Acylation Pattern Research Articles

Exploiting membrane vesicles derived from avian pathogenic Escherichia coli as a cross-protective subunit vaccine candidate against avian colibacillosis

Avian pathogenic Escherichia coli (APEC) is a notable pathogen that frequently leads to avian colibacillosis, posing a substantial risk to both the poultry industry and public health. The commercial vaccines against avian colibacillosis are primarily inactivated vaccines, but their effectiveness is limited to specific serotypes. Recent advances have highlighted bacterial membrane vesicles (MV) as a promising candidate in vaccine research. How to produce bacterial MVs vaccines on a large scale is a significant challenge for the industrialization of MVs. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in lipopolysaccharides (LPS). Here, we evaluated the immunoprotective efficacy of MVs derived from the LPS low-expressed APEC strain FY26ΔmsbB, which was an APEC mutant strain with a deletion of the msbB gene. The nitrogen cavitation technique was employed to extract APEC MVs, with results indicating a significant increase in MVs yield compared to that obtained under natural culture. The immunization effectiveness was assessed, revealing that FY26ΔmsbB MVs elicited an antibody response of laying hens and facilitated bacterial clearance. Protective efficacy studies demonstrated that immunization with FY26ΔmsbB MVs conferred the immune protection in chickens challenged with the wild-type APEC strain FY26. Notably, LPS low-carried MVs recovered from the mutant FY26ΔmsbB also displayed cross-protective capabilities, and effectively safeguarding against infections caused by O1, O7, O45, O78, and O101 serotypes virulent APEC strains. These findings suggest that MVs generated from the LPS low-expressed APEC strain FY26ΔmsbB represent a novel and empirically validated subunit vaccine for the prevention and control of infections by various APEC serotypes.

Acylated anthocyanins from Dendrobium officinale Kimura & Migo: Structural characteristics, antioxidant and hypoglycemic activities

Dendrobium officinale Kimura & Migo (D. officinale) has been widely used as Chinese medicine and functional food. In present study, the structural characteristics of anthocyanins in D. officinale were investigated by ultra-performance liquid chromatography with diode array detector (UPLC-DAD) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS/MS). Totally, 14 anthocyanins were detected and identified, and 13 of them were first reported in D. officinale. Results showed that the vast majority of anthocyanins had multi-glycosylated cyanidin core, with variable acylation pattern mainly comprising phenolic acids. The composition and content of anthocyanins in D. officinale stems with different cultivation modes and years have been compared. The anthocyanins showed potent antioxidant activity in terms of radicals scavenging capacity and reducing power, as well as superior α-amylase and α-glucosidase inhibitory activity. The results provided a complete profile of anthocyanins in D. officinale and laid a foundation for further utilizing them as functional foods.

Lipase-catalyzed synthesis of laurate esters from puerarin and its β-D-fructofuranosyl-(2→6)-puerarin derivative

This study investigates the transesterification of isoflavones puerarin and β-D-fructofuranosyl-(2→6)-puerarin to enhance their lipophilicity for specific pharmacological applications. The process achieved high yields (80-100%) in the isoflavones conversion when commercial lipases from Candida antarctica (CALB) and Thermomyces lanuginosus (TL IM) were used. The acylation patterns between CALB and TL IM varied depending on the substrate; both enzymes yielded principally monoacylated products from puerarin and β-D-fructofuranosyl-(2→6)-puerarin. However, TL IM also produced di- and triacylated derivatives of β-D-fructofuranosyl-(2→6)-puerarin in substantial amounts. Structural characterization by NMR identified puerarin-6ʹʹ-O-laurate and β-D-fructofuranosyl-(2→6)-puerarin-6ʹʹʹ-O-laurate as the primary products in CALB and TL IM reactions. These acyl derivatives presented improved lipophilicity (Log P from −1.0 − 0.16 to 3.26 − 3.58) and antioxidant activity (67.2-97.3 to 102.5-131.4 µM TE/mmol). Therefore, these compounds have potential pharmacological applications.

Total Syntheses of Phorbol and 11 Tigliane Diterpenoids and Their Evaluation as HIV Latency-Reversing Agents.

Tigliane diterpenoids possess exceptionally complex structures comprising common 5/7/6/3-membered ABCD-rings and disparate oxygen functionalities. While tiglianes display a wide range of biological activities, compounds with HIV latency-reversing activity can eliminate viral reservoirs, thereby serving as promising leads for new anti-HIV agents. Herein, we report collective total syntheses of phorbol (13) and 11 tiglianes 14-24 with various acylation patterns and oxidation states, and their evaluation as HIV latency-reversing agents. The syntheses were strategically divided into five stages to increase the structural complexity. First, our previously established sequence enabled the expeditious preparation of ABC-tricycle 9 in 15 steps. Second, hydroxylation of 9 and ring-contractive D-ring formation furnished phorbol (13). Third, site-selective attachment of two acyl groups to 13 produced four phorbol diesters 14-17. Fourth, the oxygen functionalities were regio- and stereoselectively installed to yield five tiglianes 18-22. Fifth, further oxidation to the most densely oxygenated acerifolin A (23) and tigilanol tiglate (24) was realized through organizing a 3D shape of the B-ring. Assessment of the HIV latency-reversing activities of the 12 tiglianes revealed seven tiglianes (14-17 and 22-24) with 20- to 300-fold improved efficacy compared with prostratin (12), a representative latency-reversing agent. Therefore, the robust synthetic routes to a variety of tiglianes with promising activities devised in this study provide opportunities for advancing HIV eradication strategies.

Amino acid and protein specificity of protein fatty acylation in Caenorhabditis elegans

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S-acyl moieties differ from N- and O-fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S-acylated proteins and 510 hydroxylamine-resistant N- or O-acylated proteins. Subsets of S-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

Thermal degradation of red cabbage (Brassica oleracea L. var. Capitata f. rubra) anthocyanins in a water model extract under accelerated shelf-life testing

Red cabbage (RC) represents a source of anthocyanins acylated with hydroxycinnamic acids (HCA) that are described to enhance their stability. Nevertheless, data about their thermal degradation are still controversial. Our aim was to comprehensively analyse the degradation kinetics of individual RC anthocyanins in a model aqueous extract treated at 40 °C × 30 days to simulate severe but realistic storage conditions. Free anthocyanins and radical-scavenging capacity showed different kinetics. The results confirm the high stability of RC anthocyanins (t1/2: 16.4–18.4 days), although HPLC analyses of each molecule displayed distinct kinetics with t1/2 from 12.6 to 35.1 days. In particular, the sinapoyl acylation negatively affected the stability of the anthocyanins, while the forms monoacylated with glycosyl p-coumaric and ferulic acids exhibited higher stability. In conclusion, our results indicate that acylation is not a prerogative of stability, as this is instead more dependent on specific acylation patterns and the glycosylation of HCA.

Targeted Metabolome and Transcriptome Analyses Reveal the Molecular Mechanism of Color Variation between Sepals and Petals in Fuchsia hybrida

The sepal color of Fuchsia hybrida is colorful instead of green and usually varies from the petal colors, which greatly increases its ornamental value and attract customers’ preference. However, the potential molecular mechanism underlying the color variation between sepals and petals remains unclear. The present study collected F. hybrida with red sepals and purple petals to explore the key pigments and genes involved in color development using a targeted metabolome and transcriptome. A total of 43 metabolites with diverse hydroxylation, glycosylation, methylation and acylation patterns were isolated and identified by UPLC-MS/MS. The quantification analysis showed that peonidin-3,5-O-diglucoside and malvidin-3,5-O-diglucoside were the most abundant anthocyanins accumulating in the sepals and petals, respectively. Then, six libraries from the sepals and petals were constructed for the transcriptome and 70,135 unigenes were generated. The transcript level of FhF3′H was significantly higher in the sepals, while Fh3′5′H showed more abundant expression in the petals, which can account for the abundant peonidin and malvidin accumulation in the sepals and petals, respectively. The subsequent multiomics analysis showed that both the differentially accumulated anthocyanins and expressed unigenes were enriched in the anthocyanin biosynthesis pathway. Additionally, FhMYBs potentially regulating anthocyanin biosynthesis were screened out by correlation analysis and protein interaction prediction. These findings help to elucidate the molecular mechanisms underlying the color variation between the sepals and petals in F. hybrida.

Expanding Knowledge of Methylotrophic Capacity: Structure and Properties of the Rough-Type Lipopolysaccharide from Methylobacterium extorquens and Its Role on Membrane Resistance to Methanol.

The ability of Methylobacterium extorquens to grow on methanol as the sole carbon and energy source has been the object of intense research activity. Unquestionably, the bacterial cell envelope serves as a defensive barrier against such an environmental stressor, with a decisive role played by the membrane lipidome, which is crucial for stress resistance. However, the chemistry and the function of the main constituent of the M. extorquens outer membrane, the lipopolysaccharide (LPS), is still undefined. Here, we show that M. extorquens produces a rough-type LPS with an uncommon, non-phosphorylated, and extensively O-methylated core oligosaccharide, densely substituted with negatively charged residues in the inner region, including novel monosaccharide derivatives such as O-methylated Kdo/Ko units. Lipid A is composed of a non-phosphorylated trisaccharide backbone with a distinctive, low acylation pattern; indeed, the sugar skeleton was decorated with three acyl moieties and a secondary very long chain fatty acid, in turn substituted by a 3-O-acetyl-butyrate residue. Spectroscopic, conformational, and biophysical analyses on M. extorquens LPS highlighted how structural and tridimensional features impact the molecular organization of the outer membrane. Furthermore, these chemical features also impacted and improved membrane resistance in the presence of methanol, thus regulating membrane ordering and dynamics.

Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner.

Posttranslational modifications add tremendous complexity to proteomes; however, gaps remain in knowledge regarding the function and regulatory mechanism of newly discovered lysine acylation modifications. Here, we compared a panel of non-histone lysine acylation patterns in metastasis models and clinical samples, and focused on 2-hydroxyisobutyrylation (Khib) due to its significant upregulation in cancer metastases. By the integration of systemic Khib proteome profiling in 20 paired primary esophageal tumor and metastatic tumor tissues with CRISPR/Cas9 functional screening, we identified N-acetyltransferase 10 (NAT10) as a substrate for Khib modification. We further showed that Khib modification at lysine 823 in NAT10 functionally contribute to metastasis. Mechanistically, NAT10 Khib modification enhances its interaction with deubiquitinase USP39, resulting in increased NAT10 protein stability. NAT10 in turn promotes metastasis by increasing NOTCH3 mRNA stability in an N4-acetylcytidine-dependent manner. Furthermore, we discovered a lead compound #7586-3507 that inhibited NAT10 Khib modification and showed efficacy in tumor models in vivo at a low concentration. Together, our findings bridge newly identified lysine acylation modifications with RNA modifications, thus providing novel insights into epigenetic regulation in human cancer. We propose that pharmacological inhibition of NAT10 K823 Khib modification constitutes a potential anti-metastasis strategy.

Investigations on Kaempferol, Quercetin and Other Flavonoids in Aquatic Plants of Iraqi Marshlands-I

Aquatic plants are versatile source of phytochemicals that are of significant biological influences particularly, the flavonoids and their glycosidic derivatives distributed in different plant parts including fruits, seeds, leaves, flowers, stem, roots and rhizomes. Most of the reported biological influences are reported to be expressed by quercetin, kaempferol, isorhmnatin, and their glycosidic derivatives, particularly, rutin. In this survey, we summarized the reported flavonoids types, content and factors affecting their extracts contents of three aquatic plants Nuphar, Najas and Nymphoides detected in the Iraqi central marshlands, in provenance of Thi-Qar. Surveying the phytochemical investigations regarding Nuphar species of reported abundance of polyphenolic compounds content including identified and quantified phenolic acids, flavonoids like flavonols and flavonones, particularly, in their leaves. However, flavonoids/their derivatives types and quantities in various parts extracts are not reported to the extent of our knowledge although they are presumed to contribute to the reported biological influence of the plant on one hand. On the other hands, regarding Najas species, few reports have identified the existence of flavonoids as well as other phenolic compounds, although, total quantification to the phenolic compounds, flavonoids and tannins are reported rather than identifying the individual flavonoid in the plant extracts. Only single report demonstrates the total phenolic, flavonoids and tannin contents of ethyl acetate extract of Najar minor according to our survey. Regarding Nymphoides species, the extraction level flavonoids as lipophilic compounds as well as their much polar glycosidic derivatives is depending on the solvent polarities. In general, in most of Nymphoides species the ethanolic extract querectin, methylquercetin and kaempferol flvonoides/their derivatives are identified individual flavonoids/quantified to be more than one fold of that in the aqueous extracts. Finally, the manner/position of glycosylation as well as glycosides sugar resides acylation vary among the Nymphoides species. In general, single 3-O- or di 3,4’-O- glycosylation pattern is noticed in kaempferol, quercetin and isorhamnetin. The multiply diverse glycosylation, methylation as well as acylation pattern of Nymphoides species flavonoids may explains their identification in various plant part/extracts besides the variation in their biological activities potentials. 

A Copper-Responsive Two-Component System Governs Lipoprotein Remodeling in Listeria monocytogenes.

Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Structural determination of the lipid A from the deep-sea bacterium Zunongwangia profunda SM-A87: a small-scale approach

Zunongwangia profunda SM-A87 is a deep-sea sedimentary bacterium from the phylum Bacteroidetes, representing a new genus of Flavobacteriaceae. It was previously investigated for its capability of yielding high quantities of capsular polysaccharides (CPS) with interesting rheological properties, including high viscosity and tolerance to high salinities and temperatures. However, as a Gram-negative, Z. profunda SM-A87 also expresses lipopolysaccharides (LPS) as the main components of the external leaflet of its outer membrane. Here, we describe the isolation and characterization of the glycolipid part of this LPS, i.e. the lipid A, which was achieved by-passing the extraction procedure of the full LPS and by working on the ethanol precipitation product, which contained both the CPS fraction and bacterial cells. To this aim a dual approach was adopted and all analyses confirmed the isolation of Z. profunda SM-A87 lipid A that turned out to be a blend of species with high levels of heterogeneity both in the acylation and phosphorylation pattern, as well as in the hydrophilic backbone composition. Mono-phosphorylated tetra- and penta-acylated lipid A species were identified and characterized by a high content of branched, odd-numbered, and unsaturated fatty acid chains as well as, for some species, by the presence of a hybrid disaccharide backbone.

Coupling between high-resolution mass spectrometry and focalized data-analysis methods provides the identification of new putative glycosidic non-anthocyanic flavonoids in grape.

The biochemical diversity of flavonoids is based on glycosylation, methylation, acylation, and many other modifications of the flavonoid backbone. Liquid chromatography coupled to high-resolution mass spectrometry demonstrated to be a powerful approach to gain new insights into the flavonoid composition of many plant species, including grapes. Among different metabolomic approaches, suspect screening analysis relies on the construction of a specific database and on ultra-high performance liquid chromatography/quadrupole time-of-flight (UHPLC/QTOF) analysis to find new compounds of oenological interest. A homemade database containing mass data information retrieved from the literature specific for plant flavonoid derivatives (GrapeFlavMet) was constructed. Tandem mass spectrometry analysis of V. vinifera and hybrid grape extracts was performed, and MS/MS fragmentation allowed to assign the putative flavonoid chemical structure to various identification levels, as established by the Metabolomics Standard Initiative. By this approach, putative flavonoid derivatives with different glycosylation and acylation patterns were identified. They include three pentoside derivatives of tetrahydroxy-flavone, tetrahydroxy-flavanone and myricetin isomers, a putative dihydrorhamnetin hexoside derivative, three cinchonain isomers (phenylpropanoid-substituted flavan-3-ols with antidiabetic properties), and two syringetin isomer derivatives (acetyl- and p-coumaroyl-hexoside). Two acetyl-hexoside derivatives of dihydrorhamnetin and pentahydroxy-methoxy-flavanone, and three derivatives of tetrahydroxy-dimethoxy-flavanone (acetyl, p-coumaroyl, and caffeoyl-hexoside) were tentatively annotated. Most of the compounds were identified in grape for the first time, while two putative syringetin derivatives previously proposed in the literature were confirmed. These findings deepen the current knowledge on grape flavonoids, suggesting more connections at the biochemical level.

Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants.

TLR4 is a key pattern recognition receptor that can sense pathogen‐ and danger‐ associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro‐inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species‐specific, and barely predictable structure‐activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan‐based lipid A‐mimicking molecules that can activate human and murine TLR4‐mediated signaling with picomolar affinity. Exploiting crystal structure ‐ based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)‐linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non‐reducing βGlcN(1↔1′)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β‐1,1′ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β‐1,1′‐linked diglucosamine‐derived glycolipids entailing multiple long‐chain (R)‐3‐ acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D‐molecular shape and conformational rigidity of unnatural β,β‐1,1′‐linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4‐mediated signaling in a species‐independent manner.

Structure of the lipopolysaccharide O-antigens from Fusobacterium nucleatum strains SB-106CP and HM-992 and immunological comparison to the O-antigen of strain 12230

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structures of the lipopolysaccharide O-chain (OPS) of two strains of F. nucleatum, SB-106CP and HM-992, both isolated from cancerous tissues. These strains elaborate the same sugar chain, differing only by their N-acylation pattern:-6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NABuAc- SB-106CP-6-α-D-GlcNAc-4-β-D-GlcNHBu3NABuA-3-β-D-QuiNAc4NAc- HM-992ABu = (R)-3-amino-butyrylAbuAc = (R)-3-N-acetyl-3-aminobutyrylHBu = (R)-3-hydroxy-butyrylAll monosaccharides are in the pyranose form.Previously we published the structure of the OPS from F. nucleatum 12230, a transtracheal isolate, which had similar sugar chain, differing by replacement of GlcNAc with Glc and a different acylation pattern:-6-α-d-Glc-4-β-d-GlcNHBu3NHBuA-3-β-d-QuiNAc4NABu-A mouse monoclonal antibody specific for the 12230 O-antigen did not cross react with the LPS of strains SB-106CP and HM-992 confirming the structural differentiation.

Total Synthesis of Euonymine and Euonyminol Octaacetate.

Euonymine (1) and euonyminol octaacetate (2) share the core structure of euonyminol (3), the most hydroxylated member of the dihydro-β-agarofuran family. In 2, eight of the nine hydroxy groups of 3 are acetylated, and 1 has six acetyl groups and a 14-membered bislactone comprising a pyridine dicarboxylic acid with two methyl groups. The different acylation patterns provide distinct biological activities: 1 and 2 display anti-HIV and P-glycoprotein inhibitory effects, respectively. The 11 contiguous stereocenters and 9 oxygen functionalities of the ABC-ring system of 1 and 2 represent a formidable challenge, which is further heightened by the macrocyclic structure of 1. Here we disclose an efficient synthetic strategy for enantioselective total synthesis of 1 and 2. Starting from (R)-glycerol acetonide, we constructed the B-ring by an Et3N-accelerated Diels-Alder reaction, the C-ring by intramolecular iodoetherification, and the A-ring by ring-closing olefin metathesis. The 10 stereocenters were installed through a series of substrate-controlled stereoselective C-C and C-O bond formations by exploiting the three-dimensional structures of judiciously designed substrates. These newly developed reaction sequences led to protected euonyminol 5, which served as a common intermediate for assembling 1 and 2. Global deprotection of 5 and subsequent acetylation produced 2. Alternatively, the discriminative protective groups of 5 allowed for site-selective bis-esterification to generate bislactone. Combining [3 + 2]-cycloaddition and reductive desulfurization introduced the last remaining stereocenters of the two methyl groups on the macrocycle. Finally, deprotection and acetylation gave rise to fully synthetic 1 for the first time.

Ruminal Lipid A Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli.

Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues.

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

Impact of octreotide counterion nature on the long-term stability and release kinetics from an in situ forming depot technology

The generation of acylated impurities has represented an important hurdle in the development of long acting injectables for therapeutic peptides using biocompatible polymers with a polyester moiety. We investigated here an in situ forming depot (ISFD) technology that uses polyethylene glycol - polyester copolymers and a solvent exchange mechanism to promote depot formation. This technology has shown promise in formulating small molecules as well as therapeutic proteins. In the present work, using the well-known somatostatin analog octreotide acetate (OctAc) as a model molecule, we evaluated this delivery platform to release therapeutic peptides. Peptide acylation was found to be pronounced in the formulation, while it was very limited once the depot was formed and during the release process. The octreotide acylation pattern was fully characterized by LC-MS/MS. Moreover, it was demonstrated that exchanging the acetate anion with more hydrophobic counterions like pamoate or lauryl sulfate allowed to greatly improve the peptide stability profile, as well as the formulation release performance. Finally, the in vivo evaluation through pharmacokinetics studies in rat of these new octreotide salts in ISFD formulations showed that octreotide was quantifiable up to four weeks post-administration with a high bioavailability and an acceptable initial burst.