Volume 4 Number 6 June 1977
Nucleic Acids Research .M
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-l18at position 04 of the pyrimidine nucleus regults 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 94%, and in uricil was 98%. The method is experimentally simple and the incorporation is readily monitored by mass spectrometry.
INTRODJCTION 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 interest in preparing 180 labeled nucleosides, we were attracted to the relatively simple procedure of Wang and collaborators in which 180 was exchanged into uracil and its analogs by heating with H218o and catalytic amounts of SOC12 (6). Analysis of the products by mass spectrometry showed incorporation primarily at position 04 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 Miaterials. H2 180 containing 99 atom percent 18 0 was purchased from Koch Isotopes, Inc., Cambridge, Massachusetts. All nucleosides and bases were of commercial origin.
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
Nucleic Acids Research Reaction conditions. Approximately 1 mg of pyrimidine base or nucleoside was dissolved in 5 p1 of H 180 in a melting point capillary and 0.5 p1 of conc. HCl was then added. The tube was sealed, heated for 18 hours (or other times as indicated) at 1000 and then opened. Solvent was removed under reduced pressure in the presence of P205 in a vacuum dessicator. 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 2500 and ionizing electron energy of 70 eV. The distribution of 180 at o2 vs. 04 was made from the m/e 69 fragmentation product from the general reaction
(8,9):
11
R'
11
-e
m/e 69
(R1=R2=H)
(R1=H, R2=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,
:0 HX) I) 0
10 N 180A18 180
0
2076
~~~~10180
R A
R B
R C
R D
a
b
c
d
Nucleic Acids Research and M or M - H20 abundances in the case of nucleosides. Labeling contributions at 02 and 04 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 Incorporation of 180 by exchange with H2 180 in acid proceeds through equilibria involving addition of water and dehydration. The principal intermediate is presumably derived from protonation of 04 (11) and formation of a tetragonal intermediate (6,12) which produces a product whose 180 content reflects the isotopic content of the aqueous solvent. Assuming the
o u
HN
~~~~~+180 o0 "H30H )HN H8
A
~~~~~R
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 94% 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 exchange level can be predicted by examination of the 180 incorporation curve during early stages of the reaction. Examination of the molecular ion and M - 18 regions of the mass spectra 18 of all eight nucleosides revealed evidence of incorporation of a single 80 atom. The location of 180 is presumed to be solely at position 04 in each case based on two points of evidence provided by mass spectrometry. (1) Ion 2077
Nucleic Acids Research Table 1
Incorporation of
Oxygen-18 into Nucleosides After 18 Hoursa
Compound
Uridine 5-Methyluridine 5-Fluorouridine 5-Hydroxyurdine
5-Chlorouridine 5-Bromouridine 5-Iodouridine Pseudouridine
mole %
180
90 90 77 70 67 58 46 38
aCorrected for presence of 160 in solvent, primarily from conc. HC1, by multiplication of the experimentally found value by 1.11.
Figure 1. Q18 incorporation rate curve (upper) and pseudo-firstorder kinetic plot (lower) for uridine in lN acid solution. Slope = 6.96 x 10-2 hrs.
Ao
-
At
-
A -
2078
concentration of 0l8 species at zero time, (Ao = 0). concentration of 0l8 species at tine t. concentration of 0l8 species at equilibrium.
Nucleic Acids Research 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 02 would be eligible for labeling by the major route at O4 thus giving rise to some doubly-labeled molecules, which were not observed. In the case of pseudouridine, location of the 180 label at position 04 was made using the fragment ion of m/e 98 (base + CHI20 - HNCO (13)), which contains the 04 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-S 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-(S-D-lyxofuranosyl) and l-(S-D-arabinofuranosyl)uracil have been shown to be stable to 1N HC1 for one month at 800 (14). However, some reaction of both 5-iodouridine (forms uridine) and 5-hydroxyuridine (hydrolyzes to 5hydroxyuridi,eandribose) in acid was observed, but the extent of degradation was less than 10% after 30 hours. The situation is different with pseudouridine which has been shown to be hydrolyzed to a mixture of a- and 0-ribofuranosides and a- and S-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 a-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 180, while the purine hypoxanthine underwent no exchange. In the absence of a sugar substituent at N-1, 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
2079
Nucleic Acids Research Table 2
180
Exchange in Nucleic Acid Bases After 72 Hours.
Compound
Total Incorporation of 180a
Uracil Thymine Hypoxanthine
98% 95% 0%
% at C2 6 3
% at C4
% at C2 and C4
83 83
11 10
aCorrected for presence of 160 in solvent, primarily from conc. 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). REFERENCES 1. "Proceedings of the First International Conference on Stable Isotopes in Chemistry, Biology, and Medicine," May, 1973, Argonne, Ill., Eds. Klein, P. D., and Peterson, S. V., AEC CONF-730525, 1974, National Technical Information Service, U. S. Dept. of Comnerce, Springfield, Va. 2. "Proceedings of the Second International Conference on Stable Isotopes, October, 1975, Oak Brook, Ill., Eds. Klein, E. R. and Klein P. D., ERCA CONF-751027, 1976, National Technical Information Service, U. S. Dept. of Commerce, Springfield, Va. 3. Caprioli, R., and Rittenberg, D. (1969) Biochemistry, 8, 3375-3384. 4. Caprioli, R., and Rittenberg, D. (1968) Proc. Nat. Acad. Sci. (U.S.), 60, 1379-1382. 5. Caprioli, R., and Rittenberg, D. (1968) Proc. Nat. Acad. Sci. (U.S.), 61, 1422-1427. 6. Wang, S. Y., Hahn, B. S., Fenselau, C., and Zafiriou, 0. C. (1972) Biochem. Biophys. Res. Commun., 48, 1630-1635. 7. Cadet, J., and Teoule, R., (1975) Bull. Chim. Soc. France, 879-884. 8. Rice, J. M., Dudek, G. O., and Barber, M. (1965) J. Am. Chem. Soc., 87, 4569-4576. 9. Ulrich, J., Teoule, R., Massot, R., and Cornu, A. (1969) Org. Mass Spectrom., 2, 1183-1199.
2080
Nucleic Acids Research 10. 11. 12. 13. 14. 15. 16.
17.
Biemann, K., and McCloskey, J. A. (1962) J. Amer. Chem. Soc., 84, 20052006. Parry, E. P., Hern, D. N. and Burr, J. G. (1969) Biochim. Biophys. Acta, 182, 570-572. Hausworth, W., Hahn, B. S. and Wang, S. Y. (1972) Biochem. Biophys. Res. Connn., 48, 1614-1621. McCloskey, J. A. (1974) Basic Principles in Nucleic Acid Chemistry, Vol. I, pp. 209-309, Ed. P. 0. P. Ts'o, Academic Press, New York. Garrett, E. R., Seydel, J. K., and Sharpen, A. J. (1966) J. Org. Chem., 31, 2219. Pfitzner, K. E., and Moffatt, J. G. (1964) J. Org. Chem., 29, 1508. Chambers, R. W., Kurkov, U., and Shapiro, R. (1963) Biochemistry, 2, 1192. Cohen, W. (1962) Biochemistry, 1, 490.
2081
Nucleic Acids Research .M
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-l18at position 04 of the pyrimidine nucleus regults 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 94%, and in uricil was 98%. The method is experimentally simple and the incorporation is readily monitored by mass spectrometry.
INTRODJCTION 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 interest in preparing 180 labeled nucleosides, we were attracted to the relatively simple procedure of Wang and collaborators in which 180 was exchanged into uracil and its analogs by heating with H218o and catalytic amounts of SOC12 (6). Analysis of the products by mass spectrometry showed incorporation primarily at position 04 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 Miaterials. H2 180 containing 99 atom percent 18 0 was purchased from Koch Isotopes, Inc., Cambridge, Massachusetts. All nucleosides and bases were of commercial origin.
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
Nucleic Acids Research Reaction conditions. Approximately 1 mg of pyrimidine base or nucleoside was dissolved in 5 p1 of H 180 in a melting point capillary and 0.5 p1 of conc. HCl was then added. The tube was sealed, heated for 18 hours (or other times as indicated) at 1000 and then opened. Solvent was removed under reduced pressure in the presence of P205 in a vacuum dessicator. 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 2500 and ionizing electron energy of 70 eV. The distribution of 180 at o2 vs. 04 was made from the m/e 69 fragmentation product from the general reaction
(8,9):
11
R'
11
-e
m/e 69
(R1=R2=H)
(R1=H, R2=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,
:0 HX) I) 0
10 N 180A18 180
0
2076
~~~~10180
R A
R B
R C
R D
a
b
c
d
Nucleic Acids Research and M or M - H20 abundances in the case of nucleosides. Labeling contributions at 02 and 04 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 Incorporation of 180 by exchange with H2 180 in acid proceeds through equilibria involving addition of water and dehydration. The principal intermediate is presumably derived from protonation of 04 (11) and formation of a tetragonal intermediate (6,12) which produces a product whose 180 content reflects the isotopic content of the aqueous solvent. Assuming the
o u
HN
~~~~~+180 o0 "H30H )HN H8
A
~~~~~R
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 94% 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 exchange level can be predicted by examination of the 180 incorporation curve during early stages of the reaction. Examination of the molecular ion and M - 18 regions of the mass spectra 18 of all eight nucleosides revealed evidence of incorporation of a single 80 atom. The location of 180 is presumed to be solely at position 04 in each case based on two points of evidence provided by mass spectrometry. (1) Ion 2077
Nucleic Acids Research Table 1
Incorporation of
Oxygen-18 into Nucleosides After 18 Hoursa
Compound
Uridine 5-Methyluridine 5-Fluorouridine 5-Hydroxyurdine
5-Chlorouridine 5-Bromouridine 5-Iodouridine Pseudouridine
mole %
180
90 90 77 70 67 58 46 38
aCorrected for presence of 160 in solvent, primarily from conc. HC1, by multiplication of the experimentally found value by 1.11.
Figure 1. Q18 incorporation rate curve (upper) and pseudo-firstorder kinetic plot (lower) for uridine in lN acid solution. Slope = 6.96 x 10-2 hrs.
Ao
-
At
-
A -
2078
concentration of 0l8 species at zero time, (Ao = 0). concentration of 0l8 species at tine t. concentration of 0l8 species at equilibrium.
Nucleic Acids Research 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 02 would be eligible for labeling by the major route at O4 thus giving rise to some doubly-labeled molecules, which were not observed. In the case of pseudouridine, location of the 180 label at position 04 was made using the fragment ion of m/e 98 (base + CHI20 - HNCO (13)), which contains the 04 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-S 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-(S-D-lyxofuranosyl) and l-(S-D-arabinofuranosyl)uracil have been shown to be stable to 1N HC1 for one month at 800 (14). However, some reaction of both 5-iodouridine (forms uridine) and 5-hydroxyuridine (hydrolyzes to 5hydroxyuridi,eandribose) in acid was observed, but the extent of degradation was less than 10% after 30 hours. The situation is different with pseudouridine which has been shown to be hydrolyzed to a mixture of a- and 0-ribofuranosides and a- and S-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 a-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 180, while the purine hypoxanthine underwent no exchange. In the absence of a sugar substituent at N-1, 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
2079
Nucleic Acids Research Table 2
180
Exchange in Nucleic Acid Bases After 72 Hours.
Compound
Total Incorporation of 180a
Uracil Thymine Hypoxanthine
98% 95% 0%
% at C2 6 3
% at C4
% at C2 and C4
83 83
11 10
aCorrected for presence of 160 in solvent, primarily from conc. 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). REFERENCES 1. "Proceedings of the First International Conference on Stable Isotopes in Chemistry, Biology, and Medicine," May, 1973, Argonne, Ill., Eds. Klein, P. D., and Peterson, S. V., AEC CONF-730525, 1974, National Technical Information Service, U. S. Dept. of Comnerce, Springfield, Va. 2. "Proceedings of the Second International Conference on Stable Isotopes, October, 1975, Oak Brook, Ill., Eds. Klein, E. R. and Klein P. D., ERCA CONF-751027, 1976, National Technical Information Service, U. S. Dept. of Commerce, Springfield, Va. 3. Caprioli, R., and Rittenberg, D. (1969) Biochemistry, 8, 3375-3384. 4. Caprioli, R., and Rittenberg, D. (1968) Proc. Nat. Acad. Sci. (U.S.), 60, 1379-1382. 5. Caprioli, R., and Rittenberg, D. (1968) Proc. Nat. Acad. Sci. (U.S.), 61, 1422-1427. 6. Wang, S. Y., Hahn, B. S., Fenselau, C., and Zafiriou, 0. C. (1972) Biochem. Biophys. Res. Commun., 48, 1630-1635. 7. Cadet, J., and Teoule, R., (1975) Bull. Chim. Soc. France, 879-884. 8. Rice, J. M., Dudek, G. O., and Barber, M. (1965) J. Am. Chem. Soc., 87, 4569-4576. 9. Ulrich, J., Teoule, R., Massot, R., and Cornu, A. (1969) Org. Mass Spectrom., 2, 1183-1199.
2080
Nucleic Acids Research 10. 11. 12. 13. 14. 15. 16.
17.
Biemann, K., and McCloskey, J. A. (1962) J. Amer. Chem. Soc., 84, 20052006. Parry, E. P., Hern, D. N. and Burr, J. G. (1969) Biochim. Biophys. Acta, 182, 570-572. Hausworth, W., Hahn, B. S. and Wang, S. Y. (1972) Biochem. Biophys. Res. Connn., 48, 1614-1621. McCloskey, J. A. (1974) Basic Principles in Nucleic Acid Chemistry, Vol. I, pp. 209-309, Ed. P. 0. P. Ts'o, Academic Press, New York. Garrett, E. R., Seydel, J. K., and Sharpen, A. J. (1966) J. Org. Chem., 31, 2219. Pfitzner, K. E., and Moffatt, J. G. (1964) J. Org. Chem., 29, 1508. Chambers, R. W., Kurkov, U., and Shapiro, R. (1963) Biochemistry, 2, 1192. Cohen, W. (1962) Biochemistry, 1, 490.
2081
