WO2002095044A2

WO2002095044A2 - Lipase catalyzed esterification, transesterification, and hydrolysis of arylthiols and aryl-thioesters

Info

Publication number: WO2002095044A2
Application number: PCT/US2002/015994
Authority: WO
Inventors: Hjalti Skulason
Original assignee: Molecular Electronics Corporation
Priority date: 2001-05-21
Filing date: 2002-05-21
Publication date: 2002-11-28
Also published as: WO2002095044A3; AU2002316148A1

Abstract

A method for catalyzing reactions of aryl-thiols and aryl-thioesters with water, alcohol or carboxylic acid using an enyzme or microorganism leading to the hydrolysis, esterification or trans-esterification or these compounds, which is particularly useful as a step in anchoring a self-assembled monolayer to a metal substrate.

Description

LIPASE CATALYZED ESTERIFICATION, TRANSESTERIFICATION, AND

HYDROLYSIS OF ARYLTHIOLS AND ARYL-THIOESTERS

This application claims the benefit of Provisional Patent Application, Serial

No. 60/292,750, filed May 21, 2001, entitled "Lipase Catalyzed Esterification,

Transesterification, And Hydrolysis Of Arylthiols And Aryl-Thioesters".

FIELD OF THE INVENTION

This invention covers the use of lipases in esterification, trans-esterification

and hydrolysis of aryl-thiols and aryl-thioesters of the general structure

shown below:

I II

where Ar is an aryl or substituted aryl group and R is an alkyl, aryl, substituted

alkyl or substituted aryl group.

BACKGROUND OF THE INVENTION

A variety of advances have recently been made in the field of molecular

electronics. It has been established that it is possible to fabricate individual

molecules or molecular-scale devices that perform functions identical or

comparable to functions performed by current microcircuit devices. Individual molecules have been shown to function as diodes and to conduct and switch

electric current as well as to store information.

The most common method for organizing the electronically active molecules

inside a device structure is to use the high affinity of thiols for gold, platinum,

palladium, or other coinage metal surfaces. Self-assembled monolayers (SAMs) of

alkyl- and aryl-thiols on the above mentioned metal surfaces have been studied

extensively by multiple research groups. It has been verified that among various

sulfur-containing functional groups, such as thiols, disulfides, thio-acetates, bunte

salts, and thio-ethers, that thiols form the most highly ordered SAMs. This is of

great importance when fabricating electronic devices based on SAMs, since the

device characteristics may depend strongly on the arrangement of the molecules on

the metal surface.

The most common molecular structures in molecular electronics involve

aryl-thiols as an anchor group to the metal surface. Due to their susceptibility

towards oxidation they are commonly synthesized and stored as aryl-thioesters.

Before self-assembly the acetates are then cleaved off to yield the free thiol.

Currently, cleavage of aryl-thioesters (and aryl-esters) is most commonly

accomplished by reactions with base (NaOH, NH4OH), acid (HBr, CF3COOH),

LiAlH₄, NaBH₄ and related compounds (see Jerry March in Advanced Organic

Chemistry, Fourth Edition; 1992: John Wiley & Sons, Inc.; New York). However,

these methods use highly reactive chemicals, which can react with other parts of

the aryl-thioester molecule, in addition to cleaving the aryl-thioester. For example, this shortcoming arises when cleaving certain aryl-thioacetates, such as compounds

III-V, containing two nitro groups, using NH₄OH in solvents such as ethanol,

dichloromethane and mixtures of the two. In comparison, base promoted cleavage

was not found to interfere with molecules of structure VI.

Figure III

Figure IV

Figure V

Figure VI

If a method could be found to cleave aryl-esters and aryl-thioesters without

reaction with other parts of the molecule, an important and highly useful advance

in the art would be at hand.

SUMMARY OF THE INVENTION

Cleavage of aryl-thiols and aryl-thioesters by lipase and other enzymes in

accordance with the present invention is accomplished under very mild conditions,

such as room temperature (operable temperature ranges between -10°C and 80°C), neutral pH (operable pH ranges between 1 and 12) and in the absence of highly

reactive chemicals. These conditions make this method suitable for a wide

collection aryl-thioesters in a variety of applications such as but not limited to,

molecular electronics, molecular based sensors and synthesis of new

pharmaceutical compounds.

Lipases have been widely used in organic chemistry for esterification, trans-

esterification and hydrolysis of alkyl-esters. Several alkyl-thioesters have been

used as substrates for use with lipases but only for a few aryl-esters (regular

O-esters, not thioesters). No reports have been found on the use of aryl-thioesters

with lipases. A possible reason for this may be the rarity of aryl-thioesters in

nature and their structural difference from the natural substrates of lipases

(triglycerides) which makes them an unlikely candidate to be catalyzed by a lipase.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions apply throughout this application.

The term "alkyl" used herein refers to substituted or unsubstituted straight,

branched, or cyclic chain carbon groups of 1 to 30 carbon atoms, preferably

between 1 to 12 carbon atoms.

The term "aryl" as used herein refers to monocyclic, bicyclic, tricyclic,

tetracyclic, or pentacyclic aromatic groups containing from 6 to 50 carbon or other

atoms in the ring portion, such as phenyl, pyridyl, naphthyl, and substituted phenyl, pyridyl or naphthyl, containing substituents such as nitro, amino, halogen,

hydroxy or alkyl on the aromatic ring.

The enzymatic process of the present invention has the advantage that it

performs the desired chemical transformation without the addition of highly

reactive chemicals. The present process can be carried out in an aqueous solvent

(hydrolysis reactions), organic solvent or in mixtures thereof. Typical solvents

suitable for use in the present process include, but are not limited to,

dichloromethane, chloroform, tetrahydrofuran, benzene, toluene, cyclohexane,

hexane, ethanol, methanol, de-ionized water, suitable aqueous buffer solutions and

mixtures of these organic and aqueous solvents.

The enzymes and microorganisms that may be used in the practice of this

invention must have the ability to hydrolyse, esterify or trans-esterify aryl-thiols or

aryl-thioesters of general formulas I and II (see above). Various enzymes, such as

esterases, lipases, and proteases, regardless of origin or purity, are suitable for use

in the present invention, so long as they meet the criterion. The enzymes can be in

the form of a mixture of animal and plant enzymes, cells of microorganisms,

crushed cells or extracts of cells.

Typical genuses of microorganisms suitable as sources of hydrolyzing,

esterifying or trans-esterifying enzymes include microorganisms with the ability to

hydrolyse, esterify or trans-esterify aryl-thiols or aryl-thioesters of general

formulas I and II from among the following: Mucor, Escherichia, Staphylococcus,

Agrobacterium, Rhizopus, Aspergillus, Nocardia, Streptomyces, Trichoderma, Candida, Rhodotorula, Torulopsis, Bacillus, Alcaligene, Pseudomonas,

Brevebacterium, Enterobacter, Chromobacterium, Arthorbacter, Mircobacterium,

Mycobacterium, Saccharomyces, Penicillium, Botrytis, Chaetomium, Ophiobolus,

Cladosporium, Candidfa, Geotrichum and similarly used lipases.

To carry out the process in the present invention the enzyme and the

reactants (aryl-thioester, aryl-thiol, water, alcohol, carboxylic acid, ester, or a

combination thereof) are added to the desired solvent. Frequently, the enzyme is

adsorbed onto a suitable carrier, i.e. the enzyme is immobilized on the carrier. This

helps in removing the enzyme from the solution by filtration after the reaction is

complete. The reaction solution is typically between about O.lmM and about 10M

in the aryl-thiol or aryl-thioester, but preferably between about 1 and about

lOOmM. In the case of an organic solvent, the reaction solution is typically between

about 0.1 and about 50M in water (hydrolysis), alcohol (trans-esterification),

carboxylic acid (esterification or trans-esterification), or ester (esterification or

trans-esterificaiton) but preferably between about 5 and about 25M.

The general lipase (discussed below) catalyzed hydrolysis reaction of an

aryl-thioester is:

The general lipase catalyzed esterification reaction of an aryl-thiol with a

carboxylic acid is:

Other acids could be used but would have to be screened depending on the lipase

being used.

The general lipase catalyzed esterification of an aryl-thiol with an ester is:

The general lipase catalyzed trans-esterification reaction of an aryl-thioester

with an alcohol is:

Other alcohols could be used but would have to be screened depending on the lipase

being used.

The general lipase PS catalyzed trans-esterification reaction of an aryl-

thioester with an acid is:

where R, Ri and R₂ denote, alkyl, aryl, substituted alkyl or substituted aryl group,

and Ar denotes an aryl or a substituted aryl group. Below are additional examples of molecules subject to esterification, trans-

esterification and hydrolysis in accordance with the present invention:

The present invention will now be illustrated by the following examples,

however, it should be understood that the invention is not meant to be limited by

the details therein.

Example 1

To a solution of lmL of dichloromethane and lmL of ethanol was added

3mg of compound VI and lOOmg of lipase PS "Amano" (from Amano

Pharmaceutical Co., Ltd., Nagoya, Japan, contains diatomaceous earth as filler) in

an amber screw cap vial. The vial was put on a shaker for 48h, after which the

lipase was filtered off. A drop of the solution was dried on a salt plate and the

infrared spectrum of the mixture recorded. This spectrum (Figure VIII) was

compared to the spectrum of the uncleaved compound (Figure VII, in KBr pellet).

The spectra, which are shown below, show that the carbonyl stretch of the acetate

at ~1700 cm.-¹ is almost gone and instead a S-H stretch of the free thiol is present at

2560 cur¹. From these spectra it was determined that approximately 95% of the thioacetate had been converted to the free thiol during the 48h reaction time.

Additionally, the ultraviolet-visible absorption spectrum (Figure IX below)

revealed that the product, the free thiol, had the same absorption characteristics as

the free thiol obtained with base (NH₄OH) promoted cleavage, yet it had different

absorption than the reactant, aryl-thioacetate.

Example 2

To a solution of lmL of tetrahydrofuran and lmL of ethanol were added

2mg of compound III and lOOmg of lipase PS-C "Amano" II (lipase PS immobilized

on ceramic particles) in an amber screw cap vial. The vial was put on a shaker for

24h, after which the immobilized lipase was filtered off. The ultraviolet-visual

spectrum (shown below) of the solution before and after the reaction revealed that

its absorption characteristics had changed considerably. Furthermore, the product

from the lipase-catalyzed trans-esterification had different absorption

characteristics in the UV-Vis range than when the same compound was cleaved

using NH4OH. The most important observation in Figure X below, is that for the

base cleaved solution the absorbance at 330nm is absent which is believed to

indicate some loss of conjugation in the molecule. This absorbance peak is still

present in the solutions cleaved by the lipase. This loss of conjugation during the

base promoted cleavage is thought to be a result of (an unknown) side reaction

between compound III and the base. This demonstrates the advantages this invention has over the prior art of

cleaving aryl-thioesters to yield the free thiol.

Example 3

To 2ml of ethanol were added 2mg of compound III and 200mg of lipase PS

"Amano" in an amber screw cap vial. The vial was put on a shaker for 4h, after

which the lipase was filtered off. Based on the infrared spectrum of the solution, it

was estimated that approximately 60% of the thioacetate had been converted to the

free thiol.

Example 4

If to a solution of 1.5mL of tetrahydrofuran and 0.5mL of water lOmg of

phenyl-thioacetate and 200mg of lipase PS-D "Amano" I were added, in an amber

screw cap vial, and the vial is put on a shaker for 48h after which the lipase is

filtered off, the phenyl-thiol in high yield would result.

Example 5

If to 2mL of vinyl-acetate are added 5mg of phenyl-thiol and lOOmg of lipase

PS "Amano" in a round bottom flask fitted with pressure regulating device under

inert atmosphere, and the solution is stirred vigorously for 48h after which the

lipase is filtered off, the phenyl-thioacetate in high yield would result. Example 6

If to a solution of lmL tetrahydrofuran and lmL of butyric acid are added

20mg of phenyl-thioacetate and lOOmg of lipase PS-C "Amano" II, in an amber

filtered off, the phenyl-thiobutyrate in high yield would result.

Example 7

If to a solution of lmL of vinyl-acetate is added 20mg of benzene-thiol and

lOOmg of lipase PS-C "Amano" I, in an amber screw cap vial, and the vial is put on

a shaker for 48h after which the lipase is filtered off, the phenyl-thioacetate is high

yield would result.

Example 8

To a solution of lmL of dichloromethane and lmL of ethanol was added

3mg of compound VI and lOOmg of lipase AY "Amano" (from Amano

an amber screw cap vial. The vial was put on a shaker for 48h, after which the

lipase was filtered off. Analysis of the solutions by IR and UV-Vis revealed that

lipase AY did not catalyze the trans-esterification of that specific aryl-thioester

with ethanol. While the present invention is described above in connection with preferred

or illustrative embodiments, these embodiments are not intended to be exhaustive or

limiting of the invention. Rather, the invention is intended to cover all alternatives,

modifications and equivalents included within its spirit and scope, as defined by the

appended claims.

Claims

CLAIMSWhat I claim is:

1. A method for cleaving aryl-thiols or aryl-thioesters of formula I and

II below:

I II

without reaction with non-aryl-thioester and non-aryl-thiol parts of the molecule,

alkyl or substituted aryl group, comprising reacting the aryl-thiol or aryl-thioester

with water, alcohol, or acid in the presence of an enzyme or a microorganism

having the ability to catalyze the hydrolysis, esterification or trans-esterification

reaction between the reactants.

2. The method of claim 1 in which the enzyme is chosen from the group

consisting of esterases, lipases, and proteases.

3. The method of claim 1 in which the enzyme is lipase PS.

4. The method of claim 1 in which the enzyme is in the form of a

mixture of animal and plant enzymes, cells of microorganisms, crushed cells or

extracts of cells.

5. The method of claim 1 in which the micro-organism is chosen from

the group consisting of Mucor, Escherichia, Staphylococcus, Agrobacterium,

Rhizopus, Aspergillus, Nocardia, Streptomyces, Trichoderma, Candida,

Rhodotorula, Torulopsis, Bacillus, Alcaligene, Pseudomonas, Brevebacterium,

Enterobacter, Chromobacterium, Arthorbacter, Mircobacterium, Mycobacterium,

Saccharomyces, Penicillium, Botrytis, Chaetomium, Ophiobolus, Cladosporium,

Candida, and Geotrichum.

6. The method of claim 1 in which the enzyme is immobilized on a

carrier before beginning the reaction.

7. The method of claim 1 in which the aryl-thiol or aryl-thioester is

present in the reaction solution at a level of about O.lrnM to about 10M.

8. The method of claim 1 in which the aryl-thiol or aryl-thioester is

present in the reaction solution at a level preferably between about ImM to

about lOOmM.

9. The method of claim 1 in which the reaction is carried out in an

organic solvent with water to hydrolyze the aryl-thioester and the reaction solution

is present at a level of about 0.1M to about 50M in water.

10. The method of claim 1 in which the reaction is carried out in aqueous

solution to hydrolyze the aryl-thioester.

11. The method of claim 1 in which the reaction is carried out in an

organic solvent with a carboxylic acid to esterify or trans-esterify the aryl-thiol or

aryl-thioester and the the carboxylic acid in the solution is present at a level of

about 0.1M to about 50M.

12. The method of claim 1 in which the reaction is carried out in neat

carboxylic acid to esterify or trans-esterify the aryl-thiol or aryl-thioester.

13. The method of claim 1 in which the reaction is carried out in an

organic solvent with an alcohol to trans-esterify the aryl-thioester and the alcohol

in the reaction solution is present at a level of about 0.1M to about 50M.

14. The method of claim 1 in which the reaction is carried out in neat

alcohol to trans-esterify the aryl-thioester.

15. The method of claim 1 in which the reaction is carried out in an

organic solvent with an ester to esterify the aryl-thiol and the ester in the reaction

solution is present at a level of about 0.1M to about 50M.

16. The method of claim 1 in which the reaction is carried out in the neat

ester to esterify the aryl-thiol.

17. The method of claim 9 in which the water in the reaction solution is

present at a level of between about 5 and about 25M.

18. The method of claim 11 in which the carboxylic acid in the reaction

solution is present at a level of between about 5 and about 25M.

19. The method of claim 13 in which the alcohol in the reaction solution is

present at a level of between about 5 and about 25M.

20. The method of claim 15 in which the ester in the reaction solution is

present at a level of between about 5 and about 25M.

21. The method of claim 1 in which the aryl-thiol or aryl-thioester is

chosen from among:

22. A method for cleaving aryl-thiols or aryl-thioesters of formula I and

II below:

I II

with lipase PS in the presence of sufficient water, alcohol or acid to hydrolyse,

esterify or trans-esterify the aryl-thiols or aryl-thioesters.

23. In the method of making a self-assembled monolayer anchored to a

metal surface using an aryl-thioester or an aryl ester to anchor the self-assembled

monolayer, in which the molecules are stored as aryl thioesters and aryl esters, the

improvement comprising cleaving the aryl thioesters and aryl esters by reacting

these compounds with an enzyme or microorganism having the ability to

hydrolyse, esterify or trans-esterify aryl-thiols and aryl-thioesters before initiating

the process of forming the self -assembled monolayer.

24. The method of claim 23 in which lipase PS is used to cleave the aryl

thioesters and aryl esters.