Functionalization of chlorin e6 trimethylester towards potential amphiphilic photosensitizers for photodynamic therapy

Article abstract of DOI:10.1142/S1088424618501183

Chlorins (dihydroporphyrins) are considered, due to their ideal photophysical properties, as attractive photosensitizers for photodynamic therapy (PDT) of cancer and other therapeutic and diagnostic applications. Chlorophyll a, as a naturally occurring chlorin, forms an almost unlimited renewable resource for preparation of potential biologically active chlorin photosensitizers and fluorescence markers. To achieve amphiphilic photosensitizers which might be selectively enriched in tumor cells, we addressed linkage of per se lipophilic chlorophyll derivatives with carbohydrate based hydrophilic aminopolyols.

Full text of DOI:10.1142/S1088424618501183

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2019; 23: 1–8
DOI: 10.1142/S1088424618501183
Published at http://www.worldscinet.com/jpp/
Functionalization of chlorin e trimethylester towards
6
potential amphiphilic photosensitizers for photodynamic
therapy
a
b
c
a
Daniela Bauer , Hai Vu Nghiem , Doan Duy Tien , Johannes Stelten
a ◊
and Franz-Peter Montforts *
a
Institute of Organic and Analytical Chemistry, FB 2, University of Bremen, Leobener Str. NW2/C, D-28359
Bremen, Germany
Present address: Institute of Technology and Science, Ministry of Public Security, An Ninh industrial area,
b
Lai Yen, Hoai Duc, PO Box 13220, Hanoi, Vietnam
c
Present address: Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet,
Cau Giay, Hanoi, Vietnam
Received 17 November 2018
Accepted 18 December 2018
ABSTRACT: Chlorins (dihydroporphyrins) are considered, due to their ideal photophysical properties,
as attractive photosensitizers for photodynamic therapy (PDT) of cancer and other therapeutic and
diagnostic applications. Chlorophyll a, as a naturally occurring chlorin, forms an almost unlimited
renewable resource for preparation of potential biologically active chlorin photosensitizers and
fluorescence markers. To achieve amphiphilic photosensitizers which might be selectively enriched in
tumor cells, we addressed linkage of per se lipophilic chlorophyll derivatives with carbohydrate based
hydrophilic aminopolyols.
KEYWORDS: chlorin e , aminopolyols, photosensitizer, PDT, spirulina.
6
ideal renewable feedstock for production of chlorophyll
INTRODUCTION
a­derived chlorin photosensitizers [3,4]. Based on
Nature provides chlorophyll a 1 as a pigment of
previous procedures for isolation of chlorophyll a from
photosynthesis, the most prominent and widespread
representative among the class of natural and artificial
chlorins (Fig. 1) [1, 2]. Plants produce mixtures of chloro­
Spirulina, we improved the extraction technique and
made methyl pheophorbide a 2 available on a larger scale.
Transformation of methyl pheophorbide a 2 into chlorin
phyll a and b as pigments of photosynthesis. Isolation of
e trimethlylester 3 (Chl e tme) provides a robust key
6
6
pure chlorophyll a requires chromatographic separations.
In contrast, cyanobacteria like Spirulina platensis form
only chlorophyll a 1 as a photosynthesis pigment. The
possibility to obtain only chlorophyll a from Spirulina and
widespread cultivation plants for Spirulina in subtropical
and tropical areas around the Pacific Ocean form an
intermediate for further functionalization [2b]. To date,
studied chlorophyll a derived amphiphilic photosensi­
tizers like mono­L­aspartyl chlorin e were linked with
6
additional hydrophilicity generating carboxylic acid
functions to achieve overall amphiphilicity. Transport
and tissue enrichment of this type of photosensitizers
are substantially influenced by pH values of biological
targets [5]. Amphiphilic chlorins with pH­independent
hydrophilic moieties should overcome the problem of
pH influence. In cell experiments with diglucosylated
artificial chlorins, we demonstrated advantages of these
“neutral” amphiphilic photosensitizers [6].
◊SPP full member in good standing
*
Correspondence to: Franz­Peter Montforts, Institute of Organic
and Analytical Chemistry, FB2, University of Bremen, PO Box
3
30440, D­28334 Bremen, Germany, tel.: +49 421 218 63110,
email: mont@uni­bremen.de.
Copyright © 2019 World Scientific Publishing Company

2
D. BAUER ET AL.
Fig. 1. Chlorophyll a 1 and its direct transformation product methyl pheophorbide a 2
RESULTS AND DISCUSSION
Spirulina platensis which was cultivated in Vietnam
proved to be an effective source for extraction of pure
chlorophyll a 1. It was then further transformed via
methyl pheophorbide a 2 into chlorin e trimethyl ester 3.
6
Prior to extraction, the chlorophyll a content of Spirulina
biomass was determined by a procedure reported by
Lichtenthaler for determination of chlorophyll content of
higher plants [7]. Only Spirulina biomass batches of more
than 0.4% chlorophyll content were considered for the
extraction procedure. With Soxhlet thimbles of 325 mm
length and 80 mm in diameter, 700 g of Spirulina biomass
could be extracted to yield 4.2 g of methyl pheophorbide
a 2 according a 0.6% chlorophyll a content (Scheme 1).
Prior to extraction, the biomass was filled into the Soxhlet
thimble. The filled thimble was then placed into a Dewar
container of appropriate size, treated with liquid nitrogen
and subsequently transferred to the Soxhlet extractor.
The procedure simplifies manipulation of the biomass.
Scheme 1. Extraction of chlorophyll a 1 from Spirulina platensis
and its transformation into chl e tme 3. (a) (i) Dry spirulina mass,
6
acetone, N (liq.), ca. 60 min, then warm up to rt; (ii) acetone,
2
reflux, 24 h. (b) H SO conc., MeOH, Ar, rt, 20 h. (c) 1 M KOH
2
4
in MeOH, THF/CHCl , Ar, rt, 30 min (73%) or KOMe, MeOH,
3
acetone, Ar, rt, 30 min, (85%)
1
As expected and determined by HPLC and H­NMR
spectroscopy (see Supporting information), methyl pheo­
applications chlorin e of high purity is required. To
6
2
2
phorbide a 2 consisted of a mixture of 13 R/13 S
diastereomers in a 89:11 ratio. The mixture is formed in a
deprotonation–protonation equilibrium of the b­ketoester
moiety of isocyclic ring E. In a retro­Claisen condensation,
methyl pheophorbide a 2 was transformed into chlorin
e6 trimethylester [2b,8]. The reaction was performed
achieve a certain purity of chlorin e 4 it is necessary to
6
perform a reversed phase chromatography which is cost­
intensive on a technical scale. To avoid reversed phase
chromatography we considered preparation of chlorin
e6 13­monomethylester 5 as an alternative. Preparation
of chlorin e monomethylester 5 among different di­
6
with potassium hydroxide in MeOH/THF/CHCl to give
and mono­esters of chlorin e was reported by Conant
3
6
chlorin e trimethylester 3 in 73% yield. A higher yield
and Armstrong. [9]. Conant’s work demonstrated that a
6
of 85% could be achieved with potassium methoxide in
MeOH/acetone. On a larger scale the first variant might
chlorin e monomethylester could be formed by alkaline
6
hydrolysis from chl e tme 3 in low yield in a mixture with
6
be easier to perform. With chlorin e trimethyl ester 3, a
chl e 4 and other components. An unequivocal structure
6
6
robust crystalline key intermediate is available for further
determination of Conant’s chlorin e monomethylester
6
transformations. In numerous studies chlorin e 4 was
used for functionalization with respect to preparation of
sensitizers for PDT [5].
is missing due to at­that­time unavailable methods for
structure elucidation. In our case, hydrolysis of chl e6
6
tme 3 gave chlorin e 13­monomethylester 5 in high yield
6
Alkaline hydrolysis of chl e tme 3 yielded chlorin e
in high yield (Scheme 2). However, for pharmaceutical
under acidic reaction conditions. Without any chromato­
graphic purification the crude reaction product could be
6
6
4
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 2–8

FUNCTIONALIZATION OF CHLORIN e TRIMETHYLESTER TOWARDS POTENTIAL AMPHIPHILIC PHOTOSENSITIZERS
3
6
Scheme 2. Formation of chl e 4 and chl e mme 5 by selective hydrolysis. (a) KOH (15%) in MeOH, acetone, Ar, 40°C, 40 min,
6
6
then reflux, 2.5 h (99%). (b) 5 M HCl, H O, 90°C, 30 min (82%)
2
crystallized isothermally from CHCl / petroleum ether to
to the reduced reactivity of 13­carboxylic acid function.
A similar observation was also made before [11].
3
1
1
give chl e mme 5 of high purity. Comprehensive H, H­
6
homonuclear NMR­ (COSY­, NOESY­correlations) and
1
13
H, C­heteronuclear NMR­spectra (HSQC­, HMBC­
correlations) of chl e mme 5 (for details see Supporting
information) revealed unambiguously its constitution
with a methylester group in the 13 position.
6
CONCLUSION
Solubility of amphiphilic photosensitizers in water
plays a crucial role for their intravenous application as
aqueous solutions. Though chlorin polyoldiamides 7
and 8 are not soluble in pure water, solubility in aqueous
solution could be achieved by addition of 0.3% DMSO.
For medical applications this does not present any
problems because DMSO supplements are quite common
in medicine. In contrast to chlorin diamides 7 and 8,
The harsh alkaline and prolonged reaction conditions
for hydrolysis of chlorin e trimethelester 3 cleaved all
6
three ester groups to yield chlorin e 4, whereas under
6
carefully optimized acidic reaction conditions, only
5 and 17 ester functions were hydrolyzed to yield
chlorin e6 13­mono methylester 5 selectively. The
reactivity of the 13 carboxylic group is significantly
reduced by its conjugation with the electron donating
chlorin chromophore.
1
chlorin e polyoldiamide 6 exhibits good solubility in pure
6
water, obviously due to the free 13­carboxylic acid group.
With chlorin e 4 and chlorin e mme 5 in hand, the
synthesisofamphiphilicchlorinderivativeswasenvisaged
Scheme 3). Commercially available glucamine and L­1­
The absorption spectrum of chlorin e 13­methylester­
15 ,17 ­glucodiamide 7 (Fig. 2) shows the Soret band
6
6
6
2
3
(
at 401 nm and the long wavelength Q band at 663 nm.
y
amino­1­deoxy­arabitinol were chosen as hydrophilic
moieties to be linked to chlorin chromophores. L­1­
amino­1­deoxy­arabitinol was prepared from L­arabinose
according to a literature procedure [10]. After activation
of free carboxylic acid groups of chl e 4 and chl e mme
The spectrum of 7 is also characteristic for the other
chlorins which were investigated here. Comprehensive
photophysical studies were performed and are reported
elsewhere [12,13]. The most important photophysical pro­
perties for PDT in addition to long wavelength absorption
are fluorescence and singlet oxygen formation for tumor
6
6
5
, the subsequent reaction with glucose­ and arabinose­
based aminopolyols formed chlorin diamides 6, 7 and 8.
Linkage between chlorins and polyols via amide bonds
should stabilize the amphiphilic sensitizers towards
localization and tumor destruction respectively. For Chl e
6
2
3
mme 5, Chl e 13­methylester 15 ,17 ­glucodiamide 7 and
6
2
3
Chl e 13­methylester­15 ,17 ­arabodiamide 8, wavelength
6
possible hydrolysis in biological environments. Chlorin e
of fluorescence, quantum yields of fluorescence (F ) and
6
f
2
4
4
yielded 15 , 17 ­diamide, which could be expected due
singlet oxygen formation (F ) are summarized in Table 1.
D
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 3–8

4
D. BAUER ET AL.
Scheme 3. Formation of polyol amide derivatives of chl e 4 and chl e mme 5. (a) (i) iBuOCOCl, NEt , THF, ­15°C, ca. 15 min;
6
6
3
(
ii) glucamine, NEt , EtOH/H O, rt, 15 h (88%). (b) (i) iBuOCOCl, NEt , THF, ­15°C, 1 h; (ii) glucamine, NEt , EtOH/H O, rt, 15 h
3
2
3
3
2
(59%). (c) (i) iBuOCOCl, NEt , THF, ­15°C, 30 min; (ii) 1­amino­1­deoxy­arabitinol, NEt , EtOH/H O, rt, 15 h (61%)
3
3
2
mme diarab 8 incubation followed by irradiation with red
2
light (20 J/cm ) [12].
EXPERIMENTAL
General
Starting materials were either prepared according
to literature procedures or were purchased from Fluka,
Merck, Acros Organics or Sigma Aldrich and used
without further purification. All solvents were purified
and dried by standard methods. All reactions were
carried out under argon. Melting points are not
corrected. TLC: Silica gel plates (Macherey & Nagel,
Polygram SIL G/UV ) and aluminum oxide plates
2
3
Fig. 2. UV­vis spectrum of chlorin e 13­methylester­15 ,17 ­
diglucamide 7 in MeOH
6
254
As required for PDT photosensitizers, chlorins 5, 7 and
(Macherey & Nagel, Polygram Alox N/UV ). Column
254
8
show fluorescence at the Q band and exhibit sufficient
chromatographic separations were performed on silica
gel (32–63 mm, 60 Å, ICN), aluminum oxide (activity
grade II–III, neutral, ICN), Matrix silica gel (20–45 mm,
60 Å, Amicon) and silica gel 100, C18­reversed phase
(0.015–0.035 mm, Fluka). HPLC: Knauer HPLC
instrument with pump 64, two­channel potentiometer
y
values for quantum yield of singlet oxygen formation of
ca. F = 0.5.
Preliminary experiments with human colon
adenocarcinoma cells (HCT116p53) demonstrate IC50
values for phototoxicity of 0.22+/­0.03 mMol for chl e6
D
Table 1. Selected photophysical data of chlorin photosensitizers 5, 7 and 8
em
lf [nm]
Ff
FD
Ref.
[11]
[11]
[12]
5
7
8
Chl e mme
664 (MeCN)
666 (MeCN)
674 (DMSO)
0.14 (MeCN) 0.6 (CH Cl )
6
2
2
Chl e mme diglu
0.04 (MeCN) 0.5 (CH Cl )
2 2
6
Chl e mme diarab
0.25 (EtOH)
0.63 (EtOH)
6
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 4–8

FUNCTIONALIZATION OF CHLORIN e TRIMETHYLESTER TOWARDS POTENTIAL AMPHIPHILIC PHOTOSENSITIZERS
5
6
BBC Metrawatt Servogor 120 recorder and Knauer UV
spectrometer. UV­Vis: Varian Cary 50 spectrophotometer
and HP 8453 spectrophotometer diode array detection.
IR: Perkin–Elmer Paragon 500 FT­IR spectrometer.
NMR spectra: Bruker DPX­200 AVANCE, Bruker AM
acetone (15:1)] can be applied.) Elution with n­hexane
gaveayellow/redfractionofcarotenoids.Moreofayellow/
red fraction was eluted with n­hexane/CH Cl (1:1). After
2
2
elution with CH Cl and then with CH Cl /acetone (30:1)
2
2
2
2
the green main fraction of methyl pheophorbide a was
obtained. A final fraction eluted with MeOH was treated
with H SO /MeOH and it yielded after work up and
3
60 spectrometer and Bruker AMX 600 spectrometer.
All chemical shifts were referenced to TMS lock signal.
2
4
1
Exact assignment of proton signals in H NMR spectra
chromatography a minor amount of methyl pheophor­
bide a. The combined methyl pheophorbide a fractions
were crystallized from CH Cl /MeOH (ca. 1.5:10) to
was achieved by two dimensional COSY, NOESY,
HSQC and HMBC experiments. MS: EI: Finnigan MAT
2
2
8
222 [E (70 eV), 200°C] and ESI: Bruker ESQUIRE­LC
give dark green crystals of methyl pheophorbide a 2 as
2
2
[
3.8kV, 2 mL/min].
a 89:11 mixture of 13 R: 13 S diastereomers. Yield 4.2 g
(0.6% related to biomass), mp 226–228°C. lit. 206°C
[3a], 228°C [14]. TLC (cellulose, n­heptane/pyridine,
7:3) R = 0.75; (silica gel, CCl /acetone, 5:1) R = 0.41.
Isolation of methyl pheophorbide a 2 as an 89:11
2
2
mixture of 13 R:13 S diastereomers from Spirulina
platensis. Dry spirulina biomass (700 g) was filled into
a Soxhlet thimble (length 32.5 cm, diameter 8 cm) and
placed in a Dewar container of appropriate size. (To ensure
effective production of methyl pheophorbide a, only
spirulina biomass batches of more than 0.4% chlorophyll
content were considered for extractions.) Therefore prior
to extraction, the chlorophyll a content of the spirulina
biomass was determined according the procedure
described by Lichtenthaler [7]. Acetone (200 mL,
technical grade) was added, followed by slow addition
of liquid nitrogen (ca. 2 L). After complete addition
of liquid nitrogen, the Soxhlet thimble was transferred
into a Soxhlet extractor and warmed to 10–20°C. One
part of acetone (300 mL) was added to the extractor and
another part (1.5 L) was added into a 2 L separating flask
connected to the extractor. The solvent was refluxed
under an Ar atmosphere for ca. 24 h. During this time, 70
to 75 extraction cycles yielded a deep green chlorophyll
extract. After extraction was completed, the color of the
extraction thimble had changed from deep green to light
yellow. The acetone extract was filtered through cotton
wool to remove traces of biomass and the solvent was
removed in a rotavapor. The water­containing residue was
dissolved in CH Cl (200 mL) and the water was removed
f
4
f
HPLC [LiChrosorb Si 60 Å (Merck Milipore), CH Cl /
2
2
EtOAc, 92.5:7.5, 1.5 mL/min, UV detect. at 405 nm] R
t
2
2
(13 S) = 7.6 min, R (13 R) = 8.4 min. UV­vis (THF):
t
lmax, nm (e) 274 (15700), 322 (19700), 411 (106000),
470 (4300), 505 (11800), 535 (10600), 560 (3060), 609
­1
(8250), 668 (51100). IR (KBr): n, cm 2960 (m, CH),
1732 (s, CO ester), 1700 (s, CO keto), 1621 (m), 1498
(m), 1297 (m), 1203 (m), 1164 (s), 1035 (m), 991 (m).
+
MS (EI, 70 eV, 200°C): m/z (%) 606 (100) [M ], 548
(29) 459 (17), 236 (10). MS (ESI, positive, CH Cl /
2
2
+
+
MeOH, 1:10): m/z 607 [M + H] , 629 [M + Na] , 645
[M + K] . MS (ESI, negative, CH Cl /MeOH, 1:10): m/z
+
2
2
­
605 [M–H] .
Methyl 17S,18S-[8-ethyl-17-(2-methoxycarbonyl-
ethyl)-15-methoxycarbonylmethyl-2,7,12,18-tetra-
methyl-3-vinyl-17,18-dihydro-21H,23H-porphyrin-
13-yl]carboxylate (chlorin e trimethylester) (3). Variant
6
A: Methyl pheophorbide a 2 (1.7 g, 2.8 mmol was
dissolved under an Ar atmosphere in degassed dry THF
(20 mL) and CHCl (100 mL) was added followed by
3
1 M KOH in MeOH (10 mL). The mixture was stirred
with exclusion of light for 30 min at room temperature.
The reaction mixture was poured into ice water (400 mL)
and extracted twice with diethyl ether (100 mL
portions). The combined organic layers were washed
four times with water (200 mL portions) and dried by
filtration through warm cotton wool. The solvent was
evaporated in a rotavapor and the residue purified by
column chromatography [200 g silica gel, 32–63 mm,
60 Å, CH Cl /petroleum ether/acetone (10:10:1)]. After
2
2
by azeotropic distillation in a rotavapor. The residue was
carefully dried in vacuo using an oil pump, suspended in
dry MeOH (500 mL) and concentrated H SO (25 mL)
was cautiously added. The mixture was stirred for 20 h
at room temperature under an Ar atmosphere with
exclusion of light. To the mixture were added CH Cl
2
4
2
2
(
400 mL), H O (800 mL), saturated aqueous solution of
2
2
2
NaCl (200 mL) and a 10% aqueous solution of NaHCO3
300 mL). The organic layer was separated and the
aqueous layer extracted three times with CH Cl (200 mL
removal of the eluent, the residue was crystallized from
acetone/MeOH to give black­blue crystals of chlorin e6
trimethylester 3. Yield 1.3 g (73%), mp (acetone/MeOH)
210°C, lit. 211°C [15].
Variant B: Methyl pheophorbide a 2 (151.6 mg,
0.25 mmol) was dissolved under an Ar atmosphere in
THF (10 mL) and then MeOH (100 mL) was added
(
2
2
portions). The combined organic extracts were washed
twice with water (500 mL portions), dried by filtration
through cotton wool and evaporated in a rotavapor. The
red crude extract was carefully dried in vacuo using an oil
pump, dissolved in a small amount of CH Cl (maximum
followed by KOCH in MeOH (2 mL 35% solution). The
2
2
3
4
0 mL) and subjected to column chromatography (300 g
mixture was stirred with exclusion of light for 30 min at
room temperature. The reaction was monitored by TLC.
After the reaction was complete, the mixture was poured
into water (400 mL) and extracted twice with diethyl
silica gel, 32–63 mm, 60 Å). (Alternatively, column
chromatography with Alox [400 g Alox N, activity II–
III; eluent: n­hexane to n­hexane/CH Cl (1:1) to CH Cl /
2
2
2
2
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 5–8

6
D. BAUER ET AL.
ether (100 mL portions). The combined organic extracts
were washed four times with water (200 mL portions) and
dried by filtration through cotton wool. The solvent was
removed in a rotavapor and the crude product purified
by column chromatography [40 g silica gel, 32–63 mm,
(e) 286 (14320), 401 (139600), 502 (11450), 607 (4294),
1
661 (40380). H­NMR (600 MHz, CDCl /pyridine­D )
3
5
d, ppm ­0.9, ­1.8 (2s, 2H, 21­NH, 23­NH), 1.73 (m, 4H,
2
1
2
8 ­CH , 17 ­CH), 2.28 (m, 2H, 17 ­CH ), 2.67 (m, 1H,
3
2
1
17 ­CH), 3.29 (s, 3H, 7­CH ), 3.50 (s, 3H, 2­CH ), 3.63
,
3
3
1
6
0 Å, CH Cl /EtOAc (7:1). After removal of the eluent,
(s, 3H, 12­CH ), 3.8 (q, J = 7.2 Hz, 2H, 8 ­CH ), 4.62 (dd,
2
2
3
2
1
crystallization of the fraction from acetone/MeOH
2H, 17­CH, 18­CH), 5.5 (q, 2H, J = 7.8 Hz, 15 ­CH ),
2
2
gave black­blue crystals of chlorin e trimethylester 3.
6.15, 6.17 (dd, 2H, 3 = CH ), 8.19, 8.21, 8.24, 8.25 (dd,
6
2
1
Yield 135 mg (85%), mp (acetone/MeOH) 207–208°C.
1H, 3 ­CH), 9.05 (s 1H, 20­CH), 9.67 (s, 1H, 5­CH), 9.8
TLC (silica gel, CH Cl /EtOAc, 7:1): R = 0.59. HPLC
(s, 1H, 10­CH). MS (ESI, positive, MeOH): m/z 597 [M +
2
2
f
+
­
[
LiChrosorb Si 60 Å (Merck Milipore), CH Cl /EtOAc,
H] . MS (ESI, negative, MeOH): m/z 595 [M–H] .
2
2
9
2.5:7.5, 1 mL/min, UV detect. at 405 nm]: R = 5.85 min.
Methyl 17S,18S-[8-ethyl-17-(2-carboxy-ethyl)-15-
carboxymethyl-2,7,12,18-tetramethyl-3-vinyl-17,18-
dihydro-21H,23H-porphyrin-13-yl]carboxylate (chlorin
e6 monomethylester) (5). Chlorin e6 trimethylester
3 (200 mg, 0.31 mmol) was suspended under an Ar
atmosphere in 5 M aqueous HCl (50 mL) and heated
to 90°C for 30 min. After cooling to room temperature,
ice water (200 mL) was added to the mixture and it was
exhaustively extracted with EtOAc (total 1.5 L). The
combined organic extracts were washed three times with
water (200 mL portions), dried by filtration through cotton
wool and evaporated in a rotavapor. The crude product
t
UV­vis (MeOH): lmax, nm (e) 303 (9540), 400 (162620),
00 (20510), 605 (11570), 660 (51100). UV­vis (THF):
lmax, nm (e) 303 (9540), 401 (123500), 500 (11260), 529
4680), 558 (1690), 609 (4520), 665 (43600). IR (KBr):
5
(
­
1
n, cm 3296 (w, NH), 2950 (m, CH), 2914 (w), 1731
s, CO ester), 1601 (m), 1440 (m), 1240 (m), 1165 (m),
(
1
1
064 (m). H­NMR (200 MHz, CDCl ): d, ppm ­1.42
3
(m, br, 21­NH, 23­NH), 1.61, 1.72, 1.77, 1.81 (m, 7H,
2
1
1
8
1
­CH , 18­CH , 17 ­CH), 2.16–2.61 (m, 3H, 17 ­CH,
3 3
2
7 ­CH ), 3.32 (s, 3H, 7­CH ), 3.48 (s, 3H, 2­CH ), 3.59
2
3
3
(s, 3H, 12­CH ), 3.65 (s, 3H, 17­COOCH ), 3.79 (s, 3H,
3
3
1
2
6
1
5­COOCH ), 4.26 (s, 3H, 13­COOCH ), 4.4–4.49 (t,
was isothermally crystallized from CHCl /petroleum
3
3
3
1
H, 17­CH, 18­CH), 5.19–5.43 (q, 2H, 15 ­CH ), 6.15–
ether to give chlorin e monomethylester 5 as dark violet
2
6
2
1
.41 (q, 2H, 3 = CH ), 8.0–8.15 (q, 1H, 3 ­CH), 8.8 (s,
H, 20­CH), 9.6 (s,1H, 5­CH), 9.75 (s, 1H, 10­CH). MS
crystals.Yield 155 mg (82%), mp (CHCl /petroleum ether)
2
3
> 230°C. UV­vis (DMSO): lmax, nm (e) = 403 (113250),
+
(EI, 70 eV, 200°C): m/z (%) 638 (100) [M] , 579 (13)
501 (10200), 530 (4170), 560 (2160), 608 (4090), 663
+
+
­1
[M­COOCH ] , 565 (26) [M – CH COOCH ] , 479 (20),
(26900). IR (KBr): n, cm 3420 (m), 2960 (m, C­H),
3
2
3
2
89 (7), 236 (7). MS (DCI, negative, NH 8 mA/sec): m/z
2920 (m, C­H), 2860 (m, C­H), 1720 (s, CO), 1600 (s,
3
,
­
(%) 641 (3) 640 (15), 639 (52), 638 (100) [M] . MS (ESI,
CO), 1440 (m), 1380 (m), 1255 (m), 1215 (m), 1065 (m),
+
1
positive, CH Cl /MeOH, 1:10): m/z 639 [M + H] . MS
800 (w), 730 (w), 670 (w). H­NMR (600 MHz, CDCl /
2
2
3
­
3
2
(ESI, negative, CH Cl /MeOH): m/z 637 [M–H] .
pyridine­D ): d, ppm 1.53 (t, J = 7.2 Hz, 3H, 8 ­CH ),
2
2
5
3
2
1
7S,18S-[8-Ethyl-17-(2-carboxy-ethyl)-15-carboxy-
methyl-2,7,12,18-tetramethyl-3-vinyl-17,18-dihydro-
1H,23H-porphyrin-13-yl]carboxylate (chlorin e ) (4).
1.61 (d, J = 8.0 Hz, 3H, 18­CH ), 1.67, 2.21 (2s, 2H,
3
1
2
17 ­CH ), 2.16, 2.50 (m, 2H, 17 ­COCH ), 3.10 (s, 3H,
2
2
2
7­CH ), 3.26 (s, 3H, 2­CH ), 3.39 (s, 3H, 12­CH ), 3.59 (q,
6
3 3 3
2
1
Chlorin e trimethylester 3 (147 mg, 0.23 mmol) was
dissolved under an Ar atmosphere in degassed acetone
J = 7.2 Hz, 2H, 8 ­CH ), 4.07 (s, 3H, 13­COOCH ), 4.36
3
6
2
3
(q, J = 8.0 Hz, 1H, 18­CH), 4.50 (d, 1H, 17­CH), 5.24,
3
1
(10 mL) and degassed 15% aqueous KOH (10 mL)
5.31 (q, J = 7.8 Hz, 2H, 15 ­CH ), 5.94, 6.16 (dd, 2H,
2 1
2
was added. The mixture was stirred at 40°C for 40 min
under an Ar atmosphere with exclusion of light and then
refluxed for 2.5 h. After cooling to room temperature,
the mixture was poured into water (90 mL) and 2 M
HCl (12 mL) and stored overnight in a refrigerator. The
formed micro­crystalline precipitate was separated by
centrifugation. The precipitate was suspended three times
in water (10 mL portions) and centrifugated. Water was
completely removed by freeze drying and a dark green
3 = CH ), 7.89 (dd, 1H, 3 ­CH), 8.62 (s, 1H, 20­CH), 9.41
13
2
(s, 1H, 5­CH), 9.51 (s, 1H, 10­CH). C­NMR (90 MHz,
CDCl /pyridine­D ): d, ppm 10.5 (7­CH ), 11.4 (2­CH ),
3
5
3
3
1
11.5 (12­CH ), 16.8 (8­CH ), 18.9 (8 ­CH ), 22.5 (18­
CH ), 29.4 (17 ­CH ), 31.1 (17 ­COCH ), 38.4 (15 ­CH ),
3
3
2
1
2
1
3
2
2
2
48.8 (18­CH), 52.3 (13­OCH ), 52.5 (17­CH), 93.2 (20­
3
CH), 97.8 (5­CH), 101.1 (10­CH), 103.2 (15­C), 121.0
2
1
(3 ­CH ), 123.3 (13­C), 128.9 (11­N=C), 129.0 (3 ­CH),
2
129.8 (2­C), 133.9 (3­C), 134.4 (4­N=C), 135.2 (14­N=C),
135.3 (7­C), 135.8 (12­C), 138.5 (1­N=C), 144.5 (8­C),
148.2 (9­N=C), 153.9 (6­N=C), 167.6 (16­N=C), 169.2
(13­C=O), 169.2 (19­N=C), 174.7 (15­COOH), 175.4 (17­
COOH). MS (ESI, positive, CH Cl /MeOH, 1:10): m/z
powder of quite pure chlorin e 4 was obtained. Yield
6
125 mg (90%), mp > 230°C. For further transformation
the purity of 4 is sufficient. To achieve chlorin e of higher
purity, a tedious MPL chromatography is required [Silica
6
2
2
+
gel RP­8 (Merck#11447, 40–63 mm, L 240 mm x diameter
611 [M + H] .
10 mm), MeOH/CH Cl /H O (4:3:1), 2 mL/min]. HPLC
17S,18S-{8-Ethyl-17-[2-(glucitol-1-yl-carbamoyl)-
ethyl]-15-(glucitol-1-yl-carbamoyl)-methyl-2,7,12,18-
tetramethyl-3-vinyl-17,18-dihydro-21H,23H-porphyrin-
2
2
2
[LiChrosorb 100 RP­18 (Merck Milipore), MeOH,
Bu NH PO (2.5 mmol/L) H O, 95:5, 1 mL/min, UV
4
2
4
2
detect. at 405 nm]: R = 4.8 min. UV­vis (MeOH) lmax, nm
13-yl}-carboxylate (chlorin e glucodiamide) (6). Chlorin
t
6
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 6–8

FUNCTIONALIZATION OF CHLORIN e TRIMETHYLESTER TOWARDS POTENTIAL AMPHIPHILIC PHOTOSENSITIZERS
7
6
9
10
e6 4 (45 mg, 0.075 mmol) was dissolved in dry THF
20 mL) and NEt (0.36 mL) was added. The solution
3.30 (s, 3H, 7­CH ), 3.35/3.53 (m, 4H, 15 ­OCH , 17 ­
3
2
5
8
5
9
(
OCH ), 3.40–3.70 (m, 8H, 15 –15 ­OCH, 17 –17 ­OCH),
3
2
was cooled to ­15°C and i­BuOCOCl (0.3 mL, 2.3 mmol)
in dry THF (5 mL) was slowly added. The mixture was
stirred at ­15°C for 15 min until the anhydride had
formed completely. Glucamine (150 mg, 0.83 mmol) in
EtOH (1.6 mL), NEt (1.0 mL) and H O (1.0 mL) were
3.52 (s, 3H, 2­CH ), 3.54 (s, 3H, 12­CH ), 3.81 (q, 2H,
3
3
1
5
8 ­CH ), 4.19 (s, 3H, 13­OCH ), 4.20–4.70 (m, 10H, 15 ­
2
3
9
6
10
15 ­OH, 17 –17 ­OH), 4.35 (d, 1H, 17­CH), 4.57 (m, 1H,
18­CH), 5.08 (s, 2H, 15 ­CH ), 6.19/6.46 (2d, 2H, 3 =
CH ), 7.71 (t, 1H, 15 ­NH), 7.79 (t, 1H, 17 ­NH), 8.31 (dd,
1H, 3 ­CH), 9.08 (s, 1H, 20­CH), 9.69 (s, 1H, 5­CH), 9.77
(s, 1H, 10­CH). C­NMR (90 MHz, DMSO­D ): d, ppm
10.8 (7­CH ), 11.7 (12­CH ), 11.9 (2­CH ), 17.6 (8 ­CH ),
18.6 (8 ­CH ), 22.8 (18­CH ), 30.4 (17 ­CH ), 32.5 (17 ­
CH ), 39.0 (15 ­CH ), 41.8 (15 ­NCH ), 42.4 (17 ­NCH ),
48.3 (18­CH), 52.7 (17­CH), 53.1 (13­OCH ), 63.1 (15 ­
1
2
2
3
4
3
2
2
1
added dropwise and the mixture was stirred at room
temperature for 15 h. The mixture was concentrated in a
1
3
6
2
rotavapor and the residue dissolved in H O (ca. 10 mL).
2
3
3
3
3
1
1
2
After washing the aqueous phase with EtOAc (ca. 10 mL),
water was removed by lyophilization. The residue was
purified by reversed phase column chromatography [RP­
2 3 2
1
4
5
2
2
2
2
10
3
1
0
5
8
6
9
1
8 SiO (Fluka), MeOH/H O (4:3) — MeOH]. After
OCH , 17 ­OCH ), 69.4–74.4 (15 –15 ­OCH, 17 ­17 ­
2
2
2 2
removal of the eluent in a rotavapor and in vacuo using
OCH), 94.1 (20­CH), 98.2 (5­CH), 101.4 (10­CH), 122.2
2
1
an oil pump, chlorin e glucodiamide 6 was obtained as
(3 = CH ), 129.3 (3 = CH). MS (ESI, positive, MeOH):
6
2
+
+
a green powder. Yield 61 mg (88%), mp. > 230. UV/vis
m/z 959 [M + Na] , 975 [M + K] . (ESI, negative, MeOH):
m/z 971 [M + Cl] .
­
(
(
(
MeOH): lmax, nm (e) = 400 (102000), 499 (9200), 530
­1
5500), 607 (4800), 662 (29300). IR (KBr): n, cm 3410
Methyl 17S,18S-{8-ethyl-17-[2-(arabitol-1-yl-carba-
moyl)-ethyl]-15-(arabitol-1-yl-carbamoyl)-methyl-
2,7,12,18-tetramethyl-3-vinyl-17,18-dihydro-21H,23H-
s, br, OH, NH), 2965 (m, C­H), 2935 (m, CH), 2850 (m,
C­H), 1690 (m, C=O carboxyl), 1620 (s, C=O amide),
540 (m, C=C), 1470 (m, CH), 1430 (m), 1265 (m,
C­O), 1160 (w), 1080 (m), 1030 (w), 725 (m). MS (ESI,
1
porphyrin-13-yl}-carboxylate (chlorin e monomethyl-
6
eseter arabodiamide) (8). Chlorin e monomethylester 5
6
+
+
positive, MeOH): m/z 872 [M + H] , 899 [M + Na] , 915
(39 mg, 0.063 mmol) was dissolved in dry THF (10 mL)
+
­
[
M + K] ; (ESI, negative, MeOH): m/z 911 [M + Cl] .
Methyl 17S,18S-{8-ethyl-17-[2-(glucitol-1-yl-carba-
and NEt (0.35 mL) was added. The solution was cooled to
3
­15°C and i­BuOCOCl (0.25 mL, 1.9 mmol) in dry THF
(5 mL) was slowly added. The mixture was stirred at ­15°C
for 30 min until the anhydride had formed completely.
L­1­amino­1­deoxy­arabitinol [10] (200 mg, 1.9 mmol)
in EtOH (0.8 mL), NEt (0.5 mL) and H O (0.6 mL)
moyl)-ethyl]-15-(glucitol-1-yl-carbamoyl)-methyl-
2
2
,7,12,18-tetramethyl-3-vinyl-17,18-dihydro-21H,
3H-porphyrin-13-yl}carboxylate (chlorin e monome-
6
thylester glucodiamide) (7). Chlorin e monomethylester
6
3
2
5
(20 mg, 0.033mmol) was dissolved in dry THF (5 mL)
were added dropwise and the mixture was stirred at room
temperature for 15 h. The mixture was concentrated in a
and NEt (0.12 mL) was added. The solution was cooled
3
to ­15°C and i­BuOCOCl (0.2 mL, 1.5 mmol) in dry THF
rotavapor and the residue dissolved in H O (ca. 10 mL).
2
(
­
5 mL) was slowly added. The mixture was stirred at
15°C for 1 h until the anhydride had formed completely.
After washing the aqueous phase with EtOAc (ca. 10 mL),
water was removed by lyophilization. The residue was
purified by reversed phase column chromatography [RP­
18 SiO (Fluka), MeOH/H O (1:1) – MeOH/H O (2:1)
Glucamine (60 mg, 0.33 mmol) in EtOH (0.5 mL) and H O
2
(0.5 mL), NEt (0.5 mL) and H O (0.5 mL) were added
3
2
2
2
2
dropwise and the mixture was stirred at room temperature
for 15 h. The mixture was concentrated in a rotavapor
– MeOH]. After removal of the eluent in a rotavapor and
in vacuo using an oil pump, chlorin e monomethylester
6
and the residue was dissolved in H O (ca. 10 mL). After
arabodiamide 8 was obtained as a green powder. Yield
34 mg (61%), mp. > 230°C. UV/vis (MeOH): lmax, nm
(e) 400 (103000), 501 (9400), 532 (5700), 607 (5000), 663
2
washing the aqueous phase with EtOAc (ca. 10 mL),
water was removed by lyophilization. The residue was
purified by reversed phase column chromatography [RP­
­1
(29000). IR (KBr): n, cm 3395 (s, br, OH, NH), 2945 (m,
CH), 2910 (m, CH), 2850 (m, CH), 1710 (m, C=O ester),
1600 (s, C=O amide), 1540 (m, C=C), 1440 (m, C­H), 1385
1
8 SiO (Fluka), MeOH/H O (1:1) – MeOH/H O (2:1) –
2 2 2
MeOH]. After removal of the eluent in a rotavapor and
1
in vacuo using an oil pump, chlorin e monomethylester
(m), 1240 (m, CO), 1070 (s), 725 (m). H­NMR (200 MHz,
6
glucodiamide 7 was obtained as a green powder. Yield
DMSO­D ): d, ppm ­1.59/­1.83 (2s, 2H, 21­NH, 23­NH),
6
1
2
1
8 mg (59%), mp. 132–133°C. UV/vis (MeOH): lmax
,
1.65/2.14 (t, 2H, 17 ­CH ), 1.65 (m, 6H, 18­CH , 8 ­CH ),
2
3
3
2
4
nm (e) = 401 nm (102000), 500 (9300), 531 (5800), 606
2.84 (t, 2H, 17 ­CH ), 3.03 (m, 2H, 15 ­NCH ), 3.16 (m,
2
2
­1
5
8
(
5100), 663 (28000). IR (KBr): n, cm 3395 (s, br, O­H,
2H, 17 ­NCH ), 3.19 (s, 3H, 7­CH ), 3.47 (m, 4H, 15 ­
2
3
9
5
7
5
N­H), 2945 (m, C­H), 2910 (m, C­H), 2850 (m, C­H),
OCH , 17 ­O­CH ), 3.40–3.60 (m, 6H, 15 –15 ­OCH, 17 –
2
2
8
1
710 (m, C=O ester), 1670 (s, C=C), 1540 (m, C=C),
17 ­OCH), 3.49 (s, 3H, 2­CH ), 3.52 (s, 3H, 12­CH ), 3.75
3
3
1
1
440 ( C­H), 1385 (m), 1240 (m, C­O), 1070 (s), 725 (m).
(q, 2H, 8 ­CH ), 4.09 (s, 3H, 13­OCH ), 4.20–4.50 (m, 8
2 3
1
5
8
6
9
H­NMR (600 MHz, DMSO­D ): d, ppm ­1.58/­1.81 (2s,
H, 15 –15 ­OH, 17 –17 ­OH), 4.35 (d, 1H, 17­CH), 4.45
6
1
1
2H, 21­NH, 23­NH), 1.58/2.11 (t, 2H, 17 ­CH ), 1.67 (m,
(d, 1H, 18­CH), 5.02 (m, 2H, 15 ­CH ), 6.20/6.49 (2d, 2H,
2
2
2
2
2
3
4
6H, 18­CH , 8 ­CH ), 2.08/2.42 (m, 2H, 17 ­CH ), 3.02/
3 = CH ), 7.77 (t, 1H, 15 ­NH), 7.83 (t, 1H, 17 ­NH), 8.31
3
3
2
2
4
5
2
3.24 (m, 2H, 15 ­NCH ), 3.09/3.40 (m, 2H, 17 ­NCH ),
(dd, 1H, 3 = CH), 9.06 (s, 1H, 20­CH), 9.69 (s, 1H, 5­CH),
2
2
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 7–8

8
D. BAUER ET AL.
9
.75 (s, 1H, 10­CH). MS (ESI, positive, MeOH): m/z 872
4. Ryan AA and Senge MO. Photochem. Photobiol.
Sci. 2015; 14: 638–660.
5. (a) Bonnett R. Chemical Aspects of Photodynamic
Therapy, Gordon and Breach Science Publishers:
Amsterdam, 2000. (b) Nymann ES and Hynninen
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+
+
+
[
M + H] , 899 [M + H] , 915 [M + K] ; (ESI, negative,
­
MeOH): m/z 911 [M + Cl] .
Acknowledgments
We thank Dr. Thomas Dülcks, Dipl.­Ing Doris
Kemken, (Institute of Organic and Analytical Chemistry,
Laboratory Prof. Dr. D. Leibfritz, University of Bremen)
for Mass spectrometry measurements. In particular we
are indebted to Mrs. A. Lincke for performing analytical
and preparative HPLC separations. A generous gift of
Spirulina platensis (Vietnam) was provided by Hidumi
Pharma, Hamlet 6, Quynh Luong, Quynh Luu District,
Nghe An Province, Vietnam. This work was supported by
the FIRST program FIRST/2b1/Hidumi/C03.
(
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1768: 2748–2756. (i) Luciano M and Brückner C.
Molecules 2017; 22: 980/1–980/47.
Comprehensive NMR data for 2, 3, 4, 5 and 7 are given
in the supplementary material. This material is available
free of charge via the Internet htpp://www.worldscinet.
com/jpp/jpp.shtml.
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J. Porphyrins Phthalocyanines 2019; 23: 8–8