Influence of amino acid and N-terminal protection residue structures on peptide p-nitroanilide adsorption on polystyrene-based support

Polystyrene-based support Bio-Beads® SM-2 was employed for desalting peptide-p-nitroanilides from Oxone®. Neither tosyl, 9-fluorenyl(methoxycarbonyl), p-nitroanilide groups nor indolyl or p-hydroxyphenyl side-chains of Trp and Tyr ensured an efficient adsorption of peptide-p-nitroanilides onto Bio-Beads® SM-2. Only unsubstituted phenyl-containing protection groups (carbobenzoxy or benzoyl) and Phe residues provided the adsorption of peptides on Bio-Beads® SM-2 and their efficient desalting. This support is well suitable for multiple parallel phenyl group-containing peptide derivative separations and high-throughput screenings.


Introduction
Amino acid and peptide p-nitroanilides (pNA) are widely used for kinetic research, activity detection and substrate specificity scanning of peptide hydrolases. p-Nitroaniline that liberates upon the substrate hydrolysis has yellow color and is detected by absorbance at 405 nm (Erlanger et al. 1961;Leung et al. 2001;Zabłotna et al. 2004) allowing both time-point and continuous registrations. Such substrates are still used in biochemical, biotechnological and clinical research because of their cheapness, high accessibility of photometer equipment, visibility of hydrolysis with the naked eye and simplicity of kinetic measurements (Duarte de Almeida et al 2021; Filippova et al. 2020;Mechri et al. 2022;Zhang et al 2020). Peptide-pNAs can be made by solution (Peterson et al. 2010;Rijkers et al. 1995), enzyme-catalyzed (Semashko et al. 2014) and solid-phase peptide synthesis (SPPS) (Abbenante et al. 2000;Alsina et al. 1999;Bernhardt et al. 1997;Burdick et al. 1993;Hojo et al. 2000;Kaspari et al. 1996;Kwon et al. 2004;Leung et al. 2001). Use of 5-nitro-2-aminobenzoic acid as an analog of pNA simplified SPPS and enhanced peptide-pNA solubility (Zabłotna et al. 2004;Wysocka et al. 2007); however, a free COOHgroup may influence substrate binding in an enzyme active site. The simplest and cheapest peptide-pNA SPPS procedure is based on the synthesis of peptide-p-aminoanilides (pAAs), which are further mildly oxidized to pNAs by an equivalent mixture of potassium monosulfate and potassium monopersulfate (Oxone ® ) (Abbenante et al. 2000). It would allow parallel synthesis of peptide-pNA libraries if a rapid and simple procedure for the oxidizer removal from peptide-pNAs was developed. Abbenante et al. (2000) used a reversed-phase HPLC purification together with the oxidizer removal. However, such procedure decreases the column lifetime, influences the stability of the HPLC system because of high salt and oxidizer contents and is not suitable for a multiple parallel format. Recently, we separated benzoylpeptide-pNAs from Oxone ® by a batch-wise adsorption onto Bio-Beads ® SM-2 (Chistov et al. 2018). This polystyrenebased support can also be packed into columns with a rapid flow-through and is well suited for multiple parallel procedures. Bio-Beads ® SM-2 is employed for detergent and dye removal from biomolecule samples (Horigome and Sugano 1983;Spack et al. 1986;Varhac et al. 2009); its application for peptide separations is not described. The similarity of chemical structures of Bio-Beads ® SM-2 and Porapak Q, used for removing Phe-containing peptides (Vogel and Altstein 1977), suggests the ability of Bio-Beads ® SM-2 of adsorbing phenyl group-containing peptides and separating them from other molecules. Our successful desalting of N-α-benzoyl-peptide-pNAs using Bio-Beads ® SM-2 confirms this hypothesis (Chistov et al 2018). However, the contribution of various protecting groups and amino acid side-chains to peptide adsorption onto Bio-Beads ® SM-2 is unclear. Here, we studied the influence of the most frequently used N-terminal protecting groups (carbobenzoxy (Z), tosyl (Tos), pNA and aromatic amino acid residues on the peptide-pNA adsorption on Bio-Beads ® SM-2.
After the reaction, acetonitrile was evaporated. Peptide-pNA water solution was applied to the column (2 mL Econo-Column, Bio-Rad Laboratories) with 1 g Bio-Beads ® SM-2 swollen in water. Peptide-pNA precipitate, left in the reaction tube, was washed with several portions of deionized hot (50°C) water until SO 2− 4 ions were not detected with saturated BaCl 2 solution. Combined washings were applied to the column (about 20 mg peptide per 1 g Bio-Beads ® SM-2). The column was washed with water until SO 2− 4 ions were not detected, and peptide-pNA was eluted with 1 mL acetonitrile followed by 2 mL isopropanol and 2 mL DCM (for protected peptide-pNAs). The eluted peptides were combined with the corresponding precipitates. Peptides Z-AFR-pNA, Tos-AFR-pNA, and glut-ALR-pNA were fully dissolved in water/acetonitrile (1:1, v/v), precipitated with cold ether and dried in vacuo in a desiccator with KOH. Side-chain protected peptide-pNAs containing Y(tBu), W(Boc) and R(Pbs) were totally deprotected, precipitated by ether and dried in vacuo.
Determination of Bio-Beads ® SM-2 capacity for peptide-pNAs and Fmoc-Ala is described in Supplementary information.

Results and discussion
A set of peptide-pNAs X 1 -Ala-X 2 -Arg-pNA, where X 1 -an α-amino-protecting group (Z, Tos, or glut) and X 2 -Phe, Tyr, Trp or Leu, was selected for testing a contribution of two phenyl-containing N-terminal blocking groups (Z and Tos), pNA moiety and aromatic/hydrophobic side-chains to the adsorption onto Bio-Beads ® SM-2. All peptide-pNAs were synthesized by SPPS on p-phenylene diamine-modified Trt resin as peptide-pAAs and oxidized to peptide-pNAs by Oxone ® . To avoid the Scheme 1 Oxidation of peptide-pAAs to peptide-pNAs by Oxone ® (X1-N-terminal protection group, see above) Tyr and Trp oxidation, Tyr-and Trp-containing peptide-pAAs were oxidized as side-chain protected peptides. All obtained peptide-pAAs were reasonably pure according to LC-MS (Suppl., Fig. S1-S9). Since all obtained pAAs were poorly soluble in water, the oxidation was performed in a water:acetonitrile mixture, from 19:1 (v/v, side-chain deprotected peptides) to 1:4 (protected peptides). Acetonitrile was evaporated after the oxidation for ensuring the peptide adsorption onto Bio-Beads ® SM-2. This resulted in partial peptide-pNA precipitations; however, washing with hot water for the oxidizer removal caused a partial dissolution of these precipitates, confirmed by an absorbance at 322 nm. Recovery of peptide-pNAs from combined washings with the simultaneous desalting was performed via adsorption onto Bio-Beads ® SM-2 with further elution of peptide-pNAs with organic solvents. Table 1 contains the yields of peptide-pNAs prepared from peptide-pAAs after the oxidation and desalting onto Bio-Beads ® SM-2.
Yields of Z-or Phe-containing peptide-pNAs were the highest after the oxidation and desalting on Bio-Beads ® SM-2 procedures. Z-group and Phe residue seemed to favor the peptide-pNA adsorption onto Bio-Beads ® SM-2, while Tos group, pNA, Tyr and Trp residues did not. The direct determination of Bio-Beads ® SM-2 adsorbing capacity for Z-AFR-pNA (24 mg per 1 g sorbent, with 83% recovery; see Suppl. for details) and Tos-AY(tBu) R(Pbs)-pNA (75 µg/1 g sorbent, recovery not determined) confirmed our hypothesis and corresponded to an efficient Phe-, but not Tyr-containing peptide adsorption to Porapak Q (Vogel and Altstein 1977). Fmoc-protecting group that could be used for N-terminal blockage in peptide-pNAs did not favor for the adsorption onto Bio-Beads ® SM-2, as well: the adsorbing capacity for Fmoc-Ala was about 40 µg/1 g sorbent.

Concluding remarks
Phenyl groups without substitutions in the aromatic ring (Z and benzoyl (Chistov et al. 2018) as examples) greatly favor the adsorption of peptides to Bio-Beads ® SM-2. Other hydrophobic moieties (Tos, Fmoc, hydroxyphenyl, indole) cannot contribute to the adsorption on Bio-Beads ® SM-2, probably because of steric hindrances. Desalting of phenyl group-lacking peptides can be achieved by lowand medium-pressure chromatography on alkyl-modified or unmodified silica gel; these procedures are more timeconsuming and better suitable for large-scale rather than multiple parallel preparations Adsorption chromatography on Bio-Beads ® SM-2 can represent a simple and efficient procedure for peptide extraction, desalting and purification, provided that either peptides or their impurities contain phenyl groups without aromatic ring substituents. Fast flowthrough makes this chromatographic medium highly suitable for multiple parallel purifications and use in high-throughput screening experiments.
Funding The work was supported by the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) (Theme No. 122030100170-5).
Data availability All data generated or analyzed in this study are presented in the article and the Supplementary information.