Tyrosine modified analogues of the α4β7 integrin inhibitor biotin-R8ERY prepared via Click Chemistry: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2012.02.035

Our continuing programme aiming at developing inhibitors of integrin α4β7, a key mediator of various inflammatory diseases, led us to synthesise a library of cell-permeable peptides based on the biotin-R ₈ERY* template, wherein the tyrosine residue has been modified by using the CuAAC reaction. The 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. Two of the synthesised peptidomimetics, analogues 11 and 14, are more active than our previously reported lead compound biotin-r₉YDRREY at concentrations of 100 and 50 μM, with 14 exhibiting an IC₅₀ of less than 10 μM.

Full text of DOI:10.1016/j.bmc.2012.02.035

Bioorganic & Medicinal Chemistry 20 (2012) 2638–2644
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tyrosine modified analogues of the
a
4b7 integrin inhibitor biotin-R8ERY
prepared via Click Chemistry: Synthesis and biological evaluation
Stefanie Papst a, Anaïs 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:
Our continuing programme aiming at developing inhibitors of integrin
a4b7, a key mediator of various
Received 19 December 2011
Revised 31 January 2012
Accepted 13 February 2012
Available online 22 February 2012
inflammatory diseases, led us to synthesise a library of cell-permeable peptides based on the biotin-
R8ERY⁄ template, wherein the tyrosine residue has been modified by using the CuAAC reaction. The pep-
tidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn2+-activated adhesion of
mouse TK-1 T cells to mouse MAdCAM-1. Two of the synthesised peptidomimetics, analogues 11 and
14, are more active than our previously reported lead compound biotin-r9YDRREY at concentrations of
Keywords:
Integrin inhibitors
Peptidomimetics
100 and 50 lM, with 14 exhibiting an IC50 of less than 10 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(MLNs) and the lamina propria (LP).10 In contrast, the
which is formed by association of the b7 subunit with the integrin
E subunit, mediates the binding of intestinal intraepithelial lym-
phocytes (iIEL) to E-cadherin expressed on gut enterocytes.11
The two b7 integrins 4b7 and Eb7 play key roles in regulating
aEb7 receptor
Integrins are a widely expressed family of cell surface adhesion
receptors that play a major role in cell attachment to extracellular
matrices, and also in the mediation of important cell–cell adhesion
events.1
a
a
a
gut immunity and are involved in the pathogenesis and progres-
sion of inflammatory bowel diseases, such as Crohn’s disease,
ulcerative colitis,12–14 and intestinal graft-versus-host diseases
The
meric cell surface receptors expressed on most leukocytes2,3 and
consist of noncovalently-associated and b transmembrane sub-
units with comparatively large extracellular and short cytoplasmic
domains. The most important ligands of the 4 integrin subfamily
a4 integrins a4b7 (LPAM-1) and a4b1 (VLA-4) are heterodi-
a
(GVHD).11,15 Moreover,
a4b7 integrins contribute to the infiltration
of leukocytes into the islets of Langerhans in type 1 diabetes16 and
the central nervous system in demyelinating diseases such as mul-
tiple sclerosis.17 Clearly appropriate control of b7 integrins is cru-
cial in many disease states and for this reason, the development
of selective drugs that preclude the homing of lymphocytes to
chronically inflamed tissues but with minimal effects on normal
immune surveillance would be highly desirable. To date, several
pharmaceutical companies have produced a number of anti-
a
include fibronectin (Fn), vascular cell adhesion molecule-1
(VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAd-
CAM-1).4–6 Integrin
a4b1 preferentially recognises VCAM-1,
whereas MAdCAM-1 is an exclusive ligand for
a
4b7 under physio-
logical conditions.3,7–9 Lymphocytes bind via
a
4b7 to MAdCAM-1
expressed on high endothelial venules, enabling their extravasation
from blood to gut mucosal tissues of gut-associated lymphoid tis-
sues (GALT) such as Peyer’s patches, mesenteric lymph nodes
inflammatory therapies based on small molecule
4b7 antagonists that prevent the binding of integrins to their
extracellular ligands.7,18–20
a4b1 and/or
a
A very different strategy for inhibiting b7 integrin mediated
Abbreviations: CuAAC, copper catalyzed azide-alkyne cycloaddition; FAK, focal
adhesion kinase; FBS, fetal bovine serum; GALT, gut-associated lymphoid tissues;
GnHCl, guanidine hydrochloride; GVHD, graft-versus-host disease; HBSS, Hank’s
buffered salt solution; HMP, 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
extravasation of leukocytes was developed by Krissansen and co-
workers in 2006. They demonstrated that the L- and D-enantiomers
of biotin-r9YDRREY inhibited adhesion of Mn2+-activated T cells to
b7 integrin ligands. The hexapeptide YDRREY corresponds to resi-
dues 735–740 of the cytoplasmic tail of the b7 subunit,
a
anti-inflammatory drugs; PBS, phosphate buffered saline; r9, (D-Arginine)9; SPPS,
solid phase peptide synthesis; src, sarcoma; TFA, trifluoroacetic acid; VCAM-1,
vascular cell adhesion molecule-1; VLA-4, very late activating antigen-4.
polyarginine tag and a biotin moiety were appended to the N-ter-
minus in order to minimize their effect on the biological activity of
the hexapeptide. The polyarginine tag serves as a cationic carrier
enabling its penetration into the cytoplasm of activated
⇑
Corresponding author.
E-mail address: m.brimble@auckland.ac.nz (M.A. Brimble).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.035

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 2638–2644
2639
T cells,21–23 whereas the biotin facilitates detection of peptide up-
6 (Y⁄ = O-propargyl-
L-tyrosine), which, we trusted, would further
take by cells as required. Biotin-r9YDRREY is presumably acting as
a competitive substrate for src, FAK, and other tyrosine kinases and
signalling molecules.24 It is thereby proposed to interrupt the nor-
mal phosphorylation of the intracellular b7 subunit, and the asso-
improve the activity. The choice of azide component was dictated
by the availability of certain azides in our laboratory. It was hoped
that the range of azides incorporated into the peptide scaffold
would lead to an effective cell adhesion inhibitor.
ciation of
cytoskeletal elements. It suppresses MAdCAM-1-induced cluster-
ing of 4b7 receptors on the surface of T cells and thereby disrupts
a
4b7 with intracellular signalling molecules and
Click chemistry was introduced by Sharpless and co-workers in
200125 and subsequently they and Meldal et al. independently dis-
covered a dramatic acceleration of reaction rate between terminal
alkynes and azides by the addition of catalytic amounts of Cu(I)
one year later.26,27 Consequently, although several types of reac-
tions fall under the term click chemistry,28 the CuAAC has assumed
a particularly important role in organic synthesis over the last dec-
ade owing to the stability of azides under a large number of reac-
tion conditions.29–31 Additionally, the Cu(I)-catalysed cyclisation
unites azides and terminal alkynes regiospecifically, giving only
1,4-disubstituted 1,2,3-triazoles. The CuAAC reaction can be easily
combined with peptide chemistry as the reaction remains unaf-
fected by the presence of other functional groups such as those
present on the peptide side-chains.
a
cell adhesion (Fig. 1).
Recently, we showed that biotin-R8ERY which displays a shorter
active unit, namely tripeptide ERY, exhibited similar activity to
that of the biotin-r9YDRREY analogue. We initially synthesised a
small library of biotin-R8ERY⁄ peptides using a number of commer-
cially available tyrosine analogues and we established that the
analogue biotin-R8ERY⁄ (Y⁄ = 4-chlorophenylalanine) possessed
activity approaching that of the biotin-r9YDRREY. We therefore
envisaged using the well-known Cu(I)-catalysed azide–alkyne
‘click’ cycloaddition (CuAAC) reaction to generate a new library
of complementary biotin-R8ERY⁄ analogues derived from peptide
Figure 1. An hypothesis: Interruption of cell adhesion of lymphocytes to MAdCAM-1 by competitive binding of biotin-R8ERY⁄ to FAK and src.

2640
S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 2638–2644
O
CO2H
(i)
O
NHBoc
NHBoc
HO
O
O
O
(ii)
(iii), (iv)
NH2 · HCl
O
CO2H
NHFmoc
O
Scheme 1. Reagents and conditions: (i) K2CO3, propargyl bromide, DMF, 18 h, rt; (ii) AcCl, MeOH, 4 h, 0 °C to rt; (iii) NaOH, MeOH, 1.5 h, HCl, H2O, 4 °C, 18 h; (iv) NaHCO3,
FmocOSu, dioxane, H2O.
O
O
HO
OH
PS
PS
HMP linker
NH2
HOBt, DIC,
OH
20% DMF in DCM, 2h
HO2C
NHFmoc
O
4
Fmoc SPPS
(Tribute peptide synthesiser)
i) 20% piperidine in DMF, 3 mL, 2·5 min
ii) HBTU, NMM, DMF, amino acid, 45 min
iii) Repeat step i and ii ten times
O
NHFmoc
X-RRRRRRRRERY*
O
PS
HMP linker
O
5: X = NH2
iv) biotinylation
v) cleavage from resin
6: X = biotin
Scheme 2. Fmoc SPPS of the propargylated peptide on amino methylated PS resin.
We have synthesised nine analogues of peptide biotin-R8ERY⁄6
(Y⁄ = O-propargyl-tyrosine) by using the CuAAC reaction. The con-
stituent azido-substrates were reacted with the propargylated
peptide in solution phase and the activities of the resulting prod-
ucts were evaluated in a cell adhesion assay.
method of Essen et al.33 to afford the desired building block
Fmoc-O-propargyl-L-tyrosine 4 for Fmoc SPPS (Scheme 1).
The monopropargylated peptide R8ERY⁄ 5 was synthesised on a
TributeTM peptide synthesiser under standard Fmoc SPPS condi-
tions, using compound 4. The N-terminus of peptide 5 was deriva-
tised with biotin and cleaved from resin to afford the desired
product 6 in 47% yield (99% purity) after RP-HPLC (Scheme 2).
2. Results and discussion
The required azido components 2-acetamido-2-deoxy-b-
galactopyranosyl azide (7a),34 1-b-azido-2,3,4,6-tetra-O-acetyl-
galactose (8a),35 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
D
D
-
-
-
2.1. Chemistry
galactopyranosylazide (9a),34 30-azido-10-propyl-2-acetamido-2-
First of all, the key propargylated tyrosine derivative 4 was pre-
-galactopyranoside (10a),36,37 1,3,4,6-tetra-O-acetyl-2-
-galactose (11a),38 1-deoxy-b-
-galactopyranosyl
-Ala(N3)-OH
pared. Precursor H-
ing to the procedure published by Schultz et al.32 from Boc-
tyrosine-OH 1 and subsequently Fmoc-protected according to the
L-Tyr(O-propargyl)-OH was synthesised accord-
deoxy-
azido-2-deoxy-
azide (12a),39,40 2-azidoacetic acid (13a),41 Fmoc-
a-D
L
-
D
D
L

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 2638–2644
2641
HN
NH2
HN
NH2
HN
O
HN
O
N3-R
(7a-15a)
H
N
H
N
H
N
H
N
CO2H
CO2H
N
H
X
N
H
X
O
O
CO2H
CO2H
O
O
biotin-R8ERY*
biotin-R8ERY*
N
R
N
N
6
7-15
Scheme 3. CuAAC reactions on peptide 6.
Table 1
biotin-R8ERY⁄ peptides and their percentage of inhibition in a cell adhesion assay relative to biotin-r9YDRREY
No
Y⁄=
Yield (mg, %) (purity)
MS (4+ ion) calcd/obsdb
496.1/496.0
% Inhibition (50
39.77
lM)
% Inhibition (100
39.75
lM)
a
6
Fmoc-O-propargyltyrosine
8.20 mg, 47% (99%)
HO
OH
O
R =
7
8
9
0.80 mg, 15% (99%)
1.46 mg, 27% (94%)
2.78 mg, 93% (93%)
557.6/557.6
28.03
47.38
40.02
39.63
52.46
46.53
OH
OAc
OAc
AcHN
AcO
O
617.9/617.8 (+TFA)
617.7/617.6 (+TFA)
R =
R =
AcO
AcO
OAc
O
OAc
HO
AcHN
OH
OH
O
10
11
R =
2.46 mg, 46% (99%)
1.36 mg, 25% (96%)
600.7/600.6 (+TFA)
617.9/617.8 (+TFA)
41.47
42.36
NHAc
O
AcO
OAc
OAc
OH
OH
O
104.50
104.76
R =
AcO
HO
R =
O
12
13
14
1.64 mg, 32% (86%)
2.53 mg, 52% (98%)
2.12 mg, 39% (96%)
547.4/547.3
521.4/521.3
584.2/584.1
60.17
56.14
62.73
34.85
HO
O
R =
R =
OH
114.48
110.55
FmocHN
CO2H
O
OH
P
R =
15
2.09 mg, 42% (99%)
533.9/533.8
60.30
65.39
OH
a
Purified yield was based on calculated resin loading, see Supporting information for further details.
ESI-MS, see Supporting information for further details.
b
(14a),42,43 and 2-azidoethylphosphonic acid (15a)44 were synthes-
ised according to published procedures.
diation, 80 °C, 120 W). Changing the reaction conditions by adding
more catalyst, modifying the solvent system (H2O/tBuOH/DCM) or
allowing longer reaction times also proved to be unsuccessful.
Previous studies of click reactions in our group34,36 involving
unprotected peptides and azido sugars suggested a need for great-
er than equimolar amounts of Cu(I) species per propargyl group,
possibly due to chelation of Cu ions by the peptide. Lee et al.36 re-
cently published a much milder strategy for Cu(I)-catalysed click
With the propargylated peptide 6 and nine different azides
(7a–15a) in hand, attention focused on developing suitable condi-
tions for the Cu(I)-catalysed click reaction in solution phase. Initial
attempts to carry out the CuAAC reaction using catalytic amounts
of CuSO4 and sodium ascorbate in a H2O/tBuOH solvent mixture
were unsuccessful, even at elevated temperatures (microwave irra-

2642
S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 2638–2644
160
140
120
100
80
60
40
20
0
Figure 2. Percent of inhibition relative to biotin-r9YDRREY of biotin-R8ERY⁄ peptides on Mn2+-activated TK-1 cell adhesion to mouse MAdCAM-1-Fc (100
lM concentration).
160
140
120
100
80
60
40
20
0
Figure 3. Percent of inhibition relative to biotin-r9YDRREY of biotin-R8ERY⁄ peptides on Mn2+-activated TK-1 cell adhesion to mouse MAdCAM-1-Fc (50
lM concentration).
reactions between unprotected propargylated peptides and sugar
azides, using tris(carboxyethyl)phosphine (TCEP) as reducing
agent, CuSO4 in excess and a 6 M GnHCl/0.2 M Na2HPO4 solution.
Applying these reaction conditions to the present case, propargy-
lated peptide 6 was fully converted into click analogues 7–15 with
the nine azides 7a–15a within 30 min at 60 °C (microwave irradi-
ation, 20 W) (Scheme 3). All products were obtained in sufficient
purity and yield for biological studies after purification by RP-HPLC
as shown in Table 1.
independently of a4b1. In the present work, the synthetic b7
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
2 and 3).
lM (Figs.
Peptides 7, 9 and 10 containing protected and unprotected
galactosamine moieties showed the lowest activity in the cell
adhesion assay at both concentrations and are less potent inhibi-
tors than the unmodified biotin-R8ERY sequence. Peptide 13,
synthesised by the addition of 2-azidoacetic acid exhibited incon-
2.2. Biology
sistent results: at a concentration of 100
lM it was the least active
compound, whereas at 50 M it showed activity superior to 7–10.
Compounds 8 and 12, bearing a protected and unprotected galact-
l
The
a
4b7+TK-1 T cell line is unique in that it does not express b1
integrins and, hence can be used to measure binding to MAdCAM-1
ose moiety respectively, and peptide 15 exhibited greater activity

S. Papst et al. / Bioorg. Med. Chem. 20 (2012) 2638–2644
2643
120
100
80
60
40
20
0
0
1
5
10
ERY*click product 14 (µM)
Figure 4. Dose response assays of peptides 11 and 14.
25
50
100
0
1
5
10
25
50
100
ERY*click product 11 (µM)
2. Boer, J.; Gottschling, D.; Schuster, A.; Semmrich, M.; Holzmann, B.; Kessler, H. J.
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tin-r9YDRREY at both concentrations. Finally, peptides 11 and 14
exhibited higher inhibition activity in the cell adhesion assay than
all other compounds tested, with peptide 11 demonstrating a 5%
higher level of inhibition than biotin-r9YDRREY and approximately
60% greater activity than peptide 8 at 50 lM. The marked differ-
ence in activities of 8 and 11 appears to be due to the different
points of attachment (C1 and C2 respectively) of the glycoside ring.
The most active peptide 14 in this series, carrying an Fmoc pro-
tected alanine, displayed an improvement over biotin-r9YDRREY
by 15%. This is consistent with our previous SAR studies showing
that large, non-polar residues connected to the 4-position of the
tyrosine phenyl ring increase activity.
Dose response assays for the two most active peptides 11 and
14 have been carried out and indicate the IC50 for 11 is 50 lM
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3. Conclusion
A series of nine novel cell-permeable peptidomimetics incorpo-
rating the biotin-R8ERY⁄ motif were prepared from propargylated
peptide 6 using the CuAAC reaction. The click analogues 7–15 were
then tested for a4b7 antagonism in a lymphocyte cell-adhesion as-
say. Five of the peptidomimetics displayed potency greater than
that of the unmodified biotin-R8ERY peptide at a concentration of
100 lM and six at 50 lM. Two of the analogues, 11 and 14, exhib-
ited slightly higher potency than the biotin-r9YDRREY at both con-
centrations tested, a dose response assay of the most active peptide
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Acknowledgment
The authors thank Auckland Uniservcies Ltd. for financial
support.
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