This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ~30 working days.
Total chemical synthesis provides a unique approach for the access to uncontaminated, monodisperse, and more importantly, post-translationally modified membrane proteins. In the present study we report a practical procedure for expedient and cost-effective synthesis of small to medium-sized membrane proteins in multimilligram scale through the use of automated Fmoc chemistry. The key finding of our study is that after the attachment of a removable arginine-tagged backbone modification group, the membrane protein segments behave almost the same as ordinary water-soluble peptides in terms of Fmoc solid-phase synthesis, ligation, purification, and mass spectrometry characterization. The efficiency and practicality of the new method is demonstrated by the successful preparation of Ser64-phosphorylated M2 proton channel from influenza A virus and the membrane-embedded domain of an inward rectifier K(+) channel protein Kir5.1. Functional characterizations of these chemically synthesized membrane proteins indicate that they provide useful and otherwise-difficult-to-access materials for biochemistry and biophysics studies.
Via hydrothermal synthesis, the self-assembly of M(II) ions, H3BTC or H2NDC with three structure-related flexible bis(imidazole) ligands, L1, L2, and L3, generated six metal−organic polymers (M = Co, Ni, Zn; BTC = 1,3,5-benzenetricarboxylate, NDC = 1,2-benzenebicarboxylate, L1 = 1,4-bis(imidazol-1-ylmethyl)benzene, L2 = 1,1′-(1,4-butanediyl)bis(imidazole), L3 = 1,1′-(1,4-hexanediyl)bis (imidazole)): {Co3(L1)3(BTC)2(μ-H2O)3·2H2O} n (1), {Zn2(L2)(HBTC)2·2H2O} n (2), {Co(L3)(HBTC)} n (3), {Co(L1) (NDC)} n (4), {Ni(L2)(NDC)} n (5), and {Co(L3) (NDC)} n (6). The structure of 1 is the rare 4-connected self-penetrating metal−organic framework (MOF) with the (63)2(64·82)2(62·84) topology notation; polymers 2 and 6 are two-dimensional (2D) (3,4)-connected and 4-connected nets, respectively. If the O−H…O/2.631 Å hydrogen bonds between HBTC2− dianions are not accounted for, then polymer 3 is a 2D (3,5)-connected net; contrarily, it is a pillar-layered three-dimensional supramolecular framework characterized by (4,5)-connected (42·62·82)(42·68) topology. 4 and 5 show 4-connected MOFs with 65·10 and 65·8 CdSO4-type topology, respectively. Furthermore, their thermal properties are studied by thermogravimetric analysis.
The chemical synthesis of proteins provides synthetic chemists with an interesting challenge and supports biological research through the generation of proteins that are not produced naturally. Although it offers advantages, studies of solid phase peptide synthesis have established limits for this technique: researchers can only prepare peptides up to 50 amino acids in length in sufficient yields and purity. Therefore, researchers have developed techniques to condense peptide segments to build longer polypeptide chains. The method of choice for chemical synthesis of these longer polypeptides is convergent condensation of unprotected protein fragments by the native chemical ligation reaction in aqueous buffer. As researchers apply this strategy to increasingly difficult protein targets, they have needed to overcome diverse problems such as the requirement for a thiol-containing amino acid residue at the ligation site, the difficulty in synthesizing thioester intermediates under mild conditions, and the challenge of condensing multiple peptide segments with higher efficiency. In this Account, we describe our research toward the development of new thioester equivalents for protein chemical synthesis. We have focused on a simple idea of finding new chemistry to selectively convert a relatively "low-energy" acyl group such as an ester or amide to a thioester under mild conditions. We have learned that this seemingly unfavorable acyl substitution process can occur by the coupling of the ester or amide with another energetically favorable reaction, such as the irreversible hydrolysis of an enamine or condensation of a hydrazide with nitrous acid. Using this strategy, we have developed several new thioester equivalents that we can use for the condensation of protein segments. These new thioester equivalents not only improve the efficiency for the preparation of the intermediates needed for protein chemical synthesis but also allow for the design of new convergent routes for the condensation of multiple protein fragments.
In this work, the growth kinetics of thiol-capped PbS nanoparticles was studied. Two-stage growth process was observed, which was controlled first by oriented attachment (OA) mechanism and then by the hybrid Ostwald ripening (OR) and OA mechanism. Different from the NaOH-ZnS system, where OA will occur between any two multilevel nanoparticles, an OA kinetic model only considering the attachment related to original particles was fitted well with the experimental results. Analysis reveals that this model may be a universal one to describe the OA crystal growth process of nanocrystals capped with easily destroyed ligands, such as thiol-ZnS in the previous report. The OA crystal growth characteristics determined by the surface agent were discussed and compared. We propose that with stronger surface capping, the OR growth of nanocrystals is hindered, which facilitates the size controlling via OA kinetics during nanosynthesis.
The conversion of sugars to 5-hydroxymethylfurfural (HMF) over solid acids in water, represents an environmentally and separation-friendly route to an important platform molecule. In particular, the conversion of sucrose attracts increasing attention because it is cheaper and more widely available than glucose and fructose. Sucrose can undergo rapid hydrolysis to the two monosaccharides, however conversion mechanisms and interactions with solid acids remain unclear. Here, it is shown that niobium oxides possess Brønsted acid (BA) and Lewis acid (LA) sites of tunable quantity and strength, dependent on their structure and morphology. By systematically studying these acid catalysts, it is revealed for the first time that both acid type and strength are significant for the sugars conversion: Fructose reaction is catalyzed by BA, with weaker BA sites being more selective towards HMF. Glucose conversion to HMF involves an additional isomerization step to fructose, which can be catalyzed by both LA and strong BA but LA is more efficient. Sucrose is shown to be easily hydrolyzed into glucose and fructose under the reaction conditions and HMF is formed from the further conversion of the two sugars. It is demonstrated that mesoporous niobium oxide gives the highest HMF yield for sucrose conversion amongst all niobium oxides due to balanced BA and LA sites with appropriate acid strengths.
Bioremediation of Cr(VI) through reduction relies on the notion that the produced Cr(III) may be precipitated or efficiently immobilized. However, recent reports suggest that soluble organo-Cr(III) complexes are present in various chromate-reducing bacterial systems. This work was designed to explore the factors that affect the immobilization of Cr(III) in the Ochrobactrum anthropi system. X-ray absorption fine structure analysis on the cell debris clearly verified that coordination of Cr(III) occurs on the surfaces via the chelating coordination with carboxyl- and amido-functional groups. However, competitive coordination experiments of Cr(III) revealed that the small molecules such as amino acids and their derivatives or multicarboxyl compounds hold stronger coordination ability with Cr(III) than with cell debris. We speculate that it is the preferential coordination of Cr(III) to the soluble organic molecules in the bacterial culture medium that inhibits effective immobilization of Cr(III) on the cells. On the basis of this understanding, a strategy with two-step control of the medium was proposed, and this achieved successful immobilization of Cr(VI) as Cr(III) by O. anthropi and Planococcus citreus in 5-50 L pilot-scale experiments.
Water-soluble mercaptoacetic acid-coated 3.1 nm CdS quantum dots (QDs) with two concentrations were selected for studying the correlation between the photoluminescence and the crystal growth mechanism. By achieving the classic Ostwald ripening mechanism and oriented attachment (OA) growth mechanism, we have shown that the evolution of the emission spectra were obviously different. The change in both the surface and internal defects during OA crystal growth were responsible for the specific variation of the photoluminescence of CdS QDs. Strategies for obtaining QDs with different luminescent properties are suggested.
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