Special Issue Article Received: 7 March 2014
Revised: 16 April 2014
Accepted: 30 April 2014
Published online in Wiley Online Library: 28 May 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2659
Cotton functionalized with peptides: characterization and synthetic methods‡ Andrea Orlandin, Fernando Formaggio, Antonio Toffoletti and Cristina Peggion* Three approaches for the chemical ligation of peptides to cotton fibers are described and compared. This investigation was encouraged by the need to create peptide-decorated natural textiles, furnished with useful properties (e.g. antimicrobial activity). IR absorption spectroscopy is proved to be an easy and fast method to check the covalent anchorage of a peptide to cotton, whereas for a quantitative determination, a UV absorption method was employed. We also analyzed the usefulness of electron paramagnetic resonance spectroscopy to characterize our peptide-cotton conjugates. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: peptides; functionalized textiles; EPR; dendrimers
Introduction
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Materials and Methods Fmoc and Boc (Fmoc, fluorenyl-9-methoxycarbonyl, Boc, tert-butyloxycarbonyl) protected amino acids were Bachem products (Bubendorf, Switzerland). All other chemicals and reagents for the peptide synthesis were purchased from Sigma-Aldrich (St. Louis, MO). 2-Chlorotrityl resin was purchased from Iris Biotech (Marktredwitz, Germany). The cotton used in our experiments was a commercially available tissue, ready for garment manufacture. For each reaction on textiles, a piece of fabric cotton of about 2 cm2 was accurately weighed, washed with methanol (MeOH), DMF, and MeOH, and air-dried. In one case, we employed a hydrophilic cotton wool sample, accurately weighed and washed as just described. Fmoc-NH-CH2CH2-NH2, prepared by treating Fmoc N-hydroxysuccinimide ester (3 mmol) with 1,2-diaminoethane (20 mmol) in CH2Cl2, was isolated by extraction (CH2Cl2/water) and precipitation (CH2Cl2/petroleum ether). * Correspondence to: Cristina Peggion, Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy. E-mail: [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the 1st International Conference on Peptide Materials for Biomedicine and Nanotechnology, Sorrento, October 28-31, 2013, edited by Professor Giancarlo Morelli, Professor Claudio Toniolo and Professor Mariano Venanzi. ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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New materials based on biodegradable molecules are of great interest to reduce adverse effects on the environment. The need to develop new materials for a variety of applications is greatly promoting academic and industrial research. In particular, textile manufacturers are looking at the production of fabrics made of natural fibers but opportunely functionalized for specific technical applications [1]. Among the many polymeric materials available, cellulose fibers are particularly attractive, being naturally occurring, and easy to functionalize [2–4]. In this connection, it is worth mentioning that cotton-based garments, functionalized with antimicrobial compounds, are already commercially available [1,5]. Protective and safe textiles are of fundamental importance for health care workers and for immuno-compromised or debilitated people who need to be protected from infections [6–8]. The antimicrobial textiles currently on the market are in general made of synthetic polymers that contain silver salts or silver nanoparticles [9–12] (for which toxicity on skin has not been completely made clear [13]), quaternary ammonium salts, triclosan, chitosan, dyes, and regenerable N-halamine compounds [14,15]. Most of the time, the active compounds are simply adsorbed or electrostatically linked [16] to the cotton surface. Therefore, they are usually lost after a few cycles of washing. There is a great demand for durable antimicrobial textiles based on nontoxic bioactive compounds. To contribute to this topic, we started a research program that heavily relies on our expertise in the field of antibacterial peptides [17–20]. The main advantage of using amino acids and peptides comes from their presence in nature. Thus, they should be well tolerated by our body. Recently, a cysteine residue linked to a cellulosic support showed antibacterial properties [16]. A 9-mer peptide was immobilized on cotton fibers giving a material able to inhibit the growth of bacterial strains [21]. Usually, cellulose derivatization involves the hydroxyl groups of cotton on which esters [22] and ethers (ethyl cellulose) can be prepared. This derivatization, in turn, has allowed the use of cotton as a substrate for the
‘solid phase’ synthesis of peptides (SPPS) [23,24] and for combinatorial syntheses [25–29]. This study has three main goals: (i) to compare and discuss synthetic methods to efficiently link peptides to cotton fibers, (ii) to enhance peptide concentration on the cotton fibers through the anchorage of peptide dendrimers, and (iii) to characterize and quantify the peptides linked to cellulosic supports.
ORLANDIN ET AL. Cotton Functionalization with Epibromohydrin and 1,2-Diaminoethane (C1) A cotton piece (0.3 g) was incubated with a 60% HClO4 solution (0.1 ml) in DMF and epibromohydrin (1 ml) for 4 h. Then, the sample was washed with DMF and MeOH and air-dried. This brominated cotton fiber was then immersed in a solution of Fmoc-NH-CH2CH2-NH2 (5 mmol) and triethylamine (5 mmol) in DMF (2 ml) for 12 h. Finally, the functionalized cotton sample was treated for 2 h with a 20% solution of Ac2O in DMF (10 ml) in the presence of triethylamine (1 ml). Fmoc removal was achieved with 20% piperidine solution in DMF. The sample was washed with DMF and MeOH and air-dried.
2-(tritylthio)ethylamine (640 mg, 2 mmol) in DMF in the presence of the N-ethyl, N′-[3-(dimethylamino)propyl]carbodiimide/HOAt coupling mixture. Removal of the triphenylmethyl group was achieved with 1% TFA solution in dichloromethane. Pure peptides 1 and 4 were obtained by crystallization from ethyl acetate/diethyl ether. The peptides were characterized by analytical RP-HPLC on a Jupiter Phenomenex (Torrance, CA) C18 column (4.6 × 250 mm, 5 μ, 300 Å) using an Agilent (Santa Clara, CA) 1200 HPLC instrument. The binary elution system used was A, 0.05% TFA in H2O; B, 0.05% TFA in CH3CN/H2O (9:1 v/v), flow rate 1 ml/min; and spectrophotometric detection at λ = 216 nm. ESI-MS was performed by using a PerSeptive Biosystem Mariner instrument (Framingham, MA). The amino acid sequence, mass spectral data, and HPLC retention time of peptides 1 and 4 are as follows:
Cotton Functionalization with Allyl Bromide (C2) A cotton piece (0.3 g) was immersed in a solution of NaH (200 mg) in 5 ml tetrahydrofuran (THF) and fluxed with nitrogen for 1 h at 0 °C. Then, allyl bromide was added (1.5 ml), and nitrogen flux was continued for 2 h. The reagents were removed by washing with THF.
Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)-NH(CH2)2NH2 (1) HPLC: Rt = 7.30 min (30–70% B in 20 min); mass: calculated 880.45; found 881.52 [M + H]+, 781.52 [M 1Boc + H]+, 391.23 [M 1Boc + 2H]2+. Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2SH (4) HPLC: Rt = 13.4 min (50–100% B in 20 min); mass: calculated 755.36; found 756.40 [M + H]+.
Synthesis of the Linear Peptides and Characterization Linear peptides (Table 1) were synthesized by SPPS, performed manually by means of filter-equipped syringes on a 0.8 mmol scale. For peptide 1, 2-chlorotrityl resin (500 mg, loading 1.6 mmol/g) was treated with a 20% 1,2-diaminoethane solution in DMF for 2 h under nitrogen flux. Then, Fmoc-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-Ala-OH, and Fmoc-Lys(Boc)-OH were added step-by-step using the Fmoc strategy. Each time, 3 eq. (2.4 mmol) of protected amino acid were activated with 3 eq. of HOAt/HATU and 6 eq. of DIPEA using DMF as the solvent. Deprotection of the Fmoc group was achieved with 20% piperidine solution in DMF (2× 15 min). Removal of the peptide from the resin was achieved with 5% TFA in dichloromethane (2× 15 min). The solutions were pooled and concentrated in vacuo (after addition of few milliliters of acetonitrile to prevent Boc removal) to a crude solid. Peptide 4, with two residues of Fmoc-Lys(Boc)-OH (3 eq., 4.8 mmol), was also built on a 2-chlorotrityl resin (1 g, loading 1.6 mmol/g) as described earlier. The free C-terminus peptide obtained (700 mg, 1.2 mmol) was added to a solution of Table 1. List of the peptides and peptide-cotton samples prepared Preparation method 1 1a 2a 3a 1b 2b 3b
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4 4a
Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-NH2 Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-NR-cotton (R Ac or H) [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys-NH-CH2-CH2-N(Ac)-cotton {[Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys}2-Lys-NH-CH2-CH2-N(Ac)-cotton TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-N(Ac)-cotton [TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys-NH-CH2-CH2-N(Ac)-cotton {[TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys}2-Lys-NH-CH2-CH2-N(Ac)-cotton Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2-SH Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2-S-cotton
SPPS M1 and M2 M1 M1 M1 M1 M1 SPPS M3
Peptide Synthesis on Modified Cotton Fibers and Characterization of the Conjugates Cotton samples (C1) of 0.3 g (about 2 cm2) were dunked in DMF. For sample 1a (Table 1), the peptide was built step by step by adding the required Fmoc amino acid (1.5 mmol) activated with HOAt/HATU (1.5 mmol each) and DIPEA (4.5 mmol) in DMF and letting the reaction to proceed for 3 h. Fmoc removal was achieved with a 20% piperidine solution in DMF (2× 15 min). For dendrimer 2a, Fmoc-Lys(Fmoc)-OH (1.5 mmol) was first coupled to aminofunctionalized cotton. After Fmoc removal, the SPPS was carried on as for 1a using double quantities for each reagent. For dendrimer 3a, Fmoc-Lys(Fmoc)-OH (1.5 mmol) was first linked to the functionalized cotton by activation with HOAt/HATU (1.5 mmol each) and DIPEA (4.5 mmol) in DMF. After Fmoc removal, Fmoc-Lys (Fmoc)-OH was reacted again (double quantities). Finally, after Fmoc deprotection, the peptide was built on the cotton as for 1a, using quadruple quantities of each reagent. Samples 1b, 2b, and 3b were prepared from 1a, 2a, and 3a, respectively. 1a was treated with a 20% piperidine solution for Fmoc removal. Then, it was reacted with succinic acid (177 mg, 1.5 mmol) in the presence of HOAt/EDC (1.5 mmol each) in DMF. After washing with DMF, the sample was incubated with 4-hydroxy2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) (260 mg, 1.5 mmol) and HOAt/HATU (1.5 mmol each) in DMF, for 4 h. 2a and 3b were treated as described for 1a using double and quadruple reagent quantities, respectively, for each reaction. For the preparation of 4a, a solution of peptide 4 (450 mg, 0.5 mmol) in THF, containing 2,2-dimethoxy-2-phenylacetophenone (1 wt.%), was added to an allylated cotton sample (C2). Then, the mixture was irradiated at 350 nm for 1 h in a Royonet Photochemical Reactor. All cotton samples were washed (2× DMF and 2× MeOH) and dried under vacuum. Each reaction step was checked by the Kaiser test [30] (before and after Fmoc deprotection). To this aim, a thread of cotton was washed with DMF, MeOH, and acetone; dried; and treated with the three monitor solutions heating at 100 °C for 5 min. For yield evaluation, quantitative Fmoc removal and UV absorption determination were used (see Results and Discussion Section). The UV absorption spectra of the Fmoc removal solution were recorded with a Shimadzu model UV-2501 spectrophotometer.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 547–553
PEPTIDES ON COTTON TEXTILES Cotton-peptide samples were characterized by FT-IR absorption in the solid-state (KBr disk) by means of a Perkin-Elmer model 1720X FTIR spectrophotometer. To prepare the KBr disk, a small fiber piece was reduced to powder and mixed with the KBr matrix. The most informative FT-IR bands are reported in the following text. Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)-NH(CH2)2NH2-cotton (1b) IR (KBr): ν = 1653, 1543, 1249 cm 1. [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2-Lys- NH(CH2)2NH2-cotton (2b) IR (KBr): ν = 1655, 1540, 1260 cm 1. [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]4-(Lys)2-Lys-NH(CH2)2NH2-cotton (3b) IR (KBr): ν = 1674, 1539, 1256 cm 1. EPR Measurements Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker ER 200D X-band spectrometer (~9.4 GHz). Samples of 1b, 2b, and 3b with about 1 cm2 area were inserted in EPR quartz tubes (inner diameter = 3 mm). As a control, we used the cotton fabric without any functionalization. A 0.1 mM toluene solution of 4-hydroxy-TEMPO was used as nitroxide reference solution. Temperature of the samples was regulated using a nitrogen flow cryostat (Bruker BVT 2000).
Results and Discussion For this study the sequences, -Lys-Ala-D-Ala-Lys- [31,32] and -LysLys- [33] >were selected as model peptides. These peptides were chosen because they are short (easy synthesis) and possess antibacterial properties when suitably functionalized at the N-terminus. Indeed, the follow-up of this work will be the preparation of textiles with antimicrobial properties. The compounds prepared in this study are summarized in Table 1. Textile Surface Modification The textile used in this study is a cotton fabric. Three approaches for covalently linking peptides to the cotton surface were explored. First, we examined the step-by-step synthesis of the peptide directly on the cotton surface [21] used as a support. In this case, the cotton surface needed to be opportunely modified. In a second approach, we tested the ‘one pot’ attachment of an entire peptide, via amide bond formation, to a modified cotton textile. Finally, we exploited the thiol-ene chemistry [34]. The first (M1) and second (M2) approach share the cellulose functionalization step with epibromohydrin as shown in Scheme 1.
Method M1 In the first step (i, Scheme 1), hydroxyl groups of the cellulosic support, soaked in DMF, react with epibromohydin in the presence of HClO4 [21]. The epoxide opens forming an ether bond and introducing a halogen at the same time. Bromine is then substituted by reaction with Fmoc-NH-CH2CH2-NH2 followed by acetylation of the newly formed secondary amine. The procedure adopted allows an easier functionalization of the cotton surface as (i) we introduce a spacer, thus walking away from the crowded glucose moieties, and (ii) we make available an amino group, a better nucleophile as compared with an alcoholic function. The cotton can then be used as a support to build the peptide according to standard SPPS procedures (step-by-step addition of the Fmoc amino acid of interest). Each reaction step was checked by taking out a small cotton thread on which a Kaiser test [30] was run (before and after Fmoc deprotection). To go to completion, couplings required 3 h and three equivalents of Fmoc-residue, less than what was reported in the literature [21]. Finally, the Fmoc Nα-protecting group was removed with a piperidine solution in DMF. The cotton was washed at each step with DMF to completely eliminate the coupling reagents. As we cannot increase the number of the cellulose alcoholic functions reacting with epibromohydrin, under these conditions, we decided to verify if peptide loading on the cotton can be boosted by the dendrimer approach. Therefore, small Lys dendrimers [35–38], with two or four tetrapeptide units (Table 1), were also grown on cotton according to the M1 procedure (Scheme 2). The synthesis was shown to be feasible, although a yield improvement is required in the case of the sterically hindered dendrimer 3a (see the discussion on the ‘loading’ section). Method M1 was also used to prepare the cotton-peptide samples functionalized with a free radical probe, useful for EPR investigations (Scheme 3). Compounds 1b, 2b, and 3b were prepared from 1a, 2a, and 3a, respectively. After Fmoc removal and coupling with succinic acid, the nitroxyl moiety was introduced by a HATU/HOAt mediated esterification of 4-hydroxy-TEMPO with the free carboxylic function of succinic acid. Method M2 Method M1 requires uncommon and repetitive manipulations of a tissue in order to carry out the numerous synthetic steps. To this aim, a dedicated glass apparatus was purposely built. However, to assess if M1 is an effective procedure, we synthesized sample 1a also according to a more traditional procedure. In this approach, the peptide is prepared beforehand with the 1,2-diaminoethane spacer linked to its C-terminus. The synthesis of peptide 1 was performed on a 2-chlorotrityl resin. First, the
J. Pept. Sci. 2014; 20: 547–553 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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Scheme 1. Reaction conditions: (i) epibromohydrin, 60% HClO4 in DMF; (ii) (M1) Fmoc-NH-CH2CH2-NH2, triethylamine in DMF; Ac2O, triethylamine in DMF; 20% piperidine in DMF; (iii) step-by-step SPPS: Fmoc-AA-OH, HOAt/HATU, DIPEA in DMF; then, piperidine in DMF, except after the last coupling; and (iv) (M2) DIPEA in DMF.
ORLANDIN ET AL.
Scheme 2. Reaction conditions for the synthesis of 2a and 3a: (i) Fmoc-Lys(Fmoc)-OH, HOAt/HATU, and DIPEA in DMF, then piperidine in DMF; and (ii) step-by-step SPPS with the conditions of (i) but no Fmoc deprotection after the last coupling.
Scheme 3. Introduction of the paramagnetic label in the peptide-cotton samples. (i) EDC/HOAt in DMF and (ii) HATU/HOAt in DMF.
1,2-diaminoethane spacer was linked to the resin by nucleophilic substitution of the chlorine atom on the resin support. Then, each Fmoc N-protected amino acid residue was coupled in the presence of the excellent activating agents HOAt/HATU and DIPEA, using DMF as solvent. Deprotection of the Fmoc group was achieved with piperidine. Removal of the peptide from the resin was performed with 5% TFA in CH2Cl2. The evaporation of this peptide solution was carried out in the presence of acetonitrile in order to preserve the Boc protection on the Lys side chains. The pure peptide 1, obtained by recrystallization, was linked to the brominated cotton surface by nucleophilic substitution under nitrogen flux (Scheme 1, step iv). Synthetic methods M1 and M2 gave similar results in terms of peptide loading on the tissue (see the discussion on ‘loading’ section). Therefore, both approaches can be exploited depending on the requirements of a synthetic design. However, the one pot method M2 allows for a reduced number of operations on the tissue and, in addition, permits to better check the identity and purity of the pre-synthesized peptide.
chemoselective [40]. Indeed, upon photoactivation, the thiol group reacts specifically with the alchene, while other potentially reactive moieties do not interfere [41]. As model peptide, in this case, we chose the simpler peptide 4, synthesized by SPPS on a 2-chlorotrityl resin. It was removed from the resin as described earlier and amidated with 2-(tritylthio)ethylamine. Removal of triphenylmethyl group with 1% TFA afforded peptide 4. The cellulosic support was functionalized by treatment with allyl bromide in the presence of NaH, thus introducing a double bond. Then, peptide 4 was linked to the double bond in the presence of a light source (350 nm) and a catalytic amount of the photocatalyzer 2,2-dimethoxy-2-phenylacetophenone to give sample 4a. The final peptide loading (determined via UV spectroscopy and described hereafter) is much lower as compared with that obtained with methods M1 and M2. This low functionalization is probably due to the minor reactivity of allyl bromide as compared with epibromohydine. Further investigations and optimization of this procedure are planned in our laboratory.
Method M3 We analyzed the feasibility of a third synthetic method as well, based on the thiol-ene click chemistry [34,39] (Scheme 4). It is a one pot procedure as M2, and it has the advantage of being
Peptide-Cotton Characterization and Loading Determination The FT-IR absorption spectroscopy (KBr pellet) was applied in order to detect the effective anchoring of the peptide on the
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Scheme 4. Functionalization of the hydroxyl cellulosic group via thiol-ene chemistry (M3). (i) Allyl bromine, NaH in THF and (ii) peptide-SH, 1% DMPA in THF, UV lamp (350 nm), 2 h.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 547–553
PEPTIDES ON COTTON TEXTILES cotton surface. In particular, we compared the FT-IR spectrum of pure cotton with those of the cotton-peptide samples 1a, 2a, and 3a. The presence of the peptide was revealed by the presence of the amide I band (C O stretching) near 1650 cm 1 and the amide II band (C–N stretching and N–H bending) near 1540 cm 1 [42,43] (Table 2). As shown in Figure 1, for pure cotton and sample 3a, the amide bands are relatively weak and broad, but they are absent in the pure cotton. Therefore, we are confident that our peptides are definitely linked to the cotton fibers. For a quantitative evaluation of the peptide bound, we relied on a UV-based method frequently used in SPPS. It exploits the Fmoc Nα-protecting group. After linking the N-terminal amino acid (M1) or the entire Fmoc protected peptide (M2) to the cotton, the Fmoc group was removed and the solution collected. The absorption at 301 nm of this solution gave an exact quantification of the dibenzofulvene chromophore (ε = 7800 cm 1 M 1) formed upon Fmoc removal. For the dendrimers, we need to take the number of peptide units (two for compound 2a, four for 3a) into account. The results are summarized in Table 3. We can notice that for sample 1a, the peptide loadings deriving from M1 and M2 are similar (about 0.82 mmol/gcotton), thus suggesting that both synthetic methods are equally efficient. For dendrimer 2a, we expected a value double as that obtained for 1a. Actually, we found a value (1.47 mmol/gcotton) that is slightly lower. The same trend is observed for dendrimer 3a, with a value of 2.85 mmol/gcotton instead of the expected 3.3 mmol/gcotton. We attribute these deviations from the ideal values both to experimental errors and to a slightly lower coupling yield when the peptide chain is branched and long. The type of cotton used is expected to have a high content of cellulose and thus of derivatization sites. To verify this assumption, we repeated the synthesis of sample 1a by using a commercially available, hydrophilic, cotton wool sample (1a′, Table 3). The peptide loading observed (0.85 mmol/g cotton) is comparable with that of the fabric cotton employed in all other syntheses.
Table 2. FT-IR bands found for peptide-cotton samples (m, medium; br, broad; w, weak) Sample 1a 2a 3a
1
1
Amide I (cm )
Amide II (cm )
1653 (m) 1655 (m) 1674 (m)
1543 (br, w) 1540 (br, w) 1539 (br, w)
Table 3. Loading values found for the peptide-cotton samples obtained with a UV absorption method Loading mmol/gcotton 1a 1a′ 2a 3a 4a
0.86 (M1), 0.79 (M2) 0.85 (M1) 1.47 (M1) 2.85 (M1) 0.07 (M3)
The synthetic methods used are in parenthesis.
To confirm the data of the UV analysis and to further characterize our hybrid systems, we explored the potentiality of EPR spectroscopy on the TEMPO-functionalized samples 1b, 2b, and 3b. In Figure 2, we compare the EPR spectra of 4-hydroxy-TEMPO in toluene solution and that of compound 1b at room temperature. The solution spectrum (black line) is about 30 Gauss wide. It shows three components, because of the hyperfine coupling with the 14N nucleus (hyperfine constant aN = 15.4 Gauss), with equal line widths, as expected for a nitroxide species free to rotate in a nonviscous solvent (fast motional regime, i.e. rotational correlation time τ r < 10 9 s) [44]. On the contrary, the spectrum of peptide-cotton 1b (red line) is wider (≈70 Gauss) and shows contributions from two spin probe populations. The main one is in the slow motion regime with rotational correlation time τ r = 16.5 ns, whereas the marginal population is characterized by τ r = 2 ns. This mobility difference, with respect to 4-hydroxy-TEMPO in liquid toluene, reflects the lower rotational freedom of the textile-linked nitroxide probe and confirms the actual covalent linking of the peptide to the cotton surface. Multiple contributions to the EPR spectra coming from 4hydroxy-TEMPO in different motional regimes have been reported even for a spin probe adsorbed into the cotton (not covalently bound) [45]. With the aim of better understanding the features of nitroxide spin probes on cotton, we started a preliminary investigation on the effect of temperature on the EPR spectra of sample 1b (Figure 3). At 170 K, the spectrum is about 70 Gauss wide, and its aspect is typical of a nitroxide probe immobilized in the timescale of X-band EPR [44]. Indeed, the
4
J. Pept. Sci. 2014; 20: 547–553 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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Figure 1. FT-IR spectra (KBr disc) of cotton and cotton sample 3a.
Figure 2. EPR spectra of 4-hydroxy-TEMPO solution (10 M) in toluene (black line) and of cotton-peptide 1b (red line) at T = 290 K. Calculated electron paramagnetic resonance spectrum of cotton-peptide 1b (dashed black line) obtained as the sum of two contributions with τ r1 = 16.5 ns and τ r2 = 2 ns.
ORLANDIN ET AL. about 70 Gauss and had the typical shape of slow moving nitroxide probes, even if double slow contributions are present in the traces. Conversely, the EPR spectrum of 3b is narrower, covering only ~40 Gauss, and has a line shape (three hyperfine lines) that resembles much that of 1b at high temperature. Once more, the EPR spectrum of 3b shows that there have been contributions from slow moving spin labels but also substantial contributions from fast moving nitroxides. This finding indicates a more mobile spin label. Indeed, in sample 3b, two Lys residues move the peptides, and the nitroxyl probes farther from the cotton surface.
Conclusions Figure 3. Electron paramagnetic resonance spectra of 1b at temperatures ranging from 170 K to 320 K (solid lines). Calculated electron paramagnetic resonance spectra of cotton-peptide 1b (dashed black lines) obtained with the parameters reported in Table 4. Spectra are shifted vertically for clarity.
170 K simulation (Figure 3) is obtained using τ r = 84 ns (Table 4). In this case, the spectral shape is due to both isotropic (constant aN) and anisotropic hyperfine interactions and to the anisotropy of the g tensor. As the temperature is raised, the spectrum shrinks as expected. In fact, the nitroxide label gains mobility that averages the anisotropic contributions, thus reducing their effect on the spectrum. Anyway, for T ≥ 270 K, the spectra show two contributions coming from two different spin-label populations (Table 4). One of them reaches at 320 K, the fast motion regime, while the other one is in the slow motion regime at all temperatures. At 320 K, the averaging of the anisotropic magnetic interaction is therefore almost complete only for the population of nitroxides in the fast motion regime. Its spectrum extends for about 30 Gauss and shows three well-resolved hyperfine lines, separated by aN = 16 Gauss. The other population gives still a slow motion spectrum (τ r = 7.4 ns), which contributes to about one half of the total intensity. Figure 4 reports the EPR spectra of the three spin-labeled peptides at 290 K. The spectra of 1b and 2b extend through
In this paper, we report and compare three different protocols with covalently link peptides to natural cotton textiles. Both M1 (stepby-step synthesis directly on the cotton) and M2 (anchorage of a pre-synthesized peptide) are valuable methodologies. Thus, the appropriate method can be chosen according to the nature of the peptide to be bound to the cotton. Notably, M1 allowed also the synthesis of Lys dendrimers, a useful expedient to increase peptide concentration on a tissue and, hopefully, antibacterial bioactivity [46]. Method M3, based on the thiol-ene chemistry, is a promising alternative in view of its chemoselectivity. However, this approach needs further improvements to increase the yield of peptide anchorage. For a quantitative determination of the peptide bound to cotton, the UV method commonly used in SPPS (absorption of the dibenzofulvene-piperidine adduct from Fmoc removal) appears to be reliable. EPR proved to be a highly informative spectroscopic technique, provided that a paramagnetic probe is available on the samples to be investigated. The present study illustrates a few synthetic pathways and analytical procedures that should allow the precise design of peptide-decorated cotton tissues. In particular, the syntheses of antimicrobial textiles for medical environments are currently in progress in our laboratory. Acknowledgements
Table 4. Rotational correlation times τ r used to calculate the EPR spectra reported in Figure 3
320 K 270 K 170 K
τ r1 (ns)
τ r2 (ns)
7.4 (slow) 17 (slow) 84 (slow)
0.7 (fast) 2 (fast)
The authors are grateful to the Italian Ministry of Research (PRIN 2010, 2010NRREPL) and CARIPARO Foundation (Progetto Eccellenza 2011/2012 and PhD scholarship to A. O.) for the financial support. They also wish to thank Dr Renato Schiesari for the FT-IR measurements and the Romanian and Italian authorities that sponsored an Exchange Project (RO13MO6/M00301) on the development of antibacterial textiles.
References
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Figure 4. Electron paramagnetic resonance spectra of cotton-peptide samples 1b, 2b, and 3b.
1 Gugliuzza A, Drioli E. A review on membrane engineering for innovation in wearable fabrics and protective textiles. J. Membrane Sci. 2013; 446: 350–375. DOI: 10.1016/j.memsci.2013.07.014. 2 Heinze T, Liebert T. Unconventional methods in cellulose functionalization. Prog. Polym. Sci. 2001; 26: 1689–1762. DOI: 10.1016/S0079-6700(01) 00022-3. 3 Edwards JV, Goheen SC. Performance of bioactive molecules on cotton and other textiles. RJTA 2006; 10: 19–32. 4 Carlmark A, Larsson E, Malmström E. Grafting of cellulose by ring-opening polymerisation – a review. Eur. Polym. J. 2012; 48: 1646–1659. DOI: 10.1016/j.eurpolymj.2012.06.013. 5 Gupta D, Bhaumik S. Antimicrobial treatments for textiles. Indian J. Fibre Text. Res. 2007; 32: 254–263. 6 Borkow G Gabbay J Biocidal textiles can help fight nosocomial infections. Med. Hypotheses 2008; 70: 990–994. DOI: 10.1016/j. mehy.2007.08.025.
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PEPTIDES ON COTTON TEXTILES 7 Coman D, Oancea S, Vrînceanu N. Biofunctionalization of textile materials by antimicrobial treatments: a critical overview. Rom. Biotech. Lett. 2010; 15: 4913–4921. 8 Sun G. Prevention of hospital and community acquired infections by using antibacterial textiles and clothing (Chapter 6). In Polymeric Materials with Antimicrobial Activity: From Synthesis to Applications, The Royal Society of Chemistry: Cambridge, UK, 2014; 139–155. 9 Monteiro DR, Gorup LF, Takamiya AS, Ruvollo-Filho AC, Camargo ERd, Barbosa DB. The growing importance of materials that prevent microbial adhesion: antimicrobial effect of medical devices containing silver. Int. J. Antimicrob. Ag. 2009; 34: 103–110. DOI: 10.1016/j.ijantimicag.2009.01.017. 10 Stobiea N, Duffyb B, Hinderc SJ, McHaled P, McCormack DE. Silver doped perfluoropolyether-urethane coatings: antibacterial activity and surface analysis. Colloid Surf. B. 2009; 72: 62–67. DOI: 10.1016/j. colsurfb.2009.03.014. 11 Gouveia IC. Nanobiotechnology: a new strategy to develop non-toxic antimicrobial textiles. In Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Formatex: Badajoz, Spain, 2010; 407–414. 12 Kim SS, Park JE, Lee J. Properties and antimicrobial efficacy of cellulose fiber coated with silver nanoparticles and 3-mercaptopropyltrimethoxysilane (3MPTMS). J. Appl. Polymer Sci. 2011; 119: 2261–2267. DOI: 10.1002/ app.32975. 13 Liu Z, Ren G, Zhang T, Yang Z. Action potential changes associated with the inhibitory effects on voltage-gated sodium current of hippocampal CA1 neurons by silver nanoparticles. Toxicology 2009; 264: 179–184. DOI: 10.1016/j.tox.2009.08.005. 14 Gao Y. Cranston R Recent advances in antimicrobial treatments of textiles. Text. Res. J. 2008; 78: 60–72. DOI: 10.1177/0040517507082332. 15 Xiao H, Qian L. Water-soluble antimicrobial polymers for functional cellulose fibres and hygiene paper products (Chapter 4). In Polymeric Materials with Antimicrobial Activity: From Synthesis to Applications, The Royal Society of Chemistry: Cambridge, UK, 2014; 75–96. 16 Caldeira E, Piskin E, Granadeiro L, Silva F, Gouveia IC. Biofunctionalization of cellulosic fibres with L-cysteine: assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae. J. Biotechnol. 2013; 168: 426–435. DOI: 10.1016/j.jbiotec.2013.10.021. 17 Peggion C, Biondi B, De Zotti M, Oancea S, Formaggio F, Toniolo C. Spectroscopically labeled peptaibiotic analogs: the 4-nitrophenylalanine infrared absorption probe inserted at different positions into Trichogin GA IV. J. Pept. Sci. 2013; 19: 246–256. DOI: 10.1002/psc.2475. 18 De Zotti M, Biondi B, Peggion C, Park Y, Hahm K-S, Formaggio F, Toniolo C. Synthesis, preferred conformation, protease stability, and membrane activity of heptaibin, a medium-length peptaibiotic. J. Pept. Sci. 2011; 17: 585–594. DOI: 10.1002/psc.1364. 19 Peggion C, Coin I, Toniolo C. Total synthesis in solution of alamethicin F50/5 by an easily tunable segment condensation approach. Biopolymers (Pept. Sci.) 2004; 76: 485–493. DOI: 10.1002/bip.20161. 20 Peggion C, Formaggio F, Crisma M, Epand RF, Epand RM, Toniolo C. Trichogin: a paradigm for lipopeptaibols. J. Pept. Sci. 2003; 9: 679–689. DOI: 10.1002/psc.500. 21 Nakamura M, Iwasaki T, Tokino S, Asaoka A, Yamakawa M, Ishibashi J, Development of a bioactive fiber with immobilized synthetic peptides designed from the active site of a beetle defensin. Biomacromolecules 2011; 12: 1540–1545. DOI: 10.1021/bm1014969. 22 Blankemeyer-Menge B, Nimtz M, Frank R. An efficient method for anchoring fmoc-anino acids to hydroxyl-functionalised solid supports. Tetrahedron Lett. 1990; 31: 1701–1704. DOI: 10.1016/S0040-4039(00)88858-9. 23 Lebl M. Solid-phase synthesis on planar supports. Biopolymers (Pept. Sci.) 1998; 47: 397–404. DOI: 10.1002/(sici)1097-0282(1998)47:53.0.co;2-u. 24 Hilpert K, Volkmer-Engert R, Walter T, Hancock REW. High-throughput generation of small antibacterial peptides with improved activity. Nat. Biotech. 2005; 23: 1008–1012. DOI: 10.1038/nbt1113. 25 Frank R, Döring R. Simultaneous multiple peptide synthesis under continuous flow conditions on cellulose paper discs as segmental solid supports. Tetrahedron 1988; 44: 6031–6040. DOI: 10.1016/S0040-4020 (01)89791-X.
26 Rinnova M, Jezek J, Malon P, Lebl M. Comparative multiple synthesis of 50 linear peptides – evaluation of cotton carrier vs T-bag benzhydrylamine resin. Pept. Res. 1993; 6: 88–94. 27 Kramer A, Schuster A, Reineke U, Malin R, Volkmer-Engert R, Landgraf C, Schneider-Mergener J. Combinatorial cellulose-bound peptide libraries: screening tool for the identification of peptides that bind ligands with predefined specificity. Methods 1994; 6: 388–395. DOI: 10.1006/meth.1994.1039. 28 Mutulis F, Tysk M, Mutule I, Wikberg JES. A simple and effective method for producing nonrandom peptide libraries using cotton as a carrier in continuous flow peptide synthesizers. Comb. Chem. 2002; 5: 1–7. DOI: 10.1021/cc0200078. 29 Volkmer R. Synthesis and application of peptide arrays: quo vadis SPOT technology. Chem. Bio. Chem. 2009; 10: 1431–1442. DOI: 10.1002/cbic.200900078. 30 Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 1970; 34: 595–598. DOI: 10.1016/0003-2697 (70)90146-6. 31 Makovitzki A, Avrahami D, Shai Y. Ultrashort antibacterial and antifungal lipopeptides. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15997–16002. DOI: 10.1073/pnas.0606129103. 32 Vallon-Eberhard A, Makovitzki A, Beauvais A, Latge JP, Jung S, Shai Y. Efficient clearance of Aspergillus fumigatus in murine lungs by an ultrashort antimicrobial lipopeptide, palmitoyl-Lys-Ala-DAla-Lys. Antimicrob. Agents Chemother. 2008; 52: 3118–26. DOI: 10.1128/ AAC.00526-08. 33 Makovitzki A, Baram J, Shai Y. Antimicrobial lipopolypeptides composed of palmitoyl di- and tricationic peptides: in vitro and in vivo activities, self-assembly to nanostructures, and a plausible mode of action. Biochemistry 2008; 47: 10630–10636. DOI: 10.1021/bi8011675. 34 Hoyle CE, Bowman CN. Thiol-ene click chemistry. Angew. Chem. Int. Edit. 2010; 49: 1540–1573. DOI: 10.1002/anie.200903924. 35 Tam JP. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5409–5413. 36 Tam JP, Lu Y-A, Yang J-L. Antimicrobial dendrimeric peptides. Eur. J. Biochem. 2002; 269: 923–932. DOI: 10.1046/j.0014-2956.2001.02728.x. 37 Young AW, Liu Z, Zhou C, Totsingan F, Jiwrajka N, Shi Z, Kallenbach NR. Structure and antimicrobial properties of multivalent short peptides. Med. Chem. Comm. 2011; 2: 308–314. DOI: 10.1039/c0md00247j. 38 Kowalczyk W, Monsó M, de la Torre BG, Andreu D. Synthesis of multiple antigenic peptides (MAPs) – strategies and limitations. J. Pept. Sci. 2011; 17: 247–251. DOI: 10.1002/psc.1310. 39 Ringot C, Sol V, Granet R, Krausz P. Porphyrin-grafted cellulose fabric: new photobactericidal material obtained by ‘Click-Chemistry’ reaction. Mater. Lett. 2009; 63: 1889–1891. DOI: 10.1016/j. matlet.2009.06.009. 40 Zhao G-L, Hafrén J, Deiana L, Córdova A. Heterogeneous ‘organoclick’ derivatization of polysaccharides: photochemical thiol-ene click modification of solid cellulose. Macromol. Rapid Comm. 2010; 31: 740–744. DOI: 10.1002/marc.200900764. 41 Kade MJ, Burke DJ, Hawker CJ. The power of thiol-ene chemistry. J. Polym. Sci. A1 2010; 48: 743–750. DOI: 10.1002/pola.23824. 42 Volkenstein MV. Molecular Biophysics, Academic Press: London,1977. 43 Doh S, Lee J, Lim D, Im J. Manufacturing and analyses of wet-laid nonwoven consisting of carboxymethyl cellulose fibers. Fibers Polym. 2013; 14: 2176–2184. DOI: 10.1007/s12221-013-2176-y. 44 Fajer PG. Electron spin resonance spectroscopy labeling in peptide and protein analysis. In Encyclopedia of Analytical Chemistry, John Wiley & Sons, Ltd, 2006; 5725–5761. 45 Frantz S, Hübner GA, Wendland O, Roduner E, Mariani C, Ottaviani MF. Batchelor SN Effect of humidity on the supramolecular structure of cotton, studied by quantitative spin probing. J. Phys. Chem. B 2005; 109: 11572–11579. DOI: 10.1021/jp050791x. 46 Mintzer MA, Dane EL, O’Toole GA, Grinstaff MW. Exploiting dendrimer multivalency to combat emerging and re-emerging infectious diseases. Mol. Pharmaceut. 2012; 9: 342 354. DOI: 10.1021/ mp2005033.
553 J. Pept. Sci. 2014; 20: 547–553 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
Revised: 16 April 2014
Accepted: 30 April 2014
Published online in Wiley Online Library: 28 May 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2659
Cotton functionalized with peptides: characterization and synthetic methods‡ Andrea Orlandin, Fernando Formaggio, Antonio Toffoletti and Cristina Peggion* Three approaches for the chemical ligation of peptides to cotton fibers are described and compared. This investigation was encouraged by the need to create peptide-decorated natural textiles, furnished with useful properties (e.g. antimicrobial activity). IR absorption spectroscopy is proved to be an easy and fast method to check the covalent anchorage of a peptide to cotton, whereas for a quantitative determination, a UV absorption method was employed. We also analyzed the usefulness of electron paramagnetic resonance spectroscopy to characterize our peptide-cotton conjugates. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: peptides; functionalized textiles; EPR; dendrimers
Introduction
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Materials and Methods Fmoc and Boc (Fmoc, fluorenyl-9-methoxycarbonyl, Boc, tert-butyloxycarbonyl) protected amino acids were Bachem products (Bubendorf, Switzerland). All other chemicals and reagents for the peptide synthesis were purchased from Sigma-Aldrich (St. Louis, MO). 2-Chlorotrityl resin was purchased from Iris Biotech (Marktredwitz, Germany). The cotton used in our experiments was a commercially available tissue, ready for garment manufacture. For each reaction on textiles, a piece of fabric cotton of about 2 cm2 was accurately weighed, washed with methanol (MeOH), DMF, and MeOH, and air-dried. In one case, we employed a hydrophilic cotton wool sample, accurately weighed and washed as just described. Fmoc-NH-CH2CH2-NH2, prepared by treating Fmoc N-hydroxysuccinimide ester (3 mmol) with 1,2-diaminoethane (20 mmol) in CH2Cl2, was isolated by extraction (CH2Cl2/water) and precipitation (CH2Cl2/petroleum ether). * Correspondence to: Cristina Peggion, Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy. E-mail: [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the 1st International Conference on Peptide Materials for Biomedicine and Nanotechnology, Sorrento, October 28-31, 2013, edited by Professor Giancarlo Morelli, Professor Claudio Toniolo and Professor Mariano Venanzi. ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
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New materials based on biodegradable molecules are of great interest to reduce adverse effects on the environment. The need to develop new materials for a variety of applications is greatly promoting academic and industrial research. In particular, textile manufacturers are looking at the production of fabrics made of natural fibers but opportunely functionalized for specific technical applications [1]. Among the many polymeric materials available, cellulose fibers are particularly attractive, being naturally occurring, and easy to functionalize [2–4]. In this connection, it is worth mentioning that cotton-based garments, functionalized with antimicrobial compounds, are already commercially available [1,5]. Protective and safe textiles are of fundamental importance for health care workers and for immuno-compromised or debilitated people who need to be protected from infections [6–8]. The antimicrobial textiles currently on the market are in general made of synthetic polymers that contain silver salts or silver nanoparticles [9–12] (for which toxicity on skin has not been completely made clear [13]), quaternary ammonium salts, triclosan, chitosan, dyes, and regenerable N-halamine compounds [14,15]. Most of the time, the active compounds are simply adsorbed or electrostatically linked [16] to the cotton surface. Therefore, they are usually lost after a few cycles of washing. There is a great demand for durable antimicrobial textiles based on nontoxic bioactive compounds. To contribute to this topic, we started a research program that heavily relies on our expertise in the field of antibacterial peptides [17–20]. The main advantage of using amino acids and peptides comes from their presence in nature. Thus, they should be well tolerated by our body. Recently, a cysteine residue linked to a cellulosic support showed antibacterial properties [16]. A 9-mer peptide was immobilized on cotton fibers giving a material able to inhibit the growth of bacterial strains [21]. Usually, cellulose derivatization involves the hydroxyl groups of cotton on which esters [22] and ethers (ethyl cellulose) can be prepared. This derivatization, in turn, has allowed the use of cotton as a substrate for the
‘solid phase’ synthesis of peptides (SPPS) [23,24] and for combinatorial syntheses [25–29]. This study has three main goals: (i) to compare and discuss synthetic methods to efficiently link peptides to cotton fibers, (ii) to enhance peptide concentration on the cotton fibers through the anchorage of peptide dendrimers, and (iii) to characterize and quantify the peptides linked to cellulosic supports.
ORLANDIN ET AL. Cotton Functionalization with Epibromohydrin and 1,2-Diaminoethane (C1) A cotton piece (0.3 g) was incubated with a 60% HClO4 solution (0.1 ml) in DMF and epibromohydrin (1 ml) for 4 h. Then, the sample was washed with DMF and MeOH and air-dried. This brominated cotton fiber was then immersed in a solution of Fmoc-NH-CH2CH2-NH2 (5 mmol) and triethylamine (5 mmol) in DMF (2 ml) for 12 h. Finally, the functionalized cotton sample was treated for 2 h with a 20% solution of Ac2O in DMF (10 ml) in the presence of triethylamine (1 ml). Fmoc removal was achieved with 20% piperidine solution in DMF. The sample was washed with DMF and MeOH and air-dried.
2-(tritylthio)ethylamine (640 mg, 2 mmol) in DMF in the presence of the N-ethyl, N′-[3-(dimethylamino)propyl]carbodiimide/HOAt coupling mixture. Removal of the triphenylmethyl group was achieved with 1% TFA solution in dichloromethane. Pure peptides 1 and 4 were obtained by crystallization from ethyl acetate/diethyl ether. The peptides were characterized by analytical RP-HPLC on a Jupiter Phenomenex (Torrance, CA) C18 column (4.6 × 250 mm, 5 μ, 300 Å) using an Agilent (Santa Clara, CA) 1200 HPLC instrument. The binary elution system used was A, 0.05% TFA in H2O; B, 0.05% TFA in CH3CN/H2O (9:1 v/v), flow rate 1 ml/min; and spectrophotometric detection at λ = 216 nm. ESI-MS was performed by using a PerSeptive Biosystem Mariner instrument (Framingham, MA). The amino acid sequence, mass spectral data, and HPLC retention time of peptides 1 and 4 are as follows:
Cotton Functionalization with Allyl Bromide (C2) A cotton piece (0.3 g) was immersed in a solution of NaH (200 mg) in 5 ml tetrahydrofuran (THF) and fluxed with nitrogen for 1 h at 0 °C. Then, allyl bromide was added (1.5 ml), and nitrogen flux was continued for 2 h. The reagents were removed by washing with THF.
Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)-NH(CH2)2NH2 (1) HPLC: Rt = 7.30 min (30–70% B in 20 min); mass: calculated 880.45; found 881.52 [M + H]+, 781.52 [M 1Boc + H]+, 391.23 [M 1Boc + 2H]2+. Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2SH (4) HPLC: Rt = 13.4 min (50–100% B in 20 min); mass: calculated 755.36; found 756.40 [M + H]+.
Synthesis of the Linear Peptides and Characterization Linear peptides (Table 1) were synthesized by SPPS, performed manually by means of filter-equipped syringes on a 0.8 mmol scale. For peptide 1, 2-chlorotrityl resin (500 mg, loading 1.6 mmol/g) was treated with a 20% 1,2-diaminoethane solution in DMF for 2 h under nitrogen flux. Then, Fmoc-Lys(Boc)-OH, Fmoc-D-Ala-OH, Fmoc-Ala-OH, and Fmoc-Lys(Boc)-OH were added step-by-step using the Fmoc strategy. Each time, 3 eq. (2.4 mmol) of protected amino acid were activated with 3 eq. of HOAt/HATU and 6 eq. of DIPEA using DMF as the solvent. Deprotection of the Fmoc group was achieved with 20% piperidine solution in DMF (2× 15 min). Removal of the peptide from the resin was achieved with 5% TFA in dichloromethane (2× 15 min). The solutions were pooled and concentrated in vacuo (after addition of few milliliters of acetonitrile to prevent Boc removal) to a crude solid. Peptide 4, with two residues of Fmoc-Lys(Boc)-OH (3 eq., 4.8 mmol), was also built on a 2-chlorotrityl resin (1 g, loading 1.6 mmol/g) as described earlier. The free C-terminus peptide obtained (700 mg, 1.2 mmol) was added to a solution of Table 1. List of the peptides and peptide-cotton samples prepared Preparation method 1 1a 2a 3a 1b 2b 3b
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4 4a
Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-NH2 Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-NR-cotton (R Ac or H) [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys-NH-CH2-CH2-N(Ac)-cotton {[Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys}2-Lys-NH-CH2-CH2-N(Ac)-cotton TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)NH-CH2-CH2-N(Ac)-cotton [TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys-NH-CH2-CH2-N(Ac)-cotton {[TEMPO-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2Lys}2-Lys-NH-CH2-CH2-N(Ac)-cotton Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2-SH Fmoc-Lys(Boc)-Lys(Boc)-NH(CH2)2-S-cotton
SPPS M1 and M2 M1 M1 M1 M1 M1 SPPS M3
Peptide Synthesis on Modified Cotton Fibers and Characterization of the Conjugates Cotton samples (C1) of 0.3 g (about 2 cm2) were dunked in DMF. For sample 1a (Table 1), the peptide was built step by step by adding the required Fmoc amino acid (1.5 mmol) activated with HOAt/HATU (1.5 mmol each) and DIPEA (4.5 mmol) in DMF and letting the reaction to proceed for 3 h. Fmoc removal was achieved with a 20% piperidine solution in DMF (2× 15 min). For dendrimer 2a, Fmoc-Lys(Fmoc)-OH (1.5 mmol) was first coupled to aminofunctionalized cotton. After Fmoc removal, the SPPS was carried on as for 1a using double quantities for each reagent. For dendrimer 3a, Fmoc-Lys(Fmoc)-OH (1.5 mmol) was first linked to the functionalized cotton by activation with HOAt/HATU (1.5 mmol each) and DIPEA (4.5 mmol) in DMF. After Fmoc removal, Fmoc-Lys (Fmoc)-OH was reacted again (double quantities). Finally, after Fmoc deprotection, the peptide was built on the cotton as for 1a, using quadruple quantities of each reagent. Samples 1b, 2b, and 3b were prepared from 1a, 2a, and 3a, respectively. 1a was treated with a 20% piperidine solution for Fmoc removal. Then, it was reacted with succinic acid (177 mg, 1.5 mmol) in the presence of HOAt/EDC (1.5 mmol each) in DMF. After washing with DMF, the sample was incubated with 4-hydroxy2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) (260 mg, 1.5 mmol) and HOAt/HATU (1.5 mmol each) in DMF, for 4 h. 2a and 3b were treated as described for 1a using double and quadruple reagent quantities, respectively, for each reaction. For the preparation of 4a, a solution of peptide 4 (450 mg, 0.5 mmol) in THF, containing 2,2-dimethoxy-2-phenylacetophenone (1 wt.%), was added to an allylated cotton sample (C2). Then, the mixture was irradiated at 350 nm for 1 h in a Royonet Photochemical Reactor. All cotton samples were washed (2× DMF and 2× MeOH) and dried under vacuum. Each reaction step was checked by the Kaiser test [30] (before and after Fmoc deprotection). To this aim, a thread of cotton was washed with DMF, MeOH, and acetone; dried; and treated with the three monitor solutions heating at 100 °C for 5 min. For yield evaluation, quantitative Fmoc removal and UV absorption determination were used (see Results and Discussion Section). The UV absorption spectra of the Fmoc removal solution were recorded with a Shimadzu model UV-2501 spectrophotometer.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 547–553
PEPTIDES ON COTTON TEXTILES Cotton-peptide samples were characterized by FT-IR absorption in the solid-state (KBr disk) by means of a Perkin-Elmer model 1720X FTIR spectrophotometer. To prepare the KBr disk, a small fiber piece was reduced to powder and mixed with the KBr matrix. The most informative FT-IR bands are reported in the following text. Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)-NH(CH2)2NH2-cotton (1b) IR (KBr): ν = 1653, 1543, 1249 cm 1. [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]2-Lys- NH(CH2)2NH2-cotton (2b) IR (KBr): ν = 1655, 1540, 1260 cm 1. [Fmoc-Lys(Boc)-Ala-D-Ala-Lys(Boc)]4-(Lys)2-Lys-NH(CH2)2NH2-cotton (3b) IR (KBr): ν = 1674, 1539, 1256 cm 1. EPR Measurements Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker ER 200D X-band spectrometer (~9.4 GHz). Samples of 1b, 2b, and 3b with about 1 cm2 area were inserted in EPR quartz tubes (inner diameter = 3 mm). As a control, we used the cotton fabric without any functionalization. A 0.1 mM toluene solution of 4-hydroxy-TEMPO was used as nitroxide reference solution. Temperature of the samples was regulated using a nitrogen flow cryostat (Bruker BVT 2000).
Results and Discussion For this study the sequences, -Lys-Ala-D-Ala-Lys- [31,32] and -LysLys- [33] >were selected as model peptides. These peptides were chosen because they are short (easy synthesis) and possess antibacterial properties when suitably functionalized at the N-terminus. Indeed, the follow-up of this work will be the preparation of textiles with antimicrobial properties. The compounds prepared in this study are summarized in Table 1. Textile Surface Modification The textile used in this study is a cotton fabric. Three approaches for covalently linking peptides to the cotton surface were explored. First, we examined the step-by-step synthesis of the peptide directly on the cotton surface [21] used as a support. In this case, the cotton surface needed to be opportunely modified. In a second approach, we tested the ‘one pot’ attachment of an entire peptide, via amide bond formation, to a modified cotton textile. Finally, we exploited the thiol-ene chemistry [34]. The first (M1) and second (M2) approach share the cellulose functionalization step with epibromohydrin as shown in Scheme 1.
Method M1 In the first step (i, Scheme 1), hydroxyl groups of the cellulosic support, soaked in DMF, react with epibromohydin in the presence of HClO4 [21]. The epoxide opens forming an ether bond and introducing a halogen at the same time. Bromine is then substituted by reaction with Fmoc-NH-CH2CH2-NH2 followed by acetylation of the newly formed secondary amine. The procedure adopted allows an easier functionalization of the cotton surface as (i) we introduce a spacer, thus walking away from the crowded glucose moieties, and (ii) we make available an amino group, a better nucleophile as compared with an alcoholic function. The cotton can then be used as a support to build the peptide according to standard SPPS procedures (step-by-step addition of the Fmoc amino acid of interest). Each reaction step was checked by taking out a small cotton thread on which a Kaiser test [30] was run (before and after Fmoc deprotection). To go to completion, couplings required 3 h and three equivalents of Fmoc-residue, less than what was reported in the literature [21]. Finally, the Fmoc Nα-protecting group was removed with a piperidine solution in DMF. The cotton was washed at each step with DMF to completely eliminate the coupling reagents. As we cannot increase the number of the cellulose alcoholic functions reacting with epibromohydrin, under these conditions, we decided to verify if peptide loading on the cotton can be boosted by the dendrimer approach. Therefore, small Lys dendrimers [35–38], with two or four tetrapeptide units (Table 1), were also grown on cotton according to the M1 procedure (Scheme 2). The synthesis was shown to be feasible, although a yield improvement is required in the case of the sterically hindered dendrimer 3a (see the discussion on the ‘loading’ section). Method M1 was also used to prepare the cotton-peptide samples functionalized with a free radical probe, useful for EPR investigations (Scheme 3). Compounds 1b, 2b, and 3b were prepared from 1a, 2a, and 3a, respectively. After Fmoc removal and coupling with succinic acid, the nitroxyl moiety was introduced by a HATU/HOAt mediated esterification of 4-hydroxy-TEMPO with the free carboxylic function of succinic acid. Method M2 Method M1 requires uncommon and repetitive manipulations of a tissue in order to carry out the numerous synthetic steps. To this aim, a dedicated glass apparatus was purposely built. However, to assess if M1 is an effective procedure, we synthesized sample 1a also according to a more traditional procedure. In this approach, the peptide is prepared beforehand with the 1,2-diaminoethane spacer linked to its C-terminus. The synthesis of peptide 1 was performed on a 2-chlorotrityl resin. First, the
J. Pept. Sci. 2014; 20: 547–553 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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Scheme 1. Reaction conditions: (i) epibromohydrin, 60% HClO4 in DMF; (ii) (M1) Fmoc-NH-CH2CH2-NH2, triethylamine in DMF; Ac2O, triethylamine in DMF; 20% piperidine in DMF; (iii) step-by-step SPPS: Fmoc-AA-OH, HOAt/HATU, DIPEA in DMF; then, piperidine in DMF, except after the last coupling; and (iv) (M2) DIPEA in DMF.
ORLANDIN ET AL.
Scheme 2. Reaction conditions for the synthesis of 2a and 3a: (i) Fmoc-Lys(Fmoc)-OH, HOAt/HATU, and DIPEA in DMF, then piperidine in DMF; and (ii) step-by-step SPPS with the conditions of (i) but no Fmoc deprotection after the last coupling.
Scheme 3. Introduction of the paramagnetic label in the peptide-cotton samples. (i) EDC/HOAt in DMF and (ii) HATU/HOAt in DMF.
1,2-diaminoethane spacer was linked to the resin by nucleophilic substitution of the chlorine atom on the resin support. Then, each Fmoc N-protected amino acid residue was coupled in the presence of the excellent activating agents HOAt/HATU and DIPEA, using DMF as solvent. Deprotection of the Fmoc group was achieved with piperidine. Removal of the peptide from the resin was performed with 5% TFA in CH2Cl2. The evaporation of this peptide solution was carried out in the presence of acetonitrile in order to preserve the Boc protection on the Lys side chains. The pure peptide 1, obtained by recrystallization, was linked to the brominated cotton surface by nucleophilic substitution under nitrogen flux (Scheme 1, step iv). Synthetic methods M1 and M2 gave similar results in terms of peptide loading on the tissue (see the discussion on ‘loading’ section). Therefore, both approaches can be exploited depending on the requirements of a synthetic design. However, the one pot method M2 allows for a reduced number of operations on the tissue and, in addition, permits to better check the identity and purity of the pre-synthesized peptide.
chemoselective [40]. Indeed, upon photoactivation, the thiol group reacts specifically with the alchene, while other potentially reactive moieties do not interfere [41]. As model peptide, in this case, we chose the simpler peptide 4, synthesized by SPPS on a 2-chlorotrityl resin. It was removed from the resin as described earlier and amidated with 2-(tritylthio)ethylamine. Removal of triphenylmethyl group with 1% TFA afforded peptide 4. The cellulosic support was functionalized by treatment with allyl bromide in the presence of NaH, thus introducing a double bond. Then, peptide 4 was linked to the double bond in the presence of a light source (350 nm) and a catalytic amount of the photocatalyzer 2,2-dimethoxy-2-phenylacetophenone to give sample 4a. The final peptide loading (determined via UV spectroscopy and described hereafter) is much lower as compared with that obtained with methods M1 and M2. This low functionalization is probably due to the minor reactivity of allyl bromide as compared with epibromohydine. Further investigations and optimization of this procedure are planned in our laboratory.
Method M3 We analyzed the feasibility of a third synthetic method as well, based on the thiol-ene click chemistry [34,39] (Scheme 4). It is a one pot procedure as M2, and it has the advantage of being
Peptide-Cotton Characterization and Loading Determination The FT-IR absorption spectroscopy (KBr pellet) was applied in order to detect the effective anchoring of the peptide on the
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Scheme 4. Functionalization of the hydroxyl cellulosic group via thiol-ene chemistry (M3). (i) Allyl bromine, NaH in THF and (ii) peptide-SH, 1% DMPA in THF, UV lamp (350 nm), 2 h.
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PEPTIDES ON COTTON TEXTILES cotton surface. In particular, we compared the FT-IR spectrum of pure cotton with those of the cotton-peptide samples 1a, 2a, and 3a. The presence of the peptide was revealed by the presence of the amide I band (C O stretching) near 1650 cm 1 and the amide II band (C–N stretching and N–H bending) near 1540 cm 1 [42,43] (Table 2). As shown in Figure 1, for pure cotton and sample 3a, the amide bands are relatively weak and broad, but they are absent in the pure cotton. Therefore, we are confident that our peptides are definitely linked to the cotton fibers. For a quantitative evaluation of the peptide bound, we relied on a UV-based method frequently used in SPPS. It exploits the Fmoc Nα-protecting group. After linking the N-terminal amino acid (M1) or the entire Fmoc protected peptide (M2) to the cotton, the Fmoc group was removed and the solution collected. The absorption at 301 nm of this solution gave an exact quantification of the dibenzofulvene chromophore (ε = 7800 cm 1 M 1) formed upon Fmoc removal. For the dendrimers, we need to take the number of peptide units (two for compound 2a, four for 3a) into account. The results are summarized in Table 3. We can notice that for sample 1a, the peptide loadings deriving from M1 and M2 are similar (about 0.82 mmol/gcotton), thus suggesting that both synthetic methods are equally efficient. For dendrimer 2a, we expected a value double as that obtained for 1a. Actually, we found a value (1.47 mmol/gcotton) that is slightly lower. The same trend is observed for dendrimer 3a, with a value of 2.85 mmol/gcotton instead of the expected 3.3 mmol/gcotton. We attribute these deviations from the ideal values both to experimental errors and to a slightly lower coupling yield when the peptide chain is branched and long. The type of cotton used is expected to have a high content of cellulose and thus of derivatization sites. To verify this assumption, we repeated the synthesis of sample 1a by using a commercially available, hydrophilic, cotton wool sample (1a′, Table 3). The peptide loading observed (0.85 mmol/g cotton) is comparable with that of the fabric cotton employed in all other syntheses.
Table 2. FT-IR bands found for peptide-cotton samples (m, medium; br, broad; w, weak) Sample 1a 2a 3a
1
1
Amide I (cm )
Amide II (cm )
1653 (m) 1655 (m) 1674 (m)
1543 (br, w) 1540 (br, w) 1539 (br, w)
Table 3. Loading values found for the peptide-cotton samples obtained with a UV absorption method Loading mmol/gcotton 1a 1a′ 2a 3a 4a
0.86 (M1), 0.79 (M2) 0.85 (M1) 1.47 (M1) 2.85 (M1) 0.07 (M3)
The synthetic methods used are in parenthesis.
To confirm the data of the UV analysis and to further characterize our hybrid systems, we explored the potentiality of EPR spectroscopy on the TEMPO-functionalized samples 1b, 2b, and 3b. In Figure 2, we compare the EPR spectra of 4-hydroxy-TEMPO in toluene solution and that of compound 1b at room temperature. The solution spectrum (black line) is about 30 Gauss wide. It shows three components, because of the hyperfine coupling with the 14N nucleus (hyperfine constant aN = 15.4 Gauss), with equal line widths, as expected for a nitroxide species free to rotate in a nonviscous solvent (fast motional regime, i.e. rotational correlation time τ r < 10 9 s) [44]. On the contrary, the spectrum of peptide-cotton 1b (red line) is wider (≈70 Gauss) and shows contributions from two spin probe populations. The main one is in the slow motion regime with rotational correlation time τ r = 16.5 ns, whereas the marginal population is characterized by τ r = 2 ns. This mobility difference, with respect to 4-hydroxy-TEMPO in liquid toluene, reflects the lower rotational freedom of the textile-linked nitroxide probe and confirms the actual covalent linking of the peptide to the cotton surface. Multiple contributions to the EPR spectra coming from 4hydroxy-TEMPO in different motional regimes have been reported even for a spin probe adsorbed into the cotton (not covalently bound) [45]. With the aim of better understanding the features of nitroxide spin probes on cotton, we started a preliminary investigation on the effect of temperature on the EPR spectra of sample 1b (Figure 3). At 170 K, the spectrum is about 70 Gauss wide, and its aspect is typical of a nitroxide probe immobilized in the timescale of X-band EPR [44]. Indeed, the
4
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Figure 1. FT-IR spectra (KBr disc) of cotton and cotton sample 3a.
Figure 2. EPR spectra of 4-hydroxy-TEMPO solution (10 M) in toluene (black line) and of cotton-peptide 1b (red line) at T = 290 K. Calculated electron paramagnetic resonance spectrum of cotton-peptide 1b (dashed black line) obtained as the sum of two contributions with τ r1 = 16.5 ns and τ r2 = 2 ns.
ORLANDIN ET AL. about 70 Gauss and had the typical shape of slow moving nitroxide probes, even if double slow contributions are present in the traces. Conversely, the EPR spectrum of 3b is narrower, covering only ~40 Gauss, and has a line shape (three hyperfine lines) that resembles much that of 1b at high temperature. Once more, the EPR spectrum of 3b shows that there have been contributions from slow moving spin labels but also substantial contributions from fast moving nitroxides. This finding indicates a more mobile spin label. Indeed, in sample 3b, two Lys residues move the peptides, and the nitroxyl probes farther from the cotton surface.
Conclusions Figure 3. Electron paramagnetic resonance spectra of 1b at temperatures ranging from 170 K to 320 K (solid lines). Calculated electron paramagnetic resonance spectra of cotton-peptide 1b (dashed black lines) obtained with the parameters reported in Table 4. Spectra are shifted vertically for clarity.
170 K simulation (Figure 3) is obtained using τ r = 84 ns (Table 4). In this case, the spectral shape is due to both isotropic (constant aN) and anisotropic hyperfine interactions and to the anisotropy of the g tensor. As the temperature is raised, the spectrum shrinks as expected. In fact, the nitroxide label gains mobility that averages the anisotropic contributions, thus reducing their effect on the spectrum. Anyway, for T ≥ 270 K, the spectra show two contributions coming from two different spin-label populations (Table 4). One of them reaches at 320 K, the fast motion regime, while the other one is in the slow motion regime at all temperatures. At 320 K, the averaging of the anisotropic magnetic interaction is therefore almost complete only for the population of nitroxides in the fast motion regime. Its spectrum extends for about 30 Gauss and shows three well-resolved hyperfine lines, separated by aN = 16 Gauss. The other population gives still a slow motion spectrum (τ r = 7.4 ns), which contributes to about one half of the total intensity. Figure 4 reports the EPR spectra of the three spin-labeled peptides at 290 K. The spectra of 1b and 2b extend through
In this paper, we report and compare three different protocols with covalently link peptides to natural cotton textiles. Both M1 (stepby-step synthesis directly on the cotton) and M2 (anchorage of a pre-synthesized peptide) are valuable methodologies. Thus, the appropriate method can be chosen according to the nature of the peptide to be bound to the cotton. Notably, M1 allowed also the synthesis of Lys dendrimers, a useful expedient to increase peptide concentration on a tissue and, hopefully, antibacterial bioactivity [46]. Method M3, based on the thiol-ene chemistry, is a promising alternative in view of its chemoselectivity. However, this approach needs further improvements to increase the yield of peptide anchorage. For a quantitative determination of the peptide bound to cotton, the UV method commonly used in SPPS (absorption of the dibenzofulvene-piperidine adduct from Fmoc removal) appears to be reliable. EPR proved to be a highly informative spectroscopic technique, provided that a paramagnetic probe is available on the samples to be investigated. The present study illustrates a few synthetic pathways and analytical procedures that should allow the precise design of peptide-decorated cotton tissues. In particular, the syntheses of antimicrobial textiles for medical environments are currently in progress in our laboratory. Acknowledgements
Table 4. Rotational correlation times τ r used to calculate the EPR spectra reported in Figure 3
320 K 270 K 170 K
τ r1 (ns)
τ r2 (ns)
7.4 (slow) 17 (slow) 84 (slow)
0.7 (fast) 2 (fast)
The authors are grateful to the Italian Ministry of Research (PRIN 2010, 2010NRREPL) and CARIPARO Foundation (Progetto Eccellenza 2011/2012 and PhD scholarship to A. O.) for the financial support. They also wish to thank Dr Renato Schiesari for the FT-IR measurements and the Romanian and Italian authorities that sponsored an Exchange Project (RO13MO6/M00301) on the development of antibacterial textiles.
References
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Figure 4. Electron paramagnetic resonance spectra of cotton-peptide samples 1b, 2b, and 3b.
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