From the definition of K ( 1 ) :
K,
Log K j b= Pob X Log K j a -I- VjoMab where obviously:
= Y,”/Y,’
where y”, and y’, are the activity coefficients of a solute in each of two phases. Then for regular solutions of molecules of comparable dimension, the activity coefficient of solute j in the solvent p is:
where 6, is the solubility parameter function, but the volume fraction of the solute p, is sufficiently small to be neglected under normal analytical chromatographic conditions and therefore combining Equations 6 and 7 leads directly to:
Notice, Pub and Mub are dependent only on solvent compositions. Also Equation 2 and Equation 12 are identical provided the molar volume (V,O) remains constant. Such is certainly the case for the series of steroids studied and gives a primary condition to satisfy when another “family” of compounds is considered. In consequence, Equation 2 is of proper validity and thus may be applied in the general sense.
CONCLUSION Although interfacial tension is sufficient to characterize the polarity of liquid systems, it is not explicit in the equation for calculating selectivity. The variation of the partition coefficient of a solute with the composition of the liquid-liquid system can be predicted accurately enough to enable optimization of analytical conditions. If more accurate results are needed, it is better to use the Huber factorial analysis method ( 4 ) which requires more detailed analytical data than necessary for this approximative model.
I f a characterizes a certain solvent system, let:
and:
ACKNOWLEDGMENT Then Equation 8 may be transformed:
In K,“ = ZV,’6,f(a) + V,’’d(a)
(11)
Similarly, accepting that In K I b for solvent system b maintains, it can be seen that:
We are very grateful to C. E. Roland Jones for fruitful discussions.
RECEIVEDfor review November 12, 1973. Accepted February 26,1974.
Structure-Retention Relationships in the Gas Chromatography of Nucleosides S. E. Hattox and James A. McCloskey‘ lnstitute for Lipid Research and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025
Structure-retention time correlations have been studied for 32 nucleoside trimethylsilyl derivatives. Methylene unit (MU) values were measured with a precision of f 0 . 0 2 MU covering the range from 23 to >38 MU. Lower silyl derivatives [e.g., (TMS)3 vs. (TMS)4] exhibited longer retention times on OV-17 while the reverse was true using less polar SE-30 liquid phase. Higher MU values resulted from bet. . eroatom methylatlon ( e.g., 2’-O-methyl), or by replacement of 0 by N or by s.plots of MU values from ov-17 vs. ~ ~ - 3 yielded linear correlations having approximately the same slope but different intercepts for different groupsof structurally Similar compounds (e.g., purine pyrimidine nudeom sides). Perdeuteration Of the nucleoside skeletons Of the four major ribonucleosides decreased retention times by 0.01-0.08 MU, a change - which is insufficient to interfere with nucleoside characterization.
Author t o whom inquiries should be addressed. 1378
ANALYTICAL CHEMISTRY, VOL.
Mainly because of their high polarity, nucleosides as a class represent one of the more difficult applications of gas chromatography. The earliest report is that of Miles and Fales ( I ) , who employed acetylation, methylation and 0isopropylidine formation for enhancement of volatility. As first demonstrated by Hancock ( 2 ) ,trimethylsilylation has subsequently found greatest use for nucleosides (3-18). Ex(1)H. T. Miles and H. M. Fales, Anal. Chem., 34,860(1962). 0(2) R. L. Hancock and D.L. Coleman, Anal. Biochem., I O , 365 (1965). (3) . . Y. Mizuno, N. Ikekawa, T. itoh, and K . Saito, J. erg. Chem., 30, 4066 ( 1965). (4)R. L. Hancock, J. Gas Chromatogr., 4, 363 (1966). (5)y. Sasaki and T. Hashizume, Anal. Biochem., 16, 1 (1966). (6)C. W. Gehrke, D. L. Stalling, and C. D. Ruyle. Biochem. Biophys. Res. Commun., 28,869 (1967). (7) M. Jacobson, J. F. O’Brien, and C. Hedgcoth, Anal. Biochem., 25, 363 11968) I
(8)B. H. Most, J. C. Williams, and K. J. Parker, J. Chromatogr. Scb, 38, 136 (1968). (9)R. L. Hancock. J. Gas ChrQmatogr.. 6, 431 (1968). (10)C. W. Gehrke and C. D. Ruyie, J. Chromatogr., 38,473 (1968). (11)R. L. Hancock. J. Cbromatogr. Scl.,7, 366(1969).
46, NO. 11, SEPTEMBER 1974
2’-0-methylcytidine was isolated by thin layer chromatography [ethyl acetate:l-propano1:water (8:4:2)] from 400 pg of 2’-O-methylcytidylyl-3’,5’-cytidine(Sigma Chemical Co.), after hydrolysis by snake venom phosphodiesterase for 16 hours a t pH 8.6 and 37”. Uridine-5,6,1’,2’,3’,4’,5’,5’dg, cytidine -5,6,1’,2’,3’,4’,5’,5’-ds, adenosine-2,8,1’,2’,3’,4’,5’,5’-ds, and guanosine-I’,2’,3’,4’,5’,5’-ds were isolated from deuterated E. coli, strain B, mid-log harvest, which was grown on special order by Merck, Sharp & Dohme of Canada, Ltd., Montreal, Canada. From 500 mg of lyophilized cells, RNA and DNA were isolated by phenol extraction, following closely the procedure of Caprioli and Rittenberg (25). The mixed precipitate was incubated with deoxyribonuclease I (pH 7.2, 1 hour, 37’), to remove DNA. The hydrolysate was extracted with buffersaturated (pH 7.2) phenol and centrifuged (15,000 X g, 20 min, 0’). The aqueous layer was removed, the phenol layer was reextracted, and the aqueous layers were combined. RNA was precipitated by addition of 0.1 volume of 20% potassium acetate (pH 5.0) followed by three volumes of cold 95% ethanol. After cooling a t -20’ overnight, the RNA was centrifuged (15,000 X g, 0’) for 20 minutes and then washed with cold 70% ethanol. The RNA was then desalted by dialysis against deionized water for 24 hours and lyophilized to yield 7.5 mg of labeled material. Five milligrams were hydrolyzed to nucleosides by incubation with snake venom phosphodiesterase and E. coli alkaline phosphatase (26, 27). Individual nucleosides were separated using Dowex l - X 8 resin following a procedure similar to that outlined by Hori (28), using in the present case a linear ammonium formate gradient (0.1-3.OM) for elution. The buffer was removed from each fraction by vacuum sublimation; final desalting was effected by elution with water from a 0.9X 100-cm Bio-Gel P-2 column (Bio-Rad Laboratories, Richmond, Calif.). Yields as measured by UV absorbance a t 254 nm were: uridine-&, 0.67 mg; cytidine-& (with traces of pseudouridine-dv), 0.86 mg; adenosine-&, 1.8 mg; guanosine-&, 1.4 mg. Preparation of Trimethylsilyl Derivatives. After drying over P205, 50-100 pg of nucleoside was heated with 100 p1 of N,O-bis(trimethylsily1)trifluoroacetamide and 1.5 p l of trimethylchlorosilane a t 80” for 1 hour in a glass screw-capped vial with a Teflonlined cap. In most cases, heating was continued for an additional hour a t 130” to ensure complete derivatization, but was generally EXPERIMENTAL found unnecessary. No further systematic study of reaction conditions was made. Materials. The following nucleosides were purchased from comMeasurement of Methylene Unit Values. MU values were demercial sources. Sigma Chemical Co., St. Louis, Mo.: adenosine, 1termined relative to even-numbered normal chain C22-Cs6 hydromethyladenosine, P-methyladenosine, W,N6-dimethyladenosine, carbons as internal standards (23). Initial column temperature dihydrouridine, guanosine, W-methylguanosine, hrz,W-dimethylprior to temperature programming was set 20’ lower than the eluguanosine, 8-bromoguanosine, 7-methylinosine, and 9-P-D-ribotion temperature of the most volatile standard. The resulting califuranosylpurine. Calbiochem, Los Angeles, Calif.: uridine, a-pseubration curve was slightly nonlinear, as shown in Figure 1. Nucleodouridine, pseudouridine, 1-P-D-ribofuranosylthymine,cytidine, side MU values were measured by (nonlinear) graphic interpolaand inosine. Cyclo Chemical, Los Angeles, Calif.: 5-methylcytidine, tion. Replicate measurements were made to ensure that MU values 1-methylguanosine, and 7-methylguanosine. Terra-Marine Bioresearch, La Jolla, Calif.: cu-uridine, 1-0-D-arabinofuranosyluracil, could be reproduced within f0.02 MU unit. 2,2’-anhydro-l-(a-D-ribofuranosyl)uracil, 2,2’-anhydro-l-(P-D-ara- Instrumentation. Gas chromatography was carried out using a Barber-Colman 5000 instrument, with hydrogen flame ionization binofuranosy1)uracil. The following samples were obtained from and a Keithley 417 electrometer for detection. Silanized glass Uthe sources indicated. 4’-Thioadenosine and N6-(3-methyl-2-butube columns were employed, packed with acid-washed and silanteny1)adenosine: Cancer Chemotherapy National Service Center of ized 100-120 mesh Gas Chrom P (Applied Science Laboratories, the National Institutes of Health, Bethesda, Md.; 2-methyladenoInc.) coated with 1%OV-17 or 1%SE-30. Separations were carried sine: A. Yamazaki, Ajinomoto Co., Kawasaki, Japan; N6-(3out with Nz carrier flow rates of 167 ml/min (OV-17) or 68 ml/min methyl-2-buteny1)- 2-methylthioadenosine:’ N. J. Leonard, Univer(SE-30), temperature programmed a t 2”/min. sity of Illinois, Urbana, Ill.; 2’-O-methyluridine, M. Honjo, Takeda Mass spectra were recorded using an LKB 9000 instrument, Chemical Co., Osaka, Japan; 4-thiouridine, T. Hashizume, Kyoto with sample introduction through the gas chromatographic inlet University, Kyoto, Japan. 2’-0-Methyladenosine was previously using columns similar to those described above. Ion source and prepared in this laboratory (24). carrier gas separator temperatures were 250°, and the ionizing energy was 70 eV. (12) D. F. Babcock and R. 0. Morris, Biochemistry, 9, 3701 (1970). (13) W. C. Butts, J. Chromatogr. Sci.,8, 474 (1970). RESULTS AND DISCUSSION (14) T. Hashizume, in “Modern Gas Chromatography,’’Vol. 111, W. Funasaka and N. Ikekawa, Ed.. Hirokawa Publ. Co., Tokyo, 1971, Chap. 6. Structures of Derivatives. The number of trimethylsil(15) S. N. Alam and R. H. Hall, Anal. Biochem., 40, 424 (1971). yl groups incorporated in each case was determined mass (16) W. C. Butts, Anal. Biochem., 48, 187 (1972). (17) S. Tsuboyama and J. A. McCloskey, J. Org. Chem., 37, 166 (1972). spectrometrically by the characteristic molecular ion (M) (18) I. A. Muni, C. H. Attschuler, and J. C. Neicheril, Anal. Biochem., 50, 354
amples and leading references to the gas chromatography of trimethylsilylated purine and pyrimidine bases are given in references 19-21, and for nucleotides in (10) and (22). The earliest practical application of nucleoside gas chromatography was by Mizuno and his collaborators for the characterization of synthetic nucleoside analogs (3).Other work has dealt with the major natural nucleosides and their derivatives (4-6, 10, l l , 13, 14, 16-18), including cytokinins (8: 12, 15), and a limited amount of work on nucleic acid hydrolysates (7,9,12). The most potentially useful aspect of nucleoside gas chromatography is for structural characterization, particularly in direct combination with mass spectrometry (22). The establishment of gas chromatographic structure correlations requires accurate and precise measurement of retention characteristics. Reports of retention times or temperatures have been made in most earlier studies but were often compromised by poor peak shapes, the presence of anomalous peaks, or uncertainty as to the structures of the derivatives under investigation. Significant progress was made by Butts (13, 16) who employed inherently precise methylene unit (MLJ)values (23) from sixteen nucleosides using both OV-1 and OV-17 liquid phases. We report here the results of a systematic study of the retention characteristics, measured as MU values, of 33 trimethylsilyl nucleoside derivatives. The structural parameters investigated include methylation, heteroatom substitution, changes in sugar conformation, and deuterium substitution in the nucleoside skeleton. In addition, the differences in retention behavior between two liquid phases (OV17 and SE-30) have been studied as a function of variations in nucleoside structure.
(1972). (19) V. Pacakova, V. Miller, and I. J. kernohorsky, Anal. Biochem., 42, 549 (1971). (20) D. B. Lakings and C. W. Gehrke. J. Chromatogr.,62,347 (1971). (21) J. E. Mrochek, W. C. Butts, W. T. Rainey, Jr., and C. A. Burtis, Clin. Chem.. 17, 72 (1971). (22) J. A. McCloskey. A. M. Lawson, K. Tsuboyama, P. M. Krueger, and R. N . Stillwell. J. Amer. Chem. SOC.,90, 4182 (1968). (23) C. E. Dalgliesh. E. C. Horning, M. G. Horning, K. L. Knox. and K. Yarger, Biochem. J., 101, 792 (1966). (24) S. J . Shaw, D. M. Desiderio, K. Tsuboyama, and J. A. McCloskey, J. Amer. Chem. Soc., 92,2510 (1970).
and M-CHB peaks ( 2 2 ) .The present and earlier work (22, 29) has shown that silyl incorporation occurs at sugar hydroxyls and at enolizable carbonyls and amino groups in
(25) R . Caprioli and D. Rittenberg, Biochemistry, 8, 3375 (1969). (26) R. H. Hall, in “Methods in Enzymology,” Vol. XII, Part A, L. Grossman and K. Moldave, Ed., Academic Press, New York, N.Y., 1967, Chap. 37. (27) F. Harada, F. Kimura, and S. Nishimura, Biochemistry, I O , 3269 (1971). (28) M. Hori, in “Methods in Enzymology,” Vol. XiI, Part A, L. Grossman and K. Moldave. Ed., Academic Press, New York, N.Y.. 1967, Chap. 44. (29) A. M. Lawson, R . N. Stillwell, M. M. Tacker, K. Tsuboyama, and J. A. McCloskey, J. Amer. Chem. SOC.,93, 1014 (1971).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
1379
7-Methyl -8-oxoguanosine (TMS14
2
Ilo[ 8 Z
0 t-
1
z W w I-
i 6-
E
l
:
N2, N2-Dirnethylguanosine ( T M S l 4
8- Bromoguanosine ( T M S ) g N e , N6-Dimethyladenosine (TMS)3
7 - M e t h y l - 8 - oxoinosine ( T M S ) 3
Uridine (TMS)4
4-Thiouridine (TMS)4
W
5
W E
41 I9 $-D-Ribofuran l
I -4-D-Ribofuranosylthyrnine
(TMS)4
I
2’
6 f t I% SE-30 TP 2 Y M i N (140’)
I
22 00
I
I
24 00
26 00 METHYLENE
I
I
I
2800
30 00
32 00
UNITS
Figure 1. Methylene unit (MU) calibration curve showing the elution positions of some representative trimethylsilylated nucleosides
the base. Under the conditions employed, monosilylation occurs on amino functions but is blocked by alkylation, e.g., at N6 in W-methyladenosine. When small amounts of lower derivatives are formed, mass spectrometry shows that incomplete silylation has occurred in the base rather than the sugar. 4-Thiouridine formed the 0-2’,3’,5’-tris(trimethylsilyl) derivative, with no silyl group introduced into the base. Mass spectrometry is clearly the most useful means of establishing the structure of the derivative whose chromatographic properties are under study. Correct identification is particularly important in cases in which the compound may have undergone degradative changes, as for example in earlier work with sugar nucleotides (30) and nicotinamide adenine dinucleotide (NAD) ( 4 ) . Structure-MU Value Correlations. Figure 1 shows the slightly bowed shape of the methylene unit calibration curve, along with the elution positions of some representative nucleosides. Both the OV-17 and SE-30 calibration curves were very nearly linear, and although graphic interpolation was employed, linear interpolation was estimated to result in a maximum difference of only 0.05 MU on the columns which were used. Reproducibility on the same column on different days was generally within f0.02 MU. MU values determined from the present study are given in Table I. Most values refer to OV-17, which generally shows better separation than SE-30 and is used for most nucleoside work in this laboratory. SE-30 values were studied for certain comparisons, as discussed in a later section. Thirteen of the OV-17 values (footnote, Table I) can be (30)F. Eisenberg, Jr.. and A. H. Bolden, Anal. Biochem.,29, 284 (1969).
1380
compared with Butts’ previous work (16) to give some estimate of interlaboratory reproducibility. The earlier data temwas collected from a 3% OV-17 column (us. our l%), perature programmed at 10°/minute (us. ZO/min), with a carrier gas flow rate of 80 ml/min (us. 165 ml/min). Most values are within 0.5 MU, with Butts’ numbers tending to be lower. The most notable difference is shown by cytidine and 5-methylcytidine, our values being 2.21 and 2.87 MU higher, respectively. Structures of derivatives in Butts’ work were evidently not established, but it seems likely that his data refer to the tetrasilyl derivative, as ours does. The trisilyl derivative would be expected to elute earlier, thus increasing the difference between the two sets of results, while formation of the pentasilyl derivative seems chemically unlikely (two silyl groups at N4).The reason for the discrepancy is therefore not clear. In agreement with earlier work (5-7), the order of elution of the major nucleosides is pseudouridine, uridine, adenosine, guanosine, and then cytidine. The greater volatility of pseudouridine over uridine results from the conversion of the carbonyl group at C-2 into a less polar trimethylsilyloxy group, which is made possible by the different site of attachment of the glycosidic bond (C-5 us. N-1). The observed MU value of pseudouridine may also reflect contributions from the C-C glycosidic bond, but assessment of the importance of that structural feature independent of the extent of silylation must await availability of data from other C-nucleosides. As shown by adenosine and guanosine (OV-17), and uridine and adenosine (SE-30), lower derivatives [;.e., (TMS)3] tend to be more retarded on OV-17 then the higher derivatives [ie.,(TMS)*],while the reverse is true on SE-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, S E P T E M B E R 1974
~~
~
Table I. MU Values of Nucleoside Trimethylsilyl Derivatives Parent nucleoside
Mol wt
a-Pseudouridine Pseudouridine 9-B-D-ribofuranosylpurine
1-0-D-Arabinofuranosyluracil Dihydrouridine a-Uridine Uridine 2'-O-Methyluridine 1-0-D-ribofuranosylthymine
Inosine 2-Methyladenosine Adenosine 4-Thiouridine 7-Methy linosine
N6,W-Dimethyladenosine 2'-O-Methyladenosine W-Methyladenosine 4 '-Thioadenosine Guanosine 7-Methylguanosine 1-Methyladenosine 2,2 '-Anhydro-1- (6-D-arabinofuranosyl)uracil N*,N2-Dimethylguanosine N2-Methylguanosine 2 '-0-Methylguanosine Cytidine N6- (3-methyl-2-buteny1)adenosine
2 '-0-Methylcytidine 5 '-Methylcytidine 2,2'-Anhydro-1- ( a-D-arabinofuranosyl) uracil Ne- (3-methyl-2-butenyl)-2-methylthioadenosine 1-M ethylguanosine
No. SiMea groups
604 604 468 460 534 462 532 532 460 474 546 556 569 497
5 5
555
4 3 4 4 3 3 3 4 3
483 548 586 511 497 497 571 499 643 571 673 497 370 599 585 585 531 551 473 545 370 597 513
3 4 4 3 4 4 3 3 4 4 4 3
5
4 5 3 2 4 4 4 4 3 3 4 2 3 3
MU
_ _ OV-17
5e-30
24.09 24, 63a 25.14 25.91 25. 9FjU 26.30 26.24 26.34"
23.64 24.09 23.67 24.36 24.05
26.35 26, 68a 27,57& 27.92 28.62 28. 07u 28.34 28.28 28.40 28,671~ 28.67 28. 73a 29.51 29.84 29. 88a 30.31 30.23 30.52c1 30.57 30.66 30.72 30.88 31,73* 31. 97a 31.99 32,27" 32.30 35.87 >38.00
24.53 24.94 24.62 25.03 26,04 26.35 26.09 26.36 26.95 26.35 26.39 26.26 27.34 28.33 28.76 26.67 28.62 28.43 29.91 29.48 30.47
'' Values from this liquid phase also reported in ref. (16).
30. In the lower derivative, the more polar unprotected amino or hydroxyl group is therefore relatively more retarded by the more polar OV-17 liquid phase than in the case of SE-30 (16). Similar influences can be observed due to alkylation on heteroatoms (e.g., N6-methyladenosine, N2-methylguanosine). N,N-dimethylamino substituents produce shorter retention times than their monomethyl analogs because of decreased hydrogen bonding opportunities from the amino hydrogen. Methylation of amino groups results in higher MU values than with the unmethylated nucleoside, which shows that the methyl group is less effective than trimethylsilyl in enhancing volatility. Methylated nucleosides are therefore easily separated from their unmodified counterparts. This is an interesting contrast to the N,O-per(methyl-&) derivatives of nucleosides (31), which elute a t essentially the same position as analogs methylated at heteroatoms, because of the fact that isotopic rather than gross structural differences are involved. Methylation at the 1-position in purines (adenosine, guanosine), on the other hand leads to very much higher MU values (16), which is attributed to the unusual ionic character of 1methylpurine nucleosides (32).7-Methylpurine nucleosides uniquely undergo conversion in high yield to the corresponding 7-methyl-8-oxo compounds by incorporation of dissolved atmospheric oxygen during silylation (33);there(31)D. L. von Minden and J. A. McCloskey, ./. Amer. Chem. Soc.. 95, 7480 (1973). (32)J. B. Macon and R. Wolfenden, Biochemistry, 7, 3453 (1968). (33) D. L. von Minden. R. N. Stillwell, W. A. Koenig, K. J. Lyman, and J. A. McCloskey, Anal. Biochem., 50, 110 (1972).
fore the MU values shown in Table I do not reflect the high polarity associated with the betaine character of the free nucleoside (34). Heteroatom substitution led to systematic and marked differences. Replacement of oxygen by nitrogen leads to higher polarity and, therefore, longer retention times, as shown by the pairs uridine-cytidine (AMU = 5.39), inosine-adenosine (AMU = 0.50), and xanthosine-guanosine (AMU = 2.75, using Butts' OV-17 value for xanthosine (16)). Substantially longer retention times are also exhibited by thiated nucleosides: adenosine-4'-thioadenosine (AMU = 1.44); N6-( 3-methyl-2-buteny1)adenosine-N6(3methyl-2-butenyl)-2-methylthioadenosine(AMU = 3.90); and uridine-4-thiouridine (AMU = 1.74 on SE-30 phase). The magnitude and direction of this effect correlates well with earlier data from the silylated bases uracil, cytosine, hypoxanthine, xanthine and their thio analogs (16). Data in Table I also show the effects of changes in sugar conformation upon chromatographic retention. The alpha anomer of pseudouridine elutes before the beta anomer, in confirmation of earlier reports (11, 13, 35), and was the most volatile nucleoside derivative studied. A similar difference is observed for uridine and its alpha anomer (AMU = 0.10 MU) although the difference is less. A less pronounced structural change, inversion of the 2'-hydroxyl, leads on the other hand to a greater change in retention, (34)P. 0.P. Ts'o, N. S. Kondo, R. K. Robins, and A. D. Broom. J. Amer. Chem. SOC.,91, 5625 (1969). (35)P. M. Krueger and J. A. McCloskey, Anal. Chem., 41, 1930 (1969).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
1381
r
cients of 0.998 and 0.999,respectively, with approximately the same slopes but different intercepts. Purine nucleosides which are monoalkylated a t heteroatom sites form the basis for a separate line, with a relative intercept on the ordinate which reflects their greater retention on the more polar phase (OV-17). The most striking difference is exhibited by a- and @-pseudouridine.However, since only two carbon-linked nucleosides were examined, additional data will be needed to evaluate the validity of this method for characterization of C nucleosides. Correlations of the type shown in Figure 2 also permit the prediction of nucleoside MU values on one column using data obtained on the other, since the slopes of the plots are approximately 1.0. Effects of Deuterium Substitution upon Chromatographic Retention. The successful cultivation of algae in DzO more than a decade ago (38, 39) has led to a steady inI 1 1 1 1 I I I 1 crease in the availability of highly deuterated natural prod24 26 28 30 32 ucts, and a parallel interest in their chromatographic sepaMU 1% OV-17 ration by various means. In view of a number of earlier studies which show that deuterium substitution decreases Figure 2. Comparisons of nucleoside retention from OV-17 and SE30 phases gas chromatographic retention time (e.g., 40-42), we have examined deuterated nucleosides to determine the impor(0) Purine nucleosides: (1) 9-P-~-ribofuranosylpurine-(TMS)~;(2) inosinetance of the effect from an analytical viewpoint. (TMS)4; (3) 2-methyladeno~ine-(TMS)~; (4) aden~sine-(TMS)~; (5) 7-methyl-8The four major nucleosides I-IV were isolated from E. oxoinosine-(TMS)4; (6) guanosine-(TMS)s; (7) 7-methyl-8-oxoguanosine(TMS)s. ( 0 )Pyrimidine nucleosides: (8) l-P-c-arabinofuranosyluracil-(TMS)4; coli which had been grown in perdeuterated media. Solu(9) dihydrouridine-(TMS)4; (IO) a-uridine-(TMS)4; (1 1) uridine-(TMS).; ( 1 2) 1tion in H20 during isolation ensured that labile hydrogens, @-rib~furanosylthymine-(TMS)~; (13) 4-thio~ridine-(TMS)~;(14) cytidineas well as the acidic H-8 of guanosine were in the protium (TMSh; (15) 5-methyI~ytidine-(TMS)~. (A)0- or NAikylated purine nucleoform, leaving only skeletal labels. Deuterium content was sides: (16) 2'-Omethyladenosine-(TMS)3;(17) N6-methyladenosine-(TMS)3; measured mass spectrometrically from the M-CH3 ion (22) (18) N*-methylg~anosine-(TMS)~;(19) N6-(3-methyl-2-butenyl)adenosineafter sample introduction by direct probe. Deuterium con(TMS)3. (0) C-nucleosides: (20) a-pseudouridine-(TMS)s: (21) pseudouridinetent and MU values for the trimethylsilyl derivatives of I(TMS)s IV are given in Table 11. In agreement with other classes of with 1-@-D-arabinofuranosyluracileluting 0.43 MU earlier compounds (e.g., 40-421, the deuterated analogs exhibit than uridine. Cyclization between the sugar and base reshorter retention times. The extent of the isotopic shift is sults in considerable lengthening of retention time, as clearly related more to structure than to the deuterium shown qualitatively by the elution temperatures reported content as shown by the different shifts of Uridine-ds and earlier (36) for various silylated cyclonucleosides. In the present study, 2,2'-anhydro-l-(a-D-ribofuranosyl)uracil cytidine& derivatives. elutes 5.96 MU later than uridine, reflecting the very high XH. 0 polarity of the pyrimidine cyclonucleosides. Comparison of MU Values from Two Columns. The reliability of identification or characterization is increased through comparison of MU values from two columns having different retention characteristics. The present study employed OV-17, which is moderately polar, and SE-30 which is nonpolar. Previous reports of quantitative nucleoside retention values were made using 3% OV-1 (slightly polar) and 3% OV-17 (16), and also on 5% SE-30 (13). If HO OH Hi) OH mass spectrometry is used in direct combination with gas 1 chromatography, then the need for data from a second colUridine-d, Adenosine-d, umn is generally precluded. Although OV-17 appears to be most generally useful for nucleoside separations, SE-30 may be advantageous from the viewpoint of column bleed (especially with mass spectrometry) if temperatures in the region of 260-300' are required. An additional and useful graphic interpretation of twocolumn data (37) was demonstrated by Hashizume (14), who plotted log retention time on OV-1 us. log retention time on Apiezon L or OV-17 and obtained parallel straight lines for purine us. pyrimidine nucleosides, and ribonucleosides us. 2'-deoxyribonucleosides. We have extended this approach by subjecting a larger number of compounds to IV 111 analysis, by the equivalent method of plotting MU values. Guanosine-d, Cytidine-d, The result, shown in Figure 2 reveals notable consistent differences on the basis of certain structural features. In (38) H. L. Crespi, S. M. Archer. and J. J. Katz, Nature (London), 184, 729 (1959). agreement with Hashizume (14), purine and pyrimidine (39) W. Chorney, N. J. Skully, H. L. Crespi, and J. J. Katz, Biocbim. Biopbys. nucleosides produce linear plots having correlation coeffiActa. 37, 280 (1960).
e
(36) S. Tsuboyama and J. A. McCloskey, J. Org. Cbem., 37, 166 (1972). (37) S. D. Nogare and R. S. Juvet, Jr., "Gas-Liquid Chromatography," lnterscience, New York. N.Y., 1962, D 243.
1382
(40) N. C. Saha and C. C. Sweeley, Anal. Cbem., 40, 1628 (1968). (41) G. Wendt and J. A. McCloskey, Biochemistry, 9, 4854 (1970). (42) T. Gaumann and R. Bonzo, Helv. Cbem. Acta, 56, 1165 (1973).
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 11. SEPTEMBER 1974
Table 11. MU Values of Deuterated Nucleoside Trimethylsilyl Derivatives No. SiMea groups
Maximum deuteration, %
Distribution of deuterium label
Uridine-&
4
96.5
Adenosine-dg
4
98.1
Guanosinede
5
97. l b
4
96.2
74.9% Dg; 2 2 . 5 % DiH; 2 . 6 % DcH, 86.4% De; 11.6% D,H; 1 . 9 % DSH, 85.7% Ds: 1 1 , 0 % DSH: 3 . 3 % D4H2 73.8% DE;21.8% D7H; 4.4% DoH2
Parent nucleoside
a
1% OV-17.
MU value'l
26.33 28.01 29.82 31.65
Based on absence of deuterium a t C-8.
OSi(CD,),
A
N'
(CD,).SiO OSi(CDI),, V Uridine-(TMS-d,),
The difference in MU values between uridine and uridine-& is within experimental error (f0.02 MU) but can be more accurately examined by gas chromatography-mass spectrometry using the accelerating voltage switching technique (43). Figure 3 shows the chromatographic profile from the M minus methyl ions resulting from simultaneous injection of uridine ( A ) , uridine-& ( B ) , and for further comparison, the trimethylsilyl-dg derivative (V) of uridine (curve C). Retention time differences under the conditions shown were measured as A-B (do us. d8) 2 seconds, A-C (do us. &), 14 seconds. The isotope effect therefore depends on the position of deuteration in agreement with earlier work on hydrocarbons ( 4 4 ) ,and is quite pronounced in the case of the silyl-clg derivative. These data show that skeletal labeling in nucleosides would probably not cause a sufficient retention time decrease to result in misidentification of the nucleoside if characterization were based only upon accurate measurement of retention time of a gas chromatographically introduced compound. However, if the mass spectrum is used to establish deuterium content, some errors could result from partial fractionation if the scan is not taken on the apex of the total ion current peak. If deuterated nucleosides are used as internal standards for quantitation by gas chromatography-mass spectrometry ( 4 5 ) , maximum accuracy re(43) C. C. Sweeley, W. H. Elliott. I. Fries, and R. Ryhage, Anal. Chem., 38, 1549 (1966). (44) G. Schomburg and D.Henneberg, Chromatographia,1, 23 (1968). (45) U. Axen, K. Green, P. Horlin, and B. Samuelsson, Biochem. Biophys. Res. Commun., 45, 519 (1971).
*#, C
Figure 3. Chromatographic profiles of ( A ) uridine-(TMS)4; (4uridined8-(TMS)4, ( C ) uridine-(TMS-d& (g-ft, 1 % SE-30, 210') Top: total ion current recorded during accelerating voltage switching. Bottom: multiple ion detection response for components A, B, and C
quires that peak areas be measured (e.g., Figure 3). In the latter case when the mass spectrometer is used as a chromatographic detector, additional errors due to differences in ionization cross section and extent of fragmentation may also become significant.
ACKNOWLEDGMENT The authors are indebted to K. J. Lyman for technical assistance and to R. Caprioli for helpful discussions. We are grateful to Harry B. Wood, Jr. (N.I.H.), A. Yamasaki, N. J. Leonard, M. Honjo and T. Hashizume for generous gifts of nucleosides.
RECEIVEDfor review February 15, 1974. Accepted May 6, 1974. Financial assistance was provided by the Robert A. Welch Foundation (Q-125) and the National Institutes of Health (GM-13901). S.E.H. was recipient of' a National Institutes of Health Predoctoral Fellowship.
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