Pseudouridimycin (1), a potent antibiotic against both Gram-positive and Gram-negative bacteria including multi-drug-resistant strains with a new mode of action isolated from Streptomyces sp., was synthesized by a convergent strategy from 5'-amino-pseudouridine 5 and N-hydroxy-dipeptide 26 in 23% total yield. The key intermediate 26 was synthesized by hydroxylaminolysis of the nitrone derived from glutamine and subsequent glycylation with glycine chloride. The synthetic method provides an efficient and practical way for the synthesis of N-hydroxylated peptidyl nucleoside.