Biocatalysis in tailored ionic liquids

Neil C Bruce

CNAP

Centre for Novel Agricultural Products

Department of BiologyUniversity of York

Outline

• Ionic liquids

• Biocatalysis of opiates

• Ionic liquids tailored for biocatalysis-first generation ILs-second generation ILs

• Natural product extraction using ILs

Ionic liquids• Ionic liquids are compounds composed entirely of ions, with melting points below room temperature.

• Although most ionic materials (e.g. NaCl) are high-melting solids, disruption of the stacking ability and delocalization of the charges reduces lattice energy and thus melting point.

•A very limited number of imidazolium-based ionic liquids such as BMIm PF6have been widely studied as representatives of the class, but a vast number of ionic liquids can exist.

NaCl, m.p. 801 ºC

PyrHCl, m.p. 146 ºC

BMIm PF6, m.p. –79 °C

Ionic liquids - advantages

• Zero vapour pressure; non-flammable.

• High solvation capacities; can dissolve proteins, DNA, cellulose and even coal and rocks, but may be selected to be completely compatiblewith glass, metal or plastic containers.

• No hydrolysis.

• Environmentally benign and recyclable.

• Vast number of possible permutations - “designer solvents”.

Ionic Liquids

EnergyBatteries

Fuel cells

Photovoltaics

Heat storage

Super caps

OthersLight-emitting electrochemical cells

Liquid crystals

Nanoparticles

Artificial muscles

Oils/advanced fluids

Chemical EngineeringExtraction

Separation

Membranes

Extractive distillation

Biotechnology

?Limited investigations into biocatalysis in biphasic systems.

CoatingMetal deposition

Analytical

Lubricants

Surfactants

ChemistryOrganic synthesis

Chiral synthesis

Polymerization

Catalysis

Ionic liquids - applications

Biotransformation of morphinans

N

O

HCH3

O

HO

N

O

OHCH3

O

H3CO

N

O

CH3

H3CO

HO

CH3HO

CH3

N

O

OH

O

HO

CH2

N CH3

CH3

H3C

O

H3C

R'R N CH3R"

N

O

HCH3

O

O

H3C

O

H3C

O

Hydromorphone(potent analgesic)

OxycodoneEtorphine(animal tranquiliser)

Naloxone(narcotic antagonist) Methadone

(synthetic narcotic)

Basic structure requiredfor activity at opiate receptor.

Heroin(analgesic and euphoriant)

Variations on a theme

Fermentation of P. putida M10 with morphine

ONCH3

R

HO

Dihydromorphine R=HODihydrocodeine R=CH3O

Hydromorphone R=HOHydrocodone R=CH3O

ONCH3

R

O

ONCH3

R

HOOH

14-Hydroxymorphine R=HO14-Hydroxycodeine R=CH3O

ONCH3

R

HO

Morphine R=HOCodeine R=CH3O

O

RO

HO

N CH3

OH

O

RO

O

N CH3

OH

O

RO

O

N CH3

OH

O

RO

O

N CH3

O

RO

HO

N CH3O

RO

HO

N CH3

O

RO

O

N CH3

MorphineCodeine

MorphinoneCodeinone 14β-Hydroxymorphinone

14β-Hydroxycodeinone

HydromorphoneHydrocodone

HydromorphineHydrocodeine

OxymorphoneOxycodone

Noroxymorphone etc.

MR/NADH

MR/NADH

MDH/NADPMDH/NADPH MDH/NADPH MDH/NADPHMDH/NADP MDH/NADP

Hydroxylase

N-demethylation via P4 5 0 etc.

R = H, read upperR = CH3, read lower

14β-Hydroxymorphine14β-Hydroxycodeine

Biotransformation of morphinans

Morphine R=HOCodeine R=CH3O

ONCH3

R

HO

Morphinone R=HOCodeinone R=CH3O

ONCH3

R

O

Hydromorphone R=HOHydrocodone R=CH3O

ONCH3

R

O

NADP NADPH NADH NAD

Morphinedehydrogenase

Morphinone reductase

Recombinant biocatalyst for hydromorphoneproduction

• Coexpressed the genes for MDH and MR in E. coli

• Hydromorphone yields >85%

Classical organic solvents -

High substrate concentrations achievable without solvolysis.

Enzymes can exhibit unusual activities.

Often highly flammable, hazardous to health and environment.

Expensive disposal.

Poor solvents for most enzymes - low activity if anhydrous, denaturing if aqueous.

Multi-component biosystems not generally possible.

Biocatalysis in non-aqueous media

Biocatalysis in ionic liquids

• Ionic liquids offer:

• Improved substrate and product solubility (reactor downsizing).

• Suppression of solvolysis.

• Possibility of novel transformations.

• Biphasic aqueous systems well studied.

• Same limited enzyme range as in organic solvents.

• No attempt to “tailor” ILs for biocatalytic applications.

0

10

20

30

40

50

60

70

80m

g/m

L

Water Diethyl ether Chloroform BMIm PF6

Solubility of morphine free base in various solvents.

Morphine dehydrogenase

0

5

10

15

20

25

30

35

0 10 20 30 40 50

Time (hours)

Cod

eino

ne/n

eopi

none

(% to

tal

initi

al m

orph

inan

con

tent

)

O

N

O

H

O

H3C

CH3

NADPH

NADP+

P. putida M10 MDHO

N

O

H

HO

H3C

CH3

ADH (system i)GDH (system ii)

Acetone (system i)Gluconolactone (system ii)

2-Propanol (system i)Glucose (system ii)

50mM KH2PO4 (aq.)

Biphasic BMIm PF6/H2O

BMIm PF6 (< 100 ppm H2O)

• Enzyme rates are very low in BMIm PF6

• Homogenous redox biocatalysis is not possible in BMIm PF6- enzymes have evolved to function in an aqueous environment

- physicochemical solvation of water environment is characterised by a high degree of polar coordination, strong solvent-solvent and solvent-solute H bonding and dissociative proticity

- BMIm PF6 is an ordered, non-coordinating lattice, H bonding and van der Waals interactions between solvent and solute are minimal

- the structure of BMIm PF6 is remarkably devoid of labile protons

BMIm PF6 vs water

First generation ionic Liquids

0

5

10

15

20

25

0 4 8 12 16 20 24

Time (hours)

Cod

eino

ne/n

eopi

none

(% to

tal

mor

phin

an c

onte

nt)

NNPF6

-

NN OHPF6

-

NNCH2OHCO2

-

NN OHCl

-

+

+

+

+

50mM KH2PO4

Oxidation of codeine by MDH in ionic liquids (< 100 ppm H2O) and aqueous solution.

Reduction of codeinone by MDH in ionic liquids

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Time (hours)

Cod

eine

(% to

tal i

nitia

l m

orph

inan

con

tent

)

BMIm PF6

3-HOPMIm PF63-HOPMIm Cl

BMIm gly

Hydroxylated cations improve biocatalyst performance at very low water levels.

Strongly nucleophilic anions inhibit enzyme activity, even when only trace amounts present.

Cofactor recycling facilitated at < 100 ppm bulk H2O.

Best results from combination of hydrophilic, hydrogen-bonding cation and hydrophobic, non-coordinating anion.

Choice of ions and purity both of paramount importance.

Biocatalysis in hydroxylated ionic liquids

Ionic liquids – practical obstacles

Economic, legislative, safety, environmental and practical reasons to move away from “standard” imidazolium-based ionic liquids.

New classes of IL under development.

Designed for: - optimum biocatalyst performance.

- reduced viscosity.

- low cost.

- ease of synthesis and purification.

- minimal toxicity and environmental impact.

- recyclability and biodegradability.

Second Generation ionic Liquids

HONH2

OH+

HONH3

+

HONH

R+

R'

HON

R+

R'

R''

HONH

OH+

R

HON

OH+

R'R

• Based on alkanolammonium cations

• 1•, 2•, 3• or 4• substituted

• Combinable with any anion, e.g. by direct neutralization.

• Very easily synthesized from bulk precursors.

• Facile purification – no halide anions involved.

• Cost is significantly less than conventional ILs.

• UV/Vis transparent.

• Viscosity easily tunable.

• Recyclable & biodegradable.

• Ideal templates for incorporation of biocompatible functionalities.

• Over 300 novel ILs now synthesized, purified and characterized.

Properties

Ionic liquids for ADH

DiethanolammoniumDiethanolammonium BisBis((trifluoromethyltrifluoromethyl))sulfonimidesulfonimide MethanesulfonateMethanesulfonate(DEA)(DEA) (BST)(BST) (MS)(MS)

OH OH

NH2

+

DimethylethanolammoniumDimethylethanolammonium TrifluoromethanesulfonateTrifluoromethanesulfonate TrifluoroacetateTrifluoroacetate(DMEA)(DMEA) (TFMS)(TFMS) (TFA)(TFA)

ButyldiethanolammoniumButyldiethanolammonium GlycolateGlycolate AcetateAcetate(BDEA)(BDEA) (GLY)(GLY) (ACE)(ACE)

OH OH

NH2

+

OH OH

NH2

+

OH OH

NH2

+

F

S

O

O

O-

F

F

F

S

O

O

O-

F

F

F

S

O

O

O-

F

F

F

S

O

O

O-

F

F

S

O

O

O-

CH 3 S

O

O

O-

CH 3 S

O

O

O-

CH 3 S

O

O

O-

CH 3

CH3

OH

NH

CH3

CH3

OH

NH

CH3

CH3

OH

NH

CH3

CH3

OH

NH

CH3

OH

O-

O

OH

O-

O

OH

O-

O

OH

O-

O

---N

F

S

O

O

F

F

F

O

O

F

F

F

O

O

F

F

F

O

O

F

F

O

O

O

O

O

O

S

O

O F

FFFF

FFFF

F

F

F

O-

O

O-

O

CH3

CationsCations AnionsAnions

OH OH

NH+

CH3

Ionic liquids for ADH

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

0.550.6

0.650.7

buffe

rde

a ms

dea a

cede

a gly

dea t

fade

a tfm

sde

a cl

dmea

bst

dmea

ace

dmea

gly

dmea

tfadm

ea tfm

sbd

ea bs

tbd

ea m

sbd

ea ac

ebd

ea gl

ybd

ea tfa

bdea

tfms

[NA

DH

] (m

ol/L

) x 1

0-5

ethanolmethanol

Activity and Circular Dichroism of ADH

• Ionic liquids chosen DEA MS, DMEA GLY and DEA CL• CD - 2mg/ml ADH in each concentration of ionic liquid• Use CD to scan far UV abs 190-260nm• Shows secondary structure• Different patterns of absorbance indicate different types

of structure

Activity of ADH

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60 70 80 90 100% ionic liquid (v/v) in buffer

[NA

DH

] (m

ol/L

) x 1

0-5

DMEA GLYDEA MSDEA CL

-8

-4

0

4

8

190 200 210 220 230 240 250 260

Wavelength (nm)

CD

val

ue

Buffer10% IL20% IL30% IL40% IL50% IL60% IL70% IL80% IL90% IL100% IL

ADH in DEA MS

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60 70 80 90 100

% ionic liquid (v/v) in buffer[N

AD

H] (

mol

/L) x

10-

5

DEA MS

ADH in DEA CL

-20

-15

-10

-5

0

5

210 215 220 225 230 235 240 245 250 255 260

Wavelength (nm)

CD

val

ue

buffer10% IL20% IL30% IL40% IL50% IL60% IL70% IL80% IL90% IL100% IL

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60 70 80 90 100

% ionic liquid (v/v) in buffer

[NA

DH

] (m

ol/L

) x 1

0-5

DEA CL

Varying ethanol concentration• Activity studied in buffer and a selection of ionic liquids for varying

concentrations of ethanol

0

0.02

0.04

0.06

0.08

0.1

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26 0.28 0.3

Concentration ethanol (mM)

Initi

al ra

te v

(Abs

/min

)

bufferdmea acedea msbdea msdmea gly

Natural product extraction

• Artemisinin - leading anti-malarial drug• Currently extracted from Artemisia annua using boiling hexane• The trioxane ring is unstable

H3CNH+

CH3

OH

NH+

OH

CH3

H3C

NH+

OH

CH3

H3C

H2+

NOO

H3C CH3

-O

O

(CH2)6CH3

-O

O

(CH2)6CH3

-O

O

CH2CH3N-(SO2CF3)2

2 DMEA oct

3 DEEA pro

4 DEEA oct

5 BMOEA bst

0

0.2

0.4

0.6

0.8

1

0 60 120 180 240

Time (mins)

mg/

mL

arte

mis

inin

(2)

(3)

(4)

(5)

Hexane

Extraction of artemisinin

Bioniqs

• Bioniqs designs and sells novel ionic liquids (ILs)

• based in state of the art facilities in the York Biocentre

• spinout from CNAP in Dec 04

• Initial investment from IP2IPO, University of York

• based around IP developed at Universities of Cambridge and York

• manufacturing alliance with Merck KGaA

• further information www.bioniqs.com

Summary

• Ionic liquids are not a panacea, but can offer major benefits in certain key areas of the biosciences:-

• Can now compete on comparable cost footing with conventional solvents for bulk applications.

- Reduced pollution and waste- Improved process safety- Enhanced efficiency of reaction and extraction processes

• Facilitate ex aqua bioprocesses.- Smaller bioreactors; more economical- Improved turnover rates and yields- Stabilization of sensitive biomolecules- Application of biocatalysis to water-insoluble substrates

Hazel Housden

Emma Chalmers

Heather Stubley

Francesco Falcioni

Anton LeBrun

Andrew Leech

Seishi Shimizu

Adam Walker

Acknowledgements