Photosensitized Cross-Linking of Tryptophan and Tyrosine Derivatives

Article pubs.acs.org/joc

Cite This: J. Org. Chem. 2018, 83, 10835−10844

Photosensitized Cross-Linking of Tryptophan and Tyrosine Derivatives by Rose Bengal in Aqueous Solutions Lucie Ludvíkova,́ † Peter Š tacko,† Jonathan Sperry,‡ and Petr Klań *,† †

Department of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic School of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland, New Zealand

‡

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S Supporting Information *

ABSTRACT: Photosensitized cross-linking of N-acetyl derivatives of tryptophan and tyrosine by rose bengal in phosphate buffer saline was studied by steady-state absorption and emission spectroscopy, nanosecond transient absorption spectroscopy, and chemical analyses. These amino acids undergo cross-linking to form dimeric and higher oligomeric products under anoxic conditions with rose bengal as a sacrificial oxidant, whereas they react predominantly with singlet oxygen produced by rose bengal in aerated solutions. This investigation provides a comprehensive view into the first steps of rose bengal photosensitized cross-linking of these two amino acids. The results can be helpful for future applications of rose bengal and related dyes, such as photochemical tissue bonding or visible light photocatalysis.

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INTRODUCTION Proteins can be covalently cross-linked under direct irradiation or by photosensitization.1−5 These processes are biomedically important and can be involved in pathological processes, such as photoaging of skin,6 initiation of cataracts,4,7 and degradation of insulin.1 They can also play a role in photodynamic therapy (PDT) as a promising clinical treatment for several types of cancer, psoriasis, and vitiligo,8 in preparation of biomaterials for surgical implants,9 and in a light-activated method for tissue repair called photochemical tissue bonding (PTB).10−12 Recently, peptides cross-linking was also used for the preparation of mechanically robust nanostructures.5 The mechanism of photosensitized protein cross-linking of peptides is still not well understood. It has been proposed that reactive species, such as singlet oxygen or hydroxyl radicals, formed by photosensitization react with oxidizable protein amino acid residues to give intermediates which can further cross-link with other protein parts.2,3,7,13,14 The other suggested mechanism is electron transfer from an amino acid to the excited state of a photosensitizer followed by the reactions of the radical species formed.3,15,16 These processes can also be investigated by laser flash photolysis.17−19 Unlike tyrosine, the occurrence of tryptophan in proteins is relatively rare;20 however, it is abundant in fibrinogen and fibrin21,22 involved in blood clotting. Tryptophan is also a part of the translocation mechanism of proteins through the membrane and protein anchoring.23,24 Covalent cross-links of amino acids can be found in collagen, an abundant connective tissue protein.25,26 Cross-linked tyrosine dimers have been identified in many structural proteins, such as resilin, silk fibroin, collagen, or elastin.27,28 Tryptophan has been reported © 2018 American Chemical Society

to form covalent bonds with lysine and tryptophan side chains of streptococcal bacteria proteins.29 Rose bengal (RB) is a well-known visible light absorbing dye (λmax = 550 nm),30,31 often used as a photosensitizer in PDT,32,33 PTB,10−12 or solar cell construction.34,35 It can sensitize reactions via electron36,37 or energy transfer.38,39 For example, dimerization of histidine13 and tyrosine7,40 mediated by singlet oxygen has been observed in the presence of rose bengal as a singlet oxygen mediator. Rose bengal has also been used in visible light photocatalysis applications in organic synthesis.41,42 Here we report a comprehensive investigation of the crosslinking mechanism of N-acetyl-L-tryptophan (1) and N-acetylL-tyrosine (2) used as model amino acids for photosensitization by rose bengal in an aqueous buffer. The course of the reactions was studied by steady-state spectroscopy, the major photoproducts were characterized, and the corresponding kinetic parameters were determined using nanosecond transient absorption spectroscopy.

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RESULTS AND DISCUSSION Nanosecond Transient Absorption Spectroscopy. Nanosecond time-resolved transient absorption measurements of degassed aqueous PBS (phosphate-buffered saline) buffer solutions (pH = 7.4, I = 0.1 M) of rose bengal dianion (RB2−) in the presence of either N-acetyl-L-tryptophan (1) or Nacetyl-L-tyrosine (2) (Scheme 1) were carried out (λexc = 532 nm; RB2− possesses a strong absorption band at ∼550 nm30). Received: June 19, 2018 Published: August 7, 2018 10835

DOI: 10.1021/acs.joc.8b01545 J. Org. Chem. 2018, 83, 10835−10844

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The Journal of Organic Chemistry Scheme 1. Photochemistry of Rose Bengal

aqueous PBS solutions of 1 and 2 only at higher RB2− concentrations (c = 4.5 × 10−5 M). Similar results were obtained for RB2− in PBS solutions containing N-acetyl-Ltryptophan methyl ester, N-acetyl-L-tyrosine ethyl ester, or phenol (Figures S1 and S2). One-electron oxidation potentials of tryptophan (1.015 V; 0.97 V for indole) and tyrosine (0.93 V; 0.86 V for phenol) versus NHE at pH 7 in aqueous solution45 are much lower than that of aliphatic carboxylates (1.58 V versus NHE, 1.38 V versus Ag/AgCl, obtained for 10-undecenoic acid in acetonitrile46). The reduction potentials of tryptophan and tyrosine (recalculated for SCE) and rose bengal (E0 (RB2−/ RB•3−) = −0.78 V in aqueous solution44) and the triplet-state energy of rose bengal (176 kJ mol−1 31) were used for estimation of the electron transfer driving force, ΔGeT ∼ −26.3 kJ mol−1 (−0.27 eV) for 1 and ΔGeT ∼ −35.8 kJ mol−1 (−0.37 eV) for 2. Therefore, we assume that only the indole and phenol groups can act as electron donors (Scheme 1g). This premise was also supported by measuring the transient absorption spectra and kinetics of the excited rose bengal in the presence of N-t-Boc-L-valine (a nonaromatic amino acid with a free carboxylic group) which did not exhibit any changes when compared to those measured for aqueous solutions of RB2− alone. Unfortunately, the oxidized forms of 1 and 2 (the cation radicals 1•+ and 2•+) were not directly observed in our transient spectroscopy experiments. Such species have 2 orders of magnitude lower molar absorption coefficients47,48 than those of RB•3− and RB2− (ground-state bleach); thus, their signals are too weak and probably overlap with other stronger signals. The kinetics of 3RB2− and RB•3− formed in the presence of 1 were studied. The observed decay rate constants of 3RB2− were obtained at 620 nm (Figure S3a), and its kinetic traces were fitted with a first-order rate law. The pseudo-first-order rate constant determined from the concentration dependence of 1 vs the observed triplet-state decay (Figure S3b) was k1 = (1.1 ± 0.1) × 109 s−1 M−1, which is faster than the rate constant reported elsewhere (5.74 × 108 s−1 M−1).36 At 420 nm, the fast triplet-state decay of 3RB2− and the slow decay of RB•3− were observed (Figure S4). The formation of RB•3− could not be directly observed because of its weaker absorption compared to that of the triplet state at 420 nm (Figure 1, black dotted and red lines). Such a weak absorption (and thus the concentration; the molar absorption coefficient of RB•3− is 1

Difference absorption spectra of the excited triplet state of rose bengal were observed in all samples a short time after the excitation (from nanoseconds to microseconds), possessing the absorbance maxima at 380, 465, and 590 nm, whereas a ground-state bleach appeared at ≈550 nm (Figure 1, black

Figure 1. Transient absorption spectra of rose bengal solutions in aqueous PBS buffer (c = 1.5 × 10−5 M, recorded 10 ns and 100 μs after a 532 nm flash: black dotted and black solid lines, respectively) and those containing either 1 (c = 6.0 × 10−3 M, red line; recorded 30 μs after a 532 nm flash) or 2 (c = 6.0 × 10−3 M, blue line; recorded 100 μs after a 532 nm flash) with a 5 ns integration window (Eflash = 240 mJ).

dotted line), as previously described in the studies of rose bengal laser flash photolysis.31,43 The triplet state of rose bengal (3RB2−) exhibits a complex chemistry (Scheme 1).31,36,43 It can undergo electron transfer reactions with either the second triplet excited- or ground-state RB2− molecule to give the reduced (RB•3−) and oxidized (RB•−) forms (Schemes 1c and e). Indeed, these two species were observed in degassed aqueous PBS buffer solutions of rose bengal in 100 μs after laser flash (RB•3− and RB•− possess the absorption bands at 420 and 470 nm, respectively; Figure 1, black line) and were assigned using the previously reported laser flash photolysis and pulse radiolysis results.31,43,44 In the presence of the compounds 1 or 2 (Scheme 1), only the signals for the reduced rose bengal form (RB•3−, detected at 30 and 100 μs after flash for 1 and 2, respectively) were observed at 420 nm at longer delays after the excitation (Figure 1, red and blue lines). The oxidized form, RB•−, was not detected under these conditions. The RB•3− species were observed in aerated 10836


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The Journal of Organic Chemistry order of magnitude higher than that of the triplet state) of RB•3− indicates that the quenching efficiency of the triplet state via electron transfer is low. Based on the observed fast triplet-state quenching and low concentrations of RB•3−, we assume that the rate constant of triplet-state quenching by 1 (k1) is the sum of two rate constants for electron transfer (keT) and nonproductive quenching of the triplet state of rose bengal by 1 (kq; Scheme 1h). The corresponding keT value can thus be roughly estimated from the efficiency of electron transfer process Φ = (1.9 ± 0.2) %, calculated from the concentrations of 3RB2− and RB•3− (keT = 2.0 × 107 s−1 M−1 and kq = 1.1 × 109 s−1 M−1), estimated from their maximal absorption (the maximal amplitude obtained from the fit of the RB•3− decay). The observed decay rate constant of RB•3− was kRB = (6.0 ± 0.8) × 102 s−1, which corresponds to the lifetime of 1.66 ms. In contrast, the first-order rate law gave a poor fit to the decay traces of the rose bengal triplet state measured in the presence of 2 as a quencher (Figure S5). The traces obtained in the presence or absence of 2 are compared in Figure S6, showing that quenching of the triplet state is not significantly pronounced by the addition of 2. Therefore, other deactivation processes of the triplet state of RB, such as triplet−triplet annihilation (Scheme 1d), must still be involved. The rate constant of triplet-state quenching by 2 via electron transfer, keT = (3.1 ± 0.3) × 105 s−1 M−1, was obtained by global fit of the differential rate equations involving all photochemical processes of RB, extended by a term describing quenching by 2 (Supporting Information, page S5). A detailed description of the fitting procedure has been reported elsewhere.31 This global fit of the measured data revealed that 2 also quenches the triplet state of rose bengal by a nonproductive pathway (Scheme 1h; kq = (2.8 ± 0.3) × 106 s−1 M−1). The sum of the rate constants of electron transfer and nonproductive quenching (kq and keT) by 2 is comparable to the published value of 5.12 × 106 s−1 M−1 obtained from fit by a first-order rate law.36 The observed decay rate constant of RB•3− was kRB = (4.5 ± 1.6) × 103 s−1 (τ ≈ 0.22 ms) in the presence of 2. Both amino acid derivatives quench 3RB2− through electron transfer, although with a low efficiency and rather slow rate constants. The small rate constants could be related to another efficient quenching of the triplet state or reverse electron transfer, possibly via an encounter complex. Moreover, the free energy of back electron transfer obtained from the difference in the reduction potentials of the amino acids and RB2− and the electrostatic work required to bring reactants and products together is ΔGb = −1.47 and −1.56 eV for tryptophan and tyrosine, respectively.36,45 To the best of our knowledge, the rate constants of electron transfer from both 1 and 2 to the triplet excited state of rose bengal separated from nonproductive quenching rate constants are reported for the first time in this work. Steady-State Spectroscopy of Irradiated Solutions. Degassed aqueous (freeze−pump−thaw) PBS buffer solutions of RB2− (c = 3.5 × 10−5 M) and 1 (c = 1.0 × 10−4 M; pH = 7.4, I = 0.1 M) were irradiated by LEDs at 545 nm (Figure 2) or a pulsed Nd:YAG laser with repetition rate of 0.5 Hz at 532 nm (see Supporting Information Figure S8). Absorption spectra of the reaction mixtures (the PBS buffer solution of RB2−, Figure 2, green dotted line, was used in the reference cell) were recorded for 46 h of irradiation (Figure 2). The initial absorption spectrum of 1 (Figure 2, black line) with a characteristic band at 280 nm gradually disappeared, and a new band with λmax at 290 nm and tailing to 360 nm was formed

Figure 2. Absorption spectra of 1 (c = 1.0 × 10−4 M, black line) and photoproduct(s) (red line) formed in the presence of RB2− (c = 3.5 × 10−5 M) in a degassed PBS solution measured during LEDs irradiation at 545 nm. Absorption spectrum of RB2− (c = 3.5 × 10−5 M, green dotted line) which was used as a blank.

(Figure 2, red line). During this experiment, RB2− decomposition, observed by increasing the negative absorption at 549 nm in the spectra, was nearly completed during irradiation (Figure S9), and it caused a distortion of the monitored spectra. This result suggests that RB2− acts as a sacrificial photosensitizer. Such an analogous photochemical reduction of RB2− in the presence of an electron donor to form different deiodination products has also been shown to compete with triplet sensitization of oxygen in water.49 The emission spectra obtained during irradiation showed additional emission maxima, one at 360 nm (λfexc = 280 nm, assigned to 1;50 Figure 3, black line) and another one at 400

Figure 3. Fluorescence spectra of 1 (c = 1.0 × 10−4 M, λfexc = 280 nm, black line, left y-axis) and photoproduct(s) (λfexc = 320 nm, red line, right y-axis) with RB2− (c = 3.5 × 10−5 M) in a degassed PBS solution measured during LEDs irradiation at 545 nm at 23 ± 1 °C.

nm, assigned to a photoproduct (Figure 3, red line; excited at λfexc = 320 nm where the new species absorb, Figure 2). The emission maximum of RB2− appeared at 567 nm,31 and the product of its degradation showed a broad emission from 500 to 600 nm (Figure S10). The fluorescence excitation spectra recorded at λfem = 430 nm during irradiation revealed that 1 (see Figure S11 for the fluorescence excitation spectrum of 1 at λfem = 330 nm for comparison) was transformed to a 10837


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case of 1). In addition, a new fluorescence signal with a maximum at 405 nm (Figure 5, blue line) was observed. This signal was bathochromically shifted by 100 nm when compared to the emission maximum of 2 at 305 nm50 (Figure 5, black line). The corresponding fluorescence excitation spectrum of an irradiated solution also exhibits a broader band from 290 to 350 nm of the photoproduct (Figure S16, blue line) when compared to that of 2 (Figure S16, black line), and it roughly corresponds to the signal using absorption spectroscopy (Figure 4). We deduced from this first analysis that such a signal is in the range of the absorption and emission maxima of a tyrosine dimer 5b cross-linked at the C-3 phenol positions, prepared by the oxidation of tyrosine using horseradish peroxidase (Figure 6).27,57 Moreover, the analogous dimer 5a has also been prepared by flavin mononucleotide-mediated photosensitization (type I sensitizer) via electron transfer under aerobic conditions.3 In contrast to the photochemistry of 1, similar absorption and emission signals of products were recorded for solutions of 2 in both degassed and aerated PBS solutions upon irradiation to low conversions (Figures S17 and S18), suggesting that identical or very similar photoproducts were formed. The formation of the oxidation product, 3α-hydroxy-6-oxo-2,3,3α,6,7,7α-hexahydro-1H-indol-2-carboxylic acid (Figure S34), by the reaction of tyrosine with singlet oxygen produced by rose bengal sensitization has also been reported,40,58 and we assume that this compound (or its analogues) is also formed in our prolonged experiments. However, we have not studied this reaction in greater detail. We concluded at this moment that the reaction product(s) formed upon irradiation of degassed solutions of 1 and 2 with RB2− exhibit bathochromically shifted both absorption and emission bands when compared to those of the staring material and can be related to π-extended chromophores, such as dimers or oligomers. Therefore, the HPLC−HRMS analyses using some authentic analytical standards were performed on the irradiated samples. Analyses of the Photoproducts. Degassed aqueous PBS solutions of RB2− and 1 were irradiated by either a continuous LED light source at 545 nm for up to 90 h or a Nd:YAG pulsed laser with a repetition rate of 0.5 Hz at 532 nm for 1.5 h. The reaction conversions were kept below 35% to prevent secondary reactions of the primary photoproducts. The HPLC−HRMS analyses of all irradiated solutions gave evidence for the formation of several products with m/z (M + H+) = 491.19, which were eluted as seven distinct peaks (A− G; Figures 7 and S19). This mass corresponds to the calculated mass for a dimer of 1 and based on the spectroscopic findings discussed above, where the monomers of 1 are most probably covalently linked at various positions of the indole ring. All absorption spectra of the dimers obtained for the individual chromatogram peaks (Figure S20) were slightly bathochromically shifted compared to that of 1. Only the absorption spectrum of the dimer G practically matches that of 1 (Figure S20d); unfortunately, its detection was close to the detection limit of our HPLC analysis. Some dimer spectra nearly overlap (A and C; B and D; E and F), but because their retention times are considerably different, we do not assign them to pairs of diastereomers, as suggested by Tsentalovich and co-workers in the case of the analogous photosensitization of 1 using kynurenic acid.59 Furthermore, dimers with similar absorption spectra differed in their fragmentation pattern in the HRMS analyses (Figures 7 and S19) as well as in the

photoproduct possessing a broad excitation signal from 250 to 350 nm (Figure S12). In contrast, the formation of analogous photoproducts was not observed for aerated PBS solutions of 1 and RB2− upon irradiation by LEDs. Both absorption and emission spectroscopies revealed rapid degradation of 1 to give product(s) with emission close to that assigned51−53 previously to N-acetyl-Nformylkynurenine (λfem = 450 nm, Figures S13 and S14). Other products of oxidation by singlet oxygen generated by RB, such as a hexahydropyrrolo[2,3-b]indole-2-carboxylic acid derivative (the structures are shown in Figure S34),54−56 may have been generated but were hidden in the broad band envelopes. For N-acetyl-L-tyrosine (2), the steady-state irradiation of a mixture of 2 and RB2− in degassed PBS solutions by either 545 nm LEDs (Figures 4 and 5) or a pulsed Nd:YAG laser at 532

Figure 4. Absorption spectra of 2 (c = 2.0 × 10−4 M, black line, left yaxis) and a photoproduct (blue line) formed in the presence of RB2− (c = 3.5 × 10−5 M) in a degassed PBS solution upon LEDs irradiation at 545 nm. Absorption spectra of RB2− (c = 3.5 × 10−5 M, green dotted line, right y-axis) which was used as a blank.

Figure 5. Fluorescence spectra of 2 (c = 2.0 × 10−4 M, λfexc = 265 nm, black line, left y-axis) and a photoproduct (λfexc = 320 nm, blue line, right y-axis) formed in a degassed PBS solution upon LEDs irradiation at 545 nm at 23 ± 1 °C.

nm (repetition rate of 0.5 Hz, Figure S15) was performed, and the resulting mixture was analyzed by absorption and fluorescence spectroscopy. The absorption spectrum of 2 (Figure 4, black line) changed to a broader and slightly bathochromically shifted signal (Figure 4, blue line) upon irradiation (degradation of RB2− was also observed as in the 10838


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Figure 6. Dimers and their analogues.

Figure 8. HPLC chromatograms of a degassed sample flashed of 1 with Nd:YAG laser (λmax = 532 nm, E = 200 mJ) with the repetition rate of 0.5 Hz for 1.5 h: the absorption signals detected at 250 nm (black solid line, left y-axis) and the fluorescence signals with the excitation/detection at 250/320 (red dotted line, right y-axis), 250/ 405 (blue dotted line), or 320/405 (black dotted line) nm. HPLC chromatograms of a degassed LEDs-irradiated sample for 67 h: the absorption signals at 250 nm (red solid line, left y-axis) and the fluorescence signals with the excitation and detection at 250/405 nm (green dotted line, right y-axis). HPLC chromatogram of 3a detected at 300 nm (magenta dashed line, left y-axis).

Figure 7. Chromatograms showing the absorbance at 250 nm (blue line, right y-axis) and MS intensities of ions with m/z 491.19 (black line, left y-axis) and m/z 245.09 (red line) during the HPLC-MS analysis of 1 (c = 5.0 × 10−4 M) and RB2− (c = 5.0 × 10−5 M) in degassed PBS buffer solution after 1.5 h flashing with Nd:YAG laser (λmax = 532 nm, E = 200 mJ) with the repetition rate of 0.5 Hz.

HPLC analyses with fluorescent detection: the D and F dimers were fluorescent at 320 and 405 nm, respectively (Figure 8, dotted lines), whereas B and E were not. To identify the dimeric products, some of the anticipated dimers or their analogues 3a and 4b were synthesized and used for comparison. The synthesis of dimers of 1 is not trivial.60 The 2,2′-biindole derivative 3a was synthesized using the published procedure,61,62 and the 1,1′-biindole 4b was prepared as described previously63 to study the chromophore of the anticipated photoproduct 4a. The absorption and fluorescence spectra of both compounds are bathochromically shifted when compared to those of 1 (Figure S21). The compound 3a is strongly fluorescent with an emission maximum at 380 nm (Figure S21b, dark blue solid line), whereas 4b has a weaker emission at 360 nm (Figure S21b, green solid line). Unfortunately, the comparison of the HPLC and spectroscopic data for 3a (Figure 8, magenta dashed line) and all dimers clearly showed that this compound was not formed in LEDs-irradiated mixtures. However, the analysis of the sample irradiated by the laser showed that the eluted fluorescent species H matches the retention time of the standard 3a (Figure 8), but its concentration was too low to be

detected by a UV−vis detector. Several observed dimeric products exhibited the absorption and emission bands in the same region as the 1,1′-dimer 4b (Figures S20 and S21). Small deviations in the spectra may result from changes on substituents of the 1,1′-biindole core of 4. In view of the observed emission and fragmentation to m/z = 245.09 (a symmetrical fragmentation; vide infra), F (m/z = 490.19) could be the anticipated compound 4a (Figures 7 and 8). In addition, the signals of ions with m/z = 245.09 were observed during HPLC−HRMS analyses, and they overlapped with some of the dimers’ signals (Figures 7 and S19, red and magenta lines; A, C, F). These ions were most likely formed by the fragmentation59 of dimers during the acquisition of the MS spectra, and they can be attributed to ions of 1 with the loss of two hydrogen atoms. These dimers can be more unstable under electrospray ionization, which could be related to weaker N−N or C−N bonds (167 and 305 kJ mol−1, respectively) compared to that of the C−C bond (346 kJ mol−1).64 10839


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blue-shifted) compared to those of the conjugate bases.27,57 Both K and L exhibited bathochromically shifted fluorescence (λf ≈ 405 nm) compared to that of 2 (λf ≈ 320 nm; Figure 10,

Different irradiation techniques were chosen to compare different conditions of the coupling reactionwhen either a very high (a high-intensity pulsed laser) or relatively low (LEDs) concentration of the reaction intermediates is generated. As expected, higher amounts of dimers were detected under laser irradiation even at shorter irradiation times (Figures S19 and 7). Several additional products formed under continuous LEDs irradiation were detected by HPLC. Two species with the retention times of 11.2 and 13.7 min (Figure 7, red solid line, marked with arrows and Figure S22) could not be identified as they were not ionized under ESI or APCI conditions. The latter one was fluorescent at 405 nm (Figure 7, green dotted line), and this signal is probably responsible for broader emission spectra of the reaction mixtures irradiated by LEDs when compared to that obtained under laser irradiation (Figures 3 and S8). The masses corresponding to trimers (m/z 735.28) and tetramers (m/z 979.36) of 1 were also detected in the MS spectra in several individual peaks (Figure S28). Aerated aqueous PBS solutions of RB2− and 1 irradiated by a laser resulted in the formation of small concentrations of dimers (Figure S23a, red lines), i.e., when the concentration of reactive intermediates was very high, and dimerization was a competing process to their reaction with oxygen. As we anticipated from our spectroscopic analysis (see above), the major species with the retention time of 6.0 min was formed under both irradiation conditions (Figure S23), and it was assigned to N-acetyl-N-formylkynurenine.51,56 Degassed solutions of 2 and RB2− were irradiated under the same conditions as those of 1 and analyzed by HPLC−HRMS to maximum conversions of 22%. Product ions with m/z (M + H+) = 445.16 corresponding to the mass of the dimers of 2 were eluted as four distinct peaks (as signed I−L; Figures 9

Figure 10. HPLC chromatograms of degassed sample flashed for 1.5 h with a Nd:YAG laser (λmax = 532 nm, E = 200 mJ) with the repetition rate of 0.5 Hz detected by the absorption at 300 nm (black solid line, left y-axis) and by the fluorescence with excitation/ detection at 280/405 nm (blue dotted line, right y-axis) and 280/320 (black dotted line) nm. HPLC chromatograms of a degassed sample irradiated at 545 nm by LEDs for 67 h, detected by the absorption at 300 nm (red solid line) and by fluorescence with excitation/detection at 280/405 nm (green dotted line).

dotted lines). On the other hand, the dimers I and J were not fluorescent. All four dimers were considerably populated in the samples irradiated by LEDs, whereas the compound K was clearly the major product formed by laser irradiation. In the previously reported studies of tyrosine or tryptophan cross-linking by direct or sensitized photolysis, the dimers 5a and 6a were identified as the major photoproducts.3,65 We used 2,2′-dihydroxybiphenyl (5c) and diphenyl ether 6b as the model chromophores to compare their absorption and emission spectra with those of the dimers I−L. The absorption and fluorescence spectra of 5c resembled the shape of both fluorescence excitation and emission spectra of some photoproducts formed during the reaction (Figure S26, dark blue solid and blue dotted lines). In addition, the spectrum of 5c in an aqueous 0.1% formic acid solution (Figure S26a, orange solid line) resembles those of the signals K and L recorded during the HPLC analysis (Figure S25b). The shapes of the emission spectra were not affected by the pH decrease. Similarly, the absorption and emission spectra of 6b were compared to the spectra obtained during the reaction. The absorption/emission spectra of the unsubstituted diphenyl ether chromophore 6b must differ from those of anticipated substituted dimers, especially in methanol (Figure S26, green solid line), which was used because of its low solubility in an aqueous solution. The compound 6b absorbs in the same region as the dimers K and L, whereas its intense fluorescence is slightly blue-shifted as compared to that of 2 (Figure S26b, green solid line). An ion with m/z (M + H+) = 666.22 was found in both irradiated solutions, and it corresponds to a trimer of 2 (Figure S24). The noticeable enhanced formation of trimers under LEDs irradiation can be explained simply by longer reaction times of irradiation. Additional weak signals of the products with a retention times of approximately 10 min could be assigned to higher oligomers (Figures 10 and S24).

Figure 9. Chromatograms showing the absorbance at 300 nm (blue line, right y-axis) and the intensities of MS signals for ions with m/z = 445.16 (black line, left y-axis) during the HPLC−MS analysis of 2 (c = 5.0 × 10−4 M) and RB2− (c = 5.0 × 10−5 M) in a degassed PBS solution after 1.5 h flashing with a Nd:YAG laser (λmax = 532 nm, E = 200 mJ) with the repetition rate of 0.5 Hz.

and S24). Their broad absorption bands recorded for the individual peaks (Figure S25) were bathochromically shifted compared to that of 2, signifying the π-extension of the chromophore. The major signals K and L possessed were similar in shape. The phenolic oxygen of 2 (and dimers) is protonated under the acidic conditions of HPLC analyses (0.1% formic acid) which changes the absorption spectrum significantly (the signal of the protonated form is narrower and 10840


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The Journal of Organic Chemistry Scheme 2. Complex Photosensitized Cross-Linking of 1

Scheme 3. Complex Photosensitized Cross-Linking of 2

complex reaction regioselectivity. They can recombine to produce dimers at the positions 1−3 but also react with the starting material (Scheme 2). Some structures of the tryptophanyl dimers have been proposed before. For example, the formation of N1−C3 dimer has been suggested to be formed by the catalysis of photochemically or enzymatically generated carbonate radical.73,74 The N1−C3 and C3−C3 dimers have been detected upon direct pulse radiolysis of peptides or their reaction with bovine superoxide dismutase.75 The results obtained in our work show that N1−N1 dimers are very likely among the observed products, whereas C2−C2 products are produced only in traces. Therefore, the recombination of 1• seems to be a plausible major reaction pathway. In contrast, dimerization is essentially inhibited in the presence of oxygen because of rapid oxidation processes. Under continuous prolonged irradiation, products of singlet oxygen oxygenation can also be formed.40,55 An analogous, although less complex, chemistry can be proposed for the photosensitization of 2. The primary radical cation 2•+ formed upon electron transfer to the triplet state of rose bengal undergoes deprotonation to afford the neutral radical 2•.48,69,70 These radicals can recombine to give the

In contrast to the photochemistry of 1, the dimers of 2 were found also in aerated samples irradiated by both laser (Figure S27a, red line) and LEDs (Figure S27b, red line). The additional new signals are assigned to oxidation products. The Mechanism of Cross-Linking. We can now postulate the mechanisms for the observed photosensitized cross-linking reactions of 1 and 2. Upon irradiation, the rose bengal singlet excited state efficiently intersystem crosses (Φisc ≈ 1 in water66) to give the triplet excited state 3RB2−. This species accepts an electron from the amino acid derivative to form a rose bengal anion radical (RB•3−) and cation radicals of 1 or 2 (1•+ or 2•+), and RB•3− is directly detected by nanosecond transient spectroscopy. The cation radicals can then deprotonate to give the neutral radicals 1•47,67,68 or 2•48,69,70 (Schemes 2and 3). In this work, at least six different dimer regioisomers (and higher oligomers) were detected during the photosensitization reaction of the tryptophan derivative 1, and we can draw several reaction pathways involving both intermediates 1•+ and 1•. The species 1•+ has been shown to have high spin density at the C-2, whereas higher spin densities in 1• have been identified at the N1 and C3 atoms,71,72 which can explain the 10841


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fragmentor 60 V for 2. The MS spectra were collected in the range of m/z 50−1000. Irradiation Procedures. A stirred solution of an amino acid (1 or 2, c = 0.1−2.0 × 10−3 M) with rose bengal (c = 35 × 10−6 M) in PBS (pH = 7.4, I = 0.1 M) in a 1 cm matched quartz cuvette was irradiated using LEDs (λmax = 545 nm) or a pulsed Nd:YAG laser (λmax = 532 nm, Figures S30 and S31). The formation of the cross-linked product was simultaneously monitored by UV−vis spectrometry in the course of the reaction; a solution of rose bengal was used as a control. UV− vis spectra were obtained on a diode-array spectrophotometer. Control experiments with each amino acid derivative in a PBS solution (in the absence of RB) were also performed under the same irradiation conditions; no photoproducts were formed. A PBS solution was used to mimic the conditions of a PTB treatment and to keep a neutral pH in the course of the reaction. Synthesis of 3a. N-Acetyl L-tryptophan (300 mg, 1.21 mmol) was dissolved in trifluoroacetic acid (10 mL) purged by bubbling with N2 (15 min). The mixture was left stirring for 16 h at room temperature. Afterward, the volatiles were evaporated under reduced pressure, the residue was redissolved in 1,4-dioxane (10 mL), and DDQ (414 mg, 1.83 mmol) was added. The mixture was stirred for 2 h at room temperature and diluted with ethyl acetate (40 mL). It was then washed with sat. aq. NaHCO3 (2 × 20 mL); the organic layer was dried with MgSO4 and concentrated under reduced pressure to give the crude product. Trituration with acetone (3 × 10 mL) at room temperature provided the pure desired product as a pale brown solid (186 mg, 62%). Mp 238−239 °C. 1H NMR (500 MHz, d6-DMSO): δ (ppm) 12.48 (brs, 1H), 11.06 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.13 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 1H), 7.04 (dd, J1 = 7.2 Hz, J2 = 7.2 Hz, 1H), 4.49 (dd, J1 = 14.4 Hz, J2 = 7.8 Hz, 1H), 3.29 (dd, J1 = 14.5 Hz, J2 = 6.3 Hz, 1H), 3.13 (dd, J1 = 14.5 Hz, J2 = 8.0 Hz, 1H), 1.62 (s, 3H). 13C NMR (125 MHz, d6-DMSO): δ (ppm) 174.4, 169.2, 136.2, 128.0, 127.97, 121.5, 118.9, 118.7, 111.2, 109.5, 52.7, 27.1, 22.2. HRMS (ESI+): calcd for C26H27N4O6+ [M + H+] 491.1925, found 491.1838.

biphenyl or diphenyl ether dimeric structures (Scheme 3), which were for example also found by flavin mononucleotide photosensitization of 2.3 Such a radical recombination process has been proposed in many studies on tyrosine reactivity.3,5,40,57,65,76 The other two minor dimers of 2 can also be formed from the recombination of 2• in different positions.

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CONCLUSION The dimerization of 1 and 2 photosensitized by rose bengal as a sacrificial oxidant (type I sensitizer) takes place in the absence of oxygen or at low oxygen concentrations. In aerated solutions, both 1 and 2 form dimers only when under irradiation with a powerful light source and when a high concentration of the reactive species is produced. We therefore hypothesize that under the conditions for the photochemical tissue bonding,10,11,77 the peptide cross-linking can proceed via an initial electron transfer step if the tryptophan or tyrosine residues are present. The combination of an intense laser irradiation, saturated solutions of rose bengal, and the viscous tissue media, where the diffusion of oxygen is slower and rose bengal is strongly bound to the proteins by electrostatic interactions,12,78 enhances the probability that amino acid residues will undergo coupling.

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EXPERIMENTAL PART

Material. Rose bengal disodium salt (RB; >95%), N-acetyl-Ltryptophan (98%), N-acetyl-L-tryptophan methyl ester (95%), Nacetyl-L-tyrosine (99%), N-acetyl-L-tyrosine ethyl ester monohydrate (95%), phenol (99.5%), N-t-Boc-L-valine (98%), and acetonitrile (HPLC grade) were used as purchased. Water was purified on a reverse osmosis purification system. Nanosecond Transient Laser Flash Photolysis (LFP). The LFP setup was operated in a right-angle arrangement of the pump and probe beams. Laser pulses of ≤170 ps duration at 532 nm (240 mJ) were obtained from an Nd:YAG laser. The laser beam was dispersed onto a 4 × 1 cm modified fluorescence cuvette held in a horizontal arrangement. An overpulsed xenon arc lamp was used as the source of the probe light. Kinetic traces were recorded using a photomultiplier tube. Transient absorption spectra were obtained by an ICCD camera equipped with a spectrograph. The measurements were performed at ambient temperature (20 ± 2 °C). The samples were degassed by three freeze−pump−thaw cycles under reduced pressure (0.06 Torr). Kinetic traces were fitted using the Levenberg−Marquard algorithm in the MATLAB software. Fluorescence Measurements. Fluorescence and excitation spectra were measured on a fluorescence spectrometer in a 1 cm quartz fluorescence cuvette at 23 ± 1 °C. The sample concentrations were set to keep the absorbance in the range of 0.05−0.50 at the corresponding excitation wavelengths. Each sample was measured three times, and the spectra were averaged. Emission and excitation spectra were normalized and corrected by the photomultiplier sensitivity function using correction files supplied by the manufacturer. HPLC with UV−Vis and Fluorescence Detection. Solutions of an amino acid (1 or 2, c = 5.0 × 10−4 M, 50 μL) with rose bengal (c = 5.0 × 10−5 M) in PBS (pH = 7.4, I = 0.1 M) were irradiated by LEDs or a pulsed Nd:YAG laser. The samples were injected into an HPLC system equipped with a Nucleosil-5C-18 column (250 × 4.6 mm), and 0.1% formic acid in water (A) and methanol (B) was used as eluent (flow rate 1 mL min−1 with different gradients). HPLC−HRMS. The MS analyses were performed using an accurate-mass TOF LC−MS system with electrospray ionization in the positive mode. The HPLC separation was operated using the same column and under the same conditions as described above. The MS instrumental conditions were as follows: nitrogen flow 7 L min−1, temperature 325 °C, nebulizer pressure 45 psig, capillary voltage −2500 V, and fragmentor 40 V for 1 and nitrogen flow 8 L min−1

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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.joc.8b01545.

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Steady-state and transient spectra; a kinetic model of the reaction of 2; kinetic traces for 1 and 2; HRMS and NMR spectra; emission spectra of the irradiation sources (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +420-54949-4856. Fax: +420-54949-2443 ORCID

Jonathan Sperry: 0000-0001-7288-3939 Petr Klán: 0000-0001-6287-2742 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this work was provided by the Ministry of Health of the Czech Republic (NV18-07-00342) and by the Ministry of Education, Youth and Sports of the Czech Republic (LO1214). We thank Lubos Jilek, Miroslava Bittová, and Marek Martinek (Masaryk University) for their help with the experiments. 10842


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(20) Carugo, O. Amino Acid Composition and Protein Dimension. Protein Sci. 2008, 17, 2187−2191. (21) Ishida, Y.; Takiuchi, H.; Matsushima, A.; Inada, Y. Functional ́ Consequences of tTryptophan Modification in Human Fibrinogen. Biochim. Biophys. Acta, Protein Struct. 1978, 536, 70−77. (22) Matsushima, A.; Takiuchi, H.; Saito, Y.; Inada, Y. Significance of Tryptophan Residues in the d-Domain of the Fibrin Molecule in Fibrin Polymer Formation. Biochim. Biophys. Acta, Protein Struct. 1980, 625, 230−236. (23) Schiffer, M.; Chang, C.-H.; Stevens, F. J. The Function of Tryptophan Residues in Membrane Proteins. Protein Eng., Des. Sel. 1992, 5, 213−214. (24) de Jesus, A. J.; Allen, T. W. The Role of Tryptophan Side Chains in Membrane Protein Anchoring and Hydrophobic Mismatch. Biochim. Biophys. Acta, Biomembr. 2013, 1828, 864−876. (25) Tanzer, M. L. Crosslinking of Collagen. Science 1973, 180, 561−566. (26) Knott, L.; Bailey, A. J. Collagen Crosslinks in Mineralizing Tissues: A Review of their Chemistry, Function, and Clinical Relevance. Bone 1998, 22, 181−187. (27) Kungl, A. J.; Landl, G.; Visser, A. J. W. G.; Breitenbach, M.; Kauffmann, H. F. l-Dityrosine: A Time-resolved Fluorescence Investigation. J. Fluoresc. 1992, 2, 63−73. (28) Amadò, R.; Aeschbach, R.; Neukom, H. Dityrosine: In Vitro Production and Characterization. Methods Enzymol. 1984, 107, 377− 388. (29) Schramma, K. R.; Bushin, L. B.; Seyedsayamdost, M. R. Structure and Biosynthesis of a Macrocyclic Peptide Containing an Unprecedented Lysine-to-Tryptophan Crosslink. Nat. Chem. 2015, 7, 431−437. (30) Neckers, D. C. Rose Bengal. J. Photochem. Photobiol., A 1989, 47, 1−29. (31) Ludvikova, L.; Fris, P.; Heger, D.; Sebej, P.; Wirz, J.; Klan, P. Photochemistry of Rose Bengal in Water and Acetonitrile: A Comprehensive Kinetic Analysis. Phys. Chem. Chem. Phys. 2016, 18, 16266−16273. (32) Dini, L.; Inguscio, V.; Tenuzzo, B.; Panzarini, E. Rose Bengal Acetate Photodynamic Therapy-Induced Autophagy. Cancer Biol. Ther. 2010, 10, 1048−1055. (33) Uppal, A.; Jain, B.; Gupta, P. K.; Das, K. Photodynamic Action of Rose Bengal Silica Nanoparticle Complex on Breast and Oral Cancer Cell Lines. Photochem. Photobiol. 2011, 87, 1146−1151. (34) Roy, M. S.; Balraju, P.; Kumar, M.; Sharma, G. D. DyeSensitized Solar Cell Based on Rose Bengal Dye and Nanocrystalline TiO2. Sol. Energy Mater. Sol. Cells 2008, 92, 909−913. (35) Sayyed, S. A. A. R.; Beedri, N. I.; Kadam, V. S.; Pathan, H. M. Rose Bengal Sensitized Bilayered Photoanode of Nano-Crystalline TiO2−CeO2 for Dye-Sensitized Solar Cell Application. Appl. Nanosci. 2016, 6, 875−881. (36) Lambert, C. R.; Kochevar, I. E. Electron Transfer Quenching of the Rose Bengal Triplet State. Photochem. Photobiol. 1997, 66, 15−25. (37) Sarna, T.; Zaja̧c, J.; Bowman, M. K.; Truscott, T. G. Photoinduced Electron Transfer Reactions of Rose Bengal and Selected Electron Donors. J. Photochem. Photobiol., A 1991, 60, 295− 310. (38) Lee, P. C. C.; Rodgers, M. A. J. Laser Flash Photokinetic Studies of Rose Bengal Photodynamic Interactions of Nucleotides and DNA. Photochem. Photobiol. 1987, 45, 79−86. (39) Kochevar, I. E.; Redmond, R. W. Photosensitized Production of Singlet Oxygen. Methods Enzymol. 2000, 319, 20−28. (40) Jin, F.; Leitich, J.; von Sonntag, C. The Photolysis (λ = 254 nm) of Tyrosine in Aqueous Solutions in the Absence and Presence of Oxygen. The Reaction of Tyrosine with Singlet Oxygen. J. Photochem. Photobiol., A 1995, 92, 147−153. (41) Zhang, M. J.; Schroeder, G. M.; He, Y. H.; Guan, Z. Visible Light-Mediated Decarboxylative Amination of Indoline-2-Carboxylic Acids Catalyzed by Rose Bengal. RSC Adv. 2016, 6, 96693−96699.

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