Reactions of β-propiolactone 1 REACTIONS OF BETA-PROPIOLACTONE WITH NUCLEOBASE ANALOGUES , NUCLEOSIDES AND PEPTIDES : Implications for the inactivation of viruses

Background: β -Propiolactone is applied for inactivation of viruses. Results: A systematic overview of β propiolactone modifications on the building blocks of nucleic acids and proteins. Conclusion: Proteins are more extensively modified by than nucleic acids during inactivation of viruses with β-propiolactone. Significance: The study provides detailed knowledge that can be utilised to elucidate the chemical modifications occurring in viruses after inactivation with β-propiolactone.

. According to the literature, formaldehyde reacts primarily with proteins (19,20), whereas β-propiolactone modifies mainly DNA or RNA (21)(22)(23).Therefore, it is expected that βpropiolactone will maintain a high immunogenicity during the inactivation of viruses.β-Propiolactone is a hazardous compound which may cause cancer (24)(25)(26)(27)(28)(29)(30).It consists of an almost planar highly strained four-membered ring (Figure 1).Due to this strain, β-propiolactone reacts readily with nucleophiles.Extensive studies have been performed in the past to determine the reactivity with many nucleophiles, e.g.water, inorganic salts, amines, alcohols, thiols, carboxylic acids (31)(32)(33)(34)(35)(36)(37)(38).The reaction of β-propiolactone is a typical example of an S N 2 reaction which rate of disappearance depends on the concentrations of a particular nucleophile and βpropiolactone according to equation 1: In aqueous media, water is an important nucleophile for β-propiolactone, which has at room temperature a half-life of about 3-4 hours (39).The half-life can be decreased in the presence of other nucleophiles.Both the concentration and nature of the nucleophile are important for the conversion rate (40).When a mixture of nucleophiles is used, the rate of disappearance of β-propiolactone equals the summation (41,42): The situation becomes more complicated in the case of multiple nucleophilic sites present in one molecule, e.g. in a DNA or protein molecule (43,44).The reactivity of a particular nucleophilic group in such molecules is influenced by global parameters, e.g.pH and temperature, and by local circumstances, e.g. the accessibility and non-covalent interactions (45,46).β-Propiolactone can react with nucleophiles in two ways, resulting in alkylated and acylated products (Figure 1).According to the Hard Soft Acids Bases (HSAB) theory, hard nucleophiles, such as primary amino groups (RNH 2 ), will react with the hard acyl carbon while soft nucleophiles, such as thiol groups (RSH), will give alkylated products (47).Hardness or softness of a nucleophile is also influenced by local parameters.For example, hydrogen bonding can increase softness of primary amino or thiol groups which may result in increased alkylation.Also pH plays a role: when a thiol group becomes deprotonated, its softness decreases.The same holds for water, which becomes a harder nucleophile upon deprotonation.Generally, the microenvironment of the nucleophile influences the hardness and by that, the reactivity of the nucleophilic group (48).Not all reactions with β-propiolactone will lead to inactivation of a virus.The main reaction during inactivation will be the reaction with water to give a non-toxic compound: 3-hydroxypropionic acid.Also, salts and buffer components have a large effect on the conversion of β-propiolactone (34).Virus inactivation will only occur after reaction of β-propiolactone with viral constituents e.g.DNA/RNA and protein (23,38,(49)(50)(51)(52)(53).Although a number of chemical modifications induced by β-propiolactone have been elucidated, the nature of all possible reactions is still largely unknown.Current knowledge is not sufficient to develop an accurate inactivation procedure with βpropiolactone to produce a viral vaccine that preserves the highest immunogenicity.
The purpose of the present work was to elucidate by using model compounds the reactions which occurs during inactivation of viruses with β-propiolactone.
The reactivity of β-propiolactone with (i) plain buffers, (ii) with nucleobase analogues and nucleosides and (iii) with amino acid residues were investigated in detail.The reaction conditions used in this study were based on inactivation processes applied for viral vaccines, such as influenza and rabies vaccines (10,54,55).Frequently used buffers during inactivation of viruses are phosphate, citrate, and HEPES.The kinetics of hydrolysis of β-propiolactone was examined in these buffers.Also products formed during the reaction with buffer components were identified by nuclear magnetic resonance spectroscopy (NMR).Secondly, a systematic study was performed to determine the reactivity of particular reactive sites in nucleotides.A set of nucleobase analogues and nucleosides was used to identify and quantify by NMR the different chemical modifications that occur during β-propiolactone treatment.Furthermore, the reaction of βpropiolactone was investigated with various amino acid derivatives and synthetic peptides.The peptides synthesised had an amino acid sequence Ac-VTLXVTR-NH 2 in which one amino acid residue (X) varies.The reaction of amino acid derivatives was determined by NMR, whereas the conversion of peptides was monitored by tandem reversed-phase liquid chromatography mass spectrometry (LC/MS).
In this paper, we give an overview of the major conversion products that can be formed during inactivation of viruses by β-propiolactone.

Half-life of β-propiolactone in buffer solutions
The inactivation of viruses is normally performed with β-propiolactone concentrations between 0.025 and 1% (4-160 mM).In this experiment we determined the half-life of 16 mM β-propiolactone in water or buffers containing citrate, phosphate, PBS and HEPES (Table 1).The half-life of βpropiolactone in water was 225 minutes at 25°C, which is in good agreement with the value of 3.5 hours reported previously (39).An example of NMRspectra is given in Figure 2, showing the conversion of 16 mM β-propiolactone in a citrate buffer (pH 7.8, 125 mM).β-Propiolactone (NMR-peaks at 4.3 and 3.6 ppm) forms 3-hydroxypropionic acid (2.45 and 3.8 ppm) and at least two citrate adducts (AB-systems at 2.9-2.75 and 2.75-2.6 and signals at 4.35-4.25 ppm).
Figure 3 shows the kinetics of the degradation of βpropiolactone in the citrate buffer and the formation of β-propiolactone products.The half-life of βpropiolactone in citrate buffer was short if compared to other buffers, but independent of the tested pH values (Table 1).In contrast, β-propiolactone does not react with HEPES: its half-life is equal to BPL in water (Table 1) and no reaction products were detected by NMR.Phosphate buffers showed intermediate behaviour with an effect of pH.βpropiolactone had a half-life of almost 3 hours at a pH value of 6.6, whereas at a pH of 7.8 this was reduced to little over 2 hours.When the reaction was performed in phosphate buffer, monoalkylated phosphate molecules were also observed.NMR spectra showed a characteristic pseudo quartet at δ3.94.In PBS buffers, the presence of chloride anions (at 150 mM) causes the formation of 3chloropropionic acid (characteristic NMR triplets at 2.78 and 3.78 ppm).In conclusion, the choice of the buffer and pH is important for availability of βpropiolactone to inactivate viruses.

Conversion and structural analyses of nucleobase analogues treated with β-propiolactone
Many nucleophilic sites with an unknown reactivity are present in DNA and RNA which might react with β-propiolactone.Randerath et al. demonstrated by using 32 P-postlabeling fifteen distinct adducts in DNA after treatment with high concentration of βpropiolactone (51).However, their chemical structures are not identified.As a model for nucleic acids, a set of nucleobase analogues and nucleosides was used in this study to determine the reactivity with β-propiolactone The conversion of the analogues revealed that principles e.g., electron delocalisation and tautomerisation, affect the reactivity of βpropiolactone with particular nucleophilic sites in nucleobases.
Pyrrole and pyridine -In heterocyclic compounds, such as pyrrole and pyridine, reactivity with βpropiolactone is influenced by aromaticity.In case of pyrrole, the electron pair on the nitrogen atom is needed to sustain aromaticity.Therefore this electron pair is less available for the reaction with βpropiolactone.Indeed, no reaction products were observed by NMR when treating an aqueous solution of pyrrole with β-propiolactone.According to theory, pyrrole will react at strong conditions with βpropiolactone at the C-3 carbon atom, but not at the nitrogen atom (58).On the other hand, the lone pair on the pyridine nitrogen reacted fairly readily with βpropiolactone, because this electron pair is not involved in the aromaticity of pyridine.Sixty five percent of the pyridine molecules was converted into  2.In case of indole, like pyrrole, no reaction products were observed after treatment of indole with βpropiolactone, because the single electron pair of indole is needed for aromaticity (Table 2).As expected, the pyridine like nitrogen atom N-7 in 7azaindole did react with β-propiolactone, whereas the pyrrole like nitrogen atom N-1 was not modified (Table 2).
Imidazole -In contrast to pyrrole, imidazole was alkylated by β-propiolactone, giving mono-and bisalkylated products (Table 2).This result is reported previously in the literature (59).Both nitrogen atoms of imidazole, N-1 and N-3, were involved in the alkylation reaction.As a result of tautomerisation, the nitrogen N-1 is deprotonated after alkylation at the nitrogen N-3.The electron pair at N-1, which becomes available in the mono-alkylated imidazole, reacted with β-propiolactone to form the bis-alkylated product.Moreover, acylation of imidazole occurred which resulted in 3-hydroxypropionyl imidazole.
However, the acylated compound became hydrolysed in water providing 3-hydroxypropionic acid and imidazole.This phenomenon is the basis of the catalytic effect of imidazole increasing the rate of hydrolysis of β-propiolactone (Figure 4).When the reaction of imidazole with β-propiolactone ( From the NMR-spectra the concentrations of βpropiolactone and its reaction products with imidazole and water were obtained (Figure 4).Using a model depicted in Figure 4, reaction rate constants (k1-k5) were calculated with k 1 = 5.6*10 ).The primary amino group of aniline was converted substantially after the reaction, but no acylated product was observed (Table 2).The same products were found after treatment of aniline with 3iodopropionic acid.2-Aminopyridine reacted with βpropiolactone to gave two distinct products in a 2:1 ratio (  2. In conclusion, nitrogen atoms in a pyridone or pyrimidinone ring can react with β-propiolactone, but to a lesser degree than pyridine and pyrimidine.

Reaction of β-propiolactone with nucleosides
Based on the reactions with nucleobase analogues we predicted that position N-7 and N-3 react with βpropiolactone, whereas, the nitrogen atom N-1 and the exocyclic nitrogen C2N will probably hardly react.
The nitrogen atoms N-1 and C2N have homology with a pyridone ring or with the primary amino group of 2-aminopyrimidine, respectively.In case of deoxyguanosine, additional signals were observed for the 1'H proton at 6.4 (t, 1H) and the H-8 proton at 8.88 ppm, indicating alkylation at position N-7 (Figure 5).This product was also identified by Roberts et al. (23).At prolonged reaction times, additional H-8 signals at 7.92, 7.96, and 8.00 ppm were observed, indicating an unexpected product derived from the N-7 adduct.The proposed structure of the product is given in Figure 6 in which the imidazole ring is hydrolysed.As was shown previously, alkylation at N-7 of guanosine stimulates opening of the imidazole ring during the reaction of chloroethyl ethyl sulphide with 2'-deoxyguanosine (60).No indications were found for modifications at positions N-1, N-3 and C2N.For deoxyadenosine modifications were expected at four positions i.e., N-

Reaction of β-propiolactone with peptides
A set of twelve synthetic peptides (Ac-VTLXVTR-NH 2 , in which X = A, C, D, E, H, K, M, N, Q, S, W or Y) was used to investigate the reactivity of βpropiolactone with specific amino acid residues (Table 3).The reactions were performed in phosphate buffers with a pH of 3, 5, 7 or 9. Peptide 1 was designed to be inert for β-propiolactone.Indeed, LC/MS analyses revealed that peptide 1 was not modified after incubation with β-propiolactone in buffers adjusted to a pH of 3, 5 or 7.However, a minor conversion was observed at pH 9 (Table 3).LC/MS 2 analyses showed that peptide 1 gave two products with a mass increment of 72 Da located on either the residue Thr 2 or the Thr 6 (Figure 7).Other peptides containing a cysteine (peptide 2), aspartic acid (peptide 3), glutamic acid (peptide 4), lysine (peptide 6), methionine (peptide 7), serine (peptide 10) or tyrosine residue (peptide 12) were found to be modified by β-propiolactone.The conversion of these peptides was depending on the pH (Table 3): the higher the pH the higher the conversion of the peptides, except for the methionine residue.Substantial conversion of the methionine residue was measured at pH 3.This latter result is in agreement with data published previously (52).Peptides containing asparagine, glutamine or tryptophan residues (peptide 8, 9, 11) did not react with βpropiolactone.Based on the LC/MS analyses, the conversion rate of other amino acid residues was at pH 7 in a decreasing order: cysteine > methionine > histidine > aspartic or glutamic acid > tyrosine > lysine > serine > threonine.

Structural analysis of converted amino acid residues
Cysteine -The treatment of cysteine residues (peptide 2) with β-propiolactone resulted in four reaction products, which could be separated chromatographically (Figure 8).LC/MS revealed two adducts with a mass increment of 72 Da (product I and II) and two adducts with an increase of 144 Da (product III and IV).LC/MS 2 analysis was used to verify that adducts of β-propiolactone were attached to cysteine residue, although the interpretation of MS 2 spectra is rather complex (Figure 8).Fragmentation of products I and II started with a characteristic neutral loss of 106 Da (C 3 H 6 O 2 S) followed by the fragmentation of the peptide backbone ( The two products with a mass increment of 72 Da can be explained as acylation (product I) and alkylation (product II) of the cysteine residue.The assignment of both peaks was based on the results obtained from the reaction of peptide 2 with 3-iodopropionic acid.This compound can only alkylate reactive amino acid residues.Alkylated products of β-propiolactone and 3-iodopropionic acid have the same structure.As expected treatment with 3-iodopropionic acid resulted in only one product with a mass increment of 72 Da.The retention time of this product was comparable with product II of the peptide 2 treated with βpropiolactone.MS 2 analysis confirmed that the cysteine residue of peptide 2 was modified by 3iodopropionic acid and the MS 2 spectrum was exactly the same as the MS 2 spectrum of the product in product II (Figure 8).In addition, the treatment of peptide 2 with 3-iodopropionic acid gave two products with an increase of 144 Da.The retention times on the column, mass increments and MS 2 spectra of these two products were the same as for products III and IV from β-propiolactone-treated peptide 2. A mixture of β-propiolactone and 3iodopropionic acid-treated peptide 2 was prepared (in 1:1 molar ratio) and analysed by LC/MS.The measurement confirmed that the products II-IV were identical because of the elution times and masses (Supplemental Figure S1).In a different experiment, the reaction of β-propiolactone with a cysteine analogue (Ac-Cys-OMe) was monitored by NMR.  5).The formation of acylated product is somewhat unexpected, because HSAB theory predicts alkylated product (thiol group is considered to be a prototypically soft nucleophile (47)).When ten times excess (10x) of β-propiolactone was used, bisalkylated product was also formed.The proposed structures of modified cysteine residues are given in Table 5.
Cystine -According to the literature β-propiolactone reacts also with disulphide groups (62)(63)(64).However, this finding could not be supported in the present study.Peptide 2 containing a cysteine residue was treated with iron(III)chloride to oxidise peptide 2. LC/MS analysis revealed that the oxidation resulted in the formation of a disulphide bridge between two peptide molecules (product MH + =1661.92±0.02Da).
Based on the peak areas, 97% of peptide 2 was converted into a dipeptide containing a disulphide bridge.Subsequently, the oxidised peptide was treated with β-propiolactone.The expected products (MH + =1733.9 and 1806.0Da) were not detected by LC/MS, whereas the residual amount of peptide 2 (thiol) was substantially converted by β-propiolactone (MH + =904.5 Da).A second experiment confirmed that disulphide groups do not react with βpropiolactone.A cystine derivative (Ac-Cys-OMe) 2 was treated with β-propiolactone and monitored by NMR at regular intervals.No reaction products were generated.We conclude that cystine residues do not react with β-propiolactone.
Aspartic and glutamic acid -The reaction of βpropiolactone with peptides containing an aspartic or glutamic acid residue (peptides 3 and 4, respectively) resulted in a major product with a mass increment of 72 Da and a minor component with an increase of 144 Da (conversions of about 8% and 0.1%, respectively).LC/MS 2 analyses confirmed that one or two βpropiolactone molecules were attached to the aspartic or glutamic acid residue (Supplemental Figure S2).The same products were obtained albeit in low yields by treating peptides 3 and 4 with 3-iodopropionic acid.Retention times, mass increments and MS 2 spectra were identical for either the β-propiolactone or 3-iodopropionic acid modified peptides (peptides 3 or 4).Based on these results, we conclude that aspartic or glutamic acid residues are probably alkylated by the reaction with β-propiolactone.The proposed structures of β-propiolactone-modified aspartic or glutamic acid residues are given in Table 5.
Histidine -Two reaction products were found after incubation of a peptide 5 (containing a histidine residue) with β-propiolactone.Two products of peptide 5 were chromatographically separated and detected in significant amounts by LC/MS with a mass increment of 72 Da and 144 Da (Figure 9).Two distinct products with a mass increase of 72 Da were expected, because it has been postulated that histidine can be alkylated at position N-1 and N-3 of the imidazole group (65).However LC/MS revealed only one chromatographically separated product with a mass increment of 72 Da.If histidines were alkylated at position N-1 or at position N-3, the peptides eluted simultaneously.LC/MS 2 analysis showed that βpropiolactone (ΔM of 72 Da) was present at the histidine residue (Figure 9).Unfortunately, LC/MS 2 analysis cannot reveal which nitrogen of the histidine residue is modified: position N-1 and/or N-3.The product with mass increase of 144 Da showed poor fragmentation during MS 2 analysis.Observed neutral loss fragments of 17, 44 and 61 Da were ascribed as truncation of the arginine side chain.Multistage activation performed on fragment ions revealed the peptide sequence and the modified histidine residue (Figure 9).Treatment of peptide 5 with 3iodopropionic acid did not result in significant conversion of the histidine residue.The experiment with 3-iodopropionic acid did not support that alkylation of histidine residues occurs during the reaction with  β-propiolactone.In addition, the reaction of a histidine derivative (Ac-His-NHMe) with β-propiolactone was recorded by NMR.The reaction resulted in mono-alkylation and bisalkylation (NMR: δ 6.8, s, 1H, 7.6, s, 1H and 7.3, s, 1H, 7.7, s, 1H, respectively).Also NMR demonstrated the presence of only one mono-alkylated product, which is in accordance with the results obtained from LC/MS analysis.Because the nitrogen atom N-1 of histidine is less accessible than the nitrogen atom N-3, we assume that the histidine residues will be monoalkylated at position N-3.The proposed structures of β-propiolactone-modified histidine residues are given in Table 5.
Lysine -Two reaction products with only one attachment of β-propiolactone (ΔM of 72 Da) were detected on peptide 6.Both products were widely separated by chromatography (Figure 10).MS 2 measurement confirmed the modification of the lysine residue.The two products can be explained by alkylation and acylation reactions of β-propiolactone (Table 5).Both structures were described previously as N-(2-carboxyethyl)lysine and N-(3hydroxypropionyl)lysine (66).Alkylation of the lysine side chain forms a secondary amine group which is protonated at a low pH.The polarity of the modified peptide and retention times on the column will not change drastically if compared to the non-modified peptide.Therefore, product I is probably peptide 6 with an alkylated lysine residue (Figure 10), because the peptide was detected as a doubly charged ion with slightly longer retention time (Δt = ~0.5 min).In the case of acylation of lysine residues, an amide (peptide) bond is formed which resulted in neutral moiety at a low pH.Therefore, acylation of peptides reduces the polarity and increases the retention times.Indeed product II mainly consist of singly charged ions with a prolonged retention time (Δt = ~2.5 min).The reaction of β-propiolactone with a lysine derivative (Ac-Lys-OMe) resulted in both acylated and alkylated products (Table 5).The alkylated product showed characteristic triplets for the secondary amine group (-CH 2 NHCH 2 -) at δ  3.18 and 3.20 ppm, whereas the acylated product gave triplets for -N(CO)CH 2 -at δ 2.57 and 3.84 ppm.
Methionine -LC/MS analysis revealed two adducts with one or two β-propiolactone molecules attached to peptide 7 (Figure 11).The product with a single attachment of β-propiolactone (ΔM of 72 Da) eluted 5 minutes earlier than the native peptide, indicating strongly increased polarity.The adduct containing two β-propiolactone molecules (ΔM of 144 Da) eluted 0.5 minutes later than the product with a single attachment.The structure of a single attachment to methionine has been described in the literature (52) and explains the increased polarity because of the positively charged sulphur atom.MS 2   11) with a truncated methionine side chain (ΔM of -48 Da = 72-120 Da).The reaction of 3iodopropionic acid with peptide 7 resulted in two reaction products with the same mass increments, retention times and MS 2 spectra as the products of βpropiolactone-treated peptide 7, indicating that βpropiolactone only alkylates methionine residues.A mixture of β-propiolactone and 3-iodopropionic acidtreated peptide 7 (in 1:1 ratio) demonstrated by LC/MS the same elution times and masses for the products I and II (Supplemental Figure S3).NMR analysis of Ac-Met-Ome treated with β-propiolactone revealed that an alkylated product was formed (Table 5).In the NMR spectrum, two characteristic singlets were observed, corresponding to the diastereomers (around 2.95 ppm) of the S-methyl group, together with additional signals of the alkylated product (NMR: δ 4.65, m, 1H, 3.58-3.32,m, 4H, 2.75, dt, 2H, 2.08, s, 3H).When the reaction was carried out with a 10 fold excess of β-propiolactone, another sulphonium adduct was observed (NMR: δ 2.97, ds, 3H, 3.04, dt, 2H, 4.41, t, 2H).The NMR data obtained are in accordance with a bis-carboxyethoxylated methionine.Both MS and NMR analyses confirmed that methionine residues become alkylated by reaction of β-propiolactone.The proposed structures of both products are given in Table 5.
Serine and Tyrosine -Peptides containing a serine or tyrosine residue (peptide 10 and 12, respectively) were slightly converted at pH 7 to a product with a mass increment of 72 Da, corresponding to a single attachment of β-propiolactone (Table 5).MS 2 analysis confirmed that β-propiolactone was attached to the serine or tyrosine residue (Supplemental Figures 4  and 5).β-propiolactone is reacting probably with the hydroxyl group of serine or tyrosine.The MS 2 spectrum of the peptide 10 with a modified serine residue showed a typical fragment ion with a neutral loss of 90 Da (C 3 H 6 O 3 ).A fragment ion with a similar neutral loss of 90 Da was not observed for peptide 12 with a modified tyrosine residue (Table 4).From the literature it remains unclear if serine, threonine and tyrosine residues are acylated or alkylated by β-propiolactone.Determann and Joachim found only an acid labile product from tyrosine and concluded that acylation occurred (67).This was in contrast with results obtained by Gresham et al. (68).They observed alkylation of the phenol when reacting with β-propiolactone, although under acid catalysis they found acylated phenol.Also, theoretical predictions made by Zhang and Yang indicate that alkylation of tyrosine residue will occur by treatment of β-propiolactone (69).For unequivocal identification of the reaction products three different experiments were performed.Peptides 1, 10 and 12 treated with β-propiolactone were also incubated at an increased pH (~12).The modified serine, threonine and tyrosine residues were completely hydrolysed, indicating that they were acylated by β-propiolactone instead of alkylated.The hydrolysis of these modified peptides resulted in the reversion to the original peptides.Also treatment of peptide 10 and 12 with 3iodopropionic acid did not result in significant alkylation of the serine and tyrosine residues.In addition a tyrosine derivative (Ac-Tyr-NH 2 ) was treated with β-propiolactone and the reaction was monitored by NMR, confirming that tyrosine is acylated by β-propiolactone (NMR: δ 7.32 and 7.1 (AB, 4H), 3.86, t, 2H, 2.63, t, 2H).Based on the results, we conclude that serine, threonine and tyrosine residues are acylated during the reaction with β-propiolactone (proposed structures are given in Table 5).

CONCLUSIONS
β-Propiolactone is extensively used for the production of inactivated viral vaccines against influenza and rabies.Despite decades of large scale use of these vaccines, the chemical modifications which occur during viral inactivation were only partially known.Here we present a detailed and systematic overview of β-propiolactone modifications on the building blocks of nucleic acids and proteins: nucleobase analogues, nucleosides, and amino acid residues.The nucleosides deoxyadenine, cytosine, and deoxyguanine were modified by β-propiolactone, alkylating on positions N-1 of deoxyadenine, on exocyclic amino group of cytosine and on position N-7 of deoxyguanine.Furthermore, nine amino acid residues were shown to react with β-propiolactone (Table 3).In many cases the amino acid residues were alkylated, but serine, threonine and tyrosine residues were exclusively acylated.Cysteine and lysine residues were partially acylated and alkylated.Unexpectedly, it was shown that disulphide groups in cystine residues do not react with β-propiolactone.On the other hand highest conversions were observed for cysteine and methionine residues.Less − but significant − conversions were determined for the nucleosides deoxyadenosine, cytidine and deoxyguanosine.The results indicate that proteins are more extensively modified by than nucleic acids during inactivation of viruses with β-propiolactone.The amount and nature of modifications in viral components, i.e., proteins, DNA or RNA, will be dependent on concentration of β-propiolactone, type of buffer, pH of the mixture, and intrinsic reactivity of individual nucleophilic groups.Conditions for inactivation should be optimised to result in a complete inactivation and a sustained high immunogenicity of the virus.In this study the relative reactivity of nucleosides and amino acid residues for β-propiolactone was elucidated.The data can be utilised to predict or to elucidate through LC/MS or NMR studies the chemical modifications occurring in viral components during the inactivation of viruses with βpropiolactone.

1 )
Conversion is the sum of all products compared to the residual amount of the nucleobase analogue or nucleoside.

Figure 8 .
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.MS 2 spectra I -IV revealed the peptide sequence with a truncated cysteine side chain (-34 Da).Fragmentation during MS 2 analysis resulted in a neutral loss fragment of 106 Da (C 3 H 6 O 2 S) or 178 Da (C 6 H 10 O 4 S), if one or two β-propiolactone molecules were attached to the cysteine residue respectively.

Figure 9 .
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.MS 2 analyses revealed (I) the peptide sequence and the attachment of β-propiolactone (+72 Da).(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 .
Figure10.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.MS 2 analyses revealed (I) the peptide sequence of both products and the β-propiolactone modification of lysine residue.Product I is probably peptide 6 with an alkylated lysine residue and product II with an acylated lysine residue.

Figure 11 .
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 MS 2 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) MS 2 spectrum and (Ib) multistage fragmentation spectrum.

Hydrolysis of β-propiolactone-modified residues.
(Breda, The Netherlands).The reaction was started by adding 1 μl β-propiolactone to 990 µl of a buffer.The 2 O, 40 μl of 1.0 M potassium phosphate pH 9.3.The pH of the samples was adjusted to 9.3 using sodium hydroxide of 1.0 M. (The high pH was chosen to increase the amount of reaction products.)The reaction was started by adding 2 μl of β-propiolactone.After mixing, the solution was incubated for 16 h at 22 °C.Samples were stored at 4°C before analysis by NMR.Reactions of β-propiolactone with amino acid derivatives.The amino acid analogues were dissolved in D 2 O to final concentrations of 20 mM.A reaction mixture was prepared by successively adding 1500 μl of an amino acid solution, 460 μl D 2 O, 40 μl of 1.0 M potassium phosphate pH 9.0 and 2 μl of βpropiolactone.After the addition of potassium phosphate and β-propiolactone, the solution was homogenised by gentle mixing.A sample of 500 μl was immediately taken and mixed 200 μl of TMSP. 1 H-NMR spectra were recorded at 735 second intervals during 6-15 hours.

Table 2
1H, and 8.78, dd, 1H, and two aliphatic of CH 2 groups at 2.79 and 4.39).No conversion was observed of the primary amino group in 2-aminopyrimidine.

Table 2
(23,51)pected from reactions with nucleobase analogues, deoxythymidine did not react with β-propiolactone.If compared to other studies(23,51), the reactions were preformed with relatively low concentrations of β-propiolactone (16 mM) which might also explain the little number of modifications observed.The concentration of βpropiolactone was comparable with the concentrations normally used for viral inactivation.

Table 1 .
Reaction rate constants and half-life of β-propiolactone in different buffer systems

Table 2 .
Products of nucleobase analogues and nucleosides obtained by the reaction with β-propiolactone