J. Phys. Chem. 1989, 93, 3595-3599
3595
Mechanism of I R and UV Laser Desorption of Nucleosides: A Study by Fourier Transform Mass Spectrometry M. Paul Chiarelli and Michael L. Gross* Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588 (Received: July 26, 1988; In Final Form: November 28, 1988) Laser desorption coupled with Fourier transform mass spectrometry (LD/FTMS) was employed to study the desorption characteristics of adenosine, guanosine, thymidine, and uridine at wavelengths of 266 and 1064 nm. At 266 nm near threshold, the two pyrimidines, thymidine and uridine, yield disodiated molecular ions, (M - H + 2Na)+, as the most abundant ions whereas guanosine gives the sodiated base, (B + Na)’. No evidence of excited-state proton transfer was found to explain disodiated molecular ion formation of the pyrimidines. Instead, formation of the disodiated ions is driven by a shift in the lactim-lactam equilibrium toward the lactim form in the T I state, a feature not characteristic of the purine nucleosides. Cationization in the dilute gas phase is not a major means of forming these ions upon IR irradiation, unlike the desorption of sucrose. A direct desorption mechanism is proposed whereby molecular ions are formed by dissociation of clusters of nucleosides that are bonded by means of the base ring systems.
Introduction Much attention has been given to assessing the capability of laser desorption (LD) and particularly LD coupled with Fourier transform mass spectrometry (FTMS) for desorbing biomolec~ l e s l -and ~ polymer^.^ The research reported here is a continuation of those efforts and is concerned with the understanding of the ionization5and desorption mechanisms involved in LD in an attempt to enhance its utility. The desorption characteristics of nucleosides as a function of wavelength is the particular focus of this investigation. To date, studies of the laser desorption of nucleosides have been limited to wavelengths in the IR. Studies done in this laboratory at a wavelength of 1064 nm showed that simple nucleosides give spectra similar to those obtained by using fast atom bombardment (FAB).’ Glycosidic bond cleavage and charge retention on the base almost always account for the most abundant ion when negative ions are desorbed. The protonated base is the most abundant positive ion, giving way to the monosodiated molecular ion when NaCl is added in equal weight. Studies of more complex and polyphosphorylated nucleotides with a C 0 2 laser (10.6 pm) showed that similar fragmentations occur and that ions are formed by successive cleavages of phosphate groups.6 The extent of fragmentation exhibited by desorbing molecules was interpreted as evidence for different desorption mechanisms.’ Thermal desorption8 is favored when low laser power densities at wavelengths in the IR are used. Under these conditions, molecules exhibit extensive fragmentation. The absence of fragment ions at power densities much larger than those employed for thermal desorption is cited as evidence for a shockwave me~hanism.~ Desorption at wavelengths in the UV (principally 266 nm), where aromatic molecules undergo electronic transitions, has recently become of Both the wavelength of the laser (1) Ijames, C. F.; Wilkins, C. L. J . Am. Chem. SOC.1988, 110, 2687. (2) Coates, M. L.; Wilkins, C L. Anal. Chem. 1987, 59, 197. (3) McCrery, D. A.; Gross, M. L. Anal. Chim. Acfa 1985, 178, 91. (4) Brown, R. S.; Weil, D. A.; Wilkins, C. L. Macromolecules 1986, 19, 1255. ( 5 ) Chiarelli, M. P.; Gross, M. L. Int. J . Mass Spectrom. Ion Processes 1981, 78, 3 1 . (6) Nuwaysir, L.; Wilkins, C. L. Presented at the 38th Annual Pittsburgh Conference, Atlantic City, NJ, March 9-13, 1987. (7) Hillenkarnp, F. In Ion Formofionfrom Organic Solids; Benninghoven, A., Ed.; Springer Series in Chemical Physics 25; Psringer Verlag: Berlin, 1983. (8) Cotter, R. J.; Van Breeman, R. B.; Snow, M. In?. J . Mass Spectrom. Ion Phys. 1983, 49, 35. (9) Linder, B.; Seydel, V. Anal. Chem. 1985, 57, 895. (IO) (a) Karas, M.; Bachmann, D.; Hillenkarnp, F. Anal. Chem. 1985,57, 2935. (b) Spengler, B.; Karas, M.; Buhr, V.; Hillenkarnp, F. J . Phys. Chem. 1987, 91, 6502. (1 1) Hillenkamp, F.; Karas, M.; Holtkarnp, D.; Kliisener, P. Int. J . Mass Specfrom.Ion Processes 1986, 69, 265.
0022-3654/89/2093-3595$01.50/0
and the sample were varied to study the effects of absorptivity.1° Mono- and dipeptides that are resonant with the desorbing wavelength have lower power density thresholds for desorption, proportional to their absorptivities, and a larger working power density range for desorption at 266 nm. The authors proposed that desorption is induced by a collective lattice disintegration initiated by exciton-phonon coupling. In addition to the amino acids and their derivatives, anthracene was also a subject of studies of resonant desorption.12 Laser desorption experiments were also conducted to distinguish “random protonation reactions” from functional group specific protonation at 266 nm for resonant amino acids.13 Selective deuteration of functional groups and side chains showed that protonation by side chains is not significant even at power densities large enough to strip completely the groups attached to carbon atoms and form C+. Evidence for resonant desorption was also obtained for condensed methanol at IR wavelengths accessible with a C 0 2 1 a ~ e r . l ~ It was found that desorption proceeds only when the laser wavelength coincides with a vibrational resonance and only after energy has randomized in the lattice. The goal of this investigation is the extension of these mechanistic studies to nucleosides. It is worth noting that the chromophore for the nucleosides is either a pyrimidine or purine ring system, a moiety very different from the symmetrical benzene ring incorporated in the resonant amino acids. The structural differences may lead to different chemistry and perhaps different desorption characteristics.
Experimental Section Laser desorption spectra were obtained with a Quanta-Ray DCR-2 Nd:YAG laser and a Fourier transform mass spectrometer constructed in this l a b ~ r a t o r y ’and ~ interfaced to a Nicolet 1000 data system. The magnetic field strength was 1.2 T. The cell was a cubic design of 5.08 cm dimensions. The laser probe entered the analyzer cell through a 6.35-mm hole between the excitation and transmitter plates. The laser beam was admitted to the cell along the opposing diagonal, striking the probe perpendicular to its ~ u r f a c e . ~ The nucleosides employed in this investigation were adenosine, guanosine, thymidine, and uridine. Solutions containing 1 mg/mL of NaCl and an equal weight of nucleoside were prepared in methanol, and they were either electrosprayed, as described p r e v i ~ u s l y , ~to~give J ~ a coverage of approximately 10 pg/cm2 or (12) Antonov, V. S.; Letokhov, V. S.; Shibanov, A. N. Appl. Phys. 1981, 25, 71. (13) Parker, C. D.; Hercules, D. M. Anal. Chem. 1986, 58, 25. (14) Mashni, M.; Hess, P. Chem. Phys. Letf. 1981, 77, 541. (15) Ledford, Jr., E. B.; White, R. L.; Ghaderi, S.; Wilkins, C. L.; Gross, M. L. Anal. Chem. 1980, 52, 2450. (16) McCrery, D. A,; Gross, M. L. Anal. Chim. Acfa 1985, 178, 105.
0 1989 American Chemical Society
3596
The Journal of Physical Chemistry, Vol. 93, No. 9, 1989
pipetted in 10-pL aliquots, yielding an average coverage of 120 pgfcm2. The copper probe was cleaned by etching in nitric acid for the nucleoside study. For 1-naphthoic acid and 2-naphthol studies, the probe was cleaned by facing it off in a lathe, removing a 0.05-mm depth of copper and leaving a bare surface. Following each cleaning, the copper probe was sanded down with 30- and 3-pm polishing paper successively, producing a smooth probe surface. The 2-naphthol (Merck) used in these experiments was sublimed in vacuum and then recrystallized in ethanol. All other reagents were used without further purification. Single shot laser spectra were acquired at wavelengths of 266 and 1064 nm. The spot size was 0.5-0.6 mm in diameter. The pulse length was approximately 5 ns at 266 nm and either 10 ns or 140 ps at 1064 nm. The laser energy and power density used in a particular experiment are specified in the following text. Four spectra (four laser shots) were taken from each probe loading. Mass spectra were acquired for spatially separated nucleosides and NaCl by desorption at 1064 nm in the non-Q switched mode. The probe and strategy employed for desorption in this mode were previously discussed in detaiLs Electron micrographs of 1-naphthoic acid and 2-naphthol separately and in a 4:3 mixture were acquired with a Cambridge Stereoscan S4-10 scanning electron microscope.
Chiarelli and Gross
1 i 265 t
1
w
_1
LJJ, 4 149
150 ,
1QD
...
,
'
I . .
'
MASS
200 I " ' '
IN
'
"
'
!I, j,.I 250
21,
300
A . 1.4, U.
Figure 1. LD/FTMS of thymidine and NaCl electrosprayed on a copper probe taken at a wavelength of 266 nm at energies of (A) 5.0 mJ and (B) 6.5 mJ. The ion of m / z 287 is (M + 2Na - H)+ and of m / z 265 is (M + Na)C.
Results and Discussion This study is focused on the laser desorption characteristics of adenosine, guanosine, thymidine, and uridine (see structures)
150
HO OH
odcnoshr
(MW~Z67l
HO OH
HO H
puonosinr
t hyvnk5i-d
(MW.283)
(MW.2421
200
250
300
Hod HO OH
ulbna lMW=244)
under conditions of excitation at 266 and 1064 nm. Mixtures of 2-naphthol and 1-naphthoic acid were desorbed at 266, 532, and 1064 nm because these compounds have well-known excited-state acidfbase properties. Results from these latter studies should reveal the role of excited-state acid/base chemistry in laser desorption at resonant wavelengths. Desorption from a split probe in which the nucleoside and the cationizing agent (NaC1) are spatially separated was also investigated at 1064 nm to test whether the formation of (M + Na)+ and (M- H + 2 Na)' occurs in the gas phase. The major difference between desorption of guanosine (a purine) and of thymidine and uridine (pyrimidines) is the propensity of the two pyrimidines to form doubly sodiated molecules, (M - H 2Na)+, near the desorption threshold when 266-nm photons are used. For example, the most abundant desorbed ions from thymidine and uridine are the (M - H 2Na)+ ions of m / z 287 and 289, respectively. On the other hand, guanosine yields a (B + Na)+ of m / z 174, whete B is the base (guanine), as the most abundant desorbed ion. Ions of lesser abundance that are desorbed near threshold at 266 nm are (M + Na)+ of m / z 265, 267, and 306 for thymidine, uridine, and guanosine, respectively. The first two nucleosides also desorb to give low abundance (B Na)+ ions of m / z 157 and 159, respectively. Desorption of guanosine gives small abundances of (M - H 2Na)+ and (B - H + 2Na)+ ions of m / z 328 and 196. As the power density is increased at 266 nm, the two pyrimidines desorb to yield dominant (M + Na)+ ions. On the other hand, the (B - H + 2Na)+ fragment overtakes the (B + Na)+
+
+
+
+
(17) McNeal, C. J.; MacFarlane, R. D. Anal. Chem. 1979,51, 2036
Figure 2. LD/FTMS of guanosine and NaCl electrosprayed on a copper probe taken at a wavelength of 266 nm at energies of (A) 2.0 mJ and (B) 4.0 mJ. The ion of m / z 328 is (M 2Na - H)+ and of m / z 306 is (M Na)+. The fragments (B Na)+ and (B - H 2Na)+ are at m / z 174 and 196, respectively.
+
+
+
+
and becomes the most numerous ion desorbed from guanosine at higher photon densities. When the desorption wavelength is changed to 1064 nm, the guanosine desorption characteristics remain almost the same. A major exception is that the (B + Na)+ is always the most abundant ion regardless of laser energy. Near onset, the pyrimidines show the (B Na)+ as most abundant, but as power density is increased by the factor of 2, the (M + Na)+ ions dominate the spectra. Adenosine gives very simple and nearly identical LD spectra at both 266 and 1064 nm. Near onset, both (B Na)+ and (M + Na)+ ions are observed at m f z 156 and m f z 290, respectively. As the power density is increased, the ( M + Na)' ion becomes dominate at both wavelengths (266 and 1064 nm). Negative ion spectra are consistent with those obtained prev i o ~ s l yand , ~ no significant differences can be easily discerned if a wavelength of 266 nm is used instead of 1064 nm. The dipeptide glycyltyrosine was analyzed under resonant desorption conditions to check whether laser desorption with FTMS detection gives comparable results to the previous LAMMA desorption experiment.1° AS the irradiance is increased, the abundances of (M - H + 2Na)+ ions increase with respect to the abundances of (M + Na)+ and (M + H)+ ions. Thus, the trends obtained with this FTMS experiment are consistent with those obtained under LAMMA conditions even though higher irradi-
+
+
IR and UV Laser Desorption of Nucleosides
The Journal of Physical Chemistry, Vol. 93, No. 9, 1989 3597 TABLE I: Laser Desorption of Thymidine, Uridine, and Guanosine Mixed with an Equal Weight of NaCP
(M compound
- H t 2Na)'/ (M
+ Na)'
thymidine uridine
5.0 mJ 1.7 f 0.03 1.5 f 0.02
6.5 mJ 0.19 f 0.03 0.40 f 0.04
guanosine
2.0 mJ 1.0 f 0.1
4.0 mJ 0.81 f 0.08
(B - H
+ 2Na)'/
(B + Na)'
2.0 mJ 0.63 f 0.08
4.0 mJ 1.1 f 0.1
'Power density of (1-5) X lo8 W/cm2. TABLE II: Abundance Ratios of the (M - H)-Ions from 2-Naphthol and 1-NaDhthoic Acid ( m / z 143/m/z 171) desorbed as mixtureb
pmr
wavelength, nm 266 532 1064
desorbed separately' 0.79 f 0.06c 1.1 i 0.1 0.65 f 0.07
pred 1.o 1.4 0.86
obsd 0.11 f 0.02 0.50 f 0.06 0.41 f 0.08
'Equal weights were applied to the probe. bMixture was 4:3 in weight 2-naphthol to 1-naphthoic acid. C N= 8.
ances were used in the LD/FTMS study (Figures 1-3). Of course, it is possible that the mechanism of desorption of nucleosides would be different at the lower irradiances used in ref 10. Role of Intermolecular Hydrogen Transfer. The facile formation of ( M - H 2Na)+ ions for thymidine and uridine near the desorption onset at a wavelength of 266 nm must be dominated by a hydrogen loss mechanism not common to amino acid del One possible mechanism sorption under resonant is hydrogen loss driven by the enhanced acidity of the imide nitrogen in an excited singlet state. Forster cycle calculations indicate that the nucleic acid bases thymine and uracil undergo a reduction in pKa of 6 orders of magnitude from the ground to the SIstate.Is Excited-state acid/base characteristics were also ~ i t e d ' ~as* 'a~possible driving force in the laser desorption of amino acids and other peptide derivatives, but no systematic investigation of this question has been undertaken to date. 2-Naphthol and 1-naphthoic acid were desorbed independently and in admixture at 266, 532, and 1064 nm to test for excited-state acid/base chemistry. These model compounds were chosen because their excited-state acid/base chemistry is well-known.ls 2-Naphthol is typical of phenols; its pKa decreases from 9 to 3 in the first excited singlet state relative to the ground state. 1-Naphthoic acid is a typical aromatic carboxylic acid; its pK, increases slightly from 6 to 7 in the first excited singlet state. The two model compounds should be miscible as solids so that desorption occurs from a homogeneous mixture. This is a reasonable expectation given their similar structures, but no binary phase diagram could be found in the literature to support this. Scanning electron microscopy (SEM) was used to examine the surface morphology of the crystals resulting from the evaporation of the methanol from a mixed solution. SEM was used previously to probe the sample surface prior to laser de~orption.'~Micrographs of 1-naphthoic acid, 2-naphthol, and the 4:3 mixture were acquired. The morphology of the pure compounds is consistent with the crystalline forms,*O and the 4:3 mixture gives crystals unlike either of the two, but which are homogeneous over the surface of the probe. This provides evidence that both compounds
+
(18) Ireland, J. F.; Wyatt, P. A. H. In Advances in Physical Organic Chemistry; Gold, V., Bethell, D., Eds.; Academic Press: London, 1976; Vol. 12. ( 1 9) Schueler, B.; Feigl, P.; Kruger, F.; Hillenkamp, F. Org. Mass Spectrom. 1981, 16, 502. (20) Weast, R. C., Ed. CRC Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 1981. (21) KlBning, V. K.; Goldschrnidt, Ch.R.; Ottolenghi, M.; Stein, G.J . Chem. Phys. 1973, 59, 1753.
are incorporated into the same solid solution after deposition from methanol solvent. The abundance ratios of the (M - H)- ions are used to test for excited-state chemistry (Table 11). A 4:3 mixture of 2-naphthol and 1-naphthoic acid was used for the mixture analysis at the three wavelengths so that an abundance ratio of 1 would be observed for the (M - H)- ions generated at 266 nm if the two compounds desorb independently of each other. A ratio greater than unity is evidence of an excited-state acid/base reaction. A ratio less than 1 is indicative of ground-state acidity, and indeed this is what was observed. Thus, there is no evidence to support the proposal that disodiated ions are formed via the excited state in which the acidity is considerably enhanced. The lifetimes of the excited singlet states are too short to permit this state to play a role in desorption chemistry. Fluorescence studies of 2-naphthol19showed that proton transfer is adiabatic. Therefore, the negative ion generated in the desorbing pair, RCOOH,+/RCOO- would be quenched by proton transfer itself. The solution lifetime of the fluorescent SI state, however, is approximately lo-* s. Even though there are fewer collisions per unit time in the selvedge than in solution, the ion pair NpCOOH2/Np0- (Np = naphthenyl) has insufficient time to separate before the 2-naphtholate anion decays to the ground state. The singlet lifetime of the nucleosides in solution is even shorter than that of 2-naphthol (being on the order of 10-9-10-10 s ) . ~ ~ Role of Intramolecular Hydrogen Transfer. If the hydrogen associated with the pyrimidine ring does not exchange in an intermolecular acid/base reaction, then an intramolecular rearrangement of hydrogen atoms(s) may be the key to formation of disodiated ions near the threshold of desorption at 266 nm. Intramolecular hydrogen transfer often occurs by tautomerization. The pyrimidine ring of thymidine and uridine can exist in a lactam (I) or lactim (11) form (see eq 1). Quantum me-
H
H
I
I1
chanical calculations indicate that the pyrimidine bases favor the lactim (enol) form in the S1state whereas the purine bases exist in the lactam form in both t h e ground and excited states.23 (22) Eisinger, J.; Lamola, A. A. In Excited States of Proteins and Nucleic Acids; Steiner, R. F., Weinryb, I., Eds.; Plenum Press: New York, 1971. (23) Kochetkov, N. K.; Budovskii, E. I., Eds. Organic Chemistry ofthe Nucleic Acids; Plenum Press: London, 1971; Part B.
3598
The Journal of Physical Chemistry, Vol. 93, No. 9, 1989
Chiarelli and Gross
TABLE 111: Results of Test for Gas-Phase Cationization of Thymidine and Sucrose" ion abundance ratios
(M - H
system
thymidine + NaCl on probe, no mesh thymidine on probe, mesh unloaded thymidine on probe, NaCl on mesh sucrose + NaCl on probe, no mesh sucrose on probe, mesh unloaded sucrose on probe, NaCl on mesh "At (1-5)
X lo5 W/cm2, 150
+ 2Na)'
(M
0.67 f 0.17 0.07 f 0.01 0.08 f 0.02 (M
+ Na)+
0.20 f 0.03 ND 0.29 f 0.04
+ Na)+
(B - H
+ 2Na)'
1.0 f 0.27 0.16 f 0.03 0.09 f 0.01
0.18 f 0.02
(F, + N a ) + c
(F, - H,O + Na)+ 0.54 f 0.15
1.0 f 0.17 ND 0.16 f 0.03
(B + Na)+ 0.48 f 0.05 ND
N D ~ ND
no. of determn
ND
12 8 12 14 8 12
ND 0.11 f 0.03
mJ. Separation between probe and mesh was 30 fim. *Not detected. Detection limit = 0.05 at S/N = 2. c F 1 =
C6H1206.
Experimental evidence from luminescence studies of compounds having structures that are locked into those of the two base tautomers indicates that the enol form luminesces whereas the keto form of the nucleic acid base does not. Nl,N2-Dimethyluracil (111) shows no fluorescence at room temperature, only weak fluorescence in the same glass at 77 K, and no phosphorescence at all.24 2,4-Dimethoxypyrimidine (IV) exhibits strong fluorescence at 298 K and phosphorescence in ethylene glycol-water glass at 77 K.
SCHEME I: Mechanism of Formation of M Wavelength of 266 nm"
7 9
bH3 I11
No'
O ,AY
R2
IV
From these observations, it may be concluded that excitation to the SI state is accompanied by structural rearrangement to the lactim tautomer for the pyrimidine nucleosides. Scheme I describes a mechanism by which sodiation of the imide nitrogen may proceed at power densities close to threshold. When a photon is absorbed, tautomerism occurs as the imide hydrogen shifts to one of the carbonyl oxygens. A sodium ion then bonds to the imide nitrogen. After sodiation occurs, the nucleoside relaxes to the ground state and thereby holds the sodium ion more tightly, blocking remigration of the proton to the imide nitrogen. This would then force proton transfer to another molecule. Additional support comes from a study of adenosine, a purine nucleoside with no ring N-H group, which gives no detectable (M - H + 2Na)+ ion upon laser desorption. Because the singlet lifetime is so short, it is possible that the triplet state is responsible for the formation of disodiated molecular ions from thymidine and uridine at 266 nm. The longer lifetime associated with the triplet states would allow for the sodium ion attachment to the imide nitrogen. The lifetimes of the thymidine and uridine triplet in solution at 298 K are 25 and 2 ps, respect i ~ e l y . * ~ These lifetimes parallel the ratios of (M - H 2Na)+/(M + Na)+ obtained for thymidine and uridine near threshold (see Table I). The ratio obtained for thymidine is larger than that obtained for uridine at 5.0 mJ desorption energy. If one considers the number of molecules irradiated (ca. lOI4), the intersystem crossing yield (ca. 0.01 5 for both pyrimidine nucleosides, ref 25), and the probability of photon absorption as the ratio of the absorption cross section to the hard-sphere cross section (calculated from X-ray data to be ca. 0.6),26it can be seen that approximately 1Olo triplets will be generated by the laser pulse. Many will be quenched by collisions and may not desorb. If one considers, however, a simple lifetime distribution, 5% of those excited states will have a lifetime in excess of 3 times the mean half-life of the triplet state, allowing for sufficient time for the chemistry to occur prior to desorption. Furthermore, phos-
+
(24) Longworth, J. W.; Rahn, R. 0.f Shulman, R. G. J . Chem. Phys. 1966, 45, 2930.
(25) Salet, C.; Bensasson, R.; Becker, R. S. Photochem. Photobiol. 1979, 30, 325. (26) Green, E. A.; Rosenstien, R. D.; Shiono, R.; Abraham, D. J.; Trus, B. L.; Marsh, R. E. Acta Crystallogr. Sect. B 1975, 31, 102.
- H + Na at a
, N h 5 R ' -
HoA' RZ
"For thymidine R1 = CH, and R2 = 2'-deoxyribose; for uridine R, = H and R, = ribose.
phorescence of guanosine, thymidine, and uridine in the ethylene glycol-water glass increases markedly as the pH is raised.24 This means that th,: intersystem crossing efficiency is increased when the nucleosides are deprotonated. The more facile desorption of the ( M + Na)+ ions of the pyrimidine nucleosides in the IR or at higher power densities in the UV can be explained in terms of an onset of the shockwave mechanism described earlier.9 It should be noted, however, that previous results indicating shockwave conditions were observed only in cases where the sample supporting substrate had been irradiated from the backside (Le., no direct irradiation could reach the sample). Test for Gas-Phase Cationization. We also tested for gas-phase formation of the mono- and disodiated molecular ions from the nucleosides under thermal desorption conditions (( 1-5) X los W/cm2 at 1064 nm) in the manner described previously for suc r o ~ e . The ~ nucleosides (thymidine, uridine, guanosine, and adenosine) were electrosprayed onto a smooth copper substrate and a NaCl solution was electrosprayed onto a tungsten mesh. The test experiment consisted of mounting the tungsten mesh above the copper probe (30 pm separation of reagents), keeping both substrates in line with the laser beam and then monitoring the abundances of the (M Na)' and (M - H + 2Na)+ ions generated in the laser beam. To test for gas-phase cationization, the abundances of the ions generated in the test experiment were compared to the abundances of (M + Na)+ and (M - H + 2Na)+ generated in a control experiment where no NaCl was deposited on the tungsten mesh before desorption. The results of the nucleoside experiments are the same for all nucleosides tested, and for brevity only those concerning thymidine are given in Table 111. An experimental sequence involving sucrose was executed concurrently to assure the comparability of the results of the nucleoside experiments with those of previous sucrose studies (also see Table III).s The test experiments indicate that more sodiated sucrose at m / z 365 is produced from the split probe than from the single probe when NaCl and sucrose are mixed together under conditions where the desorption energy is reduced from previous experiment (1 50 mJ here versus 225 mJ used in ref 5). The enhanced abundance
+
-he Journal of Physical Chemistry, Vol. 93, NO. 9, 1989 3599
IR and UV Laser Desorption of Nucleosides TABLE IV: Absorptivities of Nucleosides and Naphthalene Derivatives at 266 nm
compound
€a
1.2 x 1.1 x 9.8 x 9.2 x 2.3 x 3.4 x
adenosine guanosine thymidine uridine 1-naphthoic acid 2-naphthol
104 104 103 103 103 103
‘Taken from the Sadtler compilation of standard ultraviolet spectra.
of the m l z 365 ion desorbed from the split probe is consistent with other experimental facts. TOF analysis has demonstrated that neutral desorption dwarfs ion desorption.8 Near desorption onset, fragment ions are always more abundant than molecular ions (compare abundances of ions at m / z 365,203, and 185). In the split probe experiment, the sodium ions most likely to cationize neutral sucrose molecule have little translational energy. Thus, the adducts formed in the split probe experiment have little energy available to induce fragmentation as is supported by the large reduction in the relative abundance of the fragment ions compared to the molecular ions. Comparison of the abundances of the m l z 287 and m l z 265 generated in the control and test experiment shows that no detectable gas-phase formation of the mono- or disodiated thymidine occurs. Furthermore, the abundance of the (M + Na)+ ion desorbed from a mixture of thymidine and NaCl is 2.7 times greater than the abundance of the (M Na)’ ion desorbed from a mixture of sucrose and NaCl. These observations are evidence that condensed-phase cationization is nearly the exclusive pathway for ( M Na)’ formation under IR desorption conditions. Gas-phase cationization of the nucleosides at a wavelength of 266 nm was not tested but it seems unlikely because no sodium ions desorb from the tungsten mesh at 2-4 mJ of energy, sufficient 2Na)+ ions from the to produce ( M Na)’ and ( M - H nucleoside admixed with NaCl on the copper probe alone. Therefore, Na+ ions do not desorb independently of the nucleosides at this wavelength, consistent with the resonant desorption mechanism proposed earlier.I0 Mechanism for Desorption and Cationization. Some additional insight into the mechanism of desorption and cationization of nucleosides may be obtained by considering how they exist in the solid state and the differences in energy deposition between the two wavelengths employed in this study. Nucleosides show a marked tendency to self-associate by ring-stacking whereby one chromophore (base) sits above the other with the ribosyl functions alternating about the stack to minimize repulsi~n.~’This stacking of base units may be interrupted by sodium ions because the nucleic acid portion of the growing part of a stack will be attractive both to Na’ and to another nucleoside. The sodium interruption of the base stack is supported by the fact that no cationized sugar units are formed upon laser d e s ~ r p t i o n ; ~ + ~ a only cationized bases are seen. Moreover, evidence for direct laser desorption of cationized “stacks” of nucleosides was obtained at 483 nm with quadrupole/time-of-flight detectionz8 Desorption of a “freshly applied” (electrosprayed) solution containing an equimolar amount of KI and guanosine showed TOF distributions of ions with masses in the range of 600-1800 amu. The clusters were proposed to be of the form [K (M),]’, where M is guanosine and n is the number of units. Clusters corresponding to
+
+
+
+
+
(27) Broom, A. D.; Schweizer, M. P.; Ts’o, P. 0.P.J . Am. Chem. SOC. 1967, 89, 3612.
(28) Hardin, E. D.; Vestal, M. L. Anal. Chem. 1981, 53, 1492.
+
n = 2 and n = 3 could be clearly resolved, but no ( M K)+ ions were observed.28 Decompositions of these clusters clearly occur on a microsecond time scale and, therefore, would not be seen on the millisecond time scale of FTMS. Further understanding may be gained by contrasting some physical properties of nucleosides with those of sucrose. Vapor pressure osmometry was employed to characterize the nucleoside association described above.27 Sucrose is employed as a standardizing agent in this technique because the change in water vapor pressure retains Raoult’s law ideality over a much wider concentration range of sucrose than of many other compounds. The deviation of vapor pressure from ideality is evidence for the presence of dimers, trimers, etc. in solution. This means that sucrose has less tendency to self-associate compared to other compounds (recall that the most favored stacking arrangement in the solid state is one that minimizes ribosyl repulsion for the nucleosides). Gas-phase cationization of desorbed nucleoside clusters is unlikely because it is the ribosyl functions that present themselves to Na+ whereas the base moiety is buried within. Some interesting contrasts are seen between the nucleosides in this study and the amino acids studied by TOF/MS.’O Simple amino acids and dipeptides have been the only other class of compounds that have been studied in any detail under resonant desorption conditions. The four nucleosides here all have similar absorptivities (Table IV) but do not desorb at the same threshold as would be predicted from the amino acid results. The power density of the laser at 266 nm needed to induce desorption of resonant amino acids and dipeptides is proportional to their So to SI transition probabilities at 266 nm. For stronger transitions, lower energy is needed to induce There are two factors that may cause the differences in threshold behavior. First, in the stacking configuration described above, the nucleoside chromophores are only separated by 3-4 and self-association may change their absorptivities. Second, amino acids are known to exist in a head-to-tail configuration in which they are associated by their carboxyl groups, an association quite different from that of the nucleosides. These two classes of compounds should have different activation energies for desorption. Furthermore, the power densities used in the TOF study are lower than those used here, suggesting that instrumental differences may account for some of the desorption characteristics. Conclusion The most striking contrast in the desorption characteristics of the purine and pyrimidine nucleosides is the tendency of the latter to form doubly sodiated molecular ions near the desorption threshold at a wavelength of 266 nm. This is not accounted for by an increase in acidity in an excited state. Instead, a change in the lactim-lactam equilibrium in the TI state is presented as the driving force for ( M - H + 2Na)+ formation from the pyrimidines. This tautomerism does not occur for purines, and the abundances of ( M + 2Na - H)+ ions are reduced. No evidence of gas-phase formation of the (M + Na)+ or (M - H + 2Na)+ ions from the nucleosides was found at a wavelength of 1064 nm by using a split probe, quite unlike the desorption of sucrose. Instead a cluster desorption mechanism driven by the nucleosides’ self-association is proposed. Acknowledgment. This work was supported by the Midwest Center for Mass Spectrometry (NSF Grant CHE-8620177). We thank Dr. Kit Lee for his assistance in acquiring the scanning electron micrographs. Registry No. Adenosine, 58-61-7; guanosine, 118-00-3; thymidine, 50-89-5; uridine, 58-96-8; 1-naphthoic acid, 86-55-5; 2-naphthol, 135-
19-3.
