Thomas V. Lee

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.

The cerium (III) mediated reaction of trimethylsilylmethyl magnesium chloride with esters and lactones: The efficient synthesis of some functionalised allylsilanes of use in annulation reactions

Abstract The use of cerium (111) chloride alters the chemoselectivity of the reaction of trimethylsilylmethyl magnesium chloride with ester-acetals and also greatly improves the efficiency of reaction with lactones. In addition it gives improved preparations of the useful intermediates ( 7 ), ( 13 ), ( 18 ),( 20 ) and ( 22 ) and gives direct access to the valuable functionalised allylsilanes ( 1 ) to ( 3 ) of use in annulation reactions.

The γ-alkylation of enones using silyl dienol ethers: The effect of changing the electrophile and of changing the silyl group

Phenylthiomethyl chloride (3) and the trimethylsilyl dienol ether (2) of crotonophenone react to give the lowest level of γ-attack of a range of carbon electrophiles and trimethylsilyl dienol ethers; the γ-selectivity for this reaction can be raised from 45:55 to 85:15 by changing the silyl group from trimethylsilyl to triphenylsilyl.

Oxidation Adjacent to Oxygen of Alcohols by Activated DMSO Methods

The nucleophilic nature of the sulfinyl oxygen of sulfoxides has been used to good effect in synthetic chemistry, most notably in the oxidation of primary and secondary alcohols to aldehydes and ketones using dimethyl sulfoxide (DMSO). The first report of the use of dimethyl sulfoxide in such an oxidation was due to Pfitzner and Moffatt,1 although Kornblum had previously demonstrated the use of dimethyl sulfoxide as an oxidant for the conversion of alkyl halides to aldehydes.2 There are now many examples of how advantageous the use of dimethyl sulfoxide can be, most notably in the oxidation of primary alcohols to aldehydes without overoxidation to carboxylic acids. Despite much activity in this area, resulting in a plethora of activated dimethyl sulfoxide reagents which behave as an oxidant, the method is still basically that originally described by Pfitzner and Moffatt. However, considerable practical improvements have been made by a number of groups, most notably that of Swern, who utilized oxalyl chloride as the activator, and the ‘Swern oxidation’ is now a well-established synthetic method.

Enantioselective reduction of β,χ-unsaturated α-keto acids usingbacillus stearothermophilus lactate dehydrogenase: A new route to functionalised allylic alcohols

Abstract The enantioselective reduction of α-keto acids, catalysed by lactate dehydrogenase, has been extended bo β,χ-unsaturated substrates, providing functionalised allylic alcohols in high optical purity.

VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice

Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF(164) isoform, and the antiangiogenic VEGF(165)b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF(164) overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF(164) double transgenic mice. The consequences of simultaneous overexpression of VEGF(164) and VEGF(165)b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF(164)/nephrin-VEGF(165)b mice. Persistent VEGF(164) overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF(164) levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF(164)-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF(164) were not present in triple transgenic VEGF(164) and VEGF(165)b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF(164) overexpression. VEGF(164) overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF(165)b is capable of preventing VEGF(164)-induced changes in glomerular permeability and ultrastructure in vivo.

Novel annulation reactions: A simple preparation of spirocycles and an allylsilane based bifunctional acceptor-donor annulating reagent

Abstract A simple, one pot, stereoconvergent synthesis of spirocycles has been achieved in excellent yield. Further studies on one step bifunctional annulating reagents have resulted in the development of a new allylsilane based cyolopentannulation reaction.

The preparation and reactions with an o-silylated enolate of an allylsilane bifunctional [3 + 2] annulating reagent

Abstract The preparation and reactions of allylsilance-acetal (13) with various silyl enol ethers are described. This results in the one-pot synthesis of fused and spiro cyclopentanes by using a 1,3-bifunctional annulating reagent in which the two reactive centres are activated by one set of conditions .

Bifunctional annulating reagents: The use of novel allylsilanes for the one-pot preparation of six and seven-membered rings

Abstract Preliminary details of a new strategy for the synthesis of five, six, and seven-membered rings by use of novel allylsilane reagents are described.

The total synthesis of 10-(R,S)-C30 botryococcene and botryococcane and a new synthesis of a general intermediate to the botryococcene family

Abstract The first synthesis of a C-30 botryococcene has been achieved by using a modified Julia reaction for the key coupling of fragments (2) and (3). The same strategy has been used in a new synthesis of a known synthetic precursor to the entire botryococcene family.