The 5,6-di- O-tosylated derivative of l-ascorbic acid was synthesized by selective protection and deprotection of 2,3- and 5,6-dihydroxy functional groups involving 5,6-ditosylation in the final step, while the novel 6-acetoxy, 6-hydroxy, and 6-chloro derivatives of 4,5-didehydro- l-ascorbic acid were obtained by reaction of ditosylated compound with nucleophilic reagents. The analysis of 3 J H-4–H-5 homonuclear coupling constants shows that all l-ascorbic acid derivatives except for epoxy and 4,5-didehydro compounds exist in high population as gauche conformers across C-4–C-5 bonds, while 3 J C-3–H-5 heteronuclear coupling constants in 4,5-didehydro derivatives indicate cis geometry along C-4–C-5 double bond. The X-ray crystal structure analysis of 2,3-di- O-benzyl-5,6-epoxy- and 5,6-isopropylidene- l-ascorbic acid shows that the oxygen atoms attached at positions 2 and 3 of the lactone ring are disposed in a synperiplanar fashion. Besides that, the dioxolane ring adopts half-chair conformation. The molecules of epoxy derivative are joined into infinite chains by one weak hydrogen bond of C–H⋯O type. Two O–H⋯O, and C–H⋯O hydrogen bonds link the molecules of 5,6-di- O-isopropylidene compound into two-dimensional network. 6-Chloro derivative of 2,3-di- O-benzyl- l-ascorbic acid showed the best cytostatic effects against all tested malignant tumor cells (IC 50: ∼18 μM).