Cambrex.

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Chiral chemistry

Aminotransferase for commercial

Dr Gerard Scarlato of Cambrex shares two case studies in a key chiral technology

C
ambrex has been involved in the production of chiral
95%
amines and the use of aminotransferase (transaminase)
enzymes for the past 15 years. This involvement has
obviously paralleled the fact that the demand for APIs contain-
ing a chiral amine moiety is on the rise.
This article describes some fundamental properties of our
aminotransferase enzymes and how they are employed in two
5%
case studies. These examples provide a clear demonstration of
the commercial capability and implementation of aminotrans-
ferase at Cambrex within its facilities in the US and Europe.
One primary feature of using these enzymes is that they can
be used in two different modes: synthesis and resolution (Figure
1). Synthesis mode, which was used in both of the case studies
discussed here, is a more direct and productive route to the chi-
ral amine and is generally more desirable in cases where the
starting material ketone is expensive.
Resolution mode, by contrast, is best used when the starting
material ketone or racemic amine is inexpensive and readily
available. This process competes directly with classical resolution
methods and has some analogous limitations to the traditional
classical resolution.
However, resolution mode is probably most powerfully
used in a process that Cambrex calls enantiomeric enrich-
ment or polishing. In this, the ee of the desired chiral amine Figure 2 - (top) Example of
is increased by converting the undesired amine enantiomer in enantiomeric enrichment
the mixture to the corresponding pro-chiral ketone; thus
Figure 3 - (above)
enhancing the remaining presence of the desired amine Manufacture of
enantiomer. dextroamphetamine using
Figure 2 gives an example of enantiomeric enrichment. aminotransferase
technology
Here, pyruvic acid acts as an exclusive amine acceptor to the
minor (5%) benzylamine isomer present in the mixture, thus
generating the pro-chiral, p-methoxyphenyl methyl ketone.
One of the key advantages to this purification process over
classical resolution is that the undesired enantiomer is eliminat-
ed. This method could potentially make it possible to obtain
product with higher chiral purity than competitors products,
thus potentially establishing a higher quality API and petitioning
for tighter specification for chiral purity in USP and EP mono-
graphs.
After fermentation, the enzyme is supplied as a whole-cell,
spray-dried powder and exhibits general activities typically in
the range of 20-30 moles/minutes/gram. Using a whole-cell
powder instead of a highly purified enzyme has the advantage Figure 1 - Synthesis (a) &
of supplying the enzyme in a cost-efficient manner. resolution (b) modes
Synthesis mode
NH2 R-enzyme O S-enzyme NH2
a
R1 R2 R1 R2 R1 R2
Resolution mode
NH2 R-enzyme NH2 S-enzyme NH2
b The amine donor in the reaction is isopropylamine, an inex-
R1 R2 R1 R2 R1 R2
+ +
O O
R1 R2 R1 R2
NH2 95% NH2
O O
Aminotransferase
90% ee Pyruvic acid 100% ee
NH2 5% O
O O
100% ee
O NH2
Phenylacetone d-amphetamine
Aminotransferase
H2N O
Isopropylamine Acetone
Cambrex provides a range of enzymes that can convert
ketones nearly quantitatively to either amine enantiomer in a
one-step process. The enzyme activity is determined by quanti-
fying the rate of formation of a product, such as the conversion
of (S)--methylbenzylamine to acetophenone.
This value is critical for the optimisation of the reaction per-
formance and yield. As such, activity-based enzyme charges
have been employed to minimise enzyme use and allow for
more consistent product yields and a reduced cost of manufac-
turing. In addition to these properties and techniques and, in
the absence of any clever molecular biological technology,
Cambrex has been able to optimise the ability of these
enzymes using standard molecular biological tools.
The first case study (Figure 3) concerns the commercial man-
ufacture of dextroamphetamine, (d-amphetamine) using this
biocatalytic process. This amine product is formulated as the
sulphate or saccharate salt.
Both d-amphetamine and its ketone starting material, pheny-
lacetone, are Schedule II controlled substances subject to DEA
regulation and tight control over inventories. The product is
commercially manufactured on a multi-tonne scale under
cGMP conditions and is isolated as a purified liquid with titra-
tion assays of 100% while exhibiting ees of >98%.
The process is one-step in synthesis mode, with no racemi-
sation occurring during the reaction or work-up. No resolution
is required to increase the chiral purity of the final product
which minimises equipment, process time and the yield loss
associated with the resolution, the epimerisation of an unde-
sired isomer and the recycling of these streams back into a
process in order to increase yields.
pensive and readily available amine that is converted to acetone
in the process. Reaction concentrations in excess of 1.5 M iso-
propylamine help drive the equilibrium reaction to near com-
pletion. Both isopropylamine and the resultant ketone (acetone)
are non-hazardous and readily tolerated in wastewater treat-

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 Cambrex.qxp 3/6/09 13:50 Page 57

Chiral chemistry

mercial chiral amine manufacture

ment facilities. The resultant product is distilled from the bio-
O O O NH2
mass and isolated in good yields.
The second case study (Figure 4) concerns the production of
(S)-7-methoxy-2-aminotetralin (7-MAT), an intermediate in sev-
eral commercially important APIs. Cambrex has manufactured 7-methoxy-2-tetralone 7-methoxy-2-aminotetralin
it at multi-tonne scale using the aminotransferase technology Aminotransferase
and it is isolated as the HCl salt, with ees of >99%.
The transformation is a single step reaction executed in
water. The process is run at elevated temperature (~55C) to H2N O
accelerate the reaction. A more thermostable enzyme was
developed through standard error-prone PCR techniques for Isopropylamine Acetone
this process in order to achieve this elevated, temperature-sta-
ble condition in the enzyme. Both distillation and extraction Figure 4 - Production of 7- these two series of compounds. Cambrex plans to produce
processes were used to isolate the product, which is isolated in MAT using aminotransferase other APIs for commercial use that are amenable to this tech-
technology
approximately 70% yield as an HCl salt. nology for products of their own as well as in conjunction with
The utility of this class of enzymes is not limited to the sub- clients products through contract services.
strates of amphetamines and aminotetralins. These substrates
tolerate a wide range of diversity from pure aliphatic ketones to For more information, please contact:
heteroatom-substituted aromatic ring systems. In many cases, Dr Gerard R. Scarlato
>99% ees were observed upon analysis by chiral HPLC. Associate Director Sales & Business Development
Cambrex Pharmaceutical Products & Services
Conclusion 1 Meadowlands Plaza
The use of aminotransferases is demonstrated at commercial East Rutherford, NJ 07073, USA
Tel: +1 858 481 3565
scale in two examples for the commercial manufacture of tonne
E-mail: gerald.scarlato@cambrex.com
quantities of amphetamines and aminotetralins. The diversity of Website: www.cambrex.com
the enzyme substrates accepted are varied and not limited to

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