Cyclic Decapeptide Research Articles

Gramicidin S Derivatives Containing cis‐ and trans‐Morpholine Amino Acids (MAAs) as Turn Mimetics

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D-Phe-Pro two-residue turn motifs in the rigid cyclic beta-hairpin structure of GS was replaced with morpholine amino acids (MAA 2-5), differing in stereochemistry and length of the side-chain. The conformational properties of the thus obtained GS analogues (6-9) was assessed by using NMR spectroscopy and X-ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8, containing the dipeptide isostere trans-MAA 4, has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and -negative bacterial strains is better than the derivatives 6, 7 and 9.

Higher-Rank Correlation NMR Spectra with Spectral Moment Filtering

Higher-rank correlation spectroscopy is introduced as an alternative to 3D Fourier-transform (FT) NMR spectroscopy for resonance assignment and molecular structure determination. The method combines standard 2D FT spectra that share a common frequency dimension, such as a 2D (13)C-(1)H HSQC and a 2D (1)H-(1)H TOCSY spectrum, and constructs higher-rank correlation spectra with ultra-high spectral resolution. Spectral overlap along a common dimension, in particular the (1)H dimension, is addressed by a spectral filtering method, which identifies mismatches between the 1(st) and 2(nd) moments of cross-peak profiles. The method, which provides a substantial speed-up over traditional 3D FT spectroscopy while effectively suppressing false peaks, is demonstrated for the triple-rank (13)C-(1)H HSQC-TOCSY spectrum of a cyclic decapeptide with different mixing times. Higher-rank correlation spectroscopy is usefully applicable to the analysis of a wide range of NMR spectra of synthetic and natural products.

Synthesis of a highly hydrophobic cyclic decapeptide by solid-phase synthesis of linear peptide and cyclization in solution

A general method was described to synthesize a highly hydrophobic cyclic peptide, cyclo[ LW LW LW LW LQ] where underlines indicate d-configuration of the amino acid, by a two-step solid-phase/solution synthesis strategy. The linear decapeptide was assembled by standard Boc chemistry on solid-phase and subsequently cyclized in solution with high efficiency and reproducibility. In subsequent purification by semi-preparative HPLC, 50% (v/v) DMF/H 2O was employed as the solvent to overcome the difficulty of solubilization for the hydrophobic cyclic decapeptide and achieved a total yield of 30–35% with a purity of over 98%.

Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI‐MSMS and ESI‐nozzle‐skimmer‐MSMS

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions.

Bisphosphine‐Functionalized Cyclic Decapeptides Based on the Natural Product Gramicidin S: A Potential Scaffold for Transition‐Metal Coordination

The natural product Gramicidin S is a promising scaffold for novel oligopeptide-based bisphosphine ligands, combining the advantageous rigid chiral backbone with the close proximity of phosphine substituents. The required unnatural, phosphine-containing, amino acid building blocks were synthesized by means of a novel protocol that involves the enantioselective alkylation of a chiral nickel Schiff base template. Three Ni complexes were prepared with different alkyl chains between the phosphine group and the alpha-carbon atom of the incorporated glycine; the absolute stereochemistry of two of them was determined by single-crystal X-ray structure analysis. By detaching the template, enantiopure L-phosphine amino acids resulted enabling the solid-phase, stepwise construction of a linear sequence of the phosphine-modified oligopeptides. On cyclization three bisphosphine-substituted Gramicidin S analogues resulted, differing only in the size and shape of the linkage between the phosphine groups and the oligopeptides backbone. Their crystal structures suggest these species to have potential as chelating ligands.

Anti-listerial activity and structure–activity relationships of the six major tyrocidines, cyclic decapeptides from Bacillus aneurinolyticus

Six major tyrocidines, purified from the antibiotic tyrothricin complex produced by Bacillus aneurinolyticus, showed significant lytic and growth inhibitory activity towards the Gram+ bacteria, Micrococcus luteus and Listeria monocytogenes, but not against the Gram− bacterium, Escherichia coli. The isolated natural tyrocidines were in particular more active against the leucocin A (antimicrobial peptide) resistant strain, L. monocytogenes B73 -MR1 , than the sensitive L. monocytogenes B73 strain . Remarkably similar structure–activity trends toward the three Gram+ bacteria were found between growth inhibition and different physicochemical parameters (solution amphipathicity, theoretical lipophilicity, side-chain surface area and mass-over-charge ratio).

Factors affecting conformations of cyclic polypeptides in the crystalline state

Criteria for geometrical parameters, such as bond lengths, bond angles and torsional angles, are needed for building models of polypeptides based on spectral data and/or minimum energy calculations. Some assumptions that are made frequently are: that the most stable form of the peptide unit is planar and in the trans conformation; that the maximum number of hydrogen bonds will be formed: that peptides dissolved in nonpolar solvents will turn their polar groups toward the interior of the molecule (and form intramolecular hydrogen bonds), whereas those dissolved in polar solvents will turn their polar groups toward the exterior. The validity of such assumptions has been examined by crystal structure analyses of a variety of cyclic peptides crystallized under varying conditions. The cyclic backbone in telrapeptides, both natural and synthetic, has been found to assume four different conformations, cis trans cis trans with a center of inversion or with a twofold rotation axis, and trans trans trans trans with a center of inversion or with a twofold rotation axis. A pair of similar cyclic pentapeptides have been found to be rotational isomers, where the backbone conformation (all trans) remains the same, but the side chains progress by one peptide unit around the ring. A hexapeptide analogue crystallized from (CH3)2SO/(CH3)2CO and from H2O/(CH3)2CO crystallizes in different space groups. Four molecules of the hexapeptide aggregate by intermolecular hydrogen bonding to form an almost identical large cavity in each case; however, in one crystal the cavity is filled with water molecules, which make extensive hydrogen bonds to the polar groups of the surrounding peptide molecules, whereas in the other case, the cavity contains (CH3)2SO that participates in only one hydrogen bond to the peptide and otherwise presents a lipophilic surface toward the polar lining of the cavity. A similar phenomenon has been observed in the large-diameter channels formed by antamanide molecules (cyclic decapeptide) where the channel is occupied either by many water molecules in definite sites and hydrogen bound to the peptide molecules, or by nonpolar disordered n-hexane molecules. In the latter two examples the character of the solvent does not influence the conformation of the peptide.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a β-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 μM in the intact cells.

The Antimicrobial Peptide Gramicidin S Permeabilizes Phospholipid Bilayer Membranes Without Forming Discrete Ion Channels

We examined the permeabilization of lipid bilayers by the β-sheet, cyclic antimicrobial decapeptide gramicidin S (GS) in phospholipid bilayers formed either by mixtures of zwitterionic diphytanoylphosphatidylcholine and anionic diphytanoylphosphatidylglycerol or by single zwitterionic unsaturated phosphatidylcholines having various hydrocarbon chain lengths, with and without cholesterol. In the zwitterionic bilayers formed by the phosphatidylcholines, without or with cholesterol, the peptide concentrations and membrane potentials required to initiate membrane permeabilization vary little as function of bilayer thickness and cholesterol content. In all the systems tested, the GS-induced transient ion conductance events exhibit a broad range of conductances, which are little affected by the bilayer composition or thickness. In the zwitterionic phosphatidylcholine bilayers, the effect of GS does not depend on the polarity of the transmembrane potential; however, in bilayers formed from mixtures of phosphatidylcholines and anionic phospholipids, the polarity of the transmembrane potential becomes important, with the GS-induced conductance events being much more frequent when the GS-containing solution is positive relative to the GS-free solution. Overall, these results suggest that GS does not form discrete, well-defined, channel-like structures in phospholipid bilayers, but rather induces a wide variety of transient, differently sized defects which serve to compromise the bilayer barrier properties for small electrolytes.

Comparison of the Structure and Dynamics of the Antibiotic Peptide Polymyxin B and the Inactive Nonapeptide in Aqueous Trifluoroethanol by NMR Spectroscopy

The structure and dynamics of polymyxin B (PxB), an N-acylated cyclic decapeptide that displays antimicrobial activity against Gram-negative bacteria, is characterized by NMR and compared to results for the inactive nonapeptide, which is missing the N-terminal amino acid along with the attached acyl chain. Aqueous trifluoroethanol (TFE) was chosen as the solvent since the overall structure of PxB in TFE is similar to the structure when bound to vesicles. No differences were observed between the two peptides for (1)H H(alpha) chemical shifts or patterns of cross peaks in NOESY spectra, indicating that the overall structures are quite similar. The sign and intensity of NOESY spectra obtained at different temperatures were used to assess the relative mobility of the peptides. For both peptides, differential mobility is observed in different parts of the molecule, with greater mobility observed for the linear portion than the ring and faster motion seen for the side chains than the peptide backbone. However, all motion is faster in the nonapeptide, indicating that the presence of the N-terminal acyl chain restricts the mobility of PxB compared to the nonapeptide, which lacks this structural feature. For both peptides, differential mobility is also observed within the cyclic portion of the peptide. This supports a proposed model whereby the more rigid residues serve as pivot points, allowing the ring conformation to change in response to different binding partners. However, conformational flexibility within the cyclic ring is not sufficient for antimicrobial activity since both the active and inactive peptides exhibit the same flexibility. The N-terminal acyl chain on PxB, which is essential for activity, exhibits rapid, independent motion, and this flexibility may facilitate penetration of the outer membrane.

Utilizing the Charge Field Effect on Amide 15N Chemical Shifts for Protein Structure Validation

Of all the nuclei in proteins, the nuclear magnetic resonance (NMR) chemical shifts of nitrogen are the theoretically least well understood. In this study, quantum chemical methods are used in combination with polarizable-continuum models in order to show that consideration of the effective electric field, including charge screening due to solvation, improves considerably the consistencies of statistical relationships between experimental and computed amide (15)N shifts between various sets of charged and uncharged oligopeptides and small organic molecules. A single conversion scheme between shielding parameters from first principles using density functional theory (DFT) and experimental shifts is derived that holds for all classes of compounds examined here. This relationship is then used to test the accuracy of such (15)N chemical shift predictions in the cyclic decapeptide antibiotic gramicidin S (GS). GS has previously been studied in great detail, both by NMR and X-ray crystallography. It adopts a well-defined backbone conformation, and hence, only a few discrete side chain conformational states need to be considered. Moreover, a charge-relay effect of the two cationic ornithine side chains to the protein backbone has been described earlier by NMR spectroscopy. Here, DFT-derived backbone amide nitrogen chemical shifts were calculated for multiple conformations of GS. Overall, the structural dynamics of GS is revisited in view of chemical shift behavior along with energetic considerations. Together, the study demonstrates proof of concept that (15)N chemical shift information is particularly useful in the analysis and validation of protein conformational states in a charged environment.

A Novel Conformationally Constrained Parallel G Quadruplex

Guanine-rich DNA sequences are known to form highly ordered structures called G quadruplexes. These structures play an important role in many relevant biological processes, such as telomere stabilization, oncogene activation, and the regulation of the immunoglobulin switch region. The G-quadruplex motif is based on the association of planar G quartets of four guanine residues that are held together by eight Hoogsteen-type hydrogen bonds (Figure 1A). The G-quadruplex motif requires monovalent cations, such as Na and K , for stabilization. A wide variety of topologies can be adopted depending on the number of strands involved in the structure, the strand direction, as well as variations in loop size and sequence (Figure 1). The structure of parallel-stranded as well as antiparallel-stranded quadruplexes have been extensively studied by using different methods, such as NMR spectroscopy, X-ray diffraction and circular dichroism, but the exact conformation present in vivo is still under discussion. The design of small molecules that can bind to G quadruplexes has thus received attention because these nucleic acid motifs represent valuable pharmaceutical targets. For this purpose, a large number of small molecules has been evaluated for their binding with these particular DNA structures. However, as mentioned, the G quadruplex can adopt different topologies that can confuse the study of recognition phenomena. The design of a system that is able to mimic a well-defined conformation of G quadruplex is thus of great interest to precisely study the molecular interactions that can occur with small organic molecules. In 1985, Mutter proposed the TASP concept (template-assembled synthetic proteins) for the design of folded proteins. These pioneering works described the use of a cyclodecapeptide that allows the preparation of artificial proteins with a predetermined three-dimensional structure. Despite a large number of examples that use this template, to our knowledge, it has not been applied for the design of a specific folded structure of nucleic acid. With this in mind, we investigated the use of a peptidic scaffold as a topological template that directs the intramolecular assembly of covalently attached oligonucleotides into a single characteristic folding topology of G quadruplex. We anticipated that the scaffold should permit the preorganization of the DNA strands and the stabilization of the quadruplex structure. We report herein the synthesis and characterization of the novel water-soluble peptidic scaffold–oligonucleotide conjugate 1 that mimics the parallel-stranded conformation of G quadruplex (Scheme 1). We demonstrate that the use of the scaffold allows the precise control of the conformation of the quadruplex and dramatically increases the stability of the motif—all the more so as the formation of the quadruplex motif is possible even without the addition of any monovalent cations, such as K . We also show that mimic 1 can be used for surface functionalization, and this permits the study of the molecular interaction with G-quadruplex ligands by using surface plasmon resonance (SPR). The scaffold used for the synthesis of mimic 1 is a cyclic decapeptide with two independently functionalizable faces, which are due to the orientation of the lysine side-chains. On one side, the four oligonucleotides derived from the human telomeric sequence d(TTAGGGT) were anchored by using oxime bond formation, and a biotin residue was incorporated on the other side for attachment to streptavidin-immobilized surfaces. Earlier work from our laboratory has demonstrated that the oxime coupling strategy allows the efficient preparation of peptide–oligonucleotide conjugates. Figure 1. G-quartet motif and possible folded structures of the G quadruplex. A) G quartet; B) intermolecular parallel form; C) intramolecular parallel form; D) intramolecular antiparallel form.

The antimicrobial peptide gramicidin S permeabilizes phospholipid bilayer membranes without forming discrete ion channels

We examined the permeabilization of lipid bilayers by the β-sheet, cyclic antimicrobial decapeptide gramicidin S (GS) in phospholipid bilayers formed either by mixtures of zwitterionic diphytanoylphosphatidylcholine and anionic diphytanoylphosphatidylglycerol or by single zwitterionic unsaturated phosphatidylcholines having various hydrocarbon chain lengths, with and without cholesterol. In the zwitterionic bilayers formed by the phosphatidylcholines, without or with cholesterol, the peptide concentrations and membrane potentials required to initiate membrane permeabilization vary little as function of bilayer thickness and cholesterol content. In all the systems tested, the GS-induced transient ion conductance events exhibit a broad range of conductances, which are little affected by the bilayer composition or thickness. In the zwitterionic phosphatidylcholine bilayers, the effect of GS does not depend on the polarity of the transmembrane potential; however, in bilayers formed from mixtures of phosphatidylcholines and anionic phospholipids, the polarity of the transmembrane potential becomes important, with the GS-induced conductance events being much more frequent when the GS-containing solution is positive relative to the GS-free solution. Overall, these results suggest that GS does not form discrete, well-defined, channel-like structures in phospholipid bilayers, but rather induces a wide variety of transient, differently sized defects which serve to compromise the bilayer barrier properties for small electrolytes.

Gramicidin S identified as a potent inhibitor for cytochrome bd-type quinol oxidase

Gramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd-type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC 50 of 3.5 μM by decreasing V max and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics.

Synthesis of a .BETA.-Tetrapeptide Analog as a Mother Compound for the Development of Matrix Metalloproteinase-2-Imaging Agents

Matrix metalloproteinase-2 (MMP-2) is an attractive target for the diagnosis of cancer and atherosclerosis in nuclear imaging. A cyclic decapeptide, cCTTHWGFTLC (cCTT), has been used as the mother compound for the development of MMP-2-imaging agents with high potency and selectivity. Most of radiolabeled derivatives of cCTT currently developed for in vivo studies of MMP-2, however, suffer from low accumulation in the target tissues, such as tumors. For enhanced in vivo stability and tissue penetration, we designed a linear beta-tetrapeptide analog, H-beta 3-Phe-beta-Ala-beta 3-Trp-beta 3-His-OH (1), to mimic cCTT. The component beta-amino acids were prepared by reduction of N-protected alpha-amino acid methyl esters to the alcohols, followed by conversion into the cyanides, and subsequent hydrolysis. Compound 1 was obtained from these beta-amino acids by the conventional solution method. In MMP-2 inhibition assay, compound 1 displayed desirably significant inhibition, which was comparable to cCTT. These findings suggest that compound 1 may serve as a mother compound in the design and development of in vivo MMP-2-imaging agents.

A cyclodecapeptide ligand to vitamin B12

Libraries of cyclic decapeptides were screened with vitamin B(12) derivatives to give cyclic peptide ligands incorporating histidine and cysteine as coordinating residues and negatively charged amino acids. Two hits, cyclo-(HisAspGluProGlyIleAlaThrProdGln) and cyclo-(ValAspGluProGlyGluAspCysProdGln) were resynthesized in good yields for solution experiments. The peptides bind aquocobalamin with coordination of His or Cys to the cobalt with high affinities (K(a) approximately 10(5) M(-1)). Additional interactions between the peptide side chains and the vitamin B(12) corrin moiety were determined by studying the (1)H NMR solution structure. The cyclopeptide-cobalamin complex with the histidine residue showed enhanced stability towards cyanide exchange, demonstrating the shielding effect of the ligand on the metal center.

Solid‐Phase Total Synthesis and Antimicrobial Activities of Loloatins A–D

The first total synthesis of the decapeptide antibiotics loloatins A-D (1-4), originally isolated from the marine bacterial isolate MK-PNG-276A, possibly in the genus Bacillus, was accomplished by solid-phase peptide synthesis (SPPS), followed by 'head-to-tail' cyclization of the activated linear precursors, without protection of nucleophilic side-chain functions, on a safety-catch resin. The synthetic peptides were equally active as the natural products isolated from the bacterial source and found to possess similar bacterial selectivity as other members in the family of amphipathic antimicrobial cyclic decapeptides.

Development of Tyrocidine A analogues with improved antibacterial activity

The development of new antibacterial therapeutic agents capable of halting microbial resistance is a chief pursuit in clinical medicine. Classes of antibiotics that target and destroy bacterial membranes are attractive due to the decreased likelihood that bacteria will be able to generate resistance to this mechanism. The amphipathic cyclic decapeptide, Tyrocidine A, is a model for this class of antibiotics. Tyrocidine A is composed of a hydrophobic and a hydrophilic face, allowing for insertion into bacterial membranes, creating porous channels and destroying membrane integrity. We have used a combination of molecular modeling and solid phase synthesis to prepare Tyrocidine A and analogues 1– 8. The minimum inhibitory concentrations (MICs) of these compounds were determined for a host of gram positive species and E. coli as a representative gram negative bacterium. Analogues 2 and 5 demonstrated moderate 2- to 8-fold increases in antibacterial activity over the parent Tyrocidine A for a variety of pathogenic microbes (best MICs for E. coli 32 μg/mL and 2 μg/mL for most gram positives). Examination of the structure– activity relationship between the analogues demonstrated a preference for increased amphipathicity but did not show a clear preference for increasing hydrophilicity versus hydrophobicity in improving antibacterial activity. Of note, movement of positively charged lysine residues or neutral pentafluorophenyl residues to different positions within the cyclopeptide ring system demonstrated improvements in antibacterial activity.

Chemical design of a radiolabeled gelatinase inhibitor peptide for the imaging of gelatinase activity in tumors

Since elevated levels of gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are associated with a poor prognosis in cancer patients, these enzymes are potential targets for tumor imaging. In the present study, a cyclic decapeptide, cCTTHWGFTLC (CTT), was selected as a mother compound because of its selective inhibitory activity toward gelatinases. For imaging gelatinase activity in tumors, we designed a CTT-based radiopharmaceutical taking into consideration that (1) the HWGF motif of the peptide is important for the activity, (2) hydrophilic radiolabeled peptides show low-level accumulation in the liver and (3) an increase in the negative charge of radiolabeled peptides is effective in reducing renal accumulation. Thus, a highly hydrophilic and negatively charged radiolabel, indiun-111-diethylenetriaminepentaacetic acid ((111)In-DTPA), was attached to an N-terminal residue distant from the HWGF motif ((111)In-DTPA-CTT). In MMP-2 inhibition assays, In-DTPA-CTT significantly inhibited the proteolytic activity in a concentration-dependent fashion. When injected into normal mice, (111)In-DTPA-CTT showed low levels of radioactivity in the liver and kidney. A comparison of the pharmacokinetic characteristics of (111)In-DTPA-CTT with those of other CTT derivatives having different physicochemical properties revealed that the increase in hydrophilicity and negative charge caused by the conjugation of (111)In-DTPA reduced levels of radioactivity in the liver and kidney. In tumor-bearing mice, a significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of (111)In-DTPA-CTT and gelatinase activity. These findings support the validity of the chemical design of (111)In-DTPA-CTT for reducing accumulation in nontarget tissues and maintaining the inhibitory activity of the mother compound. Furthermore, (111)In-DTPA-CTT derivatives would be potential radiopharmaceuticals for the imaging of gelatinase activity in metastatic tumors in vivo.

Chemical Conversion of Natural Polymyxin B and Colistin to Their N-Terminal Derivatives

Abstract The chemical conversions of natural polymyxin B and colistin, which are fatty-acylated cyclic decapeptides, to polymyxin (2–10) and colistin (2–10) derivatives were examined. The Nα-free and side chain Nγ-protected nonapeptides, i.e., tetrakis(Nγ-trifluoroacetyl)–polymyxin B (2–10) and tetrakis(Nγ-trifluoroacetyl)–colistin (2–10), were prepared by trifluoroacetylation of polymyxin B and colistin, followed by chemical cleavage with 50% methanesulfonic acid to remove Nα-alkanoyl–Nγ-trifluoroacetyl–Dab–OH. The Nγ-protected nonapeptides were useful starting materials for the semi-synthesis of N-terminal derivatives by selective Nα-acylation at Thr2, followed by the removal of the Nγ-trifluoroacetyl protecting group with aqueous piperidine. Further, myristoyl–polymyxin B (2–10) and myristoyl–colistin (2–10) retained their antimicrobial activity with an MIC of 2–4 nmol mL−1 against Escherichia coli, Salmonella Typhimurium, and Pseudomonas aeruginosa. They also retained their high lipopolysaccahride (LPS) binding activity. Acetyl–polymyxin B (2–10) and acetyl–colistin (2–10) exhibited very low biological activities, except for a high bactericidal activity specifically against Pseudomonas aeruginosa with an MIC of 2 nmol mL−1. The distinct sensitivity of three Gram-negative bacteria tested toward acetyl-nonapeptides suggested that the N-terminal hydrophobic character of the fatty-acylated polymyxin peptides was necessary for the bactericidal activity against Escherichia coli and Salmonella Typhimurium, but not against Pseudomonas aeruginosa.