Mechanisms of acetoin toxicity and adaptive responses in an acetoin-producing species, Lactococcus lactis .

Acetoin, 3-hydroxyl,2-butanone, is extensively used as a flavor additive in food products. This volatile compound is produced by the dairy bacterium Lactococcus lactis when aerobic respiration is activated by haem addition, and comprises ∼70% of carbohydrate degradation products. Here we investigate the targets of acetoin toxicity, and determine how acetoin impacts L. lactis physiology and survival. Acetoin caused damage to DNA and proteins, which related to reactivity of its keto group. Acetoin stress was reflected in proteome profiles, which revealed changes in lipid metabolic proteins. Acetoin provoked marked changes in fatty acid composition, with massive accumulation of cycC19:0 cyclopropane fatty acid at the expense of its unsaturated C18:1 fatty acid precursor. Deletion of the cfa gene, encoding the cycC19:0 synthase, sensitized cells to acetoin stress. Acetoin-resistant transposon mutagenesis revealed a hot spot in the high affinity phosphate transporter operon pstABCDEF , which is known to increase resistance to multiple stresses. This work reveals the causes and consequences of acetoin stress on L. lactis , and may facilitate control of lactic acid bacteria production in technological processes. Importance Acetoin, 3-hydroxyl,2-butanone, has diverse uses in chemical industry, agriculture, and dairy industries as a volatile compound that generates aromas. In bacteria, it can be produced in high amount by Lactococcus lactis when it grows under aerobic respiration. However, acetoin production can be toxic and detrimental for growth and/or survival. Our results showed that it damages DNA and proteins via its keto group. We also showed that acetoin modifies membrane fatty acid composition with the production of cyclopropane C19:0 fatty acid at the expense of an unsaturated C18:1. We isolated mutants more resistant to acetoin than the wild-type strain. All of them mapped to a single locus pstABCDEF operon, suggesting a simple means to limit acetoin toxicity in dairy bacteria and to improve its production.

β-Ketophosphonates with Pentalenofuran Scaffolds Linked to the Ketone Group for the Synthesis of Prostaglandin Analogs

β-Ketophosphonates with pentalenofurane fragments linked to the keto group were synthesized. The bulky pentalenofurane skeleton is expected to introduce more hindrance in the prostaglandin analogues of type III, greater than that obtained with the bicyclo[3.3.0]oct(a)ene fragments of prostaglandin analogues I and II, to slow down (retard) the inactivation of the prostaglandin analogues by oxidation of 15α-OH to the 15-keto group via the 15-PGDH pathway. Their synthesis was performed by a sequence of three high yield reactions, starting from the pentalenofurane alcohols 2, oxidation of alcohols to acids 3, esterification of acids 3 to methyl esters 4 and reaction of the esters 4 with lithium salt of dimethyl methanephosphonate at low temperature. The secondary compounds 6b and 6c were formed in small amounts in the oxidation reactions of 2b and 2c, and the NMR spectroscopy showed that their structure is that of an ester of the acid with the starting alcohol. Their molecular structures were confirmed by single crystal X-ray determination method for 6c and XRPD powder method for 6b.

Selective cyclization modes of methyl 3′-heteroarylamino-2′-(2,5-dichlorothiophene-3-carbonyl)acrylates. Synthesis of model (thienyl)pyrazolo- and triazolo[1,5-α]pyrimidines

Interaction of methyl 3-ethoxy-2-(2,5-dichloro-3-thenoyl)acrylate (I) with 3-aminopyrazole and 3-amino-1,2,4-triazole generated the respective pyrazolo[1,5-α]pyrimidine (4) and triazolo[1,5-α]pyrimidine (7). The formation of 4 entails selective and consecutive displacement of the 3-ethoxy and methoxy (ester) anions in I by 3-NH2 and 1-NH of 3-aminopyrazole. On the other hand, the formation of 7 implies selective displacement of 3-ethoxy in I by the ring-NH followed by cyclocondensation involving the keto group in I and 3-NH2 of aminotriazole. This latter selective displacement sequence is also followed by 3-amino-5-hydroxypyrazole in its reaction with I. The structures of the new compounds are supported by microanalytical and spectral data.

A comparison of a ketogenic diet with a LowGI/nutrigenetic diet over 6 months for weight loss and 18-month follow-up

Background Obesity and its related metabolic disturbances represent a huge health burden on society. Many different weight loss interventions have been trialled with mixed efficacy, as demonstrated by the large number of individuals who regain weight upon completion of such interventions. There is evidence that the provision of genetic information may enhance long-term weight loss, either by increasing dietary adherence or through underlying biological mechanisms. Methods The investigators followed 114 overweight and obese subjects from a weight loss clinic in a 2-stage process. 1) A 24-week dietary intervention. The subjects self-selected whether to follow a standardized ketogenic diet (n = 53), or a personalised low-glycemic index (GI) nutrigenetic diet utilising information from 28 single nucleotide polymorphisms (n = 61). 2) After the 24-week diet period, the subjects were monitored for an additional 18 months using standard guidelines for the Keto group vs standard guidelines modified by nutrigenetic advice for the low-Glycaemic Index nutrigenetic diet (lowGI/NG) group. Results After 24 weeks, the keto group lost more weight: − 26.2 ± 3.1 kg vs − 23.5 ± 6.4 kg (p = 0.0061). However, at 18-month follow up, the subjects in the low-GI nutrigenetic diet had lost significantly more weight (− 27.5 ± 8.9 kg) than those in the ketogenic diet who had regained some weight (− 19.4 ± 5.0 kg) (p < 0.0001). Additionally, after the 24-week diet and 18-month follow up the low-GI nutrigenetic diet group had significantly greater (p < 0.0001) improvements in total cholesterol (ketogenic − 35.4 ± 32.2 mg/dl; low-GI nutrigenetic − 52.5 ± 24.3 mg/dl), HDL cholesterol (ketogenic + 4.7 ± 4.5 mg/dl; low-GI nutrigenetic + 11.9 ± 4.1 mg/dl), and fasting glucose (ketogenic − 13.7 ± 8.4 mg/dl; low-GI nutrigenetic − 24.7 ± 7.4 mg/dl). Conclusions These findings demonstrate that the ketogenic group experienced enhanced weight loss during the 24-week dietary intervention. However, at 18-month follow up, the personalised nutrition group (lowGI/NG) lost significantly more weight and experienced significantly greater improvements in measures of cholesterol and blood glucose. This suggests that personalising nutrition has the potential to enhance long-term weight loss and changes in cardiometabolic parameters. Trial registration NCT04330209, Registered 01/04/2020, retrospectively registered.

An Insight into the Interaction Between α-Ketoamide-Based Inhibitor and Coronavirus Main Protease: A Detailed in Silico Study

<div> <p>The search for therapeutic drugs that can neutralize the effects of COVID-2019 (SARS-CoV-2) infection is the main focus of current research. The coronavirus main protease (M_pro) is an attractive target for anti-coronavirus drug design. Further, α-ketoamide is proved to be very effective as a reversible covalent-inhibitor against cysteine proteases. Herein, we report on the non-covalent to the covalent adduct formation mechanism of α‑ketoamide-based inhibitor with the enzyme active site amino acids by QM/SQM model (QM= quantum mechanical, SQM= semi-empirical QM). To uncover the mechanism, we focused on two approaches: a concerted and a stepwise fashion. The concerted pathway proceeds <i>via</i> deprotonation of the thiol of cysteine (here, Cys₁₄₅ SgH) and simultaneous reversible nucleophilic attack of sulfur onto the α-ketoamide warhead. In this work, we propose three plausible concerted pathways. On the contrary, in a traditional two-stage pathway, the first step is proton transfer from Cys₁₄₅ SgH to His₄₁ Nd forming an ion pair, and consecutively, in the second step, the thiolate ion attacks the a-keto group to form a thiohemiketal. In this reaction, we find that the stability of the tetrahedral intermediate oxyanion/hydroxyl hole plays an important role. Moreover, as the α-keto group has two faces <i>Si</i> or <i>Re</i> for the nucleophilic attack, we considered both possibilities of attack leading to S- and R-thiohemiketal. We computed the structural, electronic, and energetic parameters of all stationary points including transition states <i>via</i> ONIOM methodology at B3LYP/6-31G(d):PM6 level. Furthermore, to get more accurate results, we also calculated the single-point dispersion-corrected energy profile by using ωB97X-D/6-31G(d,p):PM6 level. Additionally, to characterize covalent, weak noncovalent interaction (NCI) and hydrogen-bonds, we applied NCI-reduced density gradient (NCI-RDG) methods along with Bader’s Quantum Theory of Atoms-in-Molecules (QTAIM) and natural bonding orbital (NBO) analysis.</p> </div> <br>

An Insight into the Interaction Between α-Ketoamide-Based Inhibitor and Coronavirus Main Protease: A Detailed in Silico Study

<div> <p>The search for therapeutic drugs that can neutralize the effects of COVID-2019 (SARS-CoV-2) infection is the main focus of current research. The coronavirus main protease (M_pro) is an attractive target for anti-coronavirus drug design. Further, α-ketoamide is proved to be very effective as a reversible covalent-inhibitor against cysteine proteases. Herein, we report on the non-covalent to the covalent adduct formation mechanism of α‑ketoamide-based inhibitor with the enzyme active site amino acids by QM/SQM model (QM= quantum mechanical, SQM= semi-empirical QM). To uncover the mechanism, we focused on two approaches: a concerted and a stepwise fashion. The concerted pathway proceeds <i>via</i> deprotonation of the thiol of cysteine (here, Cys₁₄₅ SgH) and simultaneous reversible nucleophilic attack of sulfur onto the α-ketoamide warhead. In this work, we propose three plausible concerted pathways. On the contrary, in a traditional two-stage pathway, the first step is proton transfer from Cys₁₄₅ SgH to His₄₁ Nd forming an ion pair, and consecutively, in the second step, the thiolate ion attacks the a-keto group to form a thiohemiketal. In this reaction, we find that the stability of the tetrahedral intermediate oxyanion/hydroxyl hole plays an important role. Moreover, as the α-keto group has two faces <i>Si</i> or <i>Re</i> for the nucleophilic attack, we considered both possibilities of attack leading to S- and R-thiohemiketal. We computed the structural, electronic, and energetic parameters of all stationary points including transition states <i>via</i> ONIOM methodology at B3LYP/6-31G(d):PM6 level. Furthermore, to get more accurate results, we also calculated the single-point dispersion-corrected energy profile by using ωB97X-D/6-31G(d,p):PM6 level. Additionally, to characterize covalent, weak noncovalent interaction (NCI) and hydrogen-bonds, we applied NCI-reduced density gradient (NCI-RDG) methods along with Bader’s Quantum Theory of Atoms-in-Molecules (QTAIM) and natural bonding orbital (NBO) analysis.</p> </div> <br>

Crystal structures of (E)-5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one and [3,4-bis(phenylethynyl)cyclobutane-1,2-diyl]bis(pyridin-2-ylmethanone)

Recrystallization of (E)-5-phenyl-1-(pyridin-2-yl)pent-2-en-4-yn-1-one at room temperature from ethylene glycol in daylight afforded [3,4-bis(phenylethynyl)cyclobutane-1,2-diyl)bis(pyridin-2-ylmethanone], C32H22N2O2 (3), while (E)-5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one, C17H13NO (2), remained photoinert. This is the first experimental evidence that pentenynones can be photoreactive when fixed in nearly coplanar parallel positions. During the photoreaction, the bond lengths and angles along the pentenyne chain changed significantly, while the disposition of the pyridyl ring towards the keto group was almost unchanged. The cyclobutane ring adopts an rctt conformation.

New β-ketophosphonates for the synthesis of prostaglandin analogues. 2 Phosphonates with bicyclo[3.3.0]octene and bicyclo[3.3.0]octane scaffolds linked to the β-keto group

β-Ketophosphonates, with the keto group linked to a bicyclo[3.3.0]oct(a)ene fragment, were synthesized starting from two diacids.