Spiroisoxazoline Inhibitors of Acetylcholinesterase from Pseudoceratina verrucosa. Quantitative Chiroptical Analysis of Configurational Heterogeneity, and Total Synthesis of (±)-Methyl Purpuroceratate C.

Examination of the MeOH extract of the sponge, Pseudoceratina cf. verrucosa, Berquist 1995 collected near Ningaloo Reef, Western Australia for selective acetylcholinesterase (AChE) inhibitors, yielded five new bromotyrosine alkaloids, methyl purpuroceratates A and B (1b and 2b), purpuroceratic acid C (3a), and ningalamides A and B (4 and 5). The structures of 1-4 share the dibromo-spirocyclohexadienyl-isoxazoline (SIO) ring system found in purealidin-R, while ketoxime 5 is analogous to ianthelline and purpurealidin I. The planar structures of all five compounds were obtained from analysis of MS, 1D and 2D NMR data, and the absolute configuration of the spiroisoxazoline (SIO) unit was assigned by electronic circular dichroism (ECD) and comparison with standards prepared by total synthesis of methyl purpuroceratate C, (±)-3b. Compound 4 is the most complex SIO described, to date. The configuration of the homoserine module (C) in 4 was ascertained, after acid hydrolysis, by derivatization of an l-tryptophanamide derivative based on Marfey's reagent. Chiral-phase HPLC, with comparison to synthetic standards, revealed that most SIOs isolated from P. cf. verrucosa were configurationally heterogeneous; some, essentially racemic. Chiral-phase HPLC, with UV-ECD detection, is demonstrated as a superlative method for configurational assignment and quantitation of the enantiomeric composition of SIOs. Two SIOs─aerophobin-1 and aplysinamisine II─emerged as selective inhibitors of AChE over butyrylcholinesterase (BuChE, IC50 ratio >10), while aplysamine-2 moderately inhibited both cholinesterases (ChEs, IC50, (AChE) 0.46 µM; IC50, (BuChE) 1.03 µM). SIO alkaloids represent a potential new structural manifold for lead-discovery of new therapeutics for treatment of Alzheimer's disease.

Detection of gut microbiota and pathogen produced N-acyl homoserine in host circulation and tissues.

Recent studies suggest that quorum-sensing molecules may play a role in gut microbiota-host crosstalk. However, whether microbiota produces quorum-sensing molecules and whether those molecules can trans-kingdom transport to the host are still unknown. Here, we develop a UPLC-MS/MS-based assay to screen the 27 N-acyl homoserine lactones (AHLs) in the gut microbiota and host. Various AHL molecules are exclusively detected in the cecal contents, sera and livers from conventionally-raised mice but cannot be detected in germ-free mice. Pathogen-produced C4-HSL is detected in the cecal contents and sera of Citrobacter rodentium (C. rodentium)-infected mice, but not found in uninfected controls. Moreover, C. rodentium infection significantly increases the level of multiple AHL molecules in sera. Our findings demonstrate that both commensal and pathogenic bacteria, can produce AHLs that can be detected in host bodies, suggesting that quorum-sensing molecules could be a group of signaling molecules in trans-kingdom microbiota-host crosstalk.

Sensing of autoinducer-2 by functionally distinct receptors in prokaryotes.

Autoinducer-2 (AI-2) is a quorum sensing signal that mediates communication within and between many bacterial species. However, its known receptors (LuxP and LsrB families) are not found in all the bacteria capable of responding to this signaling molecule. Here, we identify a third type of AI-2 receptor, consisting of a dCACHE domain. AI-2 binds to the dCACHE domain of chemoreceptors PctA and TlpQ of Pseudomonas aeruginosa, thus inducing chemotaxis and biofilm formation. Boron-free AI-2 is the preferred ligand for PctA and TlpQ. AI-2 also binds to the dCACHE domains of histidine kinase KinD from Bacillus subtilis and diguanylate cyclase rpHK1S-Z16 from Rhodopseudomonas palustris, enhancing their enzymatic activities. dCACHE domains (especially those belonging to a subfamily that includes the AI-2 receptors identified in the present work) are present in a large number of bacterial and archaeal proteins. Our results support the idea that AI-2 serves as a widely used signaling molecule in the coordination of cell behavior among prokaryotic species.

A novel, quorum sensor-infused liposomal drug delivery system suppresses Candida albicans biofilms.

In contrast to the plethora of antibacterial agents, only a handful of antifungals are currently available to treat Candida albicans biofilm-associated infections. Additional novel antibiofilm strategies to eliminate C. albicans biofilm infections are needed. This study aims to improve the efficacy of a widely used azole, fluconazole by co-delivering it with a Pseudomonas aeruginosa quorum sensing molecule (QSM), N-3-oxo-dodecanoyl-L-homoserine lactone (C12AHL) in a liposomal formulation. C12AHL is known to inhibit C. albicans' morphological transition and biofilm formation. Four different formulations of liposomes with fluconazole (L-F), with C12AHL (L-H), with fluconazole and C12AHL (L-HF), and a drug-free control (L-C) were prepared using a thin-film hydration followed by extrusion method, and characterised. The effect of liposomes on colonising (90 min-24 h) and preformed (24 h) C. albicans biofilms were assessed using a standard biofilm assay. Biofilm viability (XTT reduction assay), biomass (Safranin-O staining) and architecture (confocal laser scanning microscopy, CLSM) were determined. Similar efficiencies of fluconazole entrapment were noticed in L-HF and L-F (11.74% vs 10.2%), however, L-HF released greater quantities of fluconazole compared to L-F during 24 h (4.27% vs 0.97%, P < 0.05). The entrapment and release of C12AHL was similar for L-H and L-HF liposomes (33.3% vs 33% and 88.9% vs 92.3% respectively). L-HF treated colonising, and preformed biofilms exhibited >80%, and 60% reduction in their respective viabilities at a fluconazole concentration as low as 5.5 µg/mL compared to 12% and 36%, respective reductions observed in L-F treated biofilms (P < 0.05). CLSM confirmed biofilm disruption, lack of hyphae, and reduction in biomass when treated with L-HF compared to other liposomal preparations. Liposomal co-delivery of C12AHL and fluconazole appears to suppress C. albicans biofilms through efficacious disruption of the biofilm, killing of constituent yeasts, and diminishing their virulence at a significantly lower antifungal dose. Therefore, liposomal co-formulation of C12AHL and fluconazole appears to be a promising approach to improve the efficacy of this common triazole against biofilm-mediated candidal infections.

Genetic and Biochemical Diversity for N-acylhomoserine Lactone Biosynthesis in the Plant Pathogen Pectobacterium carotovorum subsp. carotovorum.

The plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) regulates the expression of virulence factors by N-acylhomoserine lactone (AHL)-mediated quorum sensing. The LuxI family protein, ExpI, catalyzes AHL biosynthesis in Pcc. The structure of the predominant AHL produced by ExpI differs among Pcc strains, which may be divided into two quorum-sensing classes (QS classes) based on the AHL produced. In the present study, AHL produced by 282 Pcc strains were extracted and identified by LC-MS/MS. Seventy Pcc strains produced N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) as the predominant AHL and were categorized into QS class I. Two hundred Pcc strains produced N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) as the predominant AHL, and were categorized into QS class II-1. Twelve Pcc strains produced only small amounts of 3-oxo-C6-HSL, and were categorized into QS class II-2. The phylogenetic analysis revealed that the amino acid sequences of ExpI may be divided into two major clades (I and II). The Pcc strains categorized into ExpI clades I and II entirely matched QS classes I and II, respectively. A multiple alignment analysis demonstrated that only 6 amino acid substitutions were observed among ExpI from QS classes II-1 and II-2. Furthermore, many amino acid substitutions between QS classes I and II were concentrated at the C-terminal region. These amino acid substitutions are assumed to cause significant reductions in 3-oxo-C6-HSL in QS class II-2 or affect the substrate specificity of ExpI between QS classes I and II.

Crystal structure and biochemical characterization of O-acetylhomoserine acetyltransferase from Mycobacterium smegmatis ATCC 19420.

Mycobacterium smegmatis is a good model for studying the physiology and pathogenesis of Mycobacterium tuberculosis due to its genetic similarity. As methionine biosynthesis exists only in microorganisms, the enzymes involved in methionine biosynthesis can be a potential target for novel antibiotics. Homoserine O-acetyltransferase from M. smegmatis (MsHAT) catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine. To investigate the molecular mechanism of MsHAT, we determined its crystal structure in apo-form and in complex with either CoA or homoserine and revealed the substrate binding mode of MsHAT. A structural comparison of MsHAT with other HATs suggests that the conformation of the α5 to α6 region might influence the shape of the dimer. In addition, the active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HATs.

Combining Protein and Metabolic Engineering Strategies for High-Level Production of O-Acetylhomoserine in Escherichia coli.

O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.

The crystal structure of homoserine dehydrogenase complexed with l-homoserine and NADPH in a closed form.

Homoserine dehydrogenase from Thermus thermophilus (TtHSD) is a key enzyme in the aspartate pathway that catalyses the reversible conversion of l-aspartate-ß-semialdehyde to l-homoserine (l-Hse) with NAD(P)H. We determined the crystal structures of unliganded TtHSD, TtHSD complexed with l-Hse and NADPH, and Lys99Ala and Lys195Ala mutant TtHSDs, which have no enzymatic activity, complexed with l-Hse and NADP+ at 1.83, 2.00, 1.87 and 1.93 Å resolutions, respectively. Binding of l-Hse and NADPH induced the conformational changes of TtHSD from an open to a closed form: the mobile loop containing Glu180 approached to fix l-Hse and NADPH, and both Lys99 and Lys195 could make hydrogen bonds with the hydroxy group of l-Hse. The ternary complex of TtHSDs in the closed form mimicked a Michaelis complex better than the previously reported open form structures from other species. In the crystal structure of Lys99Ala TtHSD, the productive geometry of the ternary complex was almost preserved with one new water molecule taking over the hydrogen bonds associated with Lys99, while the positions of Lys195 and l-Hse were significantly retained with those of the wild-type enzyme. These results propose new possibilities that Lys99 is the acid-base catalytic residue of HSDs.

Incorporating LsrK AI-2 quorum quenching capability in a functionalized biopolymer capsule.

Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation, and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are "biofabricated"-they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the "artificial cell" capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans.

Identification of AI-2 Quorum Sensing Inhibitors in Vibrio harveyi Through Structure-Based Virtual Screening.

Quorum sensing (QS) is a cell-to-cell communication system that regulates gene expression as a result of the production and perception of signal molecules called autoinducers (AIs). AI-2 is a QS autoinducer produced by both Gram-negative and Gram-positive bacteria, in which it regulates intraspecies and interspecies communication. The identification of QS inhibitors is considered a promising strategy for the development of anti-virulence drugs with reduced selective pressure for resistance. Here we describe a high-throughput virtual screening approach to identify AI-2 quorum sensing inhibitors on the basis of Vibrio harveyi LuxPQ crystal structure. Seven potent inhibitors with IC50 values in the micromolar range were selected with no effect or low effect on V. harveyi growth rate.

A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC12-HSL). Experimental results show that 3OC12-HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC12-HSL in the range of 0.1-1µM, and the detection limit for 3OC12-HSL was calculated to be 0.094µM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production.

Structural Insights into Substrate Specificity of Cystathionine γ-Synthase from Corynebacterium glutamicum.

Cystathionine γ-synthase (MetB) condenses O-acetyl-l-homoserine (OAHS) or O-succinyl-l-homoserine (OSHS) with cysteine to produce cystathionine. To investigate the molecular mechanisms and substrate specificity of MetB from Corynebacterium glutamicum (CgMetB), we determined its crystal structure at 1.5 Å resolution. The pyridoxal phosphate cofactor is covalently bound to Lys204 via a Schiff base linkage in the deep cavity. Superposition with the structure of MetB from Nicotiana tabacum in complex with its inhibitor dl-(E)-2-amino-5-phosphono-3-pentenoic acid revealed that Thr347 from the ß10-ß11 connecting loop, located at the entrance of the active site, is speculated to be a main contributor for stabilization of the acetyl group of OAHS. Moreover, on the basis of structural comparison of CgMetB with EcMetB utilizing OSHS as a main substrate, we propose that the conformation of the ß10-ß11 connecting loops determines the size and shape of the acetyl- or succinyl-group binding site and ultimately determines the substrate specificity of MetBs toward OAHS or OSHS.

X-ray crystal structures of the pheromone-binding domains of two quorum-hindered transcription factors, YenR of Yersinia enterocolitica and CepR2 of Burkholderia cenocepacia.

The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and some of them require AHLs for folding and stability, and for protease-resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. One such protein is YenR of Yersinia enterocolitica, which is antagonized by N-3-oxohexanoyl-l-homoserine lactone (OHHL). This pheromone is produced by the OHHL synthase, a product of the adjacent yenI gene. Another example is CepR2 of Burkholderia cenocepacia, which is antagonized by N-octanoyl-l-homoserine lactone (OHL), whose synthesis is directed by the cepI gene of the same bacterium. Here, we describe the high-resolution crystal structures of the AHL binding domains of YenR and CepR2. YenR was crystallized in the presence and absence of OHHL. While this ligand does not cause large scale changes in the YenR structure, it does alter the orientation of several highly conserved YenR residues within and near the pheromone-binding pocket, which in turn caused a significant movement of a surface-exposed loop.

Asymmetric regulation of quorum-sensing receptors drives autoinducer-specific gene expression programs in Vibrio cholerae.

Quorum sensing (QS) is a mechanism of chemical communication that bacteria use to monitor cell-population density and coordinate group behaviors. QS relies on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. Vibrio cholerae employs parallel QS circuits that converge into a shared signaling pathway. At high cell density, the CqsS and LuxPQ QS receptors detect the intra-genus and inter-species autoinducers CAI-1 and AI-2, respectively, to repress virulence factor production and biofilm formation. We show that positive feedback, mediated by the QS pathway, increases CqsS but not LuxQ levels during the transition into QS-mode, which amplifies the CAI-1 input into the pathway relative to the AI-2 input. Asymmetric feedback on CqsS enables responses exclusively to the CAI-1 autoinducer. Because CqsS exhibits the dominant QS signaling role in V. cholerae, agonism of CqsS with synthetic compounds could be used to control pathogenicity and host dispersal. We identify nine compounds that share no structural similarity to CAI-1, yet potently agonize CqsS via inhibition of CqsS autokinase activity.

A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.

Structure based virtual screening for identification of potential quorum sensing inhibitors against LasR master regulator in Pseudomonas aeruginosa.

Inter and intracellular communication in bacteria, which is known as quorum sensing (QS), is mediated by small diffusible signaling molecules known as autoinducers. QS regulates various virulence factors responsible for pathogenesis. Increasing resistance of microorganisms against traditional antibiotics has turned the focus towards the QS as it exerts less selective pressure preventing development of resistance among microorganisms. LasR, a transcription factor that controls QS in Pseudomonas aeruginosa, is an attractive therapeutic target for inhibitors. This study aimed to screen natural compounds as potential inhibitors of LasR. About 2603 compounds from ZINC database were virtually screened against the structure of LasR. Then after qualifying compounds were filtered on the parameters of Lipinski's rule and ADME. Six novel potential QS inhibiting compounds were selected on the basis of binding energy. Structures of LasR-ligand complexes were analysed to have insight of binding between inhibitors and target. It is pertinent to mention here that all the molecules are structurally different from 3-oxo-C12HSL,a native autoinducer of LasR, that play key role in formation of LasR dimer which is an active form of the protein to facilitate QS.

Perfluoro-tert-butyl Homoserine Is a Helix-Promoting, Highly Fluorinated, NMR-Sensitive Aliphatic Amino Acid: Detection of the Estrogen Receptor·Coactivator Protein-Protein Interaction by 19F NMR.

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by 19F NMR and the absence of native 19F signals in biological contexts. However, the potential applications of 19F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has nine equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) coactivator peptides. A peptide containing perfluoro-tert-butyl homoserine at position i + 3 of the ER coactivator LXXLL motif exhibited a Kd of 2.2 µM for the estradiol-bound estrogen receptor, similar to that of the native ligand. 19F NMR spectroscopy demonstrated the sensitive detection (5 µM concentration, 128 scans) of binding of the peptide to the ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine in probing protein function and protein-protein interfaces in complex solutions.

Methoxinine - an alternative stable amino acid substitute for oxidation-sensitive methionine in radiolabelled peptide conjugates.

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen-bonding properties. We report here the use of methoxinine (Mox), a non-canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met15 by Mox15 and Nle15 in the binding sequence of a radiometal-labelled human gastrin derivative [d-Glu10 ]HG(10-17), named MG11 (d-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ). A comparison of the physicochemical properties of 177 Lu-DOTA[X15 ]MG11 (X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC50 ), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation-stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Homoserine as an Aspartic Acid Precursor for Synthesis of Proteoglycan Glycopeptide Containing Aspartic Acid and a Sulfated Glycan Chain.

Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is the first time that a glycopeptide containing aspartic acid and an O-sulfated glycan was synthesized.