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Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

ABSTRACT: Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high-performance photoswitches with near-quantitative, fully reversible back-and-forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4-fold turn-off factor, demonstrating the potential for optochemical control of biological processes.

SUBMITTER: Kolmar T

PROVIDER: S-EPMC8049081 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

Kolmar Theresa T Büllmann Simon M SM Sarter Christopher C Höfer Katharina K Jäschke Andres A

Angewandte Chemie (International ed. in English) 20210303 15

Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This a ...[more]

PMID: 33476096

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Project description:Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

Project description:The present study proposed modification of 5-FU by conjugation with an acyl chloride and a 5-membered heterocyclic ring to improve its in-vitro cytotoxicity and metabolic stability. XYZ-I-71 and XYZ-I-73 were synthesized by introducing a tetrahydrofuran ring on 5-fluorocytosine (a precursor of 5-FU) and conjugation with octanoyl chloride and lauroyl chloride, respectively. The structure of the synthesized compounds was validated using NMR and micro-elemental analysis. The antiproliferative activity of the analogs was determined against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. The analog's stability in human liver microsomes was quantified by HPLC. We found that the XYZ-I-73 (IC50 3.6 ± 0.4 μM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3 ± 1.7 μM), GemHCl (IC50 24.2 ± 1.3 μM), Irinotecan (IC50 10.1 ± 1.5 μM) and 5-FU (IC50 13.2 ± 1.1 μM). The antiproliferative effects of this analog in Miapaca-2 cells is evident based on it having a 7-fold,3-fold, and 4-fold increased cytotoxic effect over Gem-HCl, Irinotecan, and 5-FU, respectively. On the other hand, XYZ-I-71 exhibited a 2-fold increased cytotoxic effect over Gem-HCl but a comparable cytotoxic effect to 5-FU and Irinotecan in MiaPaCa-2 cells. A similar trend of higher XYZ-I-73 inhibition was observed in PANC-1 and BxPC-3 cultures. For 48-h MiaPaCa-2 cell migration studies, XYZ-I-73 (5 μM) significantly reduced migration (# of migrated cells, 168 ± 2.9), followed by XYZ-I-71(315±2.1), Gem-HCl (762±3.1) and 5-FU (710 ± 3.2). PARP absorbance studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU, GemHCl, and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU, GemHCl, and XYZ-I-71. In-vitro, metabolic stability studies showed that 80 ± 5.9% of XYZ-I-71 and XYZ-I-73 remained intact after 2 h exposure in liver microsomal solution compared to 5-FU. The XYZ-I-73 analog demonstrated a remarkable cytotoxic effect and improved in-vitro metabolic stability over the selected standard drugs and may have potential anticancer activity against pancreatic cancer.

Dataset Information

Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

Publications

Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

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