Microliter-Volume NMR The Wellplate and Microvial as an Efficient Medium of Sample Transport Among Automated Methods of Analysis

Workflow Diagram for the Natural Product/Metabolite/Medicinal Chemist DAD/ELSD MS or bioactivity NMR vacuum centrifuge 2

Workflow Diagram for the Natural Product/Metabolite/Discovery Chemist Crude Samples/Mixtures Purification High Sensitivity Detection Fraction Collection or Purified Samples LC DAD/ELSD MS or Low Sensitivity Detection bioactivity NMR Prep Stage Report vacuum centrifuge 3

NMR Measurement is Aided by Good Chromatography! What we don’t care about What we care about Mixture X LC Chromatogram Pure Component LC Fraction

Copyright © 2006 by Protasis Corporation, All Rights Reserved Analytical Measurement Challenges Budget Management: Mass, Money, Space, Time LC ELSD GC ICP FT-IR NMR MS UV-VIS DAD Copyright © 2006 by Protasis Corporation, All Rights Reserved

Copyright © 2006 by Protasis Corporation, All Rights Reserved Wellplate-Inclusive Workflow Budget-Effective Utilization of Analytical Instruments LC ELSD ICP GC UV-VIS FT-IR MS 6 NMR Copyright © 2006 by Protasis Corporation, All Rights Reserved

The Advantages of Going Tubeless… Incredible Time Savings No shimming between samples No washing tubes Minimal sample transfer Efficient storage Disposable Economical Don’t underestimate the efficiency and cost savings that can be achieved by switching to standard sample formats. The CapNMR probe flow-cell is effectively a single tube. As long as you use the same solvent shimming and locking is unnecessary between samples. When solvents switch, One-Minute NMR automatically loads the shims for that solvent from a file. For high-throughput applications the burden of transferring samples to tubes is prohibitive. For open-access applications walkup users have to transfer their sample in and out of an NMR tube themselves, and the time required for sample transfer and dissolution is a hidden cost. In many cases samples are already in a vial or plate because they have been submitted for Mass Spec or other analysis. The ability to use these samples directly on your NMR eliminates the wasted effort of tube use. Plates Make It Easy 7 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Protasis One Minute NMR Automation Wellplate Sample Return Stacked Sample Drawers Web-based Software Easy Parameter Setup Microliter Precision Solvent Conditioning Near Zero Dead Volume Wash Bays 8 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Principles of Injection Easy, Effective, Efficient NMR Sample is easily dispensed from syringe to probe Sample Vials Simple Plumbing To learn more about Direction Injection Loading, see the Protasis/MRM Technical Bulletin G0020 in the Tech Support Knowledge Base at www.microNMR.com

Quality Results Start with a Quality Sample Step 1: Add sample (5 – 500 mg) Microfilter Vial Step 4: Discard microfilter Spatula Microfilter Sample Solvent Step 2: Add solvent (5 – 15 mL) Step 5: Place vial on OMNMR Step 3: Spin down (~ 1 min.) 10 Copyright © 2006 by Protasis Corporation, All Rights Reserved

School of Pharmaceutical Sciences, University of Geneva, Switzerland Injection 5 µl Push volume 8 µl Courtesy J.-L. Wolfender and A. Thiocone, Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences, University of Geneva, Switzerland 11

Samples drying by speed-vac Dissolution with 6.5 µl of deuterated solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 12

Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 13

Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 14

Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 15

Analyte Signals Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 16

Analyte Signals Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 17

Sample recovery Sample Solvent HOD MeOD 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY Sample Solvent Sample recovery Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 18

MicroFlow NMR Performance Advantages of Size Scale Sensitivity Economy Simplicity Enhanced Mass Sensitivity Faster… Reliable Robotics Superior Fluidics Efficient Sample & Solvent Utilization Use Less: Dispose of Less  $$ Teflon FEP Flowpaths Simple to Use, Easy to Shim! 19 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Copyright © 2006 by Protasis Corporation, All Rights Reserved CapNMR Performance Get an independent assessment of performance from a major NMR vendor who has used our probe - Download 30-page specifications document at www.jeolusa.com/nmr/nmrprods/cap_probe.html Data Courtesy of Mike Frey, JEOL USA, Peabody, MA USA 20 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Great Sensitivity, Easy Operation, Excellent Spectral Quality 600 MHz 5 min Sensitivity Economy Simplicity 2 hr 6 hr 21 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Metabolite Identification Novatia, LLC, Princeton, NJ or SepNMR HPLC Labconco Centrivap DAD OMNMR ELSD Michrom HPLC Mass Spec 22

Buspirone Metabolites Metabolite Production 1.4 mg of Buspirone in 3mL with HLM Reduce volume (vacuum centrifuge) to 400 uL Method Column: Waters SymmetryShield RP8, 4.6X150 mm Mobile Phases: A: 0.1% TFA in water, B:0.1% TFA in ACN Method (1 mL/min) 0-13min at 4-63%B 13-14 min 88%B 14-20min equilibrate Collect fraction every 30 seconds (500 ul), or SepNMR 23 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Buspirone Metabolites: Single Injection MW 385 MW 401 Parent Absorption (240 nm) 14 24 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Buspirone Metabolite MS-MS 25 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Buspirone Metabolite: 1H NMR 26 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Metabolomics University of Geneva, Switzerland Microfractionation in 96 well plates Metabolomics University of Geneva, Switzerland Split 1:100 Micro- fractionation Large injection 5-50 mg Column 250 x 9mm i.d. High sensitivity Semi-prep Column HPLC split DAD UV MS Microfractionation Triggered by MS Complementary targeted MS experiments Low sensitivity 1D & 2D NMR Bioassays CAP-NMR

Example of a CAP-NMR 1H spectrum 6 8 2’ 3’ 6’ 1’’ 1’’’ 2’’ 3’’ 4’’ 6’’ 2’’’ 3’’’ 4’’’ 6’’’ NT=4 Spectrum recorded in CD3OD No solvent suppression Line width <1Hz Solvent consumption (60 µl) rutin (80 µg) H-1’’’ CH3-6’’’ H-8 H-1’’ H-6 H-2’’ – H-6’’ H-2’’’ – H-5’’’ H-6’ H-3’ H-2’

On-flow LC/NMR sensitivity Swertiamarin (MW 374) On-flow LC/NMR sensitivity 20 µg 16 transients 10 µg 20 µg 40 µg 80 µg 60 µg 80 µg 100 µg 150 µg 150 µg 200 µg 60 ml flow probe 500 MHz 8 7 6 5 4 3 8 7 6 5 4 3

Proton sensitivity of CAP-NMR Swertiamarin (MW 374) Proton sensitivity of CAP-NMR On flow LC/NMR 20 mg NT 16 MeCN:D2O 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 CAP-NMR 20 mg NT 16 CD3OD S/N improved by a factor of 8 same S/N 60 times less NT 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) CAP-NMR 5 mg NT 16 CD3OD

Solvent suppression in CAP-NMR Swertiamarin (MW 374) Solvent suppression in CAP-NMR WET SOLVENT SUPPRESSION 2 mg NT 128 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) water methanol CAP-NMR 2 mg NT 128

2D CAP-NMR capabilities COSY 1 x 512 11 min 2D CAP-NMR capabilities 7 11 4 6 5 3 9 8 1 10 Sweroside 100 mg (MW 358) COSY G-HSQC 8 x 256 1H30 min *10 1 1’ 3’ 5’’ 2’ 4’ *6’ *7 9 5 *6 solvent 1,8 10 7 6a 6b 5, 2’-5’ 6 G-HMBC 64 x 256 6H30 min 3 8 1’ 1 9 5 4 10 2’-5’ 1,8 7 6a 6b 5, 2’-5’ TOTAL ANALSIS TIME 8 HOURS

The model plant: Arabidopsis thaliana Genome Proteome Metabolome Transcriptome

HPLC microfractionation of the enriched flavonoid extract of A HPLC microfractionation of the enriched flavonoid extract of A. thaliana C44 C45 C46 C47 C48 Injection 10 mg Column phenyl (250 x 9 mm i.d.) Flow 2 ml/min Fractions 1 ml/min

LC/UV/MS of the chromatogram used for microfractionation 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 10 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance [M-H]- 739,3 477,5 445,6 740,8 421,6 216,2 741,8 506,6 446,7 921,7 313,3 775,8 609,8 281,3 863,8 593,7 922,9 738,8 253,3 567,7 404,9 387,6 776,8 468,5 364,9 957,7 742,9 670,4 515,2 650,8 832,2 902,0 997,5 956,1 LC/UV/MS of the chromatogram used for microfractionation m/z 739 TIC UV (254nm)

1H Cap-NMR of adjacent microfractions RT: 21,21 - 25,16 SM: 3B 21,5 22,0 22,5 23,0 23,5 24,0 24,5 25,0 Time (min) 2 4 6 8 uAU 20 40 60 80 100 m/z 755 m/z 477 m/z 739 m/z 445 m/z 369 UV trace Single ion traces (LC/ES-MS neg. ions) Each fraction 1ml = 30 sec of peak elution C44 C45 C46 C47

On LC/NMR vs CAP-NMR MW 740 -> agl 286 On flow NMR NT=512 CAP-NMR of fraction C47 NT=16

Cap-NMR gCOSY spectrum of fraction C47 NT=1 NI=512 Time=12 min

Cap-NMR TOCSY spectrum of fraction C47 NT=2 NI=512 Time=24 min

Cap-NMR gHSQC spectrum of fraction C47 3’ 4’ 1’’’’ 2’ 6’’’ 6’’’’ 8 7 9 5’ 1’ 6’ 6’’ 10 1’’’ 6 5 3 1’’ 6’’’ 6’’’ 6’’’’ 6’’’’ Sugars H2’-H5’ solvent *6’’ 1’’’ H1’’’ 1’’’’ H1’’’’ H1’’ 1’’’ H6 H8 6 H3’ 8 6’ H2’ NT=32 NI=256 Time= 5H00 2’

Cap-NMR gHMBC of fraction C47 3’ 4’ 1’’’’ 2’ 6’’’ 6’’’’ 8 7 9 5’ 1’ 6’ 6’’ 10 1’’’ 6 5 3 1’’ 4’’’ 5’’’ 6’’’ 6’’’’ 4’’’’ 5’’’’ 6’’’ Sugars H2-H5 6’’ 6’’’’ 1’’’ H1’’’ H1’’’’ 7 H1’’ H6 10 8 7 H8 7 H3’ 10 6 1’ 1’ 3’ H2’ 1’ 2 NT=256 NI=256 Time= 24H00

Some representative constituents of A Some representative constituents of A. thaliana identified at line by MS and CAP-NMR

Search for stress induced metabolites that triggers defense gene expression Presence of unknown low molecular mass regulators ? Clustered Display of cDNA Microarray Analysis of Gene Expression after Mechanical Wounding Reymond, P., et al. Plant Cell 2000;12:707-720

PCA of total crude extract MS spectra TOF MS spectra CONTROL WOUNDED C12H17O3 209.1256 spiked Total crude extract TOF MS spectra CONTROL RANKED LIST OF INDUCED IONS WOUNDED

UPLC-TOF-ES-MS of A. thaliana CAP-NMR Multiple collection triggered by MS m/z 209.12 (+/- 10 ppm) m/z 209.12 (+/- 0.5 Da) Induced by wounding

CAP-NMR of ion m/z 209 CAP-NMR ca 1 mg NT=5000 12 10 9 7 3,2,8 11,5 4 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm) 12 10 9 7 3,2,8 11,5 4 Standard 50 mg 11 8 5 3 2 1 CAP-NMR ca 1 mg NT=5000

Recently Published: Lambert et al., Anal. Chem. 2007, 79, pp. 727-735. 47

Accelerate Data Acquisition Cornell University < 1 mg crude extract 50 fireflies ~ 200 µg/vial 11 Semi-purified fractions Mass Spec ~ 40 nmol/cmpd 48 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Natural Products Firefly Steroids NMR spectral analysis allowed for complete structural characterization of all major components in each LC fraction, leading to the identification of 13 new steroids dqf-COSY HMQC HMBC NOESY

Recent Publications Cornell University M. Gronquist, J. Meinwald, F. C. Schroeder, JACS 2005, 127, pp. 10810-10811 F.C. Schroeder and M. Gronquist, Angewande Chem. Int. Ed. 2006, 45, pp.7122-7131 50 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI Wellplate-Centric Workflow An Example from the University of Canterbury 100’s µg crude extract Dionex HPLC ISCO Foxy Jr. Fraction Collector C18 column with d-ACN/D20 gradient with flow of 1 mL/min Eluant collected in 96-well conical bottom polypropylene plate Receiving wells changed every 15 seconds (250 mL/well) Daughter plates assayed for activity Dionex HPLC Foxy Jr DAD ELSD ~ 5 µg/well Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at www.microNMR.com)

Workflow Example: Natural Products Dereplication ~ 5 µg/well Evaporation using Jouan RC10-22 centrifugal vacuum evaporator Plate enclosed with a piercible silicone/Teflon cover Mass Spec NMR < 10 mL/well solvent Trace amount injected into Mass Spec Remainder available for additional analyses 52

Workflow Example: Natural Products Dereplication Active Peak(s) Bioactivity profile C18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection 53 Copyright © 2006 by Protasis Corporation, All Rights Reserved

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 54 Copyright © 2006 by Protasis Corporation, All Rights Reserved

only 1 hit

Copyright © 2006 by Protasis Corporation, All Rights Reserved Workflow Example: Natural Products Dereplication Informed Decision-Making 500 MHz (2 min) 30 mg in 6 mL CD3OD Unknown Peptide, MW=684 Rapid 1D NMR enables real-time decision-making Potentially novel compounds can be further analyzed using 2D experiments TOCSY ? 56 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Copyright © 2006 by Protasis Corporation, All Rights Reserved 57 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Copyright © 2006 by Protasis Corporation, All Rights Reserved Protasis Capillary NMR Enhanced productivity and Accelerated Decision-Making Bring more “axes of information” to decision-making up-front Incorporate NMR earlier, maximize information-per-unit-time NMR MS ELSD UV FT-IR Answer NMR vs. 58 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Enhancing Your NMR Investment Get the most from your instruments Easy Open Access Web login for vials and plates reduces time spent in the lab. Automated System Suitability and Maintenance provides assurance of good spectra. Microgram Samples avoids return trips to the lab and enables scale-down upstream in your workflow. Plate and Vial Submission simplifies reformatting and sample preparation. Closed-Loop Instrument Control enhances NMR error reporting Integrated ACD/Labs and One-Moon Scientific back-end software eliminates manual processing steps. 59 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Everyone wants a magic wand! Yield? What is it? Did I make it? You Simply Log In Samples Leap/CTC PAL Liquid Handler Information Flow E-Mail Or Web Site One-Minute NMR Manages The Details Some scientists have a passion for NMR spectroscopy, but most people just want the answers it provides. The people who need the answers would rather use a magic wand if they knew the answers were correct. By combining your NMR spectrometer with efficient sample management, intelligent instrument control, and the latest post-processing software, researchers get the answers they need in minutes. Let us show you how the One-Minute NMR system manages everything from sample loading to concise reporting of structure confirmation results in a way that can easily fit your budget. CapNMR Probe NMR Spectrometer ACD/Web Librarian ACD/Automation Server 60 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Sample Log-in Use CapNMR

Walk away… Let One-Minute NMR run your sample

Copyright © 2006 by Protasis Corporation, All Rights Reserved Seconds Later… Click… In your InBox Your magnet isn’t a magic wand, but the information flows the same way. As soon as all your samples are complete One-Minute NMR sends an HTML- formatted e-mail displaying a plate map and tabulated results for each experiment with different symbols for confirmed and rejected samples. No other system provides this level of integration and ease of use. Our e-mail and web-based system sends an HTML link to the ACD data-based and interpreted results directly to you, so you can have the information not only when you need it, but where you need it, and in the most concise format available. Automated structure confirmation provides incredible value by shortening or eliminating the time lost to processing NMR data, interpreting spectra, storing results, and formatting data for print or publication. Our integration with ACD/Labs software is unique. By choosing to work cooperatively, Protasis, ACD/Labs, and our collaborators in industry, are forcing a revolution in NMR by bringing the power of its information to everyone who needs it. Hot-linked E-mail report Integrated ACD/Web Librarian 63 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Copyright © 2006 by Protasis Corporation, All Rights Reserved System Maintenance System Suitability Check Certified and Sealed standard solutions in your solvent Run before every Nth sample or at startup Checks Signal / Noise and Line Shape to assure good results. Stops if out of spec. 67 67 Copyright © 2006 by Protasis Corporation, All Rights Reserved 67

Copyright © 2006 by Protasis Corporation, All Rights Reserved System Maintenance Probe Clean Can be executed before, after, or during a scheduled run. Injects a designated cleaning solution to ensure that flowpath walls are clear. Programmable to accommodate a wide range of sample conditions. Solvent Rinse Similar to Probe Clean, system can be programmed to inject additional solvent for flowpath rinsing when using problematic samples. 68 68 Copyright © 2006 by Protasis Corporation, All Rights Reserved 68

Copyright © 2006 by Protasis Corporation, All Rights Reserved System Maintenance Reverse Rinse Provides broad filter face for elimination of particulates Collects particulates during injection Eliminates particulates during reverse rinse ……….. injection Injection port Particulates collect on filter face needle filtered sample raw …. reverse rinse Particulates removed from filter face Clean solvent flushed to waste 69 69 Copyright © 2006 by Protasis Corporation, All Rights Reserved 69

High Pressure Transducer System Maintenance Pressure Monitoring / Pressure History Sensor monitors backpressure during sample injection Provides indication of proper system function Identifies leaks quickly Identifies blockages and shuts system down safely Indicates advance need for filter change High Pressure Transducer Pressure vs. Time Trace Copyright © 2006 by Protasis Corporation, All Rights Reserved 70 70 70 70

Copyright © 2006 by Protasis Corporation, All Rights Reserved System Maintenance Solvent Management Vacuum Degasser Helium Sparging / Blanketing Protects valuable deuterated solvents from atmospheric contamination 71 71 71 Copyright © 2006 by Protasis Corporation, All Rights Reserved 71

Copyright © 2006 by Protasis Corporation, All Rights Reserved System Maintenance Sample Management Temperature -controlled drawer stacks can handle shallow or deep-well plates or vials Plate covers and vial caps maintain dry samples that are free from atmospheric contamination 72 72 72 Copyright © 2006 by Protasis Corporation, All Rights Reserved 72

Current One-Minute NMR Installations 22 Systems Total 17 Varian 5 Bruker 1 Custom Automated Structure Confirmation Systems (VNMR) 2 Turn-Key Automated Structure Confirmation Systems (VNMR/VNMRJ) 3 Walkup Open Access Systems Customer overview 73 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Protasis Corporation For More Information, Visit: Sensitivity Economy Simplicity Protasis Corporation For More Information, Visit: www.protasis.com (product details) www.microNMR.com (technical support) 600MHz capillary NMR, ns,64 10 g/10  L Graphics on this page complements of: Mark O’Neil-Johnson, Sequoia Sciences Wolfgang Peti, Brown University Brad Bendiak, U. Colorado Health Sciences Center Dave Detlefsen, Novatia Jane Strouse, UCLA Kirk Gustafson, NCI and John Blunt, U. Canterbury Protasis technology has been advanced though the support of: NIH RR14392, RR11755, HL56546, RR14661, RR16387, EB001626-01, RR15384, RR020239, NSF DMI-9523220