Difficult Peptide Research Articles

2-Methoxy-4-methylsulfinylbenzyl: a backbone amide safety-catch protecting group for the synthesis and purification of difficult peptide sequences.

The use of 2-methoxy-4-methylsulfinylbenzyl (Mmsb) as a new backbone amide-protecting group that acts as a safety-catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid-mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi-unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH4 I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the "difficult peptide" sequence H-(Ala)10-NH2 was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac-(RADA)4-NH2 sequence was chosen to validate this methodology. The improvements in solid-phase peptide synthesis combined with the enhanced solubility of the quasi-unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac-(RADA)4-NH2. To extend the scope of the approach, the challenging Aβ(1-42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.

Abstract 4588: Polylysine linker for successful manufacturing of hydrophobic peptides

Abstract Hydrophobic peptides are common to be used in cancer vaccinations. Hydrophobic peptides, continue to present challenges for large scale production in peptide industry. Experimental Procedures: Full sequence prepared by Solid phase Synthesis:The peptide was synthesized with Fmoc-Lys(Boc)-Wang resin by using standard Fmoc/tBu chemistry. Protecting groups used for amino acids are: tBu for Thr, Boc for Trp and Lys and Boc-Ser- OH was used as a hydrolysis-labile ester provider. Fmoc-Leu-OH was coupled on the side chain of Ser by DIC/DMAP (Amino acid/DIC/DMAP, 3/1.5/0.3 eq). The other amino acids were assembled by repetitive removal of the Fmoc protecting group by treating with 2% DBU/2% piperidine in DMF 30 minutes and coupling of protected amino acid by using DIC/HOBt/NMM (4:4:4 eq) in 180 minutes to overnight. Ninhydrin test was performed after each coupling to check the coupling efficiency. After the last coupling,resin was washed and dried, then treated with reagent K for cleavage and removal of the side chain protecting groups. Crude peptide was precipitated from cold ether and collected by filtration. Table.1 Summary of 4 synthesize results Syn. NoLinker StructurePurity of CrudeDKP deletion: Product Hydrolysis YieldOverall Yield1No Linker61.4%12.3%2(O)-Ser56.5%1:252.8%12.5%3(O)-Ac-Ser40.8%1:370.6%28.2%4HMBA78.1%<1:2451.0%61.2% Conclusion: American Peptide demonstrated the utility of a hydrophilic peptide ‘tail’ in purification of a hydrophobic peptide on both small (20-30 mmol) research scale and large (800 mmol) cGMP scales to provide the peptide in hundreds-of-grams quantities. The approach described allowed for both time-and cost-competitive manufacturing of this difficult peptide for clinical trials. Citation Format: Firuz Shakoori, Archana Gangakhedkar. Polylysine linker for successful manufacturing of hydrophobic peptides. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2014-4588

O-acyl isopeptide method: development of an O-acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1-42 isopeptide.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Rapid Total Synthesis of DARPin pE59 and Barnase

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.

ChemInform Abstract: Solid‐Phase Peptide Synthesis Using Microwave Irradiation

AbstractReview: 26 refs.

ChemInform Abstract: Chemical Synthesis of Integral Membrane Proteins: Methods and Applications

AbstractReview: 83 refs.

Multivariate Analysis of Deep Ultraviolet Resonance Raman Spectra: Secondary Structure Determination

Studies pertaining to protein or peptide structure, dynamics or function fundamentally require a method for secondary structure measurement. There are four structurally sensitive protein vibrational modes in deep-ultraviolet resonance Raman (DUVRR) spectra, the amide I, II, III and S modes, making it a potentially powerful tool for studying secondary structure changes in even the most difficult peptide samples. Experimental studies reveal that the position and intensity of the four amide modes in UVRR spectra of proteins are correlated with the varying fractions of α-helix, β-sheet and unordered structures of proteins. This relationship has been applied to the prediction of protein secondary structure.Specifically, multivariate methods have been employed to decompose protein spectral data sets into their underlying pure secondary structure Raman spectra (PSSRS). Ideally, the resolved PSSRS could then be used for prediction of the secondary structure content of unknown proteins from their UVRR spectra. However, previous studies have not examined the performance of multivariate methods on prediction of the structural composition of an unknown test protein. A comparative study of the ability of classical least squares (CLS), principal component regression (PCR), partial least squares (PLS) and multivariate curve resolution- alternating least squares(MCR-ALS) to resolve the pure secondary structure Raman spectra (PSSRS) and predict the structural composition of unknown test proteins will be presented.

Chemical Synthesis of Integral Membrane Proteins: Methods and Applications

AbstractThe development of efficient chemical methods for total synthesis or semisynthesis of integral membrane proteins is an important challenge at the interface between chemistry and biology. This review outlines the recent advances in the synthesis of integral membrane proteins, with particular focus on the methods for difficult peptide synthesis, purification, and enhancement of peptide solubility under the ligation conditions. The applications of these methods to the synthesis of integral membrane proteins with one or multiple transmembrane domains are also described.

Conventional and microwave‐assisted SPPS approach: a comparative synthesis of PTHrP(1–34)NH2

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW-assisted SPPS of 1-34 N-terminal fragment of parathyroid hormone-related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin-bound peptide, monitoring was conducted by performing MW-assisted mini-cleavages and analyzing them by UPLC-ESI-MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages.

Rational development of a strategy for modifying the aggregatibility of proteins

The conversion of peptide and proteins from their soluble state into well-organized aggregates, together with the accompanied oxidation of methionine residue, presents a significant challenge to human health, to the manufacture of protein therapeutics, and to the synthesis of proteins and glycoproteins. Despite their fundamental importance, little is known about the molecular basis of these two side reactions and their control. Here, using chemical peptide synthesis, we further confirmed the importance of the balance between hydrophobic interactions and electrostatic repulsive forces in inducing and inhibiting aggregation and methionine oxidation. Most importantly, through extending the established principle, we are able to effectively stabilize the problematic peptide fragment through the attachment of cleavable arginine tags. Future applications of our approach are expected to facilitate the synthesis and study of difficult peptides, proteins, and glycoproteins and will provide more opportunities for the optimization of protein biopharmaceuticals and for the development of cell-permeable biomolecules.

Hexafluoro-2-propanol as a potent cosolvent for chemical ligation of membrane proteins

The study on membrane proteins is an important challenge mainly because of their very poor solubility in various solvents. The traditional recombinant expression strategy and the native chemical ligation method both have difficulty in generating sufficient amounts of desired proteins with high efficiency. Previous studies have shown that multiply fluorinated alcohols exhibit good ability to dissolve difficult peptide sequences, especially hexafluoro-2-propanol (HFIP). In the present study we systematically studied the capability of solvents containing different percentage of HFIP in dissolving transmembrane peptides. Through both HPLC and UV analyses we concluded that 60% HFIP/8 M urea constituted a good solvent system. In this solvent system we also optimized conditions to perform native chemical ligation (NCL). Under the optimized conditions we successfully achieved NCL’s for both dipeptide formation and the synthesis of a model protein (Trifolitoxin). These results suggested that HFIP was a potential cosolvent that could be used in the ligation of poorly soluble peptides for the generation of membrane proteins.

Improved Fmoc‐based solid‐phase synthesis of homologous peptide fragments of human and mouse prion proteins

The synthesis of difficult peptide sequences has been a challenge since the very beginning of SPPS. The self-assembly of the growing peptide chains has been proposed as one of the causes of this synthetic problem. However, there is an increasing need to obtain peptides and proteins that are prone to aggregate. These peptides and proteins are generally associated with diseases known as amyloidoses. We present an efficient SPPS of two homologous peptide fragments of HuPrP (106-126) and MoPrP105-125 based on the use of the PEGA resin combined with proper coupling approaches. These peptide fragments were also studied by CD and TEM to determine their ability to aggregate. On the basis of these results, we support PEG-based resins as an efficient synthetic tool to prepare peptide sequences prone to aggregate on-resin.

Automated Fmoc-Based Solid-Phase Synthesis of Peptide Thioesters with Self-Purification Effect and Application in the Construction of Immobilized SH3 Domains

Peptide thioesters are important building blocks in the total synthesis of proteins and protein domains via fragment ligation. However, synthetic access of peptide thioesters still is a bottleneck of this powerful ligation chemistry. The commonly used methods for the Fmoc-based synthesis of peptide thioesters involve nonautomated solution steps that have to be performed after the solid-phase assembly of the peptide. Usually, HPLC purification is required. Herein, a method that enables crude peptides to be used in divergent native chemical ligations reactions is described. We present an Fmoc-based solid-phase synthesis of peptide thioesters with self-purification which facilitates access to these important building blocks, since the often cumbersome HPLC purification can be avoided. Fmoc-protected amino acids are coupled on a safety catch sulfonamide resin. The self-purifying effect is achieved through the combination of (a) N-terminal coupling of a cleavable cyclization linker and subsequent backbone-to-side chain cyclization, (b) activation of the sulfonamide linkage by alkylation, (c) thiolysis for the selective detachment of truncation products, and (d) TFA cleavage for the liberation of the desired peptide thioester in unprotected form. We have previously shown a method wherein cyclization was performed after carboxymethylation of the sulfonamide. However, the automation of this method was difficult and side reactions at methionine residues hampered the general applicability. The new design involves peptide synthesis on a modified carboxy-functionalized sulfonamide linker, a substantially milder activation of the sulfonamide bond and the use of monomethoxytrityl as well as 2-phenyl-isopropyl protecting groups. This approach solved the problems with methionine containing peptides and enabled the complete automation of the self-purifying synthesis of peptide thioesters. The study also addressed problems in the synthesis of difficult peptides. Aggregated truncation products can resist extraction and contaminate full-length thioesters obtained after TFA cleavage. It is shown that significant enhancements of the purity were achieved when mild acidic extractions were included in the wash protocols after thiolysis. The potential of the method was demonstrated in the parallel synthesis of 20-40 amino acid long peptide thioesters, which were obtained in excellent purities. The thioesters and cysteinyl peptides were used without purification in the assembly of immobilized SH3 protein domains of SHO1 in yeast. A cysteine scan by native chemical ligation suggested single amino acid to cysteine substitutions that (a) confer useful ligation yields, (b) support correct folding, and (c) sustain the function of the folded protein domain. The chemical synthesis of the SH3-domain of SHO1 succeeded in highest yields when cysteine placements at positions S23, F24, and E36 were avoided. The synthetic SH3 mutants were examined in a binding assay, which indicated that N27C, L30C, and D34C mutations provide functional SH3-domain.

Automated ‘X‐Y’ robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains

Precise microwave heating has emerged as a valuable method to aid solid-phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the beta-amyloid 1-42 peptide. The instrument is built around a valve-free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an 'X-Y' robotic microwave-assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve-free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficult peptide sequences were achieved.

The depsipeptide method for solid‐phase synthesis of difficult peptides

After about one century of peptide chemistry, the main limitation to the accessibility of peptides and proteins via chemosynthesis is the arising of folding and aggregation phenomena. This is true not only for sequences above a critical length but also for several biologically relevant substrates that are relatively short yet form either highly folded structures (e.g. WW domains) or fibrils and aggregates after final deprotection (beta-amyloid peptide). Such so-called difficult sequences may be more easily obtained via their corresponding depsipeptides (O-acyl isopeptides), ester isomers that are often easier to assemble and purify, and are smoothly converted to the parent amides under mild conditions. The depsipeptide method is the most recent technique to improve the outcome of difficult syntheses, applicable to sequences containing residues of serine or threonine. A brief overview is presented about chemical aspects of the method, the steps that have been undertaken for its optimization, and the evaluation of its efficiency. Further applications of analogous principles to other critical topics in peptide synthesis such as condensation of peptide segments and solid-phase synthesis of naturally occurring cyclodepsipeptides are addressed as well.

Direct Solid-Phase Synthesis of the β-Amyloid (1−42) Peptide Using Controlled Microwave Heating

Standard linear Fmoc/t-Bu solid-phase synthesis of the 42-mer beta-amyloid (1-42) peptide was achieved under controlled microwave conditions at 86 degrees C using inexpensive DIC/HOBt as coupling reagent on ChemMatrix resin. In order to avoid racemization of the sensitive amino acids, the coupling of the three His residues in the difficult peptide sequence was performed at room temperature. The desired peptide was obtained within 15 h overall processing time in high yield and purity (78% crude yield).

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions.

MCR-ALS analysis of two-way UV resonance Raman spectra to resolve discrete protein secondary structural motifs

The ability of ultraviolet resonance Raman (UVRR) spectroscopy to monitor a host of structurally sensitive protein vibrational modes, the amide I, II, III and S regions, makes it a potentially powerful tool for the visualization of equilibrium and non-equilibrium secondary structure changes in even the most difficult peptide samples. However, it is difficult to unambiguously resolve discrete secondary structure-derived UVRR spectral signatures independently of one another as each contributes an unknown profile to each of the spectrally congested vibrational modes. This limitation is compounded by the presence of aromatic side chains, which introduce additional overlapping vibrational modes. To address this, we have exploited an often overlooked tool for alleviating this spectral overlap by utilizing the differential excitability of the vibrational modes associated with alpha-helices and coil moieties, in the deep UV. The differences in the resonance enhancements of the various structurally associated vibrational modes yields an added dimensionality in the spectral data sets making them multi-way in nature. Through a 'chemically relevant' shape-constrained multivariate curve resolution-alternating least squares (MCR-ALS) analysis, we were able to deconvolute the complex amide regions in the multi-excitation UVRR spectrum of the protein myoglobin, giving us potentially useful 'pure' secondary structure-derived contributions to these individual vibrational profiles.

Solid-Phase Synthesis of Difficult Peptide Sequences at Elevated Temperatures: A Critical Comparison of Microwave and Conventional Heating Technologies

The Fmoc/t-Bu solid-phase synthesis of three difficult peptide sequences (a 9-mer, 15-mer, and 24-mer) was performed using N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on polystyrene, Tentagel, and ChemMatrix resins. In order to obtain an insight into the specific role of the elevated temperature and/or the electromagnetic field for peptide syntheses carried out using microwave irradiation, peptide couplings and Fmoc-deprotection steps were studied under microwave and conventionally heated conditions at the same temperature. While room temperature couplings/deprotections generally produced the difficult peptides in rather poor quality, excellent peptide purities were obtained using microwave heating at a temperature of 86 degrees C for both the coupling and deprotection steps in only 10 and 2.5 min reaction time, respectively. While for most amino acids no significant racemization was observed, the high coupling temperatures led to considerable levels of racemization for the sensitive amino acids His and Cys. It was demonstrated for all three peptide sequences that when performing the coupling/deprotection steps at the same reaction temperature using conventional heating, nearly identical results in terms of both peptide purity and racemization levels were obtained. It therefore appears that the main effect of microwave irradiation applied to solid-phase peptide synthesis is a purely thermal effect not related to the electromagnetic field.

Overview of Solid Phase Synthesis of “Difficult Peptide” Sequences

Solid-phase peptide synthesis has contributed immeasurably to the understanding of the chemistry and biology of peptides by enabling their ready preparation in small quantities up to the ton scale. The advantages of the technology, including its simplicity, ease of operation, and general efficiency have far outweighed its limitations. However, despite the general effectiveness of the solid phase synthesis methodology, some peptides are resistant to efficient assembly and are known as "difficult peptides." Such sequences can present serious challenges to the peptide researcher and have been the subject of considerable investigation. This phenomenon is described together with modern approaches designed to minimize or overcome this hitherto long-standing and vexing problem.