Reagent for mass spectrometry
11442046 · 2022-09-13
Assignee
Inventors
- Thomas CARELL (Krailling, DE)
- Uwe Kobold (Penzberg, DE)
- Dieter Heindl (Penzberg, DE)
- Silvia Baecher (Penzberg, DE)
- Andreas Leinenbach (Penzberg, DE)
- Martin Rempt (Penzberg, DE)
- Toni Pfaffeneder (Munich, DE)
- Angie Kirchner (Munich, DE)
- Olesea Kosmatchev (Augsburg, DE)
- Rene Rahimoff (Munich, DE)
- Sarah Schiffers (Munich, DE)
- Markus Muller (Munich, DE)
Cpc classification
C07D249/04
CHEMISTRY; METALLURGY
G01N2400/00
PHYSICS
G01N2458/15
PHYSICS
International classification
C07D249/04
CHEMISTRY; METALLURGY
Abstract
The present invention relates to reagents suitable in the mass spectrometric determination of analyte molecules such as carbohydrates as well as adducts of such reagents and analyte molecules and applications of said reagents and adducts. Further, the present invention relates to methods for the mass spectrometric determination of analyte molecules.
Claims
1. Use of a compound of the general formula (I):
X-L.sub.1-Y(-L.sub.2-Z).sub.r wherein X is a reactive group capable of reacting with an analyte molecule, whereby a covalent bond with the analyte molecule is formed, L.sub.1 is a bond or a spacer, Y is a neutral ion loss unit, which itself is neutral and which, under conditions of mass spectrometry, is capable of fragmentation, whereby a neutral species is released, and wherein Y comprises a 4-, 5-, or 6-membered heterocyclic moiety, L.sub.2 is a bond or a spacer, Z is a charge unit comprising (i) at least one positively charged moiety having a pK.sub.a of 10 or higher, or (ii) at least one negatively charged moiety having a pK.sub.b of 10 or higher, r is 1, including any salt thereof, or of a composition or kit comprising at least one compound (I) for the mass spectrometric determination of an analyte molecule.
2. The use of claim 1, wherein the reactive group X is a carbonyl-reactive group, a dienophilic group, a carboxylate reactive group, a phenol reactive group, an amino reactive group, a hydroxyl reactive group, or a thiol reactive group.
3. The use of claim 1, wherein the reactive group X is a carbonyl-reactive group selected from the group consisting of (i) a hydrazine group, (ii) a hydrazone group, (iii) a hydroxylamino group, and (iv) a dithiol group.
4. The use of claim 1, wherein the reactive group X is a haloacetyl group.
5. The use of claim 1, wherein the reactive group X is an amino-reactive group selected from the group consisting of an active ester group, a hydroxybenzotrialzole (HOBt) ester and 1-hydroxy-7-azabenzotriazole (HOAt) ester group.
6. The use of claim 1, wherein neutral ion loss unit Y is capable of fragmentation by a reverse cycloaddition reaction, and wherein neutral ion loss unit Y comprises a cyclic azo compound or a 5-membered heterocyclic moiety having at least 2 heteroatoms adjacent to each other.
7. The use of claim 1, wherein the neutral species is an inorganic molecule selected from the group consisting of SO, SO.sub.2, CO, CO.sub.2, NO, NO.sub.2 and N.sub.2.
8. The use of claim 1, wherein the charge unit Z comprises (i) at least one positively charged moiety selected from the group consisting of a primary, secondary, tertiary or quaternary ammonium group and a phosphonium group having a pK.sub.a of 10 or higher, or (ii) at least one negatively charged moiety selected from the group consisting of a phosphate, sulphate, sulphonate and carboxylate group having a pK.sub.b of 10 or higher.
9. The use of claim 1, wherein the charge unit Z comprises or consists of one permanently positively charged moiety.
10. The use of claim 1, wherein said compound (I) further, under conditions of mass spectrometry, is capable of an alternative fragmentation, whereby a second neutral species different from the first neutral species is released.
11. The use of claim 1, wherein said compound (I) is of the general formula (Ia) or (Ib):
X-L.sub.1-Y-L.sub.2-Z (Ia),
X.sup.1-L.sub.1-Y.sup.1(-L.sub.2-Z).sub.r (Ib) wherein X, L.sub.1, L.sub.2, Y, Z and r are as defined in claim 1, X.sup.1 is a carbonyl-reactive group, and Y.sup.1 is a neutral ion loss unit comprising (i) a 4-, 5- or 6-membered heterocyclic moiety which, under conditions of mass spectrometry, is capable of fragmentation, whereby a first neutral species is released, and (ii) optionally a moiety, which under conditions of mass spectrometry, is capable of an alternative fragmentation, whereby a second neutral species different from the first neutral species is released.
12. The use of claim 1, wherein said compound (I) is of the general formula (Ic): ##STR00019## wherein R is in each case independently H or C.sub.1-4 alkyl and A is an anion.
13. The use of claim 1, wherein compound (I) is an isotopologue comprising at least one isotope selected from D, .sup.13C, .sup.15N and/or .sup.18O.
14. A method for the mass spectrometric determination of an analyte molecule comprising the steps: (a) covalently reacting the analyte molecule with a compound of general formula (I) as defined in claim 1, whereby an adduct of the analyte molecule and the reagent is formed, and (b) subjecting the adduct from step (a) to a mass spectrometric analysis, wherein the mass spectrometric analysis step (b) comprises: (i) subjecting an ion of the adduct to a first stage of mass spectrometric analysis, whereby the ion of the adduct is characterised according to its mass/charge (m/z) ratio, (ii) causing fragmentation of the adduct ion, whereby a first neutral species is released and a daughter ion of the adduct is generated, wherein the daughter ion of the adduct differs in its m/z ratio from the adduct ion, and (iii) subjecting the daughter ion of the adduct to a second stage of mass spectrometric analysis, whereby the daughter ion of the adduct is characterized according to its m/z ratio, and/or wherein (ii) may further comprise alternative fragmentation of the adduct ion, whereby a second neutral species different from the first neutral species is released and a second daughter ion of the adduct is generated, and wherein (iii) may further comprise subjecting the first and second daughter ions of the adduct to a second stage of mass spectrometric analysis, whereby the first and second daughter ions of the adduct are characterised according to their m/z ratios.
15. A reagent, which is a compound of formula (Ia)
X-L.sub.1-Y-L.sub.2-Z wherein X is a carbonyl reactive group, dienophilic group, a carboxylate reactive group, a phenol reactive group, an amino reactive group, a hydroxyl reactive group, or a thiol reactive group, and wherein X is no acrylester, L.sub.1 is a bond or a spacer, Y is a neutral ion loss unit, which itself is neutral and which, under conditions of mass spectrometry, is capable of fragmentation, whereby a neutral species is released, and wherein Y consists of a 4-, 5-, or 6-membered heterocyclic moiety, L.sub.2 is a bond or a spacer, Z is a charge unit comprising at least one permanently positively charged moiety selected from the group consisting of a primary, secondary, tertiary or quaternary ammonium group and a phosphonium group, wherein the overall molecule has a pK.sub.a of 10 or higher, including any salt thereof, or a composition or kit comprising at least one compound (Ia).
16. The reagent of claim 15, wherein Y consists of a cyclic azo compound or a 5-membered heterocyclic moiety having at least 2 heteroatoms adjacent to each other and, under conditions of mass spectrometry, is capable of fragmentation by reverse cycloaddition reaction, whereby a neutral species is released.
17. The reagent of claim 15, which is of formula (Ic): ##STR00020## wherein R is in each case independently H or C.sub.1-4 alkyl and A is an anion.
18. A composition or kit comprising a plurality of isotopically different reagents of claim 15.
19. Use of a covalent adduct formed by reaction of the compound of general formula (I) as defined in claim 1 and an analyte molecule, for the mass spectrometric determination of an analyte molecule, wherein the covalent adduct is a compound of the general formula (II):
T-X′-L.sub.1-Y(-L.sub.2-Z).sub.r wherein T is an analyte molecule, X′ is a moiety resulting from the reaction of a group X on the compound (I) with an analyte molecule and L.sub.1, Y, L.sub.2, Z and r are as defined in claim 1.
20. The use of claim 19 as a calibrator and/or as a standard.
Description
FIGURE LEGENDS
(1)
(2) A protocol for the quantitative determination of an analyte molecule comprises reacting the analyte molecules in a sample with a reagent (R) thereby forming covalent analyte adducts. Addition of a standard adduct comprising an isotopologue reagent (R*) allows quantitative determination of the analyte molecule by LC and subsequent MS.
(3)
(4) A protocol for the parallel quantitative determination of multiple analytes comprises reacting the analyte molecules in multiple samples with isotopically different reagents (R, R*, R**) in each sample thereby forming covalent analyte adducts. Addition of a standard adduct comprising a further isotopologue reagent (R***) allows a parallel quantitative determination of each analyte molecule in each sample by LC and subsequent MS.
(5)
(6) a Chemistry of base excision repair (BER) with formation of AP- and βE sites.
(7) b Overview of epigenetic modifications at dC and possible removal of fdC and cadC through BER.
(8) c Depiction of reagent 1 and of reaction products that are formed when 1 reacts with AP- and βE-sites.
(9)
(10) (i) TBTU, DIPEA, DMF, rt, 16 h, 90%;
(11) (ii) Trt-Cl, NEt.sub.3, pyridine, rt, 22 h, 74%;
(12) (iii) propargylamine, TBTU, DIPEA, DCM, rt, 15 h, 92%;
(13) (iv) 7+4a/b, CuBr.SMe.sub.2, H.sub.2O/DCM (1:1), rt, 16 h, 77%;
(14) (v) 6M HCl, DCM/H.sub.2O (1:1), rt, 1 h, quant.;
(15) (vi) H.sub.2O, 30° C., 16 h, HPLC, 15%;
(16) (vii) 1) DIBAL-H, DCM, −78° C. to rt, (2) DMP, DCM, 0° C. to rt, o/n, 47% over two steps;
(17) (viii) (1) 1a/b, CHCl.sub.3/H.sub.2O (1:1), rt, 16 h, 68%, (2) pTSA.H.sub.2O, H.sub.2O, 25° C., o/n, HPLC (2×), 14%.
(18)
(19) Fragmentation patterns of AP (9)- and βE-site (10) standards in the MS/MS experiment yielding highly sensitive signals.
(20)
(21) a General workflow for the derivatization and analysis of AP- and βE-sites.
(22) b Feeding of mESC cultures with labelled nucleosides results in the formation of ribose-labelled AP- and βE-site products 9 and 10 which are 5 mass units heavier than unlabelled products.
(23) c Quantitative data of the different labelled adducts and DNA-modifications.
(24)
(25) Calibration curve for a AP-(9) and b βE-site (10).
(26)
(27) a Obtained UV-signals of an ODN with an abasic site and reverse strand before (black lines) and after derivatization with 1a.
(28) b Normalized UV signals of an ODN+1 after specific time points.
(29)
(30) Derivatization of gDNA with 1 shows that the derivatization reaction is fast and does not artificially generate abasic sites.
(31)
(32) Scheme of the reaction of an acrylamide reagent with a thiole such as glutathione (GSH) and N.sub.2-loss in MS analysis.
(33)
(34) a Mol peak of the single-charged species is indicated with .diamond-solid.. Fragment formed after N.sub.2 loss is not detectable.
(35) b Precursor ion scan shows loss of N.sub.2.
(36) c The double-charged species shows N.sub.2 loss directly.
(37)
(38)
(39) MS experiments show the molecule peak at 632.5 and the fragments at 604.4 after N.sub.2 loss as well as the second fragment at 192.1
(40)
(41)
(42) The following peaks are shown:
(43) a testosterone (110 amol)
(44) b testosterone (22 amol)
(45) c adduct N.sub.2 loss (0.12 amol)
(46) d adduct phenyl loss qualifier (0.12 amol)
(47) e adduct N.sub.2 loss (3.6 amol)
(48) f adduct phenyl loss qualifier (3.6 amol)
(49) g testosterone (11 fmol)
(50)
(51) UHPLC-triple/quadrupole MS spectrum of the dopamine adduct. Clearly visible is the molecule peak at m/z=553 and the N.sub.2 loss signal at m/z=525.
(52) Further, the present invention shall be described in more detail by the following Examples:
EXAMPLES
1. Introduction
(53) Base excision repair (BER) is a major DNA maintenance process that allows cells to remove damaged bases from the genome (1, 2). The process requires the action of specific DNA glycosylases, which recognize the non-canonical base (
(54) Current approaches to measure BER intermediates such as the commercially available aldehyde reactive probe are chemical probes containing an hydroxylamine, which reacts with the open chain aldehydic form of the AP-site (
(55) Recently it was discovered that BER removes not only damaged bases from the genome, but also the epigenetically relevant bases 5-formyl-cytosine (fdC)(4) and 5-carboxy-cytosine (cadC) can be cleaved by thymine DNA glycosylase (Tdg)(5). Both are formed by oxidation of 5-methyl-cytosine (mdC) via 5-hydroxymethyl-cytosine (hmdC) with the help of β-ketoglutarate dependent Tet oxygenases (
(56) Here we report the development of a new reagent that in combination with highly sensitive UHPLC-triple quadrupole mass spectrometry and isotope feeding allows exact quantification of AP- and βE-sites with a limit of detection that goes down to 100 intermediates per genome. This new technology allowed us to uncover that both types of intermediates do not accumulate at pyrimidines showing that BER at epigenetic sites is not the expected harmful event in the genome of stem cells.
2. Results and Discussion
(57) The basis for the new technology is reagent 1 (
(58) The synthesis of the reagent is shown in
(59) O-(Carboxymethyl)hydroxylamine 5 was in parallel Trt(trityl)-protected to 6.sup.8 and 6 was reacted with propargyl amine using again TBTU as the coupling reagent to give the alkyne 7. Reaction of 4 with 7 via a Cu(I) catalyzed azide-alkyne click reaction furnished the triazole 8. Cleavage of the Trt-group under harsh acidic conditions provided the reagents 1 in a light (CH.sub.3, a) and a heavy version (CD.sub.3, b), with a Δm/z=9) in just 5 steps with a total yield of 47%. The reagent was purified two times by reversed phase HPLC to ensure purities of >99.9% as needed for the study.
(60) For the planned exact mass spectrometric quantification, the expected AP- and βE-site reaction products were prepared as normal (a) and heavy isotope (b) labelled compounds for the generation of calibration curves and as internal standards. We therefore reacted reagent 1a/b with ribose to obtain the expected AP-site reaction product 9a/b. In order to prepare the needed β-elimination products 10a/b, we reduced the acetonide protected methylester 11 with DIBAL-H to the allyl alcohol, which was selectively oxidized to aldehyde 12 using the Dess-Martin reagent. Reaction of 12 with reagent 1a/b and final cleavage of the acetal protecting group furnished the desired compound 10a/b, again in a light and heavy form, respectively. Compounds 9a/b and 10a/b were finally purified by reversed phase HPLC to purities >99.9%.
(61) We next developed the mass spectrometry based AP- and βE-site detection procedure using an UHPLC-ESI-triple quadrupole (QQQ) machine (
(62) We next performed a dilution experiment with the synthetic AP-site reaction product 9a, and monitored the MS-signal. We were able to detect the AP-site 9a/b in the attomolar (LOD of AP-site=110 amol) range. For the quantification of global AP and βE-levels we needed only 5 μg of genomic DNA per sample, which provides a sensitivity that is three orders of magnitude higher compared to previously published methods (9). The sensitivity gained with reagent 1 enabled also the detection of the βE-intermediates formed by bifunctional glycosylases. Here again the detectability extended into the attomolar range (LOD of βE-site=110 amol).
(63) We then started to quantify the intermediates of BER. During embryonic development, BER in combination with the removal of fdC and cadC by the monofunctional DNA glycosylase Tdg was reported to be a major process (4, 5, 10, 11). For the study, naïve cultures of mouse embryonic stem cells (mESCs) were grown under priming conditions (FBS/LIF) conditions for five days. We subsequently isolated genomic DNA using a standard protocol (cf. infra) and incubated it with reagent 1a.
(64) To show that the derivatization reaction of genomic DNA with 1a does not introduce abasic sites artificially and that the quantified levels are endogenous, we added 1a to stem cell DNA and stopped the reaction at several time points using our established protocol (cf. infra). Quantification of the obtained AP-sites in the mixture showed that the reaction was already complete after only one minute and even extended incubation time for up to 60 minutes showed no increase in the amount of abasic sites. Subsequently, the reagent was allowed to react for 40 minutes at 37° C. for further studies to ensure full derivatization of abasic sites. The DNA was afterwards digested with a mixture of nuclease S1, antarctic phosphatase and snake venom phosphodiesterase to the single nucleoside level (cf. infra). In order to ensure that the enzymes are able to fully carve out the reaction products, we prepared DNA with a single dU base, added to the DNA the dU-cleaving glycosylase Udg to introduce a defined abasic site followed by incubation with reagent 1a and quantified the amounts of generated AP-sites (cf. infra). We measured exactly the expected levels of AP-sites, showing that our reagent reacts quantitatively in mild conditions and that the reaction product is completely isolated by the enzymatic digestion protocol.
(65) For exact quantification of the BER intermediates in stem cell DNA we again added 9b and 10b as internal standards after DNA digestion and injected the obtained mixture into the UHPLC-QQQ system. Next to the expected signals from the canonical bases we saw two additional signals from the natural AP- and βE-site reaction products 9a and 10a. This shows that the reagent and the MS method is able to detect and quantify these key BER intermediates directly in genomic DNA.
(66) Exact quantification with the help of the isotope standards 9b and 10b allowed us to determine the global steady state levels of AP- and βE-sites to 8.8×10.sup.−7 and 1.7×10.sup.−6 per dN, respectively. This is a very low level, but due to the high sensitivity of the method it is well within the limits of quantification (cf. infra).
(67) In order to study, if these BER adducts are indeed the endogenously present steady state levels or if the adducts are for example formed during DNA isolation and sample preparation (which is hardly possible for the βE-adducts), we systematically increased the incubation time with the reagent up to one hour. Exact quantification, however, showed no increase of the values arguing against this possibility (cf. infra). We also repeated the study with dU-containing DNA and increased both the incubation time with Udg and the handling time afterwards. We saw no increase of the AP-levels, showing that DNA isolation and handling does not increase the levels of BER intermediates.
(68) The exact quantification data obtained with genomic DNA show consequently that both βE-sites and AP-sites are present at low steady state levels (
(69) We next wanted to decipher the repair processes at individual DNA bases. To this end we prepared three different mESC cultures and added either isotopically labelled dC*, dG* or dT* nucleosides, in which all the C-atoms of the ribose were exchanged against .sup.13C and the in-ring N-atoms against .sup.15N as shown in
(70) At dT, which is known to be a rather stable base, both the levels of the AP- and βE-sites are below 10.sup.−8 per dN, which amounts to less than 100 BER intermediates per genome. This level is slightly below the lower level of quantification (LLOQ) (
(71) We next studied the BER processes at dC and at epigenetically dC-derived bases. The dC base is known to deaminate to some extend in the genome, which gives dU:dG mismatches (16). In addition, dC is methylated to mdC by DNA methyl transferases (Dnmts), which also deaminates to give dT:dG mismatches (17). Both of these mismatches are known to be repaired by the monofunctional glycosylases Ung2, Smug1, Tdg and Mbd4, which should give detectable AP-sites (18, 19). Furthermore, the dC derivatives fdC and cadC were found to be cleaved by the monofunctional glycosylase Tdg (4, 5). In order to study all these BER events, we fed mESCs, grown under FBS/LIF conditions with 100 μM dC* for 5 days. After DNA isolation, we added reagent 1a, digested the DNA and measured again the levels of labelled AP- and βE-sites. We indeed detected intermediates in contrast to the experiments with dT, but in agreement with the idea that the dC base is less stable due to deamination. Surprisingly, however, we could measure only labelled βE-intermediates generated by bifunctional repair glycosylases, while formation of labelled AP-sites was below LLOQ (
(72) This result is in perfect agreement with data from a recent study showing that Tdg might act in a tight complex with the enzymes Neil1-2 (20). The Neil proteins are supposed to bind to the generated AP-sites to catalyse quick β- and δ-elimination reactions in order to keep the steady state levels of AP-sites low and hence bearable.
(73) Further quantification revealed a level of βE-sites at dC* of 1.3×10.sup.−7 per dN. In order to relate the number to the levels of in principle repairable fdC and cadC, we quantified these bases as well. For fdC we measured a level of 2.1×10.sup.−6 and for cadC 1.0×10.sup.−7 was detected. The steady state fdC levels are consequently 1 order of magnitude higher than the βE-site levels, showing that the high levels of fdC do not translate into significant amounts of BER-intermediates, which is in line with the reported stability of fdC.
(74) In order to gain further insight into repair at epigenetic dC sites we studied mESCs lacking either Tdg or both Neil1 and Neil2 proteins and fed both cell lines with dC*. The obtained data are depicted in
3. Conclusion
(75) In conclusion, reagent 1 in combination with a new mass spectrometry based technology (UHPLC-MS) and isotope feeding allows quantification of central BER intermediates at the different canonical bases with unprecedented precision and sensitivity of 100 BER intermediates per genome. Evidence for the BER removal of fdC and cadC in the framework of an active demethylation pathway could not be obtained. Most BER repair processes were detected at dG sites, likely because of oxidative damage at dG.
4. Methods
4.1 Chemical Synthesis Procedures
(76) Unless noted otherwise, all reactions were performed using oven dried glassware under an atmosphere of nitrogen. Molsieve-dried solvents were used from Sigma Aldrich and chemicals were bought from Sigma Aldrich, TCI, Carbolution and Carbosynth. Isotopically labelled trimethylamino glycine was obtained from Eurisotop. For extraction and chromatography purposes, technical grade solvents were distilled prior to their usage. Reaction controls were performed using TLC-Plates from Merck (Merck 60 F254), flash column chromatography purifications were performed on Merck Geduran Si 60 (40-63 μM). Visualization of the TLC plates was achieved through UV-absorption or through staining with Hanessian's stain. NMR spectra were recorded in deuterated solvents on Varian VXR400S, Varian (nova 400, Bruker AMX 600, Bruker Ascend 400 and Bruker Avance III HD. HR-ESI-MS spectra were obtained from a Thermo Finnigan LTQ FT-ICR. IR-measurements were performed on a Perkin Elmer Spectrum BX FT-IR spectrometer with a diamond-ATR (Attenuated Total Reflection) unit. HPLC purifications were performed on a Waters Breeze system (2487 dual array detector, 1525 binary HPLC pump) using a Nucleosil VP 250/10 C18 column from Macherey Nagel, HPLC-grade MeCN was purchased from VWR. For HPLC purifications of compounds 1a/b, 9a/b and 10a/b a buffer system of 0.25 mM ammonium formate in H.sub.2O (referred to as buffer A) and 0.25 mM ammonium formate in 80% MeCN/H.sub.2O (referred to as buffer B) was used.
4.2 Synthesis of Hydroxylamine 1 and Internal Standards 9a/b and 10a/b
4.2.1 (N-Tritylaminooxy)acetic Acid (6)
(77) ##STR00004##
(78) (N-Tritylaminooxy)acetic acid was synthesized according to Kojima et al. (8)
4.2.2 N-(prop-2-ene-1-yl)-2-((tritylamino)oxy)acetamide (7)
(79) ##STR00005##
(80) N-trityl protected aminooxyacetic acid 6 (2.50 g, 7.50 mmol, 1.0 eq) was suspended in DCM (40 mL) and was subsequently charged with TBTU (2.89 g, 9.00 mmol, 1.2 eq), DIPEA (1.60 mL, 9.00 mmol, 1.2 eq) and propargylamine (1.40 mL, 22.6 mmol, 3.0 eq). The suspension was stirred at rt (room temperature), whereas after 15 hours a clear yellowish solution was formed. The mixture was diluted with EtOAc (300 mL), the organic phase was washed with NH.sub.4Cl (300 mL) and NaHCO.sub.3 (300 mL) and then dried over Na.sub.2SO.sub.4. Volatiles were finally removed in vacuo and the crude mixture was purified through column chromatography (10% EtOAc-->40% EtOAc/iHex). 7 (2.57 g, 6.93 mmol, 92%) was yielded as a colourless solid.
(81) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ/ppm=7.37-7.22 (m, 15H, (C.sub.6H.sub.5).sub.3C), 6.59 (s, 1H, (C.sub.6H.sub.5).sub.3C—NH—O), 5.81 (bs, 1H, O═C—NH), 4.25 (s, 2H, O—CH.sub.2C═O), 3.85 (dd, .sup.3J=5.5 Hz, .sup.4J=2.6 Hz, 2H, HN—CH.sub.2), 2.15 (t, .sup.4J=2.6 Hz, 1H, C≡C—H). .sup.13C-NMR (75 MHz, CDCl.sub.3): δ/ppm=169.1 (C═O), 143.9 (3C, 3×O—NH—C—C), 129.0 (6C, C.sub.Ar—H), 128.2 (6C, C.sub.Ar—H), 127.4 (3C, C.sub.tert—H), 79.3 (C≡C—H), 74.6 (C(C.sub.6H.sub.5).sub.3), 73.4 (O—CH.sub.2), 71.7 (C≡C—H), 28.8 (NH—CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.24H.sub.22N.sub.2NaO.sub.2 [M+Na].sup.+: 393.1573; found: 393.1571. IR (ATR): v.sup.˜ (cm.sup.−1)=3288 (w), 3222 (w), 3056 (w), 2913 (w), 2359 (w), 2339 (w), 1635 (m), 1542 (m), 1489 (m), 1065 (m), 996 (m), 763 (m), 747 (m), 707 (s), 697 (s), 685 (s), 627 (s). Melting Range: 157-158° C.
4.2.3 2-((4-Azidophenyl)amino-N,N,N-trimethyl-2-oxoethaneaminium Chloride (4a)
(82) ##STR00006##
(83) Betaine 3a (0.30 g, 2.56 mmol, 1.0 eq) was first dried on high vacuum at 180° C. for 20 minutes. After cooling to rt, the colourless solid was suspended in DMF (25 mL). 4-Azidoaniline hydrochloride 2 (0.54 g, 3.17 mmol, 1.2 eq), TBTU (0.99 g, 3.07 mmol, 1.2 eq) and DIPEA (1.10 mL, 6.32 mmol, 2.4 eq) were added whereas a yellow brownish solution formed gradually. After stirring for one hour at rt all solids were dissolved and the reaction was further stirred at rt over night. DMF was then removed in vacuo and the crude mixture was purified by column chromatography (DCM/MeOH/H.sub.2O/7N NH.sub.3 in methanol=90:10:0.6:0.6) and 4a was yielded as the corresponding triazolate salt. The salt was then redissolved in H.sub.2O (50 mL) and was acidified to pH=1. The aqueous phase was then extracted with Et.sub.2O until TLC analysis of the organic phase fractions showed no UV absorption anymore. The aqueous layer was then neutralized with conc. NH.sub.3 and the chloride salt of 4a (0.62 g, 2.30 mmol, 90%) was yielded as a brownish powder.
(84) .sup.1H-NMR (300 MHz, DMSO d.sup.6): δ/ppm=11.22 (s, 1H, NH), 7.69 (d, .sup.3J=8.9 Hz, 2H, CH═C—NH), 7.12 (d, .sup.3J=8.4 Hz, 2H, CH═C—N.sub.3), 4.42 (s, 2H, CH.sub.2), 3.30 (s, 9H, N(CH.sub.3).sub.3). .sup.13C-NMR (101 MHz, dmso d.sup.6): δ/ppm=162.0 (C═O), 135.0 (NH—C═CH), 134.9 (N.sub.3—C═CH), 121.2 (2C, NH—C═CH), 119.5 (2C, N.sub.3—C═CH), 64.3 (CH.sub.2), 53.4 (3C, N(CH.sub.3).sub.3). HRMS (ESI.sup.+): calc. for C.sub.11H.sub.16N.sub.5O.sup.+[M.sup.+]: 234.1349; found: 234.1348. IR (ATR): v.sup.˜ (cm.sup.−1)=3348 (w), 2983 (w), 2118 (s), 2083 (m), 1692 (s), 1676 (m), 1615 (m), 1549 (m), 1508 (s), 1287 (s), 1256 (m), 1050 (s), 1038 (s), 922 (s), 833 (s). Melting Range: 144-146° C.
4.2.4 N,N,N-trimethyl-2-oxo-2-((4-(4-((2-((tritylamino)oxy)acetamido) methyl)-1H-1,2,3-triazole1-yl)phe-nyl)amino)ethanaminium Chloride/Bromide (8a)
(85) ##STR00007##
(86) First, a mixture of DCM and H.sub.2O (à 5 mL) was freeze-pump-thaw degassed (3×) and then azide 4a (0.18 g, 0.65 mmol, 1.0 eq), alkyne 7 (0.24 g, 0.65 mmol, 1.0 eq) and CuBr.SMe.sub.2 (40 mg, 0.20 mmol, 0.3 eq) were added. The suspension was stirred vigorously over night at rt whereas a colourless emulsion formed. The mixture was then concentrated under reduced pressure and purified by column chromatography using a short plug of silica (DCM/MeOH/H.sub.2O/7N NH.sub.3 in methanol=80:20:0.6:0.6). 8a was yielded as a slightly yellow brownish solid (0.32 g, 0.50 mmol, 77%).
(87) .sup.1H-NMR (300 MHz, DMSO d.sup.6): δ/ppm=11.67 (s, 1H, NH—C.sub.6H.sub.4), 8.55 (s, 1H, CH.sub.2—C═CH—N), 8.34 (s, 1H, Ph.sub.3C—NH), 8.32 (t, .sup.3J=5.8 Hz, 1H, O═C—NH—CH.sub.2), 7.91-7.84 (m, 4H, C.sub.6H.sub.4), 7.34-7.19 (m, 15H, C(C.sub.6H.sub.5).sub.3), 4.53 (s, 2H, (CH.sub.2—N(CH.sub.3).sub.3), 4.45 (d, .sub.3J=5.8, 2H, NH—CH.sub.2), 3.85 (s, 2H, N—O—CH.sub.2), 3.33 (s, 9H, N(CH.sub.3).sub.3). .sup.13C-NMR (101 MHz, dmso d.sup.6): δ/ppm=169.7 (O═C—NH—CH.sub.2), 162.4 (O═C—CH.sub.2—N), 146.0 (CH.sub.2—C═C), 144.1 (3C, O—NH—C—C), 138.1 (N—C═CH—CH), 132.6 (N—C═CH—CH), 128.9 (6C, C.sub.Ar—H), 127.6 (6C, C.sub.Ar—H), 126.7 (3C, C—H), 121.0 (CH.sub.2—C═CH—N), 120.5 (4C, N—C═CH—CH═C—N), 73.7 (C(C.sub.6H.sub.6).sub.3), 73.2 (O—CH.sub.2), 64.4 (CH.sub.2—N(CH.sub.3).sub.3), 53.4 (N(CH.sub.3).sub.3), 33.8 (NH—CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.36H.sub.38H.sub.7O.sub.3+ [M.sup.+]: 604.3031; found: 604.3026. IR (ATR): v.sup.˜ (cm.sup.−1)=3387 (w), 3054 (w), 2923 (w), 1685 (m), 1613 (m), 1558 (m), 1519 (s), 1490 (m), 1446 (m), 1413 (m), 1312 (m), 1265 (m), 1224 (m), 1192 (m), 1085 (m), 1045 (m), 1002 (m), 990 (m), 948 (m), 922 (m), 876 (m), 838 (m), 757 (s), 698 (s), 627 (s). Melting Range: 142-152° C.
4.2.5 2-((4-(4-((2-(Aminooxy)acetoamido)methyl)-1H-1,2,3-triazole-1-yl)phenyl-amino)-N,N,N-trimethyl-2-oxoethanaminium Formate (1a)
(88) ##STR00008##
(89) Trityl protected compound 8a (0.24 g, 0.38 mmol) was dissolved in DCM (6 mL) and 6M HCl (6 mL) was added. The mixture was rigorously stirred at rt for one hour until phase separation was visible. The aqueous phase was then extracted with DCM (5×5 mL) until TLC analysis of the organic phase fractions showed no UV absorption anymore. The pH was adjusted to 9-10 using 2M NH.sub.3 and the aqueous phase was removed in vacuo. 75 mg of 1a were then further purified by preparative HPLC (0%-->20% buffer B) and yielded 23 mg (0.05 mmol, 26%) of X as the colourless formiate salt.
(90) .sup.1H-NMR (400 MHz, D.sub.2O): δ/ppm=8.40 (s, 1H, CH.sub.2—C═CH—N), 8.21 (s, 1H, HCOO), 7.58 (d, .sup.3J=9.2 Hz, 2H, CH—CH═C—N.sub.3), 7.52 (d, .sup.3J=9.2 Hz, 2H, CH—CH═C—NH), 4.53 (s, 2H, N—O—CH.sub.2), 4.27 (s, 2H, NH—CH.sub.2), 4.22 (s, 2H, CH.sub.2—N(CH.sub.3).sub.3), 3.36 (s, 9H, N(CH.sub.3).sub.3). .sup.13C-NMR (101 MHz, D.sub.2O): δ/ppm=169.7 (O═C—NH—CH.sub.2), 162.4 (O═C—CH.sub.2—N), 146.0 (CH.sub.2—C═C), 144.1 (3C, O—NH—C—C), 138.1 (N—C═CH—CH), 132.6 (N—C═CH—CH), 128.9 (6C, C.sub.Ar—H), 127.6 (6C, C.sub.Ar—H), 126.7 (3C, C—H), 121.0 (CH.sub.2—C═CH—N), 120.5 (4C, N—C═CH—CH═C—N), 73.7 (C(C.sub.6H.sub.6).sub.3), 73.2 (O—CH.sub.2), 64.4 (CH.sub.2—N(CH.sub.3).sub.3), 53.4 (N(CH.sub.3).sub.3), 33.8 (NH—CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.16H.sub.24N.sub.7O.sub.3.sup.+ [M.sup.+]: 362.1935; found: 362.1935. IR (ATR): v.sup.˜ (cm.sup.−1)=3130 (m), 3037 (s), 2807 (m), 2649 (m), 2363 (w), 1684 (s), 1610 (m), 1556 (s), 1517 (s), 1487 (m), 1475 (m), 1442 (m), 1403 (s), 1312 (m), 1262 (m), 1193 (m), 1128 (w), 1083 (w), 1048 (m), 991 (m), 967 (w), 921 (s), 837 (s).
4.2.6 N,N,N-Trimethyl-2-oxo-2-((4-(4-((2-((((3S,4R)-3,4,5-trihydroxy-pentyliden)amino)oxy)acet-amido)-methyl)-1H-1,2,3-triazol-1-yl)phenyl)-amino)ethanaminium Formate (9a)
(91) ##STR00009##
(92) 1a (50.0 mg, 0.12 mmol, 1-0 eq) and 2′-desoxyribose (182 mg, 1.36 mmol, 11.8 eq) were dissolved in H.sub.2O (2.7 mL) and incubated over night at 30° C. and 1400 rpm in a Eppendorf comfort thermomixer. The mixture was filtered over a 0.2 μm syringe filter and was subsequently purified by HPLC twice (0-->15% buffer B). Pure product 9a (9.1 mg, 17 μmol, 15%) was obtained as a colourless foam. The compound was present as a mixture of E/Z isomers in aqueous solution that were not assigned.
(93) .sup.1H-NMR (600 MHz, D.sub.2O): δ/ppm=8.46 (s, 1H, HCOO), 8.34 (s, 1H, CH.sub.2—C═CH—N), 7.79 (d, J=9.0 Hz, 2H, CH—CH═C—N.sub.3), 7.74-7.71 (m, 8H, CH—CH═C—NH, C1′—H.sup.A), 7.08 (t, .sup.3J=5.4 Hz, 1H, C1′—H.sup.B), 4.67 (s, 2H, NO—CH.sub.2.sup.B), 4.63 (s, 2H, NH—CH.sub.2), 4.62 (s, 2H, N—O—CH.sub.2.sup.A), 4.35 (s, 2H, CH.sub.2—N(CH.sub.3).sub.3), 3.92-3.87 (m, 1H, C3′-H.sup.B), 3.85-3.80 (m, 1H, C3′—H.sup.A), 3.78-3.69 (m, 1H, C5′-H), 3.66-3.53 (m, 2H, C5′-H, C4′-H), 3.42 (s, 9H, N(CH.sub.3).sub.3), 2.79-2.69 (m, 2H, C2′-H.sup.B), 2.58-2.54 (m, 1H, C2′-H.sup.A), 2.41-2.35 (m, 1H, C2′-H.sup.A). .sup.13C-NMR (150 MHz, D.sub.2O): δ/ppm=172.4 (O═C—NH—CH.sub.2), 170.9 (HCOO), 162.7 (O═C—CH.sub.2—N), 153.5 (C1′.sup.A), 153.1 (C1′.sup.B), 145.1 (CH.sub.2—C═C), 136.8 (N—C═CH—CH), 133.5 (N—C═CH—CH), 122.5 (2C, CH═C—NH), 122.3 (CH.sub.2—C═CH—N), 121.9 (2C, CH═C—N.sub.3), 74.2 (C4′), 74.0 (C4′), 71.7 (NO—CH.sub.2.sup.B), 71.5 (N—O—CH.sub.2.sup.A), 69.0 (C3′.sup.A), 68.8 (C3′.sup.B), 65.1 (CH.sub.2—N(CH.sub.3).sub.3), 62.3 (C5′), 54.3 (N(CH.sub.3).sub.3), 34.1 (NH—CH.sub.2), 32.4 (C.sub.2.Math.A), 29.2 (C2′B). HRMS (ESI.sup.+): calc. for C.sub.21H.sub.32N.sub.7O.sub.6+[M]+: 478.2409; found: 478.2404.
4.2.7 (S,E)-3-(2,2-Dimethyl-1,3-dioxolan-4-yl)acrylaldehyd (12)
(94) ##STR00010##
(95) Methyl (2E)-3-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]prop-2-enoate 11 (0.20 g, 1.08 mmol, 1.0 eq) was dissolved in DCM (2.0 mL) and cooled to −78° C. DIBAL-H (diisobutylaluminium hydride) (2.20 mL, 2M in toluene, 2.1 eq) was added and the yellowish mixture was slowly warmed to rt. After 90 minutes, DCM (5.0 mL) and H.sub.2O (4.0 mL) and NaOH (2M, 2.0 mL) were added. After stirring for an additional hour at rt, the organic phase was separated from the aqueous and dried over Na.sub.2SO.sub.4. Volatiles were removed under reduced pressure and the allylic alcohol was yielded in quantitative yield and used without further purification.
(96) .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=5.88 (dt, .sup.3J=15.4 Hz, .sup.4J=5.0 Hz, 1H, 5′-H) 5.65 (dd, .sup.3J=15.6 Hz, .sup.4J=7.6 Hz, 1H, 5′-H), 4.47 (q, .sup.3J=7.3 Hz, 1H, 4′-H), 4.08 (d, .sup.3J=5.1 Hz, 2H, 1′-H), 4.30 (dd, .sup.3J=8.2 Hz, .sup.4J=6.1 Hz, 1H, 3′-H), 3.53 (t, .sup.3J=7.9 Hz, 1H, 2′-H), 2.34 (br s, 1H, CH.sub.2—OH), 1.36 (s, 3H, O—C(CH.sub.3)(CH.sub.3)—O), 1.32 (s, 3H, O—C(CH.sub.3)(CH.sub.3)—O).
(97) The allylic alcohol was dissolved in DCM (2.0 mL) and cooled to 0° C. and was charged with Dess-Martin-periodinan (0.45 g, 1.08 mmol, 1.0 eq). The milky suspension was slowly warmed to rt and stirred over night. After the addition of saturated Na.sub.2SO.sub.4 (10 mL) and a solution of Na.sub.2S.sub.2O.sub.3 (171 mg, dissolved in 10 mL H.sub.2O), the mixture was extracted with DCM (3×15 mL) and dried over Na.sub.2SO.sub.4. Organic solvents were removed in vacuo and the crude mixture was purified via column chromatography (2.5% MeOH/DCM). Aldehyde 12 (80 mg, 0.51 mmol, 47%) was isolated as a colourless oil.
(98) .sup.1H-NMR (400 MHz, CD.sub.2Cl.sub.2): δ/ppm=9.50 (d, .sup.3J=7.6 Hz, 1H, 1′-CHO), 6.70 (dd, .sup.3J=15.6 Hz, .sup.4J=5.3 Hz, 1H, 3′-H), 6.23 (dt, .sup.3J=15.6 Hz, .sup.4J=5.8 Hz, 1H, 2′-H), 4.73 (q, .sup.3J=6.8 Hz, 1H, 4′-H), 4.18 (dd, .sup.3J=8.4 Hz, .sup.4J=6.8 Hz, 1H, 5′-H), 3.67 (dd, .sup.3J=8.4 Hz, .sup.4J=6.8 Hz, 1H, 5′-H), 1.39 (s, 3H, O—C(CH.sub.3)(CH.sub.3)—O), 1.35 O—C(CH.sub.3)(CH.sub.3)—O). .sup.13C-NMR (101 MHz, CD.sub.2Cl.sub.2): δ/ppm=193.0 (—CHO), 153.4 (3′-C), 132.1 (2′-C), 110.3 (C), 74.9 (4′-C), quart, 68.7 (5′-C), 26.2 (O—C(CH.sub.3)(CH.sub.3)—O), 25.4 (O—C(CH.sub.3)(CH.sub.3)—O). HRMS (EI): calc. for C.sub.8H.sub.11O.sub.3. [M-H]′: 155.0708; found: 155.0707.
4.2.8 2-((4-(4-((2-((((1E,2E)-3-((S)-2,2-Dimethyl-1,3-dioxolane-4-yl)-allylidene)-amino)oxy)acetamido)me-thyl)-1H-1,2,3-triazole-1-yl)phenyl)-amino)-N,N,N-trimethyl-2-oxoethane-1-aminium Formate (10a)
(99) ##STR00011##
(100) Aldehyde 12 (20 mg, 0.13 mmol, 9.0 eq) was dissolved together with hydroxylamine 1a in a 1:1 mixture of H.sub.2O and CHCl.sub.3 (à 2.5 mL) and was stirred at rt. The course of the reaction was monitored by HPLC (0-->30% buffer B) whereas it was determined that after one hour the reaction was done. The aqueous phase was then washed with DCM (3×10 mL) and concentrated in vacuo. 13a (5.10 mg, 9.50 μmol, 68%) was yielded as a brownish viscous oil that was used without further purification.
(101) .sup.1H-NMR (400 MHz, D.sub.2O): δ/ppm=8.31 (s, 1H, HCOO), 8.12 (s, 1H, CH.sub.2—C═CH—N), 7.84 (d, 1H, 1′-H), 7.59-7.53 (m, 4H, CH—CH═C—N.sub.3, CH—CH═C—NH), 6.20-6.03 (m, 2H, 2′+3′-H's), 4.54-4.52 (m, 1H, 4′-H), 4.50 (s, 2H, NO—CH.sub.2), 4.47 (s, 2H, NH—CH.sub.2), 4.20 (s, 2H, CH.sub.2—N(CH.sub.3).sub.3), 4.02-3.98 (m, 1H, 5′-H), 3.45-3.50 (m, 1H, 5′-H), 3.27 (s, 9H, CH.sub.2—N(CH.sub.3).sub.3), 1.26 (s, 3H, O—C(CH.sub.3)(CH.sub.3)—O), 1.24 (s, 3H, O—C(CH.sub.3)(CH.sub.3)—O). HRMS (ESI.sup.+): calc. for C.sub.24H.sub.34N.sub.7O.sub.5+[M.sup.+]: 500.2616; found: 500.2617.
(102) Deprotection of acetonide 13a (4.00 mg, 7.50 μmol, 1.0 eq) was dissolved in MeOH and PTSA.H.sub.2O (1.40 mg, 7.50 μmol, 1.0 eq) was added. The mixture was incubated in a Eppendorf comfort thermomixer (1300 rpm, 25° C.) over night and the solvent was removed in vacuo by lyophylization. The crude product was finally purified by preparative HPLC (0-->35% buffer B in 45 minutes) and pure 10a was yielded as a colourless foam.
(103) .sup.1H-NMR (400 MHz, D.sub.2O): δ/ppm=8.53 (s, 1H, HCOO), 8.30 (s, 1H, CH.sub.2—C═CH—N), 8.01 (s, d, .sup.3J=8.9 Hz, 1H, 1′-H), 7.78-7.70 (m, 4H, CH—CH═C—N.sub.3, CH—CH═C—NH), 6.31-6.32 (m, 2H, 2′+3′-H's), 4.63 (s, 2H, N—O—CH.sub.2), 4.62 (s, 2H, NH—CH.sub.2), 4.35-4.32 (m, 3H, CH.sub.2—N(CH.sub.3).sub.3+4′-H), 3.61 (dd, .sup.1J=11.7 Hz, .sup.3J=4.4, 1H, 5′-H), 3.51 (dd, .sup.1J=11.7 Hz, .sup.3J=6.5, 1H, 5′-H), 3.22 (s, 9H, CH.sub.2—N(CH.sub.3).sub.3). .sup.13C-NMR (101 MHz, D.sub.2O): 15/ppm=172.3 (O═C—NH—CH.sub.2), 170.9 (HCOO), 162.7 (O═C—CH.sub.2—N), 153.7 (1′-C), 144.5 (CH.sub.2—C═C), 142.8 (3′-C), 136.9 (N—C═CH—CH), 133.6 (N—C═CH—CH), 123.0 (2′-C), 122.5 (2C, CH═C—NH), 122.3 (CH.sub.2—C═CH—N), 122.0 (2C, CH═C—N.sub.3), 72.0 (N—O—CH.sub.2) 71.6 (4′-C), 64.4 (5′-C), 65.1 (CH.sub.2—N(CH.sub.3).sub.3), 54.3 (N(CH.sub.3).sub.3), 34.1 (NH—CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.21H.sub.30N.sub.7O.sub.5.sup.+ [M].sup.+: 460.2303; found: 460.2305.
4.2.9 2-((4-Azidophenyl)amino-N,N,N-tri(methyl-d3)-2-oxoethaneaminium Chloride (4b)
(104) ##STR00012##
(105) 4b was synthesized analogous to 4a whereas [d.sub.11]-betaine (98% deuterium, Euriso-Top GmbH) was used to introduce isotopic labels. Deuterium labels from the methylene group were not stable under the reaction conditions and a complete D/H exchange was observed. Thus, a [d.sub.9]-labelled product was obtained.
(106) .sup.1H-NMR (600 MHz, D.sub.2O): δ/ppm=7.39 (d, .sup.3J=8.6, 2H, CH—CH═C—NH), 7.04 (d, .sup.3J=8.5, 2H, CH—CH═C—N.sub.3), 4.18 (s, 2H, CH.sub.2). .sup.13C-NMR (150 MHz, D.sub.2O, ppm): δ/ppm=162.7 (C═O), 137.5 (NH—C═CH), 132.5 (N.sub.3—C═CH), 123.5 (2C, NH—C═CH), 119.6 (2C, N.sub.3—C═CH), 65.0 (CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.11H.sub.7D.sub.9N.sub.5O.sup.+ [M].sup.+: 243.1914; found: 243.1916.
4.2.10 2-((4-(4-((2-(Aminooxy)acetoamido)methyl)-1H-1,2,3-triazole-1-yl)phenyl-amino)-N,N,N-tri(methyl-d3)-2-oxoethanaminium Formate (1b)
(107) ##STR00013##
(108) Isotopologue 1b was synthesized according to 1a with the slight modification that the trityl protected intermediate 8b was not isolated and deprotected without further purification.
4.2.11 N,N,N-Tri(methyl-d3)-2-oxo-2-((4-(4-((2-((((3S,4R)-3,4,5-trihydroxy-pentyliden)amino)oxy)acet-amido)-methyl)-1H-1,2,3-triazol-1-yl)phenyl)-amino)ethanaminium Formate (9b)
(109) ##STR00014##
(110) Internal standard 9b was synthesized analogous to 9a, whereas a mixture of (E)/(Z)-isomers was obtained (depicted as A and B).
(111) .sup.1H-NMR (600 MHz, D.sub.2O): δ/ppm=8.46 (s, 2H, HCOO), 8.35 (s, 1H, CH.sub.2—C═CH—N), 7.81 (d, .sup.3J=7.5 Hz, 2H, CH—CH═C—N.sub.3), 7.75-7.73 (m, 8H, CH—CH═C—NH, C1′-H.sup.A), 7.08 (t, .sup.3J=5.4 Hz, 0.1H, C1′-H.sup.B), 4.67 (s, 2H, N—O—CH.sub.2.sup.B), 4.64 (s, 2H, NH—CH.sub.2), 4.62 (s, 2H, N—O—CH.sub.2.sup.A), 4.34 (s, 2H, CH.sub.2—N(CH.sub.3).sub.3), 3.93-3.87 (m, 1H, C3′-H.sup.B), 3.86-3.79 (m, 1H, C3′-H.sup.A), 3.78-3.69 (m, 1H, 1×C5′-H.sub.2), 3.67-3.53 (m, 2H, 1×C5′-H.sub.2, C4′-H), 2.80-2.68 (m, 2H, C2′-H.sub.2.sup.B), 2.59-2.54 (m, 1H, C2′-H.sup.A), 2.43-2.34 (m, 1H, C2′-H.sup.A). .sup.13C-NMR (150 MHz, D.sub.2O, ppm): δ/ppm=172.4 (O═C—NH—CH.sub.2), 170.9 (HCOO), 162.8 (O═C—CH.sub.2—N), 153.5 (C1′.sup.A), 153.1 (C1′.sup.B), 145.1 (CH.sub.2—C═C), 136.8 (N—C═CH—CH), 133.6 (N—C═CH—CH), 122.6 (2C, CH═C—NH), 122.4 (CH.sub.2—C═CH—N), 122.0 (2C, CH═C—N.sub.3), 74.2 (C4′), 74.0 (C4′), 71.7 (N—O—CH.sub.2.sup.B), 71.5 (N—O—CH.sub.2.sup.A), 69.0 (C3′.sup.A), 68.8 (CP), 64.9 (CH.sub.2—N(CD.sub.3).sub.3), 62.3 (05′), 53.3 (N(CD.sub.3).sub.3), 34.1 (NH—CH.sub.2), 32.4 (C2′A), 29.2 (C2′B). HRMS (ESI.sup.+): calc. for C.sub.21H.sub.23D.sub.9N.sub.7O.sub.6.sup.+ [M].sup.+: 487.2973; found: 487.2967.
4.2.12 2-((4-(4-((2-((((1E,2E)-3-((S)-2,2-Dimethyl-1,3-dioxolane-4-yl)-allylidene)-amino)oxy)acetamido)me-thyl)-1H-1,2,3-triazole-1-yl)phenyl)-amino)-N,N,N-tri(methyl-d3)-2-oxoethane-1-aminium Formate (10b)
(112) ##STR00015##
(113) 10b was synthesized according to 10a.
(114) .sup.1H-NMR (800 MHz, D.sub.2O): δ/ppm=8.47 (s, 1H, HCOO), 8.32 (s, 2H, CH.sub.2—C═CH—N), 8.02 (d, .sup.3J=9.4 Hz, 1H, 1′-H), 7.80-7.71 (m, 4H, CH—CH═C—N.sub.3, CH—CH═C—NH), 6.34-6.26 (m, 2H, 2′+3′-H's), 4.65 (s, 2H, N—O—CH.sub.2), 4.63 (s, 2H, NH—CH.sub.2), 4.35-4.32 (m, 3H, CH.sub.2—N(CH.sub.3).sub.3+4′-H), 3.63 (dd, .sup.1J=11.7 Hz, .sup.3J=4.4, 1H, 5′-H), 3.53 (dd, .sup.1J=11.7 Hz, .sup.3J=6.5, 1H, 5′-H). .sup.13C-NMR (150 MHz, D.sub.2O): δ/ppm=172.3 (O═C—NH—CH.sub.2), 170.9 (HCOO), 162.8 (O═C—CH.sub.2—N), 153.7 (1′-C), 145.2 (CH.sub.2—C═C), 142.8 (3′-C), 136.9 (N—C═CH—CH), 133.6 (N—C═CH—CH), 123.0 (2′-C), 122.5 (2C, CH═C—NH), 122.3 (CH.sub.2—C═CH—N), 122.0 (2C, CH═C—N.sub.3), 72.1 (N—O—CH.sub.2) 71.6 (4′-C), 64.4 (5′-C), 65.1 (CH.sub.2—N(CH.sub.3).sub.3), 53.3 (N(CD.sub.3).sub.3), 34.1 (NH—CH.sub.2). HRMS (ESI.sup.+): calc. for C.sub.21H.sub.21D.sub.9N.sub.7O.sub.5.sup.+ [M].sup.+: 469.2868; found: 469.2874.
4.3 Cell Culture
(115) DMEM high glucose containing 10% FBS (PAN Biotech), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1×MEM Non-essential Amino Acid Solution and 0.1 mM β-mercaptoethanol (Sigma Aldrich) was used as basal medium for mESC (mouse embryonic stem cell) cultures. The mESC lines were maintained in naïve state on gelatin coated plates by supplementing basal medium with 1000 U/mL LIF (ORF Genetics), 3.0 μM GSK3 inhibitor CHIR99021 and 1.0 μM Mek inhibitor PD0325901 (2i; Selleckchem). Metabolic labelling experiments with isotope-labelled nucleosides were performed by plating mESCs in priming conditions, consisting of basal mESC medium supplemented with 1000 U/mL LIF. Labelled nucleosides (B.A.C.H. UG) were added to the culture medium at the following concentrations: dG [.sup.15N.sub.5;.sup.13C.sub.10], 100 μM for three days, followed by treatment with 200 μM labelled dG for two days; dC [.sup.15N.sub.3;.sup.13C.sub.9] and dT [.sup.15N.sub.2;.sup.13C.sub.10] were both used at a concentration of 100 μM for five days in total. Dnmt TKO J1 mESCs were described in Tsumura et al. (21). and J1 wild type mESCs were obtained from the 12954/SvJae strain (22). For Tdg+/− and the Tdg−/− cell lines reported in Cortazar et al. (11) were used.
4.4 Cell Lysis and DNA Isolation
(116) Isolation of genomic DNA was achieved using the QIAamp DNA Mini Kit from Qiagen. All mESC samples were washed with PBS (Sigma) and directly lysed in the plates by adding G2 buffer containing 400 μM of 2,6-di-tert-butyl-4-methylphenol (BHT) and desferoxamine mesylate (DM). DNA was sheared by bead milling in a microfuge tube using one 5 mm diameter stainless steel bead per tube and MM400 bead mill (Retsch) at 30 Hz for for one minute and subsequently centrifuged at 15000 rpm for ten minutes. Depending on the amount of genomic DNA to isolate, the cell lysate was treated with proteinase K (25 μL for genomic tips 20G or 100 μL for genomic tips 100G) and RNase A (2.0 μL/20G, 10 μL/100G) at 50° C. for one hour. After 30 minutes, additional RNase A (2.0 μL or 10 μL, respectively) was added to the mixture. Genomic tip columns were then equilibrated with loading buffer QBT (1.0 mL/20G or 4.0 mL/100G) and the lysate, which was vortexed for one minute, was applied on the columns. After the entire liquid had entered the column, washing steps were carried out with buffer QC (2.0 mL/20G or 2×7.5 mL/100G) and the genomic DNA was finally eluted with QF buffer (2.0 mL/20G or 5.0 mL/100G) supplemented with 400 μM BHT (butylated hydroxyl-toluene). Precipitation was then achieved through addition of i-PrOH (1.4 mL/20G or 3.5 mL/100G, 70% Vol) and the resulting genomic DNA pellet was centrifuged (15 minutes, 6000 g, 4° C.). The supernatant was discarded washing steps were carried out using 70% EtOH (5.0 mL, 15 minutes, 6000 g, 4° C.). Finally, the pure DNA pellet was resuspended in 1.0 mL 70% EtOH and centrifuged (10 minutes, 15000 rpm, 4° C.). Next, the supernatant was removed and the pellet was re-dissolved in ddH.sub.2O (50-100 μL) with 20 μM BHT. The concentration was determined with a NanoDrop (ND 1000, Peqlab).
4.5 Derivatization of Genomic DNA with 1a
(117) Derivatization of abasic sites (5.0 μg for unlabelled gDNA, 20 μg for labelled gDNA) with 1 was carried out in a total volume of 20 μL, whereas the solution was buffered with HEPES (20 mM, pH=7.5) and Na.sub.2EDTA (0.1 mM). A stock of 1 in H.sub.2O (23.8 mM) was added to the buffered solution (final concentration of 1=1.5 mM) and the reaction was started by vortexing the mixture for 5 seconds. The gDNA was incubated for 40 minutes at 37° C./1400 rpm in an Eppendorf comfort thermomixer. The reaction was stopped through addition of 1-naphthylaldehyde (66.7 μL, 2M in i-PrOH) to quench excess of 1 and incubated again for 10 minutes at 37° C./1400 rpm. Derivatized DNA was then precipitated through addition of NaOAc (3.3 μL, 3M), vortexing and incubation at 37° C./1400 rpm for another 5 minutes. Absolute i-PrOH (66.7 μL) was added, the tubes were inverted several times and then centrifuged (60 minutes, 10° C., 15000 rpm). The supernatant was removed and washing steps were carried out (1×75% i-PrOH, 10° C., 15000 rpm, 30 minutes; 2×75% cold EtOH, 4° C., 15000 rpm, 30 minutes), whereas after each washing step the supernatant was carefully removed. The resulting DNA pellet was finally re-dissolved in 35 μL of ddH.sub.2O and then enzymatically digested to the nucleoside level.
4.6 Enzymatic Digestion of Derivatized Genomic DNA
(118) For enzymatic digestion of genomic DNA (5.0 μg for unlabeled gDNA or 20 μg for labelled gDNA in 35 μL H.sub.2O) we used an aqueous solution of 480 μM ZnSO.sub.4 and incubated the mixture at 37° C. for 3 h. The solution consisted of 5 U Antarctic phosphatase (New England BioLabs), 42 U nuclease S1 (Aspergillius oryzae, Sigma-Aldrich) and specific amounts of labeled internal standards for accurate quantification of DNA-modifications and derivatised abasic sites. In the second digestion round we added 0.2 U snake venom phosphodiesterase I (Crotalus adamanteus, USB corporation) in 7.5 μl of a 520 μM [Na].sub.2-EDTA and incubated the mixture further 3 h or overnight at 37° C. After digestion, the sample was stored at −20° C. and filtered by using an AcroPrep Advance 96 filter plate 0.2 μm (0.20 μm Supor, Pall Life Sciences) before LC-MS/MS analysis (39 μg injection volume at 4° C.).
4.7 LC-ESI-MS/MS Analysis of DNA Samples
(119) For the LC-MS/MS studies we used triple quadrupole mass spectrometer Agilent 6490 and Agilent 1290 UHPLC system with an UV detector. Based on earlier published work (23-27), we developed a new method and coupled it with isotope dilution technique, which allowed us exact quantification of derivatized abasic sites, all canonical nucleoside and cytosine modifications in one single analytical run.
(120) The chromatographical separation was performed over a Poroshell 120 SB-C8 column (Agilent, 2.7 μm, 2.1 mm×150 mm). Eluting buffers were water and MeCN, each containing 0.0085% (v/v) formic acid, at a flow rate of 0.35 ml/min at 30° C. The gradient was: 0.fwdarw.5 min; 0.fwdarw.3.5% (□/□) MeCN, 5.fwdarw.6.9 min; 3.5.fwdarw.5% MeCN, 6.9.fwdarw.13.2 min; 5.fwdarw.80% MeCN, 13.2.fwdarw.14.8 min; 80% MeCN; 14.8.fwdarw.15.3 min; 80.fwdarw.0% MeCN, 15.3.fwdarw.17 min; 0% MeCN. The eluent up to 1.5 min and after 12.2 min was diverted to waste by a Valco valve.
(121) By the direct injection of synthesized internal standards we optimized the source-dependent parameters, which were as follow: gas temperature 50° C., gas flow 15 l/min (N.sub.2), nebulizer 30 psi, sheath gas heater 275° C., sheath gas flow 11 l/min (N.sub.2), capillary voltage 2500 V (positive mode) and −2250 V (negative ion mode), nozzle voltage 500 V, the fragmentor voltage 380 V, Δ EMV 500 (positive mode) and 800 (negative mode). Compound-dependent parameters which gave highest intensities during method development are summarized in Table 1.
(122) TABLE-US-00001 TABLE 1 Compound-dependent LC-MS/MS-parameters used for the analysis of genomic DNA. CE: collision energy, CAV: collision cell accelerator voltage, EMV: electron multiplier voltage. The nucleosides were analyzed in the positive ([M + H)].sup.+ species) as well as the negative ([M − H].sup.− species) ion selected reaction monitoring mode (SRM). Dwell Precursor MS1 Product MS2 time CE CAV compound ion (m/z) Resolution ion (m/z) Resolution [ms] (V) (V) Polarity Time segment 1.5-4.0 min [.sup.15N.sub.2]5cadC 274.08 wide 158.03 wide 170 5 5 Positive 5cadC 272.09 wide 156.04 wide 170 5 5 Positive [.sup.15N.sub.2, D.sub.2]5hmdC 262.12 enhanced 146.07 enhanced 40 27 1 Positive 5hmdC 258.11 enhanced 142.06 enhanced 40 27 1 Positive [D.sub.3]5mdC 245.13 enhanced 129.09 enhanced 30 60 1 Positive 5mdC 242.11 enhanced 126.07 enhanced 30 60 1 Positive dC 228.12 enhanced 112.05 enhanced 25 5 5 Positive [.sup.13C.sub.9, .sup.15N.sub.3]dC 240.12 enhanced 119.06 enhanced 25 5 5 Positive Time segment 4.0-5.5 min [D.sub.2]5hmdU 259.09 wide 216.08 wide 48 7 5 Negative 5hmdU 257.08 wide 214.07 wide 48 7 5 Negative [.sup.15N.sub.2]5fdU 257.06 wide 213.05 wide 48 6 5 Negative 5fdU 255.06 wide 212.06 wide 48 6 5 Negative Time segment 5.5-8.1 min [.sup.15N.sub.5]8oxodG 289.08 wide 173.04 wide 90 9 7 Positive 8oxodG 284.1 wide 168.05 wide 90 9 7 Positive dG 268.1 wide 152.06 wide 75 45 3 Positive [.sup.13C.sub.10, .sup.15N.sub.5] dG 283.12 wide 162.06 wide 75 45 3 Positive [.sup.15N.sub.2]5fdC 258.09 wide 142.04 wide 50 5 5 Positive 5fdC 256.09 wide 140.05 wide 50 5 5 Positive Time segment 8.1-12.2 min 1-Naphthyl-Oxime 500.24 wide 472.23 wide 5 19 5 Positive 9b_1 487.3 wide 459.29 wide 38 19 5 Positive 9b_2 487.3 wide 201.18 wide 38 40 5 Positive [.sup.13C.sub.5]9a_1 483.26 wide 455.25 wide 38 19 5 Positive [.sup.13C.sub.5]9a_2 483.26 wide 192.13 wide 38 40 5 Positive 9a_1 478.24 wide 450.23 wide 38 19 5 Positive 9a_2 478.24 wide 192.13 wide 38 40 5 Positive 10b_1 469.29 wide 441.28 wide 38 19 3 Positive 10b_2 469.29 wide 201.18 wide 38 33 3 Positive [.sup.13C.sub.5]10a_1 465.23 wide 437.22 wide 38 20 3 Positive [.sup.13C.sub.5]10a_2 465.23 wide 192.13 wide 38 34 3 Positive 10a_1 460.23 wide 432.22 wide 38 20 3 Positive 10a_2 460.23 wide 192.13 wide 38 34 3 Positive 1b 371.25 wide 343.24 wide 5 19 5 Positive 1a 362.19 wide 334.19 wide 5 19 5 Positive dT 243.1 enhanced 127.05 enhanced 35 40 3 Positive [.sup.13C.sub.10, .sup.15N.sub.2]dT 255.12 wide 130.07 wide 50 8 5 Positive
4.8 Method Validation and Data Processing
(123) Method validation and data processing were performed as described in earlier published work (24). In order to obtain calibration curves each standard (5-8 standard concentrations) was analyzed as technical triplicate and linear regression was applied using Origin® 6.0 (Microcal™). Therefore, the ratio of the area under the curve (A/A*) of the unlabelled derivatized abasic site 9a and 10a to the internal standard (*) was plotted against the ratio of the amount of substance (n/n*) of the unlabelled derivatized abasic site 9a and 10a, respectively, to the internal standard (*) (see
(124) The lower limit of detection (LOD) was defined as thrice the response of the MS-signal of the respective compound obtained at a blank. The lower limit of quantification (LLOQ) was defined as the lowest concentration fulfilling the requirements of accuracy and precision and achieving a response higher than the LOD. A compilation of LLOQs and LOD is shown in Table 2.
(125) TABLE-US-00002 TABLE 2 Compilation of absolute lower limits of quantification [fmol] (LLOQ and relative LLOQs [per dN] depending on the amount of DNA digested. The relative LLOQs were calculated by generating ratios of the absolute LLOQ [fmol] to the total amount of nucleosides (N; [fmol]) in the respective amount of DNA [μg]. The total amount of nucleosides were obtained by using the average molar mass of 308.91 g mo1.sup.−1 for the monomeric DNA entity by taking the G-content (21% G) in mESC into account. Relative Relative Absolute Absolute LLOQ LLOQ DNA LOD LLOQ [per dN] [per dN] amount [fmol] [fmol] 5 μg 20 μg 9a 0.11 1.02 6.3E−08 1.57E−8 10a 0.11 1.01 6.3E−08 1.56E−8
4.9 Preparation of a Synthetic 13-Mer Oligonucleotide with Defined Abasic Site
(126) Oligonucletides (5′-GTA ATG UGC TAG G-3′ and 3′-CAT TAC ACG ATC C-5′, à 15 nmol, Metabion) were incubated in UDG-buffer (150 μL, 20 mM Tris-HCl, pH=8.0, 1 mM DTT, 1.0 mM EDTA, New England Biolabs) at 95° C. for 5 minutes and then slowly cooled to rt. UDG (5.0 μL, 25 units, New England Biolabs) was added, carefully mixed and the mixture was incubated for 2 hours at 37° C. The oligonucleotide was then isolated through chloroform/phenol extraction as described in the following paragraph. A CHCl.sub.3/phenol solution (200 μL, Roti Phenol) was added, vortexed for 30 seconds and centrifuged for 3 minutes at rt and 13400 rpm. The aqueous phase was removed carefully and CHCl.sub.3/phenol treatment was repeated twice. After addition of NaOAc (20 μL, 3M), the oligonucleotide was precipitated with i-PrOH (600 μL). The resulting DNA pellet was centrifuged at rt for 30 (15000 rpm), washed with cold EtOH (300 μL) and centrifuged at 4° C. and 15000 rpm for another 30 minutes. The washing step was repeated once more, the supernatant removed and the pellet was dried on air for five minutes before the oligonucleotide was re-dissolved in ddH.sub.2O (150 μL). The identity was finally confirmed by MALDI-TOF analysis.
4.10 Reaction Kinetics on Synthetic Oligo with Defined Abasic Site
(127) In a total reaction volume of 20 μL, of the oligonucleotide (300 pmol) was buffered with HEPES buffer (20 mM, pH=7.5) and Na.sub.2EDTA (0.1 mM) and 1 (1.26 μL of 23.8 mM stock) was added. The reaction (37° C., 800 rpm, Eppendorf comfort thermomixer) was started after vortexing the mixture for 5 seconds and after specific time points (t=15 s, 30 s, 45 s, 90 s, 120 s, 150 s, 180 s, 4 min, 6 min, 8 min, 15 min, 20 min) stopped through addition of acetone (200 μL) and freezing the aliquots in liquid nitrogen. Excess of acetone was removed on a speed vac (RVC-2-33 IR, Christ) and was filtered on a AcroPrep Advance 96 filter plate (0.20 μm Supor, Pall Life Sciences). 75 pmol of DNA were subsequently injected into a Dionex micro HPLC system and reaction products were separated using a Zorbax SB-C.sub.18 column (0.55×250 mm, 5.0 μm pore size) with a flow rate of 350 μL/min. The analysis was run at a column temperature of 60° C. and a gradient of 0%->20% buffer B in 45 min (whereas buffer A=10 mM TEAB, pH=7.5 in H.sub.2O and buffer B=10 mM TEAB, pH=7.5 in 80% MeCN/H.sub.2O). Integration of the obtained UV signals (
4.11 Efficiency of Enzymatic Digestion
(128) In order to verify if the bulky derivatized abasic site can be excised by the enzyme cocktail described above, we quantified the amount of abasic sites that were formed with the oligo mentioned in the section above. An aliquot of the oligo that was reacted with reagent 1a for 40 minutes was diluted 1/4000, a certain amount of labelled internal standard was added and the mixture digested to the nucleoside level. We determined a total amount of 136 pmol of abasic sites. The amount of dG of the same oligo was quantified by its UV trace and was accounted to 762 pmol. Since there are six dG bases in the double stranded construct, one would expect an amount of abasic sites that would constitute to ⅙ of the amount of dG (127 pmol) showing that the digest was complete and the hydrolytic enzymes were not hindered by the abasic site adduct.
4.12 Reaction Kinetics on Abasic Sites in Genomic DNA
(129) Reactions were carried out by derivatizing 5 μg of gDNA with 1 using the same conditions as mentioned above (Derivatization of genomic DNA with 1). The reaction was stopped through the addition of 1-naphthylaldehyde (66.7 μL, 2
5. Synthesis of a Thiol-Reactive Probe and Use in MS Analysis of Glutathione (GSH)
(130) As an example for a probe capable of reacting with a thiol-functional group on an analyte molecule, an acrylamide reagent was synthesized and tested in the MS analysis of glutathione (GSH). The reaction scheme is shown in
5.1 Synthesis of the Acrylamide Probe
(131) ##STR00016##
(132) First, hydroxylamine probe (20 mg, 0.044 mmol, 1.0 eq) was dissolved in a mixture of EtOAc/H.sub.2O (2:1, 3.0 mL), Na.sub.2CO.sub.3 (19 mg, 0.176 mmol, 4.0 eq) was added and cooled to 0° C. Acyloyl chloride (120 μL, 1.47 mmol, 33 eq) was added under vigorous stirring and the reaction mixture was kept at 0° C. for 30 minutes. The volatiles were removed through lyophylization and the crude mixture was purified via semi preparative HPLC (0%-->40% buffer B in 40 minutes, buffer A: 25 mM NH.sub.4HCOO, pH=4.3 in H.sub.2O, buffer B: 20% buffer A in MeCN) using a Nucleodur C18ec column from Machery & Nagel. Fractions containing the desired compound were finally lyophylized to yield the product (6.0 mg, 0.013 mmol, 30%) of a brownish oil.
(133) .sup.1H-NMR (400 MHz, D.sub.2O), δ (ppm): 8.42 (s, 1H), 8.33 (s, 1H), 7.76 (d, J=9.0 Hz, 2H), 7.69 (d, 9.0 Hz), 6.16 (d, J=16.6 Hz, 1H), 6.10 (d, J=10.4 Hz, 1H), 5.76 (d, J=10.7 Hz), 4.61 (s, 2H), 4.50 (s, 2H), 4.31 (s, 2H), 3.37 (s, 9H).
(134) .sup.13C-NMR (121 MHz, D.sub.2O), δ (ppm): 170.8, 170.4, 162.8, 144.8, 136.8, 128.9, 125.8, 122.5, 121.9, 74.5, 65.1, 54.3, 34.0.
5.2 Synthesis of the GSH Adduct
(135) ##STR00017##
(136) The acryl amide probe (3.0 mg, 0.007 mmol, 1.0 eq) was dissolved in NaHCO.sub.3/Na.sub.2CO.sub.3 buffer (2.0 mL, 60 mM, pH=10). Glutathion (2.0 mg, 0.007 mmol, 1.0 eq) was added and the mixture was incubated at 50° C. for three hours. Volatiles were removed via lyophylization and the crude residue was purified through semipreparative HPLC (0%-->30% buffer B in 40 minutes, buffer A: 25 mM NH.sub.4HCOO, pH=4.3 in H.sub.2O, buffer B: 20% buffer A in MeCN) using a Nucleodur C18ec column from Machery & Nagel. GSH-adduct was yielded as a colourless solid (4 mg, 0.005 mmol, 74%).
(137) .sup.1H-NMR (400 MHz, D.sub.2O), δ (ppm): 8.42 (s, 1H), 8.34 (s, 1H), 7.76 (d, J=9.0 Hz, 2H), 7.67 (d, 9.0 Hz), 4.60 (s, 2H), 4.47 (s, 2H), 4.39 (dd, J=8.9 Hz, 4.9 Hz, 1H), 4.31 (s, 2H), 3.72 (t, J=6.3 Hz, 1H), 3.67-3.66 (m, 2H), 3.37 (s, 9H), 2.84 (dd, J=14.1 Hz, 5.0 Hz, 1H), 2.67-2.64 (m, 3H), 2.44 (dd, J=8.6 Hz, 6.7 Hz, 2H), 2.38 (t, J=6.7 Hz, 2H), 2.12-2.05 (m, 2H).
(138) .sup.13C-NMR (121 MHz, D.sub.2O), δ (ppm): 176.0, 174.7, 173.8, 171.6, 171.2, 170.9, 179.4, 162.7, 144.8, 136.9, 133.4, 122.4, 122.4, 121.7, 74.5, 65.1, 54.3, 54.0, 52.9, 43.2, 34.0, 32.5, 32.0, 31.3, 26.9, 26.1.
(139) HRMS: calc. for C.sub.29H.sub.43N.sub.10O.sub.10S.sup.+, [M].sup.+=723.2879, found: 723.2873.
5.3 MS Analysis of the GSH Adduct
(140) The MS analysis shows N.sub.2 loss of the single-charged species in the mass spectrometer in the precursor ion scan. The double-charged species shows the N.sub.2 loss directly. The results are shown in
6. Use of a Ketone-Reactive Probe in MS Analysis of Testosterone
(141) As a probe capable of reacting with a ketone-functional group on an analyte molecule, a hydroxylamine reagent was used and tested in the MS analysis of testosterone. The reaction scheme is shown in
6.1 Synthetic Procedure
(142) Testosterone (20 mg, 0.069 mmol, 1.0 eq) was dissolved in a mixture of H.sub.2O/MeCN/MeOH (1:1:1, 1.0 mL containing 0.05% formic acid). The hydroxylamine reagent (28 mg, 0.069 mmol, 1.0 eq) was added and the mixture was incubated at 40° C. over night. Volatiles were removed in vacuo and the crude product was finally purified via semipreparative HPLC (50% MeCN to 80% MeCN in H.sub.2O+0.05% formic acid in 40 minutes, 0.5 mL/min). The adduct was yielded as a colourless solid (5 mg, 0.009 mmol, 13%).
(143) Note: The adduct was yielded as a 53/46 mixture of the E/Z isomers which were not assigned.
(144) .sup.1H-NMR (400 MHz, CD.sub.3CN): δ (ppm)=8.67 (s, 2H), 8.11 (s, 1H), 8.09 (s, 1H), 7.96-7.94 (m, 4H), 7.76-7.73 (m, 4H), 7.10 (s.sub.t, J=5.55 Hz, 1H), 7.00 (s.sub.t, J=5.68 Hz, 1H), 6.39 (s, 1H), 5.69 (s, 1H), 4.57 (s, 4H), 4.46 (s, 4 h), 4.44 (s, 4H), 3.32 (s, 18H), 1.79-1.20 (m, 30H), 1.07 (s, 3H), 1.06 (s, 3H), 1.03-0.73 (m, 10H), 0.71 (s, 3H), 0.68 (s, 3H).
(145) .sup.13C-NMR (121 MHz, CD.sub.3CN): δ (ppm)=170.5, 170.4, 168.3, 163.0 (2×C), 162.4, 158.8, 158.2, 156.0, 146.7, 146.3, 139.7 (2×C), 133.6, 121.7, 121.6, 121.4, 121.3, 121.1, 116.6, 110.5, 81.3, 81.0, 73.2, 72.9, 65.9, 54.7, 54.5, 51.0, 50.9, 43.1, 43.0, 39.4, 38.4, 37.1, 36.9, 36.6, 36.1, 36.0, 35.0, 34.7, 34.6, 33.1, 32.6, 32.5, 32.2, 30.3, 30.2, 24.8, 23.6, 21.3, 21.1, 19.9, 17.9, 17.7, 11.1, 11.0.
(146) HRMS (ESI): calculated for C.sub.35H.sub.50N.sub.7O.sub.4.sup.+ [M.sup.+]: 632.3919, found: 632.3915.
6.2 Details of Mass Spectrometry
(147) The testosterone adduct was dissolved in water/acetonitrile (1:1) in a stock concentration of 2.1 mM. A serial dilution with the same solvent mixture finally gave a solution with a concentration of 0.12 pM. The adduct was subjected to UHPLC-QQQ-MS using a flow-rate of 0.35 mL/min with a gradient starting from 50% buffer B (Acetonitrile with 0.0075% formic acid), whereas an injection of 1.0 μL of this solution equalled 0.12 amol. This amount was still detectable in the mass spectrometer with mass transition of 632.4.fwdarw.604.4 and the qualifier transition of 632.4.fwdarw.192.1.
(148) In comparison, pure testosterone was dissolved in EtOH/acetonitrile (1:1) in a stock concentration of 11.04 mM. A serial dilution was performed with the same solvent mixture and gave a final concentration of 11 μM. This solution was again subjected to UHPLC-QQQ-MS with a flow-rate 0f 0.35 mL/min using a gradient starting from 50% buffer B (Acetonitrile with 0.0075% formic acid), whereas an injection volume of 2.0 μL of this stock equalled 22 amol. This amount was still detectable in the mass spectrometer with a mass transition of 289.2.fwdarw.109.2. Injection of only 1.0 μL did not show a signal above background noise.
(149) In summary, the adduct can be detected with 185 times higher sensitivity than the underivatized testosterone.
(150) The results are shown in
7. Use of a Reactive Ester Probe in MS Analysis of Dopamine
(151) The following adduct of dopamine and a reactive ester compound was prepared
(152) ##STR00018##
and characterized by HR-MS and UHPLC-MS/MS.
(153) HR-MS: calculated for C.sub.26H.sub.33N.sub.8O.sub.6.sup.+: 553.2518, found: 553.2518.
(154) The adduct was dissolved and diluted in MeOH/Acetonitrile (1:1) and subjected to UHPLC-MS/MS. Performance of a product ion scan identified the nitrogen loss with a peak at 525.2 resulting from fragmenting the molecule ion of a m/z of 553.4.
LIST OF REFERENCES
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