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Photosynth Res (2018) 135:309–317 DOI 10.1007/s11120-017-0413-8

ORIGINAL ARTICLE

Self-aggregation of synthetic zinc methyl 20-substituted 3-hydroxymethyl-pyropheophorbides as models of bacteriochlorophyll-c

Ayaka Wada1 · Hitoshi Tamiaki1 

Received: 5 January 2017 / Accepted: 8 June 2017 / Published online: 22 June 2017 © Springer Science+Business Media B.V. 2017

AbbreviationsBChl BacteriochlorophyllCD Circular dichroismChl Chlorophylldppf 1,1′-Bis(diphenylphosphino)ferroceneFWHM Full width at half maximumTHF Tetrahydrofuran

Introduction

Photosynthetic green bacteria possess main, huge, and extramembranous light-harvesting antennae called chlo-rosomes (Blankenship et  al. 1995; Blankenship and Ma tsuura 2003; Orf and Blankenship 2013). Chlorosomes are unique antenna systems whose core part is constructed of self-aggregates of specific chlorophyll pigments (Holz-warth et  al. 1992; Tamiaki 1996; Ganapathy et  al. 2009; Pšenčík et  al. 2014), while the other antennae are built from the interaction of chlorophylls with peptides (Cogdell et  al. 2012; Fleming et  al. 2012; König and Neugebauer 2012; Croce and van Amerongen 2014; Senge et al. 2014; Dall’Osto et al. 2015). Chlorosomal chlorophylls (Chls) are bacteriochlorophyll(BChl)-c, d, e, and f molecules which are characterized by the presence of a hydroxy group at the 31-position and the absence of the 132-methoxycarbonyl group (Fig. 1, left; Scheer 1991; Smith 1994; Tamiaki et al. 2007; Tsukatani et al. 2013; Ryan and Senge 2015). BChl-c is  often found in natural chlorosomes and its 20-demeth-ylated derivative BChl-d is less available (Maresca et  al. 2004; Scheer 2006; Bryant et  al. 2012; Bryant and Liu 2013). This situation is ascribable to the fact that BChl-c is grown in the culturing environments of green bacteria more than BChl-d. Self-aggregates of BChl-c have more bathochromically shifted absorption bands to harvest

Abstract Zinc 3-hydroxymethyl-131-oxo-chlorins bear-ing a variety of primary alkyl groups at the 20-position were prepared as models of bacteriochlorophyll-c by chem-ical modification of naturally occurring chlorophyll-a. The synthetic chlorophyll-a derivatives self-aggregated in an aqueous Triton X-100 solution to afford large oligomers whose Soret and Qy bands were red-shifted and broadened, compared with the bands of their monomers in tetrahydro-furan. The oligomeric bands are similar to those of bacte-riochlorophyll-c self-aggregates in chlorosomes, the main light-harvesting antennae of photosynthetic green bacteria. The 20-alkylation led to bathochromic shifts of the visible Soret maxima in J-type self-aggregates of the synthetic models, while elongation of the 20-alkyl group decreased the chlorosomal Qy maxima due to an increase in steric hindrance. Considering the light-harvesting and energy-transferring processes in a chlorosome, the 20-methyla-tion in bacteriochlorophyll-c would be more suitable for efficient culturing of green bacteria than the 20-ethyla-tion and propylation as well as the 20-unsubstitution in bacteriochlorophyll-d.

Keywords Bacteriochlorophyll · Chlorosome · Self-aggregation · Photosynthetic green bacterium · Visible absorption spectroscopy

Electronic supplementary material The online version of this article (doi:10.1007/s11120-017-0413-8) contains supplementary material, which is available to authorized users.

* Hitoshi Tamiaki tamiaki@fc.ritsumei.ac.jp

1 Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan

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sunlight and transfer the excited energy to the acceptor pigments in a chlorosomal envelope more efficiently than those of BChl-d (Saga and Tamiaki 2004; Orf and Blan-kenship 2013). The 20-methylation effect is observed in BChl-e and its 20-demethylated BChl-f, and the latter has not yet been found in natural chlorosomes due to the less photosynthetic activity (Tamiaki et al. 2011; Harada et al. 2012; Vogl et al. 2012).

Chlorosomal Chls are composed of various homologs prepared by methylation at the 82- and 121-positions: ethyl, propyl, isobutyl, and neopentyl groups for the 8-substitu-ent, and methyl and ethyl groups for the 12-substituent (Fig.  1, left; Chew et  al. 2007; Mizoguchi et  al. 2015). These methylations affect the visible absorption bands of their chlorosomal self-aggregates (Tamiaki 2005), simi-larly as in the 20-methylation. Elongation of alkyl groups at the 8- and 12-positions is observed in natural pigments: 8-ethyl to 8-propyl and 12-methyl to 12-ethyl. In contrast, the 20-methyl group is available for BChls-c/e, but no other alkyl groups have been found in natural chlorosomal pigments.

Here, we report the synthesis of BChl-c models bear-ing an ethyl or propyl group at the 20-position (Fig.  1, right) and their self-aggregation in an aqueous Tri-ton X-100 micelle using visible absorption and circular dichroism (CD) spectroscopies. We have already reported that zinc methyl 3-hydroxymethyl-pyropheophorbide-a

(Zn-1a) was prepared (Tamiaki et al. 1996) and its self-aggregates in an aqueous micelle solution are good struc-tural and functional models of BChl-d self-aggregates in a chlorosome (Miyatake and Tamiaki 2010). Therefore, we used Zn-1b–f substituted at the 20-position of Zn-1a as models of BChl-c and its analogs further substituted at the 201-position. The visible absorption spectra of the J-type self-aggregates of the present 20-homologs are compared, and the reason why the 20-methylated pig-ment is preferable for the chlorosomal Chls is discussed.

Materials and methods

General

Visible absorption and CD spectra were measured with a Hitachi U-3500 spectrophotometer (Tokyo, Japan) and a Jasco J-720W spectropolarimeter (Hachioji, Japan), respectively.

Triton X-100 was purchased from Nacalai Tesque (Kyoto, Japan) and used as received. For optical spec-troscopy, tetrahydrofuran (THF) and distilled water were purchased from Nacalai Tesque as reagents prepared spe-cially for HPLC.

Fig. 1 Molecular structures of naturally occurring chlorosomal chlorophylls (left) and their synthetic models (right)

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Synthesis of compounds

The synthetic procedures of zinc chlorins Zn-1a–f are described in the electronic supplementary material.

Preparation of samples

An appropriate volume of zinc chlorin Zn-1 in THF was obtained from its stock solution and the solvent was evapo-rated. The residue containing Zn-1 (ca. 40 nmol) was dissolved in a 2.5% (wt/v) THF solution (20  μl) of Triton X-100 and diluted with distilled water (1.98 ml) at room temperature to give its micelle solution of Zn-1 self-aggregates: [Zn-1] = ca. 20 μM, [Triton X-100] = 0.025% (wt/v), and [THF] = 1% (v/v).

An aliquot aqueous 10% (wt/v) solution of Triton X-100 was added to the above Zn-1 self-aggregate solution. The titra-tion afforded monomeric species of Zn-1 in aqueous micelles.

Results and discussion

Synthesis of zinc methyl 20-substituted 3-hydroxymethyl-pyropheophorbides-a Zn-1b–f

Methyl 3-hydroxymethyl-pyropheophorbide-a (1a) pre-pared by modification of chlorophyll-a was brominated

at the 20-position according to the reported procedures (Tamiaki et  al. 2014) to give 20-bromo-chlorin 1g in an 85% yield [step (i) of Scheme  1]. Bromide was treated with methylboronic acid (10  eq.) in the presence of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride (Pd(dppf)Cl2, 0.1  eq.) and cesium carbonate (10 eq.) in THF [step (ii) of Scheme 1] (Sasaki et al. 2007). After refluxing the mixture under nitrogen in the dark for 2.5 h, the starting bromide was consumed and the products were obtained by silica gel column chromatography. Two products were identified by their 1H NMR, visible, and mass spectra: debrominated 1a (minor) and methylated 1b (major). It is noted that no palladium was inserted into the chlorin center. Desired 20-methyl-chlorin 1b was difficult to separate from 20-unsubstituted chlorin 1a using conven-tional chromatographic methods. The mixture was applied for the following zinc metallation. To a dichloromethane solution of the mixture was added a methanol solution of zinc acetate dihydrate and stirred in the dark under nitrogen at room temperature for 1 h [step (iii) of Scheme 1]. After complete disappearance of the above free bases, the corre-sponding zinc complexes were purified by HPLC to give pure Zn-1b (a 27% isolated yield over the two steps) sepa-rated from Zn-1a.

Similar to the above methylation, the palladium-cata-lyzed reaction of 1g with ethylboronic acid gave a mixture

Scheme  1 Synthesis of zinc methyl 20-(un)substituted 3-hydroxy-methyl-pyropheophorbides-a (Zn-1a–f): (i) C5H5NH+Br3

−/CH2Cl2; (ii) RB(OH)2 [R = Me/Et/Pr/CH(CH2)2 for 1b/c/d/f], Pd(dppf)Cl2,

Cs2CO3/THF (reflux); (iii) Zn(OAc)2·2H2O/MeOH, CH2Cl2; (iv) CH2=CHCH2SnBu3, Pd(PPh3)4/THF (reflux); (v) H2, Rh-Al2O3/Me2CO

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of desired 20-ethyl-chlorin 1c (major) and debrominated product 1a (minor) as well as the corresponding 20-vinyl-chlorin 1h (trace). Although the formation mechanism of 1h is unclear at present, dehydrogenation of the ethyl spe-cies would proceed during the coupling reaction (vide infra). After zinc metallation of the mixed products and purification with HPLC, a pure sample of zinc 20-ethyl-chlorin Zn-1c was obtained in a 9% yield over the two steps.

Similar cross-coupling of 1g with propylboronic acid followed by zinc metallation gave a trace amount of zinc 20-propyl-chlorin Zn-1d. The yield was so low that Zn-1d was detected by its mass and visible spec-tra but could not be assigned by its 1H NMR. Moreo-ver, under the present conditions, no dodecylated product was obtained. Introduction of long alkyl chains by Suzuki–Miyaura coupling at the 20-posi-tion of such a chlorin moiety was difficult, probably due to the steric effect. In contrast, Stille coupling of 1g with allyltributylstannane in the presence of tetrakis(triphenylphosphine)palladium(0) (Tamiaki et  al. 2013) was effective for its 20-allylation to give 1e in a 38% isolated yield [step (iv) of Scheme 1]. It is noteworthy that desired 1e was readily separated from a small amount of debrominated 1a as well as unre-acted bromide 1g (39% recovery) by conventional col-umn chromatography on silica gel. The hydrogenation of the 20-allyl group in 1e was performed in a rhodium catalyst on alumina to afford 1d (58%) [step (v)]. Simi-lar catalytic hydrogenation of the 20-vinyl group in 1h prepared by Stille coupling of 1g with tributylvinylstan-nane (Tamiaki et al. 2014) was examined, but no desired 20-ethyl-chlorin 1c was obtained. This is ascribable to the fact that the 20-vinyl group is highly sterically demanded by the neighboring 2- and 18-methyl groups in a molecule and is less reactive toward the hydrogena-tion, which is consistent with the previously reported result (Tamiaki et  al. 2013). 20-Propyl- and allyl-chlo-rins 1d/e were smoothly zinc metallated [step (iii)] to Zn-1d/e in about 90% yields.

Suzuki–Miyaura cross-coupling of 1g with cyclopro-pylboronic acid (vide supra) successfully gave 20-cyclo-propyl-chlorin 1f (46%) after separation from a small amount of 1a with silica gel column chromatography. The relatively good yield can be ascribed to the superior migration of the cyclopropyl group over primary alkyl chains in the coupling reactions. The cyclopropyl group at the 20-position was not transformed to a(n) (iso)propyl moiety by standard hydrogenation. Both 1f and 1a were zinc metallated to nearly quantitatively afford Zn-1f and Zn-1a, respectively.

Self-aggregation of zinc methyl 20-(un)substituted 3-hydroxymethyl-pyropheophorbides-a Zn-1a–d in an aqueous micelle solution

Zinc 31-hydroxy-131-oxo-chlorins possessing methyl Zn-1b, ethyl Zn-1c, and propyl groups Zn-1d as well as a hydrogen atom Zn-1a were readily dissolved in THF to give monomeric species with a THF molecule as an axial ligand. The concentrated THF solution showing sharp vis-ible absorption bands (blue lines of Fig. 2a–d, upper) was diluted with 99-fold water in the presence of a nonionic detergent, Triton X-100, to give J-type self-aggregates with red-shifted and broadened bands (red lines of Fig.  2a–d, upper). Their visible absorption spectra in an aqueous micelle solution were similar to those of chlorosomal BChl self-aggregates (Saga and Tamiaki 2004; Orf and Blanken-ship 2013; Tsukatani et al. 2013).

In THF, 20-unsubstituted Zn-1a exhibited Soret and Qy maxima (λmax) at 424 and 647 nm, respectively, which were shifted to 449 and 741  nm in an aqueous 0.025% (wt/v) Triton X-100 solution (Fig.  2a, upper). The red-shifted value (Δ) of the Qy maximum was 1970 cm−1 (Table 1), to be consistent with the previously reported data (Miyat-ake et al. 1999; Kunieda et al. 2010). Both the bands were broadened by the present J-type aggregation and especially the full width at half maximum (FWHM) of the Qy band increased from 316 to 575 cm−1.

20-Methylated Zn-1b in THF gave 430- and 657-nm peaks, characteristic of its monomeric species (blue line of Fig. 2b, upper). These values are larger than those of Zn-1a (Table 1). The bathochromic shifts were due to steric repul-sion of the 20-methyl group with the neighboring 2- and 18-methyl groups. Distortion of the chlorin π-system by the 20-methylation led to such shifts, and the steric effect has already been reported (Kureishi and Tamiaki 1998; Tami-aki et al. 1998). In aqueous Triton X-100 micelles, Zn-1b self-aggregated to form J-aggregates with red-shifted and broadened absorption bands (red line of Fig.  2b, upper). The red-shifted Qy maximum, λmax(Qy), at 736 nm is com-parable to that of the Zn-1a self-aggregates, while the Soret peak λmax(Soret) at 463 nm moves to a longer wavelength than that of (Zn-1a)n (Table  1). The red-shift values of the Qy and Soret maxima Δ(Qy/Soret) in the Zn-1b self-aggregates are almost identical (1650/1660  cm−1), which are smaller and larger than the corresponding values in the Zn-1a self-aggregates, respectively (Table 1). The less shifts of the Qy maxima can be explained by the fact that the 20-methylation partially disturbs the self-aggregation of zinc chlorin π-systems along the molecular y axis (Fig. 1). Since the Soret band is composed of Bx and By bands, the greater shifts of the Soret maxima can be attributed to much larger supramolecular interaction along the x axis. The FWHM value of the Qy band of monomeric Zn-1b in THF

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Fig. 2 Visible absorption (upper) and CD spectra (lower) of Zn-1a–f (a–f, ca 20 μM) in THF (blue) and an aqueous 0.025% (wt/v) Triton X-100 solution containing 1% (v/v) THF (red)

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(311 cm−1) is almost the same as that of Zn-1a (316 cm−1), but that of the Zn-1b self-aggregates (722 cm−1) is larger by about 30% than that of (Zn-1a)n (575  cm−1, Table  1). This may be also due to partial disturbance of self-aggre-gation along the y axis by the 20-methylation, resulting in the formation of various supramolecules (inhomogeneous self-aggregation).

Insertion of one methylene group at the 20-position of Zn-1b to Zn-1c slightly affected the visible absorp-tion spectra in THF. Substitution of a methyl group with an ethyl group at the 20-position did not alter the chlorin π-system to give almost the same monomeric bands (blue lines of Fig. 2b, c; upper). 20-Ethylated Zn-1c self-aggre-gated in an aqueous Triton X-100 solution to afford supra-molecules with red-shifted and broadened Qy/Soret bands, similarly as in Zn-1a/b. In the spectrum, apparent mono-meric peaks were observed at 667 and 435 nm (red line of Fig. 2c, upper). The appearance of these peaks shows that Zn-1c self-aggregated less than Zn-1a/b under the present conditions and that the 20-ethyl group partially suppressed such J-aggregation. The λmax(Qy) of Zn-1c self-aggregates is situated at 724 nm and blue-shifted from the λmax(Qy) of Zn-1b. As a result, the Δ(Qy) value (1410 cm−1) of Zn-1c is smaller than that of Zn-1b (Table 1), indicating that the sterically bulky 20-ethyl group would disturb self-aggrega-tion along the y axis more than the 20-methyl group.

The visible absorption spectrum of 20-propylated Zn-1d in THF resembles that of Zn-1c (blue lines of Fig.  2c, d; upper). The simple alkyl chains including a methyl group at the 20-position show almost the same effects on the mono-meric absorption bands, because they have the same steric factor for the chlorin moiety in a molecule. In aqueous Tri-ton X-100 micelles, self-aggregates of Zn-1d gave λmax(Qy/Soret) at 725/462 nm, which are almost identical to those of (Zn-1c)n (red lines of Fig. 2c, d; upper). In contrast, a smaller amount of residual monomer was observed in Zn-1d than

Zn-1c and the Qy band of the Zn-1d self-aggregates was sharper than that of the Zn-1c aggregates: FWHM(Qy) = 558 [(Zn-1d)n] < 725 cm−1 [(Zn-1c)n] (Table 1). Both Zn-1d and Zn-1c form similar self-aggregates, but the 20-propyl group stabilizes them more than the 20-ethyl group, and Zn-1d may self-aggregate more homogeneously than Zn-1c.

In THF, all the monomeric species of Zn-1a–d show the same negative and positive CD bands at their Qy and Soret maxima, respectively (blue lines of Fig. 2a–d, lower). Self-aggregates of Zn-1a–d in an aqueous Triton X-100 solution gave a larger CD couplet in the region of the red-shifted and broadened Qy band than their monomeric negative bands, while another CD couplet was observed at around the Soret region of their self-aggregates and the intensity of its positive peak was comparable to that of the monomeric positive band (Fig. 2a–d, lower). The large CD bands in the Qy region mean that the supramolecular structures of the J-type self-aggregates are ordered along the y axes of the composite molecules in an excitonically coupled manner. 20-Unsubstituted and methylated Zn-1a/b exhibit S-shaped bands in the Qy and Soret regions (red lines of Fig. 2a, b; lower). Typically, in the Qy region at around 740 nm, the S-shaped CD spectral features were similar in spite of the differences in their intensities. An additional small dip was observed at the red side of the CD band of Zn-1a, support-ing a small difference of supramolecular structures between the self-aggregates of Zn-1a/b mentioned above. Almost the same CD spectra with reverse S-shaped bands were observed in the self-aggregates of 20-ethylated and pro-pylated Zn-1c/d (red lines of Fig. 2c, d; lower). The narrow-ing Qy absorption band by substitution of an ethyl group with a propyl group at the 20-position as in Zn-1c → Zn-1d led to suppress a small dip at the blue side of the CD bands in the Qy region. This observation also indicates that Zn-1c/d have partially different supramolecular structures in their self-aggregates (vide supra).

Table 1 Absorption maxima λmax of Zn-1a–f in a solution and red-shift values Δ of the maxima by their self-aggregation

a In an aqueous 0.025% (wt/v) Triton X-100 solution containing 1% (v/v) THF. The value after ≈ indicates the position of the absorption shoulderb Δ = [1/λmax(THF) − 1/λmax(aq. micelle)] × 107

Compound λmax/nm (FWHM/cm−1) Δb/cm−1

THF Aq. micellea

Soret Qy Soret Qy Soret Qy

Zn-1a (20-H) 424 647 (316) 449 741 (575) 1310 1970Zn-1b (20-CH3) 430 657 (311) 463 736 (722) 1660 1650Zn-1c (20-CH2CH3) 430 657 (324) ≈460 724 (725) – 1410Zn-1d (20-CH2CH2CH3) 430 657 (327) 462 725 (558) 1590 1420Zn-1e (20-CH2CH = CH2) 429 656 (320) 459 724 (587) 1500 1420Zn-1f [20-CH(CH2)2] 430 658 (322) ≈445 683 – 580

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Self-aggregation of isomeric zinc 20-allyl/cyclopropyl-chlorins Zn-1e/f in aqueous micelles

Dehydrogenation of a propyl group at the 20-position of Zn-1d to the 20-allyl group of Zn-1e hardly affected the visible absorption spectra of their monomeric species in THF (blue lines of Fig. 2d, e; upper). This observation is ascribable to the fact that the oxidation did not change any steric repulsion around the 20-substituents to give almost the same conformer in the chlorin π-system. Self-aggre-gates of Zn-1e in an aqueous Triton X-100 solution show a nearly identical absorption spectrum to that of (Zn-1d)n (red lines of Fig. 2d, e; upper). Therefore, both the propyl and allyl groups interacted similarly with any other moie-ties in a molecule as well as in a supramolecule of the self-aggregates. The resemblance of CD spectral features in the self-aggregates of Zn-1d/e (red lines of Fig. 2d, e; lower) supports the formation of their similar supramolecules.

In THF, 20-cyclopropyl-chlorin Zn-1f gave visible absorption maxima at longer wavelengths by 1–2 nm than those of isomeric 20-propyl-chlorin Zn-1e (Table  1). The small red-shifts mean that the cyclopropyl group is steri-cally larger near the 20-position than the allyl group and more greatly disturbs the chlorin moiety due to steric repul-sion (vide supra). In an aqueous Triton X-100 solution, Zn-1f shows a Qy absorption maximum at 683 nm and the red-shift from the monomeric peak is 580  cm−1 (Fig.  2f, upper). The shift in Zn-1f is less than half of the value in Zn-1e (1420 cm−1). The 20-cyclopropyl group greatly sup-pressed the formation of large self-aggregates observed in the present other zinc chlorins Zn-1a–e. The conformation-ally rigid cyclopropyl group sterically disturbed the π–π stacking of the chlorin moieties during chlorosomal aggre-gation. Comparing reported data (Uehara and Olson 1992; Causgrove et al. 1993; Tamiaki et al. 1994, 2004; Umetsu et al. 2002), this small red-shift indicates that Zn-1f would dimerize in an aqueous micelle. The difference in the CD spectra between the isomers in an aqueous solution (red lines of Fig.  2e, f; lower) supports the large and small J-aggregations of Zn-1e and Zn-1f molecules, respectively.

Disassembly of self-aggregates of synthetic zinc chlorins Zn-1a–f in aqueous micelles

In an aqueous 0.025% (wt/v) Triton X-100 solution, Zn-1a–f self-aggregated to give red-shifted and broadened absorption bands accompanied by a small amount of residual monomeric bands, as mentioned above. Addition of Triton X-100 to the solution decreased the number of aggregated species and concomitantly increased the blue-shifted and sharpened bands ascribable to the monomeric species (see Fig. S1). The titration experiments showed that an increase of the concentration of Triton X-100 resulted

in disassembly of the self-aggregates of Zn-1a–f in an aqueous solution. Large volumes of Triton X-100 were necessary for complete deaggregation, and the amounts were dependent on the self-aggregates: 0.6% (wt/v) (for Zn-1e) ≈ 0.7 (Zn-1f) < 0.9 (Zn-1c) ≈ 1.0 (Zn-1d) < 1.5 (Zn-1a) < 3.3 (Zn-1b). The molar ratio of Triton X-100 to Zn-1 was about 20 in the initial stage of [Triton X-100] = 0.025% (wt/v) = ca. 0.4  mM and [Zn-1] = ca. 20  μM. Therefore, 500- to 3000-fold molar amount of Triton X-100 were required for complete disassembly of the Zn-1 self-aggre-gates in an aqueous solution. Zinc 20-methyl-chlorin Zn-1b was more tolerant to deaggregation than zinc 20-ethyl/pro-pyl-chlorins Zn-1c/d, supporting that the former self-aggre-gated more tightly with π–π stacking and that the 20-sub-stituents of the latter partly disturbed their self-aggregation (vide supra). It is noted that the self-aggregates of 20-meth-ylated Zn-1b disassembled less in an aqueous Triton X-100 solution than those of 20-unsubstituted Zn-1a.

Effect of 20-alkylation on chlorosomal self-aggregation

As mentioned in the “Introduction”, self-aggregates of BChl-c molecules in a chlorosome show a more bathochro-mic Soret band than chlorosomal 20-unsubstituted BChl-d aggregates. The same situation was observed for the 20-methylation in the present models: λmax(Soret) = 449 (Zn-1a) → 463  nm (Zn-1b) in an aqueous Triton X-100 solution. Further methylation and ethylation at the 201-position of Zn-1b to Zn-1c and Zn-1d also gave the Soret bands at around 460  nm in the aqueous micelles, indicating that substitution with primary alkyl groups at the 20-position did not affect the Soret bands of the chlo-rosomal self-aggregates. The bathochromic shifts due to these 20-alkylations are benefcial for efficient absorption of sunlight, because the shifted region is more intense in solar radiation in green bacteria culturing environments (Chen and Scheer 2013; Ruban 2015).

Chlorosomal self-aggregates have red-shifted and broadened Qy bands (Introduction). The 20-methylation of BChl-d to BChl-c moves the Qy band to a longer wave-length in natural chlorosomes, similarly as in the Soret bands. In contrast, the 20-methylation of Zn-1a to Zn-1b slightly blue-shifted the chlorosomal Qy maxima in an aqueous Triton X-100 solution. The 20-ethylation and propylation led to much larger hypsochromic shifts of the chlorosomal Qy bands. These bathochromically shifted and broadened Qy bands are advantageous for singlet excited energy transfer to the BChl-a pigment in baseplates as the initial energy acceptor in a chlorosome whose absorption band is situated at around 800  nm, because Förster-type energy transfer is effective for a large overlap of donor emission and acceptor absorption bands (Orf et al. 2013). The model systems showed that large alkyl groups at the

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20-position suppressed the red-shifted values of the chlo-rosomal Qy maxima. Such a 201-alkylation would be unfavorable for efficient energy transfer in a chlorosome. Based on the above light-harvesting and energy-transfer-ring processes in chlorosomes, the 20-methylation is use-ful for chlorosomal pigments, while the other alkylations including the 20-ethylation by additional methylation at the 201-position are less effective. Therefore, BChl-c is often biosynthesized in green bacteria rather than BChl-d despite the cost of the 20-methylation and the corresponding 20-eth-ylated pigment has never been found in natural systems.

Acknowledgements This work was partially supported by JSPS KAKENHI Grant Number JP24107002 in Scientific Research on Innovative Areas “Artificial Photosynthesis (AnApple).”

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