Suzuki–Miyaura Cross-Coupling of Bromotryptophan Derivatives at Ambient Temperature

Article abstract of DOI:10.1002/chem.202002454

Mild reaction conditions are highly desirable for bio-orthogonal side chain derivatizations of amino acids, peptides or proteins due to the sensitivity of these substrates. Transition metal catalysed cross-couplings such as Suzuki–Miyaura reactions are highly versatile, but usually require unfavourable reaction conditions, in particular, when applied with aryl bromides. Ligand-free solvent-stabilised Pd-nanoparticles represent an efficient and sustainable alternative to conventional phosphine-based catalysts, because the cross-coupling can be performed at considerably lower temperature. We report on the application of such a highly reactive heterogeneous catalyst for the Suzuki–Miyaura cross-coupling of brominated tryptophan derivatives. The solvent-stabilised Pd-nanoparticles are even more efficient than the literature-known ADHP-Pd precatalyst. Interestingly, the latter also leads to the formation of quasi-homogeneous Pd-nanoparticles as the catalytic species. One advantage of our approach is the compatibility with aqueous and aerobic conditions at near-ambient temperatures and short reaction times of only 2 h. The influence of different N^α-protecting groups, boronic acids as well as the impact of different amino acid side chains in bromotryptophan-containing peptides has been studied. Notably, a surprising acceleration of the catalysis was observed when palladium-coordinating side chains were present in proximal positions.

Full text of DOI:10.1002/chem.202002454

Accepted Article
Accepted Article
Title: Suzuki-Miyaura Cross-Coupling of Bromotryptophan Derivatives
at Ambient Temperature
Authors: Steffen Dachwitz, Dario H. Duwe, Yating Hong Wang, Hendrik
Gruß, Yvonne Hannappel, Thomas Hellweg, and Norbert
Sewald
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.202002454
Link to VoR: https://doi.org/10.1002/chem.202002454
01/2020

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
SUZUKI-MIYAURA Cross-Coupling of Bromotryptophan Derivatives
at Ambient Temperature
Steffen Dachwitz,[a] Dario H. Duwe,[a] Yating Hong Wang,[a] Hendrik Gruß,[a] Yvonne Hannappel,[b] Thomas Hellweg,[b]
and Norbert Sewald[a]*
[a]
[b]
S. Dachwitz, D. Duwe, Y. Hong Wang, Dr. H. Gruß, Prof. N. Sewald
Department of Chemistry
Organic and Bioorganic Chemistry
Bielefeld University, Universitätsstraße 25, 33615 Bielefeld (DE)
E-mail: norbert.sewald@uni-bielefeld.de
Dr. Y. Hannappel, Prof. Dr. T. Hellweg
Department of Chemistry
Physical Chemistry
Bielefeld University, Universitätsstraße 25, 33615 Bielefeld (DE)
Supporting information for this article is given via a link at the end of the document.
Abstract: Mild reaction conditions are highly desirable for bio-
orthogonal side chain derivatizations of amino acids, peptides or
proteins due to the sensitivity of these substrates. Transition metal
catalyzed cross-couplings such as Suzuki-Miyaura reactions are
highly versatile, but usually require unfavorable reaction conditions,
in particular, when applied with aryl bromides. Ligand-free solvent-
stabilized Pd-nanoparticles represent an efficient and sustainable
alternative to conventional phosphine-based catalysts, because the
cross-coupling can be performed at considerably lower temperature.
We report on the application of such a highly reactive heterogeneous
catalyst for the Suzuki-Miyaura cross-coupling of brominated trypto-
phan derivatives. The solvent-stabilized Pd-nanoparticles are even
more efficient than the literature-known ADHP-Pd precatalyst. Inter-
estingly, the latter also leads to the formation of quasi-homogeneous
Pd-nanoparticles as the catalytic species. One advantage of our
approach is the compatibility with aqueous and aerobic conditions at
near-ambient temperatures and short reaction times of only 2 hours.
The influence of different Nα-protecting groups, boronic acids as well
as the impact of different amino acid side chains in bromotrypto-
phan-containing peptides has been studied. Notably, a surprising
acceleration of the catalysis was observed when palladium-
coordinating side chains were present in proximal positions.
tion, and the use of different halogenated aromatic compounds
such as bromoaryls is recommended despite their inferior reac-
tivity. Hence, bromotryptophan is an interesting starting material
for Pd-mediated functionalization as it is readily available on a
gram scale by enzymatic halogenation via FAD-dependent
halogenases[7] or by using Trp synthase.[8] Due to their electron-
rich aromatic system, halotryptophans are more challenging
substrates for Pd-mediated cross-couplings than halophenylala-
nines. A Suzuki-Miyaura reaction of unprotected halotrypto-
phans in water was first described by Deb Roy et al. who em-
ployed an air-sensitive Pd-catalyst, namely trisodium 3,3',3’’-
phosphinetriyltribenzenesulfonate (TPPTS). The use of a water-
soluble phosphine ligand enabled the arylation of 5-bromo-
tryptophan in fair yields at 40 °C, while other substrates like
7-bromotryptophan required higher reaction temperatures.[9] The
functionalization of halophenylalanine- and halotryptophan-
containing dipeptides in a Suzuki-Miyaura reaction was demon-
strated by Willemse et al. who investigated different catalytic
systems to assess the compatibility between catalysts and ami-
no acid side chain functionalities. They were able to arylate the
base-sensitive Fmoc-protected iodophenylalanine at 40 °C
without deprotection, albeit with only 80 % conversion after
24 h.[10] Frese et al. and the group of Micklefield independently
combined enzymatic halogenation and cross-coupling in a multi-
step one-pot reaction without isolation of the halogenated spe-
cies.[11,12] Based on the investigations of the fluorescence prop-
erties of arylated tryptophans by Deb Roy et al.,[9] this approach
was used by Schnepel et al. to design a high-throughput fluo-
rescence assay for screening halogenase activity in directed
evolution.[13] Gruß et al. utilized a Pd-mediated Mizoroki-Heck
cross-coupling of unprotected bromotryptophan and styrene
derivatives to further improve the fluorogenic properties by red-
shifted excitation wavelength.[14] Kemker et al. were able to use
the Suzuki-Miyaura cross-coupling for fluorescence labelling or
side chain-to-side chain cyclisation of bromotryptophan contain-
ing RGD peptides, thereby increasing stability and selectivity.[15]
The Goss group reported on in vivo Suzuki-Miyaura cross-coup-
ling of bromotryptophan and bromopacidamycin at 37 °C using
(2-dimethylamino)-4,6-dihydroxypyrimidine (DMADHP) and tri-
methyl guanidine (TMG) Pd-precatalysts which had first been
described by the Davis group.[6,16,17] They also investigated the
limitations of the cross-coupling at low temperatures due to
coordination of the α-amino group of unprotected bromotrypto-
Introduction
The Suzuki-Miyaura cross-coupling is a versatile tool for the
selective formation of carbon-carbon bonds.[1] The stability of the
used organoboron compounds under aqueous conditions and
their high tolerance towards different functional groups[2] makes
the Suzuki-Miyaura cross-coupling a suitable reaction for bio-
orthogonal late-stage derivatization of halogenated natural sub-
strates.[3] Latest investigations have demonstrated the applicabil-
ity of the Suzuki-Miyaura reaction for functionalization of halo-
genated or boronated amino acids, peptides and proteins.[4] The
modifications of complex natural molecules such as proteins are
particularly challenging because gentle reaction conditions (mild
temperatures, pH 6-8, aerobic and aqueous conditions) are
mandatory to avoid side reactions and degradation.[5] Chalker et
al. developed an approach to arylate p-iodophenylalanine in
proteins under biocompatible conditions using a Pd-pyrimidine
precatalyst system.[6] However, aryl iodides are prone to oxida-
1
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
phan to palladium. Dumas et al. reported poly(
D
,L-lactide-glyco-
The Davis catalyst consists of Pd-nanoparticles
lide)-block-poly(ethylene glycol) copolymer Pd-nanoparticles
(PLGA-PEG-Pd-NPs) as a catalyst for the Suzuki-Miyaura reac-
tion of Nα-Boc-protected halophenylalanine under mild condi-
tions in water. With these nanoparticle assemblies in hand, they
were able to achieve 98 % conversion at 37 °C after 18 h.[18]
Since biocompatible catalysts for Suzuki-Miyaura cross-
couplings, such as 2-amino-4,6-dihydroxypyrimidine Pd-
precatalysts (ADHP-Pd), have already been reported by the
Davis group,13 we started our catalytic studies by comparing the
ligand-free Pd-nanoparticles with one of those (Scheme 1). The
same reaction conditions were used as reported by the Davis
group. Due to the inhibition of the ADHP-Pd by the free amino
group of tryptophan,[16] we decided to use Nα-Boc- (1) or Fmoc-
Results and Discussion
(2) protected L-7-bromotryptophan as benchmark system. We
We embarked on the search for a more reactive, air-stable
catalyst system to extend the biocompatibility of the Suzuki-
Miyaura reaction. As Pd-nanoparticle catalyzed Suzuki-Miyaura
reactions are promoted by oxygen,[19] ligand-free solvent-
stabilized Pd-nanoparticles described by Kurscheid et al.[20]
seemed to be promising candidates for our studies. Here we
report on compatibility studies of the ligand-free Pd-nanoparticle
catalyzed Suzuki-Miyaura reactions and an array of Nα-protected
7-bromotryptophans or 7-bromotryptophan containing pentapep-
tides under mild, aerobic, and aqueous conditions. Our goal was
to test a peptide library containing different side chain moieties
to make possible predictions for the suitability of Pd-
nanoparticles as a bio-orthogonal catalyst system.
investigated the influence of inert conditions on the reactivity of
the reference catalyst system compared to ligand-free Pd-
nanoparticles (Figure 2a).
L-7-Bromotryptophan was generated applying RebH-
containing combined cross-linked-enzyme-aggregates (combi-
Synthesis and characterisation of solvent-stabilized Pd-
nanoparticles
Scheme 1. SUZUKI-MIYAURA reaction of Nα-protected
L-7-bromotryptophan
with both biocompatible ADHP-Pd and solvent-stabilised Pd-nanoparticles.
Ligand-free solvent-stabilized Pd-nanoparticles were prepared
according to the protocol by Kurscheid et al.[20] Stirring a palladi-
um dichloride suspension in 2-propanol at room temperature for
16 days gave a red suspension of solvent-stabilized nanoparti-
cles. We performed the nanoparticle synthesis either under
aerobic or inert conditions, while no major difference in catalytic
activity could be observed. The Pd-nanoparticles were charac-
terized by transmission electron microscopy (TEM). The TEM
image (Figure 1a) shows the result of the nanoparticle synthesis.
In fact, the formation of bigger clusters of smaller nanoparticles
can be observed, but their initial shape is still identifiable. Clus-
tering might occur as a side effect of the sample preparation
during the TEM imaging. The amount of dissolved palladium
was quantified by atomic absorption spectroscopy (AAS) show-
ing that 65 % of the Pd was in solution. This is in accordance
with the results of Kurscheid et al. The Pd-nanoparticle stock
solution was stored in a 2 ml microcentrifuge tube at 6 - 8 °C
without any effort to exclude oxygen. The particles retained
catalytic activity even after storing for 15 months.
CLEAs) followed by Fmoc- or Boc-protection.[11] The Pd-
nanoparticles show a slightly higher catalytic activity than the
ADHP-Pd (Figure 2). We expected the reactivity of the Pd-
nanoparticles under inert conditions to be decreased compared
to oxygen-promoted catalysis,[19] while the ADHP-Pd precatalyst
system should remain unaffected or even show an increase in
activity. Notably, both catalysts were affected in their catalytic
activity to a similar extent. Under inert conditions, both catalysts
showed nearly the same (lower) catalytic activity compared to
aerobic conditions. This phenomenon is usually only observed
for nanoparticle-catalyzed reactions (Figure 2a). Heating to
80 °C led to cleavage of the Fmoc-protecting group regardless
of the catalyst used. At ambient temperatures Pd gets coordi-
nated by the free amino group of unprotected tryptophan.[16]
At higher temperatures the effect of this coordination obvi-
ously becomes negligible. Therefore, studies at increased tem-
perature (100 °C) were performed using unprotected L-7-bromo-
tryptophan (Figure 2b). The almost identical response of both
catalysts to alterations of the reaction conditions indicates that
both catalytic systems might involve a similar reactive species.
We therefore hypothesized that the ADHP-Pd precatalyst forms
ligand-stabilized Pd-nanoparticles, too. Hence, during the Suzu-
ki-Miyaura reaction the guanidyl group of the ADHP-ligand inhib-
its the catalytically active Pd species due to steric demands of
the ligand, by blocking binding sites of the Pd (Figure 4b), as
already reported for the amino group of unprotected trypto-
phan.[16]
Therefore, ligand-free solvent-stabilized nanoparticles in 2-
propanol catalyze the reaction without steric hindrance by lig-
ands. This makes these particles much more reactive than the
ADHP-Pd. The ligand ADHP was added to pre-formed Pd-nano-
Figure 1. (a) Transmission electron microscopy (TEM) image of suspended
solvent-stabilised Pd-nanoparticles; (b) Fivefold magnification.
2
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
Figure 2. Conversion as function of time in the Suzuki-Miyaura reaction (a) of Nα-Fmoc-L-7-bromotryptophan (20 °C) and phenylboronic acid catalysed by
ADHP-Pd (red) or Pd nanoparticles (Pd-NP) (blue) under aerobic or inert conditions and (b) of Nα-Boc-L-7-bromotryptophan (40 °C) or unprotected L-7-
bromotryptophan (100 °C) and phenylboronic acid catalysed by ADHP-Pd (red), Pd-NP (blue) or Pd-NP + ADHP (grey).
particles to investigate its influence on the performance of the
ligand-free Pd-nanoparticles. After addition of ADHP, the reactiv-
ity of the Pd-nanoparticles decreased drastically. The conver-
sions of the ADHP-Pd precatalyst and the Pd-nanoparticles with
catalytically active Pd-nanoparticles. On the other hand, the
ADHP-Pd kept their catalytic activity at 40 °C after 24 h, where-
as conversion in case of the Pd-nanoparticles with added ADHP
stagnated at only 30 % (Figure 2b red and black curve). This
ADHP were close to equal during the first 5 hours of the reaction. can be explained by different size distributions of the nanoparti-
However, the conversions start to diverge at different reac-
tion temperatures. At 100 °C the reaction catalyzed by ADHP-Pd
stagnated at almost 80 % conversion and formation of palladium
black was observed, while the Pd-nanoparticles with added
ADHP maintained catalytic activity (Figure 2b dotted red and
grey curve). We suggest that the presence of 2-propanol, in
which the nanoparticles were suspended, stabilizes the nano-
particles and prevents the agglomeration to Pd-black even at
elevated temperatures.[21] Additionally, 2-propanol reduces Pd(II)
species to Pd(0),[22] resulting in a continuous regeneration of
cles. TEM images of the Davis catalyst (ADHP-Pd) show clearly
defined nanoparticles (Figure 3). This provides final evidence
that the ADHP-Pd precatalyst does not only consist of a single
molecular species but also of a heterogeneous, so called quasi-
homogeneous catalytic species.
Optimization of reaction conditions
The reaction conditions for the nanoparticle catalyzed Suzuki-
Miyaura reaction were optimized using commercially available 5-
bromoindole and phenylboronic acid as a benchmark system.
Best results were achieved in water, using 5 equiv. of boronic
acid and potassium phosphate each and a catalyst loading of
5 mol-% (Scheme 2). This led to a final 2-propanol concentration
of 0.5 %, since the added catalyst was dissolved in 2-propanol.
Scheme 2. Suzuki-Miyaura cross-coupling of 5-bromoindole (6) and phenyl-
boronic acid (3) catalyzed by ligand-free solvent-stabilized Pd-nanoparticles
(Pd-NP).
The optimized reaction conditions were then used for the ar-
ylation of unprotected bromotryptophan: A solution of
L-7-
bromotryptophan (8), boronic acid and K3PO4 in water (pH = 8 -
9) was warmed to 40 °C. Once all compounds had been dis-
solved, the catalyst was added. Noteworthy, no effort was taken
to exclude oxygen from the reaction-vessel. The reaction pro-
Figure 3. Transmission electron microscopy (TEM) image of the Davis cata-
lyst (ADHP-Pd).
3
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
Table 2. Pd-nanoparticle catalyzed Suzuki-Miyaura reaction of Nα-protected
gress was monitored by reversed-phase high-performance liquid
chromatography (RP-HPLC) at 220 nm. Using unprotected L-7-
L
-7-bromotryptophans with different boronic acids under aqueous and aerobic
bromotryptophan (8) at 40 °C in comparison to Nα-protected
tryptophan, inhibition of the catalyst was observed and full con-
version could not be achieved (Table 1).
conditions.
Table 1. Pd-nanoparticle catalyzed Suzuki-Miyaura reaction of unprotected
L-7-bromotryptophan (8) with different boronic acids under aqueous and
aerobic conditions.
entry
R1
boronic acid
#
conv. (%)[a]
yield (%)[b]
1
2
3
Ac
Boc
Fmoc
16
4
5
82
97
83
67
51
36
4
5
6
Ac
Boc
Fmoc
17
21
25
>99
>99
>99
63
60
64
entry
boronic acid
conversion (%)[a]
yield (%)[b]
40°C (120 h)
80°C (2 h)
55
H
7
8
9
Ac
Boc
Fmoc
18
22
26
80
>99
>99
74
83
97
N
B(OH)2
1
2
3
4
5
52
68
78
56
22
65
69
42
36
41
O
59
72
95
42
10
11
12
Ac
Boc
Fmoc
19
23
27
97
>99
10[c]
73
82
n.d.[d]
H
N
B(OH)2
O
13
14
15
Ac
Boc
Fmoc
20
24
28
72
86
62
40
75
55
B(OH)2
HOOC
B(OH)2
[a] determined by HPLC (220 nm); [b] Isolated yields after full conversion and
purification; [c] did not reach full conversion; [d] not determined.
HOOC
For example, applying the Suzuki-Miyaura cross-coupling of
halogenated amino acids under mild conditions in solid-phase
peptide synthesis (SPPS). In particular, the base sensitive pro-
tecting group Fmoc is of great relevance. In addition, the reactiv-
[a] determined by HPLC (220 nm); [b] isolated yields after full conversion at
80 °C and purification.
These results are in accordance with the observations of
ADHP-Pd inhibition by unprotected bromotryptophan as reported
by the Goss group.[16] Heating to 80 °C increased the conversion,
likely due to the weaker coordination of Pd by the free amine at
higher temperatures. The reaction was monitored by RP-HPLC
and the products were purified by preparative RP-HPLC once
the reaction had reached completion at 80 °C. Adequate yields
of all aryltryptophans were obtained (Table 1). As expected, best
conversions at 40 °C were observed for non-nucleophilic and
electron-rich boronic acids, e.g. para-tolylboronic acid and 3-
acetamidophenylboronic acid, giving 68 % and 78 % conversion
after 5 days (Table 1, entries 2 and 3). At higher temperatures
the electron-richer 3-aminophenylboronic acid gave the best
conversion of 95 % after 2 hours at 80 °C (entry 4). Remarkably,
no precipitating Pd (Pd-black) was observed in presence of
unprotected tryptophan, due to the coordination of Pd by amines.
Using the electron-rich boronic acids 3-acetamidophenylboronic
acid and 3-aminophenylboronic acid, homocoupling was ob-
served independently of the atmosphere used.
ities of Nα-Boc- and Nα-acetyl protected
L-7-bromotryptophan
and various functionalized boronic acids were evaluated. Suzu-
ki-Miyaura reactions of Nα-protected bromotryptophans gave
promising results at 40 °C and no further heating was necessary
to reach full conversion (Table 2). A black precipitate (Pd-black)
was observed almost instantly when the Pd-nanoparticles were
added to the reaction. Exceptions occured using 3-amino-
phenylboronic acid or 4-carboxyphenylboronic acid as cross-
coupling partners, where no precipitate was observed. Since
amines and carboxylic acids coordinate to the surface of the Pd-
nanoparticles, agglomeration of the particles is hindered. The
solutions turned slightly yellow without any precipitate.
Nα-Acetyl-
L-7-bromotryptophan (15) gave best conversions with
para-tolylboronic acid (99 %) and 3-aminophenylboronic acid
(97 %) even after 2 hours (Table 2, entries 4 and 10).
Nα-Boc-protected bromotryptophan (1) gave the best results
among all Nα-protected bromotryptophans, reaching almost full
conversion combined with every tested boronic acid in less than
2 hours (Table 2). Even with the electron-poor 4-carboxyphenyl-
boronic acid 86 % conversion was attained after 2 hours (en-
try 14). Interestingly, catalytic inhibition due to coordination of Pd
by 3-aminophenylboronic acid seems to be negligible in case of
Nα-acetyl- and Boc-protected bromotryptophans, since both
reactions showed good conversions (Table 2, entry 10 and 11).
Since nanoparticles need to be centrifuged off before samples
can be purified by preparative RP-HPLC, hydrophobic precipi-
Screening of Nα-derivatized bromotryptophans
The investigation of different Nα-protection groups is of interest
beyond bio-orthogonal reactions which do not require protecting
groups, because it broadens the range of available orthogonal
protection strategies.
4
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
tated products and those coordinating to palladium might get lost,
leading to reduced yields. Under the mild conditions of this na-
noparticle catalysis the Fmoc-protecting group is stable and
Fmoc-protected arylated tryptophans were obtained in good
Table 3. Pd-nanoparticle catalyzed Suzuki-Miyaura reaction of L-7-bromo-
tryptophan-containing pentapeptides to investigate the influence of different
amino acid side chains.
yields (Table 2). Surprisingly, Nα-Fmoc-
L-7-bromotryptophan (2)
and 3-aminophenylboronic acid were converted to only 10 %
after 2 h at 40 °C (entry 12) while 3-acetamidophenyl boronic
acid showed excellent conversion >99 % (entry 9). Repetition of
the experiment led to the same result. This may be assigned to
the steric influence of ligands coordinating the Pd-nanoparticles.
To reduce the coordination of the Pd by amines the reaction
temperature was elevated to 60 °C, just leading to a fast cleav-
age of the Fmoc-group. Hence, 3-aminophenylboronic acid does
not seem to have enough steric hindrance to inhibit the cross
couplings with Nα-acetyl- or Boc- protected tryptophan (entries
10 and 11), while the sterically more demanding Fmoc-protec-
ting group exerts strong hindrance (entry 12) (Figure 4a).
entry
Xaa
conversion (%)[a]
yield (%)[b]
40 °C (2 h)
80 °C (2 h)
93
1
2
Ala
Arg
47
88
50
86
>99
0
3
Cys
n.d.
14[c]
traces[c]
86
0
4
Gln
72
39 [d],[f]
n.d.
53
O
O
HO
5
His
20[c]
>99
n.d.
n.d.
>99
>99
>99
HO
R
NH
NH
OH
B
6
Lys
O
OH
ONa
NH
NaO
NH
7
Lys(Boc)
Met
54
70
N
Br
N
NH2
Br
NH2
8
>99[e]
56
65[e]
77
Pd
Pd
9
Phe
Pro
Pd-nanoparticle
surface
Pd-nanoparticle
surface
a
b
10
11
61
65
Tyr
58
73
Figure 4: Possible steric hindrance of Pd-nanoparticles by (a) 3-aminophenyl-
boronic acid or (b) guanidyl-ligand ADHP.
[a] determined by HPLC (220 nm); [b] isolated yields after full conversion at
40 °C and purification; [c] did not reach full conversion; [d] corrected accord-
ing to 1H NMR; [e] full conversion after 5 minutes; [f] isolated yield after full
conversion at 80 °C and purification.
Influence of different amino acid side chains in bromotryp-
tophan containing pentapeptides
1
(HPLC) to 72 % after 2 hours. The H NMR of the isolated aryl-
ated peptide at high temperatures shows three different species
in a ratio of 7:1:1, which could be identified by LC-MS as the
arylated Gln-pentapeptide (30Gln), the arylated Glu-penta-
peptide (30Glu) and a cyclized species showing a mass shift -17
Da (nominal mass), which could be the pyroglutamate or glutar-
imide side product (Figure 5).
The Suzuki-Miyaura cross-coupling in water at 80 °C with differ-
ent 5-bromotryptophan containing dipeptides has been reported
by Willemse et al. Apart from bromotryptophan, aliphatic and
aromatic side chains had been introduced into the peptides.[4,10]
An array of L-7-bromotryptophan containing pentapeptides were
designed to investigate the compatibility of ligand-free Pd-
nanoparticles with complex substrates such as peptides. The
influence of all major functional groups present in a protein
should be evaluated. Since the nanoparticles are stored in
2-propanol and the compatibility of carboxylic acids with the
nanoparticles had already been verified (Table 1-2), a sequence
containing serine and aspartic acid was designed to ensure
sufficient water solubility of the peptides (Table 3). Presence of
aliphatic or aromatic side chains did not considerably interfere
with the reaction and the cross-coupled peptides were obtained
in good isolated yields after purification by RP-HPLC (Table 3;
entry 1 and 9 – 11). Willemse et al. had already described the
problems of a Suzuki-Miyaura reaction in the presence of prima-
ry amide (asparagine) and imidazole (histidine).[10] For imid-
azoles, the formation of N-azolyl palladium complexes were
postulated by Düfert et al.[23] This might lead to poisoning of the
Pd-nanoparticles by histidine in the pentapeptide sequence
resulting in only traces of cross-coupling product after 2 hours at
40 °C (entry 5). Using Pd-nanoparticles with the Gln containing
pentapeptide (29Gln) gave 14 % conversion after 2 hours at
40 °C. Increasing the temperature to 80 °C improved conversion
HO
O
O
H2N
O
O
NH
O
NH
O
OH
OH
OH
OH
O
O
H
N
H
N
H
N
H
N
N
H
N
N
H
N
H
N
H
N
H
H
O
O
O
O
O
O
OH
OH
O
O
30Gln
30Glu
NH
O
NH
O
OH
OH
O
O
O
O
H
N
H
N
H
N
O
O
N
OH
OH
N
N
H
N
H
OH
N
H
N
H
OH
N
H
O
O
O
O
O
O
O
O
30<Glu
30Glutarimide
Figure 5. Product and side products of the Suzuki Miyaura cross coupling
using a Gln containing pentapeptide.
The species were inseparable by preparative HPLC giving a
corrected isolated yield of 30Gln of 39 % (entry 4). The ob-
served cyclisation is a possible side reaction under basic condi-
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
tions at elevated temperatures, which occasionally is observed
in solid-phase peptide synthesis.[24] Similar observations had
been made by Willemse et al. in Suzuki-Miyaura reactions using
ADHP-Pd.[10,25] Inhibition of catalysis was expected for pen-
tapeptides comprising lysine or arginine, due to coordination of
palladium by amino or guanidino groups.[26] Surprisingly, the
catalytic activity increased compared to peptides containing
aliphatic or aromatic side chains (entry 1, 9-11) giving 88 % and
86 % HPLC conversion after 2 hours at 40 °C (entry 2 and 6).
This might occur in virtue of a pre-coordination of the peptide on
the surface of the nanoparticles. Through this pre-coordination,
the proximity of the aryl halide to the palladium could favor oxi-
dative addition (Figure 6).
protected methionine as a ligand (5 mol%) or in stochiometric
amounts (1.0 eq.) to the Suzuki-Miyaura reaction of Nα-Boc-
L-
7-bromotryptophan with phenylboronic acid did not lead to an
acceleration but effected inhibition of the catalysis.
We chose Nα-Boc-bromotryptophan (1), Nα-Fmoc-bromotryp-
tophan (2) and the pentapeptide 29Lys(Boc) as benchmark
candidates to prove the applicability of Pd-nanoparticles for
Suzuki-Miyaura cross-coupling at room temperature. The cross-
coupling of 1 with phenylboronic acid reached full conversion
after 6 h, while full conversion of 2 needed 20 h, giving the aryl-
ated tryptophans 4 in 76 % and 5 in 74 % yield after purification
by RP-HPLC. 29Lys(Boc) reached full conversion after 96 h at
room temperature giving 30Lys(Boc) in 67 % yield.
O
O
OH
OH
H
O
O
O
O
O
O
H
N
H
H
H
H
Conclusion
N
N
N
N
N
N
N
OH
OH
N
N
OH
OH
H
H
H
H
O
O
O
O
O
O
HN
HN
Br
Ligand-free solvent-stabilized Pd-nanoparticles were established
as a highly efficient catalyst for a biocompatible Suzuki-Miyaura
cross-coupling of different bromotryptophan derivatives under
mild, aerobic and aqueous conditions at ambient temperature. In
comparison to the commercially available biocompatible Davis-
catalyst (ADHP-Pd) the ligand-free nanoparticles displayed
increased catalytic activity. With these catalyst systems it is
possible to perform cross-couplings of sensitive substrates at
ambient temperature. The catalytically active species of the
Davis-catalyst could be identified as guanidyl-stabilized quasi-
homogeneous nanoparticles. Evaluation of different Nα-pro-
tecting groups for 7-bromotryptophan identified the Boc-group as
the most favorable one. Under the chosen mild conditions, even
the base-labile Fmoc-group was stable and compatible with the
Pd-catalyst. Screenings of different 7-bromotryptophan contain-
ing pentapeptides proved the compatibility of the nanoparticles
to many amino acid side chain functionalities. Only catalyst-
poisoning moieties like the cysteine thiol or the histidine imidaz-
ole compromised the reaction. Surprisingly, some coordinating
side chains (lysine, arginine or methionine), which were ex-
pected to hinder the reaction, accelerated the catalysis due to a
proposed pre-coordination of the molecule on the nanoparticle
surface. Furthermore, the accompanying electron-donation
seemingly promotes the oxidative addition.
HN
H2N
H2N
NH
Br
Pd
Pd
Pd-nanoparticle
surface
Pd-nanoparticle
surface
b
a
O
H
O
OH
OH
N
O
OH
H
N
N
O
O
H
H
N
N
O
H
O
HN
Br
S
Pd
Pd-nanoparticle
surface
c
Figure 6. Possible pre-coordination of Pd-nanoparticles by certain side chain
functionalities of -7-bromotryptophan-containing pentapeptides.
L
Since amide formation prevents the coordination to the pal-
ladium (Table 2), an Nε-Boc-protected lysine was introduced into
the pentapeptide sequence to prove whether amine-mediated
pre-coordination of the peptide could be a reason for the accel-
erated catalytic activity. The Boc-protected pentapeptide
(29Lys(Boc)) showed a conversion (HPLC) of 54 % after 2
hours at 40 °C (entry 7). This value is within the range of the
pentapeptides containing aliphatic or aromatic side chains (entry
1, 9-11), letting us draw the conclusion, that a basic, non-
aromatic side chain in the peptide sequence may possibly pre-
coordinate the peptide on the surface of the Pd-nanoparticles
favoring the Suzuki-Miyaura cross-coupling. Surprisingly, the
thioether moiety of methionine accelerates the catalysis as well,
causing full conversion at 40 °C after just 5 minutes (Table 3;
entry 8). At the same time, cysteine poisons the catalyst as
expected (entry 3). The thioether provides high electron density
to the palladium, thus promoting the rate-limiting oxidative addi-
tion step and thereby drastically accelerating the reaction. Add-
ing a methionine to the peptide sequence in the position of the
aspartic acid or switching the positions of aspartic acid and
methionine led to no conversion of the pentapeptide at all. Thus,
poisoning the Pd catalyst. Further investigations on the influence
of the position of the thioether moiety to the bromotryptophan for
the Suzuki-Miyaura coupling are ongoing. Addition of Boc-
Experimental Section
Analytical HPLC was performed on a Shimadzu NexeraXR 20A System
with autosampler, degasser, column oven, diode array detector and a
Phenomenex Luna C18 column (2.9 µm, 50 ꢀ 2.1 mm) with a gradient (in
5.5 min from 5 % B to 95 % B, 0.5 min 95 % B and back to 5 % B in 3
min, total run time 9 min) at a flow rate of 650 μL/min and column oven
temperature of 40 °C. HPLC solvent A consists of 99.9% water and
0.1 % TFA, solvent B of 99.9 % acetonitrile and 0.1 % TFA.
Analytical LC-MS was performed on an Agilent 6220 TOF-MS with a
Dual ESI-source, 1200 HPLC system with autosampler, degasser, binary
pump, column oven, diode array detector and a Hypersil Gold C18 col-
umn (1.9 µm, 50 ꢀ 2.1 mm) with a gradient (in 11 min from 0 % B to
98 % B, back to 0 % B in 0.5 min, total run time 15 min) at a flow rate of
300 μL/min and column oven temperature of 40°C. HPLC solvent A
consists of 94.9 % water, 5 % acetonitrile and 0.1 % formic acid, solvent
B of 5 % water, 94.9 % acetonitrile and 0.1 % formic acid. ESI mass
spectra were recorded after sample injection via 1200 HPLC system in
6
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
extended dynamic range mode equipped with a Dual-ESI source, operat-
ing with a spray voltage of 2.5 kV.
remaining binding sites were capped by adding MeOH (15 eq.). Fmoc-
deprotection was performed by addition of 20 % piperidine and 100 m
M
HOBt in DMF to the resin and shaking for 15 minutes at room tempera-
ture; this procedure was repeated two times. After deprotection, the resin
was washed with DMF, DCM, DMF (3 x 1 min each). Natural Fmoc-
protected amino acids were coupled to the Nα-deprotected peptide by
addition of a mixture of amino acid (4 eq.), TBTU (4 eq.) and DIEA (8 eq.)
in DMF to the resin and shaking for 2 hours at room temperature. After
coupling, the resin was washed with DMF, DCM, 2-propanol and MTBE
(3 x 1 min each) and dried in vacuo. Full conversion was verified by
NMR spectra were recorded on a Bruker Avance III 500 HD (1H:
500 MHz, 13C: 126 MHz, 19F: 471 MHz) or Avance 600 (1H: 600 MHz,
13C: 151 MHz). Chemical shifts δ [ppm] are reported relative to residual
solvent signal (DMSO-d6, 1H: 2.50 ppm, 13C: 39.5 ppm). 2D spectra
(COSY, HMQC, HMBC) and DEPT-135 spectra were used for signal
assignment.
Kaiser-test.
L-Fmoc-7-bromotryptophan (1.1 eq.) was coupled to the
High resolution ESI mass spectra were recorded using an Agilent 6220
time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA,
USA) in extended dynamic range mode equipped with a Dual-ESI source,
operating with a spray voltage of 2.5 kV. Nitrogen served both as the
nebulizer gas and the dry gas. Nitrogen was generated by a nitrogen
generator NGM 11. Samples are introduced with a 1200 HPLC system
consisting of an autosampler, degasser, binary pump, column oven and
diode array detector (Agilent Technologies, Santa Clara, CA, USA) using
a C18 Hypersil Gold column (length: 50 mm, diameter: 2.1 mm, particle
size: 1,9 μm) with a short isocratic flow (60 % B for 5 min) at a flow rate
of 250 μL/min and column oven temperature of 40°C. HPLC solvent A
consists of 94.9 % water, 5 % acetonitrile and 0.1 % formic acid, solvent
B of 5 % water, 94.9 % acetonitrile and 0.1 % formic acid. The mass axis
was externally calibrated with ESI-L Tuning Mix (Agilent Technologies,
Santa Clara, CA, USA) as calibration standard. The mass spectra are
recorded in both profile and centroid mode with the MassHunter Work-
station Acquisition B.04.00 software (Agilent Technologies, Santa Clara,
CA, USA). MassHunter Qualitative Analysis B.07.00 software (Agilent
Technologies, Santa Clara, CA, USA) was used for processing and
averaging of several single spectra.
Nα-deprotected peptide on resin with HATU (1.1 eq.) and DIEA (2.2 eq.)
in DMF at room temperature for 2 hours. Full conversion was verified by
a test cleavage and analytical LC-MS. Before final cleavage, the peptide
was N-acetylated by addition of a solution of acetic anhydride (10 eq.)
and pyridine (10 eq.) in DMF to the resin. Cleavage and side chain
deprotection were performed by addition of a mixture of TFA/H2O/TIS
(95:2.5:2.5) to the resin (2 x 1.5 h) followed by peptide precipitation
overnight in MTBE at –20 °C. This mixture was spun down (4000 rpm;
4 °C; 5 min), the MTBE layer discarded, the residue dissolved in water
and freeze dried. If necessary, the peptide was purified by RP-HPLC.
Acknowledgements
We acknowledge financial support from Deutsche Forschungs-
gemeinschaft (SE 609/16-1). The TEM equipment was funded
by Deutsche Forschungsgemeinschaft (INST215/444-1).
Transmission electron microscopy (TEM) was performed on carbon
coated copper grids (mesh 200, Science Service GmbH) which were
treated with an oxygen plasma before use (Zepto, Diener electronix
GmbH). Pictures were generated on a Philips CM100 PW6021 with a
Tungsten Emission source and a voltage of 80 kV or on a JOEL 2200FS
with a cold field emission source and a voltage of 200 kV.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Bio-orthogonality; Suzuki-Miyaura reaction; hetero-
geneous catalysis; halotryptophan; Pd-nanoparticles; oxy-
gen-promoted cross-coupling; late-stage derivatisation
Preparation of ligand-free solvent-stabilized Pd-nanoparticles:
Stirring a 100 mM suspension of PdCl2 in 2-propanol for 16 days under
air at room temperature gave a dark red solution with a brown to black
precipitate. This stock-solution was stored at 6 – 8 °C. Before use the
solution was mixed thorough.
[1]
[2]
[3]
a) N. Miyaura, A. Suzuki, Chem. Rev. 1995, 2457–2483; b) A. Suzuki,
Angew. Chem. Int. Ed. 2011, 50, 6722–6737;
A. J. J. Lennox, G. C. Lloyd-Jones, Chem. Soc. Rev. 2014, 43, 412–
443.
Preparation of ADHP-Pd: On benchtop, 2-amino-4,6-dihydroxy-
pyrimidine (2.0 eq.) was dissolved in an aqueous 100 mM NaOH solution
(4.0 eq.) and heated to 65 °C over 5 minutes followed by addition of
Pd(OAc)2 (1.0 eq.). The mixture was stirred for 30 minutes, cooled to
room temperature followed by addition of water giving an orange solution
a) T. O. Ronson, R. J. K. Taylor, I. J. S. Fairlamb, Tetrahedron 2015, 71,
989–1009; b) A. Deb Roy, S. Grüschow, N. Cairns, R. J. M. Goss, J.
Am. Chem. Soc. 2010, 132, 12243–12245; c) W. Runguphan, S. E.
O'Connor, Org. Lett. 2013, 15, 2850–2853; d) L. J. Durak, J. T. Payne,
J. C. Lewis, ACS Catal. 2016, 6, 1451–1454; e) H. Gruß, N. Sewald,
Chem. Eur. J. 2020, 26, 5328-5340.
with a final palladium concentration of 10 mM.
[4]
[5]
T. Willemse, W. Schepens, H. Vlijmen, B. Maes, S. Ballet, Catalysts
2017, 7, 74–106.
General procedure for Suzuki-Miyaura cross coupling using ligand-
free Pd-nanoparticles on a preparative scale (GP1): Cross-couplings
were performed on benchtop in a flask equipped with a stirring bar.
Therefore, aryl halide (50 µmol, 1.0 eq.), boronic acid (5.0 eq.) and
K3PO4 (5.0 eq.) were dissolved in water (5.0 ml), giving a final aryl halide
a) C. D. Spicer, B. G. Davis, Nat. Commun. 2014, 5, 4740–4753; b) E.
M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974–
6998; c) A. J. de Graaf, M. Kooijman, W. E. Hennink, E. Mastrobattista,
Bioconjugate Chem. 2009, 20, 1281–1295;
concentration of 10 m
The Pd-nanoparticles dissolved in 2-Propanol (25 µl; 100 m
M
and heated to the desired reaction temperature.
; 5 mol%)
[6]
[7]
[8]
[9]
J. M. Chalker, C. S. C. Wood, B. G. Davis, J. Am. Chem. Soc. 2009,
131, 16346–16347.
M
were added, giving a final 2-Propanol concentration of 0.5 % (v/v). The
reaction progress was monitored by RP-HPLC at 220 nm. After comple-
tion the Pd-nanoparticles were removed by centrifugation (10000 rpm; 10
min) and the reaction mixture was directly purified by preparative RP-
HPLC.
a) M. Frese, N. Sewald, Angew. Chem. Int. Ed. 2015, 54, 298–301; b)
C. Schnepel, N. Sewald, Chem. Eur. J. 2017, 23, 12064–12086;
D. R. M. Smith, T. Willemse, D. S. Gkotsi, W. Schepens, B. U. W. Maes,
S. Ballet, R. J. M. Goss, Org. Lett. 2014, 16, 2622–2625.
A. D. Roy, R. J. M. Goss, G. K. Wagner, M. Winn, Chem. Commun.)
2008, 4831–4833.
[10] T. Willemse, K. van Imp, R. J. M. Goss, van Vlijmen, H. W. T., W.
Schepens, B. U. W. Maes, S. Ballet, ChemCatChem 2015, 7, 2055–
2070.
General procedure for Solid-Phase Peptide Synthesis (GP2): All
peptides were synthesized on 2-chlorotrityl chloride resin using the
Fmoc/tBu-strategy. The resin was loaded with Fmoc-Ser(tBu)-OH (4 eq.)
and DIEA (8 eq.) in DCM at room temperature, shaken for 2 hours and
7
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
[11] M. Frese, C. Schnepel, H. Minges, H. Voß, R. Feiner, N. Sewald,
ChemCatChem 2016, 8, 1799–1803.
[12] J. Latham, J.-M. Henry, H. H. Sharif, B. R. K. Menon, S. A. Shepherd,
M. F. Greaney, J. Micklefield, Nat. Commun. 2016, 7, 11873.
[13] C. Schnepel, H. Minges, M. Frese, N. Sewald, Angew. Chem. Int. Ed.
2016, 55, 14159–14163.
[14] H. Gruß, C. Belu, L. M. Bernhard, A. Merschel, N. Sewald, Chem. Eur.
J. 2019, 25, 5880–5883.
[15] a) I. Kemker, R. C. Feiner, K. M. Müller, N. Sewald, ChemBioChem
2019; b) I. Kemker, C. Schnepel, D. C. Schröder, A. Marion, N. Sewald,
J. Med. Chem. 2019, 62, 7417–7430;
[16] S. V. Sharma, X. Tong, C. Pubill-Ulldemolins, C. Cartmell, E. J. A.
Bogosyan, E. J. Rackham, E. Marelli, R. B. Hamed, R. J. M. Goss, Nat.
Commun. 2017, 8, 229.
[17] Z. Gao, V. Gouverneur, B. G. Davis, J. Am. Chem. Soc. 2013, 135,
13612–13615.
[18] A. Dumas, A. Peramo, D. Desmaële, P. Couvreur, Chimia 2016, 70,
252–257.
[19] C. Liu, X. Li, Chem. Rec. 2016, 16, 84–97.
[20] B. Kurscheid, L. Belkoura, B. Hoge, Organometallics 2012, 31, 1329–
1334.
[21] a) S. R. Chowdhury, P. S. Roy, S. K. Bhattacharya, Nano-Structures &
Nano-Objects 2018, 14, 11–18; b) G. Cárdenas-Triviño, R. A. Segura, J.
Reyes-Gasga, Colloid Polym. Sci. 2004, 282, 1206–1212;
[22] M. A. Düfert, K. L. Billingsley, S. L. Buchwald, J. Am. Chem. Soc. 2013,
135, 12877–12885.
[23] a) N. Sewald, Peptides: Chemistry and Biology, 1st ed., Wiley-VCH,
Weinheim, 2002; b) Y. Yang in Side reactions in peptide synthesis,
Elsevier/Academic Press, London, 2016, 119-161.
[24] T. Michels, R. Dölling, U. Haberkorn, W. Mier, Org. Lett. 2012, 14,
5218–5221.
[25] M. S. Khalaf, S. H. Oakley, M. P. Coles, P. B. Hitchcock, Dalton Trans.
2010, 39, 1635–1642.
8
This article is protected by copyright. All rights reserved.

10.1002/chem.202002454
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents
Research Article
Gentle conditions: Ligand-free
solvent-stabilized Pd-nanoparticles
serve as a highly efficient catalyst at
ambient temperature for a sustaina-
ble and biocompatible Suzuki-
Miyaura cross-coupling between aryl
bromides and boronic acids under
mild, aerobic and aqueous condi-
tions.
Steffen Dachwitz, Dario H. Duwe,
Yating Hong Wang, Hendrik Gruß,
Yvonne Hannappel, Thomas Hell-
weg, and Norbert Sewald*
Page No. – Page No.
Suzuki-Miyaura Cross-Coupling of
Bromotryptophan Derivatives at
Ambient Temperature
Twitter: @Sewald_group
9
This article is protected by copyright. All rights reserved.