The triphenylphosphine-sulfur trioxide adduct as a coupling reagent in peptide synthesis

Several adducts of sulfur trioxide with electron-donor molecules have been prepared. Of these the air-stable triphenylphosphine-SO₃ complex has been shown to be the most useful in the synthesis of peptides. It has been demonstrated that a variety of solvents may be used for couplings, but that in order for reaction to occur the solvent must itself be able to form an adduct. This behaviour is due to the fact that SO₃ is transferred from the triphenylphosphine-SO₃ complex to the solvent, thus forming an intermediate complex which is responsible for activation of the carboxylate unit. The carboxyl component is generally present as its tetramethylguanidinium salt. The method has been used to synthesize several model peptides and to prepare a fully biologically active sample of methionine-enkephalin.     

Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis

Benzotriazole‐based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low‐cost coupling reagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole coupling reagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole‐based HBTU and HCTU for use in in situ neutralization Boc‐SPPS. Difficult sequences, such as ACP(65–74), Jung–Redeman 10‐mer, and HIV‐1 PR(81–99), were used as model target peptides on polystyrene‐based resins, as well as polyethylene glycol‐based resins. Coupling yields obtained using fast in situ Boc‐SPPS cycles were determined with the quantitative ninhydrin test as well as via LC‐MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene‐based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.     

Synthesis of sterically hindered peptide analogs using diphenyl phosphite as the coupling reagent

Some model sterically hindered peptide analogs were synthesized with various yields using diphenyl phosphite as the coupling reagent under mild conditions. The experimental procedure is straightforward and the products are easily isolated. This method may be convenient and efficient for the synthesis of hindered peptides.     

Dipentafluorophenyl carbonate--a reagent for peptide synthesis

Dipentafluorophenylcarbonate, belonging to transesterifiying reagents, has been prepared and used for the synthesis of pentafluorophenyl esters of amino acids. In contrast to many other reagents of the kind, its preparation is simple, it is highly reactive and at the same time stable upon storage.     

Search for the optimal linker in tandem hairpin polyamides

In order to target specific DNA sequences >or=10 base pairs in size by minor groove binding ligands, a search for the optimal linker in dimers of hairpin polyamides was initiated. Two series of tandem polyamides ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-L](HN)gamma-PyPyPy-beta-Dp (1a-e), where L represents a series of 4-8 carbon long aliphatic amino acid linkers, and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-L](HN)gamma-PyPyPy-beta-Dp (2a-e), where L represents a series of 2-6 carbon long aliphatic amino acid linkers, were synthesized and characterized by quantitative DNase I footprinting. beta, gamma and Dp represents beta-alanine, gamma-aminobutyric acid, and 3-(dimethylamino)propylamine, respectively. It was found that the five-carbon 5-aminovaleric acid (delta), is suitable to span one base-pair (bp) of DNA when incorporated into a tandem polyamide. ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-delta](HN)gamma-PyPyPy-beta-Dp (1b) binds the 10 bp binding-site 5'-AGTGAAGTGA-3' with equilibrium association constant K(a)=3.2 x 10(10) M(-1) and ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPyIm-delta](HN)gamma-PyPyPy-beta-Dp (2d) binds the 11 bp binding-site 5'-AGTGATAGTGA-3' with K(a)=9.7 x 10(9) M(-1). Tandem 1b also bind the 11 bp site but with lower affinity affording a 15-fold specificity for the shorter binding site. Replacing a methylene group in the amino acid linker with an oxygen atom to form tandem polyamide ImPyIm-(R)[ImPyIm-(R)(H2N)gamma-PyPyPy-E](HN)gamma-PyPyPy-beta-Dp (4) where E represents the ether linker, resulted in that an 80-fold specificity for the 10 bp binding site over the 11 bp site.     

A potent peptide affinity reagent for the opiate receptor

The synthesis and characterization of a novel enkephalin analogue, Tyr-D-Ala-Gly-Phe-Leu-chloromethyl ketone, is described. The biological potency of the compound in various assays has been determined to be very high. The compound is an alkylating affinity reagent and irreversibly inactivates a defined population of enkephalin receptors in rat brain membrane preparations, as well as irreversibly inhibiting electrically stimulated contractions in the mouse vas deferens tissue preparation.     

A novel reagent, dialkylphosphite, for peptide synthesis

The same dialkylphosphite reagent can be used for both N protection and C activation of amino acids. Two N-diisopropyloxyphosphinyl (Dipp) tripeptide esters were prepared, and nine N-Dipp-dipeptide acids were synthesized through the activated amide intermediate. The positive ion FAB-MS of N-Dipp-tripeptide showed novel cleavage patterns in that only the N-phosphoryl fragment ions gave intense peaks while the C-terminal series ions did not appear. This novel character might be useful for peptide sequence analysis. In addition, dialkyloxyphosphinyl group can be examined by 31P-NMR for peptide conformational analysis and inspection for the degree of racemization during the coupling reaction.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Search for optimal scheme for administering live allantoic influenza vaccine

The effect of vaccine dilution and composition on indices of vaccination activity was studied in a live allantoic influenza vaccine administered intranasally to adults. The vaccine was shown to be usable in a 1:1 (instead of 1:5) dilution without risk of its reactogenicity rising. An inhibition of component A(H3N2) was observed in a bivalent vaccine obtained by combining directly before use two monovalent vaccines, A(H1N1) and A(H3N2). The conclusions and recommendations based on the results of the present study have practical implications in influenza control.     

High-throughput peptide quantification using mTRAQ reagent triplex

Background

Continuing research into the global multiple sequence alignment problem has resulted in more sophisticated and principled alignment methods. Unfortunately these new algorithms often require large amounts of time and memory to run, making it nearly impossible to run these algorithms on large datasets. As a solution, we present two general methods, Crumble and Prune, for breaking a phylogenetic alignment problem into smaller, more tractable sub-problems. We call Crumble and Prune meta-alignment methods because they use existing alignment algorithms and can be used with many current alignment programs. Crumble breaks long alignment problems into shorter sub-problems. Prune divides the phylogenetic tree into a collection of smaller trees to reduce the number of sequences in each alignment problem. These methods are orthogonal: they can be applied together to provide better scaling in terms of sequence length and in sequence depth. Both methods partition the problem such that many of the sub-problems can be solved independently. The results are then combined to form a solution to the full alignment problem.

Results

Crumble and Prune each provide a significant performance improvement with little loss of accuracy. In some cases, a gain in accuracy was observed. Crumble and Prune were tested on real and simulated data. Furthermore, we have implemented a system called Job-tree that allows hierarchical sub-problems to be solved in parallel on a compute cluster, significantly shortening the run-time.

Conclusions

These methods enabled us to solve gigabase alignment problems. These methods could enable a new generation of biologically realistic alignment algorithms to be applied to real world, large scale alignment problems.     

(1H-benzotriazol-1-yloxy)-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid-phase peptide synthesis.

A HOBt-based immonium-type compound,(1H-benzotriazol-1-yloxy)-N,N-dimethyl methaniminium hexachloroantimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt-based phosphonium- and uronium-type coupling reagents. Moreover, its racemization was lower than that of other HOBt-derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu-enkephalin both in solution and in the solid-phase.     

Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic

The method for searching for ligands exerting an adjuvant effect is described. The method involves isolation of polysomes using an immobilized peptide mimetic of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) — RN-peptide. After the affinity chromatography and washing, RN-peptide complexes with the target sequences were dissociated with guanidine hydrochloride. The obtained mRNA was used for cDNA synthesis and subsequent cloning in an expression vector. Further studies showed the effectiveness of this method. Clones interacting with the peptide were selected using biotinylated RN-peptide. It was found that all clones encode a sequence identical to the protein YB-1. Recombinant antibodies against protein YB-1 were selected from a phage display human scFv library. Using these antibodies, we determined the binding constant of RN-peptide to protein YB-1. Competitive analysis showed that RN-peptide and GMDP compete for the same portion of YB-1 at molar ratio 1: 12.     

2-Chloro-1-methylpyridinium iodide. A suitable reagent for peptide synthesis

2-Chloro-1-methylpyridinium iodide is a coupling reagent for peptide synthesis. By its use different peptides were synthesized with protected di- and trifunctional amino acids. While urethan-protected amino acids react free of racemization, the fragment condensation needs N-Hydroxysuccinimide, as shown by the Young test.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of beta-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent

A heterobifunctional linking reagent containing a masked aldehydo group and acyl hydrazide was synthesized for coupling of glycopeptides and other amino-containing compounds to proteins. After conversion to acyl azide, the reagent reacts with the amino group of a glycopeptide, and the modified glycopeptide is deacetalized with a weak acid to unmask the aldehydo group, which is then conjugated to bovine serum albumin (BSA) by reductive alkylation with pyridine-borane. The overall reaction scheme proceeds under relatively mild conditions. When the protein amino group was in a large excess (greater than 6-fold) of the aldehyde reagent, the efficiency of conjugation was as high as 88% even at submicromole levels. As a test case for application of this reagent, 6-aminohexyl beta-D-galactopyranoside (Gal-AH) was attached to the linking reagent and conjugated to BSA at various aldehyde-to-protein molar ratios ranging from 25 to 200. The level of O-galactosyl residue incorporated into BSA by this reagent far exceeded that observed in a similar reductive alkylation involving S-galactoside reagents [Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. By use of the present conjugating procedure, as many as 112 mol of Gal-AH residues were incorporated per mole of BSA, which represents near total modification of the amino groups. Some binding characteristics of the new BSA derivatives were studied in the mammalian hepatic galactose/N-acetylgalactosamine specific lectin system along with other types of BSA derivatives (containing S-galactosyl residues). In general, the behavior of the new derivatives was similar to that of other types. For instance, the affinity increased exponentially at low sugar substitution levels (up to 30 mol of galactosyl residues/mol of BSA), and the slope of exponential increase and affinity at a given sugar substitution level was similar to those of other types.