Investigation of the Protection of the C4 Hydroxyl Group in Macrobicyclic Kdo Donors

Chemical synthesis of 3-deoxy-d-manno-2-octulosonic acid (Kdo)-containing glycans, such as bacterial lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs), is in high demand for the development of vaccines against pathogenic bacteria. We have recently achieved the complete α-stereoselective glycosidation of Kdo using a macrobicyclic donor tethered at the C1 and C5 positions. In this study, to expand the scope of Kdo glycosidation, we sought to protect the 4-OH group, thereby shortening the reaction time and ensuring the conversion of the glycosyl acceptor via its selective removal. The protection of the 4-OH group influenced the reactivity of the Kdo donor, and the triisopropylsilyl (TIPS) group acted as a selectively removable booster. The 4-O-TIPS donor allowed the synthesis of the α(2,4)-linked dimeric Kdo sequence, which is widely found in bacterial LPSs.


Introduction
3-Deoxy-D-manno-2-octulosonic acid (Kdo) constitutes bacterial glycans, such as lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs), which are closely associated with the survival and virulence of pathogenic bacteria [1][2][3]. To understand and harness the functions of Kdo-containing LPSs and CPSs in bacterial infectious diseases, their synthesis is required. The chemical synthesis of structurally defined Kdo-containing glycans and their functionalized probes is highly profitable owing to their poor availability from natural sources. In particular, there has been growing interest in the field of vaccine development. However, the chemical synthesis of Kdo-containing glycans is challenging [4,5]; during the glycosylation of Kdo (referred to as Kdosidation), the stereocontrol is difficult, owing to the absence of a hydroxyl group at the position adjacent to the anomeric center. Previous studies on Kdosidation devised α-selective Kdo donors, such as those protected with bulky groups at the position diagonal to the anomeric position [6][7][8][9][10][11], appended with an auxiliary group at the C3 [12][13][14] and C5 [15] positions, and 2,3-ene derivatives as the precursor of the C3-appended intermediate [16][17][18][19][20]. Recently, DMF-assisted α-selective Kdosidation using a per-acetyl Kdo donor has been demonstrated [21].
Full α-selective Kdosidation was achieved using a macrobicyclic Kdo donor with α-configuration, which completely blocked the glycosidation at the β-face by the tether moiety between the C1 and C5 positions ( Figure 1) [22]. In principle, this method does not produce any β-isomers and can achieve high yields using various acceptors, thereby facilitating the synthesis of α-Kdo-containing glycans. However, glycosidation using donor 1 required more time compared to glycosidation using a non-bicyclic Kdo donor [22]. In To tune the reactivity of the macrobicyclic Kdo donor, we used the effect of the hydroxyl protecting group. Based on the enormous results of the reactivities of the glycosyl donors [23,24], we presumed that the protection of the C4 and C7 positions would influence the stability of the oxocarbenium ion of Kdo. Considering the higher efficiency of the protection of the C4 hydroxyl group than that at the C7, we examined the effect of protecting groups at the C4 position. The original 4-O-acetyl (Ac) Kdo donor 1 was used as the benchmark, and the protection at the hydroxyl was diversified with selectively removable triisopropylsilyl (TIPS), chloroacetyl (CAc), 2,2,2-trichloroethoxycarbonyl (Troc), and benzyloxycarbonyl (Cbz) groups, which were expected to exert different substituent effects, affording the macrobicylic Kdo donors 2-5. Additionally, the installation of an orthogonal protecting group at this position would be useful for the synthesis of various Kdo-embedded glycans.

Synthesis of Macrobicyclic Kdo Donors Diversified in the 4-OH Protection
To efficiently synthesize the macrobicyclic donors 2-5, the TIPS-protected donor 2 was used as a common intermediate for donors 3, 4, and 5 (Scheme 1a). To establish the α-ethylthioglycoside of Kdo, we exploited the reported intramolecular α-selective glycosidation using a dithioketal derivative to produce 6 [17]. Following the procedures reported by our group [22], compound 6 was converted into the 4,5-diol derivative 7. The TIPS group was then regioselectively introduced at 4-OH, affording 8 in an excellent yield. Next, for macrolactonization, 5-OH was esterified with the tethering unit 9 [22] by Shiina esterification to afford compound 10 in 92% yield. Treatment with 80% aqueous acetic acid followed to selectively remove the TBS protection of the tether moiety in the presence of the TIPS group, yielding 11. Compound 11 was then converted into the carboxylic acid derivative 12 via selective de-methylation using Ph3SiSH [25]. Finally, compound 12 underwent macrolactonization via the Mitsunobu reaction [22,26] to yield the TIPS-protected Kdo donor 2 in 93% yield. To tune the reactivity of the macrobicyclic Kdo donor, we used the effect of the hydroxyl protecting group. Based on the enormous results of the reactivities of the glycosyl donors [23,24], we presumed that the protection of the C4 and C7 positions would influence the stability of the oxocarbenium ion of Kdo. Considering the higher efficiency of the protection of the C4 hydroxyl group than that at the C7, we examined the effect of protecting groups at the C4 position. The original 4-O-acetyl (Ac) Kdo donor 1 was used as the benchmark, and the protection at the hydroxyl was diversified with selectively removable triisopropylsilyl (TIPS), chloroacetyl (CAc), 2,2,2-trichloroethoxycarbonyl (Troc), and benzyloxycarbonyl (Cbz) groups, which were expected to exert different substituent effects, affording the macrobicylic Kdo donors 2-5. Additionally, the installation of an orthogonal protecting group at this position would be useful for the synthesis of various Kdo-embedded glycans.

Synthesis of Macrobicyclic Kdo Donors Diversified in the 4-OH Protection
To efficiently synthesize the macrobicyclic donors 2-5, the TIPS-protected donor 2 was used as a common intermediate for donors 3, 4, and 5 (Scheme 1a). To establish the α-ethylthioglycoside of Kdo, we exploited the reported intramolecular α-selective glycosidation using a dithioketal derivative to produce 6 [17]. Following the procedures reported by our group [22], compound 6 was converted into the 4,5-diol derivative 7. The TIPS group was then regioselectively introduced at 4-OH, affording 8 in an excellent yield. Next, for macrolactonization, 5-OH was esterified with the tethering unit 9 [22] by Shiina esterification to afford compound 10 in 92% yield. Treatment with 80% aqueous acetic acid followed to selectively remove the TBS protection of the tether moiety in the presence of the TIPS group, yielding 11. Compound 11 was then converted into the carboxylic acid derivative 12 via selective de-methylation using Ph 3 SiSH [25]. Finally, compound 12 underwent macrolactonization via the Mitsunobu reaction [22,26] to yield the TIPSprotected Kdo donor 2 in 93% yield. The TIPS group of 2 was selectively removed using a fluoride anion to retrieve a free hydroxyl group at the C4 position (Scheme 1b) and synthesize the other donors. When compound 2 was treated with TBAF and acetic acid in THF, the migration of the ester groups at the O5 and O7 positions occurred randomly to give a complex mixture, whereas the use of HF·pyridine provided the 4-OH derivative 13 in high yield by decreasing the migrated byproduct. Next, the 4-OH, CAc, Troc, and Cbz groups were introduced to afford donors 3, 4, and 5, respectively, in excellent yields.

Examination of α-Glycosidation Using the Macrobicyclic Kdo Donors
The glycosylation of the 6-OH of the glucosyl acceptor 14 was examined to compare the reactivities of the macrobicyclic Kdo donors ( Table 1). The protection of the group at the C4 position affected the reactivity of the Kdo donors. Among the tested donors, the TIPS group boosted the reactivity of the donor most significantly. In all cases, equimolar amounts of donor and acceptor were reacted in the presence of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), and 3 Å molecular sieves in CH2Cl2 at −80 °C. The reaction was quenched upon complete consumption of the donor or halt of the reaction, confirmed by TLC analysis. The previously reported result of the glycosidation of 4-O-Ac Kdo donor [22] is shown in Entry 1 as the benchmark for this study. Among the tested groups, only the TIPS group was able to shorten the reaction time (by a factor of ~0.62), achieving a 76% yield of the product 16, comparable to that of Entry 1. However, the introduction of acyl protecting groups slowed down the reaction (Entries 3-5). The result of Entry 3 showed the significant effect of the electron-withdrawing CAc group on the reactivity of the macrobicyclic Kdo donor, in which the reaction time became much longer (96 h) by a factor of ~2.7. In this case, apart from the 2,3-ene derivative, the reaction produced complex side products of the donor, including a 4-OH derivative. Glycoside 17 was obtained in 17% yield, and the unreacted donor was recovered in 35% yield, indicating the significantly reduced reactivity of donor 3. Likewise, the introduction of carbonate groups, such as Troc and Cbz, retarded the reaction to some extent (Entries 4 and 5). A comparison of Troc and Cbz revealed sensitivity to the electron-withdrawing group. The TIPS group of 2 was selectively removed using a fluoride anion to retrieve a free hydroxyl group at the C4 position (Scheme 1b) and synthesize the other donors. When compound 2 was treated with TBAF and acetic acid in THF, the migration of the ester groups at the O5 and O7 positions occurred randomly to give a complex mixture, whereas the use of HF·pyridine provided the 4-OH derivative 13 in high yield by decreasing the migrated byproduct. Next, the 4-OH, CAc, Troc, and Cbz groups were introduced to afford donors 3, 4, and 5, respectively, in excellent yields.

Examination of α-Glycosidation Using the Macrobicyclic Kdo Donors
The glycosylation of the 6-OH of the glucosyl acceptor 14 was examined to compare the reactivities of the macrobicyclic Kdo donors ( Table 1). The protection of the group at the C4 position affected the reactivity of the Kdo donors. Among the tested donors, the TIPS group boosted the reactivity of the donor most significantly. In all cases, equimolar amounts of donor and acceptor were reacted in the presence of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH), and 3 Å molecular sieves in CH 2 Cl 2 at −80 • C. The reaction was quenched upon complete consumption of the donor or halt of the reaction, confirmed by TLC analysis. The previously reported result of the glycosidation of 4-O-Ac Kdo donor [22] is shown in Entry 1 as the benchmark for this study. Among the tested groups, only the TIPS group was able to shorten the reaction time (by a factor of~0.62), achieving a 76% yield of the product 16, comparable to that of Entry 1. However, the introduction of acyl protecting groups slowed down the reaction (Entries 3-5). The result of Entry 3 showed the significant effect of the electron-withdrawing CAc group on the reactivity of the macrobicyclic Kdo donor, in which the reaction time became much longer (96 h) by a factor of~2.7. In this case, apart from the 2,3-ene derivative, the reaction produced complex side products of the donor, including a 4-OH derivative. Glycoside 17 was obtained in 17% yield, and the unreacted donor was recovered in 35% yield, indicating the significantly reduced reactivity of donor 3. Likewise, the introduction of carbonate groups, such as Troc and Cbz, retarded the reaction to some extent (Entries 4 and 5). A comparison of Troc and Cbz revealed sensitivity to the electron-withdrawing group. Although the reactions provided similar yields as those of the Kdo glycosides (20% for 18 and 28% for 19) and 2,3-ene derivatives as major side products (75% for 23 and~70% for 24), 4-O-Troc donor 4 took much longer (68 h) than the 4-O-Cbz donor 5 (42 h). Substitution at the C4 position may affect the electron density of the sulfur atom of the leaving group and the stability of the oxocarbenium ion intermediate, through an unknown mechanism. Therefore, donors with electron-withdrawing groups required a longer period and readily underwent 1,2-elimination rather than glycosidation to produce 2,3-ene side products. Although the reactions provided similar yields as those of the Kdo glycosides (20% for 18 and 28% for 19) and 2,3-ene derivatives as major side products (75% for 23 and ~70% for 24), 4-O-Troc donor 4 took much longer (68 h) than the 4-O-Cbz donor 5 (42 h). Substitution at the C4 position may affect the electron density of the sulfur atom of the leaving group and the stability of the oxocarbenium ion intermediate, through an unknown mechanism. Therefore, donors with electron-withdrawing groups required a longer period and readily underwent 1,2-elimination rather than glycosidation to produce 2,3-ene side products.

Application of the 4-O-TIPS Kdo Donor 2 to the Synthesis of α(2,4)-Linked Dimeric Kdo
The synthesis of an α(2,4)-linked dimeric Kdo was attempted, which is widely embedded in the core regions of bacterial LPSs [1], using the 4-O-TIPS Kdo donor 2 (Scheme 2). Disaccharide 16 was used as a model of Kdo glycoside and was synthesized following the aforementioned reaction conditions. First, 16 was treated with HF·pyridine in pyridine at room temperature. The reaction successfully removed TIPS without any sequential migration of acyl groups, such as those at 5-, 7-, and 8-OH, affording the disaccharide acceptor 25 in 97% yield. Subsequently, acceptor 25 was glycosylated with Kdo donor 2. As expected, upon activation with NIS-TfOH in CH2Cl2 at −80 °C, the reaction proceeded smoothly and ended in 22 h, yielding the dimeric Kdo-containing trisaccharide 26 in 70% yield.

Application of the 4-O-TIPS Kdo Donor 2 to the Synthesis of α(2,4)-Linked Dimeric Kdo
The synthesis of an α(2,4)-linked dimeric Kdo was attempted, which is widely embedded in the core regions of bacterial LPSs [1], using the 4-O-TIPS Kdo donor 2 (Scheme 2). Disaccharide 16 was used as a model of Kdo glycoside and was synthesized following the aforementioned reaction conditions. First, 16 was treated with HF·pyridine in pyridine at room temperature. The reaction successfully removed TIPS without any sequential migration of acyl groups, such as those at 5-, 7-, and 8-OH, affording the disaccharide acceptor 25 in 97% yield. Subsequently, acceptor 25 was glycosylated with Kdo donor 2. As expected, upon activation with NIS-TfOH in CH 2 Cl 2 at −80 • C, the reaction proceeded smoothly and ended in 22 h, yielding the dimeric Kdo-containing trisaccharide 26 in 70% yield.

Chemicals
All chemicals were purchased from commercial suppliers and were used as received, unless otherwise noted. The molecular sieves were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and were pre-dried at 300 • C for 2 h in a muffle furnace followed by drying in a flask at 250 • C for 2 h under vacuum, prior to use. The dry solvents used for the reaction media (CH 2 Cl 2 , toluene, THF, MeCN, DMF, and pyridine) were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan) and were used as received. Other reaction media solvents were dried over molecular sieves and used without purification.

TLC Analysis
TLC analyses were performed on Merck TLC plates (silica gel 60F 254 on glass plate) (Darmstadt, Germany). Compounds were detected either by exposure to UV light (253.6 nm) or by soaking in a H 2 SO 4 solution (10% in EtOH) or phosphomolybdic acid solution (20% in EtOH) followed by heating.

Chromatograhic Purification
Flash column chromatography separations were performed by using silica gel (80 mesh and 300 mesh; Fuji silysia Co. (Aichi, Japan)) or Biotage Isolera TM (Uppsala, Sweden) equipped with Biotage SNAP Ultra Silica Cartridges (10 g, 25 g, 50 g, 100 g, and 340 g) (Uppsala, Sweden) or Biotage Sfär Silica HC D Cartridges (10 g, 25 g, 100 g, 200 g, and 350 g) (Uppsala, Sweden). The quantity of silica gel was typically 100 to 200 times the weight of the crude sample. Cytiva Sephadex LH-20 (Tokyo, Japan) was used for size-exclusion chromatography. Solvent systems for chromatography are specified as v/v ratios.

Structural Analysis and Acquisition of Physical Data
1 H and 13 C NMR spectra were recorded on an Avance III 500 spectrometer (Bruker, Billerica, MA, USA). The chemical shifts in the 1 H NMR spectra are expressed in ppm (δ), relative to the Me 4 Si signal (0.00 ppm). The chemical shifts in the 13 C NMR spectra are expressed in ppm (δ), relative to the Me 4 Si (0.00 ppm), CDCl 3 (77.16 ppm), and adjusted CD 3 OD (49.00 ppm) signals. The data are presented as: chemical shift, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, quint = quintet, dd = doublet of doublets, td = triplet of doublets, and m = multiplet and/or multiple resonances), integration, coupling constant in hertz (Hz), and position of the corresponding proton. COSY, HMBC, and HMQC methods were used to assign the NMR signals. Highresolution mass spectrometry (ESI-TOF MS) data were obtained using a micrOTOF, Bruker mass spectrometer. Optical rotations were measured with a high-sensitivity polarimeter (SEPA-500, Horiba (Kyoto, Japan)). The copies of 1 H and 13 C NMR spectra of the new compounds are provided in the Supplementary Material.

General Procedure for Chemical Reaction
All operations were carried out in a fume hood. All reactions were carried out under a positive pressure of argon unless otherwise noted. Evaporations and concentrations were carried out in vacuo. All heated reactions were carried out in an oil bath. All reactions below 0 • C were carried out in a low temperature reaction tank (PSL-1820, EYELA (Tokyo, Japan)).

Pretreatment for Glycosidation Reaction
A glycosyl donor (1.0-2.0 equiv.) and a glycosyl acceptor (1.0 equiv.) were mixed in a pear-shaped flask, and then residual water was azeotropically removed with dry toluene. The mixture was exposed to high vacuum for 3 h. 3Å molecular sieves (100 mg was used for 1 mL of reaction solvent) were pre-dried at 300 • C for 2 h in a muffle furnace. The pre-dried molecular sieves were added to a two-necked, round-bottomed flask and heated at 250 • C for 2 h in vacuo.

General Procedureof the Glycosidation Reaction
In a pear-shaped flask, a glycosyl donor and a glycosyl acceptor were dissolved in the reaction solvent (50 mM), and the mixture was then transferred via a cannula to a twonecked flask containing the pre-dried molecular sieves. After stirring for 1 h at −80 • C, the promoters were added to the mixture at the same temperature. The progress of the reaction was monitored using TLC analysis and/or mass spectrometry (MALDI-TOF performed with Autoflex, Bruker; matrix: CHCA). The reaction mixture was then quenched, filtered, subjected to an aqueous work-up, and concentrated. The resulting residue was purified by column chromatography. The yields of the isolated coupled products are reported. To a solution of 13 (60.3 mg, 116 µmol) in pyridine (1.2 mL), CAc 2 O (59.5 mg, 348 µmol) and DMAP (1.4 mg, 12 µmol) were added at 0 • C. After stirring for 30 min at room temperature and while the reaction was monitored by TLC (EtOAc/n-hexane = 1/2), MeOH was added at 0 • C. The mixture was co-evaporated with toluene; diluted with CHCl 3 ; and washed with 2 M HCl aq., water, 5% NaHCO 3 aq., and brine. The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel, using EtOAc in n-hexane (20%) as the eluent, to afford 3 (63. 6   To a solution of 13 (60.4 mg, 117 µmol) in pyridine (1.2 mL), TrocCl (47.1 µL, 351 µmol) and DMAP (1.4 mg, 12 µmol) were added at 0 • C. After stirring for 30 min at room temperature, TrocCl (47.1 µL, 351 µmol) was added, and the mixture was stirred for a total of 1.5 h as the reaction was monitored by TLC (EtOAc/n-hexane = 1/2). MeOH was added to the reaction mixture at 0 • C. The mixture was co-evaporated with toluene; diluted with CHCl 3 ; and washed with 2 M HCl aq., water, satd. NaHCO 3 aq., and brine. The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel, using EtOAc in n-hexane (20%) as the eluent, to yield 4 (76.1 mg, 94%)   179 µmol) were added after 7, 10, and 11 h. The mixture was stirred for a total of 12.5 h as the reaction was monitored by TLC (EtOAc/n-hexane = 1/2), and MeOH was added to the reaction mixture at 0 • C. The mixture was co-evaporated with toluene; diluted with CHCl 3 ; and washed with 2 M HCl aq., water, satd. NaHCO 3 aq., and brine. The organic layer was dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel, using EtOAc in n-hexane (30%) as the eluent, to