One- and Two-Photon Uncaging: Carbazole Fused o

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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One- and Two-Photon Uncaging: Carbazole Fused o‑Hydroxycinnamate Platform for Dual Release of Alcohols (Same or Different) with Real-Time Monitoring Yarra Venkatesh,† Hemant Kumar Srivastava,‡ S. Bhattacharya,§ Muneshwar Mehra,‡ P. K. Datta,§ S. Bandyopadhyay,⊥ and N. D. Pradeep Singh*,† †

Department of Chemistry, ‡Advanced Technology Development Centre, §Department of Physics, ⊥Department of E & ECE, Indian Institute of Technology Kharagpur, 721302 Kharagpur, West Bengal, India S Supporting Information *

ABSTRACT: A one- and two-photon activated photoremovable protecting group (PRPG) was designed based on a carbazole fused o-hydroxycinnamate platform for the dual (same or different) release of alcohols. The mechanism for the dual release proceeds through a stepwise pathway and also monitors the first and second photorelease in real time by an increase in fluorescence intensity and color change, respectively. Further, its application in staining live neurons and ex vivo imaging with two-photon excitation is shown.

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two-photon excitation (1PE and 2PE). Despite these interesting features, the o-hydroxycinnamate derivatives have not been explored for their dual uncaging ability. Herein, we report for the first time carbazole with an inbuilt ohydroxycinnamate moiety on both sides as an efficient PRPG for the dual release of alcohols (same or different) upon oneand two-photon excitation with real-time monitoring ability. Dual caged carbazole esters of the same (5a−i) and different (9) alcohols were synthesized, as outlined in Scheme 1. First, 9-

wo-photon uncaging has become a useful and noninvasive method for the delivery of bioactive molecules.1 The above approach provides advantages, such as (i) spatiotemporal control over the release, (ii) deeper penetration into biological tissue, and (iii) reduction of the illumination duration, thus mitigating the harmful effects associated with the light on living tissues. To date, several two-photon sensitive photoremovable protecting groups (PRPGs) have been developed based on chromophores such as o-nitrobenzyl,2−6 nitroindoline,7 coumarin,8,9 quinoline,10 and o-hydroxycinnamic acid derivatives.11 The two main limitations of using the aforementioned twophoton activated PRPGs in the area of drug delivery are they (i) can cage only one bioactive molecule and (ii) lack the ability to provide information regarding the release of the bioactive molecule in real time. In the literature, there is only one example in which two-photon sensitive o-nitrobenzyl PRPG was utilized for the dual release of a single substrate.12 Therefore, we intend to design a two-photon activated PRPG which can release two identical or different substrates (with the same functional group) and monitor the dual release in real time by a noninvasive fluorescent technique. The o-hydroxycinnamate derivatives are well-known PRPGs which were first introduced by Porter et al. to cage alcohols and amines. The photorelease mechanism of the o-hydroxycinnamate is known to proceed through its singlet state upon irradiation. From the singlet state, it undergoes rapid trans−cis photoisomerization, followed by thermally driven lactonization leading to release of the caged molecule along with the formation of photoproduct coumarin.13 The salient features of o-hydroxycinnamate based PRPGs include (i) clean and rapid uncaging, (ii) structural simplicity, (iii) direct caging of alcohols and amines without carbonate or carbamate linkages which are prone to be labile under physiological conditions, (iv) the resulting photoproduct coumarin serving as a fluorescent reporter, and (v) sufficient sensitivity toward both one- and © XXXX American Chemical Society

Scheme 1. Synthesis of Dual (Same or Different) Caged Carbazole Esters (5a−i and 9)

ethyl-2,7-dimethoxy-9H-carbazole (1) was prepared by using the literature procedure.14 Next, 2 was obtained from 1 via Vilsmeier−Haack reaction, followed by methoxy deprotection in the presence of AlCl3. Now, the dual caged esters 5a−i of the same alcohols were obtained by the Wittig reaction between 2 and alkyltriphenylphosphoranylideneacetate (4a−i) of corresponding alcohols (3a−i). Finally, we synthesized dual caged ester 9, by caging two different alcohols (3a and 3e) Received: February 18, 2018

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DOI: 10.1021/acs.orglett.8b00090 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

excitation. We irradiated the solution of caged esters 5a−i and 9 individually in acetonitrile/water (3:7 v/v) with a 125-W medium-pressure Hg lamp (λ ≥ 365 nm) through a 1 M CuSO4 solution as the UV cut−off filter. Irradiation of 5a−i and 9 for 120 min resulted in the release of the corresponding alcohols 3a−i in high chemical (87−92%) and moderate quantum (0.043−0.048) yields, as shown in Table 2.

independently in a sequential manner by the similar synthetic protocol used for caging same alcohols as shown in Scheme 1. All the synthesized dual caged carbazole esters (5a−i and 9) were characterized by 1H, 13C NMR and mass spectral analysis (see Figures S1−S23 in the Supporting Information (SI)). The UV/vis absorption and emission spectra of 5a and 9 (1 × 10−4 M) in DMSO were recorded. The absorption spectra of 5a and 9 show an intense peak centered at 373 and 372 nm, respectively. In the emission spectrum, the emission maximum of 5a and 9 was centered at about 490 and 488 nm, respectively, as shown in Figure 1. Further, the absorption,

Table 2. Photochemical Properties of 5a−i and 9 caged ester 5a 5b 5c 5d 5e 5f 5g 5h 5i 9

Figure 1. Normalized UV/vis absorption (blue line) and emission spectra (red line) of 5a (a), 9 (b). Excitation wavelength: 370 nm.

% of deprotectiona 92 89 91 87 89 92 88 91 90 87 89

(3a) (3b) (3c) (3d) (3e) (3f) (3g) (3h) (3i) (3a) (3e)

Φub 365 nm

(ε × Φu)c M−1 cm−1

δu (GM) 750 nmd

0.047 0.044 0.045 0.046 0.045 0.048 0.043 0.046 0.045 0.044 0.046

296.1 276.3 284.0 289.8 283.1 301.0 270.0 290.3 282.6 276.7 289.3

0.418 0.391 0.400 0.409 0.400 0.427 0.383 0.409 0.400 0.391 0.409

a

% of the alcohols released as determined by 1 H NMR; Photochemical quantum yield of (E) to (Z) photoisomerization after one-photon excitation at λ ≥ 365 nm (error limit within ±10%). c Action cross section for (E) to (Z) photoisomerization with onephoton excitation at λ ≥ 365 (error limit within ±5%). dTwo photon uncaging cross section for (E) to (Z) photoisomerization with twophoton excitation at 750 nm. b

emission maxima, Stoke’s shift, and fluorescence quantum yield of 5a−i and 9 are summarized in Table 1. The fluorescence quantum yields (Φf) of caged esters were calculated using 9,10diphenylanthracene as the standard (Φf = 0.95 in ethanol). Initially, the light-induced uncaging ability of dual caged carbazole esters (5a−i and 9) was analyzed with one-photon

As a representative example, we irradiated a solution of 5a in DMSO-d6 at regular intervals of time followed by analysis by 1 H NMR spectroscopy (Figure 2). The 1H NMR spectrum at 0

Table 1. Synthetic Yields and Photophysical Properties of 5a−i and 9

Figure 2. 1H NMR study of 5a in DMSO-d6 during photolysis at regular interval of time (0−120 min, time interval = 60 min).

min shows characteristic peaks at 8.41 ppm (singlet) and 8.06 ppm (doublet) corresponding to the aromatic proton (Ha) and olefinic proton (Hb) of 5a, respectively. After 60 min of irradiation, we observed a decrease in the peak intensity at 8.41 and 8.06 ppm, which indicates the photodecomposition of 5a. On the other hand, we noted three new peaks, one at 3.44 ppm (quartet) corresponding to the methylene protons (Hg) of released ethanol (3a) and the other two new peaks at 8.54 and 8.13 ppm corresponding to the newly formed intermediate coumarin carbazole conjugate (Cou-Cbz) (11). Upon continuation of irradiation up to 120 min, we noticed the appearance of two new peaks at 8.51 and 8.21 ppm, indicating the formation of the final photoproduct the coumarin carbazole coumarin conjugate (Cou-Cbz-Cou) (12) with the second

a

Based on the isolated yield of the last step. bMaximum absorption wavelength. cMolar absorption coefficients at 365 nm. dMaximum emission wavelength. eDifference between maximum absorption wavelength and maximum emission wavelength. fFluorescence quantum yield (error limit within ±5). B

DOI: 10.1021/acs.orglett.8b00090 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters release of ethanol (see expanded 1H NMR spectra Figure S24 in the SI). The intermediate 11 and photoproduct 12 were confirmed by 1H NMR and mass spectroscopy analysis (see Figures S25−S28 in the SI). Finally, the % of released ethanol was calculated using 1,2dichloroethane as an internal standard (see Figure S29 in the SI). The 1H NMR study clearly shows that 5a releases ethanol and photoproduct 12 in a 2:1 stoichiometric ratio through the probable intermediate 11. We also monitored the photolysis of 5a in ACN/PBS buffer (3:7 v/v) by UV/vis absorption spectroscopy (see Figure S30 in the SI). Further, we examined the release of two different alcohols from dual caged ester 9 in DMSO by irradiating for 120 min and monitoring by RP-HPLC (see Figure S31 and Scheme S1 in the SI). Figure S31 indicates that dual uncaging of 9 occurs in a stepwise manner. The % of alcohols released, 3a (87%, Φp = 0.044) and 3e (89%, Φp = 0.046), was calculated by 1H NMR using 1,2 dichloroethane as an internal standard. The quantum yield (Φu) of uncaging with one-photon excitation was calculated using potassium ferrioxalate as an actinometer.15 After one-photon excitation, we were interested in calculating the two-photon uncaging cross section (δu) of our dual caged carbazole esters (5a−i and 9). To calculate δu, the nonlinear optical measurements of 5a were carried out by a single beam zscan technique as described in the literature procedure.16 The open aperture (OA) z-scan measurements were performed with a laser pulse of 100 fs at the 1 kHz repetition rate at the wavelength 750 nm. An OA z-scan curve has been fitted using the two-photon absorption phenomenon (see Figure S32 in the SI).16 The two-photon absorption cross section (δa) was calculated to be 8.9 GM at 750 nm (1 GM = 10−50 cm4 s/ photon). Then, we calculated the two-photon uncaging cross section (δu) of 5a−i and 9 using the formula δu = Φuδa, and the results are shown in Table 2. The results show that δu values of our caged compounds are larger than the limit of 0.1 GM, ensuring their suitability for biological applications. From the literature13 and 1H NMR study, we suggest a possible stepwise mechanism for the dual release of alcohols from caged esters 5a−i as shown in Scheme 2. As a

excited to its singlet state and release of the second caged alcohol, along with the formation of final photoproduct CouCbz-Cou (12) by following a similar mechanism of the first release. Further, the proposed mechanism was supported by highresolution mass spectrometry (HRMS) by analyzing the photolysis mixture of 5a after 60 min of irradiation. It was found that the possible intermediate 11 was present in the reaction mixture, along with the final photoproduct 12 (see Figure S34 in the SI). Interestingly, our designed dual caged PRPG monitors the first and second photorelease in real time by an increase in fluorescence intensity and fluorescence color change, respectively (see Figure 3).

Figure 3. (a) Emission spectra of 5a (1 × 10−4 M, ACN/PBS buffer (3:7), pH = 7.4) recorded during photolysis at regular interval of time (0−120 min). (b) Comparison of the fluorescent spectral profile at different time intervals 0 min (light green line), 60 min (dark green line), and 120 min (blue line). Excitation wavelength: 370 nm.

At 0 min, the excitation of 5a at λmax = 370 nm produced an emission band at λmax = 504 nm (green fluorescence). Upon a gradual increase in the irradiation time (0−60 min), we noted the gradual increase in the emission intensity at λmax = 498 nm, indicating the formation of Cou-Cbz (11, Φf = 0.21, and δa = 8.7). The increase in the fluorescence intensity of Cou-Cbz compared to 5a is because, in Cou-Cbz, ICT17 (internal charge transfer) occurs between one donor (carbazole) and one acceptor (ester carbonyl), whereas, in 5a, ICT exists between one donor (carbazole) and two acceptors (ester carbonyl) (see Figure 3). Notably, after 120 min of irradiation, we observed a gradual decrease in the emission intensity at λmax = 498 nm with a new blue-shifted emission band at 470 nm. The blue fluorescence is due to the absence of ICT in the newly formed photoproduct Cou-Cbz-Cou (12, Φf = 0.16, and δa = 8.4). To confirm the observed fluorescence color change is due to the dual uncaging process, we recorded both 1P and 2P absorption and emission spectra of 11 and 12, independently (see Figures S35 and S36 in the SI). The results reveal that the emission maximum of intermediate 11 and photoproduct 12 is similar to the case of the photolysate recorded at 60 and 120 min, respectively. Further, we carried out the in vitro cytotoxicity assay of dual caged esters by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay on the normal cell line (NIH 3T3) shown in Figure S37 in the SI. Briefly, normal cells were incubated with 20 μM of 5a−i and 9 for 72 h. After 72 h of incubation, it was revealed that greater than 75% cell viability remains. Henceforth, it could be said that designed dual caged esters were biocompatible at the studied concentration. Next, we demonstrated the application of designed caged esters by performing ex vivo two-photon imaging in live slices of a mouse brain. Neurons in acutely prepared slices from the motor cortex (Figure 4a, bright field) were loaded with caged

Scheme 2. Possible Photorelease Mechanism for Dual Release

representative example, the caged ester 5a is excited to its singlet state upon irradiation (supported by the fluorescence quenching study in the presence of benzophenone in Figure S33 in the SI). From the singlet state, it undergoes rapid trans− cis photoisomerization to give 10, followed by thermally driven lactonization leading to the release of ethanol (3a) and formation of the Cou-Cbz conjugate (11). The Cou-Cbz (11) formed in situ and then absorbs light followed by becoming C

DOI: 10.1021/acs.orglett.8b00090 Org. Lett. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS We thank DST (SERB) for financial support and DST (SR/ FST/CSII-026/2013) for 500 MHz NMR. S.B. thanks SRIC Challenge Grant Scheme (IIT Kharagpur) and Wellcome Trust/DBT India Alliance for financial support. Y.V. is thankful to the Indian Institute of Technology Kharagpur for the fellowship.

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Figure 4. (a) Bright field image of the brain slice showing motor cortex region in P48 mouse. (b and c) Two-photon excited images of 5a labeled motor neurons of P2 and P48 days old mouse, respectively. White arrows indicate labeled neuronal processes.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00090. Experimental procedures and spectroscopic data; 1H, 13C NMR and HRMS spectra (PDF)

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ester 5a and imaged at 810 nm using an InSight DS (SpectraPhysics). The cytoplasmic regions of the neurons (Figure 4b and c) were clearly stained by 5a, as observed by the fluorescence. The slices were imaged for more than 6 h, showing the suitability of use in neuronal morphology tracing. Two-photon excitation of the caged ester 5a showed that the compound is neuronal membrane-permeant and is also capable of labeling neuronal processes (dendrites, arrows Figure 4b and c) especially at younger ages (Postnatal day 2, P-2) as shown in Figure 4b. Our compound reliably labels neuronal population in mature neurons at older ages (Postnatal day 48, P-48) as shown in Figure 4c (region of the bright field marked in Figure 4a), which offers an advantage over most of the reported dyes and indicators that have yielded satisfactory results in immature brains. In conclusion, we have developed a carbazole-fused ohydroxycinnamate platform for the dual release of alcohols. Our designed PRPG releases not only the same two alcohols but also different alcohols with high chemical and moderate quantum yield upon one- and two-photon excitation. The mechanism for the dual release proceeds through a stepwise pathway. Our PRPG has the ability to monitor both the first and second release in real time by an increase in fluorescence intensity and fluorescence color change, respectively. Further, the dual caged esters exhibited excellent properties, such as good biocompatibility, staining live neurons, and ex vivo imaging with two-photon excitation. However, the limitation of our system is that the photoproduct formed can act as an inner filter. We intend to use our PRPG to design a two-photon activated single component combinatorial drug delivery system with real-time monitoring ability.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yarra Venkatesh: 0000-0002-4478-1553 N. D. Pradeep Singh: 0000-0001-6806-9774 Notes

The authors declare no competing financial interest. D

DOI: 10.1021/acs.orglett.8b00090 Org. Lett. XXXX, XXX, XXX−XXX