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derived substrate 6t. In contrast to 6l, its unsaturated
derivative 7l was not converted by AsqJ (Scheme 3a). It
can therefore be precluded that the newly discovered
enzymatic reaction pathway of AsqJ proceeds via an initial
desaturation reaction, unlike the known natural reaction
cascade.
Of particular interest was the turnover of glycine-derived
substrate 6j, which likewise was converted by AsqJ following
the quinazolin-4(3H)-one pathway to give 9j, thus providing
evidence that the new reaction proceeds without any mech-
anistic involvement of a side chain (Scheme 3a). This
indicated that the mechanism starts with radical formation
at the C3-position. Indeed, evaluation of 6u (synthesis shown
in SI, chapter 2.1 and 2.3) with a dimethyl substitution at C3
that precludes radical formation at this position as substrate
for AsqJ did not show any conversion. This led to the
assumption that initial radical formation at C3 ultimately
2
=
leads to excision of C O from the ring structure during
enzymatic conversion. To gain further evidence for this
proposal, a 13C-labelled derivative 13C-6k was synthesized
starting from N-Fmoc-l-alanine-1-13C (13C-10k) by SPPS
according to Scheme 1a and subjected to reaction with AsqJ.
This experiment corroborated that exactly this carbon is
excised to transform the 6.7- into the 6.6-membered bicycle
(Scheme 3a).
Based on these combined results, the mechanism of the
quinazolinone formation may proceed via one of the path-
ways shown in Scheme 3b. The reaction sequence is assumed
to begin with a hydrogen radical abstraction at C3 of substrate
6 by the hypervalent iron(IV) oxo species in the active site of
AsqJ, thus generating a carbon-centered radical I2. From this
intermediate, two different pathways (P1, P2) are conceiv-
able. In pathway P1, a second radical hydrogen abstraction at
the nitrogen followed by bond formation between the two
resulting radical centers leads to formation of an a-lactam
intermediate I3. Hydrolytic cleavage of the amide bond
delivers the enzyme-bound carboxylic acid I4; subsequent
deprotonation, addition of O2 to the AsqJ iron center, and
elimination of CO2 yields the observed product 9 (cf. Fig-
ure S127 for mechanistic details). In pathway P2, the new N1-
C3-bond is directly formed at the expense of the amide bond,
resulting in formyl radical I5. Hydroxyl abstraction from the
AsqJ iron center by the formyl radical unites the two
proposed routes at carboxylic acid I4 (cf. Figure S127).
Alternatively, pathway P2 may proceed directly from radical
I2 to carboxylic acid I4 through concomitant amide bond
homolysis and hydroxyl abstraction from the AsqJ iron
center. Elimination of CO2, as described for P1 above, again
yields the observed product 9.
To determine the feasibility of each proposed mechanistic
route leading to 9, we undertook density functional theory
calculations. Utilizing a previously-validated representative
system for AsqJ,[13] we constructed a model of the catalytic
iron moiety and calculated the energy surface for R = H (6j)
at the uB3LYP-D3(BJ)/def2-TZVP//uB3LYP-D3(BJ)/def2-
SVP level of theory (Scheme 3c).[14–18] These calculations
demonstrate that the formation of the formyl radical I5 is
strongly disfavored, exhibiting an unsurmountable transition
state barrier of 57.2 kcalmolÀ1 leading to a product > 20 kcal
Scheme 2. Evaluation of the substrate specificity of AsqJ. a) Substrate-
dependent product selectivity of AsqJ. b) Divergent enzymatic conver-
sion of substrates 6b, h, i, all bearing aromatic side chains.
likewise converted to the corresponding quinazolin-4(3H)-
ones 9h and 9i, despite their aromatic substituent R
(Scheme 2b). AsqJ thus exclusively converts substrates with
a methylene-bridged aromatic substituent R following the
naturally observed pathway to the quinolone structural motif,
hence corroborating the published mechanistic preconditions
for this rearrangement.[7,9] All other substrates are converted
to the newly uncovered product class, raising questions on the
molecular mechanism leading to these quinazolinones.
Elucidating the Novel Reaction Pathway
For the two accepted cyclic aliphatic substrates 6m,
n delivering quinazolinones 9m, n, the respective dehydro-
genated derivatives 7m, n were also observed. In addition, the
alkene and alkyne substrates 6r and 6s, respectively, exclu-
sively delivered the corresponding desaturated intermediates
7r and 7s (see SI, Figures S39, S40, S44, S45), raising the
question if these unsaturated enzymatic products are central
intermediates in both the known and the novel enzymatic
pathway. To test this possibility, the unsaturated analog 7l was
synthesized. This compound was the substance of choice,
because its corresponding substrate precursor 6l is efficiently
converted to quinazolinone 9l by AsqJ. Synthetic 7l was
obtained as a mixture of E/Z-isomers (2:1, see SI, chapter 2.4)
t
by NaOtBu-induced elimination of BuOH from threonine-
8300 www.angewandte.org ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 8297 –8302