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So, you want to know about
siderophore synthesis
Presented by:
Steven Backues
Brooks Maki
and
Donnie Berkholz “The Invisible Man”
Hydroxamic Acid Groups
 N-Alkylation of O-substituted Hydroxamic
acid.
 Formation of an oxime from an aldehyde and
a hydroxylamine. Followed by reduction and
acylation
 These derivatives allowed synthesis of
several siderophores and their analogues
Alternative Methods
 Oxidation of Lysine and OrnithineWith
DMD (dimethyldioxarine)
 Conversion of primary amines to imines,
with oxidation of imines to oxaziridines.
Hydrolysis leads to hydroxylamines.
 D-Ferrichrome synthesized by this method
Danoxamine
 Composed of a linear series of three
hydroxamic acids.
 Composed of two major groups linked by
succinic acid.
Biosynthesis of Siderophores
How it’s really done.
Hydroxamate Siderophores
Step 1: Ornithine N5Oxygenase
 The formation of the N-O bond is the first
committed step in hydroxamate synthesis
 Ornithine, an amino acid used in the urea
cycle, is reacted with O2 and NADPH to give
an N-O bond at the end of its side chain.
Step 2:
5N Transacylase
 The nitrogen modified in this way is
additionally attached to an acyl group carried
by coenzyme A
 This completes the hydroxamate prosthetic
group
The Hydroxamate prosthetic group:
HO
O
NH 3+
H
N
COO-
H3C
Step 3: Non-ribosomal Peptide
Synthetase
 This synthetase is a large complex with many
subdomains, including an adenylation
domain, a thiolation domain, and a
condensation domain.
Adenylation
 First, the hydroxamate is is activated by
addition of an adenylate group at its C
terminus
 The source of the adenylate group is ATP,
and the reaction occurs with production of
pyrophosphate
Thiolation
 The hydroxamate group is then transferred to
the enzyme through the formation of a
thioester linkage with displacement of the
adenylate group.
Condensation
 Finally, the hydroxamate group is attached to
another molecule (perhaps another
hydroxamate group, or else a growing chain)
by the nucleophilic attack of an OH or NH
from the chain on the S-linked carbonyl,
displacing the sulfur.
Cathechols: Vibriobactin
 Vibriobactin is a siderophore used by Vibrio
cholerae
 Its synthesis also involves a large, nonribosomal peptide synthetase, and follows
many of the same pathways as the synthesis
of hydroxamate siderophores outlined above.
Vibriobactin
HO
HO
O
N
Me
O
O
NH
Me
N
NH
N
OH
O
OH
OH
O
OH
The Cathechol Prosthetic
Group
 The cathechol prosthetic group is 2,3dihydroxybenzoic acid, which is formed
from chorismic acid
Nonribosomal Peptide
Synthetase
 2,3-dihydrobenzoic acid then acts as a
substrate for Vibirobactin Syntetase
 It is first activated by adenylation, then
transferred to the enzyme with formation of a
thioester
Transfer to Norspermidine
 This thioester complex then undergoes
nucleophilic attack by a primary amine on
norspermidine.
 The norspermidine/cathechol complex goes
on to react with two more cathechol
prosthetic groups (these, however, attached
by threonine derived linkages) to form the
final siderophore
Norspermidine+cathecol
Me
O
NH
N
O
OH
OH
H
N
NH 2
Yersiniabactin
S
S
Me Me
S
Me
N
N
H
N
OH
OH
COOH
Synthesis
 Although it has neither hydroxamate nor
cathechol groups, Yersiniabactin follows
some of the same synthesis pathways, using
a nonribosomal peptide sythetase that has
clear homologies with, for example,
vibriobactin sythetase
Beginnings
 Synthesis begins with salicylic acid (2hydroxy-benzoic acid)
 This is activated by the attachment of an
adenylate group, then loaded onto the
enzyme by the formation of a thioester, as
before
Elongation
 At the same time, two cystines are also activated
then loaded onto the same enzyme, also via a
thioester linkage
 Then, in the condensation/cyclization domain, the
salicyate group is transferred onto one of the
cystines, which is then cyclized.
 This cyclization is an unusual property of this
particular synthetaes
Completion
 A second cystine is added, and also cyclized,
and the resulting molecule undergoes the
addition of a malonyl group, Sadenosylmethionine, and an additional
cystine to complete the synthesis
Overview:
 The use of a large, multidomain nonribosomal
peptide synthetase was a common element of all of
these syntheses.
 All of these processes included the activation of a
substrate by adenylation and the transfer to a
thioester linkage with the enzyme, followed by
condensation to form a longer chain. This is similar
to the process followed in biosynthesis of fatty
acids.
References
 Roosenberg, J.M. and Miller, M.J. Total Synthesis of the Siderophore
Danoxamine. J. Org. Chem. 2000 Vol. 65 No. 16. 4833 – 4838.
 Lin, Y. and Miller, M.J. Synthesis of Siderophore Components by and
Indirect Oxidation Method. J. Org. Chem. 1999 Vol. 64 No. 20.
7451 – 7458.
 Gaspar, M., Grazina, R., Bodor, A., Farkas, E., and Santos, M.A.
Macrocyclic tetraamine tris(hydroxamate) ligand. J. Chem Soc.
1999 799 – 806.
 Duhme, A.K. Synthesis of two dioxomolybdenum complexes of a
siderophore analogue. J. Chem. Soc. 1997 773 – 778.
 Atkinson, A. Bacterial Iron Transport. Biochemistry. 1998. 15965 15973