Chlorophyll-Inspired Red-Region Fluorophores: Building Block Synthesis and Studies in Aqueous Media

Fluorophores that absorb and emit in the red spectral region (600–700 nm) are of great interest in photochemistry and photomedicine. Eight new target chlorins (and 19 new chlorins altogether)—analogues of chlorophyll—of different polarities have been designed and synthesized for various applications; seven of the chlorins are equipped with a bioconjugatable tether. Hydrophobic or amphiphilic chlorins in a non-polar organic solvent (toluene), polar organic solvent (DMF), and aqueous or aqueous micellar media show a sharp emission band in the red region and modest fluorescence quantum yield (Φf = 0.2–0.3). A Poisson analysis implies most micelles are empty and few contain >1 chlorin. Water-soluble chlorins each bearing three PEG (oligoethyleneglycol) groups exhibit narrow emission bands (full-width-at-half maximum <25 nm). The lifetime of the lowest singlet excited state and the corresponding yields and rate constants for depopulation pathways (fluorescence, intersystem crossing, internal conversion) are generally little affected by the PEG groups or dissolution in aqueous or organic media. A set of chlorin–avidin conjugates revealed a 2-fold increase in Φf with increased average chlorin/avidin ratio (2.3–12). In summary, the chlorins of various polarities described herein are well suited as red-emitting fluorophores for applications in aqueous or organic media.


Introduction
Nature's "advanced functional dyes"-chosen by the fine comb of evolution [1][2][3][4][5][6]-are the chlorophylls and their analogues. The structures of chlorophyll a and chlorophyll b are shown in Figure 1 [7]. Chlorophylls a and b differ only in the nature of a single substituent at the 7-position but otherwise contain common features: (1) an 18π-aromatic macrocycle; (2) three pyrrole rings and one reduced, "pyrroline" ring; (3) a fifth, "isocyclic" ring spanning positions 13 and 15 and containing a β-ketoester unit; (4) a full complement of substituents at the β-sites of the pyrrole or pyrroline rings; (5) a centrally coordinated divalent magnesium ion; (6) a phytyl tail; (7) trans-configuration of the vicinal alkyl substituents at the β-sites of the pyrroline ring; and (8) vinyl and keto groups in conjugation with the π-system and disposed along the Q y axis. Chlorophylls a and b absorb strongly in the blue (and near-ultraviolet) and in the red spectral regions; green light is absorbed with weaker intensity, hence the characteristic color of the compounds in thin films (e.g., a leaf) or dilute solutions, and the verdant landscapes of Earth [8,9]. A longstanding objective in photochemistry, artificial photosynthesis and allied disciplines has been to create synthetic chlorophyll-like molecules as a means to examine the essential structural features in the native macrocycles and to tailor the molecules for diverse applications. In this regard, the phytyl group, which constitutes about 1/3 of the overall mass [8], does not contribute to the core photophysical features of chlorophylls. Chlorins lacking the 3-vinyl and 13-keto substituents still exhibit chlorin-like spectral features; the red-region absorption is certainly diminished versus that of chlorophylls but still enhanced by at least 5-10-fold versus that of a porphyrin [9]. The central magnesium affords a more reducing macrocycle versus the zinc chelate and even more so than the free base macrocycle, yet in broad perspective, the magnesium, zinc and free base systems have similar photophysical features. Hence, the phytyl tail, isocyclic ring, and central magnesium can often be elided in creating synthetic chlorins without loss of photochemical function [9].
The chief methods of synthesis of chlorins include derivatization of porphyrins [9,10], semisynthetic tailoring of chlorophylls [11], and de novo routes [8,12]. We have been working over the years to learn to create chlorins in de novo fashion, inspired by the roles of chlorophyll in photosynthesis, and with an eye toward exploiting the resulting synthetic chlorins in fundamental photophysical studies and diverse applications ranging from the materials sciences to biomedicine [8]. A key design element is the inclusion of a geminal-dialkyl (typically a gem-dimethyl) group in the pyrroline ring (Figure 1), thereby securing the chlorin π-system from adventitious dehydrogenation upon routine handling in aerobic environments; chlorin itself (lacking any substituents) is susceptible in this regard, the consequence of which is formation of the corresponding porphyrin and loss of the desired red-region absorption intensity. In this paper, we describe the de novo synthesis of a set of gem-dimethylsubstituted chlorins aimed primarily toward studies in the life sciences. A longstanding objective in photochemistry, artificial photosynthesis and allied disciplines has been to create synthetic chlorophyll-like molecules as a means to examine the essential structural features in the native macrocycles and to tailor the molecules for diverse applications. In this regard, the phytyl group, which constitutes about 1/3 of the overall mass [8], does not contribute to the core photophysical features of chlorophylls. Chlorins lacking the 3-vinyl and 13-keto substituents still exhibit chlorin-like spectral features; the red-region absorption is certainly diminished versus that of chlorophylls but still enhanced by at least 5-10-fold versus that of a porphyrin [9]. The central magnesium affords a more reducing macrocycle versus the zinc chelate and even more so than the free base macrocycle, yet in broad perspective, the magnesium, zinc and free base systems have similar photophysical features. Hence, the phytyl tail, isocyclic ring, and central magnesium can often be elided in creating synthetic chlorins without loss of photochemical function [9].
The chief methods of synthesis of chlorins include derivatization of porphyrins [9,10], semisynthetic tailoring of chlorophylls [11], and de novo routes [8,12]. We have been working over the years to learn to create chlorins in de novo fashion, inspired by the roles of chlorophyll in photosynthesis, and with an eye toward exploiting the resulting synthetic chlorins in fundamental photophysical studies and diverse applications ranging from the materials sciences to biomedicine [8].
A key design element is the inclusion of a geminal-dialkyl (typically a gem-dimethyl) group in the pyrroline ring (Figure 1), thereby securing the chlorin π-system from adventitious dehydrogenation upon routine handling in aerobic environments; chlorin itself (lacking any substituents) is susceptible in this regard, the consequence of which is formation of the corresponding porphyrin and loss of the desired red-region absorption intensity. In this paper, we describe the de novo synthesis of a set of gem-dimethyl-substituted chlorins aimed primarily toward studies in the life sciences.
The first three target synthetic chlorins are shown in Figure 2. Chlorins H 2 C3, H 2 C6 and H 2 C7 each contain a 10-mesityl group and one appended group including a 1 • amine, a β-ketoester and a carboxylic acid-substituted chalcone, respectively. Chlorins H 2 C3 and H 2 C6 are amphiphilic given the presence of a hydrophobic core and an ionizable terminal functional group, whereas chlorin H 2 C7 is relatively hydrophobic and lacks an ionizable functionality. This work extends our prior research toward amphiphilic chlorins [13] and draws on prior routes for installation of the β-ketoester unit [14]. All three chlorins can be regarded as building blocks given the presence of a single derivatizable group attached to the macrocycle. The photophysical properties of all three chlorins have been examined in toluene, whereas chlorins H 2 C3 and H 2 C6 have been further examined in DMF and in aqueous micellar media.
Molecules 2018, 23,130 3 of 29 The first three target synthetic chlorins are shown in Figure 2. Chlorins H2C3, H2C6 and H2C7 each contain a 10-mesityl group and one appended group including a 1° amine, a β-ketoester and a carboxylic acid-substituted chalcone, respectively. Chlorins H2C3 and H2C6 are amphiphilic given the presence of a hydrophobic core and an ionizable terminal functional group, whereas chlorin H2C7 is relatively hydrophobic and lacks an ionizable functionality. This work extends our prior research toward amphiphilic chlorins [13] and draws on prior routes for installation of the β-ketoester unit [14]. All three chlorins can be regarded as building blocks given the presence of a single derivatizable group attached to the macrocycle. The photophysical properties of all three chlorins have been examined in toluene, whereas chlorins H2C3 and H2C6 have been further examined in DMF and in aqueous micellar media. Five other target synthetic chlorins are shown in Figure 3. Free base chlorins H2C10-PEG6, H2C12-PEG6, and H2C15-PEG6 as well as zinc chlorins ZnC12-PEG6 and ZnC15-PEG6 each bear a single carboxylic acid and a tri-PEGylated aryl group attached to the chlorin 10-position. This work extends prior research in preparing PEGylated chlorins [15]; alternative approaches for installation of PEG groups in hydroporphyrins-containing arrays have been described recently by Meares et al. [16]. Each PEG group consists of six ethyleneoxy units and is terminated with a methyl group. The PEG moieties are introduced via copper(I)-mediated azide-alkyne click chemistry [17] and hence are linked via a triazole unit. Two of the PEG groups project above/below the face of the macrocycle. Such facial encumbrance (illustrated in Figure 3) is a viable strategy for suppressing π-π aggregation of tetrapyrrole macrocycles [18]. The photophysical properties of the five chlorins have been examined in aqueous solution. One chlorin (H2C12-PEG6) has been converted to the N-hydroxysuccinimidyl ester for potential use in bioconjugation processes. Taken together, the molecular design, syntheses, and photophysical studies illustrate the ability to create analogues of chlorophyll tailored for specific applications.

Reconnaissance
We previously prepared a set of chlorins containing PEG groups for water-solubilization ( Figure 3). Chlorins I and II each contain three PEG groups, which were installed by click chemistry with a trialkynyl-bearing chlorin. This design was attractive to us, and our goal here was to incorporate a tether containing a carboxylic-acid terminus for bioconjugation. In prior studies, chlorin III was converted to the N-hydroxysuccinimidyl (NHS) ester, but the latter was found to undergo rapid hydrolysis, so fast as to prohibit the bioconjugation. Five other target synthetic chlorins are shown in Figure 3. Free base chlorins H 2 C10-PEG 6 , H 2 C12-PEG 6 , and H 2 C15-PEG 6 as well as zinc chlorins ZnC12-PEG 6 and ZnC15-PEG 6 each bear a single carboxylic acid and a tri-PEGylated aryl group attached to the chlorin 10-position. This work extends prior research in preparing PEGylated chlorins [15]; alternative approaches for installation of PEG groups in hydroporphyrins-containing arrays have been described recently by Meares et al. [16]. Each PEG group consists of six ethyleneoxy units and is terminated with a methyl group. The PEG moieties are introduced via copper(I)-mediated azide-alkyne click chemistry [17] and hence are linked via a triazole unit. Two of the PEG groups project above/below the face of the macrocycle. Such facial encumbrance (illustrated in Figure 3) is a viable strategy for suppressing π-π aggregation of tetrapyrrole macrocycles [18]. The photophysical properties of the five chlorins have been examined in aqueous solution. One chlorin (H 2 C12-PEG 6 ) has been converted to the N-hydroxysuccinimidyl ester for potential use in bioconjugation processes. Taken together, the molecular design, syntheses, and photophysical studies illustrate the ability to create analogues of chlorophyll tailored for specific applications.

Reconnaissance
We previously prepared a set of chlorins containing PEG groups for water-solubilization ( Figure 3). Chlorins I and II each contain three PEG groups, which were installed by click chemistry with a trialkynyl-bearing chlorin. This design was attractive to us, and our goal here was to incorporate a tether containing a carboxylic-acid terminus for bioconjugation. In prior studies, chlorin III was converted to the N-hydroxysuccinimidyl (NHS) ester, but the latter was found to undergo rapid hydrolysis, so fast as to prohibit the bioconjugation. In this regard, PEG-O-CH2CO2-NHS is reported to have a very short half-time (0.75 min) under mild conditions in aqueous solution (pH 8, 25 °C) as measured by liberation of the NHS group, to be compared with 16.5 or 23.3 min for the structure with one or two additional methylene units (PEG-O-CH2CH2CO2-NHS or PEG-O-CH2CH2CH2CO2-NHS), respectively, where "PEG" lengths here are unspecified [19]. The poor results we encountered with the aryloxyacetic acid tether prompted exploration of other designs: we first examined a tether derived from succinic acid and a p-arylamine, and subsequently turned to an arylpropionic acid tether.
The synthetic approach to chlorins relies on the reaction of a tetrahydrodipyrrin (Western half) and a 1,9-disubstituted dipyrromethane (Eastern half). The overall synthetic approach is shown in Scheme 1. Acid-catalyzed condensation followed by zinc(II)-mediated cyclization with accompanying dehydrogenation affords the corresponding zinc chlorin. Dipyrromethanes bearing 1-formyl-9-bromo substitution patterns afford the 5-unsubstituted chlorin, whereas the 1-aroyl substituted dipyrromethanes lead to the corresponding 5-arylchlorins. Each chlorin prepared herein bears a 10aryl unit. Substitution at the 13-position is achieved by carrying through a bromo substituent in the Eastern half whereas substitution at the chlorin 15-position is accomplished by bromination of the free base 18,18-dimethylchlorin, which proceeds regioselectively at the 15-position [8]. In both cases, subsequent Pd-mediated substitution is employed to install the desired substituent at the perimeter of the macrocycle. In this regard, PEG-O-CH 2 CO 2 -NHS is reported to have a very short half-time (0.75 min) under mild conditions in aqueous solution (pH 8, 25 • C) as measured by liberation of the NHS group, to be compared with 16.5 or 23.3 min for the structure with one or two additional methylene units (PEG-O-CH 2 CH 2 CO 2 -NHS or PEG-O-CH 2 CH 2 CH 2 CO 2 -NHS), respectively, where "PEG" lengths here are unspecified [19]. The poor results we encountered with the aryloxyacetic acid tether prompted exploration of other designs: we first examined a tether derived from succinic acid and a p-arylamine, and subsequently turned to an arylpropionic acid tether.
The synthetic approach to chlorins relies on the reaction of a tetrahydrodipyrrin (Western half) and a 1,9-disubstituted dipyrromethane (Eastern half). The overall synthetic approach is shown in Scheme 1. Acid-catalyzed condensation followed by zinc(II)-mediated cyclization with accompanying dehydrogenation affords the corresponding zinc chlorin. Dipyrromethanes bearing 1-formyl-9-bromo substitution patterns afford the 5-unsubstituted chlorin, whereas the 1-aroyl substituted dipyrromethanes lead to the corresponding 5-arylchlorins. Each chlorin prepared herein bears a 10-aryl unit. Substitution at the 13-position is achieved by carrying through a bromo substituent in the Eastern half whereas substitution at the chlorin 15-position is accomplished by bromination of the free base 18,18-dimethylchlorin, which proceeds regioselectively at the 15-position [8]. In both cases, subsequent Pd-mediated substitution is employed to install the desired substituent at the perimeter of the macrocycle.
Attempted use of H 2 C10-PEG 6 -NHS in bioconjugation processes led to poor yields; subsequent LC-MS analysis showed a small peak due to H 2 C10-PEG 6 -NHS and a preceding peak of~2.5 times greater intensity ( Figure S1, panels A and B). Peak 1 gave m/z = 1716 whereas peak 2 gave the expected m/z = 1831 for H 2 C10-PEG 6 -NHS ( Figure S1, panels C and D). The lower mass value is consistent with loss of N-hydroxysuccinimide (115 Da). The loss is attributed to cyclization of the activated carboxyl group (NHS-ester) to form a succinimidyl moiety at the p-aryl position (Scheme 5). Following this observation, we found literature reports of similar facile reactions with the methyl ester of simple anilide substrates (e.g., 3-(methoxycarbonyl)propionanilide) [37,38]. To avoid this side reaction, alternative designs for bioconjugation were considered such as use of an isothiocyanate as employed with tetrapyrroles by Sutton et al. [39]. Ultimately, because we wished to limit synthetic manipulations on the intact macrocycles, designs with a propionic acid directly attached to the aryl ring were pursued. a 3-column chromatographic approach [33] including adsorption chromatography, size-exclusion chromatography (SEC), and adsorption chromatography again (to remove materials that leach from the SEC resin) afforded H2C9-PEG6 in 88% yield for the two synthetic steps. Suzuki coupling partner 6 was prepared by reaction [34] of 2-(4-aminophenyl)-1,3-dioxa-4,4,5,5-tetramethylborolane and succinic anhydride. Pd-mediated Suzuki coupling [35,36] of H2C9-PEG6 and 6 yielded target bioconjugatable PEGylated chlorin H2C10-PEG6 in 74% yield. Attempted use of H2C10-PEG6-NHS in bioconjugation processes led to poor yields; subsequent LC-MS analysis showed a small peak due to H2C10-PEG6-NHS and a preceding peak of ~2.5 times greater intensity ( Figure S1, panels A and B). Peak 1 gave m/z = 1716 whereas peak 2 gave the expected m/z = 1831 for H2C10-PEG6-NHS ( Figure S1, panels C and D). The lower mass value is consistent with loss of N-hydroxysuccinimide (115 Da). The loss is attributed to cyclization of the activated carboxyl group (NHS-ester) to form a succinimidyl moiety at the p-aryl position (Scheme 5). Following this observation, we found literature reports of similar facile reactions with the methyl ester of simple anilide substrates (e.g., 3-(methoxycarbonyl)propionanilide) [37,38]. To avoid this side reaction, alternative designs for bioconjugation were considered such as use of an isothiocyanate as employed with tetrapyrroles by Sutton et al. [39]. Ultimately, because we wished to limit synthetic manipulations on the intact macrocycles, designs with a propionic acid directly attached to the aryl ring were pursued.

Heptynoic Acid and Tris(PEGylation) at the Respective 13-and 10-Position
Bromination of formyl dipyrromethane 4 [15] using 2 equivalents of NBS at −78 °C for 1 h afforded a mixture of 8,9-dibromo-1-formyldipyrromethane 4-Br2 and 9-bromo-1-formyldipyrromethane 4-Br [42] (Scheme 7). The mixture could not be separated given the instability [15] and similar polarity (upon analysis by TLC) of the two products. The resulting mixture was used directly in the condensation with Western half 2 for 30 min in the presence of p-TsOH·H2O. Zinc-mediated cyclization in CH3CN under reflux in the presence of air for 16 h gave a mixture of zinc chlorins with or without a bromo group at the 13-position of the macrocycle, as determined upon examination by matrix-assisted laser-desorption mass spectrometry (MALDI-MS) and 1 H-NMR spectroscopy.

Heptynoic Acid and Tris(PEGylation) at the Respective 13-and 10-Position
Bromination of formyl dipyrromethane 4 [15] using 2 equivalents of NBS at −78 • C for 1 h afforded a mixture of 8,9-dibromo-1-formyldipyrromethane 4-Br 2 and 9-bromo-1-formyldipyrro-methane 4-Br [42] (Scheme 7). The mixture could not be separated given the instability [15] and similar polarity (upon analysis by TLC) of the two products. The resulting mixture was used directly in the condensation with Western half 2 for 30 min in the presence of p-TsOH·H 2 O. Zinc-mediated cyclization in CH 3 CN under reflux in the presence of air for 16 h gave a mixture of zinc chlorins with or without a bromo group at the 13-position of the macrocycle, as determined upon examination by matrix-assisted laser-desorption mass spectrometry (MALDI-MS) and 1 H-NMR spectroscopy. The 1 H-NMR spectral properties of 13-substituted chlorins have been described previously [20]. The two zinc chlorins could not be separated using preparative chromatography. Therefore, the mixture was demetalated using TFA, and the resulting free base chlorins were separated by column chromatography. Chlorin H2C13 bears a bromo substitution at the 13-position and was obtained in 12% overall yield for four steps. Zincation of H2C13 gave zinc chlorin ZnC13 in 97% yield. Copper(I)catalyzed click reaction between ZnC13 and PEG-azide followed by demetalation in the presence of TFA afforded the corresponding PEGylated chlorin H2C14-PEG6 in 93% yield for the two steps. The palladium-mediated copper-free Sonogashira coupling reaction [43][44][45] of H2C14-PEG6 and tert-butyl 6-heptynoate (9) [46] followed by removal of the tert-butyl group using TFA afford H2C15-PEG6 in 76% yield. Zincation of H2C15-PEG6 gave the corresponding zinc chlorin ZnC15-PEG6 in 96% yield.

Photophysical Characterization
Photophysical studies were carried out with several chlorins. Chlorin H2C10-PEG6 was examined for ground-state absorption and singlet-excited-state properties (fluorescence, intersystem-crossing and internal-conversion) in solvents of diverse polarity including toluene, DMF, and water. Amphiphilic chlorins H2C2, H2C3 and H2C6 were examined for absorption, fluorescence and excited-state characteristics in toluene, DMF and aqueous micellar solution. Finally, chlorin H2C12-PEG6-NHS was coupled with the protein avidin to make chlorin-avidin conjugates, which were examined for absorption and fluorescence properties in aqueous solution. The studies illustrate the utility of molecular design and synthesis in tailoring chlorins for compatibility with diverse environments.

Spectral and Photophysical Properties of PEGylated Chlorins
Chlorin H2C10-PEG6 was characterized by static absorption and emission spectroscopy at room temperature in three media of diverse polarity: toluene, DMF and water. The absorption and fluorescence spectra are shown in Figure 4. The spectral characteristics are summarized in Table 1.

Photophysical Characterization
Photophysical studies were carried out with several chlorins. Chlorin H 2 C10-PEG 6 was examined for ground-state absorption and singlet-excited-state properties (fluorescence, intersystem-crossing and internal-conversion) in solvents of diverse polarity including toluene, DMF, and water. Amphiphilic chlorins H 2 C 2 , H 2 C3 and H 2 C6 were examined for absorption, fluorescence and excited-state characteristics in toluene, DMF and aqueous micellar solution. Finally, chlorin H 2 C12-PEG 6 -NHS was coupled with the protein avidin to make chlorin-avidin conjugates, which were examined for absorption and fluorescence properties in aqueous solution. The studies illustrate the utility of molecular design and synthesis in tailoring chlorins for compatibility with diverse environments.

Spectral and Photophysical Properties of PEGylated Chlorins
Chlorin H 2 C10-PEG 6 was characterized by static absorption and emission spectroscopy at room temperature in three media of diverse polarity: toluene, DMF and water. The absorption and fluorescence spectra are shown in Figure 4. The spectral characteristics are summarized in Table 1.
The prominent fluorescence emission band in each case is sharp, as assessed by the full-width-at-half maximum (fwhm). The prominent fluorescence emission band in each case is sharp, as assessed by the full-width-at-half maximum (fwhm).  The absorption spectra of all five PEGylated chlorins in water are given in Figure 5. The absorption spectra show the Qy (S0 → S1) absorption in the red region, the Qx (S0 → S2) band in the green, and overlapping Bx and By (S0 → S3 and S0 → S4) features in the near-UV. Each of these bands has one or more vibronic satellite features to higher energy. The fluorescence spectrum is approximately mirror symmetric to the Qy absorption manifold and contains the prominent (0, 0) band at ~650 nm and the weaker (1, 0) feature at ~710 nm.   The absorption spectra of all five PEGylated chlorins in water are given in Figure 5. The absorption spectra show the Q y (S 0 → S 1 ) absorption in the red region, the Q x (S 0 → S 2 ) band in the green, and overlapping B x and B y (S 0 → S 3 and S 0 → S 4 ) features in the near-UV. Each of these bands has one or more vibronic satellite features to higher energy. The fluorescence spectrum is approximately mirror symmetric to the Q y absorption manifold and contains the prominent (0, 0) band at~650 nm and the weaker (1, 0) feature at~710 nm.  Table 1 also summarizes the spectral and excited-state properties for the two PEGylated chlorins H2C12-PEG6 and H2C15-PEG6 in water as well as the analogous zinc chelates ZnC12-PEG6 and ZnC15-PEG6 in water, toluene and DMF. Figure 6A shows transient absorption data for H2C12-PEG6 in aqueous solution, illustrating the measurement of τS and Φisc (for all the chlorins). The absorption difference spectrum at 0.3 ns contains features expected for the first S1 excited state. These features include bleaching of the near-UV (Soret) ground-state absorption (combined S0 → S3 and S0 → S4) at 405 nm and a feature at 640 nm that contains bleaching of the S0 → S1 absorption along with S1 → S0 emission stimulated by the white-light probe pulse. The spectrum at 60 ns can be assigned to the lowest triplet excited state (T1). At this time, the stimulated emission contribution has disappeared and the magnitudes of S0 → Sn bleachings have decreased because some molecules have returned to the ground state (by S1 → S0 fluorescence and internal conversion) in parallel with formation of T1. The yield of S1 → T1 intersystem crossing is obtained by comparing the bleaching magnitudes (referenced to the relatively weak featureless excited-state absorption) at long times (due to T1) compared to immediately after the excitation flash (due to S1). The value for H2C12-PEG6 in aqueous solution is Φisc = 0.70 (Table 1). Figure 6B shows the decay of the S1 excited state probed at three representative wavelengths, which together with global fitting of the entire date set afford τS = 7.6 ns.  Table 1 also summarizes the spectral and excited-state properties for the two PEGylated chlorins H 2 C12-PEG 6 and H 2 C15-PEG 6 in water as well as the analogous zinc chelates ZnC12-PEG 6 and ZnC15-PEG 6 in water, toluene and DMF. Figure 6A shows transient absorption data for H 2 C12-PEG 6 in aqueous solution, illustrating the measurement of τ S and Φ isc (for all the chlorins). The absorption difference spectrum at 0.3 ns contains features expected for the first S 1 excited state. These features include bleaching of the near-UV (Soret) ground-state absorption (combined S 0 → S 3 and S 0 → S 4 ) at 405 nm and a feature at 640 nm that contains bleaching of the S 0 → S 1 absorption along with S 1 → S 0 emission stimulated by the white-light probe pulse. The spectrum at 60 ns can be assigned to the lowest triplet excited state (T 1 ). At this time, the stimulated emission contribution has disappeared and the magnitudes of S 0 → S n bleachings have decreased because some molecules have returned to the ground state (by S 1 → S 0 fluorescence and internal conversion) in parallel with formation of T 1 . The yield of S 1 → T 1 intersystem crossing is obtained by comparing the bleaching magnitudes (referenced to the relatively weak featureless excited-state absorption) at long times (due to T 1 ) compared to immediately after the excitation flash (due to S 1 ). The value for H 2 C12-PEG 6 in aqueous solution is Φ isc = 0.70 (Table 1). Figure 6B shows the decay of the S 1 excited state probed at three representative wavelengths, which together with global fitting of the entire date set afford τ S = 7.6 ns.   (Table 1). It is also interesting that the rate constants for S1 → S0 internal conversion for ZnC12-PEG6 and ZnC15-PEG6 are greater than those for the free base analogues H2C12-PEG6 and H2C15-PEG6. Generally one expects the opposite to be true because of the involvement of N-H vibrations associated with the central free base protons to enhance the Franck-Condon factor for this non-radiative decay process. Here, internal conversion may be facilitated by structural effects (e.g., out-of-plane-distortions) or vibrational effects of water molecules coordinated to the central zinc ion. However, internal conversion has approximately the same rate and yield for the two zinc chelates in toluene or DMF as in water (Table 1). Perhaps axialcoordination effects occur but involve instead interaction of the central zinc of one chlorin with the PEG groups of an adjacent zinc chlorin.

Amphiphilic Chlorins in Various Solvents Including Micellar Solution
Chlorins H2C2, H2C3 and H2C6 were characterized by static absorption and emission spectroscopy and transient absorption spectroscopy at room temperature in three media. The media included toluene, DMF and aqueous micellar solution; the latter entailed cetyl trimethylammonium bromide (CTAB) at 10 mM in aqueous sodium phosphate buffer (pH 7, 0.1 M). The absorption and fluorescence spectra of H2C3 and H2C6 in the three media are shown in Figure 4. The spectra of H2C2 are nearly very similar to those for H2C2; the two molecules differ only in the nature of the terminal amine (-NHBOC vs. -NH2). The spectral properties of the three chlorins are summarized in Table 2 along with values for τS, Φf, Φisc and Φic in toluene, DMF and aqueous CTAB micelle solution. Noteworthy  Figure 6C,D give analogous transient absorption data for the analogous zinc chelate ZnC12-PEG 6 . The data differ from that for H 2 C12-PEG 6 in aqueous solution in several respects (Table 1): a shorter excited-state lifetime (τ S = 1.9 ns vs. 7.6 ns), a larger triplet yield (Φ isc = 0.80 vs. 0.70) and a smaller fluorescence yield (Φ f = 0.060 vs. 0.23). These differences can be traced primarily to the heavy atom (zinc) effect on spin-orbit coupling and thus the rate constant for S 1 → T 1 intersystem crossing [k isc = (2.5 ns) −1 vs. (11 ns) −1 ]. Similar comparisons can be made concerning the photophysical properties of ZnC15-PEG 6 . The data differ from that for H 2 C15-PEG 6 in water (Table 1). It is also interesting that the rate constants for S 1 → S 0 internal conversion for ZnC12-PEG 6 and ZnC15-PEG 6 are greater than those for the free base analogues H 2 C12-PEG 6 and H 2 C15-PEG 6 . Generally one expects the opposite to be true because of the involvement of N-H vibrations associated with the central free base protons to enhance the Franck-Condon factor for this non-radiative decay process. Here, internal conversion may be facilitated by structural effects (e.g., out-of-plane-distortions) or vibrational effects of water molecules coordinated to the central zinc ion. However, internal conversion has approximately the same rate and yield for the two zinc chelates in toluene or DMF as in water (Table 1). Perhaps axial-coordination effects occur but involve instead interaction of the central zinc of one chlorin with the PEG groups of an adjacent zinc chlorin.

Amphiphilic Chlorins in Various Solvents Including Micellar Solution
Chlorins H 2 C2, H 2 C3 and H 2 C6 were characterized by static absorption and emission spectroscopy and transient absorption spectroscopy at room temperature in three media. The media included toluene, DMF and aqueous micellar solution; the latter entailed cetyl trimethylammonium bromide (CTAB) at 10 mM in aqueous sodium phosphate buffer (pH 7, 0. 1 M). The absorption and fluorescence spectra of H 2 C3 and H 2 C6 in the three media are shown in Figure 4. The spectra of H 2 C2 are nearly very similar to those for H 2 C2; the two molecules differ only in the nature of the terminal amine (-NHBOC vs. -NH 2 ). The spectral properties of the three chlorins are summarized in Table 2 along with values for τ S, Φ f , Φ isc and Φ ic in toluene, DMF and aqueous CTAB micelle solution. Noteworthy observations include the bathochromic and hyperchromic effects on the Q y absorption of H 2 C6 relative to H 2 C3 (Figure 7). These coupled effects on wavelength and intensity of the S 0 → S 1 (Q y ) transition are consistent with the four-orbital model, as we have described previously for the effects of auxochromic substituents on chlorin spectra [47]. The larger intensity of the Q y absorption of H 2 C6 versus H 2 C3 is accompanied by an increase in radiative rate constant k f for spontaneous emission (Table 1) because of the relationship of the Einstein coefficients for the two processes [48]. observations include the bathochromic and hyperchromic effects on the Qy absorption of H2C6 relative to H2C3 (Figure 7). These coupled effects on wavelength and intensity of the S0 → S1 (Qy) transition are consistent with the four-orbital model, as we have described previously for the effects of auxochromic substituents on chlorin spectra [47]. The larger intensity of the Qy absorption of H2C6 versus H2C3 is accompanied by an increase in radiative rate constant kf for spontaneous emission (Table 1) because of the relationship of the Einstein coefficients for the two processes [48].

Poisson Distribution of H2C3 and H2C6 in CTAB Micelle Solution
Understanding the properties of chromophores in aqueous micellar media requires estimation of the number of chromophores per micelle. If the distribution of chromophores is random and not affected by attraction (e.g., aggregation) or repulsion, the Poisson equation (Equation (1)) [49,50] provides an appropriate description for the distribution of molecular species in the micelles. Here, m is the mean occupancy number of chromophore per micelles, and k takes on integer values for the number of chromophores per micelle (k = 0, 1, 2…):

Poisson Distribution of H 2 C3 and H 2 C6 in CTAB Micelle Solution
Understanding the properties of chromophores in aqueous micellar media requires estimation of the number of chromophores per micelle. If the distribution of chromophores is random and not affected by attraction (e.g., aggregation) or repulsion, the Poisson equation (Equation (1)) [49,50] provides an appropriate description for the distribution of molecular species in the micelles. Here, m is the mean occupancy number of chromophore per micelles, and k takes on integer values for the number of chromophores per micelle (k = 0, 1, 2 . . . ): The average aggregation number for 10 mM CTAB in aqueous solution at room temperature is about 95 [51]. The mean occupancy number varies depending on the concentration of micelles; given a critical micelle concentration (cmc) of 1 mM for CTAB [52] and chlorin concentration of 3.1 µM (H 2 C3) or 6.4 µM (H 2 C6), the mean occupancy of H 2 C3 or H 2 C6 per micelle is very low: 0.033 or 0.067, respectively.
The Poisson distribution histograms of H 2 C3 and H 2 C6 in 10 mM CTAB micelle solution are shown in Figure 8. Inspection shows that most micelles are empty, and among those that contain a chlorin, relatively few (1.8% or 3.2% for H 2 C3 or H 2 C6, respectively) contain more than one chlorin. Prior treatments of Poissonian distributions of tetrapyrroles in micelles include bacteriochlorophyll a [53] or chlorophyll a [54] in Triton-X100 micelles; octaalkylporphyrins in cetyl trimethylammonium chloride or sodium dodecyl sulfate (SDS) [55]; chlorophyll a (and derivatives thereof) [56] in Triton-X100, sodium bis(2-ethylhexyl)sufosuccinate (AOT) or SDS [57]; and synthetic chlorins and bacteriochlorins in Triton-X100, CTAB or SDS [13]. The topic of porphyrins in membranes has been reviewed [58,59]. The distributions of tetrapyrroles in micelles should not be regarded as static, given that hydrophobic solutes typically undergo extensive exchange among micelles; for example, substrates such as dodecylpyrene undergo extensive exchange in minutes-hours among SDS micelles [60]. The exchange of solutes between micelles [55,[61][62][63][64][65] can entail exchange through the aqueous solution, fusion-fission of micelles, and/or micellar fission. The prevalence of a given mechanism depends on the micellar surface charge, aqueous ionic strength, and solute polarity.
The average aggregation number for 10 mM CTAB in aqueous solution at room temperature is about 95 [51]. The mean occupancy number varies depending on the concentration of micelles; given a critical micelle concentration (cmc) of 1 mM for CTAB [52] and chlorin concentration of 3.1 µM (H2C3) or 6.4 µM (H2C6), the mean occupancy of H2C3 or H2C6 per micelle is very low: 0.033 or 0.067, respectively.
The Poisson distribution histograms of H2C3 and H2C6 in 10 mM CTAB micelle solution are shown in Figure 8. Inspection shows that most micelles are empty, and among those that contain a chlorin, relatively few (1.8% or 3.2% for H2C3 or H2C6, respectively) contain more than one chlorin. Prior treatments of Poissonian distributions of tetrapyrroles in micelles include bacteriochlorophyll a [53] or chlorophyll a [54] in Triton-X100 micelles; octaalkylporphyrins in cetyl trimethylammonium chloride or sodium dodecyl sulfate (SDS) [55]; chlorophyll a (and derivatives thereof) [56] in Triton-X100, sodium bis(2-ethylhexyl)sufosuccinate (AOT) or SDS [57]; and synthetic chlorins and bacteriochlorins in Triton-X100, CTAB or SDS [13]. The topic of porphyrins in membranes has been reviewed [58,59]. The distributions of tetrapyrroles in micelles should not be regarded as static, given that hydrophobic solutes typically undergo extensive exchange among micelles; for example, substrates such as dodecylpyrene undergo extensive exchange in minutes-hours among SDS micelles [60]. The exchange of solutes between micelles [55,[61][62][63][64][65] can entail exchange through the aqueous solution, fusion-fission of micelles, and/or micellar fission. The prevalence of a given mechanism depends on the micellar surface charge, aqueous ionic strength, and solute polarity.

H2C12-PEG6-NHS Avidin Loading Experiment
To gain insight into how fluorescence quantum yields of chlorin-protein conjugates vary with the chlorin/protein ratio, we carried out exploratory studies of chlorin-avidin conjugates. The conjugates were prepared with increasing equivalents of chlorin/avidin. Avidin is a 68,000 Da tetrameric protein from egg white and has high binding affinity for biotin (KD = 10 −15 M) [66][67][68][69][70]. There are nine lysine residues in each subunit of avidin (36 lysines for tetrameric avidin) for possible bioconjugation with an activated carboxyl group [71]. All subsequent usage of "avidin" here refers to the tetramer. The conjugation was carried out by reaction of a solution of H2C12-PEG6-NHS (0.42 mg, 0.84 mg, or 1.26 mg for 30, 60 or 90 equiv.) with avidin (0.5 mg, 7.9 nmol) in 0.1 M sodium phosphate buffer (100 µL total volume, pH 7.6) for 16 h at room temperature. The chlorin-avidin conjugate in each case was purified by (1) passage through a gel permeation chromatography column (molecular weight cut-off at 5000 Da) to obtain the chlorin-avidin conjugate free from the excess chlorin (which remains

H 2 C12-PEG 6 -NHS Avidin Loading Experiment
To gain insight into how fluorescence quantum yields of chlorin-protein conjugates vary with the chlorin/protein ratio, we carried out exploratory studies of chlorin-avidin conjugates. The conjugates were prepared with increasing equivalents of chlorin/avidin. Avidin is a 68,000 Da tetrameric protein from egg white and has high binding affinity for biotin (K D = 10 −15 M) [66][67][68][69][70]. There are nine lysine residues in each subunit of avidin (36 lysines for tetrameric avidin) for possible bioconjugation with an activated carboxyl group [71]. All subsequent usage of "avidin" here refers to the tetramer.
The conjugation was carried out by reaction of a solution of H 2 C12-PEG 6 -NHS (0.42 mg, 0.84 mg, or 1.26 mg for 30, 60 or 90 equiv.) with avidin (0.5 mg, 7.9 nmol) in 0.1 M sodium phosphate buffer (100 µL total volume, pH 7.6) for 16 h at room temperature. The chlorin-avidin conjugate in each case was purified by (1) passage through a gel permeation chromatography column (molecular weight cut-off at 5000 Da) to obtain the chlorin-avidin conjugate free from the excess chlorin (which remains bound to the top of the column); and (2) concentration of the chlorin-avidin conjugate and further liberation from any residual excess chlorin by mass-selective centrifugation through a filter (molecular weight cut-off at 30,000 Da) at 4000 rpm for 5 min. The resulting chlorin-avidin conjugate was analyzed by absorption spectroscopy and fluorescence spectroscopy (vide infra). Analysis of the absorption spectrum in each case revealed the number of chlorins attached per avidin. For the reactions of 30, 60, or 90 equiv. of H 2 C12-PEG 6 -NHS relative to avidin, the loading (number of chlorins attached per avidin) to be 2.3, 6.2, or 12, respectively ( Table 3). The loading values are estimates, yet the trend of increased chlorin/avidin with increased equiv of chlorin-NHS ester reactants is unmistakable (see Figure 9), as observed in the relative ratio of the intensities of absorption in the Soret band (where the chlorin absorbs exclusively) versus that at 280 nm (where the molar absorption coefficient of avidin exceeds that of the chlorin). Finally, it warrants emphasis that the observed loadings are average values, and as in the case of bacteriochlorin-myoglobin conjugates [36], represent a mixture composed of conjugates wherein the number of chlorins/avidin spans a very broad range. bound to the top of the column); and (2) concentration of the chlorin-avidin conjugate and further liberation from any residual excess chlorin by mass-selective centrifugation through a filter (molecular weight cut-off at 30,000 Da) at 4000 rpm for 5 min. The resulting chlorin-avidin conjugate was analyzed by absorption spectroscopy and fluorescence spectroscopy (vide infra). Analysis of the absorption spectrum in each case revealed the number of chlorins attached per avidin. For the reactions of 30, 60, or 90 equiv. of H2C12-PEG6-NHS relative to avidin, the loading (number of chlorins attached per avidin) to be 2.3, 6.2, or 12, respectively ( Table 3). The loading values are estimates, yet the trend of increased chlorin/avidin with increased equiv of chlorin-NHS ester reactants is unmistakable (see Figure 9), as observed in the relative ratio of the intensities of absorption in the Soret band (where the chlorin absorbs exclusively) versus that at 280 nm (where the molar absorption coefficient of avidin exceeds that of the chlorin). Finally, it warrants emphasis that the observed loadings are average values, and as in the case of bacteriochlorin-myoglobin conjugates [36], represent a mixture composed of conjugates wherein the number of chlorins/avidin spans a very broad range.  Attempts to use MALDI-MS to characterize the chlorin-avidin conjugates were not successful using various matrices including α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid, and the recommended sinapinic acid with ammonium citrate at pH 7 [72]). We attribute the failure in this case to the large number of PEG groups in the sample, given that (1) native avidin was successfully analyzed by MALDI-MS [72]; and (2) MALDI-MS data were readily obtained of bacteriochlorinmyoglobin conjugates wherein each bacteriochlorin was substituted with four carboxylic acid groups [36]. We note that characterization of the products of dye-protein conjugation often is quite limited; regardless, given the available data, the identity of the chlorin-avidin conjugates prepared herein must be regarded as provisional. Attempts to use MALDI-MS to characterize the chlorin-avidin conjugates were not successful using various matrices including α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid, and the recommended sinapinic acid with ammonium citrate at pH 7 [72]). We attribute the failure in this case to the large number of PEG groups in the sample, given that (1) native avidin was successfully analyzed by MALDI-MS [72]; and (2) MALDI-MS data were readily obtained of bacteriochlorin-myoglobin conjugates wherein each bacteriochlorin was substituted with four carboxylic acid groups [36]. We note that characterization of the products of dye-protein conjugation often is quite limited; regardless, given the available data, the identity of the chlorin-avidin conjugates prepared herein must be regarded as provisional.
The absorption and emission spectra of the conjugates are shown in Figure 9. The absorption spectra were normalized at the near-UV (Soret or B) maximum of the chlorin. As the chlorin loading increases, the normalized spectra show an apparent decrease in the UV protein feature near 290 nm (relative to the chlorin features). However, the chlorin Q y absorption and fluorescence features do not change shape. The only change in the chlorin absorption is a small increase on the short-wavelength side of the Soret band, potentially reflecting some modest dipole-dipole interaction among nearby chlorins. With increased loading over the~5-fold range explored here, the fluorescence quantum yield Φ f increases by~2-fold. Note that the 2-fold increase pertains to the Φ f value, not the emission across constant protein concentrations; hence, the relative brightness per protein (given by the number of chlorins/avidin × Φ f per chlorin) increases by~12-fold. Further studies are required to explore the origin of this unexpected increase in brightness with increased loading. Figure 10 compares the absorptance (1 − T, where T is transmittance) spectra and fluorescence excitation spectra for a PEGylated chlorin (H 2 C10-PEG 6 ) and a non-PEGylated benchmark chlorin (H 2 C 6 ) in DMF. There is a near-perfect match between the two types of spectra for each compound. These data reflect the high purity of the compounds, and indicate that the fluorescence originates from the target compound and is not affected by the presence of any highly fluorescent trace impurities. The absorptance (and absorption) spectra show no signs of broadening or peak shifts relative to the excitation spectrum that could arise from aggregation for either compound. Note that aggregates with strictly face-to-face interactions (e.g., H-type dimers) generally would not result in extra features in the fluorescence excitation not already present in the absorptance spectra. Such aggregates are typically non-fluorescent or weakly fluorescent because the exciton splitting for each state will have the lower-exciton component dipole-forbidden and the upper exciton component dipole-allowed. In this regard, the spectra in Figure 10 show no evidence for aggregates or other contaminants. The absorption and emission spectra of the conjugates are shown in Figure 9. The absorption spectra were normalized at the near-UV (Soret or B) maximum of the chlorin. As the chlorin loading increases, the normalized spectra show an apparent decrease in the UV protein feature near 290 nm (relative to the chlorin features). However, the chlorin Qy absorption and fluorescence features do not change shape. The only change in the chlorin absorption is a small increase on the short-wavelength side of the Soret band, potentially reflecting some modest dipole-dipole interaction among nearby chlorins. With increased loading over the ~5-fold range explored here, the fluorescence quantum yield Φf increases by ~2-fold. Note that the 2-fold increase pertains to the Φf value, not the emission across constant protein concentrations; hence, the relative brightness per protein (given by the number of chlorins/avidin × Φf per chlorin) increases by ~12-fold. Further studies are required to explore the origin of this unexpected increase in brightness with increased loading. Figure 10 compares the absorptance (1 − T, where T is transmittance) spectra and fluorescence excitation spectra for a PEGylated chlorin (H2C10-PEG6) and a non-PEGylated benchmark chlorin (H2C6) in DMF. There is a near-perfect match between the two types of spectra for each compound. These data reflect the high purity of the compounds, and indicate that the fluorescence originates from the target compound and is not affected by the presence of any highly fluorescent trace impurities. The absorptance (and absorption) spectra show no signs of broadening or peak shifts relative to the excitation spectrum that could arise from aggregation for either compound. Note that aggregates with strictly face-to-face interactions (e.g., H-type dimers) generally would not result in extra features in the fluorescence excitation not already present in the absorptance spectra. Such aggregates are typically non-fluorescent or weakly fluorescent because the exciton splitting for each state will have the lower-exciton component dipole-forbidden and the upper exciton component dipole-allowed. In this regard, the spectra in Figure 10 show no evidence for aggregates or other contaminants. Figure 10. Comparison of absorptance (1 − T, where T is transmittance) spectra (solid cyan) and fluorescence excitation spectra (dashed red) for H2C10-PEG6 (A) and H2C6 (B) in DMF, normalized at the near-UV (Soret) maximum. A very small Rayleigh light-scattering correction was applied to the absorptance spectra. For each compound, the fluorescence was detected in the Qy(0,1) feature at 700-710 nm, although the same spectrum was obtained at other detection wavelengths.

General Methods
All chemicals obtained commercially were used as received unless otherwise noted. Reagent-grade solvents (CH2Cl2, hexanes, methanol, toluene, ethyl acetate) and HPLC-grade solvents (toluene, CH2Cl2, hexanes) were used as received. THF was freshly distilled from sodium/benzophenone ketyl Figure 10. Comparison of absorptance (1 − T, where T is transmittance) spectra (solid cyan) and fluorescence excitation spectra (dashed red) for H 2 C10-PEG 6 (A) and H 2 C6 (B) in DMF, normalized at the near-UV (Soret) maximum. A very small Rayleigh light-scattering correction was applied to the absorptance spectra. For each compound, the fluorescence was detected in the Q y (0,1) feature at 700-710 nm, although the same spectrum was obtained at other detection wavelengths.

Photophysical Properties
Photophysical studies were carried out on dilute (µM) argon-purged samples at room temperature, typically on samples containing ambient O 2 . Static emission spectra were acquired using a Nanolog (Spex-Horiba, Edison NJ, USA) or QuantaMaster (Photon Technology International-Horiba, Edison, NJ, USA) spectrofluorimeter with 2-4 nm excitation and detection bandwidths and corrected for instrument spectral response. Fluorescence quantum yields were obtained relative to meso-tetraphenylporphyrin (Φ f = 0.070 in nondegassed toluene) [74] or 18,18-dimethyl-5-p-tolyl-10-mesitylchlorin in toluene (Φ f = 0.22 in nondegassed toluene) [35]. Singlet excited-state lifetimes were determined by using transient absorption spectroscopy employing~100 fs excitation flashes from an ultrafast laser system (Spectra Physics, Santa Clara, CA, USA) and acquisition of difference spectra (360-900 nm) using a white-light pulsed laser (~1 ns rise time) in 100-ps time bins with variable pump-probe spacing up to 0.5 ms (EOS, Ultrafast Systems, Sarasota, FL, USA). The same transient absorption studies provided the yield of S 1 → T 1 intersystem crossing by comparing the extent of bleaching of the ground-state absorption bands due to T 1 at the asymptote of the S 1 decay versus the extent due to S 1 immediately after the excitation flash. Measurements in the Q y region account for the contribution of both S 0 bleaching and S 1 stimulated emission to the S 1 feature.

Conclusions
The present work illustrates the design and synthesis of stable chlorins spanning a range of polarity and bearing diverse substituents. Eight target chlorins (19 new chlorins in total) have been synthesized and characterized with regards to photophysical properties. Five PEGylated chlorins were designed for aqueous solubility, wherein the narrow emission bands (fwhm ≤ 20 nm) and moderate Φ f and τ S values characteristic for analogues in organic solvents also were observed. Two amphiphilic chlorins bearing amino-or carboxylic acid-substitution have been examined in aqueous micellar media, where again spectroscopic features resembling those in organic solvents were observed. Loading of avidin with an average of 2-12 bioconjugatable chlorins afforded an approximate~2-fold increase in Φ f value across this span. One attraction of the de novo synthesis approach is the ability to achieve wavelength tunability across a family of chlorins, but doing so requires considerable synthetic effort. A less synthetically demanding route to a PEGylated bioconjugatable chlorin (without wavelength tunability) entails derivatization of a porphyrin [75]. While the design features for a palette of wavelength-tuned chlorins are now known to some extent, and approaches toward a number of desired architectural embodiments also have been established, an unsolved challenge entails how to create diverse chlorins in a concise and facile synthesis amenable to non-specialists. Accomplishing this objective is desirable for broad practical utilization of Nature's chosen chromophores.