Evaluation of bicinchoninic acid as a ligand for copper(I)-catalyzed azide-alkyne bioconjugations.

The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.

Design of protein congeners containing ß-cyclopropylalanine.

The non-canonical amino acid (ncAA) analogue of methionine (Met), ß-cyclopropylalanine (Cpa), was successfully incorporated into recombinant proteins expressed in Escherichia coli in a residue-specific manner. Proteins substituted in this way are congeners because they derive from the same gene sequence as the parent protein but contain a fraction of ncAAs. We have expressed congeners using parent and mutant gene sequences of various proteins (lipase, annexin A5, enhanced green fluorescent protein, and barstar) and found that Cpa incorporation is highly dependent on the protein sequence composition. These results indicate that the global amino acid composition of proteins might be a crucial parameter that influences the outcome of unnatural translation. In addition, we could also demonstrate that the chemical nature of the second residue could be essential for successful ncAA incorporation.

Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds.

A combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions delivered a chemically modified barstar protein with one or four carbohydrates installed at specific residues. These protein conjugates were employed in multivalent binding studies, which support the use of proteins as structurally defined scaffolds for the presentation of multivalent ligands.

Blue fluorescent amino acids as in vivo building blocks for proteins.

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.

Azatryptophans endow proteins with intrinsic blue fluorescence.

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 approximately 130 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.

Convenient syntheses of homopropargylglycine.

An improved classic Strecker synthesis was elaborated leading to racemic homopropargylglycine (Hpg) in 61% overall yield, while an asymmetric Strecker reaction produced Hpg and the higher homolog 2-aminohept-6-ynoic acid in significantly higher yields and over 80% ee.

Electrospray ionization mass spectrometric studies of nucleophilic carbenes derived from pyrazolium and indazolium carboxylates.

Pyrazolium-3-carboxylate and indazolium-3-carboxylate, which belong to the class of pseudo-cross-conjugated mesomeric betaines and which represent the electronically relevant partial structures of the betaine alkaloid Nigellicin, were examined by electrospray ionization mass spectrometry. These compounds decarboxylate to pyrazol-3-ylidene and indazol-3-ylidene. The formation of adducts of these new nucleophilic carbenes under the measurement conditions was examined.