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Selective and efficient cysteine conjugation by maleimides in the presence of phosphine reductants Maik Henkel, Niels Röckendorf, and Andreas Frey Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00371 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Bioconjugate Chemistry

Selective and Efficient Cysteine Conjugation by Maleimides in the Presence of Phosphine Reductants Maik Henkel¶, Niels Röckendorf, Andreas Frey* Division of Mucosal Immunology & Diagnostics, Priority Area Asthma & Allergy, Research Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany

ABSTRACT Sulfhydryl functions of thiol-containing amino acids are prime attachment sites for conjugation of labels, ligands or drugs to proteinaceous compounds. Usually the thiol is offered a xenobiotic electrophilic moiety from the molecule to be attached such as a maleimido function. As sulfhydryls tend to oxidize into disulfides they must be reduced before conjugation. A popular thiol reduction reagent in biosciences is the substituted phosphine tris(2-carboxyethyl)phosphine (TCEP). Yet, phosphines are nucleophilic, too, and thus potentially compete with thiols for the electron-poor alkene moiety of maleimide resulting in complex product mixtures. To overcome this shortcoming we developed a method to eliminate excess reducing agent in the reaction mixture by selective oxidation of the phosphine with azidobenzoic acid before coupling. This results in a selective and efficient labeling of cysteines by maleimides.

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Equipping proteinaceous compounds with labels, ligands or drugs for tracking or site specific delivery requires the compound to bear chemical functionality via which conjugates can be created without harming its biological activity. Preferred attachment sites among canonical amino acids are lysine and cysteine.1 In case of cysteine the reactive moiety is the sulfhydryl group in the side chain. This thiol is highly reactive, due to its nucleophilic character especially at physiological pH, where the side chain amine of lysine is protonated. Consequently, cysteine reacts faster and more selectively than lysine, rendering coupling via the cysteine side chain a highly attractive method.2 In peptide chemistry, for instance, entire ligation technologies such as the peptomer system rely on this coupling route.3 Moreover, the resulting S-cysteinyl alkyl ethers can safely be scavenged via the mercapturic acid pathway

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and usually evade immune recognition.5,6 The most popular thiol-

reactive conjugation handles are maleimides, because they couple specificly and efficiently with S-

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alkyl thiols.7 Yet, a free thiol is rarely available in proteins and if so it is biologically important and ought not to be modified. As thiols are prone to oxidation they usually form disulfides, not only in proteins upon folding but also in synthetic peptides during workup. To revert disulfides into their sulfhydryl precursors mild reducing agents such as dithiothreitol (DTT) and phosphines - the most commonly applied one being tris(2-carboxyethyl)phosphine (TCEP) - can be used.8 Thiol-based reducers would compete with the protein’s / peptide’s cysteine thiol in the subsequent maleimide coupling reaction, but also TCEP was - in contrast to earlier findings 9- reported to react with maleimides.7,10,11 Consequently, the reducing agent must either be removed under anaerob conditions, e.g. by dialysis or chromatography in deoxygenated solvents, or the maleimide-carrying conjugation partner must be used in excess of the reducing agent which in turn has to be used in surplus to the disulfides to be reduced. Thus, maleimide couplings can become wasteful procedures. Considering the limited availability of many conjugation partners and the expenditure of an additional purification step, an improved conjugation procedure which avoids excess maleimide and potential side reactions is highly desirable. To address this problem we analyzed the phosphine maleimide interaction and developed a method to eliminate the reactive phosphine species in the reaction mixture. As the reactivity of substituted phosphines with maleimides is still subject of debate we first wanted to know whether or not excess phosphine consumes maleimide under cysteine conjugation conditions. For that we chose three phosphines: tris(2-carboxyethyl)phosphine (TCEP), tris(3hydroxypropyl)phosphine (THP) and triphenylphosphine-3,3',3"-trisulfonic acid (TPPTS) as literature known agents for disulfide reduction

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and the maleimides N-ethylmaleimide (NEM)

and 3-maleimidopropionic acid (NPAM) as model phosphine reaction partners (Fig. 1).

Figure 1. Structure and exact mass of the phosphines and maleimides used in this study Beyond their function as reductive agent (disulfide reduction, Wittig reaction) phosphines are also known to act as nucleophilic catalysts in organic synthesis. In all cases a zwitterionic species is


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formed where the phosphonium lends high stability to the neighboring anionic center.14 In our case the phosphine could add to the alkene function of the maleimide moiety thereby creating a zwitterionic adduct whose carbanion is stabilized by the phosphonium group in beta- and the carbonyl/amide function in alpha position. Depending on the reaction conditions, the phosphine substituents and the reaction partners available, different products can be formed under release of the phosphine which again may enter the reaction cycle. In our case hydroxylation of the maleimide is conceivable

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due to the aqueous environment (Scheme 1a), as well as the addition of another

maleimide in a Rauhut-Currier-type manner (Scheme 1b).16 In both variants the phosphine would be fully recycled and thus remain available for disulfide reduction whereas the maleimidic target for the reduced disulfide could be substantially diminished .

Scheme 1. Possible reactions of phosphines with maleimides in aqueous buffers. Proposed reaction mechanisms are adapted from reference 15 and 16. Thus in an aqueous mixture of phosphine, disulfide and maleimide three principal reaction patterns are conceivable: i) reduction of the disulfide without the formation of any phosphine maleimide adduct; ii) formation of a phosphine-maleimide adduct, and iii) phosphine-catalyzed conversion of the maleimide into undesired side products. In order to investigate those possibilities we performed a ACS Paragon Plus Environment


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series of tests with the phosphines, the two maleimides and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman´s reagent) as model disulfide. In the course of the regular reaction of Ellman´s reagent with a phosphine the yellow product 2-nitro-5-thiobenzoate is formed which allows a simple photometric monitoring of the reduction reaction. Interfering side reactions between phosphine and maleimide will correspondingly affect the color formation (Scheme 2).

Scheme 2. Conceivable reaction mechanisms for phosphine interference with thiol maleimide coupling reactions We performed the test reactions in two steps, where in the first part the phosphine was allowed to react with an excess of maleimide (reaction part 1 in Scheme 2) before DTNB was added. A second addition of excess phosphine (reaction part 2 in Scheme 2) was used to check for any remaining unreacted DTNB. All combinations of phosphines and maleimides used in our test yielded the same coloring of reaction mixtures in the respective steps, indicating that the reaction mechanism was identical for all of them (see absorbance readouts in Fig. 2.): In each case, no color was formed during the first part of the reaction. This rules out that a phosphine-catalyzed conversion of the maleimides, either in a


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Rauhut-Currier manner (Scheme 2, iii) or by reaction with water, occurred under the conditions applied, since in this case, the remaining phosphine would have reduced the added DTNB to generate a colored product. As the addition of extra phosphine in reaction part 2 in our experiments resulted in strong color formation, we conclude that the reaction mechanism Scheme 2 i can also be ruled out. In this case, the maleimide would have remained untouched by the phosphine and would have reacted with the phosphine-reduced Ellman's reagent to form the colorless sulfide in step 1, which would have remained unchanged – and colorless - by further addition of phosphine (Scheme 2, i). In fact, the disulfide reduction of DTNB and formation of a colored product only occurred after addition of excess phosphine. Consequently the initially added phosphine had been consumed by the excess maleimide under zwitterion formation (Scheme 2, ii). Or, vice versa, the phosphine consumed the maleimide under our reaction conditions.

Figure 2. Reduction of Ellman's reagent by phosphines in the presence of maleimides. Absorbance readout at 412 nm is a measure for the amount of reduced Ellmann's reagent after incubation of DTNB with the reaction mixtures as indicated. "Expected" absorbance values are theoretically established estimates for the different reaction mechanisms depicted in Scheme 2. These conclusions could also be confirmed exemplarily via mass spectrometry for the reaction of TCEP with NEM or NPAM in phosphate-buffered saline, where the zwitterionic adduct of maleimide was clearly detected (see supplementary information). Although maleimide consumption was not quantitative in our setting, possibly due to different reaction kinetics for thiolate/thioether formation and zwitterion formation, a loss of more than 50% of the maleimide employed is hardly acceptable. Consequently, any excess phosphine ought to be removed or inactivated before maleimide is added in order to obtain a clean labeling of thiol-containing biomolecules after phosphine reduction.



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We therefore seeked a method to suppress the unwanted phosphine-maleimide side reaction. A promising approach appeared to be the oxidation of phosphines with organic azides, known as Staudinger reaction (Scheme 3).

Scheme 3. Oxidation of phosphines with 4-azidobenzoic acid to phosphine oxide

Azides are compatible with the functional groups that are normally present in proteinaceous biomolecules and the amine as well as the phosphine oxide that result from this reaction should not interfere with the Michael-type addition of thiols to maleimide. For our purpose, we chose 4azidobenzoic acid (ABA) as oxidant for the phosphines, because this compound was described to be very reactive towards phosphines (≥ 98 yield in less than 30 min).17 As read-out system for the phosphine oxidation we again used Ellman´s reagent; as phosphines TCEP and THP were employed. In our hands, i.e. under physiological conditions in PBS at RT, the reaction of the phosphines with excess ABA proceeded so rapidly that after 10 min reaction time no reducing agent remained (Fig. 3).

Figure 3. Deactivation of the phosphines THP and TCEP by azidobenzoic acid. Each phosphine was incubated with 100 equivalents of ABA. The phosphine's remaining ability to reduce Ellman´s reagent was monitored in a time frame of 80 min. After 10 min reaction time no significant amount of the reduced product is detectable (no difference to DNTB solution without reducing agent added; p > 0.05, Bonferroni with post-hoc Tukey`s multiple comparison-test) The resulting p-aminobenzoic acid is a naturally occuring, rather nontoxic substance with an LD50 of 6 g/kg body weight.18 As tris(2-carboxyethyl)phosphine (TCEP) and 4-azidobenzoic acid (ABA) display similar LD50s of 3.5 g/kg 19 and 0.98 g/kg 20 body weight, respectively, the chemicals used in


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this protocol do not pose a major toxicological risk to the experimentator and the environment; it may not even be necessary to remove those compounds from the reaction mixture before using the conjugate in biological assays. To test our method of annihilation of unreacted phosphine by azidobenzoic acid in a typical setup, we set out to label a cysteine-containing peptide with maleimide after reduction with TCEP. For this purpose we dimerized the sample peptide CWPQQQPFPRPQ via disulfide bond formation to generate a model disulfide-linked biomolecule. This disulfide was reduced by addition of excess TCEP to form the free thiol compound which was then allowed to react with the maleimide NPAM, either in presence of excess unreacted phosphine or after oxidation of the remaining phosphine reagent with ABA (10 eq. to the initial amount of TCEP) (Scheme 4).

Scheme 4: Maleimide labeling of a peptide after reductant oxidation with 4-azidobenzoic acid

All reaction mixtures were analyzed by mass spectrometry (Fig. 4). The MS spectra of the peptide labeling reaction without oxidative destruction of excess phosphine (Fig. 4c) reveals a complex product mixture which contains residual excess TCEP (251.01 [M+H]+), the NPAM-TCEP adduct (420.07 [M+H]+), NPAM-labeled peptide (840.78 [M+2H]2+; 560.54 [M+3H]3+) and unlabeled peptide (756.28 [M+2H]2+; 504.54 [M+3H]3+). This shows that labeling of the peptidic thiol compound with maleimide in the presence of a phosphine reductant leads to numerous side reactions, and the consumption of maleimido label by phosphine adduct formation obviously reduces the yield of the desired maleimide-labeled peptide. In contrast, when excess phosphine is destroyed after peptide reduction by azidobenzoic acid before addition of the maleimide (Fig. 4d), the TCEP signal (251.01 [M+H]+) is completely converted into a TCEP-oxide signal (267.01 [M+H]+). The subsequent addition of maleimide now leads to the labeling of the entire peptide which results in almost pure NPAM-peptide conjugate (840.78 [M+2H]2+; 560.54 [M+3H]3+; Fig. 4e) that shall be suited without further purification for biological assays.



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Fig. 4: MS-spectra of cysteinyl peptide maleimide labeling reactions a) peptide before reduction: peptide dimer (756.08 [M+4H]4+; 605.27 [M+5H]5+); b) peptide after reduction with TCEP: peptide monomer (756.29 [M+2H]2+; 504.53 [M+3H]3+); TCEP (251.01[M+H]+); c) reaction mixture of NPAM-labeling after peptide reduction with TCEP, without azidobenzoic acid: TCEP (251.01[M+H]+), NPAM-TCEP adduct (420.07 [M+H]+), NPAM-labeled peptide (840.78 [M+2H]2+; 560.54 [M+3H]3+) unlabeled peptide (756.28 [M+2H]2+; 504.21 [M+3H]3+); d) peptide reduction with TCEP and elimination of the reactive phosphine by oxidation with azidobenzoic acid: oxidized TCEP (267.00 [M+H]+), peptide monomer (756.28 [M+2H]2+; 504.53 [M+3H]3+); e) selective labeling of the peptide with NPAM after reduction with TCEP and elimination of the reactive phosphine by oxidation with ABA: oxidized TCEP (267.00 [M+H]+); NPAM-labeled peptide (840.77 [M+2H]2+; 560.86 [M+3H]3+) . In summary, we show that the quenching of maleimides by phosphines is a significant side reaction during thiol-maleimide coupling. To overcome this side reaction we eliminated the excessive phosphine in the reaction mixture before addition of maleimide by selective oxidation of the phosphine with azidobenzoic acid. We show that this approach allows a selective and efficient labeling of thiolated peptides without previous purification.

ASSOCIATED CONTENT Supporting Information Supporting Information: Experimental procedures; MS-spectra of TCEP-maleimide adduct formation

AUTHOR INFORMATION * Corresponding author: Andreas Frey, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany; e-mail: [email protected] ¶ Present address: Jerini Bio Tools GmbH, Berlin, FRG

Notes The authors declare having no competing financial interest with the outcomes of this study.



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ACKNOWLEDGEMENTS The authors want to thank Jürgen Sarau for expert technical assistance. This work was funded in part by the German Federal Ministry of Education and Research (BMBF) in the context of the German Center for Lung Research (DZL) (grant 82DZL00101).

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Figure 1. Structure and exact mass of the phosphines and maleimides used in this study 29x5mm (600 x 600 DPI)


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Scheme 1. Possible reactions of phosphines with maleimides in aqueous buffers. 103x151mm (600 x 600 DPI)



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Scheme 2. Conceivable reaction mechanisms for phosphine interference with thiol maleimide coupling reactions 97x62mm (600 x 600 DPI)


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Figure 2. Reduction of Ellman's reagent by phosphines in the presence of maleimides. Absorbance readout at 412 nm is a measure for the amount of reduced Ellmann's reagent after incubation of DTNB with the reaction mixtures as indicated. "Expected" absorbance values are theoretically established estimates for the different reaction mechanisms depicted in Scheme 2. 49x29mm (600 x 600 DPI)



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Scheme 3. Oxidation of phosphines with 4-azidobenzoic acid to phosphine oxide 15x2mm (600 x 600 DPI)


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Figure 3. Deactivation of the phosphines THP and TCEP by azidobenzoic acid. Each phosphine was incubated with 100 equivalents of ABA. The phosphine's remaining ability to reduce Ellman´s reagent was monitored in a time frame of 80 min. After 10 min reaction time no significant amount of the reduced product is detectable (no difference to DNTB solution without reducing agent added; p > 0.05, Bonferroni with posthoc Tukey`s multiple comparison-test) 48x35mm (600 x 600 DPI)



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Scheme 4: Maleimide labeling of a peptide after reductant oxidation with 4-azidobenzoic acid 29x6mm (600 x 600 DPI)


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Fig. 4: MS-spectra of cysteinyl peptide maleimide labeling reactions a) peptide before reduction: peptide dimer (756.08 [M+4H]4+; 605.27 [M+5H]5+); b) peptide after reduction with TCEP: peptide monomer (756.29 [M+2H]2+; 504.53 [M+3H]3+); TCEP (251.01[M+H]+); c) reaction mixture of NPAM-labeling after peptide reduction with TCEP, without azidobenzoic acid: TCEP (251.01[M+H]+), NPAM-TCEP adduct (420.07 [M+H]+), NPAM-labeled peptide (840.78 [M+2H]2+; 560.54 [M+3H]3+) unlabeled peptide (756.28 [M+2H]2+; 504.21 [M+3H]3+); d) peptide reduction with TCEP and elimination of the reactive phosphine by oxidation with azidobenzoic acid: oxidized TCEP (267.00 [M+H]+), peptide monomer (756.28 [M+2H]2+; 504.53 [M+3H]3+); e) selective labeling of the peptide with NPAM after reduction with TCEP and elimination of the reactive phosphine by oxidation with ABA: oxidized TCEP (267.00 [M+H]+); NPAMlabeled peptide (840.77 [M+2H]2+; 560.86 [M+3H]3+) 224x286mm (300 x 300 DPI)



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ToC - Graphic 26x7mm (600 x 600 DPI)


Selective and Efficient Cysteine Conjugation by ... - ACS Publications

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