Functional group modification

The multiple hydroxyl groups of a monosaccharide exhibit similar reactivity, making regioselective functionalization and coupling essentially impossible for carbohydrates in non-enzymatic processes. Therefore, protecting group (PG) manipulations are one of the fundamental reactions in carbohydrate synthesis. Multiple hydroxyl groups, usually present in unprotected monomers and oligosaccharides, have similar properties. Hence, to form glyosidic bonds at a desired oxygen position, other hydroxyl groups have to be protected with protecting groups. After the desired glycosidic bond formation, the final oligosaccharide can be deprotected.

The synthesis of the protected monosaccharide requires long reaction times and multiple reaction steps, involving multiple purifications and workup procedures. Kawakami et al. accelerated this process, synthesizing monosaccharide 27 by combining glycosylation and fluorous phase extraction by continuous microreaction technology (Figure 1.11).33 First, a peracylated glucose derivative was coupled with a perfluorinated hydrocarbon glycerol ether moiety. The tag was sufficient to pull the target into a fluorous solvent, separating the product from all non-fluorinated byproducts and the excess reagents that remained in the organic phase. Using this strategy, the authors performed six individual transformations in Teflon tubing. Following the reactor, the fluorous solvent was added along with an organic solvent for quenching and separation. The biphasic stream exited into a separatory funnel, where the fluorous phase was removed and evaporated to yield clean, crude product to be used in the next step. A six-step synthesis was realized of C4-OH protected glucose 27 in 11%, with no intermediate purifications and only one final column chromatography (Scheme 1.5).

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Scheme 1.5: synthesis of monosaccharide unit 27 with a fluorinated hydrocarbon chain.

Benzyl ethers and benzylidene acetals are some of the most common protecting groups in the field of carbohydrate chemistry for non-reaction sites.30 Their wide usage is mainly due to their stability towards various reaction conditions and facile deprotection by mild reaction conditions such as hydrogenolyses.15 However, batch reactors are commonly used for such deprotection reactions that require long reaction times, posing significant disadvantages associated with slow and laborious optimization for identifying proper reaction conditions. Hence, continuous flow systems can provide an efficient alternative to provide a faster reaction time and facile optimization of reaction conditions. The use of continuous flow systems for deprotection of several carbohydrate derivatives containing benzylidine and benzyl protecting groups was carried out by using a continuous flow (CF) hydrogenation reactor.34 _{The CF hydrogenator consists of a water reservoir to produce}

hydrogen from electrolysis, which is an alternative to the batch system where a pressurized hydrogen gas bottle is necessary. This makes the CF process inherently safer. The sample in the hydrogenator is pumped via HPLC pump and, after mixing with hydrogen gas, is passed through a catalyst packed bed (Figure 1.12). This also essentially removes the catalyst recovery step which is necessary in the batch system.

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Figure 1.12: Basic diagram of H-Cube hydrogenations via the on-demand generation of H2 via hydrolysis of water.23

Another advantage of the CF hydrogenator is the facile process of changing temperature and hydrogen pressure which can be used to rapidly screen reaction conditions for the deprotection reaction. Ekholm et al. found that global deprotection of benzyls and benylidines can be achieved selectively in the presence of both silyl and acyl protecting groups in high yield (>90%,Table 1.4). All reactions were completed using a 30 mm Pd/C prepacked cartridge reactor with a flow rate of 1 ml/min and 40 bar H2-pressure at 80 °C.35

Table 1.4: Deprotection of benzyl/benzylidene protected carbohydrates.

Entry Substrate Product Yield (%)

1 95

2 95

3 95

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Reductive ring openings of 4,6-O-benzylidene acetals are also important deprotections, as these acid and/or hydride mediated reactions are among the key transformations in carbohydrate chemistry. However, these reactions are often exothermic and it becomes imperative to prevent the subsequent acid-catalyzed hydrolysis reaction of the benzylidene groups by carefully optimizing the rate at which the acid is added in these reactions. Furthermore, the yields obtained in such reaction are often irreproducible and vary widely with scale. Often hydrolyzed byproducts such as 4, 6-diols are formed in larger scale systems. In order to improve the overall efficiency of the reaction, it is critical to precisely control temperature and mixing. Reductive opening of 4,6-O-benzylidene acetals was performed in a continuous microfluidic environment for fast optimization of reaction conditions (Table 1.5).36

Table 1.5: Reductive opening of benzylideneacetals under microfluidic conditions.

Entry Substrate Reducing

agent solvent product

Yield (%, microfluidic)a Yield (%, batch)a,b 1 Et3SiH DCM 93c ₅₈ (1.0 M) 2 BH3·Et2NH DCM 100 90 (0.5 M)

19 3 Et3SiH DCM 91 83 (1.0 M) 4 BH3·Et2NH DCM 100 86 (0.5 M) 5 BH3·Et2NH ACN 100 NAd (0.5 M) 6 Et3SiH DCM 91 62 (1.0 M)

a _{Isolated yields.} b _{Reaction was performed at 100-500 mg scale.} c _{4-O-Benzyl derivative was obtained in 5%} yield. d _{60-70% yields for the case of corresponding N-Troc derivative.} e _{PNP: p-nitrophenol.}

The formation of byproducts during protection and deprotection reactions of oligosaccharides poses another challenge in the field of carbohydrate chemistry. The installation of the trityl group as a protecting group is important for the formation of 1,6- glycosidic bonds, e.g. in the synthesis of β-glucans.37 Under batch conditions acetyl migration takes place right after the trityl group is deprotected from the 6-position of 28 due to the attack of the carbonyl group at 4-position acetyl group by the 6-position hydroxyl group (Scheme 1.6).

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To prevent such migrations, control of reaction time, temperature, and flow rate becomes necessary. Attaining such reaction conditions in batch is often not feasible. This dictates the usage of continuous flow microreactor for carrying out such transformations. Using continuous microfluidics, deprotection reactions of trityl protecting group for 28 were carried out screening reaction time, substrate concentration, and flow rate were optimized (Scheme 1.7). With these optimized reaction parameters, the deprotection could be successfully carried out in the microreactor system with the final deprotected product yield of 90%.

Scheme 1.7: Deprotection of the trityl group of 28 in microflow reaction system.