Chemical reactions

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

 Chemical reactions of nucleic acids

The bases, sugars, and phosphodiester groups of DNA and

RNA are susceptible to a variety of chemical reactions
Protonations
 Phosphodiester proton dissociation

pKa = 0
+ H+
H

The phosphodiester group is predominantly negatively charged at

physiological pH
 Base proton associations

pKa = 3.5 - 4.2

+ H+ adenine

pKa = 4.3 - 4.5

+ H+ cytosine

The first proton associations occur at N1-adenine and N3-cytosine

But bases are predominantly neutral at physiological pH
Gillingham et. al. Chem. Soc. Rev. 45, 2637 (2016)
 Base proton dissociations

pKa = 9.2 - 9.5

+ H+ guanine

pKa = 9.5 - 10
+ H+ thymidine

The first proton dissociations occur at N1-guanine and N3-thymidine

But bases are predominantly neutral at physiological pH
Gillingham et. al. Chem. Soc. Rev. 45, 2637 (2016)
Hydrolysis reactions
 Hydrolysis reactions

glycosidic
bonds

exocyclic
amino groups
phosphodiester
bonds

DNA and RNA are susceptible to hydrolysis reactions at a variety

of locations
 Hydrolysis of DNA phosphodiester bonds

3´-hydroxyl 3´-phosphate

5´-phosphate 5´-hydroxyl
DNA

Hydrolysis of phosphodiester bonds in DNA is thermodynamically

favorable (ΔGo´ = -5.3 kcal/mol)
But is extremely slow under physiological conditions (t1/2 = 3 x 107 yrs)

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Hydrolysis of RNA phosphodiester bonds

2´, 3´- cyclic phosphate

O B
O
O B O B
O O -OH
O O O
P B O B
O O H -OH O O H - O O
OH O O
O P O O P O +
O B O B + O OH OH O
O O O P O O P O
HO B
O O O
O OH O OH
O OH 3´-phosphate 2´-phosphate
RNA 5´-hydroxyl

Hydrolysis of phosphodiester bonds in RNA occurs readily (especially

under basic conditions), due to the presence of the 2´-OH group
 Hydrolytic deamination of DNA bases

Deamination occurs most readily at cytosine residues

Adenine and guanine deamination occurs at 2-3% the

rate of cytosine deamination

Thymine and uracil do not have exocyclic amino groups

and therefore do not undergo deamination reactions
 Hydrolytic deamination of cytosine

ammonia

H2O

tetrahedral
cytosine Intermediate uracil

Reaction may be base-catalyzed (attack of hydroxide on the neutral base)

or acid-catalyzed (attack of water on the N3-protonated base)

Converts cytosine to uracil (a mutagenic reaction)

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Hydrolytic deamination of DNA bases

H2O
cytosine uracil

H2O
adenine hypoxanthine

H2O
guanine xanthine

H
 Hydrolysis of glycosidic bonds

Hydrolysis of glycosidic bonds results in the removal of

the base from the deoxyribose (deglycosylation)

Creates an abasic site in the DNA

Occurs more rapidly at purine bases (guanine and adenine)

than at pyrimidine bases (cytosine, thymine)

Estimated that spontaneous deglycosylation results

in 1̴ 0,000 abasic sites per cell per day
 Deglycosylation reaction mechanism

Hydrolysis of glycosidic bonds in 2´-deoxypurines occurs

by an acid-catalyzed reaction mechanism

Equilibrium protonation increases the leaving group ability

of the base

Facilitates unimolecular rate-limiting C-N bond cleavage

that generates the free base and an oxocarbenium ion

The oxocarbenium ion reacts with a molecule of water

to yield an abasic site
 Acid-catalyzed deglycosylation of guanine

released
base

oxocarbenium
ion

guanine protonation
(pKa ̴ 2.3)

H+

abasic site

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Abasic sites

Abasic sites exist as an equilibrium mixture of the

ring-closed hemiacetal and the ring-opened
aldehyde forms

Abstraction of the acidic α-proton from the aldehyde

leads to β-elimination of the phosphate group on the
3´-side of the abasic site

Results in a breakage of the DNA strand at that point

Abasic sites generated by deglycosylation are cytotoxic

and mutagenic
 Strand breakage at abasic sites

H
5´-phosphorylated
hemiacetal aldehyde fragment

abasic site strand break

 Reactions of DNA with small molecules

Most reactions of DNA with bioactive molecules

(drugs, toxins, mutagens) fall into two general
categories:

Alkylation of a nucleophilic site in DNA with

an electrophilic agent

Reaction of a C-H bond or π-bond in DNA with

a radical species

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

Alkylation reactions
 Reaction of alkylating agents with DNA

DNA-Nu + R-X DNA-Nu-R + X

DNA alkylating alkylation leaving
nucleophile agent product group

Nucleophilic sites in DNA reacts with alkylating agents to

yield alkylation products (DNA adducts)
 Potential nucleophilic sites in DNA

exocyclic oxygens

exocyclic nitrogens

phosphodiester
ring nitrogens
oxygens

Preferred sites of reaction depend on the specific alkylating agent

 SN2 mechanism: substitution nucleophilic bimolecular

electrophile transition state substitution

product

Reaction is very sensitive to the nucleophilicity of the attacking

species
Stronger nucleophiles will react more rapidly than weaker
nucleophiles
SN1 mechanism: substitution nucleophilic unimolecular

substitution
products

carbocation
electrophile

Reaction is relatively insensitive to the nucleophilicity of the

attacking group
Both stronger and weaker nucleophiles may react rapidly
 Dimethylsulfate

Dimethylsulfate reacts with nucleophiles by an SN2 mechanism

O _ O

= =
= =

Nu CH3-O-S-OCH3 Nu-CH3 O-S-OCH3

O O
dimethylsulfate methyl adduct methylsulfate ion

Reaction occurs preferentially at the more nucleophilic sites:

N7G >> P-O > N3A >> N1A ̴ N7A ̴ N3G ̴ N3C >> O6G
 Methyldiazonium ion

Methyldiazonium ion reacts with nucleophiles by an SN1 mechanism

+
CH3-N≡N: CH3+ Nu Nu-CH3
methyldiazonium ion methyl cation methyl adduct

:N≡N:
molecular nitrogen
(N2)

Reaction at the less nucleophilic sites is observed:

P-O >> N7G > O2T > O6G > N3A ̴ O2C > O4T > N3G ̴ N3T ̴ N3C ̴ N7A
 Alkylation of phosphodiester groups

R- R = alkyl group

Alkylation of the phosphodiester group yields a phosphotriester

derivative
Phosphotriesters are relatively stable under physiological conditions
 Alkylation of exocyclic amino groups

R R

N2G N6A N4C

Alkylation at the exocyclic amino groups generally yields

chemically stable adducts
 Alkylation of exocyclic oxygens

R R

O6G O4T

Alkylation at the exocyclic oxygens generally yields chemically

stable adducts
But locks these bases in the enol state (rather than keto state)
 Pairing of O6-methylguanine with thymine

H3C
H3C

O6-methyl G:C O6-methyl G:T

O6-methylguanine can form a base pair with cytosine (correct)

or thymine (incorrect)

Is a pro-mutagenic modification
 Alkylation of ring nitrogens

R
R R

N7G N3G N1G N3C

R
R R

N7A N3A N1A N3T

 N7-guanine

The N7 position of guanine is the most nucleophilic site in DNA

N7G

Is the favored site of reaction for most small, freely-diffusible

alkylating agents
 Chemically-labile DNA alkylation adducts

Alkylation at some ring nitrogens can destabilize the

base

N7G, N7A, N3G, N3A, N3C

Can lead to deglycosylation and ring-opening reactions

 Deglycosylation of N7-alkylguanine residues

R
released
alkylated base
R

oxocarbenium abasic site

N7-alkylguanine ion

Deglycosylation of N7-alkylguanine residues occurs by a mechanism

analogous to that for the acid-catalyzed deglycosylation reaction

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

Ring-opening of N7-alkylguanine residues

Attack of water (or hydroxide) can occur at the

C8 position of an N7-alkylguanine residue

Initiates a ring-opening reaction

Yields the a 5-(alkyl)formamidopyrimidine (FAPy)

derivative as a product
 Ring-opening of N7-alkylguanine residues

R
R R 5

N7-alkylguanine 5-(alkyl)formamidopyrimidine
(FAPy) derivative

In general, the ring-opening reaction is slow compared to the

competing deglycosylation reaction
However, the glycosidic bond of alkyl-FAPy residues is stable
at neutral pH
Kent, Chem. Res. Toxicol. 22, 1747 (2009)
Radical reactions
 Reaction of radicals with DNA

Reaction of a C-H bond or a π-bond with a radical species

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

Reactive oxygen species

dianion

Biotek.com
 Abstraction of hydrogen atoms by radicals

Reactive radical species can abstract hydrogen atoms from

the deoxyribose groups of DNA

radical DNA radical

Best characterized reactions are those resulting from hydrogen

abstraction at the C1´ and C4´-positions of deoxyribose
 Abstraction of the C1´-hydrogen atom

1´
H
radical

deoxyribose C1´ deoxyribose

radical

Radical species can abstract a hydrogen atom from the C1´position

to yield a C1´ deoxyribose radical
 Reaction of C1´ radical with molecular oxygen

Molecular oxygen reacts with the C1´ radical to yield the

peroxyl radical

The peroxyl radical eliminates a superoxide radical anion

to generate a carbocation

The carbocation is attacked by water to give a C1´-hydroxyl

intermediate

Loss of the DNA base yields a deoxyribonolactone abasic

site
 Reaction of C1´ radical with molecular oxygen

superoxide
radical anion
B B
B
carbocation

·O-O·

C1´ radical peroxyl radical

B
- BH
C1´ hydroxyl

deoxyribonolactone
abasic site

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Strand breakage at deoxyribonolactone sites

Deoxyribonolactone undergoes an α,β-elimination

reaction which releases the 3´-phosphate group

Results in the breakage of the DNA strand and produces

a 5´-phosphorylated DNA fragment

The resulting ene-lactone intermediate can then

undergo a γ,δ-elimination reaction which releases the
5´-phosphate group

Produces a methylene lactone and a 3´-phosphorylated

DNA fragment
 Strand breakage at deoxyribonolactone sites

3´-phosphorylated
fragment

δ
strand break

H H γ
β α
H ene-lactone methylene lactone

-
deoxyribonolactone

5´-phosphorylated
fragment
 Abstraction of the C4´-hydrogen atom

4´
H

deoxyribose C4´ deoxyribose

radical

Radical species can abstract a hydrogen atom from the C4´position

to yield a C4´ deoxyribose radical
 Reaction of C4´ radical with molecular oxygen

Reaction of C4´ radical with molecular oxygen yields

a peroxyl radical

Reaction of the peroxyl radical with thiol yields a

hydroperoxide intermediate

Hydroperoxide undergoes rearrangement, hydrolysis,

and fragmentation

Results in 5´-phosphate fragment, 3´-phosphoglycolate

fragment, and base propenal as the products of the
reactions
 Reaction of C4´ radical with molecular oxygen

thiol
·O-O·
C4´ radical peroxyl radical hydroperoxide

- OH-

3´-phosphoglycolate

fragmentation hydrolysis product rearrangement

and strand breakage
base propenal

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Addition of radicals to DNA bases

A variety of reactions of bases with radical species have been

characterized

These occur most commonly by addition of hydroxyl radicals

to the π-bonds of the bases
Oxidatively-modified bases

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 Addition of hydroxyl radical to guanine (oxidation)

oxidation

H-
8
H - H+

guanine 8-oxo-guanine

Addition of a hydroxyl radical to the C8 position of guanine under

oxidizing conditions can lead to the formation of 8-oxo-guanine

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

 8-oxo-guanine base pairing - cytosine

8-oxo-G:C 8-oxo-G:A
correct

8-oxo-guanine can form a correct base pair with cytosine

when it is in the normal anti conformation
 8-oxo-guanine base pairing - adenine

8-oxo-G:C 8-oxo-G:A
correct pro-mutagenic

But 8-oxo-guanine can also adopt the syn conformation and

form an incorrect base pair with adenine
 Addition of hydroxyl radical to guanine (reduction)

reduction

H-
8
H H

guanine

5
5-formamidopyrimidine H
(FAPy) derivative

Addition of a hydroxyl radical to the C8 position of guanine under

reducing conditions can lead to the formation of a FAPy derivative

Kent, Chem. Res. Toxicol. 22, 1747 (2009)

Photochemical reactions
 Photochemical reactions

Pyrimidines form cyclobutane dimers upon irradiation

at 260-300 nm

UV

adjacent pyrimidines cyclobutane derivative

Occurs by a cycloaddition reaction between adjacent

pyrimidines
 Thymine/thymine photodimer formation

5 5 > 254 nm
6 6
< 254 nm

thymine thymine thymine dimer

Thymine/thymine dimers are formed upon irradiation at > 254 nm

Dimerization is reversed by irradiation at < 254 nm
 Cis-syn thymine/thymine photodimers

> 254 nm

< 254 nm

adjacent thymines cis-syn thymine dimer

Photodimers formed between adjacent thymines in duplex

DNA have the cis-syn stereochemistry
Hariharan et. al. Photochem. Photobiol. Sci. 13, 266 (2014)
Stereoisomers of thymine/thymine photodimers
 Thymine/thymine (6,4)-photoproduct formation

> 254 nm

oxetane
< 254 nm intermediate

adjacent thymines

6
(6,4)-photoproduct

Irradiation also results in the formation of (6,4)-photoproducts

Hariharan et. al. Photochem. Photobiol. Sci. 13, 266 (2014)
 Thymine/thymine photoproducts

thymine/thymine thymine/thymine
dimer (6,4)-photoproduct

Hariharan et. al , Photochem. Photobiol. Sci. 13, 266 (2014)

 Thymine/cytosine (6-4)-photoproduct formation

> 254 nm
6
4

thymine cytosine (6-4)-photoproduct

-NH3

The most common (6-4)-photoproduct is 6

formed between thymine and cytosine 4