Mark B. Frampton

The adaptive significance of unproductive alternative splicing in primates

Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase β as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase β transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing.

Biocatalytic synthesis of silicone polyesters.

The immobilized lipase B from Candida antarctica (CALB) was used to synthesize silicone polyesters. CALB routinely generated between 74-95% polytransesterification depending on the monomers that were used. Low molecular weight diols resulted in the highest rates of esterification. Rate constants were determined for the CALB catalyzed polytransesterifications at various reaction temperatures. The temperature dependence of the CALB-mediated polytransesterifications was examined. A lipase from C. rugosa was only successful in performing esterifications using carboxy-modified silicones that possessed alkyl chains greater than three methylene units between the carbonyl and the dimethylsiloxy groups. The proteases alpha-chymotrypsin and papain were not suitable enzymes for catalyzing any polytransesterification reactions.

A Comparison of Protease Active Sites and their Ability to Process Silicon-Based Substrates

Many enzymes have been identified that can participate in the hydrolysis of alkoxysilanes; each with a different degree of specificity. Our working hypothesis was that the nature of the active site of the enzyme (i.e., the compatibility of binding pockets with the substrate) could have a direct effect on the rate of catalysis. This communication reports our experiments on the relative rates of hydrolysis of a model alkoxysilane, phenyltrimethoxysilane (PTMS), by three proteases: trypsin, α-chymotrypsin, and pepsin. Trypsin which typically accepts amino acids bearing positively charged basic residues was not particularly proficient for the hydrolysis of PTMS. On the other hand, both α-chymotrypsin and pepsin, each of which contains a binding pocket, or two in the case of pepsin, suitable for accommodating aromatic residues, were more suitable for mediating hydrolysis. This report provides some preliminary data to support the hypothesis that the architecture of the enzyme active site is important in determining the proficiency with which an enzyme will process a given organosilicon substrate.

Chain length selectivity during the polycondensation of siloxane-containing esters and alcohols by immobilized Candida antarctica lipase B.

We have examined the chain length selectivity for a series of acyl donors by lipase B from Candida antarctica (CalB). CalB accepted aliphatic diesters of C4, C6 and C12 chain lengths equally. The introduction of a carbon-carbon double bond into the C4 esters dramatically lowered the rate constant associated with polymerization highlighting the role of geometry in catalysis; fumarate esters were polymerized at a reduced rate compared to the succinate esters, while the maleate esters were not polymerized above 5% over the course of 24h. A disiloxane-containing diester impeded catalysis by CalB. We examined a series of vinyl siloxane esters and alcohols, and learned that the Z arrangement around the double bond stalled esterification by CalB completely. The distance between the ester carbonyl and the dimethylsiloxy group was shown to be an important factor in mediating catalysis. The rate constants were similar when the methylene spacer was 3, 4, or 5 units in length; beyond 6 methylene units, the rate constants increased. This has been tentatively attributed to the local reduction on the steric bulk when the larger siloxane moiety lies outside of the active site of the enzyme.

Cyclotetrasiloxane frameworks for the chemoenzymatic synthesis of oligoesters

Immobilized lipase B from Candida antarctica (Novozym® 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65–80% consumption of starting materials in 24–48 h. Longer reaction times, 72–96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw = 11400, 13100, and 19400 g mol−1 for the substrate pairs of C7D4 ester/octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw = 86800 g mol−1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.

Macrocyclic Oligoesters Incorporating a Cyclotetrasiloxane Ring

Macrocyclic oligoester structures based on a cyclotetrasiloxane core consisting of tricyclic (60+ atoms) and pentacycylic (130+ atoms) species were identified as the major components of a lipase-mediated transesterification reaction. Moderately hydrophobic solvents with log P values in the range of 2-3 were more suitable than those at lower or higher log P values. Temperature had little effect on total conversion and yield of the oligoester macrocycles, except when a reaction temperature of 100 °C was employed. At this temperature, the amount of the smaller macrocycle was greatly increased, but at the expense of the larger oligoester. For immobilized lipase B from Candida antarctica (N435), longer chain length esters and diols were more conducive to the synthesis of the macrocycles. Langmuir isotherms indicated that monolayers subjected to multiple compression/expansion cycles exhibited a reversible collapse mechanism different from that expected for linear polysiloxanes.

Analysis of Trisiloxane Phosphocholine Bilayers

We have synthesized unique siloxane phosphocholines and characterized their aggregates in aqueous solution. The siloxane phosphocholines form nearly monodisperse vesicles in aqueous solution without the need for secondary extrusion processes. The area/lipid, lipid volume, and bilayer thickness were determined from small-angle X-ray scattering experiments. The impetus for the spontaneous formation of unilamellar vesicles by these compounds is discussed.

Characterization of self-assembled hybrid siloxane-phosphocholine bilayers

We have synthesized six new hybrid siloxane phosphocholines (SiPCs) and examined their self-assembly behaviour in aqueous dispersions. Employing small angle X-ray scattering we have characterized SiPC bilayers. SiPCs exhibit differential self-assembly behaviour that results from the interplay between the siloxane fatty acid in the sn-2 position and the differing chain length fatty acids in the sn-1 position. SiPCs that possess a fatty acid chain of a C8-C14 chain length in the sn-1 position form unilamellar vesicles. Extending the fatty acid chain length to C16 and C18 allows for the formation of both unilamellar and multilamellar vesicles. We propose that the preferential formation of unilamellar vesicles is the result of an enhanced hydrophobic effect imparted by siloxane chains at the termini of lipid tails.

Protease-Mediated Hydrolysis and Condensation of Tetra- and Trialkoxysilanes

Silica is a common material with uses in a diverse range of products. New methods for producing silica are always being developed. In the past 15 years biological and bio-inspired approaches for silica production have gained momentum. One of the challenges in designing new materials is developing new methods for their production and gaining a complete understanding of all of the processing parameters. We have been studying the interaction(s) between enzymes and organically modified alkoxysilanes in an attempt to determine the kinetics of hydrolysis and condensation, as well as to determine a general reaction mechanism. We believe that the nature of the enzyme (i.e. mode of catalysis, active site, and secondary interactions) contributes to the ability of any given enzyme to act as a catalyst for the production of silica-based materials.

Enzymatic Modification and Polymerization of Siloxane-Containing Materials

Enzymatic catalysis is finding a true home in the field of polymer science. Lipases are of particular interest and have been used to produce a wide range of polyesters and polyamides. We have been focused on using enzymes to mediate chemical transformations of organosilicon compounds, most notably siloxane polymers. This overview describes some of our work using N435 to modify disiloxanes and to polymerize siloxane-containing diols and diesters.