The appended sugar residues of many natural products from Actinomyces are important for their biological activities. Many of these unusual sugar biosynthetic gene clusters have been isolated and many glycosyltransferases from various antibiotic-producing organisms have been identified. The increasing knowledge about these sugar biosynthetic pathways opens up the possibility of generating novel bioactive glycosylated compounds through combinatorial biosynthesis. The work described in this dissertation focuses on the investigation of the biosynthetic pathway of a rare nitro-containing sugar, D-kijanose, from an antibiotic, kijanimicin, and the glycosyltransferase helper proteins involved in the glycosylation of macrolide antibiotics. D-Kijanose, especially its nitro group, plays an important role in conferring the biological activities of the parent antibiotics. Cloning and sequencing of the kijanimicin biosynthetic gene cluster have allowed the proposal of the biosynthetic pathway of D-kijanose. The functions of the enzymes encoded by each open-reading frame in the cluster were also assigned based on sequence comparison with known enzymes found in other biosynthetic reactions. In this thesis, the functions of KijB1, a TDP-4-keto-6-deoxy-hexose 2,3-dehydratase, and KijD2, a TDP-hexose C-3 aminotransferase, were verified. The TDP-3-amino-4-keto-2,3,6-trideoxyhexose produced as an intermediate in the early stage of D-kijanose biosynthesis was also identified. In the second part of this dissertation, the in vivo protein-protein interaction between D-desosaminyl glycosyltransferase, DesVII, and its auxiliary protein, DesVIII, was established by yeast two-hybrid assay. The complex formation between these two proteins was also demonstrated by in vitro binding assay. Several strategies were tried to overexpress the D-mycaminosyl glycosyltransferase and its auxiliary protein, TylM2 and TylM3, although none of them were successful. A two-plasmid in vivo glycosylation system was also developed to test the competence of various DesVIII homologues to serve as the helper protein for glycosyltransferase DesVII, MycB and NbmD. In summary, the work in this dissertation has provided important information on the biosynthesis of D-kijanose and also significant insight into the function of the helper proteins of macrolide glycosyltransferases. These results could be useful for future studies of natural product biosynthesis and exploitation of glycodiversification. / text

Twenty different macrolide and ketolide antibiotics were analyzed by reversed phase high performance liquid chromatography on an ODS-2 cartridge column. Each of these compounds was uniquely separated and purified by varying the flow rate. Retention times of the individual drugs were proportional to the flow rate of the mobile phase. Recovery of antimicrobial activity for most of the drugs was greater than 90% based on a microbiological assay of material recovered from the column. Retention times were related to structural differences between these antimicrobial agents.

Bacterial resistance is a formidable 21st-century global public health threat. If left unaddressed, we risk moving toward a “post-antibiotic era.” While resistance is a natural consequence of antibiotic use, the rate at which pathogenic bacteria have evaded multiple classes of drugs has markedly outpaced the introduction of new ones. New antibiotics are desperately needed to fill this void. Macrolides are one of the safest and most effective drug classes in medicine; however, resistance has compromised efficacy. To date, three generations have been developed with only the lattermost targeting bacterial resistance. Single next-generation macrolides will not keep pace with the inevitable onset of resistance; thus, there is a critical need to greatly accelerate the procurement of multiple future-generation antibiotics to tackle both current and future resistance mechanisms. My research is to meet this need by designing, synthesizing, and evaluating a novel, future-generation macrolide antibiotics that will serve as an armamentarium to be individually deployed on demand. In the previous research in Andrade group, we synthesized and evaluated various desmethyl ketolide analogs. The fact that 4-desmethyl telithromycin was fourfold less potent than telithromycin against A2058G mutants indicated replacing the 4-Me with hydrogen (i.e., desmethylation) to avoid a steric clash with the 2-amino group of G2058 was insufficient in rescuing bioactivity. Guided by MD simulation, we concluded a logical, superior alternative strategy was the replacement of the 4-Me group with one possessing a smaller vdW radius and capable of establishing favorable interactions with both wild-type and A2058G mutant ribosomes. Specifically, we reasoned that 4-fluoro solithromycin would be ideal candidate. The hypothesis was that the 4-fluoro moiety would engage in dipole-dipole interactions (C-F---H) with the exocyclic 2-amino group of guanine, which is based on accumulated evidence that strategic placement of organofluorine can strongly impact potency, selectivity, and physicochemical properties. In addition, the axially disposed of 4-fluorine would provide conformational stabilization from a gauche effect with the vicinal O5 group. The novel synthetic routes to unexplored desosamine analogs at the C3’-amino substituent to the macrolide antibiotic would play a role in bioactivity and resistance. Hofmann reaction was employed to execute the same 2,3-epoxide ring opening method without removing desosamine and re-glycosylating. This markedly reduces the steps, time, and cost involved in preparing novel desosamine-modified analogs. Significantly, this route enables the first synthesis of N,N’-disubstituted desosamine analogs from an epoxide, which was utilized to prepare novel analogs of clarithromycin. The application of in situ click chemistry toward the discovery of novel macrolide antibiotics first required the synthesis of suitable azide and aryl alkyne reactants. Alkyne partners were procured by commercial vendors or chemical synthesis. We targeted two logical, validated positions to tether the side chains, specifically N11 on the macrolactone and N3’ of desosamine. The first (N11) has been the most utilized. Moreover, extensive structure-activity relationships have revealed a four-carbon tether is ideal. Based on the solithromycin−E.coli X-ray structure, I designed, synthesized, and evaluated dehydro solithromycin, which possesses an (E)-alkene in the side-chain. The use of an unsaturated side chain would conformationally preorganize the bi-aryl side chain in order to pay the entropic penalty and thus favorably contribute to the overall binding. An insightful observation made from MD simulationed ribosomes bound with to solithromycin revealed that the interaction of the side-chain includes H-binding as well as π-stacking. The hypothesis was that employing tethered side-chains bearing motifs that maximize H-bonding and π-stacking would be superior antibiotics for treating resistant bacterial strains bearing erm¬-mediated N6 methyl and dimethylated ribosomes. To test this hypothesis, we developed various analogs with different alkynes by introducing different functional groups at the 3 and 5 positions on the aromatic ring. Another desosamine sugar modification is bis-azide. To date, the use of a two side chain strategy has not been reported. To access the requisite bis-azides, we employed a tactic the oxidative demethylation and alkylation of desosamine to afford bis-click solithromycin analogs. / Chemistry

<p>Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations.</p><p>The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. </p><p>Another example of stable resistance can also be seen in sulfonamide resistant <i>Streptococcus pyogenes</i>. Sequence determinations of the dihydropteroate synthase (<i>dhps</i>) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213.</p><p>The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (<i>hppk</i>). In the malaria parasite <i>Plasmodium falciparum</i> this enzyme is part of a polyfunctional entity, also encoding <i>dhps</i>. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to <i>P. falciparum</i>. Both are crucial for enzyme activity.</p><p>To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.</p>

Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations. The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. Another example of stable resistance can also be seen in sulfonamide resistant Streptococcus pyogenes. Sequence determinations of the dihydropteroate synthase (dhps) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213. The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (hppk). In the malaria parasite Plasmodium falciparum this enzyme is part of a polyfunctional entity, also encoding dhps. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to P. falciparum. Both are crucial for enzyme activity. To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.

Synoicum adareanum is a colonial tunicate commonly found on the benthos around Palmer Station on Anvers Island, Antarctica. A comprehensive chemical investigation of the lipophilic extract of the frozen tunicates gave a new series of polyketide macrolides, palmerolides A-E and H. The structure elucidation of these compounds was accomplished by extensive NMR and mass spectral studies.The palmerolides are unusual 20-membered macrolides displaying functionality more commonly found in sponges or cyanobacteria. Palmerolide A, the major member of the group, shows significant and selective in vitro cytotoxicity against melanoma with three orders of greater sensitivity relative to other cell lines tested, in the National Cancer Institute (NCI) 60 human cancer cell-line panel. In addition, it displays potent cytostatic activity against several other cancer cell lines. Based on NCI's COMPARE analysis, palmerolide A was investigated as a V-ATPase inhibitor and shown to bind the V0 subunit with 2 nM inhibition.Austrodoris kerguelenensis is a common Antarctic nudibranch widely distributed in the High Antarctic and Sub Antarctic Zone. It is characterized by the presence of terpenoid glyceryl esters which are supposed to be involved in defense. Chemical investigations of several specimens of A. kerguelenensis collected near Palmer station Antarctica afforded hitherto undescribed series of clerodane diterpenoid glycerides. The structure elucidation of three major compounds of this series, palmadorin A, B and C was accomplished.

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My Ph.D. research consists of two components: discovery of novel macrolide antibiotics and methodology development of N-sulfinyl metallodienamines. To tackle bacterial resistance, new antibiotics are desperately needed. My research objective is to address this need by designing, synthesizing, and evaluating novel macrolide antibiotics based on the best-in-class drug candidate, solithromycin. The drug discovery including following three projects: 1) Synthesis of solithromycin analogs wherein the desosamine sugar has been replaced with an acyclic amino alcohol surrogate; 2) In cellulo Click chemistry wherein the bacterial cell serves as the reaction vessel and the ribosome catalyzes the formation of triazole cycloadducts by testing different combinations of azide and alkyne fragments. One of the mechanisms of resistance to macrolide antibiotics is exemplified by methylation of A2058 by the methyltransferase encoded in erm genes. Methylation or dimethylation of A2058 leads to a steric clash with the macrolides and reduces the affinity of the macrolide for the ribosome. Thus, the bacterial resistant can be relieved via disrupting steric clashes between desosamine and A2058 residues. In 2017, I started a new project looking at the scope of the acyclic domino Michael/Mannich reaction to prepare chiral cyclohexenes developed by a previous group member, Dr. Vijay Chatare. This reaction is highly regioselective and stereoselective. Recent research showed that this reaction could be utilized on acroleins, acrylates and unsaturated ketones. Thus, we applied this useful methodology towards the concise total synthesis of (+)-ibogamine. / Chemistry