Peptide Bond Research Articles

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

Chasing weakly-bound biological water in aqueous environment near the peptide backbone by ultrafast 2D infrared spectroscopy

There has been a long-standing debate as to how many hydrogen bonds a peptide backbone amide can form in aqueous solution. Hydrogen-bonding structural dynamics of N-ethylpropionamide (a β-peptide model) in water was examined using infrared (IR) spectroscopy. Two amide-I sub bands arise mainly from amide C=O group that forms strong H-bonds with solvent water molecules (SHB state), and minorly from that involving one weak H-bond with water (WHB state). This picture is supported by molecular dynamics simulations and ab-initio calculations. Further, thermodynamics and kinetics of the SHB and WHB species were examined mainly by chemical-exchange two-dimensional IR spectroscopy, yielding an activation energy for the SHB-to-WHB exchange of 13.25 ± 0.52 kJ mol‒1, which occurs in half picosecond at room temperature. Our results provided experimental evidence of an unstable water molecule near peptide backbone, allowing us to gain more insights into the dynamics of the protein backbone hydration.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

Hordatines, dimerised hydroxycinnamoylagmatine conjugates of barley (Hordeum vulgare L.): an appraisal of the biosynthesis, chemistry, identification and bioactivities

AbstractHydroxycinnamoylamides are specialised metabolites widely distributed in the plant kingdom. These are phenolic moieties covalently linked to mono- or polyamines through amide bonds. Their oxidative coupling (dimerisation) leads to neolignanamides, a group of compounds showing high chemical, structural and functional diversity. Typical to barley, dehydro dimers of hydroxycinnamoylagmatines, hordatines are primarily found in germinated seeds and at the seedling stage. The first step in the biosynthesis of hordatines is catalysed by acyl-coenzyme A-dependent N-hydroxycinnamoyltransferases, and lead to the formation of hydroxycinnamoylagmatines (HCAgms). The oxidative homo- or hetero-dimerisation of the latter results in different hordatines (A, B, C or D). Hordatines can also undergo various types of conjugation and form hydroxylated, methylated or glycosylated derivatives. Although the research on the bioactivities of the hordatines is still nascent, the in planta antifungal properties have long been recognised. While hordatines are naturally and uniquely synthesised in barley plants, these molecules or lead compounds derived therefrom, also exhibit medicinal and pharmaceutical uses important for human health, stimulating research into the utilisation of biotechnology in alternative production hosts and to enhance agricultural yields and value-added production. This review summarises the older and recent knowledge about hordatines and derivatives and may serve as a springboard for future research on this intriguing class of secondary plant metabolites.

A General Method to Access Underexplored Ylideneamino Sulfates as Interrupted Beckmann-Type Rearrangement Intermediates.

The Beckmann rearrangement of ketoximes to their corresponding amides, using a Brønsted acid-mediated fragmentation and migration sequence, has found wide-spread industrial application. We postulated that the development of a methodology to access ylideneamino sulfates using tributylsulfoammonium betaine (TBSAB) would afford isolable Beckmann-type intermediates and competent partners for subsequent rearrangement cascades. The ylideneamino sulfates generated, isolated as their tributylammonium salts, are sufficiently activated to undergo Beckmann rearrangement without additional reagent activation. The generation of sulfuric acid in situ from the ylideneamino sulfate giving rise to a routine Beckmann rearrangement and additional amide bond cleavage to the corresponding aniline was detrimental to reaction success. The screening of bases revealed inexpensive sodium bicarbonate to be an effective additive to prevent classic Brønsted acid-mediated fragmentation and achieve optimal conversions of up to 99%.

Proteolytic cleavage of the TGFβ co-receptor CD109 changes its conformation, resulting in protease inhibition via activation of its thiol ester, and dissociation from the cell membrane.

The glycosylphosphatidylinositol (GPI)-anchored protein cluster of differentiation 109 (CD109) is expressed on many human cell types and modulates the transforming growth factor β (TGF-β) signaling network. CD109 belongs to the alpha-macroglobulin family of proteins, known for their protease-triggered conformational changes. However, the effect of proteolysis on CD109 and its conformation are unknown. Here, we investigated the interactions of CD109 with proteases. We found that a diverse selection of proteases cleaved peptide bonds within the predicted bait region of CD109, inducing a conformational change that activated the thiol ester of CD109. We show CD109 was able to conjugate proteases with this thiol ester and decrease their activity toward protein substrates, demonstrating that CD109 is a protease inhibitor. We additionally found that CD109 has a unique mechanism whereby its GPI-anchored macroglobulin 8 (MG8) domain dissociates during its conformational change, allowing proteases to release CD109 from the cell surface by a precise mechanism and not unspecific shedding. We conclude that proteolysis of the CD109 bait region affects both its structure and location, and that interactions between CD109 and proteases may be important to understanding its functions, for example, as a TGF-β co-receptor.

RNA-Templated Peptide Bond Formation Promotes L-Homochirality.

The world in which we live is homochiral. The ribose units that form the backbone of DNA and RNA are all D-configured and the encoded amino acids that comprise the proteins of all living species feature an all-L-configuration at the α-carbon atoms. The homochirality of α-amino acids is essential for folding of the peptides into well-defined and functional 3D structures and the homochirality of D-ribose is crucial for helix formation and base-pairing. The question of why nature uses only encoded L-α-amino acids is not understood. Herein, we show that an RNA-peptide world, in which peptides grow on RNAs constructed from D-ribose, leads to the self-selection of homo-L-peptides, which provides a possible explanation for the homo-D-ribose and homo-L-amino acid combination seen in nature.

Enhancing osteoblast proliferation and bone regeneration by poly (amino acid)/selenium-doped hydroxyapatite

Among various biomaterials employed for bone repair, composites with good biocompatibility and osteogenic ability had received increasing attention from biomedical applications. In this study, we doped selenium (Se) into hydroxyapatite (Se-HA) by the precipitation method, and prepared different amounts of Se-HA-loaded poly (amino acid)/Se-HA (PAA/Se-HA) composites (0, 10 wt%, 20 wt%, 30 wt%) by in-situ melting polycondensation. The physical and chemical properties of PAA/Se-HA composites were characterized by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and their mechanical properties. XRD and FT-IR results showed that PAA/Se-HA composites contained characteristic peaks of PAA and Se-HA with amide linkage and HA structures. DSC and TGA results specified the PAA/Se-HA30 composite crystallization, melting, and maximum weight loss temperatures at 203.33 °C, 162.54 °C, and 468.92 °C, respectively, which implied good thermal stability. SEM results showed that Se-HA was uniformly dispersed in PAA. The mechanical properties of PAA/Se-HA30 composites included bending, compressive, and yield strengths at 83.07 ± 0.57, 106.56 ± 0.46, and 99.17 ± 1.11 MPa, respectively. The cellular responses of PAA/Se-HA composites in vitro were studied using bone marrow mesenchymal stem cells (BMSCs) by cell counting kit-8 assay, and results showed that PAA/Se-HA30 composites significantly promoted the proliferation of BMSCs at the concentration of 2 mg ml−1. The alkaline phosphatase activity (ALP) and alizarin red staining results showed that the introduction of Se-HA into PAA enhanced ALP activity and formation of calcium nodule. Western blotting and Real-time polymerase chain reaction results showed that the introduction of Se-HA into PAA could promoted the expression of osteogenic-related proteins and mRNA (integrin-binding sialoprotein, osteopontin, runt-related transcription factor 2 and Osterix) in BMSCs. A muscle defect at the back and a bone defect at the femoral condyle of New Zealand white rabbits were introduced for evaluating the enhancement of bone regeneration of PAA and PAA/Se-HA30 composites. The implantation of muscle tissue revealed good biocompatibility of PAA and PAA/Se-HA30 composites. The implantation of bone defect showed that PAA/Se-HA30 composites enhanced bone formation at the defect site (8 weeks), exhibiting good bone conductivity. Therefore, the PAA-based composite was a promising candidate material for bone tissue regeneration.

Natural anti-neuroinflammatory inhibitors in vitro and in vivo from Aglaia odorata

Fifty compounds including seven undescribed (1, 13, 18–20, 30, 31) and forty-three known (2–12, 14–17, 21–29, 32–50) ones were isolated from the extract of the twigs and leaves of Aglaia odorata with anti-neuroinflammatory activities. Their structures were determined by a combination of spectral analysis and calculated spectra (ECD and NMR). Among them, compounds 13–25 were found to possess tertiary amide bonds, with compounds 16, 17, and 19–21 existing detectable cis/trans mixtures in 1H NMR spectrum measured in CDCl3. Specifically, the analysis of the cis–trans isomerization equilibrium of tertiary amides in compounds 19–24 was conducted using NMR spectroscopy and quantum chemical calculations. Bioactivity evaluation showed that the cyclopenta[b]benzofuran derivatives (2–6, 8, 10, 12) could inhibit nitric oxide production at the nanomolar concentration (IC50 values ranging from 2 to 100 nM) in lipopolysaccharide-induced BV-2 cells, which were 413–20670 times greater than that of the positive drug (minocycline, IC50 = 41.34 μM). The cyclopenta[bc]benzopyran derivatives (13–16), diterpenoids (30–35), lignan (40), and flavonoids (45, 47, 49, 50) also demonstrated significant inhibitory activities with IC50 values ranging from 1.74 to 38.44 μM. Furthermore, the in vivo anti-neuroinflammatory effect of rocaglaol (12) was evaluated via immunofluorescence, qRT-PCR, and western blot assays in the LPS-treated mice model. The results showed that rocaglaol (12) attenuated the activation of microglia and decreased the mRNA expression of iNOS, TNF-α, IL-1β, and IL-6 in the cortex and hippocampus of mice. The mechanistic study suggested that rocaglaol might inhibit the activation of the NF-κB signaling pathway to relieve the neuroinflammatory response.

Tf2O‐Promoted Synthesis of Ureas, Carbamates and Thiocarbamate via Lossen Rearrangement: A Mechanistic Insight

AbstractHerein, we report triflic anhydride mediated synthesis of ureas starting from hydroxamic acids. The scope of triflic anhydride was also expanded to encompass carbamates. In addition, this reaction is applicable to activation of hydroxamic acid derivative of dipeptide giving α‐uriedopeptidomimetics in good yield. The mechanistic study of reaction was carried out by DFT. This study is important because it demonstrates that Tf2O has the potential to be effective when used on a substrate that possesses amide bond. A broad substrate scope and mild conditions render this protocol a promising approach to the synthesis of diverse types of ureas.

Toward More Selective Antibiotic Inhibitors: A Structural View of the Complexed Binding Pocket of E. coli Peptide Deformylase.

Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.

Comparative assessment of radical scavenging potential of A1 and A2 cow's milk casein pre- and post-enzymatic hydrolysis

This paper aimed to explore the antioxidant activity of A1 and A2 casein hydrolysates and compare their effectiveness. Enzymatic hydrolysis of casein produces bioactive peptides that offer various health benefits, including antioxidant, antihypertensive, antidiabetic, antithrombotic, and immunomodulatory properties. In this study, A1 and A2 casein were hydrolyzed using trypsin at different time intervals (2, 4, and 6 h). The degree of hydrolysis (DH) was determined to assess the extent of peptide bond cleavage during proteolysis. The hydrolysates were then evaluated for their radical scavenging potential using DPPH and ABTS assays, as well as their reducing power. The results indicate that both A1 and A2 casein can be effectively hydrolyzed with trypsin, with 6 h of hydrolysis found to be optimal in terms of DH and scavenger activity. Both A1 and A2 casein hydrolysates demonstrated antioxidant activity against DPPH radical and ABTS radical cation. However, at a high concentration (5% casein), A2 hydrolysate exhibited significantly higher antioxidant activity compared to A1. These findings offer insights into the potential use of A1 and A2 casein hydrolysates as natural antioxidants in functional foods and nutraceuticals, providing a safer and more sustainable alternative to synthetic antioxidants.

Exploring bi-carbazole-linked triazoles as inhibitors of prolyl endo peptidase via integrated in vitro and in silico study

A serine protease called prolyl endopeptidase (PEP) hydrolyses the peptide bonds on the carboxy side of the proline ring. The excessive PEP expression in brain results in neurodegenerative illnesses like dementia, Alzheimer’s disease, and Parkinson's disease. Results of the prior studies on antioxidant activity, and the non-cytotoxic effect of bi-carbazole-linked triazoles, encouraged us to extend our studies towards its anti-diabetic potential. Hence, for this purpose all compounds 1–9 were evaluated to reveal their anti-prolyl endo peptidase activity. Fortunately, seven compounds resulted into significant inhibitory capability ranging from 26 to 63 µM. Among them six compounds 4–9 exhibited more potent inhibitory activity with IC50 values 46.10 ± 1.16, 42.30 ± 1.18, 37.14 ± 1.21, 26.29 ± 0.76, 28.31 ± 0.64 and 31.11 ± 0.84 µM respectively, while compound 3 was the least active compound in the series with IC50 value 63.10 ± 1.58 µM comparing with standard PEP inhibitor bacitracin (IC50 = 125 ± 1.50 µM). Moreover, mechanistic study was performed for the most active compounds 7 and 8 with Ki values 24.10 ± 0.0076 and 23.67 ± 0.0084 µM respectively. Further, the in silico studies suggested that the compounds exhibited potential interactions and significant molecular conformations, thereby elucidating the structural basis for their inhibitory effects.

Antioxidant and Prooxidant Effects of Curcumin, Piperine and Semi-synthetic Amides with Antimycobacterial Activity on the Lipid Oxidation Process

Free radical oxidation is considered an important factor in the treatment of tuberculosis. Drugs or treatments with antioxidants are reflected in the literature but the choice of those used for tuberculosis is quite limited. Selected natural and synthetic compounds (hybrid molecules consisting of fenchane scaffold and cinnamoyl like residues attached through amide bond), possessing antimycobacterial activity were studied for their capacity to accelerate or decelerate lipid oxidation process. Three of the compounds under study showed prooxidant activity. To the best of our knowledge, this is the first report about the prooxidant activity of piperine under spontaneous autoxidation conditions. The presence of fenchane scaffold together with the 1,3-benzodioxole fragment, positively affects the activity but we can only speculate about the role of the amide part. Effect of equimolar binary mixtures with curcumin has also been studied. These new results may contribute to the search for efficient bio-antioxidant mixtures in the prevention and elimination of adverse reactions of antibacterial drugs.

Highly efficient oxidative degradation of organic dyes by using magnetically recyclable nanocatalyst based on manganese-porphyrin

This study reports the attachment of meso-tetrakis-(4-carboxyphenyl)porphyrinato manganese(III) acetate (Mn(TCPP)OAc) to the surface of amine-functionalized iron oxide magnetic nanoparticle (Fe3O4@SiO2@NH2) by amide bonds. To characterize the structure and morphology of the prepared nanocatalyst (Fe3O4@SiO2@NH2@(Mn(TCPP)OAc)), various techniques such as FT-IR, ultraviolet–visible spectroscopy, flame atomic absorption spectroscopy, X-ray powder diffraction, scanning electron microscopy, vibration sample magnetometry and thermogravimetric analysis were applied. In the following, the catalytic efficiency of the prepared nanocatalyst were also investigated for the oxidative degradation of various organic dyes with hydrogen peroxide (as a green oxidant) in water and the effect of various parameters which may affect the catalytic efficiency were optimized. In addition, the prepared nanocatalyst showed little deactivation with time, was easily recovered by an external magnet, and could be reused at least five times with no significant loss in the activity.

Imidazole Bioisostere Activators of Endopeptidase Neurolysin with Enhanced Potency and Metabolic Stability.

The peptidase neurolysin (Nln) has been validated as a potential target for developing therapeutics for ischemic stroke (IS). Overexpression of Nln in a mouse model of IS provides significant cerebroprotection, leading to reduced infarction size and edema volume. Pharmacological inhibition of Nln in the post-stroke brain worsens neurological outcomes. A virtual screen identified dipeptide small-molecule activators of Nln. Optimization studies resulted in a class of peptidomimetic compounds with promising activity. However, these compounds still possessed an amide bond that compromised their stability in plasma and the brain. Herein, we report the synthesis and characterization of a series of amide bioisosteres based on our peptidomimetic leads. Imidazole-based bioisosteres afford scaffolds with increased potency to activate Nln combined with enhanced mouse plasma stability and significantly better brain permeability over the original dipeptide hits.

Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

Total Synthesis of Alternatamides B and D

AbstractThe total synthesis of alternatamines B and D is outlined. Alternatamine B demonstrates moderate antibacterial activity across a broad spectrum of bacteria. Both alternatamines B and D feature an intriguing 5,6‐dibromoindole skeleton, derived from a four‐step sequence starting with 3,4‐dibromoaniline and achieving a total yield of 48 %. The key steps involve: (a) functional group modifications, including iodination, of 3,4‐dibromoaniline with two bromine substituents to obtain the Sonogashira precursor; (b) indole synthesis through Sonogashira coupling and 5‐endo‐dig cyclization; (c) carbon chain extension and introduction of the nitrogen atom at the C3 of the indole. Subsequently, the tryptamine analogue was coupled with an L‐leucine derivative through amide bond formation after deprotection, yielding alternatamide D. The first total synthesis of alternatamine B was also achieved by selectively introducing a bromine substituent at the C2 of the indole, employing optimal bromination conditions.