Progress in Kdo-glycoside chemistry.

Glycosylation chemistry of 3-deoxy-D-manno-oct-2-ulosonic acid units has been considerably developed within the last decade. This review covers major achievements with respect to improved yields and anomeric selectivity as well as suppression of the elimination side reaction via selection of dedicated protecting groups and appropriate activation of the anomeric center.


Introduction
3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a non-mammalian higher-carbon saccharide which occurs in the cell wall glycans of Gram-negative bacteria, but has also been found in plants and algae.1-3 Kdo residues are of particular biomedical importance as constituents of bacterial lipopolysaccharides (LPS) as well as capsular polysaccharides.4 Due to their nonmammalian origin, Kdo units are recognized by components of the native and adaptive immune system as demonstrated by binding studies and crystal structures of liganded Kdo units in the binding sites of germ-line encoded antibodies and in complex with Toll-like receptor 4/MD-2.5 In LPS, Kdo is part of the structurally conserved inner core region, which interlinks the lipid A region to the outer core sugars and the O-antigenic polysaccharide. Figure 1 illustrates the most relevant Kdo-structures in the inner core of Gram-negative bacterial LPS. 4 The synthesis of these oligosaccharide ligands and neoglycoconjugates derived therefrom is thus regarded as a highly relevant task for development of glycoarrays, immunoreagents and vaccines.
The synthesis of glycosides of Kdo shares several features with the structurally related 5-Nacetyl neuraminic acid (Neu5Ac), albeit with the difference that Kdo glycosides have been found in both anomeric configurations. Most of the synthetic work on Kdo chemistry in the past had been directed to the preparation of α-glycosides. Glycosylation reactions of Kdo donors are impaired by facile formation of a 2,3-dehydro product (glycal ester), the strong deactivation of the anomeric position by the adjacent carboxylic group and the absence of a stereodirecting group next to the anomeric center which limits stereochemical control of product formation. In addition, the increased steric load being present across a ketosidic linkage and the instability of the ketoside under acidic conditions has to be considered ( Fig  2). Glycosylation reactions of Kdo monosaccharide derivatives with primary alcohols and reactive glycosyl acceptors, however, are usually doable in good yields and anomeric selectivities.
The unfavorable properties, however, are much more demanding in the assembly of Kdo oligomers such as those depicted in Figure 1, resulting in many cases in poor to modest yields only and requiring substantial efforts in product purification. A number of reviews covering the synthesis of Kdo and important Kdo-containing bacterial oligosaccharides have been published previously and the reader should consult these for further information.6 The present review summarizes and highlights recent accomplishments, which have been reported in the past decade in the field of Kdo-oligosaccharide synthesis with a focus on αketosides, but will also cover selected examples for β-Kdo glycosides. The synthesis of the Kdo monosaccharide is beyond the scope of this review, but the reader is referred to a past review and two reports for multigram-preparation of Kdo.7-9

Kdo halide donors
Kdo bromide donors have mostly been used in the 1980ies and 1990ies under Koenigs-Knorr and Helferich conditions with varying success in the synthesis of Kdo oligosaccharides. The donors are easily available by reaction of the anomeric acetate in HBr/ AcOH or TiBr 4 in CH 2 Cl 2 and are fairly stable when stored at low temperature. The use of toxic heavy-metal based promoters, the modest stereoselectivities and facile elimination side reaction leading to glycal ester byproducts, however, limits the use of Kdo bromide donors, in particular for preparation of oligosaccharides in a larger scale.
Kdo fluoride donors are accessible from the respective hemiketal precursors such as 1 and 3 by reaction with DAST, which leads to preferential formation of the α-anomeric fluorides, while reaction of 3 with N,N-diethyl-α,α-difluoro-(m-methylbenzyl)amine (DFMBA) mainly gives the β-anomeric product 4.10 Both reactions proceed with concomitant formation of glycal ester byproducts 5 and 6. Another option is the conversion of the anomeric acetate 7 with HF in pyridine to yield the α-fluoride 8 (Scheme 1).11 The groups of Kusumoto and Fukase have to be credited for developing these Kdo fluoride donors into a methodology, which is suitable for efficient coupling to protected lipid A derivatives. As a highlight, the total synthesis of Re-LPS from Escherichia coli is to be mentioned, wherein the Kdo fluorides served for constructing the α-(2→6)-linkage to the glucosamine disaccharide backbone as well as introduction of the lateral Kdo unit in an α-(2→4)-linkage. 12 The introduction of bulky silyl substituents, or alternatively, the locking of the pyranose ring in a skew/boat conformation by an isopropylidene group at O-4 and O-5, allowed for significant improvement of the α-selectivity in the glycosylation reaction, presumably by sterically shielding the β-face. 13 In general, however, an excess of donor and promoter has to be employed and significant amounts of glycal ester byproducts 5 and 6 were formed. A useful feature of the fluoride donors is the option of a glycodesilylation approach induced by liberation of HF from the BF 3 ·Et 2 O promoter. Two recent examples are illustrated in schemes 2 and 3.
In 2007, Fukase et al succeeded in synthesizing an LPS fragment from Helicobacter pylori. 10 Coupling of the 6-O-triethylsilyl protected disaccharide acceptor 9 was performed using 10 equivalents of promoter and 2.6 equivalents of fluoride donor 4 to give a high yield of the α-(2→6)-linked trisaccharide 10. Molecular sieves 5Å had been added to scavenge HF, which was formed in the reaction (Scheme 2). In 2008, the group of Boons synthesized a trisaccharide corresponding to a Kdo-lipid A fragment of Neisseria meningitidis LPS along similar lines (Scheme 3).14 Coupling of donor 2 to 3'-O-acylated disaccharide acceptor 11 was achieved with only a slight excess of promoter (relative to 11) and produced a 9:1 α/β mixture of trisaccharide 12. Separation of the anomeric mixture was achieved after 4'-O-phosphotriester formation with Watanabe reagent, which gave 13 in 88% yield.
The reactivity of these Kdo donors, however, also depends on their protecting group patterns. "Disarming" substituents introduced at the side chain lead to decreased yields in glycosylation reactions. In 2011, Ichiyanagi et al employed the 7,8-di-O-benzoyl protected donor 14 for coupling reactions to 4-OH and 8-OH Kdo acceptors (Scheme 4).15 Again, a large excess of promoter was employed and triethylamine was added to capture released HF and to increase the product yield. While coupling to the 4-OH group of diol acceptor 15 produced a good yield of the disaccharide 16 and in fair anomeric selectivity, the α-(2→8)linked disaccharide 18 was obtained in a rather poor yield only. The authors did not disclose the amount of elimination product 19 formed.

3-Iodo-Kdo fluoride donors
In order to work around the lack of stereochemical control, auxiliary stereodirecting groups at C-3 such as 3-iodo, 3-phenylselenyl and 3-thio groups had previously been used for Kdoglycoside formation, but also in the field of neuraminic acid chemistry.17,18 Recently the favorable stereodirecting effect of a 3-iodo substituent has been combined with a fluoride leaving group. In contrast to fluoride donors 2, 4 and 14, 3-iodo-Kdo donors do not require bulky or acetal type protecting groups at O-4 and O-5, which tend to increase the formation of the elimination products. Furthermore, these donors are easily accessible in good overall yields from Kdo glycal esters via an acetoxyiodination reaction to give mainly the 2,3-transdiaxial products followed by treatment with HF-pyridine (Scheme 5).19,20 This way, fluoride 22 was obtained as single anomer and was bench-stable at room temperature for several weeks.

Kdo glycal donors
Kdo glycal ester derivatives can also directly serve as glycosylating agents of reactive glycosyl acceptor derivatives upon suitable activation, mostly using triflic acid as promoter.

Kdo thioglycoside donors
Kdo thioglycoside donors allow for various activation protocols using thiophilic promoters, but are susceptible to pronounced elimination reactions with less reactive glycosyl acceptors. These donors have mainly been applied for the preparation of spacer glycosides, in particular of those with a β-anomeric configuration. 32 in a respectable yield. The glycosylation of the β-allyl acceptor 94 was explored using both promoter systems. Again, the TBPA activation was superior in the glycosylation of the "armed" but still unreactive acceptor 94, as seen in the lower product yield of the NIS/TfOH promoted reaction and conversely, the higher amount of isolated elimination product 92. In view of the difficult and challenging formation of the α-(2→5)-linkage, this is an excellent result.
Eventually the authors also applied these new donors for the preparation of a protected trisaccharide corresponding to the inner core of Burkholderia and Proteus LPS (Scheme 17).
The primary alcohol 96 generated from 86 upon DDQ-oxidation was coupled with 4amino-4-deoxy-L-arabinosyl donor 97 in very high yield but poor anomeric selectivity to give disaccharide 97.37,38 Subsequent removal of the 4-O-benzoyl group was followed by coupling of 98 with donor 81 in the presence of TBPA to give a good yield of the branched trisaccharide 99.
A similar trisaccharide had previously been obtained with minimized protecting group manipulation via coupling of alcohol 100 with 97 to give an anomeric mixture of 101 and eventually an isolated overall yield of 36% for the 4,5,7-triol 102. 38 Regioselective glycosylation of 102 was then achieved with 1 equivalent of the Kdo bromide donor 103 to give trisaccharide 104 in low yield. ring-closure of olefin 115. 42 Thus, NIS-treatment of 115 afforded an epimeric mixture of 2iodo-2-deoxy-C-glycosides 116, which were converted into the corresponding pmethoxyphenyl glycal 117 under basic conditions (Scheme 20). Subsequent reaction with a series of alcohols in the presence of NIS proceeded via preferred formation of 2-iodo βglycosides 118. The selective formation of the β-glycosides was rationalized on stereoelectronic grounds and on the skewed conformation enforced by the 4,5-Oisopropylidene group, thereby preventing the NIS attack from the top face and eventually leading to a pseudo trans-diaxial arrangement of the iodo-group and the anomeric OR group (see also Scheme 13). Radical deiodination of the methyl glycoside gave 119 and was followed by oxidative removal of the aromatic appendix to generate the carboxylic function leading to 120.

Conclusions and Outlook
As illustrated in this review, remarkable progress has been achieved in the past in the synthesis of α-glycosides of Kdo. In particular, locking of the pyranose ring conformation by 4,5-O-isopropylidene or 5,7-O-TDBS groups provides mainly the α-ketosides, albeit at the cost of a more pronounced elimination side reaction. The 3-iodo-Kdo fluoride donors have been proven to be versatile donors in α-Kdo oligomer assembly with options for block and regioselective synthesis, but have some limitations in selection of protecting groups. As a main very recent accomplishment the first syntheses of α-(2→5)-linked Kdo disaccharides should be mentioned. The stereodirecting temporary 3-iodo-group has also been of value in the construction of β-anomeric Kdo glycosides. Still, the chemistry of β-Kdo units needs to be further developed in the future and presently lags behind its α-anomeric counterparts. Numbering scheme for Kdo and structures of selected Kdo oligosaccharides from pathogenic bacteria. Kosma Page 10