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

Abstract

Chemoselective reactions have played a crucial role in the development of high-molecular-weight (>3000 Da) macromolecules with a branched architecture, particularly as peptides and epitope-based vaccines have emerged as promising tools for drug development. Based on this, in this study, we designed and synthesized the peptide macromolecule CH₃CO-[K-Aib-C(3,9-CH₂NHCOCH₃; 6,12-DFLC(CH₂NHCOCH₃)KKESEK)]₄-NH₂ [Ac-K-Aib-C(3,9-Acm); 6,12-epitope)]₄-NH₂] using chemoselective thioether bond formation between the peptide carrier CPSOC (3,9 Acm) and the IAc-DFLC_(Acm)KKESEK-NH₂ peptide epitope. The conjugate was purified via RP-HPLC and characterized via HR-ESI-MS. The epitope D¹²⁸FLCKKESEK¹³⁷ derives from the lethal toxin, ammodytoxin A, from the V. ammodytes snake species, and it was synthesized using the SPPS Fmoc/tBu methodology and characterized via HR-ESI-MS and NMR techniques.

1. Introduction

Peptide antibodies have emerged as a promising tool in immunology, offering a wide range of applications due to their numerous advantages [1,2,3,4]. Peptide epitopes are usually linked to a carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) in order to elicit a strong immune response leading to high-antibody titers [5,6]. Our lab has extensively studied and designed a variety of Sequential Oligopeptide Carriers (SOCs) suitable for binding epitopes that could maintain their spatial arrangement, leading to the production of antigens with great specificity and sensitivity [7,8]. Further research resulted in modifying the initial repetitive moiety Lys-Aib-Gly (Aib: 2-aminoisobutyric acid) to include cysteine residues (CPSOC carrier), allowing for chemoselective reactions such as thioether linkage (Figure 1) [9].

In addition, in snake antivenom research, various studies have shown that using purified toxins as antigens leads to antibodies with higher titers and affinity compared to total venom. Building on this concept, several attempts have been made to use peptide epitopes from the most lethal toxins to neutralize their pharmacological activity and serve as an alternative to whole-venom immunization [10,11,12].

Based on these insights, this study presents the synthesis of [Ac-K-Aib-C(3,9-Acm; 6,12-epitope)]₄-NH₂] (Figure 2) using chemoselective thioether bond formation between the slightly modified CPSOC carrier (CPSOC (3,9 Acm), (Acm: acetamidomethyl) and the iodoacetylated peptide epitope D¹²⁸FLCKKESEK¹³⁷ (Figure 3). The latter corresponds to the C-terminal segment of the basic phospholipase A₂, ammodytoxin A, from the V. ammodytes snake species [13]. The peptide was synthesized according to the SPPS Fmoc/tBu strategy, purified via RP-HPLC, and characterized via HR-MS and NMR spectroscopy. This synthesis represents the initial step towards employing multiple peptide conjugates to induce a robust immune response in hens, leading to the production of IgY antibodies with broad-spectrum antivenom activity.

2. Results and Discussion

2.1. Peptide and Conjugate Synthesis

The desired conjugate was successfully synthesized employing the solid-phase peptide synthesis (SPPS) and chemoselective reaction for thioether bond formation [9,14,15]. The characterization of both peptide and conjugate was accomplished via mass spectrometry (Supplementary Materials, Figures S1 and S4). HR-ESI-MS, positive (m/z): C₅₈H₉₄IN₁₅O₁₉S (IAc-D¹²⁸FLC_(Acm)KKESEK¹³⁷-NH₂): found 488.86; calc. 489.14 for the [C₅₈H₉₄IN₁₅O₁₉S]⁺³; found 732.78; calc. 733.21 for the [C₅₈H₉₄IN₁₅O₁₉S]⁺²; found 1464.57; calc. 1464.43 for the [C₅₈H₉₄IN₁₅O₁₉S]⁺¹. HR-ESI-MS, positive (m/z): C₁₇₆H₂₉₇N₄₉O₅₃S₆ [Ac-K-Aib-C(3,9-Acm; 6,12-epitope)]₄-NH₂] found 1035.78; calc. 1035.98 for the [C₁₇₆H₂₉₇N₄₉O₅₃S₆]⁺⁴; found 828.81; calc. 828.98 for the [C₁₇₆H₂₉₇N₄₉O₅₃S₆]⁺⁵; found 690.68; calc. 690.98 for the [C₁₇₆H₂₉₇N₄₉O₅₃S₆]⁺⁶. The synthesis yield was 72% for the peptide and 58% for the conjugate.

2.2. 1D and 2D NMR Spectroscopy

To further characterize the synthesized peptide, we conducted NMR spectroscopy. The ¹H (δ, ppm) chemical shifts of the peptide epitope IAc-D¹²⁸FLC_(Acm)KKESEK¹³⁷-NH₂ can be found in Table S1 of the Supplementary Materials. The 1D and 2D NMR spectra are shown in the Supplementary Materials, Figures S5–S8. Analysis of the NOESY spectrum indicates that the peptide does not adopt a specific conformation as there are no NOE signals between protons of distant amino acids. Additionally, there are no observed interactions between the side chains of amino acids, such as lysine, and protons of distant amino acids.

2.3. Purification

Both the peptide and conjugate were purified via semi-preparative RP-HPLC, and their purity was monitored via analytical RP-HPLC (Supplementary Materials, Figures S2 and S3). The purification yields were 39% for the peptide and 11% for the conjugate.

3. Materials and Methods

Fmoc amino acid derivatives, Rink amide AM resin and 1-hydroxybenzotriazole (HOBt), were acquired 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). Acetonitrile (HPLC grade) and n-hexane were sourced from Labscan (Dublin, Ireland). Acetic acid and iodoacetic acid were obtained from Sigma-Aldrich (Steinheim, Germany), and triisopropylsilane (TIS) was obtained from Acros Organics (Waltham, MA, USA).

3.1. Peptide Synthesis

The peptide synthesis was performed using the step-by-step solid-phase peptide synthesis on the Rink Amide AM resin (substitution 0.58 mmol/g) employing the Fmoc/tBu methodology (Fmoc: 9-fluorenylmethyloxy-carbonyl). N_α-Fmoc-protected amino acids were dissolved in DMF/DCM (1/1, v/v) using a molar ratio of Fmoc–amino acid/DIC/HOBt/resin: 3/3/3/1. The Fmoc protecting groups were removed using a solution of 20% (v/v) piperidine in DMF. Both the coupling reactions and Fmoc-deprotection steps were monitored via the Kaiser test. To bind the peptide to the carrier, an iodoacetyl group was introduced to the peptide’s N-terminus. For this purpose, iodoacetic acid (ICH₂COOH) was dissolved in DMF/DCM (1/1, v/v) and added to the vessel in a ratio of ICH₂COOH/DIC/resin: 10/10/1. The reaction was carried out in the dark for 3 h [9].

3.2. Peptide Cleavage from the Resin

The peptide cleavage and deprotection of side groups were performed using a solvent mixture of 95% TFA, 2.5% TIS, and 2.5% DMB. The reaction was conducted in the dark at 25 °C. After 3 h, the resin was filtered, and a mixture of DCM/hexane (1:1, v/v) was added to the solution. The mixture was concentrated using a flash evaporator. Afterwards, the peptide was precipitated with cold diethyl ether, dissolved in 2N acetic acid, lyophilized, and stored at 4 °C.

3.3. Thioether Bond Formation

The thioether bond was formed between the previously developed CPSOC carrier [9], slightly modified (CPSOC (3,9 Acm)), and the antigenic epitope IAc-DFLC_(Acm)KKESEK-NH₂. Iodoacetyl-peptide (0.0018 mmol/mL) was dissolved in a mixture of H₂O/AcN (1:1, v/v), and the pH was adjusted to 8.2 using diisopropylethylamine (DIPEA). The carrier was added gradually, in four portions, at a 20 min interval and in solid form. The reaction was carried out under inert conditions (N₂ atmosphere) for 4 h and was terminated by formic acid until pH reached 2–3.

3.4. High-Performance Liquid Chromatography

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

3.5. Mass Spectroscopy

A concentration of 2 ppm in H₂O (0.1 formic acid) of the peptide and conjugate was injected into the HR-ESI-MS (Thermo Scientific LTQ Orbitrap XL™ system, Thermo Fisher Scientific, Bremen, Germany) to acquire the mass spectra.

3.6. NMR Spectroscopy

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

4. Conclusions

In conclusion, we successfully developed the branched macromolecule Ac-[K-Aib-C(3,9-Acm; 6,12-epitope)]₄-NH₂ by conjugating two copies of the peptide epitope IAc-DFLC_(Acm)KKESEK-NH₂ to the CPSOC (3,9 Acm) carrier. The chemoselective reaction of thioether bond formation was chosen to synthesize the conjugate by linking the peptide epitope, by its modified N-terminus, in two copies to the two free Cys residues of the carrier. Both the peptide epitope and the conjugate were synthesized in satisfactory yields and were of high purity, which underscores the robustness of the synthetic strategy employed.

The present work forms the basis for producing antibodies with broad-spectrum antivenom activity, given that similarly synthesized macromolecules have been successfully used for inducing the immune response with high-titer antibodies and for neutralizing the pharmacological effects of snake toxins [12].