The organic layer was extracted and washed with a saturated solution of NaHCO3 and water, dried (MgSO4), filtered and the filtrate was concentrated = 13.88 Hz, 6H, CH3-Si-CH3), 0.70 (s, 12H, TDS-(CH3)2C-C(CH3)2), 0.94 (d, = 6.4 Hz, 3H, Fuc CH3), 1.47 (t, = 6.7 Hz, 1H, TDS-CH), 1.61 (dd, = 11.3, 5.4 Hz, 6H, 2x COCH3), 1.74 (d, = 8.5 Hz, 6H, 2x COCH3), 1.89 (s, 3H, COCH3), 3.23C3.28 (m, 3H, Gal H-5, GlcN H-2, GlcN H-5), 3.58C3.76 (m, 5H, GlcN H-3, GlcN H-6a, Fuc H-2, GlcN H-4, Gal H-6a), 3.80C3.86 (m, 2H, Gal H-6b, Gal H-2), 4.10 (dd, = 10.5, 4.8 Hz, 1H, GlcN H-6b), 4.43C4.55 (m, 4H, Fuc H-5, C= 7.9 Hz, 1H, Gal H-1), 4.83 (dd, = 9.7, 3.2 Hz, 1H, Gal H-3), 5.00 (d, = 3.0 Hz, 1H, Gal H-4), 5.08 (d, = 2.9 Hz, 1H, GlcN H-4), 5.17C5.21 (m, 2H, Fuc H-1, Fuc H-3), 5.46 (s, 1H, Naphthylidene-H), 7.16 (d, = 8.3 Hz, 1H, aromatic CH), 7.24C7.31 (m, 4H, 4x aromatic CH), 7.40 (d, = 8.5 Hz, 1H, aromatic CH), 7.49 (s, 1H, aromatic CH), 7.56C7.70 (m, 7H, 7x aromatic CH). of glycosylations. The trisaccharide-Hyp moiety was employed for the preparation of the glycopeptide using microwave-assisted solid phase peptide synthesis. Enzyme kinetic studies revealed that trisaccharide-Hyp and trisaccharide-peptide are poorly recognized by AgtA, indicating the importance of context provided by the native Skp1 protein for engagement with the active site. The trisaccharide-peptide was a potent immunogen capable of generating a rabbit antiserum that was highly selective toward the trisaccharide isoform of full-length Skp1. was established by metabolic incorporation of [3H]Fuc, and confirmed WEHI-9625 by compositional analysis of the protein showing the presence of and studies stress the importance of the glycan in promoting interactions WEHI-9625 with F-box proteins, which may represent the mechanism by which hydroxylation and glycosylation of Skp1 mediate or modulate O2-sensing during development. Studies on O2-dependent hydroxylation of Skp1 in cells, relative stability of Skp1 isoforms in cells, and competitive interactions of Skp1 isoforms with binding partners, have all been made possible by isoform-specific antibodies that differentiate unmodified, hydroxylated, and GlcNAcylated forms of Skp1 from each other and all other isoforms.[10C11] We reasoned that availability of an antibody specific for the trisaccharide form would be similarly exploitable for related studies and to investigate the contingency of Skp1 glycosylation in response to stress and during development. The failure of available anti-blood group (type 1) antibodies to react with Skp1 necessitates the generation of new Abs. We report here the chemical synthesis of a glycopeptide derived from Skp1 that is modified by a blood group H type 1 trisaccharide, which was conjugated to KLH and employed for antibody production in rabbits. A synthetic strategy was developed in which a trisaccharide was synthesized that was modified by Hyp to give, after a number of chemical manipulations, a glycosylated amino acid that could be employed for glycopeptide synthesis. 2-Naphthylmethyl (Nap) ethers were employed as permanent protecting groups to allow late stage installation of the Hyp moiety. It was found that tuning glycosyl donor and acceptor reactivities was critical for achieving high yields and anomeric selectivities of the glycosylations. The suitability of the novel glycopeptide for probing AgtA acceptor substrate preference and to induce an isoform specific antibody is presented. Results and Rabbit polyclonal to Adducin alpha Discussion The target glycopeptide 1 was synthesized by first preparing trisaccharide 5, which was modified by an appropriately protected Hyp residue to give, after WEHI-9625 a number of protecting group manipulations, glycosylated amino acid 6, which was used in solid phase peptide synthesis (Figure 1). Late stage installation of the Hyp moiety was attractive because this amino acid can adopt cis/trans configurations complicating NMR analysis. It was anticipated that trisaccharide 5 could readily be prepared from monosaccharide building blocks 2, 3 and 4. The non-participating azido moiety of 5 would allow the installation of the Hyp moiety as an -glycoside. Furthermore, Nap ethers were selected as permanent protecting groups because they can be readily removed by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to give, after standard anomeric selectivity. Alternatively, it may promote the formation of an -nitrilium ion which upon displacement by a sugar alcohol will give a -glycoside. Disaccharides 7aCc were converted to glycosyl acceptor 8 by removal of the acetyl or dFBz protecting groups of 7a and 7b, respectively by using Zempln conditions, whereas the Lev ester of 7c was cleaved using hydrazine acetate. Table 1 Optimizing galactosylation mutants that accumulate the indicated Skp1 glycoforms. Panels were probed with either pAb UOK104 or UOK77 (pan-specific for all Skp1 isoforms) at 1:1000 dilution. Note: An irregularity during placement of the gel on the blot membrane resulted in distortion WEHI-9625 of relative mobilities of the Skp1 isoforms. (B) The glycan specificity of UOK104 was tested against BSA conjugates of LNFP-I and LNFP-III, which were adsorbed in solutions containing the indicated pmol of glycan equivalents in 50 L to wells of a 96-well ELISA plate. Coated wells were probed with UOK104 (1:500) or mAb ab3355 (1:10), with specificity for the blood group H type 1 antigen present in LNFP-I/BSA. To test the basis for specific recognition of FGGn-Skp1, pAb UOK104 was tested for binding of a BSA-conjugate modified by lacto-substrate dependence of AgtA revealed that galactose was transferred to trisaccharide-Hyp 23, however it was a poor substrate and the extended glycopeptide 1 was even less active. These results are consistent with folding of the acceptor hydroxyl of Fuc into a trisaccharide-dependent glycan conformation that is not recognized by AgtA. The underlying hydroxyproline WEHI-9625 or glycopeptide may stabilize this unfavorable conformation leading to even.