Enzyme-like regiodivergent behavior of a flavopeptide catalyst in aerobic baeyer-villiger oxidation

Article abstract of DOI:10.2533/chimia.2018.866

We recently developed a flavopeptide immobilized on polystyrene resin, Fl-Pep-PS, that could realize the first N5-unmodified neutral flavin (Fl)-catalyzed aerobic oxygenation reactions under non-enzymatic conditions. Although a key active species is assumed to be the corresponding 4a-hydroperoxyflavin (Fl_4aOOH) from the unprecedented activity and unique chemoselectivity, further circumstantial support would be helpful to be sure since spectroscopic evidence is difficult to obtain due to the compound's insolubility. In this article, we report that the aerobic Baeyer-Villiger oxidation of a fused cyclobutanone, (±)-cis-bicyclo[3.2.0]hept-2-en-6-one (1), can be promoted with Fl-Pep-PS in a FMO-like chemoselectivity and regiodivergent manner via Fl-related catalytic intermediates, which delivers strong evidence of the involvement of Fl_4aOOH as an active species in Fl-Pep-PScatalyzed aerobic oxygenation reactions.

Full text of DOI:10.2533/chimia.2018.866

866 CHIMIA 2018, 72, No. 12
doi:10.2533/chimia.2018.866
Chemistry ties Linking Japan to switzerLand
Chimia 72 (2018) 866–869 © Swiss Chemical Society
Enzyme-like Regiodivergent Behavior
of a Flavopeptide Catalyst in Aerobic
Baeyer-Villiger Oxidation
Ken Yamanomotoa, Hazuki Kitaa, Yukihiro Arakawaa*, Keiji Minagawaab and Yasushi Imadaa*
Abstract: We recently developed a flavopeptide immobilized on polystyrene resin, Fl-Pep-PS, that could realize
the first N5-unmodified neutral flavin (Fl)-catalyzed aerobic oxygenation reactions under non-enzymatic condi-
tions. Although a key active species is assumed to be the corresponding 4a-hydroperoxyflavin (Fl4aOOH) from the
unprecedented activity and unique chemoselectivity, further circumstantial support would be helpful to be sure
since spectroscopic evidence is difficult to obtain due to the compound’s insolubility. In this article, we report
that the aerobic Baeyer-Villiger oxidation of a fused cyclobutanone, (±)-cis-bicyclo[3.2.0]hept-2-en-6-one (1), can
be promoted with Fl-Pep-PS in a FMO-like chemoselectivity and regiodivergent manner via Fl-related catalytic
intermediates, which delivers strong evidence of the involvement of Fl4aOOH as an active species in Fl-Pep-PS-
catalyzed aerobic oxygenation reactions.
Keywords: Aerobic oxygenation · Baeyer-Villiger oxidation · Biomimetic catalyst · Flavin · Peptide
Yukihiro Arakawa
received his BEng for Encouragement of Research in Polymer flavin-3-acetic acid residue, Scheme 1),
(2004), MEng (2006) Science; The Society of Polymer Science, consisting of Fl, a tripeptide linker, and
and PhD (2009) from Japan 2016.
polystyrene (PS) resin.[4] We calculated
ToyohashiUniversity the lowest energy conformation of Fl4aOOH
of Technology un- that could be stabilized by the conjugated
peptide through intramolecular hydrogen
bonds, and demonstrated that Fl-Pep-PS
Enzymes are specific and efficient in could efficiently catalyze the electrophilic
Itsuno native organic reactions; therefore, their sulfoxidation of thioanisole as well as the
organic synthesis. He is a recipient ofAward Ado-NH-PS (Fl-Pep-PS; 3-FlC2 = lumi-
der the supervision 1. Introduction
of Prof. Koichi Ito
(BEng) and Prof.
Shinichi
(MEng and PhD). During his PhD stud- catalytic functions have often served as nucleophilic Baeyer-Villiger oxidation of
ies, he worked as a JSPS research fellow guides for the design of highly active, se- 3-phenylcyclobutanone using O2 as the
(DC2, 2007–2009) on the development of lective, and green artificial catalysts and terminal oxidant, in which the resin could
polymeric chiral catalysts for organic reac- reactions. Among diverse classes of en- play a crucial role probably as hydropho-
tions in aqueous media. He was employed zymes, oxidoreductases employing the bic microenvironment in stabilizing the
at Universität Basel (2009–2011) and ETH
isoalloxazine ring system Fl (Scheme 1), corresponding Fl4aOOH. Although spectro-
Zürich (2011–2013) as a postdoctoral fel- found in flavin cofactors as an active cen- scopic evidence is not available due to the
low to work with Prof. Helma Wennemers ter, are called flavoenzymes, which are re- insolubility of the resin, the involvement of
on the development of immobilized peptide sponsible for various oxidative metabolic Fl
as well as the non-involvement of a
catalysts and other bio-inspired catalysts for processes in nature.[1] A notable series of pe4raaOcOiHd as the active species were support-
asymmetric reactions. He has been an as- flavoenzymes is flavin-containing mono- ed by the unique chemoselectivity of Fl-
sistant professor at Tokushima University
oxygenases (FMO) that metabolize xeno- Pep-PS, which was similar to that of FMO.
since 2013, and his current research inter- biotic substrates through the activation of For example, 3-phenylcyclobutanone was
ests are polymeric catalysts, biomimetic or- molecular oxygen (O ) followed by the do- exclusively oxidized into β-phenyl-γ-
ganocatalysis, and environmentally benign nation of an oxygen 2atom to the substrate butyrolactone in the presence of other re-
in mammalian liver. A key active species active substrates such as thioanisole and
for the monooxygenation has been recog- cyclooctene under suitable conditions with
nized to be 4a-hydroperoxy adducts of Fl Fl-Pep-PS, whereas such FMO-like che-
(Fl4aOOH) and the catalytic cycle has long moselectivity was not observed under typi-
been well understood (Scheme 1, lower cal mCPBA-based oxidation conditions.[4]
cycle).[2] Nevertheless, Fl as a simple non-
In this brief communication, we de-
enzymatic organocatalyst had never been scribe how the aerobic Baeyer-Villiger
successfully employed for simulating the reaction of a fused cyclobutanone, (±)-cis-
aerobic oxygenation ability of FMO due bicyclo[3.2.0]hept-2-en-6-one (1), can be
to the lability of Fl4aOOH, which readily promoted with Fl-Pep-PS in a FMO-like
decomposes into Fl and H2O2 under apo- chemoselectivity and regiodivergent man-
enzyme-free conditions (Scheme 1, up- ner, which can be strong evidence of the
per cycle).[3] Recently, this long-standing involvement of Fl4aOOH as a key active spe-
challenge was overcome at last using our cies in Fl-Pep-PS-catalyzed aerobic oxy-
*Correspondence: Prof. Y. Arakawaa
E-mail: arakawa.yukihiro@tokushima-u.ac.jp
aDepartment of Applied Chemistry
Tokushima University
Minamijosanjima, Tokushima 770-8506, Japan
bInstitute of Liberal Arts and Sciences
Tokushima University
designed catalyst, 3-FlC2-Pro-Tyr-Asp-
genation reactions.
Minamijosanjima, Tokushima 770-8502, Japan

Chemistry ties Linking Japan to switzerLand
CHIMIA 2018, 72, No. 12 867
H2O2
O
N
N
except FMO and Fl-Pep-PS
N
R2
N
R2
N
O
NH
N
O
O
O
Z+
N
O
O
N
ZH + H+
O
O2
8
7
CO2H
3 N
N
4a
5
N
HN
FlH2
R1
N
H
R1
O
O
O
NH
O
H
O
Fl
Fl4aOOH
N
H
only FMO or Fl-Pep-PS
11
OH
Fl4aOH
3-FlC2-Pro-Tyr-Asp-Ado-NH-PS (Fl-Pep-PS)
Sub
SubO
H2O
Scheme 1. General catalysis of neutral flavins (Fl) under aerobic oxygenation conditions.
1
washed with DMF (5×), DMF/CH2Cl2 by H NMR spectroscopy of the crude
(4:1) (5×), and CH2Cl2 (3×) to give mixture with reference to the published
H-Asp(OtBu)-Ado-NH-PS. According to spectral data of 2 and 3,[8] in which the
the coupling and Fmoc-deprotection pro- yields of 2 and 3 were estimated from the
cedures, H-Asp(OtBu)-Ado-NH-PS was integration of peaks assignable to methyl
2. Experimental
2.1 Materials
(Aminomethyl)polystyrene
(70–90
mesh, 1% cross-linked, the N loading
was determined by elemental analy-
sis to be 1.24 mmol g–1) was purchased
from Sigma-Aldrich. 3-Methyllumiflavin
(LFl),[5] lumiflavin-3-acetic acid,[6] 5-eth-
further converted into H-Pro-Tyr(tBu)-
protons of dodecane at 0.88 ppm and that
Asp(OtBu)-Ado-NH-PS via H-Tyr(tBu)- assignable to a proton of the olefin moi-
Asp(OtBu)-Ado-NH-PS. Subsequently, ety either at 5.58 ppm (for 2) or at 5.66
a solution (prepared with a minimum ppm (for 3).
amount of DMF) of lumiflavin-3-acetic
yl-3-methyllumiflavinium
perchlorate
(LFlEt+ClO4–),[5] and 3-FlC2-NH-PS[4]
were prepared according to the literature
procedures. Zinc dust was treated with 2N
HCl aq. under ultrasonication for 15 min-
utes, washed with H O and acetone, and
dried in vacuo to acti2vate prior to use. All
other reagents were purchased from com-
mercial supplies and used without purifi-
cation.
acid (244 mg, 0.78 mmol), HCTU (321
mg, 0.78 mmol), and DIPEA (301 mg, 3. Results and Discussion
2.3 mmol) was added to H-Pro-Tyr(tBu)-
Asp(OtBu)-Ado-NH-PS pre-swollen in
DMF, and the mixture was agitated for 2 Villiger oxidation of the fused cyclobu-
h. The suspension was washed with DMF tanone 1 by means of Fl-Pep-PS as a
We explored the aerobic Baeyer-
repeatedly until the solution layer be- catalyst under conditions that were pre-
comes colorless, then with DMF/CH2Cl2 viously developed by our group for the
(4:1) (5×), and with CH2Cl2 (3×) to give oxidation of 3-phenylcyclobutanone.[4]
3-FlC2-Pro-Tyr(tBu)-Asp(OtBu)-Ado-
NH-PS. Finally, 3-FlC2-Pro-Tyr(tBu)- 1 atm of O , 20 equivalents of H2O, and
Asp(OtBu)-Ado-NH-PS pre-swollen in 3.5 equival2ents of zinc dust in a mixed
In the presence of 5 mol% of Fl-Pep-PS,
2.2 Synthesis of Fl-Pep-PS
To (aminomethyl)polystyrene (NH2-
CH Cl was treated with a mixture of TFA/
solvent of acetonitrile, toluene, and ethyl
PS, 250 mg, 0.31 mmol) pre-swollen
in DMF was added a solution of Boc-
Ado-OH (244 mg, 0.78 mmol), O-(1H-
6-chlorobenzotriazole-1-yl)-1,1,3,3-
CH22Cl22 (2:1) twice (the first time: 1 h, the acetate (8:4:1), the desired oxidation re-
second time: 20 min), and washed with actions were found to proceed smoothly
CH2Cl2 by means of Soxhlet extractor and with 76% conversion of 1 in 7 h to afford
dried in vacuo to give 453 mg of 3-FlC2- the corresponding cis-lactones 2 and 3 in
tetramethyluronium
hexafluorophos-
Pro-Tyr-Asp-Ado-NH-PS
(Fl-Pep-PS) 35% yield and 29% yield, respectively,
as an orange-colored resin. The coupling without undesired oxidation to epoxides
reactions were monitored by qualitative (Table 1, entry 1). By contrast, the use of
Kaiser test[7a] and chloranil test (second- LFl instead of Fl-Pep-PS as a catalyst
phate (HCTU, 321 mg, 0.78 mmol), and
N-ethyldiisopropylamine (DIPEA, 301
mg, 2.3 mmol) and the heterogeneous
mixture was shaken for 1.5 h. The insolu-
ary amine).[7b] The Fl loading of Fl-Pep-
under identical conditions resulted in no
ble resin (Boc-Ado-NH-PS) was washed
with DMF (5×), DMF/CH2Cl2 (4:1) (5×),
and CH2Cl2 (3×), and then treated with a
PS used in this study was determined as conversion of 1 (entry 2), indicating that,
previously reported[4] to be 0.50 mmol g–1. as expected, such a simple Fl molecule
has no catalytic activity due to the gen-
mixture of TFA/CH2Cl (2:1) twice (the
first time: 1 h, the secon2d time: 20 min) to
remove the Boc group, and washed with
CH2Cl2 (3×), 5% v/v DIPEA in CH2Cl2
2.3 Aerobic Baeyer-Villiger Oxid- eral lability of the corresponding Fl4aOOH
ation of 1 Catalyzed by Fl-Pep-PS The same result, no conversion of 1, was
To an acetonitrile–toluene–ethyl ac- obtained with 3-FlC2-NH-PS as a catalyst
.
etate mixed solvent (8:4:1, 0.9 ml) was (entry 3), and Ac-Pro-Tyr-Asp-Ado-NH-
added Fl-Pep-PS (10 mg, 5 µmol) and PS containing no Fl was also totally inac-
zinc dust (22.9 mg, 0.35 mmol), and the tive (entry 4) even in the presence of LFl
(3×), and CH2Cl (6×). The resulting res-
in, H-Ado-NH-P2S, was mixed with a solu-
tion (prepared with a minimum amount of
mixture was sonicated for 2 min before (entry 5). In addition, only trace amounts
DMF) of Fmoc-Asp(OtBu)-OH (319 mg,
adding H O (36 µl) and a 1 M stock solu- of the products were formed when the
0.78 mmol), HCTU (321 mg, 0.78 mmol),
and DIPEA (301 mg, 2.3 mmol), which
tion of 1 2(0.1 ml, 0.1 mmol) in the same reaction was performed with 3-FlC2-
mixed solvent containing 10 mol% of Pro-Tyr-Asp-Ado-NH2 having no PS as a
dodecane as an internal standard, which catalyst (entry 6). Naturally, no reaction
was stirred at 35 °C for 7 h under an occurred without any catalysts (entry 7).
atmosphere of oxygen while protected These results show that all components of
was agitated for 1.5 h for coupling, washed
with DMF (5×), DMF/CH Cl (4:1) (5×),
and CH2Cl (3×), then treat2ed2with a 20%
v/v solutio2n of piperidine in DMF for 15
min twice for Fmoc-deprotection, and
from light. The reaction was evaluated
Fl-Pep-PS are essential for its catalytic

868 CHIMIA 2018, 72, No. 12
Chemistry ties Linking Japan to switzerLand
Table 1. Aerobic Baeyer-Villiger oxidation of 1 with flavin catalysts.
activity and they should be arranged prop-
erly with each other. On the other hand,
LFlEt+ClO4 , one of the most common
–
catalyst (5 mol%)
O
O
O2 (1 atm), Zn (3.5 equiv)
conventional pseudo-flavin catalysts,[3]
promoted the reaction as efficiently as Fl-
Pep-PS to furnish 2 and 3 in 36% yield
and 27% yield, respectively (entry 8), as
expected from our previous reports on the
Baeyer-Villiger oxidation with such arti-
ficial cationic flavins.[9]
O
O
+
O
H2O (20 equiv)
CH3CN—toluene—EtOAc (8:4:1)
35 °C, no light, 7 h
1
2
3
O
N
N
N
N
O
N
N
N
O
O
O
N
O
N
Provided that the above reaction is car-
ried out under the typical Baeyer-Villiger
oxidation conditions, the formation of 2
via migration of the adjacent more sub-
stituted carbon in the Criegee intermedi-
ate should be kinetically favored. Indeed,
the normal lactone 2 was preferentially
obtained under mCPBA-based oxidation
conditions in 78% yield along with the ab-
normal lactone 3 as well as an epoxidized
by-product in 10% and 12% yield, respec-
tively (Scheme 2a). Interestingly, such
electronic limitations can be overcome
under FMO-mediated enzymatic condi-
tions, providing ‘normal’ and ‘abnormal’
lactones in a ratio of nearly 1:1,[10] and this
regiodivergent behavior can be rational-
ized by fixation of Criegee intermediates
as the corresponding 4-hydroxy-1,2,5-
N
N
N
PS
O
NH
HN
N
H
N
O
O
O
O
CO2H
O
LFl
3-FlC2-NH-PS
NH
PS
O
O
N
H
11
N
N
N
OH
Fl-Pep-PS
O
ClO4
O
N
N
N
–
LFlEt+ClO4
O
NH
O
NH
HN
N
O
O
O
O
N
CO2H
O
CO2H
HN
O
NH
NH
PS
O
O
N
H
NH2
11
11
OH
OH
3-FlC2-Pro-Tyr-Asp-Ado-NH2
Ac-Pro-Tyr-Asp-Ado-NH-PS
entry catalyst
conversion [%]
yield [%]
trioxane adducts formed from the ketone
2
3
29
0
[11]
and Fl4aOOH
.
Thus we propose that the
regioselectivity of Fl-Pep-PS (Table 1, en-
1
2
3
4
5
Fl-Pep-PS
LFl
76
0
35
0
try 1) may also come from such FMO-like
cyclic transition states including one that
gives rise to abnormal migration (Scheme
2, the right transition state model) as much
3-FlC2-NH-PS
0
0
0
Ac-Pro-Tyr-Asp-Ado-NH-PS
0
0
0
as normal migration (Scheme 2, the left
transition state model), in which the cor-
Ac-Pro-Tyr-Asp-Ado-NH-PS
0
0
0
responding Fl4aOOH should be necessarily
+ 5 mol% LFl
involved as a key precursor. In other words,
the present study provides strong evidence
for the effective use of Fl4aOOH as the ac-
tive species in the Fl-Pep-PS-catalyzed
aerobic catalytic oxygenations (Scheme 1,
lower cycle).
6
7
8
3-FlC2-Pro-Tyr-Asp-Ado-NH2
4
<1
<1
none
0
0
0
LFlEt+ClO4–
66
36
27
a)
O
mCPBA (1.2 equiv)
NaHCO3 (1.0 equiv)
R
favored
1
1
2
+
3
O O
O
CH2Cl2, r.t., 24 h
78% (2), 10% (3)
7.8
:
1
H
b)
N
N
O
O
N
N
O
O
Fl-Pep-PS, O2
2
+
3
N
N
N
H
Table 1, entry 1
35% (2), 29% (3)
N
H
O
O
2
3
O
O
~1.2
:
1
O
O
[10]
with FMO, O2
~1
1
:
Scheme 2. The aerobic Baeyer-Villiger oxidation of 1 under conditions with (a) mCPBA and (b) Fl-Pep-PS, and plausible origins of each regioselec-
tivity.

Chemistry ties Linking Japan to switzerLand
CHIMIA 2018, 72, No. 12 869
[1] a) T. C. Bruice, Acc. Chem. Res. 1980, 13, 256;
Y. Imada, T. Naota, Chem. Rec. 2007, 7, 354; e)
4. Conclusion
b) C. Walsh, Acc. Chem. Res. 1980, 13, 148; c)
D. P. Ballou, ‘Flavoprotein Monooxygenases’
in ‘Flavins and flavoproteins’, Eds. V. Massey,
C. H. Williams, Elsevier: NewYork, 1982; d)
F. G. Gelalcha, Chem. Rev. 2007, 107, 3338; f)
J.-E. Bäckvall in ‘Modern oxidation methods’,
Ed. J.-E. Bäckvall, Wiley-VCH: Weinheim,
2004.
A flavopeptide catalyst, Fl-Pep-PS, re-
cently designed by our group[4] was found
to be effective for the aerobic Baeyer-
Villiger oxidation of the fused cyclobuta-
none 1, which could provide the normal
lactone 2 and the abnormal lactone 3 in a
nearly equal ratio via FMO-like Fl-related
catalytic intermediates. All components of
Fl-Pep-PS were demonstrated to be essen-
tial for the catalysis by some control exper-
‘Chemistry and biochemistry of flavoenzymes’, [4] Y. Arakawa, K. Yamanomoto, H. Kita, K.
Ed. F. Müller, CRC Press: Boston, 1991; e) R.
B. Silverman, Acc. Chem. Res. 1995, 28, 335; f)
Minagawa, M. Tanaka, N. Haraguchi, S. Itsuno,
Y. Imada, Chem. Sci. 2017, 8, 5468.
N. M. Kamerbeek, D. B. Janssen, W. J. H. van [5] Y. Imada, H. Iida, S. Ono, Y. Masui, S.-I.
Berkel, M. W. Fraaije, Adv. Synth. Catal. 2003, Murahashi, Chem. Asian. J. 2006, 1, 136.
345, 667; g) ‘Flavins–photochemistry and [6] H. Ikeda, K. Yoshida, M. Ozeki, I. Saito,
photobiology’, Eds. E. Silva, A. M. Edwards, Tetrahedron Lett. 2001, 42, 2529.
Royal Society of Chemistry: Cambridge, 2006; [7] a) E. Kaiser, R. L. Colescott, C. D. Bossinger,
h) M. W. Fraaije, D. B. Janssen, ‘Biocatalytic
scope of Baeyer-Villiger Monooxygenases’
in ‘Modern biooxidation: enzymes, reactions [8] S. Xu, Z. Wang, X. Zhang, K. Ding, Eur. J. Org.
and applications’, Eds. R. D. Schmid, V. B. Chem. 2011, 110.
Urlacher’, Wiley-VCH: Weinheim, 2007; i) M. [9] a) S.-I. Murahashi, S. Ono, Y. Imada, Angew.
P. I. Cook, Anal. Biochem. 1970, 34, 595; b) T.
Vojkovsky, Pept. Res. 1995, 8, 236.
iments. These brief but significant results
have led us to conclude that Fl4aOOH is the
active species in the Fl-Pep-PS-catalyzed
aerobic oxygenation system.
Insin´ska-Rak, M. Sikorski, Chem. Eur. J. 2014,
20, 15280.
[2] a) L. L. Poulsen, D. M. Ziegler, J. Biol. Chem.
Chem. Int. Ed. 2002, 41, 2366; b) Y. Imada, H.
Iida, S.-I. Murahashi, T. Naota, Angew. Chem.
Int. Ed. 2005, 44, 1704.
Acknowledgements
1979, 254, 6449; b) V. Massey, P. Hemmerich, [10] a) V. Alphand, R. Furstoss, J. Org. Chem. 1992,
This
work was supported by JSPS
KAKENHI (Grant-in-Aid for Scientific
Research (C), no. 18K05108).
Biochem. Soc. Trans. 1980, 8, 246; c) N. B.
Beaty, D. P. Ballou, J. Biol. Chem. 1980, 255,
3817.
57, 1306; b) F. Petit, R. Furstoss, Tetrahedron:
Asymmetry 1993, 4, 1341; c) M. D. Mihovilovic,
B. Müller, P. Stanetty, Eur. J. Org. Chem. 2002,
[3] a) H. Iida, Y. Imada, S.-I. Murahashi, Org.
3711.
Biomol. Chem. 2015, 13, 7599; b) R. Cibulka, [11] D. R. Kelly, Tetrahedron: Asymmetry 1996, 7,
Received: July 25, 2018
Eur. J. Org. Chem. 2015, 915; c) G. de Gonzalo,
M. W. Fraaije, ChemCatChem 2013, 5, 403; d)
1149.