Solid Support Oligosaccharide Synthesis

260 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

O

i, TMSOTf; ii, AcOH (80%); iii, Zn, Ac2O, AcOH, THF; Ac2O, pyridine; iv, TFA (95%)

Scheme 13.3 Synthesis of the core 4 building block.

trisaccharide conjugate 9. Its azido function and the Troc-protected amino functions were simultaneously converted into the acetamido functions. Cleavage of the tert-butyl group using TFA gave core 4 building block 10 suitable for solid-phase glycopeptide syntheses. The synthesized building blocks were incorporated at the different positions in the syntheses of two partial sequences of the repeating units of the two human intestinal mucins MUC2 and MUC3 (positions indicated by * in Scheme 13.4) by multiple-column solid-phase synthesis (MCPS) in a manual 20-column multiple synthesizer.16 Wang resin17 served as the polymer support. Coupling of the glycosylated amino acids was performed using O-(1-benzotriazole- 1-yl)- N,N,N′,N′-tetramethylammonium tetrafluoroborate (TBTU)18 as coupling reagent. The nonglycosylated amino acids were applied as their Fmoc/Pfp esters using Dhbt-OH as activating agent which allows a visual control of peptide bond formation.19 Side chains of threonine and serine were protected as their tert-butyl ethers; those of glutaminic acid, as their tert-butyl esters; and histidine was tert-butyloxycarbonyl-protected. Final detachment of the synthesized glycopeptides from the resin using TFA proceeded with simultaneous removal of the acid-sensitive side-chain protecting groups. It was followed by removal of the O-acetyl and -benzoyl protecting groups by transesterification with sodium methanolate/methanol.

13.2 BUILDING-BLOCK APPROACH 261

Scheme 13.4 Partial sequences of MUC2 and MUC3.

13.2.1.2 Glycopeptides Carrying Tumor-Associated Antigens In cancer cells, glycoproteins show glycosylation patterns different from those of glycoproteins in normal cells. The carbohydrate side chains are incompletely developed. Different tumor-associated carbohydrate structures such as the TN (GalNAcα-O-Ser/Thr), TF or T(Gal(β1→3)GalNAcα-O-Ser/Thr), SialylTN(NeuNAc(α2→6)GalNAcα-O- Ser/Thr), and the two regioisomeric SialylT Gal(β1→3)[NeuNAc(α2→6)]GalNAcα-O- Ser/Thr) and NeuNAc(α2→3)[Gal(β1→ 3)]GalNAcα-O-Ser/Thr antigens have been identified.20 They are the result of premature completion of glycosylation in cancer cells, due to changes in the expression and reactivity of glycosyltransferases.21

The tumor-associated TN antigen structure was used in the synthesis of glycopeptides that then were coupled to carrier proteins in order to obtain synthetic antigens.9b As an example, a multiple-antigen glycopeptide (MAG) was synthesized on solid phase and subjected to immunological evaluation.22 The presentation of peptidic epitopes to the immune system can be achieved by means of a polylysine core.23 To introduce an efficient antibody response, a conjugate of a B-cell epitope (TN antigen) and a T-cell epitope, the T-cell epitope of the VP1 protein of polio virus type 1,24 was presented by the multiple antigen glycopeptide 11 (Scheme 13.5).

The synthesis of the TN antigen building block 16 was performed starting from tri-O-acetyl-D-galactal 12 (Scheme 13.6).25 Glycosylation of Fmoc serine tert-butyl ester 14 with the azidogalactosyl chloride 13 using Ag2CO3/AgClO426 as promotors was followed by reduction and acetylation of the 2 position to give compound 15. Cleavage of the tert-butyl ester was performed in formic acid, and the acetyl groups of the sugar moeity were removed by NaOMe/MeOH.

For the synthesis of the multiple antigen 11, a β-alanyl spacer was attached to Wang resin17 and the lysine core was constructed by coupling successively two levels of FmocLys(Fmoc)OH using TBTU as the carboxyl activating agent. The conjugate was sequentially elongated at the four amino functions in order to assemble the T-cell epitope sequence. Finally, Fmoc-protected glycosyl serine 16, deprotected in its carbohydrate portion, was coupled to the N termini of this construct. After Fmoc removal, the conjugate was detached from the resin using TFA/water/ethanedithiol 95/2.5/2.5 with simultaneous cleavage of the acid-labile amino acid side chain protecting groups. Preliminary immunological tests showed that the MAG system 11 is recognized by the monoclonal anti-TN antibodies 83D4 (IgM) and MSL128 (IgG).22

The synthesis of a glycopeptide from the homophilic recognition domain of epithelial cadherin 1 (E-CAD1) carrying the TN structure was performed using the novel condensing reagent N,N-N′,N′-bis(tetramethylene)-O-pentafluorophenyl- uronium hexafluorophosphate PfPyU 18.27 The cadherins constitute a family of about 30 cell surface glycoproteins that are involved in the Ca2+-dependent adhesion of

13.2 BUILDING-BLOCK APPROACH 263

cells.28 They play an important role in the morphogenesis of cells and are found on every tissue-forming cell type. First attempts to synthesize the E-CAD1 sequence SHAVSSNGEAVE using TBTU as the coupling reagent gave only small amounts of the target compound, presumably because of a backfolding effect of the β-sheet-forming sequence. Comparative coupling reactions of Fmoc-Ala-OH to H-Val-OtBu showed that the coupling rate induced by PfPyU is 8 times higher than that induced by TBTU. The synthesis of a partial sequence of the homophilic recognition region of E-CAD1 containing α-GalNAc at serine within the turn structure using PfPyU as acylating reagent and β-alanyl-TentaGel S29 loaded with the allylic HYCRON30 anchor conjugate of Fmoc-Glu(OtBu)-OH as the solid support gave a high yield of desired compound 19 (Scheme 13.7).

19

OH HO

a) morpholine/DMF; 3 eq. Fmoc-Xaa-OH, PfPyU, sym-collidine, iPr2NEt, NMP; b) cat. Pdo(PPh3)4, N-methylaniline,

Scheme 13.7 Solid-phase synthesis of a glycopeptide from the homophilic recognition domain of epithelial Cadherin 1 using the new coupling reagent PfPyU.

The PfPyU/HYCRON methodology also proved efficient in the synthesis of a lipoglycopeptide of the E-CAD1 recognition region sequence. The synthesis of the glycododecapeptide amphiphile was performed using a convergent solid-phase approach.31 A fragment of the N-terminal tetrapeptide with the lipophilic tail group was synthesized first (Scheme 13.8).

264 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

Fmoc-Val-HYCRON-bAla-AMPS

Scheme 13.8 Synthesis of a lipoglycopeptide by means of fragment condensation methodology.

The allylic HYCRON anchor conjugate 20 of Fmoc-Val-OH and β-alanyl- polystyrene was used as the starting material, and the polymer-bound SHAV tetrapeptide was synthesized according to the Fmoc methodology. However, the second amino acid alanine was coupled as the Boc-protected derivative in order to minimize diketopiperazine formation on the stage of the amino-deprotected resin-linked dipeptide. Polymer-bound tetrapeptide was then coupled with the lipophilic succinylglutaminic ester derivative.32 Owing to the very mild cleavage of the HYCRON anchor by Pd(0)-catalyzed allyl transfer to morpholine leaving acidand base-sensitive protecting groups unaffected, the fully protected conjugate 21 was

13.2 BUILDING-BLOCK APPROACH 265

obtained in pure form. Fragment 21 was now coupled to the E-Cadherin octapeptide fragment H-S(tBu)S(αAc3GalNAc)N(Trt)GE(OtBu)AVE(OtBu) linked to the HYCRON resin 22 using a 1.7-fold excess and PfPyU/sym-collidine/ diisopropylethylamine as the activating reagent. After cleavage of the acid-labile protecting groups using TFA/H2O/triisopropylsilane (95:2.5:2.5), the obtained lipoglycopeptide was detached from the resin by Pd(0)-catalyzed allyl transfer to morpholine as trapping agent. Final removal of the O-acetyl groups from the carbohydrate moiety by Zemplén transesterification yielded the target compound 23. The synthesis demonstrates both the versatility of the allylic HYCRON system, which can be used to generate protected peptide fragments for fragment condensation reactions, and the efficiency of PfPyU as condensing reagent.

13.2.1.3 Glycopeptides Containing Sialic Acid The sialyl-TN structure is considered to be one of the most important tumor-associated antigens of epithelial tumors.33,34 It is found on mucins35 on glycophorin20a,36 and on the envelope glycoprotein gp120 of the human immunodeficiency virus HIV.37,38 The solid-phase synthesis of a sialyl-TN glycoundecapeptide of the MUC1-repeating unit has been described.39 The sialyl-TN-threonine building block 28 was obtained by a stereoand regioselective sialylation of galactosamine threonine conjugate 24 with the sialyl xanthate 2540 and methylsulfenyl triflate as the promoter in a yield of 32% (Scheme 13.9).

FmocHN

O

26 R1 = H, R2 = tBu

27 R1 = Ac, R2 = tBu

28 R1 = Ac, R2 = H

Scheme 13.9 Synthesis of the sialyl-TN building block 28.

O-Acetylation and subsequent cleavage of the tert-butyl ester with TFA gave building block 28. Solid-phase glycopeptide synthesis was performed using an aminomethylpolystyrene (AMPS) resin modified with the HYCRON anchor. Coupling of the Fmoc amino acids was performed with TBTU/HOBt and a 4.2-fold excess of the corresponding N-protected amino acids. After final N-acetylation of the terminal amino acid, the completely protected glycopeptide was cleaved from the resin by Pd(0)-catalyzed allyl transfer to morpholine. Cleavage of the acid-labile

268 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

Scheme 13.13 Synthesis of a type II collagen analog using acid-labile carbohydrate protection groups.

related to the immunodominant fragment of type II collagen was achieved employing acid-labile silyl and isopropylidene protecting groups (Scheme 13.13).48,49 The solid-phase synthesis was performed using crosslinked polystyrene grafted with poly(ethylene glycol) chains (TentaGel S)29 carrying an acid labile 4-alkoxybenzyl alcohol linker as the solid support. Coupling reactions were performed using DIC/HOBt for the unglycosylated amino acids and DIC/HOAt for the glycosylated amino acid.

The detachment of the glycopeptide from the resin and deprotection of the amino acid side chains as well as the removal of the acid-labile carbohydrate protecting groups were simultaneously carried out using TFA/H2O/thioanisole/ethanedithiol (87.5:5:5:2.5). This treatment proceeded without affecting the glycosidic bonds and furnished the target molecule 38.

13.2.2 N-Glycopeptides

13.2.2.1 N-Glycopeptides Carrying the Sialyl Lewis A Structure The regioisomeric tetrasaccharide structures sialyl Lewis A (sLea) (NeuAcα(2→3)Galβ(1→3) [Fucα(1→4)]GlcNAc) and sialyl Lewis X (sLex)50 (NeuAcα(2→3)Galβ(1→4)