Highly Reactive and Tracelessly Cleavable Cysteine-Specific Modification of Proteins via 4-Substituted Cyclopentenone

Article abstract of DOI:10.1002/anie.201804801

A rapid and cysteine-specific modification of proteins using 4-substituted cyclopentenone via a Michael addition tandem elimination reaction was developed. Compared to the classical method, this reaction featured fast kinetics with a stable product. More importantly, this conjugation could be tracelessly removed by exchange with a Michael addition donor. The conjugation and regeneration process not only exhibited little change to the structures or conformations of the proteins but also exhibited little disturbance to their biological functions, such as their enzymatic activities.

Full text of DOI:10.1002/anie.201804801

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Accepted Article
Title: Highly Reactive and Tracelessly Cleavable Cysteine-Specific
Modification of Proteins via 4-Substituted Cyclopentenone
Authors: Jian Yu, Xiaoyue Yang, Yang Sun, and Zheng Yin
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804801
Angew. Chem. 10.1002/ange.201804801
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Highly Reactive and Tracelessly Cleavable Cysteine-Specific
Modification of Proteins via 4-Substituted Cyclopentenone
Jian Yu, Xiaoyue Yang, Yang Sun and Zheng Yin*
Abstract: A rapid and cysteine-specific modification of proteins
using 4-substituted cyclopentenone via a Michael addition tandem
elimination reaction was developed. Compared to the classical
method, this reaction featured fast kinetics with a stable product.
More importantly, this conjugation could be tracelessly removed by
exchange with a Michael addition donor. The conjugation and
regeneration process not only exhibited little change to the
structures or conformations of the proteins but also exhibited little
disturbance to their biological functions, such as their enzymatic
activities.
modification. Compounds having low electrophilicity with only
one electron withdrawing group (EWG) such as iodoacetamide
(1a, IAA) or vinyl sulfone (2b)[16] only exhibited relatively mild
activity towards cysteine at physiological conditions. Increasing
the pH may accelerate the reaction but consequently may result
in side products.[17] The electrophilicity was enhanced with more
EWGs on alkenes such as maleimide (2a) or -cyano-,-
unsaturated carbonyl compounds (8), which surely resulted in
better reactivity but caused more side effects including poor
selectivity or the occurrence of the instability of both reagents
and products. For example, maleimides tend to undergo ring-
opening hydrolysis yielding maleamic acids[18] and may also
possibly contaminate other functionalities such as the amino
group. Alkene 9 with two EWGs on one side showed a
reversible reaction, and similar molecules such as Bardoxolone
methyl or MCE-23[19] were used as a reversible covalent
inhibitor/candidate/drug against cysteine-containing proteins.
The non-cleavable nature of the ligation prevents any
possibility for regenerating the unmodified protein through the
disassembly of the conjugate.[20] Cleavable or reversible
conjugations that are biological compatible have become useful
tools for biological research[21] since cleavable conjugations are
required in many research fields such as protein immobilization,
Chemical modification of proteins is an important tool for
monitoring, modulating, and tracking proteins in living systems
for biological research.[1-4] Among the natural 20 amino acids,
cysteine is perhaps one of the most attractive and convenient
targets for site-specific chemical modification due to its high
nucleophilicity, relatively low natural abundance[5] and the ease
of its introduction into
a
specific site by site-directed
mutagenesis.[6] Electrophiles including α-halocarbonyls (1) and
Michael acceptors (2) were classical approaches for the
modification of cysteine (Scheme 1). Reagents and approaches
recently developed for cysteine-specific modifications include
thiol-alkene (3a),[7-8] thiol-alkyne (3b),[9] allenamide (4),[10]
drug development,[22-23] and proteomics.[24-25] Only
a few
organometallic palladium (5)[11]
,
bis-alkylation (6)[12] and
methods are available for cleavable cysteine-specific
modification, including Ellman's reagent (10),[26-27] bromo-
maleimides (11a),[28] bromo-pyridazinediones (11b),[29] electron
deficient acetylenes (12),[30] and recently reported 5-methylene
pyrrolones (13).[31]
functional transformation to dehydroalanine (7)[13] or aldehyde
(8).[14]
Herein, the cysteine-specific protein modification with 4-
substituted cyclopentenone was reported. Compared to many
previous reports,[32] this ligation could achieve high reactivity
(with a rate constant over 100 M-1s-1 at the peptide level) and
stable product at physiological conditions with little impact on the
function and structure of the protein. More importantly, this
modification could be tracelessly cleavable to regenerate the
protein with a Michael addition donor.
Scheme 1. Reagents for the modification of cysteine.
4-Acetoxy cyclopentenone (14) (Scheme 2) could be deemed
as the predecessor of cyclopentadienone (16),[33-34] an extremely
unstable antiaromatic electrophile, which had a high tendency to
dimerize.[35] The literature survey showed that 14 was
significantly more stable than common -acetoxy ketone and
Despite various methodologies in the toolbox, in general, the
expectation of a rapid, highly efficient, highly selective, and
biocompatible reaction for protein modification has not yet been
fully met. In many cases, a high concentration of reagent (1~100
mM) and long incubation time (hours to overnight) are required
for protein modification, which limit the further practical
application of these reactions in biological studies at the cellular
level, in organisms and in living animals.[15] The electrophilicity of
the reagent is one of the important parameters for cysteine
resistant
to
-elimination.[33]
Hypothetically,
4-acetoxy
cyclopentenone (14) could be used for cysteine protein
modification through the mechanism of Michael addition (15a)
with rapid elimination (15b) that is similar to reported small
molecule reactions.[36-38] -Elimination could occur rapidly on 15a
only after Michael addition due to the unfavored antiaromatic
nature of 16, which would provide selectivity for cysteine. The
conjugation product (15b) is expected to be very stable due to
the same reason. In addition, the modified protein could
potentially be regenerated (15c) in the presence of nucleophilic
reagents.
[a]
J. Yu, X. Yang, Y. Sun and Prof. Z. Yin
Center of Basic Molecular Science (CBMS)
Department of Chemistry, Tsinghua University
Beijing100084, China
E-mail: yinzheng@mail.tsinghua.edu.cn
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10.1002/anie.201804801
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peptide modification to be a pseudo-first-order Michael addition
and measured the rate constant accordingly (Table S5).
Subsequently, we sought to apply this reaction to the
modification of proteins. UBXD (Supporting information part
5.1), containing one cysteine and 6 lysines, was selected as a
model protein. The reaction was quantitatively analyzed[40] using
the protein mass spectrum (Q-Exactive orbitrap). As a result, the
cysteine at UBXD could be rapidly modified in the conditions of
10 M protein and 0.5 mM 14 for 60 min at room temperature in
PBS with little by-product (Figure 2AB). The excess of 14 was
likely to be stable without any reaction in the reaction media
(Figure S8). Equivalent 14 reacting with peptide in 50 M could
finally consumed in stoichiometric fashion (Figure S9). In
comparison, 17 had no reaction and IAA only gave 21% yield
(Table 1). Accordingly, rate constants of relevant reagents for
modification of UBXD were messured (Table S6). LC−MS2
analysis of the resulting peptide fragments from trypsin digestion
showed that UBXD modification by 14 occurred only on cysteine
with high selectivity, and no modification was observed on lysine
or the N-terminal amine (Figure S7). The modification had little
impact on the structure or conformation of UBXD according to
the UV-vis and CD (circular dichroism) spectra (Figure S6).
Various buffers were studied for their impact. The reaction was
hardly affected by buffers under the physiological environment.
However, when the pH was lower than 6, the reaction could not
proceed efficiently (Table S7).
Scheme 2. (A) Possible mechanism of the modification and removal reaction.
(B) 14 could be deemed as a predecessor of the highly unstable compound
(16). (C) Model study of small molecules.
The initial study was performed using a cysteine derivative
(18) (Supporting information part 3) that reacted with 4-
acetoxy cyclopentenone (14). The reaction of 18 and 14 in a
concentration of 10 mM in PBS with no other additives
completed in 10 minutes at room temperature. This reaction was
much faster than common unsaturated carbonyl compounds
such as cyclopent-2-enone.[39] The overall reaction was
confirmed to be “substitution” instead of addition. The product
was identified as 1:1 diastereomers (19).
A model peptide with a sequence of GTSWCYNQKRHDGP
(20) was used for further studies (Figure 1A). Peptide (20)
contains all natural amino acids with nucleophilic functionalities
for the purpose of the selectivity study. The cysteine was rapidly
modified by 14 in PBS buffer (pH = 7.4) at room temperature to
afford the sole adduct with a precise molecular weight increase
corresponding to cyclopentendione (+80.026) (Figure 1C).
Intermediate (15a) was observed (Figure 1B) at 2 min right after
the beginning of the reaction, followed by rapid elimination to
afford the product, which matched the possible mechanism.
Figure 2. Deconvolouted spectra of UBXD (Please see full mass spectrum in
the Figure S4). (A) Unmodified UBXD observed with 9507.187; (B) completely
modified UBXD by 14 observed with 9587.202; (C) t = 10 min; modified UBXD
with a mercaptoethanol Michael adduct intermediate observed with 9665.713;
(D) t = 180 min; regenerated UBXD observed 9507.087.
After the chemical modification of UBXD, the small molecule
was removed via spin concentration, and the product was stored
in PBS buffer at room temperature. Cyclopentenone (17)- and
bromo-maleimide (11a)-modified UBXD were used as controls.
Similar to the previous publication,[18] maleimide-modified UBXD
tended toward ring-opening hydrolysis with considerable speed
(Figure S10), while UBXD modified by 17 slowly decomposed
via reverse Michael addition. As expected, the product of UBXD
ligated with 14 was stable without noticeable degradation.
Despite maleimide showed higher rate constant than 14, the
disadvantages such as instability were clearly observed.
Figure 1. Mass spectrum of peptide (5 M) modified by 14 (50 M) in PBS
buffer (pH = 7.4) at room temperature. The reaction was monitored by LCMS.
(A) t = 0 min; (B) t = 2 min; (C) t = 12 min.
Further study showed that 4-acetoxy cyclopentenone (14) had
much higher reactivity than cyclopent-2-enone (17) in the
reaction with peptide (20). The screening of substituted
cyclopentenones illustrated that the leaving groups were vital to
the activity. (Supporting Information part 4.1). Further
mechamism investigation suggested that the high activity of 14
was derived from the antiaromatic property of 16. As
intramolecular elimination was fast enough, we presumed the
Several cysteine-containing proteins were used to study the
application potential of this reaction, while IAA was chosen as
the control. 14 could achieve higher activity than IAA for the
modification of UBXD (Table 1), which completed modification
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with 0.5 mM 14 in 60 min at room temperature in PBS. HCP1
was a protein with one hindered cysteine, which had low activity.
14 achieved 19% and 36% modification at one hour and two
hours, respectively, while IAA had low yield (2.5% in 2 hours).
EV 71 3C protease, which contained an active cysteine and a
non-active cysteine, was employed for the study. Both cysteines
were modified by 14 with satisfactory yield, while IAA gave low
activity toward the non-active cysteine. Lastly, MERS 3C-like
protease containing 8 cysteines with different activities was
studied. After modification by 0.5 mM small molecules for 2
hours, multiple modifications on the cysteines of IAA were
observed from 2 to 5 modifications, while 14 gave 4 to 7
cysteine modifications. Please see the deconvolouted spectra in
Figure S5.
2 15c). After complete modification of UBXD by 14, excess
Michael donor, mercaptoethanol (BME), was added. As shown
in Figure 2, a Michael adduct intermediate (Figure 2C) was
observed (Figure S12). Cyclopentenone was tracelessly
removed in 3 hours (Figure 2D). Other Michael addition donors
such as glutathione (GSH) or Edaravone could achieve the
similar result. The regeneration kinetic curve and1/2 (half
protein regeneration time) are given in (Figure S13).
Table 1. Various proteins were modified by 0.5 mM 14 or IAA in PBS at room
temperature.
Conversion / 1 h
Conversion / 2 h
Protein 10 M, 25 °C, PBS
0.5 mM IAA
UBXD[a]
21%
>95%
ND[c]
19%
37%
0.5 mM 14
>95%
2.5%
36%
Figure 3. SDS-PAGE gel analysis of four proteins: myoglobin with no cysteine
as
a control; modified UBXD; EV 71 3C protease and BSA emitted
0.5 mM IAA
HCP1
fluorescence (excitation wavelength: 365 nm), which were modified by a one-
pot click reaction.
0.5 mM 14
0.5 mM IAA
(+1) 44% (+2) ND
(+1) 34% (+2) 23%
(+1) 71% (+2) 5%
(+1) 35% (+2) 51%
EV 71 3C
protease[b]
0.5 mM 14
EV 71 3C protease was a cysteine protease and could be
assayed according to our previous report.[42] The activated
cysteine at the catalytic domain was modified by 14 or IAA,
which inactivated 3C protease. After BME was added, EV 71 3C
protease was revived in exchange of cyclopentenone, while IAA
modified 3C protease still remained inactivated. The process of
ligation and the regeneration protocol not only demonstrated
little change in the structure or conformation but also had little
disturbance on the enzymatic activity (Supporting information
part 9.2).
The modification of 14 was irreversible in the presence of
other cysteine conjugation compounds such as IAA or 2a.
Therefore, we proposed that cyclopentenone, could have further
application as a protecting group for cysteine. Many Michael
acceptors could modify the N-terminal amine of proteins at pH =
6, which was obviously incompatible with cysteine. In such
cases, 14 could well serve as protecting group for cysteine in the
process of the N-terminal modification by 2a. It was investigated
by UBXD as a model protein. After complete modification of the
cysteine in UBXD by 14 (Figure 5A), 20 mM 2a was added for
N-terminal modification in PB (pH = 6.0). The cyclopentenone
was not found to be exchanged by 2a. After complete
modification of the N-terminal of UBXD by 2a (Figure 5B), BME
was added (Figure 5C) to tracelessly remove the
cyclopentenone (Figure 5D). This exemplary application
illustrated that this method could serve as a good cysteine
protecting group.
(+1) 12% (+2) 38%
(+3) 35% (+4) 15%
(+3) 4% (+4) 48%
(+5) 38% (+6) 10%
(+2) 11% (+3) 35%
(+4) 47% (+5) 7%
(+4) 22% (+5) 41%
(+6) 27% (+7) 10%
0.5 mM IAA
MERS
3C-like
protease
0.5 mM 14
[a] The modification of UBXD and HCP1 was quantified by LCMS. [b] The
modification of EV 71 3C protease and MERS 3C-like protease was quantified
by protein deconvolution software. [c] Not detected.
A one-pot click reaction[41] was studied using this methodology
to demonstrate its application in protein imaging. Terminal
alkynes were introduced into 14 to give reagent 21, which was
used for the modification of cysteine in proteins including UBXD,
EV 71 3C protease and BSA with different molecular weights
(9.5 kD, 22.3 kD and 66.4 kD). Myoglobin, which has no
cysteines, was used as a control. IAA-yne (22) was studied in
parallel to 21 for comparison. After modification by 21 and 22
respectively (1.0 mM, 60 min, room tempurature), all modified
proteins further proceeded through a one-pot click reaction,
which conjugated with an azide rhodamine derivative. The SDS-
PAGE gel analysis was shown in Figure 3. Only the modified
cysteine-containing proteins emitted fluorescence. As expected,
the imaging capability of 21 was better. The stability of UBXD-
20-dye in PBS and humam plasma was determined
(Supporting information part 8.4). The modified UBXD
remained stable in PBS and slowly decomposed in human
plasma which might be caused by the existence of nucleophile
in human plasma.
As discussed in Scheme 2a, an unsaturated ketone was
newly formed in the product of the Michael addition tandem
elimination reaction (15b). It was envisaged that the Michael
addition tandem elimination reaction could be used to remove
the modification on cysteine and regenerate the protein (Figure
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Conclusions
In this study, 4-acetoxy cyclopentenone (14) was developed
as a reagent to achieve rapid, cysteine-specific modification of
proteins. This reaction featured fast kinetics with a stable
product. In addition, this conjugation could be tracelessly
removed by exchange with a Michael donor. The conjugation
and regeneration procedure not only had little effect on the
structure or conformation of the protein but also had little
disturbance on the enzymatic activity. Several applications of
this methodology were successfully demonstrated.
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This paper is dedicated to Prof. Jin-Pei Cheng on the occasion
of his 70th birthday. The financial support of National Natural
Science Foundation of China (NSFC Grant No. 21572116),
National Key Research and Development Program of China
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Keywords: protein modification; cysteine specific; tracelessly
cleavable
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Entry for the Table of Contents
COMMUNICATION
Jian Yu, Xiaoyue Yang, Yang Sun and
Zheng Yin*
Page No. – Page No.
Title
This manuscript described a rapid and cysteine-specific modification of proteins
using 4-substituted cyclopentenone via a Michael addition tandem elimination
reaction. The reaction was featured as fast kinetic and stable adduct. More
importantly, this conjugation could be tracelessly removed by exchange with a
Michael addition donor. The conjugation and regeneration process exhibited little
disturbance to the structures/conformations and biological functions of the proteins.
This article is protected by copyright. All rights reserved.