Mirjam Nilsson​

PART 5
 1. Cell Wall Biosynthesis

. .. ... .. ... ... ..

Growing cell wall
Vancomycin

.
b-Lactams Crosslinking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Bacitracin Carrier
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala-D-Ala

Amino acid
Cycloserine

•Building block partially constructed in cytoplasm

•Transported across cell membrane and completed
•Constructed from 2 sugars (NAM, NAG) and a peptide chain
•Linked to growing cell wall by enzyme (transglycosidation)
•Final crosslinking reaction catalysed by transpeptidase enzymes
 2. D-Cycloserine

. .. ... .. ... ... ..

Growing cell wall
Vancomycin
b-Lactams
.
Crosslinking

... ... ... ... ... ... ... ... ...

Transglycosidation
.
Gly NAG

Bacitracin Carrier
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala-D-Ala

Amino acid
Cycloserine

•Natural product produced by Streptomyces garyphalus

•Inhibits L-alanine racemase and D-Ala-D-Ala ligase
•Blocks biosynthesis of D-Ala-D-Ala
•Mimics the structure of D-Ala
2. D-Cycloserine

H HO
N
O O
O Me
H
H
N H2
NH 2

D- C y c D- l A o l s a e n r i i n n e e

D-Cycloserine D-Alanine
3. Bacitracin

. .. ... .. ... ... ..

Growing cell wall
Vancomycin
b-Lactams
.
Crosslinking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Carrier
Bacitracin
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala-D-Ala

Amino acid
Cycloserine

•Polypeptide produced by Bacillus subtilis

•Binds to the carrier lipid
•Prevents the carrier lipid from transporting the NAM -
pentapeptide building block across the cell membrane
4. Vancomycin and vancomycin analogues

. .. ... .. ... ... ..

Growing cell wall

.
Vancomycin
b-Lactams Crosslinking

... ... ... ... ... ... ... ... Transglycosidation

... .

Gly NAG

Carrier
Bacitracin
lipid
Cell
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala-D-Ala

Amino acid
Cycloserine

•Narrow spectrum bactericidal glycopeptide

•Produced by Streptomyces orientalis
•Blocks transglycosidation
4. Vancomycin and vancomycin analogues
HO CH2OH
Me
H3N
HO O
HO
Vancomycin
O
O O Cl
Me O O

HO OH H3C CH3
Cl
O O
H H H H O
O N O
N N N
H H H Peptide chain
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH

•Important antibacterial agent

•Caps the building block used in the synthesis of the bacterial cell wall
•Contains a peptide chain which forms hydrogen bonds to the target
•Vancomycin acts as a receptor for the building block
4. Vancomycin and vancomycin analogues
Biosynthesis of vancomycin
Glycosidations

Chlorination Chlorination

H OH H
Hydroxylation
O O Tyr Hydroxylation
Tyr

O O O
H H H
HO2C N N N NHMe
N N N
H H H
O O O
H2NOC
Asn Val

HO OH
OH

Oxidative couplings

•Derived from a flexible hexapeptide

•Cyclisations result in a rigid structure
•Peptide backbone is held in a fixed conformation
4. Vancomycin and vancomycin analogues
Mechanism of Vancomycin
inhibition
Building
block

Cell membrane

Notes
•Vancomycin provides binding pocket for the biosynthetic building block
•Vancomycin binds to the tail of the building block’s peptide chain
•Caps the building block
•Disguises the building block from the transglycosidation enzyme
4. Vancomycin and vancomycin analogues
Mechanism of
inhibition
.. ..
. .. Vancomycin
dimer

Growing cell wall

.. . . Building
block

•Dimerisation occurs
•Dimer is highly stable
•Large vancomycin molecule acts as a steric shield
4. Vancomycin and vancomycin analogues
Mechanism of HO CH2OH

inhibition H N 3
Me
HO O
HO

O
O O Cl
Me O O

HO OH H3C CH3
Cl
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH

H-bonding interactions between

the peptide backbone of
O H O
H Me vancomycin and the biosynthetic
N building block
Cell wall building block N O
H
Me H
O
L-Lys-D-Ala-D-Ala ‘tail’
4. Vancomycin and vancomycin analogues
Binding interactions H Me O
in dimer O
N
H
N
Cell wall building block D-Ala-D-Ala-L-Lys- tail

Me H
O H O
R7

H R3 H
O R5 O2C N
NH2Me H N H
N N N
Heptapeptide R O O N O
1
backbone H
O R2 R4 H O R6

R6 O H R4 R2 O
H O O
O N R1
N N N Heptapeptide
backbone
R5 O H N H NH2Me
N CO2
H R3 H

R7

O H O
H Me
N
Cell N O L-Lys-D-Ala-D-Ala tail
Cellwall
wallbuilding
buildingblock
block H
O Me H
 4. Vancomycin and vancomycin analogues
Drug resistance
•Vancomycin-resistant Staphylococcus aureus (VRSA) (1996)
•Vancomycin-resistant enterococci (VRE) (1989)
•Resistance due to mutation in pentapeptide chain of cell wall building block
•Terminal D-alanine replaced by D-lactate

O H Me O O H Me O
H Mutation
N O
Cell wall building block N O Cell wall building block N O
H H
O Me H O Me H
L-Lys-D-Ala-D-Ala tail L-Lys-D-Ala-D-Lactate tail

•Peptide link replaced by ester link

•Loss of NH (HBD)
•Weakens binding affinity of vancomycin with ‘tail’
•Lactate acts as a leaving group in cell wall
synthesis
4. Vancomycin and vancomycin analogues
Teicoplanin
HO CH2OH
O
Alkyl anchor
HO O

OH N O
H Cl
HO NHAc O O
C D E
HO O Cl OH
O
O O
H H H H O
O N O NH3
Heptapeptide backbone N N N H
H H
O H N H
N CO2
H
H
B
HO OH
A
O
HO OH
O
HO
O
OH
HO
OH
4. Vancomycin and vancomycin analogues
Teicoplanin
•Isolated from a soil micro-organism
•Does not dimerise
•Alkyl chain anchors the antibiotic to the outer surface of the cell membrane
•Less toxic than vancomycin

Teicoplanin

Building
block

Alkyl chain anchor

Cell membrane
4. Vancomycin and vancomycin analogues
Eremomycin - naturally occurring glycopeptide

H3N Me
HO HO CH2OH

Me O HO O

O
H3N Me O Cl
HO O O

O OH H3C CH3
H
Me O
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH
4. Vancomycin and vancomycin analogues
Oritavancin- analogue of eremomycin
Biphenyl hydrophobic tail

Me CH2 NH2 Me
HO HO CH2OH

Me O HO O

O
H3N Me O Cl
HO O O

O OH H3C CH3
Cl
Me O
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH

1000 x more active than vancomycin

4. Vancomycin and vancomycin analogues
Telavancin - analogue of eremomycin

HN
NH2 Me
HO HO CH2OH

Me O HO O

O
O Cl
O O

Hydrophobic tail HO OH H3C CH3

Cl
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH
O
Approved in 2009 for skin infections
N P
H OH
OH
4. Vancomycin and vancomycin analogues
Simplification
•Simplified structures capable of binding D-Ala-D-Ala or D-Ala-D-Lac
•Lead compounds for further development

OH
O

H3C CH3

O
H H O O
N
AA1 AA2 AA3 N N
N H
O H H H NH2Me
H

CONH2