Enantioselective Total Synthesis of the Marine Macrolides Salarins A and C.

Here we report the first total synthesis of the marine macrolide salarin C, a potent anticancer agent, and demonstrate the biomimetic oxidation-Wasserman rearrangement to access salarin A. This synthesis relies on L-proline catalysis to install a chlorohydrin function that masks the sensitive C16-C17 epoxide and potentially mimics the biosynthesis of these compounds where a related chlorohydrin may yield both THF- and epoxide-containing salarins. Additional and key features of the synthesis include (i) macrocycle formation via ring-closing metathesis, (ii) macrocyclic substrate-controlled epoxidation of the C12-C13 allylic alcohol, and (iii) a late-stage Julia-Kocienski olefination to install the side chain. Importantly, this work provides a platform for the synthesis of other salarins and analogues of these potentially important anticancer natural products.

Synthesis of Enantiopure (S)-Atenolol by Utilization of Lipase-Catalyzed Kinetic Resolution of a Key Intermediate.

(S)-Atenolol ((S)-2-(4-(2-Hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide) has been synthesized in >99% enantiomeric excess (ee) with the use of Candida antarctica lipase B from Syncozymes (Shanghai, China), in a kinetic resolution of the corresponding racemic chlorohydrin. A catalytic amount of base was used in deprotonation of the phenol building block. The enantiopurity of the chlorohydrin building block remained unchanged upon subsequent amination to yield the final drug. All four steps in the synthesis protocol have been optimized compared to previously reported methods, which makes this new protocol more sustainable and in accordance with green chemistry principles. The overall yield of (S)-atenolol was 9.9%, which will be further optimized.

Phospholipid chlorohydrins as chlorine exposure biomarkers in a large animal model.

Chlorine is a toxic industrial chemical that has been used as a chemical weapon in recent armed conflicts. Confirming human exposure to chlorine has proven challenging, and there is currently no established method for analyzing human biomedical samples to unambiguously verify chlorine exposure. In this study, two chlorine-specific biomarkers: palmitoyl-oleoyl phosphatidylglycerol chlorohydrin (POPG-HOCl) and the lipid derivative oleoyl ethanolamide chlorohydrin (OEA-HOCl) are shown in bronchoalveolar lavage fluid (BALF) samples from spontaneously breathing pigs after chlorine exposure. These biomarkers are formed by the chemical reaction of chlorine with unsaturated phospholipids found in the pulmonary surfactant, which is present at the gas-liquid interface within the lung alveoli. Our results strongly suggest that lipid chlorohydrins are promising candidate biomarkers in the development of a verification method for chlorine exposure. The establishment of verified methods capable of confirming the illicit use of toxic industrial chemicals is crucial for upholding the principles of the Chemical Weapons Convention (CWC) and enforcing the ban on chemical weapons. This study represents the first published dataset in BALF revealing chlorine biomarkers detected in a large animal. Furthermore, these biomarkers are distinct in that they originate from molecular chlorine rather than hypochlorous acid.

Combination of gold and redox enzyme catalysis to access valuable enantioenriched aliphatic ß-chlorohydrins.

The synthesis of enantioenriched ß-chlorohydrins is highly appealing due to their relevance as building-blocks in organic synthesis. However, the approximation to aliphatic derivatives is particularly challenging due to the difficulties to get access to the α-chloroketone precursors. Herein, we propose a straightforward and scalable approach combining in a concurrent manner gold(I) and redox enzyme catalysis through a hydration-bioreduction cascade. A total of nine aliphatic ß-chlorohydrins bearing different functional groups were obtained with very high yields (63-88%) and stereoselectivities (>99% ee).

Synthesis of Optically Active syn- and anti-Chlorohydrins through a Bienzymatic Reductive Cascade.

A bienzymatic cascade has been designed and optimized to obtain enantiopure chlorohydrins starting from the corresponding 1-aryl-2-chlorobut-2-en-1-ones. For the synthesis of these α-chloroenones, a two-step sequence was developed consisting of the allylation of the corresponding aldehyde with 3-dichloroprop-1-ene, followed by oxidation and further isomerization. The selective cooperative catalytic system involving ene-reductases (EREDs) and alcohol dehydrogenases (ADHs) afforded the desired optically active chlorohydrins under mild reaction conditions in excellent conversions (up to >99%) and selectivities (up to >99:1 diastereomeric ratio (dr), >99% enantiomeric excess (ee)).

Biocatalytic hydrogen-transfer to access enantiomerically pure proxyphylline, xanthinol, and diprophylline.

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) have been widely used for the reversible redox reactions of carbonyl compounds (i.e., aldehydes and ketones) and primary or secondary alcohols, often resulting in optically pure hydroxyl products with high added value. In this work, we report a concise chemoenzymatic route toward xanthine-based enantiomerically pure active pharmaceutical ingredients (API) - proxyphylline, xanthinol, and diprophylline employing various recombinant short-chain ADHs with (R)- or (S)-selectivity as key biocatalysts. By choosing the appropriate ADH, the (R)- as well as the (S)-enantiomer of proxyphylline was prepared in excellent enantiomeric excess (99-99.9% ee), >99% conversion, and the isolated yield ranging from 65% to 74%, depending on the used biocatalyst (ADH-A from Rhodococcus ruber or a variant derived from Lactobacillus kefir, Lk-ADH-Lica). In turn, E. coli/ADH-catalyzed bioreduction of the carbonylic precursor of xanthinol and diprophylline furnished the corresponding (S)-chlorohydrin in >99% ee, >99% conversion, and 80% yield (in the case of Lk-ADH-Lica); while the (R)-counterpart was afforded in 94% ee, 64% conversion, and 41% yield (in the case of SyADH from Sphingobium yanoikuyae). After further chemical functionalization of the key (S)-chlorohydrin intermediate, the desired homochiral (R)-xanthinol (>99% ee) was obtained in 97% yield and (S)-diprophylline (>99% ee) in 90% yield. The devised biocatalytic method is straightforward and thus might be considered practical in the manufacturing of title pharmaceuticals.

Structure Revision of Trichomide D by Total Synthesis.

A structure revision of trichomide D has been achieved by its total synthesis. The sterically hindered peptide sequence was successfully prepared using not only a conventional amidation with EDCI but also coupling with an Fmoc-protected amino acid chloride derivative. The cyclization precursor was synthesized by coupling of a tetrapeptide with an acylproline derivative and subsequent removal of silyl groups at the N- and C-termini. Macrolactonization using MNBA/DMAPO followed by preparation of a chlorohydrin moiety furnished the proposed structure of trichomide D, whose spectra were not identical to those of the natural product. Finally, we succeeded in the elucidation of the true structure of trichomide D by its total synthesis, and the absolute configuration of the chlorohydrin moiety was revised to be S. The cytotoxicities of the natural product and its synthetic derivatives against MCF-7 and HeLa S3 cells were evaluated by the MTT method, revealing that the configuration of the chlorohydrin moiety is a pivotal factor for exhibiting potent cytotoxicity against cancer cells.

Formation of Oleic Acid Chlorohydrins in Vegetables during Postharvest Chlorine Disinfection.

High chlorine doses (50-200 mg/L) are used in postharvest washing facilities to control foodborne pathogen outbreaks. However, chlorine can react with biopolymers (e.g., lipids) within the produce to form chlorinated byproducts that remain in the food. During chlorination of micelles of oleic acid, an 18-carbon alkene fatty acid, chlorine added rapidly across the double bond to form the two 9,10-chlorohydrin isomers at a 100% yield. The molar conversion of lipid-bound oleic acid to 9,10-chlorohydrins in chlorine-treated glyceryl trioleate and produce was much lower, reflecting the restricted access of chlorine to lipids. Yields from spinach treated with 100 mg/L chlorine at 7.5 °C for 2 min increased from 0.05% (0.9 nmol/g-spinach) for whole leaf spinach to 0.11% (2 nmol/g) when shredding increased chlorine access. Increasing temperature (21 °C) and chlorine contact time (15 min) increased yields from shredded spinach to 0.83% (22 nmol/g) at 100 mg/L chlorine and to 1.8% (53 nmol/g) for 200 mg/L chlorine. Oleic acid 9,10-chlorohydrin concentrations were 2.4-2.7 nmol/g for chlorine-treated (100 mg/L chlorine at 7.5 °C for 2 min) broccoli, carrots, and butterhead lettuce, but 0.5-1 nmol/g for cabbage, kale, and red leaf lettuce. Protein-bound chlorotyrosine formation was higher in the same vegetables (5-32 nmol/g). The Chinese hamster ovary cell chronic cytotoxicity LC50 value for oleic acid 9,10-chlorohydrins was 0.106 mM. The cytotoxicity associated with the chlorohydrins and chlorotyrosines in low masses (9-52 g) of chlorine-washed vegetables would be comparable to that associated with trihalomethanes and haloacetic acids at levels of regulatory concern in drinking water.

A phase 1 study to assess the safety, pharmacokinetics, and anti-tumor activity of the androgen receptor n-terminal domain inhibitor epi-506 in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: EPI-506 is the first of a new class of drugs targeting the N-terminal domain (NTD) of the androgen receptor (AR), potentially overcoming known resistance mechanisms to androgen receptor pathway inhibitors (ARPIs) among men with metastatic castration resistant prostate cancer (mCRPC). METHODS: Patients with mCRPC who had progressed on prior ARPI were enrolled in this phase 1 open-label, adaptive 3 + 3 dose escalation study. The primary outcome was safety and tolerability of oral EPI-506. Secondary objectives included determination of the maximal tolerated dose (MTD), pharmacokinetic profile, and antitumor efficacy. RESULTS: 28 mCRPC patients were enrolled into 7 dose cohorts of EPI-506 ranging from 80-3600 mg given once daily and 1800 mg given twice daily. Six DLTs occurred in 4 patients; Grade 4 elevated amylase; Grade 3 abdominal pain; Grade 3 elevated ALT and Grade 3 elevated AST; Grade 2 nausea and Grade 1 vomiting which resulted in study drug intake of < 75% of the expected dose during the DLT assessment period. The most common drug-related adverse events included diarrhea, nausea and fatigue. Six patients had a PSA decline not meeting PSA response criteria. The study was terminated prior to reaching the MTD due to poor oral bioavailability. CONCLUSIONS: This phase 1 trial established the safety of EPI-506 and provides proof of concept for targeting the AR NTD. Next generation compounds with improved bioavailability and potency are in clinical development.

Synthesis of Difluorinated Halohydrins by the Chemoselective Addition of Difluoroenolates to α-Haloketones.

α-Haloketones are valuable intermediates in the synthesis of pharmaceuticals and natural products because they display two electrophiles. Although chemoselective additions to each of these functional groups are known, the use of fluorinated nucleophiles has not been characterized, except for the dimerization of fluorohalomethyl ketones. Our studies demonstrate the use of difluoroenolates to create difluorinated bromohydrins and chlorohydrins from α-haloketones without any cyclization or rearrangement due to the mild conditions.

Simulation of Ligand Transport in Receptors Using CaverDock.

Interactions between enzymes and small molecules lie in the center of many fundamental biochemical processes. Their analysis using molecular dynamics simulations have high computational demands, geometric approaches fail to consider chemical forces, and molecular docking offers only static information. Recently, we proposed to combine molecular docking and geometric approaches in an application called CaverDock. CaverDock is discretizing enzyme tunnel into discs, iteratively docking with restraints into one disc after another and searching for a trajectory of the ligand passing through the tunnel. Here, we focus on the practical side of its usage describing the whole method: from getting the application, and processing the data through a workflow, to interpreting the results. Moreover, we shared the best practices, recommended how to solve the most common issues, and demonstrated its application on three use cases.

Preparation and Uses of Chlorinated Glycerol Derivatives.

Crude glycerol (C3H8O3) is a major by-product of biodiesel production from vegetable oils and animal fats. The increased biodiesel production in the last two decades has forced glycerol production up and prices down. However, crude glycerol from biodiesel production is not of adequate purity for industrial uses, including food, cosmetics and pharmaceuticals. The purification process of crude glycerol to reach the quality standards required by industry is expensive and dificult. Novel uses for crude glycerol can reduce the price of biodiesel and make it an economical alternative to diesel. Moreover, novel uses may improve environmental impact, since crude glycerol disposal is expensive and dificult. Glycerol is a versatile molecule with many potential applications in fermentation processes and synthetic chemistry. It serves as a glucose substitute in microbial growth media and as a precursor in the synthesis of a number of commercial intermediates or fine chemicals. Chlorinated derivatives of glycerol are an important class of such chemicals. The main focus of this review is the conversion of glycerol to chlorinated derivatives, such as epichlorohydrin and chlorohydrins, and their further use in the synthesis of additional downstream products. Downstream products include non-cyclic compounds with allyl, nitrile, azide and other functional groups, as well as oxazolidinones and triazoles, which are cyclic compounds derived from ephichlorohydrin and chlorohydrins. The polymers and ionic liquids, which use glycerol as an initial building block, are highlighted, as well.

Role of androgen receptor splice variants, their clinical relevance and treatment options.

PURPOSE: In this review, we summarize the importance of AR variants with a particular focus on clinically relevant members of this family. METHODS: A non-systematic literature review was performed based on Medline and PubMed. RESULTS: Endocrine therapy represents the central paradigm for the management of prostate cancer. Eventually, in response to androgen ablation therapy, several resistance mechanisms against the endocrine therapy might develop that can circumvent the therapy approaches. One specific resistance mechanism that has gained increasing attention is the generation of alternatively spliced variants of the androgen receptor, with AR-V7 being the most prominent. More broadly, AR-V7 is one member of a group of alternatively spliced AR variants that share a common feature, the missing ligand-binding domain. These ΔLBD androgen receptor variants have shown the capability to induce androgen receptor-mediated gene transcription even under conditions of androgen deprivation and to drive cancer progression. CONCLUSION: The methods used for detecting AR-Vs, at least on the mRNA level, are well-advanced and harbor the potential to be introduced into clinical diagnostics. It is important to note, that the testing, especially of AR-V7 has its limitations in predicting treatment response. More promising is the great number of active clinical trials aimed at reducing the AR-Vs, and using this to re-sensitize CRPC towards endocrine treatment might provide additional treatment options for CRPC patients in the future.

Single-molecule insights into the temperature and pressure dependent conformational dynamics of nucleic acids in the presence of crowders and osmolytes.

In this review we discuss results from temperature and pressure dependent single-molecule Förster resonance energy transfer (smFRET) studies on nucleic acids in the presence of macromolecular crowders and organic osmolytes. As representative examples, we have chosen fragments of both DNAs and RNAs, i.e., a synthetic DNA hairpin, a human telomeric G-quadruplex and the microROSE RNA hairpin. To mimic the effects of intracellular components, our studies include the macromolecular crowding agent Ficoll, a copolymer of sucrose and epichlorohydrin, and the organic osmolytes trimethylamine N-oxide, urea and glycine as well as natural occurring osmolyte mixtures from deep sea organisms. Furthermore, the impact of mutations in an RNA sequence on the conformational dynamics is examined. Different from proteins, the effects of the osmolytes and crowding agents seem to strongly dependent on the structure and chemical make-up of the nucleic acid.

Heterogeneous Chlorination of Squalene and Oleic Acid.

Washing with chlorine bleach leads to high mixing ratios of gas-phase HOCl. Using two methods that are sensitive to surface film composition-attenuated total reflection fourier transform infrared (ATR-FTIR) spectroscopy and direct analysis in real time mass spectrometry (DART-MS)-we present the first study of the chlorination chemistry that occurs when gaseous HOCl reacts with thin films of squalene and oleic acid. At mixing ratios of 600 ppbv, HOCl forms chlorohydrins by adding across carbon-carbon double bonds without breaking the carbon backbone. The initial uptake of one HOCl molecule occurs on the time scale of a few minutes at these mixing ratios. For oleic acid, ester formation proceeds immediately thereafter, leading to dimeric and trimeric chlorinated products. For squalene, subsequent HOCl uptake occurs until all six of its carbon-carbon double bonds become chlorinated within 1-2 h. These results indicate that chlorination of skin oil, which contains substantial carbon unsaturation, is likely to occur rapidly under common cleaning conditions, potentially leading to the irritation associated with chlorinated bleach. This chemistry will likely also proceed with cooking oils, in the human respiratory system which has unsaturated surfactants as important components of lung fluid, and with organic components of the sea surface microlayer.

Combined N-terminal androgen receptor and autophagy inhibition increases the antitumor effect in enzalutamide sensitive and enzalutamide resistant prostate cancer cells.

INTRODUCTION AND OBJECTIVES: Multiple androgen receptor (AR)-dependent and -independent resistance mechanisms limit the efficacy of current castration-resistant prostate cancer (CRPC) treatment. Novel N-terminal domain (NTD) binding AR-targeting compounds, including EPI-001 (EPI), have the promising ability to block constitutively active splice variants, which represent a major resistance mechanism in CRPC. Autophagy is a conserved lysosomal degradation pathway that acts as survival mechanism in cells exposed to anticancer treatments. We hypothesized, that promising NTD-AR treatment may upregulate autophagy and that a combination of NTD-AR and autophagy inhibition might therefore increase antitumor effects. METHODS: AR-expressing prostate cancer cell lines (LNCaP, LNCaP-EnzR) were treated with different concentrations of EPI (10, 25, 50 µM) and in combination with the autophagy inhibitors chloroquine (CHQ, 20 µM) or 3-methyladenine (3-MA, 5 mM). Cell proliferation was assessed by WST-1-assays after 1 and 7 days. Ethidium bromide and Annexin V were used to measure viability and apoptosis on day 7 after treatment. Autophagosome formation was detected by AUTOdot staining. In addition, autophagic activity was monitored by immunocytochemistry and Western blot (WES) for the expression of ATG5, Beclin1, LC3-I/II and p62. RESULTS: Treatment with EPI resulted in a dose-dependent reduction of cell growth and increased apoptosis in both cancer cell lines on day 7. In addition, EPI treatment demonstrated an upregulated autophagosome formation in LNCaP and LNCaP-EnzR cells. Assessment of autophagic activity by immunocytochemistry and WES revealed an increase of ATG5 and LC3-II expression and a decreased p62 expression in all EPI-treated cells. A combined treatment of EPI with autophagy inhibitors led to a further significant reduction of cell viability in both cell lines. CONCLUSIONS: Our results demonstrate that NTD targeting AR inhibition using EPI leads to an increased autophagic activity in LNCaP and LNCaP-EnzR prostate cancer cells. A combination of NTD AR blockage with simultaneous autophagy inhibition increases the antitumor effect of EPI in prostate cancer cells. Double treatment may offer a promising strategy to overcome resistance mechanisms in advanced prostate cancer.

Differential proteome expression analysis of androgen-dependent and -independent pathways in LNCaP prostate cancer cells.

Prostate cancer (PC) is one of the leading causes of cancer death in men. It commonly develops in older males, but the number of younger men diagnosed with the disease has increased in recent years. Hormone therapies, such as chemical and surgical methods that inhibit androgen synthesis or androgen receptor (AR) activation, have been used for advanced disease. However, castration-resistant PC (CRPC), which exhibits androgen-independent mechanisms for activating AR, develops after a few years of such treatment and no therapy is available. In this study, we examined differences in the proteomes associated with the androgen-dependent (DHT treatment) and -independent (FSK, forskolin treatment) AR signaling conditions in LNCaP prostate cancer cells. Moreover, we used EPI-001, which inhibits AR-mediated transcriptional activity, to examine whether the observed differences in protein expression were directly affected by AR-mediated mechanisms. A total of 213 protein spots were matched in our 2-dimensional gel electrophoresis (2DE) analysis and 8 proteins with significant expression changes in our 5 different treatment groups were identified by mass spectrometry. Among these proteins, expression levels of PEPCK-M, catalase, tubulin alpha chain, alpha-enolase, and endoplasmic reticulum resident protein 29 were significantly altered by DHT and the levels of HSP 90 and EF-Tu were changed by FSK. These changes were blocked by EPI-001 in DHT-regulated proteins, PEPCK-M, catalase, and tubulin alpha chain and FSK-regulated EF-Tu protein. The results from our immunohistochemical analysis using in vivo xenograft models were consistent with the 2DE data. We therefore report the first identification of differences in proteins that are significantly regulated under androgen-dependent and -independent AR signaling conditions. Our findings could suggest a possible molecular mechanism through which AR is activated in the absence and/or presence of androgen, and might help explain the inhibitory action of EPI-001 on CRPC.

Efficient conversion of alkenes to chlorohydrins by a Ru-based artificial enzyme.

Artificial enzymes are required to catalyse non-natural reactions. Here, a hybrid catalyst was developed by embedding a novel Ru complex in the transport protein NikA. The protein scaffold activates the bound Ru complex to produce a catalyst with high regio- and stereo-selectivity. The hybrid efficiently and stably produced α-hydroxy-ß-chloro chlorohydrins from alkenes (up to 180 TON with a TOF of 1050 h-1).

Elimination of N,O-bis(trimethylsilyl)trifluoroacetamide interference by base treatment in derivatization gas chromatography mass spectrometry determination of parts per billion of alcohols in a food additive.

A novel base treatment followed by liquid-liquid extraction was developed to remove the interference of excess derivatization reagent BSTFA [N,O-Bis(trimethylsilyl)trifluoroacetamide] and its byproducts for trace determination of 1-chloro-2-propanol and 2-chloro-1-propanol in a food additive. The corresponding trimethylsilyl derivatives were analyzed by gas chromatography mass spectrometry (GC/MS) detection in selective ion monitoring mode. Due to a large volume splitless injection needed for achieving the required sensitivity, excess BSTFA in the derivatization sample solution interfered with the trimethylsilyl derivatives of the analytes of interest, making their quantitation not attainable. Efforts were made to decompose BSTFA while keeping the trimethylsilyl derivatives intact. Water or aqueous sulfuric acid treatment converted BSTFA into mainly N-trimethylsilyltrifluoroacetamide, which partitions between aqueous and organic layers. In contrast, aqueous sodium hydroxide decomposed BSTFA into trifluoroacetic acid, which went entirely into the aqueous layer. No BSTFA or its byproduct N-trimethylsilyltrifluoroacetamide or trifluroacetamide was found in the organic layer where the derivatized alcohols existed, which in turn completely eliminated their interference, enabling accurate and precise determination of parts per billion of the short-chain alcohols in the food additive. Contrary to the conventional wisdom that a trimethylsilyl derivative is susceptible to hydrolysis, the derivatized short-chain alcohols were found stable even in the presence of 0.17N aqueous sodium hydroxide as the improved GC/MS method was validated successfully, with a satisfactory linearity response in the concentration range of 10-400ng/g (regression coefficient greater than 0.999), good method precision (<4%), good recovery (90-98%), and excellent limit of detection (3ng/g) and limit of quantitation (10ng/g).

Synthesis of Natural O-Linked Carba-Disaccharides, (+)- and (-)-Pericosine E, and Their Analogues as α-Glucosidase Inhibitors.

Pericosine E (6), a metabolite of Periconia byssoides OUPS-N133 was originally isolated from the sea hare Aplysia kurodai, which exists as an enantiomeric mixture in nature. The enantiospecific syntheses of both enantiomers of Periconia byssoides OUPS-N133 has been achieved, along with six stereoisomers, using a common simple synthetic strategy. For these efficient syntheses, highly regio- and steroselective processes for the preparation of bromohydrin and anti-epoxide intermediates were applied. In order to access the unique O-linked carbadisaccharide structure, coupling of chlorohydrin as a donor and anti-epoxide as an acceptor was achieved using catalytic BF3·Et2O. Most of the synthesized compounds exhibited selectively significant inhibitory activity against α-glycosidase derived from yeast. The strongest analog showed almost 50 times the activity of the positive control, deoxynojirimycin.