174-177 °C. 1  Acetyl chloride (1.8 mL, d = 1.104 g/mL at 25 °C, 25 mmol) was added dropwise over 5 min to a stirred solution of 7a (0.175 g, 0.379 mmol) in anhydrous MeOH and anhydrous THF (1:1 mixture, 8 mL) at room temperature under Ar.Bubbles of HCl gas were observed during the addition.The resulting colorless clear solution was stirred at room temperature for 4h (completion of the reaction was confirmed by 1 H NMR analysis).The solvent was removed in vacuo to obtain an off-white solid (0.161 g).The crude solid product was purified by gravity column chromatography (silica gel, initially 2:98 MeOH:CH2Cl2 followed by 5:95 MeOH:CH2Cl2) followed by trituration using MeOH and CH2Cl2 (1:99, 2 × 5 mL) to afford the title compound as an off-white solid (0.132 g, 83%).Dec: 141-152 °C. 19 were added all at once to a stirred solution of 6 (0.354 g, 1.07 mmol) in anhydrous CH2Cl2 (50 mL) at −45 °C under Ar.A solution of (2-methanesulfonyl)benzenesulfonyl chloride (0.414 g, 1.63 mmol) in anhydrous CH2Cl2 (20 mL) was added to the resulting clear solution in one portion at −45 °C and the reaction mixture was allowed to stir at −40 °C for 30 min before being allowed to warm to room temperature and stirred for 16 h (completion of the reaction was confirmed by 1 H NMR spectroscopy).The resulting pale yellow mixture was passed through a short silica plug using an eluent mixture of CH2Cl2 and EtOAc (4:1, 300 mL).The solvent was removed in vacuo to afford a yellow solid (0.624 g).The crude product was purified by flash column chromatography (silica gel, 2:98 MeOH: CH2Cl2) to afford the title compound as a white solid (0.353 g, 60%).Mp: 174-177 °C. 1  Acetyl chloride (1.8 mL, d = 1.104 g/mL at 25 °C, 25 mmol) was added dropwise over 5 min to a stirred solution of 7a (0.175 g, 0.379 mmol) in anhydrous MeOH and anhydrous THF (1:1 mixture, 8 mL) at room temperature under Ar.Bubbles of HCl gas were observed during the addition.The resulting colorless clear solution was stirred at room temperature for 4h (completion of the reaction was confirmed by 1 H NMR analysis).The solvent was removed in vacuo to obtain an off-white solid (0.161 g).The crude solid product was purified by gravity column chromatography (silica gel, initially 2:98 MeOH:CH2Cl2 followed by 5:95 MeOH:CH2Cl2) followed by trituration using MeOH and CH2Cl2 (1:99, 2 × 5 mL) to afford the title compound as an off-white solid (0.132 g, 83%).Dec: 141-152 °C. 19 Acetyl chloride (1.8 mL, d = 1.104 g/mL at 25 • C, 25 mmol) was added dropwise over 5 min to a stirred solution of 7a (0.175 g, 0.379 mmol) in anhydrous MeOH and anhydrous THF (1:1 mixture, 8 mL) at room temperature under Ar.Bubbles of HCl gas were observed during the addition.The resulting colorless clear solution was stirred at room temperature for 4 h (completion of the reaction was confirmed by 1 H NMR analysis).The solvent was removed in vacuo to obtain an off-white solid (0.161 g).The crude solid product was purified by gravity column chromatography (silica gel, initially 2:98 MeOH:CH 2 Cl 2 followed by 5:95 MeOH:CH 2 Cl 2 ) followed by trituration using MeOH and CH 2 Cl 2 (1:99, 2 × 5 mL) to afford the title compound as an off-white solid (0.132 g, 83%).Dec: 141-152 • C. 19  Acetyl chloride (0.80 mL, d = 1.104 g/mL at 25 °C, 11 mmol) was added dropwise over 3 min to a stirred solution of 7b (0.069 g, 0.17 mmol) in an anhydrous mixture of MeOH/THF (1:1, 6 mL) at room temperature under Ar.The resulting clear solution was stirred for 3 h at room temperature before the solvent was removed in vacuo.The resulting crude white solid was triturated with CH2Cl2 to afford the title compound as a white solid (0.049 g, 80%).Dec: 187-195 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H, Ar-OH), 10.32 (s, 1H, NH), 7.93 (s, 1H), 6.92 (s, 1H), 6.37 (s, 1H), 5.14 (s, 2H), 3.07 (s, 3H). 13

N-[(6-Bromo-7-hydroxycoumarin-4-yl)methoxy]-2-methanesulfonylbenzenesulfonamide (8c)
Acetyl chloride (1.8 mL, d = 1.104 g/mL at 25 °C, 25 mmol) was added dropwise over 5 min to a stirred suspension of 7c (0.203 g, 0.370 mmol) in a mixture of anhydrous THF and anhydrous MeOH (1:1, 6 mL) at room temperature under Ar.The suspension turned into a clear solution after complete addition of the acetyl chloride and the reaction mixture was allowed to stir at room temperature.After 2h, a white precipitate with reduced volume of the solvent was observed and an additional amount of anhydrous MeOH (5 mL) was added to the suspension to increase the solvent volume before stirring for an additional 1.5 h.The reaction suspension was filtered, and the resulting white solid was washed with a mixture of petroleum ether and CH2Cl2 (10:1, 11 mL) and dried in vacuo to afford a white flaky solid (0.144 g, 77%).Dec: 182-186 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (br.s, 1H, Ar-OH), 10.37 (br.s, 1H, NH), 8.31-8.25 (m, 1H), 8.23-8.15(m, 1H), 8.08-8.00(m, 2H), 7.87 (s, 1H), 6.89 (s, 1H), 6.32 (br s, 1H), 5.17 (br s, 2H), 3.47 (s, 3H). 13 In addition, to minimize the number of synthetic steps en route to intermediate 5, an alternative approach was explored via intermediate 11.Compound 11 was prepared in high yield from 2 (see above for the preparation of 2).However, SN2 displacement of Acetyl chloride (1.8 mL, d = 1.104 g/mL at 25 • C, 25 mmol) was added dropwise over 5 min to a stirred suspension of 7c (0.203 g, 0.370 mmol) in a mixture of anhydrous THF and anhydrous MeOH (1:1, 6 mL) at room temperature under Ar.The suspension turned into a clear solution after complete addition of the acetyl chloride and the reaction mixture was allowed to stir at room temperature.After 2 h, a white precipitate with reduced volume of the solvent was observed and an additional amount of anhydrous MeOH (5 mL) was added to the suspension to increase the solvent volume before stirring for an additional 1.5 h.The reaction suspension was filtered, and the resulting white solid was washed with a mixture of petroleum ether and CH 2 Cl 2 (10:1, 11 mL) and dried in vacuo to afford a white flaky solid (0.144 g, 77%).Dec: 182-186 Acetyl chloride (0.80 mL, d = 1.104 g/mL at 25 °C, 11 mmol) was added dropwise over 3 min to a stirred solution of 7b (0.069 g, 0.17 mmol) in an anhydrous mixture of MeOH/THF (1:1, 6 mL) at room temperature under Ar.The resulting clear solution was stirred for 3 h at room temperature before the solvent was removed in vacuo.The resulting crude white solid was triturated with CH2Cl2 to afford the title compound as a white solid (0.049 g, 80%).Dec: 187-195 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H, Ar-OH), 10.32 (s, 1H, NH), 7.93 (s, 1H), 6.92 (s, 1H), 6.37 (s, 1H), 5.14 (s, 2H), 3.07 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.17The suspension turned into a clear solution after complete addition of the acetyl chloride and the reaction mixture was allowed to stir at room temperature.After 2h, a white precipitate with reduced volume of the solvent was observed and an additional amount of anhydrous MeOH (5 mL) was added to the suspension to increase the solvent volume before stirring for an additional 1.5 h.The reaction suspension was filtered, and the resulting white solid was washed with a mixture of petroleum ether and CH2Cl2 (10:1, 11 mL) and dried in vacuo to afford a white flaky solid (0.144 g, 77%).Dec: 182-186 °C. 1  N,N-Diisopropylethylamine (DIPEA, 7.90 mL, d = 0.742 g/mL at 25 °C, 45.4 mmol) was added all at once to a stirred reddish solution of 2 (5.24 g, 18.1 mmol) in anhydrous THF (120 mL) at 2 °C under Ar.Upon addition, the color of the solution immediately turned yellow.Chloromethyl methyl ether (MOMCl, 3.20 mL, d = 1.06 g/mL at 25 °C, 42.1 mmol) was added to the yellow solution dropwise over 20 min at 1-2 °C.At this point, the solution turned a reddish color and was stirred at 1-2 °C for 30 min before stirring at room temperature overnight (20 h).After 20 h stirring, the solvent was removed in vacuo to obtain a pale yellow solid, which was re-dissolved in CHCl3 (180 mL).The organic solution was washed with H2O (3 × 200 mL) and brine (200 mL) and dried over anhydrous Na2SO4.Filtration and concentration in vacuo afforded the title compound as an off-white solid (5.547 g, 92%). 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.17 A solution of 11 (4.67 g, 14.0 mmol) in anhydrous DMF (60 mL) was added in one portion to a stirred reddish solution of N-hydroxyphthalimide (2.51 g, 15.4 mmol) and DIPEA (2.6 mL, d = 0.742 g/mL at 25 °C, 15.12 mmol) in anhydrous DMF (30 mL) at room temperature under Ar.The reaction mixture was heated to 70 °C with stirring until the reaction had proceeded to completion (7 h, checked by TLC).The reaction mixture was allowed to cool to room temperature, diluted with EtOAc (200 mL) and washed with H2O (200 mL) to remove substantial amounts of the DMF reaction solvent.A white suspension was observed in between the layers that was not soluble even upon addition of EtOAc (50 mL).The organic layer along with the white suspension was separated from the aqueous layer.Filtration of the organic layer led to the isolation of the white solid, which was identified as phthalimide by 1 H NMR spectroscopy.The aqueous layer was extracted with EtOAc (2 × 150 mL) and the combined organic extracts were washed with water (2 × 400 mL) and dried over MgSO4.Filtration and concentration in vacuo afforded a reddish solid (5.15 g).The crude product was purified by trituration with EtOH to afford a pale yellow solid containing minor impurities.The pale yellow solid was triturated with EtOAc followed by washing with EtOH to afford the title compound as an off-white solid (1.415 g, 22%) that contained a small impurity (~ 6%). 1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.89-7.87(m, 2H), 7.81-7.79(m, 2H), 7.17  N,N-Diisopropylethylamine (DIPEA, 7.90 mL, d = 0.742 g/mL at 25 °C, 45.4 mmol) was added all at once to a stirred reddish solution of 2 (5.24 g, 18.1 mmol) in anhydrous THF (120 mL) at 2 °C under Ar.Upon addition, the color of the solution immediately turned yellow.Chloromethyl methyl ether (MOMCl, 3.20 mL, d = 1.06 g/mL at 25 °C, 42.1 mmol) was added to the yellow solution dropwise over 20 min at 1-2 °C.At this point, the solution turned a reddish color and was stirred at 1-2 °C for 30 min before stirring at room temperature overnight (20 h).After 20 h stirring, the solvent was removed in vacuo to obtain a pale yellow solid, which was re-dissolved in CHCl3 (180 mL).The organic solution was washed with H2O (3 × 200 mL) and brine (200 mL) and dried over anhydrous Na2SO4.Filtration and concentration in vacuo afforded the title compound as an off-white solid (5.547 g, 92%). 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.17 (s, 1H), 6.45 (t, 1H, J = 0.8 Hz), 5.33 (s, 2H), 4.60 (d, 2H, J = 0.8 Hz), 3.52 (s, 3H).5.6.14.2-[(6-Bromo-7-O-(methoxymethyl)coumarin-4-yl)methoxy]isoindoline-1,3-dione (5) A solution of 11 (4.67 g, 14.0 mmol) in anhydrous DMF (60 mL) was added in one portion to a stirred reddish solution of N-hydroxyphthalimide (2.51 g, 15.4 mmol) and DIPEA (2.6 mL, d = 0.742 g/mL at 25 °C, 15.12 mmol) in anhydrous DMF (30 mL) at room temperature under Ar.The reaction mixture was heated to 70 °C with stirring until the reaction had proceeded to completion (7 h, checked by TLC).The reaction mixture was allowed to cool to room temperature, diluted with EtOAc (200 mL) and washed with H2O (200 mL) to remove substantial amounts of the DMF reaction solvent.A white suspension was observed in between the layers that was not soluble even upon addition of EtOAc (50 mL).The organic layer along with the white suspension was separated from the aqueous layer.Filtration of the organic layer led to the isolation of the white solid, which was identified as phthalimide by 1 H NMR spectroscopy.The aqueous layer was extracted with EtOAc (2 × 150 mL) and the combined organic extracts were washed with water (2 × 400 mL) and dried over MgSO4.Filtration and concentration in vacuo afforded a reddish solid (5.15 g).The crude product was purified by trituration with EtOH to afford a pale yellow solid containing minor impurities.The pale yellow solid was triturated with EtOAc followed by washing with EtOH to afford the title compound as an off-white solid (1.415 g, 22%) that contained a small impurity (~ 6%). 1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.89-7.87(m, 2H), 7.81-7.79(m, 2H), 7.17  A solution of 11 (4.67 g, 14.0 mmol) in anhydrous DMF (60 mL) was added in one portion to a stirred reddish solution of N-hydroxyphthalimide (2.51 g, 15.4 mmol) and DIPEA (2.6 mL, d = 0.742 g/mL at 25 • C, 15.12 mmol) in anhydrous DMF (30 mL) at room temperature under Ar.The reaction mixture was heated to 70 • C with stirring until the reaction had proceeded to completion (7 h, checked by TLC).The reaction mixture was allowed to cool to room temperature, diluted with EtOAc (200 mL) and washed with H 2 O (200 mL) to remove substantial amounts of the DMF reaction solvent.A white suspension was observed in between the layers that was not soluble even upon addition of EtOAc (50 mL).The organic layer along with the white suspension was separated from the aqueous layer.Filtration of the organic layer led to the isolation of the white solid, which was identified as phthalimide by 1 H NMR spectroscopy.The aqueous layer was extracted with EtOAc (2 × 150 mL) and the combined organic extracts were washed with water (2 × 400 mL) and dried over MgSO 4 .Filtration and concentration in vacuo afforded a reddish solid (5.15 g).The crude product was purified by trituration with EtOH to afford a pale yellow solid containing minor impurities.The pale yellow solid was triturated with EtOAc followed by washing with EtOH to afford the title compound as an off-white solid (1.415 g, 22%) that contained a small impurity (~6%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.28 (s, 1H), 7.89-7.87(m, 2H), 7.81-7.79(m, 2H), 7.17 (s, 1H), 6.51 (t, 1H, J = 1.2 Hz), 5.33 (s, 2H), 5.29 (d, 2H, J = 1.2 Hz), 3.54 (s, 3H).

Figure 2 .
Figure1ashows the19 F NMR spectrum obtained upon completely photolyzing 8a in anaerobic 80:20 v/v CD 3 CN:phosphate buffer (pH 7.0, 0.1 M).Upon photolysis, 8a released CF 3 SO 2 − (94%, −88.1 ppm); that is, almost stoichiometric release of (H)NO.In addition, a small amount of CF 3 SO 2 NH 2 (6%, −80.5 ppm) was generated via competing photoinduced O-N cleavage of 8a (Scheme 2, Pathway 3).The chemical shifts of both fluorinated photoproducts were confirmed using authentic samples of these compounds.No formation and subsequent decomposition of CF 3 SO 2 N(H)O(H) was observed, in line with results found on related systems[22][23][24].If CF 3 SO 2 N(H)O(H) was an intermediate, we would have expected to observe it in the19 F NMR spectra during data collection since the half-life (t 1/2 ) for decomposition of this compound is ~13 min in pH 7.4 buffer solution[41].Hence, CF 3 SO 2 − can be presumed to form exclusively via Pathway 1, Scheme 2. The aromatic photoproducts of 8a were characterized after ~90% photodecomposition using 1 H NMR spectroscopy.Figure1bshows the 1 H NMR spectrum of a partially irradiated sample (1.0 min) of 8a.The 1 H NMR spectra of authentic samples of BHC-CHO(12), BHCM-OH (3), (E)-BHC-oxime (13a), and an irradiated sample of (E)-BHC-oxime (13a) (which isomerizes to give (Z)-BHC-oxime (13b), Section S4, SI) were also recorded for comparison purposes.The chemical shifts of the main photoproduct at 8.32, 8.17, 6.62 and 6.16 ppm can be assigned to (E)-BHC-oxime (13a), with the smaller peaks at 7.59, 7.56, 6.60 and 6.34 ppm attributable to its isomer (Z)-BHC-oxime (13b).Small 1 H NMR peaks from BHC-CHO(12) and BHCM-OH (3) are observed at 9.93, 7.97, 6.68 and 6.54 ppm, and 7.65, 6.72, 6.22 and 4.66, respectively.Both BHC-CHO(12) and BHCM-OH (3) were found to be photostable.Small unknown peaks from secondary photoproducts (6.88, and 8.43 ppm) were also observed.The effect of the solvent composition on the photoproducts obtained from 8a is shown in Figure 2. The percentage of the desired pathway (Pathway 1, Scheme 2) increases as the volume percentage of CD 3 CN in the CD 3 CN/phosphate buffer (0.10 M, pH 7.0) increases, with 97% CF 3 SO 2 − (and (H)NO) generated in 95:5 v/v CD 3 CN:phosphate buffer.However, in pure CD 3 CN only ~36% CF 3 SO 2 − is produced.A dramatic decrease in the percentage of the (H)NO-generating pathway in pure CD 3 CN compared to solvent mixtures of CD 3 CN and phosphate buffer (pH 7.0) was also observed for the (6-hydroxylnapthylen-2-yl)methyl-ON(H)SO 2 CF 3 system, with only O-N bond cleavage observed in pure CD 3 CN [23,24].The photoproducts obtained upon the irradiation of 8b were also determined as a function of solvent composition in phosphate buffer (30 mM, pH 7.0)/CD 3 CN solvent mixtures.8b solutions were irradiated using the Rayonet photoreactor (350 nm).The photoproducts were characterized by recording the 1 H NMR spectra of fully and partially photodecomposed samples.Products were assigned by recording the 1 H NMR spectra of authentic samples in the same solvent mixture.

Figure 1 . 28 Figure 2 .
Figure 1.(a) 19 F NMR spectrum of 8a (1.0 mM) in 80:20 v/v CD 3 CN:phosphate buffer (pH 7.0, 0.1 M) after complete photodecomposition (irradiation for 5.30 min).A Rayonet photoreactor (eight 4 W lamps, 350 nm) was used as the light source.The estimated error in the percentage photoproducts is 2%.(b) Comparison of the 1 H NMR spectrum of a partially irradiated sample (60 s duration) of 8a (1.0 mM) in 80:20 v/v CD 3 CN:phosphate buffer (pH 7.0, 0.1 M) with (E)-BHC oxime (13a) and (Z)-BHC oxime (13b).Small peaks attributable to BHCM-OH (3) are observed at 7.65 and 6.72, with the remaining two peaks (6.22, 4.66 ppm) overlapping with the peaks of other compounds.The minor peaks at 6.88 ppm and 8.43 ppm could not be assigned.The peak at 5.27 ppm is an impurity in the CD 3 CN solvent.Molecules 2024, 29, x FOR PEER REVIEW 6 of 28

Figure 2 .
Figure 2. Percentage of CF 3 SO 2 − released versus the % volume of CD 3 CN for the photolysis of 8a under anaerobic conditions in CD 3 CN/phosphate buffer (0.10 M, pH or pD 7.0) solvent mixtures.The estimated error in each value is 2-3%.

Figure 3 .
Figure3ashows the 1 H NMR spectrum of a completely photodecomposed sample of 8b in the 2-5.5 ppm region in 60:40 v/v, CD 3 CN to 30 mM phosphate buffer, pH 7.0.CH 3 SO 2 NH 2 was observed, consistent with O-N bond cleavage occurring.A very small amount of an unknown species was also observed at 3.21 ppm, and a trace of CH 3 SO 2 NHOH.Figure3bshows the 1 H NMR spectrum of a partially irradiated (16 s) sample of 8b.The peaks at 9.93, 7.90, 6.51 and 6.38 ppm can be assigned to BHC-CHO (10) by comparison with an authentic sample of 12. BHC-CHO was found to be photostable under the irradiation conditions.There was no evidence for (E)-BHC-oxime (13a; 8.30, 8.27, 6.56 and 6.18 ppm), (Z)-BHC-oxime (13b; 7.57, 7.67, 6.54 and 6.37 ppm) or BHCM-OH (3; 7.62, 6.51, 6.08 and 4.67 ppm) in the photoproduct mixture, consistent with essentially only O-N bond cleavage occurring.Peaks which could not be assigned were observed at 8.41 (1H, s), 6.46 (1H, s), 6.14 (2H, s) and 5.98 (2H, s) ppm, and a major peak with m/z values consistent with the parent molecule being C 10 H 5 BrO 5 was observed by LC-MS.Further experiments were not undertaken to fully characterize this species since it is a photoproduct(s) of the undesired O-N bond cleavage pathway.Molecules 2024, 29, x FOR PEER REVIEW 7 of 28
H NMR spectra of 8b were recorded with the pD of the aqueous component of the D 2 O/CH 3 CN (8% v/v CH 3 CN, I = 1.0 M, CF 3 SO 3 Na) solvent mixture varying from pD 2.52-11.03.A control experiment showed that 8b does not decompose under the pH(D) conditions of these experiments, Section S9, SI.Selected 1 H NMR spectra are shown in Figures S13 and S14, SI.As seen for compound 8a (see above), the increase in electron density on the aromatic ring upon deprotonation of the O(H) substituent of 8b leads to shielding of all three aryl protons, with resonance effects leading to greater shielding for protons b and c.The chemical shift of protons a, b and c were plotted as a function of pD (Figure 8a-c).Fitting the data to Equation (1) gave pKa (proton a) = 6.28, pKa (proton b) = 6.42, pKa (proton c) = 6.38 (I = 1.0 M, NaCF 3 SO 3 ).Averaging the values from the two experiments where substantial changes in chemical shift were seen (protons b and c) gave an average pKa value of 6.40 ± 0.03.This pKa value is assigned to deprotonation of the OH substituent of 8b.The chemical shift of protons a, b and c were unchanged from pD 2.52 to 4.90 and from pD 10.30 to 11.03 ppm.Protons e (the CH 3 group) are 6 bonds away from the aromatic group, and as expected, their chemical shift is unchanged in this region within experimental error (pD 2.52-9.75).

SO 2 −
and ~5% CH 3 SO 3 − ), at pH 10.0 and 10.7 the percentage of C-O/N-S bond cleavage increased 15-fold (~50% CH 3 SO 2 − , 2-8% CH 3 SO 3 − ), consistent with deprotonation of the N(H) of the sulfonamide playing a key role in favoring C-O/N-S cleavage over O-N bond cleavage during the photodecomposition even for 8b.Note that the percentage of C-O/N-S bond cleavage is even higher for 8b than that observed for 8a in a highly basic solvent mixture (48-54% CH 3 SO 2 − versus 19% CF 3 SO 2 − in 10:90 % v/v CD 3 CN:aqueous pH 10.0 buffer).Hence, it is clear that the electron-withdrawing effect of the CF 3 SO 2 group of 8a promotes O-N bond cleavage, while the concomitant lowering of the pKa of the proximal NH proton by the same group favors C-O/N-S cleavage over O-N cleavage.Under more neutral photolytic conditions, this latter effect dominates to favor C-O/N-S cleavage over O-N cleavage for 8a (where the NH is deprotonated), while for 8b the neutral N(H)SO 2 CH 3 under these conditions favors O-N bond cleavage.Nakagawa et al. recently reported the synthesis of (7-diethylaminocoumarin-4-yl)methyl photocaged derivatives of the Piloty's acid derivatives (2-Br)PhSO 2 NHOH and (2-NO 2 )PhSO 2 NHOH [21].HNO generation was confirmed indirectly by observation of N 2 O formation by GC-MS, observing the formation of the sulfinamide and disulfide products from the reaction of HNO with N-acetylcysteine, and an enhancement in fluorescence upon binding to an HNO-specific fluorescence probe.The corresponding oxime compound was also observed upon irradiation of the photocaged 2-nitro derivative of Piloty's acid and the authors speculated that the HNO-generating pathway proceeds via C-O bond cleavage to generate the carbocation intermediate and the anion of the parent N-hydroxysulfonamide.

Table 1 .
Effect of solvent ratio on the photoproducts derived from the N-hydroxysulfonamide moiety for photodecomposition of 8b (1.0 mM).

pH Percentages of Photoproducts CF 3 SO 2 − CF 3 SO 2 NH 2
a Phosphate buffer, b acetate buffer, and c carbonate buffer.

Table 3 .
Effect of the pH of the aqueous component of the solvent on the photoproducts derived from the N-hydroxysulfonamide for photodecomposition for 8b (1.0 mM) in 10:90 v/v CD 3 CN:aqueous buffer (30 mM).
Aqueous component of solution: a,c phosphate buffer, b acetate buffer, and d,e carbonate buffer.
6,2-HNM-ON(H)SO 2 CF 3 and 8a with a non-ionizable group (the methoxymethyl ether (MOM) protecting group) does lower the amount of O-N bond cleavage in this system.We hypothesize that the main roles of H 2 O are (i) to promote H 2 O-assisted deprotonation of the methylene carbon in the excited state molecule, leading to O-N bond cleavage and generation of RSO 2 NH 2 , and (ii) to deprotonate the N center (especially in 8a) which will favor the desired C-O/N-S cleavage pathway versus the O-N bond cleavage pathway.The presence of a neutral strongly electron-withdrawing RSO 2 N(H)O-group will assist in weakening the C-H methylene bond to facilitate O-N cleavage, while the deprotonated RSO 2 N − O-group will be less effective in weakening this C-H bond and so will presumably disfavor O-N bond cleavage.