Purification Of Intermediates Research Articles

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.

Facile Access to 1,4-Disubstituted Pyrrolo[1,2-a]pyrazines from α-Aminoacetonitriles

An efficient and practical synthetic protocol for the synthesis of 1,4-disubstituted pyrrolo[1,2-a]pyrazine derivatives is described that originates from α-substituted pyrroloacetonitriles which, in turn, are readily available from aryl and alkyl aldehydes. The α-pyrroloacetonitriles were subjected to a Friedel–Crafts acylation with methyl chlorooxoacetate followed by reduction of the nitrile group under Pd-catalyzed hydrogenation conditions and finally aromatization with DDQ leading to the desired pyrrolo[1,2-a]pyrazine derivatives. This method was generalized and successfully applied to various aryl, heteroaryl, and alkyl substrates. The developed protocol provides direct and convenient access to 1,4-disubstituted ring systems in moderate to good overall yields (51–68%) without the need for purification of the intermediates. Further functionalization via the stepwise halogenation (bromination, iodination) and nitration was also demonstrated. In addition, the potential of the ester functionality for elaboration was demonstrated by manipulating into heterocyclic ring systems, exemplified by conversion into benzoxazole derivatives.

Emerging Trends in Polymerization-Induced Self-Assembly

In this Perspective, we summarize recent progress in polymerization-induced self-assembly (PISA) for the rational synthesis of block copolymer nanoparticles with various morphologies. Much of the PISA literature has been based on thermally initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. Herein, we pay particular attention to alternative PISA protocols, which allow the preparation of nanoparticles with improved control over copolymer morphology and functionality. For example, initiation based on visible light, redox chemistry, or enzymes enables the incorporation of sensitive monomers and fragile biomolecules into block copolymer nanoparticles. Furthermore, PISA syntheses and postfunctionalization of the resulting nanoparticles (e.g., cross-linking) can be conducted sequentially without intermediate purification by using various external stimuli. Finally, PISA formulations have been optimized via high-throughput polymerization and recently evaluated within flow reactors for facile scale-up syntheses.

Facile manufacture of cellulose nanoparticles in high yields by efficient cleavage of hydrogen bonds via mechanochemical synergy

The use of bamboo for the fabrication of cellulose nanoparticles (CNs) in a facile and mild process is limited because of the existence of strong hydrogen bonding between the cellulose chains. Inspired by the synergistic effects of mechanochemistry, efficient cleavage of the strong hydrogen bonds and green manufacture of CNs with a high yield of 90.8% were realized through a shearing-assisted solvent pretreatment and a subsequent mechanochemical synergy process. In the one-pot tandem reaction, chemical action, thermodynamic interaction, and mechanical force induced by mechanochemistry created synergy to accelerate the dissociation of hydrogen bonds and ultimately regulate the disintegration of cellulose into nanofibers. The CNs showed special granular morphology and cellulose II crystal form. This method of process intensification diminished laborious intermediate purification steps and improved effectively the reaction efficiency. Thus, the study provides a facile and green approach for the large-scale production of CNs, and is expected to carry significant benefits in terms of economy and sustainability.

Exploitation of the Nanoreactor Concept for Efficient Synthesis of Multiblock Copolymers via MacroRAFT-Mediated Emulsion Polymerization.

Multiblock copolymers are a class of polymeric materials with a range of potential applications. We report here a strategy for the synthesis of multiblock copolymers based on methacrylates. Reversible addition-fragmentation chain transfer (RAFT) polymerization is implemented as an emulsion polymerization to generate seed particles as nanoreactors, which can subsequently be employed in sequential RAFT emulsion polymerizations. The segregation effect allowed the synthesis of a high molar mass (>100,000 g·mol-1) decablock homopolymer at a high polymerization rate to an extent not previously achieved. A heptablock copolymer containing seven different 100 unit blocks was also successfully prepared, demonstrating how the strategy can be employed to precisely control the polymer composition at a level hitherto not accessible in environmentally friendly aqueous emulsion polymerization. Importantly, the methodology is a batch process without any intermediate purification steps, thus, rendering industrial scale up more feasible.

Evaluation of In-Batch and In-Flow Synthetic Strategies towards the Stereoselective Synthesis of a Fluorinated Analogue of Retro-Thiorphan.

A stereoselective synthetic strategy for the preparation of trifluoromethylamine mimics of retro-thiorphan, involving a diastereoselective, metal-free catalytic step, has been studied in batch and afforded the target molecule in good yields and high diastereoselectivity. A crucial point of the synthetic sequence was the catalytic reduction of a fluorinated enamine with trichlorosilane as reducing agent in the presence of a chiral Lewis base. The absolute configuration of the key intermediate was unambiguously assigned by X-ray analysis. The synthesis was also investigated exploiting continuous flow reactions; that is, an advanced intermediate of the target molecule was synthesized in only two in-flow synthetic modules, avoiding isolation and purifications of intermediates, leading to the isolation of the target chiral fluorinated amine in up to an 87:13 diastereoisomeric ratio.

Synthesis of the Sex Pheromone of the Oleander Scale ( Aspidiotus nerii).

A total synthesis of the oleander scale [ Aspidiotus nerii (Bouche)] sex pheromone, the unique sesquiterpenoid containing a cyclobutane moiety of this class of compounds, has been developed. In order to implement this sex pheromone as a new environmentally friendly tool to manage this pest, a more cost-effective, multigram synthesis was required. This new synthetic route, having a Blaise reaction, iron-catalyzed carbon-carbon coupling, and [2 + 2] photocycloaddition reactions as key steps, provides a general access to 4-alkyl lactones as well as a robust access to the target sex pheromone. Starting from readily available compounds as 3-hydroxypropanenitrile, ethyl bromoacetate, and 2-acetyl butyrolactone, the synthetic sequence afforded the A. nerii sex pheromone with minimum intermediate purification and good overall yield in nine linear steps.

A Novel and Efficient Continuous-Flow Route To Prepare Trifluoromethylated N-Fused Heterocycles for Drug Discovery and Pharmaceutical Manufacturing

Continuous-flow processing has become one of the fastest-growing research areas in chemistry in the last 10 years. Herein we disclose an automated and scalable continuous-flow route for the quick introduction of trifluoromethyl groups on a variety of heterocycles, with application in drug discovery and manufacturing. This involves the direct alkylation-cyclization of amines in the presence of trifluoroacetic acid or anhydride, cheap and readily available CF3-containing building blocks. Compared to traditional batch reactions involving an intermediate purification step, the continuous-flow reactions occurred quickly and at mild conditions, with high yield and broad functional-group tolerance. The practical utility of the method was demonstrated by a gram-scale synthesis and by the estimation of modern green metrics.

Scalable Aqueous Reversible Addition–Fragmentation Chain Transfer Photopolymerization-Induced Self-Assembly of Acrylamides for Direct Synthesis of Polymer Nanoparticles for Potential Drug Delivery Applications

The synthesis of polymer nanoparticles for potential drug delivery applications via photoinduced electron transfer–reversible addition–fragmentation chain transfer aqueous dispersion polymerizations employing Eosin Y/triethanol amine as catalytic system is described. A scalable synthesis procedure for the polymerization of poly(dimethyl acrylamide)-b-(poly(diaceteone-stat-dimethyl acrylamide)) is reported under visible light (λmax = 530 nm). A variety of solid contents and block lengths for the solvophilic and solvophobic blocks are investigated, expanding the compositional space of worms, jellyfish, and vesicles using flow chemistry. A simple two-step process using a coupled flow reactor setup is used without the need for intermediate purification.

Efficient Syntheses of New Super Lewis Basic Tris(dialkylamino)-Substituted Terpyridines and Comparison of Their Methyl Cation Affinities.

Syntheses of very electron-rich dialkylamino-substituted 2,2':6',2''-terpyridines (TPYs) were adapted to moderate scale preparation without tedious purification of intermediates. The key 4'-bromo-6,6''-dimethyl-2,2':6',2''-terpyridine-4,4''-diyl bisnonaflate is now available in gram quantities. Its nucleophilic aromatic substitution with dimethylamine provided mixtures of 4'-bromo-substituted 4,4''-bis(dimethylamino)-TPY and the tris(dimethylamino)-TPY. The bromo compound was used in a Buchwald-Hartwig amination to provide the tris(dimethylamino)-TPY in excellent yield. The 4'-bromo substituent was reductively removed to furnish the bis(dimethylamino)-TPY. The same sequence of reactions with pyrrolidine as nucleophile leads to the hitherto unknown pyrrolidino-TPYs. Calculations at the MP2(FC)/6-31+G(2d,p)//B98/6-31G(d) level predict very high methyl cation affinities for compounds of this type, with the 4,4',4''-tri(pyrrolidin-1-yl)-TPY being the most Lewis basic TPY synthesized to date. The efficiently prepared electron-rich TPYs should be excellent ligands for many applications.

Seven‐Step Continuous Flow Synthesis of Linezolid Without Intermediate Purification

AbstractHerein, the blockbuster antibacterial drug linezolid is synthesized from simple starting blocks by a convergent continuous flow sequence involving seven (7) chemical transformations. This is the highest total number of distinct reaction steps ever performed in continuous flow without conducting solvent exchanges or intermediate purification. Linezolid was obtained in 73 % isolated yield in a total residence time of 27 minutes, corresponding to a throughput of 816 mg h−1.

Seven-Step Continuous Flow Synthesis of Linezolid Without Intermediate Purification.

Herein, the blockbuster antibacterial drug linezolid is synthesized from simple starting blocks by a convergent continuous flow sequence involving seven (7) chemical transformations. This is the highest total number of distinct reaction steps ever performed in continuous flow without conducting solvent exchanges or intermediate purification. Linezolid was obtained in 73 % isolated yield in a total residence time of 27 minutes, corresponding to a throughput of 816 mg h-1 .

Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5 cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

Green Access to α-Haloalkyl and α-Halobenzyl Esters, Versatile Intermediates for the One-Pot Two-Step Synthesis of O,O′-Diacyl Acetals Using Zinc-Based Ionic Liquid Catalyst

α-Haloalkyl and α-halobenzyl esters have been synthesized using a zinc-based ionic liquid catalyst, BMIZnCl3. These esters can then react with acids without intermediate purification to obtain mixed O,O′-diacyl acetals in a one-pot process.

Ultra-Fast Synthesis of Multivalent Radical Nanoparticles by Ring-Opening Metathesis Polymerization-Induced Self-Assembly.

We report the straightforward, time-efficient synthesis of radical core-shell nanoparticles (NPs) by polymerization-induced self-assembly. A nitroxide-containing hydrophilic macromolecular precursor was prepared by ring-opening metathesis copolymerization of norbornenyl derivatives of TEMPO and oligoethylene glycol and was chain-extended in situ with norbornene in ethanolic solution, leading to simultaneous amphiphilic block copolymer formation and self-assembly. Without any intermediate purification from the monomers to the block copolymers, radical NPs with tunable diameters ranging from 10 to 110 nm are obtained within minutes at room temperature. The high activity of the radical NPs as chemoselective and homogeneous, yet readily recyclable catalysts is demonstrated through oxidation of a variety of alcohols and recovery by simple centrifugation. Furthermore, the NPs show biocompatibility and antioxidant activity in vitro.

Chromatographic purification of recombinant human erythropoietin.

Recombinant human erythropoietin is a valuable therapeutic protein used in the treatment of several serious diseases. It exists in different isoforms and is produced by genetically modified mammalian cells such as Chinese hamster ovary or human embryonic kidney cells. As for other biopharmaceutical drugs, a key factor for its successful industrial production is to achieve a high degree of purity and to decrease the content of critical impurities to trace amounts. This goal is achieved in the separation sequence which substantial part is formed by chromatographic steps. Therefore, downstream processing forms an essential part of production costs. This review presents the overview of published separation sequences and, analyzes the use of different types of chromatographic media such as affinity, ion-exchange, reversed-phase, hydrophobic interaction, multimodal, and size-exclusion chromatography adsorbents. Their application is discussed with regard to their place in the purification stages generally denoted as capture, intermediate purification and polishing.

Improved one-pot synthetic conditions for synthesis of functionalized fluorescent coumarin-thiophene hybrids: Syntheses, DFT studies, photophysical and thermal properties

We have synthesized coumarin-thiophene hybrids using stepwise and one-pot methods under MWI and CM conditions, and then report the findings and conclusions: one-pot is better than stepwise in terms of high yields and fastness of getting products, and MWI is better than CM in terms of purity of compounds, better yields, and reaction period. Even though both protocols were found to be efficient, the microwave-assisted irradiation reactions were identified to be better, environmentally friendly, cost-effective, mild, more efficient, less time consuming but with high purity. The two protocols were employed for the synthesis of the target coumarin-thiophene hybrids via the Gewald reaction in stepwise and in one-pot three-component. The dyes 3a-j were prepared in good to excellent yields. Although, we recorded better yields via the stepwise protocol (80–96%), for the synthesis of the target molecules, as compared to the one-pot (82–90%), the stepwise condition is fraught with many changes such as long reaction conditions, extra use of solvents for purification of intermediates, and long reaction durations. Photophysical activities of all the target derivatives were investigated using a combination of UV–vis and fluorescence spectroscopies in different solvents. The umbelliferon-like derivative 3g was further investigated for possible use as a pH sensor via deprotonation (using TBAOH) and reverse protonation (using TFA), and was found to be applicable as pH sensor both in organic medium (DMSO) and partial aqueous binary (DMSO/H2O, v/v, 9:1) medium. Again, the ground and the excited state properties of the umbelliferon-like derivative 3g and its deprotonated form were also studied using the quantum chemical calculations. Finally, the thermal properties of all the target compounds were investigated using TGA in order to test their potency and applicability as optical dyes. All the dyes 3a-j have high thermal stability therefore they can be applicable as optical dyes.

A Consolidated and Continuous Synthesis of Ciprofloxacin from a Vinylogous Cyclopropyl Amide.

Ciprofloxacin is a broad-spectrum antibiotic that is recognized as one of the World Health Organization's Essential Medicines. It is particularly effective in the treatment of Gram-negative bacterial infections associated with urinary, respiratory, and gastrointestinal tract infections. A streamlined and high yielding continuous synthesis of ciprofloxacin has been developed, which employs a chemoselective C-acylation step that precludes the need for intermediate isolations, extractions, or purifications. The end-to-end process has a residence time of 4.7 min with a 15.8 g/h throughput at laboratory scale and an overall isolated yield of 83%.

A Scalable, One-Pot Synthesis of 1,2,3,4,5-Pentacarbomethoxycyclopentadiene.

1,2,3,4,5-Pentacarbomethoxycyclopentadiene (PCCP) is a strong organic acid and a precursor to useful organocatalysts, including chiral Brønsted acids and silicon-based Lewis acids. The synthetic route to PCCP, first reported in 1942, is inconvenient for a number of reasons. The two-step synthesis requires the purification of intermediates from intractable side-products, high reaction temperatures, and extensive labor (3 days). We have developed an improved procedure that delivers PCCP efficiently in 24 hours in one pot at ambient temperature and without isolation.