Synthesis of dynamic riboflavin derivatives and the study of their ability to urease photoinactivation

Abstract

Introduction. In one patents demonstrated the riboflavin ability to inactivate vaccine strains of viruses and bacteria, as well as cancer cells during the production of vaccines . DNA and RNA within living organisms have specific aptamers or flavin places where Riboflavin and its derivatives are able to selectively join . Upon further blue light irradiation 440-450 nm or ultraviolet light at 280-350 nm such complexes between nucleic acids and riboflavin observed selective nucleolysis nucleic acids in the field of riboflavin attachment .Research aim was to synthesize a series of riboflavin derivatives, including dynamic derivative having greater photodynamic activity (higher sensitivity to light) in the visible region spectrum at lower concentrations. Materials and methods. For gradient HPLC analysis were used: acetonitrile and lithium perchlorate,  perchloric acid in a kit for chromatograph BD2003. For fixing the UV / spectrum of the synthesized compounds was used spectrophotometer Gene-quant - 1300. To analyze riboflavin succinyl-dynamic systems was used HPLC- system Milichrome A-02. To study the ability of riboflavin derivatives for urease’s photo-inactivation reagents were used: test-system Urease-U and spectrophotometer Gene-quant – 1300 for fixing end of reaction.  In addition, we used a set of chromatography conditions for analysis of riboflavin derivatives: gradient separation of acetonitrile (from 0% to 100%) / 0.05 M lithium perchlorate buffer + 0.01 M perchloric acid at 40 ⁰ C, and fraction detection in UV region. As a comparison, we used substance riboflavin (I). Results and discussion. The main difference of (I) from dynamic riboflavin (IV) is much higher water solubility (up to 2%) for (IV). To compare the efficiency photoinactivation by riboflavin (I) and dynamic riboflavin (IV) was studied photoinactivation efficiency of urease as a model of microbial toxin and its transformation into a toxoid. The photoinactivators effectivity criterion is the ability to inactivate urease by disrupt performance of the active center of the enzyme. In our case - to slow down or completely block the urease ability to catalyze the hydrolysis of urea. Full succinylated derivative (V) acts in minimum effective concentration 0,8 ± 0.1 µg/mL. Dynamic derivative (IV) with an immediate showed activity at a concentration  0.2 ± 0.05 µg/ml and after 24 hours also not changed. The starting riboflavin (I) was very active, regardless to storage time and they active concentration in solution was 1.2 ± 0.2 µg/mL. The effective concentration statistically differed between compounds (I), (IV) and (V) at p≤ 0,05.  Conclusion. The first compound obtained is combinatorial - maleylated / succinylated riboflavin (IV). The synthesized compound (IV) and (V) retain photodynamic activity. The minimum effective concentration of the compound (IV) with an immediate application amounted to 0.2 ± 0.05 µg/mL, whereas for the (V) this value was 0.8 ± 0.1 µg/mL. Active dynamic structure (IV) and even exceed the original riboflavin activity at immediate use after dissolution, it is still unstable and gradually hydrolyzed. The most stable activity possessed source riboflavin (I), its active concentration is not dependent on the storage time of the aqueous solution and was 1.2 ± 0.2 µg/mL.