Multifunctional sphingomyelin and ceramide

Abstract

The invention introduces novel chemical modifications to sphingomyelin and ceramide molecules, making them readily available for targeted staining and detection techniques, such as click chemistry.

Description

[0001] Multifunctional Sphingomyelin and Ceramide

[0002] Field of the invention

[0003] The invention introduces novel chemical modifications to sphingomyelin and ceramide molecules, making them readily available for targeted staining and detection techniques, such as click chemistry.

[0004] Background of the invention

[0005] Sphingolipids are an essential part of cellular membranes in almost all vertebrates. Major human diseases, such as cancer, diabetes and neurodegenerative disorders and infectious diseases are associated with the sphingolipid metabolism. In addition, dysregulation in the sphingolipid metabolism is responsible for some rare fatal diseases (cf. NPL11).

[0006] In recent years it has been shown that complex sphingolipids like sphingomyelin have a variety of functions in the cell, they are not only important to maintain cell membrane structure, but they also affect transmembrane receptors (cf. NPL12), cell-cell interactions (cf. NPL13) and cellular signaling (cf. NPL14 and NPL15). Smaller sphingolipids, like ceramides, were also shown to be components in different signaling pathways and thereby, like sphingomyelin, play important roles in regulating cell growth, cell death, senescence, apoptosis, adhesion, inflammation and angiogenesis (cf. NPL11, and NPL16 to NPL18).

[0007] To study the role of sphingomyelin and ceramides in different cellular settings, reliable staining and fixing methods are an essential prerequisite. However, common staining methods for example with an antibody coupled to a fluorescent dye have its limitations when it comes to the detection of lipids. In addition, pre-attached bulky fluorophores to sphingomyelin might influence the location of the sphingomyelin in the cell and the activity of some enzymes or are no longer accepted as substrate. To overcome these problems, small clickable tags can be added to the lipid molecules. This is a valuable tool, commonly known as "click chemistry" (cf. NPL19 and NPL20). In click chemistry, the molecule of interest is modified by functional groups, like a terminal -alkyne or -azide group. Notably, these modified molecules are expected to localize to the right endogenous cellular position. They are taken up by the cell and are metabolized by relevant enzymes. They can be stained either in living cells or after fixation. For fixation an additional amino group can be added to the alkyl-side chain of the molecule.

[0008] So far, click chemistry could only be applied to some other molecules of the sphingolipid metabolism, such as sphinganines or sphingosine. As far as ceramides are concerned, ceramides with terminal azide groups or intramolecular amino groups have been synthesized although that modification was limited to ceramides with short alkyl side chains (C6 versus C16) (cf. NPL19 and NPL20).

[0009] The newly synthesized trifunctional sphingomyelins presented here are valuable tools to monitor intracellular sphingomyelin metabolism, trafficking and localization. Several human diseases are associated with the malfunctioning of sphingolipid metabolism. Enzymatic reactions in diseased cells and tissue can be monitored on molecular level and give important clues to further elucidate pathogenic processes. The artificial tags allow the molecules to be coupled to different conjugation partners (e.g. biotin, fluorescent dyes, quencher molecules) and visualized in combination with modern microscopic techniques such as confocal microscopy, fluorescence lifetime imaging microscopy (FLIM), expansion microscopy and Forster resonance energy transfer (FRET). Notably, the molecules are still accepted by specific enzymes (e.g. ASM) thereby allowing the assessment of enzymatic activity.

[0010] Non-patent literature

[0011] NPL 1: J. Lang, P. Bohn, H. Bhat, et al., Nat. Commun. 2020, 11, 1338.

[0012] NPL 2: T. Pinkert, D. Furkert, T. Korte, et al., Angew. Chem. Int. Ed. 2017, 56, 2790-2794.

[0013] NPL 3: T. Yamamoto, H. Hasegawa, T. Hakogi, et al., Org. Lett. 2006, 8, 5569-5572.

[0014] NPL 4: R. Yang, X. Bi, F. Li, et al., Chemical Communications 2014, 50, 7971-7974.

[0015] NPL 5: R. M. Borzilleri, X. Zheng, R. J. Schmidt, et al., J. Am. Chem. Soc. 2000, 122, 8890-8897.

[0016] NPL 6: J. G. Mina, J. A. Mosely, H. Z. Ali, et al., Org. Biomol. Chem. 2011, 9, 1823-1830.

[0017] NPL 7: W. Qu, K. Ploessl, H. Truong, et al., Bioorg. Med. Chem. Lett. 2009, 19, 3382-3385.

[0018] NPL 8: 0. Zenasni, M. D. Marquez, A. C. Jamison, et al., Chem. Mater. 2015, 27, 7433-7446.

[0019] NPL 9: J. Fink, J. Seibel, Biol. Chem. 2018, 399, 1157-1168.

[0020] NPL 10: G. Kokotos, J. Padron, C. Noula, et al., Tetrahedron: Asymmetry 1996, 7, 857-866.

[0021] NPL 11: Y. A Hannun and L.M. Obeid Nat. Rev. Mol. Cell Biol. 2018,19, 175-191.

[0022] NPL 12: U. Coskun, M. Grzybek,D. Drechsel et al., Proc. Natl Acad. Sci. 2011, USA 108, 9044-9048.

[0023] NPL 13: C. Eich, C. Manzo, S. de Keijzer, et al. Sci. Rep., 2016, 6, 20693. NPL 14: P. Haberkant, 0. Schmitt, F.-X. Contreras, et al., J. Lipid Res. 2008, 49, 251-262.

[0024] NPL 15: D. Russo, S. Parashuraman and G. D'Angelo, Int. J. Mol. Sci. 2016, 17, E1732.

[0025] NPL 16: Y.A. Hannun and L.M. Obeid, Nat. Rev. Mol. Cell Biol. 2008, 9(2):139-50.

[0026] NPL 17: A.H. Futerman and Y. A. Hannun, EMBO 2004, Rep. 5, 777-782.

[0027] NPL 18: B.M. Quinville, N.M. Deschenes, A.E. Ryckman, et al., UMS 2021, 22, 5793.

[0028] NPL 19: E. Izquierdo and A. Delgado A., Chemistry Physics of Lipids 2018, 215; 71-83.

[0029] NPL 20: D. Jamecna and D. Hoglinger, J. Cell Science 2024, 137, jcs261388.

[0030] Summary of the invention

[0031] The present invention relates to a compound of the following formula (I): wherein

[0032] A1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2 substituent,

[0033] A3is selected from C1.5 alkyl optionally substituted with one or two substituent(s) selected from Group X, wherein at least two of A1, A2and A3have the one or two substituent(s) selected from Group X, and wherein the compound of formula (I) contains at least two substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci-ealkyl- diazirinyl and tetrazinyl.

[0034] The present invention also relates to a compound of the following formula (II): wherein

[0035] A1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2substituent, one of A1and A2have the one or two substituent(s) selected from Group X, and if A1has a -OH or -NH2 substituent, the compound of formula (II) contains at least one, preferably two or three, more preferably two, substituents selected from Group X, if A1does not have a -OH or -NH2substituent, the compound of formula (II) contains at least two, preferably three, substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci.6alkyl- diazirinyl and tetrazinyl.

[0036] In addition, the present invention also relates to a conjugate obtainable by reacting a compound of formula (I) or (II) with one, two or three further compound(s) comprising an alkyne (such as a -C=CH group or a cycloalkyne), cycloalkene (such as transcyclooctenyl), tetrazinyl and / or -N3, wherein

[0037] (i) when the compound according to any one of the preceding claims comprises an alkyne group (such as a -C=CH group or a cycloalkyne); and

[0038] (ii) when the compound according to any one of the preceding claims comprises an azide group; and

[0039] (iii) when the compound according to any one of the preceding claims comprises a cycloalkene (such as transcyclooctenyl) group; and

[0040] (iv) when the compound according to any one of the preceding claims comprises a tetrazinyl group.

[0041] The present invention also concerns the conjugate for use in diagnostics, preferably in relation to a disease selected from cancer, diabetes, a neurodegenerative disease and an infectious disease.

[0042] Description of the Figures

[0043] Fig, 1:XH NMR spectrum (400 MHz, CD3OD) of target molecule TFSM 1 Fig. 2:13C NMR spectrum (100 MHz, CD3OD) of target molecule TFSM 1

[0044] Fig. 3:XH NMR spectrum (400 MHz, CD3OD) of target molecule TFSM2

[0045] Fig. 4:13C NMR spectrum (100 MHz, CD3OD) of target molecule TFSM2

[0046] Fig. 5:XH NMR spectrum (400 MHz, CDCI3) of target molecule Cerl6 2

[0047] Fig. 6:13C NMR spectrum (100 MHz, CDCI3) of target molecule Cerl6 2

[0048] Fig. 7:XH NMR spectrum (400 MHz, CDCI3) of target molecule Cerl6 1

[0049] Fig. 8:13C NMR spectrum (100 MHz, CDCI3J of target molecule Cerl6 1

[0050] Fig. 9: Fixation and enzymatic activity determination of sphingomyelases Fig. 10: Illustration of FRET, and absence of FRET in the case of cleavage Fig. 11: Trifunctional sphingomyelins (TFSMs) are taken up and metabolized by human cells: a Sphingomyelinase Forster resonance energy transfer (FRET) probe is cleaved by recombinant human acid sphingomyelinase (rhASM) but not bacterial sphingomyelinase (bSMase). rhASM or bSMase were incubated in the presence of FRET probe and probe conversion was monitored by measurement of FITC fluorescence in a microplate reader, n = 3. b, c TFSMs are incorporated into human cells. HEK293T cells were treated with TFSM 1 or TFSM 2 for 24 h at 37 °C. After cell lysis, amounts of TFSMs, ceramide (Cer), and sphingosine (Sph) were detected by LC-MS / MS. Internal standards were used for each metabolite type, and their peak areas were used to normalize the peak area of the corresponding TFSM metabolite, n - 3. d Cellular uptake of TFSMs is affected by fetal bovine serum (FBS). HeLa cells were treated with TFSM 1 in the presence of 1 or 10% FBS for 2 h. Samples were fixed and clicked with AlexaFluor™ (AF)488-DBCO (backbone) or Cy5-azide (headgroup). n = 1. Scale bars: 50 pm. e Glutaraldehyde (GA) fixation of TFSMs. HeLa cells were incubated with BODIPY-FL-Ci2-sphingomyelin (BODIPY-FL-C12-SM), TFSM l or TFSM 2 for 2 h, bSMase-treated, detached, fixed, and permeabilized with TX-100 (as indicated). Fixation was done either with paraformaldehyde (PFA) or PFA and GA. TFSM then were BODIPY-FL-DBCO-stained and analyzed by flow cytometry, n = 3 (BODIPY-FL-C12-SM, TFSM 1 PFA and TFSM 2 PFA), n = 6 (TFSM 1 GA + PFA, TFSM 2 GA + PFA). Statistics: Two-way ANOVA and Sidak's (b) or Tukey's (e) multiple comparisons. Two-sided unpaired Student's t test (c). Bars represent means ± SD. n corresponds to biological replicates.

[0051] Fig. 12: Trifunctional sphingomyelins (TFSMs) are incooperated in cellular compartments and can be used to determined bacterial sphingomyelinase (bSMase) activity: a, b TFSMs are enriched within the Golgi and mitochondria. HeLa cells were treated with TFSM 1 for 2 h (a) or 24 h (b). Samples were fixed and clicked to BODIPY-FL-DBCO (backbone) or Atto647N-azide (headgroup). Golgi and mitochondria were visualized by an anti-Golgi matrix protein 130 (GM130) and anti-Peroxiredoxin (Prx3) antibody, respectively, n - 1. Scale bars: 25 pm (a) or 5 pm (b). c, d FRET measurement of TFSM conversion. HeLa or human umbilical vein endothelial cells (HuVEC) were incubated with TFSM 1, TFSM 2, or BODIPY-FL-Cu-SM for 2 h. Then, the compounds were removed, and cells were either treated with bSMase or left untreated for 3 h. Samples treated with TFSMs were fixed, clicked with BODIPY-FL-DBCO (backbone) and AlexaFluor™546 (AF)546-azide (headgroup) and FRET efficiency was determined by acceptor bleaching. Cells treated with BODIPY-FL-Cu-SM were detached, and lipids were extracted by CHC^MeOH. The proportion of unmetabolized SM was determined by thin-layer chromatography. Scale bars: 25 pm. n = 3. Statistics: Two-way ANOVA and Sidak's multiple comparisons (d). Bars represent means ± SD. n corresponds to biological replicates.

[0052] Fig. 13: Trifunction sphingomyelins (TFSMs) can be used for expansion microscopy (ExM): a TFSMs are compatible with ExM. HeLa cells were incubated with TFSM 1 for 24 h in the presence of 1% FBS. Samples were fixed, stained with BODIPY-FL-DBCO as well AlexaFluor™ (AF)546- azide, and then either imaged by conventional CLSM or 4-fold expanded, n = 3. Scale bars: 50 pm (~ 12.5 pm with 4-fold expansion factor), b, c Forster resonance energy transfer (FRET) can be measured in expanded samples. HeLa cells were incubated with TFSM l for 24 h in the presence of 1% FBS. Then, the compound was removed, and cells were treated with 2.5 pg / ml bacterial sphingomyelinase (bSMase) or left untreated for 24 h. Samples were fixed, stained, and expanded as described in (a) and analyzed via acceptor photobleaching to determine FRET efficiency, n = 4. d-f Lysosomes possess high SM content. HeLa cells were treated as described in (a), and lysosomal-associated membrane protein 1 (LAMP1) was stained by an anti-LAMPl primary and a CF568 secondary antibody. Images recorded in the FRET channel were multiplied by factor 2 and divided by the BODIPY-FL (donor) channel resulting in images that describe the metabolic state of the molecule, n = 1. Scale bars: 50 pm (~ 12.5 pm with 4-fold expansion factor) and 12 pm for zoomed images (~ 3 pm with 4-fold expansion factor). Statistics: Two-sided unpaired Student's t test (c). Bars represent means ± SD. n corresponds to biological replicates.

[0053] Fig. 14: Chlamydia inclusions are enriched with the metabolized trifunctional sphingomyelins (TFSMs): a Chlamydia inclusions possess a higher proportion of metabolized TFSM 1 than host cell membranes. HeLa cells were infected with C. trachomatis at a multiplicity of infection (MOI) 1 in the presence of 10 pM TFSM 1 and 1% FBS. After 24 h of treatment, cells were fixed, stained with BODIPY-FL-DBCO (backbone) and AlexaFluor™ (AF)546-azide (headgroup), and samples were imaged with a confocal microscope. The metabolic state of TFSM is described by the ratio of 3x Forster resonance energy transfer (FRET) channel vs. donor channel (BODIPY-FL). Chlamydial inclusions are indicated with white circles. n = 5. Scale bars: 10 pm, zoomed: 5 pm. b, c Chlamydial inclusions possess lower FRET efficiency than host cell membranes. Samples were prepared as described in (a), and FRET efficiency was determined by acceptor bleaching. The acceptor fluorophore (AF546) was bleached in an area either containing a chlamydial inclusion (white line) or host cell membranes. Then, donor fluorescence before (pre) and after (post) bleaching was compared to determine FRET efficiency. In (b), samples incubated with TFSM 2 are depicted. Scale bars: 10 pm. TFSM 1: n = 5, TFSM 2 n = 4. d Overview fluorescence lifetime imaging microscopy (FLIM) images of infected, TFSM 1-stained HeLa cells click-labeled with BODIPY-FL-DBCO (left) as well as BODIPY-FL-DBCO and AF546-azide (right) measured by confocal imaging at an irradiation intensity of 0.5 kW cm’2. Chlamydia inclusions are marked with white lines. No intensity threshold was applied. n = l. Scale bars, 20 pm. (e) Average fluorescence decays from 9 individual FLIM images of single-cell measurements, n = 1. Statistics: Mixed effects analysis (REML) and Sidak's multiple comparisons test (c). Bars represent means ± SD. n corresponds to biological replicates.

[0054] Fig. 15: Elementary bodies possess higher proportion of metabolized TFSMs than reticulate bodies: a TFSM can be used to visualize chlamydial inclusion via 4xExM. HeLa cells were infected with C. trachomatis at MOI 1 in the presence of TFSM l for 24 h, fixed and stained with AlexaFluor™ (AF)546-azide (headgroup) and BODIPY-FL-DBCO (backbone). Chlamydia was stained with an anti-chlamydial heat-shock protein 60 (HSP60) anti-body and an AF405 secondary antibody. Samples were 4x expanded and imaged with CLSM. n = 3. Scale bars: 8 pm (with 4-fold expansion factor ~ 2 pm), b Periphery of inclusions rather possess nonmetabolized TFSM compared to their center. The image depicted in (a) was zoomed, and the metabolic state of TFSM was calculated by determining the ratio of 3-times Forster resonance energy transfer (FRET) vs. donor (BODIPY-FL) signal. White arrows indicate intermediate chlamydial developmental forms, n = 3. Scale bars: 8 pm (with 4-fold expansion factor ~ 2 pm), c, d Inclusions possess lower FRET efficiency than host cell membranes. Chlamydia-infected HeLa cells stained with TFSM 1 (BODIPY-FL: Donor / AF546: Acceptor) were 4-fold expanded. FRET efficiency in regions containing an inclusion (white circles) or host cell membranes was determined by acceptor photobleaching, n = 3. Scale bars: 20 pm (with 4-fold expansion factor ~ 5 pm), e, f Sphingolipid composition of EBs and RBs differ. The area indicated by a white line in b.) was magnified, and the intensity profile was measured in Fiji. The resulting gray values were scaled to the highest and lowest values detected in individual channels, n = 3. Scale bar: 4 pm (with 4-fold expansion factor ~ 1 pm), g Identification of high metabolized TFSM levels in EBs by FLIM imaging of 4-fold expanded TFSM 1-stained chlamydial inclusion labeled with BODIPY-FL-DBCO (left) as well as BODIPY-FL-DBCO and AF546-azide (middle, right). Zoomed image (right) of BODIPY-FL-DBCO and AF546-azide labeled TFSM shows higher fluorescence lifetimes of EBs in comparison to RBs, indicating a higher proportion of metabolized TFSMs. n = l. Scale bars: 10 pm (with 4x expansion factor ~ 2.5 pm). Statistics: Two-sided unpaired Student's t test (d). Bars represent means ± SD. n corresponds to biological replicates.

[0055] Fig. 16: Bacterial sphingomyelinase (bSMase) treatment enhances signal of backbone fluorophores: a, b bSMase affects backbone fluorophores FRET-independently. HeLa cells were incubated with TFSM 1 in the presence of 1% FBS. The molecule was removed, and cells were bSMase- treated, fixed, and stained with Cy5-azide and AlexaFluor™ (AF)488. Fluorescence intensities in Cy5 and AF488 channels were measured, and Cy5 / AF488 ratios were calculated. Scale bars: 50 pm. n = 3. c Quantification of backbone fluorescence by flow cytometry. HeLa cells were incubated with TFSM 1 in the presence of 1% FBS. TFSM 1 was removed, and cells were bSMase-treated, detached, fixed, BODIPY-FL-DBCO-stained, and analyzed by flow cytometry, n = 3. d bSMase treatment enhances the backbone fluorophore of a FRET probe. HeLa cells were incubated with the FRET probe in the presence of 1% FBS. After probe removal, cells were bSMase-treated, detached, and analyzed by flow cytometry for FRET and BODIPY-TR (backbone) fluorescence. Mean BODIPY-TR fluorescence (left y-axis) and FRET / BODIPY-TR ratios (right x-axis). n = 4. e Comparison of bSMase treatment effects on backbone fluorophores in different SM derivatives. HeLa cells were incubated with TFSM 1, the FRET probe, or BODIPY-FL-Cu-SM. Molecules were removed, and cells were bSMase- treated and detached. Cells incubated with TFSM 1 were fixed stained with BODIPY-FL-DBCO, and backbone fluorophores were analyzed by flow cytometry (BODIPY-FL for TFSM 1; BODIPY-FL-C12-SM and BODIPY-TR for the FRET probe). Mean fluorescence in bSMase-treated samples was normalized to untreated controls (100%). n = 3. f, g bSMase-induced changes in backbone fluorophores are absent in expanded samples. Samples were incubated with TFSM 1 for 24 h. Then, cells were washed, treated with bSMase, and either embedded in Mowiol or in the hydrogel, which was left unexpanded or 4x expanded. Scale bars: 40 pm. The mean fluorescence was measured within cell-containing areas (g), and fold changes were calculated (green bars, right y-axis). n = 5 for gelated + 100 ng / ml bSMase and gelated + expanded + 100 ng / ml bSMase, otherwise n = 6. Statistics: Two-sided unpaired Student's t test (b, c). Two-way ANOVA and Sidak's multiple comparisons test (d), two-sided one-sample t test (e), mixed effects analysis, and Sidak's multiple comparisons test (f, g). Bars represent means ± SD. n corresponds to biological replicates.

[0056] Fig. 17: Airyscan images of COS 7 cells incubated with 5 pM Cerl6-1 for 16 h. A) Unexpanded cell. B) Cell expanded fourfold. C) and D) Magnified sections of the cell in slide B. Detailed description of the invention

[0057] The present invention relates to a compound of the following formula (I): wherein

[0058] A1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2 substituent,

[0059] A3is selected from C1-5 alkyl optionally substituted with one or two substituent(s) selected from Group X, wherein at least two of A1, A2and A3have the one or two substituent(s) selected from Group X, and wherein the compound of formula (I) contains at least two substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci-ealkyl- diazirinyl and tetrazinyl.

[0060] It is to be understood that the compound of the formula (I) can also be present as a pharmaceutically acceptable salt or solvate. The salt is preferably a trifluoroacetic acid salt. Preferred solvates include hydrates.

[0061] Preferably all three of A1, A2and A3have the one or two substituent(s) selected from Group X.

[0062] Preferably, the compound of formula (I) contains (at least) three substituents selected from Group X. More preferably, the compound of formula (I) contains (at least) four substituents selected from Group X.

[0063] Preferably, the compound of formula (I) contains (at least) two substituents selected from Group X and A1furthermore has a -OH or -NH2substituent. More preferably, the compound of formula (I) contains (at least) three substituents selected from Group X and A1furthermore has a -OH or -NH2 substituent. In the compound of formula (I), typically, A1is C4-23 alkyl or alkenyl (preferably C6-21 alkyl or alkenyl, more preferably Cg-is alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably Cu, C12, C13 or C14 alkyl or alkenyl, such as CM alkyl or alkenyl). The alkyl or alkenyl is preferably substituted with one substituent selected from Group X.

[0064] The alkyl or alkenyl in A1is preferably alkyl. Thus, A1is preferably C2-25 alkyl or A1is C4-23 alkyl (preferably C6-2i alkyl, more preferably Cs-is alkyl, even more preferably C10-15 alkyl, most preferably Cu, Cu, C13 or CM alkyl, such as CMalkyl). The alkyl is preferably substituted with one substituent selected from Group X.

[0065] A1has preferably at least one terminal substituent of Group X, wherein the terminal substituent is preferably azide.

[0066] A1optionally has a substituent -OH, oxo or -NH2. Preferably, A1optionally has a substituent -OH or - NH2. More preferably, A1has an -NH2substituent. The -OH or -NH2substituent is preferably present at the carbon atom which is bound to the carbonyl group to which A1is bound. The -OH or -NH2substituent is thus preferably present in alpha position to the carbonyl group. More preferably, there is a -NH2substituent present in alpha position to the carbonyl group in A1.

[0067] A1typically has one further substituent of Group X, wherein the substituent is preferably diazirinyl or Ci-ealkyl-diazirinyl, more preferably Ci-salkyl-diazirinyl.

[0068] A2is typically C4.23 alkyl or alkenyl, preferably C6-2ialkyl or alkenyl, more preferably Cg-is alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably Cu, Cu, C13 or CM alkyl or alkenyl, such as C13 alkyl or alkenyl. The alkyl or alkenyl is preferably substituted with one substituent selected from Group X.

[0069] Also in A2, the alkyl or alkenyl is preferably alkyl. Thus, A2is typically C4-23 alkyl, preferably Ce-zi alkyl, more preferably C8-i8 alkyl, even more preferably C10-15 alkyl, most preferably Cu, Cu, C13 or CM alkyl, such as C13 alkyl. The alkyl is preferably substituted with one substituent selected from Group X.

[0070] A3is typically Ci-2alkyl substituted with one substituent selected from Group X, wherein the substituent of Group X is preferably alkyne (preferably with a terminal CC triple bond), more preferably a -C=CH group. Preferably, A3is Ci-2alkyl, more preferably methyl. It is preferred that the compound of formula (I) contains at least one diazirinyl group, preferably as a substituent in A1.

[0071] The members of Group X are preferably selected from — transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci.6alkyl-diazirinyl and tetrazinyl. The substituents of Group X are preferably present in terminal positions. Thus, it is preferred that the carbon atoms to which the substituents of Group X are bound are carbons having two hydrogen substituents and one substituent of Group X.

[0072] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-2o alkyl), A2is -C3-20 alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs- 15 alkyl), A2is -C8-20 alkylene-N3and A3is — (C1-3 alkylene)-CnCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-IO alkyl), A2is -C10-18 alkylene-N3and A3is -(C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-7 alkyl), A2is -Cn-17 alkylene-Ns and A3is -(C1-3 alkylene)-CHCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-6 alkyl), A2is —Cn-15 alkylene-Ns and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-5 alkyl), A2is -C12-14 alkylene-N3and A3is -(C1-3 alkylene)~C=CH.

[0073] In one embodiment of the compound of formula (I), A1is -C(NH2)(C8-2o alkyl), A2is -C8.20alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CIO- 18 alkyl), A2is -Cio -is alkylene-Ns and A3is — (C1-3 alkylene)— C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn-i7 alkyl), A2is -Cn-17 alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn-i5 alkyl), A2is -Cn-15 alkylene-Ns and A3is - (C1-3 alkylene)-CHCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Ci2-i4 alkyl), A2is -Ci2-i4 alkylene-N3and A3is -(C1-3 alkyleneJ-CnCH.

[0074] In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-2o alkylene-Ns), A2is -C3-20 alkyl and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs- 15 alkylene-Ns), A2is -C8-2o alkyl and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CS IO alkylene-Ns), A2is -Cio-is alkyl and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-7 alkylene-Ns), A2is -Cn-17 alkyl and A3is - (Ci-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-6 alkylene- Ns), A2is -Cn-15 alkyl and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-5 alkylene-Ns), A2is -C12-14 alkyl and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cg.2o alkylene-N3), A2is -C8-2o alkyl and A3is -(C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CIO- 18 alkylene-N3), A2is -Cio-is alkyl and A3is - (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cu-i7 alkylene-N3), A2is -Cn-17 alkyl and A3is -(C1-3 alkylene)-OCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn i5 alkylene-N3), A2is -Cn-15 alkyl and A3is -(Ci-3alkyleneJ-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Ci2-i4 alkylene-N3), A2is -C12-14 alkyl and A3is — (C1-3 alkylene)-C=CH.

[0075] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-2o alkylene-(Group Y)), A2is -C3-2o alkylene-N3and A3is — (C1-3 alkylene)-CECH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-i5 alkylene-(Group Y)), A2is -Cs-2o alkylene-N3and A3is -(C1-3 alkyleneJ-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-IO alkylene-(Group Y)), A2is -Cio-is alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-7 alkylene-(Group Y)), A2is -Cn-17 alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-6alkylene-(Group Y)), A2is -CH-IS alkylene-N3and A3is — (Ci 3 alkyleneJ-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-5 alkylene-(Group Y)), A2is -C12-14 alkylene-N3and A3is -(Ci-3alkylene)-C=CH. Group Y is selected from Group X, except alkynyl and N3.

[0076] In one embodiment of the compound of formula (I), A1is -C(NH2)(C8-2o alkylene-(Group Y)), A2is -C8-2o alkylene-N3and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is — C(NH2)(Cio i8 alkylene-(Group Y)), A2is -Cio-is alkylene-N3and A3is -(C1.3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn-i7 alkylene-(Group Y)), A2is -Cu-17 alkylene-N3and A3is -(C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is — C(NH2)(Cii-i5 alkylene-(Group Y)), A2is -Cu-15 alkylene-N3and A3is -(C1-3 alkylene)-CECH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Ci2-i4 alkylene-(Group Y)), A2is -C12-14 alkylene-N3and A3is -(C1-3 alkylene)-CECH. Group Y is selected from Group X, except alkynyl and N3.

[0077] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-2o alkylene-N3), A2is -C3-20 alkylene-(Group Y) and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-i5 alkylene-N3), A2is -Cs-io alkylene-(Group Y) and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-io alkylene-N3), A2is -Cio-is alkylene- (Group Y) and A3is — (C1-3 alkylene)-CECH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-7 alkylene-N3), A2is -Cu-17 alkylene-(Group Y) and A3is — (Ci-3alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-6 alkylene-N3), A2is -Cu-i5alkylene- (Group Y) and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-5 alkylene-N3), A2is -C12-M alkylene-(Group Y) and A3is — (Ci-3alkylene)-C=CH. Group Y is selected from Group X, except alkynyl and N3.

[0078] In one embodiment of the compound of formula (I), A1is -C(NH2)(Cs-2o alkylene-N3), A2is -C8-2o alkylene-(Group Y) and A3is - (Ci 3alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cio-i8 alkylene-N3), A2is -Cio-is alkylene-(Group Y) and A3is — (C1-3 alkylene)-CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CU-I7alkylene-N3), A2is -Cn-17 alkylene- (Group Y) and A3is -(Ci.3alkylenej-CnCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cu-i5 alkylene-N3), A2is -Cu-is alkylene-(Group Y) and A3is — (C1-3 alkylene)-C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Ci2-i4 alkylene-N3), A2is -CIM4alkylene- (Group Y) and A3is - (C3 3alkylene)-C=CH. Group Y is selected from Group X, except alkynyl and N3.

[0079] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.2o alkyl), A2is -C3.20alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-i5alkyl), A2is -C8-2o alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-IO alkyl), A2is -Cio-is alkylene-N3and A3is -CH2CsCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C37 alkyl), A2is -Cn-n alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-6alkyl), A2is -Cn-is alkylene-N3and A3is -CH2CECH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.5alkyl), A2is -Ci2-i4 alkylene-N3and A3is -CH2CHCH.

[0080] In one embodiment of the compound of formula (I), A1is - C(N H2)(C8-2o alkyl), A2is -C8.2o alkylene-N3and A3is -CH2CHCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CIO-IS alkyl), A2is -Cio-is alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cin7 alkyl), A2is -Cn-i7 alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(l\IH2)(Cn-i5 alkyl). A2is -Cn-15 alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CI2-M alkyl), A2is -Ci2-i4 alkylene-N3and A3is -CH2C=CH.

[0081] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.2o alkylene-N3), A2is -C3.2o alkyl and A3is -CH2CHCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-i5 alkylene- N3), A2is ~C8.2o alkyl and A3is -CH2CsCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-1Oalkylene-N3), A2is -Cio-is alkyl and A3is -CH2CHCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-7alkylene-N3), A2is -Cn-17 alkyl and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-6 alkylene-N3), A2is-Cn-is alkyl and A3IS -CHZCECH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.5alkylene-N3), A2is -CU-M alkyl and A3is -CH2C=CH.

[0082] In one embodiment of the compound of formula (I), A1is -C(NH2)(C8-2o alkylene-N3), A2is -Cs-zo alkyl and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cio-28 alkylene- N3), A2is -C10-18 alkyl and A3is -CHZCHCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cn-i7 alkylene-N3), A2is -Cu-17 alkyl and A3is -CH2CSCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn-is alkylene-N3), A2is -Cn-15 alkyl and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Ci2 i4 alkylene-N3), A2is -C 12-14 alkyl and A3is -CH2CECH.

[0083] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-2o alkylene-(Group Y)), A2is -C3-2o alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3. 15 alkylene-(Group Y)), A2is-C8-2o alkylene-N3and A3is-CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-IO alkylene-(Group Y)), A2is -Cio-is alkylene-N3and A3is -CH2OCH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-7 alkylene-(Group Y)), A2is -Cu-17 alkylene-N3and A3is -CHzC^CH. In one embodiment of the compound of formula (I), A1is -C(NHz)(C3- 6 alkylene-(Group Y)), A2is -Cn-15 alkylene-N3and A3is-CfhCECH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.5alkylene-(Group Y)), A2is -Cu-w alkylene-N3and A3is -CH2CECH. Group Y is selected from Group X, except alkynyl and N3.

[0084] In one embodiment of the compound of formula (I), A1is -C(NH2)(C8-zo alkylene-(Group Y)), A2is -C8-2o alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is - C(N H2)(CIO- 18 alkylene-(Group Y)), A2is -Cio-is alkylene-N3and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NK2)(Cii-i7 alkylene-(Group Y)), A2is -Cn.17 alkylene-N3and A3is - CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cii.i5 alkylene-(Group Y)), A2is -Cn-15 alkylene-N3and A3is -CH2CsCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Ci2-i4 alkylene-(Group Y)), A2is -C12-14 alkylene-N3and A3is -CH2C=CH. Group Y is selected from Group X, except alkynyl and N3.

[0085] In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.2o alkylene-N3), A2is -C3-2o alkylene-(Group Y) and A3is -CH2CHCH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-I5alkylene-N3), A2is -Cs-2o alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3-I0alkylene-N3), A2is -Cio-is alkylene-(Group Y) and A3is - CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.7 alkylene-N3), A2is - Cn-17 alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(C3-6 alkylene-N3), A2is -Cn-15 alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(C3.5alkylene-N3), A2is -Ci2-Malkylene-(Group Y) and A3is - CH2C=CH. Group Y is selected from Group X, except alkynyl and N3.

[0086] In one embodiment of the compound of formula (I), A1is -C(NH2)(C8-2o alkylene-N3), A2is -Cg.2o alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is - C(NH2)(Cio-i8 alkylene-N3), A2is -Cio is alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(Cn-i7 alkylene-N3), A2is -CH- 17 alkylene-(Group Y) and A3is -CH2C=CH. In one embodiment of the compound of formula (I), A1is -C(NH2)(CH.IS alkylene-N3), A2is -CH-15 alkylene-(Group Y) and A3is -CH2CECH. In one embodiment of the compound of formula (I), A1is — C(NH2)(Ci2-i4 alkylene-N3), A2is -C12-14 alkylene-(Group Y) and A3is -CH2CECH. Group Y is selected from Group X, except alkynyl and N3.

[0087] In the "alkylene-(Group Y)", the Group Y substituent may be present either in a terminal or a nonterminal position. Ci.6alkyl-diazirinyl is preferably present in a non-terminal position, whereas the other types of substituents of Group Y are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0088] In one embodiment of the compound of formula (I), A1is -(C3.2o alkylene-Group Z), A2is -C3.2o alkylene- N3and A3is — (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is -(C3.is alkylene- Group Z), A2is - C8.20alkylene-N3and A3is — (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is ~(C3-io alkylene-Group Z), A2is -Cio-is alkylene-N3and A3is — (Ci-3alkyl). In one embodiment of the compound of formula (I), A1is — (C3-7 alkylene-Group Z), A2is -Cn.17 alkylene-N3and A3is -(Ci.3alkyl). In one embodiment of the compound of formula (I), A1is -(C3.6alkylene-Group Z), A2is -Cu.15 alkylene-N3and A3is — (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is -(C3.5alkylene-Group Z), A2is -C12-14 alkylene-N3and A3is -(Ci.3alkyl). Group Z is selected from Group X, except N3. Group Z is preferably diazirinyl or Ci-ealkyl-diazirinyl. In one embodiment of the compound of formula (I), A1is -(C8-2o alkylene-Group Z), A2is -C8-2o alkylene-N3and A3is — (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is -(Cio-is alkylene-Group Z), A2is -Cio-is alkylene-N3and A3is — (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is -(Cn-i7 alkylene-Group Z), A2is -Cn-17 alkylene-N3and A3is — (Ci-3alkyl). In one embodiment of the compound of formula (I), A1is - (Cu.15 alkylene-Group Z), A2is-Cn-is alkylene-N3and A3is - (C1-3 alkyl). In one embodiment of the compound of formula (I), A1is -(C12-14 alkylene-Group Z), A2is -C12-14 alkylene-N3and A3is — (Ci-3alkyl). Group Z is selected from Group X, except N3. Group Z is preferably diazirinyl or Ci-ealkyl-diazirinyl. In the "alkylene-(Group Z)", the Group Z substituent may be present either in a terminal or a non-terminal position. Ci-galkyl-diazirinyl is preferably present in a non-terminal position, whereas the other types of substituents of Group Z are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0089] N=N

[0090] In one embodiment of the compound of formula (I), A1is -(Ci-is alkylene- V (-Co-6alkylene)H), A2is - C3-20 alkylene-N3and A3is -(Ci.3alkyl). In one embodiment of the compound of formula (I), A1is - (Ci-i8

[0091] N=N alkylene- V (-C0-6alkylene)H), A2is -C8.2o alkylene-N3and A3is -(C1-3 alkyl). In one embodiment of the

[0092] N=N compound of formula (I), A1is -{Ci-is alkylene- V (-Co-6alkylene)H), A2is -Cio-is alkylene-N3and A3is -

[0093] N=N

[0094] (Ci-3alkyl). In one embodiment of the compound of formula (I), -(Ci-is alkylene- V (-Co-6alkylene)H), A2is -Cn-i7 alkylene-N3and A3is -(C1-3 alkyl). In one embodiment of the compound of formula (I), A1is

[0095] N=N

[0096] -(Ci-is alkylene- V (-C0-6alkylene)H), A2is -Cu-is alkylene-N3and A3is -(C1-3 alkyl). In one embodiment

[0097] N=N of the compound of formula (I), A1is -(Ci-is alkylene- V (-Co-6alkylene)H), A2is -C^-w alkylene-N3and A3is — (C1-3 alkyl). The (-Co-6alky lene) is preferably methylene or ethylene, more preferably methylene.

[0098] The Group X substituents may be present either in a terminal or a non-terminal position. Ci-galkyl- diazirinyl is preferably present in a non-terminal position, whereas the other types of substituents of Group X are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0099] The compound of formula (I) is preferably a compound having the following formula (la) wherein R1, R2and R3are each independently selected from -H and Group X;

[0100] R4is selected from H, diazirino, -oxo, -OH and -NH2; wherein at least one of R1and R4is not hydrogen; and wherein at least one of R2and R3is not hydrogen; n is an integer from 0 to 20; m is an integer from 0 to 20; u is an integer from 0 to 20; wherein the sum of u and m is in the range of from 2 to 20.

[0101] In formula (la) R4is typically selected diazirino and -NHz, more preferably R4is -NHz.

[0102] Preferably neither R2nor R3is hydrogen.

[0103] The members of Group X are preferably selected from transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci-ealkyl-diazirinyl and tetrazinyl.

[0104] It is preferred that R4is -NH2. Furthermore, u is preferably 0.

[0105] In one specific embodiment R4is diazirino, m is preferably 0, u is an integer of from 1 to 18 (preferably 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 3, 1 or 2, or even 2), wherein R1is preferably hydrogen.

[0106] R2and R3are each independently preferably selected from Cz-io alkynyl, C5-io cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Cj.galkyl-diazirinyl and tetrazinyl; More preferably, R2and R3are each independently selected from — :^==, transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci-ealkyl- diazirinyl and tetrazinyl.

[0107] In one specific embodiment, R1or R2is -N3, R3is Cz-io alkynyl and R4is NHz or diazirino.

[0108] Typically, n is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11.

[0109] It is preferred that m is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11.

[0110] Typically, u is an integer of from 0 to 17, preferably 0 to 14, more preferably 0 to 11, even more preferably 0 to 8, still more preferably 0 to 5, still even more preferably 0 to 2, most preferably 0.

[0111] It is furthermore preferred that the sum of m+ u is an integer of from 2 to 30, preferably 5 to 25, more preferably 7 to 22, even more preferably 10 to 18.

[0112] The present invention furthermore relates to a compound of the following formula (II): wherein

[0113] A1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2substituent, at least one of A1and A2has the one or two substituent(s) selected from Group X, and if A1has a -OH or -NH2substituent, the compound of formula (II) contains at least one, preferably two or three, more preferably two, substituents selected from Group X, if A1does not have a -OH or -NH2substituent, the compound of formula (II) contains at least two, preferably three, substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci-ealkyl- diazirinyl and tetrazinyl.

[0114] Preferably both A1and A2have the one or two substituent(s) selected from Group X.

[0115] The compound of formula (II) preferably contains three substituents selected from Group X.

[0116] It is to be understood that the compound of the formula (II) can also be present as a pharmaceutically acceptable salt or solvate. The salt is preferably a trifluoroacetic acid salt. Preferred solvates include hydrates.

[0117] In the compound of formula (II), typically, A1is C423 alkyl or alkenyl (preferably C6-2ialkyl or alkenyl, more preferably Cs-is alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably CH, C12, C13 or C14 alkyl or alkenyl, such as CMalkyl or alkenyl). The alkyl or alkenyl is preferably substituted with one substituent selected from Group X.

[0118] The alkyl or alkenyl in A1is preferably alkyl. Thus, A1is preferably C2-25 alkyl.

[0119] A1has preferably at least one terminal substituent of Group X, wherein the terminal substituent is preferably azide. A1optionally has a substituent -OH, oxo or -NHz. Preferably, A1optionally has a substituent -OH or - NH2. More preferably, A1has an -NH2substituent. The -OH or -NH2substituent is preferably present at the carbon atom which is bound to the carbonyl group to which A1is bound. The -OH or -NH2substituent is thus preferably present in alpha position to the carbonyl group.

[0120] A1typically has one further substituent of Group X, wherein the substituent is preferably diazirinyl, Ci-6alkyl-diazirinyl, more preferably Ci-ealkyl-diazirinyl.

[0121] A2is typically C4.23alkyl or alkenyl, preferably Ce-2ialkyl or alkenyl, more preferably C8-is alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably Cn, Ci2, Ci3or CM alkyl or alkenyl, such as C13 alkyl or alkenyl. The alkyl or alkenyl is preferably substituted with one substituent selected from Group X.

[0122] Also in A2, the alkyl or alkenyl is preferably alkyl.

[0123] The members of Group X are preferably selected from transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci-ea Ikyl-d iazirinyl and tetrazinyl.

[0124] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-2O alkyl), A2is -C3.2o alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3.IS alkyl), A2is -C8.2o alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3.io alkyl), A2is -Cio-is alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-7 alkyl), A2is -Cn-17 alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3.6alkyl), A2is -Cn-15 alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3.s alkyl), A2is -Ci2.i4alkylene-N3.

[0125] In one embodiment of the compound of formula (II), A1is -C(NH2)(C8.2o alkyl), A2is -C8.20alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(Cio i8 alkyl). A2is -Cio-is alkylene-N3.

[0126] In one embodiment of the compound of formula (II), A1is -C(NH2)(Cn-i7 alkyl), A2is -Cn-17 alkylene-N3.

[0127] In one embodiment of the compound of formula (II), A1is -C(NH2)(Cn.is alkyl), A2is -Cn.15 alkylene-N3.

[0128] In one embodiment of the compound of formula (II), A1is -C(NH2)(Ci2.i4alkyl), A2is -Ci2-M alkylene-N3.

[0129] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-2o alkylene-N3), A2is -C3.2o alkyl.

[0130] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3is alkylene-N3), A2is -C8.2o alkyl.

[0131] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-IO alkylene-N3), A2is -Cio-is alkyl. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-7alkylene-N3), A2is -Cn.17 alkyl.

[0132] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-6 alkylene-N3), A2is -Cn-15 alkyl.

[0133] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-s alkylene-N3), A2is -C12-14 alkyl.

[0134] In one embodiment of the compound of formula (II), A1is -C(NH2)(C8-2o alkylene-N3), A2is -C8.2o alkyl. In one embodiment of the compound of formula (II), A1is -C(NH2)(C10-i8 alkylene-Ns), A2is -Cio-is alkyl. In one embodiment of the compound of formula (II), A1is -C(NH2)(Cu.i7alkylene-N3), A2is -Cn-i7alkyl. In one embodiment of the compound of formula (II), A1is -C(NH2)(Cu-i5alkylene-N3), A2is -Cn-15 alkyl. In one embodiment of the compound of formula (II), A1is -C(NH2)(Ci2.i4alkylene-N3), A2is -Ci2-i4 alkyl.

[0135] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-2o alkylene-N3), A2is -C3-20 alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(C3 is alkylene- N3), A2is — Cs-2o alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is - C(NH2)(C3.IO alkylene-N3), A2is -Cio-is alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(C3.7alkylene-N3), A2is -Cn-i7alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-6 alkylene-N3), A2is -Cn-15 alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-s alkylene-N3), A2is -C12-M alkylene- (Group Y). Group Y is selected from Group X, except alkynyl and N3.

[0136] In one embodiment of the compound of formula (II), A1is -C(NH2)(C8-2o alkylene-N3), A2is -C8-2o alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(Cio i8alkylene- N3), A2is -Cio-is alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is - C(NH2)(CH-I7alkylene-N3), A2is -Cn-i7alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(Cu.i5alkylene-N3), A2is -Cu.15 alkylene-(Group Y). In one embodiment of the compound of formula (II), A1is -C(NH2)(Ci2 i4alkylene-Ns), A2is -CU-M alkylene-(Group Y). Group Y is selected from Group X, except alkynyl and N3.

[0137] In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-2o alkylene-(Group Y)), A2is -C3- 20 alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-is alkylene-(Group Y)), A2is — C8-2o alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3 10 alkylene-(Group Y)), A2is -Cio-is alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-7alkylene-(Group Y)), A2is -Cn-i7alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-6 alkylene-(Group Y)), A2is -Cu-15 alkylene-N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C3-5 alkylene-(Group Y)), A2is -CU M alkylene-Ns. Group Y is selected from Group X, except alkynyl and N3. In one embodiment of the compound of formula (II), A1is -C(NH2)(C8-2o alkylene-(Group Y)), A2is -Cs- 20 alkylene-N3. In one embodiment of the compound of formula (II), A1is-C(NH2)(Cw-i8 alkylene-(Group

[0138] Y)), A2is -Cio-is alkylene-Ns. In one embodiment of the compound of formula (II), A1is -C(NH2)(CU-i7 alkylene-(Group Y)), A2is -Cn.17 alkylene-Ns. In one embodiment of the compound of formula (II), A1is -C(NH2)(Cu-i5 alkylene-(Group Y)), A2is -Cn-is alkylene-Ns. In one embodiment of the compound of formula (II), A1is -C(NH2)(Ci2-i4 alkylene-(Group Y)), A2is -C 12-14 alkylene-N3. Group Y is selected from Group X, except alkynyl and N3.

[0139] In the "alkylene-(Group Y)", the Group Y substituent may be present either in a terminal or a nonterminal position. Ci-6alkyl-diazirinyl is preferably present in a non-terminal position, whereas the other types of substituents of Group Y are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0140] In one embodiment of the compound of formula (II), A1is -(C3-20 alkylene-Group Z), A2is -C3-20 alkylene- Ns. In one embodiment of the compound of formula (II), A1is -(C3-15 alkylene-Group Z), A2is -Cs-zo alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(C3-10 alkylene-Group Z), A2is -Cio-is alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(C3-7 alkylene-Group Z), A2is -Cn.17 alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(C3-6alkylene-Group

[0141] Z), A2is -Cu-is alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(C3-5 alkylene- Group Z), A2is -C12-14 alkylene-Ns. Group Z is selected from Group X, except N3. Group Z is preferably diazirinyl or Ci-ealkyl-diazirinyl. In the "alkylene-(Group Z)", the Group Z substituent may be present either in a terminal or a non-terminal position. Ci-ealkyl-diazirinyl is preferably present in a nonterminal position, whereas the other types of substituents of Group Z are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0142] N=N

[0143] In one embodiment of the compound of formula (II), A1is -(Ci-is alkylene- V (-Co ealkylene)H), A2is -

[0144] N=N

[0145] C3-20 alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(Ci-is alkylene- V (-Co-

[0146] 6alkylene)H), A2is -Cs-zo alkylene-Ns. In one embodiment of the compound of formula (II), A1is -(Ci-is

[0147] N=N alkylene- V (-C0-6alkylene)H), A2is -Cio-is alkylene-N3. In one embodiment of the compound of

[0148] N=N formula (II), -(Ci-is alkylene- V (-Co-6alkylene)H), A2is -Cn.17 alkylene-Ns. In one embodiment of the

[0149] N=N compound of formula (II), A1is -(Ci-is alkylene- V (-Co-6alkylene)H), A2is -Cu-is alkylene-N3. In one embodiment of the compound of formula (II), A1is -(Ci-is alkylene- V (-Co-6alkylene)H), A2 alkylene-N3. The (-C0-6alkylene) is preferably methylene or ethylene, more preferably methylene.

[0150] The Group X substituents may be present either in a terminal or a non-terminal position. Ci-ealkyl- diazirinyl is preferably present in a non-terminal position, whereas the other types of substituents of Group X are preferably present in a terminal position. Diazirinyl is generally in a terminal position.

[0151] The compound of formula (II) preferably has the following formula (Ila) wherein R1, R2and R3are each independently selected from -H and Group X;

[0152] R4is selected from H, diazirino, oxo, -OH and -NH2; wherein at least one of R1and R4is not hydrogen; wherein it is preferred that R2is not hydrogen; n is an integer from 0 to 20; m is an integer from 0 to 20; u is an integer from 0 to 20; wherein the sum of u and m is in the range of from 2 to 20.

[0153] R4is preferably selected from diazirino and -NH2, more preferably R4is -NH2.

[0154] The members of Group X are preferably selected from transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Chalky l-diaziriny I and tetrazinyl.

[0155] It is preferred that R4is -NH2. Furthermore, u is preferably 0.

[0156] In one specific embodiment R4is diazirino, m is preferably 0, u is an integer of from 1 to 18 (preferably 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 3, 1 or 2, or even 2), wherein R1is preferably hydrogen. R2and R3are each independently preferably selected from C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci-ealkyl-diazirinyl and tetrazinyl; More preferably, R2and R3are each independently selected from — transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci-6a Ikyl- diazirinyl and tetrazinyl.

[0157] In one specific embodiment, R1or R2is -N3, R3is C2-10 alkynyl and R4is NH2 or diazirino.

[0158] Typically, n is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11.

[0159] It is preferred that m is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11.

[0160] Typically, u is an integer of from 0 to 17, preferably 0 to 14, more preferably 0 to 11, even more preferably 0 to 8, still more preferably 0 to 5, still even more preferably 0 to 2, most preferably 0.

[0161] It is furthermore preferred that the sum of m+ u is an integer of from 2 to 30, preferably 5 to 25, more preferably 7 to 22, even more preferably 10 to 18.

[0162] The present invention most preferably concerns a compound selected from the following compounds:

[0163]

[0164] It is to be understood that these compounds can also be present as a pharmaceutically acceptable salt or solvate. The salt is preferably a trifluoroacetic acid salt. Preferred solvates include hydrates.

[0165] The compounds described herein allow a determination of the metabolization status at a specific site during the investigation. The amino group or the diazirine group enables the conjugation of the molecule to the matrix. The alkyne or azido group enables the detection of the molecule at the matrix since one function is located in the acyl side chain and at least one functional group in the main chain.

[0166] The present invention also relates to a conjugate which is obtainable by reacting a compound as defined herein (typically a compound of formula (I) or (II) or any of the more specific formulae, such as (la), (Ila), etc.) with one, two or three further compound(s) (preferably one compound) comprising an alkyne (such as a -CECH group or a cycloalkyne), cycloalkene (such as transcyclooctenyl), tetrazinyl and / or -N3, wherein the conjugate preferably comprises a diazirine group, wherein

[0167] (i) when the compound according to any one of the preceding claims comprises an alkyne group (such as a -C=CH group or a cycloalkyne), one of the further compound(s) preferably contains an azide group; and (ii) when the compound according to any one of the preceding claims comprises an azide group, one of the further compound(s) preferably contains an alkyne group (such as a -C=CH group or a cycloalkyne); and

[0168] (iii) when the compound according to any one of the preceding claims comprises a cycloalkene (such as transcyclooctenyl) group, one of the further compound(s) preferably contains a tetrazinyl group; and

[0169] (iv) when the compound according to any one of the preceding claims comprises a tetrazinyl group, one of the further compound(s) preferably contains a cycloalkene (such as transcyclooctenyl) group.

[0170] The conjugate which is preferably obtainable by reacting a compound as defined herein (typically a compound of formula (I) or (II) or any of the more specific formulae, such as (la), (Ila), etc.) with one compound comprising an alkyne (such as a -C=CH group or a cycloalkyne), cycloalkene (such as transcyclooctenyl), tetrazinyl and / or -N3.

[0171] The conjugate preferably comprises a diazirine group. The alkyne, cycloalkene, and cycloalkyne, are preferably -C=CH, transcyclooctenyl, -and cyclooctinyl, respectively.

[0172] It is to be understood that the conjugate encompasses any pharmaceutically acceptable salts or solvates.

[0173] The present invention furthermore relates to a or a composition comprising the compound of the present invention or the conjugate of the present invention. This composition may also be referred to as an imaging composition. The term "comprising the compound of the present invention" indicates that it may comprise one or more of the compounds described herein, in particular one or more of the compounds of formula (I) or (II) or any of the more specific formulae, such as (la), (Ila), etc.

[0174] The compound of the present invention, or the conjugate, or a composition comprising the compound or conjugate of the present invention, is preferably for use in diagnostics. The diagnostics can, e.g., be in relation to a disease selected from cancer, diabetes, a neurodegenerative disease and an infectious disease.

[0175] The compound of the present invention, or the conjugate, or a composition comprising the compound or conjugate of the present invention, is preferably for use in the imaging of sphingomyelin and / or ceramide metabolism. Thus, the compound of the present invention, or the conjugate, or a composition comprising the compound or conjugate of the present invention, is typically used to identify abnormalities in the sphingomyelin and / or ceramide metabolism. These abnormalities may be related to a disease selected from cancer, diabetes, a neurodegenerative disease and an infectious disease.

[0176] For example, the compound of the present invention, or the conjugate, or a composition comprising the compound or conjugate of the present invention, can be used in relation to a disease selected from cancer, diabetes, a neurodegenerative disease and an infectious disease.

[0177] The present invention also concerns the use of a compound as set out herein, or a conjugate, or a composition comprising the compound or conjugate, as an analytical reference.

[0178] The present invention also concerns the use of a compound as set out herein, or a conjugate, or a composition comprising the compound or conjugate, as an in vitro screening tool.

[0179] The present invention also relates to the compounds disclosed herein having the mentioned functional groups wherein different tags are conjugated to molecules (e.g., Biotin, fluorescent dyes).

[0180] The compounds disclosed herein can be used with modern staining methods like confocal microscopy, Fluorescence Lifetime Imaging Microscopy (FLIM), expansion microscopy, Forster resonance energy transfer (FRET).

[0181] The compounds disclosed herein can be used for research and diagnosis of sphingomyelin and ceramide metabolism dysregulations in an illness, such as cancer, diabetes and neurodegenerative diseases and infectious diseases.

[0182] For example, the compounds disclosed herein can be used in staining methods for research and diagnosis of lysosomal storage diseases (LSD) such as acid sphingomylinase deficiency (ASMD), e.g. Nieman-Pick disease.

[0183] Alternatively, the compounds disclosed herein can be used for research studies on life cycles and proliferation mechanisms of intracellular viral or bacterial infections, e.g. Chlamydia-infections.

[0184] The present invention furthermore preferably relates to one of more of the following methods A to C: A) A method of collecting data for the diagnosis of a disorder associated with sphingomyelin and / or ceramide metabolism in a sample or a patient comprising:

[0185] (a) bringing a sample or a specific body part or body area suspected to contain an anomality in sphingomyelin and / or ceramide content into contact with a compound or conjugate as defined herein;

[0186] (b) allowing the compound or conjugate as defined herein to bind to the components of the sample;

[0187] (c) detecting the compound or conjugate as defined herein bound to the components of the sample, and

[0188] (d) optionally correlating the presence or absence of compound or conjugate as defined herein binding with the components of the sample with the presence or absence of an anomality in sphingomyelin and / or ceramide content in the sample or specific body part or body area.

[0189] B) A method of collecting data for monitoring residual disorder in a patient suffering from a disorder associated with sphingomyelin and / or ceramide metabolism who has been treated with a medicament, wherein the method comprises:

[0190] (a) bringing a sample or a specific body part or body area suspected to contain anomality in sphingomyelin and / or ceramide content into contact with a compound or conjugate as defined herein;

[0191] (b) allowing the compound or conjugate as defined herein to bind to components of the sample to form a complex;

[0192] (c) detecting the formation of the complex;

[0193] (d) optionally correlating the presence or absence of the complex with the presence or absence of anomality in sphingomyelin and / or ceramide content in the sample or specific body part or body area; and

[0194] (e) optionally comparing the amount of the complex to a normal control value.

[0195] C) A method of collecting data for predicting responsiveness of a patient suffering from a disorder associated with sphingomyelin and / or ceramide metabolism and being treated with a medicament comprising:

[0196] (a) bringing a sample or a specific body part or body area suspected to contain an anomality in sphingomyelin and / or ceramide content into contact with a compound or conjugate as defined herein;

[0197] (b) allowing the compound or conjugate as defined herein to bind to components of the sample to form a complex;

[0198] (c) detecting the formation of the complex;

[0199] (d) optionally correlating the presence or absence of the complex with the presence or absence of an anomality in sphingomyelin and / or ceramide content in the sample or specific body part or body area; and (e) optionally comparing the amount of the complex to a normal control value.

[0200] The term "associated with sphingomyelin and / or ceramide metabolism" preferably means "associated with an anomality of sphingomyelin and / or ceramide metabolism".

[0201] It is to be understood that any definitions (including any preferred definitions) provided for A1, A2and A3for one formula are also applicable to A1, A2and A3, respectively, in any other formula provided herein, where one or more of A1, A2and A3appear. Furthermore, it is to be understood that any definitions (including any preferred definitions) provided for R1, R2and R3for one formula are also applicable to R1, R2and R3, respectively, in any other formula provided herein, where one or more of R1, R2and R3appear.

[0202] The following definitions apply throughout the present specification and the claims, unless specifically indicated otherwise.

[0203] The term "hydrocarbon group" refers to a group consisting of carbon atoms and hydrogen atoms.

[0204] The term "alicyclic" is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.

[0205] As used herein, the term "alkyl" refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an "alkyl" group does not comprise any carbon- to-carbon double bond or any carbon-to-carbon triple bond. A "Ci-5alkyl" denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term "alkyl" preferably refers to CM alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.

[0206] As used herein, the term "alkenyl" refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term "C2-5 alkenyl" denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-l-en-l-yl, prop-l-en-2-yl, or prop-2-en-l-yl), butenyl, butadienyl (e.g., buta-1,3- dien-l-yl or buta-l,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term "alkenyl" preferably refers to C2-4 alkenyl. As used herein, the term "alkynyl" refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term "C2-5 alkynyl" denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term "alkynyl" preferably refers to C2-4 alkynyl.

[0207] As used herein, the term "alkylene" refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A "C1.5 alkylene" denotes an alkylene group having 1 to 5 carbon atoms; the term "Co s alkylene" indicates that a covalent bond (corresponding to the option "Co alkylene") or a C1-5 alkylene is present. Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2- CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2-CH2-CH2-CH2-). Unless defined otherwise, the term "alkylene" preferably refers to alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.

[0208] As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). "Cycloalkyl" may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, "cycloalkyl" preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3.7cycloalkyl. A particularly preferred "cycloalkyl" is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, particularly preferred examples of a "cycloalkyl" include cyclohexyl or cyclopropyl, particularly cyclohexyl.

[0209] As used herein, the term "cycloalkenyl" refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. "Cycloalkenyl" may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, "cycloalkenyl" preferably refers to a C3-u cycloalkenyl, and more preferably refers to a C3.7cycloalkenyl. A particularly preferred "cycloalkenyl" is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds. As used herein, the term "cycloalkynyl" refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and preferably does not comprise any carbon-to-carbon double bond. N. s n> — ?■

[0210] As used herein, the term diazirinyl refers to , The term "Ci-ealkyl-diazirinyl" refers to N wherein the hydrogen is replaced by a Ci-galkyl group, preferably a Ci.3alkyl, more preferably by a methyl or ethyl group, most preferably by a methyl group. Thus, the term "Ci-ealkyl-diazirinyl"

[0211] N. s n / > — <• preferably refers to N wherein the hydrogen is replaced by a methyl group.

[0212] The term "oxo" indicates the presence of a C=O group. Thus, the term "R4is oxo" means that R4together with the carbon atom to which it is bound forms a C=O group. It will be understood that in this case, the hydrogen atom at the carbon atom to which R4is bound becomes obsolete. In other words, the term "R4is oxo" indicates that is replaced by O .

[0213] N. n>

[0214] The term "diazirino" indicates the presence of a N group. Thus, the term "R4is diazirino" means hk n> that R4together with the carbon atom to which it is bound forms a N group. It will be understood that in this case, the hydrogen atom at the carbon atom to which R4is bound becomes obsolete. In other words, the term "R4is diazirino" indicates that is replaced by N=N .

[0215] The terms "bond" and "covalent bond" are used herein synonymously, unless explicitly indicated otherwise or contradicted by context. As used herein, the terms "optional", "optionally" and "may" denote that the indicated feature may be present but can also be absent. Whenever the term "optional", "optionally" or "may" is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y" (or "X may be substituted with Y") means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional", the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

[0216] Various groups are referred to as being "optionally substituted" in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the "optionally substituted" groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.

[0217] A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, preferred attachment positions for the various specific substituent groups are as illustrated in the examples.

[0218] As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a", "an" and "the" are used interchangeably with "one or more" and "at least one". Thus, for example, a composition comprising "a" compound of formula (I) can be interpreted as referring to a composition comprising "one or more" compounds of formula (I).

[0219] It is to be understood that wherever numerical ranges are provided / disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.

[0220] As used herein, the term "comprising" (or "comprise", "comprises", "contain", "contains", or "containing"), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia", i.e., "containing, among further optional elements, In addition thereto, this term also includes the narrower meanings of "consisting essentially of" and "consisting of". For example, the term "A comprising B and C" has the meaning of "A containing, inter alia, B and C", wherein A may contain further optional elements (e.g., "A containing B, C and D" would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C" and the meaning of "A consisting of B and C" (i.e., no other components than B and C are comprised in A).

[0221] The scope of the present invention embraces all pharmaceutically acceptable salt forms of the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as / V,A / -dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p- toluenesulfonate (tosylate), 2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. A pharmaceutically acceptable salt of the compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) is preferably not a hydroiodide salt. Preferred pharmaceutically acceptable salts of the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, an oxalate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) is a hydrochloride salt. Accordingly, if a compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), including any one of the specific compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) described herein, is provided in the form of a pharmaceutically acceptable salt, it is preferred that the respective compound is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, an oxalate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that it is in the form of a hydrochloride salt.

[0222] The present invention also specifically relates to the compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), including any one of the specific compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) described herein, in non-salt form.

[0223] Moreover, the scope of the invention embraces the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds of the formula (I) are likewise embraced by the invention.

[0224] Furthermore, the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis / trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto / enol tautomers or thione / thiol tautomers). All such isomers of the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures / racemates). The racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present invention further encompasses any tautomers of the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these). It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms. The formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.

[0225] The scope of the invention also embraces compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more atoms are replaced by a specific isotope of the corresponding atom. For example, the invention encompasses compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e.,2H; also referred to as "D"). Accordingly, the invention also embraces compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) which are enriched in deuterium. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 (1H) and about 0.0156 mol-% deuterium (2H or D). The content of deuterium in one or more hydrogen positions in the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) can be increased using deuteration techniques known in the art. For example, a compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) or a reactant or precursor to be used in the synthesis of the compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) can be subjected to an H / D exchange reaction using, e.g., heavy water (D2O). Further suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11- 12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy. Unless specifically indicated otherwise, it is preferred that the compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or1H hydrogen atoms in the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) is preferred.

[0226] The present invention also embraces compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g.,1SF,UC,13N,15O,76Br,77Br,120l and / or124l. Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET). The invention thus includes (i) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by18F atoms, (ii) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced bynC atoms, (iii) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by13N atoms, (iv) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by15O atoms, (v) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by76Br atoms, (vi) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by77Br atoms, (vii) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by120l atoms, and (viii) compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by124l atoms. In general, it is preferred that none of the atoms in the compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) are replaced by specific isotopes.

[0227] The compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these), as well as the conjugates of the present invention may be administered as compounds per se or may be formulated as compositions. The compositions, in particular pharmaceutical compositions, preferably for imaging, may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and / or solubility enhancers.

[0228] The pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including polyethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor’ HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, a-cyclodextrin, £- cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin, hydroxypropyl-P-cyclodextrin, hydroxyethyl-y-cyclodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl-p-cyclodextrin, sulfobutylether-P-cyclodextrin, sulfobutylether-y-cyclodextrin, glucosyl-a-cyclodextrin, glucosyl-p- cyclodextrin, diglucosyl-P-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-p-cyclodextrin, maltosyl-y- cyclodextrin, maltotriosyl-p-cyclodextrin, maltotriosyl-y-cyclodextrin, dimaltosyl-P-cyclodextrin, methyl-p-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.

[0229] The pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.

[0230] The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy", Pharmaceutical Press, 22ndedition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include tablets - in particular coated and uncoated tablets -, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.

[0231] The compounds of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) or the above described pharmaceutical compositions comprising a compound of any one of the formulae shown herein (in particular formulae (I) and (II) and any formulae encompassed by these) may be administered to a subject by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.

[0232] The present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Particularly preferred routes of administration are oral administration or parenteral administration. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject being imaged.

[0233] A proposed, yet non-limiting dose of the compounds according to the invention for oral administration to a human (of approximately 70 kg body weight) may be 0.05 to 2000 mg, preferably 0.1 mg to 1000 mg, of the active ingredient per unit dose. The unit dose may be administered, e.g., 1 to 3 times per day.

[0234] The subject or patient to be imaged in accordance with the present invention may be an animal (e.g., a human or non-human animal). Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, the subject / patient to be imaged in accordance with the invention is a human.

[0235] It is to be understood that the present invention specifically relates to each and every combination of features described herein, including any combination of general and / or preferred features. In particular, the invention specifically relates to each combination of meanings (including general and / or preferred meanings) for the various groups and parts of compounds described herein.

[0236] In this specification, a number of documents including patent applications, scientific literature and manufacturers' manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0237] The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates. An aspect of the present invention may also be summarized by the following items 1 to 10, which use a self-contained definition of R, R', R1and R2that is separate from the definition of these groups used outside of the following items: Item 1: Two new trifunctional sphingomyelin derivatives, called TFSMl and TFSM2, which are characterized by a terminal alkyne in the phosphoryl choline group, an azide in either the sphingoid backbone or the acyl side chain and a primary amino group in the a-position of the acyl chain.

[0238] TFSM 1. R = N3; R' = H

[0239] TFSM 2: R = H; R' = N3

[0240] Item 2: Two new bifunctional ceramide derivatives, defined as Cerl6-1 and Cerl6-2, the molecules have a sphingoid backbone composed of 18 carbon atoms and a side chain composed of 16 carbon atoms, wherein an azido function is either at the end of the backbone or the side chain and a primary amino group is located at the a-position of the side chain.

[0241] Item 3: A sphingomyelin derivate wherein the molecule has only one or two of the afore mention three modifications (terminal -alkin, terminal -azid, intramolecular Amino-group).

[0242] Item 4: A ceramide derivate wherein the molecule has only one of the two afore mentioned modifications (terminal azid, or intramolecular amino group).

[0243] Item 5: The compounds according to items 1-4 which have the afore mentioned functional groups and wherein different tags are conjugated to molecules (e.g., Biotin, fluorescent dyes). Item 6: Chemical synthesis of the afore mentioned molecules (item 1-4), described in detail in Schemes 1 and 2.

[0244] Item 7: The compounds according to items 1-6, wherein the molecules are used with modern staining methods like confocal microscopy, Fluorescence Lifetime Imaging Microscopy (FLIM), expansion microscopy, Forster resonance energy transfer (FRET).

[0245] Item 8: The compounds according to items 1-6, used in staining methods according to item 7 for research and diagnosis of sphingomyelin and ceramide metabolism dysregulations in illness, such as cancer, diabetes and neurodegenerative diseases and infectious diseases.

[0246] Item 9: The compounds according to items 1-6, used in staining methods according to item 8 for research and diagnosis of lysosomal storage diseases (LSD) such as acid sphingomylinase deficiency (ASMD), e.g. Nieman-Pick disease.

[0247] Item 10: The compounds according to items 1-6, used in staining methods according to item 8 for research studies on life cycles and proliferation mechanisms of intracellular viral or bacterial infections, e.g. Chlamydia-infections.

[0248] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

[0249] EXAMPLES

[0250] The compounds / examples described in this section are defined by their chemical formulae and their corresponding chemical names. In case of conflict between any chemical formula and the corresponding chemical name indicated herein, the present invention relates to both the compound / example defined by the chemical formula and the compound / example defined by the chemical name, and particularly relates to the compound / example defined by the chemical formula.

[0251] Synthesis of multifunctional sphingomyelin and ceramide derivates

[0252] Multifunctional sphingomyelin and ceramide derivatives

[0253] Sphingomyelin derivatives:

[0254] Two new trifunctional sphingomyelin derivatives, called TFSM1 and TFSM2, were synthesized (see Scheme 1). The molecules are characterized by a terminal alkyne in the phosphoryl choline group, an azide in either the sphingoid backbone or the acyl side chain and a primary amino group in the a- position of the acyl chain.

[0255] Ceramide derivatives: Two new bifunctional ceramide derivatives, defined as Cerl6-1 and Cerl6-2, were synthesized (see Scheme 2). The molecules have a sphingoid backbone composed of 18 carbon atoms and a side chain composed of 16 carbon atoms. An azido function is either at the end of the backbone or the side chain and a primary amino group is located at the a-position of the side chain.

[0256] Synthesis of trifunctional sphingomyelin derivatives

[0257] 15: R = N3; R' = H TFSM 1: R = N3; R' = H 16: R = H; R' = N3TFSM 2: R = H; R' = N3

[0258] Scheme 1: Synthesis of trifunctional sphingomyelin derivatives TFSM1 and TFSM2.

[0259] Synthesis of bifunctional ceramide derivatives

[0260] Scheme 2: Synthesis of bifunctional ceramide derivatives Cerl6-1 and Cerl6-2. Abbreviations anh: anhydrous; ar: aromatic; Boc: tert-butyloxycarbonyl; calcd: calculated; CH: cyclohexane; DCM: dichloromethane, DIPEA: A / ,A / -diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMF: / V, / V-dimethylformamide, EtOAc: ethyl acetate; EtOH: ethanol; eq. / equiv.: equivalents; HATU: 1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium-3-oxide hexa-fluorophosphate; iPrOH: iso-propanol; MeCN: acetonitrile; MeOH: methanol; MOM: methoxymethyl; TBAF: tetra-n-butylammonium fluoride; TBS: tert-butyldimethylsilyl; TFA: trifluoroacetic acid; THF: tetra hydrofuran; TLC: thin layer chromatography; TsCI: tosyl chloride.

[0261] General Information for Chemical Synthesis Air- and moisture-sensitive reactions were performed under nitrogen atmosphere using Schlenk techniques. Commercially available reagents were purchased from Acros Organics, Alfa Aesar, BACHEM, Sigma Aldrich, TCI, Thermo Scientific, Apollo Scientific, Merck and used without further purification. All solvents were purified by distillation prior to use. Anhydrous DMF, THF and DCM were obtained by a solvent purification system (PureSolv MD 5 by Inert) or purchased from Sigma Aldrich. Distilled water was used for aqueous workup. Analytical thin layer chromatography was performed on aluminium plates {ALUGRAM Xtra SIL G / UV254) from Macherey-Nagel coated with silica gel. The plates were stained with aqueous KMnCU solution (1.50 g KMnO4, 10.0 g K2CO3 and 100 mg NaOH in 200 mL distilled H2O) or ninhydrin solution (600 mg, ninhydrin, 200 mL 1-butanol, 6.0 mL cone, acetic acid) for visualization.

[0262] Column chromatography was performed on silica gel (Macherey-Nagel, Silica 60, particle size 0.040- 0.063 mm) in self-packed glass columns. Eluents were used as described in the respective procedure. In some cases, deactivated silica gel was used: Silica gel and 10 wt% K2CO3 were suspended in MeOH and the mixture was heated under reflux for 6 h. The mixture was filtered and the silica gel was thoroughly washed with methanol and dried under reduced pressure.

[0263] NMR spectra were measured at room temperature on a Bruker AVANCE III 400 FT-NMR spectrometer. Chemical shifts are reported in parts per million (ppm, 6-scale) referring to the residual solvent peak fH: CDCI3: 6 = 7.26 ppm, CD3OD: 6 = 3.31 ppm;13C: CDCI3: 6 = 77.1 ppm, CD3OD: 5 = 49.0 ppm). Analysis followed first order and data is reported in the following order: chemical shift (in ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, br dd = broad doublet of doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublet of doublets, ddt = doublet of doublets of triplets, dt = doublet of triplets, dtd = doublet of triplet of doublets, t = triplet, td = triplet of doublets, q = quartet, m = multiplet, br m = broad multiplet), coupling constant(s) (in Hz), integration. The stated atom numbers of C- / H-atoms for the attribution of NMR signals are not conform to IUPAC nomenclature and were chosen for clarity reasons. Signal assignment was accomplished with additional information from DEPT135,1H,1H-COSY,1H,13C-HSQC and1H,13C-HMBC measurements.

[0264] Mass spectra were recorded with a Bruker Daltonics micrOTOF and micrOTOF-Q III spectrometer by electrospray ionization (ESI).

[0265] Chemical Synthesis tert-Butyl-((2S,3R,E)-18-azido-l-((tert-butyldimethylsilyl)oxy)-3-(methoxymethoxy)octadec-4-en-2- yl)-carbamate (5) The starting material of this reaction was prepared in a multi-step synthesis according to a literature procedure (cf. NPL1). tert-Butyl-((2S,3R,E)-18-azido-l-((tert-butyldimethylsilyl)oxy)-3-hydroxyoctadec-4-en-2-yl)-carbamate (1.39 g, 2.50 mmol, 1.00 equiv.) was dissolved in anh. DCM (20 mL). The solution was cooled to 0 °C and MOM-CI (3.81 mL, 50.1 mmol, 20.0 equiv.) and DIPEA (8.52 mL, 50.1 mmol, 20.0 equiv.) were added. The solution was stirred for 4 h, and then diluted with DCM (40 mL). The organic phase was washed with sat. aq. NaHCOs solution (40 mL), sat. aq. NH4CI solution (30 mL), brine (30 mL) and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CH / EtOAc = 25 / 1) to yield the title compound (1.28 g, 2.14 mmol, 85%) as a highly viscous colorless oil.

[0266] JH NMR (400 MHz, CDCI3): 6 = 5.69 (dt,3J = 15.2 Hz,3J = 6.8 Hz, 1H, H-5), 5.32 (dd,3J = 15.2 Hz,3J = 8.3 Hz, 1H, H-4), 4.69 (d,2J = 6.5 Hz, 1H, H-l", superimposes NH signal), 4.51 (d,2J = 6.5 Hz, 1H, H-l"), 4.07 (br dd,3J = 7.7 Hz,3J = 7.7 Hz, 1H, H-3), 3.88-3.80 (m, 1H, H-l), 3.72-3.61 (m, 2H, H-2, H-l), 3.35 (s, 3H, H-2"), 3.25 (t,3J = 7.0 Hz, 2H, H-18), 2.03 (td,3J = 6.8 Hz,3J = 6.8 Hz, 2H, H-6), 1.64-1.55 (m, 2H, H-17), 1.42 (s, 9H, H-3'), 1.38-1.23 (m, 20H, H-7-16), 0.89 (s, 9H, H-3a), 0.05 (2 x s, 6H, H-la) ppm;13C NMR (100 MHz, CDCI3): 6 = 155.5 (C-l'), 137.0 (C-5), 126.8 (C-4), 93.6 (C-l"), 79.1 (C-2'), 76.0 (C-3), 61.7 (C-l), 55.6 (C-2"), 54.9 (C-2), 51.6 (C-18), 32.4 (C-6), 28.9 (C-17), 28.5 (3C, C-3'), 29.73, 29.69, 29.67, 29.62, 29.56, 29.3, 29.24, 29.20, 26.8 (10C, C-7-16), 26.0 (3C, C-3a), 18.3 (C-2a), -5.3, -5.4 (2C, C-la) ppm; HRMS (ESI+): m / z calcd for C3iH62N4NaO5Si [M+Na]+621.4382, found 621.4368, | Am / z| = 2.2 ppm. tert-Butyl-((2S,3R,E)-18-azido-l-(hydroxy-3-(methoxymethoxy)octadec-4-en-2-yl)-carba- mate (7) tert-Butyl-((2S,3R,E)-18-azido-l-((tert-butyldimethylsilyl)oxy)-3-(methoxymethoxy)octadec-4-en-2- yl)-carbamate (5, 253 mg, 422 pmol, 1.00 equiv.) was dissolved in anh. THE (9 mL) and the solution was cooled to 0 °C. TBAF solution (1 M in THE, 591 pL, 591 pmol, 1.40 equiv.) was added dropwise and the solution was stirred for 2 h. Then, H2O (50 mL) was added and the aqueous phase was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (50 mL) and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CH / EtOAc = 4 / 1) to yield the title compound (182 mg, 411 pmol, 97%) as a colorless oil.3H NMR (400 MHz, CDCI3): 6 = 5.75 (dt,3J = 15.4 Hz,37 = 7.0 Hz, 1H, H-5), 5.34 (ddt,3J = 15.5 Hz,3J = 8.0 Hz,4J = 1.3 Hz, 1H, H-4), 5.23 (d,3J = 7.2 Hz, 1H, NH), 4.66 (d,2J = 6.6 Hz, 1H, H-l"), 4.52 (d,2J = 6.6 Hz, 1H, H-l"), 4.23 (dd,3J = 7.7 Hz,3J = 4.7 Hz, 1H, H-3), 3.94 (dd,2J = 11.3 Hz,3J = 3.5 Hz, 1H, H- 1), 3.66 (dd,2J = 11.5 Hz,3J = 3.3 Hz, 1H, H-l), 3.66-3.59 (m, 1H, superimposes H-l signal, H-2), 3.37 (s, 3H, H-2"), 3.25 (t,3J = 7.0 Hz, 2H, H-18), 2.04 (dt,3J = 6.8 Hz,3J = 6.8 Hz, 2H, H-6), 1.63-1.54 (m, 2H, H-17), 1.44 (s, 9H, H-3'), 1.39-1.24 (m, 20H, H-7-16) ppm;13C NMR (100 MHz, CDCI3): 6 = 156.0 (C-l'), 137.1 (C-5), 126.0 (C-4), 93.9 (C-l"), 79.6 (C-2'), 78.6 (C-3), 62.5 (C-l), 55.8 (C-2"), 55.0 (C-2), 51.6 (C- 18), 32.4 (C-6), 28.9 (C-17), 28.4 (3C, C-3'), 29.70, 29.67, 29.65, 29.60, 29.55, 29.5, 29.2, 29.1, 26.8 (IOC, C-7-16) ppm; HRMS (ESI+): m / z calcd for C25H48N4NaO5 507.3517, found 507.3500, |Am / z| = 3.3 ppm. tert-Butyl-((2S,3R,E)-18-azido-l-(((2-bromoethoxy)(hydroxy)phosphoryl)oxy)-3-(methoxy-methoxy)- octadec-4-en-2-yl)carbamate (9)

[0267] The introduction of the phosphate ester was performed following a modified procedure by Pinkert et al (cf. NPL2). beta-Bromoethylphosphoryl dichloride (11.0 pL, 85.8 pmol, 4.00 equiv.) was dissolved in anh. DCM (0.3 mL) and the solution was cooled to 0 °C. Pyridine (10.4 pL, 129 pmol, 6.00 equiv.) was added and the mixture was stirred for 5 min. Then, tert-butyl ((2S,3R,E)-18-azido-l-hydroxy-3- (methoxymethoxy)octadec-4-en-2-yl)carbamate (7, 10.4 mg, 21.5 pmol, 1.00 equiv.), dissolved in anh. DCM (0.3 mL), was added dropwise and the reaction was stirred for further 4 h at 0 °C. Sat. aq. NaHCO3solution (2 mL) was added and the solution was stirred vigorously at rt for 20 min. CHCI3(20 mL), MeOH (5 mL) and H2O (15 mL) were added. The organic phase was separated and the aqueous phase was extracted with CHCI3(20 mL). The combined organic layers were dried over MgSO4and the crude product was purified by column chromatography (DCM / MeOH = 15 / 1 -> 10 / 1 -> 4 / 1) to yield the title compound (11.8 mg, 17.6 pmol, 82%) as a colorless waxy solid.

[0268] 3H NMR (400 MHz, CD3OD): 6 = 5.74 (dt,3J = 15.1 Hz,3J = 6.8 Hz, 1H, H-5), 5.33 (ddt,3J = 15.4 Hz,3J = 8.7 Hz,AJ = 1.4 Hz, 1H, H-4), 4.68 (d,2J = 6.6 Hz, 1H, H-l"), 4.51 (d,2J = 6.6 Hz, 1H, H-l"), 4.20-4.12 (m, 2H, H-la), 4.11-4.02 (m, 2H, H-l, H-3), 4.02-3.93 (m, 1H, H-l), 3.81-3.73 (m, 1H, H-2), 3.59 (t,3J = 6.3 Hz, 2H, H-2a), 3.36 (s, 3H, H-2"), 3.28 (t,3J = 6.8 Hz, 2H, H-18), 2.07 (td,3J = 6.8 Hz,3J = 6.7 Hz, 2H, H-6), 1.63-1.54 (m, 2H, H-17), 1.44 (s, 9H, H-3'), 1.41-1.28 (m, 20H, H-7-16) ppm;13C NMR (100 MHz, CD3OD): 6 - 158.0 (C-l'), 138.5 (C-5), 127.8 (C-4), 94.6 (C-l"), 80.2 (C-2'), 77.7 (C-3), 66.7 (d, 7C-P = 5.0 Hz, C-la), 65.8 (d, JC-P - 5.2 Hz, C-l), 56.1 (C-2"), 55.6 (d, JC-P = 7.8 Hz, C-2), 52.4 (C-18), 33.5 (C-6), 32.0 (d, JC-P = 8.1 Hz, C-2a), 30.77, 30.74, 30.71, 30.67, 30.65, 30.63, 30.33, 30.29, 27.8 (10C, C-7-16), 29.9 (C-17), 28.9 (3C, C-3') ppm; HRMS (ESI-): m / z calcd for C27H5iBrN4O8P [M-H]“ 669.2633, found 669.2635, | Am / z| = 0.2 ppm.

[0269] (2S,3R,E)-18-Azido-2-((tert-butoxycarbonyl)amino)-3-(methoxymethoxy)octadec-4-en-l-yl (2-

[0270] (dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (11) tert-Butyl-((2S,3R,E)-18-azido-l-(((2-bromoethoxy)(hydroxy)phosphoryl)oxy)-3-(methoxy- methoxy)octadec-4-en-2-yl)carbamate (9, 11.8 mg, 17.6 pmol, 1.00 equiv.) was dissolved in CHCI3 (0.3 mL), MeCN (0.3 mL) and iPrOH (0.3 mL). A / ,A / -Dimethylprop-2-yn-l-amine (0.3 ml) was added and the reaction mixture was stirred for 20 h at rt, then for 21 h at 45 °C. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CHCI3 / MeOH / H2O - 55 / 45 / 3) to yield the title compound (7.40 mg, 11.0 pmol, 63%) as a colorless waxy solid.

[0271] XH NMR (400 MHz, CD3OD): 6 = 5.74 (dt,3J = 15.2 Hz,3J = 6.8 Hz, 1H, H-5), 5.32 (ddt,3J = 15.3 Hz,3J = 8.6 Hz,4J = 1.4 Hz, 1H, H-4), 4.68 (d,2J = 6.6 Hz, 1H, H-l"), 4.51 (d,2J = 6.6 Hz, 1H, H-l"), 4.44 (d,4J = 2.4 Hz, 2H, H-4a), 4.33-4.25 (m, 2H, H-la), 4.10-3.96 (m, 3H, H-l, H-3), 3.78-3.68 (m, 3H, H-2, H-2a), 3.57 (t,4J = 2.5 Hz, 1H, H-6a), 3.36 (s, 3H, H-2"), 3.28 (s, 6H, H-3a), 3.28 (t,3J = 6.8 Hz, 2H, H-18, superimposed by H-3a signal), 2.07 (dt,3J = 6.8 Hz,3J - 6.8 Hz, 2H, H-6), 1.64-1.54 (m, 2H, H-17), 1.43 (s, 9H, H-3'), 1.40-1.27 (m, 20H, H-7-16) ppm;13C NMR (100 MHz, CD3OD): 6 = 158.0 (C-l'), 138.6 (C- 5), 127.9 (C-4), 94.5 (C-l"), 83.4* (C-5a), 80.1 (C-2'), 77.6 (C-3), 72.0* (C-6a), 65.9 (d, JC-P = 5.4 Hz, C-l), 65.3 (d, JC-P = 7.3 Hz, C-2a), 60.2 (d, JC-p = 5.0 Hz, C-la), 56.4 (C-4a), 56.1 (C-2"), 55.6 (d, JC.P= 7.9 Hz, C- 2), 52.4 (C-18), 52.0 (2C, C-3a), 33.4 (C-6), 30.76, 30.73, 30.70, 30.66, 30.64, 30.61, 30.32, 30.28, 30.27, 27.8 (10C, C-7-16), 29.9 (C-17), 28.9 (3C, C-3') ppm; HRMS (ESI+): m / z calcd for C32H6oN5Na08P [M+Na]+696.4072, found 696.4075, | Am / z| = 0.5 ppm.

[0272] *H-6a is exchanged by deuterium. This leads to a very low intensity of the alkyne signals of C-5a and C-6a. The signal of H-6a is only detected upon immediate measurement after dissolving the sample in CD3OD.

[0273] Perfluorophenyl (S)-2-((tert-butoxycarbonyl)amino)hexanoate (13)

[0274] A / -Boc-i_-norleucin (200 mg, 865 nmol, 1.00 equiv.) and pentafluorophenol (239 mg, 1.30 mmol, 1.50 equiv.) were dissolved in anh. DCM (8 mL). The solution was cooled to 0 °C and EDC hydrochloride (249 mg, 1.30 mmol, 1.50 equiv.) and DMAP (10.6 mg, 86.5 pmol, 0.10 equiv.) were added. The reaction mixture was warmed to rt and stirred for 16 h. The mixture was diluted with DCM (40 ml) and the organic phase was washed with HCI solution (1 M, 2 x 40 ml), then with sat. aq. NaHCCh solution (2 x 40 mL). The organic layer was dried over MgSO4and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (CH / EtOAc = 20 / 1) to afford the title compound (212 mg, 533 pmol, 62%) as a colorless oil.

[0275] Hi NMR (400 MHz, CDCI3): 6 = 4.99 (d,3J = 8.1 Hz, 1H, NW), 4.73-4.56 (m, 1H, H-2), 2.04-1.90 (m, 1H, H-3), 1.89-1.74 (m, 1H, H-3), 1.47 (s, 9H, H-3"), 1.45-1.35 (m, 4H, H-4-5), 0.94 (t,3J = 7.1 Hz, 3H, H-6) ppm;13C NMR (100 MHz, CDCI3): 5 = 169.4 (C-l), 155.2 (C-l"), 141.1 (m, 2C, Car), 139.7 (m, Car), 138.0 (m, 2C, Car), 124.9 (m, Car), 80.6 (C-2"), 53.6 (C-2), 32.0 (C-3), 28.3 (3C, C-3"), 27.3, 22.3 (C-4-5), 13.8 (C-6) ppm; HRMS (ESI+): m / z calcd for Ci7H2oF5NNa04 [M+Na]+420.1205, found 420.1213, | Am / z| = 1.9 ppm.

[0276] (2S,3R,E)-18-Azido-2-((S)-2-((tert-butoxycarbonyl)amino)hexanamido)-3-hydroxyoctadec-4-en-l-yl (2- (dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (15)

[0277] (2S,3R,E)-18-Azido-2-((tert-butoxycarbonyl)amino)-3-(methoxymethoxy)octadec-4-en-l-yl (2-(dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (11, 23.1 mg, 34.3 pmol, 1.00 equiv.) was dissolved in anh. MeOH (2 mL). HCI solution (4 M in 1,4-dioxane, 214 pL, 857 pmol, 25.0 equiv.) was added and the solution was stirred for 2 h at rt. The solution was purged with nitrogen for 10 min and the solvent was removed under reduced pressure. TLC and crudeXH NMR confirmed successful deprotection of the MOM and Boc group. The crude product was subjected to the following reaction conditions without further purification.

[0278] Crude (2S,3R,E)-2-amino-18-azido-3-hydroxyoctadec-4-en-l-yl-(2-(dimethyl(prop-2-yn-l-yl)- ammonio)ethyl) phosphate and pentafluorophenyl (S)-2-((tert-butoxycarbonyl)amino)-hexanoate (13, 20.4 mg, 51.4 pmol, 1.50 equiv.) were dissolved in anh. DCM (2 ml). DIPEA (11.9 mL, 68.6 pmol, 2.00 equiv.) was added and the solution was stirred for 18 h at rt. The solvent was removed under reduced pressure and the crude product was purified by column chromatography on deactivated silica gel (CHCI3 / MeOH / H2O = 85 / 15 / 1) to yield the title compound (21.6 mg, 29.1 pmol, 85%) as a colorless oil.XH NMR (400 MHz, CD3OD): 6 = 7.84 (d,3J = 8.9 Hz, NH), 6.65 (d,3J = 8.3 Hz, NH), 5.72 (dt,3J = 15.2 Hz,3J = 6.8 Hz, 1H, H-5), 5.45 (dd,3J = 15.3 Hz,3J = 7.3 Hz, 1H, H-4), 4.44 (d,4J = 2.3 Hz, 2H, H-4a), 4.36- 4.25 (m, 2H, H-la), 4.18-4.00 (m, 3H, H-l, H-3, H-2'), 4.00-3.89 (m, 2H, H-l, H-2), 3.79-3.70 (m, 2H, H- 2a), 3.56 (t,4J = 2.5 Hz, 1H, H-6a), 3.28 (s, 6H, H-3a), 3.28 (t,3J = 6.8 Hz, 2H, H-18, signal superimposed by H-3a signal), 2.03 (td,3J = 7.2 Hz,3J = 6.9 Hz, 2H, H-6), 1.80-1.67 (m, 1H, H-3'), 1.64-1.50 (m, 3H, H- 17, H-3'), 1.45 (s, 9H, H-3"), 1.42-1.26 (m, 24H, H-7-16, H-4'-5'), 0.93 (t,3J = 6.5 Hz, 3H, H-&) ppm;13C NMR (100 MHz, CD3OD): 5 = 175.0 (C-l'), 157.7 (C-l"), 135.2 (C-5), 130.9 (C-4), 83.2 (C-5a), 80.4 (C- 2"), 72.4 (C-6a), 72.3 (C-3), 65.7 (d, JC-P = 5.3 Hz, C-l), 65.3 (d, JC.P= 7.5 Hz, C-2a), 60.3 (d, JC-P = 4.8 Hz, C-la), 56.4 (C-4a), 56.1 (C-2'), 55.3 (d, JC-p = 7.5 Hz, C-2), 52.4 (C-18), 52.0 (2C, C-3a), 33.9 (C-3'), 33.5 (C-6), 30.79, 30.74, 30.68, 30.6, 30.5, 30.28, 30.25, 29.2, 27.8, 23.5 (12C, C-7-16, C-4'-5'), 29.9 (C-17), 28.8 (3C, C-3"), 14.4 (C-6) ppm; HRMS (ESI+): m / z calcd for C35H67N6NaO8P [M+Na]+765.4650, found 765.4669, |Am / z| = 2.4 ppm.

[0279] (2S,3R,E)-2-((S)-2-ammoniohexanamido)-18-azido-3-hydroxyoctadec-4-en-l-yl-

[0280] (2-(dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate 2,2,2-trifluoroacetate (TFSM 1)

[0281] (2S,3R,E)-18-azido-2-((S)-2-((tert-butoxycarbonyl)amino)hexanamido)-3-hydroxyoctadec-4-en-l-yl (2- (dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (15, 4.70 mg, 6.33 pmol) was dissolved in DCM (1.5 mL). The solution was cooled to 0 °C and TFA (0.5 mL) was added. The reaction mixture was stirred for 2 h at this temperature. The solvent was removed under reduced pressure to yield the title compound (quant., TFA salt) as a colorless oil.

[0282] XH NMR (400 MHz, CD3OD): 6 = 8.33 (d,3J = 9.0 Hz, NH), 5.76 (dt,3J = 15.0 Hz,3J = 6.8 Hz, 1H, H-5), 5.45 (ddt,3J = 15.3 Hz,3J = 7.7 Hz,4J = 1.4 Hz, 1H, H-4), 4.44 (d,4J = 2.4 Hz, 2H, H-4a), 4.34-4.27 (m, 2H, H- la), 4.14-4.01 (m, 3H, H-l, H-3), 4.01-3.91 (m, 1H, H-2), 3.79 (dd,3J = 6.8 Hz,3J = 6.8 Hz, 1H, H-2'), 3.77-3.72 (m, 2H, H-2a), 3.57 (t,4J = 2.5 Hz, 1H, H-6a), 3.28 (s, 6H, H-3a), 3.28 (t,3J = 6.8 Hz, 2H, H-18, signal superimposed by H-3a signal), 2.04 (td,3J = 7.0 Hz,3J = 7.0 Hz, 2H, H-6), 1.88-1.67 (m, 2H, H-3'), 1.63-1.54 (m, 2H, H-17),

[0283] 1.43-1.29 (m, 24H, H-7-16, H-4'-5'), 0.96 (t,3J = 6.9 Hz, 3H, H-6') ppm;13C NMR (100 MHz, CD3OD): 6 = 170.1 (C-l'), 135.7 (C-5), 131.1 (C-4), 83.2 (C-5a), 72.4 (C-6a), 72.0 (C-3), 65.8 (d, JC-P = 5.7 Hz, C-l), 65.3 (d, JC-P= 7.7 Hz, C-2a), 60.3 (d, JC.P= 5.0 Hz, C-la), 56.4 (C-4a), 55.7 (d, JC.P= 6.6 Hz, C-2), 54.7 (C-2'),

[0284] 52.4 (C-18), 52.0 (2C, C-3a), 33.5 (C-6), 32.4 (C-3'), 29.9 (C-17), 30.8, 30.73, 30.66, 30.65, 30.6, 30.5,

[0285] 30.3, 28.1, 27.8, 23.5 (12C, C-7-16,

[0286] C-4'-5'), 14.1 (C-6') ppm; HRMS (ESI+): m / z calcd for C3iH6oN606P [M+H]+643.4312, found 643.4343, |Am / z| = 4.8 ppm. tert-Butyl-((2S,3R,E)-l-((tert-butyldimethylsilyl)oxy)-3-(methoxymethoxy)octadec-4-en-2-yl)- carbamate (6)

[0287] The starting material of this reaction was prepared in a multi-step synthesis according to a literature procedure (cf. NPL3). tert-Butyl ((2S,3R,E)-l-((tert-butyldimethylsilyl)oxy)-3-hydroxyoctadec-4-en-2-yl)carbamate (1.27 g, 2.47 mmol, 1.00 equiv.) was dissolved in anh. DCM (20 mL) and the solution was cooled to 0 °C. MOM- CI (3.75 mL, 49.4 mmol, 20.0 equiv.) was added, followed by the dropwise addition of anh. DIPEA (8.41 mL, 49.4 mmol, 20.0 equiv.). The solution was allowed to warm to rt and stirred for 4 h. The reaction mixture was diluted with DCM (30 mL), washed with sat. aq. NaHCO3solution (30 mL), sat. aq. NH4CI solution (30 mL) and brine (30 mL).

[0288] The organic layer was dried over MgSCU and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (CH / EtOAc = 25 / 1) to afford the title compound (1.02 g, 1.82 mmol, 74%) as a colorless oil.

[0289] XH NMR (400 MHz, CDCI3): 6 = 5.68 (dt,3J = 15.2 Hz,3J = 6.8 Hz, 1H, H-5), 5.32 (dd,3J = 15.4 Hz,3J = 8.5 Hz, 1H, H-4), 4.69 (d,2J = 6.6 Hz, 1H, H-l" and m, 1H, NH), 4.50 (d,2J = 6.6 Hz, 1H, H-l"), 4.13- 3.99 (m, 1H, H-3), 3.90-3.77 (m, 1H, H-l), 3.74-3.52 (m, 2H, H-2, H-l), 3.34 (s, 3H, H-2"), 2.03 (td,3J = 6.8 Hz,3J = 6.8 Hz, 2H, H-6), 1.42 (s, 9H, H-3'), 1.39-1.18 (m, 22H, H-7-17), 0.89 (s, 9H, H-3a), 0.87 (t,3J = 7.0 Hz, 3H, H-18), 0.05 (2 x s, 6H, H-la) ppm;X3C NMR (100 MHz, CDCI3): 8 = 155.5 (C-l'), 137.1 (C-5), 126.7 (C-4), 93.6 (C-l"), 79.1 (C-2'), 76.0 (C-3), 61.7 (C-l), 55.6 (C-2"), 54.9 (C-2), 32.4 (C-6), 32.0, 29.77, 29.76, 29.74, 29.69, 29.6, 29.4, 29.3, 29.2, 22.8 (11C, C-7-17), 28.5 (3C, C-3'), 26.0 (3C, C-3a), 18.3 (C-2a), 14.2 (C-18), -5.3, -5.4 (2C, C-la) ppm; HRMS (ESI+): m / z calcd for C3iH63NNaO5Si [M+Na]+580.4368, found 580.4377, | Am / z| = 1.6 ppm. tert-Butyl ((2S,3R,E)-l-hydroxy-3-(methoxymethoxy)octadec-4-en-2-yl)carbamate (8) tert-Butyl-((2S,3R,E)-l-((tert-butyldimethylsilyl)oxy)-3-(methoxymethoxy)octadec-4-en-2-yl)- carbamate (6, 966 mg, 1.73 mmol, 1.00 equiv.) was dissolved in anh. THF (35 ml) and the solution was cooled to 0 °C. TBAF solution (1 M in THF, 2.08 mL, 2.08 mmol, 1.20 equiv.) was added and the mixture was stirred for 30 min at 0 °C and for further 30 min at rt. H2O (30 mL) was added and the aqueous phase was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CH / EtOAc = 4 / 1) to afford the title compound (602 mg, 1.36 mmol, 78%) as a waxy white solid.

[0290] XH NMR (400 MHz, CDCI3): 6 = 5.75 (dt,3J = 15.4 Hz,3J = 6.8 Hz, 1H, H-5), 5.34 (ddt,3J = 15.5 Hz,3J = 7.9 Hz,4J = 1.4 Hz, 1H, H-4), 5.23 (d,3J = 7.5 Hz, 1H, NH), 4.66 (d,2J = 6.6 Hz, 1H, H-l"), 4.51 (d,2J = 6.6 Hz, 1H, H-l"), 4.23 (dd,3J = 7.7 Hz,3J = 4.8 Hz, 1H, H-3), 3.98-3.88 (m, 1H, H-l), 3.70-3.58 (m, 2H, H-l, H-2), 3.37 (s, 3H, H-2"), 2.77 (br d,3J = 6.9 Hz, 1H, OH), 2.04 (td,3J = 6.8 Hz,3J = 6.8 Hz, 2H, H-6), 1.44 (s, 9H, H-3'), 1.37-1.22 (m, 22H, H-7-17), 0.87 (t,3J = 6.9 Hz, 3H, H-18) ppm;13C NMR (100 MHz, CDCI3): 6 = 156.0 (C-l'), 137.1 (C-5), 126.0 (C-4), 93.9 (C-l"), 79.6 (C-2'), 78.6 (C-3), 62.5 (C-l), 55.8 (C-2"), 55.0 (C-2), 32.4 (C-6), 32.0, 29.76, 29.74, 29.73, 29.67, 29.5, 29.4, 29.2, 29.1, 22.8 (11C, C-7-17), 28.4 (3C, C-3'), 14.2 (C-18) ppm; HRMS (ESI+): m / z ealed for CzsH^NNaOs [M+Na]+466.3503, found 466.3508, |Am / z| = 1.0 ppm.

[0291] The spectroscopic data agree with those reported in the literature (cf. NPL1). tert-Butyl-((2S,3R,E)-l-(((2-bromoethoxy)(hydroxy)phosphoryl)oxy)-3-(methoxy-methoxy)-octadec-4- en-2-yl)carbamate (10)

[0292] 6-Bromoethyl phosphoryldichloride (643 pL, 5.02 mmol, 4.00 equiv.) was dissolved in anh. DCM (20 mL) and the solution was cooled to 0 °C. Pyridine (607 pL, 7.53 mmol, 6.00 equiv.) was added and the mixture was stirred for 5 min. Then, a solution of tert-Butyl ((2S,3R,E)-l-hydroxy-3- (methoxymethoxy)octadec-4-en-2-yl)carbamate (8, 557 mg, 1.26 mmol, 1.00 equiv.) in anh. DCM (15 mL) was added and the reaction was stirred for 4.5 h. Sat. aq. NaHCOs solution (50 mL) was added and the biphasic mixture was stirred vigorously for 30 min. The mixture was diluted with H2O (30 mL) and brine and the aqueous phase was extracted with CHCI3 (4 x 75 mL). The combined organic layers were washed with brine and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (DCM / MeOH = 30 / 1 -> 10 / 1) to afford the title compound (444 mg, 704 pmol, 56%) as a colorless oil.

[0293] *H NMR (400 MHz, CD3OD): 6 = 5.74 (dt,3J = 15.1 Hz,3J = 6.9 Hz, 1H, H-5), 5.33 (dd,3J = 15.4 Hz,3J = 8.6 Hz, 1H, H-4), 4.68 (d,2J = 6.6 Hz, 1H, H-l"), 4.51 (d,2J = 6.6 Hz, 1H, H-l"), 4.20-4.11 (m, 2H, H-la), 4.11-4.02 (m, 2H, H-l, H-3), 4.02-3.92 (m, 1H, H-l), 3.81-3.65 (m, 1H, H-2), 3.58 (t,3J = 6.3 Hz, 2H, H-2a), 3.36 (s, 3H, H-2"), 2.07 (td,3J = 6.8 Hz,3J = 6.7 Hz, 2H, H-6), 1.43 (s, 9H, H-3'), 1.52-1.22 (m, 22H, H-7-17), 0.90 (t,3J = 6.9 Hz, 3H, H-18) ppm;13C NMR (100 MHz, CD3OD): 6 = 158.0 (C-l'), 138.5 (C-5), 127.8 (C-4), 94.6 (C-l"), 80.2 (C-2'), 77.7 (C-3), 66.7 (d, JC-P= 5.1 Hz, C-la), 65.8 (d, JC-P = 5.6 Hz, C-l), 56.1 (C-2"), 55.6 (d, JC.P= 7.9 Hz, C-2), 33.5 (C-6), 32.0 (d, 7C-P = 8.2 Hz, C-2a), 33.1, 30.81, 30.79, 30.78, 30.7, 30.6, 30.5, 30.33, 30.29, 23.8 (11C, C-7-17), 28.9 (3C, C-3'), 14.5 (C-18) ppm; HRMS (ESI+): m / z calcd for C27H53BrNNaOEP [M+Na]+652.2584, found 652.2597, |Am / z| = 1.9 ppm.

[0294] (2S,3R,E)-2-((tert-butoxycarbonyl)amino)-3-(methoxymethoxy)octadec-4-en-l-yl (2-(dimethyl(prop-2- yn-l-yl)ammonio)ethyl) phosphate (12) tert-Butyl-((2S,3R,E)-l-(((2-bromoethoxy)(hydroxy)phosphoryl)oxy)-3-(methoxymethoxy)-octadec-4- en-2-yl)carbamate (10, 401 mg, 636 pmol, 1.00 equiv.) was dissolved in a mixture of MeCN (4.5 mL), iPrOH (4.5 mL), CHCI3(4.5 mL) and A / ,A / -dimethylprop-2-yn-l-amine (4.5 mL) and the solution was stirred for 6 d at rt. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CHCU / MeOH / HzO = 55 / 45 / 3) to afford the title compound (279 mg, 441 pmol, 69%) as a white foam.

[0295] XH NMR (400 MHz, CD3OD): 6 = 6.74 (d,3J = 9.4 Hz, NH), 5.74 (dt,3J = 15.2 Hz,3J = 6.8 Hz, 1H, H-5), 5.32 (ddt,3J = 15.4 Hz,3J = 8.7 Hz,4J = 1.3 Hz, 1H, H-4), 4.68 (d,2J = 6.6 Hz, 1H, H-l"), 4.51 (d,2J = 6.6 Hz, 1H, H-l"), 4.45 (d,4J = 2.4 Hz, 2H, H-4a), 4.36-4.24 (m, 2H, H-la), 4.10-3.96 (m, 3H, H-3, H-l), 3.79-3.66 (m, 3H, H-2a, H-2), 3.57 (t,4J = 2.5 Hz, 1H, H-6a), 3.36 (s, 3H, H-2"), 3.28 (s, 6H, H-3a), 2.07 (td,3J = 6.8 Hz,3J = 6.7 Hz, 2H, H-6), 1.43 (s, 9H, H-3'), 1.50-1.21 (m, 22H, H-7-17), 0.90 (t,3J = 6.9 Hz, 3H, H-18) ppm;13C NMR (100 MHz, CD3OD): 5 = 158.0 (C-l'), 138.6 (C-5), 127.9 (C-4), 94.5 (C-l"), 83.2 (C-5a), 80.1 (C-2'), 77.6 (C-3), 72.4 (C-6a), 65.9 (d, JC-p = 5.5 Hz, C-l), 65.3 (d, JC-P= 7.4 Hz, C-2a), 60.2 (d, Jc-p = 5.0 Hz, C-la), 56.4 (C-4a), 56.1 (C-2"), 55.6 (d, JC P= 8.0 Hz, C-2), 52.0 (2C, C-3a), 33.4 (C-6), 33.1, 30.80, 30.78, 30.78, 30.7, 30.6, 30.5, 30.33, 30.27, 23.8 (11C, C-7-17), 28.9 (3C, C-3'), 14.5 (C-18) ppm; HRMS (ESI+): m / z calcd for C^Hei^NaOgP [M+Na]+655.4058, found 655.4063, | Am / z | = 0.8 ppm. Perfluorophenyl N -(tert-butoxycarbonyl)-N -diazo-L-lysinate (14)

[0296] The starting material of this reaction was synthesized according to a protocol by Yang et al (cf. NPL4). A / 2-(tert-Butoxycarbonyl)-A / 6-diazo- / .-lysine (154 mg, 566 pmol, 1.00 equiv.) and 2,3,4,5,6-pentafluorophenol (156 mg, 848 pmol, l.SO equiv.) were dissolved in anh. DCM (4 mL) and the solution was cooled to 0 °C. EDC hydrochloride (163 mg, 848 pmol, 1.50 equiv.) and DMAP (6.91 mg, 56.6 pmol, 0.10 equiv.) were added, the solution was allowed to warm to rt and stirred overnight. DCM (30 mL) was added and the mixture was washed with HCI (1 M, 2 x 20 mL), then with sat. aq. NaHCCh solution (2 x 20 mL). The organic phase was dried over MgSO4and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (CH / EtOAc = 20 / 1) to afford the title compound (160 mg, 365 pmol, 64%) as a colorless oil.

[0297] XH NMR (400 MHz, CDCI3): 6 = 5.13-4.83 (m, 1H, NH), 4.72-4.34 (m, 1H, H-2), 3.32 (t,3J = 6.6 Hz, 2H, H-6), 2.08-1.94 (m, 1H, H-3), 1.92-1.78 (m, 1H, H-3), 1.74-1.62 (m, 2H, H-5), 1.61-1.50 (m, 2H, H-4), 1.46 (s, 9H, H-3') ppm;13C NMR (100 MHz, CDCI3): 6 = 169.1 (C-l), 155.2 (C-l'), 141.1 (m), 139.8 (m), 138.0 (m), 124.8 (m) (6C, C-l'-4'), 80.8 (C-2'), 53.3 (C-2), 51.1 (C-6), 32.0 (C-3), 28.4 (C-5), 28.3 (3C, C-3'), 22.6 (C-4) ppm; HRMS (ESI+): m / z calcd for Ci7Hi9F5N4NaO4[M+Na]+461.1219, found 461.1218, |Am / z| = 0.0 ppm.

[0298] (2S,3R,E)-2-((S)-6-Azido-2-((tert-butoxycarbonyl)amino)hexanamido)-3-hydroxy-octadec-4-en-l-yl (2- (dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (16)

[0299] (2S,3R,E)-2-((te / t-Butoxycarbonyl)amino)-3-(methoxymethoxy)octadec-4-en-l-yl(2-(dimethyl-(prop- 2-yn-l-yl)ammonio)ethyl) phosphate (12, 17.6 mg, 27.8 pmol, 1.00 equiv.) was dissolved in anh. MeOH (2 mL). HCI solution (4 M in 1,4-dioxane, 0.3 mL) was added and the reaction was stirred for 3.5 h. The solvent was removed under reduced pressure to give the crude amine. Successful deprotection was confirmed by TLC andJH NMR. The intermediate was immediately subjected to the following reaction conditions without further purification. The crude amine, perfluorophenyl A / 2-(tert-butoxycarbonyl)-A / 6-diazo-L-lysinate (14, 18.3 mg, 41.7 pmol, 1.50 equiv.) and DIPEA (75.0 pL, 55.6 pmol, 2.00 equiv.) were dissolved in anh. DCM (2 mL) and stirred for 3 d at rt. MeOH was added and the solution was stirred for further 2 h. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (CHCI3 / MeOH / H2O / NH3(25% aq.) = 85 / 15 / 1 / 0.1%) to afford the title compound (17.5 mg, 23.6 pmol, 85%) as a colorless waxy solid.

[0300] XH NMR (400 MHz, CD3OD): 6 = 7.86 (d,3J = 9.2 Hz, 1H, NH), 5.73 (dt,3J = 14.8 Hz,3J = 6.9 Hz, 1H, H-5), 5.45 (dd,3J = 15.4 Hz,3J = 73 Hz, 1H, H-4), 4.45 (d,4J = 2.4 Hz, 2H, H-4a), 4.37-4.25 (m, 2H, H-la), 4.17- 4.02 (m, 3H, H-3, H-2', H-l), 4.02-3.91 (m, 2H, H-l, H-2), 3.80-3.71 (m, 2H, H-2a), 3.56 (t,4J = 2.5 Hz, 1H, H-6a), 3.36 (m, superimposed by solvent signal, 2H, H-6'), 3.29 (s, 6H, H-3a), 2.10-1.97 (m, 2H, H-6), 1.83-1.69 (m, 1H, H-3'), 1.69-1.51 (m, 3H, H-3', H-5'), 1.45 (s, 9H, H-3"), 1.44-1.25 (m, 24H, H-7- 17, H-4'), 0.90 (t,3J = 6.8 Hz, 3H, H-18) ppm;13C NMR (100 MHz, CD3OD): 5 = 174.8 (C-l'), 157.7 (C-l"), 135.2 (C-5), 130.8 (C-4), 83.2 (C-5a), 80.5 (C-2"), 72.4 (C-6a), 72.3 (C-3), 65.7 (d, JQ.P= 5.4 Hz, C-l), 65.3 (d, Jc-p = 7.5 Hz, C-2a), 60.3 (d, JC P= 4.9 Hz, C-la), 56.5 (C-4a), 55.9 (C-2'), 55.4 (d, JC-P = 7.8 Hz, C-2), 52.3 (C-6'), 52.0 (2C, C-3a), 33.6 (C-3'), 33.5 (C-6), 29.6 (C-5'), 28.8 (3C, C-3"), 33.1, 30.82, 30.80, 30.78, 30.77, 30.67, 30.5, 30.3, 24.2, 23.7 (12C, C-7-17, C-4'), 14.5 (C-18) ppm; HRMS (ESI+): m / z calcd for CaeHezNeNaOsP [M+Na]+765.4650, found 765.4617, | Am / z| = 4.4 ppm.

[0301] (2S,3R,E)-2-((S)-2-Ammonio-6-azidohexanamido)-3-hydroxyoctadec-4-en-l-yl-(2-(dimethyl-(prop-2- yn-l-yl)ammonio)ethyl) phosphate 2,2,2-trifluoroacetate (TFSM 2)

[0302] (2S,3R,E)-2-((S)-6-Azido-2-((tert-butoxycarbonyl)amino)hexanamido)-3-hydroxy-octadec-4-en-l-yl (2- (dimethyl(prop-2-yn-l-yl)ammonio)ethyl) phosphate (16, 11.0 mg, 14.8 pmol) was dissolved in DCM (1.5 ml) and the solution was cooled to 0 °C. TFA (0.3 mL) was added and the reaction was stirred for 2.5 h. The solvent was removed under reduced pressure and remaining TFA was removed by coevaporation with MeOH. The product was dried under high-vacuum to afford the title compound (quant., TFA salt) as a colorless oil.

[0303] XH NMR (400 MHz, CD3OD): 5 = 5.77 (dt,3J = 15.3 Hz,3J = 6.6 Hz, 1H, H-5), 5.46 (dd,3J = 15.3 Hz,3J = 7.5 Hz, 1H, H-4), 4.45 (d,4J = 2.4 Hz, 2H, H-4a), 4.40-4.25 (m, 2H, H-la), 4.17-3.88 (m, 4H, H-3, H-l, H-2), 3.82 (t,3J = 6.7 HZ, 1H, H-2'), 3.79-3.70 (m, 2H, H-2a), 3.56 (t,4J = 2.5 Hz, 1H, H-6a), 3.35 (t,3J = 6.7 Hz, 2H, H-6'), 3.29 (s, 6H, H-3a), 2.12-1.97 (m, 2H, H-6), 1.97-1.71 (m, 2H, H-3'), 1.69-1.57 (m, 2H, H-5'), 1.53-1.43 (m, 2H, H-4'), 1.41-1.26 (m, 22H, H-7-17), 0.90 (t,3J = 6.8 Hz, 3H, H-18) ppm;13C NMR (100 MHz, CD3OD): 6 = 170.0 (C-l'), 163.1 (q, JC-F = 34.0 Hz, C-lb), 135.7 (C-5), 131.0 (C-4), 118.3 (q, JC-F = 292.8 Hz, C-2b), 83.2 (C-5a), 72.4 (C-6a), 72.1 (C-3), 65.7 (d, JC.P= 5.7 Hz, C-l), 65.2 (d, JC-P - 7.8 Hz, C-2a), 60.3 (d, JC-p = 4.9 Hz, C-la), 56.4 (C-4a), 55.7 (d, JC.P= 6.7 Hz, C-2), 54.5 (C-2'), 52.01 (C-6'), 52.00 (2C, C-3a), 33.5 (C-6), 32.2 (C-3'), 29.6 (C-5'), 33.1, 30.80, 30.79, 30.77, 30.68, 30.50, 30.48, 30.4, 23.7 (11C, C-7-17), 23.3 (C-4'), 14.4 (C-18) ppm; HRMS (ESI+): m / z calcd for CaiHeoNeOeP [M+H]+643.4312, found 643.4314, | Am / z| = 0.3 ppm.

[0304] Methyl (S,f)-16-(benzyloxy)-2-((tert-butyloxycarbonyl)amino)hexadic-4-enoate (18)

[0305] The starting material of this reaction was prepared in a multi-step synthesis according to a literature procedure (cf NPL8 to NPL8).

[0306] Under dry N? atmosphere methyl 2-((tert-butyloxycarbonyl)amino)-L-pent-4-enoate (17, 149 mg, 651 pmol, 1.00 eq.), benzyl-tridec-12-enyl-ether (603 mg, 2.09 mmol, 3.20 eq.), and Grubbs II (14.9 mg, 17.5 pmol, 0.03 eq.) were dissolved in dry DCM (10 mL). The reaction mixture was heated to 45 °C for 4.5 h, cooled to room temperature, and the solvent removed in vacuo. The crude product was purified by column chromatography (cyclohexane / EtOAc, 20:1) to afford the title compound (214 mg, 437 pmol, 67 %) as a colorless oil.aH NMR (400 MHz, CDCI3): 6 = 7.35-7.32 ( 7.27 (m, 1H, H-5'"), 5.52 (dt, 1H,

[0307] 3J = 7.2 Hz,3J = 14.7 Hz, H-5), 5.26 (dt, 1H,3J = 7.3 Hz,3J = 14.8 Hz, H-4), 5.00 (d, 1H,3J = 7.8 Hz, NH), 4.50 (s, 2H, H-l'"), 4.33 (dd, 1H,3J = 5.8 Hz,3J = 13.8 Hz, H-2), 3.73 (s, 3H, H-l"), 3.46 (t, 2H,3J = 6.8 Hz, H-16), 2.49-2.37 (m, 2H, H-3), 1.98 (dd, 2H,3J = 7.3 Hz,3J = 14.0 Hz, H-6), 1.61 (dt, 2H,3J = 6.9 Hz,3J = 14.2 Hz, H-15), 1.44 (s, 9H, H-3'), 1.37-1.30 (m, 4H, H-7, H-14), 1.27-1.25 (m, 12H, H8-13) ppm;13C NMR (100 MHz, CDCI3): 6 = 172.9 (C-l), 155.4 (C-l'), 138.9 (C-2'"), 135.8 (C-5), 128.5 (2C, C-4'"),

[0308] 127.8 (C-5'"), 127.6 (2C, C-3'"), 123.4 (C-4), 80.0 (C-2'), 73.0 (C-l'"), 70.7 (C-16), 53.3 (C-2), 52.3 (C-l"),

[0309] 35.8 (C-3), 32.7 (C-6), 29.9 (C-15), 29.7-29.3 (6C, C-8-13), 28.5 (3C, C-3'), 27.1-26.3 (2C, C-7, C-14) ppm; HRMS (ESI+): m / z calcd for Cjg^yNNaOs [M+Na]+512.3346, found 512.3335, | Am / z| = 2.2 ppm.

[0310] Methyl (S)-2-((tert-butyloxycarbonyl)amino)-16-hydroxyhexadecanoate (19) Methyl (S,E)-16-(benzyloxy)-2-((tert-butyloxycarbonyl)amino)hexadic-4-enoate (18, 50.0 mg,

[0311] 107 pmol, 1.0 eq.) was dissolved in EtOAc / EtOH (5.2 mL, 1:1) and sonicated for 12 min. 10 wt.% Pd / C (5.80 mg, 5.45 nmol, 0.05 eq.) was added and H2led through the reaction mixture. The reaction was filtered over celite after 22 h, washed with EtOAc, and the solvent was removed in vacuo. The crude product was purified by column chromatography (cyclohexane / EtOAc, 3:1) to obtain the title compound (33.3 mg, 82.9 pmol, 82 %) as a colorless oil. 5.01 (s, 1H, NH) 4.26 (s, 1H, H-2), 3.72 (s, 3H, H-l"), 3.62 (t, 2H,3J = 6.7 Hz, H-16), 1.80-1.72 (m, 1H, H-3a), 1.68 (s, 1H, OH), 1.62-1.58 (m, 1H, H-3b), 1.55 (ddd, 2H,3J = 7.0 Hz,3J - 7.0 Hz,3J = 14.3 Hz, H-15), 1.43 (s, 9H, H-3'), 1.34-1.23 (m, 22H, H-4-14) ppm;13C NMR (100 MHz, CDCb): 6 = 173.7 (C-l), 155.5 (C-l'), 79.9 (C-2'), 63.1 (C-16), 53.6 (C-2), 52.3 (C-l"), 32.9 (C-3), 32.9 (C-15), 29.7-29.3 (9C, C-5-13), 28.4 (3C, C-3'), 25.9 (C-14), 25.4 (C-4) ppm; HRMS (ESI+): m / z calcd for C22H43NNaO5[M+Na]+424.3033, found 424.3022, | Am / z| - 2.8 ppm.

[0312] Methyl (S)-2-((tert-butyloxycarbonyl)amino)-16-(tosyloxy)hexadecanoate (20)

[0313] Under dry N2atmosphere methyl (S)-2-((tert-butyloxycarbonyl)amino)-16-hydroxyhexadecanoate (19, 102 mg, 254 pmol, 1.0 eq.) was dissolved in dry DCM (3 mL). Dry pyridine (53.8 pL, 667 pmol, 2.6 eq.), DMAP (4.67 mg, 38.2 pmol, 0.2 eq.), and TsCI (87.7 mg, 460 pmol, 1.8 eq.) were added. The reaction mixture was stirred at room temperature for 2.5 h, then diluted with DCM (10 mL) and washed 1 M aq. HCI (75 mL). The aqueous layer was extracted with DCM (3 x 75 mL), the combined organic layers successively washed with a sat. NaHCO3solution (25 mL) and brine (25 mL), dried over Na2SO4, and the solvent removed in vacuo. The crude product was purified by column chromatography (cyclohexane / EtOAc, 5:1) to afford target compound (108 mg, 194 pmol, 77 %) as a colorless oil. 7.78 (dt, 2H,3J = 8.5 Hz,4J = 1.8 Hz, H-3"'), 7.33 (dd, 2H,3J = 8.6 Hz,4J = 0.6 Hz, H-2'"), 4.98 (d, 1H,3J = 8.0 Hz, NH), 4.27 (dd, 1H,3J = 7.9 Hz,3J = 13.0 Hz, H-2), 4.00 (t, 2H,3J = BA Hz, H-16), 3.72 (s, 3H, H-l"), 2.44 (s, 3H, C-5'"), 1.78-1.74 (m, 1H, H-3a), 1.58-1.65 (m, 3H, H-3b, H-15), 1.43 (s, 9H, H-3'), 1.31-1.27 (m, 4H, H-4, H-14), 1.25-1.20 (m, 18H, H-5-13) ppm;UC NMR (100 MHz, CDCI3): 6 = 173.7 (C-l), 155.5 (C-l'), 144.7 (C-4'"), 133.3 (C-l'"), 129.9 (2C, C-2'"), 128.0 (2C, C-3'"), 79.9 (C-2'), 70.8 (C-16), 53.6 (C-2), 52.3 (C-l"), 32.9 (C-3), 29.8-29.0 (9C, C-5-13), 28.9 (C-15), 28.4 (3C, C-3'), 25.4 (2C, C-4, C-14), 21.8 (C-5'") ppm; HRMS (ESI+): m / z calcd forCzsH^NNaOzS [M+Na]+578.1329, found 578.3122, |Am / z| = 1.2 ppm. Methyl (S)-16-azido-2-((tert-butyloxycarbonyl)amino)hexadecanoate (21)

[0314] Methyl (S)-2-((tert-butyloxycarbonyl)amino)-16-(tosyloxy)hexadecanoate (20, 30.0 mg, 54.0 pmol, 1.0 eq.), NaN3(11.4 mg, 175 pmol, 3.3 eq.), and "BmNBr (2.04 mg, 6.33 pmol, 0.1 eq.) were dissolved in DMF (1 mL) and heated to 90 °C. After 20.5 h, the reaction was cooled to room temperature, diluted with water (10 mL), and extracted with EtOAc (3 x 15 mL). The combined organic layers were washed with brine (15 mL), dried over NazSO4, and the solvent was removed in vacuo. The crude product was purified by column chromatography (cyclohexane / EtOAc, 7:1) to obtain target compound (14.9 mg, 34.9 pmol, 65 %) as a colorless oil.

[0315] XH NMR (400 MHz, CDCI3): 6 = 4.98 (d, 1H,3J = 8.2 Hz, NH), 4.28 (dd, 1H,3J = 7.8 Hz,3J = 13.3 Hz, H-2), 3.73 (s, 3H, H-l"), 3.25 (t, 2H,3J = 7.0 Hz, H-16), 1.82-1.73 (m, 1H, H-3a), 1.64-1.57 (m, 1H, H-3b), 1.59 (ddd, 2H,3J = 7.2 Hz,3J = 7.2 Hz,3J = 14.6 Hz, H-15), 1.44 (s, 9H, H-3'), 1.39-1.32 (m, 2H, H-14), 1.32- 1.27 (m, 4H, H-4, H-13), 1.27-1.24 (m, 16H, H-5-12) ppm;13C NMR (100 MHz, CDCI3): 6 = 173.7 (C-l), 155.5 (C-l'), 79.9 (C-2'), 53.6 (C-2), 52.3 (C-l"), 51.6 (C-16), 32.9 (C-3), 29.7-29.3 (8C, C-5-12), 29.3 (C-13), 29.0 (C-15), 28.5 (3C, C-3'), 26.9 (C- 14), 25.4 (C-4) ppm; HRMS (ESI+): m / z calcd for C22H42N4NaO4[M+Na]+449.3098, found 449.3097, |Am / z | = 0.4 ppm.

[0316] (S)-16-Azido-2-((tert-butyloxycarbonyl)amino)hexadecanoic acid (22)

[0317] Methyl (S)-16-azido-2-((tert-butyloxycarbonyl)amino)hexadecanoate (21, 20.1 mg, 47.1 pmol, 1.0 eq.) and LiOH (2.28 mg, 95.2 pimol, 2.0 eq.) were dissolved in THF / MeOH (0.58 mL, 1:1). The reaction process was monitored by TLC and after 5.5 h the solvent removed in vacuo. The residue was dispensed in water (5 mL) and washed with Et2O (2 x 5 mL). The aqueous layer was acidified with 1 M aq. NaHSO4to pH = 2 and extracted with EtOAc (5 x 5 mL). The combined organic layers were dried over MgSO4and the solvent removed in vacuo. The crude product was purified by column chromatography (DCM / MeOH, 20:1) to afford the target molecule (14.0 mg, 33.9 pmol, 72 %) as a colorless oil. 8.26 (s, 1H, COOH), 4.99 (d, 1H,3J = 7.4 Hz, NH), 4.28 (dd, 1H,3J = 6.9 Hz,3J = 12.1 Hz, H-2), 3.25 (t, 2H,3J = 7.0 Hz, H-16), 1.87-1.80 (m, 1H, H-3a), 1.69-1.59 (m, 1H, H-3b), 1.59 (dt, 2H,37 = 7.2 Hz,3J = 14.3 Hz, H-15), 1.44 (s, 9H, H-3'), 1.40-1.34 (m, 4H, H-4, H-14), 1.31-1.25 (m, 18H, H-5-13) ppm;13C NMR (100 MHz, CDCI3): 6 = 177.8 (C-l), 155.8 (C-l'), 80.3 (C-2'), 53.6 (C-2), 51.6 (C-16), 32.5 (C-3), 29.8-29.3 (9C, C-5-13), 29.0 (C-15), 28.5 (3C, C-3'), 26.9 (C-14), 25.5 (C-4) ppm.; HRMS (ESI-): m / z calcd for C21H39N404[M-Naf 411.2977, found 411.2965, |Am / z| = 3.0 ppm. tert-Butyl ((S)-16-azido-l-(((2S,3 / ?)-l,3-dihydroxy-(f)-octadec-4-ene-2-yl)amino)-l-oxohexadecan-2- yl)carbamate (27)

[0318] Under dry N2atmosphere (S)-16-azido-2-((tert-butyloxycarbonyl)amino)hexadecanoic acid (22, 20.5 mg, 49.6 pmol, 1.1 eq.) was dissolved in dry DMF (1.3 mL) and cooled to 0 °C. At this temperature DIPEA (23.6 pL, 135 ptmol, 3.0 eq.) and HATU (20.9 mg, 55.0 pmol, 1.2 eq.) were added, stirred for 20 min, and then sphingosine (24, 134 mg, 45.1 pmol, 1.0 eq.) in dry DMF (2 mL). The reaction was warmed to room temperature after 1.5 h, quenched with water (5 mL) and a sat. NH4CI solution (15 mL) after 19 h. The aqueous layer was extracted with EtOAc (3 x 20 mL), the combined organic layers washed with brine (20 mL), dried over Na2SO4, and the solvent removed in vacuo. The crude product was purified by column chromatography (neutralized SiO2, DCM / MeOH, 20:1) to obtain the target molecule (27.5 mg, 39.6 pmol, 88 %) as a colorless solid.

[0319] XH NMR (400 MHz, CDCI3): 5 = 6.84 (d, 1H,3J = 8.0 Hz, NH), 5.79 (dtd, 1H,3J = 6.9 Hz,3J = 15.2 Hz,AJ = 1.1 Hz, H-5), 5.51 (ddt, 1H,3J = 6.1 Hz,3J = 15.5 Hz,AJ = 1.3 Hz, H-4), 5.06 (d, 1H,3J = 6.6 Hz, NHBoc), 4.36 (dd, 1H,3J = 4.5 HZ,3J = 4.5 HZ, H-3), 3.98 (dd, 1H,3J = 6.5 Hz,3J = 6.8 Hz, H-2’), 3.94 (dd, 1H,2J = 8.0 Hz,3J = 3.7 Hz, H-la), 3.84 (ddd, 1H,3J = 3.5 Hz,3J = 7.0 Hz,3J = 7.0 Hz, H-2), 3.71 (dd, 1H,2J = 11.5 HZ,3J = 3.1 HZ, H-lb), 3.25 (t, 2H,3J = 7.0 Hz, H-16'), 3.19 (s, 2H, 2 x OH), 2.05 (ddd, 2H,3J = 7.1 Hz,3J = 7.1 Hz,3J = 7.1 Hz, H-6), 1.83-1.76 (m, 1H, H-3'a), 1.59 (dt, 3H,3J = 7.2 Hz,3J = 14.5 Hz, H-3'b, H-15'), 1.43 (s, 9H, H-3"), 1.38-1.31 (m, 6H, H-7, H-4', H-14'), 1.31-1.25 (m, 38H, H-8-17, H-5'- 13'), 0.87 (t, 3H,3J = 6.9 Hz, H-18) ppm;13C NMR (100 MHz, CDCI3): 5 = 172.8 (C-l'), 156.3 (C-l"), 134.1 (C-5), 128.7 (C-4), 80.7 (C-2"), 74.1 (C-3), 61.9 (C-l), 55.7 (C-2'), 54.6 (C-2), 51.6 (C-16'), 32.5 (C-6), 32.3 (C-3'), 32.1 (C-16), 29.8-29.3 (17C, C-8-15, C-5'-13'), 29.3 (C-7), 29.0 (C-15'), 28.4 (3C, C-3”), 26.9 (C-14'), 25.9 (C-4'), 22.8 (C-17), 14.3 (C-18) ppm; HRMS (ESI+): m / z calcd for C39H75N5NaO5 [M+Na]+716.5666, found 716.5636, |Am / z| = 4.2 ppm.

[0320] (S)-2-Amino-16-azido-M-((2S,3 / ?)-l,3-dihydroxy-(f)-octadec-4-ene-2-yl)hexadecanamide (Cerl6-2)

[0321] Under dry N2atmosphere tert-butyl ((S)-16-azido-l-(((2S,3R)-l,3-dihydroxy-(£)-octadec-4-ene-2- yl)amino)-l-oxohexadecan-2-yl)carbamate (27, 27.5 mg, 39.6 pmol, 1.0 eq.) was dissolved in dry DCM (2.1 mL) and cooled to 0 °C. TFA (90.0 pL, 1.17 mmol, 30 eq.) was added and the reaction stirred for 2 h at this temperature. The reaction was neutralized with water (6 mL) and 1 M aq. NaOH (13 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over NazSCU, and the solvent removed in vacuo. The crude product was purified by column chromatography (neutralized SiO2, DCM / MeOH, 30:1) and preparative TLC (neutralized SiOz, DCM / MeOH 30:1) to afford the target compound (5.70 mg, 9.60 pmol, 24 %) as a colorless solid.

[0322] *H NMR (400 MHz, CDCI3): 5 = 7.87 (d, 1H,3J = 5.0 Hz, NH), 5.78 (ddd, 1H,3J = 7.3 Hz,3J = 7.3 Hz,3J = 14.9 Hz, H-5), 5.51 (dd, 1H,3J = 6.2 Hz,3J = 15.5 Hz, H-4), 4.31 (dd, 1H,3J = 4.7 Hz,3J = 4.7 Hz, H-3), 3.90 (dd, 1H,2J = 11.2 Hz,3J = 4.0 Hz, H-la), 3.85 (s, 1H, H-2), 3.73 (d, 1H,2J = 9.7 Hz, H-lb), 3.44 (s, 1H, H-2'), 3.25 (t, 2H,3J = 6.9 Hz, H-16'), 2.46 (s, 4H, 2 x OH, NH2), 2.05 (dd, 2H,3J = 7.1 Hz,3J = 14.2 Hz, H-6), 1.87-1.79 (m, 1H, H-3'a), 1.59 (dt, 2H,3J = 7.0 Hz,3J = 14.3 Hz, H-15'), 1.54-1.48 (m, 1H, H-3'b), 1.35-1.34 (m, 6H, H-7, H-4’ , H-14'), 1.31-1.25 (m, 38H, H-8-17, H-5 -13'), 0.88 (t, 3H,3J = 6.7 Hz, H-18) ppm;13C NMR (100 MHz, CDCI3): 6 = 175.7 (C-l'), 134.6 (C-5), 128.6 (C-4), 74.2 (C-3), 62.7 (C-l), 55.4 (C-2'), 55.3 (C-2), 51.6 (C-16'), 35.0 (C-3'), 32.5 (C-6), 32.1 (C-16), 29.9-29.5 (16C, C-9-15, C-5'- 13'), 29.4 (C-7), 29.3 (C-13'), 29.0 (C-15'), 26.9 (C-14'), 26.0 (C-4'), 22.9 (C-17), 14.3 (C-18) ppm; HRMS (ESI+): m / z calcd for ^HesNsCh [M+H]+594.5317, found 594.5324, | Am / z| = 1.2 ppm. tert-Butyl ((S)-l-(((2S,3R)-18-azido-l,3-dihydroxy-(E)-octadec-4-ene-2-yl)amino)-l-oxohexadecan-2- yl)carbamate (26)

[0323] The starting materials of this reaction were prepared in a multi-step synthesis according to a literature procedure (cf. NPL9 and NPL10).

[0324] Under dry N2atmosphere (S)-2-((tert-butyloxycarbonyl)amino)hexadecanoic acid (28, 16.8 mg, 45.2 pmol, 1.1 eq.) was dissolved in dry DMF (1 mL) and cooled to 0 °C. At this temperature HATU (19.4 mg, 51.0 pmol, 1.2 eq.) and DIPEA (21.5 pL, 123 pmol, 3.0 eq.) were added, stirred for 40 min, and then w-azidosphingosine (29, 14.0 mg, 41.1 pmol, 1.0 eq.) in dry DMF (1.5 mL). The reaction was warmed to room temperature after 1.5 h, quenched with water (5 mL) and a sat. NH4CI solution (15 mL) after 4 h. The aqueous layer was extracted with EtOAc (3 x 20 mL), the combined organic layers were washed with brine (20 mL), dried over Na2SO4, and the solvent removed in vacuo. The crude product was purified by column chromatography (CHCh / MeOH, 60:1) to obtain the target compound (27.7 mg, 39.9 pmol, 97 %) as a colorless solid.

[0325] XH NMR (400 MHz, CDCI3): 6 = 6.97 (d, 1H,3J = 8.0 Hz, NH), 5.77 (dtd, 1H,3J = 6.9 Hz,3J = 15.2 Hz,4J = 0.9 Hz, H-5), 5.49 (ddt, 1H,3J = 6.1 Hz,3J = 15.4 Hz,4J = 1.3 Hz, H-4), 5.22 (d, 1H,3J = 6.7 Hz, NHBoc), 4.31 (dd, 1H,3J = 4.6 Hz,3J = 4.6 Hz, H-3), 4.01 (dd, 1H,3J = 7.2 Hz,3J = 13.5 Hz, H-2'), 3.90 (dd, 1H,2J = 11.4 Hz,3J = 3.8 Hz, H-la), 3.85 (ddd, 1H,3J = 3.7 Hz,3J = 3.7 Hz,3J = 11.1 Hz, H-2), 3.69 (dd, 1H,2J = 11.3 Hz,3J = 3.0 Hz, H-lb), 3.24 (t, 2H,3J = 7.0 Hz, H-18), 2.03 (dt, 2H,3J = 6.9 Hz,3J = 7.2 Hz, H-6), 1.80-1.73 (m, 1H, H-3'a), 1.62-1.55 (m, 3H, H-17, H-3'b), 1.42 (s, 9H, H-3"), 1.36-1.30 (m, 6H, H-7, H-16, H-4'), 1.28-1.24 (m, 38H, H-8-15, H-5 -15'), 0.86 (t, 3H,3J = 6.9 Hz, H-16') ppm;13C NMR (100 MHz, CDCI3): 6 = 172.9 (C-l'), 156.3 (C-l"), 134.0 (C-5), 128.8 (C-4), 80.5 (C-2"), 73.9 (C-3), 61.9 (C-l), 55.5 (C-2'), 54.7 (C-2), 51.6 (C-18), 32.6 (C-3'), 32.5 (C-6), 32.0 (C-14'), 29.8-29.3 (17C, C-8-15, C-5'-13'), 29.3 (C-7), 29.0 (C-17), 28.4 (3C, C-3"), 26.8 (C-16), 25.8 (C-4'), 22.8 (C-15), 14.2 (C-16) ppm; HRMS (ESI+): m / z calcd for C39H75N5NaO5[M+Na]+716.5660, found 716.5686, | Am / z| = 3.5 ppm.

[0326] (S)-2-Amino-A / -((2S,3 / ?)-18-azido-l,3-dihydroxy-(E)-octadec-4-ene-2-yl)hexadecanamide (Cerl6-1)

[0327] Under dry N2 atmosphere tert-butyl ((S)-l-(((2S,3R)-18-azido-l,3-dihydroxy-(E)-octadec-4-ene-2- yl)amino)-l-oxohexadecan-2-yl)carbamate (26, 49.0 mg, 70.6 pmol, 1.0 eq.) was dissolved in dry DCM (2.5 mL) and cooled to 0 °C. TFA (160 pL, 2.08 mmol, 29 eq.) was added and the reaction stirred for 1 h at this temperature. The reaction was neutralized with water (7 mL) and 1 M aq. NaOH (8 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, and the solvent removed in vacuo. The crude product was purified twice by column chromatography (neutralized SiO2, DCM / MeOH, 15:1) and preparative TLC (neutralized SiO2, DCM / MeOH, 20:1) to afford the target molecule (8.40 mg, 14.1 pmol, 20 %) as a colorless solid.

[0328] XH NMR (400 MHz, CDCI3): 6 = 7.85 (d, 1H,3J = 9.2 Hz, NH), 5.78 (dtd, 1H,3J = 6.9 Hz,3J = 15.2 Hz,AJ = 1.0 Hz, H-5), 5.51 (ddt, 1H,3J = 6.4 Hz,3J = 15.4 Hz,4J = 1.4 Hz, H-4), 4.31 (dd, 1H,3J = 5.4 Hz,3J = 5.4 Hz, H-3), 3.91 (dd, 1H,2J = 11.3 Hz,3J = 4.4 Hz, H-la), 3.84 (ddd, 1H,3J = 4.2 Hz,3J = 7.7 Hz,3J = 7.7 Hz, H-2), 3.73 (dd, 1H,2J = 11.3 Hz,3J = 3.3 Hz, H-lb), 3.40 (dd, 1H,3J = 4.7 Hz,3J = 8.1 Hz, H-2'), 3.25 (t, 2H,3J = 7.0 Hz, H-18), 2.36 (s, 4H, 2 x OH, NH2), 2.05 (dt, 2H,3J = 7.1 Hz,3J = 7.1 Hz, H-6), 1.87- 1.79 (m, 1H, H-3'a), 1.59 (ddd, 2H,3J = 7.2 Hz,3J = 7.2 Hz,3J = 14.6 Hz, H-17), 1.55-1.47 (m, 1H, H-3'b), 1.40-1.33 (m, 6H, H-7, H-16, H-4'), 1.33-1.25 (m, 38H, H-8-15, H-5 -15'), 0.88 (t, 3H,3J = 6.9 Hz, H-16') ppm;13C NMR (100 MHz, CDCI3) : 6 = 175.9 (C-l'), 134.5 (C-5), 128.9 (C-4), 74.3 (C-3), 62.7 (C-l), 55.4 (C-2'), 55.2 (C-2), 51.6 (C-18), 35.2 (C-3'), 32.5 (C-6), 32.1 (C-14'), 29.9-29.5 (16C, C-9-15, C-5'- 13'), 29.4 (C-4'), 29.3 (C-8), 29.0 (C-17), 26.9 (C-16), 26.0 (C-7), 22.8 (C-15'), 14.3 (C-16') ppm; HRMS (ESI+): m / z calcd for CsAHgyNsNaOs [M+Na]+616.5136, found 616.5114, | Am / z| = 3.5 ppm. evaluation of compounds TFSM1 and TFSM2

[0329] In the first derivative (TFSM 1), the azide was placed in the sphingoid backbone. In the second derivative (TFSM 2), the acyl chain carries the azide, while the backbone was not altered. The metabolic products of TFSM 1 and TFSM 2 are thus also distinguishable, which might be an advantage for mass spectrometry-based lipidomics in certain research questions. Alos the alkyne function can be detected with Raman spectroscopy. The azide in the acyl chain might be better accessible than in the backbone and aimed for characterization and comparison of both derivatives.

[0330] Metabolic acceptance of the substrate

[0331] The structural modifications in TFSM 1 and TFSM 2 are less demanding than the ones in the probe with pre-attached fluorophores. HEK293T cells were incubated with 1 pM of the derivatives for 24 h and performed lipid extraction followed by LC-MS / MS analysis. The TFSM molecules were detected within the cellular lipid extracts, suggesting the uptake of the compounds by the cells (Fig. lib). The presence of the respective ceramide metabolites confirmed for both derivatives that they are accepted as substrates by SMases. For TFSM 1, the sphingosine metabolite indicating processing by ceramidases was also measured. The sphingosine content shown for TFSM 2 (Fig. lib) represents the sum of intrinsic sphingosine and sphingosine supposedly originating from TFSM 2. For cells treated with TFSM 2, a slight increase in sphingosine levels compared to the solvent control (normalized peak area: 0.652 vs 0.489) was found.

[0332] In addition, higher amounts of the TFSM 2 ceramide metabolite than for TFSM 1 were found, suggesting that TFSM 2 is metabolized more efficiently by SMases (Fig. 11c). Visualization of cellular membranes

[0333] Next, a protocol for staining cellular membranes with TFSMs was established. First, it was tested whether the cellular uptake of the molecules is sufficient for detection in fluorescence microscopy. Therefore, HeLa cells were treated with 10 pM TFSM 1 in the presence of 10% fetal bovine serum (FBS), which is a concentration that is commonly used in cell culture. The cells were fixed, permeabilized, and clicked with AlexaFluor™ 488-DBCO as well as Cy5 azide in the presence of Cu(l). A weak fluorescence signal (Fig. lid; 10% FBS) was detected. HeLa cells were treated in the presence of varying FBS concentrations and measured BODIPY-TR fluorescence, which correlates to probe uptake, via microscopy or flow cytometry. It was found that FBS reduces probe uptake in a concentration-dependent manner. Next, HeLa cells were treated with TFSM 1 in the presence of 1% FBS, fixed and permeabilized, and clicked with AlexaFluor™488-DBCO and Cy5 azide. The staining of cellular membranes by the molecule (Fig. lid; 1% FBS) was enhanced compared to a sample that was treated in the presence of a higher FBS concentration (Fig. lid; 10% FBS).

[0334] The retention of TFSMs and BODIPY-FL-C12-SM within samples upon permeabilization was also investigated. Therefore, HeLa cells were incubated with BODIPY-FL-C12-SM and either analyzed the cells alive, fixed, or fixed + permeabilized for BODIPY-FL fluorescence via flow cytometry (Fig. lie). While retention of the lipid was not affected by fixation, permeabilization drastically reduced BODIPY- FL fluorescence. Next, HeLa cells were incubated with TFSM 1 and TFSM 2, fixed either with glutaraldehyde (GA) + paraformaldehyde (PFA) or only with PFA, permeabilized, clicked with BODIPY- FL-DBCO and analyzed by flow cytometry (Fig lie) or microscopy. High retention of both compounds was obtained when fixed with GA + PFA, while BODIPY-FL fluorescence was markedly reduced in samples solely fixed with PFA.

[0335] Enrichment of TFSM 1 and TFSM 2 fluorescence was observed at distinct sites within the cells. TFSM 1-enriched sites thereby colocalized with the Golgi matrix protein (GM) 130 (Fig. 12a), suggesting a high abundance of the compounds within the Golgi apparatus. It was next tested, whether TFSMs would colocalize with other membrane-rich organelles and stained the mitochondrial matrix protein Peroxiredoxin 3 (Prx3). Colocalization of TFSM l and TFSM 2 with mitochondria in HeLa and primary human umbilical vein endothelial cells (HuVEC; Fig. 12b) was observed.

[0336] Measuring the activity of a neutral bSMase on human cells

[0337] To test whether cleavage of the TFSMs could be visualized by the staphylococcal neutral bSMase 0- toxin, HeLa were incubated with TFSM 2 for 2 h, removed the molecule, and treated the cells with bSMase for 3 h. The samples were fixed, and BODIPY-FL-DBCO was clicked to the backbone while AlexaFluor™546-azide was clicked to the headgroup of the molecule. FRET acceptor bleaching was conducted in selected regions of interest (ROI; Fig. 12c) and measured donor fluorescence pre- and post-bleaching in the respective area to determine the FRET efficiency. For both compounds, a FRET efficiency of ~ 30% was determined, which was markedly reduced upon treatment with bSMase (Fig. 12d). Thus, it was concluded that the bSMase cleaved off the modified phosphocholine headgroup, thereby abolishing FRET with the fluorescence acceptor in the TFSMs. The results were compared with a conventional method, where HeLa were incubated with BODIPY-FL-C12-SM, extracted the lipid, and determined the proportion of remaining unmetabolized BODIPY-FL-C12-SM via thin-layer chromatography (Fig. 12d). The amount of SM was reduced upon bSMase treatment in the same fashion as observed for TFSMs.

[0338] Visualizing the cleavage of TFSMs with subcellular resolution by 4xExM

[0339] Next, TFSM turnover was monitored by ExM. Therefore, HeLa cells were incubated for 24 h with 10 pM TFSM 1 in the presence of 1% FBS. Samples were fixed, clicked with BODIPY-FL-DBCO as well as AlexaFluor™546-azide, and were 4x expanded. Confocal fluorescence microscopy demonstrated the staining of cellular membranes. Clearly, 4-fold expansion enabled the visualization of cellular membranes at higher spatial resolution (Fig. 13a), thus enabling imaging of the mitochondrial envelope.

[0340] Acceptor photobleaching of the samples demonstrated an increase of donor fluorescence intensity in the bleached ROIs (Fig. 13b), resulting in a FRET efficiency of ~ 33%, which decreased upon treatment with bSMase (Fig. 13c). Thus, TFSM can be used to monitor cleavage by SMases. Spots with enhanced FRET signals were observed in the samples, suggesting that these sites contain a high proportion of non-processed TFSM. Since lysosomes are the major site of SM degradation, the sample was immunostained for lysosomal-associated membrane protein 1 (LAMP1) and observed colocalization (Fig. 13e, f). Moreover, ratio images were calculated by dividing the FRET and donor channels to estimate the metabolic state of the TFSM derivative with high spatial resolution (Fig. 13d). These results demonstrate the presence of non-metabolized TFSM in lysosomes (Fig. 13e, f).

[0341] TFSMs enable visualization of sphingolipid metabolization during chlamydial infection

[0342] The obligate intracellular pathogen C. trachomatis forms large inclusions within its host cells and, thereby, is highly dependent on host metabolites, such as sphingolipids. To evaluate sphingolipid metabolism and uptake into chlamydial inclusions, HeLa were infected cells with C. trachomatis at MOI 1. To avoid direct contact of bacteria with TFSMs, the medium was changed 3 h post-infection (p.i.), and the TFSMs were added subsequently. At 27 h p.i., cells were fixed, stained with BODIPY-FL- DBCO and AlexaFluor™546-azide, and analyzed via conventional CLSM. a high BODIPY-FL signal was observed within inclusions (Fig. 14a; white lines), whereas only a moderate FRET signal was detected compared to host cell areas. To visualize the metabolic state of the molecule, the FRET were divided by the donor (BODIPY-FL) channel. An increased proportion of metabolized molecules was found in chlamydial inclusions compared to host cell areas. Next, the FRET efficiency was measured either in an area containing host cell membranes or a chlamydial inclusion (Fig. 14b). A FRET signal could be detected within the inclusion, suggesting the presence of the non-metabolized TFSMs. However, the FRET efficiency was significantly higher in areas containing host cell membranes, indicating that inclusions possess a higher proportion of TFSM metabolites (Fig. 14c).

[0343] To further confirm these observations, fluorescence lifetime imaging microscopy (FLIM) was conducted with Ch / omyd / o-infected samples. A reduced fluorescence lifetime of the donor fluorophore BODIPY-FL was observed in the presence of the acceptor clicked to TFSM 1 (Fig. 14d, e), indicating FRET between the fluorophores. Furthermore, higher fluorescence lifetimes were detected in chlamydial inclusions compared to regions containing host cell membranes (Fig. 14d), again suggesting a high proportion of metabolized TFSM within inclusions.

[0344] To elucidate the distribution of the molecule within an inclusion, 4xExM was conducted with a Ch / amyd / o-infected sample and stained TFSM 1 with BODIPY-FL-DBCO and AlexaFluor™546-azide. Moreover, chlamydial HSP60 were stained to visualize the inclusion (Fig. 15a). As previously observed with unexpanded samples, the inclusion possesses a lower proportion of native molecule, which can be visualized by the metabolic state (ratio FRET vs. donor / BODIPY-FL channel, Fig. 15b). This was further confirmed by acceptor photobleaching of chlamydial inclusions or regions containing host cell membranes (Fig. 15c). Thereby, a lower FRET efficiency was observed within the inclusion area, suggesting the presence of a high proportion of the metabolized molecule (Fig. 15d).

[0345] Nevertheless, a FRET signal was detected within the inclusion membranes as well as RBs that localize to the periphery of the inclusion. Although the FRET signal in RBs was lower compared to host cell membranes, it was clearly higher than the unspecific background signal. Hence, TFSM is also present in the non-metabolized state within C. trachomatis, albeit to a lower extend, as in host cell membranes. In contrast to RBs [Fig. 15e, f; i.)], the smaller, less metabolic-active EBs, which redifferentiate from RBs during infection, predominantly possessed a higher proportion of donor signal, whereas the FRET signal was comparable [Fig. 15e, f; ii.)] or even lower [iii.)] than in RBs. This indicates a particularly high content of metabolized TFSM 1 within EBs. Particles that featured characteristics of RBs and EBs (Fig. 15b, white arrows) were only rarely found. These bacteria owned a comparably high proportion of non-metabolized TFSMs as found in RBs, while their size rather resembled EBs. Similarly, a higher proportion of metabolized TFSM 2 was detected within EBs.

[0346] Next, FLIM was conducted of 4-fold expanded samples that were infected with Chlamydia. Thereby, reduced fluorescence lifetimes were observed in areas containing host cell membranes, whereas lifetimes within chlamydial inclusions were longer, even though the differences were less pronounced as previously seen in unexpanded samples. Again, the decreased FRET signal indicates a lower proportion of native non-metabolized TFSM 1 within the inclusions. The highest fluorescence lifetimes within inclusions were measured in EBs (Fig. 14g), again indicating that EBs predominantly contain metabolized TFSM.

[0347] Membrane-integral fluorophores are affected by their lipid environment

[0348] Next, it was tested if metabolization of TFSMs can be detected by ratiometrically analyzing two fluorophores without using a FRET system. Therefore, HeLa cells were incubated with TFSM 1, treated with bSMase, and stained with AlexaFluor™488-DBCO and Cy5-azide, two fluorophores that do not interact via FRET (Fig. 16a). As expected, the fluorescence intensity of Cy5, clicked to the modified phosphocholine head group, was reduced upon bSMase treatment. For quantification, we measured the fluorescence intensity of both Cy5 as well as AlexaFluor™488 channels and calculated the ratio (Cy5 vs. AlexaFluor™488). In principle, the obtained ratios describe the metabolic conversion of the TFSMs, whereby a high ratio indicates a large proportion of the original non-metabolized TFSM 1 (Fig. 16b). As expected, upon bSMase treatment, the ratio was decreased compared to untreated controls, suggesting enzymatic turnover. Hence, in principle, bSMase treatment can be monitored ratiometrically.

[0349] However, an enhanced AlexaFluor™488 signal was observed in samples treated with bSMase (Fig. 16a), even though the presence of Cy5 should not affect AlexaFluor™488 fluorescence. To further investigate this phenomenon, a flow cytometry-based readout was established for TFSMs. HeLa was incubated with TFSM 1 for 2 h, removed the molecule again, and treated the cells with lOO ng / ml bSMase or left them untreated. Then, the cells were detached and the backbone of TFSM 1 stained with BODIPY-FL DBCO, while no other dye was clicked to the headgroup. Subsequently, BODIPY-FL mean fluorescence was analyzed via flow cytometry (Fig. 16c). Again, bSMase-treated cells possessed higher BODIPY-FL signals. Since there was no fluorophore clicked to the headgroup of TFSM 1, the observed change in fluorescence intensity cannot be due to intramolecular FRET.

[0350] Next, it was tested whether this phenomenon can be observed with other SM derivatives and repeated the experiment with the visible-range FRET probe with pre-attached fluorophores. An increased BODIPY-TR fluorescence was detected upon bSMase treatment, whereas the FRET system was not affected, thereby confirming that the FRET probe is not metabolized by the bSMase (Fig. 16d). A similar but less pronounced increase of fluorescence was also observed, when the experiments were conducted with a commercially available BODIPY-FL-C12-SM analog (Fig. 16e). Thus, it can be concluded that the enhanced signal of membrane-associated fluorophores of the SM derivatives does not necessarily require metabolization of the molecules themselves.

[0351] Since close proximity of fluorophores can result in self-quenching, ExM was performed on the samples, which physically enlarged the spatial distance between molecules within the specimen. Hence, selfquenching should be reduced in expanded samples. HeLa cells were therefore incubated with TFSM 1 for 24 h, the molecule removed, and the cells treated with bSMase. Subsequently, the backbone was clicked with BODIPY-FL-DBCO, and samples were either embedded in Mowiol or a hydrogel. The gelated samples were either left unexpanded or were expanded. An increase in BODIPY-FL fluorescence was observed upon bSMase treatment in images obtained in Mowiol and Gel-embedded unexpanded samples (Fig. 15f). In contrast, there were no differences between bSMase-treated samples and untreated controls after 4-fold expansion (Fig. 15g). It can be concluded that the enhanced signal of membrane-integral fluorophores upon bSMase treatment is due to a larger intermolecular distance between membrane-integral fluorophores.

[0352] Methods

[0353] In vitro SMase activity assay

[0354] To determine ASM as well as the bSMase p-toxin activity towards a visible range FRET probe (Kappe, C., Mohamed, Z. H., Naser, E., Carpinteiro, A. & Arenz, C. A novel visible range FRET probe for monitoring acid sphingomyelinase activity in living cells. Chem. Eur. J. 26, 5780-5783 (2020)), the probe was incubated with 0.25 mU / ml recombinant human ASM (rhASM, R&D Cat. No. 5348-PD) or recombinant p-toxin (Sigma, Cat. No. S8633) in a volume of 200 pL / well in a 96 well plate (Corning, black / clear flat bottom; Ref. 3603) at 37 °C under orbital shaking in a Tecan Mplex microplate reader. The reaction was carried out in acidic buffer [200 mM sodium acetate pH 5 (Roth, Cat. No. 6773.2), 500 mM NaCI (VWR, Cat. No. 27810.364), 500 pM ZnCI2(Sigma, Cat. No. 208086) 0.2 % Nonidet P-40 (AppliChem, Cat. No. A1694.0250)] for ASM and neutral buffer [200 mM HEPES pH 7 (Roth, Cat. No. 6763.3), 0.05% Nonidet P-40] for P-toxin. Buffer composition was taken from a previous study (Muhle, C. & Kornhuber, J. Assay to measure sphingomyelinase and ceramidase activities efficiently and safely. J. Chromatogr. A 1481, 137-144 (2017)). FITC (ex. 488 ± 9 nm, em. 520 ± 20 nm) and BODIPY-TR (ex. 550 ± 9 nm, em. 620 ± 20 nm) was measured every 20 min. The ratio FITC vs. BODIPY-TR was calculated to determine the conversion of the FRET probe.

[0355] Cell culture

[0356] HeLa cells (ATCC CCL-2™) were cultured in RPMI+GlutaMAX™ medium (Gibco™, Cat. No. 72400054) containing 10% (v / v) heat-inactivated (56 °C at 30 min) fetal bovine serum (FBS, Sigma Aldrich. Cat. No. F7524) and 1 mM sodium pyruvate (Gibco™, Cat. No. 11360088).

[0357] HeLa229 cells (ATCC CCL-2.1™) were cultured in RPMI + GlutaMAX™ medium containing 10% (v / v) heat-inactivated FBS. Hela229 cells were confirmed to be free from Mycoplasma contamination using PCR. As the HeLa229 cell line was authenticated by ATCC, it was not further validated by our laboratory.

[0358] Human umbilical vein endothelial cells (HuVEC, Gibco™, Cat. No.C01510C) were cultured in MCDB131 medium (Gibco™, Cat. No. 10372019) complemented with microvascular growth supplement (Gibco™, Cat. No. S00525), 2 mM GlutaMAX™ (Gibco™, Cat. No. 35050061), 5 % (v / v) heat-inactivated (56 °C at 30 min) FBS, 2.76 pM hydrocortisone (Sigma Aldrich, Cat. No. H0888), O.Ol ng / ml human epidermal growth factor (Pep Rotech, Cat. No. AF- 100-15) and lx Penicillin-Streptomycin (Gibco™, Cat. No. 15140122).

[0359] Cells were seeded either one day (HeLa and HeLa229) or two days (HuVEC) prior to the experiment either in a 12-well plate (1 x 105cells per well) or 24-well plates (0.5 x 105cells per well). Standard tissue culture procedures were used to maintain the cells. All cells were cultured in a humidified atmosphere with 5 % CO2(v / v) at 37 °C.

[0360] Chlamydia trachomatis culture

[0361] The L2 / 434 / Bu (ATCC VR-902B) serovar of Chlamydia trachomatis was used for this study. Chlamydia trachomatis was expanded in HeLa229 cells at MOI 1 for 48 h. Cells were disrupted and mechanically lysed using glass beads (2.85-3.45 mm, Roth, Cat. No. A557.1). Cells were centrifuged for 10 min at 755 x g at 4 °C and supernatant was centrifuged for 30 min at 40,000 x g at 4 °C to collect bacteria. The pellet was washed once with 1 x sucrose-phosphate-glutamic acid (SPG) buffer [7.5% sucrose (Roth, Cat. No. 4621.2), 0.052% KH2PO4(Roth, Cat. No. 3904.1), 0.122% Na2HPO4(Roth, Cat. No. P030.2), 0.072% L-glutamine (Gibco, Cat. No. 25030081)]. To singularize the Chlamydia, the pellet was resuspended in 1 x SPG buffer and passed through G20 (B. Braun, Cat. No. 612-0141) and G18 (B. Braun, Cat. No. 612-0147) hollow needles. Aliquoted bacteria were stored at -80 °C until further use. MOI 1 was determined by titration. Chlamydia was confirmed to be free from Mycoplasma contamination using PCR. The preparation of Chlamydia was based on a previously published protocol (https: / / www.nature.com / articles / s41467-024-51874-w).

[0362] Analysis of lipid derivatives by flow cytometry

[0363] HeLa cells were incubated with 10 pM TFSM 1, 10 pM visible range FRET probe, or 1 pM BODIPY™-FL- C12-SM (Thermo Fisher, Cat. No. D7711) in treatment medium [MCDB131 medium containing 1% (v / v) heat-inactivated FBS and GlutaMAX™] for 2 h. Then, cells were washed thrice with DPBS (Gibco™, Cat. No. 14190169) and treated with 100 ng / ml p-toxin / bSMase for 3 h.

[0364] Consecutively, cells were washed thrice with DPBS, detached with 300 pL / well trypsin [TrypLE™, (Gibco™, Cat. No. 12604039)] for 5 min at 37 °C and resuspended in 300 pL 2% FBS in DPBS. For TFSM 1, samples were centrifuged (1.100 x g, 5 min, 4 °C) and fixed with 0.2% GA (Sigma, Cat. No. G5882) in 4% PFA in DPBS (Morphisto, Cat. No. 11762) for 30 min / RT. Cells were washed twice in 2% FBS in DPBS and then, permeabilized with 0.2% Triton X-100 (Roth, Cat. No. 6909) in PBS (AppliChem, Cat. No. A0964,9100). Next, samples were washed twice with 2% FBS in DPBS and stained with 2 pM BODIPY- FL-DBCO (Jena Biosciences, Cat. No. CLK-040-05) in 500 pL / sample Hanks' buffered saline (HBSS, Gibco™, Cat. No. 14025-100) for 1 h at 37 °C. Subsequently, cells were washed thrice with 2% FBS in DPBS. If not indicated otherwise, cells were centrifuged at 5.000 x g / 5 min / 4 °C between washing steps. Fixation, permeabilization, and staining were carried out in an end-over-end rotator. Then, cells were analyzed in an Attune NxT flow cytometer (Thermo Fischer) for BODIPY-FL (ex. 488 nm / em. bandpass 530 / 30 nm).

[0365] For samples treated with BODIPY-FL-C12-SM and the visible range FRET probe, living cells were directly analyzed for BODIPY-FL / FITC (ex. 488 nm / em. bandpass 530 / 30 nm), FRET (ex. 488 nm / em, band pass 695 / 40 nm) and BODIPY-TR (ex.: 561 nm / em. bandpass 695 / 40 nm) with an Attune NxT flow cytometer. Analysis of lipid derivatives by confocal laser scanning microscopy (CLSM)

[0366] HeLa cells were incubated with 10 pM TFSM 1 or TFSM 2 in treatment medium for 2 h (unexpanded samples) or 24 h (ExM). If indicated, cells were washed thrice with DPBS and treated with 100 ng / ml bSMase for 3 h (unexpanded samples) or 2.5 pg / ml bSMase for 24 h (ExM). After treatment, samples were washed thrice with DPBS and fixed with 0.2% GA (Sigma, Cat. No. 10333) / 4% PFA in DPBS for 30 min RT. Then, cells were washed thrice and permeabilized with 0.2 % Triton X-100. Subsequently, the samples were washed thrice with DPBS and stained with 2 pM (unexpanded) or 4 pM (ExM) BODIPY-FL-DBCO or AlexaFluor™ 488-DBCO (Jena Biosciences, Cat. No. CLK-1278) in HBSS for 1 h / 37 °C. Cells were washed five times with DPBS and stained with 10 pM (unexpanded) or 20 pM (ExM) AlexaFluor™546-azide (Jena Biosiences, Cat. No. CLK-1283), 20 pM Atto647N-azide (Atto-Tec, Cat.No. AD647N) or 2 pM Cy5-azide (Wolf, N., Kersting, L., Herok, C., Mihm, C. & Seibel, J. High-yielding water-soluble asymmetric cyanine dyes for labeling applications. J. Org. Chem. 85, 9751-9760 (2020)) in click reaction buffer [50 pM CuSO4, 2.5 mM sodium ascorbate (Sigma, Cat.No. A4034), 250 pM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA; Sigma, Cat. No. 762342) in PBS] for 1 h / 37 °C. Then, samples were either mounted in Mowiol [24 g glycerol (Roth, Cat. No. 3783.2), 9.6 g Mowiol® 4-88 (Roth, Cat. No. 0713.2), 48 ml 0.2 M TRIS-HCI pH 8.5 (Sigma T1503), 24 ml Millipore H2O] or used for ExM. Imaging was performed with a TCS SP5 confocal laser scanning microscope (Leica, Wetzlar, Germany). Images were adjusted for brightness and contrast. A median filter was applied to the images.

[0367] For determining the metabolic state, gray values in the FRET channel were multiplied by factor 2 (LAMPl-stained samples) or 3 (Ch / omyd / o-infected samples) and then divided by the donor (BODIPY- FL) channel. The resulting ratio images were color-coded with the "Fires" look-up table (LUT) in Fiji.

[0368] For visualization of mitochondria, images were recorded on a Zeiss LSM900 confocal microscope with Airyscan 2, operating in the super-resolution (SR) imaging mode. An apochromatic oil immersion objective (40x / NA 1.3, Zeiss) was used for all images. The ZEN2 blue software (Zeiss, version 3.5) was used to select the optimal filter settings and excitation wavelengths for the various fluorescent dyes used in the experiment, utilizing the integrated preset configurations provided by the software. All acquired images were processed in standard strength Airyscan mode ensuring consistent and optimal data analysis across the sample set. Images were adjusted for brightness and contrast. A median filter was applied to the images.

[0369] FRET acceptor bleaching FRET acceptor bleaching was conducted with a Leica TCS SP5 microscope and the built-in FRET AB wizard. Thereby, images in donor and acceptor channels were recorded. Then, a region of interest (ROI) was selected in which the acceptor fluorophore was bleached by recording 20 frames with a high laser intensity. Subsequently, donor and acceptor channels were recorded, and FRET efficiency was determined by comparing donor and acceptor signal pre- and post-bleaching according to Eq. 1.).

[0370] ( (Donor,,.,.. - Donorn„)

[0371] FRETeff. = - - .100 (1)

[0372] \ Donorpost

[0373] Detecting bSMase activity with BODIPY-FL-C12-SM

[0374] HeLa cells were incubated with 1 pM BODIPY-FL-C12-SM in treatment medium for 2 h in a 12-well plate. BOIDPY-FL-C12-SM was removed, cells were washed thrice, and further incubated with 100 ng / ml 0- toxin / bSMase for 3 h or left untreated. Then, cells were washed thrice, detached with 300 pL / well TrypLE, resuspended with 300 pL / well 2% FBS in PBS, and stored on ice. Samples were centrifuged 2000 x g / 5 min / 4 °C, pellets were resuspended in 250 pL 2:1 MeOH (Roth, Cat. No. 8388.6) / CHCIB (Roth, Cat. No. 3313.2), thoroughly vortexed and again centrifuged 13.000 x g / 3 min / RT. 50 pLof the lower organic phase containing the lipids were taken and evaporated in a SpeedVac 5301 concentrator (Eppendorf) at 45 °C. Samples were resuspended in 10 pl 2:1 MeOH / CHCI3and spotted in 2.5 pl steps onto a thin-layer chromatography (TLC) plate (Alugram, Xtra Sil G / UV254, 0.2 mm / silica gel 60; VWR, Cat. No. 552-1006). TLC was developed with 80:20 MeOH / CHCh and scanned with a Typhoon RGB scanner (Amersham). Intensities of fluorescent SM and ceramide bands were evaluated in Fiji to determine the proportion of unmetabolized BOIDPY-FL-C12- SM.

[0375] Antibody staining

[0376] After TFSMs were stained with DBCO and azide dyes, samples were blocked for 1 h / RT with 10% FBS in DPBS. Then, cells were incubated with 4 pg / ml anti-LAMP-1 antibody (SantaCruz, Cat. No sc- 18821, clone H5G11, Lot. #H2118), 2.5 pg / ml anti-GM130 antibody (Becton Dickinson, Cat. No. 610823, Lot. 7163670), 1:50 anti-Prx3 antibody (OriGene, Cat. No. TA322472) or 4 pg / ml anti-chlamydial HSP60 antibody (SantaCruz, Cat. No. sc-57840, Lot. #K122O) in blocking buffer overnight at 4 °C or for 1 h at RT. Then, cells were washed thrice with DPBS and incubated with 20 pg / ml CF™568 (Sigma, Cat. No. SAB4600082, Lot. 16C1017) or 20 pg / ml AlexaFluor™405 (ThermoFisher, Cat. No. A48255, Lot. YA353687) secondary antibody in blocking buffer at 4 °C overnight or for 1 h at RT. Expansion microscopy of TFSMs

[0377] Stained samples were post-fixed with 0.2% GA in DPBS for 15 min / RT and washed thrice with DPBS. Then, the glass slide was turned upside down in a drop of 80 pl (015 mm slide) or 56 pl (012 mm slide) monomer solution [8.625% sodium acrylate (Sigma, Cat. No. 408220), 2.5% acrylamide (Sigma, Cat. No. A9926), 0.15% N,N'-methylene bisacrylamide (Sigma, Cat. No. 146072), 2 M NaCI (Sigma, Cat. No. S5886) in PBS] that was freshly complemented with 0.2% (w / v) ammonium persulfate (Sigma, A3678) and 2% (v / v) tetramethyl ethylene diamine. Polymerization was carried out for 90 min at RT, and gels then were digested with 8 U / ml proteinase K (Sigma, Cat. No. P4860) in digestion buffer [50 mM Tris pH 8.0, 1 mM EDTA (Sigma, Cat. No. ED2P), 0.5% Triton X-100 and 0.8 M guanidine HCI (Sigma, Cat. No. 50933)] for 30 min / RT. Consecutively, gels were expanded in excess of Millipore water, whereby water was exchanged every hour until maximum expansion was reached. The expanded gels were cut in pieces of 1-2 cm and transferred into a Lab-Tek™ II chamber (VWR, Cat. No. 734-2055), which was coated with 0.01% poly-L-lysine solution (Sigma, Cat. No. A-005-C). Gels were imaged with a Leica TCS SP5 microscope. This protocol was adapted from a previous study (Chozinski, T. J. et al. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485-488 (2016)).

[0378] Infection with Chlamydia trachomatis and treatment with lipid derivatives

[0379] HeLa229 cells were seeded a day prior to the experiment in a 12-well plate (1 x io5cells per well). The next day, the medium of the cells was exchanged for fresh RPMI + GlutaMAX™ medium containing 10% heat-inactivated FBS. The cells were infected with C. trachomatis at MOI 1. 3 h after infection, the medium was exchanged to RPMI + GlutaMAX™ medium containing 1% heat-inactivated FBS. 10 pM TFSM 1 or TFSM 2 was added for 24 h. The cells were fixed with 0.2% GA / 4% PFA in DPBS for 15 min RT and further processed for confocal laser scanning microscopy as described above.

[0380] FLIM-FRET measurements and analysis

[0381] Fluorescence lifetime measurements were performed on a MicroTime200 (PicoQuant, Berlin, Germany) time-resolved confocal fluorescence microscope setup equipped with a FLIMbee galvo scanner (PicoQuant, Berlin, Germany), an Olympus 1X83 microscope including an oil-immersion objective (60 x, NA 1.45; Olympus), two single-photon avalanche photodiodes (SPADs) (Excelitas Technologies, 75154 K3, 75154 L6), and a TimeHarp300 dual-channel board. A white-light laser (NKT Photonics, SuperK extreme) was used for pulsed excitation at 488 nm with a repetition rate of 19.5 MHz and was coupled into the MicroTime200 system via a glass fiber (NKT Photonics, SuperK FD PM, A502-010-110). For all measurements, a 100 pm pinhole was used. The emission light was split onto the SPADs using a 50:50 beamsplitter (PicoQuant). Two identical bandpass filters (ET525 / 50 M, Chroma) were installed in front of the SPADs to filter out after-glow effects as well as scattered and reflected light. Measurements were performed and analyzed with the SymPhoTime64 software (PicoQuant, Version 2.8). All measurements were performed with a pixel dwell time of 25 ps. All unexpanded samples were measured with an irradiation intensity of ~ 0.5 kW cm~2in T3 mode with 25 ps time resolution, whereas all measurements of expanded samples were measured with ~ 7 kW crrr2in T3 mode. For single-cell measurements of unexpanded samples, images of 250 pixels * 250 pixels (100 nm * pxx) were taken with a frame frequency of 0.3 Hz, and five frames were averaged to get the final image. For the overview image of unexpanded samples, images of 1000 pixels * 1000 pixels (150 nm * px-1) were measured with a frame frequency of 0.013 Hz, and two frames were averaged to get the final image. For all expanded samples, five frames were averaged, consisting of 500 pixels * 500 pixels (180 nm * pxx) at a frame frequency of 0.1 Hz to get the final image.

[0382] For analyzing the fluorescence lifetime of all images, the decay parameters were determined by leastsquares deconvolution, and their quality was judged by the reduced x2 values and the randomness of the weighted residuals (x2 of roughly 1)( Thiele, J. C., Nevskyi, O., Helmerich, D. A., Sauer, M. & Enderlein, J. Advanced data analysis for fluorescence-lifetime single-molecule localization microscopy. Front. Bioinform. 1, 740281 (2021)). A multiexponential model was used to fit the decay (Eq. 2).

[0383] (2)

[0384] For visualizing FLIM images, we encoded the lifetime and intensity using a color map and the brightness, respectively. Isoluminescent and perceptual uniform color maps were used to prevent an overlap of intensity and fluorescence lifetime using the TrackNTrace Lifetime Edition(Stein, S. C. & Thiart, J. TrackNTrace: A simple and extendable open-source framework for developing singlemolecule localization and tracking algorithms. Sci. Rep. 6, 37947 (2016); Thiele, J. C. et al. Confocal fluorescence-lifetime single-molecule localization microscopy. ACS Nano 14, 14190-14200 (2020)).

[0385] Statistics & reproducibility

[0386] The sample size was determined empirically based on similar experiments performed previously. No statistical method was used to predetermine the sample size. No data were excluded from the analyses. ROIs during microscopy were selected randomly. Otherwise, the experiments were not randomized. For recording and evaluation of the FLIM measurements, investigators were blinded. Otherwise, the Investigators were not blinded to allocation during experiments and outcome assessment. Statistical analysis was performed with GraphPad Prism (10.1.2).

[0387] Further information on the testing of compounds TFSM1 and TFSM2 are disclosed in Ruhling, M., Kersting, L., Wagner, F. et al. Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy. Nat Common 15, 7456 (2024). https: / / doi.org / 10.1038 / s41467-024-51874-w. This document, including the "Supplementary information" and the "Source data" relating to this document (available at https: / / www.nature.com / articles / s41467-024-51874-w), is incorporated herein in its entirety by reference.

[0388] Airyscan imaging

[0389] Airyscan images of COS 7 cells incubated with 5 pM Cerl6-1 for 16 h where obtained. Fig. 17A shows an unexpanded cell. Fig. 17B show the cell expanded fourfold. Fig. 17C and 17D represent magnified sections of the cell in slide B.

[0390] Based on the above, it can be seen that the present application shows that the new classes of molecules can detect the enzymatic activity of sphingomyelinase and, at the same time, allow the precise localization of the molecule to be identified through so-called expansion microscopy. This has not been possible with previously available and developed sphingolipids.

Claims

CLAIMS1. A compound of the following formula (I):whereinA1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2 substituent,A3is selected from C1.5 alkyl optionally substituted with one or two substituent(s) selected from Group X, wherein at least two, preferably all three, of A1, A2and A3have the one or two substituent(s) selected from Group X, and wherein the compound of formula (I) contains at least two, preferably three, substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyi, Ci- 6alkyl-diazirinyl and tetrazinyl.

2. The compound of formula (I) according to claim 1, wherein(i) A1is C4-23 alkyl or alkenyl (preferably C6-2i alkyl or alkenyl, more preferably C8-is alkyl or alkenyl, even more preferably Cw-is alkyl or alkenyl, most preferably Cu, C12, Ci3or CM alkyl or alkenyl, such as CM alkyl or alkenyl) wherein the alkyl or alkenyl is preferably substituted with one substituent selected from Group X, and / or(ii) the alkyl or alkenyl in A1is alkyl, and / or(iii) wherein A1has at least one terminal substituent of Group X (which is preferably azide), and / or(iv) wherein A1has a -OH or -NH2 substituent (preferably -NH2 substituent), wherein the OH or - NH2substituent is preferably present at the carbon atom which is bound to the carbonyl group to which A1is bound, and / or(v) wherein A1has one further substituent of Group X (which is preferably diazirinyl), and / or(vi) A2is C4-23 alkyl or alkenyl (preferably Ce-2i alkyl or alkenyl, more preferably Cs-i8 alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably Cu, C12, C13 or C14 alkyl or alkenyl, such as C13 alkyl or alkenyl) wherein the alkyl or alkenyl is preferably substituted with one substituent selected from Group X, and / or(vii) the alkyl or alkenyl in A2is alkyl, and / or(viii) A3is C1-2 alkyl substituted with one substituent selected from Group X, wherein the substituent of Group X is preferably alkyne (preferably with a terminal CC triple bond), more preferably a -C=CH group, and / or(ix) A3is C1-2 alkyl, preferably methyl, and / or(x) wherein the compound of formula (I) contains at least one diazirinyl group, preferably as a substituent in A1.

3. The compound according to claim 1 or 2, wherein the compound has the following formula (la)wherein R1, R2and R3are each independently selected from -H and Group X;R4is selected from H, diazirino, -oxo, -OH and -NH2; preferably wherein R4is selected from diazirino and -NH2, more preferably wherein R4is -NH2; wherein at least one of R1and R4is not hydrogen; and wherein at least one of R2and R3is not hydrogen, wherein it is preferred that neither R2nor R3is hydrogen; n is an integer from 0 to 20; m is an integer from 0 to 20; u is an integer from 0 to 20; wherein the sum of u and m is in the range of from 2 to 20.

4. A compound of the following formula (II):whereinA1and A2are each independently selected from C2-25 alkyl or alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or two substituent(s) selected from Group X, wherein A1optionally furthermore has a -OH, oxo or -NH2substituent, one, preferably both, of A1and A2have the one or two substituent(s) selected from Group X, and if A1has a -OH or -NH2substituent, the compound of formula (II) contains at least one, preferably two or three, more preferably two, substituents selected from Group X, if A1does not have a -OH or -NH2 substituent, the compound of formula (II) contains at least two, preferably three, substituents selected from Group X, wherein group X consists of C2-10 alkynyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci- 6alkyl-diaziriny I and tetrazinyl.

5. The compound of formula (II) according to claim 4, wherein(i) A1is C4-23 alkyl or alkenyl (preferably C6-2ialkyl or alkenyl, more preferably C8-is alkyl or alkenyl, even more preferably Cio-is alkyl or alkenyl, most preferably CH, C12, C13 or CM alkyl or alkenyl, such as CM alkyl or alkenyl) wherein the alkyl or alkenyl is preferably substituted with one substituent selected from Group X, and / or(ii) the alkyl or alkenyl in A1is alkyl, and / or(iii) wherein A1has at least one terminal substituent of Group X (which is preferably azide) , and / or(iv) wherein A1has a -OH or -NH2substituent (preferably -NH2substituent), wherein the OH or - NH2 substituent is preferably present at the carbon atom which is bound to the carbonyl group to which A1is bound, and / or(v) wherein A1has one further substituent of Group X (which is preferably diazirinyl), and / or(vi) A2is C4-23 alkyl or alkenyl (preferably C6-2i alkyl or alkenyl, more preferably Cs-is alkyl or alkenyl, even more preferably C10-15 alkyl or alkenyl, most preferably CH, C12, C13 or CM alkyl oralkenyl, such as C13 alkyl or alkenyl) wherein the alkyl or alkenyl is preferably substituted with one substituent selected from Group X, and / or(vii) the alkyl or alkenyl in A2is alkyl, and / or(vii) wherein the compound of formula (II) contains at least one diazirinyl group, preferably as a substituent in A1.

6. The compound according to claim 4 or 5, wherein the compound has the following formula (Ila)wherein R1, R2and R3are each independently selected from -H and Group X;R4is selected from H, diazirino, oxo, -OH and -NH2; preferably wherein R4is selected diazirino and — NH2, more preferably wherein R4is -NH2; wherein at least one of R1and R4is not hydrogen; wherein it is preferred that R2is not hydrogen; n is an integer from 0 to 20; m is an integer from 0 to 20; u is an integer from 0 to 20; wherein the sum of u and m is in the range of from 2 to 20.

7. The compound according to any one of claims 1 to 6, wherein the members of Group X are selected from - ^=, transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci.6alkyl-diazirinyl and tetrazinyl.

8. The compound according to any one of claims 3, 6 and 7, wherein R4is -NH2and u is preferably0.

9. The compound according to any one of claims 3, 6 and 7, wherein R4is diazirino, m is preferably 0, u is an integer of from 1 to 18 (preferably 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 3, 1 or 2, or even 2), wherein R1is preferably hydrogen.

10. The compound according to any one of claims 3 and 6 to 9, wherein R2and R3are each independently selected from C2-10 alky nyl, C5-10 cycloalkenyl, C5-10 cycloalkynyl, -N3, diazirinyl, Ci- ealkyl-diazirinyl and tetrazinyl; preferably wherein R2and R3are each independently selected from transcyclooctenyl, -N3, cyclooctinyl, diazirinyl, Ci-galkyl-diazirinyl and tetrazinyl.

11. The compound according to any one of claims 3 and 7 to 10, wherein R1or R2is -N3, R3is C2-10 alkynyl and R4is NH2or diazirino.

12. The compound according to any one of claims 3 and 6 to 11, wherein one or more of the following are fulfilled:(i) n is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11; and / or(ii) m is an integer of from 2 to 18, preferably 4 to 18, more preferably 6 to 16, even more preferably 8 to 14, still more preferably 9 to 13, still even more preferably 10 to 12, most preferably 11; and / or(iii) u is an integer of from 0 to 17, preferably 0 to 14, more preferably 0 to 11, even more preferably 0 to 8, still more preferably 0 to 5, still even more preferably 0 to 2, most preferably 0; and / or(iv) the sum of m+ u is an integer of from 2 to 30, preferably 5 to 25, more preferably 7 to 22, even more preferably 10 to 18.

13. The compound according to any one of claims 1 to 12, wherein the compound is selected from the following compounds:

14. A conjugate obtainable by reacting a compound according to any one of the preceding claims with one, two or three further compound(s) (preferably one compound) comprising an alkyne (such as a -C=CH group or a cycloalkyne), cycloalkene (such as transcyclooctenyl), tetrazinyl and / or -N3, wherein the conjugate preferably comprises a diazirine group, wherein (i) when the compound according to any one of the preceding claims comprises an alkyne group(such as a -C^CH group or a cycloalkyne), one of the further compound(s) preferably contains an azide group; and(ii) when the compound according to any one of the preceding claims comprises an azide group, one of the further compound(s) preferably contains an alkyne group (such as a -C=CH group or a cycloalkyne); and(iii) when the compound according to any one of the preceding claims comprises a cycloalkene (such as transcyclooctenyl) group, one of the further compound(s) preferably contains a tetrazinyl group; and(iv) when the compound according to any one of the preceding claims comprises a tetrazinyl group, one of the further compound(s) preferably contains a cycloalkene (such as transcyclooctenyl) group, wherein the alkyne, cycloalkene, and cycloalkyne, are preferably -OCH, transcyclooctenyl, -and cyclooctinyl, respectively.

15. The conjugate according to claim 14 for use in diagnostics, preferably for use in the imaging of sphingomyelin and / or ceramide metabolism, preferably to identify abnormalities in the sphingomyelin and / or ceramide metabolism, preferably in relation to a disease selected from cancer, diabetes, a neurodegenerative disease and an infectious disease.