One of the most important challenges in synthetic chemistry is the rapid and high-yield synthesis of complex molecules from simple building blocks. Many of these syntheses require the highly reactive aldehyde functional group, which can be used for (asymmetric) aldol, and Mannichtype reactions as well as Grignard additions, Wittig reactions, oxidations and reductions. Aldehydes are not only essential building blocks for the synthesis of pharmaceuticals, they also play an important role in food and flavor industry. Aldehydes are mainly synthesized by chemical oxidation of primary alcohols, by ozonolysis or the reduction of activated carboxylic acid derivatives. However, these systems are not environmentally friendly. Over the last decade, novel methodologies were developed, especially in the field of biocatalysis. Indeed, aldehydes can be synthesized in a completely green way by enzymatic means. In nature, several enzymes produce aldehydes: alcohol oxidases and alcohol dehydrogenases that convert alcohols to aldehydes; carboxylic acid reductases and aldehyde oxidoreductases that reduce carboxylates to aldehydes; amine oxidases that catalyze the oxidative deamination of primary amines. At present, none of these enzymes have been used for semi-preparative or preparative aldehyde synthesis.
Amine oxidases, the copper containing enzymes extracted from Leguminosae seedlings, appeared particularly attractive because they show a rather broad substrate specificity and a remarkable stability profile. On this basis, we will develop an enzymatic green method for gram-scale synthesis of aldehydes starting from a variety of beta-substituted ethylamines using Lathyrus cicera (chick pea) amine oxidase (LCAO). To this purpose, we will set up a new purification method involving cross flow ultrafiltration and a series of immobilization strategies to allow for enzyme recovery and reutilization. Evaluation of LCAO activity will be done also in Natural Deep Eutectic Solvents (NADES).
Green synthesis of aldehydes is taking center stage both in industry and research. Given the high-value applications and large markets for several aldehydes, commercial focus on microbial aldehyde synthesis has surged in recent years. Since aldehydes are inherently unstable and potentially toxic, they do not accumulate in microorganisms making large-scale purifications particularly challenging. A valid alternative to produce aldehydes in an environmentally sustainable way is represented by biocatalysis.
Aldehydes can be synthesized starting from the corresponding alcohols or aminoacids. Aldehydes synthesis starting from alcohols can be indeed performed enzymatically by using alcohol oxidases. These enzymes, which are commonly used in synthetic biotransformations, are active on a broad range of alcohols. However, they also show propensity towards over-oxidation of aldehydes to carboxylic acids and are not active on functionalized beta-ethylalcohols. Aldehydes can also be produced starting from aminoacids by using transaminases, but their use has a limited scope because only a small number of different aldehydes can be produced. As an alternative, aldehydes can be produced from the corresponding amines. This reaction can be performed by amine oxidases, as it indeed occurs in polyamine metabolism and monoamine degradation pathways.
Currently, our research team is engaged in the enzymatic synthesis of complex secondary plant metabolites which needs aldehydes as intermediates. For this purpose, a plant amino oxidase from Lathyrus cicera (LCAO), capable of producing aldehydes from primary amines, has been identified. This enzyme has a broad substrate specificity associated with good stability. Until now, engineered amine oxidases have been mainly used both in developing biosensors and in deracemization reactions for asymmetric synthesis of pharmaceuticals and natural products.
The innovation of our research consists in using this enzyme for synthetic purposes to obtain high value-added aldehydes on, at least, semi-preparative scale.
To achieve these objectives, it will be necessary to optimize the reaction conditions to allow the enzyme to process large quantities of substrate. For example, it will be necessary to design reactors which maintain high concentrations of oxygen in solution; it will be necessary to remove the hydrogen peroxide, a reaction by-product, which at high concentrations can inactivate the enzyme itself compromising the entire biocatalytic process. This would already represent a great step-forward compared to the current state of knowledge. In this project we also propose to develop a protocol for the immobilization of the enzyme, which would allow to improve its operational fitness and recycling in an environmentally friendly perspective.
To date, the reactivity of this enzyme in media other than water has not been described. We aim to test its activity in eutectic solvents which have the characteristic of being able to solubilize even substrates that are typically not soluble in water. Since NADES are natural compounds, they represent an environmentally sustainable alternative to organic solvents.
In addition to the biosynthetic applications, LCAO may also be used for the development of quantitative assays for the analysis of aldehydes. Current analytical methods to quantify aldehydes rely on time consuming GC and HPLC methods rather than on fast photometric assays. Commercial kits for the colorimetric detection of small aldehydes in micromolar ranges in proprietary buffer solutions and serum samples are available. However, those kits are costly and have not been established for whole cell systems.