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Silylation-mediated oxidation of dihydropyrimidine bases and nucleosides
ANALYTICAL
BIOCHEMISTRY
103, 203-213 (1980)
Silylation-Mediated Oxidation of Dihydropyrimidine Bases and Nucleosidesl JAMES A. KELLEY,MOHAMED Drug
M. ABBASI,*ANDJOHN
Design and Chemistry Section, Laboratory of Medicinal Chemiswy Therapeutics Program, Division of Cancer Treatment. National National Institutes of Health, Bethesda, Maryland
A. BEISLER
and Biology, Developmental Cancer Institute, 20205
Received September 6, 1979 Vigorous trimethylsilylation of dihydropyrimidine and dihydro-s-triazine bases and nucleosides results in their dehydrogenation to the analogous aromatic derivatives. This transformation, which cannot be effected by conventional dehydrogenating reagents, has been studied in detail using gas chromatographic and mass spectrometric methods. For 5,6-dihydro-5azacytidine (ta) the oxidation is quantitative, requires molecular oxygen as well as reagents capable of both N- and O-silylation, is accelerated by heat and selected free radical initiators, and is inhibited by diethyldithiocarbamate and galvinoxyl free radical. This anomalous silylation reaction occurs on both an analytical and preparative scale. Silylation-mediated oxidation also exhibits a substantial deuterium isotope effect with preferential retention of deuterium at the new site of unsaturation. The potential of this new reaction for the synthesis of aromatic systems and for the insertion of nonexchangeable isotopic labels in suitable reduced precursors is illustrated.
Silylation is defined as the introduction of a trimethylsilyl group into a molecule in substitution for an active hydrogen or in replacement of the metal component of a salt (1). As such, it is probably the most widely employed technique for the derivatization of polar compounds of biomedical interest for subsequent analysis by gas chromatography and combined gas chromatography-mass spectrometry (2). Nucleosides (3-9, peptides (6), steroids (7), and sugars (8) are but a few of the classes of compounds that have been rendered volatile enough for gas phase analysis. The popularity of trimethylsilylation rests with the simplicity of the procedure itself, its versatility and wide range of applicability, and ’ Presented in part at the 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, D. C., May 1977. ’ Present address: National Research Center, Medicinal Chemistry, Chemotherapy Laboratory, Dokki, Cairo, Egypt. 203
the usually straightforward formation of derivatives. Out of all the compounds derivatized by silylation and reported in the literature, there have only been a few instances of the formation of what might be called abnormal silylation products. Interestingly enough, these examples all involved oxidation of the compound being silylated. The TMS3 enol ethers of suitable keto-steroids 3 Abbreviations used: TMS, trimethylsilyl: 5-AC, 5-azacytidine; Am-S-AC, I-/3-~arabinofuranosyl-S-azacytosine; AraU, I-/3-o-arabinofuranosyl-uracil; BSA, N ,O-bis(trimethylsilyI)-acetamide; BSTFA, N ,O-bis(trimethylsilyl)-trifluoroacetamide: DDQ, 2,3-dichloroS,&iicyano-l,4-benzoquinone; gc, gas chromatography, gc/ms, gas chromatography-mass spectrometry; H,S-AC, 5,6-dihydro-5-azacytidine; H,-5-AC-d,, 5,6[6-2H]dihydro-5-azacytidine; H,-Ara-5-AC, I-p-o-arabinofuranosyl-5,6-dihydro-5-azacytosine; H,-Ara-5 AC-d,, I-p-o-arabinofuranosyl-5,6-[6-‘Hldihydro-5azacytosine; H,-AraU, I-P-o-arabinofuranosyl-5,6-dihydrouracil; HZ-U, 5,6-dihydrouridine; TMSDEA, trimethylsilyldiethylamine; TMCS, trimethylchlorosilane: TSIM, trimethylsilylimidazole. OOO3-2697/8O/O60203-11$02.OO/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
204
KELLEY,
ABBASI,
were found to form the corresponding cr-silyloxy ketones (9). This reaction was catalyzed by ultraviolet light or dibenzoyl peroxide and was presumed to be free radical in nature (10). Persilylation of norethynodrel and 5( lO)-estrene-17/3-ol-3-one with TSIM was reported to aromatize the A-rings of these compounds (11). Trimethylsilylation of 7-methylpurine nucleosides on a microgram scale resulted in molecular oxygen incorporation as well as derivatization to form the corresponding 7-methyl-8-oxo nucleosides (12). We now add to this short list of anomalous silylation reactions by describing here the silylation-mediated oxidation of 5,6-dihydropyrimidine and 5,6dihydro-s-triazine nucleosides and their respective aglycones to the corresponding aromatic derivatives. This report defines the silylation conditions required to effect this conversion and describes studies undertaken to elucidate the mechanism of this transformation. METHODS Materials
BSA, BSTFA, TMSDEA, and 1% TMCS in BSTFA were purchased from Pierce Chemical Company, Rockford, Ill. Acetonitrile, which was “distilled in glass” grade from Burdick and Jackson, Muskegon, Mich., was dried by distillation from P,O, and stored over preextracted and activated 3-pm molecular sieves. Pyridine was redistilled from KOH after a 24-h reflux. Methylene chloride was refluxed with calcium chloride overnight, filtered and distilled from PZOs, and then likewise stored over molecular sieves. Trifluoroacetic anhydride (99% +, Gold Label), DDQ, m-chloroperbenzoic acid, and galvinoxyl free radical were obtained from Aldrich Chemical Company, Milwaukee, Wis.; Aldrich was also an alternate source of BSTFA. Sodium diethyldithiocarbamate, dibenzoyl peroxide, and MnO, were purchased from Fisher Scientific Company, Fairlawn, N. J., while di-f-butyl
AND
BEISLER
peroxide was obtained from Pfaltz and Bauer, Stamford, Connecticut. Chloranil was supplied by Eastman Organic Chemicals, Rochester, N. Y., and 30% hydrogen peroxide by J. T. Baker, Phillipsburg, N. J. Uridine, HZ-U, 5 &dihydro-2,4dihydroxypyrimidine (dihydrouracil), and 4,5(5,6)dihydro-5-methyl-2,Cdihydroxypyrimidine (dihydrothymine) were purchased from Sigma Chemical Company, St. Louis, Missouri, while AraU was obtained from Pfanstiehl Laboratories, Waukegan, Illinois. 5-AC was obtained from the Division of Cancer Treatment, NCI, NIH while HZ-5-AC, H,-5-AC-dl, 5,6-dihydro-5-azacytosine, and H,-Ara-5-AC were synthesized as previously described (13,14). H,-AraU was prepared by the room temperature and atmospheric pressure hydrogenation of an aqueous solution of 500 mg (2.0 mmol) of AraU over 300 mg 5% Rh/C catalyst. Workup after cessation of hydrogen uptake (50 min, 2.8 mmol) and recrystallization from methanol afforded 180 mg (36%) of slightly hygroscopic crystals, mp 173174°C; NMR (DzO) S 5.97 (d,J = 5.5 Hz, 1, C,,H), 4.33 (t,J = 6 Hz, 1, C,,H), 3.4-4.2 (complex multiplet, 6H), 2.68 (t,J = 6.5 Hz, 2, C,W. Anal. Calcd for C,H,,N,OB: C, 43.90; H, 5.73; N, 11.38. Found: C, 43.71; H, 5.49; N, 11.14. Gas Chromatography Spectrometry
and Mass
Trimethylsilyl derivatives of nucleosides and their aglycones were analyzed on a Varian 2740 gas chromatograph equipped with a flame ionization detector and linear temperature programmer. A 1.83 m x 2 mm id. glass column packed with either 3% SE-30 or 3% OV-17 on 100/120 Gas Chrom Q was operated isothermally in the range 120-140°C for aglycones and in the range 220-230°C for nucleosides. Quantitative analysis of oxidation mixtures was accomplished with temperature programming from 220 to 250°C at 2”Clmin. Typical gc
SILYLATION-MEDIATED
operating conditions employed injector and detector temperatures of 260°C and flow rates of approximately 30 ml/minute for both the helium carrier gas and hydrogen, and 300 ml/minute for air. Peak areas and retention times were obtained on a Beckman Auto-Pro 30 computational integrator and isothermal gas chromatographic retention indices were calculated using the appropriate n-alkanes as internal standards (15, 16). Low-resolution electron impact mass spectra were obtained on a DuPont 2 I-492B gas chromatograph-mass spectrometer interfaced to a VG 2040 data system. Mass spectra were obtained with sample introduction either by direct probe (trifluoroacetyl derivatives) or via a Varian 2740 gc (trimethylsilyl derivatives) coupled to the mass spectrometer by a single-stage glass jet separator. Gas chromatography operating conditions were the same as those above. Standard mass spectrometer operating conditions were: transfer line and jet separator, 250°C; ion source, 255°C; electron energy, 75 eV; ionizing current 250 PA; and scan speed, 2 s/decade. Preparation
of Derivatives
Trimethylsilyl derivatives were prepared on a microscale by the room temperature reaction of l-2 mg of the appropriate nucleoside or aglycone with 0.15 ml of silylating reagent and 0.30 ml of redistilled solvent in a 3.5-ml screw-cap glass vial equipped with a Teflon-lined rubber septum. Sonication was routinely used to speed solution and accelerate derivatization. The silylation mixture was allowed to stand at room temperature for a minimum of 15 min after solution and l- to ~-PI aliqouts of the sample were taken for analysis. When required, the tightly sealed silylation mixtures were heated in a thermostated heating block maintained at the desired temperature. Trifluoroacetyl derivatives were prepared on the same scale using trifluoroacetic anhydride and following the procedure of Koenig et al. (17).
205
OXIDATION
Kinetic
Studies
Methanol stock solutions of approximately 1 mg/ml concentration were made of uridine and HZ-S-AC. A 1.O-ml aliquot of each stock solution was added to a 3.5-ml glass screw cap vial and the methanol solution evaporated to dryness under a stream of nitrogen. The residue was dried in vacua and silylated in the same manner as above. After gc analysis to ensure derivatization of both substrate (HZ-j-AC) and internal standard (U), the sample was placed in the heating block at the desired temperature between 60 and 100°C and aliquots were withdrawn for gc analysis at periodic intervals. Both the peak height and area ratios of trimethylsilylated HZ-j-AC (substrate) and 5-AC (product) to internal standard were calculated, and the logarithm of these ratios was plotted versus time. A standard curve was also constructed for 5-AC in the range 0. l- 1.5 mg and used to determine the absolute yield of product. Silylation
in the Absence
of Oxygen
A I.O-ml aliquot each of H,-5-AC and uridine stock solution was added to a 10 x loo-mm vacuum reaction tube equipped with an adjustable Teflon plunger (Pierce Chemical Co.) and evaporated to dryness in the same manner as above. BSTFA and acetonitrile were degassed by a freezevacuum-thaw cycle and stored under nitrogen. To the residue in the vacuum reaction tube was added 0.20 ml degassed BSTFA and 0.40 ml degassed acetonitrile and the freeze-vacuum-thaw cycle was repeated three times. The tightly sealed reaction mixture was then sonicated for 20 min at room temperature before being placed in a heating block. A control sample was prepared and silylated in an identical manner except degassed reagents were not used and no attempt was made to exclude oxygen. Where the kinetics of this oxidation were being studied, an identical but separate degassed sample was prepared for each
206
KELLEY,
ABBASI,
analysis time, since it was impossible to repeatedly sample a reaction mixture without introducing oxygen. Silylation in the Presence Initiators and Inhibitors
of Radical
Samples of dihydro-s-triazine nucleoside substrates and internal standard were prepared and silylated in 3.5 ml glass screw-cap vials as described above. Solutions of sodium diethyldithiocarbamate (4.5 mg/ml), dibenzoyl peroxide (10 mg/ml), and meta-chloroperbenzoic acid (10 mg/ml) were prepared in acetonitrile to facilitate addition to the silylation mixture. For these reagents the volume corresponding to 0.25-2.0 molar eq of substrate was calculated, and for each sample a corresponding adjustment was made in the amount of acetonitrile added to the BSTFA so that the final ratio of BSTFA:acetonitrile would still be 2: 1. Di-t-Butyl peroxide, galvinoxyl free radical, and 30% hydrogen peroxide were added directly to the silylation mixture in amounts corresponding to approximately 1 molar eq of substrate. After an initial gc analysis of the room-temperature reaction mixture, the tightly sealed samples were placed in a heating block at either 66 or 70°C and analyzed periodically by gc. The peak height and area ratios of substrate to internal standard were determined and used to calculate a percentage conversion relative to control. Determination
of Isotope
Ratios
To determine the amount of deuterated to nondeuterated isomer selected reaction mixtures of both HZ-S-AC-d, and H,-Ara-5AC-d, were analyzed by limited scan (m/z 480-550) gc/ms. The mass spectrometer was scanned repetitively every 5 s at a scan rate of 10 s/decade over the gc peak of interest, and the collector slits were opened to give flat-topped peaks with greater than unit resolution. After the A + 1 isotopic contributions of the Mt (m/z 532) and M-CH, (m/z 517) ions of 5-AC-TMS, had
AND
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been corrected for, the relative amounts of monodeuterated and unlabeled 5-AC were determined from these mass peaks, and an average was obtained for all the scans over the gc peak. Attempted
Oxidation
with Other Reagents
To 100 mg (0.4 mmol) of HZ-j-AC in 20 ml methanol was added 500 mg (2.20 mmol) DDQ. The progress of the reaction was monitored using tic systems that have been previously described (13). The solution showed no change after standing at room temperature for 10 h or after reflux for 34 h. Only starting material could be isolated upon workup and recrystallization. Attempted dehydrogenation of Hz-j-AC by refluxing with chloranil in ethanol for 24 h or by heating with MnO, in hexamethylphosphoramide for 6 h at 60°C was likewise unsuccessful. RESULTS
AND DISCUSSION
5-Azacytidine (l), a nucleoside antimetabolite that is effective in the clinical treatment of acute myelogenous leukemia, was reduced with sodium borohydride to form 5,6-dihydro-5-azacytidine (2a) in order to improve the former’s hydrolytic stability yet retain its antitumor activity (13). The gc/ms analysis of 2a to determine purity and to confirm structure required quantitative formation of a volatile derivative. However, microscale silylation of 2a according to standard procedures with BSTFA in CH,CN at 100°C for 2 h (4) did not produce the expected pentatrimethylsilyl derivative 3a, but silylated 5-AC (4a) instead (Fig. 1). The expected derivative 3a was not observed until silylation was carried out under much milder conditions at room temperature. Moreover, when monodeuterated HZ-j-AC (2b) was vigorously silylated, the S-AC that was formed preferentially retained deuterium to give a 3: 1 mixture of 4b and 4a. These initial observations prompted a more comprehensive study of the apparent
SILYLATION-MEDIATED
;’
2
HO
OH
1
,;;;2;gc;;ii$:TM\M~5 2
HO
OH
2a,R=H 2b. R = D
FIG. 1. Synthesis and silylation-mediated
TMSO
OTMS
OTMS
TMSO
3a,R=H 3b, R = D
4a,R=H 4b,R=D
oxidation of 5,6-dihydro-5-azacytidine.
dehydrogenation reaction that was occurring during trimethylsilylation. For the reliable analysis of 2a and other reduced nucleosides by gc and gclms it was important to define the scope of this oxidation and determine whether it could be blocked. Synthetically, this reaction offered a potential means of obtaining an aromatic system from a reduced precursor and also incorporating a nonexchangeable label into a suitable compound. Kinetics
207
OXIDATION
&-5AC
-
B~FA~~~~~NwI,
S-AC 2 hr
at 76.5+l”
of Oxidation
The microscale conversion of H,-5-AC to 5-AC during silylation was monitored by gc analysis using an internal standard (Fig. 2). Gas chromatography was an ideal method for this analysis since silylation was required for oxidation (see below) and the derivatized nucleosides could be well separated on an OV-17 column (Table 1). Uridine was chosen as an internal standard because of its favorable retention index and because its structural similarity as a persilylated nucleoside would hopefully allow it to act as a carrier for the less stable derivatives of interest (e.g., 3a). The above method of analysis required that the sample be silylated at room temperature before controlled heating to initiate the silylation-mediated oxidation reaction. Complete and reproducible silylation of the uridine internal standard could be accomplished without heating; sonication of the silylation mixture for 20 min followed by standing at room temperature for an
220 I 0
230 I 5
240 I 10
250 iso 250 DC II I 15 20 min
FIG. 2. Quantitative gas chromatographic analysis of a silylation-mediated oxidation reaction mixture of 5,Qdihydro-5azacytidine that was heated at 76.5 + 1°C for 2 h. A 1.83 m x 2 mm-i.d. glass column packed with 3% OV-17 on 100/120 mesh Gas Chrom Q was temperature programmed from 220 to 250°C at 2Wmin and then maintained isothermally at 250°C for 5 min. See under Methods for additional details of the analysis.
208
KELLEY,
ABBASI, TABLE
AND BEISLER 1
ISOTHERMAL RETENTION INDICES OF BASE AND NUCLEOSIDE TRIMETHYLSILYL DERIVATIVES I” No. TMS groups
M,
SE-3W
OV-17*
Uracil Dihydrouracil Thymine Dihydrothymine 5-Azacytosine Dihydro-5-azacytosine
2 2 2 2 2 3
256 258 270 272 256 330
1335(A) 1470(A) 1392(B) 1475(B) 1477(B) 1671(B)
1453(A) 1636(A) 1504(B) 161 l(B) 1634(C) 1774(C)
Uridine Dihydrouridine l-/3-D-Arabinofuranosyluracil 1-/3-D-Arabinofuranosyldihydrouracil 5Azacytidine Dihydro-5-azacytidine I-p-D-Arabinofuranosyl-5-azacytosine I-P-o-Arabinofuranosyldihydro-5-azacytosine
4 4 4 4 4 5 4 5
532 534 532 534 532 606 532 606
2469(D) 2420(D) 2505(D) 2462(D) 2622(D) 2470(D) 2648(D) 2538(D)
2625(D) 2539(D) 2613(D) 2560(D) 2823(E) 2530(E) 2808(E) 2564(E)
Parent base or nucleoside
n Retention indices were measured with a precision of +3 units. * Letter in parentheses indicates isothermal temperature: (A) = 120°C; (B) = 130°C; (C) = 140°C; (D) = 220°C; (E) = 230°C.
2.ooo
L-1 H&AC Uridine
‘h = -
degassed reagents, N, BSTFA/CH,CN (1:2), 76.5?1’
0, excluded 0
,200
complete cO”“erSlOn
,100
cl
I 1
I 2
I 3 TIME
/,I I’ 21
4 I 22
Ihrl
FIG. 3. Kinetics of the silylation-mediated oxidation of 5,6-dihydro-5-azacytidine using degassed reagents under conditions of oxygen exclusion and using untreated reagents under normal conditions (control). See under Methods for additional details.
additional 90 min with occasional shaking gave the same results as the recommended optimum conditions of heating for 15 min at 150°C (5). In an independent check of both methods the area ratio of uridine to a pyrene internal standard did not vary more than the expected precision (2 5%) of the gc method. When l-2 mg of H,-5-AC was silylated with 0.45 ml of 1:2 BSTFA and CH&N (a 25-50 M excess of silyl groups) and then oxidized by heating, the reaction was pseudo first order with respect to silylated H,-5-AC (see Fig. 3). Since oxidation was markedly accelerated by heating, most experiments were conducted at moderate temperatures so that the reaction would not be too fast to follow. A half-life of 2.02 h was observed at 76°C and the reaction was complete within 22 h; no silylated reduced nucleoside was observed by gc. Raising the temperature slightly to 80°C resulted in a somewhat shorter half-life of 1.68 h. The conversion of silylated H,-5-AC to derivatized
SILYLATION-MEDIATED
S-AC was quantitative after 24 h at this temperature with no formation of side products and no trace of starting material. An absolute gc yield of 98.0 +- 1.6% based on underivatized reduced nucleoside 2a was measured. Effect of Varying Substrate and Reaction Conditions
209
OXIDATION TABLE
3
MICROSCALE SILYLATION-MEDIATED OXIDATION OF VARIOUS SUBSTRATES (7’ = 70°C)
Product”
Substrate” H,-5-AC H,-Ara-S-AC H,-U 5,6-Dihydro-.5azacytosine 5,6-Dihydrouracil 5,6-Dihydrothymine
5-AC Ara-5-AC U
Conversion completed 00” 22’ 84 >84”
Strong silylating reagents such as BSTFA 20 5-Azacytosine or BSA easily effected oxidation while Uracil 45 weaker silyl donors such as TMSDEA or TSIM gave poor yields (see Table 2). These Thymine 160 latter reagents do not completely derivatize amines so it appears that silylation of both a As TMS derivative. b Derivatized substrate was no longer detectable by oxygen and nitrogen is required. SiIylationmediated oxidation was also observed for gc or gcims. c T = 76°C. other reduced pyrimidine nucleosides as d Reaction not followed to completion. well as their aglycones (see Table 3). The arabinosyl analog of dihydro-5azacytidine (H,-Ara-5-AC) could be aromatized to the milder conditions employed here likewise previously unattained arabinosyl derivative caused the eventual aromatization of diof 5-AC (14), but the rate of conversion hydrouracil to uracil as well as the much was approximately four times slower than slower transformation of dihydrothymine to that of the ribosyl derivative. Dihydrothymine during silylation. Other nucleoside uridine and its arabinosyl analog could also derivatization methods such as trifluorobe oxidized to the corresponding uridine acetylation (17) caused no oxidation of the derivatives, but the rate of conversion was reduced derivatives even with vigorous much slower than for either of the dihydroheating. Dihydro-5-azacytidine smoothly s-triazine nucleosides. The aromatization of formed a tetratrifluoroacetyl derivative whose dihydrouracil to uracil during vigorous molecular weight (by mass spectrometry) silylation was previously observed by White was 2 amu higher than that of similarly et al. (18) but not further investigated. The derivatized 5-azacytidine. Dehydrogenating and aromatizing reagents such as DDQ, chloranil and MnOz were also ineffective TABLE 2 in carrying out this transformation on the EFFECT OF VARIOUS REAGENTS ON SILYLATIONunderivatized reduced nucleosides. MEDIATED OXIDATION
Relative gc yield of S-AC (15 h, 80°C)
Reagents BSTFA/CH,CN (1:2) BSTFAipyridine (1:2) BSTFA, 1% TMCS/CH,CN BSA/CH,CN (1:2) TMSDEAKH,CN (1:2) TSIM/CH,CN (1:2)
OF H,-5-AC
(1:2)
100 100 96 84 19 0
Deuterium
Isotope Effect
A substantial deuterium isotope effect was observed upon gc/ms analysis of 6-deutero5,6-dihydro-5-azacytidine (2b) that had undergone silylation-mediated oxidation. The 5-AC that was formed consisted of both d, and d, derivatives in an approximate ratio of 3: 1. Limited scan gc/ms analysis gave a more precise value of 2.9 + 0.2 for the d,:d,
210
KELLEY,
ABBASI,
ratio. The M-CHB peak (m/z 517) rather than the molecular ion peak (m/z 532) was used to determine d,/d, because of its much greater relative intensity (0.8 versus 0.1%). Silylation-mediated oxidation of monodeuterated H,-Ara-5-AC resulted in a product di:d,, ratio of 2.8 ? 0.3, the same value within experimental error, even though this reaction was much slower. Therefore, at least in the case of the above s-triazine nucleosides, the rate-determining step of silylation-mediated oxidation involves breaking the C-H bond at the 6-position.
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BEISLER
gc analysis, to the atmosphere followed by heating for a sufficient length of time showed complete conversion to 5-AC. Figure 3 compares the silylation-mediated oxidation of a deoxygenated sample of H,-5-AC versus that of a control. Even after 22 h at 76.5 + 1°C the deoxygenated sample showed only trace conversion to the aromatized nucleoside, which was probably due to incomplete removal of oxygen. In contrast the control exhibited complete conversion while following good pseudo first-order kinetics. The conversion of dihydrouracil to uracil during silylation with BSTFA and CH,CN at 150°C for 1 h was also blocked when the reagents were degassed.
Oxygen Requirement It was possible that the silylating reagent alone was not responsible for the aromatizaEffect of Free Radical Initiators and tion of these reduced pyrimidine nucleosides. Inhibitors Trace analyses of the various silylating The requirement for molecular oxygen reagents showed that there were no unusual suggested that silylation-mediated oxidation concentrations (> 10 ppm) of metals such as might be occurring by a free radical mechFe, Cu, or Zn which could conceivably anism. Thus the effect of adding various catalyze an oxidation.4 Certain silylating reagents can contain appreciable amounts of free radical initiators and inhibitors to a dissolved oxygen (12). The requirement of silylation mixture of H,-5-AC was quantitatively measured (Table 4). Of the initiators molecular oxygen for silylation-mediated dibenzoyl peroxide was the most effective oxidation was demonstrated in a series of in promoting oxidation at room temperature. parallel experiments where identical samples Normally several days at room temperature of reduced nucleoside 2a were silylated with were required before any 5-AC could be degassed BSTFA and CH,CN or with detected. However, increasing amounts of reagents that had been degassed and redibenzoyl peroxide up to a maximum charged with either oxygen or nitrogen corresponding to a molar equivalent of subbefore heating at 70°C for 4 h. The results strate proportionately increased the yield of were compared to a control, silylated with silylated 5-AC. Heating this silylation mixture, untreated BSTFA, that had been heated for however, did not markedly increase the the same length of time. No aromatization yield of aromatized nucleoside with respect to derivatized 5-AC was observed when to control. A possible reason for this was silylation was conducted with completely the increased formation of by-products upon degassed reagents or with degassed reagents in a nitrogen atmosphere. However, the heating since a compound characterized as a by mass degassed silylating reagent that had been benzoylated dihydro-5-azacytidine was isolated in about 30% recharged with oxygen showed a 91% spectrometry yield from a preparative scale reaction to conversion to 4a relative to the control. Exposure of a degassed sample, which had which dibenzoyl peroxide had been added as an accelerator. Even more interesting been analyzed previously by quantitative was the result of silylation-mediated oxida4 Trace analyses were performed in duplicate by tion of monodeuterated Hz-5-AC in the Galbraith Laboratories, Inc., Knoxville, TN 37921. presence of 1.0 eq of dibenzoyl peroxide
SILYLATION-MEDIATED TABLE EFFECT
OF RADICAL
INITIATORS
AND
INHIBITORS
211
OXIDATION 4
ON THE
SILYLATION-MEDIATED
Relative conversion to 5-AC (%)
OXIDATION
OF
H,-5-AC
Relative amount H,-5-AC remaining (%)*
Reagent
Initial (RT)
4 h (66 ? 1°C)
Initial (RT)
4 h (66 k 1°C)
Control Dibenzoyl peroxide (1 .O eq) H,O, (30%) m-Chloroperbenzoic acid Di-t-butyl peroxide Diethyldithiocarbamate Galvinoxyl
0 58 4 0 2 10 27
70 74 72 63 71’ 13 15’
100 0 52 65 52 100 58
I1 0 0 21 6’ 87 61’
a One hundred percent conversion is defined as a control which has been allowed to react to completion. A control is a silylation mixture of HZ-S-AC to which initiators or inhibitors have not been added. b One hundred percent remaining is defined as a control at room temperature. c 70 c 1°C.
at room temperature. Here the dI:d,, ratio in the SAC that was produced was 1.8 2 0.3, significantly less than that observed under control conditions. Other compounds added as radical initiators did not markedly promote the room temperature oxidation of silylated HZ-SAC. Addition of aqueous hydrogen peroxide or meta-chloroperbenzoic acid had little effect at room temperature or upon heating relative to the control. The same was true for di-t-butyl peroxide, but this may be due to the necessity of temperatures of 120- 150°C to generate the t-butoxy radicals. Galvinoxyl, a free radical scavenger (19,20), seemed to act as an initiator at room temperature, but showed strong inhibition after heating for 4 h at 70°C. About 60% of the silylated starting material remained after heating. The most effective inhibitor of oxidation was sodium diethyldithiocarbamate. Although a small initial conversion to aromatized derivative was seen, this did not increase significantly with heating and the reduced starting material could be detected in undiminished yield. Mechanism Although there is insufficient data at present to formulate a detailed mechanism
for this reaction, the requirement for molecular oxygen and the effect of certain radical initiators and inhibitors strongly suggests that any possible mechanism should be free radical in nature (see Fig. 4). A trimethylsiloxy radical has been invoked as the reactive species in other oxidations that have occurred during silylation (10) and circumstantial evidence suggests that an oxy radical might likewise participate in this oxidation. Recent studies have also shown that the behavior of trialkylsiloxy radicals parallels that of alkoxy radicals, so they are capable of facile hydrogen abstraction (2 1). The difference in deuterium retention at the 6-position of thes-triazine ring after addition of dibenzoyl peroxide might be explained by a difference in reactivity of oxy radicals with the benzoyloxy radicals being more reactive and less discriminating. The accelerating effect of heating could be due to either enhanced radical formation or increased decomposition of an oxygenated intermediate to an aromatized product. An oxygenated intermediate has not been observed during gc/ms analysis, but one would expect it to be relatively unstable. Any mechanism postulating an oxygenated intermediate would also require either a stoichiometric amount of oxygen or some means of re-
212
KELLEY, Messi-a-0.SMe3
ABBASI,
-*
2 Mep-0
.
NHTMS A. N/
A N/
[.
OSiMe3
N-TMS
OANA
7
TMWd
TMSO
NHTMS
N-TMS
OAN&
AND BEISLER
TMSOd
OTMS
’
+ MepoH
OTMS
TMSO
OSiMe3 1 NHTMS NJ-/
HNTMS NAP
N--Me,
,kN$OSMe3
b H
TMSO
)
,&,A
-Me3SiOS~Me3 TMSO
TMti
~TMS
TMS6
OTMS
FIG. 4. A possible mechanism for silylation-mediated oxidation. The fishhook half-arrow indicates transfer of a single electron, while the conventional doubly barbed arrow indicates transfer of an electron pair.
generating oxy radicals. Preparative scale silylation-mediated oxidations of dihydros-triazine nucleosides generally required longer reaction times for complete aromatization than did comparable microscale silylation. Since a much larger excess of reagent was used during microscale silylations, the larger proportion of dissolved oxygen might account for the faster rate. Indeed, the preparative scale reactions could be accelerated by bubbling oxygen through the silylation mixture. Analyticaf
and Synthetic Considerations
Silylation-mediated oxidation is important from an analytical standpoint since trimethylsilylation is usually the method of choice for making volatile derivatives of nucleosides and their bases for subsequent gc and gclms analysis. Thus one would want to block, or at least be aware of the occurrence of oxidation, when susceptible compounds such as HZ-U, a minor com-
ponent of some tRNAs (22), or H,-5-AC, a new antitumor agent, are derivatized for gas phase analysis. Indeed, resolution of the question whether Hz-5-AC is a prodrug of 5-AC (13) is invariably complicated by its ease of aromatization upon derivatization for analysis. The trimethylsilyl moiety is also widely used as a protecting group for pyrimidine and purine bases in variations of the Hilbert-Johnson and fusion syntheses of nucleosides (23). Since forcing conditions may be used during either silylation of the base or coupling the base to the sugar, the potential for oxidation of susceptible compounds also occurs. The synthetic usefulness of silylationmediated oxidation has already been demonstrated in the synthesis of the previously unattained nucleoside Ara-5-AC (14). This reaction provides a mild and simple method of obtaining an aromatic system from a suitable reduced precursor. The appreciable deuterium isotope effect also makes this
SILYLATION-MEDIATED
reaction attractive for the microscale synthesis of nonexchangeable deuterated or tritiated derivatives in a simple one-pot procedure from an appropriately labeled precursor. Although the labeled molecule would not be 100% isotopically pure, one would not be faced with a total synthesis but simply a reduction and oxidation to obtain the target compound. Even in those cases where other labels are desired, aromatization through silylation-mediated oxidation has the potential to simplify the synthetic procedure. A recent approach to the synthesis of an isotopically labeled pyrimidine nucleoside utilized a four-step conversion of dihydrouracil to uracil before its silylation and subsequent reaction with I- 0 - acetyl - 2,3,4 - tri - 0 - benzoyl - /YI- D ribofuranoside to form the corresponding uridine (24). The required bis-o-trimethylsilyl uracil could be produced in one step through silylation-mediated oxidation. Work is currently in progress on the above approaches to isotopically labeled molecules. ACKNOWLEDGMENT The authors thank Dr. John S. Driscoll of this section for his encouragement and many helpful discussions during the course of this work.
6. Krutzsch, H. C., and Pisano, J. J. (1975) in Peptides: Chemistry, Structure and Biology (Walter, R., and Meienhofer, J., eds.), pp. 985990, Ann Arbor Science Publishers, Ann Arbor, Mich. 7. Chambaz, E. M., and Horning, E. C. (1968) Anal.
Biochem.
30, 7-24.
8. DeJongh, D. C., Radford, T., Hribar, J. D., Hanessian, S., Bieber, M., Dawson, G., and Sweeley, C. C. (1969) J. Amer. Chem. Sot. 91, 1728- 1740. 9. Chambaz, E. M., Maume, G., Maume, B., and Horning, E. C. (1968) Anal. Lett. 1, 749-761. 10. Maume, G. M., and Horning, E. C. (1969) Tetrahedron
11. Thompson, 12. 13. 14. 15. 16. 17. 18. 19. 20.
REFERENCES 1. Pierce, A. E. (1968) Silylation of Organic Compounds, p. 1, Pierce Chemical Co., Rockford, III. 2. Poole, C. F. (1977) in Handbook of Derivatives for Gas Chromatography (Blau, K., and King, G., eds.), pp. 152-200, Heyden, London. 3. Butts, W. C. (197O)J. Chromatogr. Sri. 8,474-476. 4. Hattox, S. E., and McCloskey, J. A. (1974) Anal. Chem. 46, 1378-1383. 5. Gehrke, C. W., and Pate], A. B. (1976) J. Chromatogr. 123, 335-345.
213
OXIDATION
21.
22. 23. 24.
Lett.,
343-346.
R. M., and Horning, E. C. (1973) Steroids Lipids Res. 4, 135- 142. Von Minden, D. L., Stillwell, R. N., Koenig, W. A., Lyman, K. J., and McCloskey, J. A. (1972) Anal. Biochem. 50, 110-121. Beisler, J. A., Abbasi, M. M., Kelley, J. A., and Dtiscoll, J. S. (1977) J. Med. Chem. 20, 806-812. Beisler, J. A., Abbasi, M. M., and Driscoll, J. S. (1977) Biochem. Pharmacol. 26, 2469-2472. Kovats, E. (1958)Helv. Chim. Acta 41, 1915- 1932. Ettre, L. S. (1964) Ann/. Chem. 36, 31A-41A. Koenig, W. A., Smith, L. C., Crain, P. F., and McCloskey, J. A. (1971) Biochemistry 10, 3968-3979. White, E., Krueger, P. M., and McCloskey, J. A. (1972) J. Org. Chem. 37, 430-438. Greene, F. D., Adam, W., and Cantrill, J. E. (1961) J. Amer. Chem. Sot. 83, 3461-3468. Greene, F. D., and Adam, W. (1963) J. Org. Chem. 28, 3550-3551. Cookson, P. G., Davies, A. G., Fazal, N. A., and Roberts, B. P. (1976) J. Amer. Chem. Sot. 98, 616-618. Zachau, H. G. (1969) Angewl. Chem. Int. Ed. Engl. 8, 711-727. Goodman, L. (1974) in Basic Principles in Nucleic Acid Chemistry (T’so, P. 0. P., ed.), Vol. 1, pp. 100- 108, Academic Press, New York. Roberts, J. L., and Poulter, C. D. (1978) J. Org. Chem. 43, 1547- 1550.
BIOCHEMISTRY
103, 203-213 (1980)
Silylation-Mediated Oxidation of Dihydropyrimidine Bases and Nucleosidesl JAMES A. KELLEY,MOHAMED Drug
M. ABBASI,*ANDJOHN
Design and Chemistry Section, Laboratory of Medicinal Chemiswy Therapeutics Program, Division of Cancer Treatment. National National Institutes of Health, Bethesda, Maryland
A. BEISLER
and Biology, Developmental Cancer Institute, 20205
Received September 6, 1979 Vigorous trimethylsilylation of dihydropyrimidine and dihydro-s-triazine bases and nucleosides results in their dehydrogenation to the analogous aromatic derivatives. This transformation, which cannot be effected by conventional dehydrogenating reagents, has been studied in detail using gas chromatographic and mass spectrometric methods. For 5,6-dihydro-5azacytidine (ta) the oxidation is quantitative, requires molecular oxygen as well as reagents capable of both N- and O-silylation, is accelerated by heat and selected free radical initiators, and is inhibited by diethyldithiocarbamate and galvinoxyl free radical. This anomalous silylation reaction occurs on both an analytical and preparative scale. Silylation-mediated oxidation also exhibits a substantial deuterium isotope effect with preferential retention of deuterium at the new site of unsaturation. The potential of this new reaction for the synthesis of aromatic systems and for the insertion of nonexchangeable isotopic labels in suitable reduced precursors is illustrated.
Silylation is defined as the introduction of a trimethylsilyl group into a molecule in substitution for an active hydrogen or in replacement of the metal component of a salt (1). As such, it is probably the most widely employed technique for the derivatization of polar compounds of biomedical interest for subsequent analysis by gas chromatography and combined gas chromatography-mass spectrometry (2). Nucleosides (3-9, peptides (6), steroids (7), and sugars (8) are but a few of the classes of compounds that have been rendered volatile enough for gas phase analysis. The popularity of trimethylsilylation rests with the simplicity of the procedure itself, its versatility and wide range of applicability, and ’ Presented in part at the 25th Annual Conference on Mass Spectrometry and Allied Topics, Washington, D. C., May 1977. ’ Present address: National Research Center, Medicinal Chemistry, Chemotherapy Laboratory, Dokki, Cairo, Egypt. 203
the usually straightforward formation of derivatives. Out of all the compounds derivatized by silylation and reported in the literature, there have only been a few instances of the formation of what might be called abnormal silylation products. Interestingly enough, these examples all involved oxidation of the compound being silylated. The TMS3 enol ethers of suitable keto-steroids 3 Abbreviations used: TMS, trimethylsilyl: 5-AC, 5-azacytidine; Am-S-AC, I-/3-~arabinofuranosyl-S-azacytosine; AraU, I-/3-o-arabinofuranosyl-uracil; BSA, N ,O-bis(trimethylsilyI)-acetamide; BSTFA, N ,O-bis(trimethylsilyl)-trifluoroacetamide: DDQ, 2,3-dichloroS,&iicyano-l,4-benzoquinone; gc, gas chromatography, gc/ms, gas chromatography-mass spectrometry; H,S-AC, 5,6-dihydro-5-azacytidine; H,-5-AC-d,, 5,6[6-2H]dihydro-5-azacytidine; H,-Ara-5-AC, I-p-o-arabinofuranosyl-5,6-dihydro-5-azacytosine; H,-Ara-5 AC-d,, I-p-o-arabinofuranosyl-5,6-[6-‘Hldihydro-5azacytosine; H,-AraU, I-P-o-arabinofuranosyl-5,6-dihydrouracil; HZ-U, 5,6-dihydrouridine; TMSDEA, trimethylsilyldiethylamine; TMCS, trimethylchlorosilane: TSIM, trimethylsilylimidazole. OOO3-2697/8O/O60203-11$02.OO/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
204
KELLEY,
ABBASI,
were found to form the corresponding cr-silyloxy ketones (9). This reaction was catalyzed by ultraviolet light or dibenzoyl peroxide and was presumed to be free radical in nature (10). Persilylation of norethynodrel and 5( lO)-estrene-17/3-ol-3-one with TSIM was reported to aromatize the A-rings of these compounds (11). Trimethylsilylation of 7-methylpurine nucleosides on a microgram scale resulted in molecular oxygen incorporation as well as derivatization to form the corresponding 7-methyl-8-oxo nucleosides (12). We now add to this short list of anomalous silylation reactions by describing here the silylation-mediated oxidation of 5,6-dihydropyrimidine and 5,6dihydro-s-triazine nucleosides and their respective aglycones to the corresponding aromatic derivatives. This report defines the silylation conditions required to effect this conversion and describes studies undertaken to elucidate the mechanism of this transformation. METHODS Materials
BSA, BSTFA, TMSDEA, and 1% TMCS in BSTFA were purchased from Pierce Chemical Company, Rockford, Ill. Acetonitrile, which was “distilled in glass” grade from Burdick and Jackson, Muskegon, Mich., was dried by distillation from P,O, and stored over preextracted and activated 3-pm molecular sieves. Pyridine was redistilled from KOH after a 24-h reflux. Methylene chloride was refluxed with calcium chloride overnight, filtered and distilled from PZOs, and then likewise stored over molecular sieves. Trifluoroacetic anhydride (99% +, Gold Label), DDQ, m-chloroperbenzoic acid, and galvinoxyl free radical were obtained from Aldrich Chemical Company, Milwaukee, Wis.; Aldrich was also an alternate source of BSTFA. Sodium diethyldithiocarbamate, dibenzoyl peroxide, and MnO, were purchased from Fisher Scientific Company, Fairlawn, N. J., while di-f-butyl
AND
BEISLER
peroxide was obtained from Pfaltz and Bauer, Stamford, Connecticut. Chloranil was supplied by Eastman Organic Chemicals, Rochester, N. Y., and 30% hydrogen peroxide by J. T. Baker, Phillipsburg, N. J. Uridine, HZ-U, 5 &dihydro-2,4dihydroxypyrimidine (dihydrouracil), and 4,5(5,6)dihydro-5-methyl-2,Cdihydroxypyrimidine (dihydrothymine) were purchased from Sigma Chemical Company, St. Louis, Missouri, while AraU was obtained from Pfanstiehl Laboratories, Waukegan, Illinois. 5-AC was obtained from the Division of Cancer Treatment, NCI, NIH while HZ-5-AC, H,-5-AC-dl, 5,6-dihydro-5-azacytosine, and H,-Ara-5-AC were synthesized as previously described (13,14). H,-AraU was prepared by the room temperature and atmospheric pressure hydrogenation of an aqueous solution of 500 mg (2.0 mmol) of AraU over 300 mg 5% Rh/C catalyst. Workup after cessation of hydrogen uptake (50 min, 2.8 mmol) and recrystallization from methanol afforded 180 mg (36%) of slightly hygroscopic crystals, mp 173174°C; NMR (DzO) S 5.97 (d,J = 5.5 Hz, 1, C,,H), 4.33 (t,J = 6 Hz, 1, C,,H), 3.4-4.2 (complex multiplet, 6H), 2.68 (t,J = 6.5 Hz, 2, C,W. Anal. Calcd for C,H,,N,OB: C, 43.90; H, 5.73; N, 11.38. Found: C, 43.71; H, 5.49; N, 11.14. Gas Chromatography Spectrometry
and Mass
Trimethylsilyl derivatives of nucleosides and their aglycones were analyzed on a Varian 2740 gas chromatograph equipped with a flame ionization detector and linear temperature programmer. A 1.83 m x 2 mm id. glass column packed with either 3% SE-30 or 3% OV-17 on 100/120 Gas Chrom Q was operated isothermally in the range 120-140°C for aglycones and in the range 220-230°C for nucleosides. Quantitative analysis of oxidation mixtures was accomplished with temperature programming from 220 to 250°C at 2”Clmin. Typical gc
SILYLATION-MEDIATED
operating conditions employed injector and detector temperatures of 260°C and flow rates of approximately 30 ml/minute for both the helium carrier gas and hydrogen, and 300 ml/minute for air. Peak areas and retention times were obtained on a Beckman Auto-Pro 30 computational integrator and isothermal gas chromatographic retention indices were calculated using the appropriate n-alkanes as internal standards (15, 16). Low-resolution electron impact mass spectra were obtained on a DuPont 2 I-492B gas chromatograph-mass spectrometer interfaced to a VG 2040 data system. Mass spectra were obtained with sample introduction either by direct probe (trifluoroacetyl derivatives) or via a Varian 2740 gc (trimethylsilyl derivatives) coupled to the mass spectrometer by a single-stage glass jet separator. Gas chromatography operating conditions were the same as those above. Standard mass spectrometer operating conditions were: transfer line and jet separator, 250°C; ion source, 255°C; electron energy, 75 eV; ionizing current 250 PA; and scan speed, 2 s/decade. Preparation
of Derivatives
Trimethylsilyl derivatives were prepared on a microscale by the room temperature reaction of l-2 mg of the appropriate nucleoside or aglycone with 0.15 ml of silylating reagent and 0.30 ml of redistilled solvent in a 3.5-ml screw-cap glass vial equipped with a Teflon-lined rubber septum. Sonication was routinely used to speed solution and accelerate derivatization. The silylation mixture was allowed to stand at room temperature for a minimum of 15 min after solution and l- to ~-PI aliqouts of the sample were taken for analysis. When required, the tightly sealed silylation mixtures were heated in a thermostated heating block maintained at the desired temperature. Trifluoroacetyl derivatives were prepared on the same scale using trifluoroacetic anhydride and following the procedure of Koenig et al. (17).
205
OXIDATION
Kinetic
Studies
Methanol stock solutions of approximately 1 mg/ml concentration were made of uridine and HZ-S-AC. A 1.O-ml aliquot of each stock solution was added to a 3.5-ml glass screw cap vial and the methanol solution evaporated to dryness under a stream of nitrogen. The residue was dried in vacua and silylated in the same manner as above. After gc analysis to ensure derivatization of both substrate (HZ-j-AC) and internal standard (U), the sample was placed in the heating block at the desired temperature between 60 and 100°C and aliquots were withdrawn for gc analysis at periodic intervals. Both the peak height and area ratios of trimethylsilylated HZ-j-AC (substrate) and 5-AC (product) to internal standard were calculated, and the logarithm of these ratios was plotted versus time. A standard curve was also constructed for 5-AC in the range 0. l- 1.5 mg and used to determine the absolute yield of product. Silylation
in the Absence
of Oxygen
A I.O-ml aliquot each of H,-5-AC and uridine stock solution was added to a 10 x loo-mm vacuum reaction tube equipped with an adjustable Teflon plunger (Pierce Chemical Co.) and evaporated to dryness in the same manner as above. BSTFA and acetonitrile were degassed by a freezevacuum-thaw cycle and stored under nitrogen. To the residue in the vacuum reaction tube was added 0.20 ml degassed BSTFA and 0.40 ml degassed acetonitrile and the freeze-vacuum-thaw cycle was repeated three times. The tightly sealed reaction mixture was then sonicated for 20 min at room temperature before being placed in a heating block. A control sample was prepared and silylated in an identical manner except degassed reagents were not used and no attempt was made to exclude oxygen. Where the kinetics of this oxidation were being studied, an identical but separate degassed sample was prepared for each
206
KELLEY,
ABBASI,
analysis time, since it was impossible to repeatedly sample a reaction mixture without introducing oxygen. Silylation in the Presence Initiators and Inhibitors
of Radical
Samples of dihydro-s-triazine nucleoside substrates and internal standard were prepared and silylated in 3.5 ml glass screw-cap vials as described above. Solutions of sodium diethyldithiocarbamate (4.5 mg/ml), dibenzoyl peroxide (10 mg/ml), and meta-chloroperbenzoic acid (10 mg/ml) were prepared in acetonitrile to facilitate addition to the silylation mixture. For these reagents the volume corresponding to 0.25-2.0 molar eq of substrate was calculated, and for each sample a corresponding adjustment was made in the amount of acetonitrile added to the BSTFA so that the final ratio of BSTFA:acetonitrile would still be 2: 1. Di-t-Butyl peroxide, galvinoxyl free radical, and 30% hydrogen peroxide were added directly to the silylation mixture in amounts corresponding to approximately 1 molar eq of substrate. After an initial gc analysis of the room-temperature reaction mixture, the tightly sealed samples were placed in a heating block at either 66 or 70°C and analyzed periodically by gc. The peak height and area ratios of substrate to internal standard were determined and used to calculate a percentage conversion relative to control. Determination
of Isotope
Ratios
To determine the amount of deuterated to nondeuterated isomer selected reaction mixtures of both HZ-S-AC-d, and H,-Ara-5AC-d, were analyzed by limited scan (m/z 480-550) gc/ms. The mass spectrometer was scanned repetitively every 5 s at a scan rate of 10 s/decade over the gc peak of interest, and the collector slits were opened to give flat-topped peaks with greater than unit resolution. After the A + 1 isotopic contributions of the Mt (m/z 532) and M-CH, (m/z 517) ions of 5-AC-TMS, had
AND
BEISLER
been corrected for, the relative amounts of monodeuterated and unlabeled 5-AC were determined from these mass peaks, and an average was obtained for all the scans over the gc peak. Attempted
Oxidation
with Other Reagents
To 100 mg (0.4 mmol) of HZ-j-AC in 20 ml methanol was added 500 mg (2.20 mmol) DDQ. The progress of the reaction was monitored using tic systems that have been previously described (13). The solution showed no change after standing at room temperature for 10 h or after reflux for 34 h. Only starting material could be isolated upon workup and recrystallization. Attempted dehydrogenation of Hz-j-AC by refluxing with chloranil in ethanol for 24 h or by heating with MnO, in hexamethylphosphoramide for 6 h at 60°C was likewise unsuccessful. RESULTS
AND DISCUSSION
5-Azacytidine (l), a nucleoside antimetabolite that is effective in the clinical treatment of acute myelogenous leukemia, was reduced with sodium borohydride to form 5,6-dihydro-5-azacytidine (2a) in order to improve the former’s hydrolytic stability yet retain its antitumor activity (13). The gc/ms analysis of 2a to determine purity and to confirm structure required quantitative formation of a volatile derivative. However, microscale silylation of 2a according to standard procedures with BSTFA in CH,CN at 100°C for 2 h (4) did not produce the expected pentatrimethylsilyl derivative 3a, but silylated 5-AC (4a) instead (Fig. 1). The expected derivative 3a was not observed until silylation was carried out under much milder conditions at room temperature. Moreover, when monodeuterated HZ-j-AC (2b) was vigorously silylated, the S-AC that was formed preferentially retained deuterium to give a 3: 1 mixture of 4b and 4a. These initial observations prompted a more comprehensive study of the apparent
SILYLATION-MEDIATED
;’
2
HO
OH
1
,;;;2;gc;;ii$:TM\M~5 2
HO
OH
2a,R=H 2b. R = D
FIG. 1. Synthesis and silylation-mediated
TMSO
OTMS
OTMS
TMSO
3a,R=H 3b, R = D
4a,R=H 4b,R=D
oxidation of 5,6-dihydro-5-azacytidine.
dehydrogenation reaction that was occurring during trimethylsilylation. For the reliable analysis of 2a and other reduced nucleosides by gc and gclms it was important to define the scope of this oxidation and determine whether it could be blocked. Synthetically, this reaction offered a potential means of obtaining an aromatic system from a reduced precursor and also incorporating a nonexchangeable label into a suitable compound. Kinetics
207
OXIDATION
&-5AC
-
B~FA~~~~~NwI,
S-AC 2 hr
at 76.5+l”
of Oxidation
The microscale conversion of H,-5-AC to 5-AC during silylation was monitored by gc analysis using an internal standard (Fig. 2). Gas chromatography was an ideal method for this analysis since silylation was required for oxidation (see below) and the derivatized nucleosides could be well separated on an OV-17 column (Table 1). Uridine was chosen as an internal standard because of its favorable retention index and because its structural similarity as a persilylated nucleoside would hopefully allow it to act as a carrier for the less stable derivatives of interest (e.g., 3a). The above method of analysis required that the sample be silylated at room temperature before controlled heating to initiate the silylation-mediated oxidation reaction. Complete and reproducible silylation of the uridine internal standard could be accomplished without heating; sonication of the silylation mixture for 20 min followed by standing at room temperature for an
220 I 0
230 I 5
240 I 10
250 iso 250 DC II I 15 20 min
FIG. 2. Quantitative gas chromatographic analysis of a silylation-mediated oxidation reaction mixture of 5,Qdihydro-5azacytidine that was heated at 76.5 + 1°C for 2 h. A 1.83 m x 2 mm-i.d. glass column packed with 3% OV-17 on 100/120 mesh Gas Chrom Q was temperature programmed from 220 to 250°C at 2Wmin and then maintained isothermally at 250°C for 5 min. See under Methods for additional details of the analysis.
208
KELLEY,
ABBASI, TABLE
AND BEISLER 1
ISOTHERMAL RETENTION INDICES OF BASE AND NUCLEOSIDE TRIMETHYLSILYL DERIVATIVES I” No. TMS groups
M,
SE-3W
OV-17*
Uracil Dihydrouracil Thymine Dihydrothymine 5-Azacytosine Dihydro-5-azacytosine
2 2 2 2 2 3
256 258 270 272 256 330
1335(A) 1470(A) 1392(B) 1475(B) 1477(B) 1671(B)
1453(A) 1636(A) 1504(B) 161 l(B) 1634(C) 1774(C)
Uridine Dihydrouridine l-/3-D-Arabinofuranosyluracil 1-/3-D-Arabinofuranosyldihydrouracil 5Azacytidine Dihydro-5-azacytidine I-p-D-Arabinofuranosyl-5-azacytosine I-P-o-Arabinofuranosyldihydro-5-azacytosine
4 4 4 4 4 5 4 5
532 534 532 534 532 606 532 606
2469(D) 2420(D) 2505(D) 2462(D) 2622(D) 2470(D) 2648(D) 2538(D)
2625(D) 2539(D) 2613(D) 2560(D) 2823(E) 2530(E) 2808(E) 2564(E)
Parent base or nucleoside
n Retention indices were measured with a precision of +3 units. * Letter in parentheses indicates isothermal temperature: (A) = 120°C; (B) = 130°C; (C) = 140°C; (D) = 220°C; (E) = 230°C.
2.ooo
L-1 H&AC Uridine
‘h = -
degassed reagents, N, BSTFA/CH,CN (1:2), 76.5?1’
0, excluded 0
,200
complete cO”“erSlOn
,100
cl
I 1
I 2
I 3 TIME
/,I I’ 21
4 I 22
Ihrl
FIG. 3. Kinetics of the silylation-mediated oxidation of 5,6-dihydro-5-azacytidine using degassed reagents under conditions of oxygen exclusion and using untreated reagents under normal conditions (control). See under Methods for additional details.
additional 90 min with occasional shaking gave the same results as the recommended optimum conditions of heating for 15 min at 150°C (5). In an independent check of both methods the area ratio of uridine to a pyrene internal standard did not vary more than the expected precision (2 5%) of the gc method. When l-2 mg of H,-5-AC was silylated with 0.45 ml of 1:2 BSTFA and CH&N (a 25-50 M excess of silyl groups) and then oxidized by heating, the reaction was pseudo first order with respect to silylated H,-5-AC (see Fig. 3). Since oxidation was markedly accelerated by heating, most experiments were conducted at moderate temperatures so that the reaction would not be too fast to follow. A half-life of 2.02 h was observed at 76°C and the reaction was complete within 22 h; no silylated reduced nucleoside was observed by gc. Raising the temperature slightly to 80°C resulted in a somewhat shorter half-life of 1.68 h. The conversion of silylated H,-5-AC to derivatized
SILYLATION-MEDIATED
S-AC was quantitative after 24 h at this temperature with no formation of side products and no trace of starting material. An absolute gc yield of 98.0 +- 1.6% based on underivatized reduced nucleoside 2a was measured. Effect of Varying Substrate and Reaction Conditions
209
OXIDATION TABLE
3
MICROSCALE SILYLATION-MEDIATED OXIDATION OF VARIOUS SUBSTRATES (7’ = 70°C)
Product”
Substrate” H,-5-AC H,-Ara-S-AC H,-U 5,6-Dihydro-.5azacytosine 5,6-Dihydrouracil 5,6-Dihydrothymine
5-AC Ara-5-AC U
Conversion completed 00” 22’ 84 >84”
Strong silylating reagents such as BSTFA 20 5-Azacytosine or BSA easily effected oxidation while Uracil 45 weaker silyl donors such as TMSDEA or TSIM gave poor yields (see Table 2). These Thymine 160 latter reagents do not completely derivatize amines so it appears that silylation of both a As TMS derivative. b Derivatized substrate was no longer detectable by oxygen and nitrogen is required. SiIylationmediated oxidation was also observed for gc or gcims. c T = 76°C. other reduced pyrimidine nucleosides as d Reaction not followed to completion. well as their aglycones (see Table 3). The arabinosyl analog of dihydro-5azacytidine (H,-Ara-5-AC) could be aromatized to the milder conditions employed here likewise previously unattained arabinosyl derivative caused the eventual aromatization of diof 5-AC (14), but the rate of conversion hydrouracil to uracil as well as the much was approximately four times slower than slower transformation of dihydrothymine to that of the ribosyl derivative. Dihydrothymine during silylation. Other nucleoside uridine and its arabinosyl analog could also derivatization methods such as trifluorobe oxidized to the corresponding uridine acetylation (17) caused no oxidation of the derivatives, but the rate of conversion was reduced derivatives even with vigorous much slower than for either of the dihydroheating. Dihydro-5-azacytidine smoothly s-triazine nucleosides. The aromatization of formed a tetratrifluoroacetyl derivative whose dihydrouracil to uracil during vigorous molecular weight (by mass spectrometry) silylation was previously observed by White was 2 amu higher than that of similarly et al. (18) but not further investigated. The derivatized 5-azacytidine. Dehydrogenating and aromatizing reagents such as DDQ, chloranil and MnOz were also ineffective TABLE 2 in carrying out this transformation on the EFFECT OF VARIOUS REAGENTS ON SILYLATIONunderivatized reduced nucleosides. MEDIATED OXIDATION
Relative gc yield of S-AC (15 h, 80°C)
Reagents BSTFA/CH,CN (1:2) BSTFAipyridine (1:2) BSTFA, 1% TMCS/CH,CN BSA/CH,CN (1:2) TMSDEAKH,CN (1:2) TSIM/CH,CN (1:2)
OF H,-5-AC
(1:2)
100 100 96 84 19 0
Deuterium
Isotope Effect
A substantial deuterium isotope effect was observed upon gc/ms analysis of 6-deutero5,6-dihydro-5-azacytidine (2b) that had undergone silylation-mediated oxidation. The 5-AC that was formed consisted of both d, and d, derivatives in an approximate ratio of 3: 1. Limited scan gc/ms analysis gave a more precise value of 2.9 + 0.2 for the d,:d,
210
KELLEY,
ABBASI,
ratio. The M-CHB peak (m/z 517) rather than the molecular ion peak (m/z 532) was used to determine d,/d, because of its much greater relative intensity (0.8 versus 0.1%). Silylation-mediated oxidation of monodeuterated H,-Ara-5-AC resulted in a product di:d,, ratio of 2.8 ? 0.3, the same value within experimental error, even though this reaction was much slower. Therefore, at least in the case of the above s-triazine nucleosides, the rate-determining step of silylation-mediated oxidation involves breaking the C-H bond at the 6-position.
AND
BEISLER
gc analysis, to the atmosphere followed by heating for a sufficient length of time showed complete conversion to 5-AC. Figure 3 compares the silylation-mediated oxidation of a deoxygenated sample of H,-5-AC versus that of a control. Even after 22 h at 76.5 + 1°C the deoxygenated sample showed only trace conversion to the aromatized nucleoside, which was probably due to incomplete removal of oxygen. In contrast the control exhibited complete conversion while following good pseudo first-order kinetics. The conversion of dihydrouracil to uracil during silylation with BSTFA and CH,CN at 150°C for 1 h was also blocked when the reagents were degassed.
Oxygen Requirement It was possible that the silylating reagent alone was not responsible for the aromatizaEffect of Free Radical Initiators and tion of these reduced pyrimidine nucleosides. Inhibitors Trace analyses of the various silylating The requirement for molecular oxygen reagents showed that there were no unusual suggested that silylation-mediated oxidation concentrations (> 10 ppm) of metals such as might be occurring by a free radical mechFe, Cu, or Zn which could conceivably anism. Thus the effect of adding various catalyze an oxidation.4 Certain silylating reagents can contain appreciable amounts of free radical initiators and inhibitors to a dissolved oxygen (12). The requirement of silylation mixture of H,-5-AC was quantitatively measured (Table 4). Of the initiators molecular oxygen for silylation-mediated dibenzoyl peroxide was the most effective oxidation was demonstrated in a series of in promoting oxidation at room temperature. parallel experiments where identical samples Normally several days at room temperature of reduced nucleoside 2a were silylated with were required before any 5-AC could be degassed BSTFA and CH,CN or with detected. However, increasing amounts of reagents that had been degassed and redibenzoyl peroxide up to a maximum charged with either oxygen or nitrogen corresponding to a molar equivalent of subbefore heating at 70°C for 4 h. The results strate proportionately increased the yield of were compared to a control, silylated with silylated 5-AC. Heating this silylation mixture, untreated BSTFA, that had been heated for however, did not markedly increase the the same length of time. No aromatization yield of aromatized nucleoside with respect to derivatized 5-AC was observed when to control. A possible reason for this was silylation was conducted with completely the increased formation of by-products upon degassed reagents or with degassed reagents in a nitrogen atmosphere. However, the heating since a compound characterized as a by mass degassed silylating reagent that had been benzoylated dihydro-5-azacytidine was isolated in about 30% recharged with oxygen showed a 91% spectrometry yield from a preparative scale reaction to conversion to 4a relative to the control. Exposure of a degassed sample, which had which dibenzoyl peroxide had been added as an accelerator. Even more interesting been analyzed previously by quantitative was the result of silylation-mediated oxida4 Trace analyses were performed in duplicate by tion of monodeuterated Hz-5-AC in the Galbraith Laboratories, Inc., Knoxville, TN 37921. presence of 1.0 eq of dibenzoyl peroxide
SILYLATION-MEDIATED TABLE EFFECT
OF RADICAL
INITIATORS
AND
INHIBITORS
211
OXIDATION 4
ON THE
SILYLATION-MEDIATED
Relative conversion to 5-AC (%)
OXIDATION
OF
H,-5-AC
Relative amount H,-5-AC remaining (%)*
Reagent
Initial (RT)
4 h (66 ? 1°C)
Initial (RT)
4 h (66 k 1°C)
Control Dibenzoyl peroxide (1 .O eq) H,O, (30%) m-Chloroperbenzoic acid Di-t-butyl peroxide Diethyldithiocarbamate Galvinoxyl
0 58 4 0 2 10 27
70 74 72 63 71’ 13 15’
100 0 52 65 52 100 58
I1 0 0 21 6’ 87 61’
a One hundred percent conversion is defined as a control which has been allowed to react to completion. A control is a silylation mixture of HZ-S-AC to which initiators or inhibitors have not been added. b One hundred percent remaining is defined as a control at room temperature. c 70 c 1°C.
at room temperature. Here the dI:d,, ratio in the SAC that was produced was 1.8 2 0.3, significantly less than that observed under control conditions. Other compounds added as radical initiators did not markedly promote the room temperature oxidation of silylated HZ-SAC. Addition of aqueous hydrogen peroxide or meta-chloroperbenzoic acid had little effect at room temperature or upon heating relative to the control. The same was true for di-t-butyl peroxide, but this may be due to the necessity of temperatures of 120- 150°C to generate the t-butoxy radicals. Galvinoxyl, a free radical scavenger (19,20), seemed to act as an initiator at room temperature, but showed strong inhibition after heating for 4 h at 70°C. About 60% of the silylated starting material remained after heating. The most effective inhibitor of oxidation was sodium diethyldithiocarbamate. Although a small initial conversion to aromatized derivative was seen, this did not increase significantly with heating and the reduced starting material could be detected in undiminished yield. Mechanism Although there is insufficient data at present to formulate a detailed mechanism
for this reaction, the requirement for molecular oxygen and the effect of certain radical initiators and inhibitors strongly suggests that any possible mechanism should be free radical in nature (see Fig. 4). A trimethylsiloxy radical has been invoked as the reactive species in other oxidations that have occurred during silylation (10) and circumstantial evidence suggests that an oxy radical might likewise participate in this oxidation. Recent studies have also shown that the behavior of trialkylsiloxy radicals parallels that of alkoxy radicals, so they are capable of facile hydrogen abstraction (2 1). The difference in deuterium retention at the 6-position of thes-triazine ring after addition of dibenzoyl peroxide might be explained by a difference in reactivity of oxy radicals with the benzoyloxy radicals being more reactive and less discriminating. The accelerating effect of heating could be due to either enhanced radical formation or increased decomposition of an oxygenated intermediate to an aromatized product. An oxygenated intermediate has not been observed during gc/ms analysis, but one would expect it to be relatively unstable. Any mechanism postulating an oxygenated intermediate would also require either a stoichiometric amount of oxygen or some means of re-
212
KELLEY, Messi-a-0.SMe3
ABBASI,
-*
2 Mep-0
.
NHTMS A. N/
A N/
[.
OSiMe3
N-TMS
OANA
7
TMWd
TMSO
NHTMS
N-TMS
OAN&
AND BEISLER
TMSOd
OTMS
’
+ MepoH
OTMS
TMSO
OSiMe3 1 NHTMS NJ-/
HNTMS NAP
N--Me,
,kN$OSMe3
b H
TMSO
)
,&,A
-Me3SiOS~Me3 TMSO
TMti
~TMS
TMS6
OTMS
FIG. 4. A possible mechanism for silylation-mediated oxidation. The fishhook half-arrow indicates transfer of a single electron, while the conventional doubly barbed arrow indicates transfer of an electron pair.
generating oxy radicals. Preparative scale silylation-mediated oxidations of dihydros-triazine nucleosides generally required longer reaction times for complete aromatization than did comparable microscale silylation. Since a much larger excess of reagent was used during microscale silylations, the larger proportion of dissolved oxygen might account for the faster rate. Indeed, the preparative scale reactions could be accelerated by bubbling oxygen through the silylation mixture. Analyticaf
and Synthetic Considerations
Silylation-mediated oxidation is important from an analytical standpoint since trimethylsilylation is usually the method of choice for making volatile derivatives of nucleosides and their bases for subsequent gc and gclms analysis. Thus one would want to block, or at least be aware of the occurrence of oxidation, when susceptible compounds such as HZ-U, a minor com-
ponent of some tRNAs (22), or H,-5-AC, a new antitumor agent, are derivatized for gas phase analysis. Indeed, resolution of the question whether Hz-5-AC is a prodrug of 5-AC (13) is invariably complicated by its ease of aromatization upon derivatization for analysis. The trimethylsilyl moiety is also widely used as a protecting group for pyrimidine and purine bases in variations of the Hilbert-Johnson and fusion syntheses of nucleosides (23). Since forcing conditions may be used during either silylation of the base or coupling the base to the sugar, the potential for oxidation of susceptible compounds also occurs. The synthetic usefulness of silylationmediated oxidation has already been demonstrated in the synthesis of the previously unattained nucleoside Ara-5-AC (14). This reaction provides a mild and simple method of obtaining an aromatic system from a suitable reduced precursor. The appreciable deuterium isotope effect also makes this
SILYLATION-MEDIATED
reaction attractive for the microscale synthesis of nonexchangeable deuterated or tritiated derivatives in a simple one-pot procedure from an appropriately labeled precursor. Although the labeled molecule would not be 100% isotopically pure, one would not be faced with a total synthesis but simply a reduction and oxidation to obtain the target compound. Even in those cases where other labels are desired, aromatization through silylation-mediated oxidation has the potential to simplify the synthetic procedure. A recent approach to the synthesis of an isotopically labeled pyrimidine nucleoside utilized a four-step conversion of dihydrouracil to uracil before its silylation and subsequent reaction with I- 0 - acetyl - 2,3,4 - tri - 0 - benzoyl - /YI- D ribofuranoside to form the corresponding uridine (24). The required bis-o-trimethylsilyl uracil could be produced in one step through silylation-mediated oxidation. Work is currently in progress on the above approaches to isotopically labeled molecules. ACKNOWLEDGMENT The authors thank Dr. John S. Driscoll of this section for his encouragement and many helpful discussions during the course of this work.
6. Krutzsch, H. C., and Pisano, J. J. (1975) in Peptides: Chemistry, Structure and Biology (Walter, R., and Meienhofer, J., eds.), pp. 985990, Ann Arbor Science Publishers, Ann Arbor, Mich. 7. Chambaz, E. M., and Horning, E. C. (1968) Anal.
Biochem.
30, 7-24.
8. DeJongh, D. C., Radford, T., Hribar, J. D., Hanessian, S., Bieber, M., Dawson, G., and Sweeley, C. C. (1969) J. Amer. Chem. Sot. 91, 1728- 1740. 9. Chambaz, E. M., Maume, G., Maume, B., and Horning, E. C. (1968) Anal. Lett. 1, 749-761. 10. Maume, G. M., and Horning, E. C. (1969) Tetrahedron
11. Thompson, 12. 13. 14. 15. 16. 17. 18. 19. 20.
REFERENCES 1. Pierce, A. E. (1968) Silylation of Organic Compounds, p. 1, Pierce Chemical Co., Rockford, III. 2. Poole, C. F. (1977) in Handbook of Derivatives for Gas Chromatography (Blau, K., and King, G., eds.), pp. 152-200, Heyden, London. 3. Butts, W. C. (197O)J. Chromatogr. Sri. 8,474-476. 4. Hattox, S. E., and McCloskey, J. A. (1974) Anal. Chem. 46, 1378-1383. 5. Gehrke, C. W., and Pate], A. B. (1976) J. Chromatogr. 123, 335-345.
213
OXIDATION
21.
22. 23. 24.
Lett.,
343-346.
R. M., and Horning, E. C. (1973) Steroids Lipids Res. 4, 135- 142. Von Minden, D. L., Stillwell, R. N., Koenig, W. A., Lyman, K. J., and McCloskey, J. A. (1972) Anal. Biochem. 50, 110-121. Beisler, J. A., Abbasi, M. M., Kelley, J. A., and Dtiscoll, J. S. (1977) J. Med. Chem. 20, 806-812. Beisler, J. A., Abbasi, M. M., and Driscoll, J. S. (1977) Biochem. Pharmacol. 26, 2469-2472. Kovats, E. (1958)Helv. Chim. Acta 41, 1915- 1932. Ettre, L. S. (1964) Ann/. Chem. 36, 31A-41A. Koenig, W. A., Smith, L. C., Crain, P. F., and McCloskey, J. A. (1971) Biochemistry 10, 3968-3979. White, E., Krueger, P. M., and McCloskey, J. A. (1972) J. Org. Chem. 37, 430-438. Greene, F. D., Adam, W., and Cantrill, J. E. (1961) J. Amer. Chem. Sot. 83, 3461-3468. Greene, F. D., and Adam, W. (1963) J. Org. Chem. 28, 3550-3551. Cookson, P. G., Davies, A. G., Fazal, N. A., and Roberts, B. P. (1976) J. Amer. Chem. Sot. 98, 616-618. Zachau, H. G. (1969) Angewl. Chem. Int. Ed. Engl. 8, 711-727. Goodman, L. (1974) in Basic Principles in Nucleic Acid Chemistry (T’so, P. 0. P., ed.), Vol. 1, pp. 100- 108, Academic Press, New York. Roberts, J. L., and Poulter, C. D. (1978) J. Org. Chem. 43, 1547- 1550.