Electrospray tandem mass spectrometry of 3 '-azido-2 ' , 3 ' dideoxythymidine and of a novel series of 3 '-azido-2 ' , 3 ' , 4 '-trideoxy-4 '-thio-5-halogenouridines and their respective u-anomers

Electrospray mass spectrometry has aided the structural characterization of 3'-azido-2',3'-dideoxythymidine (AZI) and the novel series of 3'-azido-2',3',4'trideoxy-4'-thio-S-(bromo, chi oro or fluoro )uridine nucleosides and their respective 0.anomers. Low energy CAD MSIMS analysis of the protonated molecules [M+H]+ confmned the predicted fragmentation route for AZf and a series of related 4' -thio-S-halogenouridines. This MSIMS study also provided characteristic fmgetprint patterns which permitted differentiation of anomers within the series of 3'-azido2' ,3' ,4' -trideoxy-4' -thio-S-halogenouridine nucleosides.


Introduction
The human immunodeficiency virus (HIV) has been accepted as being the etiological agent of acquired immunodeficiency syndrome (AIDS) [1].Nucleoside analogues such as 3 '-azido-2',3'-dideoxythymidine (AZT) [2] and other 2',3'_ dideoxynucleosides [3] are potent inhibitors of this retrovirus in vitro and in vivo.There is an ongoing search for novel nucleosides which are effective against the AIDS virus.Systematic structure-reactivity relationship studies of AZT analogues have shoml a pronounced enhancement of the anti-IllY activity when the 5-position of the pyrimidine ring is substituted by halogens such as Br or I [4].It has also been shoml that nucleoside analogues in which the 0-4' has been replaced by either a methylene group or sulfur atom are more resistant to nucleoside phosphorylase cleavage [5,6].Also, modifications of this type have conferred such analogues with either or both antiviral and anticancer activities.
Fenn and coworkers have shown that electrospray ionization (ES) is a powerful means of producing peptide and protein ions suitable for study by mass spectrometry [10,11].Recently, it has also been shown that the electrospray ionization process is sufficiently mild to detect non-covalent complexes involving a protein and specific ligands [12][13][14].
This approach has been applied to the study of receptor-drug interactions, proteinprotein interactions [15] and either covalent or noncovalent intennediates formed in enzyme catalyzed reactions [16][17][18].Furthennore, electrospray MS has also been used to structurally characterize and differentiate relatively small, single charged amphiphillic regioisomeric esters of sucrose [19].As a continuation of our interest in the study of carbohydrates by ESMS and ES tandem mass spectrometry, we now report the structwal characterization of AZT 1 and the novel series of anomeric pairs of 3'-azido-2',3',4'-trideoxy-4'-thio-5-(bromo, chloro and fluoro )uridines 2-7 (see Figure 1) using ESMS.We also report structural information deduced from the low energy tandem mass spectral analysis of the protonated molecules.

Mass Spectrometry:
Conventional mass spectra were obtained by scanning the mass spectrometer from 50 to 1000 Da in 10 s.Assisted nebulized electrospray mass spectra were obtained using an API III triple quadrupole mass spectrometer (SCIEX, Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization (API) source operated in the ion spray mode (also known as assisted nebulized electrospray).For the sake of convenience, in this rationale, no distinction was made between the tenns ion spray and electrospray which, nowadays, normally includes the assisted nebulizer notion.A Macintosh IIX computer was used for data acquisition and data processing.Samples were introduced to the mass spectrometer by infusing a solution at a flow rate of 5 J.1Umin.The voltage of the ion spray needle was maintained at 5 kV, while the orifice voltage was typically 100V.A 0,6 Umin flow of high purity air was used as the nebulizing gas.
Tandem mass spectrometry experiments were also done using the API III instrument.Fragment ion spectra of mass-selected ions were induced by collisions with argon in the second (RF only) quadrupole.The resulting fragments were mass analyzed by the third quadrupole.The target thickness was typically 3 x 10 14 atoms/cm 2 and collision energies of approximately 100 eV (laboratory frame) were used in all MSIMS experiments.Precursor ion scans were obtained by scanning the fITSt quadrupole while selecting a given m/z value with the third quadrupole.
The low energy CID MSIMS spectrum for the [M+H]+ precursor ion at mlz 268 is shown in Figure 2b and summarized in Table 2.The fragmentation route for the [M+H]+ parent-ion has been rationalized using the systematic nomenclatme for nucleoside fragmentation in F AB-MS proposed by Crow ~ al [20].Therefore, the main fragment-ion [BH2]+ at mlz 127 appears to be initiated by intramolecular transfer of a hydrogen atom from the sugar to the base, which is already protonated (Fig. 2b).The only other ion produced was the less abundant fragment-ion [S]+ at mlz 142 obtained by cleavage of the glycosidic bond with charge retention on the sugar moiety (A-type cleavage) [20].The electrospray mass spectra of the pair, 3 '-azido-2', 3 ',4'-trideoxy-4'-thio-5bromouridine 2 and its a-anomer 3 are summarized in Table 3.It can be seen that ESMS for this anomeric pair are very similar.As expected, each of the anomers of 4'-thio-5bromouridines 2 and 3 fonned the protonated molecules [M+H]+ at mlz 350 and 348 respectively in almost equal ratio and is in accordance with the isotopic abtmdance of bromine (ie.79J3r and SIBr).An interesting fmding was the presence of clusters of [2M+H]+ at mlz 699 and 695 corresponding to [2(CJIloNs03SSIBr)+H]+ and [2(CJIloNs03 S79J3r )+H]+, respectively.Equally noteworthy was the presence of an intense mixed cluster of [2M+H]+ fonned by combination of the two bromine isotopes [CJIloNs03SSIBr + CJIloNs03S79J3r + H]+ at mlz 697.The presence of these peaks permitted the determination of the relative masses of this anomeric pair.It should be mentioned that some fragmentation of the protonated molecules and clusters occurred during the conventional ESMS and the same characteristic fragmentations, due to the loss of bromine atoms, were observed.
The protonated molecules [C9HloNs03S79Brl-H]+ at nv'z 348 were selected for the recording of the low-energy collision induced (CID) mass analyzed ion kinetic energy (MIKE) spectra in order to probe further the electrospray fragmentation behaviour and also to see whether it would be possible to obtain discriminating differences, between the two related anomers 2 and 3, by the metastable analysis .The low energy CID MSIMS of the precursor [M+H]+ ions at mlz 348 are summarized in Table 4 and tentatively depicted in figure 3. Examination of Table 4 pennitted us to deduce the fate of the parent ions and their dissociations into product ions.Interestingly, the product ions obtained, which are of diagnostic importance for the elucidation of this anomeric pair, were not formed in the conventional electrospray MS.As expected, the diagnostic product ions result from the sugar moiety [S]+ product ion at mlz 158.We noted three ions originating from the product ion [S]+ at nv'z 115, 97 and 85, which were obtained by elimination of a molecule of hydrogen azide to from the [S-HN3]+ ion at mlz 115, by consecutive losses of molecules of hydrogen azide and water to fonn the [S-HN3-H20]+ ion at m/z 97 and by consecutive elimination of molecules of hydrogen azide and formaldehyde to fonn the [S-HN3-HCHO]+ ion at mlz 85.These last three product ions seem only to be present for all the members of the 4'-thio-5-halogenouridine nucleoside series and are absent in the case of AZT 1.

Characteristic ion
Type of ion  Comparison between the low energy CID MSIMS analyses for this anomeric pair 2 and 3 (Table 4) showed small differences in the intensities of almost all product ions with the exception of the [S]+ and [S-HN3-HCHO]+ product ions at m/z 158 and 85 respectively which showed pronounced differences in their relative intensities.These MSIMS characteristics allowed discrimination to be made between the two related anomers 2 and 3.
In a similar fashion, 3'-azido-2',3',4'-trideoxy-4'-thio-5-chlorouridine 4 and its respective a-anomer 5 were also studied by electrospray.The characteristic ions obtained for this pair of anomers are presented in Table 5 and Figure 4. Comparison of these data shows very distinct spectral differences between the two anomers.Indeed, the only common ions for this anomeric pair are the protonated molecules [M+H]+ at m/z 306 and 304 with different relative abundances (each anomer with the correct abundances for the isotopes of chlorine atoms).The exclusive presence of the diagnostic ammoniated adducts [CJ-IloN503S37CI+Nllt]+ and [CJ-IloN503S35CI+Nllt]+ can be seen, for the J3-anomer only, at m/z 323 and 321 respectively.While the presence of these adducts, in the case of the J3-anomer 4, can be tentatively rationalized by the fonnation of a complex involving the ammoniwn ion held by H-bonding between 4'-S-and 5'-0-atoms of the sugar moiety and 2-carbonyl oxygen atom of the pyrimidine base, such a complex could not be fonned in the case of the a-anomer 5.However, for this anomer the exclusive fonnation of [3M+H]+ cluster ions can be observed, resulting from the different pennutation of the two isotopes of chlorine atoms to yield respectively: either the cluster ion [2(CJ-IloN503S35CI) -K4IloN503S37CI+H]+ at m/z 912 containing two 35CI and one 37CI atoms or the related cluster ion [3(CJ-IloN503S35CI) + H]+ at m/z 910 containing three 35CI atoms.Similarly, the fonnation of the [2M + H]+ cluster ions at mlz 609 and 607 are fonned exclusively for the a-anomer 5.In order to further establish the structural elucidation of this pair of anomeric nucleosides 4 and 5 , the protonated molecules [CJ-IloN503S35CI+H]+ at m/z 304 were selected for recording by metastable MSIMS analysis.The low energy CAD MSIMS analysis of the precursor ions at mlz 304 are presented in Table 6 and Figure 5.Comparison between these two sets of data is in agreement with a fragmentation route typical of that which is expected for such kinds of 4'-thio-nucleosides.Thus, for both anomers, we observed the typical [S]+ and [BH2]+ product ions, respectively, at m/z 158 and 147.As in the case of the anomeric pair 2 and 3, identification can be made of the three fragment-ions at m/z 115, 97 and 85, originating from the sugar moiety product ions [S]+, respectively obtained by either the loss of a molecule of hydrogen azide or "consecutive and perhaps concerted losses" of either both molecules of hydrogen azide and water or both molecules of hydrogen azide and formaldehyde.In this context, please note that "consecutive or concerted losses" of many of such product ions, in MSIMS experiments, simply means that they are both lost at the same time and within the same reaction region inside the tandem mass spectrometer.It is difficult to deduce the order of elimination reaction involving "consecutive or concerted losses" under the condition of MSIMS.
Examination of Table 6 permits the identification ofpronolUlced and discriminating characteristic differences to be made between the relative abundances of the common product ions [S]+, [BH2]+' [S-HN3]+ and [S-HN3-H20]+ respectively at m/z 158, 147, 115 and 97.Such distinct differences makes possible the clear differentiation of the pair of anomeric nucleosides 4 and 5.The low energy CID MSIMS analysis of the chlorine-37 isotope of the ammoniated adduct [M+Nllt]+ at m/z 321 of the 3'-azido-2',3',4'-trideoxy-4'thio-5-chlorouridine J3-anomer was also recorded and is presented in Table 7.In addition, Table 7 identifies the same type of product ions observed for the collision induced decomposjtion of the protonated lllolecuies of [M+H]+ precursor ions presented in Table 6.Finally, the electrospray mass spectra of the anomeric pair of 3'-azido-2',3',4'-trideoxy-4'thio-5-fluorouridine 6 and 7 were also recorded.The characteristic ions, obtained for this pair of anomers, are presented in Table 8 which shows very distinct spectra.It can be noted that for both anomers the protonated molecules and the [2M+H]+ cluster ions, at mlz 288 and 575 respectively, have different relative abundances.As in the case of 4'-thio-5chlorouridine 4 (f}-anomer), the f}-anomer 6 also shows the presence of two diagnostic ammoniated adducts, namely the [M+NI--l.]+and [2M+NI--l.]+ions at mlz 305 and 592 respectively.The fonnation of these ions can be rationalized by complexation with an . .
ammoruum Ion.The low energy CID MS/MS spectra for the [M+H]+ ions of the anomers 6 and 7 are summarized in Table 9. Examination of this table showed pronounced differences in the intensities of particular fragment-ions, which permitted discrimination between these two anomers.Thus, relative intensities of the product ions [S]+ and [S-HN3-HCHO]+ respectively at mlz 158 and 85, proved to be of diagnostic value in permitting discrimination between the f}-anomer 6 and the corresponding a-anomer 7.
MS/MS spectra of this novel series of 4'-thio-5-halogenouridines, obtained using low energy collision activation, permitted differentiation between the various anomeric pairs.The difference in the relative abundance of same product ions obtained in the low energy CID MSIMS analyses reflects the inequalities of the chemical free energy, and

Table 2
Low energy CAD MSIMS spectrum of the precursor ion [M+H]+ at mlz 268 obtained for AZf 1.