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WO1996005511A1 - Procedes biocatalytiques pour synthetiser et identifier des composes biologiquement actifs - Google Patents

Procedes biocatalytiques pour synthetiser et identifier des composes biologiquement actifs Download PDF

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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
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Prior art keywords
reaction
compounds
library
biocatalysts
biocatalytic
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PCT/US1995/001759
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English (en)
Inventor
J. Wesley Fox
Jonathan S. Dordick
Douglas S. Clark
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Enzymed, Inc.
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Publication date
Priority claimed from PCT/US1994/009174 external-priority patent/WO1995005475A1/fr
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/fr

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    • 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.

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Abstract

Cette invention concerne des procédés permettant de réaliser une collection de composés de départ modifiés, en appliquant des réactions biocatalytiques au composé de départ, et d'identifier le composé de départ modifié ayant l'activité optimale souhaitée. L'invention concerne des composés de départ et des composés modifiés qui sont libres en solution. Ce procédé est utile pour produire des composés pharmaceutiques modifiés avec l'activité spécifique souhaitée.
PCT/US1995/001759 1994-08-12 1995-02-13 Procedes biocatalytiques pour synthetiser et identifier des composes biologiquement actifs WO1996005511A1 (fr)

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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 (fr) * 1997-04-04 2008-09-24 Caliper Life Sciences, Inc. Analyseurs biochimiques en boucle fermée

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* Cited by examiner, † Cited by third party
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WO1991012331A1 (fr) * 1990-02-14 1991-08-22 Receptor Laboratories, Inc. Procede de production et de tri de peptides utiles

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WO1991012331A1 (fr) * 1990-02-14 1991-08-22 Receptor Laboratories, Inc. Procede de production et de tri de peptides utiles

Non-Patent Citations (2)

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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 (fr) * 1997-04-04 2008-09-24 Caliper Life Sciences, Inc. Analyseurs biochimiques en boucle fermée

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