Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B₁₂ to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B₁₂ was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B₁₂ conjugates; namely the vitamin B₁₂ azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B₁₂ and PNA were tested, including a disulfide bond. We found that vitamin B₁₂ transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)₃K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B₁₂ as a carrier of PNA oligonucleotides into bacterial cells.

The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B₁₂–PNA conjugates. Vitamin B₁₂ was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA–vitamin B₁₂ conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B₁₂ and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF)₃K. We found that vitamin B₁₂–PNA conjugates inhibit E. coli growth at a concentration of 5 μM, similar as (KFF)₃K–PNA. We also showed that vitamin B₁₂–PNA conjugates are stable in the presence of biological media. This study provides the foundation for improving and developing PNAs conjugated to vitamin B₁₂ as antibacterials.

Resonance Raman optical activity (RROA) possesses all aspects of a sensitive tool for molecular detection, but its measurement remains challenging. We demonstrate that reliable recording of RROA of chiral colorful compounds is possible, but only after considering the effect of the electronic circular dichroism (ECD) on the ROA spectra induced by the dissolved chiral compound. We show RROA for a number of model vitamin B₁₂ derivatives that are chemically similar but exhibit distinctively different spectroscopic behavior. The ECD/ROA effect is proportional to the concentration and dependent on the optical pathlength of the light propagating through the sample. It can severely alter relative band intensities and signs in the natural RROA spectra. The spectra analyses are supported by computational modeling based on density functional theory. Neglecting the ECD effect during ROA measurement can lead to misinterpretation of the recorded spectra and erroneous conclusions about the molecular structure.