Hydrolysis of 5′,5′-Tri- or Tetraphosphate-mRNA 5′-cap Analogs Promoted by Cu²⁺ or Zn²⁺ Metal Ions

Abstract

Kinetics of the hydrolysis of a P¹-(7-methylguanosinyl-5′) P³-(guanosinyl-5′) triphosphate (m⁷GpppG), P¹-(7-methylguanosinyl-5′) P⁴- (guanosinyl-5′) tetraphosphate (m⁷GppppG), diadenosine-5′,5‴-P¹,P³-triphosphate (ApppA), and diadenosine-5′,5‴-P¹,P⁴-tetraphosphate (AppppA) promoted by Cu²⁺ or Zn²⁺ has been investigated. Time-dependent products distributions at various metal ion concentrations have been determined by CZE and HPLC-RP. The results show that in acidic conditions, in the presence of metal ion, the predominant hydrolytic reaction is the cleavage of 5′,5′-oligophosphate bridge. The 5′,5′-oligophosphate bridge of the dinucleotides studied is hydrolyzed by Cu²⁺ more efficiently than by Zn²⁺. At the catalyst concentration of 2 mM the cleavage of the 5′,5′-triphosphate bridge of m⁷GpppG was ∼3.6 times faster, and that of the tetraphosphate bridge of m⁷GppppG ∼2.3-fold faster in the presence of Cu²⁺ compared to the Zn²⁺ ion, applied as catalysts. Dependence of the rates of hydrolysis on the catalyst concentration was in some instances not linear, interpreted as evidence for participation of more than one metal ion in the transition complex.

Keywords:

• mRNA 5′-cap analogs
• dinucleotides
• hydrolysis
• kinetics
• rate constants

Acknowledgments

The authors thank Prof. Edward Darżynkiewicz (Warsaw University) and Prof. Harri Lönnberg (University of Turku) as well as their research groups for synthesis of the cap analogs; Prof. Harri Lönnberg (University of Turku) and Prof. A. Kuncewicz (University of Warmia and Mazury in Olsztyn) for providing access to HPLC and CE apparatus. Thanks is also extended to Dr. Jacek Wierzchowski (University of Warmia and Mazury in Olsztyn) for very fruitful scientific discussions and for editorial corrections.

Notes

*The ionic strength was adjusted to I = 0.1 M with sodium nitrate or sodium chlorate at 60°C. Numbers in parenthesis indicate standard errors.

**0.1 M acetate buffer; pH 4.4; I = 0.15 M.