Enzymatic synthesis of nucleoside analogues from uridines and vinyl esters in a continuous-flow microreactor

We achieved the effective controllable regioselective acylation of the primary hydroxyl group of uridine derivatives catalyzed by Lipase TL IM from Thermomyces lanuginosus with excellent conversion and regioselectivity. Various reaction parameters were studied. These regioselective acylations performed in continuous flow microreactors are a proof-of-concept opening the use of enzymatic microreactors in uridine derivative biotransformations.

add more and better biological traits to existing candidates have been reported. [18][19][20] The most common introduced substituents on sugar moiety of nucleosides include halogen, N 3 , CF 3 , CN, alkyl, alkenyl, alkynyl, aryl, thio and seleno groups. Regioselective acylation of sugar moiety of nucleosides is another way to get nucleoside analogues. [21][22][23][24][25] But as we can see, the sugar moiety of nucleosides contains at least three hydroxyl groups, which make regioselective acylation of nucleosides more difficult, traditional chemical synthesis routes oen require complex protection-unprotection procedures and harsh conditions. 20,26-28 Therefore, it is important to nd a new, simple and environmentally friendly method for the preparation of nucleosides analogues.
Enzymes are the most procient catalysts, offering much more competitive processes compared to chemical catalysts, such as mild reaction conditions, high efficiency and selectivity. In the past few years, several works about the enzyme-catalyzed synthesis of uridine esters were reported. [29][30][31][32][33][34][35][36] Some lipase such as CAL-B 22 and pseudomonas cepacia lipase 29 have been applied to the synthesis of uridine esters, but it requires a longer reaction time (24 h) to achieve the desired result. Flow chemistry especially catalyst/substrate conditions in continuous-ow systems can increase the reactivity and selectivity. 37,38 In the interest of developing highly efficient method for the synthesis of uridine esters, we envisaged modifying our procedure to achieve a continuous ow microreactor protocol for the synthesis of uridine esters. Specically, we directed our attention towards the development of an enzymatic microreactor strategy involving lipase TL IM from Thermomyces lanuginosus as catalysts (Scheme 1). The aim of this paper is to investigate, under a continuous ow microreactor, the effect of various reaction parameters on the reaction yield. What's more, we want to quickly build the related compound library through the new synthesis method for the next drug screening.
The enzymatic synthesis of uridine esters from uridine derivatives and vinyl esters in a continuous-ow microreactor is described in Fig. 2. We rst examined whether the reaction can be performed in continuous ow microreactors. The device was composed of a syringe pump, coil reactor and Y-shaped mixer (4 ¼ 1.8 mm; M). A syringe pump (Harvard Apparatus PHD 2000) was used to introduce two separate feed streams to a 3.1 mL PFA coil reactor (2.0 mm I. D.). Reagent feed 1 (10 mL) with the uridine dissolved in DMSO and tert-amyl alcohol mixed solvent, reagent feed 2 (10 mL) with the vinyl esters dissolved in tertamyl alcohol. Two feed streams were mixed into a single PFA tubing and fed a Y-shaped mixer. The coil reactor was lled with Scheme 1 Enzymatic synthesis of uridine esters from uridine derivatives and vinyl esters in continuous flow microreactors.  lipozyme TL IM (catalyst reactivity: 250 IUN g À1 ) and submerged into a thermostatic water bath to control the temperature. Aer initial optimization, it was found that the target uridine esters (3a-3l) could be obtained, aer a thirty minutes residence time, in excellent yield (80-99%) aer separation and purication (Table 1). We began to explore our research by screening some reaction parameters on the enzymatic synthesis of nucleoside esters from uridines and vinyl esters in a continuous-ow microreactor. Solvent is an important factor on the enzymatic reaction, in order to nd the optimum reaction medium, we rstly chose uridine and vinyl laurate as the model reaction and tried several solvents such as pyridine, THF, DMF, DMSO and DMSO/ tert-amyl alcohol and found that when the reactions were conducted in these solvents, the reactions were hard to occur except in the mixed solvent of DMSO/tert-amyl alcohol. In order to reduce the use of DMSO in the reaction, the volume ratio of DMSO/tert-amyl alcohol from 1 : 9 to 1 : 16 was studied. As we can see from the Fig. 3, the decrease of DMSO had a positive impact on the reaction conversion, with DMSO/tert-amyl alcohol ¼ 1 : 14 gave the best result.
The molar ratio (uridine/vinyl laurate) on the uridine esters synthesis reaction is another factor affecting the reaction. It involves the atomic economy and the conversion of the products. The inuence of molar ratio (uridine/vinyl laurate) was investigated from 1 : 1 to 1 : 13 (Fig. 4). According to the Fig. 4, the reaction conversion was 65% when the substrate molar ratio (uridine/vinyl laurate) was 1 : 5, with the increase of vinyl laurate, the conversion also gradually increase. Considering the optimal reactant economy and the best reaction conversion, we decided to choose uridine : vinyl laurate ¼ 1 : 9 as the optimum substrate molar ratio.
The reaction temperature has an important effect on the enzymatic reaction, especially when the reaction performed in continuous ow microreactors. So we continued to nd the best reaction temperature on the enzymatic uridine esters synthesis under continuous ow microreactors. The reactions were carried out from 15 C to 50 C and the results are shown in Fig. 5, when the temperature was 15 C, the conversion was less than 60%. And with the increase of temperature, the conversion rate of the reaction is obviously increased too. Considering the safety and controllability of the reaction, we chose 30 C as the optimal reaction temperature for the following experiment.
The effect of reaction time/ow rate on the conversion was also investigated. Reaction time/ow rate play a signicant role in the enzymatic synthesis of uridine esters in continuous ow microreactors. We performed the reaction from 20 minutes to 35 minutes, the results were shown in Fig. 6. The best conversion was reached for 30 minutes and at a ow rate of 20.8 mL min À1 . Therefore, we chose 30 minutes (ow rate 20.8 mL min À1 ) as the optimum reaction time for the following experiment about the scope of application.
When the optimum reaction conditions were obtained, we continued to investigate the substrate structure effect on the enzymatic uridine esters synthesis reaction under continuous ow microreactors. The effect of different substituted groups on the uridine was studied and the results were shown in Fig. 7. We found that the reaction of 5-uorouridine (1c) to vinyl laurate (2b) can get a higher yield (99%, entry 10) compared with uridine (98%, entry 6) under the same reaction conditions, it indicated that electron-withdrawing group could improve the acylation reactivity of uridine. Oppositely, the reaction of 5methyluridine (1a) with electron-donor group proceeded more slowly (entries 1-4). That is to say, under the same condition, the uridine esters synthesis of uridine to vinyl laurate was more rapid than that using 5-methyluridine as the reactant, while lower than that using 5-uorouridine as the donor.    We have also investigated the acceptor structure effect on the enzymatic uridine esters synthesis. We chose uridine as the donor, and found that the longer the vinyl esters carboxyl group chain, the higher the conversion (Fig. 7). The yield from 5-uorouridine to vinyl palmitate (99%, entry 9) was almost equal to the vinyl laurate. Meanwhile, the yield of 5-uorouridine to vinyl acetate was only 85%.
Finally, we explored the scope and limitations of this controllable enzymatic regioselective acylation of uridine derivatives in continuous ow microreactors. Three uridine derivatives, 5-methyluridine (1a), uridine (1b), 5-uorouridine (1c), and four vinyl carboxylates, vinyl palmitate (2a), vinyl laurate (2b), divinyl adipate (2c), vinyl acetate (2d) were subjected to the general reaction conditions, using both a single-mode shaker reactor and a continuous ow microreactor processing. For shaker experiments, the reaction times need to be about 24 h to obtain ideal conversion (method A in Table 1). Nevertheless, employing the enzymatic regioselective synthesis of uridine esters under a continuous-ow microreactor, 12 compounds synthesized in parallel in a single experiment at the same ow rate 20.8 mL min À1 (method B in Table 1). The results were better under continuous ow microreactors than with the single-mode shaker ( Table 1, entry 1-12).

Conclusions
In conclusion, we have developed a novel and efficient approach of controllable regioselective acylation of uridine derivatives catalyzed by lipozyme TL IM in continuous ow microreactors. The reaction conditions including solvent volume ratio, substrate molar ratio, reaction temperature, reaction time/ow rate and the substrate structure effect on the reaction were examined. The scope of the reaction was tested by varying the uridine derivatives and vinyl esters. Comparing with the traditional methods, the salient features of this method include reducing the amount of DMSO, mild reaction condition (30 C), short reaction time (30 min), high yields and high regioselectivities that make our methodology a valuable contribution to the eld of nucleoside analogues synthesis. What's more, we can continue our research to quickly build the related compound library for the next drug screening.

Conflicts of interest
There are no conicts to declare.