Automation Highlights from Literature

Salt Selection and Simultaneous Polymorphism Assessment via High-Throughput Crystallization: The Case of Sertraline

Salt selection is a strategy that is commonly employed to improve properties of pharmaceutical compounds. Crystalline salts can confer useful attributes, including improved aqueous solubility, chemical stability, and higher bioavailability compared to those of the free base or acid of the active drug substance. However, gathering information about the best salt forms and crystallization conditions can often be a tedious task. Therefore, high-throughput approaches are of special interest. Julius F. Rememar and colleagues from Transform Pharmaceuticals performed high-throughput crystallization experiments with sertra-line free base in the presence of a large variety of acids (Org. Proc. Res. Dev. 2003, 7, 990). Over 3600 crystallization trials were conducted in 96-well aluminum blocks holding borosilicate tubes containing mixtures of sertraline, acid, and solvents. These individual mixtures, devised using proprietary design software (Architect), were prepared by combinatorially dispensing the salt formers and solvents using a Cartesian SynQuad 32-channel dispenser. As a result, 18 crystalline salt forms were identified and characterized. Four salt forms were found to exist as monomorphic materials. Unlike the HCl salt in the marketed drug product, the HBr salt appears resistant to polymorphism, crystallizing as a single form from over 140 discrete trials. This study highlights the importance of coupling salt selection studies with simultaneous polymorph screening to gain a more comprehensive understanding of solid form diversity as part of the form selection process for pharmaceutical development.

Automated Parallel Solid-Phase Synthesis and Anticancer Screening of a Library of Peptide-Tethered Platinum(II) Complexes

The automated parallel solid-phase synthesis of a 36-member library of peptide-tethered platinum(II) complexes as potential anticancer drugs is reported by J. H. van Boom et al. from Leiden University (J. Comb. Chem. 2003, 5, 821). Solid-phase synthesis was carried out in parallel on a LaMosse (Labotec Modular Organic Synthesis System) robot synthesizer, and a robust solid-phase platination methodology was developed. Although the prepared platinum peptide complexes showed no promise as cytotoxic agents, this work clearly demonstrates the utility of automated solid-phase synthesis of platinum drugs, as the whole process takes considerably less time than that required by conventional synthesis and testing.

Tablets of Functionalized Polystyrene Beads Alone and in Combination with Solid Reagents or Catalysts. Preparation and Applications in Parallel Solution and Solid-Phase Synthesis

T. Ruhland et al. (H. Lundbeck A/S, Denmark) have developed a novel methodology for the preparation of tablets consisting of neat functionalized polystyrene beads and tablets of non-polymer-bound solid reagents or catalysts in combination with polystyrene beads (J. Comb. Chem. 2003, 5, 842). The methodology described produces mechanically robust tablets without requiring the use of other additives. The efficiency of the tablets was demonstrated in parallel Mitsunobu and acylation reactions in a 96-reactor microblock. Dosing and distribution of reagents and catalysts as tablets is more convenient and faster as compared to conventional methods and is also compatible with automation. Application of tablets, especially in parallel synthesis, significantly reduces the exposure of toxic, hazardous, and dusty materials to the working environment. This novel methodology has a significant potential to speed up parallel solution and solid-phase synthesis.

Quantified MS Analysis Applied to Combinatorial Heterogeneous Catalyst Libraries

A high-throughput screening system for secondary catalyst libraries has been developed by Z. Liu et al. (Chinese Academy of Sciences, Dalian) by incorporating an 80-pass reactor and a quantified multistream mass spectrometer screening (MSMSS) technique (J. Comb. Chem. 2003, 5, 802). A uniform reaction temperature could be obtained in the multi-stream reactor with maximum temperature differences of less than 1 K at 673 K. Quantification of the results was realized by combination of a gas chromatogram with the MSMSS, which could provide the product selectivities of each catalyst in a heterogeneous catalyst library. The reaction results of a promising catalyst selected from the library could be reasonably applied to further scale-up the system. The aldol condensation of acetone, with obvious differences in the product distribution over different kinds of catalysts, was selected as a model reaction to validate the screening system. By using this technique, it is possible to test catalysts under identical conditions with a throughput of 400 catalysts per week.

Application of Visual Basic in High-Throughput Mass Spectrometry-Directed Purification of Combinatorial Libraries

Manual transfer and handling of library data is often tedious, time-consuming, and erroneous. B. Li and E. C. Y. Chan (S*BIO Pte Ltd., Singapore) present an approach to customize the sample submission process for high-throughput purification of combinatorial libraries using preparative liquid chromatography electrospray ionization mass spectrometry (J. Comb. Chem. 2003, 5, 834). Visual Basic programs were developed and subsequently applied for the seamless electronic submission and handling of data for high-throughput purification. Functions were incorporated into these programs, allowing medicinal chemists to perform online verification of the purification status and on-line retrieval of postpurification data. The authors state that the application of these user-friendly and cost-effective programs has greatly increased their work efficiency by reducing paper work and manual manipulation of data.

Millisecond Kinetics on a Microfluidic Chip Using Nanoliters of Reagents

H. Song and R. F. Ismagilov (University of Chicago) describe a microfluidic chip for performing kinetic measurements with better than millisecond resolution (J. Am. Chem. Soc. 2003, 125(47), 14613). Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. Here, a droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. From fluorescent images integrated for 2 to 4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.

On-Chip Separation of Peptides Prepared within a Microreactor

Over the past five years, there has been a rapid growth in the development of microreactor technology exploiting the technique of electroosmotic flow. Recent research has demonstrated that a selection of gas and liquid phase reactions can be successfully performed within microreactors where the products are inherently produced in higher yield and purity in a much shorter time compared with traditional batch reactions. Stephen J. Haswell et al. (University of Hull, Hull, United Kingdom) now report for the first time that peptides prepared within a microreactor may be electrophoretically separated from unreacted reagents within an integrated microreactor (Chem. Commun. 2003, 2886). By applying the reverse electrokinetic flow technique, they received the pure peptide in high quality without any traces of the reagents or starting materials.

Solution-Phase Synthesis of Esters within a Microreactor

Stephen J. Haswell et al. (University of Hull) describe a range of techniques for the solution-phase synthesis of esters at room temperature within an electroosmotic flow-based borosilicate glass microreactor, including the use of mixed anhydrides and the in situ preparation of acyl halides (Tetrahedron 2003, 59(51), 10173). The reactions were carried out using a 4-channel borosilicate glass microreactor due to its compatibility with organic solvents, high mechanical strength, temperature resistance, and optical transparency. The channel dimensions were 350 μm (wide) x 52 μm (deep) x 2.5 cm (long). Excellent conversions were achieved in the reactions investigated.

A Practical Approach to Continuous Processing of High Energetic Nitration Reactions in Microreactors

Continuous processing in microreactors represents a novel way for the safe and expedient conduct of high energetic reactions and potentially hazardous chemistry. Apart from handling benefits (such as minimized problems in the scale-up process), reactions in microreactors proceed under precisely controlled conditions providing improved yields and product quality compared to the batch procedure. S. Taghavi-Moghadam (CPC-Cellular Process Chemistry Systems GmbH, Frankfurt/Main) demonstrates the potential of this technology in the crucial nitration of the pharmaceutically relevant intermediate 1-methyl-3-propyl-1H-pyrazole-5-carboxylic acid (Synthesis 2003, 18, 2827). Further fundamental nitration examples demonstrate the unproblematic handling of hazardous H₂SO₄/HNO₃ mixtures for the nitration of 2-methylindole and pyridine-N-oxide or even the explosive acetyl nitrate Ac₂O/HNO₃ for the nitration of toluene in the CYTOS microreactor.

Chemical and Physical Processes for Integrated Temperature Control in Microfluidic Devices

Microfluidic devices are a promising new tool for studying and optimising chemical reactions that require accurate temperature control. Rosanne M. Guijt et al. describe a new temperature control system for microfluidic devices (Lab on a Chip 2003, 3(1), 1). The temperature control system she presents uses chemical and physical processes to locally regulate temperature. The functionality uses endothermic and exothermic processes. The thermal effect is initiated by mixing two components. The extent of the resulting thermal effect can be controlled by either the flow ratio of the two components or by the selection of the components based on their chemical or physical properties. It is also dependent on the external ambient temperature.

In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool the microchannel. In addition, heating of the microchannel was achieved by dissolution of concentrated sulphuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allowed control of the position and the magnitude of the thermal effect. In both heating and cooling experiments, temperature ramps of about 1 °C were obtained.

The integration of the temperature control system into microfluidic devices is considered simple nowadays and does not require additional microfabrication steps. Because the chemical or physical processes take place in a microchannel, integration of a temperature control system does not dramatically increase the footprint of a device. Multiple cooling and heating systems could be integrated along a single reaction channel, allowing thermocycling of compounds migrating or being pumped through this channel. The small feature size of the cooling system also allows multiple temperature control units on a microdevice where multiple endothermic and exothermic processes occur in parallel. In addition, the driving force of the system is vacuum, resulting in low power consumption during cooling or heating. The cooling system described might also be attractive for cooling microelectronic devices.

Microchip-Based Synthesis and Analysis: Control of Multicomponent Reaction Products and Intermediates

A miniaturized-SYNthesis and Total Analysis System (mSYNTAS) was used for the solution-phase synthesis and on-line analysis (TOF-MS) of Ugi multicomponent reaction (MCR) products and intermediates (Analyst, 2001, 126(1), 24). This approach provides an unusually high degree of control of the MCR and delivers detailed, novel information about reaction intermediates in real time.

The microreactor used for all experiments operates on the principle of distributive mixing. The microstructure is a two-layer device made up of a glass/silicon/glass sandwich. It has an internal volume of ∼600 nL and measures 2 × 5 × 10 mm. Two inlet flows are split into a series of separate multichannel streams (16 partial flows). This is achieved by repeated splitting of the channels in such a way that an array of symmetrical elements results. Wafer-through nozzles connecting the two fluidic layers allow the two liquid streams to converge and mix. Channels are then sequentially combined in a reverse network until all partial flows are united in one broad outlet channel. The extremely large diffusional surface areas created within the device allow for rapid, efficient mixing. This micromixer is coupled to a TOF-MS via an electrospray unit.

The high level of structural information provided by the continuous-flow μSYNTAS provides a means for determining mechanistic characteristics of even highly complex reaction systems. Work is currently underway in this laboratory toward the dynamic optimization of reaction products by manipulation of other system parameters in real time. In comparison with solid-phase technologies, the automation of the cycle involving parameter manipulation and on-line analysis may be developed to a much higher degree in a solution-phase system. Consequently, the use of μSYNTAS for optimization of compound library syntheses may be a useful addition to the combinatorial chemistry toolkit.

Knoevenagel Condensation Reaction in a Membrane Microreactor

The advances in the design and fabrication of micromixers, microseparators, and microreactors bring closer the realization of desktop miniature factories and micropharmacies. They represent an inexpensive alternative for the production of speciality chemicals and pharmaceuticals by a continuous process, allowing simpler process optimization, rapid design implementation, better safety, and easier scale-up through replication. This enables rapid product deployment to the marketplace and thus ensures a significant competitive edge.

Sau Man Lai et al. describe a multichannel membrane reactor that has been fabricated and tested for Knoevenagel condensation (Chemical Communication 2003, 218-219). This work clearly demonstrates the potential use of the micro-reactor for the production of fine chemicals and pharmaceuticals. It also shows the benefits available from using a membrane microreactor for reactions that are constrained by unfavorable thermodynamics. Supra-equilibrium conversion is achieved at higher product purity through selective removal of water byproduct from the condensation reaction. It is conceivable that pure product could be obtained from the membrane microreactor with better catalyst formulation.

The Application of Microreactors to Synthetic Chemistry

A good overview of the fundamental characteristics and emerging applications of microtechnology in the field of synthetic chemistry is available from Stephen J. Haswell (Chemical Communication 2001, (5), 391). Despite the many advances made in synthetic chemistry over recent decades, the basic practical methodology used remains unchanged. This situation arises primarily because reactions tend to be carried out on a bulk scale using a batch approach that chemists feel comfortable manipulating. At the molecular level, however, it makes little difference fundamentally whether a reaction takes place in a 10-mL or 10-pL container. By applying technology developed for the electronics industry, it is now possible to produce reactors in which one can manipulate and analyze materials on a micron to nanometer scale. The author believes that so-called microreactor technology can do for synthetic chemistry what the solid-state transistor has done for computing, vastly increasing the versatility and the amount of chemical information that a single person can generate.

A microreactor is generally defined as a series of interconnecting channels (10-300 μ in diameter) formed in a planar surface in which small quantities of reagents are manipulated. The reagents can be brought together in a specified sequence, mixed, and allowed to react for a specified time in a controlled region. The product may then be analytically monitored and, if necessary, separated for further steps in a reaction or collected for analysis or testing.

The microreactor confers many advantages over conventional scale chemistry. The decrease in linear dimensions allows heat transfer coefficients to exceed those of conventional heat exchangers by an order of magnitude. Micromixers can reduce mixing times to milli- or nanoseconds. The increased surface-to-volume ratio in microreactors (10.000 to 50.000 m²m^-3, compared to 1.000 m²m^-3 in conventional laboratory vessels) has implications for surface-catalysed reactions.

The inherent benefits of microreactors, namely, the rapid generation of small but detectable quantities of reaction products, efficient heat transfer and fluidic control, are now being applied successfully to synthetic chemistry. In theory, these factors might give a research worker using a micro-reactor the ability to greatly increase the rate at which new compounds are produced. The work demonstrates how some of the initial findings obtained by research groups developing microreactor systems could be applied to high-throughput synthesis. There are also some operating characteristics of the microreactor environment that result in fundamental differences in chemistry. Of more immediate and perhaps significant impact to the research community is the opportunity microreactors offer in terms of performing a large number (many hundreds) of reactions to explore and optimize a single reaction or a series of chemical reactions. For example, the ability to generate information about reaction conditions, kinetics, and product selectivity is now readily accessible using microreactors, an option not easily available using conventional methodology.

A commercially available chemical synthesiser using microreaction technology is produced by IMM Mainz. It consists of a pumping module, a microreactor that results in very efficient mixing of reagents, followed by a capillary to allow time for the reaction to go to completion. The outflow is then collected for further manipulation by the user. This could be just the first step along a road that will see the integration of automated reagent manipulation, reaction monitoring and product purification into a single instrument containing several interconnected microreactors, or possibly a single microreactor device. In common with microelectronic chips, once the facilities to fabricate microreactors are in place, they become progressively less expensive to produce in quantity. This should make the production of chemicals in massive parallel arrays of reactors an economic possibility. It is likely that some of the peripheral equipment required will still represent a considerable cost, but this should be set against the potential increase in productivity per research worker. In addition, the effective production of molecules in terms of energy, safety, and environmental impact will emerge as important factors in the future exploitation of microreactor technology. One of the underlying features of any future commercially available automated synthesis system must be versatility. Research is now moving toward a “plug and play” approach in which the reaction and detection configurations can be customized. The next few years will undoubtedly see significant development in this area of the technology.

A Microchip-Based Proteolytic Digestion System Driven by Electroosmotic Pumping

The development of a proteolytic digestion system for proteomic analysis is described by Lian Ji Jin (Lab on a Chip, 2003, 3(1), 11). Microchip-based proteomic analysis requires proteolytic digestion of proteins in microdevices. Enzyme reactors in microdevices, fabricated in glass, silicon, and PDMS (polydimethylsiloxane) substrates, have recently been demonstrated for model protein digestions. The common approach used for these enzyme reactors is employment of a syringe pump(s) to generate hydrodynamic flow, driving the proteins through the reactors.

The author of this article presents a novel approach that uses electroosmotic flow (EOF) to electrokinetically pump proteins through a proteolytic system. The existence of EOF in the proteolytic system packed with immobilized trypsin gel beads was proven by imaging the movement of a neutral fluorescent marker. Digestions of proteins were subsequently carried out for 12 min, and results were comparable to those attained after an 18-h water bath digestion at 37 °C. The tryptic peptides were analyzed independently using capillary electrophoresis (CE) and MALDI-TOF mass spectrometry (MS). The results from CE analysis of the tryptic peptides from the EOF-driven proteolytic system and a conventional water bath digestion were comparable. MALDI-TOF MS was used to identify the parent protein and the tryptic peptides using MS-Fit database searching. The potential utility of the EOF-driven proteolytic system was demonstrated by direct electroelution of proteins from an acrylamide gel into the proteolytic system, with elution and tryptic digestion achieved in a single step. The EOF-driven proteolytic system provides a simple way to integrate protein digestion into an electrophoretic micro total-analysis system for protein analysis and characterization.

The electroosmotic pumping may provide additional mechanisms for protein digestion, such as inducing conformational change of protein tertiary structure to expose more peptide bonds to enzymatic cleavage. Eventually, the EOF approach should prove to be useful in integrating an enzyme reactor with separations on microchips, moving one step closer toward the goal of building an integrated electrophoretic micro total-analysis system for protein analysis and characterization. A practical application of the current method, protein gel elution and digestion in a single step, already looks promising. Other areas that need to be addressed include optimization of the microchamber reactor (in terms of chamber size), surface adsorption issues, as well as mechanisms to reduce the digestion time and increase sensitivity.

Out of the Oil Bath and into the Oven: Microwave-Assisted Combinatorial Chemistry Heats Up

The application of microwave irradiation to expedite solid-phase organic reactions could be the tool that allows combinatorial chemistry to deliver on its promise of providing rapid access to large collections of diverse small molecules. A different microwave application in the combinatorial chemistry is described by Helen E. Blackwell (Org. Biomol. Chem. 2003, 1, 1251). Herein, several different approaches to microwave (MW)-assisted solid-phase reactions and library synthesis are introduced, including the use of solid-supported reagents, multicomponent coupling reactions, solvent-free parallel library synthesis, and spatially addressable library synthesis on planar solid supports. The future impact of MW-assisted organic reactions on solidphase and combinatorial chemistry could prove to be immense, and methods for further improvement of this strategic combination of technologies are highlighted.

The familiar domestic MW oven has seen the most use in synthesis so far due to its low cost and ready availability. However, the current trend is the use of dedicated, commercial MW instruments, as these provide more homogeneous heating, reaction temperature control, built-in magnetic stirring, and significantly improved safety features. There are two types of commercial MW reactors: multimodal and monomodal systems. The multimodal system is most similar to the domestic MW oven. The MWs enter into the relatively large reaction chamber and are reflected by the reactor walls. The reactions of the waves generate a three-dimensional stationary pattern of standing waves in the cavity, which are called modes. The reaction vessel is commonly rotated in the reactor cavity so that it experiences a homogeneous field.

In the other type of MW reactor, the monomodal system, the electric field is focused with a waveguide into a small reactor cavity where the reaction vessel sits. This cavity is sized such that only a single mode is present, which is believed to yield a more homogeneous distribution of energy within the cavity. Despite this purported benefit, multimodal reactors do have the advantage that numerous reactions can be performed simultaneously (i.e., in parallel) within the reaction chamber, whereas each reaction is typically performed sequentially in the smaller monomodal units.