WO2000006707A1 - Procede de criblage permettant d'identifier l'activite catalytique a l'aide de composes chromogenes et fluorogenes - Google Patents

Procede de criblage permettant d'identifier l'activite catalytique a l'aide de composes chromogenes et fluorogenes Download PDF

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Publication number
WO2000006707A1
WO2000006707A1 PCT/US1999/017348 US9917348W WO0006707A1 WO 2000006707 A1 WO2000006707 A1 WO 2000006707A1 US 9917348 W US9917348 W US 9917348W WO 0006707 A1 WO0006707 A1 WO 0006707A1
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group
ketone
reactant
aldol
antibody
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PCT/US1999/017348
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English (en)
Inventor
Carlos F. Barbas, Iii
Benjamin List
Richard A. Lerner
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The Scripps Research Institute
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Priority to AU52472/99A priority Critical patent/AU5247299A/en
Publication of WO2000006707A1 publication Critical patent/WO2000006707A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones

Definitions

  • the field of this invention is detection chemistry More particularly, the present invention pertains to chromogemc and fluorogenic compounds that are precursors to chromophores or fluorescent molecules and the uses of such compounds in screening assays
  • detectable markers in chemistry and biology is well known. Such markers are used to determine the presence or absence of a particular chemical compound or conjugate (e.g., polypeptide, polynucleotide, antigen-antibody complex, hybridization complexes, and the like) Classes of detectable markers include chromophores and fluorescent markers.
  • markers can be used to label or tag specific compounds of interest by designing the marker to bind or otherwise attach to that compound.
  • An example of such a use for fluorescent markers is the use of fluorescent hybridization probes that hyb ⁇ dize to specific nucleotide sequences.
  • Detection markers can also be used to monitor the rate of chemical reactions.
  • the disappearance or consumption of a reactant in a chemical reaction can be measured by using a reactant labeled with a fluorescent marker and determining the disappearance or reduction of fluorescence.
  • the accuracy and precision of using detectable markers to measure the rate of a chemical reaction by momto ⁇ ng the disappearance of a reactant is compromised by the existence in the reaction mixture of any conditions that serve to non-specifically quench the visible light or fluorescent signal. Such non-specific quenching would artificially reduce the detected signal and falsely indicate an increased rate of reactant disappearance.
  • the present invention provides a solution to the problems of the prior art in the form of precursors to chromophores and fluorescent markers that serve as reactants for a variety of chemical reactions and which precursors can be tuned to produce light at any particular wavelength.
  • the present invention provides a process for forming a detectable marker.
  • the process comprises the step of reacting a compound of the formula A-Y, where A is
  • X is hydrogen or a first electron donor group
  • R is N or CR 1 where R 1 is hydrogen, C,-C 8 alkyl or a second electron donor group
  • Y is a ketone-forming group, with a reactant that converts Y to a ketone group.
  • At least one X is a first electron donor group.
  • the first and second electron donor groups are preferably each independently halogen, NR 2 R ⁇ alkylamine, alkoxy or SR 2 , where R 2 and R 3 are each independently hydrogen or C,-C 8 alkyl.
  • An especially preferred first electron donor group is methoxy or N(CH 3 ) 2 .
  • each R is CH.
  • the reactant is a hydrolytic reactant, a redox reactant, or a retro-aldol reactant.
  • the reactant is a catalyst such as, for example, an enzyme.
  • An especially prefe ⁇ ed catalyst is a catalytic antibody.
  • Exemplary and prefe ⁇ ed ketone-forming groups are -CH(OH)R 5 , -C(OH)(R 5 )CH 2 COR 5 , -C(OR 6 ) 2 , -C(OR 6 )OR 7 , -CNH(R 5 )COR 5 , or -COCOR 5 , where R 5 is hydrogen or R 6 , R 5 is C r C g alkyl, C r C 8 alkenyl or C,-C 8 aryl and R 7 is an oligosaccharide containing from 1 to about 10 saccharides.
  • Especially prefe ⁇ ed ketone-forming groups are CH(OH)CH 2 COCH 3 or C(OH)(CH 3 )CH 2 COCH 3 .
  • a process of this invention comprises reacting a compound of the formula A'-Y with a reactant as set forth above.
  • Y is the same as defined above for A-Y.
  • A' has the structure
  • R 4 is C,-C 8 alkyl or C,-C 8 alkenyl and Y' is a group capable of releasing a heteroatom directly bonded to the ring, which may also form a double bond to the ring carbon.
  • the reactant is the same as set forth above.
  • a process of this invention uses a compound of the formula A-Y' or A'-Y', where A is defined above, Y' is a heteroatom-releasing group and the reactant is a reactant that catalyzes a tandem retr ⁇ -aldol-retr ⁇ -Michael reaction.
  • a prefe ⁇ ed reactant is a catalytic antibody.
  • An exemplary and preferred heteroatom-releasing group is HetCH 2 CH 2 C(OH)R 4 CH 2 COR 4 where the heteroatom or heteroatom group (Het) may be O, S, or NR 4 .
  • the present invention further provides compounds of the formulae A-Y, A'-Y, A-Y' and A'-Y', as set forth above.
  • FIG 1 shows a process of making fluorogemc compounds.
  • FIG 2 shows va ⁇ ous pathways of forming ketone groups
  • FIG 3 shows Chromogenic compounds
  • FIG 4 shows Fluorogemc compounds.
  • FIG 5 shows antibody catalyzed retro-Michael reaction.
  • (2) The antibody catalyzed tandem retro-aldol-retra-Michael reaction.
  • FIG 6 shows Synthesis of mol-red and antibody catalyzed tandem retro-aldol-retro-Michael reaction to give red fluorescent resorufm
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight- chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group may have 1 to 12 carbons, or may have 3 to 9 carbons.
  • alkenyl refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond, including straight-chain, branched-cham, and cyclic groups.
  • the alkenyl group may have 2 to 12 carbons, or may have 3 to 9 carbons.
  • alkynyl refers to an unsaturated hydrocarbon group containing at least one carbon-carbon t ⁇ ple bond, including straight-cham, branched-cham, and cyclic groups.
  • the alkynyl group may have 2 to 12 carbons, or may have 3 to 9 carbons.
  • An “alkoxy” group refers to an "-O-alkyl” group, where "alkyl" is defined as desc ⁇ bed above.
  • aryl group refers to an aromatic group which has at least one ⁇ ng having conjugated pi electron system and includes carbocyc c aryl, heterocyc c aryl and biaryl groups, all of which may be optionally substituted.
  • the substituents of the aryl groups may be hydroxyl, cyano, alkoxy, alkyl, alkenyl, alkynyl, amino, or aryl groups.
  • alkylaryl group refers to an alkyl (as desc ⁇ bed above) covalently bonded to an aryl group (as desc ⁇ bed above).
  • Carbocychc aryl groups are groups wherein the ⁇ ng atoms on the aromatic ⁇ ng are carbon atoms The carbon atoms are optionally substituted.
  • Carbocychc aryl groups include monocychc carbocychc aryl groups and optionally substituted naphthyl groups
  • Heterocychc aryl groups are groups having from 1 to 3 heteroatoms as ⁇ ng atoms in the aromatic ⁇ ng and the remainder of the ⁇ ng atoms are carbon atoms. Suitable heteroatoms may include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, py ⁇ dyl, py ⁇ olyl, N-lower alkyl py ⁇ olo, py ⁇ midyl, pyrazmyl, imidazolyl and the like, all optionally substituted.
  • a “carbalkoxy” group refers to a COOX group, wherein "X" is an lower alkyl group.
  • amide refers to an -C(O) -NH-R, where R may be alky, aryl, alkylaryl or hydrogen.
  • a “thioamide” refers to -C(S) -NH-R, where R may be alkyl, aryl, alkylaryl or hydrogen.
  • esters refers to an -C(O) -OR', where R' may be alkyl, aryl, or alkylaryl.
  • An “armne” refers to a -N(R")R'", where R" and R'", may be independently hydrogen, alkyl, aryl, or alkylaryl.
  • a thioether refers to R-S-R, where R is either alkyl, aryl, or alkylaryl.
  • An ether refers to R-O-R, where R is either alkyl, aryl, or alkylaryl
  • the present invention provides chromogenic or fluorogemc compounds and the use of those compounds in forming a detectable marker and the use of those compounds in screening and identifying agents having a desired biological and/or chemical activity.
  • a compound of this invention contains an electron donor, a mono-, bi- or t ⁇ cyc c ⁇ ng system and a ketone forming group (Y) or a heteroatom-releasing group (Y').
  • the ketone group is a carbonyl (CHO) group.
  • the heteroatom of the heteroatom-releasing group is oxygen.
  • the electron donor component of the compound is any such donor group well known m the art to interact with a carbonyl and produce a fluorescent molecule.
  • exemplary and prefe ⁇ ed such donor groups are alkoxy, halogen, NR 2 R ⁇ alkylamine, and SR 2 , where R 2 and R 3 are each independently hydrogen or C,-C 8 alkyl.
  • An especially prefe ⁇ ed donor group is methoxy or dimethylamine.
  • each particular electron donor group when associated with a carbonyl group, produces fluorescence with a unique excitation/emission spectrum.
  • methoxy and carbonyl fluoresce with a blue wavelength exc ⁇ tat ⁇ on-287 nm; emission 450 nm
  • an association of dimethylamine and carbonyl fluoresce with a green wavelength e.g., excitation 304 nm; emission 531 nm.
  • a compound of this invention can, therefore, be designed or tuned to operate at any given wavelength.
  • the ring system component of a present compound contains one, two or three unsaturated six-member rings.
  • the ring atoms can all be carbon or one or more ring atoms of each ring can be a heteroatom such as nitrogen or oxygen.
  • the ring carbon atoms can be unsubsituted or substituted with an alkyl having from one to eight carbon atoms. It is understood that each ring of the depicted structures can contain one or more ring nitrogen (N) or oxygen (O) atoms.
  • One ring atom of the mono-, bi- or tricyclic component is attached to a ketone- forming group that is converted to a ketone group or hydroxyl as a result of hydrolysis, an oxidation-reduction reaction, or a retro-aldol reaction.
  • one ring atom of the mono-, bi-, or tri-cyclic component is attached to a heteroatom group that is released in its unbound form, which may or may not be ionizable as a result of a tandem retr ⁇ -aldol-retroMichael reaction. The compound so released is then capable of fluorescence.
  • the heteroatom group is the end art of the heteroatom releasing group. One of skill in the art can readily determine structures that form as result of such chemical reactions.
  • Exemplary and prefe ⁇ ed ketone-forming groups are -CH(OH)R 4 ,
  • Especially prefe ⁇ ed ketone- forming groups are CH(OH)CH 2 COCH 3 or C(OH)(CH 3 )CH 2 COCH 3 .
  • Groups having the structure -C(C(R 4 ) 2 )N(H)COR 4 , or -C(C(R 4 ) 2 )OCOR 4 form a ketone upon hydrolysis.
  • a group having the structure CH(OH)CH 2 COCH 3 forms a ketone via an oxidation-reduction reaction.
  • a group having the structure C(OH)(CH 3 )CH 2 COCH 3 forms a ketone via a retro- aldol reaction.
  • An exemplary and prefe ⁇ ed heteroatom-releasing group is HetCH 2 CH 2 C(OH)R 4 CH 2 COR 4 where the heteroatom or heteroatom group (Het) may be O,
  • the location of the ketone or heteroatom-releasing group forming group m the ⁇ ng component is dependent upon the location of the electron donor group. The only limitation is that the relative locations of the ketone forming group or heteroatom-releasing group and electron donor group allows for transfer of an electron from the donor.
  • a compound of this invention can contain more than one electron donor group so long as at least one of those groups is situated relative to the ketone-forming group or heteroatom-releasing group as set forth above. Where there is more than one electron donor group, those groups can be the same or different.
  • a compound of this invention co ⁇ esponds to the formula A-Y, A'-Y , A-Y', or A'-Y' set forth above.
  • a compound of this invention can be made using standard synthetic procedures well known m the an.
  • the synthesis of methoxy-tertiary aldols can proceed via standard LDA technology from commercially available methoxy-napthaldehyde precursors.
  • Tertiary aldols (with a va ⁇ ety of electron donor groups) can be made indirectly using G ⁇ gnard addition to commercially available esters followed by oxidative cleavage of the formed homoallylic alcohols.
  • a detailed desc ⁇ ption of the preparation of the preparation of compounds of this invention is set forth hereinafter in the Examples and shown schematically in FIGs 1-4.
  • a compound of this invention has many uses related to its ability to be converted into a chromophore or a fluorescent molecule.
  • the compound is used to form a fluorescent molecule.
  • a compound of the present invention is treated such that the ketone-forming group of the compound is converted to a ketone group.
  • that reactant can act via hydrolysis, oxidation/reduction, a retro-aldol reaction or a tandem retro-aldol-retro Michael reaction.
  • conversion is accomplished using a a catalyst: a compound of this invention is a substrate for the catalytic reaction. The catalyst recognizes the structure of the ketone-forming group and catalyzes the conversion of that group to a ketone.
  • the catalyst can be a naturally occur ⁇ ng or a synthetic molecule.
  • An exemplary naturally occur ⁇ ng catalyst is an enzyme.
  • the particular enzyme used in the process will depend upon the structure of the ketone-forming group. In prefe ⁇ ed embodiments, the enzyme has aldolase, esterase, armdase or acetylase catalytic activity depending upon the nature and structure of the ketone-forming group or heteroatom-releasing group.
  • Figure 2 schematically illustrates the relationship between exemplary compounds of the present invention and the reactions used to convert those compounds to fluorescent compounds having a ketone group or heteroatom-releasing group.
  • the reactant is a synthetic catalyst such as a catalytic antibody
  • Catalytic antibodies are well known in the art (See, e.g., United States Patent Nos
  • Catalytic antibodies are made by immunizing an animal with an immunogen that contains a stable analog of a catalytic (e.g , hydrolytic) transition state molecule (e.g., an enzyme) The method starts by immunizing an animal with an immunogen that includes the analog and a hapten. Then the animal is maintained for a time pe ⁇ od sufficient for it to secrete antibodies that immunoreact with the haptemc analog.
  • a catalytic e.g , hydrolytic
  • transition state molecule e.g., an enzyme
  • genes that encode antibody molecules or molecules containing antibody combining site portions are transfe ⁇ ed from antibody-producing cells of the maintained, immunized animal into host cells to form hyb ⁇ d cells
  • the hyb ⁇ d host cells contain genes from at least two sources.
  • the formed hyb ⁇ d cells have two characte ⁇ stics, viz., ( ⁇ ) they produce antibody molecules or molecules containing antibody combining site portions from the transfe ⁇ ed genes when cultured and (n) they can be cultured substantially indefinitely
  • the hyb ⁇ d cells are cultured m an approp ⁇ ate culture medium for a time pe ⁇ od sufficient for them to produce antibody molecules or molecules containing antibody combining site portions.
  • antibody molecules or molecules containing antibody combining site portions are recovered from the cultured hyb ⁇ d cells Then, the obtained antibody molecules or molecules containing antibody combining site portions are screened for catalytic activity directed to a specific reaction and finally, clones are grown of the identified hyb ⁇ d cell that produces antibody molecules or molecules containing antibody combining site portions that catalyze the reaction.
  • Prefe ⁇ ed hyb ⁇ d cells are hyb ⁇ doma cells.
  • An example of the use of such a catalytic antibody to form a fluorescent molecule is set forth hereinafter in the Examples.
  • the compounds can be used in a screening method to identify agents or compounds having a particular catalytic activity such as, for example, aldolase activity.
  • the process begins with provision of a test sample that contains one or more compounds suspected of having the desired catalytic activity.
  • the test sample is contained in an aqueous medium.
  • Compounds in the test sample can be derived from any source.
  • the test sample is a combinatorial library that contains synthetically produced compounds.
  • the test sample is culture fluid from tranformed cells engineered to produce one or more catalytic agents.
  • a screening process can be used to screen hybridomas to identify those that produce a particular monoclonal antibody.
  • the test sample containing the compound or compounds of interest is reacted with a compound of the structure A-Y or A'-Y or A-Y' or A'-Y' as set forth above.
  • the reaction is maintained for a period of time and under conditions sufficient for any catalytic compound to convert the structure to a detectable product.
  • Reaction conditions and times are readily determinable by a skilled artisan and depend, as is known in the art, on the particular activity being tested for.
  • the reaction conditions include a pH of from about 6.0 to about 8.0, a temperature of from about 25 °C to about 50 °C, an osmolality of from about 200 mOsm to about 400mOsm and a reaction time of from about 1 minute to about 24 hours, depending upon the temperature.
  • Specific reaction conditions for an aldolase reaction are set forth hereinafter in the Examples.
  • the structure of the ketone-forming group is specifically designed to be converted to a ketone by the catalytic activity of interest.
  • a ketone-forming group of the structure C(C(R 4 ) 2 )OCOR 4 can be converted to a ketone by a catalyst having esterase or acetase activity and a ketone-forming group of the structureC(C(R 4 ) 2 )N(H)COR 4 can be converted to a ketone by the action of a catalyst having amidase activity.
  • a ketone-forming group of the present invention has the structure C(OH)(R 4 )CH 2 COR 4 and is converted to a ketone by a catalyst having aldolase activity.
  • Catalytic antibodies that catalyze the aldol reaction are generated by immunization with a reactive compound that covalently traps a Lysine (Lys) residue in the binding pocket of the antibody by formation of a stable vmylogous amide, i.e., a covalent antibody/hapten complex.
  • the catalytic mechanism for these catalytic antibodies is disclosed to mimic the catalytic mechanism employed by natural class I aldolase enzymes.
  • the same reaction mechanism employed to form the covalent antibody/hapten complex is also employed to catalyze the aldol reaction.
  • the antibodies use the ⁇ -amino group of Lys to form an enamine with ketone substrates and then use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond.
  • Such catalytic antibodies are characte ⁇ zed by their broad substrate specificity and their ability to control the diastereofacial selectivity of the reaction in both Cram-Felkm and anti-Cram-
  • Particularly prefe ⁇ ed catalytic antibodies having aldolase activity are those secreted by hyb ⁇ doma 38C2, having ATCC accession number HB 12005 or by hyb ⁇ doma 33F12, having ATCC accession number HB 12004 (See United States Patent No. 5,733,757).
  • a compound of this invention can be used to screen for gene expression of genes encoding catalytic antibodies.
  • the recombinant production of catalytic antibodies is disclosed m United States Patent No. 5,733,757.
  • Cells transformed to produce and secrete catalytic antibodies e.g., hyb ⁇ doma cells
  • a compound of this invention having a ketone-forming group or heteroatom-releasing group that is known to be catalytically converted by the antibody to a ketone group or heteroatom- releasing group
  • compounds of this invention can be tuned to fluoresce at different wavelengths by selection of an approp ⁇ ate electron donor group and designed to interact with different reactants through the use of different ketone-forming groups or heteroatom-releasing group.
  • a screening process can utilize more than one compound in a single assay system and high-throughput screening of large number of agents for a va ⁇ ety of catalytic activities can be accomplished simultaneously (determining fluorescence of the same population at a va ⁇ ety of wavelengths)
  • 6-methoxyacteonaphthone (10, 801 mg, 4 mmol, 1 eq) in 10 mL of dry THF was treated at -78°C with 2-methylallylmagnes ⁇ um chlo ⁇ de (10 mL of a 0.5 M solution in THF, 5 mmol, 1.25 eq) and sti ⁇ ed for 5 mm.
  • the mixture was warmed to -30°C and treated with 0.75 mL of a saturated ammonium chlo ⁇ de solution and 15 mL of ether, warmed to r.t., d ⁇ ed (MgSO 4 ), filtered over silica, and concentrated to give a colorless product which was used without further pu ⁇ f ication in the next step.
  • PROD AN ® (5) (11, 200 mg, 0.88 mmol, 1 eq) in 15 mL of dry THF was treated at -78°C with 2-methylallylmagnes ⁇ um chlo ⁇ de (2.64 mL of a 0.5 M solution in THF, 1.32 mmol, 1.5 eq) and sti ⁇ ed for 5 min.
  • the mixture was warmed to -30°C and treated with 0.75 mL of a saturated ammonium chlo ⁇ de solution and 15 mL of ether, warmed to r.t., d ⁇ ed (MgSO 4 ), filtered, and concentrated to give a colorless product which was used without further pu ⁇ fication in the next step.
  • the product was dissolved in 10 mL of acetone and treated with 0.66 mL of a 50% NMO solution in water and 0.65 mL of a 2.5% OsO 4 solution in z-PrOH. After 1 h the mixture was treated with saturated sodium metabisulfite solution and extracted 5 times with EtOAc. After evaporation of the d ⁇ ed organic layers, 218 mg (0.688 mmol, 78%) of the t ⁇ ol were isolated.
  • Aldols 1, 2, 5-8 and aldol 14 in acetonit ⁇ le (5 mM) were diluted with PBS to the required concentration and then treated with antibody 38C2 (67 mM in PBS, pH 7 4, commercially available from Ald ⁇ ch, #47,995-0) to give a final antibody concentration of
  • Aldol compounds from Example 1 were assayed for their ability to detect the catalytic activity of catalytic antibody 38C2, prepared in accordance with the procedures of United States Patent No. 5,733,757.
  • Aldols 1 and 2 from example 1 in acetonit ⁇ le (5 mM) were diluted with PBS to the required concentration (10 ⁇ M to 4 rnN) and then treated with antibody 38C2 (67 ⁇ M in PBS, pH 7.4) usually to give a final antibody concentration of 500 nM.

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Abstract

Procédé permettant de produire un marqueur détectable à partir d'un composé précurseur. Le composé précurseur contient un groupe donneur d'électrons, un constituant bicyclique ou tricyclique et un groupe formeur de cétone ou un groupe libérant des hétéroatomes attaché au constituant d'anneau. De nouveaux précurseurs sont également présentés. Le procédé et les composés sont utiles pour rechercher et dépister une activité catalytique.
PCT/US1999/017348 1998-07-29 1999-07-29 Procede de criblage permettant d'identifier l'activite catalytique a l'aide de composes chromogenes et fluorogenes WO2000006707A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU52472/99A AU5247299A (en) 1998-07-29 1999-07-29 Screening process for identifying catalytic activity using chromogenic and fluorogenic compounds

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US9451498P 1998-07-29 1998-07-29
US60/094,514 1998-07-29

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARBAS C.F. ET AL: "Immune Versus Natural Selection: Antibody Aldolases with Enzymic Rates But Broader Scope", SCIENCE, vol. 278, 19 December 1997 (1997-12-19), pages 2085 - 2092, XP002922025 *
HAWORTH R.D. ET AL: "Synthesis of Alkylphenanthrenes. Part VII. 7-Hydroxy-1 : 2-dimethylphenanthrene", JOURNAL OF THE CHEMICAL SOCIETY, 1934, pages 864 - 867, XP002922024 *
HOFFMANN T. ET AL: "Aldolase Antibodies of Remarkable Scope", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 120, no. 12, 7 March 1998 (1998-03-07), pages 2768 - 2779, XP002922026 *
LIST B. ET AL: "Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 95, December 1998 (1998-12-01), pages 15351 - 15355, XP002922027 *

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