The influence of a bulky 5-position substituent on the amide isomer equilibrium N-terminal to proline has been explored via the synthesis and analysis of N-(acetyl)proline N′-methylamide (1) and its respective cis-and trans-5-tert-butylproline amide diastereomers 2 and 3. The relative populations of the amide cis-and trans-isomers as well as the energy barriers for amide isomerization of 1-3 in D 2 O were ascertained using NMR with coalescence and magnetization transfer experiments. The relative populations of free C-terminal amide and hydrogen-bonded amide in the γ-turn conformation were also estimated by integrating the N-H stretch absorbances in the FT-IR spectra of 1-3 in CHCl 3 and CCl 4 . In the prolyl peptides, the 5-tert-butyl substituent was found to exhibit profound effects on the amide isomer equilibrium, on the energy barrier for amide isomerization, and on the stability of the γ-turn conformation. Steric interactions between the 5-position substituent and the N-acetyl group disfavor the amide trans-and augment the cis-isomer population: 25% in 1, 48% in 2, and 66% in 3. In the case of cis-5-tert-butylproline 2, the energy barrier for amide isomerization is observed to be 3.9 kcal/mol lower than that of 1. On the other hand, the amide isomerization barrier for trans-5-tert-butylproline 3 is similar to that for 1. Only a single amide N-H stretch band is observed at 3454 cm -1 in the FT-IR spectrum of 3 in CHCl 3 and indicates that the NH group is free of intramolecular hydrogen bonding. Hence, trans-5-tert-butylproline amide 3 does not adopt a seven-membered γ-turn conformation, which is a favored conformer for 1 and 2 in CHCl 3 . Maps, in which the ψ-and ω-dihedral angles are plotted at 30°intervals against the calculated energy of the local minimum conformation, predict qualitatively and display clearly all of the observed effects of the 5-tert-butyl substituent on the amide isomer in the N-(acetyl)-proline N′-methylamides. The results of this study suggest the use of 5-tert-butylprolines to prepare both X-Pro cis-amide isomers and twisted amide surrogates for examining prolyl residue conformations in bioactive peptides.
While nature exploits folded biopolymers to achieve molecular recognition and catalysis, comparable abiological heteropolymer systems have been difficult to create. We synthesized and identified abiological peptoid heteroploymers capable of binding a dye. Using combinatorial synthesis, we constructed a library of 3400 amphiphilic 15-mer peptoids on an ultra-high-capacity beaded support. Individual macrobeads, each containing a single peptoid sequence, were arrayed into plates, cleaved, and screened in aqueous solution to locate dye binding heteropolymer assemblies. Resynthesis and characterization demonstrated the formation of defined helical assemblies as judged by size-exclusion chromatography, circular dichroism, and analytical ultracentrifugation. Inspired by nature's process of sequence variation and natural selection, we identified rare abiological sequence-specific heteropolymers that begin to mimic the structure and functional properties of their biological counterparts.
-Amyloid peptides (A) that form the senile plaques of Alzheimer disease consist mainly of 40-and 42-amino acid (A 40 and A 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of A involves -secretase and ␥-secretase activities and is regulated by membrane trafficking of the proteins involved in A production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks A 40 and A 42 production but does not impact sAPP␣ levels and does not inhibit -secretase. Rather, EHT 1864 modulates APP processing at the level of ␥-secretase to prevent A 40 and A 42 generation. This effect does not result from a direct inhibition of the ␥-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces A 40 and A 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting A formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease. Alzheimer disease (AD)2 is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterized by the presence of amyloid plaques (1), intracellular neurofibrillary tangles, and pronounced cell death. The -amyloid peptide (A) (2) is the main constituent of senile plaques found in AD brains. Furthermore, extracellular A 42 appears toxic to neurons in vitro and in vivo (reviewed in Ref.3). A is generated by proteolysis of an integral membrane protein, the amyloid precursor protein (APP), via at least two post-translational pathways. The amyloidogenic cleavage of APP is a sequential processing of APP initiated by -secretase (BACE), which cleaves APP within the luminal domain or at the cell surface, generating the N terminus of A (4). This cleavage generates several membrane-bound proteolytic C-terminal fragments (CTFs), such as the 99-residue -CTF (also called C99), as well as the secreted APP ectodomain sAPP. The C terminus of A is subsequently generated by intramembranous cleavage of CTFs by ␥-secretase, producing either A 40 or A 42. The cleavages at residues 40 -42 are referred to as ␥-cleavage, and the cleavages at residues 49 -52 are referred to as ⑀-cleavage (5). The nonamyloidogenic cleavage of APP, which precludes A generation, is mediated by ␣-secretase, a disintegrin and metalloproteinase 10, and a disintegrin and metalloproteinase 17, in a reaction believed to occur primarily on the plasma membrane. This proteolytic cleavage by ␣-secretase occurs within the A region and produces soluble APP (sAPP␣), the dominant processing product, and the residual membrane-bound 10-kDa CTF (CTF␣, also called C83). Like C99, C83 is a subs...
Steric effects on the isomer equilibrium of amides N-terminal to proline can be explored with 5-alkylprolines having bulky 5-position substituents. Enantiopure 5-tert-butylprolines were thus synthesized from glutamic acid via an acylation/diastereoselective reductive amination sequence. Double deprotonation of γ-methyl N-(PhF)glutamate (2) with LiN(SiMe 3 ) 2 and C-acylation with pivaloyl chloride provided β-keto ester 3, which upon γ-ester hydrolysis and decarboxylation gave δ-oxo-R-[N-(PhF)amino]heptanoic acid (4). Syntheses of (2S,5R)-and (2R,5S)-N-(BOC)-5-tertbutylprolines ((2S,5R)-1 and (2R,5S)-1) were accomplished by catalytic hydrogenation of their respective (2S)-and (2R)-methyl δ-oxo-R-[N-(PhF)amino]heptanoates ((2S)-5a and (2R)-5a) in methanol with di-tert-butyl dicarbonate followed by chromatography and ester hydrolysis with potassium trimethylsilanolate. The 5-tert-butylproline cis-diastereomers were proven to be of >99% enantiomeric purity after their conversion to diastereomeric R-methylbenzylamides 10. Good diastereoselectivity in favor of the trans-diastereomer was observed when (2S,5S)-5-tert-butylproline was synthesized from (2S)-δ-oxo-R-[N-(PhF)amino]heptanoate ((2S)-4) by solvolysis of the PhF group in trifluoroacetic acid and subsequent reduction of 5-tert-butyl-∆ 5 -dehydroproline (11) with tetramethylammonium triacetoxyborohydride; however, imino acid 11 was shown to be configurationally labile and racemized under acidic conditions. 5-tert-Butyl-∆ 5 -dehydroproline N′methylamide 15 was configurationally stable in acid, yet preliminary attempts to reduce 15 favored cis-diastereomer 16. Alternatively, enantiopure trans-diastereomer, (2R,5R)-methyl N-(BOC)-5tert-butylprolinate (9) was prepared by epimerization of (2S,5R)-9. In summary, this synthetic methodology now provides access to all four enantiopure 5-tert-butylproline isomers from inexpensive L-and D-glutamate as chiral educts.
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