dna-templated organic synthesis - chemistry
Post on 18-Dec-2021
15 Views
Preview:
TRANSCRIPT
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
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
Introduction
DNA-Templated Oligonucleotide LigationSelenium-Mediated AutoligationPhotoreversible Ligation
DNA-Templated Small Molecules SynthesisOne-Step SynthesisMultistep SynthesisOne-Pot Reaction
Summary
Outline
Introduction
DNA-Templated Oligonucleotide LigationSelenium-Mediated AutoligationPhotoreversible Ligation
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.
Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000, 122, 5646.
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.
Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000, 122, 5646.
Outline
Introduction
DNA-Templated Oligonucleotide LigationSelenium-Mediated AutoligationPhotoreversible Ligation
DNA-Templated Small Molecules SynthesisOne-Step SynthesisMultistep SynthesisOne-Pot Reaction
Summary
Outline
Introduction
DNA-Templated Oligonucleotide LigationSelenium-Mediated AutoligationPhotoreversible Ligation
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=
Gartmer, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961.
One-Step Synthesis
Gartmer, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961.
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
Gartmer, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961.
(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
Gartmer, Z. J.; Kanan, M.W.; Liu, D. R. J. Am. Chem. Soc. 2002, 124, 10304.
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
Gartmer, Z. J.; Kanan, M.W.; Liu, D. R. J. Am. Chem. Soc. 2002, 124, 10304.
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
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
Gartmer, Z. J.; Kanan, M.W.; Liu, D. R. J. Am. Chem. Soc. 2002, 124, 10304.
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
Gartmer, Z. J.; Kanan, M.W.; Liu, D. R. J. Am. Chem. Soc. 2002, 124, 10304.
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
Gartmer, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961.
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-
TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG
TGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG
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)
TGGTGCGGAGCCGCCGTGACGGGTGATACCACCTCCGAGCCGAGCCAGGAGCCG
TGGTGCGGAGCCGCCGNCNANCNNGATACCACCTCCGAGCCGAGCCAGGAGCCG
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 !
top related