Production of Q. Saponaria saponins

The extract from Q. saponaria is very useful in multiple applications. Table 2 shows the list of commercial products of saponin extract from Q. saponaria.

Table 2. List of commercial saponin products from Q. saponaria.

Producer Commercial name Composition declared by producer

Desert King International, USA

QY-150 Liquid, non-refined QE

Natural Response, Chile QP-1000 Spray-dried powder, non-refined QE

Producer 1, USA BF 3399 Liquid, non-refined QE

Quest, Ireland Saponin 5012 Spray-dried powder, non-refined QE

Maruzen Pharmaceuticals, Japan

Quillajanin C-100 Liquid, partially refined QE

QE: Quillaja extracts

To satisfy the demand of Quillaja saponin, there has been a great ecological impact by felling and debarking about 60,000 old trees every year by the year 1998. To reduce the number of felling trees and augment the efficacy of extractions, a new production process (Figure 4) was introduced in 1996 which allowed to extract desired saponins from not only the bark but also the whole biomass of Q. saponaria. As a result, 25,000 trees were survived every year and less organic solvent was used through this ultrafiltration technique.¹⁹ Even through this method was efficient, it remained a need to provide greener

method to obtain Quillaja saponin.

Figure 4. Production process of saponin extracts from Q. saponaria.

1.1.5. Quil-A^®

Quil-A^®, a purified Q. saponaria extracts, is a saponin vaccine adjuvant product of Brenntag Biosector. In 1974, it was first prepared from the aqueous extract from Q.

saponaria by using gel exclusion, dialysis, and ion exchange chromatography.²⁰ It was demonstrated to have antitumor potential. Although the results revealed that the survival rate of mice with leukemia was improved, high toxicity was presented in high dosage in the clinical practice.^21-22 Nowadays, Quil-A^® was mainly applied as adjuvant in veterinary vaccines, active component of ISCOM, nanoparticle adjuvants, raw material for QS-21 and other saponin fractions.²³

Q. saponaria was ever considered as an ideal substance for vaccine development

due to its strong immunoadjuvant activity in the reports for Quil-A^®.^24-25 However, the

wood

heterogeneous mixture of saponins contributed to its toxicological effects.²⁴ Therefore, efforts have been taken to find purified fractions of Q. saponaria extract, or even a single pure analogues from Quil-A^® .

1.1.6. QS-21 and its purified analogues

Quillaja saponins have been extensively used alone and mixed with other adjuvants

since that saponins were known to enhance antibody responses, helper and cytotoxic T-cell responses.²⁶ Using reverse-phase chromatography (RP-HPLC), a series of fractions from Q. saponaria bark were purified by Kensil et al.(Figure 5), with QS-7 (B in Table 3), QS-17 (C in Table 3), Q-18 (D in Table 3) and QS-21 (E in Table 3). Among these component, QS-7 and QS-21 were found to be less toxic (Table 3).²⁷ Between, QS-21 has been studied extensively and evaluated in over 100 clinical trials of vaccines against cancers and infectious diseases saponin adjuvant due to its higher abundance compared to QS-17.²⁸

Figure 5. HPLC chromatogram of an aqueous bark extract. (cited from J. Immunol 1991, 146, 433.)²⁷

Table 3. Lethality of saponins to CD-1 mice^a Dose (μg) Quil-A QS-7 QS-18 QS-21

125 1/5 0/5 4/5 0/5

250 2/5 0/5 5/5 0/5

500 4/5 0/5 5/5 1/5

aNumber of deaths per group of mice within 72 hours after intradermal injection of saponins.

QS-21 was a particular fraction from HPLC separation, and it contains two major isomeric molecular. Both of these saponins incorporate quillaic acid as a central triterpene aglycon core, with a branched trisaccharide residue is linked to the C-3 hydroxyl group, and a linear tetrasaccharide attached to the C-28 through an ester bond. The fourth component is a glycosylated pseudo-dimeric acyl chain linked to the fucose moiety via a

labile ester conjugation. The major and minor components differ in the constitution of the terminal sugar residue of the tetrasaccharide, which incorporate either an β-D -apiofuranose (Api) (65%) or a β-D-xylopyranose (Xyl) (35%), respectively.^{24, 29}

Although QS-21 has been proven to be an exceedingly powerful adjuvant in immunotherapy, its tolerated dose in patients was limited typically to below 100 µg. To overcome its drawback, QS-7 was found not only negligible toxicity in mice, but also significant stand-alone adjuvant activity.^30-31 QS-7 differs from QS-21 at the 4-O position of the fucopyranosyl unit that connects to the C-28 carboxyl group of quillaic acid (Table 4), that is, a small acetyl instead of characteristic long chiral acyl side chain. Furthermore, compared with QS-21, it has two extra monosaccharide units in the C-28 oligosaccharide domain. It was reported that the 3- and/or 4-O acetyl groups of the fucosyl unit might play an important role in tuning the adjuvanticity of the QS-7 analogs.³¹

Besides the most extensively studied QS-21, other fraction of the extracts has drawn the interest of research which pursued a reduced toxicity QS saponin-based immunostimulants, still retained or advanced adjuvant activities. QS-17 and the most abundant saponin in the extract, QS-18, were found to be the valuable leads. Comparing to QS-21, QS-17/18 has one additional β-D-glucopyranosyl (Glc) unit linked to the α-L -rhamnopyranosyl (Rha) unit at its 3-O position. QS-17 and QS-18 has a different sugar moiety at the end position of the acyl side chain, that is, disaccharide unit and

monosaccharide unit, respectively (Table 4).³²

Conclusively, the RP-HPLC fractions of the Quil-A^® mixture was studied by Kensil et al in 1991, who found the fractions QS-7, QS-17, QS-18 and QS-21 to be particularly

potent. QS-17 and QS-18 were the major component of Q. saponaria but suffered highly toxic in mice. On the other hand, QS-7 and QS-21 demonstrated far less toxicity and gained more extensive studies. To pursue the adjuvant with favorable immune responses, these compounds and their further developed analogues have been extensively studied.

Table 4. Natural saponins from of Quillaja saponaria bark.

Compound R¹ R² R³ R⁴ R⁵

1.2. Vaccine adjuvant

Vaccine immunology has been well developed since last century. To combat against a specific disease by vaccine, an innocuous form of disease agent such as killed or weakened bacteria or viruses was usually employed to stimulate antibody production and activate cellular immunology. Many vaccines such as smallpox, measles, and polio in most cases have been proven very effective in human protection.^33-34 For example, reported cases of wild polio virus was reduced to less than 100 cases globally in 2015.³⁵ However, vaccines often come with risks of adverse events and large-scale production of vaccine is very challenging.³⁶ Therefore, there is a growing need for a potentiator, so called adjuvants, which incorporate in therapeutic vaccine formation to enhance, modulate and prolong the immune response.

An adjuvant is a substance that added to vaccines to increase the immunogenicity and protection against infection. The word “adjuvant” means “to aid” which comes from the Latin word adjuvare. Adjuvants help activating the immune system by regulating the humoral or cellular immunity according to the objective of vaccination. Combining with adjuvants, human vaccines based on weakened or inactivated pathogens could elicit robust protective immune responses. For example, adjuvanted H5N1 pandemic influenza vaccines showed improved immune responses compared to un-adjuvanted vaccines in animal models.³⁷ Therefore, adjuvants have several important benefits in vaccine

immunology, especially reducing the amount of required antigen and reducing the number of vaccine doses.

1.2.1.

Approved vaccine adjuvants

Table 5 lists series of adjuvants that have been licensed in use with human vaccines to date.

Various of adjuvants have been used in human vaccines, such as mineral salts, oil in water, liposome, and TLR agonist. The development of adjuvants has been very slow.

Until the early 1990’s, aluminum salts were the only licensed adjuvants used in human vaccines in the United States.³⁸ It is also known as Alum, generally considered as a stimulator of Th2 type immune response, and is the most widely used adjuvants in practical human vaccination.^39-40 After decades, oil-in-water MF59 and AS03 adjuvants were used in pandemic influenza and avian influenza vaccines to improve the humoral and cell-mediated immunity.⁴¹ Both MF59 and AS03 contain squalene, but they have different compositions. AS03 has a component of α-tocopherol to modulate innate immune response.⁴² HBV vaccines were registered with TLR agonist class adjuvants AS04 and CpG ODN which were licensed in 2005 and 2018, respectively. Recently, liposome class adjuvant AS01 with the components of MPL and QS-21 were approved to be used in Herpes zoster vaccines. It is worthy mentioning that QS-21 is the second

non-endogenous substrate that was approved by FDA as an adjuvant.

Table 5. Licensed vaccine adjuvants, their class, components and registered vaccine.

Adjuvants

MPL; aluminum hydroxide Hepatitis B virus;

human

Liposome MPL; QS-21 Herpes zoster

CpG ODN (2018)

TLR9 agonist 1018ISS Hepatitis B virus

1.2.2. QS-21 adjuvanted vaccine

QS-21 is one of the most potential adjuvant with favorable immune responses by promoting high antigen-specific antibody responses.^43-44 Unlike aluminum hydroxide, QS-21 promotes a balanced of both IgG1 and IgG2 production.^{27, 45-46} Further studies showed that QS-21 induces IL-2, IFN-γ, and Th1 bias immunity in vaccine responses.^43,

47-48

Besides of being used solely as an adjuvant , the amphiphilic property of QS-21 allowed it to be formulated into a liposome form (AS01) or an oil-in-water emulsion (AS02) to induce humoral and cellular immunity against cancers and pathogens (Table 6).⁴⁹ Some of them had been proceeded to clinical trial, SHIGRIX^®, a combination of varicella-zoster virus glycoprotein gE and AS01, is the only approved vaccine to prevent herpes in 2017. Both Maria vaccine Mosqurix^® and OBI-822/QS-21 are being evaluated in phase III clinical trial for the intervention of malaria and triple negative breast cancer (NCT03562637, NCT03608878) respectively.^50-51 The phase III trial of GM2/QS-21 was terminated due to low efficacy.⁵² In addition, QS-21 has also been applied with ACC-001 in phase II trial of early Alzheimer’s disease.^53-54

Table 6. QS-21 adjuvanted vaccines.

Combination (brand name) Intervention Status

VSV gE/AS01 (SHIGRIX^®) Herpes zoster Approved (Oct. 2017) RTS, S/AS01 (Mosqurix^®) Malaria Phase III

GM2-KLH/QS-21 Stage II melanoma Phase III (terminated) OBI-822/QS-21 Triple negative breast cancer Phase III

ACC-001/QS-21 Early Alzheimer’s disease Phase II (completed)

1.2.3. Plausible mechanism of QS-21

Although the potency of QS-21 had been investigated in over 100 clinical trials, the

exact mechanism of action of QS-21 in vaccination still remains unknown. It is generally agreed that QS-21 does not directly interact with Toll-like receptors (TLR), and does not operate by depot effect.⁵⁵

Since QS-21 has the potential ability to increase T-cell response, it was proposed that the C4-aldehyde moiety could form a Schiff base with amino groups on T-cell surface receptors to activate T-cell.⁵⁶ To investigate its importance in activating immune responses Fernández-Tejada et al. synthesized QS-21 analogues by removing the C4-aldehyde group. However, the results revealed that either reducing the C4-C4-aldehyde to alcohol or alkyl group, saponins demonstrated the similar immunoresponses in mouse vaccination with GD3-KLH, MUC1-KLH, and OVA.⁵⁷ Thus, the hypothesis of the Schiff base mechanistic hypothesis remains as question.

DC-SIGN, a receptor on DC, usually binds to fucopyranosyl residues and biases its response toward Th2 immunity.⁵⁸ Since QS-21 induce both Th1 and Th2 immunity, The fucose residue of QS-21 was proposed to take responsibility to be an internal moiety binding to DC-SIGN to induce both Th2 immunity.⁵⁹

The amphiphilic property of QS-21 has been postulated that is important since the deacylated saponin (DS-1, Figure 6) lost the capacity to stimulate Th1 immunity.⁶⁰ The

lipophilic acyl chain might facilitate the delivery of exogenous antigens into the APC and further inducing immunity in T-cells.⁶¹ In addition, triterpene’s high affinity for cholesterol could make QS-21 facilitates the endosomal escape of antigens, leading to DC activation and antigen cross-presentation by collecting the endosomes-lysosomes and destabilizing them.^62-63

1.3. Development of QS-21

1.3.1.

Challenges of using QS-21

Despite the success of QS-21, there are several limitations while employing QS-21 in cancer vaccine or adjuvant. Firstly, it was limited to only 50 μg of dosage for the usage in healthy human due to its inherent toxicity.⁶⁴ Secondly, QS-21 is chemically unstable, it was easily decomposed in either high temperature or pH ≥ 7.4 environment, which made it more difficult to store.⁶⁵ Finally, the isolated amount of QS-21 from the soap bark extract was limited (below 0.5%) and it greatly impacts on the sustainable cultivation of Q. saponaria.^66-67 Together, these disadvantages might prevent QS-21 for a wider use in clinic. Thus, to overcome these obstacles developing of synthetic approaches toward QS-21 and its analogues would allow us to understand the structure activity relationship and might also help to explore their mechanism.

1.3.2. Synthesis of QS-21

QS-21api and its analogues were firstly synthesis by D. Gin’s group in 2005 (Scheme 1).^68-69 B(PhF5)3 was applied to build the most challenging linkage between glucuronate 2 and quillaic ester 3. After a series of modifications on glucuronide 4, the acylated tetrasaccharide 6 was attached to C-28 position under the catalysis of BF3·OEt2 at -78 °C to obtain beta-selective glycosidic bond. The similar procedures were also applied on the synthesis of other analogues such as QS-21xyl and QS-7 by the same group.^{31, 70} Briefly, they achieved total synthesis of QS-21 in 70 steps. Although this synthetic methods was still lacked of efficiency, their pioneer studies provided valuable synthetic methods for constructing complex triterpenoid glucosides.

Scheme 1. Synthesis of QS-21.⁶⁸

1.3.3. GPI-0100

To overcome the drawbacks of QS-21 and QS-7, a novel semisynthetic saponin, GPI-0100, has been developed from 7. By deletion of the unstable acyl linker of QS-21 and installation of a dodecyl long chain at the glucuronic acid domain through a more stable amide bond (Scheme 2), GPI-0100 demonstrated higher stability in aqueous solution and lower toxicity in mice. It was 10 times less toxic than QS-21 with a lethal

dose of 5 mg in mice.^71-72 However, clinical trial showed that only 20 times dosage of GPI-0100 could achieve the same efficacy of QS-21, which under such dose, it led to hepatoxicity.⁷³

Scheme 2. Semisythesis of GPI-0100.⁷²

1.3.4. Synthesis of QS-21 analogues inspired by GPI-0100

The chemically unstable acyl side of QS-21 had been hydrolyzed to give DS-1 (Figure 6) by Lui et al..⁷⁴. Besides, DS-1 was further conjugated with aliphatic chain to give RDS-1, which also known as an isolated GPI-0100. These derivatives demonstrated

significant reduction of hemolytic affect than QS-21. However, RDS-1 needed higher dose to induce comparable IgG1 titer and the IgG2a antibody secretion and DS-1 lost adjuvantivity. These results revealed that the deacylated saponin was a poor immunostimulator, but the aliphatic chain-bearing saponin restored the adjuvant activity.

Figure 6. Deacylated saponin (DS-1) and reacylated saponin (RDS-1).

Another series of GPI-0100 inspired synthetic analogues was developed by Wang et al.^75-76 In their studies the acyl side chain of QS-21 was completely removed, instead a long alkyl chain was attached on the glucuronide domain through a stable amide bond (Figure 7). Evaluation of the adjuvant activity of the four analogues with different terminal-functionalized long chains have been demonstrated. RDS-1 had poor water solubility while the glycosylated 7 and 8, which were designed to improve the solubility,

were poor in inducing Th1 immunity. Among these, only the carboxylated 9 demonstrated comparable IgG titer and IgG2a sub-type switching comparable to that of GPI-0100.

Figure 7. Synthetic GPI-0100 analogues.

1.3.5. Truncation of glucuronide and 28-O-linked tetrasaccharide

The influence of adjuvant activity by modifying glucuronic acid site of QS-21 have been investigated by Gin’s group. The results showed that conjugation of glycine,

ethylamine or ethylene diamine on glucuronic site retained similar effect on humoral and cellular immunity induction but no effect on the physical properties.⁵⁶ Furthermore, compound 10 (Figure 8), structure of removal of C-3 glucuronide trisaccharide and modification on acyl side chain, surprisingly demonstrated comparable immunological response to natural QS-21.⁵⁷

Figure 8. Structure of 10.

To investigate the function of C-28 linked moieties, the 28-O-linked tetrasaccharide

was then truncated by Gin’s group to trisaccharide, disaccharide, and monosaccharide

(Figure 9).⁵⁶ As a result, the adjuvant activity was declined along with the decrease of the sugar number.⁵⁶ Thus, trisaccharide in compound 12 was concluded to be the simplest moiety at C-28 to retain comparable immune stimulating effect to QS-21.

Figure 9. Truncation of 28-O-linkage tetrasaccharides.⁵⁶

1.3.6. Development of GPI-0100 analogues in our lab

We have developed a concise chemical approach towards a variety of GPI-0100 analogues. These analogues consisted of truncated sugar moieties at C-28 domain and a series of diversified alkyl and p-substituted phenyloctyl chains were attached to glucuronic acid via an amide linkage (Figure 10). Murine immunology studies demonstrated that phenyloctyl chain-bearing saponin variants 15e-h possessed promising activities in antigen specific CD8 T-cell immunity.

Figure 10. Our preliminary work on the modification of GPI-0100 analogues.

2. Motivation

According to the development of QS-21 as immunostimulators previously, a blueprint to make a potent and safe saponin-based immunostimulator was proposed. We have developed a series of immunity-favorable GPI-0100 analogues by chemical synthesis. In this study, we still faced some obstacles, 1) quillaic acid was expensive (USD $4000 for 5 g) making the synthesis of final compounds not cost-effective; 2) glucuronidation with quillaic acid was low yield; 3) our compounds 15a-h were a bit poor water solubility, analogue with higher solubility is demanded.

To address these obstacles, two objectives were proposed (Table 7). First objective:

6-N-glucosyl group at C-3 position was introduced, it could form a “reverse” amide moiety of our targets 15a-h. So far no such compounds have been proposed, according to the best of our attention in the literature. Second objective: Since most of quillaic acids

were obtained from the Q. saponaria bark extract through several hydrolysis process, we proposed to extract 2′-D-galactosyl-3′-D-xylosyl glucuronyl 3-quillaic acid directly from

Ultra Q-100. This would allow us to skip the glucuronidation step and also would have intact trisaccharide at C-3 position which might help to improve water solubility. Actually, such strategy was disclosed in the patent for the preparation of GPI-0100,⁷⁷ however, the extraction process from Ultra Q-100 remained to be fine-tuned .

Upon obtaining the desired compounds from both objectives, they would be further

conjugated the trisaccharide at C-28 position and then attached the arylalkyl moiety at 6-N-glucosyl or 6-position of glucuronyl through “reversed amide” bond formation or

amide to give final compounds A and B, respectively.

Table 7. Modification of our targets.

Modifications R¹ R² R³

Target A Target B

H H Reversed amide linkage

β-D-Galp β-D-Xylp Amide linkage

3. Results and discussion

3.1． Retrosynthetic analysis

The target A & B were divided into three parts, including lipophilic long chain domain, trissaccharide region, and aglycon (Scheme 3). For the convenience of diverting various long carbon chain, the first disconnection was the amide bond to achieve late stage modifications. Secondly, the preferred protecting groups on the saponins were benzyl, carbamoyl, acetyl, and triethylsilyl groups, which could be removed under mild conditions. Thirdly, construction of 21 would be achieved through (2+1) pathway, which was glycosylation of 24 and 25 to give disaccharide 22 and then conjugated it with 23.

Tricloroacetimidate 21 would be conjugated with nature extracted trisaccharide-saponin or 6-N-glucosyl bearing quillaic acid and then global deprotection to give our target saponins A and B.

Scheme 3. Retrosynthesis analysis of targets A and B

3.2． Part 1: Synthesis of building blocks and trisaccharide

The starting material L-rhamnose was proceeded under acetylation and thio-glycosylation to afford thio-rhamnoside 26 (Scheme 4). The acetyl groups on 26 were then removed by the treatment of 26 with sodium methoxide. Then, the 2,3-cis-diol was protected by isopropylidene to afford acetonide 24.

D-Xylosyl imidate donor 25 was prepared by the starting material D-xylose underwent

acetylation and then selectively removal of 1-O-acetyl group by ethylene diamine/AcOH to give hemiacetal 27. Followed by trichloroacetimidate formation, D-xylosyl donor 25 was readily available to conjugate with rhamnose building block.

D-Fucose was subjected to Fisher glycosylation with benzyl alcohol and its 3,4-cis-diol was protected by isopropylidene to afford protected 23.

The allylic group was introduced to the C-28 carboxylic acid to afford quillaic ester 3 by allylic bromide in 84% yield (Scheme 4).

Scheme 4. Synthesis of four building blocks.

The coupling of rhamnose 24 with xylose 25 under catalytic amount of BF3·OEt2

The coupling of rhamnose 24 with xylose 25 under catalytic amount of BF3·OEt2

在文檔中 半合成GPI-0100衍生物暨合成其葡萄醣醛酸類源物作為免疫活化劑 (頁 17-0)