STRUCTURE Side chain varies depending on carboxylic acid present in fermentation medium

Shape of Penicillin G Folded envelope shape

Penicillin inhibits final cross-linking stage of cell wall synthesis

It reacts with the transpeptidase enzyme to form an irreversible covalent bond

Inhibition of transpeptidase leads to a weakened cell wall

Cells swell due to water entering the cell, then burst (lysis)

Penicillin possibly acts as an analogue of the L-Ala-g-D-Glu portion of the pentapeptide chain.

Mechanism of action - bacterial cell wall synthesis

Mechanism of action - bacterial cell wall synthesis

Mechanism of action - bacterial cell wall synthesis

Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell wallThe b-lactam ring is involved in the mechanism of inhibitionPenicillin becomes covalently linked to the enzymes active site leading to irreversible inhibitionMechanism of action

Characteristics of secondary metabolitesSecondary metabolites (idiolites) are produced from substrates provided by primary metabolism.

They are not essential for growth and reproduction.

Their formation is extremely dependent on growth conditions.

It is possible to get dramatic overproduction of secondary metabolites.

ProductionAll commercially available -lactams are initially produced through the fermentation of bacteria.

Bacteria assemble the penicillin molecule mainly using L-amino adipic acid, L-valine, and L-cysteine in three steps in the presence of specific enzymes.

Modern recombinant genetic techniques have allowed the over expression of the genes which code for these three enzymes, allowing much greater yields of penicillin than in the past.

Penicillin Bio-synthetic Pathway

COMMERCIAL PRODUCTION OF PENICILLIN Originally used Penicillium notatum , now use Penicillium chrysogenum.

Initially produced via surface mat culture. Now fermentors used

The bicyclic nucleus of 6-APA is synthesized by the fungus using amino acids in enzyme catalyzed process

The side chains (for different Penicillins) are added intact from the media constituents

The basic starting material 6-APA can be utilized for synthesizing structural analogues of Fermentor-Derived Penicillins

Problems! Inefficient, slow penicillin synthesis and contamination.

DEEP LIQUID CULTURE

Inoculum prepared until it represents ~ 5-10% of the volume of the fermentor. About 3-5 tons of wet mycelial mass will be used to inoculate a 50,000 litre fermentor.

Production phases:1.Trophophase: Rapid mycelial growth (30-40 hrs)Idiophase: Penicillin production via fed batch fermentation (5-7 days).

3. Carbon and nitrogen sources are depleted, antibiotic production ceases, the mycelia lyse releasing ammonia and the pH rises.

Fermentor cooled by internal coils or external jackets (25-27oC).

The pH is maintained between 6.8-7.4 by the automatic addition of H2SO4 or NaOH as necessary.

Oxygen added and mixed with mycelium.

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CULTURE MEDIUMComposition of early media%Corn steep liquor (cotton seeds, peanut, Linseed or soybean meals)2-4Lactose, glucose or beet molasses2-4CaCO3 or phosphates (buffer)0.5-1Precursor0.1-0.5

Precursors of the appropriate side-chain are added to the fermentation. Thus if benzyl penicillin (penicillin G) is desired, phenylacetic acid is added (added continuously to avoid inhibition of fungus growth). High yielding strains of P. chrysogenum resistant to the precursors have therefore been developed.

Extraction of penicillin after fermentationThe broth is transferred to a settling tank. Penicillin is highly reactive and is easily destroyed by alkali conditions (pH 7.5-8.0) or by enzymes. So cooled rapidly to 5-10C. The separation of penicillin is based on the solubility, adsorption and ionic properties of penicillin. Since penicillins are monobasic carboxylic acids they are easily separated by solvent extraction.The fermentation broth is filtered with a rotary vacuum filter to remove mycelia and other solids and the resulting broth is adjusted to about pH 2 using a mineral acid. It is then extracted with a smaller volume of an organic solvent such as amyl acetate or butyl acetate, keeping it at this very low pH for as short a time as possible. The aqueous phase is separated from the organic solvent usually by centrifugation.

The organic solvent containing the penicillin is then typically passed through charcoal to remove impurities, after which it is back extracted with a 2% phosphate buffer at pH 7.5. The penicillin is then acidified once again with mineral acid (phosphoric acid) and the penicillin is again extracted into an organic solvent (e.g. amyl acetate). The product is transferred into smaller and smaller volumes, the penicillin becomes concentrated several times over, up to 80-100 times. The penicillin may be converted to a stable salt form in one of several ways which employ the fact that penicillin is an acid:

(a) it can be reacted with a calcium carbonate slurry to give the calcium salt which may be filtered, lyophilized or spray dried. (b) it may be reacted with sodium or potassium buffers to give the salts of these metals which can also be freeze or spray dried; (c) it may be precipitated with an organic base such as triethylamine.

NATURAL PENICILLINSThey are destroyed by acid in the stomach.

Sensitive to the enzyme penicillinase

Effective against Gram +ve bacteria only.

Semi-Synthetic PenicillinsThe acyl side chain of the penicillin molecule can be cleaved using enzyme or chemical methods to produce 6-APA, which can further be used to produce semi-synthetic penicillins or cephalosporins

75% of the penicillin produced is modified in this manner.

6-Aminopenicillanic Acid (6-APA)

Raw material for production of new semisynthetic penicillins for Fewer side effects Diminished toxicity Greater selectivity against pathogens Broader antimicrobial range including G- -ve Improved pharmacological properties Gastric acid stability & oral absorbability Resistance to beta-lactamases

6-Aminopenicillanic Acid (6-APA)

Chemical method: Use of hazardous chemicals - pyridine, phosphorous pentachloride, nitrosyl chloride Enzymatic method: Mild reaction conditions (pH 7.5, 37 oC) Enzymatatic process is cheaper by 10%

Enzymes: Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium, Streptomyces lavendulae Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium, Pseudomonas acidovorans

SEMISYNTHETIC PENICILLINS

Penicillin G6 - APASide Chain ModificationAmoxycillinAUGMENTINClavulanic acidPenicillinase (E.coli)b-lactamase resistant

Penicillins- NaturalNatural penicillins are those which can be obtained directly from the penicillium mold and do not require further modification. Many species of bacteria are now resistant to these penicillins.Penicillin G

not orally active

Penicillin G in Acidic ConditionsPenicillin G could not be administered orally due to the acidic conditions of the stomach.

Penicillin VPenicillin V is produced when phenoxyacetic acid rather than phenylacetic acid is introduced to the penicillium culture. Adding the oxygen decreases the nucleophilicity of the carbonyl group, making penicillin V acid stable and orally viable.

-Lactamase Resistant PenicillinsPenicillins which have bulky side groups can block the -Lactamases which hydrolyze the lactam ring. These lactamases are prevalent in S. aureus and S. epidermidis, and render them resistant to Penicillin G and V. This necessitated the development of semi-synthetic penicillins through rational drug design.Methicillin was the first penicillin developed with this type of modification, and since then all bacteria which are resistant to any type of penicillin are designated as methicillin resistant (MRSA- methicillin-resistant S. aureus)Methicillin is acid sensitive and has been improved upon by adding electron withdrawing groups, as was done in penicillin V, resulting in drugs such as oxacillin and nafcillin. Due to the bulky side group, all of the anti-staphylococcal drugs have difficulty penetrating the cell membrane and are less effective than other penicillins.

Broad spectrum Penicillins- AminopenicillinsIn order to increase the range of activity, the penicillin has been modified to have more hydrophilic groups, allowing the drug to penetrate into Gram (-) bacteria via the porins. These penicillins have a wider range of activity than natural or anti-staphylococcal drugs, but without the bulky side groups are once again susceptible to attack by -lactamasesThe additional hydrophilic groups make penetration of the gut wall difficult, and can lead to infections of the intestinal tract by H. pylori

Ampicillin R=Ph, Amoxicillin R= Ph-OH

Penicillins- Extended SpectrumExtended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amineLike the aminopenicillins the extended spectrum drugs have an increased activity against Gram (-) bacteria by way of the import porins. These drugs also have difficulty penetrating the gut wall and must be administered intravenously if not available as a prodrug.These are more effective than the aminopenicillins and not as susceptible to -lactamases

SAR of PenicillinsPosition 1 When the sulfur atom of thethiazolidinering is oxidized to asulfoneor sulfoxide, it improves acid stability, but decreases the activity of the agent.Position 2 No substitutions allow at this position, any change will lower activity. The methyl groups are necessary.Position 3 The carboxylic acid of thethiazolidineis required for activity. If it is changed to an alcohol or ester, activity is decreased.Position 4 The nitrogen is a must for antibacterial activity.Position 5 No substitutions allowed.Position 7 The carbonyl on theb-lactamring is a must.

Postion6 Substitutions are allowed on the side chain of the amide.An electron withdrawing group added at this position will give the compound better acid stability because this substitution will make the amide oxygen lessnucleophillic.A bulky group added close to the ring will make the compound more resistant tob-lactamases.Sterichindranceprovides protection to the-lactam ring.The spectrum of activity becomes broader when a polar group is added to this position allowing the compound to pass through theporinsof the Gram negative bacteria cell wall.These structural activity relationships have helped scientist design more effectivepenicillinsto combat many different prokaryotes.

SAR of Penicillins

CephalosporinsCephalosporins were discovered shortly after penicillin entered into widespread product, but not developed till the 1960s.

Cephalosporins are similar to penicillins but have a 6 member dihydrothiazine ring instead of a 5 member thiazolidine ring.

7-aminocephalosporanic acid (7-ACA) can be obtained from bacteria, but it is easier to expand the ring system of 7-APA because it is so widely produced.

CephalosporinsUnlike penicillin, cephalosporins have two side chains which can be easily modified. Cephalosporins are also more difficult for -lactamases to hydrolyze.

Mechanism of CephalosporinsThe acetoxy group (or other R group) will leave when the drug acylates the PBP.

Cephalosporins- ClassificationCephalosporins are classified into four generations based on their activity. Later generations generally become more effective against Gram (-) bacteria due to an increasing number of polar groups (also become zwitterions.)Ceftazidime (3rd gen) in particular can cross blood brain barrier and is used to treat meningitis. Later generations are often the broadest spectrum and are reserved against penicillin resistant infections to prevent the spread of cephalosporin resistant bacteria.

Cephlasporins

Cephalosporins are the second major group of -lactam antibiotics to be discovered.

Cephalosporins contain a b-lactam ring, like the penicillins. The main difference being that while the latter contain a 5-membered thiazolidene ring, cephalosporins have a 6-membered dihydrothiazine.

Their mode of action against bacteria is essential the same as that of penicillins.These molecules impede the transpeptidase enzymes, causing bacteria to constructfaulty cell walls which rupture, causing death.

the structural moieties of cephalosporins that are critical forantimicrobial activity. They include the b-lactam ring, the dihydrothiazine ring containing a C3-C4 double bond, the presence of a C4-carboxyl and a C7-amino side chain. The only features that can be varied are the 7-acylamino side chain, the 3- acetoxymethyl side chain, and additional substitution on C7.

First-generation cephalosporinsThe names and structures of three first generation cephalosporins are shown in Fig21 above. In general, these drugs have a lower antibiotic activity than penicillins, buta greater range. While their best activity is against Gram-positive bacteria, they alsodisplay better activities against Gram-negative infections. They show poorerbioavailability than penicillins and are generally administered intravenously.Cephalothin can be deactivated by host esterases which cleave the acetyl group atC3, leaving an unreactive primary alcohol. Replacing the ester with an esteraseresistant pyridinium group gives cephaloridine. The pyridine is an excellent leavinggroup, but as it contains a positive charge on the nitrogen (making the molecule azwitterion) it is very poorly absorbed through the GI tract, and must therefore beinjected.Th