Decoration of Coiled-Coil Peptides with N-Cysteine Peptide

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/OrgLett

Decoration of Coiled-Coil Peptides with N‑Cysteine Peptide Thioesters As Cyclic Peptide Precursors Using Copper-Catalyzed Azide−Alkyne Cycloaddition (CuAAC) Click Reaction W. Mathis Rink† and Franziska Thomas*,†,‡ †

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Institute of Organic and Biomolecular Chemistry, Georg-August Universität Göttingen, Tammannstraße 2, 37077 Göttingen, Germany ‡ Center for Biostructural Imaging of Neurodegeneration, von-Siebold-Straße 3a, 37075 Göttingen, Germany S Supporting Information *

ABSTRACT: The development of a copper-catalyzed azide−alkyne cycloaddition (CuAAC) protocol for the decoration of coiled coils with N-cysteine peptide thioesters as cyclic peptide precursors is presented. The reaction conditions include tertbutanol/PBS as the solvent and CuSO4/THPTA/ascorbate as the catalytic system. During these studies, partial formylation of N-terminal cysteine peptides is observed. Mechanistic analysis leads to identification of the formyl source and, hence, to the development of reaction conditions, under which the undesired side reaction was suppressed.

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peptides. In particular, cyclic peptides are of interest as they show good biostability and bioavailability. Our synthetic approach toward such CC-based cyclic peptide libraries includes the synthesis of N-cysteine peptide thioesters as cyclic peptide precursors, attachment to the CC scaffold by CuAAC, and subsequent cyclization by native chemical ligation (NCL).26 This has the advantage that the coil tag not only mediates immobilization but also significantly improves solubility of the often hydrophobic cyclic peptide precursors. In this report, we present the development of a generally applicable CuAAC protocol for the in vitro conjugation of peptide acids and thioesters including N-terminal cysteine (Cys) peptide thioesters to coil peptides. Two limitations of this well-studied and widely applied CuAAC click reaction will be addressed, namely, the often time-consuming optimization of the procedure and a significant side reaction on N-terminal Cys peptides. We elucidate the mechanism of this side reaction and identify reaction conditions, under which it is suppressed. Finally, more as a perspective, we present CuAAC of a cysteine peptide thioester and a coiled coil followed by on-coil cyclization.

he copper-catalyzed azide−alkyne cycloaddition (CuAAC) is a reaction between an azide and a terminal or internal alkyne to give a 1,2,3-triazole.1−4 In natural systems, this reaction is highly chemoselective and therefore belongs to a class of bioorthogonal reactions, which we term click reactions.5 By definition, click reactions are fast and irreversible and proceed under mild conditions. They show broad functional group tolerance and, hence, furnish no side products.6 CuAAC does fulfill these criteria, yet it also reveals some limitations. First, reaction times vary from minutes to days. The major drawback, however, is the formation of reactive oxygen species (ROS), which can result in undesired side products.7−9 To minimize this issue, sacrificial ligands have been added or the reaction has been performed in copper flow reactors.7,10 Nonetheless, the impact of the CuAAC and the copper-free strain-promoted azide−alkyne cycloaddition is vast and reflected in the high number of publications on applications in chemical biology and biomaterials design.5,11−15 We envisaged the development of a fast and efficient approach for the decoration of heterodimeric coiled-coil peptides (CC) with cyclic peptide precursors using CuAAC click chemistry. Heterodimeric CCs are α-helical bundles, which can be reliably designed de novo16−18 and have been widely used as tools in biomimetic systems and as affinity tags for labeling and peptide library design.19−25 We plan to exploit the CC scaffold to develop screening platforms for therapeutic © XXXX American Chemical Society

Received: October 12, 2018

A

DOI: 10.1021/acs.orglett.8b03261 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

copper(I) stabilizing and water-soluble tris((1-hydroxy-propyl1H-1,2,3-triazol-4-yl)methyl)amine (THPTA) led to an almost quantitative yield within 2 h (Table S2, entry 5). As a side note, coiled coils are also folded under these reaction conditions (Figure S2). Next, we explored the scope of our synthesis protocol. Alkyne peptide acids 2d to 2i and typical alkyne-modified fluorescence and affinity labels (2j−m) were tested using the optimized reaction conditions (Table 1). The sequences were

In our study, we used the parallel heterodimeric CC peptide N-A3B3 (for sequences see Table S1).17,18 We chose N-A3 as the carrier strand for the cyclic peptide precursor and introduced either azido lysine or azido glycine to the Nterminus to give the required azido peptide 1. The core sequence of our model alkyne peptides (2a−i) is derived from the minimal sequence of the osteogenic growth peptide.27 As alkyne moiety propargyloxycarbonyl lysine (Lys(Proc)) was introduced as the second to last amino acid. All peptides were synthesized using Fmoc-based solid-phase peptide synthesis protocols.28,29 Generally, CuAAC in the presence of chelating moieties such as N-terminal cysteine (Cys) is challenging, although a high functional group tolerance has been reported. Only two reports describe the successful CuAAC click reaction in the presence of a free N-terminal Cys, when high concentrations of either copper sulfate and reducing agent or copper(I) stabilizing ligand were used.30,31 Initially, we avoided the challenge of CuAAC in the presence of N-terminal Cys and performed optimization of the reaction conditions with azide peptide 1 and alkyne peptide thioester 2a (Scheme 1, Reaction

Table 1. Reaction Scope of Optimized CuAAC Protocol

Scheme 1. CuAAC of Alkyne Peptide Thioesters and Azido Coil Peptide N-A3

entry

alkyne

product

yielda (%)

1 2 3

H-AK(Proc)YGFGG-SBn (2a) H-CK(Proc)YGFGG-SBnb (2b) Thz-K(Proc)YGFGG-SBnb (2c)

3a 3b 3c

92 ∼38 ∼37

4 5 6 7 8 9

H-AK(Proc)YGFGG-OH (2d) H-AK(Proc)YGHGG-OH (2e) H-AK(Proc)YGCGG-OH (2f) H-AK(Proc)YGEGG-OH (2g) H-AK(Proc)YGRGG-OH (2h) H-AK(Proc)YGMGG-OH (2i)

3d 3e 3f 3g 3h 3i

95 90 82 96 91 88

10 11 12 13

biotin propargylamide (2j) 5-FAM propargylamide (2k) 6-TAMRA propargylamide (2l) 1-ethynylpyrenec (2m)

3j 3k 3l 3m

87 92 92 81

a

Yields are based on integration of HPLC traces and related to the conversion of azide 1. bNo baseline separation of 3b and 3c. cAfter 4 h.

very similar to 2a except for substitution of the phenylalanine residue with histidine (His), Cys, glutamate (Glu), arginine (Arg), and methionine (Met). For His, Cys, Arg, and Met, side reactions due to oxidation have previously been described in the context of CuAAC.7 Therefore, the reactions were performed in degassed solvents. Using our protocol, full conversion was achieved within 2 h in almost all cases and conjugation products 3d to 3i were obtained in excellent yields and quality. Undesired oxidation of amino acid side chains has only been detected in trace amounts by electrospray-ionization mass spectrometry (ESI-MS, Figure S3). The CuAAC of biotin propargylamide (2j) and fluorescent dyes 5-carboxyfluorescein (5-FAM, 2k) propargylamide, 6-carboxytetramethyl-rhodamine (6-TAMRA, 2l) propargylamide, as well as 1ethynylpyrene (2m) was also successful; however, CuAAC with 2m only gave 81% of the conjugation product after 4 h. This is presumably due to the reduced reactivity of the conjugated alkyne moiety. With a widely applicable CuAAC protocol in hand, we took the next step and tested our actual peptide of interest, Ncysteine peptide thioester 2b (Scheme 1, Reaction B). As anticipated, the reaction was completed within 2 h; yet under the formation of two major products, the desired CuAAC conjugation product 3b and a product with a mass difference of plus 14 in a ratio of 1.2:1 (Figure 1A). Consequently, we tried CuAAC with peptide thioester 2c, in which the N-

A). The conditions described by Lee et al. served as a starting point for our studies.31 The peptides were deployed in 1 mM concentration in 70% acetonitrile in phosphor-buffered saline (PBS) in the presence of CuSO4 and reducing sodium ascorbate. The latter were used in 6.7 mM and 10 mM concentration, respectively. However, under these conditions only minor conversion of 1 and 2a was observed (Table S2, entry 1, Figure S1A). Therefore, we changed from acetonitrile/ PBS to tert-butanol/PBS as the solvent system (Table S2, entries 2−5).3 This led to elevated conversions. Deploying the alkyne moiety in a small excess of 1.2 equiv and adding B


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Figure 2. CuAAC of N-cysteine peptide thioester 2b to azide N-A3 1 in tert-butanol-d10 and in the presence of 13.4 mM THPTA. (A) HPLC trace of the reaction after 2 h. (B) High-resolution ESI mass spectra of conjugation product 3b and (C) formylated conjugation product 3c.

Figure 1. HPLC traces of CuAAC of N-cysteine peptide thioester 2b (A, C−D) and N-Thz peptide thioester 2c (B) to azide N-A3 1 in the presence of 13.4 mM THPTA (A−B), 33.5 mM THPTA (C), and 3.4 mM THPTA (D).

(CuSO4/THPTA (1:5)). In accordance to the literature, CuAAC was significantly decelerated. However, much to our surprise, the ratio of nonformylated (3b) and formylated conjugation product (3c) changed in favor of the undesired 3c (Figure 1C), which indicated that the ROS formation had not been fully suppressed. Consequently, we performed the same reaction with significantly reduced amounts of THPTA (3.4 mM). Remarkably, the starting material was almost fully consumed within 2 h and desired 3b was furnished in preparative useful 74% yield, whereas only 9% of formylated 3c was formed (Figure 1D). Based on these experiments, we hypothesize the following reaction mechanism for the formylation of free N-terminal cysteine during CuAAC in aqueous tert-butanol (Scheme 2).

terminal Cys was masked as thiazolidine (Scheme 1, Reaction C).32 As already described by Lee et al., we could confirm partial cleavage of the thiazolidine under CuAAC conditions, giving the conjugation product with the free N-terminal Cys 3b in 53% yield.31 Interestingly, the HPLC traces of the CuAAC reactions of 2b and 1 (Reaction B) or 2c and 1 (Reaction C) appeared very similar (Figure 1B). The major byproduct in Reaction B showed the same retention time as 3c in Reaction C and identical mass. This led us to conclude that somehow, under the conditions of our CuAAC protocol, the free Nterminal Cys has been formylated. The equilibrium reaction of cysteine and formaldehyde under the formation of thiazolidine is well-known.33,34 To identify the formyl source, we had a closer look at the redox reactions taking place during CuAAC. The reduction of copper(II) to copper(I) by ascorbate results in the formation of trace amounts of hydrogen peroxide, which can further disproportionate under a copper(I)-mediated Fenton-like reaction to give hydroxyl radicals (Scheme S1).35 tert-Butanol is a potent hydroxyl radical scavenger,36,37 of which decomposition to acetone and methyl radicals under influence of UV light and hydrogen peroxide or ozone is well documented.38,39 The methyl radical can further be oxidized to methyl peroxy, which, in turn, disproportionates to methanol, oxygen, and formaldehyde. 40 To prove our hypothesis, we performed the CuAAC of 2b and 1 in fully deuterated tert-butanol under otherwise identical conditions and characterized the reaction products by high resolution ESIMS (Figure 2). In comparison to the CuAAC in nondeuterated tert-butanol, the mass of the side product was increased by 2 (Figure 2C). This confirms that deuterated formaldehyde was incorporated and that tert-butanol is indeed the formyl source. From our mechanistic considerations we concluded that formaldehyde formation should be suppressed by adding hydrogen peroxide scavengers, which consequently would enable CuAAC of N-cysteine peptides. Extensive studies by Finn et al. identified THPTA as a sacrificial ligand, which is partially decomposed during CuAAC click reaction under consumption of hydrogen peroxide.7,41,42 Hence, we increased the amount of 3 from 13.4 mM to 33.5 mM concentration

Scheme 2. Mechanistic Hypothesis for the Formylation of N-Cysteine Peptides in CuAAC in Aqueous tert-Butanol

Formaldehyde (5) is formed upon oxidation of tert-butanol (4) by ROS and subsequent decomposition of the oxidized species. Initial 1,2-addition of the sulfhydryl group to the carbonyl group of formaldehyde followed by elimination of water gives the thionium ion 10. This is a highly reactive electrophile, which undergoes nucleophilic addition with the terminal amino group furnishing the thiazolidine species 8. Thiazolidine cleavage, however, is catalyzed by copper(II) (7), which is complexed by the ring nitrogen and the carbonyl C


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Organic Letters group of the amide bond in 12.43,44 This destabilizes the N,Sacetal moiety and strongly promotes the back reaction toward cysteine 6. The THPTA ligand (L) stabilizes and enriches copper(I) and reduces the amount of copper(II); hence, thiazolidine cleavage is significantly slower at increased amounts of THPTA. However, it was also reported that THPTA significantly modulates the copper(II)/copper(I) redox potential and, hence, the Fenton chemistry (ROS).45 Although, our results support the hypothesized mechanism, we cannot exclude a correlation between the amount of ligand and an increase in ROS, which consequently results in formaldehyde formation. The adjustment of the synthesis protocol allowed us to combine CuAAC and NCL in one synthesis protocol to generate cyclic peptides presented on CC scaffolds. As stated in the introduction, the coil tag not only enables immobilization but also improves solubility of the cyclic peptide and its precursor. Exemplarily, we demonstrated the cyclization of 2b and 3b (Figure 3) in aqueous buffer. Whereas 3b almost reacts

challenging. We observed formylation of Cys under our reaction conditions, which, so far, has not yet been described in the context of CuAAC. As a formyl source, tert-butanol was identified, which decomposes to formaldehyde, methanol, and oxygen upon oxidation by ROS. We found that formylation of Cys to thiazolidine is in equilibrium with copper(II)-mediated thiazolidine cleavage and that this equilibrium can be influenced by the amount of copper(I) stabilizing THPTA ligand. Using a CuSO4−THPTA ratio of 2:1 under otherwise similar conditions furnished the desired condensation product in 74% yield. To the best of our knowledge, only two reports describe CuAAC on peptides with N-terminal Cys. However, high amounts of copper(II) and/or ligand as well as long reaction times were required.30,31 Our approach is fast and proceeds under mild conditions. We plan to apply this protocol in the synthesis of coiled-coil cyclic peptide libraries. However, we are confident that these conditions are broadly applicable to a range of peptides and proteins beyond the coiled coil.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03261.

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General procedures, additional tables, figures and schemes, and characterization of the compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Franziska Thomas: 0000-0002-1176-7018 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS W.M.R. and F.B.T. are grateful for funding from the German Research Foundation (DFG, TH 2008/2-1) and the FCI (Fonds der Chemischen Industrie). Furthermore, we thank Dr. Alexander Breder (Georg-August Universität Gö ttingen, Germany) for fruitful discussions and Prof. Dr. Ulf Diederichsen (Georg-August Universität Göttingen, Germany) for general scientific support.

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Figure 3. Cyclization of N-cysteine peptide thioesters 2b and 3b by NCL. (A−B) HPLC monitoring of the cyclization reactions of 2b (A) and 3b (B).

REFERENCES

(1) Huisgen, R. Proc. Chem. Soc. 1961, 333, 357. (2) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67 (9), 3057. (3) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41 (14), 2596. (4) Ahmad Fuaad, A.; Azmi, F.; Skwarczynski, M.; Toth, I. Molecules 2013, 18 (11), 13148. (5) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48 (38), 6974. (6) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40 (11), 2004. (7) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int. Ed. 2009, 48 (52), 9879. (8) Li, S.; Cai, H.; He, J.; Chen, H.; Lam, S.; Cai, T.; Zhu, Z.; Bark, S. J.; Cai, C. Bioconjugate Chem. 2016, 27 (10), 2315.

spontaneously to the desired cyclic peptide 14, cyclization of 2b to cyclic 13 proceeds significantly slower. After 6 h only 30% of 2b was converted, which is mainly due to the reduced solubility. This example clearly emphasizes the importance of the identification of robust conditions for CuAAC, which allow flexible use of CuAAC and NCL in one synthesis protocol. In summary, we have presented an efficient protocol for CuAAC click chemistry on coiled-coil peptides. This protocol uses tert-butanol and PBS as solvents, CuSO4 and THPTA in a 1:2 ratio, and reducing ascorbate as the catalytic system. These conditions are tolerant to all amino acids as well as common tags and fluorescent labels. N-terminal Cys is, however, D


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Organic Letters (9) Saito, F.; Noda, H.; Bode, J. W. ACS Chem. Biol. 2015, 10 (4), 1026. (10) Hatit, M. Z. C.; Reichenbach, L. F.; Tobin, J. M.; Vilela, F.; Burley, G. A.; Watson, A. J. B. Nat. Commun. 2018, 9 (1), 1. (11) Tang, W.; Becker, M. L. Chem. Soc. Rev. 2014, 43 (20), 7013. (12) Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39 (4), 1302. (13) Lallana, E.; Riguera, R.; Fernandez-Megia, E. Angew. Chem., Int. Ed. 2011, 50 (38), 8794. (14) Li, L.; Zhang, Z. Molecules 2016, 21 (10), 1393. (15) Pickens, C. J.; Johnson, S. N.; Pressnall, M. M.; Leon, M. A.; Berkland, C. J. Bioconjugate Chem. 2018, 29 (3), 686. (16) Lupas, A. N.; Bassler, J. Trends Biochem. Sci. 2017, 42 (2), 130. (17) Thomas, F.; Boyle, A. L.; Burton, A. J.; Woolfson, D. N. J. Am. Chem. Soc. 2013, 135 (13), 5161. (18) Groth, M. C.; Rink, W. M.; Meyer, N. F.; Thomas, F. Chem. Sci. 2018, 9, 4308. (19) Woolfson, D. N. Subcell. Biochem. 2017, 82, 35. (20) Rink, W. M.; Thomas, F. Chem. - Eur. J. 2018, DOI: 10.1002/ chem.201802849. (21) Tripet, B.; Yu, L.; Bautista, D. L.; Wong, W. Y.; Irvin, R. T.; Hodges, R. S. Protein Eng., Des. Sel. 1996, 9 (11), 1029. (22) Zacco, E.; Anish, C.; Martin, C. E.; v. Berlepsch, H.; Brandenburg, E.; Seeberger, P. H.; Koksch, B. Biomacromolecules 2015, 16 (7), 2188. (23) Zacco, E.; Hütter, J.; Heier, J. L.; Mortier, J.; Seeberger, P. H.; Lepenies, B.; Koksch, B. ACS Chem. Biol. 2015, 10 (9), 2065. (24) Chao, H.; Bautista, D. L.; Litowski, J.; Irvin, R. T.; Hodges, R. S. J. Chromatogr., Biomed. Appl. 1998, 715, 307. (25) White, S. J.; Johnson, S.; Szymonik, M.; Wardingley, R. A.; Pye, D.; Giles Davies, A.; Wälti, C.; Stockley, P. G. Nanotechnology 2012, 23 (49), 495304. (26) Dawson, P. E.; Muir, T. W.; Kent, S. B. H. Science 1994, 266 (November), 776. (27) Chen; Muhlrad, A.; Shteyer, A.; Vidson, M.; Bab, I.; Chorev, M. J. Med. Chem. 2002, 45 (8), 1624. (28) Flemer, S. J. Pept. Sci. 2009, 15 (11), 693. (29) Mende, F.; Seitz, O. Angew. Chem., Int. Ed. 2011, 50 (6), 1232. (30) Richardson, J. P.; Chan, C. H.; Blanc, J.; Saadi, M.; MacMillan, D. Org. Biomol. Chem. 2010, 8 (6), 1351. (31) Lee, D. J.; Mandal, K.; Harris, P. W. R.; Brimble, M. a.; Kent, S. B. H. Org. Lett. 2009, 11 (22), 5270. (32) Bang, D.; Kent, S. B. H. Angew. Chem., Int. Ed. 2004, 43 (19), 2534. (33) Ratner, S.; Clarke, H. T. J. Am. Chem. Soc. 1937, 59 (1), 200. (34) Kallen, R. G. J. Am. Chem. Soc. 1971, 93 (23), 6227. (35) Pham, A. N.; Xing, G.; Miller, C. J.; Waite, T. D. J. Catal. 2013, 301, 54. (36) Piechowski, M. Von; Thelen, M.-A.; Hoigné, J.; Bühler, R. E. Ber. Bunsenges. Phys. Chem. 1992, 96 (10), 1448. (37) Teton, S.; Mellouki, a.; Le Bras, G. Int. J. Chem. Kinet. 1996, 28 (4), 291. (38) Cater, S. R.; Stefan, M. I.; Bolton, J. R.; Safarzadeh-Amiri, A. Environ. Sci. Technol. 2000, 34 (4), 659. (39) Garoma, T.; Gurol, M. D. Environ. Sci. Technol. 2004, 38 (19), 5246. (40) Müller, J.-F.; Liu, Z.; Nguyen, V. S.; Stavrakou, T.; Harvey, J. N.; Peeters, J. Nat. Commun. 2016, 7 (May), 13213. (41) Semenov, S. N.; Belding, L.; Cafferty, B. J.; Mousavi, M. P. S.; Finogenova, A. M.; Cruz, R. S.; Skorb, E. V.; Whitesides, G. M. J. Am. Chem. Soc. 2018, 140 (32), 10221. (42) Hong, V.; Steinmetz, N. F.; Manchester, M.; Finn, M. G. Bioconjugate Chem. 2010, 21 (10), 1912. (43) Dondoni, A.; Marra, A.; Perrone, D. J. Org. Chem. 1993, 58 (1), 275. (44) Gielzak-Kocwin, K.; Wojciechowski, W. Transition Met. Chem. 1996, 21, 312. (45) Kennedy, D. C.; McKay, C. S.; Legault, M. C. B.; Danielson, D. C.; Blake, J. A.; Pegoraro, A. F.; Stolow, A.; Mester, Z.; Pezacki, J. P. J. Am. Chem. Soc. 2011, 133 (44), 17993. E


Decoration of Coiled-Coil Peptides with N-Cysteine Peptide

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