Pro-fluorescent ethynylthiophene-based o-nitrobenzyl photolabile protecting group for hydroxamic acid synthesis

Abstract

Photolabile protecting groups (PPGs) that enable real-time monitoring of uncaging processes are highly sought after for tracking product release during and after photolysis. Few PPGs facilitate direct detection of uncaging events through a fluorescence signal, with o-nitrobenzyl (o-NB) PPG derivatives being the only known examples exhibiting pro-fluorescent properties. In this study, we broaden the scope of accessible pro-fluorescent o-NB PPGs for direct monitoring of product release by reporting two new pro-fluorescent, ethynylthiophene-based, and visible light-absorbing o-NB PPGs, referred to as NPETs 1 and 2. UV-Vis spectroscopy confirmed the complete cleavage of hydroxamic acid (HA) derivatives from NPETs 1 and 2, as evidenced by a blue shift and reduced absorbance intensity. This step likely proceeds through an aci-nitro intermediate, supported by both spectroscopic and computational examinations. We also assessed the released HA products by monitoring the corresponding increase in fluorescence intensity, which corresponds to the co-generated nitrosoketone by-product. The 4-fold and 3-fold increase in fluorescent intensity for NPETs 1 and 2, respectively, was easily observable with the naked eye. Time-course ¹H NMR experiments revealed that NPET 2 exhibits greater stability than NPET 1, showing only minor degradation after 30 days at ambient conditions. (TD)-DFT calculations revealed that the nitrosoketone by-product emission occurs from the S₂ singlet excited state, violating Kasha’s rule. This study highlights the efficacy of pro-fluorescent, ethynylthiophene-based o-NB PPGs in facilitating precise photoreactions under mild acidic conditions. Their pro-fluorescence response and minimal degradation under ambient conditions indicate their potential for application in releasing synthetically difficult-to-synthesize functional groups.

Introduction

In chemistry and materials research, selectively protecting and deprotecting functional groups are critical, especially for lengthy synthetic routes where the number of functional groups requiring protecting groups (PGs) typically increases. Conventional methods such as strongly acidic, strongly basic, reductive, and oxidative conditions, are generally used to remove traditional PGs, which can potentially degrade the protected molecule. However, this drawback is circumvented by the alternative use of photolabile protecting groups (PPGs), which allow selective removal of PGs using light as the activator. This light-triggered functionality enables selective, spatially controlled release of molecules of interest under mild conditions.^1–7 Such light-triggered functionalities have paved the way for transformative advances, ranging from fine-tuned drug delivery systems to the controlled construction of biomolecular architectures.⁸

The first example of PPGs, the ortho-nitrobenzyl (o-NB) group containing a C–H bond at the ortho-position, was developed in 1966 by Barltrop et al. as a more desirable approach to addressing the issue of potential degradation of the protected molecule during the deprotection of PGs using conventional methods.^9–13 This approach was later improved by incorporating a methyl (–CH₃) group at the benzylic position orthogonal to the −NO₂ group in response to the formation of an unwanted nitrobenzaldehyde by-product during PPG deprotection. This detrimental by-product underwent a side reaction with amine-release products, resulting in low yields of released products.^9,14,15 Since Barltrop’s discovery, chemists have been working on developing new pro-fluorescent PPGs and increasing their uncaging efficiency.^8,16–25 Formation of a fluorescent by-product is advantageous because it provides a direct method for monitoring and quantifying the released products during and after photolysis.^26,27

There are currently limited examples of PPGs, such as cinnamate-based^28–31 or thiochromone-based molecules,^31–34 that enable the detection of uncaging events through the appearance of a fluorescence signal. Similarly, we know of only one series of o-NB PPG with pro-fluorescent properties from the literature: the 1-(2-nitrophenyl)-2-phenylethan-1-ol PPG derivatives developed by Specht et al.^35,36 Therefore, it is important to expand the range of available o-NB PPGs to include those with pro-fluorescent properties capable of directly monitoring and quantifying the released products of photo-cleavage. In this study, we are reporting the first and sole example of a pro-fluorescent, ethynylthiophene-based o-NB PPG that absorbs light in the near-visible region of the electromagnetic spectrum for the synthesis of hydroxamic acids (HAs) in high yields. It also co-generates a readily detectable fluorescent by-product that can be used to indicate the formation of HA products. HAs, a class of organic compounds, have garnered extensive attention due to their diverse applications as metal chelators for the removal of toxic metals from seawater, precursors of several anti-cancer drugs, dyes, optoelectronic devices, and polymer architectures.^37–45 However, the synthesis of HAs is widely recognized as challenging, and their purification can be difficult because HAs are highly reactive and often form a mixture of poly-substituted by-products under conventional reaction conditions.^46–48 In this report, we present the design, synthesis, and application of two new ethynylthiophene-based o-NB PPGs, referred to as NPETs 1 and 2, with a nitrosoketone pro-fluorescent by-product as a more promising approach to addressing the issues associated with HA synthesis and their purification. These findings will contribute to the development of future ethynylthiophene-based o-NB PPGs with pro-fluorescent uncaging properties, capable of photo-releasing other difficult-to-synthesize functional groups such as carboxylic acids bonded to poor leaving groups like alcohols, phenols, and thiols, wherein the initially photo-cleaved carbonic or thiocarbonic acid would be unstable and undergo decarboxylation, resulting in the more readily obtainable free alcohols or thiols.

Results and discussion

Design

Designing the 2(2-(4-nitrophenyl)ethynyl)thiophene (NPET) PPG to absorb near the visible region of the electromagnetic spectrum involved three important considerations: (1) the core of the PPG should possess a coplanar geometry and be composed of an extended conjugated π-system; (2) a push–pull character needs to exist throughout the PPG by incorporating electron-donating and/or -withdrawing groups; and (3) the conformation of the PPG should be rigid by regulating a linker between the electron-donating and -withdrawing groups, hindering free rotation throughout the PPG scaffold. This last requirement can be achieved by incorporating alkyne or alkene moieties as linkers.^49–51

The planarity of the PPGs can significantly impact the flow and overall distribution of π-electrons, influencing PPGs’ performance. To achieve this desired geometry, we incorporated an alkyne group (pink in Fig. 1) as a bridge between the thiophene and the o-NB moieties. Additionally, we used a −NO₂ group (red in Fig. 1) as an electron-withdrawing moiety at the para-position to the electron-rich thiophene (orange in Fig. 1) to create a push–pull effect throughout the conjugated π-system. This combination has been shown to lead to important optical properties, such as increased absorption and emission wavelengths.⁵² Incorporating a non-carbon atom (sulfur in our case) into the scaffold of the PPG not only enhanced its structural integrity but also lead to significance optical properties such as red-shifting both the maxima of absorption and emission.^52,53 Thiophenes are particularly valuable for manipulating the electronic properties of various organic molecules, making them an ideal choice for controlling the absorbance and the photo reactivity of our novel PPGs.⁵³

Fig. 1.

Molecular structure of NPET PPG scaffold.

Syntheses

The NPET PPGs were synthesized in two steps. Initially, the NPET PPG scaffold, which contained a hydroxylamine moiety, was synthesized (Scheme 1A). Subsequently, this scaffold was reacted with two carboxylic acid derivatives to generate NPETs 1 and 2, respectively (Scheme 1B).

Scheme 1.

Synthetic route to synthesize NPETs 1 and 2. (A) Reagents and conditions to synthesize hydroxylamine 7: (i) 1-(5-bromo-2-nitrophenyl)ethan-1-one, PdCl₂(PPh₃)₂, CuI, Et₃N, N₂, (85 °C, 2 h, 79%); (ii) NaBH₄, 5 : 1 THF : MeOH, (−78 °C – rt, 15 min, 79%); (iii) N-hydroxyphthalimide, PPh₃, DIAD, THF, (−5 °C – rt, 14 h, 94%); (iv) NH₂NH₂·H₂O, MeOH (rt, 4 h, 81%). (B) Reagents and conditions to synthesize NPETs 1 and 2 from hydroxylamine 7: (v) 4-ethylmorpholine, PyBOP, DMF (rt, 24 h, 58% and 87%, respectively).

The multistep synthesis began with a simple Sonogashira coupling reaction between 3 and 1-(5-bromo-2-nitrophenyl)ethan-1-one, catalyzed by copper iodide (CuI), yielded 4 in good yields.⁵⁴ This ketone was then reduced to alcohol 5, which was subsequently coupled to N-hydroxyphthalimide to produce 6. Finally, 6 was hydrolyzed with hydrazine hydrate, facilitating deprotection and the formation of hydroxylamine 7.

The hydroxylamine 7 served as the starting material for the synthesis of the two new NPETs 1 and 2, which are functionalized with an alkyl 8⁵⁵ and phenyl group 9⁵⁶ respectively. Notably, compound 7 can also be coupled with any number of carboxylic acid derivatives to generate any HA derivatives, highlighting the versatility of the hydroxylamine 7. The structures of previously unreported compounds and NPETs 1 and 2 were confirmed by 1H, 13C{¹H} NMR spectroscopies, 2D NMRs (COSY, DEPT 135, HSQC, HMBC) for NPETs 1 and 2, high resolution mass spectrometry, LCMS for NPETs 1 and 2, and FT-IR (see SI). All compounds were stable under ambient conditions.

Photophysical and photochemical characterization

After successfully synthesizing and fully characterizing NPETs 1 and 2, their photolytic release properties were investigated by irradiating both samples in a 4/1 (v/v) acetonitrile/1 M HCl mixture, and the absorbance and emission wavelengths were observed to change over time using UV-Vis and emission spectroscopies. Acetonitrile (MeCN) was selected as the solvent for photolysis due to its relatively inertness under photolysis conditions.⁵⁷ It is also well established in the literature that polar aprotic solvent like MeCN stabilize charged species and promote intramolecular charge transfer, leading to a red-shift in emission,^52,58 which is particularly beneficial when photolysis involves charge separated intermediates like NPETs 1 and 2. To promote hydrolysis during photolysis, 1 M HCl was added to the sample, as the HAs were not fully cleaving and the reaction was stalling at intermediate D.

Before irradiation (at time = 0 second), an absorbance scan was taken with a λ_max of 340 nm (ε = 2.12 × 10⁴ M⁻¹ cm⁻¹) corresponding to the caged NPET 1 (Fig. S41) and 345 nm (ε = 2.38 × 10⁴ M⁻¹ cm⁻¹) corresponding to the caged NPET 2 (Fig. 2A), respectively. The samples were then irradiated with a 365 nm UV lamp (30 watt 4-core LED) at 5-seconds interval, followed by 1-minute intervals for 8 minutes while recording the λ_max and relevant absorbances.

Fig. 2.

(A) Photolysis absorbance spectra for NPET 2 in 4 : 1 (v/v) MeCN : 1 M HCl after intervals of irradiation at 365 nm (concentration = 10⁻⁶ M). (B) Emission spectra for NPET 2 during and after irradiation at 365 nm showing an increase in fluorescence intensity (concentration = 10⁻⁵ M).

After the initial 5 seconds, a new red-shifted absorbance band with a λ_max of 400 nm (ε = 2.14 × 10⁴ M⁻¹ cm⁻¹) was observed for NPET 1 (Fig. S41) and 395 nm (ε = 2.38 × 10⁴ M⁻¹ cm⁻¹) for NPET 2 (Fig. 2A), indicating the formation of the aci-nitro intermediate (intermediate B). It is well-established in the literature that the primary photoreaction of 2-nitrobenzyl compounds involves an intramolecular hydrogen atom-transfer, leading to the formation of aci-nitro tautomers, which are easily identified by their strong absorption near 400 nm.^59,60 These tautomers are also known to represent the rate-limiting step in product release, as their decay is commonly used to estimate the release kinetics of the protected species.^59,60 Generation of this intermediate was monitored, with a gradual decrease in absorbance intensity until remaining constant after 8 minutes of constant irradiation, indicating complete cleavage of the N-hydroxy-4-(((tetrahydro-2H-pyran-2-yl)oxy) methyl)benzamide from the NPET 2 and benzyl (5-(hydroxyamino)-5-oxopentyl)carbamate from NPET 1 (Fig. S41). The resulting by-product is a nitrosoketone with a λ_max of 305 nm (ε = 8.91 × 10³ M⁻¹ cm⁻¹) and 310 nm (ε = 7.32 × 10³ M⁻¹ cm⁻¹) (Fig. S41 and 2A, respectively). The presence of an isosbestic point at 330 nm (the intermediate photoproduct and nitrosoketone by-product) indicates that a clean, degradation-free photo-cleavage occurred with stable released photoproducts.

The proposed mechanism for the photolysis of NPET PPGs is provided in Scheme 2. We hypothesize that the photolysis mechanism of NPETs 1 and 2 is similar to that of o-NB deprotection, which has been extensively explored and reviewed.^6,61–71 In brief, when exposed to irradiation, the NPET PPG molecule is excited from its ground state to an excited state (Scheme 2A). Subsequently, the −NO₂ group from this excited species abstracts a hydrogen intramolecularly, forming the aci-nitro intermediate (Scheme 2B). The aci-nitro intermediate then undergoes irreversible cyclization to produce the cyclic intermediate (Scheme 2C). Following ring-opening, the hemiacetal intermediate (Scheme 2D) is formed, releasing the nitrosoketone by-product and ultimately resulting in the HA products.^59,60

Scheme 2.

Proposed mechanism for the cleavage of NPET PPGs.

We also monitored the photo-induced changes in fluorescence emission changes for NPETs 1 and 2 in a 4/1 (v/v) MeCN/1 M HCl mixture. The photolysis emission spectra for NPET 1 are shown in Fig. S42 of the SI. Prior to photolysis, both NPET PPGs displayed no fluorescence, with an emission λ_max at 447 and 443 nm for NPETs 1 and 2, respectively. These emissions were not detectable with the naked eye (see inset photographs in Fig. 2B and S42). Upon 5 seconds of irradiation, NPET 1 exhibits an increase in emission intensity which continued to rise and then plateaued after 3 minutes with an emission λ_max of 448 nm. At 5 minutes, the emission intensity began to decrease and remand stable through 8 minutes with an emission λ_max of 448 nm (QY = 0.20%). This final emission band is attributed to the nitrosoketone by-product, showing a 4-fold increase in emission intensity. A similar trend was observed for NPET 2, which showed a 3-fold increase in emission after 8 minutes of irradiation, also with an emission λ_max of 449 nm (QY = 1.30%) corresponding to the same nitrosoketone by-product (Fig. 2B). A side-by-side visual comparison of caged and uncaged NPET 2 is displayed in the inset photo in Fig. 2B, highlighting the fluorescent enhancement. Initially, both NPET PPGs are non-emissive to the naked eye, however, upon full photolysis, they yielded a distinctly fluorescent nitrosoketone by-product, demonstrating the pro-fluorescent quality of these PPGs (Fig. 2B and S42).

To investigate the formation of the HA derivatives and the nitrosoketone fluorescent by-product, ¹H NMR spectroscopy analysis was conducted on NPETs 1 and 2 (see Fig. 3 for NPET 2 and Fig. S27 for NPET 1). The structures of N-hydroxy-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzamide and the nitrosoketone fluorescent by-product derived from NPET 2 were confirmed by acquiring ¹H NMR in CD₃CN : 1 M HCl (4 : 1) before and after 8 minutes of irradiation with a 365 nm UV lamp (30-watt 4-core LED).

Fig. 3.

¹H NMR overlay photolysis of NPET PPG 2 in CD₃CN : 1 M HCl (4 : 1) before (t = 0 min) and after irradiation (t = 8 min) with a 365 nm UV lamp (30-watt 4-core LED). (A) 1H NMR cleavage trace from 8.5 ppm–5.00 ppm and (B) ¹H NMR cleavage trace from 3.00 ppm–0.5 ppm. (NPET 2 concentration = 5.14 mM).

Consistent with the UV-vis spectroscopy results, the HA from NPET 2 underwent complete cleavage after 8 minutes, as evidenced by the disappearance of the H₇ proton signal at 5.57 ppm, directly bonded to the benzylic carbon (red) in the position ortho to the nitro group. Before photolysis, this signal displayed a quartet splitting at 5.57 ppm since it is directly bonded to the methyl group. Following 8 minutes of photolysis, the signal at 5.57 ppm disappeared, confirming the formation of the ketone in the nitrosoketone by-product. Additionally, the H₈ proton from the methyl group, which originally resonated at 1.61 ppm, became deshielded and shifted from a doublet to a singlet at 2.62 ppm, further supporting the formation of the ketone in the nitrosoketone fluorescent by-product.

A similar trend was observed for the ¹H NMR analysis of NPET 1 post-irradiation (see Fig. S27). The formation of benzyl (5-(hydroxyamino)-5-oxopentyl)carbamate and the nitrosoketone fluorescent by-product was confirmed by 1H NMR in CD₃CN : 1 M HCl (4 : 1) before and after irradiation with a 365 nm UV lamp (30-watt 4-core LED) for 8 minutes. Complete HA release from NPET 1 was indicated by the disappearance of the benzylic proton in the position ortho to the nitro group. Before photolysis, this signal displayed a quartet splitting at 5.41 ppm. After photolysis and the formation of the nitrosoketone by-product, the signal disappeared due to the loss of that hydrogen. Additionally, the hydrogen from the methyl group at 1.53 ppm deshielded and shifted to 2.62 ppm as a singlet. The ¹H NMR experiment revealed that photo-conversion of NPETs 1 and 2 resulted in a quantitative release of HA (>95%). FT-IR spectra further confirmed the presence of key functional groups (Fig. S55 and S56).

Stability testing of NPET PPGs 1 and 2

Following the completion of photolysis studies for NPETs 1 and 2, we assessed the stability of both PPGs using time-course ¹H NMR experiments in CD₃CN to evaluate their shelf life under ambient conditions. NPET 1 showed minimal degradation after one day, with progressive degradation observed over 30 days (Fig. S29). A similar stability study was conducted for NPET 2 in CD₃CN (Fig. 4), which revealed minimal degradation after one day. Notably, even after thirty days, NPET 2 exhibited negligible degradation, with all proton signals in the ¹H NMR spectra remaining clearly identifiable. These results demonstrate that NPET 2 remain stable under ambient conditions for at least 30 days. Time-course ¹H NMR data indicates that NPET 2 exhibits greater stability than NPET 1, likely due to the aromatic phenyl ring directly attached to the HA moiety. Aromatic rings are known to absorb UV/visible light without undergoing decomposition and dissipate absorbed energy through non-destructive relaxation pathways, thereby helping prevent photodegradation.^72–74

Fig. 4.

¹H NMR spectral overlay (in CD₃CN, 400 MHz) of NPET 2, stability test over thirty days under ambient conditions.

Computational investigation of the reactant, product, and possible intermediates of the NPET PPG cleavage

To investigate the origin of the ~310 nm, ~345 nm, and ~400 nm bands in Fig. 2A and S41 spectra, we conducted TD-DFT calculations on the putative reactant, product, and some intermediates of the photo-cleavage reaction. For the intermediates, we model B, C, and D from Scheme 2 and their deprotonated forms. We focused on NPET 2 and associated products, assuming that the R group of the HA does not strongly influence the absorption wavelengths. Calculations were carried out using the wB97X-D functional and the ccpVDZ basis set and a continuum solvation model to account for acetonitrile. The choice of functional, as well as tests using a different functional are included in the SI Computational details section (Table S57). All calculations were carried out using Q-Chem 6.0.⁷⁵ Table 1 presents the vertical excitation energies (VEEs) for some of the low-lying excited states.

Table 1.

TD-DFT computed vertical excitation energies (VEEs) for the first three singlet excited states computed with wB97X-D in acetonitrile PCM. Energies are provided in units of eV (and in nm in parentheses). The associated oscillator strength for each excitation is also shown. The truncated model of NPET 2 replaces the R group of the NPET PPG in Scheme 2 with a methyl group. The same truncation was applied for computing intermediates B, C, and D. Hydroxamic acid 2 is the photo-cleavage product for NPET 2

Molecule	S1, eV (nm)	Strength (f)	S2, eV (nm)	Strength (f)	S3, eV (nm)	Strength (f)
NPET PPG 2	3.78 (328)	0.84	4.11 (302)	0.26	4.52 (274)	0.03
NPET PPG – truncated acid	3.78 (328)	0.80	4.12 (301)	0.29	4.51 (275)	0.03
Hydroxamic acid 2	5.20 (238)	0.01	5.25 (236)	0.40	5.45 (227)	0.04
Intermediate B	2.97 (418)	0.25	4.07 (305)	1.29	4.35 (285)	0.01
Intermediate B – deprotonated	2.61 (476)	0.13	3.94 (314)	0.94	4.05 (306)	0.30
Intermediate C	4.11 (302)	1.07	4.92 (252)	0.01	5.17 (240)	0.01
Intermediate C – deprotonateda	3.06 (405)	0.08	3.12 (398)	1.36	3.97 (312)	0.01
Intermediate D	1.51 (822)	0.00	3.60 (345)	1.05	4.25 (292)	0.05
Intermediate D – deprotonated	1.72 (719)	0.01	3.06 (406)	0.10	3.68 (337)	0.01
Nitrosoketone product	1.53 (812)	0.00	3.57 (347)	0.91	3.90 (318)	0.15

^aThe deprotonated form of C could be optimized in vacuo but was unstable when optimized in PCM (it rearranged to form deprotonated intermediate D). Therefore, this TD-DFT calculation was carried out using PCM for a structure that was optimized in the gas phase.

First, we simulate the UV/visible excitation energy for the full NPET 2 model. We obtain a first excited state at 328 nm, in reasonably good agreement with the experimental λ_max (340–345 nm). We then truncated the model by replacing the R group of the NPET PPG in Scheme 2 with a methyl group and repeated the calculation. When it was verified that such a truncation has a limited effect on the low-lying excitation energies, we applied the same truncation for modeling intermediates B, C, and D. We also computed the excitation energies of the hydroxamic acid to verify that it has no absorption in the near-UV or visible range, as expected for a molecule that contains a single aromatic ring.

The appearance of an intermediate state with an absorption at 395 nm after irradiation warrants further investigation, so we computed the low-lying excited states of intermediates B, C, and D, and their deprotonated forms. Out of all the intermediates, only a few present a potentially red-shifted absorption compared to the nitrosoketone product. Intermediate B, the aci-nitro tautomer, has a first excited state (S₁) predicted computationally at near 418 nm, which would be in reasonable agreement with the experimental wavelength observed experimentally (395–400 nm). Previously reported transient absorption studies also find that such an intermediate has an absorption close to 400 nm.⁷¹ However, in the nitroaromatic compounds studied, the intermediate B was found to be very short-lived. Here, it could be that electronic factors contribute to the stability of intermediate B, allowing it to be longer lived. That said, the aci-nitro group is likely to be deprotonated to the nitronate form in the presence of water, but calculations indicate that this will give rise to a further redshift in the absorption.

TD-DFT calculations on the cyclic intermediate C indicate that it does not absorb near 395 nm, although the S₁ and S₂ states of its deprotonated form might (similar energy absorption wavelengths: ~405 and 398 nm, respectively). When attempting to optimize the deprotonated form of intermediate C in PCM, the structure spontaneously rearranged to deprotonated D form, which is energetically downhill (a frequency calculation on the two structures indicates that intermediate D is lower by 1.34 kcal mol⁻¹ than intermediate C). Intermediate D, also a nitroso compound, has similar absorption properties to the nitrosoketone fluorescent product. The computations predict that the protonated form of intermediate D has a similar absorption wavelength as the nitrosoketone. The deprotonated form has a red-shifted and bright S₂ state compared to the nitrosoketone and may explain the experimentally red-shifted band at 395 nm, but this would involve deprotonation of the hemiacetal hydroxy group, which is not expected to be acidic.

There is a possibility that the 310 nm band and shoulder may be explained by intermediate D; while the energies of the S₂ and S₃ states resemble those of the nitrosoketone product, the oscillator strength of the S₂ state is larger while S₃ is smaller.

This is consistent with intermediate D having absorption bands at the same position of the nitrosoketone absorption spectrum (Fig. 2A), but with different intensities. This explanation would also be consistent with the experimental observation that the hemiacetal is typically the long-lived intermediate in these photoreactions⁷¹ and that D is the last intermediate before photo-cleavage is completed.

We next analyzed the excited states of the nitrosoketone product. The TD-DFT calculations using both functionals revealed a very low-lying excited state (S₁). This state has a very low oscillator strength, but if it were bright, its absorption would have appeared at around 812 nm in the UV/visible spectrum. Instead, we find that the experimentally measured spectral peak at ~310 nm and shoulder spanning ~350–450 nm (Fig. 2A and S41) are due to absorption to the third and second singlet excited states (S₃ and S₂), respectively. While the calculations are again not in exact quantitative agreement with the experiment, they correctly predict a blue-shifted band and a red-shifted shoulder on either side of the reactant absorption band. We also note that the calculations indicate that S₂ is more intense than S₃, while experimentally the S₃ band at 310 nm appears more intense. However, oscillator strength calculations can often be strongly influenced by the solvent model and nature of the transition.^76,77 The same TD-DFT calculation carried out in the gas phase, for instance, gives a slightly more intense S₃ band (f = 0.50) than S₂ (f = 0.40).

Together, the TD-DFT calculations on the intermediates indicate that the short-lived observed experimentally with a λ_max of ca. 395 nm is not likely to be the acetal C, but may be either intermediate B or D.

To provide further detail on the electronic nature of these transitions for the nitrosoketone product, we computed Natural Transition Orbitals (NTOs) (Fig. 5). We find that the S₁ state can best be described as a (n,π*) excitation of a lone pair on the nitroso group. S₂ and S₃, on the other hand are both (π,π*) states involving orbitals delocalized across the thiophene and aromatic nitroso moieties.

Fig. 5.

The most representative natural transition orbitals (NTOs) of the first three excited states of the nitrosoketone product. The contribution of each NTO pair to the excitation is indicated as a percentage.

Experimentally, the nitrosoketone fluorescence shows a relatively large Stokes shift with an absorbance at ~310 nm and emission at ~475 nm. To better understand the nature of this transition, the geometries were optimized using the gradients of the first and second excited states. The S₁ state optimization led to a diminishing energy gap and convergence errors consistent with the presence of a non-adiabatic crossing with the ground state. Surprisingly, we find that the S₂ state is the one responsible for the emission; upon optimization of S₂ and calculation of the vertical emission energy, we obtain 2.73 eV with wB97X-D, corresponding to 454 nm and in good agreement with the observed fluorescence wavelength in Fig. 2B. These results clearly indicate that this fluorescent dye violates Kasha’s rule, which instead predicts that fluorescence typically occurs from the lowest excited state (S₁). In the nitrosoketone, the separation between S₁ and S₂ is large enough to prevent fast internal conversion. These calculations agree with previous studies reporting a violation of Kasha’s rule in aromatic nitroso compounds.⁷⁸

Conclusions

Photolabile protective groups (PPGs) are particularly desirable because they offer a simple method for real-time monitoring of the cleavage process of main and by-products. To address this need, we designed, synthesized, and characterized two new pro-fluorescent ethynylthiophene-based o-NB PPGs, NPETs 1 and 2. These PPGs provide an appealing strategy for directly monitoring photolysis through changes in fluorescence intensity, which correlates with the release of photo-cleavage products: hydroxamic acids (HAs) and a pro-fluorescent nitrosoketone by-product. This method addresses the conventional challenges in synthesizing and purifying HA derivatives by facilitating the efficient release of HAs alongside a readily detectable nitrosoketone by-product.

We demonstrate the complete release of HA derivatives from NPETs 1 and 2 using UV-Vis spectroscopy, as indicated by a decrease in absorbance intensity accompanied with a blue shift in wavelength. Fluorescence spectroscopy studies further revealed that the cleavage of HA derivatives from NPETs 1 and 2 can be directly monitored and observable to the naked eye. Upon photolysis, NPET 1 exhibits a 4-fold increase in fluorescent intensity, NPET 2 displays a 3-fold increase, both corresponding to the formation of a new emissive nitrosoketone by-product. TD-DFT calculations indicate that this product fluoresces from its second excited singlet state, S₂.

¹H NMR investigations on NPETs 1 and 2 and their photolysis products confirmed the quantitative release of HA (>95%) and the generation of nitrosoketone by-product. FT-IR analysis of irradiated samples for NPETs 1 and 2 revealed a new −NvO band at 1524 cm⁻¹ with corresponding disappearance of the −NO₂ band, indicating formation of nitroso species, and a new broad absorption band at 3337 cm⁻¹ corresponds to O–H stretch of benzyl (5-(hydroxyamino)-5-oxopentyl)carbamate for cleaved PPG 1, while a broad absorption band at 3467 cm⁻¹ corresponds to the O–H stretch of N-hydroxy-4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzamide for cleaved PPG 2 (see Fig. S55 and S56). Additionally, ¹H NMR time-course investigations revealed that NPET 2 exhibits greater stability than NPET 1, showing only minor degradation after 30 days at ambient conditions.

This study highlights the effectiveness of pro-fluorescent, ethynylthiophene-based o-NB PPGs in facilitating precise photoreactions under mild acidic conditions. These findings represent a significant advancement in the development of o-NB PPGs and establish a promising framework for future investigations targeting hard-to-synthesize but spectroscopically well-suited functional groups like carboxylic acids, sulfonates, and phosphates in derivatization targets for the development of absorption-tuned PPGs.

Data availability

The authors declare that the main data supporting the findings of this study, including experimental procedures and compound characterization, are available within the article and its SI files, or from the corresponding author upon request.

Experimental section: synthetic protocols, characterizations (¹H NMR, ¹³C{¹H} NMR, 2D NMRs (COSY, DEPT 135, HSQC, HMBC)) for NPETs 1 and 2, high resolution mass spec, LCMS for NPETs 1 and 2, FT-IR spectra, UV-visible and emission spectra of NPETs 1 and 2 in various solvents. See DOI: https://doi.org/10.1039/d5ob00859j.