Fmoc-O-tert-butyl-D-serine

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp and Nalpha-Fmoc-Thr-[Ac4-beta-D-Gal-(1-3)-Ac2-alpha or beta-D-GalN3]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. Nalpha-Fmoc-Ser-OPfp or Nalpha-Fmoc-Thr-OPfp) with protected beta-D-Gal(1-3)-D-GalN3 synthon mediated by silver salts resulted in only alpha- and/or beta-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO4 at -40 degrees C which afforded a-isomer in a quantitative yield (alpha:beta = 5:1). On the other hand, the beta-isomer was formed exclusively when the reaction was performed in the presence of Ag2CO3/AgClO4 at room temperature. A complete assignment of 1H resonances to individual sugar ring protons and the characteristic anomeric alpha-1 H and beta-1 H in Ac4Galbeta(1-3)Ac2GalN3 alpha and/or beta linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned alpha- and beta-isomers of the O-glycosylated amino acid building blocks carrying protected beta-D-Gal(1-3)-D-GalN3, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O-linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polymer-bound glycopeptides before the cleavage step. After cleaving the glycopeptide from the resin, the acetyl groups used for sugar OH-protection were removed with sodium methoxide in methanol. Finally, the glycopeptides were purified by reversed-phase high-performance liquid chromatography and their integrity was confirmed by proton NMR as well as by mass spectral analysis. Secondary structure analysis by circular dichroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.

A synthetic strategy to prepare peptide-polymer conjugates with precise sites of attachment is described. Amino acids modified with atom transfer radical polymerization (ATRP) initiators for the polymerization of styrenes and methacrylates were prepared. Fmoc-4-(1-chloroethyl)-phenylalanine (5) was synthesized in four steps from Fmoc-tyrosine. HATU-mediated amidation with glycine-OMe resulted in dipeptide (6). The initiator was effective for Cu(I)/bipyridine mediated bulk polymerization of styrene. Kinetic studies indicated a controlled polymerization, with high conversion (97%), and a polydispersity index (PDI) of 1.25. Fmoc-O-(2-bromoisobutyryl)-serine tert-butyl ester (10) was synthesized from Fmoc-Ser(OTrt)-OH in three steps. This initiator was employed in the ATRP of 2-hydroxyethyl methacrylate (HEMA), and kinetic studies indicated a controlled polymerization. Different monomer to initiator ratios resulted in poly(HEMA) of different molecular weights and narrow PDIs (1.14-1.25). Conversions were between 70 and 99%. HEMA modified with N-acetyl-D-glucosamine (GlcNAc) was also polymerized to 84% conversion and the resulting PDI was 1.19. The t-butyl ester protecting group of 10 was removed, and the resulting amino acid (11) was incorporated into VM(11)VVQTK by standard solid-phase peptide synthesis. Polymerization resulted in the glycopolymer-peptide conjugate in 93% conversion and a PDI of 1.14.

A synthetic strategy for the preparation of two orthogonally protected methyl esters of the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to the p-toluensulfonyl (tosyl, Ts) or acid-labile tert-butyloxycarbonyl (Boc) moieties. The synthetic approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols, which are obtained via reductive amination of an aldehyde prepared from the commercial amino acid Nα-Fmoc-O-tert-butyl-d-serine, used as the starting material. Reductive amination is carried out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(OiPr)4. The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols bearing the tosyl or benzyl protecting group on the 3-NH2 site. The procedure can easily be applied using the crude product obtained after each step, minimizing the need for chromatographic purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout all synthetic steps.