Peptide dendrimers based on polyproline helices

We present a new family of peptide dendrimers based on polyproline helices and cis-4-amino-L-proline as a branching unit. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational transition between polyproline type I helix and polyproline type II helix was observed by circular dichroism in branched polyproline building blocks with more than 14 proline residues and in the resulting dendrimers. Both linear and dendritic polyprolines were found to be actively internalized by rat kidney cells. Preliminary results show that the antibiotic ciprofloxacin form complexes with branched polyproline chains in 99.5% propanol.     

Peptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications

Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ~ 5,000 Da). Larger peptide dendrimers (MW ~ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat) ~ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobial peptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.     

Convergent Synthesis of Repeating Peptides (Val-X-Leu-Pro-Pro-Pro)(8) Adopting a Polyproline II Conformation

The N-terminal domain of maize gamma-zein has a repetitive structure (Val-His-Leu-Pro-Pro-Pro)(8) that has recently been shown to adopt an amphipathic polyproline II type conformation in aqueous solution. We report here the synthesis and conformational analysis of three model peptides (Val-X-Leu-Pro-Pro-Pro)(8) (X = Ala (1), Glu (2), Lys (3)). The three compounds have been synthesized in a very efficient way using a convergent solid-phase strategy. Circular dichroism shows unequivocally that the three model peptides adopt polyproline II (PPII) type conformations under a variety of experimental conditions and that neither the presence of histidine nor amphipathicity of the peptide is an absolute requirement for adopting the native conformation. These results open the door for the de novo design of compounds with PPII conformations and must be taken into account in the structure prediction of protein structures from sequence data banks.     

Triple‐Helix‐Stabilizing Effects in Collagen Model Peptides Containing PPII‐Helix‐Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C₂ bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Peptide‐Decorated Dendrimers and Their Bioapplications

Peptide‐decorated dendrimers (PDDs) are a class of spherical, regular, branched polymers that are modified by peptides covalently attached to their surface. PDDs have been used as protein mimetics, novel biomaterials, and in a wide range of biomedical applications. Since their design and development in the late eighties, poly‐l ‐lysine has been a preferred core structure for PDDs. However, numerous recent innovations in polymer synthesis and ligation chemistry have re‐energized the field and led to the emergence of well‐defined peptide dendrimers with more diverse core structures and functions. This Minireview highlights the development of PDDs driven by significantly improved ligation chemistry incorporating structurally well‐defined peptides and the emerging use of PDDs in imaging and drug development.     

Conformational preferences of proline oligopeptides

A conformational study on the terminally blocked proline oligopeptides, Ac-(Pro)(n)()-NMe(2) (n = 2-5), is carried out using the ab initio Hartree-Fock level of theory with the self-consistent reaction field method in the gas phase and in solutions (chloroform, 1-propanol, and water) to explore the preference and transition between polyproline II (PPII) and polyproline I (PPI) conformations depending on the chain length, the puckering, and the solvent. The mean differences in the free energy per proline of the up-puckered conformations relative to the down-puckered conformations for both diproline and triproline increases for the PPII-like conformations and decreases for the PPI-like conformations as the solvent polarity increases. These calculated results indicate that the PPII-like structures have preferentially all-down puckerings in solutions, whereas the PPI-like structures have partially mixed puckerings. The free energy difference per proline residue between the PPII- and PPI-like structures decreases as the proline chain becomes longer in the gas phase but increases as the proline chain becomes longer in solutions and the solvent polarity increases. In particular, our calculated results indicate that each of the proline oligopeptides can exist as an ensemble of conformations with the trans and cis peptide bonds in solutions, although the PPII-like structure with all-trans peptide bonds is dominantly preferred, which is reasonably consistent with the previously observed results. In diproline Ac-(Pro)(2)-NMe(2), the rotational barrier to the cis-to-trans isomerization for the first prolyl peptide bond increases as the solvent polarity increases, whereas the rotational barrier for the second prolyl peptide bond does not show the monotonic increase as the solvent polarity increases. When the rotational barriers for these two prolyl peptide bonds were compared, it could be deduced that the conformational transition from PPI with the cis peptide bond to PPII with the trans peptide bond is initiated at the C-terminus and proceeds to the N-terminus in water. This is consistent with the results from NMR experiments on polyproline in D(2)O but opposite to the results from enzymatic hydrolysis kinetics experiments on polyproline.     

A 10-A spectroscopic ruler applied to short polyprolines

Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1-6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short F?rster radius (R0 ca. 9 A), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n > or = 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2-72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor-acceptor distances between ca. 8 A (n = 1) and ca. 16 A (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30-70 A in water and 220 +/- 40 A in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor-acceptor distances extracted by FRET are correlated with the F?rster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in F?rster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation.     

结合丝组二肽的树状多肽对λDNA的切割

以Fmoc手工固相合成法合成了以多聚赖氨酸为骨架, 表面结合丝组二肽的四分支和二分支树状多肽, 以高效液相色谱提纯, 电喷雾电离质谱表征, 并通过凝胶电泳法研究了其对λDNA的切割活性.     

Characteristic fragmentations of peptide derivatives containing proline in negative-ion fast atom bombardment mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters containing proline were compared with those of the corresponding peptide derivatives not containing proline in negative-ion fast atom bombardment mass spectrometry. The fragment ion [M – 109]^? due to loss of the benzyloxy group followed by dehydrogenation from the peptide molecule was the base peak in the negative-ion mass spectra for the peptides not containing proline, whilst it was a very weak fragment ion or not observed at all in those for the peptides containing proline. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational difference of the peptide derivatives owing to the existence of proline in the derivative.     

Conformational manifold of alpha-aminoisobutyric acid (Aib) containing alanine-based tripeptides in aqueous solution explored by vibrational spectroscopy, electronic circular dichroism spectroscopy, and molecular dynamics simulations

Replacement of the alpha-proton of an alanine residue to generate alpha-aminoisobutyric acid (Aib) in alanine-based oligopeptides favors the formation of a 3(10) helix when the length of the oligopeptide is about four to six residues. This research was aimed at experimentally identifying the structural impact of an individual Aib residue in an alanine context of short peptides in water and Aib's influence on the conformation of nearest-neighbor residues. The amide I band profile of the IR, isotropic and anisotropic Raman, and vibrational circular dichroism (VCD) spectra of Ac-Ala-Ala-Aib-OMe, Ac-Ala-Aib-Ala-OMe, and Ac-Aib-Ala-Ala-OMe were measured and analyzed in terms of different structural models by utilizing an algorithm that exploits the excitonic coupling between amide I' modes. The conformational search was guided by the respective 1H NMR and electronic circular dichroism spectra of the respective peptides, which were also recorded. From these analyses, all peptides adopted multiple conformations. Aib predominantly sampled the right-handed and left-handed 3(10)-helix region and to a minor extent the bridge region between the polyproline (PPII) and the helical regions of the Ramachandran plot. Generally, alanine showed the anticipated PPII propensity, but its conformational equilibrium was shifted towards helical conformations in Ac-Aib-Ala-Ala-OMe, indicating that Aib can induce helical conformations of neighboring residues positioned towards the C-terminal direction of the peptide. An energy landscape exploration by molecular dynamics simulations corroborated the results of the spectroscopic studies. They also revealed the dynamics and pathways of potential conformational transitions of the corresponding Aib residues.     

Proline editing: a divergent strategy for the synthesis of conformationally diverse peptides

[reaction: see text] Strong conformational biases in peptides and proteins can be achieved with 4-substituted proline residues (cis-, trans-, or disubstituted fluoroproline or hydroxyproline). The practical, divergent synthesis of peptides containing these residues, via postsynthetic modification of a peptide containing an internal trans-hydroxyproline residue, is described. Significant differences in the conformations of the peptides Ac-TYXN-NH2 were observed, including K(trans/cis) values, which varied from 1.5 (X = cis-fluoroproline) to 7.0 (X = trans-fluoroproline).     

Direct observations of conformational distributions of intrinsically disordered p53 peptides using UV Raman and explicit solvent simulations

We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27S mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond time scale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection.     

The conformation of tetraalanine in water determined by polarized Raman, FT-IR, and VCD spectroscopy

The present article reports the conformation of cationic tetraalanine in aqueous solution. The determination of the dihedral angles of the two central amino acid residues was achieved by analyzing the amide I' band profile in the respective polarized visible Raman, Fourier transform-IR, and vibrational circular dichroism (VCD) spectra by means of a novel algorithm which utilizes the excitonic coupling between the amide I modes of nearest neighbor and second nearest peptide groups. It is an extension of a recently developed theory (Schweitzer-Stenner, R. Biophys. J., 2002, 83, 523-532). UV electronic circular dichroism (ECD) spectra of the peptides were used to validate the results of the structure analysis. The analyses yielded the dihedral angles (phi(12), psi(12)) = (-70 degrees, 155 degrees ) and (phi(23), psi(23)) = (-80 degrees, 145 degrees ). The obtained values are very close to the Ramachandran coordinates of the polyproline II helix (PPII). The data suggest that this is the conformation predominantly adopted by the peptide at room temperature. This notion was corroborated by the corresponding electronic circular dichroism spectrum. Tetraalanine exhibits a higher propensity for PPII than trialanine for which a 50:50 mixture of polyproline II and an extended beta-strand-like conformation was obtained from recent spectroscopic studies (Eker et al., J. Am. Chem. Soc. 2002, 124, 14330-14341). The temperature dependence of the CD spectra rule out that any cooperativity is involved in the strand if PPII transition. This led to the conclusion that solvent-peptide interactions give rise to the observed PPII stability. Our result can be utilized to understand why the denaturation of helix-forming peptides generally yields a PPII rather than a heterogeneous random conformation.     

Conformational analysis of XA and AX dipeptides in water by electronic circular dichroism and 1H NMR spectroscopy

We measured the temperature-dependent electronic circular dichroism (ECD) spectra of AX, XA, and XG dipeptides in D2O. The spectra of all XA and AX peptides indicate a substantial population of the polyproline II (PPII) conformation, while the ECD spectra of LG, KG, PG, and AG were found to be quantitatively different from the alanine-based dipeptides. Additional UV absorption data indicate that the ECD spectra of the XG peptides stem from electronic coupling between the peptide and the C-terminal group, and that spectral differences reflect different orientations of the latter. We also measured the 1H NMR spectra of the investigated dipeptides to determine the 3JHalphaNH coupling constants for the C-terminal residue. The observed temperature dependence of the ECD spectra and the respective room-temperature 3JHalphaNH coupling constants were analyzed by a two-state model encompassing PPII and a beta-like conformation. The PPII propensity of alanine in the XA series is only slightly modulated by the N-terminal side chain, and is larger than 50%. As compared to AA, XA peptides containing L, P, S, K V, E, T, and I all cause a relative stabilization of the extended beta-strand conformation. The PPII fractions of XA peptides varied between 0.64 for AA and 0.58 for DA, whereas the PPII fractions of AX peptides were much lower. From the investigated AX peptides, only AL and AQ showed the expected PPII propensity. We found that AT, AI, and AV clearly prefer an extended beta-strand conformation. A quantitative comparison of AA, AAA, and AAAA revealed a hierarchy AAAA > AAA approximately AA for the PPII population, in agreement with predictions from MD calculations and results from Raman optical activity studies (McColl et al. J. Am. Chem. Soc. 2004, 126, 5076).     

A new strategy for synthesis of branched cyclic peptide by Asn side-chain hydrazide ligation

Here, we report a new strategy for rapid synthesis of branched peptide by side-chain hydrazide ligation at Asn. The hydrazide was converted to thioester at Asn side chain by NaNO₂ and thiol reagent, and sequential ligation with an N-terminus Cys-peptide efficiently afforded the branched peptide. A branched cyclic peptide was successfully synthesized by side-chain ligation with a two-Cys-peptide and formation of a disulfide bond. This approach provides a new way for expeditious synthesis of branched peptides and facilitates the design of neopeptides as functional bio-mimics.     

High stability of the polyproline II helix in polypeptide bottlebrushes

Polymer bottlebrushes with monodisperse oligoproline side chains were efficiently synthesized, and the conformation of the peptide side chains in different solvents was investigated. Polymers with number-average degrees of polymerization (DPn) of 89 and 366 were obtained by polymerization of the macromonomer in iPrOH/MeCN (1:1) and hexafluoroisopropanol, respectively. Circular dichroism (CD) spectra of the bottlebrush polymers in the neutral and charged states reveal that the oligoproline side chains attain stable polyproline II (PPII) helical conformations not only in aqueous solution, but also in aliphatic alcohol solutions. Dense attachment of oligopeptides onto a linear polymer chain did not lead to an increase in helix content. The possible effects of the main-chain length on the conformational stability were examined. The switching between the polyproline I (PPI) and PPII helical conformations for the oligoproline side chains in aliphatic alcohol solutions is believed to be inhibited by the overcrowded structure in the polymer bottlebrushes.     

Unfolding the conformational behavior of peptide dendrimers: insights from molecular dynamics simulations

We present here the first comprehensive structural characterization of peptide dendrimers using molecular simulation methods. Multiple long molecular dynamics simulations are used to extensively sample the conformational preferences of five third-generation peptide dendrimers, including some known to bind aquacobalamine. We start by analyzing the compactness of the conformations thus sampled using their radius of gyration profiles. A more detailed analysis is then performed using dissimilarity measures, principal coordinate analysis, and free energy landscapes, with the aim of identifying groups of similar conformations. The results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviors were found: one of the dendrimers displayed mostly compact conformations clustered into distinct basins (rough landscape), while the remaining dendrimers displayed mainly noncompact conformations with no significant clustering (downhill landscape). This study brings new insight into the conformational behavior of peptide dendrimers and may provide better routes for their functional design. In particular, we propose a yet unsynthesized peptide dendrimer that might exhibit enhanced ability to coordinate aquocobalamin.     

Ring-opening cross-metathesis (ROCM) as a novel tool for the ligation of peptides

The development of ring-opening cross-metathesis (ROCM) as a novel tool for the site-specific ligation of peptide units is reported. The resulting structural units at the site of ligation resulting from ROCM resemble proline as well as other known beta-turn stabilising structural units. ROCM under mild reaction conditions between a variety of peptides bearing a cyclic olefin with amino acids or peptides results in high yields. The peptidic cross-partners for metathesis are equipped with double bonds via the N and the C terminus and the side chain, respectively, to allow the synthesis of linear as well as non-linear and branched peptides. The ligation in this manner succeeds with low catalyst loadings, with no need for any excess of one reaction partner and with a high compatibility with a wide range of functional groups. Furthermore, the stereochemical outcome of the ROCM can easily be controlled by using a Hoveyda-type chiral catalyst. Fluorescence labelling of peptides is possible in the same manner when using a cyclic olefin equipped with a fluorescence marker.     

Synthesis of homopolypeptides with PPII structure

Polyprolines are attractive polymers because of their folding property into polyproline II (PPII) structure, their significance in protein/protein interactions, and their potential as new therapeutic targets. Silaproline (Sip) is an analogue of proline, which exhibits similar conformational properties. The presence of dimethylsilyl group confers to Sip a higher lipophilicity as well as an improved resistance to biodegradation. Enantiomerically pure Sip was available in gram quantities from resolution of the enantiomers by chiral high performance liquid chromatography. This study describes the first synthesis of Sip N‐carboxyanhydride (NCA) and shows preliminary results on comparison of polymerization of (l )Pro‐NCA and (d )Sip‐NCA to obtain homopolypeptides with PPII structure, polyproline, and polysilaproline polymers. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3103–3109