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
afford disaccharide 28 in 90% yield (Scheme 5). The disaccharide 28 was then proceeded under removal of isopropylidene group by 80% AcOH(aq), followed by acetylation to obtain thio-disaccharide 22 in 33% yield over two steps. Thio-disaccharide 22 was subsequently coupled with fucose 23 to furnish desired trisaccharide 23 in 74%. Finally, after hydrogenation and trichloroacetimidate formation of trisaccharide 29, penta-acetylated trisaccharide donor 21 was readily available.
Scheme 5. Synthesis of trisaccharide donor 21
3.3． Part 2: Synthesis of azido-glucose donor
The starting material diacetone D-glucose was proceeded to benzylation with benzyl
alcohol, followed by deacetonization then acetylation to afford 30 (Scheme 6). After the 1-O-Acetyl group was selectively replaced by thiotoluene group, the remaining acetyl groups was then hydrolyzed by the treatment of sodium methoxide. Then, 4-O and 6-O position were temporary masked by benzylidene and followed by benzylchloride (BzCl) installation on 2-O position to afford 33. By the treatment of anhydrous Borane in THF under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf), the benzylidene group was hydrolyzed and selectively free 6-O while 4-O was still under protection of benzyl group.
To prepare the 6-azido deoxy-glucose 35, glucose 34 was first treated with methanesulfonyl chloride, then the mesylate group was replaced by azide group under the suspension with sodium azide in DMF at 80 °C. Finally, followed by trichloroacetimidate formation, glucosyl donor 20 was available to conjugate with quillaic ester 3.
Scheme 6. Synthesis of azido-glucose donor 20.
3.4． 3-O-Glycosylation of Quillaic ester
The conjugation of quillaic ester 3 with azido-glucose 20 was successfully resulted in 71% yield of product 36 (Scheme 7). Interestingly, this result revealed the selectivity of 3-O glycosylation over 16-O position.
Scheme 7. 3-O-Glycosylation of quillaic ester 36.
3.5． Synthesis of tetrasaccharide saponin
With the glycoside 36 in hand, further modifications had been conducted to unmask the C-28 carboxylic acid (Scheme 8). First, the benzoyl group was hydrolyzed under basic condition at elevated temperature. Surprisingly, 28-O-allyl ester was not affected under this harsh environment. The resulting azido-glycoside was then proceeded under triethylsilylation, reduction of azide group to amine and protection of amine group by benzyl carbamate formation to afford fully-protected quillaic ester 37.
The O-allyl ester 38 was hydrolyzed by the catalysis of Pd(OAc)2 under mild acidic
environment to give glycoside acceptor 39. Under the catalysis of Lewis acid at -78 °C, the monoacid 39 was conjugated with trisaccharide 21 to obtain 40 in 88% yield (α/β = 1/10).
Scheme 8. Synthesis of fully-protected tetrasaccharide saponin 40.
40 was proceeded to global deprotection prior to amide bond formation. The fully protected saponin was suspended with Pd/C in THF under H2 atmosphere to hydrolyze the amino protecting carbamoyl group of 6-N and the benzyl groups on 3-O and 4-O of glucose. However, there was no reaction under this mild condition. After attempting with a series of reagent and conditions (Table 8), the hydrogenation was furnished by suspending with Pd(OH)2 in THF/MeOH under 55 psi of H2 for 36 hours (Entry 3), but the TES and acetyl protecting group were hydrolyzed as well under this condition. Upon complement of acidic hydrolysis and basic methanolysis, 41 was obtained in 11% yield
after HPLC purification
Table 8. Global deprotection of 40 and conditions of hydrogenation.
Entry Catalysis reagent Solvent Concentration of H2 Results
1 10% Pd/C THF 1 atm No reaction in 24 h
Subjection of fully deprotected 41 with amide formation condion might appear side reactions, such as forming ester linkage coupling with 10-(4-(4-fluorophenoxy)phenyl)decanoic acid 17 or (E)-10-(4-(4-fluorophenoxy)phenyl)dec-9-enoic acid 16, the procedures were reversed by arranging the aidic and basic deprotections to the last step. After debenzylation of 40 which was confirmed mass spectrometry, the
amide bond formation was successively carried out with HBTU/DIPEA coupling system to afford conjugate amides (Scheme 9). After that, the amides were proceeded acidic hydrolysis and methanolysis to remove all the remaining protections to give compounds 42a and 42b.
Scheme 9. Amide formation then global deprotection
3.6． Part 3: Semisynthesis of hexasaccharide saponins
With the available mixture of Ultra Q-100, the semisynthesis of quillaic acid saponin was carried out with the hydrolyzation and protecting installation (Scheme 9). First, the ester bond conjugation at C-28 was hydrolyzed under basic condition at elevated temperature. To control the deacylation process in this harsh environment, the reaction mixture was monitored by HPLC chromatography following the gradient conditions as shown in Table 9 to monitor the consumption of starting materials and the yield of deacylated products. The results (Figure 11) showed that the starting material was completely hydrolyzed after 7 hours of reaction time in this basic condition The crude resulting mixture was then proceeded to triethylsilylation and selectively hydrolysis under mild condition to obtain diacid 44.
To selectively protect the carboxylic acid group on glucuronide with benzyl group, diacid 44 was treated with benzylchloroformate and the bulky tri-t-butylpyridine as a base to avoid to protect the carboxylic acid group at C-28. The monoacid 45 with 65% of yield was available to conjugate with trisaccharide. Finally, coupling of trisaccharide-imidate 21 with monoacid 45 using BF3•OEt2 brought 46 in 87%.
Scheme 10. Semisynthesis of fully-protected hexasaccharide saponin 46.
Table 9. HPLC gradient profile to monitor deacylation of QA saponin.
Time (min) % H2O %ACN Flow (mL/min)
0 90.0 10.0 1.0
30.0 60.0 40.0 1.0
40.0 30.0 70.0 1.0
42.0 90.0 10.0 1.0
50.0 90.0 10.0 1.0
Column: Vydac 214TP C4 5μ UV Detector: Wavelength = 210 nm
Figure 11. Change of %concentration of saponin starting material and product.
3.7． Global deprotection and amide formation of hexasaccharide saponins
With compound 46 in hands, our initial strategy to finish saponin target B was to conjugate with amines at final stage before removing all the protections (Table 10). The fully protected 46 was proceeded under hydrogenolysis of benzyl group, then followed by amide coupling. However, the resulting residue was a mixture of saponins with different numbers of deprotection of TES groups. Furthermore, the mixture became even more messy after conjugating with amine on TLC analysis (Table 10, Entry 2).
-20
Table 10. Global deprotection and amide formation steps.
With the experience of entry 2, the deprotection step was arranged prior to the amide
bond formation step (Table 10, Entry 3). After the debenzylation, the resulting residue was proceeded under acidic hydrolysis the isopropylidene, triethylsilyl groups, and then methanolysis the remaining acetyl groups. Because C-28 ester bond was easily to be hydrolyzed, the concentration of TFA was adjusted from 80% to 75%, and the yield was improved from 13% to 28% for three steps. Followed by HPLC purification and concentration, compound 50 was obtained in 28% ready for late stage amine conjugation with 4-phenoxyphenyl-octanoic acid and 4-methoxyphenyl-octanoic acid by using HBTU/DIPEA in DMA to give final compound 51a (80%) and 51b (30%), respectively (Scheme 11).
Scheme 11. Global deprotection then amide formation.