Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 29;11(5):657.
doi: 10.3390/biom11050657.

Green Production of Cladribine by Using Immobilized 2'-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

Cintia Wanda Rivero  1   2 , Natalia Soledad García  1 , Jesús Fernández-Lucas  3   4 , Lorena Betancor  5 , Gustavo Pablo Romanelli  6 , Jorge Abel Trelles  1   2
Affiliations

Green Production of Cladribine by Using Immobilized 2'-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

Cintia Wanda Rivero et al. Biomolecules. .

Abstract

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

Keywords: antineoplastic drug; biomimetic silica; calcium alginate; entrapment; enzyme immobilization; glutaraldehyde.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the combined immobilization procedure, carried out in this work to obtain cladribine, through the development of a highly stabilized biocatalyst. TMOS: tetramethylorthosilicate, PEI: Polyethyleneimine and LdNDT: Recombinant NDT from Lactobacillus delbrueckii.
Figure 2
Figure 2
Overall representation of surface exposed Lys residues (atom colored sticks) in LdNDT active site complexed with Thd (atom colored sticks). The cartoon representation is used for simplicity and ease of visualization. The catalytic loop region (amino acids 48 up to 62) is highlighted in blue. The figure has been prepared with PyMOL [34].
Figure 3
Figure 3
Study of several parameters using SiGPEI25000-LdNDT derivative for cladribine biosynthesis. (A) Effect of pH on reaction time course, (?) 25 mM sodium acetate buffer, pH 5.0; (•) 25 mM tris-HCl buffer, pH 7.0; (▲) 25 mM tris-HCl buffer, pH 9.0. (B) Effect of temperature on reaction time course, (•) 30 °C, (?) 50 °C, and (▲) 60 °C.
Figure 4
Figure 4
Thermal inactivation of SiGPEI25000-LdNDT (A) and SiGPEI25000-LdNDT-Alg (B) at different temperatures (▲) 30 °C, (□) 50 °C, and (○) 60 °C. Effect of pH on the stability of SiGPEI25000-LdNDT (C) and SiGPEI25000-LdNDT-Alg (D). pH 5.0 (▲), pH 7.0 (□), and pH 9.0 (○).
Figure 5
Figure 5
Surface representation of hexamericLdNDT. The different chains (A–F) are highlighted in different colors. N-terminus residues are yellow colored.
Figure 6
Figure 6
SEM and EDX analysis of SiGPEI25000-LdNDT (A,C) and SiGPEI25000-LdNDT-Alg (B,D) biocatalysts.
Figure 7
Figure 7
Storage stability at 4 °C (A) and reusability (B) of (▲)SiGPEI25000-LdNDT-Alg, (□) SiGPEI25000-LdNDT, and (○) CNBr-LdNDT.
See this image and copyright information in PMC

References

    1. Spurgeon S., Yu M., Phillips J.D., Epner E.M. Cladribine: Not just another purine analogue? Expert Opin. Investig. Drugs. 2002;18:1169–1181. doi: 10.1517/13543780903071038. - DOI - PubMed
    1. Biernacki T., Sandi D., Bencsik K., Vécsei L. Medicinal chemistry of multiple sclerosis: Focus on cladribine. Mini Rev. Med. Chem. 2020;20:269–285. doi: 10.2174/1389557519666191015201755. - DOI - PubMed
    1. Peng Y. A practical synthesis of 2-chloro-2’-deoxyadenosine (cladribine) from 2’-deoxyadenosine. J. Chem. Res. 2013;37:213–215. doi: 10.3184/174751913X13618878756705. - DOI
    1. Fernández-Lucas J., Camarasa M.J., editors. Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives. John Wiley & Sons; Hoboken, NJ, USA: 2019. - DOI
    1. Lapponi M.J., Rivero C.W., Zinni M.A., Britos C.N., Trelles J.A. New developments in nucleoside analogues biosynthesis: A review. J. Mol. Catal. B Enzym. 2016;133:218–233. doi: 10.1016/j.molcatb.2016.08.015. - DOI

Publication types

LinkOut - more resources