Catalytic Modification of Dehydroalanine in Peptides and Proteins by Palladium-Mediated Cross-Coupling

Article abstract of DOI:10.1002/chem.201802846

Dehydroalanine (Dha) is a remarkably versatile non-canonical amino acid often found in antimicrobial peptides. Herein, we present the catalytic modification of Dha by a palladium-mediated cross-coupling reaction. By using Pd(EDTA)(OAc)₂ as water-soluble catalyst, a variety of arylboronic acids was coupled to the dehydrated residues in proteins and peptides, such as Nisin. The cross-coupling reaction gave both the Heck product, in which the sp²-hybridisation of the α-carbon is retained, as well as the conjugated addition product. The reaction can be performed under mild aqueous conditions, which makes this method an attractive addition to the palette of bio-orthogonal catalytic methods.

Full text of DOI:10.1002/chem.201802846

A Journal of
Accepted Article
Title: Catalytic modification of dehydroalanine in peptides and proteins
via palladium mediated cross coupling
Authors: Dowine de Bruijn and Gerard Roelfes
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
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
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201802846
Link to VoR: http://dx.doi.org/10.1002/chem.201802846
Supported by

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
Catalytic modification of dehydroalanine in peptides and proteins
via palladium mediated cross coupling
A. Dowine de Bruijn[a] and Gerard Roelfes[a]*
Abstract: Dehydroalanine (Dha) is a remarkably versatile non-
canonical amino acid often found in antimicrobial peptides. Here we
present the catalytic modification of Dha via a palladium mediated
cross coupling reaction. Using Pd(EDTA)(OAc)2 as water soluble
catalyst,
a variety of arylboronic acids was coupled to the
dehydrated residues in proteins and peptides such as nisin. The
cross coupling reaction yields both the Heck product, in which the
sp2-hybridisation of the α-carbon is retained, as well as the
conjugated addition product. The reaction can be performed under
mild aqueous conditions, which makes this method an attractive
addition to the palette of bio-orthogonal catalytic methods.
Scheme 1. Schematic representation of palladium catalysed cross coupling on
dehydrated amino acids in peptides and proteins
modification is that the reaction has to take place under
physiological conditions (e.g. in water at neutral pH and at 37oC).
Therefore, we sought a water soluble palladium complex which
can carry out this transformation. Here, we present the
palladium catalysed cross coupling reaction for the site-selective
modification of Dha with arylboronic acids in peptides and
proteins by a complex based on ethylene-diamine-tetraacetic
acid (EDTA), a commonly used water soluble metal chelator.[13]
Introduction
Dehydroalanine (Dha) is a remarkably versatile non-canonical,
yet naturally occurring α,β-unsaturated amino acid that features
a unique sp2 hybridised α-carbon. The resulting planar structure
provides different structural properties and reactivity than
conventional sp3 hybridised amino acids.[1] In nature, dehydrated
amino acids are installed via posttranslational dehydration of
serine and threonine, and used to create lanthionine bridges
found in lantipeptides,[2] and piperidine moieties found in
thiopeptides.[3] Most of these peptides possess antimicrobial or
antitumor activity,[4] which make them interesting targets for new
antibiotics and medicines. Yet, modification of these peptides via
bio-engineering,[5] or total synthesis[6] is challenging and is thus
preferably done by late-stage site-selective chemical
modification.[7] The residual Dha residues in these peptides are
excellent reactive sites for such transformations. Michael
additions[8], 1,3-dipolar cycloadditions,[9] radical carbon-carbon
bond formations,[10] and catalytic arylation of peptides in organic
solvents have been reported.[11] In all these transformations the
sp2 configuration of the α-carbon is lost, which may be of
importance to preserve the structure and biological activity of the
proteins and peptides. Palladium mediated Heck-type[12] cross
coupling could leave the sp2 hybridisation intact, although
competition of the conjugate addition product is also to be
expected for the conjugate alkene in Dha.[12] Choosing a water
soluble organometallic complex contributes to the versatility of
the approach: a requirement for protein modification over
peptide
Results and Discussion
Initial studies focused on the reaction of Dha monomer (1), with
4-methoxyphenylboronic acid (2a) (figure 1a). Neutral to slightly
basic conditions (pH 7-8) proved necessary to obtain conversion
of the Dha monomer, as was determined by 1H-NMR. Two
products were obtained, and identified to be the Heck product
(HP) and the conjugate addition product (CAP). A mixture of
these products is commonly observed for cross coupling of
conjugated alkenes, and is difficult to avoid.[12] The Heck product
was found to be the main product of the reaction (ratio HP:CAP
80:20). Carrying out the reaction under oxygen atmosphere did
not improve the conversion, which means ambient atmosphere
provides enough molecular oxygen for the Pd(0) to Pd(II)
oxidation to occur, thereby closing the catalytic cycle. The
highest conversion was obtained with 10 mol% catalyst, an
o
excess of arylboronic acid (2 eq), in phosphate buffer at 37 C.
Interestingly other commonly used water soluble palladium
complexes did not result in any conversion of Dha (table S1).
The reaction conditions for the modification of the Dha monomer
were not further optimized since the main focus is on
modification of Dha in proteins and peptides. The
Pd(EDTA)(OAc)2 catalyst, an excess of arylboronic acid, and
phosphate buffer pH 7 were selected for our subsequent studies
on protein and peptide modification.
[a]
A.D. de Bruijn MSc, Prof. Dr. G. Roelfes
Strating Institute for Chemistry, University of Groningen
Nijenborgh 4, 9747 AG Groningen (the Netherlands)
E-mail: j.g.roelfes@rug.nl
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
We focused on the palladium mediated cross coupling reaction
of the lantipeptide nisin.[5e] Nisin naturally contains three
dehydrated amino acids: Dhb-2 (dehydrobutyrine), Dha-5 and
Dha-33, a maximum of three modifications is thus expected. The
peptide is hydrophobic in nature, which gives rise to solubility
problems in aqueous solution, and nisin is less stable at pH >
5.[14] Moreover, conjugate addition of water to Dha, and
hydrolytic cleavage at this site are well-known degradation
reactions.[15] Despite the potential of nisin as an antibiotic, to the
best of our knowledge, no catalytic methods for modification
have been reported and stoichiometric chemical modifications
are scarce.[9, 16]
Nisin was reacted with phenylboronic acid (2b) using
Pd(EDTA)(OAc)2 as catalyst (figure 1b). The crude reaction
mixture was analysed directly by UPLC/MS. When more than
one equivalent of palladium catalyst was used, no peptide signal
was observed in the UPLC/MS chromatogram (figure S5). This
was attributed to non-specific coordination of the palladium
catalyst to the backbone or side chains of the peptide, a
frequently observed limitation of palladium mediated protein
reactions.[17] This was addressed by addition of 3-
mercaptopropanoic acid (3-MPA), a commonly used palladium
scavenger, prior to mass analysis. To overcome the loss of
catalyst due to unspecific coordination, a 50-fold excess of the
catalyst was used, together with a 50-fold excess of arylboronic
acid. Subsequent scavenging with 3-MPA gave 3b as a mixture
of singly- and doubly modified nisin (figure 1c). However,
purification of the peptide from the in situ formed palladium-[3-
MPA]-complex proved difficult. The formed palladium complex is
>2 kDa, making removal by size exclusion chromatography or
dialysis inefficient.
Therefore, alternative scavengers for the palladium
catalyst were investigated, which included a variety of water
soluble thiols, as well as resin-based scavengers (table S3). In
most cases, these gave rise to either insufficient scavenging or
purification difficulties similar to what was encountered with 3-
MPA. Good results were obtained with methylthioglycolate
(MTG), and ammonium pyrrolidine dithiocarbamate (APDTC)
since these form insoluble palladium-complexes,[18] which
precipitate from the solution. The precipitate is readily removed
by centrifugation, or filtration over 0.45 μm pore filters. Using this
method, 99% of the palladium was removed, as measured by
Inductively Coupled Plasma Optical Emission Spectrometry
(ICP-OES) (Supplementary Information S3.5). Purification from
starting materials and byproducts was then achieved by size
exclusion column chromatography (PD Minitrap G25) or rp-
HPLC. In this way, modified nisin, as a mixture of 48% singly
modified, 46% doubly modified and 3% triply modified peptide,
was obtained. Control experiments where either the arylboronic
acid or palladium catalyst were omitted from the reaction
mixture, resulted in no reaction, which demonstrates that the
reaction is indeed mediated by the palladium catalyst
(Supplementary Information S3.6).
Figure 1: Cross coupling reaction on Dha a) General reaction scheme on Dha
monomer; b) General reaction scheme, optimised conditions: nisin (40 μM),
boronic acid (2 mM) and Pd(EDTA(OAc)2 (2 mM in 25 μL buffer (50 mM
NaH2PO4 pH 7 2.2% DMF) shaken 16 hours at 37oC. Prior to mass analysis 3
eq (w.r.t. Pd) 3-MPA, MTG or APDTC are added; c) Representative MALDI-
TOF spectrum of reaction mixture 3b. I: degraded (degr.) nisin(Ph); II: degr.
nisin(Ph)2; III: nisin(Ph); IV: nisin(Ph)(H2O); V: nisin(Ph)2; VI: nisin(Ph)2(H2O);
VII: nisin(Ph)3.
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
that the reaction partly followed the conjugated addition
pathway, similar to the reaction on the Dha monomer.
Interestingly, an excess of L-Phe was observed. Since the
Pd(EDTA)(OAc)2 catalyst is not chiral, the enantiomeric excess
must be induced by the chirality of the peptide (i.e. substrate
control). Furthermore, Dhb is also subjected to the cross
coupling reaction as the product of conjugate addition of 2b to
Dhb derivatised with FDAA was also observed in the LC/MS
chromatogram (Figure S8).
Marfey’s reagent does not reveal the presence of the
dehydrophenylalanine (i.e. the Heck product), since unprotected
dehydrated amino acids equal a primary enamine, and therefore
quickly tautomerise, followed by hydrolysis to their
corresponding α-keto-acid, i.e. phenylpyruvic acid (PhPA).
Therefore, the other half of the hydrolysate was treated with
dansylhydrazine, which reacts with α-keto-acids to form
hydrazones.[20] The reaction usually yields two isomers (E/Z),
which separate during LC. Analysis with LC/MS and comparison
with a sample the hydrazine formed between dansylhydrazine
and PhPA, confirmed the presence of PhPA in the hydrolysate
of 3b nisin (figure 2b). Also the α-keto-acid of the product of
Heck reaction on Dhb was detected (Figure S9). So, the Heck
pathway is also followed in the palladium mediated cross
coupling reaction of peptides. Thus, product 3b has partially
maintained its sp2 hybridised α-carbon and, as a result, its
unique structural properties.
Figure 2: Analysis of the site selectivity of cross coupled nisin, and
determination of Heck product and conjugated addition product. a) Analysis of
introduced phenylalanine using Marfey’s method: (I) EIC of [M+H] = 418 Da
corresponding to D/L phenylalanine derivatised with FDAA; (II) EIC of
hydrolysate of 3b derivatised with FDAA; (III) EIC of hydrolysate of nisin
derivatised with FDAA. b) Analysis of introduced dehydrophenylalanine using
hydrazine formation: (I) Extracted ion chromatogram (EIC) of [M+H] = 412 Da
corresponding to phenylpyruvic acid derivatised with dansylhydrazine; (II) EIC
of hydrolysate of 3b derivatised with dansylhydrazine; (III) EIC of hydolysate of
nisin derivatised with dansylhydrazine.
To determine whether the cross coupling reaction takes
place at the expected dehydrated amino acids, and to determine
whether for nisin besides the Heck product also the conjugated
addition product is formed, modified nisin (3b) was hydrolysed in
a microwave oven in 6 M HCl(aq) and the individual amino acids
were identified. Cross coupling reaction at a Dha residue with 2b
results in either dehydrophenylalanine (the Heck product), or
phenylalanine (the conjugate addition product), which should be
detectable in the hydrolysate. One half of the hydrolysate was
therefore derivatised with Marfey’s reagent (1-fluoro-2,4-
dinitrophenyl-5-L-alanine amide (FDAA)) which will react with
phenylalanine to give FDAA-Phe.[19] Analysis with LC/MS and
comparison with FDAA derivatised D/L-phenylalanine samples
showed the presence of both enantiomers of phenylalanine in
the hydrolysate of 3b (figure 2a). Since nisin naturally does not
contain phenylalanine, the presence of 3b proves the cross
coupling indeed takes place at a Dha residue and, moreover,
Figure 3: Scope of arylboronic acids in cross coupling reaction with nisin.
Single modification (*), double modification (**) and triple modification (***) is
observed. In (..) conversion is displayed based on integration of EIC of
corresponding product. Conversion is calculated based on integration of the
EIC of the corresponding product divided by sum of the areas of all
compounds, assuming that ionisation is similar for all products, which are
structurally very similar.[21]
In an attempt to increase the rate of the reaction, the cross
coupling reaction was performed at pH 8 (table S4). Although an
increased amount of double cross coupled product was obtained,
also a higher amount of degraded nisin was observed due to the
competing water addition to the Dha. The competition of the
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
cross coupling reaction with the spontaneous water addition in
nisin might explain the predominant formation of single cross
coupled product. Nevertheless, using this method it is possible
to introduce a variety of different arylgroups containing diverse
functional groups to nisin (figure 3). This includes an azide
functionality (3e) which can subsequently be modified via
alkyne-azide click reactions to conjugate the peptide further, and
an carboxylic acid functionality (3g) which may enhance the
water solubility of such peptides. Finally, the generality of
palladium mediated cross coupling reaction was investigated by
using the reaction for protein modification. SUMO (Small
Ubiquitin-like MOdifier, ~11kDa)[22] containing
a chemically
introduced Dha residue[8d, 23] was used as substrate. The Dha
residue was introduced at two different positions: near the C-
terminus of protein, to minimise steric effects on the reaction
(SUMO_G98Dha), and in one of the solvent exposed loops
(SUMO_M60Dha) (Supplementary Information S3.11-13).
Treatment of the protein with 20 eq Pd(EDTA)(OAc)2 catalyst
and 100 eq of arylboronic acid showed full conversion to the
cross coupled product for p-toluylboronic acid (figure 4). Control
experiments performed on SUMO_G98A, which lacks the Dha
moiety, resulted in no reaction, which demonstrates that the
reaction is also in proteins specific at the Dha residue (figure
S16). Reactions with phenyl-, d5-phenyl-, and methoxyphenyl
substituted boronic acids (5a-d) resulted full conversion of the
cross coupled product too. Carboxylic acid-, fluorine-, and
amine-substituted phenylboronic acids were coupled as well,
although not with full conversion (5f-h). Neither an increase of
palladium catalyst, nor an increase in arylboronic acid resulted in
full conversion being achieved. Attempts to cross couple dansyl
substituted arylboronic acid 5i, or a pyrene boronic acid 5j did
not result in any conversion. Most likely this is due to the poor
water solubility of these reagents. Azide substituted arylboronic
acid 5e was cross coupled successfully, albeit that a fraction of
the azide moieties was reduced to the corresponding amine
during the treatment with the palladium scavenger. The azide
was subsequently reacted in a copper(I) catalyzed Azide-Alkyne
Cycloaddition (CuAAC) with an alkyne substituted bodipy (12)
(figure S17).
SUMO_M60Dha showed a similar trend when applied in
the cross coupling reaction: full conversion was achieved with
deuterium-, p-methyl- and p-methoxy-substituted phenylboronic
acids (table S6b), while 4-fluorophenylboronic acid did not give
rise to full conversion.
Further investigation of the modified protein by microwave
assisted hydrolysis of 5c and subsequent derivatisation with
Marfey’s reagent or dansylhydrazine revealed that the cross
coupling on proteins also follows both the conjugate addition and
Heck pathways, as both p-toluylalanine as p-toluylpyruvic acid
were observed (figure S18).
Figure 4: Pd(EDTA)(OAc)2 catalysed cross coupling reaction on SUMO. a)
General reaction scheme for the chemical introduction of Dha in SUMO;[8d] b)
General reaction scheme, optimised conditions: protein (45 μM), boronic acid
(4.5 mM) and Pd(EDTA)(OAc)2 (0.9 mM) in 22 μL buffer (50 mM NaH2PO4 pH
7 2.2% DMF) shaken 16 hours 37 oC. Prior to UPLC/MS analysis 3 eq (w.r.t.
Pd) 3-MPA, MTG or ADPTC are added; c) Representattve UPLC/MS
spectrum of reaction mixture 5c and deconvoluted spectrum; d) Scope of
arylboronic acids in cross coupling reaction. Between (..) the conversion is
given if the reaction did proceed in full conversion. Conversion is calculated
based on integration of the EIC of the corresponding product divided by sum
of the areas of all compounds, assuming that ionisation is similar for all
products, which are structurally very similar.[21]
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
[1]
D. E. Palmer, C. Pattaroni, K. Nuami, R. K. Chadba, M. Goodman, T.
Wakamiya, K. Fukase, S. Horimot, M. Kitazawa, H. Fujita, A. Kubo, T.
Shiba, J. Am. Chem. Soc. 1992, 114, 5645.
Conclusions
In conclusion, here we have introduced the Pd(EDTA)(OAc)2
catalysed cross coupling reaction as
[2]
[3]
P. J. Knerr, W. A. van der Donk, Annu. Rev. Biochem. 2012, 81, 479.
R. Liao, L. Duan, C. Lei, H. Pan, Y. Ding, Q. Zhang, D. Chen, B. Shen,
Y. Yu, W. Liu, Chem. Biol. 2009, 16, 141-147.
a method for the
modification of the non-canonical amino acid dehydroalanine in
proteins and peptides. While no full conversion was achieved for
nisin, it has to be emphasized that such a late stage modification
approach is far more efficient than the alternatives, such as total
synthesis.[7] Detailed analysis of the individual amino acids of the
product shows the cross coupling reaction is specific for the
dehydrated residues, and follows two mechanistic pathways
yielding the Heck product and the conjugate addition product. In
the Heck product the sp2-hybridisation of the α-carbon in the
Heck product is maintained, thus leaving the geometry of the
backbone of the biomolecules intact, which may be of particular
importance for natural Dha/Dhb containing compounds.
Although an excess of the catalyst is necessary to obtain high
conversions, purification by precipitation of the palladium
catalyst with methylthioglycolate or pyrrolidine dithiocarbamate
as novel scavengers removes up to 98-99% of the catalyst. The
unique product of the reaction on Dha, combined with the fact
that the reactions can be performed under mild, aqueous and pH
neutral conditions at 37 oC, makes this method an attractive
addition to the palette bio-orthogonal catalytic methods.
[4]
[5]
a) J. M. Shin, J. W. Gwak, P. Kamarajan, J. C. Fenno, A. H. Rickard, Y.
L. Kapila, J. Appl. Microbiol. 2016, 120, 1449-1465; b) X. Just-Baringo,
F. Albericio, M. Alvarez, Mar. Drugs 2014, 12, 317-351.
a) Y. Shi, X. Yang, N. Garg, W. A. van der Donk, J. Am. Chem. Soc.
2011, 133, 2338-2341; b) L. Zhou, J. Shao, Q. Li, A. J. van Heel, M. P.
de Vries, J. Broos, O. P. Kuipers, Amino Acids 2016, 48, 1309-1318; c)
N. Garg, L. M. A. Salazar-Ocampo, W. A. v. d. Donk, PNAS 2013, 110,
7258-7263; d) X. Just-Baringo, F. Albericio, M. Alvarez, Angew. Chem.
Int. Ed. 2014, 53, 6602; Angew. Chem. 2014, 126, 6720; e) L. M.
Repka, J. R. Chekan, S. K. Nair, W. A. van der Donk, Chem. Rev. 2017,
5457.
[6]
[7]
[8]
a) M. K. K. Fukase, A. Sano, K. Shimbo, H. Fujita, S. Horimoto, t.
Wakamiya, T. Shiba, Tetrahedron Lett. 1988, 29, 795-798; b) K. C.
Nicolaou, Angew. Chem. Int. Ed. 2012, 51, 12414-12436; Angew.
Chem. 2012, 124, 12582.
a) L. R. Malins, Cur. Opin. Chem. Biol. 2018, 46, 25-32; b) J. M.
Chalker, Metal-mediated Bioconjugation. In Chemoselective and
Bioorthogonal Ligation Reactions: Concepts and Applications (Eds.: I.
Medintz, W. R. Algar, P. Dawson), Wiley, 2017, p. 231.
a) U. Schmidt, E. Öhler, Angew Chem Int Ed Engl 1976, 15, 42; Angew.
Chem. 1979, 88, 54; b) Y. Zhu, W. A. van der Donk, Org. Lett. 2001, 3,
1189-1192; c) D. P. Galonic, W. A. van Dder Donk, D. Y. Gin, Chem.
Eur. J. 2003, 9, 5997-6006; d) J. M. Chalker, S. B. Gunnoo, O.
Boutureira, S. C. Gerstberger, M. Fernández-González, G. J. L.
Bernardes, L. Griffin, H. Hailu, C. J. Schofield, B. G. Davis, Chem. Sci.
2011, 2, 1666; e) A. M. Freedy, M. J. Matos, O. Boutureira, F. Corzana,
A. Guerreiro, P. Akkapeddi, V. J. Somovilla, T. Rodrigues, K. Nicholls,
B. Xie, G. Jimenez-Oses, K. M. Brindle, A. A. Neves, G. J. L. Bernardes,
J. Am. Chem. Soc. 2017, 139, 18365.
Experimental Section
General procedure of catalysis on Nisin
Catalysis was performed in 50 mM NaH2PO4 buffer pH 7 or pH 8 with a
final concentration of 40 μM peptide, 2 mM boronic acid and 2 mM
catalyst. A typical catalysis reaction was set up as follows: Nisin (2 nmol
in 30 μL buffer) and 10 μL of 10 mM boronic acid stock solution were
combined. 10 μL of 10 mM catalyst stock solution was added. The vial
was shaken for 16 hours at room temperature. 5 μL of 250 mM
methylthioglycolate stock solution was added to scavenge the palladium,
the reaction mixture turned yellow instantly. The reaction mixture was
shaken at 37 oC for an additional hour. The precipitate was removed by
centrifugation for 10 minutes at 13.4k rpm. The supernatant was
analysed by UPLC/MS TQD and purified by rp-HPLC.
[9]
M. R. Aronoff, B. Gold, R. T. Raines, Org. Lett. 2016, 18, 1538.
[10] a) T. H. Wright, e. al., Science 2016, 354, aag1465; b) A. Yang, S. Ha,
J. Ahn, R. Kim, S. Kim, Y. Lee, J. Kim, D. Söll, H. Y. Lee, H. S. Park,
Science 2016, 354, 623.
[11] a) C. J. Chapman, A. Matsuno, C. G. Frost, M. C. Willis, Chem.
Commun. 2007, 3903-3905; b) C. J. Chapman, J. D. Hargrave, G. Bish,
C. G. Frost, Tetrahedron 2008, 64, 9528-9539; c) H. M. Key, S. J. Miller,
J. Am. Chem. Soc. 2017, 139, 15460.
[12] a) R. F. Heck, J. Am. Chem. Soc. 1968, 90, 5518-5526; b) A. L.
Gottumukkala, J. F. Teichert, D. Heijnen, N. Eisink, S. van Dijk, C.
Ferrer, A. van den Hoogenband, A. J. Minnaard, J. Org. Chem. 2011,
76, 3498-3501; c) M. E. Ourailidou, J. Y. van der Meer, B. J. Baas, M.
Jeronimus-Stratingh, A. L. Gottumukkala, G. J. Poelarends, A. J.
Minnaard, F. J. Dekker, ChemBioChem 2014, 15, 209-212.
[13] D. N. Korolev, N. A. Bumagin, Tetrahedron Lett. 2005, 46, 5751-5754.
[14] H. S. Rollema, O. P. Kuipers, P. Both, W. M. d. Vos, R. J. Siezen, Appl.
Environ. Microbiol. 1995, 2873.
Acknowledgements
The authors thank Prof. Dr. Adri Minnaard and Prof. dr. Frank
Dekker for advice on palladium catalysis, Prof. Dr. Oscar
Kuipers and Dr. Manuel Montalbán López for useful discussion
and for providing the initial batches of nisin. Financial support
from the Netherlands Ministry of Education, Culture, and
Science (Gravitation program no. 024.001.035) and the
Netherlands Organisation for Scientific Research (NWO, vici
grant 724.013.003) is gratefully acknowledged.
[15] J. W. M. H.S. Rollema, P. Both, O.P. Kuipers, R.J. Siezen, Eur. J.
Biochem. 1996, 241.
[16] T. Koopmans, T. M. Wood, P. t Hart, L. H. Kleijn, A. P. Hendrickx, R. J.
Willems, E. Breukink, N. I. Martin, J. Am. Chem. Soc. 2015, 137, 9382-
9389.
[17] a) J. M. Antos, M. B. Francis, Curr. Opin. Chem. Biol. 2006, 10, 253-
262; b) C. D. Spicer, B. G. Davis, Chem. Commun. 2011, 47, 1698-
1700.
[18] a) Y. Yamashina, Y. Kataoka, Y. Ura, Inorg. Chem. 2014, 53, 3558-
3567; b) W. P. Gallagher, A. Vo, Org. Proc. Res. Dev. 2015, 19, 1369-
1373.
Keywords: bio-orthogonal catalysis • dehydroalanine •
palladium • nisin • cross coupling
[19] P. Marfey, Carlsberg Res. Commun. 1984, 49, 591.
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
[20] F. J. Hidalgo, J. L. Navarro, R. M. Delgado, R. Zamora, Food. Chem.
2013, 140, 183-188.
Chem., 75, 2003; c) C. J. Thibodeaux, T. Ha, W. A. van der Donk, J.
Am. Chem. Soc. 2014, 136, 17513.
[21] a) L. M. Hicks, S. E. O'Connor, M. T. Mazur, C. T. Walsh, N. L. Kelleher,
Chem. Biol. 2004, 11, 327-335; b) H. Z. W. Wang, H. Lin, S. Roy, T.A.
Shaler, L. R. Hill, S. Norton, p. Kumar, M. Anderle, C.H. Becker, Anal.
[22] a) F. Melchior, Annu. Rev. Cell. Dev. Biol. 2000, 16, 591-626; b) W.
Sheng, X. Liao, Prot. Sci. 2002, 11, 1482-1491.
[23] J. M. Chalker, B. G. Davis, Curr. Opin. Chem. Biol. 2010, 14, 781-789.
This article is protected by copyright. All rights reserved.

10.1002/chem.201802846
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
FULL PAPER
A. Dowine de Bruijn and Gerard
Roelfes*
Page No. – Page No.
Catalytic modification of
dehydroalanine in peptides and
proteins via palladium mediated
cross coupling
Dehydroamino acids are conveniently modified in Pd catalyzed bio-orthogonal
cross-coupling reaction.
This article is protected by copyright. All rights reserved.