Resolution racemates

Max Planck Institute Magdeburg

Investigation of an integrated racemization/separation process for the production

of enantiomerically pure mandelic acid

García-Rivera M.1, Wrzosek K.2, Renken A.1, Seidel-Morgenstein A.2,3

MAX-PLANCK-INSTITUTDYNAMIK KOMPLEXER

TECHNISCHER SYSTEMEMAGDEBURG

1EPFL Swiss Federal Institute of Technology, Lausanne/Switzerland2Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg/Germany

3Chair for Process Systems Engineering, Otto-von-Guericke University Magdeburg/Germany

August 2015 1

Max Planck Institute Magdeburg 2August 2015

1. Introduction

2. Integrated racemization-separation process

3. Enzymatic racemization

4. Application on a fixed-bed reactor

5. Experimental

6. Results

7. Conclusions and outlook

Contents


Introduction1.

Enantiomer formulationsFine-chemicals industry

• Limited quantities vs bulk chemicals• High-value products• Selectivity, separation, purification• High production costs

• Food, agrochemicals, pharmaceuticals• Syntheses precursors: flavourings,

essences, drugs• Single enantiomers enantiomerically

pure compounds

CephalosporinSertraline Penicillin

(S)-mandelic acid (R)-mandelic acid

O

OHOH

O

OHOH

Duloxetine

• Skin treatments, urinary antiseptic• Precursors for vanillin production and thrombin inhibitors• Resolution of other racemates

Mandelic acid and derivatives


Introduction

Pure enantiomers

from

through through

Classical resolution

Kinetic resolution

Dynamic kinetic resolution

� 50% yield

� 50% yield

�100% yield

Crystallization

Chromatography

L-L extraction

Membrane separation

Non-synthetic Synthetic

Chiral or achiral compounds Racemates

Resolution techniques

Asymmetric catalysts

Biocatalysts

Chiral auxiliaries

Chiral pool

Enantioselective synthesis

1.

Enantiopure compounds production[1,2]

Integrated processes


Integrated racemization-separation process

Integrated processes[3]

• Overcome 50% yield 100%• Cost-effective methods• Racemization, chromatography and crystallization• Versatility in operation configurations• Suitable conditions for all the operations

2.

Enzymatic racemization

Enantioselective chromatography

R-enantiomer

R -enantiomer S- enantiomerRacemate

R+S

Selected integrated process

Integrated process

Suitable conditions

Max Planck Institute Magdeburg 6

Enzymatic racemization

August 2015

Integrated racemization-separation process2.

Enantioselective chromatography

• Mandelate racemase• Well studied and characterized• Broad spectrum of mandelic acid

derivatives as substrate• Mild reaction conditions• Recombinant expression in E. coli [4]

• Immobilized for easy reuse

• Chirobiotic® T• Teicoplanin/silica gel as stationary

chiral phase

• Commercially available• Highly enantioselective• Variety of enantiomers

• Proper selection of a compatible mobile phase for both operations

Octamer with ~ 39 kDa identical monomers

Macrocyclic glycopeptide

Complex chiral environment

Hydrogen bonding sites

Active site within a hydrophobic cavity


Enzymatic racemization3.

(R)-mandelic acid (R)-mandelic acidcomplex

Planar enolateintermediate

(S)-mandelic acidcomplex

(S)-mandelic acid

O

OHOH

O

OHOH

NNH

His 297NH3

+

Lys 166

H

O

O

Glu 317

NH3+

Lys 164

Mg2+

H

O O-

OH Asn 197

: NH+

NH

His 297

NH3+

Lys 166

H

O

O

Glu 317

NH3+

Lys 164

Mg2+

H

O-

O-

O Asn 197

Racemization mechanism[5,6]

NH+

NH

His 297NH2

Lys 166

H

O

O

Glu 317

NH3+

Lys 164

Mg2+

H

O O-

O

HAsn 197

:

Active site in hydrophobic cavity [7]



1 U amount of enzyme that catalyzes 1 µmol of substrate in 1 minute under determined conditions

0 20 40 60 80 100 120 140 160 180 200

CR,0 [mM]

Initi

al re

actio

n ra

te [U

]

First-order reaction

Zero-order reaction

Michaelis-Menten kinetics[8] (irreversible)

R + E C S + Ek-R

kR

kS

−𝑑𝐶𝑅

𝑑𝑡 =𝑽𝒎𝒂𝒙𝐶𝑅

𝐶𝑅+𝑲𝑴 𝑽𝒎𝒂𝒙=𝑘𝑆𝐶𝐸

Enzymatic activity, in U1

• Maximum catalytic potential• Determined temperature, pH and initial substrate concentration• Initial reaction rate used in kinetics



Reversible Michaelis-Menten kinetics [8,9]R + E C S + E

k-R

kR

kS

k-S

−𝑑𝐶𝑅

𝑑𝑡 =𝑉𝑚𝑎𝑥 (𝐶𝑅−𝐶𝑆 )𝐾𝑀+𝐶𝑅+𝐶𝑆

Simplified model

Substrate inhibition

−𝑑𝐶𝑅

𝑑𝑡 =𝑽 ′𝒎𝒂𝒙 (𝐶𝑅−𝐶𝑆 )

𝑲 ′𝑴+𝐶𝑅+𝐶𝑆+𝐶𝑅

𝑲 𝒊(𝐶𝑅+𝐶𝑆)

𝑘𝑆=𝑘𝑅 𝑘−𝑆=𝑘−𝑅

𝑽𝒎𝒂𝒙=𝑘𝑆𝐶𝐸

R + E C S + EkR

k-S

RC

+R

Ki

k-RkS

No distinction between enantiomers: and

Ineffective binding at high substrate concentration

𝑽 ′𝒎𝒂𝒙=𝑘𝑆𝐶𝐸


Application on a fixed-bed reactor4.

Enzyme immobilization

• Solid porous carrier• Easy reuse• Versatility of reactor configuration• Mass transfer effects• Apparent kinetic parameters:

Eupergit® CM

• Acrylic copolymers beads • Particle size: 50 – 300 mm• Bearing epoxy groups• Covalent multipoint attachment• Mostly via amine residues

Eupergit® CM

Mandelate racemase

..

Carrier with Immobilized mandelate racemase

O

OHOH

O

OHOH

O

OHOH

O

OHOH

𝑽𝒎𝒂𝒙𝒂𝒑𝒑𝑲𝑴

𝒂𝒑𝒑 𝑉𝑚𝑎𝑥❑𝐾𝑀

❑


Application on a fixed-bed reactor4.

Plug-flow reactor (PFR)

𝐶𝑅(𝜏 )=12 𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0 (1+𝑒𝑒𝑅 , 0𝑒

− 2𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙, 𝑃𝐹𝑅𝝉 ) 𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,𝑃𝐹𝑅=𝑽𝒎𝒂𝒙

𝒂𝒑𝒑

𝑲𝑴𝒂𝒑𝒑+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0

𝐷𝑃𝐹𝑅

(2𝑲 ′𝑴𝒂𝒑𝒑+2𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0

2

𝑲 𝒊 ) ln ( 2𝐶𝑅 (𝜏)−𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0

2𝐶𝑅 , 0−𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0)+2(𝐶𝑅(𝜏 )−𝐶𝑅 ,0)=−

4𝜏𝑽 ′𝒎𝒂𝒙𝒂𝒑𝒑 𝑲 𝒊

𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0

With substrate inhibition:

Prediction equation:

𝜏=𝜀𝑉 𝑟

��𝑑𝐶𝑅

𝑑𝜏 =∑𝑗

𝑛

𝑣𝑅𝑗𝑟 𝑗

= 𝐷𝑃𝐹𝑅=𝑚𝑐𝑎𝑟𝑟𝑖𝑒𝑟

𝑉 𝑟 ,𝑃𝐹𝑅𝑒𝑒𝑅 , 0=

𝐶𝑅 , 0−𝐶𝑆 ,0

𝐶𝑅 ,0+𝐶𝑆, 0

Max Planck Institute Magdeburg 12

Experimental 5.

• Cell growth • Extraction• Immobilization• Storage

• Acquisition in a batch reactor

• Chromatographic conditions

• Evaluation at steady state

• Chromatographic conditions

Enzyme production

August 2015

Determination of kinetic parameters

Evaluation of a fixed-bed reactor

Place for chromatographic column

PFR setupBatch reactor setup

Carrier with immobilized enzyme

Cell growth


Transformation1 Single colony Pre-culture First culture Main culture

250 mL60 mL5 mL

•LB medium•1% ampicillin•T = 37°C

•Minimal medium•Microelements•T = 37°C

• Streaking• LB3

medium• 1%

ampicillin

•pET-52b(+) plasmid2

•Stable cell line•Stored - 20°C

1 Analysis and Redesign of Biological Networks, MPI Magdeburg2 Dalhousie University, Halifax, Canada

Fermentation•T = 37 18 °C• IPTG4 induction

Extraction•Cell disruption•Clarification

Immobilization

5 L

Storage

•Free enzyme•Immobilized

enzyme

Wet carrier

Eluent

Adsorbent

Extract

•Minimal medium•Microelements•T = 37°C

Experimental 5.

Enzyme production

3 Lysogeny medium4 Isopropyl-b-D-1-thiogalactopyranoside


Experimental

Fermentation conditions

1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

5.

Storage conditions

• 250 mM HEPES pH = 7.5• Three temperatures: 20°C, 4°C and -20°C

Free enzyme

Immobilized enzyme

• Storage temperature: 4 °C• Typical buffer: 150 mM Tris HCl 0.05% sodium azide pH = 8.2• Optimal buffer: 50 mM HEPES 3.3 mM MgCl2 pH = 7.5• Dry form: lyophilized at - 50°C

Immobilization conditions

• 1 M HEPES1 pH = 8.2• Incubation: 70 h• Low stirring• Room temperature

• Minimum medium: 5L• Ampicillin: 100 mg/mL• Glucose: 0.5%• Thiamine: 5 mg/L• IPTG: 200 mM• T = 37 18 °C


Experimental5.

Batch reactor set upa)

b)

c)

a)

b)

c)d)

HPLC station

PFR setup• Steady state• Outlet concentration at different residence times• CR,0 = 20 g/L at 100%ee , 60%ee and 40%ee• CS,0 = 20 g/L • CR,0 = 40 g/L • 20 mM HEPES 3.3 mM MgCl2 20% MeOH

pH = 6.8

• Initial reaction rate at different initial substrate concentrations

• CR,0 = 1, 3, 5, 8, 10, 20, 30, 40 g/L • 20 mM HEPES 3.3 mM MgCl2 20% MeOH pH

= 6.8


Experimental

• Continuous measurement of rotation angle• High time resolution (every second)• Measuring enzymatic activity• Measuring final/outlet concentration

5.

Polarimetry method

4 mL substrate

40 mL free enzyme

Polarimeter

0 5 10 15 20 250

500

1000

1500

2000

2500

3000

3500

Time (min)

Subs

trat

e co

nsum

ptio

n (m

mol

/mgp

rote

in)

initial rate enzymatic activity

Free enzyme Batch reactor PFR

• Static volume• Reaction solution• Measured every second

• Continuous flow (8 mL/min)• Filtered reaction solution • Measured every 2 seconds

3 mL solution

Polarimeter

• Static volume• PFR outlet (non-reacting)• Measured only once

Constant inlet

Polarimeter

Constant outlet


Experimental

• Immobilized enzyme storage• Sampling at 2, 4, 6, 8, 10 and 12 min• Reaction stopped by filtering and heating up to 90 °C• Analytical HPLC measurement: Chirobiotic® T

5.

8 mL substrate

60 mg wet carrier

0 2 4 6 8 10 12 140

500

1000

1500

2000

2500

3000

3500

Time (min)

Subs

trat

e co

nsum

ptio

n (m

mol

/gca

rrie

r)

initial rate enzymatic activity

HPLC method

Enzymatic activity

Free enzyme Immobilized enzyme

Polarimetry method Enzyme characterization Kinetics from batch reactor

HPLC method - Storage stability

Protein concentration Bradford method[10] Mass balance


Experimental

Cell growth

5.

Protein identification


5 L culture 20 g dry adsorbent 60 g wet carrier

= 3x weight of dry adsorbent

337 mg protein

+

Enzyme production

Results6.

Immobilization yields

• Immobilized protein recovery: • Activity recovery:

Remaining protein in eluent

Total available protein1 -100 * ( ) = 50% Activity in total mass of wet carrier

Activity in total volume of extract100 * ( ) = 48%

Max Planck Institute Magdeburg

Free enzyme storage

20August 2015

Results

Immobilized enzyme storage(measured by polarimetry method) (measured by HPLC method)

6.

Activity at time t

Initial activity)

Activity retention = 100 * (

• Best storage at -20°C• Good activity retention after 60 days

• Best storage with Tris HCl buffer• Good activity retention after 70 days


Results6.

Initial substrate concentration, CR,0 [mM]

Initial substrate concentration, CR,0 [g/L]

Initi

al re

actio

n ra

te,

[U/g

carr

irer]

ExperimentalNonlinear fit r2 = 0.9355

ExperimentalNonlinear fit r2 = 0.8999

Initial substrate concentration, CR,0 [mM]

Initial substrate concentration, CR,0 [g/L]

−𝑑𝐶𝑅

𝑑𝑡 |0=𝑉𝑚𝑎𝑥

𝑎𝑝𝑝 𝐶𝑅 , 0

𝐾𝑀𝑎𝑝𝑝+𝐶𝑅 , 0

−𝑑𝐶𝑅

𝑑𝑡 |0=

𝑉 ′𝑚𝑎𝑥𝑎𝑝𝑝 𝐶𝑅 ,0

𝐾 ′𝑀𝑎𝑝𝑝+𝐶𝑅 ,0+𝐶𝑅 , 0

2

𝐾 𝑖

Kinetics without substrate inhibition(measured by polarimetry method) (measured by polarimetry method)

Kinetics with substrate inhibition


Results6.

Kinetics without substrate inhibition(measured by polarimetry method) (measured by polarimetry method)

Kinetics with substrate inhibition

Kinetic model at chromatographic conditionsa or or Ki r2 b

No substrate inhibition 169.46 ± 5.07 U/gcarrier 8.59 ± 1.24 mM - 0.9355

Substrate inhibition 330.09 ± 17.13 U/gcarrier 30.61 ± 4.78 mM 95 mM 0.8999a 20 mM HEPES 3.3 mM MgCl2 20%v MeOH pH=6.80b Correlation coefficient for nonlinear fit. For best fit r2 ~1

−𝑑𝐶𝑅

𝑑𝑡 |0=𝑉𝑚𝑎𝑥

𝑎𝑝𝑝 𝐶𝑅 , 0

𝐾𝑀𝑎𝑝𝑝+𝐶𝑅 , 0

−𝑑𝐶𝑅

𝑑𝑡 |0=

𝑉 ′𝑚𝑎𝑥𝑎𝑝𝑝 𝐶𝑅 ,0

𝐾 ′𝑀𝑎𝑝𝑝+𝐶𝑅 ,0+𝐶𝑅 , 0

2

𝐾 𝑖


Results6.

PFR prediction without substrate inhibition

PFR prediction with substrate inhibition

(2𝑲 ′𝑴𝒂𝒑𝒑+2𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0

2

𝑲 𝒊 ) ln ( 2𝐶𝑅 (𝜏)−𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0

2𝐶𝑅 , 0−𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0)+2(𝐶𝑅(𝜏 )−𝐶𝑅 ,0)=−

4𝜏𝑽 ′𝒎𝒂𝒙𝒂𝒑𝒑 𝑲 𝒊

𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0

𝐶𝑅(𝜏 )=12 𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0 (1+𝑒𝑒𝑅 , 0𝑒

− 2𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙, 𝑃𝐹𝑅𝝉 )

𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,𝑃𝐹𝑅=𝑽𝒎𝒂𝒙

𝒂𝒑𝒑

𝑲𝑴𝒂𝒑𝒑+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,0

𝐷𝑃𝐹𝑅

Inlet concentration [g/L]

Enantiomeric excess

koverall,PFR [1/min]

20 100%eeR

1.6720 60%eeR

20 40%eeR

20 100%eeS

40 100%eeR 0.86

𝐷𝑃𝐹𝑅=¿1512 gcarrier/Lsolution


Results6.

PFR prediction without substrate inhibition(measured by polarimetry method) (measured by polarimetry method)


(R)-m

ande

lic a

cid

conc

entr

atio

n, C

R [g

/L]

Residence time, τ [min] Residence time, τ [min]

• More accurate prediction of CR

• Good performance for all %ee

• Deviation in 100%ee prediction• Good performance for 60 and 40%ee

Coverall,0 = 20 g/L Coverall,0 = 20 g/L


Results6.



Subs

trat

e co

ncen

trat

ion,

CR o

r CS [

g/L]

Residence time, τ [min] Residence time, τ [min]

• More accurate prediction of CR

• Incapability for prediction of CS

• Simplified model considers only R→S

• Slower reaction rate due to inhibition• Matches with S→R reaction• No accurate prediction of any

Covereall,0 = 20 g/L Coverall,0 = 20 g/L


Results6.



Subs

trat

e co

ncen

trat

ion,

CR o

r CS [

g/L]

Residence time, τ [min]

• 100%ee of R-mandelic acid• Faster reaction rate predicted• 40 g/L is under substrate inhibition

• 100%ee of R-mandelic acid• Slower reaction rate predicted• Overestimation of inhibition

Coverall,0 = 40 g/L


Conclusions and outlook7.

• Immobilized mandelate racemase presented improved storage stability in comparison with the free-enzyme form

• Good operational stability and ease of reuse when used in different reactor configurations

• Effective prediction of fixed-bed reactor behavior with no-inhibition model up to CR,0 = 20 g/L

• Accurate prediction even with different initial enantiomeric excesses

Conclusions

Outlook

• Non-simplified kinetic model: consideration of kinetic parameters for both reaction directions R → S and S → R

• Non-ideality of the fixed-bed reactor: external and internal mass transfer effects, residence time distribution deviations

• Feasibility for coupling with crystallization: solubility of pure enantiomers in mobile phase


References

1. Todd, M.H., Separation of enantiomers. Synthetic methods. 2014: Wiley-VCH.

2. Lorenz, H. and A. Seidel-Morgenstern, Processes To Separate Enantiomers. Angewandte Chemie International Edition, 2014. 53(5): p. 1218-1250.

3. Kaspereit, M., S. Swernath, and A. Kienle, Evaluation of Competing Process Concepts for the Production of Pure Enantiomers. Organic Process Research & Development, 2012. 16(2): p. 353-363.

4. Narmandakh, A. and S.L. Bearne, Purification of recombinant mandelate racemase: Improved catalytic activity. Protein Expression and Purification, 2010. 69(1): p. 39-46.

5. Bearne, S.L. and R.J. Spiteri, Reduction of intrinsic kinetic and thermodynamic barriers for enzyme-catalysed proton transfers from carbon acid substrates. Journal of Theoretical Biology, 2005. 233(4): p. 563-571.

6. Nagar, M., et al., Potent Inhibition of Mandelate Racemase by a Fluorinated Substrate-Product Analogue with a Novel Binding Mode. Biochemistry, 2014. 53(7): p. 1169-1178.

7. Lietzan, A.D., et al., Structure of Mandelate Racemase with Bound Intermediate Analogues Benzohydroxamate and Cupferron. Biochemistry, 2012. 51(6): p. 1160-1170.

8. Cornish-Bowden, A., Fundamentals of Enzyme Kinetics, ed. P. Press. 2004, London.

9. Illanes, A., Enzyme biocatalysis. Principles and applications. 2008: Springer Science and Business Media B.V.

10. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976. 72(1–2): p. 248-254.


Free and immobilized enzyme kinetics

−𝑑𝐶𝑅

𝑑𝑡 |0=

𝑽𝒎𝒂𝒙𝒂𝒑𝒑 𝐶𝑅 ,0

𝑲𝑴𝒂𝒑𝒑+𝐶𝑅 ,0

Kinetics free enzyme(measured by polarimetry method) (measured by polarimetry method)

Kinetics immobilized enzyme

Enzyme form ConditionsVmax or

[U/gcarrier]Vmax or

[U/mgprotein]KM or

[mM]

Free-enzyme1 Optimal buffer conditions1,2 - 220.6 ± 2.54 3.87 ± 2.54

Immobilized enzyme

Optimal buffer conditions1,2 307.99 ± 4.54 107.61 ± 1.61 11.29 ± 0.98

Chromatographic conditions3 (without substrate inhibition)

169.46± 5.07 71.21 ± 2.13 8.59 ± 1.24 1 from previous results, not measured in this study2 50 mM HEPES 3.3 mM MgCl2 pH=7.503 20 mM HEPES 3.3 mM MgCl2 20%v MeOH pH=6.80

−𝑑𝐶𝑅

𝑑𝑡 |0=𝑽𝒎𝒂𝒙

❑ 𝐶𝑅 ,0

𝑲𝑴❑ +𝐶𝑅 , 0

Without substrate inhibition


BR and PFR comparison

Reactor Rector volume [mL]

Carrier mass [gcarrier]

Dosage [g/L]

koverall,BR or koverall,PFR [1/min] for 20 g/L

BR (Eq. 2.15) 60.0000 0.6002 10.0033 0.0121PFR (Eq. 2.28) 0.2300 0.3560 1511.9109 1.8284

(R)-m

ande

lic a

cid

conc

entr

atio

n, C

R [g

/L]

Residence time or real time, τ or t [min]

𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,𝐵𝑅=𝑉𝑚𝑎𝑥

𝐾 𝑀+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0𝐷𝐵𝑅

𝑘𝑜𝑣𝑒𝑟𝑎𝑙𝑙 ,𝑃𝐹𝑅=𝑉𝑚𝑎𝑥

𝐾 𝑀+𝐶𝑜𝑣𝑒𝑟𝑎𝑙𝑙 , 0𝐷𝑃𝐹𝑅

BR prediction

PFR prediction


Feasibility for crystallization coupling

• Isothermal mode, T = 24 °C• Addition method• Fixed volume• Fixed buffer concentration• pH correction till 6.8• Increasing (R)-mandelic acid mass • Increasing 10 N NaOH volume

Add a known mass of mandelic acid

Stirring until dissolved

pH ≥ 6.8

Add a known volume of NaOH 10 N

Completely dissolved

Add a known volume of NaOH 10 N

V<Vtotal

V=Vtotal

Adjust final pH = 6.8

Fix a new Vtotal

No

No

Yes

Yes

No

Yes

No

Yes

MeOH: 0.2Vtotal

Buffer: 0.4 VtotalH2O: 0.1 Vtotal

pH=6.8

Fix a Vtotal

First hints

C (%w)

T (°C)

9.9%

23%

24 °C

Solubility in water

Solubility in mobile phase

Iterative method

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Resolution racemates