synthesis, characterization and iron-acquisition of hydroxamic acid derivatives -functional studies...
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Synthesis, Characterization and Iron-Acquisition of Hydroxamic Acid Derivatives -Functional studies based on the subunits of amphiphilic siderophores. Linda M. Johnson University of North Carolina at Chapel Hill Class of 2003 Dr. John T. Groves/ Minkui Luo Princeton University May 6, 2004 - PowerPoint PPT PresentationTRANSCRIPT
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
ONH3+
HNO
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
NO
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
ONH3+
HNO
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
uisi
tion
Rat
es /
s-1
AcinetoferrinRhizobactinSAF
NH
NH
O
O
N
N
HOO
OHO
OH
O
OH
NH
NH
O
O
N
N
HOO
OHO
OH
O
OH
NH
NH
O
O
N
N
HOO
OHO
OH
O
OH
Apo-Acinetoferin
Apo-Rhizobactin
Apo-SAF ][/1][
][ FACkKFAC
FACkk pp
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
O NH
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
Iron-
acqu
isiti
on 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
ONO
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!