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Transformation of Sugars into Nucleosides of Biological Importance

#B-1640


Chemo-Enzymatic Transformation of Sugars into Nucleosides of Biological Importance

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-DRG/Drug Discovery/Medicine

Status
8 Project completed

Registration date
20.05.2008

Completion date
21.01.2013

Senior Project Manager
Savinova N V

Leading Institute
Institute of Bioorganic Chemistry, Belarus, Minsk

Supporting institutes

  • Institute of Bioorganic Chemistry, Russia, Moscow

Collaborators

  • University of Osnabrück; Faculty of Biology and Chemistry; Institute of Chemistry, Germany, Niedersachsen \nUniversität Osnabrück / Department of Neurobiology, Germany, Osnabrück\nUniversity of Helsinki / Faculty of Pharmacy / Drug Discovery and Development Technology Center, Finland, Helsinki

Project summary

The purpose of the project consists in the R&D of extended versatile methodology for the preparation of analogues of natural nucleosides of biological importance encompassing the transformation of pentofuranoses into nucleosides employing the recombinant ribokinase (RK), phosphopentomutase (PPM), and nucleoside [uridine (UP), thymidine (TP), and purine (PNP)] phosphorylases placing special emphasis on the mechanism of enzyme functioning and applied aspects as well. The search for the feasible chemical synthesis of D(L)-pentofuranose 1-phosphates – key intermediate of the methodology will be also undertaken.

State of the art: Analogues of natural nucleosides are one of the most important classes of antiviral drugs and a number of representatives of this family are extensively used in the treatment of a variety of cancers. A number of base and sugar modified nucleosides are very useful components of oligonucleotides of biological and medicinal importance, e.g., siRNA, aptamers and aptazymes. The latter demonstrate a high potential in biotechnology, diagnostic, and medicine.

Diverse chemical synthetic pathways to this family of biologically important nucleosides have been elaborated. However, the preparation of many antiviral and anticancer drugs and modified constituents of oligonucleotides as well remains a challenging problem resulting in a high price of drugs and, consequently, preventing from their broad application. An important drawback of chemical methods consists in the use of vast volume of organic solvents and dangerous reagents that are harmful for the environment. Therefore, the search for environmentally-friendly methods, R&D toward “green chemistry” is of great importance.

Transfer of a pentofuranosyl moiety of commercially available nucleosides or prepared by chemical methods to purine or pyrimidine bases catalyzed by nucleoside phosphorylases (“transglycosylation reaction”) was demonstrated to be a very efficient methodology for the synthesis of plenty of analogues of natural nucleosides of biological and pharmaceutical importance [1]. The bacterial nucleoside phosphorylases, PNP, TP and UP, reversibly catalyze (i) the phosphorolysis of nucleosides with an intermediary formation of α-D-pentofuranose 1-phosphate, and (ii) the synthesis of new nucleosides in the presence of external heterocyclic bases.

During recent years, the use of D-pentofuranose 1-phosphates as substrates of an enzymatic synthesis of nucleosides attracts attention [1]. The R&D in this area consists of two lines of investigation, viz., (i) biochemical (microbial, enzymatic) retro-synthesis of 2’-deoxyribonucleosides, and (ii) chemical synthesis of D-pentofuranose-1-phosphates followed by the enzymatic condensation with heterocyclic bases [1]. Analysis of the results points to a main serious bottleneck of this approach consisting in the laborious and low-yielding preparation of the sugar phosphates.

The impact of the proposed project on the progress in this field consists in the R&D of more extensive versatile methodology for the preparation of analogues of natural nucleosides of biological importance encompassing the transformation of pentofuranoses into nucleosides. This advanced methodology allows (i) preparing efficiently a number of sugar and base modified nucleosides of biological and medicinal importance making them available for the broad biological testing and synthesis of prodrugs, and (ii) producing for the first time valuable precursors of structural building blocks for automated oligonucleotide synthesis employing recombinant nucleoside phosphorylases.

All leaders of the participating institutions are established scientists in the field of (i) modern methodology of overproduction of proteins [2], (ii) modern chemistries of the enzymatic and chemical syntheses [3], and (iii) physico-chemical methods and theoretical analysis of the results [3]. This application emerged from the cooperation of the participating institutions as an extension and intensification of our joint studies performed during a couple of recent years (e.g., [2,3]), on the one hand, as well as from discussions on the prospective lines of investigations leading to promising practical applications, on the other.

Expected results of the suggested project:

  • preparation of recombinant RK, PPM, UP, TP and PNP on a practical level and application them for the synthesis of sugar and base modified nucleosides;
  • R&D of feasible chemical synthesis of D(L)-pentofuranose-1-phosphates and their C2 or/and C3 modified counterparts;
  • elucidation of the role of structural (electronic) factors of heterocyclic bases and PF-1P’s in the synthesis of nucleosides employing recombinant nucleoside phosphorylases as biocatalysts in order to recognize the scope and limitations of an enzymatic synthesis of nucleosides and to provide a rational design of new nucleosides of biological and/or medicinal potential.

scientific – extension and deepening of the knowledge of mechanism of functioning of the RK, PPM, UP, TP and PNP, of the role of different factors (structural, electronic, stereochemical) in the glycoside bond formation. It should be stressed that a better understanding of enzyme mechanisms is very high on the priority list to employ them as biocatalysts.

practical – search for (i) biocatalysts that will be more cost-effective and/or yielding more desirable products, and (ii) efficient “Green methods” for the synthesis of pentofuranose 1-phosphates (PF-1P’s) and nucleosides of biological and medicinal potential as well as their derivatives with improved bioavailability and biodistribution.

Desired area of application – “Green production” of the known and new high value modified nucleosides and their prodrugs.

The suggested project addresses the ISTC goals and objectives, namely,

  • presumes to give a support for redirecting the scientific potential of the weapon’s participants to peaceful activities, towards the R&D of products of biological and medicinal value;
  • strengthens the scientific cooperation of weapon’s scientists by means of (i) elaboration of joint research plans combining the experience of collaborators, (ii) discussion of the results of studies, and (iii) preparation of joint publications and patent applications;
  • gives support for basic and applied research of the participants within the “Green Chemistry” as well as for development of new biotechnological approaches to the products of biological and medicinal value;
  • gives an opportunity for the participants to carry into effect the results of practical value.

There are the following main scopes of activities [IBCH (Moscow), IBOCh (Minsk)]:
  • project duration - 36 months; total project effort (person*days): 5820 (Minsk – 2910; Moscow – 2910);
  • preparation of recombinant enzymes, study of their substrate specificity and R&D of practical synthesis of base and sugar modified nucleosides;
  • search for feasible chemical synthesis of D(L)-pentofuranose-1-phosphates and their C2 and/or C3 modified counterparts aiming at their use in R&D of practical synthesis of base and sugar modified nucleosides;
  • study of the role of structural (electronic) factors of heterocyclic bases and PF-1P’s in the synthesis of nucleosides employing recombinant nucleoside phosphorylases as biocatalysts in order to recognize the scope and limitations of an enzymatic synthesis of nucleosides and to provide a rational design of new nucleosides of biological and/or medicinal potential.

The role of foreign collaborators consists in (i) conducting some chemical and physico-chemical experiments, testing the prepared nucleoside for their biological activity, selection of the most promising compounds from a viewpoint of their activity and preparation of their prodrugs, and testing the activity of the prodrugs vs the parent nucleosides, (ii) discussion of the results obtained and reports, discussion of possible area(s) of implementation, (iii) the preparation of the respective materials (patent applications, manuscripts, conference presentations etc).

Technical Approach and Methodology:

  • the modern methodology of overproduction of proteins, their purification and obtaining in easily applicable form will be used for the preparation of all enzymes;
  • the modern chemistries of the enzymatic and chemical syntheses and purification of sugars, nucleosides, oligonucleotides, peptides and nucleoside-peptide conjugates will be used;
  • detailed analysis of the structure (stereochemistry) and electronic properties of compounds under investigation will be performed with the reasonable application of the modern NMR and CD spectroscopy, X-ray analysis and the modern MacroModel and HyperChem programs.
  1. Mikhailopulo, I.A. Biotechnology of Nucleic Acid Constituents – State of the Art and Perspectives. Current Org. Chem. 2007, 11, 317.
  2. Chuvikovsky, D.V.; Esipov, R.S.; Skoblov, Y.S.; Chupova, L.A.; Muravyova, T.I.; Miroshnikov, A.I.; Lapinjoki, S.; Mikhailopulo, I.A. Bioorg. Med. Chem. 2006, 14, 6327; Roivainen, J.; Elizarova, T.; Lapinjoki, S.; Mikhailopulo, I.A.; Esipov, R.S.; Miroshnikov, A.I. Nucleosides, Nucleotides & Nucl. Acids 2007, 26, 905.
  3. Seela, F.; Becher, G.; Rosemeyer, H.; Reuter, H.; Kastner, G.; Mikhailopulo, I.A. Helv. Chim. Acta 1999, 82, 105; Mikhailopulo, I.A.; Sokolov, Y.A.; He, J.; Chittepu, P.; Rosemeyer, H.; Seela, F. Nucleosides Nucleotides & Nucl. Acids 2005, 24, 701.


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