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Synthesis, Characterization and Iron-Acquisition of Hydroxamic
Acid Derivatives -Functional studies based on the subunits of amphiphilic
siderophores
Linda M. JohnsonUniversity of North Carolina at Chapel
HillClass of 2003
Dr. John T. Groves/ Minkui LuoPrinceton University
May 6, 2004Mercer County Community College
N
OHO
NH O
NHO
OH
NH
ONHO
HN
O
NH3+
HN
O
NHN
H
OH
OHHN
NH2
O
O
HO O
HO+
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.
Lack “iron” in such an iron-abundant planet ?
The Paradox of the Limited Abundance of Iron
•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.
Albrecht-Gary A. M.; Crumbliss A. L. Metal Ions in Biological Systems 1998, 35, 239
N
O
R R'
OHFe3+(aq) N
O
R R'
OFe
H+
Fe3+(aq) 2H+
HO OH O OFe
HO
R
OHFe3+(aq) 2H+
O
O
R
O
O
Fe
R R
+ +
1
+ +
2
+ +
N
OHO
NH O
NHO
OH
NH
ONHO
HN
O
NH3+
HN
O
NHN
H
OH
OHHN
NH2
O
O
HO O
HO+
Pseudobactin
A Common Strategy of Bacteria --- Iron-Delivery Shuttle, Siderophore
Water-phase Iron-acquisition Kinetic of Citrate-based Amphiphilic
Siderophore
0
0.03
0.06
0.09
0.12
0.15
0 1 2 3 4 5
FAC Concentration / mM
Iron
-acq
uis
itio
n R
ates
/ s-1
AcinetoferrinRhizobactinSAF
NH
NH
O
O
N
N
HOO
O
HO
OH
O
OH
NH
NH
O
O
N
N
HOO
O
HO
OH
O
OH
NH
NH
O
O
N
N
HOO
O
HO
OH
O
OH
Apo-Acinetoferin
Apo-Rhizobactin
Apo-SAF][
/1][
][FACk
KFAC
FACkk p
p
mobs
Purpose of Experiment
• To examine and compare the structures of siderophores’ sub-units-hydroxamic acids.
• To understand how hydroxamic acids facilitate the iron chelating process of siderophores by measuring their iron-acquisition rate.
Synthesis of N1-BOC propane diamine
NH2H2N NH2HN10% TEA/MeOH
(BOC)2O
O O
NH
HN
O O
OBz2 equi. BPO
NaOH/MeOH
NH
HN
O O
OH
Compound 1 Compound 2
Compound 3
• Synthesized Boc-protected 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.
Synthesis of Compound II & III
Five substrates used to react with the amine:
• Acetyl Chloride
• Cinnamoyl Chloride
• Butyryl Chloride
• Crotonyl Chloride
• Hydrocinnamoyl Chloride
O
Cl
O
Cl
Cl
O
Cl
O
O
Cl
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:
• 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.
OHN
O
N
O
OH
O
Cl
CH2Cl2/Reflux
tert-butyl 3-(hydroxyamino)propylcarbamate
OHN
O
NH
OH
Equations for Other Hydroxamic Acid Analogs
With Crotonyl Chloride o
With Hydrocinnamoyl Chloride o
With Butyryl Chloride o
Cl
O
CH2Cl2/ Reflux
O
NH
O
O
NH
O
NH
O
NH
OH
Cl
O
CH2Cl2/ Reflux
O
NH
O
O
NH
O
NH
O
NH
OH
Cl
O
O
NH
ONH
O
NH
O
NH
OH
CH2Cl2/ Reflux
Reaction Between Amine and Cinnamoyl Chloride
• Equation:
• 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.
OHN
O
N
O
OH
OHN
O
N
O
OHO
Cl
Reflux
OHN
O
HN
OH
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.
1H-NMR of OHN
O
N
O
OH
Four Final Analogs Used for Iron Acquisition Measurement
OHN
O
N
O
OH
OHN
O
N
O
OH
OHN
O
N
O
OH
OHN
O
N
O
OH
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
0
0.05
0.1
0.15
0.2
0 1 2 3 4 5
Fac Concentration / mM
Iro
n-a
cq
uis
itio
n R
ate
/ s
-1
HC ACCR BT
Results of iron acquisition of hydroxamate analogs
OHN
O
N
O
OH
OHN
O
N
O
OH
OHN
O
N
O
OH
OHN
O
N
O
OH
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 StudiesAdjust conditions to successfully react the following:
To continue to further examine the chelating process of iron acquisition due to hydroxamic acid structure.
NH2H2N NH2HN10% TEA/MeOH
(BOC)2O
O O
NH
HN
O O
OBz2 equi. BPO
NaOH/MeOH
CH2Cl2/ Reflux
NHN
O
O
OBz
O
Cl
O
OHN
O
N
O
OH
AcknowledgementsDr. John T. Groves
Princeton University Department of Chemistry Minkui Luo
Dr. Groves’ Research Team
Mercer County Community CollegeProf. Helen TanziniFamily and Friends
THANK YOU ALL FOR YOUR SUPPORT!
