High Resolution Mass Spectrometry With Automated Data Analysis to Support Late Stage Functionalization Yong Liu1; Fabien Fontaine2; Bella Yao1; Ismael Zamora2; Roy Helmy1; Shane Krska1; Kevin P. Bateman1
1Merck
& Co., Inc., Kenilworth, NJ, USA; 2Molecular Discovery, Barcelona, Spain
Introduction Late stage functionalization (LSF) refers to the use of C-H activation chemistries for the incorporation of structural diversity into a final product or an advanced intermediate. This approach has become a very useful synthetic tool that provides rapid access to otherwise difficult to obtain chemical entities. Its utility spans multiple areas, including discovery chemistry, drug metabolism, and discovery biology. Given the complex structure of the final product or advanced intermediate, LSF chemistry often generates mixtures with multiple products that are regioisomers. Furthermore, LSF employs high throughput synthesis techniques that give rise to many samples for analysis. In this study, we describe an analytical approach for LSF reaction analysis. A rapid liquid chromatography high resolution mass spectrometry (LC-HRMS) workflow was developed for sample analysis. MSe data acquisition and a modified version of Massmetasite were adapted for automated data analysis, with final data review and browsing enabled through WebMetabase.
UPLC-HR-MS/MS data acquisition UPLC: The chromatographic separation is performed by Acquity UPLC system. The mobile phases consist of solvent A (100% water, 0.1% formic acid ), and solvent B (100% acetonitrile, 0.1% formic acid). Column: Acquity UPLC HSS T3 column (1.8 µm, 2.1X100 mm)
Results summary
Results archive and sharing by WebMetabase
HR-MS/MS: The experiments were performed on Waters Premier QTof mass spectrometer. MSe method was developed with low collision energy as channel one to detect intact molecules and high collision energy ramp, as channel two to obtain pseudo MS/MS data.
XIC summary Chromatogram
What is LSF? •• The incorporation of chemical diversity into a final product or advanced intermediate •• A way to quickly access a suite of analogues for a compound of interest R
LSF C-H borylation, oxidation, halogenation, etc.
Biologically relevant molecule HTS hit
R
•• Archive results in a central place; allows chemists view results via Web brower Markush structure
R
R
Lead pharmacophore in a medchem project
Rapid SAR data set –– Hit validation –– On/off-target SAR –– ADME optimization
New scaffold from CM&I
Molecular probes
Applications •• Block metabolically active sites, metabolite synthesis •• Increase potency, improve selectivity, and reduce off-target liabilities
Scan 1: Q1 nonresolving, collision energy 5 eV, TOF m/z 100-1000, 0.2s scan time, 0.05 s inter-scan delay
HR-MS/MS data analysis
Correlation of fragment ions between substrate and product
Product identity summary
Provide location of substrate and product HRMS/MS data
Microscale reaction screening (HTE)
Automated data processing
Analytical challenge • Multiple isomers • Low product conversion • Large sample quantity
Structure elucidation
Correlation of structures between substrate and product
Data analysis by MetaSite
Select chemical transformation
LC-MS HR-LC-MS/MS
Mass-directed preparative HPLC
Results summary II: MS/MS fragmentation interpretation
Accurate mass and MS/MS data are acquired in one run.
•• Expand access to SAR vectors, enhance IP position Work flow of LSF and major analytical challenge
Results archive and sharing by WebMetabase II
Scan 1: Q1 nonresolving, collision energy ramp 20-40 eV, TOF m/z 100-1000, 0.1s scan time, 0.05 s inter-scan delay
Import substrate structure
•• Generate molecular probes (eg, install radiolabels)
XIC summary
Chromatogram
Registration Distribution Bioassays
Results review
Conclusions ● A general UPLC-HR-MS/MS method was
● MS/MS data analysis to propose possible
● Massmetasite was customized to adopt process
● The data analysis cycle, which is the main
developed to support all LSF chemistries
chemical transformations from late stage chemical transformations
structures is conducted in automated fashion bottleneck for the analytical support of LSF chemistry screening, is greatly shortened
● Data analysis results were deposited into
WebMetabase (centralized online searchable database)
● WebMetabase is a knowledge database based on
the chemical transformations stored, which allows prediction of LSF chemistry for new substrates
Copyright © 2015 Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. All Rights Reserved.
liuyng_186316-0001_ASMS_Poster_V1 05/26/2015 ASMA, Output Size: 44”x92” Scale: 200%