W. Kroutil et al.
lett Packard 6890 equipped with FID and a HP Mass Selective Detector
5
973 attached with a HP 5 MS capillary column (30 m, 0.25 mm, 0.25 mm
film) and on an Agilent 7890A equipped with FID and a Mass Selective
Detector 5975C attached with an Agilent 19091S-433 capillary column
(
30 m, 0.25 mm, 0.25 mm film). Helium was used as a carrier gas. CD
spectra were recorded on a Jasco J-715 spectropolarimeter. Reactions
[
20]
2
under O pressure were performed as previously described.
Substrates and enzymes: Substrates and reference materials were pur-
chased from Aldrich, Lancaster and Acros with the highest purity avail-
able. The peroxidases tested were: 1) Novozyme 51004, 2) Peroxidase
from Coprinus cinereus (produced by Aspergillus oryzae), 3) Novozymes
OON00008, 4) Baylase assist, peroxidase from Coprinus cinereus, Biester-
feld Chemiehandel GmbH & Co, Germany, 5) P8250 Peroxidase Typ II
from horseradish, Sigma–Aldrich, 031K74711, 6) P8125–5KU Peroxidase
Figure 6. pH profile for the alkene cleavage of t-anethole (1a) with
hemine. t-Anethole: 6 gLÀ1, hemin chloride: 0.28 mmol%, 228C,
1
70 rpm, 5 h.
Typ I
from
horseradish,
Sigma–Aldrich,
031K7465,
7) HRP:
POD10814407001, Roche, lot.:93396221, 8) HRP: POD10108090001,
Roche, lot.:93350720, 9) HRP: peroxidase SP 502 Batch PPX 3829, No-
vonordisk A/S, 10) Lactoperoxidase from bovine milk, l-2005, Sigma–Al-
drich, lot. 16H38311, 11) Chloroperoxidase from Caldaromyces fumago,
25810, Biochemika, 12) Bromoperoxidase from Corallina officinalis,
B2170, Sigma–Aldrich, 123 K3783 and 13) Lignin peroxidase, Fluka, lot.
ly only a single functional group of the enzyme is required,
namely in this case the prosthetic group—the hemin. Al-
though we have clearly shown for HRP that it catalyses the
alkene cleavage with promiscuous activity, the observed pro-
miscuous activity does not benefit from the peptide back-
bone. Therefore, we propose to define such promiscuous ac-
tivity as ꢀostensible enzyme promiscuity’. Thus, we call an
activity that is catalysed by an enzyme ꢀostensible enzyme
promiscuity’ if the reactivity can be tracked back to a single
catalytic site, which on its own can already perform the reac-
tion equally well as in the absence of the peptide backbone.
&
fillingcode: 1239384.32506 171.42603. Experiments were performed in
general in triplet.
General procedure for the catalytic alkene cleavage with peroxidases:
The enzyme (3 mg solid preparation or 20 mL liquid preparation) was
transferred into the corresponding reaction vessel (riplate LV). Buffer
(900 mL) and substrate (6 mL, 0.04 mmol) were added. The samples were
placed into an O
was flushed with pure O
2
-pressure reactor in an upright position. The reactor
and then the pressure was adjusted to 2 bar.
2
After 24 h at 170 rpm and 228C, the content of the reaction vessels was
transferred to Eppendorf tubes (2 mL) and the aqueous solutions were
extracted with AcOEt (2ꢃ500 mL). The combined organic phases were
2 4
dried with Na SO anhydrous and analysed by GC analysis.
Conclusion
General procedure for the catalytic alkene cleavage with hemin chloride:
A stock solution of hemin chloride (3.5 mg, 0.0054 mmol) was prepared
in DMSO (2.5 mL) to give a homogeneous solution (2 mm). The hemin
chloride solution (50 mL) was transferred into the corresponding vessel
It was shown that peroxidases are able to cleave selected C=
C double bonds adjacent to activated phenyl moieties at the
expense of molecular oxygen at an acidic pH with promiscu-
ous activity. We could unambiguously prove that exclusively
the hemin moiety present in the enzyme is responsible for
this activity; thus, the peptide backbones of the peroxidases
did not have any additional benefit for the reaction. We pro-
pose that such an activity should be called ꢀostensible
enzyme promiscuityꢁ. Such a classification allows one to dis-
tinguish between a promiscuous activity due to enzyme fea-
tures, such as the collaboration of various functional
(
riplate LV). Buffer (950 mL) and substrate (6 mL, 0.04 mmol) were
added. Reactions were run, worked-up, and analysed according to the
procedure with enzymes.
pH optimization: Conversion of t-anethol (1a) versus pH was determined
for HRPs, CiP and LiP by using the general procedure for the alkene
cleavage by enzymes. The different buffers employed were Bis-Tris
(
pH 7, 50 mm), Bis-Tris (pH 6, 50 mm), NaOAc/HOAc (pH 5, 50 mm),
NaOAc/HOAc (pH 4, 50 mm), NMe /HCO (pH 3, 20 mm), NMe
HCO H (pH 2, 20 mm) and HCO H (pH 1, 20 mm).
pressure: Dependence of conversion versus dioxygen pressure was de-
3
2
H
3
/
2
2
O
2
termined for HRP, CiP and LiP by following the general procedure for
the alkene cleavage by enzymes. The reactions were run at different pres-
sure (1, 2, 3, 4 and 6 bars).
[4,8]
groups
and promiscuous activity just due to the presence
of a single active moiety in the enzyme. Since enzymes con-
sist of an amino acid backbone, various activities described
in organocatalysis can be expected to be ꢀcatalysedꢁ by such
a peptide backbone too. Therefore, we think that a classifi-
cation such as ꢀostensible enzyme promiscuityꢁ is required,
to indicate that a single catalytic moiety on its own can al-
ready perform the reaction equally well as in the absence of
the peptide backbone.
Reaction in organic solvents: Experiments were performed with HRP
and CiP, by using the general procedure for the catalytic alkene cleavage
À1
by enzymes. Different organic solvents (17 mL, 1.8%vv ) were added
before starting the reaction.
Particularly, stability of HRP was tested with DMSO. Solutions (1 mL) of
buffer NMe
3
/HCOOH (pH 2, 20 mm) with increasing amounts of DMSO
À1
were prepared (0–5–10–15–20–30–40–50–60–80–100%vv ), and then re-
actions were run by following the general procedure.
Time study: Experiments were performed with HRP, by using the general
procedure for the biocatalytic alkene cleavage and taking samples follow-
ing this time schedule (0–1–2–3–4–5–6–8–11–14–22.5–25.5–28.5–31.5–
34.5–46.5–50.5 h).
Experimental Section
3
CD spectra: The CD spectra of HRP was recorded in buffer NMe /
General: GC analyses were carried out on a Varian 3800 gas chromato-
graph equipped with FID and a DB 1701 capillary column (30 m,
HCOOH (pH 2, 20 mm) and in buffer Bis-Tris (pH 6.5, 50 mm). Blank
CD spectra at pH 2 and 6.5 were also acquired. Spectra were compared
and analysed with the software DICHROWEB.
0
2
.25 mm, 0.25 mm film, N ). GCMS analyses were carried out on a Hew-
14146
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 14142 – 14148