Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible Thiol–Ene Click Chemistry

Article abstract of DOI:10.1002/anie.202010217

Bioconjugation methods using visible-light photocatalysis have emerged as powerful synthetic tools for the selective modification of biomolecules under mild reaction conditions. However, the number of photochemical transformations that allow successful protein bioconjugation is still limited because of the need for stringent reaction conditions. Herein, we report that a newly developed water-compatible fluorescent photosensitizer Q_PEG can be used for visible-light-induced cysteine-specific bioconjugation for the installation of Q_PEG by exploiting its intrinsic photosensitizing ability to activate the S?H bond of cysteine. The slightly modified Q_CAT enables the effective photocatalytic cysteine-specific conjugation of biologically relevant groups. The superior reactivity and cysteine selectivity of this methodology was further corroborated by traceless bioconjugation with a series of complex peptides and proteins under biocompatible conditions.

Full text of DOI:10.1002/anie.202010217

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Visible-Light-Induced Cysteine-Specific Bioconjugation:
Biocompatible Thiol-Ene Click Chemistry
Authors: Hangyeol Choi, Myojeong Kim, Jeabong Jang, and Sungwoo
Hong
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202010217
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Angewandte Chemie International Edition
DOI: 10.1002/anie.200((will be filled in by the editorial staff))
Photochemistry
Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible
Thiol-Ene Click Chemistry **
Hangyeol Choi,a,b Myojeong Kim,a,b Jeabong Jang*,b and Sungwoo Hong*,b,a
Abstract: Bioconjugation methods using visible-light photocatalysis
have emerged as powerful synthetic tools for the selective
modification of biomolecules under mild reaction conditions.
However, the number of photochemical transformations that allow
successful protein bioconjugation is still limited because of the need
for stringent reaction conditions. Herein, we report that a newly
developed water-compatible fluorescent photosensitizer QPEG can be
used for visible-light-induced cysteine-specific bioconjugation for the
installation of QPEG by exploiting its intrinsic photosensitizing ability
to activate the S–H bond of cysteine. In addition, the slightly modified
QCAT enables the effective photocatalytic cysteine-specific
conjugation of biologically relevant groups. The superior reactivity
and cysteine selectivity of this methodology was further corroborated
by traceless bioconjugation with a series of complex peptides and
proteins under biocompatible conditions.
aqueous solutions, and therefore many of the photochemical methods
developed for biomolecule-material conjugation only occur in
organic media, which limit the biochemical applications. In this
regard, the development of the water-compatible and additive-free
protocol is highly desirable for the visible-light-mediated.
Introduction
The chemical modification of biomolecules has become crucial for
the advancement of chemical biology, molecular biology, and drug
development. In particular, a hybrid of proteins and synthetic
molecules (i.e., drugs and probes) provides a powerful tool for protein
research, pharmaceutical chemistry, and biotechnology. For example,
the fluorescent labeling of proteins enables the analysis of protein
function, structure, dynamics, and trafficking pathways as well as the
development of biological assays for drug screening.[1] In addition,
the successful development of antibody-drug conjugates has fueled
the development of reliable methods amenable to the bioorthogonal
and chemoselective protein modification.[2] For the development of
more diverse conjugation techniques, continued efforts have been
made to modify natural amino acids by precisely controlling the
position and number of molecules on proteins.[3] Recently, visible-
light-induced bioconjugation has an attractive feature in terms of
environmental sustainability and mild reaction conditions, as
illustrated in Figure 1a.[4-6] This strategy proves effective in
generating reactive radical species that can participate in a unique
bond-forming process, leading to novel strategies in biomolecule
functionalization. Despite the elegant merits, photoredox-catalyzed
biorthogonal reactions are still complicated by some challenging
problems. One of the major obstacles is the use of stoichiometric
amounts of base or oxidant, which is often incompatible with the
delicate nature of proteins and the tolerability of amino acid residues.
Another limitation is the poor solubility of catalysts and reagents in
Figure 1. Visible-light mediated site-specific and chemoselective bioconjugations.
a) Current strategies for visible-light-induced site-specific conjugation. b) A new
strategy for visible-light-induced cysteine-specific bioconjugation of
photosensitizing fluorophore QPEG and QCAT-catalyzed cysteine-specific
conjugation. EWG = electron withdrawing group.
Cysteine is a crucial residue for the chemical modification in
selectively stitching two molecules together, and it has been exploited
in various fields such as bioconjugate chemistry, medicinal chemistry,
and polymer chemistry.[7] In traditional cysteine bioconjugation, the
most employed strategy is the thio-Michael addition to maleimide
acceptors by utilizing excellent nucleophilicity of the thiol group.
Although this method has been widely investigated, there are
significant concerns of a lack of chemoselectivity in the presence of
nucleophilic amino acids and the inherent instability of the resulting
thiosuccinimide linkage.[8] For the last few years, several remarkable
studies provided newly developed reagents and efficient cysteine
bioconjugation methods for the improved stability of protein
[]
H. Choi, M. Kim, Dr. J. Jang, Prof. Dr. S. Hong
aDepartment of Chemistry, Korea Advanced Institute of
Science and Technology (KAIST), Daejeon 34141 (Korea)
bCenter for Catalytic Hydrocarbon Functionalizations, Institute
for Basic Science (IBS), Daejeon 34141 (Korea)
E-mail: hongorg@kaist.ac.kr, jaebong.jang@gmail.com
[] Supporting information for this article is available on the
WWW under http://www.angewandte.org.
1
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10.1002/anie.202010217
Angewandte Chemie International Edition
conjugates.[9] Among them, radical thiol-ene reaction for C–S bond
formation is one of the most promising cysteine modification
methods, because radical insertion does not compete with other
nucleophilic residues and leads to more stable adducts.[7b,10] However,
highly oxidizing conditions often hamper the stability of oxidation-
labile residues and cause detrimental effects on proteins a result of
concurring side reactions.[10b,11] Moreover, direct chemical
modification of natural amino acids in native proteins requires
biocompatible reaction conditions (i.e., mild temperature,
physiological pH, aqueous buffered solutions, and high dilution) and
functional group tolerance to preserve protein structures, which
makes challenging the development of photomediated bioconjugation
means. In this context, the chemoselective generation of thiyl radicals
under mild and water-compatible conditions represents a significant
challenge to accomplish efficient cysteine bioconjugation via
photomediated radical thiol-ene reaction.
Recently, our group has developed an organic photocatalyst Q1,
based on a quinolinone chromophoric unit and successfully achieved
diverse visible-light-induced functionalization of pyridines under
mild and metal-free conditions.[12] This photocatalyst Q1 exhibits blue
fluorescence (λabs = 380 nm; λem = 448 nm) with a quantum yield of
0.28 in 1:1 (v/v) dimethylformamide/phosphate buffer (20 mM, pH
7.5) solvent mixture (see Figure 2b). Drawing inspiration from these
observations, we hypothesized that incorporating a suitable pendant
alkene moiety onto the photosensitizing fluorophore Q1 backbone
would enable the direct assembly of fluorophore-labeled
biomolecules by functioning bioconjugation process at the cysteine
residues. The Q1-derived water-compatible photosensitizers, QPEG,
and QCAT are capable of performing direct hydrogen-atom transfer
(HAT) upon excitation with blue LEDs and selectively generating
radicals on cysteine residues for biologically relevant cysteine-
conjugation, as shown in Figure 1b. Herein, we report a novel
visible-light-induced cysteine bioconjugation for the installation of a
fluorophore, QPEG by exploiting its intrinsic photosensitizing ability
in aqueous media under metal-, oxidant- and external photocatalyst-
free conditions. In addition, the use of water-compatible photocatalyst
QCAT allowed cysteine conjugations of biologically relevant groups
enabled by photocatalytic radical thiol-ene reaction under
biologically ambient conditions. The convenience of this protocol
was further illustrated by conjugating an important class of cysteine-
detectable modifications occurred on other amino acids (see the
Supporting Information for details, Figure S2). The orthogonal
reactivity, along with compatibility with various functional groups,
highlighted possible applications of Q1 towards cysteine-specific
bioconjugation and modification of peptides and proteins.
Based on the encouraging results, we reasoned that Q1-
photochemistry could be applied to the fluorescent labeling of
biomolecules, in which the Q1-derivative serves as both an organic
photosensitizer and a fluorophore. Indeed, QENE, a Q1-analog
possessing a terminal alkene linker, promoted the desired reaction
with 1 in dilute concentration of acetonitrile (10 mM), leading to the
formation of the desired QENE-cysteine conjugate in 77% yield after
an hour (see SI for details, Table S2, entry 2). Whereas this
preliminary result is encouraging, QENE was visibly insoluble when
increasing the ratio of phosphate buffer in the reaction solvent, and
therefore the desired conjugation was completely unreactive (Table
S2, entries 4 and 5). In this context, from the outset of our studies, the
identification of suitable photosensitizers that enable aqueous
bioconjugation processes was a priority (Table S2, entries 7-10).
Through modulation of water-soluble groups directly attached to Q1,
QPEG (Figure 2b), a derivative bearing octa-ethylene glycol (PEG-8),
was found to be visibly soluble in phosphate buffer with 10% v/v
acetonitrile, and was capable of producing conjugated adduct 4 in
71% yield under the blue LED irradiation at room temperature for 3
h (Table S2, entry 11). Importantly, water-compatible fluorophore,
QPEG exhibited blue fluorescence (λabs = 378 nm; λem = 450 nm) with
a high quantum yield (F = 0.4) in aqueous media (Figure 2b and
Table S4).
100
90
80
70
60
50
40
30
20
10
0
Q1
Eosin Y
Mes-Acr
containing peptides and proteins. Remarkably, the obtained QPEG
-
conjugates provide dual functionality for producing
photoluminescence and singlet molecular oxygen (1O2) under visible-
light irradiation, highlighting potential applications in image-guided
photodynamic therapy.
Additives
Results and Discussion
We initially examined the photocatalyzed thiol-ene reaction between
N-acetyl-L-cysteine
1 and alkene 2 using various organic
photocatalysts (Q1, Eosin Y, and Mes-Acr+) for exploring the
proposed concept, as shown in Figure 2a. When Q1 was irradiated
with blue LEDs, we were pleased to observe the formation of the
desired conjugate 3 in 86% yield. Remarkably, an external oxidant
was not required in this protocol, thereby potentially avoiding side
reactions of peptides bearing oxidation-sensitive residues. Because
the presence of various nucleophilic functional groups found in
biomolecules can significantly limit the scope and efficiency, we next
investigated the chemoselectivity of the current method by adding
amino acid additives such as Tyr, His, Ser, Asp, and Lys bearing
multiple reactive functional groups. Importantly, the cysteine-
specific conjugation process using Q1 was proceeded in high
conversion yields regardless of adding other amino acids, and no
Figure 2. Visible-light induced thiol-ene reactions with cysteine. a) Survey of
tolerance for the cysteine-specific thiol-ene reaction in the presence of competing
nucleophilic amino acid additives. Reaction conditions: 1 (100 mM), 2 (100 mM),
photocatalyst (2 mol%), and amino acid additive (100 mM) with irradiation by a
blue LED (440 nm, 8.5 W) at rt under N2 for 2 h. Yields were determined by 1H
NMR spectroscopy. b) Development of QPEG
methylacridinium Perchlorate. Boc = tert-butoxycarbonyl.
. = 9-Mesityl-10-
Mes-Acr+
As a result of modification of fluorophore, QPEG was selected as the
photosensitizing fluorophore in our study, and we further investigated
the optimization of the reaction conditions as outlined in Table 1. The
irradiation with blue-light for 3 h promoted the formation of the
2
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10.1002/anie.202010217
Angewandte Chemie International Edition
desired product 4 with yields of 87% under phosphate buffer with alkyl radical A abstracts hydrogen from the intermediate QPEG–H
10% dimethylformamide (entry 1). Among the co-solvents screened, through a reverse hydrogen atom transfer (RHAT) event to furnish
acetonitrile and dimethyl sulfoxide were slightly inferior to the product and regenerate the ground-state QPEG to complete the
dimethylformamide (entries 2 and 3). The use of pyridinium salt as catalytic cycle, as shown in Figure 3.
an oxidant, a key component in Q1-mediated reactions,[12] was not
required for this transformation (entry 4). The use of 2 equiv of 1
dramatically improved the formation of the desired product with a
yield of 95% (entry 5), but we kept using 1 as a limiting substrate for
applying this technique in bioconjugation. We next investigated the
effect of pH on the reaction because solubility and conjugation of
substrates can be pH-dependent. To our delight, the desired adduct 4
was generated in comparable yields regardless of the pH (pH 3.5
NaOAc buffer, pH 5.5 HEPES buffer, or pH 8.8 Tris buffer) (entries
6-8). The necessity of light for product formation was confirmed by
a
control experiment (entry 9). The addition of 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO) completely inhibited the
reaction, suggesting that a radical pathway is operational in this
conjugation reaction (entry 10).
Figure 3. Proposed mechanism for the cysteine-specific conjugation of QPEG
HAT = hydrogen-atom transfer. RHAT = reverse hydrogen-atom transfer.
.
Table 1: Optimization of the reaction conditions[a]
With the optimized conditions in hand, we next examined the
applicability of this photocatalytic methodology for an endogenous
tripeptide, glutathione (GSH) to investigate the tolerability of two
carboxylic acids and free-amine functionalities that are present at the
C- and N-termini of proteins, as shown in Table 2. We were pleased
to observe the conjugation reaction of QPEG with GSH proceeded
smoothly in 73% yield. Encouraged by this result, the scope of the
method and functional group tolerance were next investigated with
peptide substrates bearing naturally occurring amino acids.
Remarkably, cysteine conjugation reactions were efficiently achieved
with QPEG under pH 7.5 at room temperature. Both hydrophobic (Gly,
Ala, Leu, Ile, Val, Met, and Pro; 6a-6g) and hydrophilic (Ser, Thr,
Asp, Glu, Asn, Gln, Lys, and Arg; 6h-6o) side chains of natural amino
acids were well tolerated in these conjugation reactions. Moreover,
our results demonstrated that the tetrapeptides containing challenging
amino acids such as Ser, Lys, Trp, His, and Tyr that often exhibit the
deleterious effects on the bioconjugation outcome, were able to
efficiently participate in the desired conjugation with QPEG at pH 7.5
(6h, 6n, 6p, 6q, and 6r). It had been previously reported that lowering
the pH to 3.5 was required for improving the photoredox conjugation
of peptides containing Lys, His, and Tyr residues to prevent oxidation
of these sensitive residues.[5a] Any conjugates arising from the
additions of other nucleophilic residues were not detected despite the
presence of multiple nucleophiles in the peptide sequence, as
confirmed by LCMS analysis, demonstrating the excellent selectivity
for the cysteine conjugation. Further evaluation of the generality of
the reactions with tetrapeptides containing cysteine residues at C- or
N-termini, revealed that QPEG could be efficiently conjugated with
cysteine regardless of its position in the sequence. We next applied
the optimized conditions to more complex peptides such as fragments
of endogenous proteins derived from insulin (6u, octapeptide) and Src
kinase (6v, dodecapeptide). Both peptides were successfully
conjugated with QPEG under our optimized reaction conditions in
yields of 70% and 62%, respectively. For 6s and 6v, the conjugation
reactions were performed in an acidic NaOAc buffer (pH 3.5) due to
the insolubility of the substrates in a neutral phosphate buffer (pH 7.5).
In nature, most cysteine residues in endogenous proteins create
disulfide bonds that contribute to the formation of a stable three-
dimensional structural fold of the proteins.[14] As expected, QPEG was
not able to react with a disulfide bond under optimal conditions,
exhibiting that the QPEG-mediated C–S bond formation is highly
Entry
Deviations from the standard conditions
none
Yield (%)[b]
1
2
87
71
MeCN instead of DMF
3
DMSO instead of DMF
70
4
pyridinium salt (1 equiv)
65
5
2 equiv of 1
>95
81
6
NaOAc buffer (pH = 3.5) instead of PB buffer
HEPES buffer (pH = 5.5) instead of PB buffer
Tris buffer (pH = 8.8) instead of PB buffer
no light
7
78
8
76
9
NR
NR
10
addition of TEMPO (2 equiv)
[a] Reaction conditions: 1 (10 mM), and QPEG (10 mM) with irradiation by a blue
LED (440 nm, 8.5 W) at rt under N2. [b] Yields were determined by 1H NMR
spectroscopy using 1,1,2,2,-tetrabromoethane as a standard. DMSO = Dimethyl
sulfoxide. DMF
=
dimethylformamide. pyridinium salt
=
N-ethoxy-2-
methylpyridinium tetrafluoroborate. PB = phosphate buffer. HEPES = (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid.
To elucidate the reaction pathway, we further conducted several
mechanistic studies. Stern-Volmer fluorescence quenching
experiments (see Figure S18) did not show any meaningful quenching
of QPEG along with increasing concentrations of 1, indicating that
classical single electron transfer (SET) and energy transfer pathways
may not participate in the reaction mechanism. The evaluation of the
excited state reduction potential of QPEG from the cyclic voltammetry
(see Figure S19) and the absorption spectra (see Figure S17) analysis,
could exclude the possibility involving the SET process with a thiol
group of cysteine. Therefore, we speculated that the triplet excited
state of QPEG might serve as a direct HAT reagent to activate a labile
S–H bond of cysteine, forming the corresponding thiyl radical in the
reaction media. Thus, the thiyl radical generated through a direct
HAT process[13] of QPEG under blue LED irradiation, acts as an
electrophile towards the alkene of QPEG, leading to the formation of
the C–S bond and the generation of the alkyl radical adduct A. In the
chain pathway supported by the calculated reaction quantum yield
(ΦR = 4.13), the alkyl radical A abstracts a hydrogen atom from
another thiol group to deliver the conjugation product 4 and release
the thiyl radical that can start a new chain pathway. Alternatively, the
3
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10.1002/anie.202010217
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Table 2: Scope and functional group tolerance in polypeptides[a]
[a] Conjugation of free-cysteine-containing polypeptides. Reaction conditions: 5a-v (10 mM) and QPEG (10 mM) with irradiation by a blue LED (440 nm, 8.5 W) at rt
under N2 for 3 h. [a] NaOAc buffer (pH 3.5) was used instead of phosphate buffer. [b] Conjugation of disulfide-containing molecules. Reaction conditions: 7 (6.7 mM),
QPEG (13.4 mM), and TCEP (6.7 mM) in phosphate buffer (10% DMF, pH 7.5) were stirred at rt under N2 for 2 h and with irradiation by a blue LED (440 nm, 8.5 W)
for 3 h. [b] 7c (2 mM), QPEG (4 mM), and TCEP (2 mM) in NaOAc buffer (10% DMF, pH 3.5). Yields were determined by 1H NMR spectroscopy. The isolated yields by
HPLC are provided in parentheses. TCEP = tris(2-carboxylethyl)phosphine.
4
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10.1002/anie.202010217
Angewandte Chemie International Edition
selective to the free-thiol of cysteine over the disulfide (Table 2b).To
further demonstrate the applicability of the current method, additional
experiments were designed to determine if the process was
compatible with the protein disulfide reducing agent tris(2-
carboxyethyl)phosphine (TCEP)[15] and could enable conjugation of
disulfide-containing molecules. Indeed, QPEG efficiently underwent
reaction with N-acetyl-L-cystine 7a, and L-glutathione oxidized 7b
under the presence of TCEP, leading to the formation of the
corresponding C–S bond in good yields (4, 81% and 6a, 79%).
Moreover, neurohyphophysial nonapeptide hormone oxytocin 7c
could efficiently be conjugated with two QPEG fluorophores (8, 60%),
even in highly diluted concentration (2 mM). Taken together, this
protocol could offer a general strategy for the bioconjugation of a
photosensitizing fluorophore.
Next, we speculated that the QPEG could be used as a water-
compatible photocatalyst for the cysteine conjugation of biologically
relevant groups in aqueous media. For this purpose, we slightly
modified QPEG by removing its olefin group and assessed the
photocatalytic ability by evaluating conjugation of peptides with a
variety of important biologically relevant molecules such as affinity
tag (biotin), bioorthogonal handle (azide), crizotinib (ALK inhibitor),
and sugar. As illustrated in Table 3, we were pleased to observe that
QCAT was shown to be competent in photocatalytic cysteine
conjugations, affording the corresponding adducts under the standard
reaction conditions. The current photocatalytic strategy, therefore,
presents a valuable and versatile platform for selective cysteine
conjugation of biologically relevant groups as well as fluorophore
QPEG under biologically ambient conditions.
Combining properties of a fluorophore and a singlet oxygen (1O2)
photosensitizer can provide a unique opportunity for image-guided
therapeutic applications.[16] We reasoned that the obtained
photosensitizing fluorophore QPEG-conjugates could enable the
photosensitized production of singlet oxygen upon light irradiation.
In this regard, we evaluated the 1O2 generation ability of 6a in
aqueous media, as shown in Figure 4. The photogeneration of singlet
oxygen by 6a was confirmed by the production of oxidation product
TEMPO, which was detected by electron paramagnetic resonance
(EPR) spectroscopy (unique triplet signal with aN = 16.9 G and g =
2.00056, Figure 4b and Figure S24).[17] Next, to quantify singlet
oxygen production, singlet oxygen sensor green (SOSG) was used as
an indicator, which displayed excellent singlet oxygen generation
ability of 6a under visible-light irradiation (Figure 4c and Figure
S25).[18]
Table 3: Photocatalytic conjugation of diverse biological substrates[a]
[a] Reaction conditions: 5 (10 mM), 10 (11 mM), and QCAT (5 mol%) with
irradiation by a blue LED (440 nm, 8.5 W) at rt under N2 for 3 h. [b] QCAT (10 mol%)
was used. [c] NaOAc buffer (pH 3.5) was used instead of phosphate buffer.
To test the applicability of our method to tertiary structural proteins,
the compatibility of QPEG and QCAT in protein bioconjugation was
evaluated using ubiquitin K63C (Ub K63C)[19] and bovine serum
albumin (BSA), as illustrated in Figure 5. Native ubiquitin (Ub) is a
small size globular protein of 76 residues (8.6 kDa) found in most
tissues. Serum albumin is a globular protein found in blood plasma,
and fluorophore-labeled albumin conjugates have been widely used
for biological imaging.[20] BSA contains only one exposed surface
cysteine (Cys34) and 17 conserved disulfides. We aimed to link the
photosensitizing fluorophore QPEG directly to cysteine residues in
these proteins using the developed visible-light-induced protocol. For
this purpose, Ub K63C was mixed with QPEG in 10% DMSO in
phosphate buffer and incubated under blue light irradiation for 6 hr.
Pleasingly, an analysis of the mixture using LCMS showed a mass
corresponding to the conjugate of Ub K63C–QPEG (Figure 5a and
Figure S8). The labeled Ub K63C–QPEG was clearly observed after
trypsin digestion by peptide mapping ESI-MS spectrometry to
identify the site of conjugation. With a similar protocol utilizing QPEG
Figure 4. Singlet oxygen generation by QPEG-conjugated GSH 6a. a)
Photoinduced 1O2 generation by 6a. b) Photogenerated TEMPO from TEMP and
EPR spectra of the TEMPO radical. c) Fluorescence detection of singlet oxygen
generation using SOSG. Reaction conditions: SOSG (2.5 µM) and 6a (25 µM) in
aerated phosphate buffer (pH 7.5, 10% v/v dimethylformamide) with irradiation
by
a blue LED (415 nm) under air for 30 min. TEMP = 2,2,6,6-
tetramethylpiperidine.
5
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10.1002/anie.202010217
Angewandte Chemie International Edition
200
500
800
1100
1400
1700
2000
Figure 5. Visible-light-induced site-specific bioconjugation on proteins. a) Functionalization of Ub K63C with QPEG. Ub K63C (50 µM) and QPEG (5 mM) in phosphate
buffer (10% DMSO, pH 7.5) with irradiation by a blue LED (440 nm, 8.5 W) for 6 h. Deconvoluted LCMS mass spectrum for the Ub K63C-QPEG conjugate (Product:
9321 m/z; [M+781]+), MS/MS spectrum of the tryptic peptide TLSDYNIQCESTLHLVLR, modification at the cysteine residue of Ub K63C-QPEG. b) Functionalization of
BSA with QPEG. BSA (100 µM) and QPEG (2 mM) in phosphate buffer (10% DMF, pH 7.5) with irradiation by a blue LED (440 nm, 8.5 W) at rt under N2 for 6 h. MS/MS
spectrum of the tryptic peptide GLVLIAFSQYLQQCPFDEHVK, modification at the cysteine residue of BSA-QPEG. c) Functionalization of BSA with QCAT and 11a. BSA
(100 µM), QCAT (2 mM) and 11a (2 mM) in phosphate buffer (10% DMF, pH 7.5) with irradiation by a blue LED (440 nm, 8.5 W) at rt under N2 for 6 h. MS/MS spectrum
of the tryptic peptide GLVLIAFSQYLQQCPFDEHVK, modification at the cysteine residue of BSA-biotin.
and QCAT, Cys34 of BSA was readily labeled with QPEG and biotin,
respectively (Figure 5b and Figure 5c). These results revealed that Ub
K63C and BSA were successfully tagged with a single QPEG or biotin
in the presence of 50 to 100 μM protein, a concentration commonly
used for the labeling of proteins.
external oxidant and base is critical to suppress undesired reaction of
the peptide and protein substrates. This metal-free and operationally
simple conjugation proceeds with high efficiency, chemoselectivity,
and functional group tolerance, providing great promise in many
chemical disciplines for the preparation of fluorescent
photosensitizer- or biomolecule-bound peptide and protein
conjugates.
Conclusion
Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))
In summary, we have discovered a fluorescent photosensitizer QPEG
and photocatalyst QCAT capable of mediating visible-light-induced
cysteine-specific conjugation under biocompatible reaction
conditions. By exploiting the intrinsic photosensitizing capacities of
QPEG and QCAT, a fluorophore QPEG and biologically relevant groups
could be installed chemo- and regioselectively into a series of
complex peptides and proteins. In the process, the exclusion of
Acknowledgements
This research was supported financially by Institute for Basic Science
(IBS-R010-A2).
Conflict of interest
6
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10.1002/anie.202010217
Angewandte Chemie International Edition
I. Kim, G. Kang, S. Shin, D. Kang, M.-H. Baik, S. Hong, Nat. Commun.
2019, 10, 4117; d) K. Kim, H. Choi, D. Kang, S. Hong, Org. Lett.
2019, 21, 3417; e) I. Kim, G. Kang, K. Lee, B. Park, D. Kang, H. Jung,
Y.-T. He, M.-H. Baik, S. Hong, J. Am. Chem. Soc. 2019, 141, 9239; f)
Y. Kim, K. Lee, G. R. Mathi, I. Kim, S. Hong, Green Chem. 2019, 21,
2082.
The authors declare no conflict of interest.
Keywords: photosensitizer • cysteine • bioconjugation • photocatalysis •
thiol-ene click chemistry
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10.1002/anie.202010217
Angewandte Chemie International Edition
Photochemistry
Hangyeol Choi, Myojeong Kim, Jaebong
Jang and Sungwoo Hong __________
Page – Page
Visible-Light-Induced Cysteine-Specific
Bioconjugation: Biocompatible Thiol-Ene
Click Chemistry
Visible-light-induced cysteine-selective bioconjugation has been achieved using fluorescent
photosensitizer QPEG and photocatalyst QCAT. By exploiting the intrinsic photosensitizing
capacities of QPEG and QCAT, a fluorophore QPEG and biologically relevant groups could be
installed chemo- and regioselectively into a series of complex peptides and proteins under
biocompatible mild conditions.
8
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