Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous‐flow, vortex fluidic device (VFD). Fused His_n‐tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi‐aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this “reaction‐ready” system demonstrated excellent stability during five days of continuous‐flow processing. Towards multi‐step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous‐flow biosynthesis.     

Precise Polymer Synthesis by Autonomous Self‐Optimizing Flow Reactors

A novel continuous flow system for automated high‐throughput screening, autonomous optimization, and enhanced process control of polymerizations was developed. The computer‐controlled platform comprises a flow reactor coupled to size exclusion chromatography (SEC). Molecular weight distributions are measured online and used by a machine‐learning algorithm to self‐optimize reactions towards a programmed molecular weight by dynamically varying reaction parameters (i.e. residence time, monomer concentration, and control agent/initiator concentration). The autonomous platform allows targeting of molecular weights in a reproducible manner with unprecedented accuracy (<2.5 % deviation from pre‐selected goal) for both thermal and light‐induced reactions. For the first time, polymers with predefined molecular weights can be custom made under optimal reaction conditions in an automated, high‐throughput flow synthesis approach with outstanding reproducibility.     

Using Anilines as Masked Cross‐Coupling Partners: Design of a Telescoped Three‐Step Flow Diazotization,Iododediazotization, Cross‐Coupling Process

The conversion of commercially available anilines into biaryl and biarylacetylene products was realized by using a telescoped, three‐reactor flow diazotization/iododediazotization/cross‐coupling process. The segmented flow stream created by off‐gassing during the Sandmeyer sequence was restored to a continuous column of reaction solution by using a specially designed continuous‐flow unit controlled by custom software created in‐house. The resultant aryl iodide was then combined with a stream of cross‐coupling solution that fed into the final reactor. The system proved versatile as modifications to the diazotization/iododediazotization sequence could be made rapidly to account for any substrate specificity (e.g., solubility problems), leading to a wide substrate scope of Suzuki–Miyaura and Sonogashira cross‐coupled products.     

Continuous Consecutive Reactions with Inter‐Reaction Solvent Exchange by Membrane Separation

Pharmaceutical production typically involves multiple reaction steps with separations between successive reactions. Two processes which complicate the transition from batch to continuous operation in multistep synthesis are solvent exchange (especially high‐boiling‐ to low‐boiling‐point solvent), and catalyst separation. Demonstrated here is membrane separation as an enabling platform for undertaking these processes during continuous operation. Two consecutive reactions are performed in different solvents, with catalyst separation and inter‐reaction solvent exchange achieved by continuous flow membrane units. A Heck coupling reaction is performed in N,N‐dimethylformamide (DMF) in a continuous membrane reactor which retains the catalyst. The Heck reaction product undergoes solvent exchange in a counter‐current membrane system where DMF is continuously replaced by ethanol. After exchange the product dissolved in ethanol passes through a column packed with an iron catalyst, and undergoes reduction (>99 % yield).     

Multistep Flow Synthesis of 5‐Amino‐2‐aryl‐2H‐[1,2,3]‐triazole‐4‐carbonitriles

1,2,3‐Triazole has become one of the most important heterocycles in contemporary medicinal chemistry. The development of the copper‐catalyzed Huisgen cycloaddition has allowed the efficient synthesis of 1‐substituted 1,2,3‐triazoles. However, only a few methods are available for the selective preparation of 2‐substituted 1,2,3‐triazole isomers. In this context, we decided to develop an efficient flow synthesis for the preparation of various 2‐aryl‐1,2,3‐triazoles. Our strategy involves a three‐step synthesis under continuous‐flow conditions that starts from the diazotization of anilines and subsequent reaction with malononitrile, followed by nucleophilic addition of amines, and finally employs a catalytic copper(II) cyclization. Potential safety hazards associated with the formation of reactive diazonium species have been addressed by inline quenching. The use of flow equipment allows reliable scale up processes with precise control of the reaction conditions. Synthesis of 2‐substituted 1,2,3‐triazoles has been achieved in good yields with excellent selectivities, thus providing a wide range of 1,2,3‐triazoles.     

Continuous‐Flow Synthesis of CdSe Quantum Dots: A Size‐Tunable and Scalable Approach

In recent years, continuous‐flow/microreactor processing for the preparation of colloidal nanocrystals has received considerable attention. The intrinsic advantages of microfluidic reactors have opened new opportunities for the size‐controlled synthesis of nanocrystals either in the laboratory or on a large scale. Herein, an experimentally simple protocol for the size‐tunable continuous‐flow synthesis of rather monodisperse CdSe quantum dots (QDs) is presented. CdSe QDs are manufactured by using cadmium oleate as cadmium source, selenium dioxide as selenium precursor, and 1‐octadecene as solvent. Exploiting selenium dioxide as selenium source and 1‐octadecene as solvent allows execution of the complete process in open air without any requirement for air‐free manipulations using a glove box or Schlenk line. Continuous‐flow processing is performed with a stainless steel coil of 1.0 mm inner diameter pumping the combined precursor solution through the reactor by applying a standard HPLC pump. The effect of different reaction parameters, such as temperature, residence time, and flow rate, on the properties of the resulting CdSe QDs was investigated. A temperature increase from 240 to 260 °C or an extension of the residence time from 2 to 20 min affords larger nanocrystals (range 3–6 nm) whereas the size distribution does not change significantly. Longer reaction times and higher temperatures result in QDs with lower quantum yields (range 11–28 %). The quality of the synthesized CdSe QDs was confirmed by UV/Vis and photoluminescence spectroscopy, small‐angle X‐ray scattering, and high‐resolution transmission electron microscopy. Finally, the potential of this protocol for large‐scale manufacturing was evaluated and by operating the continuous‐flow process for 87 min it was possible to produce 167 mg of CdSe QDs (with a mean diameter of 4 nm) with a quantum yield of 28 %.     

Batch‐ and Continuous‐Flow Aerobic Oxidation of 14‐Hydroxy Opioids to 1,3‐Oxazolidines—A Concise Synthesis of Noroxymorphone

14‐Hydroxymorphinone is converted to noroxymorphone, the immediate precursor of important opioid antagonists, such as naltrexone and naloxone, in a three‐step reaction sequence. The initial oxidation of the N‐methyl group in 14‐hydroxymorphinone with in situ generated colloidal palladium(0) as the catalyst and molecular oxygen as the terminal oxidant constitutes the key transformation in this new route. This oxidation results in the formation of an unexpected oxazolidine ring structure. Subsequent hydrolysis of the oxazolidine under reduced pressure followed by hydrogenation in a packed‐bed flow reactor using palladium(0) as the catalyst provides noroxymorphone in high purity and good overall yield. To overcome challenges associated with gas–liquid reactions with molecular oxygen, the key oxidation reaction was translated to a continuous‐flow process.     

Low‐abundant protein extraction from complex protein sample using a novel continuous aqueous two‐phase systems device

The present work describes the application of a novel continuous aqueous two‐phase system prototype for the recovery of biomolecules. The prototype is an alternative platform for protein recovery and α‐amylase from soybean extracts was used as a model system. The system was selected as an example of low‐abundant protein present in complex mixtures. Compared with batch systems, continuous operation in this prototype seems to increase partition coefficient with higher recovery efficiencies. Processing time is reduced at least three times in the continuous system when compared to batch mode, while hold up (volumetric quantity of the opposing phase in a determined phase sample) decreases with decreasing phases flow. Furthermore, similar partition coefficient (Kp > 4) with a higher top phase enzyme recovery (81%) is also obtained in this system probably due to better contact surface between phases, compared with that obtained in batch (79%). A continuous aqueous two‐phase system process with purification factor 40‐fold higher than batch experiments was achieved. These preliminary results exhibit the potential of continuous systems for the recovery of low‐abundant proteins from complex mixtures. The promising performance of this prototype can raise the attention of the industry for the adoption of aqueous two‐phase system processes.     

Continuous‐Flow Synthesis and Derivatization of Aziridines through Palladium‐Catalyzed C(sp3)−H Activation

A continuous‐flow synthesis of aziridines by palladium‐catalyzed C(sp³)?H activation is described. The new flow reaction could be combined with an aziridine‐ring‐opening reaction to give highly functionalized aliphatic amines through a consecutive process. A predictive mechanistic model was developed and used to design the C?H activation flow process and illustrates an approach towards first‐principles design based on novel catalytic reactions.     

4‐(N,N‐Dimethylamino)pyridine‐Embedded Nanoporous Conjugated Polymer as a Highly Active Heterogeneous Organocatalyst

We report herein for the first time the incorporation of a versatile organocatalyst, 4‐(N,N‐dimethylamino)pyridine (DMAP), into the network of a nanoporous conjugated polymer (NCP) by the “bottom‐up” approach. The resulting DMAP‐NCP material possesses highly concentrated and homogeneously distributed DMAP catalytic sites (2.02 mmol g^?1). DMAP‐NCP also exhibits enhanced stability and permanent porosity due to the strong covalent linkage and the rigidity of the “bottom‐up” monomers. As a result, DMAP‐NCP shows excellent catalytic activity in the acylation of alcohols with yields of 92–99 %. The DMAP‐NCP catalyst could be easily recovered from the reaction mixture and reused in at least 14 consecutive cycles without measurable loss of activity. Moreover, the catalytic acylation reaction could be performed under neat and continuous‐flow conditions for at least 536 h of continuous work with the same catalyst activity.     

Rapid and Efficient Copper‐Catalyzed Finkelstein Reaction of (Hetero)Aromatics under Continuous‐Flow Conditions

A general, rapid, and efficient method for the copper‐catalyzed Finkelstein reaction of (hetero)aromatics has been developed using continuous flow to generate a variety of aryl iodides. The described method can tolerate a broad spectrum of functional groups, including N‐H and O‐H groups. Additionally, in lieu of isolation, the aryl iodide solutions were used in two distinct multistep continuous‐flow processes (amidation and Mg–I exchange/nucleophilic addition) to demonstrate the flexibility of this method.     

On‐Line Monitoring of Chemical Reactions by using Bench‐Top Nuclear Magnetic Resonance Spectroscopy

Real‐time nuclear magnetic resonance (NMR) spectroscopy measurements carried out with a bench‐top system installed next to the reactor inside the fume hood of the chemistry laboratory are presented. To test the system for on‐line monitoring, a transfer hydrogenation reaction was studied by continuously pumping the reaction mixture from the reactor to the magnet and back in a closed loop. In addition to improving the time resolution provided by standard sampling methods, the use of such a flow setup eliminates the need for sample preparation. Owing to the progress in terms of field homogeneity and sensitivity now available with compact NMR spectrometers, small molecules dissolved at concentrations on the order of 1 mmol L^?1 can be characterized in single‐scan measurements with 1 Hz resolution. Owing to the reduced field strength of compact low‐field systems compared to that of conventional high‐field magnets, the overlap in the spectrum of different NMR signals is a typical situation. The data processing required to obtain concentrations in the presence of signal overlap are discussed in detail, methods such as plain integration and line‐fitting approaches are compared, and the accuracy of each method is determined. The kinetic rates measured for different catalytic concentrations show good agreement with those obtained with gas chromatography as a reference analytical method. Finally, as the measurements are performed under continuous flow conditions, the experimental setup and the flow parameters are optimized to maximize time resolution and signal‐to‐noise ratio.     

Automated Generation and Reactions of 3‐Hydroxymethylindoles in Continuous‐Flow Microreactors

An automated sequential approach for the generation and reactions of 3‐hydroxymethylindoles in continuous‐flow microreactors is described. Consecutive halogen–magnesium exchanges of four 3‐iodoindoles followed by addition to three aldehydes provided twelve 3‐hydroxymethylindoles in a multi‐microreactor setup. The synthetic flow strategy could be coupled with an in line continuous liquid–liquid extraction workup protocol for each reaction. Further elaboration of each of these indoles within the fluidic setup was achieved by acid‐catalysed nucleophilic substitutions with allyltrimethylsilane and methanol used as nucleophiles. Overall, a set of four 3‐iodoindoles was converted into thirty‐six indole derivatives by a range of transformations including iodo–magnesium exchange/electrophile trapping and acid‐catalysed nucleophilic substitution in a fully automated sequential fashion.     

Pd(0)‐ S‐propyl‐2‐aminobenzothioate immobilized onto functionalized magnetic nanoporous MCM‐41 as efficient and recyclable nanocatalyst for the Suzuki,Stille and Heck cross coupling reactions

The present work describes the use of Pd(0)‐ S‐propyl‐2‐aminobenzothioate Complex immobilized onto functionalized magnetic nanoporous MCM‐41(Fe₃O₄@MCM‐41@Pd‐SPATB) as efficient and recyclable nano‐organometallic catalyst for C–C bond formation between various aryl halides with phenylboronic acid (Suzuki reaction), aryl halides with triphenyltin chloride (Stille reaction), and aryl halides with n‐butyl acrylate (Heck reaction). All the reactions were carried out in PEG‐400 as green solvent with short reaction time and good to excellent yields. This catalyst was characterized by FT‐IR spectroscopy, XRD, TGA, VSM, ICP‐OES, TEM, EDX and SEM techniques. Ease of operation, high efficiency, recovery and reusability for five continuous cycles without significant loss of its catalytic activities or metal leaching are the noteworthy features of the currently employed heterogeneous catalytic system.     

Photochemical Dual‐Catalytic Synthesis of Alkynyl Sulfides

A photochemical dual‐catalytic cross‐coupling to form alkynyl sulfides via C(sp)−S bond formation is described. The cross‐coupling of thiols and bromoalkynes is promoted by a soluble organic carbazole‐based photocatalyst using continuous flow techniques. Synthesis of alkynyl sulfides bearing a wide range of electronically and sterically diverse aromatic alkynes and thiols can be achieved in good to excellent yields (50–96 %). The simple continuous flow setup also allows for short reaction times (30 min) and high reproducibility on gram scale. In addition, we report the first application of photoredox/nickel dual catalysis towards macrocyclization, as well as the first example of the incorporation of an alkynyl sulfide functional group into a macrocyclic scaffold.     

Preparation of a novel polymer monolith with functional polymer brushes by two‐step atom‐transfer radical polymerization for trypsin immobilization

Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]‐co‐glycidyl methacrylate} brushes were fabricated via two‐step atom‐transfer radical polymerization and used as a trypsin‐based reactor in a continuous flow system. This is the first time that atom‐transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface‐initiated atom‐transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.     

Integrated Micro Flow Synthesis Based on Sequential Br–Li Exchange Reactions of p‐, m‐, and o‐Dibromobenzenes

A micro flow system consisting of micromixers and microtube reactors provides an effective method for the introduction of two electrophiles onto p‐, m‐, and o‐dibromobenzenes. The Br–Li exchange reaction of p‐dibromobenzene with nBuLi can be conducted by using the micro flow system at 20 °C, although much lower temperatures (p‐bromophenyllithium was allowed to react with an electrophile in the micro flow system at 20 °C. The p‐substituted bromobenzene thus obtained was subjected to a second Br–Li exchange reaction followed by reaction with a second electrophile at 20 °C in one flow. A similar transformation can be carried out with m‐dibromobenzene by using the micro flow system. However, the Br–Li exchange reaction of o‐dibromobenzene followed by reaction with an electrophile should be conducted at ?78 °C to avoid benzyne formation. The second Br–Li exchange reaction followed by reaction with an electrophile can be carried out at 0 °C. By using the present method, a variety of p‐, m‐, and o‐disubstituted benzenes were synthesized in one flow at much higher temperatures than are required for conventional batch reactions.     

Solid‐supported synthesis of well‐defined amphiphilic block copolymer from methacrylates

A new facile method for preparation of an amphiphilic block copolymer via a one‐pot sequential atom transfer radical polymerization (ATRP) of methyl methacrylate (MMA) and 2‐hydroxyethyl methacrylate (HEMA) on solid support was developed. As a model homopolymerization for the solid‐supported block copolymerization, ATRPs of MMA and HEMA in toluene and in 2‐butanone/1‐propanol solvent system were carried out, respectively. Crosslinked polystyrene beads bearing 2‐bromoisobutyrate moieties successfully initiated the polymerizations of MMA and HEMA in controlled manner. On the basis of the successful results, the one‐pot synthesis of amphiphilic block copolymer by changing the reaction medium was performed. After the ATRP of MMA in toluene at 90 °C for 1 h, the poly(MMA) formed on the beads were washed by continuous flow of 2‐butanone/1‐propanol under nitrogen with the aid of a glass filter in a U‐shaped glass vessel. Then, 2‐butanone/1‐propanol, copper chloride (I), 2,2′‐bipyridyl, and HEMA were added and heated at 50 °C for 48 h with shaking the vessel, followed by treatment with trifluoroacetic acid to isolate the well‐defined amphiphilic block copolymer, poly(MMA‐b‐HEMA). These demonstrated the feasibility of the present strategy for well‐defined synthesis of amphiphilic block copolymers via a one‐pot procedure. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1990–1997, 2008     

A Fast‐Initiating Ionically Tagged Ruthenium Complex: A Robust Supported Pre‐catalyst for Batch‐Process and Continuous‐Flow Olefin Metathesis

In this study, a new pyridinium‐tagged Ru complex was designed and anchored onto sulfonated silica, thereby forming a robust and highly active supported olefin‐metathesis pre‐catalyst for applications under batch and continuous‐flow conditions. The involvement of an oxazine–benzylidene ligand allowed the reactivity of the formed Ru pre‐catalyst to be efficiently controlled through both steric and electronic activation. The oxazine scaffold facilitated the introduction of the pyridinium tag, thereby affording the corresponding cationic pre‐catalyst in good yield. Excellent activities in ring‐closing (RCM), cross (CM), and enyne metathesis were observed with only 0.5 mol % loading of the pre‐catalyst. When this powerful pre‐catalyst was immobilized onto a silica‐based cationic‐exchange resin, a versatile catalytically active material for batch reactions was generated that also served as fixed‐bed material for flow reactors. This system could be reused at 1 mol % loading to afford metathesis products in high purity with very low ruthenium contamination under batch conditions (below 5 ppm). Scavenging procedures for both batch and flow processes were conducted, which led to a lowering of the ruthenium content to as little as one tenth of the original values.     

Continuous‐Flow Electrosynthesis of Benzofused S‐Heterocycles by Dehydrogenative C−S Cross‐Coupling

Reported herein is the synthesis of benzofused six‐membered S‐heterocycles by intramolecular dehydrogenative C?S coupling using a modular flow electrolysis cell. The continuous‐flow electrosynthesis not only ensures efficient product formation, but also obviates the need for transition‐metal catalysts, oxidizing reagents, and supporting electrolytes. Reaction scale‐up is conveniently achieved through extended electrolysis without changing the reaction conditions and equipment.