2-Hydroxyphenacyl ester: A new photoremovable protecting group

Abstract

2-Hydroxyphenacyl moiety absorbing below 370 nm is proposed as a new photoremovable protecting group for carboxylates and sulfonates. Laser flash photolysis and steady-state sensitization studies showed that the leaving group is released from a short-lived triplet state. In addition, DFT-based quantum chemical calculations were performed to determine the key reaction steps. We found that triplet excited state intramolecular proton transfer represents a major deactivation channel. Minor productive pathways involving the triplet anion and quinoid triplet enol intermediates have also been identified.

Introduction

The 4-hydroxyphenacyl (1, pHP) photoremovable protecting group (PPG)^1,2 has received considerable attention due to its fast and efficient substrate release along with the major byproduct, 4-hydroxyphenylacetic acid (Scheme 1).^3–6 A recent study has shown that various substituents on the pHP chromophore affect only slightly the overall efficiency and the release rates.⁷ The presence of water is essential for this reaction; the photorearrangement is completely suppressed at low water concentrations.^8,9 The pHP derivatives have been utilized as PPGs in a variety of fields, such as neurobiology, enzyme catalysis, or biochemistry.¹⁰ Release of the protected substrate from the pHP chromophore (Scheme 1) is believed to occur via a water-assisted adiabatic triplet biradical extrusion followed by formation of a putative ground state spirodienedione (2), reminiscent of the Favorskii rearrangement intermediate.

Scheme 1.

Photochemistry of pHP esters

We have recently demonstrated that benzofuranones are formed by irradiation of the substituted 2-hydroxyphenacyl (oHP) benzoate 3 in the presence of water (Scheme 2).⁹ It was suggested that the products are formed via the photo-Favorskii-type rearrangement through a putative spirodienedione intermediate.

Figure 6.

Triplet-triplet absorption spectra of pure xanthenone (black circles) and xanthenone in 6.2 × 10⁻⁴ M (black), 9.4 × 10⁻⁴ M (blue), and 1.6 × 10⁻³ M (red) CH₃CN solutions of 4. A spectrum of ³q4* (red triangles) obtained from the 5 × 10⁻⁴ M 4 solution is shown for comparison. The spectra were recorded in a 50-ns time window after a delay of 19 ± 2 ns following the laser flash (λ_exc = 355 nm). The concentration of xanthenone was c = 0.37 mM in all samples except for pure 4.

The 2-hydroxyphenyl ketones are known to undergo a very fast (hundreds of fs) and efficient singlet excited-state intramolecular proton transfer (ESIPT) between the two neighboring oxygen atoms in the gas phase.^11–16 Fluorescence and phosphorescence spectroscopy of oHP derivatives confirmed that the singlet ESIPT occurs in non-polar solvents while hydrogen bonding of the 2-hydroxy moiety with protic solvent molecules prevents the singlet ESIPT and subsequently leads to the triplet state.¹⁵ However, time-resolved EPR study of 2-hydroxyacetophenone (oHA) in ethanol suggested that singlet ESIPT precedes ISC to the triplet state.¹⁴

In this work, we report on the synthesis of oHP derivatives and their photochemical behavior. A series of laser flash photolysis experiments and DFT-based quantum chemical calculations were performed to determine the key reaction steps. We show that the oHP chromophore has interesting properties as a photoremovable protecting group for carboxylic and sulfonic acids.

Results

Synthesis

2-Hydroxyphenacyl esters (3a–c) were synthesized in a two-step procedure by bromination of 2-hydroxyacetophenone 4 to give 5 and subsequent nucleophilic displacement of the bromine atom by benzoate, acetate or methanesulfonate (Scheme 3). 2-Methoxyphenacyl acetate (6) was prepared using the same reaction sequence from 2-methoxyacetophenone (7) and subsequently the bromide 8. The overall chemical yields were approximately 50% (Supporting Information).

Scheme 3.

Synthesis of oHP esters

Absorption spectra

Figure 1 shows the absorption spectra of 2-hydroxyacetophenone (4; λ_max = 248 and 325 nm), the corresponding anion 4⁻ (λ_max = 222, 255, and 360 nm) and the protonated form 4⁺ (λ_max = 283 and 372 nm). The ground state pK_a (4 → 4⁻; H₂O/CH₃CN (98:2, v/v); 10 mM phosphate buffer) = 10.3 ± 0.3 was determined by a spectrophotometric titration using a global fit to the data.

Figure 1.

Ground state absorption spectra of 4 (black, dashed), 4⁻ (red, solid), and 4⁺ (green, dash–dot) obtained in H₂O/CH₃CN (95:5, v/v), aq NaOH (1 mM, pH = 11)/CH₃CN (95:5, v/v), and 70% aq HClO₄, respectively.

The absorption spectra of 3b in H₂O/CH₃CN (95:5, v/v) (λ_max = 210, 254, and 325 nm) and 3b⁻ in aq solution of sodium hydroxide (1 mM; λ_max = 240, 260, and 365 nm) are shown in Figure 2. The ground state pK_a (3b, H₂O/CH₃CN (98:2, v/v); 10 mM phosphate buffer) = 10.2 ± 0.3 was determined using the same method as that used for 4. The carboxylates 3a,b were found to be stable for over 24 h in buffered (pH = 7.4; c = 1 mM) and non-buffered aqueous (pH ≤ 10; c = 3 mM) solutions in the dark; they hydrolyzed at pH ≥ 11. The hydrolysis products were also detected after 48 h in both buffered and non-buffered solutions. 3c decomposed in the solutions of pH ≥ 8, which prevented the determination of its pK_a. The maximum aqueous solubility of 4, 3b and 3c was determined to be approximately 3 × 10⁻³ M.

Figure 2.

Ground state absorption spectra of 3b (black, dashed) and 3b⁻ (red, solid) obtained in H₂O/CH₃CN (95:5, v/v) and aq NaOH (1 mM, pH = 11)/CH₃CN (95:5, v/v), respectively.

Photochemistry

Solutions of the phenacyl ester (3a–c; c ~10 mM) in various solvents or solvent mixtures were irradiated until more than 95% of the starting material had disappeared. All esters released the corresponding acids (HX) in very high yields (Scheme 4; Table 1). The 2-hydroxyphenacyl moiety was transformed into two photoproducts, benzofuran-3(2H)-one (9) and minor amounts of benzofuran-2(3H)-one (10). The disappearance quantum yields of 3a–c in H₂O/CH₃CN (1:1 or 7:3, v/v) were determined (Table 2).

Scheme 4.

Photochemistry of 3a–c

Table 1.

Photoproducts from irradiation of 3a–c

compound	solventa	chemical yields / %b
		9	10	HXc
3a	benzene	n.r.d
	CH3OH	77 ± 2	14 ± 2	89 ± 2
	CH3CN	n.r.d
	H2O/CH3CN (1:1)	62 ± 3	11 ± 2	71 ± 2
	acetone	n.r.d
	H2O/acetone (1:9)	95 ± 2	tracese	96 ± 1
3b	H2O/CH3CN (1:1)	76 ± 4	12 ± 4	88 ± 3
3c	H2O/CH3CN (1:1)	75	15	92 ± 4
	H2O/acetone (1:4)	90	tracese	n.d.f

^aCommercial perdeuterated solvents were used. The ratios of the solvent mixtures are given as v/v.

^bIrradiated at λ_exc = 300 or 313 nm in non-degassed solutions to >95% conversion; the chemical yields were determined by ¹H NMR; the isolated chemical yields given were lower (by 3–8%) but preserved the same product ratios. The standard deviation is given when at least two independent measurements were carried out.

^cHX = HO₂CPh (3a), HO₂CCH₃ (3b), HOSO₂CH₃ (3c).

^dNo reaction detected.

^ePhotoproducts not detected by ¹H NMR of the reaction mixture.

^fNot determined.

Table 2.

Degradation quantum yields of 3a–c

compound	solvent	Φa
3a	H2O/CH3CN (1:1, v/v)	0.10 ± 0.02
3b	H2O/CH3CN (7:3, v/v)	0.21 ± 0.02
3c	H2O/CH3CN (7:3, v/v)	0.34 ± 0.02

^aIrradiated at λ_exc = 313 nm in aerated solutions (c ~3 mM) to <15% conversion. 2-Nitrobenzaldehyde (Φ = 0.41 ± 0.02 in CH₃CN)¹⁷ and valerophenone (Φ = 0.30 in cyclohexane)¹⁸ were used as the actinometers. HPLC (3a) and ¹H NMR (3b–c) were used to determine the ester concentrations. The results are based on three independent measurements; the standard deviations of the mean are shown.

The dihydrobenzofuran 11 was formed by irradiation of 2-methoxyphenacyl acetate 6 (~10 mM) in dry CH₃CN through a Pyrex filter (λ ≥ 280 nm); the acetate group was not released (Scheme 5). The reaction efficiency (Φ) was below 0.01.

Scheme 5.

Photochemistry of 6

The role of dissolved oxygen was evaluated by obtaining the ratios of the disappearance quantum efficiencies of 3b, c in samples purged with either gaseous oxygen (Φ_O2) or nitrogen (Φ_N2) (Table 3). One sample Student’s t-tests indicated that the means of the ratios measured were significantly different from unity at the 0.95-confidence level, signifying the importance of oxygen presence on the phototransformation efficiency. Also, a two-sample t-test showed a statistically significant difference between the means given in entries 1 and 2 of Table 3 at the 0.95-confidence level.

Table 3.

The role of dissolved oxygen in photochemistry of 3b, c

entry	compound	solvent	ΦO2/ΦN2a
1	3b	H2O/CH3CN (95:5, v/v)	0.796 ± 0.074 (11)
2	3b	aq 1 M malonate, pH = 2.5	0.911 ± 0.043 (6)
3	3c	aq HCl, pH = 1.5	0.911 ± 0.064 (6)

^aThe ratio of the quantum efficiencies of 3b, c (~0.2–0.5 mM) disappearance in the samples purged with O₂ (Φ_O2) and N₂ (Φ_N2) for 15 min. Irradiated at λ = 313 nm; the conversions were followed by HPLC using methyl benzoate (2 × 10⁻⁴ M) as an internal standard. The 95% confidence interval is given; the number of independent measurements is given in the parentheses for each set of experiments.

Irradiation (λ_exc = 366 nm) of 3b in the presence of 2-naphthalene sulfonate (0.2 M; triplet quencher, E_T < 61 kcal mol⁻¹; this value is estimated with respect to E_T = 61 kcal/mol for naphthalene¹⁹ which does not absorb at 366 nm) in D₂O/CD₃CN (1:1, v/v) led to the formation of both 9 and 10 with approximately the same efficiency as that obtained in the absence of a quencher (see Table 2).

Triplet sensitization of 3c with xanthenone (0.1 M) in D₂O/CD₃CN (1:1, v/v) (λ_exc = 366 nm; ε(xanthenone) / ε(3c) was 5 : 1 at this wavelength) led to the same distribution of photoproducts as that shown in Table 1.

Isotope labeling

The incorporation of oxygen from water as a solvent to the products 9 and 10 during irradiation of 3a (Scheme 4) was studied. Irradiation of 3a in D₂¹⁸O/CD₃CN (1:4, v/v) gave the same photoproduct distribution (9/10) as that found in D₂¹⁶O/CD₃CN (1:4, v/v); no ¹⁸O incorporation was observed according to the HRMS analyses. However, the –CH₂– group protons in both 9 and 10 can be exchanged by deuterons in D₂O solutions. This process has a half-life in the order of hours.

In contrast, irradiation of 4-hydroxyphenacyl (1, pHP) benzoate (~10 mM) in D₂¹⁸O/CD₃CN (1:4, v/v) to 90% conversion gave, along with the release of benzoic acid, two photoproducts, 4-hydroxyphenacylacetic acid (2, 80%) and 4-(hydroxymethyl)phenol (8%), which had one ¹⁸O atom incorporated (Scheme 6). This result is in accord with the current knowledge about the photo-Favorskii reaction mechanism (Scheme 1).

Scheme 6.

Photochemistry of 1 in D₂¹⁸O/CD₃CN

Nanosecond laser flash photolysis: 2-Hydroxyacetophenone

Laser flash photolysis (LFP) of 4 in various solvent systems was carried out using the fourth harmonic of a Nd:YAG laser at 266 nm for excitation. Analogous to our work on the triplet state tautomerization of 4-hydroxyacetophenone,²⁰ we hypothesized that four excited triplet forms of 4 or 3a–c may persist under various conditions (Chart 1; q = quinoid enol). The assignments of the transient suggested in this section are explained in the Discussion.

Chart 1.

Anticipated triplet state transients formed from 3 and 4

LFP of 4 in acetonitrile and n-hexane provided a transient absorption with λ_max = 415 nm, which was assigned to the quinoid triplet enol ³q4* (Chart 1, Figure 3). Excitation of the ground state anion 4⁻ (Figure 1) in 1 mM NaOH in acetonitrile at 266 nm gave a broad transient absorption band with λ_max ~ 490 nm (Figure 3), assigned to the triplet anion ³4⁻*.

Figure 3.

Normalized triplet-triplet absorption spectra of ³q4* in acetonitrile (blue) and n-hexane (blue empty circles), and ³4⁻* in aq NaOH (1 mM)/CH₃CN 95:5 (v/v; red). All spectra were recorded 15 ns after excitation of 4 solutions (5 × 10⁻⁴ M) with a laser pulse at 266 nm.

Transient absorption spectra with a maximum at λ_max = 420 nm and a shoulder at 380 nm were observed in aqueous non-buffered solutions of 4 (pH = 6.2, measured with a glass electrode; Figure 4). The common decay rate constants at both 420 nm and 380 nm were k_d ~ 1 × 10⁶ and 4 × 10⁴ s⁻¹ in aerated and degassed solutions, respectively. The transient was assigned to ³q4* (Chart 1; vide infra). After the ³q4* signal disappeared, a weak band with λ_max = 380 nm and k_d = (7.2 ± 0.3) × 10³ s⁻¹ was observed. It was assigned to the ground state anion 4⁻ (Figure 1). The tail of the absorption band of 4⁻, perturbed by ground state 4 bleaching, was observed in the given spectral window. The transient spectra in acetate buffer solutions (pH = 3.3, I = 0.1 M) were similar to those of aqueous solutions showing ³q4*. The spectra with λ_max = 460 nm obtained in NaOH (1 mM)/CH₃CN 95:5 (v/v) solutions were assigned to ³4⁻*; those observed in 70% aqueous HClO₄ solutions to ³4⁺* (λ_max = 430 nm) (Figure 5).

Figure 4.

Transient absorption spectra recorded 50 ns (blue) and 5 µs (red) after a 266 nm flash in a 50-ns time window in non-buffered H₂O/CH₃CN (95:5, v/v) solutions of 4 (4 × 10⁻⁴ M; pH = 6.2).

Figure 5.

Normalized transient absorption spectra of ³q4* in aqueous (blue) and acetate buffer (5% CH₃CN; pH = 3.3, I = 0.1 M, blue circles) solutions of 4 (5 × 10⁻⁴ M), ³4⁻* in aq NaOH (1 mM, pH = 11)/CH₃CN (95:5, v/v; red) and ³4⁺* in aq HClO₄ (70%; green) solutions of 4. All spectra were recorded in a 50-ns time window after a delay of 15 ns following a 266-nm flash.

We found that 4 in CH₃CN quenches the triplet state of xanthenone giving rise to the absorption band at λ_max = 415 nm which is assigned to ³q4* (Figure 6).

Nanosecond laser flash photolysis: 2-Hydroxyphenacyl esters

LFP studies (λ_exc = 266 nm) of 3b and 3c solutions in various solvent systems were also carried out. Compounds 3b and 3c in pure acetonitrile or n-hexane gave transient absorption spectra with maxima at ~420 nm (Figure S5) which were assigned to the quinoid triplet enols ³q3b* and ³q3c*, respectively, analogous to the transients formed from 4 (Chart 1).

Flash photolysis of 3b in unbuffered H₂O/CH₃CN (95:5, v/v) and in the same solvent system with pH adjusted to 2 (0.01 M HClO₄) provided transient absorption spectra with maxima at 425 nm (and a shoulder at 380 nm; Figures S6a and S8) which were assigned to ³q3b*. Excitation of 3b in 1 mM NaOH in H₂O/CH₃CN (95:5, v/v, pH = 11.2) gave a transient spectrum with a maximum at 460 nm (Figure S7) assigned to the triplet anion ³3b⁻*.

LFP of 3c in H₂O/CH₃CN (95:5, v/v) provided transient absorption spectra with a maximum at 460 nm (Figure S6b) assigned to ³3c⁻*. In weakly acidic solutions (acetate buffer, pH = 5.5, I = 0.01 M), 3c provided an absorption band at 460 nm assigned to ³3c⁻* (Figure S9); an absorption with λ_max = 425 nm observed at pH = 1 (aq HClO₄) was assigned to ³q3c*. The kinetic traces of the transients formed by LFP of 4, 3b and 3c were measured in unbuffered 2% acetonitrile aqueous solution and were fitted by monoexponential functions. The corresponding rate constants (k_d) are summarized in Table 4. In addition, we found that quenching of ³3c⁻* (λ_exc = 355 nm) in water by potassium sorbate was nearly diffusion-controlled (Figure S1), and that both 3b and 3c were sensitized by energy transfer from xanthenone triplet in water/acetonitrile 1:1 (v/v) (Figures S2 and S3).

Table 4.

The observed decay rate constants (k_d) of triplet species formed from 4, 3b and 3c in H₂O/CH₃CN (98:2, v/v) upon excitation at λ_exc = 266 nm

compound (observed transient)	λobs / nm	kd (aerated) / 106 s−1	kd (degassed) / s−1
4 (3q4*)	380	(1.0 ± 0.3)	(4.3 ± 0.2) × 104
	425	(1.2 ± 0.3)	(5.3 ± 0.2) × 104
	500	(1.6 ± 0.2)	(3.9 ± 0.3) × 104
3b (3q3b*)	380	(1.0 ± 0.1)	(2.4 ± 0.2) × 105
	425	(1.3 ± 0.2)	(2.6 ± 0.2) × 105
	500	(1.3 ± 0.4)	(3.3 ± 0.2) × 105
3c (33c−*)	460	(1.7 ± 0.2)	(5.0 ± 0.4) × 105

Quantum chemical calculations

The relative energies of various conformers of 4 and q4 in their singlet ground and lowest triplet states are summarized in Figure S11. The solvation effects were modeled within a polarizable continuum model (PCM) to account for long range solvent effects or a hybrid approach, combining the PCM with explicit solvation by a small number of water molecules to include specific interactions. Two water molecules were used in the case of a hybrid approach, each connecting an oxygen atom of 4, q4 or 4⁻ to hydrogen atom in the water molecule via a hydrogen bond or connecting the proton on the oxygen atom of 4 or q4 via a hydrogen bond with the oxygen atom of the water molecule.

No minimum was found for a conformer possessing an intramolecular hydrogen bond (Figure S11) of q4 in the ground state; proton transfer to 4 thus occurred spontaneously during the optimization procedure suggesting on a very small barrier for this process. The same behavior was observed for a ³4* conformer possessing an intramolecular hydrogen bond in the triplet state which leads to the formation of a corresponding ³q4* conformer. The triplet-triplet absorption spectra calculated for the most stable conformers of ³4*, ³q4* and ³4⁻* in PCM employing the linearized harmonic reflection principle²⁰ are shown in Figure 7. The two major vertical electronic transitions in the spectrum of ³q4* with two explicit water molecules within a simple TD-DFT model are shown as blue bars. Obviously, there are two important transitions which broaden the signal observed for the spectrum of ³q4*.

Figure 7.

The triplet-triplet absorption spectra employing the linearized harmonic reflection principle calculated for the most stable conformer of the 4 triplet species – neutral ³4* (black), quinoid enol ³q4* (blue), and anion ³4⁻* (red). The blue bars denote the two major electronic transitions of ³q4*.

Discussion

The major advantages of the p-hydroxyphenacyl (1, pHP) photoremovable protecting group (PPG) is the solubility of their cages in aqueous solutions and ultrafast release rates with high efficiencies of decaging forming chemically and biologically benign byproducts. Thus, pHP proved to be superior among a variety of known PPGs in neurobiology, enzyme catalysis, and other biochemical applications.¹⁰ A major disadvantage of the pHP chromophore is the necessity to use rather short-wavelength UV light for irradiation, which can be destructive to biological material. Excitation of derivatives of 1 is followed by ultrafast intersystem crossing (ISC) to the triplet state (~3 ps)⁸ and a subsequent cascade of intermediates formed in steps, in which water plays a crucial role. The reaction, termed the photo-Favorskii rearrangement due to its obvious similarity with the base-catalyzed transformation of α-haloketones,²¹ finally gives p-hydroxyphenylacetic acid as the major rearrangement product (Scheme 1).

Recently, we reported that benzofuranones are formed in high yields upon irradiation of 2-hydroxy-4,6-dimethylphenacyl esters (3) in solvents containing an appreciable amount of water (>10%; Scheme 2).⁹ This phototransformation, which led to the concomitant release of a carboxylic acid, appears to parallel the photo-Favorskii pathway of the pHP chromophore. In contrast to the photorearrangement of 1, the decarbonylation product (benzyl alcohol) was not formed by the photolysis of 3. In this work, we decided to explore the photochemical behavior of o-hydroxyphenacyl derivatives (3, oHP) as a novel PPG.

Scheme 2.

Photochemistry of substituted oHP derivatives

The UV spectrum of the parent chromophore of compounds 3, 2-hydroxyacetophenone (4, oHA), is significantly red-shifted with appreciable absorption above 350 nm (Figure 1) compared to p-hydroxyacetophenone.²⁰ We determined the pK_a of 4 by spectrophotometric titration to be 10.3 ± 0.3, which is shifted by more than two orders of magnitude compared to that of p-hydroxyacetophenone (pK_a = 7.9 ± 0.1 ²⁰). It is well known that the conjugate bases of pHP derivatives display a significant decrease in their quantum yields of the leaving group release at pH above their pK_as.⁷ Thus the pK_a of 4 suggests that derivatives of 3 may not suffer from this drawback. The conjugate acid of 4, 4⁺, requires very acidic conditions to exist in appreciable amounts.

The synthesis of the oHP esters 3a–c was straightforward and parallels a well-known synthetic approach to derivatives of 1. Thermal stability of 3a,b (at pH <10) and 3c (at pH <8) in aqueous solutions holds promise for their application at physiological conditions. Irradiation of 3 in water-containing solvents led to the formation of the two benzofuranones 9 and 10, accompanied by release of the corresponding acids in high chemical yields (Table 1). This photoreaction was suppressed when dry solvents were used, which is also known from the photochemistry of 1.^9,22 However, water obviously does not trap any reactive intermediates, which was demonstrated for 3a by isotopic labeling experiments (Scheme 6 and the related text in the Results).

The o-hydroxy group is essential for the observed transformation. A different although inefficient reaction channel opens when the hydroxy group is replaced by a methoxy group in 6. The dihydrobenzofuran 11, formed as the sole product, retains the acetate group (Scheme 5). Such photoinduced δ-hydrogen abstraction reactions have been studied before^23–25 and were recently applied in the synthesis of cyclophanes.²⁶

The quantum yields of 3a–c degradation were lower by a factor of 2 than those of the corresponding pHP esters 1.^7,10 The product formation was only slightly reduced by the presence of a triplet quencher (2-naphthalene sulfonate). The triplet energy (E_T) of 4, estimated from our DFT calculations, was ~ 72 kcal mol⁻¹. The triplet energy of p-hydroxyacetophenone is approximately 70.5 kcal mol⁻¹.²⁷ We found that triplet-excited xanthenone (E_T = 74 kcal mol^{−1 28}) sensitized the reaction of 3c resulting in the formation of both 9 and 10. As a result, the triplet state of 3, ³3*, is most likely the reactive state responsible for the release of the leaving group. Provided that energy transfer from ³3* to 2-naphthalene sulfonate in 95% water/acetonitrile (v/v) is a diffusion controlled process (k_diff ~ 4 × 10⁹ M⁻¹ s⁻¹),²⁹ the lifetime of ³3* must be on the order of 1 ns or less, comparable to those of pHP carboxylates.^10,22

The o-hydroxyaryl compounds, such as oHP, are known to undergo ultrafast and very efficient excited state intramolecular proton transfer (ESIPT).^30–32 Although the ISC rate of ¹3* can be comparable to that of ¹1* (~3 ps),⁸ the singlet ESIPT can effectively compete (600 fs in molecular beam)¹¹ provided that the rate constants do not differ in gas phase and aqueous solutions. We cannot unambiguously claim at the moment that ³3* is obtained by direct irradiation of 3 followed by subsequent ISC of ¹3*. ESIPT that leads to the formation of ¹q4* would be feasible if the intramolecular hydrogen bond between the o-hydroxy group and the carbonyl oxygen assuring an ideal ESIPT geometry is achieved. It has been reported that polar protic solvents such as methanol or ethanol prevent the intramolecular hydrogen bond to be established in the ground state and, consequently, diminish the efficiency of the singlet ESIPT. This enabled Nagaoka and coworkers to measure the phosphorescence spectra of the triplet intermediates.¹⁵ Hoshimoto and co-workers used time-resolved EPR to study ESIPT in oHA in ethanol and concluded that the formation of ³q4* is consistent with the singlet state ESIPT followed by ISC to give the triplet excited state.¹⁴

In this work, we performed nanosecond LFP supported by quantum chemical calculations based on the DFT to find whether the triplet excited oHP intermediates are responsible for the group's release and to reveal the subtleties of the triplet ESIPT. Analogous to our recent study of triplet state tautomerization processes of a related system, the p-hydroxyacetophenone,²⁰ four species were anticipated to co-exist in the triplet manifold of 4 (Chart 1). DFT calculations on these species predicted that a minimum which would correspond to conformer of ³4* retaining an intramolecular hydrogen bond does not exist. The hydroxy group proton was spontaneously transferred to the carbonyl oxygen to give ³q4* during the optimization process. This suggests that there is very little, if any, barrier for the formation of ³q4* except for rotational barriers of the hydroxy or acetyl groups. Any conformer of ³4* is destabilized compared to the most stable conformer of ³q4* by more than 24 kcal mol⁻¹ (Figure S11). Thus the equilibrium constant of ³4* ⇌ ³q4* is pK_E < −17. The equilibrium is markedly shifted toward ³q4*, which should be formed rapidly from ³4*. For comparison, the equilibrium constant pK_E for p-hydroxyacetophenone equals only to −2.1.²⁰

LFP of 4 provided a band at ~ 400 nm in all solvents used at neutral pH. The rise of this band was not resolved within the time resolution of our LFP setup (~ 5 ns). Its lifetime was prolonged ~ 20-times upon degassing which confirms a triplet nature of the transient. Adjusting pH to 3.3 did not influence its shape or lifetime. Thus, we assigned this band to ³q4* with the help of our TD-DFT calculations (Figure 7). A new band with a maximum at ~450 nm appeared for all derivatives 3 and 4 in the presence of a base (Figures S7 and Figure 5) and, according to the kinetics observed for aerated and degassed solutions and quantum chemical calculations, it was assigned to the triplet conjugate bases ³3c⁻* and ³4⁻*. In highly acidic solutions, a band with a maximum at 425 nm was attributed to ³4⁺*. No signal, which would correspond to ³4* (a calculated spectrum in Figure 7), was observed under any experimental condition within the response time of our apparatus. These results are summarized in Scheme 7.

Scheme 7.

Triplet-state proton transfer equilibria of 4

The most striking observation came from a comparison of the decay rate constant changes found for ³q3b* and ³q4* in aerated and degassed solutions (Table 4). The rate constants measured for ³q4* in degassed solutions were nearly an order of magnitude smaller than those of ³q3b*, while they did not differ (within the experimental error) in the presence of oxygen. This led us to anticipate that ³q3b* may partially be responsible for the acetate release.

Leaving group release from the quinoid triplet enol ³q1* (Scheme 8) of pHP cages is a matter of considerable debate.^20,33,34 It has been suggested that it is the low triplet energy of ³q1* (for ³q1* (X = H): E_T ~48 kcal mol⁻¹)²⁰ that prevents the release. Because the calculated equilibrium constant for ³4* ⇌ ³q4* is pK_E < −17, a decrease in the triplet energy of ³q4* can be expected. The DFT calculations found that the triplet energy of ³q4* is E_T ~41 kcal mol⁻¹ and that ³q4* should not be quenched by typical triplet quenchers such as naphthalenes or sorbate. As a result, the release of acetate from ³q3b* was experimentally confirmed by measuring the relative quantum efficiencies of a series of nitrogen- and oxygen-saturated solutions of 3b under the same conditions at which appreciable amounts of ³q3b* are formed upon excitation. The triplet ³3b* cannot be effectively quenched by oxygen due to its short lifetime (τ ≤ 1 ns). We were able to observe a statistically significant (at 95% confidence level, Table 3) decrease of the quantum yield in the oxygen-saturated solutions in all samples in which only ³q3* is present (Figure S8; Table 3, entries 2 and 3). In addition, we found an even greater, statistically significant, decrease in the quantum yield in oxygen-saturated solutions at neutral pH (Table 3, entry 1) where both ³3b⁻* and ³q3b* were present (Figure S6a). This suggests that the acetate release took place from both ³3b⁻* and ³q3b* regardless of a very low triplet energy of the latter species. The release rate constant from ³q3b* was estimated from our LFP measurements to be ~2 × 10⁵ s⁻¹ provided that the ISC rate constants of ³q3b* and ³q4* are comparable. The oxygen concentration in non-degassed water/acetonitrile solutions amounts to ~10⁻³ M,³⁵ sufficiently high to interfere with the release step. Inability to unambiguously confirm that ³q1* is not responsible for the leaving group release originates from the ISC rate k_ISC = 5 × 10⁶ s⁻¹ found in degassed aqueous solutions for ³q1* (X = H),²⁰ which is higher than that of ³q4* by two orders of magnitude (Table 4). Therefore, it is not its low triplet energy but the release step itself that, provided its rate constant equals to that calculated for ³q3b*, cannot efficiently compete with ISC.

Scheme 8.

Release of the leaving group from ³q1*

These results show that the adiabatic tautomerization to ³q3b* overwhelms any attempt of photorelease from ³3b*, while ³q3b* is not capable of efficient releasing of the leaving group. The quantum yields of the photoreaction of 3b, c are half of those reported for the pHP derivatives 1. The steady-state sensitization experiment clearly showed that triplet energy transfer to 3 leads to the photoproducts. It must be ³3* that is populated by the energy transfer because none of the diastereomers of q3 is populated in the ground state (Figure S11). However, the LFP spectra measured upon excitation of xanthenone followed by diffusion-controlled triplet energy transfer (~3.8 × 10⁹ M⁻¹ s⁻¹, Supporting Information) do not contain any traces of ³3*. Therefore, triplet ESIPT has occurred fully within 20 ns (Figure 6). In addition, ³q4* (and not ³4*) is detected within the response time of our apparatus (5 ns) upon direct irradiation of 4. This, together with the results of our quantum chemical calculations, suggests that the decrease in the quantum yields, compared to pHP cages, is a consequence of very fast ESIPT, which is under investigation in our laboratories at the moment. The proposal of the photo-Favorskii-like reaction mechanism of oHP derivatives is presented in Scheme 9.

Scheme 9.

Proposed reaction mechanism

Conclusions

In conclusion, the oHP group can be utilized as a photoremovable protecting group for mediocre and good leaving groups. The major advantages are its simple synthesis, ground state pK_a > 8, and bathochromically shifted absorption spectrum with appreciable molar absorption coefficients above 350 nm compared to pHP derivatives. The deprotection quantum yield is most probably lowered by competing, nonproductive adiabatic tautomerization reactions, in which the ESIPT represents a dominant energy wasting pathway. We found that the leaving group is released predominately from a short-lived triplet state (³3*) but inefficiently also from both the ³3⁻* and ³q3* intermediates.

Experimental

Quantum yields measurements

The quantum yield measurements were performed on an optical bench consisting of a 40-W medium pressure mercury lamp equipped with a 313 nm band-path filter. The concentration of the sample solutions was in the interval of 5 × 10⁻⁴ and 1 × 10⁻³ M. Hexadecane and ethyl benzoate (1 × 10⁻³ M) were used as GC and HPLC internal standards, respectively. A sealed capillary containing TMS in CD₃CN was used as an internal standard for NMR measurements. 2-Nitrobenzaldehyde in acetonitrile¹⁷ and valerophenone in cyclohexane¹⁸ were used as the actinometers. The reaction conversions were always kept below 15% to avoid the interference of photoproducts. The relative standard deviations of the mean for multiple (at least three) samples were found below 10% in all analyses.

Laser Flash Photolysis

The nanosecond laser flash photolysis setup was operated in a right angle arrangement of the pump and probe beams. Laser pulses of ≤700 ps duration at 355 nm (120–200 mJ) or 266 nm (30–80 mJ) were obtained from a Nd:YAG laser and were dispersed over the 4-cm optical path of a quartz cell by a cylindrical concave lens. The absorbance of the sample solution was adjusted to 0.3–0.5 per cm at the wavelength of excitation. A pulsed 75-W xenon lamp was used as the source of probe white light. Kinetic (photomultiplier) and spectrographic detection (ICCD camera) of the transient absorption were available. Measurements were performed at ambient temperature (20 ± 2 °C). Oxygen was removed from the sample solutions by repeated freeze-thaw cycles under reduced pressure (5 Pa).

Synthesis

2-(2-Hydroxyphenyl)-2-oxoethyl benzoate (3a)

2-Bromo-o-hydroxyacetophenone (5, 2.56 g, 11.9 mmol; Supporting Information) in acetone (5 mL) and TEA (1.6 mL, 11.9 mmol) were added to a solution of benzoic acid (1.45 g, 11.9 mmol) in acetone (30 mL) at 20 °C. The reaction mixture was then refluxed. When the conversion reached >98% (GC; ~ 2 h), the mixture was cooled to 20 °C, filtered, and the solvent was removed under reduced pressure. The resulting solid was dissolved in ethyl acetate (20 mL) and water (20 mL) was added. The aqueous layer was separated and washed with ethyl acetate (20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over MgSO₄, filtered, and the solvent was removed under reduced pressure to give the crude title product. The compound was purified using flash column chromatography (ethyl acetate/n-hexane, 1:4, v/v). Yield: 1.92 g (63%); yellowish crystals; mp 103–105 °C (lit. 104–105 °C).^{36 1}H NMR (200 MHz, CDCl₃): δ (ppm) 5.65 (s, 2H), 6.90–7.10 (m, 2H), 7.48–7.70 (m, 4H), 7.74 (dd, 1H, J₁ = 8.0 Hz, J₂ = 1.4 Hz), 8.18 (distorted d, 2H), 11.71 (s, 1H, –OH) (Figure S12). ¹³C NMR (75.5 MHz, CDCl₃): δ (ppm) 65.5, 117.5, 119.1, 119.5, 128.6, 128.8, 129.4, 130.2, 133.7, 137.3, 162.6, 166.2, 197.5 (Figure S13). MS (EI, 70 eV): m/z = 256 (8), 225 (4), 134 (15), 121 (70), 105 (100), 93 (12), 77 (35), 65 (12), 51 (11). FTIR (KBr, cm⁻¹): 3049, 2936, 1730, 1663, 1617, 1583, 1493, 1451, 1415, 1273, 1210, 1123, 1017, 1113, 1017, 968, 860, 798, 755, 712. UV-Vis: ε₃₁₃ (2 % (v/v) CH₃CN in H₂O) = 3050 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (2 % (v/v) CH₃CN in H₂O) = 400 dm³ mol⁻¹ cm⁻¹ (Figure S14). Anal. calcd for C₁₅H₁₂O₄: C, 70.31; H, 4.72, found C, 70.44; H, 4.74. HRMS (HPLC-APCI-MS): calcd for C₁₅H₁₁O₄ (M – H⁺) 255.0657, found 255.0659. This compound has also been characterized elsewhere.³⁷

2-(2-Hydroxyphenyl)-2-oxoethyl acetate (3b)

This compound was synthesized from 5 and acetic acid according to the same procedure as 3a. Yield: 50%; white crystals; mp 59.0–59.8 °C (lit. 58–59 °C).^{36 1}H NMR (300 MHz, CDCl₃): δ (ppm) 2.26 (s, 3H), 5.37 (s, 2H), 6.95 (ddd, 1H, J₁ = 7.0 Hz, J₂ = 7.0 Hz, J₃ = 1.0 Hz), 7.03 (dd, 1H, J₁ = 8.6 Hz, J₂ = 1.0 Hz), 7.53 (ddd, 1H, J₁ = 7.3 Hz, J₂ = 7.3 Hz, J₃ = 1.3 Hz), 7.63 (dd, 1H, J₁ = 8.3 Hz, J₂ = 1.3 Hz) 11.64 (s, 1H, –OH) (Figure S15). ¹³C NMR (75.5 MHz, CDCl₃): δ (ppm) 20.0, 64.7, 116.9, 118.4, 119.0, 128.3, 136.7, 161.9, 170.0, 197.4 (Figure S16). MS (EI, 70 eV): m/z = 194 (7), 163 (2), 152 (20), 134 (13), 121 (100), 105 (6), 93 (10), 77 (5), 65 (12), 43 (22). FTIR (KBr, cm⁻¹): 3064 (br), 2988, 2940, 1740, 1658, 1613, 1577, 1491, 1426, 1385, 1292, 1279, 1245, 1204, 1158, 1083, 974, 868, 790, 753, 730, 667, 646, 518, 506, 449. UV-Vis: ε₃₁₃ (CH₃CN) = 2770 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (CH₃CN) = 250 dm³ mol⁻¹ cm⁻¹; UV-Vis: ε₃₁₃ (50 % (v/v) CH₃CN in H₂O) = 2870 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (50 % (v/v) CH₃CN in H₂O) = 240 dm³ mol⁻¹ cm⁻¹; UV-Vis: ε₃₁₃ (2.5 % (v/v) CH₃CN in H₂O) = 2660 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (2.5 % (v/v) CH₃CN in H₂O) = 370 dm³ mol⁻¹ cm⁻¹ (Figure S17). HRMS (HPLC-APCI-MS): calcd for C₁₀H₉O₄ (M – H⁺) 193.0501, found 193.0500. This compound has also been characterized elsewhere.³⁷

2-(2-Hydroxyphenyl)-2-oxoethyl methanesulfonate (3c)

A suspension of 2-bromo-o-hydroxyacetophenone (5, 1.45 g, 6.73 mmol), silver (I) oxide (2.34 g, 10.1 mmol) and methanesulfonic acid (0.88 mL, 13.5 mmol) in acetonitrile (150 mL) was vigorously stirred overnight at 60 °C. After the starting material disappeared (TLC), the reaction mixture was cooled to 20 °C, filtered through a Celite pad, and the solvent was removed under reduced pressure. The compound was purified from the residue using flash column chromatography (ethyl acetate/n-hexane, 1:2, v/v). Yield: 0.95 g (62%); white crystals; mp 78.0–80.2 °C. ¹H NMR (300 MHz, CDCl₃): δ(ppm) 3.29 (s, 3H), 5.53 (s, 2H), 6.96 (ddd, 1H, J₁ = 7.3 Hz, J₂ = 7.3 Hz, J₃ = 1.3 Hz), 7.09 (dd, 1H, J₁ = 8.7 Hz, J₂ = 1.0 Hz), 7.56–7.63 (m, 2H), 11.49 (s, 1H, –OH) (Figure S18). ¹³C NMR (75.5 MHz, CDCl₃): δ (ppm) 39.3, 69.1, 116.8, 119.2, 119.8, 119.9, 128.4, 137.8, 162.6, 196.2 (Figure S19). MS (EI, 70 eV): m/z = 230 (6), 214 (<1), 196 (<1), 185 (<1), 151 (10), 134 (20), 121 (100), 105 (16), 93 (14), 77 (10), 65 (16), 50 (4). FTIR (KBr, cm⁻¹): 3433 (br), 3037, 3016, 2952, 1656, 1614, 1488, 1447, 1349, 1291, 1207, 1167, 1056, 975, 957, 782, 753, 714, 523, 504. UV-Vis: ε₃₁₃ (CH₃CN) = 4050 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (CH₃CN) <50 dm³ mol⁻¹ cm⁻¹; ε₃₁₃ (H₂O/CH₃CN, 9:1, (v/v)) = 2900 dm³ mol⁻¹ cm⁻¹; ε₃₆₆ (H₂O/CH₃CN, 9:1, (v/v)) 420 dm³ mol⁻¹ cm⁻¹ (Figure S20). HRMS (HPLC-APCI-MS): calcd for C₉H₁₁O₅S (M + H⁺) 231.0322, found 231.0322.

Photochemical experiments

Irradiation in NMR tubes (a general procedure)

A solution of the corresponding phenacyl derivative (3a–c, 6 or 1 (X = benzoate), ~2 mg) in a deuterated solvent or solvent mixture (0.50–0.75 mL) was irradiated either in a Rayonet reactor at λ = 300 ± 20 nm or with a 40-W medium-pressure mercury lamp equipped with a 313 nm band-pass filter. NMR cuvettes also act as a Pyrex filter (λ_irr ≥ 280 nm). ¹H NMR spectra were measured in the corresponding time intervals to follow the course of the reaction.

Preparative-scale irradiation

Irradiation was done in two different ways. (a) A large test tube containing a sample solution (c = 0.01–0.02 M, 70–100 mL) was fitted with a rubber septum and the sample was degassed by bubbling with argon for 10 min. The test tube was then irradiated in an immersion well. (b) For a larger-scale reaction (c = 0.01–0.02 M in 250 mL), photolysis was carried out in an immersion well equipped with a quartz cooling jacket and a condenser. A 450-W medium-pressure mercury arc lamp with a Pyrex filter (λ > 290 nm) was used as a light source.

Characterization of the photoproducts

Identification of the photoproducts was based on NMR analyses and a comparison of their mass spectra (GC-MS) and the GC retention times with those obtained from authentic samples.

Benzofuran-3(2H)-one (9)

The title compound was separated from the irradiated solution of 3a–c in the corresponding solvent (Table 1) using flash column chromatography (dichloromethane/ethyl acetate/n-hexane, 2:1:20, v/v). Orange powder; mp 84–85.5 °C. ¹H NMR (200 MHz, CDCl₃): δ (ppm) 4.67 (s, 2H), 7.09–7.20 (m, 2H), 7.73–7.61 (m, 2H). ¹³C NMR (50 MHz, CDCl₃): δ (ppm) 74.8, 113.7, 121.3, 122.1, 124.2, 137.9, 174.1, 199.9. MS (EI, 70 eV): m/z = 134 (100), 105 (80), 76 (60), 63 (8), 50 (30). FTIR (KBr, cm⁻¹): 3535, 2935, 1725, 1613, 1469, 1405, 1318, 1195, 1155, 1107, 991, 842, 767, 667. HRMS (ESI⁻): calcd for C₈H₅O₂ (M – H⁺) 133.0295, found 133.0292. This compound has also been characterized elsewhere.³⁸

Benzofuran-2(3H)-one (10)

The title compound was separated from the irradiated solution of 3a–c in the given solvent (Table 1) using flash column chromatography (dichloromethane/ethyl acetate/n-hexane, 2:1:20, v/v). White crystals; mp 37–40 °C. ¹H NMR (200 MHz, CDCl₃): δ (ppm) 3.78 (s, 2H), 7.12–7.20 (m, 2H), 7.30–7.38 (m, 2H). ¹³C NMR (50 MHz, CDCl₃): δ (ppm) 33.1, 110.9, 123.1, 124.2, 124.7, 129.0, 154.8, 174.2. MS (EI, 70 eV): m/z = 134 (90), 106 (55), 89 (2), 78 (100), 63 (10), 51 (25). FTIR (KBr, cm⁻¹): 3447, 2962, 1803, 1616, 1462, 1386, 1329, 1296, 1230, 1202, 1118, 1057, 961, 891, 822, 761, 673. HRMS (ESI⁻): calcd for C₈H₅O₂ (M – H⁺) 133.0295, found 133.0293. This compound has also been characterized elsewhere.³⁹

(±)-3-Hydroxy-2,3-dihydrobenzofuran-3-methylenyl acetate (11)

This product was isolated by flash column chromatography (ethyl acetate/n-hexane, 1:3 to 1:1, v/v) from the irradiated solution of 2-(2-methoxyphenyl)-2-oxoethyl acetate 6 in dry CH₃CN to a >90% conversion. Yield: 30%, colorless viscous liquid. ¹H NMR (300 MHz, CDCl₃): δ (ppm) 2.13 (s, 3H), 2.57 (s, 1H, –OH), 4.35–4.55 (m, 4H), 6.89 (d, 1H, J = 7.9 Hz), 6.97 (dt, 1H, J₁ = 7.6 Hz, J₂ = 0.7 Hz), 7.30 (dt, 1H, J₁ = 7.9 Hz, J₂ = 1.0 Hz), 7.36 (dd, 1H, J₁ = 7.6 Hz, J₂ = 0.7 Hz) (Figure S24). ¹³C NMR (75.5 MHz, CDCl₃): δ (ppm) 21.0, 68.2, 79.8, 80.6, 111.1, 121.4, 124.1, 128.1, 131.4, 160.7, 170.9 (acetate carboxyl C_q was observed in ¹H–¹³C HMBC NMR) (Figures S25 and S26). MS (EI, 70 eV): 208 (10), 190 (2), 148 (8), 135 (100), 119 (1), 107 (30), 91 (6), 79 (35), 77 (25), 63 (4), 51 (5), 43 (25). HRMS (APCI⁺) calcd for C₁₁H₁₁O₄ [M − H⁺] 207.0663, found 207.0658.