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Advances in instrumentation, as well as the development of powerful crystallographic software have significantly facilitated the collection of high-resolution diffraction data and have made X-ray powder diffraction (XRPD) particularly useful for the extraction of structural information; this is true even for complex molecules, especially when combined with synchrotron radiation. In this study, in-line with past instrumental profile studies, an improved data collection strategy exploiting the MYTHEN II detector system together with significant beam focusing and tailored data collection options was introduced and optimized for protein samples at the Material Science beamline at the Swiss Light Source. Polycrystalline precipitates of octreotide, a somatostatin analog of particular pharmaceutical interest, were examined with this novel approach. XRPD experiments resulted in high angular and d-spacing (1.87 Å) resolution data, from which electron-density maps of enhanced quality were extracted, revealing the molecule's structural properties. Since microcrystalline precipitates represent a viable alternative for administration of therapeutic macromolecules, XRPD has been acknowledged as the most applicable tool for examining a wide spectrum of physicochemical properties of such materials and performing studies ranging from phase identification to complete structural characterization.

Thioether containing peptides were obtained following three synthetic routes. In route A, halo acids esterified on 2-chlorotrityl(Cltr) resin were reacted with N-fluorenylmethoxycarbonyl (Fmoc) aminothiols. These were either cleaved from the resin to the corresponding (Fmoc-aminothiol)carboxylic acids, which were used as key building blocks in solid phase peptide synthesis (SPPS), or the N-Fmoc group was deprotected and peptide chains were elongated by standard SPPS. The obtained N-Fmoc protected thioether containing peptides were then condensed either in solution, or on solid support, with the appropriate amino components of peptides. In route B, the thioether containing peptides were obtained by the reaction of N-Fmoc aminothiols with bromoacetylated peptides, which were synthesized on Cltr-resin, followed by removal of the N-Fmoc group and subsequent peptide elongation by standard SPPS. In route C, the thioether containing peptides were obtained by the condensation of a haloacylated peptide synthesized on Cltr-resin and a thiol-peptide synthesized either on 4-methoxytrityl(Mmt) or trityl(Trt) resin.

Natural or artificially manufactured peptides attract scientific interest worldwide owing to their wide array of pharmaceutical and biological activities. X-ray structural studies are used to provide a precise extraction of information, which can be used to enable a better understanding of the function and physicochemical characteristics of peptides. Although it is vulnerable to disassociation, one of the most vital human peptide hormones, somatostatin, plays a regulatory role in the endocrine system as well as in the release of numerous secondary hormones. This study reports the successful crystallization and complete structural model of octreotide, a stable octapeptide analogue of somatostatin. Common obstacles in crystallographic studies arising from the intrinsic difficulties of obtaining a suitable single-crystal specimen were efficiently overcome as polycrystalline material was employed for synchrotron and laboratory X-ray powder diffraction (XPD) measurements. Data collection and preliminary analysis led to the identification of unit-cell symmetry [orthorhombic,P212121,a= 18.5453 (15),b= 30.1766 (25),c= 39.798 (4) Å], a process which was later followed by complete structure characterization and refinement, underlying the efficacy of the suggested (XPD) approach.

Human gene 2 relaxin (RLX) is a member of the insulin superfamily and is a multi-functional factor playing a vital role in pregnancy, aging, fibrosis, cardioprotection, vasodilation, inflammation, and angiogenesis. RLX is currently applied in clinical trials to cure among others acute heart failure, fibrosis, and preeclampsia. The synthesis of RLX by chemical methods is difficult because of the insolubility of its B-chain and the required laborious and low yielding site-directed combination of its A (RLXA) and B (RLXB) chains. We report here that oxidation of the Met(25) residue of RLXB improves its solubility, allowing its effective solid-phase synthesis and application in random interchain combination reactions with RLXA. Linear Met(O)(25)-RLX B-chain (RLXBO) reacts with a mixture of isomers of bicyclic A-chain (bcRLXA) giving exclusively the native interchain combination. Applying this method Met(O)(25)-RLX (RLXO) was obtained in 62% yield and was easily converted to RLX in 78% yield, by reduction with ammonium iodide. Copyright (C) 2010 European Peptide Society and John Wiley & Sons, Ltd.

N alpha-9-Fluorenylmethoxycarbonyl-N epsilon-4=methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N epsilon-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5-1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr1-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM-TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions.

Nα-Trityl amino acid 1-hydroxybenzotriazole active esters, in contrast to HOBt esters of other protected amino acids, exhibit high hydrolytic stability and can be isolated in excellent yields and purity. The derivatives occur in three isomeric forms (an ester=I, two amides=II and III). All active ester forms react at high concentrations within 2 h with various alkyl amino acid esters to produce the corresponding protected dipeptides in high yields. Amorphous active esters on prolonged storage at — 20° rearrange partially towards form III without any sign of decomposition.

Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55–65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys16, 28(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys6–Cys14 and Cys22–Cys39 linkages, followed by iodine oxidation to form the Cys16–Cys28 bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

Diamines derived from naturally occurring aminoacids were inserted into peptide chains by the reaction of the monophthaloyl diamines with amino acid 1-benzotriazolyl esters, bound through their amino functions onto trityl-type resins. The phthaloyl group was removed and peptide chains using N -Fmoc amino acids, were assembled on the liberated amino function. The peptidyl diamides obtained, were cleaved from the resins with t Bu-side chain protection remaining intact, or fully deprotected.

Aminothiols were attached through their thiol group onto the 4,4′-dimethoxytrityl (Dmt)-, 4-methoxytrityl (Mmt)-, 4-methyltrityl (Mtt)-, trityl (Trt)- and 2-chlorotrityl (Clt)-resins. The new resins were used in the solid-phase synthesis of aminothiol containing peptides utilizing N -Fmoc amino acids. The synthesized peptides were cleaved from the resins by treatment with trifluoroacetic acid (TFA) solutions using triethylsilane (TES) or ethanedithiol (EDT) as scavengers.

Monophthaloyl diamines derived from naturally occurring amino acids were attached through their free amino functions to resins of the trityl type. The phthaloyl groups were removed by hydrazinolysis, and peptide chains were assembled using Fmoc/tBu-amino acids on the liberated amino functions. The peptidyl aminoalkyl amides obtained were cleaved from the resins by mild acidolysis, with the tBu-side chain protection remaining intact. Copyright  2002 European Peptide Society and John Wiley & Sons, Ltd.

2-Aminobenzenethiol, bound through its thiol function to the 2-chlorotrityl (Clt)-, trityl (Trt)-, 4-methyltrityl (Mtt)- and 4-methoxytrityl (Mmt)-resins, was acylated at the amino-function by aliphatic and aromatic acids. The obtained 2-N-acyl-aminobenzenethiols were cleaved from the resin by treatment with trifluoroacetic acid solutions in dichloromethane. The 2-N-acyl-aminobenzenethiols released from the resin were cyclised to the corresponding 2-substituted benzothiazoles, by standing in a solution of dithiothreitol in DMF or methanol for 1–3 h at room temperature.

Histidine, histamine and urocanic acid were attached through the N im -function on resins of the trityl type. The conditions of the cleavage of these derivatives from the resin were determined. Resin bound histamine was used in the solid phase synthesis of carcinine.

Besides linear solid phase peptide synthesis, segment condensation in solution and chemical ligation, convergent peptide synthesis (CPS) was developed in order to enable the efficient preparation of complex peptides and small proteins. According to this synthetic strategy, solid phase synthesized and suitably protected peptide fragments corresponding to the entire peptide/protein-sequence are condensed on a solid support or in solution, to the target protein. This review summarizes CPS performed utilizing the mild 9-fluorenylmethyloxycarbonyl/tbutyloxycarbonyl-based protecting scheme for the amino acids.

The effective purification of protected peptide fragments of medium polarity using glass columns packed with RP-8 and RP-18 materials of 40–63 μm and eluted with mixtures of acetonitrile and water under isocratic conditions is described. Starting the purification from protected fragments of 80–97% purity, eight out of ten of the peptides were obtained in >99% purity and 53–86% purification yield.

Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc- or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl- and tert-butyl protecting groups remain unaffected.

Mercapto acids were attached through their thiol group onto 2-chlorotrityl (Clt)-, trityl (Trt)-, 4-methyltrityl (Mtt)-, 4-methoxytrityl (Mmt)- and 4,4′-dimethoxytrityl (Dmt)-resins. The new resins were used in the solid-phase synthesis of small mercaptoacylamino alcohols. Cleavage from the resins was performed by treatment with trifluoroacetic acid (TFA) solutions in dichloromethane (DCM) using triethylsilane (TES) as scavenger.

Synthesis of Calcitonin Derivatives by Fragment Condensation in Solution and on 2-Chlorotrityl Resin Ser2,Val8,Gly33)-human calcitonin (13) was synthesized in 45% yield by sequential assembly of protected fragments on 2-chlorotrityl resin. An analogous synthesis by fragment condensation in solution yielded (Ser2,Val8)-human calcitonin (21) in 10% yield.

4-Methoxytrityl (Mmt)-mercapto acids were obtained either by the reaction of mercapto acids with Mmt-chloride or by the reaction of halo acids with Mmt-thiol. The derivatives obtained were used in the solid-phase synthesis of small libraries of mercaptoacylamino acids and mercaptoacyl peptides. The removal of the Mmt-group was performed by treatment with trifluoroacetic acid (TFA) in dichloromethane (DCM) using triethylsilane (TES) as scavenger.

8 páginas, 3 figuras, 2 tablas -- p. 63re3, To infect host cells, most enveloped viruses must insert a hydrophobic fusion peptide into the host cell membrane. Thus, fusion peptides may be valuable targets for developing drugs that block virus entry. We have shown previously that a natural 20-residue fragment of α1-antitrypsin, designated VIRus-Inhibitory Peptide (VIRIP), that binds to the gp41 fusion peptide of HIV-1 prevents the virus from entering target cells in vitro. Here, we examine the efficacy of 10-day monotherapy with the optimized VIR-576 derivative of VIRIP in treatment-naïve, HIV-1–infected individuals with viral RNA loads of ≥10,000 copies per ml. We report that at the highest dose (5.0 grams per day), intravenous infusion of VIR-576 reduced the mean plasma viral load by 1.23 log10 copies per ml without causing severe adverse effects. Our results are proof of concept that fusion peptide inhibitors suppress viral replication in human patients, and offer prospects for the development of a new class of drugs that prevent virus particles from anchoring to and infecting host cells, Part of this work was supported by the CONSOLIDER CSD2009-00088 from the Ministry of Science and Innovation, the State of Lower Saxony, and the Deutsche Forschungsgemeinschaft. D.M.-O. is supported by Bundesministerium für Bildung und Forschung, the European Aids Treatment Network (NEAT), Innovationsfond des Landes Niedersachsen, and the Helmholtz-Zentrum für Infektionsforschung (IG-SCID-TwinPro02). M.S. received funding from Boehringer-Ingelheim, GlaxoSmithKline, Roche, and Tibotec. Author contributions: W.-G.F. planned and supervised the study. Y.-H.T. recruited and Tibotec

Introduction The tumor suppressor protein p53 plays a pivotal role in DNA damage recognition, signal transduction, and initiation of mechanisms of apoptosis and repair. Mdm2 (murine double minute 2) interacts with p53, inhibits its function and promotes its degradation through ubiquitination (programmed protein degradation). In the ubiquitin pathway, Mdm2 plays the role of an E3 ubiquitin ligase. High levels of Mdm2 are responsible for cancer initiation and therefore it is characterized as oncogenic protein. It has been found that the RING domain of Mdm2 (Fig. 1) located at the C-terminus of the protein is necessary for E3 activity and therefore for the regulation of p53. For these reasons the RING domain of Mdm2 is an important target for studying its interaction with small anticancer drug candidates. Due to the presence of multiple cysteine residues, the synthesis of such small proteins is challenging and very difficult to be performed using recombinant techniques. Therefore, chemical syntheses are attractive and in two examples the ligation method was used [1,2]. As an alternative method, we studied the synthesis of the 48mer RING finger domain of Mdm2 (sequence 435-482) by the sequential condensation of protected fragments on 2-chlorotrityl resin.

Besides linear solid phase peptide synthesis, segment condensation in solution and chemical ligation, convergent peptide synthesis (CPS) was developed in order to enable the efficient preparation of complex peptides and small proteins. According to this synthetic strategy, solid phase synthesized and suitably protected peptide fragments corresponding to the entire peptide/protein-sequence are condensed on a solid support or in solution, to the target protein. This review summarizes CPS performed utilizing the mild 9-fluorenylmethyloxycarbonyl/tbutyloxycarbonyl-based protecting scheme for the amino acids.

Human gene 2 relaxin (RLX) is a member of the insulin superfamily and is a multi-functional factor playing a vital role in pregnancy, aging, fibrosis, cardioprotection, vasodilation, inflammation, and angiogenesis. RLX is currently applied in clinical trials to cure among others acute heart failure, fibrosis, and preeclampsia. The synthesis of RLX by chemical methods is difficult because of the insolubility of its B-chain and the required laborious and low yielding site-directed combination of its A (RLXA) and B (RLXB) chains. We report here that oxidation of the Met(25) residue of RLXB improves its solubility, allowing its effective solid-phase synthesis and application in random interchain combination reactions with RLXA. Linear Met(O)(25)-RLX B-chain (RLXBO) reacts with a mixture of isomers of bicyclic A-chain (bcRLXA) giving exclusively the native interchain combination. Applying this method Met(O)(25)-RLX (RLXO) was obtained in 62% yield and was easily converted to RLX in 78% yield, by reduction with ammonium iodide.

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 339–349, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Zusammenfassung The use of 4-methoxytrityl (monomethoxytrityl, Mmt) and 4-methyl-trityl (Mtt) groups of N1m-protection of Fmoc-His is described. Both groups being unaffected by base, e.g. piperidine and diethylamine, can be quantitatively removed under very mild conditions, in comparison with trityl groups. The solid phase peptide synthesis of megainin I (fragment 1–10) on 2-chlorotrityl resin showed the excellent suitability of the above derivatives of histidine for peptide synthesis.

Zusammenfassung The preparation of the N-Fmoc-O-Trt derivatives of serine, threonine and tyrosine is described. Their usefulness in peptide synthesis has been determined in the successful solid phase preparation of the partially protected ACTH (fragment 1–10) and peptide T 12 . The latter, having six hydroxy amino acid side chains protected with Trt groups, can be quantitatively cleaved from the applied 2-chlorotrityl resin with simultaneous side chain deprotection.

Multiple disulfide bonds between cysteine residues form disulfide frameworks, which represent an important structural feature in many peptides and proteins secreted from cells. Among these, numerous peptide hormones, cytokines, protease inhibitors and toxins are found. Starting with chemically synth...

The S-Mmt bond can be cleaved by treatment with 1-3% TFA in DCM within 5-30 min at room temperature. In contrast, S-Trt and S-2-chlorotrityl (Clt) remain stable under these conditions. Similar acid stabilities will expose thiols to resins of the Trt-type. We used compounds which contained S-Mmt and were bound to Trtor Clt-resin through a carboxy-, hydroxyl, aminoor imino-group. Taking advantage of the acid lability, these compounds were treated with dilute TFA soltuions. The liberated thiol function reacted with the resin-trityl cations formed during the acidic treatment. A >95% reattachment of the compound onto the resin occurred through the thiol-function. Similarity, acidic treatment of thiols bound to the very acid sensitive Mmtand dimethoxytrityl (Dmt)resins caused the replacement of the resin by a group of the Trt-type. Aromatic amines bound through an O-thiol, phenoxy or amine-function on trityl-type resins yield the corresponding benzothiazolyl, oxazolyl and imidazolyl compounds after solid-phase acylation of the free amino-function. In the key step of the synthesis, acidic treatment of the resin affects quantitative cleavage of the linear compound. Then, a cyclization occures by the nucleophilic attack of the liberated nucleophilic function on the neighbor amide bond, followed by water elimination. The reaction is facilitated by the aromatic character of the resulting heterocycles, and provides long peptidyl-benzothiazoles in high yield and purity. In Fig. 1 the synthesis of a small aminoalky-benzothiazole library is presented. Starting from aminothiols attached through their thiol-function onto the Mmtor the Dmt-resin, peptide-aminothiols were assembled on solid-phase. These were used in the synthesis of cyclic S-containing peptides (Fig. 2) and in the convergent synthesis of S-containing linear peptides.