lc-ms+nmr building high throughput natural products libraries bob albrecht protasis corporation

LC-MS+NMRLC-MS+NMR

Building High Throughput Building High Throughput Natural Products LibrariesNatural Products Libraries

Bob AlbrechtProtasis Corporation

Copyright (c) 2008 by Protasis CorCopyright (c) 2008 by Protasis Corporation, All Rights Reservedporation, All Rights Reserved

22

LC / Mass Spectrometry AloneLC / Mass Spectrometry Alone

First choice for compound First choice for compound identificationidentification

Very sensitive and fast, but…Very sensitive and fast, but… Not enough information to elucidate Not enough information to elucidate

novel compoundsnovel compounds Often ambiguous Often ambiguous Ionization problems complicate Ionization problems complicate

identificationidentification


33

NMR on LC-MS SamplesNMR on LC-MS Samples(No Fraction Collection)(No Fraction Collection)

LC/MSLC/MS Sample ReturnedSample Returned

Interesting?

NMRNMR

Easy for very-high-purity compoundsEasy for very-high-purity compounds


44

Isolation, De-Replication, ElucidationIsolation, De-Replication, Elucidation

1.1. Extract / Prepare SampleExtract / Prepare Sample2.2. Analytical LC/MS With Fraction CollectionAnalytical LC/MS With Fraction Collection3.3. De-replicate with MSDe-replicate with MS4.4. Exchange solvent, concentrateExchange solvent, concentrate5.5. Run 1D NMR on unidentified or Run 1D NMR on unidentified or

ambiguous fractionsambiguous fractions6.6. De-replicate with 1D NMRDe-replicate with 1D NMR7.7. NMR Structure Elucidation Suite (HMBC, NMR Structure Elucidation Suite (HMBC,

13C, 15N, …) 13C, 15N, …) 8.8. ACD/Labs Structure Elucidator HelpsACD/Labs Structure Elucidator Helps


55

CapNMR From Purified FractionsCapNMR From Purified Fractions SimpleSimple

– No tricky SPE methodNo tricky SPE method– No stop-flowNo stop-flow– No HPLC solvent suppression No HPLC solvent suppression – No re-shimmingNo re-shimming

InexpensiveInexpensive No interferencesNo interferences = easier elucidation = easier elucidation Complete NMR elucidationComplete NMR elucidation enabled by enabled by

having more timehaving more time Better match for de-replicationBetter match for de-replication libraries libraries

withwith NMR in deuterated solventsNMR in deuterated solvents Best NMR SensitivityBest NMR Sensitivity vs. online LC-NMR or vs. online LC-NMR or

LC-SPE-NMRLC-SPE-NMR Largest fraction capacityLargest fraction capacity vs. LC-SPE-NMR vs. LC-SPE-NMR


66

Auto-sampler

HPLC Column

Splitter

MS

FractionCollectorUV

CapNMR

H11D, HMBC, 13C. 15N, …

LC-MS+NMRLC-MS+NMR

ACD/LabsStructure Elucidator

HPLC


77

Micro-plateMicro-plateFraction CollectionFraction Collection

CapNMR is designed foranalytical-scale separations.CapNMR is designed foranalytical-scale separations.


88

MS isn’t enough for MS isn’t enough for de novode novo structure structure

determination, but…determination, but… CapNMR Has The PowerCapNMR Has The Power

MS + 1D Proton NMR in deuterated solvent MS + 1D Proton NMR in deuterated solvent is often enough for de-replicationis often enough for de-replication

CapNMR enables full structure elucidation CapNMR enables full structure elucidation of unknowns of unknowns – 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY, 13C, 15N, HSQC, HMBC, HMQC, COSY, TOCSY,

ROESY, NOESY without changing probes!ROESY, NOESY without changing probes! One-Minute NMR keeps the system One-Minute NMR keeps the system

running non-stoprunning non-stop


99

Chromatographic Purification Chromatographic Purification StrategiesStrategies

Time / VolumeTime / Volume UVUV MSMS MS + UVMS + UV


1010

Time / Volume Directed PurificationTime / Volume Directed Purification

1.0 2.0 3.0 4.0Time (min)

• Fractions can contain 2+ compounds• Less mass of purified material• Many fractions• Collects everything

9.0 9.2 9.4 9.6 9.8 10.0 10.2Time (min)


1111

UV-Directed PurificationUV-Directed Purification

0.70 0.80 0.90 1.00 1.10Time (min)

• More pure compound• Few impure fractions• Too many worthless fractions• No chromaphore, no fraction


1212

MS-Directed PurificationMS-Directed Purification

0.70 0.80 0.90 1.00 1.10Time (min)• Pure fractions

• MS picks valuable fractions• “Worthless” fractions may actually have value• Poor ionization can waste valuable fractions• Unknown mass range a problem


1313

MS + UV PurificationMS + UV Purification

0.70 0.80 0.90 1.00 1.10Time (min)

• Poor ionization, fractions still collected• No chromaphore, fractions still colleted• Mass range can screen-out unlikely hits• NMR on fractions with interesting MS spectra

• Poor ionization, fractions still collected• No chromaphore, fractions still colleted• Mass range can screen-out unlikely hits• NMR on fractions with interesting MS spectra


1414

C18 HPLC of extract frommarine-derived Cephalosporium sp.

DAD detection

Bioactivity profile

Workflow Example: Natural Products DereplicationActive Peak(s)

14


1515

500 MHz8 g in 6 L CD3ODPRESAT 1.5 min

Recognisable features: 5 CH3 groups, of which 1 methoxyl,3 singlet CH3-C, 1 doublet CH3-C; 1 aldehyde

15


1616

Purification ScalePurification Scale UPLC™UPLC™ (2.1x150mm 1.7 (2.1x150mm 1.7µµ) )

– < ~2 mg sample, plenty for MS (.1< ~2 mg sample, plenty for MS (.1µµg) + CapNMR (50g) + CapNMR (50µµg)g)– Amazing chromatographic resolutionAmazing chromatographic resolution– highest purity fractionshighest purity fractions– CapNMR on CapNMR on 2.5%2.5% of total fraction mass of total fraction mass

AnalyticalAnalytical – (4.6x150mm 3-5 – (4.6x150mm 3-5µµ))– < ~7mg sample< ~7mg sample– Good chromatographic resolutionGood chromatographic resolution– CapNMR on CapNMR on 0.7%0.7% of total fraction mass of total fraction mass

Semi-PrepSemi-Prep– 50mg to grams of sample50mg to grams of sample– Longer runsLonger runs– Less resolution, more impure fractionsLess resolution, more impure fractions– Typical for compound librariesTypical for compound libraries– CapNMR on CapNMR on 0.1%0.1% of total fraction mass of total fraction mass


1717

Why Scale DownWhy Scale DownPurification + NMR?Purification + NMR?


1818

Is This Your NextIs This Your NextNatural Products Expedition?Natural Products Expedition?


1919

Use One-Minute NMR to Use One-Minute NMR to Do more with less sampleDo more with less sample

Solve Solve structures structures with with minimum minimum massmass

Most Most samples samples are not hitsare not hits

Only scale- Only scale- up when up when neededneeded

Fixed-Budget Throughput vs. Sample Mass

UPLC

Analyt

ical

Semi-P

rep

Sample MassCost / SampleThroughput


2020

Chromatographic Resolution Chromatographic Resolution MattersMatters

3.70 3.80 3.90 4.00 4.10 4.20 4.30Time (min)

There are three compounds under this peak.The MS sees them, but you will not getthe purity you need for NMR elucidation.

There are three compounds under this peak.The MS sees them, but you will not getthe purity you need for NMR elucidation.


2121

Why notWhy not scale-down purification? scale-down purification?It Is a Trade-OffIt Is a Trade-Off

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Time (min)

Next Blockbuster Drug (50ug)

You still need to scale-down NMR toElucidate the 0.1% fraction.You still need to scale-down NMR toElucidate the 0.1% fraction.


2222

EquipmentEquipment

One-Minute NMR Automation One-Minute NMR Automation Integrates:Integrates:– NMR with CapNMR ProbeNMR with CapNMR Probe– LC-MSLC-MS– ACD/Labs Structure Elucidation and ACD/Labs Structure Elucidation and

DatabasingDatabasing


2323

One-Minute NMROne-Minute NMRManages Samples

fromPlates and Vials

Interfaces with:• People (Web, e-mail)• NMR • LC-MS• ACD/Labs• OneMoon• Custom

Software

Interfaces with:• People (Web, e-mail)• NMR • LC-MS• ACD/Labs• OneMoon• Custom

Software


2424

One-Minute NMR SoftwareOne-Minute NMR Software• Web / E-Mail enabled

• Import Fraction Lists

• Run NMR Experiments

• Maintain NMR Quality

• Archive and Process Data

• Customizable


2525

CapNMRCapNMRSpecifically Designed for NMR of LC PeaksSpecifically 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

Excellent NMR Results from:

• 20 – 200µg samples

• 0.1% fraction for semi-prep

• 0.7% fraction for analytical scale

• 2.5% fraction for UPLCCapNMR TXI Runs all of your NMRexperiments for structure elucidationwithout 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…


2626

Dual-flow-cell CapNMRDual-flow-cell CapNMR

1.1. Quickly de-replicate with Quickly de-replicate with 1H1D1H1D

2.2. Elucidate unknown Elucidate unknown structures with 13C, structures with 13C, NOESY, gHMBC, gHSQCNOESY, gHMBC, gHSQC


2727

1200 Series Fraction Collector AS >

6100 Series Quadrupole LC/MS System

Agilent

AS = Analytical Scale


2828

Waters AutoPurification LC(/MS)

Semi-PrepSemi-Prep


2929

SFCSFC Normal Phase Chromatography with CO2 Normal Phase Chromatography with CO2

solventsolvent Fractions auto-concentrate Fractions auto-concentrate Semi-prep scale availableSemi-prep scale available TharSFC has a high quality systemTharSFC has a high quality system


3030

Fraction ConcentrationFraction Concentration

Vacuum Centrifuge


3131

Don’t Forget QuantitationDon’t Forget Quantitation You can only know how active your You can only know how active your

compound is if you know how much you compound is if you know how much you reallyreally have have

CapNMR of purified fractions is ideal for CapNMR of purified fractions is ideal for quantitationquantitation

One-Minute NMR Is the Ultimate System One-Minute NMR Is the Ultimate System For QuantitationFor Quantitation– No tuning between tubes = no recalibration No tuning between tubes = no recalibration

with standardwith standard– Consistent robotic injectionConsistent robotic injection– ERETIC signal for a non-interfering internal ERETIC signal for a non-interfering internal

standardstandard


3232

Isolated Model Compound NMR Isolated Model Compound NMR

5 Model Compound Mixture5 Model Compound Mixture– CapNMR at 600 MHzCapNMR at 600 MHz– 64 Scans (4 minutes)64 Scans (4 minutes)– 5 ug of 5 components5 ug of 5 components

Isolated proparacainIsolated proparacain CapNMR at 600 MHzCapNMR at 600 MHz 16 Scans (1 minute)16 Scans (1 minute) 5 ug of 1 component on 5 ug of 1 component on

columncolumnData Courtesy Novatia, LLCData Courtesy Novatia, LLC


3333

Online LC-NMR vs. Fractionation + CapNMROnline LC-NMR vs. Fractionation + CapNMR

On flow NMR

NT=512

CAP-NMR of fraction C47

NT=16

O

OH

O

O

OH

O

OO

OH

OHHO

CH3

OH

OHHO

O

O

HO

OH

OHH3C

MW 740 -> agl 286

Data Courtesy of

Jean-Luc Wolfender

MetabolomicsUniversity of Geneva, Switzerland


3434

LC/UV/MSLC/UV/MSof the chromatogram of the chromatogram

used for micro-fractionationused for micro-fractionation

RT: 0,00 - 60,08 SM: 7B

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

0

1

2

3

4

5

6

7

8

9

uAU

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

Inte

nsity

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

22,97

33,32

29,3728,63 39,4435,25 40,6930,1822,56 42,88 46,775,23 49,61 53,3618,587,25 16,99 56,304,28 12,809,04 59,211,45

31,3325,6319,0537,72

26,95

22,60

6,4837,3216,82

8,81

21,89 38,5044,54

46,36

35,9615,50 49,6128,77 40,28

40,7957,7514,595,97 11,69 57,0451,973,401,85

32,2127,7922,908,5725,13

21,72

33,2534,61

16,6820,23 37,18

6,58

38,36

18,88 39,8815,77 45,7241,73 47,3410,78 15,10 51,805,84 53,16 58,972,79

NL:6,95E5

m/z= 739,5-740,5 F: - c ESI Full ms [ 140,01-1000,02] MS exfl10mg

NL:1,68E7

Base Peak F: - c ESI Full ms [ 140,01-1000,02] MS exfl10mg

NL:9,90

Channel A UV exfl10mg

exfl10mg #676-690 RT:22,87-23,34 AV:15 SB: 77 21,55-22,66, 23,61-25,02 NL:5,92E5F: - c ESI Full ms [ 140,01-1000,02]

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

[M-H]-

739,3

477,5

445,6

740,8

421,6216,2741,8

506,6446,7921,7313,3 775,8609,8

281,3 863,8593,7 922,9738,8253,3 567,7404,9387,6 776,8468,5364,9 957,7742,9670,4515,2 650,8 832,2 902,0 997,5956,1

nm200 250 300 350 400 450 500 550

mAU

-200

0

200

400

600

800

1000

1200

DAD1, 21.878 (1449 mAU, - ) of FL10MG.D

m/z 739

TIC

UV (254nm)

Data Courtesy of

Jean-Luc Wolfender



3535

Cap-NMR gCOSY spectrum of fraction C47Cap-NMR gCOSY spectrum of fraction C47

O

OH

O

O

OH

O

OO

OH

OHHO

CH3

OH

OHHO

O

O

HO

OH

OHH3C

NT=1 NI=512 Time=12 min

Data Courtesy of

Jean-Luc Wolfender



3636

LC-MS+CapNMR for Natural LC-MS+CapNMR for Natural Products is:Products is:

Higher performance analytical scale Higher performance analytical scale chromatographychromatography

Fast online MS detection, Fast online MS detection, fractionation, and de-replicationfractionation, and de-replication

Maximum performance CapNMR to Maximum performance CapNMR to de-replicate and elucidate de-replicate and elucidate de novode novo structures structures


3737

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 natureJean-Luc Wolfender, Emerson Ferreira Queiroz, Kurt HostettmannExpert 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

Further ReadingFurther Reading


3838

ThanksThanks

Jean-Luc Wolfender, University of Geneva

David Detlefsen, Novatia

Get your Natural Products LC-MS+CapNMR system from Protasis Corporation.http://protasis.com508-481-4163

lc-ms+nmr building high throughput natural products libraries bob albrecht protasis corporation

Documents

nmr slide

copyright c

fraction slide

purification fractions

minute nmr

nmr easy

online lcnmr

ambiguous fractions