Synthesis of a Glycosylphosphatidylinositol Anchor Bearing Unsaturated Lipid Chains

Abstract

A GPI anchor bearing unsaturated fatty acid lipid chains (1) was synthesized by a highly convergent strategy employing the para-methoxybenzyl group for permanent hydroxyl protection. The final global deprotection was achieved by an efficient three-step, one-pot procedure to give an 81% isolated yield of the target structure.

Glycosylphosphatidylinositols (GPIs) are a class of complex glycolipids that are ubiquitously expressed by eukaryotic cells and are operative in many biological processes, most notably the anchorage of extracellular molecules, such as surface proteins and glycoproteins, to the cell membrane.¹ Over 30 GPI anchors have been identified since the T. brucei variant surface glycoprotein GPI anchor was first characterized by Ferguson and co-workers in 1988.² All GPIs identified so far contain the following conserved core: H₂NEt-(P)-6-Manα(1→2)Manα(1→6)Manα(1→4)GlcNH₂α (1→6)myo-inositol-1-(P)-glycerolipid. Structural diversity among GPI anchors arises from additional phosphoethanolamine and carbohydrate moieties linked to the various locations of the core, as well as modifications in the lipid chains, which may contain unsaturated bonds that are crucial to bioactivity.³

In order to probe the effects of such structural modifications and better understand the scope and mechanism of GPI anchoring, it is necessary to have access to pure and structurally well-defined samples of GPI anchors, GPI analogues, and functionalized GPIs. Advances in chemical synthesis have enabled the preparation of GPI anchors by several research groups,⁴ including ours,⁵ but limitations associated with current synthetic strategies prevent the inclusion of some important functional groups. For example, most reported GPI syntheses have employed the benzyl group for permanent hydroxyl protection, which precludes the incorporation of alkene, alkyne, azide, thiol, thioether, and other functionalities that are intolerant to Pd-catalyzed hydrogenolysis. To address this issue, Nikolaev and co-workers⁴ⁱ utilized the benzoyl group for permanent protection to achieve the first synthesis of GPI anchors containing unsaturated lipid chains. However, the usage of acyl groups for hydroxyl protection complicates the presence of other esters and/or peptides/glycopeptides in the target molecule due to sensitivity to deprotection with sodium methoxide. For instance, the reported deacylation step in Nikolaev’s synthesis gave ≤ 40% yield, likely a result of degradation of the ester-linked lipid chains.⁴ⁱ Ideally, such a precarious and low-yielding step should be avoided, particularly when dealing with valuable and complex late-stage intermediates. Therefore, the efficient synthesis of GPI anchors containing unsaturated lipids and other useful functional groups has essentially remained an unsolved problem.

In this work, we sought to develop a new strategy for the synthesis of functionalized GPIs using the para-methoxybenzyl (PMB) group for permanent hydroxyl protection. PMB ethers can be cleaved under mild acidic or oxidative conditions, which we anticipated would provide the flexibility necessary for the incorporation of a wide range of functional groups in target molecules. As a demonstration of the value and feasibility of this methodology in the preparation of functionalized GPIs, as well as other glycoconjugates, we set out to synthesize a GPI anchor 1, which bears unsaturated fatty acid lipid chains. Scheme 1 depicts the retrosynthesis of target molecule 1. A convergent strategy was designed for the assembly of the target GPI from trimannose 2, pseudodisaccharide 3, and phosphorylating reagent 4. Fragment 3 was stitched together with mannose building blocks 5–7, while 4 was synthesized from inositol derivative 8, glucosaminyl donor 9, and phosphoglycerolipid precursor 10. For the glycosylations, we mainly utilized the Schmidt trichloroacetimidate method,⁶ which can be achieved under relatively mild conditions.

Scheme 1.

Retrosynthesis of GPI anchor 1

Mannose building blocks 5–7 were prepared from common mannose derivative 11 (Scheme 2). Thioglycoside alcohol 5 was obtained from known diol 12⁷ via a three-step sequence including stannylene acetal-directed regioselective 3-O-methoxybenzylation, 2-O-allylation, and regioselective para-methoxybenzylidene ring opening using DIBAL-H. Compound 6 was derived from 11 via orthoester 15,⁸ which after deacetylation-methoxybenzylation was treated with acetic acid to open the orthoester ring, providing a hemiacetal that was then converted to the corresponding imidate. The synthesis of 7 started with BF₃-promoted glycosylation of 11 with allyl alcohol. Subsequent deacetylation and differentiation of the 6-O-position by reaction with para-methoxytrityl (PMTrt) chloride afforded triol 16. Methoxybenzylation of the 2-, 3-, and 4-O-positions was followed by swapping the PMTrt group for a tert-butyldimethylsilyl (TBS) group to produce 17. Finally, Pd-catalyzed deallylation revealed the anomeric hydroxyl group, which was transformed into imidate 7.

Scheme 2.

Synthesis of mannose building blocks 5–7^a

^a Conditions: (a) Bu₂SnO, toluene, reflux; PMBCl, CsF, DMF. (b) NaH, AllBr, DMF, 68% (two steps). (c) DIBAL-H, CH₂Cl₂, 82%. (d) NaOMe, MeOH. (e) NaH, PMBCl, DMF, 76% (two steps). (f) AcOH-H₂O (1:1), 70%. (g) Cl₃CCN, DBU, CH₂Cl₂, 77%. (h) AllOH, BF₃·OEt₂, MS 4 Å, CH₂Cl₂, 66% (two steps from D-mannose). (i) NaOMe, MeOH. (j) PMTrtCl, pyridine, 66% (two steps). (k) NaH, PMBCl, DMF, 73%. (l) AcOH, H₂O, CH₂Cl₂, 94%. (m) TBSCl, pyridine, 88%. (n) PdCl₂, AcOH, NaOAc, CH₂Cl₂, H₂O, 82%. (o) Cl₃CCN, DBU, CH₂Cl₂, 73%.

With monosaccharides 5, 6, and 7 in hand, the construction of fragment 2 commenced (Scheme 3). The glycosylation of 5 by imidate 6 proceeded smoothly using catalytic TMSOTf in CH₂Cl₂ at 0 °C. The reaction generated only the α-product 18, which was deacetylated with K₂CO₃/MeOH to give 19 in 66% yield over two steps. Reaction of 19 with imidate 7 under the same conditions gave trimannose 20 in a 5:1 α/β ratio, a figure that was improved to α-only (76% yield) upon switching the solvent from CH₂Cl₂ to Et₂O. Compound 20 was designed as a versatile key intermediate for the synthesis of various GPIs containing a substituent at the Man-I 2-O-position. For the synthesis of GPI anchor 1, however, the allyl group of 20 was replaced with a TES group to insure later compatibility with the lipidic unsaturated bonds. Thus, 20 was subjected to Cha’s deallylation protocol,⁹ which proved to be the most effective after attempting several methods. The resulting alcohol then underwent protection by TESOTf to give 21, which was converted to imidate 2 in two steps, including NIS/AgOTf-promoted hydrolysis of the thioacetal and then treatment of the resulting hemiacetal with Cl₃CCN and DBU.

Scheme 3.

Synthesis of trimannose 2^a

^a Conditions: (a) TMSOTf (cat.), MS 4 Å, CH₂Cl₂, 0 °C. (b) K₂CO₃, MeOH, 66% (two steps). (c) 7, TMSOTf (cat.), MS 4 Å, Et₂O, 0 °C, 76%. (d) Ti(OiPr)₄, cyclopentylmagnesium chloride, THF, Et₂O, 82%. (e) TESOTf, 2,6-lutidine, CH₂Cl₂, 90%. (f) NIS, AgOTf, TTBP, wet CH₂Cl₂, 74%. (g) Cl₃CCN, DBU, CH₂Cl₂, 88%.

En route to optically pure inositol 8 (Scheme 4), racemate (±)-22 was generated from myo-inositol using a reported procedure.¹⁰ Here, we protected both the 1-O- and 6-O-positions as allyl ethers to give (±)-23, which underwent acid-catalyzed methanolysis of the ketals followed by methoxybenzylation, resulting in (±)-25. Deallylation using Cha’s method⁹ was again effective, providing diol (±)-26 in 85% yield. Positions 1 and 6 were differentiated at this stage using stannylene acetal-directed selective allylation to protect the 1-O-position (72%), and the free 6-O-position of (±)-8 was then used as an anchoring point for chiral resolution. After esterification with (1S)-(−)-camphanic chloride, the diastereomeric products were separated by preparative HPLC to afford optically pure (−)-27, which was saponified to give inositol alcohol 8 in 44% yield over two steps, including the enantiomeric resolution (maximum yield 50%).¹¹

Scheme 4.

Synthesis of inositol 8^a

^a Conditions: (a) cyclohexanone dimethyl ketal, TsOH, DMF, 38%. (b) NaH, AllBr, DMF, 81%. (c) AcCl, CH₂Cl₂, MeOH, 98%. (d) NaH, PMBCl, DMF, 73%. (e) Ti(OiPr)₄, cyclohexylmagnesium chloride, THF, Et₂O, 85%. (f) Bu₂SnO, toluene, reflux; AllBr, CsF, DMF, 72%. (g) (1S)-(−)-camphanic chloride, DMAP, Et₃N, CH₂Cl₂, 46%. (h) 1M NaOH, MeOH, THF, 95%.

Preparation of glucosaminyl donor 9 (Scheme 5) began with the transformation of known sugar 28,¹² which contained a non-participating azido group at the 2-position, to allyl glycoside 29. Deacetylation with NaOMe formed a triol that was reacted with para-anisaldehyde dimethyl acetal to afford 30. A PMB group was installed at the 3-O-position, and then regioselective opening of the para-methoxybenzylidene ring using HCl and NaBH₃CN provided 32. After usage of TBSOTf to protect the 4-O-position, the anomeric allyl ether was removed by a protocol involving Ir-catalyzed isomerization to the corresponding vinyl ether and subsequent hydrolysis using Hg(II).¹³ The resulting hemiacetal 33 was readied for glycosylation by conversion to imidate 9.

Scheme 5.

Synthesis of glucosamine 9^a

^a Conditions: (a) AllOH, SnCl₄, MS 4 Å, CH₂Cl₂, 81%. (b) NaOMe, MeOH. (c) anisaldehyde dimethyl acetal, CSA, DMF, 77% (two steps). (d) NaH, PMBCl, DMF, 97%. (e) NaBH₃CN, dry HCl in Et₂O, MS 4 Å, THF, 71%. (f) TBSOTf, 2,6-lutidine, CH₂Cl₂, 85%. (g) [Ir(COD)(PMePh₂)₂]PF₆, H₂, THF; then HgCl₂, HgO, acetone, H₂O, 85%. (h) Cl₃CCN, DBU, CH₂Cl₂, 83%.

The glycosylation of inositol derivative 8 with glucosaminyl imidate 9 was achieved with catalytic TMSOTf in CH₂Cl₂ at 0 °C to form pseudodisaccharide 34 (Scheme 6). While the conversion rate was very good (84%), stereoselectivity slightly favored the undesired β anomer (α/β 1.0:1.6). However, when Et₂O was used as the solvent, a moderately improved stereoselectivity was obtained (α/β 1.2:1.0). After the anomeric mixture was separated by preparative HPLC (separation by silica gel column following the next step was also possible), the Ir/Hg deallylation protocol was utilized to expose the inositol 1-O-position, giving 35 in 96% yield. Next, the unsaturated phospholipid was installed by reaction with freshly prepared phosphoramidite 10 under the influence of tetrazole. The intermediate phosphite was selectively oxidized in situ to a phosphate using tert-butyl hydroperoxide at −40 °C. Exposure of the product to Et₃N·3HF for 5 days removed the presumably hindered 4-O-TBS group, which gave compound 3 as a 1:1 diastereomeric mixture, originating from the stereogenic phosphorus atom, in 56% yield over two steps. The resulting mixture was separated by preparative HPLC to facilitate the characterization of 3 and subsequent complex intermediates.

Scheme 6.

Synthesis of lipidated pseudodisaccharide 3^a

^a Conditions: (a) TMSOTf (cat.), MS 4 Å, Et₂O, 44%. (b) [Ir(COD)(PMePh₂)₂]PF₆, H₂, THF; then HgCl₂, HgO, acetone, H₂O, 96%. (c) 10, tetrazole, CH₃CN, CH₂Cl₂; then t-BuOOH, −40 °C. (d) Et₃N·3HF, THF, CH₃CN, 56% (two steps).

The key step in the final stage of the synthesis was to couple the trimannose and peusdodisaccharide fragments (Scheme 7). Trimannose imidate 2 reacted smoothly with 3 in the presence of catalytic TMSOTf to afford the desired α-pseudopentasaccharide in 64% yield. Overnight treatment of the product with Et₃N·3HF removed the TBS and TES groups to provide diol 36, of which the anomeric J_CH coupling constants confirmed α stereochemistry for all mannose units.¹⁴ Installation of the phosphoethanolamine group by treatment with phosphoramidite 4 for a short period (1 h) was selective for the Man-III 6-O-position to give 37.¹⁵ To obtain the target GPI 1, a three-step, one-pot deprotection protocol was developed to efficiently remove all of the protecting groups from 37 in 4 h: (i) Zn-mediated reduction of the azide; (ii) removal of the base-labile Fmoc and cyanoethoxy groups with DBU; (iii) hydrolysis of all PMB ethers with 10% TFA. The target GPI anchor 1 was finally obtained in 81% yield after purification with a Sephadex LH-20 column and was characterized with ¹H NMR spectroscopy and MALDI-TOF MS.

Scheme 7.

Assembly of GPI anchor 1^a

^a Conditions: (a) TMSOTf (cat.), MS 4 Å, CH₂Cl₂, 64%. (b) Et₃N·3HF, THF, CH₃CN, 70%. (c) 4, tetrazole, CH₃CN, CH₂Cl₂; then t-BuOOH, −40 °C, 50% for 37 (61% BRSM). (d) Zn, AcOH, CH₂Cl₂, 2 h; DBU, CH₂Cl₂ 1 h; CH₂Cl₂-TFA (9:1), 1 h, 81% (three steps).

In summary, a GPI anchor containing unsaturated lipid chains was efficiently synthesized using the PMB group for hydroxyl protection. This represents a potentially generally useful strategy for the design and synthesis of uniquely functionalized GPIs, as well as other complex oligosaccharides, which may not be easily accessible by means of traditional protection methods, such as using benzyl, acetyl, or benzoyl groups. We are currently using this synthetic strategy to prepare various biofunctionalized GPI anchors aimed at probing cell surface GPIomics.