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 !