Roger A. Sheldon

Delft University of Technology

&

CLEA Technologies

Green Bio-based Solutions for a Sustainable Economy

www.cleatechnologies.com 1

Green chemistry efficiently utilises

(preferably renewable) raw materials,

eliminates waste and avoids the use

of toxic and/or hazardous solvents

and reagents in the manufacture and

application of chemical products.

Green (Clean) Chemistry

Sheldon, Arends and Hanefeld , Green Chemistry

and Catalysis, Wiley, New York, 2007

Anastas & Warner, Green Chemistry : Theory

& Practice ,Oxford Univ. Press,New York,1998

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Green (Clean) Chemistry

• Eliminating (minimizing) waste • Avoiding toxic / hazardous substances • Conserving raw materials

Definition of Waste ?

Meeting the needs of the present

generation without compromising

the needs of future generations to

meet their own needs

Sustainability

Brundtland Report, ‘Our Common Future’, 1987

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The Bio-based Economy

Renewable Biomass

Biocatalysis

Chemocatalysis

Bio-based Products

Utilising crops and waste biomass which don’t compete with food

2nd generation:

Tonnage E Factor

Oil Refining 106-108 <0.1

Bulk Chemicals 104-106 <1 - 5

Fine chemical Industry 102-104 5 - >50

Pharmaceutical Industry 10-103 25 - >100

R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12

E Factor = kg waste/kg product

“Another aspect of process development mentioned by all pharmaceutical process chemists who spoke with C&EN is the need for determining an E Factor”. A. N. Thayer, C&EN, August 6, 2007, pp. 11-19

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What about process water? Only counts if it needs to be treated?

Major Sources of Waste • Stoichiometric Reagents

- Acids & Bases

- Oxidants & reductants

• Solvent losses ( 85% of non-aqueous mass)

- Air emissions & aqueous effluent

• Multistep syntheses

The Solution :

Atom & step economic catalytic processes

in alternative reaction media

(the best solvent is no solvent)

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The Environmental Impact EQ

EQ = E(kg waste) × Q

Q = Unfriendliness Multiplier

e.g. NaCl : Q = 1 ( arbitrary)

Cr salts : Q = 1000?

R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12

There are many shades of green!

Biocatalysis 9

Biocatalysis is Green & Sustainable • Enzymes are derived from renewable resources and are biodegradable (even edible sometimes) • Avoids use of (and product contamination by)

scarce precious metals • Mild conditions: ambient T & P in water

• High rates & highly specific : substrate, chemo-, regio-, and enantiospecific • Higher quality product • No special equipment needed

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Reduced environmental footprint

Two Types of Biotransformations • Free enzymes - isolated (purified) - whole cells (not growing) - can be very high STY • Fermentations (growing microbial cells) - less expensive (no enzyme isolation needed) - often dilute solution / low STY - water footprint /energy intensive - byproducts from enzyme impurities

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Lord, I fall upon my knees And pray that all my syntheses May not be inferior To those conducted by bacteria

J.B.S. Haldane

E factors of Fermentations

Product E factor Citric acid - excluding H2O (75% is CaSO4 ) 1.4 - including H2O 17 Bioethanol - excluding H2O and CO2 1.1 - including H2O and CO2 42a Recombinant Human Insulin - excluding H2O 6600b - including H2O 50.000

b 1692 kg urea, 1346kg HOAc, 968 kgs HCO2H, 713 kg H3PO4 ,

445 kg guanidine-HCl, 432 kg glucose, 430 kg NaCl, 424 kg MeCN

a 10000 ton/day lignocellulose feedstock produces 870 ton/day bioethanol

and generates 32x106 ltr/day of wastewater (eq. to 300.000 inhabitants)

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Enzymatic VS Chemical Process for 6-APA

1. n-BuOH, -40 oC

2. H2O, 0 oC

Process Chemical Enzymatic

Reagents (kg/ kg 6-APA)

Me3SiCl (0.6) PCl5 (1.2) PhNMe2 (1.6)

n-BuOH (8.4 ltr), NH3 (0.2)

Pen acylase (1-2) NH3 (0.09)

Solvent (ltr/kg 6-APA)

CH2Cl2 (8.4) H2O (2)

pen acylase H 2O

1. Me3SiCl

2. PCl5 /

PhNMe 2/

CH2Cl2oo

37 C-40 C

6-APA

penicillin G

H2N

N

O

S

CO2H

Cl

N

N

O

S

CO2SiMe3

H

O

N

N

O

S

CO2H

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Key improvements: enhanced enzyme production and immobilization

Reactions used for Drug Candidate Molecules

“A common synthetic sequence is the

conversion of an ester to an acid, activation of

the acid and conversion to the amide.”

Enzymatic amidation :

Lipases/esterases, Amidases, Proteases

“Three synthetic steps and multiple stoichiometric

components to accomplish a simple dehydration.”

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Carey, Laffan, Thomson, Williams

Org. Biomol. Chem. 2006, 4, 2337

Enzymatic Amidation

de Zoete, Kock van Dalen, van Rantwijk and Sheldon, J.Mol.Cat.B, 2, 19 and 141 (1996)

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van Pelt, Teeuwen, Janssen, Sheldon, Dunn, Howard, Kumar, Martínez, Wong, Green Chem. 13 (2011) 1791-1798

broadening the scope of enzymatic amidation

Protein Engineering 16

Production of Lipitor Intermediates

NC

OH O

OEt N C

O O

O R

O

Lipitor (Pfizer)

Sales in 2009: $14 bio

H N

O

N C O2 N a

O H O H

F

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Enzymatic Synthesis of Lipitor Intermediate

KRED = keto reductase ; GDH = glucose dehydrogenase HHDH = halohydrin dehalogenase (non-natural nucleophile)

OEt Cl

O O

N A D P H N A D P

OEt Cl

OH O

OEt Cl

OH O

HHDH OEt

NC

OH O

KRED

g l u c o s e g l u c o n a t e

GDH

a q . N a C N , p H 7

(99.8% ee)

> 9 9 % e e

R.J.Fox, S.C.Davis,R.A.Sheldon, G.W.Huisman, et al Nature Biotechnology, 25 (2007) 338-344

2006

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• high enantioselectivity

• mild (ambient) conditions

• no metal catalysts required

• no need for dedicated equipment

• low productivities

Directed Evolution for Improved Performance

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Features of the Wild-Type Enzymes:

Productivities of all three enzymes improved by

directed evolution using gene shuffling technology

W.P.C.Stemmer,Nature,370,389-391,1994

Waste Quantity

( kg per kg HN)

% contribution to E

(excluding water)

% contribution to E

(including water)

ECAA losses (8%) 0.08 <2% <1%

Triethanolamine 0.04 <1% <1%

NaCl and Na2SO4 1.29 22% ca. 7%

Na-Gluconate 1.43 ca. 25% ca. 9%

BuOAc

(85%recycle)

0.46 ca. 8% ca. .3%

EtOAc

(85%recycle)

2.50 ca. 43% ca. 14%

Enzymes 0.023 <1% <1%

NADP 0.005 0.1% <0.1%

Water 12.25 - 67%

E Factor 5.8 (18)

E factor of the Codexis Three-Enzyme Process

R. A. Sheldon, G. Huisman et al, Green Chem. 2010, 12, 81-86

Presidential Green Chemistry Challenge Award 2006

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Biocatalyst Engineering

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The Challenge

Disadvantages of Enzymes

Low operational stability & shelf life

Cumbersome recovery & re-use

Product contamination

Allergic reactions to proteins

Non viable biocatalytic applications

Costs are too high

Not practical

The Solution: Immobilization

Cross-Linked Enzyme Aggregates (CLEAs)

Precipitation X-linking + Co-polymerization

• “Simple” & Broadly Applicable

• Cost-effective (no need for pure enzyme)

• Short Time-to-Market (low development costs)

• Scalable Protocols

e.g. (NH4)2SO4

or tert-butanol

Advantages of CLEAs

1. Improved properties

• Better storage and operational stability

• Hypoallergenic

• No leaching of enzyme in aqueous media

2. Cost-effective

• No need for pure enzyme (crude cell lysate sufficient)

• Easy recovery and recycle (easier DSP)

• High productivities (kg product/kg enzyme)

3. Broad scope & short time to market

0

20

40

60

80

100

120

0.0 5.0 10.0 15.0 20.0

Exposure to 21oC (days)

Re

sid

ual

Act

ivit

y (%

)

Free enzyme (%) Whole cell (%) Nitrile Hydratase CLEA (%)

4 months storage no decrease in activity!

Dramatic stability enhancement of a NHase

Dramatic Stability Enhancement of a NHase

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Instability due to dissociation of multimeric enzyme

hindered by CLEA formation

van Pelt, Quignard, Kubac, Sorokin, van Rantwijk and Sheldon, Green Chem. 10 (2008) 395-400

Mitsubishi process: acrylonitrile to acrylamide > 200000 tpa

Cross-Linked enzyme Aggregates

AlcalaseCLEA commercial scale manufacture

- Free flowing powder

- Tunable particle size

- Low (no) allergenicity

- Excellent thermal stability 26

Nuijens et al, Advan. Synth. Catal. 2010, 352, 2399 – 2404.

Peptide synthesis with alcalase CLEA (DSM)

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Cross-Linked enzyme Aggregates

Protease CLEAs as

Antifouling Agents in Paint

Cross-linked enzyme aggregates (CLEAs) of proteases were tested

in artificial seawater (ASW) both as it is and as a component of the

paint. It is found that all CLEAs have tolerance to xylene and have

great stability in dried paint.

The maximal increase in relative activity was found for CLEA B. licheniformis.

CLEA B. licheniformis have shown 900% activation during storage in ASW. In

the paint, non-modified subtilisin lost more that 90% of activity in 28 days.

S. Shipovskov, et al, J. Mater. Chem., 2010, 20, 7626–7629

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The stone age didn’t end when there were no more stones left

Utilisation of Biomass for Sustainable Fuels & Chemicals

CM 0903 (UBIOCHEM)

The Plant Bottle

PET bottles

Crude oil

naphtha

ethylene

ethylene glycol

p-xylene

p-terephthalic acid

polyethylene terephthalate

Lignocellulosic biomass

cellulose

hemicellulose

C5 & C6

sugars

ethanol

ethylene

bio-ethylene glycol

isobutanol

fermentation

bio-p-xylene

APR

interm.

GEVO VIRENT

bio-formate

bio-p-xylene

Up to 30% plant-based

100% plant-based

same polymer advantages/disadvantages

An alternative biopolymer: Avantium process

C5 & C6 sugars

ethanol

ethylene

bio-ethylene glycol

ROH / chemocatalyst

PEF

Not the same polymer

Advantages /disadvantages: - Reduced carbon footprint - Better gas barrier properties - Recyclable - Not biodegradable

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Take Home Message

Green chemistry, biocatalysis & valorization

of waste biomass:

Green Bio-based Solutions

for a Sustainable Economy

“A good company delivers excellent products and services. A great company does all of this and strives to make the world a better place”

William Ford Jr.

Thank you for your attention 33

and Sustainable