Letter pubs.acs.org/OrgLett
Bimane: A Visible Light Induced Fluorescent Photoremovable Protecting Group for the Single and Dual Release of Carboxylic and Amino Acids Amrita Chaudhuri, Yarra Venkatesh, Krishna Kalyani Behara, and N. D. Pradeep Singh* Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, 721302 West Bengal, India S Supporting Information *
ABSTRACT: A series of ester conjugates of carboxylic and amino acids were synthesized based on bimane fluorescent photoremovable protecting group (FPRPG). The photorelease of single and dual (same as well as different) carboxylic and amino acids is demonstrated from a single bimane molecule on irradiation with visible light (λ ≥ 410 nm). The detailed mechanistic study of photorelease revealed that the release of two caged acids is simultaneous but in a stepwise pathway.
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visible region, and good water solubility and is useful for many biological purposes like labeling of human red cell,18 thiol sensing,19 etc. Herein, we report bimane as a FPRPG for caging (shown in Scheme 1) single and dual (same and different) carboxylic and
hotoremovable protecting groups (PRPGs) have attracted much attention, as their release step is driven by light unlike the conventional protecting groups where deprotection requires harsh chemical reagents and conditions.1 The temporal and spatial resolution afforded by light along with wavelength control over deprotection gives PRPGs a distinct advantage over conventional protecting groups in the fields like biology, biochemistry, and physiology to release various types of active molecules. Examples of some well-known PRPGs are onitrobenzyl,2 phenacyl,3 benzoyl,4 quinolinyl,5 anthracenyl,6 and coumarinyl7 derivatives. Unfortunately, except for few PRPGs,8−12 most of the well-known PRPGs absorb light in the UV region, which limits their usage for biomedical applications, as UV irradiation is constrained by its toxicity and very low tissue penetration power. Moreover, PRPGs with strong fluorescent properties help us to visualize, quantify, and follow the spatial distribution, localization, and depletion of the released molecule.13,14 The aforementioned PRPGs can deprotect only one active compound at a time on exposure to light. It will be highly beneficial if we can release two different active compounds simultaneously from a single fluorescent photoremovable protecting groups (FPRPG) at a time on exposure to light. Such FPRPGs have a wider scope of application in combination therapy for delivering two different synergistic biologically active compounds simultaneously.15 Therefore, we have taken up the challenge of developing FPRPGs, which will be able to cage two active compounds and release them on exposure to visible light. Recently, our group has reported acetyl carbazole-based FPRPG to release two similar and different carboxylic acids from single chromophore on exposure to UV light and have shown its applicability as a dual drug delivery system (DDS).16 This has prompted us to design a FPRPG based on bimane chromophore, which can cage two different or same active molecules and release them simultaneously on visible light irradiation (λ ≥ 410 nm). Choice of the bimane moiety is based on its exciting properties like strong fluorescent nature,17 prominent absorption in the © XXXX American Chemical Society
Scheme 1. Synthesis of Single and Dual (Same and Different) Component Caged Esters of Bimane
amino acids, which release the caged acids simultaneously on exposure to UV (λ ≥ 365 nm) and visible light (λ ≥ 410 nm) irradiation. First, we synthesized fluorescent bimane molecule 1 by using the described procedure.20 Next, single FPRPG 2 and dual FPRPG 5 were obtained by bromination of 1 with one and two equivalents of bromine (Br2), respectively. Single component caged esters 4a−h were obtained by treating FPRPG 2 with one equivalent of carboxylic or amino acid in the presence of K2CO3/ KI, and dual component esters 7a−g were obtained by treating the FPRPG 5 with two equivalents of carboxylic or amino acids. Received: February 10, 2017
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DOI: 10.1021/acs.orglett.7b00416 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Finally, we synthesized dual component caged ester, which can release two different substrates. Dual caged ester 12 was synthesized by refluxing 5 with sodium formate in ethanol to get the dialcohol 8, treating 8 with one equivalent of ochlorobenzoyl chloride (9) gave 10, and further treatment of 10 with p-methoxy benzoyl chloride (11) afforded compound 12. All the synthesized caged esters were characterized by 1H, 13C, and mass spectral analysis (see Figures S1−S16 in Supporting Information (SI)). UV absorption and emission spectra of 1 × 10−5 M solution of caged esters (4a−h, 7a−g, and 12) in absolute ethanol were recorded. The normalized absorption and emission spectra of caged esters 4a and 7a in absolute ethanol were shown in Figure 1a,b, respectively. The absorption spectrum of 4a and 7a shows
Table 1. Synthetic Yields and Photophysical Data for Caged Esters (4a−h, 7a−g, and 12)
Figure 1. UV absorption and emission spectra of (a) single component caged ester 4a and (b) dual component caged ester 7a.
an intense peak centered at 377 and 381 nm, respectively. In the emission spectrum, the emission maxima of 4a and 7a were redshifted to about 461 and 459 nm, respectively. We noticed that the Stoke’s shift in the case of single component caged esters varies between 84 and 85 nm, and for dual component, it is in between 70 and 79 nm (Table 1). The absorption, emission maxima, Stoke’s shift, and fluorescence quantum yield of all the caged esters are summarized in Table 1. The fluorescence quantum yields (Φf) of single component caged esters (4a−h) and dual component caged esters (7a−g and 12) in absolute ethanol (EtOH) at room temperature were in the range of 0.630 ≤ Φf ≤ 0.641 and 0.715 ≤ Φf ≤ 0.842, respectively (Table 1). The fluorescence quantum yield of all the caged esters (4a−h, 7a−g, and 12) were calculated using 9,10-diphenylanthracene as standard (Φf = 0.95 in ethanol).21 To explore the photorelease ability of bimane caged ester, we irradiated degassed solution of all caged esters (4a−h, 7a−g, and 12) (1 × 10−4 M) individually in ACN/H2O (7:3 v/v) using 125 W medium pressure Hg lamp at two different irradiation wavelengths (≥365 and ≥410 nm). The progress of the photocleavage reactions was monitored by RP-HPLC and 1H NMR spectroscopy. We found the corresponding carboxylic and amino acids were released in good chemical (75−85%) and moderate quantum (0.06−0.07) yields (Table 2). The photochemical quantum yield (Φp) was calculated by using potassium ferrioxalate as an actinometer22 (see pages 40−41 in the SI). Further, the sensitivity to the photolysis of our caged esters (4a− h, 7a−g, and 12) was calculated (see Table S1 in SI). Using 4a as a representative example, we have shown the photorelease of single component caged esters (4a−h) by reverse phase (RP) HPLC at different intervals of irradiation time (Figure 2). The HPLC shows a gradual decrease of the peak at retention time (tR) 3.06 min with an increase in irradiation time, indicating the photolysis of caged ester 4a. In addition, we also noted gradual increase of two new peaks at tR 2.74 and 3.24
a
Based on isolated yield. bMaximum absorption wavelength. cMolar absorption coefficient at maximum absorption wavelength. dMaximum emission wavelength. eDifference between maximum absorption wavelength and maximum emission wavelength. fFluorescence quantum yield (error limit within ±5%).
min, which corresponds to released photoproduct, 3-(hydroxymethyl)-2,5,6-trimethylpyrazolo[1,2-a]pyrazole-1,7-dione, and carboxylic acid, i.e., p-anisic acid (3a), respectively. The corresponding photoproducts were confirmed by injecting authentic sample and also by isolation and characterization using 1H, 13C, and mass spectral analysis (see Figure S17 in SI). To understand the solvent effect on the rate of photorelease of our FPRPG, we irradiated caged ester 4a in a different ratio of acetonitrile−water solvent systems at two different wavelengths (≥365 and ≥410 nm). We noticed that photocleavage efficiency increases with increasing amount of water in acetonitrile (Table 3). To examine the suitability in a biological system, we carried out the photolysis of 4a in O2 saturated ACN/HEPES buffer (1:19 v/v) system at physiological pH 7.4. The results indicated that our FPRPG released carboxylic acid 3a in good chemical (87%) and moderate quantum yield (Φp = 0.084, ≥410 nm). The hydrolytic stability of the caged esters (4a−h, 7a−g, and 12) was also tested by keeping them individually under dark in acetonitrile−water (1:1 v/v) solvent (of pH = 7) for a period of 30 days. The course of hydrolysis of all caged esters was B
DOI: 10.1021/acs.orglett.7b00416 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 2. Photochemical Data of Caged Esters (4a−h, 7a−g, 12) in ACN/H2O (7:3 v/v) Solvent 365 nm
Table 3. Photorelease Data of Caged Ester 4a in Different Ratio of ACN/H2O Solvent System
410 nm
365 nm
caged ester
% of deprotectiona
quantum yield (φp)b
% of deprotectionc
quantum yield (φp)d
4a 4b 4c 4d 4e 4f 4g 4h 7a 7b 7c 7d 7e 7f 7g 12
85 83 82 84 85 86 85 82 84 85 83 82 84 83 82 84 (9) 80 (11)
0.061 0.059 0.059 0.060 0.061 0.062 0.061 0.059 0.060 0.061 0.059 0.059 0.060 0.059 0.059 0.030 0.029
80 77 77 79 76 78 76 76 78 79 77 79 78 76 77 80 (9) 78 (11)
0.077 0.074 0.074 0.076 0.073 0.075 0.073 0.07 0.072 0.076 0.071 0.073 0.072 0.073 0.074 0.039 0.0377
solvent system ACN ACN/H2O (9:1) ACN/H2O (4:1) ACN/H2O (7:3) ACN/H2O (3:2) ACN/H2O (1:1) MeOH ACN/HEPES (1:19)
410 nm
% of deprotectiona
quantum yield (φp)b
% of deprotectionc
quantum yield (φp)d
40 81
0.029 0.058
30 78
0.029 0.075
83
0.060
80
0.077
85
0.061
81
0.078
87
0.063
83
0.08
88
0.063
84
0.081
70 88
0.05 0.063
68 87
0.066 0.084
a
% of deprotection of caged ester with respect to initial concentration after 90 min irradiation time. bPhotochemical quantum yield at λ ≥ 365 nm (error limit within ±5%). c% of deprotection of caged ester with respect to initial concentration after 360 min irradiation time. d Photochemical quantum yield at λ ≥ 410 nm (error limit within ±5%).
a
% of deprotection of caged ester with respect to initial concentration after 90 min irradiation time. bPhotochemical quantum yield at λ ≥ 365 nm (error limit within ±5%). c% of deprotection of caged ester with respect to initial concentration after 360 min irradiation time. d Photochemical quantum yield at λ ≥ 410 nm (error limit within ±5%).
Scheme 2. Possible Photorelease Mechanism of Bimane Caged Esters
protonation to yield the corresponding carboxylic acid. Further, the photolysis in methanol gave 3-(methoxymethyl)-2,5,6trimethylpyrazolo[1,2-a]pyrazole-1,7-dione as photoproduct (see Figure S19 in SI), which supports the formation of a zwitterion-like intermediate during the photorelease mechanism. After successful demonstration of bimane as a FPRPG, to evaluate the deprotection efficiency of dual component caged esters, we irradiated 7a−g in ACN/H2O (7:3 v/v) mixture (at λ ≥ 365 and ≥410 nm), individually (Table 2). The photocleavage reactions were monitored by 1H NMR spectroscopy. In every case, we observed that the released carboxylic acids (6a−g) and photoproduct 3,5-bis(hydroxymethyl)-2,6-dimethylpyrazolo[1,2-a]pyrazole-1,7-dione (see Figure S18 in SI) were only formed. As an illustrative example, we have shown the photorelease of 7a by 1H NMR study (Figure S21 in SI). Similarly, irradiation of 12 (the FPRPG attached with the two different type of carboxylic acids) resulted in the simultaneous release of two different carboxylic acids 9 and 11 along with the formation of photoproduct 3,5-bis(hydroxymethyl)-2,6dimethylpyrazolo[1,2-a]pyrazole-1,7-dione which was monitored by 1H NMR spectroscopy (Figure S22 in SI). From 1H NMR, we found that 12 releases carboxylic acid 9 in 80% (Φp = 0.039) and 11 in 78% (Φp = 0.038) (λ≥ 410 nm). Based on the literature,16,24 we proposed a stepwise pathway (Scheme 3 and S1 in SI) for releasing two same and different
Figure 2. HPLC profile for the photolysis of the caged ester 4a (1 × 10−4 M) in ACN/H2O (7:3 v/v) at different interval of time (0−90 min). Irradiation wavelength: λ ≥ 365 nm.
calculated using 1H NMR. We observed only 5−8% decomposition of the ester conjugates (Table S2 in SI). Based on the literature,23 photolysis quenching experiment by using naphthalene as singlet state quencher (see Figure S20 in SI) and solvent effect studies on photorelease, we suggest a possible mechanism for the photolysis of bimane caged esters (4a−h, 7a−g, and 12) as shown in Scheme 2. After absorption of a photon by bimane caged ester (4a), relaxation to the lowest excited singlet state (S1) (1[4a]*) takes place. Deactivation of 1 [4a]* occurs by fluorescence and nonradiative processes that compete with heterolytic C−O bond cleavage forming the singlet ion pair in the initial reaction step. Recombination of ion pair leads back to ground state (S0), and product formation is suggested to happen in two steps: (i) formation of solvent separated ion pair and (ii) the cation part reacts with water to yield the photoproduct alcohol and anion part undergoes C
DOI: 10.1021/acs.orglett.7b00416 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
(9) Jana, A.; Atta, S.; Sarkar, S. K.; Singh, N. D. P. Tetrahedron 2010, 66, 9798−9807. (10) Jana, A.; Ikbal, M.; Singh, N. D. P. Tetrahedron 2012, 68, 1128− 1136. (11) Goswami, P. P.; Syed, A.; Beck, C. L.; Albright, T. R.; Mahoney, K. M.; Unash, R.; Smith, E. A.; Winter, A. H. J. Am. Chem. Soc. 2015, 137, 3783−3786. (12) Šebej, P.; Wintner, J.; Müller, P.; Slanina, T.; Al Anshori, J.; Antony, L. A. P.; Klán, P.; Wirz, J. J. Org. Chem. 2013, 78, 1833−1843. (13) Politz, J. C. Trends Cell Biol. 1999, 9, 284−287. (14) Schade, B.; Volker, H.; Schmidt, R.; Herbrich, R.; Krause, E.; Eckardt, T.; Bendig, J. J. Org. Chem. 1999, 64, 9109−9117. (15) Lehár, J.; Krueger, A. S.; Avery, W.; Heilbut, A. M.; Johansen, L. M.; Price, E. R.; Rickles, R. J.; Short, G. F., III; Staunton, J. E.; Jin, X.; Lee, M. S.; Zimmermann, G. R.; Borisy, A. a. Nat. Biotechnol. 2009, 27, 659− 666. (16) Venkatesh, Y.; Rajesh, Y.; Karthik, S.; Chetan, A. C.; Mandal, M.; Jana, A.; Singh, N. D. P. J. Org. Chem. 2016, 81, 11168−11175. (17) Kosower, N. S.; Newton, G. L.; Kosower, E. M.; Ranney, H. M. Biochim. Biophys. Acta, Protein Struct. 1980, 622, 201−209. (18) Kosower, N. S.; Kosower, E. M.; Newton, G. L.; Ranney, H. M. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 3382−3386. (19) Montoya, L. A.; Shen, X.; McDermott, J. J.; Kevil, C. G.; Pluth, M. D. Chem. Sci. 2015, 6, 294−300. (20) Kosower, E. M.; Pazhenchevsky, B. J. Am. Chem. Soc. 1980, 102, 4983−4993. (21) Morris, J. V.; Mahaney, M. A.; Huberr, J. R. J. Phys. Chem. 1976, 80, 969−974. (22) Dema, J. N.; Bowman, W. D.; Zalewsk, E. F.; Velapold, R. A. J. Phys. Chem. 1981, 85, 2766−2771. (23) Schmidt, R.; Geissler, D.; Hagen, V.; Bendig, J. J. Phys. Chem. A 2007, 111, 5768−5774. (24) Jana, A.; Nguyen, K. T.; Li, X.; Zhu, P.; Tan, N. S.; Ågren, H.; Zhao, Y. ACS Nano 2014, 8, 5939−5952.
Scheme 3. Stepwise Photorelease of Bimane Caged Ester with Two Different Carboxylic Acids (12)
carboxylic acids from bimane caged esters (7a−g, 12), which was supported by high-resolution mass spectroscopy (HRMS). After photoirradiation for 60 min, the reaction mixture was subjected to HRMS, and it was found that all the possible intermediates for the stepwise mechanism were present in the reaction mixture along with released carboxylic acids (see Figures S23 and S24 in the SI). In summary, we have successfully synthesized single and dual (same and different) component caged esters based on bimane for carboxylic and amino acids with interesting photophysical and photochemical properties. The FPRPGs unmasked their corresponding carboxylic and amino acids on exposure to visible light (≥410 nm). Moreover, the release of two different carboxylic acids from single FPRPG was achieved, which will be useful in various applications. In the future, we wish to design a dual drug delivery system using our FPRPG, which will enable us to release two different drugs simultaneously by visible light.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00416. Synthesis details, characterization data, and other experimental details (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
N. D. Pradeep Singh: 0000-0001-6806-9774 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank DST (SERB) for financial support and DST-FIST for 600 and 400 MHz NMR. A.C. is thankful to UGC-New Delhi for fellowship. Y.V. is thankful to IIT Kharagpur for fellowship.
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REFERENCES
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DOI: 10.1021/acs.orglett.7b00416 Org. Lett. XXXX, XXX, XXX−XXX
