Steven M. Heilmann

Immobilization of Protein A at high density on azlactone-functional polymeric beads and their use in affinity chromatography

Abstract This paper presents the results of the use of highly cross-linked, porous, hydrophilic copolymer beads with protein immobilized on their surface for affinity chromatography. Copolymer beads composed of vinyldimethyl azlactone (oxazolone) and methylene-bis-acrylamide in various ratios, with up to 3/5 mequiv./g azlactone functionality, will undergo nucleophilic attack by amines, as well as by thiols and alcohols. The ring-opening reaction of a nucleophile-containing ligand (e.g., a protein) resulted in covalent attachment to the support. The reaction was rapid, half-complete in about 5 min, yielding proteins immobilized at very high densities, recombinant Protein A at 397 mg/g, and human immunoglobulin G at 225 mg/g. The reaction proceeded at significant levels from pH 4 to 9. There was a marked enhancement in the amount of protein coupled, its rate of reaction, and its biological activity when Protein A was made to react in the presence of high concentrations of sodium sulfate. Evaluatioin of affinity columns, prepared with Protein A immobilized at over 200 mg/g, gave molar ratios f immunoglobulin G to immobilized Protein A of 1:1 or greater. Up to 56 mg of immunoglobulin G was recovered per ml of column bed volume. The support combined high flow-rates with low back-pressures and nobed-volume changes upon changing mobile phases, including highly ionic aqueous solvents and ethanol.

Phosphorus reclamation through hydrothermal carbonization of animal manures.

Projected shortages of global phosphate have prompted investigation of methods that could be employed to capture and recycle phosphate, rather than continue to allow the resource to be essentially irreversibly lost through dilution in surface waters. Hydrothermal carbonization of animal manures from large farms was investigated as a scenario for the reclamation of phosphate for agricultural use and mitigation of the negative environmental impact of phosphate pollution. Hydrothermal reaction conditions were identified for poultry, swine, and cattle manures that resulted in hydrochar yields of 50-60% for all three manures, and >90% of the total phosphorus present in these systems was contained in the hydrochars as precipitated phosphate salts. Phosphate recovery was achieved in yields of 80-90% by subsequent acid treatment of the hydrochars, addition of base to acid extracts to achieve a pH of 9, and filtration of principally calcium phosphate. Phosphate recovery was achieved in yields of 81-87% based on starting manures by subsequent acid treatment of the hydrochars, addition of base to acid extracts to achieve a pH of 9, and filtration of principally calcium phosphate. Swine and cattle manures produced hydrochars with combustion energy contents comparable to those of high-end sub-bituminous coals.

Reaction of O-trimethylsilylated cyanohydrins with α-bromoesters: a new synthesis of tetronic acids

Abstract Tetronic acids and β-keto-γ-butyrolactones are easily prepared by the zinc induced reaction of O-trimethylsilylated cyanohydrins and α-bromoesters.

Addition of Grignard reagents to O-trimethylsulylated cyanohydrins: Synthesis of acyloins

Abstract Grignard reagents have been found to react readily with O-trimethylsilylated cyanohydrins to afford, after acid hydrolysis of intermediates, good yields of acyloins.

Crosslinked, hydrophilic, azlactone-functional polymeric beads : a two-step approach

Abstract Crosslinked, hydrophilic, azlactone-functional polymeric beads are readily prepared via a two-step approach involving: (1) reverse phase suspension copolymerization of N-acryloyl-amino acids with water-soluble crosslinkers and, optionally, dimethylacrylamide, followed by (2) cyclodehydration of pendant acylamino acid groups to azlactones using acetic anhydride. Azlactone functionalities of 0.3 to 3.0 meq/g (typically greater than 70% of the theoretical value) were achieved by this procedure. The azlactone-functional group in these beads was found to be quite reactive towards amine nucleophiles, even in aqueous solution where little competition from hydrolysis was observed. Rapid, covalent coupling of protein could be accomplished from aqueous media under mild conditions, and indicated a potential for extremely high coupling densities (up to 245 mg protein/g of beads).

Industrial symbiosis: Corn ethanol fermentation, hydrothermal carbonization, and anaerobic digestion

The production of dry‐grind corn ethanol results in the generation of intermediate products, thin and whole stillage, which require energy‐intensive downstream processing for conversion into commercial animal feed products. Hydrothermal carbonization of thin and whole stillage coupled with anaerobic digestion was investigated as alternative processing methods that could benefit the industry. By substantially eliminating evaporation of water, reductions in downstream energy consumption from 65% to 73% were achieved while generating hydrochar, fatty acids, treated process water, and biogas co‐products providing new opportunities for the industry. Processing whole stillage in this manner produced the four co‐products, eliminated centrifugation and evaporation, and substantially reduced drying. With thin stillage, all four co‐products were again produced, as well as a high quality animal feed. Anaerobic digestion of the aqueous product stream from the hydrothermal carbonization of thin stillage reduced chemical oxygen demand (COD) by more than 90% and converted 83% of the initial COD to methane. Internal use of this biogas could entirely fuel the HTC process and reduce overall natural gas usage. Biotechnol. Bioeng. 2013;110: 2624–2632.

The chemistry of 2-alkenyl-5(4H)-oxazolones. VIII acid-catalyzed reaction with alcohols

Abstract The acid-catalyzed reaction of 4,4-dimethyl-2-vinyl-5(4H)-oxazolone with primary alcohols proceeded with almost equal frequency at both CC (Michael addition) and CO (ring opening) groups; reaction with secondary and tertiary alcohols resulted in a modest elevation in Michael addition. Michael addition was not observed in reactions with 4,4-dimethyl-2-isopropenyl-5(4H)-oxazolone.

Synthesis of β-Substituted-α-[(trimethylsilyl)oxy]acrylonitriles from α, β-Unsaturated Aldehydes

Abstract The condensation of trimethylsilylcyanide with α, β-unsaturated aldehydes, followed by DBU catalyzed isomerization of the intermediate 0-silylated cyanohydrins, constitutes a facile synthesis of β-substituted- α-[(trimethylsilyl)oxy]acrylonitriles.

Iodotrimethylsilane Reduction of Benzoins: Synthesis of Deoxybenzoins and Tetraarylfurans

Abstract Benzoins react readily with iodotrimethylsilane to produce, in many cases, mixtures of deoxybenzoins and tetraarylfurans.

A One-Pot Transformation of Ketones Into N-(Meth)Acryloyl-2-Amino Acids

Abstract A synthetic sequence for the preparation of N-(meth)acryloyl-2-amino acids is outlined which involves transformation of a ketone successively into an aminonitrile and a (meth)acrylamidonitrile, followed by selective hydrolysis of the nitrile function. All reactions are performed in aqueous media, in one reaction vessel, and in a stepwise manner without isolation of any intermediate products.