Solid‐phase synthesis of peptides containing the spin‐labeled 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a nitroxide spin‐labeled, achiral C^α‐tetrasubstituted amino acid recently shown to be not only an effective β‐turn and 3₁₀/α‐helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid‐phase synthesis in an automated apparatus.     

Synthesis of cyclic peptides from unprotected precursors using removable Nα‐(1‐(4‐methoxyphenyl)‐2‐mercaptoethyl) auxiliary

Abstract: A new method to cyclize unprotected peptides is presented. The method involves the use of a 1‐phenyl‐2‐mercaptoethyl derivative on the N‐terminal glycine. This template acts as an auxiliary thiol‐containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high‐yield cyclization of the peptide GSPYSSDTTPA is described.     

Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

Amino acid–azetidine chimeras: synthesis of enantiopure 3‐substituted azetidine‐2‐carboxylic acids

Abstract: Azetidine‐2‐carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3‐position have been synthesized by modification of 1‐9‐(9‐phenylfluorenyl) (PhF)‐3‐allyl‐Aze tert‐butyl ester (2S,3S)‐ 1 . 3‐Allyl‐Aze 1 was synthesized by regioselective allylation of α‐tert‐butyl β‐methyl N‐(PhF)aspartate 13 , followed by selective ω‐carboxylate reduction, tosylation, and intramolecular N‐alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor‐leucine–Aze chimera 2 . Regioselective olefin hydroboration of (2S,3S)‐ 1 produced primary alcohol 23 , which was protected as a silyl ether, hydrogenated and N‐protected to give 1‐Fmoc‐3‐hydroxypropyl‐Aze 26 . Enantiopure (2S,3S)‐3‐(3‐azidopropyl)‐1‐Fmoc‐azetidine‐2‐carboxylic acid tert‐butyl ester 3 was prepared as a Lys–Aze chimera by activation of 3‐hydroxypropyl–Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an α‐aminoadipate–Aze chimera by oxidation of alcohol 26 . These orthogonally protected amino acid–Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

Synthesis and X‐ray study of the 6‐(N‐pyrrolyl)purine and thymine derivatives of 1‐aminocyclopropane‐1‐carboxylic acid

Abstract: The novel purine and pyrimidine derivatives of 1‐aminocyclopropane‐1‐carboxylic acid 1 and 2 were obtained by alkylation of 6‐(N‐pyrrolyl)purine and thymine with methyl 1‐benzamido‐2‐chloromethylcyclopropanecarboxylate. X‐ray crystal structure analysis shows that the cyclopropane rings in 1 and 2 posses Z‐configuration. The cyclopropane ring atoms and attached atoms of the benzamido and methoxycarbonyl moiety of both molecules are disposed perpendicularly to each other. The carbonyl oxygen of the methoxycarbonyl moiety adopts in both compounds a synperiplanar conformation with respect to the midpoint of the distal bond of the cyclopropane ring. The torsion angles φ and ψ for the 1‐aminocyclopropane‐1‐carboxylic acid residue in 1 and 2 correspond to a folded conformation, while the torsion angles ω define antiperiplanar conformation. Intermolecular hydrogen bonds connect the molecules of 1 into dimers. Each dimer is hydrogen‐bonded with four ethanol molecules, thus forming discrete unit. On the contrary, intermolecular hydrogen bonds link the molecules of 2 generating three‐dimensional network.     

Improved synthesis of 5‐hydroxylysine (Hyl) derivatives*

Abstract: The synthesis of 5‐hydroxylysine (Hyl) derivatives for incorporation by solid‐phase methodologies presents numerous challenges. Hyl readily undergoes intramolecular lactone formation, and protected intermediates often have poor solubilities. The goals of this work were twofold: first, develop a convenient method for the synthesis of O‐protected Fmoc‐Hyl; secondly, evaluate the efficiency of methods for the synthesis of O‐glycosylated Fmoc‐Hyl. The 5‐O‐tert‐butyldimethylsilyl (TBDMS) fluoren‐9‐ylmethoxycarbonyl‐Hyl (Fmoc‐Hyl) derivative was conveniently prepared by the addition of tert‐butyldimethylsilyl trifluoromethanesulfonate to copper‐complexed Hyl[?‐tert‐butyloxycarbonyl (Boc)]. The complex was decomposed with Na⁺ Chelex resin and the Fmoc group added to the α‐amino group. Fmoc‐Hyl(?‐Boc, O‐TBDMS) was obtained in 67% overall yield and successfully used for the solid‐phase syntheses of 3 Hyl‐containing peptides. The preparation of Fmoc‐Hyl[?‐Boc, O‐(2,3,4,6‐tetra‐O‐acetyl‐β‐d ‐galactopyranosyl)] was compared for the thioglycoside, trichloroacetimidate and Koenigs–Knorr methods. The most efficient approach was found to be Koenigs–Knorr under inverse conditions, where Fmoc‐Hyl(?‐Boc)‐OBzl and peracetylated galactosyl bromide were added to silver trifluoromethanesulfonate in 1,2‐dichloroethane, resulting in a 45% isolated yield. Side‐reactions that occurred during previously described preparations of glycosylated Hyl derivatives, such as lactone formation, loss of side‐chain protecting groups, orthoester formation, or production of anomeric mixtures, were avoided here. Research on the enzymology of Lys hydroxylation and subsequent glycosylation, as well as the role of glycosylated Hyl in receptor recognition, will be greatly aided by the convenient and efficient synthetic methods developed here.     

Synthesis of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

An effective organic solvent system for the dissolution of amino acids

Dimethylformamide containing strong acids (CF₃ COOH, HBF₄. TosOH, etc.) and an excess of tertiary base with p^K≤6 is an effective solvent system for the dissolution of amino acids and their derivatives. The preferable base is pyridine, which forms a system with an apparent pH of 5.3. Amino acids dissolved in this solvent system readily interact with acylating reagents (BOC₂O, ZOSu, Fmoc-OSu and activated derivatives of N-protected amino acids). A number of BOC-, Z-, Fmoc-amino acids, as well as several dipeptides, were synthesized using this solvent system with 80-99%) yields. © Munksgaard 1996.     

Synthesis and characterization of human α‐defensins 4‐6

Abstract: Human α‐defensins are small, Cys‐rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high‐quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human α‐defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N‐diisopropylethylamine (DIEA) in situ neutralization/2‐(1 H‐benzotriazolyl)‐1,1,3,3‐tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid‐phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10–16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony‐counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.     

3‐Azido‐aspartic acid derivatives – orthogonally protected precursors for the stereoselective incorporation of 2,3‐diaminosuccinic acid into peptide structures

Abstract: The stereoselective synthesis of orthogonally protected 3‐azido aspartic acid derivatives is described. The convenience of their application as 2,3‐diaminosuccinic acid in peptide chemistry was demonstrated by the incorporation of the nonproteinogenic diamino diacid as a cystine‐substitute into the core structure of somatostatin.     

Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

3‐(3′‐Fluorenyl‐9′‐oxo)‐l‐alanine: a novel photoreactive conformationally constrained amino acid

Abstract: Photoaffinity scanning of the ligand–G‐protein‐coupled receptor bimolecular interface is a direct approach to mapping the interactions of ligands and receptors. Such studies are an important first step toward generating an experimentally based model of the ligand–receptor complex. The synthesis and spectroscopic characterization of Boc‐3‐(3′‐fluorenyl‐9′‐oxo)‐l ‐alanine and 9‐fluorenone‐3‐carboxylic acid are described. Incorporation of these two photophores into the parathyroid hormone (PTH) molecule yields potent agonists. These photoreactive analogs cross‐link specifically with the recombinant human PTH1 receptor stably expressed in human embryonic kidney cells. The availability of the 9‐fluorenone (a conformationally constrained derivative of benzophenone, the abundantly used photophore) for photoaffinity scanning provides an important tool to probe the effect of conformational flexibility of the photophore on the selection of the cross‐linking site in the macromolecular acceptor.     

Synthesis of a carbon‐14 labeled 1‐(indole‐6‐carbonyl‐D‐phenylglycinyl)‐4‐(1‐methylpiperidin‐4‐YL)piperazine‐[carbonyl‐14C], LY517717‐[14C], a factor Xa inhibitor

Human Factor Xa is a trypsin‐like serine protease, which serves a critical role in blood coagulation events. LY517717 is currently under clinical investigation as a Factor Xa inhibitor. To support the ADME studies, LY517717 was synthesized using D ‐phenylglycine with a carbon‐14 labeled carboxyl moiety. This key component, D ‐phenylglycine‐[carboxyl‐¹⁴C], was synthesized by a Strecker synthesis on benzaldehyde with potassium [¹⁴C]cyanide, followed by a resolution of DL ‐phenyl‐glycine methyl ester‐[carbonyl‐¹⁴C] with (+)‐tartaric acid in the presence of benzaldehyde. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of deuterium‐labeled 3‐O‐methyldopa and 4‐O‐methyldopa

Concise methods for the synthesis of 4‐hydroxy‐3‐[²H₃]‐methoxyphenylalanine (3‐O‐[²H₃]‐methydopa) and 3‐hydroxy‐4‐[²H₃]‐methoxyphenylalanine (4‐O‐[²H₃]‐methydopa) are described. The 3‐O‐[²H₃]‐methydopa is a valuable internal standard for the tandem MS quantification of 3‐O‐methyldopa, a metabolite of value in the diagnosis of aromatic l‐amino acid decarboxylase (AADC) deficiency. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of carbon‐14–labelled peptides

Carbon‐14 (¹⁴C)–labelled active pharmaceutical ingredients (APIs) and investigational medicinal products (IMPs) are required for phase 0/I to phase III mass balance and micro‐dosing clinical trials. In some cases, this may involve the synthesis of ¹⁴C‐labelled peptides, and the analysis can be performed by accelerated mass spectrometry (AMS). The ¹⁴C‐peptide is typically prepared by the solid‐phase peptide synthesis (SPPS) approach using custom‐made glassware for the key coupling steps. Further modification of the purified ¹⁴C‐peptide can then be performed.     

Synthesis and Selective Inhibitory Activity Against Human COX‐1 of Novel 1‐(4‐Substituted‐thiazol‐2‐yl)‐3,5‐di(hetero)aryl‐pyrazoline Derivatives

Novel 1‐(4‐ethyl carboxylate‐thiazol‐2‐yl)‐3,5‐di(hetero)aryl‐2‐pyrazoline derivatives were obtained by reacting 3,5‐di(hetero)aryl‐1‐thiocarbamoyl‐2‐pyrazolines with the ethyl ester of α‐bromo‐pyruvic acid. The synthesized compounds were confirmed by spectroscopic data and assayed to evaluate their in vitro ability to inhibit both isoforms of human cyclooxygenase (hCOX). Some derivatives (compounds 5 , 6 , 13 , 16 , and 17 ) displayed promising selectivity against hCOX‐1 in the micromolar range and were shown to have a selectivity index similar or better than the reference drugs (indometacin, diclofenac). The introduction of a phenyl or a 4‐F‐phenyl ring on the C5 associated with a 4‐substituted phenyl or a heteroaryl group on the C3 of (4‐substituted‐thiazol‐2‐yl)pyrazoline derivatives improved the activity against hCOX‐1. Thanks to these preliminary results it could be possible to extend our knowledge of the pharmacophoric requirements for the discovery of new pyrazoline‐based hCOX‐1 inhibitors.     

Regioselective synthesis of 3‐(1H‐indol‐3‐yl)‐5‐(1H‐indole‐3‐carbonyl)‐4‐hydroxyfuroic acids: route to hydroxyfuroic acid‐based insulin receptor activators

A new class of insulin receptor activator with a hydroxyfuroic acid in place of a hydroxyquinone moiety is reported. The synthesis of 3‐(1H‐indol‐3‐yl)‐5‐(1H‐indole‐3‐carbonyl)‐4‐hydroxyfuroic acids ( 26 – 30 ) requires seven major steps. Key elements in the syntheses include (1) sequential preparation of two 4‐(N‐protected indole)‐3‐methoxy‐furoic 2,5‐dicarboxylic esters ( 4 and 6 ); (2) regioselective conversion of the furoic diacid 8 into its C‐5 methyl ester 10 with methyl chloroformate; and (3) acylation of 10 by a 7‐substituted indole under a mild condition. This study demonstrates a feasible route of synthesizing insulin receptor activators with a hydroxyfuroic acid scaffold. Among those hydroxyfuroic acid compounds, compound 28 demonstrates insulin receptor activation potential comparable to Merck's compound 2 with a dihydroxybenzoquinone scaffold. Drug Dev Res 72: 247–258, 2011. © 2010 Wiley‐Liss, Inc.