Two helical conformations from a single foldamer backbone: "split personality" in short alpha/beta-peptides.
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Amino Acids↗
Biomedical subjects
Samuel H Gellman
Publications and source records attributed to Samuel H Gellman.
Selective binding of TAR RNA by a Tat-derived beta-peptide.
[structure: see text] The interaction between the HIV-1 Tat protein and the TAR RNA element in the nascent viral genomic transcript is required for viral replication. An 11-residue beta-peptide (1), an all-beta homologue of the Arg-rich region Tat 47-57, binds TAR RNA with K(d) = 29 +/- 4 nM. A control beta-peptide (2) in which all Arg side chains are replaced by Lys side chains shows increased affinity but decreased specificity for wild-type vs bulge-deleted TAR RNA, as do the alpha-peptide analogues of 1 and 2.
Amino Acid Sequence↗
Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells.
Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural beta-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue beta-peptide, beta-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (beta-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and beta-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.
Active Transport, Cell Nucleus↗
Molecular architecture with functionalized beta-peptide helices.
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Circular Dichroism↗
Preparation of protected syn-alpha,beta-dialkyl beta-amino acids that contain polar side chain functionality.
We report the synthesis of syn-alpha,beta-dialkyl beta-amino acid derivatives suitably protected for solid-phase synthesis that give rise to residues containing positively charged lysine-like side chains. These amino acids, as well as syn-alpha,beta-dialkyl beta-amino acids that contain diverse hydrophobic side chains, are prepared in good de and ee. The key step in this route involves Davies's protocol for the conjugate addition of a chiral lithium amide to alpha,beta-unsaturated tert-butyl esters (Davies, S. G.; Ichihara, O.; Walters, I. A. S. J. Chem. Soc., Perkin Trans. 1 1994, 9, 1141). syn-alpha,beta-Dialkyl beta-amino acids are interesting building blocks because of their sheet-forming propensity and because of their presence in bioactive compounds.
Amino Acids↗
Secondary structural preferences of 2,2-disubstituted pyrrolidine-4-carboxylic acid oligomers: beta-peptide foldamers that cannot form internal hydrogen bonds.
We examine a new class of beta-peptides, 2,2-disubstituted pyrrolidine-4-carboxylic acid oligomers, and show that they manifest discrete conformational preferences despite the impossibility of internal hydrogen bonding. Numerous beta-peptide families have been described that display specific secondary structural preferences, but all of the conformations characterized in detail so far have contained internal hydrogen bonds. Internal hydrogen bonding is observed within the most common secondary structures of conventional peptides as well. Identifying foldamers in which shape control is independent of hydrogen bonding is significant in two ways. At a fundamental level, foldamers in this small but growing class are interesting because their shapes are controlled by distinctive networks of noncovalent forces. At a practical level, non-hydrogen bonded foldamers may be useful in biomedical applications because the low intrinsic polarity of their backbones may promote bioavailability.
Carboxylic Acids↗
Accommodation of alpha-substituted residues in the beta-peptide 12-helix: expanding the range of substitution patterns available to a foldamer scaffold.
Beta-amino acid oligomers composed exclusively of homochiral trans-2-aminocyclopentanecarboxylic acid (ACPC) residues and/or related pyrrolidine-based residues are known to favor a specific helical secondary structure that is defined by 12-membered ring C=O(i)- -H-N(i+3) hydrogen bonds ("12-helix"). The 12-helix is structurally similar to the familiar alpha-helix and therefore represents a source of potential alpha-helix-mimics. The 12-helix will be most useful in this regard if this conformational scaffold can be employed to arrange specific sets of protein-like side chains in space. Here we examine whether the 12-helix tolerates insertion of acyclic beta-amino acid residues bearing a substituent in the alpha-position ("beta(2)-residues"). Seventeen homologous beta-peptide heptamers have been prepared in which one to four beta(2)-residues reside among ACPC and/or pyrrolidine residues. Circular dichroism comparisons suggest that beta(2)-residues have a lower 12-helical propensity than do residues preorganized by a five-membered ring, as expected, but that beta-peptides containing beta(2)-residues at one or two of the seven positions retain a significant preference for 12-helix formation. These results indicate that a limited number of beta(2)-residues can be used to introduce side chains at specific positions along the surface of a 12-helix.
Circular Dichroism↗
Parallel sheet secondary structure in beta-peptides.
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Models, Molecular↗
Environment-independent 14-helix formation in short beta-peptides: striking a balance between shape control and functional diversity.
We report a significant and unanticipated advance in the study of beta-amino acid-based foldamers: a small proportion of highly preorganized residues can impart high stability to a specific helical secondary structure in water. Most of the residues in these beta-peptides (2 and 3) are intrinsically flexible. Flexible beta-amino acids can be readily and enantiospecifically prepared in functionally diverse forms, but preorganized residues with side chains are rare and challenging to synthesize. Our findings demonstrate that interspersing a few copies of an unfunctionalized but rigid residue among a larger number of flexible residues with diverse side chains is a viable strategy for creating beta-peptides that adopt the 14-helix conformation and therefore display side chains in a predictable spatial arrangement. These results are significant because they enhance the prospects of developing beta-peptides with useful activities.
Circular Dichroism↗
Catalytic transamidation under moderate conditions.
The carboxamide group is generally inert, except under harsh conditions or in the presence of highly evolved enzymes. We have identified several metal complexes that efficiently catalyze transamidation reactions of amide/amine mixtures under moderate conditions. This unprecedented reactivity represents an important step toward our long-term goal of using dynamic combinatorial chemistry to generate new amide-based molecules with interesting structures and functions.
Amides↗
Efficient synthesis of enantiomerically pure beta2-amino acids via chiral isoxazolidinones.
We report a practical and scalable synthetic route for the preparation of alpha-substituted beta-amino acids (beta(2)-amino acids). Michael addition of a chiral hydroxylamine, derived from alpha-methylbenzylamine, to an alpha-alkylacrylate followed by cyclization gives a diastereomeric mixture of alpha-substituted isoxazolidinones. These diastereomers are separable by column chromatography. Subsequent hydrogenation of the purified isoxazolidinones followed by Fmoc protection affords enantiomerically pure Fmoc-beta(2)-amino acids, which are useful for beta-peptide synthesis. This route provides access to both enantiomers of a protected beta(2)-amino acid.
Amino Acids↗
Influence of strand number on antiparallel beta-sheet stability in designed three- and four-stranded beta-sheets.
We describe experiments that probe whether antiparallel beta-sheet secondary structure becomes more stable as the number of strands increases. Several groups, including ours, have explored this issue with peptides designed to adopt three-stranded beta-sheet conformations, but the conclusions have not been consistent. In this study, we examine the effect on conformational stability of beta-sheet lengthening perpendicular to the strand direction via analysis of designed peptides that adopt three-stranded or four-stranded antiparallel beta-sheet conformations in aqueous solution. The findings reported here, along with the context provided by earlier studies, suggest that antiparallel beta-sheet does, in general, become more stable when the number of strands is increased from two to three. We show that this conclusion is not influenced by the rigidity of the loop segment used to link adjacent beta-strands (D-Pro-Gly versus Asn-Gly). We show that further extension, from three strands to four, leads to a further increase in antiparallel beta-sheet stability.
Nuclear Magnetic Resonance, Biomolecular↗
Interactions of the antimicrobial beta-peptide beta-17 with phospholipid vesicles differ from membrane interactions of magainins.
We have studied the interaction of beta-17, a potent synthetic antimicrobial beta-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, beta-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of membrane interaction relative to magainin II, but both leakage and membrane binding show that beta-17, like magainin II, has strong affinity for membranes containing anionic lipids. This is likely to be an important factor contributing to the antimicrobial specificity of the beta-peptide.
Anti-Bacterial Agents↗
Reinvestigation of the proposed folding and self-association of the Neuropeptide Head Activator.
The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (K(d) approximately 1 nM). This dimerization was proposed to occur via antiparallel beta-sheet formation between the Lys(7)-Phe(11) segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by (1)H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys(7)-Phe(11) fragment to form antiparallel beta-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable beta-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular beta-sheet, albeit only weakly, when linked by the highly beta-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.
Animals↗
Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency.
Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.
Anti-Bacterial Agents↗
Stereoselective synthesis of 3-substituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into short 12-helical beta-peptides that fold in water.
A stereoselective synthetic route is reported for the introduction of side chains at the 3-position of trans-2-aminocyclopentanecarboxylic acid (ACPC). Ring opening of the aziridine 2-benzyloxymethyl-6-azabicyclo[3.1.0]hexane with selected nucleophiles occurs in a regioselective manner and provides ACPC precursors with functional groups at the 3-position, trans to the 2-amino group. Oligomers composed of the 3-substituted ACPC residues maintain the 12-helical conformation displayed by the nonsubstituted analogues, as shown by their similar circular dichroism signatures. The added diversity of the new residues provides good dispersion of NMR signals, allowing the assignment of nearly all the NOE signals of a selected hexamer in aqueous solution. The NOEs between protons on nonadjacent residues are characteristic of the 12-helix. 3-Substituted ACPC residues allow one to arrange specific functional groups in a geometrically defined fashion, which should facilitate the design of beta-peptides for biological applications.
Circular Dichroism↗
Design of non-cysteine-containing antimicrobial beta-hairpins: structure-activity relationship studies with linear protegrin-1 analogues.
Protegrins are short, cationic peptides that display potent, broad-spectrum antimicrobial activity. PG-1, the first of the five natural analogues discovered, forms a rigid antiparallel two-stranded beta-sheet that is stabilized by two disulfide bonds. The two strands of the sheet are linked by a short two-residue loop segment. Removal of the disulfide bridges (e.g., in Cys --> Ala analogues) is known to cause marked loss of antimicrobial activity. We have used basic principles of beta-hairpin design to develop linear analogues of PG-1 that lack cysteine but nevertheless display PG-1-like activity. Our most potent reengineered molecules contain three essential design features: (i) the four cysteine residues of PG-1 are replaced by residues that have high propensity for beta-strand conformation, (ii) D-proline is placed at the i + 1 position of the reverse turn to promote a type II' beta-turn, and (iii) amino functionality is incorporated at the gamma-carbon of the D-proline residue to mimic the charge distribution of the natural beta-hairpin. Structural studies revealed that the antimicrobial potency of the non-disulfide-bonded peptides can be correlated to the stability of the beta-hairpin conformations they adopt in aqueous solution. The presence of 150 mM NaCl was found to have little effect on the antimicrobial activity of PG-1, but one of our linear analogues loses some potency under these high salt conditions. Despite this discrepancy in salt sensitivity, NMR and CD data indicate that neither PG-1 nor our linear analogue experiences a significant decrease in beta-hairpin conformational stability in the presence of 150 mM NaCl. Thus, salt inactivation is not due to destabilization of the beta-hairpin conformation. Furthermore, our results show that beta-sheet design principles can be used to replace conformation-stabilizing disulfide bridges with noncovalent conformation-stabilizing features.
Amino Acid Sequence↗
Synthesis and 12-helical secondary structure of beta-peptides containing (2R,3R)-aminoproline.
[structure: see text] (2R,3R)-Aminoproline, a pyrrolidine-based beta-amino acid, was synthesized and incorporated into hexa-beta-peptide 4. This residue confers water solubility when the ring nitrogen is protonated and allows for 12-helix formation in aqueous solution. Circular dichroism spectra display the 12-helical signature, and 12-helical structure was confirmed by 2D NMR analysis.
Circular Dichroism↗