DNA-Templated Organic Synthesis

DNA-Templated Organic Synthesis Jinsong Yang Michigan State University March 31, 2004 Outline ¾ Introduction ¾ DNA-Templated Oligonucleotide Ligati...
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DNA-Templated Organic Synthesis

Jinsong Yang Michigan State University March 31, 2004

Outline ¾ Introduction ¾ DNA-Templated Oligonucleotide Ligation ¾ Selenium-Mediated Autoligation ¾ Photoreversible Ligation

¾ DNA-Templated Small Molecule Synthesis ¾ One-Step Synthesis ¾ Multistep Synthesis ¾ One-Pot Reaction

¾ Summary

Nature’s Approach to Discovery

Ph

O

O N

HN H

Selection, amplification, diversification

O

S

OH

Rate Acceleration of Glycoside Hydrolysis

OH O

HO HO

HO HO

OPh

OH

OH

Rate of hydrolysis

H

H O

O OH

Acid-catalyzed hydrolysis

Kobs = 1.9 × 10-6 s-1 in 0.1 M HCl

Intramolecular catalysis

Kuni = 1.4 × 10-3 s-1

β−galactosidase

Kcat = 40 s-1

O

O

Enz HO

OH O

HO

OH

O H

O

OPh

Bugg, T. An Introduction to Enzyme and Coenzyme Chemisty; Blackwell Science, Inc.: Cambridge, MA, 1997.

How Do Enzymes Work?

A

enzyme

+

B

+

enzyme

enzyme

A

enzyme

A B

A B

p ro d u c ts

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

¾ 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?

¾ Is it possible to mimic enzyme-catalyzed chemical reactions? ¾ Is it possible to use Nature’s approach to devise the desired product?

Template Reactions Br

Br

O

OH

O

O O

MeO

K O

O O

O

K O

O

O

O K

O

O

O

O O

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

EM Temp

EM Untemp

Na+

K+

Rb+

Cs+

14

123

48

22

0.08

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

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

¾ Organizes an assembly of atoms in a specific spatial arrangement. ¾ Favors the formation of a single product. ¾ Promotes attractive interaction.

What Makes DNA a Good Template?

PCR: Polymerase Chain Reaction

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

PCR: Polymerase Chain Reaction

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

PCR: Polymerase Chain Reaction

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

Outline 9 Introduction ¾ DNA-Templated Oligonucleotide Ligation ¾ Selenium-Mediated Autoligation ¾ Photoreversible Ligation

¾ DNA-Templated Small Molecules Synthesis ¾ One-Step Synthesis ¾ Multistep Synthesis ¾ One-Pot Reaction ¾ Summary

Outline 9 Introduction ¾ DNA-Templated Oligonucleotide Ligation ¾ Selenium-Mediated Autoligation ¾ Photoreversible Ligation

¾ DNA-Templated Small Molecules Synthesis ¾ One-Step Synthesis ¾ Multistep Synthesis ¾ One-Pot Reaction ¾ Summary

Why is Ligation Important? replication

Two reasons:

DNA

RNA

transcription

1.

All reactions in the cycles are ligations.

2.

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

Protein

translation

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

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

Minimal Scheme for a Catalytic Template Directed Ligation Substrates A′

A

B

+

1

A

B

Template 2

Template 3

Product

+

Scheme of template-directed ligation with steps 1, molecular recognition; 2, ligation; 3, product dissociation.

Selenium-Mediated Autoligation

DNA or RNA template

3'

5'

5' O O P O X-

-

O O 1

X: Se

3' O I

pH 7.0, 37 oC 24 h

O 2

5'

3' 5' O O

O OP O X

3' O

3

B: nucleobase

Yield:70%

Highly sensitive to the sequence of the target nucleic acid.

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

O

Selenium-Mediated Autoligation

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.

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

Photoreversible Ligation NH2

O NH

N O

N

N

O

ODN 2

ODN 1 ODN 3

96 %, 12 h

ODN : oligodeoxynucleotides NH2

HH

N O

O N

NH

H H N

ODN 4

O

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

Photoreversible Ligation

4

1, 2

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.

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

Summary of DNA Ligation ¾ Highly efficient. ¾ Sequence specific. ¾ First DNA-templated carbon-carbon bond forming reaction. ¾ Possible application in ligation of other nucleic acids.

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

Outline 9 Introduction 9 DNA-Templated Oligonucleotide Ligation 9 Selenium-Mediated Autoligation 9 Photoreversible Ligation

¾ DNA-Templated Small Molecules Synthesis ¾ One-Step Synthesis ¾ Multistep Synthesis ¾ One-Pot Reaction ¾ Summary

Outline 9 Introduction 9 DNA-Templated Oligonucleotide Ligation 9 Selenium-Mediated Autoligation 9 Photoreversible Ligation ¾ DNA-Templated Small Molecules Synthesis ¾ One-Step Synthesis ¾ Multistep Synthesis ¾ One-Pot Reaction ¾ Summary

DNA-Templated Small Molecule Synthesis

DNA-bound reagent

DNA template

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

Preparation of DNA-Linked Reagents O N O

5∩ -NH2

O pH 7.2, 25

R

O

oC

5∩ -NH

1h

O

O

R=

I S

O Br

N H

O

O

O N

O

O N O

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

R

One-Step Synthesis

Reaction Conditions O

pH 8.5, 0.25 M NaCl

O NH2

Product

N H

O I

N H

37 ºC, 16 h, 60 nM template and reagent

O NH

SN2 Reaction SH

Br

N H

N H

O SH

pH 7.5, 0.25 M NaCl,

O

O

O O

Conjugate addition

NH2

O N H

O

O SH

O

25 ºC, 75 min, 60 nM template and reagent

S O

O

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

S O

O O

NH

N

N H

O

O

pH 7.5, 0.25 M NaCl

N H

O N

N H

pH 8.5, 0.25 M NaCl N

S

N H

N H

25 ºC, 10 min, 60 nM template and reagent

N

N H

37 ºC, 16 h, 60 nM template and reagent pH 7.5, 0.25 M NaCl

O

O

O

S

N H

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

S O

O

Generality of DNA-Templated Reaction Reaction Condition

Product

Yield (%)

O

Reductive Amination

NaBH3CN, pH 6.0,

HN

NH2

H

HN

0.5 M NaCl, 25 ºC, 1.5 h

Nitro -Aldol

HN

O

NO2

O

Wittig Olefination

pH 8.5, 0.3 M NaCl,

O

H

_

HN

O

Ph P+ Ph

O

Me

O

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

HN

O

H N

HN

>97

O

I

2) 0.075 M NaCl, 25 ºC, 2 h

O

O N

25 ºC, 22 h

N+

O

N

O

pH 7.5, 2.8 M NaCl,

H

HN

O

Heck Reaction

45

HO

CO2H

O N

25 ºC, 12 h

55 ºC, 1.5 h

O

1,3-Dipolar Cycloaddition

HN

pH 8.0, 1 M NaCl,

H

HN

NH

NO2 O

O

HN

81

O

O O

NH

O O

N

Me H N

53

O

NH N O

O

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

54

Sequence-Dependence I

NH SH

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

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

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

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

Multistep Synthesis?

Challenges: ¾ How to remove DNA used to direct reagents in the former steps? ¾ How to purify and isolate intermediates and final product?

Linker Strategies The solution to remove reagent-directing DNA :

¾ Scarless linker ¾ Useful scar linker ¾ Autocleaving linker

Scarless Linker Sulfo-NHS O S

Ph

O

HO

N H

O

O

O

1

O

reagent

NH

template

NH2 3

Ph HN

R

EDC, sulfo-NHS

79%

template

O

O

O

O O S

N

O

O N H

O O S

O

H 95%

O O

pH 11.8

HN 4

reagent

2

Ph template

NH

NH2

EDC Me ClH N+ (H2C)3 Me

O

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

N=C=N

C2H5

Useful Scar Linker Sulfo-NHS O

O

OH O

H N

HO

reagent

NH O

Ph

S

5

N

O

O R

O

OH EDC, sulfo-NHS

77%

template

NH2 3

O template

HN

OH O

H N O

Ph

OH

95%

6 EDC

NaIO4 O

template

reagent

NH

HN 7

H N

O H

Me ClHN+ (H2C)3 Me

O Ph

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

N=C=N

C2H5

Autocleaving Linker O

R1

reagent

NH

Ph Ph + P O

O 95%

NH

H

template

O

O R1

NH

template

O

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

Purification by Biotin-Avidin

¾ Biotinylated molecules will bind to the streptavidin magnetic beads. ¾ Non biotinylated molecules can be removed by washing with buffer.

Multistep Small Molecule Synthesis Programmed by DNA Templates

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

Multistep Small Molecule Synthesis Programmed by DNA Templates

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

Multistep Small Molecule Synthesis Programmed by DNA Templates

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

Synthesis of Non-Natural Tripeptide template bases 21-30 template biotin

EDC, Sulfo-NHS DNA-templated amide formation (step 1, 82%)

5∩ -NH2 O HN

O

O O S

13

O O

template bases 11-20 template biotin

HN

OH

O

O

O N H

O

O O S

O

H

NH2

anneal second reagent

Ph H N

template

O O

O

capture with avidininked beads, elude with pH 11.8 buffer

O

O O O S

N H

template

Ph

N H

Ph H N O

Ph

O

14

HN

OH O

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

NH2

biotin NH

Synthesis of Non-Natural Tripeptide template bases 11-20 template biotin

HN

Ph H N O O

NH2

O O O S

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

template

HN

OH

template bases 1-10

OH I

O

O

NH

NH2 O

16

NH2

NH

anneal third reagent

I

NH

O

15

O

O

template

Ph

O O

Ph H N

H N

O N H

template

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

biotin

HN

O O

NH Ph S O O

NH2 O H N

O O

O OH I OH I

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

Multistep Small Molecule Synthesis Programmed by DNA Templates

¾ 3% overall yield was achieved for three bond-forming reactions, three purification steps and three linker cleavages. ¾ 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 O N O

NH

TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG

one template

O O N O

NH

TGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG

mixture of 1024 templates

O

template-directed translation of DNA library into synthetic compounds

O HN

HS HS

biotin

CACTGCCCACCNGNTNGNNC-

one reagent mixture of 1024 reagents

N S

biotin CACTGCCCAC-

TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG

HN

1025 total reagents

O O

O

1025 total starting material

one product

O N O

S

CNGNTNGNNCTGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG

mixture of 1024 product

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

1025 presumed products out of 1,050, 625 theoretical products

One-Pot Reaction O HN

O N O

S

biotin CACTGCCCAC-

TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG

O HN

one product

O N O

1025 presumed productsout of 1,050, 625 theoretical products

S

CNGNTNGNNCTGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG

mixture of 1024 product

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)

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

One-Pot Reaction O N

R2 SH 16

H N R5∩

R6

N H

O

20

O

22

R2∩

12 reactants in one solution R4∩

HO 25

O O

R1 NH2

R3

15

17

H R3 N

18

H

H N R6∩ O

NH R3∩

N O O2N

R4

R5 NH2

NO2

EDC, Sulfo-NHS, NaBH3CN

O

O

O N H

R4∩

O R5 N H

H N R5∩ O

O

O

26

N H

O

CO2H

O

N R2∩ H

N

R2 S

O H N

O

O

O Ph Ph P+

O

N

N R1∩ H

N

R1 HN

19

O

21

O

24

R4 NH2

H N R1∩

O

H

O

O

O N O

NH 23

O

R3∩

R6 N H

H N R6∩ O

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)

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

One-Pot Reaction

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

Summary Present Multiple Generality

One-Pot

Future

?

Past DNA-Templated Synthesis

Base pair

Proximity

Ligation

Limitations

¾ Need to prepare DNA-linked reagents. ¾ Final product is still bound to DNA. ¾ Restricted to aqueous, DNA-compatible chemistry. ¾ PCR can not amplify the desired small molecule.

Proposed Solutions to Limitations Cleavage Final Product from DNA by Photolabile Linker

O

O

O

O

hv

Me

R O

Me

R

O

e

HN

.

O

NH

.

OH

H+

R

O I OH Ph HN

NH O

I

O NH

NH2 O

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

.

NH2

Proposed Solutions to Limitations Multiple-Release A Biotin Biotin

Biotin Biotin

B

C D

Photocleavable linker Product 1 Mild acid cleavable

Product 2

Strong acid cleavable Product 3 Enzyme cleavable

Product 4

Release A: Photolysis Release B: 0.5%TFA/ CH2Cl2 Release C: 50% TFA/ CH2Cl2 Release D: Enzyme

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

.

Acknowlegement ¾ Professor John Frost ¾ Dr. Karen Frost

¾ Group Members: Jihane

Wei

Jiantao

Xiaofei

Justas

Kit

Ningqing

Wensheng

Mapitso

Dongming

Sing

Heather

¾ Wife: Zhiqiu

Thank You for Your Attentions !