Radical addition of H3PO2 to N-/C-protected vinyl glycine led to the corresponding H-phosphinic acid in excellent yield. The non-nucleophilic H-phosphinic acid was converted to a nucleophilic P(III) species, RP(OTMS)2, which was used in two approaches to the target phosphinic acid containing pseudopeptide. New methodology was developed that led to excellent yields in the reaction of RP(OTMS)2 with unactivated electrophiles, including an acyclic homoallylic bromide. However, en route to the target pseudopeptide, Arbuzov reaction of RP(OTMS)2 with a cyclic homoallylic bromide, (R)-3-(bromomethyl)-cyclopent-1-ene, led to a rearranged allylic phosphinic acid rather than the desired homoallylic derivative, a putative glutarate surrogate. Conjugate addition of RP(OTMS)2 to alpha-methylene glutarate containing a chiral auxiliary resulted in only modest diastereoselectivity. Purification by flash chromatography provided protected derivatives of both diastereomers of the pseudopeptide. Following global deprotection, coupling of (S)-H-Glu-gamma-[Psi(P(O)(OH)(CH2))]-(S)-Glu-OH and (S)-H-Glu-gamma-[Psi(P(O)(OH)(CH2))]-(R)-Glu-OH to (4-amino-4-deoxy-10-methyl)pteroyl azide led to the target compounds for biochemical study as inhibitors of the ATP-dependent ligase, folylpoly-gamma-glutamate synthetase.

Several routes to a complex phosphinate phosphapeptide analogous to the gamma-glutamyl peptide Glu-gamma-Glu have been investigated. Formation of gamma-phosphono glutamate derivatives via addition of a phosphorus-based radical to protected vinylglycine was found to be of limited value because of the elevated temperatures required. Alkylation and conjugate addition reactions of trivalent phosphorus (P(III)) species were investigated. In situ generation of bis-trimethylsilyl esters of phosphinous acids proved to be an effective route to phosphinates of modest structural complexity. However, this chemistry could not be extended to the incorporation of an amino acid moiety at the N-terminal side of the desired phosphinate. A successful synthesis of the target phosphinate phosphapeptide was effected using P(III) chemistry and dehydrohalogenation to yield an alpha,beta-unsaturated phosphinic acid ester, following which conjugate addition of diethylacetamido malonate and acid-mediated hydrolysis afforded the desired phosphinate phosphapeptide. Coupling of the unprotected phosphinate phosphapeptide with two acyl azides derived from folic acid and methotrexate led to the corresponding pteroylphosphapeptides of interest as possible mimics of tetrahedral intermediates in the reaction catalyzed by folylpolyglutamate synthetase.

Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, nocardicin A has a tripeptide core of nonribosomal origin. Paradoxically, the nocardicin A gene cluster encodes two nonribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in nocardicin A biosynthesis, unless module skipping or other non-linear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-L-phenylglycine (L-pHPG) into positions 1, 3, and 5 and L-serine into position 4. No prediction could be made for position 2. Multi-domain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. Based on these analyses, the predicted product of the NocA+NocB NRPSs is L-pHPG–L-Arg–D-pHPG–L-Ser–L-pHPG, a pentapeptide. Despite being flanked by nonproteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C-terminus of the L-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of nocardicin A, a rare but precedented editing event in the formation of nonribosomal natural products which is supported by the identification of trypsin-encoding genes in N. uniformis.

No abstract

Phosphorus-containing pseudopeptides, racemic at the C-terminal α-carbon, are potent mechanismbased inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(γ-L-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R f pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH 4 Pte) heterocycle is most potent (K is = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R f species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., (LGlu-γ-L-Glu). Unexpectedly, the low R f (presumed L-Glu-γ-D-Glu) species are only slightly less potent (<30-fold) less potent than their diastereomers. Further study of this phenomenon comparing L-Glu-γ-L-Glu and L-Glu-γ-D-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial D-Glu precursors by L-Glu may give misleading information if L-Glu-γ-L-Glu substrates have low K m values.