FT-IR spectral evidence is presented which indicates that intramolecular hydrogen-bonding occurs in chloramphenicol.Chloramphenicol is a broad-spectrum antibiotic which inhibits (K, = l-2 x 10 Me6) protein biosynthesis at the level of the peptidyl transferase center of the 50s ribosomal subunit.' The naturally occurring configuration of chloramphenicol is D-threo (Ia). In 1952, Dunitr published the results of an x-ray diffraction study of the dibromoacetyl analog of chloramphenicol which determined that the antibiotic exists in a preferred conformation (Ib) where the two hydroxyl groups are within 2.74 angstroms of one another.' Because of the close proximity of these two groups, Dunitz proposed that an intramolecular hydrogen bond was the force responsible for stabilizing the Ib-conformer. Other investigations of chloramphenicol using such spectroscopic techniques as IR, Raman and 'H NMR supported Dunitz's hypothesis.'e6 In particular, an early IR investigation showed two broad bands at 3630 and 3500 cm-' which are characteristic of both free, and intramolecular hydrogen-bonded, O-H stretches.5The existence of an intramolecular hydrogen-bond in chloramphenicol was questioned by Bustard et al. in their study involving high-dilution IR, 'H NMR and potential energy calcu1ations.J They postulated that the Ib-conformer mightbe stabilized through minimal polar and/or solvent interactions. The existence of intramolecular hydrogen-bonding was further discounted by a study which posited that the Ib-conformer is stabilized solely by dipolar attractive forces between the carbonyl oxygen and the p-nitroaromatic ring.' This concept has precedent in the conformational stabilization of o-nitrophenyl esters of Boc amino acids.gThe opposing hypotheses of Dunitz and Bustard have heretofore remained unresolved. We decided to reinvestigate chloramphenicol with the aid of the powerful technique of FT-IR. Our findings are reported herein.