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Hung-wen Liu - One of the best experts on this subject based on the ideXlab platform.

  • Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-Desosamine.
    Journal of the American Chemical Society, 2009
    Co-Authors: Ping Hui Szu, Mark W. Ruszczycky, Sei Hyun Choi, Feng Yan, Hung-wen Liu
    Abstract:

    d-Desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae. It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of Desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-d-glucose (8) to TDP-3-keto-4,6-dideoxy-d-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His6-tagged DesII, characterization of its [4Fe-4S] cluster using UV−vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH to reduce the [4Fe-4S]2+ cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of ...

  • engineered biosynthesis of macrolide derivatives bearing the non natural deoxysugars 4 epi d mycaminose and 3 n monomethylamino 3 deoxy d fucose
    Journal of the American Chemical Society, 2007
    Co-Authors: Charles E Melancon, Hung-wen Liu
    Abstract:

    Previous pathway engineering work demonstrated that the Desosamine biosynthetic pathway in Streptomyces venezuelae could be converted to an efficient mycaminose biosynthesizing pathway by replacement of DesI with the hexose 3,4-ketoisomerase Tyl1a. In this work, FdtA, a ketoisomerase homologous to Tyl1a which catalyzes conversion of the Tyl1a substrate to the C-4 epimer of the Tyl1a product, was used to replace DesI. The ability of Desosamine pathway enzymes DesV, DesVI, DesVII, and DesVIII to accept substrates with inverted C-4 stereochemistry in the mutant expressing FdtA resulted in formation of macrolide derivatives bearing 4-epi-d-mycaminose, a sugar heretofore unobserved in Nature. Interestingly, minor glycosylated macrolides bearing another non-natural sugar, 3-N-monomethylamino-3-deoxy-d-fucose, were also produced by this mutant. An explanation for the formation of these unexpected new compounds is presented, and the implications of this work for combinatorial biosynthesis of new antibiotics are d...

  • Manipulating nature's sugar biosynthetic machineries for glycodiversification of macrolides : Recent advances and future prospects
    Pure and Applied Chemistry, 2007
    Co-Authors: Christopher J. Thibodeaux, Hung-wen Liu
    Abstract:

    Changing the sugar structures and glycosylation patterns of natural products is an effective means of altering the biological activity of clinically useful drugs. Several recent strategies have provided researchers with the opportunity to manipulate sugar structures and to change the sugar moieties attached to these natural products via a biosynthetic approach. In this review, we explore the utility of contemporary in vivo and in vitro methods to achieve natural product glycodiversification. This study will focus on recent progress from our labo- ratory in elucidating the biosynthesis of D-Desosamine, a deoxysugar component of many macrolide antibiotics, and will highlight how we have engineered the D-Desosamine biosyn- thetic pathway in Streptomyces venezuelae through targeted disruption and heterologous ex- pression of the sugar biosynthetic genes to generate a variety of new glycoforms. The in vitro exploitation of the substrate flexibility of the endogenous D-Desosamine glycosyltransferase (GT) to generate many non-natural glycoforms will also be discussed. These experiments are compared with recent work from other research groups on the same topics. Finally, the sig- nificance of these studies for the future prospects of natural product glycodiversification is discussed.

  • TDP-mycaminose biosynthetic pathway revised and conversion of Desosamine pathway to mycaminose pathway with one gene.
    Journal of the American Chemical Society, 2005
    Co-Authors: Charles E Melancon, Hung-wen Liu
    Abstract:

    Analysis of the tylosin gene cluster in Streptomyces fradiae uncovered an ORF, tyl1a, homologous to a hexose 3,4-isomerase found in Aneurinibacillus thermoaerophilus. Inclusion of the tyl1a gene along with other mycaminose biosynthetic genes (tylB, tylM1, tylM2, tylM3) identified in previous studies in an in vivo expression system successfully reconstituted the mycaminose pathway. Expression of tyl1a alone in the S venezuelae KdesI mutant converted a Desosamine pathway to a mycaminose pathway. These results strongly support the role of Tyl1a as a TDP-4-keto-6-deoxy-d-glucose 3,4-isomerase.

  • β-Glucosylation as a Part of Self-Resistance Mechanism in Methymycin/Pikromycin Producing Strain Streptomyces venezuelae†
    Biochemistry, 2003
    Co-Authors: Lishan Zhao, Noelle J. Beyer, Svetlana A. Borisova, Hung-wen Liu
    Abstract:

    In our study of the biosynthesis of d-Desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire Desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to β-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of Desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemica...

Goran Kragol - One of the best experts on this subject based on the ideXlab platform.

  • regioselective 2 imino 1 3 thiazolidine vs 2 imino 1 3 oxazolidine formation from the vicinal sec amino alcohol of Desosamine
    European Journal of Organic Chemistry, 2013
    Co-Authors: Zorica Marušić Ištuk, Ines Vujasinovi, Ana Cikos, Goran Kragol
    Abstract:

    In order to optimize Mukaiyama reagent-induced cyclization of vicinal sec-amino alcohols of Desosamine origin towards exclusive formation of N′-substituted-2-imino-1,3-thiazolidines via a thiocarbamoyl intermediate, the influence of reaction conditions was studied. A novel, mild, one-pot, two-step method was developed, and the formation of N′-substituted-2-imino-1,3-oxazolidines as side products was minimized. The inversion of configuration at C-2′ was unambiguously established using NMR-based conformational analysis. A reaction mechanism was proposed. A test series of novel Desosamine-modified 14- and 15-membered macrolides, bearing N′-alkyl-2-imino-1,3-thiazolidines fused to the Desosamine sugar were prepared.

  • Regioselective 2‐Imino‐1,3‐thiazolidine vs. 2‐Imino‐1,3‐oxazolidine Formation from the Vicinal sec‐Amino Alcohol of Desosamine
    European Journal of Organic Chemistry, 2013
    Co-Authors: Zorica Marušić Ištuk, Ines Vujasinovi, Ana Čikoš, Goran Kragol
    Abstract:

    In order to optimize Mukaiyama reagent-induced cyclization of vicinal sec-amino alcohols of Desosamine origin towards exclusive formation of N′-substituted-2-imino-1,3-thiazolidines via a thiocarbamoyl intermediate, the influence of reaction conditions was studied. A novel, mild, one-pot, two-step method was developed, and the formation of N′-substituted-2-imino-1,3-oxazolidines as side products was minimized. The inversion of configuration at C-2′ was unambiguously established using NMR-based conformational analysis. A reaction mechanism was proposed. A test series of novel Desosamine-modified 14- and 15-membered macrolides, bearing N′-alkyl-2-imino-1,3-thiazolidines fused to the Desosamine sugar were prepared.

  • Novel Desosamine-modified 14- and 15-membered macrolides without antibacterial activity.
    Bioorganic & medicinal chemistry letters, 2012
    Co-Authors: Ivana Palej Jakopović, Mirjana Bukvić Krajačić, Maja Matanović Škugor, Vlado Štimac, Dijana Pesic, Ines Vujasinović, Sulejman Alihodžić, Hana Čipčić Paljetak, Goran Kragol
    Abstract:

    Novel modifications of the Desosamine sugar of 14- and 15-membered antibacterial macrolides, in which the Desosamine was fused with N-substituted-1,3-oxazolidin-2-ones, were developed in order to completely suppress antibacterial activity and make them promising agents for other biological targets. The synthesis of such bicyclic Desosamine derivatives, especially 1,3-oxazolidin-2-one formation, was optimized and conducted under mild conditions without a need for protection/deprotection steps for other functional groups. A focused series of novel Desosamine-modified macrolide derivatives was prepared and their antibacterial activities tested. It was shown that these macrolide derivatives do not possess any residual antibacterial activity.

  • novel tandem reaction for the synthesis of n substituted 2 imino 1 3 oxazolidines from vicinal sec or tert amino alcohol of Desosamine
    European Journal of Organic Chemistry, 2011
    Co-Authors: Ines Vujasinovic, Antun Hutinec, Zorica Marusic Istuk, Samra Kapic, Mirjana Bukvic Krajacic, Ivica đilovic, Dubravka Matkoviccalogovic, Goran Kragol
    Abstract:

    Two one-pot methods, sequential and tandem, for the preparation of N'-substituted 2-imino-1, 3-oxazolidines from the vicinal (sec- or tert)-amino alcohol of Desosamine via intermediary alkyl-, aryl-, heteroaryl-, and heteroalkyl-thiourea moieties are described. Particularly interesting is the novel one-pot tandem reaction of the vicinal tert-amino alcohol that involves dealkylation, thiourea formation, and a final cyclization to yield 2-imino-1, 3-oxazolidine structures. The yields of both one-pot methods are comparable to the yield of the sequential reaction. A small library of a new class of Desosamine-modified 14- and 15-membered macrolides was prepared to demonstrate the variety of substituents that can be easily introduced and thus enable a huge variation of the physicochemical and hence biological properties of these new molecules.

  • Novel Tandem Reaction for the Synthesis of N′-Substituted 2-Imino-1,3-oxazolidines from Vicinal (sec- or tert-)Amino Alcohol of Desosamine
    European Journal of Organic Chemistry, 2011
    Co-Authors: Ines Vujasinović, Mirjana Bukvić Krajačić, Zorica Marušić Ištuk, Samra Kapić, Antun Hutinec, Ivica Đilović, Dubravka Matković-Čalogović, Goran Kragol
    Abstract:

    Two one-pot methods, sequential and tandem, for the preparation of N'-substituted 2-imino-1, 3-oxazolidines from the vicinal (sec- or tert)-amino alcohol of Desosamine via intermediary alkyl-, aryl-, heteroaryl-, and heteroalkyl-thiourea moieties are described. Particularly interesting is the novel one-pot tandem reaction of the vicinal tert-amino alcohol that involves dealkylation, thiourea formation, and a final cyclization to yield 2-imino-1, 3-oxazolidine structures. The yields of both one-pot methods are comparable to the yield of the sequential reaction. A small library of a new class of Desosamine-modified 14- and 15-membered macrolides was prepared to demonstrate the variety of substituents that can be easily introduced and thus enable a huge variation of the physicochemical and hence biological properties of these new molecules.

Pingsheng Lei - One of the best experts on this subject based on the ideXlab platform.

Han-young Kang - One of the best experts on this subject based on the ideXlab platform.

  • Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the Macrolide Antibiotic YC‐17
    Advanced Synthesis & Catalysis, 2015
    Co-Authors: Pramod B. Shinde, David H Sherman, Yeon Hee Ban, Eunji Kim, Hyemin Choi, Kris Rathwell, Inho Yang, Dong Gun Lee, Han-young Kang
    Abstract:

    YC‐17 is a 12‐membered ring macrolide antibiotic produced from Streptomyces venezuelae ATCC 15439 and is composed of the polyketide macrolactone 10‐deoxymethynolide appended with D‐Desosamine. In order to develop structurally diverse macrolactam analogues of YC‐17 with improved therapeutic potential, a combined approach involving chemical synthesis and engineered cell‐based biotransformation was employed. Eight new antibacterial macrolactam analogues of YC‐17 were generated by supplying a novel chemically synthesized macrolactam aglycone to S. venezuelae mutants harboring plasmids capable of synthesizing several unnatural sugars for subsequent glycosylation. Some YC‐17 macrolactam analogues were active against erythromycin‐resistant bacterial pathogens and displayed improved metabolic stability in vitro. The enhanced therapeutic potential demonstrated by these glycosylated macrolactam analogues reveals the unique potential of chemoenzymatic synthesis in antibiotic drug discovery and development

  • Total synthesis of pikromycin.
    The Journal of organic chemistry, 2011
    Co-Authors: Han-young Kang
    Abstract:

    The total synthesis of pikromycin (6), the first isolated macrolide antibiotic, was achieved. The target macrolide was retrosynthetically divided into two parts, pikronolide (6a) (aglycon) and d-Desosamine. The aglycon was synthesized using key reactions such as an asymmetric aldol reaction, Yamaguchi esterification, and ring-closing metathesis. The aglycon was coupled successfully with the trichloroacetimidate derivative of d-Desosamine under Lewis acidic conditions to afford pikromycin. Narbomycin (5) was also synthesized from narbonolide (5a) under identical conditions.

  • Total synthesis of methymycin
    Organic & biomolecular chemistry, 2009
    Co-Authors: Richeng Xuan, Han-young Kang
    Abstract:

    Methynolide and 10-epi-methynolide were synthesized from the necessary segments, which were prepared by the addition of Grignard reagents to the corresponding alpha-alkoxyketones utilizing 1,2-stereochemical selection based on Cram chelation control. Ring-closing metathesis, as the key reaction, was carried out to combine the segments for the synthesis of methynolide and 10-epi-methynolide. The total synthesis of methymycin was also achieved by the glycosylation of methynolide with the trichloroimidate derivative of D-Desosamine.

David H Sherman - One of the best experts on this subject based on the ideXlab platform.

  • Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the Macrolide Antibiotic YC‐17
    Advanced Synthesis & Catalysis, 2015
    Co-Authors: Pramod B. Shinde, David H Sherman, Yeon Hee Ban, Eunji Kim, Hyemin Choi, Kris Rathwell, Inho Yang, Dong Gun Lee, Han-young Kang
    Abstract:

    YC‐17 is a 12‐membered ring macrolide antibiotic produced from Streptomyces venezuelae ATCC 15439 and is composed of the polyketide macrolactone 10‐deoxymethynolide appended with D‐Desosamine. In order to develop structurally diverse macrolactam analogues of YC‐17 with improved therapeutic potential, a combined approach involving chemical synthesis and engineered cell‐based biotransformation was employed. Eight new antibacterial macrolactam analogues of YC‐17 were generated by supplying a novel chemically synthesized macrolactam aglycone to S. venezuelae mutants harboring plasmids capable of synthesizing several unnatural sugars for subsequent glycosylation. Some YC‐17 macrolactam analogues were active against erythromycin‐resistant bacterial pathogens and displayed improved metabolic stability in vitro. The enhanced therapeutic potential demonstrated by these glycosylated macrolactam analogues reveals the unique potential of chemoenzymatic synthesis in antibiotic drug discovery and development

  • Directing Group-Controlled Regioselectivity in an Enzymatic C–H Bond Oxygenation
    2015
    Co-Authors: Solymar Negretti, Larissa M Podust, Alison Rh Narayan, Karoline C. Chiou, Petrea M. Kells, Jessica L. Stachowski, Douglas A. Hansen, John Montgomery, David H Sherman
    Abstract:

    Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the Desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C–H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing Desosamine with varied synthetic N,N-dimethyl­amino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group Desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C–H functionalization in biocatalysis

  • Directing Group-Controlled Regioselectivity in an Enzymatic C-H Bond Oxygenation.
    Journal of the American Chemical Society, 2014
    Co-Authors: Solymar Negretti, Larissa M Podust, Alison Rh Narayan, Petrea M. Kells, Jessica L. Stachowski, Douglas A. Hansen, John Montgomery, Karoline C. Chiou, David H Sherman
    Abstract:

    Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the Desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C–H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing Desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation...

  • Biocatalytic Synthesis of Pikromycin, Methymycin, Neomethymycin, Novamethymycin, and Ketomethymycin
    Journal of the American Chemical Society, 2013
    Co-Authors: Douglas A. Hansen, Yeo Joon Yoon, Alison Rh Narayan, Christopher M. Rath, Eli B. Eisman, Jeffrey D. Kittendorf, Jonathan D. Mortison, David H Sherman
    Abstract:

    A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of d-Desosamine and final C–H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.

  • Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Mani Raj Chaulagain, Larissa M Podust, John Montgomery, Allison R. Knauff, David H Sherman
    Abstract:

    Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique Desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the Desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.