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One-Minute NMR Adding Integrated Data Processing to Open-Access, High-Throughput NMR Automation • • • 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 1

Everyone wants a magic wand! You Just Log In Samples e d Di I Everyone wants a magic wand! You Just Log In Samples e d Di I k ma d? l Yie t is it? ha W Leap/CTC PAL Liquid Handler E-Mail Or Web Site One-Minute NMR Manages The Details ACD/Web Librarian ACD/Automation Server Copyright © 2006 by Protasis Corporation, All Rights Reserved Cap. NMR Probe NMR Spectrometer 2

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

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

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

One-Minute NMR Software Web-Based Open Access Sample Queue Management • Secure Log-In • Individual One-Minute NMR Software Web-Based Open Access Sample Queue Management • 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 6 Copyright © 2006 by Protasis Corporation, All Rights Reserved

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

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

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

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

Safe Solvent Management • Vacuum Degasser – Smooth Micro. Flows SYRINGE The Deuterated Solvent Safe Solvent Management • Vacuum Degasser – Smooth Micro. Flows SYRINGE The Deuterated Solvent Conditioning Module mounts conveniently in the door of the Discovery Tower • Helium Sparging / Blanketing NEEDLE V 3 Vacuum Helium – Protect Expensive Deuterated Solvents SAMPLES 12 Copyright © 2006 by Protasis Corporation, All Rights Reserved

High Throughput Sample Loading High Pressure PUMP LOOP WASTE SYRINGE SOLV. A Cap. NMR High Throughput Sample Loading High Pressure PUMP LOOP WASTE SYRINGE SOLV. A Cap. NMR PROBE V 2 INJECT NEEDLE Sample Return Option SAMPLES 13 Copyright © 2006 by Protasis Corporation, All Rights Reserved

Spectrometer Interfaces • Varian – Sun Sockets TCP/IP • Bruker – Windows Mapping – Spectrometer Interfaces • Varian – Sun Sockets TCP/IP • Bruker – Windows Mapping – SGI / Linux Sockets RSH(a. shim. File) RPAR(a. experiment, "all") SETCURDATA XCMD("gpro") RGA STOREPAR("NS", a. scans) STOREPAR("SW", a. sweep. Width) STOREPAR("o 1 p", a. center. Of. Spectrum) exptime = Calc. Exp. Time(); TCP/IP • Others Easy instrument integration Router Copyright © 2006 by Protasis Corporation, All Rights Reserved Drive 14

Challenges of Isolation and Elucidation • Costly Sample Collection • Challenging Sample Matrix • 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 15

Purification-NMR Mismatches • Temporal – HPLC fast, NMR slow • Concentration – HPLC Fractions Purification-NMR Mismatches • Temporal – HPLC fast, NMR slow • Concentration – HPLC Fractions too dilute for good NMR • Solvent – Protonated – Gradients – Salts H 2 O ACN 16

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

Analytical Scale Purifications A Right-sized for Cap. NMR Scrape off a spot into filter. Analytical Scale Purifications A Right-sized for Cap. NMR Scrape off a spot into filter. Add DMSO-d 6, spin. Automatic Dissolution with DMSO-d 6 Collect fractions at analytical scale HPLC 18

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

Precious Sample Analysis Natural Products Dereplication ~ 5 µg/well 700 µg HPLC Courtesy John Precious Sample Analysis Natural Products Dereplication ~ 5 µg/well 700 µg HPLC Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at www. micro. NMR. com) 20

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

Step 1: Chromatography u u Dionex HPLC u Foxy Jr u u DAD ELSD Step 1: Chromatography u u Dionex HPLC u Foxy Jr u u DAD ELSD u Dionex HPLC ISCO Foxy Jr. Fraction Collector 700 ug crude extract from culture of fungus Cladobotryum sp. C 18 column with d-ACN/D 20 gradient with flow of 1 m. L/min Eluant collected in 96 -well conical bottom polypropylene plate Receiving wells changed every 15 seconds (250 u. L/well) Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at www. micro. NMR. com) 22

Step 2: Dry Down u u Evaporation using Jouan RC 10 -22 centrifugal vacuum Step 2: Dry Down u u Evaporation using Jouan RC 10 -22 centrifugal vacuum evaporator Plate enclosed with a piercible silicone/Teflon cover 23

Step 3 a: Sample Reconstitution u u Wells chosen to match peaks on ELSD Step 3 a: Sample Reconstitution u u Wells chosen to match peaks on ELSD data Reconstitution with 16 u. L of methanol-d 4 in each well of interest, followed by mixing step prior to sample injection 24

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

One-Minute NMR™ Open Access, High Throughput NMR Platform Exclusive Attributes • • Secure Open-Access One-Minute NMR™ Open Access, High Throughput NMR Platform Exclusive Attributes • • Secure Open-Access Web Interface Integrated LEAP/CTC Liquid Handler • Safe Solvent Management • High-Throughput Sample Loading and Analysis – Multiple vial/plate sample formats – Degassing, Sparging, Blanketing – Solvent Selection – 1 -minute loading, 1 -scan 1 D proton • Closed-Loop Spectrometer Control • Automated Structure Confirmation • E-Mail reporting – Run -> Idle ->Run – Error handling – Integrated 1 D+2 D ACD/Labs – Automated/adaptive experimental design – HTML Plate Maps – Error Alerts – Forgotten Passwords Copyright © 2006 by Protasis Corporation, All Rights Reserved 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 26

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