Steven Marc Schwartzman

A Shortened Synthesis of Adjuvant Dipeptide (MDP)

N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP), has been shown to be the minimal structure necessary for adjuvant activity. This compound can replace whole mycobacteria in Freunds complete adjuvant. In our continuing investigation of bacterial cell wall fragments of biological and immunotherapeutic interest, the necessity of obtaining MDP analogs of varying structure has proven to be of primary importance. We have found that the published routes to MDP could be effectively shortened to four steps starting from commercially available starting materials. As an example of this scheme, synthesis of the seryl analog will be detailed. gamma-benzyl glutamic acid could be elaborated in one step to the N-tertiary butoxycarbonyl seryl-gamma-benzyl isoglutamine. Deprotection of the Boc group followed by condensation with 1-O-benzyl-4, 6-O-benzylidine N-acetyl muramic acid provided the protected MDP. Deprotection of this product by hydrogenolysis gave the final product. Physical chemical and biological data as proof of structure is presented. Utility of these compounds in the line-10 tumor system is demonstrated.

TUMOR REGRESSION CAUSED BY ENDOTOXINS COMBINED WITH TREHALOSE DIMYCOLATE

ABSTRACT The synergistic antitumor activity of two adjuvants, endotoxic glycolipid extracted from Re mutants of gram-negative bacteria and trehalose mycolate isolated from mycobacteria, against guinea pig syngeneic line-10 tumor was abrogated after peptidic substances accompanying the endotoxic extracts had been removed. This activity could be restored by combining peptide-free endotoxin with either cell wall skeleton from Bacillus Calmette-Guerin, a polymeric mycolic acid-arabinogalactan-mucopeptide, or with a combination of two separate components, trehalose dimycolate and synthetic N-acetyl-muramyl-L-alanyl-(L-seryl)-D-isoglutamine (MDP). The tumor regressive activity could also be restored to the refined endotoxin trehalose mycolate mixture by the addition of an essentially nontoxic lipoid side fraction recovered during the isolation of endotoxic glycolipids, which contained a small amount of peptidic substances, or by the addition of nontoxic Brauns lipoprotein known to contain covalently bound MDP moieties. Highly endotoxic lipopolysaccharide (LPS) extracted from wild type Enterobacteriaceae so far tested failed to cause tumor regression. However, acid-hydrolysis of wild type Serratia marcescens LPS led to a residual peptide-containing fraction, designated RESI, which serologically cross-reacted with endotoxins from Re mutant Salmonellae and which, in combination with trehalose dimycolate, provided a cure rate of 90%. This RESI was essentially nonpyrogenic and was about 100 times less toxic than a typical potent endotoxin, suggesting that there was no correlation between the antitumor property and endotoxicity.