2-chlorotrityl Chloride Resin Research Articles

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII-Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558-565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa-Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.

English

Naturally isolated cyclic pentapeptide Longicalycinin A, which showed cytotoxicity to Hep G2 cancer cell line with an IC50 value 13.52 μg/mL, has been successfully synthesized by solid-phase methodology with Fmoc/t-Bu protecting schemes via solution-phase macrocyclization. 2-chlorotrityl chloride resin was used as solid support. Solution phase macrocyclization of linear pentapeptide precursor was carried out by two different routes with mild and severe acidic conditions correspondingly and found that percentage yield of Longicalycinin A under mild acidic condition (33%) was better than severe acidic conditions (13.7%). In addition, the cytotoxicity against Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) cell lines with CTC50 values were found to be 2.62 and 6.17 µM respectively.   Key words: Longicalycinin A, solid phase synthesis, 2-chlorotrityl chloride, solution-phase macrocyclization, cytotoxicity.

Solid-phase synthesis of Biotin-S-Farnesyl-l-Cysteine, a surrogate substrate for isoprenylcysteine Carboxylmethyltransferase (ICMT)

Inhibition of isoprenylcysteine Carboxylmethyltransferase (ICMT) is of particular interest as a potential target for the development of cancer chemotherapeutic agents. Screening for inhibitors of ICMT utilises a scintillation proximity assay (SPA) in which Biotin-S-Farnesyl-l-Cysteine (BFC) acts as a surrogate substrate. A solid-phase synthesis protocol for the preparation of BFC using 2-chlorotrityl chloride resin as a solid support has been developed to provide sufficient supply of BFC for high throughput screening (HTS) and subsequent chemistry campaigns to target inhibitors of ICMT. The BFC prepared by this method can be produced quickly on large scale and is stable when stored at −20°C as a solid, in solution, or on the resin.

Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser558-Gln565 region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa-FIXa interaction. Based on this, a series of linear peptides, analogs of the 558-565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.

Facile Synthetic Method of Alkanethiol Spacer for Biointerface

An alkanethiol spacer which is suitable for biointerface was synthesized on 2-chlorotritylchloride (CTC) resin using solid-phase synthesis method. The triethylene glycol (TEG) unit containing building block was immobilized on CTC resin followed by a coupling alkanethiol building block. After dithiothreitol (DTT) treatment and cleavage of the resulting alkanethiol spacer from the resin, the spacer was obtained in a pure form without any purification steps. The TEG-alkanethiol spacer was applied for the DNA hybridization assay on a gold surface, and proved to work well when compared to the commercial spacer.

Synthesis, biological activity and conformational analysis of head‐to‐tail cyclic analogues of LL37 and histatin 5

LL37 and histatin 5 are antimicrobial peptides. LL37 exhibits killing activity against a broad spectrum of pathogens, whereas histatin 5 is primarily an antifungal agent. Head-to-tail cyclization of histatin 5 did not affect its antimicrobial and haemolytic activity. The cyclic LL37 exhibits identical antifungal and haemolytic activity as does LL37. Its antimicrobial activity varied in one dilution depending on the kind of bacteria. The structure of cyclic peptides was studied by circular dichroism spectroscopy. Both peptides undergo a conformational change leading to stabilisation of their α-helical structure in the presence of negatively charged sodium dodecyl sulfate micelles. However, with cyclic histatin 5, the presence of Zn(2+) ions is also necessary to fuse the peptide to the micelle. The specific action of the Zn(2+) ions is attributed to the presence of a zinc-binding motif, His-Glu-X-X-His. It has been speculated that this zinc complexing may be related to the well-established anticandidal activity. In the case of cyclic LL37, also the presence of a zwitterionic dodecylphosphocholine micelle induces formation of the helical structure. A microwave-assisted procedure for the cleavage of a peptide from the 2-chlorotrityl chloride resin was, for the first time, successfully used to obtain protected peptide fragments that can be applied to the preparation of head-to-tail cyclopeptides or to condensation of peptidic fragments.

Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins

Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences for synthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93-231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during the synthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.

Use of Oxyma as pH modulatory agent to be used in the prevention of base‐driven side reactions and its effect on 2‐chlorotrityl chloride resin

The presence of low pKa N-hydroxylamines is beneficial in peptide chemistry as they reduce some base-mediated side reactions. Here we evaluated the applicability and buffering capacity of Ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma) in the prevention of aspartimide/piperidide formation and Pro-based overcoupling and compared it with the performance of HOBt and HOAt. In addition, the compatibility of these additives with the highly acid-labile 2-chlorotrityl chloride resin is examined.

Giant macrolactams based on β-sheet peptides.

This paper reports the use of natural amino acids, the tripeptide β-strand mimic Hao, and the β-turn mimic δ-linked ornithine to generate water-soluble 54-, 78-, and 102-membered-ring macrolactams. These giant macrocycles were efficiently prepared by synthesis of the corresponding protected linear peptides, followed by solution-phase cyclization and deprotection. The protected linear peptide precursors were synthesized on 2-chlorotrityl chloride resin by conventional Fmoc-based solid-phase peptide synthesis. Macrocyclization was typically performed using HCTU and N,N-diisopropylethylamine in DMF at ca. 0.5 mM concentration. The macrocycles were isolated in 13-45% overall yield after HPLC purification and lyophilization. 1D, 2D TOCSY, and 2D ROESY (1)H NMR studies of the 54- and 78-membered-ring macrolactams establish that these compounds fold to form β-sheet structures in aqueous solutions.

Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation

The immobilization of alcohols onto 2-chlorotritylchloride resin using microwave irradiation was studied. Three different Fmoc-aminoalcohols were tested: the phenol-like Fmoc-tyramine, the primary alcohol Fmoc-ethanolamine, and the secondary alcohol Fmoc-4-hydroxypiperidine. Several reaction conditions were evaluated: different bases, reaction times, temperatures, and concentrations. Microwave immobilization resulted is effective in binding to the resin all three types of alcohols with loadings which were superior or comparable to the ‘classical’ methods in shorter time and without employing toxic and racemizing reagents. This method resulted also useful for the immobilization, through the hydroxyl group, of FmocTyrOAll, FmocSerOAll, and FmocThrOAll, important building blocks for the synthesis of cyclic peptides.

Synthesis of MUC1 glycopeptide thioesters and ligation via direct aminolysis

MUC1 is a cell-surface, epithelial glycoprotein that forms an essential protective barrier of cells and serves to modulate intercellular communication. During cancer progression, the dysregulation of glycosyltransferases, which serve to elongate cell-surface glycans, leads to aberrant gIycosylation patterns on this glycoprotein. This results in the presentation of well-characterized, tumor-associated carbohydrate antigens (TACAs) which represent important biomarkers for a range of epithelial cancers. The synthesis of well-defined, multivalent MUC1 glycopeptide constructs bearing TACAs therefore represents a viable opportunity for the development of cancer vaccine candidates. We report herein the synthesis of a glycopeptide thioester, comprising the full eicosapeptide variable number tandem repeat (VNTR) region of MUC1, which was prepared bearing multiple copies of the cancer-associated TN antigen. The glycopeptide thioester was prepared on the solid-phase using two different methods on 2-chlorotrityl chloride and sulfamylbutyryl resins. The solid-phase assembly on 2-chlorotrityl chloride resin, followed by thioesterification in solution, afforded the desired thioester in 4% yield over 41 linear steps. Likewise, the glycopeptide thioester was successfully prepared on sulfamylbutyryl resin using an activation and thiol-release strategy in a 9% isolated yield. The resulting peptide thioester was successfully ligated to a deprotected MUC1 glycopeptide using the direct aminolysis ligation to afford a 5.8 kDa, 40 amino acid MUC1 glycopeptide bearing 10 copies of the T(N) antigen. This work represents an important step toward the immunological evaluation of multivalent MUC1 constructs.

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60-110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60-110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys(77)-Cys(109) bond, followed by iodine oxidation to form the Cys(67)-Cys(99) bond.

Solid-Phase Synthesis of Prenylcysteine Analogs

Prenylcysteine derivatives are of interest for a variety of different biological reasons, including probing the CaaX protein processing pathway. A solid-phase synthesis protocol for the preparation of prenylcysteines using 2-chlorotrityl chloride resin as a solid support has been developed. A series of novel amide-modified farnesylcysteine analogs were synthesized in both high purity and yield under mild conditions. The farnesylcysteine analogs were evaluated using human isoprenylcysteine carboxyl methyltransferase as a biological target, and several new inhibitors, one with significantly enhanced potency, were identified.

Synthesis of cyclic peptides via O– N-acyl migration

We describe here a novel and convenient synthesis of head-to-tail cyclic peptide avoiding racemization. Linear depsipeptides including a serine residue as the key element for ester bond formation and acyl transfer were synthesized on 2-chlorotrityl chloride resin. After cleavage from the resin, intramolecular head-to-tail cyclization was performed in solution by C-terminal activation of urethane protected O-acyl serine residue. After removal of the N α-serine protecting group, the final step consisted in O– N-acyl migration reaction on the ‘switch’ or ‘click’ element to restore native cyclic peptides.

Solid-Phase Synthesis ofN-Nosyl- andN-Fmoc-N-Methyl-α-amino Acids

We report here a convenient and simple solid-phase synthesis of N-nosyl-N-methyl-alpha-amino acids and N-Fmoc-N-methyl-alpha-amino acids, important building blocks for the synthesis of conformationally restricted and protease-resistant natural peptides and peptide analogues. The methodology involves the use of 2-chlorotrityl chloride resin to temporarily protect the carboxylic group of alpha-amino acids and of diazomethane as the reagent to methylate the sulfonamidic function. The approach developed is particularly efficient also with alpha-amino acids bearing appropriately protected functionalized side chains.

A new method for the preparation of 2-chlorotrityl resin and its application to solid-phase peptide synthesis

2-Chlorotritylchloride (2-CTC) resin was prepared efficiently from 1% DVB-crosslinked polystyrene resin and 1-chloro-2-(dichloro(phenyl)methyl)benzene, which was easily obtained from 2-chlorobenzophenone. This 2-CTC resin showed excellent properties as a support for solid-phase peptide synthesis. Four peptide fragments were obtained in high purity using the resin.

Focused Libraries of 16‐Substituted Estrone Derivatives and Modified E‐Ring Steroids: Inhibitors of 17ß‐Hydroxysteroid Dehydrogenase Type 1

17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), an oxidoreductase which has a preferential reductive activity using NADPH as cofactor, converts estrone to estradiol and is expressed in many steroidogenic tissues including breast and in malignant breast cells. As estradiol stimulates the growth and development of hormone-dependent breast cancer, inhibition of the final step of its synthesis is an attractive target for the treatment of this disease. The parallel synthesis of novel focused libraries of 16-substituted estrone derivatives and modified E-ring pyrazole steroids as new potent 17beta-HSD1 inhibitors is described. Substituted 3-O-sulfamoylated estrone derivatives were used as templates and were immobilised on 2-chlorotrityl chloride resin to give resin-bound scaffolds with a multi-detachable linker. Novel focused libraries of 16-substituted estrone derivatives and new modified E-ring steroids were assembled from these immobilised templates using solid-phase organic synthesis and solution-phase methodologies. Among the derivatives synthesised, the most potent 17beta-HSD1 inhibitors were 25 and 26 with IC50 values in T-47D human breast cancer cells of 27 and 165 nm, respectively. Parallel synthesis resulting in a library of C5'-linked amides from the pyrazole E-ring led to the identification of 62 with an IC50 value of 700 nM. These potent inhibitors of 17beta-HSD1 have a 2-ethyl substituent which will decrease their estrogenic potential. Several novel 17beta-HSD1 inhibitors emerged from these libraries and these provide direction for further template exploration in this area. A new efficient diastereoselective synthesis of 25 has also been developed to facilitate supply for in vivo evaluation, and an X-ray crystal structure of this inhibitor is presented.

Polystyrene resins cross-linked with di- or tri(ethylene glycol) dimethacrylates as supports for solid-phase peptide synthesis

Polystyrene resins cross-linked with di(ethylene glycol) dimethacrylate (DEGDMA) and tri(ethylene glycol) dimethacrylate (TEGDMA), DEGDMA–PS and TEGDMA–PS, were synthesized by suspension copolymerization. Four functionalized resins, chloromethyl resin, 4-hydroxymethylphenoxymethyl resin (Wang resin), 4-methylbenzhydrylamine resin (MBHA resin) and 2-chlorotrityl chloride resin, were prepared from DEGDMA–PS and TEGDMA–PS. DEGDMA–PS and TEGDMA–PS showed high reactivity in the functionalization reactions in comparison with Merrifield resin (polystyrene cross-linked with divinylbenzene, DVB–PS). DEGDMA–PS–Wang resin and TEGDMA–PS–Wang resin were used as the solid-phase support for the synthesis of a difficult sequence, the fragment of acyl carrier protein 65–74. The yields of the crude peptide synthesized using DEGDMA–PS–Wang resin, TEGDMA–PS–Wang resin and DVB–PS–Wang resin were 92.3%, 91.6% and 78.8%, respectively. The purities of the crude peptides were 85.7%, 88.1% and 73.3%, respectively.

An Efficient, Convenient Solid-Phase Synthesis of Amino Acid-Modified Peptide Nucleic Acid Monomers and Oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.