Insights into ß-ketoacyl-chain recognition for ß-ketoacyl-ACP utilizing AHL synthases.

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

High-efficiency solid phase peptide synthesis (HE-SPPS).

A series of improvements to the standard solid phase peptide synthesis (SPPS) process allowing for significant gains in product purity along with only a 4 min standard cycle time and a 90% reduction in total waste produced is reported. For example, syntheses of the well-known (65-74)acyl carrier protein (ACP) and (1-42)ß-amyloid peptides were accomplished with 93 and 72% purity (UPLC-MS) in only 44 and 229 min, respectively.

Microwave-assisted solid-phase peptide synthesis based on the Fmoc protecting group strategy (CEM).

Microwave-assisted peptide synthesis has become one of the most widely used tools by peptide chemists for the synthesis of both routine and difficult peptide sequences. Microwave technology significantly reduces the synthesis time while also improving the quality of the peptides produced. Microwave energy allows most amino acid couplings to be completed in just 5 min. The Fmoc removal can also be accelerated in the microwave decreasing the reaction time from at least 15 min to only 3 min in most cases. Common side reactions such as racemization and aspartimide formation are easily controllable with optimized methods that can be applied routinely. This protocol outlines the detailed procedure for performing both manual and automated microwave-assisted peptide synthesis of two difficult peptide sequences, ACP (65-74) and ß-amyloid, in high purity and yield.

Fast conventional Fmoc solid-phase peptide synthesis: a comparative study of different activators.

The ability to speed up conventional Fmoc solid-phase peptide synthesis (SPPS) has many advantages including increased productivity. One way to speed up conventional Fmoc SPPS is the choice of activator. Recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times. It was found that HDMC, PyClock, COMU, HCTU, and HATU worked well at shorter reaction times (2 × 1 min), but PyOxim and TFFH only worked well at longer reaction times. The performance of PyBOP at shorter reaction times was poor only for more difficult sequences. These results are important for selecting an appropriate activator for fast SPPS applications.

Solid-phase peptide synthesis using microwave irradiation.

Since the advent of solid-phase peptide synthesis (SPPS) in the late 1950s, numerous advancements in the underlying chemistry (i.e., orthogonal protection strategy, coupling reagents, and solid support matrices) have greatly improved the efficiency of the technique. More recently, application of microwave radiation to SPPS has been found to reduce reaction time and/or increase the initial purity of synthetic peptide products. In this protocol, conditions are described to accomplish rapid peptide coupling and 9-fluorenylmethoxycarbonyl (Fmoc) removal reactions under temperature-controlled conditions in either a manual or automated synthesis format using a microwave reactor. These microwave-assisted peptide synthesis procedures have been used to rapidly prepare a "difficult" peptide sequence from the acyl carrier protein, ACP(65-74), in less than 3 h and the reduced, linear precursor to human hepcidin, in high initial purity.

Synthesis of acyl carrier protein using a new heterocyclic carboxyl activating group, 3-mercapto-5,6-diphenyl-1,2,4-triazine through SPPS strategy.

A new, effective and easily accessible carboxyl activating group , 3-mercapto-5, 6 - diphenyl-1, 2, 4 - triazine was developed and the effectiveness was investigated using acyl derivatives, chemically by the preparation of respective amides, in addition to monitoring spectrophotometrically exploiting the UV- visible spectra. The carboxyl activating nature of the above mercapto heterocycle was further confirmed by solid phase peptide synthesis. Here CLPSER resin as polymeric support was applied. We synthesized Acyl Carrier Protein and smaller peptides by replacing this thiol for other conventional carboxyl activating reagents. These peptides synthesized were purified by HPLC and characterized by molecular weight method.

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

Synthesis of retro acyl carrier protein (74-65) fragment on a new glycerol based polystyrene support.

Retro-ACP (74-65) fragment was synthesized on a novel 4% tri-(propylene glycol) glycerolate diacrylate cross-linked polystyrene (PS-TRPGGDA) support. The peptide is grown from the functional site present in the cross-linker, which makes it unique and cost-effective among other styrene based polymer supports. A comparative study with Merrifield resin indicates high yield and purity of the peptide synthesized on the novel support.

Chain cleavage and sulfoxidation of thiastearoyl-ACP upon reaction with stearoyl-ACP desaturase.

The fatty acid analogues 9- and 10-thiastearate were converted to acyl-ACP derivatives by in vitro enzymatic synthesis and reacted with the reconstituted soluble stearoyl-ACP Delta9 desaturase complex. Electrospray ionization mass spectral analysis of the acyl chains purified from the reaction mixtures showed that 10-thiastearoyl-ACP was converted to the 10-sulfoxide as the sole product. In the presence of (18)O(2), the sulfoxide oxygen was found to be derived exclusively from O(2). This result confirms the ability of the soluble stearoyl-ACP desaturase to catalyze O atom transfer in the presence of the appropriate substrate analogue. Inhibition studies showed that 10-thiastearoyl-ACP was a mixed-type inhibitor of 18:0-ACP, with an apparent K(I) of approximately 10 microM. Comparable reactions of the stearoyl-ACP desaturase complex with 9-thiastearoyl-ACP gave the 9-sulfoxide as approximately 5% of the total products, with the O atom again exclusively derived from O(2). The remaining 95% of the total products arose from an acyl chain cleavage reaction between S-9 and C-10. Matrix-assisted laser desorption ionization time-of-flight mass spectral analysis showed that 9-thiastearoyl-ACP had a mass of 9443 amu while the acyl chain cleavage product had a mass of 9322 amu, corresponding to the calculated mass of 8-mercaptooctanoyl-ACP. Recovery of the acyl chain from the ACP product gave the disulfide of 8-mercaptooctanoate (mass of 349.1 amu), arising from the dimerization of 8-mercaptooctanoate during product workup. Gas chromatography-mass spectral analysis also showed the accumulation of nonanal in sealed reaction vials, accounting for the other product of the acyl chain cleavage reaction. The reactivity at both the 9 and 10 positions of the thia-substituted acyl-ACPs is consistent with the proximity of both positions to the diiron center oxidant in the enzyme-substrate complex. Moreover, the differential reactivity of the 9- and 10-thiastearoyl-ACPs also suggests position-dependent consequences of the reaction within the Delta9D active site. Mechanisms accounting for both sulfoxidation and acyl cleavage reactions by the stearoyl-ACP Delta9 desaturase are proposed.

Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules.

6-Deoxyerythronolide B synthase (DEBS) is the modular polyketide synthase (PKS) that catalyzes the biosynthesis of 6-deoxyerythronolide B (6-dEB), the aglycon precursor of the antibiotic erythromycin. The biosynthesis of 6-dEB exemplifies the extraordinary substrate- and stereo-selectivity of this family of multifunctional enzymes. Paradoxically, DEBS has been shown to be an attractive scaffold for combinatorial biosynthesis, indicating that its constituent modules are also very tolerant of unnatural substrates. By interrogating individual modules of DEBS with a panel of diketides activated as N-acetylcysteamine (NAC) thioesters, it was recently shown that individual modules have a marked ability to discriminate among certain diastereomeric diketides. However, since free NAC thioesters were used as substrates in these studies, the modules were primed by a diffusive process, which precluded involvement of the covalent, substrate-channeling mechanism by which enzyme-bound intermediates are directly transferred from one module to the next in a multimodular PKS. Recent evidence pointing to a pivotal role for protein-protein interactions in the substrate-channeling mechanism has prompted us to develop novel assays to reassess the steady-state kinetic parameters of individual DEBS modules when primed in a more "natural" channeling mode by the same panel of diketide substrates used earlier. Here we describe these assays and use them to quantify the kinetic benefit of linker-mediated substrate channeling in a modular PKS. This benefit can be substantial, especially for intrinsically poor substrates. Examples are presented where the k(cat) of a module for a given diketide substrate increases >100-fold when the substrate is presented to the module in a channeling mode as opposed to a diffusive mode. However, the substrate specificity profiles for individual modules are conserved regardless of the mode of presentation. By highlighting how substrate channeling can allow PKS modules to effectively accept and process intrinsically poor substrates, these studies provide a rational basis for examining the enormous untapped potential for combinatorial biosynthesis via module rearrangement.

Solid-phase peptide synthesis at elevated temperatures: a search for and optimized synthesis condition of unsulfated cholecystokinin-12.

A systematic investigation of solid-phase peptide synthesis at elevated temperatures using the well-known aggregating peptide acyl carrier protein (65-74) and the unsulfated cholecystokinin-8 as models is presented. The main goal of the investigation was the determination of an optimized experimental condition for the synthesis of unsulfated cholecystokinin-12. Of the elevated temperatures used, 60 degrees C was the most appropriate. The efficiency of N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) in 25% dimethyl sulfoxide (DMSO)/toluene at this temperature was similar to that of 2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU). Interestingly, this coupling reagent was more efficient than TBTU, benzotriazol-1-yl oxy-tris(dimethylamino)phosphonium and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in N-methylpyrrolidone. 25% DMSO/toluene proved to be suitable for the swelling of the resins phenylacetamidomethyl, methylbenzhydrylamine, hydroxymethylphenoxy, 4-(benzyloxy)-2',4'-dimethoxybenzhydrylamine, 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy and (4-succinylamido-2',2',4'-trimethoxy)benzhydrylamine. Those polymeric supports were fully compatible with the approach. Under the optimized synthesis condition found in these studies (temperature of 60 degrees C, DIC/HOBt as coupling reagent and 25% DMSO/toluene as solvent), no difficulties related to the aggregation phenomenon were encountered. These data confirm the usefulness of solid-phase peptide synthesis at elevated temperatures and extend its applicability.

Peptide synthesis on Sepharose beads.

NHS-activated Pharmacia HiTrap Sepharose was modified with 1, 3-diaminopropane to give an amino-functionalized support suitable for solid-phase peptide synthesis. The amide linker p-[(R1S)-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]- 2, 4-dimethoxybenzyl]phenoxyacetic acid was incorporated and the acyl carrier protein sequence 65-74 was synthesized manually on this support by the Fmoc procedure under controlled conditions with monitoring of the coupling reactions. The performance of the support in automated multiple synthesis in open reactors, with an Abimed AMS 422 according to standard protocols, was evaluated by the synthesis of the acyl carrier protein sequence 65-74 and two other 15-mer and 18-mer peptides. The quality of the resulting crude peptides was determined by HPLC and MALDI-MS, and compared with the same sequences synthesized in parallel on the commercial peptide synthesis resin TentaGel S RAM. The modified HiTrap material was found to be particularly suited for Fmoc solid-phase peptide synthesis and should be advantageous for the utilization of immobilized peptides and peptide libraries in biological assays.

In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).

Photoaffinity cross-linking of acyl carrier protein to Escherichia coli membranes.

Acyl Carrier Protein (ACP) is a small acidic protein which interacts with the various enzymes implicated in the biosynthesis of fatty acids in E. coli. It also interacts with the inner membrane proteins implicated in the biosynthesis of phospholipids. Samples of radioactive ACP were prepared with high specific activities and bearing photoactivable aryl azide derivatives. Two photoactivable reagents were used: para azido phenacyl bromide (pAPA) which reacts with the SH of the ACP prosthetic group and the N-hydroxysuccinimide ester of 4-azido salicylic acid (NHS-ASA) which reacts with the amino groups of the protein. Various methods were used to demonstrate that ACP could be cross-linked specifically to an inner membrane protein of E. coli, most probably to the glycerol-3-phosphate acyl transferase (GPAT). This covalent link should provide a powerful tool for further analysis of the structure of GPAT and its role in phospholipid biosynthesis. These photoactivable aryl azide derivatives of ACP could also be very useful for studying the interaction of ACP with the soluble enzymes implicated in fatty acid biosynthesis.