Protonated Phenanthroline Catalyzed Stereoselective Glycosylation: Synthesis and Evaluation of Saponin Adjuvant Lablaboside F and Variants Leading to Simplified Vaccine Adjuvants

Abstract

Vaccine adjuvants are substances that are coadministered with antigens to enhance the immune response. Lablaboside F, a triterpene oleanolic acid β-linked to two trisaccharide chains on its eastern and western sides, emerged as a promising adjuvant candidate due to exhibiting robust adjuvanticity and low toxicity . We developed a β-stereoselective glycosylation method using a [PhenH]⁺[BF₄]⁻ catalyst to glycosylate oleanolic acid with C2-branched oligosaccharides, synthesizing lablaboside F and structural derivatives (S1–S8) as a result of constructing the target oligosaccharide. This approach simplifies the synthetic process when varying sugars are used and provides a platform for structure–activity relationship studies. Lablaboside F and S1–S8 were tested in vivo and in vitro for adjuvant activity, toxicity, and cytokine production. The entire branched trisaccharide on the western side of lablaboside F is not essential for adjuvant activity. Six derivatives showed low toxicity, while four increased pro-inflammatory cytokine levels. Notably, compound S5, composed of oleanolic acid and the eastern trisaccharide, demonstrated a favorable safety profile in vitro and higher antigen-specific total IgG levels than lablaboside F in vivo, without eliciting IgE production that can lead to allergic reactions. These findings advance our understanding of saponin structure–function relationships and provide a pathway for developing nontoxic adjuvants.

Introduction

Vaccine development has evolved significantly, shifting from traditional live-attenuated microbes to the more sophisticated subunit vaccines. Traditional vaccines often utilize whole organisms, which can cause side effects and pose risks, especially to young, elderly, and immunocompromised individuals. − Subunit vaccines utilize purified antigens derived from proteins or peptides, which are less reactogenic and safer for use in individuals with weaker immune systems. − However, a key limitation is that subunit vaccines provide a shorter immunity duration, thus requiring larger and more frequent dosing. ,

Adjuvants enhance subunit vaccines by boosting immune responses, extending immunity, and minimizing the need for repeat vaccinations. However, safe and effective adjuvants for use in human vaccines are limited. − Vaccine adjuvants encapsulate a multitude of molecules and are not limited to one specific class of compounds. Saponins are a class of natural products derived from plants. They consist of a hydrophobic steroid or triterpene backbone and can be classified based on the number of attached glycan chains. The amphiphilic nature of saponins predisposes them toward a variety of applications in the food, cosmetic, and pharmaceutical industries. − Recent research has identified their many medicinal properties, including antitumor, antimicrobial, antiviral, antifungal, and immunostimulatory phenotypes, making them promising candidates for cancer treatment and as vaccine adjuvants. ,,−

To identify potent vaccine adjuvants with minimal toxicity, the Yoshikawa group screened 47 saponins and identified lablaboside F as a top candidate due to its robust adjuvant activity and low toxicity (Figure ). Lablaboside F is a bidesmodic saponin that features a triterpene oleanolic acid connected to two C2-branched trisaccharide units on its eastern and western sides. Lablaboside F and its structural variants can be extracted from Dolichos lablab (hyacinth beans). − However, extracting sufficient quantities of Lablaboside F for biological testing is inefficient and introduces potential variability between extracted samples, prompting the need for a synthetic approach to ensure purity, consistency, and efficiency. − Therefore, establishing a reliable synthesis method as well as investigating the structure–activity relationship (SAR) of lablaboside F are essential knowledge gaps. Given that minor structural variations can elicit significantly different immune responses, modifying the natural structure of lablaboside F can enhance our understanding of the role specific sugar units and stereochemical configurations play in adjuvant activity and the overall mechanism of action.

1.

Structure of the saponin lablaboside F.

Here, we present the strategy toward the synthesis of lablaboside F as well as eight novel derivatives (S1–S8), achieving high yields and excellent stereoselectivity. One major challenge in synthesizing lablaboside F is achieving stereoselective control over the formation of β-glycosidic bonds when glycosylating the C2-branched western and eastern trisaccharide units with oleanolic acid at C3 and C28, without the use of a C2-neighboring group (Figure ). To address this challenge, we employ a protonated phenanthroline [PhenH]⁺[BF₄]⁻ catalyst, previously developed by our group, to efficiently construct β-C2-branched oligosaccharides without a C2-acetyl neighboring group. [PhenH]⁺[BF₄]⁻ demonstrated remarkable efficiency in glycosylating a C2-branched trisaccharide on either side of the oleanolic acid backbone with excellent β-glycosidic bond selectivity. We tested the synthesized compound lablaboside F (S9), eight derivatives (S1–S8), and the commercially available oleanolic acid backbone (S10) for their adjuvant activity and safety both in vivo and in vitro. Our results demonstrate that most of the compounds displayed favorable safety profiles in vitro. Notably, two truncated analogs of lablaboside F (S1 and S5) exhibited robust adjuvant activity in vivo, as shown by their effectiveness in eliciting antigen-specific total IgG production, surpassing the parent compound S9. Additionally, our modifications to the natural structure of lablaboside F allowed us to identify the essential monosaccharide units and stereochemistry required for enhancing adjuvant activity. Overall, this work provides a thorough evaluation of the potential of our newly derived compounds as vaccine adjuvants and establishes a framework for rapid synthesis of a variety of saponin derivatives. Furthermore, these findings contribute to the development of safer and more effective vaccine adjuvants and establish a foundation for future clinical applications.

Results and Discussion

Retrosynthetic Analysis

Lablaboside F has a triterpene core made of oleanolic acid, with its C3-hydroxyl group and C28-carboxylic acid group linked to two C2-branched trisaccharides via β-1,2-trans glycoside bonds (Figure ). The eastern trisaccharide has a β-ester linkage between d-glucose and oleanolic acid. The glucose is then attached to two α-linked L-rhamnoses at the C2 position. The western trisaccharide has a β-ether linkage between d-glucuronic acid and oleanolic acid. Similarly, glucuronic acid’s C2 holds a β-linked d-galactose and α-linked-L-rhamnose disaccharide. Since the eastern and western trisaccharides are branched at the C2 position, accessing the β-1,2-trans linkage through C2-neighboring group participation to control β-selectivity is not suitable. We envisioned that this challenge could be potentially overcome by employing our recently developed catalyst, [PhenH]⁺[BF₄]⁻, to direct the β-stereoselective glycosylation of hydroxyl groups with oligosaccharide trichloroacetimidate electrophiles. Therefore, we investigate the feasibility of this catalyst to promote the coupling of the C3-hydroxyl of oleanolic acid with the western trisaccharide and the C28-carboxylic acid of oleanolic acid with the eastern side (Figure ). This methodology is preferred over the application of a traditional haloglycoside donor, since purification of these donors is difficult and their stability is lacking compared to trichloroacetimidate donors. However, the extension of [PhenH]⁺[BF₄]⁻ catalyst to carboxylic acids remains unknown due to the difference in reactivity and acidity between alcohol and carboxylic acid. The second challenge lies in understanding how the order of attachment of the eastern and western trisaccharides to oleanolic acid influences the overall synthesis. Therefore, this approach requires us to investigate the application of [PhenH]⁺[BF₄]⁻ to promote the glycosylation of the sterically hindered C3-hydroxyl and C28-carboxylic acid of oleanolic acid with the low-reactivity trisaccharide units.

2.

Retrosynthetic analysis of Lablaboside F.

One of our goals is to efficiently prepare truncated analogs of lablaboside F for biological evaluation (Figure ). Therefore, we also explored a [1 + 1 + 1] glycosylation strategy, utilizing a C2-neighboring group on the glucuronic acid, glucose, and galactose units to ensure the formation of a β-1,2-trans linkage. The [1 + 1 + 1] approach enables the installation of one monosaccharide unit to oleanolic acid at a time due to necessary C2 participation, facilitating the preparation of analogs S1–S8 (Figure ). The truncated analogs S1, S5, S7, and S8 of lablaboside F (S9) differ in the number of sugar units, allowing us to assess the impact of each sugar monosaccharide on adjuvant capability, identifying the essential sugars. By truncating lablaboside F, we can identify essential monosaccharide units for adjuvant activity. Derivitives S2, S3, S4, and S6 vary in the position and stereochemistry of the monosaccharides, helping to clarify the role of the galactose-1,2 glycosidic bond (S2, S3, and S6 vs. S7, S8, and S9 respectively)­and the differences between a free hydroxyl and carboxylic acid (S5 vs S4).

3.

Structures of lablaboside F, eight structural derivatives, and oleanolic acid.

The β-ester linkage in lablaboside F deters [1 + 1 + 1] glycosylation on the eastern side, since this linkage can be hydrolyzed during the deacetylation of glucose’s C2-acetyl group when coupling with rhamnose. To overcome this challenge, we plan to synthesize the eastern branched trisaccharide separately and then glycosylate it with oleanolic acid’s C28-carboxylic acid. However, we anticipate difficulties, as the bulky C2-branched trisaccharide may reduce β-selectivity and have poor reactivity. This concern has led us to consider the [PhenH]⁺[BF₄]⁻ catalyst for our synthesis.

Synthesis

In the initial stage of the synthesis, Glc-11 was glycosylated with allyl-protected oleanolic acid 2, using B­(C₆F₅)₃ as a Lewis acid to activate the trichloroacetimidate leaving group. This reaction successfully produced glycoconjugate 3 in good yield (Scheme ). Notably, the C2-acetyl group in Glc-11 serves as the participatory group, effectively directing the β-selectivity of the coupling process. From there, compound 3 was deprotected in three steps to give the lablaboside F analog S1. Compound 3 was also deacetylated to yield the C2-hydroxyl product 4. The glycosylation of the galactosyl donors, Gal-4-OAc and Gal-4, was subsequently investigated.

1. Initial Synthesis of Lablaboside F and Selected Derivatives .

^a Reaction conditions: (a) B­(C₆F₅)₃, DCM, rt, 63%; (b) K₂CO₃, MeOH, rt, 76%; (c) NIS, AgOTf, 4Ä MS, DCM, 0 °C → rt, 7: 87%, 11: 84%; (d) NaOH solution (1 N), dioxane, 50 °C then Cs₂CO₃, MeOH, rt, yield over two steps 5: 92%, 8: 93%; (e) Pd­(PPh₃)₄, pyrrolidine, DCM, rt, 74%; (f) Pd/C, H₂, MeOH, rt, 73%; (g) TfOH (0.3 equiv), DCM, −45 °C → 0 °C, 68%. (h) varies, see Supporting Information; (i) BnBr, K₂CO₃, DMF, 94%; (j) Pd­(PPh₃)₄, pyrrolidine, DCM/MeOH, 35 °C, 10: 86%, 12: 91%; (k) Pd/C, H₂, MeOH/THF, rt, S7: 71%, S8: 66%.

Initially, we explored the coupling of 4 with the galactosyl diacetate, Gal-4-OAc. This electrophile was synthesized through a one-step reaction involving D-galactal and PhI­(OAc)₂, thereby facilitating its direct preparation for subsequent applications. However, glycosylating Gal-4-OAc under triflic acid conditions with compound 4 led to the undesired α-disaccharide conjugate 7a, even in the presence of the C2-β-directing acetyl group . It is hypothesized that the observed α-selectivity may stem from the formation of an orthoester, which rearranges to yield the α-conjugate 7a. To leverage the availability of α-conjugate 7a, we functionalized it to create analogs S2 and S3 (Scheme ). This approach allows us to investigate the effect of stereochemical modification on adjuvant activity, establishing their potential as vaccine adjuvants.

To minimize the formation of α-product 7a, we screened various coupling conditions and leaving groups. We found that replacing the anomeric acetate with phenylthiol­(Gal-4) led to a successful NIS/AgOTf-mediated reaction with 4, resulting in the desired β-disaccharide conjugate 7 in 87% yield (Scheme ). The hydrolysis of 7 resulted in the removal of both the C2-acetyl and the C6-methyl ester to produce 8. Following this step, the carboxylic acid was benzylated to produce 9, allowing for a one-step global deprotection. Subsequent deprotection steps enabled the synthesis of S7. To complete the synthesis of the western trisaccharide, the benzylidene acetal Rha-6, known for promoting α-selectivity, was glycosylated with 9. This reaction successfully produced trisaccharide conjugate 11, which was then used to prepare S8 through hydrogenation.

Due to the anticipated challenges in glycosylating the sterically hindered C28-carboxylic acid of oleanolic acid 13 with the sterically hindered eastern trisaccharide ET-5, we proposed using protonated phenanthroline [PhenH]⁺[BF₄]⁻ as the catalyst (Figure ). This method was recently developed in our group and promotes β-stereoselective glycosylation of a variety of hydroxyls, phenols, and anilines with α-trichloroacetimidates, all while avoiding C2-neighboring group participation. This cooperative catalysis operates through an S_N2-like transition state, where the trichloroacetimidate (=NH) participates in a hydrogen-bond interaction with the phenanthrolinium-NH (NH···NH). Previously unrecognized the trichloroacetamide-NH₂ by product produced in the reaction also engages in a hydrogen bond with the phenanthroline nitrogen (NH···N). In addition, a hydrogen bond interaction occurs between the carbonyl oxygen of the trichloroacetamide and the hydroxyl of the alcohol or phenol, or the NH of the aniline nucleophile (CO···HO-R/HNAr). These three hydrogen bond interactions work in concert to position the nucleophile for an S_N2-like top-face attack, generating the β-product.

4.

Proposed transition state for the glycosylation of oleanolic acid 13 with α-trichloroacetimidate trisaccharide ET-5 catalyzed by [PhenH]⁺[BF₄]⁻.

Trichloroacetimidates were selected as the leaving group, as they are commonly used in oligosaccharide synthesis. − Glycosylation with α-trichloroacetimidate has been reported to proceed through S_N2-like mechanisms, allowing for stereospecific displacement of the trichloroacetimidate leaving group. ,,, Based on the proposed mechanism in Figure , we hypothesize that the carboxylic acid of oleanolic acid 13 would form a strong hydrogen bond with the carbonyl oxygen of the trichloroacetamide group. As a result, if the coupling of oleanolic acid 13 with the α-trichloroacetimidate trisaccharide ET-5 proceeds through this synergistic catalysis, product 14 would be formed with excellent β-selectivity. To validate our hypothesis, we initially utilized 5 mol % of [PhenH]⁺[BF₄]⁻ in the reaction (Table ), resulting in 14 in 26% yield with excellent β-selectivity (α:β = 1:16, entry 1). Increasing the catalyst loading led to incremental improvements in both yield and β-selectivity, ultimately achieving a yield of 56% and α:β > 1:25 at 15 mol % [PhenH]⁺[BF₄]⁻ (entries 2 and 3). However, further increasing the catalytic loading to 20 mol % resulted in a slight decrease in yield (entry 4). As a comparison, we examined whether other established methods are suitable for the glycosylation of sterically hindered oleanolic acid 13 (Table ). We first tested 5 mol % of BF₃·OEt₂ as a Lewis acid. , This resulted in product 14 with a yield of 71% and an inseparable α:β mixture of 1:2 (entry 5). Using 5 mol % of B­(C₆F₅)₃ also resulted in a similar mixture with a yield of 78% and α:β ratio of 1:2 (entry 6). The uncatalyzed reaction reported by Schmidt yielded 14 with excellent β-selectivity but only a 6% yield after 72 h (entry 7).

1. Optimization of the Glycosylation of the Eastern Trisaccharide,

entry	lewis acid/catalyst	loading (mol %)	temperature (°C)	time (h)	yield (%)	α:β ratio
1	[PhenH]+[BF4]−	5	25 °C	48	26	1:16
2	[PhenH]+[BF4]−	10	25 °C	48	51	1:18
3	[PhenH]+[BF4]−	15	25 °C	48	56	>1:25
4	[PhenH]+[BF4]−	20	25 °C	48	52	>1:25
5	BF3·OEt2	5	–78 to 0 °C	1	71	1:2
6	B(C6F5)3	5	0 °C	1	78	1:2
7	none	0	25 °C	72	6	>1:25

^aConditions: 1.0 equiv of 13, 1.2 equiv of ET-5.

^bIsolated yield.

^cα:β ratio was determined by ¹H NMR.

^dCrystal structure of [PhenH]⁺[BF₄]⁻.

The successful synthesis of 14 enabled the synthesis of truncated lablaboside S5 after removal of the protecting groups (see SI). This achievement has led us to apply [PhenH]⁺[BF₄]⁻ for β-selective glycosylation of oleanolic acid with the western trisaccharide WT-5 (Scheme ). Initially, a methyl-protected glucuronic acid was employed; however, this electrophile was too electron-withdrawing to react effectively with the pseudo-neo-pentane-like C3-hydroxyl group of oleanolic acid 2. As a result, a benzylidene-protected glucose was utilized instead, which could be selectively deprotected and oxidized in a later step. Accordingly, the western trisaccharide WT-5 was glycosylated with the allyl-protected oleanolic acid 2 using 15 mol % of [PhenH]⁺[BF₄]⁻. This reaction yielded trisaccharide conjugate 15 in good yield (57%) and excellent β-selectivity (α:β > 1:20). In this reaction, THF was used as the solvent rather than Et₂O to enhance solubility.

2. Attempted Coupling of Oleanolic Acid Nucleophiles with Western Trisaccharide Catalyzed by Protonated Phenanthroline .

^a Reaction conditions: (a) 15 mol % [PhenH]⁺[BF₄]⁻, THF, 25 °C; (b) TBAF, AcOH, THF, 25 °C, 30 min, 96%. Glycoconjugate 16 was converted into truncated analog S4 by removing the protective groups (see SI).

To further demonstrate the effectiveness of the [PhenH]⁺[BF₄]⁻ catalyst, we investigated the coupling of TIPS-protected oleanolic acid 2a with the eastern trisaccharide ET-5. The glycoconjugate product 16 was obtained in 58% yield with α:β = 1:20 and was then deprotected to give derivative S4 (Scheme ). However, when attempting to couple WT-5 with eastern trisaccharide-oleanolic acid conjugate 17, we found that 17 remained intact, WT-5 decomposed, and the desired product was not observed. This suggests that the steric hindrance from the eastern trisaccharide significantly obstructs the approach of WT-5 to the C3-hydroxyl of 17. These findings enhance our understanding of structural interactions and highlight the need for further optimization in glycoconjugate synthesis.

Having determined the order that the eastern and western trisaccharides should be attached, the allyl-protected western trisaccharide-oleanolic acid conjugate 15 was deprotected to yield the free carboxylic acid product 18. Compound 18 was then glycosylated with eastern trisaccharide ET-5 using 15 mol % of [PhenH]⁺[BF₄]⁻ to give 19 in 59% yield with excellent β-selectivity α:β > 1:20 (Scheme ). The synthesis was completed by selectively cleaving the benzylidene acetal to form the C6 hydroxyl group, which was oxidized to the carboxylic acid giving product 20. Global deprotection of 20 led to the successful isolation of lablaboside F (S9). The [PhenH]⁺[BF₄]⁻ catalyzed stereoselective glycosylation was also utilized for the total synthesis of lablaboside F analog S6 (Scheme ). In this synthesis, the free carboxylic acid compound 22, derived from 21, was reacted with eastern trisaccharide ET-5 using 15 mol % of [PhenH]⁺[BF₄]⁻ to give the fully protected lablaboside analog 23 in 57% yield with excellent β-selectivity α:β > 1:20. Subsequent global deprotection afforded the unnatural lablaboside derivitive S6, demonstrating the efficiency of this catalytic method for complex molecule preperation.

3. Total Synthesis of Lablaboside F and Analog S6 Using Protonated Phenanthroline Catalyst .

^a Reaction conditions: (a) Pd­(PPh₃)₄, pyrrolidine, DCM/MeOH, 40 °C, 18: 82% and 22: 89%; (b) 15 mol % [PhenH]⁺[BF₄]⁻, THF, 25 °C; (c) BH₃·THF, Cu­(OTf)₂, 25 °C, 84%; (d) PhI­(OAc)₂, TEMPO, H₂O/DCM, 25 °C, 78%; (e) H₂/Pd, MeOH/THF, 25 °C, lablaboside F (S9): 74% and S6: 77%.

In Vitro Evaluation of Cytotoxicity

Following the successful synthesis of lablaboside F and its eight derivatives (S1–S8), we evaluated the potential toxicity of each compound in vitro (Figure ). QS-21 is an FDA-approved saponin-based adjuvant that was selected as a positive control for the in vitro analyses due to its known high toxicity and overall similarity to our compounds (see SI for structure). , Human THP-1 Dual cells were selected for their ability to study the effect of the compounds on the innate immune system functions of monocytes and macrophages. These cells are derived from a patient with acute monocytic leukemia and can be differentiated into either macrophages or dendritic cells. , THP-1 Dual cells specifically express secreted embryonic alkaline phosphatase (SEAP) and luciferase reporter genes, which allow for the indirect measurement of NF-κB and type 1 interferon pathway activation, respectively.

5.

In vitro compound safety analyses. Human THP-1 Dual cells were stimulated with PMA, plated at 3 × 10⁵ cells/well, and then administered the indicated treatments for 24 h. Each compound was administered at 100 μg/mL. DMSO was administered at 100 μg/mL. MPLA was administered at 5 μg/mL. QS-21 was administered at 50 μg/mL. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release assay (A), while THP-1 Dual cell metabolic activity was determined by Alamar Blue assay (B). Data are depicted as the pooled mean ± SEM from three independent experiments (n = 9). Statistical significance was determined using a Kruskal–Wallis test with a Dunn’s post hoc test (A) and a one-way ANOVA with Dunnett’s posthoc test (B), comparing treatment groups to the media-only controls. *p < 0.05, **p < 0.01, ***p < 0.001.

Our findings indicate that compounds S1, S7, S8, S10, and QS-21 significantly increased the release of lactate dehydrogenase (LDH) by THP-1-Dual cells as compared to the media alone control group (Figure A). In addition, the impact of compound treatment on cell metabolic activity was also analyzed. Only S1 and QS-21 significantly decreased THP-1-Dual cell metabolic activity 24 h post-treatment as compared to the media alone control (Figure B). Our data demonstrate enhanced cell cytotoxicity and decreased viability following treatment with S1, indicating that S1 displays an unfavorably high cell toxicity profile in vitro. Although S7, S8, and S10 each elicit increased LDH release, they did not impact cell metabolic activity at the concentration tested. This could be attributed to cells being in the early stages of apoptosis, where metabolic activity is maintained but some loss of plasma membrane permeability has occurred. However, the discrepancy could also be explained by the differing readouts from the Alamar Blue and LDH release assays. The Alamar Blue assay measures cellular metabolism through the reduction of resazurin to resorufin, − while LDH release serves as an indicator of cell death through the measurement of intracellular LDH released after plasma membrane disruption. ,− Together, these assays offer distinct but complementary insights into cell viability. As such, the Alamar Blue and LDH data suggest that the 100 μg/mL concentration used with S7, S8, and S10 was likely sufficient to inflict enough membrane damage to trigger LDH release but not sufficient to decrease THP-1 Dual cell metabolic function. In contrast, the 100 μg/mL concentration of S1 was sufficient to decrease THP-1 Dual cell metabolic function and sufficiently damage plasma membranes enough to trigger LDH release. These data mark S1 as the most toxic of the 10 compounds examined, with its toxicity appearing on par with the toxicity observed with QS-21 treatment at 50 μg/mL. However, S2–S6 and S9 demonstrated promising safety profiles with no alterations in metabolic activity or increase in cytotoxicity observed with these compounds.

In Vitro Evaluation of Cytokine Production

Compounds S1–S10 were next evaluated in vitro for adjuvant activity by measuring the production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF. Macrophages secrete acute cytokines as part of their early innate immune response, and production of these specific cytokines serves as a positive indicator of adjuvant activity. To examine the production of IL-1β in vitro, cells must be primed to trigger the initial signal for inflammasome activation, followed by a second activation signal that leads to the subsequent release of IL-1β. − In this experiment, THP-1 Dual cells were differentiated into macrophage-like cells and were primed with MPLA. Cells were then treated with eitherS1–S10, or QS-21 for 24 h. The levels of IL-1β, IL-6, and TNF were measured by ELISA (Figure ). S1, S7, and S10 each elicited significantly increased IL-1β production as compared to the MPLA control (Figure A), correlating with the previously observed high levels of LDH release. Both IL-1β secretion and pyroptosis, an inflammatory form of cell death, occur downstream of inflammasome activation, thereby suggesting that S1, S7, and S10 can likely activate the inflammasome. − As expected, since it is known to activate the inflammasome, QS-21 also elicited significantly increased IL-1β production. Moreover, IL-6 production (Figure B) did not mirror the results observed with IL-1β. Only S3 and S10 showed significant levels of IL-6 production, with S3 producing the highest levels. At the same time, QS-21 did not produce any significant amount of IL-6. Lastly, compounds S1, S7, and S10 were the highest producers of TNF, showing levels comparable with QS-21 (Figure C).

6.

Pro-inflammatory cytokine production in vitro. Human THP-1 Dual cells expressing NF-κB and Luciferase reporter genes were stimulated with PMA and then primed with 5 μg/mL MPLA for 4 h. S1 (25 μg/mL due to low solubility), S2–S10 (100 μg/mL), and QS-21 (5 μg/mL) were administered, alongside a DMSO control (100 μg/mL). The media and DMSO controls did not receive the MPLA priming stimulation. The levels of IL-1β (A), IL-6 (B), and TNF (C) were determined by ELISA. Data represent the pooled mean ± SEM from three independent experiments (n = 9). Statistical significance was determined using a Kruskal–Wallis test with a Dunn’s post hoc test, comparing treatment groups to the MPLA controls. *p < 0.05, **p < 0.01, ***p < 0.001.

In Vivo Evaluation of Adjuvant Efficacy

The potential adjuvant activity of each compound was next evaluated in vivo. Wild-type female C57BL/6 mice were immunized intramuscularly (IM) with each compound and ovalbumin protein (OVA) using a prime-boost vaccination strategy (Figure A). Serum was collected 14 days postboost to determine OVA-specific total IgG production by ELISA. At this time point, OVA immunizations containing S1–S2, S4–S7, and S9–S10 each elicited significantly increased OVA-specific total IgG production as compared to immunization with the OVA alone control, with S5 and S1 producting the highest levels­(Figure B). However, neither S3 nor S8, elicited significantly increased OVA-specific total IgG production as compared to the OVA alone control. Therefore, these data suggest that multiple saponin-derived compounds elicit humoral immunity in vivo when administered in a prime-boost IM vaccination strategy.

7.

OVA-specific total IgG antibody production in vivo. Wild-type female C57BL/6 mice were immunized with the indicated compound containing 10 μg OVA using a prime-boost vaccination strategy (A). S10 was administered at 10 μg per injection. S1, S4, and S5 were administered at 25 μg per injection. S2, S3, S6, S7, S8, and S9 were administered at 50 μg per injection. Naive mice received PBS as a control. The OVA mice were immunized with 10 μg OVA without any compound. Serum was collected from the mice on day 42 post priming immunization and analyzed by ELISA to determine OVA-specific total IgG antibody production (B). Data is depicted as the pooled mean ± SEM from three experiments (n = 15). Statistical significance was determined using a Kruskal–Wallis test with a Dunn’s post hoc test, comparing treatment groups to the OVA-only control. *p < 0.05, **p < 0.01, ***p < 0.001.

Based on the in vitro cytokine profiles, we predicted that S1, S7, and S10 would elicit the most robust antigen-specific total IgG antibody production. The adjuvant activity of S10 was expected due to the medicinal properties of oleanolic acid. , In addition, the results obtained with S1 and S7, both truncated analogs of lablaboside F (Figure ), suggest that the western glucuronic acid and/or the galactose may likely be important for adjuvant activity. However, these results may also be attributed to the toxicity of S1, S7, and S10, which could trigger an immune response following cell death. In this context, extending the length of the sugar chain S1 → S7 → S8 appears to reduce cytotoxicity (Figure A) while subsequently diminishing adjuvant activity (Figure B) at the same time.

S5 does not significantly affect THP-1 Dual cell metabolic activity or LDH release at 100 μg/mL (Figure ), suggesting low cytotoxicity. In contrast, S1 significantly reduces cell metabolic activity and increases LDH release in vitro, suggesting high cytotoxicity. Therefore, S5 appears to be the more effective adjuvant candidate between the two derivitives. It is important to note that S5 was not administered at the intended dosage of 50 μg due to its reduced solubility, and adjuvant efficacy often correlates with dosage. , Given the significant OVA-specific antibody production observed after administering S5 at 25 μg in vivo, enhancing the solubility of S5 could further improve its effectiveness as an adjuvant by allowing a higher dosage to be used. The safety profile of S5 at 100 μg/mL (Figure A) suggests that a higher dosage could be safely used with improved solubility.

Upon examining lablaboside F derivatives (Figure ), S5 is the only compound with a free C3-hydroxyl group within the oleanolic acid framework, except for the structurally simplistic oleanolic acid S10. The significant adjuvant activity displayed by S5 in vivo (Figure B) highlights the importance of the free hydroxyl group and the placement of the eastern trisaccharide (S5 vs S4 and S10). Structural modifications to lablaboside F show that galactose stereochemistry does not affect adjuvant activity, as observed with S2 vs S7 and S6 vs S9. Additionally, we noted that lengthening the western trisaccharide negatively impacts adjuvant efficacy, as the longer oligosaccharides correlated with decreased IgG production (S1 → S7 → S8). Collectively, these findings reveal key structural elements essential for optimizing future lablaboside F derivatives.

We further examined the antibodies produced following our immunizations by analyzing the types of IgG subclass antibodies elicited (Figure ). The subclassing of IgG antibodies is a key indicator of the type of immune response elicited, which can inform vaccine efficacy. IgG2c and IgG1 were measured, which correspond to Th1 and Th2 immune responses, respectively. , IgG1 is found in humans, while IgG2c is only found in murine models. IgG2c activates innate immune cells, while IgG1 activates the humoral response. , OVA immunizations containing S1, S5, S7, and S10 resulted in significantly increased OVA-specific IgG1 production as compared to the OVA alone control group (Figure A). Only the OVA immunization containing S5 elicited significantly increased OVA-specific IgG2c production (Figure B). Overall, the adjuvant activity of these compounds appeared biased toward IgG1, with only S5 eliciting significant IgG2c production.

8.

OVA-specific subclass antibody production in vivo. Wild-type female C57BL/6 mice were immunized with the indicated compound containing 10 μg OVA using a prime-boost IM vaccination strategy. S10 was administered at 10 μg per injection. S1, S4, and S5 were administered at 25 μg per injection. S2, S3, S6, S7, S8, and S9 were administered at 50 μg per injection. Naive mice were immunized with PBS as a control. The OVA mice were immunized with 10 μg OVA without any compound. Serum was collected from the mice on day 42 post priming immunization and analyzed by ELISA to measure OVA-specific IgG1 (A), IgG2c (B), and serum IgE (C) antibody production. Data is depicted as the pooled mean ± SEM from three experiments (n = 15). Statistical significance was determined using a Kruskal–Wallis test with a Dunn’s post hoc test, comparing treatment groups to the OVA-only controls. *p < 0.05, **p < 0.01, ***p < 0.001.

Finally, the levels of serum IgE antibodies elicited following immunizations with each compound were examined as well. IgE antibodies are a component of the Th2 immune response and play a role in protecting against parasitic infections and toxins. These antibodies also play a role in the recognition of cancer by the immune system as well as during type I hypersensitivity. − Adjuvants, such as alum and low-dose QS-21, are known to elicit production of IgE, which contribute to allergic reactions in humans. − None of the ten compounds resulted in significantly increased OVA-specific serum IgE production as compared to the OVA alone control group (Figure C). These data suggest that these compounds are unlikely to induce undesired allergic responses when administered IM within a vaccine formulation.

Conclusions

Lablaboside F (S9) and its eight structural derivatives (S1–S8) were prepared using two synthetic approaches: the [1 + 1 + 1] glycosylation with C2-acetyl neighboring group participation and [PhenH]⁺[BF₄]⁻ catalyzed β-stereoselective glycosylation with C2-branched trisaccharides to control β-selectivity. The [PhenH]⁺[BF₄]⁻ catalyst methadology streamlines the synthesis process by eliminating the need for constant reconfiguration of the established synthetic route each time an alternative sugar is used. More importantly, the pathway catalyzed by [PhenH]⁺[BF₄]⁻ is stereoselective, allowing for the efficient creation of a diverse library of β-configured derivatives as potential vaccine adjuvants, without the need to meticulously define the coupling conditions or leaving groups. The compounds (S1-S10) were evaluated in vitro to establish safety profiles and potential mechanisms of action. Compounds S2, S3, S4, S5, S6, and S9 did not display significant toxicity in vitro. However, S1, S7, S8, and S10 exhibited LDH release in vitro, but only S1 had an impact on cellular metabolic activity . In addition, following (S1-S10) IM administration in vivo, OVA immunizations containing S1, S2, S4, S5, S6, S7, S9, and S10 each elicited significantly increased OVA-specific total IgG antibody production 2 weeks postboost, with S5 and S1 eliciting the highest levels. S1, S5, S7, and S10 each demonstrated significantly increased OVA-specific IgG1 antibody production, while only OVA immunizations containing S5 resulted in significantly increased OVA-specific IgG2c antibody production. Finally, none of the compounds produced significantly increased OVA-specific serum IgE antibodies in vivo 2 weeks post boost. These findings suggest that these compounds can elicit humoral immunity in a prime-boost IM vaccination strategy, but multiple mechanisms likely underpin their adjuvant activity. By unraveling the complexities of the intricate relationship between saponin structure and function, we pave the way for the development of nontoxic adjuvants, marking a major step forward in enhancing the efficacy of vaccines and therapeutics.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01046.

• The general synthesis of the key intermediates, final compounds, HRMS data, ¹H, ¹³C, HSQC, NMR spectra of the synthetic compounds, and biological methods (PDF)

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J.G. and A.B.S. contributed equally. CRediT: Jayanta Ghorai investigation, methodology, writing - review & editing; Austin Bruce Santhin investigation, methodology, writing - review & editing; Leila Almounajed investigation, methodology, writing - original draft; Stacey M Hartwig investigation, methodology; Steven M. Varga funding acquisition, investigation, methodology, supervision, writing - review & editing; Hien M. Nguyen conceptualization, funding acquisition, investigation, methodology, supervision, writing - review & editing.

The authors declare no competing financial interest.