Acyl Carrier Protein (ACP) 65-74

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 β-amyloid, in high purity and yield.

The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the "difficult" C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize "difficult couplings," rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods.

The development of protecting group-free synthesis has come to the forefront this century, as there is an increasing need to switch to greener synthetic methods. In peptide synthesis, a strategy of maximum protection offers the most efficient synthetic pathway, but minimal side chain protection is more favorable in terms of green chemistry. Here, we describe solid-phase peptide synthesis (SPPS) without hydroxy side chain protection based on an aqueous microwave (MW)-assisted method. First, we investigated the extent ofO-acylation of the hydroxy side chain of Ser, Thr, and Tyr occurring in our method, which uses 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. Under aqueous MW-assisted conditions, the coupling reaction proceeded efficiently without substantialO-acylation. Next, we applied the aqueous synthetic protocol without hydroxy side chain protection to synthesis of a laminin-related peptide, H-Tyr-Ile-Gly-Ser-Arg-NH2. HPLC analysis of the crude peptide revealed a single peak, suggesting the absence of side reactions includingO-acylation and racemization. We also succeeded in synthesizing a difficult peptide sequence, acyl carrier protein (65-74) peptide, by aqueous SPPS without hydroxy or carboxamide side chain protection. Based on the eighth criterion of the 12 principles of green chemistry, namely, "reduce derivatives", our approach without hydroxy side chain protection will provide a greener peptide synthesis.

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.