Resolutions with amidases

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Avecia identified approximately 60 microorganisms with amidase activity capable of resolving racemic amines [17, 18]. Arthrobacter species predominated in the list of microorganisms identified. The kinetic resolution of N acetyl 1 aminoindanol 35 by a freeze dried microbial sample (BH2 NI amidase) allowed access to (1S,2R) N acetyl 1 aminoindanol 35 in high enantiomeric excess (96%). This compound is a key intermediate in the synthesis of Merck s HIV protease inhibitor Crixivan 37 (indin avir) (Figure 14.12). 

Several methods to resolve racemic mixtures of a- amino acids have been worked out, including separation of diastereomeric salts by crystallization or amides by chromatography. Chiral HPLC on phases carrying d- and L-pro-line-copper complexes has been scaled up to 20 g quantities of amino acid race-mates. Resolution with immobilized amidases, which deacetylate only L-amino acid acetamides, and subsequent precipitation of the D-amino acid acetamides work on a 500-kg scale. All kinds of labels ( C, D) can thus be introduced... 

Scheme 6.7 Easy-on/easy-off resolution of amines with penicillin G amidase.

An example of a very efficient asymmetric transformation is the preparation of (W)-phcnylgly-cine amide (Scheme 7.8) (see also Chapter 25).40 This offers a good alternative to the enzymatic resolution of (fCS )-phcnylglycinc amide with the (S)-specific amidase from Pseudomoms putida.41 This amide is used in a coupling process for semi-synthetic antibiotics.42... 

DSM developed a slightly different approach towards enantiopure amino acids. Instead of performing the Strecker synthesis with a complete hydrolysis of the nitrile to the acid it is stopped at the amide stage. Then a stereoselective amino acid amidase from Pseudomonas putida is employed for the enantioselective second hydrolysis step [83], yielding enantiopure amino acids [34, 77, 78]. Although the reaction is a kinetic resolution and thus the yields are never higher than 50% this approach is overall more efficient. No acylation step is necessary and the atom efficiency is thus much higher. A drawback is that the racemisation has to be performed via the Schiff s base of the D-amide (Scheme 6.23). 

Recently it was reported that an a-amino-e-caprolactam racemase from Achro-mobacter obae can racemise a-amino acid amides efficiently. In combination with a D-amino acid amidase from Ochrobactrum anthropi L-alanine amide could be converted into D-alanine. This tour de force demonstrates the power of the racemase [84]. If racemic amide is used as a starting material the application of this racemase in combination with a d- or L-amidase allows the preparation of 100% d- or L-amino acid, a dynamic kinetic resolution instead of DSM s kinetic resolution (Scheme 6.24). 

At Bristol-Myers Squibb amidases were employed for the enantioselective preparation of new potential / -3-receptor agonists [62]. A kinetic resolution of the starting amides was achieved with very good to excellent enantioselectivities (Scheme 6.29). As already mentioned above, whole cells were used for these transformations. 

The starting material for the acylase process is a racemic mixture of N-acetyl-amino acids 20 which are chemically synthesized by acetylation of D, L-amino acids with acetyl chloride or acetic anhydride in alkaU via the Schotten-Baumann reaction. The kinetic resolution of N-acetyl-D, L-amino acids is achieved by a specific L-acylase from Aspergillus oryzae, which only hydrolyzes the L-enantiomer and produces a mixture of the corresponding L-amino acid, acetate, and N-acetyl-D-amino acid. After separation of the L-amino acid by a crystallization step, the remaining N-acetyl-D-amino acid is recycled by thermal racemization under drastic conditions (Scheme 13.18) [47]. In a similar process racemic amino acid amides are resolved with an L-spedfic amidase and the remaining enantiomer is racemized separately. Although the final yields of the L-form are beyond 50% of the starting material in these multistep processes, the effidency of the whole transformation is much lower than a DKR process with in situ racemization. On the other hand, the structural requirements for the free carboxylate do not allow the identification of derivatives racemizable in situ therefore, the racemization requires... 

Several multi-ton industrial processes still use enzymatic resolution, often with lipases that tolerate different substrates. BASF, for example, makes a range of chiral amines by acylating racemic amines with proprietary esters. Only one enantiomer is acylated to an amide, which can be readily separated from the unreacted amine. Many fine chemicals producers also employ acylases and amidases to resolve chiral amino acids on a large scale. l-Acylases, for example, can resolve acyl d,l-amino acids by producing the I-amino acids and leaving the N-acyl-l-amino acid untouched after separation, the latter can be racemized and returned to the reaction. d-Acylase forms the alternative product. Likewise, DSM and others have an amidase process that works on the same principle d,l-amino acid amides are selectively hydrolyzed, and the remaining d-amino acid amide can be either racemized or chemically hydrolyzed. 

In summary, a broad range of large-scale applicable biocatalytic methodologies have been developed for the production of L-amino acids in technical quantities. Among these industrially feasible routes, enzymatic resolutions play an important role. In particular, L-aminoacylases, L-amidases, L-hydantoinases in combination with L-carbamoylases, and /l-lactam hydrolases are efficient and technically suitable biocatalysts. In addition, attractive manufacturing processes for L-amino acids by means of asymmetric (bio-)catalytic routes has been realized. Successful examples are reductive amination, transamination, and addition of ammonia to rx,/fun-saturated carbonyl compounds, respectively. 

Enzymatic Resolution of Racemic a-Methyl Phenylalanine Amides. The chiral amino acids (22) and (23) (Fig. 6A) are intermediates for the synthesis of (33-receptor agonists (30,31). These are available via the enzymatic resolution of racemic a-methyl phenylalanine amide (24) and a-methyl-4-methoxy-phenylalanine amide (25), respectively, by an amidase from Mycobacterium neoaurum ATCC 25795 (32). Wet cells (10% wt/vol) completed the reaction of amide (24) in 75 min with a... 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

Recently we determined that two R. rhodochrous strains (A29 and A99) expressed nitrilase activity after induction. These strains were capable of enantioselectively hydrolyzing racemic 3-amino-3-phenylpropanenitrile directly to the corresponding (R)-3-amino-3-phenylpropanoic acid with >95% ee (Table 14.1) in a kinetic resolution. Various inhibitors were used, that indicated the observed hydrolytic activity was due to the presence of a nitrilase rather than a nitrile hydratase and amidase pair [47]. 

In the kinetic resolution of amino acid amides with the use of amidases, such as DAP and DaaA, it is possible to synthesize D-amino acids by kinetic resolution, selectively from racemic acid amides [16]. An Escherichia coli transformant highly expressing DAP catalyzed the synthesis of 2.5M (about 220g/l) D-alanine from 5M racemic alanine amide in a 4.5-h reaction, d-2-Amino butyric acid, D-methionine, D-norvaline, and D-norleucine were S5mthesized in a similar manner. We have been successful in the evolution of DAP [17] and DaaA by mutations [18]. 

In this chapter we describe the DSM aminoamidase processes in more detail. Three different enzymatic resolution routes have been developed for the preparation of natural and synthetic amino acids using biocatalysts from different origin, i.e.. Pseudomonas pu-tida, Mycobacterium neoaurum, and Ochrobactrum anthropi. Scope and limitations and enzyme characterization of these amidases will be presented together with some specific examples. In addition, the use of some of these amino acids in peptide sjmthesis, catalytic asymmetric synthesis, and further synthetic transformations will be given. 

A remarkable feature of the O. anthropi biocatalyst is its relaxed pH profile [52]. Although the amidase displays its highest activity at pH 8.5, still 55% of this activity is retained at pH 5.0. This enables the hydrolysis of substrates, which are only very poorly soluble at weakly alkaline conditions, by just performing the hydrolysis reaction at slightly acidic conditions (e.g., see Secs. V.A and V.B). Another attractive feature of the O. anthropi L-amidase is its very good temperature stability. Even a preincubation at 50°C for 2 h, does not lead to a decrease in activity. Therefore, the resolution reactions with O. anthropi whole cells are optimally performed at 50°C and at a pH value between 5 and 8.5. 

Naproxen, (S)-2-(6-methoxy-2-naphthyl)propanoic acid 126 is a nonsteroidal anti-inflammatory and analgesic agent first developed by Syntex [220,221]. Biologically active desired S-naproxen has been prepared by enantioselective hydrolysis of the methyl ester of naproxen by esterase derived from Bacillus subtilis Thai 1-8 [222]. The esterase was subsequently clone in Escherichia coli with over 800-fold ipcrease in activity of enzyme. The resolution of racemic naproxen amide and ketoprofen amides has been demonstrated by amidases from Rhodococcus erythropolis MP50 and Rhodococcus sp. C311 (223-226). 5-Naproxen 126 and 5-ketoprofen 127 (Fig. 44) were obtained in 40% yields (theoretical maximum yield is 50%) and 97% e.e. Recently, the enantioselective esterification of naproxen has been demonstrated using lipase from Candida cylindraceae in isooctane as solvent and trimethylsilyl as alcohol. The undesired isomer of naproxen was esterified leaving desired S isomer unreacted [227]. 

The enantiospecific Brevibacterium amidase was characterized as a typical homodimer consisting of two 49-kDa subunits with sequence homologies to other amidases, and the corresponding gene was cloned and overexpressed in E. coli [76,77]. Its additional (/ )-specific activity toward several 2-aryl- and 2-aryloxypropionamides was used for the racemic resolution of 2-(4-hydroxyphenoxy)propionamide (Fig. 23). 

Compared to a maximal bioconversion yield of 50% in racemic resolutions, asymmetric syntheses have a theoretical yield of 100%. Such an ideal yield was nearly reached with another R. rhodochrous ATCC 21197, which transformed disubstituted malononitriles such as butylmethylmalononitrile to the corresponding (7 )-amide carboxylic acid with high enantiomeric excesses and yields [80]. The reaction proceeded via a fast, nonstereospecific hydration of the starting dinitrile followed by a slow, enantioselective hydrolysis of the diamide intermediate by the amidase (Fig. 27). 

The presence of esterases or amidases in many microorganisms has also been employed to resolve racemic mixtures of the esters or amides of these drugs. In this sense, Kluyvera oxytoca SNSM 87 [74] and Bacillus subtilis [75] esterases have been successfully used in die resolution of the racemic esters, while Rhodococcus erythopolis MP50 has been applied for the resolution of the amides of (/ ,5 -2-(6-methoxy-2-naphthyl)propionic acid, yielding 48% yield of naproxen, with an enantiomeric excess more than 99% after 45 h [76]. 

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