DOI: 10.1002/chem.201504462

Communication

& Bioorganic Chemistry

Chemo- and Regioselective Ethynylation of TryptophanContaining Peptides and Proteins Morten Borre Hansen,*[a] Frantisˇek Hub‚lek,[a] Troels Skrydstrup,[b] and Thomas HoegJensen[a]

Chem. Eur. J. 2016, 22, 1572 – 1576

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Communication Abstract: Ethynylation of various tryptophan-containing peptides and a single model protein was achieved using Waser’s reagent, 1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1 H)-one (TIPS-EBX), under gold(I) catalysis. It was demonstrated by NMR that the ethynylation occured selectively at the C2-position of the indole ring of tryptophan. Further, MS/MS showed that the tryptophan residues could be modified selectively with ethynyl functionalities even when the tryptophan was present as a part of the protein. Finally, the terminal alkyne was used to label a model peptide with a fluorophore by means of coppercatalyzed click chemistry.

Modification of peptides and proteins with orthogonal functional groups is an important discipline in order to manipulate the properties of these molecules. This includes refinement of the biological properties, labeling for imaging purposes, and development of new peptide and protein-based materials.[1] A range of orthogonal functionalities can be introduced by standard solid-phase synthesis using suitably protected and modified building blocks. However, with regards to native or recombinant peptides/proteins, a variety of orthogonal functionalities is more limited, and the traditional selective functionalization of N-termini, lysine or cysteine side chains, still prevails.[2] Though less established, a chemoselective functionalization of other residues, such as methionine,[3] glutamine,[4] arginine,[5] serine/threonine (N-terminal oxidation),[6] tyrosine,[7] and tryptophan,[8] has been described. Among these residues, tryptophan is particularly interesting because of its rarity in proteins; with a natural abundance of only 1.09 %, tryptophan is the least abundant amino acid in proteins.[9] Therefore, it is likely that a protein of interest will contain only a single tryptophan accessible for conjugation or none at all, in which case it can be introduced by recombinant engineering, allowing excellent control of the conjugation position. In the most promising example on tryptophan-selective conjugation reported so far, Francis and co-workers exploited a vinyl rhodium carbenoid to functionalize tryptophan-containing proteins (Figure 1 A).[8a,b] Though this approach is selective for tryptophan, it yields two regioisomers due to functionalization of either the 1- or 2-position of the tryptophan indole ring. Hence, development of a strictly chemo- and regioselective methodology for tryptophan conjugation under mild conditions that is also amendable for production scale is still desirable. [a] Dr. M. B. Hansen, Dr. F. Hub‚lek, Dr. T. Hoeg-Jensen Novo Nordisk A/S Novo Nordisk Park, 2760 M”løv (Denmark) E-mail: [email protected] [b] Prof.Dr. T. Skrydstrup Carbon Dioxide Activation Center (CADIAC) Interdisplinary Nanoscience Center and Department of Chemistry Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C (Denmark) Supporting information and ORCID from the author for this article are available on the WWW under http://dx.doi.org/10.1002/chem.201504462. Chem. Eur. J. 2016, 22, 1572 – 1576

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Figure 1. Previous and current work on selective tryptophan (Trp) or indole modifications.

Recently, Waser and co-workers described how hypervalent iodine reagents developed in their laboratory can be used to ethynylate various heterocycles under gold or palladium catalysis (Figure 1 B).[10] Inspired by this work, we initiated a program on the regio- and chemoselective ethynylation of tryptophan residues using one of these hypervalent iodine reagents, 1[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1 H)-one (TIPS-EBX, 1; Figure 1 C). The ethynyl moieties introduced in this manner might serve as orthogonal handles in subsequent bioconjugation reactions, such as Sonogashira,[11] Glaser–Hay,[12] or alkyne–azide “click” reactions.[13] Herein, we describe our efforts towards the ethynylation of simple tryptophan derivatives and tryptophan residues in small peptides and proteins. We show that the installation of ethynyl functionalities in various peptides and in a model protein can be performed under mild and partial aqueous conditions. Furthermore, the newly introduced alkyne moiety was used in a proof-of-concept alkyne–azide click reaction to introduce a fluorescent label into a model peptide. We set out by screening various catalysts for their ability to catalyze ethynylation of the model compound Ac-l-Trp-NH2 using the Waser reagent (TIPS-EBX) (Table 1 and Tables S1 and S2 in the Supporting Information). Previously, Waser and coworkers demonstrated that the N-methylated indole could be ethynylated selectively at the C2-position when a palladium catalyst was used,[10c] whereas a gold catalyst promoted C3-selectivity.[10a] Hence, it was expected that palladium might be a suitable catalyst for the ethynylation of tryptophan, in which the C3-position is blocked. Regrettably, when [Pd(MeCN)4][BF4]2 was used as the catalyst, no conversion was observed at all (Table 1; entry 1). The rationale for this surprisingly poor conversion is not known at present, though binding of the palladium catalyst to the indole nitrogen, and thereby deactivating the catalyst, might play a role as suggested for other palladium catalyzed reactions.[14] Of all the catalysts tested here (for

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Communication Table 1. Optimization of ethynylation of Ac-l-Trp-NH2.[a]

Entry

Catalyst

Solvent

Conversion[b]

1 2 3 4 5 6 7 8 9 10 11

[Pd(MeCN)4][BF4]2 [Cu(MeCN)4][BF4] HAuCl4·3H2O AuCl [AuCl(SMe2)] [Ph3PAuNTf2] [AuCl(SMe2)] [AuCl(SMe2)] [AuCl(SMe2)] [AuCl(SMe2)] [AuCl(SMe2)]

MeCN MeCN MeCN MeCN MeCN MeCN DMSO MeOH THF CH2Cl2 toluene

0% 0% 2% 37 % 76 % 19 % 33 % 56 % 22 % 24 % 69 %

[a] Reaction conditions: TIPS-EBX (1.2 equiv), catalyst (0.1 equiv), and solvent with 1 % TFA at RT for 20 h. [b] Determined by UPLC-MS by integration of normalized peaks at 290 nm.

a more comprehensive list, see Tables S1 and S2 in the Supporting Information), only gold(I) catalysts led to the formation of the desired 2-(TIPS-ethynyl)-Ac-l-Trp-NH2 product in significant amounts (entries 4–11). In addition, a non-exhaustive evaluation of different gold(I) ligands revealed that a weakly coordinating ligand, Me2S (entry 5), was preferred over a strongly coordinating ligand, PPh3 (entry 6), or no ligand at all (entry 4). MeCN was the preferred solvent (entries 7–11). The reaction conditions were optimized one by one, thus identifying the following optimal conditions: gold catalyst (20 mol %), TFA (2 %), and TIPS-EBX (3 equiv) at 20 8C (Figure S1 in the Supporting information). These four parameters were combined and applied to Ac-l-Trp-NH2, as well as to other indole-containing model compounds (Table 2). First of all, com-

tively even with catalytic amounts of gold (entry 5). Remarkably, the quantitative ethynylation of melittin could also be achieved at larger scale (500 mg), although stoichiometric amounts of gold had to be used in this case. To shed light onto the TIPS-EBX-mediated functionalization of tryptophan residues, the Ac-l-Trp-NH2 ethynylation product was analyzed by NMR (see the Supporting Information), which revealed that only one regioisomer (2-(TIPS-ethynyl)-Ac-l-TrpNH2) was formed. Further, MS/MS analysis of the TIPS-ethynyl melittin revealed that only the single tryptophan residue in melittin had been functionalized (Figure S2 in the Supporting Information). To the best of our knowledge, this is the first example of a reaction, which allows for both chemo- and regioselective functionalization of tryptophan residues in a complex biomolecule enabling very defined conjugation of entities to these sites. Next, the TIPS-EBX reagent was tested on a more complex biomolecule, horse heart apomyoglobin 2 (myoglobin without its prosthetic heme group). Unfortunately, no functionalization was observed under strictly aqueous conditions. However, when a MeCN/water (3:1) mixture was used as solvent, excellent conversion to the mono- and di-functionalized apomyoglobin 3 (25 % and 67 %, respectively) was obtained (Figure 2 A). MS/MS analysis confirmed that only the two trypto-

Table 2. Ethynylation of various peptides.[a]

Entry

Peptide

Equivalents of [AuCl(SMe2)]

Conversion[b]

1 2 3 4 5 6 7 8 9

Ac-l-Trp-NH2 Ac-l-Ala-l-Trp-OH KHWD KGGFGGRGGFGGKW Melittin Ac-l-Trp-NH2 Ac-l-Ala-l-Trp-OH KHWD KGGFGGRGGFGGKW

0.15 0.15 0.15 0.15 0.15 1.0 1.0 1.0 1.0

95 % 85 % 25 % 60 % 99 % 99 % 99 % 99 % 82 %

Figure 2. Ethynylation of apomyoglobin: A) The two Trp residues in apomyoglobin are shown in green and the TIPS-ethynyl moiety is illustrated in red; B) analytical UPLC-MS trace at 214 nm of the reaction mixture, in which the modified apomyoglobin was identified.

[a] Reaction conditions: TIPS-EBX (3 equiv) and MeCN with 2 % TFA at RT for 20 h. [b] Conversion to the respective TIPS-ethynyl product determined by UPLC-MS by integration of normalized peaks at 290 nm.

bination of the individually optimized parameters improved the conversion of Ac-l-Trp-NH2 even further reaching up to 95 % (entry 1). When the more complex peptide models Ac-lAla-l-Trp-OH, KHWD, and KGGFGGRGGFGGKW were subjected to the same conditions, moderate conversions were achieved (entries 2–4). However, increasing the amount of gold catalyst to stoichiometric amounts led to almost quantitative conversions (entries 6–8). Interestingly, the largest peptide included in this study, melittin (2846 Da), was converted almost quantitaChem. Eur. J. 2016, 22, 1572 – 1576

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phan residues in the apomyoglobin were functionalized, and that the mono-functionalized apomyoglobin consisted of a mixture of the two regioisomers as expected (Figure S4 and S5 in the Supporting Information). Currently, the methodology is only suitable for peptides and robust proteins that tolerate organic solvents. Therefore, it would be desirable to adapt this chemistry to strictly aqueous conditions, thereby allowing ethynylation of a wider range of proteins. Having realized that TIPS-ethynyl can be installed in various peptides and proteins, we sought to demonstrate the use of this handle in conjugation reactions using TIPS-ethynyl melittin as the model compound (Figure 3). First, the TIPS group was removed from TIPS-ethynyl melittin 5 by means of polymersupported fluoride affording a free alkyne handle, as characterized by UPLC-MS. Next, dansyl-TEG-azide, a model fluorophore,

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Communication omitted at the expense of the yield) producing a clear solution. Then [AuCl(SMe2)] (52.3 mg, 117 mmol) was added and the reaction was stirred overnight at room temperature. Next day, solid NaI (approx. 100 mg) was added to the yellow solution followed by a few drops of sodium ascorbate (1 m in water). NaI was added to dissolve protein-Au-TIPS-ethynyl complexes and ascorbate were added to reduce any iodine formed. MeCN was removed in vacuo and the residue was redissolved in water/AcOH (MeCN and/or DMSO may be added if necessary to dissolve the crude product). The suspension was centrifuged for 4 min at 4500 rpm producing a black precipitate and clear supernatant, which was purified by preparative HPLC. The pure fractions were pooled and lyophilized affording the desired TIPS-ethynyl protein.

Acknowledgements Dr. Martin B. Mìnzel is acknowledged for the preparation of KGGFGGRGGFGGKW. Keywords: alkynylation · C¢H activation · chemoselectivity · gold · peptides

Figure 3. Fluorescent labeling of melittin by the ethynylation and azide– alkyne cycloaddition: A) Schematic representation of the ethynylation of melittin followed by deprotection and conjugation of the dansyl by CuAAC; B) analytical UPLC-MS at 214 nm of the reaction mixtures. Note that the 2-iodobenzoic acid is formed during the TIPS-EBX ethynylation reaction.

was clicked to the ethynyl-functionalized melittin 6 by a copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Gratifyingly, the standard CuAAC conditions[15] using a copper(I) generated in situ gave the desired dansyl-triazole-melittin conjugate 7 at a satisfactory conversion of 75 %, as confirmed by UPLC-MS (Figure 3 B). Thus, by combination of the TIPS-EBX and CuAAC, it was possible to introduce a fluorescent-labeled site specifically in melittin. In summary, we have described the first example of a methodology that allows for the chemo- and regioselective functionalization of tryptophan residues in peptides and robust proteins. This was realized by means of Waser’s recently developed hypervalent iodide reagent, TIPS-EBX, under gold catalysis leading to the installation of a TIPS-ethynyl moiety. We further demonstrated that the introduced ethynyl function could be used in a CuAAC conjugation reaction, thereby emphasizing the important potential of TIPS-EBX as a valuable tool in bioconjugation. Our ongoing work is focused at refining the methodology allowing for the reaction to be run not only under partial aqueous conditions but also under completely aqueous conditions.

Experimental Section General procedure for TIPS-ethynylation of proteins The protein (117 mmol) and TIPS-EBX (228 mg, 532 mmol) were dissolved in MeCN/water (3:1, 250 mL) with TFA (2 %; it could be Chem. Eur. J. 2016, 22, 1572 – 1576

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Chemo- and Regioselective Ethynylation of Tryptophan-Containing Peptides and Proteins.

Ethynylation of various tryptophan-containing peptides and a single model protein was achieved using Waser's reagent, 1-[(triisopropylsilyl)ethynyl]-1...
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