Immobilization of Lipase Enzyme to Catalyse Abiotic Organic Transformations

Abstract

Chapter 1, covered the lipase-catalyzed synthesis of various N-based molecules, including imidazole, pyridine, quinoline, 1,4-dihydropyridines, 1,3-oxazine, and spiro compounds, all

displayed highly promising biological properties such as anti-cancer, anti-bacterial, anti- fungal, anti-tubercular, anti-diabetic, anti-oxidant, anti-HIV, anti-inflammatory, etc. Besides,

it consists of an overview of different matrices used to immobilize lipase enzymes. Also, a summary of different ways and factors related to lipase enzyme immobilization has been covered in this chapter. Nevertheless, it should be highlighted that choosing suitable support materials is a challenging task and has a significant impact on the biocatalyst's properties. In fact, the immobilized form of lipase is more stable & resilient than pure lipase and is easy to recover and reuse. However, some research and development have been done on applying lipase in medicinal chemistry, especially in synthesizing N-based heterocycles. The overwhelming results have shown advantages like easy preparation, zero or low toxicity, high bioavailability, lower drug resistance, excellent biocompatibility, etc. Hence, there is enormous potential in N-heterocyclic cores yet to be discovered in the future because of their diverse molecular targets. We also believe this chapter will be valuable for encouraging the structural design and development of sustainable and effective nitrogen-based drugs. In Chapter 2, all the material and methods have been covered. Also, it comprises a brief overview of different characterization techniques such as FTIR, XRD, SEM, HR-TEM, TLC, etc. Also, it contains the equation used for calculating green chemistry matrices and formulas used for kinetics study. Chapter 3, comprehended the biocatalyst catalyzed Groebke-Blackburn-Bienaymé (GBB) multicomponent reaction to synthesize fused imidazo[1,2-a]pyridine derivatives using Candida antarctica lipase B (CALB) enzyme. Also, various substitutions were tolerated well during the enzymatic synthesis and provided imidazo[1,2-a]pyridine derivatives in good to excellent yield. Further, the CALB enzyme was immobilized on mesoporous silica and used as a reusable catalyst which displayed high catalytic efficiency up to many cycles and was characterized using techniques like FTIR, SEM, EDS, XRD, etc. A preliminary mechanistic study, including molecular docking and molecular dynamics (MD) simulation, revealed that Thr40 and Ser105 residues played a crucial role in catalyzing the GBB- multicomponentreaction, such as the Quantum Mechanical/ Molecular Mechanical (QM/MM) approach to get more insights about the mechanism of this transformation are

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145 | P a g e under progress in our lab. Finally, this work contributes to expanding the number of enzymatic transformations to synthesize clinically significant heterocycles. Chapter 4, covered the generation of a robust and reusable biocatalyst via immobilization of lipase (CALB) onto surface functionalized magnetic halloysite nanotubes. The magnetic character was introduced into halloysite tubes through in-situ attaching iron oxide nanoparticles

to HNTs. The magnetic catalyst was characterized using FTIR, XRD, XPS, HR-TEM, SEM- EDS, and VSM techniques. The loading of CALB on MHNTs was calculated using the

Bradford assay, which showed 322.9 mg of enzyme per gram of solid support. Next, the newly developed catalyst was employed in the multicomponent reaction of substituted aromatic aldehydes, anilines, and acetoacetate esters to synthesize piperidine derivatives with an excellent yield (45-91%). A gram scale reaction was set up to prove the synthetic utility of this protocol which provided the desired product in 91% yield. Also, the catalyst was reusable for up to ten consecutive cycles with easy magnetic separation. Additionally, immobilized CALB displayed superior stability and activity compared to free lipase, as revealed by kinetic studies even after storing it for five months at room temperature. Finally, this work expands the application of lipase enzymes in synthesizing clinically important heterocycles. It adds a new abiotic organic reaction accessible through the enzyme into the biocatalyst toolbox.

In Chapter 5, efficient preparation of highly reusable and eco-friendly nanobiocatalyst (CALB@MrGO) has been documented. The magnetic character was imported to nanobiocatalyst by anchoring Fe3O4 nanoparticles over reduced graphene oxide, onto which

Candida antarctica lipase B was immobilized. The successful functionalization and post- immobilization changes in nanobiocatalyst were characterized using FTIR, XRD, XPS, HR- TEM, SEM-EDS, and VSM techniques. The loading of lipase enzyme over MrGO was studied

using the Bradford assay, which exhibited 356 mg of enzyme per gram of solid support. Next, we employed the developed NBC (CALB@MrGO) in the one-pot synthesis of 2,3- dihydroquinazolin-4(1H)-one derivative using 2-aminobenzamide with various substituted aromatic aldehydes and obtained the corresponding products in good to excellent yields. Moreover, the magnetically separable CALB@MrGO was found stable after the completion of the reaction and reused up to ten catalytic cycles. However, the catalytic activity of synthesized NBC remained unchanged for five consecutive cycles, but afterward, a slight decrease in the catalytic efficiency was noticed. Further, the scalability of the transformation was proved by

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146 | P a g e set-up a gram scale reaction. Besides, the kinetics studies revealed that immobilized lipase, i.e., CALB@MrGO was more active than pure Candida antarctica lipase B. In Chapter 6, successful development of an operatively simple and efficient biohybrid catalyst [Pd(0)-CALB@SiO2] by immobilizing Candida antarctica lipase B and Pd(PPh3)4 within a silica framework. The dual, i.e., bio- and chemo-catalytic activity of biohybrid catalyst was verified by set-up a one-pot reaction consisting of the sequence of GBB-multicomponent and Suzuki-coupling reaction to produce aryl substituted imidazo[1,2a]pyridines. Both the entities of the hybrid catalyst were catalytically active and tolerated a number of differently substituted boronic acids to produce corresponding products in decent yields. Next, a gram-scale reaction was set up to prove the synthetic utility of this protocol which provided the desired product in 81% yield. The developed biohybrid catalyst was reusable up to five catalytic cycles; however, a decrement in the yield of product was observed after the second catalytic cycle. Besides, the developed biohybrid catalyst was stable even after storing at room temperature for up to 30- days and exhibited excellent catalytic activity.