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Synthesis of PNA-Metal Bioconjugates by "Copper-Free" and "Photo" Click Chemistry: An Approach towards the in Vitro Attachment of Organometallics to Biomolecules

English title Synthesis of PNA-Metal Bioconjugates by "Copper-Free" and "Photo" Click Chemistry: An Approach towards the in Vitro Attachment of Organometallics to Biomolecules
Applicant Gasser Gilles
Number 126404
Funding scheme Ambizione
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Inorganic Chemistry
Start/End 01.03.2010 - 28.02.2011
Approved amount 145'895.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Biochemistry

Keywords (5)

Peptide Nucleic Acid (PNA); Bioorganometallic Chemistry; Click Chemistry; Photoclick Chemistry; Luminescent Metal Complex

Lay Summary (English)

Lead
Lay summary
Peptide Nucleic Acids (PNAs) are non-natural DNA mimics, which hybridise with complementary DNA/RNA strands via Watson-Crick base pairing. Due to their neutral peptide backbone, which avoids electrostatic repulsions with negatively charged DNA/RNA strands, PNA-DNA/PNA-RNA duplexes are more stable than the usual DNA-DNA/RNA-RNA duplexes. Another important feature of PNAs is their resistance to attack by proteases and nucleases. Due to these favourable properties, PNAs have been keenly investigated for use in antisense and antigene therapies.

On another research front, bioorganometallics have found, over the last years, application in various areas of medicinal chemistry. One of the most noteworthy of this type of compounds is the ferrocene-chloroquine derivative, Ferroquine, which is currently in phase 2 of clinical trials due to its outstanding antimalarial properties. Not surprisingly, when PNAs were combined with organometallic compounds, the resulting metal-containing PNA bioconjugates obtained were found to have unique chemical/physical properties. These compounds have therefore been explored for various purposes such as biosensing, modulation of PNA-DNA duplex stability, modulation of the cellular uptake of PNAs, DNA-template metal catalysis and designing of radioactive probes.

In this application, the candidate proposes a totally novel approach to specifically and efficiently couple, within a cell, organometallics to PNA oligomers using the cutting-edge "Copper-free" or "Photo" Click Chemistry methodologies. This is unparalleled by any of the current concepts in that the in vitro coupling of any metal complex to any biomolecule has never been explored, to the best of the knowledge of the applicant. The current metal bioconjugation methods available are limited by the toxicity of the catalyst to both bacterial and mammalian cells (e.g. the copper used in the typical Click Chemistry reaction). If successful, the fundamental research described here could have significant implications not only in the bioorganometallic chemistry area but also in vast medicinal and biological areas with tremendous potential applications. For example, it could be envisaged that the specific and time-controlled attachment of an organometallic to a PNA triggers a biological event/process, such as the modification of the secondary structure of a PNA/mRNA duplex leading to a dramatic change in protein expression or modification of the polarity of PNA bioconjugate, inducing a change in its localisation within the cell.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
133568 Caged Metal Complexes as Tools in Inorganic Chemical Biology 01.03.2011 SNSF Professorships
129910 Photo-Induced Uncaging of Metal Complexes in Living Cells 01.08.2010 Project funding (Div. I-III)

Abstract

Peptide Nucleic Acids (PNAs) are non-natural DNA mimics, which hybridise with complementary DNA/RNA strands via Watson-Crick base pairing. Due to their neutral peptide backbone, which avoids electrostatic repulsions with negatively charged DNA/RNA strands, PNA-DNA/PNA-RNA duplexes are more stable than the usual DNA-DNA/RNA-RNA duplexes. Another important feature of PNAs is their resistance to attack by proteases and nucleases. Due to these favourable properties, PNAs have been keenly investigated for use in antisense and antigene therapies.On another research front, bioorganometallics have found, over the last years, application in various areas of medicinal chemistry. One of the most noteworthy of this type of compounds is the ferrocene-chloroquine derivative, Ferroquine, which is currently in phase 2 of clinical trials due to its outstanding antimalarial properties.Not surprisingly, when PNAs were combined with organometallic compounds, the resulting metal-containing PNA bioconjugates obtained were found to have unique chemical/physical properties. These compounds have therefore been explored for various purposes such as biosensing, modulation of PNA-DNA duplex stability, modulation of the cellular uptake of PNAs, DNA-template metal catalysis and designing of radioactive probes.In this application, the candidate proposes a totally novel approach to specifically and efficiently couple, within a cell, organometallics to PNA oligomers using the cutting-edge “Copper-free” or “Photo” Click Chemistry methodologies. This is unparalleled by any of the current concepts in that the in vitro coupling of any metal complex to any biomolecule has never been explored, to the best of the knowledge of the applicant. The current metal bioconjugation methods available are limited by the toxicity of the catalyst to both bacterial and mammalian cells (e.g. the copper used in the typical Click Chemistry reaction). If successful, the fundamental research described here could have significant implications not only in the bioorganometallic chemistry area but also in vast medicinal and biological areas with tremendous potential applications. For example, it could be envisaged that the specific and time-controlled attachment of an organometallic to a PNA triggers a biological event/process, such as the modification of the secondary structure of a PNA/mRNA duplex leading to a dramatic change in protein expression or modification of the polarity of PNA bioconjugate, inducing a change in its localisation within the cell. To achieve the ultimate aim of this research, the synthesis of versatile analogues of a PNA monomer that will then be inserted anywhere within a PNA sequence with an automated PNA synthesiser will be performed. The cellular uptake of the new synthon-containing PNA bioconjugates will be accessed and their location within the cells will be easily determined by fluorescence microscopy as the PNAs will be tagged with a red-region-emitting fluorescent moiety. Newly synthesised non-cytotoxic organometallics will then be transfected into cells and their uptake and cellular position will be followed by fluorescence microscopy. The metal complex will then be “copper-free clicked” or photoclicked using UV irradiation to the PNA oligomer. The success of the in vitro labelling by photoclick chemistry will be unequivocally confirmed by the appearance of a new green-region-emitting fluorescent signal due to the newly formed pyrazoline adduct. More complicated biochemical techniques (cellular disruption, extraction, purification, etc...) will be required for the assessment of the copper-free click chemistry methodology and the covalent binding will also be unambiguously verified.As a secondary exploratory project, the candidate intends to investigate the possibility of cross-linking PNA strands using the Copper-Free and the Photo Click Chemistry methodologies. If successful, this could lead to an entirely novel approach for the control of gene expression.
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