We describe a protein quantification method that exploits the subtle mass differences caused by neutron-binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins with metabolic labeling; analysis with high mass resolution (>100,000) reveals the isotopologue-embedded peptide signals permitting quantification. We conclude neutron encoding will enable high levels of multi-plexing (> 10) with high dynamic range and accuracy.

The copper-catalyzed N-arylation of amides, i.e., the Goldberg reaction, is an efficient method for the construction of products relevant to both industry and academic settings. Herein, we present mechanistic details concerning the catalytic and stoichiometric N-arylation of amides. In the context of the catalytic reaction, our findings reveal the importance of chelating diamine ligands in controlling the concentration of the active catalytic species. The consistency between the catalytic and stoichiometric results suggest that the activation of aryl halides occurs through a 1,2-diamine-ligated copper(I) amidate complex. Kinetic studies on the stoichiometric N-arylation of aryl iodides using 1,2-diamine ligated Cu(I) amidates also provide insights into the mechanism of aryl halide activation.

Two previous mechanistic studies of the amination of aryl halides catalyzed by palladium complexes of 1,1'-binaphthalene-2,2'-diylbis(diphenylphosphine) (BINAP) are reexamined by the authors of both studies. This current work includes a detailed study of the identity of the BINAP-ligated palladium complexes present in reactions of amines with aryl halides and rate measurements of these catalytic reactions initiated with pure precatalysts and precatalysts generated in situ from [Pd2(dba)3] and BINAP. This work reveals errors in both previous studies, and we describe our current state of understanding of the mechanism of this synthetically important transformation. 31P NMR spectroscopy shows that several palladium(0) species are present in the catalytic system when the catalyst is generated in situ from [Pd2(dba)3] and BINAP, and that at least two of these complexes generate catalytic intermediates. Further, these spectroscopic studies and accompanying kinetic data demonstrate that an apparent positive order in the concentration of amine during reactions of secondary amines is best attributed to catalyst decomposition. Kinetic studies with isolated precatalysts show that the rates of the catalytic reactions are independent of the identity and the concentration of amine, and studies with catalysts generated in situ show that the rates of these reactions are independent of the concentration of amine. Further, reactions catalyzed by [Pd(BINAP)2] with added BINAP are found to be first-order in bromoarene and inverse first-order in ligand, in contrast to previous work indicating zero-order kinetics in both. These data, as well as a correlation between the decay of bromobenzene in the catalytic reaction and the predicted decay of bromobenzene from rate constants of studies on stoichiometric oxidative addition, are consistent with a catalytic process in which oxidative addition of the bromoarene occurs to [Pd(BINAP)] prior to coordination of amine and in which [Pd(BINAP)2], which generates [Pd(BINAP)] by dissociation of BINAP, lies off the cycle. By this mechanism, the amine and base react with [Pd(BINAP)(Ar)(Br)] to form an arylpalladium amido complex, and reductive elimination from this amido complex forms the arylamine.

The mechanistic details of the Cu-catalyzed amidation of aryl iodides are presented. The kinetic data suggest that the diamine ligand prevents multiple ligation of the amide. The formation of an amidocuprate species external to the catalytic cycle helped to rationalize the dependence on diamine concentration and the inverse dependence on amide concentration at low diamine concentrations. The intermediacy of a Cu(I) amidate was established through both its chemical and kinetic competency.

A comparative kinetic examination of catalyst systems based on several monophosphinobiaryl ligands is reported. The bulk of the phosphine ligand controls the catalytic activity and the rate of catalyst activation with the catalyst based on 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl providing the greatest activity and fastest activation. In the case where catalyst activation is slow (i.e., use of the smaller ligands such as 2-dicyclohexylphosphino-2'-methylbiphenyl in combination with Pd(OAc)2) stirring the amine with the catalyst/base mixture prior to the commencement of the reaction increases the reaction rate along with the rate of catalyst activation. Kinetic isotope effects established that the catalyst activation process occurs through a beta-hydride elimination pathway.

A protocol for forming a highly active Pd(0) catalyst from Pd(OAc) 2 , water, and biaryldialkylphosphine ligands has been developed. This protocol generates a catalyst system, which exhibits excellent reactivity and efficiency in the coupling of a variety of amides and anilines with aryl chlorides.Over the past decade great progress has been made in improving the efficiency and applicablilty of palladium-catalyzed C-N cross-coupling reactions. 1 Despite these recent advances, many limitations of these methods remain. We have previously shown that biaryldialkylphosphines, of which 1 (Figure 1) is prototypical, 2 are excellent supporting ligands in C-N cross-coupling processes. We now disclose a procedure that helps to maximize the efficiency of these ligands when used with a Pd(II) precatalyst.The formation of an active L n Pd(0) complex is most commonly accomplished in one of the following ways: 1) use of a Pd(0) source such as Pd 2 (dba) 3 , 3 2) use of [(allyl)PdCl] 2 ,4 3) reduction of a Pd(II) salt [e.g., Pd(OAc) 2 ] using PhB(OH) 2 , 2 a tertiary amine,5 a tertiary phosphine,6 or an amine substrate, 1 or 4) employment of a single component precatalyst. 7 These methods all have deficiencies when used with biaryldialkylphosphine ligands. Although Pd 2 (dba) 3 has been shown to work well in combination with 1 in many instances,2 diminished reactivity is observed due to the coordination of the dba to the palladium; this is a well known consequence of using dba containing precatalysts. 8 Attempts to use [(allyl)PdCl] 2 have not proven to be productive with dialkylbiarylphosphine ligands. Reduction of Pd(OAc) 2 with 1 is slow due to the steric hindrance of the ligand and initial results show that the use of tertiary amines or PhB(OH) 2 as reducing agents give slow formation of the active L n Pd(0) catalyst. Lastly, reduction of Pd(OAc) 2 works well with primary or secondary amine substrates that have β-hydrogens. However, when non-reducing nucleophiles, such as anilines or amides, are used, formation of L n Pd (0) Because of these deficiencies, we set out to develop a protocol that utilized water and 1 to reduce Pd(OAc) 2 and generate the active LnPd(0) complex. This type of activation was first reported in 1992 by Ozawa and Hayashi, in which they were able to reduce Pd(OAc) 2 in the presence of 3 equivalents of BINAP. 9 They disclosed that in the absence of water the reduction did not proceed; however, by adding extra equivalents of water the rate of activation could be accelerated. This showed that water played a direct role in the formation of the Pd(0) catalyst. Amatore and Jutand further delineated a method in which water and several different tertiary phosphines were employed as reducing agents to form a Pd(0) complex that underwent oxidative addition to aryl halides (Scheme 1). 10 In their investigations they found that water converted an intermediate phosphonium salt to the corresponding phosphine oxide in the reduction of Pd(OAc) 2 .We began our studies by seeking out a fast, efficient, and p...

Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol–ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (Nε-Gly-l-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the Nε-Gly-l-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native Nε-Gly-l-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants utilize two different types of thioesterases for the production of highly active biological compounds1 , 2. Type I thioesterases (TEI) catalyze the release step from the assembly line3 of the final product where it is transported from one reaction center to the next as a thioester linked to a 4′-phosphopantetheine cofactor (4′-PP) that is covalently attached to thiolation (T) domains4 -9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo T-domains of NRPS and PKS systems11 -13. Mispriming of 4′-PP cofactors by acetyl-and short chain acylresidues interrupts the biosynthetic system. This repair reaction is very important, since roughly 80% of coenzyme A (CoA), the precursor of the 4′-phosphopantetheine cofactor, is acetylated in bacteria14. Here we report the first three-dimensional structure of a type II thioesterase free and in complex with a T domain. Comparison with structures of TEI enzymes3 , 15 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. In addition, we show that the TEII enzyme exists in several conformations of which only one is selected upon interaction with its native substrate, a modified holo-T domain. The transport of the growing chain between individual modules is achieved by small ~80 amino acid long T domains that interact with the aminoacyl-forming activation A-domain and both the up-and downstream peptide-bond-forming condensation C-domains 4-10 . In addition, some of the T domains of the Surfactin-synthetase have to interact with epimerization domains, located at the C-terminus of subunits SrfA-A and SrfA-B, or with the covalently linked type I thioesterase 3 . The type I thioesterase catalyzes the macrolactone formation between Leu (7) and the β-hydroxy fatty acid to release the mature surfactin. Modifications blocking the reactive thiol group of the 4′-PP cofactor attached to any T domain can occur with small molecules present in the cell (acetylation, succinylation and modification with fatty acids) and pose a significant challenge for the organism to keep the assembly line running.The surveillance and repair tasks for the surfactin assembly line are carried out by the standalone surfactin type II thioesterase (SrfTEII) 11-13. The importance of this enzyme has been demonstrated by genetic deletions that reduced the production of surfactin by 84%17. Due to the large variety of acylation modifications and the fact that the SrfTEII has to be able to interact with all seven T domains of the entire assembly line, this TEII has to be -in contrast to the type I thioesterase at the end of the last module -rather non-specific. At the same time, premature cleavage of the correct growing peptide chain has to be avoided. In addition to this repair function, the SrfTEII mi...