New techniques for microscale structural studies by mass spectrometry

A chiral GC/MS separation and identification of TFA derivatives of monosaccharides and methanolysis products of carbohydrates and glycoconjugates were developed using a Chirasil-Val column. Both ? and ? anomers were formed in the preparation of TFA derivatives. The ratio of ? anomers to ? anomers is characteristic for each monosaccharide which can provide additional information in compound identification. The developed methods have been successfully used to the identification of sugar moieties in modified queuosine nucleosides and modified DNA isolated from the Rhizobium bacteriophage DNA. A new HPLC separation method which is suitable for thermospray ionization was developed for the separation of mononucleotides. Thermospray LC/MS was shown to be a powerful tool in the structural elucidation of nucleotides and nucleosides. The major ions observed in the thermospray spectra of nucleotides were identified as MH+, NH2+, BH2+ and ions related to the sugar moieties, and their NH4+ cluster ions. Hydrolysis and thermolysis may play a major role in the thermospray ionization of nucleotides and nucleosides, although the ammonia chemical ionization mechanism may also be important. No spectrum difference has been observed for 5'-, 3'-, and 2'-mono-mucleotides; however, the studies of spectra cytidine 3',5'-cyclic monophosphate and cytidine 2',3'-cyclic monophosphate indicate that isomer differentiation by thermospray ionization may be possible. Combined HPLC-thermospray mass spectrometry of mono- and disaccharides, 1-0-methylglycosides and 0-permethyl mono- through tetrasaccharides have been studies to assess the potential role of thermospray ionization for microscale structural studies of saccharides and glycoconjugates and for high sensitivity detection of liquid chromatographic effluents. Using HCO2NH4 as eluant for reversed-phase HPLC, abundant MNH4+ ions are formed from monosaccharides and mono- and permethylated saccharides, and are suitable for monitoring subnanogram constituents in HPC effluents. Detection of 100 pg 1-0methylhexopyranosides with signal/noise>10 is demonstrated. Hydrolysis and thermolysis appear to be the predominant mechanisms in the thermospray ionization in intact mono-, di-, tri-, and tetrasaccharides; therefore consecutive losses of H20 and ions corresponding to hydrolysis products are the major fragment ions. For permethylated carbohydrates, a mechanism analogous to ammonia CI may play the major role. Positional isomers of methylmonosaccharides are chromatographically separable and spectroscopically distinguishable; which may imply the potential of thermospray LC/MS in the structural elucidation of carbohydrates and glycoconjugates at the microscale.

NEW TECHNIQUES FOR MICROSCALE STRUCTURAL STUDIES BY MASS SPECTROMETRY by Fong Fu Hsu A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medicinal Chemistry The University of Utah August 1986 THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Fong Fu Hsu This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. / //Chai an: Dr . Mc Cl os k ey ~., ct Jj)a-9 Dr. Chris M. Ireland Dr. C. Dale Poulter \ Dr. Martin P. Schweizer Dr. Rodger L. Foltz THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of The University of Utah: I have read the dissertation of FONG FU HSU In Its final form and have found that (1) its format, citations. and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures. tables, and charts are in place; and (3) the final manuscript is satisfactory to the Supervis­ory Committee and is ready for submission to the Graduate School. Dr James A. McCloskey C rpenon. Supervisory Committee Approved for the Major Department ~~hJ?i Dr. Arthur D. Broom Chairman I Dean Approved for the Graduate Council Copyright © Fong Fu Hsu 1986 All Rights Reserved ABSTRACT A chiral GC/MS separation and identification of TFA derivatives of monosaccharides and methanolysis products of carbohydrates and glyco­conjugates were developed using a Chirasil-Val column. Both a and B anomers were formed in the preparation of TFA derivatives. The ratio of a anomers to B anomers is characteristic for each monosaccharide which can provide additional information in compound identification. The developed methods have been successfully used in the identification of sugar moieties in modified queuosine nucleosides and modified DNA isolated from Rhizobium bacteriophage DNA. A new HPLC separation method which is suitable for thermospray ionization was develop~d for the separation of mononucleotides. Thermospray LC/MS was shown to be a powerful tool in the structural elucidation of nucleotides and nucleosides. The major ions observed in the thermospray spectra of nucleotides were identified as MH+, NH2+, BH2+ and ions related to the sugar moieties, and their NH4+ cluster ions. Hydrolysis and thermolysis may playa major role in the thermospray ionization of nucleotides and nucleosides, although the ammonia chemical ionization mechanism may also be important. No spectrum difference has been observed for 5'-, 3'-, and 2'-mono­nucleotides; however, the studies of spectra of cytidine 3',5'-cyclic monophosphate and cytidine 2',3'-cyclic monophosphate indicate that isomer differentiation by thermospray ionization may be possible. Combined HPLC-thermospray mass spectrometry of mono- and disaccharides, 1-0-methylglycosides and O-permethyl mono- through tetrasaccharides have been studied to assess the potential role of thermospray ionization for microscale structural studies of saccharides and glycoconjugates and for high sensitivity detection of liquid chromatographic effluents. Using HC02NH4 as eluant for reversed-phase HPLC, abundant MNH4+ ions are formed from monosaccharides and mono-and permethylated saccharides, and are suitable for monitoring subnanogram constituents in HPLC effluents. Detection of 100 pg 1-0-methylhexopyranosides with signal/noise>10 is demonstrated. Hydrolysis and thermolysis appear to be the predominant mechanisms in the thermospray ionization of intact mono-, di-, tri-, and tetrasaccharides; therefore consecutive losses of H20 and ions corresponding to hydrolysis products are the major fragment ions. For permethylated carbohydrates, a mechanism analogous to ammonia Cl may play the major role. Positional isomers of methylmonosaccharides are chromatographically separable and spectroscopically distinguishable; which may imply the potential of thermospray LC/MS in the structural elucidation of carbohydrates and glycoconjugates at the microscale. v CONTENTS ABSTRACT. LIST OF TABLES. LIST OF FIGURES LIST OF SCHEMES LIST OF ABBREVIATIONS AND SYMBOLS ACKNOWLEDGMENTS . . CHAPTER PART 1. CHIRAL GC/MS STUDIES OF CARBOHYDRATES AND SOME APPLICATIONS 1. INTRODUCTION 1.1 Sugar-Containing Nucleosides fron tRNAs and DNAs. . . . . 2. EXPERIMENTAL ....... . 2.1 Materials ....... . 2.2 Derivative Preparation .. 2.3 Gas Chromatography ......... . 2.4 Gas Chromatography-Mass spectrometry. 3. RESULTS AND DISCUSSION .......... . PART 2. THERMOSPRAY LIQUID CHROMATOGRAPHY­MASS SPECTROMETRY OF MONONUCLEOTIDES 4. INTRODUCTION............. 4.1 Background of Thermospray LC/MS .... . 4.2 Drawbacks of Thermospray LC/MS ..... . 4.3 The Fragmentation Pathways of Nucleotides and Nucleosides by other "Soft" Ionization Methods ........... . iv ix x xiv xv xvii 2 4 8 8 8 9 10 11 29 30 31 33 4.3.1 FAB and CI ............ . 4.3.2 FD and Fl ........ . 4.3.3 Mononucleotides ........... . 4.4 Purpose and Scope of the Study .. 5. EXPERIMENTAL ............ . 34 39 39 40 43 5.1 Materials. . . . . . . . . . . . . . .. .... 43 5.2 Preparation of Labelled Buffer Solutions. .... 43 5.3 Hydrolysis of Transfer RNA. . . . . . . . . . . . .. 44 5.4 Liquid Chromatography-Mass Spectrometry ..... 44 5.5 Preparation of Products Produced During Thermospray. . . . . . . 45 6. RESULTS AND DISCUSSION ... 6.1 Thermospray Mass Spectrometry of Nucleotide Monophosphates ........... . 6.1.1 The Fragmentation Pathways of Nucleotides in Thermospray Ionization. 6.1.1.1 MH+ ...... . 6.1.1.2 NH2+ .... . 6.1.1.3 BH2+ .... . 6.1.1.4 (BH)2H+. .. . .. 6.1.1.5 Sugar Related Ions .. . 6.1.1.5.1 (S·OH)NHf+ ... . 6.1.1.5.2 (S-H)NH4 or S+NH3 6.1.1.5.3 (S-H20+NH3)+ .... 6.1.1.5.4 S+ or (S-H)H+ .. 6.1.1.5.5 (S-H20)+ or (S-H-H20)H+ .... 6.1.2 Examination of Ionization Products Produced by Thermospray. . . . .. . ... 6.1.3 Pseudouridine 5'-monophosphate ..... 6.1.4 Cytidine 3',5'-cyclic Monophsophate and Cytidine 2',3'-cyclic Monophosphate. 6.2 Vapor Temperature Dependence Studies ..... 6.3 pH Dependence Studies . . . . . . . . 6.4 Thermospray Liquid Chromatography- Mass Spectrometry . . . . . 7. CONCLUSION ............... . PART 3. THERMOSPRAY LIQUID CHROMATOGRAPHY­MASS SPECTROMETRY OF CARBOHYDRATES 8. INTRODUCTION................. 8.1 Detection Methods of Carbohydrate in HPLC . 8.1.1 Refractive Index (RI) Detector. 8.1.2 Ultraviolet (UV) Detector .. 8.1.3 Moving-wire Detection ..... vii 47 47 47 53 54 55 55 56 64 77 85 87 90 91 94 97 102 102 106 114 117 119 120 121 121 8.1.4 Mass Detection. . . . . . . . . . 122 8.1.5 Polarimetric Detection. . . . . . . . . . 122 8.1.6 Postcolumn and Precolumn Detection. 123 9. EXPERIMENTAL........ 125 9.1 Materials. . . . . . . . .. . 9.2 Methanolysis Reaction ..... . 9.3 Permethylation of Carbohydrates ... . 9.4 Liquid Chromatography-Mass Spectrometry. 10. RESULTS AND DISCUSSION ..... . 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 Monosaccharides ...... . 1-0-methylglycopyranosides . Disaccharides, Trisaccharides and Tetrasaccharides . . . . . Permethylated Sugars ......... . Naringin ........... . Thermospray Ionization for High Sensitivity HPLC Detection ....... . HPLC Separation of 1-0-methylglycosides. Analysis of Methanolysis and Ethanolysis Products. 11. CONCLUSION APPENDIXES A. THERMOSPRAV MASS SPECTRA OF MONONUCLEOTIDES. B. THERMOSPRAV MASS SPECTRA OF CARBOHVDRATES. REFERENCES. . . . . . . . . . . . . . . . . . . . . . viii 125 125 125 128 130 130 145 149 158 163 165 168 168 176 178 185 207 LIST OF TABLES Table page 1. Separation of Sugars from Methanolysis Hydrolysate 22 2. Separation of Products from Methanolysis of Hexose-Containing Nucleosides ..... . 22 3. Ions from the Thermospray Mass Spectra of Nucleotides . · · · · · · · · · 52 4. Sugar Related Ions. . · · . . · · · · · 63 5. Sugar Related Ions from 2'-Deoxyribonucleosides and C-perdeuterated 2'-Deoxyribonucleosides. · · · · · 81 6. Sugar Series Ions from Deuterium Labelled Nucleosides . · · · · · · · · · · · · · · · 86 7. Ions from the Thermospray Mass Spectra of Glucose. · · · · · · · · · · · · · · · 134 8. Ions from the Thermospray Mass Spectra of Deuterium Labelled Glucoses · · · · · · · · · · · · 144 9. Ions from the Thermospray Mass Spectra of a- and 8-1-0-methylglycoside . · · · · .. · · · · · · · · · 146 10. Ions from the Thermospray Mass Spectra of Di-, Tri-, and Tetrasaccharides. · · · · · · · · · · · · · 149 11 . Ions from the Thermospray Mass Spectra of Permethylsaccharides . · · . . · · · · · · · · · · · · · · 161 LIST OF FIGURES Figure 1. Structures of Q, Q*(man), and Q*(gal) .... 2. Drawing of the glass capillary column-fused silica column connection ......... . 3. Separation of glycoside enantiomer-TFA derivatives 4. Separation of fucose enantiomer-TFA derivatives ... 5. Separation of 1-0-methylglycoside enantiomer-TFA derivatives .... 6. Separation of 1-0-methylfucoside enantiomer-TFA derivatives .... 7. Coating substance of Chirasil-Val ... 8. TFA derivatives of methanolysis mixture from unknown deoxyribonucleoside isolated from Rhizobium bacteriophage DNA ..... . 9. SIM chromatogram of 1-0-methylglucoside-TFA 10. 11. 12. 13. 14. derivatives ............... . Schematic diagram of prototype thermospray mass spectrometer ..... Designation of common ions . · Thermospray mass spectra of (a) pC . . · · · (b) Cp(3') · · · . . . . . . · (c) Cp(2') · · · . . . . . · Thermospray mass spectrum of pG. Thermospra 13 mass spectrum of uniformly C labelled pC .... page 5 10 12 14 16 18 21 24 26 32 34 49 50 51 57 58 15. Thermospray mass spectra of (a) cytosine · · · · (b) uracil · · (c) guanine. · · · · (d) thymine. · · · · · (e) adenine. · · · · · · · · · · 16. Thermospray mass spectra of (a) pdC. · · · · · (b) pU . · · · · · · · · · · (c) pUm. · · · · · · · · · · · 17. Thermospray mass spectrum yt pUm obtained by using H20/HC02 NH4. 18. Thermospray mass spectrum of dC obtained by using D20/HC02ND4. 19. Thermospray mass spectra of · · · ( a) pdC. . . . . . . . . . . . . . . (b) dC . • . . . . . . . . . (c) 2-deoxyribose ...... . · .. · · · ·· · · · 20. Thermospray mass spectra of 2'-O-methyluridine 21. 22. 23. 24. 25. 26. 27. obtained by using (a) H20/HC02ND4. · · · · · · (b) D20/HC02NH4. · · · · · · · Thermospray mass spectra of (a) perdeuteriothymidine . · (b) thymidine. · · · · · · · · Thermospray mass spectrum of deoxycytidine-2'I-d. · · · · SIM chromatograms of ionization products produced by thermos pray Thermospray mass spectrum of 1,S-anhydro-B-2-deoxyribose. · · Thermospray mass spectrum of p \l'. Thermospray mass spectra of (a) 2',3'-cCMP . · · · · · · (b) 3',S'-cCMP . · · · · · · Dependence of ion abundance of pA on vapor temperature . · · · · · · · · · · · · · · · · · · · · · · · · · · · 28. Ion abundance of MH+ versus vapor temperature at different pH value of buffer solution xi · · · · · · · · · · · · · ·· ·· · · · · · · · · · · · · · . · · . · · 60 60 61 61 62 67 68 69 70 72 74 75 76 79 80 83 84 87 92 93 95 99 100 103 104 29. HPLC separation of nucleoside 5'-monophosphates with Supelco 25cm x 4.6mm 5u ODS column ..... 30. HPLC separation of enzymatic hydrolysate of RNA from baker's yeast ....... . 31. HPLC separation of nucleoside 2' (3')-monophosphates, 107 108 with UV detection at 254nm . . . . . . . . . . . . . . 109 32. Mass chromatograms (reconstructed from repeated scans corresponding to Figure 31 . . . . . . . . . . . . . . 111 33. Mass chromatograms of mlz 244 and mlz 245 corresponding to Figure 31 . . . . . . . . 34. SIM chromatograms of minor components of alkaline hydrolysate from baker's yeast ......... . 35. Thermospray mass spectrum of 2-deoxyribose 36. Thermospray mass spectra of (a) N-acetylglucosamine ........ . (b) glucosamine ............ . 37. Thermospray mass spectrum of rhamnose .. 38. Thermospray mass spectrum of 1-0-methylrhamnose .. 39. 40. 41. 42. 43. 44. Themospray mass spectrum of 2,3,4,6-tetramethylglucopyranose Thermospray mass spectra of (a) D-glucose .... (b) glucose-3-d1 . (c) glucose-1-d1 . (d) glucose-6,6-d2 ..... . Thermospray mass spectrum of 6-0-methylglucopyranose ... Thermospray mass spectrum of 1-0-methylglucopyranoside .. Thermospray mass spectrum of 3-0-methylglucopyranose ... Thermospray mass spectra of (a) B-gentiobiose ..... . (b) trehalose ....... . 45. Thermospray mass spectrum of chondrosine . xii 112 113 131 133 133 137 137 139 142 142 142 142 143 145 148 151 152 155 46. Thermospray mass spectrum of maltose ..... . 47. Thermospray mass spectrum of permethylmaltose .. 48. Thermospray mass spectrum of naringin ... 49. 5IM (m/z 212) from LC/M5 of (a) 100 ng of a-1-0-methylglucopyranoside .. (b) 100 pg of a-l-0-methylmannopyranoside. 50. 5IM (m/z 198 and 240) from LC/M5 of 2 ng of glucose and maltotriose .... 51. 5IM from LC/M5 of (a) 1 ng of a-1-0-methylglycoside. (b) 200 pg of a-1-0-ethylglycoside 52. 5IM chromatogram from LC/M5 2ng of lactose of 159 160 164 167 167 167 170 170 (a) methanolytic hydrolysate . • • . . . . . . . 172 (b) ethanolytic hydrolysate. . . . . . . . . . . 172 53. 5IM chromatogram from LC/M5 2ng of naringin of (a) methanolytic hydrolysate . . . . . . . . . . . . . 175 (b) ethanolytic hydrolysate. . . . . . . . . . . 175 xiii LIST OF SCHEMES Page Scheme 1. 53 Scheme 2. 65 Scheme 3. 87 Scheme 4. 94 Scheme 5. 101 Scheme 6. 116 Scheme 7. 136 Scheme 8. 138 Scheme 9. 140 Scheme 10 144 Scheme 11 147 Scheme 12 154 Scheme 13 156 Scheme 14 162 Scheme 15 166 CI CID/MIKE Cp(2') Cp(3') 2',3'-cCMP 3' ,5' -cCMP dC DCI DMSO DNA dpC EI FAB FD FI GC/MS HPLC LC/MS M.W •. LIST OF ABBREVIATIONS AND SYMBOLS Chemical ionization Collision induced decomposition/mass-analyzed ion kinetic energy Cytidine 2'-monophosphate Cytidine 3'-monophosphate Cytidine 2',3'-cyclic monophosphate Cytidine 3',5'-cyclic monophosphate 2'-deoxycytidine Desorption chemical ionization Dimethylsulfoxide Deoxyribonucleic acid 2'-deoxycytidine 5'-monophosphate Electron impact Fast atom bombardment Field desorption Field ionization Gas chromatography-mass spectrometry High performance liquid chromatography Liquid chromatography-mass spectrometry Molecular weight m/z MBTFA Nth pA pC pG pGm pU pUm Plf' RI RIC RNA SIM SIMS TFA TIC TMS tRNA TSP UV Mass to charge ratio N-methyl-(bis)trifluoroacetamide Theoretical plate Adenosine 5'-monophosphate Cytidine 5'-monophosphate Guanosine 5'-monophosphate 2'-O-methylguanosine 5'-monophosphate Uridine 5'-monophosphate 2'-O-methyluridine 5'-monophosphate Pseudouridine 5'-monophosphate Refractive index Reconstructed ion chromatogram Ribonucleic acid Selected ion monitoring Secondary ion mass spectrometry Trifluoroacetyl Total ion current Trimethylsilyl Transfer ribonucleic acid Thermospray Ultraviolet xvi ACKNOWLEDGEMENTS I would like to express my gratitude to the people who made a contribution to the production of this thesis. Firstly, I thank Dr. James A. McCloskey for the privilege of working under his supervision, for his guidance and patience throughout the course of my research and writing. Special thanks to Dr. C. G. Edmonds for his enormous efforts in maintaining the LC/MS instrument which was so essential for my experiments. I am also grateful to the researchers in this mass spectrometry groups, in particular, Dr. D. L. Smith, Dr. D. Phillipson, Dr. P. F. Crain, Dr. C. H. Hocart and Mr. C. C. Nelson, for their help. The financial support for this research was provided by the National Institutes of Health (grant No. GM 21584). Finally, my thanks to Dr. A. D. Broom for providing the pleasant departmental atmosphere for my graduate studies. PART 1 CHIRAL GC/MS STUDIES OF CARBOHYDRATES AND SOME APPLICATIONS CHAPTER 1 INTRODUCTION The application of capillary gas chromatography (GC) to the analysis of enantiomeric composition has been known by its sensitivity, precision, reproducibility, and in particular, high resolution. l Two methods have been used in the resolution of enantiomeric mixtures: (a) the conversion of enantiomers to diastereomeric derivatives by chemical reaction with an enantiomerically pure, chiral resolving agent and subsequent gas chromatographic separation of the resulting diaste­reomers on an achiral stationary phase. 2 (b) Direct resolution of the enantiomers on a chiral stationary phase containing an auxiliary resolving agent of high enantiomeric purity.3 In method (a), the diastereomers are isolated before chromatographic separation whereas in method (b), the resolution is effected via the rapid and reversible diastereomeric interaction between the racemic solute and the optically active stationary phase. Of the two approaches to resolution, the direct method has proved to be more reliablel and has been widely used in the identification of the configurational constituents of peptides and proteins. 4-9 Carbohydrates occur in both enantiomeric forms in nature; in most cases they are D-enantiomers. lO Determination of their configuration may be time consuming, as measurement of their optical rotation is sufficient only for single components. This would imply the isolation of a sugar component from a mixture before its optical rotation could be determined. A simple gas chromatographic procedure, as already available for amino acids,4-9 would be useful. A capillary gas chroma­tographic analysis of derivatized, diastereoisomeric glucosides obtained from enantiomeric monosaccharides using chiral butanol (2-butanol)11,12 or a chiral octanol (2-octanol) as the derivatizing reagent13 has been used in the determination of the absolute configuration of sugars; however, a complete separation of the diastereomers was not obtained. Schweer14 separated enantiomeric sugars as diastereomeric trifluoroactylated (-)-bornyloximes on a 50 meter OV-225 capillary achiral column. Although fewer peaks were produced during derivatization, resolution was less than optimal. Besides, the reagents must be of high enantiomeric purity, and because they are not commercially available, they must be synthesized and resolved prior to use. 3 Konig et al. 1S-18 used a number of different chiral L-valine derivatives as the stationary phase coupled to slightly modified OV-225 and XE-60 liquid phases. The resulting chiral columns successfully resolved trifluoroacetyl (TFA) derivatives of enantiomeric aliphatic amines, hydroxy acids and carbohydrates. Leavitt and Sherman19 reported the separation of heptafluorobutyl derivatives of D- and L-arabinose, -fucose, -xylose and -mannose using a commercially available glass capillary column (Chirasil-Val) coated with a copolymer of N-tert-butyl-L-valinamide and an organosiloxane through a-amide linkage. 20 ,21 Recently, well deactivated glass chiral capillary columns enabled the resolution of simple hydroxy.compounds without derivatization. 22 Carbohydrates are not volatile enough to be gas chromatographed without derivatization however, and therefore cannot be included in this category. 4 The Chirasil-Val column has seen much use in the chiral separation of optical isomers. 23 The introduction of N-methyl-bis(trifluoro­acetamide) (MBTFA) reagent in the preparation of fluoroacyl derivatives has become a widely used and reliable technique for the production of volatile compounds in the analysis of carbohydrates by GC.24 Therefore, a chiral separation of TFA derivatives of carbohydrates using Chirasil-Val column was investigated. Sugar moieties other than ribose and deoxyribose have been found in tRNA25 and DNA nucleo­sides. 26 ,27 However, a chiral identification of the optical properties has never been made. Herein I report a sensitive and convenient method which allows the separation of volatile derivatives of carbohydrate enantiomers at the nanogram level. An application of this method to the identification of side chain sugar constituents of the modified nucleosides isolated from transfer RNA (tRNA) of starfish and rabbit liver, and trisaccharides attached to a modified deoxynucleoside isolated from Rhizobium bacteriophage DNA are also reported. 1.1 Sugar-containing Nucleosides from tRNAs and DNAs The queuosine (1) (designated as Q) is located in the first position of the anticodons of E. coli tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp.28 The hexose-containing queuosine derivative designated as Q*, which is isolated from rabbit liver tRNA consists of two components-Q*{man) (Z) and Q*{gal) (~) (Figure 1). The mannose or galactose is attached to the oxygen atom on C-4 of the cyclopentenediol by a B-glycosidic linkage. 29 These Q* nucleosides appear to be present only in specific tRNAs. The tRNAAsp and tRNATyr contain Q*{man) and Q*{gal) respectively.30 Quantitative analyses of the contents of Q and Q* in tRNA isolated from different sources have been reported by Kasai et al .. 29 Q* was found in a variety of animals, such as Brachiopoda, Echinoderma and Chordata in addition to Vertebrata. The content of Q* differs in different organs of the animals examined, e.g., Q* is higher in rat liver than in kidney. Rat ascites hepatoma contained more Q* than normal rat liver. 29 The biological function of queuos;ne in tRNA has been reviewed;31 however, the biological significance of Q* in tRNA remains unclear. HO5J QI ' RO • ,s Z •• HN 1, Z, 3, R:H R:S-D-mannosyl HO H R:S-D-galactosyl (~2S') OH Figure 1. Structures of Q (1), Q*(man) (Z), and Q*(gal) (~). 5 Nishimura31 suggested that the bulky side chain of Queuosine and attachment of mannose and galactose to Queuosine may constitute a suitable site for specific interaction with protein or other cell compounds, if tRNA functions in regulation with mechanisms other than protein synthesis. The modified bases in bacteriophage DNAs are defined that the major proportion of one of the four bases which are commonly found in DNA is replaced by a modified base. It does not include the methylated bases that are found in small amounts in DNA. Modified bases have been found in a variety of bacteriophages. The presence of modified bases can affect the chemical stability of a DNA, DNA conformation and function. Such functions include protecting the DNAs from host and phage nucleases, serving as signals for transcriptions and replication of the DNAs, and facilitating the packaging or injection of the DNAs.32 In some phages, the modified base may carry further substitutes. 6 All of the hydroxymethyl groups in T4 DNA are substituted--70 % of them with a-glucosidic, 30 % of them with B-glucosidic groups.26 In SP 15 DNA, the 4-hydroxyls in the d1hydroxypentyl side chains carry three glucose molecules, as maltose and glucose-l-phosphate. 27 The gluco­sylation protects the viral DNA from the action of one or more host nucleases in vivo. 33 -35 Glucosylated T4 DNA is also resistant to cleavage by many sequence-specific restriction endonucleases from other prokaryotes. 36-38 The Rhizobium bacteriophage RL38JI DNA was observed to be resistant to in vitro cleavage by 7 different site-specific endonucleases. An unusual base which results in the anomalous behavior was found. 39 It is believed that a new modification which replaces all the DNA deoxycytidine residues was present. 7 CHAPTER 2 EXPERIMENTAL 2.1 Materials The carbohydrates purchased as reference materials were obtained from Sigma (St. Louis, MO.), N-Methyl-bis(trifluoroacetamide) (MBTFA) was purchased from Pierce'Co. (Rockford, IL.). Acetonitrile and methanol (Fisher scientific, Pittsburgh, PA.) were dried by distil­lation before use. The modified nucleosides, Q*(man) and Q*(gal), were isolated from tRNA of rabbit liver and starfish testis27 by Dr. Nishimura et al. of National Cancer Center Research Institute of Japan. The sugar-containing deoxycytidine residue in Rhizobium bacteriophage RL385JI DNA39 was isolated by Dr. Hattman et al. of the University of Rochester. 2.2 Derivative Preparation The 2 N methanolic hydrochloric acid was prepared by slowly purging hydrochloric acid gas into 50 ml dry methanol. After 30 minutes, the solution was saturated (ca. 13 N by titration) and then diluted to 2 N with dry methanol. Trifluoroacetylated derivatives of carbohydrates were obtained by dissolving 200 ng samples in 8 ul of acetonitrile and reacting with 2 ul of MBTFA. The reactant mixture was sealed in a capillary tube and heated at 1000C for 1 hour. The methylglycosides of aldoses were prepared by heating 200 ng carbo­hydrate samples in 30 ul of 2 N methanolic hydrochloric acid at IOOoC for 5 hours. After removal of excess of reagents in a Savant Speed-Vac concentrator (Hicksville, N.Y.), the residues were submitted to trifluoroacetylation as described above. The products were analyzed by gas chromatography/mass spectrometry (GC/MS). The queuosine derivatives Q*(man) and Q*(gal), and modified deoxycytidine isolated from Rhizobium bacteriophage DNA were first subjected to methanolysis and then submitted to trifluoroacetylation using the same procedures described above. 2.3 Gas Chromatography A Varian (Palo Alto, CA.) model 3700 gas chromatograph equipped with a solid injection system of the dropping needle type40 was used in this analysis. A 25 meter (0.30 mm i.d.) glass capillary column coated with Chirasil-Val (Applied Science Inc. Deerfield, 11.) was used. The injector and Flame Ionization Detector (FlO) temperature were set at 2000C and 2500C respectively. The helium flow through the column was 30 cm/sec. The temperature program for the separation consisted of an isothermal interval at 900C for 3 min. followed by a temperature increase from 900C to I650C at a rate of 30C per min. Aliquots repre­senting 25 ng of sample were injected. Since the Varian 3700 Gas Chro­matograph is designed for the installation of fused silica capillary columns, a special adaptation (Figure 2) for the connection of a glass 9 Tt FlO detector fused s il i ca ....-.. -capillary column ---1_­( 10cm x .25mm i.d.) 10 fTO dropping needle injector. glass tube (Scm x .5mm i.d. 1.2mm o.d.) p ..... --heat shrink teflon tube 1lI1041----g1ass capillary column (25m x .34mm i.d.) Figure 2. Drawing of the glass capillary column-fused silica capillary column connection. capillary column to the system was made in order to minimize dead volume. 2.4. Gas Chromatography-Mass Spectrometry Gas chromatography and mass spectrometry were carried out with an LKB 9000S GC/MS system. A solid injection system of the dropping needle type40 was installed. A 15 meter (0.24 mm i.d.) fus~d silica capillary column coated with SE-52 was used in the selected ion monitoring (SIM) studies of TFA derivatives of methanolysis hydroly­sates of carbohydrates. Column temperature for separation was 1000C isothermally. Electron impact (EI) mass spectra using SIH was obtained at an ionization energy of 70 eVe Ion source and separator Tempera­tures were set at 2500C. CHAPTER 3 RESULTS AND DISCUSSION As shown in Figure 3 and Figure 4, the trifluoroactylation of carbohydrates results in a mixture of a and B pyranosides, and in most instances, a and B furanosides are also formed. It has been reported that isomerization of aldohexose and aldopentose,41-45 and their methylglycosides46 ,47 occurs due to anomerization when they are dissolved in solvents,42,43 e.g., H20, CH30H, and DMSO. The compo­sition of the mixture at equilibrium has been reported. 41 -47 This information could be used for the peak assignment in the chromatogram of the mixture. In the case of the glycoside, the order of elution is different for the different stereoisomers. For mannose and glucose, the D-enantiomers are retained on the column longer than the L­enantiomers. A reversed order of elution was observed for galactose and fucose. The elution order of the four 1-0-methylglycosides (Figures 5 and 6) which were examined was found to be analogous to the elution order for the glycosides. The B configurational isomers were retained in column longer than a configuration for all isomers. The assignments of a and B anomers have been scrutinized by injection of the derivatives of authentic compounds and retention times were compared. It has been reported48 that mlz 407 (M - TFAO-CH2-CH-OTFA), 379 (M - (TFAO-CH2-CH-OTFA + CO», and 293 (M - (TFAO-CH2-CH-OTFAOH + Figure 3. Separation of glycoside enantiomer-TFA derivatives. (1),(6),(lO)-mannose, (2),(5),(8)-glucose, (3),(4),(7),(9)­galactose. P=pyranoside, F=furanoside, *designation uncer­tain. For separation conditions see text. 13 0 (0 L) d-S 0, M (6) d-S '0 (8) d-9 0 , ==== r.n ... asol:>~L~o'O N (L) :1-9 '0-===2 (9) :I 0, (S) :I '0 0 N ? ::::J' JJ -z -:E (L) (Z~ d-~ 0 J a-'O J----~~~~==~~ ... asol:>eLeo , + a -----==:==~ r.n 1.1.1 -:E t- Z -0 t- Z .1..1...1 1.1.1 c::: 0 __ --____________ ilo Figure 4. Separation of fucose enantiomer-TFA derivatives. P=pyranoside, F=furanoside. For separation conditions see Figure 3. d a 1 a 1 + a o N o 15 Figure 5. Separation of l-O-methylglycoside enantiomer-TFA deriva­tives. (1),(5)-mannose, (2),(4)-glucose, (3),(6)-galactose P=pyranoside, F=furanoside, *designation uncertain. For separation conditions see Figure 3. (9) d-9 (v) d-9 o 1 (£) d-D 10 (2) d-D 0 1 .asO~jele6 1 + 0 ( L) d-D 0 1 ( 1 } 0 (S) d-9 01 J j J J ~ -' 1 < --_ .. _---- ._), . o M Ll') N ~ ~ .. ~ ~ ~ - Ll') L-________ - ..... ---------------------------------- 0 17 -Z -:E w -:E ~ z .0- ~ z w ~ w c:: Figure 6. Separation of 1-0-methylfucoside enantiomer-TFA deriva­tives. P=pyranoside, F=furanoside. For separation condi­tions see Figure 3. ------------------------------------------------~----~o Q d '0 --- .;j o '-=~ '-===a!E.. N -z: ~ LU -O!..;.. z: .-..0.. z: U...J. LU IX L-______________________________________________________ ~o 19 TFAOH» ions are observed only in glycopyranoside-TFA derivatives; while mlz 533 (M - TFAO-CH2), 404 (M - (TFAOH + TFAO-CH=O», and 390 (M - (TFAOH + TFAO-CH2-CH=O» ions are the characteristic ions for glycofuranoside-TFA derivatives. Hence, the assignment of the peaks of Figure 3 based on the above observations was then made. Similarly, the identification of the furanoside-TFA derivatives of fucose was determined by the characteristic ions of m/z 407 (M - TFAO-CH-CH3) and 293 (M - (TFAOH + TFAO-CH-CH3» which were not found in pyranoside-TFA derivatives. 48 At the present stage, however, differentiation of a and B anomers has not been made, since standards are not available (Figure 6). Although trimethylsilyl (TMS) derivatives are the most widely utilized derivatives which have been applied to the GC separation of carbohydrates since Sweeley et al. 49 introduced them in 1963, the enantiomeric derivatives are not separable. 50 It is conceivable that the polar trifluoroacetyl group supports the formation of diastereo­meric association complexes with the stationary phase, whereas the large TMS groups prevent the interacting molecules from coming into close contact with the chiral liquid phase, which is necessary for the formation of association complexes. Geometric considerations require that a combination of TI-TI interactions, hydrogen bonding, and dipole-dipole interactions occur simultaneously to achieve chiral recognition. 51-54 To be resolvable, the analyte must contain func­tionality complementary to that of the chiral stationary phase so that the analyte is capable of undergoing the essential interactions. The 20 21 fluid polymeric phase (Figure 7), referred to as Chirasil-Val shows a chiral center and carbonyl groups. Therefore the separation of optical isomers of the sugar derivatives may be as a result of the dipole stacking of the polar trifluoroacetyl groups and the chiral stationary phase. 55 Methanolysis rather than acid hydrolysis has been used in the elucidation of carbohydrate composition due to the simplicity of the hydrolysate56 -57 (e.g., Figure 5 is simpler than Figure 3). Therefore, the application of methanolysis and chiral separation was successfully used to identify the sugar moieties in the Q* nucleosides from tRNA of rabbit liver and testis of starfish. Table 1 shows the retention time and elution temperature of standard 1-0-methylglycoside-TFA deriva­tives. The released a and B sugar mixture from Q*(man) and Q*{gal) indicate the same retention time and elution temperature as the authen­tic D-mannose and D-galactose (Table 2). The analysis described above provided the first conclusive evidence that the sugars were the D-enan­tiomers. The formation of a and B anomers provide a more complicated Me Me I I ~i I CIH 2 MM~ I O=C O=C-NHtBu I *1 HN--C--H I iPr Figure 7. Coating substance of Chirasil-Val. Table 1 Separation of Sugars from Methanolysis hydrolysatea Sugar TFA-methyl- K' Elution glycoside (capacity factor) Temperature (OC) alB O-glucose a-P 10.54 125 1.5 B-P 10.84 142 O-mannose o.-P 9.49 120 7.4 B-P 16.02 147 D-galactose a-p 11.38 128 2.2 B-P 16.26 148 F 9.92 123 P=pyranoside; F=furanoside. aGas chromatography on a 25 meter Chirasil-Val column. For separation conditions see text. Nucleoside Q*(man) Q*(gal) Rhizobium bacteriophage DNA Table 2 Separation of Products from Methanolysis of Hexose-Containing Nucleosidesa TFA-methyl- K' Elution glycoside (capacity factor) Temperature a-P 9.51 120 B-P 15.95 147 a-P 11.40 128 B-P 16.20 148 F 9.98 123 a-p 10.60 125 B-P 14.78 142 -P 11.42 128 B-P 16.30 148 F 9.93 123 P=pyranoside; F=furanoside. (OC) aGas chromatography on a 25 meter Chirasil-Val column. Separation conditions same as Table 1. alB 8.0 2.3 1.6 2.2 22 23 chromatogram, nevertheless, additional conformation of the sugar identification is also provided. Since the ratio of the abundance of the a and B anomers is different for different sugar derivatives (column 5, Tables 1 and 2),46,47 it could be used in the reconfirmation of the sugar entities. The chiral separation of the Q* nucleosides methanolysis products simultaneously assigns both the stereoisomeric and optical properties of the sugars. The determination of the sugar moiety from modified deoxycytidine nucleoside from Rhizobium bacteriophage RL385JI DNA carried out with the same procedure shows the appearance of D-glucose and D-galactose (Figure 8). The ratio is 2 to 1. The Fast Atom Bombardment (FAB) mass spectrum of the permethylated derivative and the EI mass spectrum of trimethylsilylated derivative of the nucleoside indicate the attachment of a trisaccharide to the nucleoside. 58 The method identified the sugars which are present in the molecule and determined their optical properties. However, the sequence and the nature of the linkages could not be elucidated with the above method. The chiral separation of the methanolysis products of glycosides is a convenient, sensitive, and reliable method in the analysis of sugars. Organic contaminants such as those used in a buffer system during purification may not interfere with the methanolysis and tri­fluoroacetylation steps,57 but may produce peaks in important areas of the chromatogram. This potential problem could be easily solved by GC/MS selected ion monitoring (SIM) experiments. An example shown in Figure 9 is the SIM chromatogram of mlz 177 (M - 3TFA - C2H402) and 170 of TFA derivative of a- and B-1-0-methylglucopyranosides. a and B Figure 8. TFA derivatives of methanolysis mixture from unknown deoxy­ribonucleoside isolated from Rhizobium bacteriophage DNA. (a) D-I-O-methylgalactofuranoside, (b) a-D-I-O-methylgluco­pyranoside, (c) a-D-I-O-methylgalactopyranoside, (d) B-D-I-O-methylglucopyranoside, (e) B-O-I-O-methylgalacto­pyranoside. For separation conditions see Figure 3. ..c o M o N Ln C L-________________________________________________________ O 25 -z -~ I.I.J -~ I-Z -0 I-Z I.I.J I-I. I.J 0::: UJ V') z: o Q... V') UJ ~ c::: o to­W UJ to­UJ o , ..... t ! 0 26 a ~ 1 ~ ~ I I I I , I b J .!. .J. A. I~I.L \ ru _1. ....Ji~ ... - 170.0 177.0 2 ,[ (, RETENTION TIME (MIN.) Figure 9. SIH chromatogram of l-O-methylglucoside-TFA derivatives. Separation was carried out by a 18m SE-S2 column at 1000 C, isothermally. 10 ng were injected. (a) a-1-0-methylgluco­pyranoside. (b) B-I-O-methylglucopyranoside. 27 anomers have been observed to give a different ratio of m/z 177 to 170 in their EI mass spectra. 59 This ratio (e.g., for a- and B-I-0-methyl­glucopyranosides, the ratio is 2.6 and 4.3 respectively) could be used as additional confirmatory information in the structural assignment of a glycoside. Additionally, a chromatogram free of interferences from extraneous materials is much more likely to result. Moreover, the tremendous sensitivity, versatility, and selectivity of mass spectro­metry could give the most structural information for a very limited amount of sample. PART 2 THERMOSPRAY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY OF MONONUCLEOTIDES CHAPTER 4 INTRODUCTION Biemann and McCloskey first demonstrated the usefulness of electron ionization mass spectrometry as an aid in the structure elucidation of the so-called low volatility nucleosides in 1962. 61 Since then various new ionization methods have been employed by McCloskey62 and others63 in the continuing effort to extend mass spectrometry to intact and underivatized nucleotides. One commonly used technique for molecular weight determination has been field desorption (FD).63-65 Following on from the use of secondary ion mass spectrometry (SIMS),66 the recently introduced desorption ionization method, fast atom bombardment (FAB) mass spectrometry has already been shown to give useful spectra of nucleotides67 -69 and nucleosides. 69 - 71 But perhaps the most exciting recent development in the area of mass spectrometry is the liquid chromatographic mass spectrometric thermospray technique which produces spectra of underivatized nucleosides,72,73 mono- and dinucleotides. 72 The development72 ,74-76 of the thermospray technique as a means of combining liquid chromatography with mass spectrometry (LC/MS) has overcome some of the basic incompatibilities of the two techniques. A major attraction of the method is its simplicity, in that, unlike other approaches to LC/MS interfacing, the thermospray technique plays a dual role. It performs both in the role of a conventional interface, permitting the introduction of high flow rates of liquid directly into the mass spectrometer, and also the role of an ion source, yielding ions of a solute directly from the solvent without the need for the intervention of external ionization devices. The method depends on 30 the generation of a spray of charged solvent droplets, from which solvent and analyte ions may be directly observed. It is by this means that low volatility compounds may be introduced into the mass spectro­meter without the requirement that they be independently thermally volatilized, and consequently with reducing possibility of thermal decomposition. A number of applications of the thermospray technique, demonstrating its ability to handle involatile materials have been reported. Early examples include small peptides72 ,76 underivatized amino acids72 and some penicillins. 75 More recent examples include the analysis of glucuronides,78,79 steroidal sulfates,80 nucleosides73 and nucleobases. 73 4.1 Background of Thermospray LC/MS The thermospray (TSP) technique is based on the rapid heating of the capillary to supply sufficient heat to the effluent from the HPLC to produce a supersonic jet of vapor which consists of mist of charged particles and droplets. The means of heating the capillary has evolved from the original use of a focused 50-watt C02 laser,81 through oxy-hydrogen torches,74,75 to the use of simple electrical cartridge heaters embedded in a copper block brazed to the end of the capillary.82 Most recently, direct electric heating has been effected by passing an electric current through the capillary tube. 76 A schematic diagram of the most recent version of the LC/MS interface which has been installed in this laboratory is shown in Figure 10. The mechanisms whereby a spray of charged droplets may be generated by thermal means have been discussed by Vestal. 82 ,83 A two stage mechanism for the formation of TSP mass spectra has been proposed. 82 -84 The formation of ions in the first stage is believed to be out of the charged microdroplets produced by the vaporizer. The charge on the droplets results from statistical fluctuations in the distribution of the positive and negative ions in the droplets. Once the microdroplets are charged, field-induced ion evaporation occurs after solvent evaporation has shrunk the droplet to sufficiently small size and gaseous ions are formed. 82 ,85-87 The second stage, in which gas-phase ion/molecule reactions may remove the primary TSP ions and produce new product ions, has been recognized as potentially important. 82 ,85 Recent results have been reported by Bursey et al. 8S and Alexander et al. 89 for the gas-phase ion/molecule reactions of thermospray mass spectra. 4.2 Drawbacks of Thermospray LC/MS 31 In spite of the increasing use of TSP LC/MS some drawbacks have been found which limit its application. The first important limitation as pOinted out by Vestal 90 is its inability to generate electron-impact spectra due to the high vapor pressure in the ion source. The power of EI for qualitative identification and structural elucidation with ANALYSER DIFFUSION ...-.. PUMP (280 lis' CHROMATOGRAPH ---, EFFLUENT . , . , . , IOcm - I I 11 I ELECTRON MULTIPLIER VAPORIZER PROBE " \ "''Yl ",-.f ,.!,!./ .. "li1!'!.II·t, tt;:.,;.,.·"\Ht".t\, .. ~I .~.' j".4J.1t ~ •• \ .'~"'. ·I'~·.· ~ MAIN DIFFUSION PUMP (1250 lis J II TO TrlAP 8 M[CII/lNlCAl PUMP SOURCE IIWCK Figure 10. Schematic diagram of prototype thermospray mass spectrometer. W N and without the use of libraries, has been well established. Although this ionization technique often provides fragmentation patterns that yield some structural information, these patterns rarely are as extensive or as reproducible as with electron impact ionization. The second drawback of the technique is the limitation of the use of buffers. It is generally suggested90 that a volatile buffer such as ammonium acetate, ammonium formate, or trifluoroacetic acid be used. The use of alkali ions is not recommended because of deposition in the TSP capillary tube. These place limitations on the ability of the HPLC in achieving high resolution separation of nucleotide mixtures. Even when only volatile buffers are used, the probe blockage still occurs after long term operation. 33 The most serious problem is that of data reproducibility. It is known that at least two ionization mechanisms, direct ion evaporation and ion/molecule reactions are operating to produce parent and fragment ions,88,89 and because the relative contributions of the two mechanisms are dependent on operating conditions, the reproducibility of mass spectral data requires a very careful control of experimental parameters. 4.3 The Fragmentation Pathways of Nucleotides and Nucleosides by Other "Soft" Ionization Methods A detailed review of the studies of the fragmentation pathways of nucleosides and their derivatives by mass spectrometry has been reported by Pang in her Ph.D. dissertation in 1982. 91 More than 70 related references have been cited to which the reader is referred for detail. Recent reviews of mass spectra of underivatized nucleosides and nucleotides by FAB and other soft ionization method and a comparison of mass spectra of nucleosides by FAB with EI and CI has been reported by Slowikowski and Schram. 70 ,92 The common ions of nucleotides and nucleosides observed in mass spectrometry which will be used throughout the text are designated as shown in Figure 11. M= molecular ion, B= base fragment from cleavage of the glycosidic bond, BH= nucleobase, N= nucleoside fragment from cleavage of the 0-5'-P bond, NH= nucleoside, S= sugar fragment from the corresponding nucleoside NH. 4.3.1 FAB and CI A close relationship of fragmentation pathways has been observed between FAB and CI. The major feature of the positive ion FAB and CI spectra61 ,70 of both ribosides and deoxyribosides, is the simplicity I I I I 9~ HO-P~O II I O~ I t M Figure 11. Designation of common ions 34 of the spectra and the predominance of the MH+ and BH2+ ions. The mechanism of formation of the BH2+ ion from MH+ for nucleosides in CI61 is also thought to be operating in the FAB mode70 (Equation 1). Although the FAB spectra of nucleosides are dominated by the MH+ and BH2+ ions, other fragment ions of diagnostic value are also observed. The ions (B + 44)+, from ribosides and (B + 28)+, from 2'-deoxy­ribosides are base containing fragments which are important in determining the site of sugar modification in nucleosides of unknown structure. They represent a loss of carbons 3' through 5' with retention of a hydroxy hydrogen (Equation 2). Equation 1. ForTation of the (B+2H)+ ion from MH+ for adenosine in CI6 and FAB92. HO R MH+ R=OH H .. B+,H CH II H~ (B+44t (B+2SY Equation 2. Formtt;on of the (B+44)+ and (B+28)+ ions from MH+ in CI.6 Analogous processes are suggested to occur during FAB33 ionization. 35 The decomposition of the protonated molecular ion produced by Cl occurs by successive charge site initiated cleavages, identical in the ribosides and deoxyribosides, as indicated in Equation 2. 61 Fragmentation of the sugar with concomitant loss of water results in generation of the (B + 30)+ ion. 92 Essentially the same mechanism is thought to be operating in FAB and is illustrated in Equation 3. The (B + 30)+ ion is present in the spectra of all nucleosides examined using FAB ionization. 71 ,92 Retention of charge by the ribose moiety following glycosidic bond rupture results in the formation of sugar ions by FAB and CI. A plausible mechanism for the formation of sugar ions in CI involves sugar protonation leading to glycosidic bond cleavage with concomitant loss of the base molecule. 61 The same mechanism was proposed70 as indicated in Equation 4. A successive loss of water is also observed. + BH r CH II o (B+30)+ Equation 3. Formation of the (B+30)+ ion from MH+ in CI.61 36 OHR Equation 4. Formation of sugar ions from MH+ in C161 and FAB.92 Pseudouridine is a typical C-nucleoside. The base peak in C1 spectrum61 is the (8 + 44)+ ion at mil 155. The (B + 30)+ ion in C1(CH4)' appearing at mil 141 with a relative intensity of 19%, however, is relatively insignificant in the FAB spectrum. 92 The (B + 44)+ ion, although present as the second most intense peak, is only 29% relative intensity. The consecutive loss of water from the protonated molecular ion results in the series of ions: (MH+ - H20) 37 and (MH+ - 2H20). The (MH+ - 2H20) ion at mil 209 (Equation 5) is significantly greater in the C1 spectrum (54%)61 compared to the FAB mode. 92 Decomposition of the mil 209 ion ;n C1 and FAB mode may occur by at least two possible pathways. The loss of an additional water molecule from mil 209 gives a highly conjugated fragment ion at mil 19192 that may undergo a ring expansion reaction to produce an aromatic pyrillium lon, as indicated in Equation 6, similar to the well-documented tropylium ;on. 93 A second fragmentation pathway available to the mil 209 ion ;s loss of formaldehyde to form the mil 179 ion as shown in Equation 7. 61 38 4J HO~\j ~.= uracil-C-5 linkage --_a__ 9"'0 qJ O~ HOCa,+ .. m/z 209 Equation 5. Formation of m/z 209 ion in CI.61 + H,C m/z 191 Equation 6. Proposed rearrangement l reaction for m/z 191 in pseudouridine with CI6 and FAB.92 m/z 209 mlz 179 Equation 7. Formation of ion at m/z 179 from m/z 209 in CI61 and FAB92 of pseudouridine. 4.3.2 FD and FI The FD-MS of nucleosides64 ,65 are similar to their FI spectra,94 displaying prominent molecular peaks (MH+) and smaller but important fragmentation peaks which yield useful information about the sugar (5+) and base moieties (BH2+) comprising the molecules. A remarkable exception is guanosine which has been reported to give no signal at all for the molecular ion in its FI spectrum. 94 4.3.3 Mononucleotides The positive ion FAB mass spectra of a number of nucleotides have been reported and all of the spectra are dominated by an intense protonated or cationized molecular ion. The nature of the molecular ion species and the extent of proton replacement by counter ions, 39 e.g., Na+, K+ or Li+ depends on the origin of the sample, the procedure used to prepare the sample for analysis and in some cases, the base moiety of the nucleotides. 69 Commercially available mononucleotide disodium salts may exhibit the following series of ions: MH+, MNa+, (M - H + 2Na)+ and (M - 2H + 3 Na)+.69 The corresponding ions repre­senting protonated or cationized nucleosides due to loss of the phosphate moiety are not present. 69 The most abundant fragment ion present in the positive ion FAB spectra of mononucleotides is the BH2+ ion69 ,70 arising from cleavage of the glycosidic bond with hydrogen transfer from the sugar to the aglycone. Ions at m/z 164 and 178 corresponding (B + 30)+ and (B + 44)+ respectively were observed in the spectrum of the adenosine containing samples. 70 The fragmentation pathways were believed to proceed by the same mechanisms as for the nucleosides in FAB-MS.70 FD mass spectra of free nucleotides allows the following basic structural information to be deduced. 65 ,66 The molecular weight is indicated by the quasi-molecular ion MH+. The base signal (BH2+) and corresponding nucleoside (N + H)+ ions are major ions. Although no peak for the sugar fragment is observed, two significant ions at m/z 60 and 61, representing (CHOH)2+ and ((CHOH)2 + H)}+ respectively are present. A distinct ion at m/z 99 is observed which is due to (H3P04 + H)+ ion, the protonated orthophosphoric acid. 4.4 Purpose and Scope of the Study Directly combined gas or liquid chromatography-mass spectrometry offers a powerful means for initial examination of individual components of DNA or RNA hydrolysates without the necessity of isolating the individual components. Volatile derivatives of most of the nucleic acid bases and many of the simply-modified nucleosides and mononucleotides are amenable to gas chromatography. However, a number of the hypermodified nucleosides (e.g., queuosine) as well as most of the nonmethylated modified nucleotides are excluded from this approach. 95 Combined high performance LC/MS in general constitutes a more useful approach than GC/MS when crude mixtures such as nucleic acid hydrolysates are to be examined. Other LC introduction methods have been studied. 96 ,97 The thermospray technique is most useful because of its applicability to polar compounds and to aqueous, buffered reversed phase HPLC systems in which the entire effluent can 40 be passed into the mass spectrometer. 84 The necessity for deriva­tization is obviated, and the wide range of polarities encountered in modified constituents from RNA and DNA can be accommodated. 41 Traditionally, nucleotide identification has relied heavily on high performance liquid chromatography (HPlC); however, an unambiguous identification which depends solely on retention time is hard to obtain. The use of thermospray lC/MS can playa dual role in nucleo­tide identification; both the retention time and structural information can be provided in a single experiment. Thermospray lC/MS has been known for the limitations the technique places on the use of buffers. 90 A volatile buffer, e.g., ammonium formate is required. Although nucleotides have been successfully chromatographed by ion exchange,98- 101 reversed-phase chromatographyI02-I06 and reversed-phase ion-pair chromatography,107-ll8 a separation which utilized the volatile buffer solution has never been reported. The first objective in this study was the development of a reversed-phase separation of mononucleotides using a volatile buffer pH gradient providing the necessary compati­bility between LC separation and mass spectrometry ionization and detection. The selected ion monitoring detection of the nucleotides from an alkaline hydrolysate of tRNA was examined to evaluate the potential of thermospray lC/MS in the study of nucleotide structure. Modified nucleosides (or nucleotides) frequently occur only once per molecule of tRNA. More than 80 different bases or nucleosides are presently known to occur in RNA and DNA.119-I2l Isolating enough of an unknown modified nucleoside or nucleotide for structure characte­rization is a time consuming and difficult task. Therefore, it is important to maximize the amount of structural information from a limited amount of sample. This can only be achieved by a thorough understanding of the fragmentations of these compounds. The studies of the fragmentation pathways of nucleosides and nucleotides by thermospray mass spectrometry is my second objective in a continuing effort to extend mass spectrometry to intact and underivatized nucleotides and nucleosides. 42 CHAPTER 5 EXPERIMENTAL 5.1 Materials Uridine-2'-d, uridine-3'-d, uridine-5'-d and deoxycytidine-2'1-d were synthesized in this laboratory by Dr. K. H. Schram* in 1977-1978. Uniformly C-13 labeled and deuterium labeled tRNA and DNA were isolated by Dr. P. F. Crain. Water was prepared for LC/MS analysis using a Millipore (Bedford, MA) purification system. Analytical-grade ammonium formate was purchased from EM Science (Cherry Hill, NJ). All of the nucleotides, tRNAs and enzymes were used as obtained from Sigma Chemical Co. (St. Louis, MO). 5.2 Preoaration of Labelled Buffer Solutions Buffer solution of 0.1 M HC0215NH4 was prepared as follows: N-15 labeled ammonia gas (95% 15N, purchased from Monsanto Co., Miamisburg, OH) was slowly purged into water. The resultant 15NH40H solution was then used to adjust the 0.1 M formic acid solution to the desired pH value. The deuterium labeled ammonium formate was prepared by repetitive dissolution and evaporation (3 times) of ammonium formate (1.26 g) in 020 (5 ml). The final dry product was then dissolved in 020 (200 ml) to yield a 0.1 M HC02ND4/D20 buffer solution of pH 5.6. Present address: Department of Pharmaceutical Sciences, University of Arizona, Tucson, AZ 85721. 44 5.3 Hydrolysis of Transfer RNA The alkaline hydrolysis of tRNA was carried out by treating 100 ug RNA with 100 ul, 0.3 N KOH and incubating the mixture at 370C for 18 hours. The enzymatic tRNA hydrolysate was obtained by dissolving 100 ug RNA in pH 5.3, 0.02 M sodium acetate buffer. After treating the solution with 1-2ul (IU/ul) nuclease PI, the mixture was incubated at 370C overnight. Aliquots of the incubation mixture were submitted directly to LC/MS. 5.4 Liquid Chromatography-Mass Spectrometry The liquid chromatograph-mass spectrometer used in this work has evolved from an earlier prototype system,74 and was constructed from components which include a Finnigan MAT hyperbolic rod quadrupole mass analyzer, an Extranuclear quadrupole power supply, and Varian NRC vacuum pumps. Except for minor details the design is similar to the thermospray LC/MS system described previously.76,82 The mass spectrometer was controlled by a Teknivent Corp. 29K data system. Chromatography was carried out using a Beckman 322M liquid chromato­graph linked to an Altex (Berkeley, CA) model 420 system controller. A Waters 440 dual wavelength (254 and 280 nm) UV absorbance monitor was connected in series between the chromatograph and mass spectro­meter. HPLC separation of nucleoside monophosphate mixtures were obtained using a Supelco (Bellefonte, PA) 25 cm x 4.6 mm 5u LC-18-DB column and a Brownlee (Santa Clara, CA) 30 x 4.6 mm 5 u RP-18 guard column. Nucleotide separations were achieved by a linear gradient of 0.1 M ammonium formate from an initial pH 5.0 to a final pH 3.0 over a 20 min. interval at flow rates of 1.5 or 2.0 ml/min. The probe tip was maintained by electrical heating at 2400C, resulting in a vapor temperature of 240-2500C. All chromatography was performed at ambient temperature. 45 Ion intensity as a function of vapor temperature was studied by making multiple injections at various vapor temperatures and selected ion monitoring of the MH+, NH2+, and BH2+ ions. Samples were intro­duced to a 4.6 x 75 mm 3 u Ultrasphere ODS column (Beckman Instruments, San Jose, CAl. A flow of 1.5 ml/min, 0.1 M ammonium formate (pH 4.0) was used as the mobile phase. The entire LC effluent was passed into the mass spectrometer. 5.5 Preparation of Products Produced During Thermospray A pH 4.0, 0.1 M ammonium formate buffer solution with a concen­tration of 1 ng/ul of deoxycytidine and deoxyguanosine was prepared. An off-line experiment was carried out by running the above solution through the thermospray probe. Standard conditions (see section 5.4) for the thermospray ionization of nucleosides and nucleotides were applied. The temperature of the probe tip was monitored and adjusted continuously to maintain 2000C. Flow rate was set at 1.5 ml/min. The effluent through probe was condensed and collected by a cold trap (filled with dry ice). The condensed effluent (80 ml) was then transferred to a rotatory evaporator and evacuated to dryness. The final product was dissolved in 1 ml H20 and used in the lC/MS studies which followed the procedures described in section 5.4. 46 CHAPTER 6 RESULTS AND DISCUSSION 6.1 Thermospray Mass Spectrometry of Nucleotide Monophosphates 6.1.1 The Fragmentation Pathways of Nucleotides in Thermospray Ionization The ion intensities of nucleotides in the TSP ionization spectra depend on vapor temperature and the pH value of the mobile phase applied (discussed in section 6.2 and 6.3). Therefore, optimum conditions for formation of MH+ (0.1 M ammonium formate, pH 4.0, vapor temperature 2500C) were applied to accumulate the data. The principal product ions formed are BH2+' NH2+' (S - H20)+ and (S - H)NH4+ (or S+NH3). Minor ions are (S·OH)NH4+, (S - H20 + NH3)+, S+ (or (S·OH - H20)H+) and (BH)2H+. 5'-, 3'-, and 2'-ribonucleotides give similar mass spectra. An example is given in Figure 12, which shows the full scan mass spectra of 5'-, 3'-, and 2'-cytidine monophoshates. A summary of the major ions of 15 commercially available 5'-mononucleo­tides are tabulated in Table 3. The mass spectra of the mononucleo­tides listed in Table 3 are given in Appendix A of this thesis. The fragmentation pathways of the nucleotides in the thermospray ionization are summarized in Scheme 1. Figure 12. Thermospray mass spectra of (a) pC, (b) Cp(3'), and (c) Cp(2'). 100 112 BH2 +, ~- 80 .... + (S-H20) + 115 (S-H)NH 4 150 o -J 1-fIl' II' ~llIpr' y. 100 (a) pC. + NH2 + (BH) 2H 244 223 NHK+ + MNa+ MH ~1K+ 324 346 362 0.00 1.90 +=­~ 100 j 112 BH2 + --;; 80 >- .t..-.. V') 5 60 .t:.:.-.:. 1 LtJ ~ 40 t-c:( + + ~ 20~ (S-H20) (S-H)NH4 115 150 132 o ~llL.j I I ~IL, l \' I b ' 100 150 (b) Cp(3'). NH_' 1 l t . 1 200 250 m/z lOl3 J- 0.2 ~ 0.1 L- 0.0 300 350 400 19!J c..TI o 100 -!! 80 .>..­. ...... Vl E....j 60 .z....:. w > ~ 40 c:( ...J W ex: 20 112 BH2 + (S-H20 )+ (S-H)NH + 4 115 150¥ 132 + + NH2 232 NHK + J NHNa + (BH)2H 2~4 266 223 0.3 0.2 0.1 MH+ O --J1~'r III II-UI 0-, - t' t , F , Y H , f , i , , , Iii , iii , , JIi' ,i I ' iiL i , ,• , iii ii' iii , i , , , , , • iii , i , L- 0 • 0 I 90 100 150 200 (c) Cp (2 1 ) • 250 m/z 300 350 400 U1 --I 52 Table 3 Ions from the Thermospray Mass Spectra of Nucleotides Ion Observed Mass (% Relative Intensity) Compound MH+ NH2+ BH2+ (S-H20)+ pA 348(1.6) 268(37) 136(100) 115(3.2) pC 324(0.8) 244(13) 112(100) 115(25) pG 364 ( - ) 284 ( - ) 152(100) 115(16) pGm 378( - ) 298(0.9) 152(57) 129(100) pm1A 362(0.5) 282(7.8) 150(100) 115(4.3) pm6A 362( - ) 282(3.7) 150(100) 115(5.7) pm5C 338 ( - ) 258(1.1) 126(100) 115(4.8) pm7G 378( - ) 298( - ) 166(100) 115(0.6) pU 325(1.3) 245(10) 113(100) 115(53) pUm 339(1.9) 259(38) 113(50) 129(100) P'1' 325(1.3) 245(52) 113(0.5) 115( - ) pdA 332(0.2) 252(1.9) 136(100) 99(3.3) pdC 308( - ) 228( - ) 112(100) 99(1.4) pdG 348 ( - ) 268( - ) 152(100) 99(12) pdT 323(0.3) 243(3.8) 113(100) 99(17) 6.1.1.1 MH+ ------H-+- ---1.. ..... MH+ ------II-.... SUGAR RELATED IONS Scheme 1 The beauty of "soft" ionization techniques in mass spectrometry 53 is their ability to produce molecular ions of nonvolatile compounds without the need of derivatization. Unfortunately, decent molecular ion intensities have not been found through positive ion TSP ionization of nucleotides, especially the 2'-deoxyribonucleotides, methyl-modified nucleotides and purine nucleoside monophosphates. The nature of the molecular ion species and the extent of proton replacement by counter ions, e.g., NH2+, Na+, or K+ depends on the origin of the buffer solution. It;s known that the general order of stability of the four common nucleoside 5'-monophosphates is in the order of CMP,UMP > AMP,GMP.122 Polar effects are best exemplified by the series shown below in which the stability increases in the order: 123 Uracil Uracil Uracil HO HO H H 54 The replacement of any of hydroxy groups of the sugar by hydrogen has a destabilizing effect on the glycosyl bond ~ith respect to acid-catalyzed hydrolysis. 124 A complete hydrolysis of nucleotides to release sugar and bases could be carried out under mild cond;­tions. 125 ,126 Therefore, acid hydrolysis becomes the most probable mechanism (discussed in a later section) for TSP ionization. The rise of the intensity of MH+ ions from 2'-deoxyribonucleotides to ribo­nucleotides could be a reflection of the increased lability toward acid hydrolysis of the glycosidic bond of 2'-deoxynucleotides, relative to ribonucleotides. Another factor which is possibly responsible for the weak MH+ ion is the hydrolysis of 0-5'-P bond, which results in nucleoside formation (discussed in 6.1.1.2). 6.1.1.2 NH2+ Since the MH+ ion has been rarely found in the TSP spectra of nucleotides, the NH2+ ion becomes the most informative ion in the structural elucidation of nucleotides. Similar to nucleosides observed by TSP ionization,73 abundant NH2+ ions are found. The corresponding ion is not present in FAB.69 Instead of an NH2+ ion, a (N + H)+ ion which arises from the cleavage of 0-5'-P bond is observed in FD spectra. 64 ,65 Hence a different fragmentation pathway for the forma­tion of NH2+ in TSP ionization of nucleotides is suggested. It is believed127-130 that the NH2+ ion arises from the same cleavage via hydrolysis with which the corresponding nucleoside is formed and protonated (or cationized). 6.1.1.3 BH2+ As has been noted in earlier studies by Wilson and McCloskey,61 fragmentation in the CI mode proceeds from the protonated molecular ion MH+ where the proton is derived from the reagent gas with the site of protonation, on the base or sugar, determining the subsequent route of fragmentation. In a similar manner, decomposition in FAB proceeds from the protonated molecular ion but, in this case, the proton is derived from the sample matrix, usually glycerol. 92 55 The most abundant fragment ion present in positive ion TSP spectra of nucleosides73 and nucleotides (Table 3) is BH2+ which derives from the cleavage of the glycosidic bond with proton transfer, probably from the mobile phase to the nucleobases (Scheme 1). Although (B + 44)+ and (B + 30)+ ions are commonly seen in CI and FAB mass spectra of nucleosides,92 the fragment ions which contains the base plus various portions of the sugar moiety have not yet been found in TSP ionization (pseudouridine is an exception, which will be discussed in 6.).2). This would imply that a different fragmentation mechanism is op rating during TSP ionization. Indeed, an investigation of sugar related ions (discussed in section 6.1.1.5) and pseudouridine monophosphate reveals that hydrolysis and thermal decomposition rather than other ion chemistry reaction are predominant in the TSP ionization of nucleotides. 6.1.1.4 The (BH)2H+ ion is found as m/z 223 and mlz 303 in the TSP spectra of cytidine monophosphate (Figure 12) and guanosine 5'-monophosphate (Figure 13) respectively; however, a corresponding ion is not observed in adenosine or adenosine monophosphate. The TSP mass spectrum of 56 C-13 labeled cytidine 5' monophosphate (Figure 14) shows an 8 mass unit shift (from 223 to 231), which represents a cytosine dimer. Excluding adenine, the TSP mass spectra of major nucleobases (Figure 15) display the protonated dimer ion species, which are formed by the following gas phase adduct clustering reaction 131 (Equation 8). (Equation 8) It has been reported89 that at low {BH)aq' only BH2+ ion is observed; however at higher (BH)aq the (BH)2H+ ion appears, becoming dominant at the highest (BH)aq. This indicates that there are uncharged nucleo­bases present in the gas phase with which the gas phase ion/molecule reaction may occur. 6.1.1.5 Sugar Related Ions Basically there are three types of sugar moieties of all natural nucleotides (or nucleosides) derived from nucleic acids, D-ribo­furanose, 2'-deoxyribofuranose, and 2'-O-methylribofuranose. The sugar related ions are the fragment ions which arise from the sugar moiety of the mononucleotides. These sugar ions and mass shift of the labeled ion species are summarized in Table 4. Altogether there are five ions observed in this series. They are (S - H20)+, S+ (or (S - H)H+), (S - H20 + NH3)+, (S - H)NH4+ (or S+NH3), and (S·OH)NH4+. Since the sugar 152 BH; - -- J .3 >- .t...-.. V') f5 60 t.z..-. .. l-O.2 w ;:: 40 + NH+ t- (S-H)NH4 2 c:::( 284 --J ~ w 150 BHNH: I (BH)2H+ '-0.1 0:: 20 132 I 169 3~3 ).96 NHNa + 0--1. ' l'r'tJl.I , ""'" ~, i ,~, it, , ' Iii' i , i , i , Iii' i , i~ i , ,'.' iii, iii ii' i , i ' iii i ILO.O 100 150 200 250 300 350 400 m/z Figure 13. Thermospray mass spectrum of pG. 190 (J"1 "'-.J - 100 80 ~ 80 >­. I..-.. V) ~ 60 I­. Z.. .. UJ ;:: 40 I­ct: --I UJ a:: 20 o 100 + 116 BH2 (S-H20+NH3)+ \ (S-H)NH/ 137 155 150 NH + 2 253 m/z 350 Figure 14. Thermospray mass spectrum of uniformly 13C labelled pC. 0.20 0.15 0.10 0.05 . I 0.00 400 190 U'1 ex> Figure 15. Thermospray mass spectra of (a) cytosine, (b) uracil, (c) guanine, (d) thymine, and (e) adenine. 60 + 100 112 BH2 0.6 -~ ~O >- .t...-.. en z ~ 60 0.4 .z... .. UJ >..... . t- 40 ea:: -oJ UJ 0.2 c:: 20 0.0 (a) cytosine. 100 130 BHNH4 + 0.6 --~ 80 >- .t..-... en .4 Uz J 60 t- Z ...... UJ BH + .>.... . 40 2 eta-:: 113 .2 -oJ UJ c:: 20 (BH)2H+ 0 0.0 100 150 200 250 300 m/z 190 (b) uracil. 152 --CtQ >- I...-.. en z L&J I- .z.. .. L&J .>... . l-e:( ...J L&J ~ 20 150 (c) guanine. 100 127 BH + 2 -~. 80 ->- I...-.. Vl z 60 L&J I- z BHNH4..... + L&J .>... . 40 I- 144 e:( ...J L&J ~ 20 0 100 150 (d) thymine. BH + 2 200 m/z 200 m/z 250 300 (BH)2H+ 250 300 61 0.6 0.4 0.2 0.0 0.6 0.4 0.2 0.0 190 100 --~ ~o >- .t..-.. V') Z LIJ t- 60 .Z.. .. LIJ .>... . t- 40 c:( ...J LIJ IX 20 136 BH + 2 100 150 (e) adenine. 200 m,/z 250 300 62 0.8 0.6 0.4 0.2 0.0 190 63 Table 4 Sugar Related Ions R Ion H OH OCH3 (S-H20)+ 99 115 129 (S-H20+NH3)+ 116 132 146 (S-H)H+ or S+ 117 133 147 (S-H)NH4+ or S+NH3 134 150 164 (S·OH) NH4+ 152 168 182 (S-H20+15NH3)+ 117 133 147 (S-H) 15NH4+ 135 151 163 (S·OH) 15NH4+ 153 169 183 (S*-H20)+ 120 (S*-H20+NH3)+ 137 (S*-H)H+ or S*+ 138 (S*-H)NH4+ or S*+NH3 155 (S*·OH) NH4+ 173 *Ions observed from uniformly 13C labelled nucleosides moieties in nucleotides are similar to those of nucleosides, spectra of nucleosides and various deuterium labeled nucleosides (uridines and cytidines) were examined and compared. Other studies included the examination of spectra of C-13 labeled nucleotides and C-perdeuterated nucleotides. The identity of NH4+ cluster ions in the spectra were confirmed by N-15 labeled ammonium formate buffer (H20/HC0215NH4)' The deuterium labeled ammonium formate buffer (D20/HC02ND4) was utilized to investigate the mechanism and ion structure in the TSP ionization. Typical of the full scan spectra obtained as shown in Figure 16, are pdC, pU and pUm. The overall fragmentation pathways of the formation of sugar related ions are summarized in Scheme 2. 6.1.1.5.1 (S'OH)NH4+ 64 The ion found as mlz 152 in the spectra of 2'-deoxyribonucleotides (or 2'-deoxyribonucleosides) (Figure 16a), mlz 168 in ribonucleotides (or ribonucleosides) (Figure 16b), and mlz 182 in 2'-O-methylribo­nucleotides (or 2'-O-methylribonucleosides) (Figure 16c) is a sugar related ion species which is only observed in the TSP studies of nucleotides and nucleosides. No corresponding ion has been found when using other ionization methods. Comparing the mass spectra of the above compounds with those spectra obtained by using H20/HC0215NH4 buffer, a one mass unit shift was observed (Table 4, Figures 16c and 17), which indicated that NH4+ cluster ions were formed. The corresponding ion found in the spectra of C-13 labeled nucleotides indicates the presence of five labeled carbons by the shift from mlz 168 to mlz 173 (Table 4), which suggests that a ribofuranoside (!) is 65 OH R b I R=H, OH. OCHa Scheme 2. Formation of Sugar-related Ions Figure 16. Thermospray mass spectra of (a) pdC, (b) pU and (c) pUm. + c:::t + :::t: -:z:x:c: : ::t 0Vz:: :'t:): + :::t: c:::t C'\J I M :c::ot: -V') ..- C'\J ..- ..- (%) AIISN31NI 3AI1~13~ + :::t: -C'\J :::t: -co C'\J 1.0 ...... o 1.0 M o ,o... , 0 1.0 C'\J 0 0 C'\J 0 1.0 0 0 67 N ........ e U 0 a. --1'0 100-1 113 BH2 + I 1 -~ 80 >- I- ...... (S-H2O)+ V') z 60 L1J t- 115 .z....:. BHNH4+ L1J ~ 40 130 (S-H O+NH )+ cI-( ~2 3 -I L1J 0::: 132150(S-H)NH + 20 I I 4 SOHNH4+ 168 0-1 100 150 200 (b) pU. NH + 2 245 + NHNa NHK+ 267 283 250 300 m/z 0.20 1-0. 15 1-0.10 1-0.05 LO.OO 350 400 190 0"1 ex:> 100--1 1 ~9. (S-H20 ).+ + >- 164 (S-H)NH4 I .l..-... tn Lz& J BH + I- 1 ) (S-H2z BHNH O+NH3)+ 4 ...... + L&J J 146/ / ~ 4 l-e:( --' 130 L&J 0::: I S+ I 147 I SUHNH4 II. lA? 100 150 200 (c) pUm. NH + 2 259 NUM~+ I. '-u I 297 I 250 300 m/z IYIM IYUld T 339 361 350 rO. 2O 1-0.15 1-0.10 l- 0.05 0.00 400 190 m U) 100-l -~ 80 >- .t...-.. V) ~ 60 t- .z... .. w ~ 40 t-c:( ..J -1 w a:::: 20 o 1f:9 (S-H 2 O)+ 165 (S-H)*NH + BH + I 4 2 BH*NH + 113 4 Y20+*NH3)+ 147 I~ 1131 100 150 200 2~9 NH2 + 250 m/z 300 350 lo.20 ~.15 1-0.10 Figure 17. Thermospray mass spctrum of pUm obtained by using H20/HC0215NH4· 190 ""'-J a 71 produced. Additionally, a mlz 159 ion instead of mlz 152 was observed in the spectrum of 2'-deoxycytidine (Figure 18) when D20/HC02ND4 buffer was used during the spectral acquisition. In other words, the ion must possess 7 exchangeable hydrogens. A structure which is in accord with the above observations is proposed as the ribofuranoside-NH4+ ion, denoted as~. The protonated ion denoted as ~, however, is not signi­ficant. The same observation is reported by TSP spectra of sugars. 132 As discussed earlier, acid hydrolysis may playa important role in the TSP ionization. Indeed, sugar related ions do arise from the gas phase acid hydrolysis of nucleosides and nucleotides. A comparison of the spectra of deoxycytidine 5'-monophosphate (dpC), 2'-deoxy­cytidine (dC) and authentic 2-deoxyribose was made (Figure 19). It is astonishing to find that the spectra of both dpC and dC contain a 2- deoxyribose spectrum. With regard to the sugar related ions, the discrepancy factor (D)133 of dpC and dC relative to 2-deoxyribose calculated by equation k D = L: I PN - PN t ref unk n=1 k where PN - 1, N- observed masses, is 0.23 and 0.14 respectively, 1 indicating close similarity of the spectra. (For complete similarity, D is equal to 0.0; for complete dissimilarity, D is equal to 2.0.) More evidence of the acid hydrolysis mechanism in the TSP ionization of nucleosides will be discussed in section of 6.1.2 in which acid hydrolysates of deoxyribonucleosides were isolated and identified. 100 233 -~ -- 80 >- .I...-.. V') Z I..&J I- 60 .Z... .. .>I....&.. J. J 1-0.2 I- 40 <C ...J I..&J a:::: o. 1 .I 90 m/z Figure 18. Thermospray mass spectrum of dC obtained by using D 2 0/HC0 2 ND 4 . "".J N Figure 19. Thermospray mass spectra of (a) pdC, (b) dC, and (c) 2-deoxyribose. '=:t M N ,.-. 0 0 0 0 + ::J: -C\J ::J: .c...o... . + '=:t ::J: Z + - o::::t ::J: ::J: 0 + 'Z V') - ........ ::J,: N U") V') ..- ........ + C\J '=:t ::J: co N ..- ..- 0 0 0 0 0 0 co \0 '=:t N ..- (%) AIISN31NI 3AI1~13~ 0 0\ W 0 . 0 0 0 0 '=:t o U") M oo M 0 IJ"') N 0 0 N 0 U") ,-- 0 0 ,.- 74 N ........ e . U C c.. -ItS LO 0 LO 0 N N . -. - 0 0 0 0 + ¢ ::r: z + ::r: ¢ 0 ::r: V> + -Z ::rM: ::rI : NL n Z V> - + - 0 ¢ + N N ~~ ::r: + ...... C'Q V>_ N - -...- 0 0 0 0 0 co \0 ¢ ...- (%) AIISN31NI 3AIIV13~ 0 0'1 ~ LO 0 0 . 0 . 0 0 .. 0 0 N 0 0 M o Ln N 0 0 N 0 ,L..n.. 0 0 ...- 75 N ........ E U "t:J -..0 76 0 en w M. N -. 0 . 0 0 0 0 0 C1 N + 0 V N :::l: ::r:: Z I :E: + V Na. n 0 :::l: - a.n N . z: + -, CU :E: :::l: e (I) v :E: 0 ("'I') ..Q - 0 ..... s.. :::lN: -...... ~ I - 0 + CU :::l: 0 "0 :E 0 I - N --U 0 0 0 0 0 0 0 co \C V N - (%) A1ISN31NI 3All~13H 77 6.1.1.5.2 It has been reported that the consecutive expulsion of water molecules from MH+ is observed from carbohydrates °in their FO,64,6S FI9S and OCI134 spectra. Successive elimination of water molecules from MNH4+ ions is also common in TSP spectra of sugars. 132 In the spectra of pdC, pU and pUm (Figure 16), mil 134, 150, and 164 are observed respectively. The spectra of 2'-deoxycytidine (Figure 18) and 2'-0-methyluridine (Figure 20a) carried out in 020/HC02N04 buffer produced ions mil 139 and 169 which are equivalent to a removal of a 020 molecule from structures 15a and ~b respectively; although mil 159 is small and mil 189 ion is not seen. (The spectrum of 2'-0- methyluridine obtained by H20/HC02NH4 buffer is shown in Figure 20b for comparison.) c. R=H. m/z=159 b. R=OCH3. m/z=189 ( H.O~ND: 00 R 15 The above observations exclude the possibility of the C' bonded protons in the participation of water elimination. In other words, the expulsion of a 020 molecule originates from two -00 groups and Figure 20. Thermospray mass spectra of 2'-O-methyluridine obtained by using {a} 020/HC02ND4, and (b) H20/HC02NH4 buffer solution. \0 0 + N ::s:. Z C'I 0 ~ ~ 4II:t" N . 0 . 0 0 0 LN n----------------------------------~~~ -o (%) A1ISN31NI 3AI1V13~ + ~ :::r: -z :::r: I V') ~ \0 0 0 M Lon N o N 0 ....... N E -Lon o -o -to 79 0 0"1 ..... LO .q- M N . -. 0 . 0 0 0 0 0 0 M ~----------------------------------~~. N o -o o co (%) AIISN31NI 3AI1V13H o '" o 0 0 M o LO N o N 0 .......... '" E o -LO o o 80 ..c 81 results in the formation of 1,5-anhydrofuranose-ND4+ ion (12) for which there is an analogy in the condensed phase. 135 ,136 Similar ion species are observed in the TSP spectra of free sugars. 132 Similarly, the ion structure (2) shown in Scheme 2 was obtained by running with H20/HC02NH4 buffer. A H20 elimination from two hydroxy groups is found. Further evidence was obtained by the spectrum of C­perdeuterated deoxynucleosides. Sugar related ions of both 2'-deoxy­nucleosides and perdeuterated 2'-deoxynucleosides are tabulated in Table 5. The spectrum of C-perdeuterated thymidine shown in Figure 21a is a typical example ( The spectrum of thymidine is shown in Figure 21b for comparison). (OJNDt OD R 16 a R=H m/Z=139 Table 5 Sugar Related Ions from 2'-Deoxyribonucleosides and C-perdeuterated 2'-Deoxyribonucleosides Compound 2'-deoxyribo­ribonucleosides C-perdeuterated 2'-deoxyribo­ribonucleosides (S-H20)+ 99 105 Ion Observed m/z (S-H20+NH3)+ S+ (S-H) NH4+ 116 117 134 122 124 141 (S'OH) NH4+ 152 159 Figure 21. Thermospray mass spectra of (a) C-perdeuteriothymidine, and (b) thymidine. + 100 2r NH2 - ~.3 . !! 80 + 131 BH2 >- .t...-... V') z: 601 LLI t- 1-0.2 .z....:.. L&J ::- ....... 40 t- <C j 148 BHNH4 i; 1-0.1 ...J L&J 0::: 20 c+ I + 141 (S-H)NH4 t NHNa+ I '!l9 SOHNH4 + O-lt y I' i , II~ 1~T'4IJI ~ I I iii 1 , Iii t iii. i i ,.q, iii Il. if. II- 0.0 100 150 200 250 300 m/z (a) C-perdeuteriothymidine. I90 (')') w 100-1 . 1~7BH/ 243NH 2 - + t- 0,3 I ~ - 80 >- I...-... V') z: ~ 60, z t- 0.2 ...... w ::- ~ 40 < ....J ~ 203 I \.., -" I. I 11114 r-0•1 + l S 17 o-'~ l I J),l,r~~ ;?~~H~;, I. III I i I I I II. I ~~5tIH~:; I ,LOoO 100 150 200 250 300 m/z (b) thymidine. I 90 ex> ~ As expected, an 18 mass unit movement (from m/z 159 to 141) is observed. Therefore, a glycosan-NH4+ ion (denoted as 1I) is produced due to the elimination of water molecule from two hydroxy groups. ( o °JNH4+ ~---r 17 OH o m/z 141 6.1.1.5.3 Extensive studies have been made to investigate the structure and mechanism of formation of the (S -H20 + NH3)+ ion. The major ions of TSP spectra of various deuterium labeled uridine, deoxycytidine-2'I-d and I-B-O-arabinocytosine-2'-d, are summarized in Table 6; their unlabeled counterpart ions are also listed for comparison. In the earlier discussion, we proposed an 1,5-anhydrofuranoside ion (2) formation from structure~. In Table 6, column 1 and column 2 show 85 the exact mass shift of the labeled ion species which enable us to reconfirm the proposed ion structures (~ and 2). Another loss of water molecule from 2 results in the formation of m/z 116, 132, and 146 ion for deoxyribonucleotides, ribonucleotides and 2'-O-methylribo­nucleotides respectively (see Table 4). For I-B-0-arabinocytosine-2'-d and uridine-2'-d, m/z 132 instead of m/z 133 was found. That means the C'-2 attached deuterium is lost during ion (1) formation. In other words, a cis- or trans elimination of HOD from 2'-0 and 3'-OH occurs. 86 Table 6 Sugar Series Ions from Deuterium Labelled Nucleosides Ion Observed (m/z) compound (S'OH) NH4+ (S-HlNH4+ S+ or (S-H20+NH3)+ (S-H~O)+ or S NH3 (S-H)H+ or S -H2O uridine 168 150 133 132 115 uridine-5'-d 169 151 134 133 116 uridine-3'-d 169 151 134 133,132 116,115 uridine-2'-d 169 151 134 132 115 cytidine 168 150 133 132 115 1-S-0-ara- 169 151 134 132 115 binosylcy-tosine- 2'-d 2'-deoxy- 152 134 117 116 99 cytidine 2'-deoxycy- 153 135 118 116 99 tidine-2'1-d Since both m/z 133 and 132 ions are observed in the spectrum of uridine-3'-d, discrepancy arises; however, this result may not exclude the participation of 2'-OH and 3'-H in the water expulsion. In the case of uridine-5'-d only m/z 133 ion is formed; therefore, the 5'-0 is retained during water elimination. The spectrum of deoxycytidine-2'-d shown in Figure 22, is another example which exhibits both m/z 117 and 116 ions. It is clear that the release of 3'-OH accompanying with 2'2-H results in the formation of m/z 117 (loss of H20); however, if 2'1-0 participates instead, m/z 116 is produced (loss of HOO). Overall, the water elimination proceeds in a manner in which -OH and -H groups from C-2' or C-3' are expelled. .>t...­­.. V1 Z LLJ t­. Z... .. LLJ .:..:..­. t­c: t: -I LLJ a:: 112 BH; 153S0HNH4 + 0.4 0.3 0.2 O. 1 m/z Figure 22. Thermospray mass spectrum of 2'-deoxycytidine-2'1-d. 1:90 c...o... 88 The mechanism and ion structure (18) similar to 1 deduced from the above observations are shown in Scheme 3. The above observations do not rule out the possibilities of the participation of the H-l' and H-4' protons in the water elimination, although these protons have never been claimed to be responsible in other studies. 137 ,138 The formation of the S+ ion in the permethylated and TMS derivatives has been known to arise from the cleavage of the glycosidic bond with rearrangement of H-2' to the base in the mass spectra of nucleosides. 138 This may imply that H-2' is considerably labile. Indeed, the TSP spectra of nucleosides show that H-2' protons are participating in the water elimination; however, the relative contribution of both protons to the formation of dehydrated ion still remains unclear. NH~ 18 m/z 116 Scheme 3 6.1.1.5.4 S+ or (S - H)H+ The ion is observed as m/z 117, 133, and 147 for 2'-deoxyribo­nucleotides, ribonucleotides, and 2'-O-methylribonucleotides respectively. It is known that the proton affinity and polarity of the compound determines the abundance of protonated ion to its NH4+ cluster ion in TSP ionization. 90 ,132 If 1,5-anhydrofuranose formed during TSP ionization clustered with an NH4+ ion, structure 2 is formed; on the other hand, if a protonated ion species is formed instead, structure i is produced. No mass movement has been found in the HC0215NH4/H20 experiment (Table 2). This could rule out the possible formation of an NH4+ cluster ion. On the other hand, the m/z 149 ion which is present in Figure 20a, could represent the ion shown as 12. The m/z 124 ion from C-perdeuterated thymidine (Figure 21a) which shows a 7 mass unit shift of m/z 117 from thymidine (Figure 21b) gave further confirmation of structure i. More evidence could be demonstrated by the spectrum of deoxycytidine-2'I-d (Figure 22) in which one mass unit shift was observed (from m/z 117 to 118), and ion ZQ was formed. ( m/z 149 12. ( CH3 m/z 118 20 89 Although acid hydrolysis may playa major role in fragment ion formation of N-nucleotides, a gas phase ion/molecule reaction could be another route for fragment ion formation. The presence of m/z 117 (2'-deoxynucleotides), m/z 133 (nucleotides) and m/z 147 (2'-O-methyl­ribonucleotides) which are commonly found in Cl,61 El,60 and FAS69 spectra could mean the rupture of glycosidic bond in which a sugar ion (11) is formed as shown in Scheme 2, route b. A ion-molecule reaction of 11 with NH3' which is observed in ammonia Cl spectra of nucleo­sides61 results in the formation S+NH3 (lZ) ion indicating a mass equivalent to structure §. Another loss of H20 from lZ results in the formation of ~. 6.1.1.5.5 90 This series of ions has been found as m/z 99, 115, and 129 in the TSP spectra of deoxyribonucleotides, ribonucleotides, and 2'-O-methyl­ribonucleotides respectively (see Figure 16). The TSP spectra obtained using the HC0215NH4/H20 buffer show no mass shift which indicates that a protonated ion instead of NH4+ cluster ion is formed. The proposed structure (lQ) is based on the same rationale described earlier (6.1.1.5.3). Another possible pathway which is derived from the Cl mode fragmentation is the H20 expulsion from structure 11 and ion l! is formed (Scheme 2). 6.1.2 Examination of Ionization Products Produced by Thermospray The occurrence of 2-deoxyribose, cytosine and guanine in the recollected TSP ionization products is shown in Figure 23. The confirmation of the above compounds was conducted by injecting authentic substances and comparing HPLC retention times and SIM TSP spectra. Finding the sugar and nucleobases in the effluent strongly indicate that a hydrolysis process occurs in the probe during TSP ionization of nucleosides and nucleotides. A mechanism which could account for the above observation was shown in route a of Scheme 2, in which hydrolysis of the glycosidic bond occurred, and sugar and nucleobases were formed. The TSP spectrum of peak b of Figure 23 is shown in Figure 24, which may represent 1,5-anhydro-2-deoxyribose (£1). Compound £1 clustered with NH4+ ion results in the formation of mlz 134 (§); while the mlz 117 ion and mlz 139 ion represent the protonated (~) and Na+ adduct of compound II respectively. The hypothesis of structure II is based on other studies which were discussed in an earlier section (6.1.1.5). The above information gives additional evidence of thermolysis in the TSP ionization. An authentic compound, however, is required for confirmation of the structure of this compound. OH 21 91 1Gl!!] cytosine 58 ~, m/z 112 xl B 8 i ~ 5 18 1GlGlj 58 a m/z 116 x5 C SJ • i & I "I t1 'II i , I I i r:l 5 18 ~ 1GlGlj ~ 58 b m/z 117 x5 0 ~ S Ql, 1:7 I i I F" V I II • ~ I 5 18 1GlGlj a 0 ,b 51!) b m/z 133 x20 E 8 J 1 :rb-7,~ r '* ~. ,r i I r tB; era. r' • ~ tllf, 5 18 1GlGlj a b ,,=n~Q m/z 134 x5 F 51!) .f\", .• j S~ " t; f !; !if I 'Prl : , -r 11 1 '1\ 5 18 lS8j a m/z 152 x50 G 58 ~guanine Gl r~ :~~;I~.jt'!_~~I~\:i;."i~ "~!~(~ . 5 '. 1m RETENTlOO TIME (MIN.) Figure 23. SIM chromatograms of ionization products produced by thermospray. Ions which are chromatographically and spectroscopically equivalent to 2-deoxyribose (peak a), 1,5-anhydro-S-2-deoxyribose (peak b), cytosine (B), and guanine (G) were found. 92 134 100 ;; SO ~ ---1 I- 0.3 H Ul ~ 60 H J l- 0.2 ~-.40 ; , I 0.1 1:90 Figure 24. Thermospray mass spectrum of 1,5-anhydro-B- 2-deoxyribose. ~ w 94 6.1.3 Pseudouridine 5'-Monophosphate In 4.3.1, the fragmentation pathways of pseudouridine in CI and FAB were discussed. The TSP ionization of pseudouridine 5'-mono­phosphate (p~) (22) results in a spectrum which is characteristic of the C-nucleotides. Similar spectra have been observed for pseudo­uridine 3'-monophosphate (~p(3'» and pseudouridine 2'-monophosphate (~p(2'». As a example, the spectrum of p~ is shown in Figure 25. The most abundant peak is the (B + 44)+ ion at m/z 155 which is believed to be a cleavage of C-2'-C-3' and 0-4'-C-l' bonds of furanoside of MH+ (Scheme 4). The fragment ion clusters with cation ions result in the formation of m/z 172 (BNH3 + 44)+ and m/z 193 (BK + 44)+. The cleavage " '" '" r, /"/r+ + 185 rH4 202 2H+ ~--------~~~·~245 ~ -2HzO 209 l-HCOH 179 H+ ----·-325 OH~OH 1 H+ ... 155 t" •I NH4+ .. 17-2 't --K- --.-.1 93 22 Scheme 4- 100 155 ~ 80 .......... >.t...--.. ~ 60l 172 .z... .. I 209 LLI 245 NH2 + ~ 40j 179 191 193 20 185 oj, "11r-r~ I \LI"~"~~ • I 267 NHNa + I 283 NHK+ _ it 325 MH+ 1..._ .L _ """"'. iii r'ITr' I i 1'1'1' 1'1 i I 100 150 200 250 300 350 400 m/z Figure 25. Thermospray Mass Spectrum of p~. 0.15 0.10 0.05 0.00 190 \D U1 of C-S'-C-4' and C-1'-O-4' bonds of the molecule yields the protonated ion, m/z 185 (B + 74)+, and cation-cluster ion m/z 202 (BNH3 + 74)+. The second significant ion and its cation ion clusters are found at m/z 209 (NH2+ - 2H20), 226 (NHNH4+ - 2H20), and 231 (NHNa+ - 2H20) which are also observed with great intensity in CI (S4%) and FAB (7%) (seen as NH2+ -2H20).92 These ions are derived from the consecutive losses of water (Equation 7) from the NH2+ ion, however, the (NH2+ - H20) ion is much less significant. 96 Another fragmentation pathway which is also important in FAB and CI is the decomposition of m/z 209 to the formation of m/z 191 and 179 as illustrated in Equation S and Equation 7. A corresponding pro­tonated nucleoside ion (NH2+) with a 40% relative intensity is observed. As with all of the N-nucleotides examined, the very low abundance «0.5%) of MH+ in p~ may mean that the O-S'-P bond is readily hydrolyzed. The BH2+ ion which occurs as the most abundant ion in all of the N-nucleotide spectra is not found in p~. The sugar related ions, which are regarded as hydrolyzed product N-nucleotides were also unobserved; rather, (B + 44)+ (m/z 155) and (B + 74)+ (m/z 185) ions are present (Scheme 4). These results indicate that the strong C-C glycosidic bond in C-nucleotides (or C-nucleosides) has inhibited its cleavage and hydrolysis. The formation of (B + 44)+ and (B + 74)+ ions strongly indicates the similarity of TSP ionization to FAB and CI;61,92 therefore a CI-like mechanism could be proposed. The use of the TSP ionization method in the studies of nucleotides clearly reflect that fragmentation proceeds by a different mechanism, when bond linkage between nucleobases and ribose is different. 6.1.4 Cytidine 3',5'-cyclic Monophosphate and Cytidine 2',3'-cyclic Monophosphate 97 The CID/MIKE technique has been applied in the structural elucidation of 2',3'-cyclic mononucleotides and 3',5'-cyclic mononucleotides by Kingston et al. 139 The major fragmentation in both isomers is the glycosidic cleavage with production of the protonated nucleobase (BH2+)' Although the spectra are similar, there are a number of different ions present which allows unequivocal differen­tiation of the isomers. The cleavage through the sugar portion of the molecule gives fragment ions at m/z 140 and m/z 154 for cytidine 3',5'­cyclic monophosphate (3',5'-cCMP); while cytidine 2',3'-cyc1ic monophosphate (2',3'-cCMP) (23) shows a weak m/z 140 ion and m/z 154 ion is absent. A consecutive loss of the phosphate group could generate the m/z 178 peak (Scheme 5), a pathway not available to 3',5'-cCMP. The 2',3'-cCMP and 3',5'-cCMP pair were examined to evaluate the capability of the TSP ionization technique to differentiate isomers of cyclic mononuc1eotides (Figure 26). The most abundant ion found in both isomers is BH2+' The MH+ ion is also significant for both isomers (17% for 2',3'-cCMP, 35% for 3' ,5'-cCMP). The m/z 178 ion in analogy to CID/MIKE of 2',3'-cCMP (Scheme 5) is observed only in 2',3'-cCMP. Hence, a fragmentation pathway which is not available to 3',5'-cCMP has to occur and isomer differentiation becomes possible. The other major ions which could be utilized in the assignment of molecular structure are m/z 208, 212, 226, and 244 which are present in Figure 26. Thermospray mass spectra of (a) 2',3'-cCMP, and (b) 3',5'­cCMP. + N LO N ::J: ct:I N ...- ..- 0 0 o N 0 CO 0 \C o \C + N ::J: N N _("f') ::J:N ct:IN 0 ~ LO o + ::J: :IE: oo ~---..... m N ...- N 0 ...- 0 N 0 (%) AIISN31NI 3AIIV13~ o o ¢ o LO ("I"') o o ("I"') 0 LO N 0 0 N 0 \.l") ...- 0 0 ...- N ......... E c... :IE: u U I ("I"') ... 99 100~ -~80 >- t-t- f V) f5 60 t- Z t-f UJ ;:: 40 t-et: ...J UJ e:::: 20-. 112 BH2 + 1115 306 MH+ MNa + 328 MK+ fi ... I I 1IIIfl'II'I~illll~,~14tll"IIIII' O--'H-!""'/IiII,,~o\I'r 1·if;1 i If r ,II Ii I I " I I I , I I 350 400 100 15-0 250 300 m/z (b) 31 ,5 1 -ceMP. 0.3 0.2 o. 1 0.0 190 a a m/z 178 208 226 ... H + 23 Scheme 5 101 + BH2 (m/z 112) 2H1' H-t +. ----..... MH (m/z 306) 212 2',3'-cCMP. The m/z 244 ion represents protonated cytidine, and mlz 208 and mlz 226 may derive from MH+ - H3P04 and MH+ - H3P02 respectively. Although mlz 212 remains to be identified, it is believed to be derived from the glycosidic bond cleavage, in which the sugar-phosphate moiety-NH4+ ion is formed. More data using different model compounds must be obtained before proving the applicability of the TSP ionization technique to the problem of isomer identification. However, the above results indicate that an unequivocal analysis of cyclic mononucleotides by TSP ionization may be possible. 102 6.2 Vapor Temperature Dependence Studies That sample sensitivity and solvent ion intensities are a function of vaporizer temperature, has been reported by Vestal. 90 In the same report, the change in total ion intensity of adenosine as a function of power input to the probe was discussed. It is clear that vapor temperature is critical to the yield of molecular ion and fragment ions. Therefore, the effects of vapor temperature on ion abundance of nucleotides are examined. The ion intensity profiles for pA against vapor temperature are shown in Figure 27. Similar results were observed for pC. The optimal vapor temperature for observing the molecular ion is 2500C, although a higher temperature is required for the NH2+ (2800C) ion and the BH2+ (>3000C) ion. These results give another evidence that thermal decomposition occurs in the TSP ionization of nucleotides. Thermospray analytical conditions may be chosen to optimize response for the MH2+ ion for the nucleotides with modest sensitivity for favorable compounds (3xlO-9 C /mmole for pA being a typical value) or at a higher temperatures where nucleoside fragment or nucleobase fragment ions predominate for all compounds examined. 6.3 pH Dependence Studies The ion intensities against vapor temperature were plotted at pH 3, 4, and 5. Two nucleotides (pC and pAl have been used in this study. A typical example as shown in Figure 28 indicates that the optimal pH value for obtaining the MH+ ion is pH 4. At higher (pH 5) :::0 rn r » --r<nI l> [l) c: z o l> zn ITl 200 250 300 350 TEMPERATURE (OC) Figure 27. Dependence of ion abundance of pA on vapor temperature. Operated with 0.1 M HC02NH4 buffer, pH 4.0. Flow rate: 1.5 ml/min .. 103 Figure 28. Ion abundance of MH+ versus vapor temperature at different pH value of buffer solution. (a) pC, (b) pA. Operated with 0.1 M, HC02NH4 buffer. Flow rate: 1.5 ml/min .. L.&J V) z a 0- V) W ~ a MH': 324 --....... , - ........ pH 3 "­-.-................ . p H 5'," ' ... / . ..... , .... ~ .... ...... . '.......... ........ ' ....... -.... -- .......... --.~.-. 250 300 TEMPERATURE (DC) 350 LaJ V) z a 0- V) LaJ ~. 200 b pH 5 .-.~- ........ , H 3 /" ..........-'. \ " ,.p . \ V \ 0, \ \ '.' . \ '. \ '. \ '. \ \ '\, \ MH+: 348 ,,~\ 250 300 TEMPERATURE (OC) "',.\ .'", ~ '~~ 350 ~ o U'1 106 or lower (pH 3) pH value, the intensity drops at all the temperatures. It is conceivable that at pH 3, acid hydrolysis ~ecomes dominant in the ionization which results in diminished MH+ ion intensity. At pH 5, however, less protonated ion species exist in the solution; therefore a direct ion evaporization which results in MH+ formation becomes more difficult. Although the above data show the same pH effects for each compound in their TSP spectra, a more conclusive result cannot be reached without more extensive studies. 6.4 Thermospray liquid Chromatograph~ Mass Spectrometry The chromatogram with detection based on UV absorbance at 254 nm of a mixture of nucleoside 5'-monophosphates is shown in Figure 29. The separation of the enzymatic hydrolysate of tRNA from Baker's yeast is shown in Figure 30. Figure 31 illustrates the UV254 absorbance chromatogram of the alkaline hydrolysate of tRNA from Baker's yeast. The 3'-isomers eluted before the 2'-isomers for all of the nucleotides which were examined. The corresponding total ion current (TIC) chromatogram plotted from the above scan is shown in Figure 32 for comparison. A good correlation between the UV absorption and TIC response is observed. It is interesting to note that the separation efficiency of the thermospray lC/MS combination as measured by the theoretical plate count (Nth) compares well with that by UV detection (Nth= 12000 for thermospray compares with 18000 for UV response). These results indicate that chromatographic resolution is maintained by the MS interface. Reconstructed ion chromatogram (RIC) of mlz 115, LaJ U z <C ca 0:: a V) ca <C pC ~ o pU pG '--J . 10 ,.Gm n pn~A ~ ~ J\ J -T 20 30 40 RETENTION TIME (MIN.) Figure 29. HPLe separation of nucleoside 5'-monophosphates with Supelco 25cm x 4.6mm 5u ODS column. Separation conditions see text. --i a '-J Lr..I U z: ~ c:::: o V') c:c < b 0.1 AU pU I . I I IpG j . pA n . 5' 10 RETENTION TIME (MIN.) 15 Figure 30. HPLC separation of enzymatic hydrolysate of RNA from baker's yeast. Chromatographic conditions same as Figure 29. Flow rate: 2m1/min .• 108 ...., uz ce:sn:: 0: 0 eVnl tpC3'J c:s:: J Figure 31. Cp(3', GpC)1 I CpC2*) Ill:: II Ap(31 II I lUp(tl Gpl21 ApC21 10 20 30 -'0 RETENTION TIME (MIN.' HPLC separation of nucleoside 2'(3')-monophosphates, with UV detection at 254nm. Conditions same as Figure 29. 2'(3')­nucleotldes are produced by alkaline hydrolysis of RNA, which proceeds through a 2',3'-cycllc intermediate to yield the 2'(3') mixture. o \0 132, and 150 together with the TIC, shown in Figure 32, represent the common sugar ions which are observed in the thermospray mass spectra 110 of both nucleosides73 and nucleotides. The corresponding sugar ions were also found in thermospray mass spectra of 2'-deoxyribonucleosides and 2'-O-methylribonucleosides. 73 These fragment ions can be used in screening of nucleosides and nucleotides. The RIC at m/z 244 resulting from NH2+ ion of cytidine 3'-monophosphate (Cp(3'}) and cytidine 2'­monophosphate (Cp(2'}) (Figure 33a) shows the characteristic ion which was selected for compound detection. Another example, shown in Figure 33b, is m/z 245 resulting from the NH2+ ion of ~p(3'}, ~p(2'}, uridine 3'-monophosphate (Up(3')}, and uridine 2'-monophosphate (Up(2'». The selected ion recording chromatogram of the same hydrolysate with a 10 ug injection is shown in Figure 34. Minor constituents known as 3-methylcytidine 3'-monophosphate (m3Cp(3'}) and 3-methylcytidine 2' monophosphate (m3Cp(2'» (Figure 34a) with signal/noise>10 were detected. The same scan by monitoring NH2+ ion (m/z 245) of ~p(3'}, ~p(2'), Up(3'}, and Up(2'} (Figure 34b) also indicates high sensitivity in detection. The MH+ ions of some nucleotides have not been significant in thermospray LC/MS; however, fragment ions with significant abundance have been observed for these compounds (See section 6.1.1, Table 3.). In addition to the simple HPLC separation system we developed here, the use of thermospray mass spectrometer as a detector provides a sensitive and unambiguous method for nucleotide determination. 100! ~ TIC 0'-,:;· ; • :: · , : ~ • i : C; ,I L>:- +9 o m 1001 ~ m/z 115 ~ •• o0z~. :. 0 .• I I ~I'-; 11 • i 0 . (';-; .... ex:: ~ 100 1 ~ m/z 132 ~ A A .. 0-,. . I •• -;-.... ~ LoL I O , 0 ""7 . I e • f I ; ; m/z 150 20 30 4Q RETENTION TIME (MIN.) Figure 32. Mass chromatograms (reconstructed from repeated scans) corresponding to Figure 31. In some instances common sugar ions m/z 115, 132 and 150 serve equally as well as TIC. --' --' LIJ 10 8 6(} 4(} Q)(l" (pe2" ~ 201 III C o a. ~ v:r 0:: 0 10 0:: 10 ~ i Ill(]') u LIJ t- ~ 60 I 11ll(2', 401 , (2', IlLe]') P 20: 1p a m/z 244 ............... ..J"".uau.. ......... n .... M ........... _-L.LL~.L... ... . .-...---.~ • .--,---....---.-----------.. ----. ,- -.------. ..---.---, -.~--.------.--. 20 30 40 b m/z 245 Figure 33. Mass chromatograms of m/z 244 (a), and m/z 245 (b) corresponding to Figure 31. --' --' N 100 . ao· 60- 40- LaJ 20· Vl z ~ 0 ~ 0 n! 100 0::: o t; 80 hJ t­hJ C 60' 40· 20 I OJ~, .0 m/z 126 a 1'~"""'MH.1\"""1,I1M4'~~tI.",*"N.w ... }",U4 ... \~~J ... • , •• i •• l' i ...--..---,--..-....-.--...-, 10 20 30 40 m/z 245 b ~"f"'I"'''''''' ...... ".,..,.,. .......... ,. ".,....,~ .. If.,' .......... M' ............ .,....,."...,. .... "'. 10 20 30 40 RETENTION TIME (MIN.) Figure 34. SIM chromatograms of minor components of alkaline hydrolysate from baker's yeast (10 ug injection). m/z 126 (a): BH2+ ion of 2'(3')­m3Cp.; and m/z 245 (b): NH2+ ion of 2'(3')-~p and 2'(3')-Up. ............ w CHAPTER 7 CONCLUSION In 1973, McCloskey et al. 140 demonstrated a correlation between the solution hydrolysis of the glycosidic bond of nucleosides and gas phase cleavage, in the absence of solvent, of the base-sugar bond in the CI mode, that is, both liquid and gas-phase hydrolysis experiments yield the same qualitative results concerning the glycosidic bond stability of nucleosides. The relative intensity difference in sugar ions in the TSP spectra of deoxyribonucleotides and ribonucleotides may reflect the increased lability of the glycosidic bond of 2'-0- deoxyribonucleotides (or 2'-deoxyribonucleosides), relative to ribonucleotides toward acid hydrolysis. The dual role of TSP ionization behaving as a direct ion evaporation device as well as ordinary CI source is known. 90 The relative contributions to fragment ion formation are a function of vaporizer temperature, sample concentration, and reagent ions. 90 However, the above studies reveal that a third mechanism, acid hydrolysis, is more important in the fragment ion production of nucleosides and nucleotides. If nucleotides are vulnerable to hydrolysis, acid hydrolysis prevails. In the case of P , because of the strong C-C linkage between nucleobase and sugar, the CI mode becomes the predominant fragmentation mechanism. 115 In conclusion, the TSP ionization technique gives simple mass spectra of nucleotides and nucleosides. Relatively few fragment ions are observed, however they are usually easily assigned. Although some possible ways could be tried to enhance the molecular ion (e.g., negative ion TSP), our efforts reported in these studies have yielded important information which can be used in the structural elucidation of nucleotides and nucleosides. The BH2+, NH2+, and sugar related ions combined with the HPLC separation of the nucleotides could gives an unambiguous method for the analysis of known nucleotides and can be of significant benefit to the structure determination of unknown nucleotides in mixtures. PART 3 THERMOSPRAY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY OF CARBOHYDRATES CHAPTER 8 INTRODUCTION In Part 2 of this thesis, the mechanism of ion formation in the thermospray mass spectra and the applications of thermospray techniques to the analysis of some nonvolatile biochemical compounds were discussed. The capability of thermospray LC/MS has drawn more and more attention of mass spectroscopists, although some drawbacks remain to be overcome. 90 This part of the thesis is a continuation of applying thermospray LC/MS technique in the mass spectrometric study of carbohydrates. Mass spectra of carbohydrates were first reported by Reed and coworkers141 in 19S8. Since then more systematic investigation of carbohydrate derivatives were started by a number of groups.142,143 This early work has been summarized in many extensive accounts144-149 to which the reader is referred for details. One of the most important reviews which contains the methods to resolve adequately most problems and approaches in the area of carbohydrate investigations by mass spectrometry has been reviewed by Reinhold and Carr. ISO Structural studies of carbohydrates and of glycoconjugates rely heavily on chromatographic separations, which are of particular importance because of the great diversity of carbohydrates and the large numbers of isomers of similar structure. HPLC has been of 118 increasing importance. 151 Recent reports have been made on the HPLC of 01igosaccharides,152-155 and methylglycosides156-158 and in detailed reviews which have included discussions of detection methods. 151 ,159,160 However, the general applicability of HPLC to microscale structural and analytical work has been greatly restricted by limitations in detector sensitivity. Generally, a refractive index (RI) detector was used in large scale detection of carbohydrates. 151 ,159,160 Other than the traditional RI detector, postcolumn reaction methods,151,162 interferometry,163 amperometry,164 and other techniques 160 have been reported in the efforts to increase the sensitivity, stability and simplicity of carbohydrate detection (discussed in later section). A major reason for LC/MS system is that they provide a universal detection system for LC, and this is particularly important for compounds with weak or absent chromophore. Sugars are compounds of this type. Earlier LC/MS studies of both positive and negative ion spectra of mono-, di-, and trisaccharides were carried out with moving belt device97 ,165-167 in which ammonia or methylene chloride was used as the chemical ionization (CI) reagent. Although significant MNH4+ ion was found in positive CI spectra of monosaccharides, ions containing molecular information were not found in trisaccharides and not significant in disaccharides (1%). A direct liquid introduction (OLI) interface for combined LC/MS with CI mass spectrometer was used by Arpino et al. 168 in the study of the orientation of the jet of the droplets in the effects of the mass spectral fragmentation pattern of 119 underivatized monosaccharides and sucrose. However a conclusive result was not reached. A structural elucidation of polysaccharides using LC/MS/CI mass spectra of peralkylated sugars was reported by Aman et al. 169 with some success, but the sensitivity of the method was not reported. Recently, a discharge tube was installed on the desolvation chamber as alternative to the filament of the micro column LC/MS using a capillary interface. The negative CI spectrum of sucrose which contains molecular ion information has been successfully obtained. 170 Fenselau et al. 79 were first to report TSP positive ion spectra of glucuronides and negative ion spectra of stachyose by direct introduction of samples to the probe. Schmelzeisen-Redeker et al. 171 examined TSP spectra of sucrose and glucose to the studies of field effects on the charged droplets in molecular ion formation by the technique. However, a systematic mass spectrometric study of carbohydrates by TSP ionization method was not reported. Herein we are able to report the results of initial studies of the TSP mass spectra of mono- and disaccharides of various anomeric and linkage configurations, 1-0-methylhexopyranosides and permethylated mono­through tetrasaccharides, and the assessment of thermospray mass spectrometry as a selective detector for direct monitoring of HPLC effluent in amounts down to 10- 13 mole. 8.1 Detection Methods of Carbohydrate in HPLC High sensitivity is a necessity in modern chromatography. Available sample quantity is often limited and the compounds to be analyzed may be present at low concentration. The lack of a satisfactory detector was probably the greatest impediment to the development of HPLC in the analysis of carbohydrates. Other than the TSP ionization method in this report, different attempts have been made in an effort to enhance the sensitivity of carbohydrate detection. Two methods have been approached: (a) the direct methods, which includes RI, UV and moving wire and mass detection techniques are the methods without the need of derivative preparation. (b) The indirect methods which include the postcolumn and precolumn labeling techniques require the specific reagents to derivatize the compounds before they can be detected. 8.1.1 Refractive Index (RI) Detector 120 RI detection is a nondestructive method which is universally applicable in HPLC separation of carbohydrates.172-176 Only moderate sensitivity has been claimed. Palmer161 reported that 20 ug was the minimum detectable amount of sugar, whereas Cerny et al. 177 were able to detect a quantity of 3 ug at a signal to noise ratio of 5. Since this detection method is highly susceptible to changes of column temperature, solvent composition, and pressure,172-176,178,179 uses of this detector are usually limited to isocratic chromatography.172- 176,179,180 Based upon the same detection technique, a high sensitivity RI detector, Optilab interference refractometer, was developed. 181 Sugars detected at 10 ng level have been demonstrated. Another RI detector based on the combination of Fabry-Perot interferometry and a single 121 frequency laser reported by Woodruff and Yeung 163 ,182 enable detection of D-glucose, sucrose, and raffinose at ug level. In spite of the enhancement of detection sensitivity, the applicabilities of the above methods eventually are still hampered by the shortcomings commonly encountered by RI detectors. 163 ,181 8.1.2 Ultraviolet (UV) Detector Sugars can be detected directly in the range 185-195 nm; however, the choice of solvents is limited and high purity solvents are required. Hettinger and Majors182 reported that the sensitivity was approximately 10 times higher than that of RI detector for glucose and fructose. For sugars of higher molecular weight, the sensitivity decreased to the values of RI detectors. Binder178 analyzed a number of monosaccharides and found almost equal sensitivity for the two methods. More than 1000 times greater sensitivity can be achieved by detecting benzoyl esters sugars183 ,184 or nitrobenzoate sugars185 at 254 nm, however, derivatization has to be made before detection. More detailed discussion of derivatization followed by UV detection will be presented in section 8.1.6. 8.1.3 Moving-wire Detection The flame ionization technique, commonly used in gas chromatography, is sometimes used in HPLC. In this method, a clean platinum wire continuously rotates and becomes moistened by the pure eluent or with eluent containing the component to be detected. The eluent is evaporated and the residual material is combusted in a 122 pyrolysis oven and detected with a flame-ionization detector. This method is not well suited for HPLC of carbohydrates where a nonvolatile solvent, water, is used as eluent. Although Wells et al. 186 claimed it is suitable for the gradient systems, the manufacturer, Philips-Pye Unicam took it out of production in 1978. 8.1.4 Mass Detection In the mass detector, the eluent stream containing the solute is nebulized and carried by an air stream through a heated column. The eluent evaporates, leaving a fine mist of solute particles which pass through a light beam. Light scattering occurs and is detected by a photomultiplier. Except with eluents to which salts have been added, the method can also be used for sugar detection and the minimal detectable amount is about 500 ng. 160,187 The detector will only function efficiently if all the chromatographic solvent is removed by evaporation prior to detection. Thus, the noise level is dependent on both the evaporation temperature and the amount of water in the solvent. In the case of gradient elution, it is necessary to adjust the evaporation temperature during the chromatographic run. 188 8.1.5 Polarimetric Detection The development of a micro polarimeter allowed the optical activity of a solute to be the basis for its detection in HPLC, as shown by Yeung et al. 189 and Bohme. 190 The detection of 0.5 ug of fructose and raffinose at a signal to noise ratio of 3 was demonstrated. Problems arise in quantitative analysis, because the 123 anomeric forms give a different response. 160 However, the polarity of the response can give additional qualitative information. By using optically inactive eluents, a gradient elution can be applied. 8.1.6 Postcolumn and Precolumn Detection The use of postcolumn labeling makes up for the lack of chromophores and fluorophores in carbohydrates. The utilization of the reducing power of aldoses and related carbohydrates in the postcolumn labeling are the most widely used methods. 191 Reactions of carbohydrates with chromogenic reagents can give colored compounds which possess absorption maxima at 420, 480 and 640 nm, when orcinol ,192 phenol ,193 or anthrone194 are applied, respectively. Both aldoses and ketones are detected with sensitivity of ca 1 nmol. The absorption maxima which range from visible to fluorescence are selected for detection depending on the derivative prepared. An important method for the detection of nonreducing carbohydrates such as alditol 195 and aldonic acid196 is based on lutine formation by the Hantzsch reaction of formaldehyde, which is produced by the reaction of carbohydrates with periodate. The analysis of oxidizable compounds by electrochemical detection enables one to detect ascorbic acid197-199 in the picomole level. In another attempt,164 eluted reducing sugars were oxidized with a cupric salt, and the resultant cuprous ion was coupled to bis(phenanthro­line). The complex formed was detected using a glassy carbon elec­trode, with a detection limit down to 0.001 nmol. Direct detection of aldoses and alditols at 0.0015 nmol by a pulsed amperometry technique 124 has been reported by Rocklin and Pohl. 200 Other t~chniques include precolumn labeling with radioactive isotopes,201,202 precolumn labeling with chromophore reagents which result in intense absorbance in the UV region and sometimes fluorescence. 203 CHAPTER 9 EXPERIMENTAL 9.1 Materials Sophorose and kojibiose were purchased from Gezyme Corp. (Boston, MA). D-Glucose-1-d1, D-glucose-3-d1, and D-glucose-6,6-d2 were purchased from MSD Isotopes (Montreal, Canada). All other sugars were obtained from Sigma Chemical Co. (St. Louis, MO). HPLC grade water and D20/HC02ND4 buffer solution were prepared by the same procedures as described in Chapter 5. Bond-Elut C-18 (Analytichem International, Harbor City, CA) was used for the purification of permethylated sugars. 9.2 Methanolysis Reaction The same methanolysis procedure described in Chapter 2 was followed for the preparation of 1-0-methyl-, and 1-0-ethylglycosides from carbohydrates and glycoconjugates. The amount of 100 ng of lactose and naringin was treated and an aliquot of 1-2 ng of each hydrolysate mixture was injected for the SIM experiment. 9.3 Permethylation of Carbohydrates The permethylation of carbohydrates was based on the procedures of Hakomori 204 as modified by Sanford and Conrad. 205 The following 126 microgram permethylation reaction performed by Waehe et al. 206 was used with some modification. The methylsulfinyl anion was prepared as follows. In a dry 2S0 ml three-necked round bottom flask, fitted at one neck with a rubber serum cap and containing a magnetic stirring bar, was weighed 1.S g of sodium hydride. The sodium hydride was washed three times by stirring with 30 ml portion of n-hexane and decanting the wash. After the third wash the flask was fitted with a thermometer and a stoppered condenser and residual n-hexane was removed by successive evacuations with a vacuum pump through a needle inserted into the serum cap. After each evacuation, the flask was regassed with nitrogen. The stopper was then removed from the condenser and nitrogen was passed continuously through the flask via the needle. An aliquot of 15 ml dimethyl sulfoxide (distilled from calcium hydride) was injected into the flask through the cap. The flask was placed in a heating mantle and stirred with a magnetic stirrer at SOoC until the solution became clear and green and evolution of hydrogen ceased (ca 1 hr.). One ml of final reagent was titrated with 0.1 N HC1. The concentration of the anion from this preparation was about 4 M. For generation of the carbohydrate alkoxide, a microgram quantity of carbohydrate in solution was transferred to a 0.3 ml Reacti-Vial (Pierce Chemical Co., Rockford, IL) and dried in a Sped-Vac (for 100 ul of water solution, 3 hrs. was usually sufficient) A Reacti-Vial magnetic stirrer was placed in the vial and a Teflon coated septum screwed in place. Dry DMSO (0.2 ml) was added to the vial through the septum cap and magnetically stirred until the carbohydrate dissolved 127 solution (30 ul, 4 M) was also added through the septum, and the mixture was stirred at room temperature for 4-5 hours. An aliquot (5 ul) of the reaction mixture was tested with triphenylmethane207 before CH31 was added. If the test was negative, another 15 ul aliquot of anion solution was added and stirred for another two hours until a positive test with triphenylmethane was observed. For the methylation reaction, the final solution was cooled and maintained to 20-250C in an ice-water bath. Following this, 50 ul methyl iodide solution was added to the stirring mixture over a period of 10-20 seconds. (The amount of methyl iodide added was not critical as long as it was in molar excess of the base.) Within a few seconds of the addition of methyl iodide, the solution became clear, and the viscosity was markedly reduced. The mixture was removed from the water bath and stirred at room temperature overnight. The final mixture was purified by using Bond-Elut C-18 cartridge. A Bond-Elut C-18 cartridge was flushed with 20 ml of 100% methanol and centrifuged dry in the Spec-Vac before use. The methylation reaction mixture containing the per-O-methylated carbohydrate was diluted with an equal volume of HPLC-grade water to produce a 50% DMSO-water solution. This solution was transferred to the cartridge and sucked into the cartridge bed by connecting a 2 ml syringe to the outlet of the cartridge. The vial was rinsed with 1:1 (V/V) DMSO-water to ensure a quantitative transfer of residue (two aliquot, each aliquot 0.3 ml). Each of the residues was loaded onto the cartridge and sucked out as above. The sample containing cartridge was then washed 128 with five 2 ml aliquots of HPlC-grade water to elute polar contaminants in the methylation-reaction mixture, including t~e DMSO and CH3I. The per-O-methylated carbohydrate was then eluted with 5 ml methanol and collected in a test tube. The solvents were removed under reduced pressure and the dry residue was ready for thermospray lC/MS injection. The above procedure has been successfully used to permethylate 1 ug of 2'-deoxycytidine and 10 ug of disaccharides, trisaccharides, and tetrasaccharides. 9.4 liquid ChromatographY-Mass Spectrometry Thermospray mass spectra were acquired using a noncommercial quadrupole mass spectrometer described in Chapter 5 of this thesis. A Beckman 332M liquid chromatograph, connected to an Altex model 420 system controller interfaced to a Vestec Corp. (Houston, TX) TSP probe and temperature controller was used. The optimum interface temperature was determined by making multiple injections at various heater temperatures. The temperatures giving the largest molecular ion intensities were slightly different for each class of compounds studied. The probe tip was maintained by electrical heating76 at 240-2500C, resulting in vapor temperature ranging from 230 to 2600C. Samples of free sugars and 1-0-methylglycosides were introduced to a Supelco (Houston, TX) 25 cm x 4.6 mm 5u ODS column and a Brownlee (Santa Clara, CA) 30 x 4.6 mm 5u RP-18 guard column. A 30 cm x 3.9 mm Waters (Milford, MA) uBondpak NH2 column was used for introduction of permethylated carbohydrates. Ammonium formate, (0.1 M, pH 5.6), 1.5 ml/min. was used as the mobile phase. Mass spectra were acquired 129 ml/min. was used as the mobile phase. Mass spectra were acquired without regard to sensitivity limits, over the range 200 ng-5 ug. Sensitivity measurements were made in the selected ion monitoring mode. The separation of methanolysis or ethanolysis mixtures of lactose or naringin were also carried out by a similar Supelco column attached to a Brownlee guard column; however for these mixture, HC02NH4 buffer (0.1 M, pH 5.6) containing 10% CH30H was used as the mobile phase. CHAPTER 10 RESULTS AND DISCUSSION Thermospray ionization72 occurs during evaporation of microdroplets in a sonic jet of vapor formed from the HPLC effluent. 77 No chromophore or specific functional group is required for detection, and mass spectrometry offers a major advantage over other liquid chromatographic detector systems in the selectivity afforded by mass-specific detection. In the present study, use of HC02NH4 buffer as a chromatographic mobile phase was found to provide a source of NH4+ ions to form molecular adducts ions (MNH4+) suitable for determination of molecular weight, or in most cases, for selected ion monitoring. Many of the ions observed in TSP mass spectra described below are also found in ammonia C1 208,209 E1 146,150 and SIMS210 ,211 mass spectra of carbohydrates and permethylated nucleosides. 137 10.1 Monosaccharides The spectra of mono-, di-, tri-, and tetrasaccharides and their partially methylated and permethylated derivatives, and amino sugars are listed in Appendix B of this thesis. The thermospray mass spectrum of 2-deoxyribose (Figure 35) exhibits the features typically found in the hexose and pentoses examined: an abundant MNH4+ ion, a less 131 + 100 134 MNH4-H2O 0.4 --1iI'It 80 >- 152 MNH: 0.3 .f..-.. en .z.. .. 60 f- .z...:. 0.2 ..... .>... . 40 f- cc .......I. a:: o. 1 20 0.0 1 100 100 150 200 250 m/z Figure 35. Thermospray mass spectrum of 2-deoxyribose. abundant MH+ species and fragment ions arising from multiple losses of H20. The presence of nitrogen alters the relative abundance of ions. Both N-acetylglucosamine and glucosamine exhibit a more abundant MH+ ion than MNH4+ ion (Figure 36). Peaks corresponding to MNH4+ - H20 and MH+ - H20 were found in all the free sugars (Figures 35 and 36). The corresponding ions were not observed in 1-0-alkylglycosides. Instead, ions representing MNH4+ - ROH and MH+ - ROH were found (discussed in 10.2). This implies that the hemiacetal hydroxy (I-hydroxy) group is involved in the first water molecule elimination. The thermospray spectrum of glucose which was obtained by using D20/HC02ND4 buffer solution shows m/z 187 and 166 ions (Table 7). They represent MND4+ - D20 and MD+ - D20 respectively. This additional information strongly indicates that the Figure 36. Thermospray mass spectra of (a) N-acetylglucosamine, and (b) glucosamine. 100 -~ -....I ..>......-... V) z: LIJ ..... .z...:.. LIJ >......... .. cc . ...J LIJ c::: .>.......­... V) z: LIJ ..... .%.... . LIJ .>.... . ~ ...J LIJ ex: 80 60 133 MH +- H2O 204 O. 15 144-H2O 126 C4H804NH+4 MH+ 0.10 L4 MH+-2H 2O 222 186 13 0.05 m/z (a) 0 .. 25 0.20 0.15 0 .. 10 90 0.05 o....J~ ................. ~~f-YhII~..,.~~~,.,..., 0 .. 00 160 60 1 00 150 200 (b) m/z 134 Table 7 Ions from Thermospray Mass Spectra of -Glucose Ion Observed tmLz} Mobile Phase NH4+ MNH4+-H20 MNH4+-2H20 H20/HC02NH4 198 180 162 D20/HC02ND4 207 187 168,167 elimination of a water molecule from free sugars to form glycosans originates from two hydroxy groups, one of which is the hemiacetal hydroxy group. A further elimination of a water molecule from the above ion (glycosans) results in the formation of MNH4+ - 2H20 and MH+ - 2H20 ions. It is known that pyrolysis of cellulose,212,213 starch,212,214 O-glucose214 (24), B_O_glucoside,212,215-219 and disaccharides219 all give 1,6-anhydro-B-O-glucopyranose (1,6-levoglucosan, denoted as 25), as does treatment of cellulose with superheated steam at 24 to 30 mm Hg. 220 Pyrolysis of B-D-galactose also gives 1,6-anhydro-B-D-galacto­pyranose. 221 A mixture of 1,6-anhydro-B-O-glucopyranose and 1,6-anhydro-B-D-galactopyranose was obtained by pyrolysis of lactose. 222 ,223 Extensive studies by Shafizadeh et al.224-226 pointed out that heating of levoglucosan (25) results in the formation of levoglucosenone (ZQ). Curie-point pyrolysis in combination with high resolution FI mass spectrometry of polysaccharides reported by Schulten and Gortz227 suggests that probable structures for the pyrolysis products which give m/z 162 (25), m/z 144 (26) and m/z 126 (27) ions 135 are those proposed in Scheme 6. Therefore, the formation of the MNH4+ - H20 ion from glucose-NH4+ (28) (Scheme 7) may proceed via the same pathway in which thermal decomposition occurs and 1,6-levoglucosan­NH4+ (29) is formed. The consecutive expulsion of H20 from 29 results in m/z 162 (MNH4+ - 2H20) (30) and m/z 144 (MNH4+ - 3H20) (~) ions which may be represented by NH4+ ion clusters with 26 and 27 respectively. Another study of the ammonia CI mass spectrum of 1,6- anhydro-B-D-galactopyranose by Horton et al. 228 shows a major peak corresponding to MNH4+ (m/z 180), as well as m/z 162 (MNH4+ - H20) and m/z 144 (MNH4+ - 2H20) ions. The similarity of the observed ions of the above compound to the TSP spectrum of galactose suggests that an analogous CI mode ionization may play an important role in the TSP ionization of sugars. It has been reported229-231 that 1,4-anhydro compounds are formed in the pyrolysis of carbohydrates when C-6 hydroxy group is blocked. In the TSP spectrum of rhamnose (32) (Figure 37), where C6-0H is not available, a MNH4+ - H20 ion (m/z 164) is observed, which is derived from the H20 expulsion from 1-0H and 4-0H groups in which 1,4-anhydro­rhamnose (33) is formed (Scheme 8). The elimination of CH30H from MNH4+ ion of 1-0-methylrhamnose (Figure 38) undergoes the same mechanism. The 1,4-anhydrorhamnose formation via CH30H expulsion results from the participation of the 4-0H and 1-0CH3. It is known 135 - 136 that 1,4-anhydropyranose may be regarded as 1,5-anhydrofuranose (34). The blockage of 6-0H provides an alternative route to form this compound, which is analogous to the ion species found in the TSP spectra of ribofuranose and nucleotides (see Part 2 of this thesis). OH 24 OH H,OH -HzO .. OH mIl 162 25 -HzO .. Scheme 6 H'OH) NHt .. • OH mIl 198 28 Scheme 7 -~O .. 26 mIl 126 27 -H 0 2 .. ~NH: mIl 144 31 --' w m ·137 100 182 MNH: .A - 80 ~ ........ >- I- .3 ~ Cz../. '.) 60 MNH+4 -H20 I- z: 164 ~ .... .2 > 40 t-t l- c.....c.... 0: 20 •1 0 .0 1100 100 150 200 250 m/z Figure 37. Thermospray mass spectrum of rhamnose. 196 MNH: ...... 0.4 -~ >- I...-.. 0.3 V) z LLJ I-. Z.. .. .>....... .. 0.2 I- :5 + LLJ MNH4-CH3OH 0: 2 164 0.1 I 0.0 1100 100 150 200 250 mIl Figure 38. Thermospray mass spectrum of 1-0-methylrhamnose. 138 ~ CH- 01 H OH HO OH HO • ~o + ·NH4 CH3 OH 0 32 Scheme 8 34 In the case of 2,3,4,6,-tetramethylglucopyranose (Figure 39) (35) (Scheme 9) however, a m/z 236 (MNH4+ - H20) ion instead of m/z 222 (MNH4+ - CH30H) ion was observed; hence, no levoglucosan is formed. In other words, a different mechanism which is in analogy to ammonia C1 135,136 may occur in the ionization. In conclusion, thermal decomposition and CI mode ion/molecule reactions appear to be the major fragmentation pathways in the TSP ionization of carbohydrates. A ring rupture involving cleavage of the C2-C3 and CI-05 bonds of D-glucopyranose (Scheme lOa) results in the formation of the m/z 138 and m/z 120 ions (Figure 40a). The corresponding ions and TSP spectra of the deuterium labelled compounds observed under the same conditions are given in Table 8 and Figure 40. The shift of m/z 138 to m/z 140 for glucose-6,6-d2 (Figure 40d), as well as m/z 138 to