To monitor the reaction, 20–30 beads were taken from the reaction vessel and subjected to microcleavage, high-performance liquid chromatography (HPLC)–mass spectrometry (MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis.
#Stapled events series#
Different combinations of BBs 2 and 3 were considered to generate cross-linkers with sizes ranging from 4×CH 2 (highest rigidity, m = 1 and n = 1) to 10×CH 2 (highest flexibility, m = 4 and n = 4), as detailed in Figure 2B.ĭespite the similarity among the series of alkene-modified BBs, some optimizations of the glycosylations were required to guarantee complete conversion. BB 2a,b (bearing an alkene at C4) was always incorporated in the second position and BB 3a–d (alkene at C3) was incorporated in the sixth position, while BB 4a,b was introduced in the remaining positions. Two different sets of glucose BBs containing terminal alkenes of different lengths ( 2a,b and 3a–d) were synthesized (see the Supporting Information) and incorporated in specific positions within the hexasaccharide sequence. Bz-protection of the C2-OH ensured selective β-glycosylation. Glucose BBs were designed to contain a fluorenylmethoxycarbonyl (Fmoc) temporary protecting group at the C6-OH, while benzyl (Bn) ethers and benzoyl (Bz) esters served as permanent protecting groups.
The glycosylation required rigorous temperature control, −20 ☌ (10 min) → 0 ☌ (20 min) for thioglycosides and −30 ☌ (5 min) → −10 ☌ (30 min) for the glycosyl phosphates. Glycosyl phosphate activation was used for 4b in fourfold excess of BB. In most cases, the glycosylation step was based on thioglycoside activation ( 2a,b 3a–d and 4a), using a sixfold excess of the building block (BB) to ensure complete couplings. (33) Glycan elongation relied on sequential cycles of acidic wash, glycosylation, capping, and deprotection ( Figure 2A). These functional groups (a) are in close proximity, (b) permit the beneficial syn orientation, and (c) do not affect the C2-OH position that needs to be temporarily protected as an ester to ensure the desired β-glycosylation during backbone assembly.Īutomated glycan assembly (AGA) using polystyrene resin equipped with photocleavable linker 1 was performed on a 0.015 mmol scale in a home-built synthesizer.
A thorough inspection of the 3D structure clearly suggests C3-OH on Glc6 and C4-OH on Glc2 as the best stereochemical combination (see Figure 1D). Still, each monosaccharide offers three possible sites for modifications (C2-OH, C3-OH, and C4-OH), with stereochemistry playing an essential role in the orientation. In the energy-minimized structure of the example oligosaccharide (see Figure 1C,D), (5) monosaccharide residues positioned at i, i + 4 (e.g., Glc2 and Glc6) are in close proximity. Thus, it is necessary to functionalize two of the hydroxyl groups with additional side chains bearing complementary functional groups ( Figure 1D). In addition, unlike peptides, glycans have a minimal variety of functional groups that can be utilized in chemoselective transformations.
Similar spatial rules for the functionalization of glycans did not exist.
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