Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Background Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc. Methods Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2.2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values < 0.05). Results Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol.L−1 (S. aureus) and 10.10 μmol.L−1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2.2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n = 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.


Background
Inflammatory processes can be divided into two categories: septic (induced by bacteria or fungi) or aseptic (absence of microorganisms) inflammation [1]. Thus, differential diagnosis is required to determine the most suitable therapeutic approach. In some cases, such as bone inflammation, current diagnostic methods and conventional imaging techniques are not able to differentiate septic and aseptic inflammation. Therefore, alternatives must be found in order to ensure an accurate diagnosis. In this sense, scintigraphic imaging is a promising approach since it is based on physiological changes, which occur earlier than anatomical modifications [2][3][4]. In this context, radiolabeled antimicrobial peptides have been investigated as possible suitable tools, since they accumulate in infectious sites instead of in aseptic inflammatory lesions, once they preferentially bind to bacteria and fungi [5][6][7].
The cationic peptide LyeTx I was primarily isolated from Lycosa erythrognatha venom. After its purification and characterization, it was obtained by chemical synthesis. The peptide is composed of 25 amino acid residues and carries a natural carboxyl-terminal (C-terminal) carboxyamide (H-IWLTALKFLGKNLGKHLAKQQLAKL-NH 2 ). LyeTx I exhibits antimicrobial activity against microorganisms, such as Escherichia coli, Staphylococcus aureus, Candida krusei and Cryptococcus neoformans [8]. Therefore, radioisotope-labeled LyeTx I may be an interesting strategy for a specific imaging probe for infections.
The radioisotope technetium-99m ( 99m Tc) presents suitable features for its administration to patients in nuclear medicine. This radionuclide emits gamma rays of low energy (~140 keV) and has physical half-life of 6.02 h. 99m Tc exposes patients to low radiation doses whereas it is widely used for radiolabeling molecules employed as scintigraphic imaging probes. Furthermore, this radioisotope is easily obtained from a low cost molibdenium-99/technetium-99m ( 99 Mo/ 99m Tc) generator [9,10]. However, in order to use 99m Tc for radiolabeling peptides without disulfide bonds, such as LyeTx I, it is necessary to attach a chelating agent to the amino acid sequence. In this sense, 2-hydrazinonicotinamide (HYNIC) is a good option, since its carboxylic acid group reacts directly with the nitrogen-terminal (N-terminal) residue or alternatively with the lateral amino group of a lysine residue present in the peptide sequence. However, an extra lysine may be coupled to the C-terminal portion in order to maintain the peptide sequence with a minor change. Lastly, to stabilize 99m Tc binding to HYNIC, tricine and ethylenediamine-N,N'diacetic acid (EDDA) are used as co-ligands in the radiolabeling procedure [11][12][13].
Therefore, this study aimed to synthesize two peptide LyeTx I derivatives modified with the chelating agent HYNIC, to evaluate the maintenance of its antimicrobial activity and to standardize its radiolabeling with 99m Tc atoms.

Materials
Amino acid derivatives for peptide synthesis were purchased from Iris Biotech GmbH (Marktredwitz, Germany). Trifluoroacetic acid (TFA) and triisopropylsilane were obtained from Sigma-Aldrich (Saint Louis, USA). 1,3-diisopropylcarbodiimide was acquired from Fluka (Steinheim, Germany). 1-hydroxybenzotriazole was purchased from NovaBiochem-Merck (Darmstadt, Germany). N,Ndimethylformamide (DMF) and diisopropyl ether were obtained from Vetec (Duque de Caxias, Brazil). Acetonitrile (HPLC grade) was acquired from JT Baker (Center Valley, USA). If not mentioned otherwise, analytical grade solvents were used. All solvents used in reverse phase-high performance liquid chromatography (RP-HPLC) system (HPLC grade) were purchased from Tedia (Rio de Janeiro, Brazil). Ultrapure water, obtained through MilliQ® system of Millipore (Darmstadt, Germany), was used throughout. The bacterial strains of reference, S. aureus (ATCC® 6538) and E. coli (ATCC® 10536), were acquired from American Type Culture Collection -ATCC (Manassas, USA). 99m Tc was obtained from a 99 Mo/ 99m Tc generator supplied by the Nuclear Energy Research Institute -IPEN (São Paulo, Brazil). Other reagents and solvents for the radiolabeling procedure were acquired from Sigma-Aldrich (São Paulo, Brazil).
Synthesis and purification of two peptide LyeTx I derivatives modified with the chelating agent HYNIC Two peptide LyeTx I derivatives with the chelating agent HYNIC attached either to its N-terminal residue (HYNIC-LyeTx I) or to its C-terminal portion (LyeTx I-K-HYNIC) were synthesized and purified, as previously reported [14].
The crude synthetic products were purified by RP-HPLC on a C8 column (Discovery® BIO Wide Pore C8 column, 5 μm, 250.0 mm × 4.6 mm), previously equilibrated with 0.1 % (v:v) TFA in water (eluent A) and eluted by a linear gradient of 0.1 % (v:v) TFA in acetonitrile (eluent B), as specified in Table 1A.
The collected fractions were assessed by matrixassisted laser desorption ionization time of flight mass spectrometer (MALDI-ToF-MS) analysis on AutoFlex III (Bruker Daltonics®, Germany). Briefly, samples were spotted onto a sample plate (MTP 384 Anchorchip, Bruker Daltonics®, Germany) mixed with a saturated solution of α-cyano-4-hydroxycinnamic acid and allowed to dry at room temperature (dried-droplet method). The mass spectrometer (MS) spectra were acquired in the positive reflector mode with external calibration (Peptide Calibration Standard II, Bruker Daltonics®, Germany).
Purity assessment of the peptide LyeTx I derivatives modified with the chelating agent HYNIC The purified synthetic products were analyzed by RP-HPLC on a C18 analytical column (PepMap C18 TM column, 5 μm, 150.0 mm × 4.6 mm), previously equilibrated with 0.1 % (v:v) TFA in water (eluent A) and eluted by a linear gradient of 0.1 % (v:v) TFA in acetonitrile (eluent B), as specified in Table 1B. The peaks of the peptides were collected and analyzed by MALDI-ToF-MS on AutoFlex III (Bruker Daltonics®, Germany), as described in the previous section.
In vitro evaluation of the maintenance of the antimicrobial activity of the peptide LyeTx I derivatives modified with the chelating agent HYNIC The maintenance of the antimicrobial activity after peptide LyeTx I modifications with HYNIC was evaluated by microdilution test, according to the Clinical and Laboratory Standards Institute [15]. Bacterial strains of reference, S. aureus (ATCC® 6538) and E. coli (ATCC® 10536), were grown on tryptic soy agar at 37°C for 18 h. Then, 0.5 McFarland scale bacterial suspensions (10 8 CFU . mL −1 ) were prepared on tryptic soy broth (TSB). The readouts were carried by determination of minimum inhibitory concentration (MIC), defined as a reduction of 100 % in bacterial growth post-incubation with the peptide LyeTx I derivatives at 37°C for 24 h. LyeTx I obtained by chemical synthesis and without the coupled chelating agent was used as treatment control. Only TSB (no bacterial suspension and no peptide) was used as negative control. TSB plus bacterial suspension (no peptide) were used as positive control. MIC was expressed as median (n = 3). Each replicate was performed with a different bacterial colony, in duplicate.

Statistical analysis
Quantitative data were expressed as mean ± standard deviation (SD). Means were compared using Analysis of Variance (ANOVA), followed by Tukey multiple comparisons test. p-values < 0.05 were considered significant. Data were analyzed using the Prism software (version 5.0).

Results and discussion
Synthesis, purification and purity assessment of two peptide LyeTx I derivatives modified with the chelating agent HYNIC Two peptide LyeTx I derivatives were synthesized with the chelating agent HYNIC attached either to its Nterminal residue (Fig. 1a) or to the lateral amino group of an extra lysine residue coupled to its C-terminal portion (Fig. 1b). The synthetic crude products were purified by RP-HPLC (Fig. 2a, b) and the collected fractions were assessed by MALDI-ToF-MS analysis. Pure products with m/z 2966 (Fig. 2c) and m/z 3094 (Fig. 2d) were detected.
The synthetic pure products were assessed by RP-HPLC (Fig. 3). Both chromatograms exhibited single and well-defined peak of the respective peptide LyeTx I derivative in high purities: 93.36 ± 0.43 % (HYNIC-LyeTx I) and 97.13 ± 0.23 % (LyeTx I-K-HYNIC). Both peaks were collected and analyzed by MALDI-ToF-MS. Data showed similar MS spectra as those previously presented (Fig. 2c, d).
These findings indicated that both synthetic peptide LyeTx I derivatives were suitably synthesized and purified. Thus, the synthetic products were available for further evaluation of the antimicrobial activity.

In vitro evaluation of the maintenance of the antimicrobial activity of the peptide LyeTx I derivatives modified with the chelating agent HYNIC
The maintenance of the antimicrobial activity after peptide LyeTx I chemical modifications was assessed by means of microdilution test, followed by incubation with S. aureus and E. coli in TSB. Table 2 summarizes in vitro microbiological assay data.
Previous data obtained for the non-modified peptide LyeTx I showed MIC's of 3.79 μmol . L −1 and 7.81 μmol . L −1 for S. aureus and E. coli, respectively [8]. However, different assay conditions and other bacterial strains were employed, which can explain the slight differences in the MIC obtained in this study. The peptide LyeTx I-K-HYNIC derivative (C-terminal modification) maintained its antimicrobial activity, exhibiting similar MIC for S. aureus and about two-fold higher MIC for E. coli, when compared to the non-modified peptide. On the other hand, the peptide HYNIC-LyeTx I derivative (Nterminal modification) did not inhibit bacterial growth (NI: no inhibition). Thus, the N-terminal modification suppressed the antimicrobial activity of peptide HYNIC-LyeTx I derivative, suggesting that the N-terminal portion is important for the peptide interaction with bacteria. Therefore, only the peptide LyeTx I-K-HYNIC derivative was selected for further radiolabeling with 99m Tc, in order to be tested as a specific imaging probe for infectious.

Radiolabeling and radiochemical purity of LyeTx I-K-HYNIC with 99m Tc
As the peptide HYNIC-LyeTx I derivative did not exhibit any antimicrobial activity, the radiolabeling process was performed only with the peptide LyeTx I-K-HYNIC derivative.   The radiolabeling procedure with 99m Tc can generate two main radiochemical impurities, 99m TcO 2 and 99m TcO 4 − . High amounts of these entities may impair imaging data interpretation, once they accumulate in liver/spleen and in thyroid/stomach, respectively [18]. Therefore, it is important to determine and to optimize the radiochemical purity, which means the percentage of 99m Tc atoms that effectively bind the radiopharmaceutical molecules.
Herein, the radiolabeling of the peptide LyeTx I-K-HYNIC derivative with 99m Tc atoms was standardized taking into account some parameters: amount of peptide derivative, reducing agent, pH and heating (Table 3). 99m TcO 2 molecules are retained at the point of application (R f = 0.0) in both solvents, MEK and acetonitrile:water (1:1; v:v), once they form a colloid. In contrast, 99m TcO 4 − migrates to the top of silica gel strip (R f = 0.9-1.0) in both solvents. LyeTx I-K-HYNIC-99m Tc is a hydrophilic compound and thus it remains at the point of application when MEK is used as eluent and it migrates to the top of the silica gel strip with acetonitrile:water (1:1; v:v). Then, the later eluent was used to determine the amount of 99m TcO 2 , whereas the former was used to quantify 99m TcO 4 − . The radiochemical purity analysis (Table 3) showed that the radiolabeling procedure either with 10 or 20 μg of the peptide LyeTx I-K-HYNIC derivative presented a radiolabeling yield greater than that obtained when 5 μg of the peptide derivative was used. Then, the amount of 10 μg of the peptide LyeTx I-K-HYNIC derivative was selected for the next steps. Concerning to the reducing agent, SnCl 2 . 2H 2 O, no significant differences in radiochemical purity was observed between the employed quantities. However, when 250 μg of SnCl 2 . 2H 2 O was used, it was verified the lowest values of both impurities 99m TcO 2 and 99m TcO 4 − . Thus, 250 μg of SnCl 2 . 2H 2 O was selected in order to perform the other assays. Moreover, results showed that the optimal pH is between 6 and 8. Then, for in vivo experiments the pH = 7 was chosen. Finally, the radiolabeling process needed water bath heating (100°C) for at least 15 min. As a result, the optimal radiolabeling procedure (10 μg of the peptide LyeTx I-K-HYNIC derivative; 250 μg of SnCl 2 . 2H 2 O; pH = 7; heating for 15 min at 100°C) yielded a radiochemical purity of 87 ± 1 % (n = 3) and the final preparation presented a specific activity of 37 MBq/mL.

LyeTx I-K-HYNIC-99m Tc evaluation
Besides radiochemical purity analysis, LyeTx I-K-HYNIC-99m Tc was evaluated by RP-HPLC in association with radioactivity measurement of the collected fractions (Fig. 4). First, the peptide LyeTx I-K-HYNIC derivative and the co-ligands (EDDA and tricine) were separately injected and detected at 214 nm (Fig. 4a). Afterwards, radiolabeled compound was injected and its fractions were collected. The radioactivity was measured in an automatic gamma counter (Fig. 4b) and the results revealed that the radioactivity was associated with the coligands, instead of the peptide LyeTx I-K-HYNIC derivative. These data indicate instability of the radiolabeled complex, suggesting 99m Tc transchelation from the peptide LyeTx I-K-HYNIC derivative to the co-ligands  Values are expressed as 'mean ± SD' (n = 3). Different letters indicate significant differences (p < 0.05). RP radiochemical purity employed in this reaction. Although other authors had reported the importance of the co-ligands as stabilizing agents in the radiolabeling process, our findings did not show beneficial effects in this specific case [11,19,20]. Actually, it is related that EDDA is a strong chelating agent and, then, it might favor the transchelation, which is defined as the metal 99m Tc exchange from a weaker chelating agent to a stronger one [12,21]. Therefore, further studies will be necessary to improve radiolabeling conditions in order to reach a better stability of LyeTx I-K-HYNIC-99m Tc.

Conclusions
In summary, two peptide LyeTx I derivatives modified with the chelating agent HYNIC were synthesized, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification). The synthetic crude products were properly purified by RP-HPLC, as shown by MALDI-ToF-MS and RP-HPLC analyses. In vitro assay revealed that the attachment of HYNIC in the C-terminal portion of peptide LyeTx I did not compromise its antimicrobial activity and that the Nterminal portion is important for its interaction with bacteria. However, radiolabeling procedure conditions must be better investigated in order to optimize the process concerning to the binding between 99m Tc and the chelating agent HYNIC. Thus, this complex could be evaluated as a specific imaging agent to distinguish septic and aseptic inflammation.