Abstract
Despite substantial progress in glycosyl donor design, the development of mild and stereoselective glycosylation methods remains a central challenge in carbohydrate chemistry. Inspired by the native enzymatic ADP-ribosylation process, in which NAD+ serves as the glycosyl donor and nicotinamide acts as the leaving group, we developed a pyridinium-based glycosyl donor that chemically mimics this biological transformation. The donor enables the α-selective formation of O-, S-, and N-glycosides under mild activation conditions. Mechanistic studies indicate that the product configuration is independent of that of the donor and is instead governed by thermodynamic control, favoring formation of the α-configured product. Modification of the protecting groups on the donor allows selective access to β-configured products. This pyridinium donor is fully orthogonal to conventional donors, such as glycosyl thioglycosides and o-alkynylbenzoates, enabling iterative assembly of complex oligosaccharides. This bioinspired platform provides a versatile and complementary approach for the stereoselective construction of both α-and β-glycosidic linkages.
Keywords: Glycosylation reaction, NAD+ , Glycosylpyridinium, Biomimetic donor, Stereoselective glycosylation
Glycans play crucial roles in biological processes and disease progression, yet the chemical synthesis of structurally defined carbohydrates remains a major challenge. Glycosylation outcomes, including yield and stereoselectivity, are governed by multiple parameters, among which the choice of glycosyl donor is particularly decisive. Over recent decades, diverse donors such as glycosyl halides, acetates, thioglycosides, , trichloroacetimidates, trifluoroacetimidates, phosphates and ortho-alkynylbenzoate, as well as other donor classes ,− have been developed, enabling access to a wide range of glycans (Figure A). However, these systems often require strong Lewis acids, expensive metal catalysts, or cryogenic conditions for activation. By contrast, enzymatic glycosylation proceeds efficiently and stereospecifically under mild conditions, but its application is constrained by enzyme availability and limited substrate scope. − Consequently, the development of mild, efficient, and stereoselective glycosylation strategies remains a central objective in synthetic carbohydrate chemistry.
1.

Previous work and the design of this work. (A) Reported donor and activation method. (B) Biological mechanism of ADP-ribosylation, a post-translational modification, using PARP as the enzyme and NAD+ as the donor. Red bold line: PARPs. Lower dash line: Hydrogen bond. Upper dash line: π- stacking. (C) This work. A novel glycosylpyridinium donor that mimics NAD+ was designed for glycosylation reaction using TBAB/ZnI2 as activators.
ADP-ribosylation (ADPr) is a conserved post-translational modification across all kingdoms of life, − catalyzed by ADP-ribosyltransferases (ARTs, also known as PARPs) using NAD+ as the glycosyl donor. During this process, NAD+ is split into ADP-ribose and nicotinamide, and ADP-ribose is added to an acceptor (typically an amino-acid residue on a protein or to another ADP-ribose unit in a growing poly(ADP-ribose) chain). − This biological transformation highlights a key mechanistic feature: NAD+ functions as a pyridinium-based enzymatic glycosyl donor. These observations prompted us to explore whether synthetic glycosylpyridinium species could serve as effective donors in organic synthesis. Although such species were first reported in 1910, their use as glycosyl donors remain rare. More recently, glycosylpyridinium salts have been applied as a reagents in photolysis reaction , and in glycosidases studies. − Pyridine derivatives are also widely employed as stereocontrolling additives in glycosylation. − Theoretically, the pyridinium moiety itself possesses favorable leaving-group properties. − While NAD+ has been used in the synthesis of ADP-ribosylated biomolecules, − broader development of pyridine-based leaving groups for chemical glycosylation has remained largely unexplored. We therefore envisioned that pyridine derivatives could be harnessed as leaving groups in a new class of glycosyl donors capable of promoting mild and efficient glycosylation in a biomimetic fashion. However, several challenges must be addressed: (a) the demanding chemical synthesis of glycosylpyridinium donors; (b) the need for mild activation methods compatible with the intrinsic lability of donor; and (c) the control of stereoselectivity, which remains a central issue in glycosylation chemistry.
Herein, we report a pyridinium-based glycosyl donor that mimics the reactivity of NAD+ in ADP-ribosylation (Figure C). This donor can be activated under mild conditions (TBAB/ZnI2) and exhibits a broad substrate scope, enabling the efficient synthesis of O-, S-, and N-glycosides from both pentose and hexose substrates. Notably, benzyl-protected donors provide moderate to excellent α-selectivity, whereas acyl protection at the C-2 affords β-glycosides exclusively. This strategy thus offers a practical and complementary platform for the stereoselective construction of complex glycoconjugates.
To test whether NAD+ derivatives could serve as general glycosyl donors, we first synthesized a per-benzylated ribosyl pyridinium donor d1, which bears a dimethylnicotinamide leaving group (Scheme A). The synthesis of another donor d5 was also presented here (Scheme B). d5 can be prepared not only using TMSOTf as an activator but also under milder conditions employing Sc(OTf)3.
1. Synthesis of Donor (A) d1 using TMSOTf as Activator and (B) d5 Using TMSOTf or Sc(OTf)3 as Activator.
Inspired by previous studies on the chemical synthesis of cADPr, in which NAD+ was converted to cADPr in one step using sodium bromide and triethylamine as additives, , we examined the glycosylation of a1 with d1 under similar conditions (Table , Entry 1). The reaction afforded desired product 1, albeit in low yield. Based on preliminary results, we proceeded to improve both the reaction efficiency and stereoselectivity (Table ). Replacement of triethylamine with DIPEA improved the yield to 24% with an α/β ratio of 2:1 (Entry 2). The addition of TBAB further increased the yield to 41% (Entry 3), whereas the inclusion of ZnI2 resulted in no obvious improvement (Entry 4). Because Entry 4 involved several additives, we next examined which components were essential. We found that a combination of TBAB (4 equiv) and ZnI2 (2 equiv), in the absence of NaBr and DIPEA, afforded the product in 49% yield with an improved α/β ratio of 6:1 (Entry 5). We then turned our attention to donor structure. Donors d1-d6 were synthesized (Table ). As the electron-withdrawing ability of the pyridinium substituent increased from d1 to d6, the leaving-group ability and the donor reactivity were progressively enhanced. At 35 °C, d1 was unreactive, d2 afforded only trace product, d3 gave 34% yield, and d4 delivered 76% yield (Entries 6–9). Donor d5 proved to be optimal (Entry 10). Further increasing the electron-withdrawing character (d6) decreased the donor stability and reduced the yield (entry 11). Additional screening revealed that replacing TBAB with TBAI or ZnI2 with ZnBr2 diminished both the yield and selectivity (Entries 13 and 14). Moreover, the omission of either TBAB or ZnI2 led to reduced efficiency and stereoselectivity (Entries 15 and 16), indicating that both additives are required. Other common activators were also examined: AgOTf and PPh3AuNTf2 provided the product in moderate yield (Entries 18 and 19). After systematic optimization, the optimal conditions were identified as follows: a1 (1 equiv), d5 (2 equiv), TBAB (4 equiv), and ZnI2 (2 equiv) in 1,2-dichloroethane at 35 °C for 12 h, affording product 1 in 90% isolated yield with an α/β ratio of 13:1 (Table , Entry 10).
1. Optimization of the Reaction Conditions .

| Entry | Donor | Additives (equivalents) | Solvent | Temp. | Yield | α/β |
|---|---|---|---|---|---|---|
| 1 | d1 | NaBr (10 equiv), Et3N (4 equiv) | ACN | 70 °C | Trace | - |
| 2 | d1 | NaBr (10 equiv), DIPEA (4 equiv) | ACN | 70 °C | 24% | 2/1 |
| 3 | d1 | NaBr (10 equiv), DIPEA (4 equiv), TBAB (4 equiv) | ACN | 70 °C | 41% | 3/1 |
| 4 | d1 | NaBr (10 equiv), DIPEA (4 equiv), TBAB (4 equiv), ZnI2 (2 equiv) | ACN | 70 °C | 42% | 3/1 |
| 5 | d1 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 70 °C | 49% | 6/1 |
| 6 | d1 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | N.D. | - |
| 7 | d2 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | Trace | - |
| 8 | d3 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | 34% | 5/1 |
| 9 | d4 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | 76% | 11/1 |
| 10 | d5 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | 93% (90%) | 13/1 |
| 11 | d6 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | 23% | 8/1 |
| 12 | d5 | TBAB (4 equiv), ZnI2 (2 equiv) | DCE | 70 °C | Trace | - |
| 13 | d5 | TBAI (4 equiv), ZnI2 (2 equiv) | DCE | 35 °C | 76% | 4/1 |
| 14 | d5 | TBAB (4 equiv), ZnBr2 (2 equiv) | DCE | 35 °C | 69% | 3/1 |
| 15 | d5 | ZnI2 (2 equiv) | DCE | 35 °C | 43% | 2/1 |
| 16 | d5 | TBAB (4 equiv) | DCE | 35 °C | 44% | 4/1 |
| 17 | d5 | TMSOTf (1 equiv) | DCE | 35 °C | N.D. | - |
| 18 | d5 | AgOTf (1 equiv) | DCE | 35 °C | 47% | 3/1 |
| 19 | d5 | PPh3AuNTf2 (1 equiv) | DCE | 35 °C | 35% | 3/1 |
Reaction conditions: donor (0.1 mmol), a1 (0.05 mmol), DCE (1 mL) and 3Å MS (100 mg), 35 °C, 12 h.
The yield was determined by 1H NMR analysis of the crude product using p-Anisaldehyde as an internal standard.
NMR yield was 93% and isolated yield was 90%.
Not detected target product.
Calculated by 1H NMR.
With the optimized conditions established, we explored the generality of this method (Figure ). We first investigated primary alcohols (Figure a). Ribosyl donors coupled smoothly with ribosyl acceptors to give disaccharides 1-3 in high yields with excellent α-selectivity. The successful preparation of 3 demonstrated compatibility with the widely used Yu donor. Adenosine-derived alcohols also participated efficiently to afford compounds 6-8 exclusively as the α-anomer. Amino acid–based alcohols underwent clean glycosylation to afford products 9-12, with compound 10 obtained solely as the α-anomer. A primary-alcohol linker, a valuable handle for glycoconjugate synthesis, was also well tolerated (4).
2.

Scope of ribose type donor. [a]Standard conditions: acceptor substrate (0.1 mmol), donor substrate (0.2 mmol), TBAB (0.4 mmol), ZnI2 (0.2 mmol), DCE (2 mL) and 3Å MS (200 mg), 35 °C for 12 h. All the yields are the isolated yields. For every product, the blue part was from the acceptor, and the black part was from the donor.
Unprotected acceptors reacted selectively at the primary hydroxyl group to furnish a α-selective product (13). Sulfur nucleophiles were next examined (Figure d). Cysteine derivatives furnished 27 in α-configuration, while thiophenols were converted smoothly into the well-known thioglycoside donors 26. Secondary alcohols were also suitable nucleophiles (Figure b). For example, estradiol benzoate was transformed readily (20), and glycosylated amino acids (14-16) were obtained with good α-selectivity.
The formation of N-glycosides is generally challenging due to the low nucleophilicity of nitrogen-based heterocycles, particularly purines. Using pyridinium donor d5 (Figure c), purine derivatives were successfully transformed into N-glycosides 18-22. Compound 18 and 22 were isolated as single α-anomers, while 19 and 20 were obtained in high yields with excellent α-selectivity. Other N-heterocycles also reacted smoothly to afford compounds 23-25 in 81–96% yield. Compound 24 was formed as an N1/N2 regioisomeric mixture in a 2:1 ratio, as determined by HMBC (Supporting Information, Figure S172), whereas 25 was obtained as an equimolar N1/N2 mixture (Supporting Information, Figure S177).
Taken together, these results show that a broad range of O-, S-, and N-glycosides can be accessed in high yields with good α-selectivity. The reaction displays excellent functional group tolerance, accommodating benzyl (1), nitro (5), halogen (19), allyl (9), benzoyl (8), and sensitive groups such as Fmoc (16), and Boc (10). Of note, the activation mode of this donor is compatible with other leaving groups, including thioglycosides (1) and ortho-alkynylbenzoates (3), enabling orthogonal and sequential glycosylation in oligosaccharide synthesis. The robustness of the reaction was further validated on gram scale: glycosylation performed on a 2.5 mmol scale furnished product 1 in 83% isolated yield (Figure ).
Beyond the ribosyl donor, per-benzylated pyridinium-based donors derived from glucose, mannose, galactose, xylose, and arabinose also underwent TBAB/ZnI2-promoted glycosylation smoothly (Figure ). The synthesis of these donors follows the same strategy as for the ribose donors, employing OAc as the anomeric leaving group and TMSOTf as the activator (Supporting Information, Scheme S1). Glucosyl donors (Figure a) afforded disaccharides 28-30 and amino acid conjugates 31-33 in moderate to good yields. Estradiol benzoate was glycosylated efficiently to give predominantly the α-isomer (35). Galactosyl donors (Figure c) reacted with carbohydrate and amino acid acceptors, providing 38-40 mainly as α-isomers. Mannosyl donors (Figure d) coupled with glucosyl and serine derivatives yielded 41-43 with moderate α-selectivity. Xylosyl donors (Figure e) exhibited broad compatibility, giving disaccharide 45 and thioglycoside 44 in moderate yields and mainly an α-configuration. Arabinosyl donors (Figure b) also performed well and produced disaccharide 37 in high yield and N-glycoside 36 with good α-selectivity.
3.

Scopes of glucose, galactose, mannose and xylose type donor. [a]Standard condition: acceptor substrate (0.1 mmol), donor substrate (0.2 mmol), TBAB (0.4 mmol), ZnI2 (0.2 mmol), DCE (2 mL) and 3Å MS (200 mg), 35 °C for 12 h. All yields are isolated yields. For every product, the blue part was from the acceptor, and the black part was from the donor.
The above section describes the construction of α-glycosidic linkages using benzyl-protected pyridinium donors. We next turned to acyl-protected pyridinium donors to target β-glycosides formation. Fully acyl-protected donors d14-d17 were examined in the glycosylation with acceptor a1 (Table ). d14, which carries the same nicotinamide leaving group as NAD+, was first tested under conditions reported for cADPr synthesis, employing sodium bromide and triethylamine at 70 °C. , No desired product was detected, instead, the major product was the corresponding orthoester 50 (Entry 1), a known side product associated with 2-O-acetyl protected donors, likely reflecting insufficient activation. , We then applied the TBAB/ZnI2 system that proved effective for benzyl-protected donors. Under these conditions, donor d15 reacted smoothly with a1 to furnish disaccharide 48 in 52% yield as a single β-anomer (Entry 4). 60 °C is the optimal temperature, both higher and lower temperatures resulted in reduced yields (Entries 2, 3 and 5). More reactive donor d16 further improved the efficiency, with 50 °C identified as optimal (Entry 7). Notably, benzoyl-protected donor d17 exhibited the highest reactivity, affording β-glycoside 49 in 93% yield at 50 °C (Entry 9). Overall, TBAB (4 equiv) and ZnI2 (2 equiv) at 50 °C for 12 h constitute the optimal conditions. Under these conditions, acyl-protected pyridinium donors provide product in moderate to high yields with complete β-selectivity (Entries 7 and 9).
2. Condition Screening of Acyl-Protected Donors .
| Entry | Donor | Additives (equivalents) | Temp. | Yield |
|---|---|---|---|---|
| 1 | d14 | NaBr (10 equiv), Et3N (4 equiv) | 70 °C | 39% (50) |
| 2 | d15 | TBAB (4 equiv), ZnI2 (2 equiv) | 35 °C | N.D. |
| 3 | d15 | TBAB (4 equiv), ZnI2 (2 equiv) | 50 °C | Trace (48) |
| 4 | d15 | TBAB (4 equiv), ZnI2 (2 equiv) | 60 °C | 52% (48) |
| 5 | d15 | TBAB (4 equiv), ZnI2 (2 equiv) | 70 °C | 45% (48) |
| 6 | d16 | TBAB (4 equiv), ZnI2 (2 equiv) | 35 °C | 30% (48) |
| 7 | d16 | TBAB (4 equiv), ZnI2 (2 equiv) | 50 °C | 67% (48) |
| 8 | d16 | TBAB (4 equiv), ZnI2 (2 equiv) | 60 °C | 45% (48) |
| 9 | d17 | TBAB (4 equiv), ZnI2 (2 equiv) | 50 °C | 93% (49) |
Reaction conditions: donor (0.1 mmol), a1 (0.05 mmol), DCE (1 mL) and 3Å MS (100 mg), 12 h.
Isolated yield.
solvent is ACN.
Not detected target product.
We next examined the substrate scope of acyl-protected pyridinium donors (Figure ). The ribosyl donor d17 underwent smooth glycosylation with various nucleophiles to afford the corresponding β-products. Coupling with p-toluenethiol afforded thioglycoside 52 in moderate yield, whereas reaction with an N-heterocycle provided nucleoside 53 in high yield. N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc) are ubiquitous structural motifs in biologically important glycoconjugates, including N- and O-linked glycoproteins, proteoglycans, peptidoglycans and are implicated in a wide range of biological processes. − Most naturally occurring 2-acetamido-2-deoxysugars are found as β-linked glycosides. Consequently, the synthesis of 1,2-trans-β-glycosides from 2-acetamido-2-deoxysugars has predominantly relied on glycosyl donors bearing neighboring-group-participating amino protecting groups, which are subsequently converted into acetyl substituents after glycosylation. , In contrast, the glycosyl donor developed in this work enables the direct construction of β-glycosidic linkages without modification of the native C2 acetamido group. In this context, the 2-N-acetyl-D-glucosamine donor d18 proved effective, affording nucleoside 56 and disaccharides 57 and 58. Similarly, 2-N-acetyl-D-galactosamine donor d19 reacted efficiently to afford disaccharides 59 and 60. The glucose donor d20 also reacted smoothly to afford disaccharides 62 and 63 in moderate yields. Except for thioglycoside 52, which was obtained as an α/β mixture, all products were isolated exclusively as the corresponding β-anomers, underscoring the excellent β-selectivity of this transformation. The successful synthesis of compounds 54 and 58 further demonstrates the compatibility of this protocol with thioglycoside and o-alkynylbenzoate substrates, highlighting its potential for orthogonal and sequential glycosylation. Although d17 reacted smoothly with a secondary amino-acid alcohol to give 51 in moderate yield, no reaction was observed with the secondary hydroxyl group of ribose (55). Likewise, d19 was unreactive toward the secondary hydroxyl group of glucose (61), likely due to steric congestion.
4.

Scope of acyl-protected donors. [a]Standard conditions: acceptor substrate (0.1 mmol), donor substrate (0.2 mmol), TBAB (0.4 mmol), ZnI2 (0.2 mmol), DCE (2 mL) and 3Å MS (200 mg), 50 °C for 12 h. All the yields are the isolated yield. [b] 90 °C. For every product, blue part was from acceptor, black part was from donor.
The pyridinium donor enables not only the selective construction of α-glycosidic bonds but also the exclusive formation of β-glycosidic linkages through the donor protecting group manipulation. Overall, this system demonstrates a broad substrate scope and operational robustness across diverse sugar scaffolds and nucleophiles.
To demonstrate the synthetic utility of this methodology, we performed downstream transformations of the glycosylation products. Conventional glycosyl donors remain intact under TBAB/ZnI2 activation conditions. As a result, the pyridinium donors display orthogonal reactivity and can be used in tandem glycosylation sequences. For example, the ortho-alkynylbenzoate donor 54, derived from pyridinium donor d17, reacted efficiently with estradiol benzoate s5 to deliver the β-configured disaccharide 64 in high yield (Scheme A). Similarly, the thioglycoside donor 58, synthesized from pyridinium donor d18, reacted directly with a glucose derivates s7 to furnish trisaccharide 65 as a single β-isomer (Scheme B). These results underscore the compatibility of pyridinium donors with other donor classes and their suitability for sequential and orthogonal glycosylation in the synthesis of complex glycoconjugates.
2. Further Transformation of Glycosylation Product .

a (A) Transformation of ortho-alkynylbenzoate donor 54. (B) Transformation of thioglycosides donor 58.
Apart from the substrate scope and product transformations, we were also interested in the reaction mechanism. To probe the impact of donor anomeric configuration on stereoselectivity, the α-anomer of donor d5 was subjected to glycosylation with acceptor a1 (Scheme a). The reaction afforded disaccharide 1 with an α/β ratio of 13:1, consistent with the results obtained by using the anomeric mixture of d5 under standard conditions (Table , entry 10). As the β-anomer of d5 could not be isolated, configurationally pure donor d4 was employed as a model substrate. Notably, both the α- and β-anomers of d4 delivered 1 with comparable α/β ratios of 9:1 (Scheme b, c). These results indicate that the stereoselectivity is largely independent of the donor anomeric configuration.
3. Mechanism Study .
a Glycosylation between a1 with (A) α-d5, (B) α-d4, (C) β-d4.
Furthermore, the reaction was monitored in situ by 19F NMR (Supporting Information, Figures S1 and S2). No signals attributable to covalent glycosyl triflate intermediates were observed. In addition, TBAB and ZnBr2 were introduced to probe the possible formation of glycosyl bromides (Supporting Information). However, no signals corresponding to previously reported glycosyl bromides were observed (Supporting Information, Figure S3). The absence of detectable intermediates does not preclude their participation in the reaction pathway, as any such species formed at room temperature are expected to be highly transient and, thus, not observable by NMR under these conditions. In the case of d18, an oxazoline intermediate was isolated and characterized by NMR and HRMS, consistent with previous reports (Supporting Information).
Ding et al. reported a ZnI2-mediated protocol for α-glycosidic bond formation, in which glycosyl iodide intermediates were confirmed by NMR and HRMS. , Guided by these precedents and our experimental observations, we propose a plausible mechanism (Figure ). For donor d5 (Figure A), under the action of TBAB and zinc iodide, leaving group MFPC departs from d5 to generate oxocarbenium intermediate Int-I. Subsequently, Int-I undergoes conversion to β-glycosyl iodide Int-II and α-glycosyl iodide Int-III though the SN1 mechanism. Int-II is more susceptible to nucleophilic attack by acceptor a1, driving the equilibrium toward Int-II. As a result, Int-II predominantly reacts with a1 through the SN2 mechanism to afford the α-glycoside 1-α, whereas the minor β-product arises from Int-III. During this process, the proton released from the acceptor is scavenged by methyl 5-fluoropyridine-3-carboxylate (MFPC). For the pyranose donor, d18 can react via two pathways. In path a, the C2 acetamido group of d18 attacks the anomeric carbon from the α-face while the leaving group PC departs from the opposite side, forming the oxazoline intermediate Int-V. Subsequently, Int-V undergoes nucleophilic attack by acceptor s6 to give the β-configured product 58. In path b, the leaving group of d18 is displaced by iodide via an SN2 mechanism to form α-configured iodide intermediate Int-VI, which then reacts with acceptor s6 through SN2 substitution to yield β-configured glycosylation product 58. For benzyl-protected donors, whether pyranose or furanose, the α-configured halide intermediate is more stable due to the anomeric effect. For benzyl-protected donors, stereoselectivity is mainly thermodynamically controlled, leading predominantly to α-products. In contrast, for acyl-protected donors, the stereochemical outcome is primarily governed by neighboring group participation at C2, resulting in β-products.
5.

Proposed mechanism.
In summary, we have developed a glycosyl donor system inspired by ADP-ribosylation. These pyridinium-based donors are readily activated under mild conditions (TBAB/ZnI2), enabling the efficient and α-selective formation of O-, S-, and N-glycosides across a broad range of nucleophiles. Notably, by switching acyl protecting groups on the donor this platform also allows highly selective construction of β-glycosidic linkages. The method proceeds without strong acids or costly transition-metal activators. Its mild activation profile enables orthogonal tandem glycosylation with classical donors, such as thioglycosides and o-alkynylbenzoates, thereby expanding the synthetic options available for complex oligosaccharide assembly. The key features of this approach include: (a) a rationally designed pyridinium scaffold; (b) operational simplicity and mild activation; (c) orthogonality to common glycosylation systems; and (d) broad substrate scope and good to moderate stereoselectivity. A current limitation of this method is the low reactivity for secondary carbohydrate alcohols. Future efforts will focus on structural optimization of the donor and reaction conditions to further enhance the scope and stereocontrol of this biomimetic glycosylation platform.
Supplementary Material
Acknowledgments
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No. XDB1360000. We gratefully acknowledge the support from National Natural Science Foundation of China (22207114, 22477143), Shanghai Pujiang Program (22PJ1415600) and Zhongshan Municipal Bureau of Science and Technology (CXTD2022012). We gratefully acknowledge Prof. Qingju Zhang from Jiangxi Normal University for valuable discussions that contributed to this work.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c00036.
Experimental procedures, Figures S1–S260, 1H, 13C, 2D NMR spectra and HRMS data of all new compounds (PDF)
∇.
These authors contributed equally: Li Tang, Yanrong Chen. Qiang Liu conceived this project. Li Tang, Yanrong Chen, Yangdi Zhang, Huiqin Zhong, Shuyan Jiang, Zhenrong Chen and Zongxing Yu performed the compound synthesis, purification and characterization. Li Tang, Yanrong Chen and Qiang Liu collaboratively wrote the paper. CRediT: Li Tang investigation, methodology.
The authors declare no competing financial interest.
References
- Singh Y., Geringer S. A., Demchenko A. V.. Synthesis and Glycosidation of Anomeric Halides: Evolution from Early Studies to Modern Methods of the 21st Century. Chem. Rev. 2022;122:11701–11758. doi: 10.1021/acs.chemrev.2c00029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niedballa U., Vorbrüggen H.. A general synthesis of pyrimidine nucleosides. Angew. Chem. 1970;9:461–462. doi: 10.1002/anie.197004612. [DOI] [PubMed] [Google Scholar]
- Lian G., Zhang X., Yu B.. Thioglycosides in Carbohydrate research. Carbohydr. Res. 2015;403:13–22. doi: 10.1016/j.carres.2014.06.009. [DOI] [PubMed] [Google Scholar]
- Ferrier R. J., Hay R. W., Vethaviyasar N.. A potentially versatile synthesis of glycosides. Carbohydr. Res. 1973;27:55–61. doi: 10.1016/S0008-6215(00)82424-6. [DOI] [Google Scholar]
- Schmidt R. R., Michel J.. Facile Synthesis of α- and β-O-Glycosyl Imidates; Preparation of Glycosides and Disaccharides. Angew. Chem. 1980;19:731–732. doi: 10.1002/anie.198007311. [DOI] [Google Scholar]
- Yu B., Tao H. C.. Glycosyl trifluoroacetimidates. Part 1: Preparation and application as new glycosyl donors. Tetrahedron Lett. 2001;42:2405–2407. doi: 10.1016/S0040-4039(01)00157-5. [DOI] [Google Scholar]
- Plante O. J., Palmacci E. R., Andrade R. B., Seeberger P. H.. Oligosaccharide synthesis with glycosyl phosphate and dithiophosphate triesters as glycosylating agents. J. Am. Chem. Soc. 2001;123:9545–9554. doi: 10.1021/ja016227r. [DOI] [PubMed] [Google Scholar]
- Li Y., Yang Y., Yu B.. An efficient glycosylation protocol with glycosyl ortho-alkynylbenzoates as donors under the catalysis of Ph3PAuOTf. Tetrahedron Lett. 2008;49:3604–3608. doi: 10.1016/j.tetlet.2008.04.017. [DOI] [Google Scholar]
- Das R., Mukhopadhyay B.. Chemical O-Glycosylations: An Overview. Chemistryopen. 2016;5:401–433. doi: 10.1002/open.201600043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deng L. F., Wang Y. W., Xu S. Y., Shen A., Zhu H. P., Zhang S. Y., Zhang X., Niu D. W.. Palladium catalysis enables cross-coupling-like SN2-glycosylation of phenols. Science. 2023;382:928–935. doi: 10.1126/science.adk1111. [DOI] [PubMed] [Google Scholar]
- Dang Q. D., Deng Y. H., Sun T. Y., Zhang Y., Li J., Zhang X., Wu Y. D., Niu D. W.. Catalytic glycosylation for minimally protected donors and acceptors. Nature. 2024;632:313. doi: 10.1038/s41586-024-07695-4. [DOI] [PubMed] [Google Scholar]
- Kahne D., Walker S., Cheng Y., Vanengen D.. Glycosylation of Unreactive Substrates. J. Am. Chem. Soc. 1989;111:6881–6882. doi: 10.1021/ja00199a081. [DOI] [Google Scholar]
- Zhang J., Luo Z. X., Wu X., Gao C. F., Wang P. Y., Chai J. Z., Liu M., Ye X. S., Xiong D. C.. Photosensitizer-free visible-light-promoted glycosylation enabled by 2-glycosyloxy tropone donors. Nat. Commun. 2023;14:8025. doi: 10.1038/s41467-023-43786-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McArthur J. B., Chen X.. Glycosyltransferase engineering for carbohydrate synthesis. Biochem. Soc. Trans. 2016;44:129–142. doi: 10.1042/BST20150200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li W. Q., McArthur J. B., Chen X.. Strategies for chemoenzymatic synthesis of carbohydrates. Carbohydr. Res. 2019;472:86–97. doi: 10.1016/j.carres.2018.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thiem J.. Applications of Enzymes in Synthetic Carbohydrate-Chemistry. FEMS Microbiol. Rev. 1995;16:193–211. doi: 10.1111/j.1574-6976.1995.tb00166.x. [DOI] [PubMed] [Google Scholar]
- Alcántara A. R., Pace V., Hoyos P., Sandoval M., Holzer W., Hernáiz M. J.. Chemoenzymatic Synthesis of Carbohydrates as Antidiabetic and Anticancer Drugs. Curr. Top. Med. Chem. 2014;14:2694–2711. doi: 10.2174/1568026614666141215151056. [DOI] [PubMed] [Google Scholar]
- Suskiewicz M. J., Prokhorova E., Rack J. G. M., Ahel I.. ADP-ribosylation from molecular mechanisms to therapeutic implications. Cell. 2023;186:4475–4495. doi: 10.1016/j.cell.2023.08.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen M. S., Chang P.. Insights into the biogenesis, function, and regulation of ADP-ribosylation. Nat. Chem. Biol. 2018;14:236–243. doi: 10.1038/nchembio.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schuller M., Ahel I.. Beyond protein modification: the rise of non-canonical ADP-ribosylation. Biochem. J. 2022;479:463–477. doi: 10.1042/BCJ20210280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lüscher B., Bütepage M., Eckei L., Krieg S., Verheugd P., Shilton B. H.. ADP-Ribosylation, a Multifaceted Posttranslational Modification Involved in the Control of Cell Physiology in Health and Disease. Chem. Rev. 2018;118:1092. doi: 10.1021/acs.chemrev.7b00122. [DOI] [PubMed] [Google Scholar]
- Alemasova E. E., Lavrik O. I.. Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins. Nucleic Acids Res. 2019;47:3811–3827. doi: 10.1093/nar/gkz120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhine K., Odeh H. M., Shorter J., Myong S.. Regulation of Biomolecular Condensates by Poly(ADP-ribose) Chem. Rev. 2023;123:9065–9093. doi: 10.1021/acs.chemrev.2c00851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer E., Raske K.. Conjuction of acetobromglucosis and pyridine. Ber. Dtsch. Chem. Ges. 1910;43:1750–1753. doi: 10.1002/cber.19100430291. [DOI] [Google Scholar]
- Chong D., Brooksby P. A., Fairbanks A. J.. One-Step Aqueous Synthesis of Glycosyl Pyridinium Salts, Electrochemical Study, and Assessment of Utility as Precursors of Glycosyl Radicals Using Photoredox Catalysis. ChemistryOpen. 2025;14:e2500183. doi: 10.1002/open.202500183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glarner F., Acar B., Etter I., Damiano T., Acar E. A., Bernardinelli G., Burger U.. The photohydration of N-glycosylpyridinium salts and of related pyridinium N. O-acetals. Tetrahedron. 2000;56:4311–4316. doi: 10.1016/S0040-4020(00)00357-4. [DOI] [Google Scholar]
- Sinnott M. L., Withers S. G.. The β-galactosidase-catalysed hydrolyses of β-d-galactopyranosyl pyridinium salts. Rate-limiting generation of an enzyme-bound galactopyranosyl cation in a process dependent only on aglycone acidity. Biochem. J. 1974;143:751–62. doi: 10.1042/bj1430751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Padmaperuma B., Sinnott M. L.. Hydrolysis of Glycosylpyridinium Ions by Anomeric-Configuration-Inverting Glycosidases. Carbohydr. Res. 1993;250:79–86. doi: 10.1016/0008-6215(93)84156-Z. [DOI] [PubMed] [Google Scholar]
- Tanaka K. S. E., Zhu J., Huang X. C., Lipari F., Bennet A. J.. Glycosidase-catalyzed hydrolysis of 2-deoxyglucopyranosyl pyridinium salts: effect of the 2-OH group on binding and catalysis. Can. J. Chem. 2000;78:577–582. doi: 10.1139/v00-061. [DOI] [Google Scholar]
- Yu F., Li J. Y., DeMent P. M., Tu Y. J., Schlegel H. B., Nguyen H. M.. Phenanthroline-Catalyzed Stereoretentive Glycosylations. Angew. Chem. 2019;58:6957–6961. doi: 10.1002/anie.201901346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li J. Y., Nguyen H. M.. Phenanthroline Catalysis in Stereoselective 1,2-cis Glycosylations. Acc. Chem. Res. 2022;55:3738–3751. doi: 10.1021/acs.accounts.2c00636. [DOI] [PubMed] [Google Scholar]
- DeMent P. M., Liu C. L., Wakpal J., Schaugaard R. N., Schlegel H. B., Nguyen H. M.. Phenanthroline-Catalyzed Stereoselective Formation of α-1,2-cis 2-Deoxy-2-Fluoro Glycosides. ACS Catal. 2021;11:2108–2120. doi: 10.1021/acscatal.0c04381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu H. F., Schaugaard R. N., Li J. Y., Schlegel H. B., Nguyen H. M.. Stereoselective 1,2-cis Furanosylations Catalyzed by Phenanthroline. J. Am. Chem. Soc. 2022;144:7441–7456. doi: 10.1021/jacs.2c02063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Handlon A. L., Oppenheimer N. J.. Substituent Effects on the Ph-Independent Hydrolysis of 2’-Substituted Nicotinamide Arabinosides. J. Org. Chem. 1991;56:5009–5010. doi: 10.1021/jo00017a004. [DOI] [Google Scholar]
- Oppenheimer N. J.. NAD Hydrolysis - Chemical and Enzymatic Mechanisms. Mol. Cell. Biochem. 1994;138:245–251. doi: 10.1007/BF00928468. [DOI] [PubMed] [Google Scholar]
- Zhang F. J., Sih C. J.. Novel analogs of cyclic-ADP-ribose: 9-cyclic etheno-ADP-ribose and cyclic etheno-CDP-ribose. Bioorg. Med. Chem. Lett. 1996;6:2311–2316. doi: 10.1016/0960-894X(96)00428-3. [DOI] [Google Scholar]
- You Y. B., Zhu A. L., Fan D. S., Wang H. L., Li L. J.. Chemical O-ADP-Ribosylations: Synthesis and Bioconjugation of ADPr-Peptides/Proteins from NAD+ . Angew. Chem. 2025;64:e202418321. doi: 10.1002/anie.202418321. [DOI] [PubMed] [Google Scholar]
- Yamada S., Gu Q. M., Sih C. J.. Cyclic ADP-Ribose Via Stereoselective Cyclization of β-NAD+ . J. Am. Chem. Soc. 1994;116:10787–10788. doi: 10.1021/ja00102a055. [DOI] [Google Scholar]
- Nottbohm A. C., Dothager R. S., Putt K. S., Hoyt M. T., Hergenrother P. J.. A colorimetric substrate for poly(ADP-ribose) polymerase-1, VPARP, and tankyrase-1. Angew. Chem. 2007;46:2066–2069. doi: 10.1002/anie.200603988. [DOI] [PubMed] [Google Scholar]
- Szczepankiewicz B. G., Koppetsch K. J., Perni R. B.. One-Step, Nonenzymatic Synthesis of O-Acetyl-ADP-ribose and Analogues from NAD and Carboxylates. J. Org. Chem. 2011;76:6465–6474. doi: 10.1021/jo2008466. [DOI] [PubMed] [Google Scholar]
- Noti C., de Paz J. L., Polito L., Seeberger P. H.. Preparation and use of microarrays containing synthetic heparin oligosaccharides for the rapid analysis of heparin-protein interactions. Chem.Eur. J. 2006;12:8664–8686. doi: 10.1002/chem.200601103. [DOI] [PubMed] [Google Scholar]
- Nokami J., Osafune M., Ito Y., Miyake F., Sumida S., Torii S.. Efficient electrochemical N-glycosylation of silylated pyrimidines with protected arylthioriboses in the presence of a catalytic amount of NBS or Br2 . Chem. Lett. 1999;28:1053–1054. doi: 10.1246/cl.1999.1053. [DOI] [Google Scholar]
- Khanam, A. ; Mandal, P. K. . Chapter 4 - General strategy for the synthesis of N-glycosides. In Synthetic Strategies in Carbohydrate Chemistry; Tiwari, V. K. , Ed.; Elsevier: 2024; pp 139–186. [Google Scholar]
- Makarov M. V., Harris N. W., Rodrigues M., Migaud M. E.. Scalable syntheses of traceable ribosylated NAD+ precursors. Org. Biomol. Chem. 2019;17:8716–8720. doi: 10.1039/C9OB01981B. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fürstner A., Jeanjean F., Razon P., Wirtz C., Mynott R.. Total synthesis of woodrosin I -Part 2: Final stages involving RCM and an orthoester rearrangement. Chem.Eur. J. 2003;9:320–326. doi: 10.1002/chem.200390026. [DOI] [PubMed] [Google Scholar]
- Szpilman A. M., Carreira E. M.. β-Glycosidation of Sterically Hindered Alcohols. Org. Lett. 2009;11:1305–1307. doi: 10.1021/ol9000735. [DOI] [PubMed] [Google Scholar]
- Bertok T., Jane E., Hires M., Tkac J.. N-Acetylated Monosaccharides and Derived Glycan Structures Occurring in N- and O-Glycans During Prostate Cancer Development. Cancers. 2024;16:3786. doi: 10.3390/cancers16223786. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu A. M.. Loci and motifs of the GalNAcα1 → 3/O related glycotopes in the mammalian glycoconjugates and their lectin recognition roles. Glycoconjugate J. 2022;39:633–651. doi: 10.1007/s10719-022-10068-6. [DOI] [PubMed] [Google Scholar]
- Sun X. L.. Chemical Regulation of Glycosylation Processes. Trends in Glycoscience and Glycotechnology. 2018;30:E179–E193. doi: 10.4052/tigg.1306.1E. [DOI] [Google Scholar]
- Vasconcelos-dos-Santos A., Oliveira I. A., Lucena M. C., Mantuano N. R., Whelan S. A., Dias W. B., Todeschini A. R.. Biosynthetic machinery involved in aberrant glycosylation: promising targets for developing of drugs against cancer. Front. Oncol. 2015;5:138. doi: 10.3389/fonc.2015.00138. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dwek R. A.. Glycobiology: Toward understanding the function of sugars. Chem. Rev. 1996;96:683–720. doi: 10.1021/cr940283b. [DOI] [PubMed] [Google Scholar]
- Arihara R., Kakita K., Suzuki N., Nakamura S., Hashimoto S.. Glycosylation with 2-Acetamido-2-deoxyglycosyl Donors at a Low Temperature: Scope of the Non-Oxazoline Method. J. Org. Chem. 2015;80:4259–4277. doi: 10.1021/acs.joc.5b00138. [DOI] [PubMed] [Google Scholar]
- Banoub J., Boullanger P., Lafont D.. Synthesis of Oligosaccharides of 2-Amino-2-Deoxy Sugars. Chem. Rev. 1992;92:1167–1195. doi: 10.1021/cr00014a002. [DOI] [Google Scholar]
- Debenham J., Rodebaugh R., FraserReid B.. Recent advances in N-protection for amino sugar synthesis. Liebigs Annalen-Recueil. 1997;1997:791–802. doi: 10.1002/jlac.199719970503. [DOI] [Google Scholar]
- Prévost M., St-Jean O., Guindon Y.. Synthesis of 1′,2′-cis-Nucleoside Analogues: Evidence of Stereoelectronic Control for SN2 Reactions at the Anomeric Center of Furanosides. J. Am. Chem. Soc. 2010;132:12433–12439. doi: 10.1021/ja104429y. [DOI] [PubMed] [Google Scholar]
- Chavan R., Lefèbre J., Jochová K., Dvoráková H., Rademacher C., Ménová P.. Fucosyl glycosides for DC-SIGN targeting: Fucosylation strategies, synthesis and binding studies of model compounds. Bioorg. Med. Chem. 2025;123:118164. doi: 10.1016/j.bmc.2025.118164. [DOI] [PubMed] [Google Scholar]
- Zhao X. Y., Ding H., Guo A. X., Zhong X. M., Zhou S. I., Wang G. Q., Liu Y. H., Ishiwata A., Tanaka K., Cai H., Liu X. W., Ding F. Q.. Zinc(II)-mediated stereoselective construction of 1,2-cis 2-azido-2-deoxy glycosidic linkage: assembly of Acinetobacter baumannii K48 capsular pentasaccharide derivative. Chem. Sci. 2024;15:12889–12899. doi: 10.1039/D4SC03449J. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhou S. A., Zhong X. M., Guo A. X., Xiao Q., Ao J. M., Zhu W. M., Cai H., Ishiwata A., Ito Y., Liu X. W., Ding F. Q.. ZnI2-Directed Stereocontrolled α-Glucosylation. Org. Lett. 2021;23:6841–6845. doi: 10.1021/acs.orglett.1c02405. [DOI] [PubMed] [Google Scholar]
- Lemieux R. U., Hendriks K. B., Stick R. V., James K.. Halide ion catalyzed glycosidation reactions. Syntheses of.alpha.-linked disaccharides. J. Am. Chem. Soc. 1975;97:4056–4062. doi: 10.1021/ja00847a032. [DOI] [Google Scholar]
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