BIOCHEMICAL

Vol. 64, No. 2,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MECHANISM OF THE DIMROTH REARRANGEMENT IN ADENOSINE' James 0. Engel' Institute

Received

of Molecular Biology and Department University of Oregon Eugene, Oregon 97403

February

of Chemistry

3,19?5

SUMMARY: An unambiguous mechanism for the Dimroth rearrangement has been established for adenosine by observation of isotopic [15N] nitrogen exchange between the exocyclic (N6) and endocyclic (1-) positions of the adenine ring which occurs simultaneously with the well documented methyl migration (from positions l- to N6). The elucidation of this rearrangement by NMR techniques indicates that ring cleavage between the N1-nitrogen and C2-carbon atoms followed by 180' internal rotation, is the correct pathway. It (e.g.

is well tRNA,

ificance

known that

the majority

rRNA and DNA) are methylated

of such methylation

restriction

has only

enzyme reactions

of the artifact

with

of the Dimroth

was when tRNA chromatograms hydrolysates) constituent

showed (2).

mononucleotides,

problem

might

1

have special

(3)

by chemical

special (4),

were

investigators

precautions

to not

advantages

for

during

reinvestigation

that

first

introduce

the utilization

investigators

incidence

with

investigators

alkaline

tRNA

base

of these isolation

residues

now aware alkali

may

of the

of the original are

sign-

recorded

modified

some portion

acids

the modification-

interfering

as a major that

for

the

nucleic

the functional

used to develop

Although

we perceived

but

Possibly

rearrangement

necessitating

experiments.

base isolations

DNA.

(which

active

been elucidated

rearrangement

was shown

thus

and take

--in vivo,

N6-methyladenosine

It

have been produced

and control

of biologically

results

of the into

modified

of this

rearrangement

of polynucleotide

structure.

This work was supported by a Predoctoral Traineeship from USPHS Training Grant (No. 00715) and research funds from USPHS Research Grant No. GM 15792 (P.H. von Hippel, Principal Investigator).

2 This study will Oregon in partial

Copyright AN rights

be presented fulfillment

@ 1975 by Academic Press, IHC. in my fornl reserved.

of reproduction

to the Graduate School of the University of the requirement for the Ph.D. degree. 581

of

Vol. 64, No. 2, 1975

We recently

BIOCHEMICAL

completed

consequences

of amino-group

mononucleotide are

which that

level

preparing

6-amino

position

then

promote

residues.

been rigorously

the reaction

by reaction 500.

from

8 hours.

H20.

268.

ammonia

Low resolution

(Expected

cellulose

nmr spectra

I)

sweep mode locking

which

with

(Eastman

which

(taken

was filtered

C,. H13 0 4 [14N],

from

(Table

reacted

the experiments

adenosine

mass spectral

chromatograms

systems

and 6-methylamino-

rearrangement

(Ado)

outlined

with

in the presence

results

from

one spot

recorded

solvent

in

the frequency

The labeled product

=

ascending

of Ado.

to form

at

at m/e-

sample

to form a single ion

tube

in three

reference.

of hydroxide

with

two times

peak appears

Tic

were

(9)

in a sealed

an authentic

deuterium (10)

ion

yielded

XLlOO-15)

CHSI in CH3CON(CH3)2

rearranged

parent

Kodak)

on an internal

here

was synthesized

and recrystallized

[15N].)

comigrated

on a Varian

has not

(Cycle)

in 5 ml of methanol

The product

of

AND METHODS

amino

(BioRad)

we reasoned

(by addition

(A)

of this

(CH3)2S04,

(6,7), (8)

we

at the

of 0.5 g of 6-chloropurine-9-B-&-ribofuranoside

ml of [15N]

100°C for

studies,

with

l-position

at the

pathway.

MATERIALS [15N]

polymers

of adenine

adenosine,

bases

monomethylated

rearrangement

the mechanism

for

labeled

are

these

copolymer

Since

to define

Isotopically

these

at the

the Dimroth

established

performed

to extend

which

residues

form a random

(m6A)

acid

By treating

adenine

the conformational

of the nucleic

and in attempting

of adenine.

to finally

were

to examine

methylation

(5),

methylates

we could

adenine

designed

polydeoxyribonucleotides

first

base)

studies

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100 MHz

Ado

(Table

I)

N6-methyadenosine

(m6Ado). RESULTS AND DISCUSSION Nucleophilic CH3)2S04,

CHSI]

heterocyclic polynucleotides

attack

on adenosine

is directed

almost

base ring which

by certain exclusively

of Ado in both contain

to the

mononucleosides

Ado but lack

582

alkylating

guanosine

reagents

[e.g.,

l-position

of the

and -tides

(11)

(6,7,11).

Several

and in

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I

Compound

Rf in Solvent 0.5 M LiCl

1-butanol: acetic acid

water: (5:1:4)

ethanol:water (7.3)

adenosine

.56

.65

.65

[15N]-adenosine

.59

.61

.62

l-methyl-[15N]-adenosine

.96

.50

N6-methyladenosine

.70

.90

.88

N6-methyl-[15N]-adenosine

.70

.90

.77

streak:

.O to

.42

-

Figure 1. Proposed in adenosine.

studies

have shown

pathways

that

to the exocyclic

(N6)

Detailed

studies

the

kinetic

C5-C6 bond

(Figure

for

subsequent amino

group

(12,13) 1A).

the mechanism

of the Dimroth

to methylation, (in

the methyl

the presence

assumed

Upon ring

583

of base)

a mechanism closure,

rearrangement

group

to form m6Ado.

of hydroxide

the methyl

migrates

group

attack is

at

attached

Vol. 64, No. 2, 1975

at the 6-amino

BIOCHEMICAL

position

whereas

as the endocyclic

(N,)

opened"

was established

Brown

mechanism (14)

pyrimidine

showed

imino

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the exocyclic

nitrogen.

methylation

products

analogous

nitrogen

for studies

was correct

7.0

’ 6.0

II 4.0

is now reformed

this

type

3.0

of "ring-

on pyrimidines

by analysis

by mass spectrometry.

I

6.0

Precedent

from

such a pathway

amino

However,

where

of 2-aminoone can

2.0

~(PPMI pectra of: A ([15N]-amino)-adenosine* B l-methyl-['5N]-methyl-[15N$adenosine were recorded in-[CH3)2SO-d are reported as ppm downfield from HMO (K and K). -3' bbreviaS = singlet, D = doublet, Bd = tions used in spectral assignments-are: broad peak. The amino protons in A were identified by exchange with D20; the peaks at 6.86 and 7.77 ppm diqgppear. Spectral assignments are: A, H2 (8.30, S, Al), H8(8.10, S, Al), [ N]-amino (8.68 and 7.77, D, A2); E& H2 and H8 (8.70 and 8.66, both S, both Al), and l-CH3 (3.75, S, A3); C, H8 BdS, Al) and C6-NCH3 (8.29, S,. Al), H2 (8.26 and 8.09, D, Al), C6-NH (7.70, (2.96, BdD, A3). The doublet at 2.96 in spectrum C could be collapsed by homonuclear irradiation at 7.70 ppm or by deuteriuiii replacement. The impurity in the solvent is centered at 2.445 ppm.

584

VoL64,No.

BIOCHEMICAL

2, 1975

easily

envision

from

the

shown

exocyclic

that

labeled

an alternate

of using

the

dipolar

nucleus

exhibit

quite

[15N]

substituted

(instead

(m6Ado)

since

the methyl

group

at -3.

ppm; (ii)

by the methyl

a sharp

doublet

proximity

to the

mechanism

involving

would

dictate

two sets area,

of sharp

1.5);

with

(ii)

would

be split

a broad

resonance

at -7.5

(dipolar)

coupling

constant

endocyclic

nitrogen.

transfer

(i)

the methyl with

constant

a large

proton of -90

at -7.5

Hz (15)

the C2- and C8-protons

As can be seen

pathway

A is

consistent

involve

ring

opening

with and 180"

in the nmr spectra

internal

peak split 0.5)

into

due to its a

by the pathway

in

Figure

ppm would

between

be split

i.e.,

0.5

to the

a normal 2),

the pathway

and finally

ring

each)

[15N]

from

(Figure

of

them (each

(area

be undisturbed presented

1B

be comprised

is bonded

data;

rotation

proton

hand,

it

should

a [14N]

On the other

ppm would

the experimental

through

area,

constant

(since

3.),

due to the amino

(each

coupling

of the

(area

a C2-proton

at -3.

pathway

doublet

1.)

as outlined group

will

nmr spectrum

by a proton

(area

to a

nucleus)

the

methyl

ppm; and (iii)

methyl

doublets

and (iii)

Ado spectrum.

resonance

the single

a coupling

dipole);

a broad

a large

that:

expect

(i)

is

The advantage

If the reaction

(15).

to display:

is

or atoms attached

quadrupolar

one would

of a proton

communication

position.

groups

[14N]

removal

by use of Ado specifically

amino

that

nmr spectra

group

with

In this

is correct

of the normal

in lA,

involving

1B).

Ado is

outlined

product

(Figure

at the exocyclic

characteristic that

in purines

mechanism

nitrogen

final

split

group

[15N]

were

nucleus

pathway

the ring-opening

with

followed

amino

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only must

reformation.

REFERENCES (1) (2) (3) (4) (5) (6)

Arber, W. (1974) Prog. Nut. Acid Res. Mol. Biol. l4-, l-37. Littlefield, J.W., and Dunn, D.B. (1958) Biochem. J. 70, 642-W. Brookes, P., and Lawley, P.D. (1960) J. Chem. Sot., 53-545. Lautenberger, J., and Linn, S. (1972) J. Biol. Chem. 243, 6176-6182. Engel, J.D. and von Hippel, P.H. (1974) Biochem. 13, m3-4158. Lawley, P.D. (1971) in The Purines - Theory and Experiment; Proceedings

of the IV Jerusalem (7)

Jerusalem, Ramstein, 125-136.

Symposium, E.D. Bergmann and B. Pullman,

pp. 597-603. J., Helene, C.,

and Leng,

585

M. (1971)

Eur.

J. Biochem.

Eds., 21,

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(8)

Brown, D.M. (1974) in Basic Principles in Nucleic Acid Chemistry, Ts'o, P.O.P., Ed., New York, Academic Press, pp l-57. (9) Brown, G.B., and Weliky, V.S. (1953) J. Biol. Chem. 204, 1019-1024. (10) Jones, J.W. and Robins, R.K. (1963) J. Amer. Chem. Sot. B5-, 193-201. (11) Brimacombe, R.L.C., Griffin, B.E., Haines, J.A., Haslam, W.J., and Reese, C.B. (1965) Biochem. 4, 2452-2458. (12) Macon, J.B., and Wolfenden, R. (1968) Biochem. 7, 3453-3458. (13) Itaya, T., Tanaka, F., and Fujii, T. (1972) Tetrahedron 28, 535-547. (14) Brown, D.J. (1961) Nature l&, 828-829. (15) Shoup, R.R., Miles, H.T., and Becker, E.D. (1966) Biochem. Biophys. Res. Comm. 23, 194-201.

586