Enzymatic Amidation Research Articles

Enzyme Engineering for High-Yielding Amide Formation: Lipase-Catalyzed Synthesis of N-Acyl Glycines in Aqueous Media.

Amide syntheses remain a key challenging green chemistry reaction. For instance, green synthesis of N-acyl glycines as biosurfactants and therapeutics is highly desirable to replace chemical pathways using toxic phosgene. Herein, we report a novel concept for enzymatic amidation in an aqueous system via glycerol activation of fatty acids and theirsubsequent aminolysis with glycine to synthesize N-acyl glycines. We then engineer an enzyme (proRML) by reshaping its catalytic pocket to enhance its aminolysis activity and catalytic efficiency by 103-fold and 465-fold, respectively. The evolved proRML (D156S/L258K/L267N/S83D/L58K/R86K/W88V) catalyzed the amidation of a fatty acid with glycine to give N-lauroylglycine with high yield (80 %). It accepts a broad range of medium- to long-chain fatty acids (C8 -C18 ), giving high yields of N-decanoyl-, N-myristoyl-, and N-oleoylglycine. The developed amidation concept may be general, and the engineered enzyme is useful for the green synthesis of N-acyl glycines.

Enzyme Engineering for High‐Yielding Amide Formation: Lipase‐Catalyzed Synthesis of N‐Acyl Glycines in Aqueous Media

AbstractAmide syntheses remain a key challenging green chemistry reaction. For instance, green synthesis of N‐acyl glycines as biosurfactants and therapeutics is highly desirable to replace chemical pathways using toxic phosgene. Herein, we report a novel concept for enzymatic amidation in an aqueous system via glycerol activation of fatty acids and theirsubsequent aminolysis with glycine to synthesize N‐acyl glycines. We then engineer an enzyme (proRML) by reshaping its catalytic pocket to enhance its aminolysis activity and catalytic efficiency by 103‐fold and 465‐fold, respectively. The evolved proRML (D156S/L258K/L267N/S83D/L58K/R86K/W88V) catalyzed the amidation of a fatty acid with glycine to give N‐lauroylglycine with high yield (80 %). It accepts a broad range of medium‐ to long‐chain fatty acids (C8–C18), giving high yields of N‐decanoyl‐, N‐myristoyl‐, and N‐oleoylglycine. The developed amidation concept may be general, and the engineered enzyme is useful for the green synthesis of N‐acyl glycines.

Biocatalytic amide bond formation

The state-of-the-art of biocatalytic amide bond formation is discussed with the help of a manually curated database of enzymatic amidation reactions.

Streamlined Synthesis of a Bicyclic Amine Moiety Using an Enzymatic Amidation and Identification of a Novel Solid Form

Streamlined Synthesis of a Bicyclic Amine Moiety Using an Enzymatic Amidation and Identification of a Novel Solid Form

CAL-B-mediated efficient synthesis of a set of valuable amides by direct amidation of phenoxy- and aryl-propionic acids

An efficient, easy and sustainable amidation of a set of non-activated carboxylic acids with anilines, assisted by CAL-B, as biodegradable catalyst, is reported. The enzymatic amidation reactions are performed on set of nonsteroidal anti-inflammatory drugs (NSAIDs), phenoxypropionic acid and protected-prolines by direct condensation of one equivalent of carboxylic acids and two equivalents of anilines derivatives in heptane after 72 h of reaction at 80 °C. The obtained carboxylic amides are recovered with isolated chemical yields varied between moderate and excellent. Fourteen from them are reported for the first time, and an X-ray crystal is obtained for: N-(4-iodophenyl)-2-(4-isobutylphenyl)propanamide 1d.

Eco-Friendly Production of Fatty Amides Using 1-Monoacylglycerols as Acyl Donors

An efficient and environmentally friendly method to produce fatty amides by enzymatic amidation with 1-monoacylglycerols (MAGs) as acyl donors is described. When 1-monoolein was employed as a fatty donor to synthesize oleoyl ethanolamide, the product yield was up to 96% under optimum conditions. Subsequently, a comparative study using various acyl donors including 1-monoolein, oleic acid, triolein, and ethyl oleate was performed. The results displayed that 96.2, 76.4, 0, and 51.8% oleoyl ethanolamide were formed, respectively. Additionally, a two-step method was developed to prepare fatty ethanolamide with vegetable oil as a substrate. The results demonstrated that excess glycerol had a significant effect on lipase stability and a 3 h glycerolysis and a 3 h amidation resulted in the formation of 95.2% fatty ethanolamide. Last, various fatty amides were synthesized with MAGs as substrates. The results showed MAGs were equally effective reactants for the preparation of other amides. This was the first study reporting the use of MAGs as acyl donors to prepare fatty amides. MAGs are safer substrates compared to toxic fatty acid chlorides and fatty acid vinyl esters. In conclusion, a greener approach is developed for fatty amide preparation and the method is more efficient than those using other acyl donors.

Lipase-catalyzed amidation of carboxylic acid and amines

The amidation reaction is of a very particular interest, especially in the pharmaceutical industry and always requires the activation of the acid with a large excess of reactants. Therefore, a large amount of waste is generated. In order to reduce the environmental impact of such reaction, we have developed enzymatic amidation conditions which are compatible with a wide range of amines and acids, in particular with the biologically relevant lipoic acid. Water is the only by-product generated during this reaction thus a very high atom economy is obtained. In addition, we have shown that the lipase can be recovered and reused several times without a significant loss of activity.

Preparation of arachidonoyl ethanolamide by enzymatic amidation of arachidonic acid purified from microbial oil

Arachidonoyl ethanolamide (AEA) is a bioactive lipid that occurs naturally in animal and plant tissues. In this study, arachidonic acid (ARA) was enriched first from a type of microbial oil by a two-step purification, and the purified ARA concentrate was then used for the enzymatic preparation of AEA by amidation between ARA and ethanolamine. The purity of initial ARA increased from 44.4% to 85.1% after urea complexation and acetonitrile extraction. The purified ARA was subsequently used for lipase-catalyzed synthesis of AEA. The results showed that under optimized conditions (5 mmol purified ARA product, 5.5 mmol ethanolamine, 3 mL hexane, 10% Lipozyme 435, 70 °C for 2 h), fatty acid ethanolamide was obtained with 95.6% yield. The current method for making AEA by the enzymatic amidation of ethanolamine with ARA from fungal oil is effective, practical and economically feasible.

Enzymatic synthesis and in vitro evaluation of folate-functionalized liposomes.

In this study, folate–poly(ethylene glycol)3400–cholesterol conjugates (FA–PEG–Chol) were enzymatically synthesized in one step and incorporated into liposomes to prepare folate (FA)-functionalized liposomes for targeted drug delivery. The FA-functionalized liposomes loaded with betulinic acid (BA) (FA-L-BA) were prepared by thin lipid film method. The FA-L-BA was characterized by their morphology, particle size, zeta potential, encapsulation efficiency (EE), stability, cell cytotoxicity and cellular uptake. The average size of FA-L-BA was 222±8 nm. The spherical particles exhibited a negative electrical charge of −20.12±1.45 mV and high EE of 91.61%±1.16%. The liposomes were taken up selectively by HepG2 cells. FA-L-BA showed enhanced cytotoxicity (50% inhibitory concentration [IC50] =63.07±2.22 μg/mL) compared to nontargeted control normal liposomes loaded with BA (L-BA; IC50 =93.14±2.19 μg/mL) in HepG2 cells in vitro. In addition, FA-functionalized liposomes loaded with Ir-1 (FA-L-Ir-1) showed significantly higher cellular uptake in HepG2 cells compared to nontargeted control normal liposomes loaded with Ir-1 (L-Ir-1). This novel approach for the liposomes surface modified with FA by a one-step enzymatic amidation was expected to provide potential application as a drug carrier for active targeted delivery to tumor cells.

An effective method for reducing free fatty acid content of high-acid rice bran oil by enzymatic amidation

Abstract An improved method of deacidification of high-acid rice bran oil (HRBO) by an enzymatic amidation reaction between free fatty acids (FFA) and ethanolamine is described in this study. The reaction conditions were optimized to minimize the FFA content of HRBO. Under optimal reaction conditions (2% of Lipozyme 435, about 1:1 mass ratio of oil to solvent and at 76 °C), acid value of HRBO was reduced from 21.5 to 1.6 mg/g after 4 h reaction. The final oil product was rich in fatty acid ethanolamides (11.9 wt %) which are desirable bioactive lipids. Compared to esterification deacidification using glycerol or monoacylglycerols (MAG) as acyl acceptor, enzymatic deacidification by amidation can be completed in a much shorter time because amidation reaction is much more favorable than the esterification. This is the first time that ethanolamine is used as acyl acceptor to enzymatically deacidify a high-FFA oil. Such an enzymatic route is highly effective and environmentally desirable.

Lipozyme 435-catalyzed synthesis of eicosapentaenoyl ethanolamide in a solvent-free system

Eicosapentaenoyl ethanolamide (EPEA) is a lipid signaling molecule. In this study, an effective process is described to synthesize EPEA by enzymatic amidation using eicosapentaenoic acid ethyl ester (EPA-EE) as acyl donor with lipase as catalyst. The reaction conditions were optimized. When the amidation reaction was conducted at 70°C for 1h in a solvent-free system with agitation by reacting 2mmol fatty acid ethyl ester with 3mmol ethanolamine in presence of 10% Lipozyme 435 as a catalyst, fatty acid ethanolamides was formed at 62.5% molar yield. This was the first time reporting that Lipozyme 435 lipase was used as catalyst for enzymatic amidation. In addition, compared to previous methods using free fatty acid as acyl donor for fatty acid ethanolamide synthesis, study using fatty acid ester as acyl donor is limited and the use of fatty acid ester avoids ion pair formation between free fatty acid and ethanolamine. Finally, we found that Lipozyme 435 lipase had a higher tolerance toward polar ethanolamine as compared to Novozym 435 and Lipozyme RM IM.

Preventive effects of a fermented dairy product against Alzheimer's disease and identification of a novel oleamide with enhanced microglial phagocytosis and anti-inflammatory activity.

Despite the ever-increasing number of patients with dementia worldwide, fundamental therapeutic approaches to this condition have not been established. Epidemiological studies suggest that intake of fermented dairy products prevents cognitive decline in the elderly. However, the active compounds responsible for the effect remain to be elucidated. The present study aims to elucidate the preventive effects of dairy products on Alzheimer’s disease and to identify the responsible component. Here, in a mouse model of Alzheimer’s disease (5xFAD), intake of a dairy product fermented with Penicillium candidum had preventive effects on the disease by reducing the accumulation of amyloid β (Aβ) and hippocampal inflammation (TNF-α and MIP-1α production), and enhancing hippocampal neurotrophic factors (BDNF and GDNF). A search for preventive substances in the fermented dairy product identified oleamide as a novel dual-active component that enhanced microglial Aβ phagocytosis and anti-inflammatory activity towards LPS stimulation in vitro and in vivo. During the fermentation, oleamide was synthesized from oleic acid, which is an abundant component of general dairy products owing to lipase enzymatic amidation. The present study has demonstrated the preventive effect of dairy products on Alzheimer’s disease, which was previously reported only epidemiologically. Moreover, oleamide has been identified as an active component of dairy products that is considered to reduce Aβ accumulation via enhanced microglial phagocytosis, and to suppress microglial inflammation after Aβ deposition. Because fermented dairy products such as camembert cheese are easy to ingest safely as a daily meal, their consumption might represent a preventive strategy for dementia.

Enzymatic C-terminal amidation of amino acids and peptides

Herein, we describe two versatile and high yielding enzymatic approaches for the conversion of semi-protected amino acid and peptidyl C-terminal α-carboxylic acids into their corresponding amides. In the first approach, the lipase Candida antarctica lipase-B (Cal-B), and in the second approach, the protease Subtilisin A, are used, respectively. We found that by using the ammonium salt of the α-carboxylic acid instead of separate ammonia sources, the enzymatic amidation reactions proceeded much faster without side reactions and gave near to quantitative yields of products.

To protect peptide pharmaceuticals against peptidases

Introduction: The major hurdle in the application and delivery of peptide pharmaceuticals is their rapid in vivo breakdown. Methods: We here combined two approaches to stabilize peptide pharmaceuticals, introduction of d-amino acids and cyclization, by applying an innovative enzymatic method. This method yields peptides with thioether bridges between a d-amino acid and an l-amino acid. On the basis of guidelines concerning the flanking residues of serines/threonines and cysteines, a peptide of interest is designed with serine/threonine and cysteine at appropriate positions to allow their effective participation in cyclization. In Lactococcus lactis the peptide of interest is directly or via a spacer genetically fused to a lantibiotic leader peptide which induces enzyme-catalysed synthesis of a thioether-bridged peptide. The peptide is translocated via a lantibiotic transporter, analysed by mass spectrometry and the leader peptide is removed. Because of its therapeutic relevance and terminal modifications we chose the decapeptide Luteïnizing Hormone Release Hormone (LHRH) as a test case for thioether bridge introduction. The N-terminal pyroglutamate protects against aminopeptidase activity; the amidated C-terminus, which occurs in 50% of all therapeutic peptides, precludes carboxypeptidase action and is essential for optimal receptor interaction. We had Lactococcus posttranslationally introduce a thioether bridge between residues 4 and 7 of the Leu7Cys-LHRH analog QHWSYGCRPG. The N-terminal glutamine of the thioether-bridged peptide could be converted in pyroglutamate. The introduction of the thioether bridge proved to be compatible with subsequent chemical and enzymatic amidation methods. In this way biologically produced thioether LHRH was compared with LHRH isomers obtained by base-assisted sulfur extrusion. Results: Biologically produced thioether LHRH is the most stable thioether LHRH isomer with strongly enhanced proteolytic resistance compared to natural LHRH. Discussion: The data convincingly demonstrate the broad perspective of stereo- and regiospecifically generating cyclized peptide pharmaceuticals with significantly enhanced therapeutic potential.

Angiotensin II Stimulates Cardiac Adrenomedullin Production and Causes Accumulation of Mature Adrenomedullin Independently of Hemodynamic Stressin Vivo

Adrenomedullin is a potent hypotensive peptide that may act on myocytes to inhibit hypertrophy and on fibroblasts to inhibit growth in vitro induced by mechanical stretching and angiotensin II. Adrenomedullin is processed from the inactive intermediate adrenomedullin precursor with a glycine extension, which is subsequently converted to biologically active mature adrenomedullin by enzymatic amidation. Total adrenomedullin is the sum of intermediate and mature adrenomedullin. We examined the effect of a subpressor dose of angiotensin II on the production of left ventricular adrenomedullin and on protein levels of mature adrenomedullin in the left ventricle in vivo. We also investigated whether the effect is mediated by the angiotensin II type 1 receptor. Concentrations of total and mature adrenomedullin in the left ventricle and mature adrenomedullin-to-intermediate adrenomedullin ratio were significantly increased by angiotensin II infusion, regardless of pressure overload. Total and mature adrenomedullin concentrations significantly correlated with the weight of the left ventricle. Furthermore, increased adrenomedullin gene expression and protein levels were completely suppressed by a subdepressor dose of angiotensin II type 1 receptor blocker. In conclusion, angiotensin II stimulates the production of cardiac adrenomedullin and accumulates mature adrenomedullin in the left ventricle independently of hemodynamic stress. These processes are partially regulated through the angiotensin II type 1 receptor in vivo.

Different secretion patterns of two molecular forms of cardiac adrenomedullin in pressure- and volume-overloaded human heart failure

Background In the final step of production of adrenomedullin (AM), an inactive intermediate form of glycine-extended AM (AM-glycine) is converted to the active mature form of adrenomedullin (AM-mature) by enzymatic amidation. Recent studies have revealed that AM-mature and AM-glycine circulate in human plasma. In this study, we investigated the differences of the concentrations of cardiac AM between pressure-overloaded (PO) heart failure (HF) and volume-overloaded (VO)-HF in humans. Methods and results We measured AM-mature and AM-glycine by immunoradiometric assays in pericardial fluid and plasma in 38 patients who underwent valve replacement surgery (PO-HF: aortic stenosis, n = 14; VO-HF: aortic or mitral regurgitation, n = 24). Stable coronary artery disease with normal left ventricular function served as the control (n = 24). Plasma AM-mature (VO-HF: +59%, PO-HF: +65%, P<.05) and AM-glycine (VO-HF: +43%, PO-HF: +50%, P<0.05) were similarly higher in the 2 HF groups than in the control group. Interestingly, pericardial fluid AM-mature was markedly higher than that in plasma (control: +789%, VO-HF: +1050%, PO-HF: +1745%, all P<.001). Pericardial fluid AM-mature was higher in VO-HF (+106%, P<.01) than in controls and they were further increased in PO-HF (+243%, P<.05). Pericardial fluid molecular forms of AM correlated with left ventricular systolic pressure, but not with left ventricular end-diastolic volume index in PO-HF. In contrast, they correlated with left ventricular end-diastolic volume index, but not with left ventricular systolic pressure in VO-HF. Conclusion These results suggest that cardiac AM is differently regulated from plasma AM and that cardiac AM production is upregulated in both types of HF in response to each different stimulus.

Upregulation of ligand, receptor system, and amidating activity of adrenomedullin in left ventricular hypertrophy of severely hypertensive rats: effects of angiotensin-converting enzyme inhibitors and diuretic.

We investigated the pathophysiological role of the cardiac adrenomedullin (AM) system, including the ligand, receptor and amidating activity in the hypertrophied heart in severe hypertension. We studied the following four groups: control Wistar-Kyoto rats (WKY), spontaneously hypertensive stroke-prone rats (SHR-SP), 8 weeks captopril-treated SHR-SP, and 8 weeks trichlormethiazide-treated SHR-SP. AM precursor is converted to inactive glycine-extended AM (AM-Gly) and subsequently AM-Gly is converted to active mature AM (AM-m) by enzymatic amidation. We measured AM-m, AM-total (AM-T; AM-T = AM-m + AM-Gly), and atrial natriuretic peptide (ANP) in the plasma and left ventricle (LV) by immunoradiometric assay. We also measured gene expression of AM and ANP was and gene expression and protein levels of AM receptor system components such as calcitonin receptor-like receptor (CRLR), receptor-activity modifying protein (RAMP) 2 and RAMP3. At 7 weeks old, SHR-SP had higher blood pressure and ANP mRNA levels and lower plasma AM-T compared with WKY, however, there were no differences in other indices between the two groups. At 17 weeks old, SHR-SP had increased blood pressure, LV weight, plasma and LV ANP levels and mRNA levels of ANP compared with WKY. AM-m and AM-T levels in plasma (AM-m: + 31%; AM-T: + 56%) and the LV (AM-m: + 84%; AM-T: + 31%) were significantly higher in SHR-SP than in WKY. The LV tissue AM-m/AM-T ratio was significantly higher in SHR-SP (93.2%) than in WKY. The mRNA levels of AM, CRLR, and RAMP2 in the LV were significantly higher in SHR-SP than in WKY. Captopril and trichlormethiazide similarly decreased blood pressure and LV hypertrophy with the reduction of the LV AM-m and AM-T levels and mRNA abundance of AM and its receptor component. These results suggest that cardiac AM system is upregulated in the hypertrophied heart in this hypertension model. Considering that AM acts as an anti-remodeling autocrine and/or paracrine factor, upregulation of the AM system may modulate the pathophysiology in LV hypertrophy.

Direct measurement of glycine-extended adrenomedullin in plasma and tissue using an ultrasensitive immune complex transfer enzyme immunoassay in rats.

The mature form of the vasodilator peptide adrenomedullin (AM-m) is synthesized from a glycine-extended precursor (AM-Gly) by enzymatic amidation. We have developed a highly sensitive enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay; ICTEIA) that enables us to measure levels of AM-Gly in plasma and tissue directly. The detection limit of this assay is 1 amol/assay, and the intra- and inter-assay precision are 4.5-14.1% and 9.9-20.5%, respectively. Dilution curves for plasma samples showed good linearity, and the analytical recovery was 107-116.6%. Using ICTEIA, we determined that the plasma concentration of immunoreactive AM-Gly is substantially higher than that of AM-m (5.22 +/- 2.56 vs. 1.21 +/- 0.79 fmol/ml). In contrast, levels of AM-Gly were much lower than those of AM-m in the lung, heart, kidney, adrenal gland and liver. We also evaluated AM-Gly and AM-m levels in rats in a morbid state induced by intraperitoneal administration of lipopolysaccharide (LPS). In most tissues, levels of AM-m and AM-Gly were both increased by LPS; however, AM-Gly/AM-m ratios were not significantly affected, which suggests that AM-Gly is rapidly converted to AM-m in tissue.

Peptide amidation: Production of peptide hormonesin vivo andin vitro

Over half of all biologically active peptides and peptide hormones are α-amidated at their C-terminus, which is essential for their full biological activities. Amidation is accomplished through the sequential reaction of the two enzymes encoded by the single bifunctional, peptidylglycine α-amidating monooxygenase (PAM or an α-amidating enzyme). PAM catalyzes the formation of a peptide amide from peptide precursors that include a C-terminal glycine, and requires copper, molecular oxygen, and ascorbate. PAM is the only enzyme that produces peptide amidesin vivo. However, various strategies utilizing PAM, carboxypeptidase-Y enzymes, and chemical synthesis have been developed for producing peptide amidesin vitro. The growing need and importance of peptide amide drugs has highlighted the necessity for an efficientin vitro amidating system for industrial application. In recent years, recombinant systems for enzymatic amidation have received growing attention for the production of peptide hormones, like calcitonin and oxytocin. This review presents the current situation regarding amidation, with a special emphasis on the industrial production of peptide hormones.

Increased Plasma Levels of Mature Adrenomedullin in Chronic Glomerulonephritis

Adrenomedullin (AM) is a potent vasodilative and natriuretic peptide that is processed from its precursor as the intermediate form, AM-glycine-COOH (iAM). Subsequently, iAM is converted to the biologically active mature form, AM(1-52)-CONH₂ (mAM), by enzymatic amidation. Using immunoradiometric assays that recognize total AM (tAM) and only mAM, we determined the plasma and urinary levels of mAM and iAM in patients with chronic glomerulonephritis (CGN). The plasma mAM concentration was significantly higher in the patients than in the controls (1.8 ± 0.1 vs. 1.3 ± 0.1 fmol/ml, p < 0.01), whereas the plasma iAM concentration of the CGN patients did not significantly differ from that of the controls (9.4 ± 0.5 vs. 8.9 ± 0.5 fmol/ml). Levels of urinary mAM excretion in the patients did not statistically differ from those of the controls (1.6 ± 0.4 vs. 2.0 ± 0.3 fmol/mg creatinine), whereas urinary iAM excretion was significantly lower in the CGN patients (3.7 ± 0.7 vs. 5.6 ± 0.8 fmol/mg creatinine, p < 0.05). Urinary excretion levels of mAM significantly correlated with those of sodium (r = 0.47, p < 0.05), whereas those of iAM did not. In conclusion, the plasma ratio of mAM to iAM is augmented in CGN patients, and mAM appears to be involved in the regulation of sodium. Therefore, determination of the mAM in addition to the tAM concentration is essential in CGN patients.