(402f) Hydroxamic Acids Production in Reverse Micelles and Recovery By Membrane Separation Processes

Hydroxamic acids derivatives RC(O)NHOH present a high clinical potential, given that these compounds are efficient as antitumor, antituberculosis, antimalarial, anti-inflammatory agents and can be used in the treatment of diseases such as Alzheimer, AIDS and cardiovascular disorders [1]. Their synthesis and purification has been carried out through the use of chemical reactions from esters, in solution and on solid phase, but that involves complicated procedures [2]. Biocatalysis can be considered as an alternative approach, namely, when biocatalysis uses amidase (E.C. 3.5.1.4.) from Pseudomonas aeruginosacells encapsulated in reverse micelles [3]. This approach offers advantages such as high specificity, mild conditions and shorter reaction times. The use of nonconventional systems of reverse micelles using a surfactant, water and an organic solvent are applicable in the biocatalysis of synthesis reactions, where these synthesis reactions are favored over hydrolysis reactions due to reduced water content, with an additionally enhanced activity and stability comparatively to buffer media.

The approach used involves a novel membrane bioreactor using encapsulated Pseudomonas aeruginosacells for the synthesis of several hydroxamic acid derivatives using a range of different amidase substrates [4], according to the enzyme transferase activity illustrated below:

 R-CO-R’ + NH₂OH -> R-CO-NH-OH + R’H

where R’: -NH₂or –OR’’.

Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts, both in free and immobilized form in a reverse micellar system. The apparent kinetic constants for the transamidation reaction in hydroxamic acids synthesis were determined using substrates such as aliphatic, amino acid and aromatic amides and esters, in both media. In reverse micelles, Km values decreased 2–7 fold relatively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide ethyl ester. We concluded that overall the affinity of the biocatalyst to each substrate increases when reactions are performed in the reversed micellar system as opposed to the buffer system. The immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower substrates concentration relatively to the free form, which is advantageous. Additionally, the immobilization revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives. The optimization of work condition was then made using a factorial design methodology (CCD), through the study of the influence of some parameters: amide and hydroxylamine concentration, TTAB concentration (surfactant), pH and concentration of HEPES buffer. The results show that it is possible to obtain yields of 100% for all substrates.

The recovery of the hydroxamic acids derivatives produced can be achieved using membrane processes. Two pathways can be followed: i) use of solvent-resistant polymeric membranes, in this path the organic solvent/ micellar system can be fed into a flat sheet UF unit where the separation of the hydroxamic acids derivatives (in the permeate) from the micelles (retained on the concentrate) is envisaged or ii) a preliminary phase separation is carried out with an aqueous KCl solution, leading to the extraction of the hydroxamic acids into the aqueous solution, which can be then separated from residual surfactant and concentrated by membrane processes (UF and NF).

References:

[1] Codd R. (2008) Transversing the coordination chemistry and chemical biology of hydroxamic acids. Coordination Chemistry reviews 252:1387-1408.

[2] Devocelle M., McLoughlin B. M., Sharkey C. T., Fitzgerald D. J., Nolan K. B. (2003) A convenient parallel synthesis of low molecular weight hydroxamic acids using polymer-supported 1-hydroxybenzotriazole. Org. Biomol. Chem. 1:850-853.

[3] Pacheco R., Matos-Lopes M.L., Karmali A., Serralheiro M.L.M. (2005) Amidase encapsulated in TTAB reversed micelles for the study of transamidation reactions. Biocatalysis and Biotransformation 23: 407-414.

[4] Pacheco R., Karmali A., Serrallheiro M.L.M., Haris P.I. (2005) Application of FTIR spectroscopy for monitoring hydrolysis and synthesis reactions catalysed by a recombinant amidase. Analytical Biochemistry 346: 49-58.