An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Cytochromes P450 (P450s): A review of the class system with a focus on prokaryotic P450s.

Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are found in all Kingdoms of life and exhibit incredible diversity, both at sequence level and also on a biochemical basis. In the majority of cases, P450s can be assigned into one of ten classes based on their associated redox partners, domain architecture and cellular localization. Prokaryotic P450s now represent a large diverse collection of annotated/known enzymes, of which many have great potential biocatalytic potential. The self-sufficient P450 classes (Class VII/VIII) have been explored significantly over the past decade, with many annotated and biochemically characterized members. It is clear that the prokaryotic P450 world is expanding rapidly, as the number of published genomes and metagenome studies increases, and more P450 families are identified and annotated (CYP families).

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.

Analysis of single, cisplatin-induced DNA bends by atomic force microscopy and simulations.

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end-to-end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end-to-end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end-to-end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend.

Influence of temperature and pressure on cyclic carbonate synthesis catalyzed by bimetallic aluminum complexes and application to overall syn-bis-hydroxylation of alkenes.

The effect of moderate temperatures (22-100 °C) and pressures (1-10 bar) on the synthesis of cyclic carbonates from epoxides and carbon dioxide catalyzed by a combination of bimetallic aluminum complexes and tetrabutylammonium bromide is investigated. The combined bimetallic complex and tetrabutylammonium bromide catalyst system is shown to be an order of magnitude more active than the use of tetrabutylammonium bromide alone at all temperatures and pressures studied. At the higher temperatures and pressures used, disubstituted epoxides become substrates for the reaction and it is shown that reactions proceed with retention of the epoxide stereochemistry. This allowed a route for the overall syn-bis-hydroxylation of alkenes to be developed without the use of hazardous metal based reagents. At higher pressures it is also possible to use compressed air as the carbon dioxide source.

Kinetics and mechanism of the racemic addition of trimethylsilyl cyanide to aldehydes catalysed by Lewis bases.

The mechanism by which four Lewis bases, triethylamine, tetrabutylammonium thiocyanate, tetrabutylammonium azide and tetrabutylammonium cyanide, catalyse the addition of trimethylsilyl cyanide to aldehydes is studied by a combination of kinetic and spectroscopic methods. The reactions can exhibit first or second order kinetics corresponding to three different reaction mechanisms. Spectroscopic evidence for the formation of hypervalent silicon species is obtained for reaction between all of the tetrabutylammonium salts and trimethylsilyl cyanide. The reactions are accelerated by the presence of water in the reaction mixture, an effect which is due to a change in the reaction mechanism from Lewis to Brønsted base catalysis. Tetrabutylammonium thiocyanate is shown to be an excellent catalyst for the synthesis of cyanohydrin trimethylsilyl ethers on a preparative scale.

Reducing the cost of production of bimetallic aluminium catalysts for the synthesis of cyclic carbonates.

Bimetallic aluminium complexes of general formula [(salen)Al](2)O or [(acen)Al](2)O catalyse the formation of cyclic carbonates from carbon dioxide and terminal epoxides under exceptionally mild reaction conditions. To improve the potential for industrial scale application of these catalysts, the cost of their production has been evaluated and reduced significantly by optimization of the synthesis, including replacement of the most expensive chemicals by less expensive alternatives. The largest cost saving was associated with the formation of aluminium triethoxide in situ, which reduced the cost of the chemicals need for production of the catalysts by 49-87 %. Further savings were made by avoiding the use of tetrabutylammonium bromide and acetonitrile, resulting in overall cost savings of 68-93 %.

Determination of sulfur in biodiesel microemulsions using the summation of the intensities of multiple emission lines.

A method for the determination of sulfur in biodiesel samples by inductively coupled plasma optical emission spectrometry which uses microemulsion for sample preparation and the summation of the intensities of multiple emission lines has been developed. Microemulsions were prepared using 0.5 mL of 20% v/v HNO(3), 0.5 mL of Triton X-100, 2-3 mL of biodiesel sample, and diluted with n-propanol to a final volume of 10 mL. Summation of the emission intensities of multiple sulfur lines allowed for increased accuracy and sensitivity. The amounts of sulfur determined experimentally were between 2 and 7 mg L(-1), well below legislative standards for many countries. Recoveries obtained ranged from 72 to 119%, and recoveries obtained for the 182.562 nm line were slightly lower. This is most likely due to its lower sensitivity. Using microemulsion for sample preparation and the summation of the intensities of multiple emission lines for the successful determination of sulfur in biodiesel has been demonstrated.

Continuum source tungsten coil atomic fluorescence spectrometry.

A simple continuum source tungsten coil atomic fluorescence spectrometer is constructed and evaluated. The heart of the system is the atomizer: a low-cost tungsten filament extracted from a 150 W light bulb. The filament is resistively heated with a small, solid-state, constant-current power supply. The atomizer is housed in a glass chamber and purged with a 1 L/min flow of a conventional welding gas mixture: 10% H(2)/Ar. A 25 µL sample aliquot is pipetted onto the tungsten coil, the liquid is dried at low current, and then the atomic vapor is produced by applying a current in the range 3.5-5.5 A. The atomization current does not produce temperatures high enough to excite atomic emission. Radiation from a 300 W xenon lamp is focused through the atomic vapor, exciting atomic fluorescence. Fluorescence signals are collected using a hand-held charge-coupled device (CCD) spectrometer. Simultaneous determination of ten elements (Ag, Bi, Cr, Cu, Ga, In, Mg, Mn, and Tl) results in detection limits in the range 0.3 to 10 ng. The application of higher atomization currents (10 A) leads to straightforward detection of atomic emission signals with no modifications to the instrument.

One-component bimetallic aluminium(salen)-based catalysts for cyclic carbonate synthesis and their immobilization.

The development of one-component, bimetallic µ-oxoaluminium(salen) complexes as highly active catalysts for the synthesis of cyclic carbonates from terminal epoxides is described. The resulting homogeneous catalysts are used in batch reactions at room temperature and one atmosphere pressure. The catalysts have also been immobilized onto various support materials and used in either batch reactions or gas-phase flow reactions with ethylene and propylene oxides. Catalyst lifetime, deactivation and reactivation have been studied in both batch and flow reactions, and it has been shown that of the impurities present in power station flue gas, only sulfur trioxide deactivates the catalyst and at the concentrations of sulfur trioxide present in flue gas, this deactivation would require more than one years exposure of the catalyst to flue gas.

Mechanism-guided development of VO(salen)X complexes as catalysts for the asymmetric synthesis of cyanohydrin trimethylsilyl ethers.

Catalyze this! Detailed study of the mechanism of asymmetric cyanohydrin synthesis catalyzed by VO(salen)X complexes (see figure) led to the development of VO(salen)NCS, as the most active vanadium-based catalyst yet developed for this reaction.The mechanism by which oxovanadium(V)(salen) complexes(1) VO(salen)X catalyze the asymmetric addition of trimethylsilyl cyanide to benzaldehyde has been studied. The reaction kinetics indicated that the structure of the counterion (X) had a significant influence on the rate, but not on the enantioselectivity of the reaction. The less coordinating the counterion, the lower the catalytic activity; a trend that was confirmed by a Hammett analysis. Variable temperature kinetics allowed the enthalpies and entropies of activation to be determined for some catalysts, and showed that, for others, the overall reaction order changes from second order to zero order as the temperature is reduced. The order with respect to the catalyst was determined for nine of the VO(salen)X complexes and showed that the less active catalysts were active predominantly as mononuclear species whilst the more active catalysts were active predominantly as dinuclear species. Mass spectrometry confirmed the formation of dinuclear species in situ from all of the VO(salen)X complexes and indicated that the dinuclear complexes contained one vanadium(V) and one vanadium(IV) ion. The latter conclusion was supported by cyclic voltammetry of the complexes, by fluorescence measurements and by the fact that catalyst deactivation occurs when reactions are carried out under an inert atmosphere. Based on this evidence, it has been deduced that the catalysis involves two catalytic cycles: one for catalysis by mononuclear VO(salen)X species and the other for catalysis by dinuclear species. The catalytic cycle involving dinuclear species involves activation of both the cyanide and aldehyde, whereas the catalytic cycle involving mononuclear species activates only the aldehyde, thus explaining the higher catalytic activity observed for catalysts which are predominantly active as dinuclear complexes. Based on these mechanistic results, two new VO(salen)X complexes (X=F and NCS) were predicted to form highly active catalysts for asymmetric cyanohydrin synthesis. VO(salen)NCS was indeed found to be the most active catalyst of this type and catalyzed the asymmetric addition of trimethylsilyl cyanide to thirteen aldehydes. In each case, high yields and enantioselectivities were obtained after a reaction time of two hours at room temperature using just 0.1 mol % of the catalyst.

In situ formation of heterobimetallic salen complexes containing titanium and/or vanadium ions.

A combination of high-resolution electrospray mass spectrometry and (1)H NMR spectroscopy has been used to prove that when a mixture of [(salen)TiO]2 complexes containing two different salen ligands (salen and salen') is formed, an equilibrium is established between the homodimers and the heterodimer [(salen)TiO2Ti(salen')]. Depending upon the structure and stereochemistry of the two salen ligands, the equilibrium may favor either the homodimers or the heterodimer. Extension of this process to mixtures of titanium(salen) complexes [(salen)TiO]2 and vanadium (V)(salen') complexes [(salen')VO] (+)Cl (-) allowed the in situ formation of the heterobimetallic complex [(salen)TiO2V(salen')] (+)X (-) to be confirmed for all combinations of salen ligands studied except when the salen ligand attached to titanium contained highly electron-withdrawing nitro-groups. The rate of equilibration between heterobimetallic complexes is faster than that between two titanium complexes as determined by line broadening in the (1)H NMR spectra. These structural results explain the strong rate-inhibiting effect of vanadium (V)(salen) complexes in asymmetric cyanohydrin synthesis catalyzed by [(salen)TiO]2 complexes. It has also been demonstrated for the first time that the titanium and vanadium complexes can undergo exchange of salen ligands and that this is catalyzed by protic solvents. However, the ligand exchange is relatively slow (occurring on a time scale of days at room temperature) and so does not complicate studies aimed at using heterobimetallic titanium and vanadium salen complexes as asymmetric catalysts. Attempts to obtain a crystal structure of a heterobimetallic salen complex led instead to the isolation of a trinuclear titanium(salen) complex, the formation of which is also consistent with the catalytic results obtained previously.

Simple scaling laws for influenza A rise time, duration, and severity.

Simple scaling laws are developed for the severity and characteristic time scales of influenza A infection in man. The scaling laws are based on a model of the infection described by six coupled ordinary differential equations that describe the time courses of the numbers of infectious viral particles, activated cytotoxic T-lymphocytes, interferon molecules, infected cells, uninfected cells, and the subset of uninfected cells that are protected by interferon from viral infection. Computer simulations show that the disease can be regarded approximately as a two-stage process. In the first stage, the growth in the number of infected cells is determined primarily by the interferon-enhanced limitation in the available number of target cells. In the second stage, the bulk of the duration of the infection is determined mainly by the destruction of the infected cells by the cytotoxic T-lymphocytes. The severity and characteristic times of the infection are found to depend simply on the logarithm of the initial number of viruses.

Quality of life in patients undergoing segmental mandibular resection and staged reconstruction with nonvascularized bone grafts.

PURPOSE: The treatment of locally aggressive lesions of the mandible can have a profound effect on a patient's quality of life (QOL), both functionally and psychologically. The more knowledge available on this subject, the better a patient can be prepared and counseled. PATIENTS AND METHODS: Patients undergoing mandibular resection with immediate insertion of a reconstruction plate and subsequent staged reconstruction for the management of locally aggressive lesions of the mandible at the University of California at San Francisco were studied over a 6-year period from 1999 to 2005. The staged reconstruction involved bone grafting, removal of the reconstruction plate, vestibuloplasty, implant insertion, and construction of an implant-supported prosthesis. Each patient completed a modified University of Washington Quality of Life version 4 questionnaire after reaching his or her highest level of reconstruction. RESULTS: A total of 26 patients were identified as fulfilling the criteria for this study. Eight patients did not progress beyond resection, bone grafting, and subsequent removal of the bone plate. Seven patients progressed all the way through the 7 stages to an implant-supported prosthesis. The most important determinants of the patient's subsequent QOL were time interval from initial resection and stage of reconstruction; the greater the time interval from initial resection and the further the stage of reconstruction reached, the better the QOL. Resections involving the mandibular angle had a negative effect on appearance, and resections involving the parasymphysis and symphysis region carried a worse overall QOL, possibly due to difficulty with chewing and lip support. Males reached a higher overall QOL than females. Although most patients seemed satisfied with their overall QOL, 73% had some concerns regarding appearance, 42% had concerns with chewing, 42% had concerns regarding their mood, 35% felt more anxious since the surgery, 23% had some residual pain and discomfort, 19% had concerns with swallowing, and 15% had concerns with taste. CONCLUSION: These and other findings necessitate further investigation and confirmation.

Surrogate markers of health after titanium dental implant placement.

PURPOSE: We investigated the effects of dental implants on patient health, as measured by commonly used surrogates of human homeostasis, including hepatic metabolic and synthetic ability, renal regulatory functions, bone turnover, clotting, infection, and inflammation. MATERIALS AND METHODS: Patients enrolled had 1 to 3 mandibular uncoated, endosseous titanium dental implants placed and restored. Patients were evaluated serially over 6 months. Blood and urine tests were collected immediately before and 6 months after implant placement. Paired student t tests were used to compare quantified laboratory values. Nonquantified values were evaluated using the McNamara chi square analysis to assess patterns of change. RESULTS: Thirty-one patients met the inclusion criteria. A total of 53 dental implants were placed. Three values were found to be significantly different between the 2 time periods: total protein (P < .008), lactate dehydrogenase (P < .002), and hematocrit (P < .017). Total protein and lactate dehydrogenase were within the laboratory range of normal and judged to be clinically nonsignificant. On reevaluation, the significant change in hematocrit was entirely accounted for by a single outlier. This was attributed to laboratory error. CONCLUSION: No clinically significant effects on surrogate markers of health were found 6 months after placement of titanium dental implants. This series is the only one in the dental, craniomaxillofacial, and orthopedic literature showing a lack of adverse human health effects after dental implant placement, as measured by accepted surrogate measures of human homeostasis.

Reconstruction of extensive maxillary defects using zygomaticus implants.

PURPOSE: Maxillary reconstruction after maxillectomy remains a great challenge for the reconstructive oral and maxillofacial surgeon. This article is a clinical retrospective analysis of patients reconstructed with zygomaticus implants after maxillary ablation. PATIENTS AND METHODS: The design of the study was a retrospective review of 9 patients requiring near-total or total maxillectomy for pathologic reasons. Clinical records, photographs, and radiographs were studied. Financial billing statements were reviewed to determine charges for implant reconstruction and method of payment. RESULTS: Maxillary reconstruction using zygomaticus and standard endosseous implants was performed in 9 patients. Maxillary resection was performed for the following reasons: salivary gland malignancy (n = 2), squamous cell carcinoma (n = 5), maxillary mucormycosis (n = 1), and extensive maxillary atrophy and infection secondary to subperiosteal maxillary implant placement (n = 1). A total of 28 zygomaticus implants and 10 standard endosseous implants were used to reconstruct the 9 patients. Six zygomaticus implants and 3 standard endosseous implants failed. The time of zygomaticus implant placement ranged from placement at the time of resection to 3.2 years after the resection. Five patients received radiation therapy. Five patients have been reconstructed with a maxillary obturator and have been functioning with the prosthesis for a minimum of 2 years. CONCLUSION: The combination of zygomaticus and standard endosseous implants can be used to reliably reconstruct patients after extensive resection of the maxilla.