ORGANIC CHEMrSTRY
OF NUCLEIC ACIDS

Part 8


Contributors:
N. K. KOCHETKOV, E. I. BUDOVSKII, E. D. SVERDLOV, N. A. SIMUKOVA,
M. F. TURCHINSKII, AND V. N. SHIBAEV

ORGANIC CHEMISTRY
OF NUCLEIC ACIDS

Part B

Edited by N. K. Kochetkov and E. I. Budovskii
Translated from Russian by Basil Haigh
Translation edited by Lord Todd and D. M. Brown

9? PLENUM PRESS • London and New York • 1972


Plenum Publishing Company Ltd.
Davis House
8 Scrubs Lane
Harlesden
London NW10 6SE
Tel. 01-969-4727

U.S. Editioh published by
Plenum Publishing Corporation

227 West 17th Street
New York, N.Y. 10011
ISBN-13: 978-1-4684-2975-6
001: 10.1007/978-1-4684-2973-2

e-ISBN-13: 978-1-4684-2973-2

© 1972 by Plenum Publishing Company Ltd.
Softcover reprint of the hardcover 1st edition 1972
All rights reserved
No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any
form of by any means, electronic, mechanical, photocopying, microfilming, recording or
otherwise, without written permission from the publisher.
Library of Congress Catalog Card Number 77-178717

The original Russian text was first published by Khimiya Press in Moscow in 1970. The present
translation is published under an agreement with Mezhdunarodnaya Kniga, the Soviet book
export agency.

H. K. KO'leTK08,

a. 11.

Byao8cKutl,

oprAHHQECKA» XHMH» HYKnEHHOBMX KHCnOT
ORGANICHESKAY A KHIN1IY A NUKLEINOVYKH KISLOT

Set in cold type by Plenum Publishing Company Ltd



Foreword
The study of nucleic acids is one of the most rapidly developing fields
in modern science. The exceptionally important role of the nucleic
acids as a key to the understanding of the nature of life is reflected in
the enormous number of published works on the subject, including
many outstanding monographs and surveys. The pathways of synthesis and metabolism of nucleic acid,s and the many and varied
biological functions of these biopolymers are examined with the utmost
detail in the literature. Nearly as much attention has been paid to the
macromolecular chemistry of the nucleic acids: elucidation of the size
and shape of their molecules, the study of the physicochemical
properties of their solutions, and the appropriate methods to be used in
such research.
The surveys of the chemistry of nucleic acids which have been
published so far deal almost entirely with their synthesis and, in
particular, with the synthetic chemistry of monomers (nucleosides and
nucleotides) ; less attention has been paid to the synthesis of polynucleotides. There is yet another highly important aspect of the
chemistry of nucleic acids which is still in the formative stage, the
study of the reactivity of nucleic acid macromolecules and their
components. This can make an important contribution to the determination of the structure of these remarkable biopolymers and to the
correct understanding of their biological functions. Research in this
direction has begun to make rapid progress in recent years and its
scope has increased enormously. Nevertheless, this aspect of nucleic
acid chemistry has not yet been adequately reflected in those surveys
which have been published in this field, with the exception of a few
publications devoted to more or less specialized problems.
The authors of this present monograph have tried to remedy this
deficiency, while recognizing the difficulty of the task they have
undertaken. The book deals with reactions of the nucleic acids and
their components; these reactions lead to structural changes in nucleic

acids; to their chemical modification
Although the ultimate purpose of the book is to familiarize the reader
with the chemical reactions of the polynucleotides, most of the
material is nevertheless concerned with chemical conversions of the

v


VI

FORWaRD

monomeric components of nucleic acids; nucleosides and nucleotides.
The main reason for this is that a proper understanding of the reactivity
of polymers is unthinkable without knowledge of the reactions of their
monomeric components. Moreover, the bulk of the research at the
present time is still being undertaken at the monomer level, and only a
relatively few studies of the chemical modification of the biopolymers
themselves have been made. Nevertheless, the facts which are known
concerning reactions of polymers, especially in cases where the
problem has been studied in considerable depth and detail, can shed
light on the structure of nucleic acids. What is more, they can point
the way to a rational use of the corresponding reactions in the study of
the biological function of nucleic acids.
The material of this monograph is divided into two parts. Part A
(Chapters 1-4) deals with general aspects of the organic chemistry
of nucleic acids. The conformation and electronic structure of the
monomers, the reactivity of the heterocyclic bases (looking at the
problem also from the standpoint of quantum chemistry), and
the important question of noncovalent interactions in the polymer

chain of nucleic acids all receive attention. It was thought advisable to begin the first part of the book with a brief survey of the
classification and distribution of nucleic acids and the principles
governing the establishment of their primary structure.
Part B (Chapters 5-12) is concerned with the special organic
chemistry of the nucleic acids. The various types of reactions of
heterocyclic rings, and reactions of the carbohydrate residue and
the phosphate group are examined. A separate chapter is devoted
to a brief account of the photochemistry of nucleic acids.
A list of recommended symbols and abbreviations for the polynucleotides and their components and derivatives is included.
No special account of the synthetic chemistry of nucleic acids and
of their monomeric components is contained in this book. Because of
the existence of a number of monographs on this subject, and to keep
the size of this book within reasonable limits, it was decided to omit
an examination of the extensive literature on methods of synthesis of
nucleosides and nucleotides, and to give a very concise account of the
synthesis of polynucleotides only.
Nevertheless many sections of the book border very closely on
questions of synthesis, and in the authors' opinion they will be useful
to the synthetic chemist. In them he will find information on the
reactivity of functional groups in nucleosides and nucleotides and a
description of various reactions of the greatest use in synthesis.


FORWORD

VII

The authors are well aware of the weaknesses of their book: the
conventional and, sometimes, artificial manner of arrangement of the
material, the possibility that some of their personal views expressed in

it may be incorrect, and so on. They consider that its publication is
justified by the fact, already mentioned, that the organic chemical
aspect of the chemistry of nucleic acids has only very recently begun
to receive its due measure of attention, and its study is virtually in its
infancy. For this reason the authors hope that their generalization of
the existing data may prove particularly useful. They consider that their
task will have been fulfilled if this book helps to foster the further
development of research in the organic chemistry of the nucleic acids.
The authors are grateful to the following for their help in the preparation of this book: Corresponding Member of the Academy of Sciences
of the USSR D. G. Knorre, Corresponding Member of the Academy of
Sciences af the USSR M. A. Prokofev, Professor Z. A. Shabarova,
Professor Yu. S. Lazurkin, Candidate of Chemical Sciences M. A.
Kuz'min, and Candidates of Physico-Mathematical Sciences E. N.
Trifonov, M. D. Frank-Kamenetskii and V. I. Danilov.
The Authors


Contents
v

Foreword
Contents of Part A

XIII

1

Introduction

Part B

Chapter 5

Substitutions and Additions in the
Heterocyclic Rings of the Bases of Nucleic
Acids and their Derivatives
I. Introduction . . . . . . . . . .
II. Substitution and addition reactions at carbon atoms

1. Halogenation
2. Nitration
. . . . . . . ..
3. Hydroxymethylation, aminomethylation, and
chloromethylation
. . .
4. Reactions with diazonium salts. . . . .
5. Reactions with N-arylhydroxylamines and their
derivatives . . . . . . . . . .
6. Isotopic exchange of hydrogen atoms. . . .
7. Addition reactions at the C5-C6 double bond of
pyrimidine derivatives
8. Reduction. . . . . . . . . . .
9. Reactions with nucleophilic reagents without rupture
of the ring . . . . . . . .

III. Substitutions and additions reaction at the nitrogen
atoms
.
. .
.
.

.
.
.

1. Interaction with alkylating agents . . . . .
2. Interaction with reagents containing polarized C=C
bonds
. . . . . . . . ..
3. Interaction with reagents containing C=N bonds
4. Interaction with reagents containing C=O groups
5. Oxidation by peroxides .
Bibliography
IX

A·

269
269

269

278

278
280
281
282

285


289

295
309
309
328
331
333
334
339


x

CONTENTS

Chapter 6
Reactions of Exocyclic Substituents of the
Bases of Nucleic Acids and their Derivatives
I. Introduction .
. .
.
II. Substitution reactions at the nitrogen atom of an
exocyclic amino group .
1. N-Acylation
.
. .
2. Reaction with aldehydes
3. Reaction with nitrous acid. . . . . . .
4. Other substitutions in the amino group

.
.
.
III. Substitutions at exocyclic oxygen and sulphur atoms.
1. O-alkylation and the formation of cyclonucleosides .
2. S-alkylation of thiopyrimidine derivatives
3. Oxidation of thiopyrimidine derivatives .

Bibliography

349
349
350
355
362
366
370
371
371
373
376

Chapter 7
Reactions Involving the Cleavage or
Regrouping of Heterocyclic Rings of the
Bases of Nucleic acids and their Derivatives
I. Introduction . .
. .
. .
.

.
.
.
.
II. Reactions of cleavage and rearrangement of the rings by
the action of nucleophilic agents
.
.
.
.
.
1. Cleavage of the imidazole ring in purine derivatives.
2. Cleavage of the pyrimidine ring in purine derivatives
3. Rearrangement of 1-N-alkyladenine derivatives into
6-exo-N-alkyl compounds .
.
.
.
.
.
4. Opening and rearrangement of the ring in pyrimidine
derivatives
.
. .
.
.
II I. Cleavage by the action of hydrazine .
.
IV. Cleavage by the action of hydroxylamine.
.

.
.
V. Cleavage of rings by the' action of potassium permanganate and osmium tetroxide
.
. ..
VI. Cleavage by the action of peroxide derivatives .

Bibliography

381
381
381
385
393
397
401
408
412
416
420

Chapter 8
Hydrolysis of N-Glycosidic Bonds in Nucleosides,
Nucleotides and their Derivatives
I. Introduction

425


CONTENTS


XI

II. Hydrolysis of N-glycosidic bonds catalyzed by acids
1. Effect of structural factors on the kinetics of
hydrolysis of pyrimidine derivatives.
..
2. Effect of structural factors on the kinetics of
hydrolysis of purine derivatives .
.
.
.
.
3. Acid hydrolysis of N-glycoside bonds in polyn ucle 0 tides .
.
.
.
.
. ..
III. Hydrolysis of N -glycosidic bonds in pyrimidine
deoxyribonucleosides, not catalyzed by acids or bases
IV. Hydrolysis of N-glycosidic bonds in an alkaline medium
V. Other reactions leading to cleavage of glycosidic bonds

425

Bibliography

430
434

438
441
441
443
446

Chapter 9

Reactions of the Carbohydrate Residues of
Nucleic Acids
I. Introduction
.
.
.
.
.
.
.
.
.
.
.
II. Acylation of hydroxyl groups of carbohydrate residues.
1. Acylation .
2. Aminoacylation
.
.
.
.
.

.
3. Preparation of esters with inorganic acids . . .
III. Alkylation of the hydroxyl groups of carbohydrate
residues
.
.
.
.
.
1. Reaction with diazomethane
2. Reaction with alkyl halides.
.
.
3. Reaction with triarylchloromethanes
.
.
.
.
IV. Reactions of hydroxyl groups of carbohydrate residues
with vinyl esters
.
.
.
.
.
.
.
.
.
V. Reactions of hydroxyl groups of carbohydrate residues

with carbonyl compounds and with their derivatives.
VI. Oxidation of carbohydrate residues
.
.
1. Oxidation of an isolated hydroxyl group
.
.
.
2. Oxidation of the cis-glycol group in ribo-derivatives

Bibliography

449
450
450
455
457
458
458
459
460
461
463
465
465
466
471

Chapter 10
Cleavage of Phosphoester Bonds and some

other Reactions of Phophate Groups of Nucleic
Acids and their Derivatives
I. Introduction
.
.
.
.
.
.
.
II. Reactions with cleavage of P-O bonds

477
478


XII

CONTENTS

1. Hydrolysis of phosphomonoester bonds in ribonucleotides and degradation of RNA to nucleosides
2. Hydrolysis of phosphoester bonds in ribonucleoside
cyclic phosphates
.
.
.
.
.
.
.

.
3. Hydrolysis of phosphodiester bonds in polynucleotides .
.
.
.
.
.
"I. Reactions with rupture of C-O bonds.
. .
.
.
1. Cleavage of phosphoester bonds after removal of the
heterocyclic bases
.
.
.
.
.
.
.
.
2. Cleavage of phosphoester bonds in terminal com.
.
.
.
.
ponents of polynucleotides.
3. Some other reactions of nucleic acids leading to the
.
.

.
.
rupture of phosphodiester bonds
IV. Reactions leading to the formation of phosphoester
bonds
.
. .
. .
. .
.
1. Alkylation at the phosphate group.
.
.
.
.
2. Reactions of terminal phosphate groups in polynucleotides

Bibliography

478
482
488
504
504
517
522
523
523
525
527


Chapter 11
Some Reactions of Minor Components of
Nucleic Acids
I. Introduction .
. .
. .
. .
. ..
II. Reactions of 6-exo-N-isopentenyladenosine and its
derivatives.
.
.
. .
. .
. ..
III. Reactions of pseudouridine and its derivatives..
IV. Reactions of 5-hydroxymethylpyrimidine derivatives

Bibliography

533
533
535
539
541

Chapter 12
Photochemistry of the Nucleic Acids and their
Components

I Introduction .
. .
. .
. . ..
II The basic concepts and laws of photochemistry
III Absorption spectra of nucleic acids and their componen~.
.
.
. .
.
. .
.
.
.
.
IV Excited states of nucleic acids and their components.
1 Characteristics of excited states
.
.
.
.
.
2. Electronic structure of bases of the nucleic acids in
excited states

543
543
547
548
548

554


CONTENTS

V. Photochemical conversions of nucleic acids and their
components.
.
.
.
.
.
.
. .,
1. Photochemical reactions of pyrimidine derivatives
2. Photochemical reactions of purine derivatives .
VI. Photosensitized reactions .
.......
VII. The photodynamic effect
VIII. The action of visible light in the presence of iron ions
IX. Photochemical reactions induced by excitation of a
reagent.
.
.
.
.
.
.
. ..
X. Effect of chelating agents on photodimerization


XIII

558
558
595
599
602
606
607
608

Bibliography

610

Index for Parts A and B

619

Part A
Foreword
Contents of Part B

v
IX

Chapter 1
Structure of the Nucleic Acids
I. Introduction

II. Methods used to isolate DNA and to determine its
properties. The principal types of DNA
III. Methods used to isolate RNA and to determine its
properties. The principal types of RNA
IV. Structure of the polynucleotide chain.
.
V. Terminal groups of the polynucleotide chain
VI. Structure of nucleoside components of nucleic acids
1. Major components of RNA
2. Minor components of RNA.
3. Major components of DNA
4. Minor components of DNA
VII. Nucleotide composition and the determination of
identica~ nucleotide sequences in polynucleotides
VIII. Sequence of nucleotides in the polynucleotide chain
1. Partial cleavage of polynucleotides .
2. Principles of the unit method
3. Investigation of the primary structllre-ofpolynucleotides

15
18
23
29
31
35
36
37
41
42
43

48
48
55
56


XIV

CONTENTS

IX. Synthetic polynucleotides.
.
1. Chemical methods of synthesis
.
.
.
.
.
2. Principles of enzymic synthesis of oligonucleotides
and polynucleotides

Bibliography

64
64

74
84

Chapter 2

Conformation of Nucleosides and Nucleosides
I. Introduction
. .
.
. .
.
.
. ..
II. Conformation of the components of nucleic acids .
1. Conformation of heterocyclic rings.
.
2. Conformation of carbohydrate residues.
.
.
.
3. Mutual arrangement of the carbohydrate residues and
heterocyclic rings
.
.
4. Intramolecular interactions .

Bibliography

99
102
102
103
111
116
119


Chapter 3
Electronic Structure and Reactivity of the
Monomer Components of Nucleic acids
I. Introduction . .
. .
.
.
.
. .
.
.
II. Distribution of electrun density in the heterocyclj,c bases
of nucleic acids.
. .
.
1. Theoretical considerations.
.
. ..
2. Experimental data and their comparison with
calculated data .
. .
.
.
. ..
III. Energetic characteristics of the bases of nucleic acids
1. Resonance energy
...
.
.

.
.
.
2. Energy of the highest filled and lowest empty orbitals
IV. Tautomerism of the bases of nucleic acids .
1. Theoretical considerations .
2. Experimental data.
. .
.
. ..
V. Ionization constants of the bases of nucleic acids
1. General considerations . .
.
.
.
.
.
.
2. Localization of the attachment and detachment of
protons in nucleosides and nucleotides .
3. Localization of charges in the ions of bases.
.
.
4. Effect of different factors on the acid-base balance of
the bases
.
. .
.
.
.

.
.
.
.
VI. General matters concerned with reactivity of the bases
of nucleic acids.
.
. .
.
. .
1. The use of quantum chemical calculations
2. The use of correlation equations

Bibliography

121
121
122
129
131
131
132
134
135
137
148
148
149
152


1 53
165
166
172
179


CONTENTS

xv

Chapter 4
The Secondary Structure of Nucleic Acids
I. Introduction
II. General aspects of interaction between the bases of
nucleic a c i d s .
1. Pairing of complementary bases
.
. ..
III. Characteristics of interaction between the bases of
nucleic acids and their derivatives in aqueous
solutions
.
.
.
.
.
.
. .,
1. Association and self-association of bases,

. ..
nucleosides, and nucleotides.
2. Thermodynamic self-association constants of
purine and pyrimidine derivatives
.
.
.
.
3. Concentration changes in optical properties of
solutions of monomer components of nucleic acids
4. Concentration changes in NMR spectra of solutions
of bases and nucleosides.
.
.
.
5. Changes in the properties of bases when
incorporated in oligonucleotides by comparison
with monomers.
.
.
.
. ..
6. Thermodynamic characteristics of interaction
.
between bases and dinucleoside phosphates.
7. Nature of the forces stabilizing associations of bases
in aqueous solution.
.
.
.

.
.
.
.
IV. Investigation of the macrostructure of double-stranded
DNA
.
.
.
.
.
.
.
.
1. The Watson and Crick hypothesis .
.
.
.
.
2. Secondary and higher structures of circular DNAs
V. Investigation of the secondary structure of doublestranded RNAs.
.
.
.
.
.
.
.
.
.

VI. Destruction of the macromolecular structure of doublehelical molecules (denaturation)
.
.
1. Factors influencing thermal denaturation .
.
.
2. Special features of the denaturation of circular DNAs
VII. Processes leading to restoration of the double-helical
structure (renaturation).
.
.
. ..
1. Factors influencing the renaturation process
2. Intramolecular interactions in DNA.
VIII. Single-stranded polynucleotides .
1. The secondary structure of tRNA
2. The tertiary structure of tRNA .
.
. ..
3. The secondary and tertiary structures of 5S RNA

Bibliography

183
184
1 84
195

1 96
198


198

1 99
200
206
210
212
213
218
222
224
225
229
233

235

238
241
244
252

255

258


Chapter 5


Substitutions and Additions in the
Heterocyclic Rings of Nucleic Acid
Bases and their Derivatives

I. Introduction

Substitution reactions in the heterocyclic ring of the bases of nucleic
acids are characteristic of both purine and pyrimidine derivatives. They
include electrophilic replacement of protons bound to carbon or nitrogen
atoms composing the ring and also nucleophilic substitution of exocyclic amino
groups. Addition reactions at the carbon - carbon double bond of the
heterocyclic ring, which can be either electrophilic or nucleophilic, are at
present known only for pyrimidine bases, for in purines the C4 - C5 bond is
evidently very stable. Electrophilic reactions at the nitrogen atoms of the
pyridine type, which possess a free electron pair, can also be included conventionally among addition reactions, as they are in this book. As a result
of these reactions a new group appears in the heterocyclic ring without the
removal of any groups or atoms previously present in the ring before the
reaction.
All these types of reactions are examined in this chapter. Characteristically they lead to modification of the heterocyclic ring without its destruction. Certain reactions in which the addition or substitution products are
unstable and undergo further conversions are also considered incidentally in
this chapter. These reactions will be examined in more detail later in the
book.
II. Substitution and addition reactions at carbon atoms

1. Halogenation
Pyrimidine derivatives. As a result of halogenation in a nonaqueous
medium, direct substitution of the hydrogen atom at C5 of the pyrimidine ring
269



270

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

takes place. This substitution can be achieved by the action either of free
halogens or of N-haloamides.
Action of halogens. The most widely used method of obtaining 5-chloro
and 5-bromo (but not 5-iodo) derivatives of the pyrimidine bases of nucleosides and nucleotides is by the action of free halogens in a nonaqueous medium. This reaction can be represented by the following scheme, which assumes
an electrophilic attack by the positive halogen ion on the C5 atom, possessing
surplus 7r electron density (see page 127).

Ha)¢oI

to
NH

R

R denotes hydrogen atom or carbohydrate residue

Chlorination of uracil derivatives is usually achieved by addition of a
solution of chlorine in carbon tetrachloride to a solution of the original eompound in glacial acetic acid. The reaction takes place very readily and is
complete within a few hours at room temperature or a few minutes under
more vigorous conditions. In this way the compounds 5-chlorouridine [I, 2 J,
5-chlorodeoxyuridine [3 J, 5-chlorouridine-5 '-phosphate [4, 5 J, and 5-chlorouridine- 2' (3 ')-phosphate [6 J have been obtained. Under these conditions,
cytidine is not readily chlorinated, and it is impossible to obtain a satisfactory
yield of 5-chlorocytidine [7 J. This is most probably because cytidine exists
in the protonated form in glacial acetic acid, and electrophilic attack on the

cytidine cation is interfered with. However, after irradiation of a reaction
mixture containing chlorine and cytidine [7 J (or deoxycytidine [8 J) with ultraviolet light, formation of the 5-chloro derivative takes place very smoothly.
The probable reason why the reaction is thus facilitated is because an excited
cytosine ring takes part in the reaction (see Chapter 12). However, the possibility is not ruled out that irradiation changes the mechanism of the reaction,
so that instead of electrophilic, homolytic substitution takes place, through the
formation of chlOrine radicals.
Bromination of uracil derivatives by bromine in organic solvents also
takes place under very mild conditions. In dimethylformamide, for example,
on the addition of bromine, 5-bromo derivatives of uridine are formed with
virtually quantitative yields within a few minutes even at O°C [9J. In a similar
way, but under rather more vigorous conditions, 5-bromouridine has been
obtained by bromination in ethyl alcohol [10J. By the use of dimethylformamide
[9J, pyridine [11], and formamide [l1J as solvents, 5-bromo derivatives of
cytidine [9J and deoxycytidine [l1J have been obtained. By mild bromination
conditions, the halogen atom can be introduced directly into the pyrimidine
ring of such labile compounds as nucleoside triphosphates, and this property
has been used, in particular, to obtain 5-bromodeoxycytidine triphosphate


§I1l

SUBSTITUTION AND ADDITION REACTIONS AT CARBON ATOMS

271

by the action of bromine solution. in carbon tetrachloride at room temperature
on the triphosphate dissolved in formamide [12].
The presence of water in organic solvents leads to addition at the
double bo~d [9] (see page 285), but if an acid is present in the reaction mixture, 5-bromo derivatives are ultimately formed. For example, if nucleoside monophosphates in a mixture of dioxan with dilute nitric acid are brominated by a solution of bromine in CCl4, 5-bromouridine monophosphates
[4, 6] and 5-bromocytidine monophosphates [5] are formed almost quantitatively. In this case it is not clear whether the bromination takes place through

a mechanism of direct electrophilic substitution, or whether 5-bromo-6hydroxy-5,6-dihydronucleoside monophosphates are formed as intermediate
products (see page 285). Good yields of 5-bromo derivatives are also obtained
by the action of bromine with simultaneous ultraviolet irradiation [7, 8]. Just
as in the cas~ of chlorination (see page 270), the mechanism of this reaction
is not yet clearly understood.
Iodination of pyrimidine bases takes place under much more vigorous
conditions. For example, in Prusoff's method [13, 14], widely used for the
synthesis of 5-iodo derivatives of uridine and deoxyuridine, the nucleoside is
heated with a solution of iodine in chloroform in the presence of dilute nitric
acid for several hours. In modifications of this method, the concentrations of
nitric acid, the nature of the organic solvent, or the duration of the reaction
are varied. These modifications have been used to obtain 5-iodouridine [13,
15-17], 5-iododeoxyuridine [14, 16], and 5-iodo derivatives of uridine [13, 18]
and deoxyuridine [18, 19] labelled with radioactive isotopes of iodine. Good
results are also obtained by the iodination of uridine-5'-phosphate [4,20] and
uridine-2'(3')-phosphate [6] by Prusoff's method. Another method which can
be·used to obtain the 5-iodo derivative of uracil is by iodination with elementary iodine in the presence of an alkali [14, 18].
The use of Prusoff's method to obtain 5-iodo derivatives of cytidine and
deoxycytidine is relatively ineffective, because under the reaction conditions
considerable hydrolysis of the N-glycoside bond takes place [21]. If, however, the nitric acid is replaced by HI03 , nucleoside and nucleotide derivatives of 5-iodocytidine can be obtained with good yields and in mild conditions
[6, 21, 22]. It should be mentioned that if this latter method is used, a side
reaction is observed, leading to the formation of 9-10% of a compound with
saturation of the C5 - C6 double bond [21, 23]. This compound is evidently
5,5-diiodo-5,6-dihydro-6-hydroxycyclodeoxyuridine (Formula I) [23].
,

0

,)('NH


;AN~O

H~
HO

1


272

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

The formation of this product is evidently analogous to the formation of
5,5-dibromo-6-hydroxY-5,6-dihydro derivatives of uracil and cytosine during
bromination in an aqueous medium (see page 285).
Iodination takes place under much milder conditions if iodine chloride
ICI is used as the iodinating agent [24, 25]. If the reaction is conducted in
N-ethylacetamide [24,25] and dimethyl sulphoxide [24], high yields of iodine
derivatives can be obtained at room temperature. If dimethyiformamide is
used as the solvent [26], large quantities of 5-iodo-6-hydroxy-5,6-dihydropyrimidine derivatives are formed, presumably because of the great difficulty
in removing all water from this solvent. Parallel with this reaction, however, direct substitution of hydrogen takes place, with the formation of the
5-iodo derivative. The formation of iodine derivatives when iodine chloride
is used as the halogenating agent can be taken as evidence of the electrophilic
character of this reaction.
Action of N-haloamides and N-haloimides. N-haloamides and N-haloimides are effective halogenating agents replacing the hydrogen atom at C5 of
the pyrimidine ring under extremely mild conditions. The 5-chloro derivatives of uridine and cytidine are formed by the action of N-chlorosuccinimide
on uridine and cytidine in dimethyiformamide [27,28]. In the same way, by
the action of N-bromosuccinimide on trialkylammonium salts of uridine phosphates in dioxan for a few days at room temperature, virtually quantitative

yields of the corresponding 5-bromo derivatives can be obtained [29-32].
It is interesting to note that catalytic quantities of di-n-butyldisulphide
are necessary for iodination with N-iodosuccinimide in dimethyl sulphoxide
[24]; the reaction does not proceed in N-ethylacetamide as the solvent, even
in the presence of the catalyst. Di-n-butyldisulphide is perhaps necessary
for the formation of an intermediate halogenating agent, such as n-butylsulphenyl iodide [33]. Another interesting fact is that whereas the iodination of
uracil derivatives takes place readily and under mild conditions, so that 5iodouridine triphosphate can be obtained in high yield from uridine triphosphate
[24], cytosine derivatives cannot be iodinated under these conditions. The
reason for this phenomenon is not yet clear.
Purine derivatives. Whereas substitution of a hydrogen atom by a
halogen in pyrimidine bases takes place only in a nonaqueous medium, in
purines, where the competitive reaction of addition at the C4 - C5 double bond
is impossible, substitution is observed in both aqueous and nonaqueous
solvents. Moreover, as has already been pOinted out (see page 168), guanine
derivatives are halogenated much more easily than adenine derivatives.
During bromination with bromine water at room temperature, rapid
breakdown of the guanine ring takes place, leading to disappearance of absorption of guanOSine solutions in the region 250-300 nm [34-36]. Among the products formed by bromination of guanosine in water, 8-bromoguanosine (II) can
be isolated [37, 38], but it is rapidly oxidized by bromine with destruction of


§n]

273

SUBSTITUTION AND ADDITION REACTIONS AT CARBON ATOMS

both the pyrimidine and the imidazole rings [38]. lltimately ribosylurea
(lIn, ribosyloxaluric acid (IV), oxalic acid, and guanidine are formed.

X

o

Br·

~

HOCH

~
HO

I

NH

~NH2
-+

OH
II

---+-

H0IQl'HCONH' + HO~:CONH::COOH
HO

OH
III

HO


OH

NH2~NH2

+

(COOH),

IV

Nevertheless, the action of bromine water under mild conditions can be
used for the preparative synthesis of guanine derivatives [339, 340]. If, for
example, 1.2 eq of bromine water was used in buffer solution (PH 3) at room
temperature, 8-bromoguanosine-5'-phosphate was obtained; the yield was
higher than 60% [340]. Raising or lowering the pH of the medium reduced the
yield of the product.
When organic solvents are used, the chief product of the reaction between
guanosine and bromine is 8-bromoguanosine, regardless of whether the reaction is conducted in dimethylformamide [9], glacial acetic acid [39]. dioxan
[40], or a mixture of dioxan and methylcellosolve [41]. If the reaction between equivalent quantities of guanosine and bromine takes place at o· in dimethylformamide, the yield of the bromine derivative reaches 50% after 30
sec, and thereafter remains unchanged for 1 h [9]. The velocity of formation
of 8-bromoguanosine is about equal to that of 5-bromocytidine and 5-bromouridine underthe same conditions.
Bromination of adenosine does not take place in dimethylformamide at
O·C, but if the reaction is conducted at 50-60·C in glacial acetic acid, 8-bromoadenosine is formed relatively easily* [39,42].
The reasons for the difference in behaviour of adenosine in these two
solvents is not yet fully understood.
In aqueous solutions, adenine derivatives are not brominated by the
action of bromine water. However, in the presence of an alkali [43, 340, 341]
or in buffer solutions with pH 2! 3. the corresponding 8-bromo derivatives
*2',3',5'-Tri-O-acetyl derivative of adenosine [39] or 2',3'-isoptopylidene-adenosine [42].



274

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

are formed [340, 341]. At pH 2: 7. degradation of the heterocyclic base
evidently takes place during bromination. At pH < 3 the reaction is retarded,
probably through protonation of the adenine ring, which hinders the electrophilic attack. Bromination in a buffer with pH approximately equal to 4 at
room temperature has given high yields of 2 '-. 3 '-, and 5 '-phosphates of 8bromoadenosine, and also 8-bromoadenosine di- and triphosphates [43, 341].
Only guanosine is iodinated by iodine chloride in organic solvents such
as N-ethylacetamide [25] or dimethylformamide [26], and the adenine ring
remains unchanged under these conditions.
Conversions taking place with purine bases (as components of nucleosides) during halogenation with N-haloamides and N-haloimides as halogenating agents have not yet been adequately investigated. All that can be concluded
at present about the action of N-chlorosuccinimide on purine nucleosides in
dimethylformamide is that adenine is unaffected, whereas the guanine ring is
broken, probably with the intermediate formation of 8-chloroguanosine [27,
28].
By contrast, bromination of adenosine and deoxyadenosine as their
2',3' ,5 ,-tri- and 3',5 '-di-O -acetyl derivatives in chloroform by the action of
N-bromoacetamide under very mild conditions gives 8-bromo derivatives
[39]. The action of N-bromosuccinimide in water leaves adenosine unaffected,
but converts guanosine, evidently to 8-bromoguanosine [37]. N-Iodosuccinimide in water [37] reacts neither with adenosine nor with guanosine, but in
dimethyl sulphoxide, in the presence of di-n-butyldisulphide, guanosine is
iodinated under very mild conditions with the formation of 8-iodoguanosine
[24], whereas adenosine remains unchanged under these conditions.
To conclude this examination of the halogenation reactions of monomer
components of nucleic acids, the following basic rules should be noted;

a) Halogenation takes place through primary" attack by the halogen on the
C5 atom of pyrimidine and C8 atom of purine bases.
b) The halogens can be arranged in the following descending order of reactivity in halogenation: C1 2• Br2 > ICI > 12,
c) The velocity and direction of the halogenation reaction are strongly
dependent on the nature of the solvent, the presence of water in the reaction
mixture, and in the case of N-haloamides and N-haloimides of carboxylic acids - on the presence of catalysts (such as alkyldisulphides).
Characteristics of halogen derivatives. Introduction of a halogen into
the heterocyclic ring produces a bathochromic shift in the ultraviolet region
of the spectrum compared with the original nonhalogenated compound [6, 15,
39]. This effect is particularly clear in the case of pyrimidine derivatives.
The magnitude of the bathochromic shift increases in the order F- < CI< Br- < 1- (Table 5.1). The difference between the values of pK a of the nonhalogenated and halogenated nucleosides decreases in the same order.


§U]

SUBSTITUTION AND ADDITION REACTIONS AT CARBON ATOMS

275

TABLE 5.1. SpectralandAcid-Base Characteristics of Nucleoside Derivatives of Uracil and their Halogen Substitution Products [15, 44]
Compound

Amax. nm (pH
1.0-5.0)

Uridine . • . • . . . . . • .
5 -Fluorouridine ..•..
5 -Chlorouridine • . . . .
5 -Bromouridine •.•.••
5 -Iodouridine. . . . ...


262
271
278
279
291

pK a
9.25
7.75
8.20
8.20
8.50

Compound
Deoxyuridine •••••••
5 -Fluorodeoxyuridine .
5 -Chlorodeoxyuridine .
5-Bromodeoxyuridine ..
5-Iododeoxyuridine ...

"max. nm(pH
pKa
1.0-5.0)
262
271
279
280
287


9.30
7.80
7.90
7.90
8.20

As a result of the decrease in pKa on introduction of the halogen into
the heterocyclic ring, halogenated nucleosides can be separated from nonhalogenated by methods based on the difference between their charges,
notably by ion-exchange chromatography [18, 19]. Introduction of a halogen
into the heterocyclic ring of pyrimidine derivatives reduces the stability of
the N-glycoside bond of the corresponding nucleosides in an acid medium (see
page 426), and also lowers the resistance of the heterocyclic ring to alkaline
hydrolYSiS. Introduction of a halogen also Significantly affects the photochemical behaviour of pyrimidine bases and their derivatives (see Chapter 12).
The reactivity of the halogen substituent in the pyrimidine ring is relatively low. Nevertheless, reactions in which exchange of the halogen takes
place are known, and they enable 5-substituted analogues to be synthesized.
These are widely used in biological research. For example, when 5-bromouridine was boiled with morpholine, 5-morpholinouridine was obtained [45];
by the action of sodium bicarbonate in an alkaline medium, 5-hydroxy derivatives are formed from 5-bromo derivatives of uracil [46, 47]. The conversion
of 5-bromouridine [48, 49] and 5-bromodeoxyuridine [50] into 5-amino derivatives by the action of ammonia at 50-55°C under pressure has also been
described. The halogen linked with the C8 atom in purine derivatives is much
more mobile. For this reason it is possible to obtain 8-substituted analogues
of natural nucleosides and nucleotides by exchanging their halogen under the
action of nucleophilic reagents [39, 51, 52, 339-341], and to synthesize cyclonucleosides in which the oxygen of the hydroxyl group of the carbohydrate
residue is linked to the C8 atom of the purine [41, 42].
The 5-halogeno-derivativesofpyrimidine nucleosides and nucleotides are
widely used for biological investigations of various types, such as the study
of mechanisms of enzymic reactions [53] or of mutation processes [53-56].
Polynucleotides. Because polynucleotides are sparingly soluble in nonpolar solvents, their halogenation in a nonaqueous medium has not been carried out on a wide scale. Difficulties associated with their low solubility can
to some extent be avoided by using tetraalkylammonium salts of the polynucleotides, containing long alkyl radicals. For example, if solutions of
trimethylhexadecylammonium (cetavlon) salts of DNA [57] or of ribosomal



276

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

TABLE 5.2. Relative Composition of DNA Bases after
Treatment with Bromine Water (O°C, 10 min) followed by
Hydrolysis with Perchloric Acid [57]
Relative composition of bases
Number of moles bromine
Anole base
adenine guanine cytosine thytnine 8 -bromoguanine
Original DNA •.••••.•
0.25•.•.•.••.•.••.
0.5 •..•..••.•.•..
0.75 •.••.••..•.•••
1.0 •.••.••..•••••
3.0 •••.•.••••••••

1
1
1
1
1
1

0.82
0.73

0.63
0.52
0.49
0.15

0.82
0.71
0.76
0.770.74
0.50

1.09
1.04
0.97
1.08
1.06
1.22

-

0.08
0.16
0.16
0.21
0.20

and transferRNAs [58] are used in anhydrous dimethylformamide, these polynucleotides can be brominated with br.omine.
During the halogenation of ribonucleic acids (for 30 sec at O°C, by addition of different quantities of bromine to the reaction mixture) it has been
found that, just as in the case of monomers, adenine rings are not halogenated,
whereas uracil, cytosine, and guanine rings are brominated, to give the corresponding 5-bromouracil, 5-bromocytosine, and 8-bromoguanine derivatives.

The degree of halogenation increases with an increase in the concentration of
bromine in the reaction medium, and whatever the concentration of bromine
used in the reaction, the yield of 8-bromoguanine is always higher than the
yield of 5-bromouraci1; 5-bromocytosine products are formed in the smallest
amount. For example, on bromination of total tRNA from yeast, using 2
moles of bromine to 1 mole ofnucleotides, 40-60% of 8-bromoguanine, 30-'50%
of 5-bromouracil, and 10% of 5-bromocytosine were obtained as derivatives,
relative to the initial content of each of these bases in the reaction mixture.
During halogenation under these particular conditions, partial destruction of
the guanine rings obviously took place. The polynucleotide chain of tRNA was
not broken on bromination, as is clear from the results of ultracentrifugation,
gel-filtration, and ion-exchange chromatography of the specimens. When
ribosomal RNA was treated under these conditions, marked degradation of
the chain was observed: the sedimentation coefficients of both 168 and 238
components were reduced to the value 58 [58]. Bromination also leads to
disturbance of the secondary structure of tRNA to an extent which increases
with an increase in the degree of modification, as reflected in lowering of the
melting temperature and of the hypochromic effect. Guanine is also the most
reactive base during bromination of DNA (Table 5.2).
The cytosine ring undergoes a much smaller degree of modification,
while the thymine ring is unchanged. The absence of modification of thymine
as a component of DNA has not yet been explained, because thymine itself is
brominated under these same conditions to 5-bromo-6-hydroxy-5,6-dihydrothymine, which is evidently formed on account of the presence of traces of
water in the solvent.


§U]

SUBSTIruTION AND ADDITION REACTIONS AT CARBON ATOMS


277

The results given in Table 5.2 show that on bromination of DNA, just
as in the case of RNA, partial destruction of the guanine rings is observed.
Under the conditions used for bromination of DNA (and also RNA), bromination of the cytosine rings takes place practically instantaneously. Moreover,
a certain proportion of the bases is modified, after which the number of
modified components does not increase further. For example, if a proportion of 1 mole bromine to 1 mole DNA bases is used at O°C, 16% of the cytosine rings are modified during the fi:rst 10 min, and this proportion remains
unchanged during the next 30 h. For guanine, after the first rapid reaction,
the degree of modification rises more slowly, so that whereas 22% of the
guanine content is modified in the first 10 min, after 30 h the quantity of 8bromoguanine derivatives reaches 60%. Bromination of DNA, just as in the
case of tRNA, leads to a change in its secon<4lry structure, as can be judged
from the decrease in rotation and in the characteristic viscosity of the solution, and also from the decrease in Tm , weakening of the hypochromic effect,
and widening of the melting interval [59). Neither the difference in behaviour
of DNA and RNA on bromination nor the kinetic principles exhibited have yet
been explained.
Whereas guanine is the most reactive of the nucleic acids on bromination, the pyrimidine bases are most reactive during iodination by the action
of iodine chloride [25). After iodination of the trimethylhexadecylammonium
salt of tRNA by a 0.02 M solution of iodine chloride in dimethylformamide
solution for 18 h at room temperature, for example, 8.5% of the cytosine,
30% of the uracil, and 9% of the guanine components were modified (with the
formation of the corresponding 5-iodocytosine, 5-iodouracil, and 8-iodoguanine
derivatives) [25). If the cetavlon salt of preliminarily denatured DNA was
treated under the same conditions, 2% of the guanine and 14% of the cytosine
residues reacted. To explain the difference between the reactivity of the
bases, as components of nucleic acids, on bromination and iodination,· further
research is necessary. However, it should be noted that during neither the
bromination nor the iodination of nucleic acids is quantitative halogenation of
the component bases observed, even if the halogenating agent is present in
excess. This may perhaps be the result of the existence of a secondary structure of the nucleic acids in organic solvents. In fact, if the trimethylhexadecylammonium salt of DNA without preliminary denaturation is iodinated,
the degree of halogenation is much (approXimately 4 times) lower than when

previously denatured DNA is treated.
The basic process in chlorination of the trimethylhexadecylammonium
salt of ribosomal and transfer RNAs from yeast in dimethylformamide with
N-chlorosuccinimide is rupture of the guanine ring, probably with the intermediate formation of 8-chloroguanine [28). Uracil residues undergo a lesser
degree of modification (with the formation of 5-chloro derivatives), and cytosine and adenine residues are completely unaffected. The rule observed
during bromination of RNA thus also applies to chlorination: guanine and
uracil are the most reactive bases in RNA.


2'18

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

2. Nitration

Besides halogenation, several other reactions can lead to substitution at
the carbon atoms of the heterocyclic bases. However, these reactions require
much more vigorous conditions. If 2',3',5'-tri-O-(dinitrobenzoyl)-uridine (V)
is treated (at a temperature not exceeding 50°C) with a mixture of concentrated
sulphuric and nitric acids, the corresponding 5-nitro derivative (VI) is formed
with a yield of about 70%. After removal of the dinitrobenzoyl protection, 5nitrouridine (VII) was obtained from compound VI [60].

o

02N~.
~ ~H

HOIQJ 0

HO

v

VI

OH

VII

R,. P-CONO,

Under these same conditions, the unprotected uridine is nitrated with
simultaneous oxidation of the 5 '- CH20H group of the ribose, so that the reaction product is 5-nitrouracil-l-/3-~riburonic acid (VIII):

o
OINJ

t

NH

HOO~
HO

OH
VIII

The corresponding 5-amino substituted compounds can be obtained by reduction of the 5-nitro derivatives [60].
3. Hydroxymethylation, aminomethylation, and chloromethylation


Although the reaction of hydroxymethylation of the bases of nucleic aCids
evidently cannot be used for polynucelotides because of the vigorous conditions
of treatment, it nevertheless deserves examination here because it simulates
to some degree the process of biosynthesis of 5-hydroxymethyl derivatives
from formaldehyde and cytidylic and uridylic acids [61-64]. High yields of 5hydroxymethyluridine (IXa) and 5-hydroxymethyldeoxyuridine (IXb) can be obtained by boiling the nucleosides with formaldehyde in the presence of 0.5 N
hydrochloriC acid [65]. At a lower temperature (50°C), using a large excess of


§II]

SUBSTITUTION AND ADDITION REACTIONS AT CARBON ATOMS

279

formaldehyde, and with the same acidity of the medium [66], a practically
quantitative yield of 5-hydroxymethyldeoxyuridine-5'-phosphate (!Xc) is obtained from deoxyuridine-5 '-phosphate.

o

HOCJ(~
~ ,NH
CH,O(HCI)

,.

ROCHa 0

~O


~
IX

a (REH; R',. OH)
b (R=H; R'=H)
c (R=POaH2; R'= OH)

As an electrophilic reaction, hydroxymethylation must be retarded
strongly by protonation of the base; it is natural, therefore, that cytidine,
which is in a protonated form in an acid medium, does not undergo hydroxymethylation under these conditions [67]. Nevertheless, by carrying out the
conversion in an alkaline medium, hydroxymethylated cytidine and deoxycytidine monophosphates [67] can be obtained, and although the yields are
relatively low (approximately 10%), the reaction is of considerable interest
because 5-hydroxymethyl derivatives of cytosine are not readily available.
The mechanism and kinetics of this reaction have not yet been adequately investigated. Hydroxymethylation of uracil derivatives in an alkaline medium
also gives 5-hydroxymethyl-substituted compounds, but the yields of the
products are low [67], so that in order to obtain uracil derivatives it is preferable to carry out the reaction in an acid medium.
The reaction of aminomethylation of uracil derivatives takes place fairly
easily. For example, if uridine and uridine-5'-phosphate [68, 69] are heated
for 7 h at 100°C with a mixture of diethylamine and formaldehyde, the corresponding 5-diethylaminomethyl derivatives (X) are formed with yields of Up'
to 50%:

6:

ROVO~
'~'
HO

0

(C,H.I,NH; CH,O ,.


OH
a (R=H)
b (R=POaH1)

The reaction of chloromethylation is also Imown for uracil itself [70],
but it has not been used for uracil nucleosides and nucleotides.


280

SUBSTITUTION AND ADDITION REACTIONS IN HETEROCYCLIC RINGS

[CHAP. 5

5-Hydroxymethyl and 5-aminomethyl derivatives can be reduced to the
corresponding 5-methyl-substituted compounds by hydrogenation over platinum
oxide [65, 68, 69] or over rhodium on alumina. This reaction is very important
for the preparationof5-methyl-substitutednucleosides and nucleotides of the
pyrimidine series. In addition, 5-hydroxymethyl derivatives can be converted
into ethers or esters (at the hydroxymethyl group) or oxidized to the corresponding aldehydes or acids [65, 70]. The reaction of hydroxymethylation in
an alkaline medium is reversible: the 5-hydroxymethyl derivatives lose formaldehyde and are slowly converted into the original nucleosides or nucleotides [67].
4. Reactions with diazonium salts

Both nucleosides and nucleotides whose bases contain exocyclic amino
groups are able to react with diazonium salts with the formation of diazoamino
compounds (see page 368). In the case of guanosine (and, poSSibly, adenosine)
[71], the reaction with diazonium salts also leads to the formation of substitution products of the protons linked to the C8 atoms of the purine ring. For
example, by interaction between guanosine and diazotized p-sulphanilic acid
[71] at pH 9, substitution products of hydrogen on the p-sulphophenyl residue

(XI and XII) are formed with a total yield of about 50%.

o

o

H03S-o--<:JJ:
H01Qf NH,
HO

HOaS-o-{i,AH

HO~
HO

OH
XI

NHO

OH
XII

The possibility is not ruled out that 8-azo-(p-sulphophenyl)-guanosine
(XIII) is also obtained by azo-couplingj the formation of compound XII is probably the result of decomposition of the diazoamino derivative XIV.

o

H0


fI-
3 S-O--N

HO;~~~

N N

I

~
HO

OH
XIII

NH2

0

H035-o-(/;:H
HOlH~O,

N N

I ~

~
HO

fH

N-O-SOa H

OH

XIV

Reactions such as these have been used for specific labelling of guanosine in DNA [72] and RNA [72] with the object of subsequent electron-microscopic detection. Specificity of introduction of the label into guanosine in