# Improving the Phosphatase-Catalyzed Synthesis of 5'-Nucleotides: A Reaction Engineering Approach
## 1 Introduction  
Phosphate monoester motifs are widespread in nature and are present in molecules involved in many biological pathways [1]. Among them, nucleotides represent a preeminent class due to the central role they play in almost all cellular processes [2; 3]. In addition, their synthetic analogs (nucleotide analogs) are the fundamental agents of several anti-cancer and anti-viral drugs as well as the water-soluble prodrugs of pharmacologically active nucleosides [4; 5]. Finally, some natural nucleotides such as inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) are used as flavor enhancers in savory food manufacturing due to their ability to synergistically elicit the perception of umami taste in combination with monosodium glutamate (MSG) [6; 7]. For these reasons, considerable efforts have been directed toward the selective synthesis of naturally occurring nucleotides and their analogs [5; 8]. The most straightforward way to prepare nucleoside 5'-monophosphates consists of the introduction of a phosphate group at the 5'-hydroxy1 position of their parent nucleosides mainly by reaction with pentavalent phosphoryl donors such as phosphoryl chloride (POCl\({}_{3}\)) in the presence of trialkyl phosphates as solvents. Such a process is quite laborious due to the toxicity and moisture sensitivity of the reagents as well as the extensive formation of by-products. In addition, the harsh reaction conditions generally fail to preserve most acid-labilemoieties [9; 10]. Owing to these disadvantages, different phosphate donors or alternative strategies, i.e., formation of trivalent phosphite followed by oxidation [11; 12; 13], have been developed to allow milder and more selective syntheses. These methods, however, are not able to avoid drawbacks such as the need for multi-step sequences required by protecting group chemistry together with poor selectivity and moderate/low yields. Thus, more coofriendly and sustainable routes to \(5^{\prime}\)-monophosphate nucleosides are highly desirable. Biocatalytic procedures have emerged in the last few decades as viable alternatives to conventional chemical routes, matching high selectivity and mild conditions as well as generating less waste in agreement with \"green chemistry\" principles [14; 15]. Among enzymes from the nucleotide metabolism, deoxyribonucleoside kinases (dNKs, E.C. 2.7.1) catalyze the selective and irreversible phosphorylation of nucleoside \(5^{\prime}\)-position using ATP as a high-energy phosphate donor [16; 17]. However, examples of MNK-based phosphorylation are limited to date due to the relatively low stability and high cost of ATP, as well as the inhibitory effect of the accumulating ADP [18; 19]. In order to allow the use of ATP in catalytic amounts, several recycling systems have been developed to turn ADP into ATP, thus making the process less expensive [20; 21]. Alternatively, the formation of a new P-O bond can be catalyzed by phosphatases, a class of enzymes that dephosphorite in vivo a large set of phosphate esters, but that are also able to perform the reverse reaction (transphosphorylation reaction) [16; 22]. In particular, non-specific acid phosphatases (NSAPs), widely distributed among enteric bacteria, exhibit optimal catalytic activity at acidic to neutral pH values and are characterized by a very broad substrate specificity being active toward different and structurally unrelated phosphesters such as nucleotides and sugar phosphates [17; 22; 23]. In the late 1990s, an NSAP from _Morganella morganii_ NCIMB10466 strain (PhoC-_M_m_) possessing a regioselective phosphotransferase activity specific for the \(5^{\prime}\)-position of nucleosides was described by [PERSON] et al. who exploited it for the selective phosphorylation of C-\(5^{\prime}\) of inosine (I) to produce IMP, using the food additive pyrophosphate (PPi, E450 as sodium salt) as the phosphate donor [24; 25].