process development of transaminase catalyzed reactions ...ucbepad/rscbiocatpdf/truppo rsc...
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Process development of transaminase catalyzed reactions for large scale
industrial use
Matthew D. Truppo
Chiral Amines
• Useful building blocks in stereoselective synthesis• Present in a wide range of pharmaceutical intermediates (>70%)
FF
FN
OH NH2
NN
N
CF3
H3PO4 H2O
Merck’s Januvia for type 2 Diabetes$2B/yr projected sales
Chemical Methods to Produce Chiral Amines
1. Crystallization with chiral carboxylic acids (resolution of racemic amine)
½ of material wasted
R1
NH2
R2 R3
R4
O
OHR1
NH3+
R2 R3
R4
O
O-R1
NH3+
R2 R3
R4
O
O-
crystalline in solution
2. Reductive Amination
difficulties = making starting material and cleaving X
multiple step synthesis
R1
N
R2
X
R1
HN
R2
Xreduction
R1
NH2
R2
R1 R2
O
R1 R2
OH
R1 R2
OMs
R1 R2
N3
R1 R2
NH2Reduction ActivationAzide
Displacement Reduction
TRANSAMINASE
Transaminase Reactions: Challenges and Goals
R1
O
R2 R1
NH2
R2
Transaminase
N
O
HOOPO3
N
H2N
HOOPO3
NH2
O
OH
O
O
OHTransaminase
R1
O
R2 R1
NH2
R2
Transaminase
N
O
HOOPO3
N
H2N
HOOPO3
NH2
O
OH
O
O
OHTransaminase
Equilibrium
O
O
OHInhibition
Goals:
1. Evaluate transamination equilibrium and inhibition issues
2. Develop general methods for driving reactions to completion
• Novel rapid transaminase screening system
• Process development activities at 50mL scale
R1 R2
NH2
Inhibition
Use transaminases for the practical and economic industrial synthesis of chiral amines
Why Use Acetophenone as Model Substrate?
O NH2
• Representative building block for chemical synthesis
• One of the most difficult transformations:
• acetophenone substrate solubility issues
• product methylbenzylamine inhibition/deactivation issues
• substrate to product equilibrium issues
• If we can run this reaction in the synthesis direction, we should be able to run almost any substrate
Transamination Equilibrium
Driving Acetophenone Transamination Equilibrium
0
10
20
30
40
50
60
70
0 5 10 15 20 25
time (hours)
% c
onve
rsio
n 50mM alanine (1X eq.)
500mM alanine (10X eq.)
LDH, GDH system (10X eq.) • Transaminase still active
• Equilibrium strongly favors starting ketone and amine donor
• Excess amine donor isn’t enough for high productivity process
50mM acetophenone
O NH2Transaminase
NH2
O
OHO
O
OH
Equilibrium
Transaminase Inhibition
O NH2Transaminase
NH2
O
OHO
O
OH
Inhibition by Pyruvate
0
10
20
30
40
50
60
70
0 20 40 60 80 100 120
pyruvate (mM)
% in
hibi
tion
Inhibition by Methylbenzylamine
0
10
20
30
40
50
60
70
80
90
0 50 100 150MBA concentration (mM)
% in
hibi
tion
Driving Transamination ReactionsLactate Dehydrogenase System
• Pyruvate consumption drives equilibrium and eliminates pyruvate inhibition• GDH / NADH cofactor recycle = proven efficient, cost effective system• Acid formation enables reaction tracking
R1
O
R2 R1
NH2
R2
Transaminase
NH2
O
OH
O
O
OH
NAD
GlucoseDehydrogenase
GlucoseGluconolactoneGluconic Acid
NADH
Lactate DehydrogenaseOH
O
OH
Driving Transamination Reaction Using LDH System
Driving Acetophenone Transamination Equilibrium
0
10
20
30
40
50
60
70
0 5 10 15 20 25
time (hours)
% c
onve
rsio
n 50mM alanine (1X eq.)
500mM alanine (10X eq.)
LDH, GDH system (10X eq.) • Equilibrium strongly favors starting materials
• LDH system drives equilibrium and eliminates pyruvate inhibition
• reaction proceeds >20X further
NH2
Transaminase
NH2
O
OHO
O
OH
O
GlucoseDehydrogenase
Glucose Gluconolactone
NADHNAD+
LactateDehydrogenase
OH
O
OH
Colorimetric Screen of Acetophenone Transamination
0
5
10
15
20
25
30
35
40
45
50
101 103 105 107 109 111 113 115 117
transaminase (ATA#)
rate
(uM
/min
)
pH Indicator Colorimetric Assay
HPLC Assay
• Very precise quantification of transaminase activity• Directly scale this screening system up to first delivery scale
O NH2Transaminase
NH2
O
OH
O
O
OH
NAD
GlucoseDehydrogenase
GlucoseGluconolactoneGluconic Acid
NADH
Lactate DehydrogenaseOH
O
OH
pH indicator
Why not track NADH consumption?
pH Based Colorimetric Transaminase Activity Assay
Transaminase Screening System
0
10
20
30
40
50
60
70
80
0 10 20 30
time (hours)
% c
onve
rsio
n
HPLCpH indicator
Transaminase Screening System
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 10 20 30
time (hours)
abs
560n
m
pH indicator
Running Transamination with pH Indicator
• we have a precise colorimetric assay for enzyme activity and quantitative determination of product formation
• tuning buffer strength = track entire reaction profile
Microscale Process Development Using pH Indicator Assay
Transaminase Rate vs. pH (20C)
6.0
8.0
10.0
12.0
14.0
16.0
18.0
6.5 7 7.5 8 8.5 9
pH
rate
(uM
/min
)
• 100uL scale reactions run in plate spec for process development• Temperature activity/stability and pH activity profiles obtained• Inhibition studies, substrate and enzyme loading can be analyzed
• Note these profiles are for the complete ATA/LDH/GDH reaction system not just a single enzyme (great for direct scaling of process)
Transaminase Rate vs. Temperature
0
10
20
30
40
50
60
70
80
90
100
10 20 30 40 50
temperature (C)
rate
(uM
/min
)
0
1
2
3
4
5
6
7
1/2
life
(hou
rs)HPLC assay
colorimetric
stabiltyactivity
Transamination Scale-Up Directly from Screening Conditions to Reactor
• conversion tracked by automated pH control (NaOH addition) –red points show HPLC conversion
• >95% conversion of 50mM acetophenone (6g/L) with 1M alanine• Both S- and R-methylbenzylamine can be made with >99% ee
20mL Transamination of 50mM Acetophenone
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0.0 2.0 4.0 6.0 8.0 10.0
time (hours)
2M N
aOH
(mL)
0
10
20
30
40
50
60
70
80
90
100
conv
ersi
on (%
)
Methylbenzylamine Aqueous Conc. with Resins
0
50
100
150
200
250
300
350
400
450
0 10 25 50 100 200 400
resin (g/L)
Aqu
eous
MBA
con
c. (m
M)
ion exchange resinadsorbent resin
50% inhibition
80% inhibition
In-situ Product Extraction Using Resins
• Amberlite Ion Exchange Resin (IRP-69) and Polymeric Adsorbent Resin
• Ion exchange resin at 200g/L reduces a 420mM (50g/L) MBA chargeaqueous concentration to ~25mM (below the 50% inhibition barrier)
30mL Scale Resin/LDH Transamination System
• reactions run with automated pH control in Multimax reactors
• 50g/L (420mM) acetophenone reactions proceeded to >95% conversion with 200g/L resin
• isolation of product MBA requires simple dilute NaOH wash of ion exchange resin
NH2
Transaminase
NH2
O
OHO
O
OH
GlucoseDehydrogenase
Glucose Gluconolactone
NADHNAD+
O
LactateDehydrogenase
OH
O
OH
50g/L Transamination of Acetophenone
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60
time (hours)
% c
onve
rsio
nno resin100g/L resin200g/L resin
Ion-exchangeresin
Driving Transamination ReactionsAmino Acid Dehydrogenase System
• Catalytic alanine system• amino donor generated in situ• Eliminates pyruvate inhibition• Cheap ammonia is amine donor
NH2
O
OH
O
O
OH
NAD
GlucoseDehydrogenase
Glucose Gluconolactone Gluconic Acid
NADH
Amino Acid DehydrogenaseH2O Ammonia
R1
O
R2 R1
NH2
R2
Transaminase
Transamination Using Catalytic Alanine SystemAcetophenone Transamination (Catalytic Alanine
- LAADH System)
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
time (hours)
conv
ersi
on (%
)
• 50mM acetophenone (6g/L) with 25mM pyruvate reaction proceeded to complete conversion
• Slower rate than LDH system, but uses a 40X reduction in alanine
Actually used no amine donor that’s accepted by transaminase!
NH2 O
R1
O
R2 R1
NH2
R2
Transaminase
Driving Transamination ReactionsIsopropylamine System
• Single enzyme system with very cheap amino donor• Equilibrium can be driven via acetone removal• Limited to transaminases that accept isopropylamine (IPM)• Transaminases must also be stable in high IPM concentrations
Transamination of Acetophenone using Isopropylamine Amino Donor
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40time (hours)
conv
ersi
on (%
)
ATA-113ATA-117
Isopropylamine Transamination System
• Both S-methylbenzylamine (using ATA-113) and R-methylbenzylamine (using ATA-117) can be produced with >99% ee
• 50mM (6g/L) acetophenone with 1M IPM proceeds to >95% conversion
Driving Transaminations via Spontaneous Secondary Intramolecular Product Reactions
Transaminase
NH2 O
OEt
O O
OEt
NH2 O
NH
O
• transamination of ethyl 4-acetylbutyrate
• spontaneous cyclization of amine product to 6-methyl-2-piperidone product
Transaminase
NH2 O
OEtO
OEtNH2
O O
NH
O
• transamination of ethyl levulinate
• spontaneous cyclization of amine product to 5-methyl-2-2-pyrrolidinone
50g/L Ethyl 4-Acetylbutyrate Transamination
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15time (hours)
% c
onve
rsio
n
Ethyl 4-acetylbutyrate Demo at 50 mL Scale and 50 g/L
• ATA-113 and ATA-117 produce >99% ee S or R product with >95% conversion
Summary
• 3 process methods for driving transamination reactions demonstrated in 50mL scale reactors• Can produce both R- and S-amines using Codexis’transaminase library with:
>99% ee and >90% yield
(Method 2, AADH/GDH)
(Method 3, isopropylamine)
(Method 1, LDH/GDH)
NH2
Transaminase
NH2
O
OHO
O
OH
GlucoseDehydrogenase
Glucose Gluconolactone
NADH
Amino Acid DehydrogenaseNH4
+H2O
NAD+
O
LactateDehydrogenase
OH
O
OH
GlucoseDehydrogenase
Glucose Gluconolactone
NADHNAD+
NH2 OO NH2Transaminase
NH2
O
OH
O
O
OH
NAD
GlucoseDehydrogenase
GlucoseGluconolactoneGluconic Acid
NADH
Lactate DehydrogenaseOH
O
OH
• rapid high-throughput pH based colorimetric assay for transaminase activity• microscale process development that can be scaled up directly for material delivery
Screening hit = first material delivery
Rapid Screen/Micro-scale Development Process Development
(Isopropylamine System)
(LDH/GDH System)
R1
NH2
R2
Transaminase
NH2
O
OHO
O
OH
GlucoseDehydrogenase
Glucose Gluconolactone
NADHNAD+
R1
O
R2
LactateDehydrogenase
OH
O
OH
NH2
O
ion-exchangeresin
R1
N
Summary – Transamination Manufacturing Processes (> 50 g/L substrate)
• demonstrated transamination reactions at >50 g/L substrate loading• in situ product removal with an ion exchange resin to selectively bind amine product• spontaneous intramolecular secondary reaction of amine product• demonstrated on the production of >40 amines with >99% ee
Acknowledgements
Prof. Nick Turner