Abstract
Zinc 3-hydroxymethyl-pyroprotopheophorbides-a esterified with a chiral secondary alcohol at the 17-propionate residue were prepared as bacteriochlorophyll-d analogs. The synthetic zinc 31-hydroxy-131-oxo-porphyrins self-aggregated in an aqueous Triton X-100 micellar solution to give red-shifted and broadened Soret and Qy absorption bands in comparison with their monomeric bands. The intense, exciton-coupled circular dichroism spectra of their self-aggregates were dependent on the chirality of the esterifying groups. The observation indicated that the self-aggregates based on the J-type stacking of the porphyrin cores were sensitive to the peripheral 17-propionate residues. The supramolecular structures of the present J-aggregates as models of bacteriochlorophyll aggregates in natural chlorosomes were remotely regulated by the esterifying groups.
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1 Introduction
Light-harvesting antennas in phototrophs are generally composed of supramolecules of pigments with peptides. Most antenna systems in chlorophotosynthetic organisms are formed by the specific interaction of chlorophyll (Chl) or bacteriochlorophyll (BChl) molecules with oligopeptides: typically see recent reports [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. In contrast to the conventional photosynthetic antennas, green sulfur bacteria (GSB), filamentous anoxygenic phototrophs (FAP), and chloracidobacteria possess unique antenna systems, called chlorosomes, which are built up by the self-aggregation of BChl pigments without any interaction with peptides [20,21,22,23]. As chlorosomal pigments in GSB, farnesylated BChl-c or d molecules (R = farnesyl group in the left drawing of Fig. 1) are often observed [24]. Such BChls-c/d are a homologous mixture in the 8- and 12-substituents where further methylation occurs at the 82- and 121-positions and an epimeric mixture at the 31-position [25, 26]. Since the stereochemical configurations at the 17- and 18-positions are fixed to be (17S,18S)-stereochemistry, the 31-epimerization produces their diastereomeric mixtures [27, 28]. The 31-chiral position hardly affects the optical properties in their monomeric states due to its connection with the core chlorin π-system through the rotational C3–C31 bond [29, 30]. The 31-hydroxy group of one molecule is coordinated to the central magnesium atom of another molecule and hydrogen-bonded with the 13-carbonyl group of the third molecule to form the chlorosomal self-aggregates with π–π stacking of the composite chlorin chromophores [22, 23, 31]. Therefore, the 31-chirality influences the supramolecular structures of BChl-c/d self-aggregates and controls their optical properties including electronic absorption and emission data as well as circular dichroism (CD) [32, 33].
Molecular structures of natural BChls-c/d (left) and their synthetic models with chlorin 2 (middle) and porphyrin π-skeletons 1 (right)
To reveal the effect of the 31-chirality on the self-aggregation, the 17,18-didehydrogenated analogs of BChl-d, zinc (1-hydroxyethyl)oxoporphyrins, were previously prepared as enantiomeric mixtures with a single chiral center in a molecule [34,35,36]. The enantiomerically pure models were produced via the separation by high performance liquid chromatography (HPLC) and self-aggregated in non-polar organic solvents to give chlorosomal self-aggregates whose supramolecules were dependent on the chirality. As expected, the self-aggregates of the enantiomers exhibited intense CD spectra which were inverted by the epimerization. The phenomena are reasonable because the chiral carbon atom bearing the coordinating and hydrogen-bonding hydroxy group is directly connected with the porphyrin molecule which intermolecularly stacks via π–π interaction to form the aggregates. Since the esterifying group (R) in the 17-propionate residue is far from the chlorin chromophore in a molecule, we considered whether the R moiety could regulate the supramolecular structure or not.
Whereas GSB usually utilize BChls-c/d with a farnesyl group at the esterifying group (vide supra), BChl-c molecules in FAP possess a variety of the R groups including stearyl and phytyl groups [24, 37]. Recently, BChls-c with several different esterifying groups were detected in a chloracidobacterium, whose molecular structures have not been determined yet [38,39,40]. The effect of the esterifying substituents on the chlorosomal self-aggregation has not been fully disclosed, but their hydrophobic interactions among the neighboring BChl molecules or between the BChl and lipid molecules of the chlorosomal surface are proposed [22, 24]. Addition of some aliphatic alcohols to the medium for culturing a GSB species, Chlorobaculum tepidum, partially changed the esterifying groups in the produced BChl-c molecules [41,42,43,44], and alteration of the conditions for culturing a FAP species, Chloroflexus aurantiacus, varied the ratio of BChls-c with different esterifying groups [45]. The modified chlorosomes shifted their visible absorption maxima from the intact ones, indicating that the esterifying group would regulate the supramolecular structures of the self-aggregates. In vitro, BChls-c esterifying with different alcohols self-aggregated to give red-shifted and broadened electronic absorption bands, which were dependent on the esterifying groups [46,47,48]. Moreover, self-aggregates of synthetic BChl-d models 2 (Fig. 1, middle) exhibited chlorosomal absorption spectra in a variety of environments, which depended on the R groups [22, 24]. The hydrophobicity [49,50,51,52,53,54], hydrophilicity [50, 55], and fluorophilicity [56,57,58] of the R moieties in 2 controlled the stability of the self-aggregates in less polar organic solvents, aqueous solution, and fluorous solvents (or liquid carbon dioxide), respectively.
To confirm the regulation of the chlorosomal self-aggregation by the esterifying group and its participation in the supramolecular structures, we herein report on the synthesis of BChl-d analogs 1 (Fig. 1, right) possessing a porphyrin π-skeleton and a chiral esterifying group (R*) and their self-aggregation in an aqueous micellar solution. The point chirality in the R* group was amplified in the self-aggregates to give intense, exciton-coupled, and chirality-dependent CD bands, indicating that the esterifying group regulated the supramolecular structure.
2 Experimental
2.1 General
Ultraviolet–visible (UV–Vis) absorption and CD spectra were measured with Hitachi U-4100 (Tokyo, Japan) and JASCO J-1500 spectrometers (Hachioji, Japan), respectively. 1H NMR spectra were measured at room temperature with a JEOL ECA-600 (600 MHz) or AL400 (400 MHz) spectrometer (Akishima, Japan); chemical shifts (δs) are expressed in parts per million relative to residual chloroform (δ = 7.26 ppm) as an internal reference. Proton peaks were assigned using 1H–1H two-dimensional NMR techniques. High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF II spectrometer (Billerica, MA, USA): atmospheric pressure chemical ionization (APCI) and positive mode in acetonitrile. Flash column chromatography (FCC) was carried out with silica gel (Merck Kieselgel 60, 0.040–0.063 mm, Darmstadt, Germany). HPLC was performed on a packed octadecylated column (Inertsil ODS-P 5 μm, 10 mmϕ × 250 mm, GL Sciences, Tokyo, Japan) with Shimadzu LC-20AR pumps and SPD-M20A photodiode-array detector (Kyoto, Japan).
Pyropheophorbide-a (6) [59, 60] and its methyl ester (7) [61,62,63,64] were prepared according to reported procedures. All the reaction reagents and solvents were obtained from commercial suppliers and utilized as supplied. Especially, chiral methylcarbinols were purchased from TCI (Tokyo, Japan). Dry dichloromethane for esterification was obtained from FUJIFILM Wako Pure Chem. (Osaka, Japan) as reagent for organic synthesis as super dehydrated grade. Distilled water was prepared by a Yamato AutoStill WG250 system (Tokyo, Japan). Triton X-100 was purchased from Nacalai Tesque (Kyoto, Japan) and used as received. For optical spectroscopy, tetrahydrofuran (THF) was purchased from Nacalai Tesque as reagent prepared specially for HPLC.
2.2 Synthesis of pyropheophorbides-a 5aR/S–5cR/S
Carboxylic acid 6 (240.6 mg, 450 μmol) was dissolved into dry dichloromethane (30 ml) in the dark under argon at room temperature. The solution was cooled down in an ice bath, to which chiral methylcarbinol (2.25 mmol, 5 eq.), 1-[3-(N,N-dimethylamino)propyl]-3-ethylcarbodiimide hydrogen chloride (EDC·HCl, 431.3 mg, 2.25 mmol, 5 eq.), and 4-(N,N-dimethylamino)pyridine (DMAP, 549.8 mg, 4.5 mmol, 10 eq.) were added and stirred for 15 min. The mixed solution was further stirred for 15 h at room temperature. The reaction mixture was washed with an aqueous 1% hydrogen chloride solution, an aqueous solution saturated with sodium hydrogen carbonate, and brine, dried over sodium sulfate, and filtered. All the solvent was evaporated, and the residue was purified by FCC with dichloromethane and 0–3% diethyl ether to give the corresponding ester 5.
(R)-2-Butyl pyropheophorbide-a (5aR): Esterification of 6 with (R)-2-butanol (206.9 μl) gave the titled ester (194.1 mg, 329 μmol, 73%): black solid; mp 199–200 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.43), 610 (0.08), 560 (0.03), 539 (0.09), 508 (0.11), 476 (0.04), 414 (1.00), 399 (0.81), 379 (0.54), 321 (0.22); 1H NMR (CDCl3, 600 MHz) δ/ppm = 9.45 (1H, s, 10-H), 9.33 (1H, s, 5-H), 8.55 (1H, s, 20-H), 7.98 (1H, dd, J = 18, 11.5 Hz, 31-H), 6.25 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.15 (1H, dd, J = 11.5, 1 Hz, 32-H cis to C31–H), 5.27, 5.11 (each 1H, d, J = 20 Hz, 131-CH2), 4.81 (1H, sextet, J = 6 Hz, 172-COOCH), 4.50 (1H, dq, J = 2, 7 Hz, 18-H), 4.30 (1H, dt, J = 9, 2 Hz, 17-H), 3.65 (3H, s, 12-CH3), 3.64 (2H, q, J = 8 Hz, 8-CH2), 3.40 (3H, s, 2-CH3), 3.20 (3H, s, 7-CH3), 2.74–2.65, 2.58–2.49 (each 1H, m, 171-CH2), 2.35–2.22 (2H, m, 17-CH2), 1.81 (3H, d, J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 8 Hz, 81-CH3), 1.52–1.39 (2H, m, 172-COOCCH2), 1.15 (3H, d, J = 6 Hz, 172-COOCCH3), 0.78 (3H, t, J = 7 Hz, 172-COOC2CH3), − 0.09, − 2.04 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 591.3326, calcd. for C37H43N4O3: MH+, 591.3330.
(S)-2-Butyl pyropheophorbide-a (5aS): Esterification of 6 with (S)-2-butanol (206.9 μl) gave the titled ester (186.1 mg, 315 μmol, 70%): black solid; mp 200–201 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.43), 610 (0.08), 561 (0.03), 539 (0.09), 510 (0.10), 475 (0.04), 414 (1.00), 399 (0.81), 378 (0.53), 322 (0.20); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.44 (1H, s, 10-H), 9.33 (1H, s, 5-H), 8.55 (1H, s, 20-H), 7.98 (1H, dd, J = 18, 11 Hz, 31-H), 6.27 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.16 (1H, dd, J = 11, 1 Hz, 32-H cis to C31–H), 5.28, 5.12 (each 1H, d, J = 20 Hz, 131-CH2), 4.86 (1H, sextet, J = 6 Hz, 172-COOCH), 4.51 (1H, dq, J = 2, 7 Hz, 18-H), 4.30 (1H, dt, J = 7, 2 Hz, 17-H), 3.65 (3H, s, 12-CH3), 3.64 (2H, q, J = 8 Hz, 8-CH2), 3.40 (3H, s, 2-CH3), 3.20 (3H, s, 7-CH3), 2.77–2.66, 2.61–2.49 (each 1H, m, 171-CH2), 2.34–2.22 (2H, m, 17-CH2), 1.83 (3H, d, J = 7 Hz, 18-CH3), 1.67 (3H, t, J = 8 Hz, 81-CH3), 1.53 (2H, m, 172-COOCCH2), 1.15 (3H, d, J = 6 Hz, 172-COOCCH3), 0.84 (3H, t, J = 7 Hz, 172-COOC2CH3), 0.42, − 1.72 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 591.3320, calcd. for C37H43N4O3: MH+, 591.3330.
(R)-2-Octyl pyropheophorbide-a (5bR): Esterification of 6 with (R)-2-octanol (357.3 μl) gave the titled ester (203.8 mg, 315 μmol, 70%): black solid; mp 66–67 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.43), 609 (0.08), 561 (0.04), 539 (0.10), 510 (0.12), 477 (0.06), 414 (1.00), 399 (0.81), 379 (0.55), 321 (0.24); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.45 (1H, s, 10-H), 9.34 (1H, s, 5-H), 8.55 (1H, s, 20-H), 7.98 (1H, dd, J = 18, 12 Hz, 31-H), 6.26 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.16 (1H, dd, J = 12, 1 Hz, 32-H cis to C31–H), 5.28, 5.11 (each 1H, d, J = 20 Hz, 131-CH2), 4.86 (1H, sextet, J = 6 Hz, 172-COOCH), 4.50 (1H, dq, J = 2, 7 Hz, 18-H), 4.30 (1H, dt, J = 8, 2 Hz, 17-H), 3.65 (3H, s, 12-CH3), 3.65 (2H, q, J = 8 Hz, 8-CH2), 3.40 (3H, s, 2-CH3), 3.20 (3H, s, 7-CH3), 2.75–2.64, 2.58–2.47 (each 1H, m, 171-CH2), 2.35–2.20 (2H, m, 17-CH2), 1.81 (3H, d, J = 7 Hz, 18-CH3), 1.67 (3H, t, J = 8 Hz, 81-CH3), 1.51–1.41, 1.41–1.31 (each 1H, m, 172-COOCCH2), 1.26–1.18 (8H, m, 172-COOC2(CH2)4), 1.15 (3H, d, J = 6 Hz, 172-COOCCH3), 0.80 (3H, t, J = 7 Hz, 172-COOC6CH3), 0.42, − 1.73 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 647.3977, calcd. for C41H51N4O3: MH+, 647.3956.
(S)-2-Octyl pyropheophorbide-a (5bS): Esterification of 6 with (S)-2-octanol (357.3 μl) gave the titled ester (212.5 mg, 328 μmol, 73%): black solid; mp 67–68 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.44), 610 (0.08), 560 (0.03), 539 (0.09), 509 (0.10), 476 (0.04), 414 (1.00), 400 (0.81), 380 (0.54), 323 (0.20); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.46 (1H, s, 10-H), 9.35 (1H, s, 5-H), 8.56 (1H, s, 20-H), 7.99 (1H, dd, J = 18, 12 Hz, 31-H), 6.27 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.16 (1H, dd, J = 12, 1 Hz, 32-H cis to C31–H), 5.28, 5.12 (each 1H, d, J = 20 Hz, 131-CH2), 4.90 (1H, sextet, J = 6 Hz, 172-COOCH), 4.51 (1H, dq, J = 2, 7 Hz, 18-H), 4.30 (1H, dt, J = 8, 2 Hz, 17-H), 3.66 (3H, s, 12-CH3), 3.66 (2H, q, J = 8 Hz, 8-CH2), 3.41 (3H, s, 2-CH3), 3.22 (3H, s, 7-CH3), 2.75–2.65, 2.60–2.50 (each 1H, m, 171-CH2), 2.33–2.22 (2H, m, 17-CH2), 1.82 (3H, d, J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 8 Hz, 81-CH3), 1.56–1.47, 1.46–1.35 (each 1H, m, 172-COOCCH2), 1.28–1.08 (8H, m, 172-COOC2(CH2)4), 1.14 (3H, d, J = 7 Hz, 172-COOCCH3), 0.82 (3H, t, J = 7 Hz, 172-COOC6CH3), 0.44, − 1.71 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 647.3955, calcd. for C41H51N4O3: MH+, 647.3956.
(R)-1-Phenylethyl pyropheophorbide-a (5cR): Esterification of 6 with (R)-1-phenylethanol (269.5 μl) gave the titled ester (224.2 mg, 351 μmol, 78%): black solid; mp 86–87 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.44), 610 (0.08), 559 (0.03), 539 (0.09), 509 (0.10), 477 (0.04), 414 (1.00), 400 (0.81), 380 (0.54), 323 (0.21); 1H NMR (CDCl3, 600 MHz) δ/ppm = 9.50 (1H, s, 10-H), 9.39 (1H, s, 5-H), 8.54 (1H, s, 20-H), 8.01 (1H, dd, J = 18, 11 Hz, 31-H), 7.29–7.22 (5H, m, 172-COOCC6H5), 6.29 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.17 (1H, dd, J = 11, 1 Hz, 32-H cis to C31–H), 5.88 (1H, q, J = 6 Hz, 172-COOCH), 5.22, 5.05 (each 1H, d, J = 19 Hz, 131-CH2), 4.47 (1H, br-q, J = 7 Hz, 18-H), 4.27 (1H, br-d, J = 9 Hz, 17-H), 3.69 (2H, q, J = 8 Hz, 8-CH2), 3.67 (3H, s, 12-CH3), 3.41 (3H, s, 2-CH3), 3.24 (3H, s, 7-CH3), 2.72–2.67, 2.60–2.55 (each 1H, m, 171-CH2), 2.31–2.25 (2H, m, 17-CH2), 1.78 (3H, d, J = 7 Hz, 18-CH3), 1.70 (3H, t, J = 8 Hz, 81-CH3), 1.49 (3H, d, J = 6 Hz, 172-COOCCH3), 0.45, − 1.69 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 639.3325, calcd. for C41H43N4O3: MH+, 639.3330.
(S)-1-Phenylethyl pyropheophorbide-a (5cS): Esterification of 6 with (S)-1-phenylethanol (269.5 μl) gave the titled ester (253.0 mg, 396 μmol, 88%): black solid; mp 87–88 °C; VIS (CH2Cl2) λmax/nm = 668 (relative intensity, 0.44), 610 (0.08), 559 (0.03), 540 (0.09), 510 (0.10), 477 (0.04), 415 (1.00), 400 (0.80), 379 (0.53), 322 (0.21); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.46 (1H, s, 10-H), 9.35 (1H, s, 5-H), 8.52 (1H, s, 20-H), 7.99 (1H, dd, J = 18, 12 Hz, 31-H), 7.28–7.21 (5H, m, 172-COOCC6H5), 6.27 (1H, dd, J = 18, 1 Hz, 32-H trans to C31–H), 6.16 (1H, dd, J = 12, 1 Hz, 32-H cis to C31–H), 5.86 (1H, q, J = 7 Hz, 172-COOCH), 5.24, 5.07 (each 1H, d, J = 20 Hz, 131-CH2), 4.45 (1H, dq, J = 2, 7 Hz, 18-H), 4.26 (1H, dt, J = 8, 2 Hz, 17-H), 3.66 (2H, q, J = 7 Hz, 8-CH2), 3.66 (3H, s, 12-CH3), 3.39 (3H, s, 2-CH3), 3.21 (3H, s, 7-CH3), 2.71–2.63, 2.58–2.50 (each 1H, m, 171-CH2), 2.35–2.21 (2H, m, 17-CH2), 1.77 (3H, d, J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 7 Hz, 81-CH3), 1.42 (3H, d, J = 7 Hz, 172-COOCCH3), 0.43, − 1.75 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 639.3345, calcd. for C41H43N4O3: MH+, 639.3330.
2.3 Synthesis of pyropheophorbides-d 4aR/S–4cR/S
Olefin 5 (350 μmol) was dissolved into THF (50 ml) in the dark under nitrogen at room temperature, to which one piece of osmium tetroxide, distilled water (1 ml), sodium periodate (374.3 mg, 1.75 mmol), and acetic acid (1 ml) were added. After stirred for 15 h, the reaction mixture was diluted with dichloromethane (50 ml), washed with an aqueous solution saturated with sodium hydrogen carbonate and distilled water, dried over sodium sulfate, and filtered. All the solvents were evaporated, and the residue was purified by FCC with dichloromethane and 0–3% diethyl ether to give the corresponding aldehyde 4.
(R)-2-Butyl pyropheophorbide-d (4aR): Oxidation of 5aR (206.8 mg) gave the titled aldehyde (116.2 mg, 196 μmol, 56%): black solid; mp 114–115 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.80), 634 (0.09), 555 (0.16), 522 (0.14), 428 (1.00), 388 (0.84), 328 (0.28), 306 (0.29); 1H NMR (CDCl3, 600 MHz) δ/ppm = 11.48 (1H, s, 3-CHO), 10.17 (1H, s, 5-H), 9.49 (1H, s, 10-H), 8.81 (1H, s, 20-H), 5.34, 5.19 (each 1H, d, J = 19 Hz, 131-CH2), 4.82 (1H, sextet, J = 6 Hz, 172-COOCH), 4.59 (1H, dq, J = 2, 8 Hz, 18-H), 4.39 (1H, dt, J = 9, 2 Hz, 17-H), 3.74 (3H, s, 2-CH3), 3.67 (3H, s, 12-CH3), 3.66, 3.62 (each 1H, dq, J = 15, 8 Hz, 8-CH2), 3.23 (3H, s, 7-CH3), 2.76–2.70, 2.60–2.53 (each 1H, m, 171-CH2), 2.33–2.26 (2H, m, 17-CH2), 1.86 (3H, d, J = 8 Hz, 18-CH3), 1.67 (3H, t, J = 8 Hz, 81-CH3), 1.52–1.39 (2H, m, 172-COOCCH2), 1.16 (3H, d, J = 6 Hz, 172-COOCCH3), 0.77 (3H, t, J = 8 Hz, 172-COOC2CH3), − 0.27, − 2.19 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 593.3134, calcd. for C36H41N4O4: MH+, 593.3122.
(S)-2-Butyl pyropheophorbide-d (4aS): Oxidation of 5aS (206.8 mg) gave the titled aldehyde (190.9 mg, 322 μmol, 92%): black solid; mp 111–112 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.77), 636 (0.11), 555 (0.17), 523 (0.16), 427 (1.00), 387 (0.90), 329 (0.35), 307 (0.35); 1H NMR (CDCl3, 400 MHz) δ/ppm = 11.48 (1H, s, 3-CHO), 10.16 (1H, s, 5-H), 9.48 (1H, s, 10-H), 8.81 (1H, s, 20-H), 5.35, 5.19 (each 1H, d, J = 20 Hz, 131-CH2), 4.86 (1H, sextet, J = 6 Hz, 172-COOCH), 4.59 (1H, dq, J = 2, 7 Hz, 18-H), 4.38 (1H, dt, J = 9, 2 Hz, 17-H), 3.75 (3H, s, 2-CH3), 3.67 (3H, s, 12-CH3), 3.63 (2H, q, J = 8 Hz, 8-CH2), 3.23 (3H, s, 7-CH3), 2.80–2.69, 2.64–2.54 (each 1H, m, 171-CH2), 2.35–2.22 (2H, m, 17-CH2), 1.86 (3H, d, J = 7 Hz, 18-CH3), 1.67 (3H, t, J = 8 Hz, 81-CH3), 1.63–1.43 (2H, m, 172-COOCCH2), 1.15 (3H, d, J = 6 Hz, 172-COOCCH3), 0.85 (3H, t, J = 7 Hz, 172-COOC2CH3), − 0.27, − 2.20 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 593.3132, calcd. for C36H41N4O4: MH+, 593.3122.
(R)-2-Octyl pyropheophorbide-d (4bR): Oxidation of 5bR (226.4 mg) gave the titled aldehyde (165.8 mg, 256 μmol, 73%): black solid; mp 83–84 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.77), 635 (0.10), 555 (0.17), 522 (0.16), 428 (1.00), 388 (0.88), 329 (0.37), 307 (0.35); 1H NMR (CDCl3, 400 MHz) δ/ppm = 11.50 (1H, s, 3-CHO), 10.21 (1H, s, 5-H), 9.52 (1H, s, 10-H), 8.82 (1H, s, 20-H), 5.35, 5.19 (each 1H, d, J = 20 Hz, 131-CH2), 4.87 (1H, sextet, J = 6 Hz, 172-COOCH), 4.59 (1H, dq, J = 2, 7 Hz, 18-H), 4.38 (1H, dt, J = 9, 2 Hz, 17-H), 3.76 (3H, s, 2-CH3), 3.68 (3H, s, 12-CH3), 3.66 (2H, q, J = 8 Hz, 8-CH2), 3.25 (3H, s, 7-CH3), 2.78–2.68, 2.63–2.49 (each 1H, m, 171-CH2), 2.36–2.22 (2H, m, 17-CH2), 1.86 (3H, d, J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 8 Hz, 81-CH3), 1.51–1.41, 1.40–1.30 (each 1H, m, 172-COOCCH2), 1.24–1.12 (8H, m, 172-COOC2(CH2)4), 1.16 (3H, d, J = 6 Hz, 172-COOCCH3), 0.81 (3H, t, J = 7 Hz, 172-COOC6CH3), − 0.23, − 2.16 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 649.3730, calcd. for C40H49N4O4: MH+, 649.3748.
(S)-2-Octyl pyropheophorbide-d (4bS): Oxidation of 5bS (226.4 mg) gave the titled aldehyde (168.1 mg, 259 μmol, 74%): black solid; mp 89–90 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.81), 633 (0.09), 555 (0.16), 522 (0.15), 429 (1.00), 388 (0.84), 329 (0.30), 308 (0.31); 1H NMR (CDCl3, 400 MHz) δ/ppm = 11.48 (1H, s, 3-CHO), 10.17 (1H, s, 5-H), 9.49 (1H, s, 10-H), 8.82 (1H, s, 20-H), 5.35, 5.19 (each 1H, d, J = 20 Hz, 131-CH2), 4.91 (1H, sextet, J = 6 Hz, 172-COOCH), 4.59 (1H, br-q, J = 7 Hz, 18-H), 4.38 (1H, br-d, J = 8 Hz, 17-H), 3.75 (3H, s, 2-CH3), 3.67 (3H, s, 12-CH3), 3.63 (2H, q, J = 8 Hz, 8-CH2), 3.23 (3H, s, 7-CH3), 2.80–2.68, 2.64–2.52 (each 1H, m, 171-CH2), 2.35–2.22 (2H, m, 17-CH2), 1.86 (3H, d, J = 7 Hz, 18-CH3), 1.67 (3H, t, J = 8 Hz, 81-CH3), 1.56–1.48, 1.47–1.36 (each 1H, m, 172-COOCCH2), 1.29–1.16 (8H, m, 172-COOC2(CH2)4), 1.14 (3H, d, J = 6 Hz, 172-COOCCH3), 0.82 (3H, t, J = 7 Hz, 172-COOC6CH3), − 0.27, − 2.19 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 649.3728, calcd. for C40H49N4O4: MH+, 649.3748.
(R)-1-Phenylethyl pyropheophorbide-d (4cR): Oxidation of 5cR (223.6 mg) gave the titled aldehyde (172.7 mg, 270 μmol, 77%): black solid; mp 121–122 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.79), 635 (0.09), 555 (0.17), 522 (0.16), 488 (0.08), 430 (1.00), 388 (0.85), 329 (0.34), 306 (0.36); 1H NMR (CDCl3, 600 MHz) δ/ppm = 11.50 (1H, s, 3-CHO), 10.21 (1H, s, 5-H), 9.52 (1H, s, 10-H), 8.81 (1H, s, 20-H), 7.30–7.23 (5H, m, 172-COOCC6H5), 5.90 (1H, q, J = 7 Hz, 172-COOCH), 5.29, 5.13 (each 1H, d, J = 20 Hz, 131-CH2), 4.57 (1H, dq, J = 2, 7 Hz, 18-H), 4.36 (1H, dt, J = 9, 2 Hz, 17-H), 3.76 (3H, s, 2-CH3), 3.68 (3H, s, 12-CH3), 3.66, 3.64 (each 1H, dq, J = 13, 8 Hz, 8-CH2), 3.26 (3H, s, 7-CH3), 2.76–2.70, 2.65–2.60 (each 1H, m, 171-CH2), 2.34–2.25 (2H, m, 17-CH2), 1.84 (3H, d, J = 7 Hz, 18-CH3), 1.70 (3H, t, J = 8 Hz, 81-CH3), 1.52 (3H, d, J = 7 Hz, 172-COOCCH3), − 0.25, − 2.17 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 641.3101, calcd. for C40H41N4O4: MH+, 641.3122.
(S)-1-Phenylethyl pyropheophorbide-d (4cS): Oxidation of 5cS (223.6 mg) gave the titled aldehyde (168.2 mg, 262 μmol, 75%): black solid; mp 120–121 °C; VIS (CH2Cl2) λmax/nm = 695 (relative intensity, 0.81), 635 (0.09), 555 (0.16), 522 (0.15), 488 (0.06), 430 (1.00), 388 (0.84), 329 (0.30), 307 (0.31); 1H NMR (CDCl3, 400 MHz) δ/ppm = 11.49 (1H, s, 3-CHO), 10.20 (1H, s, 5-H), 9.52 (1H, s, 10-H), 8.79 (1H, s, 20-H), 7.28–7.22 (5H, m, 172-COOCC6H5), 5.87 (1H, q, J = 6 Hz, 172-COOCH), 5.31, 5.13 (each 1H, d, J = 20 Hz, 131-CH2), 4.53 (1H, dq, J = 2, 8 Hz, 18-H), 4.35 (1H, dt, J = 7, 2 Hz, 17-H), 3.74 (3H, s, 2-CH3), 3.68 (3H, s, 12-CH3), 3.66 (2H, q, J = 8 Hz, 8-CH2), 3.25 (3H, s, 7-CH3), 2.75–2.66, 2.63–2.53 (each 1H, m, 171-CH2), 2.34–2.24 (2H, m, 17-CH2), 1.81 (3H, d, J = 8 Hz, 18-CH3), 1.68 (3H, t, J = 8 Hz, 81-CH3), 1.43 (3H, d, J = 6 Hz, 172-COOCCH3), − 0.24, − 2.18 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 641.3099, calcd. for C40H41N4O4: MH+, 641.3122.
2.4 Synthesis of 3-hydroxymethyl-pyropheophorbides-a 3aR/S–3cR/S
Aldehyde 4 (200 μmol) was dissolved into dichloromethane (20 ml) in the dark under nitrogen at room temperature, to which tert-butylamine borane complex (69.6 mg, 800 μmol) was added. After stirred for 15 min, the reaction mixture was diluted with dichloromethane (50 ml), washed with an aqueous solution saturated with ammonium chloride, dried over sodium sulfate, and filtered. All the solvent was evaporated, and the residue was purified by FCC with dichloromethane and 0–2% methanol to give the corresponding alcohol 3.
(R)-2-Butyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3aR): Reduction of 4aR (118.5 mg) gave the titled alcohol (73.7 mg, 124 μmol, 62%): black solid; mp 249–250 °C; VIS (CH2Cl2) λmax/nm = 663 (relative intensity, 0.48), 605 (0.08), 557 (0.04), 535 (0.10), 505 (0.11), 471 (0.05), 410 (1.00), 397 (0.80), 376 (0.57), 317 (0.22); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.29 (1H, s, 5-H), 9.18 (1H, s, 10-H), 8.47 (1H, s, 20-H), 5.75 (2H, s, 3-CH2), 5.05, 4.93 (each 1H, d, J = 20 Hz, 131-CH2), 4.83 (1H, sextet, J = 6 Hz, 172-COOCH), 4.39 (1H, dq, J = 2, 7 Hz, 18-H), 4.12 (1H, dt, J = 9, 2 Hz, 17-H), 3.56 (2H, q, J = 8 Hz, 8-CH2), 3.45 (3H, s, 12-CH3), 3.34 (3H, s, 2-CH3), 3.17 (3H, s, 7-CH3), 2.57–2.44 (2H, m, 171-CH2), 2.27–2.17, 2.13–2.02 (each 1H, m, 17-CH2), 1.72 (3H, d, J = 7 Hz, 18-CH3), 1.61 (3H, t, J = 8 Hz, 81-CH3), 1.55–1.39 (2H, m, 172-COOCCH2), 1.16 (3H, d, J = 6 Hz, 172-COOCCH3), 0.80 (3H, t, J = 7 Hz, 172-COOC2CH3), − 0.09, − 2.04 (each 1H, br-s, NH × 2) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 595.3279 calcd. for C36H43N4O4: MH+, 595.3279.
(S)-2-Butyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3aS): Reduction of 4aS (118.5 mg) gave the titled alcohol (91.6 mg, 154 μmol, 77%): black solid; mp 217–218 °C; VIS (CH2Cl2) λmax/nm = 662 (relative intensity, 0.48), 605 (0.08), 559 (0.03), 536 (0.09), 506 (0.10), 471 (0.04), 410 (1.00), 395 (0.79), 376 (0.57), 318 (0.21); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.37 (1H, s, 5-H), 9.34 (1H, s, 10-H), 8.52 (1H, s, 20-H), 5.83 (2H, s, 3-CH2), 5.15, 5.02 (each 1H, d, J = 20 Hz, 131-CH2), 4.86 (1H, sextet, J = 6 Hz, 172-COOCH), 4.45 (1H, dq, J = 2, 7 Hz, 18-H), 4.20 (1H, dt, J = 9, 2 Hz, 17-H), 3.63 (2H, q, J = 8 Hz, 8-CH2), 3.56 (3H, s, 12-CH3), 3.39 (3H, s, 2-CH3), 3.22 (3H, s, 7-CH3), 2.67–2.46 (2H, m, 171-CH2), 2.31–2.11 (2H, m, 17-CH2), 1.77 (3H, d, J = 7 Hz, 18-CH3), 1.66 (3H, t, J = 8 Hz, 81-CH3), 1.61–1.43 (2H, m, 172-COOCCH2), 1.16 (3H, d, J = 6 Hz, 172-COOCCH3), 0.85 (3H, t, J = 7 Hz, 172-COOC2CH3), 0.10, − 1.92 (each 1H, br-s, NH × 2) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 595.3271 calcd. for C36H43N4O4: MH+, 595.3279.
(R)-2-Octyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3bR): Reduction of 4bR (129.8 mg) gave the titled alcohol (87.2 mg, 134 μmol, 67%): black solid; mp 101–102 °C; VIS (CH2Cl2) λmax/nm = 663 (relative intensity, 0.47), 605 (0.08), 557 (0.04), 536 (0.10), 505 (0.11), 470 (0.05), 410 (1.00), 397 (0.83), 377 (0.58), 319 (0.22); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.32 (1H, s, 5-H), 9.27 (1H, s, 10-H), 8.50 (1H, s, 20-H), 5.78 (2H, s, 3-CH2), 5.11, 5.00 (each 1H, d, J = 20 Hz, 131-CH2), 4.88 (1H, sextet, J = 7 Hz, 172-COOCH), 4.42 (1H, br-q, J = 7 Hz, 18-H), 4.17 (1H, br-d, J = 9 Hz, 17-H), 3.60 (2H, q, J = 7 Hz, 8-CH2), 3.51 (3H, s, 12-CH3), 3.36 (3H, s, 2-CH3), 3.19 (3H, s, 7-CH3), 2.61–2.45 (2H, m, 171-CH2), 2.26–2.09 (2H, m, 17-CH2), 1.75 (3H, d, J = 7 Hz, 18-CH3), 1.64 (3H, t, J = 7 Hz, 81-CH3), 1.53–1.44, 1.42–1.33 (each 1H, m, 172-COOCCH2), 1.25–1.13 (8H, m, 172-COOC2(CH2)4), 1.16 (3H, d, J = 7 Hz, 172-COOCCH3), 0.81 (3H, t, J = 7 Hz, 172-COOC6CH3), − 1.98 (each 1H, br-s, NH) [Another inner NH and the 31-OH signals were invisible.]; HRMS (APCI) found: m/z = 651.3879, calcd. for C40H51N4O4: MH+, 651.3905.
(S)-2-Octyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3bS): Reduction of 4bS (129.8 mg) gave the titled alcohol (113.3 mg, 174 μmol, 87%): black solid; mp 101–102 °C; VIS (CH2Cl2) λmax/nm = 662 (relative intensity, 0.48), 605 (0.08), 558 (0.03), 536 (0.09), 505 (0.10), 472 (0.04), 411 (1.00), 397 (0.78), 377 (0.56), 318 (0.21); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.38 (1H, s, 5-H), 9.37 (1H, s, 10-H), 8.53 (1H, s, 20-H), 5.85 (2H, s, 3-CH2), 5.18, 5.03 (each 1H, d, J = 20 Hz, 131-CH2), 4.91 (1H, sextet, J = 6 Hz, 172-COOCH), 4.46 (1H, dq, J = 2, 7 Hz, 18-H), 4.20 (1H, dt, J = 9, 2 Hz, 17-H), 3.64 (2H, q, J = 8 Hz, 8-CH2), 3.57 (3H, s, 12-CH3), 3.39 (3H, s, 2-CH3), 3.22 (3H, s, 7-CH3), 2.67–2.58, 2.54–2.48 (each 1H, m, 171-CH2), 2.30–2.11 (2H, m, 17-CH2), 1.78 (3H, d, J = 7 Hz, 18-CH3), 1.66 (3H, t, J = 8 Hz, 81-CH3), 1.56–1.47, 1.46–1.37 (each 1H, m, 172-COOCCH2), 1.29–1.12 (8H, m, 172-COOC2(CH2)4), 1.15 (3H, d, J = 7 Hz, 172-COOCCH3), 0.82 (3H, t, J = 7 Hz, 172-COOC6CH3), 0.12, − 1.90 (each 1H, br-s, NH × 2) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 651.3880, calcd. for C40H51N4O4: MH+, 651.3905.
(R)-1-Phenylethyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3cR): Reduction of 4cR (128.2 mg) gave the titled alcohol (124.7 mg, 194 μmol, 97%): black solid; mp 137–138 °C; VIS (CH2Cl2) λmax/nm = 663 (relative intensity, 0.48), 604 (0.08), 556 (0.03), 536 (0.09), 505 (0.10), 473 (0.04), 410 (1.00), 397 (0.80), 377 (0.56), 319 (0.21); 1H NMR (CDCl3, 600 MHz) δ/ppm = 9.44 (1H, s, 10-H), 9.41 (1H, s, 5-H), 8.53 (1H, s, 20-H), 7.29–7.21 (5H, m, 172-COOCC6H5), 5.89 (1H, q, J = 7 Hz, 172-COOCH), 5.88 (2H, s, 3-CH2), 5.15, 5.00 (each 1H, d, J = 19 Hz, 131-CH2), 4.45 (1H, dq, J = 2, 7 Hz, 18-H), 4.22 (1H, dt, J = 9, 2 Hz, 17-H), 3.67 (2H, q, J = 8 Hz, 8-CH2), 3.62 (3H, s, 12-CH3), 3.40 (3H, s, 2-CH3), 3.25 (3H, s, 7-CH3), 2.67–2.61, 2.59–2.54 (each 1H, m, 171-CH2), 2.29–2.17 (2H, m, 17-CH2), 2.18 (1H, br, 31-OH), 1.75 (3H, d, J = 7 Hz, 18-CH3), 1.68 (3H, t, J = 8 Hz, 81-CH3), 1.50 (3H, d, J = 7 Hz, 172-COOCCH3), 0.18, − 1.86 (each 1H, br-s, NH × 2); HRMS (APCI) found: m/z = 643.3265, calcd. for C40H43N4O4: MH+, 643.3279.
(S)-1-Phenylethyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (3cS): Reduction of 4cS (128.2 mg) gave the titled alcohol (124.7 mg, 194 μmol, 97%): black solid; mp 135–136 °C; VIS (CH2Cl2) λmax/nm = 663 (relative intensity, 0.48), 606 (0.08), 557 (0.03), 536 (0.09), 506 (0.10), 475 (0.04), 410 (1.00), 397 (0.80), 377 (0.56), 317 (0.20); 1H NMR (CDCl3, 400 MHz) δ/ppm = 9.37 (1H, s, 5-H), 9.36 (1H, s, 10-H), 8.49 (1H, s, 20-H), 7.28–7.22 (5H, m, 172-COOCC6H5), 5.87 (1H, q, J = 7 Hz, 172-COOCH), 5.84 (2H, s, 3-CH2), 5.13, 4.97 (each 1H, d, J = 20 Hz, 131-CH2), 4.39 (1H, dq, J = 2, 7 Hz, 18-H), 4.17 (1H, dt, J = 8, 2 Hz, 17-H), 3.64 (2H, q, J = 7 Hz, 8-CH2), 3.76 (3H, s, 12-CH3), 3.57 (3H, s, 2-CH3), 3.22 (3H, s, 7-CH3), 2.62–2.48 (2H, m, 171-CH2), 2.29–2.15 (2H, m, 17-CH2), 1.72 (3H, d, J = 7 Hz, 18-CH3), 1.67 (3H, t, J = 7 Hz, 81-CH3), 1.44 (3H, d, J = 7 Hz, 172-COOCCH3), 0.10, − 1.93 (each 1H, br-s, NH × 2) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 643.3263, calcd. for C40H43N4O4: MH+, 643.3279.
2.5 Synthesis of zinc 3-hydroxymethyl-pyropheophorbides-a 2aR/S–2cR/S
Free base 3 (180 μmol) was dissolved into dichloromethane (20 ml) in the dark under nitrogen at room temperature, to which a methanol solution saturated with zinc acetate dihydrate (10 ml) was added. After stirred for 30 min, the reaction mixture was diluted with dichloromethane (30 ml), washed with an aqueous solution saturated with sodium hydrogen carbonate and distilled water, dried over sodium sulfate, and filtered. All the solvents were evaporated to give the corresponding zinc complex 2.
Zinc (R)-2-butyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2aR): Zinc metalation of 3aR (107.1 mg) gave the titled zinc complex (106.6 mg, 162 μmol, 90%): black solid; mp > 300 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.74), 602 (0.10), 566 (0.06), 521 (0.03), 490 (0.02), 424 (1.00), 404 (0.57), 381 (0.31), 315 (0.20); 1H NMR (CDCl3–10% pyridine-d5, 400 MHz) δ/ppm = 9.47 (1H, s, 10-H), 9.29 (1H, s, 5-H), 8.23 (1H, s, 20-H), 5.76 (2H, s, 3-CH2), 5.10, 4.96 (each 1H, d, J = 20 Hz, 131-CH2), 4.69 (1H, sextet, J = 6 Hz, 171-CH2), 4.31 (1H, dq, J = 2.5, 7.5 Hz, 18-H), 4.10 (1H, dt, J = 8, 2.5 Hz, 17-H), 3.64 (2H, q, J = 7.5 Hz, 8-CH2), 3.59 (3H, s, 12-CH3), 3.19 (3H, s, 2-CH3), 3.08 (3H, s, 7-CH3), 2.52–2.41, 2.32–2.22 (each 1H, m, 171-CH2), 2.21–2.09, 1.92–1.83 (each 1H, m, 17-CH2), 1.62 (3H, d, J = 7.5 Hz, 18-CH3), 1.59 (3H, t, J = 7.5 Hz, 81-CH3), 1.36 (2H, m, 172-COOCCH2), 1.02 (3H, d, J = 6 Hz, 172-COOCCH3), 0.70 (3H, t, J = 7 Hz, 172-COOC2CH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 657.2392, calcd. for C36H41N4O4Zn: MH+, 657.2414.
Zinc (S)-2-butyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2aS): Zinc metalation of 3aS (107.1 mg) gave the titled zinc complex (109.0 mg, 166 μmol, 92%): black solid; mp 249–250 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.74), 602 (0.11), 566 (0.06), 521 (0.04), 489 (0.02), 424 (1.00), 404 (0.58). 382 (0.32), 315 (0.23); 1H NMR (CDCl3–10% pyridine-d5, 400 MHz) δ/ppm = 9.48 (1H, s, 10-H), 9.30 (1H, s, 5-H), 8.24 (1H, s, 20-H), 5.77 (2H, s, 3-CH2), 5.11, 4.97 (each 1H, d, J = 20 Hz, 131-CH2), 4.71 (1H, sextet, J = 6 Hz, 172-COOCH), 4.32 (1H, dq, J = 2, 7 Hz, 18-H), 4.11 (1H, dt, J = 8, 2 Hz, 17-H), 3.65 (2H, q, J = 8 Hz, 8-CH2), 3.61 (3H, s, 12-CH3), 3.21 (3H, s, 2-CH3), 3.09 (3H, s, 7-CH3), 2.53–2.42, 2.35–2.24 (each 1H, m, 171-CH2), 2.21–2.10, 1.93–1.83 (each 1H, m, 17-CH2), 1.63 (3H, d, J = 7 Hz, 18-CH3), 1.60 (3H, t, J = 8 Hz, 81-CH3), 1.39 (2H, m, 172-COOCCH2), 1.04 (3H, d, J = 6 Hz, 172-COOCCH3), 0.72 (3H, t, J = 7 Hz, 172-COOC2CH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 657.2389, calcd. for C36H41N4O4Zn: MH+, 657.2414.
Zinc (R)-2-octyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2bR): Zinc metalation of 3bR (117.2 mg) gave the titled zinc complex (91.3 mg, 128 μmol, 71%): black solid; mp 232–233 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.73), 602 (0.11), 566 (0.06), 520 (0.04), 488 (0.03), 424 (1.00), 404 (0.57), 381 (0.33), 315 (0.26); 1H NMR (CDCl3–10% pyridine-d5, 600 MHz) δ/ppm = 9.57 (1H, s, 10-H), 9.39 (1H, s, 5-H), 8.33 (1H, s, 20-H), 5.88, 5.84 (each 1H, d, J = 12 Hz, 3-CH2), 5.20, 5.07 (each 1H, d, J = 19 Hz, 131-CH2), 4.85 (1H, sextet, J = 6 Hz, 172-COOCH), 4.41 (1H, br-q, J = 7 Hz, 18-H), 4.20 (1H, br-d, J = 8 Hz, 17-H), 3.74 (2H, q, J = 8 Hz, 8-CH2), 3.69 (3H, s, 12-CH3), 3.29 (3H, s, 2-CH3), 3.18 (3H, s, 7-CH3), 2.59–2.53, 2.29–2.22 (each 1H, m, 17-CH2), 2.39–2.33, 2.00–1.94 (each 1H, m, 171-CH2), 1.72 (3H, d, J = 7 Hz, 18-CH3), 1.69 (3H, t, J = 8 Hz, 81-CH3), 1.51–1.45, 1.40–1.34 (each 1H, m, 172-COOCCH2), 1.30–1.16 (8H, m, 172-COOC2(CH2)4), 1.13 (3H, d, J = 6 Hz, 172-COOCCH3), 0.83 (3H, t, J = 7 Hz, 172-COOC6CH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 713.3035, calcd. for C40H49N4O4Zn; MH+, 713.3040.
Zinc (S)-2-octyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2bS): Zinc metalation of 3bS (117.2 mg) gave the titled zinc complex (91.3 mg, 128 μmol, 71%): black solid; mp 244–245 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.74), 602 (0.11), 566 (0.06), 521 (0.04), 487 (0.03), 424 (1.00), 404 (0.57), 380 (0.32), 315 (0.23); 1H NMR (CDCl3–10% pyridine-d5, 400 MHz) δ/ppm = 9.57 (1H, s, 10-H), 9.39 (1H, s, 5-H), 8.33 (1H, s, 20-H), 5.88, 5.84 (each 1H, d, J = 12 Hz, 3-CH2), 5.21, 5.06 (each 1H, d, J = 20 Hz, 131-CH2), 4.86 (1H, sextet, J = 6 Hz, 172-COOCH), 4.41 (1H, dq, J = 2, 7 Hz, 18-H), 4.20 (1H, dt, J = 8, 2 Hz, 17-H), 3.74 (2H, q, J = 8 Hz, 8-CH2), 3.69 (3H, s, 12-CH3), 3.29 (3H, s, 2-CH3), 3.17 (3H, s, 7-CH3), 2.62–2.51, 2.45–2.33 (each 1H, m, 171-CH2), 2.30–2.19, 2.03–1.91 (each 1H, m, 17-CH2), 1.72 (3H, d, J = 7 Hz, 18-CH3), 1.69 (3H, t, J = 8 Hz, 81-CH3), 1.55–1.45, 1.44–1.34 (each 1H, m, 172-COOCCH2), 1.29–1.15 (8H, m, 172-COOC2(CH2)4), 1.13 (3H, d, J = 6 Hz, 172-COOCCH3), 0.83 (3H, t, J = 7 Hz, 172-COOC6CH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 713.3052, calcd. for C40H49N4O4Zn; MH+, 713.3040.
Zinc (R)-1-phenylethyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2cR): Zinc metalation of 3cR (115.7 mg) gave the titled zinc complex (123.4 mg, 175 μmol, 97%): black solid; mp > 300 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.75), 602 (0.10), 566 (0.05), 521 (0.03), 488 (0.01), 424 (1.00), 404 (0.57), 380 (0.31), 315 (0.22); 1H NMR (CDCl3–10% pyridine-d5, 600 MHz) δ/ppm = 9.56 (1H, s, 10-H), 9.38 (1H, s, 5-H), 8.31 (1H, s, 20-H), 7.30–7.21 (5H, m, 172-COOCC6H5), 5.85 (2H, s, 3-CH2), 5.83 (1H, q, J = 7 Hz, 172-COOCH), 5.16, 5.02 (each 1H, d, J = 19 Hz, 131-CH2), 4.37 (1H, dq, J = 2, 7 Hz, 18-H), 4.17 (1H, dt, J = 8, 3 Hz, 17-H), 3.74, 3.72 (each 1H, dq, J = 15, 8 Hz, 8-CH2), 3.69 (3H, s, 12-CH3), 3.29 (3H, s, 2-CH3), 3.17 (3H, s, 7-CH3), 2.58–2.52, 2.41–2.34 (each 1H, m, 171-CH2), 2.30–2.22, 1.95–1.89 (each 1H, m, 17-CH2), 1.69 (3H, d, J = 7 Hz, 18-CH3), 1.69 (3H, t, J = 8 Hz, 81-CH3), 1.46 (3H, d, J = 7 Hz, 172-COOCCH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 705.2400, calcd. for C40H41N4O4Zn: MH+, 705.2414.
Zinc (S)-1-phenylethyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (2cS): Zinc metalation of 3cS (115.7 mg) gave the titled zinc complex (123.3 mg, 175 μmol, 97%): black solid; mp 234–235 °C; VIS (THF) λmax/nm = 646 (relative intensity, 0.73), 602 (0.11), 566 (0.06), 521 (0.04), 487 (0.03), 424 (1.00), 404 (0.58), 381 (0.33), 315 (0.26); 1H NMR (CDCl3–10% pyridine-d5, 400 MHz) δ/ppm = 9.57 (1H, s, 10-H), 9.39 (1H, s, 5-H), 8.31 (1H, s, 20-H), 7.29–7.20 (5H, m, 172-COOCC6H5), 5.86 (2H, s, 3-CH2), 5.82 (1H, q, J = 7 Hz, 172-COOCH), 5.18, 5.09 (each 1H, d, J = 20 Hz, 131-CH2), 4.36 (1H, br-q, J = 7 Hz, 18-H), 4.17 (1H, dt, J = 8, 2 Hz, 17-H), 3.73 (2H, q, J = 7 Hz, 8-CH2), 3.70 (3H, s, 12-CH3), 3.29 (3H, s, 2-CH3), 3.17 (3H, s, 7-CH3), 2.57–2.50 (1H, m, 171-CH), 2.41–2.25 (2H, m, 17-CHCH), 1.96–1.88 (1H, m, 17-CH), 1.69 (3H, t, J = 7 Hz, 81-CH3), 1.68 (3H, d, J = 7 Hz, 18-CH3), 1.42 (3H, d, J = 7 Hz, 172-COOCCH3) [The 31-OH signal was invisible.]; HRMS (APCI) found: m/z = 705.2394, calcd. for C40H41N4O4Zn; MH+, 705.2413.
2.6 Synthesis of zinc 3-hydroxymethyl-pyroprotopheophorbides-a 1aR/S–1cR/S
Zinc chlorin 2 (100 μmol) was dissolved into pyridine (2 ml) in the dark under nitrogen at room temperature, to which an acetone solution (20 ml) of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 34.1 mg, 150 μmol) was added dropwise. After stirred for 10 min, the reaction mixture was diluted with chloroform (20 ml), washed with an aqueous 5% potassium hydrogen sulfate solution, an aqueous solution saturated with sodium hydrogen carbonate, brine, and distilled water, dried over sodium sulfate, and filtered. All the solvents were evaporated, and the residue was purified by HPLC with methanol and 5% pyridine to give the corresponding zinc porphyrin 1. The isolated yields were at most 45%. Since the products were less soluble in deuterated solvents, their well-resolved proton peaks could not be obtained in 1H NMR and the data are not shown below. Their purities were confirmed by HPLC (see Fig. S1).
Zinc (R)-2-butyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1aR): Dehydrogenation of 2aR gave the titled zinc porphyrin: black solid; mp 280–281 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.11), 589 (0.04), 560 (0.06), 545 (0.04), 524 (0.03), 428 (1.00), 376 (0.13), 318 (0.13), 308 (0.12); HRMS (APCI) found: m/z = 655.2248, calcd. for C36H39N4O4Zn: MH+, 655.2257.
Zinc (S)-2-butyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1aS): Dehydrogenation of 2aS gave the titled zinc complex: black solid; mp > 300 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.10), 589 (0.03), 560 (0.05), 545 (0.03), 524 (0.02), 428 (1.00), 376 (0.12), 320 (0.09), 308 (0.12); HRMS (APCI) found: m/z = 655.2239, calcd. for C36H39N4O4Zn: MH+, 655.2257.
Zinc (R)-2-octyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1bR): Dehydrogenation of 2bR gave the titled zinc porphyrin: black solid; mp 257–258 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.11), 588 (0.03), 560 (0.05), 545 (0.03), 524 (0.02), 428 (1.00), 377 (0.12), 319(0.10), 309 (0.10); HRMS (APCI) found: m/z = 711.2866, calcd. for C40H47N4O4Zn: MH+, 711.2883.
Zinc (S)-2-octyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1bS): Dehydrogenation of 2bS gave the titled zinc porphyrin: black solid; mp 254–255 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.11), 588 (0.04), 560 (0.06), 545 (0.04), 524 (0.03), 428 (1.00), 376 (0.13), 319(0.12), 309 (0.12); HRMS (APCI) found: m/z = 711.2860, calcd. for C40H47N4O4Zn: MH+, 711.2883.
Zinc (R)-1-phenylethyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1cR): Dehydrogenation of 2cR gave the titled zinc porphyrin: black solid; mp > 300 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.11), 589 (0.03), 561 (0.05), 545 (0.03), 524 (0.02), 428 (1.00), 377 (0.12), 321(0.10), 307 (0.10); HRMS (APCI) found: m/z = 703.2254, calcd. for C40H39N4O4Zn: MH+, 703.2257.
Zinc (S)-1-phenylethyl 3-devinyl-3-hydroxymethyl-pyroprotopheophorbide-a (1cS): Dehydrogenation of 2cS gave the titled zinc porphyrin: black solid; mp > 300 °C; VIS (THF) λmax/nm = 609 (relative intensity, 0.10), 589 (0.03), 560 (0.05), 545 (0.03), 524 (0.02), 428 (1.00), 377 (0.11), 321(0.09), 309 (0.08); HRMS (APCI) found: m/z = 703.2237, calcd. for C40H39N4O4Zn: MH+, 703.2257.
3 Results and discussion
3.1 Synthesis of chiral zinc porphyrins
Zinc 3-hydroxymethyl-pyroprotopheophorbides-a 1aR/S–1cR/S bearing a chiral esterifying group in the 17-propionate residue were prepared from naturally occurring Chl-a based on reported procedures [65]. Their synthetic route is briefly described below (Scheme 1). Methyl pyropheophorbide-a (7), one of the Chl-a derivatives, was acidically hydrolyzed [step (i), ≈100% isolated yield], and the resulting carboxylic acid 6 was esterified with commercially available, chiral methylcarbinols R*OH [CH3C*H(OH)X] under Steglich esterification conditions to give 5aR/S–5cR/S [step (ii), 70% ~ 88%]. The 3-vinyl group of 5aR/S–5cR/S was transformed into the formyl group in 4aR/S–4cR/S under Lemieux–Johnson oxidation conditions [step (iii), 56% ~ 92%]. Aldehydes 4aR/S–4cR/S were selectively reduced with a mild reducing reagent (tBuNH2BH3) to produce the corresponding alcohols 3aR/S–3cR/S [step (iv), 62% ~ 97%]. Free bases 3aR/S–3cR/S were zinc-metalated by a conventional procedure to afford zinc complexes 2aR/S–2cR/S [step (v), 71% ~ 97%], which were successively 17,18-dehydrogenated by DDQ yielding zinc porphyrins 1aR/S–1cR/S [step (vi), ≈45%]. The oxidized products were purified with HPLC to give enantiomerically pure samples of 1aR/S [X = (CH2)2H], 1bR/S [X = (CH2)6H], and 1cR/S [X = C6H5] (Fig. 2) lacking their corresponding zinc chlorin precursors (Fig. S1). All the synthetic compounds were fully characterized by UV–Vis and/or 1H NMR spectroscopy as well as mass spectrometry.
Synthesis of zinc 3-hydroxymethyl-pyroprotopheophorbides-a 1aR/S–1cR/S possessing a chiral esterifying group in the 17-propionate residue by chemically modifying a Chl-a derivative, methyl pyropheophorbide-a (7): (i) aq. HCl/Me2CO; (ii) R*OH, EDC·HCl, DMAP/CH2Cl2; (iii) OsO4, NaIO4/aq. AcOH, THF; (iv) tBuNH2BH3/CH2Cl2; (v) Zn(OAc)2·2H2O/MeOH, CH2Cl2; (vi) DDQ/Me2CO, pyridine
Molecular structures of 1aR/S, 1bR/S, and 1cR/S
3.2 Self-aggregation of chiral zinc porphyrins
Zinc porphyrin 1aR bearing (R)-2-butyl ester was dissolved in THF to give a sharp UV–Vis absorption spectrum with the most intense peak at 428 nm as its Soret maximum and the second intense peak at 609 nm as its Qy maximum (see the black line in the upper panel of Fig. 3A). The spectral feature indicates that the central zinc of 1aR was axially coordinated with a THF molecule and the resulting five-coordinated species was monomeric [34]. The solution exhibited no CD bands in the region of 300–750 nm (Fig. 3A, lower, black). The CD silence is ascribable to no electronic absorbance of its chiral ester moiety (COOC*H(CH3)[(CH2)2H]) at the UV–Vis region.
UV–Vis absorption (upper) and CD spectra (lower) of 1aR/S (A, red/blue lines), 1bR/S (B, red/blue), and 1cR/S (C, red/blue) in an aqueous 0.025%(wt/v) Triton X-100 solution containing 1%(v/v) THF as well as those of 1aR in THF (A, black). The concentrations of the above samples were 10 μM
The concentrated THF solution of 1aR at 1 mM with 2.5%(wt/v) Triton X-100 was diluted with 99-fold distilled water to exhibit Soret and Qy absorption bands at ≈ 460 and 652 nm, respectively (Fig. 3A, upper, red). The absorption maxima moved to longer wavelengths than those of its monomeric species (vide supra). The red-shifted values of Soret and Qy maxima were ≈ 1600 and 1080 cm–1, respectively. In addition, the red-shifted bands were broadened from the monomeric bands. The full widths at half maxima of the red-shifted Soret and Qy bands were ≈ 4000 and 680 cm–1, respectively, to be larger than those of the monomeric bands (960 and 320 cm–1). The red-shifted and broadened bands indicated that 1aR self-aggregated via π–π stacking of the porphyrin cores in a J-type fashion similar to the chlorosomes [22]. Since 1aR could not be dissolved in water, the molecule was situated inside the aqueous micelle prepared by Triton X-100 as one of the nonionic detergents. Therefore, chlorosomal self-aggregates of 1aR were produced in the hydrophobic environment of aqueous Triton X-100 micelles. At the red-shifted Soret and Qy regions, apparent CD bands were observed (Fig. 3A, lower, red). An S-shaped CD band was visible at around 400 to 500 nm, and a less intense positive CD band was detected at the red-shifted Qy peak position. The observation is in sharp contrast to no CD band in the monomeric state. The self-aggregates of 1aR were supramolecules with a chiral π–π stacking manner. The supramolecular chirality was produced by the (R)-2-butyl group in the 17-propionate residue far from the core porphyrin π–system in a molecule. The esterifying group affected the production of the supramolecular structures along with the 31-OH, central Zn, and 13-C=O moieties on the molecular y-axis mentioned in the Introduction section.
Enantiomeric isomer 1aS possessing the (S)-2-butyl ester self-aggregated in an aqueous Triton X-100 solution to give red-shifted and broadened Soret and Qy bands being almost identical to those of the self-aggregated 1aR (Fig. 3A, upper, blue and red lines). In the aqueous micellar solution, self-aggregates of 1aS exhibited reverse S-shaped and relatively small negative CD bands at red-shifted Soret and Qy regions, respectively (Fig. 3A, lower, blue). The CD spectrum of 1aS was opposite to that of 1aR, confirming the supramolecular chirality by self-assembling enantiomerically pure 1aR/S in the aqueous micelles.
As shown in the red line in the upper panel of Fig. 3B, self-aggregates of (R)-2-octyl ester 1bR in the aqueous micellar solution showed a similar UV–Vis absorption spectrum to that of 1aR self-aggregates. The red-shifted and broadened Soret and Qy bands indicated that 1bR bearing a hexyl group (X in Fig. 2) at the asymmetric carbon atom self-aggregated similarly as 1aR possessing an ethyl group at the same chiral center. The CD spectral feature of self-aggregated 1bR also resembled that of (1aR)n, but the former intensities were larger by approximately three times than the latter ones (Fig. 3B/A, lower, red lines). The triply elongation of the oligomethylene chain [X = (CH2)2H → (CH2)6H] at the chiral center enhanced the CD intensities. The CD enhancement would be ascribable to an increase in the difference of the size between the alkyl groups (CH3 and X in Fig. 2) on the chiral center, methyl (C1) and ethyl groups (C2) in 1aR and methyl (C1) and hexyl groups (C6) in 1bR. It is noted that a small but apparent dip was observed near 700 nm at the longer wavelength side of the positive CD band in 1bR. As expected, 1bS self-aggregated in the aqueous micellar solution to give the same red-shifted and broadened UV–Vis spectrum as in 1bR and the CD spectrum of a reverse S-shaped band at the Soret region and a negative peak at the Qy region with a small damp at the redmost side which was reverse to that of 1bR.
In the aqueous micellar solution, self-aggregates of (R)-1-phenylethyl ester 1cR exhibited similar UV–Vis and CD spectra as in 1bR (Fig. 3C/B, red). The effect of the former phenyl group at the asymmetric carbon atom on these spectra was nearly identical to that of the latter hexyl group with the same six carbon atoms. Stereochemical inversion of 1cR to 1cS hardly affected the UV–Vis spectra of their self-aggregates, but reversed the CD spectra (Fig. 3C, red to blue). The stereochemistry of the chiral secondary moieties [C*H(CH3)X] as the esterifying group in 1 regulated the supramolecular chirality of their self-aggregates in the aqueous micelles.
4 Conclusion
Zinc 31-demethyl-protobacteriochlorophylls-d possessing enantiomeric esterifying groups were prepared from naturally occurring Chl-a. The synthetic zinc complexes of fully π-conjugated porphyrins self-aggregated inside the hydrophobic environment of an aqueous Triton X-100 micelle, which were comparable to the chlorosomal self-aggregates of BChls-c/d. The supramolecular structures were constructed by the specific bonding of Zn⋯O32–H⋯O=C131 and π–π stacking of composite porphyrin cores [34], exhibiting red-shifted and broadened Soret and Qy bands similarly as in the natural chlorosomal J-aggregates.
The synthetic zinc 3-hydroxymethyl-porphyrins were enantiomers due to the single chirality in the 17-propionate residue. The self-aggregates of all the (R)-enantiomers in the aqueous micelles gave S-shaped bisignate and positive CD bands at the red-shifted and broadened Soret and Qy regions, respectively, while those of the (S)-stereoisomers afforded the reverse CD signs. The relatively intense CD bands based on the exciton-coupling of the porphyrin cores were dependent on the chirality of the substituents remote from the core. The supramolecular structures of the chlorosomal J-aggregates were controlled by the peripheral 17-propionate residue. The observation is comparable to the previous reports that the chiral peripheries of synthetic porphyrins regulated their assemblies [66].
Data availability
All data in this work are available on request from the corresponding author.
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Acknowledgements
We thank Messrs. Akihiro Tsuru, Hajime Sumi, and Jun Komada of Ritsumeikan University for their experimental assistance and Dr. Shin Ogasawara of Ritsumeikan University for preparation of drawings. This work was partially supported by JSPS KAKENHI Grant Numbers 22H02203 in Scientific Research (B) and 17H06436 in Scientific Research on Innovative Areas "Innovation for Light-Energy Conversion (I4LEC)".
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Open Access funding provided by Ritsumeikan University. Japan Society for the Promotion of Science, 22H02203, Hitoshi Tamiaki, 17H06436, Hitoshi Tamiaki.
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Yasui, M., Tamiaki, H. Supramolecular chirality in self-assembly of zinc protobacteriochlorophyll-d analogs possessing enantiomeric esterifying groups. Photochem Photobiol Sci 23, 421–434 (2024). https://doi.org/10.1007/s43630-023-00528-9
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DOI: https://doi.org/10.1007/s43630-023-00528-9