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5
oxyphenylporphyrin 1. In the dark under a nitrogen atmos-
.1.4. Synthesis of [c-(6-aminohexyl)folic acid]-4-carb-
TFA was evaporated under high vacuum. The residue
was diluted in CH Cl and anhydrous potassium car-
2
2
ꢀ2
phere, to a solution of 7.8 mg of 5 (1.45 · 10 mmol)
in anhydrous DMSO (0.7 mL) and pyridine (0.3 mL)
were added N-hydroxysuccinimide activated porphyrin
bonate was added until a color change from green to
purple red. After filtration, the organic layer was con-
centrated to afford 60 mg (95%) of compound 9. This
ꢀ
2
1
6
(10.9 mg, 1.45 · 10 mmol). After been stirred at
material was used without further purification;
NMR (CDCl3):
H
8.78–8.64 (m, 8H), 8.18 (d,
ambient temperature for 24 h, the mixture was gradually
poured into a vigorously stirred solution of anhydrous
d
J = 7.6 Hz, 2H), 8.12–8.01 (m, 8H), 7.67–7.50 (m, 9H),
7.23 (br t, 1H, NH), 3.77–3.63 (m, 4H), 3.63–3.49 (m,
4H), 3.43 (t, J = 4.8 Hz, 2H), 2.78 (br s, 2H), 1.92 (br
t, 2H), ꢀ2.87 (s, 2H, NH).
Et O (20 mL) cooled to 0 ꢁC. The dark red precipitate
2
obtained was collected by filtration, washed with Et O
and CH Cl and dried under high vacuum to yield
2
2
2
1
6
8
7
.5 mg (34%) of compound 1; H NMR (DMSO-d ): d
6
.89–8.77 (m, 7H), 8.64 (s, 1H), 8.32–8.17 (m, 7H),
.90–7.78 (m, 13H), 7.66 (d, J = 8.8 Hz, 2H), 6.95 (br
5.1.8. Synthesis of {c-{N-{2-[2-(2-aminoethoxy)eth-
oxy]ethyl}folic acid}}-4-carboxyphenylporphyrin 2. In
the dark under a nitrogen atmosphere, to a solution of
s, 2H, NH), 6.64 (d, J = 8.8 Hz, 2H), 4.48 (d,
J = 6.0 Hz, 2H), 4.93–4.75 (m, 1H), 3.04 (br td, 2H),
1
7
N-hydroxysuccinimide activated folic acid 10
(10.5 mg, 0.019 mmol) in anhydrous DMSO (0.7 mL)
and pyridine (0.3 mL) was added compound 9
2.76 (d, J = 6.2 Hz, 2H), 2.08–1.60 (m, 4H), 1.56–1.18
(
m, 8H), ꢀ2.96 (s, 2H, NH); UV–vis(MeOH): 286 nm
(e = 35,025), 414 nm (e = 118,763), 512 nm (e = 5823),
(15.3 mg, 0.019 mmol). After been stirred at ambient
temperature for 24 h, the mixture was gradually poured
into a vigorously stirred solution of anhydrous Et O
545 nm
(e = 2431),
590 nm
(e = 1592),
650 nm
(e = 1266).
2
(
20 mL) cooled to 0 ꢁC. The dark red precipitate ob-
5.1.5. tert-Butyl N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-
carbamate 7. Under a nitrogen atmosphere, to a solu-
tained was collected by filtration, washed with Et O
and CH Cl and dried under high vacuum to yield
6.5 mg (28%) of compound 2; H NMR (DMSO-d ): d
2
2
2
0
1
tion of 2,2 -(ethylenedioxy)-bis-(ethylamine) (14.8 g,
1
0
6
00 mmol) in anhydrous CHCl3 (100 mL) cooled to
ꢁC was added dropwise di-tert-butyldicarbonate
8.90–8.75 (m, 7H), 8.65 (s, 1H), 8.36–8.12 (m, 7H),
7.88–7.75 (m, 13H), 7.65 (d, J = 8.8 Hz, 2H), 7.43 (t,
J = 6.2 Hz, 1H, NH), 6.94 (br s, 2H, NH), 6.60 (d,
J = 8.8 Hz, 2H), 4.49 (br s, 2H), 4.45–4.38 (m, 1H),
3.72–3.48 (m, 12H), 2.11–1.76 (m, 4H), ꢀ2.93 (s, 2H,
(
Boc O) (2.18 g, 10 mmol) in 50 mL CHCl . After been
2 3
stirred 24 h at room temperature, the solvent is evapo-
rated under vacuum. The thick oil obtained is taken
up in CH Cl (100 mL). The organic layer is successively
NH); UV–vis
(MeOH)
: 286 nm (e = 40,850), 414 nm
2
2
washed with saturated aqueous NaCl (2 · 50 mL), water
50 mL), dried over anhydrous MgSO4 and concen-
trated in vacuo to afford 2.20 g (89%) of crude 7. This
(e = 202,082), 512 nm (e = 9653), 545 nm (e = 4392),
590 nm (e = 2862), 650 nm (e = 2373).
(
1
material was used without further purification;
H
5.2. Mass spectrometry analysis
NMR (CDCl ): d 5.15 (br s, 1H, NH), 3.63–3.51 (m,
3
8
2
H), 3.31 (td, J = 5.0, 5.0 Hz, 2H), 2.88 (t, J = 4.8 Hz,
MALDI sample preparation: A saturated a-cyano-4-hy-
droxy-trans-cinnamic acid (CHCA) solution, in 50/50
EtOH/H O, 0.01% of trifluoroacetic acid (TFA), and
1
H), 1.45 (s, 9H), 1.40 (s, 2H, NH2); C NMR (CDCl3):
3
d 155.42, 78.13, 72.80, 69.63, 41.08, 39.67, 27.77; IR
2
ꢀ1
(
NaCl) (m, cm ): 3355, 2969, 2946, 2868, 1700, 1532,
1 lL of sample were spotted on the stainless steel
MALDI targets. The molar ratio of analyte to matrix
1
278, 1258, 1169, 1099.
4
was 1:10 . The solvent was evaporated prior to insertion
5.1.6. Synthesis of tert-butyl N-{2-[2-(2-aminoethoxy)eth-
oxy]ethyl carbamate}-4-carboxyphenyl porphyrin 8. In
the dark under a nitrogen atmosphere, to a solution of
in the source. Mass spectra were acquired over the range
0–3000 Da.
ꢀ
1
2
drous THF (1 mL) was added N-hydroxysuccinimide
6.2 mg (1.06 · 10 mmol) of compound 7 in anhy-
5.3. Time of flight mass spectrometer (TOFMS)
ꢀ
1
activated porphyrin 6 (160 mg, 2.12 · 10 mmol). After
been stirred at ambient temperature for 18 h, the reac-
tion mixture was adsorbed on silica gel and concen-
trated in vacuo. Column chromatography using
acetone/CH Cl /hexane:1/4/5 (v/v/v) as the eluant affor-
Analyses were performed on a Bruker Reflex IV time-
of-flight mass spectrometer (Bruker-Daltonic, Bremen
Germany) equipped with the SCOUT 384 probe ion
source. The system uses a pulsed nitrogen laser
(337 nm, model VSL-337ND, Laser Science Inc., Bos-
ton, MA) with energy of 400 lJ/pulse. The ions were
accelerated under delayed extraction conditions
(200 ns) in the positive mode with an acceleration volt-
age of 20 kV and a reflector voltage of 23 kV. The detec-
tor signals were amplified and transferred to the XACQ
program on a SUN work station (Sun Microsystems
Inc. Palo Alto, CA). Spectra were processed with the
XMass 5.1 program (Bruker Daltonics, Bremen, Ger-
many). External calibration of MALDI mass spectra
was carried out using sodic and potassic distribution
of PEG 600.
2
2
1
ded 71 mg (75%) of compound 8. H NMR (CDCl ): d
3
8
(
.81–8.67 (m, 8H), 8.22–8.02 (m, 10H), 7.72–7.53
m,9H), 6.96 (br s, 1H, NH), 4.95 (br s, 1H, NH),
3
3
.82–3.71 (m, 4H), 3.71–3.59 (m, 4H), 3.52 (br td, 2H),
.27 (br td, 2H), 1.32 (s, 9H), ꢀ2,86 (s, 2H, NH).
5
4
.1.7. Synthesis of N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-
-carboxyphenylporphyrin 9. In the dark under a nitro-
gen atmosphere, compound 8 (72.7 mg, 0.08 mmol)
was treated with trifluoroacetic acid (TFA) (2 mL).
After been stirred at ambient temperature for 2 h,