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A Chemoenzymatic Route to Unnatural Sugar Nucleotides and Their Applications and Enzymatic Synthesis of Rare Sugars with Aldolases In vitro and In vivo

Cai, Li
http://rave.ohiolink.edu/etdc/view?acc_num=osu1305641380

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Chemistry.
Carbohydrate represents a class of biomacromolecules which is quite different from polynucleotide and polypeptide, given its branching character and non-template based biosynthesis. Carbohydrates exist in cells or on cell surfaces mainly as glycoconjugates: conjugation of a sugar chain (glycan) with a protein (glycoprotein), a lipid (glycolipid) or both lipid and protein. Cell surface carbohydrates are of paramount significance in biochemical recognition processes and carbohydrate complexes have been widely used in pharmaceutical areas such as prevention of infection, neutralization of toxins and cancer immunotherapy. It is well known that nucleic acids are biosynthesized using dNTP (or NTP) and proteins using activated amino acids. Our focus on this research (Chapters 1-4) is to find a facile way to produce the key building blocks for enzymatic carbohydrate synthesis---sugar nucleotide donors. We discovered a pathway in which N-acetylglucosamine/N-acetylgalactosamine (GlcNAc/GalNAc) and their analogues were phosphorylated by a 1-kinase called NahK to generate sugar-1-phosphates in good yields. These sugar-1-phosphates were subsequently pyrophosphorylated by GlmU from bacteria or AGX1 from mammals to give the corresponding sugar nucleotide donor molecules. We thus obtained a library of UDP-GlcNAc/GalNAc analogues on a preparative scale since these involved enzymes proved to be rather promiscuous. With the modified sugar nucleotide donors in hand, we are primed to incorporate the structurally modified GlcNAc/GalNAc analogues into different carbohydrate-containing biomolecules to either investigate carbohydrate metabolic pathways or construct a glycorandomized library of new glycoforms. In addition, the high stereoselectivity of aldolases in C-C bond construction confers upon them tremendous applications as synthetic biocatalysts. Among the aldolases, dihydroxyacetone phosphate (DHAP)-dependent aldolases are particularly attractive as a set of four possible diastereomers of vicinal diols can be synthesized conferring upon them the potential to be used in the synthesis of rare sugars and other hydroxylated natural products. Unfortunately, the strict requirement for the donor substrate DHAP, a rather expensive and unstable compound, limits aldolase use in large-scale preparation. Therefore, the capability to generate DHAP from inexpensive sources could ultimately broaden the scope of aldolase reactions making it an attractive challenge. In Chapter 5, we employed DHAP-dependent aldolases in the synthesis of several rare sugars via a one-pot four enzyme reaction system in which DHAP is generated from the oxidation of cheap DL-glycerol 3-phosphate. Subsequently, the DHAP generated in situ is coupled with glyceraldehydes by aldolases to give different rare sugars. More significantly, we used engineered bacteria as ‘chemists’ to carry out the aldolase reactions in vivo. The bacteria were fed with green starting materials (e.g. glycerol or glucose) and DHAP is generated in vivo via bacterial glycolysis. Desired rare sugars could thus be produced by simple fermentation and isolated from the culture media.
Peng Wang (Advisor)
Craig Forsyth (Committee Member)
David Hart (Committee Member)
Zhongfa Liu (Committee Member)
353 p.
Biochemistry; Chemistry; Organic Chemistry
Sugar nucleotides; carbohydrates; enzymes; synthesis; glycosyltransferase; aldolase;

Recommended Citations

Citations

  • Cai, L. (2011). A Chemoenzymatic Route to Unnatural Sugar Nucleotides and Their Applications and Enzymatic Synthesis of Rare Sugars with Aldolases In vitro and In vivo [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1305641380

    APA Style (7th edition)

  • Cai, Li. A Chemoenzymatic Route to Unnatural Sugar Nucleotides and Their Applications and Enzymatic Synthesis of Rare Sugars with Aldolases In vitro and In vivo. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1305641380.

    MLA Style (8th edition)

  • Cai, Li. "A Chemoenzymatic Route to Unnatural Sugar Nucleotides and Their Applications and Enzymatic Synthesis of Rare Sugars with Aldolases In vitro and In vivo." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1305641380

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.