Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5-Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of Ϸ130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.expanded genetic code ͉ imaging ͉ optical probes ͉ protein engineering and design ͉ red-shift A n ideal optical probe for the analysis of single proteins or whole proteomes would have the following properties: it is biocompatible, well incorporated into the target protein(s) by the endogenous translational apparatus and does not require posttranslational modifications or extensive host-engineering. Also, this chromophore should be noninvasive, i.e., it introduces minimal structural and functional perturbations into the target(s). The most promising candidates for such a chromophore are Ind analogs. They are basic structures of numerous highly important biomolecules. For example, Ind as part of the side chain of the amino acid Trp is the main source of intrinsic protein fluorescence, and purine bases of nucleic acids are Ind derivatives as well (1).The canonical amino acid Trp is one of the most suitable targets for protein engineering and design owing to its low abundance in proteins and high relevance for protein stability and function (2). However, due to its complicated photophysics and the unfavorable overlap between nucleic acid and protein fluorescence emission spectra, Trp is not always qualified as a suitable optical probe (3), and alternatives are needed. Trp analogs with their Ind side chains containing a single atom exchange (''atomic mutation'') (4) would be highly desirable. These analogs do not perturb the local environment of the substituted target protein(s) but induce considerable spectral changes relative to Trp.(Aza)Trps meet the above described criteria. In these Trp isosteres, one of the endocyclic, CH, groups of Ind is substituted with nitrogen (Fig. 1A). This substitution comprises not only the smallest possible structural alteration of all known Trp analogs but also leads to dramatic changes in the photophysics of the aromatic system. (Aza)Inds r...

One‐dimensional porphyrin wires were self‐assembled by solvothermal methods. The porous framework, sustained by van der Waals and electrostatic interactions, is thermally stable up to 350 °C. Encapsulation of polyaniline (PANI) filaments in the host matrix provides a pathway for electron conduction (see picture). Moreover, it exhibits a highly reversible lithium‐ion cycle in a C/Li⋅PANI@porphyrin cell. These features make this material very attractive for secondary battery solid‐electrolyte applications.

The design and engineering of complex protein scaffolds with hydrocarbons partially or fully augmented with fluorocarbons is one of the most promising routes to create living systems with novel chemistries. Here we elaborate the first experimental steps in this direction.

Three in one: A new method has been developed for the simultaneous in vivo incorporation of two or three different synthetic amino acids in a protein in a single expression experiment using polyauxotrophic Escherichia coli strains. The picture shows the model protein ψ‐b* used and the synthetic amino acids incorporated.

In vivo expression of colored proteins without post-translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell-permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red-shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2-, 4-, 5-, 6-, and 7-azaindole) 4-azaindole has exhibited the largest Stokes shift (approximately 130 nm) in steady-state fluorescence measurements. It is also highly biocompatible and as 4-azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono- and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4-azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole-containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo- or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady-state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

The alkaloid Nigellicine possesses the indazolium-3-carboxylate ring system as electronically relevant partial structure which represents a member of the class of pseudo-cross-conjugated mesomeric betaines. Indazolium-3-carboxylate, prepared starting from indazole-3-carboxylic acid by an esterification-methylation-saponification sequence, can be converted into the isoconjugated phenyl-and 4-(nitrophenyl)-amidates and the thiocarboxylate as additional examples of pseudo-cross-conjugated systems. In accordance with results of ab initio calculations decarboxylation of indazolium-3-car-

Bulky amino acids in positions 2 and 3 of proteins protect both Met and noncanonical azidohomoalanine, introduced by the auxotrophy‐based method, from being excised by the enzymes responsible for N‐terminal Met excision. Bioorthogonal transformation enables new specific functionalization of target proteins. We validated this general concept by designing an N‐terminal glycoconjugated barstar capable of lectin binding without losing its biological activity.

A total synthesis of epothilone C (1) with concomitant formal synthesis of epothilone A is described, using immobilized reagents and scavengers to effect multistep synthetic transformations and purifications.