Synthesis of new glucosylated porphyrins bearing an α-D-linkage

Article abstract of DOI:10.1080/07328300600770527

This paper presents the synthesis of two new glucosyl tritolylporphyrins in which the carbohydrate moiety is connected through a carboxymethyl glycosidic α-D-linkage. These compounds have been obtained by reaction between porphyrins bearing an amino funct

Full text of DOI:10.1080/07328300600770527

Journal of Carbohydrate Chemistry, 25:345–360, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300600770527
Synthesis of New
Glucosylated Porphyrins
Bearing an a-D-Linkage
Vincent Sol, Alexandre Charmot, and Pierre Krausz
´
´
Laboratoire de Chimie des Substances Naturelles, Universite de Limoges-Faculte des
Sciences et Techniques, Limoges Cedex, France
´
Stephane Trombotto
´
´
`
´
Laboratoire d’Ingenierie des Materiaux Polymeres, CNRS UMR 5627, Universite
Claude Bernard Lyon 1, ISTIL, Villeurbanne Cedex, France
Yves Queneau
´
Laboratoire de Chimie Organique, INSA Lyon, (UMR 5181 CNRS Universite
´
`
´
Lyon 1-INSA Methodologies de Synthese et Molecules Bioactives), Villeurbanne Cedex
This paper presents the synthesis of two new glucosyl tritolylporphyrins in which the
carbohydrate moiety is connected through a carboxymethyl glycosidic a-D-linkage.
These compounds have been obtained by reaction between porphyrins bearing an
amino function with a lactone prepared from the available disaccharide isomaltulose.
The photocytotoxicity of these compounds against K562 human chronic myelogenous
leukemia cells has been evaluated in comparison to Photofrin II.
Keywords Porphyrins, a-D-glucose, Photodynamic therapy, Isomaltulose
INTRODUCTION
The chemistry of porphyrins and chlorins bearing glycosylated groups has
made rapid progress during the last few years owing to the development of
new photosensitizers designed for cancer photochemotherapy.[1–11] Indeed,
sugar moieties not only modulate the amphiphilicity of the photosensitizers,
but also may be involved in specific membrane interactions.[12–14] Moreover,
Received February 12, 2005; accepted May 4, 2006.
Address correspondence to Vincent Sol, Laboratoire de Chimie des Substances Natur-
´
´
elles, Universite de Limoges-Faculte des Sciences et Techniques, 123 Avenue Albert
Thomas, 87060, Limoges Cedex, France. E-mail: vincent.sol@unilim.fr
345

V. Sol et al.
346
the presence of carbohydrate moieties on porphyrins is known to increase their
plasmatic life time[15] and to allow cancer cell surface targeting through specific
binding to membrane receptors.[16] Use of synthetic carbohydrates as carriers
in directed drug delivery may be an interesting approach in cancer cell target-
ing. In the present work, which is part of an ongoing research program on gly-
cosylated porphyrins, we report the synthesis of two mono-a-D-glucosyl
porphyrins (7a,b). The synthesis (Sch. 1) involves 3,4,6-tri-O-acetyl carboxy-
methyl-a-D-glucopyranoside-2-O-lactone
5
as the carbohydrate moiety
supplier. This lactone is obtained from the available disaccharide isomaltulose
[6-O-(a-D-glucopyranosyl)-b-D-fructofuranose] in a straightforward oxidation-
acetylation sequence. It was shown to act as an efficient glucoside provider in
the synthesis of various derivatives such as surfactants and polymerisable
derivatives, as well as new pseudo-glycopeptides.[17,18]
RESULTS AND DISCUSSION
Monohydroxyphenyltritolylporphyrins 2a,b were prepared according to the
Little method[19] by condensation of pyrrole (4 equiv.) with paratolualdehyde
(3 equiv.) and ortho-or parahydroxybenzaldehyde (1 equiv.) in propionic acid
under reflux. Only o- and p-monohydroxyphenyltritolylporphyrins 2a,b were
isolated after purification, in 5% to 6% yield, respectively. The amine-Boc pro-
tected spacer arm 1 then reacted, in five fold excess, with monohydroxytritolyl-
porphyrin 2a,b to yield 3a,b (95%). Acidic removal of the Boc groups gave
compound 4a,b respectively (85%).
Coupling of lactone 5 and the primary amine of porphyrin 4a,b gave 6a,b
(95%). Finally, conventional transesterification of the acetyl groups gave com-
pounds 7a,b in 95% yield.
UV-visible absorption spectra of these compounds (7a,b) in CHCl3/MeOH
(8/2) showed the expected bands: the Soret band around 420 nm and the four
less intense visible Q bands between 520 and 650 nm; all the compounds dis-
played the etio spectra (1IV . 1III
.
II . 1I) characteristic of ꢀ free-base ꢁ
porphyrins. In THF/H2O (8/2), the molar absorption coefficients of the two
derivatives decrease as shown in Table 1 and the Soret band are broadening
and splitting. These results are due to the combination of cofacial and edge-
to-edge interaction of self-assembled aggregates.[2,7,20]
All fluorescence spectra of porphyrins 7a,b in THF were charaterized by
two emission bands in the 550 to 750-nm region. The fluorescence emission
wavelengths in H2O/THF (8/2) were identical to those obtained in THF, but
the emission was strongly quenched. This decay of fluorescence can be
explained by the formation of aggregates[21] and supports the results
observed in the UV-visible spectra.
Mass spectrometry analysis of all glucosylated porphyrins was performed
using the MALDI (matrix-assisted laser desorption ionization) technique.

Synthesis of New Glucosylated Porphyrins
347
Scheme 1: (i) DIBOC (2 eq), NEt3 (1.1 eq), CH2Cl2, rt, 1 h 30 min; (ii) EtCOOH, 1 h, reflux; (iii)
Br(CH2)3NH-Boc (5 eq.) 2, K2CO3 (20 eq.), DMF, 18 h,rt; (iv) CH2Cl2/TFA (80/20, v/v), 3 h, rt.

Table 1: UV-visible spectra of porphyrin derivatives 7a,b in various solvents.
Soret 1
1 [M21
Soret 2
1 [M21
IV
1 [M21
III
1 [M21
II
1 [M21
I
l
l
l
l
l
l
1 [M21
[nm] cm21
[nm] cm21
]
[nm] cm21
]
[nm] cm21
]
[nm] cm21
]
[nm] cm21
]
]
Derivatives
7a
Solvents
CHCl3/MeOH
(8/2)
H2O/THF (2/8)
418 361.6 ꢂ 103
516 13.4 ꢂ 103 552 7.8 ꢂ 103 590 4.3 ꢂ 103 648 3.9 ꢂ 103
418 126.9 ꢂ 103 442 167.9 ꢂ 103 519
5.6 ꢂ 103 551 4.3 ꢂ 103 592 3.4 ꢂ 103 648 2.8 ꢂ 103
7b
CHCl3/MeOH
(8/2)
H2O/THF (2/8)
418 322.4 ꢂ 103
516 12.2 ꢂ 103 552 7.1 ꢂ 103 592 4.0 ꢂ 103 648 3.1 ꢂ 103
418 110.6 ꢂ 103 449 114.6 ꢂ 103 524
2.7 ꢂ 103 555 2.0 ꢂ 103 596 1.5 ꢂ 103 653 1.3 ꢂ 103

Synthesis of New Glucosylated Porphyrins
349
Positive ion mass spectra exhibited a base peak corresponding to the intact
porphyrin, and no fragment ions were detected. The isotopic analysis indicated
the presence of a protonated species (M þ H)þ with a minor contribution of the
radical cation Mþ8, allowing the determination of the molecular mass with an
accuracy close to 0.001%.[7]
1H NMR spectra of these compounds showed the expected signals. More
precisely, analysis of the sugar moieties showed the expected H10-H20
0
coupling interaction corresponding to the awaited a anomer, with J1 -
0
0
20
¼ 3.5 Hz and J1 -2 ¼ 3.8 Hz for 6a and 6b, respectively. For the para com-
pounds 2b, 3b, 4b, 6b, and 7b, the resonances of the b-pyrrolic protons
appear as a single peak between 8.80 and 8.85 ppm. For ortho compounds
6a and 7a, all the sugar protons experience a pronounced shielding
from –0.43 ppm (H-6a0 ) to –3.02 ppm (H-2’). These nuclei are obviously
located inside the shielding cone of the porphyrin macrocycle. Moreover, the
b-pyrrole proton signals of 6a appear as four doublets (J ¼ 4.8 Hz) between
8.92 and 8.77 ppm, and one singlet at 8.90 ppm. According to the literature,[7,10]
these phenomena, that is, a shielding of ortho substituents and a splitting of
b-pyrrolic protons signals, reflect a reduced symmetry induced by the
orientation of the substituent that in turn leads to an unsymmetrical folded
structure because of steric hindrance.
In Vitro Photocytotoxicity
Experiments were conducted with K562 chronic leukemia cell line.
Figure 1 displays dead cell counts and the subsequent changes following a
further 24 h incubation in the dark. The results obtained with these synthetic
porphyrins have been compared with those observed with Photofrin. These
data show that ortho porphyrin 7a is much more active than para porphyrin
7b, although less active than Photofrin at the same ponderal concentration.
In addition, a further 24- h incubation does not result in a significant
increase in dead cell percentage, contrary to Photofrin; porphyrin 7a then
probably induces early necrotic death and the cellular damages do not lead
to secondary necrosis that could be attributed to the induction of apoptosis.
It should be noted that these results are reminiscent of those previously
observed with ortho and para b-glycosyl porphyrins.[10] Within the latter
series, photoactivity displayed by the ortho compounds was always signifi-
cantly stronger than their para counterparts. However, the structural basis
of this biological discrepancy is presently unknown.
In order to evaluate the influence of glucosyl units on cell viability, we irra-
diated K562 cells in the presence of nonglucosylated compound 3a; 120 min of
irradiation was followed by only 15% cell death. This result clearly shows that
introduction of a sugar unit as peripherical substituent improves the photoac-
tivity of porphyrin derivatives. Moreover, by comparison with porphyrin

V. Sol et al.
350
Figure 1: Phototoxicity of a-D-glucosyl porphyrins 7a and 7b. The figure shows the percentage
of dead cells (PI stained) vs. irradiation time; void bars: effect of Photofrin, hatched bars,
porphyrin 7b; solid bars, porphyrin 7a; A: Dead cell count just after irradiation; B: Dead cell
count after a further 24 h incubation in the dark. Error bars are based upon standard
deviations.

Synthesis of New Glucosylated Porphyrins
351
conjugate of comparable structure, previously synthetized in our laboratory,[10]
compound 7a shows similar in vitro photocytotoxicity. The exact role of
the sugar residue in the photosensitizing properties remains to be elucidated.
Further comparisons with similar prophyrins bearing carbohydrate moieties
having either a- or b-anomeric carboxymethyl linkages and based on various
mono-or oligosaccharides will be performed.
EXPERIMENTAL
General
All solvents and reagents were purchased from Aldrich, Prolabo, or Acros.
Pyrrole was distilled over CaH2 under reduced pressure immediately before
use. Dimethylformamide was distilled over CaH2 under reduced pressure
and stored under Argon. Methylene chloride (CH2Cl2) was distilled over
P2O5 and then CaH2. Analytical thin-layer chromatography (TLC) was per-
formed on silica gel Merck 60F254. Column chromatography was carried out
1
with silica gel (60 ACC; 15–40 mm, Merck). H NMR spectroscopy was per-
formed with a Brucker DPX-400 spectrometer. Chemical shifts are reported
as d ppm, downfield from internal TMS, and are listed according to the
standard numbering of meso-arylporphyrins. UV-visible spectra were
recorded on a Perkin Elmer Lambda 25 double-beam spectrophotometer
using 10- or 50-mm quartz cells. Infrared spectra were recorded on a
Perkin Elmer spectrum 1000 with KBr pellets. MALDI-TOF mass spectra
were recorded with a Voyager Elite (Framingham, MA, USA) time-of-flight
mass spectrometer equipped with a 337-nm nitrogen laser (VSL 337ND). It
was operated in the reflectron delayed extraction mode at an acceleration
voltage of 20 kV. Internal standards (peptides) were used to calibrate the
mass scale with the two-point calibration Software version 3.07.1 from PerSep-
tive Biosystems. One microliter of an aceton solution of matrix (a-cyano-4-
hydroxycinnamic acid) and compounds at concentrations of 0.1 M and
0.01 mM, respectively, was deposited onto the stainless steel sample slide and
dried in air.
N-Tert-butoxycarbonyl-3-bromopropylamine (1)
3-Bromopropylamine hydrobromide (1 g, 4.57 mmol, 1 equiv.) was dissolved
in 8 mL of distilled CH2Cl2. Triethylamine (0.7 mL, 5.03 mmol, 1.1 equiv.) and
diterbutyl dicarbonate (2.1 g, 9.57 mmol, 2 equiv.) were added and the
reaction was carried out under an argon atmosphere and stirring during 1 h
30 min at rt. After evaporation under reduced pressure, residue was redis-
solved in CH2Cl2 (20 mL) and washed with water (3 ꢂ 20 mL). The organic
layer was separated and dried over anhydrous magnesium sulfate, filtered,

V. Sol et al.
352
concentrated, and subjected to flash chromatography (CH2Cl2/EtOH, 100:0 to
50:50) to give compound 1, which was recrystallized in petroleum ether as
white crystals (910.2 mg, 84%). mp: 35–378C. Rf 0.48 (CHCl3/EtOH 98/2). IR
1
(KBr), n (cm21): 3370 (NH); 3000 (CH); 1686 (C55O). H NMR (400.13 MHz,
CDCl3), d 1.44 (s, 9H, CH3); 2.05 (q, 2H, J ¼ 6.5 Hz, Br–CH2–CH2–CH2–
NHCO); 3.27 (bt, 2H, J ¼ 6.5 Hz, Br–CH2–CH2–CH2–NHCO); 3.44 (t, 2H,
J ¼ 6.5 Hz, Br–CH2–CH2–CH2–NHCO); 4.64 (bs, 1H, NH).
General Procedure for the Synthesis of
Monohydroxyphenylporphyrins
These compounds were synthesized according to the Little method.[19] 2- or
4-hydroxybenzaldehyde (1 equiv.) and 4-tolualdehyde (3 equiv.) for porphyrins
2a,b were dissolved in propionic acid. The mixture was stirred under reflux for
30 min. Pyrrole (4 equiv.) was then added dropwise over the course of 5 min and
the mixture was stirred under reflux for a further 1 h 30 min. After cooling, a
mixture of meso phenylporphyrins crystallized. This mixture was filtered
and then the crude compounds 2a,b were purified by silica gel column
chromatography.
5-(2-Hydroxyphenyl)-10,15,20-Tris(4-Methylphenyl) Porphyrin (2a)
Reaction of 2-hydroxybenzaldehyde (3 mL, 28.0 mmol, 1 eq.), 4-tolualde-
hyde (10.1 mL, 85.0 mmol, 3 equiv.), and pyrrole (8.0 mL, 114.0 mmol, 4
equiv.) led to pure product 2a (940 mg, 5%). Rf 0.70 (CH2Cl2); UV-visible
spectrum in CH2Cl2: lmax, (1,1023 L mol21 cm21): 418 (339.2), 516 (14.4), 552
1
(6.7), 592 (4.5), 648 (4.1); H NMR (400 MHz, CDCl3, 258C): d ¼ 22.7 (s, 2H,
NH), 2.7 (s, 9H, CH3), 7.2 (m, 2H, H-3,5 Ar), 7.5 (d, 6H, JH,H ¼ 7.2 Hz, H-3,5
tolyl), 7.7 (m, 1H, H-4 Ar), 7.9 (m, 1H, H-6 Ar), 8.1 (d, 6H, JH,H ¼ 7.2 Hz,
H-2,6 Tolyl), 8.8 (s, 8H, Hb pyr). MS (MALDI) m/z ¼ 673.6 ([M þ H]þ monoiso-
topic calc 672.3).
5-(4-Hydroxyphenyl)-10,15,20-Tris(4-Methylphenyl) Porphyrin (2b)
Reaction of 4-hydroxybenzaldehyde (3.4 g, 28.0 mmol, 1 equiv.), 4-tolualde-
hyde (10.1 mL, 85.0 mmol, 3 equiv.), and pyrrole (8.0 mL, 114.0 mmol, 4 equiv.)
gave, after purification by column chromatography (CHCl3/petroleum ether
8/2 to 10/0), pure product 2b (1.12 g, 6%). Rf 0.33 (CH2Cl2); UV-visible
spectrum in CH2Cl2: lmax, (1,1023 L mol21 cm21): 418 (363.0), 516 (13.5),
1
552 (7.4), 592 (4.0), 648 (4.3); H NMR (400 MHz, CDCl3, 258C): d ¼ 22.76
(bs, 2H, NH), 2.69 (s, 9H, CH3), 7.15 (d, 2H, J ¼ 8.2 Hz, H-3,5 Aryl), 7.53
(d, 6H, J ¼ 7.7 Hz, H-3,5 tolyl), 8.03 (d, 2H, J ¼ 8.2 Hz, H-2,6 Aryl), 8.08
(d, 6H, J ¼ 7.7 Hz, H-2,6 tolyl), 8.84 (bs, 6H, Hb pyr), 8.93 (d, 2H,
J ¼ 1.2 Hz, Hb pyr); MS (MALDI) m/z ¼ 673.6 ([M þ H]þ monoisotopic calc
672.3).

Synthesis of New Glucosylated Porphyrins
353
General Procedure for the Synthesis of
Monoaminopropyloxyphenylporphyrins 4a,b
Porphyrins 2a and 2b (1 equiv.) were dissolved in dry DMF (10 mL) with a
large excess of K2CO3 (20 equiv.). The mixtures were stirred for 15 min at rt.
Compound 1 (5 equiv.) was added and the solutions were stirred at rt overnight
in the dark. After reaction, DMF was evaporated under vacuum and the crude
product was dissolved in CH2Cl2. The organic layer was washed several times
with water, dried (MgSO4), and then evaporated to give, after purification by
preparative TLC, the pure porphyrins 3a,b.
Tritolylporphyrin derivatives 3a or 3b were dissolved in CH2Cl2 (8 mL) and
trifluoroacetic acid (2 mL) was added. The mixture was stirred at rt for 2 h. Fol-
lowing completion of the reaction, CH2Cl2 (30 mL) was added, followed by
NaHCO3-saturated aqueous solution until pH ¼ 7. The organic layer was
washed with water and dried over magnesium sulfate. Porphyrins 4a,b were
obtained pure as seen by TLC (CHCl3/EtOH/NH3, 80/20/1).
5-(2-[N-Terbutoxycarbonyl-3-Aminopropyloxy]Phenyl)-10,15,20-Tris[4-
Methylphenyl] Porphyrin (3a)
Reaction of porphyrin 2a (113 mg, 0.17 mmol, 1 equiv.), compound 1
(200 mg, 0.84 mmol, 5 equiv.), and K2CO3 (475 mg, 3.44 mmol, 20 equiv.) led
to pure product 3a (132.3 mg, 95%). Rf 0.39 (CH2Cl2); UV-visible spectrum in
CHCl3: lmax, (1,1023 L mol21 cm21): 419 (531.8), 516 (18.8), 552 (9.1), 592
1
(5.8), 647 (4.3). H NMR (400.13 MHz, CDCl3, 258C): d ¼ 22.73 (s, 2H, NH),
1.06 (s, 9H, CH3 tertiobutyl), 1.16 (q, 2H, J ¼ 5.7 Hz, O–CH2–CH2–
CH2NHCO), 1.91 (q, 2H, J ¼ 5.7 Hz, –CH2–NHCO), 2.69 (s, 9H, CH3), 3.56
(t, 1H, J ¼ 4.5 Hz, –NH–CO), 3.92 (t, 2H, J ¼ 5.8 Hz, –O–CH2–CH2–), 7.32
(d, 1H, J ¼ 8.3 Hz, H-3 aryl), 7.35 (td, 1H, J ¼ 1.6-8.3 Hz, H-5 aryl), 7.54 (bd,
6H, J ¼ 7.8 Hz, H-3,5 tolyl), 7.70 (td, 1H, J ¼ 1.6-7.9 Hz, H-4 aryl), 8.04 (dd,
1H, J ¼ 1.5-7.3 Hz, H-6 aryl), 8.12 (bd, 6H, J ¼ 7.6 Hz, H-2,6 tolyl), 8.79
(d, 2H, J ¼ 4.4 Hz, Hb pyr), 8.83 (d, 2H, J ¼ 4.8 Hz, Hb pyr), 8.84 (s, 4H, Hb
pyr). MS (MALDI) m/z ¼ 831.31 ([M þ H]þ monoisotopic calc: 830.1).
5-(4-[N-Terbutoxycarbonyl-3-Aminopropyloxy]Phenyl)-10,15,20-Tris[4-
Methylphenyl] Porphyrin (3b)
Reaction of porphyrin 2b (100 mg, 0.15 mmol, 1 equiv.), compound 1
(178 mg, 0.74 mmol, 5 equiv.), and K2CO3 (410 mg, 2.97 mmol, 20 equiv.) led
to pure product 3b (88 mg, 82%). Rf 0.52 (CH2Cl2); UV-visible spectrum in
CHCl3: lmax, (1,1023 L mol21 cm21): 421 (259.3), 518 (7.9), 554 (4.7), 592
1
(2.5), 648 (2.3); H NMR (400.13 MHz, CDCl3, 258C): d ¼ 22.76 (s, 2H, NH),
1.49 (s, 9H, CH3 tertiobutyl), 2.03 (q, 2H, J ¼ 6.3 Hz, OCH2–CH2–CH2CO–),
2.69 (s, 9H, CH3), 3.42 (t, 2H, J ¼ 6.5 Hz, –CH2–NHCO), 4.29 (t, 2H,
J ¼ 5.5 Hz, –O–CH2–), 7.24 (d, 2H, J ¼ 8.4 Hz, H-3,5 Aryl), 7.54 (d, 6H,

V. Sol et al.
354
J ¼ 7.7 Hz, H-3,5 tolyl), 8.08 (d, 6H, J ¼ 7.7 Hz, H-2,6 tolyl), 8.10
(d, 2H, J ¼ 8.3 Hz, H-2,6 Aryl), 8.84 (s, 8H, Hb pyr). SM (MALDI)
m/z ¼ 831.22 ([M þ H]þ monoisotopic calc: 830.1).
5-(2-[3-Aminopropoxy]Phenyl)-10,15,20-Tris[4-Methylphenyl] Porphyrin (4a)
Reaction of porphyrin 3a (132 mg, 0.16 mmol) led to pure product 4a
(100 mg, 86%). Rf ¼ 0.85 (CHCl3/EtOH/NH3, 80/20/1); UV-visible spectrum
in CHCl3: lmax, (1,1023 L mol21 cm21): 419 (219.0), 516 (20.0), 552 (10.1), 590
1
(7.0), 645 (5.0). H NMR (400.13 MHz, CDCl3, 258C): d ¼ 22.74 (s, 2H, NH),
1.09, (quint, 2H, J ¼ 6.2 Hz, O–H2C–CH2–CH2–NH2); 1.68 (t, 2H,
J ¼ 6.5 Hz, O–H2C–CH2–CH2–NH2); 2.69 (s, 9H, CH3 tolyl), 3.93 (t, 2H,
J ¼ 5.8 Hz, O–H2C–CH2–CH2–NH2), 7.31 (dd, 1H, J ¼ 8.4-0.8 Hz, H-3 aryl),
7.34 (td, 1H, J ¼ 7.3 Hz, H-5 aryl), 7.53 (bd, 6H, J ¼ 7.5 Hz, H3,5 tolyl), 7.70
(td, 1H, J ¼ 7.6-1.6 Hz, H-4 aryl), 8.01 (dd, 1H, J ¼ 7.4-1.6 Hz, H-6 aryl), 8.07
(d, 2H, J ¼ 6.9 Hz, H-2,6 tolyl), 8.09 (d, 2H, J ¼ 7.0 Hz, H-2,6 tolyl), 8.11
(d, 2H, J ¼ 7.1 Hz, H-2,6 tolyl), 8.77 (d, 2H, J ¼ 4.8 Hz, Hb pyrrole);
8.82 (d, 2H, J ¼ 4.9 Hz, Hb pyrrole); 8.84 (s, 4H, Hb pyrrole). MS (MALDI)
m/z ¼ 730.95 ([M þ H]þ monoisotopic calc: 729.8).
5-(4-[3-Aminopropoxy]phenyl)-10,15,20-Tris[4-Methylphenyl] Porphyrin (4b)
Reaction of porphyrin 3b (132 mg, 0.16 mmol) led to pure product 4b
(98 mg, 85%). Rf ¼ 0.55 (CHCl3/EtOH/NH3, 80/20/1); UV-visible spectrum
in CHCl3: lmax, (1, 1023 L mol21 cm21): 420 (467.6), 517 (13.6), 554 (7.6), 592
1
(4.2), 648 (3.8). H NMR (400.13 MHz, CDCl3, 258C): d ¼ 22.77 (s, 2H, NH),
2.02 (m, 2H, O–H2C–CH2–CH2–NH2); 2,62 (m, 2H, O–H2C–CH2–CH2–
NH2); 2.67 (s, 9H, CH3 tolyl), 2.99 (bs, 2H, NH2), 7.25 (d, 2H, J ¼ 8.4 Hz,
H-3,5 aryl), 7.53 (d, 6H, J ¼ 7.7 Hz, H-3,5 tolyl), 8.08 (d, 6H, J ¼ 7.8 Hz,
H-2,6 tolyl), 8.10 (d, 2H, J ¼ 8.4 Hz, H-2,6 aryl), 8.85 (s, 8H, Hb pyrrole). MS
(MALDI) m/z ¼ 730.85 ([M þ H]þ monoisotopic calc: 729.8).
Triacetyl Lactone (5)
Lactone (5) was prepared in two steps[17,18] by treating isomaltulose with
excess aqueous hydrogen peroxide under acidic catalysis to yield carboxy-
methyl glucoside, followed by acetylation (and lactone formation). This last
step could be either performed by treatment with acetic anhydride in
pyridine or in anhydrous DMF or by acetyl chloride in a dichloromethane-
pyridine mixture.
General Procedure for Coupling of Triacetyl Lactone 5
Crude porphyrins 4a,b (1 equiv.) were dissolved in CH2Cl2 and lactone 5
(3 equiv.) and DMAP (2.5 equiv.) were added. The mixture was stirred 16 h in
the dark, at rt. The reaction was monitored by TLC (CHCl3/EtOH, 95/5).

Synthesis of New Glucosylated Porphyrins
355
The solution was concentrated in vacuo and purification of the residue by pre-
parative TLC gave pure products 6a,b.
5-[2-(3,4,6-Tri-O-Acetyl-a-D-Glucopyranosyloxymethylcarbonyl-3-
aminopropoxy)Phenyl] -10,15,20-Tritolylporphyrine (6a)
Porphyrin 4a (99 mg, 0.14 mmol, 3 equiv.) reacted with lactone 5 (15.7 mg,
0.04 mmol, 1 equiv.) and DMAP (13.8 mg, 0.11 mmol, 2.5 equiv.) in CH2Cl2
(13 mL) to afford pure product 6a (58.2 mg, 99%). Rf 0.54 (CHCl3/EtOH, 95/5);
UV-visible spectrum inCHCl3: lmax, (1,1023 L mol21 cm21): 420 (335.4), 516
(12.7), 552 (8.4), 590 (5.0), 648 (5.8). 1H NMR (CDCl3, 400.13 MHz):
d ¼ 22.87 (s, 2H, NH-pyrrole), 0.76 (dd, 1H, J ¼ 3,1-9,9Hz, H-2’), 0.89
(m, 2H, OH2C–CH2–CH2NHCO), 1.23 (s, 3H, acetyl), 1.46 (m, 2H, O–CH2–
CH2–CH2NHCO), 1.79 (s, 3H, acetyl), 1.99 (s, 3H, acetyl), 2.56 (d, 1H,
J ¼ 3.5 Hz, H-1’), 2.46 (bd, 2H, J ¼ 15.8 Hz, OC-CH2–O), 2.69 (s, 3H, CH3
tolyl), 2.70 (s, 3H, CH3 tolyl), 2.71 (s, 3H, CH3 tolyl), 3.16 (ddd, 1H, J ¼ 9.8-
4.0-1.8 Hz, H-5’), 3.61 (dd, 1H, J ¼ 1.8-12.2 Hz, H-6b’), 3.81 (m, 1H, OCH2–
CH2–CH2NH), 3,86 (dd, 1H, J ¼ 4.4-12.4 Hz, H-6a’), 3.93 (m, 1H, OCH2–
CH2–CH2NH), 4.00 (t, 1H, J ¼ 9.7 Hz, H-3’), 4.12 (t, 1H, J ¼ 9.8 Hz, H-4’),
7,29 (d, 1H, J ¼ 8.2 Hz, H-3 aryl), 7,41 (t, 1H, J ¼ 7.4 Hz, H-5 aryl), 7.56 (bs,
4H, H-3,5 tolyl), 7.63 (d, 2H, J ¼ 7.5 Hz, H-3,5 tolyl); 7.76 (td, 1H, J ¼ 7.9-
1.4 Hz, H-4 aryl), 8.01 (d, 2H, J ¼ 7.4 Hz, H-2,6 tolyl), 8.05 (d, 2H,
J ¼ 8.4 Hz, H-2,6 tolyl), 8.13 (dd, 1H, J ¼ 7.3-1.5 Hz, H-6 aryl), 8.10 (d, 2H,
J ¼ 7.9 Hz, H-2,6 tolyl), 8.77 (d, 1H, J ¼ 4.8 Hz, Hb pyrrole), 8.87 (d, 1H,
J ¼ 4.8 Hz, Hb pyrrole), 8.89 (d, 1H, J ¼ 4.8 Hz, Hb pyrrole), 8.90 (s, 4H, Hb
pyrrole), 8.92 (d, 1H, J ¼4.8 Hz, Hb pyrrole). MS (MALDI) m/z ¼ 1076.4
([M þ H]þ monoisotopic calc: 1075.4).
5-[4-(3,4,6-Tri-O-Acetyl-a-D-Glucopyranosyloxymethylcarbonyl-3-
aminopropoxy)Phenyl] -10,15,20-Tritolylporphyrine (6b)
Porphyrin 4b (67.1 mg, 0.10 mmol, 3 equiv.) reacted with lactone 5 (11.2 mg,
0.03 mmol, 1 equiv.) and DMAP (9.9 mg, 0.08 mmol, 2.5 equiv.) in CH2Cl2 (9 mL)
to afford pure product 6b (33 mg, 95%). Rf 0.47 (CHCl3/EtOH, 95/5); UV-visible
spectrum in CHCl3: lmax, (1,1023 L mol21 cm21): 420 (321.3), 516 (13.1), 552
1
(6.5), 592 (4.9), 648 (4.1). H NMR (CDCl3, 400.13 MHz): d ¼ 22.77 (s, 2H,
NH-pyrrole) 1.99 (s, 3H, acetyl), 2.00 (s, 3H, acetyl), 2.09 (s, 3H, acetyl), 2.20
(quint, 2H, J ¼ 6.3 Hz, OH2C–CH2–CH2N), 2.69 (s, 9H, CH3 tolyl), 3.65
(m, 2H, CH2N), 3.78 (dd, 1H, J ¼3.7-9.9 Hz, H-20), 4.07 (ddd, 1H, J ¼ 9.8-1.8-
4.6 Hz, H-50), 4.09 (d, 1H, J ¼ 12.5 Hz, CH2), 4.13 (d, 1H, J ¼ 14.5 Hz, CH2),
4.27 (dd, 1H, J ¼ 2.4-9.6 Hz, H-60b), 4.28 (t, 2H, J ¼ 6.3 Hz, CH2O), 4.29 (dd,
1H, J ¼ 3.3-9.6 Hz, H-6’a), 4.91 (d, 1H, J ¼ 3.8 Hz, H-10), 5.02 (t, 1H,
J ¼ 9.8 Hz, H-40), 5.30 (t, 1H, J ¼ 9.6 Hz, H-30), 7.25 (d, 2H, J ¼ 8.4 Hz, H-3,5
aryl), 7.39 (t, 1H, J ¼ 5.6 Hz, NH), 7.53 (d, 6H, J ¼ 7.7 Hz, H-3,5 tolyl), 8.08

V. Sol et al.
356
(d, 6H, J ¼ 7.8 Hz, H-2,6 tolyl), 8.10 (d, 2H, J ¼ 8.4 Hz, H-2,6 aryl), 8.85
(s, 8H, Hb pyrrole). MS (MALDI) m/z ¼ 1076.5 ([M þ H]þ monoisotopic calc:
1075.4).
General Procedure for Removal of Acetate Protective
Groups
Crude porphyrins 6a and 6b were dissolved in CH2Cl2/MeOH (80/20, v/v)
(5 mL) and then sodium methoxide (0.5M in MeOH, 3 equiv. per acetate groups)
was added. The mixture was stirred for 1 h in the dark. After reaction, resulting
porphyrins 7a and 7b were quantitatively precipitated by addition of pet-
roleum ether (30 mL) and then filtered on sintered glass disk (porosity 4) and
washed with MeOH and CH2Cl2.
5-[2-(a-D-Glucopyranosyloxymethylcarbonyl-3-Aminopropoxy)Phenyl]-
10,15,20-Tritolyl Porphyrine (7a)
37.6 mg (95%), Rf 0.68 (CHCl3/MeOH, 7/3); UV-visible spectrum in CHCl3/
MeOH (8/2,v/v): lmax, (1,1023 L mol21 cm21): 418 (361.6), 516 (13.4), 552 (7.8),
590 (4.3), 648 (3.9).1H NMR (DMSO d6, 400.13 MHz)): d ¼ 22.87 (s, 2H, NH-
pyrrole) 2.65 (s, 9H, CH3 tolyl), 2.95 (bt, 1H, J ¼ 9.3 Hz, H-30), 3.00 (dd, 1H,
J ¼ 3.6-9.6 Hz, H-20), 3.20 (bt, 1H, J ¼ 9.7 Hz, H-50), 3.35 (dd, 1H, J ¼ 5.7–
11.8 Hz, H-6a0 ), 3.52 (bt, 1H, J ¼ 2.0-12.1, H-6b0 ), 4.42 (d, 1H, J ¼ 3.6 Hz, H-10),
7.38 (t, 1H, J ¼ 7.4 Hz, H-5 aryl), 7.52 (d, 1H, J ¼ 7.5 Hz, H-3 aryl), 7.60
(d, 6H, J ¼ 7.6 Hz, H-3,5 tolyl), 7.82 (t, 1H, J ¼ 7.6 Hz, H-4 aryl), 7.96 (d, 1H,
J ¼ 7.3 Hz, H-6 aryl), 8.06 (bd, 6H, J ¼ 7.5 Hz, H-2,6 tolyl), 8.78(d, 2H,
J ¼ 4.6 Hz, H-b pyrrole), 8.79 (d, 2H, J ¼ 4.7 Hz, Hb pyrrole), 8.81 (s, 4H, Hb
pyrrole). MS (MALDI) m/z ¼ 950.3 ([M þ H]þ monoisotopic calc: 949.3).
5-[4-(a-D-Glucopyranosyloxymethylcarbonyl-3-Aminopropoxy)Phenyl]-
10,15,20-Tritolyl Porphyrine (7b)
16.4 mg (95%); Rf 0.52 (CHCl3/MeOH, 7/3); UV-visible in CHCl3/MeOH
(8/2,v/v): lmax,(1,1023 L mol21 cm21): 418 (322.4), 516 (12.2), 552 (7.1), 592
(4.0), 648 (3.1). 1H NMR (DMSO d6, 400.13 MHz): d ¼ 22.88 (s, 2H, NH),
2.11 (bt, 2H, J ¼ 5.4 Hz, OH2C22CH222CH2N), 2.65 (s, 9H, CH3 tolyl.), 3.13
(m, 1H, H-60b), 3.25 (bs, 1H, H-60a), 3.26 (bd, 1H, J ¼ 7.0 Hz, H-20), 3.46
(t, 1H, J ¼ 9.1 Hz, H-50), 3.48 (m, 2H, 22CH2N), 3.56 (bt, 1H, J ¼ 9.3 Hz,
H-3’), 3.66 (bd, 1H, J ¼ 10.2 Hz, H-40), 3.94 (d, 1H, J ¼ 16.2 Hz, CH2), 4.30
(bs, 2H, –CH2O), 4.12 (d, 1H, J ¼ 14.8 Hz, CH2), 4.77 (bs, 1H, H-10), 7.35
(d, 2H, J ¼ 7.0 Hz, H-3,5 aryl), 7.59 (d, 6H, J ¼ 5.0 Hz, H-3,5 tolyl), 8.06
(d, 6H, J ¼ 6.3 Hz, H-2,6 tolyl), 8.09 (d, 2H, J ¼ 7.0 Hz, H-2,6 aryl), 8.81 (bs,
8H, Hb pyrrole);. MS (MALDI), m/z 950.4 ([M þ H]þ monoisotopic calc: 949.3).

Synthesis of New Glucosylated Porphyrins
357
Cell Culture
Phototoxicities of the synthesized compounds against K562 chronic
leukemia cell line have been evaluated. K562 cells were suspended in
HEPES-buffered RPMI 1640 medium (Sigma, R4130) pH ¼ 7.0 +0.3 contain-
ing 2 mM L-glutamine supplemented with 2 g L21 NaHCO3, 50 U mL21 penicil-
lin, 50 mg mL21 streptomycin (Sigma, P0906), and 10% (v/v) fetal bovine
serum (Biochrom KG, Polylabo 60810). Cultures were incubated at 378C in a
fully humidified atmosphere containing 5% CO2 in air. Cells were picked
during exponential growth and washed and diluted to 106 cells mL21 with
fresh RPMI medium. This suspension was then distributed in 24 well plates
(2 mL/well). Porphyrins (final concentration 2.1026 M) or Photofrin (final con-
centration 1.25 mg mL21) were added to the wells and the plates were then
incubated 18 h in the dark before illumination; without this preincubation
these synthetic porphyrins did not display any detectable photocytotoxicity.
Moreover, none of the compounds studied exhibited cytotoxicity in the dark.
Flow Cytometry
Dead cells were identified as propidium iodide (PI) (Sigma) permeable ones
and the dead cell counts were measured by flow cytometry. Samples were
analyzed with a Coulter Epics XL System II, immediately after each illumina-
tion time (J0); then cell suspensions were incubated in the dark at 378C for 24 h
and the dead cell counts were estimated thereafter (J1). A stock solution of PI
was prepared in distilled water, filtered, sterilized, and used at 1024 M.
Partition Coefficient Measurements
Lipophylicity, often well correlated with the bioactivity of drugs, can be
indicated, for example, by the logarithm of a partition coefficient, log P, that
reflects the equilibrium partitioning of a molecule between a nonpolar and a
polar phase, such as an n-octanol/water system. For tritolylporphyrin 7a,b,
calculated log P—as log([Porphyrin]n-octanol)/[Porphyrin]water)—is .3, which
indicates that these compounds are very nearly insoluble in water. Determi-
nations were repeated three times.
Singlet Oxygen Production
In order to determine the photosensitizing properties of porphyrins 7a and
1
7b, trapping reactions of O2 with ergosterol acetate were carried out.[22–24]
Reference experiments with eosin, rose bengal, or hematoporphyrin (HP),
known singlet oxygen producers, gave ergosterol acetate epidioxide with
nearly quantitative yields. In the same experimental conditions, our porphy-
1
rins had the same efficiency for O2 production than HP.

V. Sol et al.
358
CONCLUSION
We have demonstrated a simple synthetic method for accessing porphyrins
bearing a glucosyl unit, by the way of an a-D-carboxymethyl linkage. The carbo-
hydrate moiety is connected by reaction with a bicyclic lactone prepared from
the available disaccharide isomaltulose. Preliminary biological results have
showed that the ortho compound was more active than the para porphyrin,
and significantly more active than the nonglycosylated analog. However,
activity was shown to be lower than that of Photofrin, suggesting that only
one sugar unit in 7a is not enough to increase significantly the amphiphilic
character for efficient photodynamic activity.[2,7] Similar porphyrins incorpora-
ting a- or b-carboxymethyl mono-or oligosaccharides will be studied. Such vari-
ations will allow to discriminate between specific interactions with the
carbohydrate parts of the molecules, the issue of the stability of the glycosidic
linkage allowing longer lifetime of the compounds, or physicochemical con-
siderations such as variations in their polarity. This might notably be under-
taken in light of the knowledge of the various lectins at the surface of cell
membranes and their implication in glycoconjugated dyes internalization.
ACKNOWLEDGEMENTS
Part of this work was achieved in the former CNRS Beghin Say research
facility in Villeurbanne. Financial support from these two institutions and
´
ANRT for a grant to ST as well as from the “Conseil Regional du Limousin”
are gratefully acknowledged. Dr Jean Claude Blais is also warmly acknow-
ledged for routine MS MALDI analysis.
REFERENCES
[1] Cornia, M.; Casiraghi, G.; Binacchi, S.; Zanardi, F.; Rassu, G. Facile entry to
5,10,15,20-tetra-C-glycosylporphyrins. J. Org. Chem. 1994, 59, 1226–1230.
[2] Momenteau, M.; Oulmi, D.; Maillard, P.; Croisy, A. In vitro photobiological activity
of a new series of photosensitizers: the glycoconjugated porphyrins. SPIE, Photody-
namic Therapy Cancer II. 1994, 2325, 13–23.
[3] Hombrecher, H.K.; Ohm, S.; Koll, D. Synthesis of galactopyranosyl substituted
porphyrins. Tetrahedron 1996, 52, 5441–5448.
´
[4] Sol, V.; Blais, J.C.; Bolbach, G.; Carre, V.; Granet, R.; Guilloton, M.; Spiro, M.;
Krausz, P. Toward glycosylated peptidic porphyrins: a new strategy for PDT? Tet-
rahedron Lett. 1997, 38, 6391–6394.
[5] Mikata, Y.; Onchi, Y.; Tabata, K.; Ogura, S.; Okura, I.; Ono, H.; Yano, S. Sugar-
dependent photocytotoxic property of tetra- and octa-glycoconjugated tetraphenyl-
porphyrins. Tetrahedron Lett. 1998, 39, 4505–4508.
[6] Schell, C.; Hombrecher, H.K. Synthesis, self-assembly properties and incorporation
of carbohydrate-substituted porphyrins into cell membrane models. Chem. Eur. J.
1999, 5, 587–598.

Synthesis of New Glucosylated Porphyrins
359
´
[7] Sol, V.; Blais, J.C.; Carre, V.; Granet, R.; Guilloton, M.; Spiro, M.; Krausz, P. Syn-
thesis, spectroscopy and photocytotoxicity of glycosylated amino acid porphyrins
derivatives as promising molecules for cancer phototherapy. J. Org. Chem. 1999,
64, 4431–4444.
[8] Zheng, G.; Graham, A.; Shibata, M.; Missert, J.R.; Oseroff, A.R.; Dougherty, T.J.;
Pandey, R.K. Synthesis of b-galactose-conjugated chlorins derivated by enyme
metathesis as galectin-specific photosensitizers for photodynamic therapy. J. Org.
Chem. 2001, 66, 8709–8716.
[9] Laville, I.; Figueiredo, T.; Loock, B.; Pigaglio, S.; Maillard, P.; Grierson, D.S.;
Sarrez, C.D.; Croisy, A.; Blais, J. Synthesis, cellular internalization and photody-
namic activity of glucoconjugated derivated of tri and tetra(meta-hydroxyphenyl)-
chlorins. Bioorg. Med. Chem. 2003, 11, 1643–1652.
´
[10] Sylvain, I.; Benhaddou, R.; Carre, V.; Cottaz, S.; Driguez, H.; Granet, R.;
Guilloton, M.; Krausz, P. Synthesis and biological evaluation of thioglycosylated
meso-arylporphyrins. J. Porphyrins Phthalocyanines 1999, 3, 1–4.
[11] Chaleix, V.; Sol, V.; Huang, Y-M.; Guilloton, M.; Granet, R.; Blais, J.C.; Krausz, P.
RGD-porphyrin conjugates: synthesis and potential application in photodynamic
therapy. Eur. J. Org. Chem. 2003, 1486–1493.
[12] Kieda, C.; Monsigny, M. Involvement of membrane sugar receptors and membrane
glycoconjugates in the adhesion of 3LL cell subpopulations to cultured pulmonary
cells. Invasion Metastasis 1986, 6, 347–366.
[13] Monsigny, M.; Roche, A.C.; Midoux, P.; Kieda, C.; Mayer, R. Lectins and Glyco-
conjugates in Oncolog: Structure, Function, Clinical Application. Gabius, H.J.
and Nagel, G.A. Ed: Spinger-Verlag, Heidelberg., 1988, 1.
[14] Fujimoto, K.; Miyata, T.; Aoyama, Y. Saccharide-directed cell recognition and mol-
ecular delivery using macrocyclic saccharide clusters: masking of hydrophobicity to
enhance the saccharide specificity. J. Am. Chem. Soc. 2000, 122, 3358–3359.
[15] Ono, N.; Bougauchi, M.; Maruyama, K. Water-soluble porphyrins with four sugar
molecules. Tetrahedron. Lett. 1992, 33, 1629–1632.
[16] Lee, Y.C.; Lee, R.T. Affinity enhancement by multivalent lectin-carbohydrate
interaction Glycoconjug. J. 2000, 17, 543–551.
[17] Trombotto, S.; Bouchu, A.; Descotes, G.; Queneau, Y. Hydrogen peroxide oxidation
of palatinose and trehalulose: direct preparation of carboxymethyl a-D-glucopyra-
noside. Tetrahedron Lett. 2000, 41, 8273–8277.
[18] Trombotto, S.; Danel, M.; Fitremann, J.; Bouchu, A.; Queneau, Y. Straightforward
route for anchoring a glucosyl moiety onto nucleophilic species: reaction of amines
and alcohols with carboxymethyl 3,4,6 tri-O-acetyl-a-D-glucopyranoside 2-O-
lactone. J. Org. Chem, 2003, 68, 6672–6678.
[19] Little, R.G.; Anton, J.A.; Loach, P.A.; Ibers, J-A. The synthesis of some substituted
tetraalkylporphyrins. J. Heterocycl. Chem. 1975, 12, 345–349.
[20] Furhop, J.H.; Demoulin, C.; Boettcher, C.; Koning, J.; Siggel, U. Chiral micellar
porphyrin fibers with 2-aminoglycosamide head groups. J. Am. Chem. Soc. 1992,
114, 4159–4165.
[21] Barber, D.C.; Freitag-Beeston, R.A.; Whitten, D.G. Atropisomer-specific formation
of premicellar porphyrin J-aggregates in aqueous surfactant solutions. J. Phys.
Chem. 1991, 95, 4074–4086.
[22] Ohloff, G. Singlet oxygen: a reagent in organic synthesis Pure. Appl. Chem. 1975,
43, 481–502.

V. Sol et al.
360
[23] Albro, P.W.; Corbett, J.T.; Shroeder, J.L. Doubly allylic hydroperoxide formed in the
reaction between sterol 5,7 dienes and singlet oxygen. Photochem. Photobiol. 1994,
60, 310–315.
¨
¨
[24] Bocking, T.; Barrow, K.D.; Netting, A.G.; Chilcott, T.C.; Coster, H.G.; Hofer, M.
Effect of singlet oxygen on membrane sterols in the yeast saccharomyces cerevisiae.
Eur. J. Biochem. 2000, 267, 1607–1618.