Β-hydroxyaspartic Acid Research Articles

Total Synthesis of Malacidin A by β‐Hydroxyaspartic Acid Ligation‐Mediated Cyclization and Absolute Structure Establishment

The development of novel antibiotics is critical to combating the growing emergence of drug-resistant pathogens. Malacidin A is a new member of the calcium-dependent antibiotic (CDAs) family with activity against antibiotic-resistant pathogens. Its mode of action is distinct from classical CDAs. However, the absolute structure of malacidin A has not been established. Herein, the total syntheses of malacidin A and its analogues are reported by a combination of Fmoc-based solid-phase peptide synthesis (SPPS) and β-hydroxyaspartic acid ligation-mediated peptide cyclization. The total synthesis enabled us to establish the absolute configuration of malacidin A, which is in agreement with those for natural malacidin A confirmed by advanced Marfey's analysis in our study.

Potashchelins, a Suite of Lipid Siderophores Bearing Both L-threo and L-erythro Beta-Hydroxyaspartic Acids, Acquired From the Potash-Salt-Ore-Derived Extremophile Halomonas sp. MG34.

Four new lipid siderophores bearing both L-threo- and L-erythro-β-hydroxyaspartic acids, potashchelins A-D (1-4), were isolated from the potash-salt-ore-derived extremophile Halomonas sp. MG34. The planar structures of 1-4 were elucidated on the basis of extensive 1D and 2D NMR studies and MS/MS data. Potashchelins 1-4 contain a hydrophilic nonapeptide headgroup sequentially consisting of β-hydroxyaspartic acid, serine, glycine, serine, serine, β-hydroxyaspartic acid, threonine, serine, and cyclic N(δ)-hydroxy-ornithine, which is appended by one of a series of fatty acids ranging from dodecanoic acid to tetradecanoic acid. The absolute configurations of the amino acids of potashchelins 1-4 were determined by C3 and advanced Marfey's reaction, partial hydrolysis, and bioinformatics analysis, which revealed that potashchelins 1-4 bear both L-threo- and L-erythro-β-hydroxyaspartic acid. Phylogenetic analysis showed that the stand-alone β-hydroxylase, PtcA, and the fused domain with β-hydroxylase activity in PtcB are expected to be responsible for the formation of L-erythro and L-threo diastereomers, respectively. Additionally, utilizing a comparative genomics approach, we revealed an evolutionary mechanism for lipid siderophores in Halomonas involving horizontal transfer. Bioassays showed that potashchelin A and D had weak antibacterial activity against B. subtilis CPCC 100029 with an MIC value of 64 μg/mL.

Ambiguity of NRPS Structure Predictions: Four Bidentate Chelating Groups in the Siderophore Pacifibactin.

Identified through a bioinformatics approach, a nonribosomal peptide synthetase gene cluster in Alcanivorax pacificus encodes the biosynthesis of the new siderophore pacifibactin. The structure of pacifibactin differs markedly from the bioinformatic prediction and contains four bidentate metal chelation sites, atypical for siderophores. Genome mining and structural characterization of pacifibactin is reported herein, as well as characterization of pacifibactin variants accessible due to a lack of adenylation domain fidelity during biosynthesis. A spectrophotometric titration of pacifibactin with Fe(III) and 13C NMR spectroscopy of the Ga(III)-pacifibactin complex establish 1:1 metal:pacifibactin coordination and reveal which of the bidentate binding groups are coordinated to the metal. The photoreaction of Fe(III)-pacifibactin, resulting from Fe(III) coordination of the β-hydroxyaspartic acid ligands, is reported.

β-Hydroxyaspartic acid in siderophores: biosynthesis and reactivity.

A growing number of siderophores are found to contain β-hydroxyaspartic acid (β-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic β-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl β-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of β-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for β-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.

Efficient preparation of β-hydroxy aspartic acid and its derivatives

We report an efficient and practical synthetic route to various properly-protected erythreo-β-OH-Asp compounds, which are key β-branched α-amino acid units in coralmycin A and other peptide natural products. Fmoc and cyclic ketal-protected erythreo-β-OH-Asp 7 is prepared from cheap chiral precursor l-diethyl tartrate in six steps without the need of column purification. The modified form of 7 serves as a versatile precursor to various β-alkoxyl analogs of erythreo-β-OH-Asp. In addition, we successfully performed a model study toward the total synthesis of coralmycin A, featuring a late stage installation of the side chain primary amide group of erythreo-β-OMe-Asn.

Poecillastrin H, a Chondropsin-Type Macrolide with a Conjugated Pentaene Moiety, from a Characella sp. Marine Sponge.

Poecillastrin H (1), a chondropsin-type macrolide with a conjugated pentaene moiety, was isolated from the Characella sp. marine sponge. The planar structure of 1 was elucidated by analysis of spectroscopic data. The absolute configuration of the β-hydroxyaspartic acid residue (β-OHAsp) was determined to be d- threo by Marfey's analysis, and the mode of lactone ring formation through the OHAsp residue was determined by chemical degradation. Poecillastrin H was extremely sensitive toward light and showed potent cytotoxic activity against 3Y1 cells with an IC50 value of 4.1 nM.

Poecillastrin E, F, and G, cytotoxic chondropsin-type macrolides from a marine sponge Poecillastra sp.

Poecillastrin E (1), F (2), and G (3) were isolated from a marine sponge Poecillastra sp. as the cytotoxic constituents. Their planar structures were determined by analyzing the MS and NMR spectra. They are closely related to the known poecillastrin C (4). The absolute configuration of the β-hydroxyaspartic acid (OHAsp) residue was determined to be D-threo by Marfey's analysis of the hydrolysate. The mode of lactone ring formation of OHAsp residue in 1–3 was determined by selective reduction of the ester linkage followed by acid hydrolysis.

Structure Revision of Poecillastrin C and the Absolute Configuration of the β-Hydroxyaspartic Acid Residue.

The planar structure of poecillastrin C (1) was revised through selective reduction of the ester carbon. The absolute configuration of the β-hydroxyaspartic acid (OHAsp) residue was determined to be d-threo by Marfey's analysis. The acid hydrolysate of the reduction product of 1 liberated (2R,3R)-2-amino-3,4-dihydroxybutanoic acid, demonstrating that the β-carboxyl group in poecillastrin C was esterified. The structures of poecillastrins B-D and 73-deoxychondropsin A were also revised.

Synthesis of a protected derivative of (2R,3R)-β-hydroxyaspartic acid suitable for Fmoc-based solid phase synthesis

Synthesis of d-threo-hydroxyaspartic acid, orthogonally protected and compatible with an Fmoc solid-phase peptide synthesis strategy is reported. This synthetic procedure starting from (2R,3R)-dimethyltartrate is adaptable to a multi-gram scale. 2,2-Dimethyl-5-oxo-1,3-dioxazolane formation between the β-hydroxy alcohol and the β-carboxylic functions constitutes a key step of the differentiation of the two acidic functions allowing the preparation of (2R,3R)-Fmoc-β-hydroxyaspartic α-allyl ester.

Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity.

Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa. The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [(3)H]lysine (15% of maximum activity) and [(3)H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.

Nucleophilic Additions of 2-Furyllithium to Carbonyl Derivatives of L-Serine. Formal Synthesis of (2R,3R)-β-Hydroxy Aspartic Acid

The nucleophilic addition of 2-furyllithium to esters derived from L-serine is described. The obtained furyl ketone 5 is stereoselectively reduced (ds≥95%) with sodium borohydride to afford the corresponding syn aminoalcohol 12 in enantiomerically pure form. Compound 12 was further converted into valuable α-hydroxy-β-amino acids by means of the furan-to-acid equivalence.

Regulation of glutamate in cultures of human monocytic THP-1 and astrocytoma U-373 MG cells

Glutamate, an excitatory neurotransmitter, is neurotoxic at high concentrations. Neuroglial cells, including astrocytes and microglia, play an important role in regulating its extracellular levels. Cultured human monocytic THP-1 cells increased their glutamate secretion following 18 and 68 h exposure to the inflammatory mediators zymosan, phorbol myristate acetate (PMA), lipopolysaccharide, interferon- γ, tumor-necrosis factor- α and interleukin-1 β. Cultured astrocytoma U-373 MG cells increased their glutamate secretion following similar exposure to zymosan and PMA. dl- α-aminopimelic acid, an inhibitor of the glutamate secretion system, reduced extracellular glutamate in both cell culture systems, while the high-affinity glutamate uptake inhibitors d-Aspartic acid, dl- threo- β-hydroxyaspartic acid and l- trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular glutamate in U-373 MG, but not THP-1 cell cultures. In co-cultures of THP-1 and U-373 MG cells, extracellular glutamate levels were increased significantly by the Alzheimer β-amyloid peptide (1–40) and were decreased significantly by the anti-inflammatory drug dexamethasone. These data indicate that inflammatory stimuli may increase extracellular glutamate while antiinflammatory drugs decrease it.

Characterization of metabotropic glutamate receptors in rat C6 glioma cells

Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both l-[ 3H]glutamate and [ 3H](±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ([ 3H](±)- trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a K d=1250±101 nM and B max=12.1±1.8 pmol/mg protein when the assays were performed with l-[ 3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and d l-threo- β-hydroxyaspartic acid. When [ 3H](±)- trans-ACPD was used as radioligand, the kinetic parameters obtained were K d=2605±1042 nM and B max=13.66±5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of l-[ 3H]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity l-glutamate> l-quisqualic acid>(±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid ( trans-ACPD)>ibotenic acid⋙(2 S,1′ S,2′ S)-2-(carboxycyclopropyl)glycine ( l-CCG-I). Specific binding of l-[ 3H]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the l-glutamate dose-response curve, increasing the IC 50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.

Structures and characteristics of novel siderophores from plant deleterious Pseudomonas fluorescens A225 and Pseudomonas putida ATCC 39167.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

On-line separation of phenylthiohydantoin derivatives of hydrophilic modified amino acids during sequencing

All 20 commonly occurring phenylthiohydantoin (PTH) amino acids can be separated by a new reversed-phase chromatography method without interference from breakthrough peaks that prevail in other methods, and with good resolution from the most common reagent-related background peaks. Separation is compatible with automated sequencing in a cartridge sequencer. A large chromatographic area exists between the breakthrough peaks and the first- and second-eluting common PTH-amino acids, PTH-His and PTH-Asn, and is consequently available for detection and separation of hydrophilic amino acid derivatives. Derivatives such as phosphotyrosine, β-hydroxyaspartic acid and N-glycosylated asparagine elute as readily identifiable peaks. The method uses chromatography on octadecylsilane with isocratic elution followed by a simple linear gradient from acetonitrile—methanol to isopropanol—methanol to provide complete separation in 23 min. The turnaround time of 34 min includes a period of column washing to eliminate sequencer byproducts. PTH-Amino acids are generally detected with single pmol (1 to 4 pmol) sensitivity. PTH-His and PTH-Arg are eluted earlier than conventionally with a consequent sharpening of peaks and higher practical sensitivity.

Dehydrooligopeptides. X. Useful Synthetic Method for (E)- and (Z)-Isomers of Dehydroaspartic Acids, and Their Δ1,2-Dehydrodipeptides by the Base-Catalyzed β-Elimination

Abstract Various kinds of N-benzyloxycarbonyl (E)- and (Z)-dehydroaspartic acid (Cbz–ΔAsp) derivatives were successfully synthesized by both a base-catalyzed β-elimination of β-hydroxyaspartic acid esters (HyAsp) via their β-mesyloxy Asp derivatives and several modes of the cleavages of α-(N-Cbz)-aminomaleic acid anhydride. Moreover, for high-yield synthesis of Δ1,2-dehydrodipeptides (DHP) containing a ΔAsp residue, a similar β-elimination was applied to Δ1-DHP with a C-terminal HyAsp residue. The configuration of all the new ΔAsp derivatives and their Δ1,2-DHPs were confirmed mainly on the basis of 1H NMR spectral data.

Assignment of the configurations of the amino acids in peptidic siderophores

Pyoverdins and azotobactins contain β-hydroxyaspartic acid, N δ-hydroxyornithine, citrulline and homoserine, in addition to the common protein amino acids. Configuration assignment of all of these was achieved by acid hydrolysis of the peptide, derivatization of the constituent amino acids to the N-pentafluoropropionyl amino acid esters and gas chromatographic separation of the stereoisomers on capillaries coated with Chirasil-Val. This approach is straightforward for the protein amino acids, but the less common amino acids are either partially degraded during acid hydrolysis or their derivatives exhibit unfavourable gas chromatographic properties. By judicious combination of partial and total hydrolysis and dual derivatization, these problems may be overcome.

Characterization of Pyoverdin pss , the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin(pss), in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin(pss) of approximately 50 mug/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of >/= 10 muM. Pyoverdin(pss) was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin(pss) and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin(pss) was structurally unique since amino acid analysis of reductive hydrolysates yielded beta-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin(pss) exhibited a relatively high affinity constant for Fe(III), with values of 10 at pH 7.0 and 10 at pH 10.0. Iron uptake assays with [Fe]pyoverdin(pss) demonstrated rapid active uptake of Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin(pss). The chemical and biological properties of pyoverdin(pss) are discussed in relation to virulence and iron uptake during plant pathogenesis.

Calcium binding to a human factor IXa derivative lacking γ-carboxyglutamic acid: Evidence for two high-affinity sites that do not involve β-hydroxyaspartic acid

A derivative of human blood clotting factor IXaβ lacking γ-carboxyglutamic acid (Gla) residues was prepared by limited proteolysis with chymotrypsin, and subsequently examined for its ability to bind calcium ions. By amino acid analysis, Gla-domainless human factor IXaβ contained 0.3–0.4 moles of β-hydroxyaspartic acid per mole of protein. Equilibrium dialysis experiments demonstrated that Gla-domainless human factor IXaβ retained two high-affinity calcium binding sites (K d=52 μM), a finding essentially identical to that observed for Gla-domainless bovine factor IX that contains 0.8–0.9 moles of β-hydroxyaspartic acid per mole of protein. These data strongly suggest that the β-hydroxyaspartic acid residue in these proteins does not participate in their high affinity calcium sites.

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues ( M r 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 γ-carboxyglutamic acid and one β-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.