C5 Epimer Research Articles

Structure and Polymannuronate Specificity of a Eukaryotic Member of Polysaccharide Lyase Family 14

Alginate is an abundant algal polysaccharide, composed of β-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a β-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 Å resolution and the putative substrate-binding model show that the enzyme adopts a β-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the β-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.

Concise enantioselective synthesis of (+)-sertraline and (−)-CP-52002 using proline catalysis

A short enantioselective synthesis of (+)-sertraline and its C4 epimer (−)-CP-52002 with an overall yield of 30%, respectively, as its hydrochloride has been described. The key steps are the proline catalyzed Mannich reaction of acetaldehyde and acid catalyzed intramolecular Friedel–Crafts’ alkylation reaction of olefin proceeding with high optical purities.

Biochemical and Structural Characterization of Alginate Lyases: An Update

Background: Alginates, a complex copolymer of α-L-guluronate (G) and its C5 epimer β-Dmannuronate (M), can be disassembled by alginate lyases to monosaccharides or alginate oligosaccharides, and the latter exhibits many fascinating bioactivities. A lot of new alginate lyases have been identified in many resources, especially in marine organisms. Elucidating the natural paradigms of alginate conversion will enable us to utilize algal biomass efficiently and enhance our understanding of how microorganisms evolved with plants. Crystal structures of different polysaccharide lyase (PL) families have been solved by X-ray diffraction, which will improve our understanding in molecule-level catalytic mechanisms. Methods: The researches related to alginate lyases after 2000 are reviewed, and data is sorted and analyzed. Protein sequences alignment and phylogenetic tree are employed to discuss different groups of alginate lyases. Furthermore, viewpoints about the studies of alginate lyases are proposed. Results: In this review, the latest resources of alginate lyases are summarized, progress of alginate lyases on structuralfunctional relationships are presented. The biochemical characterization, biological role, and the promising applications of alginate lyases also are discussed. Conclusion: New alginate lyases have sprung up throughout the marine and terrestrial organisms. Structure-function analyses of alginate lyases will shed light on the understanding of catalytic mechanism of alginate lyases. However, the mechanisms of bioactivities of alginates are still unclear. Extensive investigation of alginate lyases is crucial for the development of alginate applications. Besides, a standard and meaningful naming system for alginate lyases may be needed. Keywords: Alginate lyases, active site, catalytic mechanism, conservative sequence, crystal structure, polysaccharide lyase.

Comparative Analysis of Fluorine‐Directed Glycosylation Selectivity: Interrogating C2 [OH → F] Substitution in d‐­Glucose and d‐Galactose

AbstractThe influence of C2 [OH → F] substitution on the stereochemical course of chemical glycosylation was interrogated in both D‐glucose and its C4 epimer D‐galactose. Molecular editing at C2 and configurational inversion at C4 were simultaneously investigated by variable‐temperature glycosylation studies of both systems. Extrapolation of the differences in enthalpic (ΔΔHβα‡) and entropic (ΔΔSβα‡) contributions that discriminate these closely similar systems revealed that deoxofluorination at the C2 position induces an enthalpic bias that augments β‐stereoselection. Intriguingly, the enhanced stereoselectivity of the C4 epimer D‐galactose was found to be entropic in nature. Given the prominence of these scaffolds in chemical biology, delineating the factors that underpin selectivity will be critical in developing novel glycosylation methods and in rationalizing differences with the natural systems post facto.

Direct C5‐Isomerization Approach to l‐Iduronic Acid Derivatives

AbstractInspired by the biosynthesis of heparan sulfate (HS), we present a direct epimerization approach to l‐iduronic acid synthons (IdoA) from the corresponding C5 epimers, d‐glucuronic acids (GlcA). With the best result, 95 % conversion was achieved. Weak intramolecular interactions dramatically affect the equilibrium constants of this thermodynamically governed process. Based on this approach, GlcA and IdoA donors required for the synthesis of fondaparinux were prepared in 14 steps, whereas over 20 steps were required previously.

S-Ribosylhomocysteine analogs containing a [4-thio]ribose ring

The [4-thio]-S-ribosylhomocysteine (SRH) analogs containing substitution of a sulfur atom for the endocyclic oxygen were synthesized by coupling of the 4-thioribose substrates with a thiolate generated from the protected homocysteine. Coupling of the protected 1-deoxy-5-O-mesyl-S-oxo-4-thio-D-ribofuranose with homocysteinate salt gave the C4 epimers of [4-thio]-SRH at the sulfoxide oxidation level lacking a hydroxyl group at anomeric carbon. Treatment of these sulfoxides with BF3⋅Et2O/NaI affected simultaneous reduction to sulfide and global deprotection affording 1-deoxy-4-thio-SRH analog. Treatment of the protected 1-deoxy-S-oxo-4-thio-D-ribofuranose sulfoxide with DAST/SbCl3 resulted in the fluoro-Pummerer rearrangement to give 4-thio-β-D-ribofuranosyl fluoride. Mesylation of the latter at 5-hydroxyl position followed by coupling with homocysteinate salt and subsequent global deprotection with trifluoroacetic acid afforded [4-thio]-SRH thiohemiacetal.

Serum C3 epimer of 25-hydroxyvitamin D and its determinants in adults: a national health examination survey in Thais.

A high percentage have detectable C3 epimer of 25-hydroxyvitamin D3 (3-epi-25(OH)D3) in the population of Thai National Health Examination Survey IV. C3 epimers of vitamin D have recently been shown to contribute significantly to 25-hydroxyvitamin D (25(OH)D) levels in an infant population. However, the findings in the general adult population are unclear. Therefore, the purpose of the present study is to determine the percentage of the C3 epimer of 25(OH)D (3-epi-25(OH)D) and its determinants in an adult population. A subsample of 1148 sera randomly selected from the Thai National Health Examination Survey IV (2009) samples were measured for serum 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D2, and 3-epi-25(OH)D3 by LC-MS/MS method. The relative 3-epimer contribution (%) was used to express the amount of 3-epimer-25(OH)D3 as a percentage of total 25(OH)D3 (the sum of 25(OH)D3, and 3-epi-25(OH)D3). A high proportion of subjects had detectable 3-epi-25(OH)D3 that was <10% of the total 25(OH)D levels. Since the level of total 25(OH)D2 is low, only a minority of subjects had detectable 3-epi-25(OH)D2. Multivariate analysis suggested that age, male gender, and rural residence were independently related to the 3-epi-25(OH)D3/total 25(OH)D3 ratio. A high percentage of Thai adults had detectable 3-epi-25(OH)D3 that was <10% of the total 25(OH)D levels. Age, gender, and living in a rural area were associated with the relative amount of 3-epi-25(OH)D3 to total 25(OH)D3.

Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

Alginate lyases catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated.

ChemInform Abstract: Coupling of Sterically Hindered Trisubstituted Olefins and Benzocyclobutenones by C—C Activation: Total Synthesis and Structural Revision of Cycloinumakiol.

AbstractThe first total synthesis of the proposed structure of cycloinumakiol (I) and its C5 epimer (II) is presented.

Stereoselective Total Synthesis of Pinolide and Its C2 Epimer and Evaluation of Their Cytotoxic Activity

Pinolide, a naturally occurring nonenolide and its C2 epimer have been synthesized using d -mannitol and 1,2-epoxyhex-5-ene as the starting materials. The synthesis involves the Jacobsen’s hydrolytic kinetic resolution, Yamaguchi esterification, and intramolecular ring-closing metathesis as the key steps. The 2- epi -pinolide, the C2 epimer of pinolide, has been synthesized for the first time. The cytotoxic activity of the pinolide and 2- epi -pinolide has been studied.

A convergent synthesis of carbocyclic sinefungin and its C5 epimer.

A convergent synthesis of carbocyclic sinefungin 2 and its C5 epimer 3 is described. The key features in our synthesis include the use of commercial available L-methionine and readily available (1R, 4S)-4-hydroxy-2-cyclopentenyl acetate as starting materials, cross-metathesis coupling, enzymatic kinetic resolution and Staudinger reduction. The current synthesis is flexible and therefore provides convenient access to the synthesis of various carbocyclic SIN analogues for biological evaluation.

Coupling of sterically hindered trisubstituted olefins and benzocyclobutenones by C-C activation: total synthesis and structural revision of cycloinumakiol.

The first total syntheses of the proposed structure of cycloinumakiol (1) and its C5 epimer (18) are achieved in a concise and efficient fashion. Starting from the known 3-hydroxybenzocyclobutenone, 1 and 18 are obtained in nine and five steps with overall yields of 15% and 33%, respectively. The key for the success of this approach is the use of a catalytic C-C activation strategy for constructing the tetracyclic core of 1 through carboacylation of a sterically hindered trisubstituted olefin with benzocyclobutenone. In addition, the structure of the natural cycloinumakiol was reassigned to 19-hydroxytotarol (7) through X-ray diffraction analysis. This work demonstrates the potential of C-C activation for streamlining complex natural product synthesis.

Coupling of Sterically Hindered Trisubstituted Olefins and Benzocyclobutenones by CC Activation: Total Synthesis and Structural Revision of Cycloinumakiol

AbstractThe first total syntheses of the proposed structure of cycloinumakiol (1) and its C5 epimer (18) are achieved in a concise and efficient fashion. Starting from the known 3‐hydroxybenzocyclobutenone, 1 and 18 are obtained in nine and five steps with overall yields of 15 % and 33 %, respectively. The key for the success of this approach is the use of a catalytic CC activation strategy for constructing the tetracyclic core of 1 through carboacylation of a sterically hindered trisubstituted olefin with benzocyclobutenone. In addition, the structure of the natural cycloinumakiol was reassigned to 19‐hydroxytotarol (7) through X‐ray diffraction analysis. This work demonstrates the potential of CC activation for streamlining complex natural product synthesis.

3-Epi-25 hydroxyvitamin D in pregnancy

The physiological importance of the C3 epimers of vitamin D (3-epi-25OHD2/3) is uncertain and there have been limited studies determining the levels of these epimers in human populations. The aims of the current study were (1) to determine 3-epi-25OHD2/3 levels throughout non-diabetic and T1DM pregnancy, (2) to examine the relationships between 25OHD and 3-epi-25OHD, (3) to assess the impact of maternal BMI on 3-epi-25OHD and examine associations with markers of glycaemic control. An observational study of 52 pregnant controls without diabetes and 65 pregnant women with T1DM in a university teaching hospital. 25OHD and 3-epi-25OHD were measured by liquid chromatography tandem mass spectrometry. 3-Epi-25OHD was found in 90.2% of control (median 0.9nmol/L; range 0.1-5.9nmol/L), and in 94.5% of T1DM, women (median 1.4nmol/L; range 0.1-10.5nmol/L). In both control and T1DM groups, maternal and cord 3-epi-25OHD correlated significantly with 25OHD. Seasonal variation in maternal 3-epi-25OHD levels was evident in both groups; Summer levels were significantly higher than all other seasons in the control group (p<0.001) and significantly higher than Spring (p=0.003) and Winter (p<0.001) in the T1DM group. In T1DM women HbA1c was significantly negatively correlated with 3-epi-25OHD at trimesters 1 and 2 (p=0.049; p=0.001) and with cord 3-epi-25OHD (p=0.012). Maternal BMI >30kg/m(2) had a significant negative impact on 3-epi-25OHD. Maternal 3-epi-25OHD exhibits seasonal variation and, in common with cord 3-epi-25OHD, correlates with 25OHD throughout both non-diabetic and T1D pregnancy. In T1DM women 3-epi-25OHD is associated with a key marker of glycaemic control.

Investigation into the Feasibility of Thioditaloside as a Novel Scaffold for Galectin‐3‐Specific Inhibitors

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this β-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Stereoselective synthesis of Jaspine B and its C2 epimer from Garner aldehyde

Two different stereoselective routes for the synthesis of Jaspine B and its C2 epimer are presented here, starting from easily available Garner aldehyde. The key synthetic steps involved iodocyclization, organocuprate addition, HWE olefination, regioselective α-tosylation and cross metathesis reaction. This is the first report to synthesize Jaspine B involving iodocyclization.

Functional Characterization of AlgL, an Alginate Lyase from Pseudomonas aeruginosa

Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa, algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity and revealed that AlgL operates preferentially on nonacetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The k(cat)/K(m) for the reaction increased linearly with the number of residues in the substrate, from 2.1 × 10(5) M(-1) s(-1) for the substrate containing 16 residues to 7.9 × 10(6) M(-1) s(-1) for the substrate with 280 residues. Over the same substrate size range, k(cat) varied between 10 and 30 s(-1). The variation in k(cat)/K(m) with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by (1)H nuclear magnetic resonance spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.

Total Synthesis of the Proposed Structure of Mycosporulone: Structural Revision and an Unexpected Retro-Aldol/Aldol Reaction

The proposed structure of the fungal metabolite, mycosporulone 1, was prepared starting from the cyclohexenone ester 11 and the D-(R)-glyceraldehyde acetonide 12. The spectroscopic data for both 1 and its C2 epimer 1a did not match those reported for the natural product. A revised structure 29 for mycosporulone is proposed.

Synthesis of LY541850 and Its C4 Epimer

Synthesis of LY541850 and Its C4 Epimer

Total Synthesis of Isoprostanes Derived from Adrenic Acid and EPA

AbstractEnantiomerically enriched F2‐dihomo‐isoprostanes and F3‐isoprostanes have been synthesized. Such compounds are derived from the action of reactive oxygen species on the phospholipid‐bound polyunsaturated fatty acids (PUFA), adrenic acid and eicosapentaenoic acid, respectively. Of special interest are the F2‐dihomo‐isoprostanes because they could represent potential biomarkers for myelin damage as its main PUFA constituent is adrenic acid. Our strategy, based on a pivotal enantiomerically enriched intermediate, has allowed access to F2‐dihomo‐IsoP and both C5 epimers of 5‐F3t‐IsoP for the first time.