Microwave-Assisted Automated Glycan Assembly

Abstract

Automated synthesis of DNA, RNA, and peptides provides quickly and reliably important tools for biomedical research. Automated glycan assembly (AGA) is significantly more challenging, as highly branched carbohydrates require strict regio- and stereocontrol during synthesis. A new AGA synthesizer enables rapid temperature adjustment from −40 to +100 °C to control glycosylations at low temperature and accelerates capping, protecting group removal, and glycan modifications using elevated temperatures. Thereby, the temporary protecting group portfolio is extended from two to four orthogonal groups that give rise to oligosaccharides with up to four branches. In addition, sulfated glycans and unprotected glycans can be prepared. The new design reduces the typical coupling cycles from 100 to 60 min while expanding the range of accessible glycans. The instrument drastically shortens and generalizes the synthesis of carbohydrates for use in biomedical and material science.

Towards a Systematic Understanding of the Influence of Temperature on Glycosylation Reactions

“…[28] This trend is especially notable because galactose, Nacetyl glucosamine, and fucose glycosyl donors have often proven cumbersome during AGA, requiring double coupling cycles or additional equivalents to achieve acceptable coupling efficiencies. [29] Aglycon replacement to modulate the reactivity of these problematic glycosyl donors and to bring their activation barriers within range of AGA presents an opportunity to enhance synthetic efficiency and to improve access to previously challenging linkages.…”

“…[36] However, to avoid the excessive consumption of valuable building blocks, reaction conditions must be frequently adjusted in response to new synthetic challenges. Recent advances in AGA hardware, such as the addition of an internal probe to precisely measure and control the reaction temperature, [29] combined with the need to develop a model for reproducible isothermal glycosylations, motivated us to interrogate the role of temperature in complete AGA syntheses. Previously, mannoside 2 has been coupled using a standard temperature regime: five minutes at À 20 °C followed by 20 minutes at 0 °C.…”

Automated Chemical Solid‐Phase Synthesis of Glycans

“…9‐Fluorenylmethyloxycarbonyl (Fmoc) group (cleaved by piperidine) is the most widely tPG in AGA, followed by the commonly used levulinoyl (Lev) group (cleaved by hydrazine) as orthogonal tPG (Figure 4b). [ 17 ] Besides these two tPGs, chloroacetyl (ClAc; cleaved by thiourea), [ 37 ] 2‐(azidomethyl)benzoyl (Azmb; cleaved by tributylphosphine), [ 38 ] 2‐naphthylmethyl (Nap) and p ‐methoxybenzyl (PMB) groups (cleaved by 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone, DDQ) [ 39 ] have also been employed in the AGA for the synthesis of glycans. Notably, a capping step based on Ac 2 O/MsOH or Bz 2 O/DMAP can be utilized to block the unreacted hydroxyl groups of deletion sequences, improving the overall yields and simplifying the purification of AGA.…”

Zu einem Systematischen Verständnis des Einflusses der Temperatur auf Glykosylierungsreaktionen

“…Der Vergleich von Galaktosiden mit anderen Donors verdeutlicht eine Hierarchie der T A zwischen den Konfigurationen: Fucose, N‐Acetyl‐Galaktosamin<Galaktose<Glukosamin<Glukose<Mannose [28] . Dieser Trend ist besonders bemerkenswert, da sich Galaktose‐, N‐Acetylglukosamin‐ und Fukose‐Glykosyl‐Donors bei der AGA oft als schwerfällig erweisen und doppelte Kopplungszyklen oder zusätzliche Äquivalente erfordern, um akzeptable Kopplungseffizienzen zu erreichen [29] . Der Aglykon‐Ersatz zur Modulation der Reaktivität dieser problematischen Glykosyl‐Donors und zur Herbeiführung von Aktivierungsbarrieren innerhalb der AGA‐Reichweite bietet die Möglichkeit, die synthetische Effizienz zu erhöhen und den Zugang zu bisher schwierigen Verknüpfungen zu verbessern.…”

“…Temperaturuntersuchungen deuten darauf hin, dass diese AGA‐Bedingungen daher zu einer unnötigen Zersetzung von 2 bei erhöhten Temperaturen führen könnten, bei denen die anfänglichen fünf Minuten bei −20 °C für eine vollständige Kopplung ausreichen. Diese Beobachtung stimmt mit früheren AGA‐Vorbereitungen eines α‐(1,6)‐Octamannosids überein, bei denen vergleichsweise kurze Kopplungszeiten verwendet wurden (8 Minuten bei −20 °C und 5 Minuten Rampe auf 0 °C) [29] . Wir stellen die Hypothese auf, dass die Aufrechterhaltung einer Temperatur unterhalb von T A den Glykosyl‐Donor schont und zu effektiveren Glykosylierungen führt.…”