Presentation Information
[P01-017]Chemoenzymatic Synthesis of Multiply Deuterated Nucleosides for Intracellular RNA Dynamics Analysis
○Seita Ishide1, Akihiko Hatano1, Ryo Murakami1, Genki Shimane1, Reo Sakashita1, Mayuri Koga1 (1. Shibaura Institute of Technology (Japan))
Keywords:
Chemoenzymatic synthesis,Deuterated nucleosides,Metal-catalyzed deuteration,Raman imaging,RNA dynamics
[1. Objective]
Real-time visualization of RNA dynamics in living cells has attracted significant attention. C-D bonds are observed in the Raman-silent region (1800-2800 cm-1), making them suitable for real-time imaging. However, conventional spontaneous Raman scattering suffers from low sensitivity, requiring extensive deuterium incorporation. We aimed to synthesize multiply deuterated nucleosides as Raman probes for RNA dynamics analysis by combining metal-catalyzed deuteration of the uracil C-5 substituent with enzymatic base-exchange of uridine using a pyrimidine nucleoside phosphorylase.
[2. Methods]
The ribose and base moieties were deuterated independently and then coupled enzymatically. Base deuteration was performed using Pd/C or Pt/C (5 or 10 wt%) under H2 atmosphere in D2O at 110 ℃ for 24 h. Ribose deuteration was carried out with Ru/C (5 wt%) under identical conditions. Deuterium incorporation was quantified by 1H NMR. For the base-exchange reaction, pyrimidine nucleoside phosphorylase was used with deuterated bases (5 mM) and uridine (40 mM) in phosphate buffer (10 mM, pH 6.8) at 40 ℃ for 52 h. Reactions were monitored by reverse-phase HPLC.
[3. Results and Discussion]
Deuteration of the ribose moiety was achieved starting from D-ribose via 1-methoxy-D-ribose using a Ru catalyst, introducing deuterium at the 2- (D: 53%), 3- (D: 59%), and 5- positions (2D: 52%). After four subsequent steps, uridine bearing four deuterium atoms on the ribose moiety was obtained.
For the base moiety, 5-ethyluracil gave ~80% deuteration with 5% Pd/C and nearly quantitative deuteration with 5% Pt/C. 5-Propyluracil showed lower reactivity under 5% Pd/C (α 25%, β 77%, γ 0%), which improved markedly with 5% Pt/C (α 75%, β 99%, γ 50%). Under 10% catalyst conditions, nearly quantitative deuteration was achieved for both substrates, indicating strong dependence on substrate structure and catalyst loading.
The enzymatic base-exchange reaction using uridine and 5-ethynyluracil-d6 afforded the deuterated nucleoside with 78% conversion after 52 h.
[Conclusion]
Multiply deuterated nucleosides were successfully synthesized through a chemoenzymatic approach combining metal-catalyzed deuteration and enzymatic base-exchange. Future work will evaluate the C-D signal enhancement in the Raman-silent region to assess their utility as Raman probes.
Real-time visualization of RNA dynamics in living cells has attracted significant attention. C-D bonds are observed in the Raman-silent region (1800-2800 cm-1), making them suitable for real-time imaging. However, conventional spontaneous Raman scattering suffers from low sensitivity, requiring extensive deuterium incorporation. We aimed to synthesize multiply deuterated nucleosides as Raman probes for RNA dynamics analysis by combining metal-catalyzed deuteration of the uracil C-5 substituent with enzymatic base-exchange of uridine using a pyrimidine nucleoside phosphorylase.
[2. Methods]
The ribose and base moieties were deuterated independently and then coupled enzymatically. Base deuteration was performed using Pd/C or Pt/C (5 or 10 wt%) under H2 atmosphere in D2O at 110 ℃ for 24 h. Ribose deuteration was carried out with Ru/C (5 wt%) under identical conditions. Deuterium incorporation was quantified by 1H NMR. For the base-exchange reaction, pyrimidine nucleoside phosphorylase was used with deuterated bases (5 mM) and uridine (40 mM) in phosphate buffer (10 mM, pH 6.8) at 40 ℃ for 52 h. Reactions were monitored by reverse-phase HPLC.
[3. Results and Discussion]
Deuteration of the ribose moiety was achieved starting from D-ribose via 1-methoxy-D-ribose using a Ru catalyst, introducing deuterium at the 2- (D: 53%), 3- (D: 59%), and 5- positions (2D: 52%). After four subsequent steps, uridine bearing four deuterium atoms on the ribose moiety was obtained.
For the base moiety, 5-ethyluracil gave ~80% deuteration with 5% Pd/C and nearly quantitative deuteration with 5% Pt/C. 5-Propyluracil showed lower reactivity under 5% Pd/C (α 25%, β 77%, γ 0%), which improved markedly with 5% Pt/C (α 75%, β 99%, γ 50%). Under 10% catalyst conditions, nearly quantitative deuteration was achieved for both substrates, indicating strong dependence on substrate structure and catalyst loading.
The enzymatic base-exchange reaction using uridine and 5-ethynyluracil-d6 afforded the deuterated nucleoside with 78% conversion after 52 h.
[Conclusion]
Multiply deuterated nucleosides were successfully synthesized through a chemoenzymatic approach combining metal-catalyzed deuteration and enzymatic base-exchange. Future work will evaluate the C-D signal enhancement in the Raman-silent region to assess their utility as Raman probes.
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