Synthesis of Cell-Penetrating Conjugates of Calpain Activator

Bioconjugate Chem. 2007, 18, 130−137

130

Synthesis of Cell-Penetrating Conjugates of Calpain Activator Peptides Zoltań Bańoćzi,† A Ä gnes Tantos,‡ Attila Farkas,‡ Pe´ter Tompa,‡ Pe´ter Friedrich,‡ and Ferenc Hudecz*,†,§ Research Group of Peptide Chemistry, Eo¨tvo¨s L. University, Hungarian Academy of Sciences, P.O. Box 32, 1518 Budapest 112 Hungary, Institute of Enzymology, Biological Research Center, Hungarian Academic of Sciences, 1518, Budapest, P.O. Box 7, Hungary, and Department of Organic Chemistry, Eo¨tvo¨s L. University, Budapest, Hungary . Received July 5, 2006; Revised Manuscript Received October 31, 2006

Calpains, the intracellular proteolytic enzymes, play important roles in various processes in cells. The lack of calpain or its overexpression is thought to be an underlying factor in some diseases. In this study, we report the synthesis of a new group of cell-penetrating calpastatin-peptide conjugates with the activating capacity of m-calpain intracellularly. In these constructs, peptides related to the calpastatin A or C subunit with the capabiliy of activation of isolated m-calpain was covalently conjugated to the C-terminal of penetratin via amide, thioether, or disulfide bond. These conjugates were prepared by solid-phase synthesis and/or by chemical ligation and properly characterized (MS, HPLC). Our results using isolated m-calpain suggest that conjugation does not interfere with the enzyme-activating effect of the calpastatin peptides; in fact, the efficiency of the conjugates was markedly higher. The conjugates with different bonds showed essentially the same level of activation. Internalization experiments with fluorophore (4-[7-hydroxycoumaryl] acetic acid (Hca) at the N-terminal of penetratin and/or 5(6)-carboxyfluorescein (cf)) labeled conjugates show that these constructs are taken up by COS-7 cells. Using cell lysates produced after incubation with the 1:1 (mol/mol) mixture of calpastatin A and C peptide conjugates, we found a significant calpain activating effect. We also noticed that the conjugate even with a disulfide bond between the components seems to be stable and activate m-calpain after intracellular translocation under the conditions studied. To the best of our knowledge, this is the first report to describe conjugates with an m-calpain activating effect on isolated enzymes and more importantly within living cells after transmembrane delivery. Thus, these conjugates seem to be appropriate as molecular tools to activate intracellular m-calpain and to study calpain functions in living cells.

INTRODUCTION Calpains are intracellular cysteine proteases whose catalytic site is analogous to those of papain-like proteases (1, 2). In mammals, the family has 14 members showing various patterns of expression. The ubiquitous forms are the µ- and m-calpains (calpains 1 and 2, respectively). Calpains play crucial roles in cell adhesion and motility, exocytosis, signal transduction, cell cycle progression, and regulation of gene expression (3-7). Their activation leads to the limited proteolytic modification of a variety of substrate proteins. However, the overactivation of calpains is associated with the pathogenesis of a wide range of disorders (8). The activation of calpain by Ca2+ binding is mechanistically clear, but the key aspects of activation in vivo are still enigmatic. Namely, calpains require nonphysiologically high Ca2+ concentrations in vitro (9). Various mechanisms have been proposed for calpain sensitization to Ca2+ in the cell. One may be the fragmentation of calpastatin, the endogenous inhibitor of calpains. Calpastatin contains four inhibitory domains (10). Each domain contains three short, conserved sequences of about 20 amino acids, termed subdomains A, B, and C. Functional studies showed that only subdomain B is responsible for inhibition of calpains (11, 12). Subdomains A and C exert no inhibitory effect on calpain (12-14). However, peptides corresponding to * Ferenc Hudecz, Ph.D., D.Sc., Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University, Budapest 112, POB 32, H-1518 Hungary. Fax: 36-1-372-2620. Tel: 36-1-372 2828. Email: [email protected]. † Research Group of Peptide Chemistry, Eo¨tvo¨s L. University. ‡ Biological Research Center, Hungarian Academic of Sciences. § Department of Organic Chemistry, Eo¨tvo¨s L. University.

subdomain A (S12GKSGMDAALDDLIDTLGG31) or subdomain C (S86KPIGPDDAIDALSSDFTS105) significantly activate both human erythrocyte µ-calpain and rat m-calpain and increased the Ca2+ sensitivity in vitro (15). Peptides, when applied separately, activate the enzyme, but their 1:1 (mol/mol) mixture exhibited the maximal effect. On the basis of these findings, it was postulated that calpastatin might be fragmented, and the fragments could play a role in calpain activation in vivo. In the extensive studies on calpains physiological function, almost invariably calpain inhibitors were used. These, however, lack strict specificity. They may react with the proteosome, lysosomal proteinases, or even with nonproteolytic enzymes (16, 17). In general, the results obtained with calpain inhibitors cannot always be unequivocally attributed to the action of calpain. In contrast, peptides derived from calpastatins A and C are likely to be specific to calpain. In recent years, several oligopeptides of natural and/or synthetic origin have been described with cell membrane penetrating capability. The first peptide sequences came from the HIV-1 Tat (18, 19) and the antennapedia protein of Drosophila (20). These peptides can cross the cell membrane with different cargos (peptides, peptide-nucleic acid, oligonucleotide) (21). There have been efforts to prepare specific calpain inhibitors based on calpastatin and cell-penetrating peptides capable of transmembrane trafficking of covalently attached cargo (2224). The alternative strategy was to produce, by genetic manipulation, fusion constructs containing the cell-penetrating motif and the sequence derived from calpastatin (25). In these studies, calpastatin or its inhibitory region was joined with cellpenetrating peptides. Croce et al. developed an inhibitor of calpain by conjugating with amide bond the minimal inhibitory

10.1021/bc0601976 CCC: $37.00 © 2007 American Chemical Society Published on Web 12/23/2006

Cell-Penetrating Conjugates of Calpain Activator Peptides

Bioconjugate Chem., Vol. 18, No. 1, 2007 131

were built up by solid-phase peptide synthesis. For conjugates with thioether or disulfide bonds, nonprotected peptide fragments with added N- or C-terminal Cys residues were prepared, and appropriate ligation techniques were utilized in solution. Here, we report on our findings that the calpain-activating properties of calpastatin peptides were not only preserved after conjugation with penetratin but significantly increased in both cell-free (isolated enzyme) and cellular assays. Internalization experiments with fluorophore (4-[7-hydroxycoumaryl] acetic acid (Hca) at the N-terminal of penetratin and/or 5(6)-carboxyfluorescein (cf)) labeled conjugates show that these constructs are taken up by COS-7 cells We found that a conjugate even with disulfide bonds between the components seems to be stable and can activate calpain after intracellular translocation.

EXPERIMENTAL PROCEDURES

Figure 1. Outline of the structure of the penetratin-calpastatin A (ac) and penetratin-calpastatin C (d-f) conjugates with amide (a,d), thioether (b,e), or disulfide (c,f) linkage.

sequence (EKLGERDDTIPPEYRELLEKKTGV) of calpastatin to the C-terminal of the signal-sequence-based penetrating peptide, AAVALLPAVLLALLAP. Using this chimera and a set of peptidyl calpain inhibitors, with appropriate specificity controls, it was demonstrated that calpain regulates platelet degranulation, aggregation, and spreading (22). The peptide related to subdomain B of calpastatin (DPMSSTYIEELGKREVTIPPKYRELLA) was prepared with N-terminal 11-mer arginine also via amide bond by solid-phase peptide synthesis (23). The conjugated calpastatin peptide was able to inhibit calpain activity in a primary culture of rat cortical neurons. In the other study, the same calpastatin peptide with Cys at the N-terminal was coupled to penetratin containing Cys at the N-terminal position by disulfide bond (24). This conjugate was also able to penetrate LCLC 103H cells and inhibit calpain activation in vivo. Sengoku et al. fused the full-length calpastatin or calpastatin subdomain I (115-248 residues) with the Tat peptide (Tat47-57, YGRKKRRQRRR) and observed that the fusion proteins were able to enter primary rat cortical neurons and also exhibited a potent m-calpain inhibitory effect in a cellfree activity assay but did not influence the activity of intracellular calpain (25). The authors found that the fusion protein with Tat peptide was taken up by the endosomal route, which might be in relation to the loss of inhibitory property of the cargo. In the present work, our aim was to prepare a new class of calpastatin peptide conjugates in which the related peptide (calpastatin A or calpastatin C) with calpain-activating properties is conjugated with the well-known cell-penetrating peptide, penetratin. In contrast to the impermeable calpastatin A and C peptides, we expected that these contructs will possess membrane translocating properties and provide activation of calpain intracellularly. The conjugation of calpastatin A and C peptides was carried out via amide, thioether, and disulfide bond formation to study the effect of linkage between the components on both calpain activation and cellular uptake. In these constructs, penetratin (RQIKIWFQNRRMKWKK) was at the N-terminus as outlined in Figure 1. Conjugates with amide bond

All amino acid derivatives were purchased from Novobiochem (Laufelfingen, Switzerland) and Reanal (Budapest, Hungary), while p-cresol, thioanisole, 1,8-diazabicyclo[5.4.0]undec7-ene (DBU), piperidine, N,N′-diisopropylethylamine (DIEA), tris(hydroxymethyl)aminomethane (Tris), trifluoroacetic acid (TFA), triisopropylsilane (TIS), 1-hidroxybenztriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC), 5(6)-carboxyfluorescein (cf), 4-[7-hydroxycoumaryl] acetic acid (Hca), and hydrogen fluoride (HF) were Fluka (Buchs, Switzerland) products. The 4-methylbenzhydrylamine (MBHA, 1.4 mmol/g) and Rink amide resin (0.64 mmol/g) were purchased from Novobiochem (Laufelfingen, Switzerland). Solvents (DMF, DCM, diethyl ether, acetonitrile) for syntheses and purification were obtained from Reanal (Budapest, Hungary). The chloroacetic acid pentachlorophenyl ester and the 5(6)-carboxyfluorescein pentachlorophenyl ester were prepared in our laboratory. Synthesis of Calpastatin Peptides. Calpastatin A peptide with N-terminal Cys residue (C(Npys)S12GKSGMDAALDDLIDTLGG31) was synthesized manually by solid-phase methodology on MBHA resin (0.5 g, 1.4 mmol/g). The amino acid side-chain protecting groups were benzyl ether for Ser and Thr and cyclohexyl ester for Asp. The side chain of Lys was protected with tert-butyloxycarbonyl group, and of Cys was protected by 3-nitro-2-pyridinesulfenyl (Npys) group. The synthesis was carried out by the Boc/Bzl strategy up to the incorporation of Asp at position 18. Then, the Fmoc/tBu strategy was applied. The NR-Boc group was removed with 33% TFA in DCM (2 + 20 min) followed by washing with DCM (5 × 0.5 min), neutralization with 10% DIEA in DCM (3 × 1 min), and DCM washing again (4 × 0.5 min). For coupling, amino acid derivatives and DCC and HOBt dissolved in a minimal amount of DCM-DMF 4:1 (v/v) mixture were used in 3 molar excess for the resin capacity. The reaction proceeded at room temperature (RT) for 60 min. Then, the resin was washed (DMF 2 × 0.5 min, DCM 3 × 0.5 min). The efficiency of coupling was checked by ninhydrin (26) or isatin (27) tests. The NR-Fmoc group was removed with 2% piperidine plus 2% DBU in DMF (2 + 2 + 5 + 10 min) followed by washing with DMF (8 × 0.5 min). For coupling, amino acid derivatives and DIC and HOBt dissolved in DMF were used in 3-fold molar excess for the resin capacity. The reaction proceeded at RT for 60 min. Then, the resin was washed (DMF 2 × 0.5 min, DCM 3 × 0.5 min). Finally Boc-Cys(Npys) was added as the N-terminal residue. The Boc group was removed by 33% TFA in DCM in the presence of 0.5 g Met as a scavenger. The peptide was cleaved from the resin by 10 mL HF using 0.5 g p-cresol scavenger at 0 °C for 1.5 h. Crude products were precipitated with dry diethyl ether, filtered, washed, dissolved in 10% acetic acid, and freeze-dried. The NR-choloroacetylated calpastatin A peptide (ClAc-S12GKSGMDAALDDLIDTLGG31) was synthesized manually by

132 Bioconjugate Chem., Vol. 18, No. 1, 2007

solid-phase methodology on Rink amide resin (0.1 g, 0.64 mmol/ g) using the Fmoc/tBu strategy. The amino acid side-chain protecting groups were tert-butyl ether for Thr and Ser and tertbutyl ester for Asp. The side chain of Lys was protected with the tert-butyloxycarbonyl group. After the removal of the last NR-Fmoc group, a chloroacetic group was introduced to the N-terminal of the peptidyl resin using 5 molar excess chloroacetic acid pentachlorophenyl ester dissolved in DMF at RT for 10 h (28). Chloroacetylated peptide was removed from the resin by cleavage with 10 mL TFA containing 0.5 g phenol, 0.5 mL distilled water, 0.5 mL thioanisole, and 0.6 mL TIS as scavengers. The crude product was precipitated by dry diethyl ether, dissolved in 10% acetic acid, and freeze-dried. The calpastatin C peptide (Ac-C(Npys)S86KPIGPDDAIDALSSDPFTS105) was synthesized manually by solid-phase methodology on MBHA resin (0.2 g, 1.4 mmol/g) using Boc/ Bzl strategy. The amino acid side-chain protecting groups were benzyl ether for Ser and Thr and cyclohexyl ester for Asp. The side chain of Lys was protected with a 2-chlorobenzyloxycarbonyl group. After the cleavage of the last Boc group, the N-terminus was acetylated by acetic anhydride in DMF in the presence of DIEA (1:1 ) mol/mol). The acetylated peptide was cleaved from the resin by 10 mL HF using 0.5 g p-cresol scavenger at 0 °C for 1.5 h. The crude product was precipitated with dry diethyl ether, filtered, washed, dissolved in 10% acetic acid, and freeze-dried. Choloroacetylated calpastatin C peptide (ClAc-S86KPIGPDDAIDALSSDPFTS105) was prepared manually by solid-phase methodology on MBHA resin (0.15 g, 1.4 mmol/g) as described above. After the removal of the last NR-Boc group, the peptidyl resin was chloroacetylated by 5 molar excess of chloroacetic acid pentachlorophenyl ester dissolved in DMF for 10 h. The peptide was cleaved from the resin by 10 mL HF using 0.5 g p-cresol scavenger at 0 °C for 1.5 h. The crude product was precipitated with dry diethyl ether, filtered, washed, dissolved in 10% acetic acid, and freeze-dried. The fluorophore-labeled calpastatin C peptide (Ac-C(Npys)S86K(cf)PIGPDDAIDALSSDPFTS105) was prepared in solution using 5(6)-carboxyfluorescein pentachlorophenyl ester. 5 mg (2.2 µmol) calpastatin C peptide was dissolved in 0.5 mL DMF. 2.8 mg (4.4 µmol) 5(6)-carboxyfluorescein pentachlorophenyl ester and 0.7 µL (4 µmol) DIEA were added to the solution. The reaction was carried out at RT overnight. The crude peptides were purified by RP-HPLC (see below). Synthesis of Penetratin Peptides with C-Terminal Cys. Penetratin with C-terminal Cys (RQIKIWFQNRRMKWKKC) was synthesized manually by solid-phase methodology on Rink amide resin (0.5 g, 0.64 mmol/g) using the Fmoc/tBu strategy as described above. The amino acid side-chain protecting groups were trityl for Asn, Gln, and Cys and the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group for Arg. The side chain of Lys was protected with the tert-butyloxycarbonyl group. After assembly, the peptide was cleaved from the resin with 10 mL TFA using 0.75 g phenol, 0.5 mL distilled water, 0.5 mL thioanisole, and 0.25 mL ethandithiol as scavengers. The crude product was precipitated by dry diethyl ether, dissolved in 10% acetic acid, and freeze-dried. 4-[7-Hydroxycoumaryl]acetic acid (Hca)-labeled penetratin C peptide (Hca-penetratinC) was synthesized the same way, but after the cleavage of the last NR-Fmoc group, Hca was introduced into the sequence using the same coupling reagents and conditions as amino acid derivatives. Labeled penetratin C peptide was cleaved from the resin as the penetratin. The peptides were purified by RP-HPLC. Synthesis of Calpastatin Conjugates. Conjugates with an amide bond were prepared manually by solid-phase methodology using the Fmoc/tBu strategy on Rink amide resin (0.05 and

Bańoćzi et al.

0.1 g, 0.64 mmol/g). The amino acid side-chain protecting groups were trityl for Asn, Gln, and Cys and the 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl group for Arg. The side chain of Lys was protected with the tert-butyloxycarbonyl group. The amino acid side chains of Thr and Ser were protected by tert-butyl ether and that of Asp by tert-butyl ester. The peptides were cleaved from the resin by 10 mL TFA using 0.75 g phenol, 0.5 mL distilled water, 0.5 mL thioanisole, and 0.25 ethandithiol as scavengers. The crude products were precipitated by dry diethyl ether, dissolved in 10% acetic acid, and freezedried. Hca-labeled penetratin-calpastatin A conjugate with an amide bond was prepared the same way, but after the cleavage of the last NR-Fmoc group, Hca was introduced into the sequence using the same coupling reagents and conditions as amino acid derivatives. The labeled conjugate was cleaved from the resin as the unlabeled one. Conjugates with a thioether bond were prepared in solution. 15 mg penetratin C (6.4 µmol) or Hca-penetratinC (5.9 µmol) peptides were added in small portions into the buffered (0.1 M TRIS, pH 8.1) solution of 10 mg (5 µmol) chloroacetylated calpastatin A or C peptide (c ) 1 mg/mL) every 15 min. The reaction mixture was stirred at RT and followed by RP-HPLC. After 5 h, the solution was freeze-dried. The synthesis of conjugates with a disulfide bond was carried out in 0.16 M phosphate buffer (pH ) 6). 15 mg (6.4 µmol) penetratin C was added in small portions into the buffered solution of 10 mg (4.8 and 4.5 mmol) calpastatin A or C peptides (c ) 2 mg/mL) every 15 min. The reaction mixture was stirred at RT and followed by RP-HPLC. Dual-labeled Hca-penetratinCys-Cyscalpastatin(cf)C conjugate with a disulfide bond was prepared in the same way. All crude conjugates were purified by RP-HPLC as described below. Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). Analytical HPLC was performed on a Knauer (Herbert Knauer GmbH, Berlin, Germany) system using a Phenomenex SYNERGI MAX-RP column (250 × 4.6 mm i.d., 4 µm silica, 80 Å pore size) (Torrance, CA) as a stationary phase. Linear-gradient elution (0 min 0% B; 5 min 0% B; 50 min 90% B) was generated using 0.1% TFA in water as eluent A and 0.1% TFA in acetonitrile-water (80:20, V/V) as eluent B. A flow rate of 1 mL/min was applied at ambient temperature. Peaks were detected at λ ) 220 nm. The samples were dissolved in eluent B. The crude products were purified on a semipreparative Phenomenex Jupiter C18 column (250 × 10 mm i.d., 10 µm silica, 300 Å pore size) (Torrance, CA). The flow rate was 4 mL/min. The following gradients were applied: 0 min 30% B, 5 min 30% B, and 50 min 75% B for calpastatin A and its conjugates; 0 min 20% B, 5 min 20% B, and 50 min 60% B for calpastatin C, its conjugates, and penetratin. Amino Acid Analysis. The amino acid composition of peptides was determined by amino acid analysis using a Beckman (Fullerton, CA) model 6300 amino acid analyzer. Prior to analysis, the samples were hydrolyzed in 6 M HCl in sealed and evacuated tubes at 110 °C for 24 h. Mass Spectrometry. Positive-ion electrospray ionization mass spectrometric analysis was performed on a Bruker Esquire 3000 plus (Germany). The samples were dissolved in acetonitrile-water (50:50, v/v) containing 0.1% acetic acid. Activation of Calpain in Vitro. Activity of purified recombinant m-calpain was measured in a Jasco FP-6300 spectrofluorometer (Essex, U.K.) in a 3 × 3 mm quartz cuvette, continuously recording the increase of fluorescence corresponding to the cleavage of the substrate FRET (DABCYL-TPLKSPPPSPR-EDANS) (29). The excitation wavelength was 320 nm, and emission was monitored at 480 nm. Reactions were


set up in a final volume of 50 µL at room temperature. Data were recorded for 60 s. Comparison of the Calpain-ActiVating Capacity of Conjugates. Maximum activity was measured at 5 mM free Ca2+ concentration. Reaction mixtures contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride and 15 mM β-mercaptoethanol, 200 µM substrate, and 0.2 µM m-calpain. The reaction was started by the rapid mixing of CaCl2 into the sample immediately before starting the measurement. Activation by the conjugates was measured under the same conditions, with the sole difference in the free Ca2+ concentration, which was 0.1 mM in these cases. Conjugates were used at 75 µM concentration. Calpain ActiVation with the Conjugates with Amide Bond. m-Calpain activity was measured as described above. Different concentrations (0, 5 10, 20, 30, 50, and 100 µM) of the conjugates were used at a constant (0.1 mM) free Ca+ level. Uptake of Fluorescent-Labeled Penetratin-Calpastatin Conjugates. COS-7 cells were grown in Dulbecco’s modified Eagle’s medium (Sigma) at 37 °C and 5% CO2 content. Peptides or peptide conjugates were dissolved in a minimal amount of 10% (v/v) acetic acid and diluted with 0.01 M PBS (pH ) 7.4) and used at a final concentration of 330 µM. Cells were incubated for 4 h with peptides, then washed with PBS. The fluorescence of cells after fixation (3% paraformaldehyde for 10 min) or without fixation were mounted with MOWIOL 4-88 (Calbiochem) and analyzed by a Leica DMLS fluorescent microscope equipped with a Spot RT color digital camera (Diagnostic Instruments, Livingston, U.K.). According to the type of fluorophores, the Chroma UV and narrow-band GFP filters (Chroma Technology Corp, Rockingham, VT) were used. Flow Cytometry Analysis of Cellular Uptake. The stock solution of conjugate labeled with 5(6)-carboxyfluorescein dissolved in HPMI (c ) 0.4 mM) was used for the treatment of COS-7 cells. Cells were seeded in 24-well plate, and before treatment, the cells were washed with PBS, and they were incubated with the 5(6)-carboxyfluorescein-labeled compound (c ) 10 µM) for 1, 1.5, and 3 h at 37 °C. After incubation, cells were washed three times with PBS, trypsinized for 10 min, and resuspended in HPMI. The increase in COS-7 cell fluorescence following incubation was monitored by flow cytometry (BD LSR II, BD Bioscience, San Jose, CA). Data were analyzed with FACSDiVa software. Measurement of Endogenous Calpain Activity in COS-7 Cell Lysate. The COS-7 cells were treated with 330 µM conjugates at 37 °C for 4 h. Confluent cells were than washed three times with 0.01 M PBS (pH ) 7.4) and were scraped in 1 mL of ice-cold 0.01 mM PBS (pH ) 7.4). Cells were collected by centrifugation at 1500 × g at 4 °C for 2 min, resuspended in a 0.1 M Tris-HCl buffer containing 5 mM EDTA, 1 mM dithioerythritol, 5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, and 10 mM β-mercaptoethanol and sonicated (16 µm, MSE sonicator, West Sussex, U.K.) four times for 10 s with 1 min breaks. The cell lysate was than centrifuged at 15 000 × g at 4 °C for 20 min to remove the cell debris, and the supernatant was used for fluorometric calpain activity measurements as described above. In these experiments, either 1 mM EDTA containing 1 mM CaCl2 or 10 mM CaCl2 was added to 30 µL of lysates. The change in fluorescence intensity was monitored with the FRET peptide substrate (29).

RESULTS AND DISCUSSION Calpains are important proteases involved in several intracellular processes. In studying their function, mainly inhibitors have been used so far. These, however, are not specific for calpains; therefore, the results obtained cannot always be


unequivocally associated with the action of calpain (16, 17). We have demonstrated earlier that peptides corresponding to two regions of calpastatin, the protein inhibitor of calpains, exert activation on isolated calpains (15). This effect may also be utilized to study calpain function in living cells. To achieve this, we have produced conjugates containing calpastatin peptides derived from subdomains A and C (12) as well as cellpenetrating peptide, penetratin (20), in the N-terminal part for transmembrane delivery. Here, we report on the synthesis of a new set of penetratin conjugates with an amide, thioether, or disulfide bond between the components (Figure 1). The amide and thioether linkages are considered stable in the cell, while the disulfide bond could be cleaved in the reducing environment of the cytoplasm (30). These conjugates were assayed for their calpain activation properties using isolated m-calpain and also COS-7 cell lysate after incubation with the test compounds. We have demonstrated that fluorescent-labeled penetratin conjugates enter cells, and the nature of the covalent linkage between the components has no marked effect on the calpain activation and membrane translocation of the conjugates. Synthesis of Calpastatin Conjugates. Conjugates, including the 4-[7-hydroxycoumaryl]acetic acid (Hca) labeled variant of penetratin-calpastatin A, with an amide bond were prepared by solid-phase synthesis on Rink amide resin to produce peptide-amides by the Fmoc/tBu strategy. The completed conjugates were cleaved from the resin by the TFA cleavage mixture. The compounds were obtained without any difficulties, and their characteristics are summarized in Table 1. Conjugates with a disulfide or thioether bond were prepared in solution. Considering that none of the partner molecules contain the Cys residue, the original sequences were modified by the incorporation of Cys at an adequate position. First, penetratin and Hca-penetratin elongated at the C-terminal by a Cys residue was synthesized on Rink amide resin by the Fmoc/ tBu strategy and used for conjugates with either disulfide or thioether linkages. The peptides were removed from the resin by the TFA/phenol/thioanizol/EDT/water cleavage mixture. Simultaneously, calpastatin A and C peptide amides extended with a Cys at their N-terminal were also produced on the solid phase. To build up the heterodimer via disulfide bond, the 3-nitro2-pyridinesulfenyl (Npys) group was used in the calpastatin peptides as a thiol-blocking group. The Npys group was originally proposed for the protection of amino and/or hydroxyl functions during solid-phase peptide synthesis (31, 32). The greatest potential of Npys, however, has gradually been realized to lie in the side-chain protection of Cys (31). Indeed, the most attractive feature of Npys is its dual character as an S-protecting and S-activating group. The calpastatin C derivative with Npysprotected and N-acetylated Cys was synthesized on MBHA resin by the Boc/Bzl strategy. However, in the case of the calpastatin A peptide, from position Asp18, the Fmoc/tBu strategy was used. This combination of Boc/Bzl and Fmoc/tBu strategies is to avoid oxidation-related side reactions of Met. After completion of the assembly of the sequences, the HF/p-cresol cleavage mixture was used for detachment of the peptides from the resin. The calpastatin C peptide labeled with 5(6)-carboxyfluorescein (cf) at the -amino group of the Lys residue at position 87 was produced after the detachment of the peptide from the resin in DMF solution by using the active ester derivative of cf. The peptides were characterized and used in the conjugation reaction (Table 1.). For conjugate preparation, the coupling procedure between the Cys(Npys) peptide and the free SH groups of the protein or polypeptide are usually carried out in solution under acidic conditions (pH 4.5-7.0) to prevent the oxidation of the thiolpeptide (33). In our case, the Cys(Npys) containing calpastatin


Bańoćzi et al.

Table 1. Characteristics of Calpastatin Conjugates and Their Precursors compounds

code

H-C(Npys)S12GKSGMDAALDDLIDTLGG31-NH2 ClAc-SGKSGMDAALDDLIDTLGG-NH2 Ac-C(Npys)SKPIGPDDAIDALSSDFTS-NH2 Ac-C(Npys)S86K(cf)PIGPDDAIDALSSDFTS105-NH2c ClAc-SKPIGPDDAIDALSSDFTS-NH2 H-RQIKIWFQNRRMKWKKC-NH2 Hca-RQIKIWFQNRRMKWKKC-NH2 PenetratinCys-CysCalpC PenetratinCys-CysCalpA Penetratin-CalpC Penetratin-CalpA PenetratinCys(CalpC) PenetratinCys(CalpA) Hca-Penetratin-CalpA Hca-PenetratinCys(CalpA) Hca-PenetratinCys(CalpC) Hca-PenetratinCys-CysCalp(cf)C

CysCalpA ClAcCalpA CysCalpC CysCalp(cf)C ClAcCalpC PenetratinCys Hca-PenetratinCys

bond

[M + H]+ a calculated [measured]

Rt b [min]

disulfide disulfide amide amide thioether thioether amide thioether thioether disulfide

2092.4 [2092.2] 1912.5 [1913.0] 2235.0 [2235.0] 2593.5 [2593.3] 2013.0 [2013.0] 2349.0 [2349.0] 2551.9 [2551.5] 4427.3 [4427.0] 4285.1 [4285.0] 4165.0 [4165.0] 4066.5 [4067.0] 4325.0 [4325.0] 4226.0 [4226.2] 4266.8 [4267.0] 4427.0 [4427.0] 4526.0 [4526.8] 4987.7 [4988.2]

34.9 36.9 31.5 34.4 31.9 25.5 28.7 28.6 31.5 28.1 32.2 28.4 31.8 33.2 35.0 29.4 30.4

a ESI-MS analysis was carried out on a Bruker Esquire 3000 plus (Germany). The samples were dissolved in acetonitrile-water (50:50, v/v), 0.1% acetic acid. b HPLC retention time, column: Phenomenex SYNERGI MAX-RP (250 × 4.6 mm, 4 µm, 80 Å). Linear gradient elution: 0 min 0% B; 5 min 0% B; 50 min 90% B with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile-water (80:20, v/v)) was used at a flow rate of 1 mL/min at ambient temperature. Peaks were detected at λ ) 220 nm. c cf, 5(6)-carboxyfluorescein.

Scheme 1. Outline of the Synthesis of Conjugates with Disulfide (a) or Thioether (b) Bond

A or C peptide was dissolved in 0.16 M phosphate buffer (pH ) 6.0) at 2 mg/mL concentration, and penetratin with unprotected C-terminal Cys was added in small portions into this solution as outlined in Scheme 1. The advancement of the reaction was monitored by RP-HPLC, and after completion, the conjugates were purified and characterized (Table 1). Under the conditions used, we found a negligible amount of penetratin dimer. Conjugation resulting in a thioether bond is a simple method, which can be carried out even with unprotected fragments. For this reaction, penetratin with C-terminal Cys and calpastatin peptides chloroacetylated at their N-terminal were produced. The chloroacetylated calpastatin A was synthesized on Rink amide resin by the Fmoc/tBu strategy and cleaved from the resin by TFA in the presence of scavengers. It should be noted that EDT was replaced by TIS as a scavenger. Considering the differences in the sequence, the chloroacetylated calpastatin C was synthesized on MBHA resin by the Boc/Bzl strategy and cleaved from the resin by HF. It is important to notice that the chloroacetyl group is stable under the normal HF deprotection

conditions, even in the presence of appropriate thiols as scavengers (34). The peptides were properly characterized and used in the conjugation reaction (Table 1). Since the reaction of the thiol and Cl-Ac group is fast and selective enough, the protection of the side-chain functional groups in the peptide is not necessary. The thioether bond forms rapidly under mild alkaline conditions (35); therefore, the reaction was carried out in 0.1 M Tris buffer (pH 8.1) as outlined in Scheme 1. In most cases, this reaction is faster than the competing oxidation, which could lead to disulfide bridge formation. To avoid this side reaction, the choloroacetylated peptides were dissolved in the buffer, and the Cys-containing penetratin with or without the N-Hca-label was added in small portions into the mixture. We essentially did not observe formation of the oxidized product. It should be noted that this side reaction could be dependent on the position of Cys in the peptide chain (36). In Vitro Activation of Calpain. We have demonstrated earlier that the calpastatin A and C peptides can activate the isolated m-calpain in vitro (15). This activating effect was the most pronounced when a 1:1 (mol/mol) mixture of both peptides was used for incubation with calpain. Therefore, using the same assay (15), we have performed a comparative study to investigate the effect of conjugation on the enzyme activation effect of calpastatin A plus C peptides. Conjugates with different bonds were also compared for activation of isolated rat m-calpain. The activity of isolated enzyme measured at 5 mM Ca2+ concentration was taken as the positive control (100%) (Figure 2, column A), while activation observed in the presence of 0.1 mM Ca2+ concentration was considered the “negative” one (2.4%) (Figure 2, column B). At this low Ca2+ concentration, the calpain activity was tested in the presence of peptide or conjugate mixtures at equimolar concentration. The mixture of free calpastatin A and C peptides moderately increased the enzyme activity to 9.4% (Figure 2C). A more pronounced effect was observed in the case of the conjugate mixtures (Figure 2D-F). All three samples induced a much higher level of activation of isolated m-calpain (41.7-54.1%). The results show that the conjugation of the calpastatin A and C peptides with penetratin at the N-terminal increased, rather then decreased, the activating potential of the peptides. Penetratin alone had no influence on the calpain activity (data are not shown). Seemingly, the type of linkage between the calpastatin A or C peptides and penetratin had no significant influence on the calpain-activating effect. Perhaps conjugates with an amide bond possessed a slightly higher effect (54.1%). The concentration dependence of calpain



Figure 2. Activation of purified m-calpain with the mixture of calpastatin A and C peptides and with penetratin-calpastatin A and C conjugates. The m-calpain activity was measured at 5 mM CaCl2 (A), at 0.1 mM CaCl2 (B) concentration in the presence of the mixture of 75 µM calpastatin A + 75 µM calpastatin C peptides (C), at 75 µM penetartin-calpastatin A + 75 µM penetratin-calpastatin C conjugates with disulfide bond (D), at 75 µM penetratin-calpastatin A + 75 µM penetratin-calpastatin C conjugates with amide bond (E), and at 75 µM penetratin-calpastatin A + 75 µM penetratin-calpastatin C conjugates with thioether bond (F).

Figure 3. Activity of m-calpain at 0.1 mM CaCl2 using different concentrations of the mixture of penetratin-calpastatin A and C conjugates with amide bond. For details, see Experimental Procedures.

activation was further studied by the mixture of penetratincalpastatin A and C conjugates with amide bonds (Figure 3.). The m-calpain was measured in the presence of 0.1 mM Ca2+ concentration after the addition of 5-100 µM conjugate mixture. The concentration versus activity curve obtained has a sigmoidal pattern, like in the case of the mixture of free calpastatin A and C peptides (15). The effect reaches its maximum value at ∼75 µM. These data indicate that the presence of the penetratin sequence is beneficial for the activation of m-calpain perhaps by promoting the binding of calpastatin peptides to calpain. However, further studies are needed to understand the mechanism of action. Uptake of Penetratin Conjugates. We studied the membrane translocation of conjugates by COS-7 cell by fluorescent microscopy and also by FACS analysis as a function of time. In these experiments, the internalization ability of the conjugates with different bonds was tested. COS-7 cells were incubated with 330 µM Hca-penetratin-CalpA or Hca-penetratinCys(CalpC) conjugates containing the amide or thioether linkage, respectively. After 4 h at RT, the cells were examined with and without fixation by fluorescent microscopy. The Hca-related fluorescence detected showed that both kinds of conjugates could penetrate into the COS-7 cells and distribute nearly homogeneously in the cells (Figure 4). It should be noted that no effect of fixation on the translocation of Hca-penetratinCys-

Figure 4. Uptake of Hca-penetratin-calpastatin peptide conjugates by COS-7 cells with (A,B) or without (C) fixation. Hca-penetratinCalpA conjugate with amide bond (A); Hca-penetratinC-CalpC conjugate with thioether bond (B); and Hca-penetratinC-CalpC conjugate with thioether bond (C). COS-7 cells were treated with 330 µM solution of the conjugate at 37 °C for 4 h. Cells were fixed, and fluorescence was visualized on a Leica DMLS fluorescent microscope. Cell bodies and the nucleus can be distinguished, the conjugates distribute nearly homogeneously in the cells, and the nucleolus is most dense. Red arrows indicate cells, while yellow ones indicate the nucleus. The strong blue patches are aggregates of the conjugates outside the cells. Fixed and living cells provided essentially the same microscopic result, as can be seen in parts B and C.

(CalpC) conjugate was observed under the conditions studied. Similar findings could be detected with other conjugates (data not shown). To study the stability of the disulfide bond, COS-7 cells were also treated with the dual-labeled Hca-penetratinCys-CysCalp(cf)C conjugate as described above. The conjugate showed essentially the same cellular uptake and intracellular distribution as compared to conjugates with amide or thioether linkages (Figure 5). The signals derived from both fluorophores inside the cells provided practically the same pattern, suggesting similar intracellular distribution. Apparently, the disulfide bond was stable under the experimental conditions. Furthermore, these data suggest that by this construct calpastatin peptide can be efficiently transported into COS-7 cells. In order to study the viability of cells under conditions used for uptake studies, MTTbased cytotoxicity experiments were carried out. We found that, after the treatment with the conjugate for 3 h, almost 80% of the COS-7 cells were alive, indicating that the 330 µM concentration of the conjugate is not harmful.


Bańoćzi et al.

Figure 6. Uptake of the 5(6)-carboxyfluorescein (cf) labeled penetratin-calpastatin C conjugate by COS-7 cells. Cells were treated with a solution of the Hca-penetratinCys-CysCalp(cf)C conjugate with disulfide bond at c ) 10 µM for 60 min (blue), 90 min (black), and 180 min (red). The fluorescence intensity was measured by FACS analysis. The green line represents the untreated control. For details, see Experimental Procedures.

Figure 5. Uptake of double-labeled Hca-penetratinCys-CysCalp(cf)C conjugate with disulfide bond by COS-7 cells. Signal of the 5(6)carboxyfluorescein (A). Signal of the Hca (B). COS-7 cells were treated with 330 µM solution of the conjugate at 37 °C for 4 h. Then, fixed cells were visualized on a Leica DMLS fluorescent microscope. Red arrows indicate cells, while yellow ones indicate the nucleus. The conjugate distributes nearly homogeneously in the cell body; the nucleus is denser. Fixed and living cells gave essentially the same microscopic images.

Time dependence of the dual-labeled conjugate was examined by flow cytometry after 1, 1.5, and 3 h treatment of COS-7 cells with the compound under the conditions described above. Data obtained are shown in Figure 6, for a typical experiment. The internalization of the conjugate by cells was in direct proportion to the duration of the incubation time. Even after 1 h, uptake could be demonstrated (F(mean): 6), which increases after 1.5 h (F(mean): 90) and seems to be saturable since only modest elevation of fluorescence intensity was detected after 3 h (F(mean): 200) treatment. For detailed analysis and characterization of the mechanism of internalization, further studies (e.g., temperature dependence, inhibition) are in progress. Calpain Activity in COS-7 Cell Lysate. In order to study the intracellular calpain-activating potency of conjugate, COS-7 cells were treated with the 1:1 (mol/mol) mixture of penetratincalpastatin A and C conjugates containing an amide bond at 37 °C for 4 h. Cells without treatment were used as controls. After

Figure 7. Calpain activity in COS-7 cell lysate in the presence or absence (control) of the mixture of penetratin-calpastatin A plus C conjugates with amide bond. COS-7 cells were treated with the mixture of penetratin-calpastatin A and C conjugates with amide bond using c ) 330 µM (final), at 37 °C for 4 h. Cells were lysed, and calpain activity was measured in the presence of 1 mM EDTA and 1 mM CaCl2, or of 10 mM CaCl2. The activity of lysates were measured immediately (A) or after 30 min at RT (B). Each column represents the mean value of three independent measurements.

this period of time, the cells were washed and lysated, and the supernatant was used for fluorometric calpain activity measurements in the presence of 1 mM EDTA and 1 mM CaCl2, or of 10 mM CaCl2. Results are shown in Figure 7. Each column represents the mean value of three independent measurements. The activity of lysates was measured immediately (Figure 7A) or after 30 min at RT (Figure 7B). In the case of measurements immediately after cytolysis, calpain activity was only noticeable at 10 mM CaCl2 concentration. However, the calpain activity in the cell lysate treated with the conjugate mixture was clearly higher at both low and high CaCl2 concentrations as compared to the untreated control. We recorded essentially the same tendency in the case of experiments performed after 30 min. These results suggest that the calpastatin conjugates not only before cellular uptake but also after internalization possess a calpain-activating effect.

CONCLUSIONS In this study, we report the synthesis of a novel group of cell-penetrating calpastatin-peptide conjugates. In these constructs, peptides related to the calpastatin A or C subunit with in vitro calpain-activating capacity were covalently conjugated



to N-terminal penetratin via amide, thioether, or disulfide bond. Our results show that conjugation does not interfere with the m-calpain-activating effect of the calpastatin peptides; in fact, the efficiency of the conjugates was markedly higher. The conjugates with different bonds showed essentially the same level of activation of isolated m-calpain. We have demonstrated that the penetratin conjugates are internalized by COS-7 cells, and the 1:1 (mol/mol) mixture of calpastatin A and C peptides retained their calpain-activating effect even inside the cells. By the help of the dual-labeled penetratin-calpastatin C conjugate, we proved that the disulfide bond is stable enough to transport the attached cargo into the cells under the conditions studied. To the best of our knowledge, this is the first report to describe conjugates with m-calpain-activating effect on isolated enzyme and more importantly within living cells after transmembrane delivery. Thus, these conjugates seem to be appropriate as molecular tools to activate intracellular calpain and to study calpain functions in living cells.

ACKNOWLEDGMENT These studies were supported by grants from the Hungarian Research Fund (OTKA no. T-043576), from the Hungarian Ministry of Education (Medichem2, NFKP 1/A/005/2004), and from GVOP-2.2.1.-2004-04-0005/2.0.

LITERATURE CITED (1) Sorimachi, H., Ishiura, S., and Suzuki, K. (1997) Structure and physiological function of calpains. Biochem. J. 328, 721-732. (2) Sorimachi, H., and Suzuki, K. (2001) The structure of calpain. J. Biochem. (Tokyo) 129, 653-664. (3) Goll, D. E., Thompson, V., Li, F. H., Wei, W., and Cong, J. (2003) The calpain system. Physiol. ReV. 83, 731-801. (4) Glading, A., Lauffenburger, D. A., and Wells, A. (2002) Cutting to the chase: calpain proteases in cell motility. Trends Cell Biol. 12, 46-54. (5) Sato, K., and Kawashima, S. (2001) Calpain function in the modulation of signal transduction molecules. Biol. Chem. 382, 743751. (6) Rutledge, T. W., and Whiteheart, S. W. (2002) SNAP-23 is a target for calpain cleavage in activated platelets. J. Biol. Chem. 277, 37009-37015. (7) Franco, S. J., and Huttenlocher, A. (2005) Regulating cell migration: calpains make the cut. J. Cell Sci. 118, 3829-3838. (8) Huang, Y., and Wang, K. K. (2001) The calpain family and human disease. Trends Mol. Med. 7, 355-362. (9) Baki, A., Tompa, P., Alexa, A., Molnar, O., and Friedrich, P. (1996) Autolysis parallels activation of µ-calpain, Biochem. J. 318, 897901. (10) Maki, M., Takano, E., Mori, H., Sato, A., Murachi, T., and Hatanaka, M. (1987) All 4 internally repetitive domains of pig calpastatin posses inhibitory activities against calpain-I and calpainII. FEBS Lett. 223, 174-180. (11) Maki, M., Bagaci, H., Hamaguchi, K., Ueda, M., Murachi, T., and Hatanaka, M. (1989) Inhibition of calpain by a synthetic oligopeptide corresponding to an exon of the human calpastatin gene. J. Biol. Chem. 264, 18866-18869. (12) Takano, E., Ma, H., Yang, H. Q., Maki, M., and Hatanaka, M. (1995) Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains. FEBS Lett. 362, 93-97. (13) Ma, H., Yang, H. Q., Takano, E., Hatanaka, M., and Maki, M. (1994) Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interacting with calmodulin-like domain of the proteinase. J. Biol. Chem. 269, 24430-24436. (14) Maki, M., Takano, E., Osawa, T., Ooi, T., Murachi, T., and Hatanaka, M. (1988) Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli. J. Biol. Chem. 263, 10254-10261. (15) Tompa, P., Mucsi, Z., Orosz, Gy., and Friedrich, P. (2002) Calpastatin subdomains A and C are activators of calpain. J. Biol. Chem. 277, 9022-9026.

(16) Wang, K. K., and Yuen, P. W. (1994) Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. 15, 412-419. (17) Donkor, I. O. (2000) A survey of calpain inhibitors. Curr. Med. Chem. 7, 1171-1188. (18) Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55, 1189-1193. (19) Green, M., and Loewenstein, P. M. (1988) Autonomous functional domains of chemically synthesized human munodeficiency virus Tat trans-activator protein. Cell 55, 1179-1186. (20) Joliot, A., Pernelle, C., Deagostini-Bazin, H., and Prochiantz, A. (1991) Antennapedia homeobox peptide regulates neural morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 88, 1864-1868. (21) Hudecz, F., Bańoćzi, Z., and Csı´k, G. (2005) Medium-sized peptides as built in carriers for biologically active compounds. Med. Res. ReV. 25, 679-736. (22) Croce, K., Flaumenhaft, R., Riversi, M., Furie, B., Furie, B. C., Herman, I. M., and Potter, D. (1999) A. Inhibition of calpain blocks platelet secretion, aggregation, and spreading. J. Biol. Chem. 274, 36321-36327. (23) Wu, H. Y., Tomizawa, K., Matsushita, M., Lu, Y. F., Li, S. T., and Matsui, H. (2003) Poly-arginine-fused calpastatin peptide, a living cell membrane-permeable and specific inhibitor for calpain. Neurosci. Res. 47, 131-135. (24) Gil-Parrado, S., Assfalg-Machleidt, I., Fiorino, F., Deluca, D., Pfeiler, D., Schaschke, N., Moroder, L., and Machleidt, W. (2003) Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin. Biol. Chem. 384, 395-402. (25) Sengoku, T., Bondada, V., Hassane, D., Dubal, S., and Geddes, J. W. (2004) Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity. Exp. Neurol. 188, 161-170. (26) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test of detection of free amino groups in the solid phase synthesis of peptides. Anal. Biochem. 34, 595-598. (27) Kaiser, E., Bossinger, C. D., Colescott, R. L., and Olsen, D. B. (1980) Color test for terminal prolyl residues in the solid phase synthesis of peptides. Anal. Chim. Acta 118, 149-151. (28) Mezo¨, G., de Oliveira, E., Krikorian, D., Feijlbrief, M., Jakab, A., Tsikaris, V., Sakarellos, C., Welling-Wester, S., Andreu, D., and Hudecz, F. (2003) Synthesis and comparision of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII. Bioconjugate Chem. 14, 1260-1269. (29) Tompa, P., Buzder-Lantos, P., Tantos, A., Farkas, A., Szilagyi, A., Banoczi, Z., Hudecz, F., and Friedrich, P. (2004) On the sequential determinants of calpain cleavage. J. Biol. Chem. 279, 20775-20785. (30) Saito, G., Swanson, J. A., and Lee, K. D. (2003) Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. AdV. Drug DeliVery ReV. 55, 199-215. (31) Matsueda, R., and Walter, R. (1980) 3-Nitro-2-piridinesulfenyl (Npys) group. Int. J. Pept. Protein Res. 16, 392-401. (32) Matsueda, R., Kimura, T., Kaiser, E. T., and Matsueda, G. R. (1981) 3-Nitro-2-pyridinesulfenyl group for protection and activation of the thiol function of cystein. Chem. Lett. 737-740. (33) Mezo¨, G., Mihala, N., Andreu, D., and Hudecz, F. (2000) Conjugation of epitope peptides to branched chain polypeptides via Cys(Npys). Bioconjugate Chem. 11, 484-491. (34) Ivanov, B. B., and Robey, A. F. (1996) Effective use of free thiols as scavengers for HF cocktails to deprotect bromo- and chloroacetyl synthetic peptides. Pept. Res. 9, 305-307. (35) Robey, A. F., and Fields, L. R. (1989) Automated synthesis of N-bromoacethyl-modified peptides for preparation of synthetic peptide polymers, peptide-protein conjugates and cyclic peptides. Anal. Biochem. 177, 373-377. (36) Mezo¨, G., Manea, M., Jakab, A., Kapuva´ri, B., Bo¨sze, Sz., Schlosser, G., Przybylski, M., and Hudecz, F. (2004) Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches. J. Pept. Sci. 10, 701-713. BC0601976

Synthesis of Cell-Penetrating Conjugates of Calpain Activator

Recommend Documents