Steroid Biotransformation

Biotransformation (regiospecific and stereospecific bioconversion) is a biological process whereby an organic compound is modified into reversible product. These involve simple, chemically defined reactions catalyzed by enzymes present in the cell or if microorganisms are employed, it is microbial transformation.

Bioconversion has many advantages like:

•      Substrate specificity: Only one specific reaction step is normally catalyzed by an enzyme.

•  Site specificity (regiospecificity): Several functional groups of one type are present in the molecule - only one specific functional group at a specific position affected. Possible to obtain conversions at centers that are chemically unreactive.

•    Stereoselectivity: In a racemic mixture, only one specific enantiomer is converted; obtain specific conformation.

Bioconversion preferred to chemical synthesis due to

ü  Mild Reaction conditions: 

• No destruction of sensitive substrates due to the mild conditions of conversion
• less environmental hazard, as reactions occur chiefly in water.
• at ambient temperature (20^o -40^oC) and pressure normal in aqueous media.

ü  Several reactions can be combined, either in one fermentation step using an organism with suitable enzyme systems, or by step-wise conversions using different microorganisms.

ü  The number of process reaction steps are much less, usually.

ü  Mostly high yields

Disadvantages

• Specific organism required; selection of organism is a laborious job.
• Some times, process is not economical.
• Tedious/Tiresome
• The substrate concentration added bound by certain limits.
• Chemical reactions are easier to handle; less complicated equipment used.

When the transformation of the organic compounds is carried out by microorganism then the process is called as microbial transformation.

Naturally occurring steroids possess remarkable hormonal properties which are of therapeutic importance to human well-being, such as hormones of adrenal cortex (cortisone, cortisol, corticosterone), the progestational hormone (progesterone), the androgens or male sex hormones (testosterone, dihydrotestosterone) and the estrogens or female sex hormones (estradiol, estrone, etc.)

Cortisol

The pharmaceutical industry has great interest in the biotransformation of steroids for the production of steroid hormones. Steroid hormones and their derivatives have been used for a wide range of therapeutic purposes as immunosuppressive, anti-inflammatory, anti-rheumatic, progestational, diuretic, sedative, anabolic and contraceptive agents. Recent applications of steroid compounds include the treatment of some forms of cancer, osteoporosis, HIV infections and treatment of declared AIDS. Nowadays steroids represent one of the largest sectors in pharmaceutical industry.

TYPES OF STEROIDAL TRANSFORMATION

• Oxidation – Hydroxylation – Dehydrogenation. – Epoxidations – Oxidation to ketone through hydroxylation – Ring A Aromatization – Degradation of steroid nucleus– Oxidation of alcohols to ketone: 3β-OH to 3-keto – Side chain cleavage of steroids – Decarboxylation of acids • Reduction – Double bond – aldehyde and ketone to alcohol • Hydrolysis • Isomerization • Resolution of racemic mixture • Other reactions – Aminations – Enolization of carbonyl compounds – Esterification.

Commercially Important Transformations

1. Hydroxylation Certain microorganisms can introduce hydroxyl groups at any of several of the carbon atoms of the steroid molecule. Hydroxylation is the substitution of hydrogen in a particular position in the steroid molecule by hydroxyl group. 

Fungi are the most active hydroxylating microorganisms, but some bacteria particularly the Bacilli, Nocardia and Streptomyces show fair good activity. 

The hydroxylation at the 11-position of progesterone was one of the first hydroxylation described. Rhizopus nigricans can perform this

a. Steroid Nucleus   b. Progesterone to 11α- progesterone

Hydroxylation reactions are important because of higher efficacy of such compounds  and certain hydroxylations are difficult or even impossible to achieve via synthetic chemistry. 

 3. Dehydrogenation   Dehydrogenation with the simultaneous introduction of a double bond  in Ring A of the steroid nucleus is a reaction of commercial importance. Thus Cortisone converted to Prednisone & Cortisol  converted to Prednisolone. These compounds have increased anti inflammatory effect

In 1955, Charney and co-worker observed that they could greatly enhance the anti-inflammatory properties of cortisol by causing the compound to be dehydrogenated at 1^st position by Corynebacterium simplex. The resultant product, prednisolone, was 3-5 times more active than the parent compound and produced fewer side effects.

•      Dehydrogenation of cortisol at 1^st position by Corynebacterium simplex  yields Prednisolone - (3-5 times more active than the parent compound and had fewer side effects)

4. Ring A Aromatization  The microbial aromatization of suitable steroid substrates can lead to ring A aromatic compounds, particularly the estrogens which constitutes an important ingredient in oral contraceptive drugs and play important role in replacement therapy for menopause treatment

 Cell free extracts of Pseudomonas testosteroni could transform 19-nor-testosterone into estrone with small quantities of estradiol-17β.

Reaction done with Cell free extracts of Pseudomonas testosteroni

 Thousands of modified steroids produced by a combination of chemical and microbial reactions

An economically important transformation reaction is the Combined synthesis of cortisone and its 1-dehydroderivatives from diosgenin via Reichstein’s Substance S

                                

             Steroid transformations

• Batch fermentation usually due to limited concentration of substrate which can be used
• Steroid substrates not water soluble, so use of appropriate solvent systems - organic solvents
• Now, use of Immobilized cells/enzymes are carried out which help in reduced risk of contamination, simplified product recovery, shorter conversion times, use of increased substrate concentrations
• Organic solvents may be  toxic to microorganisms so use of aqueous two phase system eg., 1-dehydrogenation of cortisol to prednisolone carried out by cells of Arthrobacter simplex in a system consisiting of 25% (w/v) polyethylene glycol (PEG 8000) and 6% (w/v) dextran T 40

        The aqueous phase containing the cell material or the enzyme is overlayed with the water immiscible fluid phase in which the substrate has been dissolved. The substrate passes slowly into the aqueous phase and as the transformation reaction proceeds, the product passes back into the solvent phase. In some cases, the actual transformation occurs at the interface of the aqueous and solvent phases