Improved preparation of amyloid-beta peptides using DBU as Nalpha-Fmoc deprotection reagent.

Previous studies have shown the amyloid peptides, Abeta 1-40/42, to be exceptionally difficult to assemble by Fmoc-solid phase peptide synthesis due to the high hydrophobicity of the C-terminal segment and resulting on-resin aggregation. We found that the use of the stronger and more efficient base, DBU, at a concentration of 2% in DMF for Nalpha-Fmoc deprotection allowed substantially improved continuous flow solid phase assembly of the model peptide Abeta 29-40/42 fragments. This suggested that, at least for these sequences, incomplete deprotection was a greater problem than incomplete amino acid acylation. This base was then used during the synthesis of both Abeta 1-40 and Abeta 1-42, up to and including Ser8, from which point 20% piperidine in DMF was utilized so as to avoid potential aspartimide formation at Asp7. By this means, the deprotection efficiency through the difficult C-terminal portion of the sequence was much improved and resulted in increased availability of terminal amino groups for acylation. This simple strategy that obviates the need for special conditions significantly improved crude peptide quality and allowed considerable facilitation of subsequent purification.     

Hydroquinone-O,O'-diacetic acid ('Q-linker') as a replacement for succinyl and oxalyl linker arms in solid phase oligonucleotide synthesis.

When hydroquinone-O,Ooffiacetic acid is used as a linker arm in solid phase oligonucleotide synthesis, the time for NH4OH cleavage of oligodeoxy- or oligoribonucleotides is reduced to only 2 min. This allows increased productivity on automated DNA synthesizers without requiring any other modifications to existing reagents or synthesis and deprotection methods. The Q-linker may also be rapidly cleaved by milder reagents such as 5% NH4OH, potassium carbonate, anhydrous ammonia, t-butylamine or fluoride ion. However, the Q-linker is sufficiently stable for long-term storage at room temperature without degradation and no loss of material occurs during synthesis. The linker is also reasonably resistant to 20% piperidine/DMF, 0.5 M DBU/pyridine and 1:1 triethylamine/ethanol. The Q-linker can therefore serve as a general replacement for both succinyl and oxalyl linker arms.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

Summary By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

Substitution determination of Fmoc‐substituted resins at different wavelengths

In solid‐phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc‐substituted resins, substitution determination is often performed by suspending the Fmoc‐protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene–piperidine adduct that is quantified by ultraviolet–visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene–piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert–Beer's law, together with the substance‐specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol^?1 cm^?1 and 8100 l mol^?1 cm^?1 have been reported for the dibenzofulvene–piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Rapid conditions for the cleavage of oligodeoxyribonucleotides from cis-diol-bearing universal polymer supports and their deprotection

Two sets of deprotection conditions have been evolved for the deprotection of oligodeoxyribonucleotides and their cleavage from commercially available cis -diol group-bearing universal polymer supports. In the first case, oligodeoxyribonucleotides anchored on the universal support were subjected to one of the standard deprotection conditions followed by treatment with aqueous 0.5 M sodium chloride + 0.2 M sodium hydroxide solution for 30 min at room temperature. In the second case, oligonucleotides bound to the universal support were treated with methanolic sodium hydroxide solution under microwave radiation to obtain fully deprotected oligomers within 4 min. Under both conditions, the cleavage of oligonucleotides from the support and their deprotection occurred quantitatively without any side product formation. The cleaved oligonucleotides were found to be identical in all respects (retention time on HPLC and biological activity in PCR) to the corresponding standard oligo-nucleotides.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

The Mechanism of Cleavage Under Basic Conditions of Succinyl-Anchored Oligonucleotides

Abstract

Studies with a model compound provide direct evidence that cleavage of succinyl-anchored oligonucleotides takes place by intramolecular nucleophilic attack of the conjugate base of the succinamide at the ester carbonyl group. An N-substituted succinimide is exclusively formed with piperidine, DBU and TBAF, but when ammonia is used-this general mechanism seems to coexist with ammonolysis of the succinate ester. Cleavage with TBAF is extremely fast.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

The use of N-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite the tert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of the N-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Rapid deprotection procedures for synthetic oligonucleotides.

Two new rapid procedures for the full deprotection of synthetic oligonucleotides has been developed. We have successfully used the mixture of ethanolamine and ethanol (1:1) or pure ethanolamine for deprotection of oligonucleotides, prepared by different methods. In the case of oligonucleotides prepared by commonly used beta-cyanoethyl phosphoramidite and H-phosphonates method deprotection takes half an hour at 70 degrees C. We have found also that mixture of hydrazine, ethanolamine and methanol (1:3:3, v/v/v) can serve as a very efficient reagent for deprotection of oligonucleotides, prepared by beta-cyanoethyl phosphoramidite method with isopropoxyacetyl protecting group for cytosine residues. In this case deprotection time is 12-17 min at room temperature.     

Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives

Abstract

We have investigated the use of alkylamines as fast cleavage and deprotection reagents for the solid phase synthesis of oligonucleotides and found methylamine/ammonium hydroxide (or methylamine) as an efficient reagent. The transamination side product formed with the commonly used dC^bz has been eliminated by the use of dC^Ac phosphoramidite. This system has successfully been used in the synthesis of oligonucleotides and oligonucleoside phosphorothioates. DMT dC^Ac hydrogen phosphonate and DMT ribo C^Ac-2′-O Me phosphoramidite also have been prepared and used in the synthesis of oligonucleotides.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

Summary The use ofN-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite thetert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of theN-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Allyl deprotection of galacturonic acid derivatives: mechanistic aspects of mercuric-catalyzed prop-1-enyl acetal cleavage

Different deallylation methods were assayed for selective deprotection of allyl galactopyranosiduronic acid derivatives. A two-step procedure using DABCO and (Ph(3)P)(3)RhCl followed by mercuric-assisted cleavage gave quantitative yields. Reaction in the presence of [(18)O]water allowed us to obtain evidence about the mechanism of prop-1-enyl cleavage.     

Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis

The formation of 3-(1-piperidinyl)alanyl-containing peptides via phosphoryl β-elimination was identified from the application of Fmoc-Ser(PO₃Bzl,H)-OH in peptide synthesis as shown by RP-HPLC, ES-MS and ³¹P-NMR analysis. An N ^α -deprotection study using the model substrates, Fmoc-Xxx(PO₃Bzl,H)-Val-Glu(O^tBu)-Resin (Xxx = Ser, Thr or Tyr) demonstrated that piperidine-mediated phosphoryl β-elimination occurred in the N-terminal Ser(PO₃Bzl,H) residue at a ratio of 7% to the target phosphopeptide, and that this side reaction did not occur in the corresponding Thr(PO₃Bzl,H)- or Tyr(PO₃Bzl,H)- residues. The generation of 3-(1-piperidinyl)alanyl-peptides was also shown to be enhanced by the use of microwave radiation during Fmoc deprotection. An examination of alternative bases for the minimization of byproduct formation showed that cyclohexylamine, morpholine, piperazine and DBU gave complete suppression of β-elimination, with a 50% cyclohexylamine/DCM (v/v) deprotection protocol providing the crude peptide of highest purity. Piperidine-induced β-elimination was found only to occur in Ser(PO₃Bzl,H) residues that were in the N-terminal position, since the addition of the next residue in the sequence rendered the phosphoseryl residue stable to multiple piperidine treatments. The application of the alternative N ^α -deprotection protocol using 50% cyclohexylamine/DCM (v/v) is therefore recommended for deprotection of the Fmoc group from the Fmoc-Ser(PO₃Bzl,H) residue, with particular benefit anticipated for the synthesis of multiphosphoseryl peptides.     

Engineered polymer-supported synthesis of 3'-carboxyalkyl-modified oligonucleotides and their applications in the construction of biochips for diagnosis of the diseases

An engineered polymer support 5 has been prepared for the solid-phase assembly of 3'-carboxyalkyl-modified oligonucleotides using commonly available reagents. A two-step deprotection procedure resulted in the quantitative cleavage of oligonucleotides from the support and removal of the protecting groups from phosphodiesters and exocyclic amino groups of the nucleic bases. The fully deprotected oligomers, obtained in high yield, were desalted and analyzed on RP-HPLC. After characterization by MALDI-TOF, these carboxyalkylated oligonucleotides were immobilized onto the epoxy-functionalized glass microslides to prepare biochips. The performance of these biochips was evaluated under different sets of conditions and then successfully validated by the detection of base mismatches and human infectious disease, bacterial meningitis, caused by N. meningitidis.     

Regeneration of aged DMF for use in solid‐phase peptide synthesis

Dimethylformamide (DMF), which is still the most commonly used solvent for Fmoc‐SPPS, has the potential for degradation over time on exposure to air (and water vapour) and storage, to give dimethylamine and formic acid impurities. In particular, dimethylamine can lead to unwanted deprotection of the fluorenylmethyloxycarbonyl (Fmoc) group during, for example, the initial loading of Fmoc amino acids in SPPS, which leads reduced calculated loading values. We have found that treatment of such aged DMF by simple sparging with an inert gas (N₂), or vacuum sonication, can regenerate the DMF in order to restore loading levels back to those found for newer, fresh, DMF samples.     

The deprotection of Lys(Mtt) revisited.

The selective deprotection of Lys(Mtt)-containing peptidyl resins was successfully monitored by RP-HPLC using very short linear gradients. RP-HPLC analyses of the acidic filtrates also revealed the partial cleavage of the Trt groups and of the peptide-resin bond. The absorbance of the Mtt carbocation at 470 nm is only twice that of the Trt cation. Thus, the UV monitoring at 470 nm seems to be inappropriate, especially at the end of the deprotection, when the Mtt and the Trt levels are comparable.     

The aspartimide problem in Fmoc-based SPPS. Part III.

A newly developed Fmoc-Asp derivative, Fmoc-Asp beta-(2,3,4-trimethyl-pent-3-yl) ester, has been tried in the Fmoc-based SPPS of H-Val-Lys-Asp-Xaa-Tyr-Ile-OH, a well-established peptide model for studying base-catalysed aspartimide formation. When synthesizing the hexapeptide incorporating Gly, Arg(Pbf), Asn(Mtt), Asp(OtBu) or Cys(Acm) for Xaa, considerable amounts of aspartimide-related by-products were to be expected. The Asp(3) beta-carboxy protecting group and the duration of exposure to bases were varied. By-product formation could be reduced by incorporation of the new Asp derivative more efficiently than by introducing the less bulky Asp(OMpe). Significant improvements were observed in cases of prolonged contact with piperidine or DBU. Both beta-carboxy protecting groups were superior to the standard Asp(OtBu) which was also included in this study, but the additional stabilization gained by our new protecting group was valuable especially in syntheses of long peptides or difficult sequences.     

Convenient synthesis of thioamidated peptides and proteins

The unique physicochemical properties of a thioamide bond, which is an ideal isostere of an amide bond, have not been fully exploited because of the tedious synthesis of thionated amino acid building blocks. Here, we report a purification‐free and highly efficient synthesis of thiobenzotriazolides of Fmoc‐protected and orthogonally protected 20 naturally occurring amino acids including asparagine, glutamine, and histidine. The near‐quantitative conversion to the respective thioamidated peptides on solid support demonstrates the robustness of the synthetic route. Furthermore, the unaltered incorporation efficiency of thiobenzotriazolides from their stock solution till 48 h suggests their compatibility toward automated peptide synthesis. Finally, utilizing an optimized cocktail of 2% DBU + 5% piperazine for fast Fmoc‐deprotection, we report the synthesis of a thioamidated Pin1 WW domain and thioamidated GB1 directly on solid support.