Angewandte
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Chemie
likely exerts an electronic effect but is not required for the
substrate fit.
Since AsqJ has the capability to catalyze both desatura-
and epoxidation, with a surprising final fragmentation under
release of methyl isocyanate leading to the target heterocyclic
structures. Notably, it has been reported that three enzymes
are involved in an analogous benzodiazepine-quinolinone
pathway for the biosynthesis of viridicatin in Penicillium
cyclopium or P. viridicatum: a dehydrogenase, an epoxidase,
and an enzyme called cyclopenase, which is responsible for
the rearrangement.[18] AsqJ is so far unique in performing the
elucidated desaturation, epoxidation, and elimination/rear-
rangement sequence. The only other dioxygenase that
catalyzes a sequential dehydrogenation–epoxidation reaction
is involved in pentalenolactone sesquiterpenoid biosynthesis
in Streptomyces.[19]
The specific conformational control over reaction selec-
tivity displayed by this system is a peculiarity of enzymatic
reactions and not typically attained or imitated by metal-
complex model systems. AsqJ is a catalyst that induces
a remarkable one-pot multistep synthesis accompanied by
major structural changes and it thus represents a particularly
efficient type of synthetic reagent.
tion and epoxidation reactions, we aimed to elucidate the
relationship between the two oxidative processes. Intermedi-
ate 2 was synthesized by condensing isatoic anhydride and
sarcosine to give a benzodiazepinedione core,[10] to which an
exocyclic 4-anisylidene group was subsequently attached in
a Perkin-like condensation with 4-methoxybenzaldehyde (see
the Supporting Information).[16] Starting the AsqJ-catalyzed
reaction from 2 indeed led to the generation of both oxirane
intermediate 3 and product 4, but only in the presence of Fe2+,
aKG, and oxygen. These findings demonstrate that the
desaturation and epoxidation are decoupled operations,
with each of them requiring one molecule of aKG, as well
as oxygen. The regenerated FeII center (Scheme 3, Apo-AsqJ)
is thus ready to initiate the epoxidation once the succinate
from the dehydrogenation step has been replaced by a new
aKG co-substrate. Notably, AsqJ only showed activity with Z-
configured 2, but not towards its geometrical E isomer 2’
(Scheme 2, and Figure S7 in the Supporting Information).
Accordingly, we obtained an AsqJ complex structure solely
with 2 (Figure 2c, PDB ID: 5DAV), thus demonstrating that
the Z isomer of product 2 but not the E isomer 2’ is able to
bind into the active site cavity.
Acknowledgements
This work was supported by the 1974-01 TUM-KAUST
In the second FeII/a-ketoglutarate reaction cycle, the
ferryl FeIV-oxo species converts the desaturated intermediate
2 into epoxide 3 (Scheme 3, C1 and C2). Although a crystal
structure of the AsqJ complex was not achievable, it is most
likely that the enzyme-bound oxirane 3 adopts an identical
conformation to that observed with 1 and 2, since the protein
environment only tolerates a single extended boat config-
uration (Figure 2d). Importantly, this mandatory substrate
specification imposed by AsqJ is in direct contrast to the
“small-molecule” X-ray structure of 3’-hydroxycyclopenine
(CSD reference code POHBEV).[15] The three-dimensional
structure of the close analogue of 3 displays the molecule in
a substantially different flipped conformation, which allows
an energetically favorable intramolecular p–p interaction
between the two aromatic ring systems (Figure 2e and
Figure S3b in the Supporting Information). However, it is
this flipped spatial arrangement of 3 that is fundamental to
initiate the nucleophilic attack of the ortho carbon atom of the
arylamide moiety on the benzylic oxirane carbon atom
(Scheme 3 , D). As a result, the AsqJ-bound intermediate 3
is forced into a non-reactive extended conformation (Fig-
ure S4). Consequently, the final elimination and rearrange-
ment of 3 takes place non-enzymatically by a spontaneous
process that occurs after dissociation from the dioxygenase. In
this reaction, the tricylic core of the presumed intermediate E
fragments with elimination of methylisocyanate to yield the
keto form of 4, which eventually tautomerizes to its aromatic
enol form.[6,17] In essence, the enzymatically formed spiro
epoxide spring-loads the 6,7-bicyclic skeleton for rearrange-
ment into the 6,6-bicyclic quinolone framework of 4’-
methoxyviridicatin 4 as a result of ring strain and proper
structural preorganization.
agreement on selective C H bond activation (A.B.) and
À
SFB749 (M.G.). We thank the staff of the beamline X06SA at
the Paul Scherrer Institute, SLS, Villigen (Switzerland) for
assistance during data collection.
Keywords: 4’-methoxyviridicatin · alkaloids · AsqJ dioxygenase ·
biosynthesis · C H activation
À
How to cite: Angew. Chem. Int. Ed. 2016, 55, 422–426
Angew. Chem. 2016, 128, 432–436
[2] a) J. C. Price, E. W. Barr, T. E. Glass, C. Krebs, J. M. Bollinger, J.
[3] N. Ishikawa, H. Tanaka, F. Koyama, H. Noguchi, C. C. Wang, K.
[5] A. Ahmed, M. Daneshtalab, J. Pharm. Pharm. Sci. 2012, 15, 52 –
72.
[8] W. Aik, M. A. McDonough, A. Thalhammer, R. Chowdhury,
[9] C. T. Walsh, S. W. Haynes, B. D. Ames, X. Gao, Y. Tang, ACS
In summary, the biosynthesis of 4-arylquinolin-2(1H)-one
alkaloids involves an intricate sequence of dehydrogenation
Angew. Chem. Int. Ed. 2016, 55, 422 –426
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