LC-MS Plus NMR Building And Confirming Modern Molecular 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 verification  Ionization problems complicate identification

CapNMR From Purified Fractions  Simple –No tricky SPE method or LC solvents –No stop-flow –No HPLC solvent suppression –No re-shimming  Inexpensive  No interferences = easier elucidation  Best NMR Sensitivity vs. online LC- NMR or LC-SPE-NMR  Largest fraction capacity vs. LC- SPE-NMR (384+)

Auto-sampler HPLC Column Splitter MS Fraction Collector UV CapNMR H11D, HMBC, 13C. 15N, … LC-MS+NMR ACD/Labs Structure Elucidator HPLC

Micro-plate Fraction Collection CapNMR is designed for analytical-scale separations.

MS isn’t enough for novel structure determination, but… CapNMR Has The Power  MS + 1D Proton NMR in deuterated solvent is often enough for verification, but when it isn’t…  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

Chromatographic Purification Strategies  Time / Volume  UV  MS  MS + UV

Time / Volume Directed Purification Fractions can contain 2+ compounds Less mass of purified material Many fractions Collects everything

UV-Directed Purification More pure compound Few impure fractions Too many worthless fractions No chromaphore, no fraction

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

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

C 18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection Bioactivity profile Workflow Example: Natural Products Dereplication Active Peak(s) John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND 12

500 MHz 8  g in 6  L CD 3 OD PRESAT 1.5 min Recognisable features: 5 CH 3 groups, of which 1 methoxyl, 3 singlet CH 3 -C, 1 doublet CH 3 -C; 1 aldehyde 13 John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND

Purification Scale  UPLC™ (2.1x150mm 1.7 µ ) –< ~2 mg sample, plenty for MS (.1 µ g) + CapNMR (50 µ g) –Amazing chromatographic resolution –highest purity fractions –CapNMR on 2.5% of total fraction mass  Analytical – (4.6x150mm 3-5 µ ) –< ~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

Purification Scale-Down Saves Time and Money t ≈ $ ∆t + ∆$ = ∆productivity

Analytical Scale 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.

But… Trade-Off With Sensitivity You still need to scale-down NMR to Elucidate the 0.1% fraction. A second analytical run resolves a poorly resolved semi-prep fraction.

One-Minute NMR Software Web / enabled Import Fraction Lists Run NMR Experiments Maintain NMR Quality Archive and Process Data Customizable

One-Minute NMR Manages Samples from Plates and Vials Interfaces with: People (Web, ) NMR LC-MS ACD/Labs OneMoon Custom Software Interfaces with: People (Web, ) NMR LC-MS ACD/Labs OneMoon Custom Software

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… Dual flow cell now available!

Isolated Model Compound NMR  5 Model Compound Mixture –CapNMR at 600 MHz –64 Scans (4 minutes) –5 ug of 5 components Isolated proparacain Isolated proparacain CapNMR at 600 MHz CapNMR at 600 MHz 16 Scans (1 minute) 16 Scans (1 minute) 5 ug of 1 component on column 5 ug of 1 component on column 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

LC/UV/MS of the chromatogram used for micro-fractionation [M-H] - 739,3 477,5 445,6 740,8 421,6 216,2 741,8 506,6 446,7 921,7 313,3 775,8609,8 281,3 863,8593,7 922,9 738,8 253,3567,7 404,9387,6 776,8 468,5 364,9 957,7742,9 670,4 515,2650,8 832,2 902,0 997,5956,1 m/z 739 TIC UV (254nm) Data Courtesy of Jean-Luc Wolfender Metabolomics University of Geneva, Switzerland

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

Thanks   John W Blunt, Department of Chemistry, University of Canterbury, Christchurch NEW ZEALAND   Jean-Luc Wolfender, University of Geneva   David Detlefsen, Novatia Protasis Corporation