From thermoplastic elastomers to designed biomaterials

This highlight is about my metamorphosis from a cationic polymerization chemist to a biomaterialist (no pun intended) and some of the main events on the road. My earlier career faded away with the discovery of living cationic polymerizations, chronicled in my 1999 highlight, but it also put me on the road to designed biomaterials. My new career started with, and still focuses on, the creation of new polymeric architectures, mainly by cationic techniques, for toughened bone cements, injectable intervertebral discs, nonclogging artificial blood vessels, and amphiphilic networks for controlled drug delivery and immunoisolatory membranes. The enormous complexities of immunoisolation of pancreatic islets are now center stage, and lately we have been using all kinds of techniques to make unique membranes to correct type 1 diabetes. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2951–2963, 2005     

Dual-targeting conjugates designed to improve the efficacy of radiolabeled peptides

Radiolabeled regulatory peptides are useful tools in nuclear medicine for the diagnosis (imaging) and therapy of cancer. The specificity of the peptides towards GPC receptors, which are overexpressed by cancer cells, and their favorable pharmacokinetic profile make them ideal vectors to transport conjugated radionuclides to tumors and metastases. However, after internalization of the radiopeptide into cancer cells and tumors, a rapid washout of a substantial fraction of the delivered radioactivity is often observed. This phenomenon may represent a limitation of radiopeptides for clinical applications. Here, we report the synthesis, radiolabeling, stability, and in vitro evaluation of a novel, dual-targeting peptide radioconjugate designed to enhance the cellular retention of radioactivity. The described trifunctional conjugate is comprised of a Tc-99m SPECT reporter probe, a cell membrane receptor-specific peptide, and a second targeting entity directed towards mitochondria. While the specificity of the first generation of dual-targeting conjugates towards its extracellular target was demonstrated, intracellular targeting could not be confirmed probably due to non-specific binding or hindered passage through the membrane of the organelle. The work presented describes a novel approach with potential to improve the efficacy of radiopharmaceuticals by enhancing the intracellular retention of radioactivity.     

Novel antibiotics: macrocyclic peptides designed to trap Holliday junctions

[reaction: see text] Described are the syntheses of eight macrocyclic peptides designed to trap Holliday junctions in bacteria, thereby inhibiting bacterial growth. These macrocycles were designed from linear dimerized hexapeptides that bind to the C-2 symmetrical Holliday junction. They were synthesized from three monomers using a combinatorial-like strategy that permits elucidation of the monomer role in accumulation of Holliday junctions and antibiotic activity. These macrocycles are an important step in designing and synthesizing a new class of antibiotics.     

De novo designed peptides for biological applications

In recent years our ability to design and assemble peptide-based materials and objects de novo (i.e. from first principles) has improved considerably. This brings us to a point where the resulting assemblies are quite sophisticated and amenable to engineering in new functions. Whilst such systems could be used in a variety of ways, biological applications are of particular interest because of the demand for biocompatible, readily produced systems with potential as drug-delivery agents, components of biosensors and scaffolds for 3D cell and tissue culture. This tutorial review describes the building blocks (or tectons) that are being used in peptide assembly, highlights a range of materials and objects that have been produced, notably hydrogels and virus-like particles, and introduces a number of potential applications for the designs.     

Site-selective metal binding by designed alpha-helical peptides

It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)4G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of 113Cd, 1H NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.     

From IHF protein to design and synthesis of a sequence-specific DNA bending peptide

The design and synthesis of a small peptide that mimics the integration host factor (IHF), a major nucleoid-associated protein, is reported. IHF induces DNA compaction by sequence-specific binding that leads to significant bending of the DNA double strand. In a modular approach a small L-lysine dendrimer responsible for nonspecific charge-charge interactions was linked to a cyclopeptide. The latter was designed for specific DNA recognition in the minor groove followed by bending of the double strand.     

Novel antibiotics: second generation macrocyclic peptides designed to trap Holliday junctions

Described are the syntheses of 15 macrocyclic peptides designed to trap Holliday junctions (HJs) in bacteria during site-specific and homologous recombination. This leads to inhibiting bacterial growth. These second generation macrocycles were based on the C-2 symmetrical HJ. They were synthesized using a strategy that permits elucidation of the amino acid role in binding HJs. The syntheses of these macrocycles are an important step in the development of a new class of antibiotics.     

From fibers to micelles using point-mutated amphiphilic peptides

Biocompatible, self-assembled nanostructures are attracting ever more attention, in particular in aqueous media for biomedical applications. Here, we present the successful, solid-phase peptide synthesis (SPPS) and characterization of short amino acid sequences with amphiphilic character with the aim of gaining insight into their self-assembled, supramolecular structures. The peptide design includes three parts: (a) a charged lysine part, (b) an acetylated lysine part, and (c) a constant hydrophobic rodlike helix, based on gramicidin A (gA). By stepwise replacement of free lysine (K) with acetylated lysine (X) we generated a library of a total of 10 peptides, Ac-X(8)-gA and K(m)X(8-m)-gA (m ranging from 0 to 8). By using point mutations, we adjusted the degree of acetylation (DA) and thus the overall amphiphilicity of the peptides, which led to a change in the secondary structure in the aqueous environment from a β-sheet to an α-helix. This transition generated a significant change in the morphology of the self-assembled structures from fibers to micelles. Two different regions were observed for the conformation of the hydrophilic part of the peptide: one region, a β-sheet-like secondary structure, inducing fiber formation (high DA), the other an α-helical-like secondary structure, generating micelle formation (moderate and low DA). The micellar structures depended on the degree of acetylation, which influenced their critical micelle concentration (cmc). These morphology regions were determined by a combination of circular dichroism, dynamic light scattering, surface tension, and transmission electron microscopy, which allowed us to correlate the generated supramolecular architectures with the fine changes obtained by means of the point mutation strategy.     

From elastin peptides to neuraminidase-1-dependent lactosylceramide generation

Elastin peptides constitute a group of biologically active peptides derived from the fragmentation of insoluble elastin. These molecules, currently termed elastokines, have been shown to interact preferably with the elastin receptor complex. Recent data show that the sialidase activity of the neuraminidase-1 of this receptor is required for these peptides to induce their effects. As lactosylceramide generated at the plasma membrane by desialylation of the ganglioside GM₃ can be considered as a second messenger, we feel that this lipidic compound could mimic elastin peptides effects in physiopathological situations.     

From amino acids to heteroaromatics--thiopeptide antibiotics, nature's heterocyclic peptides

Amino acids, the building blocks of proteins, also serve as precursors to a wide range of other naturally occurring substances including alkaloids, antibiotics, and, the subject of this Review, heterocyclic peptides. Simple alpha-amino acids are converted into complex arrays of heteroaromatic rings that display interesting and potent biological activity. The thiopeptide antibiotics, with their complex molecular architectures, are wonderful examples. In this Review we show how organic chemists have developed innovative methods for the synthesis of the heterocyclic ring systems, including routes inspired by the likely biosynthetic processes, and successfully assembled such building blocks into the final target molecule by application of orthogonal protecting groups and coupling methodologies.     

An approach to sequence-specific antibody proteases

We describe here a novel strategy for the isolation of antibodies with sequence-specific protease activity: the synthesis of dipeptide haptens in which the targeted peptide bond has been replaced by a ring-strained or torsionally strained hydroxyethylene transition-state analog. Thus, the analogs mimic both a peptide bond in a distorted, reactive conformation and the transition state for peptide bond hydrolysis. In order to obtain sequence-specific antibody proteases, these analogs have been flanked with additional amino acid residues in preparation for immunization. In particular, we have synthesized peptides containing analogs such as 2-cis-amino-3-cis-hydroxycyclobutane carboxylic acid andendo-(3-amino-2-hydroxy)bicyclo[2.2.1]heptane-7-anti-carboxylic acid. We have also prepared a series of peptide derivatives containing analogs, such as 2-[3-amino-2-oxo-1-azetidinyl]-3-methylbutanoic acid, in which the targeted peptide bond has been incorporated into a β-lactam ring. Since the “peptide bond” has been left intact, these species mimic only a distorted ground state. At present, antibodies are being elicited against a number of the above peptide derivatives.     

Molecular carpentry: piecing together helices and hairpins in designed peptides

The design of a peptide that contains two distinct elements of secondary structure, helix and beta-hairpin, is described. Two designed 17-residue peptides: Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe (I) and Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Gly-Gly-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe (II) have been conformationally characterized by NMR spectroscopy. Peptides I and II contain a seven-residue helical module at the N terminus and a eight-residue beta-hairpin module at the C terminus, which are connected by a conformationally flexible Gly-Gly segment. The choice of the secondary-structure modules is based upon prior crystallographic and spectroscopic analysis of the individual modules. Analysis of 500 MHz 1H NMR data, recorded as solutions in methanol, suggests that the observed pattern of chemical shifts, 3JHN CalphaH values, temperature coefficients of the NH chemical shifts, and backbone inter-residue nuclear Overhauser effects favor helical structures for residues 1-7 and beta-hairpin structures for residues 10-17. The spectroscopic data are compatible with termination of the helical segment by formation of a Schellman motif; this restricts Gly(8) to a left-handed alpha-helical conformation. Gly(9) is the only residue with multiple conformational possibilities in phi,psi space. Possible orientations of the two secondary-structure modules are considered. This study validates the use of stereochemically rigid peptide modules as prefabricated elements in the construction of synthetic protein mimics.     

Fabrication of nanofibers with uniform morphology by self-assembly of designed peptides

Fabrication of controlled peptide nanofibers with homogeneous morphology has been demonstrated. Amphiphilic beta-sheet peptides were designed as sequences of Pro-Lys-X(1)-Lys-X(2)-X(2)-Glu-X(1)-Glu-Pro. X(1) and X(2) were hydrophobic residues selected from Phe, Ile, Val, or Tyr. The peptide FI (X(1)=Phe; X(2)=Ile) self-assemble into straight fibers with 80-120 nm widths and clear edges, as examined by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The fiber formation is performed in a hierarchical manner: beta-sheet peptides form a protofibril, the protofibrils assemble side-by-side to form a ribbon, and the ribbons then coil in a left-handed fashion to make up a straight fiber. These type of fibers are formed from peptides possessing hydrophobic aromatic Phe residue(s). Furthermore, a peptide with Ala residues at both N and C termini does not form fibers (100 nm scale) with clear edges; this causes random aggregation of small pieces of fibers instead. Thus, the combination of unique amphiphilic sequences and terminal Pro residues determine the fiber morphology.     

Syntheses of antibacterial peptides,gramicidin S analogs and designed amphiphilic oligopeptides

In order to clarify the relationship between antimicrobial activity and peptide-structure, gramicidin S analogs and cationic α-helical model peptides were designed and synthesized. Introduction of cationic side chains in hydrophilic side of gramicidin S increased antimicrobial activity against Gram-negative bacteria. Amphiphilic structures of the α-helical peptides were found to be effective to show antimicrobial activities against Gram positive bacteria. Increase in number of cationic amino acid residues in the α-helical peptides caused appreciable antimicrobial activities against Gram-negative bacteria, however, induced lower activities against Gram-positive ones.