Synthesis and reactivity of 4-oxo-5-trimethylsilanyl derived α-amino acids

A Lewis-acid promoted one-carbon homologation of an aspartic acid semialdehyde with trimethylsilyldiazomethane has led to the efficient synthesis of two silicon-containing α-amino acids. The use of trimethylaluminium or catalytic tin(II) chloride gave novel 4-oxo-5-trimethylsilanyl derived amino acids in yields of 71–88%. An investigation into the reactivity of these highly functional α-amino acids showed that selective cleavage of the C–Si bond could be achieved under mild basic conditions to give a protected derivative of the naturally occurring amino acid, 4-oxo-l-norvaline. Alternatively, Peterson olefination with aryl or alkyl aldehydes resulted in the formation of E-enone derived α-amino acids

Synthesis of Novel Silicon-Containing Amino Acids and Potential Organosilicon Dipeptidic Artificial Sweeteners

Der erste Teil der vorliegenden Arbeit beschreibt neuartige Synthesen siliciumhaltiger Aminosäuren, ausgehend von Malonsäurediethylester. Weitere Teilschritte dieser Synthesen sind eine biokatalytische enantioselektive Esterspaltung von Malonsäurediethylester-Derivaten mittels Schweineleberesterase und ein Curtius-Abbau. Es wurde in allen Schritten darauf geachtet, kostengünstige und leicht handhabbare Chemikalien zu verwenden, um eine etwaige Produktion der Aminosäuren in einem größeren Maßstab zu ermöglichen. Der zweite Teil der vorliegenden Arbeit widmet sich Versuchen, aus den im vorangehenden Abschnitt beschriebenen Aminosäuren potentielle dipeptidische, siliciumorganische Süßstoffe herzustellen. Diese werden am besten als Aspartam-Analoga beschrieben. Die Charakterisierung der Verbindungen erfolgte durch NMR-Spektroskopie (1H, 13C, 15N, 29Si), Elementaranalysen und gegebenenfalls durch Kristallstrukturnalysen.The first part of this PhD thesis describes novel syntheses of silicon-containing amino acids, starting from diethyl malonate. Other steps in these syntheses are an enzyme-catalyzed enantioselective ester cleavage of diethyl malonate derivatives and a Curtius rearrangement. Inexpensive and chemicals that are easy to handle were used in all steps with a view to facilitate a production of the amino acids on a larger scale. The second part of this PhD thesis describes efforts to obtain dipeptidic artificial sweeteners starting from the amino acids described in the previous section. These are best described as aspartame analogues. The characterization of all compounds was performed by NMR spectroscopy (1H, 13C, 15N, 29Si), elemental analyses and, where possible, by single-crystal X-ray diffraction

Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: Insight into the receptor activation switch

A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N6-position. In contrast, the adenosine-5′-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5′-substituents on ligand–target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation

The cell wall-associated mycolactone polyketide synthases are necessary but not sufficient for mycolactone biosynthesis

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules

An Investigation of the Interaction of Co-Solvent with Substrates in the Pig Liver Esterase-Catalyzed Hydrolysis of Malonate Esters

Previously, we have reported the effect of several co-solvents, including ethanol, on the enantioselective outcome of pig liver esterase (PLE) hydrolysis reactions. The greatest improvements were observed in those substrates that contained an atom capable of forming a hydrogen bond in the sidechain portion of the molecule. To further explore the interaction between substrate and ethanol, a second series of substrates were synthesized, in which the hydrogen bonding atoms of the side chain were modified, and subjected to PLE hydrolysis. Substrates containing atoms capable of forming hydrogen bonds showed the largest equilibrium constants. However, a large equilibrium constant did not always produce significant changes in enantioselectivity as hypothesized. Finally, molecular modeling experiments were performed to obtain a better understanding of the interactions. These experiments revealed that there are substrate–enzyme interactions that can be influenced by the addition of ethanol

SDS-PAGE and western immunoblot analysis of whole cell lysates and cell fractions of <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His.

<p>(A) SDS-PAGE separation and Coomassie-stained protein gel of 10 μg of whole cell lysate of <i>M. marinum</i> M with LM-M8 and <i>mlsA2</i> (TPS8313) and empty vector control (TPS8256); (B) Western immunoblot of (A) using an anti-AT domain antibody; (C) Western immunoblot of (A) using an anti-His antibody; (D) Western immunoblot of cell fractions from <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His using an anti-His antibody, showing MlsA2 is present only in the cell wall (P27) fraction. The reactivity of the anti-His antibody to a protein with a mass ∼65 kDa in panels (C) and (D) is the known cross-reactivity with the polyhistidines of mycobacterial GroEL (Hsp65) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070520#pone.0070520-Noens1" target="_blank">[37]</a>.</p

Expression analysis of recombinant <i>M. marinum</i> TPS8334.

<p>(A) Coomassie stained SDS-PAGE and (B) Western immunoblot of cell wall fractions of <i>M. marinum</i> expressing LM-M8, MlsA2_His, Mup038 and Mup045 (TPS8334) using an anti-AT domain antibody demonstrating the presence of the heterologously expressed PKSs in the cell wall fraction of this strain. The positive control is purified, recombinant acyltransferase from MlsA2.</p

SDS-PAGE and western immunoblot analysis of <i>M. ulcerans</i> 06-3844 expressing TKL constructs.

<p>(A) SDS-PAGE separation and Coomassie-stained protein of 10 μg of <i>M. ulcerans</i> 06-3844 containing <i>mlsA1</i> LM-M8 (TPS8162), LM_P-M8 (TPS8307) or empty vector (TPS8164) cell fractions (B) Western immunoblot analysis of (A) with an anti-AT domain antibody showing the presence of a ∼400 kDa protein produced by <i>M. ulcerans</i> harbouring either LM-M8 or LM_P-M8 (lane 1 and 4). Positive control is purified, recombinant acyltransferase derived from MlsA2.</p