Synthesis of modular building blocks using glycosyl phosphate donors for the construction of asymmetric N-glycans

Glycosyl phosphates are known as versatile donors for the synthesis of complex oligosaccharides both chemically and enzymatically. Herein, we report the stereoselective construction of modular building blocks for the synthesis of N-glycan using glycosyl phosphates as donors. We have synthesized four trisaccharide building blocks with orthogonal protecting groups, namely, Manβ2GlcNAc(OAc)₃β6GlcNAc (9), Manβ2GlcNAc-β6GlcNAc(OAc)₃ (15), Manβ2GlcNAc(OAc)₃β4GlcNAc (18) and Manβ2GlcNAcβ4GlcNAc(OAc) (22) for further selective elongation using glycosyltransferases. The glycosylation reaction using glycosyl phosphate was found to be high yielding with shorter reaction time. Initially, The phthalimide protected glucosamine donor was exploited to ensure the formation of β-glycosidic linkage and later converted to the N-acetyl group before the enzymatic synthesis. The selective deprotection of O-benzyl group was performed prior to enzymatic synthesis to avoid its negative interference.     

Dinucleotides containing two allyl groups by combinations of allyl phosphotriesters, 5-allyl-, 2′-O-allyl- and 2′-arabino-O-allyl uridine derivatives as substrates for ring-closing metathesis

Five different dinucleotides, each containing two allyl groups in various positions, were prepared and studied as substrates for ring-closing metathesis reactions. These dinucleotides were designed from appropriate nucleoside building blocks combining four different positions for the allyl group; the allyl phosphotriester linkage, 5-allyl-2′-deoxyuridine, and ribo- as well as arabino-configured 2′-O-allyluridine. Thus, convenient procedures for these building blocks were developed. From the dinucleotides, two new cyclic nucleotide structures were obtained; one connecting two adjacent nucleobase moieties and the other forming an unsaturated four-carbon linkage between the phosphate moiety and the adjacent pyrimidine nucleobase. The latter cyclic dinucleotide was also prepared with a saturated four-carbon linkage using a tandem ring-closing metathesis-hydrogenation procedure. This compound was found to be significantly more stable towards a nucleophilic ring-opening than its unsaturated counterpart.     

Linear synthesis of a protected H-type II pentasaccharide using glycosyl phosphate building blocks.

A linear synthesis of a fully protected H-type II blood group determinant pentasaccharide utilizing glycosyl phosphate and glycosyl trichloroacetimidate building blocks is reported. Envisioning an automated solid-phase synthesis of blood group determinants, the utility of glycosyl phosphates in the stepwise construction of complex oligosaccharides, such as the H-type II antigen, is demonstrated. Installation of the central glucosamine building block required the screening of a variety of nitrogen protecting groups to ensure good glucosamine donor reactivity and protecting group compatibility. The challenge to differentiate C2 of the terminal galactose in the presence of other hydroxyl and amine protecting groups prompted us to introduce the 2-(azidomethyl)benzoyl group as a novel mode of protection for carbohydrate synthesis. The compatibility of this group with traditionally employed protecting groups was examined, as well as its use as a C2 stereodirecting group in glycosylations. The application of the 2-(azidomethyl)benzoyl group along with a systematic evaluation of glycosyl donors allowed for the completion of the pentasaccharide and provides a synthetic strategy that is expected to be generally amenable to the solid support synthesis of blood group determinants.     

Synthesis of the fully phosphorylated GPI anchor pseudohexasaccharide of Toxoplasma gondii.

Retrosynthesis of the fully phosphorylated glycosylphosphatidyl inositol (GPI) anchor pseudohexasaccharide 1a led to building blocks 2-6, of which 5 and 6 are known. The formation of pseudodisaccharide building block 2 is based on readily available building block 7, which gave, via derivative 11 and its glycosylation with known donor 12, the desired compound 2. Building block 3, with the required access to all hydroxy groups being permitted, was prepared from mannose in five steps. From a readily available precursor, building block 4 was obtained, which on reaction with 3 gave disaccharide 23. The synthesis of the decisive pseudohexasaccharide intermediate 32 was based on the reaction of 23 with 5, then with 6, and finally with 2. To obtain high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. To enable regioselective attachment of the two different phosphorus esters, the 6f-O-silyl group of 32 was first removed and the aminoethyl phosphate residue was attached. Then the MPM group was oxidatively removed, and the second phosphate residue was introduced. Unprotected 1a was then liberated in two steps: treatment with sodium methanolate removed the acetyl protecting groups, and finally, catalytic hydrogenation afforded the desired target molecule, which could be fully structurally assigned.     

Dendrimers Based on Melamine. Divergent and Orthogonal, Convergent Syntheses of a G3 Dendrimer

Both convergent and divergent strategies are used to synthesize 1, a dendrimer comprising triazines linked by diamines. The convergent approach is orthogonal; neither protecting groups nor functional group manipulations are required using the building blocks selected.     

3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5' --> 3' photochemical DNA synthesis

[structure: see text]. The most powerful DNA microarrays would be prepared by photolithography with free 3'-ends that could be processed enzymatically. A photoremovable group that could be removed in quantitative yield would ensure high purity of the synthesized probes. We have developed new pyrimidine building blocks for 5' --> 3' DNA synthesis with high cycle yields using the NPPOC (3'-nitrophenylpropyloxycarbonyl) protecting group. These phosphoramidites were proved in automated photochemical DNA synthesis on a modified synthesizer.     

A variable concept for the preparation of branched glycosyl phosphatidyl inositol anchors

A variable concept for the synthesis of branched glycosyl phosphatidyl inositol (GPI) anchors was established. Its efficiency could be shown by the successful synthesis of the GPI anchor of rat brain Thy-1 and of the scrapie prion protein both in the water soluble 1c and lipidated form 1a. Retrosynthesis led to building blocks 2-6 of which 5 could be further disconnected to building blocks 7-9. Trichloroacetimidate 5 was built up in a straightforward manner starting from glycosyl acceptor 9 using known glycosyl donors 7 and 8. The carbohydrate backbone was then assembled by glycosylation of pseudodisaccharide acceptor 6 with donor 5. To ensure high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. Successive deprotection and introduction of the various phosphate residues gave the fully protected GPI anchors. Catalytic hydrogenation and acid-catalyzed cleavage of the Boc protecting groups afforded the target molecules, which could be fully structurally assigned.     

Fluorous linker facilitated synthesis of teichoic acid fragments

The use of perfluorooctylpropylsulfonylethanol as a new phosphate protecting group and fluorous linker is evaluated in the stepwise solution phase synthesis of a number of biologically relevant (carbohydrate substituted) glycerol teichoic acid fragments. Teichoic acid fragments, up to the dodecamer level, were assembled by means of phosphoramidite chemistry, using a relatively small excess of the building blocks and a repetitive efficient purification procedure of the protected intermediates by fluorous solid phase extraction (F-SPE).     

Synthesis of apiose-containing oligosaccharide fragments of the plant cell wall: fragments of rhamnogalacturonan-II side chains A and B, and apiogalacturonan

Fragments of pectic polysaccharides rhamnogalacturonan-II (RG-II) and apiogalacturonan were synthesised using p-tolylthio apiofuranoside derivatives as key building blocks. Apiofuranose thioglycosides can be conveniently prepared by cyclization of the corresponding dithioacetals possessing a 2,3-O-isopropylidene group, which is required for preservation of the correct (3R) configuration of the apiofuranose ring. The remarkable stability of this protecting group in apiofuranose derivatives requires its replacement with a more reactive protecting group, such as a benzylidene acetal which was used in the synthesis of trisaccharide β-Rhap-(1→3')-β-Apif-(1→2)-α-GalAp-OMe. The X-ray crystal structure of the protected precursor of this trisaccharide has been elucidated.     

Nucleotides,Part LXVI , β‐Heteroarylethyl Groups – New Phosphate‐Protecting Groups for Phosphotriester Chemistry

The β‐heteroaryl‐substituted ethanols 6 – 10 were synthesized and, together with pyridine‐2‐ethanols and pyridine‐4‐ethanols, were tested as a new type of phosphate‐protecting groups in the synthesis of oligonucleotides by the phosphotriester approach. The synthesis of 5′‐O‐(monomethoxytrityl)thymidine 3′‐(β‐heteroarylethyl 2,5‐dichlorophenyl phosphates) 13 – 17 and 21 provided useful monomeric building blocks in which the various blocking groups could be removed selectively by acid (MeOTr), oximate (2,5‐dichlorophenyl phosphate), and 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) (heteroarylethyl phosphate) treatment. The new, fully blocked dimers 38 – 41 , with β‐heteroarylethyl protecting groups in the phosphate moiety, were synthesized. The β‐heteroarylethyl groups show a broad range of stability towards base treatment in aprotic solvents depending upon the activation of the H−C(β) atoms by the heterocyclic moiety.     

Oligomeric building block approach to the synthesis of diastereomerically pure pentathymidine 3',5'-methanephosphonates

[formula: see text] A method for a large-scale synthesis of stereodefined oligo(nucleoside 3',5'-methanephosphonates) has been developed, based on transient 3'-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3' end of a stereoregular oligomer, into diastereomerically pure "oligomeric building blocks" for stereospecific coupling with the 5'-OH group of another oligonucleotide.     

Simplifying oligosaccharide synthesis: efficient synthesis of lactosamine and siaylated lactosamine oligosaccharide donors

A practical sequence is described for converting d-glucosamine into peracetylated Gal(beta-1,4)GlcNTroc(beta1-S)Ph and Neu5Ac(alpha-2,3)Gal(beta-1,4)GlcNTroc(beta1-S)Ph building blocks using a synthetic strategy based on chemoenzymatic oligosaccharide synthesis. The known trichloroethoxycarbonyl, N-Troc, protecting group was selected as a suitable protecting group for both enzymatic and chemical reaction conditions. These oligosaccharide building blocks proved effective donors for the beta-selective glycosylation of the unreactive OH-3 of a polymeric PEG-bound acceptor and for the axial OH-2 of a mannose acceptor in good yields. The resulting complex oligosaccharides are useful for vaccine and pharmaceutical applications.     

Discovery of linear receptors for multiple dihydrogen phosphate ions using dynamic combinatorial chemistry

We describe the use of dynamic combinatorial chemistry to discover a new series of linear hydrazone-based receptors that bind multiple dihydrogen phosphate ions. Through the use of a template-driven, selection-based approach to receptor synthesis, dynamic combinatorial chemistry allows for the identification of unexpected host structures and binding motifs. Notably, we observed the unprecedented selection of these linear receptors in preference to competing macrocyclic hosts. Furthermore, linear receptors containing up to nine building blocks and three different building blocks were amplified in the dynamic combinatorial library. The receptors were formed using a dihydrazide building block based on an amino acid-disubstituted ferrocene scaffold. A detailed study of the linear pentamer revealed that it forms a helical ditopic receptor that employs four acylhydrazone hydrogen-bond donor motifs to cooperatively bind two dihydrogen phosphate ions.     

Enhanced stereoselectivity in internucleotidic bond formation by the use of the chiral ribose moiety of thymidine

This paper deals with the synthesis of new cyclic thymidine 3'-phosphoramidite building blocks having a covalent linker between the trityl type 5'-hydroxyl protecting group and the phosphorus atom attached to the 3'-hydroxyl group of thymidine. The ring structures were designed to reduce the conformational freedom around the phosphorus center so that the stereoselectivity in the internucleotide linkage formation would be improved. The linkers were also designed to be removed readily by treatment with aqueous ammonia. These building blocks were synthesized in good yield by one-pot cyclization of the diol precursors with dichloro(N,N-diisopropylamino)phosphine, despite their large-membered ring. Various activators having 1H-tetrazole, imidazole, and triazole structures were investigated to find the best selectivity in the synthesis of thymidylyl(5'-3')thymidine phosphorothioate. It turned out that our cyclic phosphoramidites gave preferentially the R(p) diastereoisomer in high coupling yield applicable to the solid-phase synthesis of oligodeoxynucleotides. It should be noted that high stereoselectivity was achieved without any chiral sources other than the 2'-deoxyribose moiety itself. The mechanistic studies revealed the importance of the steric bulk and the acidity of the activators. It was also found that the steric bulk of the alcoholic nucleophile was an important factor that determined the stereoselectivity in our systems.     

Stereoselective synthesis of uridine-derived nucleosyl amino acids

Novel hybrid structures of 5'-deoxyuridine and glycine were conceived and synthesized. Such nucleosyl amino acids (NAAs) represent simplified analogues of the core structure of muraymycin nucleoside antibiotics, making them useful synthetic building blocks for structure-activity relationship (SAR) studies. The key step of the developed synthetic route was the efficient and highly diastereoselective asymmetric hydrogenation of didehydro amino acid precursors toward protected NAAs. It was anticipated that the synthesis of unprotected muraymycin derivatives via this route would require a suitable intermediate protecting group at the N-3 of the uracil base. After initial attempts using PMB- and BOM-N-3 protection, both of which resulted in problematic deprotection steps, an N-3 protecting group-free route was envisaged. In spite of the pronounced acidity of the uracil-3-NH, this route worked equally efficient and with identical stereoselectivities as the initial strategies involving N-3 protection. The obtained NAA building blocks were employed for the synthesis of truncated 5'-deoxymuraymycin analogues.     

An easy and versatile approach for the regioselective de-O-benzylation of protected sugars based on the I2/Et3 SiH combined system

The use of cheap and easy to handle reagents, such as I(2) and Et(3) SiH, at low temperature allows the regioselective removal of benzyl protecting groups from highly O-benzylated carbohydrates. The observed regioselectivity is dependent on the nature of the precursor, the least accessible carbinol often being liberated. A mechanistic investigation reveals that in situ generated HI is the promoter of the process, whereas the regioselectivity appears to be mainly controlled by steric effects. However, the presence of an electron withdrawing acyl protecting group can switch the regioselectivity to favour deprotection of the carbinol position farthest from the ester group. The protocol is experimentally simple and provides straightforward access in useful yields to a wide range of partially protected mono- and disaccharide building blocks that are valuable for the synthesis of either biologically useful oligosaccharides or highly functionalised chiral compounds. Partially protected sugars thus obtained can also be coupled in situ with a glycosyl donor, as illustrated by the one-pot synthesis of a Lewis X mimic from fully protected precursors.     

Traceless solid-phase synthesis of chiral 3-aryl beta-amino acid containing peptides using a side-chain-tethered beta-amino acid building block

[reaction: see text] A general method for the attachment of a chiral aromatic side-chain-containing beta-amino acid to a polymer support using a traceless silyl linkage strategy has been developed. Using this building block, solid-phase synthesis was carried out to obtain tripeptide analogues with the aromatic ring either unsubstituted or halogenated (Br, I) at the position of the silyl group. The building blocks could generate libraries of peptidomimetics or cyclic peptides containing beta-amino acids with nonpolar side chains.     

Glycal glycosylation and 2-nitroglycal concatenation, a powerful combination for mucin core structure synthesis

A 3,4-O-unprotected galactal derivative having bulky 6-O-TIPS protection (compound 2) could be regioselectively 3-O-glycosylated with O-(galactopyranosyl) trichloroacetimidates; depending on the protecting group pattern stereoselectively alpha- and beta-linked disaccharides were obtained. With O-(2-azido-2-deoxyglucopyransyl) trichloroacetimidate as donor (compound 10A), glycosylation of 2 and of a 6-O-unprotected galactal derivative led in acetonitrile as solvent exclusively to a beta(1-3)- and a beta(1-6)-linked disaccharide, respectively. Nitration of the galactal moieties of the saccharides followed by Michael-type addition of serine and threonine derivatives (7a,b) installed the alpha-galacto-configuration, thus readily furnishing O-glycosyl amino acid building blocks for the incorporation of core 1, core 2, core 3, core 6, and core 8 structures into glycopeptides. 2-Nitrogalactal and 2-nitroglucal derivatives could be also successfully employed in glycoside bond formation via Michael-type addition in a reiterative manner, affording the corresponding core 5, core 7, and core 6 building blocks. In this approach, highly stereoselective glycoside bond formations were based exclusively on Michael-type addition to the nitro-enol ether moiety of the 2-nitroglycals. Hence, 2-nitroglycals are versatile intermediates for base-catalyzed glycoside bond formation.     

Syntheses of anomerically phosphodiester-linked oligomers of the repeating units of the Haemophilus influenzae types c and f capsular polysaccharides

Spacer-equipped dimers and trimers of the repeating units of the capsular polysaccharide of Haemophilus influenzae type c, -4)-3-O-Ac-beta-D-GlcpNAc-(1-->3)-alpha-D-Galp-(1-OPO(3-)-, and type f, -3)-beta-D-GalpNAc-(1-->4)-3-O-Ac-alpha-D-GalpNAc-(1-OPO(3-)-, have been synthesized for use in immunological studies. H-Phosphonate chemistry was used for the formation of the interglycosidic phosphate diester linkages. Two types of building blocks, a spacer glycoside disaccharide starting monomer (15 and 22) and an anomeric monoester alpha-H-phosphonate disaccharide elongating monomer (12 and 27), were built up for each serotype structure from properly protected monosaccharide precursors using mainly thioglycosides as glycosyl donors. Stereospecificity in the formation of the alpha-linked monoester H-phosphonate was possible in type c through crystallization of the pure alpha-anomer of the precursor hemiacetal from an alpha/beta-mixture, whereas in type f, the hemiacetal was isolated directly as exclusively the alpha-anomer. Subsequent phosphonylation using triimidazolylphosphine was performed without anomerization. Formation of the anomeric phosphate diester linkages was performed using pivaloyl chloride as coupling reagent followed by I(2)/H(2)O oxidation of the formed diester H-phosphonates. Original experiments afforded no diester product at all, but optimization of the oxidation conditions (lowering the temperature and dilution with pyridine prior to I(2) addition) gave the dimers in good yields (71% and 81%) and, subsequently, after removal of a temporary silyl protecting group in the dimers, the trimers in fair yields (36% and 37%), accompanied by hydrolysis of the dimer phosphate linkage. One-step deprotection through catalytic hydrogenolysis efficiently afforded the target dimer (30 and 36) and trimer structures (32 and 39). The synthetic scheme allows for further elongation to give higher oligomers.     

Oligosaccharide synthesis with glycosyl phosphate and dithiophosphate triesters as glycosylating agents

Described is an efficient one-pot synthesis of alpha- and beta-glycosyl phosphate and dithiophosphate triesters from glycals via 1,2-anhydrosugars. Glycosyl phosphates function as versatile glycosylating agents for the synthesis of beta-glucosidic, beta-galactosidic, alpha-fucosidic, alpha-mannosidic, beta-glucuronic acid, and beta-glucosamine linkages upon activation with trimethylsilyl trifluoromethanesulfonate (TMSOTf). In addition to serving as efficient donors for O-glycosylations, glycosyl phosphates are effective in the preparation of S-glycosides and C-glycosides. Furthermore, the acid-catalyzed coupling of glycosyl phosphates with silylated acceptors is also discussed. Glycosyl dithiophosphates are synthesized and are also used as glycosyl donors. This alternate method offers compatibility with acceptors containing glycals to form beta-glycosides. To minimize protecting group manipulations, orthogonal and regioselective glycosylation strategies with glycosyl phosphates are reported. An orthogonal glycosylation method involving the activation of a glycosyl phosphate donor in the presence of a thioglycoside acceptor is described, as is an acceptor-mediated regioselective glycosylation strategy. Additionally, a unique glycosylation strategy exploiting the difference in reactivity of alpha- and beta-glycosyl phosphates is disclosed. The procedures outlined here provide the basis for the assembly of complex oligosaccharides in solution and by automated solid-phase synthesis with glycosyl phosphate building blocks exclusively or in concert with other donors.