Copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of nonfluorescent 3-azidocoumarins and terminal alkynes afforded intense fluorescent 1,2,3-triazole products. The mild condition of this reaction allowed us to construct a large library of pure fluorescent coumarin dyes. Since both azide and alkyne are quite inert to biological systems, this reaction has potential in bioconjugation and bioimaging applications. [reaction: see text]

Click conjugation of tobacco mosaic virus: As a prototype of “click” chemistry, the CuI catalyzed azide‐alkyne cycloaddition reaction in combination with diazonium‐coupling proved to be an efficient, versatile, and benign method to conjugate a wide range of compounds to phenolic groups of protein tyrosines. In particular, plant virus tobacco mosaic virus was chosen as a multivalent protein scaffold to demonstrate this bioconjugation strategy.

Playing tag: Noncanonical amino acid tagging enables the selective fluorescent visualization of newly synthesized proteins in mammalian cells (see the picture). Susceptibility to tagging is determined by the spatial and temporal character of the protein synthesis, thus providing a complement to methods which identify relevant members of the proteome.

Plasmid and Expression Hosts. The plasmid pQE30-Barstar contains a gene encoding histidine-tagged barstar under control of a T5 promoter. Briefly, PCR was used to add BamHI and Hind III sites to the barstar gene. The gene encodes two mutations (Cys53Ala, Cys95Ala) for improved stability that do not affect barnase binding 1 . We do not believe that cysteine deletion is necessary for effective labeling; we have observed labeling of many cysteine-bearing proteins in companion experiments. After digestion, the gene was inserted into pQE30 between the BamHI and HindIII restriction sites. For incorporation of homopropargylglycine (Hpg), pQE30-Barstar was transformed into the E. coli methionine auxotrophic strain M15-MA to make the expression host M15-MA [pQE30-Barstar]. 2 For incorporation of ethynylphenylalanine (Eth), pQE30-Barstar was linearized by digestion with NheI. The plasmid pQE15-PheRS* contains a mutant E. coli phenylalanyl-tRNA synthetase (A294G) under control of a modified tac promoter with an abolished lac repressor binding site for constitutive expression. 3, 4 This plasmid was digested with NheI, and a 1 kB fragment corresponding to the PheRS* cassette was isolated by agarose gel electrophoresis. This fragment was ligated into pQE30-Barstar to yield the plasmid pQE30-Barstar-PheRS*. This plasmid was transformed into the phenylalanine auxotrophic E. coli strain BL21 (DE3) After reaching an OD 600 of 1, the culture was sedimented by centrifugation for 5 minutes (6500g) at 4 ˚C. The cell pellets were washed twice with NaCl (0.9 wt %). The culture was resuspended in M9 minimal medium without methionine. The culture was divided into 35, 48, and 5 mL aliquots. For protein synthesis inhibition, tetracycline (10 mg/mL) was added to the 5 mL culture. After 15 minutes at 37 ˚C, the samples were supplemented with either methionine (0.75 mM in 35 mL medium) or Hpg (1 mM in 48 mL medium). After 10 minute incubation, protein expression was induced for 3.5 h by the addition of IPTG (1 mM). The 5 mL culture supplemented with Hpg and tetracycline was not induced.The expression of barstar from AF-IQ [pQE30-Barstar-PheRS*] was performed as above, except the M9 minimal medium was supplemented with chloramphenicol (35 mg/L) instead of kanamycin. After reaching an OD 600 of 1.0, a medium shift was performed as described. The cultures were supplemented with either phenylalanine (1.5 mM) or Eth (1.5 mM). Expression was induced for 3.5 h with IPTG (1 mM).(1) Ramachandran, S.; Udgaonkar,

Acetylenes. -9 Azidocoumarins and 24 acetylenes, both inert to biological systems, are used for the construction of a large library consisting of fluorescent 1,2,3-triazole dyes. This "click and probing" ligation protocol may have a high potential in functionalizing bionanoparticles. -(SIVAKUMAR, K.; XIE, F.; CASH, B. M.; LONG, S.; BARNHILL, H. N.; WANG*, Q.; Org. Lett. 6 (2004) 24, 4603-4606; Dep. Chem. Biochem., Univ. S. C., Columbia, SC 29208, USA; Eng.) -Lindner 13-138

Horse spleen apoferritin, the hollow protein shell derived from ferritin, a special biological nanoparticle, can be chemoselectively modified at the lysine residues, which affords a robust scaffold for further chemical reactions including Cu(i)-catalyzed azide-alkyne cycloaddition reaction and atom transfer radical polymerization reaction.

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Eine Frage der Anhänglichkeit: Durch Anhängen nichtkanonischer Aminosäuren können neu synthetisierte Proteine in Säugerzellen selektiv anhand ihrer Fluoreszenz sichtbar gemacht werden (siehe Bild). Da dieser Markierungsmodus von räumlichen und zeitlichen Gegebenheiten bei der Proteinsynthese abhängt, ergänzt er andere Methoden bei der Suche nach wichtigen Proteommitglieder.