Beta-Lactam Antibiotics

β-Lactam Antibiotics
 Introduction

β-Lactam antibiotics are the most widely produced

and used antibacterial drugs in the world, and have
been ever since their initial clinical trials in 1941.

β-Lactams are divided into several classes based on

their structure and function; and are often named by
their origin, but all classes have a common β-Lactam
ring structure.
 History

1928- Alexander Fleming discovers a mold which

inhibits the growth of staphylococcus bacteria
1940- penicillin is isolated and tested on mice by
researchers at Oxford
1941- penicillin mass produced by fermentation for use
by US soldiers in WWII
1950’s- 6-APA is discovered and semi-synthetic
penicillins are developed.
1960’s to today- novel β-lactams/ β-lactamase
inhibitors are discovered and modified from the
natural products of bacteria
 Target- Cell Wall Synthesis

 The bacterial cell wall is a cross linked polymer

called peptidoglycan which allows a bacteria to
maintain its shape despite the internal pressure
caused by osmotic pressure differences.
 If the peptidoglycan fails to crosslink the cell wall
will lose its strength which results in cell lysis.
 All β-lactams disrupt the synthesis of the bacterial
cell wall by interfering with the transpeptidase which
catalyzes the cross linking process.
 Peptidoglycan

 Peptidoglycan is a carbohydrate composed of

alternating units of NAMA and NAGA.
 The NAMA units have a peptide side chain which can
be cross linked from the L-Lys residue to the
terminal D-Ala-D-Ala link on a neighboring NAMA
unit.

 This is done directly in Gram (-) bacteria and via a

pentaglycine bridge on the L-lysine residue in Gram
(+) bacteria.
Mechanism
 Transpeptidase- PBP

 The cross linking reaction is catalyzed by a class of

transpeptidases known as penicillin binding proteins

 A critical part of the process is the recognition of the

D-Ala-D-Ala sequence of the NAMA peptide side
chain by the PBP. Interfering with this recognition
disrupts the cell wall synthesis.
 β-lactams mimic the structure of the D-Ala-D-Ala
link and bind to the active site of PBPs, disrupting
the cross-linking process.
 Mechanism of β-Lactam Drugs

 The amide of the β-lactam ring is unusually reactive

due to ring strain and a conformational arrangement
which does not allow the lone pair of the nitrogen to
interact with the double bond of the carbonyl.
 β-Lactams acylate the hydroxyl group on the serine
residue of PBP active site in an irreversible manner.
 This reaction is further aided by the oxyanion hole,
which stabilizes the tetrahedral intermediate and
thereby reduces the transition state energy.
 Mechanism of β-Lactam Drugs

The hydroxyl attacks the amide and forms a

tetrahedral intermediate.
 Mechanism of β-Lactam Drugs

The tetrahedral intermediate collapses, the amide

bond is broken, and the nitrogen is reduced.
 Mechanism of β-Lactam Drugs

The PBP is now covalently bound by the drug and

cannot perform the cross linking action.
 Bacterial Resistance

 Bacteria have many methods with which to combat

the effects of β-lactam type drugs.

 Intrinsic defenses such as efflux pumps can remove

the β-lactams from the cell. β-Lactamases are
enzymes which hydrolyze the amide bond of the β-
lactam ring, rendering the drug useless.

 Bacteria may acquire resistance through mutation at

the genes which control production of PBPs, altering
the active site and binding affinity for the β-lactam .
 Range of Activity

 β-Lactams can easily penetrate Gram (+) bacteria,

but the outer cell membrane of Gram (-) bacteria
prevents diffusion of the drug. β-Lactams can be
modified to make use of import porins in the cell
membrane.
 β-Lactams also have difficulty penetrating human
cell membranes, making them ineffective against
atypical bacteria which inhabit human cells.
 Any bacteria which lack peptidoglycan in their cell
wall will not be affected by β-lactams.
 Toxicity

 β-Lactams target PBPs exclusively, and because

human cell membranes do not have this type of
protein, β-lactams are relatively non toxic compared
to other drugs which target common structures such
as ribosomes.

 About 10% of the population is allergic (sometimes

severely) to some penicillin type β-lactams.
 Classes of β-Lactams

The classes of β-lactams are distinguished by the

variation in the ring adjoining the β-lactam ring and
the side chain at the α position.

Penicillin
 Modification of β-Lactams

β-Lactam type antibiotics can be modified at various

positions to improve their ability to:

-be administered orally (survive acidic conditions)

-be tolerated by the patient (allergies)
-penetrate the outer membrane of Gram (-) bacteria
-prevent hydrolysis by β-lactamases
-acylate the PBPs of resistant species (there are many
different PBPs)
 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.
 Production

 All commercially available β-lactams are initially

produced through the fermentation of bacteria.
 Bacteria assemble the penicillin molecule from L-
AAA, L-valine, and L-cysteine in three steps using
ACV synthase, IPN synthase, and acyltransferase.
 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 Biosynthetic Pathway
o
 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.
 Penicillins- Antistaphylococcal

 Penicillins which have bulky side groups can block

the β-Lactamases which hydrolyze the lactam ring.
 Penicillins- Antistaphylococcal

 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)
 Penicillins- Antistaphylococcal

 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

antistaphylococcal drugs have difficulty penetrating
the cell membrane and are less effective than other
penicillins.
 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.

 Ampicillin R=Ph

 Amoxicillin R= Ph-OH
 Penicillins- Aminopenicillins

 These penicillins have a wider range of activity than

natural or antistaphylococcal drugs, but without the
bulky side groups are once again susceptible to
attack by β-lactamases

 The additional hydrophilic groups make penetration

of the gut wall difficult, and can lead to infections of
the intestinal tract by H. pylori
 Penicillins- Aminopenicillins

 Due to the effectiveness of the aminopenicillins, a

second modification is made to the drug at the
carboxyl group.
 Changing the carboxyl group to an ester allows the
drug to penetrate the gut wall where it is later
hydrolyzed into the more polar active form by
esterase enzymes.
 This has greatly expanded the oral availability of the
aminopenicillin class.
 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
 Penicillins- Extended Spectrum

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 β-lactamases
 Cephalosporins

Cephalosporins were discovered shortly after penicillin

entered into widespread product, but not developed
till the 1960’s.

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 β-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 (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.
 Carbapenems

Carbapenems are a potent class of β-lactams which

attack a wide range of PBPs, have low toxicity, and
are much more resistant to β-lactamases than the
penicillins or cephalosporins.
 Carbapenems

Thienamycin, discovered by Merck in the late 1970’s, is

one of the most broad spectrum antibiotics ever
discovered.
It uses import porins unavailable to other β-lactams to
enter Gram (-) bacteria.
Due to its highly unstable nature this drug and its
derivatives are created through synthesis, not
bacterial fermentation.
 Carbapenems

Thienamycin was slightly modified and marked as

Imipenem. Due to its rapid degradation by renal
peptidase it is administered with an inhibitor called
cilastatin under the name Primaxin. Imipenem may
cause seizures or sever allergic reactions.
Other modifications of Thienamycin have produced
superior carbapenems called Meropenem and
Ertapenem, which are not as easily degraded by
renal peptidase and do not have the side effects of
Imipenem.
 Monobactams

The only clinically useful monobactam is aztreonam.

While it resembles the other β-lactam antibiotics and
targets the PBP of bacteria, its mechanism of action
is significantly different.

It is highly effective in treating Gram (-) bacteria and is

resistant to many β-lactamases
 β-Lactamases

β-Lactamases were first discovered in 1940 and

originally named penicillinases.
These enzymes hydrolyze the β-lactam ring,
deactivating the drug, but are not covalently bound
to the drug as PBPs are.
Especially prevalent in Gram (-) bacteria.
Three classes (A,C,D) catalyze the reaction using a
serine residue, the B class of metallo- β-lactamases
catalyze the reaction using zinc.
 β-Lactamase Inhibitors

There are currently three clinically available β-

lactamase inhibitors which are combined with β-
lactams; all are produced through fermentation.
These molecules bind irreversibly to β-lactamases but
do not have good activity against PBPs. The rings are
modified to break open after acylating the enzyme.
 β-Lactam/Inhibitor combinations

Aminopenicillins:
ampicillin-sulbactam = Unasyn
amoxicillin-clavulante = Augmentin

Extended-Spectrum Penicillins
piperacillin-tazobactam = Zosyn
ticarcillin-clavulanate = Timentin
 Summary
β-Lactam antibiotics have dominated the clinical
market since their introduction in the 1940’s and
today consist of nearly ¾ of the market.
Development of natural products such as penicillin G
into more potent forms through rational
modification has increased the range of activity of
these drugs, although this has led to some toxicity
problems.
Widespread use of β-lactams has led to the
development of resistant strains, new modifications
are necessary in order for β-lactams to remain viable.
Assigned reading:

Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition.

New York: Oxford University Press, 2009. 388-414. Print.
 Optional References/ Reading

Brunton, Laurence L. et al. Goodman and Gillman’s Pharmaceutical Basis

of Therapeutics 11th Edition. McGraw-Hill, 2006 1134- 52. Print.
Bush, Karen. β-Lactamase Inhibitors from Laboratory to Clinic. Clinical
Microbiology Reviews, Jan. 1988, p. 109-123. Web.
Elander, R.P. Industrial production of β-Lactam antibiotics. Journal of
Applied Microbiology and Biotechnology (2003) 61:385–392. Web.
Hauser, Alan R. Antibiotic Basics for Clinicians: Choosing the Right
Antibacterial Agent. Philadelphia: Lippincott, 2007. 18-46. Print.
Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition.
New York: Oxford University Press, 2009. 388-420. Print.
Rolinson, George N. Forty years of β-lactam research. Journal of
Antimicrobial Chemotherapy (1998) 41, 589–603. Web.
 Questions

1. What are two ways by which a bacteria could

become resistant to carbapenems?
2. How were the natural penicillins modified to be
orally available?
3. How are extended spectrum penicillins modified to
be orally available?
4. What are two ways that the β-lactam can be
protected from β-lactamases?