A series of nine L-2,4-syn-4-alkylglutamic acid analogues (1a-i) were synthesized in high yield and high enantiomeric excess (>99% ee) from their corresponding 4-substituted ketoglutaric acids (2a-i), using the enzyme aspartate aminotransferase (AAT) from pig heart or E. coli. The synthesized compounds were evaluated as potential ligands for the glutamate transporters EAAT1, EAAT2, and EAAT3 (excitatory amino acid transporter, subtypes 1-3) in the FLIPR membrane potential (FMP) assay. We found a distinct change in the pharmacological profile when the 4-methyl group (compound 1a, an EAAT1 substrate and EAAT2,3 inhibitor) was extended to a 4-ethyl group, compound 1b, as this analogue is an inhibitor at all three subtypes, EAAT1-3. Furthermore, we conclude that both large and bulky hydrophobic substituents in the 4-position of L-2,4-syn Glu are allowed by all three glutamate transporter subtypes EAAT1-3 while maintaining inhibitory activity.