BIOCATALYTIC METHODS FOR SYNTHESIZING AND IDENTIFYING BIOLOGICALLY ACTIVE COMPOUNDS

• US 20020039723A1
• Filed: 02/08/1999
• Published: 04/04/2002
• Est. Priority Date: 08/13/1993
• Status: Abandoned Application

• Current Assignee: Albany Molecular Research Incorporated
• Original Assignee: Albany Molecular Research Incorporated
• Inventors: CLARK, DOUGLAS S., DORDICK, JONATHAN S., FOX, J. WESLEY
• Application Number:
  US09/246,267
• Publication Number:
  US 20020039723A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J

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 method is useful in producing modified pharmaceutical compounds with desired specific activity.

Claims

1. 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.

2. A method for drug identification of claim 1 wherein

(a) the biocatalytic reactions are conducted with a group of biocatalysts that react with distinct structural moieties found within the structure of a starting compound,

(b) each biocatalyst is specific for one structural moiety or a group of related structural moieties;

and

(c) each biocatalyst reacts with many different starting compounds which contain the distinct structural moiety.

6. A method for drug identification according to claim 1 wherein the biocatalytic reactions are 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.

7. A method for drug identification according to claim 1 wherein the biocatalyst comprises 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.

8. A method for drug identification according to claim 1 wherein the biocatalytic reactions are used in combination with non-specific chemical reactions to produce a library of modified starting compounds.

9. A method for drug identification according to claim 1 further comprising:

(a) exposing the reaction mixture 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.

10. A method for drug identification according to claim 1 wherein the biocatalytic reaction is performed with a biocatalyst immobilized to magnetic particles forming a magnetic biocatalyst, the method further comprising

(a) initiating the biocatalytic reaction by combining the immobilized biocatalyst with substrate(s), cofactor(s) and solvent/buffer conditions used for a specific biocatalytic reaction;

(b) removing the magnetic biocatalyst from the biocatalytic reaction mixture to terminate the biocatalytic reaction, which 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 biocatalytic reaction mixture and allowing 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;

(c) conducting a second biocatalytic reaction, completely independent of the first biocatalytic reaction, by combining a second biocatalyst immobilized to magnetic particles with the second reaction vessel containing the biocatalytic reaction mixture transferred from the first reaction vessel; and

(d) repeating steps a) through c) to accomplish a sequential series of distinct and independent biocatalytic reactions and produce a corresponding series of modified starting compounds.

11. A method for drug identification according to claim 10 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.

12. A method for drug identification according to claim 1 using an automated robotic device, the automated robotic device comprised of:

(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 determined desired activities.

14. A method for rapid drug development according to claim 1 wherein the high affinity receptor is present on the surface or contained within a living cell or immobilized to a natural or artificial support.

15. A method for rapid drug development according to claim 1 wherein the biocatalytic reactions are selected from a group consisting of those listed in Table II.

16. A method for rapid drug development according to claim 1 wherein the testing of the library of modified starting compounds is selected from a group of assays consisting of those listed in Table VI.

17. A method for rapid drug development according to claim 1 wherein the biocatalytic reaction is performed with a biocatalyst immobilized to magnetic particles forming a-magnetic biocatalyst whereas:

(a) the biocatalytic reaction is initiated by combining the immobilized biocatalyst with the following to form the biocatalytic reaction mixture;

substrate(s), cofactor(s) and solvent/buffer conditions used for the specific biocatalytic reaction;

(b) the biocatalytic reaction is terminated by removing the magnetic biocatalyst from the biocatalytic reaction mixture, which 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 biocatalytic reaction mixture and allowing for the removal of the reaction mixture minus the magnetic biocatalyst to a second reaction vessel, leaving the magnetic biocatalyst in the first reaction vessel;

(c) a second biocatalytic reaction, completely independent of the first biocatalytic reaction, is initiated by adding a second biocatalyst immobilized to magnetic particles to the second reaction vessel containing the biocatalytic reaction mixture from the first biocatalytic reaction minus the magnetic biocatalyst used in the first biocatalytic reaction;

(d) the above described biocatalytic reaction cycle is repeated thus accomplishing a series of distinct and independent biocatalytic reactions producing a corresponding series of modified drug compounds.

(e) a magnetic separation block attached to the same XY table as above to separate the biocatalyst immobilized to magnetic particles from the biocatalytic reaction mixture by applying an external magnetic field causing the magnetic particles to be attracted to and concentrate at the source of the magnetic field, thus effectively separating them from the bulk of the biocatalytic reaction mixture; and

(f) a programmable microprocessor interfaced to the XYZ pipetting boom, the 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.

3. A method for drug identification comprising:

(a) conducting a series of biocatalytic reactions on a starting compound to produce a library of modified starting compounds;

(b) providing a high affinity receptor for a starting compound of desired activity and exposing the modified compounds formed after each biocatalytic reaction to the high affinity receptor;

(c) isolating the high affinity receptor-modified starting compound complex from the biocatalytic reaction mixture;

(d) dissociating the complex into its component parts; and

(e) determining the chemical composition of the dissociated modified starting compound.

4. A method for drug identification comprising:

(a) adding a high affinity receptor to a starting compound to form a biocatalytic reaction mixture at a concentration that allows for essentially all of the high affinity receptor to bind with the starting compound and about an equal molar amount of starting compound remaining free in solution and available for biocatalytic modification;

(b) producing a modified starting compound exhibiting a higher binding affinity than the starting compound resulting in the displacement of the starting compound from the high affinity receptor;

(c) forming a complex of the modified starting compound and the high affinity receptor, thereby protecting the modified starting compound from further biocatalytic reaction;

(d) isolating the complex from the biocatalytic reaction mixture;

(e) dissociating the complex into its component parts;

(f) determining the chemical composition of the dissociated modified starting compound; and

(g) repeating steps (a) through (b) to produce a library of modified starting compounds.

5. A method for drug identification according to claim 4 wherein the high affinity receptor is present on the surface or contained within a living cell or immobilized to a natural or artificial support.

13. A method for rapid drug development comprising:

(a) mixing a starting drug compound with a high affinity receptor at approximately one half the molar concentration of the starting drug compound to allow essentially all of the high affinity receptor to bind to the starting drug compound and leave an equal molar amount of starting drug compound free in solution and available for biocatalytic modification;

(b) producing a modified drug compound from the biocatalytic reaction between the starting drug compound and the receptor, the modified drug compound having a higher binding affinity than the starting drug compound;

(c) displacing the starting drug compound from the receptor with the modified drug compound;

(d) protecting the bound modified drug compound from further biocatalytic reactions;

(e) dissociating the complex of the modified drug compound and receptor into its component parts;

(f) determining the chemical composition of the dissociated modified starting drug compound; and

(g) repeating steps a) through e) and thus conduct a series of biocatalytic reactions on a starting drug compound to produce a library of modified drug compounds with higher binding affinities.