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1.
J Biol Chem ; 300(7): 107420, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38815868

RESUMEN

Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-ᴅ-manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.


Asunto(s)
Proteínas Bacterianas , Klebsiella pneumoniae , Antígenos O , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/genética , Antígenos O/metabolismo , Antígenos O/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Familia de Multigenes
2.
Carbohydr Res ; 536: 109013, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38185031

RESUMEN

Neuraminic acid (Neu5Ac, also known as sialic acid) is an important monosaccharide found in glycoproteins and glycolipids which plays a vital role in regulation of physiological functions and pathological conditions. The study of sialoglycans has benefitted from the development of glycomimetic probes and inhibitors for proteins and enzymes that interact with and modify neuraminic acid in glycan chains. Methods to access sialoside intermediates with high yield are needed to facilitate the design of new targets. Here, we report the synthesis of C5-azido thiosialosides using a mild method to deprotect the C5-acetamido functional group followed by the use of a diazo-transfer reagent. We examined two diazo-transfer strategies and compared their yields and tolerance of acetate protecting groups. The same methods and comparisons were also performed for the 2,3-dehydro-5-N-acetylneuraminic acid (DANA) scaffold which is commonly used to generate inhibitors of neuraminidase (sialidase) enzymes. We found that C5-azido derivatives of both thiosialosides and DANA could be produced in five or six steps with yields up to 76 % and 83 %, respectively. Diazo-transfer reagents compared in this study were trifluoromethanesulfonyl azide (TfN3) and imidazole-1-sulfonyl azide (ImzSO2N3). We found that both reagents were compatible with this method and showed comparable yields. Finally, we show that C5-azido derivatives can help to avoid O, N-acyl protecting group migration which was observed in C5-NHAc analogs.


Asunto(s)
Ácido N-Acetilneuramínico , Ácidos Neuramínicos , Neuraminidasa/metabolismo , Ácidos Siálicos/farmacología
3.
Org Lett ; 20(23): 7598-7602, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30427691

RESUMEN

A synthesis of the nonreducing end hexasaccharide of saccharomicin B, α-l-Eva-(1→4)-α-l-Eva-(1→4)-α-l-Dig-(1→4)-α-l-Eva-(1→4)-α-l-Dig-(1→4)-ß-d-Fuc, has been developed. Selective glycosylations of l-digitoxose (l-Dig) using AgPF6/TTBP-mediated thioether activation and l-4-e pi-vancosamine (l-Eva) using Tf2O/DTBMP-mediated sulfoxide activation produced the hexasaccharide as a single diastereomer in very good yield. This hexasaccharide is properly functionalized to serve as a glycosyl donor for the total synthesis of saccharomicin B.


Asunto(s)
Oligosacáridos/síntesis química , Conformación de Carbohidratos , Oligosacáridos/química
4.
J Colloid Interface Sci ; 498: 395-404, 2017 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-28343137

RESUMEN

HYPOTHESIS: The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. EXPERIMENTS: The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. FINDINGS: Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.


Asunto(s)
Ácidos Murámicos/química , Muramidasa/química , Sitios de Unión , Simulación del Acoplamiento Molecular , Peptidoglicano/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad
5.
Oncotarget ; 7(48): 78281-78296, 2016 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-27835876

RESUMEN

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.


Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Limoninas/farmacología , Enfermedades Neurodegenerativas/prevención & control , Fármacos Neuroprotectores/farmacología , Péptidos/metabolismo , Extractos Vegetales/farmacología , Agregación Patológica de Proteínas , Animales , Azadirachta/química , ADN/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células HCT116 , Células HEK293 , Factores de Transcripción del Choque Térmico/genética , Humanos , Limoninas/aislamiento & purificación , Limoninas/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Unión Proteica , Semillas , Factores de Tiempo , Transfección
6.
Carbohydr Res ; 434: 107-112, 2016 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-27639336

RESUMEN

Novel one-pot reaction conditions have been developed for the preparation of glycosyl thiourea derivatives directly from glycosyl azides mediated by a combination of sulfamic acid and sodium iodide. The reaction conditions were clean, non-toxic and the products were isolated in good to excellent yield.


Asunto(s)
Azidas/química , Glicósidos/síntesis química , Ácidos Sulfónicos/química , Tiourea/química , Glicósidos/química , Estructura Molecular , Yoduro de Sodio
7.
Beilstein J Org Chem ; 9: 1757-62, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24062839

RESUMEN

A straightforward synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O16 has been achieved following a sequential glycosylation strategy. A minimum number of steps was used for the synthesis of the target compound involving a one-pot glycosylation and a protecting group manipulation. All intermediate reactions afford their products in high yield, and the glycosylation steps are stereoselective.

8.
J Org Chem ; 78(6): 2680-6, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23421958

RESUMEN

A significantly fast reaction condition for the exclusive preparation ß-glycosyl thiol derivatives has been developed successfully. The reaction condition is one-step, fast, high yielding, highly stereoselective, and requires only benchtop chemicals. Further reaction of glycosyl thiol derivatives with Michael acceptors and alkylating agents furnished thioglycosides and (1,1)-thiolinked trehalose analogs.


Asunto(s)
Alquilantes/química , Tioglicósidos/química , Tioglicósidos/síntesis química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Estereoisomerismo
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