1. 1 One-Minute NMR Adding Integrated Data Processing to Open-Access, High-Throughput NMR Automation Easy Fast Efficient Economical Controllable Flexible Find the answers to… “What is this stuff? How much is there?” Copyright © 2006 by Protasis Corporation, All Rights Reserved

2. 2 What is it? Everyone wants a magic wand! Leap/CTC PAL Liquid Handler CapNMR Probe NMR Spectrometer ACD/Automation Server E-Mail Or Web Site You Just Log In Samples One-Minute NMR Manages The Details ACD/Web Librarian Did I make it? Yield? Copyright © 2006 by Protasis Corporation, All Rights Reserved

3. 3 The One-Minute NMR System Optimized Liquid Handling Application-Tuned CapNMR Probe Exclusive Spectrometer Interfaces Robust Software Collaboration With Partners and End- Users Copyright © 2006 by Protasis Corporation, All Rights Reserved

4. 4 MRM CapNMR TM Microcoil Probes 5-10 microliter flow cell = 8 – 15 microliter samples Best mass sensitivity Short acquisition times Concentration > 1mM works great (10 nanomoles in 10µL) Use CapNM R Copyright © 2006 by Protasis Corporation, All Rights Reserved

5. 5 One-Minute NMR Provides… Queue Management –Experiments, Structures, Fraction List Import Sample Loading –Vials and Plates, 5 & 10 microliter NMR Detection –Super-sensitive CapNMR Probe NMR Control –Adaptive Experiments Confirmation –ACD/Labs 1D + 2D Structure Verification Bonus: Quantitation –ERETIC Copyright © 2006 by Protasis Corporation, All Rights Reserved

6. 6 One-Minute NMR Software Secure Log-In Individual sample entry and Molecule upload.ZIP/.CSV Sample lists and molecule upload Bar-code support Open SQL Server Custom Fields LDAP Login Web-Based Open Access Sample Queue Management Copyright © 2006 by Protasis Corporation, All Rights Reserved

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8. 8 Click… Minutes Later… In your InBox Hot-linked E-mail report Integrated ACD/Web Librarian Copyright © 2006 by Protasis Corporation, All Rights Reserved

9. 9 Adaptive Experiments Structure Confirmation, Elucidation Coming… 1D Proton >= 1D Proton Acceptance Level HSQC ConfirmedRejected >= AVG(HSQC + 1H1D) Acceptance Level YES NO Copyright © 2006 by Protasis Corporation, All Rights Reserved

10. 10 LEAP PAL Fast Low Volume Pickup Low Dead Volume Syringe-Only Technology Flexible Sample Storage Dilute / Reconstitute Sample Return Copyright © 2006 by Protasis Corporation, All Rights Reserved

11. 11 The advantages of going tubeless… No shimming between samples No washing tubes Minimal sample transfer Efficient storage Disposable Economical Incredible Time Savings Copyright © 2006 by Protasis Corporation, All Rights Reserved

12. 12 Safe Solvent Management Vacuum Degasser –Smooth MicroFlows Helium Sparging / Blanketing –Protect Expensive Deuterated Solvents Helium NEEDLE V3 SAMPLES SYRINGE The Deuterated Solvent Conditioning Module mounts conveniently in the door of the Discovery Tower Vacuum Copyright © 2006 by Protasis Corporation, All Rights Reserved

13. 13 SOLV. A WASTE INJECT V2 LOOP SAMPLES NEEDLE SYRINGE High Pressure PUMP Copyright © 2006 by Protasis Corporation, All Rights Reserved CapNMR PROBE High Throughput Sample Loading Sample Return Option

14. 14 Varian –SunTCP/IP Sockets Bruker –WindowsDrive Mapping –SGI / LinuxTCP/IP Sockets Others Easy instrument integration Spectrometer Interfaces Copyright © 2006 by Protasis Corporation, All Rights Reserved Router RSH(a.shimFile) RPAR(a.experiment, "all") SETCURDATA XCMD("gpro") RGA STOREPAR("NS", a.scans) STOREPAR("SW", a.sweepWidth) STOREPAR("o1p", a.centerOfSpectrum) exptime = CalcExpTime();

15. 15 Challenges of Isolation and Elucidation Costly Sample Collection Challenging Sample Matrix Unknown and Complex Chemical Structures Quantification of Unknowns Difficult Purification-NMR Mismatches Complex NMR Spectra

16. 16 Purification-NMR Mismatches Temporal –HPLC fast, NMR slow Concentration –HPLC Fractions too dilute for good NMR Solvent –Protonated –Gradients –Salts H2O ACN

17. 17 The Smart Solution To Purification-NMR Mismatches Collect Fractions Put Dry Samples On One-Minute NMR Large Capacity Time-Optimized HPLC No Loop Monster No SPE Concentrator Concentrated Sample No Protonated Solvent Some salt OK Highest Mass S/N Automatic Dissolution No Solvent Supression No Shimming Evaporate HPLC Solvent

18. 18 Analytical Scale Purifications Right-sized for CapNMR Scrape off a spot into filter. Add DMSO-d6, spin. Collect fractions at analytical scale HPLC Automatic Dissolution with DMSO-d6 A

19. 19 Actual Use Case Academic Laboratory Precious Sample Analysis Natural Product De-replication

20. 20 Precious Sample Analysis Natural Products Dereplication HPLC 700 µg ~ 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)

21. 21 Precious Sample Analysis Well plate/Vial – centric Approach Dionex HPLC Foxy Jr DAD ELSD Other Optional Analytical Detectors (e.g. Mass Spec.) 1 2 3 4

22. 22 Step 1: Chromatography Dionex HPLC Foxy Jr DAD ELSD u Dionex HPLC u ISCO Foxy Jr. Fraction Collector u 700 ug crude extract from culture of fungus Cladobotryum sp. u C18 column with d-ACN/D20 gradient with flow of 1mL/min u Eluant collected in 96-well conical bottom polypropylene plate u Receiving wells changed every 15 seconds (250uL/well) Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at www.microNMR.com)

23. 23 Step 2: Dry Down u Evaporation using Jouan RC10-22 centrifugal vacuum evaporator u Plate enclosed with a piercible silicone/Teflon cover

24. 24 Step 3a: Sample Reconstitution u Wells chosen to match peaks on ELSD data u Reconstitution with 16uL of methanol- d4 in each well of interest, followed by mixing step prior to sample injection

25. 25 Step 3b: NMR Data Acquisition u 600MHz NMR u ICG 10uL CapNMR Probe u 15uL Sample Loop

26. 26 One-Minute NMR™ Open Access, High Throughput NMR Platform Secure Open-Access Web Interface Integrated LEAP/CTC Liquid Handler –Multiple vial/plate sample formats Safe Solvent Management –Degassing, Sparging, Blanketing –Solvent Selection High-Throughput Sample Loading and Analysis –1-minute loading, 1-scan 1D proton Closed-Loop Spectrometer Control –Run -> Idle ->Run –Error handling Automated Structure Confirmation –Integrated 1D+2D ACD/Labs –Automated/adaptive experimental design E-Mail reporting –HTML Plate Maps –Error Alerts –Forgotten Passwords Exclusive Attributes Productivity Features Priority Samples Night Samples Probe Clean Solvent Select Sample Return Sample List Importing Structure Confirmation Custom Counters Custom Fields -> NMR Dataset Parameters SQL Database Pressure Monitoring Macros Dilution Reverse Rinse Automatic instrument reboot Copyright © 2006 by Protasis Corporation, All Rights Reserved

27. 27 Help spread the word! www.protasis.com www.protasis.com 508-481-4163 Now NMR is easier and faster One-Minute NMR