Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme

Article abstract of DOI:10.1002/cbic.201500165

A bicyclo[6,1,0]nonyne-substituted 9-mesityl-10-methyl-acridinium cofactor was prepared and covalently linked to a prolyl oligopeptidase scaffold containing a genetically encoded 4-azido-L-phenylalanine residue in its active site. The resulting artificial

Full text of DOI:10.1002/cbic.201500165

DOI: 10.1002/cbic.201500165
Communications
Preparation, Characterization, and Oxygenase Activity of
a Photocatalytic Artificial Enzyme
Yifan Gu, Ken Ellis-Guardiola, Poonam Srivastava, and Jared C. Lewis*[a]
A bicyclo[6,1,0]nonyne-substituted 9-mesityl-10-methyl-acridi-
nium cofactor was prepared and covalently linked to a prolyl
oligopeptidase scaffold containing a genetically encoded 4-
azido-l-phenylalanine residue in its active site. The resulting
artificial enzyme catalyzed sulfoxidation when irradiated with
visible light in the presence of air. This reaction proceeds by
initial electron abstraction from the sulfide within the enzyme
active site, and the protein scaffold extended the fluorescence
lifetime of the acridium cofactor. The mode of sulfide activa-
tion and placement of the acridinium cofactor (5) in POP-ZA4-5
make this artificial enzyme a promising platform for develop-
ing selective photocatalytic transformations.
Photosensitizers, including [Ru(bpy)3]2+ and a range of other
coordination complexes and organic dyes, are used as catalysts
for a growing family of organic transformations.[1b] For exam-
ple, 9-mesityl-10-methylacridinium (Acr+-Mes) perchlorate has
been shown to catalyze a range of heteroatom, arene, and
olefin functionalization reactions upon irradiation with visible
light (l>450 nm).[8] This reactivity arises from the oxidizing
properties of this species in the excited state (Ered =+1.88 V vs.
SCE, PhCN).[9] Extensive evidence suggests that Acr+-Mes is
photoexcited to the singlet excited state, 1Acr+*-Mes, which
+
C
C
undergoes intramolecular electron transfer to form Acr -Mes
,
the proposed active oxidant.[9] We hoped that incorporating an
Acr+-Mes-based cofactor into a protein scaffold could generate
a photocatalytic enzyme for organic oxidation reactions. Al-
though a wide range of organic and organometallic cofactors
have been incorporated into protein scaffolds to create artifi-
cial enzymes,[10] no systems have been reported in which a visi-
ble light photocatalyst that acts directly on an organic sub-
strate is linked to a protein. We envisioned that such a system
could be used to modulate the photophysical properties of
visible light photocatalysts such as Acr+-Mes, in analogy to
natural systems,[4] and ultimately impart selectivity to reactions
catalyzed by these species. As a first step towards these goals,
we describe the preparation and detailed characterization of
an Acr+-Mes-based artificial enzyme that catalyzes sulfoxida-
tion when irradiated with visible light in the presence of air.
We recently reported that bicyclo[6,1,0]nonyne (BCN)-substi-
tuted cofactors could be covalently linked to p-azido-l-phenyl-
alanine (Z)-substituted protein scaffolds by strain-promoted
azide–alkyne cycloaddition (SPAAC).[11] This bioconjugation
method allows rapid, site-selective incorporation of cofactors
into protein scaffold. To explore the possibility of incorporating
Acr+-Mes cofactors into proteins, we prepared BCN-substituted
Acr+-Mes, 5 (Scheme 1). TBS protection of alcohol 1, followed
by lithiation and addition to N-methylacridone, in analogy to
the synthesis of Acr+-Mes,[12] provided alcohol 3. Reaction of
this alcohol with carbonate 4 provided 5.
Chemists have long sought to design catalysts that mimic the
ability of natural photosynthetic systems to harvest visible
light photons and use the energy from photon absorption to
drive chemical reactions.[1] The protein complexes that evolved
to accomplish these tasks, photosystems I and II, catalyze
transmembrane electron transport and light-driven water oxi-
dation, respectively.[2] Photosynthetic organisms use the elec-
trons transported by photosystem I (PSI) to reduce NADP+ to
NADPH, but a number of biohybrid systems capable of har-
nessing these electrons to reduce protons to H2 for solar fuels
applications have been developed by linking PSI to synthetic
catalysts.[3] These systems show that the unique photophysical
properties imparted to natural chromophores embedded
within the PSI reaction center protein scaffolds[4] can be ex-
ploited for non-natural chemical reactions.
Recently, an alternative biohybrid catalyst for light-driven
proton reduction was created by bioconjugation of a hydro-
gen-evolving di-iron complex to a protein scaffold lacking any
bound chromophores.[5] The photosensitizer [Ru(bpy)3]2+ was
added to a solution of the biohybrid to enable intermolecular
electron transfer to the di-iron complex upon irradiation with
visible light and catalytic proton reduction in the presence of
ascorbate. Prior to this work, Gray[6] and Cheruzel[7] demon-
strated that electrons supplied to a cytochrome P450 BM3 var-
iant by a covalently linked [Ru(bpy)3]2+ derivative upon irradia-
tion with visible light could be used to drive hydroxylation of
organic molecules, illustrating the potential of light-driven bio-
hybrid systems to catalyze organic transformations.
We selected a prolyl oligopeptidase (POP) from Pyrococcus
furiosus (Pfu) as
a
scaffold for bioconjugation of
5
(Scheme 2).[13] Because the structure of Pfu POP has not been
solved, a previously reported homology model[14] of this
enzyme was used to guide our engineering efforts. This model
suggested that Pfu POP possesses a large active site cavity
within a b-barrel domain that is ideally suited for cofactor en-
closure (Figure 1A). This unique structure and the high stability
of Pfu POP makes it an attractive candidate for artificial
enzyme formation, despite the lack of a crystal structure. As in
our initial report of artificial enzyme formation by SPAAC,[11] an
amber codon was introduced into the POP gene[15] to replace
[a] Y. Gu, K. Ellis-Guardiola, P. Srivastava, Prof. J. C. Lewis
Department of Chemistry, University of Chicago
5735 S. Ellis Avenue, Chicago, IL 60637 (USA)
E-mail: jaredlewis@uchicago.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500165.
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Scheme 1. Synthesis of BCN-substituted 9-mesityl-10-methylacridinium co-
factor 5 (pNP=p-nitrophenyl ester). a) TBSCl, imidazole, CH2Cl2, RT, quant.;
b) tBuLi, N-methylacridone, Et2O, À788C–RT; c) HClO4, EtOAc, RT (75%); d) 4,
TEA, DMAP, CH2Cl2, RT (73%).
Figure 1. A) Qualitative model of POP-ZA4-5 constructed in PyMOL by fusing
a DFT-optimized structure of the cofactor to a homology model of the POP
scaffold. Sites of alanine mutations are shown in magenta (inlay: expanded
view of cofactor). B) UV/Vis spectra of Acr+-Mes (c), POP-ZA4 (c), 5
(c), and POP-ZA4-5 (c). C) HR-ESI MS spectra of POP-ZA4 (c) and
POP-ZA4-5 (c).
Table 1. Efficiency and scope of POP-ZA4-5-catalyzed sulfoxidation.[a]
R1, R2
Cat.
Yield [%][b]
1[c]
2
H, Me
H, Me
H, Me
H, Me
Acr+-Mes ClO4À
Acr+-Mes ClO4À
POP-ZA4-5
POP-ZA4-5
POP
POP-ZA4-5
POP-ZA4-5
POP-ZA4-5
POP-ZA4-5
90
70
45
n.r.
n.r.
90
20
0
Scheme 2. Formation of artificial enzyme POP-ZA4-5. a) 5, 5% ACN/Tris.
3
4[d]
5
the catalytically active serine (S477) with a Z residue (Z477).
Four alanine mutations (E104A, F146A, K199A, and D202A)
were introduced to open a small pore in the POP structure[16]
and allow cofactor entry into the active site. A codon-opti-
mized POP gene was used as a template for genetic manipula-
tion, and the resulting scaffold, POP-ZA4, was expressed in
high yield in Escherichia coli with essentially quantitative Z
incorporation. Bioconjugation of POP-ZA4 with 5 was readily
monitored by using SDS-PAGE, followed by UV visualization,
and the expected mass of POP-ZA4-5 was confirmed by using
HR-ESI MS (Figure 1B, C). Moderate bioconjugation yields (ca.
50%) were obtained, due in part to decomposition of the Z
azide to the corresponding aniline.[17]
Acr+-Mes was also prepared according literature methods[12]
and used to evaluate the efficiency of POP-ZA4-5 catalysis.[18]
Although trace conversion was observed for several olefin ad-
dition reactions[11] catalyzed by Acr+-Mes in aqueous solution
irradiated with visible light, aerobic sulfoxidation[19] of thioani-
sole proceeded with relatively high and consistent yields in
both acetonitrile and acetonitrile/buffer (Tris or phosphate) sol-
utions (Table 1, entries 1 and 2). POP-ZA4-5 also catalyzed this
reaction with modest conversion (entry 3), demonstrating for
H, Me
6
7
8
9
p-OMe, Me
o-CO2Me, Me
p-NO2, Me
H, Et
26
[a] Reaction performed with 2 mm sulfide, 0.1 mm catalyst in air-saturated
20% acetonitrile (ACN)/50 mm tris buffer-HCl (pH 7.4), irradiated by
450 nm LEDs at RT. [b] GC yield. [c] In ACN. [d] Without LEDs.
the first time that a photocatalytic artificial enzyme could di-
rectly catalyze an organic transformation. The artificial enzyme
tolerated up to 20% (v/v) acetonitrile, but higher co-solvent
concentrations led to extensive enzyme precipitation over the
course of the reaction. No activity was observed in the absence
of visible light or when using POP lacking cofactor 5 (entries 4
and 5), and overoxidation (sulfone) was not observed in any
cases. A clear trend favoring sulfoxidation of electron-rich thio-
anisoles was observed (entries 3 and 6–8), and ethyl phenyl
sulfide also underwent sulfoxidation (entry 9). The sulfoxide
products of these reactions were isolated as racemic mixtures,
and the yields (entries 2 and 3) and rates (Figure S6 in the Sup-
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Communications
porting Information) for reactions catalyzed by POP-ZA4-5 were
generally lower than those catalyzed by 5. Analogous prob-
lems are often observed for initial artificial enzyme con-
structs,[10] including the aforementioned photocatalytic hydro-
gen evolution system,[5] as a result of suboptimal active site
configurations. Nonetheless, these systems offer unique start-
ing points for engineering efforts to improve catalytic efficien-
cy.[20]
significant difference in photobleaching rates and only minor
differences between the absorbance and emission spectra of
Acr+-Mes and POP-ZA4-5 (Figure 2B, Figure S1), but several no-
table differences in the fluorescence lifetimes of these species
were apparent. First, a modest reduction in the fluorescence
lifetime of Acr+-Mes was observed in the presence of air, and
further reduction occurred in aqueous solution, the conditions
used for photocatalysis (Table 2, entries 1–3). Under these
Photocatalytic oxygenation of sulfides[22] and phosphines[21]
has been proposed to proceed by two distinct, often substrate
dependent, mechanisms[19] that are initiated by photocatalysts
through either 1) 1O2 formation or 2) electron abstraction from
the organic substrate. This distinction is important for the pros-
pect of engineering selective artificial enzymes, as the sub-
strate would more likely be located within the protein scaffold
during the oxygenation process in the latter case (2). Although
most reports indicate that this pathway is typically operative
for sulfoxidation of thioanisoles,[19] we wanted to establish that
this was indeed the case for POP-ZA4-5, because in the former
case (1), the artificial enzyme could simply serve as a source of
1O2.[22]
Table 2. Fluorescent lifetimes of Acr+-Mes species.
Catalyst
Solvent
Atm.
Lifetime [ns][a]
1
2
3
4
5
Acr+-Mes
Acr+-Mes
Acr+-Mes
POP-ZA4-5
POP-Z37-5
ACN
ACN
20%ACN·Tris
20%ACN·Tris
20%ACN·Tris
N2
air
air
air
air
8.19
6.65
4.10
4.92
4.72
[a] Lifetimes were calculated by using a fractional average of two expo-
nential functions that were required to accurately model the observed
fluorescence decay data (Table S3).[27]
A series of experiments previously used to distinguish these
mechanisms[19] was therefore carried out. We observed no in-
hibition of POP-ZA4-5-catalyzed sulfoxidation of thioanisole in
same conditions, however, a longer lifetime was observed for
POP-ZA4-5 (entry 4). Very similar trends in the fluorescence life-
times of [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) dyes have
also been observed[26] and used to probe conformational
changes in the maltose ATP-binding cassette (ABC) transport-
er.[27] Specifically, the fluorescence lifetime of a maleimide-sub-
stituted DBD dye linked to a cysteine mutant of the MalF sub-
unit of the MalFGK2 complex increased from 5 to 6.3 ns when
MalF was bound with vanadate, which locks the enzyme in a
conformation that is proposed to generate a more hydropho-
bic environment[28] around the DBD dye. A similar increase in
the fluorescence lifetime of Acr+-Mes in POP-ZA4-5 (Table 2,
entry 4) relative to that in solution (entry 3) or on the outside
of the protein (POP-Z37-5, entry 5) suggests that 5 resides
within the hydrophobic POP active site of POP-ZA4-5. Although
obviously far from the dramatic effects of photosystem pro-
teins on their associated chromophores, these results provide
evidence that POP can influence the photophysical properties
of bioconjugated cofactors.
1
the presence of DABCO (0.05–0.2 equiv), a known O2 quench-
er, and no reaction of diphenyl sulfide, which is readily con-
1
verted to the corresponding sulfone by using O2 sensitizers.[23]
In addition, increased concentrations of 4-methoxy thioanisole
led to increased conversion of this substrate through sulfoxida-
tion (Figure 2). This is contrary to what is observed for sulfoxi-
1
dation reactions proceeding according to O2 mechanisms, as
1
the excess sulfide leads to O2 quenching and thus decreasing
conversion with increasing sulfide concentrations.[19] Together,
these observations suggest that 1O2 is not involved in POP-
ZA4-5-catalyzed sulfoxidation of thioanisoles. Analogous results
were obtained for reactions catalyzed by 5, indicating that the
POP scaffold did not alter the reaction mechanism.
The photophysical properties of Acr+-Mes have been exten-
sively studied[9] (and debated[24]) by using a range of spectro-
scopic methods, and significant medium effects on the fluores-
cence emission and lifetime of acridinium dyes have been re-
ported.[25] To determine if the POP scaffold has any impact on
the properties of the Acr+-Mes moiety, we also investigated
the photophysical properties of POP-ZA4-5. We observed no
This communication describes the synthesis of a BCN-substi-
tuted Acr+-Mes photocatalyst (5) that can be linked to pro-
teins containing a genetically encoded Z residue. Linking 5 to
an engineered POP scaffold led to the formation of the first re-
ported photocatalytic artificial enzyme to act directly on an or-
ganic substrate. A series of mechanistic experiments provided
evidence that sulfoxidation catalyzed by POP-ZA4-5 (and by 5
alone) proceeds by initial electron abstraction from the sulfide,
1
rather than through O2. Fluorescence lifetime measurements
were consistent with the cofactor being located within the hy-
drophobic POP active site. The apparent mode of sulfide acti-
vation and placement of 5 in POP-ZA4-5 make this artificial
enzyme a promising platform for developing selective photo-
catalytic transformations, and further studies are underway to
achieve this goal.
Figure 2. Sulfoxide formed after 8 h irradiation vs. initial concentration of 4-
methoxyphenyl methyl sulfide. POP-ZA4-5 loading: 0.05 mm. Total volume:
300 mL 20% v/v ACN·Tris. Conversions ranged from 8–40%.
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[10] J. C. Lewis, ACS Catal. 2013, 3, 2954–2975.
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Acknowledgements
We thank Professor Jeffry Madura at Duquesne University for pro-
viding us with the POP homology model used in these studies,
Professor Harold Schreier at the University of Maryland, Baltimore
County for providing the Pfu POP gene, and Dr. Justin Jureller at
the Institute for Biophysical Dynamics NanoBiology Facility (NIH
grant 1S10RR026988-01) for assistance with fluorescence lifetime
measurements. MS data were acquired on instruments purchased
with an NSF instrumentation grant (CHE-1048528). K.E.-G. is
funded by an NIH Chemistry and Biology Interface Training Grant
(T32 GM008720). This work was funded by an NSF CAREER
Award to J.C.L. (CHE-1351991).
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Keywords: artificial enzymes · oxygenases · photocatalysts ·
prolyl oligopeptidases · unnatural amino acids
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Manuscript received: March 29, 2015
Accepted article published: June 19, 2015
Final article published: July 14, 2015
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