module, and a Grace-Vydac RPC18 column (model 218TP5210)
at a flow rate of 0.3 mL min-1. Gradients were run with
buffer A (H2O–0.1% trifluoroacetic acid (TFA)) and buffer B
(90% acetonitrile–10% H2O–0.1% TFA). For analytical HPLC a
C-18 reverse phase column (Varian) was used with dimensions of
250 mm ¥ 4.6 mm. For semi-preparative HPLC a C-18 reverse
phase column (Varian) was used with dimensions of 250 mm ¥
21.2 mm. High-resolution eletrospray ionization (ESI) mass spec-
tra were obtained from a Bruker Daltonics APEXIV 4.7 T Fourier
transform ion cyclotron resonance spectrometer (FT-ICR-MS) in
the Department of Chemistry Instrumentation Facility (DCIF) at
the Massachusetts Institute of Technology. All 1H NMR spectra
(500 MHz) and 13C NMR spectra (125 MHz) were collected
in the solvents noted. Porphyrin 121 and b-D-glucopyranuronoyl
chloride22,23 were synthesized as described previously. Iron oxide
nanoparticles were obtained from the chemistry core at the Center
for Molecular Imaging Research.
proceed for 48 hours, at which time it was evaporated to dryness.
The two isomers of the osmate ester were separated by flash
chromatography (silica gel, CH2Cl2–MeOH, 96 : 4), with fractions
containing the respective isomers combined and evaporated to
dryness. Each isomer was then dissolved in CH2Cl2 (20 mL) and
H2S was bubbled through the solution for 5 min. The reaction
mixtures were stoppered and reacted for 45 min, at which time
they were blown dry with a stream of nitrogen. The crude products
were then dissolved in CH2Cl2 and passed through celite to remove
any residual solids, and were again evaporated to dryness. The
final products were obtained by flash chromatography using the
solvent systems detailed below. Alternatively, the reaction can be
monitored by UV-vis spectroscopy in order to determine the extent
of reaction, allowing for the adjustment of reaction time.
4I. Flash chromatography (silica gel, CH2Cl2–MeOH, gradi-
ent from 97.5 : 2.5 to 95 : 5). Fractions containing the product
were combined and evaporated to dryness, dissolved in a minimal
volume of CH2Cl2 and precipitated with petroleum ether to give
chlorin 4I in 8% yield (0.07 g). UV-vis (DMF) lmax (log e): 424
(5.52), 523 (4.36), 552 (4.42), 597 (4.08), 648 (4.51) nm; 1H NMR
(500 MHz, CDCl3, d) -1.87 (s, 2H), 2.12–2.24 (br m, 41H), 2.57
(br, 4H), 3.4 (br, 2H), 4.42 (br m, 3H), 5.23 (br m, 3H), 5.49
(br m, 6H), 5.96 (br m, 3H), 6.25 (br 2H), 7.90 (br m, 17 H),
8.39 (br m, 11 H) ppm; 13C NMR (125 MHz, CDCl3, d) 20.5,
20.6, 20.7, 20.8, 20.8, 33.0, 51.7, 68.8, 70.3, 71.9, 73.5, 91.6, 112.4,
117.9, 119.3, 119.8, 124.1, 127.9, 132.5, 134.4, 136.1, 140.6, 164.2,
164.3, 169.0, 169.3, 169.6, 169.7, 169.9, 170.0, 173.9 ppm; +ESI-
MS (30 V, CH3CN–0.1% TFA) m/z = 1871.2 (MH+); HR-MS
(ESI of MH+, CH3CN): m/z calc’d for C92H93N8O35: 1870.5816,
found: 1870.5810; HPLC tR = 12.52 min (using a gradient of 60%
to 0% of buffer A over 25 min).
5,10,15-Tris(4-1¢,2¢,3¢,4¢-O-acetyl-glucopyranuron-N-
phenylamide)-20-[4-(5¢-methoxy-1¢,5¢-
dioxopentyl)aminophenyl]porphyrin, 3
To 1.04 g of porphyrin 1 (1.54 ¥ 10-3 mol) in tetrahydrofuran
(300 mL) at 0 ◦C was added triethylamine (Et3N, 5 equiv, 1.08 mL),
and methyl glutaryl chloride (1.1 equiv, 233 mL). The reaction was
◦
allowed to proceed at 0 C for 2 h, at which time an additional
5 equiv of Et3N were added (1.08 mL). To this solution was then
added b-D-glucopyranuronoyl chloride (4 equiv, 2.34 g), and the
reaction mixture was allowed to warm to room temperature over
the course of 1 h. The reaction mixture was then concentrated
to approximately 50 mL and then diluted with CH2Cl2 (100 mL).
The solution was washed once with water, once with saturated
aq NaHCO3, and once with water again. The organic layer was
subsequently dried over anhydrous MgSO4 and evaporated to
dryness. The product was purified by flash chromatography (silica
gel, CH2Cl2–MeOH, gradient from 98 : 2 to 96 : 4). All fractions
containing the product were combined and evaporated to dryness,
redissolved in CH2Cl2, and precipitated with hexanes to give the
product in 33% recovered yield (0.92 g). UV-vis (DMF) lmax (log
e): 424 (5.90), 519 (4.44), 556 (4.36), 595 (3.99), 648 (4.09) nm;
1H NMR (500 MHz, CDCl3, d) -2.84 (s, 2H), 2.08–2.28 (m,
41H), 2.57 (m, 4H), 4.38 (d, 3H, J = 9.5), 5.26 (t, 3H, J =
8.0), 5.44 (t, 3H, J = 9.0), 5.50 (t, 3H, J = 9.5), 5.94 (d, 2H,
J = 8.0), 7.87 (br m, 8H), 8.14 (br m, 9H), 8.31 (s, 3H), 8.82
(br m, 8H) ppm; 13C NMR (125 MHz, CDCl3, d) 20.7, 20.7,
20.8, 21.0, 21.0, 33.0, 33.1, 36.8, 51.9, 69.1, 70.6, 72.1, 73.6, 91.8,
118.0, 118.8, 135.2, 136.4, 139.0, 164.4, 169.0, 169.1, 169.4, 169.9,
170.0, 174.1 ppm; +ESI-MS (30 V, CH3CN–0.1% TFA) m/z =
1837.2 (MH+), 919.0 (MH2+); HR-MS (ESI of MH+, CH3CN):
m/z calc’d for C92H91N8O33: 1836.5761, found: 1836.5729; HPLC
tR = 11.40 min (using a gradient of 60% to 0% of buffer A over
25 min).
4II. Flash chromatography (silica gel, CH2Cl2–MeOH, 96 : 4).
Fractions containing the product were combined and evaporated
to dryness, dissolved in a minimal volume of CH2Cl2 and
precipitated with petroleum ether to give chlorin 4II in 12% yield
(0.10 g). UV-vis (DMF) lmax (log e): 424 (5.41), 523 (4.28), 552
(4.34), 597 (4.02), 648 (4.45) nm; 1H NMR (500 MHz, CDCl3, d)
-1.90 (s, 2H), 2.15 (m, 41H), 2.54 (s, 4H), 2.96 (br s, 2H), 4.42 (m,
3H), 4.59 (m, 6H), 5.30 (m, 3H), 5.96 (m, 3H), 6.30 (br s, 2H),
7.87 (br, 17H), 8.45 (m, 11H) ppm; 13C NMR (125 MHz, CDCl3,
d) 20.6, 20.8, 33.0, 36.5, 51.7, 68.9, 70.4, 71.9, 73.6, 91.7, 118.8,
122.2, 124.3, 128.0, 132.6, 134.4, 135.5, 136.2, 137.8, 140.7, 152.9,
161.5, 164.4, 169.0, 169.3, 169.7, 169.9, 170.8, 173.9 ppm; +ESI-
MS (30 V, CH3CN–0.1% TFA) m/z = 1871.3 (MH+); HR-MS
(ESI of MH+, CH3CN): m/z calc’d for C92H93N8O35: 1870.5816
found: 1870.5889; HPLC tR = 12.61 min (using a gradient of 60%
to 0% of buffer A over 25 min).
Deprotection of chlorins 4I and 4II. A sample of 4I or 4II
(~20 mg) in THF–MeOH (1 : 1, 3 mL) was cooled to 0 ◦C. A
cold solution of LiOH in water (1.5 mL, 0.8 M) was then slowly
added. The reaction mixture was stirred at 0 ◦C. The reaction was
monitored by HPLC. Upon complete hydrolysis of the protecting
groups (~2 h) the reaction mixture was poured into 30 mL cold
water. The pH of the resulting solution was raised to 4 by slow
addition of cold 0.1 M HCl. The resulting mixture was purified by
reverse phase column chromatography (C-18 cartridge, eluted with
a gradient of 100 to 0 of buffer A) followed by preparative HPLC
General synthesis of 2,3-vic-dihydroxy-5,10,15-tris(4-1¢,2¢,3¢,4¢-O-
acetyl-glucopyranuron-N-phenylamide)-20-[4-(5¢-methoxy-1¢,5¢-
dioxopentyl)aminophenyl]chlorins, 4I and 4II
To a stirring solution of 3 (~0.8 g) in CHCl3–pyridine (4 : 1,
150 mL) was added OsO4 (2.0 equiv.). The reaction was allowed to
3434 | Org. Biomol. Chem., 2009, 7, 3430–3436
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