noncanonical nucleosides: pseudouridine and 5-methyldeoxycytidine · pdf filenoncanonical...
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Noncanonical Nucleosides: Pseudouridine and 5-Methyldeoxycytidine
Valerie ShurtleffMacMillan Group Meeting
September 3, 2015
Nucleic Acids: Genetic Polymers
■ Ribonucleic acid and deoxyribonucleic acid
OBase
O
OPOOH O
BaseO
OPO OOH
RNA
OH
OH
OBase
O
OH OH
nucleotide
OBase
HO
OH OH
nucleoside
OOH
HO
OH OH
ribose
PO
HO
OH
■ Monomeric building blocks
5'
3'4'
2'1' O
BaseO
OPOOH O
BaseO
OPO OOH
DNA
5'
3'4'
2'1'
The Canonical Nucleosides
ON
HO
OH
deoxyadenosine
N
N
N
NH2
ON
HO
OH
deoxyguanosine
N
N
NH
O
NH2O
NHO
OH
deoxythymidine/uridine
NHMe/H
O
OO
NHO
OH
deoxycytidine
N
NH2
O
H/OH
■ Watson–Crick base pairs
RN
N
NN
N HH
RN
N
MeO
O
H
adenine thymineA T
N
N
NN
O
N
RH
HH
guanine cytosineG C
NN
N
O R
HH
Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 5th ed.; W.H. Freeman and Company: New York, 2008.
The Organic Chemist's View of Modified Nucleosides
sofosbuvir
telbivudine
O
HO
O
F Me
PPhO
iPrOAlaO
N
HN O
O
Gilead, 2013
Novartis, 2006gemcitabine
Lilly, 1996
O
HO
HO
F F
N
NONH2
ribavirinICN, 1980
O
HO
HON
edoxudineUpjohn, 1969
O
HO
HON
HNO
O
Et
OH
NN
ONH2
cytarabinePfizer, 1969
O
HO
HON
NO
OH
NH2
Antiviral AgentsAnticancer Agents12 approved8 approved
O
HO
HON
HNO
O
Me
Naturally Occuring Noncanonical Nucleosides
ON
HO
OH
N
N
N
NH2
ONHO
OH
NH
O
Se
OH
■ Nomenclature for describing nucleoside modifications
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
OH
A m62Am
1
2
34
57
89
6
ON
HO
OH
N
N
N
N
O
MeMe
2' OH modification
2'
# of modifications
modification position
modification
Me
m5dC
NH
Me
mnm5se2UO
NHO
OH
N
NH
O
OH
NH2
Q
HNhypermodifiednucleosides
nucleoside core
ONHO
OH
N
NH2
O
Me
16
5
43
2
OH
OH
Nucleoside Modifications in RNA
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
many types of RNA with many functions many nucleoside modifications
over 100 modified nucleosides known in RNA
Nucleoside Modifications in RNA
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
sites of modification in a tRNA modified nucleosides in different domains
Pseudouridine: a C-Nucleoside Derived from Uridine
Charette, M.; Gray, M. W. IUBMB Life 2000, 49, 341.
■ Most prevalent of all natural modified nucleosides
■ Present in almost all classes of RNAO
HO
OH
NHHN
O
O
OH
pseudouridine (Ψ)
■ First modified nucleoside discovered in RNA
■ The "fifth nucleoside" in RNA
The Function of Pseudouridine in RNA
Charette, M.; Gray, M. W. IUBMB Life 2000, 49, 341.
■ Ψ can serve to rigidify surrounding structure
■ May stabilize complex 3D structure of RNA
O
HO
OH
NHHN
O
O
OH
pseudouridine (Ψ)
■ Unique H-bonding ability – coordination of water
ONHO
OH
NH
O
O
OH
uridine (U)
additional H-bonding site
H2O
phosphate
phosphate
Ψ
"Acylal Mechanism" for the Biosynthesis of Pseudouridine
Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270.
O
RO
OR
NHHN
O
O
OH Ψ
ONRO
OR
NH
O
O
OHU
pseudouridine synthase (ΨS)
O Enz
O
O Enz
O
O
NH
RO
OR
NH
O
O
OH
OEnz
O
O
HN
RO
OR
NH
O
O
OH
OEnz
O
net 180° rotation
"Michael Mechanism" for the Biosynthesis of Pseudouridine
Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270.
O
RO
OR
NHHN
O
O
OH Ψ
ONRO
OR
NH
O
O
OHU
pseudouridine synthase (ΨS)
O Enz
O
ONRO
OR
HN
O
O
OH
OO
OR OHO Enz
NH
HN
O
O
O Enz
O
RO
O Enz
O
ORO
OR
HN NH
O
O
OH
O
O EnzO
OR OH
HN NH
O
O
O Enz
O
RO
Evidence for the "Michael Mechanism"
Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270.
ONRO
OR
NH
O
O
OH5FU
F
N
NH
O
O
F
5FU-tRNA+
Foster, P. G.; Huang, L.; Santi, D. V.; Stroud, R. M. Nat. Struct. Biol. 2000, 7, 23.
highly conservedaspartate residue
ΨSI
isolable covalent5FU-tRNA–ΨSI
complex
ΨSI
What is the identity of the 5FU-tRNA–ΨSI covalent complex?
Evidence for the "Michael Mechanism"
Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270.
ONHO
OH
NH
O
OH5FU
ONHO
OH
HN
O
O
OH
O
O Me
O
F F
AcOHmodelsystem O
NHO
OH
HN
O
O
OH
OH
F
H2O, Δ
• Asp-60 attacks C6 (1,4-addition)
"fairly stable"in H2O at rt
isolable covalent5FU-tRNA–ΨSI
complex
digestion to
nucleosides ONHO
OH
HN
O
O
OH
OH
F
proposed product based
• protonation from opposite face• ester hydrolysis liberates pdt
retention ofconfiguration
on HPLC retention time
Proposal:
"Michael Mechanism" for the Biosynthesis of Pseudouridine
Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270.
O
RO
OR
NHHN
O
O
OH Ψ
ONRO
OR
NH
O
O
OH5FU
pseudouridine synthase (ΨS)
O Enz
O
ONRO
OR
HN
O
O
OH
OO
OR OHO Enz
NH
HN
O
O
O Enz
O
RO
O Enz
O
ORO
OR
HN NH
O
O
OH
O
O EnzO
OR OH
HN NH
O
O
O Enz
O
RO
F
F
F
F
F
×
Shouldn't we expect to observethe fluorinated C-nucleoside?
Rearrangement of 5FU by Pseudouridine Synthesis
Hoang, C.; Ferré-D'Amaré, A. R. Cell 2001, 107 , 929.
ORO
OR
HN NH
O
O
OH
O
O Enz
F
5FU-tRNA boundto ΨS TruB
ORO
OR
HN NH
O
O
OH
FOH
esterhydrolysis?
previously proposed product may have been misidentified
Revisiting the "Michael Mechanism"
Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G. J. Am. Chem. Soc. 2004, 126 , 12758.
ORO
OR
HN NH
O
O
OH
FOH
F(OH)Ψ
formation of F(OH)Ψ consistentwith proposed Michael mech.
(not definitive proof – multiple ORO
OR
HN NH
O
O
OH
O
O Enz
F
esterhydrolysis?
pathways can be envisioned)
N
NH
O
O
F
5FU-tRNA
1. incubation with TruBin 50% 18OH2 buffer
2. digestion ORO
OR
HN NH
O
O
OH
F H18O EnzOH
O
expected products from ester hydrolysis
18O content reflectsbuffer content
not observed
Possible Pathways for Formation of Observed Rearrangement Product
Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G. J. Am. Chem. Soc. 2004, 126 , 12758.
ORO
OR
HN NH
O
O
OH
FOH
F(OH)ΨO
RO
OR
HN N
O
O
OH
F
O
HN
RO
OR
NH
O
O
OH
OEnz
O
via acylal mechanism
via Michael mechanism
F
ORO
OR
HN NH
O
O
OH
O
O Enz
F
product release andhydration in solution
Asp60-mediated hydration(general base catalysis)
both mechanisms are consistent with data
Noncanonical Nucleosides: Pseudouridine and 5-Methyldeoxycytidine
Valerie ShurtleffMacMillan Group Meeting
September 3, 2015
Noncanonical Nucleosides in DNA
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
m5dC (mC)
DNA: carrier of genetic information, critical but limited function few modifications observed
ONHO
OH
N
NH2
O
Me
m4dC
ONHO
OH
N
NH
O
ON
HO
OH
N
N
N
NHMe
m6dA
Me
various functions
hm5dC (hmC)
ONHO
OH
N
NH2
O
f5dC (fC)
ONHO
OH
N
NH2
O
ca5dC (caC)
ONHO
OH
N
NH2
O
HO
H
O
HO
O
epigenetic bases
Importance of 5-Methyldeoxycytidine
Jurkowski, T. P.; Jeltsch, A. ChemBioChem 2011, 12 , 2543.
m5dC (mC)
ONHO
OH
N
NH2
O
Me
■ Some plants: up to 25% methylated cytosine
■ Human genome: ~5% methylated cytosine (up to 70%at CpG sites, often associated with promoter regions)
Biosynthesis of 5-Methyldeoxycytidine
Jeltsch, A. ChemBioChem 2002, 3, 274.
N
N
NH2
O
DNA methyltransferase
(DNMT) N
N
NH2
O
HS Enzyme
N
NH
NH2
OSEnzyme
HO2C SAdenosine
Me
NH2
N
N
NH2
OSEnzyme
Me
HS Enzyme
Me
5-Methyldeoxycytidine and Epigenetics
Miranda, T. B.; Jones, P. A. J. Cell. Physiol. 2007, 213, 384.
mC
blocks binding of transcription factors
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
DNAmethyltransferase
and/or induces chromatin remodeling
ACTIVE GENE
SILENCED GENEepigenetic modification, can be inherited by daughter cells
5-Methyldeoxycytidine and Epigenetics
Mayer, W.; Niveleau, A.; Walter, J.; Fundele, R.; Haaf, T. Nature. 2000, 403, 501.
■ Dramatic "genome reprogramming" events happen after fertilization and during development
examination of mouse zygote/proembryo after fertilization (anti-mC antibody stain, green)
3 h 6 h 8 h no replication metaphase
22 h 32 h 45 h
paternalpronucleus
maternalpronucleus
blastomeres
rapid, replication-independentloss of mC in paternal genome
gradual, replication-dependentloss of mC in maternal genome
The Role of the Epigenetic Bases
What is the mechanism of "active demethylation?"
mC
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
"active demethylation"
genome reprogramming
Possible Mechanisms of DNA Demethylation
Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13 , 7.
mC
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
"active demethylation"
genome reprogramming
■ Possible mechanism: deamination and mismatch repair
dT
ONHO
OH
NH
O
O
Me
AID/APOBECdeamination
T:G mismatch
deglycosylationTDG or MBD4
O
HO
OH
abasic site
repairbase excision
OH
5-Methyldeoxycytidine and Epigenetics
Iqbal, K.; Jin, S.-G.; Pfeifer, G. P.; Szabó, P. E. Proc. Natl. Acad. Sci. USA 2011, 108 , 3642.
■ Dramatic "genome reprogramming" events happen after fertilization and during development
examination of mouse zygote/proembryo using anti-mC and anti-hmC antibodies
anti-mC
metaphase
• high levels of hmC in paternal genome
• anti-mC antibodies do not recognize hmC
anti-hmC merged
anaphase anaphase
depletion of mC signal in previous studies may be due to oxidation of mC to hmC (not genome-wide demethylation)
• hmC persists through cell division
hmC
ONHO
OH
N
NH2
O
HO
hmC
ONHO
OH
N
NH2
O
HO
fC
ONHO
OH
N
NH2
O
H
O
caC
ONHO
OH
N
NH2
O
HO
O
Possible Mechanisms of DNA Demethylation
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
mC
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
"active demethylation"
genome reprogramming
■ Recent discoveries suggest other possible mechanisms for demethylation
initial detection, 1972renewed interest, 2009
initial detection, 2011 initial detection, 2011
Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13 , 7.
hmC
ONHO
OH
N
NH2
O
HO
fC
ONHO
OH
N
NH2
O
H
O
caC
ONHO
OH
N
NH2
O
HO
O
Possible Mechanisms of DNA Demethylation
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
mC
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
"active demethylation"
genome reprogramming
■ Possible mechanism: deglycosylation of oxidized intermediates
Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13 , 7.
O
HO
OH
OH
TET BER
TET TETTDG
AIDTDGSMUG
hmC
ONHO
OH
N
NH2
O
HO
fC
ONHO
OH
N
NH2
O
H
O
caC
ONHO
OH
N
NH2
O
HO
O
Possible Mechanisms of DNA Demethylation
Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110.
mC
ONHO
OH
N
NH2
O
Me
dC
ONHO
OH
N
NH2
O
"active demethylation"
genome reprogramming
■ Possible mechanism: decarboxylation
Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13 , 7.
TET TET
decarboxylation?
TET
Evidence for the Feasibility of a Decarboxylative Mechanism
Smiley, J. A.; Kundracik, M.; Landfried, D. A.; Barnes Sr., V. R.; Axhemi, A. A. Biochim. Biophys. Acta Gen. Subj. 2005, 1723 , 256.
T
ONHO
OH
NH
O
O
Me
U
ONHO
OH
NH
O
O
when uridine unavailable
■ Fungal thymidine salvage pathway proceeds through oxidation/decarboxylation
OH OH
conversion of thymidine to uridine
NH
NH
O
O
Me
thymine
NH
NH
O
O
iso-orotate
O
O
3 × [O] iso-orotate
decarboxylase NH
NH
O
O
uracil
Evidence for the Feasibility of a Decarboxylative Mechanism
Schiesser, S.; Hackner, B.; Pfaffeneder, T.; Müller, M.; Hagemeier, C.; Truss, M. et al. Angew. Chem. Int. Ed. 2012, 51, 6516.
15N-caC
ONHO
OH
N
NH2
O
■ Demethylation of 5-methylcytidine with nuclear extract from mouse embryonic stem cells
HO
O
no incubationwith mESC
after incubationwith mESC
decarboxylation ofcaC is possible
15N-dC observed,no decarboxylation
in absence of extract
no 15N-dC observed,
Evidence for the Feasibility of a Decarboxylative Mechanism
Schiesser, S.; Hackner, B.; Pfaffeneder, T.; Müller, M.; Hagemeier, C.; Truss, M. et al. Angew. Chem. Int. Ed. 2012, 51, 6516.
ONTBSO
OTBS
N
NH2
O
■ How would the decarboxylation occur on a molecular level?
MeO
O
model system
NaBH4
ONTBSO
OTBS
N
NH2
Odirect decarboxylation of acid unlikely(high energy carbanion intermediate)
ONTBSO
OTBS
NH
NH2
O
MeO
O
LiOH
ONTBSO
OTBS
N
NH2
O
HO
O
55% yield
98% yield
ONTBSO
OTBS
N
NH2
O
10% yield
DDQ
?
– CO2
Evidence for the Feasibility of a Decarboxylative Mechanism
Schiesser, S.; Hackner, B.; Pfaffeneder, T.; Müller, M.; Hagemeier, C.; Truss, M. et al. Angew. Chem. Int. Ed. 2012, 51, 6516.
N
N
NH2
O
■ But nature doesn't use NaBH4 or DDQ...
HO
O
RSH
N
N
NH2
O
HO
O
N
HN
RS
– CO2
biological conditions
N
N
NH2
ORS
N
N
NH2
O
– RSHN
HN
Evidence for the Feasibility of a Decarboxylative Mechanism
15N-caC
ONHO
OH
N
NH2
O
HO
O
observeddecarboxylation
HSCO2H
NH2
CO2H
NH2
N
HN
observeddecarboxylation
HSCO2H
NH2
CO2H
NH2
NH
H2N
NH
Schiesser, S.; Hackner, B.; Pfaffeneder, T.; Müller, M.; Hagemeier, C.; Truss, M. et al. Angew. Chem. Int. Ed. 2012, 51, 6516.
Evidence for the Feasibility of a Decarboxylative Mechanism
Xu, S.; Li, W.; Zhu, J.; Wang, R.; Li, Z.; Xu, G.-L.; Ding, J. Cell Res. 2013, 23, 1296.
NH
NH
O
O
uracil
NH
NH
O
O
■ Recall: decarboxylation of iso-orotate in thymidine salvage pathway
HO
O
iso-orotate
iso-orotate decarboxylase (IDCase)
iso-orotate decarboxylase from Cordyceps militarisCmIDCase
Evidence for the Feasibility of a Decarboxylative Mechanism
Xu, S.; Li, W.; Zhu, J.; Wang, R.; Li, Z.; Xu, G.-L.; Ding, J. Cell Res. 2013, 23, 1296.
NH
NH
O
O
HO
O
NH
NH
O
O5caU U NH
N
NH2
O
HO
O
NH
N
NH2
O5caC C
A naturally occuring decarboxylase is capable of converting caC to C.