CA2116652A1 - Tetraphenylporphyn derivatives - Google Patents

Abstract

A hapten formed of a metalloporphyrin cofactor bound to a residue of a substrate. The hapten is designed to mimic a transition state of a metalloporphyrin catalyst and the substrate in a reaction and is characterised in that the metalloporphyrin cofactor is of formula (II), in which: R1, R2, R2', R3, R3', R8 and R8' are as defined in the specification; R4 is a bridging group connecting the metalloporphyrin catalyst to the residue of the substrate; and M is a metal ion having a co-ordination number of at least 4. Also claimed are metalloporphyrin cofactors in which R4 is H, a linker group or a removable protecting group.

Description

WO 93/07150 Pl~/EPgl2/0;!283 TETRAPHENYLPORPHY~ DERIVATIVES

This in~ention relates generally to catalytic, antibody controlled processes in which tetraphenylporphyrin catalysts are used. The invention also relates to tetraphenylporphyrin derivativesr haptens containing them, antigens containing the haptens, and antibodies raised to the antigens, which ha~e application in the processes.
Metalloporphyrins are able to act as catalysts for many types of chemical reactions; especially oxidative transformations suZch as hydroxylati.on reactions, dealkylation reactions, epoxidation reactions, deZsaturation reactions and the like (~or example, Dixon, ~. and Webb, E.C.; Enzymes, 3rd Ed.; Academic Press, 1979; Collins, J.R.
et al; l991; J Am. Chem. Soc., ~13, 2736-2743 and Rettie, A.E. et al; 1988; J. BioZl. CheZm., 263, 13733-13738). However the selecti~ity of chemo- and/or regio-selecti~e attack (or oxidation~ of mekalloporphyrins is usually poor and cannot be predeZZtermined for any given substrate. Usually when a metalloporphyrin catalyst is used, a complex mixture of isomeZric anZ~l non-isomeric products will be obtained, which are difficult to separate.
To control product formation, it has beeZn recently proposed to use meta~lopor~hyrin~ as catalysts or cof~ct~rs in antibody mediated reactions; the antibody bringing site-salectivity to the process ~Schwabacher, A.W., Weinhouse, M.
I., Auditor, M.M a~d LZer~er, R.A., 1989; J. Am. Chem. SocZ., 111, 2344 to 2346). The authors' proposal is to immunise an animal with a complex of a substrate and a metalloporphyrin chosen to~bind to the substrate. Antibodies having binding sites that are complementary to both the pcZZrphyrin and the substxate are then isolat ed. It is then proposed to bind a porphyrin catalys~ to such an antibody so that only substrate that is correctly orientated can be bound and reacted.
Schwabacher et al managed to prepare antibodies to Fe3 35 and Co3~ complexes of synthetic meso-ketra-kist4-carboxyphenyl)porphine by coupling the complexes to keyhole ' WO93/071~0 2 11 6 6 ~ 2 PCT/EP92/02283 limpet hemocyanin (KLH) or bo~ine serum albumin (BSA) ~nd applying standard monoclonal techniques. However no further steps of the proposed process were carried out.
EP 0305870 discloses a similar concept, in general terms, in which an immunoproximity catalyst ~or chemical reactions is preparQd by selecting a hapten which coxresponds to, but is different from, a transition state complex of the reactant and a catalyst. An i~mune response is then stimulated using an antigen derived from the hapten to produce antibodies to the hapten. The antibodies are then isolated. '7Converting" hapten~ are then u~ed to covalently bind the catalyst to the antibodies to produce "modified" antibodi~s. The modifiad antibodies are then isolated for uYe. These modified antibodies catalyze cleavage of bonds in the target molecules; much in the manner of an enzyme. The antibodies are said to ~peed up the reaction and to introduce ~ite-~pecificity~
EP 0305870 ~uggests that the catalysts could be general acid-base catalysts, nucleophilic catalysts, electrophîlic catalysts and metal catalysts. No speci~ic mention is made of metalloporphyrins. Also, for many proce~ses, the isolation of a transition state complex for many of these catalysts may well prove to be difficult, if not impossible.
In any e~ent, EP 0305870 does not disclose any specific proce~ examples which illustrate that the modified antibodie~ ha~ in fact been prepared.
PCT patent publication WO 92~01781 discloses a similar proces~ in which metalloporphyrin derivati~eY are used as cofactors or catalysts. Also propo~ed in general terms are porphyrin3 derivativi~d with alkyl gxoup^~ so that the resultant antibody would have alkyl or aryl binding sites~
The derivat.iveY are u~ed to generate haptens that mimic the actual catalyst and the ~ubstrate in the relative orientation and spacing needed for the reaction to proceed.
The haptens are then used to generate antibodie3 which have binding sites complementary to the catalyst and the substrate in the correct orientation. Unlike the process ~116~2 WO93/07150 PCT/EP92f02283 described in EP 0305870, the antibodies need not have the catalyst covalently bound to them prior to useO
Unfortunately it is not a simple matter to create haptens from the metalloporphyrin derivati~es disclosed in the PCT publication. This i5 because coupling of a substrate to the porphyrin is not possible because there axe no convenient points of attachment on the porphyrin.
Accordingly it is an object of thi~ invention to provide a metalloporphyrin derivative that can be readily attached to a substrate to provide a hapten. It is also an ob~ect to pro~ide haptens containing the metalloporphyrins, antigen~ containing the haptens, antibodie~ raised to the antiyens, processes using th~ antibodies, and catalysts for use in the processes.
In one a~pect this invention pro~ide~ a compound of the formula I:
R, R2J ~z O

R, ~ R, I

R~ /~R~
R8 R3f~ 3 R~

R~ ~2 R, in which:
each R1 i5 independently selected from -H, -F, -Cl, -Br, -CH3, -COOH, -S03H, -COO-C~6-alkyl, -CH~CH-COOH, W~93/07150 2 1 1 6 6 5 2 ~CT/~P~/02283 -CH=CH-COO~C16-alkyl, -SO3-C16-alkyl, -NO2, phenyl, -NH2, and -NH-CO-C16-alkyl;
each R2 and R2' is independently selected from -H, -F, -Cl, -Br, -CH3, -COOH, -SO3H, -NO2 and phenyl;
each R3 and R3' is independently selected from -H, -F, -Cl, -Br, CH3, -O-C16-alkyl, -NO2, phenyl, -NH2, and -NH-CO-C16-alkyl, or at least one R3 or R3' is independently selected from (a) -NH-CO-alkylene-3N-imidazole or -NH-CO-alkylene 3-pyridine, i~ which each alkylene has 2 to 4 carbon atoms;
and (b~ -CO-alkylene-3N-imidazole or -CO-alkylene-3-pyridine~ in which each alkyl~ne has 3 or 4 carbon atoms;
or a pair o~ R3 and R3', on oppo~ing phenyl groups, jointly form (c) -NH-CO-alkylene-3-pyrldyl-5-alk~len~-CO-NH- or CO-alkylene-3-pyridyl-5-alkylene~CO~, in which each alkylene ~as 2 to 4 carbon atoms;
R4 is a) a ~ydrogen atom; b3 a linker group containing a reacti~e centre or group through which the compound of Formula I may be bonded to another compound; or c) a removable protecting group;
each Ra and R8' is independently -H, -F, -Cl, -Br or -CN; and acid addition salts of the compound and sodium, potas8iu~ and calcium salts of the compound.
: When R9 i9 a linker group b), it is preferably of the formula -(CH2j~-R5- (CH2) n~ (~6) ~iA in which Rs is -(CO)--, -(SO2)~ or -(POOH~-, R6 i9 - 0~ ~S~~ or -~NH~-, each of m, n and p independently is 0 or l and A is a xeactive leaving group or centre or, when p is l, A may al~o be a hydrogen atom.
More pre~erably m, n and p are 0 and A is halogen, particularly Cl or Br. A particularly preferred linker group is -COCl, which may ~asily react with a functional group such a~ -OH or -NH2 on another molecule to form the bridging gxoup -CO-~- or -CO-NH-.
When R~ is a removable protecting group c), it is 2 11 6 6 5 ~ PCT/EP92/02283 pref~rably a prctecting group which will protect the >N-NH2 group against oxidation by a reagent such as 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), and which is removable by hydrolysis under acid or alkaline conditions. A preferred S protecting group is CF3-CO-, which may be removed by mild alkaline hydrolysis.
Preferably R1 is H, a group which increases the water solubility of the compound, or a functiQnal group which p~rrnits the attachment of a carrier protein to the compound.
More preferably, at least one Rl is -NH-CO~CH2-CH2~COOH or NH2, at least one further R1 i~ -COOH, -COOCH3 or -CH=CH-COOH
and the remainder are H.
Preferably R2 and R2' are all H. Preferably one R3 or R3', or one pair of R3 and R3' are selected from groups a), b) and c) as defined above for R3 and R3'. The remaining R3 and R3' are preferably H. R~ and R8' ~re preferably H~
Compound~ of formula I in which R4 is m-C~3C6H4SO2- and O2NC6~4CO are known, but these two groups are neither linker group~ since they ha~e no reacti~ atom3 or groups for 20 binding, nor are they protecting groups which can be removed without destroying the ~N-N< bond.
The Cl6-alkyl may be any branched or unbranched alkyl group that contains up to 6 carbon atoms. Methyl i5 pre f erred .
25 The use o~ N-amino porphyrin compounds greatly facilitates the synthesi~ of hapt~ns since a linker group can be readily attached to the nitrogsn atom that has been added. Then a desired substrat~ or a functionalized derivativa of a desired substrate may be attached to th~
linker group.
The i!nvention also provide~ a compound of formula I, as . . defined above, for use in the preparation of a hapten that mimics a transition state of a metallopo~phyrin catalyst and a substrate in a reaction.
The invention also pro~ides a proce~s for the preparation of a compound of formula I, as defined above, comprising the steps of:

WO93/071~0 2 11 6 6 5 2 PCT/EP92/022 a) for a compound of formula I in which R4 is H, deprotecting a compound of formula I in which R4 is a protecting group;
b) for a compound of formula I in which R4 is a linker group, r acting a compound of formula I in which R4 is hydrogen with a precursor of the linker group that contains two reacti~e centres or groups, one of which is capable of forming a bond with the >~-NH2 group;
c) for a compound of formula I in which R~ is a protecting group~ i) protecting the >N~NH2 group of a compound of ~ormula III
R, R2~, R2' R ~,~R"' R, ; _ ~R~ III

R
R, in which Rl~ R2, R2', R3~ R3', R8 and R~' are a~ defined above;
and ii) oxidizing the compound of formula III to the corresponding compound of formula I.
¦ 15 In step a) the depxotection step will depend upon the I nature of the protecting group, but is preferably carried out by acid or alkaline hydrolysis. Where the protecting group is CF3CO-, mild alkaline hydrolysis may be used; for example using EtOH/KOH or EtOH/Ca(QH) 2 at temperatures in WO93~071~0 ? 1 1 6 6 ~ 2 PCT/EP92/02283 ... 7 the 65C to 75C.
In step b) the precursor of the linker group may be, for example, X-(C~I2)~- Rs~ (CH2) n (R6)p-A in which X is a reactive leaving group or atom, preferably Cl or Br, and the other symbols are as defined abo~e. Where the linker group is -CO~Cl-, a suitable precursor is phosgene or diphosgene.
In step c), where the protecting group is CF3-CO-, the compound o formula III in which R4 is hydxogen may be reacted with txifluoracetic anhydride in a polar non-aqueous solvent. The oxidation step ii) may be carried out using an oxidizing age~t such as DDQ in an inert solvent, for example methylene chloride.
The starting material o~ formula III may be prepared by reacting the corresponding porphyrin with O-m-toluene-lS sulphonylhydroxylamine as described in Callot, H. J.; 1979;Tetrahedron, 35, l455 6.
~ n another aspect this invention pro~ides a hapten comprising a metalloporphyrin cofactor bound to a residue of a substrate, the hapten mimicking a transition state of a metalloporphyrin catalyst and the substrate in a reaction, and in wh.ich the m~talloporphyrin cofactor iY of the ~ormula II: R, 1, R2.J~, Rz R ~ R~

~R, II

R~ R ~ 3 R~
O
R2~ R;
R, ~1166~2 WO93/071~0 PCTt~P92/0228~3 in which:
Rl, R2, R2 , R3, R3', R8, and R8' are as defined above for formula Ii R4' is a bridging group connecting the metalloporphyrin catalyst to the residue of the substrate; and M is a metal ion ha~ing a co-ordination number of at least 4;
or an acld addition salt thereof or a sodium, potassium or calcium salt thereo~.
The bridging group may be any suitable bridging group, with the proviso that it should b~ sel~ct~d such that the spacial orientation of the metallopvrphyrin cofactor with respect to the residue is as close as possible to that of the transition state ~ormed by the corresponding metalloporphyrin catalyst and the substrate during reaction.
Once the transition state has been identified and the residue of the subs~rate selected, selection of the bridging group will be routine.
Pre~erably the bridging group is of the ~ormula 20 ~ (CH2) m - Rs - (C~2) n~ (R6) p~ in which Rs is -~CO)-, -(SO2)- or (POOH3-, R6 is -O-, S-, or -(NH~-, m is 0~or l, n is 0 or l and p is 0 or l. Pre~erabLy m, n and p are'0. In a specific exampl~ the bridging group is -(SO2)-O- o~
--C ( 0)--0--. :
It will be appreciated that the hap~en has the ad~antag~ that bridging group projects axially from the cen~rally located amino group. Therefore the hapten can mo~e closely mimic the relati~e positions of the correspo~ding substrate and metalloporphyrin catalyst in the transition state.
'~ ~hen at least one R3 or ~3~ iS independently selected from (a) -NH--CO-alkylene~3N-imidazole or -NH~CO-alkylene-3-pyridine, in which each alkylene has 2 to 4 carbon atoms;
and ~b) -CO-alkylene~3N-imidazol~ or CO-alkylene-3-pyridine, in which each alkylene has 3 or 4 carbon atomsi or a pair of ~3 and R3' on opposing phenyl groups jointly form (c) -NH-CO-alkylene-3-pyridyl-5-alkylene-CO-NH- or -CO-alkylene-3-pyridyl~5-alkylene-CO-, in which each alky.Lene has 2 to 4 carbon atoms; a nitrogen atom in the heterocyclic ring acts as a fifth ligand for the metal ion M. The side of the porphyrin that has the fifth ligand is shielded and cannot come into contact with antibodies raised to antigens containing the hapten.
¦ The substrate may be any molecule upon which an oxidative transformation, such as a hydroxylation, dealkylation, epoxidatlon or desaturation reaction, is to be performed. The residue is a group that corresponds to the substrate molecule and is bound to the bridging group. The residue may differ from the substrate in that it may contain an additional functional group through which it is bound to the bridging group. Alternatively the residue may bind to the bridging group through an atom or functional group existin~ in ~he substrat~. In either case, the residue is attached to the bridging group in such a way to mimic a transition state of the ~ubstrate in a reaction pathway with a metalloporphyrin catalyst.
For example, the substrate may contain a non-activated primary, secondary or tertiary carbon atom which is to be hydroxyl~ated. The residue would then comprise the substrate molecule with a hydrogen rems:~ved from the carbvn atom or with the hydrogen replaced by a functional group that is bonded to th~ bridging group. In a specific example, in the preparation of~Ser8-cyclosporin A from cyclo~porin A (C$A), the substrate would be cyclosporin A and the residue would be S~x~-cyc1s~poxin A bonded to the bridging group though the OH of Ser~. The -O- of the hydroxy may be considered to 3Q be part of the residue or the bridging ~roup.
; ' As an alternatlve example, the residue may contain a group of the ~orMula >N-alkylene'- in which the alkylene' :
:~ : may be any branched, unbranched, substituted or un~ubstituted alkylene radical. In this case, the substrate will be a group of the formula ~N-alkyl.
: ~ specific example of such a substrate would be cyclosporin A in which the N-methyl of ~eu4 is to be ~ .
: :

WO93/07150 2 116 ~ 5 2 PCT/EP92/022~

hydroxylated to give N-hydroxymethylleucine4.
In another example, the re~idue may contain a group of the formula -O-alkylene'- in which alkylene' is as defined abo~e. The substrate would then have a group of the formula -O-alkyl and the hapten would mimic a transition state in the dealkylation and hydroxylation of the -0-.
A specific example of such a substrate would be ascomycin (which is described in EP 184 162) in which -O-alkyl corresponds to the methoxy group on the carbon atom numbered 15. The residue would then be ascomycin but with one of the hydrogen atoms of the methoxy group replaced by a bond to the bridging group. The hapten would then mimic a transition state in the replacement of an alkoxy group with a hydroxy group on carbon atom 15 of ascomycin.
In another example, the residue may contain an aromatic group of which a carbon atom is attached to the bridging group. The substrate would then also contain an aromatic group and the hapten would mimic a transition state in the hydroxylation o~ the aromatic ring.
In another example, the residue may contain the group of the formula : ~ :
3 ~ or Rl2--Rl3 25 ; ~ H
NH

(in which Rlz and~Rl3 are each independently a substituted car~on atom) ~which mimics an epoxy ring. In this case the substrate would~contain~the group Rl2=Rl3 which i~ to be epoxidated~
In a yet urther example, the residue may contain khe group ~ ~

Rl4-CH2-C 'H-R,4' in which Rl4 is H or an`~unsubstituted or substikuted alkyl :~

: ::: : :

i 2 W093/~7150 PCT/EP92/02283 group and Rl4' is an unsubstituted or substituted alkyl group. The subs~ra~e would then contain a group Rl9-CH2-CH2-R19' of which the single bond between the CH2~CH2 is to be desaturated. Plainly the substituents on the groups Rl4 and Rl4' must permit the desaturation of the C-C bond and hence the removal of a hydrogen atom from one of the carbon atoms. Specific examples would be the desaturation of dihydro-MeBmtl cyclosporin A to cyclosporin A and the desaturation of valproic acid to 4,5-dehydro-valproic acid.
In one preferred example, each of R2, R~', R3 and R3, i~
H and a least one Rl is -NH-C0-C~2-CH2-COOH or ~H2 and ~h~
others are H. The use o~ a hapten in which one Rl is -NH-CO-CHz-CH2-COOH or NH2 facilitates coupling of a carrier protein to the hapten. Also the solubility of the hapten 15 can be increa~ed. The solubility of the hapten can also be increased by substitutin~ the para-position of the phenyl groups with carboxy or ester groups.
Preferably the metal ion M is such that when it is ,.,~
q coordinated in the hapten, it is inert; particularly to 20 oxygen. For example, the metal iOI' may be Ni2', Zn2~ or Sn4 ~ .
In a further aspect, the invention provides an antigen comprising a hapten, as defined abo~e, coupled to a carrier protein that is capable of ~ausing an immunogenic response.
The carrier protein may be connected to the porphyrin 25 portion of the hapte~; especially to one of ~he Rl groups.
Alternatively the carrier protein may be connected to the rasidue portion of the hapten. The carrier protein m y be any suita~le protein such as keyhole limpet hemocyanin (KLH), bovi~e serum albu~in ~BSA) or ovalbumin.
I~ another aspect this invention provides an antibody, or a fragment thereof, that binds to a hapten as defined above. Preferably the antibody i5 produced by monoclonal : techniques. The antibody, or ragment, has the advar.tage that it has two binding pockets; one for the porphyrin portion and the other for the residue portion.
Xn another spect this invention pro~ide~ a process for - th:e production of antibodies suitable for controlling .~

.~
~ ' .

wo g~tO71~0 211 6 ~ 5 ~ ` PCT/EP92/022~3 reactions in which a substrate undergoes reaction in the presence of a metalloporphyrin catalyst to give rise to specific regioisomers or enantiomeric pure compounds, the process comprising:
providing a hapten as defined above that corresponds to a transition state of the substrate and catalyst;
stimulating an immune response in a mammal, preferably a mouse, for the production of antibodies to the hapten; and isolating and purifying those antibodies from the im~une response that are specific for the hapten.
Preferably the antibodies are monoclonal antibodies.
~ The process may further comprise the step of selecting the antibodies by binding them to haptens as defined above c that have been immobilised in chromatography columns or bound to tracer proteins.
In~another aspect this in~ention provides a process for the oxidation of a sub~trate, in the presence o* a metalloporphyrin catalyst, to produce a specific regioi~omer or enantiomer; the process comprislng:
providing an antibody as d~fined above that is specific for a hapten that mimics a transition state of the substrate and the catalyst;
providing a metalloporphyrin catalyst that binds to th~
antibody, providing an oxidizing agant, and com~ining the antibody, catalyst~ oxidizing agent and substrate to p~xmit the substrate to react.
Preferably the metalloporphyrin catalyst is coordinated with a metal ion selected from Fe3~, Cr3~ and Mn~.
The N-amino-porphyrins o~ fvrmula I may be synthesised ` I I by reacting tetraphanylporphyrin with O m-toluenesulfonyl-hydroxylamina:in a suitable sol~ent such as chloroform to ~: produce N-aminotetraphenylchlo.rin. The N-aminotetra-phenylchlorin may be isolatPd and purifiQd using chromatography. A suitable protecting group, for example a trifluoroacetyl group, may then be introduced to protect the introduced amino group and the compound oxidized to give N-WO93/07150 PCT~EP92/02283 (protecting group)amino-tetraphenylporphyrin. The protecting group may then be removed and a suitable linker or bridging group added. A similar procedure i5 described in Callot, H.J.; 1979, Tetrahedron, 35~ 1455 6 in which N-tosyl-aminotetraporphyrin i9 produced. Callot did not use a removable pxotecting group and hence did not obtain amino-tetraphenylporphyrin, but the procedure described can be readily adapted. Methods of manufacturing poxphyrins with a fifth ligand are known; for exa~ple Meunier et al; 1988;
Inor~ Cheo , 27, 1~1.
The residu~ of the substrat~ may be produced by first synthesising or providing the desired product (ie, the substrate when reacted). This may be done using classical chemical pathways or by direct hydroxylation using a porphyrin catalyst. For example, N-hydroxymethylleucine4-CsA may be produced by reacting CsA over a porphyrin catalyst in the presence of magnesium monoperoxyphthalate.
The product is then covalently bonded to the bridging group of the aminoporphyrin, for examp~e by condensation. The procedure adopted will depend upon the desired product but will be facilit~t~d by the amination of the porphyrin. The adduct formed in the condensation step may be isolated and puri~ied using chromatography.
The adduct is then complexed with a suitable metal ion, 25 for example by dissol~ing a salt of the m~tal ion .in a suitable solvent and refluxing with the adduct. The metal ion coordinates between the introduced nitrogen and the thr~e pyrrol nitrogen atoms o~ the porphyrin. The reaction scheme for the production of a D~Ser8-CsA hapten is illustrated below.

:, ....
. , .

6~52 W0 93,~'071~-,0 P~/EP92~02283 6~ H2N-0-S02-Ph-Me ~NH N~( CHC,'3 )~N N~
~N~ ~ ~N+

~3 N~F

(CP3C0)20 ~7 N~ P D~Q
DMF/DMAP ~ H CH2C12 b ~ 3 ~ ~3 N~- ~3 NH2 ~o ~) EtOH, KO~I 65 ~) ~3 ~
~CI

Diphosgezlc ~
""~ /~ ,CyA-l:)-Se~-8 DMAP, C}i2C'2, f~,r ~ HN--~ --N.^~

[~

, ., .

~1166~2 WO 93/07150 15 P~/EP92/02283 ~N- Ni(~AC)2 --~N~
=oo o o o =o ~ -C~ =
_ C~IC13, M~OH
0~ _N5~TN~ N~ 65 o= J H~TH$~
o~ o~
~ o 6 ~N--N~l N=~N' ~3~N--~ N=~3N~
~3~
,.'~

H~, /
CX~ O o o ~o H2, 10% Ptl/C O~N~TN~ ;N~
EtOH, HCI - o _ ~ oS~

~NH2 ~3 WO93/07150 2 1 1 6 6 ~ 2 PCT/EPg2/02283 Other haptens may be produced by similar methods. For example, to produce a hapten in which the residue is further functionalized so that the carrier protein can he attached to it, the following reaction scheme can be adopted:

M
H~N-O-S02Ph-Me ~ NH2 N~F ~R

(CF3C0)20 ~N=~ DDQ
A R~ HN--~R
DMF/DMAP 6J~H CH2C12 ~ HH
M

R~ R R ~R
~ EtOHUKOH
M

M

kOH Diphos8~ne ~N~b-~ l cDN oN N N~ DMAP, C~I2C1~ X) O O O ~o /

OH C~

O

WO 93/07150 2 ~ 2 P~/EPg2/02283 i ~f~ i\~Tf~'H~ /
~N N N N N --N N N N N-~ O o O =O ~C~ o o o =o O~N~2TNJ~ N~ Ni(OAc)2G;~_N~TN~ TN~ I_ ~ 'y CHC13~ MeOH ~
R~ S ~R R~ j ~R

' M M

\irH~k 5~OH

-~ 0~

R ~ _ ~R
R _ H, COOMc, COOH, COONa M ~ ~ COOMe, CO,OH~, COONa M

21166~2 WO93/07150 PCT~EP92~022B3 The antigen is produced by coupling to the hapten a carrier protein that renders the hapten/car.rier pro-tein complex immunogenic. The carrier protein may be covalently bound to the hapten by providing one of the R~ groups in the form of an amino group; which then forms a bridge between the carrier protein and the hapten. Suitable procedures are disclosed in Richards et ali 1990; C~urr.ent R seaxch in Photosynthesi~, 3, 6g5-8. The advantage o~ coupling the carrier protein to the porphyrin portion of the hapten, a~
opposed to the rssidue portion, is its general applicability since the residue portion need not bear a ~urther ~unctional group for the attachment of the carrier protein. However i~
a functional group is present in the re~idue portio~ or can be introduced by synthesis, the carrier protein can be attached to it. Other procedures ~or binding carrier proteins to haptens are disclo~ed in Harad~, A et al; 1~90;
Chemistry Letters, 917-918 and 1991; Chemistry L~tters, 953-~56.
The antigens may then be used to immunise mice. The spleen cells of the mice that give a good re~po~e are used with myeloma cells to produce hybridomas~ Those hybridomas that secrete monoclonal antibodies.~pecific to the haptens are then selected. The~e hybridoma technique~ are conventional and suitable techni~ues ar~ disclosed in, for example, Jacob, ~., Schultz, P.G., Sugasawara, R and Powell, M; 1987, J. Am. Chem. Soc~, 109, 2174 2176, K~inan, E. et al; 1990; Pure and Appl. ChemO~ 62, 2013-20~9 and Harada, A
et al; 1990; 5hemistrY Letters, 917-918.
The ~aptens may also be used to i~olate a~d purify the desired antibodie~ from the antibodies produced by the ~ ~ariousi hybridomas. This i5 a ~ignificant ad~antàge since radiolabelled antibodies that bind the desired antibody n2ed not be pr~pared. This can be done by selecting those antibodies which bind to the haptens; for example by immobilising the haptens in an affinity chromatograph column or radiolabelling them and allowing the a~tibodies to bind to them. Alternatively, con~entional techniques can be used WO93/07150 ~ 1 1 6 ~ 5 2 PCT/EP92/022~3 by raising antibodies against derivatives of the haptens and using these antibodies in radioimmunoassay procedures.
Once the desired antibodies have been isolated, it is pos~ible to determine the DN~ sequence coding for the antibody or to determine khe amino acid sequence of the antibody. Once this has been done, fra~ments or protein domains which include the antibody binding regions, can be built. Procedures for doing this are de~cribed in WO
90~07861.
The selected and purified antibodies may then h~ used in reactions to produce the desired product in a manner similar to tha~ described in WO 92/01781. A
metalloporphyr.in catalyst, which can fit inko the pocket of the a~tibody, i5 provided. The m talloporphyrin catalyst, the substrat~ and the antibodies are then combined. An oxygen ~ource is then ~dded under controlled conditions. If desired, the catalyst may be covalently bound to the antibody prior to the reaction as known in the art.
Alternatively the catalyst may be added separately ~rom the antibody and allowed to bind to the antibody during the process. The substrate will be able to enter the cavity formed by the antibody and porphyrin only if it is in th~
correct orientation to the catalyst to produre the desired product.
For example, the D-~laa of CsA may be converted to D-Ser~ by using the following p.rocedure. A catalyst, CsA and antibodi~s rai~ed to the aminoporphyrin-bridging group-D-Ser8-CsA antigen are th2n mixed in a suitable solvent. An oxygen source is then added under controlled conditions.
CsA with the correct orientation is able to ~nter the pocket of thelantibody and offer the methyl group to ~e hydroxylated to the metal-oxygen group. The hydroxylated D-Ser8 - CsA is then remo~ed. If necessary, the catalyst is removed and regenerated.
In another example, the N methylgroup o* leucine4 o~
C~A may be con~erted to 4-N-hydromethylleucine by using the following procedure. A catalyst, CsA and antibodies raised ~116~52 WO93/07150 ~CT/~P92/022~3 to the aminoporphyrin-bridging group-N-hydromethylleucine9~
CsA antigen are then mixed in a suita~le solvent. An oxygen source is then added under controlled conditions. CsA with the correct orientation is able to enter the pocket of the antibody and bond offer the N-methyl group of leucine4 to the metal-oxygen group of the porphyrin. The hydroxylated N~hydromethylleucine4-C~A i~ then removed.
Similar procedures may be used for all other reactions.
The source of oxygen atoms may be ~elected from H2O2, iodosob~nzene, mayne~ium monoperoxyphthalate, NaOCl, KHSO5 and the like.
It will be appreciated that substrates that have more than one site that can be hydroxylated, dealkylated, epoxidated, desaturated and the like can be s~lectively attacked so that only the desired site is alter~d.
Similar~y substrates that have prochiral centres that, when reacted, can foxm diast~reomers, can be selectively xeacted so that only one diastereomer forms~ Similarly single enantiomer products can be produced from substrates that, when ordinarily reacted, form racemic mixtures.

Example l: Hapten~formed from N-A~ino-5,l0,l5, ?-Tetraphenyl-21 ,23H-Porvhvrin-Derivat-ve and N-HydroxymethYlleucine4-Cyclo~porin A
:: :
1.1. M-Amino-tetraphenyl-chlorin from Tetraphenyl-porphyrin:

l0 g o~ Tetraphenylporphyrin is di~solved in 5Q0 ml warm chloroform. The solution is then cooled to 20C and 9.8 g O-mesitylsulfonylhydroxylamine is added to it. The solution is thenistirred for 20 hours at room temperature. The green reaction mixture is~then heated to 60C for l hour and 2N
sodium carbonate with chloroform added. The crystailine residue ~l0 g) is then separat~d using column chro~atography ~500 g Alox N, Activity V). After elution with chloroform, 7.5 g of an adduct is obtained. 68Q mg N amino-tetraphenyl~
chlorin is then eluted using a chloroform : Ethanol (ratio ~116~2 100 : 0.6 to 1.0) mixture.

1 . 2 . N~tri fluoroacetylamino-tetraphenyl-chlorin:

631 mg N-amino-tetraphenyl-chlorin is dissolved in 30 ml of absolute dimethyl-formamide and 2 ml pyridinc. 122 mg of 4-dimethyl-aminopyridin~ ~lmM) is then added and a solution of 231 mg trifluoro acetic acid anhydride (1.1 mM) in 3 ml ~ethylchloride at 2Q'C is added dropwise over 5 minutes. The solution is then stirred for 10 minutes. The reaction mixture is then evaporated and the residue is ~haken with 2N
sodium carbonate and chloroform and then washed o~ce with water. 850 mg of N~trifluoroacety~amino-tetraphenyl~chlorin is obtained.

1~3. N-tri~luoroacetylamino-tetraphenyl-porphyrin:

A solution of 850 mg N-tri~luoroac~tylamino-tetraphenyl-chlorin i~ 50 ml dichloromethane is mixed with ~81 mg ~3 mM) DDQ and refluxed or 5 hours. The reaction mixture is then sLaken once in 2N sodium carbonate and onc~
in water. The residue (780 mg) is then crystallised out of ethanol t650 mg) and th~n rec~ystalli~d out o~ an ch}oroform-etha~ol mixture to give 430 mg of N-trifluoroacetyl amino-tetr~phenyl-porphyrin.

1.4. N-amino-tetraphenyl-porphyrin:

A suspension of 400 mg o N-trifluoroacetylamino-tetraphenyl-porphyrin in 40 ml of ethanol is mixed with a solution of 0~5 g calcium hydroxide in 10 ml of ethanol. The mixture is then stirred for 30 mi~ute~ at 70 to 75 ~. The precipitate is then cooled to room t~mperature, filtered and washed with ethanol. The precipitate is then crystallised out of a chloroform-metha~ol mixture to give 280 mg of N-amino-tetraphenyl-porphyrin. Mass spectra peaks:-MH+ 630, [MH-NH2]H+ 615, and other peaks at 215, 237, 255, W093/07150 PCTfEP92/022 273, 289, 307, 343, 391, 419, 539, 646 and 730.

1.5. Condensation of N-hydroxymethylleucine4-Cyclosporin A
with N-amino-tetraphenyl-porphyrin:

A solution of 44.9 m~ of 97% diphosgene is mixed with 2 ml o~ dichloromethane. rrhe resulting solution is cooled to 0 to 5'C and a solution o~ 244 mg of N-hydrox~methyl-leucine4-Cyclosporin A in 5 ml dichloromethane is then added dropwise over 15 minutes. The solution is the~ stirred at O'C or lS minutes and a solution o~ 126 ~-amino-tetraphenyl-porphyrin and 25 mg of 4-dimethylaminopyridine in 1 ml pyridine and 8 ml dichloromethane i5 ~dded rapidly.
The reaction mixtur~ is allowed to react for 2 hours at room temperature and then 2N sodium carbonate and dichloromethane is added,. The residue (430 mg) is then purified using column chromatography (65 g Alox basic, activity II, chloroform)~
340 mg of the condensation product is obtained.
.

1.6. Produotion of a ~ickel-complex of the condensation : product of step 1.5.:

300 mg of the condensation product is dissolved in a solution o~ 50 ml chloroform. A solution of 0~8 g of nickel diacetatetetrahydrat~ l~n 30 ml methano~ is added and the : mixture re1uxed for an hour. The solution is khen reduced and shaken once~with chloroform and once with water. The residu~ is purified using column chromatography (56 ~ silica gel, acetone~: hexane 1:2). 190 mg of the nicke} complex is obtained and this i5: recrystallised ~ing tertiary-butylmethylether and a little petroleum ether.

W~93/07~50 ~ 11 6 6 5 2 pCT/~92/~22~3 Example 2: Hapten of N-amino-5,10,15,~0-tetraphenyl-21H,23H-porph~rin-derivatlve and Serine8-CYclosPorin A

2.1. N-amino-tetraphenyl-porphyrin is produced in the same manner as described in Example 1, steps 1.1. to 1.4.
.1 2.2. Condensation of Serine8-Cyclosporin A with N-amino-tetraphen~l-porphyrin:

244 mg of Serine3-cyclosporin A in S ml methylchloride is condensed with 126 mg of N-amino-tetr~phenyl-porphyrin in a manner totally analogous to thak set out in Example 1, step 1.5. 350 mg of the cond~n~ation product is obtained.

2.3. Production of the Nickel-complex of the condensation product~of step 2.2.:

300 mg of the condensation product is re1uxed with a solution of 0.8 g of nickel diacetatetetrahydrate in a manner totally analogous to that set out in example 1, step 1.6. 200 mg of the nickel complex is obtained and this is recrysta}lised u~ing tertiary-butylmethylether and a little petroleum ether. Mass spectra p~aks:- MH* 1873 ~nd other major peaks at 538, 600, 614, 630, 656.

~

Th~ hapten obtained from step 2.3 is activated as its benzotriazole ester in dimethyl formamide (DMF~ using bist2-oxo-3-oxazolidinyl]phosphinic chloride (BOP)/hydrvxy-!~ I benzotriazol (HOBt). This is then add~d to a solution of protein ~LH, BS~ or ovalbumin) in 2.5:1 DMSO:borate bufferat pH 8.5. A hapten:protein stoichiometry of 5:1 is used to prevent over-derivativisation and precipitation of the protein. After 4 hours, the reaction mixture is dialyzed against phospha~te-bu~fered saline to remove organic 21~52 solvents. A conjugate for each protein is obtained separately.

Example _4_ Generation of antibodies I~CFl mice are anaesthetized and the peritoneal cavities surgically opened to acc~ss the spleen. The surface of the spleen is swabbed with an ethanolic solution containing KLH conjugate obtained from example 3. The mice are immunised in a similar m~nner on day 22. Serum titres are measured on day 27 by ELISA analysis against fre~ hapten or BSA derivatives absorbed in well& of polystyrene microtitre plates. The antibodi~5 also h~ve partial reactivity with conjugate~ of the Ser8-CsA product and a nickel mono-p-amino-tetraphenylporphyrin control. This indicates that antibodi~s that recogni~e both the~substrate and the catalyst components of the hapten are prese~t in the sera.
ssA a~d KLH conjugates obtained from example 3 are aflded to Ribi adjuvant and 6 IRCl mice are immunised using i~p. injections. O~ day 14, the mice are boosted with conjugate absorbed on bentonite a~d the serum sampled on day 17. Three mice showing high serum titres are boocted with further conjugate and are sacrificed on day 31. The spleens from two mice are used i~ standard fusion protocol~ using Sp2/0 myeloma cells and PEG. A stable clone which secretes antibody specific for the BSA conjugate is isolated.
The antibodies may be selected and purified using free hapten immobilised in an ~~inity chromatograph.

Claims (18)

Claims

1. A hapten comprising a metalloporphyrin cofactor bound to a residue of a substrate, the hapten mimicking a transition state of a metalloporphyrin catalyst and the substrate in a reaction, characterised in that the metalloporphyrin cofactor is of the formula II:

in which:
each R1 is independently selected from -H, -F, -Cl, -Br, -CH3, -COOH, -SO3H, -COO-C1-6-alkyl, -CH=CH-COOH, -CH=CH-COO-C1-6-alkyl, -SO3-C1-6-alkyl, -NO2, phenyl, -NH2, and -NH-CO-C1-6-alkyl;
each R2 and R2' is independently selected from -H, -F, -Cl, -Br, -CH3, -COOH, -SO3H, -NO2 and phenyl;
each R3 and R3' is independently selected from -H, -F, -Cl, -Br, -CH3, -O-C1-6-alkyl, -NO2, phenyl, -NH2, and -NH-CO-C1-6-alkyl, or at least one R3 or R3' is independently selected from (a) -NH-CO-alkylene-3N-imidazole or -NH-CO-alkylene-3-pyridine, in which each alkylene has 2 to 4 carbon atoms;
and (b) -CO-alkylene-3N-imidazole or -CO-alkylene-3-pyridine, in which each alkylene has 3 or 4 carbon atoms;
or a pair of R3 and R3' on opposing phenyl groups jointly form (c) -NH-CO-alkylene-3-pyridyl-5-alkylene-CO-NH- or -CO-alkylene-3-pyridyl-5-alkylene-CO-, in which each alkylene has 2 to 4 carbon atoms;
each R8 and R8' is independently -H, -F, -Cl, -Br or -CN;
R4 is a bridging group connecting the metalloporphyrin catalyst to the residue of the substrate; and M is a metal ion having a co-ordination number of at least 4; or an acid addition salt thereof or a sodium, potassium or calcium salt thereof.

2. A hapten according to claim 1 in which the bridging group is of the formula -(CH2)m-R5-(CH2)n-(R6)p- in which R5 is -(CO)-, -(SO2)- or -(POOH)-, R6 is -O-, -S-, or -(NH)-, and each m, n and p independently is 0 or 1.

3. A hapten according to claim 2 in which the bridging group is -(SO2)-O- or -C(=O)-O-.

4. A hapten according to claim 1 in which each of R2, R2', R3 and R3' is H.

5. A hapten according to claim 4 in which at least one R1 is -NH-CO-CH2-CH2-COOH or NH2, at least one further R1 is -CH=CH-COOH, -COOH or -COOCH3 and the remainder are H.

6. A hapten according to claim 1 in which the metal ion is Ni2+, Zn2+ or Sn4+.

7. A hapten according to claim 1 in which the residue is a cyclosporin residue.

8. A hapten according to claim 7 which is selected from:
the nickel complex of D-Ser8-CyS-carbamato N-amino-tetraphenylporphyrin;
the nickel complex of D-Ser8-CyS-carbamato-N-amino-mono-p-nitro-tetraphenylporphyrin; and the nickel complex of D-Ser8-CyS-carbamato-N-amino-mono-p-amino-tetraphenylporphyrin.

9. A compound of formula I:

I

in which:
R1, R2, R2', R3, R3', R8 and R8' are as defined in claim 1;
R4 is a) a hydrogen atom; b) a linker group containing a reactive centre or a group through which the compound of Formula I may be bonded to another compound; or c) a removable protecting group; and acid addition salts of the compound and sodium, potassium and calcium salts of the compound.

10. A compound according to claim 9 in which R4 is a linker group of the formula -(CH2)m-R5-(CH2)n-(R6)p-A in which R5 is -(CO)-, -(SO2)- or -(POOH)-, R6 is -O-, -S-, or -(NH)-, m, n and p are each independently 0 or 1 and A is a reactive leaving group or atom, or where p is 1, A may also be hydrogen.

11. A compound according to claim 10 in which m and n are 0 and A is Cl or Br.

12. A compound according to claim 11 in which the linker group is C(=O)-Cl.

13. A compound according to claim 9 in which R4 is H.

14. A process for the preparation of a compound of formula I, as defined in claim 9, comprising the steps of:
a) for a compound of formula I in which R4 is H, deprotecting a compound of formula I in which R4 is a protecting group;
b) for a compound of formula I in which R4 is a linker group, reacting a compound of formula I in which R4 is hydrogen with a precursor of the linker group that contains two reactive atoms or groups, one of which is capable of forming a bond with the >N-NH2 group;
c) for a compound of formula I in which R4 is a protecting group, i) protecting the >N-NH2 group of a compound of formula III

III

in which R1, R2, R2', R3, R3', R8 and R8' are as defined above;
and ii) oxidizing the compound of formula III to the corresponding compound of formula I.

15. A process according to claim 14 in which, in step a), the protecting group is CF3CO- and it is removed by alkaline hydrolysis.

16. A process according to claim 14 in which, in step b), the precursor of the linker group is of formula X-(CH2)m-R5-(CH2)n-(R6)p-A in which X is a reactive leaving group or centre and A, R5, R6, m, n, and p are as defined in claim 10.

17. A process according to claim 16 in which the linker group is -CO-Cl- and the precursor is phosgene or diphosgene.

18. A process according to claim 14 in which, in step c), a compound of formula III in which R4 is hydrogen is reacted with trifluoracetic anhydride in a polar non aqueous solvent and then oxidized using an oxidizing agent such as 2,3-dichloro-5,6-dicyanobenzoquinone to provide a compound of formula I in which the protecting group is CF3-CO-.