STRUCTURE

Side chain varies depending on carboxylic acid present in fermentation medium

present in corn steep liquor
Penicillin G
CH
2
CO
2
H
Benzyl penicillin (Pen G)
R =
Phenoxymethyl penicillin (Pen V)
R =
CH
2
O CH
2
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
Penicillin V
(first orally active penicillin)
OCH
2
CO
2
H
Acyl side
chain
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
6-Aminopenicillanic acid
(6-APA)
Acyl side
chain
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
6-Aminopenicillanic acid
(6-APA)
Acyl side
chain
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
Thiazolidine
ring
6-Aminopenicillanic acid
(6-APA)
Acyl side
chain
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
Thiazolidine
ring
b-Lactam
ring
Shape of Penicillin G
Folded envelope shape
H
N
O
NH
C
O
R
H
S
CO
2
H
H
Me
Me
..
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
D-Alanine
TRANSPEPTIDASE
PENICILLIN
SUGAR
BACKBONE
NAM
L-Ala
NAG
D-Glu
L-Lys
D-Ala
D-Ala
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly L-Lys
NAG
D-Ala
D-Ala
D-Glu
L-Ala
NAM
SUGAR
BACKBONE
L-Lys Gly Gly
D-Ala
NAM
L-Ala
NAG
D-Glu
L-Lys Gly Gly Gly Gly Gly Gly Gly Gly
NAG
D-Ala
D-Glu
L-Ala
NAM
Cross linking
Mechanism of action - bacterial cell wall synthesis
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
L-Ala
D-Glu
L-Lys
Mechanism of action - bacterial cell wall synthesis
NAM NAM NAM NAG NAG
NAM NAM NAM NAG NAG
NAM NAM NAM NAG NAG
Bond formation
inhibited by
penicillin
Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in
the synthesis of the bacterial cell wall
The b-lactam ring is involved in the mechanism of inhibition
Penicillin becomes covalently linked to the enzymes active site leading to irreversible
inhibition
Covalent bond formed
to transpeptidase enzyme
Irreversible inhibition
N
S
Me
Me
H
N
H H
CO
2
H
O
C
O
R
Nu
Enz
C
H
N
C
CO
2
H
H H
Me
Me
S
HN
O
R
O
Nu-Enz
-H
N
S
Me
Me
H
N
H H
CO
2
H
O
C
H
Enz-Nu
O
R
Mechanism of action
Characteristics of secondary metabolites
Secondary 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.



Production
All 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)

2. 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 (25-27
o
C).

The pH is maintained between 6.8-7.4 by the automatic addition of H
2
SO
4

or NaOH as necessary.

Oxygen added and mixed with mycelium.
o
CULTURE MEDIUM

Composition of early media %
Corn steep liquor (cotton seeds, peanut, Linseed or soybean meals) 2-4
Lactose, glucose or beet molasses 2-4
CaCO
3
or phosphates (buffer) 0.5-1
Precursor 0.1-0.5


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.
CH
2
COOH
phenyl acetic acid
O CH
2
COOH
phenoxy acetic acid
- obtained from corn steep liquor
(Penicillin G)
(Penicillin V)
- first oral penicillin
Extraction of penicillin after fermentation
The 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 PENICILLINS
(i) They are destroyed by acid in the stomach.

(ii) Sensitive to the enzyme penicillinase

(iii) Effective against Gram +ve bacteria only.
Semi-Synthetic Penicillins
The 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
o
C)
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 G
6 - APA
Side Chain Modification
Amoxycillin AUGMENTIN
Clavulanic acid
Penicillinase (E.coli)
b-lactamase
resistant
Penicillins- Natural
Natural 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 Conditions
Penicillin G could not be administered orally due
to the acidic conditions of the stomach.
Penicillin V
Penicillin 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 Penicillins
Penicillins 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- Aminopenicillins
In 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 -lactamases
The 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 Spectrum
Extended spectrum penicillins are similar to the aminopenicillins in structure but
have either a carboxyl group or urea group instead of the amine
Like 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 Penicillins
Position 1 When the sulfur atom of the thiazolidine ring is oxidized to
a sulfone or 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 the thiazolidine is 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 the b-lactam ring is a must.




Postion 6 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 less nucleophillic.

A bulky group added close to the ring will make the compound more
resistant to b-lactamases. Steric hindrance provides 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 the porins of the Gram
negative bacteria cell wall.

These structural activity relationships have helped scientist design more
effective penicillins to combat many different prokaryotes.


SAR of Penicillins
Cephalosporins
Cephalosporins 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.



Cephalosporins
Unlike penicillin, cephalosporins have two side
chains which can be easily modified.
Cephalosporins are also more difficult for -
lactamases to hydrolyze.
Mechanism of Cephalosporins
The acetoxy group (or other R group) will leave
when the drug acylates the PBP.
Cephalosporins- Classification
Cephalosporins 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 (3
rd
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 construct
faulty cell walls which rupture, causing death.









the structural moieties of cephalosporins that are critical for
antimicrobial 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 cephalosporins
The names and structures of three first
generation cephalosporins are shown in
Fig
21 above. In general, these drugs have a
lower antibiotic activity than penicillins, but
a greater range. While their best activity is
against Gram-positive bacteria, they also
display better activities against Gram-
negative infections. They show poorer
bioavailability than penicillins and are
generally administered intravenously.
Cephalothin can be deactivated by host
esterases which cleave the acetyl group at
C3, leaving an unreactive primary alcohol.
Replacing the ester with an esterase
resistant pyridinium group gives
cephaloridine. The pyridine is an excellent
leaving
group, but as it contains a positive charge
on the nitrogen (making the molecule a
zwitterion) it is very poorly absorbed
through the GI tract, and must therefore be
injected.
Th