1
Enzymatic Synthesis of Nucleoside Analogues by Nucleoside Phosphorylases

Sarah Kamel1,*, Heba Yehia1,2,*, Peter Neubauer1, and Anke Wagner1,3

1 Technische Universität Berlin, Department of Bioprocess Engineering, Institute of Biotechnology, Ackerstraße 76, 13355 Berlin, Germany

2 National Research Centre, Department of Chemistry of Natural Products, 12622 Giza, Egypt

3 BioNukleo GmbH, Ackerstraße 76, 13355 Berlin, Germany

1.1 Introduction

1.1.1 Nucleosides and Nucleoside Analogues

Nucleosides primarily consist of a nitrogenous base (nucleobase), which is either a purine base or a pyrimidine base and a five-carbon sugar (pentose). The base and sugar are covalently linked via an N-glycosidic bond (Figure 1.1). The pentose sugar moiety of naturally occurring canonical nucleosides is either ribose or deoxy-ribose whereas the nucleobase might be either a purine (adenine, guanine) or a pyrimidine (cytosine, uracil, thymine). These nucleosides are structural subunits of nucleic acids and are involved in several cellular processes including enzyme regulation and metabolism, DNA and RNA synthesis, and cell signaling [1, 2].

Figure 1.1 Classification of nucleosides and nucleoside analogues. Canonical (unmodified) nucleosides are the building blocks of DNA and RNA. Non-canonical (naturally modified on pentose moiety, base moiety or both) are mainly occurring in RNA. Synthetic nucleosides are used in the treatment of viral and bacterial infections as well as in cancer treatment.

Naturally occurring nucleoside analogues (non-canonical nucleosides) are found in almost all types of RNA especially in tRNAs and they are crucial for RNA processing. Non-canonical analogues are nucleosides with different modifications on the pentose and/or the base [3] (Figure 1.1). There are more than 109 known post-transcriptional modifications in the three phylogenetic domains [4]. Pseudouridine is the most ubiquitous analogue and is sometimes considered as the fifth RNA-related nucleoside [5].

Non-natural nucleoside analogues are synthetic molecules that structurally mimic their physiological counterparts and also act as antimetabolites [2]. Nucleoside analogues access cells through specific nucleoside transporters. Within the cells, they are phosphorylated by nucleoside kinases, which leads to increased levels of di- and tri-phosphorylated nucleoside analogues in virus-infected or cancer cells. The first and the second phosphorylation step can also be catalyzed by viral kinases in cells infected by some DNA viruses. Owing to differences in the substrate spectrum of human and viral kinases, virus-specific drugs can be developed. The active forms of nucleoside analogues interfere with intracellular enzymes such as human and viral polymerases, kinases, DNA methyl transferase, ribonucleotide reductase, nucleoside phosphorylases (s) or thymidylate synthase [2, 6]. Furthermore, they can be incorporated into newly synthesized DNA and RNA, which may induce termination of the polymerization process, accumulation of mutations in viral progeny, or induction of apoptosis.

For more than 50?years, nucleosides and their analogues have been used as small molecule drugs for the treatment of several viral infections as well as for hematological malignancies and solid tumors. The first FDA approved antiviral nucleoside analogue was idoxuridine, which is used for the treatment of -1 (herpes simplex virus) [7]. In 1969, cytarabine was approved for the treatment of acute myeloid leukemia [2]. Since then, the interest in nucleoside analogues based drugs has tremendously grown. Currently, more than 39 approved nucleoside analogue drugs or drug combinations are approved for the treatment of seven human viral infections, which include HSV, varicella zoster virus (), hepatitis-B virus (), hepatitis-C virus (), human immunodeficiency virus (), respiratory syncytial virus (), and human cytomegalovirus () [7]. For treatment of cancer and viral infections, 50% and 20%, respectively, of all approved drugs belong to the class of nucleoside analogues [8]. Additional clinical indications for nucleoside analogues application include chronic hyperuricemia, immune suppression in organ transplant surgeries, and autoimmune disease as well as chronic obstructive pulmonary disease and asthma [2].

Emerging from the significance of nucleoside analogues, there have been continuous attempts to improve and simplify their synthesis processes. With the world moving toward green chemistry approaches, the enzymatic synthesis of nucleoside analogues offers several advantages over chemical methods, which include higher total yields, a higher regio- and stereo-selectivity, and higher product purity. This allows for more biological and clinical trials [9]. Accordingly, enzymatic strategies are considered as a step forward to a more efficient synthesis of nucleosides and their analogues.

1.1.2 Enzymes Involved in the Enzymatic Synthesis of Nucleoside Analogues

Two main classes are employed in the enzymatic synthesis of nucleosides and their analogues: NPs and N-deoxyribosyltransferases (s). In this chapter, the focus is on enzymatic approaches using NPs. NPs are of high interest as biocatalysts because of their wide substrate spectrum and abundance in almost all living organisms.

1.2 Nucleoside Phosphorylases

NPs are enzymes belonging to the transferases family (EC 2.4 and EC 2.7.7). NPs catalyze the reversible phosphorolysis of nucleosides into their respective nucleobase and pentofuranose-1-phosphate (). NPs have been extensively studied since 1911 when Levene and Medigrecenau [10, 11], and Johnes [10-13] observed the enzymatic hydrolysis of nucleosides. Later, Levene et al. isolated an enzyme (nucleosidase) from cattle's spleen, kidney, and pancreas, which catalyzed the hydrolysis of both inosine and adenosine in phosphate buffer, yielding a base and a ribose moiety [14-16]. In 1947, Kalckar demonstrated that the formed ribose was in fact ribose-1-phosphate and that the isolated enzyme was a purine nucleoside phosphorylase () [17]. Later, it was proven that Escherichia coli cells and cell extracts thereof contained enzymes that could phosphorolyze thymidine to thymine and deoxyribose-1-phosphate [18].

1.2.1 Classification and Substrate Spectra of Nucleoside Phosphorylases

Nucleoside phosphorylases are classified based either on their substrate specificity/affinity (Table 1.1) or on their structure [19]. In 2002, Pugmire and Ealick described a structure-based classification of NPs in two distinct families [19, 20]: NP-I and NP-II (Figure 1.2). They demonstrated that members of an NP-I family share the following characteristics [20]: (i) they have a single domain subunit, (ii) they share a common a/ß-subunit fold, (iii) their quaternary structure is either trimeric (mammals/higher organisms) or hexameric (bacteria/lower organisms), (iv) they accept both purine nucleosides (bacterial and mammalian PNPs) and pyrimidine nucleosides (uridine phosphorylase, ) as substrates, and (v) their substrate-binding sites are similarly arranged. Nevertheless, they are quite different in their quaternary structures, amino acid sequence, and substrate specificity. Additionally, active sites of the hexameric family members are significantly different from those of the trimeric enzymes of higher eukaryotes, which makes them attractive targets for the specific treatment of bacterial or parasitic infections.

Table 1.1 Nucleoside phosphorylases with acronyms and EC numbers.

Figure 1.2 Classification of nucleoside phosphorylases (NPs) and their substrate affinities. NPs are classified into two main families: NP-I and NP-II. NP-I family is further subdivided into hexameric and Trimeric NPs. NP-II family is subdivided into two main classes: TP and PyNP. Residues labeled in red are crucial for enzyme-substrate interaction; green and blue labeled substitutions are those accepted by enzymes.

Members of the NP-II family are characterized by (i) having two domain subunits: a...