Purifying the masses: integrating prepurification quality control, high-throughput LC/MS purification, and compound plating to feed high-throughput screening

In this paper we report using a parallel, four-channel HPLC/MUX/MS purification system, the Purification Factory, to purify thousands of compounds destined for high-throughput screening in a single month. The maximum sample throughput during this 20-workday month was 704 samples/day. Since this purification throughput exceeded the postpurification sample and data handling capabilities provided by commercial solutions, a custom-integrated solution was designed to address these shortcomings. In this paper we detail the key improvements in automation, solvent handling, and sample handling logistics implemented to sustain a mean throughput of 528 samples/day over a multimonth time period.     

Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.     

Iterative high-throughput polymorphism studies on acetaminophen and an experimentally derived structure for form III

Three crystal forms of acetaminophen were prepared and characterized using a newly developed high-throughput crystallization platform, CrystalMax. The platform consists of design software, robotic sample dispensing and handling, and high-throughput microanalytics and is capable of running thousands of crystallizations in parallel using several different methods to drive supersaturation and subsequent crystallization. Additionally, structural models of the elusive third form of acetaminophen will be discussed on the basis of powder X-ray diffraction data. One structure suggested has a bilayer motif, held together by O-H...O(H) hydrogen bonds, and helps explain the difficulty associated with preparing this form from solution.     

分子阵列研究进展

小分子化合物可以调节生物学过程,是研究活性生物大分子（特别是蛋白质）以及药物的重要工具,而高通量筛选是发现活性分子的重要方法。分子阵列是近年来新出现的一种高通量筛选技术,上面含有成千上万种组合合成的化合物以及天然产物,可以用于发现新的先导化合物,以及筛选已有的先导化合物。现在分子阵列已经成功应用于蛋白分析、先导化合物的发现等许多领域。本文综述了近年来分子阵列的构建过程,原位合成、非原位合成等各种固定化策略以及荧光免疫检测,表面等离子体共振成像技术等检测手段,并介绍了化学分子印刷阵列方法,最后总结了分子阵列的应用,并对分子阵列在我国中药发展等有方面将起到的潜在作用作了展望。     

Application of parallel synthesis and high-throughput characterization in photocatalyst discovery

The last decade has seen significant progresses in the application of combinatorial approaches and high-throughput screening in photocatalyst discovery. This paper aims at providing a comprehensive review on the parallel synthesis and high-throughput characterization of photocatalysts, including the development of instrumentation, strategy of experiment, preparation of libraries, high-throughput screening technique and data analysis. The review ends with a summary of the remaining challenges and prospects on combinatorial photocatalyst discovery.     

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.     

Multiplexed SNP genotyping using nanobarcode particle technology

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.     

A convenient microwave assisted arylzinc generation-Negishi coupling protocol

Arylzinc reagents were readily prepared from aryl iodides using a Zn-Cu couple in a microwave environment. A sequential arylzinc formation-Negishi cross-coupling protocol suitable for parallel high-throughput synthesis has been developed.     

Spectroscopically encoded resins for high throughput imaging time-of-flight secondary ion mass spectrometry

Spectroscopic barcoding was recently introduced as a new pre-encoding strategy wherein the resin beads are not just carriers for solid phase synthesis, but are, in addition, the repository of the synthetic scheme to which they were subjected. To expand the repertoire of spectroscopically barcoded resins (BCRs), here we introduce a new family of halogenated polystyrene-based polymers designed for high-throughput combinatorial analysis using not only infrared and Raman spectroscopy but also imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, we have established that (a) the halogen content of these new resins can be used as an encoding element in quantitative imaging ToF-SIMS and (b) the number of styrene monomers used to generate unique vibrational fingerprints can be significantly reduced by using monomers in different molar ratios. The combination of quantitative imaging ToF-SIMS and vibrational spectroscopy is anticipated to dramatically increase the repertoire of possible BCRs from a few hundreds to several thousands.     

High-throughput preparative process utilizing three complementary chromatographic purification technologies

A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.     

组合电化学--电化学研究中的新策略

组合电化学是一种新的电化学研究策略，通过设计和构建大量多样性的电极阵列，并对其进行高通量筛选和表征，快速、高效地实现了体系的电化学研究。本文综述了近年来进行的组合电化学研究，重点介绍了组合光学筛选方法、组合电化学合成方法以及电化学平行筛选方法，并探讨了各种方法的优势和存在的问题。     

Quantitative inhibitor fingerprinting of metalloproteases using small molecule microarrays

Current methods to identify interactions on small molecule microarrays (SMMs) introduce false positives that are difficult to dissect from the "real" binding events without tedious downstream re-evaluation. To specifically elucidate only activity-dependent ligand binding interactions, we have developed a technique that can be universally applied to present SMM systems. Our method makes use of a dual-color application strategy and is based on the simultaneous application of differentially treated samples. Overcoming the limitations of slide-to-slide variation, this method directly revealed activity-dependent interactions through a one-step application of protein samples on SMMs. Besides providing lead molecules for further development, the high-throughput screening results confer activity-dependent fingerprints for quantitative characterization and differentiation of proteins. The procedure was tested using a synthetic hydroxamate peptide library with 1400 discrete sequences permuted combinatorially across P1', P2', and P3' positions. Functional profiling across a panel of metalloproteases provided 44,800 datapoints within just eight SMM slides. These data were globally analyzed for activities, specificity, potency, and hierarchical clustering providing unique insights into inhibitor design and preference within this group of enzymes. Quantitative K(D) measurements performed on SMMs using one of the enzymes in the panel, Anthrax Lethal Factor, the toxic component of a notorious bioterror agent, unraveled several lead micromolar binders for further development. Overall, the effectiveness of the SMM platform is shown to be enhanced and extended using the strategy presented in this work.     

A novel approach to high-throughput quality control of parallel synthesis libraries

Combinatorial chemistry is a powerful tool to enhance drug discovery efforts in the pharmaceutical industry. One type of combinatorial chemistry, parallel synthesis, is now widely used to prepare numerous compounds of structural diversity. A novel high-throughput method for quality control of parallel synthesis libraries has been developed. The method uses flow injection MS, for proof of structure and estimation of purity, and a novel direct injection CLND technique for quantitation of amount. Following the synthesis of a small molecule library, compounds analyzed using this technique were characterized by mass spectrometry, and an accurate concentration of the compound was assessed by CLND. Characterization of one compound is completed in 60 s, allowing for up to 1000 compounds to be analyzed in a single day. The data is summarized using pass/fail criteria using internally developed software.     

Rapid determination of enantiomeric excess: a focus on optical approaches

High-throughput screening (HTS) methods are becoming increasingly essential in discovering chiral catalysts or auxiliaries for asymmetric transformations due to the advent of parallel synthesis and combinatorial chemistry. Both parallel synthesis and combinatorial chemistry can lead to the exploration of a range of structural candidates and reaction conditions as a means to obtain the highest enantiomeric excess (ee) of a desired transformation. One current bottleneck in these approaches to asymmetric reactions is the determination of ee, which has led researchers to explore a wide range of HTS techniques. To be truly high-throughput, it has been proposed that a technique that can analyse a thousand or more samples per day is needed. Many of the current approaches to this goal are based on optical methods because they allow for a rapid determination of ee due to quick data collection and their parallel analysis capabilities. In this critical review these techniques are reviewed with a discussion of their respective advantages and drawbacks, and with a contrast to chromatographic methods (180 references).     

基于GPU计算的并行PLS算法研究与实现

偏最小二乘算法(PLS)是与红外、近红外光谱分析结合使用最为广泛的化学计量学算法,然而当前PLS算法通常采用单线程方式实现,当校正模型数量多或样本数量大、波长点数和主成分数较多,模型需对光谱预处理和波长选择方法反复优化时,计算十分缓慢。为大幅提高建模速度,该文提出了一种基于图形处理器(GPU)的并行计算策略,利用具有大规模并行计算特性的GPU作为计算设备,结合CUBLAS库函数实现了基于GPU并行的PLS建模算法(CUPLS)。利用近红外光谱数据集进行性能对比实验,结果表明CUPLS建模算法较传统单线程实现的PLS算法,加速比可达近42倍,极大地提升了化学计量学算法的建模效率。该方法亦可用于其它化学计量学算法的加速。     

High-throughput study of poly(ethylene glycol)/ibuprofen formulations under controlled environment using FTIR imaging

Simultaneous analysis of many samples under identical conditions improves the effectiveness of research and accelerates product design. A novel spectroscopic imaging approach using a multichannel detector has been developed for parallel analysis of pharmaceutical formulations under controlled environments. Samples of formulations of ibuprofen in poly(ethylene glycol) have been prepared with ibuprofen concentrations ranging from 0 to 100% using a microdroplet deposition approach. The concentration of ibuprofen in PEG at which dimerization of ibuprofen molecules can be avoided has been determined via simultaneous measurement of all samples using in situ FTIR spectroscopic imaging. FTIR spectra from all samples have been analyzed to assess the molecular state of the drug and the degree of polymer swelling as a function of drug concentration. The effect of elevated temperature on the stability of all formulations was also studied. This high-throughput approach identified the concentration range for stable formulations and provided evidence that hydrogen bonding between ibuprofen and the polymer is responsible for enhanced stability at higher temperatures. This high-throughput imaging approach, based on a miniature sampling system, significantly reduces the experimental time by allowing many (potentially a few thousand) experiments to be run in parallel and increases the accuracy by minimizing variations between experiments.     

Multiple-sprayer tandem mass spectrometry with parallel high flow extraction and parallel separation for high-throughput quantitation in biological fluids.

In this work, an on-line four-column high-flow parallel extraction device was set up and interfaced to a four-column parallel separation system with a four-sprayer tandem mass spectrometric detector. This system allowed for the direct and simultaneous sampling, extraction, separation, and detection of four samples in biological fluids, resulting in significantly increased throughput. The performance of this system was evaluated using rat plasma samples spiked with methotrexate as a test compound. The results showed that the relative standard deviation for responses across the four parallel channels was less than 10% for a concentration range from 10 to 2500 nM, resulting in comparable sensitivity (slope of curve) for the analyte on all channels. A ruggedness test was performed using a mixed set of new and used extraction columns and the relative standard deviation in analyte response was found to be below 15%. The inter-channel crosstalk was measured to be about 0.1%. A comparison between the data generated on this system and on a conventional system for a rat pharmacokinetic study showed no more than 20% difference in measured concentrations. This parallel system is a viable tool for high-throughput bioanalysis in a drug discovery environment.     

Parallel synthesis and high throughput dissolution testing of biodegradable polyanhydride copolymers

We have demonstrated that polycondensation reactions can be carried out in a combinatorial fashion and that the polymer library can be screened at high throughput using a rapid prototyping technique to fabricate multiwell substrates. A linearly varying compositional library of 100 different biodegradable polyanhydride random copolymers that are promising carriers for controlled drug delivery was designed, fabricated, and characterized by IR microscopy within a few hours. The polyanhydride copolymer library was based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic anhydride (SA) and was characterized with infrared microspectroscopy to determine the composition within each well. Since degradation and release rates depend on copolymer composition, we also developed new high-throughput methods to investigate drug release from this library of copolymers by designing specific wells for each task. A subset of this library was chosen, and a substrate was designed and fabricated to enable the synthesis and monitoring of dye dissolution from a range of polyanhydride copolymers in a parallel fashion using a CCD camera. Multisample substrates were fabricated with a novel rapid prototyping method that consists of an organic solvent-resistant array of 10 x 10 microwells of 2-muL volume each. The libraries were deposited with a custom-built liquid dispensing system consisting of a series of computer-controlled volume-dispensing pumps and XYZ motion stages. The parallel dye dissolution study displayed a decreasing rate of release with increasing CPH content. This result agrees with previously published data for dye release from poly(CPH-co-SA) copolymers. The methodology described in this work is amenable to numerous applications in the arenas of high-throughput polymer synthesis and characterization.     

High-throughput bioassay-guided fractionation: a technique for rapidly assigning observed activity to individual components of combinatorial libraries,screened in HTS bioassays

In this paper, we describe an automated, high-throughput analytical tool for the unambiguous characterization of the active component(s) of a combinatorially derived reaction mixture. We call this technique high-throughput bioassay-guided fractionation (BGF). The novel aspects of this communication are the systematization of the BGF concept, the application of BGF to combinatorial chemistry, and the high-throughput nature of the identification technique. The identification of the active component in a well mixture is an essential step for subsequent resynthesis or isolation of the active component(s) or for removal of intractable wells from further consideration. We believe the technique described is also applicable to any mixture library, provided the expected component (or components) of each well is (are) known. Example mixture libraries would include collections of synthetic chemicals and collections of purified natural products. The mixture need not come from libraries produced using parallel synthesis. The BGF tool described herein allows full utilization of highly diverse combinatorial libraries, thereby obviating costly up-front purification or extensive prescreening characterization efforts.     

One-minute full-gradient HPLC/UV/ELSD/MS analysis to support high-throughput parallel synthesis

High-throughput parallel synthesis of library compounds for early drug discovery requires high-throughput analytical methods to confirm synthesis, identify reaction products, and determine purity. An ultrafast 1.0-min HPLC/UV/ELSD/MS method was developed and compared to our standard 2.5- and 5.0-min methods in order to determine if the faster method was appropriate to evaluate compound synthesis and determine purity. In addition to using standard test mixtures, a 400-member library produced by high-throughput parallel synthesis was used for comparing the various methods. Mass spectrometric detection was used for compound identification, while UV and ELSD data offered purity assessment. Compared to our longer separations, chromatographic separation achieved using the 1.0-min method was sufficient for compound evaluation and purity assessment. This ultrafast 1.0-min HPLC/UV/ELSD/MS method is expected to increase analytical throughput tremendously, provide important information faster, and reduce the overall cycle time from synthesis to screening.