Critical Evaluation and Rate Constants of Chemoselective Ligation

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Critical Evaluation and Rate Constants of Chemoselective Ligation Reactions for Stoichiometric Conjugations in Water Fumito Saito,† Hidetoshi Noda,† and Jeffrey W. Bode*,†,‡ †

Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH-Zürich, 8093 Zürich, Switzerland Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa, Nagoya 464-8602, Japan

‡

S Supporting Information *

ABSTRACT: Chemoselective ligation reactions have contributed immensely to the development of organic synthesis and chemical biology. However, the ligation of stoichiometric amounts of large molecules for applications such as protein−protein conjugates is still challenging. Conjugation reactions need to be fast enough to proceed under dilute conditions and chemoselective in the presence of unprotected functional groups; the starting materials and products must be stable under the reaction conditions. To compare known ligation reactions for their suitability under these conditions, we determined the secondorder rate constants of ligation reactions using peptide substrates with unprotected functional groups. The reaction conditions, the chemoselectivity of the reactions, and the stability of the starting materials and products were carefully evaluated. In some cases, the stability could be improved by modifying the substrate structure. These data obtained under the ligation conditions provide a useful guide to choose an appropriate ligation reaction for synthesis of large molecules by covalent ligation reactions of unprotected substrates in water.

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in this manner include the powerful native chemical ligation for protein synthesis,9 thiol−maleimide conjugations, and thio− ene reactions for forming protein−protein conjugates.10 Both applications of chemoselective ligations are extremely important, but it is essential to clearly separate the parameters of the two classes. For ligation reactions used for detection or labeling, it is acceptable to use a large excess of one reactant. This allows good conversions even if the rates of the ligations are slow or if one of the reaction partners is not completely stable under aqueous conditions. Ligations for synthetic applications, including larger proteins, protein−protein conjugates, antibody−drug conjugates, and the preparation or functionalization of macromolecules have a stricter set of requirements. Such reactions must have very fast rates, stable starting materials, and form covalent bonds that are chemically stable. For some synthetic applications, such as protein synthesis, the formation of natural bonds is required. Most importantly, ligations in the service of synthesis should operate in good yield at close to a 1:1 stoichiometry of the reactants, which are often both precious compounds. As noted by Raines, Dawson, and many others, the working concentrations of solutions of large molecules are dramatically lower than the small molecules typically encountered in classical medicinal chemistry.11,12 For example, while a 1 mg mL−1 solution of a MW 300 drug intermediate has a concentration of 3.3 mM, which is already considered to be

hemoselective ligation reactions have transformed the fields of organic synthesis and chemical biology by allowing specific, covalent bond forming reactions to proceed in the presence of diverse unprotected functional groups. A large number of reviews published over the past 15 years have documented the incredible advances made in both the underlying chemistry of chemoselective ligations as well as their applications for understanding biological processes.1−5 Ideally, chemoselective reactions should operate in the presence of all unprotected functional groups, tolerate an aqueous environment, proceed without reagents or catalysts, and give no or innocuous byproducts. There are two distinct applications of chemoselective ligations: the first is labeling, detection, or capture of a specific molecule containing a unique functional group. Selected examples include expression profiling of proteins containing an unnatural amino acid,6 detection of cell-surface carbohydrates bearing an azide,7 and pull downs of small molecules enzyme inhibitors.8 In such applications, the goal is usually the identification of a specific biomolecule in a soup of other natural materials, and a large excess of one of the reaction partners is typically employed. The second application of ligations is synthesis: the specific construction of large organic molecules where the ability to form specific bonds without reagents or protecting groups dramatically simplifies synthesis by obviating protection and deprotection steps. As many, but not all, chemoselective ligations are also relatively fast reactions, they provide advantages when attempting to couple two large molecules where reaction rates are paramount. Examples of ligations used © XXXX American Chemical Society

Received: August 24, 2014 Accepted: January 9, 2015

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DOI: 10.1021/cb5006728 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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extremely dilute for a typical organic transformation, the same 1 mg mL−1 solution of a 150 kDa antibody is only 6.6 μM. A 3.3 mM solution would require that the antibody be soluble in 2 μL! Such constraints require stoichiometric conjugations of large molecules to operate in the micromolar regime, necessitating second-order rate constants above 1 M−1 s−1 for acceptable conversion and >10 M−1 s−1 for >90% yield. A graphic illustration of the relationship between conversion and second-order rate constants for conjugations at 10 μM is shown (Figure 1).

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RESULTS AND DISCUSSION

Experimental Design. For most ligation reactions reported to date, most kinetic studies have been conducted with small molecules lacking unprotected functional groups commonly found in peptides and proteins. These experimental systems did not always provide comprehensive information on the chemoselectivity of ligation reactions and the stability of starting materials and products. In order to assess those factors and measure rate constants under ligation settings, we designed a peptide substrate containing thiols, carboxylic acids, and amine side chains (Figure 2a). The necessary functional groups for each ligation were introduced at the N-terminus of the peptide. The sole exception was the substrate for thiol− maleimide ligations, for which the sulfhydryl side chain of the cysteine residue was used directly. For the ligation partners, we incorporated an azo dye into a small molecule fragment to facilitate the efficient determination of rate constants (Figure 2b). The azo dye has a strong absorption at 301 nm, whereas peptides 1a, 2, 3, 4, and 5 do not absorb at this wavelength. Provided that the dye moiety is the only contributor to peaks at 301 nm in the UV trace of analytical HPLCs, we can quantify the reaction progress by integrating peak areas. We confirmed the assumption by making calibration curves with 6 and 7a as model compounds. The relationship between concentrations of the samples and integrated peak areas established that the azo dye moiety uniquely absorbed at 301 nm, and the strength of the absorption did not depend on the molecules to which the dye was attached (Supporting Information Figure 11). The Pictet−Spengler ligation was an exception for which we need to prepare calibration curves separately because the indole moiety of the substrate shows absorption at 301 nm. Calibration curves for Pictet−Spengler ligation were prepared in the same way as above using the small molecule fragment 8 (Supporting Information Figure 12). We carried out kinetic studies of 8 ligation reactions using 1:1 stoichiometry of ligation fragments (Table 1). To make the comparisons as fair as possible, the conditions for each ligation reaction, especially in terms of pH, were selected to show the fastest kinetics. We repeated the kinetic experiments at least

Figure 1. Relationship between second-order rate constants and conversion for the coupling of two reactants at 10 μM and 1:1 stoichiometry. Adapted from ref 11. Copyright 2008 American Chemical Society.

In this article, we provide a critical evaluation of 8 ligation reactions under conditions suitable for synthetic applications of precious materials. These criteria include an aqueous environment in the presence of common unprotected functional groups using a 1:1 ratio of starting materials. The reactions are evaluated on the basis of reaction rate, including the determination of second-order rate constants under realworld conditions, as well as the stability of the starting materials and products and absolute chemoselectivity. This information, obtained on identical or similar substrates, will be useful for the selection of chemoselective ligations for constructing large molecules.

Figure 2. Design of substrates. (a) Design of peptide substrates: peptides have the functional groups for ligations at their N-terminus. For thiol− maleimide ligations, the thiol group of the cysteine residue was used for the ligations. (b) Model compounds for calibration curves: dye moieties are attached to small molecule fragments and transferred to peptides after ligations. B


a

Stability of the starting materials and products. bStable in the absence of Cys.

Table 1. Summary of Kinetic Studies

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ACS Chemical Biology Table 2. Screening of Reaction Conditions for CuAAC

observed productsa

a

entry

conditions

dimer of 1a

11a

dimer of 11a

1 2 3b 4 5 6

CuSO4 (1.0 equiv), sodium ascorbate (10 equiv), TBTA (1.0 equiv) CuSO4 (1.0 equiv), sodium ascorbate (25 equiv), TBTA (5.0 equiv) CuBr (1.0 equiv), TBTA (1.0 equiv) CuSO4 (1.0 equiv), ZnBr2 (1.0 equiv), sodium ascorbate (10 equiv), TBTA (1.0 equiv) CuSO4 (1.0 equiv), TCEP (1.0 equiv), TBTA (1.0 equiv) CuSO4 (1.0 equiv), sodium ascorbate (10 equiv), 22 (1.0 equiv)

+ + + + + +

+ + − + + −

+ + − − − −

+, observed; −, not observed. bMeCN was used as an organic solvent due to the solubility of CuBr.

multiple cysteine residues. We therefore screened reaction conditions to circumvent peptide dimerization. Finn et al. showed that tris(triazolylmethyl)amine-type ligands (i.e., including TBTA) could play a sacrificial role and intercept the reactive oxygen species when used in an excess amount.20 However, they could not suppress substrate oxidation (entry 2). The reaction with the Cu(I) reagent21 exclusively formed the dimer of 1a (entry 3), and the addition of ZnBr2,22 which is supposed to bind to thiol groups instead of copper, proved to be unsuccessful (entry 4). TCEP is a convenient reducing agent to cleave disulfide bonds and can be also used for reduction of Cu(II).23 The in situ reduction of disulfide bonds, however, did not work well, and the dimer of 1a was still observed (entry 5). TBTA binds rather weakly to copper and is easily replaced by thiol groups of the cysteine residues. The coordination sites are offered to molecular oxygen, leading to oxidation of Cu(I) to Cu(II). We hypothesized that more strongly chelating ligands might prevent thiol from binding to copper and avoid substrate oxidation. The hybrid ligand 22 is one of the accelerating ligands with stronger donor arms such as benzimidazoles.24 The ligation was carried out using a stoichiometric amount of CuSO4, sodium ascorbate, and ligand 22, but we still encountered the dimerization problem (entry 6). Consequently, we decided to remove the cysteine residue from the substrate and synthesized peptide azide 1b. As expected, 1b did not dimerize under the reaction conditions, and the starting materials and product were stable. The ligation proceeded smoothly to give triazole 11b, and the second-order rate constant was determined to be 3.4 M−1 s−1 when 3 equiv of the Cu promoter and ligand was used (Supporting Information Figure 18). Staudinger Ligation. The Staudinger ligation of alkyl azides and arylphosphines was tested with 1a and phosphine 12. The reaction was not chemoselective, and we observed the formation of many unknown compounds by analytical HPLC (Supporting Information Figure 27). All of the peaks were analyzed by MALDI-TOF MS, and one of the peaks was identified as the phosphine oxide, but we could not find a peak for ligation product 13. Since the reaction did not proceed chemoselectively, we could not determine the rate constant of the Staudinger ligation. Oxime Ligation. The rate of oxime formation is known to be dependent on acidity of the reaction medium. A solution pH

twice to confirm the accuracy of each measurement. Typically, the concentration of reactants was determined in such a way that a reaction reaches approximately 20% conversion within a few hours. With the exception of the Staudinger ligation, we were able to determine the second-order rate constants of the reactions. Although these 8 reactions are representatives among other ligation reactions, many of them showed unexpected results including stoichiometry of reagents, the stability of starting materials and products, and slow reaction kinetics. The details on how we addressed these issues and the insights we obtained throughout the process are discussed below and provide a useful guide for selecting ligation reactions for synthetic applications. Strain-Promoted Azide−Alkyne Cycloaddition (SPAAC). The SPAAC reaction between peptide azide 1a and cyclooctyne 9 was chemoselective, furnishing triazole 10a and its regioisomer 10b. Cyclooctynes are often susceptible to the attack of thiol groups,13 but 9 was intact in the presence of the unprotected cysteine residue. The kinetic studies revealed the second-order rate constant 0.90 M−1 s−1 (Supporting Information Figure 13), which is similar to the reported value.14 The SPAAC reaction showed the high stability of both the starting materials and products under the reaction conditions. Cu-Catalyzed Azide−Alkyne Cycloaddition (CuAAC). In preliminary experiments, the CuAAC reaction, or Click reaction, was attempted with a catalytic amount of CuSO4 and sodium ascorbate.15 This reaction conditions, however, did not give the desired ligation product. The use of tris[(1-benzyl-1H1,2,3-triazol-4-yl)methyl]amine (TBTA), which is known to stabilize Cu(I) oxidation state,16 was not helpful. The direct exploitation of a Cu(I) catalyst in the absence of a reducing agent was also not successful. We found that a stoichiometric amount of CuSO4 was essential for triazole formation (Table 2, entry 1). However, the reaction was accompanied by dimerization of 1a. We also observed the dimerization of ligation product 11a. The Cu(II)/ sodium ascorbate reaction settings are known to produce reactive oxygen species.17 Peptides and proteins with oxidationprone amino acid residues are in danger of undergoing undesired oxidation reactions.18,19 In our peptide substrate 1a, the cysteine residue can suffer from oxidation through disulfide bond formation. This dimer could be isolated and successfully used in the CuAAC reaction, but this approach is unlikely to be suitable for larger substrates or those bearing D


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ACS Chemical Biology of 4−5 works best for the ligation.25,26 Aniline and its derivatives are frequently used to accelerate the reaction.26,27 The ligation between 2 and 14a was attempted at pH 4.6 with aniline, but we did not observe the formation of peptide oxime 15a (Figure 3a). Surprisingly, in addition to imine formation of

of 16 was observed by analytical HPLC. We attributed the slower reaction kinetics to the use of the glyoxaldehyde. A brief survey of the ligation using isobutyraldehyde showed that the ligation proceeded with 500 μM substrate concentration even at RT, and the second-order rate constant was estimated to be ∼2 M−1 s−1. The second-order rate constant of the ligation between 2 and 8 was determined to be 0.015 M−1 s−1 (Supporting Information Figure 40). We found that 8 and 16 were unstable to prolonged exposure to the reaction conditions (Supporting Information Figure 41). Thiol−Maleimide Ligation. Thiols can exist as thiolates at basic pH and are more reactive toward electron-deficient olefins of maleimides. We first tried the reaction at pH 10, but maleimide 17a was susceptible to the hydroxide addition under the strongly basic conditions (Figure 3b). We changed the reaction conditions to pH 7.4, and the addition of hydroxide to maleimide was no longer observed. Ligation product 7a was stable when independently dissolved in the buffer solution (Supporting Information Table 5), but decomposition of 7a and 17a was observed when they were co-dissolved in the reaction medium (Supporting Information Table 6). Slightly acidic reaction conditions (pH 6.2) were examined, but this offered no improvement in the stability. Hydrolysis (ring opening) of maleimides is a known decomposition pathway, and N-aryl-substituted maleimides are more susceptible to hydrolysis compared to that of their N-alkyl-substituted counterparts.30 Although we did not detect the ring-opening product, we envisioned the improved stability of maleimides and ligation product by changing the substituent on nitrogen. Alkyl maleimide substrate 17b was synthesized, and the ligation was carried out at pH 7.4. We were pleased to find that 17b and ligation product 7b were stable under the reaction conditions. The ligation was extremely fast, and the reaction kinetics was measured with 15 μM substrate, giving a secondorder rate constant of 734 M−1 s−1 (Supporting Information Figure 44), a value similar to that from a previous report.31 Native Chemical Ligation (NCL). For NCL, we chose thiophenylester 18a as a substrate to make the kinetic analysis simpler. Since peptide substrate 4 has an internal cysteine residue, thiophenol was added to reverse the unproductive transthioesterification product. Typically, 6 M guanidine hydrochloride buffer solution is used as a reaction medium in NCL. We tried to realize similar reaction settings, but the use of organic solvents to dissolve the small molecule fragment caused precipitation by addition of a small molecule-containing organic solution to peptide-containing aqueous buffer solution. Due to this solubility problem, a phosphate buffer/acetonitrile solvent system was chosen as a NCL reaction medium. Considering the instability of thioesters under strongly basic conditions, a pH value around 7 to 8 worked best to give a balance between the reaction kinetics and the stability of thioesters.32 The ligation was chemoselective, and the second-order rate constant was determined to be 0.067 M−1 s−1 (Supporting Information Figure 49). The value was much smaller than what was expected from the literature.33,34 Interestingly, Kent et al. reported that the amide bond next to the thioester moiety contributes to higher reaction kinetics through an inductive effect.35 To examine this factor, we synthesized thioester 18b that has an alanine residue next to the thioester. The ligation proceeded cleanly under identical conditions, and the second-order rate constant was 0.26 M−1 s−1 (Supporting Information Figure 50). The value was in good agreement with the estimated rate constant. Despite the larger

Figure 3. Initial design of hydroxylamine and maleimide. (a) Structure of hydroxylamine 14a and its ligation product 15a. (b) Structure of maleimide 17a and its ligation product 7a. The stability of these compounds was examined.

2 with aniline, we observed the dimerization of 2. To look closely at the behavior of 2, we carried out stability tests and found that 2 is stable at pH 4.6 in the absence of aniline but undergoes dimerization in the presence of aniline (Supporting Information Figures 28 and 29). The imine formed with aniline could react with a nucleophilic amino acid side chain in 2 (i.e., Cys or Lys), a result consistent with the recent observation by Derda on peptides containing N-terminal glyoxaldehydes.28 We concluded that aniline induced peptide dimerization. Oxime formation was successful in the absence of aniline, but some side peaks were observed in analytical HPLC, indicating decomposition during the reaction. The stability of the starting materials and product was investigated (Supporting Information Table 3). The results revealed that peptide 15a was not stable under acidic pH, whereas 2, 14a, and 15a were all stable when they independently existed in neutral buffer solution. However, 14a underwent a decomposition pathway in the presence of 2 (Supporting Information Table 4). Due to the instability of the substrate, we modified the structure of the hydroxylamine to 14b, which has a longer aliphatic chain. The ligation between 2 and 14b at pH 4.6 was fast, but the product underwent decomposition. At neutral pH, the ligation product was stable, allowing us to determine a second-order rate constant of 1.3 × 10−3 M−1 s−1 (Supporting Information Figure 30), which is in good agreement with the reported value.27 Although the second-order rate constant was successfully determined, peptide oxime 15b was gradually dimerized over the course of the reaction (Supporting Information Figure 31), presumably via the formation of disulfide bonds. It is likely that the glyoxaldehyde is not the best substrate, and the use of aliphatic or aromatic aldehydes may offer advantages in terms of substrate and product stability. Pictet−Spengler Ligation. In order to overcome the instability of oximes, Bertozzi et al. recently developed a Pictet−Spengler ligation that forms a hydrolytically stable C−C bond.29 As with oxime ligation, the reaction kinetics of the Pictet−Spengler ligation is pH-dependent, and the highest reaction rate was observed at pH 4. A second-order rate constant of 10.57 M−1 s−1 at pH 4.0 was reported using isobutyraldehyde as one of the substrates. We conducted the ligation of 2 and 8 at pH 4.0, but the reaction did not occur. When the reaction mixture was heated at 60 °C, the formation E


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products, as found in many synthetic applications. The KAT ligation offers a high reaction rate, stable starting materials, and the formation of a stable amide bond. In order to achieve a reasonably fast reaction, however, the KAT ligation requires an acidic additive, which may not be ideal for folded proteins. We are currently developing variants of the KAT ligation that proceed rapidly at neutral pH. There are a number of other emerging ligation methods that may also prove to be suitable for stoichiometric conjugations in water at low concentrations. In particular, an enormous amount of work on tetrazines as reaction partners for ligations with strained alkenes and alkynes has yielded promising results.40 For example, Wittmann et al. examined the kinetics of a carbohydrate-derived cyclopropene in pH 4.8 acetate buffer at 20 °C and determined a second-order rate constant of 1 M−1 s−1.41 The tetrazine fragments with electron-withdrawing groups tend to show high reaction kinetics but are known to be somewhat unstable.42 The stability can be improved by finetuning the substituents on the tetrazine, although the reaction rate is decreased. Conclusions. In practice, the ligations of large molecules with 1:1 stoichiometry requires dilute reaction conditions, and the reaction kinetics is undoubtedly one of the important factors in choosing an appropriate reaction. However, chemoselectivity and the stability of the starting materials and products under the ligation conditions cannot be overlooked. By designing peptide substrates with unprotected functional groups, we were also able to qualitatively evaluate this aspect of the conjugations. A number of the ligation reactions surveyed are suitable for the covalent attachment of small molecule tags and other applications including ligand immobilization. For other applications, including the covalent conjugation of large, unprotected molecules including proteins, antibodies, nucleic acids, and carbohydrates, where an excess of one reactant cannot or should not be used, it is apparent that further advances in chemoselective ligations are needed.

steric hindrance, 18b showed a higher reaction rate than that of 18a, indicating the importance of the amide bond next to the thioester moiety for promoting the high reaction rate. In both cases, decomposition of 4 was observed when TCEP was not added, probably due to disulfide bond formation. The use of TCEP was essential for clean ligations. KAT Ligation. We recently reported a rapid amide forming ligation of potassium acyltrifluoroborates (KATs) and N,Ndiethylcarbamoylhydroxylamines.36 This ligation proceeded in water with an equimolar ratio of reactants. PEGylation, lipidation, biotinylation, and introduction of azobenzene dye were demonstrated onto a 31-mer unprotected peptide, GLP-1 analog. The azo containing KAT 20 and 5, having an Nterminal hydroxylamine moiety, were selected for this study. The reaction rate of the KAT ligation varies based on the pH of the reaction. When the ligation was carried out in 0.1 M NH4OAc (pH 5.1)/tBuOH (1:1) reaction medium, the second-order rate constant was determined to be 0.39 M−1 s−1 (Supporting Information Figure 55). We found that the reaction was accelerated in the presence of oxalic acid, and a kinetic study conducted at 25 μM with a 1:1 ratio of reactants in H2O/tBuOH (1:1) revealed that the second-order rate constant of the KAT ligation was 22.4 ± 3.8 M−1 s−1 (Supporting Information Figure 56), which is consistent with the one obtained in our previous report. Evaluation of the Ligation Reactions for Synthetic Application. Most of the reactions examined were chemoselective and showed the expected reaction kinetics. The SPAAC reaction proceeded cleanly and showed good stability of the starting materials and products. The CuAAC reaction required a stoichiometric amount of copper reagents to undergo triazole formation.22,37 In our case, the primary amine and thiol groups in 1a could bind to copper and necessitate the use of a stoichiometric amount of the reagents. The thiol groups were also susceptible to oxidation under CuAAC reaction conditions. Ligations of Cys-containing peptides, therefore, might be problematic and require an extensive screening of the reaction conditions. The Staudinger ligation was not chemoselective, and the phosphine substrate was readily oxidized. It has demonstrated utility for the labeling of biomolecules but is not suitable for ligations with a 1:1 stoichiometry. The oxime ligation of a glyoxaldehyde was inhibited in the presence of aniline,38 and cysteine-containing peptide aldehyde 2 suffered from dimerization. Kinetic studies of aniline-catalyzed oxime ligations with peptide substrates containing no cysteine residues have been conducted in detail, and a clear rate acceleration in the presence of aniline was observed.27,39 In our case, oxime formation was observed in the absence of aniline, but the ligation product was readily decomposed under acidic pH. Although the hydrolysis of the oxime is a known decomposition pathway, we observed the formation of an unidentified compound. The need for neutral pH to perform the oxime ligation lowers its reaction rate and diminishes its value as a synthetic method. The stability of the substrates was also a concern of the Pictet−Spengler ligation. The glyoxaldehyde underwent ligation much slower than reported,29 and the reaction kinetics proved to be dependent on the aldehyde structure. The thiol−maleimide ligation demonstrated its methodological utility with an extremely high reaction rate and good stability of N-aliphatic-substituted maleimides, but the reaction is limited to peptides possessing a single cysteine residue. Native chemical ligation performed well, showing chemoselectivity and high stability of the reactants and

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METHODS

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ASSOCIATED CONTENT

Kinetic Experiments. A 2 mM solution of peptide azide 1a in H2O and a 2 mM solution of cyclooctyne 9 in MeCN were prepared. These solutions (25 μL each) were sequentially added to 950 μL of a H2O/MeCN (1:1) solution to make a final reactant concentration of 50 μM. The mixture (10 μL) was directly injected to analytical HPLC, and the reaction progress was monitored by the UV trace at 301 nm. The kinetic measurements for other ligation reactions were carried out in similar ways.

S Supporting Information *

Experimental procedures and spectroscopic data for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by ETH Zürich. We thank the LOC MS Service for analyses. F


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