Synthesis, optical properties and protonation of chlorophyll derivatives appending a pyridyl group in the C3-substituent

Article abstract of DOI:10.1016/j.dyepig.2015.03.006

Chlorophyll(Chl)-a derivatives possessing a 2-(2/3/4-pyridyl)ethenyl group at the C3 position were synthesized by Wittig reaction of the 3-formyl group in methyl pyropheophorbide-d. The catalytic hydrogenation of the resulting 3-CC double bond afforded the corresponding regioisomers of Chls bearing the 3-(2-pyridylethyl) group. Comparing their visible absorption spectra in dichloromethane, the longest wavelength (Qy) maxima were shifted bathochromically in the following order: C-C < CC as the linker between the chlorin and pyridyl moieties. The red shifts are ascribable to their intramolecular π-conjugation through the 3-vinylene moiety. All the Chl derivatives in a diluted solution gave an intense fluorescence emission band at the red-side region of Qy band and their fluorescence quantum yields were about 20%. The synthetic Chl-pyridine conjugates were titrated with trifluoroacetic acid in chloroform. Their visible spectral changes indicated that the appended pyridyl group was first protonated, followed by protonation of the chlorin π-system at the inner nitrogen atoms in a molecule.

Full text of DOI:10.1016/j.dyepig.2015.03.006

Dyes and Pigments 118 (2015) 159e165
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, optical properties and protonation of chlorophyll
derivatives appending a pyridyl group in the C3-substituent
Youhei Yamamoto, Hitoshi Tamiaki*
Department of Pharmacy and Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Chlorophyll(Chl)-a derivatives possessing a 2-(2/3/4-pyridyl)ethenyl group at the C3 position were
synthesized by Wittig reaction of the 3-formyl group in methyl pyropheophorbide-d. The catalytic hy-
drogenation of the resulting 3-C]C double bond afforded the corresponding regioisomers of Chls
bearing the 3-(2-pyridylethyl) group. Comparing their visible absorption spectra in dichloromethane, the
longest wavelength (Qy) maxima were shifted bathochromically in the following order: CeC < C]C as
the linker between the chlorin and pyridyl moieties. The red shifts are ascribable to their intramolecular
Received 5 January 2015
Received in revised form
27 February 2015
Accepted 5 March 2015
Available online 14 March 2015
p
-conjugation through the 3-vinylene moiety. All the Chl derivatives in a diluted solution gave an intense
Keywords:
Hydrogenation
Protonation
Substitution effect
Visible absorption spectra
Wittig reaction
fluorescence emission band at the red-side region of Qy band and their fluorescence quantum yields
were about 20%. The synthetic Chlepyridine conjugates were titrated with trifluoroacetic acid in chlo-
roform. Their visible spectral changes indicated that the appended pyridyl group was first protonated,
followed by protonation of the chlorin
p
-system at the inner nitrogen atoms in a molecule.
© 2015 Elsevier Ltd. All rights reserved.
p-Conjugation
1. Introduction
Wittig reaction of methyl pyropheophorbide-d bearing the 3-
formyl group, and the catalytic hydrogenation of the resulting 3-
ethenyl group to afford the corresponding regioisomers of
Chlepyridine conjugates linked with the 3-ethylene spacer (Fig. 1).
We discuss their optical properties in a solution and also the pro-
tonation of the 32-pyridyl group and chlorin ring by titration of
trifluoroacetic acid (TFA).
Chlorophylls (Chls) are known to play important roles in light
absorption and energy/electron transfer in natural photosynthesis
[1]. Due to their excellent photofunctional abilities, various Chl
derivatives have been prepared and their photophysical properties
have been reported [2]. For about two decades, our group has
focused on the development of Chl derivatives substituted with
several kinds of functional groups at any peripheral positions
including the C3, C7, C8, C13, C17 and C20 positions (see recent
investigation in Ref. [3] and cited therein as well as Ref. [4]). We
have already succeeded in the synthesis of Chl derivatives bearing
various substituents at the C3 position by modification of the 3-
vinyl group in methyl pyropheophorbide-a [5]. Among them, a
Chl derivative possessing an ethynyl group at the C3 position has
been used as a precursor for the preparation of novel Chls bearing
2. Experimental
2.1. General
Visible absorption and fluorescence emission spectra were
measured on Hitachi U-3500 and F-4500 spectrometers, respec-
tively. Fluorescence quantum yields were measured at room tem-
perature using a Hamamatsu Photonics absolute PL quantum yield
measurement system C9920-03G. All melting points were
measured with a Yanagimoto micro melting apparatus and were
uncorrected. 1H NMR spectra including COSY and NOESY were
recorded on a JEOL JNM-AL-400 spectrometer in CDCl3. Their
p-extended moieties by replacement of the terminal ethynyl
hydrogen atom to aromatics (Sonogashira coupling) and alkynes
(Glaser reaction) [3,6]. Recently, we reported the synthesis and
photophysical properties of Chle(oligo)pyridine conjugates with
the 3-ethynylene linker [3]. Here, we report the synthesis of Chls
possessing a 2-(2/3/4-pyridyl)ethenyl group at the C3 position by
chemical shifts (
peak:
with a Shimadzu IRAffinity-1 spectrophotometer. High resolution
mass spectra (HRMS) were recorded on a Bruker micrOTOF II
spectrometer: atmospheric pressure chemical ionization (APCI)
ds) are reported relative to the residual solvent
¼ 7.26 ppm (CHCl3). FTIR spectra in CH2Cl2 were measured
d
* Corresponding author. Fax: þ81 77 561 2890.
E-mail address: tamiaki@fc.ritsumei.ac.jp (H. Tamiaki).
http://dx.doi.org/10.1016/j.dyepig.2015.03.006
0143-7208/© 2015 Elsevier Ltd. All rights reserved.

160
Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
Fig. 1. Chlorophyll derivatives possessing (or lacking) a 2/3/4-pyridyl or phenyl group at the C32 position.
and positive mode in a methanol solution. Flash column chroma-
tography (FCC) was performed with silica gel (Merck, Kieselgel 60).
Methyl pyropheophorbide-a (H]Chl) [4a,5a], methyl pyro-
4Py]Chl or H]Chl (1 ml) and the mixed solution was diluted with
chloroform until the final volume was 5 ml ([Chl] ¼ 4.0 ꢀ 10ꢁ6 M).
After stirring at room temperature, the solution was analysed by
visible absorption spectroscopy. The association constants were
determined by reported procedures [7] using changes of the
absorbance of 2Py]Chl, 3Py]Chl, 4Py]Chl, and H]Chl at 677/
700, 673/700, and 680/700 nm, respectively. H]Chl: Vis (CHCl3)
lmax 668 (ε, 44,000), 609 (8000), 539 (9000), 507 (10,000), 414 nm
(101,000) and the corresponding spectral data of 2/3/4Py]Chls
were described in the following sections.
pheophorbide-d
[4a,5a],
methyl mesopyropheophorbide-a
(HeChl) [4a,5a], methyl trans-32-phenyl-pyropheophorbide-a
(Ph]Chl) [5b], methyl 3-(2/3/4-pyridylethynyl)-pyropheo-
phorbides-a (2/3/4Py≡Chls) [3], methyl 3-devinyl-3-ethynyl-
pyropheophorbide-a (H≡Chl) [6a], and methyl 3-devinyl-3-
phenylethynyl- pyropheophorbide-a (Ph≡Chl) [6a], were pre-
pared according to reported procedures. Commercially available
THF (Nacalai Tesque) was passed through an alumina column
before use. The other solvents and reagents were used as purchased
without further purification. Solvents for optical spectroscopy were
purchased from Nacalai Tesque as reagents prepared specially for
spectroscopy and used without further purification.
2.3. Wittig reaction
A DMF solution (50 ml) of 2/3/4-(chloromethyl)pyridine hy-
drochloride (1.5 g, 9.2 mmol) and triphenylphosphine (2.4 g,
9.2 mmol) was refluxed for 1 h under N2, and cooled down to room
temperature. The resulting precipitates were filtered and recrys-
tallized from EtOH and toluene to give the corresponding hydro-
chloride salt of 2/3/4-pyridylmethyl(triphenyl)phosphonium
chloride (2.8 g, 6.5 mmol) [8] as a Wittig pre-reagent.
2.2. Protonation experiments
A chloroform solution (2 ꢀ 10ꢁ3 to 2 M) of TFA (an appropriate
volume) was added to a chloroform solution (2 ꢀ 10ꢁ5 M) of 2/3/

Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
161
Methyl pyropheophorbide-d (60 mg, 109
m
mol) and the above
s, NH
C
ꢀ
2); HRMS (APCI) found: m/z 626.3137, calcd. for
Wittig pre-reagent (94 mg, 220 mol) were dissolved in CH2Cl2
m
39H40N5O3: MHþ, 626.3125. See also its spectral data in Ref. [9].
(7 ml), to which an aqueous solution (10 ml) of NaOH (15 mg) was
added with stirring. The mixture was stirred in the dark under N2 at
room temperature and a decrease of methyl pyropheophorbide-
d was monitored by visible spectroscopy: change of 694- to 680-nm
peak. After the disappearance of the former peak was stopped, the
reaction mixture was poured into water (80 ml) and CH2Cl2 (80 ml).
The aqueous phase was extracted with several portions of CH2Cl2
and the combined CH2Cl2 phases were washed with aq. 4% NaHCO3
and water, dried over Na2SO4, and evaporated in vacuo to dryness.
The residue was purified by FCC with 1%(v/v) MeOH and CH2Cl2.
The eluate was evaporated in vacuo and recrystallized from CH2Cl2
and hexane to give 2/3/4Py]Chl as a black solid.
2.4. Catalytic hydrogenation
2/3/4Py]Chl or Ph]Chl (20 mg, 32 mmol) was dissolved in a
10:1 mixture of acetone and EtOH (15 ml) and treated with PtO2
(30 mg,130 mol) under H2 atmosphere. The mixture was stirred in
m
the dark at room temperature and the Qy band of the starting olefin
was monitored by visible spectra. After the hypsochromic shift of
the peak was stopped, the reaction mixture was filtered over Celite-
500 and the filtrate was evaporated in vacuo. The residue was pu-
rified by FCC with 2%(v/v) MeOH and CH2Cl2. The eluate was
evaporated in vacuo and the residue was recrystallized from CH2Cl2
and hexane to give 2/3/4PyeChl or PheChl as a black solid.
2.3.1. Methyl trans-32-(2-pyridyl)pyropheophorbide-a (2Py]Chl)
Yield: 67%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 677 (relative in-
tensity, 0.47), 618 (0.09), 544 (0.10), 514 (0.11), 422 nm (1.00); VIS
(CHCl3) lmax 678 (ε, 52,000), 620 (10,000), 548 (11,000), 518
(12,000), 424 nm (110,000); IR (CH2Cl2) vmax 2963, 2927, 2855,1735
2.4.1. Methyl 32-(2-pyridyl)mesopyropheophorbide-a (2PyeChl)
Yield: 50%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 658 (relative in-
tensity, 0.50), 602 (0.11), 535 (0.11), 504 (0.11), 410 nm (1.00); IR
(CH2Cl2) vmax 2955, 2934, 2871, 1734 (ester-C]O), 1686 (keto-C]
(ester-C]O), 1693 (keto-C]O), 1621, 1097 cmꢁ1
;
1H NMR (CDCl3)
d
9.47 (1H, s, 10-H), 9.40 (1H, s, 5-H), 8.87 (1H, d, J ¼ 16 Hz, 3-CH),
O),1607, 1100 cmꢁ1; 1H NMR (CDCl3)
d 9.47 (1H, s, 10-H), 9.12 (1H, s,
8.83 (1H, d, J ¼ 5 Hz, 6-H of Py), 8.59 (1H, s, 20-H), 7.83 (1H, t,
J ¼ 8 Hz, 4-H of Py), 7.57 (1H, d, J ¼ 8 Hz, 3-H of Py), 7.56 (1H, d,
J ¼ 16 Hz, 31-CH), 7.34 (1H, dd, J ¼ 8, 5 Hz, 5-H of Py), 5.26, 5.09
(each 1H, d, J ¼ 20 Hz, 131-CH2), 4.45 (1H, dq, J ¼ 2, 7 Hz, 18-H), 4.29
(1H, dt, J ¼ 9, 2 Hz, 17-H), 3.66 (2H, q, J ¼ 8 Hz, 8-CH2), 3.66 (3H, s,
12-CH3), 3.62 (3H, s,172-COOCH3), 3.45 (3H, s, 2-CH3), 3.22 (3H, s, 7-
CH3), 2.70, 2.58 (each 1H, m, 17-CH2), 2.30 (2H, m, 171-CH2), 1.83
(3H, d, J ¼ 7 Hz, 18-CH3), 1.69 (3H, t, J ¼ 8 Hz, 81-CH3), 0.38, ꢁ1.74
(each 1H, s, NH ꢀ 2); HRMS (APCI) found: m/z 626.3112, calcd for
5-H), 8.73 (1H, d, J ¼ 4 Hz, 6-H of Py), 8.41 (1H, s, 20-H), 7.55 (1H, t,
J ¼ 8 Hz, 4-H of Py), 7.10 (1H, d, J ¼ 8, 4 Hz, 5-H of Py), 6.77 (1H, d,
J ¼ 8 Hz, 3-H of Py), 5.24, 5.09 (each 1H, d, J ¼ 20 Hz, 131-CH2), 4.45
(1H, dq, J ¼ 2, 7 Hz, 18-H), 4.36 (2H, t, J ¼ 7 Hz, 3-CH2), 4.26 (1H, dt,
J ¼ 9, 2 Hz, 17-H), 3.72 (2H, q, J ¼ 8 Hz, 8-CH2), 3.68 (3H, s, 12-CH3),
3.62 (3H, s, 172-COOCH3), 3.47 (2H, t, J ¼ 7 Hz, 31-CH2), 3.22 (3H, s,
2-CH3), 3.02 (3H, s, 7-CH3), 2.70, 2.56 (each 1H, m, 17-CH2), 2.30
(2H, m, 171-CH2), 1.83 (3H, d, J ¼ 8 Hz, 18-CH3), 1.70 (3H, t, J ¼ 8 Hz,
81-CH3), 0.89, ꢁ1.66 (each 1H, s, NH ꢀ 2); HRMS (APCI) found: m/z
628.3275, calcd. for C39H42N5O3: MHþ, 628.3282.
C
39H40N5O3: MHþ, 626.3125.
2.3.2. Methyl trans-32-(3-pyridyl)pyropheophorbide-a (3Py]Chl)
Yield: 56%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 674 (relative in-
tensity, 0.44), 618 (0.09), 543 (0.10), 513 (0.11), 420 nm (1.00); VIS
(CHCl3) lmax 677 (ε, 49,000), 620 (10,000), 547 (11,000), 517
(12,000), 423 nm (108,000); IR (CH2Cl2) vmax 2968, 2929, 2872,
2.4.2. Methyl 32-(3-pyridyl)mesopyropheophorbide-a (3PyeChl)
Yield: 55%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 659 (relative in-
tensity, 0.48), 603 (0.09), 535 (0.09), 504 (0.10), 410 nm (1.00); IR
(CH2Cl2) vmax 2967, 2931, 2872, 1732 (ester-C]O), 1693 (keto-C]
1735 (ester-C]O), 1693 (keto-C]O), 1621, 1120 cmꢁ1
;
1H NMR
9.40 (1H, s, 10-H), 9.28 (1H, s, 5-H), 8.98 (1H, s, 2-H of Py),
O),1626, 1122 cmꢁ1; 1H NMR (CDCl3)
d 9.51 (1H, s, 10-H), 9.11 (1H, s,
(CDCl3)
d
5-H), 8.56 (1H, s, 2-H of Py), 8.46 (1H, d, J ¼ 5 Hz, 6-H of Py), 8.42
(1H, s, 20-H), 7.54 (1H, d, J ¼ 7 Hz, 4-H of Py), 7.06 (1H, dd, J ¼ 7,
5 Hz, 5-H of Py), 5.25, 5.10 (each 1H, d, J ¼ 19 Hz, 131-CH2), 4.45 (1H,
dq, J ¼ 2, 8 Hz, 18-H), 4.36 (2H, t, J ¼ 7 Hz, 3-CH2), 4.12 (1H, dt, J ¼ 9,
2 Hz, 17-H), 3.72 (2H, q, J ¼ 8 Hz, 8-CH2), 3.67 (3H, s, 12-CH3), 3.62
(3H, s, 172-COOCH3), 3.47 (2H, t, J ¼ 7 Hz, 31-CH2), 3.22 (3H, s, 2-
CH3), 3.00 (3H, s, 7-CH3), 2.70, 2.56 (each 1H, m, 17-CH2), 2.30
(2H, m, 171-CH2), 1.80 (3H, d, J ¼ 8 Hz, 18-CH3), 1.71 (3H, t, J ¼ 8 Hz,
81-CH3), 0.89, ꢁ1.64 (each 1H, s, NH ꢀ 2); HRMS (APCI) found: m/z
628.3274, calcd. for C39H42N5O3: MHþ, 628.3282.
8.66 (1H, d, J ¼ 5 Hz, 6-H of Py), 8.58 (1H, s, 20-H), 8.25 (1H, d,
J ¼ 17 Hz, 3-CH), 8.07 (1H, d, J ¼ 8 Hz, 4-H of Py), 7.47 (1H, dd, J ¼ 8,
5 Hz, 5-H of Py), 7.44 (1H, d, J ¼ 17 Hz, 31-CH), 5.26, 5.11 (each 1H, d,
J ¼ 20 Hz, 131-CH2), 4.50 (1H, dq, J ¼ 2, 7 Hz, 18-H), 4.29 (1H, dt,
J ¼ 9, 2 Hz, 17-H), 3.66 (2H, q, J ¼ 8 Hz, 8-CH2), 3.65 (3H, s, 12-CH3),
3.63 (3H, s, 172-COOCH3), 3.41 (3H, s, 2-CH3), 3.20 (3H, s, 7-CH3),
2.68, 2.58 (each 1H, m, 17-CH2), 2.30 (2H, m, 171-CH2), 1.83 (3H, d,
J ¼ 7 Hz, 18-CH3), 1.70 (3H, t, J ¼ 8 Hz, 81-CH3), 0.37, ꢁ1.76 (each 1H,
s, NH
C
ꢀ
2); HRMS (APCI) found: m/z 626.3109, calcd for
39H40N5O3: MHþ, 626.3125.
2.3.3. Methyl trans-32-(4-pyridyl)pyropheophorbide-a (4Py]Chl)
Yield: 60%: mp > 300 ꢂC; VIS (CH2Cl2) lmax 678 (relative in-
tensity, 0.50), 618 (0.09), 545 (0.11), 514 (0.12), 421 nm (1.00); VIS
(CHCl3) lmax 680 (ε, 52,000), 621 (9000), 548 (9000), 518 (11,000),
424 nm (103,000); IR (CH2Cl2) vmax 2967, 2927, 2872, 1734 (ester-
2.4.3. Methyl 32-(4-pyridyl)mesopyropheophorbide-a (4PyeChl)
Yield: 50%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 659 (relative in-
tensity, 0.50), 603 (0.10), 535 (0.10), 503 (0.11), 410 nm (1.00); IR
(CH2Cl2) vmax 2958, 2928, 2857, 1734 (ester-C]O), 1696 (keto-C]
O),1625,1096 cmꢁ1; 1H NMR (CDCl3)
d 9.52 (1H, s,10-H), 9.11 (1H, s,
C]O), 1695 (keto-C]O), 1619, 1089 cmꢁ1
;
1H NMR (CDCl3)
d
9.49
5-H), 8.45 (1H, d, J ¼ 6 Hz, 2,6-H of Py), 8.42 (1H, s, 20-H), 7.11 (1H,
d, J ¼ 6 Hz, 3,5-H of Py), 5.26, 5.11 (each 1H, d, J ¼ 20 Hz, 131-CH2),
4.45 (1H, dq, J ¼ 2, 8 Hz, 18-H), 4.28 (1H, dt, J ¼ 9, 2 Hz, 17-H), 4.14
(2H, t, J ¼ 7 Hz, 3-CH2), 3.71 (2H, q, J ¼ 8 Hz, 8-CH2), 3.68 (3H, s, 12-
CH3), 3.61 (3H, s, 172-COOCH3), 3.47 (2H, t, J ¼ 7 Hz, 31-CH2), 3.22
(3H, s, 2-CH3), 3.03 (3H, s, 7-CH3), 2.70, 2.56 (each 1H, m, 17-CH2),
2.30 (2H, m, 171-CH2), 1.80 (3H, d, J ¼ 8 Hz, 18-CH3), 1.71 (3H, t,
J ¼ 8 Hz, 81-CH3), 0.87, ꢁ1.66 (each 1H, s, NH ꢀ 2); HRMS (APCI)
found: m/z 628.3271, calcd. for C39H42N5O3: MHþ, 628.3282.
(1H, s, 10-H), 9.33 (1H, s, 5-H), 8.77 (2H, d, J ¼ 6 Hz, 2,6-H of Py),
8.61 (1H, s, 20-H), 8.46 (1H, d, J ¼ 16 Hz, 3-CH), 7.64 (2H, d, J ¼ 6 Hz,
3,5-H of Py), 7.45 (1H, d, J ¼ 16 Hz, 31-CH), 5.27, 5.12 (each 1H, d,
J ¼ 20 Hz, 131-CH2), 4.50 (1H, dq, J ¼ 2, 7 Hz, 18-H), 4.30 (1H, dt,
J ¼ 9, 2 Hz, 17-H), 3.67 (2H, q, J ¼ 8 Hz, 8-CH2), 3.64 (3H, s, 12-CH3),
3.63 (3H, s, 172-COOCH3), 3.51 (3H, s, 2-CH3), 3.26 (3H, s, 7-CH3),
2.71, 2.59 (each 1H, m, 17-CH2), 2.30 (2H, m, 171-CH2), 1.83 (3H, d,
J ¼ 7 Hz, 18-CH3), 1.69 (3H, t, J ¼ 8 Hz, 81-CH3), 0.37, ꢁ1.76 (each 1H,

162
Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
2.4.4. Methyl 32-phenyl-mesopyropheophorbide-a (PheChl)
Yield: 65%; mp > 300 ꢂC; VIS (CH2Cl2) lmax 657 (relative in-
tensity, 0.42), 601 (0.09), 535 (0.10), 504 (0.11), 410 nm (1.00); IR
(CH2Cl2) vmax 2967, 2930, 2869, 1734 (ester-C]O), 1690 (keto-C]
50%, respectively. Similar catalytic hydrogenation of Ph]Chl gave
PheChl in 65% yield. The comparable chemical yields show that the
coordinatable pyridyl nitrogen atoms did not disturb the hydro-
genation. It is noted that 10% Pd/C was not useful for the present
hydrogenation of trans-olefins due to its lower activity, while it was
effective for the hydrogenation of the less sterically hindered 3-
vinyl group of H]Chl [4a]. According to reported procedures
[3,6], 2Py/3Py/4Py/Ph≡Chls were prepared by Sonogashira
coupling of H≡Chl with iodo-pyridines or bromo-benzene.
All the synthetic compounds were fully identified by their 1H
NMR, MS and visible absorption spectra and their following optical
properties and protonation were analysed just after purification by
FCC and successive recrystallization.
O), 1624, 1223 cmꢁ1; 1H NMR (CDCl3)
d 9.50 (1H, s, 10-H), 9.13 (1H,
s, 5-H), 8.44 (1H, s, 20-H), 7.25e7.21 (5H, m, Ph), 5.26, 5.10 (each 1H,
d, J ¼ 20 Hz, 131-CH2), 4.46 (1H, dq, J ¼ 2, 8 Hz, 18-H), 4.28 (1H, dt,
J ¼ 9, 2 Hz, 17-H), 4.06 (2H, t, J ¼ 7 Hz, 3-CH2), 3.69 (2H, q, J ¼ 8 Hz,
8-CH2), 3.67 (3H, s, 12-CH3), 3.63 (3H, s, 172-COOCH3), 3.42 (2H, t,
J ¼ 7 Hz, 31-CH2), 3.21 (3H, s, 2-CH3), 3.03 (3H, s, 7-CH3), 2.67, 2.59
(each 1H, m, 17-CH2), 2.30 (2H, m, 171-CH2), 1.81 (3H, d, J ¼ 8 Hz, 18-
CH3), 1.68 (3H, t, J ¼ 8 Hz, 81-CH3), 0.08, ꢁ1.65 (each 1H, s, NH ꢀ 2);
HRMS (APCI) found: m/z 627.3303, calcd. for C40H43N4O3: MHþ,
627.3330.
3.2. Optical properties of pyridyl-appending Chls
3. Results and discussion
Fig. 2 shows visible absorption spectra of Chls appending a C]C
moiety at the C3 position (2Py]Chl, 3Py]Chl, 4Py]Chl, Ph]Chl,
and H]Chl) and Table 1 summarizes their visible absorption and
fluorescence emission properties in dichloromethane. The 32-(un)-
substituted 3-ethenyl-Chls gave intense Soret and Qy(0,0) absorp-
tion bands at 414e422 and 672e686 nm, respectively, in a diluted
solution. All the absorption maxima, labs, in Soret, Qx(0,1), Qx(0,0),
Qy(0,1), and Qy(0,0) bands of four trans-olefins were shifted to
longer wavelengths than those of the corresponding 32-unsub-
3.1. Synthesis of pyridyl-appending Chls
The synthetic routes of 2/3/4Py]Chls are shown in Scheme 1.
Methyl pyropheophorbide-a (H]Chl) was prepared by modifying
natural Chl-a and the 3-vinyl group was oxidized to the 3-formyl
group in methyl pyropheophorbide-d according to reported pro-
cedures [4a,5a]. Methyl pyropheophorbide-d in dichloromethane
reacted with hydrochloride salts of 2/3/4-pyridylmethyl(triphenyl)-
phosphonium chloride in the presence of an aqueous NaOH solution
at room temperature for a few hours [5b] to give the corresponding
trans-olefins in 67% (2Py]Chl), 66% (3Py]Chl) and 60% yields
(4Py]Chl), respectively, after purification by FCC and recrystalli-
zation. It is noted that their cis-isomers could not be detected in the
1H NMR spectra of the reaction mixture.
stituted Chl (H]Chl), indicating that the 32-substituents were
p-
conjugated with chlorin moiety through the 3-C]C double bond.
The five labs values in 2/3/4Py]Chls were larger than those of Ph]
Chl. The bathochromic shifts by replacement of a CH moiety in the
32-phenyl group with a nitrogen atom in 2/3/4-pyridyl groups
would be ascribable to the electron-withdrawing effect of the N-
atom. Furthermore, the Qy(0,0) peaks of the pyridyl-appending
Chls were red-shifted in the following order: 3Py]Chl < 2Py]
Chl < 4Py]Chl. The observed shift is comparable to the previously
Hydrogenation of the 3-C]C double bonds in 2/3/4Py]Chls
was performed in the presence of PtO2 in a mixed solvent
(acetone:EtOH ¼ 10:1) under H2 atmosphere (Scheme 1). The iso-
lated yields of 2PyeChl, 3PyeChl, and 4PyeChl were 50%, 55%, and
reported
result
in
red
shifts
of
Qy(0,0)
peaks,
Scheme 1. Synthesis of 2/3/4Py]Chls by Wittig reaction and 2/3/4PyeChls by their hydrogenation as well as reported 2/3/4Py≡Chls by Sonogashira coupling: (i) NaIO4, OsO4, in
i
THFeaq. AcOH; (ii) in CH2Cl2eaq. NaOH; (iii) H2, PtO2, in Me2CO:EtOH (10:1); (iv) (MeO)2P(O)C(COMe)N2, in THF; (v) Pd2(dba)3, in Pr2NH/THF, reflux; (vi) H2, Pd/C, in Me2CO.

Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
163
Fig. 2. Visible absorption spectra of 2Py]Chl (red), 3Py]Chl (blue), 4Py]Chl (black solid), Ph]Chl (green), and H]Chl (black broken) in CH2Cl2 at room temperature:
normalized at the Qy maxima. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3Py≡Chl < 2Py≡Chl ] 4Py≡Chl [3]. The regioselective control
would be due to the resonance effect of the N-atom.
4Py≡Chl. The substitution effect was ascribable to the p-conjugation
of the chlorin skeleton with the 32-substituents through the 3-C^C
triple bond as the linker. The red-shift values were smaller than those
in the trans-olefins, indicating that the intramolecular interaction
between the chlorin and 32-substituent moieties through the 3-C^C
linker should be weaker than that via the 3-C]C spacer. The Stokes
shifts of ChleC^CeR were 2e5 nm and smaller than those of the
corresponding olefins. The suppression would be ascribable to the
former conformational rigidity of the 3-substituents over the latter.
Upon the excitation with the Soret maxima in a highly diluted
solution, all the conjugates emitted fluorescence light. These
emission peaks lem were shifted to longer wavelengths in the order
of H]Chl < Ph]Chl < 3Py]Chl < 2Py]Chl < 4Py]Chl, which is
consistent with that of the Qy(0,0) peak positions. Their Stokes
shifts were similar, 5e8 nm. The fluorescence quantum yields Fem
of 32-substituted Chls were determined to be about 25%, which was
slightly larger than that of 32-unsubstituted Chl. No apparent
substitution effect was observed in Fem of the trans-olefins.
Absorption and emission peaks of pyridyl- and phenyl-
appending Chls connected by the 3-CeC single bond were ob-
tained at blue-shifted regions, compared with those of Py]Chls
and Ph]Chl. Although the Qy maxima were slightly shifted in the
order of HeChl < PheChl < 2PyeChl < 3PyeChl ¼ 4PyeChl, all the
other labs values (Soret and Qx) were almost the same within 1 nm.
3.3. Protonation of pyridyl-appending Chls
Fig. 3a and b shows visible absorption spectral change of 4Py]
Chl by addition of TFA in chloroform. The analysis was performed
in chloroform instead of volatile dichloromethane in order to
prevent the increase of the concentration changes throughout
each measurement. In the initial stage of the titration (within
1000 eq of TFA), the absorption spectra quickly changed with a
small amount of TFA and both Qy and Qx peaks moved to longer
wavelengths (see Fig. 3a). Especially, the Qy(0,0) maxima were
red-shifted from 680 to 699 nm. With the addition of ꢃ2000
equivalents of TFA, the spectral change was observed but was less
sensitive to the additional TFA volume than the initial change (see
Fig. 3b). The Qy band was gradually blue-shifted from 699 to
669 nm.
On the other hand, the Qy(0,0) peak of H]Chl in chloroform
was observed at 668 nm and not shifted even after addition of 1000
equivalents of TFA. Much greater addition of TFA (2000e50,000 eq)
blue-shifted the maximum to 660 nm. The blue shift of Qy bands
and the red shift of Qx bands were similar to those in 4Py]Chl
(Fig. 3c vs Fig. 3b). These spectral changes were consistent with the
reported results that chlorin free bases were protonated at the in-
ner N-atom to shift their Qy and Qx bands hypsochromically and
bathochromically, respectively [10]. Therefore, under the present
titration conditions, the 4-pyridyl group of 4Py]Chl was first
protonated, followed by the additional protonation of the chlorin
moiety. The initial protonation at the peripheral substituent was
supported by the fact that the resulting cationic species showed a
similar visible spectrum as that of N-methylated 4Py]Chl [9a].
The proton association constant (Ka) of the N-atom in pyridyl
group of 4Py]Chl was determined by visible spectral analysis and
the pKa value was estimated to be 3.8. From similar spectral change
of 2/3Py]Chls by proton titration (see Fig. S1), their pKa were
evaluated to be 3.0 (2Py]Chl) and 3.2 (3Py]Chl). These pKa
values showed that the pyridyl group in the 3-substituent was
protonated more readily in the order of 4Py]Chl > 3Py]
Chl > 2Py]Chl. The regioisomeric control could be explained by
the combination of steric factor (4-, 3-pyridyl > 2-pyridyl) with
resonance electronic effect (4-, 2-pyridyl > 3-pyridyl) which was
The small or no changes are ascribable to no direct p-conjugation
between the chlorin ring and pyridyl moiety via the 3-CeC single
bond. Their Stokes shifts were 2e3 nm and smaller than those of
the above olefins. The enhancement as in the CeC to C]C linkers is
due to the flexibility of extended
p-conjugates in the latter through
the C3eC31 single bond.
Similarly as in Chls possessing the 3-C]CeR moiety, the Qy(0,0)
maxima of Chls bearing the 3-C^CeR group were shifted to a longer
wavelength in the order of H≡Chl < Ph≡Chl < 3Py≡Chl < 2Py≡Chl ¼
Table 1
Visible absorption maxima labs and fluorescence emission data of Chls in aerated
CH2Cl2 at room temperature.
Chls
labs/nm
Emissiona
Soret
Qx(0,1)
Qx(0,0)
Qy(0,1)
Qy(0,0)
lem/nm Fem
2PyeChl
3PyeChl
4PyeChl
HeChlb
410
410
410
410
410
422
420
421
414
418
504
503
503
504
504
514
513
515
508
511
515
514
516
511
513
535
535
535
534
535
544
543
545
539
542
545
543
544
540
542
602
603
603
600
601
618
618
618
610
614
625
622
622
616
620
658
659
659
656
657
677
674
678
667
672
682
680
682
675
679
660
661
661
658
660
684
682
686
672
678
687
684
685
677
681
0.17
0.16
0.16
0.20
0.17
0.25
0.24
0.25
0.21
0.25
0.24
0.25
0.23
0.22
0.26
PheChl
2Py]Chl
3Py]Chl
4Py]Chl
H]Chlb
Ph]Chl
2Py≡Chlc 418
3Py≡Chlc 418
4Py≡Chlc 419
H≡Chlb
415
419
Ph≡Chlb
a
Fluorescence emission maxima (lem) and quantum yields (Fem) excited at Soret
maxima.
b
c
Data from ref. [6b].
Data from ref. [3].

164
Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
4. Conclusion
We successfully synthesized methyl 3-devinyl-pyropheo-
phorbide-a linked with 2/3/4-pyridyl groups through an ethylene
and vinylene spacer at the C3 position of the chlorins. The visible
absorption spectra of synthetic pyridyl-appending Chl-a de-
rivatives showed extension of
with pyridyl groups, dependent
p
-conjugation of the chlorin rings
on their spacers;
CH2eCH2 < C^C < CH]CH. The protonation of 2/3/4Py]Chls and
H]Chl was analysed by visible absorption measurements with
titration of TFA. The N atom of the pyridyl moiety was first pro-
tonated to give red-shifted Qy maxima and then the chlorin ring
was done to afford blue-shifted Qy maxima. The pKa values of the
pyridyl moiety increased in the order of 3.0 (2Py]Chl) < 3.2 (3Py]
Chl) < 3.8 (4Py]Chl), while the mono-protonated species gave
almost the same Qy maxima: 699 (2Py]Chl), 700 (3Py]Chl), and
699 nm (4Py]Chl). The present Chlepyridine conjugates are
promising to optical sensors sensitive for a proton concentration in
a solution.
Acknowledgement
This work was partially supported by a Grant-in-Aid for Scien-
tific Research (A) (No. 22245030) from the Japan Society for the
Promotion of Science (JSPS).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2015.03.006.
References
[1] Blankenship RE. Molecular mechanisms of photosynthesis. 2nd ed. West
Sussex, UK: Wiley Blackwell; 2014.
[2] (a) Smith KM, Goff DA, Simpson DJ. The meso substitution of chlorophyll
derivatives: direct route for transformation of bacteriopheophorbides d into
bacteriopheophorbides c. J Am Chem Soc 1985;107:4946e54;
(b) Li G, Graham A, Chen Y, Dobhal MP, Morgan J, Zheng G, et al. Synthesis,
comparative photosensitizing efficacy, human serum albumin (site II) binding
ability, and intracellular localization characteristics of novel benzobacterio-
chlorins derived from vic-dihydroxybacteriochlorins. J Med Chem 2003;46:
5349e59;
(c) Lonin IS, Kuzovlev AS, Belyaev ES, Ponomarev GV, Koifman OI, Tsivadze AY.
3-(2-Bromovinyl)chlorins: a new approach towards chlorophyll a modifica-
tion. J Porphyr Phthalocyanines 2014;18:123e8;
(d) Shinozaki Y, Yoshikawa I, Araki K, Sugawa K, Otsuki J. Control of higher-
order structures of zinc chlorophyll coordination polymers. CrystEngComm
2014;16:9155e7.
[3] Yamamoto Y, Tamiaki H. Synthesis of chlorophyll derivatives possessing a
mono/bi/terpyridinyl group at the C3-ethynyl terminal and optical properties
of the
p-conjugates. Tetrahedron 2014;70:2731e7.
[4] (a) Tamiaki H, Machida S, Mizutani K. Modification of 3-substituents in
(bacterio)chlorophyll derivatives to prepare 3-ethylated, methylated, and
unsubstituted (nickel) pyropheophorbides and their optical properties. J Org
Chem 2012;77:4751e8;
(b) Xu M, Kinoshita Y, Tamiaki H. Synthesis of chlorophyll-f analogs possessing
the 2-formyl group by modifying chlorophyll-a. Bioorg Med Chem Lett
2014;24:3997e4000;
(c) Takai N, Morioka M, Yoshizato M, Tanaka T, Sasaki S, Ito S, et al. Synthesis
and photophysical properties of phenyl-sulfanylated chlorophyll derivatives.
Tetrahedron 2014;70:5109e13;
(d) Ogasawara S, Tamiaki H. C132-Methylation of methyl pheophorbide a and
stereoselective preparation of methyl (132R)-methylpyropheophorbide a.
Tetrahedron Lett 2014;55:3618e21;
(e) Tamiaki H, Nagai T, Mizutani K, Machida S. Synthesis of amino-analogs of
bacteriochlorophyll-d and their self-aggregation in an aqueous micelle solu-
tion. Bioorg Med Chem Lett 2014;24:1668e71;
Fig. 3. Visible absorption spectral changes of 4Py]Chl (4.0 ꢀ 10ꢁ6 M) in CHCl3 by
titration with (a) 0e1000 eq and (b) 2000e50,000 eq of TFA as well as (c) H]Chl
(4.0 ꢀ 10ꢁ6 M) by addition of 0e50,000 eq TFA.
(f) Yamamoto Y, Tamiaki H. Synthesis and optical properties of chlorophyll
dimers bridged by cis- and trans-forms of Pt(II) bis-acetylide complexes.
J Organomet Chem 2014;749:400e4;
(g) Tamiaki H, Fukai K, Shimazu H, Shoji S. Synthesis of zinc chlorophyll homo/
hetero-dyads and their folded conformers with porphyrin, chlorin and bac-
observed in their Qy(0,0) absorption maxima aforementioned. It is
noteworthy that the quantum yields of fluorescence emission were
slightly reduced by the protonation of the pyridyl group and halved
by that of the chlorin moiety (see Table S1).
teriochlorin
p-systems. Photochem Photobiol 2014;90:121e8;
(h) Tamiaki H, Kuno M, Kunieda M. Self-aggregation of a synthetic zinc

Y. Yamamoto, H. Tamiaki / Dyes and Pigments 118 (2015) 159e165
165
chlorophyll derivative possessing a 131-dicyanomethylene group as a light-
harvesting antenna model. Tetrahedron Lett 2014;55:2825e8;
(i) Kinoshita Y, Tamiaki H. Regioselective addition of amines to the tri-
(c) Sasaki S, Tamiaki H. Synthesis and optical properties of bacteriochloro-
phyll-a derivatives having various C3 substituents on the bacteriochlorin
system. J Org Chem 2006;71:2648e54;
p-
fluoromethyl-
Phthalocyanines 2014;18:471e4;
b
-diketonate moiety of
a
chlorophyll derivative.
J
Porphyr
(d) Sasaki S, Mizutani K, Kunieda M, Tamiaki H. Cycloaddition to a C3-
ethynylated chlorophyll derivative and self-aggregation of zinc chlorin-
pyrazole/triazole conjugates. Tetrahedron 2013;69:9772e8.
(j) Tamiaki H, Matsunaga S, Taira Y, Wada A, Kinoshita Y, Kunieda M. Synthesis
of zinc 20-substituted bacteriochlorophyll-d analogs and their self-aggrega-
tion. Tetrahedron Lett 2014;55:3351e4;
(k) Kitagawa Y, Takeda H, Ohashi K, Asatani T, Kosumi D, Hashimoto H, et al.
Photochemical reduction of CO2 with red light using synthetic chloro-
phyllerhenium bipyridine dyad. Chem Lett 2014;43:1383e5;
[6] (a) Sasaki S, Mizutani K, Kunieda M, Tamiaki H. Synthesis, modification, and
optical properties of C3-ethynylated chlorophyll derivatives. Tetrahedron Lett
2008;49:4113e5;
(b) Sasaki S, Mizutani K, Kunieda M, Tamiaki H. Synthesis and optical prop-
erties of C3-ethynylated chlorin and
p-extended chlorophyll dyads. Tetrahe-
(l) Tamiaki H, Kuno M, Ohata M. Self-aggregation of synthetic zinc chlorophyll
derivatives possessing 31-hydroxy or methoxy group and 131-mono- or
dicyanomethylene moiety in non-polar organic solvents as models of chloro-
somal bacteriochlorophyll-d aggregates. Photochem Photobiol 2014;90:
1277e86;
dron 2011;67:6065e72.
[7] Hirose K. A practical guide for the determination of binding constants. J Incl
Phenom Macrocycl Chem 2001;39:193e209.
[8] (a) Baker BR, Doll MH. Irreversible enzyme inhibitors. 183. Proteolytic en-
zymes. 18. Inhibitors of guinea pig complement derived by quaternization of
(m) Xu M, Tamiaki H. Transformation of natural chlorophyll-a into chlorophyll-c
analogs possessing the 17-acrylate residue. Chem Lett 2014;43:1864e6;
(n) Tamiaki H, Ariki N, Yasuda T, Miyatake T, Oba T. Synthesis of 20-
iodochlorophyll derivatives and their properties including reactivity, elec-
tronic absorption, and self-aggregation. Tetrahedron 2014;70:9768e75;
(o) Kitagawa Y, Ogasawara S, Kosumi D, Hashimoto H, Tamiaki H. Photophysical
properties of chlorophyll derivatives linked with rhenium bipyridine com-
plexes. Bull Chem Soc Jpn 2015;88:346e51;
phenylalkylpyridines with a-bromomethylbenzenesulfonyl fluorides. J Med
Chem 1971;14:793e9;
(b) Regnouf-de-Vains J-B, Lamartine R. Wittig reaction on calixarene upper
rim. Access to conjugated bipyridyl and pyridyl podands. Tetrahedron Lett
1996;37:6311e4.
[9] (a) Li JZ, Wang JJ, Yoon I, Cui BC, Shim YK. Synthesis of novel long wavelength
cationic chlorins via stereoselective aldol-like condensation. Bioorg Med
Chem Lett 2012;22:1846e9;
(p) Ogasawara S, Tamiaki H. Synthesis of methyl C132-alkylated pheophorbides
(b) Shinozaki Y, Richards G, Ogawa K, Yamano A, Ohara K, Yamaguchi K, et al.
Double helices of a pyridine-appended zinc chlorophyll derivative. J Am Chem
Soc 2013;135:5262e5.
a/a0 and their stereochemical characterization. Tetrahedron
2015;26:46e52.
e Asymmetry
[5] (a) Tamiaki H, Amakawa M, Shimono Y, Tanikaga R, Holzwarth AR,
Schaffner K. Synthetic zinc and magnesium chlorin aggregates as models for
supramolecular antenna complexes in chlorosomes of green photosynthetic
bacteria. Photochem Photobiol 1996;63:92e9;
[10] (a) Gerola AP, Tsubone TM, Santana A, Oliveire HPM, Hioka N, Caetano W.
Properties of chlorophyll and derivatives in homogeneous and micro-
heterogeneous systems. J Phys Chem B 2011;115:7364e73;
(b) Hynninen PH. Protonation-deprotonation equilibria in tetrapyrroles part
3: mono- and diprotonations of the trimethyl esters of chlorin e6 and the 71-
acetal of rhodin g7 in methanolic hydrochloric acid. J Porphyr Phthalocyanines
2012;16:1167e76.
(b) Tamiaki H, Kouraba M. Synthesis of chlorophyll-a homologs by Wittig and
Knoevenagel reactions with methyl pyropheophorbide-d. Tetrahedron
1997;53:10677e88;