Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates

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Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates Oligodendrocyte Differentiation Tobias Kieser, Nico Santschi, Luise Nowack, Gerald Kehr, Tanja Kuhlmann, Stefanie Albrecht, and Ryan Gilmour ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00002 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 26, 2018

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ACS Chemical Neuroscience

Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates Oligodendrocyte Differentiation Tobias J. Kieser,‡a,c Nico Santschi,‡a,c Luise Nowack,b Gerald Kehr,a Tanja Kuhlmann,b Stefanie Albrecht,*b Ryan Gilmour*a,c a

Institute for Organic Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, 48149 Münster, Germany Institute for Neuropathology, University Hospital Münster, Pottkamp 2, 48149 Münster, Germany c Excellence Cluster EXC 1003 “Cells in Motion”, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, 48149 Münster, Germany b

Supporting Information Placeholder ABSTRACT: Relapsing multiple sclerosis is synonymous with demyelination, and thus suppressing and or reversing this process is of paramount clinical significance. Whilst insulating myelin sheath has a large lipid composition (ca. 70-80%), it also has a characteristically large composition of the sialosylgalactosylceramide gangliosde GM4 present. In this study, the effect of the carbohydrate epitope on oligodendrocyte differentiation is determined. Whilst the native epitope had no impact on oligodendroglial cell viability, a single site OH → F substitution is the structural basis of a significant increase in ATP production that is optimal at 50 µg/ml. From a translational perspective, this subtle change increases the amount of MBP+ oligodendrocytes compared to the control studies and may open up novel therapeutic remyelination strategies. Key words: carbohydrates; fluorine; gangliosides; multiple sclerosis; myelin; oligodendrocytes

INTRODUCTION Identifying effective therapies for relapsing multiple sclerosis (MS) remains a vexatious problem in clinical neurology.1 Characterized by an auto-immunogenic degradation of the axon’s insulating myelin sheath, this disease is accompanied by a range of symptoms that include loss of muscle control, and impaired speech and vision. Current therapeutic regimes favoring intra-muscular injection of the cytokine interferon β1a (Avonex®) have been complemented with a variety of small molecule immuno-suppressants.2 Advances in the field of axon remyelination,3-4 utilizing antibodies5 and transcription factors,6 also show clinical promise. Whilst current treatment strategies effectively address the clinical symptoms of MS, identifying the causative factors of nerve cell demyelination are essential in suppressing, and ultimately reversing, the process.7 In this regard, inspection of the chemical composition of the myelin sheath has proven to be instructive. This electrically insulating bio-membrane is produced by oligodendrocytes, which cover several axons (Figure 1, A). Since these glial cells express specific carbohydrate antigens which are deposited in myelin,8 we sought to explore the effect of specific ganglioside epitopes on oligodendrocyte differentiation. Cognizant of the fact that GM4 (1) is specific to myelin9 and present in elevated concentrations,10-13 we elected to study this epitope. Furthermore, the high affinity of myelin basic protein (MBP) towards GM4 containing multi-lamellar liposomes9 provides future opportunities for the development of imaging probes rendering this approach even more appealing.

From the perspective of epitope probe design, two major editing sites were envisaged (Figure 1, B). The first was to replace the pendant sphingolipid chain with a robust, linear alkyl unit. An elegant chemo-enzymatic strategy has previously been reported.14 This would preserve the lipidic character of the unit whilst mitigating stability problems in subsequent differentiation assays. Secondly, the effect of OH → F substitution on the D-galactopyranose would be investigated.

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Figure 1. A conceptual overview of the axon and myelin sheath (A), and the GM4 ganglioside together with a fluorinated epitope (B). Fluorine incorporation has several advantages that include the ability to control β-glycosylation selectivity,15-19 the presence of a NMR active nucleus for direct analyses, and the enhanced metabolic stability of the glycosidic linkage towards chemical and enzymatic degradation.20-24 Herein, we report a chemical synthesis of the GM4 epitope (2) and its fluorinated counterpart (3) over a 10 linear step sequence, and evaluate the influence of single site OH → F substitution on oligodendrocyte differentiation.

RESULTS AND DISCUSSION The initial synthetic sequence to prepare epitope 3 began with the preparation of the sialic acid donor 4 from commercially available N-acetyl neuraminic acid (full details are provided in the SI).25 The fluorinated galactose acceptor 5 was generated from tri-acetyl D-galactal following a procedure developed previously by this laboratory.26 Glycosylation was achieved by exposing a mixture of 4 and 5 (1.85 equiv.) in MeCN at -40 °C to catalytic TMSOTf (0.3 eq.). Gratifyingly, the desired coupling event proceeded smoothly to generate a mixture of anomers favoring the desired α-linked disaccharide (α:β 2:1). Separation by standard silica gel chromatography proved difficult at this stage, and therefore the material was directly subjected to partial deprotection conditions. Treatment of the disaccharide in mixture of CH2Cl2:TFA:H2O (100:10:1)27 for 30 min. at ambient temperature affected benzylidene acetal deprotection, thereby furnishing a separable mixture of diols. Following separation on SiO2, 6 was isolated in 31% yield over two steps (Scheme 1). Ph AcO

AcO

OAc

O AcHN AcO 4

AcO

OP(OBn) 2 CO2Me

MeO 2C HO AcO OAc O O AcHN AcO 6

O

O

+

O

HO

OH O

OTIPS

F

5

1) TMSOTf, MeCN -40 °C 2) CH 2Cl2, TFA, H2 O (100:10:1) 31% (2 steps)

Figure 2. X-ray crystallographic analysis of the partially protected disaccharide 6. Thermal ellipsoids displayed at the 50% probability level. Hydrogen atoms and acetyl groups were omitted for clarity. CCDC 1570372. The relative stereochemistry of the glycosidic linkage was established by 13C NMR spectroscopy, following a procedure reported by Meguro and co-workers:28 the major αdiastereomer 6 exhibited a coupling constant 3JC1*-C3H* of 6.0 Hz, whereas the minor diastereomer was characterized by a significantly smaller value of 1.4 Hz. To unequivocally establish the configuration by X-ray crystallographic analysis, it was possible to grow crystalline sample of 6 by slow evaporation of a solution in wet MeOH. Figure 2 of the partially deprotected scaffold clearly confirms the α-selectivity (CCDC 1570372). To capitalize on the β-directing effect of the C2-fluorine substituent in D-galactose in side chain installation, weakly inductive protecting groups were introduced at C4 and C6. This is fully in line with previous observations from our laboratory pertaining to the reinforcing nature of C2 configuration and protecting group electronics in governing glycosylation selectivity. However various and repeated attempts to effectively benzylate these positons,29-31 were unsuccessful. Consequently, acetal groups were employed, specifically ethoxy methyl groups, given that they would likely satisfy the electronic requirements for β-selectivity.

OR OR O

RO

OTIPS 35% (2 steps) α: β 28:1

F

OTIPS

OEt

OEt

1) TBAF, THF 2) Cl3CCN, DBU, CH2Cl2

O EtO

O O

O

F HN

F

7, R = Ac

Scheme 1. Coupling of donor 425 and acceptor 526 followed by subsequent acetal deprotection to afford the fluorinated ganglioside core 6.

1) NaOMe, MeOH 2) ClCH2OEt DIPEA, CH2 Cl2 68% (2 steps)

8, R = CH 2OEt

OEt

OEt O EtO

O O

O

O CCl3

9

iPrOH TMSOTf CH 2Cl2, -78 °C 53% α:β 1:53

O

F 10

Scheme 2. Preparation of fluorinated, ethoxy-methyl-ether protected model substrate 9 and subsequent glycosylation with iPrOH to generate the isopropylglycoside 10.


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ACS Chemical Neuroscience To assess the suitability of this group in a streamlined synthesis of target 3, a model substrate was required to provide preliminary validation (Scheme 2). Deprotection of 7 with NaOMe (0.4 equiv.) in MeOH furnished the free triol, which was subsequently treated with chloromethyl ethyl ether (EtOCH2Cl, 6 equiv.) and Hünig’s base (9 equiv.) to generate 8 in 68% yield (2 steps). Desilylation with TBAF (2 equiv.) in THF and treatment with Cl3CCN (10 equiv.) and DBU (1 equiv.) yielded the trichloroacetimidate (TCA) donor 9 as mixture of anomers (α:β 28:1). Glycosylation with isopropanol (1.2 equiv.) in the presence of TMSOTf (0.1 equiv.) in CH2Cl2 at -78 °C gave 10 with an anomeric ratio of α:β = 1:53 as judged by 19F NMR analysis of the crude reaction mixture (only the β-anomer was isolated after chromatographic purification). This informative study confirmed that the ethoxymethyl-ether groups are compatible with fluorine-directed βglycosylation and that these units are robust towards the Lewis-acid activator. The protecting group regime was subsequently modified to accommodate this new finding. Disaccharide 6 was therefore treated with chloromethyl ethyl ether (EtOCH2Cl, 35 equiv.) and Hünig’s base (190 equiv.) in CH2Cl2 to furnish the fully protected epitope 11 in 73% yield. Desylilation with TBAF (2 equiv.) and generation of the TCA donor 12 proceeded smoothly in 94% yield (α:β 1:1). Installation of the pendant alkyl chain was achieved by glycoslation with 1-dodecanol (10 equiv.) using TMSOTf (0.1 equiv.) as activator at 0 °C. Unfortunately, a drop in glycosylation selectivity was observed giving a crude α:β-ratio of 1:3.4. This is in contrast with the model substrate 9 where a crude α:β-ratio of

1:53 was observed. Following chromatographic separation, the desired β-configured antigen 13 was isolated in (54%). Deprotection was achieved through a step wise strategy by initial acetal cleavage, followed by ester hydrolysis. It is important to note that under highly acidic conditions (TFA, HCl) considerable decomposition was observed, whilst under milder conditions (AcOH, Amberlite-120) the protecting groups proved recalcitrant. A combination of ZnBr2 (4 equiv.) and EtSH (24 equiv.) successfully cleaved the acetal groups on the Dgalactose core to liberate diol 14 in 92% yield. Full deprotection was achieved by hydrolysis using NaOMe (13 equiv.) which, after column chromatography,32 afforded 3 as a crystalline white solid in 75% yield (Scheme 3).

BnO

1-dodecanol TMSOTf CH2Cl2, -10 °C

OBn O

BnO

F O 15

CCl3

94% : 1:10

RO

OR

RO

O

OC12H25

F 16 R = Bn

NH

X-ray

17 R = H

Pd(OH)2/C, H2 MeOH 88%, : 1:10

Ph PhCH(OMe)2 p-TsOH MeCN

O O O

HO

OC12H25

F

76%

18

Scheme 4. Synthesis of the D-galactose building block 18. For crystallographic details of 17 see CCDC 1570373.

Scheme 3. Synthesis of the fluorinated epitope of GM4 (3).



In view of the erosion in α:β-selectivity observed when using the bulky glycosyl donors 12 (cf. 9), the synthetic route the control epitope 2 (X = OH) was modified. Exploiting the high glycosylation selectivities observed with monosaccharides, the sequence was re-ordered to first unify the D-galactose core with the sidechain prior to installing the sialic acid moiety (Scheme 4). To that end, the per-benzylated 2-fluoro-2-deoxy TCA donor 15, previously reported by this group,18 was coupled with 1-dodecanol (0.9 equiv.) using TMSOTf (0.1 equiv.) at -10 °C to generate 16 in a highly β-selective fashion (α:β 1:10) in 76% yield (Scheme 4). Debenzylation with Pd(OH)2/C under an atmosphere of H2 generated the free triol 17, which was subsequently processed to the 4,6-dibenzylidene acetal 18. Regrettably, the union of 4 and 18 was unsuccessful, and remains under investigation. It was however, possible to isolate crystals of 17 that were suitable for crystallographic analysis. CCDC 1570373 contains full details.

equiv.) in CH2Cl2 afforded 20 in 18% (Scheme 5). The major product of this reaction was the corresponding 3-benzylated product (40%). To complete the synthesis of the epitope, species 4 (1 equiv.) and 20 (2.13 equiv.) were exposed to TMSOTf (0.13 equiv.) in MeCN at -40 °C. Immediate deprotection of the benzylidene unit under acidic conditions gave only the α-anomer 21 in a good yield of 25% over two steps. Removal of the benzyl protecting group was achieved by stirring 21 under an atmosphere of hydrogen in MeOH over the course of 43 h, using Pd/C as a catalyst, to obtain 22 in very high yield (90%). Final deprotection of the acetate units was carried out by stirring 22 with NaOMe (13 equiv.) in MeOH, followed by addition of water to cleave the ester moiety. Compound 2 was isolated after column chromatography32 as a white crystalline solid in 84% yield (Scheme 6).

As a control compound for the envisioned oligodendrocyte differentiation studies, it was necessary to devise a route to the native epitope 2. This would allow the effect of single site molecular editing with fluorine to be probed. Since sialyl donor 4 is the common building block in both the synthesis of 2 and 3, only a suitable glycosyl acceptor was required for this endeavor. Non-fluorinated D-galactose building block 20 was selected and prepared from commercially available penta-acetylated D-galactose (Scheme 5, full details are provided in the SI). Regioselective benzylation of 19 with BnBr (1.24 equiv.), TBAI (0.2 equiv.) and aqueous NaOH (1.5

Scheme 5. Regioselective benzylation of 19 to obtain the Dgalactose core 20 for the construction of 2. Having devised robust strategies to prepare both the native and fluorinated analogs of the GM4 epitope, attention was focused on evaluating their effect on oligodendrocyte differentiation.

Scheme 6. Synthesis of the GM4 epitope (2). Oligodendrocyte Differentiation:Primary murine oligodendroglial cells were isolated and cultured as previously described.33,34 This immunopanning-based isolation method allows the generation of >95% pure oligodendroglial cultures with the possibility to analyze key features of oligodendroglial cell biology such as proliferation and differentiation. Isolated oligodendroglial precursor cells (OPC) were cultured in the presence of platelet-derived growth factor AA and neutrophine-3. To induce the differentiation into oligodendrocytes, PDGF-AA was replaced by ciliary neurotrophic factor. GM4 2 and F-GM4 3 were dissolved in DMSO and added to the cell culture medium to a final concentration of 10 µg/ml, 50 µg/ml,

75 µg/ml, 100 µg/ml or 150 µg/ml. The final DMSO concentration was less than 0.1% and served as solvent control. To investigate the influence of GM4 (2) and F-GM4 (3) on oligodendroglial cells we first determined the effect on cell viability employing the Cell Titer Glo® Luminescent cell viability assay (Promega®). This allowed the amount of ATP, an indicator of metabolically active cells, to be quantified. ATP, a nucleoside triphosphate and coenzyme, serves as cosubstrate for the luciferase in this assay which catalysis the mono-oxygenation from luciferin to oxyluciferin. The resulting luminescent signal directly correlates to the metabolic activity of the cells. Differentiation of OPCs was induced and


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cells were exposed media, containing compounds or solvent to higher differentiation rate.35 This difference in behavior (DMSO) and viability was determined after 48 h. The addition between 2 and 3 is remarkable given that it is the result of a of GM4 epitope 2 over 48 h had no impact on the cell viability single site modification in which OH has been replaced by a bioisosteric F atom. Overall the addition of GM4 has no negaof oligodendroglial cells (Figure 3 A). However, the addition of the fluorinated GM4 analog resulted in a significant increase tive influence on the cell viability of the oligodendrocytes during differentiation. in ATP production at a concentration of 50 µg/ml (Figure 3E). This may be indicative of a slightly higher energy demand due Figure 3. Influence of the GM4 (2) and F-GM4 (3) epitopes on oligodendroglial cells. (A) Quantification of oligodendrocyte viability after exposure to DMSO or different concentrations of GM4 (2) during 48 h of differentiation. (B) Quantification of mature, MBP+ cells after treatment with DMSO or different concentrations of GM4 (2) after 48 h. (C-D) Representative images of immunostaining of oligodendrocytes after 48 h treatment with DMSO or GM4 (2). MBP marking mature oligodendrocytes (red), PDGFRα staining OPCs (green) and DAPI marking all cell nuclei (blue). (E) Quantification of oligodendrocyte viability after exposure to DMSO or different concentrations of F-GM4 (3) during 48 h of differentiation. (F) Quantification of mature, MBP+ cells after treatment with DMSO or different concentrations of F-GM4 (3) after 48 h. (G-H) Representative images of immunostaining of oligodendrocytes after 48h treatment with DMSO or F-GM4 3. Mature oligodendrocytes are marked by MBP (red), OPCs by PDGFRα (green) and all cell nuclei are stained by DAPI (blue). Data are presented as SEM of replicates from three independent experiments, * p < 0.05, One-way ANOVA, Bonferroni post correction with selected pairs was performed for statistical analysis. After induction of the differentiation program, oligodendroglial precursor cells differentiate into mature oligodendrocytes within 48 h in vitro.36 During this process the cells not only change their morphologies, but also change their gene expression profile e.g. reduction of platelet-derived growth factor receptor α (PDGFRα) expression and start expressing genes encoding for myelin associated proteins like myelin basic protein (MBP).7 To analyze the impact of GM4 (2) and F-GM4 (3) on the differentiation of oligodendrocytes we quantified the maturation state in dependency of GM4 (2)/F-GM4 (3) treatment. The amount of mature oligodendroglial cells was determined based on their MBP expression. OPCs were cultivated in differentiation medium with and without GM4 (2) or F-GM4 (3) for 48 h. Oligodendrocytes were fixed and immunocytochemical stainings were performed to visualize and differentiate immature, differentiated oligodendrocytes. Addition of GM4 (2) does not alter the amount of mature, MBP+ oligodendrocytes at any concentration (Figure 3 B-D). However, 50µg/ml F-GM4 (3) increases the amount of MBP+ oligodendrocytes compared to DMSO treated control cells significantly (Figure 3 F-H). With 75 µg/ml F-GM4 (2) this promotion of oligodendroglial differentiation is not as potent as with 50µg/ml F-GM4 (3) and for higher concentrations the effectiveness of F-GM4 (3) seems to decrease further. Interestingly, this effect profile is comparable to the impact of GM3

on oligodendroglial cells isolated from neonatal rats.37 How FGM4 (3) is involved in the regulation of oligodendroglial differentiation is still unclear. From other gangliosides it is known that their interactions with e.g. growth factor receptors or receptor tyrosine kinases38-40 are able to modulate cellular functions such as migration and proliferation. Baron and coworkers have linked the ganglioside GD1a directly to the PKA-signaling pathway.41 Via this interaction, GD1 was sufficient to overcome the inhibitory fibronectin effect and promote myelination in vitro and in vivo. The fluorinated form of GM4 (3) may offer therapeutic opportunities to enhance differentiation and maturation of OPCs. Further studies should verify the mode of action of F-GM4 (3) and identify the underlying mechanism which leads to beneficial effect on oligodendroglial differentiation. Importantly, the study further underscores the strategic value of single site fluorination in diverting the natural function of carbohydrates to generate a therapeutic response.42 METHODS Animals Animal experiments were conducted according to the German Animal Welfare Act and approved by the responsible govern-



mental authorities (LANUV Nordrhein-Westfalen AZ 8.84.02.05.20.12.286). C57Bl/6 mice were obtained from the animal facility of the University Münster, Germany Primary oligodendroglial cell culture To isolate primary murine OPCs, mice pups aged between postnatal days 6 to 9 were sacrificed. OPCs were isolated from according to the immunopanning method described previously.33 Briefly, dissected cerebra were dissociated and transferred to an anti-BSL1 Griffonia simplicifolia lectin (#L-110, Vector Labs) coated plate for negative selection. After 15 min, supernatants were transferred to an anti-CD140a (#135902, Biolegend) coated plate to bind OPCs. Afterwards, cells were washed with PBS and bound OPCs were detached by scratching. After resuspension in OPC Sato media, cells were cultured in poly-L-lysine (PLL)-coated flasks (#P4707, Sigma). Isolated OPCs were propagated at 37°C/5 % CO2 with supplementation of platelet-derived growth factor AA (PDGFAA; 10 µg/ml, #100-13A, Peprotech) and neutrophine-3 (NT3, 5 ng/ml, #450-03, Peprotech). In case of differentiation induction, PDGF-AA was replaced by ciliary neurotrophic factor (CNTF, 10 µg/ml, #450-13, Peprotech). Cell culture assay

MOREOVER DATA WERE NORMALIZED TO DMSO CONDITION. ASSOCIATED CONTENT Supporting information, including experimental and analytical data, is provided as a PDF file.

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was supported by the WWU Münster, the Deutsche Forschungsgemeinschaft (DFG) (Excellence Cluster EXC 1003 ‘Cells in Motion’, AL-1887/1-1 to SA and the SFB-TR 128-B07 to TK) and the European Research Council (ERC-2013-StG Starter Grant to RG. Project number 336376-ChMiFluorS). We also thank the Swiss National Science Foundation (N.S., P2EZP2148757 and P300P2_161070).

Notes ‡

CellTiter-Glo® Luminescent Cell Viability Assay (#G7570, Promega®) OPCs were seeded on 96 well plates. After 24 h, differentiation was induced and the cells were exposed compounds or solvent (DMSO). Viability was determined after 48 h according to manufacturer’s instructions quantifying the luminescence via the Glo-Max©-Multi+ multimode reader (Promega®). Immunocytochemistry AFTER 48 H OF DIFFERENTIATION ON PLLCOATED COVER SLIPS, OLIGODENDROCYTES WERE FIXED WITH 4 % PFA/PBS FOR 15 MIN AT ROOM TEMPERATURE (RT) AND WASHED THREE TIMES WITH PBS. FOLLOWING PERMEABILISATION WITH 0.5 % TRITON-X 100/PBS FOR 10 MIN, COVER SLIPS WERE WASHED THREE TIMES AND SATURATED FOR 30 MIN WITH 5 % FCS/PBS. PRIMARY ANTIBODIES WERE DILUTED IN BLOCKING SOLUTION: RAT ANTI-MBP (AB7349, 1:200; ABCAM) AND RABBIT ANTI-PDGFRΑ (SC-338, 1:300; SANTA CRUZ BIOTECHNOLOGY) AND INCUBATED OVERNIGHT AT 4 °C. AFTER THREE WASHING STEPS WITH PBS, SECONDARY ANTIBODIES GOAT ANTI-RABBIT CY2 AND GOAT ANTI-RAT CY3 (1:500; DIANOVA) WERE APPLIED FOR 2 H AT RT. AFTER THREE WASHING STEPS WITH PBS, CELLS WERE MOUNTED USING ROTI MOUNT FLUORCARE DAPI (DAKO). BASED ON THREE RANDOMLY TAKEN PICTURES EACH FROM THREE INDEPENDENT EXPERIMENTS USING A LASER SCANNING MICROSCOPE (LSM 700, CARL ZEISS, JENA), AROUND 300 CELLS WERE QUANTIFIED PER CONDITION AND ASSESSED AS PERCENTAGE OF TOTAL DAPI-POSITIVE CELLS.

These authors contributed equally.

ACKNOWLEDGMENT We thank the analytical departments of the Institute for Organic Chemistry at the WWU Münster for technical support.

ABBREVIATIONS DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; GM4, monosialohexosylganglioside; MBP, myelin basic protein; MS, Multiple Sclerosis; OPC, oligodendroglial precursor cells; PDGFRα, platelet-derived growth factor receptor α; PFA/PBS, paraformaldehyde/phosphate buffered saline; RT, room temperature; TBAF, tetrabutylammonium fluoride; TBAI, tetrabutylammonium iodide; TFA, trifluoroacetic acid; TMSOTf, trimethylsilyl trifluoromethanesulfonate

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Single Site Fluorination of the GM4 Ganglioside Epitope Upregulates

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