Radio-analytical determination of the coating efficiency of cyclic RGD peptides

Article abstract of DOI:10.1002/hlca.200690085

Coating of artificial surfaces with ROD (=arginine-glycine-aspartate) peptides to enhance cell adhesion is an ongoing issue. Thereby, the physiological adhesion process to the extra-cellular matrix (ECM) is mimicked by the peptide coating, leading to a st

Full text of DOI:10.1002/hlca.200690085

Helvetica Chimica Acta – Vol. 89 (2006)
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Radio-Analytical Determination of the Coating Efficiency of Cyclic RGD
Peptides
by Jçrg Auernheimera), Roland Haubnerb)1), Margret Schotteliusb), Hans-Jürgen Westerb), and Horst
Kessler*a)
a) Department Chemie, Lehrstuhl II für Organische Chemie, Technische Universität München,
Lichtenbergstr. 4, D-85747 Garching (phone: 0049-89-289-13300; fax: 0049-89-289-13210;
e-mail: horst.kessler@ch.tum.de)
b) Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Univerität München,
Ismaninger Strasse 22, D-81675 München
Coating of artificial surfaces with RGD (=arginine-glycine-aspartate) peptides to enhance cell adhe-
sion is an ongoing issue. Thereby, the physiological adhesion process to the extra-cellular matrix (ECM)
is mimicked by the peptide coating, leading to a strong cell-surface contact, followed by spreading and
proliferation of the cells. For comparable cell adhesion studies, it is important to know the density of
the RGD peptides on the surface. Here, we present an approach to determine the amount of bound cyclic
RGD peptide by radio labeling with 125I of a tyrosine-containing RGD peptide on different materials sur-
faces (poly(methyl methacrylate) (PMMA), titanium, and silicon). For all surfaces, the amount of bound
peptides is in the range of pmol/cm2.
Introduction. – Coating of artificial surfaces with cell-adhesive molecules provides a
strong mechanical contact between cells and surface. Cell adhesion is mediated by
integrins [1], a class of heterodimeric transmembrane receptors that bind selectively
to different proteins of the extracellular matrix (ECM) [2]. Cellular binding sites,
like the arginine-glycine-aspartate (RGD) sequence, have been reported to play a
major role in mediating cell adhesion between extracellular matrix proteins and integ-
rins [3]. These interactions allow inside out as well as outside in signal transduction.
Many applications of coatings using cyclic RGD pentapeptides based on cyclo-
(-RGDfK-) have been reported by us and others [4]. The cyclic peptide binds with
high activity and selectivity to avb3 and avb5 integrins [5]. The av integrins are
known to adhere to vitronectin, a RGD-containing extracellular matrix protein.
Only the avb3 integrin is found in focal contacts and leads to spreading and migration
of (endothelial) cells on vitronectin [6]. Surfaces coated with cyclic RGD peptides of
the type cyclo(-RGDfK-) can be used in implantation medicine or for studying the
behavior of cells attached to surfaces. One major problem of these coatings was that
the amount of surface-bound RGD peptide could not be determined exactly. This prob-
lem is now solved by the radio-labeling approach presented here.
1
)
Current address: Universitätsklinik für Nuklearmedizin, Medizinische Universität Innsbruck,
Anichstrasse 35, A-6020 Innsbruck.
ꢀ 2006 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 89 (2006)
Results and Discussion. – To implement the radioactivity an approbate site for
labeling had to be introduced into the peptide structure. As phenols can be smoothly
labeled with 125I (t1/2 =59.4 d, 81.3 TBq/mmol) by the IodoGenꢁ (=1.3,4.6-tetra-
chloro-3a,6a-diphrenylglycoluril) method [7], we replaced the D-Phe in the cyclic pen-
tapeptide with D-Tyr and used it for labeling (Scheme 1). The linear peptide
H-D(OtBu)y(tBu)K(Z)R(Pbf)G-OH (1; Pbf=2,2,4,6,7-pentamethyldihydrobenzo-
furan-5-sulfonyl) was synthesized on trityl chloride polystyrol (TCP) resin [8] according
to the Fmoc strategy [9]. After cyclization and deprotection of the lysine side chain, the
peptide 2 was coupled with three different linker systems for three different surface
types – an acryloyl anchor for poly(methyl methacrylate) (PMMA; 3) [4a], a pentenoyl
anchor for silicon (4) [4c], and a phosphonate anchor for titanium (5) [4e]. Thereafter,
the peptides were deprotected with CF3COOH (TFA)/H2O/(i-Pr)3ACTHERSUNG iH (TIPS) and puri-
fied by HPLC. The resulting compounds, 6–8, were labeled with 125I and purified by
HPLC again (9–11; Scheme 2).
The corresponding 127I-containing reference compounds, 9C–11C, which are also
used to characterize the radioactive compounds and to modify their specific activity,
Scheme 1. Exchange of D-Phe through D-Tyr in the avb3-Selective Cyclic RGD Peptide cyclo-
(-RGDfK-) Enables the Radio-Labeling with 125I of the Surface-Adhesive Peptide.

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Helvetica Chimica Acta – Vol. 89 (2006)
were synthesized according to a reaction scheme, which needed some modifications
because of the unprotected side chain of the Fmoc-D-3-iodotyrosine [10]. For the syn-
thesis of the linear peptide H-K(ivDde)R(Pbf)GD(OtBu)y{I}-OH (12), Fmoc-D-3-
iodotyrosine was attached to the TCP resin. Solid-state peptide synthesis (SPPS) was
performed according to the standard coupling protocols. The cyclization of 12 was car-
ried out using [(1H-benzotriazol-1-yl)-oxy]tris(pyrrolidin-1-yl)phosphonium hexa-
fluorophosphate (PyBOP) instead of diphenylphosphoryl azide (DPPA), because the
use of DPPA led to quantitative formation of a diphenylphosphoric acid ester on the
unprotected tyrosine OH group. Lysine side chain had to be protected with the 4,4-
dimethyl-2,6-dioxocyclohex-1-ylidene (ivDde) group [11] instead of the (benzyloxy)-
carbonyl (Z) group, because its hydrogenolytical removal led also to a removal of
the iodine from the tyrosine. The resulting peptide 13 was then coupled with the
anchors 3–5. Deprotection with TFA/H2O/TIPS, followed by HPLC purification led
to the compounds 9C–11C (Scheme 3).
For the measurement of the amount of surface bound peptide, a reasonable specific
activity was adjusted by mixing the radioactive compounds 9–11 with their correspond-
ing non-radioactive analogs 9C–11C. The different surface types poly(methyl metha-
crylate) (PMMA), silicon, and titanium were coated using these mixtures according
to the protocols published previously [4a,c,e]. Briefly, 9/9C was coated on PMMA
discs (Ø 6 mm, 10 ml per disc) through UV irradiation, silicon was coated with 10/
10C in toluene at 808, and 11/11C was immobilized on titanium discs (Ø 1 cm, 300 ml
per disc) from a phosphate-buffered saline (PBS) solution. After rinsing all probes
thoroughly, the activity was detected in a g-counter.
The percentage of bound peptide was calculated using the ratio of bound activity
compared to the activity in the corresponding coating solution (same volume as used
for one disc; Table). The percentage of bound immobilized activity is considered to
be also the percentage of immobilized peptide; therefore, the amount of immobilized
peptide can be calculated from the amount of peptide applied to each sample. The
amount of bound peptide depends on the material and the concentration of the peptide
in the coating solution used, but is in a pmol/cm2 range for all surfaces examined
(Table). For titanium, the highest amount (278 pmol/cm2) could be reached.
Conclusions. – Replacment of D-Phe in the cyclic RGD pentapeptide with D-3-[125I]-
iodotyrosine resulted in a simple but efficient method to determine the amount of sur-
face bound peptide. The only limitation of this approach is that the anchoring group
used must be stable under the conditions of the radioiodination with IodoGenꢁ.
Experimental Part
General. Amino acids and coupling reagents were purchased from IRIS Biotech (D-Marktredwitz),
Trityl chloride polystyrol (TCP) resin from Pepchem (D-Tübingen), 3-(dibenzylphosphono)propionic
acid (DBPPA) from Novabiochem (D-Schwalbach). All other chemicals were purchased from Aldrich,
Sigma, or Fluka. NMR Spectra: Bruker DMX-250. ESI-MS measurements were performed on a Finnigan
LCQ instrument.
Fmoc-D-3-iodo-tyrosine (Fmoc-D-Tyr{I}-OH). This compound was synthesized according to Haub-
1
ner et al. [10]. M.p. 134–1378. H-NMR (DMSO, 500 MHz): 12.50 (br. s, COOH); 10.12 (s, NH); 7.87

Helvetica Chimica Acta – Vol. 89 (2006)
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Helvetica Chimica Acta – Vol. 89 (2006)
Table. Amount of Surface-Bound RGD Peptide on Different Materials. The mean value is the result of
triplicate determinations.
Surface
Compound Applied amount Ratio
Activity of Activity of Percentage Amount of
of peptide per
probe
[nmol]
cold/hot coating sol- coated
of bound
peptide
[%]
bound pep-
tide
ution
sample
[”106 Cpm] [”106
Cpm]
[pmol/cm2]
PMMA
discs Ø 6
mm
9
0.010
0.10
1.0
1.0 mg/mla)
0.035
0.36
500 :1
625 :1
500 :1
0.077
0.77
9.77
0.017
0.14
0.35
–b)
0.000442
0.00298
0.0121
0.194
22.92
17.95
3.58
–b)
1.92
0.97
0.39
1.33
8.12Æ1.23
63.6Æ6.27
127Æ32.1
138Æ23.9
0.47Æ0.23
2.47Æ0.59
9.92Æ0.89
310Æ85
Silicon
10
12800 :1 307
Titanium 11
discs Ø 1
cm
6.3 :1
4.7:1
4.7:1
0.0231
0.308
3.13
3.6
32
10.1:1 14.5
b
a) Same solution (940 ml) was used for all samples. ) In the case of silicon samples with different areas
were used; therefore, mean values can not be given.
(d, J=7.5, 2 H, FmocCH); 7.69 (d, J=8.5, 1 H, FmocCH); 7.65–7.59 (m, 3 H, PheCH); 7.39 (m, 2 H,
FmocCH); 7.30 (m, 2 H, FmocCH); 4.20–4.15 (m, 3 H, CHCH2O); 4.08 (m, Ha); 2.94 (dd, Hb); 2.72
(dd, Hb). ESI-MS: 308.0 ([M+H])+).
H-D(OtBu)y(tBu)K(Z)R(Pbf)G-OH (1). The peptide was synthesized by solid-phase peptide syn-
thesis (SPPS) on TCP resin [8] according to the Fmoc strategy [9]. The resin was loaded with Fmoc-
Gly-OH. Subsequently, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Z)-OH, Fmoc-D-Tyr(tBu)-OH, and Fmoc-
Asp(OtBu) (Pbf=2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) were coupled with O-(1H-benzo-
triazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), 1-hydroxy-1H-benzotriazole
(HOBt) in twofold excess and EtN(i-Pr)2 as base and 1-methylpyrrolidin-2-one (NMP) as solvent. The
Fmoc deprotection between the couplings and at the end of the sequence was performed with 20% piper-
idine in NMP. The peptide was cleaved from the resin by treatment with AcOH/2,2,2-trifluoroethanol/
t
CH2Cl2 3 :1:6. ESI-MS: 1024.5 ([MÀ2tBu+H])+), 1080.6 ([MÀ Bu+H])+), 1136.5 ([M+H])+), 1158.5
([M+Na])+).
Cyclo(-R(Pbf)GD(OtBu)y(tBu)K-) (2). Cyclization was performed via in situ activation with
3 equiv. diphenoxyphosphoryl azide (DPPA) and with 5 equiv. NaHCO3 as solid base under high-dilution
conditions (5 mM) in DMF for 16 h. Removal of the Z group was achieved by roughly stirring of a soln. in
t
N,N-dimethylacetamide with Pd/C as catalyst under H2 for 24 h. ESI-MS: 928.5 ([MÀ Bu+2 H]+), 984.4
([M+H]+), 1006.4 ([M+Na]+).
Acryloyl-Ahx-Ahx-OH (3). This compound was synthesized according to Pless et al. [12]. 1H-NMR
(DMSO, 250 MHz): 8.03 (m, NH); 7.70 (m, NH); 6.16 (dd, J=10, 17, 1 H, CH2=CH); 6.03 (dd, J=2.5, 17,
1 H, CH2=CH); 5.53 (dd, J=2.5, 10, 1 H, CH2=CH); 3.15–2.95 (m, 2 NCH2); 2.17 (t, J=7, CH2COOH);
2.02 (t, J=7, CH2CON); 1.55–1.15 (m, 2 (CH2)3). ESI-MS: 299.3 ([M+H]+), 321.3 ([M+Na]+).
Pent-4-enoyl-Ahx-Ahx-Ahx-OH (4). TCP Resin was loaded with Fmoc-Ahx-OH, followed by subse-
quent coupling of Fmoc-Ahx-OH, Fmoc-Ahx-OH, and pent-4-enoic acid. The linker was cleaved from
1
the resin with 50% TFA in CH2Cl2. H-NMR ((D6)DMSO, 250 MHz): 7.70 (m, 3 NH); 5.76 (m, 1 H,
CH2=CH); 4.97 (dd, J=19.2, 10.4, 2 H, CH2=CH); 2.99 (m, 3 CH2NH); 2.17 (m, =CH(CH2)2,
CH2COOH); 2.01 (m, 2 CH2CO); 1.53–1.15 (m, 3 (CH2)3). ESI-MS: 440.2 ([M+H]+); 462.1
([M+Na]+), 478.3 ([M+K]+), 879.1 ([2 M+H]+).
(DBPPA2-Lys)2-Lys-Ahx-Ahx-Ahx-OH (5). TCP Resin was loaded with Fmoc-Ahx-OH, followed
by subsequent coupling of Fmoc-Ahx-OH, Fmoc-Ahx-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Fmoc)-
OH, and DBPPA as described above. After the first lysine, fourfold excess was used, for DBPPA eight-
fold excess. ESI-MS: 1004.8 ([(M+2 H)/2]+), 1015.7 ([(M+H+Na)/2]+).

Helvetica Chimica Acta – Vol. 89 (2006)
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Coupling of the Linker to the Cyclic Peptide. The linker (1 equiv.) was activated with O-(7-azaben-
zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazol
(HOAt) and 10 equiv. of syn-collidine in DMF. After 2 h, 1 equiv. of cyclic peptide was added. After
another 24 h, the peptide was precipitated in H2O. Deprotection and purification by HPLC (Amersham
Pharmacia, YMC-RP, 40 mm”250 mm, C18; 8 ml/min; eluent A H2O (0.1% CF3COOH), eluent B
MeCN (0.1% CF3COOH), 220-nm UV detector) led to compounds 6, 7, and 8.
Cyclo(-RGDyK-)-Ahx-Ahx-Acryloyl (6). ESI-MS: 451.1 [(M+2 H)/2]+; 900.6 [M+H]+.
Cyclo(-RGDyK-)-Ahx-Ahx-Ahx-Pent-4-enoyl (7). ESI-MS: 521.8 ([(M+2 H)/2]+), 1041.8
([M+H]+), 1063.8 ([M+Na]+), 1079.8 ([M+K]+).
Cyclo(-RGDyK-)-Ahx-Ahx-Ahx-K-(K-PPA2)2 (8). ESI-MS: 942.9 ([(MÀ2 H)/2]À); 954.8 ([(MÀ3
H+Na)/2]À), 1885.9 ([MÀH]À), 1909.9 ([MÀ2 H+Na]À).
Labeling with 125I. The peptide (300 mg) was dissolved in 150 ml of PBS in an Eppendorf cap coated
with 150 mg of IodoGenꢁ. 1.32 mCi 125I as Na125I in 0.05M NaOH were added. After 30 min, the labeled
compound was separated by HPLC.
H-K(ivDde)R(Pbf)GD(OtBu)y{I}-OH (12). The peptide was obtained also by SPPS on the TCP
resin [8] according to the Fmoc strategy [9]. The resin was loaded with Fmoc-D-Tyr{I}-OH. Subsequently,
Fmoc-Asp(OtBu), Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Lys(ivDde)-OH were coupled with
TBTU, HOBt in twofold excess and EtN(i-Pr)2 as base and NMP as solvent. The Fmoc deprotection
between the couplings and at the end of the sequence was accomplished with 20% piperidine in NMP.
The peptide was cleaved from the resin by treatment with AcOH/2,2,2-trifluoroethanol/CH2Cl2 3 :1:6.
t
ESI-MS: 888.4 ([MÀ Bu+H]+), 944.3 ([M+H]+), 984.3 ([M+Na]+).
Cyclo(-R(Pbf)GD(OtBu)y(tBu)K-) (13). Cyclization was performed in DMF at a concentration of
5 mM with 1 equiv. of [(1H-benzotriazole-1-yl)oxy]tris(pyrrolidino-1-yl-phosphonium) hexafluorophos-
phate (PyBOP) and 10 equiv. syn-collidine as base. After 24 h, the solvent was removed under reduced
pressure, and the peptide was precipitated in H2O. The ivDde group was removed by treatment with 2%
t
hydrazine in DMF. ESI-MS: 998.3 ([MÀ Bu+2 H]+), 1054.3 ([M+H]+).
Cyclo(-RGDy{I}K-)-Ahx-Ahx-Acryloyl (9C). ESI-MS: 514.0 [(M+2 H)/2]+; 1026.5 [M+H]+;
1048.5 [M+Na]+.
Cyclo(-RGDy{I}K-)-Ahx-Ahx-Ahx-Pent-4-enoyl (10C). ESI-MS: 584.7 ([(M+2 H)/2]+), 1167.6
([M+H]+).
Cyclo(-RGDy{I}K-)-Ahx-Ahx-Ahx-K-(K-PPA2)2 (11C). ESI-MS: 1005.6 ([(MÀ2 H)/2]À).
Surface coating was performed as described in [4a,c,e].
The authors thank Wolfgang Linke for the help in radio-labeling. Financial support by the Deutsche
Forschungsgemeinschaft (FOR-411) and the Fonds der Chemischen Industrie is gratefully acknowledged.
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Received January 30, 2006