Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine

Article abstract of DOI:10.1021/jacs.9b08279

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji-Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.

Full text of DOI:10.1021/jacs.9b08279

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Article
Labelling and Natural Posttranslational Modification of Peptides and
Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine
Thomas Schlatzer, Julia Kriegesmann, Hilmar Schröder, Melanie Trobe, Christian Lembacher-
Fadum, Simone Santner, Alexander Kravchuk, Christian F.W. Becker, and Rolf Breinbauer
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08279 • Publication Date (Web): 30 Aug 2019
Downloaded from pubs.acs.org on August 30, 2019
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Labelling and Natural Posttranslational Modification of Peptides
and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cys-
teine
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Thomas Schlatzer,‡[a] Julia Kriegesmann,‡[b] Hilmar Schröder,‡[a] Melanie Trobe,[a] Christian Lem-
bacher-Fadum,[a] Simone Santner,[a] Alexander V. Kravchuk,[b] Christian F. W. Becker*[b] and Rolf
Breinbauer*[a]
[a] Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
[b] Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Währinger Strasse 38, A-1090 Vienna,
Austria
KEYWORDS prenylation, bioconjugation, lipidation, peptide cyclization, stapled peptides
ABSTRACT: The prenylation of peptides and proteins is an important posttranslational modification observed in vivo. We report
that the Pd-catalyzed Tsuji-Trost-allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides
and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-
native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical
probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, pep-
tides containing two cysteine residues can be stapled or cyclized using homo-bifunctional allylic carbonate reagents.
INTRODUCTION
Over the last three decades it has been recognized that post-
translational modifications (PTMs) (glycosylation, phosphory-
lation, sulfation, acylation, lipidation, etc.) play an important
role in controlling protein function and localization. Among
PTMs, prenylation is essential for associating certain proteins
to specific membranes. A particularly intriguing example for
this is the Ras superfamily of small GTPases involved in signal
transduction processes that lead to cell growth and differentia-
tion as well as in vesicular trafficking.1
For the biophysical and cell biological investigation of pro-
teins in general and of PTMs in particular, chemoselective
methods are needed that enable access to modified proteins via
synthetic manipulations at reactive side chains of proteinogenic
amino acids using either chemical reagents or a transition metal
catalyst.2-5 The formation of new covalent bonds allows the at-
tachment of affinity tags, fluorophores, click handles or PET
tracers. Cysteine represents an attractive handle for the intro-
duction of such chemical modifications, as it is the second least
frequent amino acid in proteins (1.7 %)6 and shows a very
strong inherent nucleophilicity, which makes it especially at-
tractive for reactions with electrophilic reagents. Maleimides7
and iodoacetamides8 represent the earliest electrophiles used to
alkylate Cys (Figure 1, A) and have been frequently applied to
date. More recent developments include a variety of car-
bonylacrylic reagents as well as vinyl pyridines.9 Since then nu-
Figure 1. Selected examples of Cys-modifications established to
date (A-G) in comparison with the Pd-catalyzed Cys-allylation de-
scribed in this work.
merous different bioconjugation strategies have been devel-
oped.2,10 One of them involves transformation of Cys into dehy-
droalanine (Dha) upon treatment with 2,5-dibromohexanedia-
mide (DBHDA)11 or O-mesitylenesulfonylhydroxylamine
(MSH)12 (B), which is then reacted with a thiol nucleophile (C).
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Table 1. Scope of the Pd-catalyzed allylation of small peptides.
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Figure 2. Allylic carbonate reagents prepared for the Pd-catalyzed
Cys-modification.
A disadvantage of this method is that the formation of Dha is
associated with racemization at the α-carbon because diastere-
oselectivity of the thiol addition in simple Dha peptides is re-
ported to be low.13 A more direct access to S-allylcysteine with-
out epimerization can be accomplished by selenenylsulfide re-
ductive rearrangement (D),14 followed by further derivatization
either by olefin cross-metathesis15 or Kirmse-Doyle reaction.16
Alternatively, allylic halides might be used to directly allylate
Cys (E)14,17 although these highly reactive reagents are more
difficult to handle and preclude more elaborate reagent struc-
tures. Very recently Buchwald et al. introduced arylpalladium
reagents, which can be used for the arylation of Cys-containing
peptides and proteins (F).18 This concept has been extended to
Au(III) complexes.19 Pentelute et al. reported the site-selective
Cys-conjugation with perfluoroarene reagents at the π-clamp
motif FCPF (G),20 and the ligation with cyclooctynes.21
product in high selectivity, as the corresponding i-product (re-
sulting from internal attack) would be impossible to separate on
the peptide or protein level. From a screening of a diverse set of
mono- and bidentate phosphorous ligands we identified the
bisphosphite ligand BIPHEPHOS as the by far most suitable
ligand producing the desired n-products in high selectivity. Fur-
thermore, the n/i ratio was found to increase over time even
when complete conversion was already reached, indicating the
reversibility of this reaction (Table S1).
S-Allylation of Model Substrates. With these optimized
conditions in hand we wanted to apply the Pd-catalyzed S-al-
lylation to a dipeptide substrate (P1) featuring Tyr as second
amino acid, which could give rise to O-allylation as described
by Francis et al.23 before. Importantly, with our
Pd/BIPHEPHOS catalyst system we observed exclusively S-al-
lylation of Cys as confirmed via NMR by HMBC experiments
(Figure S1). With a series of allylation reagents (Figure 2) we
could demonstrate that a diverse set of labelled peptides could
be easily prepared by this method (Table 1, entries 1-7). More-
over, we successfully subjected unprotected glutathione (P2) to
Pd-catalyzed S-prenylation in an aqueous solvent mixture (Ta-
ble 1, entry 8) indicating a broader applicability of this method
for the modification of longer peptides and proteins. This is cor-
roborated by the fact that the reaction proceeds with fast kinet-
ics. Full conversion of 10 mM Ac-Cys-OMe was observed
within 10 min upon treatment with 2 eq. Ra in the presence of
2.0 mol% Pd/BIPHEPHOS at 35 °C. Even 0.5 mol% of the cat-
alyst were found to be sufficient to obtain quantitative conver-
sion after 30 min demonstrating the high efficiency of the reac-
tion (Figure S2). However, strictly oxygen-free conditions were
crucial for the activity of the catalyst.
RESULTS AND DISCUSSION
Reaction Optimization. We envisioned that Cys could be
selectively modified using the Pd-catalyzed Tsuji-Trost reac-
tion, which would give rise to an allylthioether linkage, as pre-
sent in naturally prenylated proteins, in a single step. The Tsuji-
Trost-allylation with C-, N- or O-nucleophiles is well estab-
lished in organic synthesis22 and Francis et al.23 have impres-
sively demonstrated the site selective modification of proteins
via Pd-catalyzed O-allylation of Tyr residues using the water-
soluble phosphine ligand TPPTS. In contrast, the reaction with
S-nucleophiles has been rarely studied,24,25 as it faces intrinsic
difficulties: a) S-nucleophiles can also function as efficient lig-
ands for Pd and poison the catalyst, and b) thiols are easily ox-
idized and the reactions have to be carried out under exclusion
of air. We hoped that with a prudent choice of ligand we could
design a Pd-catalyst system suitable for the allylation of Cys-
containing peptides and proteins. In contrast to classic allylation
procedures14,17 using an excess of highly reactive allylic halides,
a Pd-mediated reaction would allow the use of easily accessible
allylic carbonates as electrophiles. These reagents are much
more versatile as they are bench stable and can contain highly
functionalized structural motifs. Furthermore, in situ activated
electrophiles could be sterically controlled by the Pd-complex
so that the nucleophilic attack is directed to the terminal end of
the 3-Pd-allyl complex intermediate to produce the n-allylation
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Figure 3. Peptide- and reagent-scope of the Pd-mediated allylation of oligopeptides. Peptide sequences containing internal, terminal and
multiple Cys residues were subjected to site-selective allylation enabling the introduction of native prenyl groups, bioconjugation handles
(azide/alkyne groups) as well as a fluorescent NBD-tag and a biotin affinity-tag. All modified peptides were purified, isolated and charac-
terized by LC-MS analysis.
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Chemoselective Peptide Modification. As a next step we
tested the Pd-catalyzed Cys-allylation on a series of more com-
plex oligopeptides. For this purpose, the substrate concentration
was reduced to 1 mM to account for the lower solubility of the
peptides and the reaction temperature was adjusted to 40 °C to
ensure peptide integrity. To compensate for slower kinetics un-
der these conditions, the amount of Pd and ligand was in-
creased, which fully restored the reactivity of the system. As a
relevant target protein for prenylation, we selected ubiquitin-
like protein 3 (UBL3) and started out with modifying its C-ter-
minal domain (peptide P3) with polyprenyl groups, bioconju-
gation handles, fluorescent and affinity tags, highlighting the
versatility of this method (Figure 3). Furthermore, we could
show that farnesylation is feasible at internal as well as terminal
Cys and that this bioconjugation strategy offers access to adja-
cent and non-adjacent di-farnesylated products, which are of
special importance in naturally occurring proteins.26 The high
chemoselectivity of this reaction was showcased on a 32aa pol-
ypeptide (P7) featuring nearly all functional group containing
amino acids, which was found to undergo farnesylation exclu-
sively on Cys as proven by tryptic digest and mass spectromet-
ric analysis (Figure S3).
product, whereas for P8i and P9i two separate peaks with the
expected mass occurred presumably due to the formation of E/Z
isomers. Although the intramolecular reaction was favored for
P10j, we observed also the dimer of P10j (approx. 10 %), con-
sisting of two peptides and two staples, as a side product. It is
worth mentioning that the stapled products provide motifs for
further functionalization by taking advantage of the double
bond and that allylated peptides with identical staple motifs
have recently been shown to function as substrates in decaging
strategies using transition metal catalysis as well.5
Figure 4. Peptide stapling/cyclization using Pd-mediated allyla-
tion. Three model peptides with various distances (i+3, i+4, i+11)
between the Cys residues were subjected to stapling/cyclization us-
ing two bifunctional reagents with different geometries.
Peptide Stapling/Cyclization. Having established a series of
highly selective monofunctional allylic carbonate reagents that
were successfully applied on a broad set of peptide substrates,
we were eager to see if our methodology can also be extended
to bifunctional allylation reagents. This would enable us to im-
plement an additional type of a peptide stapling protocol,27
which is based on Pd-mediated S-allylation. To this end, we pre-
pared two bifunctional allylic carbonates (Ri and Rj) with dif-
ferent geometries which were subjected to Pd-mediated allyla-
tion using two α-helical peptides (P8 and P9)28 with cysteine
residues spaced by i+3 and i+4 as well as peptide P10 with more
distant residues (i+11)29 (Figure 4). Reactions leading to P8j,
P9j and P10i gave only one peak corresponding to the desired
Modification of UBL3 Protein. To further evaluate the po-
tential of our method, we chose the full-length protein UBL3 as
substrate, which extends the application beyond classic reac-
tions based on alkyl halides with peptide substrates.17 UBL3 un-
dergoes posttranslational geranylgeranylation in vivo and direct
access to such membrane-bound UBL3 variants will help to elu-
cidate their so far unknown physiological role(s).30 Two vari-
ants, with one and with two C-terminal Cys, were used here
since mono- as well as di-lipidation occur in nature. In order to
find appropriate reaction conditions for the Pd-mediated protein
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Modifications of Hsp27. In order to assess more general ap-
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plications of our Pd-catalyzed protein allylation, we chose heat
shock protein 27 (Hsp27) as our next target. It represents a more
challenging protein target due to its higher molecular weight
and its buried cysteine residue but led to similar prenylation re-
sults (Figure S5). Applying reagents Re and Rf, respectively,
under conditions established above for UBL3, gave full conver-
sion into the azide- as well as the alkyne-tagged protein conju-
gates in just 2 h (Figure 6A and 6B). The peak-to-peak conver-
sion of Hsp27 is nicely illustrated by HPLC chromatograms at
t = 0 and after 2 h (Figure 6C). Both modified Hsp27 variants
were isolated in excellent yields (78 % and 81 %) and high pu-
rity (>95 %). Direct dissolution of the obtained purified Hsp27
products in 50 mM phosphate buffer at pH 7 led to correctly
folded proteins as demonstrated by CD measurements (Figure
S4B). To demonstrate the utility of Hsp27-alkyne, we carried
out a CuAAC reaction with a commercially available azido-bi-
otin reagent, which led to full conversion into the desired prod-
uct after only 10 min (Figure 6B).
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CONCLUSION
In conclusion, we have developed a chemoselective method
for the prenylation, functionalization and stapling of Cys-con-
taining peptides using Pd/BIPHEPHOS as a catalyst and readily
accessible allylcarbonates as reagents. This method was applied
to the modification of peptides and proteins for the installation
of native prenyl groups as well as artificial bioconjugation han-
dles. In contrast to many established peptide and protein modi-
fication reactions, our new Pd-catalyzed Cys-prenylation has
the advantage that it forms natural allylthioether linkages as
found in prenylated biomolecules and thus can be regarded as a
chemical in vitro posttranslational modification reaction, which
is compatible with all proteinogenic amino acids. In addition, it
is general regarding the allylic electrophiles that are applied in
minimal excess (1.2 eq.) and therefore provides an efficient tool
to introduce labels and tags as well as stabilizing staples into
peptides and proteins affording correctly folded products of
high purity.
Figure 5. Application of the Pd-mediated allylation for the modifi-
cation of ubiquitin-like protein 3 (UBL3). Both UBL3 variants with
one (A and B) and two (C) C-terminal Cys were successfully mod-
ified when treated with 1.2 eq. allylation reagent per Cys residue.
The HPLC-traces (214 nm) and mass spectra of the purified prod-
ucts are depicted for UBL3-1Cys-alkyne (A), UBL3-1Cys-Gerger
(B) and UBL3-2Cys-(Gerger)2 (C).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
allylation, we first applied our alkyne-carrying reagent Rf to
UBL3-1Cys using a 1:1 mixture of CH3CN/H2O as solvent to
reconcile protein, reagent and catalyst solubility. To our delight
we observed full conversion in 4 h to the corresponding alkyne-
tagged protein that could be isolated in 60 % yield with >95 %
purity after HPLC purification (Figure 5A).
Experimental procedures, characterization data, NMR spec-
tra, chromatography traces, mass spectra, circular dichroism
spectra.
AUTHOR INFORMATION
Corresponding Authors
Having demonstrated that our methodology is suitable for the
modification of proteins, we introduced the natively occurring
geranylgeranyl group with reagent Rd into both UBL3 variants
using similar conditions. These enabled geranylgeranylation of
both UBL3 variants with a conversion of 30 % in 4 h. A 1:1
mixture of 3 M aqueous Gdn•HCl with CH3CN was also tested
and increased the conversion of UBL3-2Cys to 60 %. After
HPLC purification both variants were obtained in high purity
(>95 %) and with isolated yields of 12 % for UBL3-1Cys and
22 % for UBL3-2Cys, respectively (Figure 5B and 5C). Dialy-
sis against a buffer containing 50 mM potassium phosphate at
pH 7 gave folded, prenylated UBL3 variants as confirmed by
CD-spectroscopy (Figure S4A).
Prof. Dr. R. Breinbauer
E-mail: breinbauer@tugraz.at
Prof. Dr. C. F. W. Becker
E-mail: christian.becker@univie.ac.at
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
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Figure 6. Attachment of azide- as well as alkyne-handles onto heat shock protein 27 (A and B), which can be employed for Click-derivati-
zation to introduce labels (biotin). HPLC traces (214 nm) of substrate and crude reaction mixtures (after 2 h) of the Hsp27 modifications
(C), as well as mass spectra of the purified Hsp27 with bioconjugation handles and crude CuAAC-product are depicted (D).
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(3) Reviews on metal-catalyzed modifications: a) Ohata, J.; Martin, S.
ACKNOWLEDGMENT
C.; Ball, Z. T. Metal‐Mediated Functionalization of Natural Peptides
and Proteins: Panning for Bioconjugation Gold. Angew. Chem. Int.
Ed. 2019, 58, 6176-6199; b) Jbara, M.; Maity, S. K.; Brik, A. Palla-
dium in the Chemical Synthesis and Modification of Proteins. An-
gew. Chem. Int. Ed. 2017, 56,10644-10655; c) Yang, M.; Li, J.;
Chen, P. R. Transition metal-mediated bioorthogonal protein chem-
istry in living cells. Chem. Soc. Rev. 2014, 43, 6511-6526; d)
Chankeshwara, S. V.; Indrigo, E.; Bradley, M. Palladium-mediated
chemistry in living cells. Curr. Opin. Chem. Biol. 2014, 21, 128-135;
e) Li, J.; Chen, P. R. Moving Pd‐Mediated Protein Cross Coupling to
Living Systems. ChemBioChem 2012, 13, 1728-1731; f) Böhrsch,
V.; Hackenberger, C. R. P. Suzuki–Miyaura Couplings on Proteins:
A Simple and Ready‐to‐use Catalytic System in Water. Chem-
CatChem 2010, 2, 243-245; g) Antos, J. M.; Francis, M. B. Transi-
tion metal catalyzed methods for site-selective protein modification.
Curr. Opin. Chem. Biol. 2006, 10, 253-262.
We gratefully acknowledge financial support by the Austrian Sci-
ence Fund (FWF) (Project P29458), University of Vienna and
NAWI Graz.
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our lab: g) Kljajic, M.; Schlatzer, T.; Breinbauer, R. Synthesis of 2-
Pyrrolidinones by Palladium-Catalyzed [3+2] Cycloaddition of Iso-
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(26) Wang, M.; Casey, P. J. Protein prenylation: unique fats make their
mark on biology. Nat. Rev. Mol. Cell Biol. 2016, 17, 110-122.
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Site-Specific Functionalization of Natural Antibodies. J. Am. Chem.
Soc. 2017, 139, 12617-12622; b) Chu, Q.; Moellering, R. E.; Hil-
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Coughlin, J. M.; Stagg, L. J.; Arold, S. T.; Ladbury, J. E.; Ball, Z. T.
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(24) Reviews: a) Liu, W.; Zhao, X. Carbon-Sulfur Bond Formation via
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(25) Selected examples for S-allylation: a) Roggen, M.; Carreira, E. M.
Enantioselective Allylic Thioetherification: The Effect of Phos-
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formations. Angew. Chem. Int. Ed. 2012, 51, 8652-8655; b) Wu, X.-
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Binuclear Fe-complexes as catalysts for the ligand-free regioselec-
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d) Gao, N.; Zheng, S.-C.; Yang, W.-K.; Zhao, X.-M. Carbon-Sulfur
Bond Formation via Iridium-Catalyzed Asymmetric Allylation of
Aliphatic Thiols. Org. Lett. 2011, 13, 1514-1516; e) Gais, H.-J.;
Jagusch, T.; Spalthoff, N.; Gerhards, F.; Frank, M.; Raabe, G. Highly
Selective Palladium Catalyzed Kinetic Resolution and Enantioselec-
tive Substitution of Racemic Allylic Carbonates with Sulfur Nucleo-
philes: Asymmetric Synthesis of Allylic Sulfides, Allylic Sulfones,
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SYNOPSIS TOC
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Labelling and Natural Posttranslational Modification of Peptides
and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cys-
teine
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Thomas Schlatzer,‡[a] Julia Kriegesmann,‡[b] Hilmar Schröder,‡[a] Melanie Trobe,[a] Christian Lem-
bacher-Fadum,[a] Simone Santner,[a] Alexander V. Kravchuk,[b] Christian F. W. Becker*[b] and Rolf
Breinbauer*[a]
[a] Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria
[b] Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Währinger Strasse 38, A-1090 Vienna,
Austria
KEYWORDS prenylation, bioconjugation, lipidation, peptide cyclization, stapled peptides
ABSTRACT: The prenylation of peptides and proteins is an important posttranslational modification observed in vivo. We report
that the Pd-catalyzed Tsuji-Trost-allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides
and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-
native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical
probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, pep-
tides containing two cysteine residues can be stapled or cyclized using homo-bifunctional allylic carbonate reagents.
INTRODUCTION
Over the last three decades it has been recognized that post-
translational modifications (PTMs) (glycosylation, phosphory-
lation, sulfation, acylation, lipidation, etc.) play an important
role in controlling protein function and localization. Among
PTMs, prenylation is essential for associating certain proteins
to specific membranes. A particularly intriguing example for
this is the Ras superfamily of small GTPases involved in signal
transduction processes that lead to cell growth and differentia-
tion as well as in vesicular trafficking.1
For the biophysical and cell biological investigation of pro-
teins in general and of PTMs in particular, chemoselective
methods are needed that enable access to modified proteins via
synthetic manipulations at reactive side chains of proteinogenic
amino acids using either chemical reagents or a transition metal
catalyst.2-5 The formation of new covalent bonds allows the at-
tachment of affinity tags, fluorophores, click handles or PET
tracers. Cysteine represents an attractive handle for the intro-
duction of such chemical modifications, as it is the second least
frequent amino acid in proteins (1.7 %)6 and shows a very
strong inherent nucleophilicity, which makes it especially at-
tractive for reactions with electrophilic reagents. Maleimides7
and iodoacetamides8 represent the earliest electrophiles used to
alkylate Cys (Figure 1, A) and have been frequently applied to
date. More recent developments include a variety of car-
bonylacrylic reagents as well as vinyl pyridines.9 Since then nu-
Figure 1. Selected examples of Cys-modifications established to
date (A-G) in comparison with the Pd-catalyzed Cys-allylation de-
scribed in this work.
merous different bioconjugation strategies have been devel-
oped.2,10 One of them involves transformation of Cys into dehy-
droalanine (Dha) upon treatment with 2,5-dibromohexanedia-
mide (DBHDA)11 or O-mesitylenesulfonylhydroxylamine
(MSH)12 (B), which is then reacted with a thiol nucleophile (C).
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Table 1. Scope of the Pd-catalyzed allylation of small peptides.
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Figure 2. Allylic carbonate reagents prepared for the Pd-catalyzed
Cys-modification.
A disadvantage of this method is that the formation of Dha is
associated with racemization at the α-carbon because diastere-
oselectivity of the thiol addition in simple Dha peptides is re-
ported to be low.13 A more direct access to S-allylcysteine with-
out epimerization can be accomplished by selenenylsulfide re-
ductive rearrangement (D),14 followed by further derivatization
either by olefin cross-metathesis15 or Kirmse-Doyle reaction.16
Alternatively, allylic halides might be used to directly allylate
Cys (E)14,17 although these highly reactive reagents are more
difficult to handle and preclude more elaborate reagent struc-
tures. Very recently Buchwald et al. introduced arylpalladium
reagents, which can be used for the arylation of Cys-containing
peptides and proteins (F).18 This concept has been extended to
Au(III) complexes.19 Pentelute et al. reported the site-selective
Cys-conjugation with perfluoroarene reagents at the π-clamp
motif FCPF (G),20 and the ligation with cyclooctynes.21
product in high selectivity, as the corresponding i-product (re-
sulting from internal attack) would be impossible to separate on
the peptide or protein level. From a screening of a diverse set of
mono- and bidentate phosphorous ligands we identified the
bisphosphite ligand BIPHEPHOS as the by far most suitable
ligand producing the desired n-products in high selectivity. Fur-
thermore, the n/i ratio was found to increase over time even
when complete conversion was already reached, indicating the
reversibility of this reaction (Table S1).
S-Allylation of Model Substrates. With these optimized
conditions in hand we wanted to apply the Pd-catalyzed S-al-
lylation to a dipeptide substrate (P1) featuring Tyr as second
amino acid, which could give rise to O-allylation as described
by Francis et al.23 before. Importantly, with our
Pd/BIPHEPHOS catalyst system we observed exclusively S-al-
lylation of Cys as confirmed via NMR by HMBC experiments
(Figure S1). With a series of allylation reagents (Figure 2) we
could demonstrate that a diverse set of labelled peptides could
be easily prepared by this method (Table 1, entries 1-7). More-
over, we successfully subjected unprotected glutathione (P2) to
Pd-catalyzed S-prenylation in an aqueous solvent mixture (Ta-
ble 1, entry 8) indicating a broader applicability of this method
for the modification of longer peptides and proteins. This is cor-
roborated by the fact that the reaction proceeds with fast kinet-
ics. Full conversion of 10 mM Ac-Cys-OMe was observed
within 10 min upon treatment with 2 eq. Ra in the presence of
2.0 mol% Pd/BIPHEPHOS at 35 °C. Even 0.5 mol% of the cat-
alyst were found to be sufficient to obtain quantitative conver-
sion after 30 min demonstrating the high efficiency of the reac-
tion (Figure S2). However, strictly oxygen-free conditions were
crucial for the activity of the catalyst.
RESULTS AND DISCUSSION
Reaction Optimization. We envisioned that Cys could be
selectively modified using the Pd-catalyzed Tsuji-Trost reac-
tion, which would give rise to an allylthioether linkage, as pre-
sent in naturally prenylated proteins, in a single step. The Tsuji-
Trost-allylation with C-, N- or O-nucleophiles is well estab-
lished in organic synthesis22 and Francis et al.23 have impres-
sively demonstrated the site selective modification of proteins
via Pd-catalyzed O-allylation of Tyr residues using the water-
soluble phosphine ligand TPPTS. In contrast, the reaction with
S-nucleophiles has been rarely studied,24,25 as it faces intrinsic
difficulties: a) S-nucleophiles can also function as efficient lig-
ands for Pd and poison the catalyst, and b) thiols are easily ox-
idized and the reactions have to be carried out under exclusion
of air. We hoped that with a prudent choice of ligand we could
design a Pd-catalyst system suitable for the allylation of Cys-
containing peptides and proteins. In contrast to classic allylation
procedures14,17 using an excess of highly reactive allylic halides,
a Pd-mediated reaction would allow the use of easily accessible
allylic carbonates as electrophiles. These reagents are much
more versatile as they are bench stable and can contain highly
functionalized structural motifs. Furthermore, in situ activated
electrophiles could be sterically controlled by the Pd-complex
so that the nucleophilic attack is directed to the terminal end of
the 3-Pd-allyl complex intermediate to produce the n-allylation
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Figure 3. Peptide- and reagent-scope of the Pd-mediated allylation of oligopeptides. Peptide sequences containing internal, terminal and
multiple Cys residues were subjected to site-selective allylation enabling the introduction of native prenyl groups, bioconjugation handles
(azide/alkyne groups) as well as a fluorescent NBD-tag and a biotin affinity-tag. All modified peptides were purified, isolated and charac-
terized by LC-MS analysis.
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Chemoselective Peptide Modification. As a next step we
tested the Pd-catalyzed Cys-allylation on a series of more com-
plex oligopeptides. For this purpose, the substrate concentration
was reduced to 1 mM to account for the lower solubility of the
peptides and the reaction temperature was adjusted to 40 °C to
ensure peptide integrity. To compensate for slower kinetics un-
der these conditions, the amount of Pd and ligand was in-
creased, which fully restored the reactivity of the system. As a
relevant target protein for prenylation, we selected ubiquitin-
like protein 3 (UBL3) and started out with modifying its C-ter-
minal domain (peptide P3) with polyprenyl groups, bioconju-
gation handles, fluorescent and affinity tags, highlighting the
versatility of this method (Figure 3). Furthermore, we could
show that farnesylation is feasible at internal as well as terminal
Cys and that this bioconjugation strategy offers access to adja-
cent and non-adjacent di-farnesylated products, which are of
special importance in naturally occurring proteins.26 The high
chemoselectivity of this reaction was showcased on a 32aa pol-
ypeptide (P7) featuring nearly all functional group containing
amino acids, which was found to undergo farnesylation exclu-
sively on Cys as proven by tryptic digest and mass spectromet-
ric analysis (Figure S3).
product, whereas for P8i and P9i two separate peaks with the
expected mass occurred presumably due to the formation of E/Z
isomers. Although the intramolecular reaction was favored for
P10j, we observed also the dimer of P10j (approx. 10 %), con-
sisting of two peptides and two staples, as a side product. It is
worth mentioning that the stapled products provide motifs for
further functionalization by taking advantage of the double
bond and that allylated peptides with identical staple motifs
have recently been shown to function as substrates in decaging
strategies using transition metal catalysis as well.5
Figure 4. Peptide stapling/cyclization using Pd-mediated allyla-
tion. Three model peptides with various distances (i+3, i+4, i+11)
between the Cys residues were subjected to stapling/cyclization us-
ing two bifunctional reagents with different geometries.
Peptide Stapling/Cyclization. Having established a series of
highly selective monofunctional allylic carbonate reagents that
were successfully applied on a broad set of peptide substrates,
we were eager to see if our methodology can also be extended
to bifunctional allylation reagents. This would enable us to im-
plement an additional type of a peptide stapling protocol,27
which is based on Pd-mediated S-allylation. To this end, we pre-
pared two bifunctional allylic carbonates (Ri and Rj) with dif-
ferent geometries which were subjected to Pd-mediated allyla-
tion using two α-helical peptides (P8 and P9)28 with cysteine
residues spaced by i+3 and i+4 as well as peptide P10 with more
distant residues (i+11)29 (Figure 4). Reactions leading to P8j,
P9j and P10i gave only one peak corresponding to the desired
Modification of UBL3 Protein. To further evaluate the po-
tential of our method, we chose the full-length protein UBL3 as
substrate, which extends the application beyond classic reac-
tions based on alkyl halides with peptide substrates.17 UBL3 un-
dergoes posttranslational geranylgeranylation in vivo and direct
access to such membrane-bound UBL3 variants will help to elu-
cidate their so far unknown physiological role(s).30 Two vari-
ants, with one and with two C-terminal Cys, were used here
since mono- as well as di-lipidation occur in nature. In order to
find appropriate reaction conditions for the Pd-mediated protein
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Modifications of Hsp27. In order to assess more general ap-
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plications of our Pd-catalyzed protein allylation, we chose heat
shock protein 27 (Hsp27) as our next target. It represents a more
challenging protein target due to its higher molecular weight
and its buried cysteine residue but led to similar prenylation re-
sults (Figure S5). Applying reagents Re and Rf, respectively,
under conditions established above for UBL3, gave full conver-
sion into the azide- as well as the alkyne-tagged protein conju-
gates in just 2 h (Figure 6A and 6B). The peak-to-peak conver-
sion of Hsp27 is nicely illustrated by HPLC chromatograms at
t = 0 and after 2 h (Figure 6C). Both modified Hsp27 variants
were isolated in excellent yields (78 % and 81 %) and high pu-
rity (>95 %). Direct dissolution of the obtained purified Hsp27
products in 50 mM phosphate buffer at pH 7 led to correctly
folded proteins as demonstrated by CD measurements (Figure
S4B). To demonstrate the utility of Hsp27-alkyne, we carried
out a CuAAC reaction with a commercially available azido-bi-
otin reagent, which led to full conversion into the desired prod-
uct after only 10 min (Figure 6B).
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CONCLUSION
In conclusion, we have developed a chemoselective method
for the prenylation, functionalization and stapling of Cys-con-
taining peptides using Pd/BIPHEPHOS as a catalyst and readily
accessible allylcarbonates as reagents. This method was applied
to the modification of peptides and proteins for the installation
of native prenyl groups as well as artificial bioconjugation han-
dles. In contrast to many established peptide and protein modi-
fication reactions, our new Pd-catalyzed Cys-prenylation has
the advantage that it forms natural allylthioether linkages as
found in prenylated biomolecules and thus can be regarded as a
chemical in vitro posttranslational modification reaction, which
is compatible with all proteinogenic amino acids. In addition, it
is general regarding the allylic electrophiles that are applied in
minimal excess (1.2 eq.) and therefore provides an efficient tool
to introduce labels and tags as well as stabilizing staples into
peptides and proteins affording correctly folded products of
high purity.
Figure 5. Application of the Pd-mediated allylation for the modifi-
cation of ubiquitin-like protein 3 (UBL3). Both UBL3 variants with
one (A and B) and two (C) C-terminal Cys were successfully mod-
ified when treated with 1.2 eq. allylation reagent per Cys residue.
The HPLC-traces (214 nm) and mass spectra of the purified prod-
ucts are depicted for UBL3-1Cys-alkyne (A), UBL3-1Cys-Gerger
(B) and UBL3-2Cys-(Gerger)2 (C).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
allylation, we first applied our alkyne-carrying reagent Rf to
UBL3-1Cys using a 1:1 mixture of CH3CN/H2O as solvent to
reconcile protein, reagent and catalyst solubility. To our delight
we observed full conversion in 4 h to the corresponding alkyne-
tagged protein that could be isolated in 60 % yield with >95 %
purity after HPLC purification (Figure 5A).
Experimental procedures, characterization data, NMR spec-
tra, chromatography traces, mass spectra, circular dichroism
spectra.
AUTHOR INFORMATION
Corresponding Authors
Having demonstrated that our methodology is suitable for the
modification of proteins, we introduced the natively occurring
geranylgeranyl group with reagent Rd into both UBL3 variants
using similar conditions. These enabled geranylgeranylation of
both UBL3 variants with a conversion of 30 % in 4 h. A 1:1
mixture of 3 M aqueous Gdn•HCl with CH3CN was also tested
and increased the conversion of UBL3-2Cys to 60 %. After
HPLC purification both variants were obtained in high purity
(>95 %) and with isolated yields of 12 % for UBL3-1Cys and
22 % for UBL3-2Cys, respectively (Figure 5B and 5C). Dialy-
sis against a buffer containing 50 mM potassium phosphate at
pH 7 gave folded, prenylated UBL3 variants as confirmed by
CD-spectroscopy (Figure S4A).
Prof. Dr. R. Breinbauer
E-mail: breinbauer@tugraz.at
Prof. Dr. C. F. W. Becker
E-mail: christian.becker@univie.ac.at
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
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Figure 6. Attachment of azide- as well as alkyne-handles onto heat shock protein 27 (A and B), which can be employed for Click-derivati-
zation to introduce labels (biotin). HPLC traces (214 nm) of substrate and crude reaction mixtures (after 2 h) of the Hsp27 modifications
(C), as well as mass spectra of the purified Hsp27 with bioconjugation handles and crude CuAAC-product are depicted (D).
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(3) Reviews on metal-catalyzed modifications: a) Ohata, J.; Martin, S.
ACKNOWLEDGMENT
C.; Ball, Z. T. Metal‐Mediated Functionalization of Natural Peptides
and Proteins: Panning for Bioconjugation Gold. Angew. Chem. Int.
Ed. 2019, 58, 6176-6199; b) Jbara, M.; Maity, S. K.; Brik, A. Palla-
dium in the Chemical Synthesis and Modification of Proteins. An-
gew. Chem. Int. Ed. 2017, 56,10644-10655; c) Yang, M.; Li, J.;
Chen, P. R. Transition metal-mediated bioorthogonal protein chem-
istry in living cells. Chem. Soc. Rev. 2014, 43, 6511-6526; d)
Chankeshwara, S. V.; Indrigo, E.; Bradley, M. Palladium-mediated
chemistry in living cells. Curr. Opin. Chem. Biol. 2014, 21, 128-135;
e) Li, J.; Chen, P. R. Moving Pd‐Mediated Protein Cross Coupling to
Living Systems. ChemBioChem 2012, 13, 1728-1731; f) Böhrsch,
V.; Hackenberger, C. R. P. Suzuki–Miyaura Couplings on Proteins:
A Simple and Ready‐to‐use Catalytic System in Water. Chem-
CatChem 2010, 2, 243-245; g) Antos, J. M.; Francis, M. B. Transi-
tion metal catalyzed methods for site-selective protein modification.
Curr. Opin. Chem. Biol. 2006, 10, 253-262.
We gratefully acknowledge financial support by the Austrian Sci-
ence Fund (FWF) (Project P29458), University of Vienna and
NAWI Graz.
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