Solid-phase synthesis of biaryl cyclic peptides by borylation and microwave-assisted intramolecular Suzuki-Miyaura reaction

Article abstract of DOI:10.1016/j.tet.2011.01.084

Miyaura borylation and Suzuki-Miyaura cross-coupling have been combined to set up an efficient strategy for the solid-phase synthesis of biaryl cyclic peptides. The Miyaura borylation was the key step in obtaining the linear peptidyl resin precursor containing both the boronate and the halogenated derivative of an aromatic amino acid. The Suzuki-Miyaura macrocyclization was performed under microwave irradiation leading to biaryl cyclic peptides of different ring sizes.

Full text of DOI:10.1016/j.tet.2011.01.084

Tetrahedron 67 (2011) 2238e2245
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Solid-phase synthesis of biaryl cyclic peptides by borylation and microwave-
assisted intramolecular SuzukieMiyaura reaction
*
*
Ana Afonso, Lidia Feliu , Marta Planas
ꢀ
ꢁ
Laboratori d’Innovacio en Processos i Productes de Síntesi Organica (LIPPSO), Departament de Química, Universitat de Girona, Campus Montilivi, 17071 Girona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Miyaura borylation and SuzukieMiyaura cross-coupling have been combined to set up an efficient
strategy for the solid-phase synthesis of biaryl cyclic peptides. The Miyaura borylation was the key step
in obtaining the linear peptidyl resin precursor containing both the boronate and the halogenated de-
rivative of an aromatic amino acid. The SuzukieMiyaura macrocyclization was performed under mi-
crowave irradiation leading to biaryl cyclic peptides of different ring sizes.
Received 17 December 2010
Received in revised form 26 January 2011
Accepted 26 January 2011
Available online 2 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cross coupling
Cyclization
Boronopeptides
Arylation
1. Introduction
advantageous because it avoids the synthesis and purification of
the amino acid boronate in solution, and is amenable to the prep-
The biaryl motif is an important substructure of many bioactive
and functional molecules and has been the focus of synthetic
chemists during the last years.1 In particular, biaryl amino acids are
valuable synthetic intermediates for the preparation of a plethora
of biologically active compounds, and their incorporation in pep-
tide and peptidomimetic drugs is considered a useful approach to
improve the biological activity of such compounds.2
Several methods have proven to be effective for the preparation
of biaryl amino acids.1 Among them, the SuzukieMiyaura cross-
coupling of an aryl halide and an aryl boron derivative has emerged
as one of the most reliable reactions to create the arylearyl bond.3
This reaction has been efficiently applied for accessing biaryl cyclic
peptides, such as arylomycins, biphenomycins, or RP-66453.4
However, up to now, the synthesis of this type of compounds has
only been conducted in solution. The solid-phase synthesis of biaryl
peptides has been scarcely reported and is restricted to the prep-
aration of linear sequences involving the coupling of a polymer-
bound halogenated aromatic amino acid and an aryl boron in
solution.5
aration of peptide boronates in a flexible manner under mild con-
ditions. Moreover, it allows the preparation of a large diversity of
biaryl peptides from a single boronopeptide intermediate because
the number of commercially available aryl halides is larger than
that of arylborons.
Within our current interest in cyclic peptides, in this work we
envisioned that this methodology could be extended to the solid-
phase synthesis of biaryl bridged macrocycles. The key steps of this
strategy would be the synthesis, via a Miyaura borylation, of the
linear peptide containing both the boronate and the halogenated
derivative of an aromatic amino acid, and the SuzukieMiyaura
macrocyclization for the formation of the arylearyl bond. Per-
forming the borylation and the macrocyclization on solid support
would benefit from the advantages inherent to the solid-phase
synthesis, and would represent a convergent and versatile ap-
proach for the preparation of biaryl cyclic compounds. Based on
these considerations, we report herein an efficient solid-phase
synthesis of biaryl-containing cyclic peptides.
Recently, we have described an alternative approach for the
preparation of biaryl peptides through solid-phase Miyaura bor-
ylation of a phenylalanine residue followed by SuzukieMiyaura
cross-coupling with a range of aryl halides.6 This approach is
2. Results and discussion
2.1. Arylation of boronopeptidyl resins with halogenated
aromatic amino acids
We first set up to examine the feasibility of the arylation of
boronopeptidyl resins with halogenated aromatic amino acids
through a SuzukieMiyaura cross-coupling. For this study we used
* Corresponding authors. Tel.: þ34 972 418274; fax: þ34 972 418150; e-mail
addresses: lidia.feliu@udg.edu (L. Feliu), marta.planas@udg.edu (M. Planas).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.01.084

A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
2239
the boronophenylalanine peptidyl resin Boc-Phe(4-BPin)-Leu-Leu-
Rink-MBHA (1), which was prepared following our recently
reported methodology (Scheme 1).6
spectrometry, and 1H and 13C NMR. 2D-COSY, HSQC, and HMBC
experiments were carried out to completely assign all the proton
and carbon signals. HPLC and NMR analysis of tetrapeptide 3a
pointed out the occurrence of two isomers. Further analysis by 1H
NMR at low temperature in MeOH-d4 confirmed that they corre-
sponded to two different conformers. For biaryl tetrapeptides 3b
and 3c, the NMR spectra showed the presence of only one stereo-
isomer. These results revealed that no epimerization occurred
during the borylation and SuzukieMiyaura cross-coupling steps.
O
O
BocHN
BocHN
Leu-Leu-Rink-MBHA
Leu-Leu-Rink-MBHA
i
O
B
O
I
2
1
2.2. Synthesis of biaryl cyclic peptides
Scheme 1. Solid-phase Miyaura borylation of a iodophenylalanyltripeptide. Reagents
and conditions: (i) B2Pin2, PdCl2(dppf), dppf, KOAc, DMSO, 80 ꢀC, 24 h.
In view of the excellent results obtained, the synthesis of biaryl
cyclic peptides via a solid-phase intramolecular SuzukieMiyaura
arylation was attempted. This approach involved first the prepa-
ration of the linear peptidyl resins containing the required boronate
and halogenated amino acid derivatives.
For this purpose, we planned the synthesis of the resins Boc-AA
(I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA (4aec)
and Boc-AA(I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (5ae
c), where AA(I) corresponds to Phe(4-I) (a), Tyr(3-I,Me) (b) or Tyr
(3-I,SEM) (c) (Scheme 2). SEM and methyl protecting groups of the
tyrosine residue served to ensure the stability of the hydroxyl func-
tion under the reaction conditions. We first assayed the preparation
of 4a starting from Boc-Phe(4-BPin)-Leu-Leu-Rink-MBHA (1).
Accordingly, resin Boc-Phe(4-I)-Leu-Leu-Rink-MBHA (2) was
treated with bis(pinacolato)diboron (B2Pin2), PdCl2(dppf), 1,10-bis
(diphenylphosphanyl)ferrocene (dppf), and KOAc in DMSO at 80 ꢀC
for 24 h. The resulting boronopeptidyl resin was then subjected to
arylation with Boc-Phe(4-I)-OMe,7 Boc-Tyr(3-I,SEM)-OMe8 or
a mixture of Boc-His(5-Br,1-SEM)-OMe and Boc-His(5-Br,3-SEM)-
OMe8 under the conditions previously optimized for the arylation
of phenylalanine boronates with aryl halides (SEM¼[2-(trime-
thylsilyl)ethoxy]methyl) (Table 1).6 The reaction was carried out
using Pd2(dba)3, P(o-tolyl)3, and KF in DME/EtOH/H2O under mi-
crowave irradiation (MWI) at 120 ꢀC for 30 min. After hydrolysis of
the methyl ester with LiOH in THF, the resulting biaryl peptidyl
resins were cleaved with TFA/triisopropylsilane (TIS)/H2O. HPLC
and ESI-MS analysis of the crude reaction mixtures showed the
formation of the expected biaryl tetrapeptides in good purities.
Cross-coupling of the boronopeptidyl resin 1 with the iodopheny-
lalanine a and the iodotyrosine b led to the biaryl peptides 3a and
3b with 92 and 79% purity, respectively. Moreover, 1 reacted with
bromohistidines c affording the biaryl peptide 3c in 42% purity. This
result is especially noteworthy because cross-couplings involving
the imidazole ring of a histidine have been shown to be challeng-
ing.8 Purification of the crude reaction mixtures by column chro-
matography afforded the corresponding biaryl peptides 3aec in
12e62% yield, and these products were fully characterized by mass
Fmoc-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-X-Rink-MBHA
i, ii
Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-X-Rink-MBHA
iii
Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-X-Rink-MBHA
iv, v
Boc-AA(I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-X-Rink-MBHA
4a-c
vi, vii
5a-c
Table 1
Solid-phase SuzukieMiyaura arylation of boronotripeptidyl resin 1 with haloge-
nated amino acid derivatives
6a-c
H-AA-Lys-Lys-Leu-Phe-X-NH2
O
O
7a-c
BocHN
H2N
Leu-Leu-Rink-MBHA
i, ii, iii
Leu-Leu-NH2
AA
Phe
Tyr(Me)
Tyr
Phe
Tyr(Me)
Tyr
X
Leu-Leu
4a, 6a
4b, 6b
4c, 6c
5a, 7a
5b, 7b
5c, 7c
O
Leu-Leu
B
O
Ar
Leu-Leu
1
3a-c
-
-
-
H2N
CO2H
HO
H2N
CO2H
H2N
N
CO2H
Ar =
Scheme 2. Solid-phase SuzukieMiyaura cyclization. Reagents and conditions:
(i) piperidine/DMF (3:7), 10 min. (ii) TrCl, DIEA, DMF, rt, 3 h. (iii) PdCl2(dppf), dppf,
B2Pin2, KOAc, DMSO, 80 ꢀC, 24 h. (iv) TFA/H2O/CH2Cl2 (0.2:1:98.8), 20 min. (v) Boc-Phe
(4-I)-OH, Boc-Tyr(3-I,Me)-OH or Boc-Tyr(3-I,SEM)-OH, ethyl cyanoglyoxylate-2-oxime,
DIPCDI, DMF, rt, 3 h. (vi) Pd2(dba)3, P(o-tolyl)3, KF, DME/EtOH/H2O, MWI, 120 ꢀC,
30 min. (vii) TFA/TIS/H2O, rt, 2 h.
HN
b
a
c
i) Pd2(dba)3, P(o-tolyl)3, ArX, KF, DME/EtOH/H2O, MWI,120 ºC, 30
min. ii) LiOH, THF/H2O, rt, 24 h. iii) TFA/TIS/H2O, rt, 2 h.
Selective removal of the Boc group with TMSOTf in the presence
of lutidine,9 followed by coupling of Fmoc-Leu-OH yielded Fmoc-
Leu-Phe(4-BPin)-Leu-Leu-NH2 (86% purity). However, elongation of
the peptide sequence by subsequent Fmoc removal and coupling
of two Fmoc-Lys(Boc)-OH residues resulted in the decomposition of
the boronate into the phenolic compound as shown by HPLC and
mass spectrometry analysis. Therefore, an alternative strategy for the
ArX
Peptide
Puritya (%)
Yieldb (%)
Boc-Phe(4-I)-OMe
3a
3b
3c
92
79
42
62
48
12
Boc-Tyr(3-I,SEM)-OMe
Boc-His(5-Br,1-SEM)-OMe and
Boc-His(5-Br,3-SEM)-OMe
a
Percentage determined by HPLC at 220 nm from the crude reaction mixture.
Isolated yield.
b

2240
A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
synthesis of 4aec was devised, which was based on the formation of
the boronate at the last steps of the synthesis (Scheme 2). In par-
ticular, this strategy consisted on (i) the preparation of Fmoc-Lys
(Boc)-Lys(Boc)-Leu-Phe(4-I)-Leu-Leu-Rink-MBHA; (ii) replacement
of the Fmoc group by a trityl to overcome its instability under the
Miyaura borylation conditions; (iii) formation of the boronate using
the conditions described above; (iv) trityl removal, and (v) coupling
of the corresponding iodophenylalanine or iodotyrosine derivative.
Acidolytic cleavage of an aliquot of the resulting resins 4aec yielded
the corresponding boronopeptides in purities ranging from 72 to
81%. This strategy was applied for the preparation of peptidyl resins
5aec and led to the expected borylated sequences in 76e89% purity.
All boronopeptides were characterized by HRMS. These results
strengthened the usefulness of our methodology for the solid-phase
borylation of peptides under Miyaura conditions.
With the properly functionalized peptidyl resins 4aec and 5aec
in hand, we set out to examine the macrocyclization by way of an
intramolecular SuzukieMiyaura reaction (Scheme 2 and Fig. 1).
Exposure of resins 4aec and 5aec to Pd2(dba)3, P(o-tolyl)3, and KF
in DME/EtOH/H2O under microwave irradiation at 120 ꢀC for
30 min provided the expected biaryl cyclic peptides 6aec and 7aec
in 72e79% purity. Pure compounds were obtained in 23e35% yield
after purification by column chromatography and were character-
ized by mass spectrometry and 1H NMR, which was consistent with
the formation of the biaryl bond.
Fig. 2. Structures of biaryl cyclic peptides 13e17.
six residues was the most troublesome, leading to the biaryl cyclic
peptide 15 in 36% purity together with a mixture of byproducts that
could not be identified. The other cyclizations gave biaryl cyclic
peptides 13, 14, 16, and 17 in purities ranging from 70 to 84%. The
structure of all biaryl cyclic peptides was confirmed by HRMS.
3. Conclusion
In conclusion, wehaveestablished the viabilityof the solid-phase
borylation and SuzukieMiyaura arylation for the synthesis of biaryl
cyclic peptides of three to eight residues. This work shows that the
SuzukieMiyaura reaction can be applied to the cross-coupling of
boronophenylalanine-containing peptidyl resins with halogenated
aromatic amino acids. Moreover, it constitutes the first example of
solid-phase intramolecular SuzukieMiyaura cross-coupling for the
formation of biaryl cyclic peptides. We expect that this methodology
could find applications in the synthesis of natural products and in
the diversity-oriented synthesis of biaryl-containing macrocycles.
4. Experimental section
4.1. General methods
Commercially available reagents were used throughout without
purification. Solvents were purified and dried by passing them
through an activated alumina purification system (MBraun SPS-
800) or by conventional distillation techniques.
Flash chromatography purifications were performed on C18-re-
versed phase silica gel 100 not endcapped (230e400 mesh, Fluka).
All compounds were analyzed under standard analytical HPLC
conditions with a Dionex liquid chromatography instrument com-
posed 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. Method A: Analysis
Fig. 1. Structures of biaryl cyclic peptides 6aec and 7aec.
Finally, we evaluated the applicability of the intramolecular
SuzukieMiyaura macrocyclization to the preparation of biaryl cy-
clic peptides of different ring sizes (Table 2, Fig. 2). Linear peptidyl
resins 8e12, which contained 3, 4, 6, 7 or 8 amino acids were
prepared and cyclized via arylearyl bond formation following the
strategy depicted in Scheme 2. Acidolytic cleavage of an aliquot of
resins 8e12 allowed the characterization by mass spectrometry of
the linear precursors. Cyclization of the peptidyl resin 10 containing
was carried out with a Kromasil 100 C18 (250 mmꢁ4.6 mm, 3.5
mm)
Table 2
columnwith a 2ꢂ100% B linear gradient over 28 min at a flow rate of
1 mL/min. Solvent A was 0.1% aq TFA, and solvent B was 0.1% TFA in
CH3CN. Method B: Analysis was carried out with a Kromasil 100 C18
Results of the synthesis of biaryl cyclic peptides 13e17
Linear peptidyl resin
Cyclic peptide
Puritya (%)
(40ꢁ4.6 mm, 3.5
mm) column with a 2e100% B linear gradient over
7 min at a flow rate of 1 mL/min.
Boc-Phe(4-I)-Leu-Phe(4-BPin)-Rink-MBHA (8)
Boc-Phe(4-I)-Lys(Boc)-Leu-Phe(4-BPin)-
Rink-MBHA (9)
Boc-Phe(4-I)-Phe-Lys(Boc)-Lys(Boc)-Leu-
Phe(4-BPin)-Rink-MBHA (10)
Boc-Phe(4-I)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-
Leu-Phe(4-BPin)-Rink-MBHA (11)
Boc-Phe(4-I)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-
Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (12)
13
14
71
70
ESI-MS analyses were performed with an Esquire 6000 ESI ion
Trap LC/MS (Bruker Daltonics) instrument equipped with an elec-
trospray ion source. The instrument was operated in the positive
ESI (þ) ion mode. Samples (5
spectrometer ion source directly through an HPLC autosampler. The
mobile phase (80:20 CH3CN/H2O at a flow rate of 100
Lminꢂ1) was
delivered by a 1100 Series HPLC pump (Agilent). Nitrogen was
15
16
17
36
84
77
mL) were introduced into the mass
m
a
Percentage determined by HPLC at 220 nm from the crude reaction mixture.

A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
2241
employed as both the drying and nebulizing gas. HRMS were
recorded under conditions of ESI with a Bruker MicroTof-Q in-
strument using a hybrid quadrupole time-of-flight mass spec-
trometer (University of Zaragoza). Samples were introduced into
the mass spectrometer ion source directly through a 1100 Series
Agilent HPLC autosampler and were externally calibrated using
sodium formate. The instrument was operated in the positive ESI
(þ) ion mode.
8.8 Hz, 1H, CH(
a
)-Phe], 4.41e4.52 [m, 2H, 2ꢁCH(
a
)-Leu], 7.39 [m,
4H, CHarom-2, CHarom-6, CHarom-30, CHarom-50], 7.62 [d, J¼8.0 Hz, 4H,
CHarom-3, CHarom-5, CHarom-20, CHarom-6] ppm. 13C NMR (100 MHz,
CD3CNþD2O):
[2ꢁCH( )-Leu], 36.28, 37.16 [2ꢁCH2(
[2ꢁCH2(
d
¼21.90, 23.25, 23.33 [4ꢁCH3(
)-Phe], 41.23, 41.36
)-Leu], 55.06 [2ꢁCH( )-Phe],
d)-Leu], 25.33, 25.56
g
b
b
)-Leu], 52.56, 53.44 [2ꢁCH(
a
a
128.28 [CHarom-3, CHarom-5, CHarom-20, CHarom-60], 131.15, 131.22
[CHarom-2, CHarom-6, CHarom-30, CHarom-50], 134.43, 134.83 [Carom-1,
Carom-40], 140.48 [Carom-4, Carom-10], 168.95, 173.19, 176.31 [4ꢁCO]
ppm. MS (ESI): m/z¼554.3 [MþH]þ. HRMS (ESI): calcd for
C30H44N5O5 554.3337; found 554.3362.
1H and 13C NMR spectra were measured with a Bruker 300 or
400 MHz NMR spectrometer. Chemical shifts were reported as
d
values (ppm) directly referenced to the solvent signal.
Microwave-assisted reactions were performed with an Ethos SEL
labstation microwave (Milestone) equipped with a dual magnetron
(1600 W). The time, temperature, and power were controlled with the
EasyControl software. The temperature was monitored through the
ATC-400FO Automatic Fiber Optic Temperature Control System im-
mersed in a standard Milestone reference vessel. This equipment
regulatesthepowertoachieveandmaintaintheselectedtemperature.
4.2.2. Linear biaryl peptide 3b. Starting from resin Boc-Phe(4-BPin)-
Leu-Leu-Rink-MBHA (1, 100 mg), the SuzukieMiyaura reaction was
performed following the general procedure using Boc-Tyr(3-I,SEM)-
OMe.8 After the reaction time, acidolytic cleavage of an aliquot of the
biaryl peptidyl resin afforded the corresponding methyl ester (82%
purity). tR¼16.47 min (method A). MS (ESI): m/z¼584.3 [MþH]þ.
After hydrolysis, the crude biaryl peptide 3b was obtained in 79%
purity. Elution with H2O/MeOH/TFA (70:30:0.2) yielded pure 3b
(12.5 mg, 48% yield). tR¼15.96 min (method A). 1H NMR (400 MHz,
4.2. General method for the arylation of linear peptides via
a solid-phase SuzukieMiyaura reaction
CD3OD):
2ꢁCH( )-Leu, 2ꢁCH2(
Phe], 4.04e4.27 [m, 2H, CH(
d
¼0.94e0.98 [m, 12H, 4ꢁCH3(
d)-Leu], 1.57e1.73 [m, 6H,
A 5 mL quartz vial was charged with the boronopeptidyl resin
Boc-Phe(4-BPin)-Leu-Leu-Rink-MBHA (1),6 the halogenated aro-
matic amino acid conveniently protected (5 equiv), Pd2(dba)3
(0.2 equiv), P(o-tolyl)3 (0.4 equiv), and KF (4 equiv). Thoroughly
degassed DME/EtOH/H2O (9:9:2, 1e1.5 mL) was then added under
nitrogen. The reaction mixture was heated at 120 ꢀC under mi-
crowave irradiation for 30 min. After this time, the resin was
washed with DMF (6ꢁ1 min), EtOH (6ꢁ1 min), CH2Cl2 (6ꢁ1 min),
and Et2O (3ꢁ1 min). An aliquot of the resulting biaryl peptidyl 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 Et2O ex-
traction, the crude peptide was dissolved in H2O/CH3CN, lyophi-
lized, and analyzed by HPLC.
The rest of the biaryl peptidyl resin was hydrolyzed by the
treatment with LiOH (5 equiv) in THF/H2O (7:1) at room tempera-
ture for 24 h. After the reaction time, the solvent was removed and
the resin was washed with DMF (3ꢁ1 min), MeOH (2ꢁ1 min), H2O
(2ꢁ1 min), DMF (3ꢁ1 min), and CH2Cl2 (3ꢁ1 min). The resulting
biaryl peptide was released from the solid support by treatment
with TFA/H2O/TIS (95:2.5:2.5) with stirring for 2 h at room tem-
perature. Following TFA evaporation and Et2O extraction, the crude
peptide was dissolved in H2O/CH3CN, lyophilized, analyzed by
HPLC, and purified by reverse-phase column chromatography.
Biaryl peptides were characterized by MS and NMR.
g
b
)-Leu], 2.99e3.30 [m, 4H, CH2(
b
)-Tyr, CH2( )-
b
a
)-Phe, CH(a)-Tyr], 4.39e4.51 [m, 2H,
2ꢁCH(
a
)-Leu], 6.90 [d, J¼7.8 Hz, 1H, CHarom-30], 7.10 [d, J¼7.8 Hz, 1H,
CHarom-40], 7.21 [s, 1H, CHarom-60], 7.33 [d, J¼7.8 Hz, 2H, CHarom-2,
CHarom-6], 7.56 [d, J¼7.8, 2H, CHarom-3, CHarom-5] ppm. 13C NMR
(100 MHz, CD3OD):
d
¼21.91, 21.98, 23.32, 23.40 [4ꢁCH3(
d
)-Leu],
)-Phe], 41.81,
)-Tyr, CH( )-
25.73, 25.84 [2ꢁCH(
g
)-Leu], 38.21 [CH2(
b)-Tyr, CH2(b
42.06 [2ꢁCH2(
b
)-Leu], 53.31 [CH(
a
)-Leu], 55.45 [CH(
a
a
Phe], 117.38 [CHarom-30], 126.88 [Carom-50], 129.74 [Carom-10], 130.21
[CHarom-2, CHarom-6], 130.68 [CHarom-40], 131.07 [CHarom-3, CHarom
-
5], 132.53 [CHarom-60], 133.98 [Carom-1], 139.39 [Carom-4], 155.07
[Carom-20],169.58,174.04 [4ꢁCO] ppm. MS (ESI): m/z¼570.3 [MþH]þ.
HRMS (ESI): calcd for C30H44N5O6 570.3286; found 570.3306; calcd
for C30H43N5NaO6 592.3106; found 592.3110.
4.2.3. Linear biaryl peptide 3c. Starting from resin Boc-Phe(4-BPin)-
Leu-Leu-Rink-MBHA (1, 100 mg), the SuzukieMiyaura reaction was
performed following the general procedure using a regioisomeric
mixture of Boc-His(5-Br,3-SEM)-OMe and Boc-His(5-Br,1-SEM)-
OMe.3 After the reaction time, acidolytic cleavage of an aliquot of the
biaryl peptidyl resin afforded the corresponding methyl ester (37%
purity). tR¼14.68 min (method A). After hydrolysis, the crude biaryl
peptide 3c was obtained in 42% purity. Elution with H2O/MeOH/TFA
(90:10:0.2) yielded pure 3c (3 mg,12% yield). tR¼14.59 min (method
A). 1H NMR (400 MHz, CD3OD):
Leu], 1.57e1.73 [m, 6H, 2ꢁCH( )-Leu, 2ꢁCH2(
1H, CH2( )-Phe], 3.27e3.35 [m, 1H, CH2( )-Phe], 3.43e3.48 [m, 2H,
CH( )-His], 4.03e4.09 [m, 1H, CH( )-His], 4.14e4.17 [m, 1H, CH( )-
Phe], 4.34 [dd, J¼5.2, 9.6 Hz,1H, CH( )-Leu], 4.47 [m,1H, CH( )-Leu],
7.44 [d, J¼7.6 Hz, 2H, CHarom-2, CHarom-6], 7.54 [d, J¼7.6 Hz, 2H,
CHarom-3, CHarom-5], 7.58 [s, 1H, CHimid], 8.74 [br s, 1H, NH] ppm. 13
NMR (100 MHz, CD3OD): )-Leu],
¼23.30, 23.39, 23.49 [4ꢁCH3(
25.74, 25.94 [2ꢁCH( )-Leu, CH2( )-His], 38.35 [CH2( )-Phe], 41.68,
41.89 [CH2( )-Leu], 55.28 [CH( )-Phe, CH( )-
)-Leu], 53.40 [2ꢁCH(
d
¼0.94e0.99 [m, 12H, 4ꢁCH3(
d)-
4.2.1. Linear biaryl peptide 3a. Starting from resin Boc-Phe(4-
BPin)-Leu-Leu-Rink-MBHA (1, 100 mg), the SuzukieMiyaura
reaction was performed following the general procedure using Boc-
Phe(4-I)-OMe.7 After the reaction time, acidolytic cleavage of an
aliquot of the biaryl peptidyl resin afforded the corresponding
methyl ester (87% purity). tR¼16.84 and 17.20 min (method A). MS
(ESI): m/z¼568.3 [MþH]þ, 590.3 [MþNa]þ. After hydrolysis, the
crude biaryl peptide 3a was obtained in 92% purity. Elution with
H2O/MeOH/TFA (80:20:0.2) yielded pure 3a (15.9 mg, 62% yield).
tR¼16.28 and 16.64 min (method A). 1H NMR (400 MHz,
g
b
)-Leu], 3.08e3.13 [m,
b
b
b
a
a
a
a
C
d
d
g
b
b
b
a
a
a
His], 129.76 [CHarom-3, CHarom-5], 131.58 [CHarom-2, CHarom-6],
169.41, 174.01 [4ꢁCO] ppm. MS (ESI): m/z¼544.2 [MþH]þ. HRMS
(ESI): calcd for C27H42N7O5 544.3242; found 544.3242; calcd for
C27H41N7NaO5 566.3061; found 566.3046.
CD3CNþD2O):
d
¼0.84e0.90 [m, 12H, 4ꢁCH3(
d
)-Leu], 1.50e1.60 [m,
6H, 2ꢁCH( )-Leu, 2ꢁCH2(
g
b
)-Leu], 3.16e3.32 [m, 4H, CH2(b
)-Phe],
4.17e4.32 [m, 4H, 2ꢁCH(
a)-Leu, 2ꢁCH(
a)-Phe], 7.33e7.37 [m, 4H,
CHarom-2, CHarom-6, CHarom-30, CHarom-50], 7.60e7.64 [m, 4H,
CHarom-3, CHarom-5, CHarom-20, CHarom-6] ppm. 1H NMR (400 MHz,
4.3. Solid-phase synthesis of cyclic biaryl peptides 6aec, 7aec,
and 13e17
CD3OD):
2ꢁCH( )-Leu, 2ꢁCH2(
Phe], 3.15 [dd, J¼8.0, 14.4 Hz, 1H, CH2(
CH2(
)-Phe], 4.03 [dd, J¼5.2, 8.0 Hz, 1H, CH(
d
¼0.91e1.00 [m, 12H, 4ꢁCH3(
)-Leu], 3.06 [dd, J¼8.8, 14.0 Hz, 1H, CH2(
)-Phe], 3.32e3.39 [m, 2H,
)-Phe], 4.17 [dd, J¼5.2,
d
)-Leu], 1.56e1.75 [m, 6H,
g
b
b
)-
b
4.3.1. General method for solid-phase peptide synthesis. Peptidyl
resins were synthesized manually by the solid-phase method using
b
a

2242
A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
standard Fmoc chemistry. Fmoc-Rink-MBHA resin (0.56 mmol/g)
was used as solid support. Couplings of the corresponding amino
acids Fmoc-Leu-OH, Fmoc-Phe(4-I)-OH, Fmoc-Phe-OH, Fmoc-Lys
(Boc)-OH (4 equiv) were performed using ethyl cyanoglyoxylate-2-
oxime (4 equiv), N,N0-diisopropylcarbodiimide (DIPCDI) (4 equiv) in
DMF at room temperature for 1 h. The completion of the reactions
was monitored by the Kaiser test.10 Fmoc group removal was ach-
ieved with piperidine/DMF (3:7, 2þ10 min). After each coupling
and deprotection step, the resin was washed with DMF (6ꢁ1 min)
and CH2Cl2 (3ꢁ1 min) and air-dried. Once the peptide sequence
was completed, the Fmoc group was removed and the trityl group
was introduced using TrCl (10 equiv) and N,N-diisopropylethyl-
amine (DIEA) (10 equiv) in DMF at room temperature for 3 h. Then,
the resin was washed with DMF (6ꢁ1 min) and CH2Cl2 (3ꢁ1 min)
and air-dried. An aliquot of the resulting peptidyl resin was cleaved
with TFA/H2O/triisopropylsilane (TIS) (95:2.5:2.5) whilst being
stirred for 2 h at room temperature. Following TFA evaporation and
Et2O extraction, the crude peptide was dissolved in H2O/CH3CN,
lyophilized, analyzed by HPLC, and characterized by MS.
HPLC analysis. tR¼5.31 min (boronic acid), 6.39 min (boronate)
(method B). MS (ESI): m/z¼330.7 [Mþ2H]2þ, 578.3 [MþH]þ, 660.3
[MþH]þ.
4.3.2.3. Tr-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA. This peptidyl
resin was prepared starting from Tr-Lys(Boc)-Leu-Phe(4-I)-Rink-
MBHA (90 mg) following the general method for solid-phase
Miyaura borylation. Acidolytic cleavage of an aliquot of this pep-
tidyl resin afforded H-Lys-Leu-Phe(4-BPin)-NH2 and H-Lys-Leu-Phe
(4-B(OH)2)-NH2. MS (ESI): m/z¼450.2 [MþH]þ, 532.3 [MþH]þ.
4.3.2.4. Tr-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA. This
peptidyl resin was prepared starting from Tr-Phe-Lys(Boc)-Lys(Boc)-
Leu-Phe(4-I)-Rink-MBHA (90 mg) following the general method for
solid-phase Miyaura borylation. Acidolytic cleavage of an aliquot of
this peptidyl resin afforded H-Phe-Lys-Lys-Leu-Phe(4-BPin)-NH2 (37%
purity) and H-Phe-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (30% purity),
resulting from partial hydrolysis of the pinacol boronate during HPLC
analysis. tR¼14.52 min (boronic acid),18.54 min (boronate) (method A).
MS (ESI): m/z¼807.5 [MþH]þ.
4.3.1.1. Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-Leu-Leu-Rink-MBHA. This
peptidyl resin was prepared following the general method for solid-
phase peptide synthesis. Acidolytic cleavage of an aliquot of this pep-
tidyl resin afforded H-Lys-Lys-Leu-Phe(4-I)-Leu-Leu-NH2 (73% purity).
tR¼6.69 min (method B). MS (ESI): m/z¼443.6 [Mþ2H]2þ, 886.4
[MþH]þ.
4.3.2.5. Tr-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-
Rink-MBHA. This peptidyl resin was prepared starting from Tr-Lys
(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-Rink-MBHA (65 mg)
following the general method for solid-phase Miyaura borylation.
Acidolytic cleavage of an aliquot of this peptidyl resin afforded H-
Lys-Phe-Lys-Lys-Leu-Phe(4-BPin)-NH2 (35% purity) and H-Lys-Phe-
Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (28% purity), resulting from partial
hydrolysis of the pinacol boronate during HPLC analysis.
tR¼14.45 min (boronic acid), 18.12 min (boronate) (method A). MS
(ESI): m/z¼853.5 [MþH]þ, 935.5 [MþH]þ.
4.3.1.2. Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-Rink-MBHA. This pept
idyl resin was prepared following the general method for solid-
phase peptide synthesis. Acidolytic cleavage of an aliquot of this
peptidyl resin afforded H-Lys-Lys-Leu-Phe(4-I)-NH2 (54% purity).
tR¼6.27 min (method B). MS (ESI): m/z¼330.6 [Mþ2H]2þ, 660.2
[MþH]þ, 682.1 [MþNa]þ.
4.3.2.6. Tr-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-
BPin)-Rink-MBHA. This peptidyl resin was prepared starting from
Tr-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-I)-Rink-MB
HA (100 mg) following the general method for solid-phase Miyaura
borylation. Acidolytic cleavage of an aliquot of this peptidyl resin
afforded H-Lys-Lys-Phe-Lys-Lys-Leu-Phe(4-BPin)-NH2 (48% purity)
and H-Lys-Lys-Phe-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (28% purity),
resulting from partial hydrolysis of the pinacol boronate during
HPLC analysis. tR¼14.10 min (boronic acid), 17.61 min (boronate)
(method A). MS (ESI): m/z¼1093.5 [MꢂHþTFA]ꢂ, 1175.5
[MꢂHþTFA]ꢂ.
4.3.2. General method for solid-phase Miyaura borylation. A 25 mL
round-bottomed flask was charged with the corresponding iodo-
peptidyl resin, bis(pinacolato)diboron (B2Pin2) (4 equiv), PdCl2(dppf)
(0.18 equiv), and 1,10-bis(diphenylphosphanyl)ferrocene (dppf)
(0.09 equiv). A thoroughly sonicated solution of KOAc (6 equiv) in
degassed anhydrous DMSO (20 mL/mg of resin) was then added, and
the mixture was heated at 80 ꢀC for 24 h. Then, the resin was washed
with DMSO (6ꢁ1 min), MeOH (6ꢁ1 min), CH2Cl2 (6ꢁ1 min), and Et2O
(3ꢁ1 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 Et2O extraction,
thecrude peptide was dissolved in H2O/CH3CN, lyophilized, analyzed
by HPLC, and characterized by MS.
4.3.3. General method for the solid-phase synthesis of the linear
precursors 4aec, 5aec, and 8e12. The boronopeptidyl resins
obtained following the procedure described in section 4.3.2 were
treated with TFA/H2O/CH2Cl2 (0.2:1:98.8, 2ꢁ1 min, 1ꢁ20 min), and
then washed with DMF (3ꢁ1 min), DIEA/DCM (1:19) (3ꢁ1 min),
and DMF (3ꢁ1 min). The corresponding halogenated amino acid
Boc-Phe(4-I)-OH, Boc-Tyr(3-I,Me)-OH or Boc-Tyr(3-I,SEM)-OH was
coupled using ethyl cyanoglyoxylate-2-oxime (3 equiv), DIPCDI
(3 equiv) in DMF at room temperature for 3 h. The resin was washed
with DMF (6ꢁ1 min) and CH2Cl2 (3ꢁ1 min) and air-dried. The
completion of the reaction was monitored by the Kaiser test. An
aliquot of the resulting peptidyl resin was cleaved with TFA/H2O/TIS
(95:2.5:2.5) whilst being stirred for 2 h at room temperature. Fol-
lowing TFA evaporation and Et2O extraction, the crude peptide was
dissolved in H2O/CH3CN, lyophilized, analyzed by HPLC, and char-
acterized by MS.
4.3.2.1. Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-
MBHA. This peptidyl resin was prepared starting from Tr-Lys(Boc)-
Lys(Boc)-Leu-Phe(4-I)-Leu-Leu-Rink-MBHA (600 mg) following the
general method for solid-phase Miyaura borylation. Acidolytic
cleavage of an aliquot of this peptidyl resin afforded H-Lys-Lys-Leu-
Phe(4-BPin)-Leu-Leu-NH2 (56% purity) and H-Lys-Lys-Leu-Phe(4-B
(OH)2)-Leu-Leu-NH2 (22% purity), resulting from partial hydrolysis
of the pinacol boronate during HPLC analysis. tR¼6.08 min (boronic
acid), 6.75 min (boronate) (method B). MS (ESI): m/z¼804.5
[MþH]þ, 886.5 [MþH]þ, 908.5 [MþNa]þ.
4.3.2.2. Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA. This
peptidyl resin was prepared starting from Tr-Lys(Boc)-Lys(Boc)-
Leu-Phe(4-I)-Rink-MBHA (400 mg) following the general method
for solid-phase Miyaura borylation. Acidolytic cleavage of an ali-
quot of this peptidyl resin afforded H-Lys-Lys-Leu-Phe(4-BPin)-NH2
(67% purity) and H-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (11% purity),
resulting from partial hydrolysis of the pinacol boronate during
4.3.3.1. Boc-Phe(4-I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-
Leu-Rink-MBHA (4a). This peptidyl resin was prepared starting
from Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA
following the general method using Boc-Phe(4-I)-OH7 as haloge-
nated amino acid. Acidolytic cleavage of an aliquot of the resulting

A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
2243
peptidyl resin afforded H-Phe(4-I)-Lys-Lys-Leu-Phe(4-BPin)-Leu-
Leu-NH2 (52% purity) and H-Phe(4-I)-Lys-Lys-Leu-Phe(4-B(OH)2)-
Leu-Leu-NH2 (25% purity), resulting from partial hydrolysis of the
pinacol boronate during HPLC analysis. tR¼6.67 min (boronic acid),
7.27 min (boronate) (method B). MS (ESI): m/z¼1077.4 [MþH]þ,
1159.4 [MþH]þ, 1181.4 [MþNa]þ.
MBHA following the general method using Boc-Phe(4-I)-OH7 as
halogenated amino acid. Acidolytic cleavage of an aliquot of the
resulting peptidyl resin afforded H-Phe(4-I)-Leu-Phe(4-BPin)-NH2.
MS (ESI): m/z¼677.1 [MþH]þ.
4.3.3.8. Boc-Phe(4-I)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA
(9). This peptidyl resin was prepared starting from Tr-Lys(Boc)-
Leu-Phe(4-BPin)-Rink-MBHA following the general method using
Boc-Phe(4-I)-OH7 as halogenated amino acid. Acidolytic cleavage of
an aliquot of the resulting peptidyl resin afforded H-Phe(4-I)-Lys-
Leu-Phe(4-BPin)-NH2 and H-Phe(4-I)-Lys-Leu-Phe(4-B(OH)2)-NH2.
MS (ESI): m/z¼723.1 [MþH]þ, 805.2 [MþH]þ.
4.3.3.2. Boc-Tyr(3-I,Me)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-
Leu-Rink-MBHA (4b). This peptidyl resin was prepared starting
from Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA
following the general method using Boc-Tyr(3-I,Me)-OH8 as halo-
genated amino acid. Acidolytic cleavage of an aliquot of the
resulting peptidyl resin afforded H-Tyr(3-I,Me)-Lys-Lys-Leu-Phe(4-
BPin)-Leu-Leu-NH2 (59% purity) and H-Tyr(3-I,Me)-Lys-Lys-Leu-
Phe(4-B(OH)2)-Leu-Leu-NH2 (13% purity), resulting from partial
hydrolysis of the pinacol boronate during HPLC analysis.
tR¼6.53 min (boronic acid), 7.11 min (boronate) (method B). MS
(ESI): m/z¼595.4 [Mþ2H]2þ, 1107.4 [MþH]þ, 1189.5 [MþH]þ.
4.3.3.9. Boc-Phe(4-I)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-
Rink-MBHA (10). This peptidyl resin was prepared starting from Tr-
Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA following the
general method using Boc-Phe(4-I)-OH7 as halogenated amino
acid. Acidolytic cleavage of an aliquot of the resulting peptidyl resin
afforded H-Phe(4-I)-Phe-Lys-Lys-Leu-Phe(4-BPin)-NH2 and H-Phe
(4-I)-Phe-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2. MS (ESI): m/z¼998.3
[MþH]þ, 1080.3 [MþH]þ.
4.3.3.3. Boc-Tyr(3-I,SEM)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-
Leu-Leu-Rink-MBHA (4c). This peptidyl resin was prepared starting
from Tr-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA
following the general method using Boc-Tyr(3-I,SEM)-OH8 as ha-
logenated amino acid. Acidolytic cleavage of an aliquot of the
resulting peptidyl resin afforded H-Tyr(3-I)-Lys-Lys-Leu-Phe
(4-BPin)-Leu-Leu-NH2 (42% purity) and H-Tyr(3-I)-Lys-Lys-Leu-Phe
(4-B(OH)2)-Leu-Leu-NH2 (39% purity), resulting from partial hy-
drolysis of the pinacol boronate during HPLC analysis. tR¼6.47 min
(boronic acid), 7.13 min (boronate) (method B). MS (ESI): m/
z¼1093.2 [MþH]þ, 1175.2 [MþH]þ.
4.3.3.10. Boc-Phe(4-I)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe
(4-BPin)-Rink-MBHA (11). This peptidyl resin was prepared starting
from Tr-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MB
HA following the general method using Boc-Phe(4-I)-OH7 as haloge-
nated amino acid. Acidolytic cleavage of an aliquot of the resulting
peptidyl resin afforded H-Phe(4-I)-Lys-Phe-Lys-Lys-Leu-Phe(4-BPin)-
NH2 (38% purity) and H-Phe(4-I)-Lys-Phe-Lys-Lys-Leu-Phe(4-B(OH)2)-
NH2 (23% purity), resulting from partial hydrolysis of the pinacol
boronate during HPLC analysis. tR¼16.81 min (boronic acid),19.62 min
(boronate) (method A).
4.3.3.4. Boc-Phe(4-I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-
MBHA (5a). This peptidyl resin was prepared starting from Tr-Lys
(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA following the general
method using Boc-Phe(4-I)-OH7 as halogenated amino acid. Acid-
olytic cleavage of an aliquot of the resulting peptidyl resin afforded
H-Phe(4-I)-Lys-Lys-Leu-Phe(4-BPin)-NH2 (54% purity) and H-Phe
(I)-Lys-Lys-Leu-Phe(B(OH)2)-NH2 (22% purity), resulting from par-
tial hydrolysis of the pinacol boronate during HPLC analysis.
tR¼5.79 min (boronic acid), 6.72 min (boronate) (method B). MS
(ESI): m/z¼851.2 [MþH]þ, 933.3 [MþH]þ, 955.3 [MþNa]þ.
4.3.3.11. Boc-Phe(4-I)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-
Leu-Phe(4-BPin)-Rink-MBHA (12). This peptidyl resin was prepared
starting from Tr-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe
(4-BPin)-Rink-MBHA following the general method using Boc-Phe
(4-I)-OH7 as halogenated amino acid. Acidolytic cleavage of an
aliquot of the resulting peptidyl resin afforded H-Phe(4-I)-Lys-Lys-
Phe-Lys-Lys-Leu-Phe(4-BPin)-NH2 (45% purity) and H-Phe(4-I)-Lys-
Lys-Phe-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (29% purity), resulting
from partial hydrolysis of the pinacol boronate during HPLC analysis.
tR¼15.83 min (boronic acid), 18.58 min (boronate) (method A). MS
(ESI): m/z¼627.8 [Mþ2H]2þ, 1254.4 [MþH]þ.
4.3.3.5. Boc-Tyr(3-I,Me)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-
Rink-MBHA (5b). This peptidyl resin was prepared starting from Tr-
Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA following the gen
eral method using Boc-Tyr(3-I,Me)-OH8 as halogenated amino acid.
Acidolytic cleavage of an aliquot of the resulting peptidyl resin
afforded H-Tyr(3-I,Me)-Lys-Lys-Leu-Phe(4-BPin)-NH2 (71% purity)
and H-Tyr(3-I,Me)-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (18% purity),
resulting from partial hydrolysis of the pinacol boronate during
HPLC analysis. tR¼6.01 min (boronic acid), 6.89 min (boronate)
(method B). MS (ESI): m/z¼881.2 [MþH]þ, 963.2 [MþH]þ.
4.3.4. General method for the cyclization via a Solid-phase Suzu-
kieMiyaura reaction. A 5 mL quartz vial was charged with the linear
precursor incorporating the borono and iodo functionalities,
Pd2(dba)3 (0.2 equiv), P(o-tolyl)3 (0.4 equiv), and KF (4 equiv). Thor-
oughly degassed DME/EtOH/H2O (9:9:2, 1e1.9 mL) was then added
under nitrogen. The reaction mixture was heated at 120 ꢀC under
microwave irradiation for 30 min. After this time, the resin was
washed withDMF(6ꢁ1 min), EtOH(6ꢁ1 min), CH2Cl2 (6ꢁ1 min), and
Et2O (3ꢁ1 min). The resulting cyclic biaryl peptidyl 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 Et2O extraction, the
crude peptide was dissolved in H2O/CH3CN, lyophilized, analyzed by
HPLC, and purified by reverse-phase column chromatography. Cyclic
biaryl peptides were characterized by MS and NMR.
4.3.3.6. Boc-Tyr(3-I,SEM)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-
Rink-MBHA (5c). This peptidyl resin was prepared starting from Tr-
Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA following the gen
eral method using Boc-Tyr(3-I,SEM)-OH8 as halogenated amino
acid. Acidolytic cleavage of an aliquot of the resulting peptidyl resin
afforded H-Tyr(3-I)-Lys-Lys-Leu-Phe(4-BPin)-NH2 (67% purity) and
H-Tyr(3-I,Me)-Lys-Lys-Leu-Phe(4-B(OH)2)-NH2 (21% purity), resu
lting from partial hydrolysis of the pinacol boronate during HPLC
analysis. tR¼5.76 min (boronic acid), 6.72 min (boronate) (method
B). MS (ESI): m/z¼867.2 [MþH]þ, 949.3 [MþH]þ.
4.3.4.1. Cyclic biaryl peptide 6a. Starting from resin Boc-Phe
(4-I)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA (4a)
(178 mg), SuzukieMiyaura cyclization followed by acidolytic
cleavage afforded the cyclic biaryl peptide 6a, which was dissolved
in H2O/CH3CN, lyophilized, analyzed by HPLC (74% purity) and
purified by reverse-phase column chromatography. Elution with
4.3.3.7. Boc-Phe(4-I)-Leu-Phe(4-BPin)-Rink-MBHA (8). This pept
idyl resin was prepared starting from Tr-Leu-Phe(4-BPin)-Rink-

2244
A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
H2O/MeOH/TFA (70:30:0.2) yielded pure 6a (15 mg, 28% yield).
tR¼17.48 min (method A). 1H NMR (400 MHz, CD3OD):
3.22e3.39 [m, 2H, CH2(
b
)-Phe], 4.03 [dd, J¼5.2, 12.0 Hz, 1H, CH(
a)-
Phe], 4.11e4.14 [m,1H, 2ꢁCH(
a
)-Lys], 4.348e4.40 [m,1H, CH( )-Leu],
a
d
¼0.93e0.98 [m, 18H, 6ꢁCH3(
d
)-Leu], 1.20e1.74 [m, 21H, 3ꢁCH(
g
)-
)-Lys],
)-Lys], 3.25e3.33 [m, 2H,
)-Phe], 4.34e4.46 [m, 5H,
)-Leu], 4.69e4.72 [m, 1H, CH( )-Phe], 7.29 [d,
4.67e4.72 [m,1H, CH(a)-Phe], 7.26 [d, 8.2 Hz, 4H, CHarom-2, CHarom-6,
Leu, 3ꢁCH2(
2.84e2.93 [m, 6H, CH2(
CH2( )-Phe], 4.05e4.11 [m, 1H, CH(
2ꢁCH(
b
)-Leu, 2ꢁCH2(
b
)-Lys, 2ꢁCH2(
g
)-Lys, 2ꢁCH2(
d
CHarom-30, CHarom-50], 7.67 [d, J¼8.2 Hz, 4H, CHarom-3, CHarom-5,
b
)-Phe, 2ꢁCH2(
3
CHarom-20, CHarom-60] ppm. 13C NMR (100 MHz, CD3OD):
d
¼21.49
)-Leu], 28.24
)-Phe], 38.69 [CH2( )-Phe],
)-Leu], 51.94 [CH( )-Leu], 54.32 [CH
)-Phe], 127.83, 127.95 [CHarom-3,
CHarom-5, CHarom-20, CHarom-60], 130.88 [CHarom-2, CHarom-6, CHarom
b
a
[CH2(
g
)-Lys], 23.50, 23.69 [2ꢁCH3(
d
)-Leu], 25.66 [CH(
g
a
)-Lys, 3ꢁCH(
a
a
[CH2(
b
)-Lys, CH2(
d
)-Lys], 38.15 [CH2(
b
b
J¼8.0 Hz, 4H, CHarom-2, CHarom-6, CHarom-30, CHarom-50], 7.67 [d,
J¼8.0 Hz, 4H, CHarom-3, CHarom-5, CHarom-20, CHarom-60] ppm. MS
(ESI): m/z¼905.6 [MþH]þ. HRMS (ESI): calcd for C48H78N10O7
453.3022; found 453.3034; calcd for C48H77N10O7 905.5971; found
905.5969; calcd for C48H76N10NaO7 927.5791; found 927.5772.
40.43 [2ꢁCH2(
3
)-Lys], 41.39 [CH(
b
a
(a
)-Phe, CH(a)-Lys], 55.77 [CH(a
-
30, CHarom-50], 131.38 [Carom], 134.53 [Carom], 137.56 [Carom], 140.90
[Carom],168.81,175.91 [CO] ppm. MS (ESI): m/z¼679.4 [MþH]þ. HRMS
(ESI): calcd for C36H56N8O5 340.2181; found 340.2167; calcd for
C36H55N8O5 679.4290; found 679.4273; calcd for C36H54N8NaO5
701.4109; found 701.4086.
4.3.4.2. Cyclic biaryl peptide 6b. Starting from resin Boc-Tyr(3-
I,Me)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA (4b)
(182mg), SuzukieMiyaura cyclization followed byacidolytic cleavage
afforded the cyclic biaryl peptide 6b, which was dissolved in H2O/
CH3CN, lyophilized, analyzed by HPLC (72% purity) and purified by
reverse-phase column chromatography. Elution with H2O/MeOH/
TFA (70:30:0.2) yielded pure 6b (12.7 mg, 23% yield). tR¼17.63 min
4.3.4.5. Cyclic biaryl peptide 7b. Starting from resin Boc-Tyr(3-
I,Me)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (5b) (167 mg),
SuzukieMiyaura cyclization followed by acidolytic cleavage affor-
ded the cyclic biaryl peptide 7b, which was dissolved in H2O/
CH3CN, lyophilized, analyzed by HPLC (75% purity) and purified by
reverse-phase column chromatography. Elution with H2O/MeOH/
TFA (90:10:0.2) yielded pure 7b (12.6 mg, 30% yield). tR¼14.56 min
(method A). 1H NMR (400 MHz, CD3OD):
d
¼0.91e0.98 [m, 18H,
)-Leu, 3ꢁCH2( )-Leu,
)-Lys], 2.70e2.72 [m, 2H
)-Lys, CH2( )-Phe], 3.13e3.19
)-Tyr], 3.78 [s, 3H, OCH3], 4.09e4.15 [m,
)-Lys], 4.18e4.49 [m, 3H, 3ꢁCH( )-Leu],
4.57e4.64 [m, 2H, CH( )-Phe, CH(
)-Lys], 7.05 [d, J¼8.6 Hz, 1H,
6ꢁCH3(
d
)-Leu], 1.16e1.71 [m, 21H, 3ꢁCH(
g
b
2ꢁCH2(
b
)-Lys, 2ꢁCH2( )-Lys, 2ꢁCH2(
g
d
CH2(
3
)-Lys], 2.83e2.93 [m, 3H, CH2(
)-Phe, CH2(
)-Tyr, CH(
3
b
(method A). 1H NMR (400 MHz, CD3OD):
d
¼0.92e0.96 [m, 6H,
)-Leu, CH2( )-Leu,
)-Lys], 2.69e2.76 [m, 2H,
)-Lys, CH2( )-Phe], 3.14e3.16
)-Tyr], 3.21 [dd, J¼2.8, 14.0 Hz, 1H, CH2( )-Phe], 3.78
[s, 3H, OCH3], 4.06e4.10 [m, 2H, CH( )-Tyr, CH( )-Lys], 4.33e4.36
[m, 1H, CH(
J¼2.8, 12.0 Hz, 1H, CH(
[m, 3H, CH2(
2H, CH(
b
b
2ꢁCH3(
d
)-Leu], 1.18e1.61 [m, 15H, CH(
g
b
a
a
a
2ꢁCH2(
b
)-Lys, 2ꢁCH2( )-Lys, 2ꢁCH2(
g
d
a
a
CH2(
3
)-Lys], 2.83e2.99 [m, 3H, CH2(
3
b
CHarom-30], 7.22 [s, 1H, CHarom-60], 7.24 [dd, J¼2.4, 8.6 Hz, 1H,
CHarom-40], 7.32 [d, J¼8.2 Hz, 2H, CHarom-2, CHarom-6], 7.49 [d,
J¼8.2 Hz, 2H, CHarom-3, CHarom-5] ppm. MS (ESI): m/z¼468.3
[Mþ2H]2þ, 935.6 [MþH]þ, 957.5 [MþNa]þ. HRMS (ESI): calcd for
C49H80N10O8 468.3075; found 468.3098; calcd for C49H79N10O8
935.6077; found 935.6057; calcd for C49H78N10NaO8 957.5896;
found 957.5875.
[m, 2H, CH2(
b
b
a
a
a
)-Leu], 4.53 [dd, J¼5.8, 8.6 Hz, 1H, CH(
a)-Lys], 4.59 [dd,
a
)-Phe], 7.05 [d, J¼8.4 Hz, 1H, CHarom-30],
7.23e7.26 [m, 2H, CHarom-40, CHarom-60], 7.30 [d, J¼8.2 Hz, 2H,
CHarom-2, CHarom-6], 7.48 [d, J¼8.2 Hz, 2H, CHarom-3, CHarom-5]
ppm. MS (ESI): m/z¼709.4 [MþH]þ. HRMS (ESI): calcd for
C37H58N8O6 355.2234; found 355.2232; calcd for C37H57N8O6
709.4396; found 709.4372; calcd for C37H56N8NaO6 731.4215;
found 731.4188.
4.3.4.3. Cyclic biaryl peptide 6c. Starting from resin Boc-Tyr(3-
I,SEM)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Leu-Leu-Rink-MBHA (4c)
(195 mg), SuzukieMiyaura cyclization followed by acidolytic cleav-
age afforded the cyclic biaryl peptide 6c, which was dissolved in
H2O/CH3CN, lyophilized, analyzed by HPLC (79% purity) and puri-
fied by reverse-phase column chromatography. Elution with H2O/
MeOH/TFA (70:30:0.2) yielded pure 6c (20.5 mg, 35% yield).
tR¼17.18 min (method A). 1H NMR (400 MHz, CD3OD):
4.3.4.6. Cyclic biaryl peptide 7c. Starting from resin Boc-Tyr(3-
I,SEM)-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (5c) (185 mg),
SuzukieMiyaura cyclization followed by acidolytic cleavage affor-
ded the cyclic biaryl peptide 7c, which was dissolved in H2O/
CH3CN, lyophilized, analyzed by HPLC (76% purity) and purified by
reverse-phase column chromatography. Elution with H2O/MeOH/
TFA (90:10:0.2) yielded pure 7c (13 mg, 30% yield). tR¼13.59 min
d
¼0.87e0.97 [m, 18H, 6ꢁCH3(
Leu, 3ꢁCH2( )-Leu, 2ꢁCH2(
2.66e2.73 [m, 2H, CH2( )-Lys], 2.81e2.91 [m, 3H, CH2(
CH2( )-Phe], 3.07e3.18 [m, 3H, CH2( )-Tyr, CH2( )-Phe], 4.07e4.14
[m, 2H, CH( )-Tyr, CH( )-Leu],
)-Lys], 4.27e4.54 [m, 3H, 3ꢁCH(
4.57e4.61 [m, 2H, CH( )-Phe, CH(
)-Lys], 6.88 [d, J¼8.0 Hz, 1H,
d
b
)-Leu], 1.45e1.70 [m, 21H, 3ꢁCH(
)-Lys, 2ꢁCH2( )-Lys, 2ꢁCH2( )-Lys],
)-Lys,
g)-
b
g
d
3
3
(method A). 1H NMR (400 MHz, CD3OD):
d
¼0.92e0.97 [m, 6H,
)-Leu, CH2( )-Leu,
)-Lys], 2.61e2.75 [m, 2H,
)-Lys, CH2( )-Phe], 3.12e3.13
)-Phe],
)-Tyr], 4.37e4.40 [m, 1H, CH( )-
)-Phe], 6.89 [d, J¼8.2 Hz,
b
b
b
2ꢁCH3(
d
)-Leu], 1.18e1.67 [m, 15H, CH(
g
b
a
a
a
a
2ꢁCH2(
b
)-Lys, 2ꢁCH2( )-Lys, 2ꢁCH2(
g
d
a
CH2(
3
)-Lys], 2.80e2.94 [m, 3H, CH2(
3
b
CHarom-30], 7.09 [dd, J¼2.0, 8.0 Hz, 1H, CHarom-40], 7.24e7.25 [m, 1H,
CHarom-60], 7.32 [d, J¼8.2 Hz, 2H, CHarom-4, CHarom-6], 7.60 [d,
J¼8.2 Hz, 2H, CHarom-3, CHarom-5] ppm. MS (ESI): m/z¼921.6
[MþH]þ. HRMS (ESI): calcd for C48H78N10O8 461.2997; found
461.3017; calcd for C48H77N10O8 921.5920; found 921.5900; calcd
for C48H76N10NaO8 943.5740; found 943.5713.
[m, 2H, CH2(
4.06e4.10 [m, 2H, CH(
Leu], 4.52e4.58 [m, 2H, CH(
b
)-Tyr], 3.21 [dd, J¼2.8, 14.0 Hz, 1H, CH2(
b
a
)-Lys, CH(
a
a
a
)-Lys, CH(a
1H, CHarom-30], 7.09 [dd, 1H, J¼2.4, 8.2 Hz, CHarom-40], 7.31 [d, 2H,
J¼8.2 Hz, CHarom-2, CHarom-6], 7.53 [br s, 1H, CHarom-60], 7.59 [d, 2H,
J¼8.2 Hz, CHarom-3, CHarom-5] ppm. MS (ESI): m/z¼695.4 [MþH]þ.
HRMS (ESI): calcd for C36H56N8O6 348.2156; found 348.2145; calcd
for C36H55N8O6 695.4239; found 695.4225; calcd for C36H54N8NaO6
717.4059; found 717.4047.
4.3.4.4. Cyclic biaryl peptide 7a. StartingfromresinBoc-Phe(4-I)-
Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (5a) (135 mg),
SuzukieMiyaura cyclization followed by acidolytic cleavage afforded
the cyclic biaryl peptide 7a, which was dissolved in H2O/CH3CN, ly-
ophilized, analyzed by HPLC (76% purity) and purified by reverse-
phase column chromatography. Elution with H2O/MeOH/TFA
(90:10:0.2)yieldedpure7a(8.3mg,25%yield). tR¼14.60 min (method
4.3.4.7. Cyclic biaryl peptide 13. Starting from resin Boc-Phe(4-
I)-Leu-Phe(4-BPin)-Rink-MBHA (8) (21 mg), SuzukieMiyaura cy-
clization followed by acidolytic cleavage afforded the cyclic biaryl
peptide 13, which was dissolved in H2O/CH3CN, lyophilized, and
analyzed by HPLC (71% purity). tR¼17.22 min (method A). MS (ESI):
m/z¼423.2 [MþH]þ. HRMS (ESI): calcd for C24H31N4O3 423.2391;
found 423.2391.
A). 1H NMR (400 MHz, CD3OD):
1.20e1.62 [m, 15H, CH( )-Leu, CH2(
Lys, 2ꢁCH2( )-Lys], 2.84e3.02 [m, 6H, CH2(
d
¼0.85e0.90 [m, 6H, 2ꢁCH3(
)-Leu, 2ꢁCH2( )-Lys, 2ꢁCH2(
)-Phe, 2ꢁCH2( )-Lys],
d
)-Leu],
g
b
b
g)-
d
b
3

A. Afonso et al. / Tetrahedron 67 (2011) 2238e2245
2245
ꢁ
4.3.4.8. Cyclic biaryl peptide 14. Starting from resin Boc-Phe(4-
I)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (9) (60 mg), Suzukie
Miyaura cyclization followed by acidolytic cleavage afforded the
cyclic biaryl peptide 14, which was dissolved in H2O/CH3CN, lyoph-
ilized, and analyzed by HPLC (70% purity). tR¼16.72 min (method A).
MS (ESI): m/z¼276.1 [Mþ2H]2þ, 551.2 [MþH]þ. HRMS (ESI): calcd for
C30H43N6O4 551.3340; found 551.3330.
are also grateful to the Serveis Tecnics de Recerca of the University
of Girona.
Supplementary data
Supplementary data HPLC, MS, and NMR spectra for peptides
3e17 and for their corresponding precursors are provided. Sup-
plementary data associated with this article can be found in online
version at doi:10.1016/j.tet.2011.01.084. These data include MOL
files and InChIKeys of the most important compounds described in
this article.
4.3.4.9. Cyclic biaryl peptide 15. Starting from resin Boc-Phe(4-
I)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA (10) (65 mg),
SuzukieMiyaura cyclization followed by acidolytic cleavage afforded
the cyclic biaryl peptide 15, which was dissolved in H2O/CH3CN, ly-
ophilized, and analyzed by HPLC (36% purity). tR¼15.23 min (method
A). MS (ESI): m/z¼413.7 [Mþ2H]2þ, 826.4 [MþH]þ. HRMS (ESI): calcd
for C45H65N9O6 413.7523; found 413.7530.
References and notes
1. Feliu, L.; Planas, M. Int. J. Pept. Res. Ther. 2005, 11, 53e97.
2. (a) Perdih, A.; Dolenc, M. S. Curr. Org. Chem. 2007, 11, 801e832; (b) Haldar, D.
Curr. Org. Synth. 2008, 5, 61e80.
4.3.4.10. Cyclic biaryl peptide 16. Starting from resin Boc-Phe
(4-I)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-MBHA
(11) (38 mg), SuzukieMiyaura cyclization followed by acidolytic
cleavage afforded the cyclic biaryl peptide 16, which was dissolved
in H2O/CH3CN, lyophilized, and analyzed by HPLC (84% purity).
tR¼16.05 min (method A). MS (ESI): m/z¼477.7 [Mþ2H]2þ, 954.5
[MþH]þ. HRMS (ESI): calcd for C51H77N11O7 477.7998; found
477.8007; calcd for C51H76N11O7 954.5924; found 954.5876.
€
3. (a) Knor, S.; Laufer, B.; Kessler, H. J. Org. Chem. 2006, 71, 5625e5630; (b) Skaff,
O.; Jolliffe, K. A.; Hutton, C. A. J. Org. Chem. 2005, 70, 7353e7363; (c) Gong, Y.;
Barbay, J. K.; Dyatkin, A. B.; Miskowski, T. A.; Kimball, E. S.; Prouty, S. M.;
Fisher, M. C.; Santulli, R. J.; Schneider, C. R.; Wallace, N. H.; Ballentine, S. A.;
Hageman, W. E.; Masucci, J. A.; Maryanoff, B. E.; Damiano, B. P.; Andrade-
Gordon, P.; Hlasta, D. J.; Hornby, P. J.; He, W. J. Med. Chem. 2006, 49,
3402e3411; (d) Capek, P.; Pohl, R.; Hocek, M. Org. Biomol. Chem. 2006, 4,
2278e2284; (e) Chalker, J. M.; Wood, C. S. C.; Davis, B. G. J. Am. Chem. Soc.
2009, 131, 16346e16347.
ꢂ
4. (a) Carbonnelle, A.-C.; Zhu, J. Org. Lett. 2000, 2, 3477e3480; (b) Bois-
Choussy, M.; Cristau, P.; Zhu, J. Angew. Chem., Int. Ed. 2003, 42, 4238e4241;
(c) Lepine, R.; Zhu, J. Org. Lett. 2005, 7, 2981e2984; (d) Roberts, T. C.; Smith,
4.3.4.11. Cyclic biaryl peptide 17. Starting from resin Boc-Phe(4-
I)-Lys(Boc)-Lys(Boc)-Phe-Lys(Boc)-Lys(Boc)-Leu-Phe(4-BPin)-Rink-
MBHA (12) (60 mg), SuzukieMiyaura cyclization followed by
acidolytic cleavage afforded the cyclic biaryl peptide 17, which was
dissolved in H2O/CH3CN, lyophilized, and analyzed by HPLC (77%
ꢀ
P. A.; Cirz, R. T.; Romesberg, F. E. J. Am. Chem. Soc. 2007, 129, 15830e15838;
(e) Dufour, J.; Neuville, L.; Zhu, J. Synlett 2008, 2355e2359; (f) Waldmann,
H.; He, Y.-P.; Tan, H.; Arve, L.; Arndt, H.-D. Chem. Commun. 2008, 5562e5564;
(g) Wang, Z.; Bois-Choussy, M.; Jia, Y.; Zhu, J. Angew. Chem., Int. Ed. 2010, 49,
2018e2022.
purity). tR¼15.19 min (method A). MS (ESI): m/z¼541.8 [Mþ2H]2þ
,
5. (a) Nielsen, T. E.; Le Quement, S.; Meldal, M. Tetrahedron Lett. 2005, 46,
7959e7962; (b) Haug, B. E.; Stensen, W.; Svendsen, J. S. Bioorg. Med. Chem. Lett.
1082.5 [MþH]þ. HRMS (ESI): calcd for C57H90N13O8 361.5673; found
361.5690; calcd for C57H89N13O8 541.8473; found 541.8498; calcd
for C57H88N13O8 1082.6873; found 1082.6829.
ꢀ
2007, 17, 2361e2364; (c) Doan, N.-D.; Bourgault, S.; Letourneau, M.; Fournier, A.
J. Comb. Chem. 2008, 10, 44e51.
ꢀ
6. Afonso, A.; Roses, C.; Planas, M.; Feliu, L. Eur. J. Org. Chem. 2010, 1461e1468.
7. Lei, H.; Stakes, M. S.; Herath, K.; Lee, J.; Shwabacher, A. W. J. Org. Chem. 1994, 59,
4206e4210.
Acknowledgements
ꢀ
8. Cerezo, V.; Amblard, M.; Martinez, J.; Verdie, P.; Planas, M.; Feliu, L. Tetrahedron
2008, 64, 10538e10545.
9. Zhang, A. J.; Russell, D. H.; Zhu, J.; Burgess, K. Tetrahedron Lett. 1998, 39,
7439e7442.
10. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal. Biochem. 1970, 34,
595e598.
A.A. is the recipient of a predoctoral fellowship from the Min-
ꢀ
isterio de Ciencia e Innovacion (MICINN) of Spain. This work was
supported by grant AGL2009-13255-C02-02/AGR from MICINN. We