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. 2020 Dec 24;1(2):137–146. doi: 10.1021/jacsau.0c00058

Ganglioside GM3 Analogues Containing Monofluoromethylene-Linked Sialoside: Synthesis, Stereochemical Effects, Conformational Behavior, and Biological Activities

Go Hirai †,‡,§,*, Marie Kato ‡,∥, Hiroyuki Koshino §, Eri Nishizawa ‡,∥, Kana Oonuma §, Eisuke Ota ‡, Toru Watanabe ‡, Daisuke Hashizume ⊥, Yuki Tamura ‡, Mitsuaki Okada ‡,∥, Taeko Miyagi #, Mikiko Sodeoka ‡,§,∥,*
PMCID: PMC8395706  PMID: 34467279

Abstract

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Glycoconjugates are an important class of biomolecules that regulate numerous biological events in cells. However, these complex, medium-size molecules are metabolically unstable, which hampers detailed investigations of their functions as well as their potential application as pharmaceuticals. Here we report sialidase-resistant analogues of ganglioside GM3 containing a monofluoromethylene linkage instead of the native O-sialoside linkage. Stereoselective synthesis of CHF-linked disaccharides and kinetically controlled Au(I)-catalyzed glycosylation efficiently furnished both stereoisomers of CHF-linked as well as CF2- and CH2-linked GM3 analogues. Like native GM3, the C-linked GM3 analogues inhibited the autophosphorylation of epidermal growth factor (EGF) receptor induced by EGF in vitro. Assay of the proliferation-enhancing activity toward Had-1 cells together with NMR-based conformational analysis showed that the (S)-CHF-linked GM3 analogue with exo-gauche conformation is the most potent of the synthesized compounds. Our findings suggest that exo-anomeric conformation is important for the biological functions of GM3.

Keywords: ganglioside GM3, organic synthesis, C-glycoside, biological activity, conformational analysis

1. Introduction

Glycoconjugates regulate a variety of biological phenomena. A representative method to analyze their functions at the cellular or in vivo level is to engineer the enzymes involved in the biosynthesis of target carbohydrates decorating proteins or lipids, which can perturb intra- or intercellular signaling by enhancing or blocking the functions of the glycoconjugates.1 The exogenous addition of carbohydrate molecules as chemical probes into cells is an important complementary approach. However, cellular metabolism affords a variety of bioactive glycoconjugates, rendering functional analysis complicated. Thus, there is a need for metabolically stable analogues of glycoconjugates as tools to dissect the roles of the conjugates at the molecular level.

Ganglioside GM3 (1, Figure 1A) is an important glycosphingolipid involved in regulating multiple biological processes2 on membrane microdomains.3 Sialidases trigger the metabolism of 1, removing sialic acid moiety to afford LacCer (lactosyl ceramide, Galβ(1–4)Glcβ(1–1)Cer).4 Upregulation of sialidase NEU3 contributes to cancer malignancy and causes impaired insulin signaling, which may suggest that the metabolism of 1 controls signal transduction.5 To analyze in detail the functions of 1, we require sialidase-resistant active analogues of 1. Here, we planned to address this issue.

Figure 1.

Figure 1

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(A) Structures of α(2,3)-sialylgalactoses (O-SiaGal), gangliosides GM3, GM4, S-sialosides, C-sialosides, and CHF-sialosides, and reported analogues. aCalculated pKa values obtained by the SPARC online calculator,12bref (10), cref (8c), dref (8d), eref (20), fref (17). (B) Estimated or predicted favored staggered conformations of sialoside linkages.

Several approaches to 2,3-SiaGal analogues with sialidase resistance have been investigated so far. A straightforward approach is replacement of the cleavable O-sialoside linkage in α(2,3)-sialylgalactose (O-2,3-SiaGal) by another linkage, keeping the structural change of the cyclic sialic acid part to a minimum.6 In the past 35 years, many S-sialosides7 and C-sialosides8,9 have been synthesized (Figure 1A). Nevertheless, only a few ganglioside analogues have been prepared (S-linked GM4 (3)10 and truncated derivatives11). Moreover, we thought that linkages used so far have not been ideal for mimicking O-sialoside. The O atom of O-sialoside defines the structure, acidity,12 and conformation of sialoglycosides (Figure 1A). Specifically, O-sialoside is basically flexible but favors exo-anomeric conformations (exo-gauche and exo-anti) for dihedral angle ϕ, where the antibonding orbital of the C2′–O bond overlaps with the lone pair of the oxygen atom of O-sialoside (Figures 1B, S1, and S2).13 The importance of exo-anomeric conformations has been suggested based on crystallographic analysis. Namely, O-2,3-SiaGal bound unexceptionally to the hemagglutinin of the influenza virus in one of the exo-anomeric conformations,14 whereas the non-exo conformer does not interact with protein σ1 in the crystal structure (Figures S3–S6).14dS-Sialosides are more flexible than O-sialosides, possessing a relatively long C–S bond, which results in reduced steric and stereoelectronic effects.15,16 Weaker inductive effects of the CH(OH) and CH2 groups increase the pKa value of sialic acid, and the absence of stereoelectronic effects favors conformational flexibility of C-sialosides.13a

We have previously developed the CF2-GM4 analogue (5), which showed comparable biological activity to native GM4 (2).17 Although the calculated pKa values seem favorable owing to the presence of the F atoms, we thought that CF2-sialoside was still not ideal in terms of its conformational behavior. The preferred conformation of the CF2-sialoside linkage is estimated to be opposite to that of O-SiaGal, because the gauche effect of the F atoms may destabilize both the exo-gauche and exo-anti conformations (Figures 1B and S1).18 To overcome this issue, we designed CHF-GM3 analogues 7 and 8 with a chiral CHF-sialoside linkage. The stereochemistry of the CHF-sialosides should influence their conformations through the gauche effect, and the (R)-isomer and (S)-isomer would favor the exo-anti and exo-gauche conformations, respectively. Since the calculated pKa value of the CHF-SiaGal analogue is reasonably low, CHF-SiaGal analogues are expected to meet the criteria for a sialoside mimic, as discussed above. It would also be interesting to know whether the difference of sialoside conformation affects the biological activity. Here we describe the synthesis and conformational analysis of CHF-sialosides, which are promising candidates for sialidase-resistant O-sialosides analogues, as well as provide a comparison of the biological activities of the unprecedented C-linked GM3 analogues (R)- and (S)-CHF-GM3, CH2-GM3, and CF2-GM3.19

2. Results and Discussion

Synthesis of CHF-2,3-SiaGal Analogues

Although stereocontrolled synthesis of CHF-sialosides is challenging due to the additional chiral center, we expected that our original strategy utilizing Ireland–Claisen rearrangement for the construction of CF2-17 and CH2-sialoside20 would be available. Considering the stereospecificity of the rearrangement, the (R)-CHF- and (S)-CHF-sialosides should be constructed from (E)-17 and (Z)-17, respectively, having a terminal fluoro-olefin at C3 of galactose (Scheme 1). Although stereoselective monofluoromethyleneation21 of ketones usually requires some ingenuity, we found that the Julia–Kocienski type olefination of 9a(20) with a standard reagent 10(22) afforded E-fluoro-olefin 11 exclusively. This result can be explained in terms of A-strain between the F atom and the TIPS group at O2. Hence, we next employed bromofluoromethylenation23 to prepare the Z-isomer, because a Br atom is larger than an F atom. Optimization of the Si group on O2 and the solvent ratio resulted in successful transformation of TBS-protected 9b into the 13 in a highly selective manner (E:Z = 10:1, Schemes S1–S3 and Figure S7). Subsequent Br–Li exchange followed by protonation of 13 provided (Z)-14 from (E)-13 without loss of the F atom, along with the vinylsilane 15, which was produced from (Z)-13 and was easily separable from (Z)-14. After removal of the Si groups, esterification of 12 with the sialic acid derivative 16(17) and removal of the 4,6-p-methoxylbenzylidene group gave the precursors 17. Ireland–Claisen rearrangement of 17 was performed with excess LiHMDS and TMSCl, and after treatment with TMS-diazomethane α-(R)-18 or α-(S)-18 was obtained α-selectively, with complete stereospecifity for the CHF carbon. It is noteworthy that the reaction temperature influenced the α:β stereoselectivity in an opposite manner for the two isomers (Scheme S4 and Figure S8) and that removal of the 4,6-p-methoxylbenzylidene group was essential for both high α-stereoselectivity of (R)-18 and further transformations.20 The stereochemistry of CHF-sialosides was determined based on HMBC analysis (Figure S9).

Scheme 1. Stereoselective Synthesis of Both Diastereomers of CHF-Sialosides (α-(R)-18 and α-(S)-18) and CHF-Linked SiaGal Units (20–22).

Scheme 1

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Introduction of the requisite functionalities at C2 and C3 was achieved starting from lactone 19, which was prepared in four conventional steps. Dihydroxylation of 19 by stoichiometric OsO4 followed by selective Burton-type deoxygenation provided the protected CHF-SiaGal 20, which was converted to the corresponding acetate CHF-21 for stereochemical assignment (Figures S10 and S11).

Glycosylation of C-2,3-SiaGal with Glucosylceramide Unit

The other major task to achieve the synthesis of C-linked GM3s was glycosylation of the four C-SiaGal units (including previously reported CH2- and CF2-SiaGal) with the GlcCer derivative (Figure 1A). We first addressed the glycosylation using CF2-SiaGal, which was considered the poorest donor, because fluorine decreases the reactivity of donors even at a remote position by destabilizing the oxonium intermediates.24 This time, we first designed trifluoroacetimidate CF2-donor 23(25) with a lactone moiety (Scheme 2A), as successful glycosylation has already been reported in the synthesis of native GM3 from a similar lactone donor.26 In the previous CF2-linked GM4 synthesis,17 we performed acid-promoted glycosylation of the similar CF2-SiaGal lactone donor (with trichloroacetoimidate instead of trifluoroacetimidates as OX at O1 and the acetyl group instead of benzoyl group at O2 on the galactose ring) with ceramide and obtained the corresponding glycoside in only 20% yield, along with the lactol in 40% (OH at O1 on the galactose ring). We considered that this unsatisfactory result was probably attributable to the instability of the trichloroacetoimidate donor and/or poor reactivity of the ceramide acceptor. Hence, this time we used the trifluoroacetimidate donor 23. However, TMSOTf-promoted glycosylation of 23 with the glucosyl ceramide acceptor 25 gave unsatisfactory results as well. The yield of 26 was low (the product contained inseparable impurities, Scheme 2A), and degradation of the acceptor unit 25 occurred prior to completion of the desired glycosylation reaction. Degradation of the ceramide part 28 (Scheme S6) was also observed during the synthesis of 25 under TMSOTf-promoted conditions, though the chemical yield of 25 was moderate (Scheme S7). These results suggested that glycosylation of 23 and 25 to provide 26 was too slow and that the prolonged reaction time resulted in the decomposition of the acceptor 25 and the product 26, probably due to the presence of the acid-sensitive allylic benzoate unit.

Scheme 2. (A) Glycosylation of 23 with GlcCer Unit 25 Promoted by TMSOTf; (B) Synthesis of GlcCer Unit 25 via Glycosylation of Glucose Donor 27 with Ceramide Unit 28 by Gold(I) Catalytic Condition; (C) Glycosylation of 30 with GlcCer Unit 25 Promoted by Gold(I) Catalytic Condition.

Scheme 2

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Yields were calculated based on recovered 25.

We then tried gold(I)-catalyzed glycosylation in order to avoid degradation by oxophilic Lewis acid.27 As expected, GlcCer unit 29 was cleanly formed in quantitative yield (Schemes 2B and S5), indicating a significant chemo-selective activation of the alkynyl group. We believe this is the one of the best methods available for the synthesis of GlcCer. This result prompted us to investigate the glycosylation of CF2-SiaGal donor with 25 under gold(I) catalysis. Because of the mild reaction conditions, there was no need to change the acid-labile benzyloxymethyl (BOM) protections to acetyl groups and donor 30 was prepared. However, the glycosylation of 30 with 25 did not provide the desired glycoside 31 but rather gave orthoester 32 in 71% yield (Scheme 2C). These results clearly indicated that direct formation of the desired glycosides from these donors is disfavored and orthoesters are formed as the kinetic product. In contrast to the case of ordinary oxophilic Lewis acid, isomerization of the orthoesters to the glycosides did not proceed under gold(I) catalysis. In fact, retreatment of 32 in the presence of 25 or ethanol did not provide the glycoside 31 or the corresponding ethanol adduct, and 32 was recovered quantitatively.

On the assumption that, in addition to the strong inductive effect of the F atoms, the conformation of the galactose unit might affect the chemoselectivity of the reaction, the new glycosyl donor 36 without the lactone ring was prepared from CF2-20 (Scheme 3). After protecting group manipulations, methanolysis of 33 followed by acetylation of the generated O-4 OH group provided 34. Cleavage of the p-methoxyphenyl group followed by acylation with 35 gave the alternative donor CF2-36. To our delight, the glycosylation of CF2-36 with 25 under Au(I) catalysis proceeded smoothly to afford CF2-37 in 76% yield without formation of the corresponding orthoester, indicating that the preferred kinetic pathway was dramatically changed by the opening of the lactone ring. Application to the other donors CH2-36, (R)-CHF-36, and (S)-CHF-36 was also successful, affording CH2-37, (R)-CHF-37, and (S)-CHF-37. It should be noted that the orthoester (S)-CHF-38 was only obtained from (S)-CHF-36 as a byproduct and no (R)-CHF-38 was detected, suggesting that not only the inductive effect but also the stereoelectronic effect of the F atom somehow influenced the kinetic reaction pathway.

Scheme 3. Synthesis of C-Linked GM3 Analogues 4 and 6–8.

Scheme 3

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Yields were calculated based on recovered 25.

Removal of all of the acyl groups and the methyl ester by methanolysis and hydrolysis, followed by Birch reduction, accomplished the synthesis of the C-linked GM3 analogues CH2-GM3 (4), CF2-GM3 (6), (R)-CHF-GM3 (7), and (S)-CHF-GM3 (8). These are the first C-linked GM3 analogues as well as the first glycoconjugate analogues with a CHF-linked disaccharide moiety.28

Biological Activity of C-Linked GM3 Analogues

We next investigated the biological activities of the C-linked GM3 analogues in vitro and in cellulo. A representative biological activity of GM3 is inhibition of epidermal growth factor receptor (EGFR) autophosphorylation.29 Like native GM3, the C-linked GM3 analogues (4 and 6–8) clearly inhibited EGFR autophosphorylation induced by EGF in the membrane fraction prepared from A431 cells (Figure S12). These results suggested that all C-linked GM3 analogues basically act as mimetics of GM3 regardless of the C-sialoside linkages, and their potency appeared to be similar at enzyme level. The advantage of the CHF-linkage is clearly illustrated by the effects on Had-1 cells. Native GM3 is known to inhibit proliferation of several cell lines including A431 and KB cells,29 which are relevant to inhibition of EGFR autophosphorylation. In contrast to these well-known effects, it was reported that cell proliferation for Had-1 cell was promoted by exogenous addition of the native GM3.30 Indeed, as reported previously, Had-1 cell proliferation was enhanced by native or synthetic O-linked GM3, but not LacCer (Figure 2). CH2-GM3 (4) and CF2-GM3 (6) showed comparable activity to synthetic O-linked GM3. In contrast, CHF-GM3 7 and 8 induced stronger enhancement of Had-1 cell proliferation than 4 or 6. Notably, (S)-CHF-GM3 (8) showed greater activity than native 1, synthetic O-linked GM3 (p = 0.054 by t test), or other C-linked GM3 including the stereoisomer (R)-CHF-GM3 (7). Although the direct target of GM3 responsible for this effect on Had-1 cells is not known, both the affinity for the target protein and the metabolic stability of the GM3 analogues would affect the biological activity. The metabolic stability of the all C-linked GM3 should be much higher than that of native GM3 due to the presence of the C-sialidase linkages. On the other hand, the effects of the CF2- or CH2-linkages on the conformational preference or acidity of sialic acid might decrease their binding ability to the target protein, and this might explain why their potency is not much greater than that of native GM3. The CHF-linked GM3 isomers 7 and 8 were expected to possess similar acidity to O-sialosides and similar physical properties, but are likely to have different conformational preferences. Thus, the observed difference of the cell proliferation activity between 7 and 8 might be due to a difference of preferred conformation.

Figure 2.

Figure 2

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Enhancement of Had-1 cell proliferation by exogenous addition of LacCer, native GM3 (1, NH4 salt, d18:1/23:0), synthetic O-linked GM3 (d18:1/18:0), or synthetic C-linked GM3 analogues (d18:1/18:0), 4 and 6–8 (final 10 μM). Bars indicate mean ± SD, n = 9. *p < 0.005, **p < 0.05.

Conformational Analysis of CHF-2,3-SiaGal by NMR and DFT Calculation

Previous conformational analyses of native O-2,3-SiaGal or its derivatives have mainly utilized the combination of MM/MD (or related method) calculations and NOE correlations between protons on the two saccharides.13a We also analyzed the conformations of the disaccharide structure of SiaGal units in water by means of NMR measurements of (R)-CHF-22 and (S)-CHF-22 (Scheme 1) instead of the GM3 analogues 7 and 8, because the SiaGal part in GM3 should be extended from the hydrophobic bilayer into the hydrophilic medium. (R)-CHF-22 and (S)-CHF-22 were obtained by methanolysis and hydrolysis of (R)-CHF-21 and (S)-CHF-21, respectively (Scheme 1). In contrast to previous reports, we used the 3J coupling constant (3JH–H, 3JC–H, 3JF–H, and 3JF–C) as well as 2JC–H values around CHF-sialosides to predict dihedral angles ϕ and ψ on the basis of a Karplus-type equation.

We compared the experimental J values of (R)-CHF-22 and (S)-CHF-22 obtained in water and calculated the J values of optimized structures generated from nine possible staggered conformations for each isomer as initial structures (the reducing ends of (R)-CHF-22 and (S)-CHF-22 (methoxyphenyl groups, MP) were replaced with methyl groups for calculation). The optimized structures are summarized in Figures S13 and S14 for the (R)-CHF-linked isomer and Figures S18 and S19 for the (S)-CHF-linked isomer. Then, calculation of J values for each isomer was performed at the DFT level (MPW1PW91/6-311+G(d,p), in water). The experimental and calculated 3JH–H, 3JC–H, 3JF–H, and 3JF–C values as well as 2JC–H values around the CHF-linkages are summarized in Figure S15 for the (R)-CHF-linked isomer and in Figure S20 for the (S)-CHF-linked isomer. The differences between the experimental and calculated J values (ΔJ values) are presented as bar graphs in Figure S16 for ϕ of the (R)-CHF-linked isomer, Figure S17 for ψ of the (R)-CHF-linked isomer, Figure S21 for ϕ of the (S)-CHF-linked isomer, and Figure S22 for ψ of the (S)-CHF-linked isomer. Representative bar graphs for the (S)-CHF-linked isomer are shown in Figure 3A.

Figure 3.

Figure 3

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(A) ΔJ values (calculated – experimental) for (S)-CHF-linked α(2,3)-SiaGal ((S)-CHF-22) concerning dihedral angles φ (C1′–C2′–CHF–C3) and ψ (C2′–CHF–C3–H3). (B) Averaged conformations of CHF-SiaGal (S)-CHF-22 and (R)-CHF-22 estimated by NMR analysis and their observed ROE correlations.

The results indicate that the major conformations of the SiaGal units are different. Figure 3A clearly indicates that (S)-CHF-22 preferentially adopts predominantly exo-gauche conformation, with little non-exo conformation in terms of the dihedral angle ϕ. On the other hand, for dihedral angle ψ, (S)-CHF-22 adopts an eclipsed conformation (ψ = around 0°) and not the typical staggered conformations (syn(−), syn(+), and anti). Similar eclipsed conformation for dihedral angle ψ was estimated in exo-gauche conformation of the native O-sialoside.13a The predicted conformations were supported by the observed Rotating frame nuclear Overhauser Effect (ROE) correlations (Figure 3B, top). In contrast, (R)-CHF-22 was not regulated to a specific conformation and would adopt exo-anti and non-exo conformations for ϕ and fluctuated conformations for ψ (although around −50° would be one of the major conformations), depending upon the dihedral angle ϕ. ROE correlations supported this view (Figure 3B, middle and bottom). Thus, the potent activity of 8 compared with 7 might be attributed to the favored exo-anomeric conformation, which may play a significant role in the biological functions of GM3.

3. Conclusion

In summary, our results indicate that CHF-sialoside is an effective mimicking linkage of native O-sialoside with increased metabolic stability. This finding may also be applicable to other glycosidic linkages, such as glucosides or fucosides. We believe CHF-linked analogues will be useful tools for clarification of the functions of native glycoconjugates and for identification of their target proteins at the cellular level.

4. Methods

Preparation of Membrane Fraction from A431 Cells

A431 cells were cultured, scraped off the plate, washed with PBS, and centrifuged at 800g for 5 min. Cell pellets were resuspended in buffer 1 (pH 7.4, 5 mM HEPES, 5 mM MgCl2, 5 mM 2-mercaptoethanol) and disrupted in a Dounce homogenizer (tight-fitting pestle). After addition of sucrose (1 M), the resulting cell lysate was centrifuged at 1200g for 10 min and the supernatant was ultracentrifuged at 100 000g for 1 h at 4 °C. The resulting precipitate was collected as the membrane fraction. The membrane fraction was resuspended in a suitable amount of buffer 2 (pH 7.4, 20 mM HEPES, 5 mM MgCl2, 100 mM NaCl) for determination of protein concentration or for use in further experiments.

Evaluation of the In Vitro Inhibitory Effect of GM3 on EGF-Induced EGFR Autophosphorylation

Aliquots of solutions containing native GM3 (Matreya, number 1503), synthetic GM3 (Nagara Science, number GM300501), LacCer (Matreya, number 1507), or synthetic C-linked GM3 analogues in chloroform/methanol were dried by means of a nitrogen stream and under vacuum. Buffer A (pH 7.4, 20 mM HEPES, 150 mM NaCl, 0.05% Triton X-100) was added to the completely dried glycolipid samples followed by sonication for 1 min. Then, the sample solution was sterilized by passage through a 0.22 μm Milipore filter.

The glycolipid samples were added to membrane fraction of A431 cells (1 μg each) in buffer A (total 30 μL) and incubated for 10 min at room temperature. EGF solution (1 μL, final 100 ng, recombinant Human EGF, R&D Systems, number 236-EG) was added to each tube, and incubation was continued for 10 min at room temperature. The reaction mixture was cooled on ice, then 5 μL of 500 μM ATP and 5 μL of 50 mM MgCl2 were added, and incubation was continued for 10 min on ice. SDS-PAGE sample buffer was added, and the sample was boiled for 5 min at 100 °C. Each sample was resolved by SDS-PAGE (6% gel), and bands were transferred to PVDF membranes (Millipore, Billerica, MA) and probed with specific antibodies. Detection was done with the ECL Western blotting detection system (Millipore) and a LAS-4000 (Fuji Film, Tokyo, Japan). Primary antibodies included anti-EGFR (Santa Cruz, number sc-03) and anti-phospho-EGFR (Tyr1068, Cell Signaling, number 2220) antibodies. The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG (sc-2004; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Conformational Analysis and Estimation of J Values

Calculation Methods

The Gaussian09 program was run on RICC (RIKEN Integrated Cluster of Clusters, 8 CPUs).

(R)- and (S)-CHF-linked SiaGal capped by a methyl group at the reducing end (p-methoxyphenyl group in (R)-CHF-22 and (S)-CHF-22, used for the NMR studies) were constructed and roughly optimized by Gaussian09 [B3LYP/6-311+G(d,p), scrf = water]. From the optimized structures, nine possible staggered conformations (for φ: exo-gauche, exo-anti, and non-exo; for ψ: syn(+), syn(−), and anti) for each isomer were generated (performed in Spartan’10). Semiempirical PM3 level calculation of all nine conformations (under vacuum conditions) was performed in Spartan’10. The resulting nine structures for each isomer were optimized by Spartan’10 [B3LYP/6-311+G(d,p), under vacuum condition]. The resulting structures were further optimized by Gaussian09 [HF/6-311+G(d,p), scrf = water]. The coupling constants for all nine conformations of each isomer were calculated by means of DFT using Gaussian09 [MPW1PW91/6-311+G(d,p), scrf = water, NMR = mixed].

NMR Methods

3JH,H and 3JH,F values were obtained from 1D 1H NMR spectra (600 MHz), and 3JF,C values were obtained from 1D 13C{1H} NMR spectra (150 MHz) using a JEOL JNM ECA600 spectrometer. 2JH,C and 3JH,C values were measured by the J-IMPEACH-MBC31 method using a scaling factor of N = 60, and 2D J-HMBC32 spectra with a constant maximum evolution time of 250 ms and/or HETLOC33 spectra with a mixing time of 60 ms. 2D ROESY spectra were obtained with a mixing time of 250 ms. Complete NMR signal assignments were confirmed by conventional 2D DQF-COSY, NOESY, HSQC, 1H–13C HMBC, and also 19F–13C HMBC experiments.

Evaluation of the In Vitro Promoting Effect of GM3 on Had-1 Cell Proliferation

Had-1 cells, Newcastle disease virus-resistant mutant cell of mouse mammary tumor FM3A cells, were cultured in ES medium (Eagle’s MEM medium (NISSUI 05900) containing 1% MEM nonessential amino acid solution (SIGMA M7149), 0.1 μM vitamin B (SIGMA V6629), and 1 mM sodium pyruvate (Invitrogen 11360-070)) supplemented with 4 mM l-glutamine and 2% fetal bovine serum in a 5% CO2 incubator at 37 °C.

When the effect of glycolipid on cell growth was to be examined, the cells were adapted to a chemically defined serum-free medium (ES medium supplemented with 2 mM l-glutamine and 1% HB 101 supplement (Irvine Scientific T151)) for 24 h. Then the cells were collected by centrifugation and the cell number was adjusted to 3 × 103 cells/per well in a 96-well plate. Glycolipid suspension was added to cells. After incubation at 37 °C for 4 days, cells were counted by using a TC20 Automated Cell Counter (No. 1450101, BIO-RAD) in the presence of 0.05% trypan blue.

Acknowledgments

This work was partially supported by PRIME (JP19gm5910018), CREST (JP18gm0710004), Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED, JSPS KAKENHI (18H04417 and 18H02097), Asian Chemical Biology Initiative, JSPS A3 Foresight Program, and RIKEN project funding.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.0c00058.

  • Supplementary figures, experimental procedures for synthesis of the compounds, and 1H and 13C NMR spectra for new compounds (PDF)

Author Contributions

G. H. performed the synthetic experiments and DFT calculations, and designed and supervised the whole project. M. K., E. N., E. O., T. W., and M. O. performed synthetic experiments under the guidance of GH and MS. H. K. performed NMR analysis for structural analysis of the synthetic intermediates and conformational analysis. K. O. and Y. T. performed biological experiments for inhibition of EGFR autophosphorylation and proliferation of Had-1 cells. D. H. contributed to structural investigation of the synthetic intermediates by X-ray analysis. T. M. contributed to the sialidase experiments and provided helpful advice. M. S. designed and supervised the research.

The authors declare no competing financial interest.

Supplementary Material

au0c00058_si_001.pdf (21.9MB, pdf)

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