(18)F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK).

PURPOSE: Oxime formation between an aminooxy-functionalized peptide and an (18)F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. MATERIALS AND METHODS: Here, the potential of using routinely produced and thus readily available [(18)F]fluorodeoxyglucose ([(18)F]FDG) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK(Aoa-(Boc)) as a model peptide. RESULTS: The use of [(18)F]FDG from routine production ([(18)F]FDGTUM) containing an excess of D: -glucose did not allow the radiosynthesis of [(18)F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [(18)F]FDG for the routine clinical synthesis of (18)F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [(18)F]FDG obtained via HPLC separation of [(18)F]FDGTUM from excess glucose, however, afforded [(18)F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 degrees C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [(18)F]FDG-RGD showed increased tumour accumulation compared to the "gold standard" [(18)F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds. CONCLUSION: These data demonstrate that chemoselective (18)F-labelling of aminooxy-functionalized peptides using n.c.a. [(18)F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of (18)F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [(18)F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [(18)F]FDG-synthesis, [(18)F]fluoroglucosylation of peptides may represent a promising alternative to currently used chemoselective one-step (18)F-labelling protocols.

Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns.

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (microCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide - even for the preparation of peptide-functionalized small surface areas.

Specific integrin labeling in living cells using functionalized nanocrystals.

We present an integrin labeling method using functionalized quantum dots (QDs). Cyclic Arg-Gly-Asp (RGD) peptides and a biotin-streptavidin linkage are used to specifically couple individual QDs to integrins of living cells. The spacer distance between the RGD sequence and the QD surface is a crucial parameter to ensure specific binding to individual alpha(v)beta(3) integrins of osteoblast cells. Despite blinking, the position of single QDs is tracked with nanometer precision and localized diffusive behavior is observed. We show that blinking events do not prevent the acquisition of quantitative parameters from the QD trajectories.

Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly.

Cell-extracellular matrix (cell-ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub-micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine-glycine-aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell-ECM interactions.

Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone.

In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage-dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant.

Chemical conjugation of linear and cyclic RGD moieties to a recombinant elastin-mimetic polypeptide--a versatile approach towards bioactive protein hydrogels.

An elastin-mimetic polypeptide, (EMM)(7), with the amino-acid sequence GRDPSS [VPGVG VPGKG VPGVG VPGVG VPGEG VPGIG](7) was used for chemical conjugation of various integrin ligands (RGD peptides) to prepare bioactive hydrogels. The chemical approach involved (1) chemical protection of lysine residues with Fmoc or Boc groups, (2) chemical ligation of a protected linear or cyclic RGD ligand, with or without a hexanoic-acid spacer to the glutamic acid residue, (3) deprotection of the lysine functionalities and the RGD moieties and (4) cross-linking to form a bioactive hydrogel. (1)H NMR spectroscopy was used to quantify the multiple steps in the reaction. The chemical protection was found to be between 65 and 93% for Fmoc and Boc, respectively. The ligands studied included linear RGD cell-binding [H-FGRGDS-OH (1-l-RGD), H-Ahx--FGRGDS-OH (2-Ahx-FGRGDS) and a cyclic -H(2)N-(CH(2))(6)COHN-cyclo(-RGDfK-) (H-Ahx-c(-RGDfK-)) peptide also with a hexanoic-acid spacer. Cell adhesion with mouse osteoblast cells was dependent on the ligand type, ligand density and the use of a spacer.

Benzylprotected aromatic phosphonic acids for anchoring peptides on titanium.

The development of biocompatible coatings is an ongoing issue. Mimicking the physiological adhesion process of osteoblasts to the extracellular matrix improves cell adhesion of osteoblasts in vitro and results in improved and earlier osseous integration of implants in vivo. Titanium, an often used material in implant surgery, can be easily coated by peptides bearing phosphonic acid groups. We report here, the synthesis of benzyl protected phosphonic acids suitable for solid-phase peptide synthesis (SPPS), which can be easily deprotected with TFA.

Titanium implant materials with improved biocompatibility through coating with phosphonate-anchored cyclic RGD peptides.

One key point for improving osseous integration of implants is to render them osteopromotive by specifically favoring the adhesion of osteoblasts. Mimicking the physiological adhesion process of osteoblasts to the extracellular matrix improves cell adhesion in vitro and results in improved and earlier osseous integration of implants in vivo. Our approach involves coating titanium implants with a tailor-made cyclic-RGD peptide, thus allowing them to bind to specific integrin receptors on the cell surface through multimeric phosphonates. The advantages of this very stable, new type of anchoring for practical application are presented.

Photoswitched cell adhesion on surfaces with RGD peptides.

Coating of surfaces by RGD peptides is well-known. Herein we describe the possibility to switch cell adhesion properties by changing the distance and orientation of the RGD peptides to the surface. A set of RGD peptides of the type cyclo(-RGDfK-) was synthesized containing the photoswitchable 4-[(4-aminophenyl)azo]benzocarbonyl central unit as spacer between the acrylamide anchor and the RGD peptide. PMMA (poly methyl methacrylate) surfaces were coated with these peptides. Control of adhesion stimulation by irradiation with 366 or 450 nm light could be achieved.