Noncanonical Nucleosides: Pseudouridine and 5-Methyldeoxycytidine

Valerie Shurtleff MacMillan Group Meeting September 3, 2015 Nucleic Acids: Genetic Polymers

■ Ribonucleic acid and deoxyribonucleic acid

O O 5' Base 5' Base 4' O 1' 4' O 1' 3' 2' 3' 2' O OH O O P O O P O Base Base OH OH O O

O OH O RNA DNA O P O O P O OH OH

■ Monomeric building blocks

HO O HO HO P Base Base OH O OH O O O

OH OH OH OH OH OH

nucleotide nucleoside ribose The Canonical Nucleosides

deoxyadenosine deoxyguanosine deoxythymidine/uridine deoxycytidine

O NH NH2 O 2 Me/H N N N NH NH N HO HO HO HO N N N O N O N N NH2 O O O O

OH OH OH H/OH OH

■ Watson–Crick base pairs H H N O H N N N H O Me N N H N R N N H N N N R N N N H O R O R H

A T G C adenine thymine guanine cytosine

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

Anticancer Agents Antiviral Agents 8 approved 12 approved

O O O H iPrOAla O H N N P N HO NH2 HO O O O O O PhO N N O N Et

HO OH HO HO F Me

cytarabine edoxudine sofosbuvir Pfizer, 1969 Upjohn, 1969 Gilead, 2013

O O NH O H N 2 N HO NH2 HO N HO O O N O N N O N Me

HO F F HO OH HO

gemcitabine ribavirin telbivudine Lilly, 1996 ICN, 1980 Novartis, 2006 Naturally Occuring Noncanonical Nucleosides

■ Nomenclature for describing nucleoside modifications

modification position Me Me NH2 N 7 6 nucleoside core 5 modification N 1 N N N 8 HO HO 2 9 N N 6 4 N A N m 2Am O 3 O 2' OH OH OH O Me # of modifications 2' OH modification

OH

NH2 O 4 OH Me 3 Me N N NH 5 H HN O HO 6 HO hypermodified 1N 2 O N Se O O nucleosides NH HO N OH OH OH N NH2 O m5dC mnm5se2U Q OH OH

Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Nucleoside Modifications in RNA

many types of RNA with many functions many nucleoside modifications

over 100 modified nucleosides known 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. Nucleoside Modifications in RNA

sites of modification in a tRNA modified nucleosides in different domains

Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Pseudouridine: a C-Nucleoside Derived from Uridine

O

HN NH ■ First modified nucleoside discovered in RNA HO O ■ Most prevalent of all natural modified nucleosides O ■ Present in almost all classes of RNA OH OH ■ The "fifth nucleoside" in RNA pseudouridine (Ψ)

Charette, M.; Gray, M. W. IUBMB Life 2000, 49, 341. The Function of Pseudouridine in RNA

O

NH phosphate H2O HO N O O

OH OH Ψ

uridine (U)

additional H-bonding site phosphate O

HN NH

HO O O ■ Unique H-bonding ability – coordination of water

OH OH ■ Ψ can serve to rigidify surrounding structure

pseudouridine (Ψ) ■ May stabilize complex 3D structure of RNA

Charette, M.; Gray, M. W. IUBMB Life 2000, 49, 341. "Acylal Mechanism" for the Biosynthesis of Pseudouridine

O O

NH HN NH pseudouridine synthase (ΨS) RO RO N O O O O U Ψ OR OH OR OH

O O

O Enz O Enz

O O

NH HN NH

N O O H RO RO net 180° rotation O O O O Enz Enz OR OH O OR OH O

Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270. "Michael Mechanism" for the Biosynthesis of Pseudouridine

O O

NH HN NH pseudouridine synthase (ΨS) RO RO N O O O O U Ψ OR OH OR OH

O O

O Enz O Enz

O O

HN HN NH

O N O O O O H O Enz Enz HN O O HN NH RO RO RO O N O RO O O O O O O O Enz O Enz OR OH OR OH OR OH OR OH

Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270. Evidence for the "Michael Mechanism"

highly conserved ΨSI aspartate residue

O F NH

RO N O O 5FU OR OH

O ΨSI F NH isolable covalent + 5FU-tRNA N O 5FU-tRNA–ΨSI complex

What is the identity of the 5FU-tRNA–ΨSI covalent complex?

Foster, P. G.; Huang, L.; Santi, D. V.; Stroud, R. M. Nat. Struct. Biol. 2000, 7, 23. Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270. Evidence for the "Michael Mechanism"

O O O F F F NH HN HN AcOH H2O, Δ HO model N O HO O N O HO O N OH system O O O 5FU O Me OH OH OH OH OH OH "fairly stable" retention of in H2O at rt configuration

O F Proposal: HN isolable covalent digestion to • Asp-60 attacks C6 (1,4-addition) 5FU-tRNA–ΨSI HO O N OH • protonation from opposite face complex nucleosides O • ester hydrolysis liberates pdt OH OH proposed product based on HPLC retention time

Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270. "Michael Mechanism" for the Biosynthesis of Pseudouridine

O O F NH HN NH pseudouridine synthase (ΨS) RO RO N O O O O 5FU Ψ OR OH OR OH Shouldn't we expect to observe the fluorinated C-nucleoside?

O O

O Enz O Enz

O O F × HN HN NH

O N O O O O H O F F Enz Enz HN O O HN NH RO RO RO O N O RO O O O O O O F O Enz O Enz OR OH OR OH OR OH OR OH

Gu. X.; Liu, Y.; Santi, D. V. Proc. Natl. Acad. Sci. USA 1999, 96, 14270. Rearrangement of 5FU by Pseudouridine Synthesis

5FU-tRNA bound O to ΨS TruB HN NH

RO O O O F O Enz OR OH

ester hydrolysis?

O

HN NH

RO O OH O F

OR OH

previously proposed product may have been misidentified

Hoang, C.; Ferré-D'Amaré, A. R. Cell 2001, 107, 929. Revisiting the "Michael Mechanism"

O O formation of F(OH)Ψ consistent ester HN NH HN NH with proposed Michael mech. hydrolysis? RO O OH RO O O F (not definitive proof – multiple O O F pathways can be envisioned) F(OH)Ψ O Enz OR OH OR OH

expected products from ester hydrolysis

O O 1. incubation with TruB F HN NH NH 18 in 50% OH2 buffer O RO O OH 5FU-tRNA N O 18 2. digestion O F H O Enz

OR OH

18O content reflects not observed buffer content

Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G. J. Am. Chem. Soc. 2004, 126, 12758. Possible Pathways for Formation of Observed Rearrangement Product

O both mechanisms are consistent with data

HN NH

O RO F product release and O O hydration in solution Enz OR OH O O O

via acylal mechanism HN N HN NH

RO O RO O OH O F O F F(OH)Ψ OR OH OR OH O

HN NH Asp60-mediated hydration (general base catalysis) RO O O O F O Enz OR OH via Michael mechanism

Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G. J. Am. Chem. Soc. 2004, 126, 12758. Noncanonical Nucleosides: Pseudouridine and 5-Methyldeoxycytidine

Valerie Shurtleff MacMillan Group Meeting September 3, 2015 Noncanonical Nucleosides in DNA

DNA: carrier of genetic information, critical but limited function few modifications observed

Me NH NH NH 2 2 Me N N HO N N HO HO HO N O N O N N O O O

6 m5dC (mC) hm5dC (hmC) OH m dA OH OH

Me NH O NH2 O NH2

N H N HO N

HO HO HO N O N O N O O O O

4 5 5 OH m dC OH f dC (fC) OH ca dC (caC)

various functions epigenetic bases

Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Importance of 5-Methyldeoxycytidine

NH2 Me N ■ Some plants: up to 25% methylated cytosine HO N O O ■ Human genome: ~5% methylated cytosine (up to 70% at CpG sites, often associated with promoter regions) OH

m5dC (mC)

Jurkowski, T. P.; Jeltsch, A. ChemBioChem 2011, 12, 2543. Biosynthesis of 5-Methyldeoxycytidine

NH2 NH2 Me N DNA methyltransferase N

N O (DNMT) N O

HS Enzyme HS Enzyme

Me

HO2C S NH2 Adenosine NH2 NH Me NH 2 N

Enzyme S N O Enzyme S N O

Jeltsch, A. ChemBioChem 2002, 3, 274. 5-Methyldeoxycytidine and Epigenetics

NH2

N

HO N O O

OH dC ACTIVE GENE

DNA methyltransferase

NH2 Me N

HO N O O SILENCED GENE mC OH epigenetic modification, can be inherited by daughter cells blocks binding of transcription factors and/or induces chromatin remodeling

Miranda, T. B.; Jones, P. A. J. Cell. Physiol. 2007, 213, 384. 5-Methyldeoxycytidine and Epigenetics

■ 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

paternal pronucleus rapid, replication-independent loss of mC in paternal genome maternal pronucleus

gradual, replication-dependent blastomeres loss of mC in maternal genome

22 h 32 h 45 h

Mayer, W.; Niveleau, A.; Walter, J.; Fundele, R.; Haaf, T. Nature. 2000, 403, 501. The Role of the Epigenetic Bases

NH2 NH2 Me N N "active demethylation" HO HO N O N O O genome reprogramming O

OH mC OH dC

What is the mechanism of "active demethylation?" Possible Mechanisms of DNA Demethylation

■ Possible mechanism: deamination and mismatch repair

NH2 NH2 Me N N "active demethylation" HO HO N O N O O genome reprogramming O

OH mC OH dC

deamination AID/APOBEC base excision repair

O Me deglycosylation NH HO TDG or MBD4 HO O OH N O O OH dT OH abasic site T:G mismatch

Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13, 7. 5-Methyldeoxycytidine and Epigenetics

■ Dramatic "genome reprogramming" events happen after fertilization and during development

examination of mouse zygote/proembryo using anti-mC and anti-hmC antibodies

anti-mC anti-hmC merged

NH2

HO N

HO N O O

OH hmC

• high levels of hmC in paternal genome

• anti-mC antibodies do not recognize hmC

• hmC persists through cell division

metaphase anaphase anaphase depletion of mC signal in previous studies may be due to oxidation of mC to hmC (not genome-wide demethylation)

Iqbal, K.; Jin, S.-G.; Pfeifer, G. P.; Szabó, P. E. Proc. Natl. Acad. Sci. USA 2011, 108, 3642. Possible Mechanisms of DNA Demethylation

■ Recent discoveries suggest other possible mechanisms for demethylation

NH2 NH2 Me N N "active demethylation" HO HO N O N O O genome reprogramming O

OH mC OH dC

NH2 O NH2 O NH2

HO N H N HO N HO HO HO N O N O N O O O O

OH hmC OH fC OH caC initial detection, 1972 initial detection, 2011 initial detection, 2011 renewed interest, 2009

Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13, 7. Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Possible Mechanisms of DNA Demethylation

■ Possible mechanism: deglycosylation of oxidized intermediates

NH2 NH2 Me N N "active demethylation" HO HO N O N O O genome reprogramming O

OH mC OH dC TET BER

HO

AID O OH TDG SMUG OH

NH2 O NH2 O NH2

HO N H N HO N HO HO HO TDG N O TET N O TET N O O O O

OH hmC OH fC OH caC

Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13, 7. Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Possible Mechanisms of DNA Demethylation

■ Possible mechanism: decarboxylation

NH2 NH2 Me N N "active demethylation" HO HO N O N O O genome reprogramming O

OH mC OH dC TET

decarboxylation? NH2 O NH2 O NH2

HO N H N HO N HO HO HO N O TET N O TET N O O O O

OH hmC OH fC OH caC

Branco, M. R.; Ficz, G.; Reik, W. Nat. Rev. Genet. 2012, 13, 7. Carell, T.; Brandmayer, C.; Hienzsch, A.; Müller, M.; Pearson, D.; Reiter, V.; Thoma, I; et al. Angew. Chem. Int. Ed. 2012, 51, 7110. Evidence for the Feasibility of a Decarboxylative Mechanism

■ Fungal thymidine salvage pathway proceeds through oxidation/decarboxylation

O O Me NH NH

HO HO N O N O O O

OH OH T OH OH U

conversion of thymidine to uridine when uridine unavailable

O O O O Me 3 × [O] iso-orotate NH O NH NH decarboxylase N O N O N O H H H

thymine iso-orotate uracil

Smiley, J. A.; Kundracik, M.; Landfried, D. A.; Barnes Sr., V. R.; Axhemi, A. A. Biochim. Biophys. Acta Gen. Subj. 2005, 1723, 256. Evidence for the Feasibility of a Decarboxylative Mechanism

■ Demethylation of 5-methylcytidine with nuclear extract from mouse embryonic stem cells

O NH2

HO N HO N O O

15 OH N-caC

no incubation after incubation with mESC with mESC

no 15N-dC observed, 15N-dC observed, no decarboxylation decarboxylation of in absence of extract caC is possible

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

■ How would the decarboxylation occur on a molecular level?

O NH2 NH2

N N MeO ? TBSO TBSO N O N O O direct decarboxylation of acid unlikely O (high energy carbanion intermediate) OTBS OTBS model system 55% yield

NaBH4 DDQ

O NH2 O NH2 NH2

MeO NH HO N N LiOH – CO2 TBSO TBSO TBSO N O N O N O O O O

OTBS OTBS OTBS 98% yield 10% yield

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

■ But nature doesn't use NaBH4 or DDQ...

O NH2 NH2

HO N N biological conditions N O N O

H H N N RSH – RSH N N

O NH2 NH2 – CO HO N 2 N

RS N O RS N O

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

O NH2

HO N HO N O O

15 OH N-caC

CO2H CO2H HS HS

NH2 NH NH2

N CO2H CO2H H2N N H HN NH2 NH2

decarboxylation decarboxylation observed observed

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

■ Recall: decarboxylation of iso-orotate in thymidine salvage pathway

O O O iso-orotate decarboxylase (IDCase) HO NH NH

N O N O H H iso-orotate uracil

CmIDCase iso-orotate decarboxylase from Cordyceps militaris

Xu, S.; Li, W.; Zhu, J.; Wang, R.; Li, Z.; Xu, G.-L.; Ding, J. Cell Res. 2013, 23, 1296. Evidence for the Feasibility of a Decarboxylative Mechanism

O O O O NH2 NH2

HO NH NH HO N N

5caU N O U N O 5caC N O C N O H H H H

A naturally occuring decarboxylase is capable of converting caC to C.

Xu, S.; Li, W.; Zhu, J.; Wang, R.; Li, Z.; Xu, G.-L.; Ding, J. Cell Res. 2013, 23, 1296.