Classifications

• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
  • A62D3/30—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by reacting with chemical agents
  • A62D3/37—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by reacting with chemical agents by reduction, e.g. hydrogenation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C17/00—Preparation of halogenated hydrocarbons
  • C07C17/23—Preparation of halogenated hydrocarbons by dehalogenation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
• • A—HUMAN NECESSITIES
  • A62—LIFE-SAVING; FIRE-FIGHTING
  • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
  • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
  • A62D2101/20—Organic substances
  • A62D2101/22—Organic substances containing halogen
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F2101/00—Nature of the contaminant
  • C02F2101/30—Organic compounds
  • C02F2101/36—Organic compounds containing halogen

Definitions

• This invention relates to a method of use of metal chelate complexes, in particular metal complexes of certain porphyrins, corrins and phthal ⁇ cyanines in the dehalogenation of organo-halogen compounds, especially organohalogen pollutants.
• the invention also relates to novel metal-porphyrin and metal-corrin complexes, including some such complexes which are suitable for use in this method.
• Organohalogen compounds present an environmental pollution problem. They may enter soil and aquatic environments by several routes and may threaten the drinking water supply. They are generated in large quantities as wastes by the chemical industry.
• wastes may be by incineration, which is expensive, or more commonly precipitation followed by landfill dumping which is cheaper but leads to high local concentrations of these pollutants.
• An important class of organohalogen compounds are insecticides, which are introduced deliberately and directly into the environment, or else may enter the environment indirectly, for example as a result of processing sheep fleeces obtained from sheep which have been dipped in solutions of insecticides.
• organochlorine insecticides are briefly mentioned below.
• DDT often consists of a mixture of (1) with several other organochlorine compounds including: 1,1,1 - trichloro -2- (0 - chlorophenyl ) - -2- (p - chlorophenyl ) ethane, 1, 1 - dichloro - 2, 2 - bis (p - chlorophenyl) ethane (DDD or TDE), 1 - (p - chlorophenyl) - 2,2,2 - trichloroethanol, 1,1,1 - trichloro - 2,2-bis (o - chlorophenyl) ethane and bis (p - chlorophenyl) sulphone.
• Degradation of commercial DDT may lead to other organochlorine compounds being present. Lindane: 1,2,3,4,5,6 - hexachlorocyclohexane (2) :
• Dieldrin 1,2,3,4,10, 10 - hexachloro - 6,7 - epoxy- -1,4,4a,
• Organohalides are often extremely persistent, ie they are not easily degraded into environmentally acceptable or biodegradable products. Consequently they may be transmitted along the food chain to the detriment of higher forms of life including man. It is therefore desirable to find cheap and effective methods of degrading organohalides, particularly those which are persistent pollutants.
• Porphyrins are effective at causing and/or accelerating the degradation of organohalides by dehalogenation.
• Porphyrins, corrins and phthalocyanines are large, cyclic, metal-chelating, amines of related structure.
• Porphyrins contain the ring system (4) :
• porphyrin ring systems exist in nature, for example the protoporphyrin (4A), haematoporphyrin (4B), uroporphyrin (4C) and coproporphyrin (4D) systems:
• Corrins appear to exist in a number of isomeric forms in which the position of double and single bonds in their 19-membered peripheral ring system may vary, as will be seen in the discussion of structures 5A-5H below.
• Phthalocyanines contain the ring system (6).
• one or more of the central, nitrogen atoms may be bonded to one or more hydrogen atoms, or all h may be co-ordinated to a central metal atomy which may Itself be additionally complexed with one or more other liga ⁇ ds, for exa ⁇ rple to form the metal-co-ordinated centre:
• haematin haem and haemin have protoporphyrin ring systems with an Fe(lIl)OH, a Fe(ll) and a Fe(lIl)Cl centre respectively.
• Chlorophyll is a magnesium centred porphyrin ring.
• Such porphyrins are frequently found as parts of larger biological molecules, for example haemoglobin contains an iron-centred porphyrin complexed with proteins.
• metal-centred corrins include the cobalt centred corrins found in the vitamin B12 series of compounds, eg vitamin B12 itself, ie cyanocobalamin, also known simply as cobalamin, (5A):
• dicyanocobalamin which has a structure analogous to cyanocobalamin but in which two CN ligands are coordinated to the central cobalt ion and the ribazole residue does not chelate with the cobalt.
• cobalt centred corrins include hydroxycobalamin,adenosylcobalaminand cobaloximes.
• cobalt-centred corrins are those described by Bonnett et al (1971) ie Neovitamin B12, a stereoisomer of (5A) in which the H and CH 2 CH 2 CONH 2 substituents at ring position 13 are transposed, and the compounds (5Bi-vi) of structure:
• R 4 NHCH 2 CH(OH)Me. ii : Neocobinamide
• R 1 NH 2
• R 2 CH 2 CH 2 CONH 2
• R 3 H
• R 4 NH CH 2 CH(OH)Me. iii : Cobyric Acid
• R 1 NH 2
• R 2 H
• R 3 CH 2 CH 2 CONH 2 ,
• R 4 OH. iv : Neocobyric Acid
• R 1 NH 2
• R 2 CH 2 CH 2 CONH 2
• R 3 H
• R 4 OH. : Heptamethyl Cobyrinate
• R 2 H
• R 3
• Stotter (1976) describes reactions between DDT and iron-centred porphyrins such as cytochromes, haemoglobin and haematin (these latter two being Fe (II) and Fe (III) complexes of proto-porphyrin respectively).
• cytochromes haemoglobin and haematin
• Fe (II) and Fe (III) complexes of proto-porphyrin respectively The ability of other metals such as chromium and zinc to degrade DDT in vitro or in enzymic reactions is also discussed (in Stotter (1976) but without reference to porphyrins containing these metals.
• Ohisa et al (1980) refers to the dehalogenation of Lindane by iron-centred cytochrome P450.
• Bieniek et al (1970) describes the dehalogenation of lindane by cyanocobalamin. Both Bieniek (op cit) and Stotter (1977) refer to the dehalogenation of dieldrin (3) by cyanocobalamin. Neither of these references gives any kinetic data for the reactions of cyanocobalamin with these organohalides. Stotter (1976 ) and Jagnow et al ( 1977 ) refer to in vivo reactions between cyanocobalamin and a number of organohalides including chloromethanes, chloral hydrate and lindane. The degradation of DDT in ⁇ yano ⁇ obalamin-rich sewage sludge has been observed (Stotter op cit). Little work has been carried out however to identify the corrins which are most effective in the dehalogenation of organoholides, or optimum conditions under which dehalogenation may take place.
• porphyrins and corrins are attractive compounds for use in the degradation of organohalide pollutants.
• novel metal-porphyrin complexes being complexes of an ion of a metal selected from osmium, ruthenium or from Group 5,6,7,9,10,11 or 12 of the periodic table with a porphyrin selected from protoporphyrin, haematoporphyrin, Uroporphyrin or coproporphyrin or acid derivatives thereof in which the metal ion may be additionally ⁇ omplexed with a Kenya provided that if the complex is a cobalt protoporphyrin complex, the cobalt is additionally complexed with a ligand.
• the invention also provides novel complexes of iron (II) or (III) or a Group 2 metal with haematoporphyrin, uroporphyrin or coproporphyrin or acid derivatives thereof, in which the iron may be additionally complexed with a ligand.
• acid derivatives herein is intended to include complexes in which one or more of the COOH groups on the porphyrin ring are replaced by COONR 1 R 2 groups where R 1 and R 2 are independently alkyl or hydrogen, or salts of the COOH group with a counter cation.
• novel porphyrin complexes of this invention may for example be prepared by the following methods.
• the porphyrin complexes of this aspect of the invention may be prepared by incubating together in aqueous solution the porphyrin and the metal ion, preferably in a equimolar ratio, and with an equimolar amount of the ligand if this is desired.
• aqueous solution preferably in a equimolar ratio
• a chloride counter anion is suitable, but for protoporphyrin a solution pH of 5.5 (100 m M sodium acetate) appears to be necessary.
• protoporphyrin is hydrated to form haematoporphyrin in aqueous mineral acid solutions, so it is possible that when the reaction conditions described above for use with protoporphyrin are used the metal ion complexes with the haematoporphyrin.
• the carboxylic acid groups on the porphyrin substitution positions may be ionised so that the complex may be present in solution as a carboxylate anion or as the free acid.
• suitable conditions are 37°C, preferably in the dark.
• concentration of porphyrin, metal and ligand (if used) do not appear to be critical, but a convenient concentration is about 0.02M each. Under these conditions useful amounts of the complex are formed in about 30 minutes.
• the complex may then be isolated from solution using conventional methods, or may be stored in solution, preferably in the dark.
• the porphyrin complex may be prepared by a microbiological process, ie by culturing a suitable micro-organism which produces or secretes the complex or a precursor and then harvesting these from the culture medium using known harvesting methods.
• the use of certain enzyme catalysts may be advantageous, for example cobalt porphyrin synthetase has been shown to catalyse the insertion of cobalt ions into porphyrins.
• the invention also provides a method for dehalogenation of a halogenated organic compound, which includes the step of causing the halogenated organic compound to react with a reducing agent in the presence of either:
• Preferred porphyrin complexes for use in the method of the invention are those of the ions of magnesium, cobalt, nickel, molybdenum, iron and variadium.
• Other suitable but less preferred ions are calcium, borium, strontium, chromium, copper, manganese and zinc
• haematoporphyrin Generally complexes of haematoporphyrin are preferred, especially with cobalt (II), iron (II) and (III), magnesium, molybdenum (III), nickel (II) and vanadium (V).
• the ligands with which the metal may be additionally complexed are preferably inorganic ligands, especially CN-, ClO 4 - , SCN-, S 2 O 3 2- , SO 3 2- , Cl- H 2 O, NO 2 - NO 3 -.
• inorganic ligands especially CN-, ClO 4 - , SCN-, S 2 O 3 2- , SO 3 2- , Cl- H 2 O, NO 2 - NO 3 -.
• option (iii) is preferred, especially the use of complexes of corrins having a formula 5I:
• a preferred isomeric form for the corrin ring in 5I is that founi in structures 5A and 5B.
• the corrin ring in 5I preferably has substituents on the ring which enhance the solubility of the complex in water or enhance its ability to be bounl to a solid substrate. Preferably. there are up to 8 substituents independently selected from carboxylic acid-, hydroxyl-, or amide- terminated substituents or -OCH 3 or -OC 2 H 5 , in one or more of the 2, 3, 7,
• carboxylic acid- and amide- terminated substituents have a formula (CH 2 ) n COX where n is 0-3 and X is respectively OH; NR 1 R 2 where R 1 and R 2 are independently H, C 1-10 alkyl or C 1-10 hyiroxy-substituted alkyl eg (CH 2 ) m CHOH(C p H 2p+1 ) where m and p are independently 1-5.
• Hydroxyl terminated substituents preferably have a formula (CH 2 ) r OH where r is 1-5.
• substitution positions on the 5I ring if occupied by other than hydrogen are preferably occupied by substituents which do not interfere by sterically hindering the approach of the organohalogen molecule to the metal M or do by causing a deleterious electron shift in the complex.
• Suitable substituents on these other positions include small (eg C 1-8 ) alkyl groups especially methyl, amino. cyano or ester groups, or monocyclic substituents such as in 5C .
• the method may also work but less satisfactorilly when these other positions are occupied by larger organic residues such as the ribazole group present in dicyanocobalamin, but these may cause some interference.
• the corrin ring system in complexes 51 may conveniently be, or be derived from, known corrin ring systems such as those in 5A - 5H above .
• ester, amide and cyano substituents in 5A - 5H may be hydrolysed to form carboxylic acid groups, which may then be converted to amide groups if desired by known methods.
• substituents such as the ribazole side chain of cyanocobalamin may be removed eg by hydrolysis of the phosphate ester link to yield cobinamide.
• Preferred metals M in these corrin complexes are those of Group 2, 5, 6, 7, 8, 9, 10, 11 or 12 of the periodic table, but especially cobalt, nickel, molybdenum, iron and magnesium, particularly cobalt.
• corrin complexes of formula 51 in which M is other than cobalt may be prepared from known cobalt- corrin complex precursors such as those described above by reaction of a solution of the cobalt corrin complex with a chelating ligand which has a stronger affinity for cobalt than the corrin residue, such as EDTA, preferably to form an insoluble cobalt-chelating ligand complex which may easily be separated.
• a metal ion M may be inserted by incubating the corrin with a solution of the metal ion in a manner similar to that used for the porphyrin complexes above. Suitable conditions are reaction of the precursor with an equimolar amount of the chelating ligand in aqueous solution.
• .Preferred ligands A and B are inorganic ligands such as ClO 4 -,
• (a + b) is preferably 2 if M is cobalt II or III, and 0 if M is Ni II.
• Preferred corrin complexes of formula 51 are the products of hydrolysis of cyanocobalamin (5A) or Neovitamin B12, eg on hydrolysis with an aqueous acid (eg HCl) or alkali (eg NaOH) on heating.
• Particularly preferred corrin-cobalt complexes of formula 5I are those of formula 5J :
• R 1 is selected from NH 2 and OH
• R 2 is selected from H, CH 2 CH 2 COOH and CH 2 CH 2 CONH 2
• R 3 is selected from H, CH 2 CH 2 COOH and
• R 4 is selected from NHCH 2 CH(OH)CH 3 , OH and NH 2 .
• Particularly preferred cobalt-corrin complexes of formula 5J are:
• Neocobyric acid 5Biv
• R 2 H
• R 3 CH 2 CH 2 COOH
• Neocobyrinic acid ie 5Bvi having
• a preferred phthalocyanine for use in this method is cobalt (II) phthalocyanine, which is a known and commercially available compound.
• the method of the invention is preferably carried out in an aqueous medium, eg in aqueous solution.
• aqueous medium eg in aqueous solution.
• pH does not appear to be too critical and a range of acid and alkaline pH can be used.
• Corrin complexes appear to be rather more pH dependent. In both cases a convenient pH is about pH 9.
• the method is found to work over a range of temperatures, and the rate of dehalogenation generally increases with increasing temperature.
• ambient temperature ca 20°C
• an upper optimum practical limit appears to be ca 80°C. In some cases illumination of the medium may be beneficial.
• porphyrin, corrin and phthalocyanine complexes used in the method of the invention may in some cases have one or more functional substituents such as amine, amide, hydroxy, azo and acid groups and may In some cases have a complex stereochemistry, they may exist in a number of ionised or proton ated forms depending upon the pH etc of the medium in which they are contained, and may also exist in a number of complexed forms or in an ionised form combined with a counter-ion eg C1.
• the method of the invention includes all such forms of the complex, and all stereoisomeric forms thereof. It is essential that the medium in which the reaction of the method takes place contains a reducing agent.
• inorganic reducing agents such as borohydride, dithionite, sulphite, phosphite, hyp ⁇ phosphite, sulphide etc.
• Organic reducing agents such as dithioerythreitol, dithiothreitol, mercaptosugars etc may also be used. Many such chemical reducing agents may be used and will be well known to those skilled in the art.
• microorganisms which create a reducing environment or are capable of carrying out reduction may also be employed as a reducing agent, and included among these are known anaerobic microorganisms such as those which are found in soil.
• the method of the invention is found to work over a wide range of concentrations of the complex, the organohalogen compound and the reducing agent.
• the medium contains at least one molar equivalent of the reducing agent per atom of halogen to be removed.
• the complex is not used up in the method, but appears to function essentially as a cafelyst assisting transfer of electrons from the reducing agent to the organohalogen compound.
• the ratio of complex to organohalide does not therefore appear to be critical.
• the kinetics of the reaction between the complex and the halogenated organic compound shows first order kinetics, and hence the rate at which dehalogenation occurs depends upon the relative concentrations of the complex and the compound. These may be varied between wide limits and will depend upon the environment or medium in which the reaction occurs.
• the method may be used at organohalide concentrations down to ppm level as experienced in many pollution situations, and experiments have shown the method to be effective at 1 ⁇ 10 -5 M concentrations of organohalide.
• the method of the invention is suitable for dehalogenation of a wide range of halogenated organic compounds, wherein the halogen (s) may be chlorine, bromine or iodine.
• the method is more effective at dehalogenating compounds in which the halogen atom is attached to an aliphatic carbon atom than those in which the halogen is attached directly to an aromatic ring.
• the compounds may for example be alkyl halides, alkenyl halides, halo-alkanes or alkenes, halogenated nitrile or nitro compounds, aryl-alkyl or aryl-alkenyl halides, halo-ethers, or other halogenated poly-cyclic aliphatic or aromatic compounds etc.
• the method is particularly attractive for the degradation by dehalogenation of halogenated organic compounds which are considered to be environmentaL pollutants, for example DDT, Lindane, dieldrin, hexachloro-butadiene (considered a priority pollutant by the European Economic Community). It is possible that in some circumstances the method may be used to degrade the pollutant dioxin.
• Other organohalogen compounds for which the method may be used will be apparent to those skilled in the art.
• the method of the invention may dehalogenate these compounds to form products which are considered to be environmentally acceptable or which may be further degraded by other processes.
• the method of the invention may be used to cause an initial didechlorination of Lindane to tetra-chlorocyclohexene, followed by a further dechlorination to monochloro-benzene.
• the method may for example be applied by simply mixing an appropriate quantity of a metal-porphyrin, -corrin or -phthalocyanine complex with an industrial waste solution containing the undesirable organohalide, ensuring that the solution is reducing, adjusting the pH and leaving the mixture to stand for a suitable length of time, during which the quantity of the organohalide may be monitored.
• Other methods of treating such industrial wastes, or polluted rivers, lakes or ground etc will be apparent to those skilled in the field.
• the complex is immobilised on an insoluble substrate and a solution containing the halogenated organic compound is brought into contact with the immobilised complex.
• the solution may contain a reducing agent or the substrate may have a reducing agent bonded or absorbed onto it.
• a solution to be dehalogenated eg an industrial or agricultural effluent
• a solution to be dehalogenated eg an industrial or agricultural effluent
• the flow rate to achieve a suitable residence time for the solution to be dehalogenated will of course depend upon the nature of the compound to be dehalogenated, the concentration etc, but experimentation of a relatively straightforward, nature can determine these parameters.
• the substrate may be any Insoluble material, preferably otherwise inert to the solution, onto which the complex can be immobilised.
• the complex is chemically bound to the substrate by means of interaction between functional groups on the porphyrin, corrin or phthalocyanine ring and appropriate functional groups on the substrate.
• preferred substrates are materials which have functional groups which are capable of combining with these on their surface.
• amine (NH 2 ) and amide groups on the substrate may react with carboxylate groups on the complex, and carboxylate groups on the substrate may react with aatLne or amide groups on the complex, to form chemical bonds.
• suitable substrates having such functional groups are known in particular the substrates commonly used for affinity chroraatography.
• derivatised polysaccliarides such as celluloses, agarose, dextrose and dextran, polyacrylamides and copolymers of these materials such as polyacrylamide-agarose gells, which may be either cross linked or non-cross linked.
• derivativised inorganic oxides such as silica, alumina, titania or zirconia, or glass beads may be used.
• Polymeric materials such as polystyrene beads or ion-exchange resins may also be used.
• Preferred substrates are derivativised forms of the known polysaccharide sepharose, particularly AH - Sepharose and
• CH - sepharose having respectively amino and carboxylate functional groups on their surface.
• reaction scheme may be as below:
• the complex may be bound directly to the substrate by standard methods, eg the use of a carbodiimide coupling agent. It is preferred to bind the porphyrin or corrin first to the substrate and then to add the metal ion. Binding of the porphyrin or corrin to the substrate may also use standard methods, eg the use of a carbodiimide, but a preferred method for binding the porphyrin and preferred corrin complexes to a substrate which consists of a derivativised polysaccharide such as AH - or CH - Sepharose is to prepare a suspension of the substrate In a solution containing the appropriate porphyrin or corrin at pH7 then rapidly adjusting the pH to 12 followed by a rapid adjustment of the pH to 2, then finally rapidly adjusting the pH back to 7.
• a derivativised polysaccharide such as AH - or CH - Sepharose
• the substrate plus immobilised porphyrin or corrin may then be filtered off, washed, and then exposed to a solution containing metal ions, to form the immobilised complex.
• the immobilised complex is prepared in this way it is preferred to use a ratio of porphyrin or corrin to substrate in which the amount of porphyrin or corrin is in excess of that required to occupy all the available binding sites on the substrate so as to reduce any subsequent problem caused by the organohalogen compound binding to the substrate. It is also preferred to use an excess of the metal ion to ensure that as far as possible all the porphyrin or corrin rings contain a cmplexed metal ion.
• a water-insoluble substrate having immobilised thereupon a porphyrinr corrin or phthalocyanine complex as defined above for use in the method for dehalogenation of a halogenated organic compound.
• Suitable and preferred forms of such a substrate and immobilised complex are as indicated above.
• Such materials may be supplied separately for use in the method of the invention as discussed above, or for example contained in a cartridge through which an effluent solution to be dehalogenated may be passed.
• a complex of the porphyrin, corrin, or phthalocyanine either in a free form or immobilised on an immobilised substrate may be mixed with a suitable quantity of a reducing agent prior to use.
• a mixture may subsequently be introduced into an environment containing a halogenated organic compound to be dehalogenated, for example used containers for thse compounds prior to disposal.
• the method of the invention is preferably carried out with the halogenated organic compound present in an aqueous solution.
• the method may readily applied to the dehalogenation of halogenated organic compounds dispersed or suspended in an aqueous medium, or when contained in a solid or in a gas such as contaminated air.
• the solid may be suspended or dissolved in an aqueous medium so that the organohalogen compound partitions into the aqueous phase, where it may be dehalogenated using the method of the invention.
• the gas may for example be bubbled through an aqueous medium containing the porphyrin, corrin or phthalocyanine complex and the reducing agent.
• the gas is passed through a column packed with a bed of a substrate having immobilised thereupon a porphyrin, corrin or phthalocyanine complex as described above, and a current of an aqueous medium containing a dissolved reducing agent is also passed through the bed.
• a current of an aqueous medium containing a dissolved reducing agent is also passed through the bed.
• the organohalogen partitions between the gas and liquid phases and is dehalogenated in the liquid phase.
• the invention also provides an apparatus for dehalogenation of a halogenated organic compound using the method of the invention.
• the apparatus uses a substrate having immobilised thereon a complex as described above and incorporates a bed of such a substrate plus complex contained within a body and being capable of having a liquid or gas containing the halogenated organic compound passed through.
• Such an apparatus may incorporate a replaceable cartridge containing the bed, in a construction similar to a conventional water softener or gas scrubber apparatus.
• Figs 1 and 2 show apparatus for dehalogenation of halogenated organic compounds according to the invention.
• Porphyrins (15 ug) containing no metal ions (protoporphyrins, coproporphyrin, uroporphyrin and haematoporphyrin), were dissolved in 100 mM Tris/HCl buffer (150 ⁇ l) , pH 9.0, prior to incubation with equimolar solutions of a range of metal ions (present as the chloride) at 37oC for 30 min in the dark.
• the chromatograph was equipped with a 1.5 m ⁇ 4 mm silanised glass column, packed with 3% SE30 on a 100/120 mesh Supelcoport with notrogen (40 ml/min) as the carrier gas.
• the injector and detector were maintained at 250oC, whilst the column oven was maintained at 190oC.
• reaction mixture 10 ml Trie HCl buffer, pH 9.0, containing 5 mM DTT and 10 mg/l Lindane.
• the reaction mixture was equilibrated at 37oC prior to the addition of 100 ul of a sample of the complex solution prepared in 1 or 2 above, and then purged with nitrogen and sealed with a butyl rubber septum and incubated at 37oC.
• a hypodermic syringe was used to withdraw 0.5 ml samples, which were extracted with 1ml hexane:diethyl ether (85:15 v:v), and 0.5 ul of this extract was analysed by GLC as described above. 1C Results
• Table 3 shows comparative data for known metal/porphyrin complexes, for uncomplexed porphyrins and for the two known compounds cobalt phthalocyanine and cobalt protoporphyrin. In Table 3 the identity of the metal centre is indicated.
• Tables 1 and 2 demonstrate that metal/porphyrin complexes may be prepared which are effective at dehalogenating Lindane.
• Table 3 shows clearly that many metal/porphyrin complexes, and porphyrins without metal centres, are very ineffective in comparison with the specific complexes identified by the invention.
• Results are expressed as nmole lindane dehalogenated per minute per mg of porphyrin. All incubations were at 37oC in dark unless indicated otherwise.
• Cobinamide (5Bi, having two CN ligands complexed to its central cobalt III ion) (10mg) was dissolved in water (10ml) prior to addition of 0.1M HCl to pH 2.0, The absorbance spectrum of the sample before and after acidification was determined between 250 and 700nm in a recording spectrophotometer. The acidified sample was freeze-dried overnight and subsequently redissolved in 100 mM Tris/HCl buffer (10ml), pH 9.0, and the pH adjusted to 9.0 by the addition of 0.01M NaOH. The absorbance spectrum of the sample was then redetermined.
• Lindane dehalogenation was assayed in the following reaction mixture: 1 ml 100 mM Iris HCl buffer, pH 9.0, containing 5 mM dithiothreit ⁇ l and 10 mg/l Lindane.
• the reaction was initiated by the addition of 10 ⁇ l of a sample of a solution of a complex as prepared in 2A above, and the reaction mixture was purged with nitrogen prior to incubation at 37oC for 30 minutes.
• the reaction mixture was extracted with 2ml hexane : diethyl ether (85 : 15 v : v) and 0.5 ⁇ l of this extract was analylsed by electron glc.
• the chromatograph was equipped with a 1.5 m ⁇ 4mrn silanised glass column packed with 3% SE30 on a 100/120 mesh Supelcoport with nitrogen (40ml/min) as the carrier gas.
• the injector anl detector were maintained at 250oC, whist the column oven was maintained at 190°C.
• the following reaction mixture was used:10ml 100mM Tris HCl buffer, pH 9.0, containing 5 mM DTT and 10 mg/1 Lindane.
• the reaction mixture was equilibrated at 37°C prior to the addition of 100 ul of a solution of the corrin complex (1 ⁇ g/10 ⁇ L).
• reaction vessel was purged with nitrogen then sealed with a butyl rubber septum and incubated at 37°C.
• a hypodermic syringe was used to withdraw 0.5 ml samples, which were extracted with 1ml hexane:diethyl ether (85:15 v:v), and 0.5 ⁇ l of this extract was analysed by GLC as described above.
• Corrin Rate of Dehalogenation adenosylcobalamin 750 cyanocobalamin-sulphite complex 760 cyanocobalamin-perchlorate complex 780 cyanocobalamin.
• 800 cyanocobalamin-thiosulphate complex 800 dicyanocobalamin ca.800 cyanocobalamin-thiocyanate complex 820 cobinamide (with two CN ligands) 6750 cobinamide (without CN ligands) 6825 cobyrinic acid (without CN ligands) ⁇ 6825
• AH-Sepharose 4B (2g) was swollen in 0.5M NaCl (20ml), and washed with this solution (400ml). The Sepharose was then washed in demineralised water adjusted to pH 4.5 (100ml). Haematoporphyrin (40mg) was dissolved in water (40ml) and added to the Sepharose. The pH of the slurry was adjusted to pH 12 by addition of NaOH (10M) and held at this pH for 2 min. The pH was then adjusted to pH 2 by addition of HCl (6M) and held at this pH for a further 2 min. The slurry was then adjusted to pH 7 by further addition of NaOH prior to washing of the gel with distilled water.
• haematoporphyrin was bound to the Sepharose by carbodiimide coupling.
• AH-Sepharose 4B (2g) was swollen in 0.5M NaCl (20ml), and washed with this solution (400ml). The Sepharose was then washed in demineralised water adjusted to pH 4.5 (100ml).
• Haematoporphyrin (40mg) was dissolved in water (40ml) and added to the Sepharose. The pH of the slurry was adjusted to pH 5.0, prior to the addition of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to a final concentration of 100mM. The slurry was stirred overnight at room temperature in the dark, prior to washing with distilled water. This ratio of haematoporhyrin to sepharose ensured an excess of porphyrin to available binding sites.
• EDC N-ethy
• the substrate . plus porphyrin was filtered off, washed, and treated with a large excess of the metal ion (Img/ml) in solution eg as the chloride, in a molar ratio ion porphyrin of 10 : 1. Excess unbound metal ion was removed by extensive washing In deionised water. 3B Immobilisation of cobalamin onto CH-Sepharose 4B
• CH-Sepharose 4B (2g) was swollen in 0.5M NaCl (20ml), and washed with this solution (400ml). The Sepharose was then washed in demineralised water adjusted to pH 4.5 (100mlK Cobalamin (5A) (40mg) was dissolved in water (40ml) and added to the Sepharose. The pH of the slurry was adjusted to pH 5.0, prior to the addition of EDC to a final concentration of 100mM. The slurry was stirred overnight at room temperature in the dark, prior to washing with distilled water. Problems were encountered in the immobilisation of cobalamin onto CH-Sepharose 4B, probably due to the presence of the carbonyl group of the amide.
• QAE-Sephadex A-50 (5g) was mixed with alkali-modified cobalamin prepared as in 2C above (4mg/ml; 100ml) for 20 minutes, and the slurry then poured into a column and washed with distilled water
• Amberlite XAD-2 (4g) was added to a solution of cobalamin (5A) (80mg) dissolved in 100mM Tris/HCl, pH 7, (20ml). The amberlite was then washed in the same buffer (200ml). It was found that washing of the substrate plus corrin with water led to a slow leaching of the cobalamin from the Amberlite.
• Hydrazide-derivatized polystyrene beads (6mm diameter) (25) were placed in a solution of alkali-modified cobalamin (4mg/ml in water; 10ml), and the pH adjusted to 5.5 prior to the addition of
• Alkylamine-derivatized polystyrene beads (25) were placed in a solution of alkali-modified cobalamin (4mg/ml in water:ethanol
• alkali-modified cobalamin (3C product) When immobilised onto AH-Sepharose, alkali-modified cobalamin (3C product) was capable of the dehalogenation of lindane. Over 99% of a 10ppm solution of lindane could be dehalogenated by this system at a residence time in the column of 5 minutes. When immobilised onto QAE-Sephadex, alkali-modified cobalamin (3D product) dehalogenated over 99% of a 10ppm solution of lindane at a. residence time of 5 minutes. Both carbon tetrachloride and dichloromethane were dehalogenated by this system: 99% of a 100ppm
• alkali-modified cobalamin When immobilised onto alkylamine-derivatized polystyrene beads (3F product), alkali-modified cobalamin again catalysed the dehalogenation of over 99% of a 10ppm solution of lindane when the solution was delivered at a flow rate of 60ml/h. In addition, 99% of 100ppm solutions of either carbon tetrachloride or dichloromethane could be dehalogenated when pumped at flow rates of 60ml/h. 5. Gas-phase dehalogenation
• alkali-modified cobalamin (2C product) immobilised onto both QAE-Sephadex and alkylamine-derivatized polystyrene beads was tested for their ability to dehalogenate dichloromethane. Air saturated with dichloromethane was pumped through a column of either QAE-Sephadex (5ml) or alkylamine-derivatized polystyrene beads (25) to which alkali-modified cobalamin had been bound, at a flow rate of 400ml/h. In addition, 500mM DTT was pumped onto the column at a flow rate of 4ml/h.
• the gas phase emerging from the column was bubbled through diethyl ether/hexane (15:85; v:v; 10ml), which was periodically assayed by electron capture GLC for the presence of dichloromethane.
• the levels of dichloromethane passing through the columns in the absence of DTT were taken as controls.
• air saturated with dichloromethane approximately 400ppm was passed through the column of alkali-modified cobalamin immobilised to QAE-Sephadex, approximately 92% of the dichloromethane was dehalogenated.
• Figs 1 and 2 show respectively apparatus for dehalogenation of aqueous or gaseous effluent.
• the apparatus comprises a body (11, 21), having at its upper end an upper closure (12, 22) and at its lower end a lower closure (12A, 22A), the seals (13, 23) between the body (11, 21) and the closures (12, 12A, 22, 22A) being releasable but watertight or airtight as appropriate.
• Each body (11, 21) is largely filled with polystyrene beads (14, 24) having immobilised thereon a corrin (eg the 3F product) retained in place in the body (11, 21) by retaining grids (15, 25).
• valves (16A), (17A) In the lower closure (12A) are connected inlets (16), (17), through which flow may be controlled by valves (16A), (17A).
• the upper closure (12) is an outlet (18) flow through which may be controlled by a valve (18A).
• inlet (28) In the upper closure (22) there is an inlet (28), flow through which may be controlled by valve (28A).
• the tube (28) communicates with a sprinkler (29) inside the closure (22).
• outlet (30) in the closure (20) There is also an outlet (30) in the closure (20), flow through which is controlled by a valve (30A) .
• An aqueous effluent to be dehalogenated is passed through inlet (16).
• a solution of a reducing agent is passed through inlet (17), mixing with the effluent.
• the mixture passes up the body (11) and contacts the beads (14), upon which dehalogenation takes place.
• Dehalogenated effluent leaves via outlet (18).
• a gaseous effluent to be dehalogenated is passed through inlet (26).
• a solution of a reducing agent is passed through inlet (28) so as to moisten all the beads (24) with the reducing solution.
• dehalogenation of the effluent occurs, and dehalogenated effluent leaves via outlet (30).
• Excess reducing agent leaves via outlet (27) and may for example be treated with the apparatus of Fig 1 or otherwise disposed of.
• the grids (15, 25) prevent escape of the beads (14, 24).
• PCT/GB89/00478 81) Designated States: AT, AT (European patent), AU, B BE (European patent), BF (OAPI patent), BG, BJ (O
• a method for dehalogenation of organohalogen compounds e.g. environmental pollutants in industrial waste.
• the organo halogen is reacted with a reducing agent in the presence of a selected metal-centred corrin, porphyrin or phthalocyanine complex.
• Preferred complexes are hydrolysis products of cyanocobalamin, of formula (I), in which R 1 is NHi or OH, R 2 is H, CH 2 CH 2 COOH or CH 2 CH 2 CONH 2 , R 3 is H, CH 2 CH 2 COOH or CH 2 CH 2 CONH 2 , and R* is NHCH 2 CH(OH)CH 3 , OH or NH 2 .
• the complex is preferably immobilised on a substrate.

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• 1989
  • 1989-05-05 EP EP19890905455 patent/EP0415963A1/de not_active Withdrawn
  • 1989-05-05 BR BR898907426A patent/BR8907426A/pt not_active Application Discontinuation
  • 1989-05-05 WO PCT/GB1989/000478 patent/WO1989010772A2/en not_active Application Discontinuation
  • 1989-05-05 AU AU35521/89A patent/AU3552189A/en not_active Abandoned
  • 1989-05-05 JP JP50515589A patent/JPH03504450A/ja active Pending
  • 1989-05-08 CA CA 598990 patent/CA1338313C/en not_active Expired - Fee Related
• 1990
  • 1990-11-07 GB GB9024249A patent/GB2242428B/en not_active Expired - Fee Related

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