Open Access

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

  • Leonardo Lima Fuscaldi1,
  • Daniel Moreira dos Santos2,
  • Natália Gabriela Silva Pinheiro3,
  • Raquel Silva Araújo1,
  • André Luís Branco de Barros1,
  • Jarbas Magalhães Resende3,
  • Simone Odília Antunes Fernandes1,
  • Maria Elena de Lima2 and
  • Valbert Nascimento Cardoso1Email author
Journal of Venomous Animals and Toxins including Tropical Diseases201622:16

https://doi.org/10.1186/s40409-016-0070-y

Received: 15 January 2016

Accepted: 12 April 2016

Published: 22 April 2016

Abstract

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.

Keywords

Septic and aseptic inflammationDifferential diagnosisAntimicrobial peptidesLyeTx I derivativesMALDI-ToF-MSRP-HPLCTechnetium-99mHYNICEDDATricine

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 [24]. 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 [57].

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-NH2). 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 (99mTc) 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. 99mTc 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 (99Mo/99mTc) generator [9, 10]. However, in order to use 99mTc 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 99mTc binding to HYNIC, tricine and ethylenediamine-N,N’-diacetic acid (EDDA) are used as co-ligands in the radiolabeling procedure [1113].

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 99mTc atoms.

Methods

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,N-dimethylformamide (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). 99mTc was obtained from a 99Mo/99mTc 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].

Both synthesis were performed by stepwise solid-phase using the N-9-fluorenylmethyloxycarbonyl (Fmoc) strategy on a rink amide resin (0.63 mmol . g−1). Side chain protecting groups were as follows: t-butyl for threonine, t-butyloxycarbonyl for lysine and tryptophan, (triphenyl) methyl for histidine, asparagine and glutamine. Couplings were performed with 1,3-diisopropylcarbodiimide/1-hydroxybenzotriazole in DMF for 60–180 min. Deprotections (15 min, twice) were conducted by piperidine: DMF (1:4; v:v). Cleavage from the resin and final deprotection were performed with TFA/water/triisopropylsilane (95.0/2.5/2.5, v:v) at room temperature during 90 min. Post-precipitation of the products with cold diisopropyl ether, the crude peptide complexes were extracted with water:acetonitrile (1:1; v:v), followed by freeze-drying.

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.
Table 1

Solvent conditions for RP-HPLC

(A) Crude synthetic product purification

(B) Purified synthetic product analysis

(C) LyeTx I-K-HYNIC-99mTc evaluation

Time

Gradient of eluent B

Time

Gradient of eluent B

Time

Gradient of eluent B

(min)

(%)

(min)

(%)

(min)

(%)

0–8.2

0

0–3.7

0

0–5.0

0

8.2–12.4

0–30

3.7–33.5

0–100

5.0–30.0

0–55

12.4–50.0

30–55

33.5–39

100

30.0–35.0

55–100

50.0–54.0

55–100

  

35.0–45.0

100

54.0–62.5

100

    

Flow = 1.0 mL.min−1. Detection = 214 nm

The collected fractions were assessed by matrix-assisted 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 C18TM 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 (108 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.

Radiolabeling and radiochemical purity of LyeTx I-K-HYNIC with 99mTc

The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc and radiochemical purity analysis were performed as previously reported elsewhere [16], with some modifications. Briefly, in a sealed vial, tricine (20 mg) and EDDA (5 mg) were solubilized in 0.9 % NaCl (w:v) solution (200 μL). Next, LyeTx I-K-HYNIC (5, 10 or 20 μg) and 1 mg.mL−1 SnCl2 . 2H2O solution (100, 200, 250 or 300 μL) in 0.25 mol.L−1 HCl were added. Then, the pH was adjusted (5, 6, 7, 8 or 9). Finally, Na99mTcO4 (37 MBq; q.s. ad = 1000 μL) was added to the vial and the final solution was heated (100 °C) in water bath (5, 15 or 30 min) or not heated. Radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc was performed by thin-layer chromatography on silica gel strips (Merck®). Methyl ethyl ketone (MEK) and acetonitrile:water (1:1; v:v) were used to determine the amount of free technetium (99mTcO4 ) and hydrolyzed technetium (99mTcO2), respectively. Radioactivity was measured using an automatic gamma counter (Wizard, Finland).

LyeTx I-K-HYNIC-99mTc evaluation

LyeTx I-K-HYNIC-99mTc was evaluated as previously described [17], by RP-HPLC on a C8 column (ACE 5 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 1C. LyeTx I-K-HYNIC, EDDA and tricine were separately injected and the detection was at 214 nm. LyeTx I-K-HYNIC-99mTc was injected, the fractions were collected and the radioactivity was measured using an automatic gamma counter (Wizard, Finland).

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 N-terminal 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.
Fig. 1

Structures of the peptide LyeTx I derivatives modified with the chelating agent HYNIC. a HYNIC-LyeTx I derivative (Mw = 2966 g . mol−1) and b LyeTx I-K-HYNIC derivative (Mw = 3094 g . mol−1). –NH2 represents the C-terminal carboxyamidation. H– represents the absence of N–terminal modification. Mw: molecular weight

Fig. 2

Purification of synthetic crude peptide LyeTx I derivatives modified with the chelating agent HYNIC, by RP-HPLC (Ettan LC, GE HealthCare, USA). Chromatograms of synthetic crude a HYNIC-LyeTx I and b LyeTx I-K-HYNIC: Discovery® BIO Wide Pore C8 column (5 μm, 250.0 mm × 4.6 mm) 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); the flow was 1.0 mL . min−1 and the detection was at 214 nm. Mass spectrometer (MS) spectra of the collected fraction (CF) of synthetic purified c HYNIC-LyeTx I and d LyeTx I-K-HYNIC: the molecular weights were 2966 Da and 3094 Da, respectively, obtained by deconvolution of the MS spectra

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).
Fig. 3

Purity assessment of synthetic purified peptide LyeTx I derivatives modified with the chelating agent HYNIC, by RP-HPLC (Ettan LC, GE HealthCare, USA). Chromatograms of synthetic purified HYNIC-LyeTx I (gray line) and LyeTx I-K-HYNIC (black line): PepMap C18TM column (5 μm, 150.0 mm × 4.6 mm) 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); the flow was 1.0 mL . min−1 and the detection was at 214 nm. PP: peak of peptide

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.
Table 2

Minimum inhibitory concentration (MIC) of LyeTx I (control), LyeTx I-K-HYNIC and HYNIC-LyeTx I against S. aureus and E. coli in TSB

Peptide

S. aureus (ATCC® 6538)

E. coli (ATCC® 10536)

LyeTx I (non-modified peptide)

5.52 μmol.L−1

5.52 μmol.L−1

LyeTx I-K-HYNIC (C-terminal modified derivative)

5.05 μmol.L−1

10.10 μmol.L−1

HYNIC-LyeTx I (N-terminal modified derivative)

NI

NI

Values are expressed as median (n = 3). NI no inhibition

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 (N-terminal 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 99mTc, in order to be tested as a specific imaging probe for infectious.

Radiolabeling and radiochemical purity of LyeTx I-K-HYNIC with 99mTc

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 99mTc can generate two main radiochemical impurities, 99mTcO2 and 99mTcO4 . 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 99mTc atoms that effectively bind the radiopharmaceutical molecules.

Herein, the radiolabeling of the peptide LyeTx I-K-HYNIC derivative with 99mTc atoms was standardized taking into account some parameters: amount of peptide derivative, reducing agent, pH and heating (Table 3). 99mTcO2 molecules are retained at the point of application (Rf = 0.0) in both solvents, MEK and acetonitrile:water (1:1; v:v), once they form a colloid. In contrast, 99mTcO4 migrates to the top of silica gel strip (Rf = 0.9–1.0) in both solvents. LyeTx I–K-HYNIC-99mTc 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 99mTcO2, whereas the former was used to quantify 99mTcO4 .
Table 3

Radiolabeling standardization of the synthetic peptide LyeTx I-K-HYNIC derivative with 99mTc

Amount of LyeTx I-K-HYNIC (SnCl2 .2H2O = 200 μg; pH = 7; Δ = 15 min)

5 μg

10 μg

20 μg

 RP (%)

73 ± 3a

83 ± 1b

82 ± 1b

Amount of SnCl2 .2H2O (LyeTx I-K-HYNIC = 10 μg; pH = 7; Δ = 15 min)

100 μg

200 μg

250 μg

300 μg

 RP (%)

82 ± 2

83 ± 1

87 ± 1

82 ± 3

 99mTcO2 (%)

9 ± 1a

9 ± 1a

8 ± 0a

14 ± 2b

 99mTcO4 (%)

8 ± 1a

8 ± 0a

5 ± 1b

4 ± 1b

pH (LyeTx I-K-HYNIC = 10 μg; SnCl2 .2H2O = 250 μg; Δ = 15 min)

5

6

7

8

9

 RP (%)

77 ± 2a

88 ± 2b

87 ± 1b

83 ± 0b

72 ± 3c

Heating (100 °C) (LyeTx I-K-HYNIC = 10 μg; SnCl2 .2H2O = 250 μg; pH = 7)

5 min

15 min

30 min

Unheated

 RP (%)

71 ± 2a

87 ± 1b

81 ± 1b

57 ± 4c

Values are expressed as ‘mean ± SD’ (n = 3). Different letters indicate significant differences (p < 0.05). RP radiochemical purity

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, SnCl2 .2H2O, no significant differences in radiochemical purity was observed between the employed quantities. However, when 250 μg of SnCl2 .2H2O was used, it was verified the lowest values of both impurities 99mTcO2 and 99mTcO4 . Thus, 250 μg of SnCl2 .2H2O 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 SnCl2 . 2H2O; 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-99mTc evaluation

Besides radiochemical purity analysis, LyeTx I-K-HYNIC-99mTc 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 co-ligands, instead of the peptide LyeTx I-K-HYNIC derivative. These data indicate instability of the radiolabeled complex, suggesting 99mTc transchelation from the peptide LyeTx I-K-HYNIC derivative to the co-ligands 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 99mTc 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-99mTc.
Fig. 4

LyeTx I-K-HYNIC-99mTc evaluation by RP-HPLC (717 Plus Autosampler, Waters, USA) associated with radioactivity determination of the collected fractions by automatic gamma counter (Wizard, Finland). a Chromatograms of EDDA (red line), tricine (green line) and LyeTx I-K-HYNIC (black line): ACE 5 C8 column (5 μm, 250.0 mm × 4.6 mm) 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); the flow was 1.0 mL . min−1 and the detection was at 214 nm. b Radiochromatogram of LyeTx I-K-HYNIC-99mTc: ACE 5 C8 column (5 μm, 250.0 mm × 4.6 mm) 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); the flow was 1.0 mL . min−1. cpm: counts per minute

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 N-terminal 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 99mTc and the chelating agent HYNIC. Thus, this complex could be evaluated as a specific imaging agent to distinguish septic and aseptic inflammation.

Abbreviations

LyeTx I: 

cationic peptide isolated from Lycosa erythrognatha venom

C-terminal: 

carboxyl-terminal

E. coli

Escherichia coli

S. aureus

Staphylococcus aureus

99mTc: 

technetium-99m

99Mo/99mTc generator: 

molibdenium-99/technetium-99m generator

HYNIC: 

2-hydrazinonicotinamide

N-terminal: 

nitrogen-terminal

EDDA: 

ethylene diamine-N,N’-diacetic acid

TFA: 

trifluoroacetic acid

DMF: 

N,N-dimethylformamide

RP-HPLC: 

reverse phase-high performance liquid chromatography

ATCC: 

American type culture collection

HYNIC-LyeTx I: 

peptide LyeTx I derivative with the chelating agent HYNIC attached to its N-terminal residue

LyeTx I-K-HYNIC: 

peptide LyeTx I derivative with the chelating agent HYNIC attached to the lateral amino group of an extra lysine residue coupled to the C-terminal portion

Fmoc: 

N-9-fluorenylmethyloxycarbonyl

v:v: 

volume per volume

MALDI-ToF-MS: 

matrix-assisted laser desorption ionization time of flight mass spectrometer

MS: 

mass spectrometer

TSB: 

tryptic soy broth

MIC: 

minimum inhibitory concentration

NaCl: 

sodium chloride

w:v: 

weight per volume

SnCl2 . 2H2O: 

stannous chloride dehydrate

HCl: 

hydrochloric acid

Na99mTcO4

sodium pertechnetate

q.s. ad: 

quantity sufficient added

MEK: 

methyl ethyl ketone

99mTcO4

free technetium

99mTcO2

hydrolyzed technetium

SD: 

standard deviation

ANOVA: 

analysis of variance

Mw

molecular weight

CF: 

collected fraction

PP: 

peak of peptide

NI: 

no inhibition

RP: 

radiochemical purity

cpm: 

counts per minute

Declarations

Acknowledgements

The authors would like to thank the undergraduate students Eduardo and Lívia for their support in some steps that were not covered in this manuscript, but were important as pilot studies. Thanks are also due to the Nuclear Medicine sector of Clinical Hospital of Federal University of Minas Gerais for the supply of 99mTc.

Funding

The authors would like to thank the Toxinology Network sponsored by the Coordination for the Improvement of Higher Education Personnel (CAPES) for the PhD fellowship, as well as National Council for Scientific and Technological Development (CNPq), the State of Minas Gerais Research Foundation (FAPEMIG) and the Office of the Dean for Research of the Federal University of Minas Gerais (PRPq/UFMG) for their grants. Thanks are also due to the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication of this paper (CAPES, grant no. 23038.006285/2011-21, AUXPE – Toxinologia – 1219/2011).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais
(2)
Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais
(3)
Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais

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Copyright

© Fuscaldi et al. 2016