Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
The present invention relates to a composi-tion useful
for the removal of metals, in particular iron, from liquid
media. The composition, can for example, be used to lower the
iron concentration of a liquid medium to less than 0.1 ~M.
Iron is an essential nutrient for all living thinys;
a large number of cellular enz~nes and other proteins require
iron in order to function properly. Although iron is amongst
the most plentiful of metals, it is difficult for biological
systems to acquire; in aerobic environments of substantially
neutral pH, iron exists as its oxidized Fe3~ form which readily
hydrates to highly insoluble Fe(OH)3 polymeric forms. To ensure
accessability of iron in their environment, aerobic and facul-
tative micro-organisms synthesize and release into their environ-
ment highly selective iron chelating agents called siderophores,
the function o which is to provide the microbes with this vital
nutrient. The siderophores release~d by the microbes solubilize
iron, putting it into a orm readily usable by them. Thus, a
free, microbial siderophore is a growth promoting substance
for those or~anisms which can utilise the particular siderophore
in ~uestion.
In accordance with the present invention it has been
determined that removal of iron (e.~ Fe3+) from a liquid
nutrient medium will substantiall~ restrict the prolieration
of microbes provided that the residual iron concentratlon in
~5 the medium is below 0.1 ~M; and this notwithstanding that the
other required nutrients may be present in amounts suEicient
for the support of microbial growth.
~hus, it would be advantageous to have compositions able to remove
iron rom a liquid, nutrient medium since the absence or limited
presence of iron will inhibit rnicrobial growth in such a medium.
For example, the speciEic removal of iron from an ophthalmic
solution will inhi.bit microbial growth and spoilage o such a
solution. l'he removal of iron from SUC}l a solu-tion would
obviate the addition of conventional microbial grow-th
~f~V~
inhibitors which can create their own problems such as toxicity,
etc.
Removal o iron from liquid media prior -to the present
invention did present problems (see Neilands, J. sacteriology
149: 830, 1982). Commercially available products (e.g. Chelex
100 sold by BioRad) have a low selec-tivi-ty for iron. Addition-
ally, other important cations (Mn2~, Mg2+) removed by such
commercial products are often desirable components of a licluid
medium. Commercial ion exchange products can also liberate
iO sodium or potassim ions into the liquid medium being treated which
may not be desirable.
Thus, it would be advantageous to be able to remove
iron from a liquid medium while at the same time Awoid:ing
liberating into the liquid medium undesirable ions.
lS In general, it would also be advantageous to be able
to remove iron ~rom a liquid medium when the presence of iron
is undesirable, e.g~ when iron is considered a contaminant at
concentrations greater than 0.1 ~M.
Free microbial siderophores cannot be used to remove
iron from a liquid medium; the nat:ural purpose of such side-
rophores is to make iron soluble and available to micro-
organisms. The addition of ree siderophores to a liquid
medium would therefore enhance the growth of microorganisms
which could utilise iron solubilized ~hereby. Additionall~
it would be e~tremely difficult at the very least, to recover
such siderophores loaded with iron.
Nevertheless, it would be advantageous to be able
to make use of the properties of siderophoric compounds such
as microbial siderophores and other organic compounds which
can provide co-ordinating groups or ligands for the chelating of
iron (ie Fe3 ).
The present inven-tion in general relates to inso-
luble compositions, which are capable oE removing metal (e.~.
selective:ly) Erom solution (e.g. Fe3 from a liquicl nu-trient
`Tr~6lt ~ 2 -
.~ ,.
medium so as to lower the Fe3 content to less than
0.1 ~M), the insoluble composi-tions comprise:
(i) a sui-table insoluble carrier
and (ii~ organic co-ordinating sites covalently fixed
to the surface of said carrier.
In accordance with the present invention the
necessary co-ordinating sites may, for example, be provided
by fixing an organic chelating cornpound such as a microbial
siderophore to a suitable carrier (infra).
Thus in accordance with one aspect of the present
invention, there is, in particular, provided a method for
inhibiting mlcrobial growth in a liquid nutrient medium
containing Fe3~ by lowering the Fe3~ content thereof to
less than 0.1 ~M characterized in that said medium is
contacted with an insoluble siderophoric composition and
thereafter said insoluble siderophoric composition loaded
with Fe3~ is separated from said medium, said insoluble
siderophoric composition comprising:
(1) one or more organic siderophoric compounds,
covalently fixed to the surface of
(2) a suitable insolublle carrier,
said oryanic siderophoric compounds possessing one or more
co-ordinating sites capable of chelating Fe3+.
An insoluble siderophoric composition , for the
purposes of this aspect of the invention, is a composition
having a chelating activity with respect to iron, and in
particular a selective chelating activity.
The organic co-ordinating sites of suitable organic
siderohporic compounds may, for example, be provided by
groups selected from the class consisting of
(a) N-substituted hydroxamate groups of formula
O OH
Il l I
- C - N ~ C -
3 -
(b) phenolate groups of formula
OH X
X being an atom of O or N-
(c) catecholate groups of formula
OH OH
~C~-
X' being an atom of O or N- and m being
1,
and (d) mixtures of two or more of the above groups;
see below.
If desired, catechol (1,2 dihydroxybenzene) may
be used as a siderophoric compouncl to provide catecholate
type co-ordinating sites.
The insoluble siderophoric composition can for
example be used to speciically remove iron from liquid
media such as water, juices, wine, beer, cider, chemical
so]utions, microbial and tissue culture media, pharma-
ceutical media , etc.
In accordance with another aspPct of the present
invention, tnere is provided an insoluble composition
comprising a member selected from the class consisting
of
(A) an insoluble composition comprising
(1) one or more organic chelating compounds,
covalently fixed to the surface oE
(2) a suitable insoluble carrier,
said organic chelating compounds possessing one or more
co-ordinating sites, said organic chelating compounds
being selected from the class consisting of microbial
siderophores and
(B) an insoluble composition comprising
~! , .
(1) catechol covalently fi~ed to the surface of
(2) a sui-table insoluble carrier,
the catechol being covalently fixed ko the surface of said
carrier at the benzene ring thereo.
The above compositions, in accordance with -this
other aspec-t of the present invention can be used to
remove Fe3 from solu-tion.
Thus this other aspect of the present inven-tion
also provides a method for removing Fe3 from solution
characterized in that the solution is contacted with
an insoluble composition as defined above. Thereafter,
the compos.ition loaded with metal may be separated from
the treated solution~ For example, the iron content of a
liquid medium may in this way be lowered to less -than
0.1 ~M. Thus an insoluble composition in accordance with
this aspect of the present invention may advantageously be
used asa siderophoric composition to remove Fe3~ from li~uid
nutrient medium .
In paxticular, this aspect of the present invention
also provides a method for inhibiting microbial growth in
a liquid nutrient medium containing Fe3+, by lowering the
Ee3 content thereof to less than 0.1 ~M characterized
in that said medium is contacted with an insoluble compo-
sition as defined above and thereaf-ter said composition
loaded with Fe3 is separated from the medium. The iron
loaded composition can, for example be recovered by filtration.
Compositions as defined above, loaded with Fe3~
may possibly be regenerated by chemical means sui-table for
the removal o the chelated metal; the so regenerated compo-
sition can thereafter be recycled for further use.
The pres~n-t invention thus provides not only a
mechanism for the removal of Fe3~ from liquid media but
also for the preservation of various liquid media through
the removal of iron thereErom, i.e. rendering liquid nu-trien-t
3~ media highl~ resistall-t to microbial grow-th since any micro-
-- 5
~1
~2V~
organism present cannot prolifera-te due to -the insuf~icient
amount of iron present.
Organic chelating compounds, e.g. siderophoric
compounds, useful in accordance with -the present invention
may also ~orm complexes with chromium ions and alpha-
emi-tting actinide ions due to the struc-tural (a-tomic~
similarities with iron; however, the complexPs are
formed at lower aEfinities than iron . Al-though
the compositions o~ the present invention can possibly be
used Eor the removal of these other metals from liquid
media, the following discussion will be directed to the
removal of Fe3+ from liquid media.
In accordance with the present invention, a general
process for the preparation of an insoluble composition
as de~ined above can be characterized in that organic
co-ordinating sites capable of chelating metal are covalently
fixed to the surface of a suitable carrier. Any suitable
means of covalently Eixing organic co-ordinatlng sites to
a carrier can be used provided that the composition obtained
has the necessary chelating activity.
If it is desired to procluce a side:rophoric compo-
sition comprising one or more organic siderophoric compounds
fixed to a suitable carrier, then the process o~ its pre-
paration may be characterized in that a sui-table carrier
is reacted with one or more organic siderophoric compounds
possessing co-ordinating sites capable of chelatin~ Fe3~
so as to covalently bond said siderophoric compounds to -the
surEace of said carrier, while maintaining the Fe3~ chela-ting
activity o~ said siderophoric compounds.
The siderophoric compounds as indicated above can
be microbial siderophores. In this particular case, the
process of preparation can be characterized in that a sui-
table carrier is reac-ted ~i-th one or more microbial sidero-
phores so as to bond said microbial siderophores -to the
surEace oE said carrier, said carrier and said microbial
",~", "
~;~(36~i7
siderophores possessing fu~c-tional groups reac-tl~e one with
the other so as to covalently bond said microbial sidero-
phores to said carrier while main-taininy the Fe3 chelating
ac-tivi-ty of said microbial siderophores.
Turning now to the chela-ting compounds, sufficient
metal coordination sites to chelate the metal ions (e.g.
Fe3~) may be provlded by a single organic chelating (e.g.
siderophoric) compound or alternatively hy two or more
such compounds; the number of compounds participa-ting in
the chelation of the metal ions being dependent upon the
number of coordinating sites which are available from a
particular compound fixed to a carrier.
The organic chelating compound used can as indi-
cated above be a microbial siderophore. ~ microbial side-
rophore may have a molecular weight of less than 2500
Daltons, e.g. a molecular weight in the range of 500
to 2500 Daltons. A microbial siderophore useful in accor-
dance with the present invention ~an also possess one or
more types of metal coordinating sites within i-ts structure.
The si-tes can be provided by groups selected from the class
of groups referred to earlier, e.y. N-substituted hydroxamate
groups, catecholate groups, etc. Siderop~ores possessing
these groups display high selec-tivity and very high affi-
nities for Fe3
A r~presen-tative list o~ microorganisms and
their siderophores is given in ~ollowing table 1: -
- 6 a -
~2C~
Table 1
COMMON NAMES OF SIDEROPHORE
ORGANISM NAME OBTAINED THEREFROM
Prokaryotes
Enteric spec.ies Enterobactin ~enterochelin),
Aerobactin
Agrobacterium tumefaciens Agrobactin
Pseudomonas species Pyochelin, Pyoverdine,
Pseudobactins, Ferribactin
Bacillus megaterium Schizokinen
Anaboena species Schizokinen
Art _obaoter species Arthrobactin
Azotobacter vinelandii a,~-bis-2,3,-dihydroxyben-
zoyllysine
Actinomy~e_ species Ferrioxamines
Mycobacterium species Mycobactins
Eukar~otes (Funqi)
Penicillium species, Ferrichromes, Copragen
A~E~ lus species/
Neur_spora, Ustillago
Rhodotorula species Rhodotorulic acids
ctomycorrhizal species Hydroxamate type
The basic chemical structure, trivial names and
possible sources oE some types of microbial ~iderophores
are listed below; the term microbial siderophore o course
includes any suitable functional de.rivatives, analogs or
enantioforms of these molecules:
a) Fe oxamlne
H-N ~ ~CONH/ CONH
('CH2)s ~CH2)2 ~C\2)5 ~CH2)2 ( \2 n R~
o d o ~ ~ - o o
Linear ferrioxamine
-- 7
Q~7
,~ ,
H--~ ~ CONH\ / CONH\ ~=
2 5 ; 12)2 (C\2)5 jCH2)2 (C ~ (t
N - N - Cl -N -
-- 0_ _ O~ ~ -- O ~ ' ,, A
C~lic ferrioxamine
wherein: ~
R = H or -COCH3; R' = CH3- or HOOC-(CH2)2-; n = 4 or 5-
For ferr:ioxamine B, R=H and R' - CH3-. The mesylate salt
of deEerrioxamine is marketed by Ciba-Geigy as Desferal
(U.S. pat. 1964 3,118,823 and 3,153,621; Can. pat. 1962
548981 and 715051.
b ) F~rr ichrome s
H Hl
~ ~C ' C ~
Q~ .. c~/ \ N /H
Fe3 ¦ I
'~~` ~C~O I -C N~
\ W--~ CH ~C H
H~N ~ C l / C
ll C ~ -H
R~
wherein:
R', R" , ~"' = H Ferrichrome
R = -CH (prototype)
-- 8 --
~L2~
c) Ci.trate Hydrox-dmate Derlvatives
wherein for:
fH3 ICH3 R n
f=O O=C schi~okinen H 2
N-OH HO-N aerobactin COOH 4
(CH2)n (CH2)n arthrobactin H 4
¦ 1l CIOOH 1l
H-C-N-C~CH - -CH - -N-C-H
I 1 2 1 2 1 1
R H OH H R
d) Rhodotorulic Acids
1l NH ~ N ~ C~-CH3 rhodotorulic acid
H3C-C- ~ ~ HO H
H H ~ r~CH20H
!l I H O ~ N ~ ~ N ~ ~ CH dim~mic
CH ~ C - N ~ ~ ~ Hl O 3 aci.d
~ H
HOCH2 H
(d) Rhodotorulic Acids (cont.)
-
CO~3 ¦¦ 2~C3
(CH2) 3 ~ / \
CO ~0 0 ) 03 copragen
CH~ / ~ \N
CEIN J 2
CH2 ,~ I
C/3C ~C~2)2
~2 - ~ NH
-- 10 --
~Z~ )5~
(e3 Mycobactins
~ IIO)~ HR5
N~ ~ 1HP4
1l IOH C\O R3
j ~N NH
R1 ~1H 2 ) 4 --CO
wherein:
R1 = methyl ethyl or alkyl or alkenyl
of 11 to 20 carbon atoms
R2 = H or methyl
R3 = H or methyl
20R~ = methyl, ~thyl, or alkyl of 15 to 18 carbon
atoms
R5 - H or methyl
(f) Fusarinines
O NH2 H0 O wherein:
HO ~ ~ ~ C (CH2) 3 N C ~H2-CH2-0 ~ H, n - 1 to 3
H
H CH3
~2C~
( g ~ Er~terobact in
OH
~OH
C--O
Hl~
C^' H-- C~
/ El '`~
O--C E~CH
~ ~
O / / \ /C\N
2 5 ~ cH O ~ \\
HO
OH
-- 12 --
(h) Agrobactirls
.N~
3 ~0~1
~ N ~ H
N
where in:
R = H,OH
-- :L3 --
s~
(i) Pseudobactin
o o
C~-C~ -CH -C
2 2 ~H H
H2N ~/
OH HO ~ \C~ )
f ~33
OH
CH~ \ / C113
C~C~ f
A detailed description of the above siderophores
is given by Neilands (Annu. Rev. Biochem. 50: 715-731, 1981),
and their coordination chemistry has been reviewed by
Raymond & Carrano (Account of Chemical Research, vol. 12,
No. 5, 1979 at pages 183-190).
Microbial siderophores can be extracted for example
from spent microbial culture media with organic solvents.
Examples of such methods are given in United States Patent
Nos. 3,118,823 and 3,153,621 as well as Canadian Patent Nos.
648,981 and 715,051. For example, siderophores possessing
hydroxamate ligands may be obtained in this fashion. Hydro-
xamate microbial siderophores are distributed widely through-
out the prokaryotic and eukaryotic microbial world, but to
date, only bacteria are known which produce typical mono- and
dihydroxybenzoic acid-bearing sidexophores.
Some microbial siderophores, their analogs and/or
their enantioforms have been chemically synthesized in the
laboratory:
(i) enterobactin, its enantioform and carboxylic,
methyl, and aromatic analogs;
(ii) Nl, N8-bis-2,3-dihydroxybenzoylspermidine;
(iii~ ferrichrome and enantio-ferrichxome.
Other processes for the preparation o~ various
siderophores are described in Canadian Patent Nos. 742,670,
746,~73, 773,540 and 775~539O
A discussion of the preparation of siderophores
by denovo synthesis can be Eound in Neilands Review 1981 and
Neilands et al J. Biol. Chem. 256 ; 3831 - 3238, 1381.
The ferrioxamine B is sold commercially under the
designation Desferal which is a trademark of Ciba-Geigy~
The microbial siderophores, ferrioxamine and
enterohactin, referred to above are prototypical natural micro-
bial siderophores and each represents the general structure
and properties of hydroxamate and catecholate-bearing sidero-
phores respectively. These particular siderophores will be
referred to below (e.g. m the exa~ les). For the purposes o ~is speciica-
tion-the expression des as it appears before ferrioxamine e-tc is to be under-
s-tood to refer to errioxamine etc wl~erein co-ordinating sites are unoccupied e.g.
-they are not iron loaded.
Turning now to carriers suitable in accordance with
the present invention, they must of course be insoluble in
the liquid medium of intended use; for example, the carrier
can be water insoluble. Desirably, the carrler is also
inert in the liquid medium of intended use. The carriers
can be in particulate or solid form.
The carrier can be an organlc or inorganic compound.
For example, the carrier may be a natural or modiflecl natural
polymer (e.g. lignin, agar, alignate, glucan, ce]lulose, dex-
tran, cellulose aceta-te, humic acid, e-tc.) a synthetic organic
polymer (e.g. a polyamide, a polyamine, a polyacrylamide, a
polyester, a polyurethane, a polyethylene, a polystyrene, a
polypropylene, a polycarbonate, a silicone, nylon, latex, a
poly1uroolefin, etc.) or an inorganic material (a ceramic,
a glass, carbon, etcO~.
As indicated above the present invention provides
a (siderophoric) composition comprising one or more microbial
siderophores which are covalen-tly immobilized or fixed on a
suitable insoluble carrier in such a way ~ha~ the microbial
siclerophores re-t~in the:ir high chela-ting affinity for metal
ions, i.e. iron.
A number of known processes are suitable for the
binding of microbial siderophores to carriers so as to preserve
the iron chelating or cornplexing properties thereof. Forexample,
the commonly used methods Eor covalently binding enzymes to
insoluble carriers can be adapted Eor the immobilization of
microbial siderophores. See, for example ~Me-thods of Enzy-
mology, XXXIV s:30 (Jakoby W.l3. Ed.) Academlc Press, New
York (197~).
Carriers which are su:Ltable for the process oE
prepc3ring slderophoric compositions using microbial siderophorcs
are ~.hose which have active surfaces; the active surfaces have
- 16 -
6~
functional groups which can bond to a cornpatib]e functional group`
of the chosen siderophoric compound. The Eunctional group can,
for example, be selected from the class consisting of
O O
2~ (CH2)n-NI12, n being 0, 1, 2 3 etc
OH
~-H~-CH2-X, X being a halogen atom, for example, Br, O-C - NH,
-OH ~ -C-XI X bein~, as defined above~-C-N3-SO3H,and ~ N2 .
Howevex, any functional group can be used which will react
with a functional group on the microbial siderophore in
question to bind it to the carrier, the microbial siderophore
retaining its iron chelating capacity.
It is possible to put some distance between a
microbial siderophore and the surface oE the carrier, e.y.
in order to limit the effect on the microbial siderophore
of a surface characteristic of the carrier. For
example, teflon may be used as a carrier. However, teflon
has a highly hydrophobic surface which is non-wetting. There-
fore, it is desirable to put some distance between ths sur-
face of the teflon and the microbial siderophore to allow
the siderophore to extend well into an aqueous liquid medium.
A spacer compound may be used to provide a spacer
group to space apart a carrier and a sideroph~re.
A suitable spacer compound is bifunctional; i.e.
it has a functional group which can react with a functional
group of the carrier to bind it thereto; and it has also a
second functional group which can react with a compatible
functional group on the chosen microbial siderophore to bind
it thereto: see the above groups. The spacer ~roup may
alternatively have a second functional group which while not
reactive with a compatible functional group on the siderophore,
may be convertible into such a group.
spacer compound can,for example, tn addition to
~- 17 -
}~7
the above referred to functional groups, include a hydrocarbon
chain, the length of which is chosen in accordance with the
distance which it is desired to place between the carrier and
the siderophore. The spacer compound used may be l-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride salt or
glutaraldehyde. However any compound can be used which will
space the microbial siderophore from the carrier, the necessary
or desired distance provided of course that it is bifunctional.
The spacer compound may be bound, to a carrier by
making use of conventional reac-tions involving the formation
of ester groups, amide groups, amino groups, diazo groups,
ether groups, sulphonamide groups, amidino groups; the
reaction may be a carbon-carbon condensation.
Thus a carrier suitable for the process of the
present invention may be represented generally by the formula
r
back t R - ~cn J
wherein n is an integer, "back" is a carrier backbone,
i'R"is a single bond or a suitable spacer group and " ~cn" is a
functional group as defined above. For example " ~cn" may
be a carboxyl group and R may be a group such as
( 2)2 C NH - (CH2)2 - NH - ~ - (CH2)n -
A useful carrier may need to have its surEace
treated in order to provide the surf ace with a suitable
Eunctional group which can bond to a microbial siderophore.
Nylon, for example, is a carrier which requires a
pretreatment to provide it with suitable Eunctional groups.
Since the nylon eontains the amide group, its surEace may be
sub~ected to partial hydrolysis uslng known techniques -to glve
Eree amino and carboxyl Eunctional groups. The aqueous metho~l
may proceed ~s below:
- 18 -
~z~c~æ~
O H O H
R - C - N-R HCl ~E~ - C - OH HN - R
, 1
Mylon (R being Modified Nylon
the rest of the chain~
Alternatively, nylon can be reacted in a non-
aqueous~edium with, for example, thionylchloride to give
rise to the functional group - C = N. If desired, an
Cl
appropriate spacer group can be readily attached through~
for example, the use of ethylene diamine or another func-
tionally equivalent species such as N02 ~ NH2 followed
by a reduction of -N02 to -NH2 suitable for generation of
diazonium salts which are then suitable for coupling to a mucrobial siderophore,e.g. enterobactin. Succinic ~nh~dride can thereafter be used as a further
extension of the spacer group; i.e. to form an amide linkage.
Teflon is another useful carrier which must be
pretreated in order to provide it with a suitable functional
-,~ group which can bond to a microbial siderophore.
Teflon~ a tradename or pol~vtetrafluoroethylene
from DuPont, is highly inert and is not readily attacked by
acids and bases. No easy displacement of the fluorine
atomes is known. Fluorine atom can,however,be displaced by
ion-radicals such as sodium or potassium napthalenP of
formula:
Ell NO
Na+
~ ~ El or
On reaction between te10n and such sodium or
potassiumnaphthalene, a sodio or potassio species of teflon
is formed of formula:
E;' E~`
--C -- C --
Na F
7-f~ r1~ ~ 19
~3LZ~t~
These organo metallic species of teflon are highly reactive
towards many organic functional groups and their general
behaviour is similar to the well known Grignard reagents.
Thus, they can be reacted with a dimethyl carbonate to give
rise to an alkoxy carbonyl substituted polytetrafluoroethylene.
This substituted ethylene can subsequently be subjected to
hydrolysis to provide a polytetrafluoroethylene with
carboxyl substituents. The carboxylated teflon thus generated,
can then be used for direct coupling to microbial siderophores
(or chelators) such as Desferal . As indicated above, it
may be desired to space the siderophore from the surface
of the teflon. If so, ethylenediamine and similar compounds
can be readily attached through the carboxyl group by standard
procedures. Since the teflon's backbone is very inert to
many organic and inorganic reagents, very vigorous reaction
conditions can be employed in further derivatization using
the carboxylic functional group. See, for example, ^-Methods
in Enæymology- Supra.
As indicated above, the microbial siderophore must
also possess compatible unctional groups which will react
with those of the carriers without interfering with the
chelating activity thereof. For example, suitable functional
group in the siderophore enterobactin is the 2, 3-dihydroxy
benzoic group which is susceptible to dia onium coupling
under neutral to almost neutral conditions. The acylamine
xequired in the generation of diazonium salts can be prepared
from aminopropylsilylated glass.
Examples of suitable functional groups on -the
microbial siderophores axe tha amino group, the carboxyl
group, the phenolate group and the cathecolate group, etc.
The previous comments relating to carriers,
spacer groups etc for microbial siderophores apply to the
use of other s:iderophoric compounds, ~or example catechol.
II cakechol i5 used ~s the ~iderophoric compound it may be
- 20 -
~ Tr~de ~
bonded to a carrier by diazo coupling or through reaction
with a functional group on the carrier such as
o
- C - H . In these latter cases -the functional yroup on the
carrier is directly reactive with -the benzene ring of the
catechol.
When using a composition in accordance with the
present in~ention, the conditions of use should of course
be such as to avoid the break--down or decomposition of the
composition; i.e. conditions such as pI-I, temperature,
pressure, etc. should be chosen so as to avoid the break-
down of the composition.
As indicated above, an insoluble (siderophoric)
composition, in accordance with the present invention,
can be used to remove iron from a liquid medium. In use,
the (sideorphoric) composition is intermixed with a
desired liquid medium for a suitable time, whieh will of
course depend upon the amount of (siderophorie) eomposi-
tion used, the initial iron eoncentrat on, the desired
final iron eoncentration, etc. The Fe in the medium
combines with the Isiderophoric) composition and can thus
ba physically sepa~ated from the medium. The affinity of
(siderophoric) eompounds for iron can be so great that
even small amounts of iron can ~e removed from a liquid
medium. The ~irst concentration of iron in a treated
nutrient medium can for example be far below that req~lired
to support mierobial growth.
In drawings which illustrate the present invention
Figure l is a yraph illustrating the inhibition oE microbial
growth due to the removal of iron from liquid
medium,
re 2 illustrates regeneration o-f a siderophoric com-
posi-tion by pH manipulation; and
FicJure 3 illustrates regerleration of a siderophoric compo-
sition by a reducing agent.
357
Figure 1 as indicated above is a graph illustra-
tive of the inhibition of microbial growth in even the most
nu-tritional - - -
- 21 a -
~Z~ ;7
solutions (e.g. bacteriological broth medla~ on removal oE
iron therefrom.
In particular, fi~ure 1 illustrates the inability
of the bacterium Neisseria menin~itidis to grow in a complex
-
highly nutritional medium (neisseria defined medium ~ NDM)
from which only iron has been extracted with ferrioxamine
immobilized OllagarOSe~ the agarose having previously been
activated by cyanogen bromide for coupling to ferrioxamine.
Thus, one gram of the above siderophoric composition was
contacted with 200 ml NDM at 22C for a time period of
20 min. before recovering the siderophoric composition.
Prior to treatment, the NDM contained about 3.6 ~M of iron;
after treatment, it contained less than 0.1 ~M iron. The
treated medium was then divided in-to two portions and FeC13
was added to one of them~ A control consisting of untreated
NDM and -the two portions were then inoculated with microbes
and maintained at a pH of 7.4 and a temperature of 37C.
As can be se~n in figure 1, unhibited growth occurs in
the control ~0)~ However, in the treated medium, (~), cells
are unable to undergo anymore than one or two divisions due
to the absence of the vital nutrient iron. On the
other hand if exogenous iron is added back to the tr~ated
medium full growth is again realized (~).
~iquid media to be treated to remove Fe3~ can
2S have, for example, a p~I in the range of 4.5 to 9. During
the contact with the siderophoric composition, the temperature
of the mixture can for example range from 1C to 50C and
the contact can occur under atmospheric pressure. Fxamples
of different media which can be treated with the composition
are listed in table 2 which follows:
- 22 -
~2~ 7
Table 2
Classes of liquid ~edia Specific example thereof
Liquid foods - fruit and vegetable ~uices, clear
meat broth (e.g. consommé), culture
media for microbial, plant and
animal cells
Breveragec - wine, beer, natural and synthetic
juices, cider, drinking water
~harmaceutica] - buffer solutions for lavage ie.g.
ophthalmic solution, peritoneal
lavage), water used in the manu-
0 facture OI various solutions and
1 preparationS, antibiotic solutions,
Cosmetics (li~uid) - those susceptible to microbial
degradation, contamination, or
spoilage
Indus-trial water and - cooling tower, process and waste
waste water water
Natural water - removal of actinides (e.g. plutonium)
and chromium
The sidephoric composition can, as indlcated above,
for example, be used to remove iron from microbial fermentation
cu]tures to stop further growth o~ microbes in the fermenter.
Thus, the siderophoric compositio~ in accordan~e with the
present invention may be used to treat wine in order to inhibit
microbial growth therein.
The composition of the present invention may also be
used to remove iron from cosmetic solutions to prevent contamina-
tion by the growth of microbes. Components for cosmetic solu-
tions are often obtained from natural sources and are susceptible
to microbial degradation.
The siderophoric composition oE the present invention
may also be used for the removal of iron from drinking water,
pharmaceutical and biological solutions, and industrial
water.
- 23 -
Al-though -the microbial siderophores are selective
~or iron, they can also bind me-tals that are classified as
ac-tinides e.y. plutonium. Thus, the present inven-tion addi-
tionally provides means for removing such hazardous metals
as plutonium ~rom contam~ated wa-ter; and a rapid means to
collect (concentrate) the radioactive metals (e.g. pluto-
nium) to determine the concentration thereof in standard
water volumes. Such metals are selectively removed from
water due to their structural similarity (i.e. atomic) to
iron.
As indicated previously, compositions in accor-
dance with the present invention~ can be regenerated for
further use by the removal of the iron therefrom by suitable
chemical means. In this way, the composi-tion can be econo-
mically used since it can be recycled ~or repeated use.
The regeneration, for example, of a composition
loaded with iron, can be carried out either through -the
manipulation of the pH of a medium surrounding -the iron-
loaded ~siderophoric) composition and/or by treating the
iron-loaded composition with a suitable reducing agent.
In either case, appropriate conditions should be chosen
which will not decompose the composition or destroy -the
iron binding capacity -thereof.
If regeneration is affected by manipulation
of the pH, the pH must be brought to or beyon~ a point at
which the iron is released.
In general, when making use of a microbial sidero-
phore, a pH oE 1 or lower should be avoided. The use o~
mineral acids should also be avoided. The pH can be manipu-
lated through the use of organic acids (for example,
acetic acid, s~ccinic acid, citric acid, isoci-tric acid,
ketomalonic acid, malic acid, oxalic acid or pyruvic
acid).
Figure 2 illustra-tes the regenera-tion oE a
siderophoric composi-tion by the manipula-tion of pH. The
2~ -
i7
designation (~) represents en-terobactin immobiliæed on a
polyacrylamide carrier whereas the designation (O) repre-
sents ferrioxamine immobilized to the same type of carrier.
The pH was lowered in the presence of 15 mM ci-tra-te and
0.05 M ~ - _
- 24 a -
0~
tris-s~di.um acetate. The lowering of the pH was accomplished
by an addi.tion of appropriate amounts of acetic acid.
Alternatively, the iron loaded siderophoric com-
position may be treated with a suitabl.e reducing agent to
release the iron. In accordance with the presen-t invention,
it is possible-to llse suitable di-thionites or ascorbates as the reducing
agents, e.g. sodium or potassium dithionite and sodium or potassium
ascorbate. The dithionites can be used for the reduction
of compositions which include hydroxamate ligands
whereas the ascorbates can be used for the reduc-tion of
siderophoric compositions containing phenolate/catecholate
group ligands. Other useful reducing agents include hydro-
xylamine and hydroquinone.
The siderophoric compositions wherein the sidero-
phore includes catecholate ligands (e.g. siderophoric compo-
sitions consisting o enterobactin fixed to glass) and
especially the siderophoric compositions containing diazo
linkages must be subjected to mild reduction conditions
such as provided using ascorbic acid. The siderophoric
compositions which include hydroxamate groups ~Desferal),
can be reduced with 1.0 molar sodium dithionite.
Other reduciny agents may possibly be used to
regenerate the sidephoric composition; however, the reducing
agent used must be chosen on the basis that it ~ill no-t
25 destroy the integrity or iron-bindi.ng capacity of the sidero-
phoric compositio~. Sodium dithionite (Na2S2O4),hydroxylamine
(including its acid addition salts) and hydroquinone are as
indicated above examples of useful reducing agents. In par-ticular
the reducing agent can be hydroxyl amine chloride.
rrhe regeneration of the composition
may take place in the presence of a suitable organic acid
that will complex with the iron that is rcleased. Suitable
acids are di or tricarboxylic organic acids that will
chelate the Iibera-ted iron ions.
- 25 -
~z~
Figure 3 illustrates the repeated regeneration of sidero-
phorlc compositions consis-ting of enterobactin ( ~ ) or ferrox~mine ( ~ )
bound to polyacrylamide carriers, the reducing agent consisting
of sodium ascorbate. The regeneration solution had a pH of
7 in the presence of 15 mM sodium citrate and 0.05 M tris-
sodlum acetate.
The insoluble composi-tions in accordance with
the present invention thus provides for -the advantageous
removal of metals (e.g. iron) from liquid media. Such media
remain essentially unchanged except for the absence of iron.
The liquid media reEerred to herein may be aqueous, organic
or mixtures thereoE.
Reference will now be made to a number of examples
which deal with embodiments of the present invention.
Example 1~ Activation of silica gel (glass)
The activation methods were analogous to those as
described by H. Weetal & A.M. Filbert, Methods of Enzymology
XXXIV B:59~72 1974.
(a) Pretreatment
-
lOO grams of silica gel designated Sigma S-4133
(sold by Si~ma Chemical Company) of 100 to 200 mesh
(70 to 140 microns) chromatographic grade and of pore
diameter of about 25 angstrom was suspended in a
- 26 -
.., :, .
"~{ ." f ~ ~
mixture of 50 ml of 70% HNO3 and 300 ml of
distilled water. The suspension was re1uxed
~ith mixing for about 1 hour.
The gel was allowed to se-ttle and the liquid layer
drawn ofE. The gel was then washed repeatedly
with distilled water until the wash water was
about neutral pH.
(b) Amine activated silica gel:
The above pretreated silica gel (wet) was used for
the following amination without drying. A 10%
solution of ~-aminopropyltriethoxysilane in
distilled water 1500 ml) was added to the above
obtained silica gel. The pH was then adjusted
to 3O45 with 6N hydrochloric acid. The suspension
was then maintained under stirring at a temperature
o 75C for about 3 hours. The gel was then
filtered and washed with 500 ml of distilled water
and dried in an oven at 100 to 110~C.
The above amination can be described graphicall~
as follows:
Et
~ OH O
glass - f ~ i-OH ~ Et-o-si-cH2-cH2-c~2-NH2 aqueO~15
O o 75C.
Et
- 27 -
I-l Et
~ I O , if desired,
glass--~ Si-O-Si-CH2-CH2-CH2-NH2 ~ >
~ b O treated to add group
H Et -Si-OH
-Si- OH
H O to block reactive group
r , I of Et.
glass ~ Si-O-Si-CH2-CH2-CH2-NH2
0 0
El -Si-
I Et = ethyl
The above obtained amineactivated silica gel will
hereinafter be ref~rred to as glass fNH2
(c) Aminoarylcarbonyl activated silica gel:
50 grams of glass JNH2 wa5 suspended in 300 ml
of ethanol Eree chloroEorm. 2~5 grams p~nitrobenzoyl
chloride ~Eastman Kodak~ was subsequently admixed therewith.
- 2~ ~
:
~2~6~
Thereafter, 30 ml of dry trieth~lamine was added and the
resultant mixture was refluxed for 20 hours. The beads were
allowed to settle and the liquid phase drawn off. The beads
were then wash repeatedly with chloroform then repeatedly with
ethanol and finally with distilled water with ethanol.
The wet water-washed gel was subjected to a treatment for the
reduction of the nitroaryl group to an aminoaryl group by
adding the gel to a solution of 50 gm of sodi~ dithionite
in 250 ml. of distilled water. The whole suspension there-
ater being refluxed with mixing for 45 min. The suspensionwas then filtered while still hot and washed repeatedly with
dilute hydrochloric acid and then washed with distilled water.
After thorough washing with distilled water, the gel was dried
in an oven at 70 to 80C; this t~pe of activated~carrier can
be used for subsequent diazotization and coupling.
The above reaction can be represented graphically
as follows ~2
/ ~ Chloroform H O
glass ~ NH2 + l J CHCl~glass J N-e- ~ No2
J ~ Triethylamine
C=O Et3N
Cl A
Reduction , R ~
2S2O4 ~ glass ~ N-C - ~ NH2
H20
29 -
S (d) Carboxyl activated s.ilica gel:
50 gm of glass ~ H2 (see above), were
suspended in 250 ml of water cold in an ice bath. 50 ml of
1 N sodium hydroxide was added followed by 15 gm.of solid
succinic anhydride. After mixing or 2 hours, the pH was
adjusted with the addition of 1 N sodium hydroxide to a
pH of 5-6. This adjustment of pH with sodium hydroxide was
repeated hourly three additional times and the mixture was
left overnight. The suspension was thexeafter filtered and
washed thoroughly with distilled water and dried at 100 to
1~0C
The above reaction can be represen~ed graphicall~
.20 as follows:
H
glass f N-H ~ CH2-CH2 NaOH _ ~ f
1 ¦ aqueous
/ CIH 2
HO-C=O
- 30 -
(e) Aldehyde activated silica gel:
20 gm of glass f NH2 ( see above) were
suspended in 50 ml of 0~1 M sodium phosphate at a p~ of 7
followed by the addition of 10 ml purified 8% glutaraldehyde.
The mixture was subjected to vacuum and mixed occasionally.
The reaction was allowed to proceed for 3 hours and the
mixture was thereafter filtered, washed thoroughly with
distilled water and dried under vacuum.
The reaction outlined above can be represented
gra~hically as follows:
H H H
glass ~ N-H ~ o=C-cH2-c~2-cH2-c=
H H
f l I
glass ~ N=C-CH2-CH2-CH2-C=O
- 31 -
~v~
Example 2: Activation of polyacrylamide (azide coupling)
60 ml of ethylene diamine activated polyacrylamide
gel (per Inman, Methods in Enzymology XXXIV
B:35 1974) was diluted with water to make up to
100 ml volume. About 1~2 y of p-nitrobenzoyl
azide was dissolved in 100 ml of tetrahydrofuran and
the obtained solution was subjected to filtration.
I'he obtained filtrate was added immediately to
the aqueous gel suspension referred to above.
1.5 ml of triethylamine was then added to the
suspension. The gel mixture was then stirred
gently for 30 min. An additional portion of
1.2 grams of p-nitrobenzoyl azide in 50 ml of
tetrahydrofuran was added -Eollowed by another
1 ml of triethylamine. Gentle stirring was
continued Eor an additional hour. The obtained
gel was filtered and washed-tho~ughly with
1:1 tetrahydrofuran: 0.2 M sodium chloride and
then resuspended in 0.2 M sodium chloride. 3 ml
of acetic anhydride was added to the suspension
which was then mixed for 1 hour, the gel being
thereafter washed withO.l M NaCl.
The obtained gel can be used directly for diazo
coupling.
The reaction can be represented graphically as
~ollows:
l3
C=O
- N-CII -CH2-~H2 + ~ - 3 ~ B f N--CH -CH2-~-C ~ NO
2 ~ triethyla~ine 2 2
N02
~ I-I H O
_ ~ B ~ N-cH2-cH2-N-c- ~ NH2
wherein:B = polyacrylamide backbone (ie carrier)
Example 3: Activati.on of agarose with cyanogen bromide.
Agarose swollen in water was mixed with
an equal volume of water. Finally divided CNsR
~50~300 m~ per ml of agarose~ was added atonce to
the stirred suspension. The pH of the suspension
was in~ediately adjustec~ and maintainad at pH 11 with
sodium hydroxide. The temperature oE the suspension
was maintained at 20C and the reaction allowed to
proceed ior about 30 mi.n. Thereafter t the gel was
then washed rapidly with a large amount of ice
cold water followed by washing with an appropriate
buffer. The obtained activated gel should be used
as soon as possible.
The chemical reactions involved in the
agarose activation can be indicated ~raphically as
~ollows:
agarose agarose
_ ~-OH e ~
+ CN Br ~ /C=N-H + H2O
_ I OH ~ O
_~ J
Cyanogen bromide, by analogous procedures,
can be used to activate other polysaccharides;
for example, alginate, glucans, cellulose/ agar,
~; dextrans, etc.
Example 4: Immobilization of ferrioxamine or enterobactin
1 mM HCl-washsd, water swollen CNBR acti~ated carrier,
obtained in accordance with Example 3,was mixed with
ferrioxamine or enterobactin (20 mg/mlP in an NaHCO3
buffer (O.1 M, pH 8.3) cc)ntaining 0.5 M NaCl.
The mixture was agitated overnight at 4C. The
gel was then ~7ashed w.ith 0.1 M acetate buE-Eer pH
4.0 containing 0.5 M NaC:L to remove excess uncoupled
ferrioxamine or enterobactin.
Example 5: Immobilization oE Des.~exal to polyacrylamide gel -
ta) Biogel P~150 polyacrylamide from ~ioRad was linked
through ethyLenediamine and then succinic anhydride
followin~ published procedures as described by
- 34 -
6~.3
Inman (John K. Inman, Covalen-t Linlcage of Functional
Groups, Ligands, and Proteins to polyacrylamide
beads in -Methods of Enzymology , XXXIV B, 30
(Jakobyr W.B. ed.) Academic Press, New York (1974)
and Biochemistry 8:4074 (1969)S (see examples 16 and 17)
infra. Unreac-ted free amino groups
in ethylene-diamine were blocked by reaction of
acetic anhydride at the end of the reaction period.
Test for presence of any free amino group was
through the TNBS ~trinitrobenzene sulfonic acid)
testn The addition of acetic anhydride was repeated
twice until TNBS ~est were negative.
This activated, extended polyacrylamide was immediately
used for coupling.
(b) Coupling of Desferal to activated polyacrylamide
gel
700 mg Desferal was dissolved in 5 ml oi deionized
water and followed by addition of163 mg ferric
chloride. A deep red solution was formed. 20 ml
activated gel, obtained above, was washed twice
wi-th ethanol by centrifigation and decantation.
The total solid shr~nk to a very small volume after
the second volume of ethanol (20 ml) was added.
The 5 ml of ferric complexed Desferal was added
at 20C -to this shrun~cen gel and the mixture was
mixed by swirling. Thè gel gradually swelled to
give a solid mass. 5 ml of H2O was added to help
disperse the gel and -the pH of the suspension
- 35 -
~2~
was adjusted to 4.2 with NaOH llN). 200 mg EDC
(l-ethyl-3-(3-dimethylamino propyl) carbodiimide),
200 mg was added in one portion and the pH of the
suspension monitored and kept at 4.3 to 4~6 by
addition of HCl (1 N), for 3 hrs. A further portion
of 200 mg EDC was added and the mixture, allowed
to stand overnight at room temperatuxe. The gel
was filtered on a sintered flass funnel and washed
with 0~2 M NaCl.
The reaction can be graphically represented as
follows:
PA
CH2 O H O O
I ~ 2 C~2 N l-C~I2~CH2-C-OH + H2N-CH2-Def ~ C2~5N=C=N
PA ~ CE13~ C~H2
~N CH2 CH2
~N3~
cle
2S PA H
H-C-C-N-CH -CH N-C-CH2-CH -C-N-CH -De~ t C2E12-N-C;O
1l t 2 2 1 11 2 2 ~3
~AO H H O ~N-CH -CH~-CH2
CH3H
cle
wherein: PA = polyacrylamide ~ackbone or carrier and
Def = desferal.
- 36 -
~z~
Example 6: Immobilization of ferrioxamine or enterobactin to
polyacrylamide carrier -
A swollen polyacrylamide gel acyl azide derivative
freshly prepared as in accordance with example 2
was suspended in a solu-tion containing the
Eollowing: 0.1 M CaC12, 0.001 N HCl acid,
ferrioxamine or enterobactin at 0.3 mg/ml (pH 4.0).
The pH was immediately adjusted to 9.0-and the
mixture was stirred for 60 min. at 0C. In the case
of enterobactin, the solutions are S0% ethanol.
The coupled gel waswashed with large volumes of
Q.05 M Tris-acetate-0.15 M citrate, pH 7Ø
Example 7: Immobiliæation of Desferal to silica yel (glass
beads) -
About 700 mg (or approximately one mMole) of
Desferal was dissolved in 25 ml of water followed
by the addition of 170 mg of ferric chloride.
The pH of the solution was then adjusted to 4.3
with one normal hydrochloric acid and to thls
was added 25 ym oE succinylated silica gel
characterized by the Eormula:
, R
ylAss ~ _ N - C - CH2 - CH2 - C-NH2
- 37 -
s~
200 mg of ~DC: HCl was added to this mixture which
was thereafter agitated for 3 houro at room
temperature. The pH of the solution was then
adjusted to 4.3 and the mixture allowed to stand
overnight. Aftex filtration, the obtained ~mpo~lte
was washed thoroughly with distilled water until
the washing water was colorless. Thereafter,
the obtained composite was dried under vacuum.
Example 8: Immobilization of enterobactin to aminoarylcarbonyl
activated silica gel-
5 gm of aminoarylcarbonyl activated silica gel
obtained as in step (c) of example 1, was mixed
with 10 ml of 1 M sodium nitrite and cooled in an
ice bath. 1 ml of concentrated hydrochloric acid
was added dropwise to the cooled solution. The
mixture was maintained in the ice bath for an
additional 45 min. to allow for complete diazoti-
zation. The mixture was then filtered in the cold,
washed with cold distilled water and, while still
cold, the gel was added to a solution of about
30 mg of enterobactin in 10 ml of ethanol including
about 2 ml of saturated borax solution. The
reaction was allowed to proceed for 1 hr in an
ice bath. The composite was then recovered by
flltration and wash~d with an ethanol water mixture
containing 0.1 normal hydrochloric acid and
thereafter with ethanol until the filtra-te obtained
- 38 -
~L2~
was colorless. The obtained composite was then
dried under vacuum.
The coupling reaction can be represented graphically
as below:
H OlNaNO2~ 1 1l
f ~ 2 HCl f ~ N2
1~ 0
+ Entexobactin~ glass-~ N-C- ~ N=
C=O ~
1 HO ~
10 i
~ OH ~f=o
OHEnterobactin
xample 9: Immobilization of cathechol to polyacrylamide gel:
diazo coupling -
20 ml of the gel obtained in accordance withExample 2, was suspended in 20 ml of 0.l normal
HCl and cooled in an ice bath. While the suspension
was maintained in contact with the ice bath, 2 ml
of l M sodium nitrite was added dropwise with
agitation ~f the suspension. The resultant
- 39 -
i7
mixture was kept in the ice bath for an additional
30 min. and centrifuged to remove the liquid phase.
The gel was then washed twice with ice-cold distilled
water and then placed in contact with 20 ml of an
ice-cold solution of 10~ catechol in saturated horax.
The reaction was allowed to proceed overnight
at 4C~ The composite was recovered by
filtration and ik was washed repeatedly with water
containing 0.1 normal HCl. The reaction can be
indicated schematically as Eollows:
PA f N- tCE12) 2-N-C-~ NH2 + NaN02 ~ HCl --~.
PA ~ ~l-(CH2)2-~-cl- ~ N~ + ~ OH
OH
PA f ~- (CH2 ) 2-N-t~ N--N~
t)l~ t)~
wherein PA is polyacrylamide carri.er or backbone.
0 EXample 10:Immobil:Lzation of catechol to aminoaryl
carbonyl activated silica gel-
20 gm of aminoarylcarbonyl activated silica gel
- 40 -
57
ob-tained in accordance with example 1 (c) was
.suspended in 20 ml of ice-cold water. 10 ml of
1 M scdium nitrite was added to the suspension
while maintaining the suspension in an ice bath.
10 ml of 2 normal hydxochloric acid (ice-cold)
was slowly added dropwise to the suspension.
The reaction was allowed to proceed at between
0 and 4C for about 1 hour and it was then
washed with ice~cold distilled water. The solid
was then added to 10 ml of 10% cathecol in
saturated borax solution (ice-cold) with mixing.
20 ml of water was added to facilitate mixing
and the reaction was allowed to proceed in an
ice bath for an additional one hour. The
composition was then allowed to settle and the
li~uid siphoned off. The composition was washed
with distilled water containing 0.1 normal
hydrochloric acid and ethanol. The composition
was rapeatedly washed until the washing water
was colorle6s. The composition was then recovered
- and dried under a vacuum.
The reactions involved with the catechol can be
represen-ted generally as below:
25 glass-~ N-C ~ NH2 2 glass ~ ~ N2
~ OH H 1i ~N- ~
OH OH
-- ~1 --
;7
Example ll: I~nobilization of catechol to aldehyde activated
silica gel -
5 gm of aldehyde activated silica gel (activated
as in example 1 ~e) abo~e) was suspended in 10 ml
of 10~ cathecol in saturated borax solution for
l hour. The mixture was then subjected to vacuum
evaporation and then heated while still under
vacuum at 70C or one hourO The mixture was
~hen cooled to room temperature and water was
added thereto, the mixture then being heated
at 70C for an additional hour. The mixture
was then ccoled to room temperature and 500 mg
of sodium borohydride was added and the mixture
was maintained at 70~C for a further hour.
The resultant reaction mixture was then cooled
in an ice bath and 5 ml of glacial acetic acid
was added dropwise with mixing. The reaction
was allowed to proceed for an additional 30 min.
and the mixture was then filtered. The recovered
composition was then washed repeatedly with
distilled water and ethanol alternately and then
dried under vacuum.
The general chemical reactions are believed to
be as follows:
- ~2 -
~2~
g f 2 CH2 CH2 C o + ~ Bora
OH
g f 2 CH2 CH2 1 ~ NaBH4
OH OH Sodium Borohydride
g f 2 CE~2 CH2 CH2 1 ~
H
OH OH
Example 12: Removal of iron from wine
The inability of wine to spoil after extraction
of iron usin~ the composition of the present
invention is illustrate!d in table 3 below~ This
protectlon from spoilage is retained even when
the wine is vigourousl~ aerated (agitated continu-
ally in open flasks at 25C). The treatment of
the commercial wine samples consisted of vigou-
rously aerating the sample after inoculating with
ac.etobacter xylinum, aeration continuing
for a period of about 30 days at 25~C.
- 43 -
Table3
. . .
TRE~TM~NT OBSERVATIONS
... ...
None By three weeks, wine was foul
smelling ~including açetic
acid smell); turbid from bacterial
growth
Filter sterilized~no bacteriaNo spoilage
(0.45 ~m pore size) added)
Iron extraction with No spoilage
siderophoric composition
of the present invention
Addition of iron ions By three weeks, wine was foul
to wine subjected to smelling (including acetic acid
iron removal by treat- smell); turbid from bacterial
ment with the sidero- growth
phoric composition of
the present invention
The following examples (i.e. 13&14) illustrate the procedure
which may be used to recycle siderophoric compositions in
accordance with the present invention i.e. recycling after
removal of bound iron. The reuse of the siderophoric
composition of the present invention makes it economically
attractive.
Example 13: Regeneration of an active iron-free siderophoric
composition comprising enterobactin fixed to
silica gel -
The siderophoric composition subjected to
regeneration can in general be represented bv
the following graphic formula:
H O
glass - ~ ~ C ~ N-N-enterobactin
The above siderophoric composition loaded with
iron, was subjected to treatment with an e~ual
volume of 0.1 M sodium ci-trate and 0.1 M ascorbic
- 44 -
~2~
acid, the treatment lasting for a period of
about 12 hours. The treatment was repeated
twice Eor a recovery of loaded iron in the range
of 95~.
Repeated loading and unloading of the siderophoric
composition with Fe3+ results in retention of
up to 95% of the original iron-binding capacity.
On average, each grarn of iron-loaded siderophoric
composition included about 212 to 232 micrograms
of iron per gram of composition. In the above
procedure, about 80-100mg. of siderophoric
composition were subjected to regeneration.
Example 14: Regeneration of a siderophoric composition
comprising a catechol fixed to a silica gel -
~a~ The siderophoric comp~osition can be xepresented
generally by the foll,owing graphic formula:
f ~ ~0
OH H
The siderophoric composition was subjected to
the same treatment as in Example 13. The iron-
loaded siderophoric composition contained from
110 micxograms to 163 micrograms of Fe per
gram of the composition. ~etention of iron-
bindlng caDacity after xegeneration was up to 9S%.
- ~5 -
~2~ i7
(b) The siderophoric composition having the
following general structure was subjected to a
reductive regenera-tion:
glass f N-CH2-CH2-CH2-b~I ~
OH OH
The iron loaded siderophoric composition contained
from 43 micrograms to 56 micrograms or iron per
gram of siderophoric composition. The sidero-
phoric composition was regenerated using 5 ml
of 0.1 M sodium dithiorlite. Treatment in this
way resulted in the recovery of 85 to 93~ of the iron-
blnding càpacity ofthe siderophoric composition.
Example 15: A siderophoric composition comprising ferrioxa-
mine fix~d to silica gel -
0The siderophoric composition was obtained in
accordance with the procedure outlined in
Example 7. The siderophoric composition had
an iron binding capacity of about 740 micro-
grams of iron per gram of siderophoric compo-
sition. 12 gm of the above composition when
exposed to 5 ml of a 500 ~M Fe solution was
able to remove or recover 79.1~ of the iron;
on being exposed to about 5 ml of a 5 ~M
Fe3 solution about 98.8% of this iron was
removed or recovered from the solution by the
siderophoric composition.
- 46 -
'1~2~6~5~l'
Example 16: Activation of Biogel P 150 with Ethylenediamine:
PA
fH2 PA
1~ 1
H-C-C-NH2 + H2N-CE~2 CH2 NH2 ~ CH2
PA H~~ --CH --OEl +NH
PA = polyacrylamide back bone:
All operations were carried out in a well
ventilated hood. 200 ml. anhydrous ethylene
diamine in a 500 ml. 3 necked round bottom
flask was heated with a heating mantle and
the final temper ture was reached and ad~usted
and maintained at 90~C ~ 2~C. The glass was
also equipped with a conden~er with outlet
protected by a dryi~g tube, a mechanical
stirrer and the third neck was used for
addition of materials and temperature monitoring.
10 gm of Biogel - P 150 was added through the
thermometer neck in one portion and the mixture
was stirred and heated at 90~C ~ 2~C for a
period of 3 to 4 hours. The solid gel swelled
to a great vol~e and the evolution of ammonia
can be a~certained by wetted pH paper at the
drying-tube outlet. At the end of the reaction,
the mixture was poured with mechanical stirring
on to a mixture oE 400 ml, ice and water (1:1).
Any gel aclhering to the flask can be washed down
- 47 -
57
by jets of watex. The gel was filtered while
the mixture was still cold. The gel was promptly
washed repeatedly ~ith 0.2 M NaCl and 0.00 1 N HCl
until the filtrate gave a negative TNBS test
(Trinitroben~enesulfonic Acid). The total gel
volume was about 170 ml. i.e. wet gel.
Example 17: Succinylation: Carboxylic arm extension:
~A PA
l~2 ~H2 IH2 ~H2 I~ ~
¦2 CH2 NH2 ~ C~ j O - ~ H f---C-N-CH2-CH2-N-H
PA IH2
~00~1
PA = polyacrylamide bacX-bone.
50 ml. wet gel (Biogel P-150~Ethylenediamine
activated) is suspendecl in 50 ml. 0.1 N NaOH
in a 250 ml. beaker. Ext~rnal cooling in an
ice bath and gentle mechanical stirring is
also provided. 1.~ gm. succinic anhydride
(10 mmole) was added in one portion and the
mixtur~ stirred in the cold for 2 hrs. A
Eurther 1 gm. portion of succinic anhydride
was added with further cooling and stirring
for an additional hr. During the addition
of the second portion of succinic anhydride,
the mixture pH is monitored lntermittently
~ ~8 -
ail;~O605i7
with a pH meter and additional amounts of
1 N NaOH were added to maintain a pH of 3.5
to 4Ø A third portion of 1 g. succinic
anhydride was added and the monitoring procedure
was same as the previous addition. The TNBS Test
showed that there were still free amino group
on khe gel and these were blocked by addition
10 ml. acetic anhydride and stirred for
30 min. The mixture was eventually washed
thoroughly with 0.1 M NaCl. TNBS Testwas negative
for the gel.
~ 49 -
