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 (k 2 2000 M−1 s−1). This fast reactivity enables protein modification at low concentration.
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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 bicyclo6.1.0non-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.
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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)-bicyclo6.1.0non-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.
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Rett syndrome (Rett) is the leading genetic cause of mental retardation in females. Most cases of Rett are caused by loss-of-function mutations in the gene coding for the transcriptional regulator ...methyl-CpG binding protein 2 (MeCP2), but despite much effort, it remains unclear how a loss of MeCP2 function generates the neurological deficits of Rett. Here we show that MeCP2 plays an essential and cell-autonomous role in homeostatic synaptic scaling up in response to reduced firing or reduced sensory drive in rat visual cortical pyramidal neurons. We found that acute RNAi knockdown of MeCP2 blocked synaptic scaling within targeted neocortical pyramidal neurons. Furthermore, MeCP2 knockdown decreased excitatory synapse number without affecting basal mEPSC amplitude or AMPAR accumulation at spared synapses, demonstrating that MeCP2 acts cell-autonomously to maintain both excitatory synapse number and synaptic scaling in individual neocortical neurons. Finally, we used a mouse model of Rett to show that MeCP2 loss prevents homeostatic synaptic scaling up in response to visual deprivation in vivo, demonstrating for the first time that MeCP2 loss disrupts homeostatic plasticity within the intact developing neocortex. Our results establish MeCP2 as a critical mediator of synaptic scaling and raise the possibility that some of the neurological defects of Rett arise from a disruption of homeostatic plasticity.
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.
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Go with the flow: A general approach for amide bond formation by way of a continuous‐flow photochemical rearrangement of nitrones was described (see scheme). Simple aryl‐alkyl amide bonds as well as ...complex peptide bonds were constructed efficiently with a residence time less than 20 minutes. A tetrapeptide was synthesized in this way and the method could be applied to peptide fragment coupling.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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 EC90 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, EC90 = 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, EC90 = 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.
Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second ...generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.
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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.
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