Study of the Structural Changes on the Antimicrobial Activity of [3.1.1.]-Bicyclics

Journal of Essential Oil Research: JEOR, Nov/Dec 2004 by Dhar, Preeti, Ayala, Ulysses, Andarge, Eleni, Morisseau, Sophia, Snyder-Leiby, Teresa

Abstract

α-Pinene, a rigid bicyclic monoterpene, was altered by functional group transformations and the antimicrobial activity of the resulting compounds was studied using thin layer chromatography-autobiographic assay to determine the effect of structure on antimicrobial activity. Except for α-pinanone (and α-pinene oxide in two cases), all oxygenated α-pinene derivatives showed greater antimicrobial activity against Escherichia coli, Staphylococcus aureus, Micrococcus and Candida albicans than α-pinene. Conformationally flexible methyl cyclohexene and some of its oxygenated derivatives were also tested to evaluate the role of carbon skeleton in determining the antimicrobial activity. The results indicate that both functional groups and carbon skeleton affect the antimicrobial activity of the compound and adding nitrogen to the carbon framework increases the antimicrobial activity. The results also confirm that introduction of oxygen function to the carbon framework increases antimicrobial activity. Key Word Index

[3.1.1.]-bicyclic monoterpenes, pinene, antimicrobial activity, autobiographic assay, structure function.

Introduction

Terpenes and terpenoids, compounds that are found in essential oils, exhibit varying degrees of antimicrobial activity (1-5). The antimicrobial activity of a compound increases with the presence of an oxygen containing functional group (6-10) indicating a relationship between structure and antimicrobial activity. To date, only one detailed study has examined the link between structure and antimicrobial properties of terpenes and terpenoids with only a few [3.1.1.]-bicyclics studied (11). The current study further examines the structure activity relationship of these compounds.

Specifically, this study focuses on the rigid [3.1.1.] bicyclic monoterpene, α-pinene, a component of pine oil that exhibits antimicrobial activity (12). The site of action of terpenes and terpenoids is the cell membrane (13-16). α-Pinene has been found to affect the structural and functional properties of artificial membranes (17) by permeating the membranes and causing them to swell, thus inhibiting respiratoiy enzymes and causing partial dissipation of the pH gradient and electrical potential (18).

The antimicrobial activity of a set of oxygenated a-pinene derivatives and structurally related compounds that are constituents of several essential oils was evaluated in vitro against four microorganisms by thin layer chromatography TLC-agar overlay assay (19). TLC-agar overlay method combines TLC with a bioassay in situ and allows localization of compounds in the silica matrix. With this method, previously chromatographed compounds were transferred from the stationary phase to the agar phase by diffusion (20). TLC bioautography is a very simple, convenient and rapid method for testing crude extracts as well as pure compounds and gives results similar to the broth dilution method. In addition, the method is visual and stability of the compound on the plate can be easily verified.

The test microorganisms included the gram positive bacteria Micrococcus luteus and Staphylococcus aureus, the gram negative bacterium Escherichia coli and the unicellular fungus Candida albicans. Comparison of the test results of different chemical classes of compounds enabled an examination of the relationship between chemical structure and antimicrobial activity.

In addition to the study of [3.1.1.]-bicyclics, conformationally flexible methyl cyclohexene and some of its oxygenated derivatives were also studied. This was prompted by a report (21) that concluded that oral bioavailability of a molecule increased with reduced molecular flexibility. Comparison of the test results of rigid bicyclic α-pinene series and conformationally flexible methyl cyclohexene series enabled an examination of the role of molecular rigidity in antimicrobial activity. A listing of the compounds studied is given in Figure 1,

Experimental

General: Silica gel 6OA 230-400 mesh ASTM from Aldrich was used for gravity column chromatography. IR spectra were recorded on a Perkin Elmer 1600 FTIR spectrophotorneter and ^sup 1^H- and ^sup 13^C-NMR spectra were recorded at 300 and 75 MHz, respectively on a Jeol NMR instrument. All ACS grade solvents and dry solvents were purchased from Aldrich and used as such. Most compounds were purchased from Aldrich/ Fluka and two were synthesized using standard chemical procedures (22). Synthesized compounds were found to have the same IR and NMR as reported in the literature. Purity of all the compounds was checked using ^sup 1^H-NMR.

Microorganisms and culture media: The microorganisms, M. luteus, E. coli and S. aureus, were clinical isolates and were received as a gift from Katherine Griner, chair of the medical technology program at Marist College; C. albicans (ATCC 90294) was purchased from Sigma. The bacterial strains and the yeast were grown in Luria Bertani (LB) broth and LB agar plates at 37°C and the yeast was maintained on Sabaraud's agar (SAB). All media were autoclaved at 120°C for 20 min.