Alexander G. Milbradt

Photocontrol of Cell Adhesion Processes: Model Studies with Cyclic Azobenzene-RGD Peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.

Binding mode of TMC-95A analogues to eukaryotic 20S proteasome

The complex thermodynamics that govern noncovalent protein–ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X‐ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC‐95A inhibits these proteolytic centers noncovalently with distinct affinities. X‐ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen‐bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid β‐type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC‐95A with the conformationally restricted (Z)‐prop‐1‐enyl group acting as P1 residue, at least for the chymotrypsin‐like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less‐rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.

A (4R)‐ or a (4S)‐Fluoroproline Residue in Position Xaa of the (Xaa‐Yaa‐Gly) Collagen Repeat Severely Affects Triple‐Helix Formation

The triple‐helical fold of collagen requires the presence of a glycine residue at every third position in the peptide sequence and is stabilized by proline and (4R)‐4‐hydroxyproline residues in positions Xaa and Yaa of the (Xaa‐Yaa‐Gly) triplets, respectively. Regular down/up puckering of these Xaa/Yaa residues is possibly responsible for the tight packing of the three peptide strands, which have a polyproline‐II‐like structure, into the supercoiled helix. (4R)‐Configured electronegative substituents such as a hydroxy group or a fluorine substituent on the pyrrolidine ring of the residue in the Yaa position favor the up pucker and thus significantly stabilize the triple helix. A similar effect was expected from the corresponding (4S)‐isomers in the Xaa positions, but the opposite effect has been observed with (4S)‐hydroxyproline, a result that has been speculatively attributed to steric effects. In this study, (4R)‐ and (4S)‐fluoroproline residues were introduced into the Xaa position and potential steric effects were thus avoided. Contrary to expectations, (4S)‐fluoroproline prevents triple‐helix formation, whereas (4R)‐fluoroproline stabilizes the polyPro II conformation, but without supercoiling of the three strands. The latter observation suggests that folding of the single chains into a polyproline II helix is not directly associated with triple helix formation and that fine tuning of van der Waals contacts, electrostatic interactions, and stereoelectronic effects is required for optimal packing into a triple helix.

Synthesis of TMC-95A analogues. Structure-based prediction of cyclization propensities of linear precursors

Cyclization of a TMC-95A related tripeptide precursor with the preformed C6-indole/phenol junction was found to produce exclusively the related dimer, whereas under identical conditions from the tripeptide precursor with the built-in natural C7-oxindole/phenol junction only the desired monomeric cyclic species was obtained. These experimental results were fully consistent with structure-based computations of the cyclization propensities. However, by extending these computations to the analogous complestatin molecule, a consistency was not observed, thus confirming the severe limitations of such predicting procedures, even when applied to homologous systems.