International Journal of Molecular Sciences Review Flow Biocatalysis: A Challenging Alternative for the Synthesis of APIs and Natural Compounds Micol Santi 1,* , Luca Sancineto 2 , Vanessa Nascimento 3 , Juliano Braun Azeredo 4, Erika V. M. Orozco 5 , Leandro H. Andrade 5, Harald Gröger 1 and Claudio Santi 2,* 1 Chair of Industrial Organic Chemistry and Biotechnology, Faculty of Chemistry, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany; [email protected] 2 Group of Catalysis Synthesis and Organic Green Chemistry, Department of Pharmaceutical Sciences, University of Perugia Via del Liceo 1, 06100 Perugia, Italy; [email protected] 3 Department of Organic Chemistry, Institute of Chemistry, Universidade Federal Fluminense, Valonguinho Campus, Niterói 24020-141, Brazil; [email protected] 4 Departament of Pharmacutical Science, UNIPAMPA, BR 472-KM 585, Uruguaiana 97501-970, Brazil; [email protected] 5 Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-900, Brazil; [email protected] (E.V.M.O.); [email protected] (L.H.A.) * Correspondence: [email protected] (M.S.); [email protected] (C.S.) Abstract: Biocatalysts represent an efficient, highly selective and greener alternative to metal catalysts in both industry and academia. In the last two decades, the interest in biocatalytic transformations has increased due to an urgent need for more sustainable industrial processes that comply with the principles of green chemistry. Thanks to the recent advances in biotechnologies, protein engineering and the Nobel prize awarded concept of direct enzymatic evolution, the synthetic enzymatic toolbox has expanded significantly. In particular, the implementation of biocatalysts in continuous flow systems has attracted much attention, especially from industry. The advantages of flow chemistry Citation: Santi, M.; Sancineto, L.; enable biosynthesis to overcome well-known limitations of “classic” enzymatic catalysis, such as Nascimento, V.; Braun Azeredo, J.; Orozco, E.V.M.; Andrade, L.H.; time-consuming work-ups and enzyme inhibition, as well as difficult scale-up and process intensifi- Gröger, H.; Santi, C. Flow cations. Moreover, continuous flow biocatalysis provides access to practical, economical and more Biocatalysis: A Challenging sustainable synthetic pathways, an important aspect for the future of pharmaceutical companies if Alternative for the Synthesis of APIs they want to compete in the market while complying with European Medicines Agency (EMA), Food and Natural Compounds. Int. J. Mol. and Drug Administration (FDA) and green chemistry requirements. This review focuses on the most Sci. 2021, 22, 990. https://doi.org/ recent advances in the use of flow biocatalysis for the synthesis of active pharmaceutical ingredients 10.3390/ijms22030990 (APIs), pharmaceuticals and natural products, and the advantages and limitations are discussed. Received: 30 December 2020 Keywords: biocatalysis; flow chemistry; immobilized enzyme; packed-bed reactor; segmented flow; Accepted: 14 January 2021 microreactors; APIs; natural products; sustainability Published: 20 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. The term “biocatalysis” refers to the use of enzymes, both as purified proteins or as whole cells, to catalyze the conversion of a substrate into a desired product, and it is currently a popular topic in synthetic research [1]. Since 1858, when Louis Pasteur reported the kinetic resolution of racemic tartaric acid through fermentation promoted by microorganisms [2], enzymes have been widely studied and exploited to catalyze organic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. chemical transformations, especially in the food industry [3]. An awakening of the interest This article is an open access article in biocatalytic applications has since occurred in the latter half of the 20th century [4], distributed under the terms and motivated by the need for greener and more sustainable chemical processes [5,6]. conditions of the Creative Commons Since the advent of the environmental movement [5–8] and the definition of green Attribution (CC BY) license (https:// chemistry and its 12 principles [9], chemical industries have undergone radical changes creativecommons.org/licenses/by/ by investing in more sustainable methodologies to minimize wastes and their ecological 4.0/). footprints. As a result, biocatalysis has begun to receive considerable attention from the Int. J. Mol. Sci. 2021, 22, 990. https://doi.org/10.3390/ijms22030990 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 990 2 of 32 chemical industry, thanks to its greener and sustainable features, which fulfill 10 out of the 12 principles of green chemistry [10,11]. The major advantage of enzymatic catalysis is its ability to promote highly chemo-, regio- and stereoselective transformations. Over thou- sands of years of evolution, nature has equipped cells with highly efficient and extremely selective catalytic systems to promote the synthesis of every kind of natural product while reducing waste generation. Moreover, “natural” enzymes have also been found to catalyze “non-natural” synthetic reactions and are capable of increasing their stability by direct evolution to better adapt themselves to “non-natural” reaction conditions [12–15]. This higher selectivity leads to more straightforward synthetic pathways with no function- alization steps needed, resulting in reduced wastes [16,17]. Additionally, enzymes are biodegradable compounds (proteins) produced by renewable resources such as plants [18] or microorganisms [19], and work under mild conditions, such as relatively low tempera- tures, atmospheric pressure and in non-toxic, inexpensive, and readily available solvents, such as water. The abovementioned benefits have contributed to the industrial use of biocatalysts for the synthesis of commercially interesting compounds such as pharmaceuticals or fine chemicals [20–24], aiming to satisfy economic demands while respecting the green chem- istry requirements [25]. Furthermore, modern advancements in biotechnology [26] and immobilization techniques [27,28], as well as recent developments in protein engineering and direct evolution [12–15,29], have helped to enhance the utilization of biocatalysis in synthesis [30] by enabling cheaper production of enzymes and providing access to a large library of versatile biocatalysts. Nevertheless, the number of industrial biocatalytic applications remains limited by difficulties in scaling up, tedious optimization processes and long reaction times, as well as enzyme instability over time and their incompatibility with other reaction condi- tions [1,4]. This incompatibility makes one-pot cascade reactions hard to perform, resulting in downstream purifications, which are usually time-consuming due to protein aggre- gations [31]. Moreover, the use of water as a sole solvent often leads to overly diluted solutions due to substrate solubility issues, resulting in high solvent usage, low product concentrations producing low productivities, as well as high energy-consumption due to the high boiling point required [4]. A practical improvement in performances was reached with the advent of continuous- flow technology and the implementation of enzymatic reactions with flow reactors [31] and was also promoted by the arrival of several commercially available tools [32,33]. The rapid growth of interest in continuous-flow biocatalysis from both academia and industry is evidenced by the rising number of publications reporting it in the past decades, as depicted in Figure1. Figure 1. Overview of the number of flow biocatalysis-related publications added to SciFinder® in the past 20 years. Int. J. Mol. Sci. 2021, 22, 990 3 of 32 This increased interest in flow biocatalysis arose from the possibility of merging the conveniences of enabling technologies and the advantages of enzymatic catalysis into versatile, greener and sustainable synthetic tools. The ongoing evolution of this research area has led to the generation of numerous comprehensive review articles [34–39], which aimed to clarify the terminology as well as discuss the state-of-the-art and future perspectives of continuous-flow biocatalysis. This review does not claim to be an exhaustive overview of all aspects of continuous flow biocatalysis but is more of an “add-on” to the existing review literature. The presented article, inspired by the opinion recently published by Martin et al. from Novartis [40], aims to complete the picture of modern “flow biocatalysis” by presenting recent applications in the synthesis of active pharmaceutical ingredients (APIs), natural products and key precursors for pharmaceutical production, highlighting its importance in the future of the pharmaceutical industry. Other aspects recently included in comprehensive reviews reported by De Santis et al. [34] and Britton et al. [36], as well as the catalysts special issue “Flow Biocatalysis” edited by Professor Paradisi [39], will be not discussed in the present article. 2. Biocatalysis in Industry It is noteworthy that biocatalysis has not only gained interest in academia but has al- ready found numerous applications over the last decades in the chemical industry [23,41,42]. In particular, production processes have been established for the large-scale manufacturing of chiral compounds that are