Enzyme-catalyzed racemic cleavage of primary amines

Details Enzyme-catalyzed racemic cleavage of primary amines Enzyme-catalyzed racemic cleavage of primary amines

2001-02-06

2002-10-15

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing nitrogen-containing organic compound

C435S198000, C435S280000

Reexamination Certificate

active

06465223

ABSTRACT:

The present invention relates to a process for resolving racemates of alkoxy-substituted primary amines by reacting with an ester in the presence of a lipase and subsequently separating the optically active amide which is formed from the unreacted optically active amine. This is followed where appropriate by hydrolysis of the optically active amide, separation of the optically active amine produced thereby from the acid from which the ester is derived, racemization and recycling of the unwanted enantiomer of the amine, and esterification and recycling of the acid.
WO95/08636 describes a process for resolving racemates of primary and secondary amines by reacting w with an ester in the presence of hydrolases, especially lipases. The preferred amines are primary arylalkylamines. WO 96/23894 describes a process for resolving racemates of primary and secondary heteroatom-substituted amines by reacting with an ester in the presence of hydrolases, especially lipases. The preferred amines are O-protected amino alcohols. The preferred esters mentioned in both applications are the C
1-4
-alkyl esters Of C
1-4
-alkoxyacetic acids.
DE 196 03 575 and DE 196 37 336 describe a process for preparing optically active amines by reacting the corresponding racemates with an ester in the presence of lipase from Candida antarctica. The preferred amines are alkoxy-substituted alkylamines, especially 2-amino-1-methoxypropane, and substituted phenylethylamines, especially 4-chlorophenylethylamine. The e preferred esters are C
1-6
-alkanoic esters and C
1-8
-alkoxyalkanoic esters, especially methyl methoxyacetate. Finally, DE 196 21 686 describes a process for preparing optically active amines by reacting the corresponding racemates with an ester in the presence of hydrolases, in which substituted phenylethylamines, especially 4-chlorophenylethylamine, and C
1-4
-haloalkanoic esters, especially ethyl chloroacetate, are preferred.
It has now been found, surprisingly, that the process described at the outset can be carried out particularly advantageously if esters with long-chain alcohol residues are used.
The process for resolving racemates of amines takes place particularly advantageously if an ester of the general formula 1
in which
m is 0 or 1,
R
1
is branched or unbranched C
6
-C
20
-alkyl or heteroalkyl having 6 to 20 backbone atoms,
R
2
is C
1
-C
8
-alkyl or phenyl,
R
3
is H or C
1
-C
4
-alkyl,
is reacted with an alkoxy or benzyloxy substituted primary amine of the general formula 2
in which
n is 0 or 1,
R
4
, R
5
are, independently of one another, H, C
1
-C
8
-alkyl or phenyl,
R
6
is C
1
-C
6
-alkyl or benzyl,
in the presence of a lipase,
resulting in amides of the general formula 3
in which
n, m, R
2
, R
3
, R
4
, R
5
and R
6
have the meanings stated above,
which comprise an excess of one optical isomer of the amide.
There is preferably formation of an (R) amide, and particularly preferably enantioselective acylation with a high enantiomeric excess of more than 50% ee, in particular more than 80% ee, especially more than 90% ee. The unreacted amine comprises an excess of (S) amine, preferably more than 50% ee, in particular more than 90% ee, especially more than 99% ee. (R) amide or (R) amine mean the optically active amines or amides having the (R) configuration at the carbon of the amino group. An analogous statement applies to (S) amine or (S) amide.
It has additionally been found that it is advantageous to dry the starting materials. This can in principle take place in any manner known to the skilled worker, e.g. by azeotropic drying or through desiccants such as sodium sulfate, magnesium sulfate, KOH, phosphorus pentoxide, molecular sieves, silica gel or alumina.
It has additionally been found that it is advantageous to use acid-free starting materials. Acids can in principle be removed in any way known to the skilled worker, e.g. by extraction or distillation, where appropriate after previous neutralization with alkali metal or alkaline earth metal hydroxides such as sodium, potassium or calciuit hydroxide, with amines such as triethylamine, tributylamine, triethanolamine, pyridine or N,N-dimethylaniline, with carbonates such as sodium, potassium or calcium carbonate or with ion exchangers.
It is possible to use a large number of lipases in the process according to the invention. Microbial lipases from bacteria are preferred, such as lipases from the genera Bacillus or Pseudomonas, e.g. Amano P or the lipase from Pseudomonas spec. DSM 8246, or from fungi such as Aspergillus, or yeasts such as Candida. Further preferred lipases are, for example, the lipases SP 523, SP 524, SP 525, SP 526 and Novozy® 435, which are obtained from fungi such as Humicola, Mucor or Candida antarctica and which are commercially available from Novo Nordisk. It is additionally possible to use the lipases Chirazyme L1, L2, L3, L4, L5, L6, L7 and L8 which are commercially obtainable from Boehringer Mannheim. The lipases can be used in native or immobilized form. The immobilized lipases can be icroencapsulated, emulsified with prepolymers and polymerized, crosslinked with bifunctional substances (oligomers, aldehydes etc.), or bound to inorganic or organic carrier materials such as Celites, Lewatit, zeolites, polysaccharides, polyamides or polystyrene resins. Particularly preferred lipases are Novozy® 435 and Chirazyme L2.
The enzyme-catalyzed racemate resolution can be carried out both in protic or aprotic solvents and without solvent. Examples of suitable solvents are hydrocarbons such as hexane, cyclohexane or toluene, ethers such as diethyl ether, dioxane, methyl tert-butyl ether, tert-amyl methyl ether or THF, nitriles such as acetonitrile, butyronitrile, alcohols such as tert-butanol, 3-methyl-3-pentanol, and halogenated hydrocarbons such as methylene chloride.
The reaction with lipase generally takes place under atmospheric pressure, where appropriate under inert gas such as nitrogen or argon. However, it can also be carried out under elevated pressure.
The temperature for the reaction of ester with the racemic alkoxy-substituted amine is normally from 0 to 90° C., preferably from 10 to 60° C., particularly preferably from 20 to 50° C.
From 0.5 to 2.0 mol, preferably 0.5 to 1 mol, of ester are used per mole of racemic amine. The amount of enzyme required depends on the activity of the enzyme preparation and the reactivity of the amine and can easily be established by preliminary tests. As a rule, from 0.1 to 10% by weight, preferably 1 to 5% by weight, of the immobilized enzyme preparation (based on racemic amine) are used. Novozym® has an activity of about 7000 U/g in the esterification of lauric acid with 1-propanol.
The course of the reaction can easily be followed by conventional methods such as GC or HPLC. When the desired conversion is reached, the reaction is preferably stopped by removing the catalyst, for example by filtering off the (carrier-bound) enzyme. The reaction can also be stopped, for example, by adding enzyme-decomposing substances such as acids or alkalis or by heating. In a continuous procedure, the conversion can be controlled via the loading of the enzyme, i.e. the amount of amine pumped through the enzyme reactor per unit time. The process can preferably be carried out continuously, but it can also be carried out batchwise or semicontinuously.
The enzyme-catalyzed racemate resolution finally results in a mixture of the acylated amine enantiomer, the unreacted amine enantiomer, the alcohol liberated from the ester during the acylation and, possibly, ester employed in excess. Distillation and extraction processes are particularly suitable for separating this mixture. Thus, low-boiling amines can be distilled out of the reaction mixture directly. The amide can subsequently be separated from the alcohol and, where appropriate, ester by distillation or extraction and can then be hydrolyzed in a conventional way with either an acid or base, for example by boiling with sulfuric acid or sodium or potassium hydroxide solution, with racemization or else without racemization. The hydrolysi

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