The first synthetic receptor for the RGD sequence

Article abstract of DOI:10.1021/ol025976q

(matrix presented) The combination of an optimized arginine receptor unit with a semirigid linker carrying a strategically placed primary ammonium group leads to the first synthetic RGD receptor. It binds to the free RGD peptide as well as to cyclo(RGDfV)

Full text of DOI:10.1021/ol025976q

ORGANIC
LETTERS
2002
Vol. 4, No. 13
2161-2164
The First Synthetic Receptor for the
RGD Sequence
Stephan Rensing and Thomas Schrader*
Fachbereich Chemie, UniVersita¨t Marburg, Hans-Meerwein-Strasse,
35032 Marburg, Germany
schradet@mailer.uni-marburg.de
Received April 5, 2002
ABSTRACT
The combination of an optimized arginine receptor unit with a semirigid linker carrying a strategically placed primary ammonium group leads
to the first synthetic RGD receptor. It binds to the free RGD peptide as well as to cyclo(RGDfV) in water with association constants around
1000 M-1. RGD mimetics such as benzamidine 6 are not recognized, rendering the new host a prototype of a new class of receptors selective
for the true RGD sequence in peptides.
Cell-cell and cell-matrix adhesion processes are vitally
important for higher organisms. These interactions control
essential body functions such as embryogenesis, cell dif-
ferentiation, angiogenesis, heamostasis, wound healing, or
immune response. One of the most important classes of
proteins involved in these adhesion processes are the
integrins, which are heterodimeric glycoproteins, anchored
inside the cell membrane.1 They interact only with proteins
carrying the Arg-Gly-Asp sequence (RGD) on a solvent
exposed loop. Proteins with such an RGD loop are fibrino-
gen,2 fibronectin,3 vitronectin,4 thrombospondin,5 osteospon-
tin,6 and a number of growth factors.
processes. Inhibiton of this complex formation strongly
influences all related events. Two alternatives may be
chosen: conformationally well-defined synthetic RGD mi-
metics may recognize specific integrins7 or small soluble
receptors may bind to the RGD sequence itself, distinguishing
between its extended or folded state. Whereas today almost
all large pharmaceutical companies hold patents for the first
pathway, virtually nothing is known about the second
alternative.
We have recently published small optimized structures for
the recognition of arginine in polar solvents (Figure 1).8 They
rely on the m-xylylene bisphosphonate recognition motif 1,
which combines electrostatic, hydrogen bond, and π-cation
interactions in its 1:1 complex with N/C-protected arginine
(800 M-1 in CD3OD). Introduction of an additional phos-
phonate group on the 5-position with the appropriate rigid
spacer led to improved binding because now the fifth NH
proton of the guanidinium moiety was also bound (3500 M-1
The specific complex formation of a relatively small
peptide fragent with its binding site on a membrane-bound
receptor thus constitutes a key element in many life
(1) Albelda, S. M.; Buck, C. A. FASEB J. 1990, 4, 2868.
(2) Gardner, J. M.; Hynes, R. O. Cell 1985, 42, 439.
(3) Pierschbacher, M. D.; Ruoslathi, E.; Sundelin, J.; Lind, P.; Peterson,
P. A. J. Biol. Chem. 1982, 257, 9593.
(4) Schvartz, I.; Seger, D.; Shaltiel, S. Int. J. Biochem. Cell Biol. 1999,
31, 539.
(5) Lawer, J.; Weinstein, R.; Hynes, R. O. J. Cell. Biol. 1988, 107, 2351.
(6) Helfrich, M. H.; Nesbitt, S. A.; Dorey, E. L.; Horton, M. A. J. Bone
Miner. Res. 1992, 7, 335.
(7) Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem. 1997, 109,
1440.
(8) (a) Schrader, T. Chem. Eur. J. 1997, 3, 1537. (b) Schrader, T.
Tetrahedron Lett. 1998, 39, 517.
10.1021/ol025976q CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/01/2002

Figure 2. (a) Arginine ester recognition with 2; note the extended
side chain. (b) Histidine ester recognition with 3; note the perfect
structural complementarity. Monte Carlo simulations have been
carried out in water (MacroModel 7.0, Amber*).
Monte Carlo simulations in water reach complex geom-
etries with an intriguing complementarity between host and
guest for the best binding partners described above.11 The
long trisphosphonate allows the arginine side chain to adopt
a thermodynamically favorable extended conformation and
still ensures a second electrostatic interaction with the
R-ammonium group (Figure 2a). By contrast, the compact
Figure 1. Basic m-xylylene recognition motif 1 for arginine in a
peptidic environment, optimized structure 2 with an additional
phosphonate arm, and carboxylate analogue 3.
in CD3OD).9 No binding, however, could be observed in
water.
In recent experiments, we titrated the most basic amino
acid esters as dicationic species with our new arginine
binders.10 Here we found that the new trisphosphonate
receptor molecule 2 displayed a pronounced arginine selec-
tivity, while a shorter carboxylate analogue turned out to be
moderately selective for histidine. Thus, arginine methyl ester
(36000 M-1 in CD3OD) was bound 18 times better than
histidine methyl ester and even 36 times better than lysine
methyl ester (Table 1). In D2O 2 can still bind the arginine
Figure 3. Schematic of the rational RGD receptor design.
Recognition of the RGD sequence is achieved by a combination
of an arginine host with an aspartate host interconnected by a rigid
optimized spacer, which prevents self-association.
Table 1. Binding Constants Resulting from NMR Titration
Experiments between Basic Amino Acid Esters and Optimized
m-Xylylene Receptor Structures 2 and 3 in Methanol and Water
at 25 °C
carboxylate can accommodate histidine’s imidazolium ring
just above its central benzene ring and still form an additional
salt bridge between its carboxylate anion and histidine’s
R-ammonium group, which is in close proximity to its side
chain (Figure 2b).
amino acid methyl
ester x 2 HCl
2 in CD3OD
2 in D2O
3 in D2O
arginine
histidine
lysine
36000 ( 51%a 800 ( 43%b
2400 ( 55%
1000 ( 22%
300 ( 20%
no saturation 500 ( 29%
no shifts no shifts
Its high arginine selectivity makes 2 an ideal candidate
for a potential RGD receptor. Since lysine is not bound at
all in water, we envisaged that an additional ammonium
group attached to the skeleton of 2 at the correct distance
via a rigid spacer could serve as the aspartate binding site.
The spacer should prevent intramolecular dimerization, while
the ammonium functionality is a bad guest for the trisphos-
phonate and should thus circumvent the unwanted inter-
molecular dimerization. Extensive modeling studies sug-
gested the introduction of a m-aminobenzyl substituent at
the amide nitrogen. This would also avoid the creation of a
stereogenic center that would arise from attachment of this
substituent at phosphonoglycine‘s methylene group.
Mild deprotonation of the amide in dipolar aprotic solution,
followed by addition of the phthaloylprotected spacer as a
a Errors are standard deviations from the nonlinear regressions. b The
large standard deviation in this case can be explained with small chemical
shift differences.
ester (800 M-1), but no saturation was found for the histidine
derivative and no shifts at all for the lysine ester. Interest-
ingly, the carboxylate 3 prefers histidine esters (500 M-1)
over arginine esters (300 M-1) at the same conditions but
cannot bind lysine esters.
(9) Rensing, S.; Springer, A.; Grawe, T.; Schrader, T. J. Org. Chem.
2001, 66, 5814. Other powerful arginine receptors: (a) Bell, T. W.;
Khasanov, A. B.; Drew, M. G. B.; Filikov, A.; James, T. L. Angew. Chem.,
Int. Ed. 1999, 38, 2543. (b) Ngola, S. M.; Kearney, P. C.; Mecozzi, S.;
Russell, K.; Dougherty, D. A. J. Am. Chem. Soc. 1999, 121, 1192.
(10) (a) Schneider, H. J.; Kramer, R.; Simova, S.; Schneider, U. J. Am.
Chem. Soc. 1988, 110, 6442. (b) Wilcox, C. S. In Frontiers in Supra-
molecular Chemistry; Schneider, H. J., Ed.; Verlag Chemie: Weinheim,
1991; p 123.
(11) MacroModel 7.0, Schro¨dinger Inc., force-field: Amber*, 1000 steps,
water.
2162
Org. Lett., Vol. 4, No. 13, 2002

benzyl bromide proceeded smoothly to give the secondary
amide 4 as an E/Z mixture of ∼4:1.12 Subsequent 3-fold
monodealkylation with LiBr13 followed by removal of the
phthalimide with hydrazine afforded the free base 5, which
was finally back-titrated with 1 equiv of HCl to give the
desired receptor molecule 5a. It is highly soluble in water
and shows no appreciable self-association in this solvent
according to a dilution experiment.
Figure 5. (a) Job plot and (b) NMR titration curve for the
complexation of free RGD peptide with 5a in water.
group are present just as in the real RGD loop of the target
proteins. To our great pleasure, a clear 1:1 stoichiometry with
a binding constant of 700 M-1 was detected for 5a.15 Again,
upfield shifts were found in the aspartate region of the
cyclopeptide. This shows that the concept of combining an
arginine with an aspartate binder is valid even in the simplest
case of an ammonium group.
Figure 4. Synthesis of 2, 3, and 5a by selective N-alkylation.
Table 2. Binding Constants Resulting from NMR Titration
Experiments between RGD-Containing Peptides or Mimetics
and Trisphosphonate Receptor Molecules 2 and 5a in Water at
25 °C
Before we examined its potential to bind RGD, we used
the trisphosphonate 2 as a model compound. Since the free
RGD tripeptide also contains a dicationic C-protected argi-
nine, the trisphosphonate might also show some affinity for
it in water. Indeed, a certain interaction was found (300 M-1),
probably weakened by the strong electrostatic repulsion
between the trisphosphonate anion and the two aspartate
carboxylate groups. However, for our new RGD binder 5a
the interaction with the free tripeptide produced a Ka value
half an order of magnitude higher than that of 2. Distinct
complexation-induced shifts were found in the aspartate
region, indicating that the additional salt bridge had indeed
been formed. Next we titrated 5a with Kessler’s cyclopeptide
cyclo(RGDfV), which is a model compound for the RGD
loop in fibronectin.14 Here, only one cationic and one anionic
RGD peptide
or mimetic
self-association
(dilution)
2 (M-1
)
5a (M-1
)
free RGD
cyclo(RGDfV)
benzamidine 6
300 ( 15%a 1300 ( 5% no shifts
700 ( 14% no shifts
no shifts
5a alone: no shifts
a Errors are standard deviations from the nonlinear regressions.
We also tried to bind the powerful mimetic 6 for the RGD
conformation in fibrinogen with 5a but failed completely.16
Not even the slightest shift was observable in any of the
host or guest protons. This could have two explanations: (a)
5a recognizes only the correct folded conformation of the
arginine and asparate side chain in fibronectin, and (b) the
benzamidine group present in the synthetic drug does not
(12) (a) Hoffmann, R. V.; Nayyar, N. K. J. Org. Chem. 1994, 59, 3530.
(b) Suezawa, H.; Tsuchiya, K.; Tahara, E.; Hirota, M. Bull. Chem. Soc.
Jpn. 1987, 60, 11, 3973.
(13) (a) Karaman, R.; Goldblum, A.; Breuer, E. J. Chem. Soc., Perkin
Trans. 1989, 765. (b) Krawczyk, H. Synth. Commun. 1997, 27, 3151.
(14) Dechantsreiter, M. A.; Plancker, E.; Matha¨, B.; Lohof, E.; Ho¨lze-
mann, G.; Jonczyk, A.; Goodmann, S. L.; Kessler, H. J. Med. Chem. 1999,
42, 3033.
(15) By means of a Job plot: (a) Job, P. Compt. Rend. 1925, 180, 928.
(b) Blanda, M. T.; Horner, J. H.; Newcomb, M. J. Org. Chem. 1989, 54,
4626.
(16) Stilz, H. U.; Guba, W.; Jablonka, B.; Just, M.; Klingler, O.; Ko¨nig,
W.; Wehner, V.; Zoller, G. J. Med. Chem. 2001, 44, 1158.
Org. Lett., Vol. 4, No. 13, 2002
2163

Figure 7. RGD mimetic 6, a fibrinogen receptor antagonist.
effects should occur with other drugs in the patient’s body.
In the future, we will combine our optimized arginine binder
with the powerful aspartate ligand introduced by the
Schmuck group17 to achieve highly efficient and selective
RGD binding in water.
Acknowledgment. We thank Prof. Kessler for a sample
of cyclo(RGDfV) and helpful discussions as well as the
Aventis Pharma GmbH for a sample of fibrinogen mimetic
6.
Supporting Information Available: Experimental pro-
cedures, NMR titration tables and curves, and Job plots. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 6. Proposed structure for the complex between the new
RGD ligand 5a and cyclo(RGDfV) in water (MacroModel 7.0,
OPLS-AA). Bottom right: Lewis structure.
OL025976Q
fit into the receptor’s binding site designed for the slim
alkylguanidinium moiety. Preliminary molecular mechanics
calculations support especially the second argument. This is
encouraging, because for a potential therapeutic use no side
(17) (a) Schmuck, C. Chem. Eur. J. 2000, 4, 709. Other powerful
aspartate receptors: (b) A¨ıt-Haddou, H.; Wiskur, S. L.; Lynch, V. M.;
Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 11296. (c) Albert, J. S.;
Goodman, M.; Hamilton, A. D. J. Am. Chem. Soc. 1995, 117, 1143.
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