Fmoc Amino Research Articles

Synthesis and evaluation of Fmoc-amino esters and amides bearing a substrate like quaternary ammonium group as selective butyrylcholinesterase inhibitors

The depletion of the neurotransmitter acetylcholine has been suggested to contribute to the reduced cognitive function observed in individuals suffering from neurodegenerative diseases such as Alzheimer’s Disease (AD). For the two major cholinesterases, butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), increased BChE activity observed in individuals with AD has been suggested to deplete acetylcholine levels. To reduce acetylcholine degradation and help restore the pool of the neurotransmitter, specific and potent BChE inhibitors are sought. Our previous findings have identified 9-fluorenylmethoxycarbonyl (Fmoc) amino acid-based inhibitors as effective BChE inhibitors. The amino acid-based compounds offered the opportunity to survey a range of structural features to enhance interactions with the enzyme active site. As enzymes interact with features of their substrates, incorporation of substrate-like features was predicted to lead to better inhibitors. Specifically, incorporation of a trimethylammonium moiety to mimic the cationic group of acetylcholine may lead to increased potency and selectivity. To test this model, a series of inhibitors bearing a cationic trimethylammonium group were synthesized, purified, and characterized. While the Fmoc-ester derivatives inhibited the enzyme, additional experiments showed the compounds acted as substrates and were enzymatically hydrolyzed. Inhibition studies with the Fmoc-amide derivatives showed that the compounds do not act as substrates and selectively inhibit BChE with IC50 values in the 0.06–10.0 µM range. Computational docking studies suggest that the inhibitors can interact with cholinyl binding site and peripheral site. Overall, the results suggest that introducing substrate-like characteristics within the Fmoc-amino acid-based background increases their potency. The versatile and ready access to amino acid-based compounds offers an attractive system to further our understanding of the relative importance of protein-small molecule interactions while guiding the development of better inhibitors.

Examining Fmoc‐amino acid derivatives as selective inhibitors of butyrylcholinesterase

The design of enzyme inhibitors to act as drugs or as substrates for biocatalysts remains a challenge in biochemistry, pharmacology, and biotechnology. To mitigate the effects of Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and the sixth leading cause of death in the United States, numerous enzyme inhibitors have been developed. Activity of the enzyme butyrylcholinesterase (BChE) increases in Alzheimer's patients, and this increase is suggested to lead to a depletion of the neurotransmitter, acetylcholine. To counter this increase in activity BChE‐specific inhibitors have been developed to manage the progression of AD. We hypothesized 9‐fluorenylmethyloxycarbonyl (Fmoc) amino acids could provide a new class of inhibitors, as these compounds features found in BChE inhibitors currently available as pharmaceuticals. The readily available chemistry makes amino acids an attractive scaffold for identifying BChE inhibitors. We found Fmoc‐Leu‐O− as an effective and selective BChE inhibitor with an inhibition constant (KI) value of 115 μM. A series of Fmoc‐Leu‐O− derivatives with side chain modifications were then evaluated. The Fmoc‐amino acids were purchased or synthesized from the corresponding amino acid. The KI value for each compound was determined using kinetics assays and UV‐absorbance spectroscopy. For the four Fmoc‐Leu‐O− derivatives tested, Fmoc‐neopentylglycine‐O− showed the greatest potency with an inhibition constant of 36 μM, ~three‐fold lower than the original compounds. A series of Fmocdi‐, tri‐, tetra‐, and pentapeptides were synthesized by solid phase peptide synthesis. The products were purified by HPLC and characterized by NMR and mass spectrometry. Our initial results identified the dipeptide (Fmoc‐Leu‐Leu‐O−) as an effective inhibitor with KI value of 28 μM. We are currently investigating if longer Fmoc‐Leu‐O− peptides and other Fmoc‐peptides are potent and selective BChE inhibitors. The results explore a new class of potent and selective BChE inhibitors and may identify features important for binding in the enzyme active site.Support or Funding InformationThis project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) under award numbers T34GM008074, UL1GM118979, and RL5GM118978, and the National Science Foundation (NSF; MRI CHE‐1337559). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curtius Rearrangement Using Ultrasonication: Isolation of Isocyanates of Fmoc-Amino Acids and Their Utility for the Synthesis of Dipeptidyl Ureas

An efficient conversion of NI±-(P-fluorenylmethytyoxy carbonyl (Fmoc) amino acid azides to the corresponding isocyanates using ultrasound is described. The Curtius rearrangement was accomplished using acid azides in toluene solution as well as solid powder at room temperature. All isocyanates synthesized have been obtained as crystalline solids and were characterized. Coupling of isocyanates with amino acid methyl ester hydrochloride salts in presence of N-methylmorpholine (NMM) resulted in Fmoc-protected dipeptidyl urea esters, which have been well characterized by 1H NMR, 13C NMR and mass spectrometry. © 2005 Springer Science+Business Media, Inc.

Evidence for Domain-specific Recognition of SK and Kv Channels by MTX and HsTx1 Scorpion Toxins

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

ChemInform Abstract: (Fluoren‐9‐ylmethoxy)carbonyl (Fmoc) Amino Acid Azides: Synthesis, Isolation, Characterization, Stability and Application to Synthesis of Peptides.

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Synthesis of azapeptides by the Fmoc/tert-butyl/polyamide technique

A new synthesis of azapeptides for use in the study of a proteolytic enzyme associated with Alzheimer's disease is described. The method utilizes fluoren-9-ylmethoxycarbonyl (Fmoc) amino acid carbazates and hydrazides in the Fmoc/tert-butyl/polyamide technique. The preparation of these compounds is presented. Reaction of Fmoc-amino acid hydrazides with an appropriate aldehyde, followed by reduction, gave fully protected amino acid carbazate dipeptide synthon. These derivatives were used to prepare aza amino acid peptide analogues by reaction with a resin-bound amino group, activated with bis-2,4-dinitrophenyl carbonate in the presence of a base. With this activation of the amino group, hydantoin is formed in a major side-reaction, but the cyclisation could be virtually eliminated by omission of the base from the activation procedure. Upon final trifluoroacetic acid-mediated cleavage of the azapeptide, trifluoroacetylation of the N-terminal serine residue was observed.

ChemInform Abstract: A Cleavage Method which Minimizes Side Reactions Following Fmoc Solid Phase Peptide Synthesis.

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.