Reactions of beta-propiolactone with nucleobase analogues, nucleosides, and peptides: implications for the inactivation of viruses

Abstract

β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses.

FIGURE 1.

Two possible reaction paths of a nucleophile (Nu) with β-propiolactone leading to (I) acylated and (II) alkylated products.

FIGURE 2.

NMR spectra after the reaction of β-propiolactone (16 mm) in citrate buffer (125 mm, pH 7. 8). Spectra were recorded (1–7) after 3, 15, 40, 89, 187, 387, and 775 min. Citrate adducts (C) and 3-hydroxypropionic acid (D) are indicated.

FIGURE 3.

Conversion of β-propiolactone in citrate buffer pH 7. 8, 125 mm at 25 °C; (⧫) β-propiolactone, (□) 3-hydroxy propionate and (△) citrate adducts.

FIGURE 4.

Reaction of imidazole with β-propiolactone. I, measured and calculated concentrations of β-propiolactone and products (+), hydroxypropionyl imidazole (⧫), 2-carboxyethyl imidazole (−), 1,3-bis(2-carboxyethyl) imidazole (○), and 3-hydroxypropionic acid (□), based on (II), the model with k₁ = 0.000056, k₂ = 0.067, k₃ = 0.0098, k₄ = 0.0000679, and k₅ = 0.064.

FIGURE 5.

NMR spectra of nucleosides treated with β-propiolactone. I, deoxyguanosine. II, deoxyadenosine. III, cytidine. Unmodified nucleosides cause off-scale peaks, whereas adducts show downfield-shifted peaks.

FIGURE 6.

Ring opening reaction of desoxyguanosine-N-7-carboxyethylated adduct.

FIGURE 7.

LC/MS chromatograms and MS² spectra were obtained from peptide 1 after treatment with β-propiolactone at pH of 9. 0. Peptide 1 was slightly converted in two reaction products with a β-propiolactone attached to Thr⁶ (I) and Thr² (II) residues, resulting in a mass increment of 72 Da. MS² spectra revealed also a characteristic fragment with a neutral loss of 90 Da (C₃H₆O₃).

FIGURE 8.

LC/MS analyses performed on peptide 2 treated with β-propiolactone. Four products (I–IV) were identified by LC/MS in which the cysteine residue was modified by β-propiolactone. MS² spectra I-IV revealed the peptide sequence with a truncated cysteine side chain (−34 Da). Fragmentation during MS² analysis resulted in a neutral loss fragment of 106 Da (C₃H₆O₂S) or 178 Da (C₆H₁₀O₄S) if one or two β-propiolactone molecules were attached to the cysteine residue respectively.

FIGURE 9.

LC/MS analyses performed on peptide 5 treated with β-propiolactone. Two products (I and II) were observed by LC/MS containing one or two β-propiolactone molecules attached to the histidine residue, respectively. MS² analyses revealed the peptide sequence and the attachment of β-propiolactone (+72 Da) (I). IIa, two dominant fragment ions (483.8 and 497.3) were observed during fragmentation of product II, which contains double attachment (+144 Da). IIb, multistage activation performed on fragment 497.3 provided full peptide sequence and the attribution of the modified histidine residue.

FIGURE 10.

LC/MS analyses performed on peptide 6 treated with β-propiolactone. Two products (I and II) were observed by LC/MS containing one attachment of β-propiolactone. MS² analyses revealed the peptide sequence of both products and the β-propiolactone modification of lysine residue (I). Product I is probably peptide 6 with an alkylated lysine residue and product II with an acylated lysine residue.

FIGURE 11.

LC/MS analyses performed on β-propiolactone-treated peptide 7. LC/MS chromatograms demonstrated two reaction products (I and II) with a single and double β-propiolactone attachment. Ia, the MS² spectrum obtained from peptide 7 with a single attachment of β-propiolactone provided only a fragment ion (406.8) with a neutral loss of 120 Da. Ib, multistage activation performed on the fragment ion (406.8) revealed the peptide sequence with a truncated methionine residue (−48 Da). The second product (II) with two attachments of β-propiolactone resulted in a comparable (Ia) MS² spectrum and (Ib) multistage fragmentation spectrum.