Protected Amino Research Articles

Synthesis and Self‐Assembly of Stimuli‐Responsive Amphiphilic Dendrimers

AbstractThe synthesis of stimuli‐responsive amphiphilic dendrimers consisting of two or three hydrophobic octadecyl groups linked to either a second or third generation hydrophilic polylysine dendron through disulfide linkage is reported. The polylysine dendrimers functionalized with four or eight tert‐butyloxycarbonyl (Boc)‐protected amino groups are characterized by 1H NMR spectroscopy and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. With the positively charged versions generated by removing the Boc groups, their ability to form spherical micelles that can be readily disassembled in the presence of glutathione (GSH) is demonstrated. The self‐assembly behavior is monitored by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The amphiphilic dendritic systems containing cleavable disulfide linkage serve as key building blocks for generating stimuli‐responsive supramolecular architectures.

Effects of replacing wet distillers grains with supplemental SoyPass in forage-based growing cattle diets.

One hundred twenty individually fed steers (initial BW 283 kg ± 32) were utilized in an 84-d growing trial to evaluate the effects of increased metabolizable lysine from non-enzymatically browned soybean meal (SoyPass) in grass hay-based diets containing wet distillers grains plus solubles (WDGS). The treatments were arranged as a 2 × 3 factorial with two levels of protein supplement as 20% (low; DL20) or 35% (high; DL35) of dietary DM using WDGS as the basal protein source, and three increments of SoyPass (SP) replacing 0%, 30%, or 60% of the WDGS DM in the protein supplement yielding six dietary treatments. Average daily gain (ADG), dry matter intake (DMI), gain:feed (G:F), and plasma urea N (PUN) data were analyzed using the MIXED procedure of SAS as a 2 × 3 factorial. Animal was the experimental unit and fixed effects included body weight block, dietary inclusion of distillers grains (DL20, DL35), dietary inclusion of SoyPass (SP) (3 levels), and DL × SP inclusion interactions. Linear and quadratic interactions between DL and SP inclusion were analyzed using covariate regression. No interactions were detected for ADG between SP and DL (P = 0.76). Additionally, SP had no effect on ADG (P = 0.49). However, ADG was increased for steers consuming the DL35 diet compared to DL20 (1.13 vs. 0.86 kg/d, respectively; P < 0.01). A DL × SP interaction was detected for DMI (P = 0.01). As SP replaced WDGS in the DL35 diet, DMI increased linearly from 8.10 to 8.93 kg/d (P = 0.02). In the DL20 diet, DMI was not different as SP replaced WDGS (P ≥ 0.11). Therefore, G:F tended to decrease linearly (P = 0.06) as SP replaced WDGS in the DL35 diet, while no difference (P ≥ 0.11) was detected in the DL20 diet, suggesting SP contained less energy than WDGS but did improve dietary lysine balance. Furthermore, plasma urea nitrogen (PUN) increased linearly as SP replaced WDGS in the DL20 diet (P < 0.01) but was not affected by SP substitution in the DL35 diet (P ≥ 0.19). When WDGS is fed at a low (20% DM) or high (35% DM) inclusion rate in a forage-based diet, replacing the distillers with a source of protected amino acids supplied through heat-treated soybean meal, did not improve performance. A more concentrated or energy-dense form of amino acids may be beneficial in forage-based growing cattle diets containing 20% distillers grains but is not needed in diets with 35% distillers grains.

Atropisomeric Properties of N-Acyl/N-Sulfonyl 5H-Dibenzo[b,d]azepin-7(6H)-ones.

The stereochemistry of N-acyl/N-sulfonyl 5H-dibenzo[b,d]azepin-7(6H)-ones (I, II) was examined in detail by freezing the conformation with a methyl group at the C-4 of dibenzoazepine. Because the two axes (axis 1, axis 2) move together concertedly, I and II exist only as a pair of enantiomers [(a1R, a2R) and (a1S, a2S)], which was confirmed by X-ray analysis of IIBc. It was elucidated that the amide derivatives I exist in equilibrium with the E/Z-amide (100:2–100:34), which means that the exocyclic bond (axis 3) is not in concert with the endocyclic axes (axis 1, axis 2). For the preparation of 5H-dibenzo[b,d]azepin-7(6H)-one, the intramolecular Friedel–Crafts acylation of N-(1,1′)-biphenyl-2-yl-glycine derivatives was revisited. It was revealed that the electron-withdrawing property of the amino-protective group was a key to the success of seven-membered cyclization.

Enzymatic Synthesis of Putative Mycobacterial L,D‐Transpeptidase Substrates

The Mycobacterium genus is diverse, with members ranging from harmless water or soil dwellers to the causative agent of the deadly disease tuberculosis. Bacterial cell wall biosynthetic enzymes are therapeutic targets, and mycobacterial cell walls are uniquely complex. The peptidoglycan (PG) layer of mycobacterial cell walls is largely and uniquely 3-3 crosslinked, and L,D-transpeptidase (Ldt) enzymes catalyze this transpeptidation reaction. Our lab has previously demonstrated that there are six mycobacterial Ldt classes, and the reason for this apparent redundancy is not well understood. The Mycobacterium tuberculosis (Mtb) class 2 Ldt, LdtMt2, is the most well studied, whereas the physiological roles of the remaining paralogs are largely unknown. The PG tetrapeptide L-Ala-D-isoGln-mesoDAPNH2-D-Ala is a known substrate for four of the five Mtb Ldts. While all mycobacterial species have at least five Ldt paralogs, only the Mtb enzymes LdtMt1, LdtMt2, LdtMt4, and LdtMt5 enzymes have confirmed 3-3 crosslinking activity. LdtMt3 does not crosslink this tetrapeptide substrate, and the transpeptidation capability of class 6 Ldts – of which there is no homolog in Mtb – have not been investigated. Orthologous Ldts are presumed to have the same activities, although this has not been biochemically confirmed, presumably because of limited access to putative substrates. Tetrapeptide (and many pentapeptide) PG substrates are not commercially available; accessing them is challenging because they contain unusual amino acids (D-isoGlu, D-isoGln, D-Ala, meso-diaminopimelic acid (mesoDAP), and amidated mesoDAP), and they are linked via the γ-carboxyl group of D-isoGln. Thus, even solid-support peptide synthesis is a challenge due to the requirement of non-commercial protected amino acids. LdtMt2 requires a fully amidated tetrapeptide substrate, yet 50% of the 3-3 crosslinked PG isolated from Mtb lacks amidation at the mesoDAP ε-carboxyl moiety of the donor stem peptide, suggesting at least one Mtb Ldt prefers a non-amidated donor substrate (or that amino groups are removed in subsequent biosynthetic steps). Further, some mycobacterial species, including Mycobacterium smegmatis, can utilize tetrapeptides with L-Lys in the third position. Thus, while the role of LdtMt2 in Mtb is known, the functions of the remaining paralogs and orthologous Ldts remain to be determined, highlighting the need for substrate specificity studies on these enzymes toward understanding their physiological roles. Here, we present our preliminary work in cloning and overexpressing PG biosynthetic enzymes toward characterizing the substrate preferences of mycobacterial Ldt enzymes. We are actively pursuing enzymatic synthesis of putative PG substrates and will be evaluating purified tetra- and pentapeptide substrates against the six classes of mycobacterial Ldt enzymes. The results from this study are expected to inform the community as to the physiological roles of the under studied Ldt paralogs.

A Diversity‐Oriented Approach to Large Libraries of Artificial Macrocycles

AbstractDiversity‐oriented approach to large artificial macrocycle libraries with a ring size of 13—18 atoms relying on the “build‐couple‐pair” strategy is disclosed. The “couple” phase included three one‐pot steps including consequent amide coupling of N‐Boc‐monoprotected vicinal diamines with two alkenyl carboxylic acids, followed by ring‐closing metathesis as the key “pair” step. The scope and limitations of the method were established for all three reagents. In particular, various acyclic, mono‐ and bicyclic aliphatic diamine derivatives with the N−C−C−N dihedral angle less than ca. 130° appeared to be suitable substrates. The proposed approach was used to construct a virtual library of 1.8 ⋅ 105 macrocycles derived from 12,283 different scaffolds. More than 40 % of members of this library contained a protected amino function and hence can be suitable for the post‐pairing modification, thus giving rise to at least a billion‐size chemical space based on the REAL‐type synthetic methodology. Validation of the approach under parallel synthesis conditions on a 383‐member subset showed a 61 % success rate over the whole 4–5‐step reaction sequence. Finally, the synthetic approach also worked on a gram scale (up to 8.0 g).

Scope and limitations of pseudoprolines as individual amino acids in peptide synthesis.

Protected 4-carboxyoxazolidines and thiazolidines (pseudoprolines) are derivatives of serine, threonine or cysteine amino acids. Such compounds are used in peptide synthesis among the other protected amino acids. They are usually practiced when a peptide sequence is readily aggregating during synthesis due to their ability to disrupt secondary structure formation. Such compounds are usually applied as dipeptides. In present work Fmoc-protected pseudoprolines were synthesized and applied in peptide synthesis not as dipeptides but as individual amino acids. Different acylation protocols and amino acids were tested to acylate pseudoprolines. Several "difficult" peptides were synthesized to confirm the efficacy of such constructions. It was shown that pseudoprolines could be easily synthesized and used in automated or manual synthesis not as dipeptides but as ordinary amino acids.

Preparation and evaluation a mixed-mode stationary phase with C18 and 2-methylindole for HPLC.

A modified C18 column (Silpr-2MI-C18) was prepared using 2-methylindole and C18 reagent. The extent of C18 hydrocarbon chain, conjugative rings and anion exchange site provided multiple retention mechanisms, including reversed-phase liquid chromatography (RPLC), π-π interaction, hydrophilic interaction liquid chromatography (HILIC) and anion exchange chromatography (AEC). The separation of protected amino acids was investigated on the commercial C18 and Silpr-2MI-C18 columns, while the chromatographic conditions, including methanol content and pH of the mobile phase, were studied. The separation arrangement of the hydrophilic amino acids was different on the Silpr-2MI-C18 column compared to the commercial C18 column under RPLC mode. Furthermore, these amino acids were separated on the Silpr-2MI-C18 column under HILIC mode. The modified C18 column was employed to separate amino acids, alkylbenzenes and polycyclic aromatic hydrocarbons under RPLC mode and inorganic anion under AEC mode. The results confirm that this new stationary phase of RPLC/HILIC/AEC has multiple interactions with different analytes. Effective retention of biological samples was found on the Silpr-2MI-C18 column by comparing the results obtained from the commercial C18 column.

Highly Stable Zr(IV)-Based Metal-Organic Frameworks for Chiral Separation in Reversed-Phase Liquid Chromatography.

Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1'-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.

Selective 1,2‐Aminoisothiocyanation of 1,3‐Dienes Under Visible‐Light Photoredox Catalysis

AbstractSelective three‐component 1,2‐diamination of 1,3‐dienes with concurrent introduction of two orthogonally protected amino groups remains unknown despite its significant synthetic potential. We report herein that reaction of conjugated dienes with N‐aminopyridinium salts and TMSNCS affords 1,2‐aminoisothiocyanation products in a highly chemo‐ and regio‐selective manner under mild photoredox catalytic conditions. Mechanistic studies indicate that the facile isomerization of allyl thiocyanates to allyl isothiocyanates under photocatalytic conditions is responsible for the selective formation of the observed products. The mild isomerization protocol is expected to find applications in the synthesis of allyl isothiocyanates in a broad sense.

Selective 1,2-Aminoisothiocyanation of 1,3-Dienes Under Visible-Light Photoredox Catalysis.

Selective three-component 1,2-diamination of 1,3-dienes with concurrent introduction of two orthogonally protected amino groups remains unknown despite its significant synthetic potential. We report herein that reaction of conjugated dienes with N-aminopyridinium salts and TMSNCS affords 1,2-aminoisothiocyanation products in a highly chemo- and regio-selective manner under mild photoredox catalytic conditions. Mechanistic studies indicate that the facile isomerization of allyl thiocyanates to allyl isothiocyanates under photocatalytic conditions is responsible for the selective formation of the observed products. The mild isomerization protocol is expected to find applications in the synthesis of allyl isothiocyanates in a broad sense.

Effects of Glutamate Arginylation on α-Synuclein: Studying an Unusual Post-Translational Modification through Semisynthesis

A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.

Synthesis of monophosphoryl lipid A using 2-naphtylmethyl ethers as permanent protecting groups

Lipid A, which is a conserved component of lipopolysaccharides of gram-negative bacteria, has attracted considerable interest for the development of immuno-adjuvants. Most approaches for lipid A synthesis rely on the use of benzyl ethers as permanent protecting groups. Due to the amphiphilic character of lipid A, these compounds aggregate during the hydrogenation step to remove benzyl ethers, resulting in a sluggish reaction and by-product formation. To address this problem, we have developed a synthetic approach based on the use of 2-naphtylmethyl ether (Nap) ethers as permanent protecting group for hydroxyls. At the end of a synthetic sequence, multiple of these protecting groups can readily be removed by oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). Di-allyl N,N-diisopropylphosphoramidite was employed to install the phosphate ester and the resulting allyl esters were cleaved using palladium tetrakistriphenylphosphine. The synthetic strategy allows late stage introduction of different fatty acids at the amines of the target compound, which is facilitated by Troc and Fmoc as orthogonal amino-protecting groups.

A Shortcut to the Synthesis of Peptide Thioesters.

Peptide thioesters serve as fundamental building blocks for the synthesis of proteins and cyclic peptides. Classically, methods to synthesize thioesters have been based on acid-labile amino-protecting groups for which final side-chain deprotection required the use of hazardous hydrogen fluoride (HF). Alternative protection schemes based on base-labile amino-protecting groups have become preferred methods but are not suitable due to the lability of thioester bonds toward bases. In this method, we employ a trifluoracetic acid/trimethylsilyl bromide (TFA/TMSBr) protocol using a hydroxymethyl resin obviating the need for HF. TFA/TMSBr is volatile enough to be easily removed yet less hazardous than HF, making it more practical for general peptide chemists. We describe optimized cleavage procedures and appropriate protecting group schemes and discuss in situ neutralization protocols. The method is relatively simple, straightforward, and easily scalable, allowing the facile preparation of alkyl and aryl thioesters.

Increasing the Functional Group Diversity in Helical β-Peptoids: Achievement of Solvent- and pH-Dependent Folding.

We report the synthesis of a series of bis-functionalized β-peptoid oligomers of the hexamer length. This was achieved by synthesizing and incorporating protected amino- or azido-functionalized chiral building blocks into precursor oligomers by a trimer segment coupling strategy. The resulting hexamers were readily elaborated to provide target compounds displaying amino groups, carboxy groups, hydroxy groups, or triazolo-pyridines, which should enable metal ion binding. Analysis of the novel hexamers by circular dichroism (CD) spectroscopy and 1H-13C heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy revealed robust helical folding propensity in acetonitrile. CD analysis showed a solvent-dependent degree of helical content in the structural ensembles when adding different ratios of protic solvents including an aqueous buffer. These studies were enabled by a substantial increase in solubility compared to previously analyzed β-peptoid oligomers. This also allowed for the investigation of the effect of pH on the folding propensity of the amino- and carboxy-functionalized oligomers, respectively. Interestingly, we could show a reversible effect of sequentially adding acid and base, resulting in a switching between compositions of folded ensembles with varying helical content. We envision that the present discoveries can form the basis for the development of functional peptidomimetic materials responsive to external stimuli.

Towards understanding the mode of action of the cytotoxic triosmium carbonyl cluster Os3(CO)10(NCCH3)2: Its reactivity with amino acids and oligopeptides

The reactions of the triosmium carbonyl cluster Os3(CO)10(NCCH3)2 (1) with a number of protected amino acids, tri- and pentapeptides were examined. The results show that reaction with the side chains follow the order: -SH > -COOH > -CONH2, and there is a slight preference for the longer side chain of glutamic acid over aspartic acid. The trends observed are retained under physiological conditions and in the peptides.

The effect of adding protected amino acids (methionine and lysine) on the performance and carcass characteristics of male Arabi lambs

This study was conducted at the Agricultural Research Station of the College of Agriculture / Basra University / Karma Ali site for the period from July 2019 to May 2020, to assess the effect of adding protected amino acids (methionine and lysine and their mixture) on the performance of Arab lambs and some characteristics of carcasses. So (16) male lambs were selected with an average weight of 27.95 kg and at the age of 5-6 months, and were randomly distributed to four transactions by four animals per treatment and were housed in separate cages for each group which; first the control treatment (without additives); the second treatment (5g) of protected methionine; the third treatment (5g) of protected lysine; the fourth treatment (5g + 5g) of protected methionine and lysine per kg of feed mixture for (90) days. The results indicated a significant increase (p&lt; 0.05) in favor of the fourth and second treatments in the final live body weight rates and the daily and total weight gain compared with&nbsp;the first treatment and there was an improvement in the amount of feed consumed and the efficiency of food conversion. Also, there was a significant increase (p&lt; 0.05) in carcass weights, clearance ratios, and weights of the shoulder and leg joints of the fourth and second treatments compared to the first treatment. There were no differences in the weights of cuts neck, loin, and ribs.

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Dedicated to a peptide chemist of few words and immense impact: Louis A. Carpino

Interaction Between Crude Protein Level and Rumen Protected Amino Acids in Starter Diet on Performance of Dairy Calves

BACKGROUND: A limited number of studies have investigated the inclusion of amino acids in calf starter. OBJECTIVES: This study was aimed to evaluate different levels of crude protein in starter and supplement- ing some protected amino acids on efficiency, health status and blood metabolites of dairy calves and to com- pare it with unprotected amino acids. METHODS: Forty-eight 3-day-old Holstein calves were randomly allocated to four treatments in a complete- ly randomized design. Dietary treatments were (1) 18% Crude Protein starter without Amino Acid, (2) 18% Crude Protein with 0.0340% protected lysine and 0.016% protected methionine, (3) 18% Crude Protein starter with 0.215% un-protected lysine and 0.012% un-protected methionine, (4) 22% Crude Protein without Amino Acid. All calves received the same amount of whole milk, weaned on day 56; the study was finished on day 70. RESULTS: Calves in treatment 4 had the greatest starter intake, weaning weight and final weight (P 0.05). Health score (eye, ear, nose and respiratory score), was not different among treatments but the fecal score revealed a significant difference (P<0.05). Starter diet with 22% Crude Protein resulted in higher serum urea concentration (P< 0.05) CONCLUSIONS: Results of the present study indicate that, the performance of calves received protected or un-protected amino acid in starter was not different. Also, higher Crude Protein levels in diet did not lead to a better gain to feed ratio.

Applying nanotechnology to increase the rumen protection of amino acids in dairy cows

The amino acid requirements of high-production dairy cows represent a challenge to ensuring that their diet is supplied with available dietary resources, and thus supplementation with protected amino acids is necessary to increase their post-ruminal supply. Lysine is often the most limiting amino acid in corn-based diets. The present study proposes the use of lipid nanoparticles as novel rumen-bypass systems and assesses their capability to carry lysine. Solid lipid nanoparticles, nanostructured lipid carriers and multiple lipid nanoparticles were considered and their resistance in a rumen inoculum collected from fistulated cows was assessed. All nanoparticles presented diameters between 200–500 nm and surface charges lower than −30 mV. Lysine encapsulation was achieved in all nanoparticles, and its efficiency ranged from 40 to 90%. Solid lipid nanoparticles composed of arachidic or stearic acids and Tween 60 resisted ruminal digestion for up to 24 h. The nanoparticles were also proven to protect their lysine content from the ruminal microbiota. Based on our findings, the proposed nanoparticles represent promising candidates for rumen-bypass approaches and should be studied further to help improve the current technologies and overcome their limitations.

3‐Carboxy‐/3‐Aminobicyclo[1.1.1]pentane‐Derived Sulfonamides and Sulfonyl Fluorides – Advanced Bifunctional Reagents for Organic Synthesis and Drug Discovery

A convenient approach to 1,3‐bifunctional sulfonyl fluorides, sulfonamides and sulfinates bearing a bicyclo[1.1.1]pentane unit attached to the sulfur atom and (protected) amino or carboxyl group is described. The method relied on photochemical decomposition of Barton [1‐hydroxypyridine‐2(1H)‐thione] esters of the corresponding carboxylic acids as the key step, followed by oxidation and cleavage of the 2‐pyridyl moiety. The title building blocks were obtained on a gram scale, and their utility was demonstrated by preparation of an isosteric analogue of classical sulfonamide anitibiotic sulfanilamide, as well as some other common chemical modifications.