Fast conventional Fmoc solid-phase peptide synthesis with HCTU.

1H-Benzotriazolium 1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate (1-),3-oxide (HCTU) is a nontoxic, nonirritating and noncorrosive coupling reagent. Seven biologically active peptides (GHRP-6, (65-74)ACP, oxytocin, G-LHRH, C-peptide, hAmylin(1-37), and beta-amyloid(1-42)) were synthesized with reaction times reduced to deprotection times of 3 min or less and coupling times of 5 min or less using HCTU as the coupling reagent. Expensive coupling reagents or special techniques were not used. Total peptide synthesis times were dramatically reduced by as much as 42.5 h (1.8 days) without reducing the crude peptide purities. It was shown that HCTU can be used as an affordable, efficient coupling reagent for fast Fmoc solid-phase peptide synthesis.     

Slightly modifying pseudoproline dipeptides incorporation strategy enables solid phase synthesis of a 54 AA fragment of caveolin‐1 encompassing the intramembrane domain

This work contributes to highlight the benefits of pseudoproline dipeptides introduction in difficult SPPS. We show how a slight modification in the positioning choice conditioned the synthesis achievement of a 54 amino acid long caveolin‐1 peptide encompassing the intramembrane domain. Furthermore, we report a side reaction correlated with the coupling steps and generating truncated fragments with a mass deviation of + 42 Da. Considering the need of structural data for membrane proteins, most of which are considered as prevalent therapeutic targets, chemical synthesis provides an interesting alternative pathway to obtain hydrophobic domains by pushing back the frontiers of conventional RP methods of purification. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Resin comparison and fast automated stepwise conventional synthesis of human SDF‐1α

Human SDF‐1α contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 × 2 min and coupling times of 2 × 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP‐HPLC column and the mass of the final product was confirmed with MALDI‐TOF mass spectrometry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of β-sheet formation of amyloid β (Aβ)(1–40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Aβ(15–20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Aβ peptide and assigned them to different secondary components. The result indicates that each intact sample of Aβ(15–20) fragment or Aβ(1–40) peptide previously incubated in distilled water at 37 °C transformed their secondary structure from 1649 (1651) or 1653 cm⁻¹ to 1624 cm⁻¹, suggesting the transformation from -helix and/or random coil structures to β-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 °C, the structural transformation was not found for Aβ(1–40) peptide after 24 h-incubation. But the β-sheet formation of Aβ(1–40) peptide after 48 h-incubation was evidenced from the appearance of the IR peak at 1626 cm⁻¹ by adding a little amount of Aβ(15–20) fragment. There was no β-sheet formation of Aβ(1–40) peptide after addition with much amount of Aβ(15–20) fragment, however, suggesting the higher amount of Aβ(15–20) fragment used might inhibit the β-sheet formation of Aβ(1–40) peptide. The more Aβ(15–20) fragment used made the more stable structure of Aβ(1–40) peptide and the less β-sheet formation of Aβ(1–40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of β-sheet formation of Aβ(1–40) peptide by a short amyloid fragment.     

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

Design, synthesis, and biological evaluation of glycine-based molecular tongs as inhibitors of Abeta1-40 aggregation in vitro

A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4′-oxydianiline spacers, were prepared and tested as inhibitors of β-amyloid peptide Aβ_1–40 aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC₅₀ values in the micromolar range. Structure–activity relationships suggested that π–π stacking and hydrogen-bonding interactions play a key role in the inhibition of Aβ_1–40 self-assembly leading to amyloid fibrils.     

The paradox between m values and deltaCp's for denaturation of ribonuclease T1 with disulfide bonds intact and broken.

Urea-induced denaturations of RNase T1 and reduced and carboxyamidated RNase T1 (RTCAM) as a function of temperature were analyzed using the linear extrapolation method, and denaturation m values, deltaCp, deltaH, deltaS, and deltaG quantities were determined. Because both deltaCp and m values are believed to reflect the protein surface area newly exposed on denaturation, the prediction is that the ratio of m values for RNase T1 and RTCAM should equal the deltaCp ratio for the two proteins. This is not the case, for it is found that the m value of RTCAM is 1.5 times that of RNase T1, while the denaturation deltaCp's for the two proteins are identical. The paradox of why the two parameters, m and deltaCp, are not equivalent in their behavior is of importance in the interpretations of their respective molecular-level meanings. It is found that the measured denaturation deltaCp's are consistent with deltaCp's calculated on the basis of empirical relationships between the change in surface area on denaturation (deltaASA), and that the measured m value of RNase T1 agrees with m calculated from empirical data relating m to deltaASA. However, the measured m of RTCAM is so much out of line with its calculated m as to call into question the validity of always equating m with surface area newly exposed on denaturation.     

The 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides: application to the synthesis of Alzheimer's disease-related amyloid beta peptide (Abeta) 1-42.

An efficient 'O-acyl isopeptide method' for the synthesis of difficult sequence-containing peptides was applied successfully to the synthesis of amyloid beta peptide (Abeta) 1-42 via a water-soluble O-acyl isopeptide of Abeta1-42, i.e. '26-O-acyl isoAbeta1-42' (6). This paper describes the detailed synthesis of Abeta1-42 focusing on the importance of resin selection and the analysis of side reactions in the O-acyl isopeptide method. Protected '26-O-acyl isoAbeta1-42' peptide resin was synthesized using 2-chlorotrityl chloride resin with minimum side reactions in comparison with other resins and deprotected crude 26-O-acyl isoAbeta1-42 was easily purified by HPLC due to its relatively good purity and narrow elution with reasonable water solubility. This suggests that only one insertion of the isopeptide structure into the sequence of the 42-residue peptide can suppress the unfavourable nature of its difficult sequence. The migration of O-acyl isopeptide to intact Abeta1-42 under physiological conditions (pH 7.4) via O--N intramolecular acyl migration reaction was very rapid and no other by-product formation was observed while 6 was stable under storage conditions. These results concluded that our strategy not only overcomes the solubility problem in the synthesis of Abeta1-42 and can provide intact Abeta1-42 efficiently, but is also applicable in the synthesis of large difficult sequence-containing peptides at least up to 50 amino acids. This synthesis method would provide a biological evaluation system in Alzheimer's disease research, in which 26-O-acyl isoAbeta1-42 can be stored in a solubilized form before use and then rapidly produces intact Abeta1-42 in situ during biological experiments.     

Non-nucleoside HIV-1 reverse-transcriptase inhibitors. Part 10. Synthesis and anti-HIV activity of 5-alkyl-6-(1-naphthylmethyl)pyrimidin-4(3H)-ones with a mono- or disubstituted 2-amino function as novel 'dihydro-alkoxy-benzyl-oxopyrimidine' (DABO) analogues

A series of structurally related 2,5-disubstituted 6-(1-naphthylmethyl)-pyrimidin-4(3H)-ones, compounds 6a-6r, were synthesized and evaluated for their in vitro anti-HIV activities in MT-4 cells. Most of the new compounds investigated showed moderate-to-good activities against wild-type HIV-1, with IC(50) values in the range 5.64-0.21 microM. Compound 6d was the most potent congener (IC(50)=0.21 microM, SI=724) in inhibiting HIV-1 replication, which is ca. 25 times more effective than the reference compound 2',3'-dideoxyinosine (DDI). Preliminary structure-activity relationship (SAR) studies revealed that both modulation of the amino function at C(2) and of the alkyl group at C(5) of the pyrimidine ring are crucial for high anti-HIV-1 activity.