Institute of Applied Synthetic Chemistry

SFC applications in synthetic chemistry

Dr. Florian Rudroff 25. Oktober, 2016

Convergence chromatography & synthetic chemistry

volatility

po

lari

ty

chiral/achiral GC HPLC/UV-MS

• Deriv. required

• Amino acids

• TCA metabolites

• Fatty acids

com

po

un

d

space

GC/UV-MS

UPC2/UV-MS

• Combination of GC & LC-MS

• No derivatization needed

• Highest sensitivity & selectivity

• Targeted & quant. analysis

• Achiral & Chiral separations

• Polar & apolar metabolites

• Reversed phase chromatography • Normal phase chromatography

• Convergence Chromatography

UPC2/UV-MS

• Organic comp.

• Chiral comp.

• Terpenes

• Fragrance comp.

• Vitamins

• Carbohydrates

• Nucleo-comp.

• Phosphoryl. comp.

Convergence chromatography & synthetic chemistry

volatility

po

lari

ty

chiral/achiral GC HPLC/UV-MS

• Deriv. required

• Amino acids

• TCA metabolites

• Fatty acids

com

po

un

d

space

GC/UV-MS

UPC2/UV-MS

• Combination of GC & LC-MS

• No derivatization needed

• Highest sensitivity & selectivity

• Targeted & quant. analysis

• Achiral & Chiral separations

• Polar & apolar metabolites

• Organic comp.

• Chiral comp.

• Terpenes

• Fragrance comp.

• Vitamins

• Carbohydrates

• Nucleo-comp.

• Phosphoryl. comp.

3 case studies:

Combination of GC & LC analysis for chemo-enzymatic cascade reactions

Analysis of flavones – regioisomers and their properties

Enzyme mechanism – into the world of chirality

Liebeskind – Srogl coupling ADH / TA reaction

chemo catalysis enzyme catalysis

ADH alcohol dehydrogenase (R- & S-selective ADH) TA transaminase (R- & S-selective TA)

Chemo-enzymatic transformations in vitro A B C D

enzymatic reduction

enzymatic transamination

FG1 FG2 FG3

FG4

FG5

Pd/CuTC

+

+

+ Step 1 Step 2

metal assisted coupling chemistry

enzyme mediated transformation

One-pot multi step reaction cascade

or

FG4 FG5 FG3 FG1

FG2

FG functional group CuTC Copper(I) thiophene-2-carboxylate

•Water as classical solvent for biotransformations / THF

Solvent

•Physiological temperature (20-37 °C) / 50 °C

Reaction temperature

• Substrate inhibition / 250 mM thioester concentration

•Enzyme inactivation / CuTC (stochiometric)

Inactivation of the biocatalyst

Compatibility issues

Liebeskind, L. S. & Srogl, J., J. Am. Chem. Soc. 2000, 122, 11260-11261.

A B C D

Pd2(dba)3/CuTC

POEt3

Chemo-enzymatic cascade reactions

Analytical issues

Liebeskind, L. S. & Srogl, J., J. Am. Chem. Soc. 2000, 122, 11260-11261.

A B C D Chemo-enzymatic cascade reactions

Pd/CuTC

+

+

Highly diverse polarity of analytes

Heterogeneous reaction conditions

Tricky work up & sample preparation

lipophilic

polar

A B C D Chemo-enzymatic cascade reactions

Conditions:

Column: Torus 1-AA, 2.1mm x 150mm, 1.7um Solvent: ACN = 100 Flow rate: 0.7 ml/min ABPR pressure: 1900 psi Gradient: 99/1 -> 75/25 (2min)

IS (Methylbenzoate)

Pd/CuTC

Runtime: 3 min

Chemo-enzymatic transformations in vitro

Simultaneous One Pot reaction • coupling reaction (100 mM TE) in water

• 1.7 eq RB(OH)2, 1.6 eq CuTC

• 20 mol% P(Oet)3, 2.5 mol% Pd2(dba)3

• ADH-A lyophilisate, Tris*HCl, 320 mM, pH 7.5

• 20% i-PrOH

• 24-48h reaction time

A B C D

PDMS membrane

biocatalytic chamber coupling reaction chamber

sampling port

ADH-a LK-ADH

R Yield [%] ee [%] Yield [%] ee [%]

Ph 65 99 (S) 81 99 (R)

4-ClPh 60 99 (S) 57 99 (R)

3-ClPh 47 99 (S) 53 99 (R)

4-BrPh 99 99 (S) 50 99 (R)

4-FPh 75 99 (S) 64 99 (R)

4CF3Ph 61 99 (S) 53 99 (R)

Conditions:

Column: Acclaim RSLC C18, 2.2 µm 2.1 x 150 mm Solvent: H2O/MeOH (22.5% in H2O) + 10% formic acid Flow rate: 0.2 ml/min Gradient: 91/9 -> 10/90 (40min)

Runtime: 40 min

Flavones (flavus = yellow), are a class of flavonoids based on the backbone of 2-phenyl-1-benzopyran-4-one

Flavone analysis – how to deal with regioisomers

Target structures:

Butein Isoliquiritigenin Sulfuretin

Conditions:

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / ACN = 80 / 20 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 80/20 -> 60/40 (0.5min)

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / ACN = 80 / 20 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 85/15 -> 60/40 (2min)

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 80/20 -> 60/40 (0.5min)

Runtime: 2.5 min

Runtime: 2.5 min

Runtime: 2.5 min

Flavone analysis – how to deal with regioisomers

Butein Isoliquiritigenin Sulfuretin

Conditions:

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / ACN = 50 / 50 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 80/20 -> 60/40 (0.5min)

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / ACN = 50 / 50 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 70/30 -> 60/40 (0.3min) Additive: 1 % formic acid

Column: Torus Diol, 2.5 µm; 2.1 x 75 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.5 ml/min ABPR pressure: 2200 psi Gradient: 65/35 -> 60/40 (0.3min) Additive: 1 % formic acid

Runtime: 2.5 min

Runtime: 1.5 min

Runtime: 1.5 min

Butein Isoliquiritigenin Sulfuretin

Flavone analysis – how to deal with regioisomers

Enzyme mechanism – how to handle stereoisomers

EREDs – Enoate reductases

Applications:

Enzyme mechanism – how to handle stereoisomers

Kinetic resolution:

ks >> kr

X

Conditions:

Column: Trefoil AMY1, 2.5 µm; 2.1 x 50 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.2 ml/min ABPR pressure: 2200 psi Gradient: 97/3 -> 60/40

Column: Trefoil AMY1, 2.5 µm; 2.1 x 150 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.0 ml/min ABPR pressure: 2200 psi Gradient: 97/3 -> 60/40

Column: Trefoil AMY1, 2.5 µm; 2.1 x 150 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.0 ml/min ABPR pressure: 2200 psi Gradient: 97/3 -> 65/35

Runtime: 2.5 min

Runtime: 2.5 min

Runtime: 5 min

Enzyme mechanism – how to handle stereoisomers

enantiomers

Conditions:

Column: Trefoil AMY1, 2.5 µm; 2.1 x 150 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.0 ml/min ABPR pressure: 2200 psi Gradient: 97/3 -> 60/40

Column: Trefoil AMY1, 2.5 µm; 2.1 x 150 mm Solvent: MeOH / iPrOH = 80 / 20 Flow rate: 1.0 ml/min ABPR pressure: 2200 psi Gradient: 97/3 -> 65/35

Runtime: 2.5 min

Runtime: 5 min

Enzyme mechanism – how to handle stereoisomers

anti enantiomers

Acknowledgements

Prof. Marko D. Mihovilovic Associate Prof. Michael Schnürch Dr. Michael Fink Nikolin Oberleitner Patricia Schaaf Thomas Bayer Ramana Pydi Sofia Milker Thomas Wiesinger FG MDM

Greifswald University Prof. Uwe T. Bornscheuer Christin Peters Maria Kadow Jan Muschiol Stefan Saß

FGPG Prof. Peter Gärtner Priv. Doz. Katharina Schröder Dr. Anna Ressmann Emanuel Sporer

FG Prof. Robert Mach Dr. Christian Derntl

E164 Prof. Günther Allmaier Prof. Martina Marchetti-Deschmann

E166 Priv. Doz. Oliver Spadiut

LK-ADH Prof. W. Hummel

C. cellulans Prof. Zhi Li

P. putida CumDO Prof. M. Fraaije

AlkJ Prof. Bruno Bühler

CAR Dr. Margit Winkler (ACIB)

Aldolases Prof. W.D. Fessner

Funding FWF – I723-N17 DFG – B01862/6-1 GEV-TOP163 TU Vienna FWF – P24483-B20 FWF – P28477-B21

E163 Florian Untersteiner Dr. Christian Hametner

Liebeskind-Srogl reaction mechanism