Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

A safety catch linker for Fmoc-based assembly of constrained cyclic peptides.

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities.     

Solid phase synthesis of an amphiphilic peptide modified for immobilisation at the C-terminus

The solid phase peptide synthesis (SPPS) of the amphiphilic peptide Ac-(Leu-Ala-Arg-Leu)(3)-linker, which is modified at the C-terminus with 1,8-diamino-3,6-dioxaoctane as linker moiety, has been investigated. Two different approaches that allow for the synthesis of C-terminally modified, side-chain protected peptides were examined. The solid phase peptide synthesis using aliphatic safety-catch resin followed by activation and aminolysis with the mono-Boc protected linker was compared with the synthesis on 1,8-diamino-3,6-dioxaoctane loaded 2-chlorotrityl resin.     

A practical approach to the synthesis of hairpin polyamide-peptide conjugates through the use of a safety-catch linker

Hairpin polyamides are high-affinity, sequence selective DNA binders. The use of a safety-catch linker for the solid phase synthesis of hairpin polyamides allows for easy preparation of derivatives ready for chemoselective ligation with unprotected peptides. Examples of ligations reported include thioether bond formation and thioester-mediated amide bond formation ('Native Chemical Ligation').     

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

Solid phase synthesis and antiprotozoal evaluation of di- and trisubstituted 5'-carboxamidoadenosine analogues

The rapid increase of resistance to drugs commonly used in the treatment of tropical diseases such as malaria and African sleeping sickness calls for the prompt development of new safe and efficacious drugs. The pathogenic protozoan parasites lack the capability of synthesising purines de novo and they take up preformed purines from their host through various transmembrane transporters. Adenosine derivatives constitute a class of potential therapeutics due to their selective internalisation by these transporters. Automated solid-phase synthesis can speed up the process of lead finding and we pursued the solid-phase synthesis of di- and trisubstituted 5'-carboxamidoadenosine derivatives by using a safety-catch approach. While efforts with Kenner's sulfonamide linker remained fruitless, successful application of the hydrazide safety-catch linker allowed the construction of two representative combinatorial libraries. Their antiprotozoal evaluation identified two compounds with promising activity: N(6)-benzyl-5'-N-phenylcarboxamidoadenosine with an IC(50) value of 0.91 microM against Trypanosoma brucei rhodesiense and N(6)-diphenylethyl-5'-phenylcarboxamidoadenosine with an IC(50) value of 1.8 microM against chloroquine resistant Plasmodium falciparum.     

Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

Random glycopeptide bead libraries for seromic biomarker discovery

Identification of disease-specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I(2)/NaBH(4) and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MS(n). As proof-of-principle, tumor -specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor-specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were resynthesized at the preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid discovery of O-glycopeptide biomarkers and the method has applicability in other types of assays such as lectin/antibody/enzyme specificity studies as well as investigation of other PTMs.     

Calcium coordination studies of the metastatic Mts1 protein

Mts1, also known as S100A4, is an 11 kDa calcium-binding protein strongly linked to metastasis. As a member of the S100 protein family, Mts1 is predicted to contain four alpha-helices and two calcium-binding loops, the second of which forms a canonical EF hand, while the first is a pseudo-EF hand, using two extra residues and principally backbone carbonyls rather than side chain oxygens to coordinate calcium. Here we follow chemical shift changes which occur in Mts1 upon titration of calcium. The results are consistent with calcium coordination by the EF hands described above. Filling of the first (pseudo) EF hand occurs at a lower calcium concentration than does filling of the second (canonical) EF hand. Concurrent with filling of site I, resonances from much of helix 4 vanish while the chemical shifts of a possibly nascent helical segment immediately C-terminal to helix 4 increase in helical character. Other smaller changes are seen, including a change in the linker joining helix 2 and helix 3. Since binding of effector molecules to S100 proteins has been shown to involve the C-terminus and linker regions, these calcium-induced changes have implications for the role of Mts1 in metastasis.     

Inhibitors of adenosine consuming parasites through polymer-assisted solution phase synthesis of lipophilic 5′-amido-5′-deoxyadenosine derivatives

Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed. Thus, a series of 20 adenosine derivatives with a large lipophilic substituent in N⁶-position were prepared in order to evaluate their potential to inhibit the chloroquine resistant Plasmodium falciparum strain K1 in vitro. The rationale for synthesis of these structures was the high probability of interactions with multiple adenosine associated targets and the assumption that a large hydrophobic N⁶-(4-phenoxy)benzyl substitution should allow the molecules to diffuse across parasite membranes. Starting from readily available inosine, the new compounds were prepared as single isomers using a polymer-assisted acylation protocol enabling the straightforward isolation of the target compounds in pure form. Heterocyclic ring systems were synthesized on-bead on Kenner’s safety-catch linker prior to acylation of the scaffold in solution. Most of the highly pure compounds displayed anti-plasmodial activity in the low micromolar or even submicromolar concentration range.     

Secondary structure and dynamics of an intrinsically unstructured linker domain

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psi dihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Secondary Structure and Dynamics of an Intrinsically Unstructured Linker Domain

Abstract

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psidihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi Xylanase Cex through NMR spectroscopic analysis

The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated ( approximately 47 kDa) and glycosylated ( approximately 51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear (1)H-(15)N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct (15)N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulose-binding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.     

Altered dynamics may drift pathological fibrillization in membraneless organelles

Protein phase transition can generate non-membrane bound cellular compartments, which can convert from liquid-like to solid-like states. While the molecular driving forces of phase separation have been largely understood, much less is known about the mechanisms of material-state conversion. We apply a recently developed algorithm to describe the weak interaction network of multivalent motifs, and simulate the effect of pathological mutations. We demonstrate that linker dynamics is critical to the material-state of biomolecular condensates. We show that linker flexibility/mobility is a major regulator of the weak, heterogeneous meshwork of multivalent motifs, which promotes phase transition and maintains a liquid-like state. Decreasing linker dynamics increases the propensity of amyloid-like fragments via hampering the motif-exchange and reorganization of the weak interaction network. In contrast, increasing linker mobility may compensate rigidifying mutations, suggesting that the meshwork of weak, variable interactions may provide a rescue mechanism from aggregation. Motif affinity, on the other hand, has a moderate impact on fibrillization. Here we demonstrate that the fuzzy framework provides an efficient approach to handle the intricate organization of membraneless organelles, and could also be applicable to screen for pathological effects of mutations.     

Physico-chemical analysis of G-quadruplex containing bunch-oligonucleotides

A growing number of evidences suggest that DNA G-quadruplex structures play an important role in many relevant biological processes. The introduction of chemical modifications in quadruplex structures could enhance the in vivo biological activity. The correlation between the physico-chemical properties and chemical modifications represents an essential step toward the de novo design of quadruplex forming oligonucleotides for biomedical applications. We report the physico-chemical characterisation of a quadruplex formed by a bunch of four d(TG4T) oligonucleotides whose 3'-ends are linked together by a tetra-branched linker. The study was performed by circular dichroism, gel electrophoresis and molecular modelling techniques. The data indicate an high stability for this kind of quadruplex and add some information on the role of the tetra-branched linker on the quadruplex stability.     

The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity

Initiation factor IF3 is responsible for the accuracy of translation initiation in bacteria, by destabilizing complexes involving non-initiator tRNA and/or nonstart codons. This proofreading is performed on the 30S subunit to which IF3 binds selectively. IF3 has an unusual architecture, with two globular domains connected by a mobile, positively charged linker. Here, we have investigated the function of this flexible tether by probing its conformation when IF3 is bound to the ribosomal RNA. Using site-directed mutagenesis of the linker region, we have also selectively modified its length, its flexibility and its chemical composition. The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro. The two isolated domains of IF3 were also co-expressed in order to test the requirement for their covalent attachment. The results indicate that the physical link between the two domains of IF3 is essential for the function of this protein, but that the exact length and chemical composition of the linker can be varied to a large extent. A model is presented in which the extended linker would act as a 'strap', triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.     

Regulation of the higher-order structure of chromatin by histones H1 and H5

Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.