Asymmetric Synthesis of Chiral Alcohols for API Production using Microorganisms - A Mini Review

 

Saravanan J¹, Suneetha V^2*

¹Anthem Biosciences Private Limited, Bangalore.

²School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu India

 

ABSTRACT:

Biotransformation of chiral intermediates using micro-organism has widely used in many industries due to green technology and cost effectiveness.  Moreover, Enzymes are highly ‘Chemo-, regio-, and enantio-selective’ in nature. From decades, bio-reduction performed using whole cells and free enzymes for the production of chiral alcohols. Some recent developments in the area of fermentative biotransformation processes for the reduction of Keto- sulfone to hydroxy sulfone and Lipitor intermediate are reviewed and importance of microbial mediated asymmetric synthesis of chiral alcohols are highlighted for the future perspective.

 

 

 

 

INTRODUCTION:

Asymmetric synthesis plays a major role in the synthesis of chiral intermediates for Active Pharmaceutical Intermediates (API) production and more than half of drug molecules have more than one chiral centers¹. Biotransformation processes are environment friendly, performed under ambient temperature and pressure conditions in aqueous medium. About 10% of the total drug synthesized globally using enzymatic processes² and the percentage will increase in the coming years. The use of microbial whole cells showed a great economic advantage over the isolated enzymes which needs expensive co-factors and co-factor recycling system. The keto precursors for the API production, used in the industries are unnatural synthetic compounds and mostly toxic compound³. The enzymatic processes using such compounds, the free enzymes tend to lose its activity sooner than the microbial cells.

 

The microbial cells, especially, the fungal species are robust and capable of withstanding the toxicity and sustaining its activity/viability for longer durations. The recent studies proved that screening of new microbial cultures will definitely a better choice to develop commercially viable biotransformation (bio-reduction) processes. Such processes will have significant advantages over the free enzyme; because it doesn’t require external cofactors, the cells can multiple and regenerate effectively during the biotransformation process, with reasonable purity and yield, also performed with environmentally less toxic materials. The noticeable disadvantages of using microbial processes are huge volumes and fermentation based impurities⁴. This article reviews some of the latest invention in the field of microbial asymmetric synthesis of chiral alcohols and highlight the importance of identifying new enzymes from diverse microbial cultures from nature.

 

Need of Biotransformation:

The regulatory requirements are becoming tougher and enantiomeric purity of 99.9% is deemed necessary by the regulatory authorities like Food and Drug Administration (FDA), International Conference on Harmonization (ICH), Medicines and Healthcare Products Regulatory Agency (MHRA) etc⁵. This is one of the biggest challenges to the chemical process, because the different enantiomers of the same compound can interact differently in biological systems. Hence the biotransformation’s (bio-reduction) are introduced in the synthesis of small pharmaceutical molecules to attain the required chirality. The identification of a specific step and application of desired bioconversion plays a vital role in the entire scheme. Most of the drugs in API industry are containing one or more chiral centers and bringing out the desired chiral center are being the major task. Many chiral reagents are reported to introduce the chiral centers but mostly doesn’t fit to the cost. Sometimes the scale up may not be feasible with that chiral agents and nature of the synthetic reaction, which can cause an impact in the environment, health and safety (EHS) and poor yields due to multiple purification steps. Now-a-days many Pharmaceutical companies starting their own biocatalytic divisions to explore the enzyme technology and coming out with new processes either using Free enzymes or whole cell processes during fermentation or using resting cells. Even some cases free enzymes with external co-factors and co-factor regeneration system are not fitting to the cost due to slashing of API price. Whole cell processes are considered to be the most feasible alternate approach in such cases. Bigger advantage in the biotransformation process will be happening during growth phase in Fermenter, since it can easily address all the regulatory requirements.

 

Screening of desired Microbial culture:

The key step in the bio-reduction process development is the screening of desired microbial strain/culture which possesses the enzyme of interest. It is very difficult to propose a rational method of screening for novel enzymes. However, there are three important general strategies: 1. Designing the process and deciding the type of enzymatic activity desired; 2. Deciding which groups of microorganism are to be selected and screened; and 3. Designing an appropriate; convenient and sensitive assay that will allow as many microbial cultures as possible to be screened⁷. It is necessary to observe the functions of microorganisms during growth in order to obtain the desired conversions and enantiomeric excess. Generally, the cultivation of microorganism plays a vital role in bio-reductions process. Selection of right carbon and nitrogen sources, pH, temperature, RPM and aeration, inorganic ions are important for screening reactions.

 

The most relevant and widely chosen classical screening method outlined below:

 

 

METHODOLOGY OF SCREENING:

The efficiency of screening tool depends on the interaction of Ketone compound with microbial enzyme. Most of the Keto compounds are insoluble in water, hence suitable co-solvent need to be screened prior to the bio-reduction trials. More likely the selected solvent will not have any adverse effect on the microbial cells. Due that reasons, the water miscible solvents are selected (-2.5 < log P <0), Such as Acetone, Ethanol, Isopropyl alcohol, Dimethyl sulfoxide are widely used up-to 2-10% depends upon the solubility of the compound and nature of microbial strain.

 

Similarly, the enzyme activity is directly proportional to the cell mass, hence the achieving a high cell density always preferred. Generally, the ketone compounds are added to the fermentation broth after attaining maximum growth or at the end of batch phase. The screening reactions are performed in 250ml Erlenmeyer flask and sampled at regular intervals of reaction and extracted in suitable solvent such as Ethyl acetate, Dichloromethane, methanol etc. The extracted samples are injected to HPLC, after simple workup procedure to check the percentage conversion and the percentage enantiomeric excess of the product.

 

After the completion of screening, a lab scale process developed in 2-10L fermenter and characterization of product carried out using NMR, LC-MS and further processing till final API.

 

Bio-reductions processes using Fungal cultures:

The asymmetric synthesis of chiral alcohols using fungal cultures are explained as below;

 

Example: 1:

Asymmetric synthesis of Trusopt intermediate (4S,6S)-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-4-ol 7,7-dioxide^8-11

 

Figure: 1

 

2a – Desired Isomer:

a.      Process developed using MTCC 5963 Penicillium decumbens with 99%ee and upto 80g/L by Biocon limited, India.

b.      Process developed using Rhodotorularubra ATCC 74283 and RhodotorulapilimanaeATCC 32762 with > 95% enantiomeric excess and titer of about 3g/L by Merck, NJ, USA

c.      Process developed using Neurospora crassa with about 90% enantiomeric excess and titer of 10g/L by Zeneca Limited London, England.

 

Process developed using whole cells with genus like Candida, Rhodotorula, Pichia, Cryptococcus etc., by Kanegafuchi, Japan

 

2b– Anti-isomer:

Process developed using MTCC 5967 Candida Sp. with 99% enantiomeric excess by Biocon limited, India

 

Trusopt (dorzolamide hydrochloride) is a carbonic anhydrase inhibitor and it reduces the ocular pressure in glaucoma patients. Dorzolamide is a drug, which possess two chiral centers. The synthesis of dorzolamide requires enantiomerically pure compound having methyl at position 6 and hydroxy at position 4 of 2a. Stereo isomeric configuration i.e. trans (4S, 6S) is therapeutically active however, it is difficult to make this compound without cis 2b (4R, 6S). A total of four diastereomers exists for this molecule but (4S, 6S) is desirable. A few biotransformation processes are reported by Zeneca Limited London, Merck and Co. New Jersey, Kanegafuchi, Japan and Biocon, India for the synthesis of (2) from (1). Many research works have been carried out for the selective keto reduction of keto-sulfone and the maximum titer claimed found to be 80g/L by Bioconusing Penicillium decumbens MTCC 5963 and also anti-isomer (4R, 6S) using MTCC 5967 Candida Sp.

 

In the process co-solvents such as Acetone and DMSO were used to dissolve the substrate and the substrate feed dosed in shots as well as continuous mode to the Fermentation broth, to a period of 3-6days. The pH of the reaction maintained in the range of3.5-5. Keto-reduction at pH values below 6 most of the free enzymes (KRED) may lose its activity and obtaining high conversion and titers will be a challenge. Interestingly, there is no free enzyme (KRED) reported for such conversion with higher enantiomeric excess and titer to the best of our knowledge. This biotransformation example will be the best to show the importance of bio-reductions using wild microbes with novel/non-infringing process.

 

Example: 2:

Asymmetric Synthesis of Atorvastatin Intermediates t-butyl-(3R, 5S)-6-chloro-3, 5-dihydroxy-hexanoate^11-15

 

Figure: 2

 

4a – Desired Isomer:

a.      Process developed using MTCC 6441Penicilliumchrysognum with 99.9% enantiomeric excess and upto35g/L by Biocon limited, India.

 

Enzymatic Process developed by Codexis, C-Lecta, Cambrex, DSM etc., widely used in many industries, well known product using enzymatic process was“Lipitor” from Pfizer.

 

 

4b– Anti-isomer:

Process developed using MTCC 5967 Candida Sp. with 98% enantiomeric excess by Biocon limited, India

 

Enzymatic synthesis of Atorvastatin intermediates was developed by many leading companies, in which the keto loading found to be about 5 to 20% of the reaction volume. The enzymes require co-factor NAD/NADP and co-factor recycling system comprises of GDH or FDH etc. the co-substrate used will be Glucose and Ammonium formate accordingly. The use of two enzyme system will always have challenges in terms of combined activity, by product production and expensive enzyme. To avoid that, most of the leading enzyme companies launched single enzyme system for reduction process as well as the co-factor regeneration using Keto reductases and IPA as co-substrate, in which Acetone will be the byproduct. The disadvantage of the process is found to be the by-product acetone, which will reduce the enzyme activity and conversion rate at later stage of the reaction. Various protein engineering approaches, including directed evolution, mutation of the enzyme are performed to increase activity, stability and coenzyme specificity of the enzyme. The intermediate for atorvastatin (Lipitor) was manufactured at very high industrial scale using free enzymes¹⁶, Codexis. Later, Protein engineering studies paved way for increasing activity and stability to 30% organic substrate (the hydroxyl-ketone substrate and a tert-butyl acetoacetate impurity, both liquids). (Codexis, US/2011/7879585; Pfizer, US/2008/0118962).

 

Recently Fermentative bio-reduction was mentioned by Biocon using wild Penicillium sp. For the keto reduction of 3. The media used found to be less expensive with considerable keto loading upto 35g/L will challenge the enzymatic process developed by leading companies. Since synthesis of recombinant KRED in E.coli system will be costlier than the classical fermentation of fungal cultures. Isolation and purification of E.coli enzymes will also add more cost for the Enzyme production.

 

Filamentous Fungi in Bio-reduction:

Filamentous fungi have been used as sources of secondary metabolites and enzymes for many years. Recent studies showed that the evaluation of fungal kingdom towards the application for the synthesis of chiral alcohol is not enough. Many fungal species are not studies extensively for the bio-reduction processes. Moreover, the Filamentous fungi are capable of withstanding the toxic effect of keto substrate used for the bio-reductions. Co-solvent tolerant level also considerably very high than bacterial cells due to their rigid cell wall. Fungi can grow in complex media such as Soya flour, Corn steep powder, Cotton seed flour and different starches. These media component are less expensive, Fermentation process developed using the said media will be highly cost effective.

 

Table: Systematics of biotechnologically relevant true fungi ^23-25

Phylum	Subphylum	Class (subclass)	Order	Family	Genus
Ascomycota	Saccharomycotina	Saccharomycetes	Saccharomycetales	Candidaceae	Candida
				Dipodascaceae	Geotrichum
					Yarrowia
				Eremotheciacea	Eremothecium
				Saccharomycetaceae	Hansenula/Pichia
					Kluyveromyces
					Saccharomyces
			Schizosaccharomycetales	Schizosaccharomycetaceae	Schizosaccharomyce
	Pezizomycotina	Eurotiomycetes	Eurotiales	Monascaceae	Monascus
				Trichocomaceae	Aspergillus
					Penicillium
					Talaromyces
		Sordariomycetes	Diaporthales	Valsaceae	Cryphonectria/ Endothia
			Hypocreales	Clavicipitaceae	Tolypocladium

 

The above table show the Phylum Ascomycota, which has widely used in the pharmaceutical industries for metabolites and enzyme production. The Genus listed in the table need to be screened for the bio-reductions studies for inventing novel bio-reduction processes using wild microbes with high pharmaceutical values.

 

CONCLUSION:

Microbial bio-reduction processes will a have significant impact in the near future. Foresaid two examples are likely enough to understand and conclude the capabilities of Fermentative bio-reduction process especially by using filamentous fungi for the asymmetric synthesis of chiral alcohols for API production. Process development and optimization of Fermentation process followed by bio-reduction requires more understanding. Correct control over the fermentation process and bio-reductions will provide high titer and high enantiomeric excess. Extensive screening required from vast Microbial flora from mother nature to overcome hazardous chemical processes involved.

 

ACKNOWLEDGEMENT:

The authors want to express their gratitude to honorable Chancellor, Dr. G. Viswanathan, Dr. Sekar Viswanathan, Mr. Sankar Viswanathan, and Mr. G.V. Selvam of VIT University for their constant encouragement and laboratory facilities from VIT University, Vellore, India to carry out this valuable work.

 

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Accepted on 27.05.2017        © RJPT All right reserved