The Synthesis of ‘acidic’ iron binders
Students: Matthew
Carroll, England; Inna Shvarts, Israel
Mentor: Michael
Meijler; Supervisor: Prof. Avi Shanzer
Dr Bessie F. Lawrence 31st
International Summer Science Institute, June 30 – July 30 1999
Department of Organic
Chemistry, Weizmann Institute of Science,
Rehovot, Israel.
Abstract
We synthesised what can
loosely be termed an ‘acidic’ iron binding tripod, purified it, and confirmed
its identity.
Introduction
Iron
is used by all living organisms. It plays a vital role in processes such as
respiration, DNA synthesis and oxygen transport in the blood. The abundance of
iron in the earth’s crust is high (around 5%), and consequently it is easy to
imagine that bacteria and other organisms would have little trouble in
fulfilling their need for iron. But on the contrary, most of the iron in the
earth’s crust is insoluble in water, and hence of little or no use to
microorganisms. As a result, biological systems have evolved which use complex
and intriguing ways of obtaining sufficient iron for the organisms to survive.
One of the more common systems used by microorganisms is the use of
siderophores. These are species specific iron-binding molecules that are
excreted by the cell, bind to any iron in the environment, and are then
reabsorbed by the cell using active transport.
Siderophores
are being studied with a number of aims in mind. These include potential
medical applications, such as the treatment of diseases like malaria, where
targeted iron deprivation of the parasite brings about its death. Also, the
same iron binding systems are being looked at for their use as molecular
switches (the technology that could eventually be used to build a molecular
computer).
The
aim of the project is to synthesise an iron binding molecule which can function
as such, even at a low pH (in acidic conditions). This is of interest because
many iron binders that have been synthesised as part of this ongoing research
area can only function in more alkali conditions, and it would eventually be
useful to produce siderophores which can enter more acidic cell compartments.
To accomplish this, we hope to first synthesise the ‘low-pH’ iron binder and
then perform in-vitro experimentation to determine the optimum pH for iron
binding with this compound.
Experimental
Shown
below is the entire reaction scheme for the synthesis of the 'low pH' iron
binder.
Abbreviations
Below
are the details for the individual steps in the synthesis, including any
results or measurements that were taken.
Step 1
The
HCl salt of the amino acid lysine (N-Cbz)-methyl ester (1.16 g, 4 mmol) was
added to ethyl acetate (50 mL) in a separating funnel and washed with 30 mL 1M
sodium carbonate (NaHCO3). The organic phase was dried over MgSO4
and the ethyl acetate removed in vacuo. The residue was weighted (1.155
g) and then dissolved in DCM (20 mL, dried over basic alumina) and stirred
under argon at -78o C (dry ice, acetone). Triphosgene (0.296 g,
1mmol) was dissolved in DCM (5 mL) and added to a suspension of activated coal
in DCM, and, after 10 min standing under argon, added to the solution of amino
acid. Directly after this 2,6-lutidine (0.70 mL, 6 mmol) was added and the
mixture was allowed to reach RT.
Step 2
A
solution of RL231 (1.12 g, 5 mmol) in DCM (10 mL) was added to the reaction
vessel and the mixture was stirred overnight. Hexane (50 mL) was added and the
mixture was washed with water (3 x 30 mL) and dried over MgSO4.
After concentration in vacuo the residue was subjected to flash
chromatography, yielding 1.15g (2.2 mmol) of the hydroxyurea.
Step 3
NaOH
1M (2 molar equivalents, 4.5 ml) was added to solution of the diester in 10 mL
of methanol and stirred at room temperature. The reaction was monitored on TLC
until no more starting material was seen. The solvent (MeOH) was removed in
vacuo and the resulting fluid was diluted with water (30 ml), acidified
with KHSO4 1M to pH 2, and extracted with ethyl acetate. The
solution was dried with magnesium sulphate, and evaporated to give 0.81 g (1.8
mmol) of the diacid.
Step 4
The diacid was dissolve in acetic anhydride (10ml) under argon atmosphere.
1M THF solution of Bu4N+F- (8.5 mL, 5 x molar
excess) was added, and the mixture was stirred over night. The acetic anhydride
was evaporated in high vacuum, and the identity of the resulting (impure)
product confirmed using NMR.
Step 5
The
product from step 4 was dissolved in MeOH (10 ml). H2SO4 was
added (0.5 ml conc) and the mixture was left to stir over night under reflux.
The solvent was removed in vacuo and the residue dissolved in ethyl
acetate (15 mL). The solution was washed with HCl (2 x 10 ml), with water (1 x
10 ml), and then dried with MgSO4. The
ethyl acetate was removed in vacuo and the residue dissolved in
chloroform with the minimum volume of MeOH. This was then subjected to column
chromatography, yielding 0.41 g (820 m mol)
of product, whose identity was then confirmed by NMR.
Step 6
The
product from step 5 was dissolved in EtOH (5 ml) and 150 mg of palladium (on
activated coal) was added. The mixture was then stirrred under hydrogen
atmosphere for 3 hours. The EtOH was then removed in vacuo and the
residue dissolved in chloroform. This solution was filtered twice through a
small layer of silica and MgSO4 to
remove the catalyst. Although some of the coal remained, analysis by TLC showed
the sample to be sufficiently clean to proceed with the next step.
Step 7
The
solvents were evaporated from the product from step 6 in vacuo, and then
the product was completely dried using
the high vacuum, giving 0.13 g of impure product. The tripod anchor was then
added (100 mg, 91 m
mol). This mixture was then dissolved in dry DCM, 10 drops of triethylamine
were added, and the solution was left to stir over the weekend. The resulting
solution was subjected to column chromatography twice, yielding 80 mg (72 m mol) of clean product.
Discussion
We
completed the synthesis and succeeded in producing the iron-binding tripod,
whose identity was confirmed using NMR and IR spectroscopy. We had planned to
perform in vitro experimentation to determine at what pH the tripod most
effectively binds with iron, but illness during the last week of the work
prevented our reaching that stage. The experiment we had planned was to examine
the UV absorption of the complex over a range of pH values and from this
determine the pH at which optimal binding takes place.
Acknowledgements
We
would like to thank our dear and lovely mentor Michael for being blonde and
just generally being such an ugly blonde a great mentor. Thank you for
all the support and ice cream, even if you didn’t take us out to dinner… yet!
We
would also like to thank all the counsellors of the summer science institute
for all their help picking money, and just for putting up with us for a month.
Don’t worry, we know the food wasn’t your fault.
Thanks
also to Josh (6) for having such an interesting life story.
Finally
thanks to everyone who made it possible for us to come to the summer science
institute – there are far too many to mention, but your help has been truly
appreciated.