sNHS-Mediated Bioconjugation

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Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates Kyle Totaro, Xiaoli Liao, Keshab Bhattacharya, Jari I. Finneman, Justin B. Sperry, Mark A Massa, Jennifer Thorn, Sa V Ho, and Bradley Lether Pentelute Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00043 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016

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Bioconjugate Chemistry

Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates Kyle A. Totaro,†, Xiaoli Liao,†, Keshab Bhattacharya,‡ Jari I. Finneman,‡ Justin B. Sperry,‡ Mark A. Massa,‡ Jennifer Thorn,‡ Sa V. Ho,‡‡ and Bradley L. Pentelute*,† †

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 ‡

Pfizer Worldwide Research and Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017

‡‡

Pfizer Worldwide Research and Development, 1 Burtt Road, Andover, MA 01810

KEYWORDS: EDC, sNHS, Bioconjugation ABSTRACT: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet to be reported side-products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show the trends established with model peptides hold for more complex systems.

INTRODUCTION Bioconjugations are a driving force behind discoveries in the life sciences, through the development of new medicines, drug targets, and diagnostics. One significant impact of bioconjugation chemistry is in the development of antibody-drug conjugates.1,2 This approach provided targeted therapies to be accessible by linking antibodies to cytotoxic drugs.1,3–5 Conjugate vaccines are also an equally important development in therapeutics prepared by bioconjugation.6,7 These antigencarrier protein molecules have been utilized in the prevention of Streptococcus pneumonia infections. Prevnar and Prevnar 13, which are currently marketed by Pfizer, are conjugate vaccines that have been prepared using this methodology.8,9 In bioconjugation reactions, robust chemistry is needed to form conjugates efficiently.10 However, little attention has focused on identifying and minimizing side products from the reaction. This is particularly true for carbodiimide bioconjugations using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sNHS). EDC and sNHS are often used for water-soluble conjugates.11 These reagents were developed to react in aqueous environments that biomolecules are soluble and can maintain their native tertiary structure.12–14 Unfortunately, the reaction efficiency is hampered for peptide and protein bioconjugates because aspartate or glutamate residues are stochastically labeled.15,16 In order to comprehend these promiscuous reactions, a detailed investigation is needed. We believe to fully explore EDC/sNHS bioconjugations, it would be beneficial to analyze the reaction profiles of: 1) the bioconjugation reagents alone, 2) the amine

nucleophile in the presence of the reagents, 3) the bioconjugation using a simple carboxylate containing peptide substrate, and 4) the bioconjugation using a complex peptide substrate. There have been few reports17 to pinpoint the side reactions of EDC bioconjugations (Figure 1). One side product that forms is “EDC-urea” (Figure 1, A). This material is also formed by hydrolysis under both basic and acidic pHs.16,18,19 Side reactions also take place when the carboxylate forms the Oacylisourea intermediate with EDC. From here, an additional molecule of carboxylate can react forming an anhydride (Figure 1, B).16,20 The O-acylisourea can also rearrange to yield a N-acylisourea (Figure 1, C) in large excesses of EDC.16 As for the activating reagent, no information exists wherein sNHS side reactions have been evidenced. However, there is only one report in which N-hydroxysuccinimide (NHS) participates in a side reaction with a carbodiimide.21 First, a carbodiimideNHS adduct will form, which sufficiently activates the carbonyls for nucleophilic attack. The material then undergoes a Lossen rearrangement to form an isocyanate and subsequently reacts with another nucleophile to yield a β-alanine side product (Figure 1, D).21–25 Although it has not yet been proven, it can be anticipated that this side product can form for sNHS due to its similar structure and reactivity in comparison to NHS.11,13,14,26

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knowledge, no systematic investigation exists on how the reagents, substrates, or conditions impact carboxylated peptide EDC and sNHS bioconjugations. This is likely due to the complexity of the substrates typically used in bioconjugations. Here, we present our efforts toward characterizing EDC/sNHS-mediated bioconjugation reactions with peptide substrates. Our studies commenced by investigating the reaction without peptide to isolate and characterize small molecule side products. We then investigated the reaction in the presence of an amine and subsequently with peptide and small protein. RESULTS AND DISCUSSION

Figure 1. EDC and NHS undergo a number of side-reactions without peptide or protein present.11,16,21 Reaction profiles have been reported for the peptide substrates in bioconjugations. Amino acids that interfere with EDC-mediated bioconjugations include tyrosine,27 histidine,28 and cysteine29 (Figure 2). Tyrosine peptides react with EDC to form a tyrosine-EDC isourea side product (Figure 2a).27 This side-adduct can be reversed by treatment with a strong nucleophile. Histidine peptides were found to cyclize with carboxylates under bioconjugation conditions (Figure 2b).28 This also occurs with lysine and other nucleophiles present in the peptide. Cysteine residues were revealed to form stable adducts with EDC (Figure 2c).29 Unfortunately, adduct removal even with strong nucleophiles is troublesome.

Figure 2. EDC and sNHS forms side products with the sidechains of certain amino acids: a) tyrosine, b) histidine, and c) cysteine.11,27–29

Investigation of Bioconjugation Reagents in the Absence of Amine and Peptide. We began by examining how the bioconjugation reagents react without peptide or amine nucleophile. In addition to side reactions explained earlier, we expected EDC and sNHS to react to form a covalent adduct, which then degrades into additional side products. To investigate, reaction conditions were designed to reflect typical bioconjugation environments. Therefore, a solution of 8.75 mM sNHS and 63.1 mM EDC in 50 mM of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH = 7.0-7.2 (“Ratio A” conditions) was used. The reaction was monitored over the course of 16 hours by HPLC. Using authentic standards and comparing the UV spectrum at 210 nm (Figure S1), we were able to identify peaks corresponding to the starting materials, but additional products were observed. Although the intensity of the observed peaks change over time, the spectrum after 2 hours illustrated all products formed throughout the course of the reaction (Figure 3). After analysis by LC-MS, the products were characterized and the results are summarized in Table 1. To confirm the identities of these structures, synthetic standards were independently prepared and characterized by LC-MS (Figure S1).

Figure 3. HPLC profile of EDC/sNHS reagent mixture after 2h reaction (UV spectrum at 210 nm displayed). HPLC conditions (Method A): ACE C18-AR Ultra-Inert HPLC Column: 4.6 x 150 mm, 3 μm, Gradient: 0-8 min 2% B, 8-10 min 2-95% B, 10-15 min 95% B, 15-20 min 2% B, flow rate: 1 mL/min, solvent A = water + 0.1% trifluoroacetic acid, solvent B = acetonitrile + 0.1% trifluoroacetic acid.

Although investigations have determined various side products, we hypothesized a few had yet to be reported. To our


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Table 1. Identified side products formed from EDC/sNHS reagent mixture. [M+H]+

[M+H]+

Retention Time

Calculated

Found

(min.)

sNHS

195.992

195.992

1.55

2

EDCBisulfite

238.123

238.125

3.44

3

EDC

156.150

156.150

3.65

4

EDC-Urea

174.161

174.161

4.74

5

EDC-sNHS

351.134

351.133

5.39

Compound

Name

1

Structure

Figure 4. Investigation of Bioconjugation Components that form EDC-Bisulfite: a) LC-MS of EDC-Bisulfite Standard, b) LC-MS of EDC and MOPS reaction mixture, c) LC-MS of EDC and sNHS reaction mixture (total ion current, TIC, is displayed for all spectra shown). Conditions (Method B): ACE C18-AR Ultra-Inert HPLC Column: 4.6 x 150 mm, 3 μm, Gradient: 0-8 min 1% B, 8-10 min 195% B, 10-12 min 95% B, 12-15 min 1% B, flow rate: 0.8 mL/min, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid. The identified side-products are formed by simple mechanisms. EDC-Urea (4) and EDC-sNHS (5) are products formed by nucleophilic addition of either water or sNHS to EDC, re-

spectively. However, it is not evident how the EDC-Bisulfite (2) side product is produced. We hypothesize sNHS is an available source of bisulfite, since thioether bonds formed



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from maleimide have been known to undergo retro addition. sNHS was ruled out based on kinetic data. A sodium bisulfite To support this hypothesis, MOPS and sNHS were reacted spiking experiment indicated that EDC reacts with bisulfite separately with EDC to characterize EDC-Bisulfite formation ion within minutes. Although we were unable to detect Nby LC-MS (Figure 4). To validate our observations, an EDChydroxymaleimide in the reaction mixture using our methods Bisulfite standard was prepared by reacting EDC and sodium (as determined by HPLC of the authentic material), these bisulfite in water at pH = 6.8 (Figure 4a). As expected, EDCresults suggest that bisulfite ion is released slowly during the Bisulfite was only detected in the reaction between sNHS and conjugation reaction followed by fast reaction with EDC. EDC (Figure 4c). The possibility of bisulfite contamination in Table 2. Identified side products from EDC/sNHS/2-(2-pyridyl)ethylamine reagent mixture. [M+H]+

[M+H]+

Calculated

Found

Retention Time (min.)

sNHS

195.992

195.992

2.17

2

EDC-Bisulfite

238.123

238.125

3.12

4

EDC-Urea

174.161

174.161

4.39

6

MOPS

210.080

210.081

2.56

7

2-(2-pyridyl)ethylamine

123.093

123.092

2.32

8

Sulfo-β-Alanine Monoamine

274.086

274.089

2.45

9

EDC-Amine

278.235

278.236

3.24

10

Sulfo-β-Alanine Diamine

422.150

422.154

5.741 and 6.081

Compound

Name

1

Structure

1

Two resolved peaks are observed for this product with identical masses, which have been determined to be a mixture of isomers

Investigation of Bioconjugation Reagents in the Presence of 2-(2-pyridyl)ethylamine without Peptide. After establishing the origins of side products in the mixture, we investigated the reaction profile in the presence of a nucleophile. In this instance, 2-(2-pyridyl)ethylamine was selected. As with sNHS and EDC, reactions were performed at the same concentrations and pH, including the addition of 68.5 mM of 2-(2pyridyl)ethylamine. LC-MS spectra obtained after reacting for 20 hours revealed that additional products were produced (Figure 5). By utilizing the mass spectra of the detected materials, identities were assigned (Table 2). Our observations show a more complex mixture of products. One easily identified product was adduct formation from addition of 2-(2pyridyl)ethylamine to EDC, which we designated “EDCAmine” (9). Furthermore, side products were detected that contain a sulfo-β-alanine moiety with either one or two molecules of 2-(2-pyridyle)ethylamine attached (8 and 10). As illustrated earlier in Figure 1, this type of product arises from N-hydroxysuccinimide moiety reacting with excess EDC, followed by a Lossen rearrangement.21–24 Although unexpected, the formation of 8 can be explained by decarboxylation of the Lossen rearrangement isocyanate intermediate. 10 is formed by nucleophilic addition of another 2-(2-pyridyl)ethylamine

molecule to the isocyanate. In both cases, this is the first time that these side products have been illustrated for sNHS bioconjugation.

Figure 5. HPLC profile of reagent mixture with the addition of 2-(2-pyridyl)ethylamine (total ion current, TIC, is displayed). LC-MS Conditions (Method B): ACE C18-AR Ultra-Inert HPLC Column: 4.6 x 150 mm, 3 μm, Gradient: 0-8 min 1% B, 8-10 min 1-95% B, 10-12 min 95% B, 12-15 min 1% B, flow rate: 0.8 mL/min, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid.


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Investigation of Bioconjugation Reagents in the Presence of 2-(2-pyridyl)ethylamine and Peptide. We next studied the reactions in the presence of various model peptides. Using flow based Fmoc-SPPS chemistry,30 we designed and synthesized model peptides with the sequence of Ac-Xaa-Leu-GlyGlu-Gly-Leu-CONH2, where the N-terminus and C-terminus were capped with an acetyl and amide group, respectively (Figure 6). Gly and Leu residues were incorporated in the model sequence to provide flexibility and hydrophobicity for the peptide, facilitating LC-MS analysis. For the “Xaa” residue, 12 amino acids were selected to represent diverse functional groups of reactive amino acids. Ala and Trp were chosen as amino acids represented aliphatic and indole side chains; Met as oxidation-sensitive hydrophobic side chains; Lys, Arg, and His as positively charged nucleophilic side chains that could react with the Glu residue when activated; Asp and Glu as groups capable of activation; Asn, Tyr, and Ser as polar uncharged side chains; Cys as a nucleophilic thiol that could also react with activated Glu. The EDC/sNHS-mediated conjugation of 2-(2-pyridyl)ethylamine to model peptides were performed using the following conditions: reaction buffer containing 50 mM MOPS and 50 mM NaCl, 63.1 mM EDC, 8.75 mM sNHS, 68.5 mM 2-(2-pyridyl)ethylamine, and 0.171 mM model peptide, with a final pH = 7.1 (Figure 7). Acid quench conditions were also determined using 3 times the total reaction volume of H2O with 0.5% trifluoroacetic acid (TFA), which halted the reaction without interfering side-products. The quenched reactions at different time points were then characterized by LC-MS. To determine the amount of product and all side products, the relative yields of each reaction were calculated based on the area of peptide species from integrated total ion current (TIC) spectra.

Figure 6. Structure of the model peptide. “X” indicates residue that was varied for the bioconjugation experiments. Most model peptides investigated furnished a glutamic acid labeled product, but also contained side products (Table 3 and Figures S2-S13). Alanine, serine, and asparagine model peptides showed clean and efficient reactions without detectable side products (Table 3). Glutamate and aspartate peptides reacted efficiently having significant amount of dual labeled products (Figure 8a-b). Fast EDC addition to the hydroxyl group was observed with the tyrosine model peptide, resulting in both starting peptide and product forming tyrosine-EDC adducts (Figure 8c). Lysine and arginine peptides showed 15% and 8% cyclized side products after 20 hours (Figure 8d, f). Alternatively, the histidine peptide showed almost equal amount of cyclized side product and desired product, along with a small degree of an unknown isomeric product (Figure 8e). For tryptophan and methionine peptides, a small amount of oxidation side product was observed (Figure 8g, h). As for the cysteine peptide, thiol addition to EDC was faster than EDC addition to tyrosine, completely reacting within half an hour (Figure S13). Additionally, the isothiourea adduct slowly underwent conversion to dehydroalanine (Fig-

ure 8i). It is noteworthy that the side products identified here have yet-to-be reported, with exception for tyrosine, histidine, and the cysteine-EDC adduct.27–29 Figure 7. Conditions for EDC/sNHS-mediated bioconjugation of 2-(2-pyridyl)ethylamine to model peptides.

Table 3. Relative yields of model peptide after 20h bioconjugation. Model Peptide (X = )

Relative Product Yield (%)

Relative Side Product Yield (%)

Serine

100

0

Asparagine

97

0

Alanine

91

0

Methionine

83

15

Tryptophan

83

2

Arginine

82

8

Lysine

68

15

Histidine

47

44

Aspartate

18

82

1

Glutamate

11

86

1

Tyrosine

2

97

2

Cysteine

0

100

3

Relative yields are calculated from the LC-MS integrated total ion current (TIC) spectrum (see Materials and Methods). Remaining percentage not reported is unreacted starting mate1 rial. Side products identified are materials in which the se2 cond carboxylic acid is also labeled. Side product is a mixture of 80% tyrosine-EDC adducts with appropriately labeled glutamate, and 17% tyrosine-EDC adduct only (see Figure S12). 3 Side product yield contains 48% of conjugation product, but with a cysteine-EDC adduct.



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Figure 8. Side products identified during model peptide bioconjugation, where X = A) aspartic acid, B) glutamic acid, C) tyrosine, D) lysine, E) histidine, F) arginine, G) methionine, H) tryptophan, I) cysteine. In terms of reaction kinetics, the model peptides displayed different labeling rates (Figure 9). Model peptides that contained single labeling sites generated appropriately conjugated product in the following order: Ser ~ Trp ~ Met ~ Asn > Ala ~ Arg > Lys > His. The slower reactions on Lys and His peptide resulted from the competing cyclization side reaction. For the Tyr and Cys peptide, almost no desired product formed due to EDC addition, leaving the EDC adducts as the major product. For the Asp and Glu peptides that contain two labeling sites, interesting trends for labeling were observed (Figures S5-S6). Overall, the Glu residue was more reactive than the Asp residue, as evidenced by the faster dual labeling of Glu peptide than that of Asp residue. Interestingly, the Asp peptide had its respective Glu residue labeled first, in comparison to the Glu model peptide, which had its N-terminal Glu residue labeled first. This indicates that residue identity and position affects labeling kinetics.

Figure 9. Kinetics of bioconjugation product formation for model peptides tested. “Relative Yield” refers to yields calculated from the LC-MS integrated TIC spectrum (see Materials and Methods). Curves written as “2x Label” refer to amide bond formation at both the desired glutamate and amino acid side chain. “EDC-Adduct” curves refer to bioconjugations products in which the amino acid forms either an isourea (threonine) or isothiourea (cysteine). A) Bioconjugations of model peptides where X = Serine, tryptophan, asparagine, and alanine. B) Bioconjugations of model peptides where X = Methionine, aspartate, glutamate, and tyrosine. C) Bioconjugations of model peptides where X = Arginine, lysine, histidine, and cysteine. Impact of Reagent Concentration and Stoichiometry on Peptide Labeling. First, we aimed to understand the effect of EDC/sNHS/2-(2-pyridyl)ethylamine concentrations on the labeling kinetics and side product formation. To compare among different concentrations, the labeling reactions were performed on 0.171 mM of alanine or serine model peptides using either the conditions used in previous experiments (Ratio A): 63.1 mM EDC, 8.75 mM sNHS, 68.5 mM 2-(2pyridyl)ethylamine, or conditions using near-equivalent concentrations of each reagent (Ratio B): 47 mM EDC, 37.3 mM sNHS, 45 mM 2-(2-pyridyl)ethylamine. The major difference between these two ratios was the amount of sNHS, where


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Ratio B utilizes a larger excess in comparison to Ratio A. With Ratio A as the starting point, the ratio or concentration of EDC/sNHS/2-(2-pyridyl)ethylamine were altered and monitored by LC-MS to survey the impact on labeling. Between the two conditions tested, Ratio A provided higher yields and faster labeling than Ratio B when the alanine model peptide was used (Figure 10). A lower sNHS concentration in comparison to EDC, as in Ratio A, led to slower consumption of EDC, which resulted in higher labeling rate and yield. This observation is consistent with work previously published.14 To further confirm these findings, excess EDC was added to the Ratio B reaction, which slightly increased the yield. Alternatively, adding additional sNHS to the Ratio A reaction decreased the rate. Following these experiments, we determined how critical each reagent was for the reaction to progress. Using the tyrosine model peptide, we observed that complete removal of sNHS from the reaction led to slow conversion of EDC adduct on glutamate into the desired product. This indicates that sNHS promotes the activation of the carboxylic acid and addition of amine (Figure S14). Lowering the Ratio A concentration of 2-(2-pyridyl)ethylamine to one-fifth of the original amount (13.7 mM) caused the reaction to slow and yield more side product. When reducing both EDC and sNHS concentrations to one-fifth (12.6 mM and 1.75 mM, respectively) the reaction was even slower, but side product formations were similar to the Ratio A (Figure S15).

Next, we studied how the fold excess of EDC, sNHS, and 2(2-pyridyl)ethylamine impacts reaction kinetics and yield. The serine model peptide was selected for these experiments to accurately determine overall yield without side product interference. Thus, fixed concentrations of EDC (63.1 mM), sNHS (8.75 mM), and 2-(2-pyridyl)ethylamine (68.5 mM) were maintained, while changing the concentration of the peptide from 0.1x to 100x the amount used previously (0.017-17.1 mM). For example, the fold excess of amine to peptide changed from 4000 times to 4 times. We observed very similar labeling kinetics in all four reactions (Figure 11a), even when the fold excess had been drastically changed by 1000 times. This result indicates a pseudo-zero-order reaction on the peptide concentration when using a fixed concentration of the reagents in excess.

Figure 11. Impact of concentration on bioconjugation kinetics for serine model peptide. A) Adjusting concentration of peptide starting material (0.1x = 0.017 mM, 1x = 0.171 mM, 10x = 1.71 mM, 100x = 17.1 mM). B) Adjusting concentration of peptide and reagents collectively (based on Ratio A concentrations).

Figure 10. Impact of A) Ratio A and B) Ratio B concentration on reaction kinetics and yield using alanine model peptide.

In a similar set of experiments, we wanted to determine the effect of molarity on the reaction. Ratio A concentrations with 1.71 mM peptide was selected as the starting point as 1.0x. The relative proportions among the three reagents and peptide were held constant while adjusting the overall scale of the reaction. Experiments were performed using 0.1x, 0.3x, 1.0x or 3.0x ratio A concentrations, which is 0.171 mM, 0.513 mM, 1.71 mM, or 5.13 mM peptides, respectively. We observed that the reaction was nearly complete in 7 hours for the 3.0x reaction, while the 1.0x, 0.3x, and 0.1x ratio A reactions yielded about 94%, 75%, and 54% of product at 18 hours, respectively (Figure 11b). We conclude, as expected,



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the rate of bioconjugation was influenced by the molarity of the reaction, but not the fold excess of reagents in relation to the model peptide. During these experiments, we have also determined the effect of reagent and peptide concentration on the formation of small molecule side products. First, the concentration of peptide and its effect on side product formation was investigated. Ratio A concentrations were used for the reagents (63.1 mM EDC, 8.75 mM sNHS, 68.5 mM 2-(2pyridyl)ethylamine), then used either 0.171 mM or 1.71 mM of serine model peptide. Both reactions revealed very similar side product profiles in comparison to prior experiments excluding model peptide. The profiles of the side reactions in the reagent mixture were correlated with peptide labeling results shown in Figure 5. Then, as in the molarity experiments, the relative ratios among these three species were retained while adjusting the molarity of all species (0.1x, 0.3x, 1.0x, or 3.0x of Ratio A concentrations). The results indicate that the concentration of reagents affected the formation of EDC-amine and sulfo-β-alanine diamine, but had little influence on the formation of EDC-bisulfite (Figure S16). Model Protein Labeling. Based on the information obtained from our model peptide studies, we analyzed labeling on a small protein to determine if similar reaction profiles would be observed in addition to trends in side product formation. We elected to work with ZHER2 affibody derived from the immunoglobulin binding protein A (Figure 12) as our model protein. This 63-amino-acid protein contains ten labeling sites (four Glu, five Asp and C terminus), and ten sites bearing potential side reaction residues (one Arg, six Lys, the N terminus, one His, and one Tyr). The affibody serves as a simple model protein for studying and confirming the side reactions and kinetics observed on the model peptides. To confirm labeling sites, trypsin or chymotrypsin was used to digest the labeled affibody for analysis by LC-MS/MS peptide mapping.

Figure 12. A) Structure and B) sequence of model affibody protein. Aspartate and glutamate residues are highlighted in red.

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In these experiments, Ratio A conditions were used to label the affibody protein and the reaction was monitored at different time points (1, 3, 6, 18 hours) by LC-MS. After acquiring spectra, the deconvoluted masses of the protein were utilized to determine the number of labeling sites and side reactions (Figure 13 and S17). The major products observed for each time points were as following: at 1 hour, 1-2 bioconjugations, with EDC adducts and cyclized products; at 3 hours, 2-3 bioconjugations with EDC adducts and cyclization; at 6 hours, 2-3 or 4-7 bioconjugations with EDC adducts and one or two cyclization products; at 18 hours, 3-10 bioconjugations together with EDC adducts and one sNHS-ester moiety (Table 4). Although we were able to characterize the side reactions occurring on our model protein, it proved difficult assigning them to particular residues. This was largely due to the complexity of EDC adducts that formed during the reaction. However, we were able to identify that EDC labeled one tyrosine residue located at Tyr19. Figure 13. Deconvoluted MS of affibody labeling by 2-(2pyridyl)ethylamine after an 18h bioconjugation reaction.

Table 4. Products identified by MS from affibody labeling by 2-(2-pyridyl)ethylamine. Letter

Mass

Product

A

7357.34

3x Label + EDC

B

7461.52

4x Label + EDC

C

7565.91

5x Label + EDC

D

7687.54

6x Label + EDC

E

7761.06

5x Label + sNHS + EDC

F

7882.96

8x Label + EDC

G

7986.93

9x Label + EDC

H

8090.32

10x Label + EDC

To evaluate the importance of the fold excess of EDC/sNHS/2-(2-pyridyl)ethylamine, the concentration of affibody was varied between 0.034 mM, 0.171 mM, and 1.25 mM while EDC/sNHS/2-(2-pyridyl)ethylamine concentrations were kept constant. Consistent with the model peptide study (Figure 11a), similar kinetics were observed on all three reactions, indicating that the amount of affibody present did not affect the kinetics. However, when the EDC/sNHS/2-(2pyridyl)ethylamine concentration was lowered to one-fifth of the Ratio A concentrations (12.6 mM EDC, 1.75 mM sNHS, and 13.7 mM 2-(2-pyridyl)ethylamine), the number of labeling sites drastically decreased. This observation is also consistent with the model peptide study where EDC/sNHS/2-(2pyridyl)ethylamine molarities were directly correlated with the labeling rates (Figure 11b).


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To further identify the labeling sites on the affibody, the protein was buffer-exchanged to a buffer containing 50 mM NaHCO3 or 100 mM Tris (for optimal digest conditions), and 10 mM CaCl2 (to prevent autolysis) then digested with trypsin or chymotrypsin, respectively, overnight at 37 °C. Based on the LC-MS/MS analysis of the digested fragments, we identified labeling sites and side products, which were similar to observations made in the model peptide study. We were also able to identify “hot spots” that were labeled before other sites, such as Asp41, Asp58, Glu52, and Glu13 (Figure S17). However, due to the complication of EDC adducts, it was hard to identify the cyclized peaks (Figure S18). Figure 14. Deconvoluted MS of affibody labeling by 2-(2pyridyl)ethylamine after an 18h bioconjugation reaction and subsequent hydroxylamine treatment.

Table 5. Products identified by MS from hydroxylamine treatment of affibody labeled by 2-(2-pyridyl)ethylamine.

CONCLUSION In conclusion, we have evaluated EDC/sNHS-mediated bioconjugations and identified components that interfere with product formation. The following results have provided validation and insight toward better understanding the reaction profile of EDC/sNHS bioconjugations: •

The bioconjugation reagents themselves react to yield EDC-sNHS and sulfo-β-alanine side products, which had yet to be identified

•

Neighboring nucleophilic residues (lysine, arginine, histidine, and cysteine) negatively affect peptide bioconjugation outcome

•

New side products were identified for methionine (oxidation), tryptophan (oxidation), and cysteine (βelimination) in model peptide bioconjugations

•

No additional side products were characterized in ZHER2 affibody protein bioconjugations that were not identified from model peptide studies.

Characterizing the intricacies of bioconjugation reactions offers information to address limitations in complex systems. Additionally, the data presented can serve as a foundation in identifying and predicting labeling trends in the preparation of protein bioconjugates.

Letter

Mass

Product

MATERIALS AND METHODS

A

7202.11

3x Label

B

7306.28

4x Label

C

7410.55

5x Label

D

7532.43

6x Label

E

7623.96

5x Label + sNHS – H2O

F

7727.99

8x Label

G

7831.94

9x Label

H

7935.56

10x Label

Materials. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), Fmoc-Gly-OH, FmocLeu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(S-Trt)OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH, FmocAsp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, FmocHis(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(OtBu)-OH, and Fmoc-Tyr(OtBu)-OH were purchased from Chem-Impex International (Wood Dale, IL). 4-methylbenzhydrylamine (MBHA) resin was obtained from Anaspec (Fremont, CA). Sulfo-NHS (sNHS), N,N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, and HPLC-grade acetonitrile were obtained from VWR International (Philadelphia, PA). All other reagents were purchased from Sigma-Aldrich and used as received unless otherwise noted.

Finally, we investigated chemical methods to remove the EDC adducts on tyrosine. The labeled affibody was bufferexchanged to 50 mM NaHCO3 and was subsequently treated with 0.15 M phosphate buffer pH 7, 0.5 M Tris pH 7.5, 0.5 M Glycine pH 7, 0.1 M hydroxylamine, or 0.5 M hydroxylamine. Consistent with literature,27 we found that hydroxylamine effectively removed the EDC adducts without affecting the desired bioconjugations (Figure 14 and Table 5). Furthermore, we were able to identify cyclized side products using the same trypsin/chymotrypsin digestion protocol and LC-MS/MS method. We identified cyclization of Lys63 with either the Cterminus or Asp58 after 20-hours, and cyclization of Lys40 with Asp41 or Asp42 after 3-hours. For His23, we found cyclization with Glu20 as the major product while in the same peptide fragment Glu29 and Glu30 were minor components. Only minor amounts of cyclization were observed at His23 and Glu29 or Glu30, confirming proximity driven cyclization (Figure S19).

HPLC and LC-MS Analysis. HPLC chromatograms were acquired using an Agilent 1200 Series HPLC. LC-MS chromatograms and associated mass spectra were acquired using an Agilent 1260 Infinity Quaternary HPLC coupled to an Agilent 6520 ESI-Q- TOF mass spectrometer. The following LC methods were used: Method A (HPLC): ACE C18-AR Ultra-Inert HPLC Column: 4.6 x 150 mm, 3 μm, Gradient: 0-8 min 2% B, 8-10 min 2-95% B, 1015 min 95% B, 15-20 min 2% B, flow rate: 1 mL/min, solvent A = water + 0.1% trifluoroacetic acid, solvent B = acetonitrile + 0.1% trifluoroacetic acid, UV detection at 210 nm. Method B (LC-MS): ACE C18-AR Ultra-Inert HPLC Column: 4.6 x 150 mm, 3 μm, Gradient: 0-8 min 1% B, 8-10 min 1-95% B, 10-12 min 95% B, 12-15 min 1% B, flow rate: 0.8 mL/min, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid.



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Method C (LC-MS): Agilent Zorbax 300SB C3 HPLC Column: 2.1 x 150 mm, 5 μm, Gradient: 0-2 min 1% B, 2-11 min 1-61% B, flow rate: 0.8 mL/min, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid. Method D (LC-MS): Agilent Zorbax 300SB C3 HPLC Column: 2.1 x 150 mm, 5 μm, Gradient: 0-2 min 5% B, 2-11 min 5-65% B, flow rate: 0.8 mL/min, solvent A = water + 0.1% formic acid, solvent B = acetonitrile + 0.1% formic acid. Samples from reagent mixture studies were analyzed using Method A and Method B, and samples from model bioconjugation studies were analyzed using Method C. All reported relative yields were determined by measuring total ion currents using LC-MS data via the Agilent MassHunter software package. First, peak areas were integrated for all relevant peptidic species on the chromatogram. Relative yields were calculated using following equation: % yield = Spro/Sall where Spro is the peak area of the desired product and Sall is the sum of all TIC peaks. The Y-axis in all chromatograms shown in supplementary figures represents total ion current (TIC); mass spectrum insets correspond to the maxima point of the TIC peak. For LC-MS/MS experiments, the following equation was used to calculate the collision energy setting for MS/MS: Slope * (m/z)/100 + Offset, where Slope = 3 and Offset = 2. The compound list shown in the supporting information was generated using the Agilent MassHunter software package. Identified compounds were manually matched to the mass of possible peptide sequences generated using Microsoft Excel. Synthesis of EDC-Bisulfite (2). First, a solution was prepared of 500 mg of sodium bisulfite dissolved in 1 mL of water and 3 mL of 1 M NaOH, leading to a final pH = 6.8. Next, 864 mg of EDC (4.51 mmol) was placed in a glass vial, then 3.75 mL of the sodium bisulfite solution (4.51 mmol) and 4.89 mL of water was added. The resulting mixture reacted at room temperature for 4 hours. The solution was frozen in liquid nitrogen and lyophilized to a white powder (1.421 g, 92% yield). The material was determined to be 91% pure by HPLC. Synthesis of EDC-Urea (4). 191.7 mg (1 mmol) of EDC was placed in a 15 mL conical centrifuge tube and was dissolved in 2 mL of water. The solution was allowed to stand overnight at room temperature before freezing in liquid nitrogen and lyophilizing to a white powder (quantitative yield). In situ Synthesis of EDC-sNHS (5). 215 mg of sNHS (0.99 mmol) is first dissolved in 3 mL of DMSO at room temperature. Stirring is required for approximately 5 minutes to provide a clear solution. Once sNHS has been fully dissolved, 190 mg of EDC (0.99 mmol) is added and subsequently analyzed by HPLC by using Method A. General Procedure for Reagent Mixture Studies. Studies involving the investigation of the bioconjugation reagents were performed on a 1 mL scale. In the initial studies without 2-(2-pyridyl)ethylamine, 100 μL of sNHS stock solution (87.5 mM in water, pH = 7.0-7.2) and 100 μL of EDC stock solution (631 mM in water) were added to a solution of 50 μL of 20X reaction buffer (1M MOPS in water, pH = 7.1) and 750 μL of water (total volume of 1 mL). The pH of the resulting solution was checked and adjusted, if needed, to a final pH between 7.0 and 7.2. The reaction was vortexed and left at room tem-

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perature (25 °C) for 16-20 hours. After the reaction completed, a 150 μL aliquot was withdrawn for analysis by HPLC (0.5 μL injection). For studies incorporating 2-(2pyridyl)ethylamine, the reaction was performed similarly, including 100 μL of 2-(2-pyridyl)ethylamine stock solution (685 mM in water, pH = 7.0) and 650 μL of water (instead of 750 μL) for a total volume of 1 mL. General Procedure for Synthesis of Model Peptides. All peptides were synthesized using published fast-flow coupling/deprotection conditions for Fmoc-SPPS chemistry.30 In summary, synthesis was performed on a 0.1 mmol scale using 4-methylbenzhydrylamine (MBHA) resin functionalized with a Rink amide linker. After synthesis was complete, peptides were simultaneously cleaved from the resin and side-chain deprotected by treatment with a cleavage solution containing TFA/H2O/EDT/TIPS (94/2.5/2.5/1 %v/v) for 2 hours at room temperature, which was evaporated by blowing a stream of nitrogen gas over its surface for 15 minutes. Peptide is then isolated by precipitation and washing with cold diethyl ether. The resulting white solid was dissolved in 1:1 acetonitrile/water containing 0.1% TFA, filtered, and lyophilized to produce a white powder. Peptide Purification. Crude peptide was first dissolved in 99% A (water + 0.1% TFA): 1% B (acetonitrile + 0.1% TFA). 6 M guanidinium hydrochloride was added to the solution if the peptide was not soluble in this mixture. The solubilized crude peptide was purified by semi-preparative RP-HPLC (Agilent Zorbax 300SB C18 column: 9.4 x 250 mm, 5 μm, gradient: 131% B over 80 min, flow rate: 5 mL/min). 1 μL of each HPLC fraction was mixed with 1 μL of alpha-cyano-4hydroxycinnamic acid matrix in 50% water + 0.1% TFA: 50% acetonitrile + 0.1% TFA, and checked for fractions with desired molecular mass by matrix-assisted laser desorption ionization (MALDI). Fractions containing only product material were confirmed by LC-MS analysis (Method C), combined, and then lyophilized (Table S1). General Procedure for Model Bioconjugation Studies. Studies involving the investigation of the bioconjugation reagents were performed on a 200 μL scale. 100 μL of a 2X stock solution mixture of both sNHS (17.5 mM in water) and 2-(2pyridyl)ethylamine (137 mM in water) was added to 33.4 μL of peptide stock solution (1.027 mM in water) in 10 μL of 20X reaction buffer (1M MOPS, 1M NaCl, pH = 7.1) and 31.4 μL of water. 25.2 μL of EDC stock solution (500 mM in water) was added last to initiate the reaction. The pH of the resulting solution was checked and adjusted, if needed, to a final pH = 7.1. The reaction was vortexed and left at room temperature (25 °C) for 20 hours. 10 μL aliquots were withdrawn at various time points and quenched with 30 μL of water + 0.5% TFA prior to analysis by LC-MS (Method C). Synthesis of Model Affibody Protein. Affibody was synthesized in three fragments using Fmoc-SPPS chemistry, ligated, and purified using procedures previously described.30 General Procedure for Affibody Bioconjugation. The bioconjugation reaction using affibody protein was performed on a 50 μL scale. 5 μL of a 10X stock solution of sNHS (87.5 mM in water) and 5 μL of a 10X stock solution of 2-(2pyridyl)ethylamine (685 mM in water) was added to 4.44 μL


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of affibody stock solution (1.926 mM in water) in 2.63 μL of 20X reaction buffer (1M MOPS, 1M NaCl, pH = 7.1) and 28 μL of water. 5 μL of 10X EDC stock solution (631 mM in water) was added last to initiate the reaction. The pH of the resulting solution was checked and adjusted, if needed, to a final pH = 7.1. The reaction was vortexed and left at room temperature (25 °C) for 20 hours. 5 μL aliquots were withdrawn at 1, 3, 6 and 18 hour time points and quenched with 15 μL of water + 0.5% TFA prior to analysis by LC-MS. Trypsin digestion of the affibody was performed by first buffer exchanging an aliquot to 50 mM NaHCO3 buffer, followed by boiling to denature the protein. 4 μL of 0.5 μg/μL trypsin was added to digest overnight at 37 °C. Chymotrypsin digestion was performed by adding 2 μL of 2 μg/μL chymotrypsin to denatured protein in a buffer containing 100 mM Tris pH 8.0, 10 mM CaCl2 for 3.5 hours at 37 °C. After digest, the peptide fragments were analyzed by LC-MS (Method C and Method D).

ASSOCIATED CONTENT Supporting Information. Supporting figures, HPLC, and LC-MS data.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions

K.A.T. and X.L. have contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

REFERENCES (1) Agarwal, P., and Bertozzi, C. R. (2015) Site-Specific Antibody–Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development. Bioconjug. Chem. 26, 176–192. (2) Lambert, J. M. (2015) Antibody–Drug Conjugates (ADCs): Magic Bullets at Last! Mol. Pharm. 12, 1701–1702. (3) Sievers, E. L., and Senter, P. D. (2013) Antibody-Drug Conjugates in Cancer Therapy. Annu. Rev. Med. 64, 15–29. (4) Kovtun, Y. V., and Goldmacher, V. S. (2007) Cell killing by antibodydrug conjugates. Cancer Lett. 255, 232–240. (5) Ducry, L., and Stump, B. (2010) Antibody-drug conjugates: Linking cytotoxic payloads to monoclonal antibodies. Bioconjug. Chem. 21, 5–13. (6) Chu, F. S., Chang, F. C., and Hinsdill, R. D. (1976) Production of antibody against ochratoxin A. Appl. Environ. Microbiol. 31, 831–5. (7) Chu, F. S., and Ueno, I. (1977) Production of antibody against aflatoxin B1. Appl. Environ. Microbiol. 33, 1125–1128. (8) Darkes, M. M., and Plosker, G. (2002) Pneumococcal Conjugate Vaccine (PrevnarTM1; PNCRM7). Pediatr. Drugs 4, 609–630. (9) Gruber, W. C., Scott, D. a, and Emini, E. a. (2012) Development and clinical evaluation of Prevnar 13, a 13-valent pneumocococcal CRM197 conjugate vaccine. Ann. N. Y. Acad. Sci. 1263, 15–26.

(10) Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal chemistry: Fishing for selectivity in a sea of functionality. Angew. Chemie - Int. Ed. (11) Hermanson, G. T. (2013) Chapter 4 - Zero-Length Crosslinkers, in Bioconjugate Techniques (Third edition) (Hermanson, G. T., Ed.), pp 259– 273. Academic Press. (12) Sheehan, J., Cruickshank, P., and Boshart, G. (1961) A Convenient Synthesis of Water-Soluble Carbodiimides. J. Org. Chem. 26, 2525–2528. (13) Staros, J. V. (1982) N-hydroxysulfosuccinimide active esters: bis(Nhydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry 21, 3950– 3955. (14) Staros, J. V, Wright, R. W., and Swingle, D. M. (1986) Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220–222. (15) Yamada, H., Imoto, T., Fujita, K., Okazaki, K., and Motomura, M. (1981) Selective modification of aspartic acid-101 in lysozyme by carbodiimide reaction. Biochemistry 20, 4836–4842. (16) Nakajima, N., and Ikada, Y. (1995) Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjug. Chem. 6, 123–130. (17) Six reports are cited from reference 11. No additional literature sources are found when searching for "EDC-bioconjugation side reactions" via SciFinder. (18) Gilles, M. A., Hudson, A. Q., and Borders, C. L. (1990) Stability of water-soluble carbodiimides in aqueous solution. Anal. Biochem. 184, 244–248. (19) Williams, A., and Ibrahim, I. (1981) A new Mechanism involving cyclic tautomers for the reaction with nucleophiles of the water-soluble peptide coupling reagent 1-ethyl-3-(3’-(dimethylamino) propyl) carbodiimide (EDC). J. Am. Chem. Soc. 103, 7090–7095. (20) Wang, C., Yan, Q., Liu, H.-B., Zhou, X.-H., and Xiao, S.-J. (2011) Different EDC/NHS Activation Mechanisms between PAA and PMAA Brushes and the Following Amidation Reactions. Langmuir 27, 12058– 12068. (21) Gross, H., and Bilk, L. (1968) Zur reaktion von N-Hydroxysuccinimid mit dicyclohexylcarbodiimid. Tetrahedron 24, 6935–6939. (22) Hoare, D. G., Olson, A., and Koshland, D. E. (1968) The reaction of hydroxamic acids with water-soluble carbodiimides. A Lossen rearrangement. J. Am. Chem. Soc. 90, 1638–1643. (23) Bauer, L., and Exner, O. (1974) The Chemistry of Hydroxamic Acids and N-Hydroxyimides. Angew. Chemie Int. Ed. English. (24) Wilchek, M., and Miron, T. (1987) Limitations of Nhydroxysuccinimide esters in affinity chromatography and protein immobilization. Biochemistry 26, 2155–2161. (25) Zalipsky, S. (1998) Alkyl succinimidyl carbonates undergo Lossen rearrangement in basic buffers. Chem. Commun. 69–70. (26) Anjaneyulu, P. S., and Staros, J. V. (1987) Reactions of Nhydroxysulfosuccinimide active esters. Int. J. Pept. Protein Res. 30, 117– 124. (27) Carraway, K. L., and Koshland, D. E. (1968) Reaction of tyrosine residues in proteins with carbodiimide reagents. Biochim. Biophys. Acta Protein Struct. 160, 272–274. (28) Cuatrecasas, P., and Parikh, I. (1972) Adsorbents for affinity chromatography. Use of N-hydroxysuccinimide esters of agarose. Biochemistry 11, 2291–2299. (29) Carraway, K. L., and Triplett, R. B. (1970) Reaction of carbodiimides with protein sulfhydryl groups. Biochim. Biophys. Acta. 200, 564–566. (30) Simon, M. D., Heider, P. L., Adamo, A., Vinogradov, A. A., Mong, S. K., Li, X., Berger, T., Policarpo, R. L., Zhang, C., Zou, Y., et al. (2014) Rapid flow-based peptide synthesis. ChemBioChem 15, 713–720.



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1 2 R" 3 H+ O 4 O C O N 5 O O R" N H+ 6 H N R" N O N 7 R" N R" H Cl H O 8 Lossen β-Alanine (D) 9 Rearrangement 10 11 R" 12 O 13 H 14 Side N O NH N 15 Reaction Cl 16 N O 17 18 O 19 20 21 N OH 22 23 O O O 24 O NHS NH H + 2 R' + 25 R' N N N R N H R OH 26 Cl H H H N N C N Cl 27 EDC Urea (A) Product 28 29 Side 30 Reactions 31 32 33 O HN 34 H NH O HN N 35 N C N R O N Cl NH 36 Cl + R O N + 37 Cl 38 O H 2O 39 40 HO R 41 42 O O O 43 O O H H 44 R N N N N N N Cl H R O R 45 H H Cl 46 47 Urea (A) Anhydride (B) N-acylurea (C) 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57


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sNHS-Mediated Bioconjugation

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