Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 9924991

Sequence-assisted peptide synthesis (SAPS).

B Due Larsen·A Holm
View original source ↗Export metadata ↓

Abstract

In solid-phase peptide synthesis (SPPS) the growing peptide chain may undergo chain aggregation which can cause serious synthetic problems. A number of investigations concerning this problem have been reported in the chemical literature. During a study of such "difficult sequences" using the Fmoc-protection strategy, we have observed that peptide-chain aggregation may be significantly reduced when certain amino acid sequences are incorporated C-terminally (1). Thus, synthesis of the difficult poly-alanine, (Ala)n, sequence (n < or = 20) has been investigated with (Lys(Boc))m (m < or = 6) and (Glu(tBu))m (m > or = 6) as pre-sequences. With m > or = 3, peptides are obtained as single, homogeneous products while a complex mixture of deletion peptides and corresponding Fmoc-protected peptides is formed (n > or = 6) without the pre-sequence. A mixed pre-sequence, (Lys(Boc)Glu(tBu))3, has a similar favorable effect on the synthetic results, but the positive effect seems confined to a rather narrow framework of amino acids and side-chain protecting groups in the pre-sequence as discussed in the article. Among other reputedly difficult sequences the synthesis of H-(Thr-Val)5-OH, H-Val-Asn-Val-Asn-Val-Gln-Val-Gln-Val-Asp-OH, the Acyl Carrier Protein (65-74) and the human insulin B-chain has been investigated. In all cases introduction of a pre-sequence gives rise to satisfactory synthetic results. In the latter case, the lysine pre-sequence may be cleaved enzymatically to give the desB30 insulin B-chain. NIR-FT Raman studies of the synthesis of the poly-alanine, (Ala)n, sequences have shown that the pre-sequence (Lys(Boc))6 shifts the conformation of the growing peptide chain from a beta-structure (n > or = 6) to a random coil conformation (1c). This result is in agreement with the general observation that SPPS proceeds optimally under random coil conditions.

Explore related subjects

Acyl Carrier ProteinAmino AcidsChromatography, High Pressure LiquidPeptide BiosynthesisJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

B Due Larsen, A Holm. 1998. Sequence-assisted peptide synthesis (SAPS).. https://doi.org/10.1111/j.1399-3011.1998.tb01251.x

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Neutralization of acidic residues in helix II stabilizes the folded conformation of acyl carrier protein and variably alters its function with different enzymes.

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

Acyl Carrier Protein↗
PubMed2006

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase.

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

Acyl Carrier Protein↗
PubMed2006

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A.

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.

Acyl Carrier Protein↗