Synthesis of Biologically Active Peptide Nucleic Acid-Peptide Conjugates by Sortase-Mediated Ligation
SCHEME 1. Reactiona
Sortase A-Mediated Transpeptidation
Stephan Pritz,* Yvonne Wolf, Oliver Kraetke, Jana Klose, Michael Bienert, and Michael Beyermann Leibniz-Institute of Molecular Pharmacology, Robert-Ro¨ssle-Strasse 10, 13125 Berlin, Germany a
[email protected] ReceiVed NoVember 13, 2006
Sortase A is a transpeptidase that cleaves at a pentapeptidemotif and subsequently transfers the acyl component to a nucleophile containing N-terminal oligoglycines. We investigate the reaction conditions of the sortase-mediated ligation and demonstrate a useful application by the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera, the reaction equilibrium of which can be shifted in favor of the product by dialyzing out the low molecular weight byproduct. The synthesized conjugate exhibits dose-dependent antisense activity. Sortases are transpeptidases found in Gram-positive bacteria. They catalyze a cell wall sorting reaction that attaches surface proteins to the cell wall envelope.1 In Staphylococcus aureus, the sortase isoform SrtA (sortase A) cleaves surface proteins 1 at a LPXTG-motif (leucine, proline, X, threonine, and glycine, where X is any amino acid) between threonine and glycine to form a threonyl thiol ester with the active site cysteine. The latter undergoes nucleophilic attack from the N-terminal amino group of the pentaglycine branch of Lipid II (2, n ) 5), resulting in amide bond formation between the threonine and the incoming glycine (Scheme 1 shows a generalized reaction scheme).2 In subsequent steps, this cell wall precursor becomes polymerized into mature peptidoglycan.3 In the absence of a glycine nucleophile, the acyl-enzyme intermediate is slowly hydrolyzed to form the corresponding peptide carboxylic acid and the free enzyme.2 (1) (a) Mazmanian, S. K.; Liu, G.; Ton-That, H.; Schneewind, O. Science 1999, 285, 760-763. (b) Paterson, G. K.; Mitchell, T. J. Trends Microbiol. 2004, 12, 89-95. (2) Frankel, B. A.; Kruger, R. G.; Robinson, D. E.; Kelleher, N. L.; McCafferty, D. G. Biochemistry 2005, 44, 11188-11200. (3) (a) Perry, A. M.; Ton-That, H.; Mazmanian, S. K.; Schneewind, O. J. Biol. Chem. 2002, 277, 16241-16248. (b) Ruzin, A.; Severin, A.; Ritacco, F.; Tabei, K.; Singh, G.; Bradford, P. A.; Siegel, M. M.; Projan, S. J.; Shlaes, D. M. J. Bacteriol. 2002, 184, 2141-2147.
Xaa ) any amino acid, n g 1.
Recently, sortase-mediated ligation4 was introduced as a new technique for peptide and protein ligation, complementing existing methods such as native chemical ligation,5 Staudinger ligation,6 and intein-mediated ligation.7 Peptides with one or more N-terminal glycines could be used as nucleophiles.4 Due to the low tolerance of sortase for deviation in the LPXTGrecognition motif, this enzymatic ligation is highly selective. In particular, the very limited occurrence of this motif in proteins makes the SrtA-mediated reaction interesting for protein modification. Here, we demonstrate the feasibility of this methodology investigating the reaction conditions with the synthesis of a peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (vide infra). As product 3 of SrtA-mediated coupling contains the crucial LPXTG recognition motif just like starting compound 1, it is also a potential substrate for ligation along with byproduct 4. It is, therefore, possible for the reaction to attain equilibrium. Although the transpeptidation mechanism is quite well understood as a ping-pong-bi-bi-reaction with a hydrolytic shunt,2 the presence of such an equilibrium and its role for the application of SrtA in synthesis has to date not been observed, with some reactions producing yields of up to 90%.4,8 This raises the question of which conditions control the reaction. To make this ligation synthetically useful, the equilibrium should be shifted to the product side. At first, we focused on optimizing the substrate to improve yields. Looking at the recognition motif, the N-terminal preceding residue (R1) should be of minor influence because it is not altered during the reaction. The major difference between the product 3 and the substrate 1 are the residues C-terminal of the LPXTG-motif R2 and G(n-1)R3, respectively (Scheme 1). To test the impact of the residues in the P2′ and P3′ positions, respectively, two peptide libraries were synthesized (subsite nomenclature according to Schechter and Berger).9 In both libraries, we used lysine in position X of the LPXTG-motif (Scheme 1) to ensure solubility in the reaction buffer and good detectability in mass spectrometry. In the first (Scheme 2), small dansyl-labeled peptides (5) with the (4) Mao, H. Y.; Hart, S. A.; Schink, A.; Pollok, B. A. J. Am. Chem. Soc. 2004, 126, 2670-2671. (5) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923960. (6) Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 21412143. (7) Muir, T. W. Annu. ReV. Biochem. 2003, 72, 249-289. (8) Kruger, R. G.; Otvos, B.; Frankel, B. A.; Bentley, M.; Dostal, P.; McCafferty, D. G. Biochemistry 2004, 43, 1541-1551. (9) (a) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157-162. (b) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1968, 32, 898-902.
10.1021/jo062331l CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/14/2007
J. Org. Chem. 2007, 72, 3909-3912
3909
SCHEME 2. LPKTGX1-Library, Optimizing the Residue in the P2′ Position in the Sortase-Mediated Ligationa
a
Dns ) dansyl.
SCHEME 3. LPKTGGX2-Library, Optimizing the Residue in the P3′ Position in the Sortase-Mediated Ligation
FIGURE 1. Yield of sortase-mediated ligation as shown by the
LPKTGX1-motif were made with the residue X1 in position P2′ representing the proteinogenic amino acids (with the exception of cysteine, because this residue is prone to oxidation under the experimental conditions used). As a nucleophile, pentapeptide 6 was chosen, replacing two hydrophilic arginines of the leaving group by two hydrophobic tryptophans. Thus, in all experiments, product 7 and substrate 5 were readily separable and quantifiable by reversed-phase HPLC with fluorescence detection. The results of the ligations from the first library clearly show an impact of the X1 residue (Figure 1). While glycine in this position gives the fastest and highest conversion (about 70% after 24 h), there are several hydrophobic (isoleucine, leucine, methionine, glutamine, valine) residues and one positively charged (lysine) residue that lead to conversions of about 50% after 24 h. All other amino acids show lower conversions, with proline as the poorest (below 5% after 24 h). Interestingly, most abundant in this position in naturally occurring substrates of sortase are aspartic acid (23%), glutamic acid (23%), serine (13%), glycine (12%), and threonine (7%), as revealed by a search in a database specific for sortase-substrates.10 Proline was found only once in the 792 hits. The impact of this residue is rarely mentioned in the literature.10b,11 We presume that the binding pocket S2′ is small, thus glycine fits best, but flexible, as the other substrates are processed as well. To test the importance of the residue in the P3′ position, a second library with LPKTGGX2-motif (9) was synthesized, that is, the pentapeptide-motif followed by glycine (the best hit from the first library) and any other amino acid. These substrates were reacted with the same nucleophile (6) as in the first library, leading to the same product 7 (Scheme 3). As can be seen from Figure 2, all reactions proceed equally efficiently after 24 h, showing a lack of dependence in reaction ratio at the P3′ position. To investigate whether the differences in the first library arise from different equilibrium constants or are due to kinetic effects, some substrates 5 (X1 ) G, D, L, or S) were reacted as before (10) (a) http://www.doe-mbi.ucla.edu/Services/Sortase/. (b) Comfort, D.; Clubb, R. T. Infect. Immun. 2004, 72, 2710-2722. (11) (a) Fischetti, V. A.; Pancholi, V.; Schneewind, O. Mol. Microbiol. 1990, 4, 1603-1605. (b) Janulczyk, R.; Rasmussen, M. Infect. Immun. 2001, 69, 4019-4026. (c) Boekhorst, J.; De Been, M. W. H. J.; Kleerebezem, M.; Siezen, R. J. J. Bacteriol. 2005, 187, 4928-4934.
3910 J. Org. Chem., Vol. 72, No. 10, 2007
conversion of substrate 5 to product 7 in dependence on the nature of residue X1 of the LPKTGX1-motif after 7 and 24 h (white and black bars, respectively). Conversions were determined by HPLC with fluorescence detection.
FIGURE 2. Yield of sortase-mediated ligation as shown by the conversion of substrate 9 to product 7 in dependence on the nature of residue X2 of the LPKTGGX2-motif after 7 and 24 h (white and black bars, respectively). Conversions were determined by HPLC with fluorescence detection.
and the product development was monitored over 72 h (Figure 3a). Surprisingly, the reactions proceed to almost the same product yield but with significantly different reaction rates. While glycine in this position gives the fastest reaction, the substrate with aspartic acid hardly reaches equilibrium within 72 h. To directly show the equilibrium position, we also investigated the reverse reaction starting with the peptides 7 and 8 (X1 ) G, Figure 3b), which were products in the forward reaction (Figure 3a, black crosses). As can be seen, independently from the starting point, the reaction ends up at the same state of equilibrium. Moreover, residues further away from the cleavage site seem to have an influence on the equilibrium. Substrates (DnsLPKTGGGX3X3-NH2) with three glycine residues and two arginine or tryptophan residues, respectively, following the cleavage site, were reacted with nucleophiles of the type H-GGGX4X4-NH2 with X4 being glycine, arginine, or tryptophan, respectively (Table 1). The reactions were allowed to
FIGURE 3. Equilibrium formation for sortase-mediated transpeptidation. (a) Four substrates 5 (Dns-LPKTGX1RR-NH2) were reacted with the same nucleophile 6: X1 ) G, black crosses; X1 ) D, blue filled circles; X1 ) L, red triangles; and X1 ) S, green filled squares. (b) Course of the reverse reaction. For all reactions, the portion of the same molecule (7), which is product in the left reaction (a) and substrate in the right reaction (b), is shown.
TABLE 1. Product/Educt Ratio for the Sortase-Mediated Ligation
SCHEME 4.
Synthesis of the PNA-MAP Conjugate 13
of Dns-LPKTGGGX3X3-NH2 with H-GGGX4X4-NH2 in Equilibrium
entry
substrate
nucleophile
% producta
product/educt ratioa
1 2 3 4
Dns-LPKTGGGRR-NH2 Dns-LPKTGGGRR-NH2 Dns-LPKTGGGWW-NH2 Dns-LPKTGGGWW-NH2
H-GGGGG-NH2 H-GGGWW-NH2 H-GGGRR-NH2 H-GGGGG-NH2
39 ( 2 61 ( 4 41 ( 2 31 ( 3
0.63 1.6 0.69 0.45
a
In equilibrium.
attain equilibrium and the product yield was determined. It is known that in the presence of a glycine nucleophile the ratedetermining step of the transpeptidation is the formation of the threonyl thiol ester,2 that is, the cleavage of the substrate, which means that a product/educt ratio * 1 is indicative for a different recognition of the substrate and the product by sortase. If, under equilibrium conditions, the product/educt ratio is 1, the substrate is cleaved more rapidly. Our results show that the reaction rate increases in the order W < R < G for the residue X3 of the substrate. A general problem that is often encountered results from poor solubility of substrates in aqueous buffers. Therefore, it could be useful to carry out the reaction in the presence of organic co-solvents. We tested whether dimethyl sulfoxide (DMSO) or polyethylene glycol (PEG) at different concentrations affect sortase activity compared to aqueous buffer (see Supporting Information). The addition of 20% (v/v) DMSO or PEG did not alter sortase activity. Concentrations of 40% organic solvent decreased formation of product, while at 60%, the activity is abolished. Thus, one can expect that addition of 20% DMSO or PEG to increase solubility of one or both of the educts will not affect ligation yields. CPPs ranging from simple cationic sequences to proteinderived and designed peptides (reviewed in ref 12) have shown to improve the delivery of PNAs into mammalian cells and enhance their biological activity.13 Previous attempts in our group to synthesize PNA-CPP conjugates were done by (12) (a) Snyder, E. L.; Dowdy, S. F. Pharm. Res. 2004, 21, 389-393. (b) Zorko, M.; Langel, U ¨ . AdV. Drug DeliVery ReV. 2005, 57, 529-545. (c) Langel, U ¨ ., Ed. Handbook of Cell-Penetrating Peptides, 2nd ed.; CRC Press: Boca Raton, 2006.
straightforward solid-phase peptide synthesis. However, this strategy is often prone to inefficient synthesis due to aggregation, purification is difficult, and the product is often contaminated with termination sequences (for a recent review concerning the synthesis of peptide conjugates of oligonucleotides see ref 14). Therefore, we used sortase-mediated ligation for the synthesis of a PNA-CPP conjugate (Scheme 4) to overcome purification problems by using purified starting materials. The PNA residues are flanked by three oxyethylene spacers, to reduce their aggregation tendency and to improve solubility, and are Cterminally extended by the optimized sortase recognition sequence LPKTGG. The 18mer PNA sequence (11) is targeted to the aberrant splice site of a mutated β-globin intron 2, which interrupts the coding sequence of a Luciferase reporter gene.15 The peptide to be attached is the well-known CPP MAP (model amphipathic peptide)16 with three additional N-terminal glycine residues (12). Ligation of PNA 11 and peptide 12 under the same conditions as for the optimized case in the library studies gave only a product formation of 38%, again showing that the recognition motif is important but not the only determinant for the reaction yield. To push an equilibrium reaction to the product side, there are several possibilities, like using an excess of the cheaper educt (13) (a) Pooga, M.; Soomets, U.; Ha¨llbrink, M.; Valkna, A.; Saar, K.; Rezaei, K.; Kahl, U.; Hao, J. X.; Xu, X. J.; Wiesenfeld-Hallin, Z.; Ho¨kfelt, T.; Bartfai, A.; Langel, U ¨ . Nat. Biotechnol. 1998, 16, 857-861. (b) Oehlke, J.; Wallukat, G.; Wolf, Y.; Ehrlich, A.; Wiesner, B.; Berger, H.; Bienert, M. Eur. J. Biochem. 2004, 271, 3043-3049. (c) Abes, S.; Williams, D.; Prevot, P.; Thierry, A.; Gait, M. J.; Lebleu, B. J. Controlled Release 2006, 110, 595-604. (d) Wolf, Y.; Pritz, S.; Abes, S.; Bienert, M.; Lebleu, B.; Oehlke, J. Biochemistry 2006, 45, 14944-14954. (14) Venkatesan, N.; Kim, B. H. Chem. ReV. 2006, 106, 37123761. (15) Kang, S. H.; Cho, M. J.; Kole, R. Biochemistry 1998, 37, 62356239. (16) Oehlke, J.; Scheller, A.; Wiesner, B.; Krause, E.; Beyermann, M.; Klauschenz, E.; Melzig, M.; Bienert, M. Biochim. Biophys. Acta 1998, 1414, 127-139.
J. Org. Chem, Vol. 72, No. 10, 2007 3911
the recognition motif is present only in few human proteins. Therefore, recombinant proteins bearing a C-terminal LPXTGmotif should serve as substrates for semisynthetic work, such as incorporation of marker molecules like fluorophors or other non-natural moieties. Experimental Section
FIGURE 4. Dose-dependence of antisense activity of the PNA-MAP conjugate 13. Activity of the PNA 11 and the PNA-MAP conjugate 13 in RLU per µg protein (black and white bars, respectively).
or removal of product. The leaving group 14 is small compared to the other reactants and should therefore be easily removable by dialysis. Indeed, reaction under dialysis conditions gave conversions of 61% and 94% at molar ratios of 1/1 and 1/5 (11/12), respectively. The product was readily separable by semipreparative reversed-phase HPLC and was homogeneous according to analytical HPLC and mass spectrometry. The biological activity of the PNA-MAP conjugate was investigated using the splicing-correction assay developed by Kole et al.15 As shown in Figure 4, attachment of the CPP MAP to the PNA led to an enhanced antisense activity (expressed in relative luminescence units (RLU)) in a dose-dependent manner, whereas the PNA alone remained ineffective in restoring the aberrant splicing. A conjugate with a scrambled PNA sequence did not show any activity confirming the sequence specificity of the observed effect of the PNA-MAP conjugate (data not shown). The results presented indicate that sortase-mediated ligation is a versatile technique for the ligation of unprotected peptides and proteins. Sortase A is a robust enzyme that can be easily expressed in E. coli and purified by immobilized metal affinity chromatography without the necessity of further refolding steps (see Supporting Information). It is stable and active without reducing agents (e.g., thiols), even in the presence of organic solvents such as 20% DMSO. Recognition of a substrate by sortase is influenced not only by the LPXTG-motif itself. While the residues at P4′ and/or P5′ positions influence the equilibrium position, the residue at the P2′ position has a remarkable impact on the time after which the equilibrium is achieved. We demonstrate that this reaction is also driven to the product side, removing a small leaving group by dialysis. Sortase is also able to process non-natural sequences as long as the restrictions concerning the recognition motif are fulfilled: at first, a substrate with LPXTGX1-motif (X ) any amino acid, X1 ) not proline, preferably glycine), and secondly, a nucleophile with N-terminal (oligo)glycine. This could be shown by ligation of a PNA to a CPP, resulting in a construct that showed dose-dependent antisense activity. A search in protein databases showed that
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General Procedure for the Sortase-Mediated Ligation of the Two Libraries. A solution containing 0.33 mM 5 or 9, 0.33 mM 6, and 4.4 µM or 6.6 µM SrtA in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5 mM CaCl2 (buffer A) was incubated at 23 °C. Aliquots (60 µL) of the reaction mixture were quenched in 20 µL of hydrochloric acid (1 N) and analyzed by HPLC on a PolyEncap A300 column using an acetonitrile/water/TFA solvent system (eluent A: 0.1% TFA, eluent B: 80% acetonitrile/0.1% TFA, linear gradient 5-55% B in 22 min) with fluorescence detection (excitation 345 nm, emission 520 nm) after 7 and 24 h, respectively. HPLC Measurements of the Rate of Sortase-Mediated Ligations. A solution containing 0.33 mM 5 (X1 ) Gly, Asp, Leu or Ser), 0.33 mM 6, and 6.4 µM SrtA in buffer A was incubated at 23 °C. Aliquots of the reaction mixture were analyzed as described above after 0, 1, 2, 3, 4, 5, 6.5, 7, 8, 24, 48, and 72 h, respectively. A solution containing 0.33 mM 7, 0.33 mM 8 (X1 ) Gly), and 6.4 µM SrtA in buffer A/5% DMSO (v/v) was analyzed accordingly (DMSO was added due to the dissolving problems of 7 in buffer). Determination of the Product Yield of the Sortase-Mediated Ligation of Dns-LPKTGGGX3X3-NH2 and H-GGGX4X4-NH2. A solution containing 0.33 mM substrate Dns-LPKTGGGX3X3NH2 (X3 ) Arg or Trp), 0.33 mM nucleophile H-GGGX4X4-NH2 (X4 ) Arg, Trp, or Gly), and 4.0 µM SrtA in buffer A was incubated at 23 °C. Aliquots of the reaction mixture were analyzed as described above showing the same results after 24 and 48 h, respectively. Enzymatic Activity in Different Solvents. A solution containing 0.20 mM 5 (X1 ) Gly), 0.20 mM 6, and 3.8 µM SrtA in buffer A with 0, 20, 40, or 60% (v/v) DMSO or PEG was incubated at 23 °C. Aliquots of the reaction mixture were analyzed as described above after 4 and 24 h, respectively. Synthesis of 13: A solution containing 0.33 mM 11, 1.67 mM 12, and 5.6 µM SrtA in buffer A was dialyzed against 1 L of the same buffer through a membrane with a molecular mass cutoff of 2000 Da (Spectrum Labs) for 24 h at ambient temperature. The resulting solution showed a conversion of 94%. The product 13 was readily separable by RP-HPLC (PolyenCap, A300, 10 µm, 250 × 8 mm i.d.), using an acetonitrile/water/TFA solvent system (eluent A: 0.1% TFA, eluent B: 80% acetonitrile/0.1% TFA, linear gradient 10-80% B in 70 min). Lyophilization gave 13, >99% pure according to HPLC analysis. The product was characterized by matrix-assisted laser desorption/ionization mass spectrometry (calcd, 8149.7 Da; found, 8150.6 Da).
Acknowledgment. We thank J. de Diego (MPI for Infection Biology) for providing a S. aureus culture. H. Lerch and D. Krause are thanked for skillful technical assistance. This work was supported by the European Commission (QLK3-CT-200201989). Supporting Information Available: Sortase expression and purification, solid-phase synthesis and characterization of peptides and PNA, and the splicing correction assay. This material is available free of charge via the Internet at http://pubs.acs.org. JO062331L
