Oligoprolines as Molecular Entities for Controlling Distance in

Article Cite This: Acc. Chem. Res. 2017, 50, 2420-2428

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Oligoprolines as Molecular Entities for Controlling Distance in Biological and Material Sciences Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Stefanie Dobitz,§ Matthew R. Aronoff,§ and Helma Wennemers* ETH Zürich, Laboratory of Organic Chemistry, D-CHAB, Vladimir-Prelog-Weg 3, CH-8093 Zürich, Switzerland CONSPECTUS: Nature utilizes large biomolecules to fulfill tasks that require spatially well-defined arrangements at the molecular level such as electron transfer, ligand−receptor interactions, or catalysis. The creation of synthetic molecules that enable precise control over spacing and functionalization provides opportunities across diverse disciplines. Key requirements of functionalizable oligomeric scaffolds include the specific control of their molecular properties where the correct balance of flexibility and rigidity must be maintained in addition to the prerequisite of defined length. These molecules must ideally be equally applicable in aqueous and organic environments, they must be easy to synthesize in a controlled stepwise fashion, and they must be easily modified with a palette of chemical appendages having diverse functionalities. Oligoproline, a peptidic polymer comprised of repeating units of the amino acid proline, is an ideal platform to meet such challenges. Oligoproline derives its characteristic rigidity and well-defined secondary structure from the innate features of proline. It is the only naturally occurring amino acid that has its side-chain cyclized to its α-amino group, generating often-populated trans and cis conformers around the tertiary amide bonds formed in proline oligomers. Oligoprolines are widely applied to define distance on the molecular level as they are capable of serving as both a “molecular ruler” with a defined length and as a “molecular scaffold” with precisely located and predictably oriented substitutions along the polymeric backbone. Our investigations focus on the use of oligoproline as a molecular scaffold. Toward this end, we have investigated the role of solvent upon helical structure of oligoproline, and the effect that substituents on the pyrrolidine ring and the oligomer termini have on the stability of the helix. We have also further explored the molecular characteristics of oligoproline through spectroscopic and crystallographic methods. All of these structural insights laid the basis for implementation of oligoproline in materials science and chemical biology. Within this Account, we highlight the value of oligoprolines for applications in distinctly different research areas. Toward materials chemistry, we have utilized oligoprolines for the size-controlled generation of noble metal nanoparticles, and to probe the role of spatial preorganization of π-systems for molecular self-assembly. Within the biological realm, we have applied oligoprolines to probe the role of distance on G-protein coupled receptor-mediated ligand uptake by cancerous cells and to investigate the effects of charge preorganization on the efficacy of cationic cell-penetrating peptides.

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INTRODUCTION Proline-rich sequences are widespread in nature.1−3 As the only proteinogenic amino acid containing a secondary amine, proline cannot donate hydrogen bonds when it is part of a peptide chain. The distinct cyclic structure and conformational rigidity of proline is beneficial for proline-rich sequences to modulate protein−protein interactions and is crucial for the structure of collagen.1,3,4 In nature proline-only peptides are found up to 30 residues long,3 and in these oligoprolines the unique conformational effects of proline are augmented to a larger molecular scale. Yet, interest in oligoproline was originally not sparked by their role in nature but arose instead from its value as a molecular tool to study how the amino acid proline behaved in a peptide bond.5 Oligoprolines were recognized already in the 1950s for their rigidity and coined as a “spectroscopic ruler” by Stryer and Haugland.6 Their research demonstrated, by means of Förster © 2017 American Chemical Society

resonance energy transfer (FRET), the value of oligoproline to act as a molecular spacer between two entities attached at the peptidic termini.6 Despite limited structural information, this work was the genesis for decades of research applying oligoprolines as “molecular rulers” where substitutions on the N- or C-termini or both are spanned at precise distances by varying the peptide length (Figure 1).7−11 With more elaborate synthetic tools, oligoprolines were later applied as “molecular scaffolds”, where functionalizations are appended to the rigid backbone through the Cγ position of the proline ring.12 Herein, work from our laboratory is highlighted that has explored the structural and conformational features of oligoprolines and has benefitted from using oligoproline as a well-defined peptidic scaffold. The research demonstrates the Received: July 7, 2017 Published: September 8, 2017 2420

DOI: 10.1021/acs.accounts.7b00340 Acc. Chem. Res. 2017, 50, 2420−2428

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Accounts of Chemical Research

the cis amide content of the peptide. Unfortunately, FRET studies could not produce a consensus regarding the persistence length of oligoproline and the amount of cis amide bonds in PPII helical conformation.6,20−24 This longstanding debate was resolved by studying a series of GdIIInitroxide spin-labeled oligoproline octadecamers with electron paramagnetic resonance (EPR) spectroscopy.25 The experiments resulted in an upper limit of 2% cis amide bonds per residue in a water/glycerol mixture, but none in TFE, and a persistence length of 3−3.5 nm in both solvents. Functionalization, an important aspect for the use of oligoproline as a molecular scaffold, can effect conformational changes upon the secondary structure of the oligoproline. One measure to probe the conformational integrity of an oligoproline molecule is the solvent-induced switch between the two secondary structures that arises from the cis/trans isomerization of the prolyl amide bonds. This switching between the two secondary structures has been analyzed using spectroscopic and spectrometric methods such as FRET, NMR, or optical rotation.13,20,23,26−29 Factors that influence the conformational switch include substitutions at Cγ of the pyrrolidine ring within an oligoproline sequence and capping of the peptide termini.30−33 Some substitutions at the Cγ of the pyrrolidine ring can be beneficial to the stability of one of the two conformations. For example, (4R)-configured proline derivatives bearing electronwithdrawing groups (e.g., azides) and (4S)-configured proline derivatives with hydrogen-bond donating electron-withdrawing groups (e.g., ammonium) stabilize the PPII helix relative to PPI.30−32 Within these proline derivatives, the substituent exerts a stereoelectronic gauche effect, and the trans is favored over the cis amide bond; therefore, the PPII is enforced over the PPI helix. Further, the dipole of an oligoproline sequence along the helix can be used to influence the equilibrium between the PPII and the PPI helix.33 Both helices have a macrodipole from the C- to the N-terminus but of different strengths. Since the macrodipole of the PPI helix is significantly stronger than that of the PPII helix, capping at the N- and Ctermini of an oligoproline sequence with neutral moieties (e.g., with an N-terminal acetyl group and a C-terminal primary amide) favors the PPII relative to the PPI helix.33 Thus, substitutions on the Cγ of the pyrrolidine ring and at the N- and C-termini of an oligoproline sequence can be utilized to tune the equilibrium between the two secondary structures. In addition to the dynamic behavior of the oligoproline secondary structures, knowledge about precise distances within

Figure 1. Oligoprolines as molecular ruler and scaffold.

versatility of oligoprolines and includes applications in diverse disciplines such as the development of supramolecular assemblies, peptide−metal nanoparticles, tumor targeting, and cell-penetrating peptides (CPPs).

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STRUCTURAL CHARACTERISTICS OF OLIGOPROLINE Oligoproline can adopt two conformationally defined secondary structures depending on the environment: the polyproline I (PPI) and the polyproline II (PPII) helix (Figure 2).1,13,14 While the PPI helix has, so far, no known biological relevance, PPII helices are one of the most abundant secondary structures adopted in proteins along with α-helices and β-sheets.2,3,15,16 The two secondary structures of oligoproline have distinctly different features. The left-handed PPII helix found in aqueous solutions has a helical pitch of ≈9.0 Å with 3.0 amino acid residues per turn, has almost ideal C3 symmetry along the central screw axis, and contains all trans amide bonds. The right-handed PPI helix is found in less polar solvents and consists of all cis amide bonds that generate a more densely packed helix with a pitch of ≈5.4 Å and 3.3 amino acid residues per turn. Oligoprolines cannot donate hydrogen bonds as only tertiary amides are formed along the backbone. Thus, the secondary structure of oligoproline is not stabilized by intramolecular hydrogen bonds but rather by secondary interactions between adjacent amide bonds, such as n → π* interactions.17−19 The PPII helical form of oligoproline is more widely used in practical applications, and thus it is important to understand the dynamic behavior between the two secondary structures. Accordingly, the mechanical stiffness of the secondary structure of oligoproline, its persistence length, is innately connected to

Figure 2. Models of PPII and PPI helical oligoproline Ac-Pro15-NH2 generated with idealized dihedral angles Ψ = 145°, Φ = −75°, and ω = 180° for PPII and Ψ = 160°,Φ = −75°, and ω = 0° for PPI. 2421


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adjacent carbonyl groups, and the highest pyramidalizations of Ci yet observed for interactions between two amides. These observations were subsequently corroborated by Hanessian and co-workers with a crystal structure of a 4,5-methanoproline tetramer.39

the secondary structures is equally valuable for the application of oligoprolines as molecular scaffolds and rulers. Powder diffraction studies in the 1950s by Cowan and McGavin established the basic structural parameters of the oligoproline PPII helix, for example, all amide bonds are in trans conformation, shortly after the first successful oligoproline synthesis.5,34,35 It is noteworthy that this low-resolution structure also revealed the configuration of the peptide linkage involving the proline nitrogen, a feature that had been hypothesized but was unknown.5 Many molecular details were, however, left unknown since the crystallization of a PPII helical oligoproline remained elusive. Sixty years after the first powder diffraction studies on oligoprolines by Cowan and McGavin, our group was able to crystallize oligoproline in a PPII conformation.17 The highresolution crystal structure of the oligoproline hexamer provided detailed insights into the dimensions and conformational properties of oligoproline in PPII helical conformation. The crystal structure (Figure 3b) showed almost ideal C3-

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THE FUNCTIONALIZATION OF OLIGOPROLINES

Chemical advancements for a facile means to functionalize oligoprolines at the Cγ-position of the pyrrolidine ring initiated the development of oligoprolines as molecular scaffolds. Alkylation of the hydroxy group of hydroxyproline has enabled functionalization via ether linkages, but more versatile functional groups such as the azide (N3−R) are easily accessible from commercially available (4R)-hydroxyproline.40,41 Functionalization of 4-azido-proline (Azp) containing oligoprolines can be performed in solution or on solid support, either through the copper(I)-catalyzed azide−alkyne [2 + 3] cycloaddition (CuAAC) or by reduction and acylation.30,32,41

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OLIGOPROLINES FOR CELL-PENETRATING PEPTIDES The cell membrane presents an important obstacle for the translocation of large or abiotic molecules. Consequently, the efficacy of therapeutics and imaging agents having intracellular targets relies on penetration of the cellular membrane. Many molecular tools have been generated for this purpose: cellpenetrating peptides (CPPs), an example inspired by strategies found in nature, apply peptide sequences that can translocate the cytoplasmic membrane.42,43 Early examples of proline-rich CPPs originated from natural peptides such as Bactenicin 7 (Bac 7) and γ-zein, which adopt PPII-like structures.44,45 Despite the moderate hydrophobicity of proline residues, proline-rich CPPs generally retain the excellent water solubility requisite for in cellulo use.46 Without modification, the solvatory effects of oligoproline enable utilization of this scaffold as a neutral, lipophilic CPP.47,48 Whether the overall effect of cellular uptake results from slow permeation or energy dependent endocytosis is unclear in these examples. The cellular uptake of oligoprolines can be potentiated by functionalizing proline at the Cγ position of the pyrrolidine ring with cationic and hydrophobic functional groups in order to predictably generate amphiphilic peptides with high uptake.43,49,50 Our own laboratory used oligoprolines to explore the effects of preorganized versus undefined cationic charge display and to examine the role of conformational rigidity versus flexibility on cellular uptake.51 Oligoproline octamers bearing guanidinium (Gdn) groups directly at the Cγ position of the pyrrolidine ring were designed to create defined locations of positive charge along the backbone of the PPII helix (Z8, Figure 4a) and were compared with peptides displaying charge in undefined positions either along the rigid PPII helix or from an unstructured polyarginine (R8 and X8, Figure 4a). Charge localization along the rigid oligoproline backbone enabled more efficient cellular entry into different cancer cell lines (HeLa, MCF7, and HT-29) in comparison to the examples with undefined charge display (Figure 4c). The initial step in the mechanism of cellular entry of cationic CPPs is their association with the anionic cell surface glycans.42 We recognized that the lateral distance between two sulfate moieties of cell surface glycans such as heparan sulfate is nearly identical to the helical pitch of the oligoproline scaffold,

Figure 3. (a) Hexaproline p-Br-C6H4-Pro6-OH. (b) Crystal structure of hexaproline p-Br-C6H4-Pro6-OH (ORTEP). (c) Segmental side view and view along the axis. (d) n → π* Interaction between adjacent prolyl amide bonds (left) and distance between Oi−1···Ci, Bürgi− Dunitz angle (θBD), and the pyramidalization of Ci (Δ, right). Adapted with permission from ref 17. Copyright 2014 American Chemical Society.

symmetry along the helical axis with a pitch of 8.98 ± 0.14 Å completing one turn. No coordinating water molecules cocrystallized with the oligoproline hexamer, indicating that hydration is not a prerequisite for PPII helices. This finding settled an intensely debated issue regarding the need for water in stabilization of PPII helical oligoproline.18,36−38 In addition, the hexaproline crystal structure provided the opportunity to analyze the secondary interactions between adjacent amide bonds (Figure 3d). Signatures of n → π* interactions19 between adjacent amide bonds were apparent including shortened Oi−1···Ci distances below the sum of their van der Waals radii, Bürgi−Dunitz trajectories of ≈104° between 2422



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OLIGOPROLINES FOR THE TARGETING OF G-PROTEIN COUPLED RECEPTORS In tumor targeting, the overexpressed extracellular transporters and receptors of cancer cells, for example, G-protein coupled receptors (GPCRs), are specifically targeted with ligands that bear a radioactive metal or cytostatic agent.52,53 In such cases, the utilization of multivalent ligands to target GPCRs is appealing because the entropic penalty for binding more than one recognition motif is minimized.54,55 Additionally, multivalent ligands with defined distances between the recognition motifs can provide information about the distance between the GPCRs, an aspect that is still not well understood.56,57 Multivalent ligands scaffolded by oligoproline offer an opportunity to precisely control the distance between recognition motifs, thus facilitating study of the role of distance. The groups of Overkleeft and Tamamura evaluated ligands bearing agonistic recognition motifs for the CXCR4 and the LHR/FSHR, respectively.58,59 Differences in binding affinity were observed depending on the distance between the recognition motifs but no correlation of the distance between the recognition motifs and the affinity for the GPCRs was observed. Our group evaluated the uptake of hybrid ligands that bear an agonistic and an antagonistic recognition motif to target the human gastrin-releasing peptide receptor (hGRP-R).60 Here, bombesin-based agonists and antagonists were covalently attached to an oligoproline scaffold in distances of 10 Å, 20 Å, and 30 Å (H10−H30, Figure 5a) in order to evaluate the role of distance for GPCR-mediated cellular uptake. Additionally, a collection of mono- and homodivalent ligands was evaluated as control compounds. In vitro studies with prostate cancer (PC-3) cells showed that hybrid ligand [177Lu]-H20 (d = 20 Å) exhibited a higher cellular uptake than all monovalent, homodivalent, and hybrid ligands, indicating the importance of a defined distance between the recognition motifs for hGRP-Rmediated cellular uptake (Figure 5b). Quantitative biodistribution studies with hybrid ligand [177Lu]-H20 in nude mice bearing PC-3 cell xenografts showed a combination of high tumor cell uptake (typical for antagonists) in combination with a low wash out (typical for agonists). This synergy renders hybrid ligands highly attractive for tumor targeting.

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OLIGOPROLINES TO CONTROL METAL NANOPARTICLE FORMATION Noble metal nanoparticles (NPs) have distinct optical, electronic, and catalytic features that highly depend on their size and shape. Thus, control over the size of NPs is key for applications in imaging, molecular electronics, and catalysis.61−64 A convenient way to form metal NPs is the reduction of metal ions in the presence of additives.65 The additive is important for (a) controlling the growth of the NPs and (b) stabilizing the NPs against coalescence.65 However, the correlation between the molecular properties of the additive and the nanoscopic properties of the resulting NPs is poorly understood. Consequently, the design of additives for the controlled formation of NPs in defined size is a tremendous challenge, and most additives are developed empirically. We reasoned that the rigidity and ease of functionalization of oligoproline is ideal to probe whether the length of a molecular scaffold additive is reflected in the nanoscopic properties of the resulting metal NPs. With this goal in mind, we functionalized a series of oligoprolines of different lengths (6-mers to 24-mers)

Figure 4. (a) Design (left), structure of CPPs (center) [CF = 5(6)carboxyfluorescein], and corresponding confocal microscopy images (right). (b) Model of a PPII helical oligoproline (top) and segment of the crystal structure of heparin (bottom). (c) Mean cellular fluorescence of HeLa cells incubated with peptides at 10 μm for 1 h at 37 °C. Tat = Tat(48−60), GRKKRRQRRRPPQ, and Pen = penetratin, RQIKIWFQNRRMKWKK. (Control cells were not treated with peptides). Adapted with permission from ref 51. Copyright 2017 WILEY-VCH Verlag GmbH & Co. KGaA.

both ≈9 Å (Figure 4b). Thus, the pendant guanidinium moieties on the oligoproline backbone align with the anionic sulfuryl groups of heparin, an interaction that likely contributes to their efficacy. This observation was corroborated by binding studies of Z8 with heparin as well as cell penetration assays with heparan-sulfate-deficient CHO-K1 cells.51 2423


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Figure 6. (a) TEM images and size distribution of AgNPs that were formed in the presence of oligoprolines with 2, 5, and 8 repeating units [scale bars = 20 nm]. (b) General structure of oligoproline series with repeating units of n = 2−8. (c) Correlation of the lengths of oligoprolines 2−8 with the average diameter of the corresponding AgNPs. Adapted with permission from ref 66. Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA.

Figure 5. (a) Hybrid ligands H10−H30. (b) Time dependent uptake of 177Lu labeled hybrid ligands H10−H30 in PC-3 cells. Adapted with permission from ref 60. Copyright 2013 American Chemical Society.

the molecular level of the additive and the nanoscopic level of the metal NPs.

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with an aldehyde moiety at every third proline residue and used them as additives to form AgNPs under Tollens conditions (Figure 6a).66 In this reaction, the aldehyde groups reduce Ag+ to Ag0 and the resulting carboxylic acid moieties coordinate the emerging AgNPs. Thus, the aldehyde-functionalized oligoproline scaffold serves to direct both the nucleation and the growth of the AgNPs. AgNPs formed with distinctly different diameters that depended on the length of the aldehyde-functionalized oligoproline additive. Most remarkably, the average diameter of the AgNPs correlated linearly with the length of the oligoproline scaffold (Figure 6b,c). This correlation is likely due to the nucleation of the AgNPs on the oligoproline scaffold as well as stabilization of the AgNPs with sizes dictated by the length of the scaffold on which the nuclei originated. This is a rare example where a direct relationship was observed between

OLIGOPROLINES FOR SUPRAMOLECULAR ASSEMBLIES Control over the order of functional building blocks (e.g., πsystems) into large, ordered systems is key to the development of new materials, for example, for organic electronics.67,68 Conjugates between π-systems and peptides with well-defined secondary structures are attractive tools toward this goal since peptides are easily accessible via modular synthetic routes and the properties of the chromophore can be tuned.69,70 Several studies used the inherent self-assembly properties of the peptidic compound via intra- or intermolecular hydrogen bonding (α-helix and β-sheet, respectively) in combination with those of the chromophore to achieve supramolecular assemblies.71−76 In contrast, oligoprolines do not self-assemble 2424


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conjugate in water/THF mixtures; from worm-like threads via bundles of fibrils to nanosheets (Figure 7). Alterations to the length of the conjugate and the stereochemistry at the Cγ of the connecting proline residues allow for changes in the supramolecular architecture.78 The combination of grazing-incidence wide-angle X-ray scattering (GIWAXS), UV−vis, and fluorescence spectroscopy of the nanostructures revealed J-aggregate formation by a ladder-like arrangement of the PMIs within the nanosheets (Figure 7b). This organization creates an inner hydrophobic region with the hydrophilic oligoprolines decorating the outside of the assembly. Building on these insights into the spatial organization, we expanded the utilization of oligoproline−chromophore conjugates into advanced topologies. We reasoned that building blocks such as A (Figure 8a) should form fibers via π−π stacking of the N- and C-terminal chromophores (Figure 8b) (such as the oligoproline−PMI conjugates from the previous studies)77 and could enable crossing points and entwining if voids were created at regular intervals. Indeed, conjugate A selfassembles in a mixture of THF and water into a triaxial weave (Figure 8c,d).79 The self-assembly, which forms by a cooperative process in solution, extends into the micrometer regime and endows the nanoscopic material, similar to macroscopic woven materials, with mechanical strength and stability. It is noteworthy that humans have relied on woven macroscopic objects since the Stone Age, but weaving on the nanoscopic scale is so difficult that only two wholly organic weaves (both with a diaxial topology)80,81 have been developed. Thus, the oligoproline scaffold allowed, through precise spatial control at the molecular level, the formation of this self-assembled organic triaxial weave that expands the scope of complex architectures at the nanoscale.

Figure 7. (a) Structure of oligoproline−PMI conjugates (left) and TEM micrographs of supramolecular assemblies deposited from THF/ H2O (30:70) solutions and staining with 2% uranyl acetate. (b) Model of the supramolecular organization of the conjugate with n = 3. Adapted with permission from ref 77. Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA.

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SUMMARY AND OUTLOOK The unique molecular characteristics of oligoprolines have enabled their usage as molecular scaffolds and rulers in diverse disciplines, as highlighted herein. All of these applications benefit from the ease of synthesis and functionalization, as well as the solubility and structural predictability engendered by the oligoproline platform. While other foldameric molecules82 may be capable of creating defined distances on the molecular level, few possess the collective molecular characteristics and depth of understanding as oligoproline, which draws from structural insight accumulated over the last 60 years. In all regards, the future holds great promise for further utilization of oligoprolines as molecular scaffolds and rulers to tackle challenges in

on their own but serve merely as a scaffold for distance control over the number and spatial preorganization of π-systems. We asked whether the covalent tethering of oligoprolines with chromophoresthat also do not self-assemblesuffices for the formation of well-defined supramolecular architectures.77,78 In initial studies, we functionalized oligoprolines in every third position with perylene-monoimides (PMIs) bearing sterically demanding isopropyl groups, which are known to obstruct π-stacking. Spectroscopic (UV−vis and fluorescence) and microscopic (TEM) studies revealed hierarchical supramolecular self-assemblies with increasing length of the

Figure 8. (a) Structure of oligoproline−PMI conjugate A. (b, c) Cartoon of the self-assembly into threads and the triaxial weave. (c, d) TEM micrographs at different magnifications of the supramolecular assemblies deposited from THF/H2O (30:70) solutions and stained with 2% uranyl acetate. Adapted with permission from ref 79. Copyright 2017 Nature Publishing Group. 2425


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chemical biology and material science, as well as other untapped territories.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Helma Wennemers: 0000-0002-3075-5741 Author Contributions §

M.R.A. and S. D. contributed equally.

Funding

We are grateful for financial support from the Swiss National Science Foundation and the Volkswagen foundation. S.D. thanks the Fonds of the Chemical Industry (Germany) for a Kekulé scholarship, and M.R.A. thanks ETH Zürich for an ETH Postdoctoral Fellowship. Notes

The authors declare no competing financial interest. Biographies Stefanie Dobitz studied chemistry at the Justus Liebig University Giessen, completing her B.Sc. and M.Sc. with Prof. P. R. Schreiner on peptide-catalyzed stereoselective oxidations. She is currently pursuing her Ph.D. at ETH Zürich with Prof. H. Wennemers on the use of oligoprolines as multivalent scaffolds for tumor targeting. Matthew R. Aronoff received his Ph.D. in chemistry and chemical biology from the University of Wisconsin−Madison in 2015 under the direction of Prof. R. T. Raines. There he developed novel chemoselective tools for biological application. He is now an ETH Zürich postdoctoral fellow in the group of Prof. H. Wennemers. Helma Wennemers studied chemistry at the Johann-WolfgangGoethe University in Frankfurt before moving to Columbia University where she received her Ph.D. with Prof. W. C. Still. Following postdoctoral studies at Nagoya University with Prof. H. Yamamoto, she joined the University of Basel as Assistant Professor. She was promoted to Associate Professor before moving to ETH Zürich in the fall of 2011 where she is Professor of Organic Chemistry. Her research focuses on the development of small molecules with functions that are fulfilled in nature by large macromolecules. This scope includes the development of bioinspired asymmetric catalysts, functionalizable collagen, the controlled formation of metal nanoparticles, and the use of molecular scaffolds for applications in supramolecular and biological chemistry.

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REFERENCES

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