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Abyssomicin CJohn TrantDepartment of ChemistryUniversity of Ottawa, 2007
Abyssomicin C—From isolation to mechanism of action
3
An introduction to the tetrahydrofolate biosynthetic pathway
The isolation identification of Abyssomicin CA brief retrosynthetic overview of Abyssomicin CK.C. Nicolaou’s synthesis: The application of Lewis-
acid catalysed self-assembling (LACASA) Diels-Alder reaction
The mechanism of action
Folic Acid: An Introduction
4
Vitamin B9 Needed for the catalysis of one-carbon transfer reactions
including dTMP from dUMP, and in the catalysis of glycine synthesis.
Not synthesised in vertebrates, but synthesised in plants, archaea, fungi, bacteria, and some lower animals.
HN
N N
N
H2N
C
NH
O
NH
CO2H
CO2HO
NH
O
ON
O
OH
OP-OO-
O
NH
O
ON
O
OH
OP-OO-
O
dUMP dTMP
COO-
OHHOOH
COO-
OOH
COO-
Aminodeoxychorismate Synthase
COO-
OOH
COO-
Glutamine Glutamic Acid
COO-
ONH3
+COO-
NH3PabA PabB
COOH
NH2
NH
NHN
N
NH2
OP
OP
HO O
HO OH
O
Dihydropteroate Synthetase
NHN
HN N
H2N
COO-
HN
DihydrofolateSynthetase
NHN
HN N
H2N
C
HN
Glutamic Acid
OHN
CO2HHO2C
H2O
DihydrofolateOxidase
ONH
N
N N
H2N
C
HN
OHN
CO2HHO2C
O
Folic Acid Dihydrofolate
Dihydropteroate
O
O
p-Aminobenzoic Acid
Dihydropteridine
ChorismateShikimic Acid
Aminodeoxychorismate Lyase
Aminodeoxychorismate
p-Aminobenzoic acid/Tetrahydrofolate pathway
5
TrimethoprimSulfa Drugs
6
Kozlowski, M.C et al. J. Am. Chem. Soc. 1995. 117 2128-2140.
CO2-
OHO CO2
-
-O2C
OH
O
CO2-
Prephenate
CO2-
OH
p-hydroxybenzoate
Chorismate mutase
Chorismatelyase
Anthranilatesynthase
p-Aminobenzoate Synthase
CO2-
OCO2
-
NH3+
CO2-
NH3+
CO2-
NH3+O CO2
-
2-amino-2-deoxyisochorismate
Anthranilate
4-amino-4-deoxychorismate
CO2-
NH3+
p-aminobenzoate
IsochorismateSynthase
CO2-
O CO2-
OH
Isochorismate
Tryptophan
Phenylalanineand Tyrosine
Ubiquinone (Coenzyme Q)
Siderophorebiosynthesis
Folate
p-Aminobenzoic acid/Tetrahydrofolate pathway
7
COO-
OHHOOH
COO-
OOH
COO-
Aminodeoxychorismate synthase
COO-
OOH
COO-
Glutamine Glutamic Acid
COO-
ONH3
+COO-
NH3PabA PabB
COOH
NH3
NH
NHN
N
NH2
OP
OP
HO O
HO OH
O
Dihydropteroate synthetase
NHN
HN N
H2N
COO-
HN
Dihydrofolatesynthetase
NHN
HN N
H2N
C
HN
Glutamic Acid
OHN
CO2HHO2C
H2O
DihydrofolateOxidase
O
HNN
NN
NH2
C
NH
O NH
HO2C CO2H
O
Folic Acid Dihydrofolate
Dihydropteroate
O
O
p-aminobenzoic acid
dihydropteridine
ChorismateShkimic AcidAminodeoxychorismate
lyase
Aminodeoxychorismate
Abyssomicin C
The Abyssomicins
8
O
OH
O
OOO
O
OH
O
OHO
O
ON O
OH
O
OOHO
DCB
Verrucosispora AB-18-032
Riedlinger, J. et al. J. Antibiotics. 2004. 57, 271-279.Bister, D. et al. Angew. Chem. Intl. Ed. 2004. 43, 2574-2576.
Abyssomicin C as a Chorismate mimic
9 Copley, A.D.; Knowles, J.R. J. Am. Chem. Soc. 1987. 109, 5008-5013.
O
OH
O
OO
O
HO O
COO-
-OOC
O
OH
COO-
CO
-OChorismate
O
OH
O
OO
O
O
OH
O
OO
O
O
OH
O
OO
O
O
OH
O
OO
O
O
OH
O
OO
O
O
OH
O
OO
O
Abyssomicin C
10
O
OH
O
OO
O
11
A Short Retrosynthetic OverviewSnider/Sorenson/Couladaros Approach to the initial disconnection
Snider, B.B.; Zou, Y. Org. Lett. 2005. 7, 4939-4941.Zapf, C.W.; Harrison, B.A.; Drahl, C.; Sorenson, E.J. Angew. Chem. Int. Ed. 2005. 44, 6533-6537.Couladouros, E.A.; Bouzas, E.A.; Magos, A.D. Tet. Lett. 2005. 62, 5272-5279.
O
OH
O
OOO
OH
O
O
OOO
MeO
O
OO
O
O
O
O
OMeOO
O
O
OLi
MeO
1
12
A Short Retrosynthetic OverviewSorenson’s Retrosynthesis of 1
Snider’s Retrosynthesis of 1
O O
1
O OPO
OO
O O
1
(MeO)2P
O
OPO
O
OO
O OP
A Short Retrosynthetic Overview
13
Rath, J.; Kinast, S.; Maier, M.E. Org. Lett. 2005. 7, 3089-3092.Zografos, A.L.; Yiotakis, A.; Georgiadis, D. Org. Lett. 2005. 7, 4512-4518.
Maier’s and Georgiadis’ Retrosynthesis of Abyssomicin C
O
OH
O
OOO
O
X
OP
O
OP
O
O
O
OH
O
O
TBSO
O
O
HO
O
OAcO OR
POTBSO
OH
AcOH
O
Nicolaou’s Retrosynthesis
14 Nicolaou, K.C.; Harrison, S.T. Angew. Chem. Int. Ed. 2006. 45, 3256-3260.Nicolaou, K.C.; Harrison, S.T. J. Am. Chem. Soc. 2007. 129, 429-440.
O
OH
O
OOO
O
OH
O
OOO
O
OH
O
O
O
AcO
OH
OO
OH
O
CH3
AcO
O
O
CH3OCH3
O
O
OH
O
OMe
15
OH
O
O
O
O
HO
O
MeO
MeOH
OH
O
OCH3
OH
O
O
O
MeOH
OH
O
MeO
MeO
OH
OMeO
OH
OMeO
AB
C
D
A:B:C:D=1.3:1.2:1.3:1.0
But...
16
Ward, D. E.; Abaee, M.S. Org .Lett. 2000. 2, 3937-3940.
120º C
17
Examples from literature
Ward, D. E.; Abaee, M.S.Org .Lett. 2000. 2, 3937-3940.
O
MeO
OH
O
MeO
OL.A.
R1B(OR2)2
OH R1BOR2
O
THF 16 h25ºC
PhCH3
R1 OHOH
60-80%endo:exo 2:1-9:1
8 examples
1)
2) Me3N(O)H2O
Batey, R.A.; Thadani, A.N.; Lough, A.J. J. Am. Chem. Soc. 1999. 121, 450-45.
R
OHOH
3 eq
AlMe3
OH
R 49-67%dr 1:1-3:12 examples
Bertozzi, F.; Olsson, R.; Frejd, T. Org. Lett. 2000. 2, 1283-1286.
OH
MgX
RR
OH
130 ºC3 h
60%dr 9:11 example
Stork, G.; Chan, T.Y. J. Am. Chem. Soc. 1995. 117, 6595-6596.
OH
MeMgBr
BrMgOO
O
O
O
95%
O
MeO
OMgBr
Benzene0-25ºC
Ward’s Solution
18 Ward, D.E.; Abaee, M.S. Org. Lett. 2000. 2, 3937-3940.
An Early Attempt at Olefination
19
O
O
O
O OHKHMDS
O2, P(OEt)3HO
O OH
LiO
O
O OH
H3O+
HO
O OH
HO
O
O OH
HO
O OH
HO
LiOH
96%
Nicolaou, K.C.; Harrison, S.T. J. Am. Chem. Soc. 2007. 129, 429-440.
A Modified Julia-Olefination Strategy
20
O
O OH1) NaSPh
2) MeOH, H2SO4
SPh
O OH
MeO
1) H2O2
2) TMSCl, NEt3, DMAP
SO2Ph
O OTMS
MeO
1) LiHMDS, CH2ICI2) Na (Hg)3) aq HCl, MeOH
O OH
MeO
71% 72%
82%
OH
Me2Zn
0 ºC 5min
HOHO
MeMgBr
0 ºC 5min
5 min
0 ºC
OO
S- BINOL
MgBrZn
O
MeO
O
O
Zn
O
O
OMg
Br
OO
O
95% yield93% ee
21Ward, D.E.; Souweha, M. S. Org. Lett. 2005. 7, 3533-3536.
The Disadvantages
22
Stoichiometric amount of enantiopure BINOL and ZnMe2 in the first step of the synthesis.
Synthesis had become lengthy (7 steps, 38% yield).
O OH
MeO
MeON
O 1.5eq PhSCH3
1.5 eq DABCO1.5 eq n-BuLi
OS
Ph
1.1 eq catecholborane
0.1 eq
NB
O
H PhPh
Me-(R)-CBSOHS
Ph
95% yield, 90% eeO
H
HPhS
O
30%
Toluene 55 ºC
MeMgBrMethyl Acrylate
81%
Asymmetric Borane Reduction
23
30%!!!!
24
Use of “sacrificial” alcohol resulted in no increase in yield.
Lewis Acid scan produced no increase of the yield (TiCl4, AlCl3, Zn(OTf)2, MgBr2•OEt2/i-Pr2NEt).
But...Remember that the enantioselective Diels-Alder Reaction was faster than the racemic version...
Bidentate Ligands
25
Entry Auxiliary Base/Metal Reaction Time1 (h)
Yield (%)
1 None MeMgBr (1.0 eq) 24 30 2 (±)-Binol MeMgBr (1.0 eq)
Me2Zn (1.0 eq) 24 <5
3 OH
OH
MeMgBr (1.0 eq) Me2Zn (1.0 eq)
36 35
4 OH
NH2
MeMgBr (2.0 eq) 24 49
5 OH
N
MeMgBr (2.0 eq) 48 55
6 2eq “ ” MeMgBr (3.0 eq) 48 70 7 3 eq “ ” MeMgBr (4.0 eq) 12 80
1) Time required for consumption of diene as monitored by NMR.
6
The Proposed Transition State
26
OHS
Ph
O
H
HPhS
O
80%
Toluene 55 ºC
MeMgBrMethyl Acrylate
O
O
OMg
Br
OMg
N
MeOH
PhS
O
O
PhSH
H
O
O
PhSH
OHLiHMDS; O2, P(OEt)3
74%
0.5 eq. dtBB9.6 eq Li
MeI, K2CO3
HOCO2Me
O
OAcO
MeO
1) t-BuOOH VO(OEt)32) Ac2O
99%
88%
1)2.5 eq LiHMDS 1 hr -78 to 25 ºC
O
OTES
O
O
3) TESCl, Imidazole DMAP97%
2) aq NH4Cl 66 ºC 2h
O
OO
MeO
O-
Julia-Type Reduction
27 dtBB
[O]
[O]
O
O
PhO2SH
OH
HO
O
H
OH
28
O
OH
O
OO
O
Synthesis of The Coupling Partner
29
HO OTBS SO3 PyridineDCM:DMSO (2:1) O OTBS
MgBr2 eqTHF -78 ºC
OTBS
OH
74% (two steps)
1) NaH, PMBCl, TBAI, DMF2) HCl, MeOH3) SO3 Pyridine, DCM:DMSO (2:1)
O
OPMB84%
O
OTES
O
O
1) t-BuLi, THF2)
O
OPMB
O
OTES
O
OOH
OPMB
61%
3 eq DDQDCM: NaHCO3 saturated (10:1)
O
OTES
O
OOH
5 mol% Grubbs 2HO
96%
O
OTES
O
OHOOH
30
Various homodimerised and polymerised by-products
5 mol% Grubbs 2
O
OTES
O
OOHHO
1 mol% HClMeOH
25 ºC, 1 h O
OH
O
OOHHO
5 mol% Grubbs 2
O
OH
O
OHOOH
IBXMnO2
Dess-MartinPDCSwern
O
OH
OO
OOH
94%
78%
O
O
O
OOO
31
Covers blue
A New Approach
32
O
OTES
O
O
AcO O
1) 1.2 eq t-BuLi2) O
OTES
O
OAcO OH
IBXDMSO 25 ºC
45 min
75%
O
OTES
O
OAcO O
90%
5 eq (CH2SH)25 eq BF3 OEt2DCM, 12 h
O
OH
O
OAcO S
S
90%
3.4 eq K2CO3MeOH 25 ºCO
OH
O
OHO S
S
90%
33
O
OH
O
OHO S
S1) IBX2)
MgBr O
OH
O
O
SSHO
5 mol% Grubbs 2
65%
85%
2.5 eq IBXDMSO
50%
3 eq PhI(OTFA)2CH3CN/H2O (10:1)
25 ºC 10 min
73%
O
OH
O
O
SS
HOH
H
O
OH
O
O
SS
OH
H
O
OH
O
OO
H
H
O
34
5.6% overall yield, 19 steps from Weinreb AmideThe NMR did not match that of the previously
isolated natural compound.After 18 hours in CDCl3 a new set of peaks
appeared.The new peaks matched those of the previously
isolated abyssomicin C.
MeON
O
Generation of Possible Atropisomers
35
O
OH
O
O
SSHO
O
OH
O
O
SS
LnRu
HO
[si]
[re]
O
OH
O
OHO
SS
H
HLnRu
O
OH
O
OHOSS
LnRuH
H
si approach re approach
O
OH
O
OHO
SS
H
H
O
OH
O
OHOSS
H
H
Ru
PCy3Cl
Cl
Ph
NNMes Mes
5 mol% Grubbs 20.002M DCM
40 ºC 1hr
36Nicolaou and Harrison J. Am. Chem. Soc. 2006, 129, 430-440.
The Differences
37
O
OH
O
OOH
O
O
OH
O
OOH
OCDCl3
21
Nicolaou and Harrison J. Am. Chem. Soc. 2006, 129, 430-440.
O
OH
O
OOH
O
O
OH
O
OOH
OCDCl3
21
Nicolaou’s Proposed Mechanism
38
O
OH
O
OOO
O
OH
O
OOO
CDCl3/HCl
O
OH
O
OOO
O
OH
O
OOO
O+
OH
O
OOO
H+
HOH
O
O
OOO
O+
OH
O
OOO
H
O
OH
O
OOO
O
OH
O
OOO
H+ O
O
OH
O
HOOO
O
OH
O
HOO
O
OH
O
OHOO
H+
O
OH
O
OOO
O
OH
O
OOO
O+
OH
O
OOO
H+
O+
OH
O
OOO
H H
O+
OH
O
OOO
H H+
39
Biosynthetic Ramifications
39 Figure modified from Nicolaou et Harrison J. Am. Chem. Soc 2007. 129, 429-440.
O
OH
O
OOO
O
OH
O
OOO
L-selectride
O
OH
O
OOO-
H
O
OH
O
O-O
O
H
Z-enolate
E-enolate
O
OH
O
OOHO
H
O
OH
O
OOHO
H
H3O+
O
OH
O
OOO
H
Abyssomicin D
Iso-abyssomicin D
atrop-abyssomicin C
abyssomicin C
L-selectride
H3O+
Biosynthetic Ramifications
40
Abyssomicin C
Figure modified from Nicolaou et Harrison J. Am. Chem. Soc. 2007. 129, 429-440.
Time (h)
Atrop-abyssomicin C
N
O
OHOH
HO
O
NH2
H H
NADH
NH
Me Me
CO2EtEtO2C
Minimum Inhibitory Concentrations for the analogues
41
O
OH
COO-
CO
-O
O
OH
O
OO
H
H
O
Abyssomicin C20 µM
O
OH
O
OO
H
H
O
atrop-Abyssomicin C15 µM
O
OAc
O
OO
H
H
O
Ac-Abyssomicin C20 µM
O
OH
O
OO
H
H
SS
Dithiane atrop-Abyssomicin C
70 µM
O
OH
O
OHO
H
H
SS
Dithiane-hydroxy Abyssomicin C
>500 µM
O
OH
O
O
H
>500 µM
O
OH
O
O
MeO
O
>500 µM
Proposed Mechanism of Action
42Figure adapted from Parsons, J. F. et. al. Biochem. 2002. 41, 2198-2208.
Narrowing the Search
43
Active Site
O
OH
COO-
CO
-O
Ser254
Ser256
Cys263Ser266 Thr270
Thr276
Ser342
Thr343
Thr345
Ser366
Thr368
Cys391
Ser393
Thr411Thr408Cys421
Ser422
Active Site
O
OH
COO-
CO
-O
Identifying the nucleophile
44Keller, H. et. al. Angew. Chem. Intl. Ed. 2007. 46, 8284-8286.
45
O
OH
O
OOO
H
HCys
O
OH
O
O-OO
Cys
O
OH
O
OHOO
Cys
O
OH
O
OHOOH
Cys
HOSH HN CO2H
SH
O
O
OH
O
OOO
H
HCys
O
OH
O
O-OO
Cys
O
OH
O
OHOO
Cys
O
OH
O
OHOOH
Cys
Minimum Inhibitory Concentrations for the analogues
46
O
OH
O
OO
H
H
O
Abyssomicin C20 µM
O
OH
O
OO
H
H
O
atrop-Abyssomicin C15 µM
O
OAc
O
OO
H
H
O
Ac-Abyssomicin C20 µM
O
OH
O
OO
H
H
SS
Dithiane atrop-Abyssomicin C
70 µM
O
OH
O
OHO
H
H
SS
Dithiane-hydroxy Abyssomicin C
>500 µM
O
OH
O
O
H
>500 µM
O
OH
O
O
MeO
O
>500 µM
In Conclusion
47
Examined the Folate Biosynthesis pathway.Examined Nicolaou’s application and modification
to Ward’s LACASA approach to Diels-Alder Reactions using an allylic alcohol diene.
Delved into Nicolaou’s Approach for the total synthesis of Abyssomicin C.
Demonstrated how Nicolaou’s synthetic work uncovered the potent inhibitor, atrop-abyssomicin C, leading to a better understanding of the abyssomicin mechanism of action.
Acknowledgements
48
Roger Tam Pawel Czechura Jennifer Chaytor Elisabeth Von Moos Tahir Rana Wendy Campbell Sandra Ferreira Ruoying “Gloria” Gong Jaqueline Tokarew Ivan Petrov
Dr. Michael SouwehaDr. Matthieu Leclere
Dr. Robert Ben
And NSERC for providing funding to make this possible
Couladouros’ Retrosynthesis of 2
49
O
O
O
OMeO
2
HO
OH
O
OMeO
OH
O
OMeO
O
I
O
OH
O
OMeO
AcOOAcO
OO
OMe
OO O
O
OMe EtO2C CO2Et
OO
OMe
3
4
5
Synthesis of Building Block 5
50
OO
MeO
OO
MeO
N
OO
MeO
N
OO
MeO
NMe3O
OMeO
(NMe2)2CHOMeNaCNBH4
MeI
NaHCO3
5
Takeda et al. J. Org. Chem. 1987, 52, 4135-4137
Synthesis of 4
51
EtO2C CO2Et
OMe
O
1) NaOEt 25ºC2) HCl, AcOH 105ºC
3) Ac2O, reflux OO O OO O
7 3:
NEt3, THF, Reflux
65% total yield
1) LiAlH4, THF 0ºC2) AcOCHCH2, Amino Lipase AK THF 0ºC
AcO OH(COCl)2, DMSO
DCM NEt3
AcO O
Putting The Pieces Together
52
OO
MeO
5AcO O
4
1) LDA, THF -100 C
2)O
OMeO
AcO OH1) IBX DMSO
2) Novozyme 435, Toluene phosphate buffer
OO
MeO
HO O
OO
MeO
HO O
CrCl2
IO
OMeO
HO OIBX DMSO
45-58% 41%
70%
Putting The Pieces Together Take 2
53
OO
MeO
5AcO O
4
1) LDA, THF -100 C
2)O
OMeO
AcO OH
45-58%O
OMeO
AcO OTBSTBSClImidazole
DMF
85%
OO
MeO
HO OTBS
78%
Guanidine HClEtOH/4M NaOH
1) IBX DMSO2) CrCl2, NiCl2 THF/DMSO
OO
MeOHO OTBS
I
1) TBAF, THF
44%
OO
MeOO O
70%
2) IBX, DMSO
I2
Toluene 100ºC
OMeO
OOO
OMeO
OOO
I
OMeO
OOO
IOMe
O
OOO
I
I-
54
OMeO
OOO
1 eq DMDOAcetone 0º-23ºC
18 hOMe
O
OOO
O
67%
10 eq. LiClDMSO50ºC
OHO
OOO
Oquantitative
O
OH
O
OOO 1.2 eq. PTSA
5 eq. LiClAcN 50ºC 2h.
50%
Synthesis of Vinyl Iodide 3
55
Entry Conditions Solvents E/Z Yield (%)
11 CrCI2, CH3I, 0°C THF 2:1 85 22 CrCI2, CH3I, 0°C THF:Dioxane; 6:1 2:1 75 33 Ph3P+(I-)CH2I,
NaHMDS, -78°C THF 1.2:1 87
1Takai’s Conditions (Takai, K. et al. J. Am. Chem. Soc. 1986, 108, 7408-7410. 2Evans’ Conditions (Evans, D.A. et al. J. Am. Chem. Soc.1993, 115, 4497-4513. 3Stork’s conditions (Stork, G. et al. Tet. Lett. 1989, 30, 2173-2174.
OI
Isomerisation Conditions
56
O
OH
O
OOH
O
O
OH
O
OOH
OCDCl3
21Entry Conditions Time (h) Ratio (1:2) 1 Unstabalised CDCl3 24 2:1 2 Xylenes 180°C 12 No isomerisation 3 TFA/DCM (1:1) 24 No isomerisation 4 1 eq. BF3• OEt2 DCM 24 No isomerisation 5 1 eq. CSA, DCM 24 No isomerisation 6 1 M aq HCl:THF (3:1) 24 No isomerisation 7 1M HCl in Et2O (0.2 eq.)/d6-THF 1 1.0:1.6 8 1M HCl in Et2O (0.2 eq.)/CDCl3 1 2.5:1.0 9 1 eq. p-TsOH, 5 eq. LiCl
CD3CN, 50°C 2 1.0:2.0
Isolation and Identification
57
Isolated in 2004 by Süssmuth from an actinomycete of genus Verrucosipora, discovered in a sediment sample from the Japanese Sea.
Investigated because of pABA inhibition identified by an agar-plate diffusion assay
Structure determination by NMR and X-Ray
Whoopdeedoo
58
Folic acid, Vitamin B12 is an essential vitamin.Not synthesised in vertebratesFungi, Bacteria, Plants, Achaea, and insects and
anthropods all synthesise it.So it has the possibility of providing broad
spectrum antibiotics.Sulfonamides and Trimethoprim are existing
antibiotics that inhibit the Folate Synthesis Pathway
Andrus’ Lactone
59
Agar Plate Diffusion Assay
60
Screened 930 extracts.
Riedlinger, J. et al. J. Antibiotics. 2004. 57, 271-279
6161 ibid
Verrucosispora AB-18-032
Borane Transition State
62
SPh
OB
Me
N O
PhPh
L2BH
A Shorter Route
63
O
OTES
O
O
AcO O
1) 1.2 eq. t-BuLi2) O
OTES
O
OAcO OH
IBXDMSO 25ºC
45 min
75%
O
OTES
O
OAcO O
90%
5 eq(CH2SH)25 eq BF3 OEt2DCM 12 h
O
OH
O
OAcO S
S
90%
3.4 eq K2CO3MeOH 25ºCO
OH
O
OHO S
S
O
OTES
O
OHO O
1.2 eq t-BuLi1.1 eq
O O
(CH2SH)2TMSOTf
76% over 2 steps
64
Kinetic Studies
O
OH
O
OOO
O
OH
O
OOO
Irreversible Inhibition: KIapp=390μM;
kinact=0.8 min-1
Keller, H et. al. Angew. Chem. Intl. Ed. 2007. 46, 8284-8286
Ward’s DA evidence
65