1,6‐hexanediol diacrylate‐crosslinked polystyrene: Preparation,characterization, and application in peptide synthesis

A copolymer of 1,6‐hexanediol diacrylate (HDODA) and styrene was prepared by a suspension polymerization method. The resin was characterized by infrared and carbon‐13 cross‐polarization magic‐angle spin (¹³C CP‐MAS) spectroscopy. The topology of the resin was examined by scanning electron microscopy (SEM). The polymer swells extensively in common solvents used for peptide synthesis. The resin exhibited chemical stability even in neat trifluoroacetic acid. The applicability of the new resin was demonstrated by synthesis of Val‐Ala‐Val‐Ala‐Ala‐Gly, Gln‐Val‐Gly‐Gln‐Val‐Glu‐Leu‐Gly, and Val‐Gln‐Ala‐Ala‐Ile‐Asp‐Tyr‐Ile‐Asn‐Gly. Comparative synthetic studies showed that the new resin is superior to divinylbenzene (DVB)‐based resin in the case of the synthesis of hydrophobic peptide sequences. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 1290–1296, 2003     

Kinetics of amide formation on 1,4‐butanediol dimethacrylate crosslinked polystyrene resin: A comparison with PS‐DVB resin

An efficient crosslinked polymer support was synthesized by introducing 1,4‐butanediol dimethacrylate crosslinker to a polystyrene network using aqueous suspension polymerization technique. The support was functionalized with aminomethyl groups using chloromethyl phthalimide. The kinetics of amide bond formation on 1,4‐butanediol dimethacrylate crosslinked polystyrene (PS‐BDODMA) polymer were carried out using the attachment of Rink amide linker as a model reaction. The efficiency of the support was tested and compared with Merrifield resin by following different steps involved in the synthesis of a 14‐residue model peptide, Lys‐Ile‐Asn‐Thr‐Asn‐Ala‐Ser‐Trp‐His‐Ala‐Asn‐Arg‐Thr‐Ala‐NH_2, under the same synthetic conditions. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2897–2903, 2003     

Model polypeptide of mussel adhesive protein. I. Synthesis and adhesive studies of sequential polypeptides (X‐Tyr‐Lys)n and (Y‐Lys)n

The sequential polytripeptides and polydipeptides, (X‐Tyr‐Lys)_n, (XGly, Ala, Pro, Ser, Leu, Ile, Phe), (Y‐Lys)_n, (YGly, Tyr), and (Gly‐Tyr)_n, which imitate a mussel adhesive protein, have been synthesized. The molecular weights of the polypeptides were estimated to be 7,200 ∼ 13,400 (19 ∼ 42 repeating units), and the polypeptides were found to have satisfactory amino acid sequences. The polypeptides were crosslinked by tyrosinase, and the optimal pH in the crosslinking reaction was 7.4 in the case of the polytripeptide, (Gly‐Tyr‐Lys)_n. The optimal tyrosinase amount for the adhesive strength of (Gly‐Tyr‐Lys)_n was 0.34 unit/mg (polypeptide) at pH 7.4. The shear adhesive strength of the polytripeptide increased with an increase in the polytripeptide concentration, and was not influenced by the third amino acid, X. The shear adhesive strengths of polytripeptides (X‐Tyr‐Lys)_n were equal to one of the synthetic polydecapeptides, (Ala‐Lys‐Pro‐Ser‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n and (Gly‐Pro‐Lys‐Thr‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n which were the model polydecapeptides for blue mussel and Californian mussel, respectively. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 929–937, 2000     

Rational Design of Tryptophan‐Rich Antimicrobial Peptides with Enhanced Antimicrobial Activities and Specificities

Trp‐rich antimicrobial peptides play important roles in the host innate defense mechanism of many plants and animals. A series of short Trp‐rich peptides derived from the C‐terminal region of Bothrops asper myothoxin II, a Lys49 phospholipase A₂ (PLA₂), were found to reproduce the antimicrobial activities of their parent molecule. Of these peptides, KKWRWWLKALAKK—designated PEM‐2—was found to display improved activity against both Gram‐positive and Gram‐negative bacteria. To improve the antimicrobial activity of PEM‐2 for potential clinical applications further, we determined the solution structure of PEM‐2 bound to membrane‐mimetic dodecylphosphocholine (DPC) micelles by two‐dimensional NMR methods. The DPC micelle‐bound structure of PEM‐2 adopts an α‐helical conformation and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates that two of the three tryptophan residues are packed against the peptide backbone and form a hydrophobic face with Leu7, Ala9, and Leu10. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that PEM‐2 interacted with negatively charged phospholipid vesicles and efficiently induced dye release from these vesicles, suggesting that the antimicrobial activity of PEM‐2 could be due to interactions with bacterial membranes. Potent analogues of PEM‐2 with enhanced antimicrobial and less pronounced hemolytic activities were designed with the aid of these structural studies.     

Key Residues in Octyl‐Tridecaptin A1 Analogues Linked to Stable Secondary Structures in the Membrane

Tridecaptin A₁ is a linear antimicrobial lipopeptide comprised of 13 amino acids, including three diaminobutyric acid (Dab) residues. It displays potent activity against Gram‐negative bacteria, including multidrug‐resistant strains. Using solid‐phase peptide synthesis, we performed an alanine scan of a fully active analogue, octyl‐tridecaptin A₁, to determine key residues responsible for activity. The synthetic analogues were tested against ten organisms, both Gram‐positive and Gram‐negative bacteria. Modification of D ‐Dab8 abolished activity, and marked decreases were observed with substitution of D ‐allo‐Ile12 and D ‐Trp5. Circular dichroism showed that octyl‐tridecaptin A₁ adopts a secondary structure in the presence of model phospholipid membranes, which was weakened by D ‐Dab8‐D ‐Ala, D ‐allo‐Ile12‐D ‐Ala, and D ‐Trp5‐D ‐Ala substitutions. The antimicrobial activity of the analogues is directly correlated to their ability to adopt a stable secondary structure in a membrane environment.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. III. Asn20-->Cys/Ala27-->Cys, Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Lys108- ->Met, Gly137-->Ala, 311-314 Loop, Tyr312-->Trp and Ser436-->Pro

Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild- type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.      

Baceridin,a Cyclic Hexapeptide from an Epiphytic Bacillus Strain,Inhibits the Proteasome

A new cyclic hexapeptide, baceridin ( 1 ), was isolated from the culture medium of a plant‐associated Bacillus strain. The structure of 1 was elucidated by HR‐HPLC‐MS and 1D and 2D NMR experiments and confirmed by ESI MS/MS sequence analysis of the corresponding linear hexapeptide 2 . The absolute configurations of the amino acid residues were determined after derivatization by GC‐MS and Marfey's method. The cyclopeptide 1 consists partially of nonribosomal‐derived D ‐ and allo‐D ‐configured amino acids. The order of the D ‐ and L ‐leucine residues within the sequence cyclo(‐L ‐Trp‐D ‐Ala‐D ‐allo‐Ile‐L ‐Val‐D ‐Leu‐L ‐Leu‐) was assigned by total synthesis of the two possible stereoisomers. Baceridin ( 1 ) was tested for antimicrobial and cytotoxic activity and displayed moderate cytotoxicity (1–2 μg mL^?1) as well as weak activity against Staphylococcus aureus. However, it was identified to be a proteasome inhibitor that inhibits cell cycle progression and induces apoptosis in tumor cells by a p53‐independent pathway.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches

Experimental and computational analyses were performed on thecorepressor (L-tryptophan) binding site of the trp-repressorof Escherichia coli to investigate the ligandprotein interactions.Gly 85, one of the residues forming the hydrophobic pocket ofthe binding site, was systematically replaced with Ala, Val,Leu and Trp by cassette mutagenesis. Biochemical characterizationshowed that all these mutations caused significant decreasesin tryptophan binding activity. Free energy perturbation calculationswere performed for the mutants and were consistent with theexperimental results. The lack of a side chain at position 85was concluded to be essential for binding the corepressor; thestructure of the binding pocket was suggested to be tight inthe vicinity of Gly85.     

The inverse type II β-turn on D-Trp-Phe, a pharmacophoric motif for MOR agonists

Herein we propose the D ‐Trp‐Phe sequence within an inverse type II β‐turn as a new kind of pharmacophoric motif for μ‐opioid receptor (MOR) cyclopeptide agonists. Initially, we observed that c[Tyr‐D ‐Pro‐D ‐Trp‐Phe‐Gly] ( 4 ), an analogue of endomorphin‐1 (H‐Tyr‐Pro‐Trp‐Phe‐NH₂) lacking the crucial protonatable amino group of Tyr 1, is a MOR agonist with 10^?8 M affinity. Molecular docking analysis suggested that the relevant interactions with the receptor involve D ‐Trp‐Phe. The bioactive conformation of this region was investigated by selected derivatives of 4 designed to adopt an inverse type II β‐turn. These efforts led to c[Tyr‐Gly‐D ‐Trp‐Phe‐Gly] ( 14 ) and to the cyclotetrapeptide c[D ‐Asp‐1‐amide‐β‐Ala‐D ‐Trp‐Phe] ( 15 ), showing improved nanomolar affinity. Both 14 and 15 selectively bind MOR, as they have negligible affinity for the κ‐ and δ‐opioid receptors. Both 14 and 15 behave as partial MOR agonists in functional assays. Conformational and docking analyses confirm the role of the inverse β‐turn in binding. These results indicate that the D ‐Trp‐Phe inverse β‐turn structure can be used for designing non‐endomorphin‐like peptidomimetic opioid agonists in general, characterized by an atypical mechanism of interaction between ligand and receptor.     

Carbon assimilation and dissimilation during growth of Geotrichum candidum on amino acids and glucose

BACKGROUND: This work examines the metabolic behaviour of amino acids during Geotrichum candidum growth, in the presence of a primary carbon source like glucose. Amino acids were characterized based on their carbon assimilation and dissimilation by G. candidum, in the presence of glucose as the limiting substrate. RESULTS: The first group (Cys, His, Phe, Thr and Trp) was only used as nitrogen sources by G. candidum, with glucose being the carbon and energy source. Glucose repression was shown for the rest of the amino acids, since only after glucose depletion amino acids from the second group (Gly, Lys, Met, Val) were dissimilated for energy supply by oxidation into CO₂, while those from the third group (Ala, Arg, Asp, Glu, Leu, Pro and Ser) were assimilated as carbon sources (and additionally used as nitrogen sources), leading to a diauxic growth. CONCLUSION: This energy‐saving response was not previously shown for the second fungus involved in ripening of soft white cheese—P. camembertii—leading to simultaneous use of some amino acids and glucose as carbon and energy sources, and hence lower growth rates than those recorded during G. candidum growth. Copyright © 2007 Society of Chemical Industry     

Euphorbia tirucalli β-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH–π Interaction between Val483 and Trp534 for Catalytic Action

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli β-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair–chair–chair–boat–boat conformation. The Ile variant, with a somewhat larger bulk, afforded β-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation–π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH–π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.     

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state k(cat) with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Cl alpha) from 1,2-dichloroethane interacts with Trp125 and the Cl beta interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2- dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in k(cat) could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The k(cat) for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Cl alpha or Br alpha) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.      

Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD

Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV‐induced cyclobutane pyrimidine dimers (CPDs) or 6–4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as “site 377”, that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light‐dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases.     

Amino acids as carbon,energy and nitrogen sources for Penicillium camembertii

Three groups of amino acids were previously characterized on their ability to be assimilated as carbon source by Penicillium camembertii. In view of a deeper understanding of their metabolic behaviour, growth of P. camembertii on glucose, the limiting substrate, and an amino acid was examined in batch culture. Amino acids from the first group (Cys, His, Lys, Met, Trp and Val) are convenient nitrogen sources, but cannot be assimilated as carbon sources. However, they are also dissimilated, namely used for energy supply by oxidation into CO₂, during stationary phase, after glucose depletion, as shown for lysine; and the corresponding nitrogen was released as ammonium. Growth exhibited diauxic behaviour for the second group of amino acids (Arg, Leu), since they can be assimilated as carbon sources, in addition to their assimilation as nitrogen sources, but only after glucose depletion, as shown for arginine. A clear differentiation between the assimilated and the dissimilated carbon was demonstrated for the third group of amino acids (Ala, Asp, Glu, Gly, Pro, Ser, Thr); it was shown that the carbon from glutamic acid was assimilated, while the carbon from glucose was dissimilated. Copyright © 2006 Society of Chemical Industry     

A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto

Bacillus subtilis subsp. natto secretes the ComX_natto pheromone as a quorum‐sensing pheromone to produce poly‐γ‐glutamate for biofilm formation. The amino‐acid sequence of the pheromone is Lys‐Trp‐Pro‐Pro‐Ile‐Glu, and the tryptophan residue is post‐translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C‐terminal end of the precursor peptide ComX_natto. Here, we report the functional analysis of ComQ_natto, which catalyzes a unique farnesyl‐transfer reaction. ComQ_natto recognizes not only full‐length ComX_natto but also N‐ and/or C‐terminal truncated ComX_natto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQ_natto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.     

Vero细胞批式培养中的氨基酸代谢

用高效液相色谱(HPLC)测定了批式培养的Vero细胞培养液中氨基酸的变化,结果表明Vero细胞在生长过程中需要消耗12种氨基酸,同时生成4种氨基酸, Vero细胞在生长期吸收Leu,Thr,Ile,Lys,Phe和Tyr较多,而在静止期这些氨基酸却消耗较少.     

Phage Display to Identify Nedd8‐Mimicking Peptides as Inhibitors of the Nedd8 Transfer Cascade

The Nedd8 activating enzyme (NAE) launches the transfer of the ubiquitin‐like protein Nedd8 through an enzymatic cascade to covalently modify a diverse array of proteins, thus regulating their biological functions in the cell. The C‐terminal peptide of Nedd8 extends deeply into the active site of NAE and plays an important role in the specific recognition of Nedd8 by NAE. We used phage display to profile C‐terminal mutant sequences of Nedd8 that could be recognized by NAE for the activation reaction. We found that NAE can accommodate diverse changes in the Nedd8 C‐terminal sequence (⁷¹LALRGG⁷⁶), including Arg and Ile replacing Leu71, Leu, Ser, and Gln replacing Ala72, and substitutions by bulky aromatic residues at positions 73 and 74. We also observed that short peptides corresponding to the C‐terminal sequences of the Nedd8 variants can be activated by NAE to form peptide～NAE thioester conjugates. Once NAE is covalently loaded with these Nedd8‐mimicking peptides, it can no longer activate full‐length Nedd8 for transfer to the neddylation targets, such as the cullin subunits of cullin‐RING E3 ubiquitin ligases (CRLs). We have thus developed a new method to inhibit protein neddylation by Nedd8‐mimicking peptides.     

Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis

A B72.3 Fab/sTn² complex was modeled from the known structureof B72.3 Fab and the dimeric Tn-serine cluster (sTn²). In thecomplex model, the side chains of 15 heavy- and light-chaincomplementarity-determining region (CDR) residues and the mainchains of two light-chain CDR residues contact the sTn² epitope.Among 15 CDR residues which contact sTn² in the model, two heavy-chainresidues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96)have been confirmed in a previous study. To test the accuracyof the computational model, further site-directed mutagenesiswas performed by alanine scanning on the remaining 12 residuesthat are predicted in the model to have side-chain interactionswith sTn². Of these 12 mutants, eight that are all from theheavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala,Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn²affinities, and four consisting of three light-chain mutations(Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation(His35Ala) retained wild-type sTn² affinity. On the whole, thisevidence suggests that the complex model, although not perfect,is correct in many of its features. In a more general vein,these results lend credibility to the computational modelingapproach for the study of the molecular basis of antigen–antibodycomplexes.     

Indoloazepinone‐Constrained Oligomers as Cell‐Penetrating and Blood–Brain‐Barrier‐Permeating Compounds

Non‐cationic and amphipathic indoloazepinone‐constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l ‐Aia‐Xxx]_n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc‐[l ‐Aia‐Gly]_2,4‐OBn oligomers 12 and 13 and Boc‐[l ‐Aia‐β³‐h‐l ‐Ala]_2,4‐OBn oligomers 16 and 17 were totally or partially disordered, Boc‐[l ‐Aia‐l ‐Ala]₂‐OBn ( 14 ) induced a typical turn stabilized by C₅‐ and C₇‐membered H‐bond pseudo‐cycles and aromatic interactions. Boc‐[l ‐Aia‐l ‐Ala]₄‐OBn ( 15 ) exhibited a unique structure with remarkable T‐shaped π‐stacking interactions involving the indole rings of the four l ‐Aia residues forming a dense hydrophobic cluster. All of the proposed FITC‐6‐Ahx‐[l ‐Aia‐Xxx]₄‐NH₂ oligomers 19 – 23 , with the exception of FITC‐6‐Ahx‐[l ‐Aia‐Gly]₄‐NH₂ ( 18 ), were internalized by MDA‐MB‐231 cells with higher efficiency than the positive references penetratin and Arg₈. In parallel, the compounds of this series were successfully explored in an in vitro blood–brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac‐[l ‐Aia‐Xxx]₄‐NH₂ oligomers in the PAMPA model, Ac‐[l ‐Aia‐l ‐Arg]₄‐NH₂ ( 26 ) showed significant permeation in the in vitro cell‐based human model of the BBB, suggesting an active mechanism of cell penetration.