+

WO1996005511A1 - Biocatalytic methods for synthesizing and identifying biologically active compounds - Google Patents

Biocatalytic methods for synthesizing and identifying biologically active compounds Download PDF

Info

Publication number
WO1996005511A1
WO1996005511A1 PCT/US1995/001759 US9501759W WO9605511A1 WO 1996005511 A1 WO1996005511 A1 WO 1996005511A1 US 9501759 W US9501759 W US 9501759W WO 9605511 A1 WO9605511 A1 WO 9605511A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
compounds
library
biocatalysts
biocatalytic
Prior art date
Application number
PCT/US1995/001759
Other languages
French (fr)
Inventor
J. Wesley Fox
Jonathan S. Dordick
Douglas S. Clark
Original Assignee
Enzymed, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1994/009174 external-priority patent/WO1995005475A1/en
Application filed by Enzymed, Inc. filed Critical Enzymed, Inc.
Priority to AU19165/95A priority Critical patent/AU1916595A/en
Publication of WO1996005511A1 publication Critical patent/WO1996005511A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00364—Pipettes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
    • G01N35/109—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane with two horizontal degrees of freedom

Definitions

  • This invention is in the field of synthesizing and identifying biologically active compounds.
  • Synthesizing and testing new compounds for biological activity which is the first step in identifying a new synthetic drug, is a time consuming and expensive undertaking.
  • compounds must by synthesized, purified, tested and quantitatively compared to other compounds in order to identify active compounds or identify compounds with optimal activity.
  • the synthesis of new compounds is accomplished for the most part using standard chemical methods. Such methods provide for the synthesis of virtually any type of organic compound; however, because chemical reactions are non ⁇ specific, these syntheses require numerous steps and multiple purifications before a final compound is produced and ready for testing.
  • Fodor S.P.A. et al (1990) Science 251. 767-773, describe methods for discovering new peptide ligands that bind to biological receptors. The process combines solid-phase chemistry and photolithography to achieve a diverse array of small peptides. This work and related works are also described in Fodor WO Patent #9,210,092, Dower WO #9,119,818, Barrett WO #9,107,087 and Pirrung WO#9,015,070.
  • peptides and oligonucleotides have poor bioavailability and limited stability in vivo, which limits their use as therapeutic agents.
  • non-biological compounds which mimic the structure of the active peptides and oligonucleotides must be synthesized based on the approximated three dimensional structure of the peptide or oligonucleotide and tested before an effective drug structure can be identified.
  • Bunin et al., J. Am. Chem. Soc. (1992) 114, 10997-10998 describe the synthesis of numerous 1,4 benzodiazapine derivatives using solid phase synthesis techniques.
  • the present invention is used to synthesize a library of non-biological organic compounds from a starting compound and identify individual compounds within the library which exhibit biological activity. Unlike peptides and.oligonucleotides, non-biological organic compounds comprise the bulk of proven therapeutic agents.
  • the invention can be used to directly identify new drug candidates or optimize an established drug compound which has sub-optimal activity or problematic side effects. This is accomplished through the use of highly specific biocatalytic reactions.
  • Enzymes are highly selective catalysts. Their hallmark is the ability to catalyze reactions with extraordinarily stereo-, regio-, and chemo-selectivities that are unparalleled in conventional synthetic chemistry. Moreover, enzymes are remarkably versatile. They can be tailored to function in organic solvents, operate at extreme pH's and temperatures, and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates.
  • Enzymes are reactive toward a wide range of natural and unnatural substrates, thus enabling the modification of virtually any organic lead compound. Moreover, unlike traditional chemical catalysts, enzymes are highly enantio- and regio-selective. The high degree of functional group specificity exhibited by enzymes enables one to keep track of each reaction in a synthetic sequence leading to a new active compound. Enzymes are also capable of catalyzing many diverse reactions unrelated to their physiological function in nature. For example, peroxidases catalyze the oxidation of phenols by hydrogen peroxide. Peroxidases can also catalyze hydroxylation reactions that are not related to the native function of the enzyme. Other examples are proteases which catalyze the breakdown of polypeptides. In organic solution some proteases can also acylate sugars, a function unrelated to the native function of these enzymes.
  • the present invention exploits the unique catalytic properties of enzymes.
  • biocatalysts i.e., purified or crude enzymes, non-living or living cells
  • the present invention uses selected biocatalysts and reaction conditions that are specific for functional groups that are present in many starting compounds.
  • Each biocatalyst is specific for one functional group, or several related functional groups, and can react with many starting compounds containing this functional group.
  • the biocatalytic reactions produce a population of derivatives from a single starting compound. These derivatives can be subjected to another round of biocatalytic reactions to produce a second population of derivative compounds. Thousands of variations of the original compound can be produced with each iteration of biocatalytic derivatization.
  • Enzymes react at specific sites of a starting compound without affecting the rest of the molecule, a process which is very difficult to achieve using traditional chemical methods.
  • This high degree of biocatalytic specificity provides the means to identify a single active compound within the library.
  • the library is characterized by the series of biocatalytic reactions used to produce it, a so called "biosynthetic history". Screening the library for biological activities and tracing the biosynthetic history identifies the specific reaction sequence producing the active compound. The reaction sequence is repeated and the structure of the synthesized compound determined. This mode of identification, unlike other synthesis and screening approaches, does not require immobilization technologies, and compounds can be synthesized and tested free in solution using virtually any type of screening assay.
  • the present invention is unique in that it involves a soluble state of the starting compound and its subsequent derivatives. This is a highly unique aspect of the present invention that has been thought to be a barrier.
  • Previous organic modifying technologies for biologically active compound identification involve starting compounds and derivatives attached to insoluble supports. This is taught in examples by Ellman (Bunin, B.S.; Ellman, J.A. "A general and expedient method for the solid-phase synthesis of 1,4- benzodiazepine derivatives", J. Am. Chem. Soc. 1992, 114 10997-10998), Gordon et al., (Gordon, E.M. ; Barrett, R.W. ; Dower, W.J.; Fodor, S.P.A.; Gallop, M.A.
  • the libraries of the instant invention can then be screened immediately without removal of the libraries from solid supports.
  • the present invention provides for useful, convenient, and efficient methods of generating and screening libraries of starting compounds and derivatives.
  • the present invention specifically incorporates a number of diverse technologies such as: (1) the use of enzymatic reactions to produce a library of drug candidates; (2) the use of enzymes free in solution or immobilized on the surface of particles, and organic compounds derivatized while dissolved in solution; (3) the use of receptors (hereinafter this term is used to indicate true receptors, enzymes, antibodies and other biomolecules which exhibit affinity toward biological compounds, and other binding molecules to identify a promising drug candidate within a library, even where such receptors are still associated with cell membranes, or intact cells); (4) the automation of all biocatalytic processes and many of the procedural steps used to test the libraries for desired activities, and (5) the coupling of biocatalytic reactions with drug screening devices which can immediately measure the binding of synthesized compounds to receptor molecules or in whole-cell assays and thereby immediately identify specific reaction sequences giving rise to biologically active compounds.
  • the present invention encompasses a method for drug identification comprising:
  • the enzymatic reactions are conducted with a group of enzymes that react with distinct structural moieties found within the structure of a starting compound.
  • Each enzyme is specific for one structural moiety or a group of related structural moieties.
  • each enzyme reacts with many different starting compounds which contain the distinct structural moiety.
  • the instant invention also provides for the systematic process of building up each reaction, an approach from the other end of the pathway.
  • a particular feature of the instant invention is the soluble state of the starting compounds and the subsequent derivatives.
  • enzymatic reactions are conducted in "reaction boxes", wherein a single enzyme or a group of enzymes which recognize the same functional group and perform the same type of enzymatic derivatization, are used.
  • a "reaction box” for acylation of an alcohol could contain numerous different lipases and esterases, for reaction with the same substrate.
  • a group of enzymes are used, the probability of achieving the desired transformation of acylation, is increased to an almost certainty, since there is more than one single enzyme present in the "reaction box”.
  • a group of enzymes together with a group of co-substrates can be used to make a library of derivatives. For example, acylation of an alcohol with a mixture of organic acids (co-substrates) can give a mixture of acylated alcohol derivatives.
  • reaction box represents a different enzymatic activity, where the different reactions are conducted separately. Accordingly there would be one “reaction box” for acylation of an alcohol, another “reaction box” for oxidation of alcohols and yet another “reaction box for the reduction of carbonyl groups. It is important that the different enzymatic reactions be conducted separately, so as to aid in the subsequent identification of a compound responsible for a specific activity.
  • reaction boxes are not only used concurrently on the same substrate, but also sequentially so as to conduct a second "iteration” of enzymatic derivatization. More specifically, an initial substrate can be first subjected to a series of "reaction boxes” which are believed to have reactivity with the known functional groups in the initial substrate, followed by subjecting the reaction product of an individual "reaction box” to a further series of "reaction boxes". By subjecting the reaction product of a "reaction box” to a further series of "reaction boxes", a second iteration of enzymatic derivatization is achieved.
  • reaction box The product of a second "reaction box” can itself be subjected to a further series of “reaction boxes", producing yet a third iteration of enzymatic derivatization.
  • a series of “reaction box” derivatizations can be conducted, to create any number of iterations, one, two, three, four, five, six, seven, eight, nine, ten, up to "n” iterations of enzymatic derivatization.
  • the present method is used to generate at least two iterations, more preferably at least three iterations and even more preferably at least four iterations.
  • the idea of iterations is exemplified in Figure 3, where the initial compound AZT is subjected to three iterations of enzymatic derivatization.
  • Each product of a "reaction box” can be analyzed to determine whether a desired activity is present within the product(s). If a "reaction box" is determined to possess the desired activity, the compound responsible for the desired activity can be identified and if desired isolated.
  • reaction box By using a series of "reaction boxes" identification of the compound responsible for a desired activity is greatly simplified. Since the reactivity of each "reaction box" is already known, one could easily conclude that the result of subjecting a compound which possessed an aldehyde group, to the sequential reaction boxes of a dehydrogenase followed by an esterase would produce a compound bearing an esterified alcohol. Therefore, knowledge of the sequence of reaction boxes which produced a product with a desired activity, would greatly facilitate structure identification. In some cases, it is not even necessary to isolate the compound with the desired activity, since the "reaction box” history will enable identification of the structure of the compound.
  • each "reaction box” can contain, in addition to a single enzyme or a group of enzymes, any co- factors, reagents and solvents necessary to conduct the desired reaction.
  • any co- factors, reagents and solvents necessary to conduct the desired reaction can be determined.
  • the present invention is also directed to a method of preparing a library of compounds, using the above-identified "reaction box” technique, as well as the library of compounds generated by such a technique.
  • a library of compounds is itself useful as it can be screened for a desired activity.
  • a library of compounds, generated by the present method can be screened for any desired activity, such as pharmaceutical, herbicidal, insecticidal and toxicological activities.
  • Figure 1 shows the starting active compound AZT with four potential sites for biocatalytic derivatization and eight possible biocatalytic reactions that can be used to produce a library of derivative compounds.
  • Figure 2 shows an automated system employing robotic automation to perform hundreds of biocatalytic reactions and screening assays per day.
  • Figure 3 illustrates the tracking of biocatalytic reactions to identify the sequence of reactions producing an active compound, which can subsequently be used to produce and identify the structure of the active compound.
  • Figure 4a illustrates biocatalytic modification of castanospermine.
  • Figure 4b illustrates biocatalytic modifications of ethotrexate.
  • Figure 1' is a chromatogra of a second iteration taxol- vinyl adipate alcohol ester
  • a starting compound such as AZT (3'-azidothymidine) , is chosen which exhibits drug activity or is believed to exhibit drug activity for a given disease or disorder.
  • the compound is analyzed with respect to its functional group content and its potential for structural modifications using selected biocatalytic reactions.
  • Functional groups which can be chemically modified using the selected biocatalytic reactions are listed in Table I. One of more of these functional groups are present in virtually all organic compounds.
  • a partial list of possible enzymatic reactions that can be used to modify these functional groups is presented in Table II.
  • a strategy is developed to systematically modify these functional groups using selected biocatalytic reactions and produce a library of derivative compounds to be screened for biological activity.
  • AZT contains four functional groups which are selected for biocatalytic modification: a primary hydroxyl, two carbonyls and a tertiary amine.
  • the biosynthetic strategy is designated in the form of biocatalytic "reaction box” numbers which correspond to specific types of biocatalytic reactions acting on specific functional groups present in the starting compound. These "reaction boxes” are listed in Table III. The following biocatalytic "reaction boxes” are selected to synthesize an AZT derivative library: A3, A10, All, C2, G6, G10 and G12. FIG. 1 illustrates the reaction of AZT with these selected biocatalytic reaction boxes. c) The biocatalytic reaction boxes are entered into an automated system which is shown in FIG 2. The system is programmed to automatically execute the aforementioned biocatalytic reactions and synthesize a library of derivative products. A single automated system in capable of performing hundreds of pre-programmed biocatalytic reactions per day.
  • Table IV details the number of potential reaction products produced in each reaction box and the resulting total number of possible compounds produced.
  • AZT up to 1.75 x 10 11 new compounds can be synthesized. It should be pointed out that this compares very favorably to peptide libraries.
  • a library of hexapeptides will contain 20 6 or 64 million compounds. This is a mere fraction, about 0.04% of the compounds that are possible using the biosynthetic approach described herein.
  • Table V lists the results of a similar analysis on eleven other starting drug compounds. As shown in this table, the biocatalytic reactions can generate huge numbers of derivative compounds for drug screening.
  • the synthesized library of new compounds is assayed using enzyme inhibition assays, receptor-binding assays, immunoassays, and/or cellular assays to identify biologically active compounds.
  • any remaining AZT present in the library is either removed or inhibited to simplify the interpretation of screening assay results. This is easily accomplished by HPLC, TLC, or the addition of a monoclonal antibody specific for the starting compound.
  • Numerous in vitro assays are available that test for anti-viral, anti-cancer, anti-hypertensive and other well known pharmacological activities. Some of these assays are listed in Table VI. Most of these assays are also performed on the automated system.
  • the reaction sequence is repeated to produce a sufficient amount of product for chemical analysis.
  • the specificity of the biocatalytic reactions also permits the accurate duplication of the reaction pathway producing the active compounds.
  • the structure of the active compound is qualitatively determined by analyzing the starting compounds, substrates and identified biocatalytic reaction sequence. The structure is then confirmed using gas chromatography, mass spectroscopy, NMR spectroscopy and other organic analytical methods. This mode of identification eliminates the need for product purification and also reduces the amount of test screening required to identify a promising new drug compound. This process dramatically reduces the time necessary to synthesize and identify new drug compounds.
  • this mode of active compound identification does not require immobilization technologies, and compounds can be synthesized and tested free in solution under in vivo like conditions using virtually any type of screening assay (receptor, enzyme inhibition, immunoassay, cellular, animal model) .
  • biocatalytic reactions are optimized by controlling or adjusting such factors as solvent, buffer, pH, ionic strength, reagent concentration and temperature.
  • the biocatalysts used in the biocatalytic reactions may be crude or purified enzymes, cellular lysate preparations, partially purified lysate preparations, living cells or intact non-living cells, used in solution, in suspension, or immobilized on magnetic or non-magnetic surfaces.
  • non-specific chemical reactions may also be used in conjunction with the biocatalytic reaction to obtain the library of modified starting compounds.
  • non-specific chemical reactions include: hydroxylation of aromatics and aliphatics; oxidation reactions; reduction reactions; hydration reactions; dehydration reactions; hydrolysis reactions; acid/based catalyzed esterification; transesterification; aldol condensation; reductive amination; amminolysis; dehydrohalogenation; halogenation; acylation; acyl substitution; aromatic substitution; Grignard synthesis; Friedel-Crafts acylation; etherification.
  • the biocatalytic reaction can be performed with a biocatalyst immobilized to magnetic particles forming a magnetic biocatalyst.
  • the method of this embodiment is performed by initiating the biocatalytic reaction by combining the immobilized biocatalyst with substrate(s) , cofactors(s) and solvent/buffer conditions used for a specific biocatalytic reaction.
  • the magnetic biocatalyst is removed from the biocatalytic reaction mixture to terminate the biocatalytic reaction. This is accomplished by applying an external magnetic field causing the magnetic particles with the immobilized biocatalyst to be attracted to and concentrate at the source of the magnetic field, thus effectively separating the magnetic biocatalyst from the bulk of the biocatalyst reaction mixture.
  • biocatalytic reactions can also be performed using biocatalysts immobilized on any surface which provides for the convenient addition and removal of biocatalyst from the biocatalytic reaction mixture thus accomplishing a sequential series of distinct and independent biocatalytic reactions producing a series of modified starting compounds.
  • the biocatalytic reactions can also be used to derivatize known drug compounds producing new derivatives of the drug compound and select individual compounds within this library that exhibit optimal activity. This is accomplished by the integration of a high affinity receptor into the biocatalytic reaction mixture, which is possible because of the compatibility of the reaction conditions used in biosynthesis and screening.
  • the high affinity receptor is added to the reaction mixture at approximately one half the molar concentration of- the starting active compound, resulting in essentially all of the receptor being bound with the starting active compound and an equal molar concentration of starting active compound free in solution and available for biocatalytic modification.
  • the biocatalytic reaction mixture produces a derivative which possesses a higher binding affinity for the receptor, which can translate into improved pharmacological performance, this derivative will displace the bound starting active compound and remain complexed with the receptor, and thus be protected from further biocatalytic conversions.
  • the receptor complex is isolated, dissociated and the bound compound analyzed. This approach accomplishes the identification of an improved version of the drug compound without the need to purify and test each compound individually.
  • the biocatalytic reactions and in vitro screening assays can be performed with the use of an automated robotic device.
  • the automated robotic device having:
  • a magnetic separation block attached to the same XY table to separate the biocatalyst immobilized to magnetic particles from the biocatalytic mixture by applying an external magnetic field causing the magnetic particles to be attracted to and concentrate at the source of the magnetic filed, thus effectively separating them from the bulk of the biocatalytic reaction mixture; and (e) a programmable microprocessor interfaced to the XYZ pipetting boom, and XYZ reaction-vessel transfer boom, the temperature block and the magnetic separation block to precisely control and regulate all movements and operations of these functional units in performing biocatalytic reactions to produce modified starting compounds and assays to determine desired activities.
  • Figure 2 illustrates the automated robotic device of this invention.
  • Mounted in the frame 1 of the system are containers for starting compounds 2., and containers for reagents 3. such as enzymes, cofactors, and buffers.
  • There are specific biosynthesis boxes 4 . which contain reagents for various classes of reactions.
  • the frame also has arrays of reaction vessels 5_, and a heating block 6 with wells 2 for conducting reactions at a specific temperature.
  • the frame has an area 8. for reagents for screening test 8_ which contains reagents used for conducting screening tests, and area 9 which contains assay vessels for conducting screening tests, the automated system uses a X-Y-Z pipetting and vessel transfer boom 0 to dispense all reagents and solutions, and transfer reaction vessels.
  • the X-Y-Z reaction-vessels transfer boom can deliver starting compounds and reagents to specific locations for making specific modified starting compounds which in turn can be delivered to specific locations for conducting assays. In this way the process of making modified starting compounds and testing for optimum activity is largely automated.
  • Figures 4a and 4b illustrate derivatization of castanospermine and methotrexate. All of these embodiments utilize the biocatalytic conversions set out in Table II and the assays set out in Table VI.
  • A. Hydroxyl Groups These groups can undergo numerous reactions including oxidation to aldehydes or ketones (1.1), acylation with ester donors (2.3, 3.1), glycosidic bond formation (2.4, 3.2, 5.3), and etherification (2.1, 3.3). Potential for stereo- and regio-selective synthesis as well as prochiral specificity.
  • Carboxyl Groups These groups can be decarboxylated (1.2, 1.5, 4.1), and esterified (3.1, 3.6).'
  • Aromatic Groups These groups can hydroxylated (1.11, 1.13, 1.14), and oxidatively cleaved to diacids (1.14).
  • Carbohydrate Groups These groups can be transferred to hydroxyls and phenols (2.4, 3.2, 5.3), and to other carbohydrates (2.4).
  • Enzvmes l.l. Dehydrogenases, Dehydtratases, Oxidases Representative Enzvmes:
  • Enzyme Classes 3.1, 3.4, 3.5, 3.6
  • Enzymes Esterases, lipases, proteases, sulfatases, phosphatases, acylases, lactamases, nucleases, acyl transferases
  • Cosubstrates/Cofactors Esters of alkyl, aryl, charged, polar/neutral groups. These acyl donors can be chosen from the class consisting of the following structural formulae:
  • R-O-CO-R' alkyl, vinyl, isopropenyl haloalkyl, aryl, derivatives of aryl (i.e., nitrophenyl) and R' can be any alkyl or aryl group with or without derivatives.
  • Such derivatives include halogens, charged functional groups (i.e., acids, sulfates, phosphates, amines, etc.), glycols (protected or unprotected) , etc.
  • Phosphorylase a Phosphorylase b Dextransucrase Levansucrase Sucrose Phosphorylase Glycogen Synthase UDP-Glucuronyltransferase Galactosyl Transferase Nucleoside Phosphorylase ⁇ - and ⁇ -Amylase Amyloglucosidase (Glucoamylase) Cellulase Dextranase Chitinase Pectinase Lysozyme TABLE II (cont. )
  • Cosubstrates/Cofactors All available sugars and their derivatives. These sugars can be onosaccharides, disaccharides, and oligosaccharides and their derivatives. 4. Etherification of primary and secondary alcohols Reaction Boxes: All, Bll
  • Cosubstrates/Cofactors alcohols of any chain length being alkyl, aryl, or their structural derivatives.
  • Beta-adrenergic receptor binding assay (bronchodilator, cardiotonic, tocolytic, anti-anginal, anti-arrhythmic, anti-glaucoma)
  • Dopamine receptor binding assay anti-migraine, anti- parkinsonian, anti-emetic, anti-psychotic
  • Product peaks were detected at 227 nm using a photodiode array detector.
  • a mixture of 55 enzymes consisting of crude lipases, crude proteases, and purified proteases (total mass of 600 mg) was added to a solution of taxol (17.0 g, 20 ⁇ mol) and vinyl butyrate (0.25 ml, 1.5 mmol) in 2.9 ml tert a yl alcohol.
  • the mixture was placed into a septum-sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C for a period of 48 h. The reaction was then stopped by removing the suspended solid catalyst by centrifugation.
  • the supernatant containing taxol esters was evaporated to dryness in vacuum and redissolved in 0.3 ml methanol.
  • An aliquot of the resultant concentrated solution (15 ⁇ l) was diluted with 250 ⁇ l methanol and analyzed by HPLC as described in 1.1.
  • the chromatogram revealed a peak with a retention time of 9.7 min (unreacted taxol), and a peak at 14.6 min (2 , -taxol butyrate, yield 7%).
  • the enzymes were then split into three groups: 26 lipases (100 mg/ml) , 26 crude proteases (100 mg/ml) , and 4 purified proteases (10 mg/ml) and the identical reaction as above performed. Only the purified proteases showed significant activity with a yield of 2'-butyrate of 15% after 48 h.
  • the purified proteases were then divided into individual enzymes (all used at a concentration of 10 mg/ml) and thermolysin (a bacterial protease from Bacillus thermoproteolyticus rokko a .k .a . thermolysin) was identified as the most active enzyme with a yield after 48 h of 24%. This approach demonstrates that active biocatalysts can be easily identified by a sequential process of eliminating unreactive biocatalysts.
  • Rapidase S-90 B subtilis Gist Brocades HT-Proteolytic 200 Solvay Opticlean M-375 Alkaline protease
  • the optimal enzyme catalyst used for the synthesis was produced by freeze-drying an aqueous solution containing thermolysin, KCl and potassium phosphate buffer adjusted to pH 7.5.
  • the solid catalyst obtained after free-drying contained 5% enzyme, 94% KCl and 1% potassium phosphate.
  • the powdered enzyme catalyst (335 mg - containing 16.75 mg thermolysin) was added to a solution of taxol (17.0 mg, 20 ⁇ mol) and vinyl caproate (straight-chain C 6 ester) (0.24 ml. 1.5 mmol) in 2.9 ml tert-amyl alcohol.
  • the mixture was placed into a septum- sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C. After 28 h the reaction mixture (including enzyme) was removed and the full contents of the mixture added to a separate solution containing vinyl propionate (0.24 ml, 2.2 mmol), vinyl acrylate (0.24 ml, 2.2 mmol), and vinyl butyrate (0.24 ml, 1.9 mmol). This second reaction was placed on the shaker and incubated at 250 rpm at 35°C.
  • reaction mixture including enzyme
  • vinyl acetate (0.25 ml, 2.6 mmol) and vinyl chloroacetate (0.24 ml, 1.8 ,mol)
  • This reaction was allowed to proceed for 24 h at 250 rpm and 35°C.
  • the sequential reaction was then stopped by removing the suspended solid catalyst by centrifugation.
  • the sequential reaction was aided by the soluble nature of the taxol and taxol derivatives.
  • the supernatant containing taxol esters was evaporated to dryness in vacuum and redissolved in 0.3 ml methanol.
  • a concentrated methanol solution of reaction products produced as described in 1.3 was applied on a preparative TLC silica plate (Whatman, 20x20 cm, silica layer thickness 500 ⁇ m, containing fluorescent marker) , and the plates were developed using a solvent mixture of chloroform:acetonitrile (4:1 v/v) . Positions of product spots were determined by irradiating the plates with ultraviolet fight.
  • the R f value of taxol is 0.16 and the R f of the taxol esters range from 0.28 to 0.71.
  • the products were removed from the TLC plate and dissolved in ethyl acetate. The products were then dried in vacuo.
  • the library of taxol derivatives described above was screened for cytotoxicity against HL-60 cells, a promyelocytic leukemia cell line, and MOLT-4 cells, a lymphoblastic leukemia cell line.
  • Cells were seeded in 96-well plates at densities of 30,000 cells/well and grown in RPMI-1640 medium containing 10% bovine fetal calf serum at 37°C for 24 h. The medium was then replaced with fresh medium containing the taxol derivatives (excluding taxol, which had been removed by preparative thin-layer chromatography) dissolved in DMSO at final concentrations ranging from 100 nM to 0.1 nM. The final concentration of DMSO in the cell medium was 0.5% (v/v) .
  • the powdered enzyme catalyst prepared as described in 1.3 above (140 mg) was added to a solution of taxol (5.5 mg, 6.5 ⁇ mol) and on individual vinyl ester (80 ⁇ l, approximately 2 mmol) in 1.0 ml tert-amyl alcohol.
  • the following vinyl esters were used as acylating agents: acetate, chloroacetate, acrylate, propionate, butyrate, and caproate.
  • Each mixture was placed into a septum-sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C. After 96 h the reaction was stopped by removing the suspended solid catalyst by centrifugation.
  • reaction products were separated from taxol by TLC.
  • Concentrated methanol solutions of reaction products produced as described in 1.6 were applied on preparative TLC silica plates (Whatman, 20x20 cm, silica layer thickness 500 ⁇ m, containing fluorescent market) , and the plates were developed using a solvent mixture of chloroform:acetonitrile (4:1 v/v) . Positions of product spots were determined by irradiating the plates with ultraviolet light. The R f values of the products are given in Table VIII. Product spots were scraped off the plates separately and scrapings were eluted with 15 ml ethyl acetate to recover the product Dry products were obtained by evaporation of ethyl acetate in vacuo.
  • the enzyme catalyst used for hydrolysis was produced by freeze-drying an aqueous solution (adjusted to pH 7.5) containing equal weight amounts of thermolysin, subtilisin Carlsberg, chymotrypsin and trypsin.
  • the enzyme catalyst (1.1 mg) was dissolved in 0.7 ml 0.1 M potassium phosphate buffer pH 7.5.
  • the aqueous enzyme solution was added to a solution of taxol (2.0 mg, 2.4 ⁇ mol) in 0.3 ml tert amyl alcohol.
  • the biphasic reaction system produced in this way was placed into a septum-sealed glass vial and put on an orbit shaker operating at 75 strokes/min and 20°C.
  • the chromatogram of the methanol solution of the dry residue obtained from the organic layer revealed a peak with retention time of 20.4 min (unreacted taxol) and a peak at 18.4 min, representing a product of enzymatic hydrolysis of taxol, as identified by the characteristic uv-absorbance scan.
  • the estimated yield of this product was 9%.
  • Product peaks were detected at 227 nm using a photodiode array detector.
  • ⁇ -glucosidase from baker's yeast as a biocatalyst.
  • the enzymes used were: ⁇ -glucosidase from brewer's yeast (pH 7.0), ⁇ -glucosidase from baker's yeast (pH 7.0), ⁇ -galactosidase from E. coli (pH 5.0), ⁇ galactosidase from A. oryzae (pH 5.0), ⁇ -glucuronidase from bovine liver (pH 5.0).
  • Example 4 The synthesis of second iteration taxol ester products.
  • Taxol is initially acylated with vinyl adipate in a first reaction box acylation to give a mixture of taxol-2'-vinyl adipate an taxol-2' ,7-divinyl adipate.
  • the mixture and individual adipate esters were then used in several second reaction boxes, each containing a mixture of alcohols or sugars to give a second iteration taxol library.
  • the mixture was supplemented with 1 vol.% each of n-butanol, n-hexanol, l,3-S(+)-butanediol and sec-phenethyl alcohol for a total of 5 vol.% of alcohols.
  • the reaction mixture was incubated at 45°C for 4 days under constant shaking at 250 rpm. After the reaction the enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program given in Table 1'.
  • FIG. 1' A representative HPLC trace is shown in Fig. 1'.
  • the individual identities of these peaks were determined by repeating the second iteration reaction with individual alcohols in the second iteration step. This is the same approach that is used to re-synthesize, or backtrack, active products from the results of screening assays. Retention times of some of the products are given in Table 2' .
  • Enzymatically synthesized taxol-2'-vinyl adipate (5 mM) in acetonitrile was reacted with a mixture of sugars (mono- and disaccharides) (50 mM) catalyzed by lipase from Candida antarctica (75 mg/ml) .
  • the reaction mixture was incubated at 45°C with 250 rpm shaking for 7 days. After the reaction, the enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program given in Table 3'.
  • Retention Yield Retention Yield, Retention Yield, Retention Yield, time, mm % time , min % time , min %
  • Taxol acylation was also extended to produce carbonates at the 2' and 7-positions of taxol. This reaction is similar to that for acylation with vinyl esters, but uses a different reaction box for vinyl carbonate co-substrates.
  • Taxol (5mM) was dissolved in hexane containing 30 vol.% tetrahydrofuran. To this solution, immobilized lipases from Candida antartica and Mucor miehei were added in concentration 50 mg/ml each. The mixture was supplemented with 1 M butyl vinyl carbonate and incubated at 45°C for 4 days under constant shaking at 250 rpm. After the reaction the enzyme was removed, the supernatant dried in vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program in Table 1'. The reaction produced a single product with a reaction time of 21.1 min and 30% yield. Preliminary structural determination by NMR indicates that this product is taxol-7-butyl carbonate.
  • Taxol (5 mM) was dissolved tert-amyl alcohol.
  • thermolysin powder (containing 95% KCl, 4% enzyme, and 1% phosphate buffer salt) was added at concentration of 1.2 mg protein/ml.
  • the mixture was supplemented with 1 M butyl vinyl carbonate or 1 M 1,3-R(- )butanediol di(vinyl carbonate) and incubated for at 45°C for 4 days under constant shaking at 250 rpm. After this time the reaction was stopped and enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program in Table 1' .
  • the third major class of reactions recently performed involved the phosphorylation of taxol.
  • the phosphorylation reaction was performed in a biphasic water-organic system.
  • the aqueous phase consisted of a solution of alkaline phosphatase from chicken intestine (1 mg/ml) in 0.2 M phosphate buffer, pH 8, whereas the organic phase was 15 mM solution of taxol in chloroform. The volume ratio of water to chloroform was 3:7.
  • the mixture was incubated at 25°C with gentle shaking for 7 days.
  • the organic phase was then separated, dried in vacuum, redissolved in methanol and analyzed by reversed phase HPLC using the gradient program in Table 6' .
  • reaction products two products with retention time 27.1 minutes (23% yield) and 29.8 minutes (21% yield) (retention time of taxol was 38.3 min). Thus, these products have retention times shorter then taxol itself, and this is indicative of more water-soluble taxol derivatives.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

This invention encompasses methods for producing a library of modified starting compounds by use of biocatalytic reactions on a starting compound and identifying the modified starting compound with the optimum desired activity. The invention encompasses starting compounds and modified compounds that are free in solution. The method is useful in producing modified pharmaceutical compounds with desired specific activity.

Description

Description
Biocatalytic Methods For Synthesizing And Identifying Biologically Active Compounds
Technical Field
This invention is in the field of synthesizing and identifying biologically active compounds.
Background Art
The prior art is replete with examples of chemically, icrobially, or enzymatically synthesizing compounds with biological activity. The goal of these efforts is the discovery of new and improved pharmaceutical compounds.
The discovery of new pharmaceutical compounds is for the most part a trial and error process. So many diverse factors constitute an effective pharmaceutical compound that it is extremely difficult to reduce the discovery process to a systematic approach. Typically, thousands of organic compounds must be isolated from biological sources or chemically synthesized and tested before a pharmaceutical compound is found.
Synthesizing and testing new compounds for biological activity, which is the first step in identifying a new synthetic drug, is a time consuming and expensive undertaking. Typically, compounds must by synthesized, purified, tested and quantitatively compared to other compounds in order to identify active compounds or identify compounds with optimal activity. The synthesis of new compounds is accomplished for the most part using standard chemical methods. Such methods provide for the synthesis of virtually any type of organic compound; however, because chemical reactions are non¬ specific, these syntheses require numerous steps and multiple purifications before a final compound is produced and ready for testing.
New biological and chemical approaches have recently been developed which provide for the synthesis and screening of large libraries of small peptides and oligonucleotides. These methods provide for the synthesis of a broad range of chemical compounds and provide the means to potentially identify biologically active compounds. The chemistries for synthesizing such large numbers of these natural and non- naturally occurring polymeric compounds is complicated, but manageable because each compound is synthesized with the same set of chemical protocols, the difference being the random order in which amino acids or nucleotides are introduced into the reaction sequence.
Fodor, S.P.A. et al (1990) Science 251. 767-773, describe methods for discovering new peptide ligands that bind to biological receptors. The process combines solid-phase chemistry and photolithography to achieve a diverse array of small peptides. This work and related works are also described in Fodor WO Patent #9,210,092, Dower WO #9,119,818, Barrett WO #9,107,087 and Pirrung WO#9,015,070.
Houghten, R.A. et al. (1991) Nature 354. 84-86, describe an approach that synthesizes libraries of free peptides along with an iterative selection process that permits the systematic identification of optimal peptide ligands. This work is also described in Appl. WO Patent #9,209,300.
Lam, K.S., et al. (1991) Nature 354. 82-84, describe a method that provides for the systematic synthesis and screening of peptide libraries on a solid-phase microparticle support on the basis of a 'one-bead, one-peptide' approach.
Cwirla, S.E., et al (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382, describe a method for constructing a library of peptides on the surface of a phage by cloning randomly synthesized oligonucleotides into the 5' region of specific phage genes resulting in millions of different hexapeptides expressed at the N terminus of surface proteins.
These methods accelerate the identification of biologically active peptides and oligonucleotides. However, peptides and oligonucleotides have poor bioavailability and limited stability in vivo, which limits their use as therapeutic agents. In general, non-biological compounds which mimic the structure of the active peptides and oligonucleotides must be synthesized based on the approximated three dimensional structure of the peptide or oligonucleotide and tested before an effective drug structure can be identified.
Bunin et al., J. Am. Chem. Soc. (1992) 114, 10997-10998 describe the synthesis of numerous 1,4 benzodiazapine derivatives using solid phase synthesis techniques.
The prior art is replete with examples showing enzymatic conversion of non-physiological substances under many conditions.
References demonstrating that enzyme specificity can be changed/tailore :
1. Zaks, A. and Klibanov, A.M. Substrate specificity of enzymes in organic solvents vs. water is reversed. Journal of the American Chemical Society 108 2767-2768, 1986.
2. Ferjancic, A., Puigserver, A. and Gaertner, H. Unusual specificity of PEG-modified thermolysin in peptide synthesis catalyzed in organic solvents. Biotechnology Letters 10 (2) 101-106, 1988.
3. Nasri, M. and Thomas, D. Increase of the potentialities of restriction endonucleases by specificity relaxation in the presence of organic solvents. Ann. N.Y. Acad. Sci. 542.255-265, 1988.
4. Stahl, M. , Mansson, M.O. and Mosbach, K. The synthesis of a D-amino acid ester in an organic media with chymotrypsin modified by a bio-imprinting procedure. Biotechnology Letters JL2. (3) 161-166, 1990.
5. Stahl, M. , Jeppsson-Wistrand, U. , Mansson, M.O. and Mosbach, K. Induced stereoselectivity and substrate selectivity of bio-imprinted a-chymotrypsin in anhydrous organic media. Journal of the American Chemical Society 113 (24) 9366-9368, 1991.
6. Gololobov, M.Y. , Voyushina, T.L. , Stepanov, V.M. and Adlercreutz, P. Organic solvent changes the chymotrypsin specificity with respect to nucleophiles. FEBS Letters 307 (3) 309-312, 1992.
7. Hertmanni, P., Pourplanche, C. and Larreta-Garde, V. Orientation of enzyme catalysis and specificity by water- soluble additives. Ann. New York Acad. Sci. (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 672. 329-335, 1992.
8. Cabezas, M.J., del Campo, C. , Llama, E. , Sinisterra, J.V. and Gaertner, H. Organic reactions catalyzed by modified enzymes. 1. Alteration of the substrate specificity of a- chymotrypsin by the modification process. Journal of Molecular Catalysis 71 (2) 261-278, 1992.
9. Nagashima, T., Watanabe, A. and Kise, H. Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity. Enzyme and Microbial Technology .14. (10) 842-847, 1992.
10. Parida, S. and Dordick, J.S. Tailoring lipase specificity by solvent and substrate chemistries, J. Org. Chem. 58. (12) 3238-3244, 1993.
11. Tawaki, S. and Klibanov, A.M. Chemoselectivity of enzymes in anhydrous media is strongly solvent dependent. Biocatalysis 8. (1) 3-19, 1993.
12. Wescott, C.F. and Klibanov, A.M. Solvent variation inverts substrate specificity of an enzyme. JACS 115 (5) 1629-1631, 1993.
References demonstrating that enzyme enantioselectivity can be changed/tailored:
1. Sakurai, T. , Margolin, A.L., Russell, A.J. and Klibanov, A. M. Control of enzyme enantioselectivity by the reaction medium. Journal of the American Chemical Society 110 (21) 7236-7237, 1988.
2. Fitzpatrick, P.A. and Klibanov, A.M. How can the solvent affect enzyme enantioselectivity? Journal of the American Chemical Society 113 (8) 3166-3171, 1991.
3. Hult, K. and Norin, T. Enantioselectivity of some lipases - control and prediction. Pure and Applied Chemistry 64 (8) 1129-1134, 1992.
4. Miyazawa, T. , Kurita, S., Ueji, S., Yamada, T. and Kuwata, S. Resolution of racemic carboxylic acids via the lipase-catalyzed irreversible transesterification using vinyl esters - effects of alcohols as nucleophiles and organic solvents on enantioselectivity. Biotechnology Letters .14 (10) 941-946, 1992.
5. Tawaki, S. and Klibanov, A.M. Inversion of enzyme enantioselectivity mediated by the solvent. Journal of the American Chemical Society 114 (5) 1882-1884, 1992.
6. Ueji, S., Fujino, R. , Okubo, N. , Miyazawa, T. , Kurita, S., Kitadani, M. and Muromatsu, A. Solvent-induced inversion of enantioselectivity in lipase-catalyzed esterification of 2-phenoxypropionic acids. Biotechnology Letters 14 (3) 163-168, 1992.
7. Terradas, F. , Testonhenry, M. , Fitzpatrick, P.A. and Klibanov, A.M. Marked dependence of enzyme prochiral selectivity on the solvent. Journal of the American Chemical Society 115 (2) 390-396, 1993.
8. Herradon, B. Biocatalytic synthesis of chiral polyoxygenated compounds: effect of the solvent on the enantioselectivity of lipase catalyzed transesterifications in organic solvents. Synlett 2. 108-110, 1993.
References demonstrating the ability of enzymes to convert unnatural substrates:
1. Bianchi, D. , Cesti, P., Golini, P., Spezia, S., Garavaglia, C. and Mirenna, L. Enzymatic preparation of optically active fungicide intermediates in aqueous and in organic media. Pure and Applied Chemistry 6 (8) 1073-1078, 1992.
2. Natoli, M. , Nicolosi, G. and Piattelli, M. Regioselective alcoholysis of flavonoid acetates with lipase in an organic solvent. Journal of Organic Chemistry 5 (21) 5776-5778, 1992. 3. Izu i, T. , Tamura, F. and Sasaki, K. Enzymatic kinetic resolution of <4>(1,2)ferrocenophane derivatives. Bulletin of the Chemical Society of Japan 65. (10) 2784-2788, 1992.
4. Miyazawa, T., Mio, M. , Watanabe, .Y. , Ya ada, T. and Kuwata, S. Lipase-catalyzed transesterification procedure for the resolution of non-protein amino acids. Biotechnology Letters 14 (9) 789-794, 1992.
5. Murata, M. , Uchida, H. and Achiwa, K. Lipase- catalyzed enantioselective synthesis of optically active mephobarbital, hexobarbital and febarbamate. Chemical- Pharmaceutical Bulletin 40. (10) 2605-2609, 1992.
6. Johnson, C.R. , Golebiowski, A. and Steensma, D.H. Enzymatic asymmetrization in organic media - synthesis of unnatural glucose from cycloheptatriene. Journal of the American Chemical Society 114 (24) 9414-9418, 1992.
7. Cruces, M.A. , Otero, C. , Bernabe, M. , Martinlomas, M. and Ballesteros, A. Enzymatic preparation of acylated sucroses. Ann. New York Acad. Sci. (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 672 436-443, 1992.
8. Tanaka, A., Fukui, T. , Uejima, A., Zong, M.H. and Kawamoto, T. Bioconversion of nonnatural organic compounds - esterification and dehydrogenation of organosilicon compounds. Ann. New York Acad. Sci (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 671 431-435, 1992.
9. Kodelia, G. and Kolisis, F.N. Studies on the reaction catalyzed by protease for the acylation of flavonoids in organic solvents. Ann. New York Acad. Sci. (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 672. 451-457, 1992.
10. Wagner, F. , Kleppe, F. , Lokotsch, W. , Zie ann, A. and Lang, S. Synthesis of uncommon wax esters with immobilized lipases. Ann. New York Acad. Sci. (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 672 484-491, 1992.
11. Patel. R.N., Howell, J.M. , Banerjee, A., Fortney, K.F. and Szarka, L. J. Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid. Ann. New York Acad. Sci (Enzyme Eng. XI, D.S. Clark, D.A. Estell, eds) 672 415-424, 1992.
12. Bergbreiter, D.E. and Momongan, M. Asymmetric synthesis of organometallic reagents using enzymatic methods. Applied Biochemistry and Biotechnology .32.1-3 55-72, 1992.
13. Carretero, J.C. and Dominguez, E. Lipase-catalyzed kinetic resolution of - hydroxy phenyl sulfones. Journal of Organic Chemistry 5J (14) 3867-3873, 1992.
14. Johnson, C.R. , Adams, J.P., Bis, S.J. , Dejong, R.L., Golebiowski, A., Medich, J.R. , Penning, T.D., Senanayake, CH. , Steensma, D.H. and Vanzandt, M.C. Applications of enzymes in the synthesis of bioactive polyols. Indian Journal of Chemistry Section B - Organic Chemistry Including Medicinal Chemistry 22 (l) 140-144, 1993.
15. Fabre, J. , Betbeder, D. , Paul, F. , Monsan, P. and Perie, J. Regiospecific enzymic acylation of butyl a-D- glucopyranoside. Carbohydrate Research 243 (2) 407-411, 1993.
16. Bianchi, D. , Cesti, P., Golini, P., Spezia, S., Garavaglia, C. and Mirenna, L. Enzymatic preparation of optically active fungicide intermediates in aqueous and in organic media. Indian Journal of Chemistry Section B - Organic Chemistry Including Medicinal Chemistry J32. (1) 176- 180, 1993.
17. Kanerva, L.T. and Sundholm, O. Lipase catalysis in the resolution of racemic intermediates of diltiazem synthesis in organic solvents. Journal of the Chemical Society - Perkin Transactions I 13 1385-1389, 1993.
18. Ikeda, I. and Klibanov, A.M. Lipase-catalyzed acylation of sugars solubilized in hydrophobic solvents by complexation. Biotechnology and Bioengineering £2. (6) 788- 791, 1993.
19. Hyun, C.K. , Kim, J.H. and Ryu, D.D.Y. Enhancement effect of water activity on enzymatic synthesis of cephalexin. Biotechnology and Bioengineering 42. (7) 800-806, 1993.
20. Panza, L. , Luisetti, M. , Crociati, E. and Riva, S. Selective acylation of 4,6-O-benzylidene glycopyranosides by enzymatic catalysis. J. Carbohydrate Chem. 12. (1) 125-130, 1993.
21. Wang, L. , Kobatake, E. , Ikariya a, Y. and Aizawa, M. Regioselective oxidative polymerization of 1,5- dihydroxynaphthalene catalyzed by bilirubin oxidase in a water-organic solvent mixed solution. Journal of Polymer Science Part A - Polymer Chemistry 3_1 (11) 2855-2861, 1993.
22. Knani, D. and Kohn, D.H. Enzymatic polyesterification in organic media. 2. Enzyme-catalyzed synthesis of lateral-substituted aliphatic polyesters and copolyesters. J. Polymer Sci., Part A - Polymer Chem. 3_l (12) 2887-2897, 1993.
23. Kanerva, L.T. and Sundholm O. Enzymatic acylation in the resolution of methyl threo-2-hydroxy-3-(4-methoxyphenyl)- 3-(2-x-phenylthio) propionates in organic solvents. Journal of the Chemical Society - Perkin Transactions I 20 2407-2410, 1993.
24. Sharma, A. and Chattopadhyay, S. Lipase catalyzed acetylation of carbohydrates. Biotechnology Letters 1J5 (11) 1145-1146, 1993.
25. Pavel, K. and Ritter, H. Enzymes in polymer chemistry. 7. Lipase-catalyzed esterification of carboxyl- terminated methacrylic oligomers and copolymers with isopropyl alcohol and 9-fluorenylmethanol. Makromolekulare Che ie- Macromolecular Chemistry and Physics 194 (12) 3369-3376, 1993.
26. Uejima, A., Fukui, T. , Fukusaki, E. , O ata, T. , Kawamoto, T. , Sonomoto, K. and Tanaka, A. Efficient kinetic resolution of organosilicon compounds by stereoselective esterification with hydrolases in organic solvent. Appl. Microbiol. Biotech. 38. (4) 482-486, 1993.
27. Mukesh, D. , Sheth, D. , Mokashi, A., Wagh, J. , Tilak, J.M. , Banerji, A.A. and Thakkar, K.R. Lipase catalyzed esterification of isosorbide and sorbitol. Biotechnology Letters 15. (12) 1243-1246, 1993.
28. Baldessari, A., Iglesias, L.E. and Gros, E.G. Regioselective acylation of 3-mercaptopropane-l,2-diol by lipase-catalyzed transesterification. Journal of Chemical Research - S 9 382-383, 1993.
29. De Goede, A.T.J.W. , Benckhuijsen, W. , van Rantwijk, F. , Maat, L. and van Bekkum, H. Selective lipase-catalyzed 6- o-acylation of alkyl a-D-glucopyranosides.using functionalized ethyl esters. Recueil Des Travaux Chimiques Des Pays Bas - Journal of the Royal Netherlands Chemical Society 112 (11) 567-572, 1993.
30. Menendez, E. and Gotor, V. Acylation and alkoxycarbonylation of oximes through an enzymatic oximolysis reaction. Synthesis - Stuttgart 1 72-74, 1993.
31. Li, Y.F. and Hammerschmidt, F. Enzymes in organic chemistry. l. Enantioselective hydrolysis of a-(acyloxy) phosphonates by esterolytic enzymes. Tetrahedron - Asymmetry 1 (1) 109-120, 1993.
32. Schlotterbeck, A., Lang, S., Wray, V. and Wagner, F. Lipase-catalyzed monoacylation of fructose. Biotechnology Letters 15 (1) 61-64, 1993.
33. Frykman, H. , Ohrner, N. , Norin, T. and Hult, K. S- Ethyl thiooctanoate as acyl donor in lipase catalyzed resolution of secondary alcohols. Tetrahedron Letters 3_4 (8) 1367-1370, 1993.
34. Oguntimein, G.B. , Erdmann, H. and Schmid, R.D. Lipase catalyzed synthesis of sugar ester in organic solvents. Biotechnology Letters 15 (2) 175-180, 1993.
35. Lopez, R. , Perez, C. , Fernandez-Mayorales, A. and Conde, S. Enzymatic transesterification of alkyl 2,3,4-tri-O- acyl-beta-D-xylopyranosides. J. Carbohydrate Chem. 12. (2) 165-171, 1993.
36. Nae ura, K. , Ida. H. and Fukuda, R. Lipase YS- catalyzed enantioselective transesterification of alcohols of bicarbocyclic compounds. Bull. Chem. Soc. Japan 6_6 (2) 573- 577, 1993.
37. Lambusta, D. , Nicolosi, G. , Piattelli, M. and Sanfilippo, C. Lipase catalyzed acylation of phenols in organic solvents. Ind. J. Chem. Section B 3.2 (1) 58-60, 1993. 38. Astorga, C. , Rebolledo, F. and Gotor, V. Enzymatic hydrazinolysis of diesters and synthesis of N-aminosuccinimide derivatives. Synthesis - Stuttgart 3.287-289, 1993.
39. Fukui, T. , Kawamoto, T. and Tanaka, A. Enzymatic preparation of optically active silylmethanol derivatives having a stereogenic silicon atom by hydrolase-catalyzed enantioselective esterification. Tetrahedron - Asymmetry 5_ (1) 73-82, 1994.
40. Vazquez-Duhalt, R. , Westlake, D.W.S. and Fedorak, P.M. Lignin peroxidase oxidation of aromatic compounds in systems containing organic solvents. Applied and Environmental Microbiology 6_0 (2) 459-466, 1994.
41. Kodelia, G. , Athanasiou, K. and Kolisis, F.N. Enzymatic synthesis of butyryl-rutin ester in organic solvents and its cytogenetic effects in mammalian cells in culture. Appl. Bioche . Biotech. 44. (3) 205-212, 1994.
42. Tsai, S.W. and Wei, H.J. Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol. Biotechnology and Bioengineering 43. (1) 64-68, 1994.
43. Athawale, V.D. and Gaonkar, S.R. Enzymatic synthesis of polyesters by lipase catalyzed polytransesterification. Biotechnology Letters 16 (2) 149-154, 1994.
44. Janssen, G.G. and Haas, M.J. Lipase-catalyzed synthesis of oleic acid esters of polyethylene glycol 400. Biotechnology Letters JL6 (2) 163-168, 1994.
45. Kitazuma, T. , Ikeya, T. and Murata, K. Synthesis of optically active trifluorinated compounds: asymmetric Michael additional with hydrolytic enzymes. Journal of Chemical Society, Chemical Communications 1331, 1986.
References demonstrating icrobial transformations:
1." Holland, H.L. Fungi as reagents in organic synthesis: preparation of some metabolites of prostanoids, steroids, and other natural products. Rev. Latinoam. Quim. , (1st Spec. Suppl . ) , 318-329 , 1990.
2. Sih, C.J. and Rosazza, J.P. Microbial transformations in organic synthesis. Tech. Chem. (N.Y.), 10 (Appl. Biochem. Syst. Org. Chem., Part 1) 69-106,1976.
3. Sih, C.J. and Chen, C.S. Microbial asymmetric catalysis-enantioselective reduction of ketones. Angew. Chem. Int. Ed. Engl. Z3 (8) 570-578, 1984.
4. Thompson, L.A. and Knowles, C.J., Linton, E.A. and Wyatt, J.M. Microbial biotransformations of nitrites. Chem. Br. 2! (9) 900, 1988.
5. Servi, S. Baker's yeast as a reagent in organic synthesis. Synthesis 1 1-25, 1990.
6. Csuk, R. and Glanzer, B.I. Baker's yeast mediated transformations in organic chemistry. Chem. Rev. 91 (1) 49- 97, 1991.
7. Roberts, S.M., Wiggins, K. and Casy, G. Whole cells and isolated enzymes in organic synthesis . Wiley, New York, 1992.
8. Ward, O.P. and Young, C.S. Reductive biotransformations of organic compounds by cells or enzymes of yeast. Enzyme Microb. Technol. 12. (7) 482-493, 1990.
Reviews:
1. Jones, J.B. Enzymes in organic synthesis. Tetrahedron 42, (13) 3351-3405, 1986.
2. Ya ada, H. and Shimizu, S. Microbial and enzymatic processes for the production of biologically and chemically useful compounds. Angew. Chem. Int. Ed. Engl. 21. (5) 622-642, 1988.
3. Roberts, S.M. Enzymes as catalysts in organic synthesis. NATO ASI Ser. , Ser. A. 178 443-463, 1989.
4. Chen, S.S. and Sih, C.J. General aspects and optimization of enantioselective biocatalysis in organic solvents: the use of lipases. Angew. Chem (Int. Ed. Engl. 28, n 6 695-707) 101 (6) 711-724, 1989. Disclosure of the Invention
The present invention is used to synthesize a library of non-biological organic compounds from a starting compound and identify individual compounds within the library which exhibit biological activity. Unlike peptides and.oligonucleotides, non-biological organic compounds comprise the bulk of proven therapeutic agents. The invention can be used to directly identify new drug candidates or optimize an established drug compound which has sub-optimal activity or problematic side effects. This is accomplished through the use of highly specific biocatalytic reactions.
Enzymes are highly selective catalysts. Their hallmark is the ability to catalyze reactions with exquisite stereo-, regio-, and chemo-selectivities that are unparalleled in conventional synthetic chemistry. Moreover, enzymes are remarkably versatile. They can be tailored to function in organic solvents, operate at extreme pH's and temperatures, and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates.
Enzymes are reactive toward a wide range of natural and unnatural substrates, thus enabling the modification of virtually any organic lead compound. Moreover, unlike traditional chemical catalysts, enzymes are highly enantio- and regio-selective. The high degree of functional group specificity exhibited by enzymes enables one to keep track of each reaction in a synthetic sequence leading to a new active compound. Enzymes are also capable of catalyzing many diverse reactions unrelated to their physiological function in nature. For example, peroxidases catalyze the oxidation of phenols by hydrogen peroxide. Peroxidases can also catalyze hydroxylation reactions that are not related to the native function of the enzyme. Other examples are proteases which catalyze the breakdown of polypeptides. In organic solution some proteases can also acylate sugars, a function unrelated to the native function of these enzymes.
The present invention exploits the unique catalytic properties of enzymes. Whereas the use of biocatalysts (i.e., purified or crude enzymes, non-living or living cells) in chemical transformations normally requires the identification of a particular biocatalyst that reacts with a specific starting compound, the present invention uses selected biocatalysts and reaction conditions that are specific for functional groups that are present in many starting compounds. Each biocatalyst is specific for one functional group, or several related functional groups, and can react with many starting compounds containing this functional group.
The biocatalytic reactions produce a population of derivatives from a single starting compound. These derivatives can be subjected to another round of biocatalytic reactions to produce a second population of derivative compounds. Thousands of variations of the original compound can be produced with each iteration of biocatalytic derivatization.
Enzymes react at specific sites of a starting compound without affecting the rest of the molecule, a process which is very difficult to achieve using traditional chemical methods. This high degree of biocatalytic specificity provides the means to identify a single active compound within the library. The library is characterized by the series of biocatalytic reactions used to produce it, a so called "biosynthetic history". Screening the library for biological activities and tracing the biosynthetic history identifies the specific reaction sequence producing the active compound. The reaction sequence is repeated and the structure of the synthesized compound determined. This mode of identification, unlike other synthesis and screening approaches, does not require immobilization technologies, and compounds can be synthesized and tested free in solution using virtually any type of screening assay. It is important to note, that the high degree of specificity of enzyme reactions on functional groups allows for the "tracking" of specific enzymatic reactions that make up the biocatalytically produced library. Many of the procedural steps are performed using robotic automation enabling the execution of many thousands of biocatalytic reactions and screening assays per day as well as ensuring a high level of accuracy and reproducibility. As a result, a library of derivative compounds .can be produced in a matter of weeks which would take years to produce using current chemical methods.
The present invention is unique in that it involves a soluble state of the starting compound and its subsequent derivatives. This is a highly unique aspect of the present invention that has been thought to be a barrier. Previous organic modifying technologies for biologically active compound identification involve starting compounds and derivatives attached to insoluble supports. This is taught in examples by Ellman (Bunin, B.S.; Ellman, J.A. "A general and expedient method for the solid-phase synthesis of 1,4- benzodiazepine derivatives", J. Am. Chem. Soc. 1992, 114 10997-10998), Gordon et al., (Gordon, E.M. ; Barrett, R.W. ; Dower, W.J.; Fodor, S.P.A.; Gallop, M.A. "Applications of Combinatorial Technologies to Drug Discovery, 1. Background and Peptide Combinatorial Libraries", J. Med. Chem. 1994, 31_ 1233-1251; and Gordon, E.M. ; Barrett, R.W. ; Dower, W.J. ; Fodor, S.P.A.; Gallop, M.A. "Applications of Combinatorial Technologies to Drug Discovery, 2. "Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions", J. Med. Chem. 1994, 3_7 1385-1401), Hobbs Dewitt et al., (Hobbs Dewitt, S.; Kiely, J.S.; Stankovic, C.J. ; Schroeder, M.C.; Reynolds Cody, D.M. ; Pavia, M.R. "Diverso ers: An approach to nonpeptide, nonoligomeric chemical diversity" P.N.A.S. (USA) 1993 90 6909-6913). In addition to synthesizing the libraries on a soluble state, the libraries of the instant invention can then be screened immediately without removal of the libraries from solid supports. Thus by maintaining the components in the soluble state, the present invention provides for useful, convenient, and efficient methods of generating and screening libraries of starting compounds and derivatives.
The present invention specifically incorporates a number of diverse technologies such as: (1) the use of enzymatic reactions to produce a library of drug candidates; (2) the use of enzymes free in solution or immobilized on the surface of particles, and organic compounds derivatized while dissolved in solution; (3) the use of receptors (hereinafter this term is used to indicate true receptors, enzymes, antibodies and other biomolecules which exhibit affinity toward biological compounds, and other binding molecules to identify a promising drug candidate within a library, even where such receptors are still associated with cell membranes, or intact cells); (4) the automation of all biocatalytic processes and many of the procedural steps used to test the libraries for desired activities, and (5) the coupling of biocatalytic reactions with drug screening devices which can immediately measure the binding of synthesized compounds to receptor molecules or in whole-cell assays and thereby immediately identify specific reaction sequences giving rise to biologically active compounds.
Specifically, the present invention encompasses a method for drug identification comprising:
(a) conducting a series of biocatalytic reactions by mixing biocatalysts with a starting compound to produce a reaction mixture and thereafter a library of modified starting compounds;
(b) testing the library of modified starting compounds to determine if a modified starting compound is present within the library which exhibits a desired activity;
(c) identifying the specific biocatalytic reactions which produce the modified starting compound of desired activity by systematically eliminating each of the biocatalytic reactions used to produce a portion of the library and testing the compounds produced in the portion of the library for the presence or absence of the modified starting compound with the desired activity; and (d) repeating the specific biocatalytic reactions which produce the modified compound of desired activity and determining the chemical composition of the reaction product.
More specifically, the enzymatic reactions are conducted with a group of enzymes that react with distinct structural moieties found within the structure of a starting compound. Each enzyme is specific for one structural moiety or a group of related structural moieties. Furthermore, each enzyme reacts with many different starting compounds which contain the distinct structural moiety. In addition to systematically eliminating each reaction, the instant invention also provides for the systematic process of building up each reaction, an approach from the other end of the pathway. A particular feature of the instant invention is the soluble state of the starting compounds and the subsequent derivatives.
According to the present invention, enzymatic reactions are conducted in "reaction boxes", wherein a single enzyme or a group of enzymes which recognize the same functional group and perform the same type of enzymatic derivatization, are used. For example, a "reaction box" for acylation of an alcohol, could contain numerous different lipases and esterases, for reaction with the same substrate. When a group of enzymes are used, the probability of achieving the desired transformation of acylation, is increased to an almost certainty, since there is more than one single enzyme present in the "reaction box". Furthermore, a group of enzymes together with a group of co-substrates can be used to make a library of derivatives. For example, acylation of an alcohol with a mixture of organic acids (co-substrates) can give a mixture of acylated alcohol derivatives.
Each "reaction box" represents a different enzymatic activity, where the different reactions are conducted separately. Accordingly there would be one "reaction box" for acylation of an alcohol, another "reaction box" for oxidation of alcohols and yet another "reaction box for the reduction of carbonyl groups. It is important that the different enzymatic reactions be conducted separately, so as to aid in the subsequent identification of a compound responsible for a specific activity.
The different "reaction boxes" are not only used concurrently on the same substrate, but also sequentially so as to conduct a second "iteration" of enzymatic derivatization. More specifically, an initial substrate can be first subjected to a series of "reaction boxes" which are believed to have reactivity with the known functional groups in the initial substrate, followed by subjecting the reaction product of an individual "reaction box" to a further series of "reaction boxes". By subjecting the reaction product of a "reaction box" to a further series of "reaction boxes", a second iteration of enzymatic derivatization is achieved. The product of a second "reaction box" can itself be subjected to a further series of "reaction boxes", producing yet a third iteration of enzymatic derivatization. Thus, a series of "reaction box" derivatizations can be conducted, to create any number of iterations, one, two, three, four, five, six, seven, eight, nine, ten, up to "n" iterations of enzymatic derivatization. Preferably, the present method is used to generate at least two iterations, more preferably at least three iterations and even more preferably at least four iterations. The idea of iterations is exemplified in Figure 3, where the initial compound AZT is subjected to three iterations of enzymatic derivatization.
Each product of a "reaction box" can be analyzed to determine whether a desired activity is present within the product(s). If a "reaction box" is determined to possess the desired activity, the compound responsible for the desired activity can be identified and if desired isolated.
By using a series of "reaction boxes" identification of the compound responsible for a desired activity is greatly simplified. Since the reactivity of each "reaction box" is already known, one could easily conclude that the result of subjecting a compound which possessed an aldehyde group, to the sequential reaction boxes of a dehydrogenase followed by an esterase would produce a compound bearing an esterified alcohol. Therefore, knowledge of the sequence of reaction boxes which produced a product with a desired activity, would greatly facilitate structure identification. In some cases, it is not even necessary to isolate the compound with the desired activity, since the "reaction box" history will enable identification of the structure of the compound.
It is to be understood, that within the context of the present invention, each "reaction box" can contain, in addition to a single enzyme or a group of enzymes, any co- factors, reagents and solvents necessary to conduct the desired reaction. Those of skill in the art would easily be able to determine the identity of the co-factors, reagents and solvents, where necessary, without undue experimentation.
The present invention is also directed to a method of preparing a library of compounds, using the above-identified "reaction box" technique, as well as the library of compounds generated by such a technique. A library of compounds is itself useful as it can be screened for a desired activity. A library of compounds, generated by the present method can be screened for any desired activity, such as pharmaceutical, herbicidal, insecticidal and toxicological activities.
Brief Description of the Drawings
Figure 1 shows the starting active compound AZT with four potential sites for biocatalytic derivatization and eight possible biocatalytic reactions that can be used to produce a library of derivative compounds.
Figure 2 shows an automated system employing robotic automation to perform hundreds of biocatalytic reactions and screening assays per day.
Figure 3 illustrates the tracking of biocatalytic reactions to identify the sequence of reactions producing an active compound, which can subsequently be used to produce and identify the structure of the active compound. Figure 4a illustrates biocatalytic modification of castanospermine.
Figure 4b illustrates biocatalytic modifications of ethotrexate.
Figure 1' is a chromatogra of a second iteration taxol- vinyl adipate alcohol ester
Best Mode for carrying Out the Invention
While the invention will be described in connection with certain preferred embodiments, it will be understood that the description does not limit the invention to these particular embodiments. The embodiments of the invention are described with AZT, and further described for taxol, only as particular embodiments of the instant invention. In fact, it is to be understood that all alternatives, modifications and equivalents are included and are protected, consistent with the spirit and scope of the inventions as defined in the appended claims.
The preferred embodiments of the invention are set forth in the following example: a) A starting compound such as AZT (3'-azidothymidine) , is chosen which exhibits drug activity or is believed to exhibit drug activity for a given disease or disorder. The compound is analyzed with respect to its functional group content and its potential for structural modifications using selected biocatalytic reactions. Functional groups which can be chemically modified using the selected biocatalytic reactions are listed in Table I. One of more of these functional groups are present in virtually all organic compounds. A partial list of possible enzymatic reactions that can be used to modify these functional groups is presented in Table II. b) A strategy is developed to systematically modify these functional groups using selected biocatalytic reactions and produce a library of derivative compounds to be screened for biological activity. AZT contains four functional groups which are selected for biocatalytic modification: a primary hydroxyl, two carbonyls and a tertiary amine.
The biosynthetic strategy is designated in the form of biocatalytic "reaction box" numbers which correspond to specific types of biocatalytic reactions acting on specific functional groups present in the starting compound. These "reaction boxes" are listed in Table III. The following biocatalytic "reaction boxes" are selected to synthesize an AZT derivative library: A3, A10, All, C2, G6, G10 and G12. FIG. 1 illustrates the reaction of AZT with these selected biocatalytic reaction boxes. c) The biocatalytic reaction boxes are entered into an automated system which is shown in FIG 2. The system is programmed to automatically execute the aforementioned biocatalytic reactions and synthesize a library of derivative products. A single automated system in capable of performing hundreds of pre-programmed biocatalytic reactions per day. We can estimate the total number of compounds that can be produced by analyzing the reaction products produced in each "reaction box" and multiplying the results. Table IV details the number of potential reaction products produced in each reaction box and the resulting total number of possible compounds produced. In the case of AZT, up to 1.75 x 1011 new compounds can be synthesized. It should be pointed out that this compares very favorably to peptide libraries. For example, a library of hexapeptides will contain 206 or 64 million compounds. This is a mere fraction, about 0.04% of the compounds that are possible using the biosynthetic approach described herein. Table V lists the results of a similar analysis on eleven other starting drug compounds. As shown in this table, the biocatalytic reactions can generate huge numbers of derivative compounds for drug screening. d) The synthesized library of new compounds is assayed using enzyme inhibition assays, receptor-binding assays, immunoassays, and/or cellular assays to identify biologically active compounds. Before assaying the library of derivative compounds, any remaining AZT present in the library is either removed or inhibited to simplify the interpretation of screening assay results. This is easily accomplished by HPLC, TLC, or the addition of a monoclonal antibody specific for the starting compound. Numerous in vitro assays are available that test for anti-viral, anti-cancer, anti-hypertensive and other well known pharmacological activities. Some of these assays are listed in Table VI. Most of these assays are also performed on the automated system. e) Libraries which test positive are further analyzed using a biocatalytic tracking protocol which quickly identifies the specific sequence of reactions responsible for the synthesis of the compound testing positive in the screening assay. The high degree of specificity exhibited by biocatalysts, i.e., the ability of a given enzyme to react with a given functional group, enables this approach to be easily performed. The library is characterized by the series of biocatalytic reactions used to produce it, a so called "biosynthetic history". Portions of the library are screened for biological activity until the specific reaction sequence producing the active compound is identified. FIG. 3 illustrates this tracking process. For example, the dark line path 15. illustrates the reaction pathway to the most active compound. The reaction sequence is repeated to produce a sufficient amount of product for chemical analysis. The specificity of the biocatalytic reactions also permits the accurate duplication of the reaction pathway producing the active compounds. The structure of the active compound is qualitatively determined by analyzing the starting compounds, substrates and identified biocatalytic reaction sequence. The structure is then confirmed using gas chromatography, mass spectroscopy, NMR spectroscopy and other organic analytical methods. This mode of identification eliminates the need for product purification and also reduces the amount of test screening required to identify a promising new drug compound. This process dramatically reduces the time necessary to synthesize and identify new drug compounds. In addition, this mode of active compound identification does not require immobilization technologies, and compounds can be synthesized and tested free in solution under in vivo like conditions using virtually any type of screening assay (receptor, enzyme inhibition, immunoassay, cellular, animal model) .
Those skilled in the pharmaceutical arts will recognize the large number of biocatalytic conversions such as those listed in Table II and Table III, as well as the in vitro drug screening assays listed in Table VI.
Those skilled in the pharmaceutical arts will recognize that biocatalytic reactions are optimized by controlling or adjusting such factors as solvent, buffer, pH, ionic strength, reagent concentration and temperature.
The biocatalysts used in the biocatalytic reactions may be crude or purified enzymes, cellular lysate preparations, partially purified lysate preparations, living cells or intact non-living cells, used in solution, in suspension, or immobilized on magnetic or non-magnetic surfaces.
In addition, non-specific chemical reactions may also be used in conjunction with the biocatalytic reaction to obtain the library of modified starting compounds. Examples of such non-specific chemical reactions include: hydroxylation of aromatics and aliphatics; oxidation reactions; reduction reactions; hydration reactions; dehydration reactions; hydrolysis reactions; acid/based catalyzed esterification; transesterification; aldol condensation; reductive amination; amminolysis; dehydrohalogenation; halogenation; acylation; acyl substitution; aromatic substitution; Grignard synthesis; Friedel-Crafts acylation; etherification.
The biocatalytic reaction can be performed with a biocatalyst immobilized to magnetic particles forming a magnetic biocatalyst. The method of this embodiment is performed by initiating the biocatalytic reaction by combining the immobilized biocatalyst with substrate(s) , cofactors(s) and solvent/buffer conditions used for a specific biocatalytic reaction. The magnetic biocatalyst is removed from the biocatalytic reaction mixture to terminate the biocatalytic reaction. This is accomplished by applying an external magnetic field causing the magnetic particles with the immobilized biocatalyst to be attracted to and concentrate at the source of the magnetic field, thus effectively separating the magnetic biocatalyst from the bulk of the biocatalyst reaction mixture. This allows for the transferral of the reaction mixture minus the magnetic biocatalyst from a first reaction vessel to a second reaction vessel, leaving the magnetic biocatalyst in the first reaction vessel. A second biocatalytic reaction is conducted completely independent of the first biocatalytic reaction, by adding a second biocatalyst immobilized to magnetic particles to the second reaction vessel containing the biocatalytic reaction mixture transferred from the first reaction vessel. Finally, these steps are repeated to accomplish a sequential series of distinct and independent biocatalytic reactions, producing a corresponding series of modified starting compounds.
The biocatalytic reactions can also be performed using biocatalysts immobilized on any surface which provides for the convenient addition and removal of biocatalyst from the biocatalytic reaction mixture thus accomplishing a sequential series of distinct and independent biocatalytic reactions producing a series of modified starting compounds.
The biocatalytic reactions can also be used to derivatize known drug compounds producing new derivatives of the drug compound and select individual compounds within this library that exhibit optimal activity. This is accomplished by the integration of a high affinity receptor into the biocatalytic reaction mixture, which is possible because of the compatibility of the reaction conditions used in biosynthesis and screening. The high affinity receptor is added to the reaction mixture at approximately one half the molar concentration of- the starting active compound, resulting in essentially all of the receptor being bound with the starting active compound and an equal molar concentration of starting active compound free in solution and available for biocatalytic modification. If the biocatalytic reaction mixture produces a derivative which possesses a higher binding affinity for the receptor, which can translate into improved pharmacological performance, this derivative will displace the bound starting active compound and remain complexed with the receptor, and thus be protected from further biocatalytic conversions. At the end of the experiment, the receptor complex is isolated, dissociated and the bound compound analyzed. This approach accomplishes the identification of an improved version of the drug compound without the need to purify and test each compound individually.
The biocatalytic reactions and in vitro screening assays can be performed with the use of an automated robotic device. The automated robotic device having:
(a) an XY table with an attached XYZ pipetting boom to add volumetric amounts of enzyme, substrate, cofactor, solvent solutions and assay reagents from reagent vessels positioned on the XY table to reaction and assay vessels positioned on the same XY table;
(b) an XYZ reaction-vessel transfer boom attached to the same XY table used to transfer reaction and assay vessels positioned on the XY table to different locations on the XY table;
(c) a temperature incubation block attached to the same XY table to house the reaction and assay vessels during reaction incubations and control the temperature of the reaction mixtures;
(d) a magnetic separation block attached to the same XY table to separate the biocatalyst immobilized to magnetic particles from the biocatalytic mixture by applying an external magnetic field causing the magnetic particles to be attracted to and concentrate at the source of the magnetic filed, thus effectively separating them from the bulk of the biocatalytic reaction mixture; and (e) a programmable microprocessor interfaced to the XYZ pipetting boom, and XYZ reaction-vessel transfer boom, the temperature block and the magnetic separation block to precisely control and regulate all movements and operations of these functional units in performing biocatalytic reactions to produce modified starting compounds and assays to determine desired activities.
Figure 2 illustrates the automated robotic device of this invention. Mounted in the frame 1 of the system are containers for starting compounds 2., and containers for reagents 3. such as enzymes, cofactors, and buffers. There are specific biosynthesis boxes 4. which contain reagents for various classes of reactions. The frame also has arrays of reaction vessels 5_, and a heating block 6 with wells 2 for conducting reactions at a specific temperature. The frame has an area 8. for reagents for screening test 8_ which contains reagents used for conducting screening tests, and area 9 which contains assay vessels for conducting screening tests, the automated system uses a X-Y-Z pipetting and vessel transfer boom 0 to dispense all reagents and solutions, and transfer reaction vessels.
In operation the X-Y-Z reaction-vessels transfer boom can deliver starting compounds and reagents to specific locations for making specific modified starting compounds which in turn can be delivered to specific locations for conducting assays. In this way the process of making modified starting compounds and testing for optimum activity is largely automated.
Figures 4a and 4b illustrate derivatization of castanospermine and methotrexate. All of these embodiments utilize the biocatalytic conversions set out in Table II and the assays set out in Table VI.
While the invention as described herein is directed to the development of drugs, those skilled in the biological arts will recognize that the methods of this invention are equally applicable to other biologically active compounds such as food additives, pesticides, herbicides, and plant and animal growth hormones .
TΑBLE I. Major Functional Groups Available for Biocatalytic Modi ication*
A. Hydroxyl Groups — These groups can undergo numerous reactions including oxidation to aldehydes or ketones (1.1), acylation with ester donors (2.3, 3.1), glycosidic bond formation (2.4, 3.2, 5.3), and etherification (2.1, 3.3). Potential for stereo- and regio-selective synthesis as well as prochiral specificity.
B. Aldehydes and Ketones — These groups can undergo selective reduction to alcohols (1.1). This may then be followed by modifications of hydroxyl groups.
C. Amino Groups — These groups can undergo oxidative deamination (1.4), N-dealkylation (1.5, 1.11), transferred to other compounds (2.6), peptide bond synthesis (3.4, 6,3), and acylation with ester donors (2.3, 3.1).
D. Carboxyl Groups — These groups can be decarboxylated (1.2, 1.5, 4.1), and esterified (3.1, 3.6).'
E. Thiol Groups — These groups can undergo reactions similar to hydroxyls, such as thioester formation (2.8, 3.1), thiol oxidation (1.8), and disulfide formation (1.8).
F. Aromatic Groups — These groups can hydroxylated (1.11, 1.13, 1.14), and oxidatively cleaved to diacids (1.14).
G. Carbohydrate Groups — These groups can be transferred to hydroxyls and phenols (2.4, 3.2, 5.3), and to other carbohydrates (2.4).
H. Ester and Peptide Groups — These groups can be hydrolyzed (3.1, 3.4, 3.5, 3.6, 3.9), and transesterified
(or interesterified) (3.1, 3.4). I. Sulfate and Phosphate Groups — These groups can be hydrolyzed (3.1, 3.), transferred to other compounds
(2.7, 2.8), and esterified (3.1). J. Halogens — These groups can be oxidatively or hydrolytically removed (1.11, 3.8), and added (1.11). K. Aromatic Amines and Phenols — These groups can be acylated (2.3, 3.1) or oxidatively polymerized (1.10, 1.11, 1.14). 'Numbers in parentheses correspond to the EC (Enzyme Commission) categorization of enzymes and-enzyme classes.
TABLE II. A Representative Subset of Biocatalytic Reactions Which May Be Used to Modify Functional Groups
1. Oxidation of primary and secondary alcohols; Reduction of Aldehydes and ketones. Reaction Boxes: Al, Bl, C2, D2
Enzyme Class: l.l. Dehydrogenases, Dehydtratases, Oxidases Representative Enzvmes:
Alcohol dehydrogenase
Glycerol Dehydrogenase
Glycerol-3-Phosphate Dehydrogenase
Xylulose Reductase
Polyol Dehydrogenase
Sorbitol Dehydrogenase
Glyoxylate Reductase
Lactate Dehydrogenase
Glycerate Dehydrogenase
/3-Hydroxybutyrate Dehydrogenase
Malate Dehydrogenase
Glucose Dehydrogenase
Glucose-6-Phosphate Dehydrogenase
3α- and 33-Hydroxysteroid Dehydrogenase
3a- , 2O/S-Hydroxys eroid Dehydrogenase
Fucose Dehydrogenase
Cytochrome-Dependent Lactate Dehydrogenase
Galactose Oxidase
Glucose Oxidase
Cholesterol Oxidase
Alcohol Oxidase
Glycolate Oxidase
Xanthine Oxidase
Fructose Dehydrogenase
Cosubstrates/Cofactors: NAD(P) (H) TABLE II (cont.) 2. Acylation of primary and secondary alcohols.
Reaction Boxes: A3, B4
Enzyme Classes: 3.1, 3.4, 3.5, 3.6
Representative Enzymes: Esterases, lipases, proteases, sulfatases, phosphatases, acylases, lactamases, nucleases, acyl transferases
Esterases
Lipases
Phospholipase A
Acetylesterase
Acetyl Cholinesterase
Butyryl Cholinesterase
Pectinesterase
Cholesterol Esterase
Glyoxalase H
Alkaline Phosphatase
Acid Phosphatase
A Variety of nucleases
Glucose-6-Phosphatase
Fructose 1,6-Diphosphatase
Ribonuclease
Deoxyribonuclease
Sulfatase
Chondro-4-Sulfatase
Chondro-6-Sulfatase
Leucine Aminopeptidase
Carboxypeptidase A
Carboxypeptidase B
Carboxypeptidase Y
Carboxypeptidase W
Prolidase
Cathepsin C TABLE II (cont. )
Chymotrypsin
Trypsin
Elastase
Subtilisin
Papain
Pepsin
Ficin
Bromelain
Rennin
Proteinase A
Collagenase
Urokinase
Asparaginase
Glutaminase
Urease
Acylase I
Penicillinase
Cephalosporinase
Creatininase
Guanase
Adenosine Deaminase
Creatine Deaminase
Inorganic Pyrophosphatase
ATPase
Choline Acetyltransferase
Carnitine Acetyltransferase
Phosphotransacetylase
Chloramphenicol Acetyltransferase
Transgluta inase γ-Glutamyl Transpeptidase TABLE II (co t. )
Cosubstrates/Cofactors: Esters of alkyl, aryl, charged, polar/neutral groups. These acyl donors can be chosen from the class consisting of the following structural formulae:
R-O-CO-R' Where R = alkyl, vinyl, isopropenyl haloalkyl, aryl, derivatives of aryl (i.e., nitrophenyl) and R' can be any alkyl or aryl group with or without derivatives. Such derivatives include halogens, charged functional groups (i.e., acids, sulfates, phosphates, amines, etc.), glycols (protected or unprotected) , etc.
3. Transglycosylation of primary and secondary alcohols.
Reaction Boxes: A10, BIO
Enzyme Class: 2.4, 3.2
Representative Enzymes: Phosphorylase a Phosphorylase b Dextransucrase Levansucrase Sucrose Phosphorylase Glycogen Synthase UDP-Glucuronyltransferase Galactosyl Transferase Nucleoside Phosphorylase α- and β-Amylase Amyloglucosidase (Glucoamylase) Cellulase Dextranase Chitinase Pectinase Lysozyme TABLE II (cont. )
Neuraminidase
Xylanase α- and β-Glucosidase α- and 3-Galactosidase α- and /0-Mannosidase
Invertase
Trahalase 3-N-Acetylglucosaminidase γ-Glucuronidase
Hyaluronidase S-Xylosidase
Hesperidinase
Pullulanase α-Fucosidase
Agarase
Endoglycosidase F
NADase
Glycopeptidase F
Thioglucosidase
Cosubstrates/Cofactors: All available sugars and their derivatives. These sugars can be onosaccharides, disaccharides, and oligosaccharides and their derivatives. 4. Etherification of primary and secondary alcohols Reaction Boxes: All, Bll
Enzyme Classes: 2.1, 3.2
Representative Enzymes:
Catechol α-Methyltransferase Aspartate Transcarbamylase Ornithine Transcarbamylase S-Adenosylhomocysteine Hydrolase Cosubstrates/Cofactors: Alcohols or ethers of any chain length. 5. Acylation of primary and secondary amines.
Reaction Boxes: E3, E4
Enzvme Classes: 2.3, 3.1, 3.4, 3.5, 3.6
Representative Enzymes:
Choline Acetyltransferase
Carnitine Acetyltransferase
Phosphotransacetylase
Chloramphenicol Acetyltransferase
Transglutaminase γ-Glutamyl Transpeptidase
Esterases
Lipases
Phospholipase A
Acetylesterase
Acetyl Cholinesterase
Butyryl Cholinesterase
Pectinesterase
Cholesterol Esterase
Glyoxylase II
Alkaline Phosphatase
Acid Phosphatase
A Variety of nucleases
Glucose-6-Phosphatase
Fructose 1,6-Diphosphatase
Ribonuclease
Deoxyribonuclease
Sulfatase
Chondro-4-Sulfatase
Chondro-6-Sulfatase
Leucine Aminopeptidase TABLE II (cont.) Carboxypeptidase A Carboxypeptidase B Carboxypeptidase Y Carboxypeptidase W Prolidase Cathepsin C Chymotrypsin Trypsin Elastase Subtilisin Papain Pepsin Ficin Bromelin Rennin Proteinase A Collagenase Urokinase Asparaginase Glutaminase Urease Acylase I Penicillinase Cephalosporinase Creatininase Guanase
Adenosine Deaminase Creatine Deaminase Inorganic Pyrophosphatase ATPase
Cosubstrates/Cofactors: See example number 2, above. -36-
TABLE II (COnt. )
6. Esterification of carboxylic acids.
Reaction Boxes: 17.
Enzvme Classes: 3.1, 3.6
Representative Enzymes:
Esterases
Lipases
Phospholipase A
Acetylesterase
Acetyl Cholinesterase
Butyryl Cholinesterase
Pectinesterase
Cholesterol Esterase
Glyoxylase II
Alkaline Phosphatase
Acid Phosphatase
A Variety of nucleases
Glucose-6-Phosphatase
Fructose 1,6-Diphosphatase
Ribonuclease
Deoxyribonuclease
Sulfatase
Chondro-4-Sulfatase
Chondro-6-Sulfatase
Inorganic Pyrophosphatase
ATPase
Cosubstrates/Cofactors: alcohols of any chain length being alkyl, aryl, or their structural derivatives.
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
TABLE IV. Reaction Box Analysis of AZT Derivatization Indicating the Total M***"t">*- of Possible Reaction Products
Reaction Box Number of Possible Products
A3 30<a> A10 30<b) All 30
C2 2 X 30<c)
C2 2 X 30(c)
G6 30
G10 30
G12 X2
Total 1.75 x 1011 distinct compounds"-1
(a) Assuming 30 different acyl donors to be added to this reaction mixture. This includes alkyl, aryl, and of different lengths, (b) Assuming 30 UDP-sugars used in this reaction box, (c) Reduction of the ketones to secondary alcohols leads to the potential acylation of the secondary alcohols and adds 30- fold more potential products; and (d) Each box's possible permutations are multiplied together to estimate the total number of compounds synthesized.
TABLE V. Reaction Box Analysis of Established Drugs Indicating the Total «"">">*• of Possible Reaction Products
Number of Functional Estimated Number of
Starting Compound GΓOUDS Derivatives
Castanospermine 4 810,000
Cyclosporin 24 billions
Gentamicin 8 billions
Haloperidol 3 120 ethotrexate 7 greater than 101'
Muscarine 2 2,400
Prazosin 6 288, 000
Prednisone 12 46,080,000
Thyroxine 3 2,160,000
Valproic Acid 1 900
Vancomycin many billion
TABLE VI. Representative Subset of screening Assays
Possible to Test for Anti-Cancer, Anti-Viral and
Anti-Hypertensive Activities
Anti-Cancer Drugs
1. KB (Eagle) cell culture assay
2. Inhibition of the growth of human breast cancer cell lines in vitro
3 . Inhibition of the growth of P388 leukemia cells in vitro 4. Inhibition of the growth of murine L1210 cells in vitro 5* Inhibition of gylcinamide ribonucleotide formyltransferase activity 6* Inhibition of ribonucleotide reductase activity 7* Inhibition of protein kinase C activity 8* Inhibition of human aromatase activity 9* Inhibition of DNA topoisomerase II activity
10. Inhibition of dihydrofolate reductase
11. Inhibition of aminoimidazole carboxamide ribonucleotide formyltransferase
Anti-AIDS Drugs
1. Inhibition of HIV virus replication devoid of cytotoxic activity
2. Inhibition of HIV protease activity
3. Soluble-formazan assay for HIV-1
4. Inhibition of HIV reverse transcriptase activity
Anti-Hypertensive Drugs
1. Inhibition of ACE activity
2. Inhibition of human plasma renin
3. Inhibition of in vitro human renin
4. Inhibition of angiotensin converting enzyme
5. Alpha 1-adrenergic receptor binding assay
6. Alpha 2-adrenergic receptor binding assay Beta-adrenergic receptor binding assay (bronchodilator, cardiotonic, tocolytic, anti-anginal, anti-arrhythmic, anti-glaucoma)
Dopamine receptor binding assay (anti-migraine, anti- parkinsonian, anti-emetic, anti-psychotic)
Example 1
l. Enzymatic esterification of taxol
1.1 Analysis of reaction products
Products of enzymatic esterification of taxol were analyzed using reversed-phase HPLC on a Waters system with a 990 photodiode array detector, and a 3.9 x 300 mm μBondapak C18 column. The solvent system used consisted of a water:acetonitrile mixture (40:15 v/v) and isopropanol. Elution was performed at 1 ml/min according to the following linear gradient program:
Time(min) Solvent Composition (v/v %)
Water/Acetonitrile Isopropanol 0-8 72 28
8-16 50 50
16-24 0 100
26-28 0 100
28-30 72 28
Product peaks were detected at 227 nm using a photodiode array detector.
1.2 Enzymatic synthesis of taxol esters - Identification of active biocatalysts (Reaction Box B-4)
A mixture of 55 enzymes (Table VII) consisting of crude lipases, crude proteases, and purified proteases (total mass of 600 mg) was added to a solution of taxol (17.0 g, 20 μmol) and vinyl butyrate (0.25 ml, 1.5 mmol) in 2.9 ml tert a yl alcohol. The mixture was placed into a septum-sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C for a period of 48 h. The reaction was then stopped by removing the suspended solid catalyst by centrifugation. The supernatant containing taxol esters was evaporated to dryness in vacuum and redissolved in 0.3 ml methanol. An aliquot of the resultant concentrated solution (15 μl) was diluted with 250 μl methanol and analyzed by HPLC as described in 1.1. The chromatogram revealed a peak with a retention time of 9.7 min (unreacted taxol), and a peak at 14.6 min (2,-taxol butyrate, yield 7%).
The enzymes were then split into three groups: 26 lipases (100 mg/ml) , 26 crude proteases (100 mg/ml) , and 4 purified proteases (10 mg/ml) and the identical reaction as above performed. Only the purified proteases showed significant activity with a yield of 2'-butyrate of 15% after 48 h. The purified proteases were then divided into individual enzymes (all used at a concentration of 10 mg/ml) and thermolysin (a bacterial protease from Bacillus thermoproteolyticus rokko a .k .a . thermolysin) was identified as the most active enzyme with a yield after 48 h of 24%. This approach demonstrates that active biocatalysts can be easily identified by a sequential process of eliminating unreactive biocatalysts.
TABLE VII. Enzymes used in the acylation of taxol Lipases and Esterases
Enzyme Source Company
CES Amano
L-10 Amano
G Penicillium sp. Amano
N Rhizopus niveus Amano
AP Aspergillus niger (acid stable) Sigma
Wheat Germ Amano
GC-20 Geotrichwn candidum Amano
AY-30 Candida rugosa Amano
P Pseudomonas cepacia Amano
AG-975 Sigma
Porcine Pancreatic Amano
APF A. niger Amano
R-10 Amano
AK Pseudomonas sp. Amano
PGE Calf tongue root Amano
D Amano
GC-4 Amano
CE Sigma
Candida rugosa Amano
FAP-15 Rhizopus sp. Amano
MAP-10 Mucor sp. Amano
Enzeco K16825 Enzeco
Lamb Pre-Gastric Esterase Quest
Calf Pre-Gastric Esterase Quest
Esterase 30,000 #3586 Gist Brocades
Lipase 80,000 #5093 Rhizopus sp. Gist Brocades TABLE VII. (cont.) Enzymes used in the acylation of taxol . Proteases Enzyme Source Company B Penicillium sp. Amano
Papain Papaya - Sigma 2A Aspergillus oryzae Amano
Proleather Bacillus sp. Amano N B . suhtilis (neutral) Amano
Acid Stable Rhizopus sp. Amano Alcalase-2T B . Licheniformis (Subtilisin
Carlsberg) Novo Bromelain Pineapple Sigma Alkaline Protease Quest Fungal Protease #9240810 Biocon Acid Protease #8221108 Biocon Neutral Protease 900,000 Biocon #6W16B HT MKC
Rapidase S-90 B . subtilis Gist Brocades HT-Proteolytic 200 Solvay Opticlean M-375 Alkaline protease
(from Bacillus sp) . Solvay Optimase M-440 Alkaline protease
(from Bacillus sp) . Solvay
Fungal Protease 60,000 Solvay
Esperase-4T Alkaline protease
(from Bacillus sp) . Novo
Peptidase A A. oryzae Amano
Prozyme 6 Aspergillus sp. Amano
Protease M A . oryzae Amano
Newlase A A. niger (acid stable) Amano
Acylase Cone. Aspergillus sp. Amano
Protease S Bacillus sp.
(thermostable) Amano
Acylase I Porcine kidney Sigma III. Purified Enzymes
Enzyme Source Company
Trypsin Bovine pancreas Sigma
Chymotrypsin Bovine pancreas Sigma
Subtilisin Carlsberg Bacillus liquefaciens Sigma
Protease X B . thermoproteolyticus rokko Sigma
1.3. Enzymatic synthesis of a mixture of taxol esters - Sequential reaction box derivatization of taxol
The optimal enzyme catalyst used for the synthesis was produced by freeze-drying an aqueous solution containing thermolysin, KCl and potassium phosphate buffer adjusted to pH 7.5. The solid catalyst obtained after free-drying contained 5% enzyme, 94% KCl and 1% potassium phosphate. The powdered enzyme catalyst (335 mg - containing 16.75 mg thermolysin) was added to a solution of taxol (17.0 mg, 20 μmol) and vinyl caproate (straight-chain C6 ester) (0.24 ml. 1.5 mmol) in 2.9 ml tert-amyl alcohol. The mixture was placed into a septum- sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C. After 28 h the reaction mixture (including enzyme) was removed and the full contents of the mixture added to a separate solution containing vinyl propionate (0.24 ml, 2.2 mmol), vinyl acrylate (0.24 ml, 2.2 mmol), and vinyl butyrate (0.24 ml, 1.9 mmol). This second reaction was placed on the shaker and incubated at 250 rpm at 35°C. After 24 h, the reaction mixture (including enzyme) was removed and the full contents of the mixture added to a separate solution containing vinyl acetate (0.25 ml, 2.6 mmol) and vinyl chloroacetate (0.24 ml, 1.8 ,mol) . This reaction was allowed to proceed for 24 h at 250 rpm and 35°C. The sequential reaction was then stopped by removing the suspended solid catalyst by centrifugation. The sequential reaction was aided by the soluble nature of the taxol and taxol derivatives. The supernatant containing taxol esters was evaporated to dryness in vacuum and redissolved in 0.3 ml methanol. An aliquot of the resultant concentrated solution (15 μl) was diluted with 250 μl methanol and analyzed by HPLC as described in l.l. The chromatogram revealed a peak with retention time 9.7 min (unreacted taxol), .a broad peak between 11 and 13 min (total estimated yield 52%), peaks at 14.6 min (yield 9%) and 17.4 min (yield 18%), and a small peak at 18.4 min (yield 0.2%). Total reaction yield was approximately 80%.
1.4 Removal of taxol from reaction mixture
A concentrated methanol solution of reaction products produced as described in 1.3 was applied on a preparative TLC silica plate (Whatman, 20x20 cm, silica layer thickness 500 μm, containing fluorescent marker) , and the plates were developed using a solvent mixture of chloroform:acetonitrile (4:1 v/v) . Positions of product spots were determined by irradiating the plates with ultraviolet fight. The Rf value of taxol is 0.16 and the Rf of the taxol esters range from 0.28 to 0.71. The products were removed from the TLC plate and dissolved in ethyl acetate. The products were then dried in vacuo.
1.5 Screening the mixture of taxol esters
The library of taxol derivatives described above was screened for cytotoxicity against HL-60 cells, a promyelocytic leukemia cell line, and MOLT-4 cells, a lymphoblastic leukemia cell line. Cells were seeded in 96-well plates at densities of 30,000 cells/well and grown in RPMI-1640 medium containing 10% bovine fetal calf serum at 37°C for 24 h. The medium was then replaced with fresh medium containing the taxol derivatives (excluding taxol, which had been removed by preparative thin-layer chromatography) dissolved in DMSO at final concentrations ranging from 100 nM to 0.1 nM. The final concentration of DMSO in the cell medium was 0.5% (v/v) . After 72 h, samples were removed for cell counts. Total cell number and viability were determined by trypan blue exclusion and manual cell counting on a hemacytometer. The dose- response data are reported in Table IX. The hemacytometer cell counts revealed that the taxol-derivative library contained at least one cytotoxic derivative.
1.6 Backtracking to identify active product(s) 1.6.1 Enzyme synthesis of individual taxol esters -
Identification of active taxol esters by building-up the reactions comprising taxol acylation in 1.3 above.
Given the predictable nature of the enzymatic acylation reactions, it is possible to identify the possible products without analyzing the chemical compositions of the mixture. The possible products were, therefore, synthesized individually, as described below, and screens on these individual ester products were then initiated as part of the backtracking process.
The powdered enzyme catalyst prepared as described in 1.3 above (140 mg) was added to a solution of taxol (5.5 mg, 6.5 μmol) and on individual vinyl ester (80 μl, approximately 2 mmol) in 1.0 ml tert-amyl alcohol. The following vinyl esters were used as acylating agents: acetate, chloroacetate, acrylate, propionate, butyrate, and caproate. Each mixture was placed into a septum-sealed glass vial, sonicated for 30 s, and put into an orbit shaker operating at 250 rpm and 35°C. After 96 h the reaction was stopped by removing the suspended solid catalyst by centrifugation. Supernatants containing taxol esters were evaporated to dryness in vacuo and redissolved in 0.3 ml methanol each. An aliquot of each resultant concentrated solutions (20 μl) was diluted with 80 μl methanol and analyzed by HPLC as described in 1.1. Results of HPLC analysis are given in Table VIII. Table VIII. Retention times (RT) and Rf values of Taxol esterification products*
Ester 2' -ester 7-eβter 2' ,7-diester Total Yield
(%)
RT R, Yield RT Rf RT Rf
(min) <%) Yield Yield
(min) (%) (min) (*)
Acetate 10.5 0.28 57 13.1 0.48 30 15.2 0.57 2 89
Acrylate 12.2 0.39 80 14.1 0.56 10 15.6 0.67 0.4 90
C loroacetate 12.5 0.32 70 15.9 0.51 3 0.71 0.71 12 85
Propionate 12.8 0.39 78 14.5 0.58 12 - - 90
Butyrate 14.5 0.41 67 15.8 0.61 11 - - 78
Hexanoate 17.3 0.47 50 18.1 0.67 7 - - 57
•Taxol: retention time 9.7 min. R,0.16
1.6.2 Recovery of reaction products by thin layer chromatography (TLC. Individual ester reaction products were separated from taxol by TLC. Concentrated methanol solutions of reaction products produced as described in 1.6 were applied on preparative TLC silica plates (Whatman, 20x20 cm, silica layer thickness 500 μm, containing fluorescent market) , and the plates were developed using a solvent mixture of chloroform:acetonitrile (4:1 v/v) . Positions of product spots were determined by irradiating the plates with ultraviolet light. The Rf values of the products are given in Table VIII. Product spots were scraped off the plates separately and scrapings were eluted with 15 ml ethyl acetate to recover the product Dry products were obtained by evaporation of ethyl acetate in vacuo.
1.6.3 Screening individual taxol esters
To determine which of the newly synthesized product(s) was active, the synthetic history of the library was "backtracked" by enzymatically synthesizing all possible ester products individually (as described above) . Following isolation of the products by TLC, each derivative was tested for cytotoxic activity as described above. The cytotoxicity experiments revealed that two derivatives, 2'- chloroacetyltaxol and 2'-acryloyltaxol, are active against both cell lines (Table IX) .
Table IX. Percent Viability of MOLT-4 and HL-60 Cells After 72 h exposure to taxol and its derivatives at various concentrations
100 nM 10 nM 1 nM 0.1 nM
MOLT-4 Taxol 1.22 11.1 92.8 92.9
Library 3.13 67.9 95.9 95.0
2 '-Chloroacetyltaxol 4.26 91.9 94.4 96.9
2'-Acryloyltaxol 2.70 61.0 95.6 91.0
HL-60
Taxol 0.00 32.6 96.1 98.3
Library 5.26 98.3 98.5 99.0
2 '-Chloroacetyltaxol 2.13 99.5 97.3 99.5
2'-Acryloyltaxol 4.69 87.1 96.9 97.5
Example 2 2. Enzymatic hydrolysis of taxol
2.l Analysis of reaction products
Products of enzymatic hydrolysis of taxol were analyzed using reversed-phase HPLC on a Waters system with a 990 photodiode array detector, with 3.9 x 300 mm μBondapak ClB column. The solvent system used consisted of water and acetonitrile. The solvents were fed into the HPLC system at 1 ml/min according to the following linear gradient program:
Time (min) Solvent Composition (V/V %)
Acetonitrile Water
0-1 25 75
1-5 45 55
5-26 58 42
26-30 100 0
30-35 25 75
Product peaks were detected at 227 nm using a photodiode array detector. 2.2 Enzymatic hydrolysis of taxol (Reaction Boxes J-8 and S- 8)
The enzyme catalyst used for hydrolysis was produced by freeze-drying an aqueous solution (adjusted to pH 7.5) containing equal weight amounts of thermolysin, subtilisin Carlsberg, chymotrypsin and trypsin. The enzyme catalyst (1.1 mg) was dissolved in 0.7 ml 0.1 M potassium phosphate buffer pH 7.5. The aqueous enzyme solution was added to a solution of taxol (2.0 mg, 2.4 μmol) in 0.3 ml tert amyl alcohol. The biphasic reaction system produced in this way was placed into a septum-sealed glass vial and put on an orbit shaker operating at 75 strokes/min and 20°C. After 26 h the organic solvent layer was separated, evaporated to dryness and redissolved in 0.2 ml methanol for HPLC analysis. The aqueous phase was diluted with 3.5 ml methanol and insoluble solids (precipitated enzymes) were removed by centrifugation. Clear supernatant was evaporated into dryness and redissolved in 0.2 ml methanol for HPLC analysis. HPLC analysis was performed as described in 2.1. Neither taxol, nor any hydrolysis products were detected in the methanol solution obtained after workup of the aqueous phase of the biphasic reaction system. On the other hand, the chromatogram of the methanol solution of the dry residue obtained from the organic layer revealed a peak with retention time of 20.4 min (unreacted taxol) and a peak at 18.4 min, representing a product of enzymatic hydrolysis of taxol, as identified by the characteristic uv-absorbance scan. The estimated yield of this product was 9%.
Example 3 3. Enzymatic glycosylation of taxol
3.1 Analysis of Reaction Products Products of enzymatic glycosylation of taxol were analyzed using reversed-phase HPLC on a Waters system with a 990 photodiode detector, with a 3.9 x 300 mm μBondapak C18 column. The solvent system used consisted of water and methanol. The solvents were fed into the HPLC system at 1 ml/min according to the following linear gradient program: Time (min) Solvent Composition (v/v %)
Methanol Water
0-5 25 75
5-37 85 15
37-40 100 0
40-42 100 0
42-44 25 75
Product peaks were detected at 227 nm using a photodiode array detector.
3.2 Enzymatic glycosylation of taxol
Glycosylation of taxol was performed using α-glucosidase from baker's yeast as a biocatalyst. For reference, the enzymes used were: α-glucosidase from brewer's yeast (pH 7.0), α-glucosidase from baker's yeast (pH 7.0), β-galactosidase from E. coli (pH 5.0), β galactosidase from A. oryzae (pH 5.0), β-glucuronidase from bovine liver (pH 5.0). These enzymes were used in two mixtures (one at pH 7.0 and one at pH 5.0) and it was determined that only the mixture at pH 7.0 was active in glycosylating taxol. This mixture was then divided into individual enzymes where it was found that α-glucosidase from baker's yeast was the active biocatalyst.
Example 4 4. The synthesis of second iteration taxol ester products.
Taxol is initially acylated with vinyl adipate in a first reaction box acylation to give a mixture of taxol-2'-vinyl adipate an taxol-2' ,7-divinyl adipate. We have separated the individual adipate products from this first acylation reaction box. The mixture and individual adipate esters were then used in several second reaction boxes, each containing a mixture of alcohols or sugars to give a second iteration taxol library.
Synthesis of derivatives based on taxol vinyl adipate esters -
Second Iteration .
4.1. Acylation of alcohols bv taxol-vinyl adipate esters Enzymatically synthesized taxol-2'-vinyl adipate (2 mM) and taxol-2' ,7-divinyl adipate (2 mM) were dissolved in hexane containing 30 vol.% tetrahydrofuran. To this solution, immobilized lipases from Candida antarctica and Mucor miehei were added in concentrations 100 mg/ml and 75 mg/ml, respectively. The mixture was supplemented with 1 vol.% each of n-butanol, n-hexanol, l,3-S(+)-butanediol and sec-phenethyl alcohol for a total of 5 vol.% of alcohols. The reaction mixture was incubated at 45°C for 4 days under constant shaking at 250 rpm. After the reaction the enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program given in Table 1'.
Table 1'. Reversed-Phase HPLC Gradient Elution Program for Taxol-Vinyl Adipate Alcohol Esters
Time, Water, Acetonitrile, Isopropanol, min % % %
0 52.4 19.6 28.0
8 52.4 19.6 28.0
16 36.4 13.6 50.0
24 20.4 7.6 72.0
26 0 0 100.0
28 0 0 100.0
30 52.4 19.6 28.0
A representative HPLC trace is shown in Fig. 1'. The individual identities of these peaks were determined by repeating the second iteration reaction with individual alcohols in the second iteration step. This is the same approach that is used to re-synthesize, or backtrack, active products from the results of screening assays. Retention times of some of the products are given in Table 2' .
Table 2' . Retention Times of Various Second Iteration Reaction Products Determined by Repeating Individual Second Step Reactions
Retention Estimated Product of time, yield, % min*
11.6 14 taxol-2'-vinyl adipate + 1,3 S(+) butanediol
14.7 13 taxol-2' ,7-divinyl adipate + 1,3 S(+) butanediol
20.1 19 taxol-2'-vinyl adipate + n-butanediol
20.4 taxol-2'-vinyl adipate + sec-phenethyl alcohol
20.8 12 taxol-2',7-divinyl adipate + n- butanediol
22.4 taxol-2'-vinyl adipate + n-hexanol
23.0 taxol-2' ,7-divinyl adipate + n-hexanol
♦Retention times for standards: taxol, 8.8 min; taxol-2'- vinyl adipate, 17.4 min; taxol-2' ,7-divinyl
4.2 Acylation of sugars by taxol-2'-vinyl adipate
Enzymatically synthesized taxol-2'-vinyl adipate (5 mM) in acetonitrile was reacted with a mixture of sugars (mono- and disaccharides) (50 mM) catalyzed by lipase from Candida antarctica (75 mg/ml) . The reaction mixture was incubated at 45°C with 250 rpm shaking for 7 days. After the reaction, the enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program given in Table 3'.
Table 3' . Reversed-Phase HPLC Gradient Elution Program for Taxol-Vinyl Adipate Sugar Esters
Time, Water, Acetonitrile, min % %
0 75 25
5 55 45 26 42 58
30 0 100
35 75 25
As with the alcohols, a mixture of sugar-based taxol vinyl adipates was obtained. The individual identities of the products in the mixture were determined by repeating the second iteration reaction with individual sugars in the second iteration step. Retention times of some of the products are given in Table 4' .
Table 4' . Retention Times of Various Second Iteration Reaction Products Based on Sugars Reacting in this Second Step
Sugar Major Secondary product products
Retention Yield, Retention Yield, Retention Yield, time, mm % time , min % time , min %
Lactose 24 .17 95 _ - - -
Mannoβe 24.30 86 17.47 10 - -
Galactose 24.30 95 - - - -
Cellobiose 24 .30 95 - - - -
Sucrose 24 . 37 95 - - - -
Fructose 24 .27 74 17. 6 6 18 16
Maltose 24 .30 95 - - - -
Note : re stention tin- e of start :ing taxol- 2 ' -vinyl « idipate is ii.
Example 5 5. Synthesis of taxol carbonates
Taxol acylation was also extended to produce carbonates at the 2' and 7-positions of taxol. This reaction is similar to that for acylation with vinyl esters, but uses a different reaction box for vinyl carbonate co-substrates.
Interestingly, preliminary results strongly suggest that the regioselectivity of acylation with the vinyl carbonates differs depending on the choice of enzyme. For example, lipases appear to prefer to catalyze carbonate formation on the 7-hydroxyl group of taxol, whereas proteases prefer to catalyze carbonate formation on the 2'-hydroxyl group of taxol. This regioselectivity of acylation toward taxol has never been reported before with any chemical catalyst, and demonstrates that high regioselective reactions are feasible with enzymes as catalysts.
Reaction using lipases: Taxol (5mM) was dissolved in hexane containing 30 vol.% tetrahydrofuran. To this solution, immobilized lipases from Candida antartica and Mucor miehei were added in concentration 50 mg/ml each. The mixture was supplemented with 1 M butyl vinyl carbonate and incubated at 45°C for 4 days under constant shaking at 250 rpm. After the reaction the enzyme was removed, the supernatant dried in vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program in Table 1'. The reaction produced a single product with a reaction time of 21.1 min and 30% yield. Preliminary structural determination by NMR indicates that this product is taxol-7-butyl carbonate.
Reaction using thermolysis: Taxol (5 mM) was dissolved tert-amyl alcohol. To this solution, thermolysin powder (containing 95% KCl, 4% enzyme, and 1% phosphate buffer salt) was added at concentration of 1.2 mg protein/ml. The mixture was supplemented with 1 M butyl vinyl carbonate or 1 M 1,3-R(- )butanediol di(vinyl carbonate) and incubated for at 45°C for 4 days under constant shaking at 250 rpm. After this time the reaction was stopped and enzyme was removed, the supernatant dried under vacuum and redissolved in acetonitrile, and the products analyzed by reversed phase HPLC using the gradient program in Table 1' . The reaction with butyl vinyl carbonate produced a single major product with a retention time of 18.0 min (43% yield) . Preliminary structural determination by NMR indicates that this product is taxol-2'-butyl carbonate. The reaction was 1,3-R(-)-butanediol di(vinyl carbonate) also produced a single major product with a retention time of 17.7 min ( 35% yield) .
Example 6 6.. Synthesis of phosphorylated taxol
The third major class of reactions recently performed involved the phosphorylation of taxol.
The phosphorylation reaction was performed in a biphasic water-organic system. The aqueous phase consisted of a solution of alkaline phosphatase from chicken intestine (1 mg/ml) in 0.2 M phosphate buffer, pH 8, whereas the organic phase was 15 mM solution of taxol in chloroform. The volume ratio of water to chloroform was 3:7. The mixture was incubated at 25°C with gentle shaking for 7 days. The organic phase was then separated, dried in vacuum, redissolved in methanol and analyzed by reversed phase HPLC using the gradient program in Table 6' .
Table 6' . Reversed Phase HPLC Gradient Elution Program for Taxol Phosphorylation
Time, min Water2, % Methanol, %
0 75 25
5 75 25
37 15 85
40 0 100
* Contaiins 0.01% trifluoroace .tic acid.
The reaction products two products with retention time 27.1 minutes (23% yield) and 29.8 minutes (21% yield) (retention time of taxol was 38.3 min). Thus, these products have retention times shorter then taxol itself, and this is indicative of more water-soluble taxol derivatives.
This application is based on U.S. Patent Application 08/106,279 filed in the U.S. Patent Office on August 13, 1993 and International Application PCT/US94/09174 filed with the U.S. receiving office on August 12, 1994, wherein the entire contents of each application are hereby incorporated by reference.

Claims

Cl ims
1. A method for drug identification comprising:
(a) reacting biocatalysts with a starting compound to produce a library of modified compounds;
(b) determine if a modified starting compound exhibiting a desired activity is present within the library; and
(c) if a modified compound exhibiting a desired activity is found, determining its identity.
(d) repeating the specific biocatalytic reactions which produce the modified compound of desired activity and determining the chemical composition of the reaction product.
2. The method for drug identification of Claim 1 wherein said biocatalysts are a group of biocatalysts that react with distinct structural moieties found within the structure of a starting compound,
(b) each of said biocatalysts is specific for one structural moiety or a group of related structural moieties; and
(c) each of said biocatalysts reacts with many different starting compounds which contain the distinct structural moiety.
3. The method for drug identification of Claim 1 wherein reacting said biocatalysts produces a reaction selected from a group consisting of:
(a) Oxidation of primary and secondary alcohols;
(b) Reduction of aldehydes and ketones;
(c) Acylation of primary and secondary alcohols;
(d) Transglycosylation of primary and secondary alcohols;
(e) Etherification of primary and secondary alcohols;
(f) Acylation of primary and secondary amines; and
(g) Esterification of carboxylic acids.
4. The method for drug identification of Claim 1 wherein said biocatalysts are in a form selected from the group consisting of crude or purified enzymes, cellular lysate preparations, partially purified lysate preparations, living cells and intact non-living cells, used in a soluble, suspended or immobilized form.
5. The method for drug identification of Claim 1 wherein biocatalytic reactions are used in combination with non¬ specific chemical reactions to produce a library of modified starting compounds.
6. The method for drug identification of Claim 1 a determination of whether a modified starting compound exhibits a desired activity is conducted by:
(a) exposing said library of compounds of step (a) to a drug screening device that measures the binding of compounds with desired activity to localized or immobilized receptor molecules or cells;
(b) correlating a positive measurement from the drug screening device with the sequence of biocatalytic reactions used to synthesize the reaction mixture and the specific reaction sequence producing the modified starting compound with desired activity; and
(c) repeating the biocatalytic reaction sequence to produce the modified starting compound of desired activity and to determine its chemical composition.
7. The method for drug identification of Claim 1 wherein said biocatalysts are immobilized to magnetic particles forming a magnetic biocatalyst.
8. The method for drug identification of Claim 1 wherein the biocatalytic reaction is performed with a biocatalyst immobilized to a particle and the biocatalyst is removed from the biocatalytic reaction mixture by centrifugation or filtration.
9. The method for drug identification of Claim 1, wherein said identification is performed in an automated robotic device, said automated robotic device comprising:
(a) an XY table with an attached XYZ pipetting boom to add volumetric amounts of enzyme, substrate, cofactor, solvent solutions and assay reagents from reagent vessels positioned on the X-Y table to reaction and assay vessels positioned on the same XY table;
(b) an XYZ reaction-vessel transfer boom attached to the same XY table used to transfer reaction and assay vessels positioned on the XY table to different locations on the X-Y table;
(c) a temperature incubation block attached to the same XY table to house the reaction and assay vessels during reaction incubations and control the temperature of the reaction mixtures;
(d) a magnetic separation block attached to the same XY table to separate a biocatalyst immobilized to magnetic particles from a biocatalytic mixture by applying an external magnetic field causing the magnetic particles to be attracted to and concentrate at the source of the magnetic filed, thus effectively separating them from the bulk of a biocatalytic reaction mixture; and
(e) a programmable microprocessor interfaced to the XYZ pipetting boom, and XYZ reaction vessel transfer boom, the temperature block and the magnetic separation block to precisely control and regulate all movements and operations of these functional units in performing biocatalytic reactions to produce modified starting compounds and assays to determined desired activities.
10. The method for drug identification comprising
(a) reacting biocatalysts with a starting compound to produce a library of modified compounds, wherein biocatalysts which recognize the same functional group and perform the same type of enzymatic derivatization are reacted in the same reaction vessel, and biocatalysts which recognize different functional groups and perform different types of enzymatic derivatization are reacted in different reaction vessels;
(b) determining if a modified starting compound exhibiting a desired activity is present within a specific reaction vessel of said library; and
(c) if a modified compound exhibiting a desired activity is found, determining its identity.
11. The drug identification method of Claim 10, further comprising reacting said library of modified compounds from step (a) with biocatalysts to produce a second iteration library of modified starting compounds wherein biocatalysts which recognize the same functional group and perform the same type of enzymatic derivatization are reacted in the same reaction vessel, and biocatalysts which recognize different functional groups and perform different types of enzymatic derivatization are reacted in different reaction vessels;
(b) determining if a modified starting compound exhibiting a desired activity is present within a specific reaction vessel of said second iteration library; and
(c) if a modified compound exhibiting a desired activity is found, determining its identity.
12. The drug identification method of Claim 11, further comprising conducting a series of up to n biocatalytic reactions on said second iteratation library, to form an nth iteration library, wherein after each iteration, determining if a modified starting compound exhibiting a desired activity is present within a specific reaction vessel of said nth iteration library.
13. The method for drug identification of Claim 10, wherein reacting of said biocatalysts is conducted in the presence of co-factors, reagents and solvents necesary to conduct the desired reaction.
PCT/US1995/001759 1994-08-12 1995-02-13 Biocatalytic methods for synthesizing and identifying biologically active compounds WO1996005511A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19165/95A AU1916595A (en) 1994-08-12 1995-02-13 Biocatalytic methods for synthesizing and identifying biologically active compounds

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1994/009174 WO1995005475A1 (en) 1993-08-13 1994-08-12 Biocatalytic methods for synthesizing and identifying biologically active compounds
USPCT/US94/09174 1994-08-12

Publications (1)

Publication Number Publication Date
WO1996005511A1 true WO1996005511A1 (en) 1996-02-22

Family

ID=22242853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/001759 WO1996005511A1 (en) 1994-08-12 1995-02-13 Biocatalytic methods for synthesizing and identifying biologically active compounds

Country Status (2)

Country Link
AU (1) AU1916595A (en)
WO (1) WO1996005511A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
EP1959255A3 (en) * 1997-04-04 2008-09-24 Caliper Life Sciences, Inc. Closed-loop biochemical analyzers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991012331A1 (en) * 1990-02-14 1991-08-22 Receptor Laboratories, Inc. Method for generating and screening useful peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991012331A1 (en) * 1990-02-14 1991-08-22 Receptor Laboratories, Inc. Method for generating and screening useful peptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANGEW. CHEM. INTERNATIONAL EDITION ENGLISH, Volume 27, issued 1988, YAMADA et al., "Microbial and Enzymatic Processes for the Production of Biologically and Chemically Useful Compounds", pages 622-642. *
CURRENT OPINION IN BIOTECHNOLOGY, Volume 2, issued 1991, WILKS et al., "Alteration of Enzyme Specificity and Catalysis by Protein Engineering", pages 561-567. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
EP1959255A3 (en) * 1997-04-04 2008-09-24 Caliper Life Sciences, Inc. Closed-loop biochemical analyzers

Also Published As

Publication number Publication date
AU1916595A (en) 1996-03-07

Similar Documents

Publication Publication Date Title
EP0713533B1 (en) Biocatalytic methods for synthesizing and identifying biologically active compounds
US11926867B2 (en) Generating capture probes for spatial analysis
Laurent et al. Enzyme catalysis on solid surfaces
Tolbert et al. Intein-mediated synthesis of proteins containing carbohydrates and other molecular probes
CN113767177B (en) Generation of capture probes for spatial analysis
Yeo et al. Expanded utility of the native chemical ligation reaction
Palomo et al. Interfacial adsorption of lipases on very hydrophobic support (octadecyl–Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases
Díaz-Mochón et al. Microarray platforms for enzymatic and cell-based assays
JPH09500007A (en) Random chemistry for new compound formation
Halling et al. Understanding enzyme action on immobilised substrates
Calleri et al. Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification
Krstenansky et al. Biocatalytic combinatorial synthesis
Plieva et al. Immobilization of hog pancreas lipase in macroporous poly (vinyl alcohol)-cryogel carrier for the biocatalysis in water-poor media
Hessel et al. Biotechnical micro-flow processing at the EDGE–Lessons to be learnt for a young discipline
CN101156062A (en) Electrode array device having an adsorbed porous reaction layer having a linker moiety
Byun et al. In-gel expression and in situ immobilization of proteins for generation of three dimensional protein arrays in a hydrogel matrix
Hu et al. Microarray: a versatile platform for high-throughput functional proteomics
Fang et al. Enzymes in organic synthesis: alteration of reversible reactions to irreversible processes
Sofia Generation of oligosaccharide and glycoconjugate libraries for drug discovery
WO1996005511A1 (en) Biocatalytic methods for synthesizing and identifying biologically active compounds
US6136961A (en) Biocatalytic methods for synthesizing and identifying biologically active compounds
Timm et al. On-chip protein biosynthesis.
KR0164050B1 (en) Method for preparing ester compound using enzyme in non-solvent
WO1997010233A9 (en) Biocatalytic methods for synthesizing and identifying biologically active compounds
Bavaro et al. Regioselective deacetylation of disaccharides via immobilized aspergillus niger esterase (s)‐catalyzed hydrolysis in aqueous and non‐aqueous media

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载