Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂

Abstract

Advances in synthetic peptide methodologies have enabled the development of macromolecules mimicking protein properties and have found applications in various fields, particularly in immunology. Furthermore, Sequential Oligopeptide Carriers (SOC_n and CPSOC) have been designed as multi-functional core molecules, to which multiple bio-cargos can be anchored, allowing the construction of high molecular weight molecules (>3000 Da) capable of inducing a strong immune response. This study presents the design and synthesis of the Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂ peptide conjugate of branched architecture. The peptide epitope S¹²⁸SNCQENSDK¹³⁷ belongs to the V. berus basic phospholipase A₂, a member of the European viper species’ most lethal protein families. The peptide epitope was synthesized according to the SPPS Fmoc/tBu strategy and characterized by HR-ESI-MS and NMR experiments, while the conjugate was purified by RP-HPLC and characterized by HR-ESI-MS.

1. Introduction

Synthetic peptides are extensively used in various fields, including diagnostics, treatment, vaccine development, and peptide antibody production, leading to a remarkable revival in peptide drug discovery over the last decades [1,2,3,4]. Furthermore, branched architecture peptide macromolecules have been employed in immunology to overcome several limitations associated with linear epitopes, such as poor antigenicity, immune response, and stability [5,6,7,8].

In addition, Sequential Oligopeptide Carriers (SOCn) with the moiety [Lys(antigen)-Aib-Gly]₄, (Aib: 2 aminoisobutyric acid), as well as the modified versions [Lys-Aib-Cys]₄ (C-SOC₄) and [Lys-Aib-Cys(3,9 Acm)]₄ (CPSOC(3,9 Acm)), have been developed for anchoring multiple copies of peptide epitopes, enhancing their antigenic/immunogenic potency [9,10,11]. These carriers allow the construction of high molecular weight molecules (>3000 Da) capable of inducing a potent immune response. Their key advantage lies in their ability to produce antibodies recognizing different regions of the same protein.

Moreover, innovative strategies for snake antivenom include the immunization of animals with chemically synthesized toxin epitopes, which mitigates several challenges associated with traditional antivenom production, such as the maintenance and handling of dangerous animals [12,13,14,15].

Based on the above, in this study, we designed and synthesized the Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂ (Acm: acetamidomethyl group) peptide conjugate of branched architecture. The peptide epitope S¹²⁸SNCQENSDK¹³⁷ belongs to the V. berus basic phospholipase A₂, one of the most lethal snake toxins. This synthesis represents a pivotal step in developing alternative antivenom production methods, such as employing multiple peptide antigenic macromolecules to produce antibodies that cross-react with heterologous snake venoms.

2. Results and Discussion

2.1. Peptide and Conjugate Synthesis

We successfully synthesized the desired peptide conjugate of branched architecture through the Solid Phase Peptide Synthesis (SPPS) and a chemoselective reaction for the thioether linkage. The characterization for both peptide (Figure 1) and conjugate (Figure 2) was accomplished via mass spectrometry (Supplementary Materials Figures S3 and S4). HR-ESI-MS, positive (m/z): C₄₅H₇₃IN₁₆O₂₂S (IAc-S¹²⁸SNC_(Acm)QENSDK¹³⁷-NH₂): found 675.20; calc. 675.56 for the [C₄₅H₇₃IN₁₆O₂₂S]⁺²; found 1349.39, calc. 1349.12 for the [C₄₅H₇₃IN₁₆O₂₂S]⁺¹. HR-ESI-MS, positive (m/z): C₁₅₀H₂₅₅N₅₁0₅₉S₆ (Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂) found 1955.35; calc. 1955.66 for the [C₁₅₀H₂₅₅N₅₁0₅₉S₆]⁺²; found 1303.91; calc. 1304.11 for the [C₁₅₀H₂₅₅N₅₁0₅₉S₆]⁺³; found 977.93; calc. 978.33 for the [C₁₅₀H₂₅₅N₅₁0₅₉S₆]⁺⁴. The crude recoveries were 70% and 64% for the peptide and the conjugate, respectively.

2.2. NMR Spectroscopy

Further characterization of the synthesized peptide was accomplished by NMR spectroscopy. 1D and 2D NMR spectra of the IAc-S¹²⁸SNC_(Acm)QENSDK¹³⁷-NH₂ peptide are shown in Figures S5–S8 (Supplementary Materials) and ¹H (δ, ppm) chemical shifts are listed in Table S1 in the Supplementary Materials. The assignment started from the terminal part of the lysine side chain, which exhibits the characteristic signal sequence of the side chain at 1.35/1.65/1.58/2.79/7.42 ppm from TOCSY/NOESY spectra and following the signals through NOE correlations between CH and NH protons. In this specific peptide, the protons of the two neighboring serine residues appear at almost identical values (Ser 1: 4.38/3.83 ppm, Ser 2: 4.38/3.82 ppm), while the protons of Ser 3 display a different chemical shift (4.46/3.84 ppm) due to the distinct chemical environment (N, D residues). The absence of NOE signals between hydroxyl or amine groups or the side chains of amino acids, such as lysine with other protons of distant amino acids, at expected values suggests that there are no intramolecular hydrogen bond interactions leading to the bending of the molecule or any particular conformation.

2.3. Purification

Both the peptide and conjugate were purified via RP-HPLC. Chromatograms from analytical RP-HPLC are shown in Figures S1 and S2 in the Supplementary Materials. Purification yields were 50% for the peptide and 15% for the conjugate.

3. Materials and Methods

3.1. Materials

Fmoc amino acid derivatives, Rink amide AM resin, and 1-hydroxybenzotriazole (HOBt) were obtained from GL Biochem (Shanghai, China). Trifluoroacetic acid (TFA) and piperidine were purchased from Honeywell, Riedel-de Haen (Seelze, Germany). Dichloromethane (DCM), Dimethylformamide (DMF), N,N′-Diisopropylcarbodiimide (DIC), and dimethylbenzene (DMB) were purchased from Fluka (Seelze, Germany), while acetonitrile (HPLC grade) and n-hexane were purchased from Labscan (Dublin, Ireland). Acetic acid and iodoacetic acid were obtained from Sigma-Aldrich (Steinheim, Germany) and TIS from Acros Organics (Waltham, MA, USA).

3.2. Peptide Synthesis

The peptide synthesis was carried out by employing the stepwise solid-phase peptide synthesis on the Rink Amide AM resin (substitution 0.58 mmol/g) using the Fmoc/tBu methodology [16,17]. Amino acids were introduced as Na-Fmoc protected and were dissolved in DMF/DCM (1/1, v/v) using a molar ratio of Fmoc-amino acids/DIC/HOBt/peptide-resin: 3/3/3/1. The Fmoc protecting group was removed using 20% (v/v) piperidine in DMF. The coupling reactions and Fmoc-deprotection steps were monitored by the Kaiser test. For the iodoacetyl group introduction to the N-terminal, after completing the desired peptide sequence, iodoacetic acid (ICH₂COOH) was dissolved in DMF/DCM (1/1, v/v) and added to the vessel. The reaction was carried out for 3 h in the dark and tested by the Kaiser test.

3.3. Peptide Cleavage from the Resin

The peptide cleavage and side groups’ deprotection were accomplished using the following mixture of solvents: 95% TFA, 2.5% TIS, 2.5% DMB. The reaction was left for 3 h, in the dark, at RT. Then, the resin was filtered, DCM/hexane (1/1 v/v) was added, and the mixture was concentrated in a flash evaporator (Heidolph, Germany). The peptide was precipitated using cold diethyl ether and finally dissolved in 2N acetic acid, lyophilized (Martin Christ, Germany), and stored at 4 °C.

3.4. Thioether Bond Formation

The thioether bonds were formed between the CPSOC (3,9 Acm) carrier and the antigenic epitope IAc-S¹²⁸SNC_(Acm)QENSDK¹³⁷-NH₂. The iodoacetyl-peptide was dissolved in H₂O/AcN (1:1, v/v) and the pH was adjusted to 8.2 using diisopropylethylamine (DIPEA). The peptide carrier CPSOC (3,9 Acm) was added to the solution in solid form in small portions with a ~20 min interval. The reaction was performed under inert conditions (N₂ atmosphere) for 4 h and was terminated by adding formic acid to the solution until pH reached 2–3 [9].

3.5. High-Performance Liquid Chromatography

The crude, lyophilized peptide and conjugate were purified by semipreparative RP-HPLC (Shimadzu, Germany, Discovery C18 column (25 cm × 10 mm)) with a flow rate of 4.7 mL/min. The wavelength was set at 214 nm and the gradient elution system was H₂O (0.1% TFA)/acetonitrile (0.1% TFA) from 90/10% to 60/40%. Peptide purity was monitored by analytical RP-HPLC associated with a C₁₈ Supelco column (25 cm × 3 mm, 5 μm), (Shimadzu, Germany).

3.6. Mass Spectroscopy

HR-ESI-MS (Thermo Scientific LTQ Orbitrap XL™system, Thermo Fisher Scientific, Bremen, Germany) was used to record the mass spectra. The pure lyophilized peptide and conjugate were dissolved in a concentration of 2 ppm in H₂O/0.1% formic acid (FA).

3.7. NMR Spectroscopy

NMR experiments for the peptide epitope were conducted on a Bruker Avance spectrometer at a frequency of 500.13 MHz. and were processed using Topspin 4.2 (Bruker Analytik GmbH, Bremen, Germany). Both 1D and 2D experiments (¹H, ¹H–¹H-TOCSY, ¹H–¹H COSY, ¹H–¹H NOESY) were acquired in a 9:1 (v/v) H₂O:D₂O solution at a concentration of 5 mM, T = 298 K).

4. Conclusions

In summary, we successfully synthesized the peptide macromolecule Ac-[K-Aib-C(3,9-Acm; 6,12-SSNC_(Acm)QENSDK)]₄-NH₂ of branched architecture by linking two copies of the peptide epitope IAc-S¹²⁸SNC_(Acm)QENSDK¹³⁷-NH₂ to the CPSOC (3,9 Acm) carrier. The conjugation was achieved through a chemoselective thioether bond formation, specifically targeting the two available cysteine residues on the carrier. Both the peptide epitope and the final conjugate were synthesized with satisfactory yields and high purity.

This synthesis serves as the first step towards the development of alternative antivenom production strategies, as this macromolecule shows potential as a candidate for generating antibodies capable of cross-reacting with venoms from various snake species.