Serine-/Cysteine-Based sp2-Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma

Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp2-iminosugar moiety in a prototypic TLR4 agonist, CCL-34, yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8+ T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model.


■ INTRODUCTION
Toll-like receptors (TLRs) are evolutionarily preserved type I transmembrane proteins that recognize conserved pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), representing the first line of host defense.The ability to sense molecular patterns makes TLRs vital regulators for innate immunity, further helping to shape the adaptive immune response by providing key signals to prime nai ̈ve CD4 + T cells toward a specific T helper (Th) profile, like cell-mediated response (Th1) or humoral immune response (Th2). 1−3 Toll-like receptor 4 (TLR4) is one of the most important members of the whole TLR family.It is responsible for the detection of lipopolysaccharides (LPS) from Gram-negative bacteria and plays a key role in the nonspecific inflammatory reaction.
The role of TLR4 in driving Th1/Th2 immune response stimulation is complex and multifaceted.TLR4 activation can induce the release of both Th1 (e.g., interferon-γ; IFN-γ) and Th2 cytokines (e.g., interleukines-4 and 13; IL-4 and IL-13) in a context-dependent manner.The precise mechanisms at work are not fully understood, but it is believed to involve the activation of several downstream signaling pathways, including the myeloid differentiation primary response 88 (MyD88)dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways.The resulting Th1/ Th2 polarization is further influenced by various factors, such as the type of TLR4 ligand, the duration and strength of TLR4 activation, and the presence of other co-stimulatory signals. 4,5−8 Both the agonistic and antagonistic aspects of TLR4 signaling pathways are being explored, and a number of clinical trials are currently in progress for a variety of diseases. 9fforts in discovering novel TLR4 agonists have mainly been directed toward the development of new vaccine adjuvants, with better properties, improved immune-stimulatory effects, and reduced toxicity, thanks to their ability to trigger a Th1mediated immune response. 10,11−23 The underlying concept is that hematopoietic cells (HPCs) and nonhematopoietic stromal or structural airway cells (SACs) express TLR4 in qualitatively differentiated patterns: TLR4 signaling in SACs instructs dendritic cells (DCs) to induce Th2 responses, whereas on HPCs, this receptor can trigger a signaling pathway that programs DCs to polarize the immune response toward Th1 states.Consistent with this view, activation by TLR4 agonists could lead to the attenuation of allergen-triggered Th2 sensitization by enhancing the Th1 response, thus providing protection from airway hyperreactivity (AHR).Although improvement in nasal symptom scores was observed, a relatively high dose (100 μg) was required, and no inhibition of nasal allergen challenge responses compared with placebo was achieved; it was not pursued to phase III.It becomes apparent that exploiting the full potential of TLR4-associated immunity requires small-molecule TLR4 ligands with a specific function such as molecular probes to highlight the specific role of TLR4 in immune system regulation.
Serine-based glycolipids, of which CCL-34 (Figure 1) is the archetype, have emerged as a new class of unnatural TLR4 agonists with a strong potential for drug development. 24onically, it was originally conceived as a structural analogue of α-galactosylceramide (α-GalCer or KRN7000; Figure 1), the prototypic ligand of the CD1d receptor in immune system cells.The corresponding CD1d−αGalCer complex binds the T-cell receptor (TCR) on invariant natural killer T cells (iNKT) to induce a strong immune response.CCL-34 comprises a galactopyranose fragment α-linked to a serinescaffolded hydrophobic lipid moiety that contains a linear C 12 acyl substituent at the amine group and an N-undecyl amide chain at the carboxylic function of the amino acid component.Compared with other known TLR4 agonists, CCL-34 has the advantages of a defined structure, easier synthesis, and less contamination with bioactive microbial components.It has been shown to modulate differentiation and maturation of myeloid DCs 25 and to elicit anticancer immunity via TLR4. 26,27Structure−activity relationship studies demonstrated that the sugar portion plays an essential role in the biological properties.The configurational pattern of the monosaccharide admitted substantial variability.Thus, the glucosyl lipid derivative CCL-34-S3 (Figure 1) exhibited a TLR4 agonist potency that was similar to that of CCL-34, suggesting that the stereochemistry of the 4-hydroxyl group is not crucial for activity.However, shifting from αto βanomeric configuration fully abolished the capability of activating TLR4-dependent signaling. 28The complications associated with the stereoselective synthesis of α-glycosides, generally requiring tedious and time-consuming procedures, 28−30 and their instability to the action of α-glycosidases represent two important hurdles that may thwart the translation of these glycolipids to the clinic.We conceived that the abovementioned difficulties could be overcome by replacing the carbohydrate moiety in CCL-34 with an sp 2 -iminosugar-type surrogate.sp 2 -Iminosugars are a unique family of glycomimetics characterized by the presence of a pseudoamide-type nitrogen (e.g., a carbamate, urea, thiocarbamate, thiourea, or guanidine nitrogen) in place of the endocyclic oxygen atom in monosaccharides.−34 Notably, different from classical iminosugars, sp 2 iminosugars can engage in O and S-glycosylation reactions, affording exclusively the corresponding α-Oor S-glycoside. 35,36This property has been previously exploited in the synthesis of specific glycosidase inhibitors and effectors, 37,38 lectin ligands, 39−42 mitogen-activated protein kinase (MAPK) regulators with anti-inflammatory, 43−46 anticancer, 47−49 and antiparasitic activities 50,51 and tumor-associated carbohydrate antigen mimics, 52,53 including a glycoconjugate-based anticancer vaccine. 54The structural resemblance of the diantennated lipid aglycone in CCL-34 derivatives with the ceramide portion of endogenous glycolipid immunomodulators may enable additional mechanisms contributing to the resolution of the inflammatory process associated with allergy.Thus, glucosylceramide and glycosphingolipids are known to promote or inhibit TLR4-mediated signal transduction in a context-dependent manner by modifying the cell membrane properties. 55,56Notably, the corresponding α-anomers can further regulate the production of pro-and anti-inflammatory mediators by iNKT cells, 57 a subset of innate-like T cells (CD1d-restricted T cells) that also contribute to allergic inflammation. 58,59With these considerations in mind, in the present study, we have synthesized serine-and cysteine-based sp 2 -iminoglycolipids (sp 2 -IGLs) featuring 5N,6O-oxomethylidene-galactonojirimycin (OGJ) and -nojirimycin (ONJ) glycone moieties, namely CCL-34-OGJ (1) and CCL-34-ONJ (2), or the S-glycoside analogues, CCL-34S-OGJ (3) and CCL-34S-ONJ (4), respectively (Figure 2).Assessment of their immunomodulatory properties showed that they represent a novel class of TLR4 agonists with the ability to counteract Th2 sensitization.Investigation of their potential in asthma immunotherapy in a murine model led to the identification of compound 2 as a promising candidate for drug development.
■ RESULTS AND DISCUSSION Synthesis of sp 2 -Iminoglycolipids.The preparation of the sp 2 -IGLs 1−4 implies the (thio)glycosylation of the corresponding serine or cysteine lipid with an appropriate OGJ or ONJ pseudoglycosyl donor.The corresponding per-Oacetates 11 and 13 were considered for this purpose.Previous syntheses of the parent sp 2 -iminosugars, limited to <100 mg batches, proved unsuitable for larger scale production, which seriously hampers access to the target conjugates in sufficient amounts to conduct the relevant in vitro and in vivo studies.Therefore, we first settled on an optimized route for the gramscale preparation of 11 and 13 from commercial D- glucurolactone as a common starting material (Scheme 1).
The preparation of the cysteine analogues 3 and 4 was successfully achieved in a two-step sequence involving thioglycosidation of 11 or 13 with the diantennated cysteine lipid 17 in the presence of BF 3 •OEt 2 (→22 and 23) followed by Zempleń deacetylation.Attempts to use the analogous diantennated serine lipid as an acceptor proved unpractical due to solubility issues.All the sp 2 -iminosugar derivatives (final compounds as well as intermediates) showed mass spectrometry and spectroscopic data ( 1 H and 13 C NMR) compatible with the proposed structures.Notably, the proton−protoncoupling constants about the six-membered piperidine ring supported that only the α-anomer was present, in agreement with the overwhelming anomeric effect in this family of compounds.The purity of the final compounds 1−4 has been confirmed to be >95% by RP−HPLC and combustion microanalysis.
In Vitro Cytokine Signature, Adjuvancy Ability, and Signaling Route.Immunological evaluation of the sp 2 -IGLs 1−4 was performed first by assessing their ability to induce the secretion of proinflammatory cytokines in splenocytes isolated from B6 and myeloid differentiation primary response protein (MyD88) KO (MyD88 −/− ) mice.Since MyD88 is an intermediator of immune response signaling by all TLRs, 67 depletion of inflammatory cytokine level expression on going from wild-type (WT) to MyD88 −/− splenocytes can be correlated with TLR involvement.
Binding of lipid A to the TLR4/MD-2 complex in immune cells triggers the production of inflammatory cytokines, such as IFNγ and IL-6. 68The serine-based glycolipid CCL-34 likewise promotes an increase in the levels of these two cytokines in splenocytes. 24We found that the four sp 2 -IGL-type mimetics synthesized in this work displayed significantly stronger IFNγ expression than the parent compound used as the control in wild-type splenocytes from B6 mice (Figure 3A).The serinebased O-glycolipid mimetics 1 and 2 further outperformed CCL-34 in enhancing IL-6 expression, whereas the cysteinebased S-glycolipid mimetics 3 and 4 were less efficient in this case (Figure 3B).We also found that MyD88 −/− mice splenocytes treated with the test compounds produced no or very low concentrations of IFNγ and IL-6, proving that their proinflammatory activity is MyD88-dependent (Figure 3A,B).TLR4-dependent signaling was next confirmed by using human embryonic kidney 293 (HEK-293; 293) cells engineered to express all components of the human TLR4 receptor complex (293/hTLR4A−MD-2−CD14), which is the key for signal transduction by ligands targeting TLR4 (Figure 3C).HEK-293 cells, which do not express any components of the TLR4 receptor complex (TLR4, MD-2, and CD14), were transfected with the genes for human TLR4, MD-2, and CD14.Nontransfected HEK-293 and HEK-293 expressing only the human MD2 and CD14 components (293/hMD-2−CD14) were used as controls.Upon treatment with compounds 1−4, human IL-8 expression was detected in 293/hTLR4A−MD-2− CD14 cells, but not in the control cells, as an unequivocal indicator of TLR4 signaling.To verify the selectivity of TLR4 over TLR2 signaling, the compounds were also tested on HEK-293 cells expressing human TLR2 (293/hTLR2), and no agonist activity (no expression of the human IL-8 reporter molecule) was detected (Figure 3C).We also established that the treatment of compounds 1−4 did not cause a significant reduction in cell viability in B6 mouse splenocytes or HEK-293 cells and was well tolerated (MTT assay; Supporting Information Figure S1).
Given the structural resemblance of CCL34 and the sp 2 -IGL-type mimetics 1−4 with αGalCer, discerning the off-target immune activation via the CD1d−TCR pathway is mandatory.By using the mouse iNKT hybridoma DN3A4−1.2 reporter system, we confirmed that none of the test compounds could activate this signaling route (no significant expression of the reporter interleukine IL-2 was detected; Figure 3D).Further, neither treatment with CCL34 nor with any of the analogues 1−4 reduced IL-2 expression levels after α-GalCer stimulation of the mouse iNKT hybridoma, supporting that they do not compete with α-GalCer for CD1d binding (Figure 3E).The serine-based glycolipid mimetic 2, exhibiting an immunostimulatory activity in vitro higher than the parent compound CCL34, was next assessed for its ability to induce the maturation of bone marrow-derived dendritic cells (BMDCs).Our results showed that compound 2 displayed a dose-dependent effect in inducing the expression of the cluster of differentiation 86 (CD86), a key mediator of T-cell activation and survival (Figure 4A) in WT mouse BMDCs. 69 parallel assay using BMDCs isolated from MyD88 KO mice evidenced much lower levels of CD86 upon treatment with 50 μM of 2, consistent with a mechanism of action implying TLR4 (Figure 4B).Similarly, the levels of IL-12p40 secretion, a chemoattractant for macrophages, in the WT BMDC culture supernatant increased in the presence of compound 2 in a dose-dependent manner, which was not the case for MyD88 −/− BMDCs (Figure 4C).
The adjuvancy function of CCL34 and compounds 1−4 was additionally evaluated in human peripheral blood mononuclear cells (PBMCs) from two different donors, A and B, via the detection of IFNγ and IL-6.Compounds 1 and 2 induced expression levels of both IFNγ (Figure 5A,C) and IL-6 (Figure 5B,D) that rivaled the performance of CCL34, supporting a Thelper 1 (Th1)-biased trait.The cysteine-based derivatives 3 and 4 exhibited lower immunostimulatory potency in this assay.
In Vivo Evaluation of the Immunostimulatory Activity.The in vivo potential of the sp 2 -IGLs 1−4 as adjuvants was initially judged by determining their capacity to induce the production of IFNγ and IL-4 as archetypical mediators of humoral and immune responses, respectively, in WT mice. 70The levels of both cytokines in serum were significantly increased (Figure 6A,B) at 2 h post-treatment (20 μg compound/g mouse weight, intraperitoneal (i.p.) administration) and remained high after 18 h.Compound 2 was found to be the most efficient in the series at increasing the mRNA level of Cd86 in the spleen, signifying its capability to promote DC maturation (Figure 6C).All compounds could also promote a rise in the mRNA level of Il-4, with compound 3 showing the strongest effect in this case (Figure 6D).
To demonstrate the adjuvant properties of the serine-based sp 2 -IGLs, we tested their ability to promote ovalbumin (OVA)-specific proliferation of CD8 + T cells in the OT-1 adoptive transfer mouse model. 71Briefly, splenocytes from this mouse model express an OVA-specific T cell receptor.Upon OVA binding, CD8 + T cell proliferation is induced, a characteristic feature of an adaptive immune response.We found that mixing OVA with either compound 1 or 2 significantly enhanced OVA-specific CD8 + T cell proliferation (Supporting Information Figures S2 and 6E) by 10-and 5-fold, respectively, supporting a strong immunostimulatory activity (Figure 6F).
In Vivo Evaluation of the Immunotherapeutic Potential against Asthma.The ability of compounds 1−4 to induce high expression levels of IFNγ in vivo through the TLR4 pathway, meaning a Th1-biased profile, suggests their potential to counteract Th2-dominated inflammatory pathologies, such as asthma. 72We have explored this notion in the OVA-induced AHR mouse model. 73After OVA sensitization, compounds 1−4 (20 μg/g mouse weight, i.p.) were used to treat mice.With the administration of increasing concentrations of methacoline, these mice developed bronchoconstriction and consequently enhanced pulmonary resistance (R L ).Interestingly, R L values were curtailed by as much as 50% upon treatment with compounds 1−4 (Figure 7A).Additionally, determination of the levels of the Th2 cytokines IL-4 and IL-13, which are considered instigators of AHR and airway inflammation, in the bronchoalveolar fluid (BALF) and in the lungs was additionally conducted.We found that all compounds reduced the expression levels of IL-4 in BALF, although data only reached significance for compounds 2−4 (Figure 7B), and a similar trend was observed in the lungs.In the case of IL-13, only the serine-based derivatives 1 and 2 were efficient at decreasing expression levels in both BALF and lungs, which agrees with their stronger Th1-biased immunomodulatory character (Figure 7C).

■ CONCLUSIONS
The results above-discussed show that (thio)glycosidation of sp 2 -iminosugar glycosyl donors with serine-or cysteine-based lipid acceptors provides an efficient entry to bioactive glycolipid mimetics, namely sp 2 -IGLs.In these compounds, the sugar portion is replaced by a piperidine−carbamate bicyclic moiety, whose hydroxylation and configurational profiles can be predetermined.Notably, the glycosylation reaction proceeds with total α-stereoselectivity, which was here exploited to synthesize sp 2 -IGL analogues of the serine-based glycolipid CCL-34, a non-LPS-related TLR4 agonist.Evaluation of the immunomodulatory properties of the new sp 2 -IGLs supports their TLR4 agonistic character both in vitro and in vivo, with a cytokine signature characterized by high expression levels of IFNγ, suggesting a Th1-biased profile.Interestingly, the compounds were found to counteract the Th2 proinflammatory context in a mouse model of OVA-induced respiratory hyperreactivity mimicking asthma, promoting crisis resolution.They possess well-defined chemical structures, are chemically stable, are expected to be metabolically inert, and can be prepared through efficient and scalable experimental procedures suitable for optimization strategies.Investigation of this novel family of immunomodulators in other immune-

Journal of Medicinal Chemistry
compromised pathologies, such as infectious diseases and cancer, is currently sought in our laboratories.
In Vitro Culture of Human PBMCs.Human PBMCs from normal volunteers were collected from whole blood and isolated by separation over a histopaque-1077 (Sigma) density gradient.All donors provided informed consent, and the respective institutional review boards approved all experimental protocols.Supernatants were collected for ELISA analysis.
In Vitro Stimulation of Splenocytes, 293 Cells, and Mouse iNKT Hybridoma DN3A4−1.2Cells.Stock solutions of the compounds were prepared in DMSO at a 20 mM concentration, and aliquots of these solutions were added to PBS to reach the desired target concentrations.The final proportion of DMSO was <5% in all cases.Mouse splenocytes (1 × 10 6 cells/well) were isolated and stimulated with the glycolipids in 96-well plates for 48 h.HEK-293 (293), 293/ hTLR2, 293/hMD2−CD14, and 293/hTLR4−MD2−CD14 cells were from InvivoGen (San Diego, CA, USA).The cells (2 × 10 5 cells/well) were stimulated in the presence of the indicated glycolipid mimetic in 96-well plates for 24 h.DN3A4−1.2,A20, and A20− CD1d cells were kindly provided by Dr. Mitchell Kronenberg (La Jolla Institute, CA) and were maintained in RPMI 1640 medium.DN3A4−1.2 cells (1 × 10 5 cells/well) were stimulated by co-culturing with A20 or A20−CD1d cells (5 × 10 4 cells/well) in the presence of the indicated glycolipids in 96-well plates for 24 h.
Mice.Eight-to ten-week-old C57BL/6 and BALB/c female mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan).OT-1 mice were kindly provided by Dr. Liao Nan-Shih (Academia Sinica, Taiwan).All animals were housed under specific pathogen-free conditions.All protocols were approved by the Academia Sinica Institutional Animal Care and Use Committee (IACUC), and all experiments were performed according to the guidelines of the IACUC and adhered to the guidelines on animal studies outlined in the ACS Ethical Guidelines.
OVA Challenge Model.For the OVA challenge, mice were sensitized intraperitoneally with 10 μg of OVA and 2% alhydrogel adjuvant (InvivoGen).Mice were challenged three times with 10 μg of OVA on day 14 post-sensitization, either daily or every other day.Mice were sacrificed the day after the last OVA challenge.
Measurement of AHR.Mice were anesthetized with 100 mg per kg body weight pentobarbital (Sigma-Aldrich), tracheotomized, and mechanically ventilated via the FinePointe RC system (Buxco Research Systems, Wilmington, NC).Lung function was assessed by measuring lung resistance and dynamic compliance in response to increasing doses of aerosolized methacholine (1.25−40 mg/mL, Sigma-Aldrich).
Collection and Analysis of BALF.Upon exposure of the trachea, the lungs were lavaged twice with 1 mL of PBS supplemented with 2% fetal calf serum (FCS).BALF was pooled, and BAL cells were pelleted by centrifugation and fixed onto cytospin slides.The slides were stained with Diff-Quik solution (Polysciences Inc), and a BAL differential cell count was performed.
Lung Cell Isolation.Whole lungs were flushed by PBS (supplemented with 2% FCS) through the right ventricle and minced prior to incubation in 3 mL of the DMEM medium with 0.1% (vol/ vol) DNase I (Worthington Biochemicals) and 1.6 mg/mL collagenase IV (Worthington Biochemicals) for 40 min at 37 °C.Tissues were filtered through a 100 μm mesh to obtain single-cell suspensions.Red blood cells were removed by incubation in ACK lysing buffer (GIBCO) for 5 min at 25 °C.Single-cell suspensions were resuspended in the appropriate buffer for further processing.
ELISA.Cytokines (mouse IL-2, IL-4, IL-12p40, IL-13, and IFN-γ; human IL-6, IL-8, and IFN-γ) in the supernatants of cell cultures, as well as in the lungs, serum, and BAL fluid of mice were analyzed with ELISA kits from Biolegend, with the exception of mouse IL-13 (eBioscience).For determination of cytokine concentrations in vivo, the lungs were flushed and minced thoroughly prior to sonication with the Bioruptor Plus sonicator (Diagenode) in RIPA buffer.Lung protein lysates were obtained by centrifugation.For determination of cytokine concentrations in vitro, supernatants from treated cells were collected after centrifugation and analyzed by capture ELISA according to the manufacturer's protocol.
Real-Time PCR.Total RNA from cultured cells and tissues was extracted using the Direct-zol MiniPrep (Zymo Research, Irvine, CA), and 2 μg of cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA).The expression levels of IL-4 and CD86 were measured by real-time qPCR using SYBR green on a TOptical 96 real-time PCR thermal cycler (Biometra, Analytik Jena, Germany).
Statistics.Data were analyzed with GraphPad Prism 6 software (GraphPad Prism software, San Diego, CA).Statistical analysis was determined using the two-way analysis of variance (ANOVA) or the Student's two-tailed t-test.All data were expressed as mean ± SEM, and p values of <0.05 were considered statistically significant.
■ ASSOCIATED CONTENT * sı Supporting Information J.M.G.F.).The CITIUS (University of Seville) is also acknowledged for technical support.We would also like to thank the staff of the IBMS Flow Cytometry Core Facility, Academia Sinica (AS-CFII111-212), for flow cytometry and cell sorting services.M.G.-C. was supported by a postdoctoral fellowship (Contrato de Acceso al Sistema Espanõl de Ciencia y Tecnología) funded by the University of Seville.

Figure 4 .
Figure 4. Compound 2-promoted BMDC maturation is dose-dependent and MyD88-dependent.(A,B) BMDCs from B6 (WT) and MyD88 KO mice were treated with compound 2, and the expression of CD86 was assessed using flow cytometry.(C) Expression of IL-12p40 in the culture supernatants of WT and MyD88 KO BMDCs was assessed.Data are shown as means ± SEMs of two independent experiments (n = 4 each).*P < 0.05.

Figure 5 .
Figure 5. Compounds 1−4 induce strong IFNγ and IL6 expression in PMBCs from different donors.PBMCs from two donors were treated with compounds 1−4.IFNγ in the culture supernatants of PBMCs from donors A (A) and B (C), and IL-6 in the culture supernatants from donors A (B) and B (D) were assessed.Data are shown as means ± SEMs of two independent experiments (n = 2−5 each).*P < 0.05; **P < 0.01.

Figure 6 .
Figure 6.Compounds 1−4 possess strong adjuvancy function and promote DC and B cell maturation in WT mice and OVA-specific CD8 + T cell proliferation in the OT-1 adoptive transfer mouse model.(A−D) B6 mice treated with compounds 1−4 (20 μg compound/g mouse weight, i.p. administration).Sera were collected at 2 and 18 h post-treatment of compounds, and IFNγ (A) and IL-4 (B) protein concentrations were measured.Spleens from treated mice were also collected, and the mRNA levels of Cd86 (C) and Il4 (D), normalized to the glyceraldehyde-3phosphate dehydrogenase (Gaphd) level, were analyzed.(E,F) Splenocytes isolated from OT-1 mice, in which OVA-specific CD8 + T cells could be expanded by the antigen OVA, labeled with CFSE dye, and adoptively transferred into B6 mice.Recipient B6 mice were then treated with OVA 257−264 peptide mixed with compound 1 or 2, and the proliferation of OVA-specific CD8 + T cells was analyzed using flow cytometry.(E) Experimental scheme.(F) Percentage of proliferating OVA-specific CD8 + T cells.Data are shown as means ± SEMs of two independent experiments (n = 3−8 each).*P < 0.05; **P < 0.01; ***P < 0.001.
C NMR) spectra were recorded at 500 (125.7)MHz with a Bruker 500 DRX magnet.1D TOCSY, 2D COSY, HMQC, and HSQC experiments were used to assist with NMR assignments.Thinlayer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F 254 Merck with visualization by UV light and by charring with ethanolic 10% H 2 SO 4 and 0.1% ninhydrin.Column chromatography was carried out on Silice 60 A.C.C. Chromagel (SDS 70−200 and 35−70 μm) for preparative purposes.