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Nucleosides (TMS Derivatives)
C H A P T E R 2 7
N U C L E O S I D E S ( T M S D E R I V A T I V E S )
Nucleosides consist of a purine or a pyrimidine base and a ribose or a deoxyribose sugar connected via a β-glycosidic linkage. These compounds are associated with structures of RNA (ribose sugars) and DNA (deoxy ribose sugars). The compounds are very polar and their analysis by GC/MS is only possible when they have been derivatized. It is possible to get fairly respectable spectra when the pure compounds are introduced into an EI source using a direct insertion probe. The structures of the nucleosides of most biological interest are shown in Figure 27.1.
27.1. DERIVATIZATION Add 0.25 mL of DMF (N,N-dimethylformamide) and 0.25 mL of TRI-Sil TBT reagent to the sample in a screw-cap septum vial. If TRI-Sil TBT reagent is not readily available, add 0.25 mL of acetonitrile and 0.25 mL of BSTFA reagent instead. Heat at 60 °C for at least 1 hour for ribonucleosides or for a minimum of 3 hours for deoxyribonucleosides. After cooling to room temperature, inject 1–2 µL of the reaction mixture directly into the gas chromatograph. The resulting derivatives have been reported to be stable for weeks if tightly capped and refrigerated [1].
27.2. GC SEPARATION NUCLEOSIDES
OF
DERIVATIZED
A. Capillary column 1. 2′-Deoxyuridine, thymidine, 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine: 10–30-m DB-17 column, 100–275 °C at 10 °C min�1; injection port at 280 °C. Gas Chromatography and Mass Spectrometry DOI: 10.1016/B978-0-12-373628-4.00027-7
� 2010 by Academic Press. Inc. All rights reserved.
369
370
Chapter 27 H2N
O NH
N
N
N NH2
HO
CH2
N
N
O
HO
OH 2′-Deoxyadenosine dA O
CH2
O
N
O
NH2
O NH
N O
HO
OH
CH2
N
O
HO
O
CH2
O
N
O
OH 2′-Deoxyuridine dU
OH 2′-Deoxycytidine dC
Thymidine dT
Figure 27.1
N
N
OH 2′-Deoxyguanosine dG
NH HO
CH2
Structures of the five biologically active nucleosides.
27.3. MASS SPECTRA
OF
TMS NUCLEOSIDES [2]
Display a mass chromatogram of m/z 103 to determine the elution time of the TMS–nucleosides. Next, determine the molecular weight by identify ing the M+• peak (which is usually observed) associated with an [M – 15]+ peak and often with [M – 90]+, [M – 105]+, and [M – 203]+ peaks. The M+• peak of the TMS derivatives of ribonucleosides is 88 m/z units higher than the TMS derivatives of the deoxyribonucleosides. If the difference between the m/z value of the M+• peak and the base peak is 260, the sugar portion is deoxyribose. A difference of 290 represents an o-methylribose, and a difference of 348 Da suggests a ribose. Nucleoside–TMS
MW
m/z Values that indicate the base
Base
2′-Deoxyuridine Thymidine 2′-Deoxyadenosine 2′-Deoxycytidine 2′-Deoxyguanosine
444 458 467 443 555
169, 183 192, 168, 280,
Uracil Thymine (5-methyluracil) Adenine Cytosine Guanine
184 207 183 295
Nucleosides (TMS Derivatives)
371
73
100
207 Si N
H N
N
103 Si O
O
50 236
45 59
81
Si
117 147165
218 264
60
Figure 27.2 MW 467 Da.
90
TMS
120 150 180 210 240 270 300
derivative
N
O
0 30
N
of
467
349 330 360
2′-deoxyadenosine,
EC:
390 420
450 480
C19H37N5O3Si3,
If necessary, the nucleosides can be hydrolyzed to the sugar and the base by heating in formic acid [2]. Kresbach et al. [3] have used pentafluorobenzyla tion combined with electron capture negative ionization for the detection of trace amounts of nucleobases. For fragmentation patterns of TMS 2′-, 3′-, and 5′-deoxynucleosides, see Reimer et al. [4] (Figure 27.2).
REFERENCES 1. Schram, K. H., McCloskey, J. L. (1979). In: Tsuji K, ed. GLC and HPLC of Therapeutic Agents. New York: Marcel Dekker. 2. Crain, P. F. (1990). In: McCloskey, J. A., ed. Methods in Enzymology, Chapter 43 (Vol. 193). San Diego, CA: Academic Press. 3. Kresbach, G. M., Annan, R. S., Saha, M. G., Giese, R. W., Vouros, P. (1988). Mass Spectrometric and Chromatographic Properties of Ring-Penta Fluorobenzylated Nucleobases used in the Trace Detection of Alkyl DNA Adducts. Proceedings of the 36th Annual Conference of the American Society for Mass Spectrometry, San Francisco, June 5–10. 4. Reimer, M. L. J., McClure, T. D., Schram, K. H. (1988). Investigation of the Fragmentation Patterns of the TMS Derivatives of 2’-, 3’-, and 5’-Deoxynucleosides. Proceedings of the 36th Annual Conference of the American Society for Mass Spectrometry, San Francisco, June 5–10.
N U C L E O S I D E S ( T M S D E R I V A T I V E S )
Nucleosides consist of a purine or a pyrimidine base and a ribose or a deoxyribose sugar connected via a β-glycosidic linkage. These compounds are associated with structures of RNA (ribose sugars) and DNA (deoxy ribose sugars). The compounds are very polar and their analysis by GC/MS is only possible when they have been derivatized. It is possible to get fairly respectable spectra when the pure compounds are introduced into an EI source using a direct insertion probe. The structures of the nucleosides of most biological interest are shown in Figure 27.1.
27.1. DERIVATIZATION Add 0.25 mL of DMF (N,N-dimethylformamide) and 0.25 mL of TRI-Sil TBT reagent to the sample in a screw-cap septum vial. If TRI-Sil TBT reagent is not readily available, add 0.25 mL of acetonitrile and 0.25 mL of BSTFA reagent instead. Heat at 60 °C for at least 1 hour for ribonucleosides or for a minimum of 3 hours for deoxyribonucleosides. After cooling to room temperature, inject 1–2 µL of the reaction mixture directly into the gas chromatograph. The resulting derivatives have been reported to be stable for weeks if tightly capped and refrigerated [1].
27.2. GC SEPARATION NUCLEOSIDES
OF
DERIVATIZED
A. Capillary column 1. 2′-Deoxyuridine, thymidine, 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine: 10–30-m DB-17 column, 100–275 °C at 10 °C min�1; injection port at 280 °C. Gas Chromatography and Mass Spectrometry DOI: 10.1016/B978-0-12-373628-4.00027-7
� 2010 by Academic Press. Inc. All rights reserved.
369
370
Chapter 27 H2N
O NH
N
N
N NH2
HO
CH2
N
N
O
HO
OH 2′-Deoxyadenosine dA O
CH2
O
N
O
NH2
O NH
N O
HO
OH
CH2
N
O
HO
O
CH2
O
N
O
OH 2′-Deoxyuridine dU
OH 2′-Deoxycytidine dC
Thymidine dT
Figure 27.1
N
N
OH 2′-Deoxyguanosine dG
NH HO
CH2
Structures of the five biologically active nucleosides.
27.3. MASS SPECTRA
OF
TMS NUCLEOSIDES [2]
Display a mass chromatogram of m/z 103 to determine the elution time of the TMS–nucleosides. Next, determine the molecular weight by identify ing the M+• peak (which is usually observed) associated with an [M – 15]+ peak and often with [M – 90]+, [M – 105]+, and [M – 203]+ peaks. The M+• peak of the TMS derivatives of ribonucleosides is 88 m/z units higher than the TMS derivatives of the deoxyribonucleosides. If the difference between the m/z value of the M+• peak and the base peak is 260, the sugar portion is deoxyribose. A difference of 290 represents an o-methylribose, and a difference of 348 Da suggests a ribose. Nucleoside–TMS
MW
m/z Values that indicate the base
Base
2′-Deoxyuridine Thymidine 2′-Deoxyadenosine 2′-Deoxycytidine 2′-Deoxyguanosine
444 458 467 443 555
169, 183 192, 168, 280,
Uracil Thymine (5-methyluracil) Adenine Cytosine Guanine
184 207 183 295
Nucleosides (TMS Derivatives)
371
73
100
207 Si N
H N
N
103 Si O
O
50 236
45 59
81
Si
117 147165
218 264
60
Figure 27.2 MW 467 Da.
90
TMS
120 150 180 210 240 270 300
derivative
N
O
0 30
N
of
467
349 330 360
2′-deoxyadenosine,
EC:
390 420
450 480
C19H37N5O3Si3,
If necessary, the nucleosides can be hydrolyzed to the sugar and the base by heating in formic acid [2]. Kresbach et al. [3] have used pentafluorobenzyla tion combined with electron capture negative ionization for the detection of trace amounts of nucleobases. For fragmentation patterns of TMS 2′-, 3′-, and 5′-deoxynucleosides, see Reimer et al. [4] (Figure 27.2).
REFERENCES 1. Schram, K. H., McCloskey, J. L. (1979). In: Tsuji K, ed. GLC and HPLC of Therapeutic Agents. New York: Marcel Dekker. 2. Crain, P. F. (1990). In: McCloskey, J. A., ed. Methods in Enzymology, Chapter 43 (Vol. 193). San Diego, CA: Academic Press. 3. Kresbach, G. M., Annan, R. S., Saha, M. G., Giese, R. W., Vouros, P. (1988). Mass Spectrometric and Chromatographic Properties of Ring-Penta Fluorobenzylated Nucleobases used in the Trace Detection of Alkyl DNA Adducts. Proceedings of the 36th Annual Conference of the American Society for Mass Spectrometry, San Francisco, June 5–10. 4. Reimer, M. L. J., McClure, T. D., Schram, K. H. (1988). Investigation of the Fragmentation Patterns of the TMS Derivatives of 2’-, 3’-, and 5’-Deoxynucleosides. Proceedings of the 36th Annual Conference of the American Society for Mass Spectrometry, San Francisco, June 5–10.