Reaction mechanisms in peptide synthesis. Part 2. Tautomerism of the peptide bond

Summary We had concluded in previous work that ring opening of a 2-alkyl-5(4H)-oxazolone by water or ammonia leads to transient high-energy imidol intermediates which instantly tautomerize to the native amides. Using the MOPAC molecular orbital program, detailed geometric and energetic characteristics of the tautomerism of a peptide bond have been determined on the AM1 level. The results demonstrate that tautomerism of a peptide bond comprises a three-stage process involving three successive transition states and a bimolecular mechanism: (i) EZ peptide bond isomerization followed by dimerization, (ii) concerted double-hydrogen exchange leading to an -hydroxyimine (imidic acid) followed by splitting of the dimer, and (iii) ZE N-methylimine inversion. While pathway (iiiiii) is predicted as a feasible route terminating in the formation of a peptide bond, the inverse route (iiiiii) is excluded as a possible initial step in the generation of a 5(4H)-oxazolone intermediate.     

Radiolabelled peptide ergot alkaloids.

Paspalic acid ( 11 ), labelled at different positions with ³H or ¹⁴C and at specific activities up to 1 Ci/mmol (³H) and 2mCi/mmol (¹⁴C), has been prepared biosynthetically in a scale of 2–5 mmol in submerged cultures of a selected strain of Claviceps paspali by incorporation of DL -[5-³H]-tryptophan, DL -[6-³H]-tryptophan, DL -[alanine-2,3-³H]-tryptophan, or DL -[alanine-3-¹⁴C]-tryptophan. Radioactive lysergic acid ( 12 ) was obtained from paspalic acid by base catalysed rearrangement. The procedures for labelling the precursors at high specific activity arc described. The ergolene carboxylic acids 11 and 12 were used as key intermediates for tlie chemical synthesis of radiolabelled peptide ergot alkaloids 14 required for pharinacokinetic and metabolic studies. Linking of the aminocyclols 13 (peptide parts) of the ergotarnine (R¹ = methyl), ergoxine (R¹ = ethyl) and ergotoxine (R¹ =isopropyl) series with a reactive derivative of either lysergic acid or its mixture with paspalic acid was accomplished by standard procedures. l-Mctliyl-[13-³H]-ergotarnine (MY 25) ( 16 ) and 2-brotno-α-ergocryptine (bromocriptine, C73 154) ( 17 ), labelled with ³H at position 12 and with ¹⁴C in position 4, were obtained by alkylating ³H-ergolamine and by ljrominating appropriately labelled α-ergocryptines. Radioactive peptide ergot alltaloitls labelled by tlic present nietliotl proved suitable for the use in biological tracer studies.     

Pharmacokinetics of brain natriuretic peptide in rats.

The pharmacokinetics of rat brain natriuretic peptide (rBNP) was compared with that of alpha-rat atrial natriuretic peptide (alpha-rANP) in rats. After intravenous infusion in rats (600 pmol min-1kg-1 for 2 min), the disappearance of plasma rBNP was 4-fold slower than that of alpha-rANP. The estimated mean plasma clearance rates for rBNP and alpha-rANP were 45.9 ml min-1kg-1 and 74.4 ml min-1kg-1, respectively. The affinity of rBNP for the clearance receptor or degradation enzyme was considered to be lower than that of alpha-rANP.     

In-source fragmentation of peptide aldehydes and acetals: influence of peptide length and charge state.

The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared.     

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

Application of capillary electrophoresis in peptide research.

Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.     

Charge promotion of low-energy fragmentations of peptide ions.

We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directing properties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.     

The oxazole-triamide rearrangement. Application to peptide synthesis

The carboxyl group of a N-acylated amino acid may be protected by conversion to an oxazole derivative which, on photoxygenation, regenerates the carboxyl group in activated (triamide) form for peptide synthesis.     

Oriented immobilization of peptide ligands on solid supports.

A synthetic procedure was developed for the direct immobilization on preactivated affinity supports of peptidic ligands requiring free alpha-amino groups to recognize their targets properly. The peptidic ligand is assembled by solid-phase peptide synthesis on an octa-branched heptalysine core through a polyglycine spacer, similar to the method developed for the production of multiple antigenic peptides. After deblocking from the resin, peptide is dialysed, lyophylized and used directly for coupling to preactivated supports. Following immobilization, only a limited number of peptide chains are covalently linked to the solid phase, leaving the remainder facing the mobile phase and sufficiently spaced to interact properly. This procedure was applied successfully to the design, synthesis and oriented immobilization of a multimeric tripeptide ligand (Met-Tyr-Phe) for affinity purification of bovine neurophysin.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.