Total chemical synthesis of fully functional Photoactive Yellow Protein

The Photoactive Yellow Protein (PYP) is a structural prototype for the PAS superfamily of proteins, which includes hundreds of receptor and regulatory proteins from all three kingdoms of life. PYP itself is a small globular protein that undergoes a photocycle involving a series of conformational changes in response to light excitation of its p-coumaric acid chromophore, making it an excellent model system to study the molecular basis of signaling in the PAS super family. To enable novel chemical approaches to elucidating the structural changes that accompany signaling in PYP, we have chemically synthesized the 125 amino acid residue protein molecule using a combination of Boc chemistry solid phase peptide synthesis and native chemical ligation. Synthetic PYP exhibits the wildtype photocycle, as determined in photobleaching studies. Planned future studies include incorporation of site-specific isotopic labels into specific secondary structural elements to determine which structural elements are involved in signaling state formation using difference FTIR spectroscopy.     

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.     

Highly enantioselective synthesis, crystal structure, and circular dichroism spectroscopy of (R)-bambuterol hydrochloride

The present article describes the asymmetric synthesis of (R)-bambuterol hydrochloride based on 1-(3,5-dihydroxyphenyl)ethanone as starting material, which was esterified by dimethylcarbamic chloride, and brominated by copper (II) bromide. Then the carbonyl group was reduced efficiently using (-)-B-chlorodiisopinocamphenylborane [(-)-DIP-chloridetrade mark] as an asymmetrical reducing agent. Followed by epoxide ring closure with NaOH and ring expansion with tert-butylamine led to the desired product (R)-bambuterol with e.e. up to 99%. The optical properties and absolute configuration of (R)-bambuterol hydrochloride were further investigated using circular dichroism spectroscopy and X-ray single crystal analysis.     

Design and synthesis of dual active neovibsanin derivatives based on a chemical structure merging method

A hybrid compound consisting of neovibsanin and trans-banglene was designed according to a structure merging method and synthesized via a sequence of key steps including a Diels–Alder cycloaddition, stereoselective alkynylation, and intramolecular oxa-Michael addition reaction. The biological activity of the synthetized acetal compound and its hemiacetal analogue was investigated in PC12 cells. These studies revealed that the designed hybrid compounds displayed neuritogenic activity. Furthermore, a relatively strong neurite outgrowth promoting activity was observed in the presence of NGF. These results suggest that the designed hybrid compound exhibited a dual activity.     

Total Synthesis of (+)-(2S,3R)-Piscidic Acid via Catalytic Asymmetric Dihydroxylation of a Trisubstituted Olefin

(+)-(2S,3R)-Piscidic acid was efficiently synthesized with high optical purity (90% e.e.) via Sharpless catalytic asymmetric dihydroxylation of a trisubstituted olefin in only 6 steps from commercially available 4-hydroxyphenylpyruvic acid as the starting material. The reaction proceeded with high optical purity by using the chiral ligands, dihydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl diether or dihydroquinidine 1,4-anthraquinonediyl diether.     

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

The 50‐residue snake venom protein L ‐omwaprin and its enantiomer D ‐omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L ‐ and D ‐omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L ‐amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L ‐ and D ‐omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2₁/c and its structure was determined at atomic resolution (1.33 Å) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high‐resolution X‐ray structures by direct methods.     

A novel cadmium(II) complex of bipyridine derivative: synthesis,X-ray crystal structure,DNA-binding and antibacterial activities

Abstract

A mononuclear cadmium(II) complex of formula [Cd(5,5′-dmbipy)₂(OAc)₂]·2H₂O (5,5′-dmbipy = 5,5′-dimethyl-2,2′-bipyridine and OAc?=?acetato ligand) has been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis and single-crystal X-ray structure analysis. The molecular structure of the complex shows a distorted tetragonal antiprism CdN₄O₄ coordination geometry around the cadmium atom, resulting in coordination by four nitrogen atoms from two 5,5′-dmbipy ligands and four oxygen atoms from two acetate anions. The interaction of this complex to FS-DNA (fish sperm DNA) has also been studied by electronic absorption, fluorescence and gel electrophoresis techniques. Binding constant (K_b), Stern–Volmer constant (K_sv), number of binding sites (n) and bimolecular quenching rate constant (k_q) have been calculated from these spectroscopic data. These results have revealed that the metal complex can bind effectively to FS-DNA via groove binding. The calculated thermodynamic parameters (ΔH°, ΔS° and ΔG°) show that hydrogen bonding and van der Waals forces have an important function in the Cd(II) complex–DNA interaction. The antibacterial effects of the synthesized cadmium complex have also been examined in vitro against standard bacterial strains: one Gram-positive (Staphylococcus aureus, ATCC 25923) and one Gram-negative (Escherichia coli, ATCC 25922) bacteria, using disk diffusion and macro-dilution broth methods. The obtained results show that the Cd(II) complex exhibits a marked antibacterial activity which is significantly better than those observed for its free ligand and metal salt for both Gram-positive and Gram-negative bacteria. However, this metal complex is a more potent antibacterial agent against the Gram-positive than that of the Gram-negative bacteria.

Communicated by Ramaswamy H. Sarma     

氯化两面针碱的提取分离、全合成及药理活性的研究进展

两面针是我国民间常用的传统中药之一,从根部提取分离到的氯化两面针碱是两面针植物多种生物活性的主要有效成分。当前,直接从植物中提取分离有效成分的方法不仅回收率低、成本高,而且氯化两面针碱存在水溶性差和生物利用度低的问题,因此大多学者致力于改造其结构以提高活性和利用度,同时探索高效经济的全合成方法以满足研究和生产的需求。该文以国内外相关文献为基础,对氯化两面针碱的提取、分离纯化、结构化合物的全合成路线以及药理活性机制的研究进展做一综述,总结目前研究中存在的不足,并对今后的研究重点做出展望,旨在为今后深入研究两面针碱及衍生物活性改造、解析作用机制、开发基于此结构的创新药物分子等提供理论依据。     

Total chemical synthesis,assembly of human torque teno virus genome

Torque teno virus(TTV)is a nonenveloped virus containing a single-stranded,circular DNA genome of approximately 3.8kb.We completely synthesized the 3808 nucleotides of the TTV(SANBAN isolate)genome,which contains a hairpin structure and a GC-rich region.More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerise chain assembly reaction(PCA),and the synthesis was completed with splicing by overlap extension(SOEing).This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.     

Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis.

The complexes Cu(OBt)2 and Cu(OAt)2, which are derived from copper(II) and HOBt and HOAt, respectively, are shown to be more effective in suppressing racemization during solid-phase peptide synthesis (SPPS) than are those compounds currently being used for this purpose. These compounds can readily be used in conjunction with the commonly applied coupling reagents in fully automated systems for solid-phase peptide chemistry.     

Total synthesis of (+)-macrosphelides A,C, E,F, and G based on enzymatic function

The total synthesis of (+)-macrosphelide A (1) (18.5% overall yield in 11 steps), (+)-macrosphelide C (2) (25% overall yield in 9 steps), (+)-macrosphelide E (3) (23.9% overall yield in 11 steps), (+)-macrosphelide F (4) (20% overall yield in 9 steps), and (+)-macrosphelide G (5) (22% overall yield in 9 steps) was achieved from a chemoenzymatic reaction product (4R,5S)-4-benzyloxy-5-hydroxy-2(E)-hexenoate 10.     

The chemical cell biology of zinc: structure and intracellular fluorescence of a zinc-quinolinesulfonamide complex

p -toluenesulfonamido-quinoline, TSQ, are potentially powerful probes of intracellular zinc chemistry; however, the structure, thermodynamics, and stoichiometry of the metal complexes, and the molecular basis of Zn(II) recognition, remain open issues. To address these, we report the first structural characterization of a Zn(II) complex of a TSQ derivative, namely 2-methyl-6-methoxy-8-p-toluenesulfonamido-quinoline (3) and describe its unusual coordination chemistry. The crystal structure of the fluorescent complex of 3 with zinc reveals a 2 : 1 stoichiometry wherein bidentate coordination of two nitrogens from each ligand gives rise to a highly distorted tetrahedral Zn(II) center. Both sulfonamido groups in the zinc complex are tilted away from zinc to make room for coordination of the amide nitrogens. Zn-O(2) and Zn-O(4) distances are essentially nonbonding (3.06 and 3.10 Å, respectively). The bond angles [N(1)-Zn-N(2) 83.5° and N(3)-Zn-N(4) 83.0°] are quite small relative to the 109° angle of an ideal tetrahedral center. This result provides an insight into the zinc-binding mode of the TSQ derivative zinquin, in which a methyl group replaces the hydrogen in the 2-position of the quinoline ring. The methyl group and sulfonamide oxygen atoms clearly hinder formation of both square planar and octahedral complexes. We also show here that the Zn(II) complex of 3 in DMSO-water (80/20 w/w) exhibits an overall binding stability (logβ ₂ = 18.24 ± 0.02) similar to zinquin. Fluorescence microscopy suggests that each of these members of this family demarks a similar set of Zn(II)-enriched compartments that are common to all eukaryotic cells examined to date, and further shows that the ester function is not required for observation of these ubiquitous Zn-loaded compartments. The combined structural, thermodynamic, and physiological results provide a basis for design of other Zn(II)-specific membrane permeant probes with a range of Zn(II) affinities and photophysical properties. Received: 8 May 1999 / Accepted: 15 September 1999     

Total chemical synthesis of the D2 domain of human VEGF receptor 1

The interaction of the vascular endothelial growth factor (VEGF) with its cellular receptors exerts a central role in the regulation of angiogenesis. Among these receptors, the VEGF receptor 1 may be implicated in pathological angiogenesis. Here, we report the first total chemical synthesis of the VEGF‐binding domain of the VEGF receptor 1. Aggregation issues were overcome by the use of a low‐substituted resin and the stepwise introduction of pseudoproline dipeptides and Dmb‐glycines. The folding of the protein was achieved by air oxidation and its biological activity was verified on ELISA‐based assays. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae (Spirulina platensis)

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2₁ and cell parameters a =108.0 Å , b = 117.0 Å , c = 184.0 Å , β = 90.2° and 12(αβ ) units in the asymmetric unit was obtained by using (NH₄)₂SO₄ as precipitant. These crystals diffract up to 2.8 Å . Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 Å , c = 40.3 Å , γ =120.0° and one(αβ ) unit in the asymmetric unit. The crystals diffract beyond 2.9 Å . The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Template synthesis, structure and spectra of the semiclathrochelate and clathrochelate 1,4-pentadienylboron-capped iron(II) oximehydrazonates

The direct template condensation of diacetylmonooxime hydrazone with the 1,4-pentadienylboronates as Lewis acids on an iron(II) ion matrix led to the 1,4-pentadienylboron-capped semiclathrochelate iron(II) oximehydrazonates. The H⁺-ion-catalyzed macrocyclization of these precursors with an excess of triethyl orthoformate resulted in macrobicyclic complexes with a single apical 1,4-pentadienyl substituent. The complexes obtained were characterized using elemental analysis, MALDI-TOF mass spectrometry, IR, UV-Vis, ¹H, ¹¹B{¹H} and ¹³C{¹H} NMR, ⁵⁷Fe Mössbauer spectroscopies, and X-ray crystallography. The X-ray diffraction and NMR data for complexes obtained showed the syn,syn,syn-orientation of all ethoxy substituents in 1,3,5-triazacyclohexane capping fragment in relation to a clathrochelate framework.     

A cell‐free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site‐specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell‐free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single‐subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N‐glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell‐free synthesized OSTs to glycosylate multiple target proteins with varying N‐glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane‐embedded OSTs from all kingdoms of life.

     

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.     

One pot synthesis of Cu(II) 2,2'-bipyridyl complexes of 5-hydroxy-hydurilic acid and alloxanic acid: synthesis, crystal structure, chemical nuclease activity and cytotoxicity

A barbiturate derivative [1,5-dihydro-5-[5-pyrimidine-2,4(1H,3H)-dionyl]-2H-chromeno[2,3-d] pyrimidine-2,4(3H)-dione] (LH₄) was allowed to react with 2,2′-bipyridyl-dinitrato-Copper(II)-dihydrate which provides two complexes, characterized as [Cu(bpy)(L1)]·3H₂O () and [Cu(bpy)(L2)]·H₂O (), where bpy = 2,2′-bipyridine, L1 = 5-hydroxy-hydurilic acid and L2 = alloxanic acid. In a separate reaction of LH₄ with Cu(NO₃)₂·H₂O another type of complex [Cu(LH₃)₂·(H₂O)₂]·4H₂O () is formed. The complexes were characterized by single crystal X-ray crystallography, physicochemical and electrochemical studies. The interaction of complexes 1 and 3 with DNA was monitored using absorption and emission titrations as well as circular dichroism spectroscopy. The complexes were found to cleave supercoiled plasmid DNA to nicked circular and linear DNA. Complexes 1 and 3 were also tested against T-cell lymphoma (Dalton lymphoma DL) and showed significant cytotoxic activity with IC₅₀ values of ~ 9.0 nM and 0.6 nM.     

Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): Total chemical synthesis of IAG

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.