Photochemistry and Photobiology, 2007, 83 1009
404 (109,400); Mass Spectra (MALDI): m ⁄ z 768 (M + H)+ HRMS
131-Mono-(L)-aspartylchlorin-e6 tetramethyl ester (6). Chlorin-e6
dimethyl ester (14) (75 mg, 0.120 mmol) was dissolved in dry dichloro-
methane with 1 mL triethylamine. O-Benzotriazol-1-yl-N,N,N’,N’-te-
tramethyluronium hexafluorophosphate (HBTU, 136 mg, 0.36 mmol)
was added and the mixture was stirred until completely dissolved. Then
aspartic acid dimethyl ester was added. The reaction mixture was heated
under reflux for 6 h (or stirred overnight at room temperature), and after
cooling it was diluted with dichloromethane and then washed with 5%
aqueous citric acid, followed by a wash with brine and with water. It was
dried over anhydrous sodium sulfate and then evaporated. The residue
was dissolved in 2% methanol ⁄ dichloromethane and purified via silica
gel column chromatography, with the same mobile phase, to yield 50 mg
(51%) of 6, C42H49N5O9: mp 159–163ꢂC. UV–Vis (dichloromethane):
kmax (e ⁄ M)1 cm)1) 660 nm, 608, 558, 530, 500, 404; Mass Spectra
1
requires 767.87; found 767.947; H-NMR (CDCl3, 500 MHz ): d 9.61
(1H, s), 9.48 (1H, s), 8.67 (1H, s), 7.98 (1H, J = 11.6, 17.8 Hz), 6.29
(1H, dd, J = 17.8, 1.3 Hz), 6.06 (1H, br s), 6.06 (1H, dd, J = 11.6,
1.3 Hz), 5.22 (2H, s), 4.74 (1H, m), 4.37 (2H, m), 4.19 (3H, s), 3.68 (3H,
s), 3.71 (2H, q, J = 7.6 Hz), 3.59 (3H, s), 3.49 (3H, s), 3.45 (3H, s),
3.40 (3H, s), 3.22 (3H, s), 2.81 (1H, m), 2.59 (1H, m), 2.21 (2H, m), 1.81
(1H, m) 1.66 (3H, d, J = 5.2 Hz), 1.61 (3H, t, J = 7.6 Hz), )1.34 (s),
)1.48 (s); Anal. Calcd for C42H49N5O9: C, 65.69, H, 6.43, N, 9.12.
Found: C, 65.30, H, 6.47, N, 8.95.
Methyl pheophorbide-a (10). Method 1: Algal extract was treated
with 5% sulfuric acid in methanol (degassed by bubbling with argon) for
12.5 h at room temperature under argon and protected from light. It was
diluted with dichloromethane, washed with water and then with 10%
saturated aqueous sodium bicarbonate. The aqueous layer was dried
over sodium sulfate, filtered and then evaporated. Recrystallization of
the residue from dichloromethane and methanol gave the title product.
Method 2: Pheophorbide-a (12) (100 mg; 0.169 mmol), was treated
with excess ethereal diazomethane. Argon was flushed through the
flask and the solution was evaporated and recrystallized from
dichloromethane and methanol to quantitatively give 102 mg of the
product (10), C36H38N4O5; mp 218–122ꢂC [lit. mp (20) 224–226ꢂC].
UV–Vis (dichloromethane): kmax (e ⁄ M)1 cm)1) 668 nm (40,700), 610
(8100), 560 (3200), 538 (9400), 506 (10,400), 412 (93,400); 1H-NMR
(CDCl3, 300 MHz): d 9.50 (1H, s), 9.36 (1H, s), 8.57 (1H, s) 8.0 (1H,
m), 6.29 (1H, m), 6.26 (1H, s), 6.16 (1H, m), 4.46 (1H, m), 4.20 (1H,
m), 3.88 (3H, s), 3.70 (2H, q, J = 7.6 Hz), 3.68 (3H, s), 3.57 (3H, s),
3.41 (3H, s), 3.25 (3H, s), 2.63 (1H, m), 2.32 (1H, m), 2.52 (1H, m), 2.2
(1H, m), 1.81 (3H, d, J = 7.3 Hz) 1.69 (3H, t, J = 7.6 Hz), 0.53 and
)1.67 (2H, br, s).
(MALDI): m ⁄ z 768 (M + H)+ 1H-NMR (CDCl3, 300 MHz) d 9.70
;
(1H, s), 9.62 (1H, s), 8.78 (1H, s), 8.08 (1H, m), 6.35 (1H, dd, J = 17.8,
1.5 Hz), 6.12 (1H, dd, J = 11.5, 1.5 Hz), 5.56 (1H, d, J = 18.8 Hz),
5.48 (1H, J = 8.1, 4.6, 4.6 Hz) 5.39 d, J = 8.1), 4.45 (2H, m), 3.96 (3H,
s), 3.77 (3H, s), 3.80 (2H, q, J = 7.7 Hz), 3.66 (3H, s), 3.62 (3H, s), 3.60
(3H, s), 3.60 (3H, s), 3.50 (3H, s), 3.33 (3H, s), 2.55–2.16(4H m), 1.74 (m),
1.70 (3H, d J = 7.2 Hz), 1.68 (3H, t, J = 7.7 Hz), )1.47 (1H, s), )1.53
(1H, s).
152-Mono-(L)-aspartylchlorin-e6 tetramethyl ester (7). Chlorin-e6
(1) (75 mg, 0.126 mmol) was dissolved in dry dichloromethane with
1 mL triethylamine. HBTU (57 mg, 0.15 mmol) was added and stirred
until completely dissolved. Then (L)-aspartic acid dimethyl ester
hydrochloride was added. The reaction mixture was allowed to stir
for 2 h. The mixture was diluted with dichloromethane and then
washed with 5% aqueous citric acid, followed by a wash with brine
and with water. It was dried over anhydrous sodium sulfate and then
evaporated. The residue was dissolved in dichloromethane and treated
with excess ethereal diazomethane. The residue was dissolved in 2%
methanol ⁄ dichloromethane and purified via silica gel column chroma-
tography with the same mobile phase to afford 60 mg (61%) of 7,
C42H49N5O9: mp 157–160ꢂC. UV–Vis (dichloromethane): kmax
(e ⁄ M)1 cm)1) 660, 608, 558, 530, 500, 404; Mass Spectra (MALDI)
m ⁄ z 768 (M + H)+; 1H-NMR (CDCl3, 300 MHz ) d 9.62 (1H, s), 9.50
(1H, s), 8.69 (1H, s), 7.95 (1H, m), 6.30 (1H, dd, J = 17.8, 1.5 Hz),
6.07 (1H, dd, J = 11.5, 1.5 Hz), 5.22 (2H, br), 4.76 (1H, ddd, J = 8.1,
4.9, 4.4 Hz), 4.40 (1H, m), 4.19 (3H, s), 3.71 (2H, q, J = 7.3 Hz), 3.50
(3H, s), 3.49 (3H, s), 3.40 (3H, s), 3.31 (3H, s), 3.22 (3H, s), 3.01 (3H,
s), 2.78 (2H, dd, J = 16.8, 4.4 Hz) 2.56 and 2.21 (2H, m), 1.66 (2H,
m), 1.65 (3H, d, J = 7.3 Hz), 1.61 (3H, t, J = 7.7 Hz), )1.34 (1H, s),
)1.48 (1H, s).
Chlorin e6 trimethyl ester (11). Methyl pheophorbide-a (10)
(102 mg, 0.168 mmol), was dissolved in dry methanol and stirred
under argon for 10 min. Thereafter, 0.35 mL of a 0.5 M sodium
methoxide solution was added to the solution and it was allowed to stir
for 2 h at 0ꢂC. The solution was diluted with H2O and extracted with
dichloromethane. The organic layer was dried with sodium sulfate,
filtered and then evaporated. The solid obtained was dissolved in
dichloromethane and chromatographed on a plug of neutral alumina
(Brockmann grade III) with the same mobile phase. Chlorin-e6
trimethyl ester (11) was eluted with dichloromethane. After evapor-
ation, 105 mg (98%) of 11, C37H42N4O6 was obtained: mp 206–210ꢂC
[lit. mp (21) 210ꢂC]. UV–Vis (dichloromethane): kmax (e ⁄ M)1 cm)1
664 nm (49,600), 608 (9900), 530 (9900), 501 (17,600), 402 (154,200);
Mass Spectra (MALDI): m ⁄ z 639 (M + H)+ 1H-NMR (CDCl3,
)
;
500 MHz): d 9.63 (1H, s), 9.45 (1H, s), 8.71 (1H, s), 7.93 (1H, dd,
J = 11.4, 17.8), 6.25 (1H, dd, J = 17.8, 1.2 Hz), 6.03 (1H, dd,
J = 11.4, 1.2 Hz), 5.30 (2H, d, J = 18.9 Hz), 4.43 (1H, m), 4.39 (1H,
m) 4.24 (3H, s) 3.76 (3H, s), 3.68 (2H, q, J = 7.6 Hz), 3.56 (3H, s),
3.55 (3H, s), 3.39 (3H, s), 3.19 (3H, s), 2.55 (1H, m), 2.19 (2H, m), 1.75
(1H, m), 1.74 (3H, d, J = 7.4 Hz), 1.66 (3H, t, J = 7.6 Hz), )1.33
(1H, s), )1.47 (1H, s); 13C-NMR (CDCl3, 500 MHz instrument): d 174,
173.1, 169.6, 169.6, 167, 154.8, 148.9, 144.8, 139.5, 136.4, 134.7, 135.9,
135.3, 135.4, 130.5, 129.3, 129.3, 123.3, 121.2, 102.2, 102.1, 99, 93.6,
53.0, 52.9, 52.1, 51.6, 49.4, 38.7, 29.1, 27.6, 22.9, 19.6, 17.7, 12.1, 12.0,
11.3.
RESULTS AND DISCUSSION
Synthesis of 152-LS-11 (5)
152-LS-11 tetramethyl ester (7) was obtained by transesteri-
fication of the phytyl ester group of pheophytin-a (9) [i.e.
demetalated chlorophyll
a obtained by extraction from
S. pacifica alga], to form methyl pheophorbide-a (10). Sub-
sequent isocyclic ring-opening and treatment with diazometh-
ane formed chlorin-e6 trimethyl ester (11). Alkaline hydrolysis
of the methyl esters yielded chlorin-e6 (1). Activation and
coupling of chlorin-e6 to di-tert-butyl-protected aspartic acid
followed by deprotection with TFA yielded 152-LS-11 (5)
(Scheme 1).
Classical isocyclic ring-opening conditions require treat-
ment of methyl pheophorbide-a (10) with an excess of sodium
methoxide in tetrahydrofuran (19). To minimize loss of
product due to partial hydrolysis of the ester groups, the
solution is subsequently treated with diazomethane (prior to
chromatography) (19). Yields are extremely variable (30–60%)
under these conditions, but starting material is recoverable and
can be recycled. An improved ring-opening synthesis was
reported in 1980; chlorin-e6 trimethyl ester (11) was obtained
in 80% yield when chlorin-e6 (1) was treated with 0.5% KOH
Chlorin-e6 173,152-dimethyl ester (14). Chlorin-e6 (1) (75 mg,
0.126 mmol) was dissolved in 5% sulfuric acid and methanol and
allowed to stir protected from light, under argon overnight. The
reaction was poured into cold saturated aqueous sodium bicarbonate
and extracted twice with dichloromethane. The extract was washed
twice with brine, dried over anhydrous sodium sulfate and filtered. The
solvent was evaporated and re-dissolved in dichloromethane. It was
then purified on a silica gel column. Elution with 6% methanol and
dichloromethane gave a major green fraction that was collected. The
solvent was removed to afford 75 mg (95%) of 14, C36H40N4O6: mp
280–290ꢂC. UV–Vis (CHCl3): kmax (e ⁄ M)1 cm)1) 666 nm (49,700), 610
(5900), 562 (2700), 523 (5900), 502 (13,200) 402 (143,400); Mass
Spectra (MALDI): m ⁄ z 625 (M + H)+
;
1H-NMR (CDCl3,
300 MHz): d 9.65 (1H, s), 9.52 (1H, s), 8.72 (1H, s), 8.06 (1H, m),
6.32 (1H, dd, J = 17.8, 1.2 Hz), 6.13 (1H, dd, J = 11.5, 1.2 Hz), 5.50
(1H J = 18.6 Hz), 5.23 (1H, d, J = 18.6 Hz), 4.45 (1H, m), 3.82 (3H,
s), 3.76 (2H, q, J = 7.6 Hz), 3.60 (3H, s), 3.59 (3H, s), 3.46 (3H, s),
3.28 (3H, s), 1.69 and 2.12 (2H, m), 2.19 and 2.56 (2H, m), 1.81 (3H, d,
J = 7.1 Hz), 1.64 (3H, t, J = 7.6 Hz).