Small and Random Peptides: An Unexplored Reservoir of Potentially Functional Primitive Organocatalysts. The Case of Seryl-Histidine

Abstract

Catalysis is an essential feature of living systems biochemistry, and probably, it played a key role in primordial times, helping to produce more complex molecules from simple ones. However, enzymes, the biocatalysts par excellence, were not available in such an ancient context, and so, instead, small molecule catalysis (organocatalysis) may have occurred. The best candidates for the role of primitive organocatalysts are amino acids and short random peptides, which are believed to have been available in an early period on Earth. In this review, we discuss the occurrence of primordial organocatalysts in the form of peptides, in particular commenting on reports about seryl-histidine dipeptide, which have recently been investigated. Starting from this specific case, we also mention a peptide fragment condensation scenario, as well as other potential roles of peptides in primordial times. The review actually aims to stimulate further investigation on an unexplored field of research, namely one that specifically looks at the catalytic activity of small random peptides with respect to reactions relevant to prebiotic chemistry and early chemical evolution.

Keywords: Ser-His; fragment condensation; organocatalysis; peptide bond formation; phosphodiester bond formation; small peptides.

Figure 1

Chemical structure of Ser-His with evidence of its several functional groups. The imidazole ring is shown in its neutral N^ϵ–H tautomeric form.

Figure 2

Mechanism of serine protease. The catalytic triad Ser/His/Asp acts in a concerted manner and cleaves the peptide bond in two steps. The acyl group is firstly transferred to Ser hydroxyl oxygen, then to water. See details in the text. The dashed lines indicate favorable interactions between the negatively-charged aspartate residue and the positively-charged histidine residue, which make the histidine residue a more powerful base. Note the ‘oxyanion hole’, which stabilizes via hydrogen bonding with amide N–H the negative oxygen of the tetrahedral intermediate [70].

Figure 3

Formation of peptidic amide bond. (a) Condensation of free amino acids in water, where the reacting groups (terminal carboxylate and terminal ammonium) are charged and unreactive. A proton transfer from the ammonium group to carboxylate would lead to a reactive couple undergoing aminolysis. However, actually, $K_{exch}$ favors the charged compounds. As $K_{cond}$ = $K_{exch}$ $K_{am}$, the main thermodynamic barrier to amino acid condensation in water is their presence as a charged state, whereas $K_{am}$ would be in favor of the amide. (b) Aminolysis of the activated acyl derivative. In contrast to free amino acid condensation, ester aminolysis is thermodynamically favored (typically, $K_{am}^{\prime }$ 50–60), but the reaction pathway may be hindered by a kinetic barrier; and if carried out in water, hydrolysis competes with aminolysis.

Figure 4

Ser-His catalyzed peptide formation [18]. N-protected and C-activated phenylalanine residue (AcPheOEt) reacts with C-protected leucine residue (HLeuNH₂) to give ethanol and the desired dipeptide (AcPheLeuNH₂) in a 20%–50% yield (depending on incubation time, up to 30 days), at pH 7–9 (Britton–Robinson buffer was used, including acetate, phosphate and borate). The reaction has been tested from 4–65 °C and runs well also in the presence of cupric ions and urea. Note that AcPheLeuNH₂ precipitates in the reaction conditions, shifting the equilibrium to the right. As a comparison, if $\alpha$-chymotrypsin is used instead of Ser-His, the reaction occurs in a few minutes. Together with aminolysis, AcPheOEt hydrolysis is observed (AcPheOH).

Figure 5

Schematic representation of the peptide-forming reaction catalyzed by Ser-His [18]. In addition to the “1 + 1” case, yielding a dipeptide, other combinations have been explored, up to the formation of tetrapeptide by a “2 + 2” condensation scheme. Moreover, by using “bifunctional” substrates, namely peptides having both an electrophile moiety and a nucleophile counterpart in the same molecules, the oligomerization of peptides was shown (H-Trp-OEt and H-Phe-OEt, up to the dimer, n = 2) and peptide nucleic acids (PNAs) units (up to a tetramer, n = 4).

Figure 6

Proposed reaction scheme. Top: The oligomerization of imidazole-activated nucleotides (1) by Ser-His (2) involves the creation of a covalent linkage between dipeptide and mononucleotide (3), thereby releasing imidazole (4). Compound 3 rapidly forms and is then slowly consumed. It can be attacked by the 3′- or 2′-hydroxyl group of another nucleotide, thereby forming dimers 9 and 6 (only 3′–5′ bonds shown). Bottom: alternatively, it can be converted into side products: pyrophosphate dimer 7, ribonucleotide 5 and an inactive, stable complex 8 are formed upon nucleophilic attack by a 5′-phosphate of 5, water or the N-terminus of dipeptide 2, respectively. Reproduced from Wieczorek et al. [19] with the permission of John Wiley and Sons.

Figure 7

Profile of the Ser-His efficiency in promoting the aminolysis reaction shown in Figure 4. The inflection point of the sigmoidal curve, as obtained by non-linear fitting of experimental data, is at pH 7.85 [18].

Figure 8

Molecular models of (a) N^δ–H His-Ser, (b) N^ϵ–H His-Ser, (c) N^δ–H Ser-His and (d) N^ϵ–H Ser-His, as obtained after 1-ns molecular dynamics simulations in explicit solvent.

Figure 9

Peptide fragment condensation (adapted from [101]). Condensation scheme of n peptides (10 amino acids long) to yield ideally $n^2$ peptides (20 amino acids long), of which only m are soluble, and undergo further random fragment condensation yielding $m^2$ peptides (40 amino acids long), of which only a few will be water soluble. By continuing this procedure, we reach long chains that are soluble and that can be seen as the product of a prebiotic molecular evolution. The asterisk indicates a catalytic center, capable of inducing peptide synthesis. The synthesis can be in principle also induced by an external catalytic peptide. Reproduced from [20] with the permission of the authors.

Figure 10

Novel folded peptide by fragment condensation [102]. (a) The sequence of a 44-mer peptide deriving from fragment condensation of four random 10-mers, whose sequence was randomly generated. Starting from the 10-mers, a library of 20-mers was constructed by Merrifield synthesis, and the soluble products were selected for the next condensation cycle. To the final 40-mer, which was partially soluble, a hydrophilic tail (DDEE) was added to the N-terminus, yielding a soluble 44-mer peptide, which was subjected to circular dichroism investigation. Spectroscopic data indicated that about 50% of the residues were in $\alpha$-helix conformation. (b) Model of the three-dimensional structure of the 44-mer peptide (obtained by the ROKKY protein structure suite). Left: global view; right: detailed view of the hydrophobic core. Reproduced from Chessari et al. [102] with the permission of John Wiley and Sons.