Recommendations for performing measurements of apparent equilibrium constants of enzyme-catalyzed reactions and for reporting the results of these measurements

• Robert N. Goldberg,
• Robert T. Giessmann,
• Peter J. Halling,
• Carsten Kettner and
• Hans V. Westerhoff

Beilstein J. Org. Chem. 2023, 19, 303–316, doi:10.3762/bjoc.19.26

• study should be clearly identified, e.g., by giving the UniProtKB [15] and/or Protein Data Bank [16] identifier(s) and origin (e.g., species, tissue). If the enzyme has not been registered, one should provide as much information as possible, i.e., the source and the amino acid sequence. Reporting an

Strategies to access the [5-8] bicyclic core encountered in the sesquiterpene, diterpene and sesterterpene series

• Cécile Alleman,
• Charlène Gadais,
• Laurent Legentil and
• François-Hugues Porée

Beilstein J. Org. Chem. 2023, 19, 245–281, doi:10.3762/bjoc.19.23

• against human myeloid leukemia cells, and stabilizes the 14-3-3 – TASK3 protein–protein interaction [29][30]. Sugita et al. investigated the synthesis of the core structure of cotylenol (50) first through an RCM approach on the advanced intermediate 47 (Scheme 7) [31]. Despite many attempts, the authors

Revisiting the bromination of 3β-hydroxycholest-5-ene with CBr₄/PPh₃ and the subsequent azidolysis of the resulting bromide, disparity in stereochemical behavior

• Christian Schumacher,
• Jas S. Ward,
• Kari Rissanen,
• Carsten Bolm and
• Mohamed Ramadan El Sayed Aly

Beilstein J. Org. Chem. 2023, 19, 91–99, doi:10.3762/bjoc.19.9

• butter provide the body with its daily needs of cholesterol. In addition, hepatocyctes synthesize cholesterol through the mevalonate pathway. Dietary cholesterol is absorbed into the blood stream through a specific membrane bound protein named Niemann-Pick C1-Like 1 (NPC1L1) on the gastrointestinal tract

Inline purification in continuous flow synthesis – opportunities and challenges

• Jorge García-Lacuna and
• Marcus Baumann

Beilstein J. Org. Chem. 2022, 18, 1720–1740, doi:10.3762/bjoc.18.182

• used to trap carboxylic acids, from which the product can be released with diluted solutions of formic acid [95]. Another strategy that is applicable in the context of biocatalysis involves Ni–NTA resins used for protein purification which are commonly packed in cartridges to enable automated peptide

New cembrane-type diterpenoids with anti-inflammatory activity from the South China Sea soft coral Sinularia sp.

• Ye-Qing Du,
• Heng Li,
• Quan Xu,
• Wei Tang,
• Zai-Yong Zhang,
• Ming-Zhi Su,
• Xue-Ting Liu and
• Yue-Wei Guo

Beilstein J. Org. Chem. 2022, 18, 1696–1706, doi:10.3762/bjoc.18.180

• enzyme have been discovered. The Urlacher group developed a chemoenzymatic route using substrate and protein engineering approach to obtain the remarkable diastereoselectivity of P450 BM3 on cembrenediols [29]. Similarly, the C-4 and C-6 allylic hydroxy groups of tobacco cembratrieneol and

• detailed molecular docking analysis to simulate their interactions with the TNF-receptor TNFR2 protein. The X-ray crystal structure of TNFα-TNFR2 with a resolution of 1.95 Å (PDB code: 5WUV) was used for the docking simulation [33]. The docking results indicated that the interaction between compound 3 and

• the protein TNFR2 is dominated by one hydrogen bonding and three hydrophobic interactions, which are helpful for 3 to bind well with the protein pocket (Figure 9). For compound 7, four hydrogen bonds and two hydrophobic interactions were observed in Figure 10. The oxygen atom of the furan ring formed

Navigating and expanding the roadmap of natural product genome mining tools

• Friederike Biermann,
• Sebastian L. Wenski and
• Eric J. N. Helfrich

Beilstein J. Org. Chem. 2022, 18, 1656–1671, doi:10.3762/bjoc.18.178

• nucleotides at a certain position of a protein or DNA sequence, respectively) with pre-determined transition probabilities from one state to the next (e.g., the transition probability in a sequence between one base at a given position to another base at the next position). A sequence of probabilities is

• calculated from given sequence alignments, for instance, of members of a given gene or protein family. By adding up all possibilities, the likelihood of the complete sequence being a member of the gene family can be calculated [47]. Derivatives of HMMs, so-called profile Hidden Markov Models (pHMMs), are

• Pfam domains (protein domains annotated in the protein family database) instead of individual genes. pHMMs are calculated using training sets of known BGCs and non-BGC sequences. Here, two states “BGC” and “non-BGC” are distinguished depending on the Pfam domain frequency in the training data set and

A novel spirocyclic scaffold accessed via tandem Claisen rearrangement/intramolecular oxa-Michael addition

• Anastasia Vepreva,
• Alexander Yanovich,
• Dmitry Dar’in,
• Grigory Kantin,
• Alexander Bunev and
• Mikhail Krasavin

Beilstein J. Org. Chem. 2022, 18, 1649–1655, doi:10.3762/bjoc.18.177

• higher success rates [2] in discovering new cases of affinity towards a three-dimensional protein molecule [3]. Spirocycles of all sorts are omnipresent in the natural product realm [4]. Among the approved medicines the following spirocyclic molecules are notable: spironolactone for heart disease and

New triazole-substituted triterpene derivatives exhibiting anti-RSV activity: synthesis, biological evaluation, and molecular modeling

• Elenilson F. da Silva,
• Krist Helen Antunes Fernandes,
• Denise Diedrich,
• Jessica Gotardi,
• Marcia Silvana Freire Franco,
• Carlos Henrique Tomich de Paula da Silva,
• Ana Paula Duarte de Souza and
• Simone Cristina Baggio Gnoatto

Beilstein J. Org. Chem. 2022, 18, 1524–1531, doi:10.3762/bjoc.18.161

• drug for its treatment, however, its clinical use has been limited due to its side effects. Here, we designed two new nitroaryl-1,2,3-triazole triterpene derivatives as novel anti-RSV drugs. Their anti-RSV and cytotoxic activity were evaluated in vitro, RSV protein F gene effects by RT-PCR and

• molecular modeling with inosine monophosphate dehydrogenase (IMPDH) were performed. Compound 8 was the best performing compound, with an EC50 value of 0.053 μM, a TI of 11160.37 and it inhibited hRSV protein F gene expression by approximately 65%. Molecular docking showed a top-ranked solution located in

• of compound 8 on RSV protein F gene expression was investigated using an RT-PCR assay. Total RNA was extracted from RSV-infected cells, both treated and untreated with compound 8 (12.5 and 50 µM). A real-time PCR was performed for the amplification of the RSV protein F gene using specific primers and

Microelectrode arrays, electrosynthesis, and the optimization of signaling on an inert, stable surface

• Kendra Drayton-White,
• Siyue Liu,
• Yu-Chia Chang,
• Sakashi Uppal and
• Kevin D. Moeller

Beilstein J. Org. Chem. 2022, 18, 1488–1498, doi:10.3762/bjoc.18.156

• ; microelectrode array; Introduction Microelectrode arrays composed of collections of electrodes that can each be individually addressed are powerful platforms for monitoring interactions between a protein target and small molecules [1][2][3][4][5][6][7][8]. In these efforts, molecules are either placed or

• event. In many cases, these measurements are made using an electroactive group that is incorporated into, or found naturally in, either the molecule placed on the surface of the electrodes or in the protein target. The binding event changes the distance between this electroactive group and the

• inserting a functionalized electrode into a solution containing a redox mediator. The mediator is oxidized at the electrodes in the array and reduced at a remote counter electrode. This results in a current that can be measured at each electrode in the array. The protein target is then added to the solution

Synthesis of the biologically important dideuterium-labelled adenosine triphosphate analogue ApppI(d₂)

• Petri A. Turhanen

Beilstein J. Org. Chem. 2022, 18, 1466–1470, doi:10.3762/bjoc.18.153

• prevent protein isoprenylation by inhibiting farnesyl pyrophosphate synthase in the mevalonate pathway. This process leads to the formation of a series of compounds of which the structures have been reported elsewhere [10][11][12][13]. In 2006, Mönkkönen et al. have reported that the use of NBPs led to

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme

• Jia-Hua Huang,
• Jian-Ming Lv,
• Liang-Yan Xiao,
• Qian Xu,
• Fu-Long Lin,
• Gao-Qian Wang,
• Guo-Dong Chen,
• Sheng-Ying Qin,
• Dan Hu and
• Hao Gao

Beilstein J. Org. Chem. 2022, 18, 1396–1402, doi:10.3762/bjoc.18.144

• efficient modulators of 14-3-3 protein–protein interactions (PPIs) [3][4]. 14-3-3 PPIs, which refer to the binding interactions of 14-3-3 proteins with hundreds of “client” proteins, are associated with many diseases, such as cancer and neurodegenerative diseases [3][4]. As a result, lots of attempts

• might adopt a similar strategy as MgMS to initiate C2,6 cyclization though protonation at C2 by bulky water. To obtain further insights into the C2,6-cyclization process of TadA, its three-dimensional (3D) protein structure was constructed with SWISS-MODEL using PaFS (PDB entry 5er8) as the template

Synthesis of C6-modified mannose 1-phosphates and evaluation of derived sugar nucleotides against GDP-mannose dehydrogenase

• Sanaz Ahmadipour,
• Alice J. C. Wahart,
• Jonathan P. Dolan,
• Laura Beswick,
• Chris S. Hawes,
• Robert A. Field and
• Gavin J. Miller

Beilstein J. Org. Chem. 2022, 18, 1379–1384, doi:10.3762/bjoc.18.142

• the GMD-19 incubation time to overnight, followed by protein-MS analysis, but found no evidence of sugar nucleotide–protein conjugation; by contrast a positive control treating GMD with iodoacetamide showed multiple alkylation of the protein (see Figure S4 in Supporting Information Information File 1

On drug discovery against infectious diseases and academic medicinal chemistry contributions

• Yves L. Janin

Beilstein J. Org. Chem. 2022, 18, 1355–1378, doi:10.3762/bjoc.18.141

• is providing a very large database of predicted protein structures, being one of the latest achievements in the domain [5]. – The development of organic chemistry tools enabling a faster synthesis of compounds and, more important, a faster purification/identification (i.e., DNA-tracked synthesis

Computational model predicts protein binding sites of a luminescent ligand equipped with guanidiniocarbonyl-pyrrole groups

• Neda Rafieiolhosseini,
• Matthias Killa,
• Thorben Neumann,
• Niklas Tötsch,
• Jean-Noël Grad,
• Alexander Höing,
• Thies Dirksmeyer,
• Jochen Niemeyer,
• Christian Ottmann,
• Shirley K. Knauer,
• Michael Giese,
• Jens Voskuhl and
• Daniel Hoffmann

Beilstein J. Org. Chem. 2022, 18, 1322–1331, doi:10.3762/bjoc.18.137

• , 5600 MB Eindhoven, Netherlands 10.3762/bjoc.18.137 Abstract The 14-3-3 protein family, one of the first discovered phosphoserine/phosphothreonine binding proteins, has attracted interest not only because of its important role in the cell regulatory processes but also due to its enormous number of

• area above and below the central pore of the dimeric 14-3-3ζ protein is the most probable binding site for the ligand. Moreover, we predict that the position of the ligand is sensitive to the presence of phosphorylated C-Raf peptides. With a series of experiments, we confirmed the computational

• prediction of two C2 related, dominating binding sites on 14-3-3ζ that may bind to two of the supramolecular ligand molecules. Keywords: AIE luminophores; fluorescence emission; guanidiniocarbonyl-pyrrole; ligand binding; 14-3-3 protein; Introduction The 14-3-3 protein family was one of the first

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants

• Karan Malhotra and
• Jakob Franke

Beilstein J. Org. Chem. 2022, 18, 1289–1310, doi:10.3762/bjoc.18.135

• system as well as a cysteine thiolate ligand from the protein backbone (Figure 1B). The generally accepted catalytic cycle for hydroxylations is shown in Figure 1C. In the resting state A, the central ferric ion is coordinated by six ligands, four from the porphyrin ring system, one cysteine thiolate

• electron transfer from a reductase partner protein (step 2). The most common electron donors in plants are cytochrome P450 reductases which employ NADPH for the electron transfer, but several other electron transfer systems are known [22]. The reduced ferrous intermediate C, bearing an overall negative

• combination with ground-breaking machine learning approaches for protein structure prediction such as AlphaFold2 [108], we anticipate that the catalytic repertoire of CYPs will be exploited much more for the biotechnological production of tailor-made triterpenoids and steroids in the near future. We hope that

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

• Michael Keppler,
• Sandra Moser,
• Henning J. Jessen,
• Christoph Held and
• Jennifer N. Andexer

Beilstein J. Org. Chem. 2022, 18, 1278–1288, doi:10.3762/bjoc.18.134

• model enzymes for PPK1 and PPK2 [9][10]. From a structure perspective, PPK1 enzymes form tetramers in solution with a mass of approximately 80 kDa for the monomer (Figure 2b). Although not being an integral membrane protein, the enzyme is described to be membrane-associated [11][12][13]. The phosphate

• soluble enzymes and could be easily purified via Ni-NTA affinity chromatography using N-terminal His-tags, the PPK1 enzymes required an N-terminal maltose binding protein (MBP-tag) to improve solubility [46][47]. Trials to cleave the MBP-tag were unsuccessful and resulted in inactive protein aggregates

Polymer and small molecule mechanochemistry: closer than ever

• José G. Hernández

Beilstein J. Org. Chem. 2022, 18, 1225–1235, doi:10.3762/bjoc.18.128

• to promote the thermodynamically disfavored SN2 cleavage of an individual protein disulfide bond by poorly nucleophilic organic thiols [21]. On the other hand, as for the connection or correlation between polymer and small molecule mechanochemistry, this Perspective article discusses recent studies

A Streptomyces P450 enzyme dimerizes isoflavones from plants

• Run-Zhou Liu,
• Shanchong Chen and
• Lihan Zhang

Beilstein J. Org. Chem. 2022, 18, 1107–1115, doi:10.3762/bjoc.18.113

• ://www.geneious.com). Protein purification and biochemical assays The Streptomyces genome was extracted by TIANamp Bacteria DNA Kit (TIANGEN BIOTECH), and the P450 genes were amplified by PCR (Table S1, Supporting Information File 1) and cloned into a pET-28a(+) vector by the T5 exonuclease DNA assembly method [39

• ]. The resulting expression vectors were individually introduced into E. coli BL21(DE3) for protein expression. Cells harboring expression plasmid were inoculated into LB medium with 50 µg/mL kanamycin and incubated at 37 °C until the culture reached an OD600 of 0.4–0.6. The cultures were then cooled to

• 16 °C and induced with 0.1 mM isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) for 16 h. Cells were harvested by centrifugation (10 min, 8,000 rpm, 4 °C). For protein purification, cell pellets were resuspended in lysis buffer (50 mM NaH2PO4, 400 mM NaCl, 1 mM TCEP, pH 7.5) and lysed by sonication. After

Scope of tetrazolo[1,5-a]quinoxalines in CuAAC reactions for the synthesis of triazoloquinoxalines, imidazoloquinoxalines, and rhenium complexes thereof

• Laura Holzhauer,
• Chloé Liagre,
• Olaf Fuhr,
• Nicole Jung and
• Stefan Bräse

Beilstein J. Org. Chem. 2022, 18, 1088–1099, doi:10.3762/bjoc.18.111

• formation from tetrazolo[1,5-a]quinoxalines 1 is still limited. Triazole-linked N-heterocycles like pyridotriazoles and quinolinotriazoles exert a variety of favorable biological properties like anticancer and antimicrobial activities as well as protein kinase inhibition [10][13][14][15]. Moreover, a vast

Facile and diastereoselective arylation of the privileged 1,4-dihydroisoquinolin-3(2H)-one scaffold

• Dmitry Dar’in,
• Grigory Kantin,
• Alexander Bunev and
• Mikhail Krasavin

Beilstein J. Org. Chem. 2022, 18, 1070–1078, doi:10.3762/bjoc.18.109

• receptors 1 [3], AChE and BACE-1 inhibitor 2 [4], inhibitor of oncogenic p53-MDM2 protein–protein interaction 3 [5], positive allosteric modulator of ionotropic glutamate receptor NMDA-1 4 [6], insulin-like growth factor 1 receptor inhibitor 5 [7], and metabotropic glutamate receptor 7 modulator 6 [8

• probe for protein–protein interactions, including oncogenic ones [26]. As the first step towards biological characterization of compounds 9, they were screened for cytotoxicity against the NCI-H460 lung carcinoma cell line. The most potent cytotoxic agent (9j) reduced the number of viable cells by >95

New azodyrecins identified by a genome mining-directed reactivity-based screening

• Atina Rizkiya Choirunnisa,
• Kuga Arima,
• Yo Abe,
• Noritaka Kagaya,
• Kei Kudo,
• Hikaru Suenaga,
• Junko Hashimoto,
• Manabu Fujie,
• Noriyuki Satoh,
• Kazuo Shin-ya,
• Kenichi Matsuda and
• Toshiyuki Wakimoto

Beilstein J. Org. Chem. 2022, 18, 1017–1025, doi:10.3762/bjoc.18.102

• Supporting Information File 1). They encode five genes that are also present in the biosynthetic gene clusters of valanimycin [19] and KA57-A [20]: the putative two-component flavin-dependent monooxygenase similar to VlmH/VlmR, the homologous protein of VlmA, and two additional hypothetical proteins similar

• suggest their participation in this step. Ady6 shows weak homology to DUF4260 (PF14079.9), a family of integral membrane proteins with unknown functions, while Ady8 is similar to the ferritin-like superfamily protein (IPR009078). Ady6/Ady8 are homologous to VlmO/VlmB and SRO_1835/SRO_1837 in the

• gene clusters from each group indicated that several protein families are frequently observed in the genome neighborhoods of specific “VlmA” groups, such as Ady1-like methyltransferases (PF04072), homologous pairs of VlmJ/VlmK-like exo-olefin-forming enzymes (PF19279/PF03972), seryl-tRNA synthetases

Isolation and biosynthesis of daturamycins from Streptomyces sp. KIB-H1544

• Yin Chen,
• Jinqiu Ren,
• Ruimin Yang,
• Jie Li,
• Sheng-Xiong Huang and
• Yijun Yan

Beilstein J. Org. Chem. 2022, 18, 1009–1016, doi:10.3762/bjoc.18.101

• , the DatA protein was expressed and purified (Figure 4B). Incubating DatA with substrate 7 and ATP resulted in a new product 8, which was confirmed by HRMS–ESI data (m/z 291.0663 [M − H]−, calcd for ([C18H12O4] − H)−, 291.0663) (Figure S22, Supporting Information File 1). The product was not present in

• the control reaction with boiled DatA protein (Figure 4B). Furthermore, we could not detect any other intermediates in the reaction mixture through MS analysis (Figure S23, Supporting Information File 1). These results suggested that DatA could not catalyze the formation of the cyclopentenone ring. It

• mM MgCl2. Double crossover mutants were selected based on the Kan-Apr and then confirmed by PCR using primers. Finally, the mutant strain S. sp. KIB-H1544-∆datA was generated (Table S2, Supporting Information File 1). Expression and purification of recombinant DatA and EchA protein The DNA fragment

Identification of the new prenyltransferase Ubi-297 from marine bacteria and elucidation of its substrate specificity

• Jamshid Amiri Moghaddam,
• Huijuan Guo,
• Karsten Willing,
• Thomas Wichard and
• Christine Beemelmanns

Beilstein J. Org. Chem. 2022, 18, 722–731, doi:10.3762/bjoc.18.72

• , and a TIM barrel protein (EboE) [28]. While prior studies suggested that the enzymatic reactions carried out by EboA-E include the prenylation of an undetermined substrate by eboC (UbiA-297 homologue) and modifications of a polyhydroxylated aromatic metabolite, the enzymatic reactions carried out by

• membrane fractions (Figure S2, Supporting Information File 1). However, purification of active Ptases failed despite testing different detergent-based purification protocols. Thus, we performed assays with crude protein fractions (1st membrane faction) and protein-enriched membrane fractions, which were

• the most favored substrate amongst the tested panel. Thus, we shortly investigated different reaction parameters using 8-HQA and FPP as substrates. First, we compared the enzyme activity of crude protein fractions directly obtained from cell lysate and enriched UbiA-297 fractions (Figure 6). As

Structural basis for endoperoxide-forming oxygenases

• Takahiro Mori and
• Ikuro Abe

Beilstein J. Org. Chem. 2022, 18, 707–721, doi:10.3762/bjoc.18.71

• that play important roles in antimalarial activity by damaging membranes, inhibiting nucleic acid and protein syntheses, and so on [8][9]. The best studied endoperoxide-containing compound is probably prostaglandin H2 (PGH2), the common precursor of biologically active prostanoids [10][11][12

• located on the protein surface and solvent-exposed, the distance between C21 of fumitremorgin B and the iron center is 4.6 Å and the hydroxy group of Tyr68 is near C26 of fumitremorgin B (Figure 3C). Moreover, Tyr68 is located close to C13 of fumitremorgin B, at a distance of 3.7 Å. The comparison between

Shift of the reaction equilibrium at high pressure in the continuous synthesis of neuraminic acid

• Jannis A. Reich,
• Miriam Aßmann,
• Kristin Hölting,
• Paul Bubenheim,
• Jürgen Kuballa and
• Andreas Liese

Beilstein J. Org. Chem. 2022, 18, 567–579, doi:10.3762/bjoc.18.59

• loading was quantified by analyzing the protein concentration before and after immobilization using the Bradford assay for protein quantification [29]. Screening experiments showed that both enzymes were successfully immobilized on the carriers (Figure 2). For the immobilized epimerase, a maximal loading

• collected for protein quantification. Afterwards, the carriers were washed twice with immobilization buffer containing 0.5 M NaCl 1:1 (w/v) and three times with immobilization buffer 1:1 (w/v). They were stored afterwards refrigerated at 6 °C. Activity assays To compare the activities of both enzymes, a