Chemically robust fluoroalkyl phthalocyanine-oligonucleotide bioconjugates and their GRP78 oncogene photocleavage activity

Article abstract of DOI:10.1039/c4cc00703d

The first representative of functionalized fluoroalkyl phthalocyanines, F₄₈H₇(COOH)PcZn, is reported. The complex generates ¹O₂ affording long-lasting photooxidation of an external substrate without self-decompo

Full text of DOI:10.1039/c4cc00703d

Volume 50 Number 48 18 June 2014 Pages 6287–6410
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Sergiu M. Gorun, David Sabatino et al.
Chemically robust fluoroalkyl phthalocyanine–oligonucleotide
bioconjugates and their GRP78 oncogene photocleavage activity

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Chemically robust fluoroalkyl phthalocyanine–
oligonucleotide bioconjugates and their GRP78
oncogene photocleavage activity†
Cite this: Chem. Commun., 2014,
50, 6309
Received 27th January 2014,
Accepted 18th March 2014
Pradeepkumar Patel,a Hemantbhai H. Patel,ab Emily Borland,ab Sergiu M. Gorun*ab
and David Sabatino*a
DOI: 10.1039/c4cc00703d
www.rsc.org/chemcomm
The first representative of functionalized fluoroalkyl phthalocyanines, in particular, are easily modified while exhibiting high thermal
F48H7(COOH)PcZn, is reported. The complex generates 1O2 affording and chemical stability, in addition to intense light absorption
long-lasting photooxidation of an external substrate without self- toward the advantageous red spectral region.4
decomposition. The carboxylic group couples with an antisense
In order to help mitigate current photosensitizers’ limitations,
oligonucleotide targeting GRP78 oncogenes, resulting in the we report the first example of a new class of functionalized
F48H7PcZn–cancer targeting oligonucleotide (CTO). The bioconjugated fluoroalkyl phthalocyanine photosensitizers, F48H7(COOH)PcZn,
fluorophthalocyanine effectively hybridizes complementary GRP78 3, in which most of the C–H bonds of the Pc macrocycle are
DNA and mRNA sequences. Piperidine cleavage assays reveal desired replaced by aromatic and aliphatic C–F bonds. The fluorinated
photochemical oligonucleotide oxidative degradation for both phthalocyanines, Fx(RF)yPcM, (Fx = fluorine, RF = perfluoro alkyl
F48H7PcZn–CTO:DNA and F48H7PcZn–CTO:mRNA hybrids. This group, x + y r 16, Pc = phthalocyanine and M = metal) constitute
new materials strategy could be extended to other functional an important class of chemically robust photosensitizers.5 The
fluorinated phthalocyanines–antisense oligonucleotide combina- fluorinated alkyl substituents, RF, in contrast to aromatic F groups
tions for long-lasting oncogene-targeting photodynamic therapy.
are expected to enhance the stability of the Pc macrocycle towards
nucleophilic attack as the y/x ratio increases, as well as the
Photodynamic therapy (PDT) uses photosensitizers to produce resistance to attack by ROS that the Pcs produce during photo-
1
reactive oxygen species, ROS (e.g. singlet oxygen, O2), that kill dynamic events.6 Thus, partial replacement of aromatic F in
tumor cells.1 Advantageously, PDT does not trigger immuno- perfluorophthalocyanine, F16PcM (M = Zn; x = 16, y = 0), with
suppressive or myelosuppressive effects that accompany surgery, iso-perfluoroalkyl groups (RF) to give octakis-(perfluoro-i-C3F7)-
chemotherapy or radiation treatments. Historically, the first (perfluoro)PcM, F64PcM (x = y = 8) was shown to improve the Pc’s
successful clinical application of PDT in oncology was demon- solubility in organic solvents, depress the HOMO–LUMO gaps,
strated over 100 years ago,2 with the first PDT drug approved for thereby extending the Q-bands absorption in the 600–900 nm NIR
bladder cancer in 1993.3 However, unfavorable photosensitizer region, while retaining high B105 molar extinction coefficients
properties that lower therapeutic index, such as: poor water favouring efficient 1O2 production.7 The RFPc macrocycle scaffold’s
solubility, limited extinction coefficients in the maximum tissue resistance to degradation prompted their use as artificial enzymes,8
penetration NIR region, poor clearance from normal cells and anti-cancer drugs,9 photosensitizers for degradation of azo dyes,10
photostability contributed to limited approval of PDT drugs for and as redox catalysts.11 However, the lack of the RFPc’s specific
clinical use. Moreover, poor tumor-targeting results in dispersion tissue distribution suggested the need for a targeting approach
in all tissue types, depressing efficiency and enhancing harmful in our PDT applications. The enhanced tissue localization of
side-effects. Most of the photosensitizers used in oncology are photosensitizers is particularly useful for potentially increasing
based on the tetrapyrrole scaffold, such as, the porphyrins, their therapeutic index. Strategies for specific tissue targeting
chlorins, corroles and phthalocyanines.1 The phthalocyanines, include anchoring protio Pcs onto bio-probes, following their
functionalization with –COOH, –NH2, –SO3H, –X (X = Cl, Br, I)
a Department of Chemistry and Biochemistry, Seton Hall University, South Orange,
NJ 07079, USA. E-mail: sergiu.gorun@shu.edu, david.sabatino@shu.edu
b Center for Functional Materials, Seton Hall University, South Orange, NJ 07079,
USA
groups12,13 that thus facilitate Pc conjugation with carbo-
hydrates,14 nucleosides,15 oligonucleotides,16 peptides17 and
proteins.18 However, these approaches offer a limited therapeutic
index for effective and long-lasting PDT due to the decomposition
of the protio Pcs by the ROS they produce.19 Moreover, chemically
less stable hydrocarbon-based Pcs coupled to short, arbitrary
† Electronic supplementary information (ESI) available: Experimental methods,
synthetic details and characterization data including NMR, IR, UV-Vis and LCMS.
See DOI: 10.1039/c4cc00703d
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 6309--6311 | 6309

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Fig. 2 (a) Photooxidation of b-citronellol in EtOH by 3, (‘‘ZnPc’’). ISC =
intersystem crossing; (b) time-dependent O2 titration at 25 1C. No reaction
occurs in the absence of either light or O2. See also Fig. S6 (ESI†). The O2
was consumed in the calculated stoichiometric amount.
Fig. 1 F48H7PcZn–GRP78 cancer-targeting oligonucleotide (CTO) bio-
conjugate, 6.
factor for avoiding the shortening of the Pc excited states lifetimes
via intermolecular interactions which lowers 1O2 production
efficiency. Importantly, 3 remains unchanged (499%) at the
end of the photooxidation reaction thus establishing its reactivity
without self-decomposition. Taken together, this data demon-
strates the ability of the functionalized fluorophthalocyanine to
exhibit single-site structural characteristics in solution and to
photooxidize an external substrate without being decomposed by
the 1O2 it produces.
The reaction obeys pseudo-first order kinetics with an initial
reaction rate of 27.8 mmol O2 minꢀ1 that corresponds to a
turnover frequency 460 mmol citronellol sꢀ1 mol Pcꢀ1. This
rate is consistent with the 33.6 mmol O2 minꢀ1 value reported
for the homoleptic F64PcZn, 4.6,26
In contrast, the hydrocarbon-based Pc-conjugates are not stable,
as exemplified by the loss of Q-bands intensities in the case of a
protio PcCo–oligonucleotide conjugate that is exposed to ROS.19a
The Pc complexes with Zn and Al are presumably active in generat-
ing singlet oxygen, but the stability of the Pc is not reported.19b
The carboxy group of the Pc catalyst, 3, was next
coupled with the amino group of the solid-supported CTO,
30-TCGCGGCCGTTCTACTTC-50-C6-NH2, 10, using conventional
amide coupling conditions27 to yield after HPLC purification the
bioconjugate F48H7PcZn–CTO, 6, in isolated purity 4 95% (see ESI†
for synthesis and characterization). The hybridization of bio-
conjugate 6 with complementary GRP78 DNA, 7 and mRNA, 8
sense strands, afforded the duplexes F48H7PcZn–CTO:DNA, 6:7,
and F48H7PcZn–CTO:mRNA, 6:8. These hybrids, soluble in phos-
phate buffers, exhibited stable (Tm = 75 and 72 1C, respectively)
temperature-dependent duplex to single-strand transitions
(Table S2, ESI†). Taken together, these results unambiguously
establish the ability of F48H7PcZn–CTO to target and bind to
complementary GRP78 DNA and mRNA, affording thermally
stable duplex structures, similar to the ones formed by the native
controls (Table S2, ESI†).
oligonucleotide sequences have been reported in the literature.19
However, to the best of our knowledge, coupling a chemically
robust fluoro Pc with an oligonucleotide vector, Fig. 1, targeting
a DNA or mRNA oncogene, has not been reported.
The cancer-targeting oligonucleotide, CTO, was selected
from the antisense DNA sequence exhibiting potent Glucose
Regulating Protein 78, GRP78 knockdown in neonatal rat
cardiomyocytes.20 GRP78 functions as a chaperone that regulates
protein folding events in the lumen of the endoplasmic reticulum
(ER) of all cell types.21
In cancer, GRP78 is overexpressed and localized on the cell
surface where it exhibits a myriad of signaling activities related
to cancer initiation, production and metastatic spread.22 The
GRP78 oncogene is thus an excellent target for the development
of potent and selective anti-cancer strategies.23
The F48H7(COOH)PcZn, 3, was prepared in 21% isolated
yield by reacting perfluoro phthalonitrile 1,24 with a carboxy
phthalonitrile 2,25 in a microwave reactor, Scheme 1. Its com-
position and purity were confirmed by 1H, 19F NMR and HRMS
(see ESI†).
Importantly, 3 generates 1O2 as evidenced by the aerobic
photo-hydroperoxidation of b-citronellol (Fig. 2a and b). The
catalyst nuclearity and stability under reaction conditions were
established via time-dependent UV-Vis spectroscopy. The solvent-
independent Q-bands intensities and linearity of the Lambert–
Beer plot for 3 (Fig. S5, ESI†) reveal its lack of aggregation, a key
Given the ability of F48H7(COOH)PcZn, 3, to generate and
1
survive O2 production, the photoreactivity of F48H7PcZn–CTO, 6,
hybridized with complementary GRP78 DNA and mRNA, i.e. 6:7 and
6:8, respectively, was explored. This reactivity is critically important
for establishing the ability of bioconjugates to generate singlet
oxygen and the capacity of the latter to oxidatively cleave the
thermally stable oligonucleotides. Thus, illumination with a 300 W
halogen light for 12 h in the presence of O2 followed by hot
piperidine treatment induced site-specific cleavage along the
Scheme 1 Microwave-assisted synthesis of F48H7(COOH)PcZn, 3.
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In conclusion, an entry point into a new family of highly
fluorinated phthalocyanines was established. A highly fluorinated,
functionalized molecule was synthesized and shown to generate
1O2 without self-decomposition, underscoring its potential in PDT1
and other applications.29 Its coupling with a cancer-targeting
oligonucleotide vector proved useful in directing GRP78 DNA and
mRNA oncogene binding, and cleavage upon aerobic illumination.
The photoactive and chemically robust fluorophthalocyanine–CTO
bioconjugates encompass a new class of potential anticancer
agents. Variations in the fluorinated phthalocyanine structure,
coupled with variations in the types of vectors may facilitate the
targeting of a broad range of oncogenes and cancer types.
The financial support from the National Science Foundation
(SMG) and Seton Hall University is gratefully acknowledged.
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