Synthesis of glycosylated β3-homo-threonine conjugates for mucin-like glycopeptide antigen analogues

Summary Glycopeptides from the mucin family decorated with tumour-associated carbohydrate antigens (TACA) have proven to be important target structures for the development of molecularly defined anti-cancer vaccines. The strategic incorporation of β-amino acid building blocks into such mucin-type sequences offers the potential to create pseudo-glycopeptide antigens with improved bioavailability for tumour immunotherapy. Towards this end, TN and TF antigen conjugates O-glycosidically linked to Fmoc-β3-homo-threonine were prepared in good yield via Arndt–Eistert homologation of the corresponding glycosyl α-amino acid derivative. By incorporation of TN-Fmoc-β3hThr conjugate into the 20 amino acid tandem repeat sequence of MUC1 using sequential solid-phase glycopeptide synthesis, a first example of a mixed α/β-hybrid glycopeptide building block was obtained. The latter is of interest for the development of novel glycoconjugate mimics and model structures for anti-cancer vaccines with increased biological half-life.


Introduction
Glycosylation is the predominant co-and post-translational modification in higher organisms responsible for tailoring and fine-tuning of the activity of proteins involved in fundamental biological recognition events of cell adhesion, cell differentiation and cell growth [1][2][3]. As a consequence, synthetic oligosaccharides and their conjugates are recognised as important tools for the expanding field of chemical biology [4]. Aberrant glycosylation of cell surface glycoproteins is associated with various pathological incidents, e.g., autoimmune and infectious diseases and cancer. In the latter case, unusual glycan structures composed of truncated O-linked oligosaccharides of carcinoma-derived mucin glycoproteins can be used as markers of the tumourigenic process and as target structures for cancer immunotherapy [5]. Over the last years, mucintype glycopeptides decorated with tumour-associated carbohydrate antigens (TACA) have been shown to trigger strong humoral immunity within molecularly defined vaccine prototypes [6][7][8][9][10]. However, the limited metabolic stability of the glycopeptide conjugates represents a major obstacle for the development of efficient carbohydrate-based vaccines. Various strategies towards the incorporation of non-natural hydrolysis resistant carbohydrate analogues into vaccine constructs have been pursued. For instance, stable TACA mimics comprising C-glycosides [11][12][13][14], S-glycosides [15][16][17][18][19] and deoxyfluoro sugars [20] have been used to circumvent hydrolytic degradation by endogenous glycosidases.
In principle, antigenicity of the artificial TACA derivatives should be enhanced by minor structural modifications assuming that the conformations remain similar to those of the natural antigens. In this respect, hybrid peptides in which β 3 -homoamino acids are used to strategically replace α-amino acids might be of interest as platforms for carbohydrate-based vaccines. That is because such mixed α/β-peptides adopt stable secondary structures closely related to those of natural α-peptides [21,22]. Moreover, inclusion of a single β-amino acid into an α-peptidic sequence already augments local and/or general stability against proteolytic degradation in vitro and in vivo; thus enabling the development of diverse peptidomimetics for an increasing number of applications [22][23][24][25]. Therefore we contemplated the use of mucin-derived α/β-hybrid glycopeptides as stable mimetics of naturally occurring glycocopeptide antigens for cancer vaccines. We were surprised to see how little precedence was available for this approach. Besides a recent report on α/δ-hybrid peptides derived from Neu2en and L-Glu [26], representing potentially immunogenic mimics of α-2,8-linked polysialic acid, only a few β-glycopeptides comprising N-acetylglucosamine [27][28][29] and N-acetylgalactosamine [30,31] (T N antigen) linked to β 3 -homo-serine are known. Despite their importance as specific tumour antigens, conjugates of Fmoc-β 3 hSer and Fmoc-β 3 hThr carrying larger TACA structures such as the Thomsen-Friedenreich antigen (TF) or its sialylated variants (α2-6sTF and α2-3sTF) have not been reported to date.
By presenting orthogonally protected T N and TF antigen conjugates of Fmoc-β 3 hThr ( Figure 1) as well as a first α/βhybrid glycopeptide analogue comprising the 20 amino acid tandem repeat sequence of the human mucin MUC1, we describe preliminary results of our synthetic efforts towards the preparation of mucin-type glycopeptide mimetics.

Results and Discussion
Initial attempts to directly link the carbohydrate entity to the β-side chain of a preformed Fmoc-β 3 hThr conjugate were unsuccessful due to rapid lactonisation [29]. Similarly, despite the use of various glycosyl donors and reaction conditions, glycosylation of the corresponding dipeptide precursor Fmocβ 3 hThr(OH)-Ala-OBn failed completely. Therefore we en- countered the strategy of Arndt-Eistert homologation in the synthesis of the target Fmoc-β 3 hThr(αAc 3 GalNAc) and Fmocβ 3 hThr(β(Ac 4 Gal(1-3))α(Ac 3 GalNAc)) conjugates 2a and 4, respectively, as reported by Norgren et al. [29]. T N antigen derivative Fmoc-Thr(αAc 3 GalNAc)-OH (1a) was prepared according to published procedures [32,33] and converted into the corresponding diazo ketone upon treatment with isobutyl chloroformate in the presence of N-methylmorpholine (NMM) and diazomethane (Scheme 1). Without further purification, the latter was subjected to a silver-promoted Wolff-rearrangement, again using NMM as the base, providing the spectroscopically pure T N antigen analogue Fmoc-β 3 hThr(αAc 3 GalNAc)-OH (2a) in 60% yield over two steps after aqueous work-up.
Compound 2a was also accessible from a direct synthetic precursor of T N derivative 1a in which the 2-acetamido substituent was masked by an azido group. Thus, upon subjection of Fmoc-Thr(αAc 3 GalN 3 )-OH (1b) [32] to the same homologation sequence as before, the corresponding β 3 hThr analogue 2b was obtained in 82% yield. Subsequent zinc-mediated reduction and acetylation led to the formation of conjugate 2a in 56% yield.
During biosynthesis T N antigen acts as an immediate precursor of the TF antigen. As a consequence, a biomimetic approach towards larger TACA structures via stepwise assembly of the glycan chain has been pursued in various antigen syntheses [33,34]. By applying chemical or enzymatic 3β-galactosylation, the 3-OH deprotected conjugate 2b could be converted into the desired antigen derivative Fmocβ 3 hThr(β(Ac 4 Gal(1-3))α(Ac 3 GalNAc))-OH (4). While this strategy certainly requires the use of optimised protecting group manipulations and glycosylation protocols, the alternative route to compound 4 via Arndt-Eistert homologation would benefit from an established and reliable synthesis of key building block 3 [32,33]. To our delight, the homologation reaction of glycosyl amino acid 3 again proceeded smoothly to afford the desired TF-β 3 hThr conjugate 4 in spectroscopically pure form and good chemical yield after aqueous work-up (Scheme 1).
To demonstrate the usefulness of the novel glycosylated β 3 hThr conjugates as antigen mimics, T N antigen analog 2a was incorporated into an α/β-hybrid glycopeptide 7 comprising a full tandem repeat sequence of the epithelial mucin MUC1 and an N-terminal non-immunogenic triethylene glycol spacer. The latter can be used for further conjugation to immunostimulants (e.g., BSA [35] or tetanus toxoid [36]) and for immobilisation onto microarray platforms [37] within functional immunological studies. The MUC1 pseudo-glycopeptide was assembled in an automated synthesiser by the Fmoc-strategy on a TentaGel S resin 5 equipped with a bulky trityl linker [38] to avoid diketopiperazine formation and pre-loaded with Fmoc-proline (Scheme 2). The first 13 amino acids of the MUC1 sequence were coupled under standard conditions using piperidine in N-methylpyrrolidone (NMP) to remove the temporary Fmoc protecting group followed by coupling of excess (10 equiv) Fmoc-amino acid activated by HBTU/HOBt [39] and diisopropylethylamine (DIPEA) in DMF. Unreacted amino acids were capped after each cycle with Ac 2 O in the presence of DIPEA and catalytic amounts of HOBt in NMP. The sterically demanding glycosylated β 3 hthreonine building block 2a (1.5 equiv), was coupled over an extended reaction time of 8 h employing the more reactive reagents HATU/HOAt [40] with N-methylmorpholine (NMM) in NMP for activation. After the final five Fmoc-amino acids of the TR-sequence were coupled according to the standard protocol, a triethylene glycol spacer 6 [41] (10 equiv) was attached using the standard coupling procedure, again. Simultaneous detachment of the glycopeptide from the resin and cleavage of the acid-labile amino acid side chain protective groups was achieved upon treatment with a mixture of TFA, triisopropylsilane and water. The resulting partially deblocked glycopeptide 7 was isolated after purification by semi-preparative RP-HPLC in a yield of 36%, based on the loaded resin 5. The final de-O-acetylation of the glycan portion was accomplished upon prolonged treatment with catalytic amounts of NaOMe in methanol at pH 9.5 to afford glycopeptide 8 in 18% yield (based on the loaded resin) after semi-preparative RP-HPLC.

Conclusion
Two novel tumour-associated carbohydrate antigen analogues with T N and TF determinants O-glycosidically linked to the side chain of Fmoc-β 3 hThr-OH have been prepared by Arndt-Eistert homologation of the corresponding glycosylated α-amino acids 1a and 1b. The resulting β 3 hThr glycoconjugates are valuable antigen mimetics with potentially enhanced chemical and metabolic stability. They might serve as precursors for the preparation of further mucin-type antigen structures, e.g., sialylated ones or those based on core2 structures. In addition, the preparation of a first MUC1 pseudo-glycopeptide comprising the modified glycosyl amino acid Fmoc-β 3 hThr(αGalNAc)-OH at position Thr15 has been accomplished using solid-phase peptide synthesis. By appropriate conjugation, the resulting α/βhybrid glycopeptide conjugate could be used as an antigen surrogate to elucidate the effects of chemically modified antibody determinants on the immunological properties of glycopeptide antigen analogues.

Experimental
General remarks: DMF (amine-free, for peptide synthesis) and NMP were purchased from Roth and Ac 2 O in p.a. quality from Acros. Fmoc-protected amino acids were purchased from General procedure for the synthesis of diazomethane in ethereal solution Caution: Diazomethane is toxic, highly-volatile, cancerogenic and explosive. Its generation and handling thus requires special precautions [42]. With regard to the free acids, 10 equiv of N-methyl-N-nitroso-urea were added to a solution of 50% KOH in H 2 O which was layered on top with Et 2 O. The organic layer was decanted and replaced by a new portion of Et 2 O until the organic layer was no longer coloured. The united organic phases were dried over KOH at −25 °C for 3 h.

General procedure (GP1) for the synthesis of diazo ketones:
The free acid (1 equiv) was dissolved in 2 mL of dry THF under an argon atmosphere. At −25 °C, 1 equiv of NMM and 1 equiv of isobutyl chloro formate were added subsequently and the resulting suspension was stirred for 20 min at this temperature. The mixture was allowed to reach 0 °C and the diazomethane solution in Et 2 O was added. The yellow solution was stirred 20 min at 0 °C before it was allowed to reach room temperature and stirred for further 16 h. Excess of diazomethane was destroyed by adding a few drops of acetic acid to the solution until no further nitrogen formation was observed. The solvents were removed under reduced pressure and the residue was dissolved in 20 mL Et 2 O. The organic layer was washed twice with saturated aqueous NaHCO 3 , saturated aqueous NH 4 Cl and brine. The organic layer was dried over Na 2 SO 4 , filtered, and the solvents were removed under reduced pressure. The resulting diazo ketones were used without further purification.

General procedure (GP2) for the Wolff-rearrangement:
The diazo ketone (1 equiv) was dissolved in a mixture of THF/H 2 O (9:1) and was cooled to 0 °C. A solution of 0.11 equiv silver trifluoroacetate in 2.3 equiv NMM was added, and the mixture was stirred for 16 h while it was allowed to warm to room temperature. After evaporation of THF, the aqueous layer was diluted with saturated aqueous NaHCO 3 and Et 2 O was added. The organic layer was extracted three times with saturated aqueous NaHCO 3 . The aqueous phases were collected, cooled to 0 °C and acidified to pH 1. The resulting suspension was extracted five times with Et 2 O. The organic layer was dried over Na 2 SO 4 and evaporated in vacuo.