Lee Speight, PhD, MBA

The world’s habits have been forever changed from the events over the past year, especially in regards to cleansing and disinfecting. While March marks the one year anniversary of a global shutdown, the 16th of March this year has another significance: the 155th anniversary of Joseph Lister’s discovery that hand washing before surgery prevented infections. While hand washing and sanitizing at regular intervals is undoubtably good for personal and public health, as it dramatically curtails the… Show more

The world’s habits have been forever changed from the events over the past year, especially in regards to cleansing and disinfecting. While March marks the one year anniversary of a global shutdown, the 16th of March this year has another significance: the 155th anniversary of Joseph Lister’s discovery that hand washing before surgery prevented infections. While hand washing and sanitizing at regular intervals is undoubtably good for personal and public health, as it dramatically curtails the spread of opportunistic pathogens, it does enact a heavy toll on individual’s skin leading to a search for
safer, milder cleansing ingredients. Show less

Consumers are becoming much more conscious of the ingredients in everyday products that they use on themselves and for their families. This has motivated cosmetic and personal care formulators to reevaluate product formulations to provide simpler, shorter ingredient lists.

The manufacture of bio-chemicals through the use of microbial fermentation and renewable feedstock has a number of well-known advantages linked to sustainability and reduced impacts on the environment. Markets for molecules produced with greener credentials are growing as consumers become more aware of what is in the formulated products they use every day. The use of solventogenic clostridia has now been re-commercialised for the production of bio-acetone and bio-n-butanol. The different… Show more

The manufacture of bio-chemicals through the use of microbial fermentation and renewable feedstock has a number of well-known advantages linked to sustainability and reduced impacts on the environment. Markets for molecules produced with greener credentials are growing as consumers become more aware of what is in the formulated products they use every day. The use of solventogenic clostridia has now been re-commercialised for the production of bio-acetone and bio-n-butanol. The different impurity profiles of these bio-based molecules compared with petro-versions results in performance advantages in downstream derivatisation chemistry, giving an added benefit alongside sustainability advantages. Advances in genome editing now enable us to take the benefits observed with clostridial fermentation and apply them to the production of the next generation of bio-molecules. Show less

The art of extractions is an intricate, yet vast and booming, industry with many variables to consider, including maximum efficiencies, cost and quality of end-product. There are many tried-and-true solvents used by the flavor and fragrance industry, but openness to explore new and better alternatives can lead to more productive and/or more economical processes. Here we explore why bio-derived n-butanol (herein referred to as bio n-butanol) is an often overlooked, yet incredibly effective, and… Show more

The art of extractions is an intricate, yet vast and booming, industry with many variables to consider, including maximum efficiencies, cost and quality of end-product. There are many tried-and-true solvents used by the flavor and fragrance industry, but openness to explore new and better alternatives can lead to more productive and/or more economical processes. Here we explore why bio-derived n-butanol (herein referred to as bio n-butanol) is an often overlooked, yet incredibly effective, and high-performing solvent for the extraction of essential oils and other lipophilic molecules. Show less

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative… Show more

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid. Show less

This article is a call for innovation and collaboration between bio-based renewable chemical producers and the petrochemical industry. The ascent—or, more appropriately—re-ascent of renewable chemistry provides great promise for the adhesives and sealants industry. However, the full promise of these innovations can only be commercially realized through collaboration between renewable innovators, synthetic chemistry providers, and downstream formulators.

A short review of the acetone-butanol-ethanol (ABE) fermentation process. The article summarizes the fate of glucose molecules being used as fermentation substrates for Clostridia bacteria all the way to the solvent products.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. It is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate and low affinities in solution… Show more

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. It is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate and low affinities in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'​-myoinositol-4'​,5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse. Show less

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here… Show more

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr and the identification of an Acd synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu3+ to monitor protein folding and binding interactions. Show less

The rapid synthesis of cis-2,6-disubstituted dihydropyrans is achieved in a three-component, one-pot cascade reaction. BiBr3-mediated addition of ketene silyl acetals or silyl enol ethers to β,γ-unsaturated cis-4-trimethylsilyl-3-butenal provides a Mukaiyama aldol adduct containing a vinylsilane moiety tethered to a silyl ether. Addition of a second aldehyde initiates a domino sequence involving intermolecular addition followed by an intramolecular silyl-modified Sakurai (ISMS) reaction… Show more

The rapid synthesis of cis-2,6-disubstituted dihydropyrans is achieved in a three-component, one-pot cascade reaction. BiBr3-mediated addition of ketene silyl acetals or silyl enol ethers to β,γ-unsaturated cis-4-trimethylsilyl-3-butenal provides a Mukaiyama aldol adduct containing a vinylsilane moiety tethered to a silyl ether. Addition of a second aldehyde initiates a domino sequence involving intermolecular addition followed by an intramolecular silyl-modified Sakurai (ISMS) reaction. Isolated yields of this one-pot reaction vary from 44 to 80% and all compounds were isolated as the cis-diastereomers (10 examples). Show less

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond - a single atom substitution of the peptide backbone - can quench fluorophores that are red-shifted from intrinsic… Show more

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond - a single atom substitution of the peptide backbone - can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetiem measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a larger number of measurements to model protein folding. Show less