Synthesis and biological evaluation of tyrosine modified analogues of the α4β7 integrin inhibitor biotin-R8ERY

Article abstract of DOI:10.1016/j.bmc.2012.07.010

The α4β7 integrin is a well-known target for the development of drugs against various inflammatory disease states including inflammatory bowel disease, type 1 diabetes and multiple sclerosis. The synthesis of a small library of cell-permeable β7 integrin inhibitors based on the peptide biotin-R₈ERY is reported, in which the tyrosine residue has been modified by using the Suzuki-Miyaura cross-coupling reaction. The synthesised peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn²⁺-activated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. All of the synthesised peptidomimetics are more active than our previously reported lead compound biotin-R₈ERY with two of the analogues, 6 and 7, exhibiting IC₅₀ values of <15 μM.

Full text of DOI:10.1016/j.bmc.2012.07.010

Bioorganic & Medicinal Chemistry 20 (2012) 5139–5149
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of tyrosine modified analogues of the
a4b7 integrin inhibitor biotin-R8ERY
Stefanie Papst a, Anaïs F. M. Noisier a, Margaret A. Brimble a, , Yi Yang b, Geoffrey W. Krissansen b
⇑
a School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland Central 1010, New Zealand
b Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The a4b7 integrin is a well-known target for the development of drugs against various inflammatory dis-
Received 9 May 2012
Revised 28 June 2012
Accepted 5 July 2012
Available online 21 July 2012
ease states including inflammatory bowel disease, type 1 diabetes and multiple sclerosis. The synthesis of
a small library of cell-permeable b7 integrin inhibitors based on the peptide biotin-R8ERY is reported, in
which the tyrosine residue has been modified by using the Suzuki-Miyaura cross-coupling reaction. The
synthesised peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn2+-acti-
vated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. All of the synthesised peptidomimetics
are more active than our previously reported lead compound biotin-R8ERY with two of the analogues,
Keywords:
Peptidomimetics
Integrins
6 and 7, exhibiting IC50 values of <15 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Mucosal T cell numbers are selectively reduced in
out mice.
aE gene knock-
As described in our previous paper,1 the
a4 integrins
a
4b7
b7 integrin adhesion plays a crucial role in regulating gut
immunity10,11 and is therefore implicated in the pathogenesis
and progression of inflammatory bowel diseases, such as Crohn’s
disease, ulcerative colitis12–14 and intestinal graft-versus-host dis-
(LPAM-1) and
tors expressed on most leukocytes.2,3 They consist of noncova-
lently-associated and transmembrane subunits with
a4b1 (VLA-4) are heterodimeric cell-surface recep-
a
b
comparatively large extracellular domains and short cytoplasmic
eases (GVHD).8,15 Furthermore,
a4b7 integrins contribute to leuko-
domains. They mediate fundamental cell-extracellular matrix and
cyte infiltration into the islets of Langerhans in type 1 diabetes16
and the central nervous system in demyelinating diseases such
as multiple sclerosis.17
cell-cell adhesion events.4 Ligands of the
a4 integrin subfamily in-
clude fibronectin (Fn), vascular cell adhesion molecule-1 (VCAM-
1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-
Currently, steroids and non-steroidal anti-inflammatory drugs
(NSAIDs) are the mainstay treatments for the above diseases, but
the long-term usage of such drugs has detrimental side-effects.
The concept of developing selective drugs that would prevent the
homing of lymphocytes to chronically inflamed tissues with mini-
mal effects on normal immune surveillance has prompted a num-
ber of pharmaceutical companies to focus on the development of
1).5–7
a4b1 preferentially recognises VCAM-1, whereas
a4b7 pref-
erentially recognises MAdCAM-1. 4b7 binds to MAdCAM-1 ex-
a
pressed on high endothelial venules, which contributes to the
homing of lymphocytes to gut-associated lymphoid tissues (GALT)
such as Peyer’s patches and the lamina propria.8 The b7 subunit
also associates with the integrin aE subunit, forming the receptor
a
Eb7 which mediates the binding of intestinal intraepithelial
a4b1 and/or a4b7 antagonists, mainly by using small molecule
lymphocytes (iIEL) to E-cadherin expressed on gut enterocytes.9
inhibitors to block the binding of the integrins to their extracellular
ligands.7,18–20
Krissansen and coworkers have previously shown that both
- and D-enantiomers of a cell-permeable peptide biotin-r9YDRREY,
residues 735–740 of the cytoplasmic tail of the b7 subunit, inhib-
ited adhesion of Mn2+-activated T cells to b7 integrin ligands. It
was demonstrated that biotin-r9YDRREY suppressed MAdCAM-1-
Abbreviations: FAK, focal adhesion kinase; FBS, fetal bovine serum; GALT, gut-
associated lymphoid tissues; GVHD, graft-versus-host disease; HBSS, Hank’s
buffered salt solution; HMP, linker 4-(hydroxymethyl)phenoxyacetic acid; iIEL,
intestinal intraepithelial lymphocytes; LPAM-1, lymphocyte Peyer’s patch adhesion
molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; NSAIDs,
non-steroidal anti-inflammatory drugs; PBS, phosphate buffered saline; R8,
L
induced clustering of
a4b7 receptors on the cell surface, poten-
(L-arginine)8; r9, (D-arginine)9; SPPS, solid phase peptide synthesis; src, sarcoma;
VCAM-1, vascular cell adhesion molecule-1; VLA-4, very late activating antigen-4.
tially by acting as a competitive substrate for src, FAK, and other
tyrosine kinases and signalling molecules. It is postulated that pep-
⇑
Corresponding author.
E-mail address: m.brimble@auckland.ac.nz (M.A. Brimble).
tide biotin-r9YDRREY thereby interrupted the association of
a4b7
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.010

5140
S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
with intracellular signalling molecules and cytoskeletal elements,
which in turn prevented the ability of the receptors to aggregate,
resulting in disruption of cell adhesion. The identity of the key
b7 integrin ligand, the exact binding mechanism and the structure
of the binding pockets of the b7 integrin ligand(s) are unknown to
date. Nevertheless, the inhibitory activity of the biotin-r9YDRREY
peptide was shown to depend on the flanking tyrosine residues,
as deletion of the tyrosines or their substitution with phenylala-
nine led to loss of activity.21 We also found that the peptide bio-
BocHN
CO2Me
2
CO2H
H2N
(i), (ii), (iii)
OTf
OH
1
Scheme 1. Reagents and conditions: (i) SOCl2, MeOH, reflux, 4 h; (ii) NEt3, (Boc)2O,
MeOH, 0 °C, 12 h, N2 (94%); (iii) pyridine, Tf2O, DCM, ꢀ15 °C, 30 min, N2 (90%).
tin-R8ERY,
a short pseudo-analogue of the biotin-r9YDRREY
peptide, has similar activity, hence it was used as a lead motif.
The tyrosine residue in the shortened tripeptide ERY was consid-
ered to be the key residue to modify, at least initially, in order to
increase inhibitory activity. A number of commercially available
tyrosine analogues were incorporated into the ERY⁄ scaffold. The
resulting trimers were fused to octa-arginine tags, which act as cat-
ionic carriers enabling crossing of the cell membrane and delivery
of the inhibitors to the cytoplasm of activated T cells.22–24 We pre-
viously demonstrated that substitution of tyrosine by 4-chlorophe-
nylalanine, 4-nitrophenylalanine or, most interestingly, 4,40-
biphenylalanine afforded analogues with activity approaching that
of the earlier-reported biotin-r9YDRREY peptide (unpublished re-
sults). Furthermore it is known that oral bioavailability can be an
Table 1
Synthetic building blocks 4 prepared by Suzuki coupling using tyrosine triflate 27,30
Compd
R=
Yield (%)
Method
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-Methylphenyla
4-Ethylphenyla
90
68
68
73
73
29
33
74
77
A
A
A
B
B
B
B
B
B
Biphenyla
2-Methoxyphenyla
2,6-Dimethoxyphenyla
3,4-Dimethoxyphenyla
3-Nitrophenyla
4-Cyanophenylb
10-Cyclohexenylb
a
Derived from requisite boronic acid.
Derived from requisite boronic acid pinacol ester.
b
issue with peptidic a4b7/a
4b1 antagonists.7 In an effort to enhance
the activity of these cell-permeable peptides by decreasing their
peptidic nature and using the 4,40-biphenylalanine scaffold, we
decided to use the versatile Suzuki-Miyaura cross-coupling reac-
tion to synthesise a small library of biaryl tyrosine building blocks
derived from tyrosine triflate 2, which were then incorporated into
the R8ERY⁄ peptide sequence by automated Fmoc SPPS.
CO2Me
BocHN
BocHN
OH
O
(i) or (ii)
R
B
or
R B
OH
O
OTf
R
The Suzuki-Miyaura reaction was first described in 1979,25 pro-
viding the synthetic community with a facile method to effect the
coupling of organoboron reagents with organo halides or pseudo
halides. Today, Pd-catalysed cross-coupling reactions are amongst
the most widely used reactions in the pharmaceutical industry.26
The development of new ligands, for example the electron rich bia-
rylmonophosphine SPhos by Buchwald27 and of Pd(II) catalysts
which exhibit greater air and moisture stability,28 has broadened
the scope of the Suzuki reaction. The coupling of less reactive sub-
strates, such as aryl chlorides, electron deficient organoboron re-
agents,29 electron rich aryl halides, or sterically encumbered
systems30 are now possible.
2
CO2Me
CO2H
FmocHN
(iii), (iv), (v)
R
4a-i
3a-i
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, K2CO3, toluene/DMF, 80 °C, 12 h,
2 (method A); (ii) Pd(OAc)2, SPhos, K3PO4, toluene/DMF, 90 °C, N2 (method B); (iii)
NaOH, MeOH/THF (1/1), rt; (iv) TFA/DCM (1/1), 30 min; (v) Fmoc-OSu, NaHCO3,
N
THF/H2O, rt, 12 h.
We have synthesised nine biaryl tyrosine analogues by using
the Suzuki cross-coupling reaction and herein describe the synthe-
sis and biological evaluation of cell permeable peptidomimetics
based on this biotin-R8ERY⁄ motif, which have potential utility as
anti-inflammatory agents for the treatment of chronic inflamma-
tory diseases.
to building blocks 4d–f, and the electron deficient boron reagents,
giving 4g and 4h, were coupled to triflate 2 using Pd(OAc)2 and
SPhos (Scheme 2, method B30).
With the building blocks 4a–i in hand, biotinylated R8ERY⁄ pep-
tides 5–14 were then prepared on a 0.1 mmol scale following stan-
dard Fmoc SPPS procedures using a TributeTM peptide synthesiser
and aminomethylated polystyrene resin41 derivatised with HMP
linker (Scheme 3). Peptides 13 and 14 were obtained using the
same building block 4i, but applying different cleavage conditions.
In order to obtain peptide 13 the cyclohexene moiety of building
block 4i was reduced to cyclohexane using 3,6-dioxa-1,8-octane-
dithiol (2.5% v/v) during cleavage from resin. The unsaturated ana-
logue 14 was obtained, albeit in low yield, performing the cleavage
with TFA/5% H2O. All products were obtained in sufficient purity
and yield for biological studies after purification by RP-HPLC (Table
2).
2. Results and discussion
2.1. Chemistry
The intermediate required for the Suzuki couplings was syn-
thesised as shown in Scheme 1. Tyrosine 1 was esterified,31 Boc-
protected32–34 then converted to the requisite triflate 2,35,36 which
was then subjected to Pd-catalysed Suzuki reactions to produce the
nine analogues shown in Table 1.
The reaction conditions were adjusted to meet the steric de-
mands imposed by the boron reagents investigated. 4-Methyl-
phenyl-, 4-ethylphenyl- and 4-biphenyl-boronic acids were
cross-coupled with triflate 2 using Pd(PPh3)4 (Scheme 2, method
A7), affording tyrosine mimetic building blocks 4a,37 4b and 4c38
for Fmoc SPPS after the appropriate deprotection/protection
steps.39,40 The more sterically demanding boron reagents, leading
2.2. Biology
The
as it is unique in that it does not express b1 integrins and hence can
a
4b7+ TK-1 T cell line was chosen as a representative T cell

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
5141
O
O
HO
OH
PS
HMP linker
PS
OH
NH2
HO2C
NHFmoc
R
4a-i
O
Fmoc SPPS
biotin-RRRRRRRRERY*
(peptides 5-14)
NHFmoc
(Tribute peptide synthesiser)
O
PS
HMP linker
1) 20% piperidine in DMF, 3 mL, 2·5 min
2) HBTU, NMM, DMF, amino acid, 45 min
3) Repeat step 1 and 2 ten times
4) biotinylation
R
5) cleavage f rom resin
Scheme 3. Fmoc SPPS of peptides 5–14 on aminomethylated PS resin.
Table 2
Biotin-R8ERY⁄ peptides and their percentage of inhibition in a cell adhesion assay
No.
Y⁄=
Yielda (%) (purity)
MS (4+ion) Calcd/Obsdb
% Inhibition (50
lM)
% Inhibition (100 lM)
5
6
7
8
9
10
11
12
4a
4b
4c
4d
4e
4f
4g
4h
4i
13% (93%)
35% (96%)
15% (97%)
47% (96%)
53% (99%)
43% (99%)
34% (98%)
23% (93%)
15% (91%)
3% (99%)
—
505.1/505.1
508.6/508.6
520.6/520.6
509.1/509.0
516.6/516.6
516.6/516.5
512.9/512.8
507.9/507.8
503.1/503.1
502.6/502.5
—
47.36
57.99
80.22
48.06
38.29
37.93
50.46
45.05
71.72
49.70
8.73
57.27
74.19
91.31
47.15
50.86
50.13
60.34
57.01
64.85
59.08
18.18
71.32
13 (Cyclohexyl)
14 (10-Cyclohexenyl)
Biotin-R8ERY
Biotin-r9YDRREY
4i
—
—
—
—
66.36
a
Purified yield was based on calculated resin loading, see experimental section for further details.
ESI-MS, see experimental section for further details.
b
be used to measure binding to MAdCAM-1 independently of
a
4b1,
When the size of the tyrosine substituent was decreased from
biphenyl (7) to 4-ethylphenyl (6) the activity decreased by 22%
and by changing to 4-methylphenyl (5) or 4-cyanophenyl (12) it
decreased by 33% and 35%, respectively.
which shares 4b7 ligands. In the present work, the synthetic b7
a
tripeptides were tested for their ability to block the adhesion of
Mn2+-activated mouse TK-1 cells to mouse MAdCAM-1 coated onto
chamber slides at a peptide concentration of 50 and 100
The inhibition assays were initially conducted at peptide con-
centrations of 50 and 100 M (Fig. 1). Analogue 7, carrying the p-
terphenyl group, exhibited the strongest inhibition of TK-1 cell
adhesion to MAdCAM-1 at both 50 and 100 M, exceeding the
activity of biotin-R8ERY by 73% (p = 0.01) at 100 M. Analogue 7
and compounds 13 and 6 exhibited the strongest inhibition in this
library, which suggests that activity can be enhanced by incorpo-
rating large non-polar groups into the Y⁄ side chain. Those modifi-
cations for compounds 7 and 6 rendered the trimeric ERY⁄ peptide,
which is considered to be the active unit of biotin-R8ERY⁄, more ac-
tive than the hexamer YDRREY, albeit the differences did not reach
l
M.
Though not statistically significant, the 4-cyclohexyl analogue
13 is slightly more active than the unsaturated 4-(10-cyclohexenyl)
analogue 14 at both concentrations and the methoxy substituted
l
analogues 8–10 showed the poorest activity at 100 lM amongst
l
the ten peptidomimetics. This is in accordance with the discovered
steric demand for the 2-methoxyphenyl analogue 8 and 2,6-dime-
thoxy analogue 9, as they lack a substituent at the 4-position of the
second phenyl ring. Why the 3,4-dimethoxyphenyl analogue 10
does not display higher activity is not clear at this stage. The rela-
tively high activity of the 3-nitrophenyl analogue 11 within this
l
series at 100 lM could be an indication that the attachment of
large aromatic substituents at the 3-position of the tyrosine ring
statistical significance [inhibition by analogue 7 at 50
lM ap-
could enhance activity. Whilst compound 13 appeared to be
proached significance (p = 0.08)]. This is also backed up by the re-
sults of the previous study where the biotin-R8ERY⁄ analogue
carrying an Fmoc protecting group in the side chain of the altered
tyrosine moiety was the most potent inhibitor (58% inhibition at
10
interactions between the peptide and the receptor (Scheme 4).
slightly more active at 50
cally significant difference between the levels of inhibition at the
two concentrations, indicating that inhibition is maximal at 50 M.
In order to investigate the ability of the peptides to inhibit T cell
adhesion at lower concentrations a dose response assay was con-
ducted for the three most active peptides 6, 7 and 13 at a wide
lM than at 100 lM there is no statisti-
l
l p–p stacking
M),1 suggesting a ‘deep’ binding pocket and

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S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
Figuer 1. Percent of inhibition of biotin-R8ERY⁄ peptides on Mn2+-activated TK-1 cell adhesion to mouse MAdCAM-1-Fc (50 and 100
experiments with duplicate wells. ⁄p 60.05, significantly more inhibitory than ERY.
lM concentration). SEM of two
HN
NH2
HN
NH2
HN
O
HN
O
X = biotin-R8-
H
N
H
N
H
N
H
CO2H
N
CO2H
X
N
H
N
H
X
O
O
CO2H
CO2H
ERY* p-terphenylalanine 7
alpha4beta7 IC50 = 7.3 uM
inhibition at 50 uM: 80.22%
ERY* 4-(4-ethylphenyl)phenylalanine 6
alpha4beta7 IC50 = 14.3 uM
inhibition at 50 uM: 57.99%
HN
NH2
HN
O
H
H
N
N
CO2H
N
H
X
O
CO2H
O
N
N
NHFmoc
N
HO2C
most potent inhibitor from previous series
alpha4beta7 IC50 < 10 uM
Scheme 4. Comparison of the structures of the two most active peptides 6 and 7 of this study to the most potent inhibitor of the series published earlier.1
range of concentrations. The assay revealed that the two most ac-
tive compounds 6 and 7 retained a good level of activity despite
lowering the concentrations (Fig. 2). All three peptides were
significantly more active at 50 M (p <0.05–0.01) and 25 lM
l

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
5143
Figure 2. Dose response assay of the three most active peptides. Percent of inhibition of selected ERY⁄ peptides on Mn2+ activated TK-1 cell adhesion to mouse MAdCAM-1-Fc.
SEM of two experiments with duplicate samples. (a) 50 M significantly different from 10 M (p <0.05), (b) 50 M significantly different from 5 M (p <0.05), (c) 50
significantly different from 5 M (p <0.01), (d) 25 M significantly different from 5 M (p <0.05), (e) 25 M significantly different from 5 M (p <0.01), (f) 10 M significantly
different from 5 M (p 60.05).
l
l
l
l
lM
l
l
l
l
l
l
l
(p <0.05–0.01) than at 5
icantly more active at 50
10 M. The dose-response analysis revealed that peptides 6, 7, and
13 had IC50s of approximately 14.3, 7.3, and 19 M. Peptide 13 lost
inhibitory activity more significantly when its concentration was
lowered.
l
l
M (p <0.05–0.01). Peptide 13 was signif-
Analytical thin layer chromatography (TLC) was performed on
0.2 mm aluminium plates of silica gel 60 F254 (Merck) and visual-
ised by UV fluorescence. Flash chromatography was performed
using DavisilÒ chromatographic silica (LC60Å 40–63 micron)
(Grace GmbH & Co. KG) with indicated solvents. Infrared spectra
were obtained using a Perkin Elmer spectrum One Fourier Trans-
form infrared spectrometer with a universal ATR sampling acces-
sory. Nuclear magnetic resonance (NMR) spectra were recorded
as indicated on either a Bruker AVANCE DRX300 (1H, 300 MHz;
13C, 75 MHz) or on a Bruker AVANCE DRX400 (1H, 400 MHz; 13C,
100 MHz) spectrometer at 298 K. Optical rotations were deter-
mined at 20 °C with a Perkin-Elmer 341 polarimeter and are given
in units of 10ꢀ1 deg cm2 gꢀ1. Melting points were determined on a
ElectrothermalÒ melting point apparatus and are uncorrected. ESI-
MS were recorded on a Thermo Finnigan Surveyor MSQ Plus spec-
trometer or a Bruker micrOTOF-Q II spectrometer. Semi-prepara-
tive RP-HPLC was performed on a Dionex Ultimate 3000 system
M (p <0.05) and 25 M (p <0.05) than at
l
l
l
3. Conclusion
Building on the previously reported library of a
4b7 inhibitors1 a
series of ten novel, cell-permeable peptidomimetics incorporating
the biotin-R8ERY⁄ motif, centered on aryl-substituted phenylala-
nine, was prepared. These tyrosine analogue building blocks were
synthesised using Suzuki-Miyaura cross-coupling methodology
then incorporated into the requisite peptide sequences, which
were then tested for a4b7 antagonism in a lymphocyte cell-bind-
ing assay. Nine of the peptidomimetics exhibited statistically sig-
nificant greater potency than the previously reported lead
using
a
Phenomenex Gemini C18 column (110 Å,
5 lM,
10 ꢁ 250 mm) eluting with a linear gradient of water/0.1% TFA
and MeOH/0.1% TFA at a flow rate of 5 mL/min. Analytical RP-HPLC
was performed on a Dionex P680 system using a Phenomenex
compound biotin-R8ERY at 50 and 100
lM and a dose response as-
say showed an IC50 of <10 M for the most active analogue 7. Based
l
on these promising results investigations to further improve the
activity of the synthetic peptidomimetics are underway.
Gemini C18 column (110 Å, 5
lM, 4.6 ꢁ 150 mm) with the same
eluents at a flow rate of 1 mL/min. Gradient systems were adjusted
according to the elution profiles and peak profiles obtained from
the analytical RP-HPLC chromatograms.
4. Experimental
4.1. Reagents and general methods
5. Syntheses of building blocks using Suzuki-Miyaura reaction
5.1. General procedure A: Suzuki cross-coupling reaction
All reagents were purchased as reagent grade from commercial
sources and used as supplied. Solvents were used as supplied un-
less otherwise stated or dried according to standard methods.42
RP-HPLC solvents were purchased as HPLC grade and used without
further purification. All amino acids were purchased as
The requisite boron reagent (2.28 mmol) and K2CO3
(4.55 mmol, 580 mg) were combined and suspended in a degassed
mixture of toluene/DMF (10:1, 15 mL). Boc–Tyr(OTf)–CO2Me 2
L-enantiomers.

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S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
(1.75 mmol, 750 mg) was dissolved in the same solvent (4 mL) and
added under N2 followed by Pd(PPh3)4 (0.18 mmol, 202 mg) and
the reaction was heated at 80 °C overnight. The black mixture
was filtered through Celite and the filtrate concentrated. The resi-
due was dissolved in EtOAc and the organic layer was washed with
water, dried over Na2SO4 and concentrated to give the crude prod-
ucts 3a–c, which were purified by flash column chromatography.
CH3/Boc), 2.95 (1H, dd, J = 6.3, 14.1 Hz, bCH2), 3.01 (1H, dd, J = 5.7,
13.9 Hz, bCH2), 3.68 (3H, s, CO2CH3), 4.52 (1H, m, CH), 5.09 (1H, d,
a
J = 7.7 Hz, NH), 6.73 (2H, d, J = 8.1 Hz, 3-CH), 6.93 (2H, d, J = 8.4 Hz,
2-CH); 13C NMR (100 MHz, CDCl3): d 28.3 (3x CH3/Boc), 37.6
(bCH2), 52.3 (CO2CH3), 54.2 (aCH), 80.3 (C/Boc), 115.5 (3-CH),
127.4 (1-C), 130.3 (2-CH), 155.2, 155.4 (C@O/Boc, 4-C), 172.7
(CO2CH3); IR tmax (cmꢀ1): 3305, 2982, 1760, 1686, 1508, 1448,
1369, 1209, 1147, 834; ESI-MS m/z: 296.1490 (M+H)+ ((M+H)+,
5.2. General procedure B: Suzuki cross-coupling reaction
C
15H22NO5 requires 296.1498), 318.1308 (M+Na)+, 240.0870
(MꢀC(CH3)3)+, 196.0970 (MꢀBoc)+.
The requisite boron reagent (2.63 mmol) and K3PO4 (5.25 mmol,
1.113 g) were combined and suspended in a degassed mixture of
toluene/DMF (10:1, 15 mL). Boc–Tyr(OTf)–CO2Me 2 (1.75 mmol,
750 mg) was added under N2, followed by SPhos (12.5 mol %,
90 mg) and Pd(OAc)2 (5 mol %, 20 mg) and the reaction was heated
at 90 °C overnight. The black mixture was filtered through Celite
and the filtrate concentrated. The residue was dissolved in EtOAc
and the organic layer was washed with water, dried over Na2SO4
and concentrated to give the crude products 3d–i, which were
purified by flash column chromatography.
5.6. Boc–Tyr(OTf)–CO2Me (2)
Boc–Tyr–CO2Me (15.5 mmol, 4.584 g) was dissolved in DCM
(50 mL), pyridine (38.8 mmol, 3.1 mL) added and the solution
was stirred under N2 at ꢀ15 °C. Triflic anhydride (18.6 mmol,
3.1 mL) was added dropwise and the red solution was stirred un-
der N2 at ꢀ15 °C for 30 min. To the reaction was added water
and the layers were separated. The organic layer was washed with
0.5 N NaOH and 5% citric acid, dried over MgSO4 and concentrated
to give the crude product 2 as a red oil, which was purified by flash
column chromatography (EtOAc/hexane 15/85, Rf 0.25) to give the
desired product as a yellowish crystalline solid (5.972 g, 90%): mp
5.3. General procedure C: Fmoc protection
The Boc-protected amino acid methyl ester 3a–i (1 equiv) was
dissolved in MeOH/THF (1:1, 6 mL) and 1 N NaOH (5 equiv) was
added. The reaction mixture was stirred at room temperature until
complete hydrolysis was observed by TLC. The solution was acidi-
fied to pH 3 with 1 N HCl and then extracted with DCM. The organ-
ic layer was washed with water, dried over MgSO4 and
concentrated. The crude product was dissolved in DCM/TFA (1:1)
and stirred at room temperature for 30 min whereupon the solvent
was removed in vacuo.
The unprotected product was then dissolved in H2O/THF, basi-
fied with NaHCO3 to pH 10. Fmoc-OSu (1.05 equiv) was added
slowly and the solution stirred at room temperature overnight.
The solution was acidified to pH 3 with 5% citric acid, the precipi-
tated product extracted into EtOAc and the organic layer was
washed with water, dried over MgSO4 and concentrated to give
the crude product 4a–i, which was purified by flash column
chromatography.
49 °C (lit.35 47–48 °C); ½a 2D0
ꢂ
+34.80 (c 0.1, CHCl3) (lit.36 +33.60 (c 1,
CHCl3)); 1H NMR (400 MHz, CDCl3): d 1.41 (9H, s, CH3/Boc), 3.04
(1H, dd, J = 6.5, 13.8 Hz, bCH2), 3.17 (1H, dd, J = 5.7, 13.9 Hz,
bCH2), 3.71 (3H, s, CO2CH3), 4.60 (1H, m,
J = 7.7 Hz, NH), 7.21 (4H, m, aromatic); 13C NMR (100 MHz, CDCl3):
d 28.2 (3x CH3/Boc), 37.9 (bCH2), 52.4 (CO2CH3), 54.2 ( CH), 80.2
(C/Boc), 117.1 (C/OTf), 121.3 (3-CH), 131.1 (2-CH), 136.9 (1-C),
148.6 (4-C), 154.9 (C@O/Boc), 171.9 (CO2CH3); IR tmax (cmꢀ1):
3382, 2984, 1733, 1690, 1502, 1517, 1424, 1248, 1129, 895; MS
(EI) m/z: 450.0809 (M+Na)+ ((M+Na)+, C16H20F3NO7SNa requires
450.0810), 372.0367 (MꢀC(CH3)3)+, 328.0467 (MꢀBoc)+.
aCH), 5.02 (1H, d,
a
5.7. Fmoc-4-(4-methylphenyl)phenylalanine (4a)
The intermediate 3a was prepared following general procedure
A, using 310 mg of 4-methylphenylboronic acid. The crude product
(brown oil) was purified by flash column chromatography (EtOAc/
hexane (1:9), Rf 0.14) to yield the desired product 3a as a white so-
5.4. NH2–Tyr–CO2Me
lid (498 mg, 77%): mp 78–79 °C (lit.36 77–79 °C); ½a D20
ꢂ
+50.00 (c 0.1,
CHCl3) (lit.36 +52.24 (c 1, CHCl3)); 1H NMR (400 MHz, CDCl3): d 1.45
(9H, s, CH3/Boc), 2.39 (3H, s, CH3), 3.09 (1H, dd, J = 6.3, 13.8 Hz,
bCH2), 3.19 (1H, dd, J = 5.6, 13.8 Hz, bCH2), 3.73 (3H, s, CO2CH3),
NH2–Tyr–CO2H 1 (16.6 mmol, 3 g) was dissolved in MeOH
(12 mL) and neat SOCl2 (18.3 mmol, 1.3 mL) added dropwise. The
clear solution was heated under reflux for 4 h then cooled and
the solvent removed in vacuo to give a white solid which was used
without further purification in the next step. 1H NMR (400 MHz,
D2O) d 3.19 (1H, dd, J = 7.4, 14.7 Hz, bCH2), 3.28 (1H, dd, J = 5.8,
14.7 Hz, bCH2), 3.85 (3H, s, CO2CH3), 4.39 (1H, dd, J = 5.8, 7.4 Hz,
4.65 (1H, m,
aCH), 5.21 (1H, d, J = 8.2 Hz, NH), 7.21 (2H, d,
J = 8.0 Hz, aromatic), 7.24 (2H, d, J = 8.1 Hz, aromatic), 7.48 (2H, d,
J = 8.1 Hz, aromatic), 7.52 (2H, d, J = 8.1 Hz, aromatic); 13C NMR
(100 MHz, CDCl3): 21.1 (CH3), 28.3 (3xCH3/Boc), 37.9 (bCH2), 52.2
(CO2CH3), 54.5 (a
CH), 79.8 (C/Boc), 126.8, 127.0 (2, 30-CH), 129.5,
a
CH).
CH), 6.92 (2H, d, J = 8.5 Hz, 3,5-CH), 7.18 (2H, d, J = 8.6 Hz, 2,6-
129.7 (3, 20-CH), 134.9, 137.0, 137.9, 139.8 (1, 4, 10, 40-C), 155.2
(C@O/Boc), 172.4 (CO2CH3); IR tmax (cmꢀ1): 3313, 2976, 1734,
1696, 1498, 1209, 1158, 809; ESI-MS m/z: 370.2016 (M+H)+
((M+H)+, C22H28NO4 requires 370.2018), 392.1835 (M+Na)+,
314.1395 (MꢀC(CH3)3)+, 270.1498 (MꢀBoc)+.
5.5. Boc–Tyr–CO2Me
NH2–Tyr–CO2Me was dissolved in MeOH (40 mL), NEt3
(24.9 mmol, 3.3 mL) added and the red solution cooled on ice un-
der N2. Boc2O (18.3 mmol, 3.985 g) was added portionwise and
the stirring continued overnight at room temperature under N2.
The solution was acidified with 1 N HCl (pH 3) and extracted with
DCM. The organic layer was dried over MgSO4, filtered and concen-
trated to give the crude product as an orange oil, which was puri-
fied by flash column chromatography (EtOAc/hexane 3/7, Rf 0.17)
to give the desired product as a white foam (4.584 g, 94% over
Intermediate 3a (1.23 mmol, 476 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product (orange oil) was purified by flash column
chromatography (DCM then EtOAc/hexane (3:7 to neat EtOAc) + 1%
acetic acid, Rf 0.35 in EtOAc/hexane (1:1) + 1% acetic acid) to yield
the desired product 4a as a white solid (557 mg, 90% over three
steps): mp 130 °C; ½a D20
ꢂ
+48.51 (c 0.1, CHCl3); 1H NMR (400 MHz,
CDCl3): d 2.39 (3H, s, CH3), 3.16 (1H, dd, J = 6.0, 13.8 Hz, bCH2),
3.25 (1H, dd, J = 5.3, 14.0 Hz, bCH2), 4.21 (1H, t, J = 7.1 Hz, CH/
Fmoc), 4.38 (1H, dd, J = 6.8, 10.4 Hz, CH2/Fmoc), 4.46 (1H, dd,
two steps): mp 108 °C (lit.32 100–102 °C); ½a 2D0
ꢂ
+43.27 (c 0.1, CHCl3)
(lit.32, +46.00 (c 1, CHCl3)); 1H NMR (400 MHz, CDCl3): d 1.41 (9H, s,
J = 7.0, 10.7 Hz, CH2/Fmoc), 4.75 (1H, m, aCH), 5.28 (1H, d,

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
5145
J = 8.1 Hz, NH), 7.20 (2H, d, J = 8.0 Hz, aromatic), 7.23 (2H, d,
J = 8.0 Hz, aromatic), 7.29 (2H, t, J = 7.4 Hz, 3-CH/Fmoc), 7.39 (2H,
t, J = 7.4 Hz, 4-CH/Fmoc), 7.45 (2H, d, J = 7.9 Hz, aromatic), 7.50
(2H, d, J = 7.9 Hz, aromatic), 7.56 (2H, d, J = 5.4 Hz, 2-CH/Fmoc),
7.76 (2H, d, J = 7.5 Hz, 5-CH/Fmoc); 13C NMR (100 MHz, CDCl3):
(3H, s, CO2CH3), 4.66 (1H, m,
aCH), 5.07 (1H, d, J = 7.0 Hz, NH),
7.23 (2H, d, J = 7.8 Hz, aromatic), 7.37 (1H, t, J = 7.1 Hz, 400-CH),
7.47 (2H, t, J = 7.4 Hz, 300-CH), 7.59 (2H, d, J = 8.1 Hz, aromatic),
7.65 (2H, d, J = 7.5 Hz, aromatic), 7.68 (4H, s, 20,30-CH); 13C NMR
(100 MHz, CDCl3): 28.3 (3x CH3/Boc), 38.0 (bCH2), 52.3 (CO2CH3),
21.1 (CH3), 37.4 (bCH2), 47.1 (CH/Fmoc), 54.7 (
a
CH), 66.9 (CH2/
54.4 (aCH), 80.0 (C/Boc), 127.0, 127.2, 127.4, 127.5, 128.9, 129.8
Fmoc), 119.9, 125.1, 125.2, 126.8, 127.0, 127.1, 127.7, 129.5,
129.9 (16x CH/aromatic BiPhe + aromatic Fmoc), 134.9, 136.9,
137.8, 139.7, 141.3, 143.8, 143.9 (8x C/aromatic BiPhe + aromatic
Fmoc), 155.8 (C@O/Fmoc), 174.1 (CO2H); IR tmax (cmꢀ1): 3347,
2924, 2497, 1723, 1689, 1529, 1254, 1216, 809, 738; ESI-MS m/z:
478.2003 (M+H)+ ((M+H)+, C31H28NO4 requires 478.2018),
500.1819 (M+Na)+.
(2, 3, 20, 30, 200, 300, 400-CH), 135.2, 139.4, 139.7, 140.1, 140.7 (1, 4,
10, 40, 100-C), 155.2 (C@O/Boc), 172.4 (CO2CH3); IR tmax (cmꢀ1):
3382, 2981, 1742, 1697, 1516, 1485, 1298, 1167, 817, 762; ESI-
+
MS m/z: 432.2182 (M+H)+ ((M+H)
, C27H30NO4 requires
432.2175), 454.2002 (M+Na)+, 376.1550 (MꢀC(CH3)3)+, 332.1648
(MꢀBoc)+.
Intermediate 3c (1.04 mmol, 331 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (1:1 to neat EtOAc) + 1% acetic
acid, Rf 0.35 in EtOAc/hexane (1:1) + 1% acetic acid) to yield the de-
sired product 4c as a white solid (383 mg, 68% over three steps):
5.8. Fmoc-4-(4-ethylphenyl)phenylalanine (4b)
The intermediate 3b was prepared following general procedure
A, using 342 mg 4-ethylphenylboronic acid. The crude product was
purified by flash column chromatography (EtOAc/hexane (7:93), Rf
0.36) to yield the desired product 3b as an off-white solid (339 mg,
mp 217 °C; ½a 2D0
ꢂ
+50.77 (c 0.1, CHCl3); 1H NMR (400 MHz, DMSO-
d6): d 2.93 (1H, dd, J = 10.8, 13.6 Hz, bCH2), 3.15 (1H, dd, J = 4.4,
13.9 Hz, bCH2), 4.15–4.29 (5H, m, CH/Fmoc + CH2/Fmo-
51%): mp 88–89 °C; ½a D20
ꢂ
+40.98 (c 0.1, CHCl3); 1H NMR (300 MHz,
CDCl3): d 1.31 (3H, t, J = 7.6 Hz, CH3), 1.47 (9H, s, CH3/Boc), 2.72
(2H, q, J = 7.6 Hz, CH2), 3.11 (1H, dd, J = 6.1, 13.7 Hz, bCH2), 3.20
(1H, dd, J = 5.6, 13.8 Hz, bCH2), 3.75 (3H, s, CO2CH3), 4.67 (1H, m,
c + aCH + NH), 7.26–7.42 (6H, m, aromatic), 7.48 (2H, t, J = 7.4 Hz,
aromatic), 7.61–7.67 (4H, m, aromatic), 7.70–7.75 (6H, m, aro-
matic), 7.79 (1H, d, J = 8.5 Hz, 400-CH), 7.87 (2H, d, J = 7.5 Hz, aro-
matic); 13C NMR (100 MHz, DMSO-d6): 46.5 (CH/Fmoc), 55.4
a
CH), 5.17 (1H, bs, NH), 7.23 (2H, d, J = 7.9 Hz, aromatic), 7.29
(2H, d, J = 8.1 Hz, aromatic), 7.54 (4H, m, aromatic); 13C NMR
(100 MHz, CDCl3): 15.6 (CH3), 28.3 (3x CH3/Boc), 28.5 (CH2), 38.0
(aCH), 65.6 (CH2/Fmoc), 120.0, 125.2, 126.3, 126.5, 126.9, 127.0,
127.1, 127.5, 127.6, 129.0, 129.8 (21x CH/aromatic TriPhe + aro-
matic Fmoc), 138.8, 138.9, 139.5, 140.6, 143.7 (9x C/aromatic Tri-
Phe + aromatic Fmoc), 156.0 (C@O/Fmoc), 173.3 (CO2H); IR tmax
(cmꢀ1): 3337, 2945, 1697, 1525, 1284, 1226, 814, 760, 738; ESI-
MS m/z: 540.2160 (M+H)+ ((M+H)+, C36H30NO4 requires
540.2175), 562.1976 (M+Na)+.
(bCH2), 52.2 (CO2CH3), 54.4 (aCH), 79.9 (C/Boc), 126.9, 127.1,
128.3, 129.7 (2, 3, 20, 30-CH), 134.8, 138.1, 139.9, 143.4 (1, 4, 10,
40-C), 155.2 (C@O/Boc), 172.4 (CO2CH3); IR tmax (cmꢀ1): 3362,
2965, 1735, 1682, 1513, 1498, 1299, 1158, 814; ESI-MS m/z:
384.2163 (M+H)+ ((M+H)+, C23H30NO4 requires 384.2175),
406.1988 (M+Na)+, 328.1531 (MꢀC(CH3)3)+, 284.1628 (MꢀBoc)+.
Intermediate 3b (0.80 mmol, 215 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (2:8) + 1% acetic acid, Rf 0.39 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
4b as a white solid (269 mg, 68% over three steps): mp 161 °C;
5.10. Fmoc-4-(2-methoxyphenyl)phenylalanine (4d)
The intermediate 3d was prepared following general procedure
B, using 399 mg of 2-methoxyphenylboronic acid. The crude prod-
uct was purified by flash column chromatography (EtOAc/hexane
(2:8), Rf 0.13 in EtOAc/hexane (2:8)) to yield the desired product
½
a 2D0
ꢂ
+45.19 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.28 (3H,
3d as a colourless foam (595 mg, 88%): ½a D20
ꢂ
+39.10 (c 0.1, CHCl3);
t, J = 7.6 Hz, CH3) 2.69 (2H, q, J = 7.6 Hz, CH2), 3.16 (1H, dd, J = 6.0,
13.8 Hz, bCH2), 3.26 (1H, dd, J = 5.3, 14.2 Hz, bCH2), 4.21 (1H, t,
J = 6.8 Hz, CH/Fmoc), 4.39 (1H, dd, J = 6.5, 10.6 Hz, CH2/Fmoc),
1H NMR (400 MHz, CDCl3): d 1.44 (9H, s, CH3/Boc), 3.09 (1H, dd,
J = 6.3, 13.9 Hz, bCH2), 3.17 (1H, dd, J = 5.6, 13.8 Hz, bCH2), 3.75
(3H, s, CO2CH3), 3.81 (3H, s, OCH3), 4.64 (1H, m,
aCH), 5.05 (1H,
4.46 (1H, dd, J = 7.0, 10.6 Hz, CH2/Fmoc), 4.75 (1H, m,
aCH), 5.29
d, J = 8.0 Hz, NH), 6.98 (1H, d, J = 8.3 Hz, 30-CH), 7.03 (1H, td,
J = 0.9, 7.5 Hz, 50-CH), 7.18 (2H, d, J = 8.0 Hz, aromatic), 7.31 (2H,
m, aromatic), 7.48 (2H, d, J = 8.2 Hz, aromatic); 13C NMR
(100 MHz, CDCl3): 28.3 (3x CH3/Boc), 38.0 (bCH2), 52.2 (CO2CH3),
(1H, d, J = 8.0 Hz, NH), 7.20 (2H, d, J = 7.9 Hz, aromatic), 7.25–7.33
(4H, m, aromatic), 7.39 (2H, t, J = 7.4 Hz, 4-CH/Fmoc), 7.46–7.51
(4H, m, aromatic), 7.57 (2H, m, aromatic), 7.76 (2H, d, J = 7.5 Hz,
5-CH/Fmoc); 13C NMR (100 MHz, CDCl3): 15.6 (CH3), 28.5 (CH2),
54.4 (a
CH), 55.5 (OCH3), 79.9 (C/Boc), 111.2 (30-CH), 120.9 (50-
37.3 (bCH2), 47.1 (CH/Fmoc), 54.6 (
a
CH), 67.1 (CH2/Fmoc), 120.0,
CH), 128.6, 129.0, 129.7 (2, 3, 40-CH), 130.2 (10-C), 130.8 (60-CH),
134.6, 137.3 (1,4-C), 155.2 (C@O/Boc), 156.5 (20-C), 172.5
(CO2CH3); IR tmax (cmꢀ1): 3367, 2976, 1743, 1711, 1487, 1237,
1161, 1025, 752; ESI-MS m/z: 386.1957 (M+H)+ ((M+H)+,
125.0, 126.9, 127.1, 127.2, 127.8, 128.3, 129.8 (16x CH/aromatic
BiPhe + aromatic Fmoc), 134.2, 138.0, 140.1, 141.3, 143.5, 143.7,
143.8 (8x C/aromatic BiPhe + aromatic Fmoc), 155.8 (C@O/Fmoc),
175.9 (CO2H); IR tmax (cmꢀ1): 3323, 2967, 1717, 1692, 1530,
1263, 1228, 816, 731; ESI-MS m/z: 492.2177 (M+H)+ ((M+H)+,
C
22H28NO5 requires 386.1967), 408.1778 (M+Na)+, 330.1336
(MꢀC(CH3)3) +, 286.1436 (MꢀBoc)+.
C
32H30NO4 requires 492.2175), 514.2001 (M+Na)+.
Intermediate 3d (1.50 mmol, 595 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (3:7) + 1% acetic acid, Rf 0.31 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
4d as a white solid (542 mg, 73% over three steps): mp 184 °C;
5.9. Fmoc–(p-terphenyl)alanine–COOH (4c)
The intermediate 3c was prepared following general procedure
A, using 451 mg of 4-biphenylboronic acid. The crude product was
purified by flash column chromatography (EtOAc/hexane (1:9–
2:8), Rf 0.24 in EtOAc/hexane (15:85)) to yield the desired product
3c as an off-white solid (450 mg, 60%): mp 185 °C (lit.37, 189–
½
a 2D0 +52.82 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 3.11 (1H,
dd, J = 6.5, 13.9 Hz, bCH2), 3.23 (1H, dd, J = 4.9, 14.0 Hz, bCH2),
ꢂ
3.70 (3H, s, OCH3), 4.16 (1H, t, J = 6.8 Hz, CH/Fmoc), 4.30–4.44
190 °C); ½a 2D0
ꢂ
+43.95 (c 0.1, CHCl3) (lit.37, + 54.81 (c 1, CHCl3)); 1H
NMR (400 MHz, CDCl3): d 1.45 (9H, s, CH3/Boc), 3.12 (1H, dd,
J = 5.9, 13.6 Hz, bCH2), 3.20 (1H, dd, J = 5.6, 13.7 Hz, bCH2), 3.76
(2H, m, CH2/Fmoc), 4.73 (1H, m, aCH), 5.56 (1H, d, J = 8.0 Hz,
NH), 6.91 (1H, d, J = 8.3 Hz, 30-CH), 6.96 (1H, t, J = 7.3 Hz, 50-CH),
7.17 (2H, d, J = 7.8 Hz, aromatic), 7.25 (4H, m, aromatic), 7.33

5146
S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
(2H, t, J = 7.4 Hz, aromatic), 7.44 (2H, d, J = 7.7 Hz, aromatic), 7.53
(2H, m, aromatic), 7.44 (2H, d, J = 7.4 Hz, aromatic); 13C NMR
matic), 7.39 (2H, d, J = 8.0 Hz, aromatic); 13C NMR (100 MHz,
CDCl3): 28.3 (3x CH3/Boc), 37.8 (bCH2), 52.2 (CO2CH3), 54.4
(100 MHz, CDCl3): 37.5 (bCH2), 47.2 (CH/Fmoc), 54.8 (
aCH), 55.5
(a
CH), 55.9 (OCH3), 79.8 (C/Boc), 110.3, 111.5 (20, 50-CH), 119.2
(OCH3), 67.2 (CH2/Fmoc), 111.4 (30-CH), 120.1, 121.0, 124.9,
125.0, 125.2, 125.3, 127.2, 127.8, 128.8, 129.2, 129.9, 130.2, 130.9
(15x CH/aromatic BiPhe + aromatic Fmoc), 134.4, 134.6, 137.5,
141.4, 143.8, 143.9 (7x C/aromatic BiPhe + aromatic Fmoc), 156.2,
156.5 (20-C, C@O/Fmoc), 177.5 (CO2H); IR tmax (cmꢀ1): 3310,
2930, 1698, 1529, 1254, 738; ESI-MS m/z: 494.1956 (M+H)+
((M+H)+, C31H28NO5 requires 494.1967), 516.1787 (M+Na)+.
(60-CH), 126.9–129.7 (2, 3-CH), 133.7, 134.6, 139.7 (1, 4, 10-C),
148.6, 149.1 (30, 40-C), 155.1 (C@O/Boc), 172.3 (CO2CH3); IR tmax
(cmꢀ1): 3383, 2993, 2969, 1742, 1688, 1504, 1254, 1170, 1145,
1018, 802; ESI-MS m/z: 416.2059 (M+H)+ ((M+H)+, C23H30NO6 re-
quires 416.2073), 438.1878 (M+Na)+, 360.1432 (MꢀC(CH3)3)+,
316.1530 (MꢀBoc)+.
Intermediate 3f (1.60 mmol, 640 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to DCM + 1% MeOH, Rf 0.25 in EtOAc/hexane
(1:1) + 1% acetic acid) to yield the desired product 4f as a white so-
5.11. Fmoc-4-(2,6-dimethoxyphenyl)phenylalanine (4e)
The intermediate 3e was prepared following general procedure
B, using 478 mg of 2,6-dimethoxyphenylboronic acid. The crude
product was purified by flash column chromatography (EtOAc/
hexane (2:8), Rf 0.18) to yield the desired product 3e as a white so-
lid (243 mg, 29% over three steps): mp 141 °C; ½a D20
ꢂ
+44.58 (c 0.1,
d 3.16 (1H, dd, J = 6.3,
CHCl3); 1H NMR (400 MHz, CDCl3):
13.9 Hz, bCH2), 3.26 (1H, dd, J = 5.3, 14.0 Hz, bCH2), 3.91 (6H, s,
OCH3), 4.20 (1H, t, J = 6.9 Hz, CH/Fmoc), 4.36 (1H, dd, J = 7.0,
10.3 Hz, CH2/Fmoc), 4.45 (1H, dd, J = 7.3, 10.7 Hz, CH2/Fmoc), 4.74
lid (248 mg, 34%): mp 65 °C; ½a D20
ꢂ
+24.60 (c 0.1, CHCl3); 1H NMR
(400 MHz, CDCl3): d 1.43 (9H, s, CH3/Boc), 3.09–3.13 (2H, m,
bCH2), 3.72 (6H, s, OCH3), 3.73 (3H, s, CO2CH3), 4.62 (1H, m,
(1H, m,
aCH), 5.29 (1H, d, J = 8.6 Hz, NH), 6.91 (1H, d, J = 8.3 Hz,
a
CH), 5.05 (1H, d, J = 7.9 Hz, NH), 6.64 (2H, d, J = 8.4 Hz, 30-CH),
50-CH), 7.05 (1H, s, 20-CH), 7.09 (1H, d, J = 8.4 Hz, 60-CH), 7.20 (2H,
d, J = 7.9 Hz, aromatic), 7.28 (2H, t, J = 7.9 Hz, 3-CH/Fmoc), 7.38
(2H, t, J = 7.4 Hz, 4-CH/Fmoc), 7.47 (2H, d, J = 7.9 Hz, aromatic),
7.55 (2H, dd, J = 3.3, 7.2 Hz, 2-CH/Fmoc), 7.75 (2H, d, J = 7.8 Hz, 5-
CH/Fmoc); 13C NMR (100 MHz, CDCl3): 37.3 (bCH2), 47.1 (CH/
7.16 (2H, d, J = 7.9 Hz, aromatic), 7.23 (1H, dd, J = 3.0, 13.4 Hz, 40-
CH), 7.28 (2H, d, J = 8.1 Hz, aromatic); 13C NMR (100 MHz, CDCl3):
28.3 (3x CH3/Boc), 38.1 (bCH2), 52.2 (CO2CH3), 54.4 (aCH), 55.9 (2x
OCH3), 79.9 (C/Boc), 104.2 (30-CH +10-C), 128.6, 128.7 (2, 40-CH),
131.2 (3-CH), 134.2 (1-C), 155.2 (C@O/Boc), 157.7 (20-C), 172.6
(CO2CH3); IR tmax (cmꢀ1): 3371, 2934, 1739, 1688, 1515, 1245,
1161, 1105, 1004, 781; ESI-MS m/z: 416.2053 (M+H)+ ((M+H)+,
Fmoc), 54.6 (aCH), 55.9 (2x OCH3), 67.1 (CH2/Fmoc), 110.3, 111.5
(20, 50-CH), 119.3, 120.0, 125.0, 125.1, 127.1, 127.8, 129.8 (13x
CH/aromatic BiPhe + aromatic Fmoc), 133.6, 134.1, 140.0, 141.3,
143.7 (7x C/aromatic BiPhe + aromatic Fmoc), 148.6, 149.1 (30, 40-
C), 155.8 (C@O/Fmoc), 175.6 (CO2H); IR tmax (cmꢀ1): 3319, 2937,
2574, 1697, 1529, 1503, 1251, 1143, 737; ESI-MS m/z: 524.2078
(M+H)+ ((M+H)+, C32H30NO6 requires 524.2073), 546.1901
(M+Na)+.
C
23H30NO6 requires 416.2073), 438.1879 (M+Na)+, 316.1546
(MꢀBoc)+.
Intermediate 3e (0.60 mmol, 248 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (3:7) + 1% acetic acid, Rf 0.34 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
4e as a white solid (227 mg, 73% over three steps): mp 116 °C;
5.13. Fmoc-4-(3-nitrophenyl)phenylalanine (4g)
½
a 2D0
ꢂ
+46.39 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 3.16 (1H,
The intermediate 3g was prepared following general procedure
B, using 438 mg of 3-nitrophenylboronic acid. The crude product
was purified by flash column chromatography (EtOAc/hexane
(1:9), Rf 0.16 in EtOAc/hexane (2/8)) to yield the desired product
dd, J = 6.4, 14.1 Hz, bCH2), 3.24 (1H, dd, J = 5.2, 14.1 Hz, bCH2),
3.67 (6H, s, OCH3), 4.21 (1H, t, J = 7.0 Hz, CH/Fmoc), 4.36 (1H, dd,
J = 6.9, 10.2 Hz, CH2/Fmoc), 4.43 (1H, dd, J = 7.5, 10.6 Hz, CH2/
Fmoc), 4.75 (1H, m,
a
CH), 5.45 (1H, d, J = 8.3 Hz, NH), 6.62 (2H, d,
3g as an off-white solid (638 mg, 91%): mp 92 °C; ½a D20
ꢂ
+34.60 (c
J = 8.4 Hz, 30-CH), 7.19 (2H, d, J = 8.0 Hz, aromatic), 7.28 (5H, m,
aromatic), 7.36 (2H, t, J = 7.4 Hz, 4-CH/Fmoc), 7.57 (2H, dd, J = 2.7,
7.1 Hz, 2-CH/Fmoc), 7.73 (2H, d, J = 7.4 Hz, 5-CH/Fmoc); 13C NMR
0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.43 (9H, s, CH3/Boc),
3.11 (1H, dd, J = 6.1, 13.7 Hz, bCH2), 3.20 (1H, dd, J = 5.6, 13.7 Hz,
bCH2), 3.76 (3H, s, CO2CH3), 4.64 (1H, m,
aCH), 5.08 (1H, d,
(100 MHz, CDCl3): 37.5 (bCH2), 47.2 (CH/Fmoc), 54.6 (
a
CH), 55.9
J = 7.9 Hz, NH), 7.27 (2H, d, J = 8.2 Hz, aromatic), 7.56 (2H, d,
J = 8.3 Hz, aromatic), 7.60 (1H, t, J = 8.0 Hz, 50-CH), 7.89 (1H, d,
J = 8.2 Hz, 60-CH), 8.18 (1H, ddd, J = 0.8, 2.2, 8.2 Hz, 40-CH), 8.43
(1H, dd, J = 2.0 Hz, 20-CH); 13C NMR (100 MHz, CDCl3): 28.3 (3x
(2x OCH3), 67.2 (CH2/Fmoc), 104.3 (30-CH), 119.0 (10-C), 120.0,
125.2, 127.1, 127.8, 128.8, 131.3 (13x CH/aromatic BiPhe + aro-
matic Fmoc), 133.1, 133.9 (1, 4-C), 141.3, 143.8, 143.9 (1, 6-C/
Fmoc), 156.0 (C@O/Fmoc), 157.6 (20-C), 177.7 (CO2H); IR tmax (cm
ꢀ1): 3340, 2948, 2647, 1696, 1523, 1246, 724; ESI-MS m/z:
524.2058 (M+H)+ ((M+H)+, C32H30NO6 requires 524.2073),
546.1877 (M+Na)+.
CH3/Boc), 38.0 (bCH2), 52.3 (CO2CH3), 54.4 (aCH), 79.9 (C/Boc),
121.7, 121.9 (20, 40-CH), 127.2, 129.7, 130.1, 132.8 (2, 3, 50, 60-CH),
136.8, 137.2, 142.3 (1, 4, 10- C), 148.7 (30-C), 155.1 (C@O/Boc),
172.2 (CO2CH3); IR tmax (cmꢀ1): 3350, 2987, 2946, 1750, 1689,
1518, 1347, 1164, 730; ESI-MS m/z: 401.1703 (M+H)+ ((M+H)+,
5.12. Fmoc-4-(3,4-dimethoxyphenyl)phenylalanine (4f)
C
21H25N2O6 requires 401.1713), 423.1524 (M+Na)+, 345.1079
(MꢀC(CH3)3)+, 301.1182 (MꢀBoc)+.
The intermediate 3f was prepared following general procedure
B, using 478 mg of 3,4-dimethoxyphenylboronic acid. The crude
product was purified by flash column chromatography (MeOH/
DCM 0 to 1% then EtOAc/hexane (2:8), Rf 0.14 in EtOAc/hexane
(2:8)) to yield the desired product 3f as a white solid (640 mg,
Intermediate 3g (1.60 mmol, 638 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (3:7) + 1% acetic acid, Rf 0.28 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
4g as a white solid (265 mg, 33% over three steps): mp 199 °C;
88%): mp 112 °C; ½a D20
ꢂ
+48.10 (c 0.1, CHCl3); 1H NMR (400 MHz,
CDCl3): d 1.33 (9H, s, CH3/Boc), 2.97 (1H, dd, J = 5.9, 13.6 Hz,
bCH2), 3.06 (1H, dd, J = 5.5, 13.7 Hz, bCH2), 3.64 (3H, s, CO2CH3),
½
a 2D0 +38.96 (c 0.1, CHCl3); 1H NMR (400 MHz, acetone-d6): d 3.14
(1H, dd, J = 9.5, 13.7 Hz, bCH2), 3.37 (1H, dd, J = 4.5, 13.9 Hz,
bCH2), 4.17 (1H, t, J = 7.1 Hz, CH/Fmoc), 4.26 (1H, dd, J = 7.1,
10.2 Hz, CH2/Fmoc), 4.33 (1H, dd, J = 7.4, 10.3 Hz, CH2/Fmoc), 4.44
ꢂ
3.80 (3H, s, OCH3), 3.84 (3H, s, OCH3), 4.52 (1H, m,
aCH), 5.03
(1H, d, J = 7.9 Hz, NH), 6.83 (1H, d, J = 8.2 Hz, 50-CH), 7.00 (1H, s,
20-CH), 7.02 (1H, d, J = 7.2 Hz, 60-CH), 7.09 (2H, d, J = 7.8 Hz, aro-
(1H, m,
aCH), 6.82 (1H, d, J = 8.6 Hz, NH), 7.30 (2H, m, aromatic),

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
5147
7.39 (2H, t, J = 7.4 Hz, 4-CH/Fmoc), 7.49 (2H, d, J = 7.9 Hz, aromatic),
7.66–7.73 (5H, m, aromatic), 7.84 (2H, d, J = 7.5 Hz, 5-CH/Fmoc),
8.01 (1H, d, J = 7.7 Hz, 60-CH), 8.19 (1H, dd, J = 1.5, 8.1 Hz, 40-CH),
8.4 (1H, s, 20-CH); 13C NMR (100 MHz, acetone-d6): 37.9 (bCH2),
13.8 Hz, bCH2), 3.71 (3H, s, CO2CH3), 4.56 (1H, m,
a
CH), 4.98 (1H,
d, J = 8.1 Hz, NH), 6.10 (1H, bs, 20-CH), 7.05 (2H, d, J = 8.1 Hz, aro-
matic), 7.30 (2H, d, J = 8.3 Hz, aromatic); 13C NMR (100 MHz,
CDCl3): 23.0, 24.8, 25.9, 27.3 (30, 40, 50, 60-CH2), 28.3 (3x CH3/Boc),
48.0 (CH/Fmoc), 56.0 (
a
CH), 67.2 (CH2/Fmoc), 120.8, 122.0, 122.7,
37.8 (bCH2), 52.2 (CO2CH3), 54.4 (aCH), 79.9 (C/Boc), 124.6,
126.1, 126.2, 127.9, 128.5, 131.0, 133.8 (16x CH/aromatic
BiPhe + aromatic Fmoc), 137.7, 139.2, 142.0, 143.2, 145.0 (7x C/
aromatic BiPhe + aromatic Fmoc), 149.8 (30-C), 156.9 (C@O/Fmoc),
173.3 (CO2H); IR tmax (cmꢀ1): 3309, 3063, 1694, 1529, 1515,
1353, 1257, 741; ESI-MS m/z: 509.1710 (M+H)+ ((M+H)+,
125.0, 129.1 (2, 3, 20-CH), 134.1, 136.1, 141.3 (1, 4, 10-C), 155.1
(C@O/Boc), 172.4 (CO2CH3); IR tmax (cmꢀ1): 3369, 2930, 1744,
1713, 1498, 1162, 732; ESI-MS m/z: 360.2166 (M+H)+ ((M+H)+,
C
21H30NO4 requires 360.2175), 382.1979 (M+Na)+, 304.1538
(MꢀC(CH3)3)+, 260.1644 (MꢀBoc)+.
C
30H25N2O6 requires 509.1713), 531.1531 (M+Na)+.
Intermediate 3i (1.63 mmol, 567 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (3:7) + 1% acetic acid, Rf 0.44 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
5.14. Fmoc-4-(4-cyanophenyl)phenylalanine (4h)
The intermediate 3h was prepared following general procedure
B, using 601 mg of 4-cyanophenylboronic acid pinacol ester. The
crude product was purified by flash column chromatography
(EtOAc/hexane (1:9 to 2:8, Rf 0.17 in EtOAc/hexane (2:8)) to yield
4i as a white foam (588 mg, 77% over three steps): ½a D20
ꢂ
+60.85 (c
0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.66 (2H, m, CH2/cyclo-
hexene), 1.76 (2H, m, CH2/cyclohexene), 2.19 (2H, m, CH2/cyclo-
hexene), 2.37 (2H, m, CH2/cyclohexene), 3.11 (1H, dd, J = 6.1,
13.9 Hz, bCH2), 3.21 (1H, dd, J = 5.2, 13.8 Hz, bCH2), 4.20 (1H, t,
J = 6.9 Hz, CH/Fmoc), 4.36 (1H, dd, J = 7.2, 10.7 Hz, CH2/Fmoc),
the desired product 3h as a colourless foam (310 mg, 48%): ½a D20
ꢂ
+37.90 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3): d 1.42 (9H, s,
CH3/Boc), 3.08 (1H, dd, J = 6.0, 13.7 Hz, bCH2), 3.19 (1H, dd,
J = 5.6, 13.7 Hz, bCH2), 3.75 (3H, s, CO2CH3), 4.63 (1H, m,
a
CH),
4.44 (1H, dd, J = 7.4, 10.5 Hz, CH2/Fmoc), 4.72 (1H, m, aCH), 4.26
5.05 (1H, d, J = 7.9 Hz, NH), 7.25 (2H, d, J = 8.1 Hz, aromatic), 7.53
(2H, d, J = 8.3 Hz, aromatic), 7.66 (2H, d, J = 8.6 Hz, aromatic),
7.72 (2H, d, J = 8.4 Hz, aromatic); 13C NMR (100 MHz, CDCl3):
(1H, d, J = 8.2 Hz, NH), 6.11 (1H, bs, 20-CH), 7.09 (2H, d, J = 7.9 Hz,
aromatic), 7.31 (4H, m, aromatic), 7.40 (2H, t, J = 7.4 Hz, 3-CH/
Fmoc), 7.56 (2H, t, J = 6.6 Hz, 4-CH/Fmoc), 7.77 (2H, d, J = 7.5 Hz,
13
28.3 (3x CH3/Boc), 38.1 (bCH2), 52.3 (CO2CH3), 54.3 (
a
CH), 80.1
aromatic); C NMR (100 MHz, CDCl3): 22.1, 23.0, 25.9, 27.3 (30,
(C/Boc), 110.8 (40-C), 118.9 (CN), 127.3, 127.6 (3, 20-CH), 130.1 (2-
CH), 132.6 (30-CH), 136.9, 137.8 (1, 4-C), 145.2 (10-C), 155.1
(C@O/Boc), 172.2 (CO2CH3); IR tmax (cmꢀ1): 3368, 2977, 2227,
1743, 1709, 1497, 1249, 1161, 1055, 1006, 817; ESI-MS m/z:
381.1813 (M+H)+ ((M+H)+, C22H25N2O4 requires 381.1814),
403.1628 (M+Na)+, 325.1197 (MꢀC(CH3)3)+, 281.1300 (MꢀBoc)+.
Intermediate 3h (0.84 mmol, 310 mg) was converted into the
corresponding Fmoc- protected building block using general proce-
dure C. The crude product was purified by flash column chroma-
tography (DCM to EtOAc/hexane (3:7) + 1% acetic acid, Rf 0.28 in
EtOAc/hexane (1:1) + 1% acetic acid) to yield the desired product
4h as a white solid (303 mg, 74% over three steps): mp 173 °C;
40, 50, 60-CH2), 37.3 (bCH2), 47.1 (CH/Fmoc), 54.6 (
aCH), 67.1 (CH2/
Fmoc), 120.0, 124.9, 125.1, 127.1, 127.8, 129.2 (13x CH/aromatic
Phe + aromatic Fmoc + 20-CH), 133.6, 136.0, 141.3, 141.5, 143.7,
143.8 (7x C/aromatic Phe + aromatic Fmoc + 10-C), 155.8 (C@O/
Fmoc), 176.3 (CO2H); IR tmax (cmꢀ1): 2941, 1708, 1515, 1487,
1450, 1408, 1234, 738; ESI-MS m/z: 468.2163 (M+H)+ ((M+H)+,
C
30H30NO4 requires 468.2175), 490.1979 (M+Na)+.
6. Solid phase peptide synthesis
The peptides were assembled on a 0.1 mmol scale using stan-
dard Fmoc solid phase peptide synthesis (SPPS) on a Tribute™ pep-
tide synthesiser (Protein Technologies, Inc.). The syntheses were
performed on aminomethylated polystyrene resin (1.0 mmol/g)
derivatised with HMP linker and all peptides were capped with
½
a 2D0 +49.25 (c 0.09, CHCl3); 1H NMR (400 MHz, CDCl3): d 3.15
(1H, dd, J = 6.5, 13.9 Hz, bCH2), 3.29 (1H, dd, J = 5.3, 14.0 Hz,
bCH2), 4.17 (1H, t, J = 6.8 Hz, CH/Fmoc), 4.36 (1H, dd, J = 6.9,
10.2 Hz, CH2/Fmoc), 4.45 (1H, dd, J = 7.3, 10.2 Hz, CH2/Fmoc), 4.76
ꢂ
D-biotin.
(1H, m,
aCH), 5.41 (1H, d, J = 8.1 Hz, NH), 7.24–7.30 (4H, m, aro-
6.1. Biotin-R8ERY⁄ (Y⁄ = 4-(4-methylphenyl)phenylalanine) (5)
matic), 7.39 (2H, t, J = 7.4 Hz, aromatic), 7.46 (2H, d, J = 7.9 Hz, aro-
matic), 7.53–7.58 (4H, m, aromatic), 7.67 (2H, d, J = 8.1 Hz,
aromatic), 7.76 (2H, d, J = 7.5 Hz, aromatic); 13C NMR (100 MHz,
The peptide chain was assembled using building block 4a.
16 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
3 mg of purified peptide (13%) as a white solid in ca. 93% purity
according to analytical HPLC. Rt 12.5 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 337.0 [M+6H+], 404.2 [M+5H+], 505.1
[M+4H+].
CDCl3): 37.5 (bCH2), 47.1 (CH/Fmoc), 54.6 (aCH), 67.1 (CH2/Fmoc),
110.8 (40-CH), 119.0 (CN), 120.1, 125.0, 125.1, 127.1, 127.4, 127.5,
127.8, 130.2, 132.6 (16x CH/aromatic BiPhe + aromatic Fmoc),
136.4, 138.0, 141.3, 143.7, 145.1 (7x C/aromatic BiPhe + aromatic
Fmoc), 155.9 (C@O/Fmoc), 177.4(CO2H); IR tmax (cmꢀ1): 3307,
2948, 2227, 1691, 1539, 1257, 736; ESI-MS m/z: 489.1819
(M+H)+ ((M+H)+, C31H25N2O4 requires 489.1814), 511.1641
(M+Na)+.
6.2. Biotin-R8ERY⁄ (Y⁄ = 4-(4-ethylphenyl)phenylalanine) (6)
5.15. Fmoc-4-(10-cyclohexenyl)phenylalanine (4i)
The peptide chain was assembled using building block 4b.
17 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
5 mg of purified peptide (35%) as a white solid in ca. 96% purity
according to analytical HPLC. Rt 23.1 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 339.4 [M+6H+], 407.0 [M+5H+], 508.6
[M+4H+].
The intermediate 3i was prepared following the general proce-
dure B, using 0.6 mL of cyclohexene-1-boronic acid pinacol ester.
The crude product was purified by flash column chromatography
(EtOAc/hexane (1:9), Rf 0.19 in EtOAc/hexane (1:9)) to yield the de-
sired product 3i as a yellow oil (567 mg, 93%): ½a D20
ꢂ
+41.00 (c 0.1,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.41 (9H, s, CH3/Boc), 1.61–
1.67 (2H, m, CH2/cyclohexene), 1.74–1.80 (2H, m, CH2/cyclohex-
ene), 2.19 (2H, m, CH2/cyclohexene), 2.37 (2H, m, CH2/cyclohex-
ene), 3.02 (1H, dd, J = 6.1, 13.9 Hz, bCH2), 3.08 (1H, dd, J = 5.8,
See supporting information for details.

5148
S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
6.3. Biotin-R8ERY⁄ (Y⁄ = (p-terphenyl)alanine) (7)
purified peptide (15%) as a white solid in ca. 91% purity according
to analytical HPLC. Rt 22.5 min (1–80% B over 30 min, 1 mL/min);
m/z (ESI-MS): 335.7 [M+6H+], 402.6 [M+5H+], 503.1 [M+4H+].
The peptide chain was assembled using building block 4c.
12 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
1 mg of purified peptide (15%) as a white solid in ca. 97% purity
according to analytical HPLC. Rt 20.0 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 347.4 [M+6H+], 416.6 [M+5H+], 520.6
[M+4H+].
6.10. Biotin-R8ERY⁄ (Y⁄ = 4-(10-cyclohexenyl)phenylalanine) (14)
The peptide chain was assembled using building block 4i.
12.6 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
0.6 mg of purified peptide (3%) as a white solid in ca. 99% purity
according to analytical HPLC. Rt 13.9 min (1–90% B over 30 min,
1 mL/min); m/z (ESI-MS): 335.4 [M+6H+], 402.2 [M+5H+], 502.5
[M+4H+].
6.4. Biotin-R8ERY⁄ (Y⁄ = 4-(2-methoxyphenyl)phenylalanine) (8)
The peptide chain was assembled using building block 4d.
16.5 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
9 mg of purified peptide (47%) as a white solid in ca. 96% purity
according to analytical HPLC. Rt 14.9 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 407.4 [M+5H+], 509.0 [M+4H+].
7. Cell assays
7.1. Cell line
6.5. Biotin-R8ERY⁄ (Y⁄ = 4-(2,6-dimethoxyphenyl)phenylalanine)
(9)
The mouse spontaneous AKR/Cum Y CD8
phoma cell line TK-1 was purchased from the American Type Cul-
ture Collection, Rockville, MD. It was cultured at 37 °C in RPMI
a a
4b7+/ 4b1ꢀ T lym-
1640 medium supplemented with 50 U/ml penicillin, 50
streptomycin, 200 g/ml -glutamine, 10% (v/v) FCS and 0.05 mM
b-mercaptoethanol.
lg/ml
The peptide chain was assembled using building block 4e.
15 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
6 mg of purified peptide (53%) as a white solid in ca. 99% purity
according to analytical HPLC. Rt 15.3 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 344.7 [M+6H+], 413.4 [M+5H+], 516.6
[M+4H+].
l
L
7.2. Recombinant MAdCAM-1
The production of soluble recombinant mouse MAdCAM-1-Fc
from insect cells using a baculovirus expression system has been
described previously.43
6.6. Biotin-R8ERY⁄ (Y⁄ = 4-(3,4-dimethoxyphenyl)phenylalanine)
(10)
7.3. Cell adhesion assay
The peptide chain was assembled using building block 4f.
14 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
5 mg of purified peptide (43%) as a white solid in ca. 99% purity
according to analytical HPLC. Rt 10.6 min (1–60% B over 20 min,
1 mL/min); m/z (ESI-MS): 344.7 [M+6H+], 413.4 [M+5H+], 516.5
[M+4H+].
Lab-Tek 16-well glass slides (Nunc) were coated with purified
mouse MAdCAM-1-Fc (10 lg/ml), and incubated at 4 °C overnight.
Slides were washed once with PBS, and blocked with FBS for 2 h at
room temperature. They were washed thrice with Hanks balanced
salt solution (HBSS) containing 10 mM Hepes. TK-1 cells were
washed thrice with HBSS containing 10 mM Hepes. They were
resuspended in HBSS containing 10 mM Hepes and different con-
centrations of the b7 peptides or no peptide, and left standing for
1 h at room temperature. Integrins on the cells were activated by
addition of MnCl2 to 2 mM. The cells were added to the MAd-
CAM-1-coated slides at 106 cells/well and left to adhere for
30 min at room temperature. Non-adherent cells were removed
by twice dipping slides gently into PBS. The adherent cells were
fixed in PBS containing 2% (v/v) glutaraldehyde for at least 3 h,
and stained with 0.1% methylene blue for 5 min, and destained
with ethanol. The slides were air-dried and the absorbance at
495 nm was read and plotted, as a measure of cell adhesion. The
adhesion assays were repeated at least thrice using duplicate
wells.
6.7. Biotin-R8ERY⁄ (Y⁄ = 4-(3-nitrophenyl)phenylalanine) (11)
The peptide chain was assembled using building block 4g.
18 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
5 mg of purified peptide (34%) as a white solid in ca. 98% purity
according to analytical HPLC. Rt 10.8 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 342.2 [M+6H+], 410.4 [M+5H+], 512.8
[M+4H+], 683.4 [M+3H+].
6.8. Biotin-R8ERY⁄ (Y⁄ = 4-(4-cyanophenyl)phenylalanine) (12)
The peptide chain was assembled using building block 4h.
17 mg of crude peptide were purified by semi-preparative HPLC
using a linear gradient of 1% B to 80% B over 60 min, yielding
4 mg of purified peptide (23%) as a white solid in ca. 93% purity
according to analytical HPLC. Rt 16.3 min (1–80% B over 30 min,
1 mL/min); m/z (ESI-MS): 338.9 [M+6H+], 406.4 [M+5H+], 507.8
[M+4H+], 714.7 [M+3H++TFA].
7.4. Statistical analysis
Results were expressed as mean standard error of the mean
(SEM), and a Student’s t-test was used to evaluate statistical signif-
icance. P values 60.05 were considered statistically significant.
Acknowledgments
6.9. Biotin-R8ERY⁄ (Y⁄ = (4-cyclohexyl)phenylalanine) (13)
We thank the Maurice Wilkins Centre for Molecular Biodiscov-
ery for supporting the peptide synthesis laboratory and Auckland
Uniservices Ltd. and the Foundation for Research, Science and
Technology, New Zealand for financial support (AN).
The peptide chain was assembled using building block 4i. 18 mg
of crude peptide were purified by semi-preparative HPLC using a
linear gradient of 1% B to 80% B over 60 min, yielding 3 mg of

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 5139–5149
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