diversity-oriented synthesis (dos): a platform for discovery in … · 2014. 12. 2. · pathway...
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Diversity-Oriented Synthesis (DOS): A Platform for Discovery in Chemical Genetics
Harvard Institute of Chemistry and Cell Biology (ICCB)
Jared Shaw – Institute Fellow (ICCB)Co-Project Leader, Harvard Center for Methodology and Library
Development (HCMLD)[email protected]
http://iccb.med.harvard.eduhttp://chembank.med.harvard.edu
http://iccb.med.harvard.edu/chemistry/shaw_group/index.htm
The Initiative For Chemical Genomics (ICG)
Daylight MUG ‘04
Harvard Institute of Chemistry and Cell BiologyThe Initiative For Chemical Genomics
• Diverse Goals of the ICCB– Chemistry - To bring the power of modern synthetic chemistry
to bear on problems in Cell Biology.• Diversity-oriented, split-pool synthesis
– Biology - To increase our understanding of fundamental mechanism in cell biology.
• Via Screening broadly for activities/phenotypes.
Information Analysis enables both of the above.(this powers our iterative approach)
• Chemical Synthesis platform - flexible, economical, scalable, integrated, enables split-pool diversity-oriented synthesis (DOS)
• Screening program - economical, high throughput and flexible.• Informatics- system that is essentially open for all.• ChemBank - fully QC’d central repository of data, technology, how to
do this, etc…
Goals • accomplished economically, efficiently in an academic setting.• Portable and Publicly accessible• We are not trying to discover drugs.
Key Enabling Technologies of the ICCB
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
80 µm tentagel100 pmol/bead
600 µm polystyreneminimally 50 nmol/bead
How we got to version 2.0…
•Not a robust/efficient Linker system•Arraying beads problematic•Enough for a single assay.•Must resynthesize every molecule!
•Silicon-based Linker system•HF/pyr cleavage system•Single Bead per well arrayer•Arrayed stock DMSO solutions (~ 4 mM) •Enough material for ‘cherry-picking’•Realistically, ~ 130 nmol/bead
Split-Pool SynthesisDiversity step
x 12
x 3
Pool step
Splitstep
9 moleculesFor ScreeningRemove 3 molecules (1 each)
for screening; Carry 9 forward
Diversity step
Splitstep
Plastic bead
6 reactions → 12 molecules
Most Libraries to Date are “Mono-skeletal”
Advantages• Short, linear synthesis-the number of reactions minimized.• First Step is enantioselective and catalytic- changing enantiomer of catalyst is an
element of diversityDisadvantages (real and potential)• Synthesis of building blocks detracts from the efficiency of this library• Lack of three-dimensional diversity could bias library toward certain targets, away
from others
*
SiO
i-Pr i-Pr
O O
O
O
R1
R3SiO O O
R1
O
ORRO O O
R1
O
NR2R3NH
PyBOP R2
R3
R = Allyl
R=H
Pd(Ph3)4thiosalisylicacid
The dihydropyran carboxamide (DHPC) library: Stavenger & Schreiber 2001
Skeletal Diversity May Offer Greater Variety of Bioactivity from Smaller Libraries
Diversity Step 1: Attach Skeletal Determinant #1 to Solid Phase
Diversity Step 2: Attach Building Block #1
Diversity Step 3: Attach Building Block #2
SkeletalAssembly
Compounds With Widely Varied Core Structures Originating from Common Precursors
Skeletal Determinant: 1) Alters Placement of Building Blocks in 3-Dimensional Space2) Prefereable if Commercialy available or synthesized in <4 steps3) 2-6 Units Necessary for 103 - 104 compounds
Buliding Block: 1) Presents functionality to biological target 2) Must be commercially available for maximum efficiency3) 20-50 units necessary for 103 - 104 compounds
Diversity Step 4: Attach Skeletal Determinant #2
Can a linear Synthetic Sequence Produce Molecules with Different 3-dimensional Structures?
NR1
O
NR1
O
NN O
R2O
R2
O
X
500-600µm Macrobead"
H R1NH2
N
X
H
R1 O
O
O
X
O
O
O X
N
X
R1O
NR3SiO
X
R1
O OH
O
X
X
R2NH2Cyclize
N NR1 R1O O
N OR2
NO R2
X = H or I...one I/cycloadduct,4 possible structures
Cleave
Three-dimensional representations of the 4 possible skeletal arrays
*
O OHR R
HO HO OH HO
OSi
i-Pri-Pr
-OR-
-OR-
Pathway Development
O
H
X
RNH2Na2SO4
N
H
XBn
NXO
O
O
OHO
OBn
+
NX
OHN
OBn
X= I or Br R= i-Pr or Bn
OMe
NH
ON
OBn
OMe
N
ON
OBn
OMe
N
ON
OBn
OMe
NH2
MeOCH2Cl2/RT
CuI (5 mol%)K2CO3
NHMe
NHMe+
HATU
85% Yield>95:5 diastereoselection
Trace Trace NOTOBSERVED
NX
OHN
OBn
OMe
Pathway Development
Buchwald Reaction: Klapars, A.; Huang, X.; Buchwald, S. L. "A General and Efficient Copper Catalyst for the Amidation of Aryl Halides." J. Am. Chem. Soc. 2002, 124, 7421-7428.
Tokuyama Reaciton: Yamada, K.; Kubo, T.; Tokuyama, H.; Fukuyama, T. "A mild copper-mediated intramolecular amination of aryl halides." Synlett 2002, 231-234.
N
H
O
O
O
NBn
O
Me
I I
MeOH
O
>95:5 Diastereoselection
+Bn N
BnO
I
Me
HNO
OMe
C6H6/RT
NH2
MeO
NBn
O
I
Me
HNO
OMe
CuI (5 mol%)/K2CO3
NHMe
NHMe(5 mol%)
CuI/CsOAc
NBn
O
MeN
OMe
O
Pathway DevelopmentNH2
MeO
NBn
O
NO
MeO
N
H
O
O
O
NBn
O
I
OHO
I
I
>95:5 Diastereoselection
+
BnN
BnO
I
NHO
I
MeO
C6H6/RT
CuI (5 mol%)/K2CO3
NHMe
NHMe(5 mol%)
CuI/CsOAc
NO REACTION
>95% conversion
DMSO/ 90 °C
NBn
O
I
NHO
I
MeO
Pathway Development
O
H
X
BnNH2Na2SO4
N
H
XBn O
+
X= I or BrR = H or CH2OSi(i-Pr)3 NH2
MeO
O
O
NX
O OH
OBn
NX
O NH
OBn
Solvent(reflux)
N
OBn
N O
RR
R
anti : syn% yieldCH2Cl2CHCl3EtOAcTHFEtOHDMFCH3CNBenzeneToluene
83 : 1783 : 1775 : 2570 : 3070 : 30
—65 : 3575 : 2590 : 10
8090888579
Decomp.929493
Solvent
NX
O OH
OBn
Ranti syn
MeO MeO
R
R
HATU
85-90% YieldX = Br, No ReactionX = I, R = H, 95% yieldX = I, R =CH2OSi(i-Pr)3, 95% yield
CuI/CsOac/DMSO
Pathway Development
*
SiO
i-Pr i-Pr
O
HR
N
H
Bn
R
NBn
O
R
NBn
OI
II I
NH
OOHO R N
NBn
BnO
O
= R 97% yield80:20 Diastereoselection
PhPh
Bn
>95% yield >95% yield
O
O
OR1
NR2
H
R1
NR2
H
O
O
OPh
R1
N+R2
H
O
O-O
R1
N+R2
H
O Ph
O
O-
R1
N+R2
H
O
O-
HO
R1
N+R2
H
O Ph
OH
O-
Carboxylate Aryl-Stabilized Enolates
R1
NR2
R1
NR2
O
O
HO2CPh
CO2H
Pathway Development
*
SiO
i-Pr i-Pr
N
H
R
NBn
O
I I
OHO
= R
R
anhydride R SM Product (anti : syn)
>95 (76 : 24)
16 84 (80 : 20)
<5 >95 (71 : 29)
<5 >95 (69 : 31)
>95 (>95 :5 )
H
OMe
Cl
CF3
NO2
O
RO
O
R
Summary
*
SiO
i-Pr i-Pr
O
H
NR1
O
X
NHO
NO2
NR1
O
Me
NHO
NR1
O
NO
R2
N
NR1
R2O
O
R2
HO
HO
HO
HO
R2
*
SiO
i-Pr i-Pr
N
H
XR1
R1NH2
O
O
OMe
O
O
O
O
O
OR2NH2
(X = H or I)
I
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
Bead Arrayer
Illustration By: Jim Horn Prof. Randy King, Les Walling
Bead Arrayer
HF DispenserHF Pump system
Stacking Unit
Self-Contained Cleavage System
2 DMSO Stock Plates @ 5mM DMF Stock Plate
Compound and Beads
Library Formatting
CellBasedAssays
SmallMoleculePrinting
Tag Cleavage&
Decoding
Master copyStorage &Reference Samples
Beads
residue
Abram Calderon, Xiaohua Li
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
C
cytoplasm nucleus
cell division,differentiation,cell death, etc.
5' 3'* 5' 3'
*inactivating mutation in gene C activating mutation in gene C
geneticapproach
chemicalgenetic
approach
MeHO
Me
OH
OOHMe
F H
H
OO
OMe
MeOMeO
MeONHCOCH 3
colchicine dexamethasone
A B
Classical Genetics/Chemical Genetics
Classical genetics
Chemical genetics
Collaborative Screening at the ICCB
patterned after forward genetics patterned after reverse genetics
phenotypic screening proteomic screening• screen collections of structurally complex and diverse small molecules in search of a particular cellular/organismal phenotype.• ICCB hosts investigators
• modify cellular/organismal function of a targeted protein with a small molecule, search broadly for the resulting phenotype.• ICCB will distribute microarrays
htp cytoblot assays htp zebrafish, worm screening-by-imaging small molecule and protein microarrays
Pin Transfer robotics
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
2 DMSO Stock Plates @ 5mM DMF Stock Plate
Compound and Beads
Library Formatting
CellBasedAssays
SmallMoleculePrinting
Tag Cleavage&
Decoding
Master copyStorage &Reference Samples
Beads
residue
Abram Calderon, Xiaohua Li
Typical GC run for decoding solution
Manual Decoding Process
Afferent SDF file ChemFinder
Chemistry Beads GC/LCMS
Visual analysis & manual data entry
Excel files
Disadvantages of Manual Decoding
• Visual analysis, manual data entry and maintenance of the data is slow and laborious
• Excel is not suited to storing data large amounts of decoding data
• The process is so time-consuming that generally only a small subset of compounds (those that hit in a screen) are actually decoded
Afferent SDF file
ChemistryBeads
GC
Automated Decoding System
Can Chemical Encoding be integrated with Library Enumeration into an Automated Process?
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
A technology platform for diversity-oriented synthesis: One bead-one stock solution (Version 2.0)
Sii-Pr i-Pr
O
substrate
Sii-Pr i-Pr
OSii-Pr i-Pr
OMe HO substrate
tag-1
tag-3
tag-2
(1) load substrate
(4) bead arraying
(5) compound release
(6) stock solution preparation
(9) "hit" decoding
(10) resynthesis and maturation
(11) database entry
(7) pin transfer robot1. forward chemical geneticspin transfer to high density wells for phenotypic assays
2. reverse chemical geneticsrecapture small molecules on microarrays for binding assays
*
*= 500-600 µm beads
0.1 mg compound/bead
linker
*(2) pathway development (diversity-oriented organic synthesis)
(3) library realization (encoding protocol)
*small
moleculeencoded library
384-well stock plates> 5 mM stock solutions(8) microarraying robotChemBank
Si
OMe
i-Pr i-Pr
= C, Si-linker-functionalized mimotope lantern;
10 mg compound/lantern
(A) (B)
AcknowledgmentsICCB Professional StaffchemistryXiaohua LiJennifer RaggioLeticia CastroKerry PierceMax NarovlyanskyHua MaioLi LaiScreeningCaroline Shamu (head)James FollenDavid HayesKatrina SchulbergInformaticsErik Brauner (head)Jermey Muhlich
Shaw LaboratoryYoshiyuki FukasePui-Yee NgCraig MasseMaria Sanchez-RoselloJudy Mitchell
NCI HCMLDNIGMS ICG
ICCB LaboratoriesClemons LaboratoryTallarico LaboratorySchreiber LaboratoryKing LaboratoryFeng LaboratoryMitchison Laboratory
Funding
Dr. John Tallarico
Dr. Rebecca WardProf. Stuart SchreiberProf. Tim Mitchison