Gas-Liquid Chromatography of Pyrimidine and Purine Nucleosides as Their A/,0-Permethyl Derivatives Andre P. D e Leenheerl and Christian F. Gelijkens Laboratoria voor Medische Biochemie en voor Klinische Analyse, Faculteit van de Farmaceutische Wetenschappen, Academisch Ziekenhuis, 135, De Pintelaan, 8-9000 Gent, Belgium
A method for the preparation on an analytical scale of the N,O-permethyl derivatives of pyrimidine and purine nucleosides, using potassium-ferf-butoxide in DMSO and methyl iodide, is presented. Reaction conditions, i.e., influence of the methylsulfinyl carbanlon and methyl iodide reaction time were optimized. For 5-fiuoro-2’-deoxyuridine (FUDR) a linear response up to 80 pg was shown. Chromatographic character. istics of the N,O-permethyl derlvatlves of ten pyrimidine and three purine nucleosides were studled on 13 different stationary liquid phases. From these SP-2250,0V-25, and STAP were found to be superior. Plots of methylene unit (MU) values from SP-2250 vs. OV-25 yielded a linear correlation for each group of compounds-pyrimldine or purine derlvativesexamined. Structural data on the N,O-permethyl derivatlves were obtained by combined gas chromatography-mass spectrometry and uv spectrometric analysis.
The application of gas-liquid chromatography to nucleoside analysis represents several problems. These are essentially due to the high polarity and low volatility of these compounds. The first experiments on gas-liquid chromatography of nucleosides were carried out by Miles and Fales ( I ) . Trimethylsilyl(2-6), acetyl (7) trifluoroacetyl(8), and isopropylidene ( I , 7) derivatives have been described. Methyl derivatives suitable for mass spectrometry have also been prepared (7,9, IO) and successfully separated by gas-liquid chromatography (9). As a related procedure, the permethyl derivative of 5fluorouracil has been analyzed by combined gas-liquid chromatography/mass spectrometry ( 1 1 ) . Their low molecular weight and high degree of stability make methyl derivatives very useful in gas-liquid chromatography. To prepare the latter, several methods have been proposed: Dolhun and Wiebers (7) used silver oxide and methyl iodide whereas von Minden and McCloskey (9) employed the methylsulfinyl carbanion (12,13),generated by heating a NaH/oil suspension in DMSO, and methyl iodide as reagents. As reported (14,25), the methylsulfinyl carbanion can also be obtained by dissolving potassium-tert- butoxide in dry DMSO. This procedure is less hazardous and yields a more stable reagent. Current interest in our laboratories in the analysis of 5fluoro-2’-deoxyuridine (FUDR) in biological samples has led to the development of gas chromatographic systems for the determination of FUDR and related natural and synthetic nucleosides as their N,O-permethyl derivatives.
EXPERIMENTAL Materials. The following nucleosides were obtained from commercial sources: 5-fluoro-2’-deoxyuridine (FUDR) (111, 2’-deoxyuridine (111), thymidine (V), uridine (VII), 2’-deoxyadenosjne (XI) (Sigma Chemical Co.); 5-bromo-2’-deoxyuridine 5-iodo-2’deoxyuridine (X) (P-L Biochemicals Inc.); 5-chloro-2’-deoxyuridine (VIII) (Calbiochem); and adenosine (XIII) (Merck A.G.). 5-Fluoro2’,3’-dideoxyuridine (I) was kindly provided by C. Heidelberger (McArdle Laboratory for Cancer Research, Madison, Wis.) while 9-P-D-arabinofuranosyladenine (XII) was supplied to us by J. Drach (Parke, Davis and Co., Ann Arbor, Mich.). 5-Fluorouridine (VI) was
(1x1,
obtained from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, Md.). The compound l-(2’-deoxy-~-D-lyxofuranosyl)-5-fluorouracil (IV) was synthesized by the method of Horwitz et al. (16). Potassium-tert-butoxide was purchased from Aldrich Europe (Beerse, Belgium); dried DMSO, Na&O3, anhyd NaZS04, methyl iodide, CDC13, CHC13, ethyl acetate, diethyl ether, and triphenylmethane were all from Merck A. G. (Darmstadt, W. Germany). The n-alkanes were purchased from Poly-Science Corp. (Evanston, Ill.) Apparatus. Gas chromatographic determinations were carried out on a Hewlett-Packard model 5830 A gas-liquid chromatograph equipped with dual flame ionization detectors and a built-in recorder/integrator ( H P 18850 A GC terminal). Spiral silanized glass columns (2.3-m X 2.0-mm id.) were packed with the following liquid phases: 1%OV-1 (methyl silicone polymer), 3% SP-2250 (methyl phenyl silicone polymer), 1or 3% OV-25 (methyl phenyl silicone), 2% QF-1 (fluoroalkyl silicone polymer), 5%XE-60 (nitrile silicone gum), 5% OV-225 (cyanopropyl methyl phenyl methyl silicone polymer), 1%FFAP (reaction product of Carbowax 20 M and m-nitroterephthalic acid), 1or 2% STAP (reaction product of Carbowax 20 M and succinic acid), 2% DEGA (diethylene glycol adipate), 3% EGSS-X (ethylene glycol succinate methyl silicone), 3% OV-275 (cyano silicone polymer), 2% ethylene glycol phthalate and 1%DEGS (diethylene glycol succinate). All liquid phases were coated by the filter/fluidizing method on Gas Chrom Q 100-120 mesh (17). Injector and detector (FID) block temperatures were 230 and 250 OC, respectively. The oven temperature was kept isothermally a t 200 “C for pyrimidine and 230 “C for purine nucleosides. Nitrogen was used as a carrier gas at a linear velocity of about 8 cm/s. The hydrogen and air flow rates were adjusted to give optimum sensitivity and good stability. Analysis by gas chromatography-mass spectrometry was performed using an LKB 9000s instrument equipped with a 1%OV-1 column (on Gas Chrom Q 100-120 mesh, 1.8-m X 2.0-mm i.d.). The carrier gas was helium and the flow rate 30 ml/min. The temperature of the column was 200 or 220 “C for pyrimidine or purine nucleosides, respectively. The temperature of the flash heater was 230 “C, whereas for the separator and ion source 270 “C was used. The electron energy was set a t 70 eV and the trap current to 60 FA. The accelerating voltage was set to 3.5 kV. NMR spectra were recorded in CDC13 with a Varian T 60A (60 MHz) instrument, using 1%tetramethylsilane (TMS) as internal standard. UV spectra were run with a Pye-Unicam SP 1800double beam recording spectrophotometer provided with 1-cm quartz cells. Methylsulfinyl Carbanion Reagent. Six grams of potassiumtert-butoxide in 100 ml of dry DMSO are heated to 70 “C with mechanical stirring. After 2 h, the solution is filtered and 10-ml portions are stored at -18 “C. This stock solution is diluted 4X with dry DMSO to yield the reagent. Prior to use, the reagent is tested by mixing a 50-pl aliquot with 100 pl of a solution containing 1 mg triphenylmethane in DMSO: an intense red color should develop immediately. Synthesis of t h e I n t e r n a l S t a n d a r d (N,O-Permethyl-2’deoxyuridine). T o a solution containing 500 mg of 2’-deoxyuridine (111) in 60 ml of DMSO is added 40 ml of methylsulfinyl carbanion concd solution. After 15 min, a volume of 10 ml of methyl iodide is added and the solution is thoroughly mixed for an additional 2 h. The reaction is then terminated by adding 15 ml of Hz0. The product is extracted with two portions of 75 ml of CHC13 and the combined extracts are washed successively with 50-ml volumes of 0.5 M NazSz03 (2X) and HzO (3x1. The organic phase is dried over anhyd Na2S04 and concentrated in a rotatory evaporator. The gum obtained was dissolved in a minimal volume of ethyl acetate and diethyl ether was added until the solution became turbid: the product crystallized upon refrigeration overnight (overall yield: 60%). TLC on silica gel F-254 and using acetone/cyclohexane: 1/1(v/v) showed the material to be
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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0
c-5 PYRIMIDINE NUCLEOSIDE
5-FLUORO-2',3'-DIDEOXYLRIDINE 5-FLUORO-2 ' -DEOXYURIDINE (FUDR) 2'-DEOXYURIDINE 1-(2'-DEOXY-u-D-LYXOFURANOSYL)IV 5-FLUOROURAC~L= THYMIDIYE V 5-FLUOROURIDINZ VI VI1 URIDINE VI11 5-CHLORO-2 ' -DEOXYURIDINE 5-BROMO-2 ' -DEOXYURIDINE IX 5-1ODO-2'-DEOXYURIDINE X I I1 I11
c-2'
c-3'
R1
R2
R3
F F H
H H H
H H O H H O H r i
F Cr13
F H C1 Br I
H
R4
H
O
1
H
H OH
OH OH
H
OH
OH
3
H
OH
H M
OH OH
H H
H
ii
2
3
4
5
10 TIME(MINUTES1
15
20
Figure 2. Derivatization: influence of the methylsulfinyl carbanion reaction time
I
A.
OH R2 0.5.
c-2 ' PURINE NUCLEOSIDE XI 2'-DEOXYADENOSINE XI1 9-fi-~-ARnBINOFURANOSYLADENINE XI11 ADENOSINE
R1
R2
H
4 I
OH
H
H
OH
Figure 1. Structural formulas of pyrimidine nucleosides
homogeneous (Rr = 0.45). The principal spectral characteristics are: mass spectrum, m/e (rel. int.): 270 (3) Ma+, 145 (100) C7H1303 or 2'-deoxyribosyl moiety, 127 (8) CsHvNzOz or base + 2H; NMR (CDC13, TMS) (IO, 18)6 (ppm):3.30 [3H, s, OCH3 a t C-3'],3.33 [3H, s, OCH3 at C-5'],3.38 [3H, s, CH3 at N-3],6.30 [lH,t, anomeric proton at C-1'1 and 7.80 [ l H , d, at C-61; uv, ' : \ :A = 262 nm ( t = 8 362 cmz/ mmol) and ':A;,f = 233 nm. Optimization of the N,O-Permethylation Reaction. Influence of the Methylsulfinyl Carbanion Reaction Time. To solutions containing each 50 pg of FUDR (11) in 100 pl of DMSO was added 50 ~l of diluted methylsulfinyl carbanion reagent. After a reaction time of 10,20, and 30 s, 1,2,5,15,and 30 min, respectively, 100 pl of methyl iodide was added and each mixture allowed to stand for an additional 1 h. The reaction was then stopped by addition of 5 ml of HzO, 50 pl of a solution containing 25 pg of IS in DMSO were added and the N,O-permethylated nucleosides extracted into 5 ml of CHC13. The organic phase was washed 3X with 5 ml of HzO, dried over anhyd NaZS04 and taken to dryness under a nitrogen stream in a water bath at 60 OC. The residue obtained was dissolved in 100 pl of CHC13 and 1pl was injected on top of the GC column with a Hamilton 701 syringe. Peak area ratios of FUDR to IS were plotted vs. methylsulfinyl carbanion reaction time. Influence of the Methyl Iodide Reaction Time. Another but similar experiment was performed as follows: the methylsulfinyl carbanion reaction time was fixed at 1min whereas after addition of methyl iodide the reaction was stopped (by adding 5 ml of HzO) a t 1,5,10, and 30 min, and 1,2, 3, and 4 h, respectively. Peak area ratios of FUDR to IS were plotted vs. methyl iodide reaction time. Procedure, An amount in the range 0-100 pg of nucleoside is treated as described in the optimization experiments but utilizing a 2204
2
1
3
L
TIME(WRS1
Flgure 3. Derivatization: influence of the methyl iodide reaction time.
methylsulfinyl carbanion reaction time of 1min and a methyl iodide reaction time of 1h. For qualitative work, the addition of IS is omitted. Determination of Methylene Unit Values. Even numbered nalkanes (CzpC36) were chromatograpbed under identical conditions as the N,O-permethyl derivatives of nucleosides. The MU value of each derivatized nucleoside was computed according to (19): 1% IR(X)- log t R G ) 1% t R ( C n + 2 ) - 1% I R ( C n ) where MUx = MU value of the N,O-permethyl derivative of nucleoside X , ~ R ( x=)adjusted retention time of the N,O-permethyl derivative of nucleoside X (rnin), t ~ ( c ,=) adjusted retention time of n-alkane with the even number carbon atoms n (min), t R ( C , + z ) = adjusted retention time of n-alkane with the even number carbon atoms n + 2 ( m i d , and ~ R ( c , )< tR(X)< ~ R ( C " + ~ ) . Linearity Experiment. An amount of 10.6 mg of FUDR is dissolved in 50.0 ml of DMSO (Le., 106 ~ g / 5 0 0pl). From this solution, dilutions were made with DMSO as to give 84.8,62.4,42.4,21.2,10.5, and 5.3 pg/500 pl. Of each dilution, a 500-pl sample was taken through the derivatization procedure. After termination of the reaction, 250 pl of a solution containing 57.5 pg of N,O-permethyl-2'-deoxyuridine (stock soln: 2.3 mg/lO ml DMSO) was added to each reaction mixture. A linearity check was done on a 1%STAP column by plotting peak area ratios of FUDR to IS vs. the amount (pg) of FUDR.
MU^ =
+
RESULTS AND DISCUSSION T h e structures of the nucleosides investigated are given i n Figure 1. T h e y are n a t u r a l occurring (111,V, VII, XI, XIII), synthetic (VIII, IX), or nucleosides of medicinal importance, Le., used in cancer chemotherapy (I, 11,IV, VI) and t r e a t m e n t of viral infections (X, XII). The study of the N , O - p e r m e t h y l
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
I ! 0 8 1 6 2 4 3 2 TIME( MINUTES)
Figure 4. GC of 5-fluoro-2’-deoxyuridine (FUDR) (II), 2’-deoxyuridine (Ill),thymidine (V), 5-chloro-2’-deoxyuridine (VIII), 5-bromo-2’-deoxyuridine (IX), and 5-iodo-2’-deoxyuridine (X) as their N,O-permhthyl derivatives on the 3 % SP-2250 column
derivatization comprised the preparation of these derivatives on an analytical scale (hg range) in a controllable and reproducible way. We preferred the methylsulfinyl carbanion reagent prepared by dissolving potassium-tert-butoxide in DMSO. In fact, this method is safer and more practical than the one employing a NaH/oil suspension and DMSO (9):no hydrogen gas is generated and the risk of reagent contamination by traces of oil is eliminated. In addition to this, we found the working reagent is more stable. The latter fact is most probably due to the existing equilibrium between the tert-butoxyl anion and the methylsulfinyl carbanion (20).The influence of the methylsulfinyl carbanion reaction time on the derivatization process is shown in Figure 2. Apparently we obtain a better yield a t reaction times less than 1 min. However, for quantitative work where reproducible results are needed, a reaction time of a t least 1 min is required. Methylsulfinyl carbanion reaction times of longer than 30 min gave no response at all. This might be attributed to deactivation of the reagent by atmospheric moisture. Results on the optimization of the second step in the derivatization, namely the methyl iodide reaction time, are visualized in Figure 3. Here, the optimum is reached after 1 h and remains constant for several subsequent hours. Utilizing former optimum conditions developed-reaction times of 1 min and 1h for methylsulfinyl carbanion and methyl iodide, respectively-a linear response was demonstrated for FUDR in the 0- to 80-18 range. For gas-liquid chromatography experiments, the best results in terms of high theoretical plate number, good peak
Figure 5. GC of 5-fIuoro-2’,3’dideoxyuridine (I),5-fluoro-2‘deoxyuridine (FUDR) (Il),2’deoxyuridine (Ill),and thymidine (V) as their N,O-permethyl derivatives on the 3 % OV-25 column
I‘ I
LO
Figure 6. GC of P’deoxyadenosine (XI), 9-/h-arabinofuranosyladenine (XII), and adenosine (XIII) as their N,O-permethyl derivatives on the 1 % STAP column
symmetry, and selectivity were obtained with 3% SP-2250,3% OV-25, and 2% STAP. Typical examples of good separations of nucleosides as their N,O-permethyl derivatives on these column systems are presented in Figures 4 to 6. Retention characteristics on 3% SP-2250 and 3%OV-25 were measured as MU values and are given as part of Table I. The average of three determinations was used and a standard deviation of 0.01 MU or less was obtained. Mass spectral characteristics are identical to those found by von Minden and McCloskey (9) and indicate the number of CH3 groups introduced by the derivatization reaction in each nucleoside. For all compounds studied, the ribosyl or 2’-deoxyribosyl moiety always under-
Table I. Retention (MU Values) and Other Structural Data of Nucleosides as Their N,O-Permethyl Derivatives
Nucleoside 5-Fluoro-2’,3’-dideoxyuridine (I) 5-Fluoro-2’-deoxyuridine (11)
2’-Deoxyuridine (111) l-(2’-Deoxy-/3-D-lyxofuranosyl)-5-fluorouracil (IV)
5-Fluorouridine (VI) Thymidine (V) Uridine (VII) 5-Chloro-2’-deoxyuridine (VIII)
5-Bromo-2’-deoxyuridine (IX) 5-Iodo-2’-deoxyuridine (X) 2’-Deoxyadenosine (XI) 9-6-D-Arabinofuranosyladenine (XII)
Adenosine (XIII)
Me+ (rel. int.)
258(3) 288(3) 270(3) 288(4) 318(3) 284(3) 300(4) 304(4) 348(1) 396(4) 307(2) 337(29) 337(5)
N,O-Permethyl derivative Number of Methylene Unit, GH3 groups SP-2250 2
3 3 3 4
3 4
3 3 3 4 5 5
23.55 24.50 24.79 24.95 25.10 25.14 25.32 26.31 21.29 28.54 28.78 29.29 29.38
(MU) values, OV-25 25.43 26.28 26.77 26.80 26.92 27.08 27.40 28.32 29.48 30.93 31.33 31.91 32.04
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
2205
I 33'
/
m
832
2
'*' .,/
y=116~-217 r.0998
29.
y:i.n~-o.93 r.0.999 27' 26.
1
24
25
26
27
28
29
30
MU 3°/nSP.22M
Flgure 7. Linear correlations of M U values of the nucleosides measured as N,O-permethyl derivatives on OV-25 vs. SP-2250
show only a very slight decrease in retention on both column systems when the OH at C-2' points to the direction above the plane of the sugar. Thus, in agreement with the observation of Hattox and McCloskey (23),the N,O-permethyl derivative of adenosine (XIII) elutes a little bit later than 9 the one of P-D-arabinofuranosyladenine(XII). However, as seen from Figure 6, former epimers are much better and readily resolved as their N,O-permethyl derivatives on the 1%STAP column. An additional phenomenon is shown in Figure 7 where the MU values on SP-2250 vs. OV-25 are linearly correlated. The N,O-permethyl derivatives of pyrimidine and purine nucleosides each possess a regression line with approximately the same slope, but different intercepts; correlation coefficients were 0.998 and 0.999, respectively.
ACKNOWLEDGMENT The authors are grateful to A. Cruyl (aspirant) from the NFWO, for running the mass spectra. LITERATURE CITED
(1) H. T. Miles and H. M. Fales, Anal. Chem., 34,860 (1962). goes complete 0-methylation. The position of the CH3 (2) R. L. Hancock and D. L. Coleman, Anal. Biochem., IO, 365 (1965). group(s) in the pyrimidine moiety is somewhat more specu(3) Y. Sasaki and T. Hashizume, Anal. Biochem., 16,1 (1966). (4)M. Jacobson, J. F. O'Brien, and C. Hedgcoth, Anal. Biochem., 25,363 lative. However, the fact that no wavelength shift is observed (1968). between the uv spectra of the pyrimidine nucleosides and their (5) C. W. Gherke and C. D. Ruyle, J. Chromatogr., 38,473 (1968). (6) W. C. Butts, J. Chromatogr. Sci., 8,474 (1970). respective N,O- permethyl derivatives affords in itself good (7) J. J. Dolhun and J. L. Wiebers, Org. Mass Spectrom., 3,669 (1970). evidence for N-3 rather than 04-methylation (18, 21). For (8)W. A. Koenig, L. C. Smith, P. F. Crain, and J. A. McCloskey, Biochemistry, adenosine nucleosides, a wavelength shift in the absorption IO, 3968 (1971). (9) D. L. von Minden and J. A. McCloskey, J. Am. Chem. SOC.,g5, 7480 maximum of 15 nm was found. The latter effect strongly 1197.11 \._._,. suggests generation of a N6,N6-dimethylgroup (22). As exR. P. Panzica, L. B. Townsend, D. L. von Minden, M. S. Wilson, and J. A. McCloskey, Biochim. Biophys. Acta, 331, 147 (1973). pected on the basis of their higher molecular weight, purines 6. L. Hillcoat, M. Kawai, P. 8. McCulloch, J. Rosenfeid, and C. K. 0. Williams, show longer retention times when compared to pyrimidine Br. J. Clin. fharm., 3, 135 (1976). nucleoside derivatives. Incorporation of one or two OH groups P. A. Leclercq and D. M. Desiderio Jr., Anal. Lett., 4,305 (1971). E. J. Corey and M. Chaykovsky, J. Am. Chem. Soc., 87, 1345 (1965). in the structure also results in a corresponding increment in J. Eagles, W. M. Laird, R. Self, and R. L. M. Synge, Biomed. Mass Specfrom., retention. For the N,O-permethyl derivatives of pyrimidine 1. 43 (1974). (15) J. I. Brauman, J. A. Bryson, D. C. Kahi, and N. J. Nelson, J. Am. Chem. SOC., nucleosides, and in particular the deoxy compounds, substi92, 6679 (1970). tution at the C-5 position affords on both column systems an (16) J. P. Horwitz, J. Chua, J. A. Urbanski, and M. Noel, J. Org. Chem., 28,942 increase in retention according to the molecular weight of the (1963). (17) E. C. Horning, E. A . Moscatelli, and C. C. Sweeley, Chem. lnd. (London), substituent (H < CH3 < C1< Br < I). However, an exception 78, 75 1 (1959). to this is fluorine substitution which decreases the retention (18) T. A. Khwaja and C. Heidelberger, J. Med. Chem., 13,64 (1970). (19) C. E. Dalgliesh, E. C. Horning, M. G. Horning, K. L. Knox, and K. Yargey, time: the derivatives of FUDR (11) and 5-fluorouridine (VI) Biochem. J., 101, 792 (1966). elute remarkably faster than those of their natural occurring (20) D. Martin and H. G.Hauthal, "Dimethyl Sulphoxide", Van Nostrand Reinhold nonfluorinated analogues 2'-deoxyuridine (111)and uridine Co. Ltd., Berkshire, England, 1975, p 349. (21) K. Kikugawa, M. Ichino, and T. Ukita, Chem. Pharm. Bull,, 17, 785 (VII), respectively. This is a most interesting phenomenon for ( 1969). N,O-permethyl derivatives which, contrary to the trimethyl (22) H. A. Sober, "Handbook of Biochemistry", 2d ed., The Chemical Rubber Co. (CRC), Cleveland, Ohio, 1973, p G-28. silyl derivatives (2) are completely separated. Inversion of the (23) S. E. Hattox and J. A. McCloskey, Anal. Chem., 46, 1378 (1974). OH at C-3' (R3 R4) apparently increases the polarity of the molecule and 1-(2'-deoxy-/3-D-lyxofuranosy~)-5-fluorourac~1 (IV) elutes later than FUDR. Therefore, both these epimers RECEIVEDfor review May 13,1976. Accepted September 13, are easily separated. Another but minor effect is noticed for 1976. This work was supported in part by the FGWO through the N,O- permethyl derivatives of purine nucleosides which grants 20007,20210, and 20452.
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