Irreversible Cysteine-Selective Protein Labeling Employing Modular Electrophilic Tetrafluoroethylation Reagents

Article abstract of DOI:10.1002/chem.201700607

Fluoroalkylation reagents based on hypervalent iodine are widely used to transfer fluoroalkyl moieties to various nucleophiles. However, the transferred groups have so far been limited to simple structural motifs. We herein report a reagent featuring a secondary amine that can be converted to amide, sulfonamide, and tertiary amine derivatives in one step. The resulting reagents bear manifold functional groups, many of which would not be compatible with the original synthetic pathway. Exploiting this structural versatility and the known high reactivity toward thiols, the new-generation reagents were used in bioconjugation with an artificial retro-aldolase, containing an exposed cysteine and a reactive catalytic lysine. Whereas commercial reagents based on maleimide and iodoacetamide labeled both sites, the iodanes exclusively modified the cysteine residue. The study thus demonstrates that modular fluoroalkylation reagents can be used as tools for cysteine-selective bioconjugation.

Full text of DOI:10.1002/chem.201700607

A Journal of
Accepted Article
Title: Irreversible Cysteine-Selective Protein Labeling Employing
Modular Electrophilic Tetrafluoroethylation Reagents
Authors: Jiří Václavík, Reinhard Zschoche, Iveta Klimánková, Václav
Matoušek, Petr Beier, Donald Hilvert, and Antonio Togni
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700607
Link to VoR: http://dx.doi.org/10.1002/chem.201700607
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COMMUNICATION
Irreversible Cysteine-Selective Protein Labeling Employing
Modular Electrophilic Tetrafluoroethylation Reagents
Jiří Václavík,+[a, b] Reinhard Zschoche,+[a] Iveta Klimánková, Václav Matoušek,** Petr Beier,*
[b]
[c]
[b]
[a]
[a]
Donald Hilvert,* Antonio Togni*
6]
Abstract: Fluoroalkylation reagents based on hypervalent iodine are
widely used to transfer fluoroalkyl moieties to various nucleophiles.
However, the transferred groups have so far been limited to simple
structural motifs. We herein report a reagent featuring a secondary
amine that can be converted to amide, sulfonamide, and tertiary
amine derivatives in the last step. The resulting reagents bear
manifold functional groups, many of which would not be compatible
with the original synthetic pathway. Exploiting this structural
versatility and the known high reactivity toward thiols, the new-
generation reagents were used in bioconjugation with an artificial
retro-aldolase, containing an exposed cysteine and a reactive
catalytic lysine. Whereas commercial reagents based on maleimide
and iodoacetamide labeled both sites, the iodanes exclusively
modified the cysteine site. The study thus demonstrates that
modular fluoroalkylation reagents can be used as tools for cysteine-
selective bioconjugation.
thioether formation,[ self-hydrolyzing maleimides,[7] exocyclic
olefinic maleimides,[8] Julia-Kocieński-like methylsulfonylated
heterocycles,[9]
arylation reagents,
S
N
Ar reagents.[12]
3-arylpropiolonitriles,[10]
11]
palladium-based
and π-clamp-mediated perfluoroarylated
[
Trifluoromethylation reagents based on hypervalent iodine (1
and 2, Figure 1) are known to transfer the trifluoromethyl group
to various S-, P-, O-, N-, and C-nucleophilic functionalities.[
Thiols, in particular, exhibit excellent reactivity and selectivity in
such fluoroalkylation reactions, which has been demonstrated by
13]
[
14]
selective S-trifluoromethylation of, e.g., coenzyme A
and
[
15]
Sandostatin using 1 and 2.
We recently reported reagents 3 and 3′ analogous to 1 and 2,
containing S-Ar, O-Ar, or Arhet motifs connected to
a
tetrafluoroethyl linker unit.[16] This feature, combined with the
high selectivity toward thiols, high reaction rate, and
irreversibility of the fluoroalkylation reaction motivated us to
develop a new family of reagents for thiol bioconjugation. In the
meantime, Waser and co-workers have reported the use of a
hypervalent iodine-based alkynylation reagent (EBX reagent,
Figure 1) bearing an azide group in cysteine-selective protein
profiling,[17] and Hansen et al. employed the EBX reagents in
Site-specific posttranslational modification of proteins is
popular tool to investigate their biological function.
a
[
1]
Complementarily, chemically modified proteins have found wide-
spread commercial use, most notably as antibody-drug
conjugates[ and PEGylated biologics. Cysteine is the most
prominent canonical amino acid used for protein labeling, owing
to its relatively low abundance and excellent nucleophilicity.[4]
The reagents typically employed in thiol bioconjugation are
haloalkane and maleimide derivatives, which undergo polar
2]
[3]
gold-catalyzed labeling of tryptophan residues in peptides and a
[18]
protein.
These two recent studies suggest that hypervalent
iodine reagents are attractive candidates for bioconjugation
reactions.
Our original example was based on a reagent containing
pyrene as fluorophore (3a, Figure 1), which irreversibly labeled a
protected cysteine.[16] However, the lengthy synthesis of 3, along
reactions (S
with cysteine thiols. However, since reactive nucleophiles other
than thiols are often encountered in proteinaceous
N
2 reactions and Michael additions, respectively)
a
with the presence of a potentially labile ester group, made
improvements in the reagent design desirable, most notably
shorter synthesis and a modular approach that would provide
access to reagents bearing functional groups (e.g. dyes, groups
suitable for bioorthogonal reactions,[19] biotin, etc.). Herein, we
disclose a versatile reagent featuring a secondary amino group.
This can be modified with a variety of biologically relevant
groups using three different linking strategies and exploits
cysteine’s ability to undergo single-electron oxidations. We
demonstrate its superiority over haloalkane and maleimide
environment, it can be difficult to achieve chemo- and
regioselectivity. Furthermore, the Michael adducts can
decompose via an undesired retro-Michael pathway. Thus, a
variety of new-generation reagents has been developed,
including unsaturated hydrocarbons that engage in radical thiol-
ene and thiol-yne reactions,[4c,5] reagents that induce oxidative
elimination of cysteine to dehydroalanine with subsequent
[
[
[
a] J. Václavík,[+] Dr. R. Zschoche,[+] Prof. Dr. D. Hilvert, Prof. Dr. A. Togni
Department of Chemistry and Applied Biosciences
Swiss Federal Institute of Technology, ETH Zürich
Vladimir-Prelog-Weg 2, 8093 Zürich (Switzerland)
E-mail: atogni@ethz.ch, hilvert@ethz.ch
b] J. Václavík,[ I. Klimánková, Dr. P. Beier
Institute of Organic Chemistry and Biochemistry, v.v.i.
Academy of Sciences of the Czech Republic
Flemingovo nám. 2, 166 10 Prague (Czech Republic)
E-mail: beier@uochb.cas.cz
+]
c] Dr. V. Matoušek
CF Plus Chemicals s.r.o.
Kamenice 771/34, 625 00 Brno (Czech Republic)
These authors contributed equally to this work.
[+
]
[
**] The CF Plus Chemicals s.r.o. (www.cfplus.cz) company, an ETH
Zurich spin-off, commercializes the reagents reported in this
publication.
Figure 1. Structures of reagents based on hypervalent iodine.
Supporting information for this article is given via a link at the end of
the document.
1
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reagents in selectively labeling an exposed thiol present in an
enzyme possessing an unusually reactive nucleophilic primary
amine.
In the synthesis of our first-generation electrophilic
tetrafluoroethylation reagents,[16] the desired functional group
Even though deprotected reagent 7 could be isolated and
characterized, it decomposed rapidly even when stored in a
freezer. For this reason, in all subsequent studies it was
generated in situ from 7-salt by addition of base.
The easy-to-handle fluorophore 1-pyrenecarboxylic acid was
selected for optimizing the reaction conditions to derivatize 7-
salt via peptide coupling to form the corresponding amide
reagents 8.
Aiming for mild reaction conditions, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) was tested as the
coupling reagent. Initially, product 8a was obtained in 26%
isolated yield, which was increased to 63% by further
2 2
attached to the CF CF block was already present at the stage
of generation of the corresponding hypervalent iodine reagent.
This implied a similar sequence of synthetic steps for every
functional group. To circumvent this drawback, we opted for a
reagent containing a secondary amino group, thus providing
several robust linking methods such as amide, sulfonamide, and
tertiary amine formation, compatible with hypervalent iodine.
The synthesis of the versatile reagent (Scheme 1) was
based on procedures developed for tetrafluoroethylation
reagents.[16] It was thus convenient to employ the readily
available and inexpensive phenol derivative N-methyltyramine
hydrochloride (NMT) as the source of the secondary amine. The
phenolic oxygen of Boc-protected NMT was deprotonated with
NaH and treated with BrCF
converted to the corresponding fluoroalkylsilane 5. An Umpolung
reaction of with fluoroiodane provided the protected
2 2
CF Br giving bromide 4, which was
5
6
fluoroalkylation reagent 7-Boc as a brown oil, which was stable
at least for six months at –20 °C. Reagent 7-Boc was
deprotected using excess trifluoroacetic acid and subsequent
precipitation with diethyl ether afforded crystalline bis-
trifluoroacetate salt (7-salt), which is fairly stable at ambient
temperature with less than 5% decomposition after 1 month.
Previous findings from our laboratory have shown that salts of
protonated reagents 1 and 2 are stable crystalline materials.[20]
Scheme 1. Synthesis of reagent 7-salt.
Scheme 2. Synthesis of reagents 8a–q.
2
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COMMUNICATION
optimization (Table S1, entries 1 and 2).[21] Addition of the
activator 1-hydroxybenzotriazole (HOBt) or using the structurally
related 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate (HATU) led to reagent
decomposition (Table S1, entries 3 and 4). Interestingly, acyl
chloride (generated with oxalyl chloride and cat. DMF) initially
afforded 8a in 78% yield (Table S1, entry 5) and 89% under
optimized conditions (Table S1, entry 6).
6-(2-bromoacetamido)fluorescein, which was coupled with 7-salt
to give reagent 10a in 39% yield (Scheme 4). In a similar fashion,
propargyl-functionalized amine reagent 10b was prepared in
56% yield (Scheme 4).
To test thiol bioconjugation, we turned our attention to a
computationally designed and laboratory evolved retro-aldolase,
RA95.5-8 S25C K210M[24] (RA-CM), containing an exposed
surface thiol (Cys25) and a nucleophilic lysine at position 83
Reagents 8b–q were synthesized mostly in good to excellent
yields by using acyl chlorides and only slightly modified
conditions, such as Hünig’s base instead of triethylamine in
some cases, (Scheme 2). The eight benzoic acid derivatives
a
(pK = 6.8). The lysine, buried in a hydrophobic pocket, is
essential for the catalytic activity of RA-CM, which is typically
monitored by retro-aldol cleavage of 4-hydroxy-4-(6-methoxy-2-
[
24a]
naphthyl)-2-butanone (methodol).
In previous studies on
(
8b–i) exemplify the remarkable scope of functional groups that
encapsulation of enzymes such as RA-CM in a protein cage,
RA-CM had to be labeled with a fluorophore to determine
enzyme loading.[24b] However, labeling with the commercially
available fluorescent probe Atto-565-maleimide (Atto-565m)
resulted in complete loss of catalytic activity as not only Cys25,
but also Lys83 underwent modification. Thus, the labeling had to
be carried out with substoichiometric quantities of Atto-565m
and in the presence of acetone and 6-methoxy-2-
naphthaldehyde (that is, the products of the retro-aldol reaction),
which gave only 20–40% labeled protein.[24b] A truly thiol-
selective labeling reagent would therefore be ideal. Because
thiols are susceptible to single-electron oxidation, the
hypervalent iodine fluoroalkylation reagents seemed to be viable
candidates for tagging RA-CM since they operate via a radical
mechanism in many cases.[13,25]
can be used for further functionalization via coupling,
cycloaddition, substitution, etc. Aside from two fluorophores
(
reagents based on coumarin 8j and fluorescein 8k), the
remaining examples comprise aliphatic carboxylic acids.
Coupling (1S)-(–)-camphanic acid (in 8l) demonstrates the
introduction of a chiral building block while reagent 8m is a
viable candidate for thiol-selective biotinylations.[22] Finally, we
synthesized reagents with tetraethylene glycol as a linker
frequently used in bioconjugation.[23] These reagents featured
groups suitable for click reactions (8n, 8o) and two examples
contaning pyrene as a fluorophore (8p, 8q).
To explore the utility of the amine functionality in 7 for the
preparation of sulfonamide reagents 9, three new compounds
were prepared starting from 4-nitrophenylsulfonyl chloride (9a),
dansyl chloride (9b), and the highly fluorescent Lissamine
Rhodamine B sulfonyl chloride (9c) (Scheme 3).
Reagents 8k, 9c and 10a were examined for labeling RA-
CM; Atto-565m and 6-(iodoacetamido)fluorescein (6IAF) served
as controls. The reagents were dissolved in DMSO or
The synthesis of a reagent bearing a tertiary amine group
was motivated by an expected increase in water solubility upon
its protonation. To this end,
commercially available 6-(2-iodoacetamido)fluorescein was
attempted. However, the iodide liberated by the S 2 reaction
acetonitrile (Table 1) and added to the enzyme in degassed
buffer. Trypsin digests of the labeled enzymes (Table S2[
)
21]
a
reaction of 7-salt with
were analyzed by mass spectrometry with MALDI ionization to
identify the site of conjugation (Figure 2).
N
reduced the hypervalent iodine species and the desired product
could not be isolated. This issue was resolved by starting from
analogous
While Atto-565m and 6IAF reacted at both Cys25 and Lys83,
Scheme 4. Synthesis of reagents 10a and 10b.
Scheme 3. Synthesis of reagents 9a–c.
3
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[
a]
Table 1. Tagging RA-CM with various fluorescent reagents and testing the
retro-aldol reaction of (±)-methodol[b]
reagents 8k, 9c and 10a exclusively labelled Cys25. In the latter
case, no modified Lys83 was detected (Figure 2). The degree of
labeling (Table 1), determined by UV-Vis absorbance using
[
21]
reported extinction coefficients (Table S3 ), exceeded the
previously reported level of 20–40% achieved with Atto-
5
65m.[24b] Since the artificial retro-aldolase slowly deactivates at
room temperature, the incubation time was reduced from 18 to 4
hours in the case of reagent 9c, resulting in the preservation of
82% enzyme activity while maintaining good labeling efficiency
(
Table 1, entry 5).
0 0
v /[E] ]
[min–1
Entry
Enzyme
Co-solvent
Labeling
efficiency [%][
Although not tested within the scope of this work, we expect
d]
2 2 2
the –CH SCF CF – moiety to be stable under physiological
conditions, because, as known from closely related SCF
3
1
RA-CM
--
--
5.15
0
compounds, only strongly oxidizing,[26] reducing[15a] or basic[15a]
reagents can lead to its decomposition.
2
RA-CM-Atto-565m
RA-CM-6IAF
RA-CM-8k
--
146
92
67
61
40
In summary, an exposed cysteine of a synthetic retro-
aldolase was selectively and irreversibly labelled with
fluorophores using fluoroalkylation reagents based on
hypervalent iodine, leaving the reactive catalytic lysine at the
active site intact. Such selectivity could not be achieved with
commercially available maleimide or iodoacetamide reagents.
The modular tetrafluoroethylation reagents thus present a new
approach to thiol bioconjugation that overcomes the drawbacks
of currently established methods, i.e. poor selectivity (especially
at basic pH) and reversibility. Relatively simple, one-step
derivatization of a common secondary amine opens expedient
access to a variety of custom-made reagents tailored to specific
needs of researchers in biochemistry and chemical biology.
3
10% MeCN
30% DMSO
30% DMSO
10% MeCN
1.08
1.89
4.25
2.20
4
5[c]
6
RA-CM-9c
RA-CM-10a
[a] Conditions for the labeling: c(RA-CM) = 25 µM, c(reagent) = 250 µM,
degassed buffer (25 mM HEPES, 100 mM NaCl, pH 7.5), 18 h at rt, quenched
with 10 mM 2-mercaptoethanol. [b] The reactions were carried out at 29 °C in
25 mM HEPES and 100 mM NaCl (pH 7.5) containing 2.7% acetonitrile. [c]
Reaction time for labeling was reduced to 4 h. [d] Labeling efficiency signifies
the average number of chromophores attached to one protein.
Figure 2. MALDI MS spectra of the peptides generated by trypsin digest of RA-CM and its labeled variants.
4
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Acknowledgements
[
This work was financially supported by ETH and the Academy of
Sciences of the Czech Republic (RVO: 61388963) and Czech
Science Foundation (17-00598S). R.Z. is grateful for
scholarship from the Stipendienfonds der Schweizerischen
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COMMUNICATION
Entry for the Table of Contents
Layout 1:
COMMUNICATION
Bioconjugation – from iodine to
sulfur: Modular fluoroalkylation
reagents based on hypervalent
iodine were used for cysteine-
selective labeling of an artificial
enzyme without reacting with its
catalytic lysine, showing a new
approach to thiol bioconjugation.
Jiří Václavík, Reinhard Zschoche, Iveta
Klimánková, Václav Matoušek, Petr
Beier, Donald Hilvert, Antonio Togni
Page No. – Page No.
Irreversible Cysteine-Selective
Labeling of a Retro-Aldolase
Employing Modular Electrophilic
Tetrafluoroethylation Reagents
6
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