linda elizabeth jewell january 22 nd, 2009 the total synthesis and structural reassignment of...
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Linda Elizabeth JewellJanuary 22nd, 2009
The Total Synthesis and Structural Reassignment of Chlorofusin
N
O
O
Cl
O
O
O
OH
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
OutlineBackgroundDiscovery and Structural AssignmentMacrocycle StudiesYao’s SynthesisBoger’s SynthesisConclusions
2
Discovery and BioactivityCancer is caused by unregulated cell
reproductionp53 is a transcription regulator
It can prevent cancer by “stalling” the cell cycle or inducing apoptosis when DNA damage is detected
Its activity is modulated by a second protein, MDM2
3 Chéne, P. Nat. Rev. Cancer 2003, 3, 102
Background
4 Adapted from Chéne, P. Nat. Rev. Cancer 2003, 3, 102
p53
Nucleus
DNA
MDM2Prevents entry into the nucleus
p53
MDM2
Tags p53 to favourdegradation
p53
MDM2
Prevents p53 from binding to DNA
Discovery and Bioactivity
5
Disordered regulation of either p53 or MDM2 can lead to the growth of tumours
Disrupting the p53-MDM2 interaction could restore normal function of p53 and halt the growth of tumours
Chlorofusin was discovered in a screening program designed to identify novel antagonists of the p53-MDM2 interactionKD = 4.7μM
Williams et al, JACS, 2001, 123, 554-560
Discovery and Bioactivity
6
Chlorofusin was isolated by extraction and purified by reverse-phase HPLC
Molecular formula determined by mass spectroscopy as C63H99O19N12Cl
Williams et al, JACS, 2001, 123, 554-560
Discovery and Bioactivity
7
Amino acids were identifying by degradation of the peptide in HCl, purification on an ion exchange resin, and analysis of the resulting fractions by spectrophotometry
Tests identified leucine (x2), asparagine or aspartic acid (x2), alanine (x1) and threonine (x2)Two nonstandard amino acids were also present
Williams et al, JACS, 2001, 123, 554-560
Discovery and Bioactivity
8
Acetylation of the unknown compound under basic conditions lead to the addition of three acetyl groupsSuggests three acidic hydroxyl groups
Attempted esterification failedSuggests absence of carboxylic acid functionalities
Compound was stable under acidic and basic conditions
Williams et al, JACS, 2001, 123, 554-560
Discovery and Bioactivity
9
COSY and TOCSY revealed 14 spin systemsA number of CH2 groups, initially obscured by
other protons, were also observedThese were assigned to the nonstandard amino
acid aminodecanoic acidOrnithine was identified as the remaining amino
acid
Williams et al, JACS, 2001, 123, 554-560
Discovery and Bioactivity
10
The macrocycle was assembled based upon key HMBC and NOESY correlations
N
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
Williams et al, JACS, 2001, 123, 554-560
SelectedNOESY Correlations
N
O
O
Cl
O
OCH3
OH
R
H
H
O
Chromophore Assignment – Key NOEs
11
8
4
10
NOESY Correlations
Williams et al, JACS, 2001, 123, 554-560
N
O
O
Cl
O
OCH3
OH
R
H
H
O
Williams’ Structural Assignment
12
Williams thus proposed that the O-substituents of the chromophore are all syn
Williams et al, JACS, 2001, 123, 554-560
N
O
O
Cl
O
O
O
OH
R
H
Williams’ Structural Assignment
13 Williams et al, JACS, 2001, 123, 554-560
N
O
O
Cl
O
O
O
OH
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
Asn
Asn
D-Leu
L-Thr
D-Leu
L-Thr
L-Ala
L-Orn
D-Ada
OutlineBackgroundDiscovery and Structural AssignmentMacrocycle StudiesYao’s SynthesisBoger’s SynthesisConclusions
14
Determination of Macrocycle Stereochemistry
15
To unambiguously determine the stereochemistry of the amino acids in the macrocycle, Boger and Searcey simultaneously completed syntheses of the macrocycle
Boger chose a solution-phase synthesis while Searcey pursued a solid-phase approach
Boger et al, OrgLett, 2003, 5,5047-5050; Searcey et al, OrgLett, 2003, 5, 50501-5054
Synthesis of D-ADA
16 Boger et al, OrgLett, 2003, 5,5047-5050
N
N
MeO
OMe C8H17In-BuLi
THF-78oC
94% yield, >96% de
N
N
MeO
OMe
(CH2)7CH3
0.5M HCl
25oCquant.
H2NOMe
O
(CH2)7CH3
BocHNOH
O
H2NOBn
O
OH
EDCl, HOAt
DMF94% yield
BocHN
HN
O
OBn
O
OH
H2Pd/C
EtOHquant.
BocHN
HN
O
OH
O
OH
Boger’s Peptide Studies
17 Boger et al, OrgLett, 2003, 5,5047-5050
D-Leu L-Thr D-Leu D-Ada L-Orn L-Thr L-Ala
Boger’s Peptide Studies
18 Boger et al, OrgLett, 2003, 5,5047-5050
D-Ada
D-Leu
L-Thr
D-Leu
D-AsnL-Asn
L-Ala
L-Orn
L-Thr
D-Ada
D-Leu
L-Thr
D-Leu
L-AsnD-Asn
L-Ala
L-Orn
L-Thr
Determination of Macrocycle Stereochemistry
19 Boger et al, OrgLett, 2003, 5,5047-5050
Bioactivity of MacrocycleInterestingly, the macrocycle alone displayed no
inhibition of the p53-MDM2 complex in either in vivo and in vitro tests
Boger also tested a number of structures including chromophore mimics unsuccessfully
20 Boger et al, OrgLett, 2003, 5,5047-5050; Searcey et al, OrgLett, 2003, 5, 50501-5054
OutlineBackgroundDiscovery and Structural AssignmentMacrocycle StudiesYao’s SynthesisBoger’s SynthesisConclusions
21
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
O
OOR
N
ClO
OH
6
R= butyrate
Yao’s Retrosynthesis
22 Yao et al, JACS, 2007, 129, 6400-6401
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
O
OOR
N
Cl HO
6
R= butyrate
Yao’s Retrosynthesis
23 Yao et al, JACS, 2007, 129, 6400-6401
Yao’s Retrosynthesis
24 Yao et al, JACS, 2007, 129, 6400-6401
O
ORO
O
Cl HO
R= butyrate
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
H2N
6
Yao’s Synthesis
25 Yao et al., Tet, 2005, 61, 11882-11886
MeO
Me
OMePOCl3
DMF60oC
90% yield
MeO
Me
OMe
CHO
MeO
Me
OMe
CH(OAc)2O2N
Cu(NO3)2 3H2OAc2O
94% yield
MeO
Me
OMe
CH2OHH2N
Raney Ni(50% in H2O)
H2
THF98% yield
MeO
Me
OMe
CH2OHH2N
NBS
CHCl392% yield
Br
MeO
Me
OMe
CH2OH
Br
PCC4A sieves
CH2Cl20oC-RT
87% yield
MeO
Me
OMe
CHO
Br
HO
Me
OH
CHO
Br
BBr3
CH2Cl2-78oC-RT94% yield
MeO
Me
OMe
CH2OHH2N
Br
1. c. HClTHF/H2ONaNO2
2. urea,50% H3PO289% yield
OAc HO
Me
OH
CHO
OAc
5 mol% Pd(PPh3)4,CuI, Et3N
DMF70oC
92% yield
Yao’s Synthesis
26 Yao et al., Tet, 2005, 61, 11882-11886
Me
HO
OH
CHO
OAc(-)-sparteine, O2,Cu(CH3CN)4PF6,
DIEA, DMAP
CH2Cl
O
OHO
OAc
OH
aq NH4Cl
CH3CN55% yield (2 steps)
O
OOH
O
AcO
O
OOR
O
AcO
n-PrCOClDMAP, pyridine
CH2Cl270% yield
R=butyrate
SO2Cl2
CH2Cl266% yield
O
OOR
O
AcO
R=butyrate
K2CO3
MeOH80% yield
Cl
O
OOR
O
HO
R=butyrate
Cl
91% ee
Yao’s Synthesis
27 Yao et al, JACS, 2007, 129, 6400-6401
R
O OH
OH
Cu
OO
Cu
NN
NNR
O OH
O
DIEA
R
O OH
O
Cu
OO
Cu
NN
NN
R
O OH
O
CuO
N
N R
OOH
O
OCu
N
N
Cu
O
O
Cu
N
N
N
N
2+
28 Porco et al, JACS, 2005, 127, 9342-9343
Asymmetric Oxidation / Cyclization
R
OO
O
OCuN
N H
OAc
OH
OOH
O
H
O
O
OOH
OAc
R
O
O
OCuN
N
OH+/H2O
R
O
O
HO
OH
NH4Cl
R
OHO
O
OCuN
N
Asymmetric Oxidation / Cyclization
29 Porco et al, JACS, 2005, 127, 9342-9343
Me
HO
OH
CHO
OAc(-)-sparteine, O2,Cu(CH3CN)4PF6,
DIEA, DMAP
CH2Cl
O
OHO
OAc
OH
aq NH4Cl
CH3CN55% yield (2 steps)
O
OOH
O
AcO
O
OOR
O
AcO
n-PrCOClDMAP, pyridine
CH2Cl270% yield
R=butyrate
SO2Cl2
CH2Cl266% yield
O
OOR
O
AcO
R=butyrate
K2CO3
MeOH80% yield
Cl
O
OOR
O
HO
R=butyrate
Cl
91% ee
Yao’s Synthesis
30 Yao et al, JACS, 2007, 129, 6400-6401
O
OOR
O
AcO
R=butyrate
SO2Cl2
CH2Cl266% yield
O
OOR
O
AcO
R=butyrate
Cl
O
R
R
S O
O
Cl
Cl
O
R
R
Cl
H
Cl
O
R
R
Cl
O
R
R
Cl
Cl
Regioselective Chlorination
31
SO2
Me
HO
OH
CHO
OAc(-)-sparteine, O2,Cu(CH3CN)4PF6,
DIEA, DMAP
CH2Cl
O
OHO
OAc
OH
aq NH4Cl
CH3CN55% yield (2 steps)
O
OOH
O
AcO
O
OOR
O
AcO
n-PrCOClDMAP, pyridine
CH2Cl270% yield
R=butyrate
SO2Cl2
CH2Cl266% yield
O
OOR
O
AcO
R=butyrate
K2CO3
MeOH80% yield
Cl
O
OOR
O
HO
R=butyrate
Cl
91% ee
Yao’s Synthesis
32 Yao et al, JACS, 2007, 129, 6400-6401
O
OOR
O
HO
R=butyrate
Cl
O
OOR
N
Cl HO
HN
HN
O
O
O
OH
HN
O
NH
R=butyrate
cyclic peptideNaHCO3
MeCN80% yield
O
OOR
O
HOCl
cyclic peptideNaHCO3
MeCN80% yield
R=butyrate
O
O
RO N
Cl HO
HN
HN
O
O
O
OH
HN
O
NH
R=butyrate
Yao’s Model Studies
33 Yao et al, JACS, 2007, 129, 6400-6401
S
R
D-AdaD-Leu
L-Thr
L-Asn
D-Asn
D-Leu
D-AdaD-Leu
L-Thr
L-Asn
D-Asn
D-Leu
Using their assignment of the 1st stereocentre, Yao synthesized the remaining diastereomers
Yao’s Model Studies
34
O
OOR
O
HO
R=butyrate
Cl
NBS
CH3CN
O
OOR
O
R=butyrate
ClO
Br
MeNH2HClaq NaHCO3
CH3CN
O
OOR
N
R=butyrate
ClO
Br
O
OOR
N
R=butyrate
ClO
OH
Ag2O
MeCN70% across 3 steps
Yao et al, JACS, 2007, 129, 6400-6401
Addition-Elimination
35
O
OOR
O
ClO
Br
NH
H
O
OOR
O
ClO
Br
NH
HO OH
O
O
OOR
O
ClO
Br
NH
O
OOR
O
Cl
O
Br
NH
O
OOR
O
Cl
OH
Br
NH
O
OOR
N
Cl
OH
Br
H
O
HO O
O
H
Addition-Elimination
36
O
OOR
N
Cl
OH
Br
OH
O
OOR
N
Cl
OH
Br
OH
O
OOR
N
Cl
OH
Br
O
OOR
N
Cl BrHO
O
OOR
N
Cl
OH
Br
H
O
O
OOR
N
Cl BrO
P.T.
O
O
OHH
2
Yao’s Model Studies
37
O
OOR
O
HO
R=butyrate
Cl
NBS
CH3CN
O
OOR
O
R=butyrate
ClO
Br
MeNH2HClaq NaHCO3
CH3CN
O
OOR
N
R=butyrate
ClO
Br
O
OOR
N
R=butyrate
ClO
OH
Ag2O
MeCN70% across 3 steps
Yao et al, JACS, 2007, 129, 6400-6401
N
O
O
Cl
O
OCH3
OH
H
O
N
O
O
Cl
O
OCH3
OH
H
O
N
O
O
Cl
O
OCH3
OH
H
O
N
O
O
Cl
O
OCH3
OH
H
O
Yao’s Stereochemical Assignment
38 Yao et al, JACS, 2007, 129, 6400-6401
A
C
B
D
S
R S
O
OOR
O
OH
Cl
R=butyrate
NBS
CH3CN
O
OOR
O
Cl
R=butyrate
cyclic peptideaq. NaHCO3then Ag2O
CH3CN25% yield
O
OOR
N
ClO
BrO
OH
HN
HN
O
O
O
OH
HN
O
NH
Yao’s Synthesis
39 Yao et al, JACS, 2007, 129, 6400-6401
Chlorofusin
D-AdaD-Leu
L-Thr
L-Asn
D-Asn
D-Leu
Yao also synthesized the Williams-proposed (S,S,S) isomer, which clearly did not fit the NMR data as well as his (S,R,S) isomer
Yao’s Synthesis
40
N
O
O
Cl
O
O
O
OH
R
N
O
O
Cl
O
O
O
OH
R
1
8
8
10
13
12
11
14
16
17
18
18
17
1613
14 12
11
10
1
Yao et al, JACS, 2007, 129, 6400-6401
Summary of Yao’s Synthesis
41
14 steps, 2.6% yield (not accounting for peptide synthesis)
NMR data suggested that a revision of chromophore stereochemistry was required
N
O
O
Cl
O
O
O
OH
R
Williams’ structure
N
O
O
Cl
O
O
O
OH
R
Yao’s structure
SS
S RS S
OutlineBackgroundDiscovery and Structural AssignmentMacrocycle StudiesYao’s SynthesisBoger’s SynthesisConclusions
42
Boger’s Retrosynthesis
43
NH
HN
O
ONH
O
HNO
O NH
OH
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
O
OOR
N
ClO
OH
6
R= butyrate
Boger et al, JACS, 2008, 130,12355-12369
NH2
HN
O
O OH
OH
O
OOR
N
Cl OH
R= butyrate
Boger’s Retrosynthesis
44
NH2
HN
O
O OH
OH
O
OOR
N
ClO
OH
R= butyrate
ONH
O
HNO
NH2
HN
O
NH2
O
NH
O
O NH
NHHO
O
NH2
O
6
OH
Standard peptide synthesisBoger et al, JACS, 2008, 130,12355-12369
Boger’s Retrosynthesis
45
O
OOR
O
Cl OH NH2
HN
O
O OH
OH
H2N
NH2
HN
O
O OH
OH
O
OOR
N
Cl OH
R= butyrate
Boger et al, JACS, 2008, 130,12355-12369
90% yield
Boger’s Total Synthesis
46
MeO
OMe
CO2H1. SOCl2
2. BtCH2TMS
3. 2,6-lutidineTf2O
86% yield
MeO
OMe
OTf
Bt1. NaOMe
2. HCl87% yield
MeO
OMe
CO2Me
i -PrMgCl
NH(OMe)Me95% yield
MeO
OMe
MeN
O
OMe
I OTIPS
t-BuLi, -78oC
MeO
OMe
O
OTIPS
HO
OH
O
IHO
OH
O
IAlCl3CH(OEt)3
-45 to -15oC75% yield
COH
TMSI
120oC, μW95% yield
Boger et al, JACS, 2008, 130,12355-12369
Boger’s Total Synthesis
47 Boger et al, JACS, 2008, 130,12355-12369
MeO
OMe
CO2H
SOCl2
MeO
OMe
O
Cl
N
N
NMeO
OMe
O
Cl
MeO
OMe
O
Bt
BtN
H
MeO
OMe
O
BtS
OS
O O O O
CF3F3C
MeO
OMe
OTf
Bt
Me3Si
SiMe3
3
90% yield
MeO
OMe
CO2H1. SOCl2
2. BtCH2TMS
3. 2,6-lutidineTf2O
86% yield
MeO
OMe
OTf
Bt1. NaOMe
2. HClMeOH
87% yield
MeO
OMe
CO2Me
i -PrMgCl
NH(OMe)Me95% yield
MeO
OMe
MeN
O
OMe
I OTIPS
t-BuLi, -78oC
MeO
OMe
O
OTIPS
HO
OH
O
IHO
OH
O
IAlCl3CH(OEt)
toluene-45 to -15oC75% yield
COH
Boger’s Total Synthesis
48
TMSI
MeCN120oC, μW95% yield
Boger et al, JACS, 2008, 130,12355-12369
HO
OH
O
I
COH
p-TsOH
Pb(OAc)4butyric acid31% yield
O
O
I
O
ORR=butyrate
AgOAc
O
O
OAc
O
ORR=butyrate
O
O
OAc
O
OR
Cl
R=butyrate
HOAc63% yield
NCS, AcOH
85% yield
Boger’s Total Synthesis
49 Boger et al, JACS, 2008, 130,12355-12369
(racemic mixture)
O
O
OAc
O
OR
Cl
R=butyrate
O
O
OAc
N
OR
Cl
butylamine
CH2Cl299% yield
K2CO3
MeOH, H2O0oC
91% yield
O
O
OH
N
OR
Cl
O
O
N
OR
Cl
R=butyrate
R=butyrate R=butyrate
I2, AgNO3
DMSO-H2O59% yield
OOH
Boger’s Chromophore Studies
50 Boger et al, JACS, 2008, 130,12355-12369
N
O
O
Cl
O
OCH3
OH
R
H
H
O
Recall: Williams’s Key NOE
51 Williams et al, JACS, 2001, 123, 554-560
N
O
O
Cl
O
O
O
OH
R
H
O
O
N
OR
Cl
R=butyrate
OOH
D
H
O
O
N
OR
Cl
R=butyrate
OOH
A
H
X-Ray Structures of Model Systems
52 Boger et al, JACS, 2008, 130,12355-12369
4.617 Å
4.854 Å
2.458 Å
2.484 Å
O
O
O
Cl
RO
R= butyrate
1. Fmoc-L-Orn-L-Thr-OBnNaHCO3
DMF-CH2Cl2
2. 1N HCl84% yield
N
O
O
Cl
OH
RO
R= butyrate
FmocHN
HN
O
OBn
O
OH
N
O
O
Cl
RO
FmocHN
HN
O
OBn
O
OH
OHO
HO
I2, AgNO3
H2O-DMSO61% combined yield
R= butyrate
Boger’s Total Synthesis
53 Boger et al, JACS, 2008, 130,12355-12369
N
O
O
Cl
O
RO
OH
NH
HN
O
O
O NHCbz
OH
HN
O
OBn
R= butyrate
Cbz-L-Ala-L-Asn-D-Asn-D-Leu-L-THr-D-Leu-D-ADAEDCl, HOAT
55% yield
R= butyrate
N
O
O
Cl
RO
H2N
HN
O
OBn
O
OH
OHO
D-AdaD-Leu
L-Thr
L-Asn
D-Asn
D-Leu
Boger’s Total Synthesis
54
FmocHN
piperidine
Boger et al, JACS, 2008, 130,12355-12369
N
O
O
Cl
O
RO
OH
HN
HN
O
O
OBn
OH
HN
O
NHCbz
R= butyrate
O
H2, Pd/C
Boger’s Total Synthesis
55
All 8 Diastereomers!
EDCl, HOAt
60% yield
D-AdaD-Leu
L-Thr
L-Asn
D-Asn
D-Leu
NH2
OHNH
Boger et al, JACS, 2008, 130,12355-12369
Syn isomer
Comparison of NMR Data
56
Natural chlorofusin
Boger’schlorofusin
Yao’schlorofusin
Williams’chlorofusin
Boger et al, JACS, 2008, 130,12355-12369
O
O
O
RO N
Cl OH
O
O
O
RO N
Cl OH
O
O
O
RO NR
Cl OH
OO
O
RO
Cl
N
OH
OH
O
O
N
Cl
O
O
RO
Boger’s Total Synthesis
57
To prove that his isomer was correct, Boger used not only NMR, but also CD spectra
CD spectra measure the different absorption of left and right-handed circularly polarized light at a number of different wavelengths
Produces a “fingerprint” resulting from the different stereocentres in a molecule
Boger et al, JACS, 2008, 130,12355-12369
Comparison of CD Spectra
58 Boger et al, JACS, 2008, 130,12355-12369
So where did Yao go wrong?
59
Yao determined the structure of the chromophore based upon oversimplified model systems
Additionally, Yao overlooked critical but subtle differences in the NMR of his compound relative to true chlorofusin
Boger also used model studies to guide his group’s synthesis, but ultimately constructed all plausible diastereomers
Conclusions
60
Chlorofusin’s ability to interrupt a protein-protein interaction makes it a valuable structure for future study
Yao correctly identified that the O-substituents were not syn, but ultimately constructed an incorrect isomer of chlorofusin
Boger unambiguously identified the true natural product after synthesizing all possible isomers
Acknowledgements
61
Current Group Members: Dr. Tony Durst Daria Klonowska Lina Chan Christine Choueiri David Ngyuen Darija Muharemagic Ana Garagun Anna Chkrebtii Asim Muhammad
Former Group Members: James Cheng Yiming Qian Melanie Waite Samira Salari Halima Mao Mohamed El-Salfiti
The DurstBunch
62
63
HO
Me
OH
CHO
OAc
Pd(PPh3)2
HO
Me
OH
CHO
Broxidativeaddition
HO
Me
HO CHO
Pd Br
PPh3
PPh3
cis/transisomerization
HO
Me
OH
CHO
PdPh3P
PPh3
R
reductiveelimination
R
Cu
HO
Me
OH
CHO
PdPh3P PPh3
R
CuIEt3N
Et3N H R
Et3N RH
HI
Circular Dichromism
64 Szilágyi, A. (2008) Electromagnetic waves and circular dichromism: an animated tutorial.http://www.enzim.hu/~szia/cddemo/edemo0.htm