Bio mimicking of siderophores for metal ion encapsulation

Mritunjay(Assistent Professor)

Dev Samaj College for Women Firozpur

Scholar of M.Sc. Chemistry, Giani Zail Singh College of Engineering and Techonolgy, (MRSPTU), Bathinda 151001.

Abstract

Under the iron stress conditions some small organic molecules are produced by microorganisms to enhance the uptake of iron , these small organic molecules are called as siderophores in the ferric form of iron is insoluable and inaccesable at physiological pH(7.35-7.40) , and this is environmental form under this conditions siderophore helps to convert that insoluble form to soluble and absobable form for the microrganism. Previously it was thought that siderophores are only produced by microrganisms but now a days some metods of synthesis are developed to synthesize that in the laboratory . Some of that methods of synthesis of Siderophore are studied in this paper.

Keywords: Biomimicking, Siderophores, Biomimicking of siderophore, Applications of siderophores, Synthesis of siderophores.

Introduction

For all plants and animals, iron is very important for their proper growth and development. Only some Lactobacilli and Borelia species are exceptions1.Typically iron required in key metabolic processes are 105 Fe-ions for a single bacterial cell. Iron is fourth most abudant element on Earth’s crust2. But it is present in areobic conditons at neutral pH in extremely insoluble form like hamatite, pyrite and goethite or as polymeric oxidehydrates, silicates and carbonates which are not easliy absorbed by living beings. To survive in that condition, microorganisms adapted their self. They produce the high-affinity iron-binding compounds called siderophores3-5. The term ‘siderphore’ had been coined from Greek word meaning “iron bearer”. The first siderophore discovered was ferrichrome produced by Ustilago sphaerogena which can work as growth factor for for others6. Siderophores captures the insoluble ferric iron from the different habitats. Siderophores are generally designed to capture Fe3+ only but will bind with other elements also for eg. Al3+,Cr3+and Rh3+ etc. But it has more stability constant with the Fe3+ complex rather than any other comples with Al3+,Cr3+and Rh3+ etc. Existing literature shows that under the iron stress condition two bacteria gram-negative and gram positive synthesized siderophore. It has been reported that Pseudomonas stutzeri which is a facultative aerobic bacterium produce siderophores in both aerobic and anaerobic conditions, but the siderophore which is produced in the anaerobic condition is little bit different from the siderophore which is produced in the aerobic condition. Under the aerobic condition, four ferrioxamine siderophore was reported to produced by Pseudomonas stutzeri. But in the anaerobic condition, none of these

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ferrioxamines siderophores were found. Siderophores firslty get tight bonded with Fe3+ ion and make a siderophore-iron complex, then that complex moves into the cell through the cell membrane with the help of siderophore receptors which are present their. In cell, generally siderophore binding protien, permeases and ATPases are involved in the transport of siderophore iron (Fe3+) complex in the cell membrane. Although the primary and main function of the siderophore is to supply the soluble iron the microorganisms for their proper growth and development, they also have number of application in the fields of agriculture, biosensor, medicine, bioremediation and in ecology also. According to literatures there are more than 500 different siderophores were reported, and only 270 from them are characterized and other remaining are uncharacterized and their function are to be determined7-8.

Here we have to study biomimcry of siderophores, now we know about siderphores as well. Now to study its biomimicry we must have an general idea and outline about it, so biomimcry is the imitation of the models, systems and elements of nature for the purpose of solving complex problems in living beings9.

Types of siderophores

There are four main categories, are on the basis of the oxygen ligand for Fe (III) coordination10, are namely,

Hydroxamate siderophores Catecholate (phenolates) siderophores Carboxylate siderophores Mixed ligands and heterocyclic chelating siderophores

Hydroxamate siderophores

Siderophores which bearing hydroxamate group are normally synthesized by the Fungi and Gram-positive filament-forming bacteria (streptomycetes). Hydroxamic acid chelating group in fungal system is commonly derived from acylated Nδ-acyl-Nδ-hydroxy-L-orrnithine. Mostly Hydroxamate group consists of C(=O)N-(OH) R, where R may be amino acid or may be a derivative of it. Hydroxamate gives its two oxygen group to form bidentate ligand with iron. Thats why, each siderophore is able to make the octaherdral complex with Fe3+. Rang of binding constant between the hydroxamates and ferric iron is 1022 to 1032 M-1. That strong binding between siderophore and ferric iron protects the complex with enzymatic degradation and hydrolysis in the evironment.

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Fig.2 Ferrioxamines Fig.1 Ferrichromes

The ferrichromes consists one large family of siderophore having hydroxamate and were isolated from many fungi by low-iron cultures. Ferrichrome is with some exceptions that, it possess backbone of cyclic hexapeptide in which one tripeptide is linked to a second tripeptide of Nδ-acyl-Nδ-hydroxy-L-orrnithine. Albomycines is a antibiotic, which has linear derivatives of the latter tripeptide. Linear derivatives of the latter tripeptide form backbones of many antibiotics also.

Actinomycetes, having a typical constituents of culture broths is Ferrioxamines, found in both linear and cyclic compounds containing 1-amino-5-hydroxypentane (N-hydroxycadaverine) and succinic acid as building blocks. Some derivatives of the ferrioxamnines shows antibiotic activity and so they have been designated as ferrimycins11-17.

There are several methods to detect the hydroxamate type of siderophores. Initially, for the detection of hydroxamate siderophore Neilands spectrophotometric assay was used. And widely used method to detect the hydroxamate siderophore Electrospray ionization mass spectrometry (ESI-MS) has been used. Here is another method which can also be used to detect hydroxamate siderophore is Modified overlaid chrome azurol S (O-CAS) assay. Csaky’s assay is another widely used to detect hydroxamate siderophores such as aerobactin of Escherichia coli18-22.

Catecholate (phenolates) siderophores

In the iron stress condition, many bacteria produce siderophores exhibiting 2,3-dihydroxybenzoate (DBH) or phenolate iron binding groups. Catecholate type of siderophores is mainly excreted by certain bacteria. Hexadentate octahedral complex is formed by the supply of two oxygen atoms by catecholate group for chelation with iron. E. Coli, Aerobactor aerogenes and Salmonaell typhimurium was first tricatechol siderophore which was isolated from the culture fluids of these bacterias. Some bacteria like Klebsiella pneumoniae produce enterochelin and this can bind to ferric ion too tightly ( K=1052M-1). To estimate very low concentraion of iron in the environment sample this strong binding between iron and enterochelin can be exploited.

Some bacterias may produce mixed siderophores (where one member is catechole also) or catecholate siderophore alone. For eg. Only catecholate siderophore can be produced by Erwinia carotovora . where some members can produce mixed siderophores having both hydroxamates and catecholate siderophores like Pseudomonas.

Enterobactin is bascially a cyclic triester of 2,3-dihydroxybenzoylserine (DHBS) having very good features like (1). For physiological reducatants Enterobactin has very low redox potential (2). Complex formation constant value is extremely high (3).a strong pH- dependence of (1) And (2), and (4). A trilactone backbone the clevage of which lowers the redox potential.

Parabactin, N4-(2,3-dihydroxybenzene-3-methyloxazoline-2-carboxamidyl)-N1,N8-bis(2,3-dihydroxybenzoyl) spermidine, is produced by Paracocus denitrificans23-27.

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Fig.3 Enterbactin Fig.4 Parabactin Fig.5 Mycobactin

There are several essays by which we can detect the catecholate type of siderophore. Neilands spectrophotomertic assay is oNe of the most reported essay. HPLC (High-performance liquid chromatography) analysis with DAD ( diode array detection), O-CAS assay and ESI-MS (electronspray ionization mass spectrometry) assays can also be used to detect the catecholate siderophores28-29.

Carboxylate siderophores

In the nineties of the last century, a new type of siderophores was detected on that time and whose members neither have hydroxamate and not phenolate ligands. Rather, FeIII iron binding is achieved exclusively by carboxylates and α-hydroxycarboxylates. The late discovery of that compounds is notably based on the fact that they are not having any colour, that’s why requiring new methods for isolation and screening (iron nutrition nioassays and chromazurole test). Carboxylate siderphores are found in bacteria and in realm of fungi also. Staphyloferrin is very much hydrophilic complex, was isolated from from Staphylococcus hyicus. Molecule is made up from two moles of citrate linked by ornithine. Besides staphyloferrin A, also staphyloferrin B, vibrioferrin, and rhizoferrin having citric acid building blocks in it30-32.

Fig.6(a) Rhizobactin Fig.6(b) Staphyloferrin

Rhizobium melilotli, can fix the nitrogen present in atmosphere when symbiotically associated with some excretes, legumes and utilizes rhizobactin DM4. Carboxylate siderophores can be detected with the help of a apectrometric test, in that test siderophore copper complex is formed and is scanned for absroption maximum between 190 and 280 nm. O-CAS assay, HPLC,and MS can be used to detect the carboxymate siderphores and their structure can also be identified by these methods33-35.

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Mixed ligands and heterocyclic chelating siderophores

Myobactin, was the first siderophore fiftyfive years ago and was isloated from the aluminium complex by its crystallization. Nocobaction from Nocardia and mycobactin from gram-positive Mycobacteria embody a series of lipid-soluble siderophore present in the rich lipid boundary layers of these bacteria. Iron binding in mycobactins is possess by a phenolate group, two hydroxymates and oxazoline nitrogen on the basis of revealed structure from the X-ray. There is a general assumption is that in thiazoline and oxazoline containing donor-deficient siderophores, the N-imine will participate in ferric ion complexation. An enzymatic cyclization of seryl, threonyl and cysteinyl side chains physiologically resulted into heterocycles. Pyochelin, yersiniabactin, acinetobactin and anguibactin are siderophores having a imine-type of Fe-coordination is common feature in them35-37.

D

Fig.7 (A) Acinetobactin (B) Anguibactin (C) Yersiniabactin (D) Maduraferrin

Synthesis

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As from the above study, we comes to know that we can produce siderophore with the help of bacteria, by putting that bacteria in the iron stress condition. But now we will talk about werther we synthesize that in our laboratory or not. So yes we can, in this synthesis we will basically focus on tripodal type. Tripodal having three arm like structure or having tris-bidentate ligands. There are to many exampels of tripodal structure some of tripodal structured siderophores are given below39.

Fig.8 Examples of Tripodals

O-TRENSOX

Preparation starts from 8-hydroxyquinoline, is cheapest precursor, which is get coverted into 7-carboxylated derivative be a kolbe procedure. Activate the carboxylic group, with the help of N,N’-carbonyldiimidazole (CDI), allows the conjugation with tripodal tren. In last step increase both acidity and aqueous solubility is done by para-sulphonation on fifth position which is regiospecifical40.

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Fig.9 Synthesis of O-TRENSOX

TRENSOSX2CAMS

The process of synthesis for the hetero-tripodal hexadentate ligands is shown in figure below. two sub units of catecholate work connected to the scaffold of tripodal by direct condensation of two equivalent of 2,3- di methoxy benzoic chloride with one equivalent of Tren (path a) . The free primary amine group was grafted by the oxinate subunit(L2) by coupling with CDI-activated 7-carboxy -8 – hydroxy quinoline. To synthesis another one (L1 ) required the protection of one arm of Tren with a trityl group (path E), while phenol groups were respectively benzylated(oxine) and methylated (catechol ) . deprotection is done with the help of BBr3 , regiospecific sulphonation in the fifth position of both oxinate and catecholate subunits afforded the ligands which are hydrosoluable41-42.

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Fig.10. Synthesis of TRENSOSX2CAMS.

1,3,5-N,N’,N”-Tris(2,3-dihydroxybenzoyl)triaminomethylbenzene

A 25ml of CH 2CL2 solution was added dropwise (15minutes) into the solution of 1,3,5-N,N’,N”-Tris(2,3-dimethoxybenzoyl)triaminomethylbenzene (2.2g,3.3mmol) and then added solution of BBr3 (3.1ml,33mmol) with vigrous stirring. This reaction vessel was immersed into an ice bath at room temperature, the solution was stirred overnight under argon , H2O (25ml) was added dropwise to hydrolyze boron compounds . the crude product was collected by filtration (which we get from additional stirring for two hours then crude washed well with H2O , dissolved in MeOH , and evaporated to dryness several times . then solid was titrated with EtOAc and clarified by filtration and several volumes of Et2O followed by low boiling petroleum ether were added to the ppt white43-45.

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Fig.11 Synthesis of 1,3,5-N,N’,N”-Tris(2,3-dihydroxybenzoyl)triaminomethylbenzene

Applications

Microbial ecology

The study of relationship between microorganisms and their suoorunding environments is called microbial ecology. The growth of unculturable microorganisms in artifical media is enhanced by siderophore. The complex microbial association in a habitat is represented by microbial community. Hbitat reveals the balanced interaction betweedn biotic and abiotic sources. Only 0.1 to 1% of the population in the microbial community can be cultivated in laboratory condition whereas most of populations are uncultivable. Various methods undertaken to collect the more information regarding the cultivation of unculturable organisms in artifical growth media under laboratoty conditions. In the course of coculture of microorganism one type of microorganism produces siderophore whoch act as a growth factor that enhance the growth of other unculturable microorgainsm. The growth of uncultivable marine bacterial species in laboratory growth medium is enhanced by the exogenous addtion of siderophores. Although it was reported many strains of unculturable organisms are not able to grow in laboratory as they are unabke to autnomously prouce the siderophore, their cahmical dependence on other neighbouring microorganisms helps in the regulation of community establishment in the environment46.

Agriculture

The plant growth is promoted by siderophore: The quantity and quality of crop production is reduces by the iron starvation. The different pseudomonas species can enhance plant growth by producing pyoverdrine siderphores. Sometimes the plant also modifies the structure of root soil microbial community and favours the growth of more siderphore secreting microbes by secreting phenolic exudates from their roots . so the soulibilty of insoublie iron and enhances plant uptake of iron via microbial siderophores get improved.

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Siderophore alsao act as potential biocontroll agent: Binding of siderophores with the iron tightly reduces the bioavaliable iron the plant pathogens and thus facilitating the killing of fighting pathogens. The bioremediation of heavy metals is enhanced by siderophores: Siderophore also play significant roles detoxifying heavy metal contanimated samples by binding to wide array of toxic metals for example- Cr3+, Al3+, Eu2+, Cu2+, Pb2+, But the principle role of siderophore is to act as chelate to ferric iron46.

Siderophore as medicines

Siderophore are also used as medicine. They have important application the medical feild to fight against antibiotic resistant bacteria and then treatment of several human diseases. Siderophores are used in antimalarial activity. Desferrioxamine B enter inside the paracite and causes the intra deplition of iron. Siderphores are also used in the transuranic elements. Siderophores such as desferol is used in the treatment of chronic Aluminium overload beacuse it mobilises and chelates Aluminium bound to tissue by forming an Aluminoxamine. It is freely soluble in water and excreted through urine and feces. Siderophore in cancer treatment: Inside the human body iron act as oxygen carrier but excess iron increases the chances of cancer due to production reactive oxygen species. The decrease in the growth aggerssive tumores in patients with neuroblastuma and lukemia is significantly reduced by the use of desferrioxamines. This desferrioxamines can inhibit the DNA replication and also reduces the viability of malignant melanoma cell significantly. Their are also some other iron chelator which are used in cancer therapy are dexrazoxane, desferriexochelins, tachpyridine and o- trensox46.

Conclusions

As we know that iron is vital element which is required by every living organisms for number of cellular processes. Under the drastic conditions in which iron is deficient in the environment the growth of microrganisms become impared. So to survive in that environmental conditions microrganisms secretes siderophores . and the main benefit of siderophore is that it holds the promise to be implimented as a potential agent in different areas including agriculture ecology and medicines . but now a days we can also prepare the siderophores in laboratory and the methods of preparations were discussed in study above.

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