Academic literature on the topic 'Branched peptide'

Redesigning linear cell penetrating peptides (CPPs) into a multi-branched topology with short dipeptide branches gave cell penetrating peptide dendrimers (CPPDs) with higher cell penetration, lower toxicity and hemolysis and higher serum stability than linear CPPs.

Here, we report the use of an enzymatic reaction to cleave the branch off branched peptides for inducing the morphological transition of the assemblies of the peptides. The attachment of DEDDDLLI sequences to the ε-amine of the lysine residue of a tetrapeptide produces branched peptides that form micelles. Upon the proteolytic cleavage of the branch, catalyzed by proteinase K, the micelles turn into nanofibers. We also found that the acetylation of the N-terminal of the branch increased the stability of the branched peptides. Moreover, these branched peptides facilitate the delivery of the proteins into cells. This work contributes insights for the development of peptide supramolecular assemblies via enzymatic noncovalent synthesis in cellular environment.

Two peptides of eight (T2) and 10 (T1) residues corresponding to the branched moiety of ubiquitinated histone H2A have been synthesized and used for raising specific antibodies in rabbits. Antisera to peptide T1 reacted in ELISA with T1 and with H2A but not with ubiquitin; antisera to peptide T2 reacted with T2 but not with H2A or ubiquitin. When tested in immunoblotting, both peptide antisera reacted with ubiquitinated H2A but not with unconjugated H2A or with ubiquitin. Sera from patients with systemic lupus erythematosus (SLE) were shown previously to react with ubiquitin in ELISA and immunoblotting. When tested for their ability to react in ELISA with synthetic peptides T1 and T2, 96% of the SLE sera (diluted 1:500) that recognized ubiquitin also reacted with peptide T2. Of the SLE sera that did not react with ubiquitin, only 13% possessed antibodies able to bind peptide T2. Antibodies from seven SLE sera, purified on a T2-immunoadsorbent column, were also able to react either with H2A, and in three cases also with ubiquitin.

The plant kingdom contains vastly untapped natural product chemistry, which has been traditionally explored through the activity-guided approach. Here, we describe a gene-guided approach to discover and engineer a class of plant ribosomal peptides, the branched cyclic lyciumins. Initially isolated from the Chinese wolfberry Lycium barbarum, lyciumins are protease-inhibiting peptides featuring an N-terminal pyroglutamate and a macrocyclic bond between a tryptophan-indole nitrogen and a glycine α-carbon. We report the identification of a lyciumin precursor gene from L. barbarum, which encodes a BURP domain and repetitive lyciumin precursor peptide motifs. Genome mining enabled by this initial finding revealed rich lyciumin genotypes and chemotypes widespread in flowering plants. We establish a biosynthetic framework of lyciumins and demonstrate the feasibility of producing diverse natural and unnatural lyciumins in transgenic tobacco. With rapidly expanding plant genome resources, our approach will complement bioactivity-guided approaches to unlock and engineer hidden plant peptide chemistry for pharmaceutical and agrochemical applications.

Here, we report a peptide aspartic acid side-chain benzyl ester as a useful precursor that can be efficiently converted into various functional groups, including acid, amide, carbonyl hydrazide, carbonyl azide, or thio ester groups, without other protection for the peptide. With this strategy, we synthesized a series of novel branched and cyclic arginylglycylaspartic acid peptides through successive peptide C-terminal ligation and side-chain ligation based on a side-chain carbonyl azide or thio ester.

The lethal and oedema toxins produced by Bacillus anthracis, the aetiological agent of anthrax, are made by association of protective antigen with lethal and oedema factors and play a major role in the pathogenesis of anthrax. In the present paper, we describe the production of peptide-based specific inhibitors in branched form which inhibit the interaction of protective antigen with lethal and oedema factors and neutralize anthrax toxins in vitro and in vivo. Anti-protective antigen peptides were selected from a phage library by competitive panning with lethal factor. Selected 12-mer peptides were synthesized in tetra-branched form and were systematically modified to obtain peptides with higher affinity and inhibitory efficiency.

Doctor of Philosophy
Department of Biochemistry
John M. Tomich
Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

RNA is essential for the transfer of genetic information, as the central dogma of biology dictates. The role of RNA, however, is not limited to serving as an information shuttle between DNA and fully functional protein. Indeed, RNA has experienced a surge of interest in the field of chemical biology for its other critical roles in biology including those in control of transcription, translation, splicing, genetic replication, and catalysis. RNA has proven to be a difficult and complex target for the design of small molecular ligands because of its structural heterogeneity and conformational flexibility. Yet, the highly folded tertiary structures of these oligomers present unique scaffolds which designed ligands should be able to selectively target. To that end, two branched peptide libraries ranging in size from 4,096–46,656 unique sequences were screened for their ability to bind HIV-1 related RNA structures, the transactivation response element (TAR) and the Rev response element (RRE). In addition to discovering a mid-nanomolar branched peptide ligand for TAR, the first branched boronic acid peptide library designed to target RNA was screened for binding to RRE. Each of these efforts resulted in the identification of selective binders to their respective RNA targets, and the unnatural branching of these compounds was demonstrated to provide a multivalent binding interaction with the RNA. Furthermore, these compounds were shown to be cell permeable and displayed little to no cytotoxicity in HeLa and A2780 cells.
Ph. D.

RNAs have gained significant attention in recent years because they can fold into well-defined secondary or tertiary structures. These three dimensional architectures provide interfaces for specific RNA-RNA or RNA-protein interactions that are essential for biological processes in a living system. These discoveries greatly increased interest in RNA as a potential drug target for the treatment of diseases. Two of the most studied RNA based regulatory systems are HIV-1 trans-activating response element (TAR)/Tat replication pathway and Rev response element (RRE)/Rev export pathway. To efficiently target TAR and RRE RNA, we designed and synthesized three generations of branched peptide libraries that resulted in medium sized molecules. The first generation of BPs were discovered from screening a one-bead one-compound library (4,096 compounds) against HIV-1 TAR RNA. One peptide FL4 displayed a binding affinity of 600 nM to TAR RNA, which is tighter than its native protein counterpart, Tat. Biophysical characterization of these BP demonstrated that "branches" in BPs impart multivalency, and they are cell permeable and non-toxic. The second generation peptides were discovered from an on-bead high-throughput screening of a 3.3.4 branched peptide boronic acids (BPBAs) library that bind selectively to the tertiary structure of RRE IIB. The library comprised of 46,656 unique sequences. We demonstrate that our highest affinity BPBA (BPBA1) selectively binds RRE IIB in the presence of competitor tRNAs as well as against six RRE IIB structural variants. Further, we show that the boronic acid moieties afford a novel binding mode towards RNA that is tunable; their Lewis acidity has critical effects on binding affinity. In addition, biophysical characterizations provide evidence that "branching" in these peptides is a key structural motif for multivalent interactions with the target RNA. Finally, RNA footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. BPBA1 is cell permeable and non-toxic. In the next generation of branched peptides, a 3.3.4 branched peptide library composed of 4,096 unique sequences that featured boronic acid and acridine moieties was designed. We chose acridine as the amino acid side chain due to its potential for π-stacking interaction that provides high binding affinity to RNA target. The library was screened against HIV-1 RRE IIB RNA. Fifteen peptides were sequenced and four contained acridine alone and/or in conjunction with boronic acid moieties displayed dissociation constants lower than 100 nM. The ribonuclease protection assays of A7, a sequence that contains both boronic acid and acridine residues, showed a similar protection pattern compared to previous peptide BPBA1, suggesting that the 3.3.4 branched peptides shared similar structural elements and contacted comparable regions of the RRE IIB RNA. The results from this research indicated that "branching" in peptides imparts multivalent interactions to the RNA, and that functional groups such as boronic acid and acridine are key structural features for efficient binding and selectivity for the folded RNA target. We demonstrated that the branched peptides are cell permeable and non-toxic.
Ph. D.

Heparan sulfate Proteoglycans (HSPGs) play a number of signaling and structural roles in tumor progression and metastasis spread. The biological function of HSPGs resides in their ability of interaction with many different types of ligands like growth factors, morphogens, chemokines and proteins of extracellular matrix (ECM). These bindings activate signaling pathways that modulate major transformations of cancer cells, leading to tumor growth, migration, invasion and metastasis. HSPGs are over-expressed on cancer cell membranes. The tetra-branched peptide NT4 binds with high selectivity to different human cancer cells and tissues. Its ability to discriminate between tumor and healthy tissues resides in the high-affinity binding to HSPGs and can be exploited by conjugating NT4 with different functional units, like chemotherapeutical drugs and tracers for cancer cell imaging and therapy. In this work, we test NT4 ability to interfere with processes mediated by HSPGs in tumor cell adhesion, migration and matrix invasiveness. Since HSPGs modulate also neo-angiogenesis, because of their expression by endothelial cells of microvessels that generate new vasculature, we focus also on NT4 role in endothelial cell proliferation, migration and tube formation, in the presence of Fibroblast Growth Factor-2 (FGF-2). Since the internalization and trafficking behaviour of HSPGs seems to reveal a clathrin and caveolin- independent, but dynamin-dependent endocytic pathway, we investigate the pathway used by NT4 to enter cells. Last, but not least, we radiolabel NT4 with 18F in order to measure tumor uptake and whole-body biodistribution in a mouse model of breast cancer, using in vivo PET imaging. NT4-HSPG interactions and consequent modulation of signaling pathways can prove the importance of NT4 peptide as a specific tool to enlighten the role of HSPG in tumor onset and progression.

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. In this thesis are repoted strategies to improve the biopharmaceutical development and manufacturing process of this peptide. First, the secondary structure of SET-M33 was investigated by NMR to fully characterize the product in the framework of preclinical studies. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, It is also reported the studies on a new salt formulation, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient to decrease the manufacturing peptide cost and did not increase the toxicity of the antimicrobial drug. In addition, to identify back-up molecules, a panel of modified versions of SET-M33 was tested in order to produce new molecules with better performance in terms of pharmaceutical profile and manufacturing costs. Amongst them, the opportunity of using SET-M33D-L-Ile and SET-M33D-Leu/Ile will allow to decrease the costs in the synthesis process and SET-M33-Gly/Ala, to eliminate the degradation site for bacterial proteases, without altering the strong antimicrobial activity of the original peptide. Finally cloning strategies, expression systems, purifications and structural characterizations of various proteins of mammalian inflammasomes performed at Boston Children's Hospital, affiliated with Harvard Medical School, are described.

Doctor of Philosophy
Department of Biochemistry and Molecular Biophysics
John M. Tomich
Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.

The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability. The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding. A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic. High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication.
Ph. D.

Les travaux de recherches de cette thèse concernent la conception et la synthèse de nouveaux matériaux hybrides basés sur un squelette polysiloxane. Pour cela, plusieurs molécules ont été silylées dans le but d’être incorporées dans une chaine de silicone multifonctionnel. En effet, au contraire des approches de post-greffages, nous avons mis au point une copolymérisation directe de ces macromonomères hybrides présentant une fonction methyldihydroxysilane avec du dichlorodimethylsilane (DCDMS). Plusieurs types de biomolécules ont été silylées : peptides, médicaments, sondes pour l’imagerie, chacune de ces molécules apportant des propriétés particulières au matériau final. Trois principales applications sont présentées : (i) la synthèse directe de film de silicone réticulés bioactifs, (ii) la préparation de nanoparticules (NPs) de silicone multifunctionnel (iii) ou des polyplexes. Des films de PDMS bioactifs (antibactériens ou promouvant l’adhésion cellulaire) réticulés ont été obtenus par copolymérisation de macromonomères hybrides peptidiques avec du DCMS et des monomères silane ou vinyl silane permettant une réticulation par hydrosilylation. Les NPs de silicone hybride sont issues de l’introduction de plusieurs macromonomères hydrophiles de 0.5 à 1mol% par rapport au DCDMS. Des ligands peptidiques ciblant les cellules cancéreuses, du PEG et du méthotrexate, tous trois silylés, ont été préparés et copolymérisés. Enfin, nous présentons les résultats préliminaires obtenus pour la préparation de polyplexes de siRNA basés sur la polymérisation in situ de peptides hybrides possédant des séquences riches en histidine et lysine
The purpose of this PhD work was the design and synthesis of new hybrid biomaterials based on a polysiloxane backbone. To do so, several biomolecules were silylated, in order to be incorporated in a multifunctional silicone backbone by a bottom-up strategy. Indeed, in contrast to post-grafting approaches, we set up the direct copolymerization of hybrid biomolecule macromonomers presenting a methyldihydroxysilyl moiety, with the dichlorodimethylsilane (DCDMS). Different types of biomolecules have been silylated: peptides, drugs and imaging probes, each of them affording specific properties to the final bioorganic silicone material. Three main applications are described: (i) the design and synthesis of bioactive PDMS cross-linked materials, (ii) silicone-based nanoparticules (NPs) and (iii) silicone-based polyplexes. PDMS materials with biological properties, either antimicrobial or cell adhesion, were obtained by copolymerization of hybrid peptide macromonomer with DCDMS, vinyl and silane reagents followed by hydrosilylation. Silicone-based NPs resulted from the introduction of several hydrophilic macromonomers at 0.5 to 1 mol% compared to DCDMS. Hybrid peptide ligands targeting cancer cell receptors, PEG and a drug model (Methotrexate) were prepared and copolymerized. At last, we investigated the preparation of siRNA polyplexes involving LysHis-based hybrid peptide macromonomers by an in situ polymerization method

Abstract 1) Newly tailored peptide nucleic acids (PNA) and PNA-modified magnetic nanoparticles for DNA targeting. 2) PEG branched polymer chains functionalization of nano-systems for biocompatibility. Newly tailored peptide nucleic acids: azaPNA1 Peptide nucleic acid (PNA) is an artificial DNA mimic introduced by Nielsen in 1991, characterized by a pseudopeptide backbone (figure 1) replacing the sugar-phosphate chain. The backbone is made of N-(2-aminoethyl)glycine (aeg) units joint in a polyamide structure, and the purine (A, G) and pyrimidine (C, T) nucleobases are linked to the β-nitrogen atom of the amino acid unit through methylene carbonyl residues. The repeating unit is made of six atoms, exactly as in DNA and RNA. The PNAs suffer of some drawbacks such as low water solubility, the tendency to selfaggregate and low cell uptake. It is possible to change in many ways the monomer structure of the classic PNA in order to improve their physical-chemical properties. Our strategy consisted in introducing polar groups, such as nitrogen atom, in the backbone of aegPNA. Our hypothesis was that this would increase their solubility in aqueous medium by increasing their hydrophilicity. At the same time, this would improve their binding affinity towards DNA thanks to more favorable interactions (e.g. through possibly additional hydrogen bonds). Therefore, first we focused our attention on the synthesis of new PNA monomers that we called azaPNA (Figure 1). In these new molecules, the substitution of the glycine CH2 with an NH group confers them new chemical-physical properties. NH N O OR O B aegPNA B = nucleobase NH N NH O OR O B azaPNA PG PG Figure 1. Aminoethylglycine and azaPNA (aeg- and azaPNA) monomers. Retrosynthetic analysis of new azaPNA monomers 4-7 and 8-11 led us to the synthesis of the two backbones 12 and 13, on with we performed the coupling of nucleobases 14- 17 (Scheme 1). X Boc N H HN NH O OCH3(Fmoc) Boc N H N NH O OCH3(Fmoc) B O HO O B 4-7; (8-11) 12; (13) 14-17 4, 8: B NH N O O 5, 9: B N N N N NH N N NH O NH N N N O NH Cbz Cbz Cbz 6, 10: B 7, 17: B + Scheme 1. Retrosynthetic scheme for azaPNA monomers. The backbone 12 (with the COOMe group on the nitrogen atom of hydrazine) was prepared following the synthetic sequence shown in Scheme 2. Boc N H HN NH O OCH3 H2N OH NH Boc OH a > 98% 21 22 NH Boc O 23 Boc N H N NH O OCH3 b 83% c 90% d 69% 12 24 Scheme 2. Reagents: (a) (Boc)2O, EtOH; (b) (i) Dess-Martin periodinane, CH2Cl2; (ii) Na2S2O3, NaHCO3, H2O, CH2Cl2; (C) H2NNHCO2CH3, PhCH3; (d) NaBH3CN, MeOH, CH3COOH. Compounds 12 and 24 are new, and were completely characterised by means of spectroscopic data. Similarly, the azaPNA backbone 13 was synthesised following the strategy shown in Scheme 3. Boc N H HN NH H2N OH NH Boc OH a > 98% 21 22 NH Boc O 23 Boc N H N NH Fmoc b 85% c 90% d >98% Fmoc 13 25 Scheme 3. Reagents: (a) (Boc)2O, EtOH; (b) (i) Dess-Martin periodinane, CH2Cl2; (ii) Na2S2O3, NaHCO3, H2O, CH2Cl2; (C) H2NNHFmoc, PhCH3; (d) NaBH3CN, MeOH, CH3COOH. This time, we have used hydrazine-Fmoc to obtain the carbazone 25, that was then reduced by means of NaBH3CN to give the backbone 13. XI Finally, the target azaPNA monomers 4-7 and 8-11 were obtained in good yield by introducing the nucleobases onto the nitrogen atom of 12 and 13, using standard coupling condition (Schema 4). Boc N H HN NH O OCH3 Boc N H N NH O OCH3 B O HO O B 12 14-17 + a Boc N H HN NH Boc N H N NH Fmoc B O HO O B 13 14-17 + b Fmoc 4: B = Thymine 70% 5: B = Cytosine (Cbz) 57% 6: B = Adenine (Cbz) 61% 7: B = Guanine (Cbz) 68% 8: B = Thymine 63% 9: B = Cytosine (Cbz) 69% 10: B = Adenine (Cbz) 89% 11: B = Guanine (Cbz) 80% Scheme 4. (a) DhbtOH, DIPEA, EDC.HCl, DMF, 30 h, rt; (b) EDC.HCl, DMF, 5-10 h, rt. The new PNA monomers 4-7 and 8-11 are the building blocks necessary for the construction of azaPNA oligomers. First, it was necessary to find the appropriate conditions for methyl ester hydrolysis for the monomers 4-7 and Fmoc deprotection for the monomers 8-11. The ester group in compound 1 was hydrolysed very efficiently but in very strong conditions by means of aq lithium hydroxide at reflux to give the corresponding decarboxylate product 1 in good yield. (scheme 5). The new azaPNA 1 is stable, and it was completely characterised by means of spectroscopic data. NH Boc N NH OCH3 O O NH N NH OH O O B Boc NH N NH2 O B Boc -CO2 4, 8-11 1: B= Thymine 18: B= Cytosine 19: B= Adenine 20: B= Guanine B Scheme 5. Compounds 5-7 can not be deprotected at the ester group, because the unexpectedly strong basic hydrolysis conditions remove first the Cbz-group from nucleobase. The Fmoc group in compounds 8-11 was hydrolysed very efficiently and easily by means of a reaction with piperidine to give the corresponding decarboxylate monomers 18-20 in high yield. Once synthesized the azaPNA monomers 1, 18-20, we proceeded by introducing one or more azaPNA monomers in a oligomer of aegPNA. First we decided to introduce one monomer of aza in the middle of sequence of a decamer aegPNA. In order to do this, XII we synthesized the tetramer of aegPNA on the resin 51. Then we coupled the aza monomer 1 to the tetramer. We tried many different reaction conditions to form the ureic bond between the aza monomer and the tetramer 51. We obtained the best results through isocyanate. We preformed the isocyanate on the γNH2 of aegPNA 51 with COCl2 in toluene and then we added 1 in THF, to obtain the pentamer 25. The synthesis was completed by adding the last five aegPNA monomers by automated synthesizer, to afford the decamer 3 after the cleavage (scheme 6). NH O N H3 COCl2 N Toluene 15 min O NH N O O H3C CF3COONH O N NH O NH N O O H3C NH O N O NH Boc NH N O O H3C NH2 N O NH Boc NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C C O (aeg(T)PNA)4 (aza(T)PNA)-(aeg(T)PNA)4 THF 12 h, DIEA NH O N NH O NH N O O H3C NH O N O NH NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C NH2 O N O NH N O O H3C (aeg(T)PNA)5-(aza(T)PNA)-(aeg(T)PNA)4 NH O N NH O NH N O O H3C O N O NH NH N O O H3C NH O N O NH N O O H3C NH O N O NH N O O H3C O N O NH N O O H3C H3C O 51 53 3 1 Scheme 6 Similarly, we synthesized the decamer (not shown) with the sequence (TCACTAGATG), containing a monomer of azaPNA 1 in the middle of the sequence. The decamers were purified by reverse phase HPLC. The purity was checked by LCMS. Then we checked the solubility of the decamer (TCACTAGATG) and the ability to recognise a complementary sequence of DNA by measuring the melting temperature. PNA-modified magnetic nanoparticles for DNA targeting 2. The first generation of PNAs suffers from some drawbacks including low cell uptake and low solubility in physiological media. To overcome these problems, and to improve their physical–chemical properties, many modified PNAs have been synthesized in recent years following different strategies. In this context, superparamagnetic iron oxide nanoparticles (MNP) attracted our attention because of their unique magnetic properties, which can be controlled rigorously and activated easily by applying an external magnetic field. In this thesis we have set up an effective synthetic platform for the development of monomer and decamer PNA-nanoconjugates, starting from synthetic PNA and nanometer-sized maghemite. The sequence-selective DNA recognition and sequestration ability of the resulting magnetic PNA (MPNA) were assessed according XIII to their capability of enhancing the T2 relaxation response in aqueous solutions under conventional hybridization conditions with complementary DNA. We have used nearly homogeneous commercial γ-Fe2O3 nanoparticles (10 ± 4 nm), approximately spherical in shape. We chose the thymine monomer as a model PNA building unit. First, we designed a small library of PNA monomer derivatives endowed with three different appropriate linkers useful for MNP conjugation. The trialkoxysilane, the carboxyl, and the propargylic groups are all suitable linkers to stably connect biomolecules to MNP through the formation of different kinds of linkages. Such linkers, however, have never been applied to PNA for MNP conjugation. aegPNA HN Si Si N3 + + + 72, 81 O O 74, 83 73, 82 Si aegPNA O HN Si EtO EtO OEt aegPNA O NH 77, 87 78, 90 79, 89 aegPNA in: NH N O NH N O O Me OMe O NH N O NH N O O Me NH2 O 10 72-74, 77-79 81-83, 87, 89-90 O HO O 37 NH N O NH N O O Me Boc 37, 76 NH O aegPNA O O aegPNA O O aegPNA 76 + aegPNA O O HO O aegPNA N N N gFe2O3 gFe2O3 gFe2O3 gFe2O3 Scheme 7 Scheme 7 illustrates the conjugation strategies exploited for the preparation of MPNA monomers and decamers. Monomers 37 and 72 were able to interact directly with nanoparticles through their carboxylate linkers. Similarly, compound 74 could be anchored to MNP effectively as a consequence of the high affinity of siloxane group for iron oxide. The functionalized nanoparticles were isolated by centrifugation and carefully washed affording 76-78. The propargyl-terminating monomer 73, was instead conjugated to the nanoparticles via a Cu(I)-catalyzed azide-alkyne click reaction with XIV a MNP-silylpropylazide adduct, affording 79. MNP-silylpropylazide was prepared according to our recently developed procedure. The organic structures of magnetic products 76-79 were completely characterized by FT-IR (bulk in KBr) and by high-resolution magic angle spinning (HR-MAS) NMR. Inductively coupled plasma spectroscopy (ICP-OES) analysis gave the content of iron isotopes in a sample of functionalized MNP, whereas EA provided quantitative data on the amount of organic material in the sample. In the same way we have prepared homo-thymine 10-mer PNA supported on NPs to afford the 87, 89 and 90. MPNA nanohybrids 87, 89 and 90 were characterized by FTIR, demonstrating the presence of PNA on nanoparticles, while EA and ICP-OES provided information on the average amount of PNA decamers 81-83 on the nanoparticles. Once a reliable, multiple-approach synthetic platform for the production of MPNA was optimized, the next objective was to determine the capability of these MPNA to recognize and bind the complementary DNA. On the basis of the fact that the formation of magnetic aggregates would result in a detectable increment in the T2 relaxation time of water protons, we decided to determine whether a variation of T2 of MPNA could occur under MPNA/DNA hybridization conditions, and could be sensitively detected by relaxometric analysis. Our preliminary results demonstrate that these MPNA maintain excellent performances in PNA/DNA hybridization events, which can be evidenced in a new way, through the measurement of variation of the T2 relaxation time of water dispersions in the presence of complementary DNA and by measurement of Tm. As mentioned, the NPs-PNA described above, is weakly soluble in water. A good solubility in water is necessary for the NPs-PNA conjugates to be used in practical domains, such as the MRI imaging or in vivo applications. In order to increases the hydrophilicity we conjugated NP-PNA with the biocompatible water soluble polyvinyl pyrrolidone carboxilic acid (PVPCOOH) polymer (4.500 Da). In particular, we used the nanoparticles cover with 3-aminopropyl silane (NPs-NH2), witch can be treated with an excess of PVPCOOH and sonicated for 30 minutes in presence of EDC chloridrate, in order to connect the polymer to the construct NP-NH2 by a covalent bond. In this way we achieved a very stable and high concentrated solution of NPs in water. We tried to repeat the reaction of functionalization of NPs-NH2 with PVP and PNA at the same time. After several attempts, we managed to find the right ratio of PVP/PNA in order to obtain a stable solution of NPs-PNA-PVP. PEG branched polymer chains functionalization of nano-systems for biocompatibility3-4. Introduction: I spent my II year Ph.D. thesis at Stanford University in the lab of Professor Hongjie Dai. I have worked on: Various novel nanomaterials have been actively pursued for biogolical and medical applications in recent years. XV Poly-γ-glutamic acid (γPGA) is a naturally occurring bio-material, produced by microbial fermentation. γPGA is water soluble, biodegradable, nontoxic, and even edible. As a result, it is promising for various applications, and has recently attracted considerable interest for biomedical applications such as drug delivery. We thus envisioned γPGA as a polymeric backbone in the synthesis of a new amphiphilic polymer capable of suspending nanostuctures of many shapes and aspect ratios. In particular, we envisioned that the free carboxylic acid of γPGA would first be used to attach lipophilic groups for robust particle interaction, while the remaining carboxylic acids would be conjugated with PEG, providing enhanced aqueous solubility and better biocompatibility. Pyrene-containing and phosfo-lipds-coating moieties have been used extensively for suspension of carbon nanotubes, gold nanoparticles and semiconductor quantum dots due to strong surface interactions via Van der Waals forces, π-π overlap, charge transfer, and/or hydrophobic interactions. Thus, in the first synthetic step (see Method), we used the free carboxylic acid of γPGA to couple 1-methylaminopyrene via EDC amidation, and in a second step, we used the remaining carboxylic acid groups of γPGA to attach primary amine-terminated poly(ethylene glycol) methyl ethers (mPEG-NH2, MW 5000). This synthetic strategy proved to be quite general. Long aliphatic amines (C18, C12) were coupled via the same procedure to obtain additional amphiphilic gPGA-based polymers. Moreover, by varying the amount of hydrophobic molecule and PEG, the properties of the polymer could be tuned to optimize aqueous stability and protein adsorption resistence. After much experimentation, the optimal polymer to obtain stable suspensions was found to contain 30% pyrene, while the remaining 70% of carboxylic acids were loaded with PEG to yield a water soluble, amphiphilic polymer. In the case of PGA with DSPE, the optimal polymer to obtain stable suspensions was found to contain 10% of DSPE, while the remaining 60% of carboxylic acids were loaded with PEG and 30% of free carboxylic acids, to yield a water soluble, amphiphilic polymer. O NH O NH O O O n HN NH O NH OO O NH O O n O 30% HN NH HN NH O O NH OO O O NH O O O n O 10% 30% O P OO O O O O O O O O n n HN 70% O 60% OH PMHC18-mPEG (118) PGA-DSPE-mPEG (114) PGA-Py-mPEG (109) Figure 2 XVI We also use the amphiphilic polymer base on poly(maleic anhydride-alt-1-octadecene) (PMHC18) like a backbone. In this case the polymer itself contein the idrofobic unit, the C18 chain, so in this case we need to do only the PEGylation step. In particular, in the first synthetic step, we used to open the anhydride with primary amine-terminated poly(ethylene glycol) methyl ethers (mPEG-NH2, 5KDa), then we used the second carboxylic acid of PMHC18 to couple another moleculer mPEG-NH2 via EDC amidation to obtain a full PEGylated polymer very high soluble in water. As Figure 3 demonstrat, following sonication, it is possible obtain direct suspension of bulk NTs stabilized in water with the polymer PGA-Py-mPEG, DSPE-PGA-mPEG and PMHC18-mPEG, even after removal of excess polymer by vacuum filtration. NT suspensions demonstrated excellent stability at pH’s ranging from 1 to 12, at 70°C overnight, and in 50% fetal calf serum for 48 h. The AFM (figure 3b) image inset in the figure shows mostly dispersed, single NTs. The UV/visible absorbance spectrum of methoxyPEGylated pyrene-γPGA NTs, methoxyPEGylated DSPE-γPGA NTs and methoxyPEGylated PMHC18 NTs are typical of well dispersed SWNTs, demonstrating van Hove singluarity resonances, as well as pyrene absorption below 400 nm. In the casa of pyrene moiety has a strong tendency to adsorb on SWNTs by π-stacking and hydrophobic interactions in aqueous media. In this way, we obtiain three robust polymers coating SWNTs. Figure 3 Also, very good suspensions of gold NPs in water were obtained through sonication for 10 min in presence of excess pyrene/PEG-γPGA or DSPE/PEG-γPGA to displace citrate. As with NTs, this suspension was observed to be stable to conditions ranging pH , at 70°C overnight, and in 50% serum for 48 h. In contrast, thiol-mPEG(5KDa), a strong and covalent passivator of gold nanoparticles, showed less stability. In particular, as shown in figure 4c, the NPs-thiol-PEG(5KDa) are stable only with the excess of thiol-PEG, indeed if the excess of the thiol-PEG is remuved by centrifugation the solution of NPs become anstable and they form agragate (purpe solution). The UV/visible spectrum in Figure 4b shows the absorbance of gold nanoparticles at 550 nm, as well as the peaks of pyrene. XVII Figure 4 We also used our polymeric amphiphile to suspend gold nanorods. The procedure gave mostly dispersed nanorods, as shown by TEM and UV. Suspensions of gold nanorods in pyrene/PEG-γPGA were stable at neutra base pHs at 70°C overnight, and in serum for 48 h. This result is important because nanorods with covalent thiol-based passivation are anstable. The UV/visible spectrum in figure 3b shows the transverse and longitudinal adsorbance of gold nanorods at 520 nm and 860 nm respectively. To further demostrate the versatility of pyrene/PEG-γPGA and DSPE/PEG-γPGA, we successfully suspended InAs/InP/ZnSe core/shell/shell quantum dots. We found that the lability of the cysteamine capping layer allowed it to be easily exchanged for pyrene/PEG-γPGA or DSPE/PEG-γPGA. InAs/InP/ZnSe core/shell/shell quantum dots were synthesized by previously reported methods in organic solvent. The particles were then transferred from chloroform to water using cysteamine, which was then replaced by γPGA surfactant by dialyzing against a 3500 MWCO membrane. This suggests that the pyrene/PEG-γPGA and DSPE/PEG-γPGA are able to displace the cysteamine coating to stabilize the quantum dots. Again, these suspensions were were stable at differents neutral-base pH , at 70°C overnight, and in serum for 48 h. This is an important result as quantum dots are susceptible to surface oxidation and instability in harsh environments. Figure 6b shows the absorption of InAs/InP/ZnSe quantum dots with excess removed and in 50% serum. No significant loss of quantum yield was observered in the quantum dots upon addition to serum. 1 Giuseppe Prencipe; Paolangelo Cerea; Anna Daghetti; Sergio Dall’Angelo; Clelia Giannini; Emanuela Licandro and Stefano Maiorana. Aza-peptide nucleic acid (azaPNA) monomers: building blocks for the construction of nitrogenenriched PNA oligomers. Manuscript in preparation. 2 Giuseppe Prencipe; Stefano Maiorana; Paolo Verdelio; Miriam Colombo; Paola Fermo; Enrico Caneva; Davide Prosperi and Emanuela Licandro. Magnetic Peptide Nucleic Acids for DNA targeting. ChemComm (2009), 40, 6017 – 6019. 3 Giuseppe Prencipe; Scott Tabakman; Kevin Welsher; Zhuang Liu; Andrew Goodwin; Li Zhang; Joy Henry and Hongjie Dai. PEG Branched Polymer for Functionalization of Nanomaterials with Ultralong Blood Circulation. Journal of the American Chemical Society (2009), 131 (13), 4783-4787. XVIII 4 Andrew P. Goodwin; Scott M. Tabakman; Kevin Welsher; Sarah P. Sherlock; Giuseppe Prencipe and Hongjie Dai. Phospholipid−Dextran with a Single Coupling Point: A Useful Amphiphile for Functionalization of Nanomaterials. Journal of the American Chemical Society (2009), 131, 289-296.

Interactions between peptides and biological lipid membranes play a crucial role in many cellular processes such as in the mechanism behind Alzheimer’s disease where amyloid-beta peptide (Abeta)is thought to be a key component. The initial step of binding between a surface active peptide and its target membrane or membrane receptor can involve a non specific electrostatic association where positively charged amino acid residues and a negatively charged membrane surface interact. Here, the use of high resolution MAS NMR provides a highly sensitive and non perturbing way of studying the electrostatic potential present at lipid membrane surfaces and the changes resulting from the association of peptides. The interaction between pharmacologically relevant peptides and lipid membranes can also involve incorporation of the peptide into the membrane core and by complementing the NMR approach with differential scanning calorimetry (DSC) the hydrophobic incorporation can be studied in a non invasive way. By using 14N MAS NMR on biological lipid systems for the first time, in addition to 31P, 2H NMR and differential scanning calorimetry (DSC), gives a full picture of the changes all along the phospholipid following interactions at the membrane interface region. Being able to monitor the full length of the phospholipid enables us to differentiate between interactions related to either membrane surface association or hydrophobic core incorporation. This approach was used to establish that the interaction between nociceptin and negatively charged lipid membranes is electrostatic and hence that nociceptin can initially associate with a membrane surface before binding to its receptor. Also, it was found that Abeta can interact with phospholipid membranes via two types of interactions with fundamentally adverse effects. The results reveal that Abeta can associate with the surface of a neuronal membrane promoting accelerated aggregation of the peptide leading to neuronal apoptotic cell death. Furthermore it is also shown that Abeta can anchor itself into the membrane and suppress the neurotoxic aggregation of Abeta.