Cyclic RGD peptides containing β-turn mimetics

Article abstract of DOI:10.1021/ja9608757

The α(v)β₃-receptor, one member of the integrin family, is implicated in angiogenesis and in human tumor metastasis. Spatial screening led to the highly active first-generation peptide c(RGDfV), which shows a βII'/γ-turn arrangement with D-Phe

Full text of DOI:10.1021/ja9608757

J. Am. Chem. Soc. 1996, 118, 7881-7891
7881
Cyclic RGD Peptides Containing â-Turn Mimetics
Roland Haubner,† Wolfgang Schmitt,† Gu1nter Ho1lzemann,‡ Simon L. Goodman,‡
Alfred Jonczyk,‡ and Horst Kessler*,†
Contribution from the Institute of Organic Chemistry and Biochemistry, TU Mu¨nchen,
Lichtenbergstrasse 4, D-85747 Garching, Germany, and Merck KGaA Preclinical Research,
Frankfurter Strasse 250, D-64271 Darmstadt, Germany
ReceiVed March 18, 1996X
Abstract: The RVâ3-receptor, one member of the integrin family, is implicated in angiogenesis and in human tumor
metastasis. Spatial screening led to the highly active first-generation peptide c(RGDfV), which shows a âII′/γ-turn
arrangement with D-Phe in the i + 1 position of the âII′-turn. Further reduction of the flexibility should be achieved
by incorporating different rigid building blocks (turn mimetics) like the (S)- and (R)-Gly[ANC-2]Leu dipeptide, the
â-turn dipeptide (BTD) and the (S,S)-spiro-Pro-Leu moiety. These distinct â-turn mimetics are introduced by replacing
the D-Phe-Val dipeptide in the lead structure c(RGDfV). In peptide analogues c(RGD“S-ANC”) (PA1), c(RGD“R-
ANC”) (PA2), and c(RGD“BTD”) (PA3) the turn mimetic does not adopt the desired position in the â-turn, instead
Gly occupies the i + 1 position of the âII′-turn. Only c(RGD“spiro”) PA4 led to the desired âII′/γ-turn arrangement
with the turn motif in the i + 1 and i + 2 position of the â-turn. These effects may arise from particular steric
effects of the cyclic pentapeptide system in combination with steric requirements of the ANC and BTD moiety.
Additional investigations on cyclic hexapeptide derivatives show that the BTD occupies the expected i + 1 and i +
2 position of a âII′-turn in these systems. Structure-activity investigations showed that the incorporation of the
rigid turn motifs could not reduce the flexibility of the RGD site (ANC and BTD) or fix a conformation which is
unable to match the receptor very well (spiro). On the other hand, recent findings that the proton of the amide bond
between Asp and the following amino acid is essential for high activity can be confirmed. Moreover, the synthesis
of c(RGD“R-ANC”) PA2 led to one of the compounds most active in inhibiting vitronectin binding to the RVâ3-
integrin.
Introduction
used as antithrombotic agents,7 a vitronectin receptor (RVâ3) is
of great interest concerning metastasis of tumor cells.8 There
is a striking difference in the expression of the â3-subunit
between tumorigenic and nontumorigenic lesions, when expres-
sion of integrins on cells in tissue sections are examined.9 In
addition to the vitronectin receptor, there is a variety of other
integrins reported to be expressed on the surface of tumor cells.10
Furthermore, it was shown that the RVâ3-receptor plays a crucial
role in the angiogenesis which is important for the development
of metastatic colonies.11 Metastasis of several tumor cell lines12
as well as tumor-induced angiogenesis13 can be inhibited by
antibodies or small, synthetic peptides acting as ligands for these
receptors.
Cell-cell and cell-matrix adhesion are important in patho-
logical processes like thrombosis,1 osteoporosis,2 and tumor
metastasis.3 One of the major cell surface receptor classes
involved in these interactions are the integrins.4 Each integrin
is an integral plasma membrane, heterodimeric glycoprotein
consisting of an R-subunit and a smaller â-subunit.5 The
specificity for ligand binding is determined by a particular
combination of different R- and â-subunits.
Besides the platelet glycoprotein IIb/IIIa (RIIbâ3), which is
involved in platelet aggregation,6 the inhibitors of which are
† Institut of Organic Chemistry and Biochemistry.
‡ Merck KGaA Preclinical Research.
X Abstract published in AdVance ACS Abstracts, August 1, 1996.
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S0002-7863(96)00875-X CCC: $12.00 © 1996 American Chemical Society

7882 J. Am. Chem. Soc., Vol. 118, No. 34, 1996
Haubner et al.
All inhibitory peptides contain the amino acid triplet Arg-
Gly-Asp (RGD), the so-called “universal cell recognition
sequence”. This sequence is found in many extracellular matrix
proteins like vitronectin, laminin, fibrinogen, and fibronectin.14
Despite this common sequence, a high substrate specificity
among the different integrins is observed. This can be explained
by particular conformations of the RGD sequence in different
matrix proteins.5c
Figure 1. Replacement of the D-Phe-Val dipeptide by several turn
motifs to fix the âII′/γ-turn arrangement. The full circle represents a
D-amino acid.
In order to define an antagonist pharmacophore, it is necessary
to determine the spatial structure of the active site with high
precision. The only known X-ray structure results from the
N-terminal A domain of an R-subunit.15 But neither the
structure of a complete integrin nor of a receptor-bound ligand
complex is known. The conformations of some proteins
containing a biologically relevant RGD sequence (γ-crystal-
line,16 tenascin,17 foot-and-mouth disease virus,18 and the 10th
type III module of fibronectin19 ) as well as of some disintegrins
like kistrin,20 echistatin,21 and flavoridine22 have been examined.
In addition, the secondary structure elements of albolabrin,
another disintegrin, are known.23 In each structure the RGD
sequence is exposed at the tip of a flexible loop or in an extended
edge-strand of a â-sheet (γ-crystalline). Recently, the structure
of the RGD-containing decorsin,24 a leech protein, was deter-
mined. In contrast to the above mentioned proteins the structure
of decorsin is well-defined in the region of the RGD sequence
and shows an extended conformation on the tip of a loop, with
the side chains of Arg and Asp orientated in almost opposite
directions. Decorsin binds to both GPIIb/IIIa and the vitronectin
receptor with high affinity. This leads to the assumption that
the well-defined RGD loop is still capable of fitting the different
receptors. This flexibility of particular RGD motifs prohibits
a determination of the bioactive conformation necessary for a
structure-based rational drug design.
space. Nevertheless, such cyclic peptides can still perform
conformational transitions.29 The introduction of rigid building
blocks should further decrease this flexibility.
Here we describe the incorporation of several known rigid
building blocks into cyclic penta- and hexapeptides containing
the RGD sequence. Structural influences and consequences
concerning the inhibition of vitronectin and fibrinogen binding
to the vitronectin receptor and platelet glycoprotein IIb/IIIa are
examined.
Strategy
The first investigations on the inhibition of fibrinogen and
vitronectin binding to the RVâ3 and RIIbâ3 receptor led to the
highly active and RVâ3 selective cyclic pentapeptide
c(RGDfV),30 which also suppresses tumor-induced angiogenesis
in a chick chorioallantoic membrane model.13 This peptide
shows a âII′/γ-turn arrangement with D-Phe in the i + 1 position
of the âII′-turn (Figure 1).31 However, especially the conforma-
tion of the γ-turn is not well-defined, and this part of the
backbone still shows a certain flexibility.29b Therefore, we
wished to reduce the accessible conformational space by
incorporation of several â-turn mimetics32 (Figure 2). This
restriction of flexibility should lead to a better insight into
structure-activity relations. If the biologically active conforma-
tion is matched by these peptides, they should exhibit a tighter
binding to the integrins for entropic reasons.27
Because of these problems we indirectly determine the
conformation of the active site using small peptides containing
the RGD sequence.25,31 Small linear peptides possess a very
high flexibility and are normally not suited for structural
analysis.26 For that reason our group27 and others28 use
cyclization as a method to reduce the accessible conformational
The RGD sequence is very sensitive to modifications, and
minor variations like the replacement of Gly with Ala lead to
a drastic loss of activity.33 Therefore, we replaced the D-Phe-
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Cyclic RGD Peptides Containing â-Turn Mimetics
J. Am. Chem. Soc., Vol. 118, No. 34, 1996 7883
BTD moiety adopts a very unfavorable position at the side or
(b) the turn mimetic occupies the i + 1 and i + 2 position of
a âII′-turn, for which it is designed, and D-Ala is shifted in the
i + 2 position of the adverse second turn.
Experimental Methods
Synthesis of the Turn Mimetics. Synthesis of the BTD (Figure 2)
follows the scheme outlined by Bach et al.37e However, it was modified
concerning the protecting group strategy and deprotection route. We
wanted to introduce the BTD moiety using solid phase peptide synthesis
with 9-fluorenylmethoxycarbonyl (Fmoc) strategy. Thus, the N-
terminal Boc-protecting group had to be exchanged by the Fmoc group.
The deprotection of the phthaloyl group with hydrazine in the presence
of the C-terminal ethyl ester results in the C-terminal hydrazide in high
amounts. Therefore, the ethyl ester was cleaved first under acidic
conditions using a 4:1 mixture of acetic acid and concentrated
hydrochloric acid. The phthaloyl group was unaffected under these
conditions, leading to Pht-BTD-OH. Subsequent hydrazinolysis of the
phthaloyl group and reprotection of the N-terminus gave the desired
Fmoc-BTD-OH.
Figure 2. The different turn mimetics: (S)- and (R)-Gly[ANC-2]Leu
dipeptide ((S)-ANC, (R)-ANC), â-turn dipeptide (BTD), and (S,S)-spiro-
Pro-Leu moiety (spiro).
Val dipeptide by several different turn mimetics (see Figure 2)
to fix the backbone conformation. The ability of the (S)- and
(R)-Gly[ANC-2]Leu dipeptide34 (S- and R-ANC) and the â-turn
dipeptide35 (BTD) as well as the (S,S)-spiro-Pro-Leu moiety36
(spiro) to introduce turns in linear or cyclic peptides has already
been demonstrated.37 In our peptide analogues these building
blocks should occupy the i + 1 and i + 2 position of the âII′-
turn (Figure 1). The distinct turn mimetics thereby should
reduce the flexibility of different backbone dihedral angles in
the cyclic peptides. The ANC moiety fixes the ψ-angle of the
amino acid in the i + 1 position of the â-turn. In contrast, the
BTD and spiro motif fix one φ- and one ψ-angle (BTD: ψi+1
and φi+2; spiro: φi+1 and ψi+1). Therefore, the latter two turn
mimetics should lead to more rigid cyclic peptides. Fixing the
peptide in the region of the D-Phe-Val dipeptide should also
influence the flexibility of the γ-turn on the opposite side.
To examine the influence of the ring size on the ability to
induce a â-turn, the BTD moiety was also incorporated into
the two cyclic pseudo-hexapeptides (c(RGD“BTD”V), PA5, and
c(RaD“BTD”V), PA6). Cyclic hexapeptides normally adopt a
conformation consistent of two opposed â-turns. It is known
that a D-amino acid in such cyclic peptides induces a âII′-turn
with the D-amino acid located in the i + 1 position.38 We were
interested to see whether the D-amino acid exhibits a higher
preference to occupy the i + 1 position of the â-turn or if the
turn motif forms the â-turn. Therefore, we chose, besides
c(RGD“BTD”V), the biologically less interesting c(Ra-
D“BTD”V) to investigate this aspect. For the latter compound
two different conformations are possible: (a) the D-amino acid
is dominant and induces a âII′-turn with D-Ala in i + 1 and the
Gly[ANC-2]Leu building blocks (Figure 2) were synthesized ac-
cording to the literature.34,39 The Boc-protecting group was exchanged
similarly as described.39 The Fmoc-protected (S,S)-4,4-spirolactam
moiety (Fmoc-(S,S)-spiro-Pro-Leu-OH, Figure 2) was purchased from
Neosystems, France.
Synthesis of the Peptide Analogues. Linear peptides were as-
sembled leaving the glycine residue at the C-terminus to prevent
racemization and steric hindrance during the cyclization step. The
synthesis was performed using Merrifield solid phase peptide synthesis40
with Tentagel41 (peptides PA1, PA2, and PA4) or o-chlorotritylchlorid42
(cTrt) resins (peptides PA3, PA5, and PA6) applying Fmoc-strategy.43
The Fmoc-protected amino acids and turn mimetics were coupled with
O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate (TBTU) and 1-hydroxybenzotriazole (HOBt) using diisopropyl-
ethylamine (DIEA) as base. The Fmoc group was cleaved with 20%
piperidine in dimethylformamide (DMF). Peptides were cleaved from
the cTrt solid support by acetic acid/2,2,2-trifluoroethanol(TFE)/
dichlormethane (1:1:3) or from the Tentagel solid support by hydro-
genation. Both procedures lead to peptides with intact side chain
protecting groups. Cyclization was performed via in situ activation
using diphenyl phosphorazidate (DPPA) in DMF with sodium bicar-
bonate as a solid base44 or N-ethyl-N,N′-(dimethylaminopropyl)-
carbodiimide (EDCI) and 4-(N,N-dimethylamino)pyridine (DMAP)
under high dilution conditions. Final deprotection was done with
trifluoracetic acid (TFA) and scavengers.
Purification by reverse phase high-performance liquid chromatog-
raphy (RP-HPLC) yielded peptide analogues, which were >95% pure.
All peptides were characterized by fast atom bombardment (FAB) mass
spectrometry and various NMR techniques (for data see supporting
information).
NMR Spectroscopy. All spectra were recorded in DMSO-d6
solution and calibration was performed with reference to the residual
DMSO signal (1H, 2.49 ppm; 13C, 39.5 ppm). The assignment of all
proton and carbon resonances followed the standard strategy as
previously described.45 Sequential assignment was accomplished by
through-bond connectivities from heteronuclear multibond correlation
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1988, 22, 189. (c) Ward, P.; Ewan, G. B.; Jordan, C. C.; Ireland, S. J.;
Hagan, R. M.; Brown, J. R. J. Med. Chem. 1990, 33, 1848-1851. (d) Hinds,
M. G.; Welsh, J. H.; Brennand, D. M.; Fisher, J.; Glennie, M. J.; Richards,
N. G. J.; Turner, D. L.; Robinson, J. A. J. Med. Chem. 1991, 34, 1777-
1789. (e) Bach, A. C., II; Markwalder, J. A., Ripka, W. C. Int. J. Pept.
Protein Res. 1991, 38, 314-323.
(44) Brady, S. F.; Paleveda, W. J.; Arison, B. H.; Freidinger, R. M.;
Nutt, R. F.; Veber, D. F. In Peptides: Structure and Function. Proceedings
of the 8th American Peptide Symposium; Hruby, V. J., Rich, D. H., Eds.;
Pierce Chemical Co.: Rockford, Illinois, 1983; pp 127-130.
(38) Kessler, H. In Trends in Drug Research; Claasen, V., Ed.; Elsevier
Science Publishers: Amsterdam, 1990; pp 73-84.

7884 J. Am. Chem. Soc., Vol. 118, No. 34, 1996
Haubner et al.
Table 1. Temperature Dependence of the Amide Proton Chemical
the four-dimensional space using the random metric matrix algorithm.54
The optimization step consists of 200-step steepest-descent minimization
followed by a short distance driven dynamic (DDD) run (5 ps with a
strong coupling to a temperature bath at 300 K and 2 ps with a weak
coupling to 1 K) using NOEs as restraints. After reprojection into the
three-dimensional space, 200 steps of steepest-descent minimization
to optimize the chiral volumes were performed. Final optimization
was achieved with a two-step distance-, and angle-driven dynamic
(DADD)55 simulation (5 ps with a strong coupling to a temperature
bath of 500 K and 2 ps with a weak coupling to 1 K) using NOEs,
vicinal coupling constants, and chiral volumes. The resulting structures
for each peptide were analyzed for convergence. RMSD values for
the superposition of the backbone C′, N, and CR atoms were in the
range of 4 to 38 pm for each compound; therefore, the structure with
the lowest total error was used as a starting structure for a subsequent
MD simulation using a modified version of the Gromos program.56
The structures were placed in a truncated octahedron which was filled
with DMSO molecules.57 This box was then energy minimized in two
steps (each with 2000 steps of steepest descent), the first one to relax
the solvent molecules while the peptide is held fixed and the second
for all atoms. A MD trajectory was recorded for 170 ps at a temperature
Shift of the Peptide Analogues in DMSOa.
amino acid
no. peptide analogue Arg1 Gly2/D-Ala2 Asp3 Mim4 b Val5
PA1 c(RGD“S-ANC”) -0.5
PA2 c(RGD“R-ANC”) -2.3
-6.3
-2.3
-6.0
-9.3
-6.7
-6.7
-7.3 -1.1
-7.3 -0.9
PA3 c(RGD“BTD”)
PA4 c(RGD“spiro”)
1.1
-9.7
-2.8
3.0
-2.6
PA5 c(RGD“BTD”V) -4.7
-3.4 -6.8 -0.7
-4.4 -5.1 -0.9
PA6 c(RaD“BTD”V)
-3.7
a The coefficients are given in parts per billion per K. b Correspond-
ing turn mimetic.
(HMBC)46 spectra. Aromatic and carbonyl resonances were also
assigned using through bond long-range correlations. As only one set
of signals for each peptide could be detected, no cis-trans isomerization
at peptide bonds occurs under measurement conditions. Proton
distances were calculated according to the isolated two-spin approxima-
tion from volume integrals of nuclear Overhauser enhancement
(NOESY)47 or rotating frame nuclear Overhauser enhancement (ROE-
SY)48 spectra. NOESY spectra were recorded with mixing times of
150 ms (for PA1 and PA3) and compensated ROESY spectra with
200 ms (for PA2, PA4, PA5, and PA6). Homonuclear coupling
constants have been measured from one-dimensional spectra and from
P.E.COSY49 crosspeaks. Determination of the heteronuclear vicinal
coupling constants followed the scheme of Titman and Keeler from a
combination of HMBC spectra with z-filtered TOCSY.50 For peptide
of 300 K using NOEs and coupling constants as restraints (kNOE
)
2000 kJ/(mol nm2); kJ ) 1 kJ/(mol rad2)) and each picosecond a
structure was stored. The first 70 ps of the individual trajectories were
disregarded to account for equilibration, the last 100 ps were analyzed
for violation of experimental data, population of hydrogen bonds, and
dihedral transitions. The structures presented in this paper have been
averaged over the last 100 ps of the MD run and minimized by 200
steps of steepest descent.
3
analogues PA5 and PA6 J(HN,Câ) coupling constants were extracted
from HETLOC experiments.51 Temperature coefficients for the amide
protons of each peptide were determined via one-dimensional spectra
in the range from 300 to 340 K with a step size of 5 K (Table 1).
Chemical shift data, proton distances and determined coupling constants
are listed in the supporting information.
Results
Structures of the Cyclic Pentapeptides. c(RGD“S-ANC”),
PA1. The structure of PA1 as determined in DMSO does not
exhibit the expected conformation. Gly occupies the i + 1
position of a distorted âII′-turn while the S-ANC moiety is
located in the i + 3 and i + 4 position. The conformation of
the â-turn is slightly distorted, the particular hydrogen bond is
not populated throughout the whole MD simulation. The amide
protons of Arg1 and of the turn mimetic show the smallest
temperature dependence of chemical shifts since Arg1-HN is
sterically shielded from the solvent and ANC4-HN is involved
in the hydrogen bond of the âII′-turn. The assignment of the
diastereotopic â-protons of Asp3 was achieved using the
3J(HR,Hâ) coupling constants in combination with the HN,Hâ
and HR,Hâ proton distances. Subsequently, the population of
the side chain rotamers of Asp3 can be calculated from the
homonuclear HR,Hâ coupling constants according to the Pachler
equations.58 The gauche (-) rotamer (ø1 ) -60°) is predomi-
nant with a value of 56%. In contrast, the side chain of Arg1
is flexible, its orientation as shown in Figure 3 is arbitrarily.
c(RGD“R-ANC”), PA2. The peptide PA2 exhibits a higher
flexibility in DMSO than PA1 and shows a lower tendency to
occupy a predominant conformation. The â-turn with Gly2 in
the i + 1 position, as found for the peptides PA1 and PA3, is
populated only to a degree of 12%. The carbonyl oxygen of
Arg1 is also involved in a γ-turn (bifurcation), as is common in
âII′-turns. The structural element with the highest invariability
is a γ-turn with R-ANC in the i and i + 1 position (populated
Computational Methods. A two-step method for the prediction
of the conformations was applied: a restrained distance geometry (DG)
calculation to explore the conformational space of the peptides, followed
by a restrained molecular dynamic (MD) simulation using explicit
solvent for the refinement of the structures. Upper and lower bounds
for proton distances were generated by adding or subtracting 10% of
the experimental NOE and ROE value. Coupling constants were used
directly as restraints.52
Distance geometry calculations were performed using the Disgeo
program.53 For each peptide 100 structures have been embedded into
(45) (a) Kessler, H.; Seip, S. in Two-Dimensional NMR-Spectroscopy;
Applications for Chemists and Biochemists; Croasmun, W. R., Carlson, M.
K., Eds.; VCH Puplishers: New York, 1994; pp 619-654. (b) Kessler, H.;
Schmitt, W. in Encyclopedia of Nuclear Magnetic Resonance; Grant, D.
M., Harris, R. K., Eds.; J. Wiley & Sons: New York, 1995; in press.
(46) (a) Bax, A.; Summers, M. T. J. Am. Chem. Soc. 1986, 108, 2093-
2094. (b) Bermel, W.; Wagner, K.; Griesinger, C. J. Magn. Reson. 1989,
83, 223-232. (c) Emsley, L.; Bodenhausen, G. J. Magn. Reson. 1989, 82,
211-221. (d) Kessler, H.; Schmieder, P.; Ko¨ck, M.; Kurz, M. J. Magn.
Reson. 1990, 88, 615-618.
(47) (a) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem.
Phys. 1979, 71, 4546-4553. (b) Wu¨thrich, K. In NMR of Proteins and
Nucleic Acids; Wiley & Sons: New York, 1986.
(48) (a) Bothner-By, A. A.; Stephensen, R. L.; Lee, J.; Warren, C. D.;
Jeanloz, R. W. J. Am. Chem. Soc. 1984, 106, 811-813. (b) Kessler, H.;
Griesinger, C.; Kerssebaum, R.; Wagner, K.; Ernst, R. R. J. Am. Chem.
Soc. 1987, 109, 607-609.
(49) Mueller, L. J. Magn. Reson. 1987, 72, 191-196.
(50) (a) Titman, J. J.; Neuhaus, D.; Keeler, J. J. Magn. Reson. 1989, 85,
111-131. (b) Titman, J. J.; Keeler, J. J. Magn. Reson. 1990, 89, 640-646.
(51) Kurz, M.; Schmieder, P.; Kessler, H. Angew. Chem. Int. Ed. Engl.
1991, 30, 1329-1330.
(54) (a) Havel, T. F. Biopolymers 1990, 29, 1565-1585. (b) Reference
53d.
(55) Mierke, D. F.; Kessler, H. Biopolymers 1993, 33, 1003-1017.
(56) (a) Hermans, J.; Berendsen, H. J. C.; Van Gunsteren, W. F.; Postma,
J. P. M. Biopolymers 1984, 23, 513-1518. (b) van Gunsteren, W. F.;
Berendsen, H. J. C. Groningen Molecular Simulations (GROMOS) Library
Manual. GROMOS user manual; Biomos B. V.: Nijenborgh 16 NL 9747
AG Groningen, 1987. (c) van Gunsteren, W. F.; Berendsen, H. J. C. Angew.
Chem. Int. Ed. Engl. 1990, 29, 902-1023.
(57) Mierke, D. F.; Kessler, H. J. Am. Chem. Soc. 1991, 113, 9466-
9470.
(58) (a) Pachler, K. G. R. Spectrochim. Acta 1963, 19, 2085-2092. (b)
Pachler, K. G. R. Spectrochim Acta 1964, 20, 581-587.
(52) (a) Kim, Y.; Prestegard, J. H. Proteins: Struct. Funct. Genet. 1990,
8, 377-382. (b) Mierke, D. F.; Kessler, H. Biopolymers 1992, 32, 1277-
1282. (c) Eberstadt, M.; Mierke, D. F.; Ko¨ck, M.; Kessler, H. HelV. Chim.
Acta 1992, 75, 2583-2592.
(53) (a) Havel, T. F.; Wu¨thrich, K. Bull. Math. Biol. 1984, 46, 673-
698. (b) Havel, T. F. DISGEO, Quantum Chemistry Exchange Program,
Exchange No. 507, Indiana University, 1988. (c) Crippen, G. M.; Havel,
T. F. Distance Geometry and Molecular Conformation; Research Studies
Press LTD., John Wiley & Sons: Somerset, England, 1988. (d) Havel, T.
F. Prog. Biophys. Mol. Biol. 1991, 56, 43-78.

Cyclic RGD Peptides Containing â-Turn Mimetics
J. Am. Chem. Soc., Vol. 118, No. 34, 1996 7885
Figure 3. Stereoplots of the averaged and energy-minimized conformations of c(RGD“S-ANC”) (PA1), c(RGD“R-ANC”) (PA2), c(RGD“BTD”)
(PA3), and c(RGD“spiro”) (PA4) resulting from restrained MD simulations in DMSO. Carbon and hydrogen atoms are white; nitrogen atoms are
black; oxygen atoms are gray; the sulfur atom of PA3 is dark gray.
to 98%). Another dominating structural feature is an inverse
γ-turn with Asp3 in the i + 1 position, which is found in half
of the structures during the MD simulation. As indicated by
the homonuclear 3J(HR,Hâ) coupling constants (7.6 and 6.5 Hz),
the side chain of Asp3 shows no predominant orientation.
c(RGD“BTD”), PA3. Analogous to the structure of PA1
in solution, Gly2 adopts the i + 1 position of a distorted âII′-
turn and the BTD moiety is located in the i + 3 and i + 4
positions, respectively. The RMSD value for the superposition
of the backbone CR, C′, and N atoms of PA1 and PA3 is only
33 pm. The experimental data were reproduced very well. Only
tion this dihedral angel fluctuates between the extreme values
of -150° and +110°. The coupling constants averaged over
the whole trajectory are much closer to the experimental values
than those calculated from the averaged and minimized structure.
The small temperature coefficient of the amide proton of Arg1
can be explained by the internal orientation and the resulting
sterical shielding from the solvent. The second amide proton
with a small temperature dependence is the BTD-HN, which is
involved in the hydrogen-bond forming the â-turn. The
orientation of the Asp3 side chain (ø1 ) -60° populated to 61%)
is similar to that of PA1 and Arg1 is flexible.
3
3
the J(HN,HR) and the J(HN,C′) coupling constants of Arg1
calculated from the φ-angle of the averaged and minimized
structure deviate more. During the restrained dynamic simula-
c(RGD“spiro”), PA4. The (S,S)-spiro-Pro-Leu moiety adopts
the desired i + 1 and i + 2 position of a âII′-turn and Gly2 is
in the i + 1 position of a γ-turn at the opposite side of the

7886 J. Am. Chem. Soc., Vol. 118, No. 34, 1996
Haubner et al.
Figure 4. Stereoplots of the averaged and energy minimized conformations of c(RGD“BTD”V) (PA5) and c(RaD“BTD”V) (PA6) resulting from
restrained MD simulations in DMSO. Carbon and hydrogen atoms are white; nitrogen atoms are black; oxygen atoms are gray; sulfur atoms are
dark gray.
molecule. Both hydrogen bonds are populated throughout the
whole MD simulation. Chemical shifts of the particular amide
protons show only a small temperature dependence (Table 1).
The homonuclear 3J(HR,Hâ) coupling constants of Arg1 (8.0 and
6.3 Hz) reflect the population of more than one conformation
of this side chain. Both other side chains (Asp3 and the isobutyl
side chain of the spiro moiety) adopt a rotamer with a value of
-60° for the ø1-angle. This rotamer of Asp3 is populated to a
degree of 62%. Due to the hydrophobic interaction of the δ1-
methyl groups with the neighboring ꢀ- and ú-methylene groups
within the spiro moiety, the population of this side chain rotamer
is much higher (88%). The conformation of this cyclic peptide
seems to be quite rigid as all experimental data are well-fulfilled
by the averaged and minimized structure.
an orientation with a ø1-angle of -60° is dominating (populated
to 63%).
Biological Data. The inhibitory capacities of the peptide
analogues on the binding of vitronectin and fibrinogen to the
isolated, immobilized RIIbâ3- and RVâ3-receptors were compared
with the activity of the c(RGDfV) and the linear standard peptide
GRGDSPK. Peptide analogues PA4 and PA6 show no activity
for either receptor. Peptide analogues PA1, PA2, PA3, and
PA5 show an increased activity compared to the linear standard
peptide GRGDSPK with respect to the inhibition of vitronectin
binding to the RVâ3-receptor. Only compound PA2 has a higher
inhibitory activity on vitronectin binding than the lead structure
c(RGDfV).
In contrast, peptide derivatives PA2 and PA5 reveal an
increased activity and compounds PA1 and PA3 a decreased
activity compared to GRGDSPK concerning the inhibition of
fibrinogen binding to the RIIbâ3-receptor. Similar to the effects
on the RVâ3-receptor, compound PA2 reveals a 10-fold higher
activity than the lead peptide c(RGDfV). Thus PA2 is the
derivative with the highest activities among all compounds of
this series, but possesses only little selectivity (10-fold higher
activity for inhibition of vitronectin binding to RVâ3 than for
fibrinogen binding to RIIbâ3). On the contrary, peptide analogue
PA1 is 10-fold less active in inhibiting vitronectin binding to
the RVâ3-receptor compared to c(RGDfV), but shows a 100-
fold higher inhibitory activity for vitronectin binding to RVâ3
Structures of the Cyclic Hexapeptides. c(RGD“BTD”V),
PA5, and c(RaD“BTD”V), PA6. The structures of both
peptides show a similar â/â-conformation, which is typical for
cyclic hexapeptides. BTD adopts the i + 1 and i + 2 position
of a âII′-turn, and Gly2 or D-Ala2 are located in the i + 2 position
of a âII-turn on the opposite side. In both compounds the amide
proton of Val5 shows the smallest temperature coefficient,
corresponding to a highly populated hydrogen bond. The larger
temperature coefficient of both Asp3-HN indicates that the
corresponding turn is less rigid. The side chain of Arg1 is freely
rotating in both compounds. In PA6 the Hâ protons of Asp3
are degenerated, indicating a flexible side chain, while in PA5

Cyclic RGD Peptides Containing â-Turn Mimetics
J. Am. Chem. Soc., Vol. 118, No. 34, 1996 7887
Figure 5. (A) Expected (left) and calculated (right) conformation of PA3. The characteristic difference of both conformations is focused on the
orientation of the amide bond between Asp3 and BTD4. (B) Superimposing of the RGD site of c(RGDfV) (gray) and c(RGD“BTD”) PA3 (black).
than for fibrinogen binding to RIIbâ3 and is thus the most
selective compound in this series.
According to these results the peptide derivatives can be
divided into four categories concerning the inhibition of
vitronectin binding to the RVâ3-receptor: (a) very active, but
less selective, c(RGD“R-ANC”) (PA2); (b) active and selective,
c(RGD“S-ANC”) (PA1); (c) active and less selective,
c(RGD“BTD”) (PA3) and c(RGD“BTD”V) (PA5); (d) not
active, c(RGD“spiro”) (PA4) and c(RaD“BTD”V) (PA6).
Discussion
Structural Aspects. Our results demonstrate that the BTD
moiety is not able to induce a â-turn at the desired position in
PA3. Glycine, which also prefers to adopt the i + 1 position
in â- or γ-turns, is part of the detected â-turn. The major
difference between the expected and the resulting structure is
the orientation of the amide plane adjacent to Asp3 (Figure 5).
Figure 6. The superimposing of PA1 (light gray), PA2 (gray) and
PA3 (black) show that the conformation of the RGD site is very similar
As both accompanying CR-atoms have the S-configuration, the
orientation found in DMSO may be caused by minimization of
gauche interactions as well as 1,3-allylic strain. The peptide
carbonyl oxygen has a strong tendency to be orientated parallel
to the C-H bond at the R-carbon atom of the following amino
acid (here BTD). Although the backbone structure differs from
that initially designed, the conformation of the important RGD-
binding site is similar to that in the lead compound c(RGDfV).
This is shown in the superposition of the RGD pharmacophore
of both structures in Figure 5.
A comparison of PA1 with the structure of the other
diastereomer PA2 shows only minor variations of the backbone
conformation (Figure 3 and 6). Both peptides possess a â-turn
with Gly2 in the i + 1 position. The most remarkable difference
is the orientation of the lactam bond of the ANC moiety. The
S-ANC differs from the R-ANC derivative by a 180° rotation
of this lactam bond. Thus the carbonyl group of the R-ANC
motif forms a hydrogen bond in a γ-turn, and the carbonyl group
in all three peptides.
of the S-ANC motif is orientated antiparallel to the correspond-
ing amide hydrogen. In addition, the value of the Gly2 φ-angle
deviates about 40° in both structures.
An analysis of the dihedral angles in the i + 3 and i + 4
position of ten cyclic RGD pentapeptides from our group59
which possess a âII′/γ-turn structure reveals angles of -140°
( 20° (φi+3), +100° ( 20° (ψi+3), 80° ( 15° (φi+4) and -60°
( 15° (ψi+4). The ANC moieties of PA1 and PA2 fix only
one ψ-angle of the backbone. This angle can achieve values
in the range of -140° ( 10° (PA1) or 140° ( 10° (PA2).37c
A
comparison with the ψi+3-angle in an ideal âII′/γ-conformation
(see above) shows that the R-ANC moiety fits also in this
position. In contrast, the S-ANC moiety is not suitable for an
(59) See refs 29c and 31.

7888 J. Am. Chem. Soc., Vol. 118, No. 34, 1996
Haubner et al.
Table 2. Inhibition of Fibrinogen (Fbg) Binding to the RIIbâ3-Receptor and Vitronectin (Vn) Binding to the RVâ3-Receptora
R
IIbâ3 (Fbg)
RVâ3 (Vn)
no.
peptide analogue
IC50 (µM)
Q
IC50 (µM)
Q
GRGDSPK
c(RGDfV)
1
5.0
1.6
1
8.3 × 10-1
2.8
2.2 × 10-3
4.0 × 10-2
8.5 × 10-4
2.8 × 10-1
na
6.1 × 10-3
1.1 × 10-2
2.3 × 10-4
7.3 × 10-2
PA1
PA2
PA3
PA4
PA5
PA6
c(RGD“S-ANC”)
c(RGD“R-ANC”)
c(RGD“BTD”)
c(RGD“spiro”)
c(RGD“BTD”V)
c(RaD“BTD”V)
8.5 × 10-3
4.8
4.9 × 10-3
2.7
nab
5.5 × 10-1
na
3.1 × 10-1
4.3 × 10-2
na
1.1 × 10-2
a Values are given as IC50 and as quotients Q ) IC50[peptide]/IC50[GRGDSPK]. b na, not active.
Table 3. Characterization of the Relative Orientation of the
Biologically Relevant RGD Sequence
ideal âII′/γ-conformation. A MD simulation in vacuo for 1 ns
for the BTD building block shows that the two restricted
dihedrals can reach values of -130° ( 50° (ψ) and -90° (
40° (φ′). We interpret our structural results as a balance between
the high steric strain in these cyclic pentapeptides and the
geometric restriction of the particular dihedral angles in the
corresponding turn mimetics. It seems as if the location of the
incorporated building blocks in the âII′-turn leads to an
unfavorable strain in our cyclic pentapeptides. Therefore,
R-ANC prefers the relative i + 3 and i + 4 position which also
fits the restricted ψ-angle. It has been shown that cyclic
pentapeptides are still quite flexible in the region of the γ-turn.29a
For that reason the adaptation of the S-ANC and the BTD moiety
in the i + 3 and i + 4 position might be energetically favored
over a location in a â-turn, although the particular dihedral
angles do not fit the ideal values. The spiro moiety in PA4
fixes the two consecutive dihedral angles φ (+75° ( 20°) and
ψ (-140° ( 10°).37c One of these two fixed angles is the
φ-angle in the i + 1 position. If the turn mimetic would occupy
the i + 3 and i + 4 position, not only the flexible γ-turn but
also the more rigid â-turn would be strongly distorted. There-
fore, PA4 prefers the desired âII′/γ-conformation in solution.
To test whether the BTD building block is able to induce
turns in cyclic peptides at all, two cyclic hexapeptides were
synthesized. To incorporate a turn mimetic into the sequence
of the lead compound c(RGDfV), two principal possibilities
exist: the substitution of either Phe or Val. The structure of
c(RGDF“BTD”) has already been described by Hann et al.60
This compound shows a conformation consistent with two facing
â-turns, where BTD adopts the i + 1 and i + 2 positions of
one turn and Gly2 is in i + 1 position at the opposite side. This
is the conformation one would expect for such a molecule
because the relative orientation of the turn mimetic and of the
Gly residue fits to their preferred positions in the reverse turns.
D-Amino acids and Gly exhibit a high tendency to occupy the
i + 1 position of â-turns.38 The sequences of PA5, and PA6
do not allow that the BTD moiety and Gly2 or D-Ala2,
respectively, occupy their preferred positions in two facing turns
simultaneously. The calculated structures of both cyclic
hexapeptides show one âII′-turn with the BTD building block
in the i + 1 and i + 2 position and Gly2 or D-Ala2 in the i +
2 position of a facing âII-turn. These results lead to the
conclusion that, in these cyclic hexapeptides, the turn-inducing
potential of BTD exceeds that of Gly2 and D-Ala2, respectively.
The strain in cyclic hexapeptides is not as high as in cyclic
pentapeptides. A comparison with the backbone dihedral angles
found for cyclic hexapeptides having a âII′/â-conformation31
shows that for the BTD building block only one location in the
turn is possible. The position at the side of the turn (i + 3 and
i + 4) is excluded by the restricted dihedral angles.
no.
PA1 c(RGD“S-ANC”)
PA2 c(RGD“R-ANC”) 618
peptide analogue CR/CR Câ/Câ µ(Arg)c ν(Asp)d æe
562
781
850
783
670
815
706
668
840
140
152
137
119
165
151
113
150
132
127
123
118
123
101
113
132
4
-5
10
-11
56
29
-6
13
PA3 c(RGD“BTD”)
PA4 c(RGD“spiro”)
PA5 c(RGD“BTD”V)
PA6 c(RaD“BTD”V)
c(RGDfV)
589
530
576
539
547
605
c(RGDFk)
a Distance between the CR atoms of Arg1 and Asp3, in pm. b Distance
between the Câ atoms of Arg1 and Asp3, in pm. c Angle formed by the
Câ-CR(Arg) vector and the CR(Arg)-CR(Asp) vector, in deg. d Angle
formed by the Câ-CR(Asp) vector and the CR(Asp)-CR(Arg) vector,
in deg. e Dihedral formed by the CR-Câ vectors of Arg and Asp, in
deg.
slightly distorted âII′-turn arrangement with Gly2 in the i + 1
position. These â-turn arrangements correspond with the
structures found for a group of active peptides with the sequence
c(RGDFx). In these peptides Gly2 also adopts the i + 1 position
of a âII′-turn.29c The analysis of the parameters determining
the relative orientation of the pharmacophoric groups (Arg and
Asp side chain) also shows that the ArgCR/AspCR and the
ArgCâ/AspCâ distances as well as the µ(Arg)-, ν(Asp)-, and
æ-angle (for definition see Table 3 and ref 61) are very similar
and correspond with the data found for the peptides with the
sequence c(RGDFx) (Table 3).
Despite these structural similarities at the RGD site, the
peptide analogues reveal very different biological activities. The
activity increases in the series PA3 < PA1 < PA2 (Table 2).
There are two possible explanations: (a) structural differences
in the region flanking the RGD sequence or (b) the calculated
conformation of the active site in solution is not in accordance
with the receptor bond conformation, and the three peptide
analogues may possess different abilities to assume conforma-
tional transitions to fit the receptor.
Structure-activity investigations on a large number of cyclic
pentapeptides of the sequence c(RGDXY) showed that a
hydrophobic amino acid in position 4 increases the activity.29c
Therefore, the low activity of PA3 could be explained by the
missing hydrophobic side chain in the region of the carbon
atoms C4 and C5 of the BTD motif, which corresponds with
position 4 in the cyclic RGD-containing pentapeptides. But the
active peptide analogues PA1 and PA2 with the ANC moiety
do not possess a hydrophobic side chain in this region, too.
Thus, the different activities of these three peptide analogues
may result from different possibilities to fit the active site of
the receptor. The different fitting abilities are based on the
(60) Hann, M. M.; Carter, B.; Kitchin, J.; Ward, P.; Pipe, A.; Broomhead,
J.; Hornby, E.; Forster, M.; Perry, C. In Molecular Recognition: Chemical
and Biochemical Problems II; Roberts, S. M., Ed.; Royal Society of
Chemistry: Cambridge 1992; pp 145-160.
(61) Mu¨ller, G.; Gurrath, M.; Kessler, H. J. Comput.-Aided Mol. Design
1994, 8, 709-730.
Structure-Activity Relationship. In PA1, PA2, and PA3
the region of the biological relevant RGD sequence has a very
similar conformation (Figure 6). All three analogues show a

Cyclic RGD Peptides Containing â-Turn Mimetics
J. Am. Chem. Soc., Vol. 118, No. 34, 1996 7889
Figure 7. Superimposing of c(RGDFk) (gray) and PA5 (black; left side) and c(RGDfVG) and c(RGDfLG) (both gray) and PA5 (black; right side).
increasing flexibility of the peptide analogues. Compound PA3
with the BTD motif fixes two backbone dihedral angles which
leads, according to the design, to a more rigid peptide derivative
than the homodetic cyclic pentapeptides. This can be seen, for
example, in well-separated temperature coefficients (Table 1).
The ANC motif in peptide analogues PA1 and PA2 fixes only
one backbone dihedral angle and, therefore, reduces the flex-
ibility of the peptides to a smaller extent. PA2 shows the highest
flexibility, which is indicated, for example, via less separated
temperature coefficients (Table 1) and a lower convergence of
the generated DG structures.
poration of the RGD sequence in cyclic pentapeptides or peptide
analogues which reduce the torsional freedom of the æ-angle
in a large amount61 still results in compounds with highly
increased activity for the RVâ3-integrin compared to the linear
standard peptide GRGDSPK.62 Thus, often a balance between
conformational restrictions and flexibility is the better approach
to develop highly active compounds, but does not lead to a better
understanding of the ligand-receptor interactions.
It is not surprising that the RaD-containing PA6 shows no
activity, as earlier investigations63 revealed that the replacement
of Gly with D-Ala leads to a drastic loss of activity. But more
interestingly, PA5 shows an activity in inhibiting the binding
of vitronectin to the RVâ3-receptor in the same range as
homodetic cyclic pentapeptides of the sequence c(RGDFx)29c
and is hence about 10-fold more active than hexapeptides with
analogous conformations31b like c(RGDfVG) and c(RGDfLG).
One explanation for this result could be that the BTD moiety
in peptide analogue PA5 is able to replace the hydrophobic side
chain of the comparably active c(RGDFk) (Figure 7) and can
interact with the same hydrophobic pocket of the receptor. By
contrast, the side chain of the phenylalanine of c(RGDfVG) and
c(RGDfLG) is too far away to adopt the same position as in
c(RGDFk) (Figure 7).
The only difference of peptide analogues PA1 and PA2 is
focused on the orientation of the carbonyl group of the ANC
moiety (see also above). Besides the high flexibility of PA2,
this different orientation of the carbonyl group could be a reason
for the extreme high activity of compound PA2, because the
carbonyl oxygen could possibly interact as a hydrogen-bond
acceptor with the receptor.
The peptide analogue PA4 shows no activity in the limits of
the test system. This is somewhat surprising as the structure
of this compound reveals the desired âII′/γ-turn arrangement
with the turn mimetic in the i + 1 and i + 2 positions of the
âII′-turn and Gly2 in the i + 1 position of the γ-turn. Especially
the region of the RGD sequence is very similar to the
conformation of the active site in the lead structure c(RGDfV),
which by contrast possesses a very high activity (Table 2). This
drastic loss of activity can be explained by the missing
hydrophobic side chain in the region of the spiro compound in
combination with the absence of an amide proton at the peptide
bond between Asp3 and the turn motif. In recent investigations
this amide proton was found to be essential for high activity.29c
In addition, the introduction of the turn mimetic to reduce the
flexibility could prevent the matching of the RGD site with the
receptor.
Conclusion
The conformational analysis of peptide analogues PA1-PA4
(pentapeptide derivatives) shows that only the incorporation of
the spiro moiety in c(RGD“spiro”) results in the desired âII′/
γ-turn arrangement with the turn motif in the âII′-turn. The
other three peptide analogues with the S- and R-ANC as well
as the BTD moiety reveal conformations in which Gly2 adopts
the i + 1 position of the âII′-turn while the turn motif is shifted
in the i + 3 and i + 4 position. These unexpected results may
be due to the adaptability of the pentapeptide system, which
allows the turn mimetics to be located in other positions.
Especially, the γ-turn region is very flexible and is therefore
predestinated to match the structural demands of the turn
mimetics.
Altogether, these results suggest that the receptor-bound
conformation of the RGD sequence differs from the structure
found in solution for peptide analogues PA1, PA2, PA3, and
PA4 and that the different analogues possess different abilities
to match the receptor due to their different flexibilities.
Moreover, these findings show again that a reduction in
flexibility can prevent activity when the pharmacophores are
fixed in a mismatched conformation. Nevertheless, the incor-
The structures of the pseudo-hexapeptide derivatives PA5 and
PA6 exhibit a âII′/âII-turn arrangement with the BTD moiety
(62) See refs 27c, 28 and 29.
(63) See refs 31a and 33.

7890 J. Am. Chem. Soc., Vol. 118, No. 34, 1996
Haubner et al.
added 1.35 mL of hydrazine hydrate. The reaction mixture was stirred
for 24 h at ambient temperature, filtered, and washed with 95% ethanol.
The filtrate was evaporated to dryness under reduced pressure: yield
1.3 g (96%); mp ) 265 °C; TLC Rf(BAW) ) 0.83; HPLC K′ ) 3.79
in the desired i + 1 and i + 2 position of the âII′-turn. These
results suggest that the turn motif adopts the position for which
it was designed in the hexapeptide system, even if there is a
competition with a likewise turn-inducing D-amino acid which
is shifted into the unfavorable i + 2 position of the â-turn.
Structure-activity investigations show inhomogenous results.
Whereas in peptide analogues PA1, PA2, and PA3 the
conformation of the active RGD site is very similar they reveal
very different activities concerning the inhibition of binding of
vitronectin to the RVâ3-receptor. In addition, peptide analogue
PA4, which possesses a âII′/γ-turn conformation similar to the
highly active lead structure c(RGDfV), shows no activity on
this receptor. These findings suggest that the structures
determined for the peptide analogues examined in solution may
differ from the receptor bound conformation, which is probably
adopted upon binding via an induced fit. The different activities
could be explained with different possibilities for conformational
transitions which allow the distinct peptide analogues to match
the receptor in different ways. These fitting abilities are
determined by the decreasing flexibility induced by the single
turn motives. Moreover, the drastic loss of activity of PA4
confirms recent findings29c which postulate that the proton at
the amide bond between Asp3 and the following residue is
essential for high activity.
1
(10-60% ACN; 30 min); H-NMR (DMSO-d6) δ 8.30 (s, 3H), 4.95
(dd, 1H), 4.85 (dd, 1H), 4.00 (m, 1H), 3.45 (dd, 1H), 3.10 (dd, 1H),
1.80-2.40 (m, 4H); FAB-MS [M + H]+ ) 217.
Fmoc-BTD-OH. To a stirred solution of 1.27 g (5.9 mmol) of
H-BTD-OH in 20 mL of water was added 2.0 g (5.93 mmol) of Fmoc-
ONSu dissolved in 20 mL of acetonitrile. The pH was maintained at
pH 8.0 using triethylamine. The solution was stirred for 30 min at
ambient temperature. The mixture was filtered and concentrated in
Vacuo. The residue was added with rapid stirring to 100 mL of 1.5 N
HCl. The crystallizing product was collected by filtration, washed with
water, and dried in Vacuo: yield 1.50 g (57%); mp ) 210 °C; TLC
1
Rf(AW) ) 0.63; HPLC K′ ) 4.83 (30-90% ACN; 30 min); H-NMR
(DMSO-d6) δ 7.87 (d, 2H), 7.67 (m, 3H), 7.28-7.43 (m, 4H), 4.85-
4.96 (m, 2H), 4.22-4.29 (m, 3H), 4.09 (dd, 1H), 3.39 (dd, 1H), 3.05
(dd, 1H), 1.79-1.97 (m, 4H); FAB-MS [M + H]+ ) 439.
Synthesis of PA1, PA2, and PA4: Peptide Synthesis. Fmoc-Gly-
Tentagel S PHB was used in the 9050 peptide synthesizer (Milligen,
Eschborn, Germany). Protected amino acids (Fmoc-Arg(Mtr)-OH,
Fmoc-Asp(OtBu)-OH) and building blocks (PA1, Fmoc-(S)-Gly[ANC-
2]Leu-OH; PA2, Fmoc-(R)-Gly[ANC-2]Leu-OH; PA4, Fmoc-(S,S)-
spiro-Pro-Leu-OH) were used in 4-fold excess. The synthetic protocol
was essentially as described by the manufacturer.
Cleavage. After completion of the coupling cycles and deprotection
of the Fmoc group of the Asp residue, the peptide was cleaved from
the resin as described previously.66 The linear protected peptides were
cyclized without purification.
Cyclization. The linear peptide was dissolved in CH2Cl2/DMF (1
mg/mL) and cooled to -10 °C. One equivalent of N-methylmorpholine
and DMAP and 2 equiv of EDCI‚HCl were added successively. The
solution was stirred for 2 h at -10 °C and overnight at room
temperature. After evaporation, 2-3 mL of DMF was added and the
concentrated solution was poured into water. The precipitate was
sucked off.
Side Chain Deprotection. Deprotection of the Mtr group at arginine
and the tert-butyl ester of asparagine was performed with TFA/10%
thioanisole.
Purification. Final purification was achieved by preparative RP-
HPLC using a Prepbar Lichrosorb RP18 (10 µm, 250 × 50 mm)
column. The gradient was 5 min 5% B in 50 min to 75% B [(A) 0.1%
TFA, (B) 0.1% TFA in water/acetonitrile 1:9]. All peptides were >95%
pure.
Although these investigations could not improve our under-
standing of ligand-receptor interactions at the RVâ3-integrin,
incorporation of the R-ANC moiety into the RGD peptide led
to one of the compounds most active in inhibiting the binding
of vitronectin to the RVâ3-receptor. This peptide analogue could
be a prospective candidate for further drug developments
targeting the interactions of integrin essential during osteoporo-
sis, angiogenesis, and tumor metastasis.
Experimental Section
Materials and Methods. All chemicals were used as supplied
without further purification. Apart from N-methylpyrrolidone (NMP),
all organic solvents were distilled before use. Fmoc amino acids were
purchased from Bachem (Heidelberg, Germany) and Novabiochem (Bad
Soden, Germany), the Fmoc-(S,S)-spiro-Pro-Leu-OH was from Neo-
systems (Strasbourg, France), cTrt resin was from either Novabiochem
or CBL Patras (Patras, Greece), Fmoc-Gly-Tentagel S PHB was from
Rapp Polymers (Tu¨bingen, Germany), and TBTU was from Richelieu
Biotechnologies (Montreal, Canada). HOBt was synthesized using the
route described by Ko¨nig and Geiger.64
Solvent systems for TLC were acetonitrile/water 4:1 (AW), n-
butanol/acetic acid/water 2:1:1 (BAW), and chloroform/methanol/acetic
acid 85:10:5 (CMA). FAB mass spectra were obtained by a Varian
MAT 311 A or a Vacuum Generator VG 70-250SE mass spectrometer
using nitrobenzyl alcohol or glycol matrices.
Analytical reverse phase HPLC was performed using columns with
Macherey-Nagel Nucleosil C-18 packing (5 µm, 250 × 4 mm) or a
Lichrosorb RP18 (5 µm, 250 × 4 mm) column. For analytical data,
given as K′, several acetonitrile (ACN)/0.1% aqueous trifluoroacetic
acid gradients are used (see supporting information).
Synthesis of the BTD Moiety: Phth-BTD-OH. To 2.30 g (6.1
mmol) of Phth-BTD-OEt65 in 130 mL of acetic acid, was added 30
mL of concentrated HCl and the solution was allowed to reflux for 90
min. The solvent was evaporated at room temperature, and the product
was obtained as a white solid: yield 2.12 g (99%) (95% pure according
to HPLC); HPLC K′ ) 5.97 (20-80% ACN; 30 min); 1H-NMR (DMSO-
d6) δ 7.85-7.93 (m, 4H), 5.05 (dd, 1H), 4.95 (dd, 1H), 4.80 (dd, 1H),
3.45 (dd, 1H), 3.10 (dd, 1H), 1.90-2.50 (m, 4H); FAB-MS [M + H]+
) 347.
Synthesis of PA3, PA5, PA6: Loading of the cTrt Resin. A 0.7
g portion of cTrt resin suspended in 10 mL of dry DCM was treated
with 0.74 g (2.5 mmol) of Fmoc-Gly-OH and 400 µL of DIEA for 1
h. After adding another 400 µL of DIEA and 3 mL of methanol the
mixture was shaken further 15 min. The solution was removed and
the resin was washed several times with DMF (2×), DCM (5×),
2-propanol (2×), methanol (5×), and ether (2×).
Peptide Synthesis. Starting with 0.8-0.9 g of Fmoc-Gly-cTrt resin
(substitution about 0.55-1.10 mmol amino acid/g resin), the synthesis
was carried out using standard Fmoc coupling protocols. Deprotection
of the N-terminal Fmoc group was accomplished using 20% piperidine
in DMF. Coupling of the amino acids or amino acid derivative was
carried out using 2.5 equiv of the appropriate amino acid or 1.5 equiv
of amino acid derivative, 2 equiv of TBTU, 2.5 equiv of HOBt, and
3-5 equiv of DIEA in 20 mL of NMP. Coupling times between 30
and 60 min provide complete couplings. Coupling reactions were
monitored by the ninhydrin test.67
Acetic Acid Cleavage. The resin bound peptide analogue was
treated with 20 mL of a mixture of acetic acid, TFE, and DCM (1:1:3)
for 1 h at ambient temperature. The resin was washed twice with 20
mL of the mixture mentioned above. The combined solution was
evaporated in Vacuo and triturated with ether, filtered, and washed three
times with ether.
H-BTD-OH. To a stirred solution of 2.12 g (6.1 mmol) of Phth-
BTD-OH in 15 mL of chloroform and 170 mL of 95% ethanol was
(66) (a) Anwer, M. K.; Spatola, A. F.; Bossinger, C. D.; Flanigan, E.;
Liu, R. C.; Olsen, D. B.; Stevenson, D. J. Org. Chem. 1983, 48, 3505-
3507. (b) Ho¨lzemann, G.; Lo¨w, A.; Harting, J.; Greiner, H. E. Int. J. Pept.
Protein Res. 1994, 44, 105-111.
(64) Ko¨nig, W.; Geiger, G. Chem. Ber. 1970, 103, 788-798.
(65) The synthesis of Phth-BTD-OEt follows the scheme of Bach et al.
(see ref 37e) and was slightly modified using EDCI‚HCl as coupling reagent
in the synthesis of the ethyltrimethylsilyl protected glutamic acid.
(67) Troll, W.; Cannan, R. K. J. Biol. Chem. 1953, 200, 803-811.

Cyclic RGD Peptides Containing â-Turn Mimetics
J. Am. Chem. Soc., Vol. 118, No. 34, 1996 7891
Cyclization. The peptide analogue was dissolved in DMF (con-
centration 5 × 10-3 mol/L), 5 equiv of NaHCO3 and 3 equiv of DPPA
were added, and the solution was stirred at ambient temperature for 24
h. After filtration of the solid NaHCO3, DMF was evaporated in Vacuo
and the residue was triturated with water, filtered, and washed with
water and ether.
Briefly, human placenta was extracted with octyl â-D-glucopyranoside
(OG) at 4 °C. The extract was cleared by centrifugation and circulated
over an LM609 antibody column72 and specifically bound material was
eluted at pH 3.1. The eluant was neutralized, dialyzed against NP-40
(0.1% in PBS), concentrated to >1 mg/mL, and stored at -70 °C.
R
IIbâ3-integrin was prepared from human platelets73 with modifica-
tions.71 Briefly, platelets were extracted with OG (50 mM). The extract
was circulated over a linear GRGDSPK-conjugated CL-4B Sepharose
column, and specifically bound material was eluted with linear
GRGDSPK. The eluant was dialyzed against NP-40 (0.1% in PBS),
concentrated to >1 mg/mL, and stored at -70 °C.
Both preparations were ∼95% pure as judged by anti-integrin ELISA
using R- and â-chain specific monoclonal antibodies and by SDS-
PAGE.
Side Chain Deprotection. The cyclic peptide analogue was treated
with 20 mL of a solution of 85.5% TFA, 5% phenol, 2% water, 5%
thioanisole and 2.5% ethanedithiol for 24 h at ambient temperature.
The mixture was filtered if necessary, evaporated in Vacuo, triturated
with ether, filtered, and washed several times with ether.
Purification. The crude, cyclic peptide analogues were purified by
RP-HPLC using a 250 mm × 21 mm column containing a Nucleosil
C-18 packing (7 µm, 100 Å pore size). Eluation from the column is
done with several linear acetonitrile/0.1% aqueous TFA gradients. All
peptides were >95% pure.
Isolated Integrin Binding Assays. Inhibitory effects of cyclic
peptides were quantified by measuring their effect on the interactions
between immobilized integrin and biotinylated soluble ligands. Purified
vitronectin or fibrinogen (1 mg/mL; pH 8.2) was biotinylated with
N-hydroxysuccinimidobiotin (100 µg/mL; 1 h, 20 °C), before dialysis
into PBS. Ninety-six-well mictrotiter plates were coated with 1 µg/
mL of purified integrin (1 h; 4 °C), blocked with BSA (1% in PBS),
and incubated [3 h at 30 µg/mL in binding buffer (0.1% BSA, 1 mM
CaCl2, 1 mM MgCl2, 10 µM MnCl2, 100 mM NaCl, 50 mM
tris(hydroxymethyl)aminomethane; pH 7.4)] in the presence or absence
of serially diluted peptides. After washing (3 × 5 min with binding
buffer), bound biotinylated ligand was detected with alkaline-phos-
phatase conjugated to goat-anti-biotin antibodies (1 µg/mL; 1 h, 37
°C), using nitro blue tetrazolium-bromochloroindolyl phosphate as
chromogen. Vitronectin binding in the absence of competitor was
defined as 100% signal, binding to blocked wells in the absence of
integrin was defined as 0%. The signal-to-noise ratio was >10.
Concentrations of peptides required for 50% inhibition of signal (IC50
values) were estimated graphically. The linear heptapeptide GRGDSPK
was included as external reference, and IC50 values within a given
experiment were normalized to the reference IC50 to give a value, ‘Q’,
which allowed IC50 between experiments to be compared.
NMR Spectroscopy. All NMR spectra were recorded on a Bruker
AMX 500 spectrometer and processed on an Aspect X32 station with
the UXNMR software (Bruker). Measurements were performed using
20 mM solutions of the peptides in DMSO-d6 at 300 K sealed under
Vacuo after three pump-and-freeze cycles. The TOCSY spectrum for
PA1 was recorded with 2048 data points in the direct dimension and
512 experiments using an 80 ms MLEV-17 spinlock. It was processed
by applying a π/2-shifted squared sine bell window function in both
dimensions and zero-filling in F1 to a final size of 2048 × 11024 data
points. For the other peptides, z-filtered TOCSY spectra with an 80
ms DIPSI spinlock and 11 different variable delays for each experiment
were recorded with 8192 data points in the direct dimension and 512
experiments using TPPI. After application of a squared sine bell
function, shifted by π/2, they were zero-filled to a final size of 8192
× 1024 points. For P.E.COSY experiments (8192 × 512 data points)
a 37° reading pulse was applied. After the subtraction of the one-
dimensional reference spectrum and multiplication with a π/2-shifted
squared sine bell, the spectra were zero-filled in F1 to a size of 8192
× 1024 data points. The heteronuclear HMQC spectra (1024 × 512
points) have been recorded using a BIRD pulse to suppress protons
bound to 12C with a recovery delay of 198 ms. HMQC-TOCSY
experiments were measured under the same conditions but with an
additional MLEV-17 spinlock with a mixing time of 80 ms. HMBC
spectra (8192 × 384 data points) with a delay of 70 ms for the evolution
of the heteronuclear long-range coupling have been folded in the F1-
dimension to increase the resolution. The first increment was adjusted
to obtain a phase correction of 180° (zero-order) and -360° (first-
order) in the indirect dimension. For the heteronuclear spectra, a
squared sine bell shifted by π/2 was applied in F2 before Fourier
transformation to a final size of 2048 × 512. For compounds PA5
and PA6 HETLOC spectra were recorded with 8192 data points in F2
and 512 experiments using a mixing time of 35 ms with the MLEV-17
spinlock. Prior to the Fourier transformation a π/2-shifted squared sine
bell window function was applied. NOESY spectra for PA1 and PA3
were recorded with mixing times of 150 ms, and compensated ROESY
spectra were recorded for the other peptides with a mixing time of 200
ms using a pulsed spinlock with 3 kHz (4096 × 512 data points). After
processing (π/2-shifted squared sine bell, zerofilling in F1 to 1024
points) the volume integrals of the crosspeaks were measured using
the integration subroutine of the UXNMR-program (Bruker). Distances
have been calculated using the isolated two-spin approximation with
reference to the distance between two geminal protons set to 178 pm.
For the volume integral values of the compensated ROESY spectra an
offset correction was performed.
Acknowledgment. Financial support by the Deutsche For-
schungsgemeinschaft and the Fonds der Chemischen Industrie
is gratefully acknowledged. The authors thank M. Nowee, H.
Kraft, and D. Hahn for excellent technical support; H. Mu¨ller
for contributing the FAB-mass spectra; and M. Kranawetter,
B. Cordes, and M. Wurl for HPLC separations.
Supporting Information Available: Additional tables with
1H- and 13C-chemical shift data for all peptide analogues,
selected homonuclear and heteronuclear 3J coupling constants,
comparisons between experimentally determined and simulated
NOE-derived distances of all peptide analogues, and analytical
data such as FAB masses, HPLC retention times, and yields of
the peptide analogues (17 pages). See any current masthead
page for ordering and Internet acess instructions.
JA9608757
(68) Yatohgo, T.; Izumi, M.; Kashiwagi, H.; Hayashi, M. Cell Struct.
Funct. 1988, 13, 281-292.
(69) Kazal, L. A.; Ansel, S.; Miller, O. P.; Tocantins, L. M. Proc. Soc.
Exp. Biol. Med. 1963, 113, 989-994.
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(72) Cheresh, D. A.; Spiro, R. C. J. Biol. Chem. 1987, 262, 17703-
Biological Assay: Protein Purification. Human plasma vitro-
nectin68 and fibrinogen69 were purified as described previously. RVâ3-
integrin was purified from human placenta70 with modifications.71
17711.
(73) Pytela, R.; Pierschbacher, M. D.; Ginsberg, M. H.; Plow, E. F.;
Ruoslahti, E. Science 1986, 231, 1559-1562.