Supplementary Material

In vitro synthesis and characterization of bacteriochlorophyll-f and its absence in bacteriochlorophyll-e producing organisms

Hitoshi Tamiaki *, Jun Komada, Michio Kunieda, Kazuhiro Fukai, Taichi Yoshitomi, Jiro Harada ‡, Tadashi Mizoguchi

Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan

‡Present address: Department of Medical Biochemistry, Kurume University School of Medicine, Fukuoka 830-0011, Japan

Scheme S1. Synthesis of 31-epimerically pure bacteriochlorophyll-f (R[EM]BChl-fF and S[EM]BChl-fF): (i) Zn(OAc)2 / CH2Cl2–MeOH; (ii) RP-HPLC separation, a 10x 250 mm ODS column with 30% aqueous MeCN (2.0 mL/min); (iii) 4% aq. HCl / acetone, farnesol–(Bu2ClSn)2O / toluene, Mg(ClO4)2 / pyridine.

Table S1

RP-HPLC retention times (TR / min)a of R[EM]BChl-xF as well as visible absorption (abs / nm)and fluorescence emission peaks (em / nm, excited at Soret maxima) of BChl-xF.b

Substituentsabs

x720TRcSoretQydeme

cMeMe20.1 (11.4)432661 (290) [0.66]66370

dMeH18.6 (10.9)425650 (310) [0.81]65370

eCHOMe16.6 (10.1)459646 (360) [0.28]64980

fCHOH15.1 (9.5)451634 (410) [0.37]63770

a RP-HPLC conditions: a 4.6x 150 mm ODS column with 15% aqueous MeCN (1.0 mL/min).

b BChl-cF, an epimeric mixture of [EE]-homolog isolated from Chl. tepidum ATCC49652; BChl-dF, epimeric mixtures of [EE]- and [PM]-homologs isolated from Chl. vibrioforme NCIB 8327 d-strain; BChl-eF, epimeric mixtures of [EE]- and [PE]-homologs isolated from Chl. phaeobacteroides 1549; BChl-fF, a 1:1 epimeric mixture of synthetic [EM]-homolog.

c Values in parentheses indicate calculated log [partition coefficient between 1-octanol and aqueous phases] (= ClogP).

d Values in parentheses and brackets indicate full widths (cm-1) at half maxima and relative intensities [= Abs(Qy) / Abs(Soret)], respectively.

e Stokes shift (/ cm-1) = [1/abs(Qy) – 1/em] x 107.

Experimental

General

Visible (Vis) absorption and fluorescence (Flu) spectra were measured with a Hitachi U-3500 and F-4500 spectrophotometer, respectively. Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra were recorded with a JEOL ECA-600 spectrometer.Chemical shifts (s) of 1H and 13C NMR spectra are expressed in parts per million relative to CHCl3 (7.26 ppm) and 13CDCl3 (77.0 ppm), respectively, as internal references. Proton and carbon-13 peaks were assigned by two-dimensional spectra including COSY, NOESY, HMBC and CHSHF. Fast atom bombardment-mass spectra (FAB-MS) were measured on a JEOL GCmate II spectrometer using m-nitrobenzyl alcohol as a matrix.Atmospheric pressure chemical ionization-mass spectra (APCI-MS) were obtained on a Shimadzu LCMS-2010EV instrument. Circular dichroism (CD) spectra were obtained with a Jasco J-720W spectropolarimeter. High performance liquid chromatography (HPLC) was performed on a packed column (Cosmosil, Nacalai Tesque) with a Shimadzu LC-10ADvp pump and SPD-M10Avp photodiode-array detector.Solvents for measurements of optical spectra were purchased from Nacalai Tesque (grade for UV-spectroscopy).

Methyl bacteriopheophorbide-fpossessing 8-ethyl and 12-methyl groups as a 1:1 mixture of 31R- and 31S-epimers was prepared according to reported procedures [Tamiaki, H., Kubo, M. and Oba, T. (2000) Synthesis and self-assembly of zinc methyl bacteriopheophorbide-f and its homolog. Tetrahedron 56, 6245–6257]. All the synthetic procedures were done in the dark.

Spectral data of methyl bacteriopheophorbide-f (R/S[EM]BPhe-dM)

1H NMR (CDCl3, 600 MHz)  = 11.079/11.075 (1H, s, 7-CHO), 10.47/10.46 (1H, s, 5-H), 9.46/9.45 (1H, s, 10-H), 8.51 (1H, s, 20-H), 6.53 (1H, q, J = 7 Hz, 3-CH), 5.22, 5.078/5.076 (each 1H, d, J = 19 Hz, 131-CH2), 4.47 (1H, br-q, J = 6 Hz, 18-H), 4.26 (1H, br-d, J = 10 Hz, 17-H), 3.94–3.84 (2H, m, 8-CH2), 3.633/3.629, 3.61, 3.60 (each 3H, s, 2-, 12-CH3, 172-COOCH3), 2.82 (1H, br-s, 31-OH), 2.74–2.55, 2.36–2.23 (each 2H, m, 17-CH2CH2), 2.154/2.149 (3H, d, J = 7 Hz, 31-CH3), 1.821/1.815 (3H, d, J = 7 Hz, 18-CH3), 1.751/1.747 (3H, t, J= 8 Hz, 81-CH3), 0.37/0.36, -1.68/-1.69 (each 1H, s, NH x 2).

Synthesis of bacteriopheophytin-f(R/S[EM]BPhe-dF)

To a solution of methyl bacteriopheophorbide-f (31R/S =1/1, 11.4 mg, 19.6 mol) in toluene (10 mL) was added farnesol (105 mg, 472 mol) and (Bu2ClSn)2O (4.2 mg, 7.6 mol). The mixture was refluxed for 3 h, cooled to room temperature and purified by silica gel flash column chromatography (Wakogel C-300, Wako, 10% Et2O–CH2Cl2) and normal-phase HPLC (retention time was 35 min, Cosmosil 5SL-II 10 x 250 mm, 10% acetone–1,2-dichloroethane) to give a 31-epimeric mixture of bacteriopheophytin-f (31R/S=1/1) as a black solid [Vis, CD and Flu spectra are shown in Fig. S1]: Vis (CH2Cl2) max = 649 (relative intensity, 0.21), 595 (0.06), 563 (0.05), 526 (0.06), 436 (1.00), 415 (0.38), 374 (0.14), 326 nm (0.15); 1H NMR(CDCl3, 600 MHz, 35°C)  = 10.95/10.94 (1H, s, 7-CHO), 10.35/10.34 (1H, s, 5-H), 9.17/9.16 (1H, s, 10-H), 8.492/8.489 (1H, s, 20-H), 6.49/6.48 (1H, q, J = 7 Hz, 3-CH), 5.25 (1H, m, F1-CH), 5.19/5.18, 5.04/5.03 (each 1H, d, J = 19 Hz, 131-CH2), 5.04, 5.03 (each 1H, m, F5-, F9-CH), 4.57 (2H, m, 172-COOCH2), 4.47/4.46 (1H, q, J = 7 Hz, 18-H), 4.25 (1H, m, 17-H), 3.72–3.60 (2H, m, 8-CH2), 3.500/3.498 (3H, s, 12-CH3), 3.444/3.438 (3H, s, 2-CH3), 2.92 (1H, s, 31-OH), 2.74–2.65, 2.29–2.22 (each 1H, m, 17-CH2), 2.64–2.54, 2.38–2.29 (each 1H, m, 171-CH2), 2.141/2.135 (3H, d, J = 7 Hz, 31-CH3), 2.08–2.02 (2H, m, F4-CH2), 2.02–1.96 (4H, m, F3-, F8-CH2), 1.94–1.89 (2H, m, F7-CH2), 1.830/1.826 (3H, d, J = 7 Hz, 18-CH3), 1.66/1.65 (3H, s, F3-CH3), 1.64/1.63 (3H, t, J= 8 Hz, 81-CH3), 1.62 (3H, s, F11-CH3cis to CF10–H), 1.55 (3H, s, F11-CH3trans to CF10–H), 1.53 (3H, s, F7-CH3), 0.13/0.12, -1.82/-1.84 (each 1H, s, NH x 2); 13C NMR (CDCl3, 150 MHz, 35°C) =195.8 (C131), 187.6 (C71), 173.2 (C19), 172.9 (C173), 163.0 (C16), 158.5 (C8), 150.40/150.38 (C6), 150.0 (C13), 146.6 (C9), 143.1 (C1), 142.80/142.78 (CF3), 142.6 (C3), 137.7 (C11), 135.83/135.79 (C4), 135.4 (CF7), 132.4 (C7), 132.22/132.19 (C2), 131.4(C12), 131.2, 131.1 (C14, CF11), 124.3 (CF10), 123.6 (CF6), 118.0 (CF2), 106.2 (C10), 105.7 (C15), 101.0/100.9 (C5), 92.8 (C20), 65.45/65.41 (C31), 61.6 (CF1), 51.9 (C17), 50.0 (C18), 47.9 (C132), 39.6 (CF8), 39.5 (CF4), 31.28/31.25 (C172), 29.7 (C171), 26.7 (CF9), 26.2 (CF5), 25.64 (C32), 25.60 (CF12), 23.08/23.05 (C181), 19.1 (C82), 18.8 (C81), 17.6 (CF111), 16.4 (CF31), 15.9 (CF71), 12.0 (C121), 11.4 (C21); MS (FAB) found: m/z 770.4. Calcd. for C48H58N4O5: M+, 770.4.

Synthesis of bacteriochlorophyll-f

To a solution of bacteriopheophytin-f in pyridine was added Mg(ClO4)2 under N2. After being stirred for 1 h at 80 °C, the mixture was diluted with CH2Cl2, washed with phosphate buffer (pH = 6.8) and distilled water, dried over anhydrous Na2SO4 and concentrated. The residue was purified by RP-HPLC (Cosmosil 5C18-ARII 10  x 250 mm, 15% H2O-CH3CN, 1 mL/min) to give an epimeric mixture of [EM]BChl-fF(31R/S=1/1): Vis (Et2O) max = 634 (relative intensity, 0.37), 587 (0.09), 451 (1.00), 429 nm (0.56); Flu (Et2O, ex= 451 nm) em= 637 (relative intensity, 1.00), 693nm (0.07).

The above 1:1 epimeric mixture was separated by RP-HPLC (Cosmosil 5C18-ARII4.6  x 150 mm, 15% H2O-CH3CN, 1 mL/min) to give epimerically pure samples. R[EM]BChl-fF: Retention time = 15.1 min; Vis (15% H2O-CH3CN) max = 639 (relative intensity, 0.32), 457 nm (1.00); MS (APCI) found: m/z 793.6. Calcd. for C48H57N4O5Mg: MH+, 793.4. S[EM]BChl-fF: Retention time = 16.0 min; Vis (15% H2O-CH3CN) max = 639 (relative intensity, 0.32), 456 nm (1.00); MS (APCI) found: m/z 793.6. Calcd. for C48H57N4O5Mg: MH+, 793.4.

Fig. S1. Visible absorption (A, black), fluorescence emission (A, red, excited at 436 nm), circular dichroism (B) and uncorrected fluorescence excitation spectra (C, emitted at 651 nm) of R/S[EM]BPhe-fF in dichloromethane.

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