Structure-dependent response of a chemiluminescence nitrogen detector for organic compounds with adjacent nitrogen atoms connected by a single bond.

High-throughput screening (HTS) of chemical libraries is indispensable for drug discovery research. However, the HTS data quality for lead discovery, lead optimization, and quantitative structure activity relationship studies has been severely compromised due to the uncertain compound concentrations in screening plates. In order to address this issue, we compared various high-throughput technologies for quantification of compounds in microtiter plate format without the need for authentic compounds as standards and identified the chemiluminescence nitrogen detector (CLND) as the method of choice at the present time. However, the structure dependence of this detector has not been well studied. A proposed rule suggested that the only exception to equimolar response is for compounds that contain adjacent nitrogen atoms. The response should be zero when the adjacent nitrogen atoms are connected by a double bond and 0.5 when they are connected by a single bond. In this investigation, we studied a broad range of compounds with isolated and adjacent nitrogen atoms. We confirmed that compounds with isolated nitrogen atoms produce an equimolar response with a 15-20% variation depending on structures and compounds with adjacent nitrogen atoms connected by a double bond giving nearly zero response. We discovered that the CLND response for compounds containing adjacent nitrogen atoms that are connected with a single bond is highly structure dependent. Substitutions on the nitrogen atoms or nearby in the molecule can increase the CLND response to approach a value higher than the predicted value 0.5 (maximal value 0.82/nitrogen atom). Without substitution, much lower values than predicted (minimal value 0.0-0.08/nitrogen atom) are obtained. Therefore, the prediction of response of 0.5/nitrogen atom for compounds with adjacent nitrogen atoms connected by a single bond should be abandoned. Compounds with similar structures should be used to generate calibration curves for quantification of this class of compounds.

High-throughput purification of combinatorial libraries II: Automated separation of single diastereomers from a 4-amido-pyrrolidone library containing intentional diastereomer pairs.

A 4-amido-pyrrolidone library that was intentionally synthesized as pairs of diastereomers was produced by solution-phase parallel syntheses and purified by an automated high-throughput purification system. A total of 2592 4-amido-pyrrolidinones were ultimately isolated as single diastereomers from a matrix of 1920 syntheses. After the four-step synthesis and HPLC purification, the average yield of a single diastereomer was 36.6%. The average chemical purity was >90%, and the average diastereomeric purity was >87%. The choice of chiral amines used to make amides with heterocyclic acid chlorides had a dramatic effect on success. Analysis of the relationship between amines used for synthesis and the diastereomeric separation showed that amides made from chiral 1,2-amino alcohols gave superior separation to amides from chiral morpholines. The presence of a hydrogen bond donor on the amide side chain seems to be required for a better diastereomeric separation.

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach.

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.