Design of peptide-containing: N 5-unmodified neutral flavins that catalyze aerobic oxygenations

Article abstract of DOI:10.1039/c7sc01933e

Simulation of the monooxygenation function of flavoenzyme (Fl-Enz) has been long-studied with N5-modified cationic flavins (FlEt⁺), but never with N5-unmodified neutral flavins (Fl) despite the fact that Fl is genuinely equal to the active center of Fl-Enz. This is because of the greater lability of 4a-hydroperoxy adduct of Fl, Fl_OOH, compared to those of FlEt⁺, FlEt_OOH, and Fl-Enz, Fl_OOH-Enz. In this study, Fl incorporated into a short peptide, flavopeptide (Fl-Pep), was designed by a rational top-down approach using a computational method, which could stabilize the corresponding 4a-hydroperoxy adduct (Fl_OOH-Pep) through intramolecular hydrogen bonds. We report catalytic chemoselective sulfoxidation as well as Baeyer-Villiger oxidation by means of Fl-Pep under light-shielding and aerobic conditions, which are the first Fl-Enz-mimetic aerobic oxygenation reactions catalyzed by Fl under non-enzymatic conditions.

Full text of DOI:10.1039/c7sc01933e

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Yamanomoto, H. Kita, K. Minagawa, M. Tanaka, N. Haraguchi, S. Itsuno and Y. Imada, Chem. Sci., 2017,
DOI: 10.1039/C7SC01933E.
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EDGE ARTICLE
Design of Peptide-Containing N5-Unmodified Neutral Flavins That
Catalyze Aerobic Oxygenations
Received 00th January 20xx,
Accepted 00th January 20xx
Yukihiro Arakawa,a Ken Yamanomoto,a Hazuki Kita,a Keiji Minagawa,a,b Masami Tanaka,c Naoki
Haraguchi,d Shinichi Itsunod and Yasushi Imada*a
DOI: 10.1039/x0xx00000x
Simulation of the monooxygenation function of flavoenzyme (Fl-Enz) has been long-studied with N5-modified cationic
flavins (FlEt+), but never with N5-unmodified neutral flavins (Fl) despite the fact that Fl is genuinely equal to the active
center of Fl-Enz. This is because of the greater lability of 4a-hydroperoxy adduct of Fl, FlOOH, compared to those of FlEt+,
FlEtOOH, and Fl-Enz, FlOOH-Enz. In this study, Fl incorporated into a short peptide, flavopeptide (Fl-Pep), was designed by a
rational top-down approach using a computational method, which could stabilize the corresponding 4a-hydroperoxy
adduct (FlOOH-Pep) through intramolecular hydrogen bonds. We report catalytic chemoselective sulfoxidation as well as
Baeyer-Villiger oxidation by means of Fl-Pep under light-shielding and aerobic conditions, which are the first Fl-Enz-
www.rsc.org/
mimetic
aerobic
oxygenation
reactions
catalyzed
by
Fl
under
non-enzymatic
conditions.
on flavin chemistry.1 A single oxygen atom is transferred from
4a-hydroperoxyflavin (FlOOH-Enz), a key active species in Fl-Enz
catalysis, to a substrate (Sub) to give an oxidized product
(SubO) and 4a-hydroxyflavin (FlOH-Enz), which eliminates H2O
to form the oxidized flavin Fl-Enz. Then, Fl-Enz is reduced with
NAD(P)H to afford the reduced flavin (FlH2-Enz), which finally
Introduction
Isoalloxazines, such as riboflavin and its analogues (Figure 1a),
show flexible redox activities as well as visible-light emission
properties due to the specific conjugated heterocyclic
structure, which are responsible for the catalytic functions of a
variety of flavoenzymes such as flavin-containing
monooxygenase (Fl-Enz, Fig. 1a), oxidase, and photolyase.1
Whereas a number of flavin-inspired catalytic reactions for
organic synthesis have been developed with artificial
isoalloxazines, N5-modified cationic flavins (FlEt+, Fig. 1b,
upper),2 there have been much less progress in developing
those with genuine isoalloxazines, N5-unmodified neutral
flavins (Fl, Fig. 1b, lower), under non-enzymatic conditions
despite their availability and the fact that nature actually
utilizes them as catalysts. Recently, Fl has received increasing
attention because of its economical as well as environmental
friendliness and appeared as thermal-redox,3a-3c photoredox,3d-
3i and photosensitizing catalysts.3j-3m However, the use of Fl as
oxygenation catalysts simulating the function of Fl-Enz has
remained unexplored.
reacts with molecular oxygen to regenerate FlOOH-Enz
.
Previsouly, we have successfully simulated this catalytic cycle
using FlEt+ and hydrazine monohydrate instead of Fl-Enz and
NAD(P)H, respectively (Fig. 1b, upper).4 For example, the
aerobic oxygenation of sulfides was feasible in the presence of
1 mol% of 5-ethyl-3-methyllumiflavinium perchlorate (Fig. 1b,
R1=R2=R3=R4=Me in FlEt+),
1
equivalent of hydrazine
monohydrate, and 1 atm of O2 in 2,2,2-trifluoroethanol (TFE),
in which TFE was crucial as a reaction solvent for predominant
oxidation of sulfides in the coexistence of readily oxidizable
hydrazine. By contrast, 3-methyllumiflavin (Fig. 1b,
R1=R2=R3=R4=Me in Fl) was sluggish as a catalyst under the
same reaction conditions, which was not surprising because of
a kind of common knowledge that there is a huge difference in
stability between the active species, 4a-hydroperoxyflavins
FlOOH-Enz, FlEtOOH, and FlOOH (Fig. 1a and 1b). While FlOOH-Enz
The catalytic cycle of the oxygenation by Fl-Enz has been
well understood (Fig. 1a) as a result of numerous early studies
can be properly stabilized by hydrogen bonds between its
5
FlOOH and peripheral proteins
(
Enz
)
and also FlEtOOH
themselves are relatively stable,6 enzyme free FlOOH are
typically so labile and readily decomposed to H2O2 and Fl. In
1988, Tamao and co-workers introduced Fl-catalyzed aerobic
Tamao-Fleming oxidation, in which the eliminated H2O2 from
FlOOH was utilized as an oxidant for the reaction.3a Very
recently, König reported Fl-catalyzed oxidative chlorination of
arenes under visible-light irradiation, in which the eliminated
H2O2 from FlOOH was utilized for converting acetic acid to
a.Department of Applied Chemistry, Tokushima University, Minamijosanjima,
Tokushima 770-8506 (Japan).
b.Institute of Liberal Arts and Sciences, Tokushima University, Minamijosanjima,
Tokushima 779-8502 (Japan).
c. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro,
Tokushima 770-8514 (Japan).
d.Department of Environmental and Life Sciences, Toyohashi University of
Technology, Toyohashi 441-8580 (Japan).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
peracetic acid that subsequently oxidizes Cl– to OCl–
, the active
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The design of Fl-Pep (Fig. 1c) was started by hypothesizing the
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DOI: 10.1039/C7SC01933E
following things: (i) Pep should be connected to the N3
position of Fl relatively close to the active site; (ii) readily
available lumiflavin-3-acetic acid (3-FlC2)10 should be used as Fl
and incorporated to the N terminus of Pep; (iii) a simple di-
(AA1-AA2) or tripeptide (AA1-AA2-AA3) should be designed as
Pep using inexpensive
L-amino acids; (iv)
L-proline residue
should be placed at AA1 to induce constrained

-turn structure
and make the active site and AA2-AA3 spatially close; (v) AA2
and/or AA3 should be filled with acidic amino acid residues
that can be expected to interact with the active site by
intramolecular hydrogen bonds. In accordance with these
design policies, we initially supposed 3-FlC2-Pro-AA2 and 3-
FlC2-Pro-AA2-AA3 as the frameworks of Fl-Pep
.
To estimate
appropriate structures for AA2/AA3 in Fl-Pep, lowest energy
conformations of several FlOOH-Pep bearing different amino
acid residues in vacuum were explored by DFT calculation at
B3LYP/6-31G* level. Stable conformations of dipeptidic FlOOH
Pep, 3-FlC24a(R)OOH-Pro-Glu-NHMe, 3-FlC24a(R)OOH-Pro-Tyr-NHMe,
and 3-FlC24a(R)OOH-Pro-Gly-NHMe had no desirable
-
intramolecular hydrogen bonds in calculation. On the other
hand, tripeptidic 3-FlC24a(R)OOH-Pro-Tyr-Glu-NHMe was
suggested to be
a promising sequence whose stable
conformation includes ideal intramolecular hydrogen bonds
between (1) CO neighboring to the nitrogen atom of Pro and
NH of Tyr (-turn), (2) C(4)O of 3-FlC2 and OH in the side chain
Fig 1. Flavin-catalyzed aerobic oxygenation reaction.
of Tyr, and (3) 4aOOH of 3-FlC2 and CO in the side chain of Glu
(Fig. 2). Such a set of hydrogen bonds was not observed when
Tyr-Glu in 3-FlC24a(R)OOH-Pro-Tyr-Glu-NHMe was replaced with
other residues, Phe-Glu, Asp-Glu, and Tyr-Ser. In addition,
species for the chlorination.3i The only relevant work on FlOOH
-
related oxygenation was reported by Yoneda and co-workers
who showed that an artificial Fl bearing a carboxyl group at C6
position could promote the oxidation of thioanisole, although
the oxidant was H2O2 and its actual active species was not
identified.7 As a result, the development of Fl-Enz mimetic
aerobic oxygenation catalyzed by Fl has never been realized.
Herein, we present the first Fl-catalyzed aerobic
oxygenation reactions under non-enzymatic conditions. To
break through this long-standing challenge, we envisioned Fl
replacement of either Pro with -Ala or 3-FlC24a(R)OOH with 3-
FlC24a(S)OOH also led to loosing effective hydrogen bonds. These
results obtained from just the above 9 calculation samples (for
more details see ESI†) led us to synthesize Fl-Pep consisting of
the sequence of 3-FlC2-Pro-Tyr-Glu.
containing
a short peptide such as di- or tripeptides,
flavopeptide (Fl-Pep), which might be stabilized in its 4a-
hydroperoxy adduct (FlOOH-Pep) by intramolecular hydrogen
bonds between FlOOH and Pep (Fig. 1c). Though peptides as
catalysts have recently become powerful tools for organic
synthesis with the advancement of combinatorial “bottom-up”
screening methods using peptide libraries, the rational “top-
down” design of peptidic catalysts from a large degree of
molecular diversity is still highly challenging.8 In this study, we
successfully designed Fl-Pep as efficient catalysts for aerobic
sulfoxidation as well as aerobic Baeyer-Villiger oxidation by a
top-down approach that simply consists of computational
estimation9 followed by experimental fine-tuning of suitable
structures.
Fig 2
. Lowest energy structure of 3-FlC24a(R)OOH-Pro-Tyr-Glu-
NHMe estimated by DFT calculation (left) and graphical
representation of remarkable hydrogen bonds (right).
The synthesis of Fl-Pep was accomplished by standard solid
phase peptide synthesis following Fmoc/tBu protocol using an
amine-functionalized polystyrene resin (NH2-PS) (see ESI†).
Aerobic Sulfoxidation Catalyzed by Fl-Pep
First of all, 3-FlC2-Pro-Tyr-Glu-Ala-NH-PS (Fl-Pep1-a, Fig. 3)
bearing the peptide sequence designed by the above
computational calculation was synthesized and tested as a
polymer-supported peptide catalyst11 for aerobic oxidation of
Results and discussion
Computational Design and Synthesis of Fl-Pep
2 | J. Name., 2012, 00, 1-3
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Table
1
Flavopeptide-catalyzed aerobic oxViidewatAirotincle Oonlfine
thioanisolea
DOI: 10.1039/C7SC01933E
Entry
Catalyst
Fl-Pep1-a
Time (h) Yield (%)b
1c
2
36
24
36
36
36
36
36
36
36
24
24
24
24
24
24
24
24
9
Fl-Pep1-a
36 (60)d
3
3-methyllumiflavin
3-FlC2-NH-PS
2
4
1
5
Fl-Pep2-a
10
6
Fl-Pep3-a
25
7
Fl-Pep3-a + 10 mol% phenol
Fl-Pep4-a
18
8
15
9
Fl-Pep4-a + 10 mol% AcOH
Fl-Pep5-a
16
10
11
12
13
14
15
16
52 (78)d
44
Fl-Pep1-b
Fig 3. Structures of flavopeptides Fl-Pep1–Fl-pep5.
Fl-Pep2-b
18
thioanisole under conditions that were previously developed
Fl-Pep3-b
18
4
by us for the reaction catalyzed by FlEt+
.
In the presence of
Fl-Pep3-b + 10 mol% phenol
Fl-Pep4-b
16
10 mol% of Fl-Pep1-a and 1 atm of O2 and 4 equivalents of
hydrazine monohydrate in TFE, 9% of thioanisole was
converted to methyl phenyl sulfoxide in 36 h (Table 1, entry 1),
which was hopeful despite its low efficiency because the
reaction did not proceed at all in the absence of the catalyst
under otherwise identical conditions. As the efficiency of an
insoluble polymer-supported catalyst can be strongly
12
Fl-Pep4-b + 10 mol% AcOH
Fl-Pep5-b
10
17
62 (99)d
a
Reactions were performed using 0.1 mmol of thioanisole, 0.4
mmol of hydrazine monohydrate in 0.5 ml of a mixture of TFE
and DCE (1:1) in the presence of 10 mol% of the catalyst under 1
atm of O2 at 25 °C. b Determined by GC analysis. c In TFE. d Value
after 36 h.
influenced by the nature of
a
reaction solvent,12 we
subsequently used the mixed solvent of TFE and 1,2-
dichloroethane (DCE) that can make polystyrene resin well
swollen. As expected, the desired reaction was smoothly
catalyzed by Fl-Pep1-a to give methyl phenyl sulfoxide in 60%
yield in 36 h without any side reactions such as overoxidation
to methyl phenyl sulfone (Table 1, entry 2). It should be noted
that no reaction occurred in the absence of either TFE, O2,
hydrazine (NH2NH2), or Fl-Pep, which indicates that all of them
are essential. In addition, light was certainly shut out during
the successful reaction, so that the involvement of singlet
oxygen could be ruled out.3j,3m Moreover, the excellent
chemoselectivity, which is one of the feature of flavin
catalyst,1,2 could leave the participation of peracid out and
suggest FlOOH-Pep as a major oxidant. Furthermore, 3-
methyllumiflavin as well as 3-FlC2-NH-PS was ineffective as a
catalyst under the same reaction conditions (Table 1, entries 3
and 4), showing the Pro-Tyr-Glu sequence in Fl-Pep1-a is
responsible for its catalytic activity.
with Fl-Pep1 in the aerobic oxidation of thioanisole (Table 1).
When Pro was replaced with Ala (3-FlC2-Ala-Tyr-Glu-Ala-
NH-PS, Fl-Pep2-a), the catalytic activity dropped considerably
(entry 5). Likewise, the replacement of Tyr with Phe (3-FlC2-
Pro-Phe-Glu-
(3-FlC2-Pro-Tyr-Gln-
decreases in reaction efficiency, respectively (entries 6 and 8),
which were not improved even if a catalytic amount of phenol
(entry 7) or acetic acid (entry 9) was used as an external
additive. Interestingly, by contrast, enhancement of activity
was observed (entry 10) when Glu was replaced with Asp (3-

Ala-NH-PS, Fl-Pep3-a), and that of Glu with Gln
Ala-NH-PS, Fl-Pep4-a led to large

)
FlC2-Pro-Tyr-Asp-

Ala-NH-PS, Fl-Pep5-a).
These results
indicate that the structure and functionality of all amino acid
residues initially designed by the computational method was
crucial for the efficient catalysis and, in particular, the
carboxylic acid functionality of AA3 could play a significant role
for fine-tuning of the activity. The same tendency on catalytic
To explore structural and functional requirements for the
catalytic activity of Fl-Pep1, we synthesized some analogues Fl-
activities of Fl-Pep1
–
Fl-Pep5 was observed by using those
Pep2–Fl-Pep5 (Fig. 3) and compared their catalytic activity
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immobilized on polystyrene resin having a longer alkyl spacer
Fl-Pep1-b
Fl-Pep5-b, Fig. 3) with rather better performance stoichiometric oxidation of sulfides withDOFI:lE10tO.1O0H39/C7SC01933E
(entries 11 17), probably because both conformational and also those of the aerobic (
1.60)4b as well as H2O2
flexibility of the immobilized Fl-Pep and its accessibility to the sulfoxidation ( This result
1.90)4b catalyzed by FlEt+
–1.54 (Fig. 5). The  value is similar to that of the
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(
–
(=–
1.47)15
–
=–

=–
.
substrate are enhanced. The best efficiency was achieved with suggests that the present oxidation of sulfides takes place
Fl-Pep5-b for the present reaction, which provided methyl electrophilically with FlOOH-Pep as the active species.
phenyl sulfoxide in 99% yield in 36 h (entry 17).13 It should be
noted that all reaction yields in Table 1 were determined by GC
Aerobic Baeyer-Villiger Oxidation Catalyzed by Fl-Pep
analysis without product isolation to precisely evaluate the
catalytic activity of each Fl-Pep 14
In addition, no methyl
.
Encouraged by the above results we turned our attention to
the Baeyer-Villiger oxidation for expanding the utility of Fl-Pep
catalyst. Previously we developed this type of reaction
catalyzed by FlEt+, which has so far been the sole example of
organocatalytic Baeyer-Villiger oxidation using O2 as a terminal
oxidant.16 The active species of nucleophilic FlEtOOH generated
in situ allowed for selective Baeyer-Villiger oxidation of
phenyl sulfone was observed in any cases.
With these results in hand, we revisited the computational
prediction of Fl-Pep to ensure its validity. We calculated 3-
FlC24a(R)OOH-Pro-Tyr-Gln-NHMe, which was proven to be an
ineffective sequence (Table 1, entries
8 and 15), and
FlC24a(R)OOH-Pro-Tyr-Asp-NHMe, which was found to be the
most effective sequence (Table 1, entries 10 and 17). In
accordance with the experimental results, an effective set of
hydrogen bonds (1), (2), and (3), similar to that highlighted in
Figure 2, were observed only in the lowest energy structure of
FlC24a(R)OOH-Pro-Tyr-Asp-NHMe (Fig. 4, for others see ESI†). It
seems obvious that the Asp-derivative (Fig. 4) has an even
better coordination than the Glu-derivative (Fig. 2) between
the carboxylic acid and the hydroperoxy moiety with an
additional interaction (4).
cyclobutanones into the corresponding -butyrolactones in the
presence of alkene or sulfide functionality that could be readily
oxidized with mCPBA, a typical oxidant for Baeyer-Villiger
oxidation. Thus, Fl-Pep has also a great potential in the
development of aerobic Baeyer-Villiger oxidation, and such
chemoselectivity will be a strong evidence for the involvement
of FlOOH-Pep as an active species.
The Baeyer-Villiger oxidation of 3-phenylcyclobutanone
into
-phenyl--butyrolactone was used as a test reaction
under conditions that were previously developed by us for the
16
reaction catalyzed by FlEt+
.
In the presence of 5 mol% of Fl-
Pep5-b, 1 atm of O2, 20 equivalents of H2O, and 3.5
equivalents of zinc dust in a mixed solvent of acetonitrile,
toluene, and ethyl acetate (8:4:1), the desired oxidation
proceeded smoothly to afford the target product in 72% yield
in 7 h (Table 2, entry 1).17 Ethanol can be used instead of both
CH3CN as a hydrophilic co-solvent and water as an essential
Fig 4
. Lowest energy structure of 3-FlC24a(R)OOH-Pro-Tyr-Asp-
proton source,16 however, toluene was crucial as
a
NHMe estimated by DFT calculation (left) and graphical
representation of remarkable hydrogen bonds (right).
hydrophobic co-solvent that could render polystyrene resin
properly swollen (see ESI†). As expected, 3-methyllumiflavin
(entry 2) as well as 3-FlC2-NH-PS (entry 3) was totally inactive
under the same reaction conditions. These results convinced
To gain an insight into active species for the oxygen
transfer, we performed a Hammett study for the present
aerobic sulfoxidation using Fl-Pep1-a
values for p-substituted thioanisoles with respect to X=H
(kX/kH) were determined, and the corresponding log(kx/kH)
versus the Hammett values were plotted to give value of
. The relative reactivity
Table
2
Flavopeptide-catalyzed aerobic Baeyer-Villiger
oxidationa
–


Entry
Catalyst
Yield (%)b
1
2
Fl-Pep5-b
72
<1
2
3-methyllumiflavin
3-FlC2-NH-PS
3
a
Reactions were performed using 0.1 mmol of 3-
phenylcyclobutanone, 0.35 mmol of zinc, 2.0 mmol of H2O in 1.0
ml of a mixture of acetonitrile, toluene, and ethyl acetate (8:4:1)
in the presence of 5 mol% of the catalyst under 1 atm of O2 at
Fig 5
. Hammett plot for aerobic oxidation of p-substituted
b
35 °C. Determined by NMR analysis using dodecane as an
internal standard.
methyl phenyl sulfides catalyzed by Fl-Pep1-a
.
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us that, as in the case of the above sulfoxidation, an large difference in structure and functionality. Given that Ac-
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appropriate peptide sequence in Fl-Pep is essential for the Pro-NHMe predominantly favours the tDraOnI:s1-0C.71039/C7SC01933E
catalysis involving the key stabilization of FlOOH-Pep as over other forms including the cis form in the gas phase and
illustrated in Figure 4.
non-polar solvents,20 it is plausible that the flavopeptide
With the appropriate conditions in hand, we then carried moiety surrounded by the strongly hydrophobic environment
out the Baeyer-Villiger oxidation of 3-phenylcyclobutanone in Fl-Pep5 also populates the -turn form as included in the
form (-turn)

catalyzed by Fl-Pep5-b in the presence of an equimolar predicted stable conformation of 3-FlC24a(R)OOH-Pro-Tyr-Asp-
amount of other reactive substrate. Cyclooctene as a NHMe (Fig 4). In fact, the catalytic activity of 3-FlC2-Pro-Tyr-
competitor remained intact during the desired conversion of Asp-Ado-NH2 was found to be much lower (7% yield in 24 h,
the ketone (eq. 1), whereas the preferential formation of see ESI†) than that of Fl-Pep5-b (Table 1, entry 17) in the
cyclooctene oxide has occurred under mCPBA-based sulfoxidation of thioanisole under the same conditions,
conditions (eq. 2). Such excellent chemoselectivity was also showing the importance of the hydrophobic support resin that
observed in a compeptitive oxygenation of the ketone and would make the flavopeptide conformationally profitable.21
thioanisole (eqs. 3 and 4). These results strongly suggest that
peracid does not participate in the Fl-Pep5 systems (eqs. 1 and
3) and, given that the ketone underwent oxidation
Mechanistic Aspects of Fl-Pep-Catalyzed Aerobic Oxygenations
Given all the above experimental facts, it is plausible to
consider that both the sulfoxidation and the Baeyer-Villiger
oxidation catalyzed by Fl-Pep occur via Fl-Enz-like mechanism
(Fig. 6). As for the sulfoxidation, since effective Fl-Pep1 and Fl-
Pep5 possess a carboxyl group that can readily react with an
predominantly, the corresponding FlOOH-Pep5 can be rather
nucleophilic as opposed to the above chemoselective
sulfoxidation.18
equivalent of NH2NH2 to be the corresponding salt (Fl-Pep
‧
NH2NH2) in situ, the catalytic cycle (Fig. 6a) can be initiated by
reducing Fl-Pep‧NH2NH2 with another molecule of NH2NH2 to
afford FlH2-Pep‧NH2NH2 and diazene (NH=NH). The resulting
NH=NH can also be used to reduce Fl-Pep
second cycle by releasing N2. Molecular oxygen can be then
inserted into the C(4a) of FlH2-Pep‧NH2NH2 to give FlOOH-Pep
NH2NH2. This hydroperoxy species may be effectively
stabilized to perform subsequent monooxygenation of a
substrate to give the corresponding 4a-hydroxy adduct (FlOH
‧NH2NH2 from the
‧
-
Pep ‧ NH2NH2), which finally undergoes dehydration to
regenerate Fl-Pep‧NH2NH2. The Hammett study (Fig. 5) shows
that the oxygen transfer from FlOOH-Pep ‧ NH2NH2 to a
substrate is a rate-determining step of the proposed catalysis
and takes place electrophilically. Although it is not trivial to
verify the generation of the FlOOH-Pep species spectroscopically
due to the insolubility of resin, for the present, it is reasonable
to understand that FlOOH-Pep can be stabilized by means of
intramolecular hydrogen bonds similar to those predicted by
the DFT calculations (Figs. 2 and 4) including a probable
Conformational Analysis of Fl-Pep
We synthesized the soluble analogue of Fl-Pep5, 3-FlC2-Pro-
Tyr-Asp-Ado-NH2, using Rink amide Resin to gain its
conformational information by NMR spectroscopy (see ESI†).
3-FlC2-Pro-Tyr-Asp-Ado-NH2 was soluble in polar solvents such
as dimethyl sulfoxide and methanol, but unfortunately, hardly
soluble in less polar solvents such as acetonitrile, acetone, and
chloroform. Thus, DMSO-d6 was inevitably used as the solvent,
although it is quite unlike the actual reaction
microenvironment that must be much less polar because of
the hydrophobic nature of polystyrene resin. The NMR
analysis showed that 3-FlC2-Pro-Tyr-Asp-Ado-NH2 forms two
different conformers in DMSO-d6 at 25 °C in a ratio of ~1.4:1,
in which the major conformer has 3-FlC2-Pro amide bond in
the trans conformation (58%), while that of the minor
conformer is cis (42%). It should be noted that the observed
trans-cis ratio for 3-FlC2-Pro-Tyr-Asp-Ado-NH2 in DMSO-d6 is
similar to that for N-acetyl-L-proline N’-methylamide (Ac-Pro-
NHMe, 65% trans)19 in the same solvent regardless of their
+
coordination between C(4a)O of 3-FlC2 and NH3NH2 to make
the hydroperoxy moiety electrophilic (Fig. 6b). The fact that
Asp instead of Glu in AA3 enhanced the catalytic activity (Table
1, entries 2 vs 10 and entries 11 vs 17) could be rationalized by
assuming such stabilization model that allows for a spatially
less-forced intervention of NH2NH2 in between the carboxyl
group and the hydroperoxy group. We consider that the
presence of +NH3NH2 is a key for stabilizing FlOOH-Pep, which is
a similar situation to FlOOH-Enz that can be stabilized by
complexation with NAD(P)+.5c Actually, an additional
experiment on the effect of equivalents of hydrazine
monohydrate for the present sulfoxidation revealed that the
larger equivalents of NH2NH2, the faster reaction rate (see
ESI†).22
On the other hand, FlOOH-Pep in the Baeyer-Villiger
oxidation can be formed via the reduction of Fl-Pep with Zn
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DOI: 10.1039/C7SC01933E
Fig 6. Proposed catalytic cycles and transition state models for Fl-Pep-catalyzed aerobic (a and b) sulfoxidation and (c and d)
Baeyer-Villiger oxidation.
and H2O into FlH2-Pep followed by the oxygen insertion to the computational design of Fl-Pep catalyst was reasonable
FlH2-Pep and stabilized by computationally predicted hydrogen although the fine-tuned Fl-Pep5 showed superior activity than
bonds (Fig. 4) including a cyclic coordination between 4aOOH the original Fl-Pep1. On the other hand, the use of zinc as an
of 3-FlC2 and COOH in the side chain of Asp, which then alternative reductant under suitable conditions was found to
selectively oxidizes the ketone into the lactone to give FlOH-Pep allow for Fl-Pep5-catalyzed aerobic Baeyer-Villiger oxidation
that finally release H2O to regenerate Fl-Pep. (Fig. 6c). The with excellent chemoselectivity. Multiple control experiment
nucleophilic activity of FlOOH-Pep can be explained by assuming as well as mechanistic experiment suggested that both types
a transition state model involving simultaneous activation of of oxygenations could proceed via Fl-Enz-like mechanism and
the hydroperoxy moiety and the keto-carbonyl moiety by the the active species could be FlOOH that had been efficiently used
COOH group (Fig. 6d), although the involvement of Zn+(OH) only in Fl-Enz so far. It is noteworthy that the electronic
instead of H+ cannot be excluded for the moment.
properties of the hydroperoxy moiety in FlOOH-Pep can be
orthogonally controlled by reductants and reaction conditions,
realizing electrophilic sulfoxidation as well as nucleophilic
Baeyer-Villiger oxidation in a highly chemoselective manner.18
We believe that the results are so important for the research
fields of both flavin chemistry and peptide chemistry, because
they not only provide new possibilities for the development of
flavin catalysts as well as the fundamental study on flavin-
containing monooxygenase but also demonstrate great
potential of computational chemistry for the rational design of
peptide-based catalysts.
Conclusion
In conclusion, the first Fl-Enz-mimetic aerobic oxygenation
reactions catalyzed by Fl under non-enzymatic conditions were
realized. We predicted the structure of Fl-Pep that could
stabilize the corresponding FlOOH-Pep by a computational
method, and synthesized the most promising Fl-Pep1 and its
analogies Fl-Pep2–Fl-Pep5 as resin-immobilized peptides.
Exploring their catalytic activity for aerobic sulfoxidation using
hydrazine monohydrate as terminal reductant revealed that
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9
A splendid work on the structural analysis of catalytic
peptides using DFT calculation tDoOgeI:t1h0e.1r03Vi9ew/wCiAt7rhStiCcl0e1XO9-n3rlia3nEye
crystallography and NMR spectroscopy was recently
reported by Miller’s group, see: A. J. Metrano, N. C. Abascal,
B. Q. Mercado, E. K. Paulson, A. E. Hurtley, S. J. Miller, J. Am.
Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research on Innovative Areas ‘Advanced Molecular
Transformations by Organocatalysts’ from MEXT.
Chem. Soc. 2017, 139, 492–516.
10 H. Ikeda, K. Yoshida, M. Ozeki, I. Saito, Tetrahedron Lett.
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Notes and references
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13 The product was racemic. To achieve not only the
stabilization of active species but also enantioselective
,
reactions, the catalysts must be designed in
a more
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elaborated perspective. However, it would be interesting to
note that the oxidation of 2-(4-methoxyphenyl)-1,3-dithiane
could also be promoted with Fl-Pep5-b under the optimal
conditions found for the sulfoxidation of thioanisole to give
2
3
(a) H. Iida, Y. Imada, S.-I. Murahashi, Org. Biomol. Chem.
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diastereoselectivity of 24:1 (trans:cis), and an
a
enantioselectivity of 3% ee (trans). This preliminary result
shows a possibility of the development of asymmetric
flavopeptidic catalysts.
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14 To demonstrate the feasibility of product isolation, we
carried out the oxidation of thioanisole with Fl-Pep5-b under
the optimized conditions in a larger reaction scale (1 mmol
of thioanisole, see ESI†). The reaction was rather less
efficient compared with the standard scale, possibly due to
varied mixing efficiency of the gas-liquid-solid triphasic
system, so that 4 equivalents of hydrazine monohydrate was
added after 14 h to the reaction mixture. After 48 h in total,
methyl phenyl sulfoxide was obtained in 94% GC yield and,
after purification by silica gel column chromatography, in
85% isolated yield (119 mg, 0.85 mmol).
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18 Similar orthogonal reactivities in catalytic oxygenations were
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transiently formed peracid of the aspartyl group unlike that
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21 Such an effect that gives rise to the unordinary catalytic
function on polystyrene resin is still unusual. For a recent
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22 Hydroxylamine can also be used as an alternative reductant,
although the reaction has required heating (60 C) to be
View Article Online
DOI: 10.1039/C7SC01933E
°
efficient (see ESI†). We suppose that hydroxylamine serves
the same role as hydrazine (Figs 6a and 6b) and the
insufficient activity is ascribable to its lower oxidation
potential than that of hydrazine. For the oxidation potentials
of hydrazine and hydroxylamine, see: J. Li, X. Lin, Sens.
Actuators B Chem. 2007, 126, 527–535.
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