Volume 4 Number 6 June 1977
Nucleic Acids Research
Incorporation of oxygen-18 into nucleosides and bases
G.Puzo, Karl H.Schram and James A.McCloskey
Department of Biopharmaceutical Sciences, University of Utah, Salt Lake City, UT 84112, USA
Received 5 April 1977
ABSTRACT
Extensive incorporation of oxygen-18gat position 0 of the pyrimidine
nucleus results from exchange between H, 0 and nucleosides or bases in IN
HC1 at 100°. The reaction is hindered By substitution at C-5 with the
greatest effect shown in pseudouridine (R = ribosyl) and the least in uridine (R = H ) . Maximum incorporation in the latter compound was 941, and in
uricil was 983. The method is experimentally simple and the incorporation
is readily monitored by mass spectrometry.
INTRODUCTION
In view of the growing role of stable isotopes in chemistry and biology
(1,2), it is interesting that relatively few studies have been directed toward the incorporation of stable isotopes of oxygen into the nucleic acid
bases and nucleosides (3-7). The availability of such suitably labeled
substrates is of potential importance in a number of fields, in particular
those which employ measurements by nmr or mass spectrometry. Based on an
1 ft
interest in preparing 0 labeled nucleosides, we were attracted to the
18
relatively simple procedure of Wang and collaborators in which 0 was ex18
changed into uracil and its analogs by heating with H 2 0 and catalytic
amounts of SOC1 2 (6). Analysis of the products by mass spectrometry showed
incorporation primarily at position 0 with yields ranging from 4 to 13 mole
percent. Unfortunately, we found their, procedure gave essentially no incorporation into uridine and related nucleosides, and so reaction conditions
were systematically modified by increasing the acid strength and varying the
heating period so as to increase the extent of protonation and therefore the
extent of oxygen exchange.
MATERIALS AND METHODS
~~~— I Q
1g
Materials. H2 0 containing 99 atom percent
0 was purchased from
Koch Isotopes, I n c . , Cambridge, Massachusetts. All nucleosides and bases
were of commercial origin.
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
Nucleic Acids Research
Reaction conditions.
Approximately 1 mg of pyrimidine base or
18
nucleoside was dissolved in 5 pi of FL 0 in a melting point capillary and
0.5 \il of cone. HC1 was then added.
The tube was sealed, heated for 18
hours (or other times as indicated) at 100° and then opened.
removed under reduced pressure in the presence of Py^s ^
Solvent was
n a vacuuin
dessi-
cator.
Measurement of oxygen-18 incorporation levels. Reaction products were
analyzed directly by mass spectrometry using direct probe introduction of
the sample into an LKB 9000S instrument at an ion source temperature of 250°
18
7
and ionizing electron energy of 70 eV. The distribution of 0 at 0 vs.
4
0 was made from the m/e 69 fragmentation product from the general reaction
(8,9):
0
II
cII
,
C-R1
m/e 69
1
2
(R1=R2=H)
(R =H, R =ribosyl or H)
In the case of the nucleosides, production of m/e 69 occurs in two steps,
through the intermediate base + H species which corresponds in mass to that
of the free base (10). In the case of uracil or uridine, the four possible
exchange products shown below will exist as mole fractions A - D which can
be measured directly from molecular ion (M) abundances in the base series,
I8
IBO0
18,
m
0
o
(r**N
!»(K* N
C
D
HN
^^N
A
1
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1
B
1
1
Nucleic Acids Research
and M or M - H90 abundances in the case of nucleosides.
O
Labeling contri-
A
butions at 0 and 0
are represented by the relative abundances of ion
species d and c, respectively, but correction must be made for overlap from
species a (overlaps with d) and b (overlaps with c).
Then,
c = (rel. abund. m/e 71) - b (rel. abund. m/e 69 + rel. abund. m/e 71)
d = (rel. abund. m/e 69) - a (rel. abund. m/e 69 + rel. abund. m/e 71)
The second term in each equation represents the fraction of the total molecular population due to unlabeled or dilabeled species, expressed in relative abundance units measured directly from the mass spectrum.
RESULTS AND DISCUSSION
IQ
Incorporation of
IQ
0 by exchange with FL 0 in acid proceeds through
equilibria involving addition of water and dehydration. The principal
intermediate is presumably derived from protonation of 0
(11) and formation
of a tetragonal intermediate (6,12) which produces a product whose
0 con-
tent reflects the isotopic content of the aqueous solvent. Assuming the
failure of Wang's method (6) to cause exchange in uridine to be a result of
insufficent acidity, substantially higher acid concentrations were used in
order to increase the proportion of protonated base species present.
The results of exchange of uridine and its derivatives for single 18
hour exchange reactions, are shown in Table 1.
In the case of uridine the equilibrium plateau is obtained after approximately 24 hours as shown in Figure 1, resulting in 941 incorporation.
A
variety of heating times and acidities were investigated, with no significant improvement over that shown. As indicated by the lower panel in Figure
1, the reaction follows pseudo-first order kinetics, and so the plateau ex18
change level can be predicted by examination of the 0 incorporation curve
during early stages of the reaction.
Examination of the molecular ion and M - 18 regions of the mass spectra
"' 8
of all eight nucleosides revealed evidence of incorporation of a single " 0
atom.
The location of
0 is presumed to be solely at position 0
case based on two points of evidence provided by mass spectrometry.
in each
(1) Ion
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Table 1
Incorporation of
Oxygen-18 into Nucleosides After 18 Hours'3
Compound
mole %
Uridine
5-Methyluridine
5-Fluorouridine
5-Hydroxyurdine
5-Chlorouridine
5-Bromouridine
5-Iodouridine
Pseudouridine
18
0
90
90
77
70
67
58
46
38
a
Corrected for presence of
O in solvent, primarily
from cone. HC1, by multiplication of the experimentally
found value by 1.11.
12
14
TIME (tours)
Figure 1. 0^8 incorporation rate curve (upper) and pseudo-firstorder kinetic plot (lower) for uridine in IK acid solution.
Slope = 6.96 x 10" 2 hrs.
A Q - concentration of Ol8 species at zero time, (AQ= °) •
Aj. - concentration of 018 species at time t.
A - concentration of ol8 species at equilibrium.
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abundance patterns of the m/e 69 ion-types showed essentially complete retention of the 180 label, although an uncertainty of several percent exists
due to the presence of minor interfering ions. (2) Any species labeled at
position 0 would be eligible for labeling by the major route at 0 thus
giving rise to some doubly-labeled molecules, which were not observed. In
the case of pseudouridine, location of the 0 label at position 0 was
made using the fragment ion of m/e 98 (base + O L O - HNCO (13)), which contains the 0 moiety.
The rates of incorporation as reflected in the 18-hour reactions (Table
1) generally show the influence of steric hindrance from the substituent at
C-5. With the exception of 5-methyluridine (thymine riboside) the order
found shows substantial dependence on substituent size, with the greatest
effect shown by the bulky ribosyl moiety at position C-5 in pseudouridine.
Based on previous studies of the acid hydrolysis of pyrimidine nucleosides (14,15), the potential problem of glycosidic bond cleavage under these
exchange conditions was considered to be generally unimportant. For example,
uridine, 5-bromouridine, 5-chlorouridine and the 5-fluoro analogs of
l-(B-D-lyxofuranosyl) and l-((3-D-arabinofuranosyl)uracil have been shown to
be stable to IN HC1 for one month at 80° (14). However, some reaction of
both 5-iodouridine (forms uridine) and 5-hydroxyuridine (hydrolyzes to 5hydroxyuridi;ieand ribose) in acid was observed, but the extent of degradation
was less than 101 after 30 hours. The situation is different with pseudouridine which has been shown to be hydrolyzed to a mixture of a- and 6-ribofuranosides and a- and B-ribopyranosides (16,17). The total ion chromatogram from gas chromatography-mass spectrometry of the trimethylsilylated reaction mixture of pseudouridine indicated the presence of four components
with one being present in large excess (>50%). The major component, was
identified by its relative retention time as B-pseudouridine. Thus, although isomerization and anomerization of pseudouridine occurs under these
conditions, the reaction is still of potential use for preparation of
labeled pseudouridine, but requires isolation from the reaction mixture.
The results of application of the same technique to several bases are
shown in Table 2. The heating time employed was found to produce maximum
(plateau) incorporation. The pyrimidines uracil and thymine show nearly
quantitative incorporation of 0, while the purine hypoxanthine underwent
no exchange. In the absence of a sugar substituent at N-l, both pyrimidines
were found to undergo minor exchange at C-2 with the result that some dilabeled species are also formed. Wang and collaborators reported .qualitively
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Table 2
Exchange in Nucleic Acid Bases After 72 Hours.
Compound
Total Incorporation
o f 18pa
Uracil
Thymine
Hypoxanthine
98%
95%
0%
% at C2
6
3
% at C4
% at C2 and C4
83
83
11
10
a
Corrected for presence of l^O i n solvent, primarily from cone.
HC1, by multiplication of the experimentally found value by 1.1.
similar labeling patterns for uracil and thymine but made no mention of
doubly labeled products (6), probably because the overall levels of incorporation they observed were relatively low.
The experimental simplicity of the exchange procedure, as well as the
more difficult alternatives for most forms of stable isotopic labeling of
the nucleic acid bases and nucleosides, speaks in favor of the present
method.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. C. D. Poulter for helpful discussion,
and to the National Institutes of Health for financial support (CA 18024,
GM 21584). KHS received fellowship support from the National Cancer
Institute (CA 02466).
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Nucleic Acids Research
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