DOI: 10.1002/cbic.201402649
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Azide-Tagged Sphingolipids: New Tools for Metabolic Flux Analysis Mara Garrido,[a] Jos Luis Abad,[a] Gemma Fabris,[a] Josefina Casas,*[a] and Antonio Delgado*[a, b] In memory of Professor Robert Bittman.
fecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.
Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, w-azidosphingolipids 1–3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)-Garner’s aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC-27 cells showed that probes 1–3 compete with the natural metabolites and are incorporated into sphingolipid pathways without af-
Introduction domics,[11, 12] as well as the use of isobaric tags (iTRAQ reagents)[13] have emerged as such procedures. On the other hand, the MS analysis of labeled sphingolipidomes for metabolic studies relies on the individual analysis of samples from cells labeled with non-natural probes.[14] The laborious preparation of individual samples and the cost of using equipment over long hours would be reduced with procedures for massive analysis, which would facilitate fluxomics studies. The recent advances in the development of bioorthogonal reactions have boosted their applications in chemical biology.[15, 16] In particular, those involving azide–alkyne cycloadditions (click chemistry) allow the selective labeling of target biomolecules within the complex environments of live cells and cell extracts, helping the study of live systems at the molecular level. In this context, although click chemistry has been extensively used in chemical biology, only a few studies on lipids have been reported. Bioorthogonal processes applied to alkyne-functionalized fatty acids[17–19] and lipids[20] have recently been described, and a very recent work addresses the application of click chemistry to introduce isobaric tags for the analysis of lactosylceramides.[21] On the basis of the above premises and our previous work on the use of wN3-sphingolipids in artificial membrane studies,[22] we envisaged the use of the wN3-sphingoid bases 1–4 as precursors of chemically taggable entities for the identification of sphingolipid populations in UPLC-MS analysis of pooled samples. Thus, probes 1, 2, and 4 can be regarded as tagged sphingosine (So), sphinganine (Sa, also known as dihydrosphingosine), and ceramide (Cer), respectively, whereas probe 3 is a tagged analogue of ketosphinganine, the first metabolic precursor in the de novo sphingolipid biosynthesis.[23] Thanks to the small size of the azide group, the probes would be expect-
Sphingolipids (SLs) are bioactive molecules that play important roles in signal transduction and as second messengers in a variety of processes including, inter alia, proliferation, differentiation, migration, apoptosis, senescence, autophagy, angiogenesis, etc.[1–3] Because of their physiological importance, the accurate spatiotemporal description of the complete SL profile—the socalled sphingolipidome—within a cell or tissue has attracted much attention in the last years. This information is valuable for correlating sphingolipid metabolites and their amounts with phenotypic changes observed under different conditions and in different experimental setups. Several analytical approaches to the quantification of SLs, including some based on GC[4] and HPLC,[5–7] have been described in the literature. In addition, thanks to advances in mass spectrometry, this technique has become, in its multiple variants, the method of choice for the quantitative measurement of SLs.[8–10] However, despite the advances in MS, alternative methods easily adaptable to high-throughput formats are still required. Shotgun lipi[a] Dr. M. Garrido, Dr. J. L. Abad, Prof. G. Fabris, Dr. J. Casas, Prof. A. Delgado Research Unit on BioActive Molecules Department of Biomedicinal Chemistry Institute for Advanced Chemistry of Catalonia (IQAC-CSIC) Jordi Girona 18–26; 08034 Barcelona (Spain) E-mail: [email protected] [email protected] [b] Prof. A. Delgado University of Barcelona (UB), Faculty of Pharmacy Department of Pharmacology and Medicinal Chemistry Unit of Pharmaceutical Chemistry (Associated Unit to CSIC) Avga. Joan XXIII s/n, 08028 Barcelona (Spain) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402649.
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Full Papers moderate yield and with excellent E selectivity, as judged from the 1H NMR spectrum of the crude reaction mixture. Treatment of 7 with excess NaN3 in DMF at 80 8C, followed by the simultaneous cleavage of the oxazolidine and N-Boc groups under acidic conditions, afforded probe 1 (RBM2–31) in excellent yield. Acylation of 1 with n-octanoic acid, with use of EDC/ HOBt as coupling system, gave probe 4 (RBM2–37). On the other hand, synthesis of probes 2 (RBM2–40) and 3 (RBM2–63) required the addition of undec-10-yn-ol (dilithium salt) to (S)-Garner’s aldehyde (5), followed by catalytic hydrogenation of the intermediate alkyne to afford the diol 8. Selective tosylation of the terminal hydroxy group in 8 and subsequent treatment of the intermediate tosylate 9 with excess NaN3 afforded azido alcohol 10. Oxidation of the secondary alcohol in 10 and acidic deprotection of the amino alcohol moiety in the resulting ketone 11 led to the expected probe 3, whereas probe 2 (RBM2–40) could easily be obtained in good overall yield by direct deprotection of 10 under acidic conditions (Scheme 1). Finally, the synthesis of tags 15–19 was carried out by acylation of a homologous series of linear alkylamines (ethylamine to n-pentylamine) with the NHS-activated precursor 14 (Scheme 2).
ed to be good competitors for the natural compounds (Figure S1 in the Supporting Information), becoming incorporated into SL metabolic pathways to afford wN3-tagged sphingolipid metabolites amenable to bioorthogonal tagging by click chemistry methods. Here we report on the synthesis of wN3tagged sphingoid bases as a proof of concept that this technology can foster high-throughput analysis of sphingolipidomes in metabolic flux experiments.
Results and Discussion Synthesis of the probes and tags
Metabolization of probes
The synthesis of the probes is described in Scheme 1. Crossmetathesis[24, 25] of 11-bromoundec-1-ene and vinyl alcohol 6, obtained from Garner’s aldehyde 5 (S)-Garner’s aldehyde (5) according to a reported protocol,[26] afforded bromide 7 in
In order to confirm the incorporation of probes into the SL metabolic pathways, HGC-27 cells were incubated with probes 1–3 at 5 mm for different time periods, and the resulting metabolites were analyzed by UPLC-TOF. Cell viability was not
Scheme 1. Reagents and conditions; a) see ref. [26]; b) 11-bromoundec-1-ene, second-generation Grubbs catalyst, CH2Cl2, 45 8C, 59 %; c) NaN3, DMF, 80 8C (12, 93 %; 10, 76 %); d) ClCOCH3, MeOH, RT, 1 h (1, 84 %; 2, 85 %; 3, 90 %); e) C7H15COOH, EDC, HOBt, Et3N, CH2Cl2, 59 %; f) undec-10-yn-1-ol, BuLi, HMPA, THF, 78 8C, 50 %; g) H2 (1 atm), Rh catalyst, MeOH (89 %); h) TsCl, DMAP, Et3N, CH2Cl2, RT, 58 %; i) PCC, CH2Cl2, 92 %.
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Scheme 2. Synthesis of tags 15–19 and their reactions with probe 4 (RBM2–37). Single regioisomers for adducts 20–24 are represented. The general reaction of the tags with wN3 metabolites is also shown. a) DIPEA, DMF, RT, n = 1–4; b) MeOH, 37 8C, 18 h.
matic reduction of azides is known to take place in the presence of monothiols (such as glutathione) at alkaline pH values. However, the reported reduction rates under physiological conditions[27, 28] are irrelevant on the timescale of our experiments. Because probes 1–3 are incorporated into SL metabolism, competition with their natural metabolic intermediates was expected. Consistently with this, cells incubated with probes 2 and 3 accumulated Sa without showing any major impact on other dhSL’s pools (Figure 2 C and D). The accumulation of Sa is consistent with wN3Sa—either exogenously added (probe 2) or formed by reduction of 3—replacing natural Sa in sphingolipid metabolism, with sphingosine kinases being a likely target (significant amounts of wN3SaP are produced from wN3Sa, Figure 1). Unexpectedly, probe 1 did not provoke the anticipated increase in natural So (Figure 2 B). However, whereas levels of Sa in vehicle-treated cells are below the detection limit (Figure 2 A), around 10 pmol of Sa are formed in cells treated with 1 at 3 and 6 h (Figure 2 B). Furthermore, probe 1 caused a 2.5to fourfold increase in dhCers over vehicle-treated cells (Figure 2 B). Increases in Sa and dhCers on treatment with sphingosine are unprecedented, and the reasons why 1 (an wN3 analogue of sphingosine) provokes Sa and dhCers buildup are so far unknown. As a possible explanation, HGC27 cells might rapidly acylate exogenous probe 1 to form the corresponding N3labeled Cer derivatives (their formation is evident as early as 1 h after treatment; see Figure 1), and this, in turn, might lead to an increase in their metabolic precursors dihydroceramides and sphinganine through a negative feedback control. Finally, probes 1–3 did not provoke any significant change in sphingolipids derived from So (Figure S4). Collectively, the above results validate the suitability of probes 1–3 as tracers for study of SL metabolism.
compromised under these assay conditions (Figure S2). As shown in Figure 1, the three wN3-bases were metabolized into the sphingoid base phosphates (wN3SoP from 1 and wN3SaP from 2 and 3), together with different N-acyl amides (wN3Cers from 1–3 and wN3dhCers from 2 and 3) and their phosphocholine derivatives (wN3SMs from 1–3 and wN3dhSMs from 2 and 3). Unlike in the cases of probes 1 and 2, the levels of probe 3 (wN3ketoSa) could not be monitored because its exact mass (285.2291 [M+H] + ) was identical, and its retention time very similar, to those of its azide-labeled sphingosine metabolite (probe 1). No peak was detected by selection of this exact mass at the expected retention time in chromatograms from cells treated with probe 3, whereas large amounts of wN3Sa were found. This result suggests that exogenous wN3ketoSa is immediately reduced to wN3Sa by 3-ketosphinganine reductase. With regard to the different N-acyl species, the three probes gave a distribution of wN3Cers resembling that of natural Cers (Figure S3). It is worth noting that wN3 monohexosyl(dihydro)ceramides were detected at very low levels (less than 5 % of total wN3 metabolites) at all time points. To rule out the possibility that the wN3-probes are poorly incorporated into the glycosylation pathway, incubations with C17:Sa were carried out. Glycosylation also proved to be very low from this precursor [mean % SD of total metabolites at 6 h (n = 3): ceramide monohexosides, 3.7 0.4; dihydroceramide monohexosides, 1.3 0.1]. The low levels of glycosphingolipids formed from the exogenous bases suggest either low glycosyltransferase (synthesis) activities, high glycosylhydrolase (recycling) activities, or a very active synthesis/recycling pathway in HGC27 cells under the experimental conditions of the assays. Importantly, no metabolites arising from the putative enzymatic reduction of the terminal azide groups were detected. The enzyChemBioChem 2015, 16, 641 – 650
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Figure 1. Metabolization of probes 1–3 in HGC7 cells. Cells were treated with the compounds (5 mm) for different time periods. SLs were extracted and analyzed as detailed in the Experimental Section. Data are given as pmol equivalents to: C17Sa for wN3Sa and wN3So, C17SaP for wN3SaP and wN3SoP, N-C12Cer for wN3dhCer and wN3Cer, and N-C12SM for wN3dhSM and wN3SM. Data each correspond to the mean SD of one representative experiment in triplicate.
Figure 2. Effects of probes 1, 2, and 3 on the dihydrosphingolipidome in HGC-27 cells treated with the compounds (5 mm) for different time periods. SLs were extracted and analyzed as detailed in the Experimental Section. Data each correspond to the mean SD of one representative experiment in triplicate.
The biocompatible Cu-free, strain-promoted alkyne–azide cycloaddition (SPAAC)[15, 29, 30] was chosen for the labeling of the wN3 metabolites with azadibenzocyclooctyne tags 15–19. The robustness of the resulting triazole systems, together with the natures of the amide groups in the above tags, offer excellent chemical stability under the alkaline conditions required for sample extraction prior to the sphingolipidome analysis (see the Experimental Section). In addition, the use of a Cu-free system allows a cleaner extraction protocol and avoids potential interference in the subsequent MS analysis. Preliminary studies: The reactivities of tags 15–19 were first examined at the analytical level with wN3ceramide 4 (RBM2– 37) as metabolite model (Scheme 2 and Table S1). Compound 4 was used at a concentration within the range of that expected in cell assays (150 pmol), tags were used at 50-fold
Applications of the w-azido probes for LC/MS analysis of sphingolipids in pooled samples Our procedure for studying SL metabolism by use of wN3 probes for metabolic labeling and further UPLC-MS analysis of tagged SL metabolites in population pools is depicted in Figure 3. The methodology involves a step consisting of metabolic labeling with the probe of choice, followed by a step consisting of differential tagging of SL from each cell population, after which the experimental groups are pooled and processed as a single sample. Although simultaneously analyzed, the sphingolipidomes corresponding to each cell population are unambiguously identified by the characteristic masses conferred by their tags. ChemBioChem 2015, 16, 641 – 650
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Full Papers (Figure 1). For an efficient click reaction, and because uptake of probes is about 1/10 of the administered amount, tags were added in excess (4 , 20 nmol) prior to lipid solubilization and allowed to react overnight at 48 8C. After lipid extraction, unreacted wN3SL metabolites turned out to be null or negligible (< 1.5 % relative to control). Moreover, wN3SLs were efficiently derivatized to the corresponding click adducts with tags 15–19, as shown in Figure 4 for the corresponding wN3C16Cer and wN3C16SM metabolites. The unambiguous profile observed for each tagged species by use of this analytical procedure should be pointed out. The retention times and the calculated and found high-resolution masses for all identified metabolites with the corresponding tags 15–19 are indicated in Table S2. Analysis of pooled samples: After the above experiments, we considered the feasibility of metabolic labeling with wN3 probes and further tagging with cyclooctynes for the analysis of pooled samples. As a proof of concept, we determined the effect of Figure 3. Simultaneous UPLC-MS analysis of SLs tagged through click reactions. T1–Tn fumonisin B1 (FB1), a validated inhibitor of ceramide represent different treatment conditions for each cell population. Theoretically, the number of cell populations can be as large as the number of available tags. synthases,[31] on the metabolization of probe 1. To this end, three populations of HGC-27 cells were independently treated with vehicle (control, populamolar excess (7.5 nmol), and long reaction times (18 h, 37 8C, tion 1) and with FB1 at 25 mm (population 2) and at 50 mm MeOH) were allowed to ensure the total consumption of the (population 3) for 24 h. After this period, probe 1 (5 mm) was starting azide. Although click reactions with tags 15–19 were added to each cell population, and incubation was carried out expected to produce 1:1 mixtures of regioisomeric 1,2,3-triafor an additional 1 h. This incubation time afforded suitable zoles, only single peaks for the triazole adducts 20–24 were detected in all cases. Additionally, no starting azide 4 was ever detected after the click reaction, an indication of its quantitative transformation. Interestingly, the triazole adducts were detected with an approximately eightfold higher sensitivity than the starting probe 4, as indicated by the relative peak areas shown in Table S1. This is of analytical interest because the sample size can be reduced. After demonstrating the occurrence of the click reaction of pure 4 to similar extents with the different tags on the lowmm scale, we next used probe 1 (5 mm, 6 h) as a model to evaluate the reaction of its w-N3SL metabolites with tags 15–19 in cell extracts. A 6 h incubation Figure 4. Formation of SL adducts with tags 15–19. Traces correspond to representative UPLC-TOF traces corretime was considered approprisponding A)–E) to C16-w-N3Cers, and F–J) to C16-w-N3SMs from cells incubated with probe 1 for 6 h and further ate in light of the abundant derivatized with tags A), F) 15, B), G) 16, C), H) 17, D), I) 18, and E), J) 19. The retention times are indicated next to metabolization observed for each peak, and the exact masses of A)–E) the C16-wN3Cer adducts, and F)–J) the C16-wN3SM adducts are shown 1 under these conditions in italics on the right. ChemBioChem 2015, 16, 641 – 650
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Figure 5. Pooled sample analysis of the effect of FB1 on the metabolism of probe 1. HGC-27 cells were incubated for 24 h without (vehicle) or with FB1 at 25 or 50 mm, followed by 1 h incubation with probe 1 (5 mm). Cells were finally treated with tags 15, 16, and 17, followed by on-plate extraction and sample pooling as detailed in the Experimental Section. A) Pooled sample UPLC-TOF traces corresponding to the C16-wN3Cer adducts with tags 15 (940.6639, control sample), 16 (954.6796 25 mm FB1 sample), and 17 (968.6952, 50 mm FB1 sample). B) Partial mass spectrum (from 935 to 975 amu) corresponding to the 6:15– 6:80 min range of the total ion chromatogram showing the presence and abundance of the C16-wN3Cer adducts with tags 15 (940.6665, control sample), 16 (954.6771), and 17 (968.6995). C), D) Amounts of produced wN3SLs adducts, given as pmol equivalents with respect to the standard mix (Experimental Section). Data correspond to the mean SD of one representative experiment (experiments performed in quadruplicate).
Conclusion
levels of wN3Cer and low metabolization to wN3SM. Cell populations were next independently treated with a specific tag (tag 15 for population 1, tag 16 for population 2, and tag 17 for population 3) and allowed to react for 2 h. Samples were then pooled, processed, and analyzed. As would be expected for FB1, wN3Cer levels in populations 2 and 3 were substantially reduced, as evidenced (Figure 5 A–C) by the lower concentrations of the corresponding click adducts with tags 16 (80 7 pmol equiv) and 17 (43 6 pmol equiv), respectively, relative to the control population, which contained wN3Cer adducts of tag 15 (249 14 pmol equiv). Similarly, wN3SM levels were also diminished, whereas those of wN3So and wN3SoP were increased, especially in population 3 (50 mm FB1; wN3So: 2973 342 pmol equiv for tag 17 adducts vs. 1549 149 pmol equiv for tag 15 adducts; wN3SoP: 1038 161 pmol equiv for tag 17 adducts vs. 625 90 pmol equiv for tag 15 adducts). A control experiment, in which all three populations were treated with vehicle, followed by probe 1, and further tagged with cyclooctynes 15, 16, and 17, was performed in parallel. Analysis of the pooled control samples showed similar levels of each of the different wN3SL metabolites adducts (Figure 5 D). These results provide the proof of concept that wN3-sphingoid bases, and probably other wN3SLs, are interesting tools for the metabolic labeling and later tagging of metabolites for pooled sample HPLC/MS analysis.
In summary, w-azidoSLs 1–3 have been synthesized and shown to be well incorporated into sphingolipid metabolic pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited for the mass spectrometric analysis of sphingolipidomes in pooled samples originating from different cell populations after individual tagging by treatment with homologous dibenzocyclooctynes. Essentially, the method is not intended to determine actual amounts of metabolites, but for use in identifying differences between different experimental groups. This is easily achieved with this methodology, which combines all the experimental groups in one sample, thus reducing the time of sample processing, the equipment costs of use, and the experimental errors originating from manipulation of individual samples. The development of a large collection of appropriate cyclooctyne tags, as well as that of computational algorithms for the automatic analysis of tagged metabolites, should allow the procedure to be expanded to pools containing large numbers of metabolite populations.
Experimental Section General remarks: Solvents were distilled prior to use and, if required, dried by standard methods. 1H and 13C NMR spectra were
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Full Papers obtained in CDCl3 solutions at 400 MHz (for 1H) and 100 MHz (for 13 C), unless otherwise indicated. Chemical shifts (d) are reported in ppm relative to the solvent (CHCl3 or CDCl3) signal. Multiplets are indicated by the chemical shift corresponding to the center of the peak pattern. All reactions were monitored by TLC on aluminum foil precoated silica gel plates. Flash column chromatography was performed with the indicated solvents and use of silica gel 60 (particle size 0.035–0.070). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Optical rotations were measured at the sodium D line (589 nm) and are reported as [a]D = [c, (g per 100 mL), solvent]. Electrospray ionization was used for HRMS analysis. HPLC was performed with a Supelcosil LC-18 column (25 cm 4.6 mm i.d., 5 mm particle size) and a HPLC system equipped with a 515 HPLC pump, a 2545 binary gradient module, a 3100 mass detector, and a 2998 photodiode array detector. Solvent A (water) and solvent B (acetonitrile) were used as eluents. The samples were eluted under the following logarithmic gradient conditions: 0.0 min, 50 % B; 6.0 min, 90 % B; followed by an isocratic elution: 6.1 to 20.0 min, 90 % B. After each run, the column was thoroughly washed with acetonitrile and equilibrated with the initial conditions. The mobile phase was degassed and pumped at a flow rate of 1.0 mL min 1. The injection volume was set at 20 mL. UPLC-TOF was carried out with a Waters Acquity UPLC system coupled to a Waters LCT Premier orthogonal accelerated time-of-flight mass spectrometer (Waters). The samples were injected onto a reversed-phase C18 column (1.7 mm C18 Acquity UPLC BEH, 2.1 100 mm, Waters) at 0.3 mL min 1. Solvent A [methanol/formic acid (99.8:0.2, v/v) with ammonium formate (1 mm)]; solvent B [water/formic acid (99.8:0.2, v/v) with ammonium formate (2 mm)]. Two different sets of gradient conditions were used: gradient 1: 0.0 min, 65 % A; 8.0 min, 90 % A; 13.0 min, 99 % A; 15.0 min, 99 % A; 18.0 min, 65 % A; gradient 2: 0.0 min, 80 % A; 3.0 min, 90 % A; 15.0 min, 99 % A; 18.0 min, 99 % A. The mass spectrometer was run in the positive electrospray ionization mode. Full scan spectra from 50 to 1500 Da were acquired, and individual spectra were summed to produce data points each 0.2 s. Mass accuracy and reproducibility were maintained by using an independent reference spray by the LockSpray interference. The column was held at 30 8C. The injection volume was set at 10 mL. Positive identification of compounds was based on accurate mass measurement with an error < 5 ppm. Azadibenzocyclooctyne 14 was purchased from Click Chemistry Tools (http://www.clickchemistrytools.com). A “standard mix” for SL quantification was prepared by mixing 200 pmol of each of the following standards: C17So, C17SaP, NC12Cer, NC12SM. HGC-27 cells were obtained from Health Protection Agency Culture Collections (Salisbury, UK).
(2S,3R)-2-Amino-14-azidotetradecane-1,3-diol (2, RBM2–40): wN3 Aminodiol 2 (RBM2–40, 79 mg, 0.27 mmol, 85 %) was obtained according to the procedure described for compound 1, as a waxy solid, from compound 10 (137 mg, 0.32 mmol) and acetyl chloride (600 mL) in MeOH (10 mL). Purification was carried out by flash chromatography (CH2Cl2/MeOH/NH3 8:1.9:0.1). [a]D = +3.9 (c = 0.9, CHCl3); 1H NMR (400 MHz, CDCl3): d = 3.70 (2 H; NH2), 3.60 (m, 1 H), 3.25 (appt t, 2 H), 2.83 (br, 1 H), 2.33 (br, OH), 1.59 (m, 2 H), 1.45 (m, 2 H), 1.40–1.24 ppm (m, 18 H); 13C NMR (101 MHz, CDCl3): d = 74.6, 63.5, 55.8, 51.6, 33.9, 29.8–29.6 (five peaks), 29.2, 28.9, 26.8, 26.2 ppm; HRMS: m/z calcd for C14H31N4O2 : 287.2442 [M+H] + ; found: 287.2442. (S)-2-Amino-14-azido-1-hydroxytetradecan-3-one hydrochloride (3, RBM2–63): Ketone 3 (RBM2–63, 30 mg, 0.09 mmol, 90 %) was obtained according to the procedure described for compound 1, as a yellow pale wax, from 11 (41 mg, 0.10 mmol) and acetyl chloride (300 mL) in MeOH (5 mL) and used without further purification. [a]D = +23.9 (c = 0.6, CHCl3); 1H NMR (400 MHz, CD3OD): d = 4.16 (t, J = 4.0, 1 H), 4.10 (dd, J = 12.0, 4.3 Hz, 1 H), 3.97 (dd, J = 12.0, 3.4 Hz, 1 H), 3.30 (t, J = 7.0 Hz, 2 H), 2.64 (t, J = 7.2 Hz, 2 H), 1.67–1.54 (m, 4 H), 1.41–1.28 ppm (m, 18 H); 13C NMR (101 MHz, CD3OD): d = 205.2, 62.2, 60.3, 52.4, 39.6, 30.6–30.5 (four peaks), 30.3, 30.1, 29.9, 27.8, 24.2 ppm; HRMS: m/z calcd for C14H29N4O2 : 285.2285 [M+H] + ; found: 285.2278. N-[(2S,3R,E)-14-Azido-1,3-dihydroxytetradec-4-en-2-yl]octanamide (4, RBM2–37): A solution of octanoic acid (65 mL, 0.4 mmol) in anhydrous CH2Cl2 (2 mL) was added dropwise to a solution of EDC (105 mg, 0.60 mmol) and HOBt (60 mg, 0.44 mmol). The mixture was stirred for 10 min under Ar and next added dropwise to a solution of amine 1 (RBM2–31, 105 mg, 0.37 mmol) and Et3N (200 mL, 1.44 mmol) in anhydrous CH2Cl2 (2 mL). The reaction mixture was stirred for 1 h under argon, diluted with CH2Cl2 (10 mL), and washed, successively, with H2O (10 mL) and brine (10 mL). The organic layer was worked up as usual to give a residue, which, after flash column chromatography (CH2Cl2/MeOH 0–5 %), afforded amide 4 (RBM2–37, 89 mg, 0.22 mmol, 59 %) as a white solid. [a]D = 3.6 (c = 1.13, CHCl3); 1H NMR (400 MHz, CDCl3): d = 6.35 (d, J = 6.5 Hz, 1 H), 5.76 (dt, J = 15.6, 6.8 Hz, 1 H), 5.51 (dd, J = 15.6, 6.4 Hz, 1 H), 4.28 (br, 1 H), 3.90 (m, 1 H), 3.88 (br, 1 H), 3.68 (br d, 1 H), 3.25 (t, 1 H; OH), 2.90 (br, OH), 2.21 (t, J = 7.6 Hz, 2 H), 2.04 (appt q, 2 H), 1.67–1.57 (m, 4 H), 1.41–1.21 (br, 22 H), 0.87 ppm (t, J = 7.2 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 174.2, 134.2, 128.9, 74.5, 62.5, 54.65, 51.6, 36.9, 32.4, 31.8, 29.5–28.9 (nine peaks), 26.8, 25.9, 22.7 ppm; HRMS: m/z calcd for C22H42N4NaO3 : 433.3149 [M+Na] + ; found: 433.3153. tert-Butyl (S)-4-[(R,E)-12-bromo-1-hydroxydodec-2-en-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (7): The second-generation Grubbs catalyst (100 mg, 0.12 mmol) was added portionwise to a degassed solution of vinyl alcohol 6[26] (730 mg, 2.80 mmol) and 11-bromoundec-1-ene (2.6 g, 11.20 mmol) in CH2Cl2 (20 mL). The reaction mixture was stirred for 5 h at reflux under Ar. The solvent was next removed in vacuo, and the resulting residue was flash chromatographed (hexane/AcOEt 0–27 %) to give 7 (770 mg, 1.66 mmol, 59 %) as a colorless oil. [a]D = 26.2 (c = 1.04, CHCl3); 1 H NMR (mixture of rotamers, 400 MHz, CDCl3): d = 5.72 (dt, J = 15.2, 7.2 Hz, 1 H), 5.44 (dd, J = 15.2, 6.0 Hz, 1 H), 4.23–3.77 (m, 4 H), 3.41 (t, J = 6.9 Hz, 2 H), 2.04 (m, 2 H), 1.85 (m, 2 H), 1.55–1.33 (m, 12 H), 1.45 (s, 6 H), 1.27 ppm (br, 9 H); 13C NMR (major rotamer, 101 MHz, CDCl3): d = 154.3, 133.4, 128.2, 94.5, 81.1, 74.1, 65.0, 62.4, 34.2, 32.9, 32.5, 29.3–28.4 (eight peaks), 28.24, 26.4, 24.7 ppm; HRMS: m/z calcd for C22H40BrNNaO4 : 484.2033 [M+Na] + ; found: 484.2037.
Chemistry (2S,3R,E)-2-Amino-14-azidotetradec-4-ene-1,3-diol (1, RBM2–31): Acetyl chloride (900 mL, 6 % volume) was added to a solution of 12 (335 mg, 0.80 mmol) in MeOH (15 mL). The reaction was complete in 1 h, as monitored by TLC (hexane/AcOEt 1:1). Methanol was removed in vacuo, and the resulting residue was purified by flash column chromatography (CH2Cl2/MeOH/NH3 9:0.9:0.1) to give wN3 aminodiol 1 (RBM2–31, 190 mg, 0.67 mmol, 84 %) as a white solid. [a]D = 1.2 (c = 0.47, CHCl3); 1H NMR (400 MHz, CDCl3): d = 5.72 (dt, J = 15.6, 6.8 Hz, 1 H), 5.44 (dd, J = 15.6, 7.1 Hz, 1 H), 4.03 (t, J = 6.2 Hz, 1 H), 3.64 (2 H; NH2), 3.24 (m, 2 H), 2.77 (br, 1 H + 2 OH), 2.05 (appt q, 2 H), 1.58 (m, 2 H), 1.41–1.22 ppm (m, 14 H); 13C NMR (101 MHz, CDCl3): d = 134.6, 129.4, 75.2, 63.8, 56.2, 51.6, 32.4, 29.5– 29.1 (five peaks), 28.9, 26.8 ppm; HRMS: m/z calcd for C14H29N4O2 : 285.2285 [M+H] + ; found: 285.2274.
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Full Papers of rotamers): d = (208.9, 208.4), (152.2, 151.6), (95.3, 94.6), (81.0, 80.7), (65.9, 65.5), (65.5, 65.3), 51.6, (39.2, 38.6), 29.7–29.4 (10 peaks), (28.9, 28.5), (26.3, 25.5), (24.9, 23.8), (23.2, 23.1 ppm); HRMS: m/z calcd for C22H40N4NaO4 : 447.2942 [M+Na] + ; found: 447.2957.
tert-Butyl (S)-4-[(R)-1,12-dihydroxydodecyl]-2,2-dimethyloxazolidine-3-carboxylate (8): Rhodium on alumina (Sigma–Aldrich, ref. 212 857, 27 mg) was added portionwise to a solution of starting compound 13 (150 mg, 0.38 mmol) in freshly degassed MeOH (10 mL). The mixture was vigorously stirred at room temperature for 6 h under H2 (1 atm) and filtered through a plug of Celite. The solid was rinsed thoroughly with MeOH (3 3 mL), and the combined filtrates were concentrated in vacuo to afford a crude mixture, which was used without further purification. [a]D = 11.0 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, mixture of rotamers): d = 4.66–3.11 (m, 4 H), 3.62 (t, J = 6.6 Hz, 2 H), 1.61–1.19 (br, 20 H), 1.52 (s, 6 H), 1.31 ppm (s, 9 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.7, 94.3, 81.1, 73.0, 64.8, 63.0, 62.4, 32.8, 29.7–29.5 (nine peaks), 28.5, 26.5, 26.2, 25.8, 24.3 ppm; HRMS: m/z calcd for C22H44NO5 : 402.3214 [M+H] + : found: 402.3219.
tert-Butyl (S)-4-[(R,E)-12-azido-1-hydroxydodec-2-en-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (12): NaN3 (310 mg, 4.78 mmol) was added portionwise to a solution of 7 (550 mg, 1.19 mmol) in anhydrous DMF (20 mL). The mixture was heated at 80 8C and stirred overnight under Ar. After the system had cooled down to room temperature, H2O was added (100 mL), and the aqueous layer was extracted with Et2O (3 50 mL). The combined organic extracts were dried with MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography (hexane/ AcOEt 0–20 %) to afford 12 (470 mg, 1.11 mmol, 93 %) as a colorless oil. [a]D = 30.86 (c = 0.93, CHCl3); 1H NMR (400 MHz, CDCl3, major rotamer): d = 5.73 (dt, J = 15.2, 7.2, 7.2 Hz, 1 H), 5.44 (dd, J = 15.2, 5.2 Hz, 1 H), 4.22–3.76 (m, 4 H), 3.25 (t, J = 7.0 Hz, 2 H), 2.04 (m, 2 H), 1.63–1.45 (m, 16 H), 1.41–1.24 ppm (m, 13 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 133.5, 128.5, 94.6, 81.2, 74.2, 65.1, 62.4, 51.6, 32.5, 29.5–29.2 (eight peaks), 29.0, 28.5, 26.8, 26.4, 24.7 ppm; HRMS: m/z calcd for C22H40N4NaO4 : 447.2942 [M+Na] + ; found: 447.2960.
tert-Butyl (S)-4-[(R)-1-hydroxy-12-(tosyloxy)dodecyl]-2,2-dimethyloxazolidine-3-carboxylate (9): A solution of 8 (136 mg, 0.34 mmol) and Et3N (47 mL, mmol) in dry CH2Cl2 (7 mL) was added dropwise under Ar to a solution of tosyl chloride (65 mg, 0.34 mmol) and DMAP (21 mg, 0.17 mmol) in dry CH2Cl2 (3 mL). After vigorous stirring at RT for 8 h, the reaction mixture was diluted by addition of CH2Cl2 (10 mL). The organic layer was washed with brine (2 10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the resulting residue by flash chromatography (hexane/EtOAc gradient 10:0 to 5:5) gave tosylate 9 (110 mg, 0.20 mmol, 58 %) as a colorless wax. [a]D = 6.7 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, mixture of rotamers): d = 7.78 (d, J = 8.2 Hz, 2 H), 7.34 (d, J = 8.0 Hz, 2 H), 4.00 (t, 2 H), 4.11– 3.69 (m, 4 H), 2.44 (s, 3 H), 1.70–1.53 (m, 6 H), 1.48 (s, 6 H + broad signal 6 H), 1.20 (s, 9 H), 1.34–1.16 ppm (m, 8 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.2, 144.7, 133.3, 129.9, 128.0, 94.3, 81.1, 73.1, 70.8, 64.8, 62.5, 32.9, 29.9–28.5 (12 peaks), 26.5, 26.2, 25.4, 24.3, 21.7 ppm; HRMS: m/z calcd for C29H50NO7S: 556.3303 [M+H] + ; found: 556.3325.
tert-Butyl (S)-4-[(R)-1,12-dihydroxydodec-2-yn-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (13): A solution of nBuLi (1.29 mL, 3.22 mmol, 2.5 m in hexanes) was added dropwise to a solution of undec-10-yn-1-ol (225 mg, 1.34 mmol) in anhydrous THF (15 mL) at 20 8C. After the system had been vigorously stirred at 20 8C for 30 min, neat HMPA (350 mL, 2.01 mmol) was added dropwise. The reaction mixture was cooled to 78 8C, followed by dropwise addition of a solution of aldehyde 5[32] (236 mg, 1.03 mmol) in anhydrous THF (2 mL). After stirring at 78 8C for 1 h, the reaction mixture was allowed to warm to 20 8C over around 2 h, and next quenched by addition of saturated aqueous NH4Cl (10 mL). After dilution by addition of water, the aqueous phase was extracted with Et2O (3 20 mL). The combined organic extracts were successively washed with HCl (0.5 n) and brine, dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography (hexane/EtOAc 10:0 to 6:4) to afford compound 13 (205 mg, 0.52 mmol, 50 %) as a colorless oil. [a]D = 30.3 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3): d = 4.50 (br, 1 H), 4.15– 3.85 (m, 3 H), 3.61 (t, J = 6.6 Hz, 2 H), 2.17 (t, J = 7.1 Hz, 2 H), 1.63– 1.51 (m, 4 H), 1.51 (s, 6 H), 1.49 (s, 9 H), 1.38–1.21 ppm (m, 10 H); 13 C NMR (101 MHz, CDCl3): d = 154.2, 95.0, 86.7, 81.3, 78.0, 65.1, 64.1, 63.0, 62.8, 32.8, 29.5, 29.4, 29.0, 28.8, 28.6–28.5 (four peaks), 25.9, 25.8, 25.5, 18.8 ppm; HRMS: m/z calcd for C22H39NNaO5 : 420.2720 [M+Na] + ; found: 420.2712.
tert-Butyl (S)-4-((R)-12-azido-1-hydroxydodecyl)-2,2-dimethyloxazolidine-3-carboxylate (10): NaN3 (52 mg, 0.80 mmol) was added portionwise under Ar to a solution of tosylate 9 (110 mg, 0.20 mmol) in anhydrous DMF (10 mL), and the resulting mixture was stirred overnight at 80 8C. After cooling to room temperature, the reaction mixture was quenched by addition of H2O (50 mL) and worked up as usual to give an oil, which was flash chromatographed (hexane/EtOAc 8:2) to afford azide 10 (65 mg, 0.15 mmol, 76 %). [a]D = 2.6 (c = 0.9, CHCl3); 1H NMR (400 MHz, CDCl3, major rotamer): d = 4.15–3.95 (m, 2 H), 3.95–3.65 (m, 2 H), 3.25 (t, J = 7.0 Hz, 2 H), 1.64–1.56 (m, 6 H), 1.52–1.40 (m, 8 H), 1.48 (s, 6 H), 1.27 (s, 9 H), 1.40–1.22 ppm (m, 8 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.2, 94.3, 81.0, 73.0, 64.8, 62.5, 51.5, 29.7–28.4 (10 peaks), 26.8, 26.5, 26.2, 26.1, 24.3 ppm; HRMS: m/z calcd for C22H43N4O4 : 427.3279 [M+H] + ; found: 427.3293.
General procedure—acylation of azadibenzocyclooctyne 14: The appropriate alkylamine (0.02 mmol) was added under Ar to a solution of commercial azadibenzocyclooctyne 14 (5 mg, 0.010 mmol) and DIPEA (5 mL, 0.025 mmol) in anhydrous DMF (2 mL), and the mixture was stirred at RT overnight. Concentration under reduced pressure gave the crude tag. Tags were flash chromatographed (CH2Cl2/MeOH 100:0 to 96:4) to give 15–19.
tert-Butyl (S)-4-(12-azidododecanoyl)-2,2-dimethyloxazolidine-3carboxylate (11): PCC (72 mg, 0.33 mmol) was added under Ar to a solution of compound 10 (55 mg, 0.13 mmol) in dry CH2Cl2 (4 mL). After vigorous stirring at room temperature overnight, the reaction mixture was filtered through a plug of Celite and concentrated in vacuo. The residue was purified by flash chromatography (hexane/EtOAc 10:0 to 9:1) to afford compound 11 (51 mg, 0.12 mmol, 92 %) as a colorless wax. [a]D = 30.0 (c = 1.0, CHCl3); 1 H NMR (500 MHz, CDCl3, mixture of rotamers): d = 4.44 (dd, J = 7.5, 2.0 Hz, 0.4 H), 4.31 (dd, J = 7.5, 2.5 Hz, 0.6 H), 4.18–4.09 (m, 1 H), 3.93 (dd, J = 9.5, 2.0 Hz, 0.4 H), 3.88 (dd, J = 9.5, 2.5 Hz, 0.6 H), 3.25 (t, J = 7.0 Hz, 2 H), 2.55–2.40 (m, 2 H), 1.60–1.47 (m, 8 H), 1.55 (s, 6 H), 1.41 (s, 9 H), 1.38–1.24 ppm (br, 10 H); 13C NMR (101 MHz, CDCl3, mixture ChemBioChem 2015, 16, 641 – 650
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N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5ethyl glutaramide (15): Compound 15 (tag 1, 3.1 mg, 74 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol), and ethylamine (1.6 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (d, J = 6.9 Hz, 1 H), 7.52–7.45 (m, 4 H), 7.41–7.32 (m, 2 H), 7.27 (dd, J = 7.4, 1.6 Hz, 1 H), 5.15 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.24–3.13 (m, 4 H), 2.51–2.41 (m, 6 H), 1.74 (m, 2 H), 1.11 ppm (t, J = 7.2 Hz, 3 H); 13C NMR (101 MHz, CD3OD): d = 175.2,
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Full Papers Cell viability: Cells were seeded at a density of 105 cells per well in 96-well plates in fresh medium (100 mL per well) and grown overnight at 37 8C under a water-saturated, 5 % CO2 atmosphere. The medium was next removed and replaced with fresh medium (100 mL) containing the appropriate test compound. Cells were incubated at 37 8C under CO2 (5 %) for 24 h. The number of viable cells was quantified by estimation of dehydrogenase activity, which reduces the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to water-insoluble formazan, which was dissolved in DMSO (100 mL per well) and measured at 570 nm with a Spectramax Plus instrument (Molecular Devices). All compounds were dissolved in EtOH, and control experiments were performed with EtOH (0.1 %). See Figure S2 for results.
173.3, 152.7, 149.4, 133.4, 130.4, 130.0, 129.7, 129.2, 128.9, 128.1, 126.5, 115.7, 100.8, 56.6, 36.7, 36.2, 36.1, 35.5, 35.2, 23.2, 14.8 ppm; HRMS: m/z calcd for C25H27N3NaO3 : 440.1945 [M+Na] + ; found: 440.1965. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5propylglutaramide (16): Compound 16 (tag 2, 3.4 mg, 80 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol), and propylamine (1.3 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CDCl3): d = 7.66 (d, J = 7.3 Hz, 1 H), 7.44–7.27 (m, 7 H), 5.14 (d, J = 13.9 Hz, 1 H), 3.71 (d, J = 13.9 Hz, 1 H), 3.27–3.17 (m, 3 H), 3.27–3.41 (m, 1 H), 2.87 (t, J = 5.6 Hz, 1 H), 2.47 (ddd, J = 16.6, 7.4, 4.0 Hz, 1 H), 2.16 (t, J = 6.8 Hz, 2 H), 2.10–1.94 (m, 3 H), 1.83 (quint, J = 7.2 Hz, 2 H), 1.54 (sext, J = 7.2 Hz, 2 H), 0.93 ppm (t, J = 7.6 Hz, 3 H); HRMS: m/z calcd for C26H29N3NaO3 : 454.2101 [M+Na] + ; found: 454.2106. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5-butylglutaramide (17): Compound 17 (tag 3, 3.3 mg, 80 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and butylamine (1.5 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (d, J = 7.0 Hz, 1 H). 7.52–7.30 (m, 6 H), 7.27 (dd, J = 7.2, 1.6 Hz, 1 H), 5.14 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.28–3.10 (m, 4 H), 2.51–2.42 (m, 1 H), 2.14–1.99 (m, 5 H), 1.74 (quint, J = 7.6 Hz, 2 H), 1.48 (quint, J = 8.0 Hz, 2 H), 1.41–1.33 (m, 2 H), 0.94 ppm (t, J = 7.2 Hz, 3 H); HRMS: m/z calcd for C27H31N3NaO3 : 468.2258 [M+Na] + ; found: 468.2277. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5pentylglutaramide (18): Compound 18 (tag 4, 3.4 mg, 73 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and pentylamine (1.8 mL, 0.02 mmol) in DMF (2 mL); 1H NMR (400 MHz, CDCl3): d = 7.66 (d, J = 7.2 Hz, 1 H), 7.44–7.27 (m, 7 H), 5.14 (d, J = 13.9 Hz, 1 H), 3.71 (d, J = 13.9 Hz, 1 H), 3.39–3.29 (m, 1 H), 3.26–3.17 (m, 3 H), 2.46 (ddd, J = 16.6, 7.3, 3.8 Hz, 1 H), 2.11–1.95 (m, 5 H), 1.82 (quint, J = 7.2 Hz, 2 H), 1.53–1.45 (m, 2 H), 1.34–1.28 (m, 4 H), 0.90 ppm (t, J = 7.2 Hz, 3 H); HRMS: m/z calcd for C28H33N3NaO3 : 482.2414 [M+Na] + ; found: 482.2412.
Click reactions between wN3 metabolites and tags 15–19 in lipid extracts Standard protocol: HGC-27 cells, prepared as above, were incubated with probe 1 (5 mm) for 6 h. Lipids were extracted from PBSwashed cell pellets by suspending them in water (100 mL), MeOH (500 mL), and CHCl3 (250 mL). The standard mix (200 pmol of a mixture of SL, “standard mix”, see above) and tags 15–19 (20 nmol) were then added. Controls were treated similarly, except that tags were not added. The resulting mixtures were dispersed by sonication, incubated at 48 8C overnight, and analyzed by UPLC-TOF (gradient 1).[33, 34] The retention times and the calculated and found high-resolution masses for all identified metabolites with the corresponding tags 15–19 are given in Figure 4 and Table S2.
N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5hexylglutaramide (19): Compound 19 (tag 5, 3.7 mg, 78 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and hexylamine (2.0 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (dd, J = 7.3, 1.2 Hz, 1 H), 7.51–7.30 (m, 6 H), 7.27 (dd, J = 7.4, 1.6 Hz, 1 H), 5.14 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.29– 3.20 (m, 1 H), 3.18–3.09 (m, 3 H), 2.51–2.41 (m, 1 H), 2.13–1.98 (m, 5 H), 1.74 (quint, J = 7.2 Hz, 2 H), 1.54–1.43 (m, 2 H), 1.38–1.27 (m, 6 H), 0.91 ppm (t, J = 6.8 Hz, 3 H); HRMS: m/z calcd for C29H36N3O3 : 474.2751 [M+H] + ; found: 474.2753. Click reactions between azido probe 4 (RBM2–37) and tags 15– 19: Probe 4 (150 pmol from 15 mL of a 10 mm MeOH stock solution) was placed in a 7 mL glass vial and treated with the appropriate tag (7.5 nmol, from 15 mL of a 500 mm MeOH solution). The resulting mixture was diluted with MeOH (470 mL), stirred at 48 8C overnight, and next transferred to a UPLC vial. After concentration under a stream of nitrogen, the resulting residue was taken up in MeOH (130 mL) and analyzed by UPLC-TOF (see Table S1)
On-plate click reaction: Cells prepared as above were treated with probe 1 (0.5 mL per well of 10 mm probe 1 solution in EtOH for a final 5 mm concentration) and incubated for 1 h at 37 8C under CO2 (5 %). Then, methanol (1 mL) was added, followed by addition of the internal standards (200 pmol each; see above) and tags 15– 17 (150 nmol). After 2 h of gentle agitation at RT, the solution was transferred into glass tubes and dried in a centrifugal evaporator. The resulting residue was extracted and analyzed as described above. A parallel experiment was performed by use of the above standard protocol. It should be pointed out that under the “onplate” conditions, unreacted w-N3SL metabolites were not detected and that recovery of tagged w-N3SL was equivalent.
Biology Cell culture: The human gastric cancer cell line HGC-27 was maintained at 37 8C under CO2 (5 %) in modified Eagle’s medium supplemented with fetal bovine serum (10 %), non-essential amino acids (1 %), glutamine (1 %), and penicillin and streptomycin (each 100 ng mL 1).
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Metabolization of probes 1–3 and effects on the sphingolipidome in HGC-27 cells: Cells were seeded at 2.5 105 cells mL 1 in 6-well plates (1 mL per well) and grown overnight at 37 8C under CO2 (5 %). The medium was gently aspirated, and fresh medium (1 mL per well) was added again. Cells were treated with a solution (10 mm, 0.5 mL per well) of probes 1–3 in EtOH (final concentration = 5 mm) and incubated for the indicated times at 37 8C under CO2 (5 %). Each compound was tested in triplicate, and EtOH was used as control. After incubation, the medium was gently aspirated, and the cells were washed twice with PBS (1 mL), harvested by trypsinization, counted, and transferred to glass vials. SL standards (200 pmol, see above) were added to the cell pellets, and SLs were extracted and analyzed by UPLC-TOF (gradient 1; see above).[33, 34] Identification of wN3-SL was based on accurate mass measurement (< 5 ppm error), and LC retention time relative to an wN3Cer-C16 standard. Quantification was carried out with the extracted ion chromatogram of each compound, with use of 50 mDa windows. The amounts of wN3SLs adducts are given as pmol equivalents relative to each specific standard (wN3So, C17So; wN3SoP, C17SaP; wN3Cer, NC12Cer; wN3SM, NC12SM).
Analysis of pooled samples: Cells prepared as above were treated with 1 mL/well of fresh medium containing either vehicle or FB1 (25 or 50 mm). After 24 h, a solution of probe 1 in EtOH (10 mm,
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[9] M. Scherer, K. Leuthuser-Jaschinski, J. Ecker, G. Schmitz, G. Liebisch, J. Lipid Res. 2010, 51, 2001 – 2011. [10] R. L. Shaner, J. C. Allegood, H. Park, E. Wang, S. Kelly, C. A. Haynes, M. C. Sullards, A. H. Merrill, J. Lipid Res. 2009, 50, 1692 – 1707. [11] X. Han, K. Yang, R. W. Gross, Mass Spectrom. Rev. 2012, 31, 134 – 178. [12] D. Schwudke, K. Schuhmann, R. Herzog, S. R. Bornstein, A. Shevchenko, Cold Spring Harb. Perspect. Biol .2011, 3, doi: 10.1101/cshperspect. a004614. [13] T. Nabetani, A. Makino, F. Hullin-Matsuda, T. A. Hirakawa, S. Takeoka, N. Okino, M. Ito, T. Kobayashi, Y. Hirabayashi, J. Lipid Res. 2011, 52, 1294 – 1302. [14] I. Sultan, C. E. Senkal, S. Ponnusamy, J. Bielawski, Z. Szulc, A. Bielawska, Y. A. Hannun, B. Ogretmen, Biochem. J. 2006, 393, 513 – 521. [15] E. M. Sletten, C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 666 – 676. [16] M. Boyce, C. R. Bertozzi, Nat. Methods 2011, 8, 638 – 642. [17] P. Haberkant, R. Raijmakers, M. Wildwater, T. Sachsenheimer, B. Brugger, K. Maeda, M. Houweling, A. C. Gavin, C. Schultz, G. van Meer, A. J. Heck, J. C. Holthuis, Angew. Chem. Int. Ed. 2013, 52, 4033 – 4038; Angew. Chem. 2013, 125, 4125 – 4130. [18] B. Zheng, M. DeRan, X. Li, X. Liao, M. Fukata, X. Wu, J. Am. Chem. Soc. 2013, 135, 7082 – 7085. [19] C. Thiele, C. Papan, D. Hoelper, K. Kusserow, A. Gaebler, M. Schoene, K. Piotrowitz, D. Lohmann, J. Spandl, A. Stevanovic, A. Shevchenko, L. Kuerschner, ACS Chem. Biol. 2012, 7, 2004 – 2011. [20] A. Gaebler, R. Milan, L. Straub, D. Hoelper, L. Kuerschner, C. Thiele, J. Lipid Res. 2013, 54, 2282 – 2290. [21] S.-H. Son, S. Daikoku, A. Ohtake, K. Suzuki, K. Kabayama, Y. Ito, O. Kanie, Chem. Commun. 2014, 50, 3010 – 3013. [22] M. Garrido, J. L. Abad, A. Alonso, F. M. Goni, A. Delgado, L.-R. Montes, J. Chem. Biol. 2012, 5, 119 – 123. [23] P. Gangoiti, L. Camacho, L. Arana, A. Ouro, M. H. Granado, L. Brizuela, J. Casas, G. Fabris, J. L. Abad, A. Delgado, A. G mez-MuÇoz, Prog Lipid Res. 2010, 49, 316 – 334. [24] A. K. Chatterjee, T. L. Choi, D. P. Sanders, R. H. Grubbs, J Am Chem. Soc 2003, 125, 11360 – 11370. [25] C. Peters, A. Billich, M. Ghobrial, K. Hogenauer, T. Ullrich, P. Nussbaumer, J. Org. Chem. 2007, 72, 1842 – 1845. [26] P. Herold, Helv. Chim. Acta 1988, 71, 354 – 362. [27] I. L. Cartwright, D. W. Hutchinson, V. W. Armstrong, Nucleic Acids Res. 1976, 3, 2331 – 2339. [28] B. P. Duckworth, Z. Zhang, A. Hosokawa, M. D. Distefano, ChemBioChem 2007, 8, 98 – 105. [29] E. Lallana, R. Riguera, E. Fernandez-Megia, Angew. Chem. Int. Ed. 2011, 50, 8794 – 8804; Angew. Chem. 2011, 123, 8956 – 8966. [30] M. F. Debets, S. S. van Berkel, J. Dommerholt, A. T. Dirks, F. P. Rutjes, F. L. van Delft, Acc. Chem. Res. 2011, 44, 805 – 815. [31] T. D. Mullen, Y. A. Hannun, L. M. Obeid, Biochem. J. 2012, 441, 789 – 802. [32] P. Garner, J. M. Park, J. Org. Chem. 1987, 52, 2361 – 2364. [33] D. Canals, D. Mormeneo, G. Fabris, A. Llebaria, J. Casas, A. Delgado, Bioorg. Med. Chem. 2009, 17, 235 – 241. [34] A. H. Merrill Jr., M. C. Sullards, J. C. Allegood, S. Kelly, E. Wang, Methods 2005, 36, 207 – 224.
Abbreviations Cer: ceramide, dhCers: dihydroceramides, dhSM: dihydrosphingomyelin, GlycodhSL: glycodihydrosphingolipids, GlycoSL: glycosphingolipids, ketoSa: ketosphinganine (ketodihydrosphingosine), LacdhCers: lactosyl dihydroceramides, MHdhCers: monohexoxyl dihydroceramides, Sa: sphinganine (dihydrosphingosine), SaP: sphinganine-1-phosphate (dihydrosphingosine-1-phosphate), SM: sphingomyelin, So: sphingosine, SoP: sphingosine-1-phosphate.
Acknowledgements The authors wish to thank Eva Dalmau and Pedro Rayo for their experimental contribution. This work was supported by grants from the Ministry of Science and Innovation (SAF2011-22444) and the Agncia de Gesti d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya (2009SGR1072) to G.F. M.G. was supported by a JAE–predoc fellowship (CSIC). Keywords: azides · bioorthogonality · click chemistry · highthroughput screening · lipidomes · sphingolipids [1] A. Morales, J. C. Fernandez-Checa, Mini-Rev. Med. Chem. 2007, 7, 371 – 382. [2] T. Kolter, Chem. Phys. Lipids 2011, 164, 590 – 606. [3] Y. A. Hannun, L. M. Obeid, Nat. Rev. Mol. Cell Biol. 2008, 9, 139 – 150. [4] C. Vieu, F. Terc, F. Chevy, C. Rolland, R. Barbaras, H. Chap, C. Wolf, B. Perret, X. Collet, J. Lipid Res. 2002, 43, 510 – 522. [5] S. Snada, Y. Uchida, Y. Anraku, A. Izawa, M. Iwamori, Y. Nagai, J. Chromatogr. 1987, 400, 223 – 231. [6] M. Previati, L. Bertolaso, M. Tramarin, V. Bertagnolo, S. Capitani, Anal. Biochem. 1996, 233, 108 – 114. [7] M. Yano, E. Kishida, Y. Muneyuki, Y. Masuzawa, J. Lipid Res. 1998, 39, 2091 – 2098. [8] T. Kasumov, H. Huang, Y.-M. Chung, R. Zhang, A. J. McCullough, J. P. Kirwan, Anal. Biochem. 2010, 401, 154 – 161.
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Azide-Tagged Sphingolipids: New Tools for Metabolic Flux Analysis Mara Garrido,[a] Jos Luis Abad,[a] Gemma Fabris,[a] Josefina Casas,*[a] and Antonio Delgado*[a, b] In memory of Professor Robert Bittman.
fecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.
Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, w-azidosphingolipids 1–3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)-Garner’s aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC-27 cells showed that probes 1–3 compete with the natural metabolites and are incorporated into sphingolipid pathways without af-
Introduction domics,[11, 12] as well as the use of isobaric tags (iTRAQ reagents)[13] have emerged as such procedures. On the other hand, the MS analysis of labeled sphingolipidomes for metabolic studies relies on the individual analysis of samples from cells labeled with non-natural probes.[14] The laborious preparation of individual samples and the cost of using equipment over long hours would be reduced with procedures for massive analysis, which would facilitate fluxomics studies. The recent advances in the development of bioorthogonal reactions have boosted their applications in chemical biology.[15, 16] In particular, those involving azide–alkyne cycloadditions (click chemistry) allow the selective labeling of target biomolecules within the complex environments of live cells and cell extracts, helping the study of live systems at the molecular level. In this context, although click chemistry has been extensively used in chemical biology, only a few studies on lipids have been reported. Bioorthogonal processes applied to alkyne-functionalized fatty acids[17–19] and lipids[20] have recently been described, and a very recent work addresses the application of click chemistry to introduce isobaric tags for the analysis of lactosylceramides.[21] On the basis of the above premises and our previous work on the use of wN3-sphingolipids in artificial membrane studies,[22] we envisaged the use of the wN3-sphingoid bases 1–4 as precursors of chemically taggable entities for the identification of sphingolipid populations in UPLC-MS analysis of pooled samples. Thus, probes 1, 2, and 4 can be regarded as tagged sphingosine (So), sphinganine (Sa, also known as dihydrosphingosine), and ceramide (Cer), respectively, whereas probe 3 is a tagged analogue of ketosphinganine, the first metabolic precursor in the de novo sphingolipid biosynthesis.[23] Thanks to the small size of the azide group, the probes would be expect-
Sphingolipids (SLs) are bioactive molecules that play important roles in signal transduction and as second messengers in a variety of processes including, inter alia, proliferation, differentiation, migration, apoptosis, senescence, autophagy, angiogenesis, etc.[1–3] Because of their physiological importance, the accurate spatiotemporal description of the complete SL profile—the socalled sphingolipidome—within a cell or tissue has attracted much attention in the last years. This information is valuable for correlating sphingolipid metabolites and their amounts with phenotypic changes observed under different conditions and in different experimental setups. Several analytical approaches to the quantification of SLs, including some based on GC[4] and HPLC,[5–7] have been described in the literature. In addition, thanks to advances in mass spectrometry, this technique has become, in its multiple variants, the method of choice for the quantitative measurement of SLs.[8–10] However, despite the advances in MS, alternative methods easily adaptable to high-throughput formats are still required. Shotgun lipi[a] Dr. M. Garrido, Dr. J. L. Abad, Prof. G. Fabris, Dr. J. Casas, Prof. A. Delgado Research Unit on BioActive Molecules Department of Biomedicinal Chemistry Institute for Advanced Chemistry of Catalonia (IQAC-CSIC) Jordi Girona 18–26; 08034 Barcelona (Spain) E-mail: [email protected] [email protected] [b] Prof. A. Delgado University of Barcelona (UB), Faculty of Pharmacy Department of Pharmacology and Medicinal Chemistry Unit of Pharmaceutical Chemistry (Associated Unit to CSIC) Avga. Joan XXIII s/n, 08028 Barcelona (Spain) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402649.
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Full Papers moderate yield and with excellent E selectivity, as judged from the 1H NMR spectrum of the crude reaction mixture. Treatment of 7 with excess NaN3 in DMF at 80 8C, followed by the simultaneous cleavage of the oxazolidine and N-Boc groups under acidic conditions, afforded probe 1 (RBM2–31) in excellent yield. Acylation of 1 with n-octanoic acid, with use of EDC/ HOBt as coupling system, gave probe 4 (RBM2–37). On the other hand, synthesis of probes 2 (RBM2–40) and 3 (RBM2–63) required the addition of undec-10-yn-ol (dilithium salt) to (S)-Garner’s aldehyde (5), followed by catalytic hydrogenation of the intermediate alkyne to afford the diol 8. Selective tosylation of the terminal hydroxy group in 8 and subsequent treatment of the intermediate tosylate 9 with excess NaN3 afforded azido alcohol 10. Oxidation of the secondary alcohol in 10 and acidic deprotection of the amino alcohol moiety in the resulting ketone 11 led to the expected probe 3, whereas probe 2 (RBM2–40) could easily be obtained in good overall yield by direct deprotection of 10 under acidic conditions (Scheme 1). Finally, the synthesis of tags 15–19 was carried out by acylation of a homologous series of linear alkylamines (ethylamine to n-pentylamine) with the NHS-activated precursor 14 (Scheme 2).
ed to be good competitors for the natural compounds (Figure S1 in the Supporting Information), becoming incorporated into SL metabolic pathways to afford wN3-tagged sphingolipid metabolites amenable to bioorthogonal tagging by click chemistry methods. Here we report on the synthesis of wN3tagged sphingoid bases as a proof of concept that this technology can foster high-throughput analysis of sphingolipidomes in metabolic flux experiments.
Results and Discussion Synthesis of the probes and tags
Metabolization of probes
The synthesis of the probes is described in Scheme 1. Crossmetathesis[24, 25] of 11-bromoundec-1-ene and vinyl alcohol 6, obtained from Garner’s aldehyde 5 (S)-Garner’s aldehyde (5) according to a reported protocol,[26] afforded bromide 7 in
In order to confirm the incorporation of probes into the SL metabolic pathways, HGC-27 cells were incubated with probes 1–3 at 5 mm for different time periods, and the resulting metabolites were analyzed by UPLC-TOF. Cell viability was not
Scheme 1. Reagents and conditions; a) see ref. [26]; b) 11-bromoundec-1-ene, second-generation Grubbs catalyst, CH2Cl2, 45 8C, 59 %; c) NaN3, DMF, 80 8C (12, 93 %; 10, 76 %); d) ClCOCH3, MeOH, RT, 1 h (1, 84 %; 2, 85 %; 3, 90 %); e) C7H15COOH, EDC, HOBt, Et3N, CH2Cl2, 59 %; f) undec-10-yn-1-ol, BuLi, HMPA, THF, 78 8C, 50 %; g) H2 (1 atm), Rh catalyst, MeOH (89 %); h) TsCl, DMAP, Et3N, CH2Cl2, RT, 58 %; i) PCC, CH2Cl2, 92 %.
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Scheme 2. Synthesis of tags 15–19 and their reactions with probe 4 (RBM2–37). Single regioisomers for adducts 20–24 are represented. The general reaction of the tags with wN3 metabolites is also shown. a) DIPEA, DMF, RT, n = 1–4; b) MeOH, 37 8C, 18 h.
matic reduction of azides is known to take place in the presence of monothiols (such as glutathione) at alkaline pH values. However, the reported reduction rates under physiological conditions[27, 28] are irrelevant on the timescale of our experiments. Because probes 1–3 are incorporated into SL metabolism, competition with their natural metabolic intermediates was expected. Consistently with this, cells incubated with probes 2 and 3 accumulated Sa without showing any major impact on other dhSL’s pools (Figure 2 C and D). The accumulation of Sa is consistent with wN3Sa—either exogenously added (probe 2) or formed by reduction of 3—replacing natural Sa in sphingolipid metabolism, with sphingosine kinases being a likely target (significant amounts of wN3SaP are produced from wN3Sa, Figure 1). Unexpectedly, probe 1 did not provoke the anticipated increase in natural So (Figure 2 B). However, whereas levels of Sa in vehicle-treated cells are below the detection limit (Figure 2 A), around 10 pmol of Sa are formed in cells treated with 1 at 3 and 6 h (Figure 2 B). Furthermore, probe 1 caused a 2.5to fourfold increase in dhCers over vehicle-treated cells (Figure 2 B). Increases in Sa and dhCers on treatment with sphingosine are unprecedented, and the reasons why 1 (an wN3 analogue of sphingosine) provokes Sa and dhCers buildup are so far unknown. As a possible explanation, HGC27 cells might rapidly acylate exogenous probe 1 to form the corresponding N3labeled Cer derivatives (their formation is evident as early as 1 h after treatment; see Figure 1), and this, in turn, might lead to an increase in their metabolic precursors dihydroceramides and sphinganine through a negative feedback control. Finally, probes 1–3 did not provoke any significant change in sphingolipids derived from So (Figure S4). Collectively, the above results validate the suitability of probes 1–3 as tracers for study of SL metabolism.
compromised under these assay conditions (Figure S2). As shown in Figure 1, the three wN3-bases were metabolized into the sphingoid base phosphates (wN3SoP from 1 and wN3SaP from 2 and 3), together with different N-acyl amides (wN3Cers from 1–3 and wN3dhCers from 2 and 3) and their phosphocholine derivatives (wN3SMs from 1–3 and wN3dhSMs from 2 and 3). Unlike in the cases of probes 1 and 2, the levels of probe 3 (wN3ketoSa) could not be monitored because its exact mass (285.2291 [M+H] + ) was identical, and its retention time very similar, to those of its azide-labeled sphingosine metabolite (probe 1). No peak was detected by selection of this exact mass at the expected retention time in chromatograms from cells treated with probe 3, whereas large amounts of wN3Sa were found. This result suggests that exogenous wN3ketoSa is immediately reduced to wN3Sa by 3-ketosphinganine reductase. With regard to the different N-acyl species, the three probes gave a distribution of wN3Cers resembling that of natural Cers (Figure S3). It is worth noting that wN3 monohexosyl(dihydro)ceramides were detected at very low levels (less than 5 % of total wN3 metabolites) at all time points. To rule out the possibility that the wN3-probes are poorly incorporated into the glycosylation pathway, incubations with C17:Sa were carried out. Glycosylation also proved to be very low from this precursor [mean % SD of total metabolites at 6 h (n = 3): ceramide monohexosides, 3.7 0.4; dihydroceramide monohexosides, 1.3 0.1]. The low levels of glycosphingolipids formed from the exogenous bases suggest either low glycosyltransferase (synthesis) activities, high glycosylhydrolase (recycling) activities, or a very active synthesis/recycling pathway in HGC27 cells under the experimental conditions of the assays. Importantly, no metabolites arising from the putative enzymatic reduction of the terminal azide groups were detected. The enzyChemBioChem 2015, 16, 641 – 650
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Figure 1. Metabolization of probes 1–3 in HGC7 cells. Cells were treated with the compounds (5 mm) for different time periods. SLs were extracted and analyzed as detailed in the Experimental Section. Data are given as pmol equivalents to: C17Sa for wN3Sa and wN3So, C17SaP for wN3SaP and wN3SoP, N-C12Cer for wN3dhCer and wN3Cer, and N-C12SM for wN3dhSM and wN3SM. Data each correspond to the mean SD of one representative experiment in triplicate.
Figure 2. Effects of probes 1, 2, and 3 on the dihydrosphingolipidome in HGC-27 cells treated with the compounds (5 mm) for different time periods. SLs were extracted and analyzed as detailed in the Experimental Section. Data each correspond to the mean SD of one representative experiment in triplicate.
The biocompatible Cu-free, strain-promoted alkyne–azide cycloaddition (SPAAC)[15, 29, 30] was chosen for the labeling of the wN3 metabolites with azadibenzocyclooctyne tags 15–19. The robustness of the resulting triazole systems, together with the natures of the amide groups in the above tags, offer excellent chemical stability under the alkaline conditions required for sample extraction prior to the sphingolipidome analysis (see the Experimental Section). In addition, the use of a Cu-free system allows a cleaner extraction protocol and avoids potential interference in the subsequent MS analysis. Preliminary studies: The reactivities of tags 15–19 were first examined at the analytical level with wN3ceramide 4 (RBM2– 37) as metabolite model (Scheme 2 and Table S1). Compound 4 was used at a concentration within the range of that expected in cell assays (150 pmol), tags were used at 50-fold
Applications of the w-azido probes for LC/MS analysis of sphingolipids in pooled samples Our procedure for studying SL metabolism by use of wN3 probes for metabolic labeling and further UPLC-MS analysis of tagged SL metabolites in population pools is depicted in Figure 3. The methodology involves a step consisting of metabolic labeling with the probe of choice, followed by a step consisting of differential tagging of SL from each cell population, after which the experimental groups are pooled and processed as a single sample. Although simultaneously analyzed, the sphingolipidomes corresponding to each cell population are unambiguously identified by the characteristic masses conferred by their tags. ChemBioChem 2015, 16, 641 – 650
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Full Papers (Figure 1). For an efficient click reaction, and because uptake of probes is about 1/10 of the administered amount, tags were added in excess (4 , 20 nmol) prior to lipid solubilization and allowed to react overnight at 48 8C. After lipid extraction, unreacted wN3SL metabolites turned out to be null or negligible (< 1.5 % relative to control). Moreover, wN3SLs were efficiently derivatized to the corresponding click adducts with tags 15–19, as shown in Figure 4 for the corresponding wN3C16Cer and wN3C16SM metabolites. The unambiguous profile observed for each tagged species by use of this analytical procedure should be pointed out. The retention times and the calculated and found high-resolution masses for all identified metabolites with the corresponding tags 15–19 are indicated in Table S2. Analysis of pooled samples: After the above experiments, we considered the feasibility of metabolic labeling with wN3 probes and further tagging with cyclooctynes for the analysis of pooled samples. As a proof of concept, we determined the effect of Figure 3. Simultaneous UPLC-MS analysis of SLs tagged through click reactions. T1–Tn fumonisin B1 (FB1), a validated inhibitor of ceramide represent different treatment conditions for each cell population. Theoretically, the number of cell populations can be as large as the number of available tags. synthases,[31] on the metabolization of probe 1. To this end, three populations of HGC-27 cells were independently treated with vehicle (control, populamolar excess (7.5 nmol), and long reaction times (18 h, 37 8C, tion 1) and with FB1 at 25 mm (population 2) and at 50 mm MeOH) were allowed to ensure the total consumption of the (population 3) for 24 h. After this period, probe 1 (5 mm) was starting azide. Although click reactions with tags 15–19 were added to each cell population, and incubation was carried out expected to produce 1:1 mixtures of regioisomeric 1,2,3-triafor an additional 1 h. This incubation time afforded suitable zoles, only single peaks for the triazole adducts 20–24 were detected in all cases. Additionally, no starting azide 4 was ever detected after the click reaction, an indication of its quantitative transformation. Interestingly, the triazole adducts were detected with an approximately eightfold higher sensitivity than the starting probe 4, as indicated by the relative peak areas shown in Table S1. This is of analytical interest because the sample size can be reduced. After demonstrating the occurrence of the click reaction of pure 4 to similar extents with the different tags on the lowmm scale, we next used probe 1 (5 mm, 6 h) as a model to evaluate the reaction of its w-N3SL metabolites with tags 15–19 in cell extracts. A 6 h incubation Figure 4. Formation of SL adducts with tags 15–19. Traces correspond to representative UPLC-TOF traces corretime was considered approprisponding A)–E) to C16-w-N3Cers, and F–J) to C16-w-N3SMs from cells incubated with probe 1 for 6 h and further ate in light of the abundant derivatized with tags A), F) 15, B), G) 16, C), H) 17, D), I) 18, and E), J) 19. The retention times are indicated next to metabolization observed for each peak, and the exact masses of A)–E) the C16-wN3Cer adducts, and F)–J) the C16-wN3SM adducts are shown 1 under these conditions in italics on the right. ChemBioChem 2015, 16, 641 – 650
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Full Papers
Figure 5. Pooled sample analysis of the effect of FB1 on the metabolism of probe 1. HGC-27 cells were incubated for 24 h without (vehicle) or with FB1 at 25 or 50 mm, followed by 1 h incubation with probe 1 (5 mm). Cells were finally treated with tags 15, 16, and 17, followed by on-plate extraction and sample pooling as detailed in the Experimental Section. A) Pooled sample UPLC-TOF traces corresponding to the C16-wN3Cer adducts with tags 15 (940.6639, control sample), 16 (954.6796 25 mm FB1 sample), and 17 (968.6952, 50 mm FB1 sample). B) Partial mass spectrum (from 935 to 975 amu) corresponding to the 6:15– 6:80 min range of the total ion chromatogram showing the presence and abundance of the C16-wN3Cer adducts with tags 15 (940.6665, control sample), 16 (954.6771), and 17 (968.6995). C), D) Amounts of produced wN3SLs adducts, given as pmol equivalents with respect to the standard mix (Experimental Section). Data correspond to the mean SD of one representative experiment (experiments performed in quadruplicate).
Conclusion
levels of wN3Cer and low metabolization to wN3SM. Cell populations were next independently treated with a specific tag (tag 15 for population 1, tag 16 for population 2, and tag 17 for population 3) and allowed to react for 2 h. Samples were then pooled, processed, and analyzed. As would be expected for FB1, wN3Cer levels in populations 2 and 3 were substantially reduced, as evidenced (Figure 5 A–C) by the lower concentrations of the corresponding click adducts with tags 16 (80 7 pmol equiv) and 17 (43 6 pmol equiv), respectively, relative to the control population, which contained wN3Cer adducts of tag 15 (249 14 pmol equiv). Similarly, wN3SM levels were also diminished, whereas those of wN3So and wN3SoP were increased, especially in population 3 (50 mm FB1; wN3So: 2973 342 pmol equiv for tag 17 adducts vs. 1549 149 pmol equiv for tag 15 adducts; wN3SoP: 1038 161 pmol equiv for tag 17 adducts vs. 625 90 pmol equiv for tag 15 adducts). A control experiment, in which all three populations were treated with vehicle, followed by probe 1, and further tagged with cyclooctynes 15, 16, and 17, was performed in parallel. Analysis of the pooled control samples showed similar levels of each of the different wN3SL metabolites adducts (Figure 5 D). These results provide the proof of concept that wN3-sphingoid bases, and probably other wN3SLs, are interesting tools for the metabolic labeling and later tagging of metabolites for pooled sample HPLC/MS analysis.
In summary, w-azidoSLs 1–3 have been synthesized and shown to be well incorporated into sphingolipid metabolic pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited for the mass spectrometric analysis of sphingolipidomes in pooled samples originating from different cell populations after individual tagging by treatment with homologous dibenzocyclooctynes. Essentially, the method is not intended to determine actual amounts of metabolites, but for use in identifying differences between different experimental groups. This is easily achieved with this methodology, which combines all the experimental groups in one sample, thus reducing the time of sample processing, the equipment costs of use, and the experimental errors originating from manipulation of individual samples. The development of a large collection of appropriate cyclooctyne tags, as well as that of computational algorithms for the automatic analysis of tagged metabolites, should allow the procedure to be expanded to pools containing large numbers of metabolite populations.
Experimental Section General remarks: Solvents were distilled prior to use and, if required, dried by standard methods. 1H and 13C NMR spectra were
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Full Papers obtained in CDCl3 solutions at 400 MHz (for 1H) and 100 MHz (for 13 C), unless otherwise indicated. Chemical shifts (d) are reported in ppm relative to the solvent (CHCl3 or CDCl3) signal. Multiplets are indicated by the chemical shift corresponding to the center of the peak pattern. All reactions were monitored by TLC on aluminum foil precoated silica gel plates. Flash column chromatography was performed with the indicated solvents and use of silica gel 60 (particle size 0.035–0.070). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Optical rotations were measured at the sodium D line (589 nm) and are reported as [a]D = [c, (g per 100 mL), solvent]. Electrospray ionization was used for HRMS analysis. HPLC was performed with a Supelcosil LC-18 column (25 cm 4.6 mm i.d., 5 mm particle size) and a HPLC system equipped with a 515 HPLC pump, a 2545 binary gradient module, a 3100 mass detector, and a 2998 photodiode array detector. Solvent A (water) and solvent B (acetonitrile) were used as eluents. The samples were eluted under the following logarithmic gradient conditions: 0.0 min, 50 % B; 6.0 min, 90 % B; followed by an isocratic elution: 6.1 to 20.0 min, 90 % B. After each run, the column was thoroughly washed with acetonitrile and equilibrated with the initial conditions. The mobile phase was degassed and pumped at a flow rate of 1.0 mL min 1. The injection volume was set at 20 mL. UPLC-TOF was carried out with a Waters Acquity UPLC system coupled to a Waters LCT Premier orthogonal accelerated time-of-flight mass spectrometer (Waters). The samples were injected onto a reversed-phase C18 column (1.7 mm C18 Acquity UPLC BEH, 2.1 100 mm, Waters) at 0.3 mL min 1. Solvent A [methanol/formic acid (99.8:0.2, v/v) with ammonium formate (1 mm)]; solvent B [water/formic acid (99.8:0.2, v/v) with ammonium formate (2 mm)]. Two different sets of gradient conditions were used: gradient 1: 0.0 min, 65 % A; 8.0 min, 90 % A; 13.0 min, 99 % A; 15.0 min, 99 % A; 18.0 min, 65 % A; gradient 2: 0.0 min, 80 % A; 3.0 min, 90 % A; 15.0 min, 99 % A; 18.0 min, 99 % A. The mass spectrometer was run in the positive electrospray ionization mode. Full scan spectra from 50 to 1500 Da were acquired, and individual spectra were summed to produce data points each 0.2 s. Mass accuracy and reproducibility were maintained by using an independent reference spray by the LockSpray interference. The column was held at 30 8C. The injection volume was set at 10 mL. Positive identification of compounds was based on accurate mass measurement with an error < 5 ppm. Azadibenzocyclooctyne 14 was purchased from Click Chemistry Tools (http://www.clickchemistrytools.com). A “standard mix” for SL quantification was prepared by mixing 200 pmol of each of the following standards: C17So, C17SaP, NC12Cer, NC12SM. HGC-27 cells were obtained from Health Protection Agency Culture Collections (Salisbury, UK).
(2S,3R)-2-Amino-14-azidotetradecane-1,3-diol (2, RBM2–40): wN3 Aminodiol 2 (RBM2–40, 79 mg, 0.27 mmol, 85 %) was obtained according to the procedure described for compound 1, as a waxy solid, from compound 10 (137 mg, 0.32 mmol) and acetyl chloride (600 mL) in MeOH (10 mL). Purification was carried out by flash chromatography (CH2Cl2/MeOH/NH3 8:1.9:0.1). [a]D = +3.9 (c = 0.9, CHCl3); 1H NMR (400 MHz, CDCl3): d = 3.70 (2 H; NH2), 3.60 (m, 1 H), 3.25 (appt t, 2 H), 2.83 (br, 1 H), 2.33 (br, OH), 1.59 (m, 2 H), 1.45 (m, 2 H), 1.40–1.24 ppm (m, 18 H); 13C NMR (101 MHz, CDCl3): d = 74.6, 63.5, 55.8, 51.6, 33.9, 29.8–29.6 (five peaks), 29.2, 28.9, 26.8, 26.2 ppm; HRMS: m/z calcd for C14H31N4O2 : 287.2442 [M+H] + ; found: 287.2442. (S)-2-Amino-14-azido-1-hydroxytetradecan-3-one hydrochloride (3, RBM2–63): Ketone 3 (RBM2–63, 30 mg, 0.09 mmol, 90 %) was obtained according to the procedure described for compound 1, as a yellow pale wax, from 11 (41 mg, 0.10 mmol) and acetyl chloride (300 mL) in MeOH (5 mL) and used without further purification. [a]D = +23.9 (c = 0.6, CHCl3); 1H NMR (400 MHz, CD3OD): d = 4.16 (t, J = 4.0, 1 H), 4.10 (dd, J = 12.0, 4.3 Hz, 1 H), 3.97 (dd, J = 12.0, 3.4 Hz, 1 H), 3.30 (t, J = 7.0 Hz, 2 H), 2.64 (t, J = 7.2 Hz, 2 H), 1.67–1.54 (m, 4 H), 1.41–1.28 ppm (m, 18 H); 13C NMR (101 MHz, CD3OD): d = 205.2, 62.2, 60.3, 52.4, 39.6, 30.6–30.5 (four peaks), 30.3, 30.1, 29.9, 27.8, 24.2 ppm; HRMS: m/z calcd for C14H29N4O2 : 285.2285 [M+H] + ; found: 285.2278. N-[(2S,3R,E)-14-Azido-1,3-dihydroxytetradec-4-en-2-yl]octanamide (4, RBM2–37): A solution of octanoic acid (65 mL, 0.4 mmol) in anhydrous CH2Cl2 (2 mL) was added dropwise to a solution of EDC (105 mg, 0.60 mmol) and HOBt (60 mg, 0.44 mmol). The mixture was stirred for 10 min under Ar and next added dropwise to a solution of amine 1 (RBM2–31, 105 mg, 0.37 mmol) and Et3N (200 mL, 1.44 mmol) in anhydrous CH2Cl2 (2 mL). The reaction mixture was stirred for 1 h under argon, diluted with CH2Cl2 (10 mL), and washed, successively, with H2O (10 mL) and brine (10 mL). The organic layer was worked up as usual to give a residue, which, after flash column chromatography (CH2Cl2/MeOH 0–5 %), afforded amide 4 (RBM2–37, 89 mg, 0.22 mmol, 59 %) as a white solid. [a]D = 3.6 (c = 1.13, CHCl3); 1H NMR (400 MHz, CDCl3): d = 6.35 (d, J = 6.5 Hz, 1 H), 5.76 (dt, J = 15.6, 6.8 Hz, 1 H), 5.51 (dd, J = 15.6, 6.4 Hz, 1 H), 4.28 (br, 1 H), 3.90 (m, 1 H), 3.88 (br, 1 H), 3.68 (br d, 1 H), 3.25 (t, 1 H; OH), 2.90 (br, OH), 2.21 (t, J = 7.6 Hz, 2 H), 2.04 (appt q, 2 H), 1.67–1.57 (m, 4 H), 1.41–1.21 (br, 22 H), 0.87 ppm (t, J = 7.2 Hz, 3 H); 13C NMR (101 MHz, CDCl3): d = 174.2, 134.2, 128.9, 74.5, 62.5, 54.65, 51.6, 36.9, 32.4, 31.8, 29.5–28.9 (nine peaks), 26.8, 25.9, 22.7 ppm; HRMS: m/z calcd for C22H42N4NaO3 : 433.3149 [M+Na] + ; found: 433.3153. tert-Butyl (S)-4-[(R,E)-12-bromo-1-hydroxydodec-2-en-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (7): The second-generation Grubbs catalyst (100 mg, 0.12 mmol) was added portionwise to a degassed solution of vinyl alcohol 6[26] (730 mg, 2.80 mmol) and 11-bromoundec-1-ene (2.6 g, 11.20 mmol) in CH2Cl2 (20 mL). The reaction mixture was stirred for 5 h at reflux under Ar. The solvent was next removed in vacuo, and the resulting residue was flash chromatographed (hexane/AcOEt 0–27 %) to give 7 (770 mg, 1.66 mmol, 59 %) as a colorless oil. [a]D = 26.2 (c = 1.04, CHCl3); 1 H NMR (mixture of rotamers, 400 MHz, CDCl3): d = 5.72 (dt, J = 15.2, 7.2 Hz, 1 H), 5.44 (dd, J = 15.2, 6.0 Hz, 1 H), 4.23–3.77 (m, 4 H), 3.41 (t, J = 6.9 Hz, 2 H), 2.04 (m, 2 H), 1.85 (m, 2 H), 1.55–1.33 (m, 12 H), 1.45 (s, 6 H), 1.27 ppm (br, 9 H); 13C NMR (major rotamer, 101 MHz, CDCl3): d = 154.3, 133.4, 128.2, 94.5, 81.1, 74.1, 65.0, 62.4, 34.2, 32.9, 32.5, 29.3–28.4 (eight peaks), 28.24, 26.4, 24.7 ppm; HRMS: m/z calcd for C22H40BrNNaO4 : 484.2033 [M+Na] + ; found: 484.2037.
Chemistry (2S,3R,E)-2-Amino-14-azidotetradec-4-ene-1,3-diol (1, RBM2–31): Acetyl chloride (900 mL, 6 % volume) was added to a solution of 12 (335 mg, 0.80 mmol) in MeOH (15 mL). The reaction was complete in 1 h, as monitored by TLC (hexane/AcOEt 1:1). Methanol was removed in vacuo, and the resulting residue was purified by flash column chromatography (CH2Cl2/MeOH/NH3 9:0.9:0.1) to give wN3 aminodiol 1 (RBM2–31, 190 mg, 0.67 mmol, 84 %) as a white solid. [a]D = 1.2 (c = 0.47, CHCl3); 1H NMR (400 MHz, CDCl3): d = 5.72 (dt, J = 15.6, 6.8 Hz, 1 H), 5.44 (dd, J = 15.6, 7.1 Hz, 1 H), 4.03 (t, J = 6.2 Hz, 1 H), 3.64 (2 H; NH2), 3.24 (m, 2 H), 2.77 (br, 1 H + 2 OH), 2.05 (appt q, 2 H), 1.58 (m, 2 H), 1.41–1.22 ppm (m, 14 H); 13C NMR (101 MHz, CDCl3): d = 134.6, 129.4, 75.2, 63.8, 56.2, 51.6, 32.4, 29.5– 29.1 (five peaks), 28.9, 26.8 ppm; HRMS: m/z calcd for C14H29N4O2 : 285.2285 [M+H] + ; found: 285.2274.
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Full Papers of rotamers): d = (208.9, 208.4), (152.2, 151.6), (95.3, 94.6), (81.0, 80.7), (65.9, 65.5), (65.5, 65.3), 51.6, (39.2, 38.6), 29.7–29.4 (10 peaks), (28.9, 28.5), (26.3, 25.5), (24.9, 23.8), (23.2, 23.1 ppm); HRMS: m/z calcd for C22H40N4NaO4 : 447.2942 [M+Na] + ; found: 447.2957.
tert-Butyl (S)-4-[(R)-1,12-dihydroxydodecyl]-2,2-dimethyloxazolidine-3-carboxylate (8): Rhodium on alumina (Sigma–Aldrich, ref. 212 857, 27 mg) was added portionwise to a solution of starting compound 13 (150 mg, 0.38 mmol) in freshly degassed MeOH (10 mL). The mixture was vigorously stirred at room temperature for 6 h under H2 (1 atm) and filtered through a plug of Celite. The solid was rinsed thoroughly with MeOH (3 3 mL), and the combined filtrates were concentrated in vacuo to afford a crude mixture, which was used without further purification. [a]D = 11.0 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, mixture of rotamers): d = 4.66–3.11 (m, 4 H), 3.62 (t, J = 6.6 Hz, 2 H), 1.61–1.19 (br, 20 H), 1.52 (s, 6 H), 1.31 ppm (s, 9 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.7, 94.3, 81.1, 73.0, 64.8, 63.0, 62.4, 32.8, 29.7–29.5 (nine peaks), 28.5, 26.5, 26.2, 25.8, 24.3 ppm; HRMS: m/z calcd for C22H44NO5 : 402.3214 [M+H] + : found: 402.3219.
tert-Butyl (S)-4-[(R,E)-12-azido-1-hydroxydodec-2-en-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (12): NaN3 (310 mg, 4.78 mmol) was added portionwise to a solution of 7 (550 mg, 1.19 mmol) in anhydrous DMF (20 mL). The mixture was heated at 80 8C and stirred overnight under Ar. After the system had cooled down to room temperature, H2O was added (100 mL), and the aqueous layer was extracted with Et2O (3 50 mL). The combined organic extracts were dried with MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography (hexane/ AcOEt 0–20 %) to afford 12 (470 mg, 1.11 mmol, 93 %) as a colorless oil. [a]D = 30.86 (c = 0.93, CHCl3); 1H NMR (400 MHz, CDCl3, major rotamer): d = 5.73 (dt, J = 15.2, 7.2, 7.2 Hz, 1 H), 5.44 (dd, J = 15.2, 5.2 Hz, 1 H), 4.22–3.76 (m, 4 H), 3.25 (t, J = 7.0 Hz, 2 H), 2.04 (m, 2 H), 1.63–1.45 (m, 16 H), 1.41–1.24 ppm (m, 13 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 133.5, 128.5, 94.6, 81.2, 74.2, 65.1, 62.4, 51.6, 32.5, 29.5–29.2 (eight peaks), 29.0, 28.5, 26.8, 26.4, 24.7 ppm; HRMS: m/z calcd for C22H40N4NaO4 : 447.2942 [M+Na] + ; found: 447.2960.
tert-Butyl (S)-4-[(R)-1-hydroxy-12-(tosyloxy)dodecyl]-2,2-dimethyloxazolidine-3-carboxylate (9): A solution of 8 (136 mg, 0.34 mmol) and Et3N (47 mL, mmol) in dry CH2Cl2 (7 mL) was added dropwise under Ar to a solution of tosyl chloride (65 mg, 0.34 mmol) and DMAP (21 mg, 0.17 mmol) in dry CH2Cl2 (3 mL). After vigorous stirring at RT for 8 h, the reaction mixture was diluted by addition of CH2Cl2 (10 mL). The organic layer was washed with brine (2 10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the resulting residue by flash chromatography (hexane/EtOAc gradient 10:0 to 5:5) gave tosylate 9 (110 mg, 0.20 mmol, 58 %) as a colorless wax. [a]D = 6.7 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3, mixture of rotamers): d = 7.78 (d, J = 8.2 Hz, 2 H), 7.34 (d, J = 8.0 Hz, 2 H), 4.00 (t, 2 H), 4.11– 3.69 (m, 4 H), 2.44 (s, 3 H), 1.70–1.53 (m, 6 H), 1.48 (s, 6 H + broad signal 6 H), 1.20 (s, 9 H), 1.34–1.16 ppm (m, 8 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.2, 144.7, 133.3, 129.9, 128.0, 94.3, 81.1, 73.1, 70.8, 64.8, 62.5, 32.9, 29.9–28.5 (12 peaks), 26.5, 26.2, 25.4, 24.3, 21.7 ppm; HRMS: m/z calcd for C29H50NO7S: 556.3303 [M+H] + ; found: 556.3325.
tert-Butyl (S)-4-[(R)-1,12-dihydroxydodec-2-yn-1-yl]-2,2-dimethyloxazolidine-3-carboxylate (13): A solution of nBuLi (1.29 mL, 3.22 mmol, 2.5 m in hexanes) was added dropwise to a solution of undec-10-yn-1-ol (225 mg, 1.34 mmol) in anhydrous THF (15 mL) at 20 8C. After the system had been vigorously stirred at 20 8C for 30 min, neat HMPA (350 mL, 2.01 mmol) was added dropwise. The reaction mixture was cooled to 78 8C, followed by dropwise addition of a solution of aldehyde 5[32] (236 mg, 1.03 mmol) in anhydrous THF (2 mL). After stirring at 78 8C for 1 h, the reaction mixture was allowed to warm to 20 8C over around 2 h, and next quenched by addition of saturated aqueous NH4Cl (10 mL). After dilution by addition of water, the aqueous phase was extracted with Et2O (3 20 mL). The combined organic extracts were successively washed with HCl (0.5 n) and brine, dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash chromatography (hexane/EtOAc 10:0 to 6:4) to afford compound 13 (205 mg, 0.52 mmol, 50 %) as a colorless oil. [a]D = 30.3 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3): d = 4.50 (br, 1 H), 4.15– 3.85 (m, 3 H), 3.61 (t, J = 6.6 Hz, 2 H), 2.17 (t, J = 7.1 Hz, 2 H), 1.63– 1.51 (m, 4 H), 1.51 (s, 6 H), 1.49 (s, 9 H), 1.38–1.21 ppm (m, 10 H); 13 C NMR (101 MHz, CDCl3): d = 154.2, 95.0, 86.7, 81.3, 78.0, 65.1, 64.1, 63.0, 62.8, 32.8, 29.5, 29.4, 29.0, 28.8, 28.6–28.5 (four peaks), 25.9, 25.8, 25.5, 18.8 ppm; HRMS: m/z calcd for C22H39NNaO5 : 420.2720 [M+Na] + ; found: 420.2712.
tert-Butyl (S)-4-((R)-12-azido-1-hydroxydodecyl)-2,2-dimethyloxazolidine-3-carboxylate (10): NaN3 (52 mg, 0.80 mmol) was added portionwise under Ar to a solution of tosylate 9 (110 mg, 0.20 mmol) in anhydrous DMF (10 mL), and the resulting mixture was stirred overnight at 80 8C. After cooling to room temperature, the reaction mixture was quenched by addition of H2O (50 mL) and worked up as usual to give an oil, which was flash chromatographed (hexane/EtOAc 8:2) to afford azide 10 (65 mg, 0.15 mmol, 76 %). [a]D = 2.6 (c = 0.9, CHCl3); 1H NMR (400 MHz, CDCl3, major rotamer): d = 4.15–3.95 (m, 2 H), 3.95–3.65 (m, 2 H), 3.25 (t, J = 7.0 Hz, 2 H), 1.64–1.56 (m, 6 H), 1.52–1.40 (m, 8 H), 1.48 (s, 6 H), 1.27 (s, 9 H), 1.40–1.22 ppm (m, 8 H); 13C NMR (101 MHz, CDCl3, major rotamer): d = 154.2, 94.3, 81.0, 73.0, 64.8, 62.5, 51.5, 29.7–28.4 (10 peaks), 26.8, 26.5, 26.2, 26.1, 24.3 ppm; HRMS: m/z calcd for C22H43N4O4 : 427.3279 [M+H] + ; found: 427.3293.
General procedure—acylation of azadibenzocyclooctyne 14: The appropriate alkylamine (0.02 mmol) was added under Ar to a solution of commercial azadibenzocyclooctyne 14 (5 mg, 0.010 mmol) and DIPEA (5 mL, 0.025 mmol) in anhydrous DMF (2 mL), and the mixture was stirred at RT overnight. Concentration under reduced pressure gave the crude tag. Tags were flash chromatographed (CH2Cl2/MeOH 100:0 to 96:4) to give 15–19.
tert-Butyl (S)-4-(12-azidododecanoyl)-2,2-dimethyloxazolidine-3carboxylate (11): PCC (72 mg, 0.33 mmol) was added under Ar to a solution of compound 10 (55 mg, 0.13 mmol) in dry CH2Cl2 (4 mL). After vigorous stirring at room temperature overnight, the reaction mixture was filtered through a plug of Celite and concentrated in vacuo. The residue was purified by flash chromatography (hexane/EtOAc 10:0 to 9:1) to afford compound 11 (51 mg, 0.12 mmol, 92 %) as a colorless wax. [a]D = 30.0 (c = 1.0, CHCl3); 1 H NMR (500 MHz, CDCl3, mixture of rotamers): d = 4.44 (dd, J = 7.5, 2.0 Hz, 0.4 H), 4.31 (dd, J = 7.5, 2.5 Hz, 0.6 H), 4.18–4.09 (m, 1 H), 3.93 (dd, J = 9.5, 2.0 Hz, 0.4 H), 3.88 (dd, J = 9.5, 2.5 Hz, 0.6 H), 3.25 (t, J = 7.0 Hz, 2 H), 2.55–2.40 (m, 2 H), 1.60–1.47 (m, 8 H), 1.55 (s, 6 H), 1.41 (s, 9 H), 1.38–1.24 ppm (br, 10 H); 13C NMR (101 MHz, CDCl3, mixture ChemBioChem 2015, 16, 641 – 650
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N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5ethyl glutaramide (15): Compound 15 (tag 1, 3.1 mg, 74 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol), and ethylamine (1.6 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (d, J = 6.9 Hz, 1 H), 7.52–7.45 (m, 4 H), 7.41–7.32 (m, 2 H), 7.27 (dd, J = 7.4, 1.6 Hz, 1 H), 5.15 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.24–3.13 (m, 4 H), 2.51–2.41 (m, 6 H), 1.74 (m, 2 H), 1.11 ppm (t, J = 7.2 Hz, 3 H); 13C NMR (101 MHz, CD3OD): d = 175.2,
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Full Papers Cell viability: Cells were seeded at a density of 105 cells per well in 96-well plates in fresh medium (100 mL per well) and grown overnight at 37 8C under a water-saturated, 5 % CO2 atmosphere. The medium was next removed and replaced with fresh medium (100 mL) containing the appropriate test compound. Cells were incubated at 37 8C under CO2 (5 %) for 24 h. The number of viable cells was quantified by estimation of dehydrogenase activity, which reduces the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to water-insoluble formazan, which was dissolved in DMSO (100 mL per well) and measured at 570 nm with a Spectramax Plus instrument (Molecular Devices). All compounds were dissolved in EtOH, and control experiments were performed with EtOH (0.1 %). See Figure S2 for results.
173.3, 152.7, 149.4, 133.4, 130.4, 130.0, 129.7, 129.2, 128.9, 128.1, 126.5, 115.7, 100.8, 56.6, 36.7, 36.2, 36.1, 35.5, 35.2, 23.2, 14.8 ppm; HRMS: m/z calcd for C25H27N3NaO3 : 440.1945 [M+Na] + ; found: 440.1965. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5propylglutaramide (16): Compound 16 (tag 2, 3.4 mg, 80 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol), and propylamine (1.3 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CDCl3): d = 7.66 (d, J = 7.3 Hz, 1 H), 7.44–7.27 (m, 7 H), 5.14 (d, J = 13.9 Hz, 1 H), 3.71 (d, J = 13.9 Hz, 1 H), 3.27–3.17 (m, 3 H), 3.27–3.41 (m, 1 H), 2.87 (t, J = 5.6 Hz, 1 H), 2.47 (ddd, J = 16.6, 7.4, 4.0 Hz, 1 H), 2.16 (t, J = 6.8 Hz, 2 H), 2.10–1.94 (m, 3 H), 1.83 (quint, J = 7.2 Hz, 2 H), 1.54 (sext, J = 7.2 Hz, 2 H), 0.93 ppm (t, J = 7.6 Hz, 3 H); HRMS: m/z calcd for C26H29N3NaO3 : 454.2101 [M+Na] + ; found: 454.2106. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5-butylglutaramide (17): Compound 17 (tag 3, 3.3 mg, 80 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and butylamine (1.5 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (d, J = 7.0 Hz, 1 H). 7.52–7.30 (m, 6 H), 7.27 (dd, J = 7.2, 1.6 Hz, 1 H), 5.14 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.28–3.10 (m, 4 H), 2.51–2.42 (m, 1 H), 2.14–1.99 (m, 5 H), 1.74 (quint, J = 7.6 Hz, 2 H), 1.48 (quint, J = 8.0 Hz, 2 H), 1.41–1.33 (m, 2 H), 0.94 ppm (t, J = 7.2 Hz, 3 H); HRMS: m/z calcd for C27H31N3NaO3 : 468.2258 [M+Na] + ; found: 468.2277. N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5pentylglutaramide (18): Compound 18 (tag 4, 3.4 mg, 73 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and pentylamine (1.8 mL, 0.02 mmol) in DMF (2 mL); 1H NMR (400 MHz, CDCl3): d = 7.66 (d, J = 7.2 Hz, 1 H), 7.44–7.27 (m, 7 H), 5.14 (d, J = 13.9 Hz, 1 H), 3.71 (d, J = 13.9 Hz, 1 H), 3.39–3.29 (m, 1 H), 3.26–3.17 (m, 3 H), 2.46 (ddd, J = 16.6, 7.3, 3.8 Hz, 1 H), 2.11–1.95 (m, 5 H), 1.82 (quint, J = 7.2 Hz, 2 H), 1.53–1.45 (m, 2 H), 1.34–1.28 (m, 4 H), 0.90 ppm (t, J = 7.2 Hz, 3 H); HRMS: m/z calcd for C28H33N3NaO3 : 482.2414 [M+Na] + ; found: 482.2412.
Click reactions between wN3 metabolites and tags 15–19 in lipid extracts Standard protocol: HGC-27 cells, prepared as above, were incubated with probe 1 (5 mm) for 6 h. Lipids were extracted from PBSwashed cell pellets by suspending them in water (100 mL), MeOH (500 mL), and CHCl3 (250 mL). The standard mix (200 pmol of a mixture of SL, “standard mix”, see above) and tags 15–19 (20 nmol) were then added. Controls were treated similarly, except that tags were not added. The resulting mixtures were dispersed by sonication, incubated at 48 8C overnight, and analyzed by UPLC-TOF (gradient 1).[33, 34] The retention times and the calculated and found high-resolution masses for all identified metabolites with the corresponding tags 15–19 are given in Figure 4 and Table S2.
N1-[3-(11-Azadibenzo[b,f]cyclooct-5-yn-11-yl)-3-oxopropyl]-N5hexylglutaramide (19): Compound 19 (tag 5, 3.7 mg, 78 %) was obtained as a colorless oil from 14 (5 mg, 0.01 mmol) and hexylamine (2.0 mL, 0.02 mmol) in DMF (2 mL). 1H NMR (400 MHz, CD3OD): d = 7.66 (dd, J = 7.3, 1.2 Hz, 1 H), 7.51–7.30 (m, 6 H), 7.27 (dd, J = 7.4, 1.6 Hz, 1 H), 5.14 (d, J = 14.0 Hz, 1 H), 3.72 (d, J = 14.0 Hz, 1 H), 3.29– 3.20 (m, 1 H), 3.18–3.09 (m, 3 H), 2.51–2.41 (m, 1 H), 2.13–1.98 (m, 5 H), 1.74 (quint, J = 7.2 Hz, 2 H), 1.54–1.43 (m, 2 H), 1.38–1.27 (m, 6 H), 0.91 ppm (t, J = 6.8 Hz, 3 H); HRMS: m/z calcd for C29H36N3O3 : 474.2751 [M+H] + ; found: 474.2753. Click reactions between azido probe 4 (RBM2–37) and tags 15– 19: Probe 4 (150 pmol from 15 mL of a 10 mm MeOH stock solution) was placed in a 7 mL glass vial and treated with the appropriate tag (7.5 nmol, from 15 mL of a 500 mm MeOH solution). The resulting mixture was diluted with MeOH (470 mL), stirred at 48 8C overnight, and next transferred to a UPLC vial. After concentration under a stream of nitrogen, the resulting residue was taken up in MeOH (130 mL) and analyzed by UPLC-TOF (see Table S1)
On-plate click reaction: Cells prepared as above were treated with probe 1 (0.5 mL per well of 10 mm probe 1 solution in EtOH for a final 5 mm concentration) and incubated for 1 h at 37 8C under CO2 (5 %). Then, methanol (1 mL) was added, followed by addition of the internal standards (200 pmol each; see above) and tags 15– 17 (150 nmol). After 2 h of gentle agitation at RT, the solution was transferred into glass tubes and dried in a centrifugal evaporator. The resulting residue was extracted and analyzed as described above. A parallel experiment was performed by use of the above standard protocol. It should be pointed out that under the “onplate” conditions, unreacted w-N3SL metabolites were not detected and that recovery of tagged w-N3SL was equivalent.
Biology Cell culture: The human gastric cancer cell line HGC-27 was maintained at 37 8C under CO2 (5 %) in modified Eagle’s medium supplemented with fetal bovine serum (10 %), non-essential amino acids (1 %), glutamine (1 %), and penicillin and streptomycin (each 100 ng mL 1).
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Metabolization of probes 1–3 and effects on the sphingolipidome in HGC-27 cells: Cells were seeded at 2.5 105 cells mL 1 in 6-well plates (1 mL per well) and grown overnight at 37 8C under CO2 (5 %). The medium was gently aspirated, and fresh medium (1 mL per well) was added again. Cells were treated with a solution (10 mm, 0.5 mL per well) of probes 1–3 in EtOH (final concentration = 5 mm) and incubated for the indicated times at 37 8C under CO2 (5 %). Each compound was tested in triplicate, and EtOH was used as control. After incubation, the medium was gently aspirated, and the cells were washed twice with PBS (1 mL), harvested by trypsinization, counted, and transferred to glass vials. SL standards (200 pmol, see above) were added to the cell pellets, and SLs were extracted and analyzed by UPLC-TOF (gradient 1; see above).[33, 34] Identification of wN3-SL was based on accurate mass measurement (< 5 ppm error), and LC retention time relative to an wN3Cer-C16 standard. Quantification was carried out with the extracted ion chromatogram of each compound, with use of 50 mDa windows. The amounts of wN3SLs adducts are given as pmol equivalents relative to each specific standard (wN3So, C17So; wN3SoP, C17SaP; wN3Cer, NC12Cer; wN3SM, NC12SM).
Analysis of pooled samples: Cells prepared as above were treated with 1 mL/well of fresh medium containing either vehicle or FB1 (25 or 50 mm). After 24 h, a solution of probe 1 in EtOH (10 mm,
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Full Papers 0.5 mL, final concentration 5 mm) was added to each well, and incubation was performed for 1 h at 37 8C under CO2 (5 %). Then, the medium was aspirated, cells were washed (PBS), and MeOH (1 mL) was added, followed by addition of the internal standards (200 pmol; see refs. [30] and [31]) and tag 15, 16, or 17 (150 nmol) to the wells containing cells treated with vehicle or with 25 mm FB1 or 50 mm FB1, respectively. After 2 h of gentle agitation, the solutions were pooled into glass tubes and dried in a centrifugal evaporator. The resulting residue was dissolved in MeOH (150 mL), and 130 mL was transferred to UPLC vials for analysis of tagged wN3SLs by UPLC-TOF (gradient conditions 2) as indicated above. In an experiment carried out in parallel, three cell populations were all treated with vehicle prior to addition of tags 15, 16, and 17 to each one. Pooled samples were prepared as detailed above.
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Abbreviations Cer: ceramide, dhCers: dihydroceramides, dhSM: dihydrosphingomyelin, GlycodhSL: glycodihydrosphingolipids, GlycoSL: glycosphingolipids, ketoSa: ketosphinganine (ketodihydrosphingosine), LacdhCers: lactosyl dihydroceramides, MHdhCers: monohexoxyl dihydroceramides, Sa: sphinganine (dihydrosphingosine), SaP: sphinganine-1-phosphate (dihydrosphingosine-1-phosphate), SM: sphingomyelin, So: sphingosine, SoP: sphingosine-1-phosphate.
Acknowledgements The authors wish to thank Eva Dalmau and Pedro Rayo for their experimental contribution. This work was supported by grants from the Ministry of Science and Innovation (SAF2011-22444) and the Agncia de Gesti d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya (2009SGR1072) to G.F. M.G. was supported by a JAE–predoc fellowship (CSIC). Keywords: azides · bioorthogonality · click chemistry · highthroughput screening · lipidomes · sphingolipids [1] A. Morales, J. C. Fernandez-Checa, Mini-Rev. Med. Chem. 2007, 7, 371 – 382. [2] T. Kolter, Chem. Phys. Lipids 2011, 164, 590 – 606. [3] Y. A. Hannun, L. M. Obeid, Nat. Rev. Mol. Cell Biol. 2008, 9, 139 – 150. [4] C. Vieu, F. Terc, F. Chevy, C. Rolland, R. Barbaras, H. Chap, C. Wolf, B. Perret, X. Collet, J. Lipid Res. 2002, 43, 510 – 522. [5] S. Snada, Y. Uchida, Y. Anraku, A. Izawa, M. Iwamori, Y. Nagai, J. Chromatogr. 1987, 400, 223 – 231. [6] M. Previati, L. Bertolaso, M. Tramarin, V. Bertagnolo, S. Capitani, Anal. Biochem. 1996, 233, 108 – 114. [7] M. Yano, E. Kishida, Y. Muneyuki, Y. Masuzawa, J. Lipid Res. 1998, 39, 2091 – 2098. [8] T. Kasumov, H. Huang, Y.-M. Chung, R. Zhang, A. J. McCullough, J. P. Kirwan, Anal. Biochem. 2010, 401, 154 – 161.
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Received: November 7, 2014 Published online on February 11, 2015
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