Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones

Article abstract of DOI:10.1021/jacs.7b00670

5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly, and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

Full text of DOI:10.1021/jacs.7b00670

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Article
Thiol Specific and Tracelessly Removable Bioconjugation
via Michael Addition to 5-Methylene Pyrrolones
Yingqian Zhang, Xiaoping Zhou, Yonghui Xie, Marc M. Greenberg, Zhen Xi, and Chuanzheng Zhou
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 Apr 2017
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Thiol Specific and Tracelessly Removable Bioconjugation via Michael
Addition to 5-Methylene Pyrrolones
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Yingqian Zhang,†, # Xiaoping Zhou,†, # Yonghui Xie,† Marc M. Greenberg,§ Zhen Xi†,‡ and Chuan-
† ‡
zheng Zhou*, ,
† State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, College of Chemistry,
Nankai University, Tianjin 300071, China
‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
§Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218
ABSTRACT: 5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools.
5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physio-
logically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addi-
tion of thiol to 5MPs occurs rapidly, cleanly and does not generate a stereocenter. The conjugates efficiently release thiols
via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs
valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein
immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.
INTRODUCTION
They react with cysteine to form disulfide linkages, which
can be cleavaged by thiols such as β-mercaptoethanol
(BME), dithiothreitol (DTT), or glutathione (GSH). Bro-
momaleimide is another reversible bioconjugation mole-
cule whose cysteine conjugate is reversed by thiols.20,21
Beyond these, dichlorotetrazine22 and 2-napthoquinone-
3-methides (oNQMs),23 have been used to release cysteine
upon photolysis of the corresponding conjugates.
Site-specific chemical modification is an invaluable ap-
proach for producing conjugated peptides and proteins,
which are widely used as therapeutics and as tools for
biochemical and biophysical studies.1 Among the 20 natu-
ral amino acids, cysteine is especially attractive for site-
specific chemical modification owing to its high nucleo-
philicity at physiological conditions and relatively low
natural abundance.2 Common reagents used for conjuga-
tion to cysteine include maleimides, iodoacetamides, al-
kyl halides, and pyridyl disulfides.3,4 Recently developed
cysteine bioconjugation reagents include dehydroa-
lanine,5,6 allyl sulfones,7 diene,8 exocyclic olefinic malei-
mides,9 perfluoroaromatic reagents,10,11 and organometal-
lic palladium reagents.12
Scheme 1
In some cases, reversible modification of a target pro-
tein is desired. For instance, once inside cells, release of
the active drug from an antibody-drug conjugate (ADC) is
necessary for the drug to fulfill its function.13,14 In another
example, enzymatic tagging and removal of epigenetic
modifications on histones is widely involved in gene ex-
pression regulation.15 Introducing cysteine by site-
directed mutagenesis followed by cysteine specific tag-
ging is a popular approach to generate epigenetic modifi-
cations.16 Development of chemically reversible bioconju-
gation reagents will help to illustrate the functions of epi-
genetic modifications.17 In addition, reversible targeting of
noncatalytic cysteines was recently shown to be an advan-
tageous strategy for developing kinase inhibitors.18
Recently, during the mechanistic studies of cleavage of
the C4'-oxidized abasic site (C4-AP) in a nucleosome, we
found that the sugar moiety of C4-AP is transferred to the
Despite the technique's potential utility, only a few
methods are available for reversible cysteine-specific bio-
conjugation. Pyridyl disulfides are commonly used.14,19
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ε-amine of lysine, resulting in a 5-methylene pyrrolone
Page 2 of 7
OH
O
OH
(5MP) modification on the histone.24,25 Here we report
that 5MPs are thiol-specific, reversible bioconjugation
reagents (Scheme 1) that are superior to commonly used,
structurally similar maleimides. 5MPs are easy to prepare,
stable under physiologically relevant conditions, and are
highly thiol-specific. The conjugates formed between
5MPs and cysteines are stable at neutral pH, but traceless-
ly regenerate native peptides/proteins in either pH 9.5
buffer or by thiol exchange.
1
2
3
4
5
6
7
8
NH2
O
N
O
O
N
N
HO
O
O
S
HO
S
6
4
5
O
NH2
H
N
O
NH2
NH2
O
HN
SH
O
7
9
8
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Highly selective conjugation of 5MPs to thiols. The
reactivity of 5MP with thiol was studied using 3a as a
model. Treatment of 3a with excess BME at pH 7.5 rapidly
produced 6 as the sole product (Figures S20-24). Fur-
thermore, reaction of 3a with a protected cysteine (7) was
also rapid and clean (Figure S27). Competitive nucleo-
philic attack between N-terminal amine of peptides or the
ε-amine of lysine with cysteine is a limitation of malei-
mides as bioconjugation reagents.29,30 However, 3a did not
react with the ε-amine of a protected lysine (8) between
pH 6.0 and 9.5 (Figure S28), suggesting that 5MPs have
superior selectivity toward cysteine compared to malei-
mides. Finally, unlike a maleimide, thiol addition to a
5MP (e.g. 3a) does not generate a stereocenter, which can
introduce complications.
RESULTS AND DISCUSSION
5-Methylene pyrrolone (5MP) preparation. 5MPs are
readily prepared by coupling primary amines with 1' in
neutral aqueous solution (Scheme 2).26 Treatment of
readily available 124,27 with 0.1 M HCl resulted in the as-
sumed intermediate 1', which after neutralization with
NaHCO3, reacted with amino acid derivative 2a at room
temperature for 1 h to give 3a in 90% yield. Other primary
amine substrates, including 2-aminoethanol (2b), propar-
gylamine (2c), biotin hydrazide (2d), 5-aminofluorescein
(2e), and anti-cancer drug doxorubicin (2f) were also effi-
ciently transformed to 5MPs 3b-f, suggesting this cou-
pling reaction is mild and compatible with a variety of
functional groups that are commonly used for protein
bioconjugation.
Scheme 2
(iii) 2a-e,
HEPES buffer,
pH 7.5
R
(i) 0.1M HCl
(ii) NaHCO3
AcO
AcO
HO
O
N
O
OMe
O
OH
MeO
3a-e
1
1'
NH2
O
OH
H2N
H2N
H2N
O
NH
H
HN
Glycinamide
Proparglymine
2c
2-Aminoethanol
H
H
N
2a 90%
2b 83%
80%
H2N
S
O
Biotin hydrazide 2d 42%
HO
O
OH
O
O
OH
O
OH
OH
O
O
O
OH
O
NH2
OH
H2N
O
5-Aminofluorescein
41%
Doxorubicin
40%
Figure 1. UPLC analysis of the reaction between peptide 9 (A)
with 3a (B) or 4 (C). Reaction conditions: 9 (0.4 mM) and 3a
or 4 (4 mM, 10 eq.) in HEPES (50 mM, pH 7.5), 100 mM NaCl,
37 °C, 1 h.
2e
2f
5-Methylene pyrrolones (5MPs) are more stable
than N-alkyl maleimides. Maleimides are chemically
unstable, especially in alkaline solutions. Ring-opening
hydrolysis yields maleamic acids, which are unreactive
toward thiols.28 For instance, N-hydroxyethyl maleimide
(4), decomposed completely via ring-opening hydrolysis
in 2 h at pH 7.5 and 9.5 (Figure S19). In contrast, no de-
composition was observed by UPLC-MS when 3a or 3b
was incubated between pH 6.0 and 9.5 for up to 72 h
(Figure S17 & 18). Hence, replacing one carbonyl with
methylene in maleimide significantly improves its re-
sistance to ring-opening hydrolysis.
The superior thiol specificity of 5MPs was further illus-
trated with reaction between 3a and peptide 9 (Figure 1B).
Peptide 9 was completely transformed to a single product
(10) after incubation with 10 eq. of 3a in HEPES buffer
(pH 7.5) for 1 h at 37 °C. The product exhibits a [MH]+ of
1906.7963 Da, which is consistent with a conjugate with
one molecule of 3a, despite the presence of other nucleo-
philic amino acids and the N-terminal amine. MS/MS
mapping of this conjugate revealed that 3a was exclusive-
ly attached to the side chain of cysteine (Figure S30). In
contrast, treating 9 with N-hydroxyethyl maleimide (4)
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ESI-MS analysis showed that 1 mM 3a yielded singly mod-
under the same conditions resulted in a complex mixture
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4
5
6
7
8
(Figure 1C). In addition to reaction at cysteine, conjugates
between the maleimide and the N-terminal amine, lysine,
as well as ring-opened hydrolysis products were also ob-
served (Figure 1D and S31).
ified ilvN, and almost completely bis-modified protein at
25 mM (Figure 3 and S39); whereas a more complex mix-
ture of modified ilvB was obtained upon incubation with
3a (Figure S40). Modification of ilvN by more hydropho-
bic and larger 3e was also efficient (Figure S41), similar to
3a yielded bis-modified protein at higher concentration
(25 mM).
To evaluate the conjugation efficiency and specificity of
5MP at different pH values, 9 was incubated with 10 eq. of
3a in acidic (pH 6.0), neutral (pH 7.5) or alkaline (pH 8.5,
9.5) buffer. UPLC-MS analysis of these reactions showed
that they were completed in 10 min, and produced a sole
product at pH 6.0–8.5 (Figure S33). At pH 9.5, the biocon-
jugation was still cysteine specific but accompanied by
formation of peptide dimer. Thus, cysteine bioconjuga-
tion using 5MPs is highly specific and efficient between
pH 6.0–8.5.
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Cysteine specific bioconjugation on proteins by
5MPs. We evaluated the reactivity and selectivity of 5MPs
with a histone H4 mutant (H4-R45C), which contains 11
lysines but only one cysteine at position 45.31 ESI-MS
analysis revealed that H4-R45C almost quantitatively re-
acted with 5MPs 3a-f in HEPES buffer (pH 7.5) in 2 h
(Figure 2 and S34). Trypsin digestion of the 3a/3b modi-
fied proteins, followed by peptide mapping using UPLC-
MS/MS showed that the 5MP reacted only on cysteine
(Figure S35 & 36). In contrast, treating H4-R45C with ma-
leimide 4 under the same conditions resulted in a mixture
containing 1-4 modifications per molecule (Figure S37).
Conjugation on lysine was identified except on the cyste-
ine (Figure S38).
Figure 3. MS analysis of the reaction between protein ilvN
with different concentrations of 3a. A +178 Da adduct (de-
noted by*) was present during the ESI-MS analysis for pro-
tein ilvN.
Traceless thiol regeneration from thiol-5MP conju-
gates. Thiol-maleimide conjugates decompose mainly via
ring-opening hydrolysis, accompanied by a small amount
of thiol exchange,34,35 which adds to the complexity of
their application in vitro and in vivo. In contrast, no trace
of ring-opening product was observed when thiol-5MP
conjugate (6) was incubated between pH 6.0 and 9.5.
However, UPLC-MS analysis revealed that 6 reverted over
time to 3a and BME (Figure S42). The half-lives of de-
composition of 6 in pH 6.0, 7.5, 8.5 and 9.5 buffers at 37
°C were 104.9, 16.9, 4.3 and 0.6 h, respectively. We pro-
pose that the thiol-5MP conjugate (e.g. 6) undergoes a
retro-Michael reaction, which is base catalyzed because of
the acidic property of the proton at position 3 in 6 (Figure
4A). The reversible reaction between thiol and 3a was fur-
ther explored by titrating 3a (1 mM) with increasing concen-
trations of BME or GSH in different buffers. Fitting the titra-
tion data provided apparent Kds18 of ~1 mM (Figure S43) over
a pH range from 6.0 to 8.5, suggesting 5MP has high activity
toward thiol addition over a rather broad pH range. At pH
9.5, it is hard to quantitatively convert 3a to adduct even in
presence of high concentration of thiol, suggesting that high
pH facilitates the addition and elimination reactions, and the
latter is more favorable.
Figure 2. MS analysis is of reactions between protein H4-
R45C and 3a-f. Reaction conditions: H4-R45C (50 ꢀM) and
3a-f (10 mM) in HEPES (10 mM, pH 7.5), 100 mM NaCl, 37 °C,
2 h.
In another example, we conjugated 5MPs to E. coli. ace-
tohydroxyacid synthase isozyme I (AHAS I) which cata-
lyzes the conversion of pyruvate to acetolactate during
the biosynthesis of branched-chain amino acids in plants
and microorganisms.32,33 AHAS I is composed of a regula-
tory subunit (ilvN) and a catalytic subunit (ilvB) that con-
tain two and nine free cysteines, respectively. These pro-
tein subunits were employed as substrates to evaluate the
efficiency of multiple cysteine bioconjugation by 5MPs.
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These results indicate that thiol-5MPs could be useful and ilvB. We immobilized ilvN to a 5MP functionalized
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6
7
8
as reversible and traceless cleavable conjugates at pH 9.5.
However, triggering the retro-Michael reaction under
alkaline conditions would be incompatible with intracel-
lular and some in vitro applications. We speculated that
the retro-Michael reaction of thiol-5MPs could be pro-
moted at neutral pH by excess thiol, through a known
thiol exchange mechanism.35 Consequently, conjugate 6
was incubated with 20 eq. (20 mM) of GSH at pH 7.5.
UPLC-MS analysis of the reaction showed that 6 was al-
most quantitatively transformed to 15 in less than 30 min
(Figure 4B & S44), demonstrating that thiols can efficient-
ly regenerate native proteins from 5MP-protein conju-
gates under mild and physiologically relevant
conditions.36 In contrast, thiol-maleimide conjugate 5 did
not release any thiol when incubated in the presence of
GSH, but instead decomposed exclusively through ring-
opening hydrolysis (Figure S45 & 46).
support and used it to fish out ilvB, with the aim to ulti-
mately releasing the complex in a controlled manner
(Figure 6A).
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Figure 5. SDS-PAGE gels showing the release of fluorescein
moiety from H4-R45C-3e in pH 7.5 buffer (A), with 10 mM
GSH (B) or 100 mM DTT (C) at 37 °C. Top gels: Fluorescence.
Bottom gels: Coomassie staining.
5MP functionalized solid supports were prepared by
treating commercially available long chain aminoalkyl
controlled porosity glass (LCAA-CPG)37 with freshly pre-
pared 1' at pH 7.5 for 4 h. Quantitation of free amines on
the support before and after the reaction using 4-chloro-
7-nitrobenzofurazan38 revealed that 5MP functionaliza-
tion proceeded essentially to completion. Mixing ilvN
with 5MP-CPG resulted in quantitative protein immobili-
zation in less than 10 min (Figure S49). Blocking the 5MP-
CPG with BME before adding ilvN substantially sup-
pressed ilvN immobilization, suggesting that protein im-
mobilization occurs through covalent bond formation
between an ilvN cysteine and a 5MP on the support. After
ilvN immobilization, excess 5MP was blocked by incubat-
ing with 5 mM BME for 10 min (Thiol exchange is negligi-
ble under these conditions). The ilvN-CPG was incubated
with a solution of ilvB at 4 °C for 1 h. SDS-PAGE analysis
of the supernatant indicated that ilvB was completely
pulled-down by the solid support (Figure S50).
Figure 4. Proposed mechanism (A) and UPLC analysis of the
thiol exchange reaction of 6 with GSH. Reaction conditions:
6 (1 mM) and GSH (20 mM, 20 eq.) in HEPES (20 mM, pH
7.5), 100 mM NaCl, 37 °C.
The stability of a 5MP-protein conjugate was examined
by incubating purified H4-R45C-3e at 37 °C in pH 7.5
buffer. Aliquots at different incubation times were ana-
lyzed by SDS-PAGE (Figure 5). Fluorescence quantifica-
tion of the protein bands indicates that the half-life for
fluorescein cleavage from protein is 28.5 h (Figure S47).
Addition of 10 mM GSH to the incubation buffer de-
creased the half-life to 16.8 h. Addition of 100 mM DTT
significantly accelerated fluorescein release from the pro-
tein (Figure 5C). The cleavage reaction was almost com-
plete in 6 h and MS analysis showed that the released
protein was intact (Figure S48), suggesting that thiol re-
lease occurs in a clean and traceless manner.
Various conditions were then testing for removing the
protein(s) from the support. For instance, incubation with
SDS-PAGE loading buffer (SDS LB: 50 mM Tris, pH 6.8,
100 mM DTT, 2% (w/v) SDS) at 95 °C for 5 min efficiently
released ilvN and ilvB (Figure 6B). Treatment with the
same buffer at room temperature (5 min) selectively elut-
ed the non-covalently bonded ilvB. Several conditions
were also examined for releasing active ilvB-ilvN complex.
SDS-PAGE analysis of supernatants following incubation
of supports using buffers of pH varying from 6.0 to 9.5 at
4 °C for 6 h revealed that the proteins were removed only
at the most alkaline pH employed (Figure 6B). Protein
elution was also examined using various BME concentra-
tions (Figure S51), and 700 mM BME at 4 °C for 2 h almost
quantitatively released ilvB-ilvN from the support (Figure
6B).
Protein immobilization and active complex pull-
down using 5MPs. The simplicity of 5MP synthesis sug-
gested that they could be readily employed to functional-
ize primary amine containing solid supports. 5MP modi-
fied supports could be useful for immobilizing proteins
and for purifying cysteine containing proteins. As an ex-
ample, the ability of 5MPs to immobilize a protein for
pull-down of an active complex was illustrated using ilvN
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Figure 6. Preparation and utility of a 5MP immobilized protein. (A) 5MP functionalized solid support used for protein immobi-
lization and pull-down of active protein complex. (B) SDS-PAGE analysis of the eluted proteins from ilvB-ilvN-CPG under differ-
ent conditions. (C) The relative activity of eluted ilvB-ilvN complex released under different conditions. (D) SDS-PAGE showing
pull-down of ilvB from cell lysate using ilvN-CPG.
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The ilvB-ilvN proteins eluted using pH 9.5 buffer and
BME were subjected to dialysis to remove salt or BME. MS
analysis revealed that the eluted ilvN and ilvB were intact
(Figure S52). Moreover, reconstituted ilvN-ilvB complex
converted two pyruvate molecules to an acetolactate in
the prescence of thiamin diphosphate (ThDP) and flavin
ade-nine dinucleotide (FAD) cofactors. The enzymatic
activity of the eluted proteins was almost the same as that
of reconstituted complex (Figure 6C).
5MP-protein conjugates decompose slowly at neutral
pH (half time ~ 1 day), but tracelessly and rapidly release
native proteins at pH 9.5 or via a thiol exchange reaction.
The slow degradation of 5MP-protein conjugates could
limit their applications in some fields, such as in vivo de-
livery conjugated drugs. However, the properties of 5MPs,
especially compared to maleimides, will make them very
valuable in cases where reversible and traceless cleavage
of bioconjugates under physiological conditions or tem-
porary thiol/cysteine protection is required.
Finally, we demonstrated that ilvN-CPG could be used
for efficiently capturing ilvB from cell lysate (Figure 6D).
Thus, 5MP functionalized solid supports are good choices
for protein immobilization and pull-down of active com-
plexes, which could be applicable in protein purification,
as well as protein-protein, and protein-nucleic acid inter-
action studies.
In addition, the mild and efficient generation of 5MPs
from primary amines could be further utilized to produce
5MPs on lysine residues and N-terminal positions of pro-
teins,39,40 which could be subjected to further functionali-
zation or to generate inter/intramolecular lysine-cysteine
cross-links. Research in this area is ongoing.
ASSOCIATED CONTENT
CONCLUSIONS
Supporting Information. General experimental methods,
spectroscopic data for all new compounds，HPLC profiles
and gel pictures. This material is available free of charge via
the Internet at http://pubs.acs.org.
The above experiments demonstrate that 5-methylene
pyrrolones (5MPs) are a new type of thiol-specific and
tracelessly removable bioconjugation tools. 5MPs have a
number of merits, including ease of synthesis. 5MPs are
prepared from primary amines through a mild coupling
reaction using readily available chemicals. 5MPs exhibit
significantly improved stability and thiol specificity com-
pared to maleimides, which are widely used for thiol bio-
conjugation. Cysteine specific bioconjugation of protein
by 5MP occurs rapidly, cleanly, and the Michael addition
does not generate a new stereocenter, which simplifies
product analysis.
AUTHOR INFORMATION
Corresponding Author
* chuanzheng.zhou@nankai.edu.cn
Author Contributions
# These authors contributed equally.
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(19) Saito, G.; Swanson, J. A.; Lee, K.-D. Adv. Drug Deliv. Rev.
2003, 55, 199-215.
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Am. Chem. Soc. 2010, 132, 1960-1965.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by NSFC (21572109, 21332004) and
Natural Science Foundation of Tianjin City (15JCYBJC53300).
C. Z. is grateful for the sponsorship from the National Thou-
sand Young Talents Program. M. M. G. is grateful to the Na-
tional Institute of General Medical Science (GM-063028) for
financial support.
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