Biocatalytic Friedel-Crafts alkylation using non-natural cofactors

Article abstract of DOI:10.1002/anie.200905095

A novel biocatalytic protocol for C -C bond formation is described and is an equivalent to Friedel-Crafts alkylation. S-Adenosyl-L-methionine (SAM), the major Chemical Equation Presentation methyl donor for biological methylation catalyzed by methyltransferases (Mtases), can perform alkylations (see scheme). These enzymes can accept non-natural cofactors and transfer functionalities other than methyl onto aromatic substrates

Full text of DOI:10.1002/anie.200905095

Communications
DOI: 10.1002/anie.200905095
Enzymatic Synthesis
Biocatalytic Friedel–Crafts Alkylation Using Non-natural Cofactors**
Harald Stecher, Martin Tengg, Bernhard J. Ueberbacher, Peter Remler, Helmut Schwab,
Herfried Griengl, and Mandana Gruber-Khadjawi*
À
The formation of C C bonds is a central aspect of
synthetic organic chemistry. However, in biocatal-
ysis only few enzymes capable to perform this
reaction are known, among which aldolases, trans-
ketolases, and hydroxynitril lyases have been
investigated thoroughly.[1] Some have even found
their way into industrial applications.[2]
Friedel–Crafts alkylation is a classic organic
reaction of great importance. However, in partic-
ular for large scale application, this transformation
is ecologically very critical and regiospecific mono-
alkylation is difficult to achieve. Therefore, an
environmentally friendly and selective alternative
would be highly desirable.
In nature methyl groups are selectively intro-
duced into reactive aromatic rings by methyltrans-
ferases (Mtases), in particular with S-adenosyl-l-
methionine (SAM) as the cofactor. Furthermore,
enzyme-catalyzed reactions are important for
access to isoprenoids. Also, prenylation of aro-
matic rings has been performed.[3] For phenyl-
alanine ammonia lyases a Friedel–Crafts-type
mechanism has been proposed.[4]
Recently, it has been shown that besides the
methyl group other alkyl, alkenyl, and alkinyl
groups can be introduced into S-adenosyl-l-homo-
Scheme 1. C-Mtases involved in the biosynthesis of the antibiotics coumermycin A1
in Streptomyces rishiriensis and Novobiocin in Streptomyces spheroides.
cystein. These modified cofactors of transferases
were used for a sequence-specific alkylation of
DNA.[5]
Having cofactor and modified cofactors in hand, we
investigated the possibility of alkylation of aromatic sub-
strates, thus transferring the biosynthesis into the laboratory
(Scheme 1).
[*] Dipl.-Ing. H. Stecher, M. Sc. M. Tengg, Dr. B. J. Ueberbacher,
Dr. P. Remler, Prof. Dr. H. Griengl, Dr. M. Gruber-Khadjawi
Kompetenzzentrum Angewandte Biokatalyse
Petersgasse 14, 8010 Graz (Austria)
Aminocoumarins are antibiotics produced by some Strep-
tomyces species and are targets for the methyl transfer from
the natural cofactor SAM. The Mtase A and B are involved in
Fax: (+43)316-873-8740
E-mail: mandana.gruber@a-b.at
[7]
the biosynthesis[6] of the antibiotics coumermycin A1 (pro-
duced by Streptomyces rishiriensis) and novobiocin[8] (pro-
duced by Streptomyces spheroides; Scheme 1).
Dipl.-Ing. H. Stecher, Dr. M. Gruber-Khadjawi
Institut fꢀr Organische Chemie, Technische Universitꢁt Graz
Stremayrgasse 16, 8010 Graz (Austria)
SAM analogues were synthesized by modifying the
ˇ
strategy published by Klimasauskas, Weinhold, and co-work-
M. Sc. M. Tengg, Prof. Dr. H. Schwab
Institut fꢀr Molekulare Biotechnologie, Technische Universitꢁt Graz
(Austria)
ers.[9] S-Adenosyl-l-homocysteine (SAH) was alkylated by
seven different alkyl bromides using formic acid as the solvent
and AgOTf as a Lewis acid activator and catalyst. We
observed quantitative conversion in less than 2 days (average
reaction time 24 h; Table 1). The chemical synthesis of SAM
analogues results in approximately 1:1 diastereomeric mix-
tures at the sulfonium center. In the first screenings the
diastereomers were separated by preparative HPLC and used
as cofactors for the alkylation of coumarin compound 3a.
Both epimers were accepted by the enzymes NovO and CouO
[**] The ꢂsterreichische Forschungsfꢃrderungsgesellschaft (FFG), the
Province of Styria, and the Styrian Business Promotion Agency
(SFG)—within the framework of the Kplus programme as well as
GASS programme within NAWI Graz—are acknowledged for
financial support, as well as Birgit Krenn for technical support.
M.G.-K. thanks Prof. Dr. Rolf Breinbauer for his fruitful contribution
to this manuscript.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905095.
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Angewandte
Chemie
Table 1: Synthesis of SAM analogues. Tf=trifluoromethanesulfonyl.
(Mtase A and B) with only slight difference in the conversion
after 24 hours. Therefore, the tedious epimer separation was
not undertaken. For all following experiments the crude
diastereomeric mixture of SAM analogues were performed
without further purification and separation of the diastereo-
mers.
The methyltransferase gene novO found in Streptomyces
spheroides[10] shows higher sequence homology to couO,
which is the methyltransferase gene in Streptomyces rishir-
iensis[11] (84% identity on protein level). These Mtases were
heterologously expressed in E. coli. Both enzymes were
purified as Strep-tagged proteins by affinity chromatography.
NovO shows a KM value of 26.7 mm for substrate 3b and the
KM value of CouO for substrate 3a is 64.4 mm. The activity
assay is based on HPLC/MS analysis of the enzymatic
transformation. For the activity screening model substrates
with high similarity to the natural substrates of these enzymes
were chosen and synthesized (e.g. 3a and 3b; see the
Supporting Information).
Alkyl halide
R1
Product
1a
1b
1c
1d
1e
1 f
CH3
CH2 CHCH2
CH3CH CHCH2
CH CCH2
CH3C CCH2
C6H5CH2
2a
2b
2c
2d
2e
2 f
=
=
ꢀ
ꢀ
formed on a larger scale (30 mg substrate, 3 mm solution) and
the products were isolated. At first coumarin derivatives were
chosen as substrates and the results are given in Table 2.
The methylation of coumarin 3a is not surprising owing to
the high structural similarity to the natural substrate. How-
ever, besides methyl other alkyl groups can be introduced
The biocatalytic transformations were performed in small
shaked vials on a 1 mL scale (1 mm substrate) using the crude
lysate of the expression in E. coli in the presence of 10%
DMSO (dimethyl sulfoxide) at 358C for 24 hours (see the
Supporting Information). Some of the reactions were per-
Table 2: Enzymatic synthesis of alkyl coumarin derivatives.
Substr. Product
NovO[a] CouO[a]
Substr. Product
NovO[a] CouO[a]
Substr. Product NovO[a] CouO[a]
2a 3a 4a:
R1 =CH3
>99
>99
42
>99
>99
38
2a 3b 4b:
R1 =CH3
>99
96
96
42
11
11
–
SAM 3c 4c:
R1 =CH3
SAM 3d 4d:
R1 =CH3
SAM 3e 4e:
R1 =CH3
SAM 3 f 4 f:
R1 =CH3
>99
>99
–
>99
>99
30
2b 3a 4a:
2b 3b 4b:
1
1
=
=
R =CH2 CHCH2
R =CH2 CHCH2
2c 3a 4a:
2c 3b 4b:
30
1
1
=
=
R =CH3CH CHCH2
2d 3a 4a:
R =CH3CH CHCH2
2d 3b 4b:
99
99
35
–
10
1
1
ꢀ
ꢀ
R =CH CCH2
2e 3a 4a:
R =CH CCH2
2e 3b 4b:
41
77
28
1
1
ꢀ
ꢀ
R =CH3C CCH2
2 f 3a 4a:
R1 =C6H5CH2
R =CH3C CCH2
2 f 3b 4b:
R1 =C6H5CH2
40
45
24
21
[a] % conversion was determined by HPLC analysis.
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9547

Communications
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possible. These transformations are interesting because they
are the first examples of biocatalytic Friedel–Crafts alkyla-
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Scheme 2. Methylation products of naphthalenediols catalyzed by
CouO (% conversion after 24 h).
There seem, however, to be quite stringent structure
requirements since naphthalenediols with a 1,2-, 1,3-, 1,4-,
1,5-, 1,6-, or 2,3-substitution pattern gave no reaction. In
addition, resorcinol and phloroglucinol were not accepted.
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modified cofactors and yield not only methylated but also
allyl, propargyl, and benzylated arenes in moderate to high
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may serve as the beginning of a “green” and selective Friedel–
Crafts alkylation.
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Received: September 11, 2009
Revised: October 7, 2009
Published online: November 6, 2009
À
Keywords: biocatalysis ·C Ccoupling·Friedel–Craftsalkylation·
S-adenosyl-l-methionine · sustainable chemistry
.
[1] a) M. Gruber-Khadjawi, T. Purkarthofer, W. Skranc, H. Griengl,
Adv. Synth. Catal. 2007, 349, 1445 – 1450; b) T. Purkarthofer, K.
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