4-Aminoproline-based arginine-glycine-aspartate integrin binders with exposed ligation points: Practical in-solution synthesis, conjugation and binding affinity evaluation

Article abstract of DOI:10.1039/b914836a

An expedient and practical in-solution synthesis of three new 4-aminoproline-based arginine-glycine-aspartate integrin binders - compounds 15, 17 and 19- is presented. Two candidates carrying exposed azide and amine functional points were further advanced to trimeric platform 21 as well as fluorescein- and DOTA-conjugates 23 and 25. The new compounds were assayed for their binding affinity towards human α_Vβ₃ and α_Vβ₅ integrin receptors. Both monomeric candidates and covalent conjugates revealed potent ligand competence for the α_Vβ₃ receptor in the one-digit nanomolar range (IC₅₀α_Vβ₃ = 0.2-8.0 nM; IC ₅₀α_Vβ₅ = 5.0-1621 nM), thus demonstrating that conjugation does not impair the exquisite binding profile of this new generation of integrin ligands. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b914836a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
4-Aminoproline-based arginine-glycine-aspartate integrin binders with
exposed ligation points: practical in-solution synthesis, conjugation and
binding affinity evaluation†
Lucia Battistini,*a Paola Burreddu,b Paola Carta,b Gloria Rassu,b Luciana Auzzas,b Claudio Curti,a
Franca Zanardi,a Leonardo Manzoni,c,d Elena M. V. Araldi,c Carlo Scolasticoc and Giovanni Casiraghi*a
Received 22nd July 2009, Accepted 8th September 2009
First published as an Advance Article on the web 6th October 2009
DOI: 10.1039/b914836a
An expedient and practical in-solution synthesis of three new 4-aminoproline-based
arginine-glycine-aspartate integrin binders – compounds 15, 17 and 19 – is presented. Two candidates
carrying exposed azide and amine functional points were further advanced to trimeric platform 21 as
well as fluorescein- and DOTA-conjugates 23 and 25. The new compounds were assayed for their
binding affinity towards human aVb3 and aVb5 integrin receptors. Both monomeric candidates and
covalent conjugates revealed potent ligand competence for the aVb3 receptor in the one-digit
nanomolar range (IC50 aVb3 = 0.2–8.0 nM; IC50 aVb5 = 5.0–1621 nM), thus demonstrating that
conjugation does not impair the exquisite binding profile of this new generation of integrin ligands.
based on a single copy (monomers) or replicates (multimers)
Introduction
of aVb3 ligands is a major trend in the current research on
Integrin proteins are comprised of an integral transmembrane
integrin receptors, especially those involved in angiogenesis.
complex, which modulates the association between the extracel-
Various scaffolds have been exploited in the design of mono-
lular matrix and the cytoskeleton.1 More than twenty different
a/b heterodimeric integrins have been recognized so far, which
result from the non-covalent combination of eighteen a and
eight b subunits. The multifaceted functional diversity of integrins
has been translated into a wide variety of biological processes,
including inflammation, innate and antigen-specific immunity,
haemostasis, wound healing, tissue morphogenesis, and regulation
of cell growth and differentiation.2 Accordingly, specific integrin
dysregulation phenomena have been linked to the pathogenesis
of many disease states, and may offer druggable opportunities
in a number of diseases.3 Within the great receptor diversity,
the vitronectin receptors aVb3 and aVb5 have received increasing
attention as interesting therapeutic targets, because of their critical
role in tumor-induced angiogenesis and metastasis formation.4
Since their discovery, the biological and pharmacological potential
of the integrin antagonists c(RGDfV)5 and c(RGDf[N-Me]V)
(alias Cilengitide, EMD121974)6 have found expression into a vast
scenario of biomedical and pharmaceutical applications, spanning
from pharmaceutical technology, material science, biomedical
engineering, and nanotechnology. The development of mono-
and multifunctional bioconjugates and supramolecular systems
and multifunctional RGD-presenting systems, such as proteins,7,8
lipids,9,10 polymers,11 and even inert, biocompatible matrices and
surfaces.12 Together with these, sophisticated supramolecular
assemblies have been elaborated as well, such as liposomes,9a,10
micelles,13 plasmids,13b and nanoparticles.14 Pioneering studies
with these systems have been useful to understand the molecular
basis of integrin targeting. For instance, mono- and especially
multivalent presentations15,16 have served as biological tools to
clarify the mechanisms underlying integrin adhesion, clustering,
internalization and endocytosis;7,17,18 generate models of cell ad-
hesion, spreading and proliferation;9,19 and elaborate biologically
inductive materials for medical engineering.20 Multifunctional
systems have been developed as molecular and supramolecular
vectors for drug and gene delivery, molecular imaging, and
biomarker detection.21
In this scenario, the major effort has been devoted to the design
and implementation of flexible chemical platforms to conveniently
graft the most diverse bioconjugates and supramolecular assem-
blies. Among the integrin-addressing motifs, only a few RGD-
containing linear and cyclic peptide building blocks adapted to
be covalently linked, are mostly represented. On the other hand,
alternative semipeptide and peptidomimetic small molecules have
been rarely considered. In this work, RGD semipeptides based
on a 4-aminoproline (Amp‡ ) scaffold were used, demonstrating
aDipartimento Farmaceutico, Universita` degli Studi di Parma, Viale G. P.
Usberti 27A, I-43100 Parma, Italy. E-mail: lucia.battistini@unipr.it,
giovanni.casiraghi@unipr.it; Fax: +39-0521-905006; Tel: +39-0521-
906040; Tel: +39-0521-905080
bIstituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, I-07040
Li Punti, Sassari, Italy
‡ Abbreviations: Amp, 4-aminoproline; Bn, benzyl; Boc, tert-butoxy-
carbonyl; Z, benzyloxycarbonyl; Mtr, 4-methoxy-2,3,6-trimethylben-
zenesulfonyl; Fmoc, 9-fluorenylmethoxycarbonyl; RGD, arginine-glycine-
aspartate; DCE, 1,2-dichloroethane; DCM, dichloromethane; DMF, N,N-
dimethylformamide; DIEA, diisopropylethylamine; DEA, diethylamine;
NMM, N-methylmorpholine; TFA, trifluoroacetic acid; MsCl,
methanesulfonyl chloride; i-BuOCOCl, isobutylchloroformate; DEAD,
diethyl azodicarboxylate; DPPA, diphenylphosphoryl azide; FmocOSu,
cCentro Interdipartimentale Studi Biomolecolari e Applicazioni Industriali,
Universita` degli Studi di Milano, Via Fantoli 16/15, I-20138 Milano, Italy
dIstituto di Scienze e Tecnologie Molecolari del CNR, Via Fantoli 16/15,
I-20138 Milano, Italy
† Electronic supplementary information (ESI) available: 1H NMR spectra
of compounds 15, 17, 19, 21, 23 and 25. See DOI: 10.1039/b914836a
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their utility in the design of either multivalent or multifunctional
presentations.
Subnanomolar cyclopeptide antagonist ligands of aVb3 and
aVb5 integrin receptors have been recently discovered by us,
bearing simple 4-aminoproline modules (Amp) grafted onto the
Arg-Gly-Asp tripeptide segment via the proline C2 carboxyl and
C4 amino functionalities.22 With this embodiment, the proline N1
site still remains untouched, exploitable for chemical ligation to
active substances and spacers en route to relevant bio-conjugates
and multimeric compositions thereof.23 Although milligrams of
these small ligands are actually accessible by conventional solid-
state peptide methodology, development of practical and scalable
synthesis would be attractive, which could provide the suitable
quantity (gram-scale) required for in-vivo multiple assays and
extensive conjugation experiments. To this goal, this paper reports
on practical, in-solution synthesis of a novel progeny of Amp-
based Arg-Gly-Asp integrin binders carrying exposed peripheral
anchorages and follow up ligation chemistry to model alkyne
and carboxylic acid frameworks. Starting from these platforms,
multivalent presentation and multifunctional systems embedding
classic detection probes were assembled, and their binding affinity
for aVb3 and aVb5 receptors compared with their monomeric
counterparts.
Scheme 1 Reagents and conditions: (a) Boc2O, Et3N, MeOH, reflux;
(b) aq Cs2CO3; then BnBr, DMF, rt; (c) DPPA, DEAD, PPh3, THF, rt;
(d) TFA, DCM, rt; (e) CHOCH2OTBS, NaBH(OAc)3, DCE, rt; (f) H2,
Pd/C, EtOH, rt; (g) FmocOSu, aq Na2CO3, THF, rt.
Results and discussion
Chemistry
To design our prototypical ligand class we considered two basic
criteria: (1) the molecules should have a peripheral, chemically
exposed and malleable anchorage to be exploited as a covalent
ligation point for diverse bioactive units and spacer systems, and
(2) the structural constitution, shape, and stereochemistry of the
newly designed ligands should mirror the overall steric disposition
of the sub-nanomolar lead candidates of our previous studies.
To meet both criteria, analogues of type cyclo-[Arg-Gly-Asp-
Amp] were designed, based on N-silyloxyethyl-substituted cis-
4-amino-L-proline module 4, in turn available from commercial
trans-4-hydroxy-L-proline (1), as shown in Scheme 1 (7 steps, 48%
overall yield on a 15 mmole scale).
Amide bond disconnection of the cyclopeptide targets into
four amino acid components–Arg, Gly, Asp, and Amp–revealed
a broad latitude in the sequence of fragment coupling. However,
capitalizing on several precedents in this field,22,24 we opted to
embody the peptide turn-inducing Amp residue between the
Asp unit and the Gly-Arg segment to forge a linear H-Asp-Amp-
Arg-Gly-OH sequence, which we hoped would participate in a
clean and productive macrocyclization. Accordingly, the assembly
of the cyclopeptide sub-target 14 (Scheme 2) commenced with in-
solution coupling of Mtr-protected N-Boc-L-arginine 5 to glycine
Scheme 2 Reagents and conditions: (a) H-Gly-OBn (6), i-BuOCOCl,
NMM, THF, rt; (b) Sn(OTf)2, DCM, rt; (c) DEPBT, DIEA, THF, rt;
(d) Et2NH, DCM, rt; (e) Z-Asp(Ot-Bu)-OH (11), DEPBT, DIEA, THF,
rt; (f) H2, Pd/C, EtOH, rt; (g) HATU, HOAt, 2,4,6-collidine, DMF, rt.
benzyl ester 6 using isobutylchloroformate/NMM activation,
leading to protected Arg-Gly dipeptide 7 in quantitative yield.
Chemoselective fission of the Arg N-Boc protective group using
tin(II) triflate in DCM then provided fragment 8 (96% yield) to
which the Fmoc-protected Amp module 4 was appended (DEPBT
activation in THF)25 to afford the Amp-Arg-Gly tripeptide
construct 9 in a rewarding 92% isolated yield.
9-fluorenylmethyl-N-succinimidyl carbonate; HATU, O-(7-azabenzo-
triazol-1-yl)-N,N,N¢,N¢-tetramethyluronium
hexafluorophosphate;
HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole;
DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one; BOP,
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoropho-
sphate; TBTU, O-(benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium
tetrafluoroborate; TIS, triisopropylsilane; DOTA, 1,4,7,10-tetraazacy-
clododecane-1,4,7,10-tetraacetic acid; DOTA(Ot-Bu)3, 4,7,10-tri-(tert-
butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecan-1-yl-acetic acid.
A number of alternative coupling agents were investigated
for this acylation reaction (e.g. HBTU/HOBt, TBTU/HOBt,
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HATU/HOAt, BOP); however, the DEPBT condensing agent
proved the best system in terms of efficiency and reliability.
Once the Fmoc blockage was dismantled to 10 (Et2NH, DCM),
embodiment of the Asp residue 11 proceeded cleanly, providing
linear tetrapeptide 12 in a 77% yield over two steps.
Next, both the chain terminals were liberated simultaneously
by catalytic hydrogen giving rise to partially protected H-Asp-
Amp-Arg-Gly-OH tetrapeptide 13 in a 88% yield. With the
exposed NH2 and CO2H functionalities in place, building of
14-membered tetrapeptide macrocycle 14 was finally addressed
by exposing a medium-dilution DMF solution (3.5 mM) of 13 to
the HATU/HOAt/collidine system at room temperature. Under
our optimized conditions, the crucial macrocyclization went to
completion in a matter of hours, providing 14 in a gratifying 66%
isolated yield after flash column chromatography. Even in this
circumstance, careful choice of the experimental conditions proved
crucial, with the HATU/HOAt/collidine system demonstrating
capabilities superior to the DPPA/NaHCO3 or DEPBT/DIEA
systems.
Overall, our synthesis to 14 provided gram quantity of the pure
macrocycle sub-target in seven steps and 40% yield for the pep-
tide construction, with only four chromatographic purifications
involved (20% overall yield for the longest linear sequence of 12
steps from commercially available trans-4-hydroxy-L-proline 1).
With a practical synthesis of 14 accomplished, the next phase of
our study was directed at chemical ligation of representative Amp-
containing azide and amine constructs to suitable model entities
bearing complementary alkyne and carboxylate anchoring points.
Utilizing 14 as the common precursor, divergent preparation
of partially protected azide 16 and amine 18, as well as fully
deprotected ligands 15, 17, and 19 was first executed, as shown
in Scheme 3. Actually, all chemical manipulations proved to be
reliable and productive, providing the targeted compounds in
excellent isolated yields (68-96%; for details, see the Experimental
section).
Scheme 3 Reagents and conditions: (a) TFA, TIS, H2O, rt; (b) TBAF,
AcOH, DMF, rt; (c) MsCl, Et3N, DCM, sonication; (d) NaN3, DMF,
reflux; (e) H2, Pd/C, EtOH.
and their structure validated by high resolution (NSI) mass
spectrometry, and extensive NMR measurements.
Complete chemical shift assignment of proton resonances of
the targeted cyclotetrapeptides 15, 17, 19, 21, 23, and 25, was
carried out for D2O and H2O/D2O (9:1) solutions, using one-
and two-dimensional NMR experiments at 600 MHz. Proton
spectra (copies are reported in the Electronic Supplementary
Information) invariably revealed well resolved peaks in the 1.4-
4.6 ppm region attributable to non-exchangeable protons of the
RGD-Amp cyclopeptide moiety. Also, the presence of sharp
signals in the 7.0-9.0 ppm region (H2O/D2O 9:1 solutions) was
indicative of exchangeable NH amide protons of the peptide
backbone. A summary of selected proton and carbon assignments
is reported in Table 1.
The NMR spectral data of trimeric compound 21 perfectly
reflected its C3-symmetry, resulting in a single resonance sys-
tem corresponding to the three equivalent cyclopeptide-triazole
appendages anchored at the common phenolic core. As for the
fluorescein conjugate 23, complete splitting of each resonance was
observed in the NMR spectra, highlighting the existence of two
inseparable, almost equimolar regioisomeric forms attributable to
the different nature of the appended fluorescein moiety.28
The chemical identity of purified, unprotected cyclic tetrapep-
tides 15, 17 and 19, and of the various intermediary compounds
in Scheme 3, was firmly ascertained by high-resolution mass spec-
trometry, extensive NMR experiments, and elemental analyses,
when applicable.
As distinctive examples highlighting the ability of the azido- and
amino-terminal analogues of this study to participate in covalent
ligation to complementary monodentate and polydentate units,
three conjugates, triazole-linked trimeric star-shaped display 21
and amide-linked fluorescein and DOTA constructs 23 and
25 were designed and assembled (Scheme 4). Thus, three-fold
conjugation of protected azide 16 with trivalent phenoxyalkyne
20 exploiting the versatile Huisgen azide-alkyne 1,3-dipolar
cycloaddition26 proved successful, which returned, after global
deprotection, multimeric presentation 21 in a nice 72% isolated
yield for the two steps. On the other hand, starting from amine
18, acylation with either fluorescein derivative 22 or tetraaza-
cyclododecane (DOTA) congener 24 was performed, by using
the standard activating HBTU/DIEA reagent pair in different
solvents. Acidic deprotection allowed us to obtain conjugates
23 and 25 in 90% and 96% isolated yields for the two steps,
respectively.27
Extensive NMR-derived in-solution structural studies carried
out on aminoproline-based cyclotetrapeptides in previous works22
were central in identifying the structural features responsible for
their integrin ligand competence. There, the ability to act as
powerful aVb3/aVb5 integrin binders was ascribed to the presence
of a highly organized arrangement of the peptide backbone, which
featured an inverse g-turn or an organized kink centered at the Asp
˚
Unprotected, water soluble conjugate cyclopeptide ligands 21,
23 and 25 were purified by preparative reverse-phase HPLC,
residue, along with Cb Arg-Asp distances in the 8.0-8.5 A range.
Now, in consideration of NMR data collected for cyclopeptides
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Scheme 4 Reagents and conditions: (a) Cu(OAc)2, Na ascorbate, H2O, t-BuOH; (b) TFA, TIS, H2O, rt; (c) HBTU, DIEA, DMF, rt; (d) HBTU, DIEA,
DCM, rt.
15, 17, 19, 21, 23 and 25, which evidenced strict analogies with
response curves for fluorescein- and DOTA-conjugate candidates
23 and 25 vs the aVb3 and aVb5 receptors are displayed in
Fig. 1.
3
previous Amp-based ligand generations (e.g. J2,3a
=
3J4,3a
=
3J4,5a = 0.0 Hz, JNH,aH Asp = 9.0-9.6 Hz, Dd H2(a)-Gly = 0.47-
0.63 ppm; compare with reference compound 26, Table 1), one
can confidently argue that a similar conformational arrangement
is here adopted, which might account for the biological activity
exerted by the novel candidates (see infra).
3
Inspection of the panel of binding affinity data reveals remark-
able nanomolar activity for all candidates towards aVb3 and aVb5
receptors, with b3 affinity reaching outstanding single-digit nM
values comparable with those of reference compounds 26 and 27.
Monomeric compounds 15, 17, and 19 showed almost equipo-
tent results in this assay, suggesting that functional group substi-
tution at the appended N1-ethyl chain (hydroxy vs azide vs amine)
does not perturb the binding capability towards the aVb3 receptor
(aVb3 preferred over aVb5). When passing from amine 19 to amide-
linked conjugates 23 and 25, the aVb3 IC50 values remained un-
changed, demonstrating wide tolerance of these structures towards
ligation with bulky fluorescein or chelating DOTA appendages. As
for the integrin binding selectivity, however, the aVb3 preference is
even more pronounced for both candidates, with aVb5/aVb3 ratios
of 235 and 469, respectively.
Triazole-linked conjugate 21, possessing three RGD-Amp cy-
clopeptide repeats, showed a nice one-order magnitude increase
in binding affinity with respect to its monomeric counterpart
17; and this highlighted that the trisubstituted phenolic core
is well tolerated and, most importantly, that the overall ligand
competence may benefit from the multivalent presentation of the
molecule.
Biology
The binding affinity of integrin ligands to isolated aVb3 and
aVb5 receptors has over the course of time been determined
in different ways such as competitive displacement assays using
either radiolabeled echistatin or biotinylated vitronectin, the latter
being their most important physiological ligand. In the present
study, monomeric ligands 15, 17, and 19, as well as conjugates
21, 23, and 25 (92-98% pure lyophilized trifluoroacetate salts),
were tested for their ability to bind to aVb3 and aVb5 human
integrin receptors subtypes29 using the biotinylated vitronectin
displacement assay, and compared to Amp-based hit compound
2622 and to known RGD high-affinity binder 27 c(RGDfV).5 The
binding affinities were expressed as IC50 values, and calculated
through a computer-assisted image processing system by nonlinear
regression analysis, as described in Experimental section, and
listed in Table 2. Moreover, representative sigmoid concentration-
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Table 1 Selected 1H and 13C spectral data of target compounds 15, 17, 19, 21, 23, and 25 as compared to prototypical cyclopeptide binder 26 obtained for
D2O or H2O/D2O (9:1) solutions at 298 K (600/150 MHz). Chemical shifts in ppm; J coupling constants in Hz; Dd in ppm. Conditions, see Experimental
section
a
Compd
Res
NH
3JNHaH
H/H2(a)
H2(b)
H/H2(g)
H2(d)
DdH2(a)
0.53
Ca
Cb
Cg
Cd
15
Amp
Arg
Gly
Asp
Amp
Arg
Gly
Asp
Amp
Arg
Gly
Asp
Amp
Arg
Gly
Asp
Amp
Arg
Gly
Asp
Amp
Arg
Gly
Asp
Amp
Arg
Gly
7.11
8.86
8.93
8.40
7.11
8.93
8.92
8.39
7.13
8.72
8.86
8.43
7.06
8.76
8.89
8.40
7.00
8.85
8.79
8.30
7.11
8.92
8.94
8.37
7.30
8.90
9.07
8.51
4.2
5.4
6.0
9.6
4.8
5.4
nd
9.0
4.8
5.4
5.4
9.0
4.2
5.4
6.0
9.6
nd
5.7
6.0
9.6
4.2
5.4
6.0
9.0
5.4
5.5
nd
4.60
4.09
3.94/3.41
4.59
4.63
4.13
3.94/3.42
4.60
4.64
4.15
3.92/3.45
4.58
4.22
3.99
3.87/3.37
4.54
4.55
4.07
3.93/3.30
4.56
4.54
2.80/2.35
1.63
4.27
1.57/1.49
3.93/3.42
3.07
66.6
56.0
44.6
49.8
65.5
56.1
44.7
49.9
66.9
56.2
44.7
50.0
66.5
56.0
44.6
49.8
65.0
55.7
44.4
49.8
65.7
56.0
44.4
49.6
59.0
58.2
47.4
nd
35.4
26.6
49.8
24.6
60.8
40.6
2.77
2.81/2.38
1.65
34.9
35.5
26.6
17
19
21
23b
25
26c
4.27
1.59/1.50
3.92/3.46
3.08
49.9
24.7
61.0
40.7
0.52
2.79
2.88/2.47
1.65
35.0
35.0
26.5
4.27
1.58/1.49
3.99/3.46
3.08
50.3
24.7
60.3
40.6
0.47
2.79
2.75/2.30
1.53
34.9
35.4
26.6
4.19
1.35-1.5
3.80/3.90
2.93
49.8
24.6
61.2
40.6
0.50
2.71
2.75/2.38
1.61
34.9
34.9
26.3
4.29
1.55/1.46
4.02/3.43
3.02
49.8
24.4
60.8
40.3
0.63
2.72
2.83/2.39
1.64
34.9
34.9
26.3
4.30
1.59/1.50
3.98/3.47
3.08
49.5
24.5
61.1
40.3
4.10
3.95/3.42
4.61
4.58
4.15
4.03/3.47
4.65
0.53
2.78
2.67/2.40
1.70
35.0
38.7
29.3
4.43
1.64/1.56
3.62/3.59
3.14
52.3
27.2
55.3
43.4
0.56
Asp
9.0
2.83
37.8
a
3JNHgH for Amp residue. b One regioisomer reported. c HCl salt at 800/200 MHz.
to those observed with closely related Amp-based cyclopeptides,22
hence confirming that the decoration of such integrin binders with
exposed ligation points and biofunctional moieties does not erode
their exquisite binding capability.
Table 2 Inhibition of biotinylated vitronectin binding to aVb3 and aVb5
receptors. High affinity integrin binders 26 and 27 were used as positive
controlsa
Conclusions
The purpose of this investigation was to establish a reliable
and scalable synthetic protocol to assemble gram quantities of
4-aminoproline-based arginine-glycine-aspartate cyclic tetrapep-
tides carrying exposed points for covalent ligation to bioac-
tive units. Also, we intended to certify that such a conjugation
would not compromise the exquisite integrin binding capabilities
of our aminoproline-based ligand progeny.
Both these goals have been accomplished. Representative
monomeric candidates 15, 17 and 19 were obtained in useful
yields and quantities (14-19% overall yields from proline 1), which
exhibited one-digit nanomolar binding affinity for the aVb3 inte-
grin receptor, and remarkable aVb3 vs aVb5 selectivity. Likewise,
conjugated constructs were easily accessed using either triazole or
amide ligation, with trivalent presentation 21 and fluorescein- and
DOTA-derivatives 23 and 25 exhibiting subnanomolar or one-
digit nanomolar aVb3 binding capability.
Entry Compound IC50 (nM) SD for aVb3 IC50 (nM) SD for aVb5
1
2
3
4
5
6
7
8
9
15
2.4 1.0
6.0 1.5
6.1 1.6
0.2 0.1
8.0 0.4
6.9 0.6
20.7 6.2
3.2 1.3
47.1 10.0
38 11
1208 373
315 137
5.0 1.6
3751 1372
1621 390
227 88
17
19
21
23
25
26b
27b
7.5 4.8
13.7 6.2
vitronectin
a IC50 values were calculated as the concentration of compound required
for 50% inhibition of biotinylated vitronectin binding, as estimated by the
Prism GraphPad program. Results are expressed as the mean compound
concentration standard deviation that inhibited 50% of biotinylated
vitronectin binding to isolated receptors. b This assay.
We anticipate that this chemistry will find use in the synthesis of
several integrin ligand-drug conjugates, such as the tumor-targeted
cyclo(Arg-Gly-Asp-Amp)-paclitaxel or doxorubicin assemblies.
This is the focus of ongoing research.30
To summarize, the new cyclopeptide representatives in this work
were shown to possess similar binding activity and aVb3-selectivity
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Synthetic procedures
4-Fmoc-[1-(tert-Butyldimethylsilyloxyethyl)Amp]-Arg(Mtr)-Gly-
OBn (9). To an ice-cooled solution of aminoproline 4 (3.68 g,
7.21 mmol) in THF (40 mL) under nitrogen atmosphere, DEPBT
(4.31 g, 14.42 mmol) and DIEA (2.51 mL, 14.42 mmol) were
added and the resulting mixture was allowed to rise to room
temperature. After 15 min, the reaction was re-cooled to 0 ◦C
and a solution of H-Arg(Mtr)-Gly-OBn (8) (5.0 g, 9.37 mmol)
in 25 mL of THF was added. The reaction mixture was stirred
overnight at room temperature. After reaction completion,
EtOAc was added and the organic phase was washed with
saturated aq NH4Cl solution, saturated aq NaHCO3 solution and
water. The organic phase was dried, filtered and evaporated in
vacuo. The crude was purified by silica gel flash chromatography
(DCM/i-PrOH, 92:8) affording tripeptide 9 (6.80 g, 92%) as a
white foam: [a]25 -9.3 (c 3.5, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d 8.11 (d, J = 8.4 Hz, 1H, NH Arg), 7.71 (m, 3H, NH
Gly and Fmoc), 7.54 (m, 2H, Fmoc), 7.2-7.4 (m, 9H, Fmoc and
Ph), 6.46 (s, 1H, CHMtr), 6.27 and 6.15 (m, 3H, NHe Arg), 5.71
(bs, 1H, NH Amp), 5.10 (m, 2H, CH2Ph), 4.62 (m, 1H, Ha Arg),
4.38 (m, 2H, CH2Fmoc), 4.1-4.3 (m, 3H, CHFmoc, H4 Amp and
Ha Gly), 3.90 (bd, J = 17.6 Hz, 1H, Ha Gly), 3.7-3.8 (m, 5H,
OMe Mtr and H2¢Amp), 3.21 (m, 2H, H2 Amp and H5 Amp),
3.15 (m, 2H, Hd Arg), 2.77 (m, 1H, H5 Amp), 2.65 (s, 3H, Me
Mtr), 2.58 (s, 3H, Me Mtr), 2.56 (m, 2H, H1¢Amp), 2.48 (m, 1H,
H3 Amp), 2.07 (s, 3H, Me Mtr), 1.93 (m, 1H, Hb Arg), 1.85 (m,
1H, H3 Amp), 1.66 (m, 1H, Hb Arg), 1.48 (m, 2H, Hg Arg), 0.86
(s, 9H, t-Bu), 0.02 (s, 3H, CH3), 0.01 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3) d 174.7 (Cq), 172.2 (Cq), 169.8 (Cq), 158.4
(Cq), 156.5 (Cq), 156.0 (Cq), 143.9 (2C, Cq), 141.2 (2C, Cq),
138.6 (Cq), 136.5 (Cq), 135.2 (Cq), 133.5 (Cq), 128.6 (2C, CH),
128.4 (CH), 128.2 (2C, CH), 127.7 (2C, CH), 127.1 (2C, CH),
125.1 (2C, CH), 124.8 (Cq), 119.9 (2C, CH), 111.7 (CH), 67.1
(CH2), 66.7 (CH2), 62.2 (CH2), 62.0 (CH), 60.1 (CH2), 57.2 (CH2),
55.4 (CH3), 51.5 (CH), 50.6 (CH), 47.2 (CH), 41.2 (CH2), 40.2
(CH2), 37.7 (CH2), 30.7 (CH2), 25.9 (3C, CH3), 25.1 (CH2), 24.1
(CH3), 18.4 (CH3), 18.2 (Cq), 11.9 (CH3), -5.4 (2C, CH3). MS
(ESI) calcd for C53H72N7O10SSi [M+H]+: 1026.48; found: 1026.6.
Anal. calcd for C53H71N7O10SSi: C, 62.02; H, 6.97; N, 9.55; found:
C, 61.83; H, 6.89; N, 9.71.
Fig. 1 Inhibition of biotinylated vitronectin specific binding to purified
human integrin proteins aVb3 (A) and aVb5 (B) by amide-linked conjugates
23 and 25. Each data point is the result of the average of triplicate wells,
and was analyzed by nonlinear regression analysis with Prism GraphPad
program. Data are representative of three separate experiments.
Experimental section
General
Preparative and semipreparative reverse-phase HPLC were per-
formed on a Prostar 210 apparatus (Varian, UV detection at 220
or 254 nm) equipped with C18-10 mm columns (Discovery BIO
Wide Pore, 21.2 ¥ 250 mm and Discovery BIO Wide Pore, 10 ¥
250 mm). Direct infusion ESI-MS spectra were recorded on API
150EX apparatus (Applied Biosystems). High-resolution mass
spectrometry measurements (HRMS) were performed on a LTQ
ORBITRAP XL Thermo equipped with an external nanoelectro-
spray ion source (NSI). Optical rotations were measured using
a Perkin-Elmer model 341 polarimeter at ambient temperature
using a 100-mm cell with a 1-mL capacity and are given in units
of 10-1 deg cm2 g-1. Elemental analyses were performed by the
Microanalytical Laboratory of the University of Parma. NMR
spectra were recorded on Avance 300 (Bruker) or Mercury Plus
MP-400 (Varian) or 600 INOVA (Varian) NMR spectrometers.
Chemical shifts (d) are reported in parts per million (ppm) with
TMS (CDCl3), CD2HOD, and HOD resonance peaks set at 0,
3.31, and 4.80 ppm, respectively. Peak assignments were performed
using conventional 1D and 2D NMR experiments, such as COSY,
TOCSY, HSQC and DEPT sequences. For NMR experiments of
compounds 15, 17, 19, 21, 23, and 25, the samples were dissolved in
D2O or H2O/D2O (90%/10%) at a concentration of 0.1-1 mM. The
H-[1-(tert -Butyldimethylsilyloxyethyl)Amp]-Arg(Mtr)-Gly-
OBn (10). Protected peptide 9 (6.80 g, 6.63 mmol) was dissolved
in 50 mL of a 1:1 DCM/DEA mixture under nitrogen atmosphere.
The resulting solution was stirred at room temperature for 5 h,
meanwhile an additional 10 mL of DCM/DEA mixture were
added. After reaction completion, the solution was concentrated
in vacuo and the residue was dissolved in 30 mL of DCM and
concentrated again. The crude was purified by silica gel flash
chromatography (EtOAc/MeOH, 80:20 to 50:50) affording pure
peptide 10 (4.90 g, 92%) as a white solid: [a]25 -71.4 (c 0.4,
D
CHCl3);1H NMR (400 MHz, CDCl3) d 8.33 (d, J = 8.8 Hz, 1H,
NH Arg), 7.62 (bs, 1H, NH Gly), 7.33 (m, 5H, Ph), 6.51 (s, 1H,
CHMtr), 6.36 and 6.31 (m, 3H, NHe Arg), 5.13 (m, 2H, CH2Ph),
4.51 (m, 1H, Ha Arg), 4.11 (dd, J = 18.0, 5.6 Hz, 1H, Ha Gly),
3.93 (dd, J = 18.0, 5.2 Hz, 1H, Ha Gly), 3.81 (s, 3H, OMe Mtr),
3.74 (dd, J = 5.6, 5.6 Hz, 2H, H2¢Amp), 3.55 (m, 1H, H4 Amp),
3.20 (m, 3H, H2 Amp and Hd Arg), 3.07 (bd, J = 10.4 Hz, 1H, H5
◦
NMR measurements were performed at 25 C with a 600 MHz
Varian-INOVA spectrometer.
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Amp), 2.80 (m, 1H, H5 Amp), 2.67 (m, 4H, Me Mtr and H1¢Amp),
2.61 (m, 4H, Me Mtr and H1¢Amp), 2.40 (m, 1H, H3 Amp), 2.32
(bs, 2H, NH2 Amp), 2.12 (s, 3H, Me Mtr), 1.93 (m, 1H, Hb Arg),
1.73 (m, 2H, H3 Amp and Hb Arg), 1.57 (m, 2H, Hg Arg), 0.87
(s, 9H, t-Bu), 0.04 (s, 3H, CH3), 0.03 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3) d 175.6 (Cq), 172.0 (Cq), 169.7 (Cq), 158.4
(Cq), 156.5 (Cq), 138.6 (Cq), 136.5 (Cq), 135.2 (Cq), 133.6 (Cq),
128.6 (2C, CH), 128.5 (CH), 128.3 (2C, CH), 124.7 (Cq), 111.7
(CH), 67.1 (CH2), 62.3 (CH), 62.2 (CH2), 57.2 (2C, CH2), 55.4
(CH3), 51.8 (CH), 50.8 (CH), 41.2 (CH2), 40.6 (CH2), 39.3 (CH2),
30.2 (CH2), 25.9 (3C, CH3), 25.2 (CH2), 24.1 (CH3), 18.3 (CH3),
18.2 (Cq), 11.9 (CH3), -5.2 (CH3), -5.3 (CH3). MS (ESI) calcd
for C38H62N7O8SSi [M+H]+: 804.41; found: 804.5. Anal. calcd for
C38H61N7O8SSi: C, 56.76; H, 7.65; N, 12.19; found: C, 56.69; H,
7.76; N, 12.03.
H-Asp(Ot-Bu)-1-(tert-butyldimethylsilyloxyethyl)Amp-Arg-
(Mtr)-Gly-OH (13). Protected tetrapeptide 12 (5.68 g,
5.12 mmol) was dissolved in EtOH (250 mL) and a catalytic
amount of 10% palladium on carbon (1.0 g) was added. The
reaction vessel was evacuated by aspirator and thoroughly purged
with hydrogen (three times). The resulting heterogeneous mixture
was stirred overnight under hydrogen atmosphere at room temper-
ature. The catalyst was filtered off and the filtrate was concentrated
in vacuo to give deprotected intermediate 13 (3.99 g, 88%) which
was used as such in the following step: white glassy solid; [a]25
D
-13.2 (c 1.3, MeOH); 1H NMR (400 MHz, CD3OD) d 6.69 (s, 1H,
CHMtr), 4.46 (m, 2H, Ha Arg and H4 Amp), 4.21 (dd, J = 8.4,
4.8 Hz, 1H, Ha Asp), 3.98 (d, J = 16.8 Hz, 1H, Ha Gly), 3.75-3.90
(m, 7H, OMe Mtr, Ha Gly, H2¢Amp and H2 Amp), 3.47 (bd, J =
10.8 Hz, 1H, H5 Amp), 3.15-3.30 (m, 3H, Hd Arg and H5 Amp),
3.08 (m, 1H, H1¢Amp), 2.9-3.0 (m, 2H, Hb Asp), 2.88 (m, 1H,
H1¢Amp), 2.76 (m, 1H, H3 Amp), 2.71 (s, 3H, Me Mtr), 2.64 (s,
3H, Me Mtr), 2.15 (s, 3H, Me Mtr), 2.03 (m, 1H, H3 Amp), 1.91
(m, 1H, Hb Arg), 1.78 (m, 1H, Hg Arg), 1.66 (m, 2H, Hg Arg
and Hb Arg), 1.49 (s, 9H, t-Bu), 0.93 (s, 9H, t-Bu), 0.11 (s, 3H,
CH3), 0.10 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD) d 174.7
(Cq), 174.5 (Cq), 171.4 (Cq), 169.9 (Cq), 160.8 (2C, Cq), 159.1
(Cq), 140.3 (Cq), 138.7 (Cq), 135.6 (Cq), 126.6 (Cq), 113.7 (CH),
84.6 (Cq), 69.1 (CH2), 63.0 (CH2), 61.6 (CH2), 59.4 (CH), 56.9
(CH3), 55.2 (CH), 51.8 (CH), 51.2 (CH), 43.2 (CH2), 39.7 (CH2),
38.3 (CH2), 38.0 (CH2), 31.3 (CH2), 29.3 (CH2), 29.2 (3C, CH3),
27.3 (3C, CH3), 25.2 (CH3), 20.0 (CH3), 19.8 (Cq), 13.0 (CH3),-4.3
(CH3), -4.4 (CH3). MS (ESI) calcd for C39H69N8O11SSi [M+H]+:
885.46; found: 885.6. Anal. calcd for C39H68N8O11SSi: C, 52.92; H,
7.74; N, 12.66; found: C, 53.06; H, 7.90; N, 12.49.
Z-Asp(Ot-Bu)-1-(tert-butyldimethylsilyloxyethyl)Amp-Arg-
(Mtr)-Gly-OBn (12). To an ice-cooled solution of Z-Asp(Ot-
Bu)-OH (11) (2.17 g, 6.71 mmol) in THF (35 mL), DEPBT
(4.02 g, 13.42 mmol) and DIEA (2.34 mL, 13.42 mmol) were
added under argon atmosphere, and the mixture was allowed to
rise to room temperature. After 15 min, this solution was added
to a pre-cooled solution of tripeptide 10 (4.90 g, 6.10 mmol) in
THF (20 mL). The mixture was allowed to stir for 24 h at room
temperature. After reaction completion, EtOAc was added and
the organic phase was washed with saturated aq NH4Cl solution,
saturated aq NaHCO3 solution and water. The organic layer was
dried, filtered and evaporated in vacuo, and the crude residue
was subjected to flash chromatographic purification (DCM/
i-PrOH, 92:8) providing 5.68 g of protected tetrapeptide 12 (84%)
as a colourless glassy solid: [a]25 -0.8 (c 10.3, CHCl3);1H NMR
cyclo[Arg(Mtr)-Gly-Asp(Ot-Bu)-1-(tert-butyldimethylsilyloxy-
ethyl)Amp] (14). To a 3.5 mM solution of tetrapeptide 13 (3.99 g,
4.51 mmol) in DMF (1290 mL), HATU (5.14 g, 13.53 mmol),
HOAt (0.6 M solution in DMF, 22.55 mL, 13.53 mmol) and 2,4,6-
collidine (1.79 mL, 13.53 mmol) were added under nitrogen and
the resulting mixture was stirred at ambient temperature for 8 h.
The reaction was then concentrated under vacuum, the residue
dissolved in EtOAc (100 mL) and the solution was washed with
saturated aq NaHCO3 solution. The organic layer was evaporated
under vacuum to afford a crude residue which was purified by flash
chromatography (EtOAc/MeOH, 80:20) furnishing the side-chain
protected cyclic tetrapeptide 14 (2.58 g, 66%) as a colourless glassy
D
(400 MHz, CDCl3) d 7.99 (d, J = 9.2 Hz, 1H, NH Arg), 7.55 (m,
1H, NH Gly), 7.2-7.4 (m, 11H, Ph and NH Amp), 6.47 (s, 1H,
CHMtr), 6.29 and 6.17 (m, 4H, NHe Arg and NH Asp), 5.10 (m,
2H, CH2Ph), 5.04 (1/2 ABq, J = 12.4 Hz, 1H, CH2Ph), 4.93 (1/2
ABq, J = 12.4 Hz, 1H, CH2Ph), 4.56 (m, 1H, Ha Arg), 4.43 (dd,
J = 14.0, 6.8 Hz, 1H, Ha Asp), 4.37 (m, 1H, H4 Amp), 4.08 (dd,
J = 17.6, 7.2 Hz, 1H, Ha Gly), 3.93 (dd, J = 18.0, 6.0 Hz, 1H,
Ha Gly), 3.77 (s, 3H, OMe Mtr), 3.71 (m, 2H, H2¢Amp), 3.21 (dd,
J = 9.6, 6.0 Hz, 1H, H2 Amp), 3.11 (m, 2H, Hd Arg), 3.06 (bd,
J = 10.4 Hz, 1H, H5 Amp), 2.7-2.8 (m, 4H, H5 Amp, Hb Asp and
H1¢Amp), 2.66 (s, 3H, Me Mtr), 2.60 (s, 3H, Me Mtr), 2.57 (m,
1H, H1¢Amp), 2.47 (m, 1H, H3 Amp), 2.09 (s, 3H, Me Mtr), 1.92
(m, 1H, Hb Arg), 1.76 (m, 1H, H3 Amp) 1.70 (m, 1H, Hb Arg),
1.54 (m, 2H, Hg Arg), 1.38 (s, 9H, t-Bu), 0.85 (s, 9H, t-Bu), 0.02
(s, 3H, CH3), 0.01 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d
174.5 (Cq), 171.9 (Cq), 170.9 (Cq), 169.9 (Cq), 169.7 (Cq), 158.3
(Cq), 156.5 (Cq), 156.2 (Cq), 138.6 (Cq), 136.5 (Cq), 136.0 (Cq),
135.2 (Cq), 133.6 (Cq), 128.6 (2C, CH), 128.5 (2C, CH), 128.4
(2C, CH), 128.3 (2C, CH), 128.2 (CH), 127.8 (CH), 124.7 (Cq),
111.6 (CH), 81.9 (Cq), 67.0 (CH2), 66.5 (CH2), 62.1 (2C, CH and
CH2), 59.5 (CH), 57.3 (CH2), 55.4 (CH3), 52.0 (CH), 51.1 (CH2),
49.0 (CH), 41.1 (CH2), 40.8 (CH2), 38.6 (CH2), 37.5 (CH2), 30.4
(CH2), 28.0 (3C, CH3), 25.9 (3C, CH3), 25.7 (CH2), 24.2 (CH3),
18.4 (Cq), 18.3 (CH3), 12.0 (CH3),-5.3 (2C, CH3). MS (ESI) calcd
for C54H81N8O13SSi [M+H]+: 1109.54; found: 1109.4. Anal. calcd
for C54H80N8O13SSi: C, 58.46; H, 7.27; N, 10.10; found: C, 58.39;
H, 7.11; N, 10.17.
solid: [a]25 +31.7 (c 3.2, CHCl3); 1H NMR (400 MHz, CDCl3) d
D
8.16 (bs, 1H, NH Gly), 7.63 (bs, 1H, NH Asp), 7.55 (bs, 1H, NH
Arg), 7.08 (bd, J = 6.6 Hz, 1H, NH Amp), 6.51 (s, 1H, CHMtr),
6.41 and 6.36 (m, 3H, NHe Arg), 4.61 (m, 1H, Ha Asp), 4.40 (m,
1H, H4 Amp), 4.08 (m, 2H, Ha Gly and Ha Arg), 3.79 (s, 3H,
OMe Mtr), 3.50-3.70 (m, 4H, Ha Gly, H2¢Amp and H2 Amp),
3.10-3.30 (m, 3H, Hd Arg and H5 Amp), 3.02 (m, 1H, H5 Amp),
2.78 (m, 2H, H1¢Amp), 2.69 (m, 2H, Hb Asp), 2.63 (s, 3H, Me
Mtr), 2.57 (s, 3H, Me Mtr), 2.25 (m, 1H, H3 Amp), 2.09 (s, 3H,
Me Mtr), 1.92 (m, 1H, H3 Amp), 1.82 (m, 2H, Hb Arg), 1.50-
1.70 (m, 2H, Hg Arg), 1.37 (s, 9H, t-Bu), 0.83 (s, 9H, t-Bu), 0.01
(s, 3H, CH3), -0.01 (s, 3H, CH3); 13C NMR (75 MHz, CD3OD)
d 176.9 (Cq), 171.9 (2C, Cq), 171.5 (Cq), 160.0 (2C, Cq), 158.3
(Cq), 139.6 (Cq), 138.0 (Cq), 135.0 (Cq), 125.8 (Cq), 112.9 (CH),
82.4 (Cq), 63.4 (CH2), 61.3 (CH2), 56.5 (3C, CH2, CH, CH), 51.1
(CH3), 50.5 (CH), 50.0 (CH), 45.8 (CH2), 42.0 (CH2), 38.5 (CH2),
4930 | Org. Biomol. Chem., 2009, 7, 4924–4935
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37.6 (CH2), 28.5 (3C, CH3), 28.3 (CH2), 27.4 (CH2), 26.6 (3C,
CH3), 24.5 (CH3), 19.4 (CH3), 19.0 (Cq), 12.3 (CH3),-4.8 (CH3),
-5.0 (CH3). MS (ESI) calcd for C39H67N8O10SSi [M+H]+: 867.45;
found: 867.3. Anal. calcd for C39H66N8O10SSi: C, 54.02; H, 7.67;
N, 12.92; found: C, 54.30; H, 7.73; N, 12.73.
1.98 (d, J = 13.8 Hz, 1H, H3 Amp), 1.68 (m, 2H, Hb Arg),
1.56 (m, 1H, Hg Arg), 1.48 (m, 1H, Hg Arg), 1.41 (s, 9H, t-Bu);
13C NMR (150 MHz, CD3OD) d 179.0 (Cq), 171.7 (Cq), 171.4
(Cq), 171.1 (Cq), 159.9 (2C, Cq), 159.0 (Cq), 139.5 (Cq), 137.9
(Cq), 134.8 (Cq), 125.7 (Cq), 112.8 (CH), 82.3 (Cq), 64.7 (CH),
61.3 (CH2), 59.9 (CH2), 56.8 (CH2), 56.3 (CH), 56.0 (CH3), 51.0
(CH), 50.7 (CH), 45.4 (CH2), 41.3 (CH2), 38.2 (CH2), 37.4 (CH2),
28.3 (3C, CH3), 27.8 (CH2), 27.3 (CH2), 24.3 (CH3), 18.8 (CH3),
12.1 (CH3).
cyclo[Arg-Gly-Asp-1-(hydroxyethyl)Amp] (15). The protected
cyclic tetrapeptide 14 (150 mg, 0.17 mmol) was treated with 8.7 mL
of a solution of TFA/TIS/H2O 95:2.5:2.5 at room temperature.
After 18 h, the solvent was evaporated under vacuum and the
residue dissolved in 10 mL of water and thoroughly washed with
Et2O (4 ¥). The aqueous phase was concentrated under vacuum
furnishing the deprotected cyclic tetrapeptide 15 (99 mg, 96%)
as a trifluoroacetate salt. The cyclic tetrapeptide was purified
by preparative RP-HPLC (RP C18-10 mm, 250 ¥ 21.2 mm)
using acetonitrile (0.05% TFA) in H2O (0.05% TFA), 0-35%
linear gradient over 25 min at room temperature. A flow rate
of 9.0 mL/min was used and detection was at 220 nm. HPLC
Rt = 18.1 min. The HPLC sample was evaporated under vacuum
A solution of hydroxyethyl intermediate (1.73 g, 2.30 mmol)
in anhydrous DCM (40 mL) was treated with MsCl (357 mL,
4.60 mmol) and Et3N (1.28 mL, 9.20 mmol) under argon. The
resulting mixture was sonicated for 4 h. After reaction completion,
the mixture was diluted with DCM and washed with saturated
aq NH4Cl solution. The organic phase was concentrated under
vacuum and the crude, dissolved in DMF (25 mL), was treated
with NaN3 (1.20 g, 18.40 mmol). The resulting mixture was heated
to reflux for 24 h and then concentrated under reduced pressure.
The residue was dissolved in DCM and washed with water and
the organic layer was evaporated under reduced pressure affording
azido-cyclopeptide 16 (1.75 g, 98% over two steps) which was used
as such in the following step: a colourless glassy solid; [a]25D +41.4
25
ready for biological assay. A colourless glassy solid; [a]D -15.3
1
(c 1.0, H2O) (HCl salt); H NMR (600 MHz, D2O) d 4.60 (m,
1H, H2 Amp), 4.59 (dd, J = 6.0, 6.0 Hz, 1H, Ha Asp), 4.27 (m,
1H, H4 Amp), 4.09 (dd, J = 7.8, 7.8 Hz, 1H, Ha Arg), 3.94 (d,
J = 13.8 Hz, 1H, Ha Gly), 3.93 (m, 1H, H5 Amp), 3.65-3.75 (m,
2H, H2¢Amp), 3.42 (m, 1H, H5 Amp), 3.41 (d, J = 13.8 Hz, 1H,
Ha Gly), 3.30-3.40 (m, 2H, H1¢Amp), 3.07 (dd, J = 7.2, 7.2 Hz,
2H, Hd Arg), 2.80 (m, 1H, H3 Amp), 2.77 (m, 2H, Hb Asp), 2.35
(d, J. = 15.0 Hz, 1H, H3 Amp), 1.63 (m, 2H, Hb Arg), 1.57 (m,
1H, Hg Arg), 1.49 (m, 1H, Hg Arg); 13C NMR (150 MHz, D2O,
HSQC-based) d 66.6 (CH, C2 Amp), 60.8 (CH2, C5 Amp), 57.0
(2C, CH2, C1¢Amp and C2¢Amp), 56.0 (CH, Ca Arg), 49.8 (2C,
CH, Ca Asp and C4 Amp), 44.6 (CH2, Ca Gly), 40.6 (CH2, Cd
Arg), 35.4 (CH2, C3 Amp), 34.9 (CH2, Cb Asp), 26.6 (CH2, Cb
Arg), 24.6 (CH2, Cg Arg). HRMS (NSI) calcd for C19H33N8O7
[M+H]+: 485.2472; found: 485.2459.
1
(c 1.0, MeOH); H NMR (300 MHz, CD3OD) d 6.69 (s, 1H,
CHMtr), 4.63 (dd, J = 5.8, 5.8 Hz, 1H, Ha Asp), 4.45 (m, 1H,
H4 Amp), 4.16 (d, J = 14.0 Hz, 1H, Ha Gly), 4.07 (dd, J =
7.2, 7.2 Hz, 1H, Ha Arg), 3.86 (s, 3H, OMe Mtr), 3.70 (d, J =
8.7 Hz, 1H, H2 Amp), 3.30-3.40 (m, 4 H, Ha Gly, H2¢Amp and
H5 Amp), 3.20 (m, 2H, Hd Arg), 3.02 (m, 1H, H5 Amp), 2.95
(dd, J = 5.8, 5.8 Hz, 2H, H1¢Amp), 2.70 (m, 2H, Hb Asp), 2.69
(s, 3H, Me Mtr), 2.63 (s, 3H, Me Mtr), 2.41 (ddd, J = 13.8,
9.2, 7.2 Hz, 1H, H3 Amp), 2.15 (s, 3H, Me Mtr), 1.98 (bd, J =
13.8 Hz, 1H, H3 Amp), 1.70 (m, 2H, Hb Arg), 1.50-1.65 (m, 2H,
Hg Arg), 1.46 (s, 9H, t-Bu); 13C NMR (75 MHz, CD3OD) d 179.1
(Cq), 176.6 (Cq), 171.9 (Cq), 171.5 (Cq), 171.1 (Cq), 165.0 (Cq),
160.0 (Cq), 139.6 (Cq), 138.0 (Cq), 135.0 (Cq), 125.8 (Cq), 113.0
(CH), 82.4 (Cq), 64.4 (CH), 60.3 (CH2), 56.4 (CH), 56.2 (CH3),
52.5 (CH2), 51.4 (CH2) 51.1 (CH), 50.3 (CH), 45.7 (CH), 41.3
(CH2), 38.9 (CH2), 37.5 (CH2), 28.5 (3C, CH3), 28.3 (CH2), 27.3
(CH2), 24.5 (CH3), 19.0 (CH3), 12.3 (CH3). MS (ESI) calcd for
C33H51N11O9SNa [M+Na]+: 800.35; found: 800.5. Anal. calcd for
C33H51N11O9S: C, 50.95; H, 6.61; N, 19.81; found: C, 50.76; H, 6.79;
N, 19.75.
cyclo[Arg(Mtr)-Gly-Asp(Ot-Bu)-1-(2-azidoethyl)Amp]
(16).
To a stirring solution of TBAF trihydrate (1.32 g, 4.20 mmol) and
AcOH (252 mg, 4.20 mmol) in 50 mL of DMF, in a plastic vial,
was added a solution of cyclic tetrapeptide 14 (2.43 g, 2.80 mmol)
in DMF (50 mL). The reaction mixture was allowed to stir at
room temperature for 24 h, meanwhile additional portions of the
TBAF/AcOH mixture (2 ¥ 4.20 mmol) were added. After reaction
completion, the mixture was poured into a saturated aq NaCl
solution (200 mL) and extracted with EtOAc (4 ¥). The combined
organic layers were washed with water, dried, filtered and
evaporated under reduced pressure. The flash chromatographic
purification of the crude residue (EtOAc/MeOH, 70:30) afforded
1.73 g of hydroxyethyl intermediate in 82% yield, as a glassy
cyclo[Arg-Gly-Asp-1-(2-azidoethyl)Amp] (17). The protected
cyclic tetrapeptide 16 (150 mg, 0.19 mmol) was treated with 10 mL
of a solution of TFA/TIS/H2O 95:2.5:2.5 at room temperature.
After 18 h, the solvent was evaporated under vacuum and the
residue dissolved in 10 mL of water and thoroughly washed with
Et2O (4 ¥). The aqueous phase was concentrated under vacuum
furnishing the deprotected cyclic tetrapeptide 17 (118 mg, 98%)
as a trifluoroacetate salt. The cyclic tetrapeptide was purified
by preparative RP-HPLC (RP C18-10 mm, 250 ¥ 21.2 mm)
using acetonitrile (0.05% TFA) in H2O (0.05% TFA), 0-35%
linear gradient over 25 min at room temperature. A flow rate of
9.0 mL/min was used and detection was at 220 nm. HPLC Rt =
16.1 min. The HPLC sample was evaporated under vacuum ready
solid: [a]25 +29.0 (c 4.2, MeOH); 1H NMR (600 MHz, CD3OD)
D
d 6.64 (s, 1H, CHMtr), 4.58 (dd, J = 7.2, 5.4 Hz, 1H, Ha Asp),
4.33 (dd, J = 5.3, 5.3 Hz, 1H, H4 Amp), 4.13 (d, J = 14.4 Hz,
1H, Ha Gly), 4.00 (dd, J = 7.8, 7.8 Hz, 1H, Ha Arg), 3.81 (s,
3H, OMe Mtr), 3.50-3.60 (m, 3H, H2 Amp and H2¢Amp), 3.29
(d, J = 14.5 Hz, 1H, Ha Gly), 3.10-3.30 (m, 2H, Hd Arg), 3.08
(dd, J = 10.2, 6.0 Hz, 1H, H5 Amp), 3.04 (bd, J = 9.6 Hz, 1H,
H5 Amp), 2.80 (dd, J = 6.0, 6.0 Hz, 2H, H1¢Amp), 2.70 (dd,
J = 16.8, 7.2 Hz, 1H, Hb Asp), 2.64 (s, 3H, Me Mtr), 2.61 (dd,
J = 16.8, 5.4 Hz, 1H, Hb Asp), 2.58 (s, 3H, Me Mtr), 2.36 (ddd,
J = 13.8, 10.2, 6.6 Hz, 1H, H3 Amp), 2.10 (s, 3H, Me Mtr),
25
for biological assay. A colourless glassy solid; [a]D -20.1 (c 1.0,
1
H2O) (HCl salt); H NMR (600 MHz, D2O) d 4.63 (m, 1H, H2
Amp), 4.60 (dd, J = 6.0, 6.0 Hz, 1H, Ha Asp), 4.27 (dd, J = 4.8,
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 4924–4935 | 4931
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4.8 Hz, 1H, H4 Amp), 4.13 (dd, J = 7.8, 7.8 Hz, 1H, Ha Arg),
3.94 (d, J = 13.8 Hz, 1H, Ha Gly), 3.92 (m, 1H, H5 Amp), 3.67
(m, 1H, H1¢Amp), 3.60 (m, 1H, H1¢Amp), 3.46 (m, 1H, H5 Amp),
3.44 (m, 1H, H2¢Amp), 3.42 (d, J = 13.8 Hz, 1H, Ha Gly), 3.36
(m, 1H, H2¢Amp), 3.08 (dd, J = 6.6, 6.6 Hz, 2H, Hd Arg), 2.81
(m, 1H, H3 Amp), 2.79 (m, 2H, Hb Asp), 2.38 (bd, J. = 15.0 Hz,
1H, H3 Amp), 1.65 (m, 2H, Hb Arg), 1.59 (m, 1H, Hg Arg), 1.50
(m, 1H, Hg Arg); 13C NMR (150 MHz, D2O, HSQC-based) d 68.0
(CH, C2 Amp), 63.5 (CH2, C5 Amp), 58.6 (CH, Ca Arg), 56.8
(CH2, C2¢Amp), 52.4 (2C, CH, Ca Asp and C4 Amp), 49.7 (CH2,
C1¢Amp), 47.2 (CH2, Ca Gly), 43.2 (CH2, Cd Arg), 38.0 (CH2,
C3 Amp), 37.5 (CH2, Cb Asp), 29.1 (CH2, Cb Arg), 27.2 (CH2,
Cg Arg). HRMS (NSI) calcd for C19H32N11O6 [M+H]+: 510.2537;
found: 510.2551.
Amp), 4.58 (dd, J = 6.0, 6.0 Hz, 1H, Ha Asp), 4.27 (dd, J = 5.4,
5.4 Hz, 1H, H4 Amp), 4.15 (dd, J = 7.8, 7.8 Hz, 1H, Ha Arg),
3.99 (bd, J = 12.0 Hz, 1H, H5 Amp), 3.92 (d, J = 13.8 Hz, 1H,
Ha Gly), 3.57 (m, 2H, H2¢Amp), 3.46 (dd, J = 12.6, 4.2 Hz, 1H,
H5 Amp), 3.45 (d, J = 13.8 Hz, 1H, Ha Gly), 3.26 (ddd, J =
13.8, 7.2, 7.2 Hz, 1H, H1¢Amp), 3.19 (ddd, J = 13.8, 7.2, 7.2 Hz,
1H, H1¢Amp), 3.08 (dd, J = 6.6, 6.6 Hz, 2H, Hd Arg), 2.88 (ddd,
J = 15.0, 11.4, 6.0 Hz, 1H, H3 Amp), 2.79 (m, 2H, Hb Asp), 2.47
(bd, J. = 15.0 Hz, 1H, H3 Amp), 1.65 (m, 2H, Hb Arg), 1.58
(m, 1H, Hg Arg), 1.49 (m, 1H, Hg Arg); 13C NMR (150 MHz,
D2O, HSQC-based) d 66.9 (CH, C2 Amp), 60.3 (CH2, C5 Amp),
56.2 (CH, Ca Arg), 51.6 (CH2, C2¢Amp), 50.3 (CH, C4 Amp),
50.0 (CH, Ca Asp), 44.7 (CH2, Ca Gly), 40.6 (CH2, Cd Arg),
35.1 (CH2, C1¢Amp), 35.0 (CH2, C3 Amp), 34.9 (CH2, Cb Asp),
26.5 (CH2, Cb Arg), 24.7 (CH2, Cg Arg). HRMS (NSI) calcd for
C19H34N9O6 [M+H]+: 484.2632; found: 484.2619.
cyclo[Arg(Mtr)-Gly-Asp(Ot-Bu)-1-(2-aminoethyl)Amp]
(18).
The protected cyclic tetrapeptide 16 (1.08 g, 1.39 mmol) was
dissolved in EtOH (50 mL) and a catalytic amount of 10%
palladium on carbon (100 mg) was added. The reaction vessel
was evacuated by aspirator and thoroughly purged with hydrogen
(three times). The resulting heterogeneous mixture was stirred
overnight under hydrogen atmosphere at room temperature. The
catalyst was filtered off and the filtrate was concentrated in vacuo
to give aminoethyl intermediate 18 (950 mg, 91%) which was
Trivalent cyclo[Arg-Gly-Asp-Amp] peptide dendrimer (21).
Protected azidopeptide 16 (520 mg, 0.67 mmol) and 1,3,5-
tris(prop-2-ynyloxy)benzene (20) (45 mg, 0.186 mmol), dissolved
in 10 mL of t-BuOH/H2O (1:1), were sequentially treated with
0.3 M solution of Cu(OAc)2·H2O (372 mL, 0.112 mmol) and 0.9 M
solution of sodium ascorbate (249 mL, 0.224 mmol). The resulting
heterogeneous mixture was sonicated for 1 h and then stirred at
room temperature for an additional 4 h. After reaction completion,
the mixture was lyophilized and the residue was purified by
flash chromatography (DCM/MeOH, 80:20 to 70:30) affording
protected trivalent peptide dendrimer (364 mg, 76%) as a glassy
solid. Protected cycloadduct intermediate (364 mg, 0.141 mmol)
was treated with 21 mL of a solution of TFA/TIS/H2O 95:2.5:2.5
at room temperature. After 18 h, the solvent was evaporated
under vacuum and the residue dissolved in 15 mL of water and
thoroughly washed with Et2O (4 ¥). The aqueous phase was
concentrated under vacuum furnishing the deprotected peptide
dendrimer 21 (283 mg, 95%) as a trifluoroacetate salt (¥ 3 TFA).
The trimeric peptide was purified by preparative RP-HPLC (RP
C18-10mm, 250 ¥ 21.2 mm) using acetonitrile (0.05% TFA) in
H2O (0.05% TFA), 0-45% linear gradient over 20 min at room
temperature. A flow rate of 9.0 mL/min was used and detection
was at 220 nm. HPLC Rt = 16.2 min. The HPLC sample was
evaporated under vacuum ready for biological assay. A colourless
used as such in the following step: a colourless glassy solid; [a]25
+24.0 (c 1.4, MeOH); H NMR (300 MHz, CD3OD) d 6.68 (s,
D
1
1H, CHMtr), 4.62 (m, 1H, Ha Asp), 4.40 (m, 1H, H4 Amp),
4.16 (bd, J = 14.5 Hz, 1H, Ha Gly), 4.06 (m, 1H, Ha Arg), 3.85
(s, 3H, OMe Mtr), 3.52 (bd, J = 9.3 Hz, 1H, H2 Amp), 3.35 (d,
J = 14.9 Hz, 1H, Ha Gly), 3.20 (m, 2H, Hd Arg), 3.05-3.15 (m,
2H, H5 Amp), 2.70-2.90 (m, 4H, H1¢Amp and H2¢Amp), 2.69
(s, 3H, Me Mtr), 2.62 (s, 3H, Me Mtr), 2.61 (m, 2H, Hb Asp),
2.40 (m, 1H, H3 Amp), 2.14 (s, 3H, Me Mtr), 1.98 (m, 1H, H3
Amp), 1.71 (m, 2H, Hb Arg), 1.50-1.65 (m, 2H, Hg Arg), 1.45
(s, 9H, t-Bu); 13C NMR (75 MHz, CD3OD) d 179.2 (Cq), 176.5
(Cq), 171.9 (Cq), 171.5 (Cq), 171.2 (Cq), 160.0 (Cq), 158.3 (Cq),
139.6 (Cq), 138.0 (Cq), 135.0 (Cq), 125.8 (Cq), 113.0 (CH), 82.4
(Cq), 64.7 (CH), 60.1 (CH2), 56.4 (CH), 56.2 (2C, CH3 and CH2),
51.2 (CH), 50.9 (CH), 45.6 (CH2), 41.4 (CH2), 41.1 (CH2), 38.5
(CH2), 37.5 (CH2), 28.5 (3C, CH3), 28.1 (CH2), 27.3 (CH2), 24.5
(CH3), 18.5 (CH3), 12.3 (CH3). MS (ESI) calcd for C33H54N9O9S
[M+H]+: 752.37; found: 752.2. Anal. calcd for C33H53N9O9S:
C, 52.71; H, 7.10; N, 16.76; found: C, 52.93; H, 7.19; N,
16.59.
glassy solid; [a]D -26.6 (c 1.0, H2O) (TFA salt); 1H NMR
25
(600 MHz, D2O) d 7.96 (s, 3H, H-triazole), 6.27 (s, 3H, H-phenyl),
5.03 (m, 6H, CH2OAr), 4.69 (m, 6H, H2¢Amp), 4.54 (dd, J = 6.0,
6.0 Hz, 3H, Ha Asp), 4.22 (bd, J = 10.8 Hz, 3H, H2 Amp), 4.19
(dd, J = 5.4, 5.4 Hz, 3H, H4 Amp), 3.99 (dd, J = 7.8, 7.8 Hz,
3H, Ha Arg), 3.80-3.90 (m, 9H, H1¢Amp and H5 Amp), 3.87
(d, J = 13.2 Hz, 3H, Ha Gly), 3.39 (m, 3H, H5 Amp), 3.37 (d,
J = 13.8 Hz, 3H, Ha Gly), 2.93 (dd, J = 7.2, 7.2 Hz, 6H, Hd
Arg), 2.75 (m, 3H, H3 Amp), 2.71 (m, 6H, Hb Asp), 2.30 (d, J. =
15.6 Hz, 3H, H3 Amp), 1.53 (m, 6H, Hb Arg), 1.35-1.50 (m, 6H,
Hg Arg); 13C NMR (150 MHz, D2O, HSQC-based) d 125.9 (3C,
CH, triazole), 96.6 (3C, CH, phenyl), 66.5 (3C, CH, C2 Amp),
61.4 (3C, CH2, CH2OAr), 61.2 (3C, CH2, C5 Amp), 56.0 (3C, CH,
Ca Arg), 54.0 (3C, CH2, C1¢Amp), 49.8 (6C, CH, Ca Asp and C4
Amp), 46.6 (3C, CH2, C2¢Amp), 44.6 (3C, CH2, Ca Gly), 40.6 (3C,
CH2, Cd Arg), 35.4 (3C, CH2, C3 Amp), 34.9 (3C, CH2, Cb Asp),
26.6 (3C, CH2, Cb Arg), 24.6 (3C, CH2, Cg Arg); HRMS (NSI)
calcd for C72H106N33O21 [M+H]+: 1768.8241; found: 1768.8273.
cyclo[Arg-Gly-Asp-1-(2-aminoethyl)Amp] (19). The protected
cyclic tetrapeptide 18 (150 mg, 0.20 mmol) was treated with 10 mL
of a solution of TFA/TIS/H2O 95:2.5:2.5 at room temperature.
After 18 h, the solvent was evaporated under vacuum and the
residue dissolved in 10 mL of water and thoroughly washed with
Et2O (4 ¥). The aqueous phase was concentrated under vacuum
furnishing the deprotected cyclic tetrapeptide 19 (112 mg, 94%)
as a trifluoroacetate salt. The cyclic tetrapeptide was purified
by preparative RP-HPLC (RP C18-10 mm, 250 ¥ 21.2 mm)
using acetonitrile (0.05% TFA) in H2O (0.05% TFA), 0-25%
linear gradient over 25 min at room temperature. A flow rate of
9.0 mL/min was used and detection was at 220 nm. HPLC Rt =
15.9 min. The HPLC sample was evaporated under vacuum ready
25
for biological assay. A colourless glassy solid; [a]D -13.3 (c 1.0,
1
H2O) (HCl salt); H NMR (600 MHz, D2O) d 4.64 (m, 1H, H2
4932 | Org. Biomol. Chem., 2009, 7, 4924–4935
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HRMS (NSI) calcd for C72H107N33O21 [M+2H]2+: 884.9159; found:
884.9185.
C44H51N10O13 [M+H]+: 927.3637; found: 927.3619. HRMS (NSI)
calcd for C44H52N10O13 [M+2H]2+: 464.1857; found: 464.1834.
DOTA-conjugated cyclo[Arg-Gly-Asp-Amp] peptide (25). To
a stirring solution of DOTA(Ot-Bu)3-derivative 24 (525 mg,
0.80 mmol) and HBTU (424 mg, 1.12 mmol) in dry DCM (20 mL),
a solution of protected cyclopeptide 18 (400 mg, 0.53 mmol) and
DIEA (463 mL, 2.66 mmol) in dry DCM (20 mL) was added
dropwise under argon atmosphere, and the resulting mixture was
maintained under stirring at room temperature for 24 h. After
reaction completion, the mixture was diluted with DCM and
washed with saturated aq NaHCO3 solution (2 ¥), saturated
aq NH4Cl solution (2 ¥) and water (1 ¥). The organic layer
was evaporated under vacuum affording crude DOTA-conjugate
intermediate as a pale yellow oil (726 mg, 98%). The protected
intermediate (726 mg, 0.52 mmol) was treated with 25 mL of a
solution of TFA/TIS/H2O 95:2.5:2.5 at room temperature. After
18 h, the solvent was evaporated under vacuum and the residue was
dissolved in 20 mL of water and thoroughly washed with Et2O (4
¥). The aqueous phase was concentrated under vacuum affording
deprotected conjugate 25 (546 mg, 98%) as a trifluoroacetate salt.
Compound 25 was purified by semipreparative RP-HPLC (RP
C18-10 mm, 250 ¥ 10 mm) using acetonitrile (0.05% TFA) in
H2O (0.05% TFA), 0-25% linear gradient over 26 min at room
temperature. A flow rate of 3.0 mL/min was used and detection
was at 220 nm. HPLC Rt = 13.8 min. The HPLC sample was
evaporated under vacuum ready for biological assay. A colourless
Fluorescein-conjugated cyclo[Arg-Gly-Asp-Amp] peptide (23).
To
a stirring solution of a 1:1 isomeric mixture of 5-
and 6-(3-carboxypropylaminocarbonyl)fluorescein (22) (368 mg,
0.80 mmol) and HBTU (424 mg, 1.12 mmol) in dry DMF
(12 mL), a solution of protected cyclopeptide 18 (400 mg,
0.53 mmol) and DIEA (463 mL, 2.66 mmol) in DMF (12 mL)
was added under argon atmosphere, and the resulting mixture
was maintained under stirring at room temperature for 24 h in
the dark. After reaction completion, the mixture was treated with
saturated aq NH4Cl solution and extracted with EtOAc (4 ¥).
The collected organic layers were dried, filtered and concentrated
to afford crude conjugate intermediate as a yellow oil (616 mg,
97%). The protected intermediate (616 mg, 0.52 mmol) was
treated with 25 mL of a solution of TFA/TIS/H2O 95:2.5:2.5
at room temperature. After 18 h, the solvent was evaporated
under vacuum and the residue was dissolved in 20 mL of water
and thoroughly washed with Et2O (4 ¥). The aqueous phase
was concentrated under vacuum affording deprotected conjugate
23 (500 mg, 93%) as a trifluoroacetate salt. Compound 23 was
purified by semipreparative RP-HPLC (RP C18-10 mm, 250 ¥
10 mm) using acetonitrile (0.05% TFA) in H2O (0.05% TFA), 0-
30% linear gradient over 20 min at room temperature. A flow rate
of 3.0 mL/min was used and detection was at 254 nm. HPLC Rt =
22.3 min. The HPLC sample was evaporated under vacuum ready
glassy solid; [a]D -12.3 (c 0.2, H2O) (TFA salt); 1H NMR
25
(600 MHz, D2O) d 4.61 (m, 1H, Ha Asp), 4.54 (d, J = 10.8 Hz,
1H, H2 Amp), 4.30 (dd, J = 6.0, 6.0 Hz, 1H, H4 Amp), 4.10 (dd,
J = 7.2, 7.2 Hz, 1H, Ha Arg), 3.98 (m, 1H, H5 Amp), 3.95 (d,
J = 13.8 Hz, 1H, Ha Gly), 3.50-3.80 (m, 8H, CH2-DOTA), 3.47
(m, 1H, H5 Amp), 3.42 (d, J = 13.8 Hz, 1H, Ha Gly), 3.30-3.40
and 3.10-3.30 (m, 20H, CH2-DOTA, H1¢Amp and H2¢Amp), 3.08
(dd, J = 6.6, 6.6 Hz, 2H, Hd Arg), 3.04 (m, 2H, H4¢¢), 2.83 (m,
1H, H3 Amp), 2.78 (m, 2H, Hb Asp), 2.39 (d, J. = 14.1 Hz, 1H,
H3 Amp), 2.16 (t, J = 7.2 Hz, 2H, H2¢¢), 1.64 (m, 4H, H3¢¢ and
Hb Arg), 1.59 (m, 1H, Hg Arg), 1.50 (m, 1H, Hg Arg); 13C NMR
(150 MHz, D2O, HSQC-based) d 65.7 (CH, C2 Amp), 61.1 (CH2,
C5 Amp), 56.0 (CH, Ca Arg), 55.7 (3C, CH2, CH2N), 50.9 (CH2,
CH2N), 50.2 (2C, CH2, CH2N), 49.6 (CH, Ca Asp), 49.5 (CH,
C4 Amp), 48.9 (2C, CH2, CH2N), 48.4 (2C, CH2, CH2N), 48.3
(2C, CH2, CH2N), 44.4 (CH2, Ca Gly), 40.3 (2C, CH2, CH2N
and Cd Arg), 38.5 (CH2, CH2N), 35.2 (CH2, CH2N), 35.0 (CH2,
Cb Asp), 34.9 (CH2, C3 Amp), 32.7 (CH2, C2¢¢), 26.3 (CH2, Cb
Arg), 24.5 (CH2, Cg Arg), 24.4 (CH2, C3¢¢). HRMS (NSI) calcd for
C39H67N14O14 [M+H]+: 955.4961; found: 955.4987. HRMS (NSI)
calcd for C39H68N14O14 [M+2H]2+: 478.2519; found: 478.2535.
25
for biological assay. A yellow glassy solid; [a]D -6.0 (c 0.4, H2O)
(TFA salt); 1H NMR (600 MHz, D2O, ca. 1:1 mixture of 5- and 6-
carboxyfluorescein regioisomeric conjugates) d 8.37 (s, 0.5H, Ar),
8.11 (d, J = 7.8 Hz, 0.5H, Ar), 8.01 (d, J = 7.8 Hz, 0.5H, Ar), 7.95
(d, J = 7.8 Hz, 0.5H, Ar), 7.49 (s, 0.5H, Ar), 7.30 (m, 1H, Ar),
7.06 (dd, J = 8.4, 2.4 Hz, 1H, Ar), 6.97 (m, 1.5H, Ar), 6.89 (s, 1H,
Ar), 6.79 (d, J = 8.4 Hz, 1H, Ar), 6.72 (d, J = 8.4 Hz, 1H, Ar),
4.56 (dd, J = 6.0, 6.0 Hz, 0.5H, Ha Asp), 4.55 (d, J = 11.4 Hz,
0.5H, H2 Amp), 4.52 (dd, J = 6.0, 6.0 Hz, 0.5H, Ha Asp), 4.47 (d,
J = 10.8 Hz, 0.5H, H2 Amp), 4.29 (dd, J = 4.8, 4.8 Hz, 0.5H, H4
Amp), 4.24 (dd, J = 5.4, 5.4 Hz, 0.5H, H4 Amp), 4.07 (dd, J =
8.4, 8.4 Hz, 0.5H, Ha Arg), 4.02 (m, 0.5H, H5 Amp), 4.00 (dd,
J = 7.2, 7.2 Hz, 0.5H, Ha Arg), 3.97 (m, 0.5H, H5 Amp), 3.93 (d,
J = 14.4 Hz, 0.5H, Ha Gly), 3.86 (d, J = 13.8 Hz, 0.5H, Ha Gly),
3.43 (m, 0.5H, H5 Amp), 3.30-3.40 (m, 4H, Ha Gly, H2¢Amp,
H4¢¢ and H5 Amp), 3.20-3.30 (m, 3.5H, Ha Gly, H1¢Amp, H4¢¢),
3.02 (dd, J = 6.6, 6.6 Hz, 1H, Hd Arg), 2.98 (dd, J = 6.6, 6.6 Hz,
1H, Hd Arg), 2.75-2.88 (m, 1H, H3 Amp), 2.72 (m, 1H, Hb Asp),
2.68 (m, 1H, Hb Asp), 2.38 (bd, J. = 15.6 Hz, 0.5H, H3 Amp),
2.34 (bd, J = 14.4 Hz, 0.5H, H3 Amp), 2.28 (t, J = 7.2 Hz, 1H,
H2¢¢), 2.17 (t, J = 7.2 Hz, 1H, H2¢¢), 1.84 (m, 1H, H3¢¢), 1.71 (m,
1H, H3¢¢), 1.61 (m, 2H, Hb Arg), 1.55 (m, 1H, Hg Arg), 1.46 (m,
0.5H, Hg Arg), 1.40 (m, 0.5H, Hg Arg); 13C NMR (150 MHz, D2O,
1 isomer reported, HSQC-based) d 130.6 (CH, Ar), 119.2 (CH,
Ar), 117.2 (CH, Ar), 117.1 (CH, Ar), 115.3 (CH, Ar), 113.0 (CH,
Ar), 112.6 (CH, Ar), 111.0 (CH, Ar), 102.8 (CH, Ar), 65.0 (CH,
C2 Amp), 60.8 (CH2, C5 Amp), 55.7 (CH, Ca Arg), 55.6 (CH2,
CH2N), 49.8 (2C, CH, C4 Amp and Ca Asp), 44.4 (CH2, Ca Gly),
40.3 (CH2, Cd Arg), 39.4 (CH2, CH2N), 35.2 (CH2, CH2N), 34.9
(2C, CH2, Cb Asp and C3 Amp), 33.0 (CH2, C2¢¢), 26.3 (CH2, Cb
Arg), 24.4 (2C, CH2, Cg Arg and C3¢¢). HRMS (NSI) calcd for
Solid-phase receptor-binding assay
Purified avb3 and avb5 receptors (Chemicon International, Inc.,
Temecula, CA, USA) were diluted to 0.5 mg/mL in coating buffer
containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl,
1 mmol/L MnCl2, 2 mmol/L CaCl2 and 1 mmol/L MgCl2. An
aliquot of diluted receptor (100 mL/well) was added to 96-well
microtiter plates (NUNC MW 96F MEDISORP STRAIGHT)
and incubated overnight at 4 ◦C. Plates were then treated with
blocking solution (coating buffer plus 1% bovine serum albumin)
for an additional 2 h at room temperature to block nonspecific
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 4924–4935 | 4933
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binding. Integrin-coated 96-well plates were incubated 3 h at room
temperature with 1 mg/mL biotinylated vitronectin in the presence
of serially diluted (10-12-10-5 M) competing compounds. Biotiny-
lation was performed with the EZ-Link Sulfo-NHS-Biotinylation
kit (Pierce, Rockford, IL, USA). After extensive washing with
blocking buffer, plates were incubated for 1 h at room temperature
with streptavidin-biotinylated peroxidase complex (Amersham
Biosciences, Uppsala, Sweden). Wells were then accurately washed
with blocking buffer and incubated 30 min with 100 mL Substrate
Reagent Solution (R&D Systems, Minneapolis, MN, USA), before
stopping the reaction by addition of 50 mL of 2M H2SO4.
Absorbance at 415 nm was immediately read in a SynergyTM HT
Multi-Detection Microplate Reader (BioTek Instruments, Inc.).
Each data point is the result of the average of triplicate wells
and was analyzed by nonlinear regression analysis with Prism
GraphPad program.
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Acknowledgements
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This work was supported by Ministero dell’Istruzione,
dell’Universita` e della Ricerca (PRIN 2006, Roma, Italy). The au-
thors thank the Centro Interdipartimentale Misure “G. Casnati”
(Universita` degli Studi di Parma) for instrumental facilities. A
postdoctoral fellowship to Dr. Paola Burreddu from Centro Inter-
dipartimentale di Studi Biomolecolari e Applicazioni Industriali
(CISI, Milano, Italy) and a fellowship to Dr. Elena M. V. Araldi
from Scuola di Dottorato in Medicina Molecolare (Universita`
degli Studi di Milano) are gratefully acknowledged. Thanks are
due to Dr. Eugenia Monferini for preliminary biological assays.
4415.
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4934 | Org. Biomol. Chem., 2009, 7, 4924–4935
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