Judith M. Rollinger

Ulrike Grienke

Preparative SFC QDa Applications in

Natural Products Research

ÖSTERREICHISCHES TECHNOLOGIE SYMPOSIUM 2016, October 25, 2016

Pharmacognosy Group, UNIVIE

Plants, fungi, microorganisms, marine organisms,

…are still the major source of new chemical entities

for drug discovery!

64% = derived/inspired

by nature

34% = natural cpds /derivatives

Sources of drug substances (small chemical entities;1981 – 2011, n = 1078): Newman & Cragg (2012) J. Nat. Prod., 75, 311-335

Research Groups

Dirsch Lab: Molecular Targets Research focus: to understand the molecular interaction of natural products with proteins/signaling molecules within cells and to provide cellular models for the identification of bioactive natural products.

Rollinger Lab: Phytochemistry & Biodiscovery We use interdisciplinary approaches (e.g. in silico guided, ethnopharmacology-driven) to identify, analyze, and purify multi-component mixtures from plant, fungal, or marine sources aiming at the characterization, the targeted isolation and discovery of bioactive secondary metabolites.

Zotchev Lab: Pharmaceutical Biotechnology Discovery of natural product from microorganisms, supported by cutting edge biotechnology-related approaches, including genome-based bioprospecting, metabolic engineering and synthetic biology.

http://pharmakognosie.univie.ac.at/

• Pharmaceutical lead and target identification from natural

resources

• Quality control of herbal medicinal products

• Sustainable production of natural products by optimized extract

procedures, chromatographic workflows and biotechnological

approaches

• Educational work and young researchers’ training

Aims

Analytic lab for metabolic profiling, dereplication… • UPLC systems with different detectors (UV/Vis, DAD, QD, ELSD, CAD) • UPLC - triple quadrupole MS with ESI and APCI; UPLC - ion trap mass spectrometer with ESI and APCI ion source • GC-MS with EI- and CI-ionization; GC with flame ionization detectors (FID) • Different HPLC systems with UV/Vis, DAD and/or ELSD for routine analysis • High-performance TLC system with application unit, TLC visualizer and evaluation unit

Extraction, fractionation, isolation • Prep. supercritical fluid chromatograph (SFC) with parallel detection units (PDA, ELSD, QDa), fraction collector • Prep. HPLC systems with autosampler, DAD and fraction collector; • High performance counter-current chromatograph (HPCCC) • Ultra-performance flash chromatograph combined with PDA and ELSD

Chem. characterization of natural compounds/metabolites • QTOF-MS with ESI and APCI ion source (with UPLC system; shared with Dep. Pharm. Chem.) • NMR spectrometers (400 MHz & 500 MHz) with autosampler and cryoprobe (shared with Dep. Pharm. Chem) • UV/VIS spectrophotometers • Attenuated total reflection (ATR) - Fourier transform infrared (FTIR) spectrophotometer

Biological characterization of natural compounds/multicomponent mixtures • Caenorhabditis elegans Screening Plattform (in vivo Model) Different wild type strains (e.g., GFP

marked) and genetically modified strains adapteto 96 well plates (for chemical genetics screens & therapeutic screen (neurodegen. diseases)

• In silico Screening Plattform: Expertise in computer-aided NP drug discovery; VS adapted NP databases (3D-molecule-DB: >120.000 entries); access to modeling software (LigandScout, ROCs,…)

See: http://pharmakognosie.univie.ac.at/research/phytochemistry-biodiscovery/equipment/

.... state-of-the-art equipment!

To complement our systems …

... our latest additions

H-Class UPLC (with PDA, ELSD, QDa)

including fraction manager SFC Prep-15 (with PDA, ELSD, QDa)

Instrumental set-up: Waters SFC Prep-15

Instrumental set-up: Waters SFC Prep-15

Instrumental set-up: Waters SFC Prep-15

N2 generator

chiller

CO2 cylinders

Pump for co-solvent and CO2

pump with different channels for 6 co-solvents

pump for CO2

(cooled with ethylene glycol)

fraction collector & autosampler: accommodates both

sample injection &

fraction collection

column oven: automated column switching and temperature control of up to 10 columns (analytical & preparative)

Oven, fraction collector, and autosampler

“Our” add-on detection configuration

QDa

Waters H-Class UPLC

MS-based triggering better collection precision by targeting only the mass of interest

Columns for the SFC Prep-15

further chemistries: Silica 2-EP Silica

4.6 x 250 mm 10 x 250 mm

4.6 x 250 mm

10 x 250 mm

4.6 x 250 mm

10 x 250 mm

4.6 x 250 mm

10 x 250 mm Silica 2-EP

particle shape: spherical, particle size: 5 µm

Dimension analytical preparative

Columns available in our lab

VIS

Vanillin/H2SO4

TLC System:

DCM : MeOH : H20

(10 : 1 : 0.25)

Application example: POLYPORES

one species

strain 1

strain 2 strain 3

Pictures: http://www.mycobank.org/

first evaluation of metabolite profile

many triterpenes difficult to detect with HPLC!

Profiling with SFC!

finding the best source material for bioactive compounds

Dresch, P. Aguanno MN, Rosam K, Grienke U, Rollinger JM, Peintner U. (2015): Fungal strain matters: Colony growth and bioactivity of the European medicinal polypores Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus. AMB Express 5:4.

different strains different metabolite profile different bioactivity!

column Viridis BEH, 5 µm 4.6 x 250 mm

flow rate 5 mL/min

make-up flow rate 3 mL/min

injection volume 5 µL

sample conc. 5 mg/mL

detection ELSD

system back pressure 120 bar

temp. 40°C

Time (min) EtOH (%)

0 5

1 5

9.5 50

10.5 50

11 5

12 5

Comparison of metabolite profiles

Strain 1

Strain 2

Strain 3

Strain 4

Strain 5

Strain 6

best bioactive starting material to isolate the target compound

EtOH

crude extract

DCM

fraction active

S1F12

active

Step 1: liquid/liquid partition

Step 2: Silica gel column

> 200 constituents 98% lanostane triterpenes highly similar molecular weight bad UV detection

Application example: POLYPORES

Purification of selected fractions of a polypore fungus

S1F12

separated with

SFC

separated with

Flash CC (RP18)

SFC

LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35° C; mobile phase: A= water; B= CH3CN; flow rate: 1.0 mL/min; detection: CAD; injection volume: 10 µL; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B.

HPLC-CAD

Highly complex triterpene mixture !

SFC method development for S1F12

• analytical column screening:

Viridis SFC columns (particle size: 5 µm, dimension: 4.6 x 250 mm)

BEH

CSH Fluoro-Phenyl

Silica 2-EP

• co-solvent screening:

MeOH

EtOH

2-propanol

CH3CN

flow rate 5 mL/min

make-up 3 mL/min

injection volume

20 µL

sample conc. 3 mg/mL

detection PDA-ELSD

system back pressure

120 bar

temp. 40°C

Time (min) co-solvent (%)

0 5

1 5

6 50

7 50

7.5 5

9 5

S1F12 best separated with EtOH as co-solvent

BEH

CSH FP

Silica 2-EP

best separation with BEH column further optimisation

SFC method development for S1F12

Scal

e-u

p

column Viridis BEH, 5 µm 4.6 x 250 mm (analyt.) 10 x 250 mm (prep.)

flow rate 5 mL/min (analyt.) 15 mL/min (prep.)

make-up flow rate

3 mL/min (analyt.) 5 mL/min (prep.)

injection volume

20 µL (analyt.) 150 µL (prep.)

sample conc.

3 mg/mL

detection PDA-ELSD

system back pressure

120 bar

temp. 40°C Time (min) EtOH (%)

0 8

9.5 8

10 50

11 50

11.5 8

12 8

Scale-up: analytical to preparative mode

good separation of single peaks

FractionLynx Method:

Development of fractionation method

Collection of 10 fractions

1

2

3

4

5

6

7

8

9

10

• split/collector delay: 9 sec

• PDA/ELSD delay: 2 sec fractionation based on timed events

S1F12 SFC separation

fractions 5, 7, 9 monitoring with RP-HPLC-CAD: good separation on Prep-15 system enriched fractions still contain a few peaks

Fraction monitoring with RP-HPLC-CAD

1

2

3

4

5 6

7 8

9 10

9

7

5

HPLC monitoring

LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35° C; mobile phase: A= water; B= CH3CN; flow rate: 1.0 mL/min; detection: CAD; injection volume: 10 µL; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B.

Pre-fractionation of S1F12 with Flash chromatography

column PuriFlash Column 15 C18 HP 6.0 g

solvents gradient: H2O/CH3CN

flow rate 5 mL/min

amount separated

~ 500 mg

detection PDA-ELSD

HPLC monitoring

LC-parameter: stationary phase: Zorbax (4.6 x 150 mm, 3.5 µm); temperature: 35° C; mobile phase: A= water; B= CH3CN; flow rate: 1.0 mL/min; detection: CAD; injection volume: 10 µL; gradient: 0 min: 45% B; 42 min: 80% B; 43 min; 95% B.

Pre-purified fractions further separation via SFC

Fraction 6

Fraction 7

Fraction 8

Separation of two co-eluting cpds

SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40°C; system back pressure: 120 bar, mobile phase: A= CO2; B= EtOH; flow rate: 15.0 mL/min; detection: ELSD; injection volume: 250 µL; gradient: 0 min: 10% B; 8 min: 10% B; 9.5 min: 50% B; 11.5 min: 50% B; 12 min: 10% B; 13 min: 10% B.

A B

A

B

HPLC monitoring

Fraction 6

Separation of two co-eluting cpds

SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40°C; system back pressure: 120 bar, mobile phase: A= CO2; B= EtOH; flow rate: 15.0 mL/min; detection: ELSD; injection volume: 250 µL; gradient: 0 min: 8% B; 9.5 min: 8% B; 10 min: 50% B; 11 min: 50% B; 11.5 min: 8% B; 12 min: 8% B.

HPLC monitoring

C C

Fraction 7

Separation of further cpds

HPLC monitoring

SFC-parameter: stationary phase: Viridis BEH, 5 µm 10 x 250 mm; temperature: 40°C; system back pressure: 120 bar, mobile phase: A= CO2; B= EtOH; flow rate: 15.0 mL/min; detection: ELSD; injection volume: 250 µL; gradient: 0 min: 8% B; 9.5 min: 8% B; 10 min: 50% B; 11 min: 50% B; 11.5 min: 8% B; 12 min: 8% B.

D

D

Fraction 8

Integration of SFC in NP workflows

+ SFC is orthogonal to C18

+ inexpensive and non-toxic carbon dioxide

+ easy handling via automated sample collection

through FractionLynx Application Manager

+ beneficial alternative to purification and analytical applications

Thank you for your attention!

Dr. Ulrike Grienke Natalie Prenner Julia Zwirchmayr

Department of Pharmacognosy, University of Vienna, AUSTRIA

Acknowledgement