Chemoselective Copper-Mediated Modification of Selenocysteines in Peptides and Proteins

Article abstract of DOI:10.1021/jacs.1c06101

Highly valuable bioconjugated molecules must be synthesized through efficient, chemoselective chemical modifications of peptides and proteins. Herein, we report the chemoselective modification of peptides and proteins via a reaction between selenocysteine residues and aryl/alkyl radicals. In situ radical generation from hydrazine substrates and copper ions proceeds rapidly in an aqueous buffer at near neutral pH (5-8), providing a variety of Se-modified linear and cyclic peptides and proteins conjugated to aryl and alkyl molecules, and to affinity label tag (biotin). This chemistry opens a new avenue for chemical protein modifications.

Full text of DOI:10.1021/jacs.1c06101

pubs.acs.org/JACS
Article
Chemoselective Copper-Mediated Modification of Selenocysteines
in Peptides and Proteins
Zhenguang Zhao, Daphna Shimon, and Norman Metanis*
Cite This: https://doi.org/10.1021/jacs.1c06101
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Highly valuable bioconjugated molecules must be synthesized through efficient,
chemoselective chemical modifications of peptides and proteins. Herein, we report the
chemoselective modification of peptides and proteins via a reaction between selenocysteine
residues and aryl/alkyl radicals. In situ radical generation from hydrazine substrates and copper
ions proceeds rapidly in an aqueous buffer at near neutral pH (5−8), providing a variety of Se-
modified linear and cyclic peptides and proteins conjugated to aryl and alkyl molecules, and to
affinity label tag (biotin). This chemistry opens a new avenue for chemical protein modifications.
INTRODUCTION
■
Protein post-translational modifications (PTMs) play a
prominent role in expanding proteins function and enable
the precise modification of proteins with a diverse range of
functional moieties.1 Although PTMs are normally facilitated
by enzymatic processes, chemists have turned their attention to
the development of regio- and chemoselective chemical
modifications of proteins based on metal or organic catalysis.
In recent years we have witnessed significant progress in the
field, as many research groups have contributed to a rich
chemical toolbox with precise and highly chemoselective
reactions,2 most of which work in aqueous solutions and under
ambient conditions.3−7 Among the 20 canonical amino acids,
cysteine (Cys) is still the most extensively targeted for
Figure 1. Modification of Sec/Cys in peptides and proteins.
chemical modifications owing to its high nucleophilicity and
low abundance (1.7% of known sequences).8−12 This includes
classical thiol−ene chemistry,13 typical nucleophilic substitu-
tion reactions with electrophiles,8−10,14,15 or transition-metal-
mediated modifications.16−19 Because these reactions require a
free Cys residue, site selectivity among multiple free Cys
residues in the protein sequence has eluded protein chemists
(Figure 1a).20 Therefore, it is important to develop
complementary protein modification methods based on other
amino acid residues that are chemoselective, even in the
presence of Cys residues.
target for chemical modification in peptides and pro-
teins.9,11,15,30,41,42 As a result, Sec is readily oxidized to
diselenide (Se−Se) or selenylsulfide (Se−S); to generate active
selenol for downstream modification, a reducing reagent, such
as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine
(TCEP), is typically needed (Figure 1a). However, both
reagents reduce disulfide cross-links as well, and TCEP is
Selenocysteine (Sec, U), the 21st natural amino acid,21 is
Cys’s isostere and shares many of its properties, with notable
differences including lower pKa22,23 and reduction poten-
tial.24−26 These differences have been explored in chemical
protein synthesis,27−29 modification,30,31 and folding.32−40
Similar to Cys, Sec’s inherent nucleophilicity makes it a good
Received: June 15, 2021
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
© XXXX American Chemical Society
A

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
known to cause undesired deselenization.27 Both outcomes
would interfere with the intended modification reactions.
Recently, Pentelute, Buchwald, and co-workers exploited the
electrophilicity of oxidized Sec, and using arylboronic acids
with either copper catalysts or electron-rich aromatic
conjugates, they showed regio- and chemoselective modifica-
tion of oxidized Sec in peptides and proteins (Figure 1b).43,44
Yet, in these reports the modification was not tested in the
presence of other reactive side chains (such as disulfide
bonds). Furthermore, it is extremely desirable to develop
highly selective bioconjugation techniques that are based on
new chemistries.
a
Table 1. Optimization of the Reaction Conditions
Here, we report a previously unexplored, chemoselective Sec
modification with a series of hydrazine compounds in the
presence of copper ions. This strategy exploits the efficient
generation of an aryl/alkyl radical from hydrazine substrates in
the presence of Cu(II),45−48 which readily reacts with Sec in
peptides and proteins to provide the desired corresponding
conjugates (Figure 1c).
Recently, we reported on the use of Cu(II) ions for the
deprotection of selenazolidine (Sez) and thiazolidine (Thz)
during chemical protein synthesis49−51 and proposed that
Cu(II) acts as a Lewis acid that binds the selenium of Sez. In
addition, Arsenyan’s group reported the use of Cu(II) to bind
and oxidize the Se atom of Ph2Se2, converting it to the
electrophilic PhSe+, which in turn reacted with triple-bond-
containing molecules.52 Almost half a century ago, it was
reported that Cu(II) (and other metal ions) can oxidize
phenylhydrazine to generate a phenyl radical, which can react
with biomolecules in vivo.45,53,54 Of note, the deselenization
reaction of Sec to Ala27,28 (and Ser)28,55 in the presence of
TCEP was proposed to go through a radical mechanism, owing
to the ability of Se atoms to form radicals under mild
conditions.56 Combining these observations, we envisioned
generating radical intermediates from hydrazine reagents with
Cu(II), enabling efficient, chemoselective modification of Sec
residues in peptides and proteins (Figure 1c).
a
b
Further details can be found in the Supporting Information. 1.0
c
mM of reduced peptide. Yields calculated according to integrated
areas of HPLC peaks (at 220 nm).
RESULTS AND DISCUSSION
■
Optimization of the Reaction Conditions. To test our
hypothesis, a Sec-containing model peptide, TFUGK-NH2
dimer (1a), was prepared by standard Fmoc-SPPS,57−59 and
3,5-dimethylphenylhydrazine (2a) was employed as the model
substrate. Combining model peptide (1a, 1.0 mM for the
selenol monomer) with 2 mM 2a and 0.5 equiv of CuSO4·
5H2O in phosphate buffer (PB, 10 mM, pH 6) at room
temperature provided the desired product 3a in 70% yield in
10 min (Table 1, entry 1). The yield increased to 95% with 1.0
equiv of CuSO4·5H2O (entry 2), but was not improved further
with more Cu(II) (entry 2, Figure S2, Supporting
Information). Expectedly, the yield of 3a decreased to 60%
(entry 4) and 80% (entry 5) when the concentration of
hydrazine 2a was decreased to 1.2 and 1.5 mM, respectively
(Figure S3). Varying pH conditions (entries 2, 6−10, Figure
S4) showed that slightly acidic conditions (pH 6, entry 2) gave
the optimized yield of 3a.
Scope of Hydrazines in the Reaction. With the
optimized reaction conditions in hand, we next investigated
the scope of hydrazine derivatives tolerated in the modification
reaction. As shown in Figure 2, we tested the reaction between
the model peptide 1a and aromatic hydrazine substrates with
electron-donating groups on the phenyl ring, such as 3,5-
dimethyl (2a) and p-methoxyl (2b) substituents (Figure 2a).
Figure 2. Substrate scope of hydrazines. 11.0 mM with respect to the
selenol monomer. (a) Arylhydrazines: The reactions for 3a−3d were
performed at room temperature, and the yields were obtained after 10
min, whereas strong electron-deficient 3e was obtained after 3 h. (b)
Heteroarylhydrazine: The reaction performed at 37 °, and the yield
was obtained after 2 h. (c) Benzyl and alkylhydrazines: The reaction
performed at 37 °C; the yields of 3g and 3h were obtained after 1 h,
and for 3i, 3j, and 3k they were obtained after 2 h. (d) Radical-clock
reaction. (e) Biotinylation of the model peptide 1a under optimized
conditions. All yields presented were determined by integrated areas
of HPLC peaks (at 220 nm), 2whereas isolated yields are in
parentheses.
Both reactions were completed within 10 min and generated
the corresponding products with excellent yields (3a and 3b,
Figures S4 and S5). Unsubstituted phenylhydrazine (2c)
reacted with model peptide 1a to form the modified product
(3c) with 95% conversion (Figure S6), implying that the
B
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
electron-donating groups on the phenyl ring are not required
for the modification of Sec residue in peptides. Furthermore, 4-
hydrazineylbenzoic acid (2d), which contained a weak
electron-withdrawing group (p-carboxyl), was also found to
be effective under optimized conditions (Figure S7). However,
when the extremely electron-poor perfluorophenylhydrazine
(2e) was used as a substrate, the strong electron-withdrawing
substituents slowed down the reaction, and only 19%
conversion of modified product (3e) was observed after
prolonged incubation (3 h, Figure S8). As a heteroarene, 4-
hydrazineylpyridine (2f) was also tolerated in this reaction
system and afforded the desired product (3f) in good yield
within 2 h (Figure 2b and Figure S9).
Given that the diversity of substrates is one of the most
challenging targets in in vitro peptide modification, we decided
to test the applicability of alkyl hydrazine substrates, which are
less reactive than aromatic hydrazine because of the unstable
radical intermediates60 in this reaction. We were delighted to
find that all tested hydrazines including benzyl, isobutyl,
isopropyl, and tert-butyl hydrazines proceeded smoothly to
form the modified products in moderate-to-good yields (52−
93%, 3g−3j) and within 1−3 h (Figure 2c and Figures S10−
S13). Notably, the reaction of the model peptide 1a with
isobutylhydrazine (2h) provided the isobutyl-modified product
3h, whereas the reaction with tert-butylhydrazine (2j) provided
the tert-butyl product 3j. This was supported by HPLC data
(Figure S13) and unequivocally confirmed with NMR analysis
(Figures S11 and S13) of the two products, 3h and 3j,
suggesting that the radical combination reaction was so rapid
that any undesired 1,2-rearrangement of isobutyl radical, which
could occur to generate stabilized tert-butyl radical,61 had not
taken place. Furthermore, (cyclopropylmethyl)hydrazine (2k,
Figure 2d) reacted with the model peptide 1a to afford the
exclusive cyclopropane-opened modified product 3k (Figure
S14), which supports the formation of radical intermediates in
this transformation.62 Lastly, the biotin affinity tag (2l, see
Scheme S2 for synthesis details) was successfully introduced to
the model peptide (1a) within 10 min by our developed
protocol (Figure 2e and Figure S15).
Mechanistic Study by Electron Paramagnetic Reso-
nance (EPR). For further support of the likely radical
mechanism, we followed the arylation reaction by EPR. The
clean formation of a DMPO-Ph spin adduct was obtained
when the model seleno-peptide TFUGK-NH2 dimer (1a)
reacted with phenylhydrazine (2c) and CuSO4 (Figure 3), as
well as when just 2c and CuSO4 were used alone (Figure S17).
The hyperfine values (g = 2.00552, aN = 15.9227 G, and aH =
24.6433 G) are consistent with those of a carbon radical being
trapped by the DMPO and agree with Hill and Thornalley,
who ascribed them to the phenyl radical.45 In addition, we
studied three peptides containing Se (TFUGK-NH2, 1a), S
(LKFCAG-NH2, 1c), and neither Se nor S (ALKFAG-NH2,
1b), first analyzing the effect of varied molar ratios of peptide/
hydrazine/CuSO4, 1:2:1 and 1:1:1 (Figure S20, top row and
middle row, respectively), in which DMPO was added last. No
obvious difference of the signal of the DMPO-Ph spin adduct
was observed in these cases. However, when the ratio of
1:0.5:1 was tested, the signal of the DMPO-Ph spin adduct was
largest with 1a (Figure 3c, green) and then with 1c (Figure 3e,
green) and was smallest with 1b (Figure 3d, green).
Furthermore, the signal intensity of the DMPO-Ph spin
adduct increased dramatically (approximately 100%) when 2c
was the last component added in the cases of ALKFAG-NH2
Figure 3. EPR experiments support the radical reaction. (a) Scheme
of the trapping of phenyl radical by DMPO. EPR spectrum of DMPO-
Ph spin adduct, formed during the reaction of the peptides 1a−1c,
phenylhydrazine 2c, and CuSO4, with a ratio of 1:2:1 (b) and 1:0.5:1
(c−e). (b) Peptide: TFUGK, 1a, black for experimental, red for
simulated. (c) Peptide: TFUGK, 1a. (d) Peptide: ALKFAG, 1b. (e)
Peptide: LKFCAG, 1c. Black lines in (c−e): Phenylhydrazine 2c was
added last. Green lines in (c−e): DMPO was added last.
(1b, Figure 3d, black) and LKFCAG-NH2 (1c, Figure 3e,
black), whereas the signal intensity increase of the DMPO-Ph
spin adduct was much smaller than that for TFUGK-NH2 (1a,
Figure 3c) (approximately 30%) when 2c was the last
component added. These results suggest that the Se atom
interacts with the formed Ph radical and perhaps partially
stabilizes it, thus increasing the radical lifetime until it is able to
react with DMPO to form the DMPO-Ph spin adduct. Sulfur is
partially able to stabilize the Ph radical, whereas when no Se or
S are present, the Ph radical lifetime is very short because of
the reactions with water and other radicals (further EPR
experiments can be found in the Supporting Information).
In light of the above results, we propose the mechanism
shown in Scheme 1. R-NHNH2 (2) is oxidized to R• by Cu2+,
which binds to the Se of Sec in the peptide.45−48,52 The formed
R• reacts with diselenide (or selenylsulfide) to generate the
intermediate trivalent selenium radical (3),63,64 which converts
to the final product (4) and another Cys peptide (3-II) or
another Sec radical (3-I), which could in turn form dimer (1)
for the next reaction.
Chemoselectivity Study. Next, we turned to study the
tolerance of this modification on unprotected amino acid side
chains. First, in the absence of Sec residue, the peptide
ALKFAG-NH2 (1b) was inert to the reaction with phenyl-
hydrazine (2c) under optimized conditions (Figure 4a and
Figure S21). Because of similar properties of sulfur and
selenium, the chemoselectivity of this reaction was tested.
Thus, peptide LKFCAG-NH2 (1c) showed 10% Cys-modified
products (Figure S22), whereas 13% of the Met modification
product was obtained for LKMAG-NH2 (1d) (Figure 4a and
Figure S23), both of which were observed only after an
extended time (2−18 h) when compared to that of the Sec
reaction (<10 min).
To establish the versatility of this methodology, the
modification of more complex peptide substrates (1e and 1f,
Figure 4b), which contained various functional groups, was
also evaluated. When the modification of peptide 1e was
conducted under standard conditions, the modified product
(4e) was obtained in 16% yield. Yet, the yield of product 4e
increased to 73% when the concentration of phenylhydrzaine
C
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
Scheme 1. Proposed Mechanism for Radical Modification of
Sec by Hydrazine in the Presence of Cu Ions
Figure 4. Systematic investigation of Sec-specific modification in the
presence of other reactive residues. The yields were determined by
HPLC, whereas the values in parentheses were the isolated yields. (a)
1
Chemoselectivity study. (b) Modification of linear peptides. 2 equiv
(2c) was increased to 5 mM, the amount of CuSO4 was
increased to 2 equiv, and the reaction was incubated for 30 min
(Figure 4b). Note that even with excess phenylhydrazine (2c)
used in this reaction, only a minor degree of arylation at the
Cys residue (∼3%) was observed (Figure S24). Moreover,
trypsin digestion of 4e confirmed the site-selective modifica-
tion of Sec residue of the peptide (Figure S28). The radical
conjugation was similarly successful in a variety of more
unusual circumstances. Because peptide 1f contains a Met
residue at its N-terminus, which can be easily oxidized (to
Met(O)), we decided to do the reaction under argon. Hence,
Cu(II) was provided in excess, to compensate for the absence
of molecular oxygen (typically required for the hydrazine
oxidation step, Scheme 1). Therefore, using 8 equiv of CuSO4,
peptide 1f reacted with phenylhydrazine (2c) to afford the
corresponding monoarylated product (4f) in 93% yield (Figure
4b and Figure S25), with no side reactions observed on other
functional groups in the peptide. Furthermore, the modifica-
tion of head-to-tail cyclic peptide 1g proceeded smoothly to
form the product (4g) in 88% yield (Figure 4c and Figure
S26), which further demonstrates the potential of this
transformation in the late-stage modification of biomolecules.
Finally, peptide 1h, which possessed a selenylsulfide between
selenocysteine and 2-thiol-5-nitropyridine (TNP), reacted
readily with phenylhydrazine (2c), giving 96% yield of Sec-
modified peptide (4h, which has the same structure as 3c)
within 10 min under our standard conditions (Figure 4d and
Figure S27).
2
of CuSO4 and 5 mM phenylhydrazine were used. 8 equiv of CuSO4
and 1.5 mM phenylhydrazine under an Ar atmosphere. (c)
Modification of cyclic peptide: 0.5 mM peptide dimer (1g), 1.0
mM with respect to the selenol monomer. (d) Modification of TNP-
Sec-containing peptide, the structure of 4h is the same as 3c. TNP =
2-thiol-5-nitropyridine.
Figure 5. Intramolecular modification. (a) Cyclization of Sec-
containing peptide (1i) bearing phenylhydrazine at the N-terminus.
(b) HPLC traces of the cyclization of 1i under different conditions (at
room temperature and after 10 min). The yields were determined by
HPLC; yield in parentheses was isolated yield. * is a side product
formed by hydrogen transfer to the phenyl radical intermediate; # is a
side product formed by self-coupling of the phenyl radical
intermediate (dimer) (Figure S30).
Intramolecular Cyclization of Sec-Contained Peptide.
With an efficient intermolecular peptide modification method
established, we envisioned applying this technique to provide a
cyclic peptide through the construction of an intramolecular
Se-phenyl linkage (Figure 5a). To this end, we further
optimized conditions for peptide cyclization with a linear
Sec-containing peptide (1i) bearing phenylhydrazine at the N-
terminus (Figure S29). The cyclization of peptide 1i (1 mM)
under the standard conditions described above (10 mM PB,
D
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
1 equiv of CuSO4, pH 6) provided the desired cyclic product
(4i) in 45% yield after 10 min. Two side products were
observed (assigned as * and #, the chromatogram in Figure 5b
and Figure S30) formed by hydrogen transfer to the phenyl
radical intermediate and self-coupling of phenyl radical
intermediate (to give the diphenyl dimer peptide, Figure
S30), respectively. Increased yield of the desired cyclization
product could be obtained when diluted peptide solutions
were used.65,66 Ultimately, this transformation was further
improved to obtain 74% yield of 4i within 10 min by using 50
μM peptide and 10 equiv of CuSO4 at slightly more acidic
conditions (pH 5, Figure S30), which decrease the percentage
of both side products.
Biotinylation of Sec-Containining Proteins. Encour-
aged by these results, we focused on expanding our method
toward larger protein domains. The ubiquitin(G47U) variant
5a was prepared by Fmoc-SPPS57−59 and native chemical
ligation (NCL)67 (Figure S33), in which the solvent-exposed
Gly47 was substituted with Sec. With ubiquitin(G47U) in
hand, the biotin-containing phenylhydrazine (2l) (Scheme S2)
was utilized for the modification of 5a. To prevent Met
oxidation in the presence of Cu(II) and O2,68,69 the reaction of
5a was carried out under an argon atmosphere. Gratifyingly,
almost quantitative biotinylation of protein 5a was observed at
100 μM concentration within 30 min in the presence of 4
equiv of CuSO4 (Figure 6 and Figure S35) and the modified
folding of the synthetic protein was accomplished in the
presence of reduced and oxidized glutathione (GSH and
GSSG, respectively) to provide the folded protein 7a,
containing two disulfide bonds and glutathionylated Sec25
(Sec25-SG)71 (Figures S40 and S70). The modification of 7a
with 2l also proceeded under an argon atmosphere to prevent
undesired oxidation at the Met residues. For the SDF1(S25U)
modification, we employed 12 equiv of 2l and 4 equiv of
CuSO4 (excess reagent was used in this case to minimize the
possible electrostatic repulsion of Cu(II) with the solvent-
exposed cluster of positive charges on the protein surface).
Within 30 min, we observed more than 70% biotinylation of
protein 7a, and the modified product 8a was isolated in 43%
yield (Figure 7 and Figures S41 and S72). Gratifyingly, the
Figure 7. Modification of disulfide-containing protein SDF1(S25U).
(a) Scheme and (b) HPLC trace of the modification of SDF1(S25U)
7a with 2l to form the modified product 8a (see Figures S76 and S77
for HR-MS data). & is a side product from hydroxylation of 2l radical
intermediate; * is a side product from hydrogen transfer to 2l radical
intermediate; and # is a side product from self-coupling of 2l radical
intermediate (dimer).
reaction was tolerant to the two disulfide bonds of SDF1-
(S25U), and all other amino acid side chains in the product 8a.
For further support of the site-selective modification at the Sec
residue, we tested the deselenization reaction of SDF1(S25U)
(7a) in comparison to the biotinylated protein 8a. Under
deselenization reaction conditions (50 mM TCEP, pH 5),
SDF1(S25U)-SG (7a) was converted into fully reduced
SDF1(S25A) (the Sec was converted into Ala, as expected,
and the two disulfide bonds were reduced to dithiols). On the
other hand, in the biotinylated SDF1(S25U) case, 8a, under
the same reaction conditions, only the two disulfide bonds
were reduced to dithiols, whereas the modified biotinylated-
Sec was unaffected. These results implied that the modification
of Sec by our approach has a negligible effect on disulfide
bonds or other functional groups in the protein.
Figure 6. Modification of ubiquitin(G47U) 5a with 2l, and the mass
spectra of 5a and modified product 6a (see Figures S73 and S74 for
HR-MS data).
product 6a was isolated in 60% yield by semiprep HPLC. In
contrast, the G47A variant of ubiquitin (5b), which was
prepared by the deselenization of 5a,27,28 was inert to the
reaction with 2l (see Figure S36 for the details).
Furthermore, we decided to confirm the reaction’s chemo-
selectivity with a Cys-rich protein, stromal cell-derived factor-1
(SDF1), which contains two disulfide bonds and all reactive
side chains.70 The chemical synthesis of SDF1(S25U), in
which the solvent-exposed Ser25 was substituted with Sec, was
accomplished by SPPS and NCL (see Figures S37−S39 for
further details). With SDF1(S25U) in hand, the oxidative
CONCLUSION
■
In summary, we have developed a widely applicable and
efficient protocol for chemoselective modification of peptides
E
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
and proteins based on a radical-mediated arylation and
alkylation of selenocysteine using Cu(II) and a corresponding
hydrazine. Mechanistic analysis, specifically EPR and radical
clock reaction, supports a radical mechanism. The reported
facile transformation can be carried out without any reducing
agent, at a near-neutral pH (5−8), in aqueous solution,
without organic cosolvents, and under mild reaction
conditions. This reaction displays a broad scope of hydrazine
substrates, including aromatic, heteroaryl, and even alkyl
hydrazines, and is highly selective, even in the presence of
other reactive amino acids. In addition, oxidized and reduced
selenocysteine both perform well in this transformation, which
greatly expands the range of application of this technique.
Furthermore, we demonstrated the high efficiency of this
strategy in the preparation of cyclic peptides and the
modification of larger and more complex peptides and proteins
(ubiquitin and SDF1) at micromolar concentrations. Although
all tested peptides and proteins were prepared by chemical
synthesis, this methodology can be applied to selenoproteins
that are prepared using expression systems.72−76 This new
approach is complementary to the reported umpolung method
by Cohen et al.,43,44 as our radical reaction is highly
chemoselective, does not require ligands for copper ions,
works in neat aqueous solutions on peptides and proteins, and
tolerates all functional groups. Further work to explore the
labeling and fine-tuning of more complex biological molecules
in vivo and in vitro with this technology is ongoing in our
laboratory.
REFERENCES
■
(1) Walsh, C. T.; Garneau-Tsodikova, S.; Gatto, G. J., Jr. Protein
Posttranslational Modifications: The Chemistry of Proteome
Diversifications. Angew. Chem., Int. Ed. 2005, 44, 7342−7372.
(2) Davis, B. G. Mimicking Posttranslational Modifications of
Proteins. Science 2004, 303, 480−482.
(3) Prescher, J. A.; Bertozzi, C. R. Chemistry in living systems. Nat.
Chem. Biol. 2005, 1, 13−21.
(4) Wright, T. H.; Bower, B. J.; Chalker, J. M.; Bernardes, G. J.;
Wiewiora, R.; Ng, W. L.; Raj, R.; Faulkner, S.; Vallee, M. R.;
Phanumartwiwath, A.; Coleman, O. D.; Thezenas, M. L.; Khan, M.;
Galan, S. R.; Lercher, L.; Schombs, M. W.; Gerstberger, S.; Palm-
Espling, M. E.; Baldwin, A. J.; Kessler, B. M.; Claridge, T. D.;
Mohammed, S.; Davis, B. G. Posttranslational mutagenesis: A
chemical strategy for exploring protein side-chain diversity. Science
2016, 354, No. aag1465.
(5) Yang, A.; Ha, S.; Ahn, J.; Kim, R.; Kim, S.; Lee, Y.; Kim, J.; Söll,
D.; Lee, H.-Y.; Park, H.-S. A chemical biology route to site-specific
authentic protein modifications. Science 2016, 354, 623−626.
(6) Spicer, C. D.; Davis, B. G. Selective chemical protein
modification. Nat. Commun. 2014, 5, 4740.
(7) Kundu, R.; Ball, Z. T. Rhodium-catalyzed cysteine modification
with diazo reagents. Chem. Commun. 2013, 49, 4166−4168.
(8) Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G.
Chemical Modification of Proteins at Cysteine: Opportunities in
Chemistry and Biology. Chem. - Asian J. 2009, 4, 630−640.
(9) Embaby, A. M.; Schoffelen, S.; Kofoed, C.; Meldal, M.; Diness,
F. Rational Tuning of Fluorobenzene Probes for Cysteine-Selective
Protein Modification. Angew. Chem., Int. Ed. 2018, 57, 8022−8026.
(10) Kasper, M. A.; Glanz, M.; Stengl, A.; Penkert, M.; Klenk, S.;
Sauer, T.; Schumacher, D.; Helma, J.; Krause, E.; Cardoso, M. C.;
Leonhardt, H.; Hackenberger, C. P. R. Cysteine-Selective Phospho-
namidate Electrophiles for Modular Protein Bioconjugations. Angew.
Chem., Int. Ed. 2019, 58, 11625−11630.
(11) Cal, P. M. S. D.; Bernardes, G. J. L.; Gois, P. M. P. Cysteine-
Selective Reactions for Antibody Conjugation. Angew. Chem., Int. Ed.
2014, 53, 10585−10587.
(12) Gunnoo, S. B.; Madder, A. Chemical Protein Modification
through Cysteine. ChemBioChem 2016, 17, 529−553.
(13) Hoyle, C. E.; Bowman, C. N. Thiol-Ene Click Chemistry.
Angew. Chem., Int. Ed. 2010, 49, 1540−1573.
(14) Zhang, Y.; Zhou, X.; Xie, Y.; Greenberg, M. M.; Xi, Z.; Zhou,
C. Thiol Specific and Tracelessly Removable Bioconjugation via
Michael Addition to 5-Methylene Pyrrolones. J. Am. Chem. Soc. 2017,
139, 6146−6151.
(15) Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.;
Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach
to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135, 5946−
5949.
(16) Deng, J. R.; Chung, S. F.; Leung, A. S. L.; Yip, W. M.; Yang, B.;
Choi, M. C.; Cui, J. F.; Kung, K. K. Y.; Zhang, Z.; Lo, K. W.; Leung,
Y. C.; Wong, M. K. Chemoselective and photocleavable cysteine
modification of peptides and proteins using isoxazoliniums. Commun.
Chem. 2019, 2, 93.
(17) Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B.
L.; Buchwald, S. L. Organometallic palladium reagents for cysteine
bioconjugation. Nature 2015, 526, 687−691.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c06101.
■
sı
Materials and methods, characterization data, HPLC,
1
LCMS, and HRMS, H- and 13C NMR spectra, and
synthesis procedures (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Norman Metanis − Institute of Chemistry, The Center for
Nanoscience and Nanotechnology, The Hebrew University of
Jerusalem, Jerusalem 9190401, Israel; orcid.org/0000-
0002-6373-9318; Email: metanis@mail.huji.ac.il
Authors
Zhenguang Zhao − Institute of Chemistry, The Hebrew
University of Jerusalem, Jerusalem 91904, Israel
Daphna Shimon − Institute of Chemistry, The Hebrew
University of Jerusalem, Jerusalem 91904, Israel
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c06101
(18) Messina, M. S.; Stauber, J. M.; Waddington, M. A.; Rheingold,
A. L.; Maynard, H. D.; Spokoyny, A. M. Organometallic Gold(III)
Reagents for Cysteine Arylation. J. Am. Chem. Soc. 2018, 140, 7065−
7069.
Notes
The authors declare no competing financial interest.
(19) Lu, X.; He, S.-J.; Cheng, W.-M.; Shi, J. Transition-metal-
catalyzed C H functionalization for late-stage modification of peptides
and proteins. Chin. Chem. Lett. 2018, 29, 1001−1008.
(20) Kuan, S. L.; Wang, T.; Weil, T. Site-Selective Disulfide
Modification of Proteins: Expanding Diversity beyond the Proteome.
Chem. - Eur. J. 2016, 22, 17112−17129.
ACKNOWLEDGMENTS
■
We thank Ms. Reem Mousa, Mrs. Ricki Notis Dardashti, and
Dr. Ahmad Masarwa for helpful discussions and input for this
article. We also thank Dr. Ilia Kaminker (Tel Aviv University)
for access and assistance with the EPR experiments. Z.Z. is
grateful for a CSC fellowship. N.M. acknowledges financial
support from the Israel Science Foundation (783/18).
(21) Stadtman, T. C. Selenocysteine. Annu. Rev. Biochem. 1996, 65,
83−100.
F
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
(22) Byun, B. J.; Kang, Y. K. Conformational preferences and pKa
value of selenocysteine residue. Biopolymers 2011, 95, 345−353.
(23) Huber, R. E.; Criddle, R. S. Comparison of the chemical
properties of selenocysteine and selenocystine with their sulfur
analogs. Arch. Biochem. Biophys. 1967, 122, 164−173.
(24) Nauser, T.; Dockheer, S.; Kissner, R.; Koppenol, W. H.
Catalysis of Electron Transfer by Selenocysteine. Biochemistry 2006,
45, 6038−6043.
(41) Yu, J.; Yang, X.; Sun, Y.; Yin, Z. Highly Reactive and Tracelessly
Cleavable Cysteine-Specific Modification of Proteins via 4-Substituted
Cyclopentenone. Angew. Chem., Int. Ed. 2018, 57, 11598−11602.
(42) Calce, E.; De Luca, S. The Cysteine S-Alkylation Reaction as a
Synthetic Method to Covalently Modify Peptide Sequences. Chem. -
Eur. J. 2017, 23, 224−233.
(43) Cohen, D. T.; Zhang, C.; Pentelute, B. L.; Buchwald, S. L. An
Umpolung Approach for the Chemoselective Arylation of Selenocys-
teine in Unprotected Peptides. J. Am. Chem. Soc. 2015, 137, 9784−
9787.
(25) Besse, D.; Siedler, F.; Diercks, T.; Kessler, H.; Moroder, L. The
Redox Potential of Selenocystine in Unconstrained Cyclic Peptides.
Angew. Chem., Int. Ed. Engl. 1997, 36, 883−885.
(44) Cohen, D. T.; Zhang, C.; Fadzen, C. M.; Mijalis, A. J.; Hie, L.;
Johnson, K. D.; Shriver, Z.; Plante, O.; Miller, S. J.; Buchwald, S. L.;
Pentelute, B. L. A chemoselective strategy for late-stage functionaliza-
tion of complex small molecules with polypeptides and proteins. Nat.
Chem. 2019, 11, 78−85.
(26) Metanis, N.; Keinan, E.; Dawson, P. E. Synthetic seleno-
glutaredoxin 3 analogues are highly reducing oxidoreductases with
enhanced catalytic efficiency. J. Am. Chem. Soc. 2006, 128, 16684−
16691.
(27) Metanis, N.; Keinan, E.; Dawson, P. E. Traceless ligation of
cysteine peptides using selective deselenization. Angew. Chem., Int. Ed.
2010, 49, 7049−7053.
(28) Dery, S.; Reddy, P. S.; Dery, L.; Mousa, R.; Dardashti, R. N.;
Metanis, N. Insights into the deselenization of selenocysteine into
alanine and serine. Chem. Sci. 2015, 6, 6207−6212.
(45) Hill, H. A. O.; Thornalley, P. J. Phenyl radical production
during the oxidation of phenylhydrazine and in phenylphydrazine-
induced haemolysis. FEBS Lett. 1981, 125, 235−238.
́
(46) Varea, T.; González-Nunez, M. E.; Rodrigo-Chiner, J.; Asensio,
̃
G. Aryl radicals by copper(II) oxidation of hydrazines: A new method
for the oxidative and reductive arylation of alkenes. Tetrahedron Lett.
1989, 30, 4709−4712.
(29) Mitchell, N. J.; Malins, L. R.; Liu, X.; Thompson, R. E.; Chan,
B.; Radom, L.; Payne, R. J. Rapid Additive-Free Selenocystine-
Selenoester Peptide Ligation. J. Am. Chem. Soc. 2015, 137, 14011−
14014.
(47) Jonnalagadda, S. B.; Nattar, K. Cu(II) catalyzed reaction
between phenyl hydrazine and toluidine bluedual role of acid. Int. J.
Chem. Kinet. 1999, 31, 271−276.
(30) Liu, J.; Chen, Q.; Rozovsky, S. Utilizing Selenocysteine for
Expressed Protein Ligation and Bioconjugations. J. Am. Chem. Soc.
2017, 139, 3430−3437.
(48) Ren, X.; Tang, S.; Li, L.; Li, J.; Liang, H.; Li, G.; Yang, G.; Li,
H.; Yuan, B. Surfactant-Type Catalyst for Aerobic Oxidative Coupling
of Hydrazine with Thiol in Water. J. Org. Chem. 2019, 84, 8683−
8690.
(31) Whedon, S. D.; Markandeya, N.; Rana, A. S. J. B.; Senger, N.
̌
A.; Weller, C. E.; Turecek, F.; Strieter, E. R.; Chatterjee, C.
(49) Zhao, Z.; Metanis, N. Copper-Mediated Selenazolidine
Deprotection Enables One-Pot Chemical Synthesis of Challenging
Proteins. Angew. Chem., Int. Ed. 2019, 58, 14610−14614.
(50) Zhao, Z.; Metanis, N. Utilizing Copper-Mediated Deprotection
of Selenazolidine for Cyclic Peptide Synthesis. J. Org. Chem. 2020, 85,
1731−1739.
(51) Naruse, N.; Kobayashi, D.; Ohkawachi, K.; Shigenaga, A.;
Otaka, A. Copper-Mediated Deprotection of Thiazolidine and
Selenazolidine Derivatives Applied to Native Chemical Ligation. J.
Org. Chem. 2020, 85, 1425−1433.
(52) Arsenyan, P.; Lapcinska, S.; Ivanova, A.; Vasiljeva, J. Peptide
Functionalization Through the Generation of Selenocysteine Electro-
phile. Eur. J. Org. Chem. 2019, 2019, 4951−4961.
(53) Misra, H. P.; Fridovich, I. The oxidation of phenylhydrazine:
superoxide and mechanism. Biochemistry 1976, 15, 681−687.
(54) Kibriya, G.; Mondal, S.; Hajra, A. Visible-Light-Mediated
Synthesis of Unsymmetrical Diaryl Sulfides via Oxidative Coupling of
Arylhydrazine with Thiol. Org. Lett. 2018, 20, 7740−7743.
(55) Malins, L. R.; Mitchell, N. J.; McGowan, S.; Payne, R. J.
Oxidative Deselenization of Selenocysteine: Applications for Pro-
grammed Ligation at Serine. Angew. Chem., Int. Ed. 2015, 54, 12716−
12721.
(56) Griffiths, R. C.; Smith, F. R.; Long, J. E.; Williams, H. E. L.;
Layfield, R.; Mitchell, N. J. Site-Selective Modification of Peptides and
Proteins via Interception of Free-Radical-Mediated Dechalcogenation.
Angew. Chem., Int. Ed. 2020, 59, 23659−23667.
Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for
Deubiquitylating Enzymes. J. Am. Chem. Soc. 2016, 138, 13774−
13777.
(32) Metanis, N.; Foletti, C.; Beld, J.; Hilvert, D. Selenoglutathione-
Mediated Rescue of Kinetically Trapped Intermediates in Oxidative
Protein Folding. Isr. J. Chem. 2011, 51, 953−959.
(33) Metanis, N.; Hilvert, D. Strategic use of non-native bridges to
steer oxidative protein folding. Angew. Chem., Int. Ed. 2012, 51, 5585−
5588.
(34) Metanis, N.; Hilvert, D. Harnessing selenocysteine reactivity for
oxidative protein folding. Chem. Sci. 2015, 6, 322−325.
(35) Mousa, R.; Notis Dardashti, R.; Metanis, N. Selenium and
Selenocysteine in Protein Chemistry. Angew. Chem., Int. Ed. 2017, 56,
15818−15827.
(36) Weil-Ktorza, O.; Rege, N.; Lansky, S.; Shalev, D. E.; Shoham,
G.; Weiss, M. A.; Metanis, N. Substitution of an Internal Disulfide
Bridge with a Diselenide Enhances both Foldability and Stability of
Human Insulin. Chem. - Eur. J. 2019, 25, 8513−8521.
(37) Muttenthaler, M.; Nevin, S. T.; Grishin, A. A.; Ngo, S. T.;
Choy, P. T.; Daly, N. L.; Hu, S.-H.; Armishaw, C. J.; Wang, C.-I. A.;
Lewis, R. J.; Martin, J. L.; Noakes, P. G.; Craik, D. J.; Adams, D. J.;
Alewood, P. F. Solving the α-Conotoxin Folding Problem: Efficient
Selenium-Directed On-Resin Generation of More Potent and Stable
Nicotinic Acetylcholine Receptor Antagonists. J. Am. Chem. Soc. 2010,
132, 3514−3522.
(38) de Araujo, A. D.; Callaghan, B.; Nevin, S. T.; Daly, N. L.; Craik,
D. J.; Moretta, M.; Hopping, G.; Christie, M. J.; Adams, D. J.;
Alewood, P. F. Total Synthesis of the Analgesic Conotoxin MrVIB
through Selenocysteine-Assisted Folding. Angew. Chem., Int. Ed. 2011,
50, 6527−6529.
(39) Gowd, K. H.; Yarotskyy, V.; Elmslie, K. S.; Skalicky, J. J.;
Olivera, B. M.; Bulaj, G. Site-Specific Effects of Diselenide Bridges on
the Oxidative Folding of a Cystine Knot Peptide, ω-Selenoconotoxin
GVIA. Biochemistry 2010, 49, 2741−2752.
(40) Mousa, R.; Hidmi, T.; Pomyalov, S.; Lansky, S.; Khouri, L.;
Shalev, D. E.; Shoham, G.; Metanis, N. Diselenide crosslinks for
enhanced and simplified oxidative protein folding. Commun. Chem.
2021, 4 (1), 30.
(57) Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis
of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149−2154.
(58) Carpino, L. A.; Han, G. Y. 9-Fluorenylmethoxycarbonyl
function, a new base-sensitive amino-protecting group. J. Am. Chem.
Soc. 1970, 92, 5748−5749.
(59) Carpino, L. A.; Han, G. Y. 9-Fluorenylmethoxycarbonyl amino-
protecting group. J. Org. Chem. 1972, 37, 3404−3409.
(60) Shirota, H.; Pal, H.; Tominaga, K.; Yoshihara, K. Ultrafast
intermolecular electron transfer in coumarin-hydrazine system. Chem.
Phys. 1998, 236, 355−364.
(61) Kobayashi, N.; Minami, T.; Tani, A.; Nakagoshi, M.; Sugahara,
T.; Takeya, K.; Ohgaki, K. Intermolecular Hydrogen Transfer in
Isobutane Hydrate. Energies 2012, 5, 1705−1712.
G
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
(62) Maillard, B.; Forrest, D.; Ingold, K. U. Kinetic applications of
electron paramagnetic resonance spectroscopy. 27. Isomerization of
cyclopropylcarbinyl to allylcarbinyl. J. Am. Chem. Soc. 1976, 98,
7024−7026.
(63) Yoshida, M.; Cho, T.; Kobayashi, M. SH2 REACTIONS OF
SELENOSULFIDES WITH CARBON RADICALS. Chem. Lett.
1984, 13 (7), 1109−1112.
(64) Ghiazza, C.; Debrauwer, V.; Monnereau, C.; Khrouz, L.;
Medebielle, M.; Billard, T.; Tlili, A. Visible-Light-Mediated Metal-
Free Synthesis of Trifluoromethylselenolated Arenes. Angew. Chem.,
Int. Ed. 2018, 57 (36), 11781−11785.
(65) Knops, P.; Sendhoff, N.; Mekelburger, H.-B.; Vögtle, F. High
dilution reactions  New synthetic applications. Top. Curr. Chem.
1992, 161, 1−36.
(66) Schiller, P. W.; Nguyen, T. M.-D.; Miller, J. Synthesis of side-
chain to side-chain cyclized peptide analogs on solid supports. Int. J.
Pept. Protein Res. 1985, 25, 171−177.
(67) Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H.
Synthesis of Proteins by Native Chemical Ligation. Science 1994, 266,
776−779.
(68) Younan, N. D.; Nadal, R. C.; Davies, P.; Brown, D. R.; Viles, J.
H. Methionine Oxidation Perturbs the Structural Core of the Prion
Protein and Suggests a Generic Misfolding Pathway. J. Biol. Chem.
2012, 287, 28263−28275.
(69) Barman, A.; Taves, W.; Prabhakar, R. Insights into the
mechanism of methionine oxidation catalyzed by metal (Cu2+, Zn2+,
and Fe3+)Amyloid beta (Aβ) peptide complexes: A computational
study. J. Comput. Chem. 2009, 30, 1405−1413.
(70) Crump, M. P.; Gong, J.-H.; Loetscher, P.; Rajarathnam, K.;
Amara, A.; Arenzana-Seisdedos, F.; Virelizier, J.-L.; Baggiolini, M.;
Sykes, B. D.; Clark-Lewis, I. Solution structure and basis for functional
activity of stromal cell-derived factor-1; dissociation of CXCR4
activation from binding and inhibition of HIV-1. EMBO Journal 1997,
16 (23), 6996−7007.
(71) Murphy, J. W.; Cho, Y.; Sachpatzidis, A.; Fan, C.; Hodsdon, M.
E.; Lolis, E. Structural and Functional Basis of CXCL12 (Stromal
Cell-derived Factor-1α) Binding to Heparin*. J. Biol. Chem. 2007, 282
(13), 10018−10027.
(72) Mukai, T.; Sevostyanova, A.; Suzuki, T.; Fu, X.; Söll, D. A
Facile Method for Producing Selenocysteine-Containing Proteins.
Angew. Chem., Int. Ed. 2018, 57 (24), 7215−7219.
(73) Welegedara, A. P.; Maleckis, A.; Bandara, R.; Mahawaththa, M.
C.; Dilhani Herath, I.; Jiun Tan, Y.; Giannoulis, A.; Goldfarb, D.;
Otting, G.; Huber, T. Cell-Free Synthesis of Selenoproteins in High
Yield and Purity for Selective Protein Tagging. ChemBioChem 2021,
22 (8), 1480−1486.
(74) Liu, J.; Zheng, F.; Cheng, R.; Li, S.; Rozovsky, S.; Wang, Q.;
Wang, L. Site-Specific Incorporation of Selenocysteine Using an
Expanded Genetic Code and Palladium-Mediated Chemical Depro-
tection. J. Am. Chem. Soc. 2018, 140 (28), 8807−8816.
(75) Thyer, R.; Filipovska, A.; Rackham, O. Engineered rRNA
Enhances the Efficiency of Selenocysteine Incorporation during
Translation. J. Am. Chem. Soc. 2013, 135 (1), 2−5.
(76) Thyer, R.; Robotham, S. A.; Brodbelt, J. S.; Ellington, A. D.
Evolving tRNASec for Efficient Canonical Incorporation of
Selenocysteine. J. Am. Chem. Soc. 2015, 137 (1), 46−49.
H
https://doi.org/10.1021/jacs.1c06101
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX