dna-templated organic synthesis - chemistry

DNA-Templated Organic Synthesis

Jinsong Yang

Michigan State University

March 31, 2004

Outline

Introduction

DNA-Templated Oligonucleotide LigationSelenium-Mediated AutoligationPhotoreversible Ligation

DNA-Templated Small Molecule SynthesisOne-Step SynthesisMultistep SynthesisOne-Pot Reaction

Summary

Nature’s Approach to Discovery

Ph O

HNN

O

H S

OHO

Selection, amplification, diversification

Rate Acceleration of Glycoside Hydrolysis

×

×O

HOHO OH

OPh

OHH

OHO

HO OHO

O OH

OHO

OHOPh

Enz

O OH

OH

OHHO

Acid-catalyzed hydrolysis

Intramolecular catalysis

β−galactosidase

Rate of hydrolysis

Kobs = 1.9 10-6 s-1 in 0.1 M HCl

Kuni = 1.4 10-3 s-1

Kcat = 40 s-1

Bugg, T. An Introduction to Enzyme and Coenzyme Chemisty;

Blackwell Science, Inc.: Cambridge, MA, 1997.

How Do Enzymes Work?

A B ++

enzyme enzymeA

enzyme

BA

enzyme

BA produc ts

The Secret to the Rate Enhancement------Proximity Effect

Proximity effect: enzymes bind their substrates so that

active functional groups are brought close together and

stay in place long enough for the reaction to proceed.

What Can We Learn from Nature?

Is it possible to mimic enzyme-catalyzed chemical

reactions?

Is it possible to use Nature’s approach to devise

the desired product?

Template Reactions

OH

OO

O

OO

Br

KO

OO

O

OO

Br

KO

OO

O

OO

KMeO

Mandolini, L.; Masci, B. J. Am. Chem. Soc. 1984, 106, 168.

0.08224812314

Cs+Rb+K+Na+

EM UntempEM Temp

Effective molarity (M) for various metal cations as templates for the synthesis of benzo-18-crown-6 at 25 º C.

Molecular Template: Definition

“ A chemical template organizes an

assembly of atoms with respect to one or

more geometric loci, in order to achieve a

particular linking of atoms”.

D. H. Busch

Busch, D. H. J. Inclusion Phenom. 1992, 12, 389.

Essential Features of Molecular Template

Organizes an assembly of atoms in a specific spatial

arrangement.

Favors the formation of a single product.

Promotes attractive interaction.

What Makes DNA a Good Template?

PCR: Polymerase Chain Reaction

http://allserv.rug.ac.be/~avierstr/principles/pcr.html

Outline

Introduction


DNA-Templated Small Molecules SynthesisOne-Step SynthesisMultistep SynthesisOne-Pot Reaction

Summary

Why is Ligation Important?

DNA

RNA Protein

The central dogma of molecular biology as formulated in 1958 with probable (solid arrows) and possible (broken arrows) reactions indicated.

replication

transcription translation

1. All reactions in the cycles are ligations.

2. Need to develop new and efficient way to detect disease in nucleotides.

Two reasons:

Crick, F. H. C. Sym. Soc. Exp. Biol. XII 1958, 138.

Minimal Scheme for a Catalytic Template Directed Ligation

Scheme of template-directed ligation with steps 1, molecular recognition;

2, ligation; 3, product dissociation.

SubstratesA B

+A′

A B

+

Template

Product

Template

1

2

3

Selenium-Mediated Autoligation

Xu, Y. Z.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 9040.

DNA or RNA template

OO O

PO O-

X-O

O

I1 X: Se 2

OO O

PO O-

XO

O

3' 5'

pH 7.0, 37 oC

24 h

Yield:70%B: nucleobase

3

5' 3'

3'

5'

5'

3'

Highly sensitive to the sequence of the target nucleic acid.

Selenium-Mediated Autoligation

Xu, Y. Z.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 9040.

1. Selenium reaction proceeded 2 times faster than sulfur counterpart

2. All-oxygen phosphate showed no ligation.

3. May be useful for direct analysis of RNAs.

N

N

O

NH2

NH

N

O

O

N

N

O

NH2

NH

N

O

O

H

H

H

H

ODN 1 ODN 2

ODN 3

ODN 4

Photoreversible Ligation

Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000, 122, 5646.

ODN : oligodeoxynucleotides96 %, 12 h

Photoreversible Ligation

Lane 1: control 12-mer;Lane 2: control 6-mer; Lane 3: ODN 1 + ODN 2, irradiation at 366 nm; Lane 4: ODN 1 + ODN 2 + ODN 3, irradiation at 366 nm, 80% yield 3 h; Lane 5: irradiation of lane 4 at 302 nm, 1 h; Lane 6: irradiation of lane 5 at 366 nm, 3 h.


4

1, 2

Summary of DNA Ligation

Highly efficient.

Sequence specific.

First DNA-templated carbon-carbon bond forming reaction.

Possible application in ligation of other nucleic acids.


Outline

Introduction


DNA-Templated Small Molecules SynthesisOne-Step SynthesisMultistep SynthesisOne-Pot Reaction

Summary

DNA-Templated Small Molecule Synthesis

DNA template

DNA-bound reagent

Gartmer, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961.

Preparation of DNA-Linked Reagents

5∩-NH2 N

O

O

O

O

R pH 7.2, 25 oC

1 h5∩-NH

O

R

NH

BrO

OI

N

O

O

N

O

O

SO O

R=


One-Step Synthesis


Reaction Conditions Product

NH2 NH

O

OI

pH 8.5, 0.25 M NaCl

37 ºC, 16 h, 60 nM template and reagent

NH

O

ONH

SN2 Reaction

SH NH

O

NH

BrO

pH 7.5, 0.25 M NaCl,

37 ºC, 16 h, 60 nM template

and reagent

NH

O

NH

SO

SH

NH

O

N

O

O

pH 7.5, 0.25 M NaCl

25 ºC, 10 min, 60 nM template and reagent

NH

O

N

O

O

S

NH2

NH

ON

O

O

pH 8.5, 0.25 M NaCl

25 ºC, 75 min, 60 nM template and reagent

NH

ON

O

O

NHConjugate addition

SH

NH

O

SO O

pH 7.5, 0.25 M NaCl

25 ºC, 10 min,, 60 nM template and reagent

NH

O

SO

O

S

Reaction Condition Product Yield (%)

Reductive Amination NH2

HN

O

H

O

NaBH3CN, pH 6.0,

0.5 M NaCl, 25 ºC, 1.5 h

HNNH

O

81

Nitro -Aldol HN

OH

O

HN

O

NO2

pH 8.5, 0.3 M NaCl,

25 ºC, 12 h

HN

O NO2

HO

NH

O

45

Wittig Olefination

HN

OH

O

HN

O

CO2H

P+Ph

Ph_

pH 8.0, 1 M NaCl,

55 ºC, 1.5 h HN

O HN

HN

O HN

HN

O HN

O >97

1,3-Dipolar Cycloaddition

N

O

O

HN

O

N+

H

Me

pH 7.5, 2.8 M NaCl,

25 ºC, 22 h N

O

O

NO MeHN

O

53

Heck Reaction N

O

O

HN

I

O

1) Na2PdCl4 with 2 eq. P(p-SO3C6H4)3

2) 0.075 M NaCl, 25 ºC, 2 h

ON

O

O

NH

54

Generality of DNA-Templated Reaction

Gartmer, Z. J.; Kanan, M. W.; Liu, D. R. Angew. Chem. Int. Ed. 2002, 41(10), 1796.

Sequence-Dependence

T-G-C-C-A-G-C-C-C-T

A-C-G-C-T-C-G-C-C-A-T-G-G-T-A-C-G-A-A-T-TC-C-A-T-C-C-T-T-A-A

O

NH SHI


(a) Hairpin templates linked to α-iodoacetamide group were reacted with thiol reagents containing 0, 1, or 3 mismatches at 25 °C.

(b) Reactions in (a) were repeated at the indicated temperature for 16 h.

Multistep Synthesis?

Challenges:

How to remove DNA used to direct reagents in the former steps?

How to purify and isolate intermediates and final product?

The solution to remove reagent-directing DNA :

Scarless linker

Useful scar linker

Autocleaving linker

Linker Strategies

Scarless Linker

Gartmer, Z. J.; Kanan, M.W.; Liu, D. R. J. Am. Chem. Soc. 2002, 124, 10304.

NHO

HOO

OS

O O

O NH

O

NHO

HNO

OS

O O

O NH

OPh

H

NH2O

HN

Ph

NH2

1

3

2

79%

reagent

template

reagenttemplate

template

95% pH 11.8

EDC, sulfo-NHS

4

PhNS

O

OO

O

R

Sulfo-NHS

EDC

N=C=N C2H5(H2C)3N+

Me

Me

HCl-

Useful Scar Linker


HOHN

NH

O

OHO

O

HNHN

O

O O

H

reagent

NH2 template

5

3

77%

reagent

template

95% NaIO4

6

7

HNHN

NH

O

OHO

O

EDC, sulfo-NHS

OH

OH

Ph

Ph

Ph

template

NS

O

OO

O

R

Sulfo-NHS

EDC

N=C=N C2H5(H2C)3N+

Me

Me

HCl-

Autocleaving Linker


P+

O

NHPh

PhO

R1

R1

O

O

NH

H

O

O

NH

reagent

template

template

95%

Purification by Biotin-Avidin

Biotinylated molecules will bind to the streptavidin

magnetic beads.

Non biotinylated molecules can be removed by washing

with buffer.

Multistep Small Molecule Synthesis Programmed by DNA Templates


Synthesis of Non-Natural Tripeptide


EDC, Sulfo-NHSDNA-templatedamide formation(step 1, 82%)

capture with avidin-inked beads, eludewith pH 11.8 buffer

anneal second reagenttemplate NH2

O

HN

Ph

14

13

biotin

templatebases 21-30

5∩-NH2

HNO

OS

O O

OO

N

OOH

Ph

H

templateN N

HOO

OS

O O

O

OPh

H

template NHbiotin

H

HN

HN

OOH

OO

S OOOHN

NH2O

Ph

templatebiotin

template bases 11-20

O

Synthesis of Non-Natural Tripeptide


1)EDC, Sulfo-NHS (step 2, 52%)2)avidin beads, then pH 11.8 buffer NH2NH

O

HN

Ph O

template

1)EDC, Sulfo-NHS (step 3, 55%)2)avidin beads, then pH 11.8 buffer

15

16

anneal third reagent

NHO

N

Ph

NHO

O

I

OHI

NH2templateH

HN

HN

OOH

OO

S OOOHN

NH2O

Ph

templatebiotin

template bases 11-20

O

template

templatebases 1-10

biotin

NHO

NH

Ph

NH2O

HN

O

OHO

O

SOO

O

O

HN

I

OHI

Multistep Small Molecule SynthesisProgrammed by DNA Templates

3% overall yield was achieved for three bond-forming reactions, three purification steps and three linker cleavages.

The final tripeptide linked to the template was characterized by MALDI mass spectrometry. (expected mass 10069 vs observed mass 10059-10075)

New Architecture Enables Two Reactions on One Template in One Step

Gartner, Z. J.; Grubina, R.; Calderon, C. T.; Liu, D. R. Angew. Chem. Int. Ed. 2003, 42(12), 1370.

One-Pot Reaction


TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG

TGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG

N OO

ONH

N OO

ONH

one template

mixture of 1024 templates

1025 total starting material

CNGNTNGNNC-

HSHS

one reagentmixture of 1024 reagents

template-directed translation of DNA

library into syntheticcompounds

1025 total reagents

biotinCACTGCCCAC-



NO

OO

HN

one product

mixture of 1024 product

1025 presumed products out of1,050, 625theoretical products

S biotinCACTGCCCAC-

CNGNTNGNNC-

NO

OO

HN S

One-Pot Reaction

Gartmer, Z. J.; Liu, D. R. J.Am. Chem. Soc. 2001, 123, 6961.

1) in vitro selection with streptavidin beads

2) PCR amplification of selected products

TGGTGCGGAGCCGCCG? ? ? ? ? ? ? ?GATACCACCTCCGAGCCGAGCCAGGAGCCG- -DNA encoding selected and amplified molecules

characterized by DNA sequencing and digestion

TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG- - primary product (1000 fold)



NO

OO

HN

one product

mixture of 1024 product

1025 presumed productsout of1,050, 625theoretical products

S biotinCACTGCCCAC-

CNGNTNGNNC-

NO

OO

HN S

One-Pot Reaction

Calderone, C. T.; Puckett, J. W.; Gartner, Z. J.; Liu, D. R. Angew. Chem. Int. Ed. 2002, 41(21), 4104.

N

O

OO

HN R1∩

R4 NH2R2 SH

R5 NH2R1 NH2 R3

O

NH

NO2

R6

CO2H

P+O

NH

PhPh

N

O

OO

NH R3∩

N

O

OO

HN R2∩

R6∩

O

H HN

O

R5∩

O

H HN

O

O

HOR4∩

1916

1815

20

17

21

22

23

25

26

2412 reactants in one solution

EDC,Sulfo-NHS,NaBH3CN

N

O

OO

NH

R1∩HNR1

N

O

OO

NH R2∩

R2 S

N

O

OO

NH R3∩R3

O

HN

O2N

O

NH

R4∩R4 R5∩NH H

N

O

R5

R6∩HN

O

O

NH

R6

One-pot reactions containing one biotinylated template (15, 16, 17, 18, 19, or 20)

+ five non-biotinylated templates (out of 15-20) + six reagents (21-26)

One-Pot Reaction

Calderone, C. T.; Puckett, J. W.; Gartner, Z. J.; Liu, D. R. Angew. Chem. Int. Ed. 2002, 41(21), 4104.

Summary

DNA-Templated Synthesis

Proximity

? Ligation

Generality

Multiple

One-Pot

Base pair

Present

PastFuture

Need to prepare DNA-linked reagents.

Final product is still bound to DNA.

Restricted to aqueous, DNA-compatible chemistry.

PCR can not amplify the desired small molecule.

Limitations

Cleavage Final Product from DNA by Photolabile Linker

Proposed Solutions to Limitations

O

NHMe R

O

HNR

O

.e

H+ NH2R

Ohv

NHO

HN

Ph

NHO

O

I

OHI

NH2

O

O

OH

Me

O

O .

Guillier, F.; Orain, D.; Bradley, M. Chem. Rev. 2000, 100, 2091.

Proposed Solutions to LimitationsMultiple-Release

Guillier, F.; Orain, D.; Bradley, M. Chem. Rev. 2000, 100, 2091.

Photocleavable linker Product 1

Mild acid cleavable Product 2

Strong acid cleavable Product 3

Enzyme cleavable Product 4

Release A: PhotolysisRelease B: 0.5%TFA/ CH2Cl2Release C: 50% TFA/ CH2Cl2Release D: Enzyme

A

B

C

D

Biotin

Biotin

Biotin

Biotin

AcknowlegementProfessor John Frost

Dr. Karen Frost

Group Members:

Jihane Wei

Jiantao Xiaofei

Justas Kit

Ningqing Wensheng

Mapitso Dongming

Sing Heather

Wife: Zhiqiu

Thank You for Your Attentions !

dna-templated organic synthesis - chemistry

Documents