Regioisomeric synthesis of chlorin-e 6 dimethyl esters and their optical properties

Article abstract of DOI:10.1142/S1088424618501043

Chlorin-e6 dimethyl esters possessing a single carboxy group at the 13-, 15^1-,or 17²-position were prepared by chemically modifying chlorophyll-A. These three synthetic regioisomers were fully characterized by their mass, NMR, and visible absorption spectra. Their molecular structures were unambiguously identified by the specific ¹H-¹³C correlation at the 13-, 15-, and/or 17-substituents in their respective HMBC spectra. Methyl esterification of 13/15¹-COOH and hydrolysis of 13/15¹-COOMe affected small shifts of the Qy absorption and fluorescence emission maxima in a diluted CH₂Cl₂ solution, while no substitution effect of 17²-COOH/Me was observed.

Full text of DOI:10.1142/S1088424618501043

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2018; 22: 1–8
DOI: 10.1142/S1088424618501043
Published at http://www.worldscinet.com/jpp/
Regioisomeric synthesis of chlorin-e dimethyl esters
6
and their optical properties
*◊
Yasunobu Nagano, Shin Ogasawara and Hitoshi Tamiaki
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Received 10 September 2018
Accepted 12 October 2018
1
ABSTRACT: Chlorin-e dimethyl esters possessing a single carboxy group at the 13-, 15 -, or
1
6
2
7 -position were prepared by chemically modifying chlorophyll-a. These three synthetic regioisomers
were fully characterized by their mass, NMR, and visible absorption spectra. Their molecular structures
1
13
were unambiguously identified by the specific H– C correlation at the 13-, 15-, and/or 17-substituents in
1
1
their respective HMBC spectra. Methyl esterification of 13/15 -COOH and hydrolysis of 13/15 -COOMe
affected small shifts of the Qy absorption and fluorescence emission maxima in a diluted CH Cl solution,
2
2
2
while no substitution effect of 17 -COOH/Me was observed.
KEYWORDS: chlorophyll-a derivative, ester protection, fluorescence emission, Qy absorption, retro-
Dieckmann condensation.
regioisomers 1a–1c and determine their molecular
INTRODUCTION
structures in their intact forms for the first time using a
Chlorin-e6 is one of the chlorophyll-a derivatives
which is readily available through the retro-Dieckmann
condensation opening of the exo-five-membered
ring bearing a b-keto-ester moiety. Chlorin-e6 and
its derivatives are widely used as photosensitizers in
medicinal [1] and material sciences [2]. Chlorin-e6
has three carboxy groups in a molecule (Fig. 1) which
are partially different in their reactivities, but their
regioselectively chemical modification is difficult.
Herein, we report on the preparation of regioselectively,
1
13
variety of H and C NMR techniques. With all the three
regioisomers in hand, their visible absorption, circular
dichroism (CD), and fluorescence emission spectra in
a diluted dichloromethane solution were systematically
compared and also their emission quantum yields and
lifetimes were evaluated. The present regioisomers 1a–1c
possess a single carboxy group in a molecule and will be
useful for the preparation of various regioisomerically
pure chlorin-e derivatives.
6
doubly methyl-esterified chlorin-e molecules 1a–1c and
6
reveal their optical properties in a solution, which are
compared with those of trimethyl ester 2.
RESULTS AND DISCUSSION
One of the regioisomers, 1a, has previously been
prepared and its molecular structure was confirmed by
chemical modification [3]. The synthetic route of another
regioisomer 1c was described [4], but its structure has not
yet been identified. Moreover, 1b has never been reported
in any papers, to the best of our knowledge. In this paper,
we mention fully synthetic procedures for obtaining
Synthesis of chlorin-e dimethyl ester regioisomers
6
Chlorophyll-a was extracted from commercially
available cyanobacterial cells, spirulina powder, by
methanol [5], readily demetalated to pheophytin-a by
treatment of the extract with an aqueous diluted hydro-
chloric acid solution (step (i) in Scheme 1) [3, 5], and
further hydrolyzed at the 17-propionate residue with
trifluoroacetic acid to give pheophorbide-a (step (ii)) [3].
It is noted that the conditions of step (ii) promoted no more
◊
SPP full member in good standing
*
Correspondence to: Hitoshi Tamiaki, fax: +81-77-561-3729,
2
email: tamiaki@fc.ritsumei.ac.jp.
hydrolysis of the 13 -methoxycarbonyl group. During the
Copyright © 2018 World Scientific Publishing Company

2
Y. NAGANO ET AL.
pheophorbide-a was converted to chlorin-e trimethyl
6
ester (2) by the same step (vi) as the aforementioned
cleavage of the E ring [3]. The resulting three methyl
esters were fully hydrolyzed with lithium iodide in
refluxing ethyl acetate (step (viii)) [9]. Chlorin-e bearing
6
the three carboxy groups which is also available from
some commercial suppliers was doubly and selectively
1
2
esterified to give its 15 ,17 -dimethyl ester 1a in a 41%
isolated yield (step (iv)) [3, 8]. The regioselectivity can
be attributed to less reactivity of the 13-carboxy group
directly conjugated with chlorin p-system.
Structural identification of chlorin-e dimethyl
6
ester regioisomers
All the three aforementioned products were purified
with chromatography and showed the identical mass
1
2
3
Fig. 1. Molecular structure of chlorin-e6 (R =R =R =H)
as well as its dimethyl esters 1a–1c and trimethyl ester 2
+
peak at m/z = 625 as MH which is identical to that of
1
2
3
(
R =R =R =Me)
chlorin-e dimethyl ester (C H N O ). The molecular
6
36 41
4
6
structures of the dimethyl ester regioisomers were
fully identified by the following NMR techniques
in chloroform-d at room temperature. All single-set
2
above procedures, the epimerization at the 13 -position
occurred and the resulting pheophorbide-a contained
about 20% of the (13 S)-epimer, pheophorbide-a′ (prime
proton signals except two methyl esters (COOCH ),
3
2
one carboxylic acid (COOH), and two inner NH peaks
1
form). The mixture of pheophorbides-a/a′ was dissolved
in chloroform and treated with 0.5% (wt/v) potassium
hydroxide in methanol at room temperature (step (iii))
were assigned by 1D and 2D H NMR spectra including
COSY and ROESY (Figs S1–S3/S6–S8/S11–S13). All
1
3
36 carbon-13 peaks were recognized by 1D C NMR
spectra including DEPT (Figs S4/S9/S14). Furthermore,
two singlet proton signals of the methyl esters and almost
1
2
[
3, 4]. The basic conditions cleaved the C13 –C13 bond
of the exo-five-membered ring (E-ring) to afford chlorin-e
6
1
13
1
13
13,15 -dimethyl ester (1c) as the methanol adduct (a retro-
all the C peaks were assignable by H– C HMQC and
Diekmann condensation product) in a 61% isolated yield
based on consumed pheophorbide-a/a′ after purification
with flash column chromatography (FCC).
HMBC spectra (Figs S5/S10/S15).
1
The 15- and 17 -CH
proton signals were characterized
2
at 5 to 6 ppm and 2 to 3 ppm, respectively. Three carbonyl
carbon-13 signals were visible in a low field (169–178
ppm, see Table 1). In the HMBC spectra, one of the
doublet proton peaks for the 15-CH
significantly correlated with one of the carbonyl
Methyl pheophorbide-a was obtained from chloro-
phyll-a via pheophytin-a (steps (i) and (iv)) according
to the reported procedures [3, 5]. As mentioned above,
(J = 19 Hz) was
2
2
13
the 13 -epimerization also occurred and about 10% of
C
2
the prime form was observed in the product. Its methyl
signals, which was assigned to the C15 peak (see Figs 2/
S16/S17). A methylene proton peak at the 17 -position
2
1
ester at the 13 -position was selectively transesterified
13
with p-methoxybenzyl alcohol by action of 2-chloro-
was clearly correlated with another carbonyl C signal
13
1
-methylpyridinium iodide (Mukaiyama condensation
reagent) and 4-(N,N-dimethylamino)pyridine (step (v),
6%) [6]. The product was subjected to similar basic
in HMBC spectra and the C peak was unambiguously
3
13
originated from the C17 . The remaining carbonyl
C
1
4
signal was ascribed to the C13 . In the HMBC spectrum
1
3
conditions as the above step (iii) and the E-ring-cleaved
product was produced in a 59% isolated yield based on
consumed pheophorbide-a derivative (step (vi)). The
prolonged reaction led to the transesterification of the
desired product to trimethyl ester 2, so it was important
to frequently check the ring-opening reaction using TLC.
The resulting p-methoxybenzyl ester was hydrolyzed
of 1b (Fig. 2), both the C13 and C17 peaks at 169.5 and
173.6 ppm were correlated with singlet proton peaks of
methyl esters at 4.19 and 3.56 ppm, respectively, while
2
the C15 peak at 176.7 ppm displayed no correlation with
proton peaks at 3 to 5 ppm. The observation indicates that
1
regioisomerically pure 1b possesses 13-COOCH
, 15 -
3
2
COOH, and 17 -COOCH
. Similarly, the HMBC spectral
3
by trifluoromethanesulfonic acid in trifluoroacetic acid
analyses indicate that 1a and 1c regioisomers have
2
1
2
(
step (vii)) to afford chlorin-e 13,17 -dimethyl ester (1b,
13-COOH/15 -COOCH
3
/17 -COOCH
15 -COOCH /17 -COOH, respectively (Figs S16/S17).
3
3
and 13-COOCH /
3
6
1
2
6
9%). It is noteworthy that no hydrolysis of methyl esters
13
was found under acidic conditions [7].
Table 1 shows the following two shifts for the C peaks
of carbonyl groups. The esterification of COOH to
1
2
Chlorin-e 15 ,17 -dimethyl ester (1a) was obtained
6
13
according to procedures reported previously [3, 8].
Its preparation is briefly described below. Methyl
COOCH moves the carbonyl C signals to higher fields
3
by 2.7–3.8 ppm and direct conjugation of the carbonyl
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 2–8

REGIOISOMERIC SYNTHESIS OF CHLORIN-e DIMETHYL ESTERS AND THEIR OPTICAL PROPERTIES
3
6
Scheme 1. Synthesis of chlorin-e dimethyl esters 1a–1c by chemical modification of naturally occurring chlorophyll-a: (i) aq.
6
+
-
HCl, rt; (ii) aq. CF COOH, rt; (iii) 0.5% KOH/MeOH, rt; (iv) 5% H SO /MeOH, rt; (v) 4-MeOC H CH OH, 2-ClC H NMe I ,
3
2
4
6
4
2
5
4
4
-Me NC H N/PhMe, reflux; (vi) 0.5 M MeONa/MeOH, CHCl , rt; (vii) 10% CF SO H/CF COOH, 0°C; (viii) LiI/AcOEt, reflux
2
5
5
3
3
3
3
groups with a chlorin p-system induces high-field shifts
redmost Qy absorption band at around 665 nm (see
Fig. 4a) showed small substitution effects. Hydrolysis
of methyl ester at the 13-position as in 2 → 1a moved
2
3
1
(
≈4 ppm) of the peaks (C15 /C17 → C13 ).
the Qy maximum labs (Qy) to a longer wavelength by
Optical properties of chlorin-e dimethyl ester
6
1
1
nm, while that at the 15 -position as in 2 → 1b did it to
regioisomers
a shorter wavelength by 1 nm (Table 2). No shift of the
Qy maximum was observed in the hydrolysis of methyl
In a diluted dichloromethane solution, chlorin-e6
dimethyl ester regioisomers 1a–1c and trimethyl ester 2
gave almost the same visible absorption and CD spectra
2
ester at the 17 -position as in 2 → 1c. The carboxy and
methoxycarbonyl groups at the 13-position are directly
(
Fig. 3). These spectra indicated that these esters were
conjugated with chlorin p-system in a molecule and
1
monomeric in the solution. A close inspection of the
those at the 15 -position are homoconjugated with it
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 3–8

4
Y. NAGANO ET AL.
1
13
Fig. 2. Specific HMBC spectrum of 1b in chloroform-d at room temperature and the H– C correlation (red arrows) in the peripheral
substituents at the 13-, 15-, 17-, and 18-positions (inset)
1
13
Table 1. Chemical shifts ds (ppm) for H and C signals of COOCH and COOH of chlorin-e
3
6
dimethyl ester regioisomers 1a–1c in chloroform-d
Compound
COOR (R=CH or H)
COOCH3
COOCH3
3
1
2
1
2
1
2
1
3-
15 -
17 -
13-
—
15 -
17 -
13-
—
15 -
17 -
1
1
1
a (13-COOH)
173.2
169.5
169.5
174.0
176.7
173.2
173.5
173.6
177.3
52.5
—
51.6
51.7
—
3.80
—
3.58
3.56
—
1
b (15 -COOH)
53.2
53.0
4.19
2
c (17 -COOH)
52.1
4.22 3.73
3
-1
image of the Qy band with approximately 150 cm
through one sp -carbon atom, so they slightly affected
the Qy absorption bands. These functional groups at the
of Stokes shift D (Fig. 4b and Table 2). A small red
shift (1 nm) of fluorescence emission maximum lem
of 1a over 1b/1c/2 is ascribable to the aforementioned
bathochromic shift of the Qy maximum labs (Qy) of the
former bearing the 13-COOH over the latter possessing
2
1
7 -position are connected with chlorin p-core via four
single C–C bonds and far from the p-system, resulting
in no effect on the visible absorption bands. These
observations are consistent with the reported data for
chlorophyll derivatives with various esterifying groups
at the 17-propionate residue [10]. It is noted that no
apparent effect was found in their Soret bands at 403 nm.
When the diluted dichloromethane solutions (ca.
the 13-COOCH . Their fluorescence emission quantum
3
yields Fem are almost the same, 16–17% (Table 2). All
the fluorescence emissions were single-exponentially
decayed to give virtually identical lifetimes tem (about
4.5 ns). Apparent substitution effects on Fem and tem are
invisible for 1a–1c and 2.
1
mM) of 1a–1c and 2 were excited at the Soret maxima,
an intense emission band was observed as the mirror
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 4–8

REGIOISOMERIC SYNTHESIS OF CHLORIN-e DIMETHYL ESTERS AND THEIR OPTICAL PROPERTIES
5
6
Fluorescence emission lifetimes excited at 403 nm
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
(
a)
1
1b
1
2
a
were determined with a Hamamatsu Photonics C7990S
1
13
system. H and C NMR spectra were recorded on
a JEOL ECA-600 (600 MHz) spectrometer; tetra-
methylsilane (0.00 ppm) was used as an internal
reference. Proton and carbon-13 peaks were assigned
c
1
1
13
1
13
using H– H COSY/ROESY, C DEPT, and H– C
HMQC/HMBC techniques. Standard mass data were
obtained using laser desorption/ionization (LDI) by
a Shimadzu AXIMA-CFR plus spectrometer. High
resolution mass spectra (HRMS) were recorded on a
Bruker micrOTOF II spectrometer: atmospheric pressure
chemical ionization (APCI) or electrospray ionization
(
b)
1
0
5
0
(
ESI) and positive mode in a methanol solution. FCC and
TLC were performed with silica gel (Wakogel C-300 and
Merck Kieselgel 60 F254). RP-HPLC was performed by
3
00
400
500
600
700
a Shimadzu LC-10AD
pump, SPD-M10A
diode-
VP
array detector, and SCL-10A
VP
system controller using
Wavelength / nm
VP
a packed ODS column (Nacalai Tesque Cosmosil 5C -
1
8
Fig. 3. Visible absorption (a) and CD spectra (b) of chlorin-e6
dimethyl ester regioisomers 1a–1c and trimethyl ester 2 in
dichloromethane (ca. 10 mM) at room temperature. All the
absorption spectra were normalized at their intense peaks at the
Soret region
AR-II, 10 mmf × 250 mm).
Chlorin-e6 [9], chlorin-e6 trimethyl ester (2) [3],
methyl pheophorbide-a/a′ [3, 5], and pheophorbide-a/a′
[3] were prepared according to reported procedures. All
the reaction reagents and solvents were obtained from
commercial suppliers and utilized as supplied. Dry
toluene was obtained by reflux with calcium chloride
and distillation. Distilled water was prepared by a
Yamato AutoStill WG250 system. All the reactions were
performed in the dark. Dichloromethane for optical
spectroscopy was purchased from Nacalai Tesque as
a reagent prepared specially for spectroscopy and used
without further purification.
EXPERIMENTAL
General
All melting points were measured with a Yanagimoto
micro melting apparatus and were uncorrected. Visible
absorption and CD spectra were measured with
Hitachi U-3500 and Jasco J-720W spectrophotometers,
respectively. The fluorescence emission spectra and
quantum yields excited at Soret maxima were obtained
by a Hamamatsu Photonics C9920-03G spectrometer.
Before measurements of optical properties, the
sample was purified by RP-HPLC with MeOH : H
CF COOH = 75 : 25 : 0.1 (1.0 ml/min). The desired
HPLC elution was diluted with dichloromethane, washed
O :
2
3
Fig. 4. Visible absorption (a), ca. 10 mM and fluorescence emission spectra (b), ca. 1 mM of chlorin-e dimethyl ester regioisomers
6
1
a–1c and trimethyl ester 2 in aerated dichloromethane at room temperature. All the spectra were normalized at their intense peaks
at the Qy region and the emission spectra were measured by excitation at the Soret maxima
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 5–8

6
Y. NAGANO ET AL.
Table 2. Optical properties of chlorin-e dimethyl ester regioisomers 1a–1c and trimethyl ester
6
a
2
in aerated dichloromethane at room temperature
-1
Compound
labs/nm
l /nm
em
D/cm
F /%
em
t /ns
em
Soret
Qy
1
1
1
a (13-COOH)
403 (2090)
403 (2040)
403 (2100)
403 (2050)
666 (490)
664 (470)
665 (480)
665 (450)
672 (480)
671 (490)
671 (470)
671 (480)
140
160
150
140
16
16
17
17
4.3
4.6
4.6
4.5
1
b (15 -COOH)
2
c (17 -COOH)
2
a
labs, intense absorption maximum; lem, emission maximum (excited at Soret maxima); D, Stokes shift =
7
[
(
1/labs (Qy) – 1/lem] × 10 ; Fem, emission quantum yield (excited at Soret maxima); tem, emission lifetime
-1
excited at 403 nm); the values in parentheses indicate full widths at half maxima of bands (cm ).
+
with distilled water, and dried over sodium sulfate. After
evaporation, theresiduewasdissolvedindichloromethane
and the solution was optically analyzed.
(ESI) found: m/z 625.3021, calcd for C H N O : MH ,
36 41 4 6
625.3021.
2
Synthesis of chlorin-e 13,17 -dimethyl ester (1b)
6
1
2
Synthesis of chlorin-e 15 ,17 -dimethyl ester (1a)
6
Methyl pheophorbide-a/a′ (7/1, 101.6 mg, 167 mmol)
was dissolved in dry toluene (10 ml), to which were
added 2-chloro-1-methylpyridinium iodide (132.2 mg,
517 mmol, 3.1 eq.), 4-(N,N-dimethylamino)pyridine
(136.4 mg, 1.116 mmol, 6.7 eq.), and 4-methoxybenzyl
alcohol (37.7 mg, 273 mmol, 1.6 eq.). The mixture was
refluxed under nitrogen for 3 h and cooled down to room
temperature:thereactionwasmonitoredbyTLC(CH Cl :
Chlorin-e (35.4 mg, 59.3 mmol) was dissolved in a 5%
6
(
v/v) methanol solution of sulfuric acid and stirred at room
temperature under argon for 14 h. After disappearance of
the starting material checked by TLC (CH Cl : MeOH =
2
2
10 : 1), the reaction mixture was poured into ice-chilled
water and extracted with dichloromethane several times.
The combined organic phases were washed with an
aqueous 4% (wt/v) sodium hydrogen carbonate and
brine, dried over sodium sulfate, and filtered. All the
solvents were evaporated and the residue was purified
with FCC (CH Cl : MeOH = 10 : 1) and recrystallization
2
2
+
Et O = 100 : 3) and mass analysis (m/z = 712 for M
2
of the product). After evaporation of all the solvent, the
residue was purified with FCC (CH Cl : Et O = 100 : 3)
2
2
2
2
to give methyl (13 R/S)-(4-methoxybenzyloxycarbonyl)
pyropheophorbide-a (10/1, 54.4 mg, 76.3 mmol, 46%):
bluish black solid; mp = 178–179°C; UV-vis (CH Cl )
2
2
from dichloromethane and hexane to give 1a (15.2 mg,
4.3 mmol) in a 41% yield (95% in [3]): bluish black
solid; mp = 155–156°C; VIS (CH Cl ) l = 666 (relative
2
2
2
lmax = 668 (relative intensity, 0.43), 611 (0.08), 539 (0.10),
2
2
max
1
intensity, 0.30), 610 (0.03), 532 (0.03), 502 (0.08), 403 nm
508 (0.11), 414 nm (1.00); H NMR (600 MHz, CDCl )
3
1
(
1.00); H NMR (600 MHz, CDCl ) d = 9.60 (1H, s, 10-H),
d(10/1) = 9.37/9.33 (1H, s, 10-H), 9.18/9.15 (1H, s, 5-H),
8.49/8.47 (1H, s, 20-H), 7.85 (1H, dd, J = 18, 12 Hz,
3
9
1
3
5
.50 (1H, s, 5-H), 8.71 (1H, s, 20-H), 8.03 (1H, dd, J = 18,
1
2
1
2
2 Hz, 3 -H), 6.33 (1H, dd, J = 18, 1 Hz, 3 -H trans to
3 -H), 7.30 (2H, d, J = 8 Hz, 2,6-H of 13 -COOCAr),
2
2
-C–H), 6.12 (1H, dd, J = 12, 1 Hz, 3 -H cis to 3-C–H),
6.78 (2H, d, J = 8 Hz, 3,5-H of 13 -COOCAr), 6.18 (1H,
2
.54, 5.26 (each 1H, d, J = 19 Hz, 15-CH ), 4.41 (1H, q,
dd, J = 18, 1 Hz, 3 -H trans to 3-C–H), 6.09 (1H, dd,
2
2
J = 7 Hz, 18-H), 4.38 (1H, d, J = 10 Hz, 17-H), 3.80 (3H,
J = 12, 1 Hz 3 -H cis to 3-C–H), 5.37, 5.28/5.32, 5.23
1
2
s, 15 -COOCH ), 3.69 (2H, q, J = 8 Hz, 8-CH ), 3.58 (6H,
(each 1H, d, J = 12 Hz, 13 -COOCH ), 4.35/4.45 (1H,
3
2
2
2
s, 12-CH , 17 -COOCH ), 3.45 (3H, s, 2-CH ), 3.25 (3H,
dq, J = 2, 7 Hz, 18-H), 3.93/3.90 (1H, dt, J = 9, 2 Hz,
3
3
3
1
2
s, 7-CH ), 2.57–2.48 (1H, m, 17 -CH), 2.20–2.10 (2H, m,
17-H), 3.74/3.72 (3H, s, 4-OCH of 13 -COOCAr), 3.65
3
3
2
1
7-CHCH), 1.76 (3H, d, J = 7 Hz, 18-CH ), 1.70 (1H, br,
(3H, s, 12-CH ), 3.55 (3H, s, 17 -COOCH ), 3.51 (2H,
q, J = 8 Hz, 8-CH ), 3.33 (3H, s, 2-CH ), 3.07/3.05 (3H,
s, 7-CH ), 2.57–2.52, 2.29–2.22 (each 1H, m, 17-CH ),
2.36–2.31, 2.17–2.12 (each 1H, m, 17 -CH ), 1.61 (3H,
t, J = 8 Hz, 8 -CH ), 1.61 (3H, d, J = 7 Hz, 18-CH ),
3
3
3
1
1
7-CH), 1.65 (3H, t, J = 8 Hz, 8 -CH ), –1.33 (1H, s, NH)
3
2
3
[the 13-COOH and one of the two NH signals could be
3
2
1
3
2
1
invisible]; C NMR (CDCl , 150 MHz) d = 174.0 (C15 ),
3
1
2
3
1
1
1
1
1
73.5 (C17 ), 173.2 (C13 ), 169.7 (C19), 167.2 (C16),
54.9 (C6), 148.8 (C9), 145.0 (C8), 139.6 (C1), 137.0,
29.3 (C11, 12), 136.1 (C14), 135.8 (C7), 135.5 (C4),
3
3
0.39, –1.78/0.53, –1.56 (each 1H, s, NH × 2); HRMS
(APCI) found: m/z 713.3330, calcd for C H N O :
4
3
45
4
6
1
+
34.8 (C3), 130.6 (C2), 129.4 (C3 ), 123.1 (C13), 121.7
MH , 713.3334.
2
(C3 ), 102.4 (C10), 102.2 (C15), 98.6 (C5), 93.6 (C20),
The above 4-methoxybenzyl ester (48.4 mg,
67.9 mmol) was dissolved in chloroform (5 ml), to which
was added methanol (10 ml) and successively a 0.5 M
methanol solution (5 ml) of sodium methoxide was
4
5
52.8 (C17), 52.5 (C15 ), 51.6 (C17 ), 49.5 (C18), 39.2
1
2
1
1
1
(C15 ), 31.1 (C17 ), 29.3 (C17 ), 22.7 (C18 ), 19.6 (C8 ),
2
1
1
1
17.6 (C8 ), 12.6 (C12 ), 12.1 (C2 ), 11.3 (C7 ); HRMS
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 6–8

REGIOISOMERIC SYNTHESIS OF CHLORIN-e DIMETHYL ESTERS AND THEIR OPTICAL PROPERTIES
7
6
dropped. The mixture was stirred at room temperature
under nitrogen for 1 h.After detection of a more movable
17-CHCH), 1.71 (3H, d, J = 7 Hz, 18-CH ), 1.70 (3H,
3
1
t, J = 7 Hz, 8 -CH ), 1.69 (1H, br, 17-CH), –1.45 (1H,
3
spot on TLC (CH Cl : Et O = 100 : 3) and a new mass
s, NH) [the 15-COOH and one of the two NH signals
2
2
2
1
3
peak at m/z = 744, the reaction mixture was quenched
with water, diluted with chloroform, washed with water,
dried over sodium sulfate, and evaporated. The residue
was purified with FCC (CH Cl : Et O = 100: 3) to give
could be invisible]; C NMR (CDCl , 150 MHz) d =
3
2 3 1
176.7 (C15 ), 173.6 (C17 ), 169.7 (C19), 169.5 (C13 ),
167.1 (C16), 154.9 (C6), 148.9 (C9), 145.1 (C8), 139.6,
130.6 (C1, 2), 136.6, 129.3 (C11, 12), 136.0 (C7),
2
2
2
2
1
1
chlorin-e 13,17 -dimethyl-15 -(4-methoxybenzyl)ester
135.4 (C4), 135.3 (C14), 134.8 (C3), 129.3 (C3 ), 123.1
6
2
(
the first fraction, 28.3 mg, 37.9 mmol, 56%) with
(C13), 121.9 (C3 ), 102.3 (C10), 101.5 (C15), 98.7
3
5
recovery of the starting material (the second fraction,
(C5), 93.6 (C20), 53.2 (C13 ), 52.8 (C17), 51.7 (C17 ),
1
2
1
2
1
0
.8 mg, 3.9 mmol, 6%): bluish black solid; mp = 109–
10°C; UV-vis (CH Cl ) l = 660 (relative intensity,
49.4 (C18), 38.4 (C15 ), 31.1 (C17 ), 29.4 (C17 ), 22.8
1
1
2
1
1
(C18 ), 19.7 (C8 ), 17.7 (C8 ), 12.4 (C12 ), 12.1 (C2 ),
2
2
max
1
.30), 610 (0.04), 531 (0.04), 502 (0.09), 403 nm
11.3 (C7 ); HRMS (APCI) found: m/z 625.3026, calcd
for C H N O : MH , 625.3021.
1
+
(
1.00); H NMR (600 MHz, CDCl ) d = 9.70 (1H,
3
36 41
4
6
s, 10-H), 9.55 (1H, s, 5-H), 8.73 (1H, s, 20-H), 8.05
1
(
2
1H, dd, J = 18, 12 Hz, 3 -H), 7.23 (2H, d, J = 9 Hz,
1
Synthesis of chlorin-e 13,15 -dimethyl ester (1c)
1
6
,6-H of 15 -COOCAr), 6.76 (2H, d, J = 9 Hz, 3,5-H
1
2
of 15 -COOCAr), 6.18 (1H, d, J = 18 Hz, 3 -H trans
Pheophorbide-a/a′ (4/1, 78.5 mg, 131.6 mmol) was
dissolved in chloroform (5 ml), to which was added a
0.5% (wt/v) methanol solution (20 ml) of potassium
hydroxide. The mixture was stirred at room temperature
under nitrogen for 10 h.After detection of a more movable
2
to 3-C–H), 6.13 (1H, d, J = 12 Hz 3 -H cis to 3-C–H),
1
5
.22, 5.14 (each 1H, d, J = 12 Hz, 15 -CH ), 4.39 (1H,
2
q, J = 7 Hz, 18-H), 4.35 (1H, d, J = 10 Hz, 17-H), 4.16
(
3
3H, s, 13-COOCH ), 3.78 (2H, q, J = 7 Hz, 8-CH ),
3
2
1
2
.74 (3H, s, 4-OCH of 15 -COOCAr), 3.60 (3H, s, 17 -
spot on TLC (CH Cl : MeOH = 10 : 1), the reaction
2 2
3
COOCH ), 3.58 (3H, s, 12-CH ), 3.46 (3H, s, 2-CH ),
mixture was quenched with an aqueous 5% (wt/v) citric
acid solution and extracted with chloroform several times.
The combined organic phases were dried over sodium
sulfate, filtered, and evaporated. The residue was purified
3
3
3
1
3
2
1
0
.29 (3H, s, 7-CH ), 2.57–2.50 (1H, m, 17 -CH), 2.20–
3
.13 (2H, m, 17-CHCH), 1.72 (3H, t, J = 7 Hz, 18-CH ),
3
1
.71 (1H, br, 17-CH), 1.65 (3H, d, J = 7 Hz, 8 -CH ),
3
.10, –1.47 (each 1H, s, NH × 2) [the corresponding
with FCC (CH Cl : MeOH = 10 : 1) and recrystallization
2 2
1
mono-benzyl ester showed a similar H NMR spectrum,
from dichloromethane and hexane to give 1c (the first
fraction, 45.1 mg, 72.2 mmol, 55%) with recovery of the
starting material (the second fraction, 8.2 mg, 13.7 mmol,
10%): bluish black solid; mp = 220–221°C; UV-vis
see [11]]; HRMS (APCI) found: m/z 745.3595, calcd
+
for C H N O : MH , 745.3595.
4
4
49
4
7
2
1
Chlorin-e6 13,17 -dimethyl-15 -(4-methoxybenzyl)
ester (8.5 mg, 11.4 mmol) was dissolved into 10% (v/v)
trifluoromethanesulfonic acid in trifluoroacetic acid
(CH
531 (0.03), 502 (0.08), 403 nm (1.00); H NMR
(600 MHz, CDCl ) d = 9.68 (1H, s, 10-H), 9.55 (1H, s,
Cl ) lmax = 665 (relative intensity, 0.30), 609 (0.03),
2
1
2
(
4 ml) and stirred at 0°C under nitrogen for 1 h. After
TLC (full disappearance of 4-methoxybenzyl ester at
R = 0.88 and appearance of carboxylic acid at R =
3
5-H), 8.73 (1H, s, 20-H), 8.05 (1H, dd, J = 18, 12 Hz,
1
2
3 -H), 6.34 (1H, dd, J = 18, 1 Hz, 3 -H trans to 3-C–H),
f
f
2
0
(
.5 in CH Cl : MeOH = 10 : 1) and mass analyses
6.13 (1H, dd, J = 12, 1 Hz, 3 -H cis to 3-C–H), 5.31,
5.26 (each 1H, d, J = 19 Hz, 15-CH ), 4.43 (1H, q, J =
2
2
detection of peak at m/z = 624), the reaction mixture
2
was diluted with dichloromethane, washed with water
three times, dried over sodium sulfate, and filtered.
The solvents were evaporated and the residue was
purified with FCC (CH Cl : MeOH = 10 : 1) and recrys-
7 Hz, 18-H), 4.42 (1H, d, J = 10 Hz, 17-H), 4.22 (3H, s,
13-COOCH ), 3.77 (2H, q, J = 8 Hz, 8-CH ), 3.73 (3H, s,
15 -COOCH ), 3.56 (3H, s, 12-CH ), 3.45 (3H, s, 2-CH ),
3.28 (3H, s, 7-CH ), 2.64–2.58 (1H, m, 17 -CH), 2.25–
3
2
1
3
3
3
1
2
2
3
tallization from dichloromethane and hexane to give
2.16 (2H, m, 17-CHCH), 1.74 (3H, d, J = 7 Hz, 18-CH
3
),
),
1
1
1
b (4.9 mg, 7.8 mmol, 69%): bluish black solid; mp =
62–164°C; UV-vis (CH Cl ) l = 664 (relative inten-
1.73 (1H, br, 17-CH), 1.70 (3H, t, J = 8 Hz, 8 -CH
3
2
–1.48 (1H, s, NH) [the 17 -COOH and one of the two NH
signals could be invisible]; C NMR (CDCl , 150 MHz)
2
2
max
1
3
sity, 0.30), 610 (0.03), 531 (0.03), 502 (0.08), 403 nm
3
1
3
2
1
(
1
1.00); H NMR (600 MHz, CDCl ) d = 9.68 (1H, s,
d = 177.3 (C17 ), 173.2 (C15 ), 169.5 (C13 ), 169.4
(C19), 166.7 (C16), 154.8 (C6), 148.9 (C9), 145.0 (C8),
139.5 (C1), 136.4, 129.3 (C11, 12), 136.0 (C7), 135.4,
3
0-H), 9.54 (1H, s, 5-H), 8.71 (1H, s, 20-H), 8.04
1
(
1H, dd, J = 18, 12 Hz, 3 -H), 6.34 (1H, dd, J = 18,
2
1
2
1
1
Hz, 3 -H trans to 3-C–H), 6.13 (1H, dd, J = 12,
135.4 (C4, 14), 134.8 (C3), 130.6 (C2), 129.4 (C3 ),
2
2
Hz, 3 -H cis to 3-C–H), 5.34, 5.23 (each 1H, d, J =
123.4 (C13), 121.8 (C3 ), 102.2, 102.2 (C10, 15), 98.7
3
4
9 Hz, 15-CH ), 4.41 (1H, q, J = 7 Hz, 18-H), 4.40 (1H,
(C5), 93.6 (C20), 53.0 (C13 ), 52.9 (C17), 52.1 (C15 ),
2
1
2
1
d, J = 10 Hz, 17-H), 4.19 (3H, s, 13-COOCH ), 3.77
49.4 (C18), 38.6 (C15 ), 30.6 (C17 ), 29.2 (C17 ), 22.9
3
2
1
1
2
1
1
(
2H, q, J = 7 Hz, 8-CH ), 3.564 (3H, s, 17 -COOCH ),
(C18 ), 19.7 (C8 ), 17.7 (C8 ), 12.4 (C12 ), 12.1 (C2 ),
2
3
1
3
7
.559 (3H, s, 12-CH ), 3.45 (3H, s, 2-CH ), 3.28 (3H, s,
11.3 (C7 ); HRMS (ESI) found: m/z 625.3021, calcd for
3
3
1
+
-CH ), 2.61–2.53 (1H, m, 17 -CH), 2.23–2.13 (2H, m,
C
36
H
41
N
4
O
6
: MH , 625.3021.
3
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 7–8

8
Y. NAGANO ET AL.
DM. Pharmaceuticals 2017; 10: 41. (g) Chen H,
Humble SW, Jinadasa RGW, Zhou Z, Nguyen
AL, Vicente MGH and Smith KM. J. Porphyrins
Phthalocyanines 2017; 21: 354–363. (h) Yaghini E,
Dondi R, Tewari KM, Loizidou M, Eggleston IM
and MacRobert AJ. Sci. Rep. 2017; 7: 6059. (i) Cao
L, Guo X, Wang L, Wang S, Li Y and Zhao W. New
J. Chem. 2017; 41: 14279–14287. (j) Otvagin VF,
Nyuchev AV, Kuzmina NS, Grishin ID, Gavry-
ushin AE, Romanenko YV, Koifman OI, Belykh
DV, Peskova NN, Shilyagina NY, Balalaeva IV
and Fedorov AY. Eur. J. Med. Chem. 2018; 144:
CONCLUSION
Three regioisomerically pure chlorin-e dimethyl esters
6
1a–1c were prepared by chemical modification of naturally
occurring chlorophyll-a. 13-Carboxylated chlorin-e6
1
2
1
5 ,17 -dimethyl ester (1a) was obtained by regioselective
1
1
methylation at the 15 - and 17 -carboxy groups of
1
chlorin-e over its 13-COOH. 15 -Carboxylated chlorin-e
6
6
2
1
3,17 -dimethylester(1b)waspreparedthroughprotection
1
of the 15 -carboxy group with p-methoxybenzyl ester.
2
1
17 -Carboxylated chlorin-e 13,15 -dimethyl ester (1c)
6
was synthesized by the E-ring cleavage of pheophorbide-a
under basic conditions. The molecular structures of the
three regioisomers were fully identified by 1D and 2D
NMR techniques including H– C HMBC. The synthetic
regioisomers showed almost the same visible absorption,
CD, and fluorescence emission spectra as well as emission
quantum yields and lifetimes in a diluted dichloromethane
solution. Small substitution effects were observed in Qy
absorption and fluorescence emission bands by methyl-
7
40–750. (k) Srdanović S, GaoY-H, Chen D-Y,Yan
Y-J, Margetić D and Chen Z-L. Bioorg. Med. Chem.
Lett. 2018; 28: 1785–1791. (l) Phuong PTT, Lee S,
Lee C, Seo B, Park S, Oh KT, Lee ES, Choi H-G,
Shin BS and Youn YS. Colloids Surf. B: Biointer-
faces 2018; 171: 123–133.
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13
2
. (a) Wang X-F and Tamiaki H. Energy Environ. Sci.
2
010; 3: 94–106. (b) Wang X-F and Kitao O. Mol-
1
esterification at the 13- and 15 -carboxy groups, but not
ecules 2012; 17: 4484–4497. (c) Wang X-F, Tami-
aki H, Kitao O, Ikeuchi T and Sasaki S. J. Power
Sources 2013; 242: 860–864.
. Hargus JA, Fronczek FR, Vicente MGH and Smith
KM. Photochem. Photobiol. 2007; 83: 1006–1015.
. Zhang L, Yao J-Z, Zheng H, Wang X-Y and Zhang
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2
at the 17 -COOH.
Acknowledgments
3
4
5
6
This work was partially supported by JSPS KAKENHI
Grant Number JP17H06436 in Scientific Research
on Innovative Areas “Innovation for Light-Energy
4
Conversion (I LEC).”
Supporting information
3
7: 4945–4948. (b) Furukawa H, Oba T, Tamiaki H
and Watanabe T. Bull. Chem. Soc. Jpn. 2000; 73:
341–1351.
Figures S1–S17 are given in the supplementary
material. This material is available free of charge via the
Internet at http://www.worldscinet.com/jpp/jpp.shtml.
1
7
8
9
. Tamiaki H, Hagio N, Tsuzuki S, Cui Y, Zouta T,
Wang X-F and Kinoshita Y. Tetrahedron 2018; 74:
4
078–4085.
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J. Porphyrins Phthalocyanines 2018; 22: 8–8