Building High Throughput Natural Products Libraries LC-MS+NMR Building High Throughput Natural Products Libraries Bob Albrecht Protasis Corporation

LC / Mass Spectrometry Alone First choice for compound identification Very sensitive and fast, but… Not enough information to elucidate novel compounds Often ambiguous Ionization problems complicate identification Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

NMR on LC-MS Samples (No Fraction Collection) Interesting? Sample Returned LC/MS NMR Easy for very-high-purity compounds Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Isolation, De-Replication, Elucidation Extract / Prepare Sample Analytical LC/MS With Fraction Collection De-replicate with MS Exchange solvent, concentrate Run 1D NMR on unidentified or ambiguous fractions De-replicate with 1D NMR NMR Structure Elucidation Suite (HMBC, 13C, 15N, …) ACD/Labs Structure Elucidator Helps Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

CapNMR From Purified Fractions Simple No tricky SPE method No stop-flow No HPLC solvent suppression No re-shimming Inexpensive No interferences = easier elucidation Complete NMR elucidation enabled by having more time Better match for de-replication libraries with NMR in deuterated solvents Best NMR Sensitivity vs. online LC-NMR or LC-SPE-NMR Largest fraction capacity vs. LC-SPE-NMR Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved LC-MS+NMR HPLC Column MS HPLC CapNMR H11D, HMBC, 13C. 15N, … Fraction Collector Auto-sampler Splitter UV ACD/Labs Structure Elucidator Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Micro-plate Fraction Collection CapNMR is designed for analytical-scale separations. Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved MS isn’t enough for de novo structure determination, but… CapNMR Has The Power MS + 1D Proton NMR in deuterated solvent is often enough for de-replication CapNMR enables full structure elucidation of unknowns 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY, ROESY, NOESY without changing probes! One-Minute NMR keeps the system running non-stop Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Chromatographic Purification Strategies Time / Volume UV MS MS + UV Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Time / Volume Directed Purification Fractions can contain 2+ compounds Less mass of purified material Many fractions Collects everything Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

UV-Directed Purification More pure compound Few impure fractions Too many worthless fractions No chromaphore, no fraction Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

MS-Directed Purification Pure fractions MS picks valuable fractions “Worthless” fractions may actually have value Poor ionization can waste valuable fractions Unknown mass range a problem Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved MS + UV Purification Poor ionization, fractions still collected No chromaphore, fractions still colleted Mass range can screen-out unlikely hits NMR on fractions with interesting MS spectra Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Workflow Example: Natural Products Dereplication Active Peak(s) Bioactivity profile C18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection Copyright (c) 2008 by Protasis Corporation, All Rights Reserved 14 14

Recognisable features: 5 CH3 groups, of which 1 methoxyl, 500 MHz 8 mg in 6 mL CD3OD PRESAT 1.5 min Recognisable features: 5 CH3 groups, of which 1 methoxyl, 3 singlet CH3-C, 1 doublet CH3-C; 1 aldehyde Copyright (c) 2008 by Protasis Corporation, All Rights Reserved 15 15

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Purification Scale UPLC™ (2.1x150mm 1.7µ) < ~2 mg sample, plenty for MS (.1µg) + CapNMR (50µg) Amazing chromatographic resolution highest purity fractions CapNMR on 2.5% of total fraction mass Analytical – (4.6x150mm 3-5µ) < ~7mg sample Good chromatographic resolution CapNMR on 0.7% of total fraction mass Semi-Prep 50mg to grams of sample Longer runs Less resolution, more impure fractions Typical for compound libraries CapNMR on 0.1% of total fraction mass Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Why Scale Down Purification + NMR? Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Is This Your Next Natural Products Expedition? Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Use One-Minute NMR to Do more with less sample Solve structures with minimum mass Most samples are not hits Only scale- up when needed Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Chromatographic Resolution Matters There are three compounds under this peak. The MS sees them, but you will not get the purity you need for NMR elucidation. Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Why not scale-down purification? It Is a Trade-Off You still need to scale-down NMR to Elucidate the 0.1% fraction. Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Equipment One-Minute NMR Automation Integrates: NMR with CapNMR Probe LC-MS ACD/Labs Structure Elucidation and Databasing Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved One-Minute NMR Interfaces with: People (Web, e-mail) NMR LC-MS ACD/Labs OneMoon Custom Software Manages Samples from Plates and Vials Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

One-Minute NMR Software Web / E-Mail enabled Import Fraction Lists Run NMR Experiments Maintain NMR Quality Archive and Process Data Customizable Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

CapNMR Specifically Designed for NMR of LC Peaks Excellent NMR Results from: 20 – 200µg samples 0.1% fraction for semi-prep 0.7% fraction for analytical scale 2.5% fraction for UPLC CapNMR TXI Runs all of your NMR experiments for structure elucidation without changing the probe! 1H, 13C, 15N, 1D, 2D: APT, INEPT, DEPT, NOE, COSY, RELAY, DQFCOSY, TQCOSY, TOCSY, HOMJ, NOESY, ROESY, INADEQUATE, PFG, gCOSY, gDQFCOS, HETCOR, HMQC, HSQC, HMBC, gHMBC, gHMQC, gHSQC… Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Dual-flow-cell CapNMR Quickly de-replicate with 1H1D Elucidate unknown structures with 13C, NOESY, gHMBC, gHSQC Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Agilent 6100 Series Quadrupole LC/MS System 1200 Series Fraction Collector AS > AS = Analytical Scale Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Waters AutoPurification LC(/MS) Semi-Prep Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved SFC Normal Phase Chromatography with CO2 solvent Fractions auto-concentrate Semi-prep scale available TharSFC has a high quality system Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Fraction Concentration Vacuum Centrifuge Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Don’t Forget Quantitation You can only know how active your compound is if you know how much you really have CapNMR of purified fractions is ideal for quantitation One-Minute NMR Is the Ultimate System For Quantitation No tuning between tubes = no recalibration with standard Consistent robotic injection ERETIC signal for a non-interfering internal standard Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Isolated Model Compound NMR 5 Model Compound Mixture CapNMR at 600 MHz 64 Scans (4 minutes) 5 ug of 5 components Isolated proparacain CapNMR at 600 MHz 16 Scans (1 minute) 5 ug of 1 component on column Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Data Courtesy Novatia, LLC

Online LC-NMR vs. Fractionation + CapNMR On flow NMR NT=512 CAP-NMR of fraction C47 NT=16 MW 740 -> agl 286 Data Courtesy of Jean-Luc Wolfender Metabolomics University of Geneva, Switzerland Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

LC/UV/MS of the chromatogram used for micro-fractionation exfl10mg # 676-690 RT: 22,87-23,34 AV: 15 SB: 77 21,55-22,66, 23,61-25,02 NL: 5,92E5 F: - c ESI Full ms [ 140,01-1000,02] 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z 5 [M-H]- 739,3 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance m/z 739 477,5 445,6 740,8 216,2 421,6 TIC 446,7 506,6 741,8 253,3 281,3 313,3 609,8 775,8 921,7 364,9 387,6 404,9 468,5 515,2 567,7 593,7 650,8 670,4 738,8 742,9 776,8 832,2 863,8 902,0 956,1 922,9 957,7 997,5 Data Courtesy of Jean-Luc Wolfender Metabolomics University of Geneva, Switzerland UV (254nm) Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Cap-NMR gCOSY spectrum of fraction C47 NT=1 NI=512 Time=12 min Data Courtesy of Jean-Luc Wolfender Metabolomics University of Geneva, Switzerland Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

LC-MS+CapNMR for Natural Products is: Higher performance analytical scale chromatography Fast online MS detection, fractionation, and de-replication Maximum performance CapNMR to de-replicate and elucidate de novo structures Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Further Reading J Biomol Tech. 2003 June; 14(2): 136–142. PMCID: Mass-Based Fraction Collection of Crude Synthetic Peptides in Analytical and Preparative Scale The importance of hyphenated techniques in the discovery of new lead compounds from nature Jean-Luc Wolfender, Emerson Ferreira Queiroz, Kurt Hostettmann Expert Opinion on Drug Discovery, August 2006, Vol. 1, No. 3, Pages 237-260 (doi: 10.1517/17460441.1.3.237 ASMS 2008 "Evaluation of Column Retentivity with large injection volume for High Sensitive and High-Throuput LC-MS/MS Quantitative Analysis." J. Watanabe, et. al. Takara Bio – Japan   Copyright (c) 2008 by Protasis Corporation, All Rights Reserved

Copyright (c) 2008 by Protasis Corporation, All Rights Reserved Thanks Jean-Luc Wolfender, University of Geneva David Detlefsen, Novatia Get your Natural Products LC-MS+CapNMR system from Protasis Corporation. http://protasis.com 508-481-4163 Copyright (c) 2008 by Protasis Corporation, All Rights Reserved