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A system has been designed for the NMR observation (at 60 M H z ) of unstable intermediates, with half-lives of 100 ms, at concentration levels above lo-* M. Rapid mixing can be achieved in less than 40 ms and a series of eight spectra acquired in less than 1.08s. Low temperature operation is straightforward. Reactive intermediates involved in the reaction of methylamine with 2,4,6,-trinitroanisole and the acid-catalysed hydrolysis of l,l-diethoxy-2,3-diphenylcycloprop-2-ene were identified, and in a kinetic application, the rate of hydrolysis of ttimethyloxonium tetrafluoroborate was measured.

A series of novel penicillins, bearing an S-aminosulfenimine (R′(R′′)NSNd) side chain at the 6-position, have been prepared by direct reaction of a penicillin ester with sulfur diimides. A set of these structures, with the thiazolidine-ring sulfur in the sulfide oxidation state, exhibited a pattern of reactivity not previously encountered in penicillin chemistry, viz., cleavage of the β-lactam ring resulted in a rapid intramolecular displacement of the S-amino moiety as R′(R′′)NH. This was found to be the exclusive reaction occurring consequent on cleavage of the β-lactam ring. The mechanism of this process was delineated in a detailed study in basic methanol. That a similar reactivity pattern held for a penicillin salt in aqueous solution was also verified. Thus the salt 5bm (R′ ) CH 3 , R′′ ) p-CH 3 C 6 H 4 SO 2 ) behaved as a moderate substrate for β-lactamase type I from Bacillus cereus (k cat /K m ) 6.26 × 10 5 M -1 min -1 ). On enzyme-catalyzed hydrolysis of this compound, displacement of N-methyl-p-toluenesulfonamide (R′(R′′)NH) was directly observed ( 1 H NMR) and found to occur faster than displacement of this group from (intact) 5bm in aqueous buffer, by a factor of at least 600. These findings identified the potential of the S-aminosulfeniminopenicillin structure type to be developed as β-lactamase substrates for use as site-specificrelease prodrugs. A degree of enzyme inhibition was also observed with this set of thiazolidinering-sulfide structures with the most potent inhibitor having the most nucleofugic S-amino moiety p-ClC 6 H 4 SO 2 N(CH 3 ), indicating that displacement of this group, at the enzyme active site, played a role in their mode of inhibition. Structures with the thiazolidine-ring sulfur in the sulfone oxidation state were considerably more potent as inhibitors, with the structure 5a 2 being the most active. As this compound bore the least nucleofugic S-amino moiety C 2 H 5 OC(O)NH, it indicated that the mode of inhibition of the sulfones was distinct from that of the thiazolidine-ring sulfides; it is probable that the sulfones reacted in a manner similar to that shown by sulbactam viz., rapid scission of the thiazolidine-sulfone ring after cleavage of the β-lactam ring. Synergy of action was observed with 5a 2 at high concentration (78 µg/mL) against Escherichia coli when combined 1:1 with penicillin G; no synergy was observed at low concentration (4 µg/mL) when combined with pipericillin, indicating poor permeation characteristics.

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The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.Host defense peptides (HDPs) are multifunctional molecular effectors of innate immunity in multicellular organisms (3,8,26). Deficiencies in their activity or expression levels can be associated with enhanced susceptibility to infections (6) and to chronic infections in conditions such as cystic fibrosis (CF) (19). While there is a sound underlying principle in compensating disease-associated deficiencies in HDPs with exogenous peptides, their potential toxicities contend with their localized administration to sites of infection (10,26). The activities of endogenous HDPs themselves can be stringently controlled (24). HDPs expressed as inactive prepropeptides are activated by proteolytic cleavage of an anionic profragment masking the net positive charge of the C-terminal mature sequence (9). Human cathelicidin hCAP18/LL-37, for example, is activated by proteinase 3, while other neutrophilic serine proteases, such as neutrophil elastase (NE), can also cleave the profragment in vitro (1, 25). As this enzyme is associated with chronic airway inflammation and infections in CF patients (13), a prodrug approach analogous to the natural control mechanism exerted on HDPs and that exploits the abnormal concentrations of NE in the CF lung is reported here.Synthetic propeptides were prepared by modification of an HDP with an anionic profragment, using L-P18, an ␣-helical, salt-resistant, cecropin A-magainin 2 hybrid sequence, identified by K.-S. Hahm's group (20, 21). Peptides were assembled by solid-phase synthesis (14), purified by reversed-phase highperformance liquid chromatography, and characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Six propeptides were synthesized by elongating the parent sequence (KWKLFKKIPKFLHLAKKF-NH 2 ) at its N terminus with a trialanine linker as an NE substrate (2) and from 2 to 7 glutamic acids to mask the net charge determinant (ϩ8) of the mature peptide's antimicrobial activity. When more than 4 glutamic acids were added, a ␤-alanine spacer was introduced between the fourth and fifth glutamates, to reduce interchain association effects (11).The antimicrobial activities of these peptides were assessed for two representative CF pathogens, Staphylococcus aureus (SH1000) and Pseudomonas aeruginosa (PAO1). MICs (Table 1) were determined by the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute. Typical MICs of L-P18 for P. aeruginosa and S. aureus were 32 M and 64 M, respectively, while MICs of all propeptides were higher than 128 M. As the activities of ␣-helical membrane-active peptides have been suggested to be independent of the stereochemistry of their constitutive amino acids (23), the enantiomeric D-P18...