Research Article: Penicillin Acylase-Catalyzed Effective and Stereoselective Acylation of 1-phenylethylamine in Aqueous Medium using Non-Activated Acyl Donor

Date Published: April , 2010

Publisher: A.I. Gordeyev

Author(s): D.T. Guranda, G.A. Ushakov, V.K. Švedas.



Until recently, the biocatalytic preparation of enantiomerically pure amines was based on
stereoselective acyl transfer in an organic medium using activated acyl donors. The possibility
of performing an effective and enantioselective enzymatic acylation of amines in an aqueous
medium without using activated acyl donors was demonstrated for the first time as the example
of direct condensation of phenylacetic acid and racemic 1–phenylethylamine. Direct
condensation of the acid and the amine took place at mild reaction conditions with a high
initial rate (3.3 µmole/(l·h)), degree of conversion (80% acylation of active amine
enantiomer), and enantioselectivity (enantiomeric excess of the product was more than 95%). The
suggested approach has remarkable advantages compared to enzymatic reactions in organic media
and is of practical value for the biocatalytic preparation of enantiomerically pure compounds
at mild conditions using readily available reagents.

Partial Text

Since enzymes demonstrate such unique characteristics as chemo–, regio– and
stereospecificity, biocatalytic processes have significant advantages as compared to
traditional organic synthesis. The advantages of enzymatic technologies are especially
noticeable during the synthesis of multifunctional or enantiomerically pure compounds. Experts
relate further possibilities of growth in the enantiomerically pure compound market (which has
shown an annual growth rate of more than 13% in the last decade [2]) mainly to the introduction of biocatalytic technologies [1]. The synthesis of enantiomerically pure amines is of
especial interest, since these compounds are important chiral synthones in the pharmaceutical
and agrochemical industries [3].

Reagents. This study used phenylacetyl chloride (Sigma, USA); phenylacetic acid
(Aldrich Chemie, Germany); (R)– and (S)–
α –phenylethylamine (Fluka, Switzerland); phenylmethylsulphonylfluoride, sodium
dodecyl sulphate (Merck, Germany); acetonitrile («CryoChrome», Russia); N
–phenylacetyl derivatives of α –phenylethylamine were synthesized according to
[15]; the penicillin acylase from Alcaligenes
faecalis was supplied by the LLC Innovations and Higher Technologies of MSU (Russia).
The concentration of penicillin acylase active sites was determined according to [16].

Stereoselective acylation is the key step in the resolution of racemic amines, and the use of
enzymes as catalysts of this process seems to be a very promising approach. However, it is
worth noting that primary amines are strongly basic compounds, and that they can be effectively
acylated only in alkaline aqueous solutions (pH approximately 10) or a water–free organic
medium, where the majority of enzymes exhibit diminished catalytic activity and low stability.
Also, conducting enzymatic reactions in organic solvents requires the use of activated acyl
donors, which can spontaneously acylate reactive amino groups in a non–stereoselective
manner, thus lowering the enantiomeric purity of the reaction product [3, 9]. We suppose that these drawbacks
can be circumvented by conducting the enzymatic acylation of amines in an aqueous medium using
the direct condensation of the carboxylic acid and the amine. In this case, thermodynamically
favorable conditions for the condensation reaction are achieved in a practically neutral medium
(pH approximately 6–8), where most enzymes are highly active and stable. Both reacting
substrates are highly soluble and stable at these conditions, which allow the use of highly
concentrated solutions of the reagents and creates favorable thermodynamics for enzymatic amine
acylation. The driving force of the reaction, which makes the whole process so efficient, is
the shift of equilibrium towards synthesis, caused by the precipitation of the poorly soluble
reaction product. Notably, direct condensation does not require activation of the acyl donor,
which simplifies the enzymatic amine acylation process and reduces costs. The wide substrate
specificity and stereoselectivity of penicillin acylases towards N–acylated derivatives
of amino compounds [16, 19, 20] provides reason to hope that
enzymatic amine acylation via direct condensation and using this family of enzymes will be

This is the first report on the possibility of effective and enantioselective enzymatic
acylation of primary amines in an aqueous medium without the use of activated acyl donors.
Direct condensation catalyzed by penicillin acylase is highly efficient, and the target product
of high enantiomeric purity can be easily isolated from the reaction mixture with a high yield.
This method can be used for preparative biocatalytic synthesis of enantiomerically pure amines
under mild reaction conditions using readily available substrates and a biocatalyst.