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Enzymes are capable of unique and selective transformations that can enable sustainable chemical production. While many industrial processes have been developed using free enzymes in aqueous solutions, immobilizing enzymes on a solid support can offer considerable advantages, including improved reaction efficiency, enzyme stability, the ability to perform reactions in non-aqueous media, and simplified separation of product from enzyme. Herein, we describe the development of a biocatalytic transaminase reaction of CyreneTM (2) utilizing an immobilized, evolved transaminase enzyme in an organic solvent to provide amine intermediate 3a en route to the Brutons tyrosine kinase (BTK) inhibitor nemtabrutinib. Enzyme immobilization is critical to facile isolation of the water-soluble product. Improved reaction kinetics and diastereoselectivity were achieved by bridging directed enzyme evolution with the selection of an optimal reaction solvent and solid support for immobilization, enabling a unified solvent system and direct isolation of 3a as a crystalline salt with dr > 50:1.

As practitioners of organic chemistry strive to deliver efficient syntheses of the most complex natural products and drug candidates, further innovations in synthetic strategies are required to facilitate their efficient construction. These aspirational breakthroughs often go hand-in-hand with considerable reductions in cost and environmental impact. Enzyme-catalyzed reactions have become an impressive and necessary tool that offers benefits such as increased selectivity and waste limitation. These benefits are amplified when enzymatic processes are conducted in a cascade in combination with novel bond-forming strategies. In this article, we report a highly diastereoselective synthesis of MK-1454, a potent agonist of the stimulator of interferon gene (STING) signaling pathway. The synthesis begins with the asymmetric construction of two fluoride-bearing deoxynucleotides. The routes were designed for maximum convergency and selectivity, relying on the same benign electrophilic fluorinating reagent. From these complex subunits, four enzymes are used to construct the two bridging thiophosphates in a highly selective, high yielding cascade process. Critical to the success of this reaction was a thorough understanding of the role transition metals play in bond formation.

Two scalable and efficient synthetic routes for the synthesis of a T-type calcium channel antagonist MK-8998 were developed from a simple pyridine building block. The key step to set the stereochemistry relied on either chiral rhodium catalyst-mediated asymmetric hydrogenation of an enamide or transamination of an arylketone that provided the corresponding product in high enantioselectivity and high yield.

Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes., Biocatalysis has developed into a mature technology for chemical and pharmaceutical synthesis as well as other areas where high selectivity and mild reaction conditions are required. This Review highlights recent achievements with a special focus on industrialized applications including the introduction of key performance indicators (KPIs) to judge the efficiency of enzymes.

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s-1 is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

Maximal efficiency from enzyme cascades Enzymes are highly selective catalysts that can be useful for specific transformations in organic synthesis. Huffman et al. combined designer enzymes in a multistep cascade reaction (see the Perspective by O'Reilly and Ryan). The approach eliminates purification steps, recycles expensive cofactors, and couples favorable and unfavorable reactions. With the target molecule islatravir, an experimental HIV drug, they optimized five enzymes by directed evolution to be compatible with unnatural substrates and stable in the reaction conditions. Stereochemical purity was amplified at every enzymatic step, and the final synthesis was both atom economical and efficient. Science , this issue p. 1255 ; see also p. 1199

A non-targeted method for classifying authentic and adulterated skim and nonfat dry milk powders (MP) by solution-state, high-field 1H NMR spectroscopy and conformity index analysis has been developed. Authentic MP samples from the global market and synthetically-adulterated MP samples involving eight adulterants were used in a pre-validation study. Adulteration was detected at the lowest concentrations (≥ 0.005 – 0.05% w/w) for samples containing nitrogen-rich, small molecules (melamine and dicyandiamide). For urea, a milk metabolite, and for sucrose and maltodextrin, the detection thresholds were higher (≥ 0.5% w/w). An NMR peak associated with ammonium sulfate was not observed; however, adulteration was indirectly detected at 5% w/w via matrix effects on milk metabolites. Adulteration by soy protein isolate and whey protein concentrate was not detected even at 5% w/w of spiking, which was attributed in part to poor protein solubility. These results provide a successful proof of concept that 1H NMR combined with conformity index analysis can support MP authentication and detect adulteration.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence

Kjeldahl and combustion (Dumas) methods are widely accepted for total protein determination but lack analytical selectivity for protein because they measure protein on the basis of sample nitrogen content. Adulteration incidents exploiting this analytical vulnerability (for example, melamine) demonstrate that these methods are no longer sufficient to protect the public health. This article explores the challenges and opportunities to move beyond total nitrogen based methods for total protein measurement. First, it explores the early history of protein measurement science, complexities of current global protein measurement activities, and ideal analytical performance characteristics for new methods. Second, it comprehensively reviews the pros and cons of current and emerging approaches for protein measurement, including their selectivity for protein, ability to detect adulteration, and practicality for routine use throughout the supply chain. It concludes that some existing highly selective methods for food protein measurement have potential for routine quality control. It also concludes that their successful implementation will require matrix-specific validation and the use of supporting reference materials. These methods may be suitable only for food ingredients that have a low degree of compositional variability and are not complex finished food products.

The higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes. We surveyed in silico modeling correlated with peptide level HDX-MS experiments to characterize and localize transaminase enzyme structural dynamics at different conditions. This approach was orthogonally correlated with a global Size-Exclusion-HDX (SEC-HDX) screen for global conformational comparison and global alpha-helical content measurements by circular dichroism. Enzymatic activity and stereo-selectivity of transaminases were compared at different reaction-solution conditions that forced protein conformational changes by increasing acetonitrile concentration. The experimental peptide-level HDX-MS results demonstrated similar trends to the modeling data showing that certain regions remained folded in transaminases ATA-036 and ATA-303 with increasing acetonitrile concentration, which is also associated with shifting stereoselectivity. HDX modeling, SEC-HDX and CD experimental data showed that transaminase ATA-234 had the highest level of global unfolding with increasing acetonitrile concentration compared to the other two enzymes, which correlated with drastically reduced product conversion in transamination reaction. The combined HDX modeling/experimental workflow, based on enzymatic reactions studied at different conditions to induce changes in enzyme conformation, could be used as a tool to guide directed evolution efforts by identifying and focusing on the regions of an enzyme required for reaction product conversion and stereoselectivity.

Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to create combinations of mutations as part of the initial design was confounded by the relatively large number of predicted mutations that were inactivating (30% and 45% for the sequence-based and structure-based methods, respectively). Despite this, combining several mutations identified within a given method produced variant lysates 7- and 9-fold more active than the wild-type lysate, highlighting the capability of mutations chosen this way to generate large advances in activity in addition to the reductions in screening.

Proton nuclear magnetic resonance spectra for 66 commercial powdered milk samples were analyzed by principal component analysis, soft independent modeling of class analogy, and pooled, crossed analysis of variance. It was found that the sample type (skim milk powder or non-fat dry milk), the supplier, the production site, the processing temperature (high, medium, or low temperature), and the day of analysis provided statistically significant sources of variation. Interestingly, inexact alignment (deviations of ±0.002 ppm) of the spectral reference peak was a significant source of variation, and fine alignment was necessary before the variation arising from the other experimental factors could be accurately evaluated. Using non-targeted analysis, the lowest detectable adulteration for dicyandiamide, melamine, and sucrose was 0.05%, the lowest detectable adulteration for maltodextrin and urea was 0.5%, the lowest detectable adulteration for ammonium sulfate and whey was 5%, and the lowest adulteration for soy protein isolate was undetectable using methods described herein. The measurement of variance and detection of adulteration were relatively unaffected by the resolution. Similar results were obtained with unbinned data (0.0003 ppm resolution) and binning of 333 data points (0.1 ppm resolution).

Food fraud, the intentional misrepresentation of the true identity of a food product or ingredient for economic gain, is a threat to consumer confidence and public health and has received increased attention from both regulators and the food industry. Following updates to food safety certification standards and publication of new U.S. regulatory requirements, we undertook a project to (i) develop a scheme to classify food fraud-related adulterants based on their potential health hazard and (ii) apply this scheme to the adulterants in a database of 2,970 food fraud records. The classification scheme was developed by a panel of experts in food safety and toxicology from the food industry, academia, and the U.S. Food and Drug Administration. Categories and subcategories were created through an iterative process of proposal, review, and validation using a subset of substances known to be associated with the fraudulent adulteration of foods. Once developed, the scheme was applied to the adulterants in the database. The resulting scheme included three broad categories: 1, potentially hazardous adulterants; 2, adulterants that are unlikely to be hazardous; and 3, unclassifiable adulterants. Categories 1 and 2 consisted of seven subcategories intended to further define the range of hazard potential for adulterants. Application of the scheme to the 1,294 adulterants in the database resulted in 45% of adulterants classified in category 1 (potentially hazardous). Twenty-seven percent of the 1,294 adulterants had a history of causing consumer illness or death, were associated with safety-related regulatory action, or were classified as allergens. These results reinforce the importance of including a consideration of food fraud-related adulterants in food safety systems. This classification scheme supports food fraud mitigation efforts and hazard identification as required in the U.S. Food Safety Modernization Act Preventive Controls Rules.

The United States Pharmacopeial Convention has led an international collaborative project to develop a toolbox of screening methods and reference standards for the detection of milk powder adulteration. During the development of adulterated milk powder reference standards, blending methods used to combine melamine and milk had unanticipated strong effects on the near-infrared (NIR) spectrum of melamine. The prominent absorbance band at 1468 nm of melamine was retained when it was dry-blended with skim milk powder but disappeared in wet-blended mixtures, where spray-dried milk powder samples were prepared from solution. Analyses using polarized light microscopy, Raman spectroscopy, dielectric relaxation spectroscopy, X-ray diffraction, and mass spectrometry indicated that wet blending promoted reversible and early Maillard reactions with lactose that are responsible for differences in melamine NIR spectra between wet- and dry-blended samples. Targeted detection estimates based solely on dry-blended reference standards are likely to overestimate NIR detection capabilities in wet-blended samples as a result of previously overlooked matrix effects arising from changes in melamine hydrogen-bonding status, covalent complexation with lactose, and the lower but more homogeneous melamine local concentration distribution produced in wet-blended samples. Techniques used to incorporate potential adulterants can determine the suitability of milk reference standards for use with rapid detection methods.

The combination of high speed autosampler technology and ultrafast chromatographic separations enables faster high throughput analysis. With an injection cycle time of 10.6 s, MISER (Multiple Injection in a Single Experimental Run) HPLC-MS analysis of a 96 well microplate can be completed in only 17min. As chromatographic separations in the sub 5s range become increasingly common, even faster autosamplers will be needed to realize further speed improvements in high throughput LC-MS analysis. Indeed with proper hardware sampling approaches, chromatographic analysis of microplates could approach speeds of spectrophotometric plate readers while maintaining the advantage of multicomponent detection and monitoring.

Kjeldahl and Dumas crude protein measurement methodologies do not distinguish between nitrogen native to milk and nitrogen in low molecular mass, nitrogen-rich adulterants. Measuring the non-protein nitrogen (NPN) content is one possible means of closing this loophole. Four methods were considered, with three selected for further research and validation: protein precipitation using trichloroacetic acid, protein precipitation using tannic acid, and molecular mass cut-off filtration. Kjeldahl assay was used on the supernatant or filtrate for all three methods. An NPN reference concentration range was established using 15 milk powder samples. This was followed by spiking experiments using seven low-molecular-weight, nitrogen-rich adulterants. Tannic acid precipitation and molecular mass cut-off filtration, both simple techniques available for routine use in food analysis laboratories, proved to be suitable for detecting the adulteration of milk powders with a variety of nitrogenous contaminants. NPN concentrations of ≥0.34% in milk powders are suspected to result from adulteration.

An enantioselective synthesis of the potent anti-HIV nucleoside EFdA is presented. Key features of stereocontrol include construction of the fully substituted 4'-carbon via a biocatalytic desymmetrization of 2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate and a Noyori-type asymmetric transfer hydrogenation to control the stereochemistry of the 3'-hydroxyl bearing carbon. The discovery of a selective crystallization of an N-silyl nucleoside intermediate enabled isolation of the desired β-anomer from the glycosylation step.

Raman spectroscopy in combination with chemometrics was explored as a rapid, non-targeted screening method for the detection of milk powder (MP) adulteration using melamine as an example contaminant. Raman spectroscopy and an unsupervised pattern-recognition method, principal component analysis (PCA), allowed for the differentiation of authentic MPs from adulterated ones at concentrations > 1.0% for dry-blended (DB) samples and > 0.30% for wet-blended (WB) ones. Soft independent modelling of class analogy (SIMCA), a supervised pattern-recognition method, was also used to classify test samples as adulterated or authentic. Combined statistics at a 97% confidence level from the SIMCA models correctly classified adulteration of MP with melamine at concentrations ≥ 0.5% for DB samples and ≥ 0.30% for WB ones, while no false-positives from authentic MPs were found when the spectra in the 600–700 cm–1 range were pre-processed using standard normal variate (SNV) followed by a gap-segment derivatisation. The combined technique of Raman spectroscopy and chemometrics proved to be a useful tool for the rapid and cost-efficient non-targeted detection of adulteration in MP at per cent spiking levels.

A practical through-process to prepare iso-Boc (S)-2-amino-8-nonenoic acid has been developed. The short synthesis utilizes a highly enantioselective, enzymatic reductive amination of an α-keto acid substrate, which is prepared via a Grignard addition to diethyl oxalate. Starting from 7-bromohept-1-ene and without isolation of any intermediates, iso-Boc (S)-2-amino-8-nonenoic acid is prepared in 60% overall yield and >99.9% ee.

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.

An asymmetric synthesis of orexin receptor antagonist MK-6096 piperidine core, ((3R,6R)-6-methylpiperidin-3-yl)methanol (3), is described. The target is synthesized in four steps and 40% overall yield from methyl vinyl ketone and diethyl malonate. The key operation is a practical crystallization-induced dynamic resolution for the conversion of a trans/cis mixture of lactam acid 17 into the desired trans-lactam acid salt in >95% de and 91% yield. The substrate lactam acid mixture was prepared via a solvent-free Michael reaction and a practical biocatalytic transamination process.

investigated by the Unites States Pharmacopeia (USP) in an effort to develop a successful non-targeted solution for milk powder. A significant challenge to be faced in this study is the normal physicochemical variability of pure milk powders. The existence of these variations will have the effect of widening the boundaries for any potential non-targeted method which may have the effect of decreasing the method’s ability to detect adulterants present at low concentrations. This problem is especially true for NIR diffuse reflectance spectroscopy, with its inherent limitations in the accurate detection of chemical components which are present below approximately 0.1% w /w in a sample matrix. In this report, the variance of NIR spectra from 41 different bovine skim milk powders and non-fat dry milk powders was explored. Spectral data were examined to identify influential sources of variance using princi pal component score trends and spectral signatures in rotated principal component loadings. Chemical analysis of samples of interest was also used to support the interpretations of the rotated principal components.

Isotopically labeled compounds have numerous applications in drug discovery and development. (R)-9-(2-Hydroxypropyl)adenine 1 is an important intermediate in the synthesis of various acyclic nucleoside phosphonates including tenofovir. We have developed a short synthetic method to prepare deuterium and tritium labeled 1 using a ketoreductase enzyme catalyzed reduction as the key step to introduce the label in a highly efficient and enantioselective way.

Forty-one samples of skim milk powder (SMP) and nonfat dry milk (NFDM) from 8 suppliers, 13 production sites, and 3 processing temperatures were analyzed by NIR diffuse reflectance spectrometry over a period of 3 days. NIR reflectance spectra (1700-2500 nm) were converted to pseudoabsorbance and examined using (a) analysis of variance-principal component analysis (ANOVA-PCA), (b) pooled-ANOVA based on data submatrices, and (c) partial least-squares regression (PLSR) coupled with pooled-ANOVA. ANOVA-PCA score plots showed clear separation of the samples with respect to milk class (SMP or NFDM), day of analysis, production site, processing temperature, and individual samples. Pooled-ANOVA provided statistical levels of significance for the separation of the averages, some of which were many orders of magnitude below 10⁻³. PLSR showed that the correlation with Certificate of Analysis (COA) concentrations varied from a weak coefficient of determination (R²) of 0.32 for moisture to moderate R² values of 0.61 for fat and 0.78 for protein for this multinational study. In this study, pooled-ANOVA was applied for the first time to PLS modeling and demonstrated that even though the calibration models may not be precise, the contribution of the protein peaks in the NIR spectra accounted for the largest proportion of the variation despite the inherent imprecision of the COA values.

Chromatographic profiles of skim milk powder (SMP) and mixtures of SMP with soy (SPI), pea (PPI), brown rice (BRP), and hydrolyzed wheat protein (HWPI) isolates were obtained by ultrahigh-performance liquid chromatography (UHPLC) with 215 nm detection. Two data analysis approaches were compared for their utility to classify samples as authentic or adulterated. The t test approach evaluated data points exceeding the 99% confidence limit of the mean authentic SMP chromatogram and used data points from the entire chromatogram. The other approach used the multivariate Q statistic from a SIMCA model of authentic samples to determine adulteration and used a selected retention window to obtain best classifications. Q-Statistic and t test correctly classified adulteration of SMP with SPI at the 1% and 3% levels, respectively, while minimizing false classifications of authentic SMP. Detection of SMP adulterated with PPI, BRP, and HWPI was possible at higher adulteration levels.

This document provided is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society, after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/jf4023433 peer-reviewed A multinational collaborative team led by the U.S. Pharmacopeial Convention is currently investigating the potential of near-infrared (NIR) spectroscopy for nontargeted detection of adulterants in skim and nonfat dry milk powder. The development of a compendial method is challenged by the range of authentic or nonadulterated milk powders available worldwide. This paper investigates the sources of variance in 41 authentic bovine skim and nonfat milk powders as detected by NIR diffuse reflectance spectroscopy and chemometrics. Exploratory analysis by principal component analysis and varimax factor rotation revealed significant variance in authentic samples and highlighted outliers from a single manufacturer. Spectral preprocessing and outlier removal methods reduced ambient and measurement sources of variance, most likely linked to changes in moisture together with sampling, preparation, and presentation factors. Results indicate that significant chemical variance exists in different skim and nonfat milk powders that will likely affect the performance of adulterant detection methods by NIR spectroscopy.

MK-6096 is an orexin receptor antagonist in clinical trials for the treatment of insomnia. Herein we describe its first kilogram-scale synthesis. Chirality on the α-methylpiperidine core was introduced in a biocatalytic transamination using a three-enzyme system with excellent enantioselectivity (>99% ee). Low diastereoselectivity of the lactam reduction was overcome by development of a camphor sulfonic acid salt formation and dr upgrade. A chemoselective O-alkylation with 5-fluoro-2-hydroxypyridine was optimized and developed. Overall, 1.2 kg of MK-6069 was prepared in nine steps and 13% overall yield.

There is a need to develop rapid tools to screen milk products for economically motivated adulteration. An understanding of the physiochemical variability within skim milk powder (SMP) and non-fat dry milk (NFDM) is the key to establishing the natural differences of these commodities prior to the development of non-targeted detection methods. This study explored the sources of variance in 71 commercial SMP and NFDM samples using Raman spectroscopy and principal component analysis (PCA) and characterised the largest number of commercial milk powders acquired from a broad number of international manufacturers. Spectral pre-processing using a gap-segment derivative transformation (gap size = 5, segment width = 9, fourth derivative) in combination with sample normalisation was necessary to reduce the fluorescence background of the milk powder samples. PC scores plots revealed no clear trends for various parameters, including day of analysis, powder type, supplier and processing temperatures, while the largest variance was due to irreproducibility in sample positioning. Significant chemical sources of variances were explained by using the spectral features in the PC loadings plots where four samples from the same manufacturer were determined to likely contain an additional component or lactose anomers, and one additional sample was identified as an outlier and likely containing an adulterant or differing quality components. The variance study discussed herein with this large, diverse set of milk powders holds promise for future use as a non-targeted screening method that could be applied to commercial milk powders.

Food ingredient fraud and economically motivated adulteration are emerging risks, but a comprehensive compilation of information about known problematic ingredients and detection methods does not currently exist. The objectives of this research were to collect such information from publicly available articles in scholarly journals and general media, organize into a database, and review and analyze the data to identify trends. The results summarized are a database that will be published in the US Pharmacopeial Convention's Food Chemicals Codex, 8th edition, and includes 1305 records, including 1000 records with analytical methods collected from 677 references. Olive oil, milk, honey, and saffron were the most common targets for adulteration reported in scholarly journals, and potentially harmful issues identified include spices diluted with lead chromate and lead tetraoxide, substitution of Chinese star anise with toxic Japanese star anise, and melamine adulteration of high protein content foods. High-performance liquid chromatography and infrared spectroscopy were the most common analytical detection procedures, and chemometrics data analysis was used in a large number of reports. Future expansion of this database will include additional publically available articles published before 1980 and in other languages, as well as data outside the public domain. The authors recommend in-depth analyses of individual incidents. Practical Application: This report describes the development and application of a database of food ingredient fraud issues from publicly available references. The database provides baseline information and data useful to governments, agencies, and individual companies assessing the risks of specific products produced in specific regions as well as products distributed and sold in other regions. In addition, the report describes current analytical technologies for detecting food fraud and identifies trends and developments.

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

An ultra performance liquid chromatography-mass spectrometric (UPLC-MS) method was developed to investigate the pharmacokinetic properties of isorhamnetin, kaempferol and quercetin from a total flavone extract of Hippophae rhamnoides L. (TFH) after single dose oral administration. Rat plasma samples were pretreated using liquid-liquid extraction, and chromatographic separation was performed on a C(18) column using a linear gradient of methanol and formic acid (0.1%). The pharmacokinetic parameters of isorhamnetin, kaempferol and quercetin from TFH in rats were quantitatively determined by UPLC with photodiode array detection (PDA). The qualitative detection of the three flavones was accomplished by selected ion monitoring in negative ion mode ESI-MS. Results of the pharmacokinetic study indicate that the three flavones in TFH were absorbed by passive diffusion in rats, and no "double-peak" phenomenon was observed in C-t curves of the three flavones from TFH except for quercetin. Results of this study indicate that the pharmacokinetic behaviors of isorhamnetin, kaempferol and quercetin when administered together in a complex herbal extract might be different than the individual behaviors of the same compounds administered in their pure forms. Results of this study also demonstrate that UPLC-MS is a rapid and practical method to determine the pharmacokinetic parameters of flavones present in an herbal extract.

Biocatalytic Boost Enzymes tend to direct reactions toward specific products much more selectively than synthetic catalysts. Unfortunately, this selectivity has evolved for cellular purposes and may not promote the sorts of reactions chemists are seeking to enhance (see the Perspective by Lutz ). Siegel et al. (p. 309 ) now describe the design of enzymes that catalyze the bimolecular Diels-Alder reaction, a carbon-carbon bond formation reaction that is central to organic synthesis but unknown in natural metabolism. The enzymes display high stereoselectivity and substrate specificity, and an x-ray structure of the most active enzyme confirms that the structure matches the design. Savile et al. (p. 305 , published online 17 June) applied a directed evolution approach to modify an existing transaminase enzyme so that it recognized a complex ketone in place of its smaller native substrate, and could tolerate the high temperature and organic cosolvent necessary to dissolve this ketone. This biocatalytic reaction improved the production efficiency of a drug that treats diabetes.

An activity-directed fractionation and purification process was used to isolate 1,1-diphenyl-2-picrylhydrazyl radical (DPPH ) scavenging components from Juglans regia kernels. Ethyl acetate and n -butanol fractions showed greater DPPH scavenging activities compared to those of water and petroleum ether fractions. The ethyl acetate fraction was subjected to purification using column chromatography. Seven phenolic compounds, pyrogallol (1), p -hydroxybenzoic acid (2), vanillic acid (3), ethyl gallate (4), protocatechuic acid (5), gallic acid (6) and 3,4,8,9,10-pentahydroxydibenzo[ b , d ]pyran-6-one (7), containing significant antioxidant activities were isolated and identified in J. regia by spectroscopic methods for the first time in this study. The relative order of DPPH scavenging capacity for these compounds was 7 > 6 ⩾ 4 ⩾ 1 > Trolox ⩾ 5 > 3 > 2. The results of this study suggested that the antioxidant activities of these phenolic compounds may be influenced by the number of hydroxyls in their aromatic rings.

This study investigated the effects of processing conditions including bran particle size, dough fermentation time, and baking time and temperature on the extractable antioxidant properties of whole-wheat pizza crust. Experiments were carried out using two different varieties of hard white winter wheat, Trego and Lakin. Antioxidant properties examined included oxygen radical absorbing capacity (ORAC), hydroxyl radical scavenging capacity (HOSC), relative 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (RDSC), cation 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging capacity, total phenolic contents (TPC), and ferulic acid contents. Results indicated that bran particle size had no effect on the antioxidant properties evaluated. Increasing dough fermentation time from 0 to 48 h had no significant influence on antioxidant properties except HOSC, which increased as much as 28%, possibly as a result of increase in soluble free ferulic acid, which increased as much as 130%. Increasing baking temperature from 204 to 288 degrees C with a 7 min bake time increased all evaluated antioxidant properties by as much as 82%. Increasing baking time from 7 to 14 min with 204 degrees C baking temperature might increase some antioxidant properties as much as 60%. The results from this study suggest that longer dough fermentation times and increased baking time or temperature may be potential approaches to increase the antioxidant availability in whole-wheat pizza crust.

The asymmetric enzymatic ketone reduction of 4,4-dimethoxytetrahydro-2H-pyran-3-one provided the (R)-α-hydroxyketal, an important chiral precursor for a pharmaceutical intermediate, with high enantioselectivity (>99% ee). An economical process including in situ NADPH-cofactor regeneration using glucose dehydrogenase has been developed to produce the desired material in high yield (96–98%). The two-enzyme process was employed at pilot-plant scale to produce 80 kg of (R)-4,4-dimethoxytetrahydro-2H-pyran-3-ol. Critical factors for scale-up were found to be pH control and agitation speed.

Cold-pressed seed flours from pumpkin, parsley, mullein, cardamom, and milk thistle were examined for total oil, fatty acid profile of the oil, total phenolic content (TPC), scavenging activities against peroxyl (ORAC), hydroxyl (HOSC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (RDSC) radicals, and antiproliferative capacity against HT-29 human colon cancer cells. The cold-pressed parsley seed flour contained a very high concentration of total oil—17.6 g/100 g flour—with primarily C18:1 fatty acid at 86.2 g/100 g fatty acids. All other flour oils had relatively high levels of saturated fats, ranging from 39.0 to 62.9 g/100 g fatty acids. The tested seed flours demonstrated significant TPC and free radical scavenging activities. Milk thistle seed flour had the highest TPC value of 25.2 mg gallic acid equivalent per g flour (GAE mg/g) followed by that of parsley seed flour at 8.1 GAE mg/g. Milk thistle seed-flour extract also had significantly higher antioxidant activities than all other extracts against all tested radicals. The milk thistle seed-flour extract had an ORAC value of 1131 μmol trolox equivalents (TE) per g flour (TE μmol/g), a HOSC value of 893 TE μmol/g, and an RDSC value of 61 TE μmol/g. Also, ORAC, HOSC, and TPC values were significantly correlated (P < 0.01) under the experimental conditions. The cold-pressed milk thistle seed flour inhibited the proliferation of HT-29 cancer cells in a dose-dependent manner. Results from this study suggest that these cold-pressed seed flours may serve as natural sources of antioxidants and may be used to improve human health.

Immobilized Candida antarctica lipase B has been shown to form the pharmaceutically important (S)-γ-fluoroleucine ethyl ester intermediate from the ring opening of an azlactone with ethanol, providing 79% yield and 78% enantiomeric excess (ee) for a stirred tank batch process demonstrated at multi kilogram scale. A kinetic analysis of the reaction system was defined which included the selective enzyme mediated dynamic kinetic ethanolysis, the background hydrolysis to acid and the non selective ethanolysis to racemic ester. This was used to generate a kinetic model which made excellent predictions of the reaction behavior. This kinetic model was then used to develop two new optimized processes. A fed batch stirred tank process was developed that increased productivity four fold compared to the original batch process, while maintaining a similar product ee value. Additionally, a plug flow column reactor process was developed that significantly improved productivity via a 20 fold reduction in the enzyme deactivation rate observed in the batch processes. As a result, the plug flow reactor generated a 20 fold reduction in enzyme to substrate ratio and an increased product yield (>90%) and ee (86%) over the initial batch process, producing an effective and economical process for large-scale production.

Ethanol extracts of Chardonnay grape and black raspberry seed flours were evaluated for their capacity to suppress lipid oxidation, preserve important fatty acids, and inhibit microbial growth. They were also tested for radical scavenging activity against DPPH and peroxyl radicals as reflected in oxygen radical absorbance capacity (ORAC), and total phenolic content (TPC). Both tested seed flour extracts suppressed lipid oxidation and rancidity development in fish oil. Black raspberry seed flour extract significantly reduced the degradation of biologically important n − 3 PUFA under accelerated oxidative conditions. Black raspberry and Chardonnay seed flour extracts at 165 and 160 μg seed flour equivalents/mL, respectively exhibited bacteriocidal activity against Escherichia coli and growth inhibition of Listeria monocytogenes under experimental conditions. Both seed flour extracts exhibited DPPH radical quenching activity and Chardonnay had the stronger ORAC of 663 μmol Trolox equivalents per gram seed flour and the higher TPC of 99 mg gallic acid equivalents/g flour. The data from this study suggest the potential for developing natural food preservatives from these seed flours for improving food stability, quality, safety, and consumer acceptance.

Two methods are reported for synthesizing a chiral allylic alcohol from the corresponding racemic α,β-unsaturated ketone. Transition metal-based transfer hydrogenation provides the desired allylic alcohol in high enantiomeric purity but low diastereomeric excess. In contrast, an enzymatic dynamic kinetic reduction proceeds with high diastereoselectivity and enantioselectivity.

Recent consumer interest in controlling and preventing chronic diseases through improved diet has promoted research on the bioactive components of agricultural products. Wheat is an important agricultural and dietary commodity worldwide with known antioxidant properties concentrated mostly in the bran fraction. The objective of this study was to determine the relative contributions of genotype (G) and growing environment (E) to hard winter wheat bran antioxidant properties, as well as correlations of these properties to growing conditions. Bran samples of 20 hard winter wheat varieties grown in two locations were examined for their free radical scavenging capacities against DPPH, ABTS cation, peroxyl (ORAC), and superoxide anion radicals and chelating properties, as well as their total phenolics and phenolic acid compositions. Results showed significant differences for all antioxidant properties tested and multiple significant correlations between these properties. A factorial designed analysis of variance for these data and pooled previously published data showed similar results for four of the six antioxidant properties, indicating that G effects were considerably larger than E effects for chelating capacity and DPPH radical scavenging properties, whereas E was much stronger than G for ABTS cation radical scavenging capacity and total phenolics, although small interaction effects (G x E) were significant for all antioxidant properties analyzed. Results also showed significant correlations between temperature stress or solar radiation and some antioxidant properties. These results indicate that each antioxidant property of hard winter wheat bran is influenced differently by genotype and growing conditions.

An efficient route to the pharmaceutically important (6S,9R)-11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene intermediate has been demonstrated via kinetic resolution of 11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene using a commercially available ketoreductase. Biocatalytics KRED 101 has been shown to selectively reduce the (6R,9S) enantiomer leaving behind the desired (6S,9R) enantiomer. This novel reaction is the first demonstration of a high yielding (44% versus 50% maximum theoretical yield) highly stereoselective (>99% ee) resolution of a bicyclic ketone, via enzymatic reduction using a commercially available ketoreductase, where the stereochemistry of the substrate is determined by a bridged ring system. Several challenges were overcome, including enhancing the selectivity of the enzyme by controlling temperature and increasing substrate solubility by employing a combination of cyclodextrin and organic co-solvent in the aqueous reaction system.

Pseudomonas fluorescens lipase was found to catalyze the asymmetric hydrolysis of racemic indole-ethyl ester 1.1 The lipase converted the undesired (S) enantiomer to its corresponding acid, while l...