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The properties of hemiacetal hydroxyl groups, which may be derivatised or displaced, are shared in many respects by the secondary hydroxyls attached to furanoid and pyranoid rings and exocyclic primary alcohol groups, which have the properties and undergo the full range of reactions common to polyhydroxylic systems. In addition to the numerous products of interaction with organic reagents, metal complexes, such as those with iron (Zamojski and Jarosz 2003 ), copper and molybdenum, are valuable as carriers of these biologically important minerals (Angyal 1989; Cucinotta et al. 1992; Geetha et al. 1995). Equally important in this respect are the products of association of carbohydrates carrying carboxyl groups with calcium and other divalent cations (Saladini et al. 2001).
The practice of protecting hydroxyl groups in carbohydrates, initiated by Emil Fischer, has been developed impressively during the second half of the century just past (Kunz and Waldmann 1991; cf. Ziegler 1998; Oscarson 2006). The general requirement for synthetic modifications at the anomeric centre, namely that the remaining hydroxyl groups should be temporarily protected in such a manner that their subsequent removal does not negate the reaction performed, is crucial. Glycoside formation, for example, normally requires prior formation of ethers, esters or acetals from non-anomeric hydroxyl groups; in addition, the course of glycosidation is materially affected by the nature of the neighbouring group adjacent to the anomeric carbon atom. The well-known principle of latent and active protecting groups (Roy et al. 1992; Cao et al. 1994; Madsen and Fraser-Reid 1996) relates to the part played by benzyl as opposed to benzoyl groups, for example. The efficiency of hydroxyl protecting groups is thus assessed on the basis of a number of criteria (Figure 13). The transformation of hydroxyl groups to esters is a means frequently adopted to bring about elimination or nucleophilic substitution reactions at the carbon atoms involved. In chromatographic analysis derivatisation is a powerful method of altering the polarity of hydroxyl groups, of labelling, and as a means of identification. Mono- or partially-substituted sugar derivatives may be required without prior, temporary protection of other hydroxyl groups (cf. Moitessier et al. 2005). Enzymes are ideal catalysts to bring this about, but it is possible to exploit the greater chemical reactivity of primary hydroxyl groups and the many techniques that have been developed to differentiate secondary hydroxyl groups on the basis of their inherently different reactivities and susceptibility to varying reaction conditions (Garegg 1984; Gridley et al. 1999). A powerful device is to form O-stannylene acetals in order to enhance the nucleophilicity of one of the oxygen atoms engaged. An alternative approach is to remove selectively from fully-derivatised material such groups as are not required (Haines 1981). Although hydrolysis or hydrogenolysis are most frequently used for de-protection, photolytic methods are applicable in cases where suitably designed protecting groups have been incorporated in the carbohydrate structure (Zehavi 1988; Binkley and Hehemann 1990).(See Figure 14). Alkoxycarbonyl protection of hydroxyls parallels that of amino groups in protein chemistry (Adinolfi et al. 2000).
(a) Esters
Hydroxyl groups in carbohydrates are very commonly converted to acetate, by heating with acetic anhydride with anhydrous sodium acetate as catalyst, or treatment with acetic anhydride (or acetyl chloride) and pyridine (or other tertiary amine) at lower temperatures. Anomeric per-O-acetates are formed as a rule, complex derivatives from keto-sugars (Lichtenthaler et al. 1995a,b), and it follows that alditols and glycosides, and their partially-substituted derivatives, give single products; these are of great importance in chromatographic/spectrographic analysis (Sellers et al. 1990). Enzyme-controlled acetylation may be used to advantage (Theil and Schick 1991), and in the drive towards "green chemistry", alternative approaches to those using organic solvents are being developed (Forsyth et al. 2002).
Analogous procedures afford trifluoroacetates, benzoates or aryl-substituted derivatives thereof, pivaloates, or carboxylates of various chain lengths; sulphates (Falshaw and Furneaux 1999), nitrates (Gavria et al. 2005), and the widely-used methane-, trifluoromethane- and p-toluenesulphonic esters, and phosphates. The use of SO3 or ClSO3H and pyridine in a solvent parallels the acetylation process, acid chlorides or anhydrides in the presence of an acid scavenger furnishing the desired ester in most cases. Bifunctional acids engaging two hydroxyl groups have varying uses, carbonates (cf. Bufali et al. 2005) and other esters of dibasic acids (Aly et al. 2001) as protecting agents, and periodates as intermediates in oxidation reactions. Removal of ester groups by saponification is achieved under conditions of varying severity, acetates with sodium methoxide in cold methanol (or under even more mild conditions with dibutyltin oxide, Liu et al. 2002, or with bound benzylamine, Johnsson and Ellervik 2005), benzoates on more vigorous treatment in alkali; sulphonates are still more resistant but are converted to alcohols with sodium and liquid ammonia, lithium aluminium hydride or Raney nickel in favourable circumstances as reduction to CH sometimes occurs. p-Toluenesulphonates are stable to base and are not easily hydrogenolysed. Methanesulphonates undergo displacement by halides, and sulphates may be converted to anhydro (ether derivatives) with alkali. A further variant of the esterification approach to hydroxyl group protection is to form trichloroacetimidates, more familiar as generators of glycosyl cations (section 4.2.1.2), which can be deprotected under mild conditions, acidic, basic or neutral (Yu et al.1999).
In the context of GLC the volatility of trifluoroacetates, boronates and carbonates counteracts problems that are experienced in carrying out the esterification process (Churms 1982). For HPLC p-nitrobenzoates and naphthoates are used pre-column in addition to the more common acetates and benzoates (Sturgeon 1990).
Regioselective esterification of carbohydrates may be achieved in part by making use of the differing reactivities of hydroxyl groups, but advances have been made enzymatically (Theil and Schick 1991) and by using Mitsunobu conditions (Bourhim et al. 1993). The acylation of keto-sugars has aroused interest on account of the abundance of available D-fructose, though considerable difficulty is experienced in the isolation of specific products (Lichtenthaler et al. 1995a,b).
Displacement reactions of ester groups are an effective means for the introduction of other substituents such as hydrogen and halogens. Apart from the applications of carbohydrate esters in synthesis surface-active esters made from sucrose and fatty acid esters of the polyols other than glycerol are among many derivatives of industrial importance (Akoh and Swanson 1990). The range of esters that may be prepared and subsequently removed with a variety of reagents is extensive (Gan and Whistler 1990).
(b) Ethers: methyl ethers
Conversion to methyl ethers of non-anomeric hydroxyl groups (Ciucanu and Kerek 1984; Churms 1982,1991) is a long established procedure used, in conjunction with ethylation and deutero-methylation, for the analysis of glycosides, oligosaccharides and polysaccharides. Methyl ethers are relatively stable, and in methylation analysis the per-O-methylated compound is normally cleft hydrolytically or reductively at glycosidic centres. The methylation pattern of the resulting monosaccharide derivatives affords information as to the positions of glycosidic linkage, and often the ring sizes, of the sugar unit incorporated in the oligomeric structure. Both identification and quantitative analysis are achieved by various chromatographic techniques (Churms 1991), notably derivatisation and GLC-MS. Methyl ethers are not normally regarded as protecting groups though they may be considered in special cases (Schürrle et al. 1991; Greene and Wuts 1991); cleavage involves heating with aqueous HI or treatment with boron halides in the cold, or oxidising conditions (Fenton's reagent). The synthesis of partially methylated sugars, which have formed the basis of quantitative chromatographic analyses of the type described above, has been accomplished using blocking groups or by utilising the inherent reactivities of the differently situated hydroxyls. Methylation methods depend on electrophilic methyl donors, the modification of hydroxyl groups to enhance their nucleophilic character, and choice of the most effective (usually aprotic and polar) solvent. Generation of dimsyl (methylsulphinylmethanide) ion for deprotonation of hydroxyl groups in DMSO solution is a highly effective method of activating a dissolved saccharide, whereupon cautious addition of the methylating agent causes rapid ether formation at a low temperature. Diazomethane activated by BF3 or a combination of MeOTf and (MeO)3PO may be used in the presence of acetate or phosphate esters as these groups are not affected. A recent modern study has demonstrated a wide range of susceptibilities to methylation of the hydroxyls in various methyl pyranosides using diazomethane together with transition metal chlorides and boric acid (Evtushenko 1999).
(c) Other alkyl ethers, and alkenyl ethers
Ethyl groups are employed in structural analysis of carbohydrates to differentiate positions of O-linkage from those labelled by methylation, and bulky substituents (such as t-butyl) in controlled syntheses. Long-chain alkyl ether groups impart lipophilic character and the products are suitable for industrial applications demanding chemical stability (see Chapter 6 and Beifuss et al.1999). On the other hand etherification of glucose at O-4 using a hexaethylene glycol donor system followed by intramolecular glycosidation results in the formation of a chiral crown ether, useful for selective complexing wih enantiomers (Miethchen and Fehring 1998). Analogous products carrying HO(CH2)3O- substituents at 2,3 and 4,6 provide useful glycosyl acceptors for diantennary oligosaccharide synthesis (Neda et al. 1999). In the past two decades the use of allyl ether groups for the temporary protection of hydroxyls in carbohydrate synthesis has assumed considerable importance on account of the stability of the group as such and its ready removal after isomerisation (Boons et al. 1996; Opatz and Kunz 2000) (See Figure 15).
(d) Benzyl and trityl ethers
The advantage of using benzyl ethers for protection of hydroxyl groups lies in the lability towards mild hydrogenolysis, catalytically or with sodium dissolved in ethanol or liquid ammonia, of the benzyl to oxygen bond (see also Lecourt et al. 2004), a property shown too by trityl (triphenylmethyl) ethers. Photochemical bromination with NBS followed by hydrolysis (Binkley and Hehemann 1990; cf. Adinolfi et al. 1999), and oxidation by chemical or electrolytic means, are also effective, while the ethers remain relatively stable under acidic and basic conditions. Benzyl and trityl ethers are bulky and if sited adjacent to an anomeric centre play important roles as non-participating groups in the stereocontrol of glycosidation procedures (see chapter 4). The many preparative methods include the use of benzyl halides under Kuhn conditions (as for methylations in DMF with BaO), BnOTf with 2,6-di-tert-butylpyridine under phase transfer conditions, Bn trichloroacetimidate catalysed by TfOH, and the Mitsunobu procedure (Hughes 1992). Tritylation of primary hydroxyl groups using trityl chloride in pyridine is one of the oldest selective alkylation processes in carbohydrate chemistry, though forcing conditions (trityl perchlorate and 2,4,6-tri-tert-butyl pyridine in dichloromethane) cause etherification of secondary hydroxyl groups, equatorial for preference. In an oxidative process benzyl trityl ether is used (Oikawa et al. 1998). Removal of bulky trityl substituents is effected by hydrogenolysis or mild acid hydrolysis, silica gel combining the properties of acidic catalyst and chromatographic adsorbent in an interesting procedure (Lehrfeld 1967). p-Methoxybenzyl ethers are cleft with relative ease by electrolytic means, or by using DDQ or CAN, and 3,4-dimethoxybenzyl ethers even more readily, by oxidation; another variant employs the p-acetyloxy benzyl substituent, removed by mild oxidation or treatment with methoxide. A systematic study has emphasised the extent to which ease of debenzylation on hydrogenolysis varies with the substituent group (Gaunt et al. 1998).
Electrophilic reagents including glycosyl cations react smoothly with trityl ethers so that the synthesis of 1-->6-linked disaccharides can be conveniently achieved. Kochetkov et al. (Kochetkov et al.1991) have extended this concept in forming other inter-sugar linkages by an SN2-type reaction, a-D-glucosyl derivatives from b-thiocyanates, for example, with regeneration of the trityl cation from the acceptor molecule. The non-participating 2-benzyl ether assists the 1,2-cis glycosylation reaction. (See Figure 16).
(e) Silyl ethers
Commencing with trimethylsilyl (TMS) ethers (Johnson 1992) an immense range of derived products has been developed in order to moderate the extreme lability of TMS ethers towards acid hydrolysis, and to increase bulk so as to permit stereoselectivity in ether formation with polyols. The widely used t-butyldimethylsilyl (TBDMS) ethers (Icheln et al. 1996) are much more stable towards acid and are moderately so in base, cleavage being effected by reaction with fluoride ion (Bu4NF in TMF) or iodine bromide (Kartha and Field 1999). t-Butyldiphenylsilyl (TBDPS) ethers are cleft similarly but are even more resistant to acid hydrolysis; desilylation may then be achieved using fluoride under pressure (Matsui et al. 2002). (See Figure 17).
The almost universal usage of TMS ethers in GLC is made possible by their thermal stability. Per-O-trimethylsilylation is conveniently carried out by reaction with Me3SiCl (chlorotrimethylsilane) and (Me3Si)2NH (hexamethyldisilazane) in pyridine (Sweeley method), DMSO or DMF for short periods in the cold or with warming to 850C, and work up (Churms 1982). Sugars can be derivatised in aqueous solution if N-(Me3Si)imidazole is used, and many other reagents are employed according to the carbohydrate to be silylated (Greene and Wuts 1991; Paquette 1995; Johnson 1992; Chung et al. 2002; Boons and Demchenko 2000).
(f) Acetals or ketals
Suitably-positioned pairs of hydroxyl groups in sugars, glycosides and polyols form cyclic derivatives with aldehydes and ketones and have properties similar in many respects to those of O-glycosides, which are mixed acetals or ketals. These protecting groups are formed by acid-catalysed reactions with carbonyl compounds, by transketalation, or by using any of a variety of substances developed for the purpose (Greene and Wuts 1991; Ley et al. 1992,1994). Some occur naturally, notably pyruvate ketals (Chapter 2), which may be synthesised (Liptak and Szabo 1988). Benzylidene derivatives are formed preferentially with 1,3-diols of which anomeric and primary hydroxyl groups are a part, and isopropylidene ketals with cis-1,2-diols; catalysis with concentrated H2SO4 or such Lewis acids as ZnCl2 usually results in high yields, particularly if a desiccant is present to remove water produced during the reaction (cf.Suzuki et al. 2003). Acetal exchange using acetal, 2,2-diethoxypropane or 1,1-dimethoxycyclohexane yields ethylidene, isopropylidene or cyclohexylidene derivatives. Protection using cyclohexane-1,2-diacetals or the related butane-2,3-diacetals represents a modified approach which has proved its worth in complex oligosaccharide synthesis (Ziegler 1994; Ley et al. 1994; Downham et al. 1995; Douglas et al. 1996). Stannylene acetals have assumed importance in stereocontrolled synthesis (Guilbert et al. 1994; Hodosi and Kovác 1996; Bredenkamp 1999; Grindley 1998; cf.Dubois and Beau 1992). Ketal (acetal) protecting groups are removed with ease under acid conditions by hydrolysis (Barone et al. 2002), or by non-aqueous transacetalation (Andrews and Gould 1992). (See Figure 18).
The ease of formation and the structures of products are a function of the regio-and stereochemistry of the hydroxyl groups and the conformation of the carbohydrate, as well as the properties of the carbonyl reagents and the catalytic conditions employed (Collins and Ferrier 1995). Generalisations are not possible, therefore, but useful derivatives have been isolated in good yield, notably the "acetonation" product 1,2-5,6-di-O-isopropylidene-D-glucose and its cyclohexylidene analogue; 1,2-3,5-di-O-isopropylidene-D-xylose; 4,6-O-ethylidene-D-glucose and the corresponding benzylidene derivative; and, from methyl a-D-mannoside the 2,3-4,6-di-O-benzylidene compound which contains both 5- and 6-membered cyclic acetals. Glycosides of glucose and galactose, converted to 4,6-O-benzylidene derivatives, normally yield single products with the phenyl group equatorial, two chair forms being possible for the galactoside. Using acetone, methyl a-D-glucopyranoside can be induced to form the 4,6-O-isopropylidene ketal.
The rate of cleavage of acetals by hydrolysis (using 90% trifluoroacetic acid, for example) varies with structure, hexafluoroacetone and anisylidene acetals being more and less stable respectively, than their unsubstituted counterparts. Electron-releasing groups facilitate the acid hydrolysis of substituted benzaldehydes, and in this respect the 9-anthraldehyde acetal has advantages as a protecting group with fluorescent-labelling potential (Ellervik 2003). Complex migrations may occur in acid.
Reduction of 4,6-O-benzylidene acetals is an important synthetic reaction as monobenzyl ethers are formed from the erstwhile diol system, at O-4 when LAH and AlCl3 is used or at O-6 with NaCNBH3 in THF acidified with dry HCl; other reagents, affording better regioselection and control, have since been developed (Oikawa et al. 1996; Sherman et al. 2003).(See Figure 19). Further differentiation can be effected with the more reactive anisylidene analogue. On the other hand oxidation of a benzylidene acetal with NBS gives the 6-brominated 4-benzoate, other oxidants being less regioselective. Photochemical ring opening occurs with o-nitrobenzylidene acetals. A controlled method of removing benzylidene groups makes use of transacetalation with 1,2-diols (Andrews and Gould 1992).
Synthetic cyclic acetals of alditols and the open-chain sugar dialkylmercaptals are known in abundance. D-Glucitol yields a 1,3-2,4-benzylidene diacetal, as expected, and a third group enters at 5,6 by default; the latter is the first of the three groups to be removed on hydrolysis in acid. For steric reasons galactitol yields only a pair of di-O-isopropylidene compounds, whereas D-glucitol and D-mannitol can form 1,2-3,4-5,6 derivatives (Collins and Ferrier 1995).
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