The use of biocatalysis in the synthesis of labelled compounds

Article abstract of DOI:10.1002/jlcr.1388

Since most biotransformations are highly chemo-, regio- and stereoselective, they offer many opportunities for the synthesis of compounds that are not easily attainable by classical organic chemistry, The catalytic properties of the cytochrome P450 (CYP45

Full text of DOI:10.1002/jlcr.1388

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 342–346.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1388
Short Research Article
The use of biocatalysis in the synthesis of labelled
compoundsy
´
´
JOHN ALLEN, DENIS M. BRASSEUR, BEATRICE DE BRUIN, MIREILLE DENOUX, SERGE PERARD, NICOLAS PHILIPPE
and SEBASTIEN N. ROY*
´
´
Isotope Chemistry and Metabolite Synthesis Department, Sanofi-Aventis Recherche et Developpement, 1 avenue Pierre Brossolette, 91385 Chilly
´
Mazarin Cedex, France
Received 25 August 2006; Revised 10 April 2007; Accepted 16 April 2007
Abstract: Since most biotransformations are highly chemo-, regio- and stereoselective, they offer many opportunities
for the synthesis of compounds that are not easily attainable by classical organic chemistry, The catalytic properties
of the cytochrome P450 (CYP450) enzymes of microorganisms can be exploited to produce suitable N-, S- or O-
dealkylated precursors, which can be realkylated with the appropriate labelled reagent. These enzymes are also well
reported for hydroxylation of activated or nonactivated carbon centres, to produce drug metabolites. Nevertheless, to
use such enzymes, a whole cell system is preferred due to the need for cofactor regeneration. Nitrile hydrolysing
enzymes also attract attention, due to their ability to selectively hydrolyse nitrile derivatives under mild conditions.
The scope of such biotransformations is discussed. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: biocatalysis; biotransformation; microorganisms; CYP450; nitrilase; nitrile hydratase; isotope labelling
Introduction
bial model for drug metabolism’.1 Biotransformations
can, therefore, be useful for the preparation of labelled
drug metabolites, when the labelled parent compound
is available.
Enzymes are well-known catalysts for specific chemical
reactions, and their reactions offer excellent regio-,
chemo-, stereo- and substrate selectivity. They are now
commonly used in organic chemistry, especially as
isolated enzymes such as the commercially available
hydrolases.
Nitrile derivatives are common precursors of 14C-
labelled compounds, but their functionalization by
hydrolysis often requires harsh conditions incompatible
with other functional groups of the molecule. In these
cases, hydrolysis under mild conditions is necessary. A
family of bacteria called Rhodoccocus have long been
known for their ability to hydrolyse nitrile-containing
compounds at neutral pH under mild, aqueous condi-
tions. This methodology allows the preparation of classes
of 14C-labelled molecules, which are otherwise difficult to
obtain under classical organic hydrolysis conditions.
However, biocatalytic systems can operate in two
ways: either as isolated enzymes or as whole cell
systems, depending on the transformation required.
For instance, if cofactor regeneration is necessary or
the enzyme involved presents a relative instability,
whole cell systems are usually preferred. In this way,
cytochrome P450 (CYP450) enzymes can be exploited to
catalyse selective N-, O- and S-dealkylation of complex
structures. These precursors can then be realkylated
with the desired labelled alkyl group.
Results and discussion
The CYP450 enzymes of microorganisms are also well
described for their metabolizing ability, as ‘the micro-
From complex substrates, selectively O-dealkylated
compounds can be interesting precursors for the
synthesis of labelled compounds. Such dealkylation
reactions can be catalysed under mild conditions by
CYP450, in a culture broth. SR121960 1 was deethy-
lated using a culture of the fungus Cunninghamella
echinulata LCP73.2203 in a fermenter (7 L broth). After
extraction of the broth with ethyl acetate and purifica-
*Correspondence to: Se´bastien N. Roy, Isotope Chemistry and Meta-
bolite Synthesis Department, Sanofi-Aventis Recherche et De´veloppe-
ment,
1 avenue Pierre Brossolette, 91385 Chilly Mazarin Ce´dex,
France. E-mail: sebastien.roy@sanofi-aventis.com
y
Proceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Copyright # 2007 John Wiley & Sons, Ltd.

USE OF BIOCATALYSIS 343
Scheme 1
tion by flash chromatography on silica gel, 1.2 g of the
desired phenol derivative 2 was produced. The yield
was 60% at 0.3 g/L as the initial concentration of
starting material 1. The phenol function was protected
with a TIPS group before coupling the benzene sulpho-
nyl chloride derivative SR122037 to afford compound 3
with 45% yield in two steps. After the removal of the
TIPS protecting group with TBAF, tetravinyltin was
used to vinylate the phenolic group and to obtain the
unsaturated tritiation precursor 4. The double bond of
precursor 4 was successfully tritiated using tritium gas
to give [3H2]-SR121463 5 with a specific activity of
56 Ci/mmol. It should be noticed that attempts to
directly dealkylate SR121463 failed (Scheme 1).
large amount of the parent drug 6 was recovered and a
desethyl amide compound was also produced during
the fermentation. Compound 7 was thus purified by
preparative HPLC after extraction of the broth with
ethyl acetate. Nevertheless the yield remained low
(15%, at 0.3 g/L as the initial concentration of the
starting material 6), but we were able to prepare this
interesting precursor 7 in one step.
Model compound 7 was successfully remethylated
using methyl iodide and sodium hydride in DMF for
10 min, in order to confirm that compound 7 could be
used as a PET precursor.
In 1974, Smith and Rosazza1 first introduced the
concept of using microorganisms as ‘microbial models
for mammalian metabolism.’ While conducting micro-
bial hydroxylation of aromatic substrates, they noticed
a similarity between microbial metabolites and those
obtained from mammalian systems. In pharmacoki-
netic and metabolism laboratories, Midazolam is used
to phenotype hepatic CYP3A activity. Formerly Mid-
azolam clearance was used as a marker, but now, the
monitoring of the formation of the major metabolite of
This dealkylation–realkylation strategy can also be
used to prepare interesting precursors for PET studies,
as illustrated in Scheme 2.
A strain of fungus Mortierella isabellina MMP108 was
selected for its ability to selectively monodemethylate
the compound SSR126768 6. Two hundred mg of the
desired phenol 7 was produced using a 7-day long fed
batch culture of the fungus in a fermenter (7 L broth). A
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 342–346
DOI: 10.1002.jlcr

344 J. ALLEN ET AL.
Scheme 2
Scheme 4
Scheme 3
several collection of microorganisms (ATCC, DSM,
NBRC, NCIMB, etc.). Furthermore, purified enzymes
are now commercially available (Biocatalytics).
Midazolam is the preferred method. So, for this mass
spectrometry-based bioanalytical assay, stable-isotope
labelled 10-hydroxymidazolam is required (Scheme 3).
Since the parent [13C4]-labelled drug 8 was available,
it was directly converted to its [13C4]-labelled metabo-
According to the literature, these enzymes can operate
in a chemo4-, regio5- and stereo6-selective way. The
chemoselective hydrolysis was successfully applied to
several substrates (10, 12, 14) containing a methyl ester
group. In the case of the hindered nitrile 16 no strain or
enzyme was able to perform the reaction (Scheme 5).
When using the whole cell system, the strain was
grown on a specific medium containing an inducer of
the activities. The cells were then harvested by cen-
trifugation and re-suspended in a phosphate buffer at
pH 7. The substrate, solubilized in a minimum amount
of ethanol, was added to the mixture and the reaction
was monitored by LC-UV. After complete conversion,
the cells were harvested by centrifugation and the
supernatant was acidified to pH 2 and extracted with
ethyl acetate. No further purification was necessary.
This methodology was successfully applied to 14C-
labelled molecule 18 (Scheme 6).
lite
9 using a culture of Beauveria sulfurescens
ATCC7159 in 30% yield.
This example demonstrates that bioconversion can
be an interesting alternative route to synthesize
labelled metabolites.
Nitrile hydrolysing enzymes
K14CN is the most commonly used radiolabelled starting
material for organic synthesis. In most cases, the synth-
esis of a radiolabelled compound requires the hydrolysis
of a CN derivative. Classical hydrolysis often requires
harsh conditions in terms of pH and temperature. Some
other functions in the molecule are often affected by this
reaction, leading to degradation or undesirable side
products. A nitrile hydrolysis system operating under
mild conditions would hence be very useful.
Conclusion
Enzyme-catalysed hydrolysis of nitriles has been
shown to proceed by either direct transformation to
carboxylic acids with a nitrilase2 or via the correspond-
ing amide mediated by a nitrile hydratase/amidase
system3 (Scheme 4). A range of nitrile hydrolysing
microorganisms has been isolated and is available from
We have demonstrated that biocatalysis is an effi-
cient tool for the preparation of a range of valuable
precursors of labelled molecules. This methodology
allows an access in one step to intermediates, which
could have been difficult or impossible to obtain in a
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 342–346
DOI: 10.1002.jlcr

USE OF BIOCATALYSIS 345
Scheme 5
2. (a) Harper DB. Biochem Soc Trans 1976; 4: 502–
504; (b) Harper DB. Biochem J 1977; 165: 309–319;
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Agric Biol Chem 1982; 46: 1175–1181.
Scheme 6
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341–344.
classical chemical approach. It is especially useful at
the small scales often required in labelled compound
synthesis.
5. (a) Yokoyama M, Sugai T, Ohta H. Tetrahedron Asym
1993; 4: 1081–1084; (b) Kerridge A, Parratt JS,
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Acknowledgement
We are grateful to Elisabeth Kauffmann for analytical
support.
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 342–346
DOI: 10.1002.jlcr