Phosphoramidation-Based Biocompatible and Bioorthogonal Reactions for Synthesis of Novel Sequence-Specific Crrna Antimicrobials Able to Harness Innate Crispr-Cas Systems in Pathogens
Description: The inappropriate administration and overuse of antimicrobials in people and animals has ignited the worldwide prevalence of diverse antimicrobial resistance which threatens an effective prevention and treatment of the drastically increasing range of infections caused by bacteria, parasites, viruses and fungi in the global society. The treatment cost of patients with resistant infections is far more expensive due to longer duration of illness, additional tests, use of more expensive drugs and more frequent patient death. It is especially dire challenge to control and treat antibiotics-resistant bacterial infections because a major share of antimicrobial resistance is attributed to pathogenic bacteria. Without effective antibiotics, the success of major surgery and cancer chemotherapy would be compromised. Thus development of novel antimicrobials for treating drug-resistant bacterial infections is in urgent need that requires intensive global research efforts immediately. Consequently, the two-year MOST grant proposal aspires to harness potential of the advanced aqueous-phase nucleic acid phosphoramidation reactions developed by my research group for effective synthesis of novel sequence-specific RNA-based antimicrobials via biorthogonal and biocompatible approaches. Development of sequence-specific antimicrobials will be facilitated by employing the noteworthy bacterial clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) mechanisms in which cleavage of specific nucleic acid sequences in bacteria is achieved by hybridization specificity in CRISPR RNAs (crRNAs). The proposal is therefore highlighted by three major aims: (1) organic synthesis of compounds essential to bioorthogonal and biocompatible conjugations of crRNAs, effective synthesis (2) of peptide-oligonucleotide conjugates (POCs) and (3) of siderophore-oligonucleotide conjugates for sequence-specific antibacterial treatments. Specifically, the first aim of the MOST grant proposal is to synthesize cyclooctyne derivatives indispensable to performing the well-established Cu-free strain-promoted 1,3-diplolar azide-alkyne cycloaddition (SPAAC) for the biorthogonal and biocompatible production of POCs. To demonstrate chemical stability of the proposed cyclooctyne derivatives, we also will synthesize several disulfide-containing fluorescence resonance energy transfer (FRET) probes to in vivo and in situ detect redox states in biological systems. In addition, the bacterial siderophore desferrioxamine B (DFOB) functionalized with cyclooctyne will be synthesized and exploited for subsequent synthesis of crRNA-siderophore conjugates by SPAAC. In the second and third research aims, we seek to effectively and efficiently assemble crRNA POCs and crRNA-FOB [the coordination compound of DFOB and iron(III)] conjugates by combining the regioselective phosphoramidation and the powerful SPAAC reactions. The tailor-made crRNA POCs and crRNA-FOB conjugates will demonstrate antimicrobial efficacy and reveal the potential as novel effective crRNA-based therapeutics for treating bacterial infections. Experiments are thus proposed to fulfill the aims described above. In the first year of the grant proposal, we will synthesize cyclooctyne derivatives, disulfide-containing FRET probes and a DFOB derivative functionalized with cyclooctyne. The indicated compounds are indispensable to performing bioorthogonal and biocompatible reactions for crRNA conjugations required in the proposal. In the proposed second year, the site-specific reactivity of the phosphoramidation reaction will work in line with SPAAC to achieve more effective synthesis of crRNA conjugates. We will explore several cell-penetrating peptides (CPPs) and conjugate the CPPs with crRNAs by the optimized advanced two-step phosphoramidation and SPAAC reactions. The approach will provide more effective synthesis of POCs and avoid nucleic acid degradation and cell death due to Cu cytotoxicity. Moreover, the DFOB derivative will be conjugated with crRNAs to obtain crRNA-FOB conjugates. In each case, the CPPs or FOB will play the role of Trojan horses to deliver crRNAs into bacterial cells. Each afforded crRNA POCs will be characterized by the MALDI-TOF MS method developed in my research group, inoculated into cultures of Escherichia coli K-12 W3110 derivate hns (JW1225) harboring active CRISPR-Cas and a kanamycin resistance gene (kan+), and demonstrated its potential as valuable novel antibacterial therapeutics by cleaving the kan gene and inhibiting E. coli K-12 hns growth through the bacterial innate CRISPR-Cas system. Finally, we will again take advantage of the optimized advanced two-step phosphoramidation and SPAAC reactions to synthesize crRNA-FOB conjugates able to enact target delivery and sever antimicrobial resistance genes in Pseudomonas aeruginosa. The acquired crRNA-FOB conjugates will also be characterized by MALDI-TOF MS and gel electrophoresis, inoculated into P. aeruginosa cultures and demonstrated the proficiency of more effective antibacterial treatments through cleavage of antimicrobial resistance genes and inhibition of growth in P. aeruginosa. Future results will be compiled and prepared as manuscripts to be timely submitted to high-impacted journals for publications. The current MOST grant proposal is well-balanced on synthetic organic chemistry and therapeutic applications of aqueous-phase nucleic acid phosphoramidation and SPAAC reactions. The outcomes of the proposed research will demonstrate broad applications of phosphoramidation and SPAAC reactions including more effective synthetic routes for POCs and other nucleic acid conjugates invaluable to therapeutic administrations in clinic.
Amount: TWD 601,000
Funding type: research
Funder scheme: 學術補助
Investigator: 王子斌
Institution: 高雄醫學大學醫藥暨應用化學系
Funded outputs count: 2
Funder award ID: MOST106-2113-M037-010
OpenAlex ID: G4292889618