Recyclable fluorous-tag assisted two-directional oligosaccharide synthesis enabled by interrupted Pummerer reaction mediated glycosylation

Herein, we report a novel fluorous-tag assisted two-directional oligosaccharide assembly strategy, which maintained the high coupling efficiency of solution-phase synthesis and featured the advantage of an easy purification process comparable to solid-phase synthesis. A well-designed fluorous tag was decorated on the latent anomeric leaving group in interrupted Pummerer reaction mediated (IPRm) glycosylation. The high efficiency of the in-solution phase glycosylation and the unique affinity of the fluorous tag towards polytetrafluoroethylene (PTFE) particles allowed flexible assembly from the reducing end to the non-reducing end and fast purification by PTFE-assisted filtration. Moreover, the fluorous-tagged latent anomeric leaving group could be activated by oxidation and cleaved by IPRm glycosylation, thus enabling the elongation of the carbohydrate chain from the non-reducing end to the reducing end as well as the recovery of the fluorous tag. The present two-directional synthetic strategy is used to assemble the repeating unit of Streptococcus pneumoniae type 14 capsular polysaccharide.


Introduction
Saccharides are one of the most abundant biomolecules in nature. 1 However, the exploration of their various physiological functions is hampered by their extraordinarily complex chemical structures and poor availabilities. Consequently, the development of efficient oligosaccharide and polysaccharide assembly strategies is of great demand. Signicant progress has been made in the past few decades 2 as exemplied by the access to many complex oligosaccharides and polysaccharides, especially the remarkable studies of Ye's 92-mer branched arabinogalactan, 3 Yu's 128-mer liner glycan in solution-phase synthesis 4 and Seeberger's 151-mer glycan in automated solidphase synthesis. 5 Despite these achievements, huge challenges are still encountered in carbohydrate synthesis, which largely encumbered the development of glycoscience. Among these challenges, one of the major obstacles is the tedious purication process. 2g Time-consuming and eco-unfriendly column chromatography is always required in conventional solution-phase synthesis ( Fig. 1A(a)). Solid-phase synthesis is one of the most representative state of the art solutions to this problem, [6][7][8] however, at the expense of consuming a large excess of carbohydrate building blocks due to the heterogeneous reaction manner, while the carbohydrate building blocks are much more expensive compared to amino acids and nucleotides in solid-phase polypeptide and polynucleotide synthesis ( Fig. 1A(b)).
As a complementary type of solution-phase synthesis and solid-phase synthesis, uorous synthesis pioneered by Zhu, 9 Horváth 10 and Curran 11 et al. began to ourish in organic synthesis from the middle 1990s. 12,13 Fluorous techniques retain the advantages of solution-phase synthesis and solid-phase purication. In this strategy (Fig. 1A(c)), a uorous tag was installed on substrates, which allowed the reaction to be performed in a homogenous system. Upon completion of the reaction, uorous solid-phase extraction (F-SPE) 14 or uorous liquid-liquid extraction (F-LLE) 11b,15 was implemented for the purication by virtue of the unique interactions between uorous-tag labeled substrates and uorous silica gel or uorous solvent, thus signicantly simplifying the purication processes. 16 This superiority has also enriched the carbohydrate synthesis. Since Curran rstly introduced a benzyl type uorous protecting group as a tag for disaccharide synthesis, 17 Seeberger, 18 Pohl, 19 Mizuno 20 and others 21 have developed diversi-ed uorous tags for carbohydrate chemical synthesis in the past few decades. To address the problem of the requirement of expensive uorous solvent and uorous silica gel in purication steps, Chai and co-workers introduced cheap and reusable polytetrauoroethylene (PTFE) particles for F-SPE, which allowed purication by simple ltration, namely PTFE assisted ltration. This process signicantly simplied the purication step and made the isolation efficiency comparable to that of Cite this: Chem. Sci., 2022, 13, 8759 All publication charges for this article have been paid for by the Royal Society of Chemistry solid-phase synthesis. 22 Among the various uorous-tag assisted carbohydrate synthesis methods, the installation of a uorous tag at the reducing end displayed exceptional advantages due to the stability and the exibility. It also facilitated the synthesis of carbohydrate microarrays 19a and automated solution-phase iterative synthesis 19c advanced by Pohl and co-workers. The nal cleavage of the uorous tags in this strategy was generally relied on alcoholysis, 21f olen metathesis, 18b,19e oxidative cleavage, 20a,b reductive hydrogenation 22a,b and so on, and consequently only allowed single directional assembly from the reducing end to the non-reducing end and very few of the cleaved uorous tags were reported to be recovered and recycled. 20a,21f, 22a We have recently developed a solution-phase oligosaccharide synthesis strategy based on interrupted Pummerer reaction mediated (IPRm) glycosylation. 23 In this strategy, we comprehensively applicated latent O-2-(2-propylthiol)-benzyl (OPTB) glycosides and its active counterpart O-2-[(propan-2-yl) sulnyl]benzyl (OPSB) glycosides as glycosyl donors in glycosylation. It enables efficient and rapid assembly of complex oligosaccharides and also allows the recovery and regeneration of the anomeric leaving group (Fig. 1B). [24][25][26][27] In view of these advantages, we envisioned that the installation of uorous side chains on anomeric OPTB and OPSB functionalities (OPTB F and OPSB F respectively) would further facilitate the reducing end to non-reducing end assembly sequence by virtue of the PTFE assisted ltration technology. Most importantly, the uorous tagged anomeric leaving group would be amenable to be cleaved and recovered by IPRm glycosylation and thus allow the non-reducing end to reducing end assembly and overall, provide a two-directional oligosaccharide synthesis (Fig. 1C).

Results and discussion
Our study commenced on the design of a uorous tagged anomeric leaving group by installing appropriate uorous chains on the anomeric PTB group at appropriate positions. Inspired by Chai's work, 22b we intended to directly x two per-uoroalkane chains on the terminal position of PTB-OH by ether linkages aimed at simplifying the synthesis and minimizing the impact of the uorous chains on the glycosylation efficiency (Scheme 1). In addition, the two peruoroalkane chains would maintain the high affinity of the extended sugar chains to the PTFE particles. To this purpose, known alcohol 1 bearing two peruoroalkyl ether chains was selected as the starting material. 22a The tosylation of alcohol 1 followed by a S N 2 displacement with methyl thiosalicylate under basic conditions furnished 3 in good yield. The subsequent reduction of the ester group of 3 produced PTB F -OH 4 in 97% yield. It's worth noting that all the purication steps in this synthetic procedure were performed by PTFE assisted ltration, that is the adsorption of the reaction residues on PTFE powder (200 mm particle size) in a solvent mixture of acetone/H 2 O (6 : 4, v/v), followed by ltration with a sand core funnel and successive washing with acetone/H 2 O and dichloromethane (Scheme 1). Each purication could be nished within half an hour and the synthesis was easy to scale up to 5 grams in one batch. This newly designed uorous-tagged PTB F -OH has great stability and is amenable to be stored on shelf at least for 10 months without detectable decomposition.
With uorous-tagged PTB F -OH in hand, we embarked on proving the concept (Scheme 2). The attachment of the uorous tag on carbohydrate was achieved by coupling of 4 with glucosyl trichloroacetimidate donor 5 in the presence of a catalytic amount of TMSOTf at À20 C. The standard PTFE assisted ltration procedure delivered OPTB F glycoside 6 in 90% yield. Further oxidation of 6 by [bis(triuoroacetoxy)iodo]benzene (PIFA) provided the OPSB F glycosyl donor 7 in 93% yield. Subsequently, the possibility of the cleavage of the uoroustagged anomeric leaving group by the IPRm glycosylation reaction was examined. Subjecting OPSB F donor 7 and acceptor 8 to a standard IPRm glycosylation procedure 23 afforded the Scheme 1 Synthesis of fluorous-tagged PTB F -OH via PTFE assisted filtration.
Scheme 2 Proof of concept. Table 1 Orthogonal glycosylations of fluorous-tagged glycosyl acceptors desired disaccharide 9 in 91% yield. In the meantime, the uorous-tag derivative 10 (PSB F -OH) was isolated in 87% yield. Moreover, PSB F -OH was found to be easily reduced to PTB F -OH (4) in the presence of thiosalicylic acid (TSA) and a catalytic amount of iodine. This regenerated PTB F -OH was able to be installed as the latent leaving group again and thus was almost completely recycled. The results indicated that the attached uorous tag is highly conducive to the IPRm glycosylation reaction and our design of the recyclable uorous tag assisted two-directional oligosaccharide synthesis is conceptually realized.
Encouraged by these results, we synthesized a series of OPTB F acceptors 12 (for details, see the ESI †) and examined their orthogonality to various classical glycosylation methods (Table 1). To our delight, the uorous-tagged acceptors glycosylated efficiently with various types of glycosyl donors under the corresponding activation conditions, and all the reactions are highly conducive to the practical PTFE assisted ltration purication procedure. The uorous tag was proved to be stable under acidic (13a-13d) and transition metal-catalyzed (13e) conditions, which are frequently used to activate the trichloroacetimidate glycosyl donor (5), 28 OPSB/SPSB glycoside donors (11a/11b), 23,29-31 n-pentenyl glycosyl donor (11d), 32,33 thioglycoside donor (11c) 34 and o-alkynylbenzoate (Abz) glycosyl donor (11e). 35,36 The high glycosylation efficiency of these reactions implied the limited restrictions of the method in the assembly of complex oligosaccharides from the reducing end to the non-reducing end, because many nice glycosylation methods are amenable to be selected to address particular coupling problems that may be encountered in the assembly sequence. In addition, the OPTB F moiety at the reducing end was able to be transformed to the active leaving group for further elongation via IPRm glycosylation. These exibilities as well as the ease of purication processes highlighted its great potential in the oligosaccharide synthesis.
Subsequently, the scope and limitations on the cleavage of the uorous tag by IPRm glycosylation and meanwhile the elongation of the sugar chains at the reducing end were examined (Scheme 3). Firstly, various glycosides 13 possessing terminal OPTB F groups were oxidized to active OPSB F glycosides 14 by PIFA in aqueous CH 3 CN. These oxidation reactions proceeded smoothly and most importantly their purication by simple aqueous workup followed by extraction delivered the OPSB F glycosides in good quality, which were pure enough for next IPRm glycosylations in most cases (for details, see the ESI †).
A variety of acceptors including simple alcohols such as MeOH, N-benzyl-N-benzyloxycarbonyl-5-aminopentanol, 3methoxyl-4-O-benzyl phenylethanol and diversied carbohydrate acceptors were found to be effective nucleophiles in this uorous assisted IPRm glycosylation. The standard IPRm glycosylation reaction conditions delivered glycosidic products 16a-16i in good to excellent overall yields. Among these examples, it is worth noting that: (1) p-methoxyphenyl group (16e), cis-alkene (16h), and other acid-sensitive groups such as tertbutyldiphenylsilyl group (16e) and acetonide (16i) were tolerated well under current conditions. (2) The thioglycoside acceptor (16g) and OPTB acceptor (16i) remained stable in glycosylation reactions and would be benecial to further orthogonal glycosylations. (3) Disaccharide 16d and trisaccharide 16h were the protected forms of dictamphenosides E and (Z) 3-hexenol glycosides, which were recently isolated from Cortex Dictamni 37 and Physalis alkekengi var. franchetii, 38 respectively. Their good glycosylation output laid the basis for more complicated oligosaccharide assembly through the established PTFE assisted two-directional synthetic protocol.
As a nal endeavor to demonstrate the utilization potential of the protocol, structurally more complex S. pneumonia type 14 (ST14) tetrasaccharide and octasaccharide were chosen as synthetic targets (Scheme 4). 39,40 The tetrasaccharide repeating unit is the smallest structure to induce ST14-specic antibody effects 41,42 and represents an important component for the development of synthetic or semi-synthetic pneumococcal vaccines. 43 Savage 44 and Seeberger 42,45 have developed glycoconjugate vaccine candidates, which are advantageous to clinically used vaccines on the basis of solution-phase synthesized tetrasaccharide fragments. Besides, Salwiczak also has synthesized ST14 tetrasaccharide and octasaccharide antigens by a solid-phase method. 41 We believe that the present twodirectional assembly strategy has great potential to provide fast access to ST14 oligosaccharides and would be benecial to vaccine development. With PTB F -OH, thioglycosides 17a and 17b, and OPSB glycosides 18a and 18b in hand, uorous-tag assisted two-directional assembly was conducted. PTB F -OH was rstly glycosylated to building block 17a and provided OPTB F monosaccharide 19. gave OPTB F tetrasaccharide 20 in 48% overall yield in seven steps. It's worth mentioning that tetrasaccharide 20 was obtained without column chromatography with the aid of PTFE assisted ltration. The existence of a levulinyl group at the nonreducing end allows the further elongation of the repeating polysaccharide chain. Additionally, the OPTB F moiety at the reducing end permits the elongation at the reducing end by IPRm glycosylation.
Simple oxidization of OPTB F tetrasaccharide 20 and coupling with 2-azidoethanol successfully provided the minimum repeating unit tetrasaccharide 22 in 82% yield with the introduction of a biological compatible tail for further elaboration in biological studies. Meanwhile the anomeric uorous tag was cleaved. The treatment of tetrasaccharide 20 with hydrazinium hydroxide solution to remove the levulinyl group furnished the tetrasaccharide OPTB F acceptor. Next, the dimerization reaction between tetrasaccharide donor 21 (2.0 equiv.) and tetrasaccharide acceptor under standard IPRm glycosylation conditions performed very well and provided octasaccharide 23 in 85% yield with PTFE assisted ltration. Finally, the oxidation and elaboration of 2-azidoethanol accomplished the synthesis of octasaccharide dimer 25 in overall eleven steps and 26% yield from mono-saccharide building blocks. Eight PTFE assisted ltrations were adopted in the whole synthesis that largely minimized the column chromatography separation steps and simplied the purication processes.

Conclusions
In conclusion, we developed a uorous-tag assisted twodirectional oligosaccharide assembly strategy. The strategy relied on our recently developed IPRm glycosylation by designing a uorous tagged OPTB anomeric leaving group. The upgraded IPRm glycosylation allowed exible, rapid and efficient oligosaccharide assembly from the reducing end to the non-reducing end with the aid of PTFE assisted ltration puri-cation technology. Upon the completion of the reducing end to non-reducing end assembly, the terminal OPTB F was able to be oxidized to the OPSB F group, which could be cleaved and recycled by another IPRm glycosylation and at the same time elongated the oligosaccharide chain at the reducing end. The efficacy was exemplied by fast access to the ST14 tetrasaccharide and octasaccharide fragments. Additionally, encouraged by recent remarkable achievements in automated uorous-assisted solution-phase oligosaccharide synthesis 46 and HPLC-based automated synthesis of glycans in solution, 47 we expect to employ the developed uorous-tag assisted synthetic strategy in an automated platform and we are currently studying on this issue.

Data availability
Experimental data including experimental procedures, characterization data, and NMR spectra for the new compounds have been uploaded as ESI material. †

Author contributions
Lei Cai and Qi Chen carried out most of the experiments, brought the project to completion and prepared the dra. Jian Guo and Zhihua Liang synthesized PTB F -OH and some uorous-tagged glycosyl acceptors. Dengxian Fu participated in the revision of the dra. Lingkui Meng contributed to the discussion of the project and revision of the dra. Qian Wan conceived the idea. Jing Zeng and Qian Wan oversaw the project, discussed the results, and edited and reorganized the manuscript.

Conflicts of interest
There are no conicts to declare.