lactacystin: an inhibitor in the ubiquitin proteasome pathway
DESCRIPTION
Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway. Ami Jun-Yee Chin February 17, 2005. Chemistry Nobel Prize 2004. Awarded to Aaron Cichanover, Avram Hershko, and Irwin Rose. . Central Dogma of Molecular Biology. DNA. TRANSCRIPTION of RNA. TRANSLATION to Protein. - PowerPoint PPT PresentationTRANSCRIPT
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Lactacystin: An Inhibitor in the Ubiquitin Proteasome Pathway
Ami Jun-Yee ChinFebruary 17, 2005
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Chemistry Nobel Prize 2004
Awarded to Aaron Cichanover, Avram Hershko, and Irwin Rose
.
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Central Dogma of Molecular Biology
RNA
PROTEIN
DNA
TRANSCRIPTION of RNA
TRANSLATION to Protein
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Protein DegradationI - Lysosomal Degradation
ProteinLysosome
Amino Acids
Activated at times of stress
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Protein DegradationII – Ubiquitin Proteasome Pathway
ProteinProteasome
Amino Acids
Housekeeping role A role in protein regulation
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Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SHO
S E2
E2 SHO
N Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Ciechanover, A. EMBO J. 1998, 17, 7151.
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Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SHO
S E2
E2 SHO
N Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Transfer of ubiquitin to a carrier protein
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Protein Tagging by UbiquitinO
OH E1 SH
OS E1
E2 SH
E1 SHO
S E2
E2 SHO
N Lys
+
E3
Activation of ubiquitin UBIQUITIN
TARGET PROTEIN
Transfer of ubiquitin to a carrier protein
Selection of target protein
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Formation of Polyubiquitin Chain
UBIQUITIN
TARGET PROTEIN
E3E2 SHE1 SH
ONH
LysO
NH
Lys
ON Lys
ONH
Lys
1.2.3.
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Protein Recognition
UBIQUITIN
TARGET PROTEIN
Ubiquitin tag is recognized
Components are recycled and reused
ONH
LysO
NH
LysO
NH
Lys
Amino Acids Recycled Ubiquitin
ONH
LysO
NH
LysO
NH
Lys
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Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
Ubiquitin Tag
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Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
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Protein Regulation
TRANSCRIPTION FACTOR
PROTEIN SYNTHESIS
INHIBITOR
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
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Biological Relevance
TF-
INFLAMMATORY PROTEINS
I-
PROTEASOME
AMINO ACIDSNUCLEUS
UBIQUITIN TAG
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Biological Relevance
INHIBITEDPROTEASOME
I-
TF-
INFLAMMATORY PROTEINS
NO
NUCLEUS
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Lactacystin
NH
O
O
SNHAc
HO2C
Me
HOHO
(+) - Lactacystin
Isolated in 1991
Initially studied as a nerve growth factor
Later found lactacystin to be a proteasome inhibitor
Omura, S., et al. J, Antibiot. 1991, 44, 113.
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Determination of Cellular Target
NH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin wasincubated withcell extract
Sample wassubjected to SDS PAGE
Sequencing showed homologyto proteasome
+ Lactacystin- Lactacystin
Schreiber, S.L. et al. Science. 1995, 268, 726.
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Retrosynthetic Analysis
O
NPh
OTBS
OHO
Me
ONH
OMe
HO O
SNHAc
HO2C
HO
H2N
HO
CO2Me
O
NPh
CO2Me
MeO2CHN
OHPh
OH
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First Total Synthesis NH
O
O
SNHAc
HO2C
Me
HOHO
O
NPh
CO2Me
H
O
O
NPh
HO
CO2Me
PhCH2BrO
HN CO2Me
MeO2CHN
OHPh
OH
O
HH2N
HO
CO2Me
LDA, LiBr
MeOH, TfOH
91%
84% 52%
51%
Strategy = Self Regeneration of Stereocenters
Corey, E.J. and Reichard, G. J. Am. Chem. Soc. 1992, 114, 10677.
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Self Regeneration of Stereocenters
HN
HO
Ph
CO2Me
R1
O
H
O
N CO2MeR1
Ph
O
NR1
OLi
OMe
Ph
R2 X
O
NR1
CO2MeR2
Ph
HN
HO
Ph
CO2MeR2
H2OAchiral aldehyde
LDA
N-benzylserine methyl ester
Seebach, D. et al. Helv. Chim. Acta. 1987, 70, 1194
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First Total Synthesis
MeO2CHN
OHPh
OH
O
NPh
OTBS
O
NPh CO2Me
OTBS
O
NPh CHO
OTBS
1. TBSCl, Im, 2. TsOH, (CH2O)n,
LiBH4/THF MeOH
Swern
85%
92%
OH
NH
O
O
SNHAc
HO2C
Me
HOHO
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First Total Synthesis
O
NPh CHO
OTBS
O
O
O
NPh
OTBS
OO
MeHO
O
NPh
OTBS
OOHO
Me
2:1LDA, THF, -78 C 48%
30%
NH
O
O
SNHAc
HO2C
Me
HOHO
Pirrung-Heathcock anti-aldol gave poordiastereoselectivitey
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Anti-Aldol Closed Transition States
O
O N OTBS
HO
O Li
HO
N O
OO Li
O
OLi
O
NPh
OTBS
OH
OTBS
O
MeHO
O
NPh
OTBS
O
O
NPh
OTBS
OOHO
Me
+
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First Total Synthesis
N O
O
Me
HOHO2C
NH
OMe
HO CO2H
SH
HS NHAc
O O
N O
O
Me
HOHO
NH
OMe
HO O
SNHAc
HO2C
HO
O
NPh
OTBS
OHO
Me
O1. H2/Pd-C2. 5% HF
1. Swern 2. NaClO2
78% 69%
BOPCl, Et3N
2. Pd(Ph3P)3 Et3N, HCO2H
1. H+95%
66%HO
HS
NH
O
O
SNHAc
HO2C
Me
HOHO
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Drawbacks to Synthesis
Poor diastereoselectivity Needed to upscale to pursue biological studies
O
NPh
OTBS
OO
MeHO
O
NPh
OTBS
OOHO
Me
NH
OMe
HO O
SNHAc
HO2C
HO
H2N
HO
CO2Me
+2:1
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Revised Aldol Reaction
O
NPh CHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
NPh
OTBS
OO
MeHO
O
NPh
OTBS
HO OMeO
Me
O
NPh
OTBS
OOHO
Me
O
NPh
OTBS
HO OMeO
Me
2:1LDA
9:1
ORIGINAL ROUTE
REVISED ROUTE
+
+
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Revised Aldol Reaction
O
NPh CHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
NPh
OTBS
OO
MeHO
O
NPh
OTBS
HO OMeO
Me
O
NPh
OTBS
OOHO
Me
O
NPh
OTBS
HO OMeO
Me
2:1LDA
9:1
ORIGINAL ROUTE
REVISED ROUTE
+
+
Corey, E.J. et al. J. Am. Chem. Soc. 1998, 120, 2330
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Magnesium Catalyzed Anti-Aldol
NO
OSi MeMe
But
O
H
MgI
BOTTOM
TOP
Top face is favoured for attack of nucleophile
Bottom face is shielded by Benzyl and OTBS
NU
NU
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Open Transition State Aldol
MgI
MgI
OO
N
OHN
OH
N
OOTBS
CO2Me
Me
HON
OOTBS
CO2MeHO
Me
SYNCLINICAL ANTIPERIPLANAR
Anti-aldol Syn-aldol9:1
Me
HO
OMe
SiMe
MeMe
MeH
OMeOSi
Me MeMe
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Improvements to Synthesis
Doubly diastereoselective aldol Synthesis of lactacystin in kilogram quantities Quantity allowed further biological investigation
NH
OMe
HO O
SNHAc
HO2C
HOH2N
HO
CO2Me
O
NPh
OTBS
HO OMeO
Me
O
NPh
OTBS
HO OMeO
Me
+ 9:1
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SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
WHAT Which parts of the target molecule is essential ?
HOW Stepwise changes are made and activity is measured
WHY
To maximize activity of target molecule
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Initial SAR Studies
NH
O
Me
O
SNHAc
HO2C
HO
NH
O
O
SNHAc
Me
HOHO
HO2C
Compound Proteasome Inhibition
Inactive
Inactive
NH
O
Me
O
SNHAc
HO2C
HO
ActiveHO
Lactacystin
OH cis to carbonylnecessary
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Initial SAR Studies
NH
O
Me
O
OH
HO
NH
O
Me
Compound Proteasome Inhibition
Inactive
More Active
NH
O
Me
O
SNHAc
HO2C
HO
ActiveHO
HO
OO OH
Lactacystin
Electrophilic carbonylessential
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Mechanistic Studies: In Vitro
Dick, L. et al. J. Biol. Chem. 1996, 271, 7273.
NH
O
HOHO
O
SNHAc
HO2CMe pH 8, H2O NH
O
HOHO
O
OHMe
HS NHAc
CO2H+
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Mechanistic Studies: In VitroA
238 (
nm)
Time (min)
NH
O
HOHO
O
SNHAc
HO2CMe pH 8, H2O NH
O
HOHO
O
OHMe
HS NHAc
CO2H+
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Mechanistic Studies: In Vitro
NH
O
HOHO
O
SNHAc
HO2CMe pH 8, H2O NH
O
HOHO
O
OHMe
HS NHAc
CO2H+
A23
8 (nm
)
Time (min)
Not First order kinetics Suggests intermediate involved
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Mechanistic Studies: Hypothesis
Is -Lactone an intermediate ? Increasing [NAC] will decrease rate of hydrolysis
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
OO
OH
NAC
HS NHAc
CO2H
NH
O
O
OH
HO
Me
HO+
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Effects of [NAC] on Rate of Hydrolysis
2.0
1.5
1.0
0.5
0.00.1 1 10
k hyd
rol y
sis
( x 1
0-4s- 1
)
[NAC] (mM)
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
OO
OH
NACHS NHAc
CO2H
NH
O
O
OH
HO
Me
HO+
Addition of NAC impedesrate of hydrolysis
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HPLC Detection of -Lactone
Abs
orba
nce
0.00
0.05
0.10
1.01.21.4
5 10 15 20 25 30Retention Time (min)
-Lactone
NH
O
Me
OO
OH
HS NHAc
CO2H
NH
O
O
OH
HO
Me
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
+
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Mechanistic Studies
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
Me
HO OHO
OH
NH
O
OH
Me
OO
INHIBITION
NO INHIBITION
INHIBITION
NO INHIBITION
pH 8
pH 6.3NO -Lactone
pH 8 and pH 6.3
pH 8 and pH 6.3
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Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HS NHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
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Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HS NHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
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Mechanistic Studies: Role of Glutathione
NH
O
OH
Me
OO
HS NHAc
CO2H
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
N-Acetylcysteine
Glutathione Lactathione
+
+
Can Glutathione react with -Lactone to give a thioester adduct ?
Dick, L. et al. J. Biol. Chem. 1997, 272, 182.
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44Retention Time (min)
Lactathione Formation In Vitro Confirmed
HS CO2H
NH2
OO NH
CO2H
NH
O
OH
Me
OO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
Glutathione Lactathione
+
Glutathione + -Lactone
Glutathione
-Lactone
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In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
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In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
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47
In Vivo Studies of Lactathione Formation
Cells
Cells Washed cells
Washed cells
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
OH
Me
OO+
?Cell lysate
HPLC
HPLCCell lysate
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48
HPLC Analysis of Cell Extract
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
NH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin
-Lactone
Lactacystin
HPLC
HPLC
HPLCNH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
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49
Fate of Lactacystin In Vivo : 2 Possibilities
CELLNH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin is impermeable to cell membrane
Lactacystin
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50
Fate of Lactacystin In Vivo : 2 Possibilities
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
Conversion in cells occurs too rapidly for lactacytin to be detected
Lactacystin Lactathione
OR
CELLNH
O
O
SNHAc
HO2C
Me
HOHO
Lactacystin is impermeable to cell membrane
Lactacystin
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51
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
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52
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
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53
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Washed cells
Cell lysate
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54
Control with Glutathione Depleted Cells
No Glutathione
HPLC
NH
O
O
SNHAc
HO2C
Me
HOHO
+
NH
O
O
SNHAc
HO2C
Me
HOHO
Results suggest that Lactacystin Is impermeable to cell membrane
Washed cells
Cell lysate
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55
Mechanism of Action: Role of -Lactone
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
Lactacystin -Lactone
-Lactone is the active inhibitor Only -Lactone is permeable to cell membrane?
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56
Hydrolysis of Lactacystin vs -Lactone
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
OHMe
HOHO
Lactacystin
Time (min)
-L
acto
ne]
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57
-Lactone] Outside the Cell
NH
O
OH
Me
OO
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
OHMe
HOHO
- Lactone
Lactacystin
Hydrolysis of -Lactone is slower when starting with Lactacystin
Time (min)
-L
acto
ne]
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58
Lactathione Accumulation in Cells
Lactathione accumulation is slower in Lactacystin treated cells
NH
O
O
SNHAc
HO2C
Me
HOHO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
CELLS
NH
O
OH
Me
OO
NH
O
O
S
Me
HOO
CO2H
NH2
O NH
CO2H
HO
CELLS
Time (min)
[Lac
tath
ione
]
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59
Mechanism of Action: Conclusions
Hydrolysis of -Lactone
- Lactone
Lactacystin
Time (min)
-L
acto
ne]
Lactathione Accumulation
- Lactone
Lactacystin
Time (min)
[Lac
tat h
ione
]
Extracellular [-Lactone] Intracellular [Lactathione]
CONCLUSION
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60
Mechanism of Action: Summary
NH
O
O
SNHAc
O2C
Me
HOHO
NH
O
OH
Me
OO
NH
O
O
OMe
HOHO
NH
O
OH
Me
OO
-GSH
+GSH
NH
O
O
S
Me
HOO
CO2
NH2
O NH
CO2H
HO
INSIDE CELL
OUTSIDE CELL
MEMBRANE
O
O
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61
SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
OH and carbonyl are cis -Lactone formation is necessary for activity
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62
SAR Studies of Lactacystin
NH
O
O
SNHAc
Me
HOHO
HO2C
NH
O
Me
OO OH
C9
C7
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63
Synthesis: C9 Analogues
MeS CO2MeMe CO2Me
CO2HCO2Me
MeSMe
NO
PMB
OHTBSOCO2MeMe
MeSNO
Me PMB
TBSOCO2Me
CHO
NO
O CO2MeHPMB
MeMeS
NO
PMB
HOCO2MeOH
MeMeS
PLE, PH 7
62%
1. (COCl)2 DMF2. PMB-NHCH2CO2Me, Et3N3. LDA,
92%
1. Formalin, DBU2. NaBH(OAc)3
86%
1. PivCl, Pyridine2. TBSOTf3. NaOMe,MeOH
77%
1. Raney Ni,2. Dess-Martin
78%
95%ee
99%ee
NH
O
Me
OO OH
Corey, E. J. et al. Angew. Chem. Int. Ed. 1998, 37, 1676.
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64
SAR of C9 Analogues
NO
PMB
CHO
MeCO2Me
TBSO
NO
PMB
R
Me
TBSOCO2Me
HO
NHO
O O
R
OH
MeRMgBr,TMSCl
4 Steps
R
OH
OH
H
OH
OH
Entry Rel. kinhibition
1
2
3
4
5
1
0.006
0.003
Inactive
OH
0.095
Isopropyl group isalready optimum
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65
SAR of C7 Analogues
O
NPh
CHOOTBS
R
OTMS
OMe O
NPh
OTBS
CO2Me
RHO
NHO
R
O OOH
+9 Steps MgI2
R
Me
H
Et
PHCH2
Entry Rel. kinhibition
1
2
3
4
5
1
0.15
2.18
2.33
0.73
nPr
Activity Increased with larger groups at C7
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66
SAR Studies of Lactacystin
NH
O
O
SNHAc
HO2C
HOHO
Larger groups
EssentialElectrophillicCarbonyl
Essential
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67
-Lactone : Important Feature for Activity
NH
O
OH
Me
OO
-Lactone
NH
O
OHOO
Salinosporamide A
Cl
Cell permeability Electrophillic carbonyl for acylation of proteasome
Isolated by Fenical, 2003 More potent inhibitor than -Lactone Cytotoxic activity
Fenical, W. et al. Angew. Chem. Int. Ed. 2003, 42, 355.
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68
Synthesis of Salinosporamide A
H2N CO2Me
MeHO
MeOO
Cl
O
ClO N
Me
CO2Me
HOOBn
PMB
MeOO
N CO2Me
Me
MeO
HN
HO
CO2Me
MeOBn
57%
1.
2. pTsOH,
1. LDA, THF-HMPA ClCH2OBn2. NaCNBH3
62%1. TMSCl2. , iPr2NEt,
3. H+
96%
NH
O
OHOO
Cl
Corey, E.J. et al. J. Am. Chem. Soc. 2004, 126, 6230
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69
Synthesis of Salinosporamide A
O N
Me
CO2Me
HOOBn
PMB
NOCO2Me
OBn
O
PMB
Me
NO
Me
CO2Me
OBn
OH
PMB
O N
Me
CO2Me
OBn
PMB
O
NO
OH
CO2Me
OBn
Me
PMB
Dess-Martin [O]
96%
Quinuclidine, DME90%
9:1
BrCH2Si(CH3)2ClEt3N, DMAP
95%Si(Me)2CH2Br
NH
O
OHOO
Cl
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70
Synthesis of Salinosporamide A
NOCO2Me
OBn
O
PMB
Me
Si(Me)2CH2Br
NOPMB
OSi
Me Me
H Me
CO2Me
OH
H
ZnCl
NOCO2Me
OBn
PMB
OSi
Me Me
H Me
NO CHOCO2Me
PMB
OSi
Me Me
H Me
Bu3SnH, AIBN,
89%
1. H2/Pd-C2. Dess-Martin
88%
NH
O
OHOO
Cl
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71
Synthesis of Salinosporamide A
NOPMB
OSi
Me Me
H Me
CO2Me
OH
1. KF, KHCO3, H2O22. CAN
HNO
OHH Me
CO2Me
OH
HO
81%
1. LiOH2. BOPCl, Py3. Ph3PCl2, MeCN, Py
65%
NH
O
OHOO
Cl
Total synthesis of Salinosporamide A was achieved in 10%over 18 steps
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72
Summary: Ubiquitin Proteasome Pathway
ONH
LysO
NH
LysO
NH
Lys
Amino Acids Recycled Ubiquitin
UBIQUITIN
TARGET PROTEIN
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73
Summary: Synthesis of Lactacystin
O
NPh CHO
OTBS
O
O
Me OMe
OTMS
MgI2
O
NPh
OTBS
OO
MeHO
O
NPh
OTBS
HO OMeO
Me
LDA
ORIGINAL ROUTE
REVISED ROUTE
NH
O
O
SNHAc
HO2C
Me
HOHO
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74
Mechanism of Action
NH
O
O
SNHAc
O2C
Me
HOHO
NH
O
OH
Me
OO
NH
O
O
OMe
HOHO
NH
O
OH
Me
OO
-GSH
+GSH
NH
O
O
S
Me
HOO
CO2
NH2
O NH
CO2H
HO
INSIDE CELL
OUTSIDE CELL
MEMBRANE
O
O
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75
Summary: Synthesis of Salinosporamide A
H2N CO2Me
MeHO
NH
O
OHOO
Cl
Synthesized in 18 steps, 10% overall yield
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76
Summary: SAR Studies and Analogs
NH
O
O
SNHAc
HO2C
Me
HOHO
Can be a larger group Essential to form -Lactone Initially optimized
NH
O
OH
Me
OO
NH
O
OHOO
- Lactone
Salinosporamide A
NH
O
OHOO
MLN-519Phase 1
Cl
Lactacystin
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77
Acknowledgements
Dr. OgilvieLivia AumandAlison LemayMathieu LemayMatt ClayMy Family and FriendsAnd You!