Fig. 3. Synthesis of mono-Arg^Rha peptide and antibody generation. (A) Work-flow of antibody generation: in the first step an Arg^Rha containing glycopeptide was synthesized via guanidyl formation, cleavage and subsequent coupling to bovine serum albumin (BSA). The resulting glycoconjugate was used to immunize rabbits and accordingly to collect crude sera containing polyclonal antibodies against Arg^Rha. Using a two-step affinity chromatography technique we finally purified a highly sensitive and specific polyclonal anti-Arg^Rha antibody. Trt = trityl; Boc = tert-butoxycarbonyl. (B) Synthesis of building block 6. Reagents and conditions: (a) acetyl chloride, r.t., 2 days, 85%; (b) KSCN, TBAI, and CH₃CN, reflux, 3 h, 70%; (c) NH₃, and THF, 1 h, 99%; (d) EtI, and MeOH, reflux, 3 h; then Boc₂O, Et₃N, and CH₂Cl₂, 75%. (C) NMR spectroscopic characterization of compounds 4, 5, 6 and 1. (D) Single crystal structure of compound 5. (E) Solid-phase synthesis of mono-Arg^Rha peptide 1. Reagents and conditions: (a) TEA, DMF, AgNO₃, and 6 (3 eq.), r.t.; (b) 5% NH₂NH₂ in DMF; (c) 5% TIPS in TFA. (F) ELISA analysis of two batches of crude anti-sera. The crude anti-sera immunized by the BSA-glycoconjugate can recognize Arg^Rha with high affinity. anti-Serum 1# and anti-serum 2# were successively diluted up to 128 000 fold and subjected to indirect ELISA experiments against the BSA-glycoconjugate. (G) ELISA analysis of purified anti-Arg^Rha. Purified anti-Arg^Rha can recognize Arg^Rha with high specificity. The purified antibody was successively diluted up to 32 000 fold and subjected to indirect ELISA experiments against the BSA-glycoconjugate (BSA-Arg^Rha) and BSA carrying the non-glycosylated peptide (BSA-Arg).