Browsing by Author "Lowell, Andrew N."

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    Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase
    Kalkreuter, Edward; Bingham, Kyle S.; Keeler, Aaron M.; Lowell, Andrew N.; Schmidt, Jennifer J.; Sherman, David H.; Williams, Gavin J. (Nature Research, 2021-04-13)
    Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.
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    Design, Synthesis, and Structure-Activity Relationship Investigation of Selective Sphingosine Kinase Inhibitors
    Li, Hao (Virginia Tech, 2019-05-08)
    Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h).
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    Development of Methods for Boron Reagents
    Gates, Ashley Michelle (Virginia Tech, 2020-03-19)
    Boron reagents are known to be valuable in the field of organic chemistry due to their abilities to undergo a variety of transformations, resulting in useful pharmaceuticals and synthetic intermediates. It has also been shown that diboron reagents can act as reaction mediators due to the unique properties of the boron atom. To that end, this dissertation discloses three novel methods of employing boron reagents. Chapter 1 describes a method of utilizing a diboron reagent mediator in the palladium-catalyzed hydrogenation of allenes. In the presence of a palladium catalyst, tetrahydroxydiboron and stoichiometric water, allene semireduction proceeds in good yield. This semireduction is regioselective for the terminal alkene and results in the selective formation of Z-alkenes when used with unsymmetrical allenes (>80:20 Z:E). It is also compatible with more sterically hindered 1,1-diarylallenes, resulting in tri-substituted alkenes in good yields (63-88%). A borylation, defluorination of alpha-trifluoromethyl-alpha,beta-unsaturated esters is described in Chapter 2. The borylation is copper-catalyzed (10 mol %) and proceeds in the presence of stoichiometric bis(pinacolato)diboron and sodium tert-butoxide. The reaction affords compounds that contain two potentially useful functional handles: boronic esters and gem-difluoroalkenes. The products are obtained in moderate to good yield (up to 75%) with a large substrate scope including compounds with electron-donating, electron-withdrawing, heteroatom, and aryl substituents. In addition, the utility of the products in further transformation is demonstrated. A proposed reaction mechanism that provides rationale for the formation of products is described along with experimental evidence. Finally, Chapter 3 describes a transition-metal-free trans hydroboration of alkynoate esters and amides. The reaction is phosphine-catalyzed and proceeds with pinacolborane to afford (E)-beta-borylacrylates and (E)-beta-borylacrylamides in good to excellent yields. The reaction products are converted into novel oxaboroles through reduction with sodium borohydride. Theoretical calculations provide mechanistic insight for the transformation. The formation of a key phosphonocyclobutene intermediate is responsible for the observed stereoselectivity.
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    Expanding the Biosynthetic Toolbox: The Potential and Challenges of In Vitro Type II Polyketide Synthase Research
    Rivers, Max A. J.; Lowell, Andrew N. (MDPI, 2024-03-07)
    Type II polyketide synthase (PKS) systems are a rich source of structurally diverse polycyclic aromatic compounds with clinically relevant antibiotic and chemotherapeutic properties. The enzymes responsible for synthesizing the polyketide core, known collectively as the minimal cassette, hold potential for applications in synthetic biology. The minimal cassette provides polyketides of different chain lengths, which interact with other enzymes that are responsible for the varied cyclization patterns. Additionally, the type II PKS enzyme clusters offer a wide repertoire of tailoring enzymes for oxidations, glycosylations, cyclizations, and rearrangements. This review begins with the variety of chemical space accessible with type II PKS systems including the recently discovered highly reducing variants that produce polyalkenes instead of the archetypical polyketide motif. The main discussion analyzes the previous approaches with an emphasis on further research that is needed to characterize the minimal cassette enzymes in vitro. Finally, the potential type II PKS systems hold the potential to offer new tools in biocatalysis and synthetic biology, particularly in the production of novel antibiotics and biofuels.
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    Synthesis, antimicrobial activity, and catalytic activity of rhodium and iridium piano stool complexes: Teaching an old dog new tricks
    Duchane, Christine Marie (Virginia Tech, 2019-06-14)
    This dissertation describes the synthesis, antimicrobial properties, and catalytic activity of a variety of eta5-ligand rhodium and iridium complexes. Cp*RM(beta-diketonato)Cl (Cp*R = R-substituted tetramethylcyclopentadienyl ligand) complexes were found to have selective activity against Mycobacterium smegmatis, with activity highly dependent upon the substituents on the Cp*R ligand as well as on the beta-diketonato ligand. These complexes were synthesized in good yield from the reaction of the chloro bridged dimers ([Cp*RMCl2]2) with the desired beta-diketonato ligand under basic conditions. All complexes were fully characterized by 1H and 13C NMR. Twenty single crystal X-ray structures were solved. The success of these syntheses led to investigation of another beta-diketonato ligand: 1,1,1,5,5,5-hexafluoroacetylacetonate (hfac). Though many metal complexes of this ligand are known, reaction with [Cp*MCl2]2 did not yield the desired Cp*M(hfac)Cl complexes. Instead, a variety of products were obtained, three of which were characterized crystallographically. The most interesting structure featured a non-coordinating trifluoroacetate (TFA) anion and a [Cp*Ir]3Na1O4 cubane structure, which is an unprecedented and highly unusual arrangement for iridium. Attempts to synthesize this cluster rationally through reactions of [Cp*IrCl2]2 with TFA yielded instead a chloro bridged [Cp*IrCl(TFA)] dimer. Reaction of [Cp*MCl2]2 with 1,1,1-trifluoroacetylacetonate (tfac) yielded the expected Cp*M(tfac)Cl complex, indicating that the problem lies with using hfac as a ligand for Cp*M(III) complexes. Finally, the indenyl effect was investigated for the oxidative annulation of 2-phenylimidazole with 1-phenyl-1-propyne catalyzed by a series of methyl-substituted [(indenyl)RhCl2] dimers. [(Ind*)RhCl2]2 was found to have significantly greater activity than [Cp*RhCl2]2 (100% vs. 51%). Two plausible catalytic cycles were proposed, one of which invokes a ring slip transition state. Though it is unclear if the "indenyl effect" is responsible for this differing activity, it is certainly apparent that using an indenyl ligand has a notable effect in this catalytic reaction. Cyclometalation was also investigated stoichiometrically for 2-phenyl-1H-imidazole and 1-phenylpyrazole and found to proceed readily for [(Ind*)RhCl2]2. Additionally, the crystallographic structure of a Rh+ /Rh– ionic pair was solved. Ionic pairs such as this are rarely found in the literature.