The ribosome builds proteins by joining together amino acids in an order determined by messenger RNA. Here, we report on the design, synthesis, and operation of an artificial small-molecule machine that travels along a molecular strand, picking up amino acids that block its path, to synthesize a peptide in a sequence-specific manner. The chemical structure is based on a rotaxane, a molecular ring threaded onto a molecular axle. The ring carries a thiolate group that iteratively removes amino acids in order from the strand and transfers them to a peptide-elongation site through native chemical ligation. The synthesis is demonstrated with ~10(18) molecular machines acting in parallel; this process generates milligram quantities of a peptide with a single sequence confirmed by tandem mass spectrometry.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

Rational drug design relies on an iterative procedure of initial protein-structure determination, followed by the design, chemical synthesis, and subsequent biological evaluations of specific compounds. However, there is still a large gap between known protein sequences and 3D structures. To date, the most successful theoretical approach to bridge this gap is homology modeling. It is possible to construct an approximate 3D model of the structural unknown protein if the sequence homology to the known 3D structure of the reference protein is higher than 40%. Such a homologymodeled structure is suitable for rational drug design.[1]Herein we describe the successful use of our recently published homology model of the integrin a5b1[2] to design potent (with activities up to the subnanomolar range) and selective ligands for the two highly similar integrin receptors a5b1 and avb3. Structural considerations were used to trigger potency and selectivity in both directions. These ligands could allow functional studies in vivo of the role of these two integrin subtypes and might be used as lead structures for antiangiogenic cancer therapy.Integrins constitute an important class of heterodimeric cell-adhesion receptors that are involved in many severe pathological processes, such as tumor metastasis, thrombosis, inflammation, and osteoporosis.[3] Therefore, they have been attractive therapeutic targets for several years.[4] Since Brooks et al. reported that various low-molecular-weight ligands (for example, our synthesized cyclopentapeptide cyclo(-Arg-GlyAsp-D-Phe-Val-) = c(-RGDfV-) [5a] ), which are recognized by the avb3 and avb5 integrins, block angiogenesis in response to growth factors in tumors,[5b] many selective avb3-ligands have been developed and some compounds have reached clinical trials.[7] As a result of our research, the cyclic Nmethylated pentapeptide c(-RGDf[NMe]V-), [5b] known as cilengitide, has entered phase II trials for patients with glioblastoma.Recent knock-out experiments showed, however, that genetically altered mice (lacking the av integrin) show extensive angiogenesis in some cases, whereas other mice (lacking the b3 or b5 integrins) show no significant effects, and as such, the idea that these two integrins are proangiogenic was seriously questioned. [8,9] On the other hand, the proangiogenic function of the a5b1 receptor has been clearly demonstrated [10,11] so that the a5b1 integrin moved into the focus of research. Although crystal structures of the extracellular domains of the avb3 and aIIbb3 integrins have been solved and provided a deep insight into the ligand binding, [12,13] very little detailed structural information about the a5b1 receptor itself or about ligand-receptor interactions have been obtained until now. [14] Furthermore, there are only a few small-molecule ligands known to bind a5b1, [15,16] which prompted us to focus our research on this integrin subtype. A first hint for the design of new a5b1 ligands came from our homology model of the a5b1 integrin in complex with a rece...

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While residual dipolar couplings (RDCs) are an established method in high-resolution biomolecular NMR, their use for structure determination of small molecules in organic solvents is limited by the alignment media available. Only recently stretched polystyrene (PS) gels were introduced for the measurement of RDCs on small compounds that allowed urgently needed free scalability of the induced anisotropy. Here, the properties of such stretched PS gels in different organic solvents as well as for different magnetic field strengths and temperatures are studied and practical NMR-spectroscopic aspects are discussed.