Diapositiva 1 - Universidad Complutense de Madrid

Reacciones- Red-ox
Dr. Sinisterra
Biotransformations Group
Faculty of Pharmacy
Universidad Complutense
www.biotransformaciones.com
Metodologías para reducir compuestos carbonílicos
E3
E4
HO H
HR
HS
O
+
N
ADPR
Alcohol S
E1
Cara re
E2
HS
O
HR
H OH
+
N
ADPR
Cara si
Cara si
Alcohol R
Método fotoquímico de regeneración de la coenzima
LUZ
Captura de la luz por la célula
H2O
Sistema de transferencia electrónica
+
NADPH
O
O2
NADP
H
OH
X
CO2
S
96-98%
e.e.
X
Ciclo de Calvin
Cianobacteria
X= o-, m-, p- Clo-, m-, p-F; o-, m-, p-Me
Nakamura et al. 2003, 14, 1659-2681
Comp. Orgánicos
Los métodos experimentales para hacer la reducción se
pueden clasificar en
I)Búsqueda del biocatalizador.
a.Screening
b.Sobre expresión
c.Evolución dirigida
d.Anticuerpos catalíticos
II)Modificación de sustratos
III)Optimización de las condiciones de reacción
a.Temperatura de reacción
b.Ingeniería del disolvente. Fluidos supercríticos,
líquidos iónicos, disolventes orgánicos,
adsorbentes
c.Tratamiento de la células: alteración de la
permeabilidad con acetona o DMSO
d.Empleo de Inhibidores enzimáticos
Sustrato
reducido
Sustrato
oxidado
Reduction of C=O
Using two enzymes
NAD(P)H/H +
NAD(P)+
Sustrato auxiliar
reducido
Sustrato auxiliar
oxidado
L
S
Reductase
O
Cl
O
Cl
O
NAD(P)H/H+
O
HO H
R
NAD(P)+
CO2 + "H2"
O
H
H OH
H OH
HO
H
H
H+
O-
Formiate dehydrogenase
HO
HO
O
OH
O
Glucose dehydrogenase
HO
HO
HO
H
H
OH
H
OH
RS
O
cara
Re
RL
RL
Grupo grande
Me Et
Rs
Grupo pequeño
Me nBu
Rs
RL
e.e.(%)
S 67%
S 82%
CF3 CH3 S > 80%
cara Si
E3
E4
HO H
HR
HS
O
+
N
ADPR
Alcohol S
E1
Cara re
E2
HS
O
HR
H OH
+
N
ADPR
Cara si
Cara si
A lcohol R
Esquema cinético de reducción cetonas
proquirales
NADH
K2
Enz-A
K1
K-1
K3
Enz-A-NADH
K-2
NADH
K-3
K'-1
NADH
K'3
K'2
Enz-A'
K'-2
NADH
K4
K-4
Enz-NAD
Enz + A
K'1
Enz-R-NAD
Enz-A'-NADH
K' -3
K'-4
K'4
Enz-S-NAD
R
Enz + NAD
S
Tabla 6. Parámetros cinéticos de las oxido-reductasas aisladas.125
KM (mM)
Kcat (s-1)
pHopt.
Tªopt. (ºC)
D-Enzima-1
0.59
-----
8.5
45
D-Enzima-2
4.54
303
6.5
45
L-Enzima-1
0.13
6.20
6.5
55
L-Enzima-2
0.15
4.66
5.5-6.0
40
Enzima
HO H
D-Enzima-2
O
Cl
OEt
S
O
Cl
O
OEt
L-Enzima-1
L-Enzima-2
H OH O
Cl
OEt
R
HO
D-Enzima-2
Cl
O
O
Cl
O
H
Naftil
S
Naftil
L-Enzima-1
L-Enzima-2
H
OH
Cl
O
R
Naftil
Tabla 5. Reducción enzimática de 4-cloro-3-oxo-butanoato de etilo.
Pm (KDa)
Act. total (U)
Config. alcohol
e.e. (%)
D-Enzima-1
25
26
S
> 99 %
D-Enzima-2
1600
1442
S
> 99 %
L-Enzima-1
32
246
R
> 99 %
L-Enzima-2
32
3108
R
> 99 %
Enzima
Nakamura y cols
Figura 1.9. Alcohol deshidrogenasa de hígado de caballo.
Protein Data Bank. www.pdb.org
His 190
NH---- Figura 1.13. En el estado APO, en el centro activo de la enzima
existen 3 moléculas de agua ligadas a
aminoácidos de la maquinaria catalítica.
HN
O
N
pKa=6.7
2.7 A
Ser 138
OH
Tyr 151
H
O
H
HO
H
O
pKa= 9-9.5
2.7 A
H
2.7 A
+H3N
H
Lys 155
O
H
Protón cedido al medio
Figura 1.12. Mecanismo de reacción
de las alcohol deshidrogenasas
Pro-47/His 47. Cesión al medio de un hidrógeno
mediante formación de una molécula de agua.
H
N
NAD+
N
R
HO
His 47
H
HO
O
O
Tyr-48
N
+
H
H2N
O
H
O
H
H
Zn+2
H
N
O
N
SH
R2
CyS-46
HS
R1
His67
Cys-178
His 190
NH----
HN
Figura 1.14. Estado del NAD+ en el sitio activo unido
a la enzima formando el complejo binario
y unido a una molécula de agua
O
N
pKa=6.7
Tyr 151
OH
Ser 138
2. 5 A
H
H
O
H
O
-O
pKa= 7.6
3.7 A
4.5 A
H2N
+N
H
NAD +
O
(R)
O
+H2N
(R)
O
R
H
Lys 155
His 190
NH----
HN
Figura 1.15. Complejo ternario Enzima-NAD+-Alcohol
O
N
pKa=6.7
Tyr 151
OH
Ser 138
R1
O
R2
Alcohol
R1>R2
O
H 2N
H
-O
H
pKa= 7.6
H
(E)
(Z)
N
H
NAD +
O
(S)
O
+H2N
(R)
O
R
H
Lys 155
NH----
HN
His 190
Figure 1.1.6- Complejo ternario Enzima -NAD + - Cetona
O
N
pKa=6.7
Al exterior del
centro activo
R1
R2
Tyr 151
OH
Ser 138
2.5 A
O
2.6 A
O
H2N
H
H
O
pKa= 10
H
(E)
(Z)
N
H
NAD +
O
(S)
O
+H2N
(R)
O
R
H
Lys 155
His 190
Figura 1.17- Centro activo después de liberar la cetona,
quedando en su lugar una molécula de agua
NH----
HN
O
N
pKa=6.7
Tyr 151
OH
Ser 138
H
H
O
HO
O
H2 N
H
pKa= 10
H
(E)
(Z)
N
H
NAD H
O
(S)
O
+H2N
(R)
O
R
H
Lys 155
Figura 1.20. Disposición estérica de los oxígenos
del Asp-150 unido al Zn(II).
HO
OH
NADH
O
N
Cys 37
NH2
His 59
HS
Hs
O
N
N
Zn(II)
HR
H
O
O-
O
-O
Asp 150
Glu 60
HO
OH
(S)
Figura 1.22. Se aprecian 2 moléculas
de agua unidas al Zn (II).
(S)
NADH
O
N
NH2
(E)
(Z)
Hs
H
O
H
Hr
O
H
O
O
H
His 67
H
Ser 48
Zn(II)
N
S
H
Cys 46
N
H
Tabla 3.5. Microorganismos seleccionados como potenciales biocatalizadores para la reducción de cetonas.
Conversiones medias reducción de ciclohexanona.
Colección
& código
Nº Ref.
Bibliográficas[1
]
especie
(género)
Origen
Gongronella butleri
(Absidia butleri)
CBS 157.25
8 (176)
Indonesia
H.
filamentoso
95
Monascus kaoliang
CBS 302.78
1 (139)
Taiwan
H.
filamentoso
86
Diplogelasinospora
grovesii
IMI 171018
1 (1)
Japón
H.
filamentoso
85
Schizosaccharomyces
octosporus
NCYC 427
4 (4090)
India
Levadura
84
Absidia glauca
CBS 100.48
27 (176)
Alemania
H.
filamentoso
79
Pyrenochaeta oryzae
IMI 195679
0 (33)
Swazilandia
H.
filamentoso
70
Neosartorya
hiratsukae
CBS 294.93
1 (48)
Japón
H.
filamentoso
66
Echinosporangium
transversale
CBS 357.67
1 (1)
Nevada
USA.
H.
filamentoso
55
Actinoplanes sp.
DSMZ
43031
(144)
-
Actinomicet
os
53
Nocardia uniformis
ATCC
21806
3 (1106)
-
Actinomicet
os
48
Cepa
a
Media de las conversiones obtenidas en los tres ensayos. (-) Procedencia desconocida
J.D. Carballeira- Tesis Doctora. Fac. Farmacia . UCM 2003
Grupo
%
conv.
a
CoMFA model for reduction of ketones
CARBALLEIRA et al. High throughput screening and QSAR-3D/CoMFA : Useful tools to design predictive models of substrate specificity
for biocatalysts Molecules 2004, 9 pp 673-693
A)
B)
The CoMFA models obtained for depict the
substrate requirements common to G. candidum (A) and S. octosporus (B)
During the evaluation of the results obtained in the reduction reactions
and the CoMFA models we concluded that the pattern of substrate
affinity was similar between the ADHs from different strains.
This fact was astonishing as far as ADHs present in microbial strains –
distant from the taxonomic point of view - were showing considerable
similarities in substrate scope. In the bibliography different genetic
and biochemical publications pointed out the structural relationships
between the different ADHs, with special homology at the active site and
even the hypothesis about the existence of a common ancestor based
in structural studies of different proteins of the same family.
The building of 3D-QSAR/CoMFA models has several potential utilities
that justify the use of this theoretical method for biotransformations
and directed evolution, especially when there is no reference structure
of the enzyme available (Carballeira et al 2006).
Indeed, when an enzyme or library of enzymes is screened against a
collection of substrates it is not easy to have a 3D overview of the capabilities
of the enzyme. CoMFA is a good option to display a comprehensive model
from the large amount of data generated that could serve as reference for the
rational interpretation of the results and furthermore for the selection a priori
of new potential substrates.
Nowadays, new biocatalysts could be created by directed evolution enhancing
the activity and selectivity of a known enzyme structure (Reetz and Carballeira 2007)
and overcoming some of the catalytic restrictions of the natural protein.
In this way CoMFA models are especially interesting when the x-ray structure
of the enzyme is not available with the substrate docked at the active site
because CoMFA models generated from different docking ways of the
ligands may help in the proper location of the substrate in the binding site.
CoMFA models are predictive. Indeed, to test this predictiveness
of the model, we tried to hypothesize the activity of the biocatalysts against
two
new cyclic substrates: pulegone and menthone (Carballeira et al 2006).
The substrates must be aligned according to the fitting model. After the
fitting
we observe the CoMFA zones occupied by these substrates.
We can see that menthone fit into the model and so, it can be reduced by
the ADHs. Indeed 52% yield of menthol – the only product - is achieved.
According to the model, pulegone would not be reduced by the strains, due
to the fitting of the double bond in an electrostatic zone where the presence
of
negative charge is not allowed (blue zone) (previous slide). Using the same
model
we can predict that acetophenone or α- bromo-acetophenone would be
reduced
by Rhodotorula sp AS 2241 giving (S)-1-phenylethanol (34.7% yield,
>99.5% e.e)
and (R )-2-bromo-1-phenylethanol (20% yield, > 99% e.e.) as predicted by
our CoMFA model.
This result indicates the evolutive conservation of active site of ADHs.
O
Pulegone
O
Menthone
Table 5- Effect of the reaction conditions on the selectivity of reduction of the
1-acetoxy-3-phenoxy`ropan-2-one by Baker’s yeast. Substrate: 500mg; sodium
phosphate buffer (0.15M), 200ml; yeast wet 12g o 2.5 g lyophilized. Co-substrate glucose: 5g. .
Ar
Baker's yeast
OAc
O
Ar
O
Yeast
Aryl group
Wet caked
O
OAc
HO H
pH
Time (h)
Yield(%)
e.e(%)
S>R
Ph
Non
controlled
3
n.r.
52
Wet caked
Ph
7
1
84
80
Wet caked
Ph
8
1
n.r
78
Lyophilized
Ph
7
1.5
n.r
64
Wet caked
2-iPrphenyl
7
1
36
28
3-Cl-phenyl
7
2
38
93
4-Cl-phenyl
7
2
65
61
2-Me-phenyl
7
2
32
65
3-Me-phenyl
7
2
66
77
4-Me-phenyl
7
4
36
52
2,6-diMe-phenyl
7
1.5
60
>95
2,4,6-dri-Mephenyl
7
6
58
95
•Egri et al Baker’s yeast mediated steoselective biotransformation of 1-acetoxy-3-aryloxypropan-2-ones. Tetrahedron: Asymmetry 9, 271-283.(1998)
Table 6.- Reduction of 1-chloro-3(1-naphthyloxy)propan-2-one using different microorganisms
. Growing or resting cells conditions (Martinez-Lagos et al 2004).
Resting cells b
Fermentor conditions a
Microorganism
Yield.
c (%)
Enantiopref.d
e.e.c
(%)
M. kaoliang
55
R>S
1
5
R>S
10
N. hiratsukae
39
R>S
6
10
R>S
90
Y. lipolytica CECT
1240
20
S>>R
99
87
S>>>R
99
P. mexicana
CECT 11015
19
R>>S
85
85
R>>>S
95
S. bayanus CECT
1969
15
R>S
52
40
R>>S
87
S. cerevisiae (Type
II)
12
S>R
70
30
S>>R
70
S. cerevisiae
CECT 1317
13
R>S
70
27
R>S
71
a Reductions
Yield. Enantiopr e.e.c
c (%)
ef.d
(%)
were carried out with growing cells (48 hrs.).
were carried out using 0.19 mmol of substrate and 4 g of sucrose in 25 mL of reaction media (0.1M sodium phosphate buffer, pH 8.0). Incubations were performed at 30 ºC for 48 h.
c Molar Conversion and e.e. determined by chiral HPLC .
d Absolute configuration of the halohydrin was assigned by specific rotation value.
b Reductions
The synthesis of (R)-3-Fluoro-4-[[hydroxyl(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl
– 2 –naphthalenyl)acetyl]amino] benzoic acid (1), a retinoic acid receptor gamma
-specific agonist with dermatological and anti-tumour activities can be performed
by stereoselective reduction of the ketoester 2 or of the ketoacid 3 or of the
ketoamide 4. These processes have been performed in
Bristol Myers Squibb Research Institute (USA)
O
OR
O
HO
microbial
reduction
OR
O
2
O
OH
microbial
reduction
HO
OH
O
HO
O
H
N
3
F
OR
O
1
microbial
reduction
O
H
N
R= H,
F
OR
O
4
O
O
•Patel et al. Enantioselective microbial reduction of 2-(1’,2’,3’,4’-tetrahydro-1’,1’,4’,4’-tetramethyl-6’-naphtalenyl)acetic acid and its ethyl ester. Tetrahedron: Asymmetry 13, 349-355 (2002).
Looking for anti-Prelog’s rule specificity in order to obtain the (R)-alcohol, precursor of 1, several interesting
microorganisms were selected by BMS The reductions were performed using glucose as second substrate (25mg/ml) in
order to regenerate the coenzyme NADPH/H+.
Aureobasidium pullutans SC13894 can reduce the keto-ester 2 and the keto-amide 4.
The keto-acid is efficiently reduced by Candida maltosa SC 16112 and Candida utilis SC 13983 that show specificity
for the keto-acid.
Finally, Hunsemula anamola SC 16158 shows a good selectivity versus 4
The cell extract of A. pullutans shows better activity than the whole cells, especially if NADPH is added as
coenzyme This positive effect can be associated to the diminution of the mass transfer through the cell wall and/or
membrane that is the rate controlling step in the whole cell catalyzed reactions.
pH=7.0, 20% w/v wet cells, 1mg/ml ketone in MeOH 25mg ml-1 of glucose. T=28ºC. Reaction time = 48hr. .
Substrate
Product yield(%)
e.e.(%)
2
98
98 -R
4
12
96-R
Candida maltosa SC 16112
3
56
99-R
Candida utilis SC 13983
3
64
98-R
Hunsemula anamola SC 16158
4
20
85-R
Aureobasidium pullutans
SC13894a
3b
98
98-R
Aureobasidium pullutans
SC13894a
3c
12
98-R
Aureobasidium pullutans
SC13894a
4b
60
96-R
Aureobasidium pullutans
SC13894a
4c
10
97-R
Microorganism
Aureobasidium pullutans
SC13894
Syntheses of the key intermediate of
34- squalene synthase inhibitor
Synthesis of β-adrenergic receptor
-137The synthesis is performed in a 8L fermentor
Pro-R / cara re
Pro-R / cara re
Pro-R / cara re
Pro-R / cara si
Pro-S / cara si
Pro-R / cara si
Cara si
H
H
R
S
O
H
O
N H
N
R
Cara re
2
S
L
H
R
S
O
N H
N
R
2