Site-Selective Functionalization of Methionine Residues via Photoredox Catalysis

Article abstract of DOI:10.1021/jacs.0c09926

Bioconjugation technologies have revolutionized the practice of biology and medicine by allowing access to novel biomolecular scaffolds. New methods for residue-selective bioconjugation are highly sought to expand the toolbox for a variety of bioconjugati

Full text of DOI:10.1021/jacs.0c09926

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Site-Selective Functionalization of Methionine Residues via
Photoredox Catalysis
Junyong Kim, Beryl X. Li, Richard Y.-C. Huang, Jennifer X. Qiao, William R. Ewing,
and David W. C. MacMillan*
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ABSTRACT: Bioconjugation technologies have revolutionized the practice of biology and medicine by allowing access to novel
biomolecular scaffolds. New methods for residue-selective bioconjugation are highly sought to expand the toolbox for a variety of
bioconjugation applications. Herein we report a site-selective methionine bioconjugation protocol that uses photoexcited lumiflavin
to generate open-shell intermediates. This reduction-potential-gated strategy enables access to residues unavailable with traditional
nucleophilicity-based conjugation methods. To demonstrate the versatility and robustness of this new protocol, we have modified
various proteins and further utilized this functional handle to append diverse biological payloads.
he development of general strategies for the selective
modification of proteins remains an important challenge
transfer (SET) events, gated by amino acid oxidation
potentials, can selectively target specific residues to yield
reactive open-shell radical intermediates, leaving other residues
intact. Crucially, the mild and highly selective nature of
photoredox catalysis, as well as its compatibility with biological
conditions, renders it an ideal platform for the development of
new bioconjugation technologies.9 Along these lines, our group
recently reported the selective C-terminal alkylation of proteins
via photoredox catalysis,10 wherein the C-terminal carboxylate
group is selectively oxidized in preference to aspartate and
glutamate residues owing to subtle differences in oxidation
potential.
In an effort to expand the reach of our photoredox
bioconjugation platform, we aim to develop new conjugation
techniques targeting other redox-active residues. Under the
traditional nucleophilicity-based reactivity paradigm, bioconju-
gation of methionine has posed a particular challenge because
of the pronounced hydrophobicity and weak nucleophilicity of
the thioether side chain.11 However, methionine is readily
oxidizable,12 rendering it potentially susceptible to functional-
ization via photoredox catalysis. Along these lines, the Chang
and Toste groups13 and the Gaunt group14 recently reported
methionine-selective bioconjugation methods that employ
chemoselective sulfur-oxidizing reagents to convert the
thioether moiety to a sulfilimine or α-azo sulfonium ion,
respectively, seminal studies that are likely to have a broad
range of pharmaceutical applications, including antibody−drug
conjugates,15 stapled peptides,16 and positron emission
tomography17 and optical18 imaging and diagnostics.
T
in chemical biology.1 Especially, the emergence of small-
molecule−protein conjugates as a new class of pharmaceuticals
has inspired a wealth of research efforts aimed at the discovery
of new methods to achieve site- and chemoselective
bioconjugation of wild-type proteins. To date, a number of
elegant strategies have been devised that permit the direct
functionalization of endogenous amino acids.2 However,
despite impressive advances in this area, there remain
significant selectivity challenges due to the inherent structural
complexity of proteins.
Traditional bioconjugation methods rely on substitution
reactions between nucleophilic amino acid residues and small-
molecule electrophiles to covalently link the biological payload
at specific sites on the protein. Early bioconjugation protocols
had been focused on highly nucleophilic residues, such as
lysine3 and cysteine,4 in order to ensure chemoselective
reactivity under the requisite aqueous conditions. Furthermore,
under a nucleophilicity-based reactivity paradigm, it is difficult
to control the structural homogeneity of the modified protein
mixture. The surface abundance of lysine residues renders site-
selective bioconjugation challenging, while cysteine-based
modifications sometimes require pretreatment of the native
protein to reduce disulfide bonds where free cysteine residues
are unavailable.4a In light of these challenges, recent efforts
have focused on achieving site selectivity through the use of
less abundant residues, such as tyrosine,5 tryptophan,6 and
histidine.7
Given the aforementioned complexities in implementing
nucleophilicity-based bioconjugation strategies, our laboratory
has been pursuing an alternative approach wherein site
selectivity is attained by exploiting differences in the inherent
reduction potentials of the native amino acid residues.
According to our photoredox-based strategy, photonic energy
from visible light is harvested by a photocatalyst and delivered
as an electrochemical potential (Figure 1).8 Single electron
Received: September 16, 2020
https://dx.doi.org/10.1021/jacs.0c09926
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© XXXX American Chemical Society
A

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Figure 1. Development of a methionine-selective bioconjugation method via photoredox catalysis.
Figure 2. Optimized conditions and proposed mechanism for the alkylation of methionine with lumiflavin photocatalyst.
Alternatively, using our photoredox-based strategy, we
envisioned achieving single-electron oxidation of sulfur by an
excited photocatalyst, with subsequent α-deprotonation to
generate a carbon-centered α-thio radical. This nucleophilic
radical species could then react with SOMOphilic alkylating
reagents (Figure 1b). In contrast to other methionine-based
methods, in which thioether modification occurs on the sulfur
atom, our proposed method involves conjugation to the
adjacent carbon to irreversibly afford a stable conjugate
product. The successful development of such a methionine
conjugation protocol would underscore the utility of photo-
redox catalysis in facilitating the mild, selective, and robust
functionalization of complex biomolecules.
B
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a
Table 1. Scope of Michael Acceptors for the Photoredox Methionine-Selective Alkylation
a
Conditions: aprotinin (50 μM), 3 (500 μM), and Michael acceptor (10 mM) in PBS (pH 7.4)/DMF (19:1) under irradiation at 440 nm for 30
min. See the Supporting Information for experimental details.
In our initial studies, we examined the conjugation of the
tetrapeptide VMFP (1) with diethyl ethylidenemalonate (2) in
phosphate-buffered saline (PBS) (pH 7.4) under blue light
(Kessil, 440 nm) in the presence of a water-soluble
photocatalyst. A number of photocatalysts were explored;
iridium- and ruthenium-based complexes, as well as organic
dyes, resulted in exclusive formation of an undesired
methionine sulfoxide product (Table S1), presumably as a
result of sluggish α-deprotonation following sulfur single-
electron oxidation.19 We thus turned our attention to the flavin
family of photocatalysts. We postulated that after quenching of
the flavin triplet excited state by methionine via SET, the
resulting reduced flavin radical anion could act as a base20 to
deprotonate the position adjacent to the sulfur radical cation,
thereby generating the desired α-thio radical species. Gratify-
ingly, lumiflavin (3) efficiently catalyzed the transformation
under the photocatalytic conditions, producing the desired
methionine conjugate product 4 with 79% conversion without
observable formation of the methionine sulfoxide byproduct
(Figure 2a and Table S2).
Information). Deprotonation at the α-position of the resultant
methionine radical cation 7 (pKa = −3.5)22 by lumiflavin
radical anion (LF−, 8) (pKa (HLF•) = 8.5)23 furnishes α-thio
radical 9 and the stable reduced form of lumiflavin (HLF•, 10).
The nucleophilic carbon-centered radical intermediate 9 then
undergoes addition to Michael acceptor 2 to afford α-acyl
radical 11. Subsequent hydrogen atom transfer (HAT)
between radical intermediate 11 and hydrogen atom donor
10 (N−H bond dissociation energy (BDE) = 59.9 kcal/mol)23
regenerates the ground-state lumiflavin 3 and furnishes the
methionine conjugate product 12.
It should be noted that tyrosine, tryptophan, and histidine
residues can also undergo sequential electron transfer−proton
transfer (ET−PT) with excited lumiflavin; however, the
corresponding radical intermediates are electrophilic in nature
and expected to favor HAT reaction with 10 to regenerate the
parent residues rather than addition to electrophilic Michael
acceptor 2.24 Accordingly, we expected this method to permit
selective modification of the methionine residue. To evaluate
amino acid selectivity in the conjugation, we subjected a wide
array of tetrapeptide substrates that did not contain
methionine residues to the reaction conditions; in each case,
the substrates were recovered (>90%) with no observable
conjugate product formation (Table S3). One caveat that
needs to be noted is that thiol radicals generated from free
cysteine residues can readily react with Michael acceptors
under the reaction conditions (see Table S4). In contrast to
The proposed mechanism of flavin-catalyzed methionine
bioconjugation is illustrated in Figure 2b. Initially, photo-
excitation of ground-state lumiflavin (1LF, 3) (λ = 444 nm)
abs
max
produces a long-lived (τ = 20 μs) triplet excited state (3LF,
6).20b This highly oxidizing excited state (Er1e/d2 [3LF/LF−] = 1.5
V vs SCE)21 is capable of undergoing SET with the methionine
thioether (Epa = 1.36 V vs SCE; see the Supporting
C
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free cysteine residues, however, cysteine disulfide bonds did
not undergo photocatalyzed modification.
With a methionine-selective bioconjugation protocol in
hand, we turned our attention to exploring the scope of the
Michael acceptor component (Table 1). Aprotinin, a serine
protease inhibitor containing one methionine residue, was
selected as the model protein substrate. Under our optimal
conditions, 2 was found to afford the monoalkylation product
with 93% conversion. Similarly, 3-methylene-2-norbornanone
(13) and phenyl vinyl sulfone (14) provided the conjugated
products with 91% and 96% conversion, respectively. Notably,
when acceptors 13 and 14 were employed, mixtures of mono-,
bis-, and trisalkylation products were obtained, presumably as a
result of subsequent α-thio radical formation. Proteomic
analysis of the products confirmed multialkylation on the
single methionine residue. Importantly, various functional
groups and biological payloads could be attached to the vinyl
sulfone acceptor and readily incorporated into the protein via
our methionine conjugation protocol. Notably, carboxylic acid
and primary amine moieties were well-tolerated under the
reaction conditions (15 and 16, >95% and 65% conversion,
respectively). Vinyl sulfones containing bio-orthogonal azide
and alkyne handles for copper-catalyzed click chemistry were
also readily employed (17 and 18, >95% and 80% conversion,
respectively). Finally, a substrate bearing a desthiobiotin
affinity tag was successfully conjugated with useful efficiency
(19, 48% conversion). Thus, the photocatalytic conjugation
can accommodate a range of functional groups on the Michael
acceptor component, allowing the introduction of useful
functional handles that are not compatible with traditional
nucleophilicity-based bioconjugation protocols.
We next sought to evaluate the generality and site selectivity
of the protocol across a diverse range of protein substrates of
varying molecular weights. As shown in Figure 3, proteins with
one methionine, including ubiquitin (20) and α-lactalbumin
(21), underwent conjugation with high conversions. Notably,
an extended reaction time of 90 min was required for the
ubiquitin conjugation, which we attributed to the hindered
placement of the methionine in the protein. In general, we
anticipated that the rate of conjugation would be mediated by
the degree of methionine surface exposure. To test this
hypothesis, we exposed substrates with multiple methionines
to the bioconjugation conditions. Following reaction, labeled
protein products were digested with trypsin and then analyzed
using liquid chromatography tandem mass spectrometry (LC−
MS/MS) to determine the relative amounts of peptides with
modified methionines. Myoglobin (22) is a muscular oxygen-
binding protein with a surface-exposed M55 residue and an
internal M131 residue. Under standard bioconjugation
conditions, myoglobin was alkylated with 90% conversion.
Proteomic analysis of modified myoglobin revealed a 5:2
alkylation ratio at positions M55 and M131. It should be noted
the alkylation ratio was based solely on the relative ion count
and does not account for possible differences in the ionization
efficiencies of different peptides.
Figure 3. Scope of proteins and site selectivity of the methionine
bioconjugation methodology. The reaction conditions are the same as
those shown in Table 1. See the Supporting Information for
experimental details.
(24), which contains no surface-exposed methionines, was
conjugated with nearly complete conversion, with an
M58:M221:M239 alkylation ratio of 6:1:15. Finally, we were
also able to label ribonuclease A (25) with greater than 43%
conversion.
We then set out to evaluate the photoredox bioconjugation
in the context of a biological system to probe whether the
protocol is capable of modifying proteins under mild
conditions without altering the tertiary structure. Enhanced
green fluorescent protein (EGFP) was selected as a model
substrate for this study. As illustrated in Figure 4a, we aimed to
install an alkyne functional group by conjugating Michael
acceptor 17 to EGFP. The alkyne would then serve as a handle
for further functionalization of the protein via copper-catalyzed
alkyne−azide cycloaddition (CuAAC). In practice, exposure of
10 μM EGFP to the standard reaction conditions resulted in
the formation of EGFP−alkyne conjugate 26 with 45%
conversion. Because EGFP is known to lose its activity as a
result of denaturation or photobleaching,25 the fluorescence
level of the product was measured to verify the integrity of
However, the ratio partly supports the hypothesis that the
surface-exposed (M55) residue should be alkylated to a higher
degree than the internal (M131) amino acid. Similar selectivity
was observed in the conjugation of recombinant human
growth hormone (23). Three methionine residues on the
proteinM14, M125, and M170were labeled in a ratio of
11:2:3, with conjugation primarily occurring on the most
surface-exposed M14 residue. Surprisingly, carbonic anhydrase
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Figure 4. Functionalization of EGFP via the photoredox methionine bioconjugation protocol. (a) EGFP was initially labeled with 17, and then
azides containing biological motifs were appended to the modified EGFP via CuAAC. (b) Azides bearing a biotin tag and a nonaarginine peptide.
(c) After functionalization of EGFP via the photoredox methionine bioconjugation protocol, the product retained 95% of the fluorescence relative
to wild-type EGFP (n = 3, mean SD). (d) Western blot images of wild type EGFP and modified EGFPs. (e) Confocal microscopy images of
SJSA-1 cells treated with EGFP, EGFP−alkyne, and EGFP−(Arg)9. Scale bars = 20 μm.
protein function. Notably, we observed 95% retention of
fluorescence relative to wild-type EGFP (Figure 4c).26 EGFP−
alkyne conjugate 26 readily underwent CuAAC, which served
to install the target biological motifs. Both biotin azide 27 and
nonaarginine azide 28 were successfully employed in this
transformation, affording the corresponding EGFP−biotin and
EGFP−(Arg)9 adducts (Figure 4b). We confirmed the
attachment of the biotin tag to EGFP by streptavidin
immunohistological staining (Figure 4d, lane 3). To verify
attachment of the nonaarginine tag, SJSA-1 cells were treated
with EGFP−(Arg)9 and visualized using confocal microscopy.
Since the nonaarginine peptide facilitates delivery of cargo
proteins into cells,27 only EGFP−(Arg)9 conjugate would be
internalized (Figure 4e). As expected, no significant internal-
ization was observed in the cells treated with unmodified
EGFP or EGFP−alkyne conjugate 26. These results highlight
the capacity of the photocatalytic bioconjugation method to
enable rapid methionine-selective conjugation and delivery of
biological payloads into cells.
Experimental procedures, NMR spectra, and CV data
(PDF)
Proteomics analysis (ZIP)
AUTHOR INFORMATION
■
Corresponding Author
David W. C. MacMillan − Merck Center for Catalysis at
Princeton University, Princeton, New Jersey 08544, United
States; orcid.org/0000-0001-6447-0587;
Email: dmacmill@princeton.edu
Authors
Junyong Kim − Merck Center for Catalysis at Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0001-7037-8621
Beryl X. Li − Merck Center for Catalysis at Princeton
University, Princeton, New Jersey 08544, United States
Richard Y.-C. Huang − Pharmaceutical Candidate
Optimization, Research and Development, Bristol-Myers
Squibb Company, Princeton, New Jersey 08542, United
States; orcid.org/0000-0002-7172-110X
Jennifer X. Qiao − Discovery Chemistry, Research and
Development, Bristol-Myers Squibb Company, Princeton,
New Jersey 08542, United States
ASSOCIATED CONTENT
■
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c09926.
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William R. Ewing − Discovery Chemistry, Research and
Development, Bristol-Myers Squibb Company, Princeton,
New Jersey 08542, United States; orcid.org/0000-0002-
9434-8053
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c09926
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful for financial support provided by the
National Institute of General Medical Sciences (NIGMS), the
NIH (under Award R35GM134897-01), the Princeton
Catalysis Initiative, and kind gifts from Merck, Janssen, BMS,
Genentech, Celgene, and Pfizer. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of NIGMS.
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nm) in the reaction mixture inhibits photoexcitation of EGFP (λ
abs
max
abs
max
=
488 nm), thereby preventing photobleaching.
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(27) Bohmova, E.; Machova, D.; Pechar, M.; Pola, R.; Venclíkova,
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K.; JanouSkova, O.; Etrych, T. Cell-Penetrating Peptides: A Useful
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https://dx.doi.org/10.1021/jacs.0c09926
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX