CHEMICAL SYNTHESIS OF GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORS

Abstract

Many eukaryotic cell-surface proteins and glycoproteins are anchored to the plasma membrane by glycosylphosphatidylinositols (GPIs), a family of glycolipids that are post-translationally attached to proteins at their C-termini. GPIs and GPI-anchored proteins play important roles in many biological and pathological events, such as cell recognition and adhesion, signal transduction, host defense, and acting as receptors for viruses and toxins. Chemical synthesis of structurally defined GPI anchors and GPI derivatives is a necessary step toward understanding the properties and functions of these molecules in biological systems and exploring their potential therapeutic applications. In the first part of this comprehensive article on the chemical synthesis of GPIs, classic syntheses of naturally occurring GPI anchors from protozoan parasites, yeast, and mammals are covered. The second part of the article focuses on recent diversity-oriented strategies for the synthesis of GPI anchors containing unsaturated lipids, “click chemistry” tags, and highly branched and modified structures.

I. Introduction

The attachment of membrane proteins and glycoproteins to the cell surface by glycosylphosphatidylinositol (GPI) anchors, a structurally diverse class of glycolipids, is ubiquitous in eukaryotic species.^1–4 To date, more than 250 eukaryotic membrane proteins have been established as GPI-anchored,⁵ and genomic investigations have suggested that as many as 0.5–1% of all eukaryotic proteins may be anchored to the cell surface by GPIs.⁶ The GPIs and GPI-anchored molecules play vital roles in numerous biological and pathological processes, such as cell recognition and adhesion,⁷ signal transduction,^8,9 pathogenic infections,^10,11 enzymatic reactions on the cell surface,¹² and functioning as cellular markers.⁴

The discovery of GPI anchorage as a unique mode of membrane-protein binding spanned the 1970s and 1980s¹ and culminated in the full characterization of the Trypanosomabrucei variant surface glycoprotein GPI anchor in 1988 by Ferguson and co-workers.¹³ Since then, numerous GPIs have been identified and characterized, and a highly conserved core structure has been established: H₂N-(CH₂)₂-(P)-6-α-Man-(1→2)-α-Man-(1→6)-α-Man-(1→4)-α-GlcN-(1→6)-myo-inositol-1-(P)-glycerolipid (Fig. 1). The protein C-terminus is covalently linked to the non-reducing end of the GPI core glycan through a phosphoethanolamine bridge, and the entire structure is anchored to the cell surface by insertion of the phosphatidylinositol (PI) fatty acid chains into the membrane bilayer. The tetrasaccharide core, whose function remains obscure, exhibits considerable structural diversity amongst known GPIs, primarily in the form of additional carbohydrate and phosphoethanolamine units linked to various positions. For example, mannose and galactose mono/oligomers are commonly appended to the O-2 position of Man-III and the O-3/4 positions of Man-I, respectively, while the O-2 position of Man-I frequently carries a phosphoethanolamine group. Additionally, the PI moiety may be palmitoylated (hexadecanoylated) at the inositol O-2 position, while its phosphoglycerolipid can undergo fatty acid remodeling,¹⁴ leading to modified lipids that can be acyl- or alkyl-linked or exhibit various chain lengths and unsaturation patterns.

FIG. 1.

GPI structure and anchoring function.

Despite impressive advances in elucidating the biosynthesis, structure, and function of GPI anchors and GPI-anchored proteins,¹⁵ many aspects of these molecules remain poorly understood. For example, the significance of structural complexity and diversity of GPI anchors, particularly in the conserved glycan core, is essentially unexplained and prompts studies on structure–activity relationships in various contexts. The use of GPI partial structures in the development of antimalarial vaccines^16,17 suggests GPIs have therapeutic value, but new technologies are needed to explore their potential more fully. Imaging of GPI anchors in vivo could provide insight into their expression, distribution, and endocytosis.¹⁸ Another emerging area of research is “GPIomics,” which seeks to develop proteomic tools for identifying proteins carrying the GPI anchor as a post-translational modification.¹⁹

To accelerate progress in these and other areas of GPI research, homogeneous and structurally defined GPIs and GPI derivatives must be accessible in sufficient purity and quantity, a requirement that can be addressed by chemical synthesis. GPIs are among the most complex of natural products, as they incorporate carbohydrate, lipid, phosphate, and inositol groups, the combination of which makes their synthesis very challenging. Difficult tasks in GPI synthesis include the preparation of enantiomerically pure and properly discriminated inositol derivatives, stereoselective formation of glycosidic bonds, and regiocontrolled introduction of side chains. A general convergent approach for GPI assembly is shown in Scheme 1, where the target GPI anchor I is typically accessed from a fully protected intermediate II bearing orthogonal protecting groups at sites for late-stage introduction of the phosphoglycerolipid and phosphoethanolamine groups. In most cases, structure II is disconnected at the glucosamine–mannose glycosidic bond, leading back to trimannose and pseudodisaccharide fragments III and IV, which are in turn generated from their corresponding monomeric subunits.

Scheme 1.

General retrosynthesis of a GPI anchor. PG = protecting group, PG′ = orthogonal protecting group, X = activatable leaving group, X′ = orthogonally activatable leaving group.

In this survey, the field of GPI-anchor synthesis iscovered chronologically, starting with the first synthesis of a GPI anchor by the Ogawa group in 1991.In the ensuing two decades, numerous impressive total syntheses of naturally occurring GPI anchors were completed by various research groups, and these works are discussed in the first section, “Classic Approaches to GPI Anchor Synthesis.” Subsequently, more effort has been focused on the development of novel synthetic strategies that have emphasized flexibility, which has enabled access to GPI anchors containing biologically important unsaturated lipids, versatile “click chemistry” tags, and branched structures. These syntheses are covered in the second section, “Diversity-Oriented Approaches to GPI Anchor Synthesis.” It is expected that this article will have some overlap with previous reviews on the subject,^20–22 but will offer coverage of new material as well as a contemporary and unique perspective.

To keep this article within reasonable constraints, detailed discussion of research involving GPI partial structures has been omitted. Notable works in this category include syntheses of non-lipidated GPIs by Martín-Lomas²³ and Schmidt,²⁴ lipophosphoglycans (LPGs) by Konradsson and Oscarson,^25,26 GPI-based antimalarial candidates by Seeberger,^17,27,28 and GPI–peptide/glycopeptide constructs by Seeberger²⁹ and Guo.^30,31 Also excluded from this discussion is recent work from the Bertozzi group using GFP-modified GPI partial structures to explore their behavior in live cells^18,32 and work from the Guo laboratory focused on chemoenzymatic ligation of GPI analogues and proteins.^33–35

II. Classic Approaches To GPI Synthesis

1. Synthesis of a T. brucei GPI Anchor by the Ogawa Group (1991)

The Ogawa group reported the chemical synthesis of a T. brucei GPI anchor in 1991,^36,37 three years after Ferguson and co-workers published its complete structural determination. Ogawa’s reports, which were preceded by synthetic studies on GPI-related pseudooligosaccharides in the Fraser-Reid,³⁸ Ogawa,³⁹ and van Boom⁴⁰ laboratories, marked the first total synthesis of a GPI anchor. In accordance with the natural structure of the T. brucei GPI anchor, an additional α-Gal-(1→6)-α-Gal-(1→3) component located at Man-I was incorporated into the GPI core structure.

Scheme 2 depicts the retrosynthesis of T. brucei GPI anchor 1.While most modern GPI syntheses involve a convergent assembly featuring formation of the mannose–glucosamine linkage as the key and final glycosidic-bond formation (see Scheme 1), Ogawa’s pioneering effort relied on a different approach. The assembly strategy had both linear and convergent components, as benzyl-protected GPI precursor 2, which contained orthogonal protection for late-stage phosphorylation with H-phosphonates 3 and 4, was disconnected to give digalactosyl fluoride 5, mannosyl halide donors 6 and 7, and pseudotrisaccharide 8.The latter compound was further disconnected into the monosaccharide and inositol building blocks 9–11. For stereoselective glycosylation reactions, the Ogawa group primarily employed the Suzuki method, using glycosyl fluoride donors,⁴¹ while also making use of Lemieux’s halide ion-catalyzed glycosylation⁴² and the classical Koenigs–Knorr reaction.⁴³ The H-phosphonate method⁴⁴ was used to introduce the phosphoglycerolipid and phosphoethanolamine groups.

Scheme 2.

Retrosynthesis of T. brucei GPI anchor 1 by the Ogawa group.

The preparation of digalactosyl fluoride 5 is shown in Scheme 3. Penta-O-acetyl-α-D-galactopyranose (12) underwent treatment with p-methoxyphenol in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloroethane (DCE) to give a p-methoxyphenyl (PMP) glycoside, which was subsequently deacetylated and selectively protected at O-6 with 4,4′-dimethoxytrityl chloride (DMTrtCl) to provide 13. After the remaining hydroxyl groups had been protected by benzyl ether groups, acid-catalyzed methanolysis of the DMTrt group was performed to give the glycosyl acceptor 14. This compound underwent coupling with the previously described 1-thiogalactoside 15⁴⁵ under Lemieux conditions,⁴² namely α-stereoselective halide ion-catalyzed glycosidation of the in situ-generated glycosyl bromide. This procedure generated α-galactoside 16 in 67% yield (the β-anomer was isolated in 10% yield), and subsequent conversion into glycosyl fluoride 5 was accomplished by oxidative hydrolysis of the anomeric PMP group with ceric ammonium nitrate (CAN), followed by treatment with diethylaminosulfur trifluoride (DAST).

Scheme 3.

Synthesis of digalactosyl fluoride 5by the Ogawa group.

The main fragment in Ogawa’s synthesis, pseudotrisaccharide 8, was synthesized from building blocks 9–11 (Scheme 4). The mannosyl thioglycoside 9 was obtained from compound 18⁴⁶ through acetylation, anomeric acetolysis, and finally treatment with methyl tributyltin sulfide in the presence of tin (IV) chloride. The azidoglucose acceptor 10 was synthesized from triol 19,⁴⁷ which required acid-catalyzed 4,6-benzylidenation with PhCH(OMe)₂, benzylation at O-3, and regioselective benzylidene ring-opening with BH₃·NMe₃ in the presence of AlCl₃.⁴⁸

Scheme 4.

Synthesis of building blocks 9–11 and their elaboration to 8 by the Ogawa group.

Preparation of the enantiomerically pure inositol derivative 11 was lengthy. The synthesis began from racemic 2,3;4,5-di-O-cyclohexylidene-myo-inositol (±)-20,⁴⁹ Garegg’s inositol derivative, a common starting point for inositol synthesis. Stannylene acetal-directed p-methoxybenzylation of (±)-20 favored the O-6 position over the O-1 position (ratio = 3:1), leaving the latter site free for protection with an allyl group. After exchanging the cyclohexylidene acetals for benzyl groups to give (±)-21, the allyl group at O-1 was removed, exposing this position for reaction with the chiral resolving reagent (1S)-(−)-camphanic chloride, which generated separable diastereomeric inositol derivatives. At this stage, the absolute structure of the separated inositols was assigned by conversion into the previously characterized compound 2,3,4,5-penta-O-benzyl-myo-inositol. Unfortunately, the specific rotations for the D and L enantiomers of this compound were incorrectly reported in 1987,⁵⁰ an error whose correction in 1991⁵¹ was undetected by the Ogawa group prior to publication of their work.^52,53 Consequently, the wrong inositol enantiomer was chosen, which led to the total synthesis of an unnatural stereoisomer of the T. brucei GPI anchor, a feat made no less spectacular by this minor mishap.

Moving forward, the inositol intermediate was converted into 11 through a series of manipulations at the 1 and 6 positions. After CAN-mediated oxidative cleavage of the 6-O-PMB group, four steps were performed consecutively without chromatographic purification, including acid-catalyzed reprotection with a 1-ethoxyethyl ether (EE) group, exchange of the (−)-CAM group for a PMB group at O-1, and finally methanolysis of the 6-EE group by use of AcOH/MeOH.

The assembly of pseudotrisaccharide 8began by the coupling of compounds 9 and 10 (Scheme 4). The glycosylation occurred efficiently under the modified Lemieux conditions previously developed by Ogawa, specifically the use of AgOTf as an additional promoterin the presence of CuBr₂/Bu₄NBr to improve reactivity with poor glycosyl acceptors such as 10.⁵⁴ The α-disaccharide was generated in 90% yield and subsequently converted into glycosyl fluoride 23 by tetrabutylammonium fluoride (TBAF)-mediated anomeric desilylation followed by treatment of intermediate hemiacetal 22 with DAST. Compound23, bearing a non-participating 2-azido group adjacent to the leaving group, then underwent glycosylation with the inositol acceptor 11 by Suzuki’s method employing Cp₂ZrCl₂ and AgClO₄⁴¹ to form the crucial 1,2-cis glycosidic bond between the glucosamine and inositol components. The α anomer 24 was obtained in 73% yield, whereas the amount of the β anomer was minor (α:β ratio = 3.7:1), probably because of solvent participation from diethyl ether.⁵⁵ To expose the O-3 position of Man-I for addition of the digalactose moiety, compound 24 was deacetylated at the O-2 and O-6 positions of Man-I with NaOMe/MeOH and selectively re-acetylated at the O-6 position of Man-I to give 8.

Pseudotrisaccharide 8 was carried forward to the fully protected GPI precursor 2 as shown in Scheme 5. First, the digalactosyl group was installed at O-3 of Man-I by reaction of 8 with the digalactosyl fluoride 5 in the presence of Cp₂ZrCl₂/AgClO₄ in diethyl ether. An α,β mixture was obtained in 76% yield (α:β ratio = 9:1), and separation of the two anomers was performed following deacetylation at O-6 of Man-I with NaOMe in MeOH to give 25 (68% over two steps). During the glycosylation, pseudotetrasaccharide 26 and 1,6-anhydro sugar 27 were also formed (Scheme 6), suggesting that a portion of glycosyl fluoride 5 underwent decomposition via ring flip, cleavage of the galactose at O-6, and intramolecular attack of the liberated primary hydroxyl group on the oxocarbenium cation. Compound 25, bearing a primary hydroxyl group at O-6 of Man-I, was coupled with mannosyl chloride 6⁵⁶ in the presence of mercury(II) bromide and mercury(II) cyanide to generate the branched pseudohexasaccharide 28 with excellent α stereoselectivity (89% yield). Deacetylation of 28 gave compound 29, which reacted with mannosyl fluoride 7 (generated from its corresponding anomeric acetate⁵⁷ by deacetylation and treatment with DAST) in the presence of Cp₂ZrCl₂/AgClO₄ in diethyl ether to provide pseudoheptasaccharide 2 in 93% yield. Because of protecting-group complications, the acetyl group at O-6 on Man-III was exchanged for a chloroacetyl (CA) group, followed by removal of the PMB group at O-1 of the inositol with TMSOTf to give compound 30, which was set up for both late-stage phosphorylation events.

Scheme 5.

Completion of T. brucei GPI anchor 1 by the Ogawa group.

Scheme 6.

Generation of pseudotetrasaccharide 26 and anhydro sugar 27in a side reaction during the coupling of 5 and 8 by the Ogawa group.

First, the H-phosphonate method⁴⁴ was used to install a diacylglycerophosphate moiety to the O-1 position of inositol by reaction of 30 with H-phosphonate 3 and pivaolyl chloride (PivCl) in pyridine, affording intermediate 31 in 64% yield. Following removal of the 6-chloroacetyl group on Man-III with thiourea, this site was then allowed to react with H-phosphonate 4 in the presence of PivCl in 40% yield. After the intermediate had been oxidized with aqueous iodine, product 33 was subjected to global deprotection by hydrogenolysis, using Pd(OH)₂/C as catalyst, which afforded the target GPI anchor 1 in 23% yield.

In summary, the Ogawa group accomplished the first synthesis of a GPI anchor by using a combined linear–convergent strategy in concert with classical methods for formation of glycosidic bonds together with H-phosphonate chemistry. Although it was an impressive starting point for GPI synthesis, future improvements were called for in the areas of assembly sequence, inositol preparation, orthogonal protecting-group selection, global deprotection efficiency, and usage of more environmentally acceptable reagents.

2. Synthesis of a Yeast GPI Anchor by the Schmidt Group (1994)

In 1994, Schmidt and co-workers reported the total synthesis of a GPI anchor from yeast (Saccharomyces cerevisia).^58,59 This anchor contains a ceramide lipid region rather than the glycerolipid commonly found in other organisms. Synthesis of the target GPI 34 made use of a fully convergent strategy (Scheme 7). As with Ogawa, Schmidt’s laboratory performed two late-stage phosphorylations on a benzyl-protected GPI intermediate (35) to install the phosphoethanolamine and ceramide components. For this purpose, the phosphoramidite method,⁶⁰ employing building blocks 36 and 37, was used as an alternative to the H-phosphonate method, allowing incorporation of fully protected phosphate groups. Compound 35 was formed in a key glycosylation between the tetramannosyl trichloroacetimidate 38 and pseudodisaccharide 39. The former compound 38 was built up from mannose derivatives 40–42, while the latter was synthesized from inositol acceptor 43 and azidoglucosyl trichloroacetimidate 44. Schmidt’s own powerful method,⁶¹ employing glycosyl trichloroacetimidate donors, was exclusively used for glycosidic-bond formation, and acyl protecting groups at O-2 on the glycosyl donors aided the α-stereoselective formation of glycosidic bonds.

Scheme 7.

Retrosynthesis of yeast GPI anchor 34 by the Schmidt group.

For synthesis of the tetramannosyl donor 38, all required building blocks (40–42) were accessed from the known orthoester 45 (Scheme 8).⁵⁶ En route to the Man-I monomer 40, orthoester 45 was treated with tert-butylchlorodiphenylsilane in the presence of imidazole to selectively protect O-6, leaving the remaining sites for protection with benzyl groups to give the 3,4-diether 46. Acid-catalyzed hydrolysis of the 1,2-orthoester was followed by acetylation and regioselective hydrolysis of the anomeric acetate with (NH₄)₂CO₃. Subsequent treatment of the hemiacetal intermediate with trichloroacetonitrile and the non-nucleophilic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave the Man-III glycosyl donor 42. This compound was also elaborated to Man-I derivative 40 in four steps, including installation of allyl groups atO-1 and O-2 followed by 6-desilylation with TBAF/AcOH. The Man-II donor 41 was accessed from orthoester 45 by successive per-O-benzylation, orthoester hydrolysis, acetylation, selective anomeric deacetylation, and trichloroacetimidation, as just described for compound 42.

Scheme 8.

Synthesis of building blocks 40–42 and their elaboration to tetramannosyl donor 38 by the Schmidt group.

Coupling of the Man-I acceptor 40 and Man-II trichloroacetimidate donor 41 in the presence of catalytic TMSOTf in Et₂O provided dimannoside 47 in 84% yield with complete α-stereoselectivity. Following removal of the temporary stereodirecting Man-II 2-O-acetyl group with NaOMe/MeOH, the acceptor 48 was allowed to react with Man-III trichloroacetimidate donor 42 under similar conditions, affording trimannoside 49 in 92% yield, again with complete α-stereoselectivity through neighboring-group participation. A final iteration of this process, using building block 41, installed the Man-IV unit in 91% yield. Conversion of the allyl tetramannoside 50 into trichloroacetimidate 38 required four steps, including double deallylation with Wilkinson’s catalyst, acetylation of the intermediate diol, regioselective anomeric deacetylation with (NH₄)₂CO₃, and treatment with trichloroacetonitrile and DBU. This sequence served to activate the anomeric center as a trichloroacetimidate and install a participating acetyl group at the O-2 position of Man-I to facilitate α-stereoselectivity in the key glycosylation.

The synthesis of pseudodisaccharide 39 featured efficient preparation of the inositol derivative 43, followed by a highly stereoselective glycosylation reaction with the azidoglucosyl donor44 (Scheme 9). Synthesis of 43 was accomplished by enantiomeric resolution of inositol diketal (±)-20,⁴⁹ which underwent treatment with bis(tributyltin)oxide followed by reaction with the chiral reagent (−)-menthyl chloroformate [(−)-MntOCOCl] to give the diastereomeric carbonates 43 and 51,⁶² which were separated by recrystallization. Compound 43 was directly glycosylated by the azidoglucosyl trichloroacetimidate 44⁶³ in the presence of TMSOTf to give exclusively the α stereoisomer 52 in 85% yield. The authors attributed the exceptional stereoselectivity observed in this coupling to an appropriate choice of protecting groups and reaction optimization, which still remains a challenging task for forming the 1,2-cis glycosidic bond between azidoglucosyl donors and inositol acceptors.

Scheme 9.

Synthesis of pseudodisaccharide 37 by Schmidt.

To expose the O-4 of the azidoglucose unit for the key glycosylation, compound 52 was converted into pseudodisaccharide 39 in three steps, each featuring crucial regiocontrol. First of all, selective deacetylation with NaOMe/MeOH formed an intermediate triol while leaving the (−)-menthyl carbonate intact. The O-6 position of the azidoglucose component was then selectively benzoylated with BzCN in the presence of triethylamine at −70 °C. Finally, selective 3-O-benzylation with BnBr promoted by Ag₂O gave the pseudodisaccharide 39 in 53% yield over three steps. Overall, the highly economical synthesis of this key intermediate constituted an impressive demonstration of regio- and stereocontrol.

Completion of the synthesis of yeast GPI anchor 34 is shown in Scheme 10. The key glycosylation reaction between tetramannosyl donor 38 and pseudodisaccharide acceptor 39 was accomplished using catalytic TMSOTf in diethyl ether, which afforded the desired α-pseudohexasaccharide intermediate in an excellent 91% yield. Unfortunately, several protecting-group changes were required to access GPI intermediate 35 prior to the two phosphorylation events, including removal of all acyl groups with KCN/MeOH (45%) and reprotection of the exposed positions with benzyl groups, as well as replacement of the 1-O-(−)-menthyl carbonate group of the inositol by an acetyl group. These late- stage protecting-group manipulations, which are best avoided on complex intermediates, were the price to be paid for a short pseudodisaccharide synthesis (Scheme 9), where appropriate protection steps were postponed.

Scheme 10.

Completion of yeast GPI anchor 34 by the Schmidt group.

Compound 35 was made ready for installation of the phosphoethanolamine group after TBAF-mediated removal of the 6-O-TBDPS group on Man-III. Formation of the phosphotriester was achieved by 1H-tetrazole-promoted phosphitylation with phosphoramidite 36, followed by in situ oxidation with t-BuOOH. Subsequent treatment with NaOMe/MeOH cleaved both the cyanoethoxyl and the inositol 1-O-acetyl groups to give phosphodiester 54, which was poised for attachment of the ceramide moiety. Ceramide phosphoramidite 37, which was prepared readily from a previously reported azido lipid,⁶⁴ reacted with intermediate 54 in the presence of 1H-tetrazole, and sequential in situ oxidation with t-BuOOH, and cyanoethoxyl removal with dimethylamine afforded GPI intermediate 55. Two steps were required for global deprotection, including acid-catalyzed alcoholysis of the acetal protecting groups (63%) and Pd-catalyzed reductive removal of the benzyl/benzyloxycarbonyl groups and reduction of the azido group (70%) to give the target GPI anchor 34.

Schmidt’s total synthesis of yeast GPI anchor 34 was highlighted by incorporation of a ceramide phospholipid, showcasing the trichloroacetimidate “Schmidt” glycosylation method, and perhaps most impressively synthesizing pseudodisaccharide 39 with high efficiency and superb control of regio- and stereoselectivity. Furthermore, global removal of the benzyl groups using Pd-catalyzed hydrogenolysis featured an improved yield (70%) as compared with Ogawa’s result in the first total synthesis of a GPI anchor (23%). The versatility of the strategy was further demonstrated by Schmidt’s use of building blocks from this synthesis in subsequent syntheses of GPIs from Toxoplasma gondii²⁴ and rat brain Thy-1 (for the latter, see Section II-5).

3. Synthesis of a Rat Brain Thy-1 GPI Anchor by the Fraser-Reid Group (1995)

The Fraser-Reid group published several synthetic studies on GPI partial structures^38,65–69 prior to the 1995 communication by Campbell and Fraser-Reid describing the synthesis of a rat brain Thy-1 GPI anchor, which constituted the first total synthesis of a mammalian GPI and the third total synthesis in general of a GPI.⁷⁰ The Thy-1 GPI glycan contains an additional phosphoethanolamine group at the O-2 position of Man-I (a common modification in mammalian GPIs), as well as GalNAc and Man components attached to the O-4 position of Man-I and the O-2 position of Man-III, respectively. These modifications, while numerous, were all effectively incorporated into the “fully phosphorylated” target molecule by Fraser-Reid.

A convergent approach was used to synthesize the Thy-1 GPI (compound 56), in which a [3 + 2 + 2] block strategy was applied (Scheme 11). Because of the additional phosphoethanolamine group present in the target, the benzyl-protected intermediate 57 would undergo three sequential phosphorylation reactions at sites bearing appropriate orthogonal protecting-groups. As with Schmidt, Fraser-Reid used the phosphoramidite method^60,66 with precursors 58 and 59 to install the phosphoethanolamine and phosphoglycerolipid groups. Compound 57 was synthesized from trimannoside 60, disaccharide 61, and pseudodisaccharide 62, which were each accessed from their corresponding sugar and inositol monomers 63–69. A combination of glycosylation methods were used for stereoselective formation of glycosidic bonds, including the Koenigs–Knorr method,⁴³ Schmidt’s trichloroacetimidate method,⁷¹ and Fraser-Reid’s own method employing the versatile n-pentenyl glycosides (NPGs).⁷²

Scheme 11.

Retrosynthesis of Thy-1 GPI anchor 56 by the Fraser-Reid group.

The synthesis of n-pentenyl trimannoside 60 relied on sequential Koenigs–Knorr glycosylations using mannose monomers 63–65, which were all prepared from the n-pentenyl orthoester⁷³ 70⁶⁸ (Scheme 12). This intermediate allowed for ready conversion into 2-O-benzoyl-protected donors, thus facilitating α-stereoselective glycosylations and flexibility with regard to choice of leaving group at the anomeric position. The Man-II component 63 was synthesized from 70 by benzylation, acid-catalyzed rearrangement of the n-pentenyl orthoester to the corresponding n-pentenyl glycoside, and then removal of the newly formed 2-O-benzoyl group with NaOMe. Compound 70 was converted into the Man-III component 64 by 6-O-silylation with TBDPSCl/imidazole, benzylation, and treatment with bromine. The Man-IV donor 65, also a glycosyl bromide, was readily generated from 70 by benzylation and treatment with bromine.

Scheme 12.

Synthesis of n-pentenyl trimannoside 60 by the Fraser-Reid group.

The Man-II acceptor 63 and Man-III donor 64 were coupled in the presence of promoter AgOTf, resulting in the formation of α-dimannoside 73 in 89% yield. Removal of the temporary stereodirecting 2-O-benzoyl group was accomplished with NaOMe to afford alcohol 74, which underwent α-stereoselective glycosylation with the Man-IV glycosyl bromide 65, again under the influence of AgOTf. The product 75 was isolated in 74% yield, and subsequently underwent two protecting-group exchanges to provide the n-pentenyl trimannoside 60. First, the stereodirecting 2-O-benzoyl group of Man-IV was replaced by a benzyl group, and then the 6-O-TBDPS group of Man-III was exchanged for a chloroacetyl group by TBAF-mediated desilylation followed by treatment with chloroacetic anhydride in the presence of triethylamine.

The β-GalNAc-Man disaccharide 61was prepared from the n-pentenyl mannoside 77⁷⁴ and tetraacetate 79,⁷⁵ as shown in Scheme 13. Compound 77, constituting the Man-I building block, was treated with PhCH(OMe)₂ in the presence of pyridinium p-toluenesulfonate (PPTS) to generate the 4,6-benzylidene acetal. Stannylene acetal-directed, regioselective 3-O-benzylation was followed by acetylation to give compound 78, which was converted in to the Man-I acceptor 66 by acid-promoted hydrolysis of the benzylidene group and selective acetylation at O-6. The 2-phthalimido galactosyl trichloroacetimidate 67 was accessed from 2-deoxy-2-phthalimido-β-D-galactose tetraacetate (79) by successive TMSOTf-promoted glycosidation with p-methoxyphenol, deacetylation, benzylation, oxidative hydrolysis of the anomeric PMP group, and finally reaction with trichloroacetonitrile and DBU.

Scheme 13.

Synthesis of β-GalNAc-Man disaccharide 61 by the Fraser-Reid group.

The TMSOTf-catalyzed Schmidt glycosylation of66by67 was completely β-stereoselective because of the participating effect of the 2-phthalimido group. The β-disaccharide product 81 was isolated in 79% yield, and afterwards the temporary 2-phthalimido group was replaced by a 2-acetamido group via sequential aminolysis and selective N-acetylation. Differentiation of the two free hydroxyl groups on Man-I was accomplished by selective O-6 chloroacetylation followed by acetylation at O-2, affording building block 61.

Fraser-Reid’s synthesis of the pseudodisaccharide fragment began with Garegg’s indispensable inositol derivative (±)-20⁴⁹ (Scheme 14), as did the previous GPI syntheses by Ogawa and Schmidt. The route was initiated by stannylene-mediated p-methoxybenzylation at O-6, which was followed by esterification of the free O-1 position by the chiral resolution reagent (1S)-(−)-camphanic chloride.⁷⁶ After separation, the correct diastereomer was subjected to saponification with LiOH followed by allylation to provide the optically active 82. After the cyclohexylidene acetals had been replaced by benzyl groups, CAN-mediated cleavage of the PMB group at O-6 furnished the inositol acceptor 68. To decrease loss of material, Fraser-Reid also developed a route to convert the 1-p-methoxybenzylated inositol regioisomer, generated from (±)-20 in the first step, into compound 68 (not shown here).⁶⁹ The 2-azido glucosyl bromide 69 was synthesized from 1,6-anhydro-2,3,4-tri-O-benzyl-β-D-mannopyranose (83) via intermediate 84 (a route developed by Hori and co-workers⁷⁷), which after trifluoroacetic acid (TFA)-promoted ring opening/acetylation was treated with TiBr₄ to afford 69.

Scheme 14.

Synthesis of pseudodisaccharide 62 by the Fraser-Reid group.

The Koenigs–Knorr-type glycosylation between inositol acceptor 68 and azido glucosyl bromide 69 proceeded with acceptable α-stereoselectivity when using AgClO₄ in diethyl ether. The inseparable α,β mixture 85, obtained in 85% yield (α:β = 3:1), was subjected to deacetylation at O-4 and O-6, after which the preponderant α-pseudodisaccharide diol 86 was separated out. The remaining task of selective benzylation at O-6 turned out to be quite problematic, as the authors were forced to perform five successive steps, namely selective 6-O-acetylation, 4-O-tetrahydropyranylation (THP), deacetylation, 6-O-benzylation, and acid-promoted methanolysis of the THP group. This sequence blemished an otherwise efficient synthesis of pseudodisaccharide fragment 62.

Assembly of the pseudoheptasaccharide from subunits 60–62using the [3 + 2 + 2] block approach was accomplished with n-pentenyl glycoside chemistry (Scheme 15). The n-pentenyl disaccharide 61 was chemoselectively activated by N-iodosuccinimide (NIS) and triethylsilyl trifluoromethansulfonate (TESOTf), and subsequent reaction with pseudodisaccharide acceptor 62 gave the α-pseudotetrasaccharide 87 as the sole isomer in 55% yield. The remainder of the mass balance was attributed to triethylsilylation of the acceptor hydroxyl group. To prepare for chain extension of the glycan, the 6-chloroacetyl group on Man-I of 87 was removed with thiourea to give 88, which reacted with n-pentenyl trimannoside 60 in the presence of NIS/TESOTf to afford α-pseudoheptasaccharide 57 in moderate (32%) yield [39% based on recovered starting material (BRSM)]. Again, triethylsilylation of the acceptor hydroxyl group accounted for the low yield, although the silylated product could be recovered and transformed back into 88 by treatment with TBAF. This recycling effort would not have been possible without the earlier protecting-group exchange at O-6 of Man-III from TBDPS to chloroacetyl (Scheme 12), which on first glance may have seemed unnecessary. Also of note, Fraser-Reid took advantage of the ability of n-pentenyl glycosides to be selectively activated with NIS/TESOTf in the presence of allyl protecting groups in this sequence.

Scheme 15.

Completion of mammalian Thy-1 GPI anchor 56 by the Fraser-Reid group.

Pseudoheptasaccharide 57 was elaborated to the target GPI (56) via three phosphorylation events and global deprotection. Phosphoramidite chemistry optimized for GPI synthesis by Fraser-Reid⁶⁶ was used to form the appropriate phosphotriester bonds efficiently. For the phosphoethanolamine group on Man-III, dechloroacetylation of 57 was followed by 1H-tetrazole-promoted phosphitylation with 58 and in situ oxidation with m-chloroperoxybenzoic acid (m-CPBA). After deacetylation of the O-2 position of Man-I, the same conditions were used to install a phosphoethanolamine group at this site. Next, compound 90 was subjected to Pd-mediated removal of the inositol O-1 allyl group to give 91, which was coupled with phospholipid precursor 59, again using the phosphoramidite method. All three phosphorylation events proceeded smoothly and in high yield (75–90%).Finally, hydrogenolytic global deprotection with Pd(OH)₂/C and H₂ provided the fully phosphorylated Thy-1 GPI 56 in 75% yield.

The in-depth synthetic studies on GPI synthesis between 1989 and 1995 by the Fraser-Reid group culminated in the first total synthesis of a mammalian GPI anchor, namely Thy-1 GPI (56). Through careful choice of assembly strategy and orthogonal protecting groups, the structural modifications present in the natural compound were successfully incorporated into the target molecule, including two sugar groups and an additional phosphoethanolamine group. Fraser-Reid’s n-pentenyl glycoside chemistry was used prominently, both in the preparation of mannose building-blocks and in the final two stereoselective glycosylations to join fragments 60–62. Where necessary, the glycosylation methods of Koenigs–Knorr and Schmidt were also used for stereoselective formation of glycosidic bonds.

4. Synthesis of a T. brucei GPI Anchor by the Ley Group (1998)

In contrast to the combined linear–convergent synthesis of the T. brucei GPI anchor 1 reported by Ogawa,³⁶ the Ley group used a highly convergent route (Scheme 16).^78,79 The target molecule 1 was accessed from a fully protected pseudoheptasaccharide 93 via sequential phosphorylation reactions with 58 and 94 using the phosphoramidite method.⁶⁰ In turn, compound 93 was formed in a key glycosylation by two highly elaborated building-blocks, namely pentasaccharide donor 95 and pseudodisaccharide 96. To synthesize these intermediates, the Ley group made elegant use of their bis(dihydropyran) chemistry for selective protection and desymmetrization procedures.^80–82 Rapid preparation of pentasaccharide 95 from building blocks 99–103 (via 97 and 98) relied on tuning of the reactivity of glycosyl donors using judicious protecting-group choices,⁸³ as well as by taking advantage of selenoglycoside and thioglycoside orthogonality.⁸⁴ Pseudodisaccharide 96 was generated from azido glucosyl bromide 104 and inositol acceptor 68, the latter of which was made chiral by using bis(dihydropyran)-based desymmetrization.

Scheme 16.

Retrosynthesis of T. brucei GPI anchor 1 by the Ley group.

Building blocks 99–103 were generated from seleno- and thio-glycosides 105–107 (Scheme 17). The Man-I building-block 99 was obtained from thioglycoside 105⁸⁵ in seven steps. Selective installation of a TBS group at O-6 was followed by cis-acetonation to protect the O-2 and O-3 positions, which readied O-4 for benzylation. After deacetonation, intermediate 108 under went trimethylsilylation at O-3, chloroacetylation at O-2, and aqueous HF-mediated desilylation to provide compound 99. The selenogalactoside 106⁸⁶ was used as a common starting point for the preparation of compounds 100 and 101. Thus, compound 106 was subjected to 2,3-butanediacetal (BDA) protection with butane-2,3-dione/CSA, selective silylation at O-6 with TBSCl, chloroacetylation of the free axial OH group at O-4, and removal of the TBS group with aqueous HF to give the glycosyl acceptor 100. Alternatively, compound 106 was readily perbenzylated to give glycosyl donor 101. Finally, selenomannoside 107⁸⁷ was readily converted into the Man-II and Man-III building blocks 102 and 103, the former being accessed by 3,4-BDA protection followed by tin-mediated chloroacetylation at O-6, and the latter by silylation at O-6 with TBSCl followed by benzylation.

Scheme 17.

Synthesis of building blocks99–103 by the Ley group.

With careful use of “armed” and “disarmed” glycosyl donors, as introduced by Fraser-Reid,⁸³ as well as by using selectively activatable selenoglycosides and thioglycosides,⁸⁴ the preparation of pentasaccharide 95 remarkably took only five steps from building blocks 99–103 (Scheme 18). First, the glycosyl acceptor 100, whose anomeric selenoacetal group was disarmed by virtue of the deactivating BDA⁸¹ and chloroacetyl groups, was coupled with the benzyl-protected, armed glycosyl donor 101 in the presence of NIS/TMSOTf. The α-digalactoside 111 was obtained in 71% yield, while the β anomer was isolated in 15% yield, and no homocoupling product was observed. Subsequent reaction of 111 with the Man-I thioglycoside acceptor 99 was facilitated by chemoselective activation of the selenoacetal with MeOTf,⁸⁸ affording the α-trisaccharide 112 in 75% yield. The α-dimannose fragment 98 was generated in 87% yield by selective coupling of selenoglycosides 102 (disarmed) and103 (armed) in the presence of NIS/TMSOTf. Finally, compound 98was combined with 97 (generated from 112 by desilylation), again relying on the higher reactivity of selenoglycosides with MeOTf as compared to thioglycosides (product generated in 75% yield). A large excess of the donor and high concentrations of reactants were used to minimize formation of an anhydro sugar resulting from intramolecular glycosidation of 97 through attack from O-6. Overall, the α-pentasaccharide 95 was efficiently prepared from compounds 99–103 by employing strategies to control anomeric reactivity.

Scheme 18.

Synthesis of pentasaccharide 95 by the Ley group.

The synthesis of pseudodisaccharide 62 shown in Scheme 19 was highlighted by Ley’s efficient preparation of inositol derivative 68 using bis(dihydropyran)-mediated desymmetrization. The known symmetrical tetraol 113⁸⁹ reacted with the chiral bis(dihydropyran) 114 in the presence of CSA to form dispiroketal 115 (ee ≥ 98%) in 88% yield. Positions 2–5 were masked with benzyl groups by deacylation and perbenzylation. The dispiroketal group was removed in a two-step process involving oxidation with m-CPBA followed by treatment with LiN(TMS)₂. The inositol derivative 68 was then formed by stannylene acetal-directed regioselective allylation at the O-1 position. The azido glucosyl bromide 104 was prepared by conventional methods beginning with thioglycoside 116,⁹⁰ which underwent benzylation at O-3, regioselective cleavage of the benzylidene ring with Et₃SiH/TFA, and exchange of the phthalimido group by an azido group using the diazo-transfer chemistry developed by Vasella and co-workers.⁹¹ This four-step procedure generated compound 117, which was silylated at O-4 with TBSCl/KHMDS and then treated with bromine to give the glycosyl bromide 104. Lemieux conditions⁴² were employed for α-stereoselective joining of inositol acceptor 68 and glycosyl bromide 104, which provided compound 96 in 65% yield. Removal of the TBS group at O-4 was accomplished by treatment with TBAF, giving the pseudodisaccharide acceptor 62.

Scheme 19.

Synthesis of pseudodisaccharide 62 by the Ley group.

Completion of the synthesis of T. brucei GPI anchor 1 by the Ley group is shown in Scheme 20. The key glycosylation between the pentasaccharide ethylthio donor 95 and pseudodisaccharide 62 was promoted by NIS/TMSOTf and generated the α-pseudoheptasaccharide 93 in 51% yield. Installation of the phosphoethanolamine group was performed by treatment with aqueous HF to expose the O-6 position of Man-III, followed by reaction at this site with the phosphoramidite 58 and in situ oxidation with m-CPBA. Intermediate 119 underwent Pd-mediated deallylation to deprotect the O-1 position of the inositol, which was followed by phospholipidation with 94 [synthesized using a bis(dihydropyran)-controlled desymmetrization of glycerol], again using phosphoramidite chemistry, to provide compound 121. The global deprotection required three sets of conditions, including Pd-catalyzed hydrogenolysis, thiourea-mediated dechloroacetylation, and finally TFA-promoted hydrolysis of the BDA groups. This sequence generated the target GPI 1 in an impressive 90% yield over the final three steps.

Scheme 20.

Completion of T. brucei GPI anchor 1 by the Ley group.

In summary, Ley and co-workers successfully completed the fourth total synthesis of a GPI anchor, and this was the second effort aimed at the GPI (1) of T. brucei, following Ogawa’s seminal work. This synthesis served as a proving ground for methods developed in the Ley laboratory, namely the usage of bis(dihydropyrans) as reactivity-tuning BDA protecting-groups and desymmetrizing chiral reagents. These chemistries were elegantly integrated with strategies for selective activation of the glycosyl donor, specifically Fraser-Reid’s armed/disarmed concept and orthogonally activatable anomeric groups (selenoglycosides and thioglycosides), culminating in a highly efficient convergent synthesis of the target molecule.

5. Synthesis of a Rat Brain Thy-1 GPI Anchor by the Schmidt Group (1999, 2003)

Following their 1994 synthesis of a yeast GPI anchor,⁵⁸ the Schmidt group reported a synthesis of the Thy-1 GPI anchor (56) of rat brain in 1999,⁹² a target that had previously yielded to total synthesis by Fraser-Reid in 1995 (see Section II-3).⁷⁰ Unsurprisingly, Schmidt’s assembly strategy closely resembled that of Fraser-Reid, particularly in the synthetic end-game. We forgo here a detailed discussion of this 1999 report, instead opting to cover a 2003 paper by Schmidt in which the Thy-1 GPI anchor was synthesized by a similar but modified strategy; it emphasized future couplings with peptides/glycopeptides at the phosphoethanolamine group of Man-III,⁹³ a strategic decision that foreshadowed looming advances in GPI–protein synthetic methods. In terms of synthetic strategy, this work was also closely tied to a 2001 report by Schmidt disclosing the synthesis of a partial structure of a non-lipidated GPI from the parasitic protozoan T. gondii.²⁴

The retrosynthetic analysis from Schmidt’s 2003 synthesis of the Thy-1 GPI anchor is shown in Scheme 21. The precursor to the target molecule, the benzyl-protected pseudoheptasaccharide 122, differed only slightly from the corresponding intermediate in Fraser-Reid’s synthesis of the Thy-1 GPI (compound 57, Scheme 11). However, late-stage phosphorylation events were designed to install orthogonally protected phosphoethanolamine groups (using the phosphoramidites 36 and 123), which would permit selective amino group deprotection and therefore the opportunity for future peptide/glycopeptide conjugation at Man-III. Intermediates 125 and 126 were employed in a highly convergent assembly strategy, for which the Schmidt group capitalized on the versatility of their previous GPI syntheses by employing some of the same building blocks. Naturally, the Schmidt trichloroacetimidate method⁶³ was used for stereoselective formation of glycosidic bonds.

The synthesis of pentasaccharide donor 125 was accomplished by stepwise elongation of the βGal-Man disaccharide 127 with mannosyl trichloroacetimidates 41 and 42 (Scheme 22). In turn, disaccharide 127was prepared from monosaccharides 129 and 131. First, the allyl mannoside 128⁹⁴ was converted into Man-I derivative 129via stannylene-mediated benzylation at O-3, p-methoxybenzylidenation at O-4 and O-6, allylation at O-2, and regioselective reductive cleavage of the acetal ring with NaBH₃CN/TFA. Preparation of the galactosyl trichloroacetimidate 131, which contained a trichloroacetamido (TCA) group to ensure β-selective glycosylation while also acting as a latent acetamido group, was initiated from the known azido galactose derivative 130.⁷¹ The anomeric position of130 was temporarily masked with a thexyldimethylsilyl (TDS) group, followed by exchange of the azide group for a trichloroacetamido group. Conversion into the glycosyl donor 131 was accomplished by anomeric desilylation with TBAF, followed by treatment with trichloroacetonitrile and DBU. Coupling of Man-I acceptor 129 and trichloroacetamido galactosyl donor 131 in the presence of boron trifluoride etherate afforded exclusively the β-linked disaccharide 132, which was treated with CAN to oxidatively cleave the PMB group to give 127.

Starting from protected disaccharide 127, consecutive mannosylations using donors 41 and 42 were effected under standard Schmidt glycosylation conditions to extend the glycan chain. Each of the three glycosylations was high yielding (92–96%) and highly α-stereoselective because of neighboring-group participation. The pentasaccharide 137 generated through this process required several manipulations to obtain donor 125, starting with radical reduction of the trichloroacetamido group to form an acetamido group, followed by deacetylation–benzylation of the O-2 position of Man-III. The Man-I component was then adjusted via double deallylation with Wilkinson’s catalyst, followed by diacylation with phenoxyacetyl (PA) chloride. Selective anomeric deacylation with (NH₄)₂CO₃ generated an intermediate hemiacetal that was treated with trichloroacetonitrile and DBU to provide the donor compound 125.

Compound 126 differed from the pseudodisaccharide building-blocks in previous GPI syntheses because N-tert-butyloxycarbonyl (Boc) protection was used rather than the traditional azido group. This was necessary to permitlate-stage selective deprotection of the Man-III phosphoethanolamine (Cbz-protected) for peptide conjugation. The preparation of 126 began from inositol derivative 11, which Schmidt generated from the optically active inositol 43 in a few straightforward steps (Scheme 23). Allylation at O-6 was assisted by Ag₂O rather than basic conditions, so as not to affect the carbonate bond. After replacement of the 1-O-menthyl carbonate with a PMB group and the cyclohexylidene acetals with benzyl groups, the allyl group ot O-6 was removed with Wilkinson’s catalyst to give 11.

Scheme 23.

Synthesis of pseudodisaccharide 126 by the Schmidt group.

Inositol acceptor 11 was then coupled with azido glucosyl trichloroacetimidate 44 in the presence of TMSOTf, which afforded pseudodisaccharide 139 in 70% yield. Numerous protecting-group manipulations were required at this point to prepare the azido glucose unit for the remainder of the synthesis. First, deacetylation with NaOMe gave an intermediate triol that was masked with a 4,6-O-benzylidene ring to give the pseudodisaccharide derivative 140, which was converted into compound 141 by benzylation at O-3, regioselective benzylidene cleavage, and phenoxyacetylation at O-4 with PACl in pyridine. Exchange of the inositol 1-O-PMB group for a benzoyl group, and dephenoxyacetylation with MeNH₂ preceded the crucial amino-protection swap, which was accomplished by propanedithiol-assisted azide reduction and treatment with Boc anhydride. The lengthy route to 126 dampened the overall efficiency of this GPI synthesis.

Completion of the Thy-1 GPI anchor synthesis by Schmidt is shown in Scheme 24. The key glycosylation between pentasaccharide donor 125 and pseudodisaccharide acceptor 126 under promotion by TMSOTf afforded the expected α-pseudoheptasaccharide 122 in 74% yield. The TBDPS group on O-6 of Man-III was removed with TBAF, making intermediate 142 ready for phosphorylation with compound 36 using the phosphoramidite method. In situ cleavage of the PA group at O-2 of Man I and cyanoethoxyl groups with MeNH₂ generated alcohol 143, which underwent installation of the second phosphoethanolamine group by reaction with 123, again using phosphoramidite chemistry. Phospholipidation of GPI intermediate 144 was accomplished by 1-O-debenzoylation of the inositol, followed by reaction with phosphoramidite 124. Concomitant in situ oxidation and aminolysis (to remove the final cyanoexothyl group) afforded the partially deprotected GPI derivative 146. At this stage, the authors demonstrated the feature of orthogonal amino-group protection by subjecting compound 146 to Pd-catalyzed hydrogenolysis to generate GPI 147 (75%), which contained a single free amino group at the Man-III phosphoethanolamine component for future coupling with peptides/glycopeptides. In addition, intermediate 146 was subjected to a two-step global deprotection, including acidic and reductive conditions, resulting in formation of the rat-brain Thy-1 GPI anchor 56 in 54% yield over the final two steps.

Schmidt and co-workers’ 2003 synthesis of the rat-brain Thy-1 GPI anchor, the second time they accomplished this feat, combined elements from their previous work in the field, including syntheses of GPI anchors (or related glycoconjugates) from yeast (1994), Thy-1 (1999), and T. gondii (2001). Thoughtfully-designed building blocks were reused throughout these projects, a testament to their versatility. The syntheses of these GPIs relied on convergent assembly tactics in combination with expert use of glycosyl trichloroacetimidate and phosphoramidite chemistries for key bond-forming events. In the 2003 Thy-1 GPI synthesis, a strategy for orthogonal amino-group protection, which would permit future peptide/glycopeptide conjugation at Man-III, was demonstrated, although this feature was not applied. The Guo group was the first team to report the chemical synthesis of GPI-anchored peptides³⁰ and glycopeptides,³¹ in 2003 and 2004, respectively (see Scheme 31), but these constructs did not contain GPI anchors of natural structures.

Scheme 31.

GPI-peptide/glycopeptide conjugates 175 and 176 chemically prepared by the Guo group.

6. Synthesis of a Human Sperm CD52 Antigen GPI Anchor Containing an Acylated Inositol by the Guo Group (2003)

The second mammalian GPI anchor to be chemically synthesized was that of the human sperm CD52 antigen by the Guo group in 2003.⁹⁵ Related synthetic studies by the Guo group included the total synthesis of CD52 GPI-peptide/glycopeptide conjugates bearing GPI partial structures (see Scheme 31).^30,31 In addition to the intriguing and therapeutically relevant biological activities^96,97 of the sperm and lymphocyte CD52 antigens, these GPI-anchored glycopeptides exhibit a short peptide chain (12 amino acids) and interesting GPI structures,^98,99 making them an ideal model for synthetic studies on GPIs and GPI-anchored molecules. Notably, the 2003 report by Xue and Guo was the first synthesis of a GPI anchor of the CD52 antigen and also the first of a GPI containing an acylated inositol, which is a structural feature present in many GPIs.¹ Acylation at O-2 of the inositol is important during GPI biosynthesis, although it is often deleted after biosynthesis is accomplished, and it also confers resistance of GPIs to cleavage by bacterial PI phospholipase C.^2,100 To develop a synthetic strategy to access these GPIs for structural and biological studies, the inositol-acylated GPI 148 was targeted for synthesis.

A convergent strategy was used for synthesizing 148 (Scheme 25), whose fully protected precursor 149 would undergo late-stage installation of phosphoethanolamine by phosphoramidite 150, which exhibited orthogonal protection of the amino group to permit future peptide/glycopeptide coupling. Uniquely, rather than perform phospholipidation at a late stage as in other reported GPI syntheses, this component was attached to the pseudodisaccharide fragment prior to the key glycosylation reaction. This decision was made because of the observation of an undesired cyclophosphitamidation reaction during the attempted phospholipidation of inositol-acylated pseudopentasaccharide 158, which in fact generated compound 161 (Scheme 26). Because such reactivity was not observed by other research groups, or on pseudodisaccharide model-systems,¹⁰¹ it was proposed that the coexistence of the 2-O-acyl group on the inositol along with the trimannose moiety forced the GPI intermediate159 into a conformation that favored the intramolecular redox cyclization reaction that was observed. Therefore, the key coupling to give 149 would take place between the trimannose thioglycoside 151 and phospholipidated pseudodisaccharide 152. The former intermediate was built up from the monosaccharide mannose derivatives 153, 6, and 154, while the latter was synthesized from the inositol derivative 155, the azido glucosyl fluoride 156, and phosphoramidite 157. Glycosyl halides were primarily used as sugar donors, and the phosphoramidite method was used for phosphotriester formation.

Scheme 25.

Retrosynthesis of GPI anchor 148of the sperm CD52 antigen by the Guo group.

The synthesis of trimannose thioglycoside 151 was straightforward and relied on the orthogonal relationship of glycosyl halides and thioglycosides (Scheme 27). The thioglycoside (153) of Man-I was obtained from methyl α-D-mannopyranoside (162). Benzylation and sulfuric acid-promoted acetolysis of 162 gave diacetate 163, which on BF₃-promoted glycosylation with ethanethiol and subsequent deacetylation at O-6 provided 153. Koenigs–Knorr glycosylation of the Man-I acceptor 153 by the Man-II glycosyl chloride 6⁵⁶ in the presence of AgOTf provided α-dimannoside 164 in 82% yield with complete stereoselectivity. Removal of the temporary stereodirecting 2-O-acetyl group on Man-II generated 165, which was further glycosylated by glycosyl bromide 154 (also formed from 163 by treatment with HBr/AcOH), under the influence of AgOTf, to give the α-trimannoside product 166 in moderate (53%) yield. Finally, the 6-O-acetyl group on Man-III was exchanged for a TBS group to furnish 151.

Scheme 27.

Synthesis of trimannose thioglycoside 151 by the Guo group.

Motivated by the typical 50% material loss when resolving inositol enantiomers, Guo devised an inositol preparation based largely on tin-mediated chemistry for utilizing both enantiomers, namely (+)-20 and (−)-20, to improve efficiency (Scheme 28). Compound (+)-20 underwent stannylene acetal-directed allylation at O-6, leaving the O-1 position free for p-methoxybenzylation. Next, the less-stable trans-cyclohexylidene ring was selectively methanolyzed under acidic conditions, freeing the O-4 and O-5 positions for benzylation. The product from this sequence (167) was treated under acidic conditions for a prolonged period to cleave the more stable cis-cyclohexylidene ring, thus generating a diol whose O-3 position was selectively benzylated by using tin chemistry. This route gave the inositol derivative 168, which was also accessed from the enantiomer (−)-20 by using the opposite sequence. Dibenzylation of (−)-20 was followed by removal of the trans-cyclohexylidene group, selective allylation at O-6, and benzylation at O-5. The product 169 converged to the common intermediate 168 by cleavage of the cis-cyclohexylidene substituent and selective p-methoxybenzylation at O-1. Transformation of 168 into the correct derivative involved esterification of the free axial alcohol with hexadecanoic (palmitic) acid in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), followed by Pd-mediated deallylation to give the 2-O-acylated inositol 155.

Synthesis of the phospholipidated pseudodisaccharide 152 was completed as shown in Scheme 29. The azido glucosyl fluoride building block 156 was prepared from the known anhydro sugar 170.¹⁰² First, acetolysis of 170 gave the diacetate 171, whose anomeric acetate was exchanged for fluoride through selective deacetylation and treatment with DAST. Deacetylation at O-6 followed by benzylation gave donor 156, which underwent coupling with 155 in the presence of Cp₂HfCl₂/AgOTf.⁴¹ An anomeric mixture was generated in 70% yield (α:β = 4:3), from which the desired α-pseudodisaccharide 172 was isolated in 40% yield. Oxidative hydrolysis of the PMB group gave comound 173, which was then phosphitylated with phosphoramidite 157 in the presence of 1H-tetrazole. In situ oxidation occurred on treatment with t-BuOOH, and subsequent Pd-mediated deallylation afforded the pseudodisaccharide phosphotriester 152. Notably, no migration of the 2-O-hexadecanoyl group on the inositol was observed during removal of the PMB group or phosphitylation.

Scheme 29.

Synthesis of phospholipidated pseudodisaccharide 152 by the Guo group.

Owing to the early installation of the alkylglycerophosphate moiety, the synthetic end-game was considerably shorter than GPI syntheses previously reported (Scheme 30). The key coupling of trimannose thioglycoside 151 and pseudodisaccharide acceptor 152 was carried out with NIS/TfOH in CH₂Cl_2, with the addion of Et₂O to improve α-stereoselectivity. Desilylation at O-6 of Man-III with boron trifluoride etherate in the same vessel afforded the desired pseudopentasaccharide intermediate 149 in 52% yield over two steps. Facile installation of the phosphoethanolamine group by using phosphoramidite chemistry generated the fully protected GPI derivative 174. An orthogonal 9-fluorenylmethoxycarbonyl (Fmoc) group was used to block the phosphoethanolamine group, a feature designed to allow future coupling with peptides/glycopeptides (see Scheme 31 for related work). In this case, the Fmoc group was selectively removed with DBU in 87% yield, and subsequent hydrogenolytic removal of the remaining protecting groups provided the target GPI 148 in 84% yield.

Xue and Guo’s first total synthesis of a GPI anchor of sperm CD52 antigen was founded on well-established synthetic methods, such as the Koenigs–Knorr and Suzuki glycosylations to form glycosidic bonds, and phosphoramidite chemistry to form phosphotriester bonds. However, problems arising from the incorporation of a 2-O-acylated inositol group necessitated the development of a novel assembly strategy involving early installation of the phospholipid. Other noteworthy aspects of the synthesis were (i) an efficient preparation of inositol 168 that employed both enantiomers of the starting material, and (ii) orthogonal protection of the Man-III phosphoethanolamine group for future peptide coupling. Concerning the latter feature, the Guo group published closely related chemical syntheses of GPI-peptide/glycopeptide conjugates 175 and 176 in 2003 and 2004 (Scheme 31).^30,31 Since they contained only GPI partial structures (without the glycerolipid moiety),they are not discussed in detail here. Current research efforts by the Guo laboratory concerning the synthesis of GPI-anchored peptides/proteins are focused on developing chemoenzymatic methods,^33–35 which likewise are not covered here.

7. Synthesis of a P. falciparum GPI Anchor Containing an Acylated Inositol by the Fraser-Reid Group (2005)

The Fraser-Reid group explored the consequences of incorporating a 2-O-acylatedinositol into GPIs in parallel with, but independent of, the Guo group. A brief 2004 communication by Jayaprakash and Fraser-Reid¹⁰³ describing the synthesis of a P. falciparum GPI “prototype” (bearing unnatural truncated lipids) was followed by a comprehensive 2005 report¹⁰⁴ that included a fully lipidated target GPI anchor. The broader implications of this work lie in the fact that some protozoan parasites, including the malaria-causing agent P. falciparum, use free GPI anchors and GPI-anchored proteins to modulate the immune system of the host. As a result, the development of synthetic GPI-based constructs as anti-parastic vaccines has emerged as a promising area of research.¹⁰⁵

Fraser-Reid’s group set out to synthesize the GPI anchor 177of P. falciparum bearing an acylated inositol (Scheme 32). In contrast to Guo’s early-stage phospholipidation to avoid a side reaction, Fraser-Reid disconnected the target to pseudopentasaccharide 178, which after introduction of phosphoethanolamine would undergo late-stage acylation at O-2 of the inositol and phospholipidation at O-1 via an intermediate orthoester. The preparation of 178 made use of a linear assembly strategy involving consecutive mannosylations of pseudodisaccharide 181 with n-pentenyl orthoester (NPOE)⁷³ building blocks 182, 72, and 183. A novel strategy was developed to enhance the α-stereoselectivity in the formation of pseudodisaccharide 181. Rather than employing an azido glucosyl donor, which often gives low stereoselectivity, the authors used the mannosyl NPOE 184, which enhanced stereoselective formation of the glycosidic bond while acting as a latent azido glucose unit via inversion at the 2-position with an azide nucleophile. The synthesis of 177 was thus centered on the versatility of NPOEs as building blocks.

Scheme 32.

Retrosynthesis of the GPI anchor 177ofP. falciparum by the Fraser-Reid group.

The mannosyl NPOEs 182–184 were prepared from the readily available precursor 70⁶⁸ (Scheme 33).The NPOE 182 of Man-I was obtained by silylation at O-6 followed by benzylation. The Man-III diacetate 183 was generated in a single flask from precursor 70 by sequential treatment with TrtCl and Ac₂O. The latent azido glucosyl building-block 184, at this stage in the form of a mannosyl NPOE, was synthesized by tin-mediated selective dibenzylation at O-3 and O-6, leaving the O-4 position available for p-methoxybenzylation.

Pseudodisaccharide 181 was prepared according to Scheme 34. De novo synthesis of the inositol building-block 185 from methyl α-D-glucopyranoside (186)was performed by using a modified version of Bender’s method.¹⁰⁶ After elaboration of the glucoside 186 to the inositol derivative 190, a sequence highlighted by stereocontrolled Hg(II)-mediated Ferrier rearrangement and ketone reduction, conversion into compound 185 was accomplished by deacetylation and cis-cyclohexylidenation. Stereoselective glycosylation of the inositol acceptor 185 by the mannosyl NPOE 184 under activation by NIS/Yb(OTf)₃ in CH₂Cl₂ afforded the desired pseudodisaccharide 191 in an impressive 98% yield. Transformation into the desired azido glucose (2-equatorial) configuration was accomplished by debenzoylation, triflation at O-2, and azide displacement by DeShong’s procedure (TMSN₃/TBAF),¹⁰⁷ although the desired product 193 was obtained in only 39% yield. Nevertheless, the concept introduced by Fraser-Reid is an attractive one, given first the notoriously low stereoselectivity of azido glucose–inositol couplings, and second the prospect that azide displacement might be optimized. Finally, pseudodisaccharide acceptor 181 was obtained by treatment of 193 with boron trifluoride etherate to cleave the PMB group.

Scheme 34.

Synthesis of pseudodisaccharide 181 by the Fraser-Reid group.

Stepwise elongation of the pseudodisaccharide by mannose building blocks took advantage of the propensity of NPOE donors to form α-configured products (Scheme 35). Thus, acceptor 181 reacted with the NPOE 182 of Man-I in the presence of NIS/BF₃·OEt₂, providing α-pseudotrisaccharide 194 in 79% yield. Protecting-group manipulations of the Man-I unit furnished compound 195, which had a free O-6 position, and this was linked to Man-II 72 under the same activation conditions to form the α-pseudotetrasaccharide 196 in excellent yield (99%). Debenzoylation at O-2 of the Man-II component gave alcohol 197, which was elongated with the NPOE 183 of Man-III, again activated by NIS/BF₃·OEt₂, to generate the all-α-linked pseudopentasaccharide 198 in 75% yield. The acyl protecting-groups on Man-III were replaced by benzyl groups, and subsequent removal of the O-6 trityl group of Man-III by mild acid gave intermediate 178, which was ready for the three phosphorylation reactions.

Completion of the GPI synthesis is shown in Scheme 36. Introduction of the phosphoethanolamine group to the Man-III O-6 position of pseudopentasaccharide 178 using the phosphoramidite method was successful. Cleavage of the 1,2-cyclohexylidene acetal group from the inositol was accomplished with CSA/ethylene glycol to give diol 199. Model studies by Fraser-Reid predicted that introduction of an acyl group at the axial O-2 position of 199 using manipulations of protecting groups would be precarious, and so an alternative approach involving intermediate cyclic orthoesters was pursued. The intermediate diol 199 was therefore allowed to react with the trimethyl orthoester 179 in the presence of CSA to give the intermediate GPI-orthoester 200, which underwent rearrangement with Yb(OTf)₃ to provide a 2:1 mixture of regioisomers in favor of the desired 2-O-acylated product 201 (isolated in 50% yield). Despite Guo’s observation that attempted late-stage phospholipidation was thwarted by the coexistence of 2-O-acylated inositol and the trimannose moiety (see Scheme 26), Fraser-Reid encountered no problems in converting compound 201 into 202 (60–70%) using phosphoramidite chemistry, suggesting that minor structural differences in the substrate and/or phosphoramidite reagent can impact the reaction outcome. Global deprotection by Pd-catalyzed hydrogenolysis, although challenging because of unanticipated solubility issues, was eventually completed to deliver the target GPI anchor 177.

Scheme 36.

Completion of the target GPI 177 by the Fraser-Reid group.

In summary, the Fraser-Reid group accomplished the second total synthesis of an inositol-acylated GPI anchor using a linear-assembly strategy in concert with NPOE chemistry. Four mannosyl NPOEs, all accessed from a single synthetic precursor (70), were used for stereoselective glycosidic-bond formation, including the glucosamine–inositol linkage. In the latter instance, a mannose-configured NPOE donor was employed to facilitate the typically difficult α-glycosylation of inositol, and after serving this purpose was converted into an azido glucose moiety by invertive introduction of an azido group at the 2-position. Also contributing to the overall efficiency of the synthesis was the use of Bender’s de novo inositol synthesis, which obviated the need for cumbersome enantiomeric resolution protocols. In the final synthetic sequence, an inositol 1,2-orthoester intermediate was used to attach regioselectively the correct (phospho)lipids to the GPI.

8. Synthesis of a P. falciparum GPI Anchor Containing an Acylated Inositol by the Seeberger Group (2005)

The Seeberger labortory became involved in the study of GPI anchors in the early 2000s, when they used chemical synthesis to construct a GPI-based anti-toxin vaccine against malaria, which was effective in a rodent model of the disease.¹⁰⁸ An intriguing aspect of this project was the application of automated solid-phase oligosaccharide synthesis for rapid generation of large quantities of GPI intermediates, which could be elaborated to conjugate vaccines.¹⁷ These exciting developments offered further support to the notion that synthetic GPIs–indeed, carbohydrates in general–can be harnessed to develop novel therapeutic approaches.¹⁰⁵ Seeberger expanded on this early work in 2005with the solution-phase total synthesis of a fully lipidated GPI anchor of P. falciparum.

The inositol-acylated GPI anchor 203 was almost identical in structure to that synthesized by Fraser-Reid (compound 177, see Scheme 32), but it contained the additional mannose unit present in natural GPIs from P. falciparum (Scheme 37). The target compound was accessed from the GPI pseudohexasaccharide 204, which had orthogonal protecting-groups, including PMB, allyl, and triisopropylsilyl (TIPS), for sequential installation of hexadecanoyl, phospholipid, and phosphoethanolamine groups. For the two phosphorylation events, the H-phosphonate method⁴⁴ was used. Intermediate 204 was formed convergently from the tetramannosyl trichloroacetimidate 207 and pseudodisaccharide alcohol 208, which were both prepared in a straightforward manner using the building blocks 41, 44, and 209–211. Schmidt’s trichloroacetimidate method,⁶³ in combination with 2-O-acyl protecting groups, was used for the stereocontrolled formation of glycosidic bonds.

Synthesis of the tetramannose fragment 207 involved extension of the Man-I acceptor 209 using mannosyl trichloroacetimidate donors (Scheme 38). Compound 209 was glycosylated by imidate 41 in the presence of TMSOTf, generating dimannoside 212, which then underwent selective deacetylation by treatment with AcCl/MeOH to give product 213 in 88% yield over two steps. This process was repeated twice more, first using the TIPS-protected Man-III donor 210 followed by reuse of donor 41 to install the Man-IV group, which afforded tetramannoside 216 (58%, three steps). Conversion of the anomeric allyl group into trichloroacetimidate 207 was accomplished by Pd-mediated deallylation and subsequent treatment with trichloroacetonitrile and DBU.

Scheme 38.

Synthesis of tetramannosyl donor207 by the Seeberger group.

The pseudodisaccharide fragment was efficiently prepared starting from the known inositol diol 217 generated from compound 190 by deacetylation/allylation (Scheme 39).^106,109 Selective protection at O-2 of 217 with a PMB group allowed for late-stage hexadecanoylation of this site. The union of the resulting product 211 and the azido glucosyl trichloroacetimidate 44⁶³ under standard activation conditions yielded the α,β mixture 218 in 89% yield with good stereoselectivity (α:β = 4:1). This inseparable mixture was carried forward by deacetylation to give a triol, which underwent successive 4,6-O-benzylidenation, benzylation, and regioselective benzylidene cleavage to afford the α anomer 208, which was separated from the corresponding β anomer at this stage.

The key glycosylation between tetramannosyl trichloroacetimidate 207 and pseudodisaccharide alcohol 208 was performed in the presence of TMSOTf, while the 2-O-benzoyl group of the donor ensured α-stereoselectivity through neighboring-group participation (Scheme 40). The reaction generated pseudohexasaccharide 204 in 94% yield, and the subsequent transformation of this to the intermediate 220 required deacylation followed by benzylation. The fully protected GPI intermediate 220 could be obtained in nearly gram quantities, an important accomplishment considering the large amount of material required for generating and testing antimalarial vaccine candidates.

Scheme 40.

Completion of GPI anchor 203of P. falciparum by the Seeberger group.

The remaining steps centered on lipidation and phosphorylation events. After oxidative removal of the PMB group at O-2 of the inositol, the axial hydroxyl group thus exposed was esterified with hexadecanoic acid in the presence of DCC and DMAP to give 221. Subsequent removal of the neighboring allyl group at O-1 of the inositol to furnish compound 222 was successfully performed by using an excess of PdCl₂without acyl migration or decomposition. This outcome was in contrast to Fraser-Reid’s previously reported difficulties in a nearly identical system. The phospholipidation step was performed by using H-phosphonate 205 in the presence of PivCl and pyridine, and this was followed by iodine-mediated oxidation, to give 223 without any side reactions involving the azido group. After the 6-O-TIPS group on Man-III had been hydrolyzed in the presence of Sc(OTf)₃ to give compound 224, the H-phosphonate method was again used to install the phosphoethanolamine group. Final deprotection of intermediate 225 was effected by Pd-catalyzed hydrogenolysis in a mixture of solvents, which provided the target GPI 203 in excellent (94%) yield.

Seeberger’s synthesis of the P. falciparum GPI anchor 203 bearing an acylated inositol group featured a logical and straightforward convergent assembly strategy based on previous GPI syntheses. Proven methods were used for the formation of glycosidic and phosphodiester bonds, while difficulties encountered by the Guo and Fraser-Reid groups in the late-stage lipidation and phosphorylation steps were avoided. Impressively, gram-scale quantities of complex pseudohexasaccharide intermediates were obtained on scale-up, a critical point given the group’s development of GPI-based antimalarial vaccines. Subsequent advances in this area by the Seeberger group include the 2008 development of synthetic GPI glycan arrays to characterize malaria-induced antibody responses,^110,111 which should aid in the design of improved vaccine candidates. Also reported by the Seeberger laboratory in 2008 was a project describing the semisynthesis of a GPI-anchored prion-protein construct, which hinged on the use of native chemical ligation to couple the GPI and protein fragments.²⁹ As these research efforts involved partial GPI structures (missing the phosphoglycerolipid), they are not covered in detail here, but the target GPI--prion conjugate 226 is shown in Scheme 41.

9. Synthesis of a T. cruzi GPI Anchor by the Vishwakarma Group (2005)

In 2000, Ferguson and co-workers discovered that GPIs from T. cruzi, the causative agent of Chagas’ disease, have potent proinflammatory activity. This leads to various pathologies and, interestingly, was attributed to unsaturated fatty acids at the sn2 position of the GPI acylalkylglycerophosphate.¹¹² In 2005, the Vishwakarma group reported the first synthesis of a T. cruzi GPI anchor,¹¹³ although the authors targeted a GPI containing saturated lipids that was more synthetically accessible and would be compatible with global benzyl-group protection (for the synthesis of T. cruzi GPIs bearing naturally occurring unsaturated lipids and a 2-aminoethyl phosphonate group, see Nikolaev’s work in section III-1).

Vishwakarma’s chemical synthesis of the GPI anchor 227adopted a convergent strategy resembling previous syntheses by other groups (Scheme 42). The key intermediate 228was obtained from compounds 230 and 231, the former of which was constructed in a [2 + 2] fashion using disaccharides 232 and 233 rather than the typical linear elongation of a Man-I building block. Notably, pseudodisaccharide 231 was prepared from the racemic inositol derivative 11 and azido glucosyl trichloroacetimidate 44, with the latter being conveniently used as a chiral auxiliary for inositol resolution, eliminating the need for lengthy routes for resolution used in previous GPI syntheses. Glycosyl NPOE⁷³ and trichloroacetimidate donors⁶³ were used for glycosylations, while both phosphoramidite⁶⁰ and H-phosphonate⁴⁴ methods were used for phosphorylations.

Scheme 42.

Retrosynthesis of T. cruzi GPI anchor 227 by the Vishwakarma group.

The [2 + 2] construction of tetramannosyl donor 230 from dimannosides 232 and 233 is shown in Scheme 43. Allyl α-D-mannopyranoside (128) was used as a common starting material for three of the four mannose building blocks. En route to 232, compound 128 was advanced by tritylation at O-6, perbenzylation at the O-2, 3, and 4 positions, and detritylation to give 234. Subsequent α-stereoselective glycosylation of 234 by the mannosyl NPOE 72 in the presence of NIS and TESOTf followed by debenzoylation furnished compound 232 (92%, two steps). The other dimannoside 233 was accessed from monomers 235 and 236, each of which arose from allyl mannoside 128, the first by successive benzylation, deallylation, and 1-O-trichloroacetimidation and the second by sequential 4,6-O-benzylidenation and selective benzylation at O-3. Compounds 235 and 236 were then joined via Schmidt glycosylation in 81% yield. A series of subsequent protecting-group manipulations were used to generate 233, including concomitant deallylation/benzylidene hydrolysis with treatment with t-Bu OK then HCl, peracetylation, selective anomeric deacetylation with MeNH₂, and finally treatment with DBU and trichloroacetonitrile. The [2 + 2] coupling of 232 and 233 under activation by TMSOTf proceeded in moderate (65%) yield, perhaps because of the lack of a participating 2-O-acyl group on the donor. Subsequent transformation into the tetramannosyl donor 230 was completed by Pd-mediated anomeric deallylation and treatment with DBU and trichloroacetonitrile.

In the preparation of pseudodisaccharide 231 (Scheme 44), the azido glucosyl group was used as an efficient chiral auxiliary, and this eliminated the yield-depleting techniques for inositol resolution that are traditionally used. The racemic mixture of inositols (±)–11 was treated with the azido glucosyl trichloroacetimidate 44 in the presence of TMSOTf to generate a 1:1 mixture of diastereomers 237 and 238. The high α-stereoselectivity achieved using donor 44, as previously reported by Schmidt and other groups, prevented the formation of a complex mixture of products. As 237 and 238 were inseparable at this stage, they were subjected to deacetylation followed by 4,6-O-benzylidenation, which produced the separable diastereomers 239 and 240. The desired isomer 239 was carried forward to compound231 by benzylation and regioselective benzylidene cleavage. This approach for resolving inositol enantiomers was efficient, and should be useful in future GPI syntheses.

Scheme 44.

Synthesis of pseudodisaccharide 231 by the Vishwakarma group.

Vishwakarma’s completion of the synthesis of GPI anchor 227 (Scheme 45) commenced with the coupling of 230 and 231, promoted by TMSOTf, to afford the α-linked pseudohexasaccharide 228 in 70% yield. The product was designed to permit orthogonal protection of the O-4 and O-6 positions of Man-III to allow first the installation of phosphoethanolamine, and second the synthesis of 4-deoxy Man-III GPI analogues for biological studies. In this synthesis, the Man-III moiety was subjected to deacetylation, 6-O-silylation, 4-O-benzylation, and finally desilylation. This lengthy sequence furnished intermediate 241, which was then treated with phosphoramidite 58 in the presence of 1H-tetrazole, followed by in situ oxidation with m-CPBA, to install the phosphoethanolamine group. Phospholipidation of the inositol was accomplished by CAN-mediated oxidative cleavage of the PMB group, followed by coupling with building block 229 using the H-phosphonate method. Global deprotection by hydrogenolysis over Pd(OH)₂/C provided the target GPI 227 in 73% yield.

Vishwakarma and co-workers thus accomplished the first chemical synthesis of a T. cruzi GPI anchor. Overall, standard building blocks, assembly strategies, and carbohydrate synthetic methods were used to access the target. Of note was the efficient preparation of the pseudodisaccharide, which was highlighted by inositol resolution using the azido glucosyl group as a chiral auxiliary.

10. Synthesis of a Fully Phosphorylated and Lipidated Human Sperm CD52 Antigen GPI Anchor by the Guo group (2007)

In 2007, Wu and Guo reported the total synthesis of a fully phosphorylated and lipidated GPI anchor of the human sperm CD52 antigen,^114,115 which followed their 2003 synthesis of a related CD52 antigen GPI that contained an acylated inositol but lacked the phosphoethanolamine group at O-2 of Man-I of the natural product (see section II-6). Thus, the target molecule 245, bearing an additional phosphoethanolamine unit, was synthesized by a similar convergent strategy (Scheme 46). To install both phosphoethanolamine groups, a late-stage double phosphorylation using phosphoramidite 58 was performed on the intermediate GPI diol 246, which in turn was accessed from trimannose thioglycoside 247 and phospholipidated pseudodisaccharide 248. The latter compound was obtained by consecutive glycosylation, lipidation, and phospholipidation of inositol 249 by building blocks 104, 250, and 251. For acylation of the inositol, the unsaturated hexadecane-9-enoyl group was used to act as either a latent aldehyde (on oxidative cleavage of the C=C bond) for conjugation chemistry, or a latent hexadecanoyl group (on reductive global deprotection).

Scheme 46.

Retrosynthesis of fully phosphorylated and lipidated human sperm CD52 antigen GPI anchor 245 by the Guo group.

The synthesis of phospholipidated pseudodisaccharide 248 is shown in Scheme 47. The required inositol derivative 249 was readily accessed from the previously reported compound 168 by acetylation followed by deallylation. Reminiscent of a previous route by Ley (see Scheme 19), glycosylation of 249 by the azido glucosyl bromide 104 under Lemieux conditions⁴² generated pseudodisaccharide 252 in moderate (56%) yield but with complete α-stereoselectivity. Deacetylation at O-2 of the inositol was followed by esterification with hexadec-9-enoic acid in the presence of DCC/DMAP to give 254, which then underwent removal of the PMB group with CAN. Phospholipidation of 255 using phosphoramidite 251 in the presence of 1H-tetrazole followed by t-BuOOH-mediated oxidation provided an intermediate phosphotriester, which was subjected to desilylation with boron trifluoride etherate to give 248. Phospholipidation was performed at this stage rather than later, so as to prevent the cyclophosphitamidation side-reaction previously discussed (see Scheme 26).

Scheme 26.

Unexpected cyclophosphitamidation reaction of the inositol-acylated GPI intermediate 159 (Guo group).

Completion of the target molecule is shown in Scheme 48. Prior to the key glycosylation, the trimannose thioglycoside 247 (synthesized according to Guo’s previous route, see Scheme 27) was converted into trichloroacetimidate 256 via NIS-mediated hydrolysis of the thioacetal followed by reaction with DBU and trichloroacetonitrile. The union of trichloroacetimidate 256 and pseudodisaccharide 248 under Schmidt glycosylation conditions formed the desired intermediate, which was immediately treated with boron trifluoride etherate to remove simultaneously the TBS and PMB groups. This sequence provided the α-pseudopentasaccharide diol 246 in moderate (40%) yield over two steps. Double phosphorylation of 246 using phosphoramidite 58, a difficult task, proceeded in 50% yield to give the fully protected GPI 257. Global deprotection was accomplished by treatment with DBU to remove the cyanoethoxyl group, followed by debenzylation using Pd-catalyzed hydrogenolysis to afford the target GPI 245 in 82% yield.

Scheme 48.

Completion of fully phosphorylated and lipidated human sperm CD52 antigen GPI anchor 245 by the Guo group.

In summary, the synthesis of 245, a fully phosphorylated and lipidated GPI, was guided by Guo’s 2003synthesis of a CD52 antigen GPI while using an orthogonal protection strategy that enabled late-stage double phosphorylation. The target molecule and related GPI derivatives were used to investigate their relative abilities to bind CAMP factor, a pore-forming toxin secreted by Streptococcus agalacticae that is thought to utilize GPI anchors as its binding partners.¹¹⁵ Initial results suggested that both the intact GPI anchor 245, and its corresponding truncated phospholipidated pseudodisaccharide, bound CAMP factor with similarly strong affinities. In general, such studies using well-characterized synthetic GPIs and GPI derivatives should prove instrumental in elucidating GPI structure–activity relationships in various contexts.

III. Diversity-Oriented Approachesto GPI Synthesis

The so-called classic GPI syntheses thus far detailed here were accomplished by strategies having limited potential for structural diversity, and this can be attributed primarily to the global protection tactics used. Traditionally, carbohydrate chemists have used benzyl groups for permanent protection of hydroxyl groups because of their ease of installation, stability under a broad range of reaction conditions, numerous choices for orthogonal protection, and mild conditions for global deprotection. However, the latter feature, deprotection via Pd-catalyzed hydrogenolysis, constitutes a considerable synthetic shortcoming given its incompatibility with many important functional groups. In this category are alkenes, alkynes, azides, thiols, thioethers, and other potentially reducible or catalyst-poisoning functionalities. Therefore, global protection by benzyl groups in GPI synthesis precludes the incorporation of the biologically important unsaturated fatty acids and indispensable functional handles such as “click” chemistry, imaging, and affinity-purification tags. Furthermore, the natural diversity of GPIs arising from core structure modifications, such as additional glycans, phosphoethanolamine groups, and variation in lipids (see Figure 1),has prompted the development of newer general methods for efficientl synthesis of a range of GPIs from common building-blocks. Research at the recent forefront of GPI synthesis has been focused on developing diversity-oriented synthetic strategies to address these drawbacks. Progress in this area by the Guo, Nikolaev, and Seeberger groups is discussed in this section.

1. Synthesis of T. cruzi GPI Anchors Containing Unsaturated Lipids by the Nikolaev Group (2006)

As already mentioned, Ferguson and co-workers identified GPIs from T. cruzi exhibiting potent proinflammatory activity that was thought to be associated with unsaturated fatty acids in the sn2 position of the GPI acylalkylglycerophosphate.¹¹² In an effort to establish rigorous structure activity relationships in this system, the Nikolaev group set out to synthesize T. cruzi GPIs containing the unsaturated fatty acids present in the natural structure.¹¹⁶ The GPI anchors 258 and 259 (Scheme 49), bearing the potentially crucial sn2-oleoyl [(Z)-octadec-9-enoyl] and linoleoyl [cis,cis-octadec-9,11-dienoyl] groups, respectively, were targeted for chemical synthesis.

Scheme 49.

Retrosynthesis of T. cruzi GPIs 258 and 259 by the Nikolaev group.

Nikolaev’s 2006 publication was the first total synthesis of GPIs containing unsaturated lipids, and accordingly required the development of a novel strategy to obviate the need for reductive conditions employed in global benzyl-group deprotection. Acyl-based permanent protection of hydroxyl groups in the form of benzoic esters was chosen, despite potential compatibility issues with the ester-linked lipid in the acylalkylglycerophosphate moiety. The authors’ hypothesis was that, upon deprotection by mild base in a polar solvent, the nascent amphiphilic GPI intermediate may undergo micelle formation, thereby protecting the bond of the fatty ester during selective removal of the benzoic esters. Further increasing the synthetic challenge, the target GPIs had a 2-aminoethylphosphonate group linked to O-6 of the glucosamine component, which is a parasite-specific modification of some GPIs.

The retrosynthesis of GPIs 258 and 259 is shown in Scheme 49. Both targets were accessed from the pseudohexasaccharide 260, which was poised to undergo a triad of phosphorylation events with building blocks 261, 262, and 263/264, to install the 2-aminoethylphosphonate, phosphoethanolamine, and phosphoglycerolipid components, respectively. Intermediate 260 was assembled convergently from the benzoyl-protected tetramannosyl donor 265 and a derivative of Schmidt’s pseudodisaccharide 52, which in turn were built up from their respective monomers. For all steps involving glycosidic-bond formation, the Schmidt method⁶¹ using glycosyl trichloroacetimidate donors was applied.

The tetramannose fragment 265 was prepared by the stepwise joining of mannose monosaccharides 267–270 (Scheme 50), a process entailing sequential stereoselective α-mannosylations, each aided by neighboring-group participation. The Man-I component (267) was coupled with the Man-II component (268) in the presence of TMSOTf, providing an α-dimannoside in 94% yield that was subjected to selective deacetylation at O-2 of the Man-II component with HCl/MeOH to give compound271. The same two-step sequence was used to extend the glycan chain with the Man-III component (269, 96% yield) and expose the O-2 position of the Man-III component. Subsequent reaction of this site with the Man-IV component 270, again promoted by TMSOTf, afforded fully α-configured tetramannose product 273 in nearly quantitative yield. The O-6 benzyl group on the Man-III precursor was then exchanged for a TBS group via Pd-catalyzed hydrogenolysis, followed by treatment with TBSOTf/Et₃N. Finally, the anomeric benzoyl group of the Man-I precursor was converted into a trichloroacetimidate by regioselective debenzoylation with ethylenediamine followed by treatment with Cl₃CCN/Cs₂CO₃.

Scheme 50.

Synthesis of tetramannosyl donor 265 by the Nikolaev group.

For the pseudodisaccharide fragment, the authors converted known compound 52, previously described by Schmidt (see Scheme 9 for its preparation),⁵⁸ into a suitably protected derivative containing only acid-labile groups whose late-stage removal would not interfere with the unsaturated lipid chains. However, rather than perform the separation of inositol diastereomers 43 and 51 as reported in the literature,^58,117 it was found that conducting the glycosylation directly on the diastereomeric mixture afforded readily separable pseudodisaccharide products 52 and 275 (Scheme 51), indicating that the azidoglucose component acted as a supplementary chiral auxiliary. Notably, this approach relied on the exceptional α-stereoselectivity observed in the glycosylation reaction previously fine-tuned by Schmidt,⁵⁸ otherwise a complex and possibly inseparable mixture of isomers would have been generated.

Scheme 51.

Synthesis of pseudodisaccharide 266 by the Nikolaev group.

From Schmidt’s pseudodisaccharide 52, several protecting-group manipulations were required to obtain 266. First, the azidoglucose component was deacetylated and then treated with PhC(OEt)₃ under acidic conditions to form a 4,6-orthoester derivative. Subsequent protection of the free O-3 position with an acid-labile 2-trimethylsilylethoxymethyl (SEM) group, using SEMCl/i-Pr₂Net, gave compound 276, which was subjected to cleavage of the inositol 1-O-(−)-menthylcarbonate group with NaOMe. Protection of the exposed hydroxyl group with a TBS ether was accomplished by treatment with TBSOTf/Et₃N to give the protected pseudodisaccharide 277. The final sequence involved removal of the 4,6-orthoester from the azidoglucose, followed by selective triethylsilylation at O-6. This synthetic route from52, while somewhat lengthy, was very efficient, providing 266 in 50% yield over seven steps.

Elaboration of the tetramannose and pseudodisaccharide fragments to the target GPI anchors is shown in Scheme 52. The key glycosylation reaction between the tetramannosyl trichloroacetimidate 265 and pseudodisaccharide 266 proceeded smoothly, giving the fully protected α-pseudohexasaccharide 260 in 71% yield. At this stage, three phosphorylation events were required to decorate the GPI with the appropriate structures found in GPIs of T. cruzi. First, the unique 2-aminoethylphosphonate group was installed at O-6 of the azidoglucose in three steps, including selective cleavage of the TES ether using AcOH-buffered TBAF, 1H-tetrazole-promoted phosphorylation with 2-azidoethylphosphonodichloridate 261 (generated from 2-bromoethylphosphonate in two steps), and methanolysis of the intermediate chlorophosphonate to give 278. Subsequent Staudinger reduction and treatment with Boc anhydride converted the azido groups into N-Boc-protected amino groups. The next transformation involved selective O-6 desilylation of the Man-III TBS group with triethylamine trihydrofluoride, which exposed this position for introduction of the phosphoethanolamine unit by using H-phosphonate derivative 262 in the presence of pivaloyl chloride, followed by in situ iodine-mediated oxidation to phosphodiester 280. Subsequent removal of the TBS from O-1 of the inositol under strong conditions, namely TBAF/AcOH at 55 °C, set the stage for installation of the unsaturated lipid-containing acylalkylglycerophosphate components. Again, the H-phosphonate method was successfully employed, resulting in the phospholipidation of 281 with precursors 263 or 264 to introduce oleic [(Z)-octadec-9-enoic] or linoleic (cis,cis-octadec-9,11-dienoic) acids, respectively, into the GPI,.

Scheme 52.

Completion of T. cruzi GPI anchors258 and 259by the Nikolaev group using acyl-based global protection.

The global deprotection of fully protected GPI intermediates 282 and 283 required three reactions. First, demethylation of the 2-aminoethylphosphonate group was effected by using PhSH and Et₃N. The second and most critical deprotection step required base-promoted debenzoylation, a process that risked cleavage of the GPI fatty esters. Nikolaev used 0.05 M NaOMe in MeOH, which successfully delivered the products, albeit in moderate (38–40%), yields, most probably a result of poor selectivity. Finally, the purified intermediates were subjected to treatment with aqueous 90% TFA to hydrolyze the remaining acetal and N-Boc protecting groups, which proceeded with high efficiency to afford, after purification by reverse-phase chromatography, the target GPIs 258 and 259.

Overall, use of benzoyl groups as an acyl-based global-protection strategy to access GPIs containing unsaturated lipids was successful, but compatibility issues with acylglycerophosphates render this approach a precarious one for synthesizing functionalized GPIs. Shortly after their 2006 publication, Nikolaev and co-workers adapted their synthetic strategy by using acetal- and silyl-based protection in lieu of acyl-based protection to address this issue.¹¹⁸ In this work, a convergent [3 + 3] strategy was used to synthesize GPI 258 from the trimannose thioglycoside 284 and pseudotrisaccharide 285, which were both permanently protected with acetal or silyl groups (Scheme 53). Briefly, these two components combined in the presence of methyl triflate and 2,6-di-tert-butyl-4-methylpyridine (DTBMP),with subsequent selective removal of the TES group using TBAF and AcOH to generate α-pseudohexasaccharide 286 in 75% yield. The 2-aminoethylphosphonate, phosphoethanolamine, and alkyloleoylglycerophosphate groups were installed as previously to arrive at the fully protected GPI 290. Final deprotection was accomplished by a two-step sequence comprising desilylation with triethylamine trihydrofluoride and TFA-promoted acetal hydrolysis. The target oleoyl [(Z)-octadec-9-enoyl] GPI 258 was obtained in enhanced (67%) yield over two steps, although the desilylation step required 18 days to complete.

Scheme 53.

Completion of T. cruzi GPI anchor 258by the Nikolaev group using acetal- and silyl-based global protection.

In summary, Nikolaev’s innovative strategies for access to diversely structured GPIs were used for the synthesis of biologically important GPIs of T. cruzi containing unsaturated lipids. Both strategies were focused on using alternative protecting groups that would not require Pd-catalyzed hydrogenolysis for their removal. Although acyl-based protection probably led to compatibility issues with the acylalkylglycerophoshphate during global deprotection with NaOMe, an improved strategy based on acetal and silyl protection was developed that featured a higher-yielding, but time-consuming, global deprotection. Synthetic GPIs 258 and 259 were subjected to a preliminary biological evaluation, which showed activity similar to that of the naturally occurring GPIs in a Toll-like receptor stimulation-assay.¹¹⁶ More detailed biological studies of these compounds are ongoing.

2. Synthesis of a GPI Anchor Containing Unsaturated Lipid Chains by the Guo group (2010)

In an effort to develop a robust approach for the synthesis GPI anchors bearing unsaturated lipids and other important functionalities, the Guo group focused on a global hydroxyl-protection strategy based on the PMB group, which can be removed under relatively mild acidic conditions, such as 5–10% TFA, or oxidation, as with CAN or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). It was expected that PMB protection would offer a major improvement in functional-group tolerance during the global deprotection, thus allowing access to highly complex and sensitively functionalized GPI anchors.

In 2010, Swarts and Guo provided a proof-of-concept for the PMB-protection strategy by synthesizing a GPI anchor containing unsaturated lipids,¹¹⁹ specifically a dioleoylglycerophosphate moiety. The retrosynthetic analysis, shown in Scheme 54,was guided by previous convergent syntheses from the Guo group. The target GPI anchor 291 was accessed from intermediate 292, which was formed in a key glycosylation reaction between the trimannosyltrichloroacetimidate donor 294 and pseudodisaccharide 295. The trimannose fragment, joined together from PMB-protected monomers 296–298, contained orthogonal silyl ether protecting-groups on the Man-I and Man-III components for late-stage phosphorylation with the phosphoethanolamine precursor 293. The pseudodisaccharide fragment, already containing the dioleoylglycerophosphate group at O-1 of the inositol, was synthesized from the optically pure inositol 299, the azido glucosyl trichloroacetimidate 300, and the phospholipid precursor 301.

Scheme 54.

Retrosynthetic analysis of a GPI anchor bearing unsaturated lipid chains by the Guo group using the PMB protection strategy.

The preparation of trimannoside 294 involved the α-stereoselective coupling of mannose building blocks 296–298, which were prepared from a common intermediate, mannose pentaacetate 302 (Scheme 55). The Man-I building block, thioglycoside alcohol 296 bearing a free 6-OH group, was obtained from diol 303¹²⁰ via sequential stannylene acetal-directed regioselective p-methoxybenzylation at O-3, allylation at O-2, and regioselective opening of the p-methoxybenzylidene ring using DIBAL-H. the Man-II component 297 was prepared from 302 via orthoester 304,⁵⁶ which after deacetylation and methoxybenzylation was treated with acetic acid to open the orthoester ring, providing a hemiacetal that was converted into the corresponding trichloroacetimidate. Synthesis of mannose derivative 298 started with a BF₃-promoted glycosidation of 302 with allyl alcohol. Subsequent deacetylation gave tetraol 305, which then underwent differentiation of the 6-position by reaction with p-methoxytrityl (PMTrt) chloride and methoxybenzylation of the 2-, 3-, and 4-positions to generate 306. After exchanging the PMTrt protecting group for a TBS group, Pd-catalyzed deallylation freed up the anomeric hydroxyl group for transformation into trichloroacetimidate 298by treatment with trichloroacetonitrile and DBU. In general, the preparation of PMB-protected mannose building-blocks was comparable to the corresponding benzyl-protected variants in terms of both strategy and efficiency.

Scheme 55.

Synthesis of PMB-protected mannose derivatives, and elaboration to the trimannosyl donor 294 by the Guo group.

With monosaccharides 296–298 in hand, the trimannosyl donor 294 was constructed by using the Schmidt glycosylation method.⁶¹ The coupling of Man-I component 296 and Man-II component 297 proceeded well using catalytic TMSOTf in CH₂Cl₂, and complete α-stereoselectivity was imparted by the participating acetyl group at O-2. Deacetylation with K₂CO₃/MeOH exposed the O-2 position, resulting in the dimannoside alcohol 308 in 66% yield over two steps. Coupling of 308 with the Man-III trichloroacetimidate 298, also under Schmidt conditions, gave trimannoside 309 in a 5:1 α:β ratio, a figure that was improved to exclusively α (76% yield) upon changing the reaction solvent to diethyl ether. The trimannose thioglycoside 309 was also used as a divergence point for the preparation of other functionalized GPI anchors (see section III-4).In this instance, compound 309 was elaborated to the trichloroacetimidate 294 in four steps, including titanium-mediated deallylation using Cha’s protocol¹²¹ and triethylsilylation to change the protecting group at O-2 of the Man-I component, followed by treatment with NIS/AgOTf/2,4,6-tri-tert-butylpyrimidine (TTBP) to hydrolyze the anomeric thioacetal, and final treatment with trichloroacetonitrile and DBU.

For the pseudodisaccharide fragment, the PMB-protected inositol derivative 299 was required (Scheme 56). The O-1 and O-6 positions of racemic diol (±)-20were simultaneously protected as allyl ethers, which were unaffected by the subsequent cyclohexylidene-group removal and p-methoxybenzylation reactions. Next, deallylation of the bis-allyl ether (±)-311 using Cha’s protocol¹²¹ provided inositol derivative (±)-312 in 85% yield. For differentiation of the PMB-protected diol (±)-312at O-1 and O-6, regioselective stannylene acetal-directed allylation was used to generate compound (±)-299in 72% yield. Enantiomeric resolution of racemate (±)-299 was achieved by acylation of the free O-6 position with the chiral reagent (1S)-(−)-camphanic chloride to yield HPLC-separable diastereomers. Finally, the desired, purified diastereomer (−)-313 was subjected to saponification to provide the enantiomerically pure inositol 299 in 44% yield over two steps (maximum yield 50%).

Scheme 56.

Synthesis of inositol derivative299 by the Guo group.

The synthesis of phospholipidated pseudodisaccharide 295 is depicted in Scheme 57. Preparation of the azido glucosyl donor 300 began with the transformation ofcompound314¹²² into the allyl glycoside 315via BF₃-promoted glycosidation with All OH, deacetylation, and formation of the 4,6-p-methoxybenzylidene acetal. A PMB group was installed at the O-3 position, which was followed by regioselective opening of the p-methoxybenzylidene ring using dry HCl and NaBH₃CN to provide compound 316. After use of TBSOTf to protect the O-4 position to afford compound 317, the anomeric allyl ether was removed by Ir(I)-catalyzed isomerization to the corresponding vinyl ether and subsequent hydrolysis by Hg(II).¹²³ The resulting hemiacetal was converted into trichloroacetimidate 300 by using trichloroacetonitrile and DBU.

Scheme 57.

Synthesis of pseudodisaccharide 295 by the Guo group.

Coupling of the inositol derivative 299 and azido glucosyl donor 300 was performed in toluene--1,4-dioxane in the presence of TMSOTf to generate the pseudodisaccharide intermediate in 80% yield as an inseparable α,β-mixture (α:β = 2.3:1). Subsequent deallylation of the mixture using the Ir–Hg protocol produced the α-pseudodisaccharide 318, which was readily separable from the accompanying β anomer. Phospholipidation of the inositol at O-1 using phosphoramidite 301, followed by in situ oxidation with t-BuOOH installed the dioleoylglycerophosphate moiety to produce compound 319.Treatment of this intermediate with triethylamine trihydrofluoride cleaved the O-4 TBS group of the azido glucose derivative, which required several days for completion, but cleanly produced the desired pseudodisaccharide 295.

The final stage of the synthesis centered on union of the trimannose and pseudodisaccharide fragments (Scheme 58). The trimannose trichloroacetimidate 294 reacted smoothly with 295 in the presence of TMSOTf to afford the desired α-pseudopentasaccharide 292 in 64% yield. Overnight treatment of 292 with Et₃N·3HF removed the TBS and TES groups to provide diol 320. Installation of the phosphoethanolamine unit by treatment with phosphoramidite 293 for a short period (1 h) was selective for the O-6 position of Man-III to give 321. Alternatively, the diol could be used to access a fully phosphorylated GPI bearing two phosphoethanolamine groups. To obtain the target GPI anchor, compound 291, a three-step, one-flask protocol was developed to efficiently remove all of the protecting groups from 321 in under 4 hours: first, Zn-mediated reduction of the azide; second, removal of the base-labile Fmoc and cyanoethoxyl groups with DBU; and third, hydrolysis of all PMB ethers with 10% TFA. The target GPI 291 was obtained in 81% yield over the final three steps.

Scheme 58.

Completion of a GPI anchor bearing unsaturated lipid chains (291) using the PMB protection strategy by the Guo group.

This proof-of-concept synthesis established the PMB-protection strategy as a general approach to the synthesis of functionalized GPIs and potentially other complex functionalized glycoconjugates. Overall, the preparation of PMB-protected monosaccharide and inositol building-blocks was conducted efficiently, and the performance of these intermediates in Schmidt glycosylation reactions was comparable to their benzyl-protected counterparts. The highlight of this convergent synthesis was the three-step, one-flask reaction that afforded the target GPI 291 in 81% yield in under 4 hours. In the next two sections, use of the PMB-protection strategy by the Guo group for access to a complex naturally occurring GPI anchor, as well as “clickable” GPI anchors, is discussed.

3. Synthesis of a Human Lymphocyte CD52 Antigen GPI Anchor Containing a Polyunsaturated Arachidonoyl Lipid by the Guo Group (2011)

The structural diversity of GPI lipids comes about by the “lipid remodeling” process of GPI-anchored proteins.^14,124–126 GPI biosynthesis begins from cellular phosphatidylinositol, which in mammalian systems typically contains a polyunsaturated arachidonoyl lipid (20:4) at the sn-2 position. The arachidonoyl [(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl] group can be replaced by other lipids through a series of transformations in the Golgi apparatus, and as a result most mature mammalian GPI-anchored proteins contain only saturated fatty acids. However, several GPI-anchored proteins bearing 2-arachidonoyl phosphatidylinositol have been identified, including a major form of the human lymphocyte CD52 antigen.^98,127 The effects of unremodeled lipids on the function of lymphocyte CD52 and other GPI-anchored proteins have not been thoroughly explored, although in general it is expected that the structure of GPI lipids has important implications on the localization and function of GPI-APs. For example, a recent study has demonstrated that saturated fatty acids resulting from lipid remodeling are required for the association of mammalian GPI-APs with lipid rafts.¹²⁸

To develop a strategy for accessing GPIs relevant to studying the effects of lipid remodeling and to continue synthetic studies of GPI anchors and the human CD52 antigen (see sections II-6 and II-10), the Guo group targeted the lymphocyte CD52 GPI anchor 322 (Scheme 59) for total synthesis.¹²⁹ The target molecule contained modifications of the GPI core glycan present in the human lymphocyte CD52 antigen, including additional phosphoethanolamine and mannose groups linked to the O-2 positions of Man-I and Man-III, respectively. Most importantly, compound 322 contained an octadecanoyl-arachidonoyl-glycerophosphate component, which is present in the lymphocyte CD52 antigen and other GPI-anchored proteins that do not undergo lipid remodeling. To achieve the synthetically challenging incorporation of a highly sensitive polyunsaturated arachidonoyl lipid in GPI322, the PMB-protection strategy was used.

Scheme 59.

Retrosynthetic analysis of human lymphocyte CD52 antigen GPI anchor 322 containing a polyunsaturated arachidonoyl lipid by the Guo group.

A convergent approach was used for the construction of GPI 322 that relied on a key glycosylation between PMB-protected coupling partners, namely the tetramannosyl donor 324 and the pseudodisaccharide acceptor 325 (Scheme 59). Silyl ether protecting-groups at the O-2 position of Man-I and O-6 position of Man-III would be selectively removed at a late stage to enable double phosphorylation of these sites with phosphoramidite 293. The glycosyl donor 324 would arise from sequential α-mannosylations of disaccharide 308 using monomers 326 and 327. Acceptor 324 could be accessed through phospholipidation of compound 318 with phosphoramidite 328, which would attach the arachidonoyl-containing phosphoglycerolipid. Guided by previous success, the Schmidt method⁷¹ was used to couple stereoselectively all PMB-protected glycosylation partners. Some PMB-protected building blocks (308, 318) from Guo’s 2010 synthesis of a GPI anchor containing unsaturated lipids were also used in this synthesis, showing their versatility.

The preparation of PMB-protected tetramannosyl donor 324 is shown in Scheme 60. To obtain the Man-III building-block 326, the orthoester 304 was subjected to consecutive deacetylation, silylation at O-6, p-methoxybenzylation at O-4 and O-6, acid-promoted regioselective opening of the orthoester ring, and finally treatment of the resulting hemiacetal with trichloroacetonitrile and DBU. The resultant compound 326 was then treated with 308 in the presence of TMSOTf to furnish the α-trimannoside 329 in 72% yield. Deacetylation of 329 gave the acceptor 330, which was glycosylated by the Man-IV donor 327 to afford tetramannoside 331 in 76% yield, although stereoselectivity for this reaction was low (α:β = 3:2). At this stage, the allyl group at O-2 of Man-I was exchanged for a TES group using Cha’s deallylation protocol,¹²¹ followed by treatment with TESOTf/2,6-lutidine to furnish compound 332. Transformation of the anomeric thioacetal of 332 to the trichloroacetimidate 324 was accomplished by treatment with NIS/AgOTf/TTBP followed by trichloroacetonitrile and DBU.

Scheme 60.

Synthesis of PMB-protected tetramannosyl donor 324 by the Guo group.

Given the documented sensitivity of arachidonoyl-containing compounds to air and light (leading to oxidation of the alkene) and heat and base (leading to rearrangements),^130–132 care was taken to avoid these conditions in the preparation of phosphoramidite 328 and throughout the completion of the synthesis (Scheme 61). Phospholipidation of the afore-described PMB-protected pseudodisaccharide 318 was performed by using the arachidonoyl-containing phosphoramidite 328 in the presence of 1H-tetrazole. In situ oxidation with t-BuOOH, followed by removal of the TBS group with triethylamine trihydrofluoride, provided compound325. The key glycosylation between acceptor 325 and the tetramannosyl donor 324 was performed under conventional Schmidt conditions, leading to the α-pseudohexasaccharide 323 in moderate (39%) yield. Subsequent desilylation to remove the Man-I TES and Man-III TBDPS groups gave diol 333, which underwent double phosphorylation with phosphoramidite 293 to provide the fully PMB-protected GPI 334. Application of the previously described three-step, one-flask global-deprotection protocol involving consecutive treatments with Zn/AcOH, DBU, and 10% TFA delivered the target GPI anchor 322 in 90% yield over the final three steps (under 3 hours of reaction time).

Scheme 61.

Completion of the human lymphocyte CD52 antigen GPI anchor 322 by the Guo group.

Construction of the lymphocyte CD52 GPI anchor 322 constituted the first total synthesis of a GPI anchor bearing unremodeled fatty acids, namely a polyunsaturated arachidonoyl lipid at the sn-2 position of the phosphatidylinositol moiety. Inclusion of the oxidation- and reduction-sensitive arachidonoyl lipid was made possible by utilizing global protection by the PMB group, which featured a mild and rapid three-step, one-flask deprotection protocol that gave the target GPI anchor in 90% yield in under 3 hours. Furthermore, two additional structural modifications in the GPI core present in the lymphocyte CD52 antigen, specifically additional phosphoethanolamine and mannose units, were incorporated into the target molecule, which further demonstrated the applicability of the PMB-protection strategy to the efficient synthesis of highly complex and sensitively functionalized GPIs.

4. Synthesis of “Clickable” GPI Anchors by the Guo Group (2011)

Given the rapid influx of techniques in chemical biology that use “click” chemistry for chemoselective conjugation, particularly the powerful [3+2] cycloaddition reactions between between azides and alkynes/cyclooctynes,^133–136 the arming of GPIs with “click” tags provides a platform for developing novel approaches for exploring their biological properties and therapeutic applications (Fig. 2). For example, “clickable” GPIs could be deployed in studies involving molecular imaging, profiling of GPI-anchored proteins (“GPIomics”), and biophysical characterization of GPIs and GPI-anchored molecules on the surfaces of live cells. However, “click” chemistry tags and other useful functionalities, including many imaging and affinity purification tags, are often incompatible with traditional protecting-group methodologies used in carbohydrate synthesis.

Fig. 2.

Probing GPI anchors and GPI-anchored molecules with “click” chemistry.

In 2011, Swarts and Guo used the PMB-protection strategy to synthesize “clickable” GPI anchors.¹³⁷ Retrosynthetic analysis (Scheme 62) suggested a convergent–divergent strategy that would optimize efficiency while enabling access to a diverse set of GPIs. Flexibility was emphasized in the synthetic design to accommodate the structural heterogeneity of naturally occurring GPIs, particularly in lipid chains, as well the ability to functionalize GPIs with “click” tags for biological studies. Accordingly, the afore-described trimannose thioglycoside 309 and pseudodisaccharide 318 were used as adaptable, PMB-protected intermediates that would allow for a choice of phosphoglycerolipid structure and functional tag in the target GPI. Using these intermediates, two generations of “clickable” GPIs were synthesized. In this article, synthesis of the second-generation Alkynyl-GPI 335 and Aazido-GPI 336, as well as subsequent chemoselective labeling of these GPIs via “click” chemistry, is discussed.

Scheme 62.

Convergent–divergent strategy for the synthesis of “clickable” GPI anchors by the Guo group.

For both of the target GPI anchors335 and 336, a distearoylglycerophosphate group was chosen as the phospholipid (Scheme 63). Toward the synthesis of Alkynyl-GPI 335, the pseudodisaccharide 318 was phospholipidated by using phosphoramidite 338 under conventional conditions, followed by treatment with triethylamine trihydrofluoride to remove the TBS group, which gave the pseudodisaccharide 339. En route to Azido-GPI 336, the azido group of pseudodisaccharide 318 was replaced by an Fmoc group to give compound337, which subsequently underwent phospholipidation–desilylation in the same manner to furnish the Fmoc-protected pseudodisaccharide 340.

Scheme 63.

Synthesis of pseudodisaccharides 339 and 340 by the Guo group.

Completion of the synthesis of Alkynyl-GPI 335 from trimannoside 309 and pseudodisaccharide 339 is shown in Scheme 64. First, the terminal alkyne “click” tag was installed in the trimannose fragment by consecutive Cha deallylation¹²¹ and propargylation of the O-2 position of Man-I. Following hydrolysis of the anomeric thioacetal using p-nitrobenzenesulfenyl chloride (PNBSCl) and AgOTf/TTBP¹³⁸ in wet CH₂Cl₂, the intermediate hemiacetal was converted into trichloroacetimidate 341 by treatment with DBU and trichloroacetonitrile. The key glycosylation reaction of 341 with pseudodisaccharide 339, promoted by TMSOTf, furnished an intermediate that was immediately subjected to removal of the TBS group on O-6 of Man-III with triethylamine trihydrofluoride to give 342. This alkyne-modified pseudopentasaccharide was isolated in good yield (82%) over two steps, and no β anomer was observed. Installation of the phosphoethanolamine group into Man-III using phosphoramidite 293 proceeded well and gave compound 343, which was finally subjected to the aforementioned three-step, one-flask global deprotection to provide the target Alkynyl-GPI 335 in excellent (93%) yield in under 3 hours (three steps).

Scheme 64.

Completion of “clickable” alkynyl-GPI 335 by the Guo group.

The synthesis of Azido-GPI 336, shown in Scheme 65, followed a similar route. First, the O-2 allyl group on Man-I of compound 309 was exchanged for a 2-azidoethyl group in five steps, including Cha deallylation,¹²¹ base-promoted alkylation using tert-butyl bromoacetate, ester reduction with LiAlH₄, mesylation of the primary hydroxyl group, and finally displacement of the mesylate with NaN₃.¹³⁹ Transformation into the trichloroacetimidate 344 was accomplished by treatment with NIS/AgOTf/TTBP in wet CH₂Cl₂, followed by DBU and trichloroacetonitrile. The TMSOTf-promoted coupling of 344 with N-Fmoc-protected pseudodisaccharide 340 gave the desired α-pseudopentasaccharide intermediate, which was desilylated with triethylamine trihydrofluoride to generate compound 345 in moderate yield (30%, 88% based on recovered 340). The remaining steps involved installation of phosphoethanolamine at Man-III as already described to give 346, followed by global deprotection, which proceeded in two steps (DBU and then 10% TFA) to afford Azido-GPI 336 in 97% yield over the final two steps.

Scheme 65.

Completion of “clickable” azido-GPI 336 by the Guo group.

To establish Alkynyl-GPI 335 and Azido-GPI 336 as “clickable” tools for the biological study of GPI anchors, they were coupled via “click” chemistry to imaging and affinity probes (Scheme 66). Alkynyl-GPI 335 was conjugated with Azide-Fluor 488 (347), a commercially available regioisomeric mixture, via Cu(I)-catalyzed [3+2] cycloaddition under standard conditions.^140,141 This Cu-catalyzed azide–alkyne cycloaddition (CuAAC) reaction led to complete conversion of the starting material into the GPI-Fluor conjugate 348, a promising tool for visualizing GPI anchors by fluorescence microscopy in cellular contexts. Azido-GPI 336 was effectively coupled via Cu-free, strain-promoted azide–cyclooctyne [3+2] cycloaddition (Cu-free “click”)to BARAC-biotin (349), a biarylazacyclooctynone-based affinity probe developed by Jewett et al.¹⁴² This step effected virtually quantitative conversion of the starting material into GPI-Biotin 350. Conjugation of GPIs to affinity tags such as biotin is a potentially useful strategy for pull-down experiments to probe cell-surface, GPI-anchored proteomics. Alkynyl-GPI 335 and Azido-GPI 336, which were not accessible by traditional tactics for carbohydrate protection, were synthesized using Guo’s PMB-protection strategy. These “clickable” GPIs were demonstrated to undergo efficient coupling reactions via both CuAAC and Cu-free “click” reactions, and therefore constitute versatile tools for the development of chemical approaches in studying the biology of GPI anchors. In addition, this work further validated the PMB-protection strategy as an alternative global-protection option, most usefully when sensitively functionalized GPI anchors are targeted for synthesis.

Scheme 66.

“Click” reactions of alkynyl-GPI 335 and azido-GPI 336 by the Guo group.

5. General Synthetic Strategy for Branched GPI Anchors by the Seeberger Group (2011)

In addition to methods focused on expanding functional-group tolerance, such as the efforts by Nikolaev and Guo already discussed, the development of strategies that address structural diversity in terms of branching sugars and other substituents is also an important goal. As already discussed (see Fig. 1 in the introduction), the conserved core-structure of GPI anchors is often decorated in a species- and cell-type dependent manner, with additional sugars, phosphoethanolamine groups, and lipid chains at various positions. In 2011, the Seeberger group developed a general method for the synthesis of GPI anchors containing, in principle, any type of structural modification that is compatible with global benzyl-group protection.¹⁴³ A general retrosynthetic analysis for this strategy is shown in Scheme 67. It was expected that diverse target GPI anchors could be accessed from orthogonally protected GPI intermediates containing first of all the desired branching sugars at Man-I and Man-III, and secondly orthogonal protecting-groups at sites for late-stage phospholipidation, O-2 acylation of the inositol, and selective installation of phosphoethanolamine. These orthogonally protected GPI intermediates could be prepared from the interchangeable mannose building-blocks 351–354 and pseudodisaccharides 62 and 208.

Scheme 67.

General retrosynthesis for branched GPI anchors by the Seeberger group.

To demonstrate the applicability of this general method, Seeberger conducted the total synthesis of T. gondii GPI anchor 355, which contains a core modification of an α-Glc-(1→4)-β-Gal-(1→4) disaccharide at Man-I. The target GPI (355) was first disconnected to the orthogonally protected GPI intermediate 356, which was properly set up to undergo late-stage introduction of the phospholipid and phosphoethanolamine groups by the H-phosphonate method.⁴⁴ The preparation of 356 centered on Man-I building block 354, which possessed the appropriate array of orthogonal protecting-groups to allow the following [3 + 2 + 2] assembly sequence: first elongation at the O-3 position with donors 357 and 358 to give a trisaccharide; next [3 + 2] coupling with donor 351; followed by subsequent [5 + 2] coupling with pseudodisaccharide 62; and finally optional installation of a phosphoethanolamine group at the O-2 position of Man-I (not performed in this synthesis). For the glycosylation steps, a combination of glycosyl phosphates¹⁴⁴ and trichloroacetimidates⁷¹ were used as donors.

Preparation of the central Man-I derivative 354 and its elaboration to trisaccharide fragment 365 is shown in Scheme 69. En route to compound 354, the previously reported allyl mannoside 359¹⁴⁵ was protected at O-4 by the oxidatively cleavable 2-napthylmethyl (Nap) group, which was followed by acid-catalyzed deacetonation and selective tin-mediated benzylation at O-3 to give compound 360. Subsequent conversion into 354 was accomplished by diisopropylcarbodiimide (DIC)/DMAP-promoted levulinoylation (Lev) at O-2, followed by oxidative cleavage of the Nap group at O-4 with DDQ. Alternatively, this sequence could be modified to expose the O-3 position to obtain the Man-I derivative 353, which would be useful for the synthesis of GPIs of T. brucei containing additional sugars at this location.

Scheme 69.

Synthesis of central trisaccharide 365 by the Seeberger group.

The galactosyl phosphate derivative357, the coupling partner of Man-I derivative 354, was synthesized from the previously reported 2-azido selenoglycoside 361¹⁴⁶ in four steps, including regioselective benzylidene cleavage, napthylmethylation at O-4, Staudinger reduction followed by N-trichloroacetylation, and finally formation of the glycosyl phosphate by treatment with NIS/dibutyl phosphate. The Man-I acceptor 354 and galactosyl phosphate donor 357 combined in the presence of TMSOTf to give the β disaccharide 362, which was treated with DDQ to remove the Nap group, furnishing compound 363 quantitatively over two steps. Subsequent glycosylation of 363 with the glucosyl phosphate donor 358 in the presence of TBSOTf and thiophene (a reported α-enhancing additive),¹⁴⁷ afforded 364 (82% yield, α:β = 6:1), which underwent desilylation by HF·pyridine to generate the central trisaccharide 365.

Scheme 70 depicts the [3 + 2 + 2] assembly of building blocks 365, 351, and 62 to generate the orthogonally protected pseudoheptasaccharide intermediate 356. TBSOTf and added thiophene were again used to promoted α-stereoselective glycosylation between the dimannosyl donor 351 and the acceptor 365, resulting in the formation of pentasaccharide 366 in 69% yield. Reductive conversion of the trichloroacetamido group to an acetamido group gave compound 367, which was converted into the trichloroacetimidate donor 368 by deallylation using the Ir–Hg protocol¹²³ and subsequent treatment with trichloroacetonitrile and DBU. Reaction of this donor with the pseudodisaccharide 62 in the presence of catalytic TMSOTf forged the key glycosidic bond, delivering compound 356 in 71% yield. The participating Lev group at O-2 ensured α-stereoselectivity in this crucial coupling reaction.

Scheme 70.

Synthesis of orthogonally protected GPI intermediate 356 by the Seeberger group.

At this stage, compound 356 was subjected to Ir–Hg deallylation to expose O-1 of the inositol, which was phospholipidated by using H-phosphonate 205 and iodine-mediated oxidation to give intermediate 369 (Scheme 71). Acid-promoted removal of the TIPS group at O-6 of Man-III prepared the intermediate for the second phosphorylation, which was accomplished by using the H-phosphonate 4 to afford 370. Hydrazinolysis of the O-2 Lev group on Man-I deprotected the 2-position of Man-I, which had the potential to undergo further phosphorylation at this site if desired. In this synthesis, intermediate 370 was taken forward to global deprotection by hydrogenolysis to provide the target GPI 355 in 62% yield.

Scheme 71.

Completion of synthesis of the T. gondii GPI anchor 355, using the general method for branched GPI synthesis by the Seeberger group.

In this work, the Seeberger group synthesized the T. gondii GPI anchor 355, which carried a branching disaccharide at Man-I, using a [3 + 2 + 2] assembly strategy. The most important aspect of this strategy is its potential applicability to the synthesis of virtually any type of GPI anchor bearing common modifications, including acyl groups on O-2 of the inositol, phosphoethanolamine on O-2 of Man-I, sugars on O-3 or O-4 of Man-I, and a sugar on O-2 of Man-III. This flexibility is a result of carefully designed building-blocks, in particular the central Man-I components 353 and 354, and a well-optimized assembly sequence. In addition, synthetic 355 was used in combination with Seeberger’s GPI microarray technology¹¹⁰ to confirm that it was indeed the immunogenic “low-molecular-weight antigen” of T. gondii responsible for antibody production in toxoplasmosis patients.

IV. Conclusions and Outlook

Starting with Ogawa’s seminal work in the early 1990s, the field of chemical synthesis of GPI anchors has enjoyed marked success in melding the knowledge and methods of carbohydrate, inositol, lipid, and phosphate chemistry into a reliable framework for constructing these complex natural products. Numerous naturally occurring GPI anchors from protozoan parasites, yeast, and mammals have been synthesized, and through this many new synthetic methods have been developed and tested. Currently, the approach to GPI synthesis has shifted toward developing novel diversity-oriented strategies that have enabled access to new classes of molecules, including GPI anchors bearing the biologically important unsaturated lipids, indispensable functionalities such as “click” chemistry tags, and highly branched and modified core structures.

In the future, a combination of such diversity-oriented strategies, with the goal of a truly general method for the synthesis of any type of target GPI anchor, would be a highly valuable contribution. Another goal of great importance is improvement of the efficiency by limiting the number of steps in a synthesis, which, taking into account preparation of the monomers, can become prohibitively large. In this context, solid-phase synthesis, one-flask oligosaccharide synthesis, and proper reactivity tuning of glycosyl acceptors and donors will all contribute toward meeting this goal. Synthetic GPIs and GPI derivatives have the potential to play vital roles in deducing structure–activity relationships of these molecules in various contexts, exploring the scope of GPI anchorage as a post-translational modification, probing therapeutic applications, and in general gaining insight into their fundamental biological functions.

Scheme 21.

Retrosynthesis ofThy-1 GPI anchor 56 by the Schmidt group (2003).

Scheme 22.

Synthesis of pentasaccharide 125 by the Schmidt group.

Scheme 24.

Completion of Thy-1 GPI anchor 56 by the Schmidt group (2003).

Scheme 28.

Synthesis of inositol derivative 155 by the Guo group.

Scheme 30.

Completion of GPI anchor 148of human CD52 bearing an acylated inositol by the Guo group.

Scheme 33.

Synthesis of mannosyl NPOEs 182–184 by the Fraser-Reid group.

Scheme 35.

Construction of pseudopentasaccharide 178 by the Fraser-Reid group.

Scheme 37.

Retrosynthesis of P. falciparum GPI anchor 203 by the Seeberger group.

Scheme 39.

Synthesis of pseudodisaccharide acceptor 208 by the Seeberger group.

Scheme 41.

GPI–prion conjugate 226 synthesized by the Seeberger group.

Scheme 43.

Synthesis of tetramannosyl donor 230 by the Vishwakarma group.

Scheme 45.

Completion of T. cruzi GPI anchor 227 by the Vishwakarma group.

Scheme 47.

Synthesis of pseudodisaccharide 248 by the Guo group.

Scheme 68.

Retrosynthetic analysis of T. gondii GPI anchor 355, using the general method for branched GPI synthesis by the Seeberger group.

ABBREVIATIONS

Ac

acetyl

All

allyl

BDA

butanediacetal

Bn

benzyl

Boc

tert-butyloxycarbonyl

BRSM

based on recovered starting material

Bz

benzoyl

CAM

camphanoyl

CAN

ceric ammonium nitrate

Cbz

benzyloxycarbonyl

CA

chloroacetyl

“Click” chemistry

[3 + 2] azide—alkyne cycloaddition

CSA

camphorsulfonic acid

CuAAC

Cu-catalyzed azide—alkyne cycloaddition

DAST

diethylaminosulfur trifluoride

DBU

1,8-diazabicyclo[5.4.0]undec-7-ene

DCC

N,N′-dicyclohexylcarbodiimide

DCE

dichloroethane

DDQ

2,3-dichloro-5,6-dicyano-1,4-benzoquinone

DIBAL-H

diisobutylaluminum hydride

DIC

N,N′-diisopropylcarbodiimide

DMAP

4-dimethylaminopyridine

DMF

N,N-dimethylformamide

DMTrt

4,4′-dimethoxytrityl

DTBMP

2,6-di-tert-butyl-4-methylpyridine

EE

1-ethoxyethyl ether

Fmoc

9-fluorenylmethoxycarbonyl

Gal

galactose

GalNAc

N-acetylgalactosamine

GFP

green fluorescent protein

Glc

glucose

GlcN

glucosamine

GPI

glycosylphosphatidylinositol

KHMDS

potassium hexamethyldisilazide

Lev

levulinoyl

LPG

lipophosphoglycan

Man

mannose

m-CPBA

m-chloroperoxybenzoic acid

Mnt

menthyl

MS

molecular sieves

NAP

2-napthylmethyl

NIS

N-iodosuccinimide

NPG

n-pentenyl glycoside

NPOE

n-pentenyl orthoester

p-TsOH

p-toluenesulfonic acid

PA

phenoxyacetyl

PBS

phosphate-buffered saline

PG

protecting group

PI

phosphatidylinositol

PIV

pivaloyl

PMB

p-methoxybenzyl

PMP

p-methoxyphenyl

PMTrt

p-methoxytrityl

PNBSCl

p-nitrobenzenesulfenyl chloride

PPTS

pyridinium p-toluenesulfonate

pyr

pyridine

SEM

2-trimethylsilylethoxymethyl

TBAF

tetrabutylammonium fluoride

TBDPS

tert-butyldiphenylsilyl

TBS

tert-butyldimethylsilyl

TCA

trichloroacetyl

TDS

thexyldimethylsilyl

TES

triethylsilyl

Tf

trifluoromethanesulfonate

TFA

trifluoroacetic acid

THF

tetrahydrofuran

THP

tetrahydropyranyl

TIPS

triisopropylsilyl

TMS

trimethylsilyl

tol

toluene

TTBP

2,4,6-tri-tert-butylpyrimidine