Synthesis of homo- and heteromultivalent carbohydrate-functionalized oligo(amidoamines) using novel glyco-building blocks

Summary We present the solid phase synthesis of carbohydrate-functionalized oligo(amidoamines) with different functionalization patterns utilizing a novel alphabet of six differently glycosylated building blocks. Highly efficient in flow conjugation of thioglycosides to a double-bond presenting diethylentriamine precursor is the key step to prepare these building blocks suitable for fully automated solid-phase synthesis. Introduction of the sugar ligands via functionalized building blocks rather than postfunctionalization of the oligomeric backbone allows for the straightforward synthesis of multivalent glycoligands with full control over monomer sequence and functionalization pattern. We demonstrate the potential of this building-block approach by synthesizing oligomers with different numbers and spacing of carbohydrates and also show the feasibility of heteromultivalent glycosylation patterns by combining building blocks presenting different mono- and disaccharides.


General experimental details
Commercial grade reagents and solvents were used without further purification. 1 H NMR and 13 C NMR spectra were measured with a Varian 400-MR and a Varian 600-MR spectrometer.
The proton signal of residual, non-deuterated solvent (δ 7.26 ppm for CHCl 3 ) was used as an internal reference for 1 H NMR spectra. For 13 C NMR spectra, the chemical shifts are reported relative to the δ 77.36 ppm resonance of CDCl 3

TEC residence time optimization
General TEC pilot optimization procedure. In order to access the reactivity of different thioglycosides during TEC, a photoreactor was set up using 2 mL (loop of FEP tubing around a Pyrex and a medium pressure Hg lamp [1,2] (see 1.2 construction and configuration of photoflow reactor for TEC). A solution of DDS 1 (1.0 equiv), acetyl protected thioglycosides 2-7 (1.5-2.0 equiv) and acetic acid (3 equiv) (total volume 2 mL, corresponding to the volume of the reactor) in degassed methanol was injected into the photoreactor. Both before and after the injected sample a plug (0.3 mL) of Argon was injected. The sample and Ar plugs were then pushed through the reactor with pure methanol. The entire reactor output was collected and evaporated under reduced pressure to afford the crude material.

Solid-phase synthesis
In a similar manner as described in [2] all solid phase reactions were performed on an automated standard peptide synthesizer at 0.02 mmol scale according to the following general 1 Experimental procedures S7 solid phase protocols. Tentagel S RAM resin (loading 0.24 mmol/g) and ethylenediamine preloaded Tentagel S Trityl resin (loading 0.20 mmol/g) were used as solid supports, and were swollen twice for 15 min in DCM before starting the initial Fmoc-deprotection or coupling protocols.
Coupling/Fmoc-deprotection protocol. In a similar manner as described in [2]  Fmoc deprotection was performed using 25% piperidine in DMF for 5 min and checked by UV monitoring for the fluorenyl piperidine adduct at 301 nm. This step was repeated until the deprotection was complete.
Final cleavage from solid support. In a similar manner as described in [2] the final cleavage was performed by adding the cleavage cocktail (50% DCM, 47.5% TFA and 2.5% TIS 1 mL/50 mg resin) to the resin and allowing it to react for 60 min using the Tentagel S RAM resin. The resin cleavage for the EDA-Trityl Tentagel resin was carried out with 10% TFA and 90% DCM for 20 min and was performed twice for complete cleavage of the oligomers.
The cleavage solution was filtered and poured onto ice cold diethyl ether. The white precipitate was collected by centrifugation and washed twice with diethyl ether to give acetyl protected compound 14-16.
Deprotection of the acetyl groups. Fully protected oligomers 14-16 were dissolved in MeOH (3 mL). Then NaOMe (0.8 mL; 10 mg/mL in MeOH) was added slowly. The reaction was monitored via RP-HPLC. After complete deprotection the reaction mixture was neutralized using a cation exchange resin (H + -form), filtered and evaporated under reduced pressure. The product was dissolved in water and lyophilized to give the final deprotected