Solid-phase synthesis of biaryl cyclic peptides containing a histidine-tyrosine linkage

Article abstract of DOI:10.1016/j.tet.2019.03.014

A solid-phase strategy for the synthesis of biaryl cyclic peptides containing a side-chain to side-chain His-Tyr linkage was developed. The key step was the macrocyclization of a linear peptidyl resin incorporating a 5-bromohistidine and a 3-boronotyrosin

Full text of DOI:10.1016/j.tet.2019.03.014

Accepted Manuscript
Solid-phase synthesis of biaryl cyclic peptides containing a histidine-tyrosine linkage
Iteng Ng-Choi, Àngel Oliveras, Marta Planas, Lidia Feliu
PII:
S0040-4020(19)30289-3
https://doi.org/10.1016/j.tet.2019.03.014
TET 30200
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 1 February 2019
Revised Date: 4 March 2019
Accepted Date: 9 March 2019
Please cite this article as: Ng-Choi I, Oliveras À, Planas M, Feliu L, Solid-phase synthesis of biaryl
cyclic peptides containing a histidine-tyrosine linkage, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.03.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Solid-phase synthesis of biaryl cyclic
peptides containing a histidine-tyrosine
linkage
Leave this area blank for abstract info.
Iteng Ng-Choi, Àngel Oliveras, Marta Planas* and Lidia Feliu*
LIPPSO, Departament de Química, Universitat de Girona, Spain

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Solid-phase synthesis of biaryl cyclic peptides containing a histidine-tyrosine linkage
Iteng Ng-Choia, Àngel Oliverasa, Marta Planasa,* and Lidia Feliua,*
aLaboratori d’Innovació en Processos i Productes de Síntesi Orgànica (LIPPSO), Departament de Química, Universitat de Girona, Maria Aurèlia Capmany 69,
17003 Girona, Spain
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A solid-phase strategy for the synthesis of biaryl cyclic peptides containing a side-chain to side-
chain His-Tyr linkage was developed. The key step was the macrocyclization of a linear peptidyl
resin incorporating a 5-bromohistidine and a 3-boronotyrosine via the formation of the biaryl
bond by means of a microwave-assisted Suzuki-Miyaura reaction. This method allowed direct
access to biaryl cyclic peptides containing a 3- or 5-amino acid ring and bearing the histidine
residue at the N- or the C-terminus, being especially conducive for analogues in which this
amino acid is located at the C-terminus. This study also served to establish a strategy for the
synthesis of biaryl cyclic peptides derived from the two hemispheres of the natural biaryl
bicyclic peptides aciculitins.
Available online
Keywords:
Cross-coupling
Cyclization
Macrocycles
Microwave chemistry
Suzuki-Miyaura
2009 Elsevier Ltd. All rights reserved
.
1. Introduction
In this context, the synthesis of biaryl cyclic peptides
containing a 5-arylhistidine is especially noteworthy because the
arylation of the 4(5)-position of the imidazole ring has proven to
be difficult. So far, several methods based on transition metal-
catalyzed reactions have been developed for the synthesis in
solution of 4(5)-arylimidazoles, but most of them provide
moderate yields and/or require drastic conditions [25-36].
Regarding 5-arylhistidine derivatives, to the best of our
knowledge, their synthesis in solution has only been described in
two reports through microwave-assisted Pd-catalyzed direct C-H
activation [37] or a Suzuki-Miyaura cross-coupling [38]. Taking
advantage of the solid supported chemistry, we extended the
latter methodology to the solid-phase preparation of 5-
arylhistidine-containing peptides via arylation of a resin-bound 5-
bromohistidine residue with commercially available arylboronic
acids [39,40]. It was observed that microwaves were crucial to
enhance the arylation of histidine, shortening the reaction time
and providing higher overall purities [38]. This strategy enabled
the first solid-phase synthesis of biaryl linear tri- and
tetrapeptides containing a 5-arylhistidine [40]. Later, we applied
this approach to the modification of lead antimicrobial peptides
with activity against plant pathogens of economic importance
[39]. The resulting 5-arylhistidine-containing peptides displayed
antibacterial and antifungal activity, and low hemolysis. This low
cytotoxicity was attributed to the presence of the imidazole ring
of histidine.
Over the last decades, much attention has been focused on
biaryl cyclic peptides containing an aryl-aryl bond between the
side chains of two aromatic amino acids. The interest in this type
of peptides has risen because they exhibit interesting biological
activities, in which the biaryl motif plays a crucial role [1].
Among these biaryl moieties, aryltyrosines are found in simple
peptides as well as in complex macrocycles, such as the
antimicrobial peptides arylomycins [2,3], the proteasome
inhibitor TMC-95 [4-7], the neurotensin antagonist RP-66453
[8,9], or the antibiotic vancomycin [10,11]. Similarly,
arylhistidines are present in the active site of heme-copper
oxidases, and in cytotoxic and antifungal marine peptides, such
as aciculitins [12-14].
Biaryl cyclic peptides are also significant from a structural
point of view, constituting an important synthetic challenge [1].
The formation of the biaryl bond has been achieved via a Suzuki-
Miyaura cross-coupling reaction, a Pd-catalyzed C-H activation
or a Cu-mediated oxidative coupling. The former reaction has
allowed the synthesis of peptides containing a Trp-Tyr [4,15], a
Phe-Phe [15-18], a Phe-Tyr [15,17,19] or a Tyr-Tyr [15,19-21]
linkage. The C-H activation reaction has been used to form the
biaryl bonds Trp-Phe and Trp-Tyr [22,23], while the Cu-
mediated oxidative phenol coupling has provided access to
peptides bearing a Tyr-hydroxyphenylglycine motif [24]. Despite
the benefits of the solid supported chemistry, most of these
reactions have been performed in solution. In fact, the solid-
phase synthesis of biaryl peptides has been scarcely reported.
This methodology has only been applied by four research groups
to obtain cyclic peptides featuring a covalent bond Phe-Phe [16-
18], Phe-Tyr [17,19], Tyr-Tyr [19], Trp-Phe or Trp-Tyr [22].

2
Tetrahedron
ACCEPTED MANUSCRIPT
by their cyclization through a microwave-assisted Suzuki-
Within our current interest on biaryl cyclic peptides, we
Miyaura cross-coupling (Scheme 1). Synthesis of the
regioisomeric peptidyl resins Boc-His(5-Br,1-SEM)-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA (1a) and
Boc-His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-
Leu-Leu-Rink-MBHA (1b) started from a Fmoc-Rink-MBHA
resin. First, the iodopeptidyl resin Fmoc-Lys(Boc)-Lys(Boc)-
Leu-Tyr(3-I,Me)-Leu-Leu-Rink-MBHA was obtained following
a standard 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu)
strategy through sequential Fmoc removal and coupling steps.
The Fmoc group was removed using piperidine/DMF (3:7) (2 +
10 min). Couplings of Fmoc-Leu-OH and Fmoc-Lys(Boc)-OH
were mediated by N,N-diisopropylcarbodiimide (DIPCDI) and
ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) in DMF for 1 h.
Fmoc-Tyr(3-I,Me)-OH (2) was anchored using 1-[(1-(cyano-2-
ethoxy-2-oxoethylylidineaminooxy)dimethylaminomorpholino)]
uronium hexafluorophosphate (COMU), Oxyma and N,N-
diisopropylethylamine (DIEA) in DMF overnight. This tyrosine
derivative was synthesized in solution from Boc-Tyr(3-I,Me)-
OMe [40] through subsequent removal of the tert-
butoxycarbonyl (Boc) group, methyl ester hydrolysis, and Fmoc
protection of the Nα-amino group.
envisioned that the above strategy devised for the preparation of
linear peptides containing a 5-arylhistidine could be applied to
the solid-phase synthesis of biaryl macrocyclic sequences
incorporating a biaryl bond between the side-chain of a histidine
and of a tyrosine. To the best of our knowledge, the synthesis of
5-arylhistidine-containing cyclic peptides has not yet been
reported. Thus, the main purpose of this work was the solid-
phase preparation of biaryl cyclic peptides I and II, which
contain a biaryl linkage between the 5-position of the imidazole
of the histidine and the 3-position of the phenyl group of the
tyrosine (Fig. 1). The histidine is located at the N-terminus in
biaryl cyclic peptides with general structure I, whereas
compounds II bear this residue at the C-terminus. The size of the
ring is changed by incorporating different combinations of
lysines and leucines, as representative cationic and hydrophobic
amino acids. The synthesis of these compounds could be
accomplished through the cyclization of a linear peptidyl resin
via the formation of the biaryl bond under the conditions of the
Suzuki-Miyaura reaction. Herein, the feasibility of this strategy
to prepare biaryl cyclic peptides I and II of different ring sizes
was studied.
Once the iodopeptidyl resin Fmoc-Lys(Boc)-Lys(Boc)-Leu-
Tyr(3-I,Me)-Leu-Leu-Rink-MBHA was obtained, the Fmoc
group was replaced by a trityl (Tr) group due to the instability of
the former to the basic Miyaura borylation conditions (Scheme
1). After Fmoc removal, the Nα-amino group of the resin was
protected by treatment with trityl chloride (TrCl) in presence of
DIEA for 4 h. An aliquot of the resulting resin Tr-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-I,Me)-Leu-Leu-Rink-MBHA
(3)
was
exposed to trifluoroacetic acid (TFA)/H2O/triisopropylsilane
(TIS) (95:2.5:2.5) for 2 h, affording H-Lys-Lys-Leu-Tyr(3-I,Me)-
Leu-Leu-NH2 in 84% HPLC purity, which was characterized by
mass spectrometry.
Fig. 1. General structure of biaryl cyclic peptides I and II.
2. Results and discussion
Next, borylation of the iodopeptidyl resin 3 led to Tr-
Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA
(4). This reaction was performed under the conditions previously
described in our group [19], which involved the use of
bis(pinacolato)diboron (B2Pin2) (4 equiv.), PdCl2(dppf) (0.18
equiv.), 1,1’-bis(diphenylphosphanyl)ferrocene (dppf) (0.09
equiv.), and KOAc (6 equiv.) in anhydrous DMSO at 80 ºC for 8
h (Scheme 1). It was observed that longer reaction times
promoted the protodeborylation of the tyrosine residue of the
boronopeptidyl resin 4. Acidolytic cleavage of an aliquot of 4
gave the boronopeptide H-Lys-Lys-Leu-Tyr(3-B(OH)2,Me)-Leu-
Leu-NH2 in 81% HPLC purity. This boronic acid resulted from
the hydrolysis of the pinacol boronic ester group during HPLC
analysis, which was confirmed by mass spectrometry.
2.1. Biaryl cyclic peptides containing a histidine residue at the
N-terminus
We first investigated the solid-phase preparation of biaryl
cyclic peptides BPC782, BPC784, and BPC786 containing a
side-chain to side-chain His-Tyr linkage with the histidine
residue located at the N-terminus (Fig. 2).
The trityl group of the resin-bound tyrosine boronic ester 4
was then selectively removed with TFA/H2O/CH2Cl2 (0.2:1:98.8)
(2×1 + 2×20 min). Finally, the regioisomeric mixture of Boc-
His(5-Br,1-SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b),
prepared as previously reported [40], was coupled using DIPCDI
and Oxyma in DMF for 3 h to yield the expected regioisomeric
peptidyl resins 1. An aliquot of 1 was treated with TFA/H2O/TIS
(95:2.5:2.5) under stirring for 3 h. H-His(5-Br)-Lys-Lys-Leu-
Tyr(3-B(OH)2,Me)-Leu-Leu-NH2 (6) was obtained in 75% HPLC
purity, and was characterized by mass spectrometry.
Fig. 2. Structure of biaryl cyclic peptides containing a His at the N-terminus.
In particular, we first assayed the synthesis of BPC786 which
comprises a 5-residue ring and a Leu-Leu spacer at the C-
terminus. The synthetic strategy involved the preparation of the
linear peptidyl resins 1, incorporating a 5-bromohistidine at the
N-terminus and a 3-boronotyrosine at the C-terminus, followed

3
O
O
H
ACCEPTED MANUSCRIPT
i, ii, i, ii,
i, iii
FmocHN
N
i, ii
Fmoc-Lys(Boc)-Lys(Boc)-Leu
Leu-Leu-Rink-MBHA
Leu-Leu-Rink-MBHA
Fmoc-Rink-MBHA
OMe
I
OMe
I
O
O
H
H
N
N
Tr-Lys(Boc)-Lys(Boc)-Leu
Tr-Lys(Boc)-Lys(Boc)-Leu
Leu-Leu-Rink-MBHA
Leu-Leu-Rink-MBHA
v
i, iv
,
OMe
I
OMe
3
4
B
O
O
O
O
H
BocHN
N
H2N
Leu-Leu-Rink-MBHA
OMe
Lys(Boc)-Lys(Boc)-Leu
N
1a
H
H2N
N
Br
N
HN
NH
B
SEM
O
O
O
O
N
NH
O
O
H
O
vi, vii
viii, ix
N
NH2
+
N
H
O
O
O
O
H
BocHN
Br
N
H2N
Leu-Leu-Rink-MBHA
OMe
Lys(Boc)-Lys(Boc)-Leu
1b
HN
OMe
NSEM
BPC786
N
B
O
O
Scheme 1. Solid-phase synthesis of the biaryl cyclic peptide BPC786. Reagents and conditions: (i) Piperidine/DMF (3:7) (2 + 10 min). (ii) Fmoc-Leu-OH or
Fmoc-Lys(Boc)-OH, DIPCDI, Oxyma, DMF, 1 h. (iii) Fmoc-Tyr(3-I,Me)-OH (2), COMU, Oxyma, DIEA, DMF, overnight. (iv) TrCl, DIEA, DMF, 4 h. (v)
B2Pin2, PdCl2(dppf), dppf, KOAc, DMSO, 80 ºC, 8 h. (vi) TFA/H2O/CH2Cl2. (0.2:1:98.8) (2×1 + 2×20 min). (vii) Boc-His(5-Br,1-SEM)-OH (5a) and Boc-His(5-
Br,3-SEM)-OH (5b), DIPCDI, Oxyma, DMF, 3 h. (viii) Pd2(dba)3, KF, SPhos or P(o-tolyl)3, DME/EtOH/H2O, MW, 140 ºC, 30 min. (ix) TFA/H2O/TIS
(95:2.5:2.5), 3 h, stirring.
With the linear peptidyl resins
1
in hand, their
7 and 8 were synthesized following the protocol described for
peptidyl resins 1. Accordingly, iodopeptidyl resins Tr-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-I,Me)-Rink-MBHA (9) and Tr-Leu-
Tyr(3-I,Me)-Rink-MBHA (10) were prepared. Acidolytic
cleavage of an aliquot of 9 and 10 provided the corresponding
iodopeptides in 87 and 72% HPLC purity, respectively.
Borylation of 9 and 10, followed by trityl group removal and
subsequent coupling of bromohistidines 5 yielded the expected
peptidyl resins 7 and 8, respectively. Treatment of an aliquot of
these resins with TFA/H2O/TIS (95:2.5:2.5) led to H-His(5-Br)-
Lys-Lys-Leu-Tyr(3-B(OH)2,Me)-NH2 (11) and H-His(5-Br)-Leu-
Tyr(3-B(OH)2,Me)-NH2 (12) in 60% HPLC purity which were
characterized by mass spectrometry.
macrocyclization was initially attempted under the conditions of
the Suzuki-Miyaura cross-coupling (Scheme 1). Thus, resins 1
were first exposed to Pd2(dba)3 (0.2 equiv.), P(o-tolyl)3 (0.4
equiv.), and KF (4 equiv.) in degassed 1,2-dimethoxyethane
(DME)/EtOH/H2O (9:9:2) under microwave irradiation at 140 ºC
for 30 min. The resulting resin was cleaved, and HPLC and ESI-
MS analysis of the crude reaction mixture revealed the formation
of the expected biaryl cyclic peptide BPC786 (30% purity)
together with the protodeborylated and debrominated derivative
H-His-Lys-Lys-Leu-Tyr(Me)-Leu-Leu-NH2 (29% purity),
a
common byproduct of this reaction [41]. The cyclization was also
assayed using SPhos [42-45] instead of P(o-tolyl)3. Giralt and co-
workers reported that the presence of Buchwald’s SPhos ligand
avoids racemization of α-amino acids such as tyrosine
derivatives in Suzuki-Miyaura reactions [42]. Under these
conditions a similar result was obtained, BPC786 was formed in
31% purity. H-His-Lys-Lys-Leu-Tyr(Me)-Leu-Leu-NH2 and the
oxidized and debrominated byproduct H-His-Lys-Lys-Leu-
Tyr(3-OH,Me)-Leu-Leu-NH2 were also detected (20 and 11%
purity, respectively). BPC786 was then isolated by reverse-phase
column chromatography, analyzed and characterized by HPLC
and mass spectrometry, being obtained in 79% purity.
Cyclization of 7 and 8 was performed through a microwave-
assisted intramolecular Suzuki-Miyaura reaction under the
conditions described above for 1 using SPhos as ligand. After
cleavage of the resin resulting from the cyclization of 7, HPLC
and mass spectrometry analysis of the crude reaction mixture
revealed the formation of the biaryl cyclic peptide BPC782 as
major product in 35% purity, together with a mixture of the
common Suzuki-Miyaura arylation byproducts. After column
chromatography purification, BPC782 was obtained in 68%
HPLC purity and was characterized by mass spectrometry.
Similarly, cyclization of 8 led to the formation of BPC784 in
30% purity, which was purified and obtained in 90% purity. Its
structure was confirmed by mass spectrometry. Thus, the
synthesis of BPC782, BPC784 and BPC786 revealed that the
cyclization is not influenced by the presence of a Leu-Leu spacer
and that similar results are obtained for the formation of a 3- or 5-
residue ring.
In view of these results, we then studied the application of this
methodology to the synthesis of biaryl cyclic peptides BPC782
and BPC784 (Scheme 2). The former is a BPC786 analogue that
does not contain the Leu-Leu spacer at the C-terminus while
BPC784 consists of a 3-residue ring. The synthesis of BPC782
required the preparation of the linear peptidyl resins Boc-His(5-
Br,1-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-Rink-
MBHA (7a) and Boc-His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-
Tyr(3-BPin,Me)-Rink-MBHA (7b) whereas the linear precursors
of BPC784 were resins Boc-His(5-Br,1-SEM)-Leu-Tyr(3-
BPin,Me)-Rink-MBHA (8a) and Boc-His(5-Br,3-SEM)-Leu-
Tyr(3-BPin,Me)-Rink-MBHA (8b). These linear peptidyl resins

4
Tetrahedron
ACCEPTED MANUSCRIPT
Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-I,Me)-Rink-MBHA (9)
Tr-Leu-Tyr(3-I,Me)-Rink-MBHA (10)
i, ii, iii
i, ii, iii
Boc-His(5-Br,1-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (7a)
Boc-His(5-Br,1-SEM)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (8a)
+
+
Boc-His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (7b)
Boc-His(5-Br,3-SEM)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (8b)
iv, v
iv, v
H2N
O
H
O
H2N
N
H
H2N
N
N
NH
HN
NH
NH2
O
N
NH
O
O
NH2
O
NH
MeO
O
O
H2N
BPC784
HN
OMe
BPC782
Scheme 2. Solid-phase synthesis of biaryl cyclic peptides BPC782 and BPC784. Reagents and conditions: (i) B2Pin2, PdCl2(dppf), dppf, KOAc, DMSO, 80 ºC, 8
h. (ii) TFA/H2O/CH2Cl2 (0.2:1:98.8) (2×1 + 2×20 min). (iii) Boc-His(5-Br,1-SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b), DIPCDI, Oxyma, DMF, 3 h.
(iv) Pd2(dba)3, KF, SPhos, DME/EtOH/H2O, MW, 140 ºC, 30 min. (v) TFA/H2O/TIS (95:2.5:2.5), 3 h, stirring.
2.2. Biaryl cyclic peptides containing a histidine residue at the
C-terminus
(TMSOTf) in presence of 2,6-lutidine (10×30 min) [46]. Peptide
elongation was carried out by sequential coupling and
deprotection steps using Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH and
Boc-Tyr(3-B(OH)2,Me)-OH (14) as amino acid derivatives.
Boronotyrosine 14 was prepared in solution through Miyaura
borylation of Boc-Tyr(3-I,Me)-OMe [40], followed by hydrolysis
of the pinacolate and saponification of the methyl ester. It should
be noticed that coupling of 14 to the solid support was mediated
by DIPCDI and Oxyma in DMF and should not exceed a 3 h
reaction time in order to avoid side products. An aliquot of the
resulting resins 13 was treated with TFA/H2O/TIS (95:2.5:2.5)
under stirring for 3 h to provide H-Tyr(3-B(OH)2,Me)-Lys-Lys-
Leu-His(5-Br)-Leu-Leu-NH2 (15) in 65% HPLC purity, which
was characterized by mass spectrometry.
We next turned our attention to the solid-phase synthesis of
biaryl cyclic peptides BPC788, BPC790 and BPC792 in which
the histidine residue was located at the C-terminus (Fig. 3).
The cyclization of the regioisomeric peptidyl resins 13 was
carried out through a Suzuki-Miyaura arylation under the
conditions used for the synthesis of biaryl cyclic peptides bearing
a histidine at the N-terminus which involved treatment with
Pd2(dba)3, SPhos, and KF under microwave irradiation at 140 ºC
for 30 min (Scheme 3). HPLC and mass spectrometry analysis of
the crude reaction mixture obtained from the acidolytic cleavage
of the resulting resin indicated the presence of the desired biaryl
cyclic peptide BPC792 as major product (55% purity) together
with the debrominated and protodeborylated byproduct H-
Tyr(Me)-Lys-Lys-Leu-His-Leu-Leu-NH2 (22% purity). BPC792
was finally purified by reverse-phase column chromatography
being obtained in 92% HPLC purity, and characterized by mass
spectrometry.
Fig.3. Structure of biaryl cyclic peptides containing a His at the C-terminus.
The preparation of BPC792, incorporating a 5-residue ring
and a Leu-Leu spacer at the C-terminus, was first investigated. A
similar strategy to that of biaryl cyclic peptides that contain the
histidine residue at the N-terminus was followed (Scheme 3). In
this case, the linear peptidyl resins Boc-Tyr(3-B(OH)2,Me)-
Lys(Boc)-Lys(Boc)-Leu-His(5-Br,1-SEM)-Leu-Leu-Rink-
Synthesis of BPC788, a BPC792 analog lacking the Leu-Leu
spacer, and of BPC790, consisting of a 3-residue ring, was
performed following the same synthetic approach used for
BPC792 (Scheme 4). The corresponding linear peptidyl resins 16
and 17 were prepared as described for 13. After acidolytic
cleavage of an aliquot of each resin, the corresponding expected
linear peptides 18 and 19 were detected in 59% and 52% HPLC
purity, respectively.
MBHA (13a) and Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-Lys(Boc)-
Leu-His(5-Br,3-SEM)-Leu-Leu-Rink-MBHA
(13b),
incorporating a 3-boronotyrosine at the N-terminus and a 5-
bromohistidine at the C-terminus, were required. First, Fmoc-
Leu-Leu-Rink-MBHA was synthesized following an Fmoc/tBu
strategy as previously described. After Fmoc removal, Boc-
His(5-Br,1-SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b)
were coupled using COMU, Oxyma and DIEA in DMF
overnight. Then, the Boc group was selectively removed under
mild conditions by treatment with trimethylsilyltriflate

5
O
ACCEPTED MANUSCRIPT
BocHN
O
BocHN
Leu-Leu-Rink-MBHA
Leu-Leu-Rink-MBHA
NSEM
i, ii, i, ii
i, iii
Fmoc-Leu-Leu-Rink-MBHA
Fmoc-Rink-MBHA
+
N
Br
Br
N
N
SEM
O
H
O
NH2
N
BocHN
Lys(Boc)-Lys(Boc)-Leu
Leu-Leu-Rink-MBHA
N
H2N
H
N
OMe
OH
Br
N
HN
NH
SEM
B
HO
O
O
iv, ii, i, v,
i, v, i, vi
O
13a
O
O
NH
NH2
vii, viii
H
N
+
N
O
O
H
O
H
N
O
BocHN
H2N
Lys(Boc)-Lys(Boc)-Leu
Leu-Leu-Rink-MBHA
NSEM
NH
N
OMe
OMe
B
Br
N
HO
OH
BPC792
13b
Scheme 3. Solid-phase synthesis of biaryl cyclic peptide BPC792. Reagents and conditions: (i) Piperidine/DMF (3:7) (2 + 10 min). (ii) Fmoc-Leu-OH, DIPCDI,
Oxyma, DMF, 1 h. (iii) Boc-His(5-Br,1-SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b), COMU, Oxyma, DIEA, DMF, overnight. (iv) TMSOTf, 2,6-
lutidine, CH2Cl2 (10×30 min). (v) Fmoc-Lys(Boc)-OH, DIPCDI, Oxyma, DMF, 1 h. (vi) Boc-Tyr(3-B(OH)2,Me)-OH (14), DIPCDI, Oxyma, DMF, 3 h. (vii)
Pd2(dba)3, KF, SPhos, DME/EtOH/H2O, MW, 140 ºC, 30 min. (viii) TFA/H2O/TIS (95:2.5:2.5), 3 h, stirring.
Fmoc-Rink-MBHA
i, ii
Fmoc-Rink-MBHA
i, ii
Boc-His(5-Br,1-SEM)-Rink-MBHA
Boc-His(5-Br,1-SEM)-Rink-MBHA
+
+
Boc-His(5-Br,3-SEM)-Rink-MBHA
Boc-His(5-Br,3-SEM)-Rink-MBHA
iii, iv, i, v, i, v, i, vi
iii, iv, i, vi
Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-Lys(Boc)-Leu-His(5-Br,1-SEM)-Rink-MBHA (16a)
Boc-Tyr(3-B(OH)2,Me)-Leu-His(5-Br,1-SEM)-Rink-MBHA (17a)
+
+
Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-Lys(Boc)-Leu-His(5-Br,3-SEM)-Rink-MBHA (16b)
Boc-Tyr(3-B(OH)2,Me)-Leu-His(5-Br,3-SEM)-Rink-MBHA (17b)
vii, viii
vii, viii
NH2
O
O
NH
HN
H2N
O
NH2
H
N
HN
NH
H2N
O
O
O
NH
NH
N
O
NH
O
OMe
NH2
H2N
BPC790
N
OMe
BPC788
Scheme 4. Solid-phase synthesis of biaryl cyclic peptides BPC788 and BPC790. Reagents and conditions: (i) Piperidine/DMF (3:7) (2 + 10 min). (ii) Boc-His(5-
Br,1-SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b), COMU, Oxyma, DIEA, DMF, overnight. (iii) TMSOTf, 2,6-lutidine, CH2Cl2 (10×30 min). (iv) Fmoc-
Leu-OH, DIPCDI, Oxyma, DMF, 1 h. (v) Fmoc-Lys(Boc)-OH, DIPCDI, Oxyma, DMF, 1 h. (vi) Boc-Tyr(3-B(OH)2,Me)-OH (14), DIPCDI, Oxyma, DMF, 3 h.
(vii) Pd2(dba)3, KF, SPhos, DME/EtOH/H2O, MW, 140 ºC, 30 min. (viii) TFA/H2O/TIS (95:2.5:2.5), 3 h, stirring.
The intramolecular Suzuki-Miyaura cross-coupling of 16 and
17 afforded the expected biaryl cyclic peptides BPC788 and
BPC790, respectively. BPC788 was detected in 32% HPLC
purity together with a mixture of the byproducts H-Tyr(Me)-Lys-
Lys-Leu-His-NH2 (15% purity) and H-Tyr(3-OH,Me)-Lys-Lys-
Leu-His-NH2 (5% purity). Interestingly, BPC790 was
successfully obtained (49% HPLC purity) and only traces of the
common byproducts were detected. Column chromatography
purification provided these biaryl cyclic peptides in 87 and 96%
HPLC purity, respectively, and they were characterized by mass
spectrometry. These results pointed out that the macrocyclization
through formation of a His-Tyr linkage is favored when the
histidine residue is located at the C-terminus.

6
Tetrahedron
ACCEPTED MANUSCRIPT
Fmoc-Rink-MBHA
i, ii, i, iii
2.3. Biaryl cyclic peptides derived from the northern and
southern hemispheres of aciculitins
Taking into account the biological properties of aciculitins as
well as their synthetic interest [12-14], we decided to extend the
above methodology to the solid-phase synthesis of analogues of
the northern and southern hemisphere of these bicyclic peptides
(Fig. 4). In particular, we focused our attention on the preparation
of the northern hemisphere analog 20 and of the southern
hemisphere derivative 21.
Boc-His(5-Br,1-SEM)- Ala-Rink-MBHA
+
Boc-His(5-Br,3-SEM)- Ala-Rink-MBHA
iv, v, i, vi, i, v, i, vii, i, viii
Boc-Tyr(3-B(OH)2,Me)-Ala-Gln(Tmob)-Gly-Gln(Tmob)-His(5-Br,1-SEM)- Ala-Rink-MBHA (22a)
+
Boc-Tyr(3-B(OH)2,Me)-Ala-Gln(Tmob)-Gly-Gln(Tmob)-His(5-Br,3-SEM)- Ala-Rink-MBHA (22b)
ix, x
O
H2N
O
H
O
O
N
HN
O
HN
HN
N
OMe
NH2
HN
O
O
NH
HN
O
O
NH2
NH2
20
Scheme 5. Solid-phase synthesis of the biaryl cyclic peptide 20. Reagents and
conditions: (i) Piperidine/DMF (3:7) (2 + 10 min). (ii) Fmoc-βAla-OH,
DIPCDI, Oxyma, DMF, 1 h. (iii) Boc-His(5-Br,1-SEM)-OH (5a) and Boc-
His(5-Br,3-SEM)-OH (5b), COMU, Oxyma, DIEA, DMF, overnight. (iv)
TMSOTf, 2,6-lutidine, CH2Cl2 (10×30 min). (v) Fmoc-Gln(Tmob)-OH,
DIPCDI, Oxyma, DMF, 1 h. (vi) Fmoc-Gly-OH, DIPCDI, Oxyma, DMF, 1 h.
Fig. 4. Structure of aciculitins A-C and of biaryl cyclic peptides 20 and 21.
Biaryl cyclic peptides 20 and 21 were designed based on
(vii) Fmoc-Ala-OH, DIPCDI, Oxyma, DMF,
1 h. (viii) Boc-Tyr(3-
commercially available
L-amino acids. In particular, the 3-
B(OH)2,Me)-OH, DIPCDI, Oxyma, DMF, 3 h. (ix) Pd2(dba)3, KF, SPhos,
DME/EtOH/H2O, MW, 140 ºC, 30 min. (x) TFA/H2O/TIS (95:2.5:2.5), 3 h,
stirring.
hydroxyglutamine, the 2,3-diaminobutyric acid and the 2-amino-
2-butenoic acid residues were replaced by a glutamine, a β-
alanine and an alanine, respectively.
the peptidyl resins Boc-His(5-Br,1-SEM)-βAla-Thr(tBu)-Tyr(3-
BPin,Me)-Ala-Gln(Tmob)-Rink-MBHA (24a) and Boc-His(5-
Br,3-SEM)-βAla-Thr(tBu)-Tyr(3-BPin,Me)-Ala-Gln(Tmob)-
Rink-MBHA (24b), containing a 5-bromohistidine at the N-
terminus and a 3-boronotyrosine at the C-terminus (Scheme 6).
To achieve this objective, we first synthesized the peptidyl resin
Tr-βAla-Thr(tBu)-Tyr(3-I,Me)-Ala-Gln(Tmob)-Rink-MBHA
(25) following the strategy used for resins 3, 9 and 10. An aliquot
of the resulting resin 25 was treated with a mixture of
TFA/H2O/TIS (95:2.5:2.5) for 2 h, affording the expected
iodopeptide H-βAla-Thr-Tyr(3-I,Me)-Ala-Gln-NH2 in 87%
purity.
We initially tested the synthesis of the biaryl cyclic peptide 20
which contains a histidine residue at the C-terminus. The β-
alanine was chosen as anchoring point to the solid support. This
peptide was synthesized according to the strategy described
above for peptides BPC788, BPC790 and BPC792. Thus, the
regioisomeric heptapeptidyl resins Boc-Tyr(3-B(OH)2,Me)-Ala-
Gln(Tmob)-Gly-Gln(Tmob)-His(5-Br,1-SEM)-βAla-Rink-
MBHA (22a) and Boc-Tyr(3-B(OH)2,Me)-Ala-Gln(Tmob)-Gly-
Gln(Tmob)-His(5-Br,3-SEM)-βAla-Rink-MBHA (22b) were
prepared (Scheme 5). An aliquot of these peptidyl resins was
acidolytically cleaved, yielding H-Tyr(3-B(OH)2,Me)-Ala-Gln-
Gly-Gln-His(5-Br)-βAla-NH2 (23) in >99% purity, which was
characterized by mass spectrometry.
Then, resin 25 was exposed to bis(pinacolato)diboron (B2Pin2)
(4
equiv.),
PdCl2(dppf)
(0.18
equiv.),
1,1’-
These resins were then subjected to the Suzuki-Miyaura
macrocyclization reaction as described above. HPLC and mass
spectrometry analysis of the crude reaction mixture showed the
presence of the desired biaryl cyclic peptide 20 in 64% purity.
Two peaks appeared in the HPLC chromatogram, both
corresponding to 20 as demonstrated by HPLC-MS analysis of
the crude reaction mixture carried out at different temperatures
(20 to 60 ºC). These two peaks coalesced at 40 and 60 ºC which
pointed out that they could be attributed to two different
conformers. This peptide was purified by column
chromatography, being obtained in 87% purity and characterized
by mass spectrometry.
bis(diphenylphosphanyl)ferrocene (dppf) (0.09 equiv.), and
KOAc (6 equiv.) in anhydrous DMSO at 80 ºC for 8 h (Scheme
4). Acidolytic cleavage of an aliquot of the resulting resin 26
gave H-βAla-Thr-Tyr(3-B(OH)2,Me)-Ala-Gln-NH2 in 62%
purity. Selective removal of the trityl group, followed by
coupling of the regioisomeric histidine derivatives 5 afforded the
expected peptidyl resins 24 which were subjected to Suzuki-
Miyaura macrocyclization. Acidolytic cleavage of these resins
rendered the expected biaryl cyclic peptide 21 in 12% purity, as
confirmed by mass spectrometry. This peptide was purified by
reverse-phase column chromatography, being obtained in 84%
HPLC purity, and characterized by mass spectrometry.
In the case of the biaryl cyclic peptide 21, bearing a histidine
at the N-terminus, the glutamine residue was selected as the
anchoring point. Thus, the synthesis involved the preparation of

7
Analysis was carried out with a Kromasil 100 C18 (4.6 mm ×
ACCEPTED MANUSCRIPT
250 mm, 5 ꢀm) column with 2–100% B over 28 min at a flow
rate of 1 mL/min. Conditions C: Analysis was carried out with a
Kromasil 100 C18 (4.6 mm × 250 mm, 5 µm) column with 2–25%
B over 3 min followed by 25-35% B over 30 min and 35-100% B
over 1 min at a flow rate of 1 mL/min. Peptides were also
analyzed with a 1260 Infinity II liquid chromatography
instrument (Agilent Technologies) composed of a Diode Array
Detector HS, a Quaternary Pump VL, a 1260 Vial sampler and
OpenLab CDS ChemStation software. Conditions D: Analysis
was carried out with a linear gradient of 2–100% B over 12 min
at a flow rate of 1 mL/min. Conditions E: Analysis was carried
out with a linear gradient of 2–100% B over 12 min at a flow rate
of 1 mL/min at 40 ºC.
Peptide purifications were performed on a CombiFlash Rf200
automated flash chromatography system using RediSep Rf Gold
reversed-phase C18 column packed with high performance C18
derivatized silica.
Scheme 6. Solid-phase synthesis of the biaryl cyclic peptide 21. Reagents and
conditions: (i) B2Pin2, PdCl2(dppf), dppf, KOAc, DMSO, 80 ºC., 8 h. (ii)
TFA/H2O/CH2Cl2 (0.2:1:98.8), (2×1 + 2×20 min). (iii) Boc-His(5-Br,1-
SEM)-OH (5a) and Boc-His(5-Br,3-SEM)-OH (5b), DIPCDI, Oxyma, DMF,
3 h. (iv) Pd2(dba)3, SPhos, KF, DME/EtOH/H2O, MW, 140 ºC, 30 min. (v)
TFA/H2O/TIS (95:2.5:2.5), 3 h, stirring.
ESI-MS analyses were performed at the Serveis Tècnics de
Recerca of the University of Girona with an Esquire 6000 ESI
ion Trap LC/MS (Bruker Daltonics) instrument equipped with an
electrospray ion source. The instrument was operated in the
positive ESI(+) ion mode. Samples (5 ꢀL) were introduced into
the mass spectrometer ion source directly through an HPLC
autosampler. The mobile phase (80:20 CH3CN/H2O at a flow rate
of 100 ꢀL/min) was delivered by a 1100 Series HPLC pump
(Agilent). Nitrogen was employed as both the drying and
nebulising gas. Nitrogen was employed as both the drying and
nebulising gas.
The synthesis of 20 and 21 confirmed that the cyclization
through the formation of a biaryl bond His-Tyr via a Suzuki-
Miyaura cross-coupling gives better results when the histidine
residue is located at the C-terminus. This study constitutes the
first approach towards the synthesis of the naturally occurring
biaryl bicyclic peptides aciculitins.
HRMS were recorded on
a Bruker MicroTof-QIITM
3. Conclusions
instrument using an electrospray ionization source at the Serveis
Tècnics de Recerca of the University of Girona. Samples were
introduced into the mass spectrometer ion source by direct
infusion using a syringe pump and were externally calibrated
using sodium formate. The instrument was operated in the
positive ion mode.
1H and 13C NMR spectra were measured with a Bruker 300 or
400 MHz NMR spectrometer at the Serveis Tècnics de Recerca
of the University of Girona. Chemical shifts were reported as δ
values (ppm) directly referenced to the solvent signal.
In summary, we describe the first solid-phase synthesis of
biaryl cyclic peptides containing a His-Tyr linkage. The key
feature of our synthetic methodology is the cyclization via the
formation of a biaryl bond between a 5-bromohistidine and a 3-
boronotyrosine. This strategy allowed the preparation of biaryl
cyclic peptides incorporating a 3- or 5-residue ring and with the
histidine residue at the N- or the C-terminus. Best results were
obtained when the histidine was at the C-terminus. In addition,
this study was extended to the synthesis of analogues of the
northern and southern hemispheres of aciculitins. We envisaged
that this methodology could be considered a useful starting point
for the total synthesis of aciculitins or of other biaryl bicyclic
peptides.
Microwave-assisted reactions were performed with a single
mode Discover S-Class labstation microwave (CEM) (0-300 W).
The time, temperature, and power were controlled with the
Synergy software. The temperature was monitored through an
infrared sensor in the floor of the cavity.
4. Experimental section
4.1. General information
4.2. Synthesis of methyl 3-iodo-4-methoxy-L-tyrosinate
Manual peptide synthesis was performed in polypropylene
syringes fitted with a polyethylene porous disk. Solvents and
soluble reagents were removed by suction. Most chemicals were
purchased from commercial suppliers Sigma–Aldrich (Madrid,
Spain), Iris Biotech GmbH (Marktredwitz, Germany), Scharlab
(Sentmenat, Spain) or Panreac (Castellar del Vallès, Spain) and
used without further purification.
Boc-Tyr(3-I,Me)-OMe [40] (4.50 g, 10.34 mmol, 1 equiv.)
was dissolved in TFA/CH2Cl2 (1:1, 30 mL) and stirred at room
temperature for 2 h. After this time, the solvent mixture was
removed under vacuum. Diethyl ether was then added and
evaporated, this process was repeated three times to completely
remove the TFA. The resulting product was dried in vacuo
overnight to afford H-Tyr(3-I,Me)-OMe as a white solid (3.25 g,
1
94% yield). tR = 6.65 min (93% purity) (Conditions A). H NMR
Peptides were analyzed under standard analytical HPLC
conditions with a Dionex liquid chromatography instrument
composed of an UV/Vis Dionex UVD170U detector, a P680
Dionex bomb, an ASI-100 Dionex automatic injector, and
CHROMELEON 6.60 software. Detection was performed at 220
nm. Solvent A was 0.1% aq. TFA and solvent B was 0.1% TFA
in CH3CN. Conditions A: Analysis was carried out with a
Kromasil 100 C18 (4.6 mm × 40 mm, 3.5 ꢀm) column with 2–
100% B over 7 min at a flow rate of 1 mL/min. Conditions B:
(400 MHz, CDCl3): δ = 7.59 [d, J = 2.2 Hz, 1 H, CH-2arom], 7.18
[dd, J = 2.2 and 8.4 Hz, 1 H, CH-6arom], 6.76 [d, J = 8.4 Hz, 1 H,
CH-5arom], 4.18 [t, J = 6.6 Hz, 1 H, CH-α], 3.84 [s, 3 H,
CO2CH3], 3.74 [s, 3 H, OCH3], 3.16 [d, J = 6.6 Hz, 2 H, CH2-β]
ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 169.47 [CO2CH3],
157.14 [C-4arom], 139.79 [CH-2arom], 130.89 [C-1arom], 128.60
[CH-6arom], 111.58 [CH-5arom], 86.37 [C-3arom], 56.44 [OCH3],
53.20 [CH-α], 52.77 [CO2CH3], 34.47 [CH2-β] ppm.

8
Tetrahedron
ACCEPTED MANUSCRIPT
4.3. Synthesis of 3-iodo-4-methoxy-
L-tyrosine
and dried over anhydrous magnesium sulphate. Removal of the
solvent gave a dark brown oil, which was purified by column
chromatography. Elution with hexane/EtOAc (4:1) afforded Boc-
Tyr(3-B(OH)2,Me)-OMe together with Boc-Tyr(3-BPin,Me)-
OMe. A solution of CH3CN/H2O (1:1) was then added and stirred
at 75 ºC for 4 h. The resulting solution was lyophilized to afford
a white solid, which was purified by column chromatography.
Elution with hexane/EtOAc (1:1) yielded Boc-Tyr(3-
B(OH)2,Me)-OMe as a white solid (450 mg, 59% yield). tR =
A solution of LiOH (1.24 g, 28.91 mmol, 3 equiv.) in water
(17 mL) was added to a solution of H-Tyr(3-I,Me)-OMe (3.23 g,
9.64 mmol, 1 equiv.) in MeOH/THF (1:1, 34 mL). The reaction
mixture was stirred at room temperature for 2 h. After this time,
the organic solvents were evaporated under reduced pressure and
water (60 mL) was added to the resulting residue. The solution
was adjusted to pH 5 by addition of glacial AcOH and the
resulting precipitate was filtered, washed with cold diethyl ether,
and dried in vacuo overnight, yielding H-Tyr(3-I,Me)-OH as a
white solid (3 g, 96% yield). tR = 6.21 min (>99% purity)
1
7.51 min (>99% purity) (Conditions A). H NMR (400 MHz,
CDCl3): δ = 7.57 [d, J = 2.8 Hz, 1 H, CH-2arom], 7.20 [dd, J = 2.8
and 8.4 Hz, 1 H, CH-6arom], 6.84 [d, J = 8.4 Hz, 1 H, CH-5arom],
5.79 [bs, 2 H, B(OH)2], 4.98 [bs, 1 H, CONH], 4.56-4.54 [m, 1
H, CH-α], 3.89 [s, 3 H, OCH3], 3.73 [s, 3 H, CO2CH3], 3.09 [dd,
J = 5.6 and 13.8 Hz, 1 H, CH2-β], 3.01 [dd, J = 6.0 and 13.8 Hz,
1 H, CH2-β], 1.41 [s, 9 H, C(CH3)3] ppm. 13C NMR (75 MHz,
CDCl3): δ = 172.53 [CO2CH3] 163.85 [C-4arom], 155.37 [CONH],
137.87 [CH-2arom], 133.75, 128.64 [CH-6arom, C-1arom], 110.84,
110.34 [CH-5arom, C-3arom], 80.30 [C(CH3)3], 55.95 [OCH3], 54.73
[CH-α], 52.41 [CO2CH3], 37.60 [CH2-β], 28.41 [(CH3)3C] ppm.
1
(Conditions A). H NMR (300 MHz, [D6]DMSO): δ = 7.64 [d, J
= 2.1 Hz, 1 H, CH-2arom], 7.24 [dd, J = 2.1 and 8.4 Hz, 1 H, CH-
6arom], 6.91 [d, J = 8.4 Hz, 1 H, CH-5arom], 3.78 [s, 3 H, OCH3],
3.31 [dd, J = 4.5 and 7.7 Hz, 1 H, CH-α], 3.00 [dd, J = 4.5 and
14.1 Hz, 1 H, CH2-β], 2.75 [dd, J = 7.7 and 14.1 Hz, 1 H, CH2-β]
ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 156.37 [C-4arom],
139.60 [CH-2arom], 132.22 [C-1arom], 130.76 [CH-6arom], 111.36
[CH-5arom], 85.98 [C-3arom], 56.33 [OCH3], 55.74 [CH-α], 35.87
[CH2-β] ppm.
4.6. Synthesis of 3-borono-N(α)-tert-butoxycarbonyl-4-methoxy-
4.4. Synthesis of N(α)-(9-fluorenylmethoxycarbonyl)-3-iodo-4-
methoxy-L-tyrosine (2)
L-tyrosine (14)
An aqueous solution of LiOH (3 mL, 4.17 mmol, 3 equiv) was
A solution of H-Tyr(3-I,Me)-OH (3 g, 9.34 mmol, 1 equiv.) in
dioxane (32 mL) was adjusted to pH 7-8 by addition of aqueous
10% Na2CO3. The reaction mixture was stirred at room
temperature for 30 min and Fmoc-OSu (3.31 g, 9.81 mmol, 1.05
equiv.) was then added. The mixture was stirred for 24 h at room
temperature and then concentrated in vacuo. EtOAc (40 mL) was
added and the organic solution was washed with 1 N HCl (30
mL) and H2O (3×30 mL). The aqueous layers were combined,
adjusted to pH 1 and extracted with EtOAc (3×40 mL). All the
organic layers were combined, washed with brine (30 mL) and
dried over anhydrous magnesium sulfate. Removal of the solvent
followed by digestion of the resulting precipitate in
pentane/diethyl ether (1:1, 50 mL) for 2 h afforded a white solid,
which was purified by column chromatography. Elution with
CH2Cl2/MeOH (95:5) gave Fmoc-Tyr(3-I,Me)-OH (2) as a white
solid (2.85 g, 57% yield). tR = 9.00 min (>99% purity)
(Conditions A). 1H NMR (400 MHz, [D6]DMSO): δ = 12.78 [bb,
1 H, CO2H], 7.88 [d, J = 7.6 Hz, 2 H, 2 CHarom-Fmoc], 7.71 [d, J
= 2.0 Hz, 1 H, CH-2arom], 7.66-7.62 [m, 2 H, 2 CHarom-Fmoc],
added to a solution of Boc-Tyr(3-B(OH)2,Me)-OMe (450 mg,
1.27 mmol, 1 equiv.) in MeOH/THF (1:1, 6 mL). The reaction
mixture was stirred at room temperature for 1.5 h. After this time,
the organic solvents were evaporated under reduced pressure and
water (25 mL) was added to the resulting residue. The solution
was adjusted to pH 5-6 by addition of 1 N HCl followed by
extraction with EtOAc (3×25 mL). The organic layers were
combined, washed with brine (25 mL), and dried over anhydrous
magnesium sulfate. Removal of the solvent afforded Boc-Tyr(3-
B(OH)2,Me)-OH (14) as a white solid (410 mg, 95% yield). tR =
1
6.84 min (>99% purity) (Conditions A). H NMR (400 MHz,
CDCl3): δ = 7.60 [s, 1 H, CH-2arom], 7.25-7.22 [m, 1 H, CH-
6arom], 6.82 [d, J = 8.8 Hz, 1 H, CH-5arom], 5.06-5.04 [m, 1 H,
CONH], 4.51-4.49 [m, 1 H, CH-α], 3.87 [s, 3 H, OCH3], 3.13
[dd, J = 4.8 and 13.6 Hz, 1 H, CH2-β], 3.03 [dd, J = 5.2 and 13.6
Hz, 1 H, CH2-β], 1.40 [s, 9 H, C(CH3)3] ppm. 13C NMR (75
MHz, CDCl3): δ = 175.45 [CO2H] 163.74 [C-4arom], 155.49
[CONH], 137.79 [CH-2arom], 134.02, 128.57 [CH-6arom, C-1arom],
110.82, 110.30 [CH-5arom, C-3arom], 80.33 [C(CH3)3], 55.70
[OCH3], 54.70 [CH-α], 37.28 [CH2-β], 28.43 [(CH3)3C] ppm.
7.43-7.39 [m, 2 H, 2 CHarom-Fmoc], 7.34-7.25 [m, 3 H, 2 CHarom
-
Fmoc, CH-6arom], 6.90 [d, J = 8.4 Hz, 1 H, CH-5arom], 4.22-4.10
[m, 4 H, CH-α, CH2-Fmoc, CH-Fmoc], 3.77 [s, 3 H, OCH3], 3.01
[dd, J = 4.4 and 13.8 Hz, 1 H, CH2-β], 2.77 [dd, J = 10.4 and 13.8
Hz, 1 H, CH2-β] ppm. 13C NMR (75 MHz, [D6]DMSO): δ =
173.20 [CO2H], 156.37 [C-4arom], 155.97 [CONH], 143.74,
140.67 [4 Carom-Fmoc], 139.46 [CH-2arom], 132.20 [C-1arom],
4.7. General method for the synthesis of the 3-iodotyrosylpeptidyl
resins 3, 9, 10 and 25
Peptidyl resins were synthesized manually by the solid-phase
method with standard Fmoc chemistry. MBHA resin (0.4
mmol/g) was used as solid support and it was swollen with
CH2Cl2 (1×20 min) and DMF (1×20 min), and washed with
piperidine/DMF (3:7, 1×5 min) and DMF (6×1 min). Then, the
resin was treated with Fmoc-Rink linker (4 equiv.), DIPCDI (4
equiv.) and Oxyma (4 equiv.) in DMF at room temperature
overnight. After this time, the resin was washed with DMF (6×1
min). Elongation of the peptide chain was performed through
sequential Fmoc removal and coupling of the corresponding
amino acids. Fmoc group removal was achieved with a mixture
of piperidine/DMF (3:7, 2 + 10 min). Coupling of the
corresponding commercially available amino acids Fmoc-Leu-
OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Tmob)-OH, Fmoc-Ala-OH,
Fmoc-Thr(tBu)-OH, and Fmoc-βAla-OH (4 equiv.) was
performed by using DIPCDI (4 equiv.) and Oxyma (4 equiv.) in
DMF at room temperature for 1 h, whereas coupling of Fmoc-
Tyr(3-I,Me)-OH (2) (2 equiv.) was carried out using COMU (2
equiv.), Oxyma (2 equiv.) and DIEA (4 equiv.) in DMF at room
130.40 [CH-6arom], 127.63, 127.10, 125.29, 120.11 [8 CHarom
-
Fmoc], 111.28 [CH-5arom], 85.73 [C-3arom], 65.69 [CH2-Fmoc],
56.27 [OCH3], 55.61 [CH-α], 46.57 [CH-Fmoc], 34.95 [CH2-β]
ppm.
4.5. Synthesis of methyl 3-borono-N(α)-tert-butoxycarbonyl-4-
methoxy-L-tyrosinate
A solution of Boc-Tyr(3-I,Me)-OMe [40] (920 mg, 2.11
mmol, 1 equiv.) in degassed anhydrous DMSO (9 mL) was added
to a solution of bis(pinacolato)diboron (B2Pin2) (1.08 g, 4.23
mmol, 2 equiv.), PdCl2(dppf) (100 mg, 0.12 mmol, 0.06 equiv.),
and KOAc (840 mg, 8.45 mmol, 4 equiv.) in degassed anhydrous
DMSO (4.5 mL). The mixture was stirred under nitrogen at 80 ºC
for 7 h. After this time, brine (50 mL) was added and the
resulting solution was extracted with EtOAc (3×50 mL). The
combined organic extracts were washed with brine (3×50 mL),

9
temperature overnight. The completion of the reactions was
monitored by the Kaiser test [47]. After each coupling and
deprotection step, the resulting resin was washed with DMF (6×1
min).
(3) (170 mg) following the general method for solid-phase
ACCEPTED MANUSCRIPT
Miyaura borylation. Acidolytic cleavage of an aliquot of this
peptidyl resin gave H-Lys-Lys-Leu-Tyr(3-B(OH)2,Me)-Leu-Leu-
NH2 (81% purity), resulting from the hydrolysis of the pinacol
boronic ester during HPLC analysis. tR = 15.87 min (Conditions
C). MS (ESI): m/z = 417.7 [M + 2H]2+, 834.6 [M + H]+.
Upon completion of the peptide sequence, the N-terminal
Fmoc group was removed and a trityl group was introduced using
TrCl (10 equiv.) and DIEA (10 equiv.) in DMF at room
temperature for 4 h. Then, the resulting resin was washed with
DMF (6×1 min) and CH2Cl2 (3×1 min), and air-dried. The
completion of this reaction was monitored by the Kaiser test [47].
An aliquot of the resulting peptidyl resin was cleaved with
TFA/H2O/TIS (95:2.5:2.5) whilst stirring for 2 h at room
temperature. Following TFA evaporation and diethyl ether
extraction, the crude peptide was dissolved in H2O/CH3CN (1:1),
lyophilized, analysed by HPLC and characterized by mass
spectrometry.
4.8.2. Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-
Rink-MBHA
This boronotetrapeptidyl resin was prepared starting from Tr-
Lys(Boc)-Lys(Boc)-Leu-Tyr(3-I,Me)-Rink-MBHA (9) (57 mg)
following the general method for solid-phase Miyaura borylation.
Acidolytic cleavage of an aliquot of this peptidyl resin gave H-
Lys-Lys-Leu-Tyr(3-B(OH)2,Me)-NH2 (69% purity), resulting
from the hydrolysis of the pinacol boronic ester during HPLC
analysis. tR = 16.85 min (Conditions B). MS (ESI): m/z = 304.7
[M + 2H]2+, 608.4 [M + H]+.
4.7.1. Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-I,Me)-Leu-
Leu-Rink-MBHA (3)
4.8.3. Tr-Leu-Tyr(3-BPin,Me)-Rink-MBHA
This boronodipeptidyl resin was prepared starting from Tr-
Leu-Tyr(3-I,Me)-Rink-MBHA (10) (90 mg) following the
general method for solid-phase Miyaura borylation. Acidolytic
cleavage of an aliquot of this peptidyl resin gave H-Leu-Tyr(3-
B(OH)2,Me)-NH2 (50% purity), resulting from the hydrolysis of
the pinacol boronic ester during HPLC analysis. tR = 14.84 min
(Conditions B). MS (ESI): m/z = 352.1 [M + H]+.
The iodohexapeptidyl resin 3 was prepared following the
general method described above. Acidolytic cleavage of an
aliquot of this peptidyl resin afforded H-Lys-Lys-Leu-Tyr(3-
I,Me)-Leu-Leu-NH2 in 84% purity. tR = 18.99 min (Conditions
B). MS (ESI): m/z = 458.7 [M + 2H]2+, 916.5 [M + H]+, 938.5 [M
+ Na]+.
4.8.4. Tr-β Ala-Thr(t Bu)-Tyr(3-BPin,Me)-Ala-
Gln(Tmob)-Rink-MBHA (26)
4.7.2. Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-I,Me)-Rink-
MBHA (9)
The iodotetrapeptidyl resin 9 was prepared following the
general method described above. Acidolytic cleavage of an
aliquot of this peptidyl resin afforded H-Lys-Lys-Leu-Tyr(3-
I,Me)-NH2 in 87% purity. tR = 4.80 min (Conditions D). MS
(ESI): m/z = 345.6 [M + 2H]2+, 690.3 [M + H]+.
This boronodipeptidyl resin was prepared starting from Tr-
βAla-Thr(tBu)-Tyr(3-I,Me)-Ala-Gln(Tmob)-Rink-MBHA (25)
(175 mg) following the general method for solid-phase Miyaura
borylation. Acidolytic cleavage of an aliquot of this peptidyl
resin gave H-βAla-Thr-Tyr(3-B(OH)2,Me)-Ala-Gln-NH2 (62%
purity), resulting from the hydrolysis of the pinacol boronic ester
during HPLC analysis. tR = 5.20 min (Conditions A). MS (ESI):
m/z = 610.3 [M + H]+, 632.3 [M + Na]+.
4.7.3. Tr-Leu-Tyr(3-I,Me)-Rink-MBHA (10)
The iododipeptidyl resin 10 was prepared following the
general method described above. Acidolytic cleavage of an
aliquot of this peptidyl resin afforded H-Leu-Tyr(3-I,Me)-NH2 in
72% purity. tR = 18.13 min (Conditions B). MS (ESI): m/z =
434.0 [M + H]+.
4.9. General method for the solid-phase synthesis of the linear
peptidyl resins 1, 7 and 8
The corresponding boronopeptidyl resins were treated with
TFA/H2O/CH2Cl2 (0.2:1:98.8, 2×1 + 2×20 min), and washed
with DMF (3×1 min), DIEA/CH2Cl2 (1:19, 3×1 min), CH2Cl2
(3×1 min), and DMF (3×1 min). Then, coupling of the
regioisomeric mixture of Boc-His(5-Br,1-SEM)-OH (5a) and
Boc-His(5-Br,3-SEM)-OH (5b) (3 equiv.) was carried out using
DIPCDI (3 equiv.) and Oxyma (3 equiv.) in DMF at room
temperature for 3 h. The resins were then washed with DMF (6×1
min) and CH2Cl2 (3×1 min), and air-dried. The completion of the
reactions was monitored by the Kaiser test [47]. An aliquot of the
resulting peptidyl resins was cleaved with TFA/H2O/TIS
(95:2.5:2.5) whilst being stirred for 3 h at room temperature.
Following TFA evaporation and diethyl ether extraction, the
corresponding crude peptide was dissolved in H2O/CH3CN (1:1),
lyophilized, analysed by HPLC, and characterized by mass
spectrometry.
4.7.4. Tr-β Ala-Thr(t Bu)-Tyr(3-I,Me)-Ala-
Gln(Tmob)-Rink-MBHA (25)
The iododipeptidyl resin 25 was prepared following the
general method described above. Acidolytic cleavage of an
aliquot of this peptidyl resin afforded H-βAla-Thr-Tyr(3-I,Me)-
Ala-Gln-NH2 in 87% purity. tR = 5.87 min (Conditions A).
4.8. General method for the solid-phase Miyaura borylation
A 2-10 mL round-bottomed flask was charged with the
corresponding 3-iodotyrosylpeptidyl resin, B2Pin2 (4 equiv.),
PdCl2(dppf) (0.18 equiv.), and dppf (0.09 equiv.). A thoroughly
sonicated solution of KOAc (6 equiv.) in degassed anhydrous
DMSO (20 µL/mg of resin) was then added, and the mixture was
heated at 80 ºC for 8 h. Upon completion of the reaction, the
resin was washed with DMSO (6×1 min), MeOH (6×1 min),
CH2Cl2 (6×1 min), and diethyl ether (3×1 min). An aliquot of the
resulting boronopeptidyl resin was cleaved with TFA/H2O/TIS
(95:2.5:2.5) whilst being stirred for 2 h at room temperature.
Following TFA evaporation and diethyl ether extraction, the
crude peptide was dissolved in H2O/CH3CN (1:1), lyophilized,
analysed by HPLC, and characterized by mass spectrometry.
4.9.1. Boc-His(5-Br,1-SEM)-Lys(Boc)-Lys(Boc)-
Leu-Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA (1a) and
Boc-His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-
Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA (1b)
Resins 1 were synthesized starting from Tr-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA (4) (100
mg) following the general method described above. Acidolytic
cleavage of an aliquot of these peptidyl resins afforded H-His(5-
Br)-Lys-Lys-Leu-Tyr(3-B(OH)2,Me)-Leu-Leu-NH2 (6) (75%
purity), resulting from the hydrolysis of the pinacol boronate
4.8.1. Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-
Leu-Leu-Rink-MBHA (4)
The boronohexapeptidyl resin 4 was prepared starting from
Tr-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-I,Me)-Leu-Leu-Rink-MBHA

10
Tetrahedron
4.10.1. Boc-Tyr(3-B(OH)2 ,Me)-Lys(Boc)-Lys(Boc)-
during HPLC analysis. tR = 16.25 min (Conditions C). MS
ACCEPTED MANUSCRIPT
(ESI): m/z = 1049.5, 1051.5 [M + H]+.
Leu-His(5-Br,1-SEM)-Leu-Leu-Rink-MBHA (13a)
and Boc-Tyr(3-B(OH)2 ,Me)-Lys(Boc)-Lys(Boc)-Leu-
His(5-Br,3-SEM)-Leu-Leu-Rink-MBHA (13b)
4.9.2. Boc-His(5-Br,1-SEM)-Lys(Boc)-Lys(Boc)-
Leu-Tyr(3-BPin,Me)-Rink-MBHA (7a) and Boc-
His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-
BPin,Me)-Rink-MBHA (7b)
Resins 7 were synthesized starting from Tr-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (50 mg) following
the general method described above. Acidolytic cleavage of an
aliquot of these peptidyl resins afforded H-His(5-Br)-Lys-Lys-
Leu-Tyr(3-B(OH)2,Me)-NH2 (11) (60% purity), resulting from
the hydrolysis of the pinacol boronate during HPLC analysis. tR =
14.98 min (Conditions B). MS (ESI): m/z = 823.3, 825.3 [M +
H]+.
Peptidyl resins 13 were prepared following the general
method described above. Acidolytic cleavage of an aliquot of
these peptidyl resins afforded H-Tyr(3-B(OH)2,Me)-Lys-Lys-
Leu-His(5-Br)-Leu-Leu-NH2 (15) in 65% purity. tR = 14.71 min
(Conditions C). MS (ESI): m/z = 1049.5, 1051.5 [M + H]+.
4.10.2. Boc-Tyr(3-B(OH)2 ,Me)-Lys(Boc)-Lys(Boc)-
Leu-His(5-Br,1-SEM)-Rink-MBHA (16a) and Boc-
Tyr(3-B(OH)2 ,Me)-Lys(Boc)-Lys(Boc)-Leu-His(5-
Br,3-SEM)-Rink-MBHA (16b)
Peptidyl resins 16 were prepared following the general
method described above. Acidolytic cleavage of an aliquot of
these peptidyl resins afforded H-Tyr(3-B(OH)2,Me)-Lys-Lys-
Leu-His(5-Br)-NH2 (18) in 59% purity. tR = 13.53 min
(Conditions B). MS (ESI): m/z = 412.1, 413.1 [M + 2H]2+, 823.4,
825.4 [M + H]+.
4.9.3. Boc-His(5-Br,1-SEM)-Leu-Tyr(3-BPin,Me)-
Rink-MBHA (8a) and Boc-His(5-Br,3-SEM)-Leu-
Tyr(3-BPin,Me)-Rink-MBHA (8b)
Resins 8 were synthesized starting from Tr-Leu-Tyr(3-
BPin,Me)-Rink-MBHA (85 mg) following the general method
described above. Acidolytic cleavage of an aliquot of these
peptidyl resins afforded H-His(5-Br)-Leu-Tyr(3-B(OH)2,Me)-
NH2 (12) (60% purity), resulting from the hydrolysis of the
pinacol boronate during HPLC analysis. tR = 15.38 min
(Conditions B). MS (ESI): m/z = 567.1, 569.1 [M + H]+.
4.10.3. Boc-Tyr(3-B(OH)2 ,Me)-Leu-His(5-Br,1-
SEM)-Rink-MBHA (17a) and Boc-Tyr(3-
B(OH)2 ,Me)-Leu-His(5-Br,3-SEM)-Rink-MBHA
(17b)
Peptidyl resins 17 were prepared following the general
method described above. Acidolytic cleavage of an aliquot of
these peptidyl resins afforded H-Tyr(3-B(OH)2,Me)-Leu-His(5-
Br)-NH2 (19) in 52% purity. tR = 14.30 min (Conditions B). MS
(ESI): m/z = 567.1, 569.1[M + H]+.
4.10. General method for the solid-phase synthesis of the linear
peptidyl resins 13, 16, 17 and 22
These peptidyl resins were synthesized manually by the solid-
phase method with standard Fmoc chemistry. MBHA resin (0.4
mmol/g) was used as solid support and it was swollen with
CH2Cl2 (1×20 min) and DMF (1×20 min), and washed with
piperidine/DMF (3:7, 1×5 min) and DMF (6×1 min). Then, resins
were treated with Fmoc-Rink linker (4 equiv.), DIPCDI (4
equiv.) and Oxyma (4 equiv.) in DMF at room temperature
overnight. After this time, resins were washed with DMF (6×1
min). Elongation of the peptide chain was performed through
sequential Fmoc removal and coupling of the corresponding
amino acids. Fmoc group removal was achieved with a mixture
of piperidine/DMF (3:7, 2 + 10 min), and then resins were
washed with DMF (6×1 min). Couplings of the corresponding
Fmoc-amino acids (4 equiv.) were performed using DIPCDI (4
equiv.) and Oxyma (4 equiv.) in DMF at room temperature for 1
h. Coupling of Boc-His(5-Br,1-SEM)-OH (5a) and Boc-His(5-
Br,3-SEM)-OH (5b) (2 equiv.) was mediated by COMU (2
equiv.), Oxyma (2 equiv.) and DIEA (4 equiv.) in DMF at room
temperature overnight. Coupling of Boc-Tyr(3-B(OH)2,Me)-OH
(14) (3 equiv.) was carried out using DIPCDI (3 equiv.) and
Oxyma (3 equiv.) in DMF at room temperature for 3 h. After
each coupling step, the resulting resins were washed with DMF
(6×1 min). The completion of the reactions was monitored by the
Kaiser test [47]. The Boc group of the 5-bromohistidine residue
was removed by treatment with a mixture of TMSOTf and 2,6-
lutidine in CH2Cl2 (final concentrations: 2.5 M TMSOTf and
3.75 M 2,6-lutidine) at room temperature (10×30 min). The
resulting resins were washed with CH2Cl2 (5×1 min), MeOH
(3×5 min) and DMF (5×1 min) [46].
4.10.4. Boc-Tyr(3-B(OH)2 ,Me)-Ala-Gln(Tmob)-Gly-
Gln(Tmob)-His(5-Br,1-SEM)-βAla-Rink-MBHA
(22a) and Boc-Tyr(3-B(OH)2 ,Me)-Ala-Gln(Tmob)-
Gly-Gln(Tmob)-His(5-Br,3-SEM)-βAla-Rink-MBHA
(22b)
Peptidyl resins 22 were prepared following the same
procedure described above. Acidolytic cleavage of an aliquot of
these peptidyl resins afforded H-Tyr(3-B(OH)2,Me)-Ala-Gln-
Gly-Gln-His(5-Br)-βAla-NH2 (23) in >99% purity. tR = 5.15 min
(Conditions A). MS (ESI): m/z = 909.1, 911.1 [M + H]+, 931.0,
933.0 [M + Na]+.
4.11. General method for the solid-phase intramolecular Suzuki-
Miyaura arylation
A 15 mL reaction vessel containing a magnetic stir bar was
charged with the corresponding linear peptidyl resins, Pd2(dba)3
(0.2 equiv.), SPhos (0.4 equiv.), and KF (4 equiv.). Thoroughly
degassed DME/EtOH/H2O (9:9:2, 0.25-0.51 mL) was then added
under nitrogen. The reaction mixture was heated at 140 ºC under
microwave irradiation for 30 min. After the reaction time, upon
cooling, the solvent was removed and the resin was washed with
DMF (6×1 min), EtOH (6×1 min), CH2Cl2 (6×1 min), and diethyl
ether (3×1 min). The resulting biaryl cyclic peptidyl resins were
cleaved with TFA/H2O/TIS (95:2.5:2.5) whilst being stirred for 3
h at room temperature. Following TFA evaporation and diethyl
ether extraction, the corresponding crude peptide was dissolved
in H2O/CH3CN (1:1), lyophilized, analysed by HPLC and mass
spectrometry. Biaryl cyclic peptides were purified by reverse-
phase column chromatography, analysed by HPLC, and
characterized by HRMS.
Upon completion of the peptide sequence, an aliquot of the
resulting peptidyl resins was cleaved with TFA/H2O/TIS
(95:2.5:2.5) whilst being stirred for 3 h at room temperature.
Following TFA evaporation and diethyl ether extraction, the
corresponding crude peptide was dissolved in H2O/CH3CN (1:1),
lyophilized, analysed by HPLC, and characterized by mass
spectrometry.
4.11.1. Biaryl cyclic peptide BPC782
Starting from resins Boc-His(5-Br,1-SEM)-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-BPin,Me)-Rink-MBHA (7a) and Boc-
His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-
Rink-MBHA (7b) (50 mg), Suzuki-Miyaura cyclization followed

11
4.11.6. Biaryl cyclic peptide BPC792
by acidolytic cleavage gave biaryl cyclic peptide BPC782 (tR =
ACCEPTED MANUSCRIPT
13.78 min, 35% purity) together with H-His-Lys-Lys-Leu-Tyr(3-
OH,Me)-NH2 (tR = 14.35 min, 11% purity) and H-His-Lys-Lys-
Leu-Tyr(Me)-NH2 (tR = 15.45 min, 12% purity) (Conditions B).
Elution with H2O/MeOH/TFA (90:10:0.2) gave BPC782 in 68%
purity (tR = 5.26 min) (Conditions A) and in 6% yield. MS (ESI):
m/z = 350.2 [M + 2H]2+, 699.5 [M + H]+. HRMS (ESI): calcd. for
C34H56N10O6 [M + 2H]2+ 350.2187, found 350.2192; calcd. for
C34H55N10O6 [M + H]+ 699.4301, found 699.4323.
Starting from resins Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-
Lys(Boc)-Leu-His(5-Br,1-SEM)-Leu-Leu-Rink-MBHA (13a)
and Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-Lys(Boc)-Leu-His(5-Br,3-
SEM)-Leu-Leu-Rink-MBHA (13b) (50 mg), Suzuki-Miyaura
cyclization followed by acidolytic cleavage gave biaryl cyclic
peptide BPC792 (tR = 14.22 min, 55% purity) together with H-
Tyr(Me)-Lys-Lys-Leu-His-Leu-Leu-NH2 (tR = 15.59 min, 22%
purity) (Conditions C). Elution with H2O/MeOH/TFA
(90:10:0.2) gave BPC792 in 92% purity (tR = 5.96 min)
(Conditions A) and in 9% yield. MS (ESI): m/z = 463.2 [M +
2H]2+, 925.6 [M + H]+, 947.6 [M + Na]+. HRMS (ESI): calcd.
for C46H79N12O8 [M + 3H]3+ 309.2042, found 309.2056; calcd. for
C46H78N12O8 [M + 2H]2+ 463.3027, found 463.3045; calcd. for
C46H77N12O8 [M + H]+ 925.5982, found 925.6021.
4.11.2. Biaryl cyclic peptide BPC784
Starting from resins Boc-His(5-Br,1-SEM)-Leu-Tyr(3-
BPin,Me)-Rink-MBHA (8a) and Boc-His(5-Br,3-SEM)-Leu-
Tyr(3-BPin,Me)-Rink-MBHA (8b) (73 mg), Suzuki-Miyaura
cyclization followed by acidolytic cleavage gave biaryl cyclic
peptide BPC784 (tR = 12.65 min, 30% purity) together with H-
His-Leu-Tyr(3-OH,Me)-NH2 (tR = 14.47 min, 11% purity), H-
His(5-Br)-Leu-Tyr(3-OH,Me)-NH2 (tR = 14.89 min, 12% purity)
and H-His-Leu-Tyr(Me)-NH2 (tR = 15.98 min, 7% purity)
(Conditions B). Elution with H2O/TFA (100:0.2) gave BPC784
in 90% purity (tR = 5.10 min) (Conditions A) and in 7% yield.
MS (ESI): m/z = 443.2 [M + H]+. HRMS (ESI): calcd. for
C22H31N6O4 [M + H]+ 443.2401, found 443.2394.
4.11.7. Biaryl cyclic peptide 20
Starting from resins Boc-Tyr(3-B(OH)2,Me)-Ala-Gln(Tmob)-
Gly-Gln(Tmob)-His(5-Br,1-SEM)-βAla-Rink-MBHA (22a) and
Boc-Tyr(3-B(OH)2,Me)-Ala-Gln(Tmob)-Gly-Gln(Tmob)-His(5-
Br,3-SEM)-βAla-Rink-MBHA (22b) (50 mg), Suzuki-Miyaura
cyclization followed by acidolytic cleavage gave the biaryl cyclic
peptide 20 (tR = 11.49 and 11.78 min, 64% purity) (Conditions
B). Elution with H2O/TFA (100:0.2) gave 20 in 87% purity (tR =
2.43 min) (Conditions E) and in 6% yield. MS (ESI): m/z = 785.5
[M + H]+. HRMS (ESI): calcd. for C34H49N12O10 [M + H]+
785.3689, found 785.3715; calcd. for C34H48N12O10Na [M + Na]+
807.3509, found 807.3533.
4.11.3. Biaryl cyclic peptide BPC786
Starting from resins Boc-His(5-Br,1-SEM)-Lys(Boc)-
Lys(Boc)-Leu-Tyr(3-BPin,Me)-Leu-Leu-Rink-MBHA (1a) and
Boc-His(5-Br,3-SEM)-Lys(Boc)-Lys(Boc)-Leu-Tyr(3-BPin,Me)-
Leu-Leu-Rink-MBHA (1b) (50 mg), Suzuki-Miyaura cyclization
followed by acidolytic cleavage gave biaryl cyclic peptide
BPC786 (tR = 15.79 min, 31% purity) together with H-His-Lys-
Lys-Leu-Tyr(3-OH,Me)-Leu-Leu-NH2 (tR = 15.50 min, 11%
4.11.8. Biaryl cyclic peptide 21
Starting from resins Boc-His(5-Br,1-SEM)-βAla-Thr(tBu)-
Tyr(3-BPin,Me)-Ala-Gln(Tmob)-Rink-MBHA (24a) and Boc-
His(5-Br,3-SEM)-βAla-Thr(tBu)-Tyr(3-BPin,Me)-Ala-
purity) and H-His-Lys-Lys-Leu-Tyr(Me)-Leu-Leu-NH2 (tR
=
18.35 min, 20% purity) (Conditions C). Elution with
H2O/MeOH/TFA (85:15:0.2) gave BPC786 in 79% purity (tR =
6.10 min) (Conditions A) and in 5% yield. MS (ESI): m/z = 925.6
[M + H]+, 947.6 [M + Na]+. HRMS (ESI): calcd. for C46H77N12O8
[M + H]+ 925.5982, found 925.5940; C46H76N12O8Na [M + Na]+
947.5801, found 947.5763.
Gln(Tmob)-Rink-MBHA (24b) (50 mg), Suzuki-Miyaura
cyclization followed by acidolytic cleavage gave the biaryl cyclic
peptide 21 (tR = 4.84 min, 12% purity) (Conditions A). Elution
with H2O/TFA (100:0.2) gave 21 in 84% purity (tR = 5.01 min)
(Conditions A) and in 7% yield. MS (ESI): m/z = 701.3 [M +
H]+, 723.3 [M + Na]+. HRMS (ESI): calcd. for C31H45N10O9 [M +
H]+ 701.3365, found 701.3369.
4.11.4. Biaryl cyclic peptide BPC788
Starting from resins Boc-Tyr(3-B(OH)2,Me)-Lys(Boc)-
Lys(Boc)-Leu-His(5-Br,1-SEM)-Rink-MBHA (16a) and Boc-
Tyr(3-B(OH)2,Me)-Lys(Boc)-Lys(Boc)-Leu-His(5-Br,3-SEM)-
Rink-MBHA (16b) (68 mg), Suzuki-Miyaura cyclization
followed by acidolytic cleavage gave biaryl cyclic peptide
BPC788 (tR = 15.46 min, 32% purity) together with H-Tyr(3-
OH,Me)-Lys-Lys-Leu-His-NH2 (tR = 15.26 min, 5% purity) and
H-Tyr(Me)-Lys-Lys-Leu-His-NH2 (tR = 16.16 min, 15% purity)
(Conditions B). Elution with H2O/TFA (100:0.2) gave BPC788
in 87% purity (tR = 5.19 min) (Conditions A) and in 8% yield.
MS (ESI): m/z = 350.2 [M + 2H]2+, 699.5 [M + H]+, 721.4 [M +
Na]+, 737.4 [M + K]+. HRMS (ESI): calcd. for C34H55N10O6 [M +
H]+ 699.4301, found 699.4305; calcd. for C34H54N10O6Na [M +
Na]+ 721.4120, found 721.4128.
Acknowledgments
Iteng Ng Choi was recipient of a predoctoral fellowship from
the MICINN of Spain. Àngel Oliveras was recipient of
predoctoral fellowship from the University of Girona. This work
was supported by grants AGL2009-13255-C02-02/AGR,
AGL2012-39880-C02-02,
AGL2015-69876-C2-2-R
(MINECO/FEDER, EU) and MPCUdG2016/038. The authors
acknowledge the Serveis Tècnics de Recerca of the University of
Girona for the NMR and mass spectrometry analysis.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at https://doi.org/xxxx.
4.11.5. Biaryl cyclic peptide BPC790
Starting from resins Boc-Tyr(3-B(OH)2,Me)-Leu-His(5-Br,1-
SEM)-Rink-MBHA (17a) and Boc-Tyr(3-B(OH)2,Me)-Leu-
His(5-Br,3-SEM)-Rink-MBHA (17b) (50 mg), Suzuki-Miyaura
cyclization followed by acidolytic cleavage gave biaryl cyclic
peptide BPC790 (tR = 12.87 min, 49% purity) (Conditions B).
Elution with H2O/CH3CN (88:12) gave BPC790 in 96% purity
(tR = 5.10 min) (Conditions A) and in 8% yield. MS (ESI): m/z =
443.1 [M + H]+. HRMS (ESI): calcd. for C22H31N6O4 [M + H]+
443.2401, found 443.2382.
References and notes
[1] L. Feliu, M. Planas, Int. J. Pept. Res. Ther. 11 (2005) 53–97.
[2] A. Holtzel, D.G. Schmid, G.J. Nicholson, S. Stevanovic, J. Schimana, K.
Gebhardt, H.P. Fiedler, G. Jung, J. Antibiot. 55 (2002) 571–577.
[3] J. Schimana, K. Gebhardt, A. Holtzel, D.G. Schmid, R. Sussmuth, J.
Muller, R. Pukall, H.P. Fiedler, J. Antibiot. 55 (2002) 565–570.
[4] A. Coste, A. Bayle, J. Marrot, J. Evano, Org. Lett. 16 (2014) 1306–1309.
[5] M. Inoue, H. Sakazaki, H. Furuyama, M. Hirama, Angew. Chem. Int. Ed.
42 (2003) 2654–2657.

12
Tetrahedron
[6]
J. Kohno, Y. Koguchi, M. Nishio, K. Nakao, M. Kuroda, R.
Shimizu, T. Ohnuki, S. Komatsubara, J. Org. ChemA. 65C(2C00E0)P99T0–E99D5. MANUSCRIPT
[7] Y. Koguchi, J. Kohno, M. Nishio, K. Takahashi, T. Okuda, T. Ohnuki, S.
Komatsubara, J. Antibiot. 53 (2000) 105–109.
[8] P.J. Krenitsky, D.L. Boger, Tetrahedron Lett. 44 (2003) 4019–4022.
[9] G. Helynck, C. Dubertret, D. Frechet, J. Leboul, J. Antibiot. 51 (1998)
51, 512–514.
[10]J. L. Pace, G. Yang, Biochem. Pharmacol. 71 (2006) 968–980.
[11]F. Van Bambeke, Y. Van Laethem, P. Courvalin, P.M. Tulkens, Drugs
64 (2004) 913–936.
[12]F. Tomson, J.A. Bailey, R.B. Gennis, C. J. Unkefer, Z. Li, L.A. Silks,
R.A. Martinez, R.J. Donohoe, R.B. Dyer, W.H. Woodruff, Biochemistry
41 (2002) 14383–14390.
[13]C.A. Bewley, H. He, D.H. Williams, D.J. Faulkner, J. Am. Chem. Soc.
118 (1996) 4314–4321.
[14]D.J. Faulkner, H. Hai-yin, M.D. Unson, C.A. Bewley, M.J. Garson,
Gazz. Chim. Ital. 123 (1993) 301–307.
[15]T. Willemse, W. Schepens, H.W.T. van Vlijmen, B.U.W. Maes, S.
Ballet, Catalysts 7 (2017) 74.
[16]A. Afonso, L. Feliu, M. Planas, Tetrahedron 67 (2011) 2238–2245.
[17F.M. Meyer, J. C. Collins, B. Borin, J. Bradow, S. Liras, C. Limberakis,
A.M. Mathiowetz, L. Philippe, D. Price, K. Song, K. James, J. Org.
Chem. 77 (2012) 3099–3114.
[18]J. García-Pindado, S. Royo, M. Teixidó, E. Giralt, J. Pept. Sci. 23 (2017)
294–302.
[19]A. Afonso, O. Cussó, L. Feliu, M. Planas, Eur. J. Org. Chem. (2012)
6204–6211.
[20]M. Bois-Choussy, P. Cristau, J. Zhu, Angew. Chem. Int. Ed. 42 (2003)
4238–4241.
[21]A.-C. Carbonnelle, J. Zhu, Org. Lett. 2 (2000) 3477–3480.
[22]L. Mendive-Tapia, S. Preciado, J. García, R. Ramón, N. Kielland, F.
Albericio, R. Lavilla, Nat. Commun. 6 (2015) 7160.
[23]L. Mendive-Tapia, A. Bertran, J. García, G. Acosta, F. Albericio, R.
Lavilla, Chem. Eur. J. 22 (2016) 13114–13119.
[24]D.S. Peters, F.E. Romesberg, P.S. Baran, J. Am. Chem. Soc. 140 (2018)
2072–2075.
[25]P. Nshimyumukiza, E. Van Den Berge, B. Delest, T. Mijatovic, R. Kiss,
J. Marchand-Brynaert, R. Robiette, Tetrahedron 66 (2010) 4515–4520.
[26]V. Mathieu, E. Van Den Berge, J. Ceusters, T. Konopka, A. Cops, C.
Bruyère, C. Pirker, W. Berger, T. Trieu-Van, D. Serteyn, R. Kiss, R.
Robiette, J. Med. Chem. 56 (2013) 6626–6637.
[27]J. Tan, Y. Chen, H. Li, N. Yasuda, J. Org. Chem. 2014, 79, 8871-8876.
[28]S. Vichier-Guerre, L. Dugué, S. Pochet, Tetrahedron Lett. 55 (2014)
6347–6350.
[29]F. Bellina, R. Rossi, Adv. Synth. Catal. 352 (2010) 1223–1276.
[30]C.F. Lee, A. Holownia, J.M. Bennett, J.M. Elkins, J.D. St. Denis, S.
Adachi, A.K. Yudin, Angew. Chem. Int. Ed. 56 (2017) 6264–6267.
[31]C.B. Bheeter, L. Chen, J.-F. Soulé, H. Doucet, Catal. Sci. Technol. 6
(2016) 2005–2049.
[32]L. Théveau, C. Schneider, C. Fruit, C. Hoarau, ChemCatChem 8 (2016)
3183–3194.
[33]D. Nandi, S.S. Siwal, K. Mallick, ChemistrySelect 2 (2017) 1747–1752.
[34]F.-M. Chen, F.-D. Huang, X.-Y. Yao, T. Li, F.-S. Liu, Org. Chem. Front.
4 (2017) 2336–2342.
[35]R. Bhaskar, A.K. Sharma, A.K. Singh, Organometallics 37 (2018) 2669–
2681.
[36]S. Mao, X. Shi, J.-F. Soulé, H. Doucet, Adv. Synth. Catal. 360 (2018)
3306–3317.
[37]A. Mahindra, N. Bagra, R. Jain, J. Org. Chem. 78 (2013) 10954–10959.
[38]V. Cerezo, A. Afonso, M. Planas, L. Feliu, Tetrahedron 63 (2007)
10445–10453.
[39]I. Ng-Choi, M. Soler, V. Cerezo, E. Badosa, E. Montesinos, M. Planas,
L. Feliu, Eur. J. Org. Chem. (2012) 4321–4332.
[40]V. Cerezo, M. Amblard, J. Martinez, P. Verdié, M. Planas, L. Feliu,
Tetrahedron 64 (2008) 10538–10545.
[41]A.J.J. Lennox, G.C. Lloyd-Jones, Chem. Soc. Rev. 43 (2014) 412–443.
[42]M. Prieto, S. Mayor, P. Lloyd-Williams, E. Giralt, J. Org. Chem. 74
(2009) 9202–9205.
[43]S. Kozuch, J.M.L. Martin, Chem. Commun. 47 (2011) 4935–4937.
[44]R. Martin, S.L. Buchwald, Acc. Chem. Res. 41 (2008) 1461–1473.
[45]T. Barder, S.D. Walker, J.R. Martinelli, S.L. Buchwald, J. Am. Chem.
Soc. 127 (2005) 4685–4696.
[46]A.J. Zhang, D.H. Russell, J.P. Zhu, K. Burguess, Tetrahedron Lett. 39
(1998) 7439–7442.
[47]E. Kaiser, R.L. Colescott, C.D. Bossinger, P. Cook, Anal. Biochem. 34
(1970) 595–598.