Benzomalvin G and H, an Atropisomeric Pair of Dimethoxylated Quinazolinobenzodiazepine Alkaloids Produced by Aspergillus fumigatiaffinis

Abstract

Aspergillus and Penicillium species produce a variety of quinazolinobenzodiazepine (QBD) alkaloids by using different biosynthetic gene clusters (BGCs). During the course of metabolome analysis of Aspergillus fumigatiaffinis IFM55214, we discovered an atropisomeric pair of QBD natural products, benzomalvin G (1) and H (2), that are produced by this fungus. The isomer 1 was favored in organic solvents, and 2 was favored in aqueous solutions. The stereochemistry and conformation of these 2 compounds were determined unambiguously by Marfey’s method and nuclear Overhauser effect (NOE) analysis, respectively. Furthermore, a putative BGC for 1 and 2 (Afben cluster), composed of 3 genes (AfbenX, AfbenY, and AfbenZ) shared between the ben cluster in Aspergillus terreus and 4 additional genes (methyltransferases and oxygenases), was identified in the genome of A. fumigatiaffinis IFM55214.

Introduction

Quinazolinobenzodiazepines (QBDs) are a class of compounds featuring a fused 6/7/6/6 tetracyclic ring system derived from 2 molecules of anthranilic acid (Ant) and 1 molecule of an amino acid. QBDs are often isolated from Aspergillus and Penicillium species: benzomalvins from Penicillium sp.,^1–3) Aspergillus terreus⁴⁾ and A. udagawae,⁵⁾ (−)-auranthine from Penicillium auranthiogriseum⁶⁾ and A. lentulus,⁷⁾ (+)-auranthine from A. lentulus,⁷⁾ novobenzomalvins from A. novofumigatus,⁸⁾ asperlicins from A. aliaceus,^9,10) and circumdatins from A. ochraceus.^11–14) The biosynthetic mechanisms of QBDs have been investigated by several research groups, and the biosynthetic gene clusters (BGCs) for benzomalvins, (−)-auranthine, (+)-auranthine, and asperlicins have been identified as ben,⁴⁾ Alnit,⁷⁾ nit,⁷⁾ and asp¹⁵⁾ clusters, respectively. Although all of these BGCs were identified in Aspergillus species, the identities of the amino acid sequences of the core genes in these BGCs are relatively low (the highest identity is only 38.2% found between AlnitA and aspA),⁷⁾ suggesting that the fungi evolved to produce various QBDs in different manners.

Our recent metabolome analysis of Aspergillus species in the section Fumigati^7,16,17) revealed that the extract of Aspergillus fumigatiaffinis IFM55214 cultured on oatmeal agar (OMA) contained a pair of unknown compounds possessing the same molecular formula (C₂₆H₂₃N₃O₄) and UV absorption (λ_max 240, 312 nm). These features resemble those of benzomalvin A/D², and we identified a ben cluster-like BGC in the genomic sequence of A. fumigatiaffinis IFM55214, indicating that the detected compounds are new benzomalvin derivatives. Herein, we report the isolation, structure elucidation, and putative BGC of new QBD alkaloids, benzomalvin G (1) and H (2).

Results and Discussion

We could detect the production of compounds 1 and 2 when A. fumigatiaffinis IFM55214 was cultured on potato dextrose agar or OMA plates, but not when cultured in liquid medium (Fig. 1a), such as potato dextrose broth. The production of 1 and 2 reached its highest level 1–2 weeks after inoculation of the fungi onto OMA plates (Fig. 1a), and compounds 1 (0.9 mg) and 2 (0.5 mg) were isolated from a 2-L culture of A. fumigatiaffinis through 3 steps of chromatography. Although the amount of 2 detected in the crude extract was greater than that of 1, the isolated amounts were reversed. This finding encouraged us to test the interconvertibility of 1 and 2. After incubation for 1 d in solutions, both 1 and 2 transformed into a mixture of 1 and 2 (Fig. 1b), indicating that these 2 compounds are tautomeric pairs. The conversion rate from 1 to 2 was higher in 50% MeOH than in MeOH, while the conversion rate from 2 to 1 was higher in MeOH than in 50% MeOH. The interconversion of these compounds was slow enough for separating 1 and 2 at 25°C (room temperature), but was accelerated at 37°C (Fig. 1b).

Fig. 1. (a) Production of Compounds 1 and 2 under Various Conditions Detected by UV Absorption at 272 nm

(b) Interconversion of 1 and 2 stocked for 1 d in solution states. The condition aqueous MeOH indicates 50% aqueous methanol. MS were monitored at m/z = 442.1761 in the positive mode.

The molecular formula of 1 and 2 was established as C₂₆H₂₃N₃O₄ based on high-resolution-electrospray ionization-MS (HR-ESI-MS) (m/z 442.1760 [M + H]⁺, Calcd 442.1761) and ¹H- and ¹³C-NMR spectra. The ¹H-NMR spectrum of 1 recorded in CD₃OD resembles that of benzomalvin A (Fig. 2), except for the presence of additional methoxy protons at δ 3.96 (3H, s) and 3.87 (3H, s) parts per million (ppm) and a reduced number of aromatic protons. These differences suggest that 1 contains additional methoxy groups attached to aromatic rings. A detailed analysis of the ¹³C, heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation spectra of 1 revealed that C-5 and C-6 in ring A are substituted with methoxy groups, and the other moieties of 1 are the same as those of benzomalvin A, consisting of a fused 6/7/6/6 tetracyclic ring system (Fig. 2, Supplementary Figs. S1–S5, and Table 1). Next, the stereochemistry at C-19 was determined with the advanced Marfey’s method.¹⁸⁾ Compound 1 was hydrolyzed in D₂O and the resulting amino acids were derivatized with Nα-(5-fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA) or d-FDLA. The retention times of the obtained FDLA derivatives on LC-MS were opposite to those of authentic samples synthesized from N-methyl-d-phenylalanine, revealing that 1 possesses the S-configuration at C-19 (Fig. 3a).

Fig. 2. The Structure of Benzomalvin A (Left), Atom Numbering (Middle), and 2D-NMR Correlations of 1/2 (Right)

DQF-COSY (bold) and key HMBC (arrow) correlations are shown.

Table 1. NMR Data of Compounds 1 and 2 in CD₃OD

Position	1				2				Benzomalvin A (in CDCl3)1
	δH (ppm), multi. (J [Hz])	δc (ppm)	HMBC	ROESY	δH (ppm), multi. (J [Hz])	δc (ppm)	HMBC	ROESY*	δH (ppm), multi. (J [Hz])	δc (ppm)
2	—	169.5			—	167.6			—	167.6
3	—	124.7			—	125.2			—	133.0
4	7.34 s	111.8	2, 3, 5, 6, 8	28	7.42 s	112.5	2, 3, 5, 6, 8	28	7.86 ddd (8.0, 1.5, 0.6)	130.3
5	—	150.7			—	151.0			7.61 ddd (8.0, 7.0, 1.4)	129.5
6	—	152.4			—	152.8			7.67 ddd (8.1, 7.0, 1.5)	131.3
7	7.21 s	112.2	3, 5, 6, 8	29	7.29 s	112.7	3, 5, 6, 8	29	7.71 ddd (8.1, 1.4, 0.6)	129.0
8	—	128.5			—	128.4			—	134.2
10	—	163.3			—	163.5			—	161.8
11	—	122.7			—	122.5			—	122.8
12	8.27 d (8.0)	128.1	10, 14, 16		8.29 ddd (8.0, 1.5, 0.5)	128.2	10, 14, 16		8.26 ddd (7.6, 1.7, 0.5)	127.9
13	7.60 m	128.8	11, 15		7.61 ddd (8.0, 7.2, 1.1)	129.0	11, 15		7.62 ddd (7.6, 7.1, 1.5)	128.2
14	7.90 m	136.2	12, 16		7.87 ddd (8.2, 7.2, 1.5)	136.4	12, 16		7.93 ddd (8.1, 7.1, 1.7)	135.6
15	7.87 m	128.8	11, 13		7.70 ddd (8.2, 1.1, 0.5)	128.3	11, 13		7.88 ddd (8.1, 1.5, 0.5)	128.5
16	—	147.5			—	147.6			—	147.1
18	—	153.9			—	155.5			—	153.7
19	5.04 dd (7.5, 7.5)	60.2	2, 18, 20, 21, 27	22	4.87 m	71.6	2, 18, 20, 21, 27	27	4.87 dd (7.5, 7.5)	59.3
20 α	3.75 dd (14.5, 7.3)	34.0	19, 21, 22/26	22, 27	2.83 dd (13.7, 6.8)	36.6	19, 21, 22/26	22	3.79 dd (14.6, 7.5)	33.7
β	3.53 dd (14.6, 7.7)		18, 19, 21, 22/26	22, 27	2.51 dd (13.7, 10.4)		18, 19, 21, 22/26	22	3.42 dd (14.6, 7.5)
21	—	138.4			—	137.5			—	138.4
22/26	7.32 br d (7.7)	130.2	20, 23/25, 24, 26	19, 20αβ	7.06 m	130.1	20, 23/25, 24, 26	20αβ	7.39 br d (7.3)	130.0
23/25	7.25 br dd (7.7, 7.7)	129.7	21, 23/25		7.30 m	129.9	21, 23/25		7.27 br dd (7.3, 7.3)	129.3
24	7.17 br dd (7.3, 7.3)	127.9	22/26, 23/25		7.23 m	128.3	22/26, 23/25		7.20 br dd (7.3, 7.3)	127.5
27	3.03 s	28.6	2, 19	20αβ	2.95 s	38.5	2, 19	19	3.03 s	27.9
28	3.96 s	56.7	5	4	4.02 s	56.8	5	4	—	—
29	3.87 s	56.7	6	7	3.93 s	56.9	6	7	—	—

^*ROESY spectrum of 2 was recorded in CDCl₃.

Fig. 3. Determination of the Stereochemistry of 1 and 2

(a) LC-MS analysis of the FDLA derivatives. MS were monitored at m/z = 474.1983 in the positive mode. (b) NOEs observed in 1 and 2.

The planar structure of 2 was elucidated to be the same as 1 based on 1D and 2D-NMR spectra, but the chemical shifts at H-19, H-20, H-22, H-27, C-19, C-20, and C-27, derived from N-methylphenylalanine, were significantly different (Fig. 2, Supplementary Figs. S7–S11, and Table 1). The stereochemistry of 2 was determined using the advanced Marfey’s method,¹⁸⁾ revealing that the configuration at C-19 is S, consistent with that of 1 (Fig. 3a). Combined with the fact that 1 and 2 interconverted, 1 and 2 were considered to be an atropisomeric pair. The same phenomenon was reported for benzomalvin A and D by Sun et al., and the conformation was estimated based on the analysis of the chemical shifts at C-19, C-20, and C-27.²⁾ The chemical shifts of 1 and 2 fit well with those of benzomalvin A and D, respectively. The conformation of benzomalvin A and D was also characterized using nuclear Overhauser effect (NOE) by Sugimori et al. in 1998¹⁹⁾; therefore, we analyzed the rotating-frame NOE spectroscopy (ROESY) spectra of 1 (Supplementary Fig. S6) and 2 (recorded in CDCl₃ to prevent water signal from overlapping with H-19; Supplementary Figs. S12–S14). An NOE was observed between H-27 and H-20 in 1, whereas an NOE was observed between H-27 and H-19 in 2, indicating that the benzyl group is equatorial in 1 and axial in 2, consistent with the conformation of benzomalvin A and D,^2,19) respectively (Fig. 3b).

The biosynthetic pathway of benzomalvin A/D reported by Clevenger et al.⁴⁾ is as follows (Fig. 4a). BenX (N-methyltransferase) and BenZ (nonribosomal peptide synthetase [NRPS]) produce an N-methyl dipeptide intermediate from l-phenylalanine (l-Phe) and Ant. This intermediate is condensed with an additional Ant unit on BenY and cyclized by the BenY-C_T domain to give benzomalvin A/D. AfBenX, AfBenY, and AfBenZ, encoded in the Afben cluster, exhibit high similarity to these enzymes (Fig. 4b and Table 2); thus, the core architecture of 1/2 is expected to be synthesized by them. ORF3 is not encoded in the ben cluster of A. terreus and is deduced to be an incomplete NRPS, suggesting that this is not necessary for the biosynthesis of 1/2. The putative functions of additional genes in the Afben cluster are O-methyltransferases (ORF1 and ORF5), flavin-dependent monooxygenase (ORF2), and P450 (ORF4), which agree with the structural differences between 1/2 and benzomalvin A/D. Moreover, the other Ant-activating BGCs identified in the genomic sequence of A. fumigatiaffinis IFM55214 lack methyltransferases or oxygenases. Therefore, the Afben cluster is deduced to correspond to the biosynthesis of 1/2.

Fig. 4. (a) BGC and Biosynthetic Pathway of Benzomalvin A/D, and (b) Putative BGC for 1/2 Found in the Genomic Sequence of A. fumigatiaffinis IFM55214

The arrows in BGCs indicate NRPS (black), N-methyltransferase (light gray), O-methyltransferase (dark gray), or oxidase (striped). Domains of NRPS genes are shown as follows: A (adenylation), T (thiolation), C (condensation), and C_T (terminal condensation) domains.

Table 2. Putative Function of the Genes in the Afben Cluster

Gene	ID	Putative function	Homolog gene (identity/similarity)
ORF1	jgi.p_Aspfumig1_153238	O-Methyltransferase	tpcA (50.8/67.4)
ORF2	jgi.p_Aspfumig1_153239	Flavin-dependent monoxygenase	cfoG (32.1/47.8)
AfbenZ	jgi.p_Aspfumig1_145120	NRPS (ATCATC)	benZ (66.5/79.2)
AfbenY	jgi.p_Aspfumig1_145122	NRPS (ATC)	benY (63.7/76.3)
AfbenX	jgi.p_Aspfumig1_145123	N-Methyltransferase	benX (63.1/77.9)
ORF3	jgi.p_Aspfumig1_145124	NRPS (TC)	chyA (40.3/56.7)
ORF4	jgi.p_Aspfumig1_153242	P450	mpaDE′ (55.5/72.4)
ORF5	jgi.p_Aspfumig1_145126	O-Methyltransferase	afvC (32.2/48.7)

Conclusion

In this study, we discovered a new atropisomeric pair of QBD natural products, benzomalvin G (1) and H (2), from A. fumigatiaffinis IFM55214. The stereochemistry of the 2 compounds was determined with the advanced Marfey’s method, and the conformation was determined based on ROESY spectra. The interconversion analysis of 1 and 2 revealed that 1 and 2 are favored in organic solutions and aqueous solutions, respectively, explaining why the isolated amount of 1 was greater than that of 2. We identified a putative BGC for 1/2 (Afben cluster), containing 3 genes (AfbenX, AfbenY, and AfbenZ) essential for constructing the benzomalvin core structure, and additional oxygenase and methyltransferase genes. Since the conformations of compounds 1 and 2 (and also benzomalvin A and D) are significantly different, of interest are the structures recognized by the additional oxygenases and methyltransferases. Detailed analysis of the biosynthesis of 1/2 is ongoing, and this question will be answered in the future.

Experimental

General Experimental Procedures

NMR spectra were obtained with a Bruker BioSpin, Billerica, MA, U.S.A. AVANCE III HD 500 MHz spectrometer (¹H 500 MHz, ¹³C 125 MHz) equipped with a cryoprobe. ¹H-NMR chemical shifts are reported in ppm using the proton resonance of residual solvent as a reference: CD₃OD δ 3.31 and CDCl₃ δ 7.26.^{20) 13}C-NMR chemical shifts are reported relative to CD₃OD δ 49.0 and CDCl₃ δ 77.16.²⁰⁾ MS were recorded with a Thermo Fisher Scientific UltiMate 3000 Q Exactive Plus LC–MS (Thermo Fisher Scientific, Waltham, MA, U.S.A.) using both positive and negative electrospray ionization (ESI). Samples were separated for analysis on an ACQUITY UPLC 1.8 µm, 2.1 × 50 mm C18 reversed-phase column (Waters, Milford, MA, U.S.A.) using a linear gradient of 5–100% (v/v) MeCN in H₂O supplemented with 0.05% (v/v) formic acid at a flow rate of 0.5 mL/min unless otherwise indicated. Optical rotations were measured on a JASCO P-2200 digital polarimeter (JASCO, Tokyo, Japan).

Strains and Analyses of DNA Sequences

Aspergillus fumigatiaffinis IFM55214 was provided by the Medical Mycology Research Center, Chiba University, with support in part from the National BioResource Project (NBRP), Agency for Medical Research and Development, Japan. The genomic sequence and structural annotation data of A. fumigatiaffinis IFM55214 (= CBS117186) were produced by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) in collaboration with the user community. These data were used for the analysis of BGCs with fungal antiSMASH version 7.1.0.²¹⁾ Homology of the amino acid sequences was calculated with EMBOSS Needle.²²⁾

Isolation of Benzomalvin G (1) and H (2) from A. fumigatiaffinis IFM55214

A. fumigatiaffinis IFM55214 was inoculated on 200 OMA plates, each containing 20 mL of OMA (total 2 L), and incubated for 16 d at 30°C. All the mycelia and the media were collected in a large beaker and extracted with acetone (500 mL, twice). Insoluble materials were removed by filtration, and the resulting supernatant was concentrated in vacuo to remove organic solvents. The resulting aqueous mixture was diluted with distilled water to 500 mL and extracted with the same volume of EtOAc 3 times. The organic phases were combined, concentrated in vacuo, and fractionated by silica gel column chromatography (Kanto Chemical, Tokyo, Japan) with CHCl₃/MeOH (100/0, 100/1, 100/2, 100/5, 100/20, and 100/100). The samples eluted with CHCl₃/MeOH (100/1) contained 1 and 2, and were purified by reversed-phase HPLC (COSMOSIL 5C₈ MS, Nacalai Tesque, Kyoto, Japan, 20 × 250 mm) using H₂O/MeCN (50/50 to 0/100 gradient for 60 min) at a flow rate of 8.0 mL/min. The fraction containing 1 (23–24 min) was further purified by reversed-phase HPLC (COSMOSIL 5C₈ MS, 10 × 250 mm) using H₂O/MeCN (60/40 isocratic) at a flow rate of 4.0 mL/min to afford 1 (0.9 mg, t_R = 47–48 min). Meanwhile, the fraction containing 2 (17–18 min) was purified by reversed-phase HPLC (COSMOSIL 5C₈ MS, 10 × 250 mm) using H₂O/MeCN (67/33 isocratic) at a flow rate of 4.0 mL/min to afford 2 (0.5 mg, t_R = 56–57 min).

Benzomalvin G (1)

See Table 1 and Supplementary Figs. S1–S6 for NMR spectra. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −9.5 (c 0.1, CH₃OH); IR (neat) cm⁻¹: 2981 (br), 1690, 1643, 1609, 1519, 1471, 1367, 1274, 1225, 1051, 1032, 1014; HR-ESI-MS: m/z 442.1760 [M + H]⁺ (Calcd for C₁₈H₂₉N₂O₈: 442.1761).

Benzomalvin H (2)

See Table 1 and Supplementary Figs. S7–S14 for NMR spectra. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +39 (c 0.01, CH₃OH); HR-ESI-MS: m/z 442.1760 [M + H]⁺ (Calcd for C₁₈H₂₉N₂O₈: 442.1761)).

Marfey’s Analysis of Compounds 1 and 2

To determine the stereochemistry of the N-methyl phenylalanine unit of compounds 1 and 2, 50 µg of each compound was hydrolyzed with 1 M HCl in D₂O containing 10% H₂O at 100°C for 12 h, then dried in vacuo. The hydrolysates were dissolved in 0.1 mL of D₂O, 50 µL of which was reacted with 10 µL of 1 M NaHCO₃ in D₂O and 50 µL of d- or l-FDLA (1% (w/v) in acetone) for 1 h at 65°C to give FDLA derivatives. The FDLA derivatives were analyzed by a Thermo Fisher Scientific UltiMate 3000 Q Exactive Focus LC–MS (Thermo Fisher Scientific) using a positive ESI monitored at m/z = 474.1983. Samples were separated for analysis on an ACQUITY UPLC HSS 1.8 µm, 2.1 × 50 mm C18 reversed-phase column (Waters) using solution A (H₂O/MeCN = 95/5 with 0.05% formic acid) and solution B (MeCN with 0.05% formic acid) with a gradient system of A/B = 90/10 (1.0 min), 90/10 to 50/50 (8.0 min), 50/50 to 0/100 (1.5 min), and 0/100 (2.5 min) at a flow rate of 0.5 mL/min (Fig. 2a).

Acknowledgements

This work was financially supported by the Japan Society for the Promotion of Science (JSPS) (K.W., 22H05121, 22H04979, 22K19158; S.K., 24K08731), the Uehara Memorial Foundation (K.W.), SECOM Science, and the Japan Agency for Medical Research.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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