ABSTRACT
Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
Subject(s)
Alkynes/chemistry , Amino Acids/chemistry , Cyclooctanes/chemistry , Fluorescent Dyes/chemistry , Tetrazoles/chemistry , Alkenes/chemistry , Amino Acids/genetics , Animals , Binding Sites , Cells, Cultured , Escherichia coli/genetics , HEK293 Cells , Humans , Recombinant Proteins/genetics , Time FactorsABSTRACT
Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.
Subject(s)
Chemistry/methods , Cyclooctanes/chemistry , Genetic Engineering/methods , Pyridines/chemistry , Amino Acids/chemistry , Catalysis , Escherichia coli/metabolism , Green Fluorescent Proteins/metabolism , Methanococcus/metabolism , Models, Chemical , Molecular Conformation , Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tyrosine-tRNA Ligase/chemistryABSTRACT
Computation was used to design a trans-cyclooctene derivative that displays enhanced reactivity in the tetrazine-trans-cycloctene ligation. The optimized derivative is an (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained 'half-chair' conformation. Toward 3,6-dipyridyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 160 times more reactive than the parent trans-cyclooctene.
Subject(s)
Cyclooctanes/chemical synthesis , Molecular Structure , StereoisomerismABSTRACT
A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 3,6-diaryl-s-tetrazines and an (18)F-labeled trans-cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.
Subject(s)
Cyclooctanes/chemistry , Fluorine Radioisotopes/chemistry , Chromatography, High Pressure Liquid , RadiometryABSTRACT
The hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The moderate efficacy along with side effects of the current pegylated interferon and ribavirin combination therapy underscores the need for more effective and safer new treatment. In an effort to improve upon our current clinical candidate, Boceprevir (SCH 503034), extensive SAR studies were performed on the P3 capping moieties. This led to the discovery of tert-leucinol derived cyclic imides as a potent series of novel P3 capping groups. Thus, the introduction of these imide caps improved the cell-based replicon EC(90) by more than 10-fold. A number of imides with various substitutions, ring sizes, bicyclic systems, and heterocyclic rings were explored. The 4,4-dimethyl substituted glutarimide emerged as the best cap as exemplified in compound 21 (K(i)* = 4 nM, EC(90) = 40 nM). Systematic optimization of different positions (P', P3, and P1) of the inhibitor resulted in the identification of the lead compound 46, which had an excellent potency (K(i)* = 4 nM, EC(90) = 30 nM) and good pharmacokinetic profile (22% and 35% bioavailability in rats and dogs, respectively). X-ray structure of inhibitor 46 bound to the enzyme revealed that there was an additional hydrogen bonding interaction between one of the imide carbonyls and Cys159.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Piperidones/chemical synthesis , Serine Proteinase Inhibitors/chemical synthesis , Urea/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Biological Availability , Crystallography, X-Ray , Dogs , Haplorhini , Hepacivirus/genetics , Hydrogen Bonding , Imides/chemical synthesis , Imides/chemistry , Leucine/analogs & derivatives , Leucine/chemical synthesis , Leucine/chemistry , Models, Molecular , Piperidones/pharmacokinetics , Piperidones/pharmacology , Rats , Serine Proteinase Inhibitors/pharmacokinetics , Serine Proteinase Inhibitors/pharmacology , Stereoisomerism , Structure-Activity Relationship , Urea/chemical synthesis , Urea/pharmacokinetics , Urea/pharmacologyABSTRACT
Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality and proceed in high yield in organic solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concentration.
