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Development of antimicrobial peptides: Methodological aspects, design, synthesis and evaluation of a new class of antimicrobial peptides
Stockholm University, Faculty of Science, Department of Neurochemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A new protective group for the indole nitrogen of the amino acid tryptophan, the Boc-sarcosinoyl-sarcosinolyl (Boc-Sar-Sar) is described. This protective group shows good stability to the reaction conditions used in solid phase peptide synthesis and can be incorporated into the peptide as Fmoc-Trp(Boc-Sar-Sar)-OH. When the peptide is cleaved from the resin with trifluororoactetic acid, the Boc group is simultaneously cleaved and the N-terminal nitrogen in the Sar-Sar group is exposed. At low pH this amino group is protonated and thereby the peptide will have higher solubility in aqueous solution. High solubility of the peptide is an important factor for the purification of peptides with reversed phase HPLC. During HPLC purification, the protonated Sar-Sar group will remain on the peptide. When the purified peptide is exposed to physiological pH, the Sar-Sar group will be cleaved and the peptide will be obtained in its fully deprotected form.

Gramicidin A (GA) is an extremely hydrophobic peptide with four tryptophan residues. To improve the solubility of GA, Fmoc-Trp(Boc-Sar-Sar)-OH was used in the solid phase synthesis of GA resulting in (H-Sar-Sar)4-GA. When this peptide was exposed to physiological pH native GA was formed.

A new class of antimicrobial peptides, represented by the enantiomers; cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] is described. Neither of the two peptides shows any significant antimicrobial activity against Bacillus megaterium. However, when both peptides are present during the experiment a potent antimicrobial activity with an IC50 value of 2 µM could be observed. The peptides also show low hemolytic activity with an HC50 value of 316 µM. The synergistic mode of action of these peptides is dependent on the presence of both enantiomers. Circumstantial evidences indicate that the antimicrobial effect is the result of formation of peptide nanotubes within the bacterial membrane.

It is also shown that the cyclic peptides described above can be modified at the lysine residue with three arginine residues in which the N-terminus is acylated with a short fatty acid. Togheter these peptides show a synergistic mode of action against Staphylococcus aureus resulting in a MIC value of 9 µM.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2012. , 75 p.
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-73366ISBN: 978-91-7447-459-6 (print)OAI: oai:DiVA.org:su-73366DiVA: diva2:504680
Public defence
2012-03-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Available from: 2012-03-08 Created: 2012-02-21 Last updated: 2015-03-06Bibliographically approved
List of papers
1. Protection of the indole nucleus of tryptophan in solid-phase peptide synthesis with a dipeptide that can be cleaved rapidly at physiological pH
Open this publication in new window or tab >>Protection of the indole nucleus of tryptophan in solid-phase peptide synthesis with a dipeptide that can be cleaved rapidly at physiological pH
2011 (English)In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 52, no 44, 5876-5879 p.Article in journal (Refereed) Published
Abstract [en]

The synthesis of a new derivative of tryptophan Fmoc-Trp(Boc-Sar-Sar)-OH is described. Fmoc-Trp(Boc-Sar-Sar)-OH is introduced into peptides by solid-phase peptide synthesis and during treatment with TFA at the end of the synthesis, the Boc group is cleaved and the peptide is obtained with the indole nucleus modified with the sarcosinyl-sarcosinyl (Sar-Sar) moiety. This modification will introduce a cationic charge that improves the solubility of the peptide during HPLC purification. The Sar-Sar moiety is cleaved upon exposure to physiological pH. The Boc-Sar-Sar group might, therefore, also find use in the design of pro-drugs of indole-containing compounds.

Keyword
Solid-phase peptide synthesis, Protecting group, Cyclization-activated prodrugs, Tryptophan, Fmoc amino acids
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-69881 (URN)10.1016/j.tetlet.2011.08.172 (DOI)000296168300039 ()
Note
authorCount :3Available from: 2012-01-18 Created: 2012-01-15 Last updated: 2017-12-08Bibliographically approved
2. Solid phase peptide synthesis of a Gramicidin A analogue with high aqueous solubility that can generate native GA upon exposure to physiological pH
Open this publication in new window or tab >>Solid phase peptide synthesis of a Gramicidin A analogue with high aqueous solubility that can generate native GA upon exposure to physiological pH
(English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3904Article in journal (Refereed) Submitted
Abstract [en]

Gramicidin A (GA) was synthesized by anchoring Fmoc-ethanolamine to a 2-chlorotrityl resin and the peptide chain was assembled using COMU activated Fmoc amino acid followed by introduction of formyl-valine at the N-terminus. In contrast to other synthesis of GA reported in the literature, all four tryptophan residues were coupled as the recently described derivative Fmoc-Trp(Boc-Sar-Sar)-OH.  The peptide was cleaved from the resin with TFA containing 1 % anisole simultaneously with the Boc groups resulting in the GA derivate; (H-Sar-Sar-)4-GA, in which all four indole nitrogens are modified by the protonated H-Sar-Sar- dipeptidyl moiety. Compared to native GA this derivative had an at least 10 000 fold higher solubility in water. Upon exposure to physiological pH, the H-Sar-Sar- groups were cleaved by an intramolecular cyclization reaction producing native GA although experimental limitations related to the solubilites of the peptide prevented us from demonstrate complete conversion of (H-Sar-Sar-)4-GA  to native GA.

National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-72969 (URN)
Available from: 2012-02-21 Created: 2012-02-17 Last updated: 2015-03-16
3. Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane
Open this publication in new window or tab >>Pharmacodynamic synergy strictly dependent on the co-operative aggregation of enantiomers of cyclic peptides in the bacterial cell membrane
2011 (English)In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 21, no 18, 5262-5265 p.Article in journal (Refereed) Published
Abstract [en]

The antimicrobial activity of the peptide enantiomers cyclo[d-Tle-d-Lys-d-Tle-l-Ala-d-Tle-l-Ala-d-Tle-l-Ala] and cyclo[l-Tle-l-Lys-l-Tle-d-Ala-l-Tle-d-Ala-l-Tle-d-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2μM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316μM.

Keyword
Antimicrobial peptides, Synergy, Enantiomers, Cyclic peptides
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-62563 (URN)10.1016/j.bmcl.2011.07.032 (DOI)000294051800028 ()21820306 (PubMedID)
Available from: 2011-09-23 Created: 2011-09-23 Last updated: 2017-12-08Bibliographically approved
4. Antimicrobial activity of enantiomeric pairs of cationic cyclic lipopeptides against Staphylococcus aureus
Open this publication in new window or tab >>Antimicrobial activity of enantiomeric pairs of cationic cyclic lipopeptides against Staphylococcus aureus
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have previously shown that the peptides cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] have a potent antimicrobial activity against Staphylococcus aureus  when combined but almost no effect as a single peptide. In this study the antimicrobial activity of the peptides cyclo L-Lys(R)-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle and cyclo D-Lys(R)-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle where R is –L-Arg-L-Arg-L-Arg-CO-(CH2)6-CH3  was investigated  against Staphylococcus aureus  in a broth microdilution assay.  At a concentration of 10 mM neither peptide showed any antimicrobial activity. However, when both peptides were present in the experiment a potent inhibition with a MIC value of 8.9 mM was observed. These results show that this class of peptides can be modified by branching of the side-chain of an amino acid residue in the cyclic peptide while retaining or possibly enhancing the antimicrobial activity. This observation will facilitate the optimization of antimicrobial activity and specificity of peptides with similar design.   

National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-72967 (URN)
Available from: 2012-02-21 Created: 2012-02-17 Last updated: 2015-03-16Bibliographically approved

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