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Chemical ionization mass spectrometry of nucleosides
BIOCHIMICA ET BIOPHYSICAACTA
623
BBA Report BBA 91317 Chemical i o n i z a t i o n mass s p e c t r o m e t r y o f nucleosides
M.S. WILSON, I. DZIDIC and JAMES A. McCLOSKEY Institute for Lipid Research and Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025 (U.S.A.) (ReceivedJune 1st, 1971) SUMMARY Basic reactions of nucleosides under conditions of chemical ionization have been investigated, using CI-I4 or NH3 as reagent gases at a pressure of 0.4 mm Hg. C2 I-L, was found to be useful for the elucidation of some mechanistic and structural details, and was augmented by high resolution techniques. Principal ions corresponded to protonated forms of the molecular ion and the free base. Fewer structural details are generaUy available from chemical ionization spectra compared with their electron ionization counterparts, although molecular ion (/.e. M + H) enhancement in the former case offers a major advantage.
Electron ionization mass spectrometry has on numerous occasions demonstrated its usefulness for the determination of nucleoside structure (for a review see ref. 1), in particular for modified components from transfer RNA. In recent years chemical ionization mass spectrometry has been explored as a highly useful method which is complementary to electron ionization techniques2 . The ion spectrum is formed not by electron bombardment but by low energy collisions with ions derived from a reagent gas such as methane, present in the ion source at relatively high pressure (approx. 1 mm Hg). The chemical ionization mass spectra of a number of biologically important classes of compounds have been examined, including alkaloids 3 , amino acids4 and peptides s . We have therefore made a preliminary investigation of the chemical ionization mass spectra of free nucleosides, with the objectives of providing further information on gaseous ion-molecule reactions involving complex molecules, and of judging the utility of the method for structural studies in comparison with conventional electron ionization techniques. This communication presents results of a survey of fourteen nucleosides, using methane or ammonia as reagent gases. The following nucleosides were obtained from commercial sources: thymidine, 2'-deoxyadenosine, uridine, adenosine, N 6 ~V~-dimethyladenosine, 1-methyladenosine, pseudouridine, 5,6-dihydrouridine, 5,6-dihydrothymidine, and 9-~-ribofuranosylpurine. 2'-O-Methyladenosine was previously prepared in this laboratory 6 ; 2'-deoxy-~-adenosine was a gift from Dr. Leon Goodman of Stanford Research Institute; N ~-(3-methyl-2-butenyl)Biochir~ Biophy& Acta, 240 (1971) 623-626
624
BBA REPORT
adenosine and 4'-thioadenosine were obtained through the Cancer Chemotherapy National Service Center of the National Institutes of Health. C2 I-I4 was purchased from Merck, Sharp and Dohme of Canada. Mass spectra were determined on a CEC 21-110B double focusing instrument, with modifications for high-pressure studies based in large part on the work of Futrell and WojcikT,~'; details will be described elsewheres . All samples (0.5-3 #g) were introduced by direct probe under the following conditions: accelerating potential, 8 kV; electron energy, 200 eV; repeller field, 30 V/cm; ion source temperatures, 120-250°; ionization chamber pressure, 0.4 mm Hg. High resolution chemical ionization spectra (methane) of thymidine, N 6 ,N 6-dimethyladenosine and 2'-O-methyladenosine were photographically recorded, with resolution set at approximately 17 000, using a mixture of perdeuterated hydrocarbons (Merck of Canada) as internal mass standards 8 . The general fragmentation behavior of nucleosides when using methane as reactant gas can be illustrated by the spectra of thymidine and adenosine (Fig. 1). In every case examined the protonated molecular ion (M + H) was of high relative abundance, a considerable advantage over the much lower molecular ions obtained from conventional electron ionization I . For example, 5,6-dihydrouridine shows an intense M + H peak (37% relative intensity, 7% Zso), but none under electron ionization9 .
10(>
',bose + 2H) 127
0 HN.~CH3 0~[_[]~N/
?5 117
81
=
M = 242
HOC@~
(sugar) 50"
- 3q. 3
99 I
- 17 (o) 155
OH
I
(M+H-
HzO)
M+H 243
>~
~I00
(bose+2H) 136
-J
95 SO
NH2 N~, %
M= 267
"~N I~N7 HOCH20 |
M+H 268
"19.8
(a) 164
HO
u~
-9
2S 121
100
150
M+CH3 ~
176
' \
...... L ! 50
L l"'c'"'/
M+G3H 5
,
. . . . . . . . . . . . . . . . .
200 m/e
250
300
Fig. l . Chemical ionization mass spectza of thymidine (top) and adenosine (bottom) using methane as the reagent gas.
~ e are giateful to Professor Futrell for detailed discussions of his modifications, and a preprint of his manuscript prior to publication. Biochim. Biophys. Acta, 240 (1971) 623-626
BBA REPORT
625
Cleavage of the glycosidic bond to produce base or sugar fragments was represented in every case by the major ion base + 2H, in general analogy to electron ionization spectra 1° . NH2
NH2
~.N~INx~ N~ HOCH2 I base ~._~-HO OH
N ~ ' Nx~ ~N?~I--N~` H H base+ 2H
Spectra produced from C2 I-I4 reactant gas* showed that base + 2H predominantly contained one hydrogen from the CHs + (or C2Hs +) donor, and one from the sugar. Although it was found that extensive exchange between labile hydrogens of the nucleoside and deuterium from methane occurred**, the number of additional hydrogens from methane in various fragment ions could usually be ascertained by careful inspection. The odd-electron sugar ion(s) was of significance only in the case of pyrimidine nucleosides, similar to electron ionization spectra 1° . Further expulsion of one or two molecules of water was evident in some cases (e.g. m/e 99, 81 in Fig. 1, top). In many cases, ions are observed which correspond structurally to adducts of M or base + H, with C2 Hs + or C3 Hs + (ion-molecule reaction products of methane 2); however, their mechanistic origins are presently not known. The base + H + C2 Hs ion is equal in nominal mass to the major fragment base + CH20 (C-I' and 0-4') in electron ionization spectra 1° . The two species, which for adenosine can be formulated as a~ (ref. 6"1and a2, can be distinguished by use of C2 I-I4 or by measurement of exact mass. In adenosine, both NH2
NH2
o~C'~H R5C2 QI bose+30 Q2 (R= H ,2H)
forms of ion a were found, while in thymidine a2 greatly predominated. The general ions M + CH3 and base + H + CH3 involving CH3 derived from methane were also observed. Among the modified nucleosides, fewer diagnostic distinguishing features were found than in the corresponding electron ionization spectra, although 2'-O-methyladenosine showed a characteristic base + H + CHCHOCH3 peak in analogy to its behavior on electron ionization6 Preliminary experiments involving adenosine and thymidine were conducted using ammonia as reagent gas. Under the conditions used, NI-I4+ and NI-h+NH3 (20:1) are the primary reactants 11 . Although the proton affinity of ammonia is quite high~2, nucleosides are sufficiently good Bronsted bases to permit proton transfer from NH4 +. The base + 2H ions are still abundant, and M + H is more intense than in the case of methane, both in the case of thymidine (16% Zso) and adenosine (30% Y-so). Since the high proton affinity of 4°l~heuse of C2H4 for mechanistic studies was independently suggested by Professor J. Futrell
~
sonal communication, December, 1970). 2 or example, the deuterium distribution in the M + H ion of adenosine, which has five exchangeable hydrogens in addition to the one initially transferred from methane, was 38% 2H 1 , 38% 2H 2, 20% 2H3, 4% 2H4, 0% 2Hs. Biochim. Biophys. Acta, 240 (1971) 623-626
626
BBA REPORT
ammonia prevents proton transfer to most, if not all, non-nitrogen containing molecules, it may be useful as a highly selective reagent gas. Further studies of its use are in progress in this laboratory. This work was supported by the Robert A. Welch Foundation (Q-125); U.S. Public Health Service Grants GM 13901 and GM 02055, and computer facilities through FR 259. The authors thank Miss P.F. Crain for technical assistance. REFERENCES 1 J.A. McCloskey, in P.O.P. Ts'o, Basic Principles in Nucleic Acid Chemistry, Academic Press, New York, in the press. 2 F.H. Field, Acc. Chem. Res,, 1 (1968) 42. 3 H.M. Fales, H.A. Lloyd and G.W.A. Milne,J. Am. Chem. Soc., 92 (1970) 1590. 4 G.W.A. Milne, T. Axenrod and H.M. Fales, J. Am. Chem. Soc., 92 (1970) 5170. 5 W.R. Gray, L.H. Wojcik and J.H. FutreU, Biochem. Biophy~ Re~ Commun., 41 (1970) 1111. 6 S.J. Shaw, D.M. Desiderio, K. Tsuboyama and J.A. McCloskey,J. Am. Chem. Sea, 92 (1970) 2510. 7 J.H. Futrell and L.H. Wojcik, Rev. Sci. Instrum., 42 (1971) 244. 8 I. Dzidie, D.M. Desiderio, M.S. Wilson, P.F. Crain and J.A. MeCloskey,AnaL Chem., in the press. 9 S.M. Hecht, A.S. Gupta and N.J. Leonard, Anal Biochem., 30 (1969) 249. 10 K. Biemann and J.A. McCloskey,J. Am. Chem. Soc., 84 (1968) 2005. 11 S.K. Searles and P. Kebarle, J. Phy£ Chem., 72 (1968) 742. 12 M.S.B. Munson, J. Am. Chem. $oc., 87 (1965) 2332.
Biochim. Biophy~ Acta, 240 (1971) 623-626
623
BBA Report BBA 91317 Chemical i o n i z a t i o n mass s p e c t r o m e t r y o f nucleosides
M.S. WILSON, I. DZIDIC and JAMES A. McCLOSKEY Institute for Lipid Research and Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025 (U.S.A.) (ReceivedJune 1st, 1971) SUMMARY Basic reactions of nucleosides under conditions of chemical ionization have been investigated, using CI-I4 or NH3 as reagent gases at a pressure of 0.4 mm Hg. C2 I-L, was found to be useful for the elucidation of some mechanistic and structural details, and was augmented by high resolution techniques. Principal ions corresponded to protonated forms of the molecular ion and the free base. Fewer structural details are generaUy available from chemical ionization spectra compared with their electron ionization counterparts, although molecular ion (/.e. M + H) enhancement in the former case offers a major advantage.
Electron ionization mass spectrometry has on numerous occasions demonstrated its usefulness for the determination of nucleoside structure (for a review see ref. 1), in particular for modified components from transfer RNA. In recent years chemical ionization mass spectrometry has been explored as a highly useful method which is complementary to electron ionization techniques2 . The ion spectrum is formed not by electron bombardment but by low energy collisions with ions derived from a reagent gas such as methane, present in the ion source at relatively high pressure (approx. 1 mm Hg). The chemical ionization mass spectra of a number of biologically important classes of compounds have been examined, including alkaloids 3 , amino acids4 and peptides s . We have therefore made a preliminary investigation of the chemical ionization mass spectra of free nucleosides, with the objectives of providing further information on gaseous ion-molecule reactions involving complex molecules, and of judging the utility of the method for structural studies in comparison with conventional electron ionization techniques. This communication presents results of a survey of fourteen nucleosides, using methane or ammonia as reagent gases. The following nucleosides were obtained from commercial sources: thymidine, 2'-deoxyadenosine, uridine, adenosine, N 6 ~V~-dimethyladenosine, 1-methyladenosine, pseudouridine, 5,6-dihydrouridine, 5,6-dihydrothymidine, and 9-~-ribofuranosylpurine. 2'-O-Methyladenosine was previously prepared in this laboratory 6 ; 2'-deoxy-~-adenosine was a gift from Dr. Leon Goodman of Stanford Research Institute; N ~-(3-methyl-2-butenyl)Biochir~ Biophy& Acta, 240 (1971) 623-626
624
BBA REPORT
adenosine and 4'-thioadenosine were obtained through the Cancer Chemotherapy National Service Center of the National Institutes of Health. C2 I-I4 was purchased from Merck, Sharp and Dohme of Canada. Mass spectra were determined on a CEC 21-110B double focusing instrument, with modifications for high-pressure studies based in large part on the work of Futrell and WojcikT,~'; details will be described elsewheres . All samples (0.5-3 #g) were introduced by direct probe under the following conditions: accelerating potential, 8 kV; electron energy, 200 eV; repeller field, 30 V/cm; ion source temperatures, 120-250°; ionization chamber pressure, 0.4 mm Hg. High resolution chemical ionization spectra (methane) of thymidine, N 6 ,N 6-dimethyladenosine and 2'-O-methyladenosine were photographically recorded, with resolution set at approximately 17 000, using a mixture of perdeuterated hydrocarbons (Merck of Canada) as internal mass standards 8 . The general fragmentation behavior of nucleosides when using methane as reactant gas can be illustrated by the spectra of thymidine and adenosine (Fig. 1). In every case examined the protonated molecular ion (M + H) was of high relative abundance, a considerable advantage over the much lower molecular ions obtained from conventional electron ionization I . For example, 5,6-dihydrouridine shows an intense M + H peak (37% relative intensity, 7% Zso), but none under electron ionization9 .
10(>
',bose + 2H) 127
0 HN.~CH3 0~[_[]~N/
?5 117
81
=
M = 242
HOC@~
(sugar) 50"
- 3q. 3
99 I
- 17 (o) 155
OH
I
(M+H-
HzO)
M+H 243
>~
~I00
(bose+2H) 136
-J
95 SO
NH2 N~, %
M= 267
"~N I~N7 HOCH20 |
M+H 268
"19.8
(a) 164
HO
u~
-9
2S 121
100
150
M+CH3 ~
176
' \
...... L ! 50
L l"'c'"'/
M+G3H 5
,
. . . . . . . . . . . . . . . . .
200 m/e
250
300
Fig. l . Chemical ionization mass spectza of thymidine (top) and adenosine (bottom) using methane as the reagent gas.
~ e are giateful to Professor Futrell for detailed discussions of his modifications, and a preprint of his manuscript prior to publication. Biochim. Biophys. Acta, 240 (1971) 623-626
BBA REPORT
625
Cleavage of the glycosidic bond to produce base or sugar fragments was represented in every case by the major ion base + 2H, in general analogy to electron ionization spectra 1° . NH2
NH2
~.N~INx~ N~ HOCH2 I base ~._~-HO OH
N ~ ' Nx~ ~N?~I--N~` H H base+ 2H
Spectra produced from C2 I-I4 reactant gas* showed that base + 2H predominantly contained one hydrogen from the CHs + (or C2Hs +) donor, and one from the sugar. Although it was found that extensive exchange between labile hydrogens of the nucleoside and deuterium from methane occurred**, the number of additional hydrogens from methane in various fragment ions could usually be ascertained by careful inspection. The odd-electron sugar ion(s) was of significance only in the case of pyrimidine nucleosides, similar to electron ionization spectra 1° . Further expulsion of one or two molecules of water was evident in some cases (e.g. m/e 99, 81 in Fig. 1, top). In many cases, ions are observed which correspond structurally to adducts of M or base + H, with C2 Hs + or C3 Hs + (ion-molecule reaction products of methane 2); however, their mechanistic origins are presently not known. The base + H + C2 Hs ion is equal in nominal mass to the major fragment base + CH20 (C-I' and 0-4') in electron ionization spectra 1° . The two species, which for adenosine can be formulated as a~ (ref. 6"1and a2, can be distinguished by use of C2 I-I4 or by measurement of exact mass. In adenosine, both NH2
NH2
o~C'~H R5C2 QI bose+30 Q2 (R= H ,2H)
forms of ion a were found, while in thymidine a2 greatly predominated. The general ions M + CH3 and base + H + CH3 involving CH3 derived from methane were also observed. Among the modified nucleosides, fewer diagnostic distinguishing features were found than in the corresponding electron ionization spectra, although 2'-O-methyladenosine showed a characteristic base + H + CHCHOCH3 peak in analogy to its behavior on electron ionization6 Preliminary experiments involving adenosine and thymidine were conducted using ammonia as reagent gas. Under the conditions used, NI-I4+ and NI-h+NH3 (20:1) are the primary reactants 11 . Although the proton affinity of ammonia is quite high~2, nucleosides are sufficiently good Bronsted bases to permit proton transfer from NH4 +. The base + 2H ions are still abundant, and M + H is more intense than in the case of methane, both in the case of thymidine (16% Zso) and adenosine (30% Y-so). Since the high proton affinity of 4°l~heuse of C2H4 for mechanistic studies was independently suggested by Professor J. Futrell
~
sonal communication, December, 1970). 2 or example, the deuterium distribution in the M + H ion of adenosine, which has five exchangeable hydrogens in addition to the one initially transferred from methane, was 38% 2H 1 , 38% 2H 2, 20% 2H3, 4% 2H4, 0% 2Hs. Biochim. Biophys. Acta, 240 (1971) 623-626
626
BBA REPORT
ammonia prevents proton transfer to most, if not all, non-nitrogen containing molecules, it may be useful as a highly selective reagent gas. Further studies of its use are in progress in this laboratory. This work was supported by the Robert A. Welch Foundation (Q-125); U.S. Public Health Service Grants GM 13901 and GM 02055, and computer facilities through FR 259. The authors thank Miss P.F. Crain for technical assistance. REFERENCES 1 J.A. McCloskey, in P.O.P. Ts'o, Basic Principles in Nucleic Acid Chemistry, Academic Press, New York, in the press. 2 F.H. Field, Acc. Chem. Res,, 1 (1968) 42. 3 H.M. Fales, H.A. Lloyd and G.W.A. Milne,J. Am. Chem. Soc., 92 (1970) 1590. 4 G.W.A. Milne, T. Axenrod and H.M. Fales, J. Am. Chem. Soc., 92 (1970) 5170. 5 W.R. Gray, L.H. Wojcik and J.H. FutreU, Biochem. Biophy~ Re~ Commun., 41 (1970) 1111. 6 S.J. Shaw, D.M. Desiderio, K. Tsuboyama and J.A. McCloskey,J. Am. Chem. Sea, 92 (1970) 2510. 7 J.H. Futrell and L.H. Wojcik, Rev. Sci. Instrum., 42 (1971) 244. 8 I. Dzidie, D.M. Desiderio, M.S. Wilson, P.F. Crain and J.A. MeCloskey,AnaL Chem., in the press. 9 S.M. Hecht, A.S. Gupta and N.J. Leonard, Anal Biochem., 30 (1969) 249. 10 K. Biemann and J.A. McCloskey,J. Am. Chem. Soc., 84 (1968) 2005. 11 S.K. Searles and P. Kebarle, J. Phy£ Chem., 72 (1968) 742. 12 M.S.B. Munson, J. Am. Chem. $oc., 87 (1965) 2332.
Biochim. Biophy~ Acta, 240 (1971) 623-626