Synthesis and utility of novel C-meso-glycosylated metalloporphyrins

Article abstract of DOI:10.1016/S0040-4020(00)00295-7

Novel hybrid porphyrins bearing two and four suitably protected glycosidic units appended at the meso positions of the central macrocycle through robust carbon-carbon bonds have been constructed and characterized. Metallation of these constructs with certain bivalent metal ions then produced a series of porphyrinato entities which had all the sugar protecting groups removed to arrive at the corresponding water soluble porphyrin-sugar hybrid species. It is noteworthy that two palladium derivatives, compounds 6 and 10, proved to be efficient reagents for the selective cleavage of double strand DNA into form II nicked circular DNA upon exposure to visible light at room temperature in aqueous media. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00295-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3977±3983
Synthesis and Utility of Novel C-meso-Glycosylated
Metalloporphyrins
Mara Cornia,a Monica Menozzi,a Enzio Ragg,b Stefania Mazzini,b Alessio Scarafoni,b
Franca Zanardic and Giovanni Casiraghic,
*
a
Á
Dipartimento di Chimica Organica e Industriale, Universita di Parma, Parco Area delle Scienze 17A, I-43100 Parma, Italy
b
Á
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, I-20133 Milano, Italy
c
Á
Dipartimento Farmaceutico, Universita di Parma, Parco Area delle Scienze 27A, I-43100 Parma, Italy
Received 29 November 1999; revised 22 March 2000; accepted 6 April 2000
AbstractÐNovel hybrid porphyrins bearing two and four suitably protected glycosidic units appended at the meso positions of the central
macrocycle through robust carbon±carbon bonds have been constructed and characterized. Metallation of these constructs with certain
bivalent metal ions then produced a series of porphyrinato entities which had all the sugar protecting groups removed to arrive at the
corresponding water soluble porphyrin±sugar hybrid species. It is noteworthy that two palladium derivatives, compounds 6 and 10, proved to
be ef®cient reagents for the selective cleavage of double strand DNA into form II nicked circular DNA upon exposure to visible light at room
temperature in aqueous media. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
noncovalent associations, bringing the photoactive
porphyrin core into the vicinity of the target and inducing
selective DNA strand breakage when irradiated. We report
herein the viability of this project and the resultant new class
of potential DNA photocleavers.
The identi®cation of materials that are able to achieve non-
random DNA strand scission proves to be an invaluable
resource in the design of potential anticancer or antiviral
drugs.1 In particular, compounds conjugating a reactive
chromophore to proper functional vectors such as peptide,2
oligonucleotide,3 and carbohydrate4 segments have
attracted special interest, these hybrids being constructed
to behave simultaneously as nucleic acid binders and degra-
dative chemical substances.5 Advantages of this approach
can be found in compounds which are not toxic in the dark
but can be rendered active by light.6 Such is the case with
certain photochemotherapeutic agents that are currently
under evaluation in the treatment of solid tumors,7 as well
as the emerging ®eld of viral photoinactivation.8
Synthesis and Characterization
Our choice was to assemble two hybrid porphyrin proto-
types, 5 and 9Ðand the corresponding deprotected free
bases 7 and 14Ðbearing two and four peripheral furanose
components, respectively, and to prepare certain neutral,
water soluble bivalent metal complexes, namely, palladium
porphyrins 6 and 10, as well as copper, zinc, and nickel
species 11, 12, and 13.
The aim of the present work was to construct porphyrin±
sugar conjugates9 and their metallo species by anchoring a
carbohydrate or a nucleoside motif to the meso positions of
the macrocycle through robust carbon±carbon bonds and to
evaluate their ability to cleave nucleic acids upon exposure
to visible light. The rationale behind these conjugates is that
the water solubilizing carbohydrate portion close to the
chromophore can potentiate the recognizing capability and
speci®city of the entire molecule for nucleic acids via
The synthesis of ligand 5 (Scheme 1), possessing alternate
uridine and p-¯uorophenyl groups at the 5, 10, 15, 20 meso
positions, was planned and executed as indicated, by
coupling of protected dipyrryluridine 3 to p-¯uorobenzalde-
hyde (4) followed by oxidation by DDQ. To assemble the
key dipyrryl nucleoside 3, condensation between readily
available aldehydo uridine 210 and pyrrole (1) was attained
by using SnCl4 as the Lewis acid promoter. The reaction
proceeded smoothly, giving the expected adduct 3 as the
sole component in 33% yield. Exposure of a 1:1 molar
mixture of 3 and p-¯uorobenzaldehyde (4) to BF3 etherate
(0.5 mol equiv.) in CH2Cl2 at room temperature, followed
by DDQ oxidative treatment, according to a MacDonald-
type [212] condensation procedure,11 allowed the assembly
Keywords: porphyrins; metalloporphyrins; nucleic acids; photodynamic
therapy.
* Corresponding author. Tel.: 139-0521-905080; fax: 139-0521-905006;
e-mail: casirag@ipruniv.cce.unipr.it
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00295-7

3978
M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
Scheme 1. Synthesis of C-meso-linked aryl-uridinyl porphyrins 5±7. (a) SnCl4, CHCl3, rt, 1 h (33%); (b) BF3´OEt2, CH2Cl2, rt, 3 h, then DDQ, sonication,
30 min (15%); (c) Pd(OAc)2, CH3OH/CHCl3 (1:1, v/v), sonication, 2 h, then 75% aq TFA, CH2Cl2, rt, 3 h (78%); (d) 75% aq TFA, CH2Cl2, sonication, 3 h
(90%).
of C-meso-linked aryl±uridinyl porphyrin hybrid construct
5 in an acceptable 15% yield over two steps.
water-soluble ligand 7 was also synthesized, by simply
exposing 5 to 75% aq TFA under ultrasonic irradiation
(90% yield).
In order for the water-soluble palladium porphyrin 6 to be
prepared, a two-step protocol was ef®ciently carried out
(78% yield), consisting of metallation of free ligand 5
[Pd(OAc)2, CH3OH/CHCl3, sonication] and subsequent
acidic full deacetonidation (75% aq TFA). In this context,
The design of tetra-C-glycosylated porphyrin 9 entails two-
component macrocyclization involving commercially
available dialdose 8 and pyrrole (1) (1:1 molar ratio), by
following essentially the Lindsey protocol (Scheme 2).12
Scheme 2. Synthesis of tetra-C-glycosylated porphyrins 9±14. (a) BF3´OEt2, CH2Cl2, rt, 3 h, then DDQ, Et3N, rt, 18 h (6%); (b) M(OAc)2, CH3OH/CHCl3 (1:1,
v/v), sonication, then 75% aq TFA, CH2Cl2, rt, 3 h (63±90%); (c) 75% aq TFA, CH2Cl2, sonication, 3 h (90%).

M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
3979
Figure 1. Agarose gel electrophoresis pattern for the porphyrin-triggered photocleavage of ds pUC18 DNA. Final porphyrin concentration, 25.7 mM;
irradiation times, 3, 6, and 9 h at 27^0.58C. pUC18 control experiment after 3, 6, and 9 h irradiation (lanes 1±3); pUC18 in the presence of 7 (lanes
4±6); 6 (lanes 7±9); 14 (lanes 10±12); 10 (lanes 13±15); linearized pUC18 (form III) (lane 16).
Indeed, direct condensation between 8 and 1 under BF3
etherate guidance followed by DDQ oxidation afforded
sugar±porphyrin assemblage 9 in 6% overall yield. As
previously described, metallation/deprotection of 9 to
water-soluble palladium porphyrin 10 was executed
[Pd(OAc)2, CH3OH/CHCl3, sonication; 75% aq TFA, 90%
yield], while similar treatment of 9 with copper(II), zinc(II),
and nickel(II) acetates followed by acidic deacetonidation
allowed the porphyrin series 11, 12, and 13 to be prepared in
87, 80, and 63% yields, respectively. In addition, fully
deprotected ligand 14 resulted from simple deprotection of
the parent porphyrin 9, as indicated (90% yield).
1027.1481; C48H35F2N8O10Pd), the D2 symmetry of the
1
parent ligand 5, as manifested in its H NMR spectrum,
was completely lost, possibly due to a ring distortion
induced by palladium ion complexation. By contrast, the
highly substituted palladium porphyrin 10 retained the D4
symmetry of its free ligand precursor 9 as indicated by its 1H
NMR spectrum which reveals a single resonance at dˆ9.65
for all the eight pyrrole protons.
DNA Photocleavage Experiments
The DNA-cleaving activities of porphyrin bases 7 and 14, as
well as the metallo derivatives 6, 10, 11, 12, and 13, were
tested under visible light irradiation (100 W tungsten lamp)
by monitoring the conversion of the supercoiled pUC18
(form I), a well characterized plasmid from E. coli (2686
base pairs), into nicked circular and linear DNA (forms II
and III).
Fully protected porphyrins 5 and 9, the corresponding water
soluble deprotected counterparts 7 and 14, and the various
novel metallated species 6, 10, 11, 12, and 13 were fully
1
characterized by H NMR analysis, high resolution mass
spectrometry, and, in some instances, elemental analyses.
Furthermore, UV±visible and circular dichroism spectro-
scopies provided immediate proof of the existence of the
porphyrin ring chromophore. As an example, uridinyl
porphyrin 5 had an exact mass of 1003.3216 m/z, as
expected, corresponding to a molecular formula [M1H]1
C54H45F2N8O10 (calculated 1003.3226 m/z). The UV±vis
spectrum showed an intense Soret band centered at
414 nm, accompanied by four less intense Q bands at 514,
The effect of light was quanti®ed by also taking into account
the amount of nicked DNA spontaneously produced by the
plasmid without added porphyrins. Under dark conditions
none of the products showed any DNA cleaving ability. The
DNA photocleavage results are reported in Fig. 1. Upon
irradiation at [porphyrin]ˆ25.7 mM, free aryl±uridinyl
porphyrin 7 and ligand 14, which bears four pendant
furanose moieties, behaved similarly, showing moderate
cleaving activity with ca. 15% nicked, circular DNA
(form II) produced within 9 h of light exposure. Strongly
enhanced activity was displayed by the palladium
complexes of the same ligands. The porphyrinato derivative
6 exhibited 19, 47, and 62% conversion to form II DNA
within 3, 6, and 9 h of irradiation, respectively, whereas the
porphyrinato counterpart 10 produced about 60% of form II
DNA at 3 h irradiation, suggesting an even greater photo-
dynamic activity.
1
549, 590, and 644 nm. The H NMR spectrum of 5 at
400 MHz indicated a D2 symmetry of the whole molecule
under the experimental conditions (258C in CDCl3), display-
ing two doublets each integrating to four protons for the
b-pyrrolic resonances and only one resonance system for
the two appended nucleoside chiral fragments.
As expected, tetrasubstituted sugar porphyrin 9 (m/z
999.4247; C52H63N4O16) showed a rather simple H NMR
1
pro®le with a single broad resonance at dˆ9.73 integrating
to eight protons. Clearly, this pattern re¯ects a high level of
molecular symmetry (D4), and this arrangement is corrobo-
rated by a single set of signals for the meso-furanose
moieties. A very intense Soret band at 419 nm in the
UV±vis spectrum (CHCl3) was indicative of some level of
deviation from planarity of the macrocycle, though this
diminished symmetry was not evident in its 1H NMR
spectrum.
Copper porphyrin 11 produced only 16% of form II after 9 h
of irradiation, showing a photodynamic activity similar to
the free porphyrin bases. In contrast, under the same con-
ditions, zinc and nickel complexes 12 and 13 had no DNA-
cleaving activity (,5%). None of the considered porphyrins
were able to produce the linear form III under the tested
experimental conditions.
All the metallated porphyrins of this study invariably
showed exact masses in line with the corresponding postu-
lated molecular formulas, with precisions within the usual
con®dence level. Clear, fully detailed 1H NMR spectra were
measured for the important palladium porphyrins 6 and 10,
showing diagnostic resonances for all the constitutional
fragments, the b-pyrrole system, the aromatic frames, and
the sugar units. For bis-uridinyl palladium porphyrin 6 (m/z
Conclusions
An easy preparation of C-meso-glycosylated porphyrin
ligands has been described, by using either a dipyrryl-
methane/aldehyde [212] cyclocondensation protocol, or a
direct pyrrole/aldehyde macrocyclization. When complexed

3980
M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
with a palladium ion, and fully liberated from the various
protecting groups, bis-uridinyl porphyrin 5 and tetra-
glycosylated porphyrin 9 gave rise to water soluble metal-
lated species, 6 and 10, which proved to be ef®cient DNA
cleavers upon exposure to visible light. These experiments
enlighten the central role played by the water-solubilizing
carbohydrate attached to the lipophilic porphyrin during
cleavage of the DNA target. Further studies aimed at a better
understanding of the basic mechanisms of recognition and
binding involving the DNA-based constructs and these
promising hybrid structures are in progress.
To this reaction mixture was added the above obtained
protected uridine (510 mg, 1.8 mmol) dissolved in 8 mL
of a 4:1 CH2Cl2/DMF solution, and the resulting mixture
was treated with acetic anhydride (0.68 mL, 7.2 mmol).
After 10 min, the reaction was quenched with EtOH
(0.9 mL) and poured into ethyl acetate (90 mL). The result-
ing mixture was ®ltered with gentle suction through a
sintered glass funnel packed, as a column, with silica topped
with a layer of anhydrous Na2SO4. After elution with ethyl
acetate, the solvent was evaporated under reduced pressure,
affording 406 mg (80% yield) of aldehyde 2, which was
used as such in the subsequent coupling step. Compound
1
2: H NMR (300 MHz, CDCl3): dˆ9.45 (s, 1H), 7.25 (d,
Jˆ7.9 Hz, 1H), 5.78 (d, Jˆ7.9 Hz, 1H), 5.49 (s, 1H), 5.22
(dd, Jˆ6.3, 1.6 Hz, 1H), 5.11 (d, Jˆ6.3 Hz, 1H), 4.56 (d,
Jˆ1.6 Hz, 1H), 1.54 (s, 3H), 1.37 (s, 3H). 13C NMR
(75 MHz, CDCl3): dˆ199.4, 163.6, 151.0, 144.3, 112.0,
103.0, 100.2, 94.2, 85.0, 83.8, 26.6, 25.0. C12H14N2O6
(282.26): calcd. C 51.07, H 5.00, N 9.92; found C 50.96,
H 5.13, N 9.84.
Experimental
General. Flash chromatography was performed on 40±
60 mm silica gel (Merck) or Florisil (100±200 mesh,
Carlo Erba), using the indicated solvent mixtures. Analyti-
cal thin-layer chromatography was performed on Merck
silica gel 60 F254 plates (0.25 mm). The compounds were
visualized by dipping the plates in a solution of cerium(IV)
1
sulfate/ammonium molybdate. H and 13C NMR spectra
Dipyrryluridine 3. To a stirring solution of pyrrole (1)
(0.20 mL, 2.88 mmol) and aldehyde 2 (406 mg, 1.44
mmol) in CHCl3 (60 mL), SnCl4 (80 mL, 0.72 mmol) was
added dropwise at room temperature under argon atmos-
phere. After being stirred for 1 h, the pale-violet reaction
mixture was quenched by addition of saturated aqueous
NaHCO3 and solid NH4Cl. The resulting slurry was
extracted with CH2Cl2 (3£25 mL), and the combined
organic layers were dried (MgSO4), ®ltered, and concen-
trated under vacuum. The crude material was puri®ed by
¯ash chromatography (75:25 CH2Cl2/acetone) to furnish
190 mg (33% yield) of pure 3 as a colorless oil.
[a]2D0ˆ2208.0 (c 0.024, CHCl3). UV±vis (CHCl3) lmax
258 nm (eˆ3440 cm21 M21). 1H NMR (400 MHz,
CDCl3): dˆ8.38 (bs, 1H), 8.37 (bs, 1H), 8.28 (bs, 1H),
6.72 (m, 1H), 6.69 (m, 1H), 6.34 (d, Jˆ8.1 Hz, 1H), 6.18
(m, 2H), 6.13 (m, 2H), 5.90 (d, Jˆ2.9 Hz, 1H), 5.61 (d,
Jˆ8.1 Hz, 1H), 4.77 (dd, Jˆ6.8, 4.6 Hz, 1H), 4.65 (dd,
Jˆ6.8, 2.9 Hz, 1H), 4.58 (dd, Jˆ4.6, 4.4 Hz, 1H), 4.56 (d,
Jˆ4.4 Hz, 1H), 1.32 (s, 3H), 1.25 (s, 3H). 13C NMR
(25 MHz, CDCl3): dˆ163.4, 162.6, 150.6, 141.9, 129.2,
129.0, 117.4, 117.3, 114.7, 108.3, 108.1, 106.7, 106.6,
102.9, 92.2, 87.8, 83.3, 81.1, 27.1, 25.3. C20H22N4O5
(398.42): calcd. C 60.29, H 5.57, N 14.06; found C 60.40,
H 5.65, N 14.00.
were obtained on a Bruker AMX-400, Bruker AC-300, or
Bruker AC-100 spectrometer, and are reported in parts per
million (d) relative to tetramethylsilane (0.0 ppm) as an
internal reference, with coupling constants in Hertz (Hz).
Optical rotations were measured on a Rudolph Research
Autopol III polarimeter and are given in units of
1021 deg cm2 g21. Circular dichroism spectra were obtained
using a Jasco J715 spectropolarimeter apparatus and molar
elipticity [u] is reported in deg cm2 dmol21. Infra-red spec-
tra were recorded on a Jasco FT/IR-300E spectrometer.
Ultraviolet±visible spectra were recorded on a Kontron
Uvicon 860 spectrophotometer. Elemental analyses were
performed by the Microanalytical Laboratory of University
of Parma. Low-resolution mass spectra were obtained on a
Finnigan 1060 6c mass spectrometer, and high-resolution
spectra were obtained on a Kratos MS08RFA mass spectro-
meter using the chemical ionization method (CH4). All the
solvents were distilled before use according to standard
methods.
Pyrrole (1), p-¯uorobenzaldehyde (4), and 1,2-O-isopropyl-
idene-3-O-methyl-a-d-xylopentodialdofuranose-(1,4) (8)
were obtained from commercial suppliers (Fluka) and
used without further puri®cation.
Porphyrin 5. To a solution of dipyrryl uridine 3 (170 mg,
0.43 mmol) in dry CH2Cl2 (60 mL) were added sequentially
4-¯uorobenzaldehyde (4) (46 mL, 0.43 mmol) and BF3´OEt2
(26 mL, 0.22 mmol), while a stream of pure argon was
passing. The dark-red solution was carefully shielded from
light and stirring was continued for 3 h. Then, dichloro-
dicyanobenzoquinone (DDQ) (159 mg, 0.7 mmol) was
added and the resulting dark reaction mixture was kept
under ultrasonic irradiation for 30 min. The solvent was
evaporated under vacuum and the product was puri®ed by
¯ash chromatography on Florisil (75:25 CH2Cl2/acetone) to
furnish 32 mg of porphyrin 5 (15% yield) as a red-brown
glassy solid. [a]2D0ˆ2675.0 (c 0.03, CHCl3). FT-IR (KBr)
Aldehyde 2. To a stirring solution of uridine (Aldrich)
(0.55 g, 2.25 mmol) in dry acetone (45 mL), a catalytic
amount of H2SO4 was added dropwise, under argon atmos-
phere at room temperature. After being stirred for 1 h, the
reaction mixture was quenched with BaCO3 until neutrali-
zation. The heterogeneous mixture was ®ltered on a celite
pad, and the ®ltrates were evaporated under reduced
pressure, to give a crude white solid which was subjected
to ¯ash chromatographic puri®cation on silica gel (90:10
CH2Cl2/acetone). A pure protected uridine intermediate
was recovered (510 mg, 80% yield) as a white solid.
To a stirring solution of CH2Cl2 (8 mL) and DMF (2 mL)
under argon were sequentially added pyridine (1.16 mL,
14.4 mmol) and CrO3 (720 mg, 7.2 mmol), and the resulting
mixture was allowed to stir at room temperature for 20 min.
3350 (N±H), 1681 (CvO), 1600, 1360, 1040, 770 cm21
.
UV±vis (CHCl3) lmax 414 nm (eˆ1.15£105 cm21 M21),
514 (eˆ5900), 549 (eˆ2810), 590 (eˆ2190), 644

M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
3981
(eˆ1350). CD (7.5£1024 M, CHCl3) [u]300
ˆ
washed with ether, and neutralized by addition of a 2 M
aqueous ammonia solution. Evaporation of the water
solvent under vacuum gave crude compound 7, that was
puri®ed by a short ¯ash chromatographic column on silica
gel (60:40 acetone/CHCl3), furnishing 8 mg (90% yield) of
2815 deg cm2 dmol21, [u]439ˆ2940, [u]540ˆ1500. CD
(6.2£1025 M, CHCl3) [u]371ˆ12470 deg cm2 dmol21,
[u]404ˆ
16930,
[u]420ˆ15140,
[u]429ˆ15260,
1
[u]602ˆ22470. H NMR (400 MHz, CDCl3): dˆ9.70 (d,
Jˆ5.0 Hz, 4H), 8.87 (d, Jˆ5.0 Hz, 4H), 8.80 (bs, 2H),
8.13 (m, 4H), 7.45 (m, 6H), 7.18 (d, Jˆ8.1 Hz, 2H), 5.76
(d, Jˆ8.1 Hz, 2H), 5.50 (bs, 2H), 5.16 (d, Jˆ6.0 Hz, 2H),
4.98 (d, Jˆ6.0 Hz, 2H), 2.63 (s, 6H), 2.17 (s, 6H), 22.67
(bs, 2H). 13C NMR (75 MHz, CDCl3): dˆ164.5 (2C), 162.5
(2C), 162.1 (2C), 161.4 (2C), 149.9 (2C), 143.1 (2C), 138.3
(2C), 135.6 (4C), 132.4 (4C), 128.5 (4C), 127.2, 119.2,
116.2 (2C), 113.7 (2C), 113.4 (2C), 103.5 (2C), 102.7
(2C), 95.7 (3C), 93.4 (2C), 88.8 (3C), 87.6 (2C), 82.5
(2C), 29.3 (2C), 29.2, 27.6. HRMS (CI, CH4) m/z:
1003.3216 [M1H]1 (calcd 1003.3226 for C54H45F2N8O10).
C54H44F2N8O10 (1003.00): calcd C 64.67, H 4.42, N 11.17;
found C 64.51, H 4.59, N 10.99.
pure free base 7 as a red-brown glassy solid. [a]2D0ˆ17.1 (c
20
0.06, MeOH). [a] ˆ115.5 (c 0.06, CHCl3). FT-IR (KBr)
546
3330 (broad), 1682 (CvO), 1600, 1360, 1040, 770 cm21
.
UV±vis (CHCl3) lmax 409 nm (eˆ1.6£104 cm21 M21), 511
(eˆ1550), 545 (eˆ670), 587 (eˆ550), 642 (eˆ280). UV±
vis (MeOH) lmax 416 nm (eˆ1260 cm21 M21), 545 (eˆ
130), 589 (eˆ90). CD (2.3£1023 M, MeOH) [u]413
ˆ
196 deg cm2 dmol21, [u]553ˆ246. 1H NMR (400 MHz,
[D6]acetone/CD3OD): dˆ9.99 (bs, 4H), 9.55 (bs, 4H),
8.35 (m, 4H), 7.93 (m, 4H), 7.30 (m, 2H), 7.22 (d,
Jˆ8.0 Hz, 2H), 6.51 (d, Jˆ8.0 Hz, 2H), 6.40 (bs, 2H),
5.51 (d, Jˆ3.5 Hz, 2H), 5.25 (m, 2H). HRMS (CI, CH4)
m/z: 923.2595 [M1H]1 (calcd 923.2600 for C48H37F2
N8O10). C48H36F2N8O10 (922.87): calcd C 62.47, H 3.93, N
12.14; found C 62.55, H 3.81, N 11.96.
Porphyrin 6. To a solution of protected porphyrin 5 (22 mg,
0.02 mmol) in 50 mL of methanol/chloroform (1:1, v/v),
Pd(OAc)2 (6.7 mg, 0.03 mmol) was added under stirring
and the resulting reaction mixture was kept under ultrasonic
irradiation until complete disappearance of the starting
porphyrin as judged by TLC analysis (2 h). The mixture
was ®ltered and the solvent was removed under reduced
pressure, furnishing a protected palladium porphyrin inter-
Porphyrin 9. To a stirring solution of pyrrole (1) (170 mL,
2.5 mmol) and dialdose 8 (500 mg, 2.5 mmol) in anhydrous
CH2Cl2 (200 mL), freshly distilled BF3 etherate (30 mL,
0.25 mmol) was added under argon atmosphere at room
temperature. The reaction vessel was carefully shielded
from light, and stirring was continued for 3 h. Triethylamine
(70 mL, 0.50 mmol) and DDQ (610 mg, 2.70 mmol) were
added, and the dark-violet reaction mixture was stirred at
room temperature for an additional 18 h. The solvent was
evaporated under vacuum and the resulting solid was
dissolved in CH2Cl2/ethyl acetate (93:7 ratio, 5 mL). Florisil
(1 g) was added and the solvent was evaporated to give a
powder which was charged at the top of a short silica gel
column. Elution with CH2Cl2/ethyl acetate (90:10) solvent
mixture furnished pure glycosylated porphyrin 9 (37 mg,
6% yield) as a dark-violet powder. [a]2D0ˆ1135.4 (c 0.01,
CHCl3). FT-IR (KBr) 3320 (N±H), 1640, 1370, 1057,
1
mediate (24 mg, 98%) as a brown glassy solid. H NMR
(400 MHz, CDCl3): dˆ10.2 (s, 2H), 9.88 (bs, 1H), 9.72
(s, 2H), 9.40 (s, 2H), 9.35 (bs, 1H), 8.6-9.0 (m, 2H), 7.5±
8.3 (m, 12H), 6.05 (d, Jˆ7.1 Hz, 2H), 5.52 (d, Jˆ1.0 Hz,
2H), 5.29 (d, Jˆ3.0 Hz, 2H), 5.08 (d, Jˆ3.0 Hz, 2H), 3.5±
3.7 (4s, 12H). This intermediate (24 mg, 0.019 mmol) was
dissolved in 15 mL of CH2Cl2 and treated with 8 mL of a
75% aqueous tri¯uoroacetic acid solution at room tempera-
ture. After stirring for 3 h, the organic phase discolored
completely, while the aqueous phase assumed a pink-
brown color. The aqueous layer was separated, washed
with ether, and neutralized by addition of a 2 M ammonia
solution. Evaporation of the water solvent under reduced
pressure gave crude palladium porphyrin 6, that was
subjected to ¯ash chromatographic puri®cation on silica
gel (80:20 CH2Cl2/acetone). Porphyrin 6 was obtained
(16 mg, 78% yield from 5) as a red-brown glassy solid.
[a]2D0ˆ23.9 (c 0.07, CHCl3). FT-IR (KBr) 3330 (broad),
1680 (CvO), 1600, 1363, 1275, 1152, 1038, 840,
770 cm21. UV±vis (CHCl3) lmax 410 nm (eˆ9110 cm21
745 cm21
.
UV±vis (CHCl3) lmax 419 nm (eˆ
1.4£105 cm21 M21), 523 (eˆ8945), 560 (eˆ1840), 595
(eˆ3100), 652 (eˆ2130). CD (CHCl3) [u]414ˆ
257745 deg cm2 dmol21 1H NMR (400 MHz, CDCl3):
.
dˆ 9.73 (bs, 8H), 7.87 (d, Jˆ3.0 Hz, 4H), 6.69 (d,
Jˆ4.0 Hz, 4H), 5.16 (d, Jˆ4.0 Hz, 4H), 4.61 (d,
Jˆ3.0 Hz, 4H), 2.48 (s, 12H), 1.90 (s, 12H), 1.60 (s,
12H), 22.62 (bs, 2H). 13C NMR (100 MHz, CDCl3):
dˆ145.4 (8C), 129.9 (8C), 112.0 (4C), 109.8 (4C), 104.9
(4C), 90.0 (4C), 88.4 (8 C), 58.8 (4C), 27.4 (4C), 26.6 (4C).
HRMS (CI, CH4) m/z: 999.4247 [M1H]1 (calcd 999.4239
for C52H63N4O16). C52H62N4O16 (999.09): calcd C 62.51, H
6.26, N 5.61; found C 62.63, H 6.23, N 5.52.
1
M21), 522 (eˆ845), 556 (eˆ270), 586 (eˆ110). H NMR
(400 MHz, [D6]acetone): dˆ10.00 (d, Jˆ5.1 Hz, 2H), 9.5±
10.1 (m, 6H), 8.80 (d, Jˆ5.1 Hz, 2H), 8.20 (m, 4H), 7.98 (d,
Jˆ8.1 Hz, 2H), 7.80 (d, Jˆ8.1 Hz, 1H), 7.63 (d, Jˆ7.1 Hz,
1H), 7.55 (m, 4H), 5.90 (m, 2H), 5.3±5.7 (m, 6H), 2.90 (bs,
4H). HRMS (CI, CH4) m/z: 1027.1481 [M1H]1 (calcd
1027.1473 for C48H35F2N8O10Pd).
Porphyrin 10. The title compound was obtained from
protected porphyrin 9 (25 mg, 0.025 mmol) and Pd(OAc)2
(45 mg, 0.2 mmol) following the two-step procedure
described for 6. After the metallation reaction (24 h), a
protected palladium porphyrin intermediate was obtained
(26 mg, 94%) as a blue-magenta glassy solid. UV±vis
(CHCl3) lmax 419 nm (eˆ3.0£104 cm21 M21), 534 (eˆ
Porphyrin 7. To a solution of protected porphyrin 5 (10 mg,
0.001 mmol) in CH2Cl2 (5 mL) a 75% aqueous tri¯uoro-
acetic acid solution (1 mL) was added under stirring at
room temperature. After 10 min, the mixture was subjected
to ultrasonic irradiation and was allowed stirring for 3 h.
The organic phase discolored completely, while the aqueous
phase assumed a green color indicative of the formation of a
porphyrin dication. The aqueous layer was separated,
1
2200). H NMR (300 MHz, CDCl3): dˆ9.67 (s, 8H), 7.73
(d, Jˆ3.0 Hz, 4H), 6.65 (d, Jˆ4.0 Hz, 4H), 5.12 (d, Jˆ
4.0 Hz, 4H), 4.54 (d, Jˆ3.0 Hz, 4H), 2.44 (s, 12H), 1.87
(s, 12H), 1.58 (s, 12H). HRMS (CI, CH4) m/z: 1103.0470

3982
M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
[M1H]1 (calcd 1103.3111 for C52H61N4O16Pd). This inter-
mediate (26 mg, 0.024 mmol) was subjected to TFA-
promoted deprotection treatment as described for compound
6. After ¯ash chromatographic puri®cation (85:15 CH2Cl2/
ethyl acetate), pure deprotected palladium porphyrin 10 was
obtained (21 mg, 90% yield from 9) as a red-brown glassy
solid. [a]2D0ˆ17.6 (c 0.02, CHCl3). FT-IR (KBr) 3330
UV±vis (CHCl3) lmax 411 nm (eˆ9900 cm21 M21), 527
(eˆ1000), 563 (eˆ610). 1H NMR (300 MHz, [D6]-
acetone/CD3OD): d ˆ9.65 (bs, 8H), 7.69 (d, Jˆ3.0 Hz,
4H), 6.62 (d, Jˆ4.0 Hz, 4H), 5.10 (d, Jˆ4.0 Hz, 4H), 4.51
(d, Jˆ3.0 Hz, 4H), 2.41 (s, 12H). HRMS (CI, CH4) m/z:
943.1850 [M1H]1 (calcd 943.1859 for C40H45N4O16Pd).
HRMS (CI, CH4) m/z: 901.2130 [M1H]1 (calcd 901.2122
for C40H45N4O16Zn).
Porphyrin 13. The nickel porphyrin 13 was obtained from 9
(10 mg, 0.01 mmol) and excess Ni(OAc)2´4H2O (100 mg,
0.4 mmol) following the two-step procedure described for
6. After the metallation reaction (10 days), a protected
nickel porphyrin intermediate was obtained (7.4 mg, 70%)
as a brown-red glass. UV±vis (CHCl3) lmax 421 nm (eˆ
(broad), 1608, 1371, 1275, 1152, 1055, 839, 745 cm21
.
1
1.0£104 cm21 M21), 547 (eˆ590), 589 (eˆ210). H NMR
(300 MHz, CDCl3): dˆ9.36 (s, 8H), 7.09 (d, Jˆ3.0 Hz, 4H),
6.48 (d, Jˆ4.0 Hz, 4H), 4.98 (d, Jˆ4.0 Hz, 4H), 4.33 (d,
Jˆ3.0 Hz, 4H), 2.52 (s, 12H), 1.75 (s, 12H), 1.51 (s, 12H).
HRMS (CI, CH4) m/z: 1054.9804 [M1H]1 (calcd
1055.3436 for C52H61N4O16Ni). This intermediate (7.4 mg,
0.007 mmol) was subjected to TFA-promoted deprotection
treatment, as described for compound 6. After ¯ash chro-
matographic puri®cation (80:20 CH2Cl2/ethyl acetate), pure
deprotected nickel porphyrin 13 was recovered (5.6 mg,
63% yield from 9) as a brown-red glass. FT-IR (KBr)
3330 (broad), 1618, 1372, 1272, 1147, 1054, 837,
Porphyrin 11. The copper porphyrin 11 was obtained from
protected porphyrin 9 (10 mg, 0.01 mmol) and Cu(OAc)2
(2.0 mg, 0.01 mmol) following the two-step procedure
described for 6. After the metallation reaction (1 h), a
protected copper(II) porphyrin intermediate was obtained
(10 mg, 95%) as a blue-magenta glassy solid. UV±vis
(CHCl3) lmax 419 nm (eˆ9.5£104 cm21 M21), 550 (eˆ
4300), 591 (eˆ1830). 1H NMR (300 MHz, CDCl3):
dˆ10.2±9.3 (m, 8H), 8.2-7.9 (m, 4H), 7.0-6.9 (m, 4H),
6.6-6.2 (m, 4H), 5.3-4.9 (m, 4H), 3.1-2.8 (2s, 12H), 2.5-
1.5 (2s, 24H). HRMS (CI, CH4) m/z: 1060.3390 [M1H]1
(calcd 1060.3378 for C52H61N4O16Cu). This intermediate
(10 mg, 0.009 mmol) was subjected to TFA-promoted
deprotection treatment, as described for compound 6.
After a short-column ¯ash chromatographic puri®cation
(80:20 CH2Cl2/ethyl acetate), pure deprotected copper(II)
porphyrin 11 was obtained (8 mg, 87% yield from 9) as a
dark red solid. FT-IR (KBr) 3330 (broad), 1602, 1370, 1271,
1148, 1058, 850, 750 cm21. UV±vis (CHCl3) lmax 414 nm
754 cm21
.
UV±vis (CHCl3) lmax 418 nm (eˆ4550
cm21 M21). 1H NMR (300 MHz, [D6]acetone/CD3OD):
dˆ9.35 (bs, 8H), 7.05 (d, Jˆ3.0 Hz, 4H), 6.45 (d, Jˆ
4.0 Hz, 4H), 5.00 (d, Jˆ4.0 Hz, 4H), 4.30 (d, Jˆ3.0 Hz,
4H), 2.50 (s, 12H). HRMS (CI, CH4) m/z: 895.2191
[M1H]1 (calcd 895.2184 for C40H45N4O16Ni).
Porphyrin 14. Deprotected free base 14 was obtained from 9
(10 mg, 0.01 mmol) following the procedure described for 7
(reaction time 3 h). After ¯ash chromatographic puri®cation
on silica gel (90:10 CH2Cl2/MeOH), pure free base 14 was
recovered (7.5 mg, 90%) as a brown-red glassy solid. FT-IR
(KBr) 3320 (broad), 1640, 1370, 1057, 745 cm21. UV±vis
(CHCl3) lmax 421 nm (eˆ4750 cm21 M21), 523 (eˆ220),
1
(eˆ6500 cm21 M21), 547 (eˆ305). H NMR (300 MHz,
1
[D6]acetone/CD3OD): dˆ9.8±9.2 (m, 8H), 8.1±7.8 (m,
4H), 7.0±6.8 (m, 4H), 6.5±6.2 (m, 4H), 5.0±4.7 (m, 4H),
3.1±2.8 (2s, 12H). HRMS (CI, CH4) m/z: 900.2132
[M1H]1 (calcd 900.2126 for C40H45N4O16Cu).
600 (eˆ100), 652 (eˆ60). H NMR (300 MHz, CD3OD):
dˆ10.0±9.6 (m, 8H), 7.7±7.4 (m, 4H), 6.4±5.6 (m, 4H),
4.65 (m, 4H), 4.43 (m, 4H), 2.55 (s, 12H). HRMS (CI,
CH4) m/z: 839.2996 [M1H]1 (calcd 839.2987 for
C40H47N4O16).
Porphyrin 12. The zinc porphyrin 12 was obtained from 9
(10 mg, 0.01 mmol) and Zn(OAc)2 (2.2 mg, 0.01 mmol)
following the two-step procedure described for 6. After
the metallation reaction (30 h), a protected zinc porphyrin
intermediate was obtained (9 mg, 85%) as a red-brown
glassy solid. UV±vis (CHCl3) lmax 425 nm (eˆ1.4£
105 cm21 M21), 561 (eˆ6500), 600 (eˆ2740), 620 (eˆ
Gel electrophoretic experiments. Plasmid pUC18 (300 ng),
dissolved in 10 mM MES (2-morpholinoethanesulfonic
acid) at pH 6.0 ([pUC18]ˆ8.6 nM), was irradiated for 3,
6, and 9 h at 27.0^0.58C with a 100 W tungsten lamp in
the absence or in the presence of ethanolic 25.7 mM solu-
tions of porphyrins 7, 6, 14, and 10. Linearized pUC18 was
obtained by digestion of 50 ng of pUC18 at 37.0^0.58C for
2 h with 5U of EcoRI restriction enzyme. Samples were
loaded on a 0.8% agarose gel in a 40 mM tris-acetate pH
7.7/1 mM EDTA buffer, submitted to electrophoresis (run at
constant 100 V), and then stained with ethidium bromide.
The DNA bands were revealed by UV light and a polaroid
photograph taken; the amounts of DNA forms were quanti-
®ed by scan densitometer with software CREAM (KEN EN
TEC), Copenhagen.
1
1830). H NMR (300 MHz, CDCl3): dˆ9.67 (s, 8H), 7.85
(d, Jˆ3.0 Hz, 4H), 6.62 (d, Jˆ4.0 Hz, 4H), 5.14 (d,
Jˆ4.0 Hz, 4H), 4.50 (d, Jˆ3.0 Hz, 4H), 2.40 (s, 12H),
1.80 (s, 12H), 1.50 (s, 12H). HRMS (CI, CH4) m/z:
1061.3382 [M1H]1 (calcd 1061.3374 for C52H61N4±
O16Zn). This intermediate (9 mg, 0.0085 mmol) was
subjected to TFA-promoted deprotection treatment as
described for compound 6. After ¯ash chromatographic
puri®cation (85:15 CH2Cl2/ethyl acetate), pure deprotected
zinc porphyrin 12 was recovered (7 mg, 80% yield from 9)
as a dark red glass. FT-IR (KBr) 3330 (broad), 1370, 1268,
1147, 1057, 838, 748 cm21. UV±vis (CHCl3) lmax 416 nm
Acknowledgements
1
(eˆ1.4£104 cm21 M21), 556 (eˆ1470), 590 (eˆ380). H
NMR (300 MHz, [D6]acetone/CD3OD): dˆ9.66 (bs, 8H),
7.85 (d, Jˆ3.0 Hz, 4H), 6.59 (d, Jˆ4.0 Hz, 4H), 5.13 (d,
Jˆ4.0 Hz, 4H), 4.48 (d, Jˆ3.0 Hz, 4H), 2.37 (s, 12H).
Á
We wish to thank the Ministero dell'Universita e della
Ricerca Scienti®ca e Tecnologica (MURST), Italy and CNR
(Comitato per le Ricerche Tecnologiche e l'Innovazione),

M. Cornia et al. / Tetrahedron 56 (2000) 3977±3983
3983
F. V.; Detmer III, C. A.; Bocarsly, J. R. J. Am. Chem. Soc. 1996,
118, 5339±5345.
Italy, for ®nancial support. The `Centro Interdipartimentale
di Misure G. Casnati', University of Parma, Italy, is grate-
fully acknowledged for instrumental facilities. M. M.
acknowledges a scholarship from Telethon Proj. N. E574.
6. Gasper, S. M.; Armitage, B.; Shui, X.; Hu, G. G.; Yu, C.;
Schuster, G. B.; Williams, L. D. J. Am. Chem. Soc. 1998, 120,
12402±12409. (b) Nakatani, K.; Shirai, J.; Sando, S.; Saito, I.
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