Advanced studies on cyclic amino acids in neurological signaling and peptide antibiotics

by Blanchard, David

L-pipecolic acid (L-PA) is the higher homolog of proline. It occurs naturally in

many organisms, including primates, as an intermediate in lysine degradation. The

pathway by which lysine is converted into L-pipecolic acid employs the enzyme Lpipecolate

oxidase (L-PO), and appears to be tissue specific to the central nervous system

(CNS). The oxidation facilitated by L-PO is the rate limiting step of lysine degradation

in the CNS. For this reason, the mechanism of action for L-PO may be useful in the

development of neuromodulation. This thesis describes efforts to probe the mechanism

of action of L-PO through the synthesis of substrate analogs as alternate substrates and

inhibitors of L-PO.

Analogs that contain heteroatoms and other functionalities at key positions have

been synthesized and analyzed as both alternate substrates and inhibitors of L-PO. The

4,5-methanopipecolic acid has been shown to be an excellent substrate for L-PO. The 5-

keto analog was not a substrate or inhibitor of the enzyme. The 5-hydroxy and 5,5-

2

difluoro analogs have been shown to be weak inhibitors of L-PO. 6S-methyl-L-pipecolic

acid (35) was shown to be a weak substrate and strong inhibitor while the enantiomeric

6R methyl-D-pipecolic acid (36) was neither a substrate nor inhibitor.

These results suggest flexibility within the binding pocket of L-pipecolate oxidase

toward analogs containing substituents at the 5-position. Additionally, these studies

demonstrate the potential to develop mechanism-based inhibitors that could be used to

control the rate of L-pipecolic acid consumption as well as the production of downstream

products.

A hallmark of peptide antibiotics are the varied and unique amino acids they

employ. Capreomycidine and enduracididine are two such examples found in the peptide

antibiotics muraymycin and enduracidin, respectively. Both are cyclic amino acids

derived from arginine.

Muraymycin is produced by Streptomyces sp. 30471 and has been shown to be

active against a number of Gram-positive bacteria including Methicillin-resistant

Staphylococcus aureus (MRSA). In an effort to further our understanding of this

antibiotic, efforts to isolate the muraymycin gene cluster have begun. A genomic library

has been constructed in the pCCFos1 Copy Control vector. Efforts to identify the genes

encoding for the enzyme involved in the conversion of L-arginine to capreomycidine are

described.

Enduracidin is another peptide antibiotic which contains a cyclized form of

arginine, enduracididine. Enduracidin has potent activity against Gram-positive bacteria

including Methicillin-resistant Staphylococcus aureus. The enduracidin gene cluster has

been cloned and sequenced by Yin and Zabriskie . Efforts to express and characterized

the genes involve in the biosynthesis of enduracididine are described. Labeled feeding

studies were also employed to determine the precursor to enduracididine. ¹³C-?-hydroxyarginine was synthesized and ?- and ?-hydroxyarginine were used in feeding

experiments to determine which, if either, would be incorporated into enduracidin.

Labeled enduracidin was isolated and characterized by LC-MS from feeding studies

using ¹³C-?-hydroxyarginine, identifying ?-hydroxyarginine as a precursor to

enduracididine.

Additional studies were performed using 3-fluoro-L-tyrosine. The fluoro-tyrosine

was converted to 3-fluoro-4-hydroxyphenylglycine through a pathway utilized for

hydroxyphenylglycine biosynthesis. The 3-fluoro-4-hydroxyphenylglycine was found to

be incorporated into multiple positions within enduracidin by LC-MS.