Serine Analogues Research Articles

Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes.

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-l-SerN3) or carbonyl (C-l-SerN3) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.

Dynamic Metabolic Response to (p)ppGpp Accumulation in Pseudomonas putida.

The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These alarmones, jointly referred to as (p)ppGpp, control gene transcription, mRNA translation and protein activity to adjust the metabolism and growth rate to environmental changes. While the ability of (p)ppGpp to mediate cell growth slowdown and metabolism adaptation has been demonstrated in Escherichia coli, it’s role in Pseudomonas putida remains unclear. The aims of this study were therefore to determine which forms of (p)ppGpp are synthetized in response to severe growth inhibition in P. putida, and to decipher the mechanisms of (p)ppGpp-mediated metabolic regulation in this bacterium. We exposed exponentially growing cells of P. putida to serine hydroxamate (SHX), a serine analog known to trigger the stringent response, and tracked the dynamics of intra- and extracellular metabolites using untargeted quantitative MS and NMR-based metabolomics, respectively. We found that SHX promotes ppGpp and pppGpp accumulation few minutes after exposure and arrests bacterial growth. Meanwhile, central carbon metabolites increase in concentration while purine pathway intermediates drop sharply. Importantly, in a ΔrelA mutant and a ppGpp0 strain in which (p)ppGpp synthesis genes were deleted, SHX exposure inhibited cell growth but led to an accumulation of purine pathway metabolites instead of a decrease, suggesting that as observed in other bacteria, (p)ppGpp downregulates the purine pathway in P. putida. Extracellular accumulations of pyruvate and acetate were observed as a specific metabolic consequence of the stringent response. Overall, our results show that (p)ppGpp rapidly remodels the central carbon metabolism and the de novo purine biosynthesis pathway in P. putida. These data represent a hypothesis-generating resource for future studies on the stringent response.

Fluorinated O-phenylserine residues enhance the broad-spectrum antimicrobial activity of ultrashort cationic lipopeptides

As a crucial structural element in many antimicrobial lipopeptides, phenylalanine poses a potential, but sensitive, point of modification in the pursuit of enhancing the antimicrobial efficacy of peptide antibiotics. We report the synthesis and application of a library of fluorinated O-phenyl serine analogues, followed by biological testing on relevant bacterial and fungal pathogens and the comparison of these analogues to commercial fluorinated phenylalanine amino acids. The synthetic aromatic residues were investigated for their enhanced potency over the original Phe when incorporated into a model ultrashort lipopeptide sequence derived from the octapeptin battacin and were found to enhance broad-spectrum antimicrobial activity across the panel of microbial species (while retaining non-hemolytic activity). While the major drawback of antimicrobial peptides is the cost of synthesis, they are some of the last antimicrobials to be majorly affected by antimicrobial resistance, making them a promising class of antibiotic for studying. Antimicrobial activity of the most potent fluorinated peptide was around 16-fold greater than the unfluorinated Phe-peptide against the fire blight pathogen, with milder increases in potency toward other pathogens such as S. aureus and E. coli. This indicates a potential future direction for these peptides to be developed as crop treatments to protect rosaceae from fire blight.

Novel mouse GABA uptake inhibitors with enhanced inhibitory activity toward mGAT3/4 and their effect on pain threshold in mice

γ-Aminobutyric acid (GABA) uptake transporters are membrane transport proteins that are involved in the pathophysiology of a number of neurological disorders. Some types of chronic pain appear to result from the dysfunction of the GABAergic system. The deficiency of mouse GAT1 transporter (mGAT1) abolishes the nociceptive response, which means that mGAT1 inhibition is an appropriate medical approach to achieve analgesia. The mGAT4 transporter is the second most abundant GAT subtype in the brain; however, its physiological role has not yet been fully understood in the central nervous system. In this study, we examined whether the combination of mGAT1 and mGAT3/mGAT4 inhibition in a single molecule might lead to potentially synergistic effects improving analgesic activity to relieve neuropathic pain. To study this hypothesis, new GABA uptake inhibitors were designed, synthesized, and evaluated in terms of their activity and subtype selectivity for mGAT1-4. Among new functionalized amino acid derivatives of serine and GABA analogs, compounds with preferential mGAT3/4 inhibitory activity were discovered. Two selected hits (19b and 31c) were subjected to in vivo tests. We found a statistically significant antiallodynic activity in the von Frey test in diabetic and oxaliplatin-induced neuropathic pain model. The novel compounds (4-hydroxybutanoic, 4-hydroxypentanoic, and 4-aminobutanoic acid derivatives and serine analogs) provide new insights into the structure–activity relationship of mGAT3/mGAT4 inhibitors and indicate a new direction in the search for potential treatment of neuropathic pain of various origin.

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae.

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi-chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division-replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.

Design of Glycosyltransferase Inhibitors: Serine Analogues as Pyrophosphate Surrogates?

Mimicking the diphosphate moiety of nucleotide diphosphate sugars with serine analogues provided modest glycosyltransferase inhibitors. The synthetic strategy employed a combination of glycosylation, amide bond formation and azide-alkyne "click" chemistry. Inhibition constants (Ki ) in the high micromolar range were obtained with a selection of five galactosyltransferases. Cocrystals of three inhibitors bound at the active site of a blood group A/B synthesizing glycosyltransferase were analysed. The structures and inhibitory patterns of the analogues demonstrate the flexibility of the enzymes which complicates the rational design of glycosyltransferase inhibitors.

N‐Substituted Glycines with Functional Side‐Chains for Peptoid Synthesis

AbstractN‐Substituted glycines (NSG) constitute mimics of natural amino acids, and bring stability to peptide analogues. We developed a method to synthesize NSG bearing reactive secondary heterofunctionality. It was shown that persilylation could be used for temporary protection of reactive groups present on the substrate, with the aim of selectively alkylating the amine groups. NSG ready for peptide coupling were obtained by C‐terminal deprotection and N‐terminal protection of the resulting amino acid derivatives. This method was applied to prepare N‐alkylated glycines as analogues of serine, cysteine, tyrosine and tryptophan. The last two derivatives were obtained in good yields and N‐homotyrosine (N‐hTyr) has been introduced into a peptide sequence of interest using automated solid‐phase peptide synthesis (SPPS).

Modulation of the extinction of fear learning.

We review recent work on extinction learning with emphasis on its modulation. Extinction is the learned inhibition of responding to previously acquired tasks. Like other forms of learning, it can be modulated by a variety of neurotransmitter systems and behavioral procedures. This bears on its use in the treatment of fear memories, particularly in posttraumatic stress disorder (PTSD), for which it is the treatment of choice, often under the name of exposure therapy. There have not been many laboratories interested in the modulation of extinction, but the available data, although not very abundant, are quite conclusive. Most studies on the nature of extinction and on its modulation have been carried out on fear motivated behaviors, possibly because of their applicability to the therapy of PTSD. A role for d-serine and the glycine site of NMDA receptors has been ascertained in two forms of extinction in the ventromedial prefrontal cortex, basolateral amygdala and dorsal hippocampus. The serine analog, d-cycloserine, has received clinical trials as an enhancer of extinction. The brain histaminergic system acting via H2 receptors, and the endocannabinoid system using CB1 receptors in the ventromedial prefrontal cortex, hippocampus and basolateral amygdala enhance extinction. Dopaminergic D1 and ß-noradrenergic receptors also modulate extinction by actions on these three structures. Isolated findings suggest roles for on serotonin-1A, dopaminergic-D2 and a- and ß-noradrenergic receptors in extinction modulation. Importantly, behavioral tagging and capture mechanisms in the hippocampus have been shown to play a major modulatory role in extinction. In addition, extinction of at least one aversive task (inhibitory avoidance) can be made state dependent on peripheral epinephrine.

Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Synthesis and Application of Enantioenriched Functionalized α-Tetrasubstituted α-Amino Acids from Biocatalytic Desymmetrization of Prochiral α-Aminomalonamides

Catalyzed by Rhodococcus erythropolis AJ270, an amidase-containing microbial whole cell catalyst in neutral phosphate buffer at 30 °C, a number of prochiral α-substituted α-aminomalonamides underwent highly efficient and enantioselective hydrolytic desymmetrization to afford functionalized α-tetrasubstituted α-amino acids in 74-98% chemical yields and 94.0 to >99.5% ee. The presence of a free α-amino (NH(2)) substituent in the substrates was deemed important to ensure high biocatalytic efficiency and enantioselectivity. The synthetic application of biocatalytic desymmetrization was demonstrated by practical chemical transformations of (R)-2-amino-2-carbamoylpent-4-enoic acid to α-substituted serine analogues and a bioactive diamino alcohol derivative.

Synthesis of analogues of ochratoxin A

Four analogues of ochratoxin A (OTA) differing for the aminoacidic moiety were synthesised using ochratoxin α (OTα) as the starting material. The condensation reaction between protected amino acids and OTα, carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC • HCl) and N-hydroxybenzotriazole (HOBt) as coupling agents, followed by deprotection and PTLC purification afforded OTA alanine, leucine, serine and tryptophane analogues in satisfactory yields (33–47%, based on OTα).

Pre-transfer Editing of Serine Hydroxamate within the Active Site of Methanogenic-type Seryl-tRNA Synthetase

Aminoacyl-tRNA synthetases (aaRSs) maintain fidelity of protein synthesis by matching only cognate amino acid-tRNA pairs. Aminoacylation occurs through activation of amino acid to yield aminoacyl-adenylate followed by transfer of acyl-moiety to tRNA. Error-prone aaRSs achieve high level of accuracy using inherent hydrolytic activities towards noncognate aminoacyl-adenylate or misacylated tRNA (pre- and post-transfer editing).Seryl-tRNA synthetases can be divided into two structurally different types: canonical and methanogenic-type. Both types have been shown to efficiently activate serine analogue serine hydroxamate (SerHX). Moreover, this analogue has been also eliminated by pre-transfer editing within the canonical synthetic site of yeast SerRS. Here we show that methanogenic-type SerRS from Methanosarcina barkeri clears misactivated SerHX similarly as the yeast enzyme: SerHX-adenylate is not expelled into solution, but is enzymatically hydrolyzed in a tRNA-independent manner. Since the enzyme lacks domain specialized in editing, this shows that methanogenic-type catalytic core is also capable to perform pre-transfer editing.

Free Energy Calculations of the Adsorption of Amino Acid Analogues at the Aqueous Titania Interface

The potential of mean constraint force approach, in partnership with atomistic molecular dynamics simulations, is used to calculate the change in free energy upon adsorption of amino acid side chain analogues at the aqueous rutile titania (110) interface. Our results indicate that both positively charged and negatively charged moieties have favorable free energy of binding to the titania surface. Stable contact, mediated via the first two solvent layers at the interface, is also indicated for the charged adsorbates. Hydrophobic adsorbates showed no appreciable binding to the titania surface. In contrast, the binding for our serine analogue (methanol) features a slight possibility to bind via hydrogen bonding to the titania surface. Our calculated results indicate very good agreement with available experimental data. In partnership with other information regarding peptide conformation and intrapeptide interactions, our findings should be helpful in building design principles for peptide sequences with pred...

ChemInform Abstract: Asymmetric Synthesis of (2R,3S)-4-Halo-3-benzyloxy-2- (N-methoxycarbonyl-N-benzylamino)butyronitriles as Precursors for the Synthesis of β-Hydroxy-α-amino Acids.

Abstract (2R,3S)-4-Halo-3-benzyloxy-2-(N-methoxycarbonyl-N-benzylamino)butyronitriles have been prepared through an efficient three-step sequence from (2R,3S)-2-benzylamino-3-benzyloxy-4-(tert-butyldimethylsilyloxy)butyronitrile, which is readily available in diastereomerically pure form by a Strecker-type reaction of the N-benzylimine, derived from selectively protected (R)-glyceraldehyde, and trimethylsilyl cyanide. These compounds enable the facile synthesis of chiral β-hydroxy-α-amino acids containing virtually any nucleophile capable of substituting the γ-halogen atom. As an illustration of their synthetic potential, the 4-bromo derivative has been successfully converted into (1R,2R)-2-benzyloxy-1-(N-methoxycarbonyl-N-benzylamino)cyclopropanecarboxamide, which is a new conformationally restricted serine analogue, in two steps: base-induced cyclisation and subsequent hydrolysis of the nitrile group.

ChemInform Abstract: Highly Stereoselective Synthesis of New α‐Amino‐β‐hydroxy Six‐Membered Heterocyclic Phosphonic Acids, Serine Analogues.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Identification of a Novel l-Serine Analog That Suppresses Osteoclastogenesis in Vitro and Bone Turnover in Vivo

Osteoclasts are multinucleated giant cells with bone resorbing activity. We previously reported that the expression of the transcription factor NFAT2 (NFATc1) induced by receptor activator of NF-kappaB ligand (RANKL) is essential for the formation of multinucleated cells. We subsequently identified L-Ser in the differentiation medium as necessary for the expression of NFAT2. Here we searched for serine analogs that antagonize the function of L-Ser and suppress the formation of osteoclasts in bone marrow as well as RAW264 cells. An analog thus identified, H-Ser(tBu)-OMe x HCl, appeared to suppress the production of 3-ketodihydrosphingosine by serine palmitoyltransferase, and the expression and localization of RANK, a cognate receptor of RANKL, in membrane lipid rafts was down-regulated in the analog-treated cells. The addition of lactosylceramide, however, rescued the osteoclastic formation. When administered in vivo, the analog significantly increased bone density in mice and prevented high bone turnover induced by treatment with soluble RANKL. These results demonstrate a close connection between the metabolism of L-Ser and bone remodeling and also the potential of the analog as a novel therapeutic tool for bone destruction.

Totally drug-resistant tuberculosis strains: evidence of adaptation at the cellular level: Fig. 1—

To the Editors: In the Eastern Mediterranean Region (EMRO), there are no accurate estimates of numbers of patients with multidrug-resistant (MDR)/extensively drug-resistant (XDR) tuberculosis (TB). In 2006, we documented the existence and transmission of XDR-TB among patients with MDR-TB 1. In a further study, we isolated more dangerous forms of bacilli, named totally drug-resistant (TDR) 2. This group of strains showed in vitro resistance to all first- and second-line drugs tested ( i.e. aminoglycosides, cyclic polypeptides, fluoroquinolones, thioamides, serine analogues and salicylic acid derivatives). TDR-TB patients remained smear- and culture-positive after 18 months’ median treatment with second-line drugs. Even changing the treatment to co-amoxyclav (625 mg per 8 h) or clarithromycin (1,000 mg·day−1), along with a high dose of isoniazid (15 mg·kg−1) led to no improvement 2. In the present study, transmission electron microscopy (TEM) was used to evaluate the differences between such deadly bacilli and MDR or susceptible ones at the ultrastructural level. Furthermore, to evaluate the genotyping patterns, the strains were subjected to spoligotyping and variable number of tandem repeats (VNTR) analysis. Susceptibility testing against first- and second-line drugs was performed using the proportional method on Lowenstein–Jensen media 3. For spoligotyping and VNTR analysis, five isolates from each group of strains …

Highly stereoselective synthesis of new α-amino-β-hydroxy six-membered heterocyclic phosphonic acids, serine analogues

The synthesis of 2-hydroxy-4-heterocyclic phosphonic acids was achieved in a six-step sequence from the appropriate ketones. Thus, 2-hydroxyheterocyclic ketone acetals were prepared and then esterified by N-Boc- l-phenylalanine, used as a chiral auxiliary. The resulting heterocyclic acetal esters gave by a one-pot reaction bicyclic ketimines. These imines underwent nucleophilic addition with phosphite to provide efficiently and stereoselectively, under kinetic control, bicyclic aminophosphonates. Cleavage of the phenylalanine moiety by oxidation followed by acidic hydrolysis of the resulting heterocyclohexylphosphonates provided the new (4-amino-3-hydroxypiperidin-4-yl)-, (4-amino-3-hydroxytetrahydro-2 H-pyran-4-yl)- and (1-amino-2-hydroxycyclohexyl)phosphonic acids.

Development of a practical mass spectrometry based assay for determining enantiomeric excess. A fast and convenient method for the optimization of PLE-catalyzed hydrolysis of prochiral disubstituted malonates

A practical mass spectrometry-based enantioselectivity assay is presented which makes use of enantiomerically enriched, but not enantiomerically pure, probe molecules readily obtained from esterase hydrolysis of prochiral malonates. The technique presented here allows us to recycle materials obtained from esterase hydrolysis which give substantial, but synthetically insufficient, enantiomeric excess as probe molecules in an enantioselectivity assay. The enantiomerically enriched products are esterified using deuterium-labelled alcohol. The enantiomeric excess is measured using mass spectrometry (LC–MS and LDI) by measuring the D 5/H 5 ratio in the resulting products obtained from an enzymatic hydrolysis. The D 5/H 5 ratio is corrected to account for the enantiomeric purity of the probe. Herein we report the results obtained from Pig Liver Esterase hydrolyses of prochiral malonate esters and outline the strengths and limitations of this approach to enantioselectivity determinations. This assay strategy was able to identify reaction conditions that led to an improvement in ee from 70% ee to >97% ee in the PLE-catalyzed hydrolysis of a prochiral malonate used to prepare unnatural serine analogues.

The Nature and Sequence of the Amino Acid Aglycone Strongly Modulates the Conformation and Dynamics Effects of Tn Antigen's Clusters

Synthetic oligosaccharide vaccines based on carbohydrate epitopes are currently being evaluated as potential immunotherapeutics in the treatment of cancer. In an effort to study the role that the amino acid moiety (L-serine and/or L-threonine residues) plays on the global shape of the resulting glycopeptides and on the dynamics of the carbohydrate moiety, diverse glycopeptides based on the Tn antigen have been synthesized and studied in aqueous solution by combining NMR spectroscopic experiments and molecular dynamics simulations. Our results demonstrate that although the effect of the clustering of Tn on the peptide backbone is not remarkable, it substantially modifies the dynamics, and thus, the presentation features of the carbohydrate moiety. In fact, the selected sequence has a crucial influence on both the orientation and flexibility of the sugar region. Thus, although a serine-threonine pair shows a well-defined spatial disposition of the Tn epitopes, its analogue sequence threonine-serine allows a certain degree of mobility that could favor the interaction with a diversity of receptors without a major energy penalty. These features can be explained by attending to the different conformational behavior of the glycosidic linkage of threonine-containing glycopeptides when compared with those of the serine analogues. On this basis, and taking into account that these carbohydrates interact with components of the immune system, these findings could have implications for further design of new cancer vaccines.