combinatorial evolution of site- and enantioselective catalysts for polyene epoxidation guillaume...
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Combinatorial evolution of site- and enantioselective catalysts for polyene epoxidation
Guillaume Pelletier Literature meeting - November 20th 2012
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Analysis of biosynthetic patways reveals functional group selectivity
H
Taxadiene
[O]
HOH
[O]
HOH
RO
ORRO
HOH
RO
ORRO OR
OAc
HRO
OAcO OH
OOH OBz
[O]
[O]
[O] = Cytochrome P450 mono-oxygenases
Taxol
Taxinine E or J
HRO
ORRO OR
OR OR
OH
2-Acetoxybrevifoliol
[O]
O
MeMe
BrMe
O
Me2Zn
Me3Al
Taxadien-5-ol
For a review, see: Clardy, J.; Walsh, C. Nature 2004, 432, 829-837.Walker, K.; Croteau, R. Phytochemistry 2001, 58, 1-7.
Mendoza, A.; Ishihara, Y.; Baran, P. S. Nature Chem. 2012, 4, 21-25.
Analysis of biosynthetic patways reveals functional group selectivity
HO
MeO
N
MeH
OH
MeO
Reticuline
HO
MeO
N
MeH
O
MeO
Salutaridine
O
RO
N
MeH
HO
H
Morphine (R = H)Codeine (R = Me)
Salutar idinesynthase
NADPH H+ O2 NADP+ 2 H2O
NADPH
NADP+
HO
MeO
N
MeH
OH
MeO
Acetyl-CoA
Salutaridinol
O
MeO
N
MeH
MeO
Thebaine
[O]/NADPH
For a review, see: Clardy, J.; Walsh, C. Nature 2004, 432, 829-837.Novak, B. H.; Hudlicky, T.; Reed, J. W.; Mulzer, J.; Trauner, D. Curr. Org. Chem. 2000, 4, 343-362.
Calderon, S. N. et al. J. Med. Chem. 1997, 40, 695.
Analysis of biosynthetic patways reveals functional group selectivity
HO
MeO
N
MeH
OH
MeO
Reticuline
HO
MeO
N
MeH
O
MeO
Salutaridine
O
RO
N
MeH
HO
H
Morphine (R = H)Codeine (R = Me)
Salutar idinesynthase
NADPH H+ O2 NADP+ 2 H2O
NADPH
NADP+
HO
MeO
N
MeH
OH
MeO
Acetyl-CoA
Salutaridinol
O
MeO
N
MeH
MeO
Thebaine
[O]/NADPH
For a review, see: Clardy, J.; Walsh, C. Nature 2004, 432, 829-837.Novak, B. H.; Hudlicky, T.; Reed, J. W.; Mulzer, J.; Trauner, D. Curr. Org. Chem. 2000, 4, 343-362.
Calderon, S. N. et al. J. Med. Chem. 1997, 40, 695.
Me
Me Me Me 2
Squalene
squalene 2,3-oxide cyclase
Me
Me
O
Me Me
Me Me Me
Me
2,3-squalene oxide70-85%
Me
Me
MeT
Me
MeMe
MeMe
H
squalene 2,3-oxide cyclase
Me
Me
MeT
Me
MeMe
MeMe
H
O
2.1-2.5%
Me
Me Me Me O
OMe Me
Me Me Me O
OHOH
OHsqualene 2,3-oxide cyclase
2-4%Farnesyl methyl ester
32610
711
Enzyme-mediated oxidation does preclude generality…
Van Tamelen, E. E.; Heys, R. J. J. Am. Chem. Soc. 1975, 97, 1252-1253.
Small synthetic molecules meets some of these challenges
Me
Me Me
OH
N
N
Ph
Ph
Ph
Ph
O
O
O V (Oi-Pr)2
(1 mol%)
TBHP (70% aq), DCM
Me
Me Me
OHO
68%, 95% ee
Me
Me Me
OH
30% aq. H2O2 (1.5 equiv)CHCl3/brine, 40 °C, 24 h
Me
Me Me
OHO
82%, 84% ee
Nb(OiPr)5 (4 mol%)ligand (5 mol%)
NH HN
OH HOArArligand :
Yamamoto
Katsuki
Zhang, W.; Basak, A.; Kosugi, Y.; Hoshino, Y.; Yamamoto, H. Angew. Chem., Int. Ed. 2005, 44, 4389-4391.Egami, H.; Oguma, T.; Katsuki, T. J. Am. Chem. Soc. 2010, 132, 5886-5895.
Small synthetic molecules meets some of these challenges
Me
Me Me
OH
N
N
Ph
Ph
Ph
Ph
O
O
O V (Oi-Pr)2
(1 mol%)
TBHP (70% aq), DCM
Me
Me Me
OHO
68%, 95% ee
Me
Me Me
OH
30% aq. H2O2 (1.5 equiv)CHCl3/brine, 40 °C, 24 h
Me
Me Me
OHO
82%, 84% ee
Nb(OiPr)5 (4 mol%)ligand (5 mol%)
NH HN
OH HOArArligand :
Yamamoto
Katsuki
Zhang, W.; Basak, A.; Kosugi, Y.; Hoshino, Y.; Yamamoto, H. Angew. Chem., Int. Ed. 2005, 44, 4389-4391.Egami, H.; Oguma, T.; Katsuki, T. J. Am. Chem. Soc. 2010, 132, 5886-5895.
Small synthetic molecules meets some of these challenges
Barlan, A. U.; Basak, A.; Yamamoto, H. Angew. Chem., Int. Ed. 2006, 45, 5849-5852.Chang, S.; Lee, N. H.; Jacobsen, E. N. J. Org. Chem. 1993, 58, 6939-6941.
Me
Me
O
Me Me
Me Me Me
Me
53%, 76% ee
Me
Me
O
94%, 66% de
Me
Me
O
82%, 76% ee
O
98%, 95% ee
MoO2(acac)2 (2 mol%)CHP (1.5 equiv)
DCM, 18-46 h, 0 °CCHP =
O
OH
N
N
OHOH
O
OCR3
CR3
R = 4-(tertbutyl)phenyl
(2 mol%)
Yamamoto
Small synthetic molecules meets some of these challenges
O
O
PhOCO2Me
3
O
O
PhOO
CO2Me3
62%, 82% ee, 8:1 trans:cis
O
CO2Me
stepsO
OCO2Me
3
1) NH3, MeOH, rt2) MnO2 (act.)
Mn(III)salen (4 mol%)aq. NaOCl (1.2 equiv)
4-PhPyr-N-Oxide (20 mol%)pH 11.3 buffer
Leukotriene A4 methyl ester
Jacobsen
Me
Me
O
Me Me
Me Me Me
Me
53%, 76% ee
Me
Me
O
94%, 66% de
Me
Me
O
82%, 76% ee
O
98%, 95% ee
MoO2(acac)2 (2 mol%)CHP (1.5 equiv)
DCM, 18-46 h, 0 °CCHP =
O
OH
N
N
OHOH
O
OCR3
CR3
R = 4-(tertbutyl)phenyl
(2 mol%)
Yamamoto
Barlan, A. U.; Basak, A.; Yamamoto, H. Angew. Chem., Int. Ed. 2006, 45, 5849-5852.Chang, S.; Lee, N. H.; Jacobsen, E. N. J. Org. Chem. 1993, 58, 6939-6941.
Template-directed internal epoxidation of polyenes
X
2
OSi
O
Br
BrO
OH
t-Bu t-Bu
X
2
OSi
O
Br
BrO
OH
t-Bu t-Bu
5 92 3
1) Im2CO, DCM, rt, 30 min2) H2O2 (dry in Et2O/EtOAc) 60 °C, DCM [0.5 mM], then 20 °C
Epoxide distr ibution
X =
Me Me
Me
3
If spacer =
O
OH
NO2
O2N
345
5-prenol selective(5:89:6 for 6:5:4)
Gnanadesikan, V.; Corey, E. J. J. Am. Chem. Soc. 2008, 130, 8089-8093.
The goal of the present study
Me
Me
Me Me
OH
Peptide A Me
Me
Me Me
OHO
Peptide B Me
Me
Me Me
OH
* *
O* *
Me
Me
Me Me
OHO* *
Peptide C
2,3-epoxyfarnesol
10,11-epoxyfarnesol
6,7-epoxyfarnesol
Combinatorial EvolutionaryCatalyst/Lead Optimization
Me
Me
Me Me
OH
Me
Me
Me Me
OHO* *
iPrO2C CO2iPr
HO OHTi(OiPr)4, 4 Å MS
t-BuOOH 50 to 20 °C
Six reports (1993-2010)83% 95% ee
Sharpless Epoxidation
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Literature precedents on small peptide mediated epoxidation
Peris, G.; Jakobsche, C. E.; Miller, S. J. J. Am. Chem. Soc. 2007, 129, 8710-8711.Kolundzic, F.; Noshi, M. N.; Tjandra, M.; Movassaghi, M.; Miller, S. J. J. Am. Chem. Soc. 2011, 133, 9104-9111.
NH
NPhth
N1-Anthr
NPhthHO
NH
O
NPhth1-Anthr
Sc(OTf)3
(1.0 equiv)
tol, reflux
35%, 98% ee
Peptide 2 (10 mol%)DMAP (5 mol%)H2O2 (1.2 equiv)
DIC (1.2 equiv)CHCl3 (0.1M)
0 °C, 20 h
Peptide 2 = N-(Boc)Asp-Pro-Val-Leu-Val(OMe)
HN
HO2C
O
N
Boc
O
HN
O
Me
Me
HN
NH
CO2MeMe
MeO
MeMe
O
O
NH
Ph
88%, 90% ee
Peptide 1 = N-(Boc)Asp-Pro-Val(N-((R)-MePh))
HN
HO2C
O
N
Boc
O
HN
O
Me
Me
HN
Me
Peptide 1 (10 mol%)DMAP (10 mol%)
UreaH2O2 (2.5 equiv)
DIC (2.5 equiv)tol (0.1M)4 °C, 33 h
O
O
NH
Ph
76%, 92% ee
Proposed catalytic cycle for the asymmetric epoxidation
Peris, G.; Jakobsche, C. E.; Miller, S. J. J. Am. Chem. Soc. 2007, 129, 8710-8711.Kolundzic, F.; Noshi, M. N.; Tjandra, M.; Movassaghi, M.; Miller, S. J. J. Am. Chem. Soc. 2011, 133, 9104-9111.
O
OBn
HN
CO2H
Boc N C N iPriPr
O
OBn
HN
Boc
O
O N
HNiPr
iPrO
OBn
HN
Boc
O
OO
H
R1R2
R1R2
O
O
OBn
HN
Boc
O
OO
O
NH
Boc
O
BnO
DIC
O
O N
HNiPr
iPr
DMAP, H2O2
or DMAP-N-Oxideor H2O2
First screening of catalysts
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
Conditions
Conditions O
OH(10 mol%)
HOBt (10 mol%)DMAP (10 mol%)DIC (1.0 equiv)
H2O2 (1.0 equiv)
Cl
O
OOH
(1.0 equiv)
Na2HPO4 (2.0 equiv)DCM/H2O
Conversion
67%
65%
2.6 2.2 1.0
2.7 2.2 1.0
Epoxide distribution
• With initial peptide catalyst screening, the authors chose to run the reactions at low conversions in order to allow a preleminary assessment of catalyst krel
Methodology employed for catalyst design
Lam, K. S.; Lebl, M.; Krchňák, V. Chem. Rev. 1997, 97, 411-448.Furka, A.; Sebestyen, F.; Asgedom. M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493.
• The one-bead-one-compound concept is based on the fact that combinatorial beads beads prepared from the « split-pool synthesis » contain single beads displaying one
type of compounds although there may be 1013 copies on a 100 μm bead
One-bead-one-compound and split-pool synthesis
Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84.
Native apitope
On-bead ligands*
Ki (nM) for monoclonal antibody (-endorphins)
Tyr-Gly-Gly-Phe-Leu (YGGFL)
Tyr-Gly-Gly-Phe-Gln (YGGFQ)
Tyr-Gly-Gly-Phe-Ala (YGGFA)
Tyr-Gly-Gly-Phe-Thr (YGGFT)
Tyr-Gly-Gly-Leu-Ser (YGGLS)
Tyr-Gly-Ala-Leu-Gln (YGALQ)
Tyr-Gly-Gly-Met-Gln (YGGMQ)
17.5 3.2
15.0 1.7
32.9 2.0
36.9 7.7
726 134
1980 303
8780 1500
* Structure of bead sequence was determined using peptide microsequencer with Edman degradation and HPLC/MS analysis
One-bead-one-compound and split-pool synthesis
Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84.
Native apitope
On-bead ligands*
Ki (nM) for monoclonal antibody (-endorphins)
Tyr-Gly-Gly-Phe-Leu (YGGFL)
Tyr-Gly-Gly-Phe-Gln (YGGFQ)
Tyr-Gly-Gly-Phe-Ala (YGGFA)
Tyr-Gly-Gly-Phe-Thr (YGGFT)
Tyr-Gly-Gly-Leu-Ser (YGGLS)
Tyr-Gly-Ala-Leu-Gln (YGALQ)
Tyr-Gly-Gly-Met-Gln (YGGMQ)
17.5 3.2
15.0 1.7
32.9 2.0
36.9 7.7
726 134
1980 303
8780 1500
* Structure of bead sequence was determined using peptide microsequencer with Edman degradation and HPLC/MS analysis
Initial screening (with parallel peptide synthesis)
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
Conditions
Conditions
Conversion
13% 1.0 1.0 3.9
Epoxide distribution
Resin bound peptide (cat.)HOBt, DIC, DMAP (0.3 equiv)
H2O2 (1.0 equiv), DCM
N-(Boc)Asp-Pro-DTyr(O-tBu)-Asn(Trt)-Leu HN
Boc
HO2C
N
O
O
NH
OtBuMe
O
HN
O
NH
NH
O
O
HN
Trt"Peptide 6"
i i + 1 i + 2 i + 3 i + 4
Initial screening (with parallel peptide synthesis)
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.Lichtor, P. A.; Miller, S. J. ACS Comb. Sci. 2011, 13, 321-326.
Split-pool optimization and synthesis of a large OBOC library (iterative approach)
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.Lichtor, P. A.; Miller, S. J. ACS Comb. Sci. 2011, 13, 321-326.
• The resulting library possess a theorical size of about 3000 unique peptide sequences
(for the first directed library)
OBOC library results towards epoxidation of farnesol
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
iConversion
13% 1.0 1.0 3.9
Epoxide distributionPeptide sequence
i + 1 i + 2 i + 3 i + 4 i + 5
N-(Boc)Asp Pro D-Tyr(O-tBu) Asn(Trt) Leu (6 IS)
13% 1.3 1.0 8.2N-(Boc)Asp Pro Asn(Trt) Thr(OBn) (7 DL1)
15% 1.2 1.0 18.0N-(Boc)Asp Pro Asn(Trt) D-Phe Thr(OBn) Asn(Trt) (8a DL2)
12% 1.0 1.0 14.6N-(Boc)Asp Pro Asn(Trt) D-Phe Pro Asn(Trt) (9a DL2)
D-Phe
***IS = Initial Screening DL1 = Directed Library #1 DL2 = Directed Library #2
Resin bound peptide (cat.)HOBt, DIC, DMAP (0.3 equiv)
H2O2 (1.0 equiv), DCM
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Identification of peptides via sequencing and HPLC/MALDI-QToF analysis
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
HN
Boc
HO2C
N
O
O
AA3
AA4
AA5
O
NH
O
HN
O
NH
HN
Boc
HO2C
N
O
O
AA3
AA4
AA5
O
NH
O
HN
O
SMe
O
BrCN 70% in TFAthen 33%H2O/MeCN
H2N+
Resynthesis and « in solution » trials with hits
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Asn(Trt)H2NSPPS
Asn(Trt)ProDPheAsn(Trt)ProBocAsp(OFm)
4:1:1DCM/TFA/AcOH
HN
O
OFm
Boc O
N
O
NH
O
O
HN
Ph
O
N
O
OHO
NHTrt
OTrtHN
HN
O
OH
Boc O
N
O
NH
O
O
HN
Ph
O
N
O
OMeO
NHTrt
OTrtHN
1) EDC, HOBtMeOH
2) Et2NH, DCM
32% overall yield
Resynthesis and « in solution » trials with hits
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
HN
O
OH
Boc O
N
O
NH
O
O
HN
Ph
O
N
O
OMeO
NHTrt
OTrtHN
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
HN
Boc
HO2C
N
O
O
NH
O
HN
NH
OOBn
O
HN
O
NHTrtO
OMe
O
NHTrt
Yield (%)Peptide ee (%) A Selectivity
8b
9b
>100
>100
82
8681%
n.d.
Peptide 8b or 9b (10 mol%)HOBt, DMAP (10 mol%)
DIC (1.0 equiv)H2O2 (2.0 equiv)
4 °C, 3 h
Substrate scope with optimized 9b catalyst
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
R1 OH
Peptide 9b (10 mol%)HOBt, DMAP (10 mol%)
DIC (1.0 equiv)H2O2 (2.0 equiv)
4 °C, 3 h
R2
R1 OH
R2
O
Me
Me
Me Me
OHO
Me
Me Me
OHO
Me
Me Me
O
OH
Me
Me
OHO
81%, 86% ee, ~100% Selectivity
79%, 93% ee, ~100% Selectivity 80%, 87% ee, ~100% Selectivity 75%, 92% ee
Biased 2nd OBOC directed at 6,7-selective epoxidation
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
iConversion
13% 1.0 1.0 3.9
Epoxide distributionPeptide sequence
i + 1 i + 2 i + 3 i + 4 i + 5
N-(Boc)Asp Pro D-Tyr(O-tBu) Asn(Trt) Leu (6 IS)
15% 1.2 1.0 18.0N-(Boc)Asp Pro Asn(Trt) D-Phe Thr(OBn) Asn(Trt) (8a DL2)
16% 1.5 2.9 1.0N-(Boc)Asp D-Pro Thr(OBn) Asn(Trt) Tyr(O-tBu) (12a DL3)
***IS = Initial Screening DL2 = Directed Library #2 T2 = Truncated Sequence (from DL2) DL3 = Directed Library #3
14% 1.3 1.0 5.0N-(Boc)Asp Pro Asn(Trt) D-Phe (8a T2)
14% 1.5 1.9 1.0N-(Boc)Asp D-Pro Thr(OBn) Leu (11a IS)
Resin bound peptide (cat.)HOBt, DIC, DMAP (0.3 equiv)
H2O2 (1.0 equiv), DCM
Resynthesis and « in solution » trials with hits
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
HN
Boc
HO2C
N
O
O
NH
O
HN
NH
O
O
12b (X = OMe)
Peptide 12b or 12d (10 mol%)HOBt, DMAP (10 mol%)
DIC (1.0 equiv)H2O2 (2.0 equiv)
4 °C, 3 h
OBn
O
NHTrt
OtBu
X
1.0 4.3 1.3
12d (X = NHGlyOMe) 1.2 8.0 1.0
(47%, 10% ee)
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
2,3-Selectivity and 6,7-Selectivity is hydroxy driven in epoxidation
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me
Me
Me Me
OMe
Me
Me
Me Me
OMe
Peptide 9b or 12d (10 mol%)HOBt, DMAP (10 mol%)
DIC (1.0 equiv)H2O2 (2.0 equiv)
4 °C, 3 h
O O
No selectivity!
Validation of both 9b and 12d catalyst with geranylgeraniol in solution
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me Me Me
OH
Me Me Me
OH
C B A
Epoxide distribution
Me
MeMe
Me
D
Cl
O
OOH
3.1 3.1 2.5 1.0
Conditions
Peptide 9b 1.0 1.0 1.0 >100
Peptide 12d 1.0 1.8 13.2 1.5
(85%, 86% ee)
(42%, ee % n.d.)
Peptide 9b or 12d (10 mol%)HOBt, DMAP (10 mol%)
DIC (1.0 equiv)H2O2 (2.0 equiv)
4 °C, 3 h
Na2HPO4 (2.0 equiv)DCM/H2O
Conditions
• Enzymes mediated approches are most often not general to a series of substrates.
• The application of diversity-based approaches may prove fruitful and may also offer analogy to the directed evolution of strategies employed by natural an bioengineered enzymatic systems.
• Peptide 9b and 12d found by one-bead-one-compound library screening are operating via a hydroxyl-mediated mechanism.
• They offer comparable selectivity to the well-known Sharpless epoxidation conditions and are amenable to new selectivity pattern.
Conclusions
Lichtor, P. A.; Miller, S. J. Nature Chem. 2012, ASAP.
Me
Me
Me Me
OH
Me
Me
Me Me
OH
C B A
Peptide
Future aspects of peptide site-selectivity
Fowler, B. S.; Laemmerhold, K. M.; Miller, S. J. J. Am. Chem. Soc. 2012, 134, 9755-9761.Pathak, T. P.; Miller, S. J. J. Am. Chem. Soc. 2012, 134, 6120-6123.
O O
O
O
HO
OHO
HO
O
NHAlloc
OHMe
Me
Cl
NHH
O
NH2
O
HN
O
Cl
OH
NH
Me
Me
NAlloc
Me
HO
O
HN
OAllylOAllyl
AllylO
NHO H
HN
O
H
AllylO2C
H
H
H
O O
O
O
HO
OHO
R1O
O
NHAlloc
OHMe
Me
Cl
NHH
O
NH2
O
HN
O
Cl
OH
NH
Me
Me
NAlloc
Me
R2O
O
HN
OAllylOAllyl
AllylO
NHO H
HN
O
H
AllylO2C
H
H
H
Cl
S
OPh (1.5 equiv)
PEMP (2.0 equiv)Peptide 11 or 14 (cat.)THF/CHCl3 (1:3, 10 L)
rt, 24 h
With peptide 11, OR1 = C(S)OPh, OR2 = H (89%, 1:21)
With peptide 14, OR1 = H, OR2 = C(S)OPh (95%, 24:1)
N
OtBuO
NN
ON O
O
N
O NH OtBu
O
OMe
Ph
H
H
Peptide 11