Direct Aminolysis Research Articles

Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis

The synthesis of constrained 12-membered rings is notably difficult. The main challenges result from constraints during the linear peptide cyclization. Attempts to overcome constraints through excessive activation frequently cause peptidyl epimerization, while insufficient activation of the C-terminus hampers cyclization and promotes intermolecular oligomer formation. We present a β-thiolactone framework that enables the synthesis of cyclo-tetrapeptides via direct aminolysis. This tactic utilizes a mechanism that restricts C-terminal carbonyl rotation while maintaining high reactivity, thereby enabling efficient head-to-tail amidation, reducing oligomerization, and preventing epimerization. A broad range of challenging cyclo-tetrapeptides ( > 20 examples) are synthesized in buffer and exhibits excellent tolerance toward nearly all proteinogenic amino acids. Previously unattainable macrocycles, such as cyclo-L-(Pro-Tyr-Pro-Val), have been produced and identified as μ-opioid receptor (MOR) agonists, with an EC50 value of 2.5 nM. Non-epimerizable direct aminolysis offers a practical solution for constrained peptide cyclization, and the discovery of MOR agonist activity highlights the importance of overcoming synthetic challenges for therapeutic development.

Carbon-Nitrogen Bond Formation Using Sodium Hexamethyldisilazide: Solvent-Dependent Reactivities and Mechanisms.

The solvent-dependent reactivity of sodium hexamethyldisilazide (NaHMDS) toward carbon-centered electrophiles reveals reactions that are poorly represented or unrepresented in the literature, including direct aminolysis of aromatic methyl esters to give carboxamides, nitriles, or amidines, depending on the choice of solvent. SNAr substitutions of aryl halides and opening of terminal epoxides are also examined. A combination of 1H and 29Si nuclear magnetic resonance (NMR) spectroscopic studies using [15N]NaHMDS, kinetic studies, and computational studies reveals the complex mechanistic basis of the preferences for simple aryl carboxamides in toluene and dimethylethylamine and arylnitriles or amidines in tetrahydrofuran (THF). A prevalence of dimer- and mixed dimer-based chemistry even starting from the observable NaHMDS monomer in THF solution is notable.

Biomimetic construction of phospholipid membranes by direct aminolysis ligations.

Construction of artificial cells requires the development of straightforward methods for mimicking natural phospholipid membrane formation. Here we describe the use of direct aminolysis ligations to spontaneously generate biomimetic phospholipid membranes from water-soluble starting materials. Additionally, we explore the suitability of such biomimetic approaches for driving the in situ formation of native phospholipid membranes. Our studies suggest that non-enzymatic ligation reactions could have been important for the synthesis of phospholipid-like membranes during the origin of life, and might be harnessed as simplified methods to enable the generation of lipid compartments in artificial cells.

The structure and role of lactone intermediates in linkage-specific sialic acid derivatization reactions

Sialic acids occur ubiquitously throughout vertebrate glycomes and often endcap glycans in either α2,3- or α2,6-linkage with diverse biological roles. Linkage-specific sialic acid characterization is increasingly performed by mass spectrometry, aided by differential sialic acid derivatization to discriminate between linkage isomers. Typically, during the first step of such derivatization reactions, in the presence of a carboxyl group activator and a catalyst, α2,3-linked sialic acids condense with the subterminal monosaccharides to form lactones, while α2,6-linked sialic acids form amide or ester derivatives. In a second step, the lactones are converted into amide derivatives. Notably, the structure and role of the lactone intermediates in the reported reactions remained ambiguous, leaving it unclear to which extent the amidation of α2,3-linked sialic acids depended on direct aminolysis of the lactone, rather than lactone hydrolysis and subsequent amidation. In this report, we used mass spectrometry to unravel the role of the lactone intermediate in the amidation of α2,3-linked sialic acids by applying controlled reaction conditions on simple and complex glycan standards. The results unambiguously show that in common sialic acid derivatization protocols prior lactone formation is a prerequisite for the efficient, linkage-specific amidation of α2,3-linked sialic acids, which proceeds predominantly via direct aminolysis. Furthermore, nuclear magnetic resonance spectroscopy confirmed that exclusively the C2 lactone intermediate is formed on a sialyllactose standard. These insights allow a more rationalized method development for linkage-specific sialic derivatization in the future.

Chemoenzymatic Synthesis and Anti-Inflammatory Activity of Fatty Acid Amides Prepared from Bertholletia excelsa (Brazil Nut) Triglycerides

This study reports the synthesis of fatty acid amides (FAA), analogous to anandamide, prepared from Brazil nut oil (BNO), using Amano lipase from Pseudomonas fluorescens as a catalyst of direct aminolysis, with a good yield (95-68%). The anti-inflammatory effects of BNO and FAA were evaluated by the carrageenan-induced paw edema method. Analysis of the formation of edema showed that these FAA, at all dose used, significantly reduced the development of edema.

Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts.

Ubiquitin (Ub)-based fluorescent reagents are crucial to explore the activity of deubiquitinases (DUBs). Ub-Rho110-G is one of the preferred tools, whereas the current synthetic route is time-consuming. Here, we report a new semisynthetic strategy to produce Ub-Rho110-G through direct aminolysis of Boc-protected Ub-Mesna using bisglycyl-rhodamine 110. We also applied this strategy to synthesize active SUMO2-Rho110-G for the first time. Biochemical analysis demonstrated that semisynthetic Ub or SUMO-Rho110-G can be effectively used for the detection of the activity of DUBs or SUMO-specific enzymes.

Functional hydrophilic highly biodegradable PCL nanofibers through direct aminolysis of PAMAM dendrimer

Polycaprolactone (PCL) was modified with second-generation poly(amidoamine) dendrimer (PAMAM-G2) in electrospinning solution through aminolysis of PCL ester bonds. Two factors affected the modification of PCL in the presence of PAMAM dendrimer. One of them was the concentration of PAMAM, and the other one was the mixing time with PCL. Therefore, PCL/PAMAM nanofiber scaffolds with the ratios of 85:15, 75:25, and 65:35 and mixing times of 1 h and 3 h were fabricated. The presence of NH2 terminal groups of dendrimers and OH groups formed after aminolysis reduced the contact angle from 98° for pure PCL to 9° for a PCL/PAMAM with a ratio of 65:35 mixed for three hours. The degradability test showed that 65:35 specimen lost 47.5% of its initial weight after 8 weeks while for pure PCL was less than 3%. To evaluate the host-guest properties of PCL/PAMAM nanofiber, curcumin was incorporated into the solution before electrospinning as a drug model. MTT assay, cell morphology study and anticancer activity of PCL/PAMAM/curcumin nanofibers against breast cancer cells (MCF-7) showed that by increasing the concentration of dendrimer from 15% to 35%, curcumin loading in the sample increased and inhibition of cancer cells increased from 31.5% to 42.4%, respectively.

Efficient semi-synthesis of ubiquitin-7-amino-4-methylcoumarin

Ubiquitin (Ub)-based probe Ub-7-amino-4-methylcoumarin is widely used to detect deubiquitinase (DUB) enzymatic activity, but its current synthesis procedures are cumbersome. In this work, we reported a new highly efficient semi-synthetic strategy to prepare Ub-AMC through two facile steps: first, preparation of Ub-thioester through hydrozinolysis-thioesterification of expressed Ub (1-G76C) mutant; second, direct aminolysis of Ub-thioester in a mixed-solvent system which exhibits very low hydrolysis.

Glycopeptide ligation via direct aminolysis of selenoester

Direct aminolysis of selenoester in aqueous media was investigated as a glycopeptide ligation strategy. This strategy allows the peptide and glycopeptide ligation to proceed smoothly (even with hindered amino acids) without the need of cysteine residue, N-terminal thiol auxiliary or coupling additive, and to afford the corresponding amide products in excellent yields. No epimerization was observed during ligation reations. In this work, the selenoester of unprotected glycopeptide was readily prepared, and the direct aminolysis of glycopeptide selenoester was successfully applied to synthesize MUC1 mucin sequence efficiently.

Coupling of sterically demanding peptides by β-thiolactone-mediated native chemical ligation.

The ligation of sterically demanding peptidyl sites such as those involving Val-Val and Val-Pro linkages has proven to be extremely challenging with conventional NCL methods that rely on exogenous thiol additives. Herein, we report an efficient β-thiolactone-mediated additive-free NCL protocol that enables the establishment of these connections in good yield. The rapid NCL was followed by in situ desulfurization. Reaction rates between β-thiolactones and conventional thioesters towards NCL were also investigated, and direct aminolysis was ruled out as a possible pathway. Finally, the potent cytotoxic cyclic-peptide axinastatin 1 has been prepared using the developed methodology.

Extremely Stable Supramolecular Hydrogels Assembled from Nonionic Peptide Amphiphiles.

Peptide hydrogels with high stability in different media are of great interest in biomedical applications. In this paper, we report an easy, fast, and scalable method for preparing a family of nonionic peptide amphiphiles (PAs) obtained by direct aminolysis of alkyl-oilgo(γ-benzyl-l-glutamate) samples, which were synthesized via the alkyl amine-initiated sequence ring-opening reaction of α-amino acid N-carboxyanhydrides. One great advantage of this method is that vast chemical diversity and large-scale yields can be achieved easily using commercially available hydramines. These PA samples can readily form a clear hydrogel without any external aid and show exceptionally enhanced gelation properties with a critical gelation concentration as low as 0.05 wt %. The hydrogels are highly stable against extreme pH values of 1 and 14 and a high salt concentration of 200 mM NaCl. These properties combined with the shear-thinning properties make these PA hydrogels ideal candidates for the new generation of injectable scaffolds.

Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation.

Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

Direct Aminolysis of Ethoxycarbonylmethyl 1,4-Dihydropyridine-3-carboxylates.

The ethoxycarbonylmethyl esters of 1,4-dihydropyridines were directly converted into carbamoylmethyl esters in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) in good to excellent yields under mild conditions. The use of TBD is crucial for the successful aminolysis of ethoxycarbonylmethyl ester of 1,4-dihydropyridines with secondary amines as without it the reaction does not proceed at all. The aminolysis reaction proceeded regioselectively, as the alkyl ester conjugated with the 1,4-dihydropyridine cycle was not involved in the reaction. Screening of other N-containing bases, such as triethylamine (TEA), pyridine, 4-(N,N-dimethylamino)pyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), imidazole, tetramethyl guanidine (TMG) and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) as catalysts revealed no activity in the studied reaction.

Facile Synthesis of 4-Oxo-4H-quinolizine-2-carboxamide Derivatives

A facile synthetic method for the construction of 2-substituted-4-oxo-4H-quinolizine-based core structure has been successfully developed. The synthesis made use of a one-pot Stobbe condensation followed by cyclization starting from the commercially available 2-pyridinecarbaldehyde. The structure of the formed 4-oxo-4H-quinolizine-2-carboxylate was fully confirmed by mass spectra, 1H NMR and 13C NMR, correlation spectrography, heteronuclear multiple bond correlation, and heteronuclear single quantum coherence (HSQC) spectra. The ethyl carboxylate moiety was then further functionalized via direct aminolysis by a range of amines to afford the corresponding 4-oxo-4H-quinolizine-2-carboxamides 4a–i in moderate to good yields.

One step synthesis of fatty acid diethanolamides and methyl esters from triglycerides using sodium doped calcium hydroxide as a nanocrystalline heterogeneous catalyst

A recyclable heterogeneous catalyst based direct aminolysis of vegetable oils.

Microwave irradiation: synthesis and characterization of α-ketoamide and bis (α-ketoamide) derivatives via the ring opening of N-acetylisatin.

BackgroundThe carbonyl group at position 2 of N-acetylisatin behaves as an amide which is more susceptible to nucleophilic attack via ring-opening in the presence of nucleophiles. Because of this behavior, in the present work we describe the microwave synthesis of a series of α-ketoamide and bis-(α-ketoamide) derivatives via the facile ring-opening of N-acylisatin with different amines and diamines. The microwave irradiation afforded the product in less reaction time, higher yield and purity. Reaction of N-acylisatin with methanol under microwave irradiation afforded the α-phenylglyoxyl methyl ester derivatives with excellent yields and purities. Aminolysis of the ester derivatives with piperidine and morpholine afforded the same α-ketoamide derivatives obtained from direct aminolysis of N-acylisatin. The structures of the synthesized compounds were confirmed by FT-IR, NMR, X-ray and elemental analysis.ResultsReaction of N-acetylisatin and N-propoionylsatin with different amines and diamines afforded a series of α-ketoamide and bis-(α-ketoamide) derivatives respectively via the ring opening of N-acylisatins. The reaction was performed under conventional condition as well as microwave irradiation. The microwave irradiation afforded the product in less reaction time, higher yield and purity. Reaction of N-acylisatin with methanol under microwave irradiation afforded the α-phenylglyoxyl methyl ester derivatives in excellent yields and purities as observed from their spectral data. A plausible mechanism involves nucleophilic attack by methanol at C2 carbonyl carbon of N-acetylisatin and subsequent ring opening to generate the α-ketoester. Aminolysis of α-ketoester with amine afforded the same α-ketoamide which is obtained by direct aminolysis of N-acylisatin. The IR, NMR spectra, microanalyses, and single crystal X-ray diffraction confirmed the structures of the synthesized compounds.ConclusionsIn conclusion, we have demonstrated that microwave irradiation could be employed efficiently for the synthesis of biologically important α-ketoamide and bis-(α-ketoamide) derivatives. The microwave irradiation has more advantageous over the classical method with regard to reaction time, solvent quantity, and product yield. Reaction of N-acylisatin with methanol under microwave irradiation afforded the α-phenylglyoxyl methyl ester derivatives with excellent yields and purities. Aminolysis of the methyl ester derivatives with amine under microwave irradiation afford the same α-ketoamide derivatives as obtained from direct aminolysis of N-acylisatins.

Direct aminolysis of triglycerides: A novel use for heterogeneous catalysts

The aminolysis of triglycerides of Passiflora edulis oil was investigated using heterogeneous catalysts previously reported for biodiesel production. High rates of conversions (88–100%) to fatty ethanolamides were obtained with all the catalysts when the described transesterification conditions of reaction were used. ZnO.La2CO5.LaOOH (2.3%, w/w) was the most efficient, reaching 100% conversion after 8h at 100°C.

Synthesis of MUC1 glycopeptide thioesters and ligation via direct aminolysis

MUC1 is a cell-surface, epithelial glycoprotein that forms an essential protective barrier of cells and serves to modulate intercellular communication. During cancer progression, the dysregulation of glycosyltransferases, which serve to elongate cell-surface glycans, leads to aberrant gIycosylation patterns on this glycoprotein. This results in the presentation of well-characterized, tumor-associated carbohydrate antigens (TACAs) which represent important biomarkers for a range of epithelial cancers. The synthesis of well-defined, multivalent MUC1 glycopeptide constructs bearing TACAs therefore represents a viable opportunity for the development of cancer vaccine candidates. We report herein the synthesis of a glycopeptide thioester, comprising the full eicosapeptide variable number tandem repeat (VNTR) region of MUC1, which was prepared bearing multiple copies of the cancer-associated TN antigen. The glycopeptide thioester was prepared on the solid-phase using two different methods on 2-chlorotrityl chloride and sulfamylbutyryl resins. The solid-phase assembly on 2-chlorotrityl chloride resin, followed by thioesterification in solution, afforded the desired thioester in 4% yield over 41 linear steps. Likewise, the glycopeptide thioester was successfully prepared on sulfamylbutyryl resin using an activation and thiol-release strategy in a 9% isolated yield. The resulting peptide thioester was successfully ligated to a deprotected MUC1 glycopeptide using the direct aminolysis ligation to afford a 5.8 kDa, 40 amino acid MUC1 glycopeptide bearing 10 copies of the T(N) antigen. This work represents an important step toward the immunological evaluation of multivalent MUC1 constructs.

ChemInform Abstract: Amidation of Esters Assisted by Mg(OCH3)2 or CaCl2.

AbstractThe title reagents facilitate the direct aminolysis of various esters by NH3 (or NH4Cl) and amine hydrochlorides.

Amidation of esters assisted by Mg(OCH3)2 or CaCl2

Abstract Magnesium methoxide (Mg(OCH3)2) and calcium chloride have been shown to facilitate the direct aminolysis of esters by ammonia to primary amides. Methyl, ethyl, isopropyl, and tert-butyl esters were converted to the corresponding carboxamides in good yields. Reactions have been run on a larger scale and without the safety liability inherent in the use of magnesium nitride (Mg3N2). Ammonium chloride and amine hydrochlorides have been used successfully in the place of ammonia with magnesium methoxide.