Article pubs.acs.org/bc
Site-Selective Lysine Modification of Native Proteins and Peptides via Kinetically Controlled Labeling Xi Chen,†,‡ Kasturi Muthoosamy,† Anne Pfisterer,§ Boris Neumann,∥ and Tanja Weil*,†,‡,§ †
Institute of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore § Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany ∥ Proteome Factory AG, Magnusstr. 11, Haus 2, 1.OG, 12489 Berlin, Germany ‡
S Supporting Information *
ABSTRACT: The site-selective modification of the proteins RNase A, lysozyme C, and the peptide hormone somatostatin is presented via a kinetically controlled labeling approach. A single lysine residue on the surface of these biomolecules reacts with an activated biotinylation reagent at mild conditions, physiological pH, and at RT in a high yield of over 90%. In addition, fast reaction speed, quick and easy purification, as well as low reaction temperatures are particularly attractive for labeling sensitive peptides and proteins. Furthermore, the multifunctional bioorthogonal bioconjugation reagent (19) has been achieved allowing the site-selective incorporation of a single ethynyl group. The introduced ethynyl group is accessible for, e.g., click chemistry as demonstrated by the reaction of RNase A with azidocoumarin. The approach reported herein is fast, less labor-intensive and minimizes the risk for protein misfolding. Kinetically controlled labeling offers a high potential for addressing a broad range of native proteins and peptides in a site-selective fashion and complements the portfolio of recombinant techniques or chemoenzymatic approaches.
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accessible disulfide bonds24,25 or the N-terminus of a protein.26 However, nonphysiological reaction conditions, lack of accessible disulfide bonds for intercalation, or N-termini not suitable for further chemical modifications often represent prevalent limitations. Frequently, protein mixtures of the native protein and the corresponding modified protein are obtained, as achieving either high conversions or facile purifications is often rather challenging. Herein, lysine residues that are abundant in native proteins and peptides are targeted site-selectively under kinetically controlled modification conditions. Following this approach, the proteins ribonuclease A (RNase A) and lysozyme C as well as the peptide hormone somatostatin (SST-14) have been successfully decorated with a single substituent in a highly siteselective fashion. In addition, the multifunctional linker biotinTEO-ethynyl-NHS (Figure 8a, 18, TEO: tetraethylene oxide) has been designed that allows site-selective introduction of a bioorthogonal substituent at the protein surface. The kinetically controlled chemical labeling strategy reported herein benefits from a convenient reaction scheme and straightforward purification and it opens up various opportunities for precisely functionalizing proteins and peptides.
INTRODUCTION In recent years, there has been an emerging interest in the siteselective functionalization of proteins.1 Major applications of modification approaches include the improvement of pharmacokinetic properties of therapeutic proteins by adding polymer chains,2,3 glycoslation of proteins,4 attachment of chromophores for studying protein dynamics5 or protein folding,6,7 fabrication of protein microarrays,8,9 and tailoring novel biohybrid materials such as protein−polymer amphiphiles.10 However, native protein labeling usually lacks site-selectivity since it is often challenging to address a single functional group only. In most cases, cysteine point mutations are introduced to allow site-specific attachment of functional groups.11−13 Recently, the incorporation of noncanonical amino acids via either selective pressure incorporation14,15 or nonsense coden suppression16 allows introduction of functional groups that facilitate bioorthogonal reactions17,18 such as click reactions,19 Staudinger ligations,20 or Sonogashira cross-coupling reactions21 on proteins. Additionally, proteins have also been functionalized site-specifically via enzymatic post-translational modifications.22 Most recently, the recombinant maltose binding protein has been modified site-specifically by applying a dirhodium metallopeptide catalyst utilizing a proximity-driven concept.23 Although biological approaches have been successful, the development of efficient and generally applicable chemical procedures toward site-specific modification of native proteins is still elusive. There have been few attempts such as targeting © 2012 American Chemical Society
Received: October 14, 2011 Revised: February 3, 2012 Published: February 17, 2012 500
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EXPERIMENTAL PROCEDURES Materials. D-biotin-amido-caproate-N-hydroxysuccinimidyl ester (Biotin-LC-NHS) (≥98%) for protein biotinylation was received from Sigma-Aldrich (Cat. No. B2643). Ribonuclease A (RNase A) and lysozyme from hen egg white (lysozyme C) were obtained from MP Biomedicals, LLC (Cat. No. 101076) and Fluka Biochemika (Cat. No. 62971), respectively. Somatostatin (SST-14) was supplied by GL Biochem (Shanghai). 10× Phosphate buffered saline, ultra pure grade, was purchased from First BASE (Cat. No. 2040). Pierce Monomeric Avidin Kit for affinity chromatography and Sephadex G-15 Gel for gel filtration were from Thermo Scientific (Cat. No. 20227) and Sigma-Aldrich (Cat. No. G15120), respectively. Bicinchoninic acid (BCA) assay kit for protein quantification was obtained from Novagen (Cat. No. 71285−3). UV analysis of protein fractions during gel filtrations and absorbance determinations for BCA assays were recorded on a BioTEK SYNERGY 4 Microplate Reader. All MALDIToF-MS spectra of the protein samples were recorded on an Autoflex MALDI-ToF (Bruker Daltonics) mass spectrometer or Applied Biosystems 4700 Proteomics Analyzer 88 using sinapinic acid solution as matrix. nanoLC-MS2 analysis of modified proteins were accomplished at Proteome Factory AG (Germany). NuPAGE Novex 4−12% Bis-Tris gel and its corresponding NuPAGE MES SDS running buffer for gel electrophoresis were purchased from Invitrogen. Baker’s yeast ribonucleic acid (S. cerensiae) (yRNA) was purchased from Sigma-Aldrich (Cat. No. R6750). SYBR Safe DNA gel stain (10 000× concentrated) in DMSO was obtained from Invitrogen Molecular Probes (USA). Ultrapure grade Tris (Cat. No. 1400) and ultrapure grade EDTA (Cat. No. 1050) were purchased from First BASE. 384-well black microtiter plates for conducting the RNase A functional assay and sterile 96-well plate were received from Greiner Bio-One Ltd.. (UK) and Nunclon Surface (Denmark), respectively. Site-Selective Biotinylation of RNase A (1) and Lysozyme C (4). Kinetically controlled site-selective biotinylation labeling includes six major steps and the procedure is briefly summarized in Scheme S1. Under minimized light exposure, 1 mL of freshly prepared protein solution (72.5 μM, 72.5 nmol) in PBS buffer (0.1 M, pH 7.2) was added into a 14 mL aluminum foil coated Epppendorf tube. Fresh biotin-LCNHS solution (16.6 μg, 36.3 nmol) in dry DMSO (50 μL) was added by a 2.5 μL pipet with portions of 0.5 μL per addition per minute and immediately followed by an intensive vortex after capping. The addition was finished within 100 min and the reaction solution was allowed to shake at RT for an additional 1 h (Step I: the bioconjugation step). Subsequently, 33.2 μL of an ethanolamine solution (2.66 mg mol−1, 1.45 μmol, 20 equiv relative to protein) were added into the reaction solution in PBS buffer (pH 7.2) in order to quench any traces of remaining unreacted biotin-LC-NHS (Step II: the quenching step). After overnight incubation, any biotin-containing small molecules were removed from the protein solution by gel filtration using Sephadex G-15 gel (1.5 kD cut off, Step III: the first biotinremoval step). The purified protein mixture solution was loaded onto a monomeric avidin resin column. Excess of native protein was first recovered by eluting the resin column with PBS buffer (pH 7.2), and then, the amount of recovered protein in the eluent was quantified by a BCA assay (Step IV: protein recovery step). Subsequently, biotinylated protein was obtained by eluting the resin column with PBS buffer (pH 7.2)
containing additional 2 mM of biotin (Step V: affinity elution step). Gel filtration via Sephadex G-15 facilitated the separation of (+)-biotin as well as desalting of the monobiotinylated protein thus yielding the pure monobiotinylated protein in solution. The protein concentration was determined by the BCA assay (Step VI: the second biotin-removal step). The calculated labeling yield for biotin-LC-(K1)RNase A and biotinLC-(K1)lysozyme C by considering the recovered native protein was determined as 92% (197 μg) and 94% (226 μg), respectively. Since biotin-LC-NHS is not readily water-soluble, minor amounts of DMSO (5% DMSO) were required. However, the functional assay of the recovered RNase A revealed that the enzymatic activity was retained completely. Site-Selective Biotinylation of SST-14 (6) at K9. A solution of biotin-LC-NHS (64.6 μg, 0.142 μmol) in DMF (0.324 mL) was added via an autosyringe pump during 200 min into a solution of SST-14 (6, 0.775 mg, 0.473 μmol) in PBS buffer (0.1 M, pH 7.2, 10 mL) at 4 °C under stirring. The resultant solution was further stirred at 4 °C overnight. The reaction solution was analyzed by MALDI-ToF-MS spectroscopy using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix, revealing the monobiotinylation of SST-14. Herein, DMF was used instead of DMSO in order to avoid the solidification of DMSO at 4 °C. Site-Selective Ethynylation of RNase A (1) Using Biotin-TEO-Ethynyl-NHS (18). The general procedure for the ethynylation of RNase A is similar to the biotinylation protocol. NHS ester (18) (6.8 mg, 5.85 μmol, 64% NHS ester content according to 1H NMR,) was dissolved in 0.5 mL dry DMSO and this solution was added into 10 mL solution of RNase A in PBS buffer (1 mg mL−1, 0.725 μmol) in 100 equal portions during 100 min. Then, the solution was stirred at RT for additional 1 h and 332 μL of an ethanolamine solution (2.66 mg mL−1, 14.5 μmol) was added to quench any unreacted NHS ester. The resultant solution was shaken at RT overnight. Due to the loading capacity limit of the Monomeric Avidin Kit, 2.6 mL of protein solution was purified sequentially by gel filtration, affinity chromatography, and gel filtration, and lyophilized to afford 323 μg of monoethynylated RNase A. 1.82 mg of native RNase A was recovered and thus 777 μg of RNase A was consumed. Therefore, the labeling yield of monoethynylated RNase A was calculated as 40%. It is noteworthy that 8 equiv of biotin-TEO-ethynyl-NHS (18) was used rather than 0.5 equiv since biotin-TEO-ethynylNHS (18) reacts more slowly than biotin-LC-NHS and, in addition, autohydrolysis takes place more rapidly most likely due to the enhanced water solubility of this reagent. This deactivation of 18 during the bioconjugation process reduces the effective amount of 18 in solution. Therefore, only a limited amount instead of an excess of 18 is available for reaction with RNase A. In order to minimize the autohydrolysis of 18, minor amounts of DMSO were used to dissolve 18 instead of choosing pure water.
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RESULTS AND DISCUSSION RNase A Is Site-Selectively Modified at K1 under Kinetically Controlled Conditions and Preservation of Enzymatic Acitivity. Ribonuclease A (RNase A, 1) is selected for site-specific modification since this enzyme is stable, wellcharacterized, and of therapeutic relevance as one of its close variants, onconase, is under clinical investigations as a potential cancer therapeutic.27 RNase A carries ten lysine residues at its surface. In order to assess whether there is a preference for a 501
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Figure 1. (a) Reaction scheme of the kinetically controlled labeling reaction of native RNase A (1) by applying biotin-LC-NHS (2); (b) the MS spectrum of native RNase A (1) (M.W. 13 681 g mol−1); the MS spectra of biotinylated RNase A solution after one-portion addition of 10 equiv, 1 equiv, or 0.5 equiv of 2 for 2 h (c), (d), and (e), respectively; the MS spectrum of biotinylated RNase A solution by adding 0.5 equiv of 2 in 100 equal portions during 100 min and reacted for further 1 h; the MS spectrum of the affinity purified monobiotinylated RNase A (3, M.W. 14 020 g mol−1).
been divided into 100 equal portions and added stepwise during 100 min into the RNase A solution in PBS buffer. Under these conditions, only 0.01 equiv of 2 is added into protein solution at each time point, ensuring that RNase A always remains in excess compared to 2. After completion of the addition of 0.5 equiv of 2, the reaction solution is further incubated for 1 h followed by a MALDI-ToF-MS analysis. This time, only monobiotinylated RNase A is formed (Figure 1f). In case larger quantities of 2 are added in such a stepwise fashion, the formation of the bis-modified byproduct becomes evident, particularly if more than 0.7 equiv of 2 are added (data not shown). Therefore, 0.5 equiv of biotin-LC-NHS (2) relative to the amount of protein represents the best compromise of selectivity and protein yield. Thereafter, the reaction solution is quenched by addition of ethanolamine solution and incubated overnight in order to deactivate any remaining traces of unreacted 2. This is an essential step since it has been found that remaining unreacted NHS ester interacts with monobiotinylated RNase A after affinity purification, which again decreases the quality of monobiotinylated RNase A due to the formation of byproducts. Such deactivated biotin-containing small molecules are removed via gel-filtration and the protein solution which exclusively contains excess native RNase A and monobiotinylated RNase A is subjected to affinity purification. Monofunctionalized RNase A (3) is isolated from the native protein based on the highly specific interaction between biotin and the monomeric egg white protein avidin. Monomeric avidin based affinity chromatography is chosen since it allows elution of proteins under mild conditions at physiological pH, thus minimizing the risk for protein denaturation.28 The isolated biotinylated RNase A exhibits a single peak with m/z 14021 [M +H]+ (Figure 1g, Δ m/z 339.7 relative to RNase A), which
single lysine residue, RNase A is reacted with the biotinylation reagent biotin-LC-NHS (2, Figure 1a) bearing an aminereactive NHS group as well as a biotin tag for affinity purification. First, 10 equiv of biotin-LC-NHS (2) is added in a single portion to RNase A in PBS buffer (pH 7.2) at RT and the reaction is stirred for 2 h. The resulting MALDI-ToF-MS spectrum reveals a typical modification pattern as typically found in normal protein labeling reactions using NHS esters in excess (Figure 1c) and a mixture of bis-, tris-, tetrakis-, pentakis-, and heptakis- biotinylated RNase A has been obtained. However, by decreasing the equiv of biotin-LC-NHS (2) relative to RNase A, a more selective labeling should be achieved since more reactive and accessible residues should react faster. Therefore, the amount of 2 is first decreased from 10 equiv to 1 equiv and is directly added into RNase A in one portion. After 2 h of reaction, aside from the unreacted native RNase A, mono-, bis-, and minor amounts of tris-biotinylated RNase A are found, suggesting an increase of selectivity (Figure 1d). Inspired by this result, the amount of biotin-LC-NHS (2) is further decreased from 1 equiv to 0.5 equiv and is added into RNase A PBS solution in one portion. Obviously, the selectivity is further increased, and apart from unreacted RNase A (1), monobiotinylated RNase A (3) is obtained as the major product, accompanied with minor amounts of bis-labeled product (Figure 1e). In order to improve the selectivity of the labeling reaction, the ratio of biotin-LC-NHS (2) relative to RNase A could be further decreased. However, such an approach would substantially decrease the labeling yield, which is not favorable since proteins are usually expensive. Therefore, the total amount of 2 was kept to 0.5 equiv, but the mode of the addition of 2 was changed. 0.5 equiv of biotin-LC-NHS (2) has 502
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matches the calculated M.W. of 14020 g mol−1. It is noteworthy that no traces of bis- or higher biotinylated RNase A proteins have been identified by applying high-resolution MALDI-ToFMS. Excessive amounts of RNase A could be easily recovered during the affinity chromatography step at physiological pH in order to avoid protein loss during modification. Briefly, after loading the native and biotinylated RNase A mixture onto the monomeric avidin resin column, the unreacted native RNase A is readily recovered by eluting the resin column with PBS buffer (0.1 M, pH 7.2) at RT. Subsequently, the biotinylated RNase A is obtained by eluting the resin column a second time with PBS buffer (0.1 M, pH 7.2) which contains additional 2 mM of (+)-biotin. According to BCA assay quantifications, out of 1 mg native RNase A, about 791 μg RNase A has been recovered and only 209 μg of RNase A (1) was consumed during the reaction. In this way, by considering the recovery of RNase A, a high yield of about 92% (197 μg) of monobiotinylated RNase A (3) has been obtained. Noteworthy, the recovered RNase A displays similar enzymatic activity, as the initial RNase A and no activity loss after recovery has been observed (Figure 4). Thereafter, the lysine residue carrying the biotin substituent is determined by nanoLC-tandem mass spectrometry in the high resolution mode to prove whether only the most reactive lysine residue has reacted site-selectively. After in-gel digestion of monobiotinylated RNase A (3) by chymotrypsin, peptide fragments are obtained and subjected to nanoLC-MS2 whereby each peptide fragment is fractionated by nanoLC and identified by MS2. The peptide fragment KETAAAKF (1−8) with m/z 1203.63 [M+H]+ carries the biotin-LC moiety since it corresponds to the calculated M.W. of 1202.62 g mol−1. Since this peptide contains two lysine residues, an additional MS2 experiment has been recorded demonstrating that K1 rather than K7 is modified due to the presence of the fragments b 1 (Figure 2, modification at K1) and y 7 (Figure 2, no modification in ETAAAKF (2−8)) (Figure 2).
chromatogram (Figure 3a, bottom), thus demonstrating that every K1 residue of KETAAAKF (1−8) has been modified. This observation together with the MALDI-ToF-MS spectrum revealing only monofunctionalized RNase A (Figure 3b) indicates that biotinylation occurs at K1 only. In this way, site-selectivity is demonstrated yielding biotin-LC-(K1)RNase A (3) under mild reaction conditions. The catalytic activities of biotinylated RNase A as well as the recovered native RNase A are investigated in a functional RNase assay using Baker’s yeast ribonucleic acid (yRNA) as substrate. yRNA forms a fluorescent complex in the presence of SYBR Safe DNA gel stain dye, and RNase A quickly degrades yRNA and destroys the fluorescent complex leading to fluorescence decay (Figure 4a). The initial speed of the fluorescence decay is proportional to the enzymatic activity and autohydrolysis of yRNA in the absence of RNase A is determined as control (green rhombic dots). According to Figure 4b, both native RNase A (brown triangular dots) and the recovered RNase A (orange rectangular dots) quickly hydrolyze yRNA at the same rate, suggesting no significant loss of enzymatic activity of the recovered RNase A. Bbiotin-LCRNase A (3) also hydrolyzes yRNA leading to a decrease of the relative fluorescence unit (RFU) in the beginning but with a slightly reduced velocity (violet foursquare dots). According to the initial slopes of the blank control (k1 −0.07), biotin-LCRNase A (k2 −0.56), and native RNase A (k3 −0.63), the relative activity of biotin-LC-RNase A in relation to native RNase A is calculated to be (k2 − k1)/(k3 − k1) = 88%. This indicates that modified RNase A still maintains significant catalytic activity, and minor activity loss might be attributed to steric hindrance due to the presence of the biotin-LC moiety. Lysozyme C (4) Is Mono-Biotinylated at K1 under Kinetically Controlled Conditions. In order to demonstrate the general applicability of this kinetic chemical labeling scheme, the protein lysozyme from hen egg white (lysozyme C) (4) as well as the peptide hormone somatostatin (SST-14) (6) are biotinylated in a similar fashion. Lysozyme C reacts with peptidoglycans and hydrolyzes the glycosidic bond that connects N-acetylmuramic acid with the fourth carbon atom of N-acetylglucosamine. It has been applied successfully as a biopharmaceutical in many aspects, such as treating bactofugation, rendering modified lysozyme C products attractive for medicinal applications. Lysozyme C contains six lysine residues in total, and to the best of our knowledge, entirely chemical approaches toward site-selective modification of lysozyme C have not been achieved before. As described previously, lysozyme C (4) is treated with biotin-LC-NHS (2) under the same conditions used for RNase A (1) labeling (Figure 5a). The MALDI-ToF-MS spectrum (Figure 5b) confirms monobiotinylation yielding biotin-LC-lysozyme C (5) with a corresponding molecular mass signal at m/z 14 644 [M+H]+ (Figure 5b, right, calculated M.W. of 5 is 14 643 g mol−1 with a difference of 339.5 relative to native lysozyme C with m/z 14 305). According to nanoLC-MS2 together with XIC analysis (Figure 5c), K1 is identified as biotinylation site of lysozyme C. Starting from 1.04 mg of native lysozyme C (4), 764 μg of native lysozyme C have been recovered and 226 μg of biotinLC-lysozyme C (5) have been isolated corresponding to a calculated yield of about 94% by considering the amount of recovered lysozyme C. SST-14 Is Mono-Biotinylated at K9 under Kinetically Controlled Conditions. SST-14 represents a cyclic peptide hormone consisting of 14 residues, featuring a single disulfide
Figure 2. Full interpretation of the MS2 spectrum of biotinylated peptide fragment KETAAAKF (1−8) demonstrating that the modification occurred at K1 rather than at K7.
It is noteworthy that peptide fragments bearing cysteine residues are not detected directly, since they are generally lost during in-gel digestion due to their Michael addition reactions with the polyacrylamide gel. In order to ensure that monobiotinylation has occurred only at K1 of KETAAKF (1−8), extracted ion chromatography (XIC) of both modified and unmodified KETAAKF (1−8) is performed (Figure 3a). The biotinylated KETAAAKF fragment exhibits a predominant peak in its XIC chromatograph at tR 32.55 min (Figure 3a, top) and no unmodified KETAAAKF (1−8) is detected in its XIC 503
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Figure 3. Demonstration of the site-selectivity. (a) The XIC chromatograph of the K1 biotinylated peptide fragment KETAAAKF (1−8) (up) and the XIC chromatograph of unmodified KETAAAKF (1−8) (down) indicate the formation of biotinylated KETAAAKF (1−8) and the absence of unmodified KETAAAKF fragments (1−8) suggesting that all K1 residues carry a single biotin group. Taking this information as well as monomodification of RNase A (Figure 3b) into account confirms the site-selectivity of the modification under kinetically controlled conditions.
evidence is obtained from the presence of the b 8 ion (proofing that A1 and K4 remain unmodified), the b 9 ion (indicating biotinylation at K9), and the y 5 ion (proof that TFTSC (10− 14) remains unmodified). Thus, the site-selective modification of the small, native SST-14 peptide has been achieved at K9. Solvent Accessibility as well as Interaction with Adjacent Amino Acids Might Contribute to the Observed Site-Selectivity of Kinetically Controlled Chemical Labeling. It has been demonstrated above that different lysine residues of proteins and peptides reveal significant alterations of their individual reactivities. In order to gain some additional insight into this phenomenon, we have carefully examined the crystal structure of RNase A and lysozyme C (Figure 7). First, the solvent accessibility29 of all lysine residues of RNase A and lysozyme C is calculated by implementing 1RCA.pdb and 2ZYP.pdb into the software package Molecular Operating Environment (MOE, Chemical Computing Group). This analysis recognizes the steric hindrance in the surrounding of the respective lysine residues that might affect their relative reactivities. In the case of RNase A, the modified K1 residue is indeed the lysine residue showing the highest solvent accessibility (Figure 7a). Interestingly, the modified K1 residue of lysozyme C does not display the highest solvent accessibility, since according to the crystal structure of lysozyme C, K97 should be more accessible (Figure 7b, bottom). After close examination of the chemical environment of K1, a hydrogen bond interaction between the hydrogen atom of the ε-amino group of K1 and the oxygen of the adjacent carboxylic acid group of E7 is recognized (Figure 7b, top). Due to this interaction, the amino group of K1 might be less positively charged and thus more nucleophilic and reactive. However, these considerations are still very premature and higher-level calculations are required to elucidate the origin of the reactivity of different lysine residues. However, such studies assessing the reactivity of different amino acid residues on the surface of proteins might be of broad interest to allow a better understanding of post-translational modifications. Kinetically Controlled Chemical Labeling Allows Introduction of an Ethynyl Group onto RNase A Facilitating Site-Specific Click Reaction. In the last step, the more sophisticated, multifunctional bioconjugation linker biotin-TEO-ethynyl-NHS (18, Figure 8a) is designed for integrating a single bioorthogonal ethynyl group onto K1 of RNase A. Biotin-TEO-ethynyl-NHS (18) bears a biotin tag for purification, an ethynyl group that allows click reactions, an amine-reactive NHS ester for protein functionalization, and a
Figure 4. Functional RNase A assay. (a) General scheme of the functional assay: Nonfluorescent SYBR dye forms a fluorescent complex with yRNA which is subsequently decomposed by active RNase, yielding the nonfluorescent SYBR dye; (b) the functional assay diagram: The green rhombic dots represent autohydrolysis of yRNA; the violet foursquare dots suggest the enzymatic activity of biotin-LCRNase A (2); the brown triangular dots and the orange rectangular dots reveal the activity of native RNase A and recovered RNase A, respectively.
bond (Csy3-Cys14), and contains three amino groups (Lys 4, Lys 9, and the N-terminus of Ala, Figure 6b). SST-14 plays an important role in many physiological processes, and its analogue, octreotide (Novartis Pharmaceuticals) with a higher stability, is in clinical use. Modification of this peptide hormone is of therapeutic interest to increase metabolic stability and it also offers the advantage of direct nanoLC-MS2-XIC analysis without the necessity for in-gel digestion. In this way, the entire modification pattern of SST-14 can be assessed. To contribute to the low stability of SST-14 and to improve site-selectivity, the labeling reaction is performed at 4 °C by adding a total amount of 0.3 equiv of biotin-LC-NHS (2) that is injected into the SST-14 solution during a time frame of 200 min (Figure 6a). Following this procedure, SST-14 has been monobiotinylated according to the MALDI-ToF-MS spectrum (Figure 6c). After reduction of the Cys3-Cys14 disulfide by TCEP (tris-(2carboxyethyl)phosphine), the linearized peptide is directly analyzed by nanoLC-MS 2-XIC. According to the XIC chromatograph, there is only a symmetric peak corresponding to monobiotinylated SST-14 (7, Figure S3). This suggests that the modification occurs at only one residue and the MS2 spectrum confirms biotinylation at K9 only (Figure 6d). Direct 504
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Figure 5. (a) Site-selective biotinylation scheme of lysozyme C (4) by applying biotin-LC-NHS (2); (b) MALDI-ToF-MS spectra of native lysozyme C (4, left) and biotinylated lysozyme C (5, right) demonstrating monomodification (the tiny peak with a slightly higher M.W. does not indicate bis-modification; see Supporting Information); (c) the XIC chromatographs of modified KVF (1−3) (above) and unmodified KVF (1−3) (bottom) suggest full modification of K1 of lysozyme C.
Figure 6. (a) Biotinylation scheme of SST-14 (6) by biotin-LC-NHS (2); (b) chemical structure of SST-14 (6) stressing all three amino groups of A1, K4 and K9, all highlighted in green; (c) MALDI-ToF-MS spectrum of the reaction mixture indicating monobiotinylation of SST-14 (6); (d) MS2 spectrum of reduced monomodified SST-14 proves that biotinylation occurs at K9.
TEO spacer to impart water solubility. The synthesis of this bioorthogonal linker is accomplished in nine reaction steps, most of them proceeding with high yields (Figure 8b). Briefly, 1 equiv of tert-butyl 2-bromoacetate (8) is treated with 2 equiv of N-propargylamine (9) under basic condition to afford tert-butyl 2-propargylaminoacetate (10), which is
subsequently acylated by succinic anhydride to give 11. In parallel, 4,7,10-trioxa-1,13-tridecanediamine (12) is protected to afford N-Boc-4,7,10-trioxa-1,13-tridecanediamine (13) and th en coupled t o biotin via EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide) condensation yielding biotin-TEO-NH(Boc) (14). Afterward, 14 is deprotected by 505
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Figure 7. (a) Crystal structure of RNase A (PDB file: 1RCA.dpb) shows the presence of ten lysine residues of RNase A. K1 is highlighted in a violet ball-and-sphere model and exhibits the highest solvent accessibility. (b) Crystal structure of lysozyme C (PDB file: 2ZYP) reveals six lysine residues as well as a hydrogen bond interaction between K1 and E7 with a typical bond length of 2.21 Å. The relative solvent accessibility of each lysine residue of both RNase A and lysozyme C is given as column length (below) (“down” refers to accessible and “up” indicates buried inside).
Figure 8. (a) Design of the ethynylation reagent (18), featuring a biotin tag, a water-soluble TEO linker, a bioorthogonal ethynyl group, and an amine-reactive NHS ester; (b) synthetic scheme toward biotin-TEO-ethynyl-NHS (18). (a) K2CO3/DMF, RT, 48 h, 66%; (b) succinic anhydride/ DMF, RT, overnight, 79%; (c) (Boc)2O, RT, overnight, 34%; (d) (+)-biotin, EDC/DMF, RT, 48 h, 73%; (e) TFA/DCM, RT, 12 min; (f) NH3 (aq), quantitative yield from 14 to 15; (g) 11, HBTU, DIEA/DMF, RT, 48 h, 86%; (h) TFA/DCM, RT, 5 h, 70%; (i) NHS, EDC/DMF, RT, 25 h, 99%.
calculated M.W. of 14 305 g mol−1 (Figure 9b, left; M.W. of biotin-TEO-ethynyl: 623.8 g mol−1). Thereafter, the modified protein (19) is in-gel digested by chymotrypsin and analyzed by nanoLC-MS2-XIC. The MS2 spectrum of KETAAAKF (1−8) confirms the modification at K1 (Figure 9c), and the XIC chromatograph of both modified and unmodified KETAAAKF (1−8) indicates full labeling at K1 (Figure S2). Hence, ethynyl(K1)RNase A has been achieved that allows further click labeling. According to BCA assay quantification, the yield of this ethynylation reaction is about 40% again considering the recovery of native RNase A. The relatively lower yield of this reaction is attributed to the monomeric avidin resin column supplied, as well as a lower reactivity of (18). Finally, 19 is reacted with fluorogenic azidocoumarin (20) via Cu (I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction to examine the accessibility of the integrated ethynyl group for further click labeling (Figure 9a, right). The application of azidocoumarin is of great advantage to prove that the click reaction has been successful since this chromophore only emits
trifluoroacetic acid (TFA) to give biotin-TEO-NH2 (15) and then coupled with 11 by HBTU (O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate) to yield 16. Subsequently, 16 is deprotected by TFA to afford biotinTEO-ethynyl-COOH (17). In the last step, 17 is activated with NHS yielding the target molecule biotin-TEO-ethynyl-NHS (18). Noteworthy, the synthesis of bioorthogonal linkers bearing alternative reactive groups could be achieved in principle by replacing N-propargylamine (9) by a corresponding primary amine, e.g., 2-azidoethylamine without the necessity to develop a novel synthetic scheme. All reaction steps and full characterizations are given in the Supporting Information. As described above, small amounts of biotin-TEO-ethynylNHS (18) are added into a solution containing RNase A in 100 equal portions over a time period of 100 min (Figure 9a). Monoethynyl RNase A (19) is successfully isolated after purification and a predominant mass peak of m/z 14306 [M +H]+ is found in the MALDI-ToF-MS spectrum matching its 506
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Figure 9. (a) Monoethynylation of RNase A using biotin-TEO-ethynyl-NHS (18) and subsequent labeling of ethynyl-(K1)RNase A (19) via Huisgen 1,3-dipolar cycloaddition reaction (click reaction) with azido-coumarin (20); (b) MALDI-ToF-MS spectrum of native RNase A (left) and ethynylated RNase A (right) indicating monomodification; (c) the MS2 spectrum of modified KETAAAKF (1−8) proves modification at K1 rather than K7; (d) the fluorescence image of the reaction solution under UV irradiation (above), the fluorescence image of the gel (middle) and the Coomassie stain image of the gel (below) demonstrate the successful click reaction.
The approach reported herein is fast (the entire process takes about 1−2 days), less labor-intensive, presumably of low cost, and it minimizes the risk of misfolding. Complementary to cysteine mutants, highly accessible and reactive lysine groups might also be introduced into the protein sequence via point mutations, thus opening up opportunities for dual-color labeling of proteins for single molecule experiments. Our results offer a high potential for addressing a broad range of native proteins and peptides in a site-selective fashion and complements the portfolio of recombinant techniques or chemoenzymatic approaches. Future studies will focus on the application of proteins as macromolecular building blocks for the formation of ordered supramolecular assemblies.
after the triazol ring has been formed. Under UV light excitation, the reaction solution reveals bright emission after about 24 h reaction time. In contrast, control solutions without azidocoumarin (Figure 9d, left) or Cu (I) catalyst (Figure 9d, middle) do not reveal any emission (Figure 9d, above). Gel electrophoresis analysis ascertains the formation of the click product coumarin-(K1)RNase A (22) due to the intensive fluorescent band at 14−15 kD corresponding to its M.W. of 14 508 g mol−1 (Figure 9d, right). This study successfully demonstrates the great potential of biotin-TEO-ethynyl-NHS (18) for the site-selective introduction of a single reactive ethynyl group into proteins. Ethynyl groups are of particular importance since they allow a broad range of site-selective posttranslational modifications such as 1,3-dipolar Diels−Alder or Sonogashira reactions.
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ASSOCIATED CONTENT
S Supporting Information *
CONCLUSIONS In summary, the synthesis of four monofunctionalized proteins/peptide, biotin-LC-(K1)RNase A (3), biotin-LC(K1)lysozyme C (5), biotin-LC-(K9)SST-14 (7), as well as biotin-TEO-ethynyl-(K1)RNase A (19), has been presented. All biomolecules are decorated with a single substituent at a defined site based on the kinetically controlled reaction scheme. The reaction proceeds at mild conditions at physiological pH and RT as demonstrated by the retention of the catalytic activity of RNase A. In addition, fast reaction speed, quick and easy purification, as well as low reaction temperatures are particularly attractive for labeling sensitive peptides and proteins such as somatostatin or antibodies. Excess of unreacted proteins can be readily recovered without activity loss under physiological pH, contributing to the high labeling yield of over 90%. Furthermore, the bioorthogonal bioconjugation reagent, biotin-TEO-ethynyl-NHS (18), has been achieved allowing the site-selective incorporation of an ethynyl group for further siteselective labeling reactions. The introduced ethynyl group is accessible for, e.g., click chemistry as demonstrated by reacting ethynyl-(K1)RNase A with azidocoumarin.
Interpretation of XIC chromatographs, click labeling of ethynyl(K1)RNase A (19), gel filtration and affinity chromatography procedures, BCA assay protocol, RNase A functional assay protocol, and the nine-step synthesis of biotin-TEO-ethynylNHS (18) including the full NMR spectra of all key intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: (+) 49-731-50-22883. Tel: + 49-731-5022870. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank J. Wilhelmi and A. Mishra for providing the functional RNase A assay protocol. This work was financially supported by the NUS start-up grant under the Grant No. 507
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WBS-R-143-000-381-133 and the German Research Foundation (DFG) under grant P3246 029 and SFB 625.
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