1316
Anal. Chem. 1986, 58, 1316-1324
(19) Cunnlngham, A. J.; Payzant, J. D.; Kebarle, P. J . Am. Chem. Soc. 1972, 94, 7627. (20) Lias, S. 0.; Liebman, J. F.; Levin, R. D. J . Phys. Chem. Ref. Data 7984, 13, 695. (21) Yamdagni, R.; Kebarle, P. J . Am. Chem. Soc. 1973, 95, 3504. (22) Barber, M.; Bordoll, R. S.; Elliot, 0. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A.
(23) Sunner, J.; Kebarle, P. J . Phys. Chem. 1981, 8 5 , 327.
RECEIVED for review October 16,1985. Accepted January 14, 1986. The Present work Was supported by a grant-from the Canadian Natural Sciences and Engineering Research Council.
Fast Atom Bombardment and Collisional Activation Mass Spectrometry as Probes for the Identification of Positional Isomers in a Series of Benzylated Guanosines Yves Tondew*' Program Resources, Inc., National Cancer Institute-Frederick
Cancer Research Facility, Frederick, Maryland 21 701
Robert C. Moschel, Anthony Dipple, and Steven R. Koepke Litton Bionetics, Inc., Basic Research Program, National Cancer Institute-Frederick Frederick, Maryland 21 701
The mass spectra of a serles of 1-, N2-, 0'-, 7-, and 8-(p-Ybenzyi)guanosines (where Y = H, CH,, CH,O, Ci, and NO2) were examined in order to ldentlfy spectral characteristics that might dlstinguish these posltlonal isomers. Field desorption and positive chemical ionlzation (Isobutane) spectra were not diagnostic, whlle both positive and negatlve ion fast atom bombardment (FAB) spectra provided sufficient informatlon to differentiate among these isomers. Abundant [M HI+ and [M HI- ions together with extensive fragmentation were obtained by uslng a glycerol-N,N-dimethylformamide (1:l v/v) mlxture as FAB matrlx. Collision-activated dlssociatlon-mass analyzed Ion kinetic energy (CAD-MIKE) spectrometry and kinetic energy release measurements provided addltional informatton for distinguishing members of this series of Isomers. Short- and long-term reproduclbllity of the relative ion abundance was excellent and demonstrated that, of the Ionization technlques examined, FAB was the most useful for isomer dlfferentiatlon.
-
+
Cancer Research Facility,
ionization techniques such as electron impact (8, 9) and chemical ionization (10, 11). However, these ionization methods often require that the sample undergo preliminary chemical derivatization (12) to increase volatility and, therefore, minimize the thermal degradation preceding ionization. Other ionization techniques, such as field desorption (FD) (13,14),desorption chemical ionization (15),thermospray ionization (16), and liquid ionization ( I 7) have been successfully used on nonderivatized nucleotides, nucleosides, and nucleoside adducts. Perhaps the most promising of these new ionization methods is the group of particle-assisted desorption ionization techniques (18)that has been recently developed. Several examples of the application of fast atom bombardment (FAB) (19-27), secondary ion mass spectrometry (28, 29), plasma desorption (30,311, and laser desorption (32) to the study of nucleic acid components have already appeared. The concomitant introduction of high-mass analyzers has even permitted the observation of high molecular weight oligonucleotides and the determination of their base sequence (33,
34). The interaction of chemical carcinogens with cellular nucleic acids is regarded as a critical event in the initiation of the carcinogenic process (1,Z). A challenging aspect of the total characterization of carcinogen-nucleic acid adducts is the determination of the site of substitution of the carcinogen on the multidentate nucleic acid components. If sample size is adequate, these structural details can usually be established through a combination of ultraviolet, nuclear magnetic resonance, and mass spectrometry (3-7). However, the amount of nucleoside adducts commonly isolated from modified polynucleotides is generally very small. Consequently, not all of the above forms of spectrometry are equally efficacious. Because mass spectrometry can provide both molecular weight and additional structural information on minute quantities of material, it is certainly one of the most valuable tools for characterization of isolated adducts. The mass spectrometric properties of nucleotides and nucleosides have been intensively studied using more traditional Present address: Environmental Research Center, University of Nevada, Las Vegas, 4505 South Maryland Parkway, Las Vegas, NV 89154. 0003-2700/86/0358-13 16$01.5010
The purpose of this investigation was to evaluate the potential of some of these modern ionization techniques for mass spectrometric discrimination among various positional isomers in a series of model carcinogen-nucleic acid adducts. The adducts selected were the series of 1-(p-Y-benzy1)guanosines (1-(p-Y-Bzl)Guo),M-(p-Y-benzy1)guanosines (w-(p-Y-Bzl)(p-Y-benzy1)guanosines(06-(p-Y-Bzl)Guo),8-(p-YGuo), 06benzy1)guanosines (8-(p-Y-Bzl)Guo), and 7-(p-Y-benzyl)guanosines (7-(p-Y-Bzl)Guo) where Y = H, CH3, CH30, C1, and NOz as illustrated in Figure 1. Of the variety of techniques examined, fast atom bombardment mass spectrometry (FABMS) provided the most useful structural information for this series of nucleosides.
EXPERIMENTAL SECTION The preparation and characterization of the benzylated guanosines used here have been described elsewhere (35-39). Mass Spectrometry. A reversed-geometry VG Micromass ZAB-2F (VG Analytical, Altrincham, UK) was used for all mass spectrometric measurements. The mass spectrometer was interfaced to a VG 2035 data system for acquisition and processing of the data. The positive chemical ionization (PCI) reagent gas was prepared by using isobutane (Matheson Gas Products, East Rutherford, NJ). The sample was introduced using glass crucibles. 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
loo
1-(p-Y -Bzl)Guo
A
31 3 M+.
N~-(~-Y-~zI)Guo
loo
B
372
[M-HI-
0
1317
CH&aHa-Y-P
ii E ,
7-(p-Y-Bzl)Guo Y
= H, CH,, CH30, CI, NO,
2
lii
Figure 1. Structures of benzylated guanosines.
The emission was 0.5 mA, and the electron energy was 100 eV. FD spectra were obtained from carbon-grown whisker emitters. Samples were loaded by the syringe technique. The optimum emitter heating current was approximately 17 mA. FAB spectra were obtained by using a fast atom beam of Xe.(8 keV, 1-mA plasma discharge current) generated by charge exchange from a modified saddle field neutral beam gun (Ion Tech, Ltd., Middlesex, UK). A mixture of glycerol and NJV-dimethylformamide (DMF) (1:l v/v) (1-2 KL)was used to dissolve the aralkylated guanosine on a stainless-steelprobe target (2 mm wide, 5.5 mm long), which was then introduced into a commercial FAB source. Total time of sample exposure to the atom beam was 9 min. All mass spectrometric analyses were performed with an 8-kV accelerating voltage and a resolution of 1000; scan rates were 1 s decade-’ for PCI and 10 s decade-’ for FD and FAB. A typical spectrum of the solvent used as matrix (i.e., glycerol-N,N-dimethylformamide) was recorded and saved for subsequent “background”subtraction from the FAB spectra. CAD-MIKE spectra were recorded on UV-sensitive oscillographic paper by scanning the electrostatic sector voltage at a rate of 33 ms eV-’; the main beam energy was 8 keV. Helium was used as the collision gas; the ion gauge pressure reading was 2 X lo-‘ torr (50% attenuation of the main beam). Kinetic energy release values were determined by use of described methods (40) and were corrected for main beam width (41); the ion gauge pressure reading for these measurements was 2 X lo4 torr in the second field free region.
RESULTS AND DISCUSSION Three different ionization procedures, i.e., PCI, FD, and positive ion FAB (+ve FAB) and negative ion FAB (-ve FAB), were employed in this investigation. Each of these procedures exhibited widely different utility for obtaining informative mass spectra of the various nucleosides (Figure 1). PCI spectra using isobutane as a reagent gas were recorded for the series 1-,I@, 06-,7-, and 8-(p-H-Bzl)Guo. These spectra were devoid of relevant information on molecular weights for these compounds presumably because the sample heating required by this ionization approach led to decomposition of the thermally labile nucleosides. Often an ion corresponding to the ribose residue ( m / z 133) was observed as the base peak in these spectra and only infrequently was a [B 2H]+ ion (where B represents the aglycone) observed. More useful spectra were obtained with each of the two other ionization techniques. In Figure 2, we illustrate this for a sample of fl-(p-H-Bz1)Guo. The FD spectrum for this nucleoside is shown in Figure 2A where an abundant molecular
+
C
242
lB+ZH]+
+ HI+
50
80
130
170
210
2&
2W mh
-
330
370
410
450
490
530
Figure 2. Mass spectra of N2-benzylguanosine: (A) field desorption spectrum, (B) negative ion FAB spectrum, (C) positive Ion FAB spectrum.
radical ion is apparent at m / z 373 but no fragment ions are observed in this case. With some of the other benzylguanosines a [B HI+peak at m / z 241 was observed. This latter ion was the base peak in the FD spectrum for 7benzylguanosine hydrobromide, but for this derivative no signal corresponding to the intact nucleoside was detected. While the FD spectra for these nucleosides provided more structural information than the PCI spectra, they did not, in our estimation, yield a sufficient number of fragment ions to make differentiation of positional isomers feasible. In contrast, both the -ve FAB spectrum (Figure 2B) and +ve FAB spectrum (Figure2C) for M-(p-H-Bzl)Guoexhibited a substantial number of fragment ions indicating that different fragmentation patterns for different positional isomers might be observable. Consequently, we focused our attention on obtaining a variety of spectra using the FAB technique. In Figure 3A-D we present representative -ve FAB spectra for the remaining N2-substituted derivatives, i.e., W-(p-CH3Bzl)Guo, fl-(p-CH30-Bzl)Guo, W-(p-Cl-Bzl)Guo, and fl(p-N02-Bzl)Guo,respectively. These spectra complement that shown in Figure 2B for fl-(p-H-Bzl)Guo, and they serve to illustrate mass shifts in certain ions brought about by substitution on the benzyl residue. For example, the mass of the
+
ANALYTICAL CHEMISTRY. VOL. 58. NO. 7. JUNE 1986
1318 IW
m
A
IU-HI-
0-6 t r e F A B
I
I
4
10
B
Figure 4. (A) NegaWe ion and (0)posiiii ion FA0 mass spectral data for Oe4p-Y-BzI)Guo.
BJ
IM
170
2,o
no
290
130
370
110
lyi
490
530
",a
Flgure 3. Negative bn FAB mass spectra of N24p-YBzl)Guo: (A) = CH,. (B) Y = CH,O. (C) Y = GI, (0) Y = NO1.
Y
molecular ion minus a proton ([M - HI-) and of the aglycone ([BII resulting from complete loss of the ribose residue or portions of the ribose residue (e.g., [M - H - 901- and [M H- 10413 differ from one another by the mass difference between the respective para substituent on the benzyl group (Figures 2B and 3A-D). The ion [M - H - 901- could also he referred to as [B + 421- (42). Ions not containing a p-Y-Bzl residue (e.g., the ribose residue [SI-, [B - p-Y-Bzl]., and [M - H - p-Y-Bzl + HI-) can be readily located because they appear a t the same m / z in the spectra of all the benzylated guanosines regardless of the structures of the benzyl moiety. The ion labeled [M + G + HIf in Figure 2C and [M + G HI- in Figures 2B and 3A-D is a glycerol adduct of the parent nucleoside. I t is of interest that the -ve FAB spectra of all
the isomeric @-ch1orohenzyl)guanosines also show glycerolchloride ion adducts a t m / r 127, 219, and in some cases at 311. Ions a t 127 and 219 appear in Figure 3C. These ions have been observed in -ve FAB spectra of other chlorinecontaining organic compounds, and they represent complexes of one, two, and three molecules of glycerol per chloride ion, respectively (43,44). In order to facilitate comparison of the +ve and -ve FAB spectra for the 25 substituted guanosines, spectral data are presented in a histogram format where for each positional isomer the relative intensities of the major ions are illustrated. Spectra for the five types of positional isomers are discussed separately. 06-(p-Y-Benzyl)puanosine8. For the 06-substituted guanosines, -ve FAR spectral data are summarized in Figure 4A. As illustrated, the relative intensity of the [M - HI- ion is less than 30% for this group of nucleosides. Dominant ions in the spectra are those resulting from loss of the ribose residue (i.e., [BI-) and the p-Y-Bzl residue accompanied by hydrogen transfer (i.e., [M - H -p-Y-Bzl + H I ) . It is of interest that the relative abundance of the [B]- ion is 100% for 06-@-YBz1)Guowhere Y = H, CH,, and CH,O, but it is less than 50% for Y = C1 and NOt. The +ve FAB spectral data for these O6 derivatives are summarized in Figure 4B. Here the relative intensity of the [M + HI+ ion is 50% or less for all the OBsubstituted compounds studied. It is significant that under these positive ion detection conditions, all the spectra exhibit an abundant [p-Y-Bzl]+ion. This was observed even for those derivatives bearing an electron-withdrawing para substituent (Le., p-C1 and p-NO,). The electron-withdrawing para substituents on the benzyl residue also increase the relative intensity of the [M + H - p-Y-Bzl + HI+ ion, the ion for unsubstituted guanine (i.e., [Gua + HI+), and the ion for the ribose residue (i.e., [S+ 2H]+). In addition to these effects,
ANALYTICAL CHEMISTRY, VOL. 58. NO. 7, JUNE 1986 C-8
N-,
-reFAB
A
-r.FAB
. [pY-BdI+
* 1319
[Y
+ H-pY-Bd+
. HI+
IHt H-1Ml'
[M+Hl+
bn and (E) positive bn FAB mass spectral data
Figure 6. (A) N w t l v e ion and (E) positive ion FAB mass spectral data for I-@-Bzl)Guo.
it should be pointed out that the p-CI and p-NOz substituents lead to very inefficient ionization of the respective 06-substituted nucleoside under either +ve or -ve FAB conditions. Reasons for this are unknown a t present. S-(p-Y-Benzyl)guanosines.The -ve FAB characteristics for the 8-substituted guanosines are presented in Figure 5A. As is illustrated, the majority of the negative ion current (-80%) is produced hy the [M - HI- and [B]. ions for these derivatives. Loss of the respective p-Y-Bzl moiety to produce either the [B - p-Y-Bzl]- or [M - H - p-Y-Bzl + HI- ion is not as significant in the spectra for the 8-substituted guanosines as i t is for the other positional isomers. This is presumably a consequence of the carbon attachment of the benzyl residue in the 8-substituted nucleosides in contrast to heteroatom attachment in the other derivatives. Under +ve FAB conditions (Figure 5B) the major ions produced by the 8-substituted guanosines are [M + HI+, [B + ZH]' and [p-Y-Bzl]+. The relative intensity of the [M HI+ ion is lowest for 8-benzylguanosine and then increases over the series of substituted derivatives in the order CH, < CH,O ^I C1 < NO,. 1-(p-Y-Benzy1)guanosines.Negative and positive FAB spectral data for the 1-substituted guanmines are summarized in parts A and B, respectively, of Figure 6. Under negative ion detection conditions (Figure 6A) the [M - HI- and [B]ions are the most abundant ions. Interestingly, a significant proportion of the total ion current in the spectrum for these isomers is contributed by the [SI- ion. Indeed, the signal for this ion produced by these derivatives is more intense than that produced by any of the other positional isomers. It is significant that the ratio of the relative intensity for the [B -p-Y-Bzll-/[S]- ions is 0.6 (relative standard deviation = 16% for all the 1-substituted derivatives). This is the lowest value observed for this ratio in the spectra of any of the benzylated guanosines.
Under positive FAB conditions (Figure 6B), the [M + HI+, [B + 2H]+, [Gua + HI+, and [p-Y-Bzl]+ions are all significant. The relative intensity of the last ion is lower in the spectrum for 1-(p-chlorobenzy1)-and I-(p-nitrobenzy1)guanosinethan for the other three 1-substituted derivatives. while these same electron-withdrawing para substituents enhance formation of the [Gua + HI+, [B 2H]+, and [M + H]+ ions. N z - ( p-Y-Benzy1)gnanosines. The distribution of ions in the -ve FAB spectra for the N k b s t i t u t e d guanosines (Figure IA) is very similar to that observed for the 1-substituted analogues (Figure 6A). However, for the Nz derivatives, the relative intensity of the [SI- ion is lower than that observed in the spectra for the 1-substituted isomers. The ratio of intensities for the [B - p-Y-Bzl]-/[S]- ions in the Nz series is 4 (relative standard deviation 22% for all NZderivatives studied). The +ve FAB data for the Nz-(p-Y-benzy1)guanosinesare summarized in Figure IB. Major ions observed under these conditions are [M + HI+, [B + 2H]+, and [p-Y-Bzl]+. 7-(p-Y-Benzyl)guanosines.Where Y = H, CH,, CH30, and NOz, the counter ion for the positively charged 74p-Ybenzy1)guanosine was bromide (35,38,39) (Figure 1,I-(p-YBzl)Guo, X = Br-). For these salts the base peak in the -ve FAB spectra was [Brl- accompanied by ions derived from clusters of glycerol with [Brl-. The number of glycerols per bromide Ton ranged from 0 to 4 in these clusters. However, [MXI-. ions for these nucleosides were observable, and the relative intensity for these was in the range of 1.2-2.9%. For the case of I-(p-chlorobenzy1)guanosine ((I-p-CI-Bz1)Guo) (Figure 1)the counterion was 1- (39).No signal corresponding to an [MX]; ion was observed in the -ve FAB spectrum for this nucleoside. Positive ion FAB spectral data for the I-substituted products are presented in Figure 8. For these products, an [M + HI+ ion is fairly intense for I-(p-chlorohenzyl). and
Flgue 5. (A) N-We for 84p-Y-BzI)Guo.
+
+
1320
.
VOL.
ANALYTICAL CHEMISTRY,
N-2
sa, NO.
7, JUNE 1986
FA6
A
twFA0
B
-1.
N-7
treFAB
Flgure 8. Positive ion FAB mass spectral data for 7-(p-Y-Bzl)Guo. N-2
f
E
t
-
5 -
50
e *
1St2HI' Ip-Y-Bzll+
+
,G".+HI' IB 2H!f IM+H-O01+ [M + H-p-Y-Bd HI* I M t H-i041* [MIHI*
+
Figure 7. (A) N~gative ion and (B) positive ion FAB mass spectral data for N~+-Y-BZI)GUO.
Table I. Effects of Sample Size on the Relative Intensity of Selected Ions in the Negative Ion FAB Mass Spectrum of N2-Benzylguanosine relative intensity at the fallowing sample size' rn/z 1 0 p g 1pg 0 . 5 0 p g 0.25pg 0.10pg
ion
372 100 100 100 100 100 282 25.2 29.2 20.2 91.7 40.0 [Bl240 51.4 35.8 b b b [B -p-Y-Bzl]149 20.5 b b b b [SI133 4.3 b b b b "Amounts are per 1 p L of glycerol-DMF (1:l) loaded an the FAB target. bInterferenee from glycerol signals prevented aceurate measurement of the relative intensity.
[M - HI[M - H - 901-
7-(p-nitrobenzyl)guanosine,but where the para substituent Y = H, CH,, and CH,O, the relative intensity of this ion is only 5% or less. Not unexpectedly, the intensity of the liberated [p-Y-Bzl]+ion is 100% for the 7-substituted nucleosides with para substituent Y = H, CH,, and CH,O. The relative intensity of this ion decreases to 76 and 22% for Y = CI and NO2, respectively. In general, the relative abundance of the [M HIf and [p-Y-Bzl]+ ions produced by ring nitrogen-
+
substituted adducts (i.e., 1-and 7-@-Y-henzyl)guanosines) is more sensitive to the electronic character of the attached para Substituent Y than is the relative abundance of the analogous ions produced by exocyclic nitrogen-substituted adducts (Le., W-(p-Y-henzy1)guanosines) (compare Figures 6B, 7B, and 8). Concentration Effects a n d Reproducibility of FAB Mass Spectra of Benzylated Guanosines. In Table I we summarize the effects of decreasing sample quantity on the detectability of various ions in the -ve FAB spectra for W-benzylguanosine. The data indicate that at least 1fig of [M sample is required for reliable detection of the [M - HI-, - H - go]-, and [B]- ions when spectra are measured by conventional magnet scanning techniques. Using sample quantities of 1 fig or more, we determined the short- and long-term reproducibility of the -ve FAB mass spectra for all the nucleosides studied here (Figure 1). Representative data are presented in Table 11. The short-term reproducibilities (represented hy error bars on the histograms of Figure 4-8) are reported in Table 11. These were obtained from seven consecutive scans and were generally within &IO% except for certain ions derived from the poorly ionized Ofi-(p-chlorobenzyl)- and Ofi-(p-nitrobenzyl)guanosine(Figure 4). Longterm spectral reproducibility was assessed by recording in triplicate the -ve FAB m a spectra for the 25 nucleosides after a 3-month interval. As indicated (Table II), the long-term reproducibility of these data is high. The condition of the ion source and the atom gun had little effect on spectral reproducihility. Additionally, although total time of sample exposure to the primary beam was 9 min (the time necessary to record 8-10 full-scan spectra followed by 4 CAD-MIKE spectra), no evidence for significant changes in the appearance of the spectra was noted suggesting that no major modifications of the spectra due to radiation damage to the sample ( 4 5 4 6 ) had occurred. CAD-MIKE Spectra. In Table I11 we present negative ion CAD-MIKE spectral data for the neutral (p-methylhenzy1)guanosines that were ionized using FAB. The ion resulting from glycosidic bond cleavage, Le., [BI-, always constitutes the base daughter ion peak in these spectra. The ratio of intensities for the [B ~p-Y-Bzl]-/[S]-daughterions
Table 11. Comparison of the Short-Term (S) and Long-Term (L) Reproducibility of the Relative Intensity of Selected Ions in the Negative Ion FAB Spectrum of Representative Benzylated Guanosines benzylated guanosine ion [M - HI-
[M - H - 901IBI-
l-@-N02-Bzl)Guo
*
98.7 3.4 (SI 98.8 + 1.2 (L) 5.1 + 0.8 (S) 4.3 1.2 (L) 45.6 5.3 (S) 97.1 I 4.2 (Ll
*
W-(p-CI-Bzl)Guo
06-(p-CH,-Bzl)Guo
100 + 0.0 (S) 100 + 0.0 (L) 4.1 1.6 (S) 4.3 f 0.8 (L)
27.1 f 4.1 (S) 25.6 f 7.1 (L) 3.6 f 0.8 (S) 2.5 1.2 (L) 100 f 0.0 (S) 100 + 0.0 (L)
+
57.2 + 2.2 (S) 64.1 6.4 (Ll
*
*
B-(p-H-Bzl)Guo 100 + 0.0 (S) 100 0.0 (L) 8.6 f 1.7 (S)
*
8.6 + 0.3 (L) 64.0 f 5.7 (SI 68.7 + 10.4 (Ll
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table 111. Relative Abundance of Ions in the Negative Ion FAB CAD-MIKE Mass Spectrum from [M ( p-Methylbenzyl)guanosinesG
1321
- HI-( m / z 386) for
(p-methylbenzy1)guanosine ion
mlz
l-(p-CH3-Bzl)Guo
[M - H - HzO][M - H - 911[M - H - 1051[M - H - 1201-
368 295 281 266 254 238 163 149 133
0.93 f 0.06 5.57 f 0.19 1.75 f 0.03 1.28 f 0.07 82.03 f 0.32 2.01 f 0.03 0.32 f 0.05 1.62 f 0.08 1.49 f 0.11
[BI-
[M - H - 1491[B - p-Y-C,H,][B - p-Y-Bzl][SI-
W - ( ~ - C H ~ - B Z ~ ) GO8-(p-CH3-Bzl)Guo UO 0.90 f 0.04 7.99 f 0.05 3.00 f 0.07 1.63 f 0.06 75.98 f 0.10 1.67 f 0.14 0.65 f 0.02 5.39 f 0.03 0.43 f 0.02
8-(p-CHg-Bzl)Guo
b 1.52 f 0.28 1.04 f 0.61 b 88.21 f 1.84 1.83 f 0.70 0.70 f 0.40 6.05 f 0.63 0.65 f 0.34
0.91 f 0.07 4.07 f 0.15 1.64 f 0.06 1.71 f 0.17 74.16 f 0.16 7.31 f 0.12 0.61 f 0.05 0.22 f 0.01 b
Not detected.
aData reported are in % f SD from three consecutive scans.
Table IV. Relative Abundance of Ions in the Positive Ion FAB CAD-MIKE Mass Spectrum from [M ( p-Methylbenzyl)guanosinesG
+ HI+( m / z 388) for
(p-methylbenzy1)guanosine ion
m/z
1@-CH,-Bzl)Guo
[M + H - HZO]' [M + H - 911' [M + H - 1051'
370 297 283 256 240 165 151 135 105
0.64 f 0.06 2.84 f 0.10 1.85 f 0.09 64.49 f 0.69 1.82 f 0.07 1.12 f 0.10 8.18 f 0.32 1.69 f 0.09 14.13 f 0.27
[B
+ 2H]'
[M + H - 1491' [ 1651'
[Gua]'
[S t 2H]' [p-Y-Bzl]'
w-
06-
8-
(p-CH,-Bzl)Guo
(p-CH3-Bzl)Guo
(p-CH3-Bzl)Guo
7(p-CH3-Bzl)GuO
0.55 f 0.08 3.65 f 0.11 1.58 f 0.05 68.44 f 0.88 2.12 f 0.13 2.24 f 0.07 2.74 f 0.10 1.83 f 0.19 13.38 f 0.31
0.43 f 0.03 2.20 f 0.09 3.00 f 0.03 59.49 f 1.38 1.46 f 0.03 1.03 f 0.03 12.06 f 0.46 2.53 f 0.11 15.03 f 0.74
0.88 f 0.10 3.00 f 0.07 1.39 f 0.04 79.59 f 0.39 4.07 f 0.08 3.32 f 0.12 0.78 f 0.04 0.69 f 0.04 2.44 f 0.07
3.09 f 0.86 3.67 f 0.36 6.08 f 0.13 44.14 f 3.77 2.18 f 0.77 2.75 f 0.27 7.04 f 1.01 1.83 f 0.18 24.90 f 0.47
Data reported are in % f SD from three consecutive scans. Table V. Relative Abundance of Ions in the Negative Ion FAB CAD-MIKE Mass Spectrum from [M ( p-Nitrobenzyl)guanosinesa
ion
mlz
l-@-NOp-Bzl)Guo
[M - H - HzO][M - H - 911[M - H - 1051[M - H - 1201-
399 326 312 296 285 268 163 149 133
4.36 f 0.30 7.18 f 0.39 2.44 f 0.09 0.92 f 0.09 63.96 f 0.92 2.33 f 0.27 0.20 f 0.07 0.90 f 0.06 0.77 h 0.09
PI[M - H - 1491[B - p-Y-C&][B - p-Y-Bzl][SIa Data
reported are in % f SD from three consecutive scans.
- HI-( m / z 417) for
(p-nitrobenzy1)guanosine W-(p-NOz-Bzl)Guo 06-(p-N0,-Bzl)Guob 4.26 f 0.71 8.75 f 1.09 3.54 f 0.58 1.74 f 0.53 60.68 f 1.34 4.21 f 1.03 0.53 f 0.05 2.80 f 0.10 0.53 f 0.05 Not recorded.
8-(p-NOz-Bzl)Guo 6.90 f 0.57 6.15 f 1.23 1.45 f 0.85 2.34 f 0.02 66.51 f 2.59 8.28 f 0.40 1.19 f 0.22 C
c
Not detected.
Table VI. Relative Abundance of Ions in the Positive Ion FAB CAD-MIKE Mass Spectrum from [M ( p -Nitrobenzyl)guanosines"
+ HI'
( m / z 419) for
(p-nitrobenzy1)guanosine
mlz
1(p-NOz-Bzl)Guo
(p-NOz-Bzl)Guo
[M + H - HzO]' [M + H - 911' [M + H - 1051'
401 328 314 286 270 165 151 135 136
1.37 f 0.62 4.55 f 0.47 1.51 f 0.47 68.12 f 0.12 3.46 f 0.25 1.40 i~ 0.27 3.90 f 0.56 4.94 h 0.45
1.82 f 0.08 4.02 f 0.16 1.82 f 0.21 70.35 f 0.84 4.49 f 0.33 1.37 f 0.24 2.46 f 0.33 3.94 f 0.16
[B + 2H]' [M+ H - 1491' [165]' [Gua] [S + 2H]' [p-Y-Bzl]+ +
a
w-
ion
C
06-
(p-NOz-Bzl)Guob
8-
7(p-NOz-Bzl)Guo
3.98 f 0.27 2.88 f 0.38
1.82 f 0.12 4.21 f 0.74 2.30 f 0.59 62.71 f 0.67 3.34 f 0.50 2.12 f 0.25 6.23 f 0.80 3.18 f 0.28
(p-NO2-Bzl)Guo
c
64.96 f 2.96 11.64 f 1.02 2.51 h 0.29 C
c
C
Data reported are in % h SD from three consecutive scans. *Not recorded.
is near 1 for the 1-substituted guanosine, while this ratio is considerablygreater than 1for the N2-,8-,and 06-substituted isomers. Positive ion FAB CAD-MIKE spectral data for the (p-methylbenzy1)guanosinesappear in Table IV. Under these
Not detected.
conditions the [B + 2H]+ daughter ion is the base peak. The [p-Y-Bzl]' ion is also fairly intense in the spectrum for the 1-, N2-,06-, and 7-substituted isomers, but it is of considerably lower intensity in the spectrum of 8-(p-methylbenzyl)-
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table VII. Relative Abundance of Ions i n t h e Negative Ion FAB CAD-MIKE Mass Spectrum from [M
- HI- ( m / z 406) for
(p-Chlorobenzyl)guanosinesa
ion
mlz
l-(p-Cl-Bzl)GuO
[M - H - HzO][M - H - 911[M - H - 1051[M - H - 1201-
388 315 301 287 274 256 163 149 133
0.87 f 0.07 6.02 f 0.49 1.92 f 0.21 1.09 f 0.09 80.03 f 1.92 0.43 f 0.08 0.38 f 0.05 1.14 f 0.09 1.49 f 0.06
PI-
[M - H - 1491[B - p-Y-C,H,][B - p-Y-Bzl][SI-
(p-chlorobenzy1)guanosine W-(p-Cl-Bzl)GuO O6-(p-C1-Bzl)GuOb
8-(p-Cl-B~l)Guo
1.21 f 0.06 8.44 f 0.22 3.46 f 0.12 1.12 f 0.05 71.86 f 0.76 0.78 f 0.06 0.60 f 0.12 2.13 f 0.11 0.19 f 0.03
Data reported are in % f SD from three consecutive scans. bNot recorded.
1.02 f 0.09 4.44 f 0.17 2.08 f 0.08 1.62 f 0.05 72.07 f 0.27 6.50 i 0.46 0.36 f 0.01 0.48 f 0.05 C
Not detected.
Table VIII. Relative Abundance of Ions i n t h e Positive Ion FAB CAD-MIKE Mass Spectrum from [M (p-Chlorobenzyl)guanosines4
ion
mlz
[M + H - HZO]' [M + H - 911' [M + H - 1051'
390 317 303 276 258 165 151 135 125
[B + 2H]+ [M + H - 1491' [165]+ [Gua]' [S + 2H]' [p-Y-Bzl]+
+ HI'
(p-chlorobenzy1)guanosine 1-(p-C1-Bz1)Guo W-(p-C1-Bz1)Guo 06-(p-C1-Bzl)Guo 8-(p-Cl-Bzl)Guo 0.58 f 0.13 2.93 f 0.41 1.27 f 0.13 65.18 f 0.97 1.37 f 0.09 1.38 f 0.14 1.92 f 0.21 2.01 f 0.15 20.12 f 1.06
0.56 f 0.08 3.31 f 0.14 1.19 f 0.33 71.92 f 1.92 1.20 f 0.09 1.47 f 0.20 1.94 f 0.03 1.85 f 0.12 13.38 f 1.86
1.45 f 0.66 3.02 f 0.32 2.40 f 0.10 66.43 f 1.32 1.70 f 0.25 1.23 f 0.17 3.02 f 0.68 1.91 f 0.38 15.98 f 1.81
1.17 f 0.49 3.24 f 0.18 1.33 f 0.06 78.10 f 1.43 3.82 f 0.39 2.35 f 0.19 1.17 f 0.21 b 5.00 f 0.29
( m / z 408) for
7-(p-Cl-Bzl)Guo 0.83 f 0.01 3.88 f 0.45 2.22 f 0.96 69.25 f 0.39 1.38 f 0.48 1.38 & 0.48 3.05 f 0.99 b 15.12 f 0.90
aData reported are in '70 f SD from three consecutive scans. bNot detected. Table IX. Relative Abundance of Ions in the Negative Ion FAB CAD-MIKE Mass Spectrum from [M Benzylguanosinesa
ion
[M - H [M - H [M - H [M - H
- HzO]- 911- 1051- 1201-
PI-
[M - H - 1491[B - P-Y-C~HJ [B - p-Y-Bzl]-
[SI-
mlz
l-(p-H-Bzl)Guo
354 281 268 252 240 225 163 149 133
0.90 f 0.03 7.87 f 0.60 2.31 f 0.36 1.10 f 0.08 82.51 f 0.81 0.53 f 0.03 0.38 f 0.10 1.52 f 0.08 1.85 f 0.08
- HI- ( m / z 372) for
benzylguanosine W-(p-H-Bzl)Guo O6-(p-H-Bzl)Gu0 1.05 f 0.03 9.71 f 0.10 3.72 f 0.11 1.52 & 0.12 75.62 f 0.10 0.93 f 0.09 0.82 f 0.11 4.54 f 0.12 0.70 f 0.02
~-(~-H-Bz~)Guo
0.29 f 0.12 2.95 f 0.17 0.37 f 0.12
1.01 & 0.13 5.54 & 0.29 1.89 f 0.26 1.72 f 0.10 76.39 f 0.61 6.03 f 0.33 0.75 f 0.09 0.35 f 0.03 b
b 91.38 f 0.23 0.51 f 0.12 0.37 f 0.13 3.76 f 0.33 0.59 f 0.13
Data reported are in % f SD from three consecutive scans. bNot detected. Table X. Relative Abundance of Ions in the Positive Ion FAB CAD-MIKE Mass Spectrum from [M Benzylguanosines" ion
mlz
[M + H - H,O]+
356 283 269 242 224 165 151 135 91
[M + H - 911' [M + H - 1051" [B + 2H]' [M + H - 1491' [165]+ [Gua]' [S + 2H]+ [p-Y-Bzl]' a
+ H]+ ( m / z374) for
benzylguanosine 1-(p-H-Bz1)Guo P-(p-H-Bzl)Guo 06-(p-H-Bzl)Guo %(p-H-Bzl)Guo 0.89 f 0.09 3.56 f 0.14 0.25 f 0.02 73.62 f 2.54 1.65 f 0.17 1.19 f 0.07 1.86 f 0.13 1.32 f 0.41 10.65 f 2.05
0.60 f 0.14 3.54 f 0.19 0.26 f 0.07 72.22 f 0.94 1.02 f 0.11 1.73 f 0.16 1.43 f 0.10 1.81 & 0.04 10.54 f 0.22
Data reported are in % f SD from three consecutive scans.
0.53 f 0.01 3.62 f 0.08 0.46 f 0.05 66.67 f 0.52 1.49 f 0.03 1.14 f 0.14 3.54 f 0.08 2.48 f 0.09 13.33 f 0.12
0.91 f 0.09 3.33 f 0.04 0.19 f 0.01 80.47 f 0.21 4.15 f 0.15 2.20 f 0.05 0.54 f 0.03 b 2.23 f 0.03
7-(p-H-Bzl)Guo 4.40 f 0.68 8.65 f 0.11 1.47 f 0.25 33.75 f 0.69 2.53 f 0.20 2.80 f 0.38 3.73 f 0.23 2.39 f 0.38 30.10 f 0.98
Not detected.
guanosine. These characteristics of the -ve and +ve FAB CAD-MIKE spectra for the (p-methylbenzy1)guanosinesare common to the CAD-MIKE spectra of the other (p-Ybenzyl)guanosines, and these additional data are presented in Tables V-XII.
Kinetic Energy Release ( T I j z Measurements. ) Tllz values accompanying the metastable glycosidic cleavage [M - H 1 - 2 [B]- in the second field free region were determined for the neutral benzylguanosines. The Tllzvalues for 1-and W-benzylguanosine were 37.1 and 37.7 f 3.4 meV ( n = 31,
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
1323
Table XI. Relative Abundance of Ions in the Negative Ion FAB CAD-MIKE Mass Spectrum from [M - HI-( m / z 402) for ( p-Methoxybenzyl)guanosinesa (p-methoxybenzy1)guanosine ion
[M - H - HzO][M - H - 911[M - H - 1051[M - H - 1201[BI[M - H - 1491[B - P-Y-CBH~I[B - p-Y-Bzl][SI-
Data reported are in %
f
mlz
l-(p-CH,O-Bzl)Guo
384 311 297 282 270 254 163 149 133
5.56 f 0.18 4.77 f 0.31 1.60 f 0.12 1.08 f 0.07 74.57 f 0.31 2.42 f 0.26 0.29 f 0.06 1.71 f 0.11 1.21 f 0.06
W-(~-CH~O-BZ~)GUO Oe-(p-CH30-Bzl)Guo ~-(~-CH~O-BZ~)GUO 9.17 f 0.54 5.69 f 0.12 2.26 f 0.19 1.68 f 0.12 64.55 f 1.07 6.40 f 0.11 0.84 f 0.08 3.40 f 0.05 0.44 f 0.08
1.30 1.43 0.59 1.32 88.97 1.24 0.21 2.54 0.21
1-
[M + H - H,O]+ [M + H - 911' [M + H - 1051' [B + 2H]+ [M + H - 1491' 11651'
[Gua]+ [S 2H]+ [p-Y-Bzl]'
+
m/z 386 313 299 272 256 165 151 135 121
b
SD from three consecutive scans. *Not detected.
Table XII. Relative Abundance of Ions in the Positive Ion FAB CAD-MIKE Mass Spectrum from [M ( p-Methoxybenzyl)guanosinesa
ion
3.66 f 0.38 4.23 f 0.10 2.27 f 0.43 1.70 f 0.01 68.11 f 0.38 8.51 f 0.19 0.52 f 0.09 0.47 f 0.01
f 0.35 f 0.18 f 0.19 f 0.25 f 0.85 f 0.15 f 0.05 f 0.33 f 0.05
w-
(p-methoxybenzy1)guanosine . OB-
(p-CH30-Bzl)Guo (p-CHgO-Bzl)Guo (p-CH3O-Bzl)Guo 0.50 f 0.12 2.28 f 0.27 1.28 f 0.03 56.15 f 0.23 2.56 f 0.22 1.42 f 0.11 2.13 f 0.04 1.56 f 0.09 26.23 f 0.13
0.84 f 0.16 2.67 f 0.30' 1.09 f 0.14 60.93 f 0.72 1.67 f 0.12 5.34 f 0.22 1.75 f 0.03 1.67 f 0.15 18.03 f 0.21
0.58 f 0.22 1.89 f 0.18 1.46 f 0.24 50.04 0.81 1.76 k 0.12 2.48 f 0.11 3.51 f 0.23 1.90 f 0.25 30.31 f 0.35
+ H]+( m / z 404) for
8-
(p-CH,O-Bzl)Guo 0.84 2.64 1.28 72.49 5.84 4.81 0.60 0.60 4.73
f 0.09 f 0.11 f 0.24 f 0.55 f 0.18 f 0.21 f 0.03 f 0.14 f 0.09
7-
(p-CH30-Bzl)G~o 0.78 f 0.04 2.62 f 0.37 1.58 f 0.08 47.79 f 0.72 2.34 f 0.12 2.09 f 0.36 4.73 f 0.24 1.05 f 0.44 29.67 f 0.29
Data reoorted are in % f SD from three consecutive scans.
respectively. The Tl,2 value for 8-benzylguanosine was 34.4 meV. Significantly, the Tl/2 for Oe-benzylguanosinewas 18.9 f 1.2 meV (n = 3). These data suggest that kinetic energy release measurements can also provide information for assigning structures in the series of aralkylated guanosines. Of the variety of ionization techniques employed in this study, FAB, notably -ve FAB, provided the most structural information for this series of substituted guanosines. Spectra were diagnostic for each of the 25 related compounds such that for one para substituent on the benzyl group, the spectra for the corresponding set of four neutral nucleosides with the same molecular weight were distinguishable. However, the spectra for the nucleosides described here are dependent on both the site of substitution on the base and the nature of the attached para substituent so that it is difficult to identify specific spectral characteristics which are always associated with a particular site of substitution. Nevertheless, some fairly general features are observable, and these could be helpful in structure determinations for unknown aralkyl guanosines. The +ve FAB spectra (Figure 8) for the 7-substituted guanosines show no obvious characteristics that would distinguish these products from many of the other substituted guanosines, but fortunately, the chromatographic properties of these derivatives are quite different from the neutral isomers examined herein (37-39). The 06-substituted derivatives, on the other hand, have somewhat distinctive spectra in that the relative intensities of the [M - HI- ions are always 30% or less. The 1-and 8-(p-Me-Bzl)Guoare the only other products for which this ion is substantially less than 100% relative intensity. Furthermore, the intensities of the [p-Y-Bzl]+ion and the [M - H - p-Y-Bzl + HI- ion are all close to 100% for the 06-substituted compounds. This is not the case for most of the other products, and these aspects of the spectra for the O6 derivatives allow us to think that these +ve and -ve FAB spectral characteristics should be fairly diagnostic for 06aralkylated guanosines.
Discrimination among the other three neutral substituted guanosines is not immediatelyobvious. Some differences that may be suggestive of structure can be discerned, however. For example, the ratio of intensities for the [B - p-Y-Bzl]-/[S]ions is less than 1for all the 1-substituted compounds, while this ratio is greater than or equal to 1 for all the 8- and W-substituted derivatives. A related characteristic of the spectra for the 1-substituted compounds is that the relative intensity of the [SI- ion is in the 20-40% range, while it tends to be less intense in the spectra for the 8- and N2-substituted guanosines. Similarly, in the negative ion FAB CAD-MIKE spectra, the intensity of the [SI- ion from the 1-substituted guanosines is greater than that from the 8- and N2-substituted isomers. Somewhat surprisingly, the spectra for the 8- and " h u b stituted guanosines do not show obvious clear-cut differences distinguishingthese two sites of substitution. However, it can be seen that the [p-Y-Bzl]+ion is of high relative intensity in the +ve FAB and +ve CAD-MIKE spectra for most of the N2 derivatives, while it is of lower intensity for most of the 8-substituted compounds. It is conceivable, therefore, that for an unknown 8- or N2-substitutedguanosine, the intensity for this [p-Y-Bzl]' ion might be suggestive of structure but would not be definitive by itself. In conclusion, we have demonstrated that FABMS of modified guanosines provides much useful structural information. This ionization method can distinguish five sets of five isomers and to some extent has allowed us to develop generalizations about the FAB spectra associated with specific sites of substitution on guanosine. Collisional activation spectrometry and the measurement of kinetic energy release can provide complementary information., Clearly, the data presented herein relate to a series of closely related benzylated guanosines,and it remains to be seen if +ve and -ve ion FAB mass spectra together with CAD-MIKE spectra will be equally valuable in identifying other modified guanine nucleosides.
1324
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Until more data are available for comparison, it would certainly be ill-advised to rely exclusively on the generalizations put forth here to deduce the structure of an unknown sample. Nevertheless, our observations should provide a useful data base for future comparisons and can be used in testing the reliability of conclusions based on other chemical or instrumental methods for structural characterization.
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