Synthesis, Characterization and
Iron-Acquisition of Hydroxamic
Acid Derivatives
-Functional studies based on the subunits of amphiphilic
siderophores
O
OH
O
NH O
N
NH2
HO
O
NH
HN
OH
N
HN +
O
HO
HO
OH
O
NH
H
O
HN
HN
O
O
NH3+
N
H
O
Linda M. Johnson
University of North Carolina at Chapel Hill
Class of 2003
Dr. John T. Groves/ Minkui Luo
Princeton University
May 6, 2004
Mercer County Community College
Iron – An Important Element
• 4th most abundant
element in the world
• An important
nutrient for the body.
• Helps with growth
and development in
the body, especially
in children.
• Iron is forever cycled
from a liver
storehouse in a
protein called ferritin.
The Paradox of the Limited
Abundance of Iron
Lack “iron” in such an
iron-abundant planet ?
•Extremely limited bioavailability.
•Bacteria battles to acquire iron to obtain nutrients
that to help contribute to growth
Siderophores
• Small weight compounds that have
a high affinity for Fe (III).
• Help with Iron Transport.
• Three subunits of siderophore’s
chelating groups.
A Common Strategy of Bacteria --- IronDelivery Shuttle, Siderophore
R'
3+
N + Fe (aq)
OH
R
O
R'
R
O
1
N
O
Fe
R
+ Fe (aq)
OH
O
HO
O
+ Fe3+(aq)
OH
Fe
O
OH
O
2H
N
HN +
O
Fe
HO
2H+
OH
O
HN
O
+
O
O
HO
NH
H
O
O
O
O
OH
HN
R
HO
NH
HN
+
NH O
N
NH2
+
2
R
H+
R
3+
HO
+
N
H
O
O
NH3+
Pseudobactin
Albrecht-Gary A. M.; Crumbliss A. L. Metal Ions in Biological Systems 1998, 35, 239
Water-phase Iron-acquisition Kinetic of
Citrate-based Amphiphilic Siderophore
O
NH N
HO
OH
O
OH
O
O
HO
NH N
-1
0.15
Apo-Acinetoferin
O
O
NH N
HO
OH
O
OH
O
O
HO
NH N
Apo-Rhizobactin
O
O
Iron-acquisition Rates / s
O
0.12
Acinetoferrin
Rhizobactin
SAF
0.09
0.06
0.03
0
NH N
HO
OH
O
OH
O
O
HO
NH N
0
1
2
3
4
FAC Concentration / mM
Apo-SAF
kobs 
km  [ FAC ]
 kp  [ FAC ]
[ FAC ]  1 / Kp
5
Purpose of Experiment
• To examine and compare the
structures of siderophores’ sub-unitshydroxamic acids.
• To understand how hydroxamic acids
facilitate the iron chelating process of
siderophores by measuring their ironacquisition rate.
Synthesis of N1-BOC propane
diamine
H2N
2 equi. BPO
10% TEA/MeOH
NH2
HN
(BOC)2O
O
NH2
HN
O
O
N
H
O
Compound 1
Compound 2
NaOH/
MeOH
HN
O
N
H
O
Compound 3
OH
OBz
Synthesis of Compound II & III
• Synthesized Bocprotected amine
• Use Flash Column
Chromatography (FCC)
to help separate
compound.
• Perform Thin Layer
Chromatography (TLC)
to help locate the final
product.
• Check purity of
compound with
1H-NMR.
Five substrates used to react
with the amine:
• Acetyl Chloride
O
Cl
Cl
• Cinnamoyl Chloride
O
• Butyryl Chloride
O
Cl
O
Cl
• Crotonyl Chloride
• Hydrocinnamoyl Chloride
Cl
O
Procedure of Analog Synthesis
• Performed test reactions with each
halide.
• Check TLC and take 1H-NMR for
each reaction.
• Once reaction is successful, upscale
amount of material for future
experiments.
Reaction Between Amine and
Acetyl Chloride
• Equation:
O
O
H
N
O
NH
Cl
H
N
O
OH
O
CH2Cl2/
Reflux
N
OH
O
tert-butyl 3-(hydroxyamino)propylcarbamate
• Results:
• 1st attempt: No distillation or either substrate nor solvent.
Some reaction occurred, but it was not complete, even
after adding 10% base to help push reaction.
• 2nd attempt: Distilled Chloride and Solvent (CH2Cl2).
Reaction completed. Rxn. time estimate: ~0.5 hr.
Equations for Other Hydroxamic Acid
Analogs
With Butyryl Chloride
o
O
OH
NH
O
NH
O
O
Cl
NH
NH
O
CH2Cl2/ Reflux
O
With Crotonyl Chloride
o
O
OH
NH
O
NH
O
Cl
NH
NH
O
CH2Cl2/ Reflux
O
With Hydrocinnamoyl Chloride
o
O
OH
O
NH
NH
Cl
O
O
NH
NH
O
CH2Cl2/ Reflux
O
Reaction Between Amine and
Cinnamoyl Chloride
• Equation:
O
H Cl
N
O
H
N
O
H
N
O
N
OH
H
N
O
OH
O
O
O
Reflux
O
N
OH
• Result: Did not completely react even after distilling
both chloride and solvent.
• 2 ways attempted to push reaction
- No heat, extra base added
- With heat and extra base added.
Procedure (Cont.)
• Once the analogs
were synthesized,
solvent was removed
with rotavapor and
oil pump
• 1H-NMR, 13C-NMR
and Mass Spec. were
taken of each
derivative to check
purity.
O
1H-NMR
of
H
N
O
N
OH
O
Four Final Analogs Used for
Iron Acquisition Measurement
O
H
N
O
O
N
OH
O
H
N
O
N
OH
O
O
H
N
O
N
OH
O
O
H
N
O
N
OH
O
Procedure for Measuring the
Iron-Acquisition Rate
• Iron stock solution was
made (.6394g/200ml
distilled water)
• 10mM Buffer solution
-Ammonium Fe(III) Citrate
(FAC) w/Hepes buffer
• Used SF 61 DX2 Mixing
Stopped-Flow Machine
• Conditions:
- pH of buffer solution:
7.4
- Temp-37oC
Results of iron acquisition of
hydroxamate analogs
O
O
0.2
H
N
O
O
N
HC
CR
OH
O
O
H
N
Iron-acquisition Rate / s -1
O
O
H
N
N
OH
AC
BT
N
OH
O
O
H
N
O
O
N
OH
0.15
0.1
0.05
0
0
1
2
3
Fac Concentration / mM
4
5
Results and Conclusions
• There was a general curve that
formulated for each analog.
• The shorter the substrate, the
higher the iron acquisition rate.
• Having a unsaturated substrate
also increased the iron-acquisition
rate.
Future Studies
Adjust conditions to successfully react the following:
2 equi. BPO
10% TEA/MeOH
H2N
NH2
(BOC)2O HN
O
NH2
HN
O
O
N
H
OBz
O
Cl
CH2Cl2/ Reflux
O
H
N
O
O
O
N
OH
NaOH/
MeOH
HN
N
O
O
OBz
O
To continue to further examine the chelating process of iron
acquisition due to hydroxamic acid structure.
Acknowledgements
Dr. John T. Groves
Princeton University Department of Chemistry
Minkui Luo
Dr. Groves’ Research Team
Mercer County Community College
Prof. Helen Tanzini
Family and Friends
THANK YOU ALL FOR YOUR SUPPORT! 