Solid-Phase Stereocontrolled Synthesis of Oligomeric P-Modified Glycosyl Phosphate Derivatives Using the Oxazaphospholidine Method

Abstract

Glycosyl phosphate repeating units can be found in the glycoconjugates of some bacteria and protozoa parasites. These structures and their P-modified analogs are attractive synthetic targets as antimicrobial, antiparasitic, and vaccine agents. However, P-modified glycosyl phosphates exist in different diastereomeric forms due to the chiral phosphorus atoms, whose configuration would highly affect their physiochemical and biochemical properties. In this study, a stereocontrolled method was developed for the synthesis of P-modified glycosyl phosphate repeating units derived from the lipophosphoglycan of Leishmania using the oxazaphospholidine approach. The solid-phase synthesis facilitated the elongation and purification of the glycosyl phosphate derivatives, while two P-modified glycosyl phosphates (boranophosphate and phosphorothioate) were successfully synthesized with up to three repeating units.

Introduction

Glycosyl phosphate repeating units exist in biomolecules of pathogens, which play several important biological and immunological roles.¹ For example, Streptococcus pneumoniae(2) and Neisseria meningitidis,(3) especially their most pathogenic serotypes, include a glycosyl phosphate repeating unit in their capsular polysaccharides (CPs). CPs contribute to the infectivity of the pathogens, as they help them avoid the host’s immune responses by masking the highly antigenic components of these bacteria.^4,5 In addition, the CPs are recognized as antigens.⁶ Glycosyl phosphate repeating units can also be found in lipophosphoglycans (LPGs) of Leishmania(7) (Figure 1), a protozoan parasite that causes fetal tropical diseases.⁸ LPGs are reported to be vital for the infection process and survival of Leishmania.(7,9) Thus, the glycosyl phosphate moieties are an attractive synthetic target as candidates for antipathogen and vaccine applications. In addition, chemically synthesized glycosyl phosphates with defined and uniform structures would contribute to the elucidation of their functions. Therefore, many efforts have focused on the chemical synthesis of these moieties.^1,10−19 Furthermore, modifications on the phosphorus (P) atoms of the intersugar phosphate groups are expected to improve the properties of the glycosyl phosphates, such as the chemical stability^20,21 and biological activity,²² thus allowing the development of glycosyl phosphate-based drugs. However, P-modified glycosyl phosphate analogs generally exist in several diastereomers states due to the chirality of the phosphorus atoms, which can highly affect their physiochemical and biological nature. Thus, the stereocontrolled synthesis of these molecules is essential to develop analogs with desired properties.

Figure 1.

(a) Structure of the glycosyl phosphate repeating units found in the LPGs of Leishmania Donovani. (b) P-Modified analogs of the repeating units.

In earlier studies, we have reported the stereoselective synthesis of glycosyl phosphate derivatives using the oxazaphospholidine method.^23,24 Oxazaphospholidine is a phosphoramidite derivative bearing a chiral auxiliary. The oxazaphospholidine method has been originally developed for the generation of stereocontrolled internucleotide linkages of oligonucleotides²⁵⁻²⁷ and allowed the successful synthesis of a wide variety of P-modified analogs with high stereoselectivity, such as phosphorothioate and boranophosphate derivatives, which bear the characteristic P–S and P→BH₃ moieties, respectively. We applied this strategy to carbohydrate chemistry, and disaccharides bearing an intersugar P-modified phosphate linkage were obtained with high stereoselectivities of chiral phosphorus atoms.²³ In particular, a stereodefined oxazaphospholidine monomer was utilized, which was subsequently coupled stereospecifically with a hydroxy group of another sugar in the presence of a non-nucleophilic acidic activator, N-(cyanomethyl) pyrrolidinium triflate (CMPT).²⁸ Then, the phosphorus atom of the resultant phosphite was modified by a stereospecific sulfurization or boronation reaction to afford the phosphorothioate and boranophosphate triester, respectively. In the final stage of the synthesis, the chiral auxiliary was removed upon treatment with a base, such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), to give the stereodefined P-chiral phosphorothioate and boranophosphate diesters. In this study, to broaden the scope of the method, we have developed a solid-phase synthetic methodology (Scheme 1). Generally, the solid-phase synthesis enables the rapid purification of the intermediates by simple washings after each reaction step, while it offers an easy access to a wide variety of glycosyl phosphates with multiple P-modified phosphate linkages in fully deprotected forms. The above-mentioned advantages were nicely demonstrated in the successful automated solid-phase synthesis of oligosaccharides by Seeberger et al.^29,30 The [→6)β-d-Gal-(1→4)-α-d-Man-(1-P)] repeating unit, which is a characteristic structure found in LPGs of Leishmania donovani,(7) was chosen as the synthetic target. Nikolaev et al. studied the activity of elongating α-mannosyl phosphate transferase, which is a unique enzyme for Leishmania, using the β-d-Gal-(1→4)-α-d-Man-(1-PO₄)⁻ derivative and its analogs including its P-modified counterparts, and reported that the P-modification substantially altered the substrate activities.²² Since these derivatives are thought to be diastereomeric mixtures, we expected that the stereocontrol of phosphorus configuration would provide a deep insight into the transferase recognition. Thus, owing to its perspective, the synthesis of the abovementioned structure was investigated in the current study.

Scheme 1. Strategy for the Stereocontrolled Synthesis of P-Modified Glycosyl Phosphate Derivatives.

Results and Discussion

Synthesis of Oxazaphospholidine Monomers

The monomer units for the solid-phase synthesis were at first synthesized. To investigate the suitable reaction conditions, two monomer types were prepared, namely, an α-mannosyl unit as a model compound (Scheme 2) and a β-d-Gal-(1→4)-α-d-Man unit (Scheme 3). The selective protection of the 6-hydroxy group of mannose with a 4-methoxy trityl (MMTr) group followed by the benzoylation of the remaining hydroxy groups and the selective removal of the anomeric benzoyl group afforded hemiacetal 2 in 69% yield over three steps (α/β = 93:7). The α-mannosyl monomer unit with the Rp configuration (Rp)-4 was obtained in 53% yield through the reaction of the anomeric hydroxy group of 2 and the phosphitylation reagent L-3 derived from L-proline.³¹ The TLC analysis of the reaction mixtures indicated an almost complete consumption of 2. However, the ³¹P NMR spectrum of (Rp)-4 after silica gel column chromatography purification indicated that some byproducts, probably an oxazaphospholidine with a cis-configuration (δ 142.1 ppm) and oxidized counterpart (δ 23.1 ppm). Multiple purifications resulted in the reduced isolated yield of (Rp)-4. The α-anomeric stereochemistry was confirmed by the relatively large ¹J_CH value (174 Hz).³² The trans stereochemistry at the phosphorus center was verified by the large ²J_PC value (34.7 Hz) of the signal at δ 47.0 ppm (8-position of the bicyclic structure).^33,34 It is noteworthy that the monomer (Rp)-4 was obtained with excellent anomeric and phosphorus stereopurity based on the ¹H NMR, ¹³C NMR, and ³¹P NMR measurements. On the other hand, some signals derived from a byproduct(s) was detected in the upfield region of ¹³C NMR spectra. The following investigation of solid-phase synthesis using (Rp)-4 revealed that the byproduct(s) did not cause crucial side reactions in the condensation step. For the synthesis of the β-d-Gal-(1→4)-α-d-Man unit, a suitably protected trichloroacetimidate galactosyl donor (5)³⁵ was reacted with the hydroxy group at the 4-position of the mannose derivative 6¹⁰ using TMSOTf as an activator, affording the disaccharide derivative 7 in good yield. The cleavage of the TBDPS group from the 6-position, the protection of the resultant free hydroxy group with an MMTr moiety, and the selective debenzoylation of the anomeric position afforded hemiacetal 10 with an anomeric ratio of α/β = 93:7. The loss of stereopurity at the anomeric position was probably attributed to an anomerization of the product under the reaction conditions and/or during the silica gel column chromatography purification. (Rp)-11 and (Sp)-11 were then obtained by the reaction of 10 with phosphitylation reagents derived from L- and D-proline, respectively.³¹ Multiple purifications and instability of (Rp)-11 and (Sp)-11 on silica gel led to moderate isolated yields. The anomeric and phosphorus stereochemistry of the (Rp)-11 and (Sp)-11 derivatives was also determined by the ¹J_CH values (172 Hz)³² and the large ²J_PC value of the signal at δ 46.9 ppm,^33,34 respectively, while the ¹H and ³¹P NMR spectra indicated that both monomers were obtained with high anomeric and phosphorus stereopurity.

Scheme 2. Synthesis of the α-Mannosyl Oxazaphospholidine Derivative (Rp)-4.

Reagents and conditions: (a) MMTrCl (1.4 equiv), pyridine, room temperature (rt), 21 h; (b) BzCl (8.0 equiv), rt, 3 h; (c) MeNH₂ (11.0 equiv), THF–MeOH (3:2, v/v), −30 °C, 23 h, 69% over three steps; and (d) L-3 (3.0 equiv), Et₃N (7.0 equiv), THF, −78 °C to rt, 1 h, 53%.

Scheme 3. Synthesis of the Disaccharide Oxazaphospholidine Monomers (Rp)-11 and (Sp)-11.

Reagents and conditions: (a) 5 (1.2 equiv), 6 (1 equiv), TMSOTf (0.3 equiv), CH₂Cl₂, 0 °C, 1 h, 76%; (b) TBAF (3.0 equiv), acetic acid (3.0 equiv), THF, 0 °C to rt, 4 h, 70%; (c) MMTrCl (3.0 equiv), pyridine, rt, 24 h, 88%; (d) MeNH₂ (11 equiv), THF–MeOH (8:1, v/v), −30 °C, 20 h, 88%; (e) L-3 (3.0 equiv), Et₃N (7.0 equiv), −78 °C to rt, 1 h, 62% ((Rp)-11); and (f) D-3 (3.0 equiv), Et₃N (7.0 equiv), −78 °C to rt, 1 h, 65%.

Solid-Phase Synthesis of Glycosyl Boranophosphates

For the solid-phase synthesis of the P-modified glycosyl phosphates with high stereopurity at phosphorus atoms, controlled pore glass (CPG) was chosen as a solid support, and the 1,4-bis(2-hydroxyethyl) hydroquinone spacer was introduced via a succinyl linker to CPG.³⁶ The spacer allows the detection of products through the UV absorbance of the hydroquinone moiety. Thus, the hydroxy group of 12 was condensed with the monomer (Rp)-4 in the presence of CMPT as an acidic activator. The resultant phosphite 13 was boronated using BH₃·SMe₂ to afford a boranophosphotriester 14, while the solid support was washed with MeCN and CH₂Cl₂. Moreover, the MMTr group on the 6-hydroxy group was cleaved under acidic conditions with trifluoroacetic acid (TFA) using Et₃SiH as the MMTr cation scavenger to prevent the side reaction on the borano group,³⁷ which afforded 15. The subsequent removal of the chiral auxiliary, the deprotection of the hydroxyl groups, and the cleavage of the succinyl linker under various conditions (Table 1) yielded the α-mannosyl boranophosphodiester 16.

Table 1. Solid-Phase Synthesis of α-Mannosyl Boranophosphate.

entry	conditions	16:17:18a
1	40% MeNH2 aq, rt, 4 h	35:23:42
2	1 M DBU in MeCN, rt, 4 h, then 40% MeNH2 aq, rt, 4 h	56:21:23
3	1 M DMANb in MeCN, rt, 1 h, then 40% MeNH2 aq, rt, 4 h	85:6:9

^aDetermined by RP-HPLC.

^b1,8-Bis(dimethylamino)naphthalene.

These three steps were initially attempted simultaneously upon treatment with an aqueous MeNH₂ solution (entry 1). An analysis of the crude mixture by reverse phase high-performance liquid chromatography (RP-HPLC) revealed that although the desired product 16 was obtained, byproducts such as the spacer 17 and its phosphorylated counterpart 18 were formed. The retention time of 17 was confirmed by injecting the authentic sample under the same conditions, while 18 was characterized by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) (calcd for 18 [M – H]⁻, 261.0533; found 261.0544). A plausible mechanism of the chiral auxiliary removal and side reactions is presented in Scheme 4. The chiral auxiliary is probably removed by the formation of an aziridine derivative.³⁸ Since MeNH₂ is highly nucleophilic, the ligand exchange reaction may occur as a competitive side reaction. The resultant phosphite would be thus hydrolyzed under basic conditions to give 17 and 18. Hence, it was assumed that a strong base with low nucleophilicity would hinder the deboronation reaction and effectively afford the desired boranophosphodiester. Therefore, treatment with DBU that prevented the deboronation side reaction in the solution-phase synthesis,²³ prior to the cleavage of the linker and deprotection of the hydroxy protecting group using an aqueous MeNH₂ solution, slightly suppressed the formation of the byproducts as indicated by the RP-HPLC analysis (entry 2). It was found that 1,8-bis(dimethylamino)naphthalene (DMAN) was a prominent reagent for the removal of the chiral auxiliary, and the ratio of the product was significantly improved with treatment of DMAN (entry 3). DMAN is known as a ″proton sponge″ and has high basicity.³⁹ On the other hand, DMAN is substantially a non-nucleophilic amine derivative.³⁹ These properties might contribute to the suppression of the side reactions.

Scheme 4. Plausible Mechanism for the Removal of Chiral Auxiliary and the Side Reactions.

To prove the proposed mechanism for the chiral auxiliary removal, the reaction mixture and washings were collected after treatment of DMAN, and the mixture was concentrated and analyzed by ¹H NMR. The NMR spectrum indicated the presence of the signals from the aziridine derivative 20 (Figure S11), which was confirmed by a comparison with reported data.⁴⁰ The result suggested that the removal of the chiral auxiliary proceeded mainly with the formation of the aziridine derivative 20.

Taking into account the abovementioned results, the solid-phase synthesis of the disaccharide 1-boranophosphate was carried out using the disaccharide monomers (Rp)-11 and (Sp)-11 under the conditions of entry 3 in Table 1 (Table 2). The RP-HPLC profiles of the crude reaction mixtures indicated that the disaccharide 1-boranophosphates (presumptive Rp)-23 and (presumptive Sp)-23 were obtained in high HPLC yields (Figure S2). The purified products (presumptive Rp)-23 and (presumptive Sp)-23, derived from the disaccharide monomers (Rp)-11 and (Sp)-11, respectively, were co-injected into HPLC, indicating that the two compounds had a different retention time (Figure S3). It has been earlier demonstrated that the condensation reaction of an oxazaphospholidine monomer with a hydroxy group leads to the inversion of the phosphorus stereochemistry.²⁷ Moreover, considering that a boronation reaction proceeds with the retention of phosphorus atom’s stereochemistry of a phosphite derivative,⁴¹ the (Rp)- and (Sp)-oxazaphospholidine monomers probably afforded the (Rp)- and (Sp)-boranophosphate linkages, respectively. Based on the HPLC profiles of the crude mixtures, it was also confirmed that the reaction proceeded with high stereoselectivity. The reaction course was previously analyzed in detail by ab initio calculations. Since a protonated oxazaphospholidine has a large LUMO orbital on the back side of the P—N bond according to a calculation,³¹ a nucleophile is expected to attack from the back side of the P—N bond, leading to the stereospecific condensation. Moreover, it was revealed that after a protonation of the nitrogen atom in the oxazaphospholidine ring by dialkyl(cyanomethyl)ammonium salt, its conjugate base promotes the C—N back side attack of a hydroxy group via the formation of a hydrogen bond.³⁸ In addition to this, since 2-cyanomethyl pyrrolidine is a virtually non-nucleophilic amine, it was experimentally proved that the presence of CMPT did not cause an epimerization of the phosphorus atom of the oxazaphospholidine.³¹ These factors contributed to the stereoselectivity of the condensation reaction. The isolated yields of (presumptive Rp) and (presumptive Sp)-23 were estimated from the UV absorbance of the hydroquinone moiety of the isolated products at 286 nm (ε = 2.22 × 10³ L mol^–1 cm^–1).³⁶

Table 2. Solid-Phase Synthesis of Disaccharide Boranophosphates (Presumptive Rp)-23 and (Presumptive Sp)-23.

entry	monomer	product	23:17:18a	Rp/Spa	isolated yield (%)b
1	(Rp)-11	(presumptive Rp)-23	95:2:3	99:1	35
2	(Sp)-11	(presumptive Sp)-23	87:5:8	1:99	29

^aDetermined by RP-HPLC.

^bEstimated from UV absorption at 286 nm after RP-HPLC purification.

Given the successful synthesis of the disaccharide 1-boranophosphate structures, the synthesis of tetrasaccharide derivatives bearing two boranophosphate linkages was investigated according to the scheme shown in Table 3. The synthesis of the tetrasaccharide derivative was first attempted by two repeated cycles that included the condensation of (Rp)-11 in the presence of CMPT, a boronation step using BH₃·SMe₂, and the removal of the MMTr group, followed by treatment with a DMAN solution (1 M) and a 40% MeNH₂ aqueous solution. The analysis of the crude reaction mixture by RP-HPLC indicated a significant formation of the disaccharide 23 that resulted in low HPLC yield of the desired tetrasaccharide (presumptive Rp)-24 (entry 1, Figure S4), probably due to the inhibition of the subsequent condensation reaction by BH₃·SMe₂ and/or its residue(s). Examining the reaction conditions, it was found that, to improve the coupling performance, the BH₃·SMe₂ concentration should be reduced, while the solid support should be washed with EtOH after the boronation step (entry 2). However, the coupling yield of the second condensation reaction was not satisfactory as indicated by the formation of the disaccharide 1-boranophosphate 23 in 13%. This could be attributed to the lower nucleophilicity of the sugar hydroxy group compared to that of the hydroxy group of the spacer. The repetition of the coupling reaction for a second time was effective in compensating for the low reactivity (entry 3). Moreover, a hexasaccharide derivative bearing three Rp boranophosphate linkages ((presumptive Rp)-25) was successfully obtained as a main product based on the same synthetic process (Table 3 entry 4, Figure 2). The relatively low isolated yields of the synthesized compounds can be attributed to the loss during the HPLC purification.

Table 3. Solid-Phase Synthesis of the Repeating Units Having Multiple Boranophosphate Linkages.

entry	monomer	BH3·SMe2 concentration [M]	washing solvents	product	24:23:17:18a	isolated yield (%)b
1	(Rp)-11	1.1	MeCN and CH2Cl2	(presumptive Rp)-24	28:60:5:7	-c
2	(Rp)-11	0.05	MeCN, EtOH, and CH2Cl2	(presumptive Rp)-24	76:13:3:8	-c
3d	(Rp)-11	0.05	MeCN, EtOH, and CH2Cl2	(presumptive Rp)-24	84:6:2:7	26
4e	(Rp)-11	0.05	MeCN, EtOH, and CH2Cl2	(presumptive Rp)-25	-c	23

^aDetermined by RP-HPLC.

^bEstimated from the UV absorption at 286 nm after RP-HPLC purification.

^cNot determined.

^dThe second condensation reaction was conducted twice.

^eThe second and third condensation reactions were conducted twice.

Figure 2.

RP-HPLC profiles of the crude mixture of (presumptive Rp)-25 with detection at 286 nm. RP-HPLC was performed with a linear gradient of 0–15% MeCN in 0.1 M ammonium acetate (AA) buffer (pH 7.0) at 30 °C for 60 min with a flow rate of 0.5 mL/min using a C18 column. (Presumptive Rp)-25 was eluted at 31 min.

Solid-Phase Synthesis of Glycosyl Phosphorothioates

The stereocontrolled synthesis of another P-modified glycosyl analog, a glycosyl phosphorothioate, was also investigated. The synthesis of the disaccharide 1-phosphorothioates 26 was carried out in five steps. In particular, a condensation reaction was initially performed, followed by the capping of the amino group of the chiral auxiliary by 9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu). Next, the sulfurization of the resultant phosphite was achieved using 3-phenyl 1,2,4-dithiazoline-5-one (POS), and the chiral auxiliary was cleaved upon treatment with DBU. Finally, the cleavage of the linker and the debenzoylation reaction were conducted simultaneously using an aqueous MeNH₂ solution (Scheme 5). Since POS affords an isocyanate residue during its reaction with a phosphite,⁴² the amino group of the chiral auxiliary would react with the residue. Thus, the capping step was crucial to prevent a side reaction between the amino group and the residue. The HPLC profiles of the crude mixtures indicated the predominant formation of the desired products (Figure 3). The obtained disaccharide 1-phosphorothioates, derived from the (Rp)- and (Sp)-oxazaphospholidine monomer, were co-injected in RP-HPLC and found to have different retention times, indicating that these were diastereomers (Figure S6). Judging from RP-HPLC profiles of the crude mixtures, the stereoselectivities of the synthesis using each of the (Rp)- and (Sp)-oxazaphospholidine monomer were estimated to be both >99:1. Also, the ¹H and ³¹P NMR spectra of products were measured, and ¹H NMR spectra indicated that the both had high stereopurities since there were some separated signals (Figure S12). We have earlier reported that the (Rp)- and (Sp)-oxazaphospholidine monomers can generate oligonucleotides bearing (Sp)- and (Rp)-phosphorothioate linkages, respectively, as proved by nuclease digestion tests.³¹ Thus, it was expected that the same applies to the disaccharide 1-phosphorothioates, namely, (presumptive Sp)- and (presumptive Rp)-26 were obtained from (Rp)- and (Sp)-11, respectively. Isolated yields of (presumptive Sp-) and (presumptive Rp)-26 were 27 and 30%. It should also be noted that the same spatial orientation of four substituents around a phosphorus atom leads to the opposite stereochemistry assignment for the boranophosphate and phosphorothioate derivatives since the assignment priority order is B < O < S.⁴³ Although, in this study, only the synthesis of a disaccharide is presented, it is expected that oligosaccharides with multiple phosphorothioate linkages could be prepared by repeating the cycles consisting of the condensation, capping of the amino group, sulfurization, and detritylation steps.

Scheme 5. Solid-Phase Synthesis of Glycosyl Phosphorothioates (Presumptive Sp)-26 and (Presumptive Rp)-26.

Figure 3.

RP-HPLC profiles of the crude mixture of (a) (presumptive Sp)-26 and (b) (presumptive Rp)-26 with detection at 286 nm. RP-HPLC was performed using a linear gradient of 0–15% MeCN in 0.1 M AA buffer (pH 7.0) at 30 °C for 60 min with a flow rate of 0.5 mL/min using a C18 column.

Chemical Stability of Glycosyl Boranophosphates

Regarding the chemical stability of the synthesized glycosyl boranophosphates, a partial decomposition of (presumptive Rp)-23 into the H-phosphonate 18 was observed. (Presumptive Rp)-23 was stored as a crude mixture after the synthesis at −30 °C for 1 year (Figure S7). In contrast, no sign of decomposition of (presumptive Sp)-23 was identified even after storage at −30 °C for 1 year as a crude mixture, which was the same condition as for (Rp)-23 (Figure S8). This phenomenon indicated that the (presumptive Sp)-glycosyl boranophosphate is more stable than the (presumptive Rp) counterpart. To elucidate the effect of the storage buffer on the stability of a glycosyl boranophosphate, an acceleration test was also conducted. In particular, the purified tetrasaccharide (presumptive Rp)-24 was dissolved in a 0.1 M ammonium acetate (AA) or triethylammonium acetate (TEAA) buffer at pH 7 and stored at 40 °C for 8 days. The solutions were then analyzed by RP-HPLC (Figure S9). The profile of the sample stored in the AA buffer solution showed a partial decomposition of (presumptive Rp)-24, whereas that of the TEAA buffer counterpart showed almost no sign of degradation. Thus, it was indicated that the chemical stability of the glycosyl boranophosphate is highly dependent on the counter cation. Notably, this is the first example to indicate the relative chemical stability of the diastereomers of a glycosyl boranophosphate.

Conclusions

In this study, an efficient solid-phase approach was developed for the synthesis of P-modified glycosyl phosphate derivatives (boranophosphate and phosphorothioate) with high stereoselectivity using the oxazaphospholidine monomers. For the synthesis of the glycosyl boranophosphates, the selection of the base was crucial for the cleavage of the chiral auxiliary without side reactions. In addition, it was revealed that washing with EtOH to eliminate the boronation reagent and/or its residues was an essential step to achieve high condensation efficiency. The phosphorothioate counterparts were successfully synthesized with an appropriate capping of the amino group of the chiral auxiliary to prevent side reactions with the residue(s) of a sulfurization reagent.

Moreover, as the solid-phase synthesis does not require tedious workup and purification processes, the developed method is expected to promote the provision of useful probes for the elucidation of biological processes and drug candidates. To that end, the biological properties of the synthesized stereodefined glycosyl phosphate analogs are currently under investigation. Yet, a direct determination of stereochemistry is a priority concern since stereochemical assignments rely on the previous studies on a stereochemical course. We currently deal with this matter and will report the details in due course.

Experimental Section

General Information

All the reactions were conducted under an Ar atmosphere. Dry organic solvents were prepared by the appropriate relevant procedures. The ¹H NMR spectra were recorded at 400 MHz with tetramethylsilane (δ 0.0 ppm) as the internal standard in CDCl₃ or CD₃CN or with ammonium acetate (δ 1.72 ppm)⁴⁴ as an internal standard in D₂O. The ¹³C NMR spectra were recorded at 100 MHz in CDCl₃ or CD₃CN, which was used as the internal standard at δ 77.0 and 1.3 ppm, respectively. COSY, HMQC, and HMBC were recorded on a 400 MHz spectrometer. The ³¹P NMR spectra were recorded at 162 MHz with H₃PO₄ (δ 0.0) as the external standard in CDCl₃ or D₂O. IR spectra were obtained using an ATR-IR spectrometer. Optical rotations were measured on a polarimeter and given in units of (deg·mL)/(g·dm). Analytical TLC was performed on commercial glass plated 0.25 mm thickness silica gel layer. Silica gel column chromatography was performed using spherical, neutral, 63–210 μm silica gel unless otherwise noted. The solid-phase synthesis was carried out manually using a glass filter (10 × 50 mm) with a stopper at the top and a stopcock at the bottom as a reaction vessel, and compounds synthesized by this method were analyzed and purified by RP-HPLC and identified by ESI MS. The detections in RP-HPLC were achieved at 286 nm at a temperature of 30 °C and a flow rate of 0.5 mL/min using a C18 column (100 Å, 3.9 × 150 mm) unless otherwise noted. The isolated yields of the synthesized oligomers were estimated by UV–vis spectroscopy using a molar absorption constant of the spacer at 286 nm (ε = 2.22 × 10³ L mol^–1 cm^–1). The quantities of the products obtained by solid-phase synthesis were too small to acquire characterization data other than HRMS.

2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-α-d-mannopyranose (2)

To a solution of d-mannose (1.80 g, 10 mmol) in dry pyridine (100 mL) was added 4-methoxytrityl chloride (MMTrCl) (4.33 g, 14 mmol), and the mixture was allowed to stir at rt for 21 h. The reaction was quenched with MeOH (10 mL), and the mixture was concentrated under reduced pressure. The obtained residue was dissolved in dry pyridine (50 mL) followed by the addition of BzCl (9.3 mL, 80 mmol). The resultant mixture was allowed to stir at rt for 3 h. The reaction was then quenched with MeOH (10 mL), and the mixture was concentrated under reduced pressure. CH₂Cl₂ (100 mL) was added to the residue, and the organic layer was washed with a saturated NaHCO₃ aqueous solution (3 × 100 mL). The aqueous layers were then combined and extracted with CH₂Cl₂ (2 × 50 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was roughly purified by silica gel column chromatography (neutral silica, 300 g) using hexane–AcOEt (5:1–3:1, v/v) as an eluent. The obtained crude mixture was dissolved in dry THF (50 mL), a solution of 2.14 M MeNH₂ in THF–MeOH (1:3.6, v/v, 51.2 mL, 110 mmol) was added dropwise to the solution over 10 min at −30 °C, and the mixture was allowed to stir at the same temperature for 23 h. The mixture was then diluted with toluene (50 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (neutral silica, 250 g) using hexane–AcOEt (5:1–4:1, v/v) as an eluent to yield 2 as colorless foam (5.28 g, 6.90 mmol, 69% yield, containing 7% β-isomer).

[α]_D²⁴ = −123.7 (c 0.53, CHCl₃); IR (neat, cm^–1) 3447, 1726, 1602, 1509, 1492, 1315, 1250, 1093, 1068, 1026, 1001, 901, 830, 794, 766, 707, 632; ¹H NMR (400 MHz, CD₃CN) δ 8.15 (dd, J = 8.3, 1.3 Hz, 2H, Ar), 7.82–7.76 (m, 4H, Ar), 7.74–7.69 (m, 1H, Ar), 7.59–7.43 (m, 8H, Ar), 7.40–7.30 (m, 4H, Ar), 7.25 (dd, J = 8.9, 2.6 Hz, 2H, Ar), 7.22–7.14 (m, 6H, Ar), 6.64 (ddd, J = 8.9, 2.7, 2.4 Hz, 2H, Ar), 6.23 (t, J = 10.3 Hz, 1H, H-4), 5.73 (dd, J = 10.3, 3.2 Hz, 1H, H-3), 5.65 (dd, J = 3.2, 1.8 Hz, 1H, H-2), 5.47–5.45 (br, 1H, H-1), 4.97 (d, J = 4.1 Hz, 1H, -OH), 4.47 (dt, 10.1 Hz, 2.5 Hz, 1H, H-5), 3.63 (s, 3H, −OCH₃), 3.39 (dd, J = 10.5, 2.1 Hz, 1H, H-6), 3.13 (dd, J = 10.5, 3.2 Hz, 1H, H-6′).

¹³C {¹H}NMR (100 MHz, CD₃CN) δ 166.4, 166.3, 166.0 (C(4), C=O), 159.5, 145.9, 145.2, 136.0 (C(4), Ar), 134.6, 134.3, 134.3, 131.3 (CH, Ar), 130.7 (C(4), Ar), 130.4, 130.3 (CH, Ar), 130.3 (C(4), Ar), 130.2, 129.9, 129.4, 129.4, 129.2, 129.0, 128.7, 128.7, 127.8, 127.8, 113.8 (CH, Ar), 92.9 (C-1, ¹J_CH = 172 Hz), 86.8 (C(4), OCAr₃), 72.3 (C-2), 72.0 (C-3), 70.5 (C-5), 67.5 (C-4), 62.6 (C-6), 55.7 (−OCH₃).

HRMS(ESI-TOF) m/z: [M + Na]⁺ calcd for C₄₇H₄₀NaO₁₀⁺, 787.2514; found 787.2518.

(Rp)-2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-α-d-mannopyranosyl Oxazaphospholidine Monomer [(Rp)-4]

Compound 2 (0.76 g, 1.0 mmol) was dried by repeated co-evaporations with dry pyridine and dry toluene and was added in dry THF (7.0 mL) and Et₃N (0.97 mL, 7.0 mmol). To the resulting solution, a 1 M solution of the 2-chloro-1,3,2-oxazaphospholidine derivative L-3³¹ in dry THF (3 mL, 3.0 mmol) was added dropwise over 5 min at −78 °C. The mixture was then warmed to rt and allowed to stir for 1 h. The mixture was diluted with CHCl₃ (200 mL) and washed with a saturated NaHCO₃ aqueous solution (3 × 100 mL), and the aqueous layers were combined and extracted with CHCl₃ (2 × 50 mL). The combined organic layers were subsequently dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography twice (NH silica gel, 20 g for the first time, 10 g for second time) using hexane–AcOEt (5:1, v/v) with Et₃N (4% for the first time and 1% for the second time) as an eluent to yield (Rp)-4 as colorless foam (0.52 g, 0.53 mmol, 53% yield).

[α]_D²⁵ = −79.9 (c 0.50, CHCl₃); IR (neat, cm^–1) 2925, 1727, 1603, 1510, 1451, 1315, 1250, 1179, 1093, 1069, 1026, 993, 954, 902, 831, 799, 754, 705, 632.

¹H NMR (400 MHz, CDCl₃), δ 8.22 (dd, J = 8.2, 1.1 Hz, 2H, Ar), 7.87 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 7.73 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 7.65–7.61 (m, 1H, Ar), 7.52–7.40 (m, 9H, Ar), 7.33–7.05 (m, 16H, Ar), 6.59 (ddd, J = 8.9, 3.1, 1.9 Hz, 2H, Ar), 6.32 (t, J = 10.3 Hz, 1H, H-4), 5.91–5.85 (m, 3H, H-1, H-3, 5-position of oxazaphospholidine), 5.70 (dd, J = 3.2, 2.1 Hz, 1H, H-2), 4.43 (ddd, J = 10.1, 2.4, 2.3 Hz, 1H, H-5), 4.08–4.01 (m, 1H, 4-position of oxazaphospholidine), 3.70–3.60 (m, 4H, −OCH₃, pyrrolidine ring), 3.41 (dd, J = 10.6, 1.9 Hz, 1H, H-6), 3.27–3.17 (m, 1H, pyrrolidine ring), 3.11 (dd, J = 10.5, 3.0 Hz, 1H, H-6′), 1.78–1.55 (m, 2H, pyrrolidine ring), 1.32–1.23 (m, 1H, pyrrolidine ring), 1.07–0.96 (m, 1H, pyrrolidine ring).

¹³C {¹H} NMR (100 MHz, CDCl₃) δ 165.6, 165.5, 164.8 (C(4), C=O), 158.2, 144.5, 143.9, 138.3 (d, ³J_PC = 3.9 Hz), 135.1 (C(4), Ar), 133.3, 132.9, 132.8, 130.3, 129.9, 129.7 (CH, Ar), 129.6 (C(4), Ar), 129.5 (CH, Ar), 129.4, 129.2 (C(4), Ar), 128.6, 128.4, 128.2, 128.2, 128.1, 127.6, 127.6, 127.4, 126.6, 126.5, 125.9, 125.5, 112.8 (CH, Ar), 91.4 (¹J_CH = 174 Hz, C-1) , 85.9 (C(4), OCAr₃), 83.6 (d, ²J_PC = 10.6 Hz, 5-position of oxazaphospholidine), 71.6 (C-2), 70.8 (C-3), 70.6 (C-5), 67.5 (d, ²J_PC = 3.9 Hz, 4-position of oxazaphospholidine), 66.4 (C-4), 61.3 (C-6), 54.9 (−OCH₃), 47.0 (d, ²J_PC = 34.7 Hz, pyrrolidine ring), 28.2 (pyrrolidine ring), 26.0 (d, ³J_PC = 2.9 Hz, pyrrolidine ring).

³¹P {¹H} NMR (162 MHz, CDCl₃) δ 146.1.

HRMS(ESI-TOF) m/z: [M + MeOH + H]⁺ calcd for C₅₉H₅₇NO₁₂P⁺, 1002.3613; found 1002.3613.

Benzoyl-O-(2,3,4-tri-O-benzoyl-6-O-TBDPS-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranoside (7)

Compound 5(35) (3.10 g, 3.54 mmol) and compound 6¹⁰ (1.76 g, 2.95 mmol) were dried by repeated co-evaporations with dry toluene and dissolved in dry CH₂Cl₂ (12.6 mL). Afterward, a solution of TMSOTf (0.16 mL, 0.89 mmol) in dry CH₂Cl₂ (5.0 mL) was added dropwise to the mixture over 5 min at 0 °C, and the mixture was allowed to stir for 1 h. The reaction was then quenched with Et₃N (1 mL), and the solution was washed with a saturated NaHCO₃ aqueous solution (3 × 50 mL). The aqueous layers were then combined and extracted with CH₂Cl₂ (2 × 10 mL), and the obtained organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (neutral silica, 200 g) using hexane–AcOEt (5:1–4:1, v/v) as an eluent to yield 7 as colorless foam (2.96 g, 2.26 mmol, 76% yield).

[α]_D²⁴ = +78.4 (c 0.52, CHCl₃); IR (neat, cm^–1) 1727, 1602, 1452, 1428, 1315, 1255, 1177, 1092, 1068, 1025, 965, 803, 742, 703, 612.

¹H NMR (400 MHz, CDCl₃), δ 8.12 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 8.02–7.91 (m, 6H, Ar), 7.84 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 7.77–7.72 (m, 4H, Ar), 7.66–7.49 (m, 9H, Ar), 7.44–7.36 (m, 6H, Ar), 7.34–7.15 (m, 11H, Ar), 7.04–6.99 (m, 1H, Ar), 6.92 (t, J = 7.8 Hz, 2H, Ar), 6.78 (t, J = 7.8 Hz, 2H, Ar), 6.47 (d, J = 2.1 Hz, 1H, Man-H-1), 5.94 (d, J = 3.2 Hz, 1H, Gal-H-4), 5.89 (dd, J = 9.6, 3.4 Hz, 1H, Man-H-3), 5.77 (dd, J = 3.3, 2.2 Hz, 1H, Man-H-2), 5.66 (dd, J = 10.4, 7.9 Hz, 1H, Gal-H-2), 5.48 (dd, J = 10.3, 3.2 Hz, 1H, Gal-H-3), 4.94 (d, J = 7.8 Hz, 1H, Gal-H-1), 4.63–4.51 (m, 3H, Man-H-4, 6, 6′), 4.17 (ddd, J = 9.8, 2.4, 2.3 Hz, 1H, Man-H-5), 3.73 (dd, J = 9.4, 5.3 Hz, 1H, Gal-H-5), 3.40–3.28 (m, 2H, Gal-H-6, 6′), 0.88 (s, 9H, C(CH₃)₃).

¹³C {¹H} NMR (100 MHz, CDCl₃) δ 165.6, 165.4, 165.0, 165.0, 164.9, 163.9 (C(4), C=O), 135.5, 135.4, 133.9, 133.5, 133.2, 133.1, 133.0 (CH, Ar), 132.7, 132.0 (C(4), Ar), 130.1, 129.9, 129.7, 129.7, 129.6, 129.6, 129.5, 129.3 (CH, Ar), 129.2, 129.1, 128.8 (C(4), Ar), 128.7 (CH, Ar), 128.6 (C(4), Ar), 128.5, 128.4, 128.4, 128.1, 128.1, 127.6, 127.4 (CH, Ar), 101.4 (Gal-C-1), 91.1 (Man-C-1), 73.3 (Gal-C-5), 72.8 (Man-C-4), 72.0 (Gal-C-3), 71.7 (Man-C-5), 70.2 (Gal-C-2), 69.8 (Man-C-3), 69.5 (Man-C-2), 66.9 (Gal-C-4), 61.9 (Man-C-6), 59.7 (Gal-C-6), 26.4 (−C(CH₃)₃), 18.7 (−C(CH₃)₃).

HRMS(ESI-TOF) m/z: [M + Na]⁺ calcd for C₇₇H₆₈O₁₈SiNa⁺, 1331.4067; found 1331.4051.

Benzoyl-O-(2,3,4-tri-O-benzoyl-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranoside (8)

Compound 7 (2.82 g, 2.16 mmol) was dissolved in dry THF (50 mL), and acetic acid (0.37 mL, 6.47 mmol) was added to the solution. A 1 M solution of TBAF in dry THF (6.45 mL, 6.45 mmol), which was dried over MS 4 Å overnight, was added dropwise to the mixture over 5 min at 0 °C. The mixture was then warmed to rt, allowed to stir for 4 h, diluted with toluene (100 mL), and washed with H₂O (5 × 100 mL). The combined aqueous layers were extracted with toluene (2 × 30 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (neutral silica, 150 g) using hexane–AcOEt (2:1, v/v) as an eluent to yield 8 as colorless foam (1.61 g, 1.50 mmol, 70% yield).

[α]_D²⁵ = +112.9 (c 0.55, CHCl₃); IR (neat, cm^–1) 3064, 1723, 1602, 1585, 1492, 1452, 1316, 1248, 1177, 1090, 1067, 1025, 964, 803, 755, 704, 617.

¹H NMR (400 MHz, CDCl₃) δ 8.13 (dd, J = 8.4, 1.3 Hz, 2H, Ar), δ 8.03–7.99 (m, 4H, Ar), 7.98–7.93 (m, 4H, Ar), 7.89 (dd, J = 8.2, 1.1 Hz, 2H, Ar), 7.74 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 7.67–7.59 (m, 4H, Ar), 7.54–7.33 (m, 11H, Ar), 7.26–7.17 (m, 6H, Ar), 6.52 (d, J = 2.3 Hz, 1H, Man-H-1), 6.01 (dd, J = 8.9, 3.4 Hz, 1H, Man-H-3), 5.84–5.79 (m, 2H, Man-H-2, Gal-H-2), 5.60 (d, J = 3.2 Hz, 1H, Gal-H-4), 5.41 (dd, J = 10.3, 3.4 Hz, 1H, Gal-H-3), 4.99 (d, J = 7.8 Hz, 1H, Gal-H-1), 4.69–4.59 (m, 2H, Man-H-4, 6), 4.50 (dd, J = 12.4, 3.0 Hz, 1H, Man-H-6′), 4.26 (dt, J = 9.6, 2.2 Hz, 1H, Man-H-5), 3.61 (t, J = 6.8 Hz, 1H, Gal-H-5), 3.19–3.12 (m, 1H, Gal-H-6), 3.10–3.02 (m, 1H, Gal-H-6, 6′), 2.27 (t, J = 7.2 Hz, 1H, −OH).

¹³C {¹H}NMR (100 MHz, CDCl₃) δ 166.5, 165.5, 165.3, 165.1, 165.1, 165.0, 163.9 (C(4), C=O), 133.9, 133.7, 133.6, 133.3, 133.3, 133.1, 130.1, 130.0, 129.7, 129.6, 129.5, 129.4 (CH, Ar), 129.4, 129.0 (C(4), Ar), 128.7, 128.6, 128.6 (CH, Ar), 128.5, 128.4 (C(4), Ar), 128.4, 128.3, 128.2 (Ar, CH), 101.4 (Gal-C-1), 91.2 (Man-C-1), 74.0 (Gal-C-5), 73.4 (Man-C-4), 71.8 (Gal-C-3), 71.5 (Man-C-5), 70.1, 70.1 (Man-C-3, Gal-C-2), 69.5 (Man-C-2), 68.4 (Gal-C-4), 61.9 (Man-C-6), 59.8 (Gal-C-6).

HRMS(ESI-TOF) m/z: [M + Na]⁺ calcd for C₆₁H₅₀O₁₈Na⁺, 1093.2889; found 1093.2866.

(2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-1,2,3,6-tetra-O-benzoyl-α-d-mannopyranoside (9)

Compound 8 (1.50 g, 1.40 mmol) was dried by repeated co-evaporations with dry pyridine and was dissolved in dry pyridine (14 mL). MMTrCl (1.30 g, 4.2 mmol) was added, and the mixture was allowed to stir at rt for 24 h. The reaction was then quenched with MeOH (10 mL) and concentrated under reduced pressure. The obtained residue was dissolved in CH₂Cl₂ (20 mL) and washed with a saturated NaHCO₃ aqueous solution (3 × 50 mL). The aqueous layers were combined and extracted with CH₂Cl₂ (2 × 10 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified twice by silica gel column chromatography (neutral, 100 g each) using hexane–AcOEt (3:2 for the first time, 3:1–3:2 for the second time, v/v) as an eluent to yield 9 as colorless foam (1.66 g, 1.23 mmol, 88% yield).

[α]_D²⁶ = +60.7 (c 0.52, CHCl₃); IR (neat, cm^–1) 3063, 1726, 1602, 1585, 1510, 1492, 1451, 1315, 1247, 1177, 1090, 1068, 1025, 967, 832, 800, 755, 704, 617.

¹H NMR (400 MHz, CDCl₃) δ 8.12 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 8.02–7.91 (m, 6H, Ar), 7.84 (dd, J = 8.5, 1.1 Hz, 2H, Ar), 7.74–7.71 (m, 4H, Ar), 7.66–7.49 (m, 6H, Ar), 7.42–7.14 (m, 17H, Ar), 7.08–7.05 (m, 6H, Ar), 6.99 (ddd, J = 8.7, 3.2, 2.0 Hz, 2H, Ar), 6.92 (t, J = 7.9 Hz, 2H, Ar), 6.52 (ddd, J = 6.9, 3.0, 2.1 Hz, 2H, Ar), 6.46 (d, J = 2.1 Hz, 1H, Man-H-1), 5.96 (d, J = 3.2, 1H, Gal-H-4), 5.86 (dd, J = 9.5, 3.3 Hz, 1H, Man-H-3), 5.80 (dd, J = 3.2, 2.3 Hz, 1H, Man-H-2), 5.63 (dd, J = 10.3, 8.0 Hz, 1H, Gal-H-2), 5.45 (dd, J = 10.4, 3.3 Hz, 1H, Gal-H-3), 4.91 (d, J = 8.0 Hz, 1H, Gal-H-1), 4.64–4.48 (m, 3H, Man-H-4, 6, 6′), 4.15 (ddd, J = 9.6, 2.4, 2.2 Hz, 1H, Man-H-5), 3.74 (dd, J = 9.2, 5.3 Hz, 1H, Gal-H-5), 3.62 (s, 3H, −OCH₃), 3.20 (dd, J = 8.8, 4.9 Hz, 1H, Gal-H-6), 3.07 (t, J = 9.2 Hz, 1H, Gal-H-6′).

¹³C {¹H} NMR (100 MHz, CDCl₃) δ 165.6, 165.4, 165.1, 165.0, 164.8, 163.9 (C(4), C=O), 158.3, 143.9, 143.0, 134.6 (C(4), Ar), 133.9, 133.5, 133.2, 133.1, 133.0, 133.0, 130.1, 129.8, 129.7, 129.6, 129.5, 129.4 (CH, Ar), 129.3, 129.2, 129.1128.8 (C(4), Ar), 128.6 (CH, Ar), 128.6 (C(4), Ar), 128.5, 128.4, 128.4, 128.2, 128.1, 128.1, 128.0, 127.6, 127.6126.8, 126.7, 112.9 (CH, Ar), 101.3 (Gal-C-1), 91.1 (Man-C-1), 86.4 (C(4), OCAr₃), 72.6 (Man-C-4), 72.4 (Gal-C-5), 72.0 (Gal-C-3), 71.8 (Man-C-5), 70.1 (Gal-C-2), 69.9 (Man-C-3), 69.4 (Man-C-2), 67.4 (Gal-C-4), 61.9 (Man-C-6), 59.5 (Gal-C-6), 54.9 (−OCH₃).

HRMS(ESI-TOF) m/z: [M + Na]⁺ calcd for C₈₁H₆₆O₁₉Na⁺, 1365.4091; found 1365.4108.

(2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranose (10)

Compound 9 (1.53 g, 1.14 mmol) was dissolved in dry THF (5.2 mL), and a 2 M solution of MeNH₂ in a THF–MeOH mixture (4:1, v/v, 6.25 mL, 12.5 mmol) was added dropwise to the solution over 5 min at −30 °C to the solution. The mixture was stirred for 20 h at the same temperature and then diluted with toluene (40 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (NH silica gel, 75 g) using hexane–AcOEt (1:1–0:1, v/v) as an eluent to yield 10 as colorless foam (1.25 g, 1.01 mmol, 88% yield, containing 7% β-d-Man isomer).

[α]_D²⁶ = +37.7 (c 0.50, CHCl₃); IR (neat, cm^–1) 3445, 1725, 1602, 1510, 1451, 1315, 1250, 1177, 1092, 1068, 1026, 832, 798, 768, 705, 617.

¹H NMR (400 MHz, CD₃CN) δ 8.00 (dd, J = 8.4, 1.3 Hz, 2H, Ar), 7.95–7.89 (m, 4H, Ar), 7.79 (dd, J = 8.2, 1.1 Hz, 2H, Ar), 7.71–7.58 (m, 7H, Ar), 7.50–7.07 (m, 23H, Ar), 6.96–6.87 (m, 4H, Ar), 6.53 (ddd, J = 8.8, 3.2, 2.2 Hz, 2H, Ar), 5.94 (d, J = 3.2 Hz, 1H, Gal-H-4), 5.63 (dd, J = 10.5, 3.4 Hz, 1H, Gal-H-3), 5.56 (dd, J = 9.7, 3.3 Hz, 1H, Man-H-3), 5.45–5.40 (m, 2H, Gal-H-2, -OH), 5.20 (dd, J = 4.4, 1.8 Hz, 1H, Man-H-1), 5.10 (d, J = 8.0 Hz, 1H, Gal-H-1), 4.85 (dd, J = 4.4, 0.9 Hz, 1H, Man-H-2), 4.61–4.48 (m, 3H, Man-H-4, 6, 6′), 4.19–4.15 (m, 1H, Man-H-5), 4.10 (dd, J = 9.4, 5.3 Hz, 1H, Gal-H-5), 3.61 (s, 3H, −OCH₃), 3.09 (dd, J = 8.5, 5.0 Hz, 1H, Gal-H-6), 2.76 (t, J = 9.0 Hz, 1H, Gal-H-6′).

¹³C {¹H} NMR (100 MHz, CD₃CN) δ 166.6, 166.2, 166.1, 166.0, 166.0, 165.9 (C(4), C=O), 159.5, 145.6, 144.3, 135.4 (C(4), Ar), 134.5, 134.4, 134.3, 134.0, 131.1 (CH, Ar), 130.9, 130.6 (C(4), Ar), 130.6, 130.5, 130.3, 130.3, 130.3, 130.2, 130.1 (CH, Ar), 129.9, 129.8 (C(4), Ar), 129.7, 129.6, 129.6, 129.5, 129.4, 129.2, 129.2, 128.8, 128.7, 128.7, 128.0, 127.8, 113.8 (CH, Ar), 101.6 (Gal-C-1), 92.4 (Man-C-1, ¹J_CH = 172 Hz), 87.1 (C(4), OCAr₃), 74.0 (Man-C-4), 73.1 (Gal-C-3), 72.7 (Gal-C-5), 72.0 (Gal-C-2), 71.4 (Man-C-2), 70.9 (Man-C-3), 70.4 (Man-C-5), 68.8 (Gal-C-4), 63.5 (Man-C-6), 60.3 (Gal-C-6), 55.7 (−OCH₃).

HRMS(ESI-TOF) m/z: [M + Na]⁺ calcd for C₇₄H₆₃O₁₈Na⁺, 1261.3828; found 1261.3839.

General Procedure for the Synthesis of the Disaccharide 1-Oxazaphospholidine Monomers (Rp)-11 and (Sp)-11

Compound 10 (1.28 g, 1.03 mmol for the synthesis of (Rp)-11 and 1.24 g, 1.00 mmol for the synthesis of (Sp)-11) was dried by repeated co-evaporations with dry pyridine and toluene followed by the addition of dry THF (6.9 mL for (Rp)-11 and 7.0 mL for (Sp)-11) and Et₃N (1.0 mL, 7.2 mmol for (Rp)-11 and 0.97 mL, 7.0 mmol for (Sp)-11). A 1 M solution of the 2-chloro-1,3,2-oxazaphospholidine derivative³¹ (L-3 for (Rp)-11 and D-3 for (Sp)-11) in dry THF (3.1 mL, 3.1 mmol for (Rp)-11 and 3.0 mL, 3.0 mmol for (Sp)-11) was then added dropwise to the solution over a specified time (5 min for (Rp)-11 and 10 min for (Sp)-11) at −78 °C, and the mixture was warmed to rt and stirred for 1 h. The obtained mixture was then diluted with CHCl₃ (50 mL) and washed with a saturated NaHCO₃ aqueous solution (3 × 50 mL). The combined aqueous layers were extracted with CHCl₃ (2 × 20 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Silica gel column chromatography (NH silica gel, 30 g each) using CH₂Cl₂–hexane (1:2, v/v) and 1% Et₃N as an eluent was repeated three times to yield (Rp)-(2,3,4-tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranosyl oxazaphospholidine monomer ((Rp)-11) as colorless foam (0.92 g, 0.64 mmol, 62% yield) and twice to yield (Sp)-(2,3,4-tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranosyl oxazaphospholidine monomer ((Sp)-11) as colorless foam (0.93 g, 0.65 mmol, 65% yield).

(Rp)-(2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranosyl Oxazaphospholidine Monomer ((Rp)-11)

[α]_D²⁶ = +30.3 (c 0.20, CHCl₃); IR (neat, cm^–1) 2934, 1727, 1602, 1510, 1451, 1315, 1252, 1177, 1093, 1069, 1026, 959, 831, 744, 706.

¹H NMR (400 MHz, CDCl₃) δ 7.97–7.90 (m, 6H, Ar), 7.79 (dd, J = 8.2, 1.1 Hz, 2H, Ar), 7.75–7.71 (m, 4H, Ar), 7.58–7.51 (m, 3H, Ar), 7.41–7.37 (m, 1H, Ar), 7.34–7.13 (m, 21H, Ar), 7.09–7.04 (m, 6H, Ar), 7.01–6.97 (m, 2H, Ar), 6.93 (t, J = 7.8 Hz, 2H, Ar), 6.55–6.50 (m, 2H, Ar), 5.95 (d, J = 2.7 Hz, 1H, Gal-H-4), 5.91 (d, J = 6.4 Hz, 1H, 5-position of oxazaphospholidine), 5.82 (dd, J = 9.6, 3.2 Hz, 1H, Man-H-3), 5.65–5.58 (m, 2H, Man-H-1, Gal-H-2), 5.57 (dd, J = 3.2, 2.3 Hz, 1H, Man-H-2), 5.45 (dd, J = 10.3, 3.4 Hz, 1H, Gal-H-3), 4.90 (d, J = 7.8 Hz, 1H, Gal-H-1), 4.59–4.47 (m, 3H, Man-H-4, 6, 6′), 4.28 (ddd, J = 9.6, 2.6, 2.2 Hz, 1H, Man-H-5), 3.95–3.87 (m, 1H, pyrrolidine, ring), 3.70 (dd, J = 9.3, 4.9 Hz, 1H, Gal-H-5), 3.61 (s, 3H, −OCH₃), 3.59–3.51 (m, 1H, pyrrolidine, ring), 3.20 (dd, J = 8.7, 4.6 Hz, 1H, Gal-H-6), 3.14–3.01 (m, 2H, Gal-H-6′, pyrrolidine ring), 1.63–1.55 (m, 2H, pyrrolidine ring), 1.20–1.12 (m, 1H, pyrrolidine ring), 0.98–0.88 (m, 1H, pyrrolidine ring).

¹³C {¹H} NMR (100 MHz, CDCl₃) δ 165.7, 165.5, 165.1, 164.8 (C(4), C=O), 158.3, 143.9, 143.0, 138.2 (d, ³J_PC = 3.9 Hz), 134.6 (Ar, C(4)), 133.2, 133.1. 133.0, 133.0, 132.8, 130.1, 129.9 (Ar, CH), 129.8 (Ar, C(4)), 129.7, 129.6, 129.5, 129.4 (Ar, CH), 129.3, 128.8, 128.7 (Ar, C(4)), 128.5, 128.4, 128.4, 128.3, , 128.1, 127.7, 127.6, 127.4, 126.8, 126.7, 126.6, 126.0, 125.5, 112.9 (Ar, CH), 101.1 (Gal-C-1), 91.4 (d, ¹J_CH = 172 Hz, ²J_PC = 5.8 Hz, Man-C-1), 86.4 (C(4), OCAr₃), 83.0 (d, ²J_PC = 9.6 Hz, 5-position of oxazaphospholidine), 73.0 (Man-C-4), 72.3 (Gal-C-5), 72.0 (Gal-C-3), 71.2 (Man-C-2), 70.3 (Gal-C-2), 70.1 (Man-C-5), 70.0 (Man-C-3), 67.4, 67.4 (4-position of oxazaphospholidine, Gal-C-4), 62.3 (Man-C-6), 59.5 (Gal-C-6), 55.0 (−OCH₃), 46.9 (d, ²J_PC = 35.6 Hz, pyrrolidine ring), 28.1 (pyrrolidine ring), 25.8 (d, ³J_PC = 2.9 Hz, pyrrolidine ring).

³¹P {¹H} NMR (162 MHz, CDCl₃), δ 149.6.

HRMS(ESI-TOF) m/z: [M + H]⁺ calcd for C₈₅H₇₅NO₁₉P⁺, 1444.4665; found 1444.4664.

(Sp)-(2,3,4-Tri-O-benzoyl-6-O-(4-methoxytrityl)-β-d-galactopyranosyl)-(1→4)-O-2,3,6-tri-O-benzoyl-α-d-mannopyranosyl Oxazaphospholidine Monomer ((Sp)-11)

[α]_D¹⁴ = +58.7 (c 0.22, CHCl₃); IR (neat, cm^–1) 2939, 1726, 1603, 1510, 1451, 1315, 1251, 1177, 1092, 1068, 1026, 830, 745, 705.

¹H NMR (400 MHz, CDCl₃) δ 7.96–7.92 (m, 4H, Ar), 7.82–7.77 (m, 4H, Ar), 7.75–7.70 (m, 4H, Ar), 7.61–7.53 (m, 3H, Ar), 7.41–7.05 (m, 28H, Ar), 6.98 (ddd, J = 8.7, 3.0, 1.9 Hz, 2H, Ar), 6.91 (t, J = 7.8 Hz, 2H, Ar), 6.53 (ddd, J = 8.7, 3.2, 1.9 Hz, 2H, Ar), 5.94 (d, J = 3.2 Hz, 1H, Gal-H-4), 5.89 (d, J = 6.4 Hz, 1H, 5-position of oxazaphospholidine), 5.81 (dd, J = 9.6, 3.7 Hz, 1H, Man-H-3), 5.62–5.55 (m, 3H, Man-H-1, H-2, Gal-H-2), 5.45 (dd, J = 10.5, 3.2 Hz, 1H, Gal-H-3), 4.84 (d, J = 7.8 Hz, 1H, Gal-H-1), 4.49–4.39 (m, 3H, Man-H4, H-6, H-6′), 4.18 (ddd, J = 9.8, 3.0, 2.3 Hz, 1H, Man-H-5), 4.04–3.96 (m, 1H, pyrrolidine ring), 3.69 (dd, J = 9.2, 5.5 Hz, 1H, Gal-H-5), 3.61 (s, 3H, −OCH₃), 3.59–3.50 (m, 1H, pyrrolidine ring), 3.24–3.12 (m, 2H, pyrrolidine ring, Gal-H-6), 3.01 (t, J = 9.2 Hz, 1H, Gal-H-6′), 1.67–1.54 (m, 2H, pyrrolidine ring), 1.27–1.16 (m, 1H, pyrrolidine ring), 1.00–0.89 (m, 1H, pyrrolidine ring).

¹³C {¹H} NMR (100 MHz, CDCl₃) δ 165.7, 165.4, 165.2, 165.0, 164.8 (C(4), C=O), 158.3, 143.9, 143.1, 138.2 (d, ³J_PC = 3.9 Hz), 134.6 (C(4), Ar), 133.3, 133.0, 132.9, 132.8, 130.1, 129.9 (CH, Ar), 129.8 (C(4), Ar), 129.7, 129.6, 129.5, 129.4 (CH, Ar), 129.3, 128.9, 128.6 (C(4), Ar), 128.5, 128.3, 128.1, 128.1, 128.1, 127.7, 127.6, 127.4, 126.8, 126.7, 126.0, 125.5, 112.9 (CH, Ar), 101.4 (Gal-C-1), 92.2 (d, ¹J_CH = 172 Hz, ²J_PC = 13.5 Hz, Man-C-1), 86.4 (C(4), OCAr₃), 82.7 (d, ²J_PC = 9.6 Hz, 5-position of oxazaphospholidine), 73.4 (Man-C-4), 72.3 (Gal-C-5), 71.9 (Gal-C-3), 71.3 (d, ³J_PC = 7.7 Hz, Man-C-2), 70.2, 70.1 (Man-C-5, Gal-C-2), 69.8 (Man-C-3), 67.4 (Gal-C-4), 67.0 (d, ²J_PC = 3.9 Hz, 4-position of oxazaphospholidine), 62.5 (Man-C-6), 59.4 (Gal-C-6), 55.0 (−OCH₃), 46.9 (d, ²J_PC = 34.7 Hz, pyrrolidine ring), 28.1 (pyrrolidine ring), 26.0 (d, ³J_PC = 3.9 Hz, pyrrolidine ring).

³¹P {¹H} NMR (162 MHz, CDCl₃) δ 155.3.

HRMS(ESI-TOF) m/z: [M+ H]⁺ calcd for C₈₅H₇₅NO₁₉P⁺, 1444.4665; found 1444.4663.

General Procedure for the Synthesis of the α-Mannopyranosyl Boranophosphate Derivative 16 (Table 1)

The CPG-loaded O-MMTr protected hydroquinone spacer (22.43 μmol/g, 0.50 μmol for entries 1 and 2, 30.76 μmol/g, 0.68 μmol for entry 3), via a succinyl linker,³⁶ was treated in a reaction vessel with 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each) and washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL). Thereafter, it was dried in vacuo for 10 min. Then, the (Rp)-α-mannosyl oxazaphospholidine monomer ((Rp)-4) (14.6 mg, 15 μmol), which was dried in vacuo overnight, was added to the reaction vessel and dried in vacuo for 3 min. A 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added under an Ar atmosphere to the reaction vessel. After 10 min, the CPG was washed with dry MeCN (3 × 1 mL) and dried in vacuo for 5 min. The resultant phosphite was boronated upon treatment with a 1.05 M solution of BH₃·SMe₂ (100 μL, 105 μmol) in dry MeCN (900 μL), and the reaction vessel was shaken for 15 min. Then, the CPG was washed with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL), and the detritylation reaction was carried out using 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). Subsequently, the CPG was washed with dry CH₂Cl₂ (4 × 1 mL) and dry MeCN (3 × 1 mL). For entry 2, a 1 M solution of DBU (150 μL, 1 mmol) in dry MeCN (850 μL) was added to the reaction vessel. For entry 3, a 1 M solution of DMAN (214 mg, 1 mmol) in dry MeCN (1.0 mL), which was dried over MS 3 Å overnight, was added to the reaction vessel. After a specified time (4 h for entry 2 and 1 h for entry 3), the CPG was washed with MeCN (3 × 1 mL). The CPG was then treated with a 40% MeNH₂ aqueous solution (4 mL) at rt for 4 h, filtered, and washed with EtOH. The filtrate and the washings were combined and concentrated under reduced pressure, and the obtained residue was analyzed by RP-HPLC, which was performed with a linear gradient of 0–20% MeCN for 60 min in a 0.1 M TEAA buffer (pH 7.0).

16: HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₁₆H₂₇BO₁₁P^–, 437.1390; found 437.1379.

Elucidation of a Mechanism for the Chiral Auxiliary Removal by DMAN

The CPG-loaded O-MMTr protected hydroquinone spacer (36.37 μmol/g, 2 μmol), via a succinyl linker,³⁶ was treated in a reaction vessel with 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each) and washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL). Thereafter, it was dried in vacuo for 10 min. Then, the (Rp)-α-mannosyl oxazaphospholidine monomer ((Rp)-4, 14.6 mg, 15 μmol), which was dried in vacuo overnight, was added to the reaction vessel and dried in vacuo for 3 min. A 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL each), which was dried over MS 3 Å overnight, was added under an Ar atmosphere to the reaction vessel twice. After 20 min, the CPG was washed with dry MeCN (3 × 1 mL) and dried in vacuo for 5 min. The resultant phosphite was boronated upon treatment with a 1.05 M solution of BH₃·SMe₂ (100 μL, 105 μmol) in dry MeCN (900 μL), and the reaction vessel was shaken for 15 min. Then, the CPG was washed with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL), and the detritylation reaction was carried out using 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). Subsequently, the CPG was washed with dry CH₂Cl₂ (4 × 1 mL) and dry MeCN (3 × 1 mL). A 1 M solution of DMAN (64.3 mg, 0.3 mmol) in dry MeCN (0.3 mL), which was dried over MS 3 Å overnight, was added to the reaction vessel. After 1 h, the solution was collected, then the CPG was washed with MeCN (3 × 1 mL). The solution and washings were combined and concentrated under reduced pressure. The residue was analyzed by ¹H NMR (Figures S10 and S11).

General Procedure for the Synthesis of the Disaccharide 1-Boranophosphate Derivatives (Presumptive Rp)-23 and (Presumptive Sp)-23 (Table 2)

The disaccharide 1-boranophosphate derivatives (presumptive Rp)-23 and (presumptive Sp)-23 were prepared following the synthetic procedure for compound 16 using the CPG-loaded O-MMTr-protected hydroquinone spacer (30.76 μmol/g, 0.68 μmol), via succinyl linker, and the disaccharide 1-oxazaphospholidine monomer ((Rp)-11 for the synthesis of (presumptive Rp)-23 or the (Sp)-11 for the synthesis of (presumptive Sp)-23, 21.7 mg, 15 μmol). In this case, DMAN was used for the suppression of the side reactions. The final obtained crude mixture of (presumptive Rp)- and (presumptive Sp)-23 was analyzed and purified by RP-HPLC with a linear gradient of 0–20% MeCN in a 0.1 M TEAA buffer (pH 7.0) for 60 min, and the purified (presumptive Rp)- and (presumptive Sp)-23 were analyzed under the same conditions. The purification was conducted with a part of the crude mixture (twenty-three over hundred-and-twenty-fifth for (presumptive Rp)-23; one-fifth for (presumptive Sp)-23)) and the quantity of the purified (presumptive Sp)-23 was estimated by the UV absorption at 286 nm. Isolated yield: 35% ((presumptive Rp)-23, 44 nmol) and 29% ((presumptive Sp)-23, 39 nmol).

(Presumptive Rp)-23: HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₂₂H₃₇BO₁₆P^–, 599.1918; found 599.1918.

(Presumptive Sp)-23: HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₂₂H₃₇BO₁₆P^–, 599.1918; found 599.1927.

Synthesis of a Tetrasaccharide Derivative Having Two Boranophosphate Linkages ((Presumptive Rp)-24) (Table 3, Entries 1–3)

The CPG-loaded O-MMTr protected hydroquinone spacer (30.76 μmol/g, 0.68 μmol for entries 1 and 2; 26.35 μmol/g, 0.50 μmol for entry 3), via succinyl linker, was treated in a reaction vessel with 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each) and washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL). Afterward, it was dried in vacuo for 10 min. The disaccharide 1-oxazaphospholidine monomer ((Rp)-11, 21.7 mg, 15 μmol), which was dried overnight in vacuo, was added, and the mixture was dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added to the reaction vessel under an Ar atmosphere. After 10 min, the CPG was washed with dry MeCN (3 × 1 mL) and dried in vacuo for 5 min. The resultant phosphite was boronated using either a 1.05 M solution of BH₃·SMe₂ (100 μL, 105 μmol) in dry MeCN (900 μL) for entry 1 or a 0.05 M solution of BH₃·SMe₂ (4.7 μL, 5 μmol) in dry MeCN (1 mL) for entries 2 and 3, and the reaction vessel was shaken for 15 min. Afterward, the CPG was washed with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL) for entry 1 or dry MeCN (3 × 1 mL), dry EtOH (3 × 1 mL), and dry CH₂Cl₂ (3 × 1 mL) for entries 2 and 3, and the detritylation was carried out using 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). Washing of the CPG with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL) and drying in vacuo for 10 min followed. Subsequently, the disaccharide 1-oxazaphospholidine monomer (Rp)-11 (21.7 mg, 15 μmol), which was dried overnight in vacuo, was added, and the mixture was dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added under an Ar atmosphere. After 10 min, the CPG was washed with MeCN (3 × 1 mL) and dried in vacuo for 5 min. For entry 3, the condensation step was repeated one more time. The resultant phosphite was then boronated upon treatment with a 1.05 M solution of BH₃·SMe₂ (100 μL, 105 μmol) in dry MeCN (900 μL, entry 1) or a 0.05 M solution of BH₃·SMe₂ (4.7 μL, 5 μmol) in dry MeCN (1 mL, entries 2 and 3), and the reaction vessel was shaken for 15 min. The obtained CPG was then washed either with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL) for entry 1 or with dry MeCN (3 × 1 mL), dry EtOH (3 × 1 mL), and dry CH₂Cl₂ (3 × 1 mL) for entries 2 and 3. The detritylation reaction was carried out using 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each), and the CPG was washed with dry CH₂Cl₂ (4 × 1 mL) and dry MeCN (3 × 1 mL). A 1 M solution of DMAN (214 mg, 1 mmol) in dry MeCN (1.0 mL), which was dried over MS 3 Å overnight, was then added to the reaction vessel, and after 1 h, the CPG was washed with MeCN (3 × 1 mL). The CPG was finally treated with a 40% MeNH₂ aqueous solution (4 mL) at 30 °C for 4 h, filtered, and washed with EtOH. The filtrate and the washings were combined and concentrated under reduced pressure. The residue was analyzed and purified by RP-HPLC, which was performed with a linear gradient of 0–15% MeCN for 60 min in a 0.1 M AA buffer (pH 7.0). The purification was conducted with one-fifth of the crude mixture, and the quantity of the purified (presumptive Rp)-24 was estimated by the UV absorption at 286 nm. Isolated yield: 26% ((presumptive Rp)-24, 26 nmol).

(Presumptive Rp)-24: HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₃₄H₆₁B₂O₂₈P₂^–, 1001.3016; found 1001.3026.

Synthesis of Hexasaccharide Derivatives Having Three Boranophosphate Linkages ((Presumptive Rp))-25) (Table 3, Entry 4)

The CPG-loaded O-MMTr protected hydroquinone spacer (26.35 μmol/g, 0.50 μmol) was treated in a reaction vessel with 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each), washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL), and dried in vacuo for 10 min. The disaccharide 1-oxazaphospholidine monomer ((Rp)-11, 21.7 mg, 15 μmol), which was dried overnight in vacuo, was then added to the reaction vessel and dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added to the reaction vessel under an Ar atmosphere. After 10 min, the CPG was washed with dry MeCN (3 × 1 mL) and dried in vacuo for 5 min. The resultant phosphite was boronated using a 0.05 M solution of BH₃·SMe₂ (4.7 μL, 5 μmol) in dry MeCN (1 mL), and the reaction vessel was shaken for 15 min. Afterward, the CPG was washed with dry MeCN (3 × 1 mL), dry EtOH (3 × 1 mL), and dry CH₂Cl₂ (3 × 1 mL), and the detritylation reaction was carried out with 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). The CPG was then washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL) and dried in vacuo for 10 min. The disaccharide 1-oxazaphospholidine monomer ((Rp)-11, 21.7 mg, 15 μmol), which was also dried overnight in vacuo, was added to the reaction vessel and dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added under an Ar atmosphere to the reaction vessel. After 10 min, the CPG was washed with MeCN (3 × 1 mL) and dried in vacuo for 5 min. The condensation step was repeated one more time. Subsequently, the resultant phosphite was boronated with a 0.05 M solution of BH₃·SMe₂ (4.7 μL, 5 μmol) in dry MeCN (1 mL), and the reaction vessel was shaken for 15 min. Afterward, the CPG was washed with dry MeCN (3 × 1 mL), dry EtOH (3 × 1 mL), and dry CH₂Cl₂ (3 × 1 mL), and the detritylation reaction was carried out with 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). Then, the CPG was washed with dry CH₂Cl₂ (4 × 1 mL), dry EtOH (3 × 1 mL), and dry MeCN (3 × 1 mL) and dried in vacuo for 10 min. The disaccharide 1-oxazaphospholidine monomer ((Rp)-11, 21.7 mg, 15 μmol), which was dried overnight in vacuo, was added to the reaction vessel and dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added under an Ar atmosphere. After 10 min, the CPG was washed with MeCN (3 × 1 mL) and dried in vacuo for 5 min. The condensation step was repeated one more time. The resultant phosphite was boronated using a 0.05 M solution of BH₃·SMe₂ (4.7 μL, 5 μmol) in dry MeCN (1 mL), and the reaction vessel was shaken for 15 min. Then, the CPG was washed with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL), and the detritylation reaction was carried out with 1% TFA in dry CH₂Cl₂–Et₃SiH (1:1, v/v) (4 × 15 s, 1 mL each). Afterward, the CPG was washed with dry CH₂Cl₂ (4 × 1 mL) and dry MeCN (3 × 1 mL). A 1 M solution of DMAN (214 mg, 1 mmol) in dry MeCN (1.0 mL), which was dried over MS 3 Å overnight, was added to the reaction vessel, and after 1 h, the CPG was washed with MeCN (3 × 1 mL). The CPG was finally treated with a 40% MeNH₂ aqueous solution (4 mL) at 30 °C for 4 h, filtered, and washed with EtOH. The filtrate and the washings were combined and concentrated under reduced pressure. The residue was analyzed and purified by RP-HPLC, which was performed with a linear gradient of 0–15% MeCN for 60 min in a 0.1 M AA buffer (pH 7.0). The purification was conducted with one-fifth of the crude mixture, and the quantity of the purified (presumptive Rp)-25 was estimated by the UV absorption at 286 nm. Isolated yield: 23% (23 nmol).

HRMS(ESI-TOF) m/z: [M – 3H]^3– calcd for C₄₆H₈₃B₃O₄₀P₃^3–, 467.1323; found 467.1311.

General Procedure for the Synthesis of Disaccharide 1-Phosphorothioate Derivatives (Presumptive Sp)- and (Presumptive Rp)-26 (Scheme 5)

The CPG-loaded O-MMTr protected hydroquinone spacer (26.35 μmol/g for the synthesis of (presumptive Sp)-26 and 20.20 μmol/g for the synthesis of (presumptive Rp)-26, 0.50 μmol), via a succinyl linker, was treated in a reaction vessel with 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each) and washed with dry CH₂Cl₂ (4 × 1 mL) and MeCN (3 × 1 mL). Afterward, it was dried in vacuo for 10 min. The disaccharide 1-oxazaphospholidine monomer ((Rp)-11 for the synthesis of (presumptive Sp)-26 or (presumptive Sp)-11 for the synthesis of (presumptive Rp)-26, 21.7 mg, 15 μmol), which was dried overnight in vacuo, was added to the reaction vessel and dried in vacuo for 3 min. Then, a 1 M solution of CMPT (39.0 mg, 150 μmol) in dry MeCN (150 μL), which was dried over MS 3 Å overnight, was added under an Ar atmosphere to the reaction vessel. After 10 min, the CPG was washed with dry MeCN (3 × 1 mL) and dried in vacuo for 5 min. To the reaction vessel, Fmoc-OSu (101 mg, 300 μmol), which was dried in vacuo overnight, and a 1 M solution of DMAN (64 mg, 300 μmol) in dry MeCN (300 μL) were added successively, and after 10 min, the CPG was washed with MeCN (3 × 1 mL). The phosphite linkage was sulfurized upon treatment with a 0.3 M solution of POS (11.7 mg, 60 μmol) in dry MeCN (0.2 mL) for 10 min. Afterward, the CPG was washed with dry MeCN (3 × 1 mL) and dry CH₂Cl₂ (3 × 1 mL). The detritylation was carried out using 1% TFA in dry CH₂Cl₂ (4 × 15 s, 1 mL each). The CPG was then washed with dry CH₂Cl₂ (4 × 1 mL) and dry MeCN (3 × 1 mL). A 1 M solution of DBU (150 μL, 1 mmol) in dry MeCN (850 μL), which was dried over MS 3 Å overnight, was added to the reaction vessel, and after 1 h, the CPG was washed with MeCN (3 × 1 mL). The CPG was ultimately treated with a 40% MeNH₂ aqueous solution (4 mL) at 30 °C for 4 h, filtered, and washed with EtOH. The filtrate and the washings were combined and concentrated under reduced pressure. The residue was analyzed and purified by RP-HPLC, which was performed using a linear gradient of 0–15% MeCN for 60 min in a 0.1 M AA buffer (pH 7.0). The purification was conducted with half of the crude mixture, and the quantity of the purified (presumptive Sp)-26 and (presumptive Rp)-26 was estimated by the UV absorption at 286 nm. Isolated yield: 27% ((presumptive Sp)-26, 67 nmol); 30% ((presumptive Rp)-26, 74 nmol).

(Presumptive Sp)-26: ¹H NMR (400 MHz, D₂O, only a part of signals was assigned) δ 7.02 (d, J = 2.3 Hz, 4H, Ar), 5.58 (dd, J = 2.1, 9.5 Hz, 1H, Man-1), 4.43 (d, J = 7.8 Hz, 1H, Gal-1), 4.24 (d, J = 4.6 Hz, 4H), 4.12 (t, J = 4.6 Hz, 2H), 4.02–3.71 (m, 12H), 3.66 (dd, J = 3.1, 10.2 Hz, 1H), 3.54 (dd, J = 7.8, 10.1 Hz, 1H, Gal-2).

³¹P NMR (162 MHz, D₂O) δ 54.9.

HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₂₂H₃₄O₁₆PS^–, 617.1311; found 617.1293.

(Presumptive Rp)-26: ¹H NMR (400 MHz, D₂O, only a part of signals was assigned) δ 7.04 (s, 4H, Ar), 5.62 (dd, J = 1.4, 10.3 Hz, 1H, Man-1), 4.45 (d, J = 7.8 Hz, 1H, Gal-1), 4.28–4.24 (m, 4H), 4.13 (t, J = 4.5 Hz, 2H), 4.06–3.89 (m, 7H), 3.85–3.72 (m, 5H), 3.67 (dd, J = 3.3, 10.0 Hz, 1H), 3.56 (dd, J = 7.4, 9.9 Hz, 1H, Gal-2).

³¹P NMR (162 MHz, D₂O) δ 55.2.

HRMS(ESI-TOF) m/z: [M – H]⁻ calcd for C₂₂H₃₄O₁₆PS^–, 617.1311; found 617.1310.

Evaluation of the Chemical Stability of (Presumptive Rp)-24

The purified (presumptive Rp)-24 (6.6 nmol) was dissolved in a 0.1 M AA buffer (pH 7, 200 μL) or a 0.1 M TEAA buffer (pH 7, 200 μL) and stored at 40 °C for 8 days. The solution was analyzed by RP-HPLC, which was performed with a linear gradient of 0–15% MeCN for 60 min in a 0.1 M AA buffer (pH 7.0).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03058.

• HPLC profiles and copies of ¹H, ¹³C, ³¹P NMR, COSY, HMQC, and HMBC spectra (PDF)

The authors declare no competing financial interest.