diversity-oriented synthesis (dos): a platform for discovery in

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)

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


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



(9) "hit" decoding


(11) database entry



*

*= 500-600 µm beads


linker



*small



Si

OMe

i-Pr i-Pr



(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


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



(9) "hit" decoding


(11) database entry



*

*= 500-600 µm beads


linker



*small



Si

OMe

i-Pr i-Pr



(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


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



(9) "hit" decoding


(11) database entry



*

*= 500-600 µm beads


linker



*small



Si

OMe

i-Pr i-Pr



(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?


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



(9) "hit" decoding


(11) database entry



*

*= 500-600 µm beads


linker



*small



Si

OMe

i-Pr i-Pr



(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

diversity-oriented synthesis (dos): a platform for discovery in

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