Stereoselective synthesis of photoactivatable Man(β1,4)GlcNAc-based bioorthogonal probes

Abstract

We report an operationally facile protocol to prepare photoactivatable probes of the bioactive mammalian disaccharide, Man(β1,4)GlcNAc. Using conformationally restricted mannosyl hemi-acetal donors in a one-pot chlorination, iodination and glycosylation sequence, β-mannosides were generated in excellent diastereoselectivities and yields. Upon accessing the disaccharide, we generated the corresponding photoactivatable probes by appending a diazirine-alkyne equipped linker via a condensation reaction between a diazirine-containing linker and C-1 and C-2 derivatized mannosylamines to furnish the desired C-1 and C-2 modified Man(β1,4)GlcNAc-based probes. This new synthetic protocol greatly simplifies the preparation of this important bioactive disaccharide to enable future work to identify its protein binding partners in cells.

Graphical Abstract

Introduction

Free oligosaccharides (fOs) are a class of structurally diverse, unconjugated (not bound to proteins or lipids) glycans that display a plethora of important biological functions. These activities range from antimicrobial and anti-inflammatory properties, to mediating signaling events and modulating the immune system.^1–3 Despite the growing awareness of the role fOs play in human health and disease, the molecular mechanisms underlying many of these observed biological functions are not well understood. The inherent transient nature of fO-protein binding events coupled with the low binding affinity characteristic of most glycan-protein interactions, limits the use of conventional characterization tools,^4–7 and requires the use of non-intrusive endogenous-like approaches to decipher such binding events in live cells.^{8, 9 10}

The mammalian disaccharide, Man(β1,4)GlcNAc, is one such example of a bioactive mammalian autogenous fO with unknown binding interactions and important immune activating properties.¹¹ As a result of aberrant hydrolysis of N-linked glycans by the oligosaccharyltransferase complex (OST), excess accumulation of Man(β1,4)GlcNAc occurs. The enhanced presence of Man(β1,4)GlcNAc in cells erroneously activates the innate immune system, manifesting phenotypes mimicking those found in autoimmunity. Man(β1,4)GlcNAc stimulates intracellular signaling pathways in a manner specific to both the glycosidic linkage (β) and the monosaccharide constituents.^{11, 12} Although the biogenesis and bioactivities of Man(β1,4)GlcNAc are known, its protein binding partners, and hence, its mechanism of action, remain elusive.^{11, 13, 14} As the aberrant hydrolysis step takes place in the cytosol, unambiguously identifying the postulated counter receptor residing in the cytosol remains a nontrivial endeavor.

The integration of photoaffinity labeling methods with mass spectrometry-based proteomic analysis provides an avenue to capture, enrich and identify the intracellular interactions between Man(β1,4)GlcNAc and its protein-based binding partners in live cells.^15–17 Recently, we reported the use of photoactivatable human milk oligosaccharide (HMO) probes appended with bifunctional linkers composed of a photoactivatable diazirine moiety for covalent entrapment of engaged proteins, and an alkyne reporter group to facilitate the analysis of HMO binding proteins via gel-based assays, various imaging techniques and mass spectrometry-based methods.¹⁸ This platform overcomes the inherent low affinity binding associated with glycan-protein interactions, and provides detailed examination of the proteome-wide binding events in situ.

Here, we describe an alternate and stereoselective synthesis of Man(β1,4)GlcNAc (1) and its corresponding C-1 and C-2 functionalized photoactivatable probes as tools to map its proteome-wide interactors and receptors. Previous synthetic protocols used to access Man(β1,4)GlcNAc suffered from poor stereoselectivity (<1:1 β:α) and low yields (< 43%).^{11, 19–22} Stereoselective synthesis of 1,2-cis-β-linkages in mannosides can be challenging due to various stereoelectronic factors, such as the anomeric effect and neighboring group participation, favoring the antithetical α anomer.^{23, 24} Established strategies to access β-mannosides include the use of 4,6-benzylidene acetals to confer conformational control,²⁵ employment of directing groups via intramolecular aglycone delivery,^{26, 27} direct anomeric O-glycosylation,²⁸ and recently a one-pot chlorination-iodination-glycosylation sequence, which was developed by McGarrigle and co-workers (Fig 1).^29–31 We hypothesized that employing this S_N2-type β-mannosylation strategy using conformationally constricted mannosyl hemi-acetals could improve the yield for glucosamine acceptors – a subtle limitation in this method.

Figure 1:

(A) Key intermediates typically leveraged to induce β-selectivity in glycosylation reactions. (B) Synergistic use of conformationally restricted mannosyl donors with a S_N2-type reaction to enhance β-selectivity.

Towards this end, we envisioned gaining access to β-linked photoaffinity probes by applying the elegant β-mannosylation protocol described by McGarrigle and co-workers to convert hemiacetal precursors in situ into reactive mannosyl iodide donors to facilitate a one-pot S_N2-like glycosylation reaction with the corresponding GlcNAc acceptor.²⁹ (Scheme 1) A brief retrosynthetic analysis of our targets provided a convergent approach to stereoselectively furnish the orthogonally protected disaccharide A from its mannosyl donor B and glucosaminyl acceptor C. Subsequent divergent orthogonal deprotection steps and functionalization sequences would give rise to our desired C-1 and C-2 modified photoactivatable probes.

Scheme 1:

Retrosynthetic analysis depicting the modular synthesis of Man(β1,4)GlcNAc and its corresponding C-1 and −2 diazirine-alkyne modified photoactivatable probes.

Results and Discussion

The preparation of the glucosylamine acceptor 7 commenced with the conversion of the amino functionality of readily available D-glucosamine to a phthalimido motif using phthalic anhydride and triethylamine. Temporary masking of all four hydroxy motifs as acetyl ester groups, followed by BF₃.Et₂O mediated conversion of the anomeric acetyl group into its corresponding benzyl glycoside, provided 4 in 73% over two steps (Scheme 2). Sequential synthetic steps comprising global removal of the acetyl protecting groups using catalytic quantities of NaOMe in anhydrous methanol accompanied by regioselective masking of the C-4 and C-6 hydroxy motifs as benzylidene acetals, and benzylation of the remaining C-3 hydroxy group afforded the fully protected glucosaminyl derivative 6 in 65% yield over 3 steps. Subsequent regioselective cleavage of the benzylidene ring using Et₃SiH and triflic acid in anhydrous CH₂Cl₂ furnished the desired 4-OH glucosaminyl acceptor 7 in 87% yield. With 7 in hand, we turned our efforts toward the preparation of the corresponding mannosyl donors.

Scheme 2:

Synthesis of C4-OH glucosaminyl acceptor.

Guided by the knowledge that 4,6-benzylidene acetalic motifs are known to enhance beta selectivity and provide conformational rigidity for the ensuing glycosylation reaction,^{32, 33} peracetylated mannoside 9 was converted into its corresponding mannosyl thioglycoside 10 in 71% yield via treatment with thiophenol and BF·₃Et₂O in anhydrous CH₂Cl₂ (Scheme 3). Removal of the temporary acetyl protecting groups via Na₂CO₃ catalyzed methanolysis, followed by regioselective masking of the C-4 and C-6 hydroxy moieties afforded the benzylidene acetal protected intermediate 12 in a quantitative yield. Using milder benzylating conditions,³⁴ 12 was converted into its corresponding mono-benzylated derivative 13 by treatment with benzyl bromide, barium oxide and barium hydroxide in anhydrous DMF. To further explore and optimize the scope of the beta mannosylation method reported by Pongener et al,²⁹ we opted to derivatize the C-2 and C-3 positions with benzyl and silyl ether groups. Accordingly, conversion of compound 13 into its di-benzylated derivative 14 was achieved by treatment with sodium hydride, tertbutyl ammonium bromide and benzyl bromide at elevated temperatures (140 °C).³⁵ In the same vein, alcohol 13 was derivatized into tertbutyldimethyl silyl ether 15 and tertbutyldiphenyl silyl ether 16 in 73% and 61% yield, respectively.

Scheme 3:

Derivatization of mannosyl thioglycosides

We hypothesized that these ‘pseudo-armed’ benzyl substituents would compensate for the deactivating effect imposed by torsional strain caused by the 4,6-benzylidene ring.^{36, 37} Bulky silyl protecting groups are also known to confer ‘super-armed’ to their respective glycosyl donors.^{38, 39} Facile removal of the silyl ether protecting at C-2 to facilitate future oligosaccharide elongation was another key impetus for silyl ether functionalization at this position. Mannosyl thioglycoside donors 14–15 were converted into their corresponding hemi-acetal analogues in yields ranging from 42% to 80% (Scheme 4).

Scheme 4:

Hydrolysis of thiophenyl glycosides

With the glucosaminyl acceptor (7) and mannosyl (17–19) donors in hand, we began exploring the feasibility of applying the elegant β-mannosylation developed by McGarrigle and co-workers using silyl ether protected 18 as our initial donor (Table 1). Conversion of 18 into its corresponding mannosyl chloride using (COCl)₂, Ph₃PO and subsequent halide methathesis by addition of LiI, diisopropylethylamine (DIPEA) and acceptor 7 at room temperature resulted in low yield and no stereoselectivity (entry 1). Elevation of reaction temperature (45 °C) coupled with increased times (24 h to 48 h) resulted in furnishing 21β in 69% with high beta stereoselectivity (15:1, β:α) (entry 3). Formation of compound 21β and subsequent was confirmed by high resolution mass spectrometry, and the anomeric configuration was verified by the presence of ¹H NMR diagnostic anomeric singlet at 5.50 ppm, as well as the ¹J_1CH = 155.1 Hz coupling between the anomeric proton and its adjacent carbon.^{40, 41} Efforts to substitute LiI to other iodination sources, such as tertbutylammonium iodide (TBAI), resulted in the loss of stereoselectivity for the desired anomer (entry 4). It should be noted at scale above 500 mg, cleavage of silyl ether protecting group occurs resulting complex mixture of products. Similarly, minor cleavage of the benzylidene acetal was observed at reaction temperature exceeding 45 °C. With the knowledge that elevated temperature and reaction duration greatly increased our desired outcome for hemiacetal 18, we employed the optimized reaction conditions for the di-benzylated and silyl ether derivatives 17 and 19. As hypothesized previously, complete β-stereoselectivity and an excellent yield was obtained with the di-benzylated derivative 17 (entry 5). Similarly, excellent stereoselectivity and yield was obtained for the ‘super-armed’ silyl ether containing compound 22β.

Table 1:

Optimization of β-mannosylation reaction

Entry	Hemiacetal	Promoter	Solvent	Temp	Time	β/α ratioa	Product Yield
1	18	Ph3PO, (COCl)2, LiI	CHCl3	rt	12 h	1:1	38% (21)
2	18	Ph3PO, (COCl)2, LiI	CHCl3	45 °C	24 h	2.5:1	61% (21)
3	18	Ph3PO, (COCl)2, LiI	CHCl3	45 °C	48 h	15:1	69%b (21β)
4	18	Ph3PO, (COCl)2, TBAI	CHCl3: CH3CN	45 °C	48 h	1:2	53% (21)
5	17	Ph3PO, (COCl)2, LiI	CHCl3	45 °C	48 h	β only	75b (20β)
6	19	Ph3PO, (COCl)2, LiI	CHCl3	45 °C	48 h	9:1	89%b (22β)

^aDetermined by ¹H NMR analysis of the crude reaction mixture.

^bIsolated yield of β-anomer.

With our desired β-configured disaccharides in hand, we began our efforts toward installing the photoactivatable diazirine tag at C-1 and C-2 position of glucosamine (Scheme 5). To this end, silyl ether cleavage and hydrogenolysis of compounds 20, 21 and 22 with Pd/C and H₂ produced the key deprotected intermediate 23 required for divergent access to the desired C-1 and C-2 probes. Thus, hemiacetal 23 was converted into its corresponding amine analogue via treatment with ammonium carbonate in anhydrous DMSO under microwave irradiation conditions.^{18, 42} Subsequent condensation with diazirine-alkyne-acid linker by conventional amide bond coupling protocol produced the desired C-1 probe, which was used in the subsequent steps without further purification. Upon removal of the N-phthalimido moiety, sequential acetylation and deacetylation sequences furnished the C-1 probe (1) in 31% yield over 4 steps. Similarly, N-phathalimido cleavage by exposure of 23 to hydrazine, followed by EDC and HOBT mediated condensation between 25 and a diazirine-alkyne-acid linker generated the corresponding C-2 diazirine-equipped Man(β1,4)GlcNAc (2) in 55% from intermediate 23.

Scheme 5:

Orthogonal deprotection of divergently functionalized disaccharides (20 – 22), and subsequent EDC and HOBT mediated amide bond formation to generate the C-1 and C-2 diazirine equipped probes.

Conclusion

In summary, we describe an operationally facile protocol for the stereoselective synthesis of Man(β1,4)GlcNAc and its corresponding C-1 and C-2-diazirine-alkyne equipped analogues. A key defining feature in our synthesis relies on the use of a previously disclosed β-mannosylation protocol coupled with the use of conformationally restricted glycosyl donors to facilitate the requisite stereoselective S_N2-type reaction. In addition to the immune activating properties displayed by Man(β1,4)GlcNAc, it is also a conserved core motif found within N-linked glycoproteins. Thus, facile access to relatively high yielding, stereoselective protocols to furnish this disaccharide is desirable. We anticipate these probes can be deployed as bioorthogonal tools to survey their respective protein binders in live cells, cell lysates, or purified recombinant proteins.