Chemistry and Biology of Siderophores from Marine Microbes

marine drugs

Review Chemistry and Biology of Siderophores from Marine Microbes

Jianwei Chen 1, Yuqi Guo 1, Yaojia Lu 1, Bixia Wang 1, Jiadong Sun 2, Huawei Zhang 1 and Hong Wang 1,*

1 College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, China; [email protected] (J.C.); [email protected] (Y.G.); [email protected] (Y.L.); [email protected] (B.W.); [email protected] (H.Z.) 2 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20878, USA; [email protected] * Correspondence: [email protected]; Tel.: +86-571-8832-0622

Received: 3 September 2019; Accepted: 29 September 2019; Published: 29 September 2019

Abstract: Microbial siderophores are multidentate Fe(III) chelators used by microbes during siderophore-mediated assimilation. They possess high aﬃnity and selectivity for Fe(III). Among them, marine siderophore-mediated microbial iron uptake allows marine microbes to proliferate and survive in the iron-deﬁcient marine environments. Due to their unique iron(III)-chelating properties, delivery system, structural diversity, and therapeutic potential, marine microbial siderophores have great potential for further development of various drug conjugates for antibiotic-resistant bacteria therapy or as a target for inhibiting siderophore virulence factors to develop novel broad-spectrum antibiotics. This review covers siderophores derived from marine microbes.

Keywords: siderophores; marine microbes; synthesis; biosynthesis

1. Introduction Marine microbial community diversity and chemodiversity lead to the generation of numerous biological secondary metabolites. These compounds increase the chances of finding valuable drug candidates [1–3]. Marine siderophores are a type of low molecular weight natural products, the functions of which are to facilitate the microbial acquisition of iron in the ocean environment [4–6]. Marine microbes typically require micromolar levels of iron for their growth, yet the concentration of iron in the surface water of the world ocean is only 0.01–2 nM [7]. In order to overcome this lack of iron, marine microbes have evolved siderophore-mediated delivery system to obtain iron. Siderophores coordinated to insoluble Fe(III) are first transported into microbial cells by membrane bound iron-siderophore receptors. Afterwards, iron is then released from siderophores, typical via reduction of Fe(III) to Fe(II) by microbe-mediated redox processes [8–10]. Based on the efficient siderophore-mediated iron acquisition mechanisms, marine siderophores may be developed as novel antimicrobials by covalently attaching clinical antibiotics to marine siderophores or novel iron chelator desferals [11–14]. Furthermore, marine siderophores have also exhibited various non-classical biological functions, for example, as agents which may interfere with quorum sensing regulation and swarming in bacteria, as mediators of mutualistic interactions, and as secreted signaling molecules regulating virulence factors of pathogens [15–17]. These wide range of activities exhibited their great potential in medicine. The present review gives a comprehensive overview of siderophores from marine microorganisms (Table1).

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Table 1. List of abbreviations.

Abbreviation Full Name CAS Chrome azurol sulfonate ED50 Median eﬀective dose IC50 Hablalf maximal inhibition concentration NRPSs Nonribosomal peptide synthetases HSC N-hydroxy-N-succinylcadaverine HSDs N-hydroxy-N-succinyl diamines ArCP Aryl carrier protein Cy Cyclisation PCP Peptidyl carrier protein NIS NRPS-independent siderophore hLys N6-hydroxy-l-lysine ahLys N6-acetyl-N6-hydroxy-l-lysine 3-DHS 3-dehydroshikimate 3,4-DHBA 3,4-dihydroxybenzoic acid 2,3-DHBA 2,3-dihydroxybenzoic acid

2. Diversity of Siderophores from Marine Microorganisms Recently,a large number of siderophores with diverse structures have been discovered in the marine microorganisms (Table2). According to their functional groups and hydrophobicity, siderophores from marine microorganisms are divided into seven types, including hydroxamates (Figure1), α-hydroxycarboxylates (Figure2), catecholates (Figure3), mixed hydroxamates /α-hydroxycarboxylates (Figure4), mixed α-hydroxycarboxylates/catecholates (Figure5), mixed hydroxamates /catecholates (Figure6), and other types of siderophores (Figure7)[18–21].

2.1. Hydroxamate-Type Siderophores The distinct characteristic of some acyl peptidic hydroxamate-type siderophores is the presence of a nonpolar fatty acid tail. Many marine bacteria have been reported to produce large suites of acyl peptidic hydroxamate siderophores. Marine bacterium Marinobacter sp. DS40M6 produces the suit of marinobactin siderophores A–F (1–6) and HG (7)[22,23]. The conversion of exogenously added 15N-labeled 1 to the bacterial culture suggests that bacteria produce 7 via the hydrolysis of the acyl amide bond of 1 rather than as a precursor to the compounds 1–6 or independent production. It is worth noting that compound 7 can still coordinate Fe(III) via three iron-coordinating moieties, as these moieties are not affected by deacylation. Marine bacterium Vibrio sp. R-10 is also reported to produce a suite of amphiphilic siderophores, amphibactins (8–17)[24]. Each amphibactin has the similar structure, including a peptidic headgroup composed of one serine residue and three ornithine residues but differs in the saturated, unsaturated, or hydroxylated acyl appendage ranging from C-14 to C-18. They are cell-associated siderophores. This association may play an important role in cell defense against siderophore diffusion in the marine environments. Moreover, several acyl peptidic hydroxamate siderophores, moanachelins (18–22)[25] from the marine bacterium, Vibrio sp. Nt1, and amphibactins U–V (23–24)[26] from two marine bacterial species (Synechococcus sp. PCC 7002 and Vibrio cyclitrophicus 1F53) have also been identified. Additionally, several macrocyclic hydroxamate siderophores were discovered in the marine bacteria, such as alcaligin (25)[27], bisucaberin (26)[28], avaroferrin (27)[29], and putrebactin (28)[30]. Alcaligin (25) could promote Fe(III) uptake in the iron limited cells. It was originally discovered from Alcaligenes denitrificans isolated from sediments of lake Biwa, Japan [31]. Recently, it was also obtained from Alcaligenes eutrophus CH34 [27]. Bisucaberin (26) was isolated from marine bacterium Alteromonas haloplanktis SB-1123 [28]. It could inhibit the growth of both invasive micropapillary carcinoma and L-1210 leukemia cells with IC50 of 12.7 and 9.7 µM, respectively, and sensitize tumor cells to macrophage-mediated cytolysis [32]. Studies of ecology showed that the increasing contents of bisucaberin (26) and acidification enhanced the solubilities and dissolution rates of ferric hydroxides. This suggested that siderophores generated by marine bacteria could grasp ferric Mar. Drugs 2019, 17, 562 3 of 28 Mar. Drugs 2019, 17 3 of 4 hydroxideshydroxides in in aeolian aeolian particles particles to to meet meet thethe growthgrowth of phytoplankton in in the the ocean ocean environment. environment. Thus, Thus, thethe production production rate rate of of marine marine siderophores siderophores maymay playplay a vital role role in in the the survival survival of of phytoplankton phytoplankton andand biogeochemical biogeochemical cyclecycle of of iron iron in in the the sea sea environment environment [33]. [As33]. an Asopen an form open of form26, a linear of 26 ,dimeric a linear dimerichydroxamate hydroxamate class siderophore, class siderophore, bisucaberin bisucaberin B (29 B) (was29) wasisolated isolated from from the the marine marine bacterium bacterium TenacibaculumTenacibaculum mesophilum mesophilumcollected collected in in the theRepublic Republic ofof PalauPalau [34]. [34]. Its Its ferric ferric ion-chelating ion-chelating activity activity was was consistentconsistent with with that that of of its its macrocyclic macrocyclic counterpartcounterpart 26. Moreover, Moreover, the the siderophore siderophore avaroferrin avaroferrin (27 (27) was) was recentlyrecently identified identified in in the the marine marine bacterium bacteriumShewanella Shewanella algae algaeB516 B516 by by heterologous heterologous expression expression of of deep-sea deep- sedimentsea sediment metagenomics metagenomics DNA [29DNA]. It [29]. is a macrocyclic It is a macrocyclic heterodimer heterodimer of N-hydroxy- of N-hydroxy-N-succinyl-putrescineN-succinyl- andputrescineN-hydroxy- andN N-succinyl-hydroxy- cadaverineN-succinyl thatcadaverine is shown that to is inhibit shown the to swarminginhibit the ofswarmingVibrio alginolyticus of Vibrio B522.alginolyticus Another B522. new Another cyclic trihydroxamate new cyclic trihydro compound,xamate compound, thalassosamide thalassosamide (30), was (30 isolated), was isolated from the marine-derivedfrom the marine-derived bacterium bacteriumThalassospira Thalassospira profundimaris profundimaris[35] and [35] showed and showed moderate moderate antibiotic antibiotic activity againstactivityEscherichia against Escherichia coli and Pseudomonas coli and Pseudomonas aeruginosa withaeruginosa the same with MIC the valuesame ofMIC 64 µvalueg/mL ofin 64 vitro μg/mL. In vivoin antibacterialvitro. In vivo assays antibacterial showed thalassosamideassays showed (thalassosamide30) could reduce (30 the) could infection reduce of P.the aeruginosa infectionwithout of P. aeruginosa without toxicity compared with untreated control animals in the murine thigh P. aeruginosa toxicity compared with untreated control animals in the murine thigh P. aeruginosa infection model. infection model. New bioactive siderophores, fradiamine A (31) and fradiamine B (32), were isolated from the New bioactive siderophores, fradiamine A (31) and fradiamine B (32), were isolated from the Streptomyces fradiae MM456M-mF7 derived from the marine sediments of Calyptogena Community, Streptomyces fradiae MM456M-mF7 derived from the marine sediments of Calyptogena Community, Sagami Bay, Japan. They contain two alkyl amines asymmetrically bonded to citrate, rarely observed Sagami Bay, Japan. They contain two alkyl amines asymmetrically bonded to citrate, rarely observed in siderophores. These molecules exhibited medium antibiotic activity against Clostridium difficile in siderophores. These molecules exhibited medium antibiotic activity against Clostridium difficile BAA-1382 of IC values of 32 and 8 µg/mL, respectively. However, their antimicrobial activities were BAA-1382 of IC5050 values of 32 and 8 μg/mL, respectively. However, their antimicrobial activities were cancelledcancelled dose-dependently dose-dependentlyunder under thethe presencepresence of Fe(III) [36]. [36]. A A novel novel albi albisporachelinsporachelin siderophore siderophore (33(33) was) was obtained obtained from from iron-depleted iron-depleted cultureculture brothsbroths ofof marinemarine actinomycete AmycolatopsisAmycolatopsis albispora albispora T WP1WP1.T. The The study study ofof iron-chelatingiron-chelating ability ability suggested suggested that that its its hydroxamate hydroxamate moieties moieties were were involved involved in iniron iron binding, binding, which which was was consistent consistent with with its its structure structure [37]. [37]. Moreover, Moreover, the the variable variable siderophore siderophore metabolomemetabolome of of the the marine marine actinomycete actinomycete SalinisporaSalinispora tropica CNB-440CNB-440 revealed revealed the the production production of ofsix six knownknown siderophores, siderophores, desferrioxamine desferrioxamine AA11,A, A2,, B, D 11,,D D22, ,and and E E (34 (34––3939),), and and one one novel novel siderophore siderophore desferrioxaminedesferrioxamine N N ( 40(40)[) 38[38].]. AmongAmong these,these, 36 has been developed developed as as an an iron iron chelator chelator desferal desferal for for treatingtreating iron iron overload overload in in man. man.

O O

H N N O O N HO HO 3, R= HO O O O H H R N N OH 4, R= N N N N O H H H O H O O O OH OH 5, R= O O 6, R= 1, R= O 2, R= 7, R=H

Figure 1. Cont.

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O O OH O N N 12, R= HO HO OH O O O 13, R= H H O N N O R N N H H 14, R= O OH OH O OH 15, R= OH O N 16, R= O O O

N N HO HO

O O H H N N O N N H H O O OH OH OH N

O 17 O O O N N HO HO 18, R1=H; R2= O O O H H 19, R1=CH3; R2= N N O O R2 N N H H 20, R1=H; R2= O R1 OH O OH 21, R1=H; R2= N O Figure 1. Cont.

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O O

N N HO HO

O O H H N N O N N H H O O OH

OH N

O 22 O O

N N OH OH

O O O H HO N N N N OH H H O O HN R OH HO O 23, R= 24, R=

OH H O HO N O HO N N N H O O OH O H N N N N O OH H O OH O 25 26

N H H O OH HO N O O O N N OH H N N N HO O O H OH N N N N O HO H O NH2 O O O OH 27 28 29 Figure 1. Cont.

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HO O

O NH O O N O OH O O HO N O H N HO O OH O N O O N H O O OH 30 OH CH3 O O OH H H HN N N O N N N N OH O O H OH m O CH3 O O N N H OH OH OH N N O OH H O H O 31, m=0 32, m=1 33 O OH O H N N N N H n O O OH o O m

N 34, m=1, n=1, o=1 H2N OH 35, m=1, n=0, o=0 36, m=1, n=1, o=1 40, m=2, n=1, o=1 O OH O H N N N N H n O O OH o O m

N O N H OH 37, m=1, n=1, o=1 O OH H N N O N H n O O HO o H N N N m OH O O 38, m=1, n=1, o=0 39, m=1, n=1, o=1 FigureFigure 1. 1.Hydroxamate-type Hydroxamate-type siderophor siderophoreses from from marine marine microbes. microbes.

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2.2. α-Hydroxycarboxylates 2.2. α-Hydroxycarboxylates Vibrioferrin (41) was originally isolated from an enteropathogenic estuarine bacterium, Vibrio parahaemolyticusVibrioferrin. Recently, (41) was it originallywas also obtained isolated from anMarinobacter enteropathogenic species collected estuarine from bacterium, Pacific andVibrio Atlanticparahaemolyticus Oceans. .41 Recently, was considered it was also as obtained one of from the Marinobacterweakest ironspecies chelators collected of known from Paciﬁc marine and siderophoresAtlantic Oceans. according41 was to considered the metal–ligand as one of binding the weakest constant iron for chelators 41–Fe(III) of known (1024.02(5) marine). However, siderophores 41– 24.02(5) Fe(III)according complex to the was metal–ligand shown to bindingbe more constant sensitive for 41to–Fe(III) photolysis (10 than). However,other marine41–Fe(III) photoactive complex siderophores,was shown tosuch be moreas aerobactin, sensitive topetrobactin, photolysis marinobactins, than other marine or aquachelins. photoactive siderophores,Taken together, such 41as photodegradationaerobactin, petrobactin, may be marinobactins, an evolutionarily or aquachelins. adaptive response Taken together, by which41 photodegradationthe marine bacteria may share be an photochemicallyevolutionarily adaptive produced response soluble by iron which with the their marine algal bacteria associates share photochemicallypossibly through produced the exchange soluble withiron algal-excreted with their algal metabolites associates [39]. possibly through the exchange with algal-excreted metabolites [39].

HOOC OH O N O N COOH H O OHO COOH

41 FigureFigure 2. 2.α-Hydroxycarboxylate-typeα-Hydroxycarboxylate-type siderophores siderophores from from marine marine microbes. microbes. 2.3. Catecholates 2.3. Catecholates Structural elucidation revealed the discovery of a novel siderophore, nigribactin (42) derived from Structural elucidation revealed the discovery of a novel siderophore, nigribactin (42) derived marine bacteria Vibrio nigripulchritudo [40]. It could enhance the expression of spa encoding a major from marine bacteria Vibrio nigripulchritudo [40]. It could enhance the expression of spa encoding a surface bound virulence factor, Protein A by inducing spa transcription. However, another potent major surface bound virulence factor, Protein A by inducing spa transcription. However, another siderophore, enterobactin failed to affect the expression of Staphylococcus aureus virulence genes. This potent siderophore, enterobactin failed to affect the expression of Staphylococcus aureus virulence suggested the influence of 42 on spa expression might be independent from its siderophore activity. genes. This suggested the influence of 42 on spa expression might be independent from its Vanchrobactin (43) from the marine bacteria Vibrio anguillarum is a dipeptide composed by arginine siderophore activity. Vanchrobactin (43) from the marine bacteria Vibrio anguillarum is a dipeptide and serine residues linked to the 2,3-dihydroxybenzoyl moiety [41–43]. Its catecholate and salicylate composed by arginine and serine residues linked to the 2,3-dihydroxybenzoyl moiety [41–43]. Its groups are two potential bidentate coordination sites of Fe3+ [44]. The marine bacterium Vibrio sp. catecholate and salicylate groups are two potential bidentate coordination sites of Fe3+ [44]. The DS40M4 was reported to produce 43, a new triscatechol amide siderophore, trivanchrobactin (44), and marine bacterium Vibrio sp. DS40M4 was reported to produce 43, a new triscatechol amide a new related siderophore, divanchrobactin (45)[45]. Among them, 43 and 45 may be derived from siderophore, trivanchrobactin (44), and a new related siderophore, divanchrobactin (45) [45]. Among the hydrolysis products of 44. All compounds were found to be non-cytotoxic against P388 murine them, 43 and 45 may be derived from the hydrolysis products of 44. All compounds were found to leukemia cell lines. In addition, a novel triscatecholate siderophore, turnerbactin (46) was isolated from be non-cytotoxic against P388 murine leukemia cell lines. In addition, a novel triscatecholate the shipworm endosymbiont Teredinibacter turnerae T7901 [46]. It is a trimer of N-(2,3-dihydroxybenzoic siderophore, turnerbactin (46) was isolated from the shipworm endosymbiont Teredinibacter turnerae acid)-l-Orn-l-Ser with three monomeric units through the Ser ester linkages. Its structure was similar T7901 [46]. It is a trimer of N-(2,3-dihydroxybenzoic acid)-L-Orn-L-Ser with three monomeric units to the catecholate siderophore 43. They have a serine backbone, 2,3-dihydroxybenzoic acid functional through the Ser ester linkages. Its structure was similar to the catecholate siderophore 43. They have moiety, a positively charged, and a hydrophilic spacer amino acid. a serine backbone, 2,3-dihydroxybenzoic acid functional moiety, a positively charged, and a Using the chrome azurol sulfonate (CAS) assay-guided isolation, three new siderophores, hydrophilic spacer amino acid. dibenarthin (47), streptobactin (48), and tribenarthin (49) were isolated from the marine actinomycete Using the chrome azurol sulfonate (CAS) assay-guided isolation, three new siderophores, Streptomyces sp. YM5-799 collected from Hokkaido in north Japan. Among these, the ED values of 47 dibenarthin (47), streptobactin (48), and tribenarthin (49) were isolated from the marine actinomycete50 and 48 were 117 µM and 156 µM, slightly stronger than that of the control, deferoxamine mesylate Streptomyces sp. YM5-799 collected from Hokkaido in north Japan. Among these, the ED50 values of (ED = 195 µM), whereas 49 exhibited a weak binding with Fe(III) (ED = 937 µM). These siderophores 47 and50 48 were 117 μM and 156 μM, slightly stronger than that of the control,50 deferoxamine mesylate might play a key role in the survival of marine-derived Streptomyces sp. YM5-799 under iron-limited (ED50 = 195 μM), whereas 49 exhibited a weak binding with Fe(III) (ED50 = 937 μM). These conditions [47]. The marine Penicillium bilaii from Port Huon, Tasmania, Australia was also reported to siderophores might play a key role in the survival of marine-derived Streptomyces sp. YM5-799 under produce a rare catechol-type siderophore, pistillarin (50)[48]. It showed potent iron chelation as well iron-limited conditions [47]. The marine Penicillium bilaii from Port Huon, Tasmania, Australia was as antioxidant activity (free radical-scavenging activity), and significantly protected DNA against the also reported to produce a rare catechol-type siderophore, pistillarin (50) [48]. It showed potent iron damage of hydroxyl radicals generated by the Fenton reaction [49]. chelation as well as antioxidant activity (free radical-scavenging activity), and significantly protected DNA against the damage of hydroxyl radicals generated by the Fenton reaction [49].

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OH HO OH NH H N N O N NH2 O H H H O N N N O NH HO OH HO OH O O OH COOH 42 43

HO OH

O NH O O HN O H H H H H N N N N N NH 2 O O 2 NH O O NH O NH HO O HO

O HN NH HO

NH2 OH 44 OH

O O HN O H H N N NH HO O 2 O NH O NH HO

O HN NH HO

NH2 OH 45 Figure 3. Cont.

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HO OH

O NH O O HN O H H H N N N NH 2 O O 2 O O NH O HO O HO

O NH 2 HO OH 46

O O HN O H H N N NH HO O 2 O NH O NH HO

O HN NH HO

NH2 OH 47

NH H N HO N NH2 H OH O O NH O

O O O O H O N NH HN N OH H O O OH H2N N O H O NH NH HO HN NH2 48 HO Figure 3. Cont.

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HO O NH O

H2N N N H H O NH OH OH O HO H HO N N N OH H H HO O HO 49 50 Figure 3. Catecholate-type siderophores from marine microbes. Figure 3. Catecholate-type siderophores from marine microbes. 2.4. Mixed Hydroxamates/α-Hydroxycarboxylates 2.4. Mixed Hydroxamates/α-Hydroxycarboxylates Many of the marine siderophores are amphiphilic, including a polar head group and a nonpolarMany fatty of the acid marine tail, which siderophores allow themare amphiphilic, to form micelles including or a to polar be tetheredhead group to theand bacteriala nonpolar outerfatty cell acid membranes tail, which to allow avoid thethem problem to form of micelles fast diff usionor to ofbe free tethered siderophores to the bacterial without bindingouter cell tomembranes iron [50]. to A avoid suite ofthe amphiphilicproblem of fast siderophores, diffusion of loihichelinsfree siderophores A–F( without51–56), arebinding isolated to iron from [50]. marineA suite bacterium of amphiphilicHalomonas siderophores,sp. LOB-5 loihichelins [51]. They A–F are (51 comprised–56), are isolated of a hydrophilic from marine headgroup bacterium (anHalomonas octapeptide sp. consisting LOB-5 [51]. of They cyclic areN( comprisedδ)-hydroxy- ofd -ornithine,a hydrophilicd-serine, headgroup dehydroamino-2-butyric (an octapeptide consisting acid, l-Nof( δ)-acetyl-cyclic NN(δ(δ)-hydroxy-)-hydroxyornithine,D-ornithine,l-serine, D-serine,l-glutamine, dehydroamino-2-butyricd-serine, and d- threoacid,-β -hydroxyasparticL-N(δ)-acetyl-N(δ)- acid),hydroxyornithine, and different length L-serine, of fatty L-glutamine, acid tail, ranging D-serine, from and decanoic D-threo- acidβ-hydroxyaspartic to tetradecanoic acid), acid. and A curious different characteristiclength of fatty of the acid loihichelins tail, ranging is their from photoreactivity. decanoic acid to However, tetradecanoic Halomonas acid. A LOB-5 curious is obtainedcharacteristic from of sedimentsthe loihichelins below a is depth their ofphotoreactivity. 1714 m, where sunlightHowever, would Halomonas not penetrate. LOB-5 is Thus, obtained the photoreactivity from sediments ofbelow the Fe(III)-loihichelins a depth of 1714 m, may where not sunlight be mainly would responsible not penetrate. for Fe(III) Thus, uptake. the photoreactivity Halomonas aquamarina of the Fe(III)- DS40M3,loihichelins isolated may from not the be sample mainly of responsible seawater under for ironFe(III) limited uptake. and Halomo pure culturenas conditionsaquamarina led DS40M3, to the biosynthesisisolated from of aquachelin the sample siderophores of seawater A–D under (57–60), iron I (61),limited and and J (62) pure [22, 23culture,52]. Among conditions them, led 61 andto the 62biosynthesis were more hydrophilic of aquachelin than siderophores 57–60. When A–D H. aquamarina (57–60), I (61), DS40M3 and J and(62) Marinobacter[22,23,52]. Among sp. DS40M6 them, 61 wereand co-cultured, 62 were more a novel hydrophilic aquachelin than siderophore 57–60. When HG H. (63) aquamarina from hydrolysis DS40M3 of acyl and aquachelins Marinobacter was sp. produced.DS40M6 However,were co-cultured, acyl amidase a novel activity aquachelin of Marinobacter siderophoresp. DS40M6 HG is(63) predominantly from hydrolysis constrained of acyl toaquachelins the cellular membrane.was produced. Thus, theHowever, acyl aquachelins acyl amidase within activity the Marinobacter of Marinobactermembrane sp. areDS40M6 mainly is responsiblepredominantly for aquachelin constrained HG (to63 )the production cellular [membrane.29]. An amphiphilic Thus, the siderophore acyl aquachelins imaqobactin within (64 the) isMarinobacter detected in themembrane arctic marine are mainly bacterium responsibleVariovorax forsp. aquachelin RKJM285 HG [53]. (63 Its) structureproduction has [29]. been An implicatedamphiphilic in the siderophore photoreduction imaqobactin of Fe(III) (64 to) is Fe(II). detected Reduction in the mayarctic occur marine through bacterium a charge Variovorax transfer sp. fromRKJM285 ligand-to-metal [53]. Its structure leading tohas the been cleavage implicated of 64 in. Metal-binding the photoreduction capabilities of Fe(III) showed to Fe(II). that Reduction64 was capablemay occur of forming through stable a charge adducts transfer with Fe(III),from ligand-to-metal Ga(III), and Al(III). leading Moreover, to the cleavage64 also appeared of 64. Metal- to bebinding reactive capabilities with gold, showed but more that studies 64 was were capable required of forming to determine stable adducts whether with this Fe(III), was a reductiveGa(III), and process.Al(III). Of Moreover, particular 64 note, also Fe(III)– appeared64 and to Ga(III)–be reactive64 salts with showed gold, nobut antimicrobial more studies activity. were However,required to 64determineshowed moderate whether antimicrobial this was a reductive activity against process.Staphylococcus Of particular warneri note,, methicillin-resistantFe(III)–64 and Ga(III)–S. aureus64 salts, vancomycin-resistantshowed no antimicrobialEnterococcus activity.and However,Proteus vulgaris 64 showedwith the moderate IC50 values antimicrobial of 28, 35, 11,activity and 14 againstµM, respectively.Staphylococcus It may warneri be attributed, methicillin-resistant to the ability S. ofaureus64 to, bindvancomycin-resistant Fe(III) or Ga(III), Enterococcus depressing microbialand Proteus proliferationvulgaris with via the cellular IC50 values iron depletion. of 28, 35, 11, and 14 μM, respectively. It may be attributed to the ability of The64 to planktonic bind Fe(III) marine or Ga(III),Vibrio depressingsp. DS40M5 microbia wasl reported proliferation to produce via cell aular mixed iron hydroxamatesdepletion. /α- hydroxycarboxylateThe planktonic siderophore, marine Vibrio aerobactin sp. DS40M5 (65)[ was54]. reported Subsequently, to produce other a marine mixed environmentalhydroxamates/α- strainshydroxycarboxylate of Vibrio also were siderophore, reported toaerobactin produce 65(65.) It [54]. was Subsequently, a shuttle molecule other recycled marine inenvironmental each cycle ofstrains transport of Vibrio [55]. Recently, also were three reported65-based to produce amphiphilic 65. It was siderophores, a shuttle molecule ochrobactins recycled A–C in (66 each–68) cycle were of discoveredtransport from [55]. the Recently, marine proteobacteriumthree 65-based amphiphilicOchrobactrum siderophsp. SP18.ores, They ochrobactins contain a citric A–C backbone, (66–68) two were lysinediscovered residues from and ditheff erentmarine fatty proteobacterium acids. This is the Ochrobactrum first example sp. of SP18. aerobactin-derivative They contain a citric siderophores backbone, withtwo two lysine different residues fatty acidand appendages.different fatty The acids. ochrobactin–Fe(III) This is the first complexes example are of photoreactiveaerobactin-derivative under visiblesiderophores sunlight with or UV two light. different Fe(II) isfatty produced acid appendages. by the photoreaction The ochrobactin–Fe(III) of ochrobactin–Fe(III) complexes [56 ].are Moreover,photoreactive three siderophores,under visible synechobactins sunlight or UV (69– 71light.) from Fe(II) the marineis produced cyanobacteria by theSynechococcus photoreactionsp. of PCCochrobactin–Fe(III) 7002 were identified [56]. by Moreover, a high throughput three siderophores, method which synechobactins is combined liquid(69–71 chromatography) from the marine cyanobacteria Synechococcus sp. PCC 7002 were identified by a high throughput method which is

Mar. Drugs 2019, 17, 562 11 of 28 Mar. Drugs 2019, 17 11 of 4 inductivelycombined coupledliquid chromatography plasma mass spectrometry inductively withcoupled high plasma resolution mass electrospray spectrometry ionization with high mass spectrometryresolution electrospray [57]. ionization mass spectrometry [57].

O O

N N O HO HO OH OH HO O O O H H H R N N N OH N N N N H H H H O O O O HO OH

H2N O O O

57, R= O 60, R= OH O

58, R= 61, R= O O 59, R= 62, R=

O O

N N OH OH O HO HO O O O OH H H H HO N N N N N N NH2 H H H O O O O HO HO

O NH2 63 Figure 4. Cont.

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O H HO N O NH OH H O N N HO H O O NH O O N N O OH HO OH O O OH 64 COOH O O N N R 65, R1=CH3; R2=CH3 H 1 OH OH 66, R1= ; R2= COOH OH H 67, R1= ; R2= N N R2 O COOH O 68, R1= ; R2=

O O OH O O

N N N N R H COOH H OH OH

69, R=

70, R= 71, R=

Figure 4. 4. MixedMixed hydroxamates/ hydroxamatesα/-hydroxycarboxylatesα-hydroxycarboxylates siderophores siderophores from from marine marine microbes. microbes. 2.5. Mixed α-Hydroxycarboxylates/Catecholates 2.5. Mixed α-Hydroxycarboxylates/Catecholates Two photoreactive siderophores, petrobactin (72) and petrobactin sulfonate (73), are produced Two photoreactive siderophores, petrobactin (72) and petrobactin sulfonate (73), are produced byby marinemarine bacterium bacterium MarinobacterMarinobacter hydrocarbonoclasticus hydrocarbonoclasticus [58–60][58.– They60]. are They composed are composed of a citrate of abis- citrate bis-spermidinespermidine skeleton, skeleton, with with two two 3,4-dihydroxy-di 3,4-dihydroxy-dihydroxybenzoylhydroxybenzoyl groups groups providing providing four fourdonor donor groupsgroups forfor Fe(III) bindin binding.g. Fe(III) Fe(III) complexes complexes with with 72 72andand 73 are73 are photoreactive photoreactive unde underr visible visible sunlight, sunlight, regulatedregulated by by the the Fe(III)–citrate Fe(III)–citrate moie moiety.ty. This reaction This reaction leads to leads decarb tooxylation decarboxylation and reduction and of reduction Fe(III) of Fe(III)to Fe(II). to Fe(II).73 is also73 isthe also first the marine ﬁrst marinesiderophore siderophore including including a sulfonated a sulfonated 3,4-dihydroxy 3,4-dihydroxy aromatic ring. aromatic ring.Alterobactin Alterobactin A (74) A and (74 )B and (75) B was (75 )originally was originally obtained obtained from Alteromonas from Alteromonas luteoviolacea luteoviolacea isolated isolatedby byoligotrophic oligotrophic and andcoastal coastal waters. waters. Alterobactin Alterobactin A (74) showed A (74) showedan exceptionally an exceptionally high affinity high for aFe(III)ﬃnity for Fe(III)[61,62]. [61 It, 62contains]. It contains two unusual two unusual amino amino acids: acids:(3R,4S (3)-4,8-diamino-3-hydroxyoctanoicR,4S)-4,8-diamino-3-hydroxyoctanoic acid and acid L- and lthreo--threo-β-hydroxyasparticβ-hydroxyaspartic acid, acid, attaching attaching to a to catechol a catechol carboxylate. carboxylate. 74–Fe(III)74–Fe(III) complex complex is stable is stable in in solutionsolution with little change change of of absorbance. absorbance. However, However, in inthe the absence absence of coordi of coordinatednated Fe(III), Fe(III), the serine the serine esterester ofof74 will74 bewill hydrolyzed be hydrolyzed to alterobactin to alterobactin B (75) siderophore. B (75) siderophore. New siderophores, New pseudoalterobactinsiderophores, A(pseudoalterobactin76) and B (77) were A ( obtained76) and B from (77) marinewere obtained bacterium fromPseudoalteromonas marine bacteriumsp. Pseudoalteromonas KP20-4 isolated fromsp. the RepublicKP20-4 isolated of Palau from [63]. the They Republic contain of a Pa catechollau [63]. and They two containβ-hydroxy-Asp a catechol residues. and two Pseudoalterobactinsβ-hydroxy-Asp residues. Pseudoalterobactins showed strong activity by a CAS assay, which was comparable to that showed strong activity by a CAS assay, which was comparable to that of desferrioxamine B and of desferrioxamine B and enterobactin. Both 76 and 77 exhibited ED50 value of 20 μM (the reduction enterobactin. Both 76 and 77 exhibited ED value of 20 µM (the reduction of the absorbance at 630 nm of the absorbance at 630 nm of the CAS solution50 by 50%), while desferrioxamine B and enterobactin of the CAS solution by 50%), while desferrioxamine B and enterobactin showed ED50 values of 500 µM showed ED50 values of 500 μM and 60 μM under the same conditions, respectively. and 60 µM under the same conditions, respectively.

Mar. Drugs 2019, 17, 562 13 of 28 Mar. Drugs 2019, 17 13 of 4

OH O H H N N N OH H OH O R COOH O H N OH N N O H H OH 72, R=H 73, R=SO3H H N NH O 2 NH NH O HO OH O N OH O H NH HN OH NH2 HO HN O O HN O H O N O O O OH OH 74

HN NH2 H N HN OH O OH OH OH O O O H H H N N N COOH N N N H H H NH O O O 2 HO COOH HO COOH 75 O

N H HO S CONH HN O 3 OH O 2 O O H H H N N N COOH HO N N N H H H OH O NH O O OH 2 HO COOH

R 76, R=CH2NH2 77, R=NHCH(NH)NH 2 Figure 5.5. Mixed α-hydroxycarboxylates-hydroxycarboxylates/catecholates/catecholates siderophoressiderophores fromfrom marinemarine microbes.microbes.

2.6. Mixed Hydroxamates/Catecholates Three unusual siderophores, lystabactins A–C (78–80), were obtained from the marine bacterium Pseudoalteromonas sp. S2B [64]. In order to further determine their stability by chelating Fe(III), the

Mar. Drugs 2019, 17, 562 14 of 28

2.6. Mixed Hydroxamates/Catecholates Three unusual siderophores, lystabactins A–C (78–80), were obtained from the marine bacterium Mar. Drugs 2019, 17 14 of 4 Pseudoalteromonas sp. S2B [64]. In order to further determine their stability by chelating Fe(III), the pFe scale was used to compare with the iron-binding stability of siderophores. pFe was the negative logarithm of aqueous, free Fe(III) at fixedfixed concentrationsconcentrations of siderophore,siderophore, Fe(III), and acidificationacidification (pH). The tighter tighter a a given given siderophore siderophore bound bound Fe( Fe(III),III), the the higher higher the the pFe. pFe. The The pFe pFe of ofcompounds compounds 78 78andand 79 were79 were calculated calculated to be to 26.0 be 26.0 and and27.5, 27.5, respectively. respectively. This Thissuggested suggested that their that theirstability stability of iron- of iron-bindingbinding was stronger was stronger than thanthe tris-hydroxamate the tris-hydroxamate siderophore, siderophore, desferrioxamine desferrioxamine B, while B, while weaker weaker than thanthe tris-catecholate the tris-catecholate siderophor siderophore,e, enterobactin. enterobactin. Anguibactin Anguibactin (81) siderophore (81) siderophore was found was in found the marine in the marinepathogen pathogen V. anguillarumV. anguillarum or Vibrioor sp.Vibrio DS40M4sp. DS40M4 [42,45]. [42 It ,was45]. Itevaluated was evaluated cytotoxic cytotoxic effects e ffonects the on P388 the µ P388murine murine leukemia leukemia cell lines cell linesand andwas wasshown shown to be to cytotoxic be cytotoxic (IC50 (IC < 5015< μ15M). M).It was It was also also identified identified as an as animportant important virulence virulence factor. factor.

H O H O

N N OH OH

OH O NH O O 2 H H HO N N O N N N R H H H OH O O O OH CONH2 78, R=H 80, R=CH 3 O HO N H O O NH N 2 H O O NH OH O HN OH H N O N H HO N H OH O NH2 O O 79 HO OH O NH N N N S OH

81 Figure 6. MixedMixed hydroxamates/catecholates hydroxamates/catecholates sidero siderophoresphores from marine microbes. 2.7. Other Types of Siderophores 2.7. Other Types of Siderophores Besides, several other types of siderophores were also discovered in the marine microorganisms. Besides, several other types of siderophores were also discovered in the marine microorganisms. A novel siderophore, piscibactin (82), was isolated from the marine fish pathogens Photobacterium A novel siderophore, piscibactin (82), was isolated from the marine fish pathogens Photobacterium damselae subsp. piscicida and V. anguillarum. The inactivation of piscibactin system would lead to a severe damselae subsp. piscicida and V. anguillarum. The inactivation of piscibactin system would lead to a loss of the virulence degree. Therefore, piscibactin had a great impact in the expression of virulence, the severe loss of the virulence degree. Therefore, piscibactin had a great impact in the expression of ability to piscibactin seemed to be sufficient to confer maximal virulence to pathogens [65–67]. Two new virulence, the ability to piscibactin seemed to be sufficient to confer maximal virulence to pathogens oxazole–thiazole siderophore compounds, tetroazolemycins A (83) and B (84), were isolated from marine [65–67]. Two new oxazole–thiazole siderophore compounds, tetroazolemycins A (83) and B (84), were Streptomyces olivaceus FXJ8.012 [68]. Their heavy metal ion-binding ability was evaluated by Cu2+, Zn2+, isolated from marine Streptomyces olivaceus FXJ8.012 [68]. Their heavy metal ion-binding ability was evaluated by Cu2+, Zn2+, Fe3+, Pb2+, Cr3+, and Mn2+. The results indicated that 82 and 83 had affinity for Cu2+, Zn2+, and Fe3+ but not affinity for Pb2+, Cr3+, and Mn2+. Further studies showed the free-state tetroazolemycins did not show antimicrobial activity. Their metal ion complexes were also inactive against pathogens E. coli, S. aureus, Mycobacterium gilvum, Bacillus subtilis, P. aeruginosa, Candida pseudorugosa, Candida albicans, Rhizoctonia solani, Aspergillus fumigatus, and Fusarium oxysporum,

Mar. Drugs 2019, 17, 562 15 of 28

Fe3+, Pb2+, Cr3+, and Mn2+. The results indicated that 82 and 83 had affinity for Cu2+, Zn2+, and Fe3+ but not affinity for Pb2+, Cr3+, and Mn2+. Further studies showed the free-state tetroazolemycins did Mar.not Drugs show 2019 antimicrobial, 17 activity. Their metal ion complexes were also inactive against pathogens15 E.of coli4 , S. aureus, Mycobacterium gilvum, Bacillus subtilis, P. aeruginosa, Candida pseudorugosa, Candida albicans, inactiveRhizoctonia against solani A/H1N1, Aspergillus influenza fumigatus virus,, and and inactiFusariumve against oxysporum human, inactive lung ad againstenocarcinoma A/H1N1 cell influenza line A549virus, and and murine inactive macrophage against human cell lung line adenocarcinoma P388D. However, cell linetheir A549 Zn2+ and complexes murine macrophage showed weakly cell line activityP388D. against However, pathogenic their Zn2 +Klebsiellacomplexes pneumoniae showed weakly with activityMICs of against 125–250 pathogenic μg/mL Klebsiellaand 125 pneumoniaeμg/mL, respectively.with MICs ofThus, 125–250 it wasµg speculated/mL and 125 thatµg/ mL,the antimi respectively.crobial Thus, activity it was of oxazol speculatede/thiazole that the siderophores antimicrobial mightactivity be closely of oxazole related/thiazole to Zn siderophores2+. might be closely related to Zn2+.

H H S S H S N N H OH N OH HOOC 82 OH CH3 O S N H N N S H O H H O H S N N H N S O CH3 HO 83 OH CH3 O S N H N N S H O H H O H S N N H N S O CH3 HO 84 FigureFigure 7. Other 7. Other types types of siderophores of siderophores from from marine marine microbes. microbes. 3. Biosynthesis of Siderophores from Marine Microorganisms 3. Biosynthesis of Siderophores from Marine Microorganisms To date, more than 80 siderophores have been isolated from marine microorganisms, however, only To date, more than 80 siderophores have been isolated from marine microorganisms, however, part of siderophore biosynthetic pathways have been reported. A large number of novel siderophore only part of siderophore biosynthetic pathways have been reported. A large number of novel biosynthetic pathways still need to be further studied. Therefore, researchers should focus more on the siderophore biosynthetic pathways still need to be further studied. Therefore, researchers should biosynthesis of siderophores from marine microorganisms to obtain structurally diverse siderophores focus more on the biosynthesis of siderophores from marine microorganisms to obtain structurally as drug candidates. diverse siderophores as drug candidates. 3.1. NRPS-Mediated Siderophore Biosynthetic Pathway 3.1. NRPS-Mediated Siderophore Biosynthetic Pathway Recently, it has become apparent that the biosynthesis of siderophores involves covalently tethered a seriesRecently, of intermediates it has become to apparent the multienzymes that the biosynthesis throughout theof siderophores assembly process involves and covalently is catalyzed tetheredby nonribosomal a series of peptideintermediates synthetases to the (NRPSs). multienzymes NRPSs throughout are responsible the forassembly assembling process structurally and is catalyzedcomplex by peptides nonribosomal from small pept buildingide synthetases blocks such (NRPSs). as carboxyl NRPSs or amino are responsible acids. Each for module assembling is highly structurallyeﬃcient dedicated complex topeptides the activation, from small modiﬁcation, building andblocks incorporation such as carboxyl of one smallor amino building acids. block Each into modulethe compound is highly andefficient harbors dedicated all key to enzymatic the activa activitiestion, modification, as specialized and domains incorporation to catalyze of one the small single buildingchemical block reaction into the (Table compound3). The de and novo harbors study all of key biosynthesis enzymatic showed activities EnzA–D as specialized (NRPSs) domains were mainly to catalyzeresponsible the single for the chemical biosynthesis reaction of siderophores,(Table 3). The bisucaberinde novo study B (29 of) andbiosynthesis bisucaberin showed (26) (SchemeEnzA–D1 ). (NRPSs)EnzA and were EnzB mainly catalyzed responsible the biosynthesis for the ofbios intermediatesynthesis of from siderophores, L-lysine to cadaverine,bisucaberin and B ( cadaverine29) and bisucaberin (26) (Scheme 1). EnzA and EnzB catalyzed the biosynthesis of intermediates from L- lysine to cadaverine, and cadaverine to N-hydroxy-cadaverine, respectively. EnzC activated the acylation of N-hydroxy-cadaverine with succinyl-CoA to produce N-hydroxy-N-succinylcadaverine (HSC) monomer. EnzD as the last step noncovalently bound to HSC and ATP to catalyze the oligomerization–macrocyclization reaction of HSC to form 29 or 26. Firstly, a HSC molecule and ATP noncovalently bound to the active site of EnzD. A second molecule of HSC also noncovalently bound

Mar. Drugs 2019, 17, 562 16 of 28 to N-hydroxy-cadaverine, respectively. EnzC activated the acylation of N-hydroxy-cadaverine with succinyl-CoA to produce N-hydroxy-N-succinylcadaverine (HSC) monomer. EnzD as the last step noncovalently bound to HSC and ATP to catalyze the oligomerization–macrocyclization reaction of HSCMar. Drugs to form 2019, 2917 or 26. Firstly, a HSC molecule and ATP noncovalently bound to the active site16 of of 4 EnzD. A second molecule of HSC also noncovalently bound to EnzD, which dissociated from the active siteto EnzD, of EnzD. which The dissociated activated carboxyl from the group active of site the of ﬁrst EnzD. molecule The activated of HSC underwent carboxyl group a base-promoted of the first addition–eliminationmolecule of HSC underwent reaction witha base-promoted the protonated addi aminotion–elimination group of the second reaction molecule with the of HSC protonated to yield theamino linear group dimer of the29 .second The activated molecule carboxyl of HSC to group yield and the linear protonated dimer amino29. The group activated of 29 carboxylcould further group undergoand protonated an intramolecular amino group base-promoted of 29 could condensation further undergo to generate an 26intramolecular[34,69,70]. In thebase-promoted biosynthetic networkcondensation of 27 ,to it generate exhibits the26 [34,69,70]. similar scheme In the ofbiosynthetic biosynthesis network with 26 ofand 27, it29 exhibits. Firstly, the MbsA–C similar catalyze scheme successiveof biosynthesis reactions, with 26 resulting and 29 in. Firstly, ornithine MbsA–C and lysine catalyze to the successiveN-hydroxy- reactions,N-succinyl resulting diamines in ornithine (HSDs). MbsDand lysine then to utilizes the N both-hydroxy-N-hydroxy-N-succinylN-succinyl-putrescine diamines (HSDs). and MbsDN-hydroxy- then utilizesN-succinyl both N cadaverine-hydroxy-N as- immediatesuccinyl-putrescine precursors and and N-hydroxy- catalyzes bothN-succinyl the oligomerization cadaverine as and immediate ﬁnal macrocyclization, precursors and leading catalyzes to theboth biosynthesis the oligomerization of 27. and final macrocyclization, leading to the biosynthesis of 27.

Scheme 1. The proposed biosynthesis of bisucaberin B (29) and bisucaberin (26). Vibrioferrin (41) is synthesized by five biosynthetic enzymes PvsA-E (NRPSs). The biosynthetic Vibrioferrin (41) is synthesized by five biosynthetic enzymes PvsA-E (NRPSs). The biosynthetic pathway starting with enzyme PvsE. PvsE has a highly homologous with 2,6-diaminopimerate pathway starting with enzyme PvsE. PvsE has a highly homologous with 2,6-diaminopimerate decarboxylase, indicating PvsE catalyzes decarboxylation from serine to ethanolamine. PvsB and PvsD decarboxylase, indicating PvsE catalyzes decarboxylation from serine to ethanolamine. PvsB and form amide bond between L-alanine and 2-ketoglutaric acid, or pentanoic acid and ethanolamine. PvsA PvsD form amide bond between L-alanine and 2-ketoglutaric acid, or pentanoic acid and is perceived to form an ester bond between ethanolamine and citrate. The final product vibrioferrin ethanolamine. PvsA is perceived to form an ester bond between ethanolamine and citrate. The final (41) is secreted by PvsC which is most likely to be a transporter involved in secreting the product from product vibrioferrin (41) is secreted by PvsC which is most likely to be a transporter involved in the cell [71]. secreting the product from the cell [71]. The biosynthetic and regulatory elements of vanchrobactin (43) in marine bacteria V. anguillarum The biosynthetic and regulatory elements of vanchrobactin (43) in marine bacteria V. anguillarum has been completely elucidated [42–44]. It proceeds through a NRPS-mediated pathway encoded by has been completely elucidated [42–44]. It proceeds through a NRPS-mediated pathway encoded by chromosomal genes to produce 43 (Scheme2). VabB and VabE–F have been identified as three NRPSs. chromosomal genes to produce 43 (Scheme 2). VabB and VabE–F have been identified as three Isochorismate synthase (VabC) first started with the conversion of chorismate into isochorismate. NRPSs. Isochorismate synthase (VabC) first started with the conversion of chorismate into Isochorismatase (VabB) and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (VabA) then catalyzed isochorismate. Isochorismatase (VabB) and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase the biosynthesis of intermediates from isochorismate to 2,3-dihydro-2,3-dihydroxybenzoic acid (VabA) then catalyzed the biosynthesis of intermediates from isochorismate to 2,3-dihydro-2,3- (2,3-dihydro-2,3-DHBA), and (2,3-dihydro-2,3-DHBA) respectively to 2,3-DHBA. Once 2,3-DHBA dihydroxybenzoic acid (2,3-dihydro-2,3-DHBA), and (2,3-dihydro-2,3-DHBA) respectively to 2,3- was synthesized, VabE (NRPS) activated 2,3-DHBA to its acyl-adenylated intermediate in an DHBA. Once 2,3-DHBA was synthesized, VabE (NRPS) activated 2,3-DHBA to its acyl-adenylated ATP-dependent manner, and then VabE (NRPS)transferred this activated intermediate to VabB. intermediate in an ATP-dependent manner, and then VabE (NRPS)transferred this activated VabB further bound with 2,3-DHBA through its ACP domain. VabF (NRPS) was involved in intermediate to VabB. VabB further bound with 2,3-DHBA through its ACP domain. VabF (NRPS) the last steps of 43 assembly. It was composed of two modules, including C-A1-PCP domain was involved in the last steps of 43 assembly. It was composed of two modules, including C-A1-PCP (condensation-adenylation-peptidyl-carrier-protein) in the N-terminal, and C-A2-PCP-TE domain domain (condensation-adenylation-peptidyl-carrier-protein) in the N-terminal, and C-A2-PCP-TE (condensation-adenylation-peptidyl-carrier-protein-thioesterase) in the C-terminal. VabF A1 domain domain (condensation-adenylation-peptidyl-carrier-protein-thioesterase) in the C-terminal. VabF A1 activated an arginine residue, and synthesized 2,3-dihydroxybenzoyl-arginine precursor through its domain activated an arginine residue, and synthesized 2,3-dihydroxybenzoyl-arginine precursor PCP domain. VabF A2 domain activated a serine residue. The TE domain as the last domain was to through its PCP domain. VabF A2 domain activated a serine residue. The TE domain as the last release 43 into solution. domain was to release 43 into solution.

Mar. Drugs 2019, 17 17 of 4

Mar. Drugs 2019, 17 17 of 4 Mar. Drugs 2019, 17, 562 17 of 28

Scheme 2. The proposed biosynthesis of vanchrobactin (43). SchemeScheme 2.2. The proposed biosynthesis of vanchrobactin vanchrobactin ( (4343).). Mechanism of anguibactin (81) biosynthesis has been characterized in V. anguillarum (Scheme 3) Mechanism of anguibactin (81) biosynthesis has been characterized in V.anguillarum (Scheme3)[ 72]. [72]. AMechanism series of NRPSs of anguibactin are involved (81) biosynthesis in V. anguillarum has been anguibactin characterized (81) inbiosynthesis, V. anguillarum such (Scheme as AngB, 3) A series of NRPSs are involved in V. anguillarum anguibactin (81) biosynthesis, such as AngB, AngD, AngD,[72]. A AngE, series ofAngM, NRPSs AngN, are involved and AngR. in V. VabD anguillarum and Va anguibactinbE are functional (81) biosynthesis, homologues such of AngDas AngB, and AngE, AngM, AngN, and AngR. VabD and VabE are functional homologues of AngD and AngE, AngE,AngD, respectively.AngE, AngM, Each AngN, protein and AngR.provides VabD impo andrtant VabE functional are functional domain(s) homologues for anguibactin of AngD and(81 ) respectively. Each protein provides important functional domain(s) for anguibactin (81) biosynthesis. biosynthesis.AngE, respectively. The adenylation Each protein (A) domainsprovides ofimpo VabErtant or likelyfunctional of AngE domain(s) activate for 2,3-DHBA anguibactin into ( 812,3-) The adenylation (A) domains of VabE or likely of AngE activate 2,3-DHBA into 2,3-DHBA-AMP [73,74]. DHBA-AMPbiosynthesis. [73,74].The adenylation An aryl carrier(A) domains protein of (ArCP) VabE or domain likely ofof AngEthe C-terminal activate 2,3-DHBA region of into AngB 2,3- is An aryl carrier protein (ArCP) domain of the C-terminal region of AngB is responsible for tethering responsibleDHBA-AMP for [73,74]. tethering An 2,3-DHBA-AMP.aryl carrier protein The (ArCP) adenylation domain (A) of domain the C-terminal of AngR region is likely of relatedAngB is to 2,3-DHBA-AMP. The adenylation (A) domain of AngR is likely related to the activation of cysteine. theresponsible activation for of tetheringcysteine. 2,3-DHBA-AMP.It is worth noting The that adenylation cyclisation (A)(Cy) domain and peptidyl of AngR carrier is likely protein related (PCP) to It is worth noting that cyclisation (Cy) and peptidyl carrier protein (PCP) domains of AngR are not domainsthe activation of AngR of cysteine. are not functional It is worth due noting to the that replac cyclisationement of(Cy) the and first peptidyl aspartic carrieracid by protein asparagine (PCP) in functional due to the replacement of the ﬁrst aspartic acid by asparagine in the Cy domain, and the thedomains Cy domain, of AngR and are the not replacement functional due of toan the essentia replaclement serine of by the alanine first aspartic in the acid PCP by domain asparagine [75,76]. in replacement of an essential serine by alanine in the PCP domain [75,76]. AngD or VabD is responsible AngDthe Cy or domain, VabD isand responsible the replacement for transferring of an essentia the phosphopantetheinyll serine by alanine in moietythe PCP to domain serine residues[75,76]. for transferring the phosphopantetheinyl moiety to serine residues located in the ArCP domain of locatedAngD orin theVabD ArCP is responsible domain of AngBfor transferring and PCP domainthe phosphopantetheinyl of AngM, making moietythem become to serine active residues forms AngBlocated and in PCP the ArCP domain domain of AngM, of AngB making and them PCP become domain active of AngM, forms making [74,77]. them The PCPbecome domain active of forms AngM [74,77]. The PCP domain of AngM is used to tether the activated cysteine, and its C domain is likely is[74,77]. used to The tether PCP the domain activated of AngM cysteine, is used and to its tether C domain the activated is likely cysteine, to catalyze and peptide its C domain bond formation is likely to catalyze peptide bond formation between cysteine from AngM and 2,3-DHBA from AngB. AngN betweento catalyze cysteine peptide from bond AngM format andion 2,3-DHBA between cysteine from AngB. from AngN AngM contains and 2,3-DHBA two Cy from domains, AngB. Cy1 AngN and contains two Cy domains, Cy1 and Cy2 that could condense and cyclize 2,3-DHBA and cysteine to Cy2contains that couldtwo Cy condense domains, and Cy1 cyclize and Cy2 2,3-DHBA that could and condense cysteine and to producecyclize 2,3-DHBA thiazoline and [78 cysteine,79]. The to C produce thiazoline [78,79]. The C domain of AngM could attach N-hydroxyhistamine to the domainproduce of thiazoline AngM could [78,79]. attach TheN-hydroxyhistamine C domain of AngM to the could dihydroxyphenyl–thiazoline–thioester attach N-hydroxyhistamine to the to dihydroxyphenyl–thiazoline–thioester to form anguibactin (81). AngT as the last step is to release formdihydroxyphenyl–thiazoline–thioe anguibactin (81). AngT as thester last to step form is to anguibactin release anguibactin (81). AngT (81 as) from the last AngM step [72 is]. to release anguibactin (81) from AngM [72]. anguibactin (81) from AngM [72].

Scheme 3. The proposed biosynthetic pathway of anguibactin (81). SchemeScheme 3.3. TheThe proposed biosynthetic pathway ofof anguibactinanguibactin ( (8181).).

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Mar. Drugs 2019, 17 18 of 4 Irp1-5 of Photobacterium damselae subsp. piscicida are mainly involved in the biosynthesis of Irp1-5 of Photobacterium damselae subsp. piscicida are mainly involved in the biosynthesis of siderophore piscibactin (82) (Scheme4)[ 65,80]. Firstly, Irp5 (salicylate-activating enzyme) initiates siderophore piscibactin (82) (Scheme 4) [65,80]. Firstly, Irp5 (salicylate-activating enzyme) initiates piscibactin assembly, and then transfers the activated salicylate to aryl carrier protein domain piscibactin assembly, and then transfers the activated salicylate to aryl carrier protein domain of Irp2 of Irp2 (Iron-regulated protein 2). The ArCP domain of Irp2 is speciﬁc for cysteine activation. (Iron-regulated protein 2). The ArCP domain of Irp2 is specific for cysteine activation. The activated The activated cysteine is transferred to PCP1 and PCP2 simultaneously. Then, Irp2 catalyzes the cysteine is transferred to PCP1 and PCP2 simultaneously. Then, Irp2 catalyzes the continuous continuous addition and cyclization of every cycle to form two thiazoline rings connected to a addition and cyclization of every cycle to form two thiazoline rings connected to a salicylate portion. salicylateIrp1 (PKS/NRPS portion. Irp1multifunctional (PKS/NRPS multifunctionalenzyme) is composed enzyme) of is 11 composed domains of including 11 domains a includingcarboxyl- a carboxyl-terminatedterminated thioesterase thioesterase domain domainand three and elongation three elongation modules. modules. The ketosynthase The ketosynthase domain domain in Irp1 in Irp1loads loads a malonyl a malonyl group group which which is linked is linked to tothe the ACP ACP domain. domain. The The PCP3 PCP3 domain domain of ofIrp1 Irp1 catalyzes catalyzes the the condensationcondensation/heterocyclization/heterocyclization of of methyl-cysteine. methyl-cysteine. Irp4 Irp4 (thioesterase) (thioesterase) hydrolyzes hydrolyzes the thioester the thioester linkage. Thelinkage. ﬁnal maturation The final maturation of piscibactin of piscibactin (82) must be(82 accomplished) must be accomplished by Irp3 (reductase) by Irp3 (reductase) which reduces which the middlereduces thiazoline the middle ring thiazoline to the thiazolidine. ring to the thiazolidine.

SchemeScheme 4. 4. TheThe proposedproposed biosynthesis of of piscibactin piscibactin (82 (82).).

3.2.3.2. NRPS-Independent NRPS-Independent Siderophore Siderophore BiosyntheticBiosynthetic Pathway NRPS-independentNRPS-independent siderophore siderophore (NIS) (NIS) synthetasessynthetases are an emerging emerging member member of of the the family family of ofnew new synthetases.synthetases. They They dodo notnot havehave thethe sequencesequence or structuralstructural similarity similarity to to NRPSs. NRPSs. However, However, they they also also playplay an an important important role role in in the the biosynthesis biosynthesis ofof siderophores.siderophores. Many Many structurally structurally diverse diverse siderophores siderophores areare biosynthesized biosynthesized by by NIS NIS synthetases synthetases (Table(Table3 3).). NISNIS synthetasessynthetases contain type type A, A, type type B, B, and and type type C C basedbased on on sequence sequence similarity similarity criteria. criteria. Type Type A enzymes A enzymes catalyze catalyze the condensation the condensation of amines of amines or alcohols or withalcohols a prochiral with a carboxylprochiral groupcarboxyl of group citric acid.of citric Type acid. B enzymesType B enzymes catalyze catalyze the condensation the condensation of amines of withaminesγ-carboxyl with groupγ-carboxyl of α -ketoglutarate.group of α-ketoglutarate. Type C enzymes Type catalyze C enzymes either the catalyze oligomerization either the and macrocyclisationoligomerization ofandω-amino-carboxylic macrocyclisation of acids, 𝜔-amino-carboxylic or an amine or alcohol acids, withor an a amine monoamide or alcohol derivative with a of citricmonoamide acid. By derivative selective gene of citric knockout acid. By and selective reconstruction gene knockout of iuc genes, and reconstruction aerobactin (65 of) biosyntheticiuc genes, pathwayaerobactin was (65 deciphered.) biosynthetic IucA–D pathway are was responsible deciphered. for IucA the–D biosynthesis are responsible of 65 for(Scheme the biosynthesis5). They of are highly65 (Scheme stereoselective. 5). They are IucB highly is confirmed stereoselective. to its lIucB-lysine is confirmedN6 monooxygenase to its L-lysine activity N6 monooxygenase [81]. Similarly, IucDactivity is also [81]. confirmed Similarly, toIucD its Nis6 -hydroxy-also confirmedl-lysine to its (hLys) N6-hydroxy- acetyltransferaseL-lysine (hLys) activity acetyltransferase [82]. IucB and IucDactivity respectively [82]. IucB catalyzeand IucD the respectively biosynthesis catalyze of intermediates the biosynthesis from of intermediatesl-lysine to hLys, from andL-lysine hLys to to N6hLys,-acetyl- andN 6hLys-hydroxy- to N6-acetyl-l-lysineN (ahLys).6-hydroxy- IucAL-lysine and (ahLys). IucC represent IucA and the IucC archetypal represent Type the archetypal A and Type CType NIS synthetases,A and Type respectivelyC NIS synthetases, [83]. IucA respectively catalyzes [83]. the IucA carboxymethyl catalyzes the group carboxymethyl of citric acid group to form of a citryl-adenylatecitric acid to form intermediate a citryl-adenylate via the intermediate activation ofvia ATP. the activation IucC then of consumes ATP. IucC 3S then,2’S-citryl-ahLys consumes 3S,2’S-citryl-ahLys intermediate in a relatively modest catalytic efficiency to synthesize 65. intermediate in a relatively modest catalytic efficiency to synthesize 65.

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SchemeScheme 5. The proposed biosyntheticbiosynthetic pathwaypathway ofof of aerobactinaerobactin aerobactin ( (65 (6565).).).

BasedBased on the NRPS-independent assembly, assembly, thethe the biosyntheticbiosynthetic schemescheme scheme ofof of petrobactinpetrobactin petrobactin ((72 (7272)) ) isis is developeddevelopeddeveloped (Scheme 66).). AsbA AsbA (type (type A ANIS NIS synthetase synthetase))) hashas has beenbeen been demonstrateddemonstrated demonstrated toto catalyzecatalyze to catalyze thethe ATP-ATP- the 8 8 ATP-dependentdependentdependent condensation condensation of N8 of of spermidineN of spermidine with citriccitric with acidacid citric toto affordafford acid toNN88-citryl-spermidine. a-citryl-spermidine.fford N -citryl-spermidine. Another Another AnotherNISNIS synthetase NIS synthetase AsbB (type AsbB C NIS (type synthetase) C NIS synthetase) catalyzes ATP-dependent catalyzesATP-dependent ATP-dependent condensationcondensation condensation ofof NN8-citryl-8-citryl- of Nspermidinespermidine8-citryl-spermidine or N1-(3,4-dihydroxybenzoyl)- or N1-(3,4-dihydroxybenzoyl)-N88-citryl-spermidine-citryl-spermidineN8-citryl-spermidine withwith spermidine.spermidine. with spermidine. AsbF,AsbF, AsbF, aa 3-3- a 3-dehydroshikimatedehydroshikimatedehydroshikimate (3-DHS) (3-DHS) dehydrat dehydratase,ase, catalyzes catalyzes thethe the conversionconversion conversion ofof of thethe the bacterialbacterial bacterial metabolitemetabolite metabolite 3-DHS 3-DHS 3-DHS tototo form formform 3,4-dihydroxybenzoic 3,4-dihydroxybenzoic3,4-dihydroxybenzoic acid acid (3,4-DHBA). (3,4-DHBA). AsbC, AsbC,AsbC, a NRPS-like aa NRPS-likeNRPS-like adenylating adenylatingadenylating enzyme, isenzyme,enzyme, responsible isis forresponsibleresponsible catalyzing for the catalyzing ATP-dependent the ATP-dependent acylation of phosphopantetheine acylacylationation ofof phosphopantetheinephosphopantetheine thiol of AsbD thiol (athiol carrier ofof AsbDAsbD protein) (a(a withcarriercarrier 3,4-DHBA. protein) AsbEwith 3,4-DHBA. as an acyltransferase AsbE as an is involvedacyltransferase in the 3,4-dihydroxybenzoyl isis involvedinvolved inin thethe 3,4-dihydroxybenzoyl3,4-dihydroxybenzoyl group transfer from thegroupgroup carrier transfer protein from AsbD the to carrierN1 and Nprotein8 of spermidine AsbD toto toNN a11 ffandandord NNN188-(3,4-dihydroxybenzoyl)-spermidine ofof spermidinespermidine toto affordafford NN1-(3,4-1-(3,4- anddihydroxybenzoyl)-spermidinedihydroxybenzoyl)-spermidineN8-(3,4-dihydroxybenzoyl)-spermidine, and N8-(3,4-dihydroxybenzoyl)-spermidine, respectively, suggesting that AsbE respectively, respectively, has relatively suggesting suggesting relaxed substratethatthat AsbEAsbE specificity has relatively [60,84 relaxed,85]. substrate specificityspecificity [60,84,85].[60,84,85].

Scheme 6. The proposed biosynthetic pathway of petrobactin (72). Scheme 6. The proposed biosynthetic pathwaypathway ofof petrobactinpetrobactin ( (7272).). 4. Synthesis and Study of Siderophores from Marine Microorganisms 4.4. SynthesisSynthesis and Study of Siderophores from Marine MicroorganismsMicroorganisms Marine siderophores, as potentially valuable drug candidates, are attracting extensive attention of Marine siderophores, as potentially valuablevaluable drugdrug candidates,candidates, areare attractingattracting extensiveextensive attention attention researchers. However, the content of siderophores from marine microbes is very low. Therefore, in order ofof researchers. However, the content of siderophores fromfrom marinemarine microbesmicrobes isis veryvery low.low. Therefore,Therefore, to further study their biological activities, mechanisms, absolute conﬁgurations, or structure–activity inin orderorder toto furtherfurther study their biological activitiesactivities,, mechanisms,mechanisms, absoluteabsolute configurations, configurations, or or structure structure–– relationships, chemical syntheses of siderophores would be crucial. activityactivity relationships, chemical syntheses of siderophores wouldwould bebe crucial.crucial.

Mar. Drugs 2019, 17, 562 20 of 28 Mar. Drugs 2019, 17 20 of 4

4.1.4.1. AlcaliginAlcaligin AlcaliginAlcaligin ((2525)) hadhad similarsimilar structuralstructural relationshiprelationship to to siderophores siderophores bisucaberin bisucaberin (26 (26) )and and nocardamine.nocardamine. BisucaberinBisucaberin (26) could inhibit the growth of of both invasive invasive micropapillary micropapillary carcinoma carcinoma µ andand L-1210L-1210 leukemialeukemia cellscells withwith ICIC5050 of 12.7 and and 9.7 9.7 μM,M, respectively, respectively, and and sensitize sensitize tumor tumor cells cells to to macrophage-mediatedmacrophage-mediated cytolysiscytolysis [[32].32]. However, this activity activity was was absent absent in in siderophore siderophore nocardamine. nocardamine. AnAn investigation investigation ofof thethe biologicalbiological propertiesproperties of 25 is particularly attractive. attractive. Therefore, Therefore, Bergeron Bergeron R.J. R.J. etet al. al [[86]86] firstfirst performed the total synthesis of alcaligin ( (2525)) (Scheme (Scheme 77).). The starting starting point point in in the the total total synthesissynthesis ofof alcaliginalcaligin waswas thethe regiospecificregiospecific N-alkylation of of ditosylate ditosylate 1a1a providingproviding monotosylate monotosylate 2a2a. . AA second second amino amino groupgroup waswas thenthen coupledcoupled to C-1 of 2a2a byby NN-alkylation-alkylation of of trifluoroacetamide trifluoroacetamide producing producing thethe diamidediamide 3a. The The primary primary amine amine 4a 4awaswas produced produced by basic by basic cleavage cleavage of 3a. ofBrief3a. exposure Brief exposure of the N of- the(tertN-butoxycarbonyl)amine-(tert-butoxycarbonyl)amine 4a to trifluoroacetic4a to trifluoroacetic acid led to acid the led bis(benzyloxy)putrescine to the bis(benzyloxy)putrescine 5a. Addition5a . Additionof 2-[[(tert of-butoxycarbonyl)oxy]imino]-2-phenylacetonitrile 2-[[(tert-butoxycarbonyl)oxy]imino]-2-phenylacetonitrile to 5a resulted to 5a inresulted the benzyloxy in the amine benzyloxy 6a. amine6a was6a next. 6a acylatedwas next with acylated succinic with anhydride succinic to anhydridegenerate tert to-butoxycarbonyl generate tert-butoxycarbonyl acid 7a. Subsequently, acid 7a . Subsequently,the condensation the condensation of 5a and 7a ofto 5aproduceand 7a theto produce (benzyloxy)amine the (benzyloxy)amine 8a. The second8a. The succinate second unit succinate was unitcoupled was coupledwith 8a to with generate8a to generatetert-butoxycarbonyltert-butoxycarbonyl acid 9a and acid 10a9a. Macrocyclicand 10a. Macrocyclic tetrabenzyl tetrabenzyl alcaligin alcaligin11a was 11aobtainedwas obtained under the under catalysis the catalysis of diphenylph of diphenylphosphorazidate.osphorazidate. Finally, Finally,the benzyl the groups benzyl of groups 11a ofwere11a removedwere removed to obtain to obtain alcaligin alcaligin (25). (25).

SchemeScheme 7.7. Chemical synthesis of of alcaligin alcaligin ( (2525).). 4.2. Vanchrobactin 4.2. Vanchrobactin In the course of the studies into vanchrobactin (43), its stereochemistry could not be elucidated by In the course of the studies into vanchrobactin (43), its stereochemistry could not be elucidated spectroscopic analysis. In order to determine the absolute configuration of vanchrobactin (43), Soengas by spectroscopic analysis. In order to determine the absolute configuration of vanchrobactin (43), R.G. et al. [87] performed the total synthesis of vanchrobactin (43) (Scheme8). 2,3-dihydroxybenzoic Soengas R.G. et al [87] performed the total synthesis of vanchrobactin (43) (Scheme 8). 2,3- acid (2,3-DHBA) was first protected by benzyl bromide to generate 1b. Subsequent saponification with dihydroxybenzoic acid (2,3-DHBA) was first protected by benzyl bromide to generate 1b. Subsequent barium hydroxide gave 2,3-dibenzyloxybenzoic acid 2b. On the other hand, Nδ-Cbz-d-ornithine-OMe saponification with barium hydroxide gave 2,3-dibenzyloxybenzoic acid 2b. On the other hand, Nδ- 3b was obtained from the Cbz analogue of d-ornithine copper complex. Coupling of 3b with Cbz-D-ornithine-OMe 3b was obtained from the Cbz analogue of D-ornithine copper complex. 2,3-dibenzyloxybenzoic acid 2b gave compound 4b. Deprotection of the benzyl groups in 5b, Coupling of 3b with 2,3-dibenzyloxybenzoic acid 2b gave compound 4b. Deprotection of the benzyl introduction of the guanidine functionality to obtain 6b and acidic hydrolysis produced vanchrobactin groups in 5b, introduction of the guanidine functionality to obtain 6b and acidic hydrolysis produced (43). Moreover, in order to further deduce structure–activity relationships of vanchrobactin (43), several vanchrobactin (43). Moreover, in order to further deduce structure–activity relationships of 43 43 vanchrobactinanalogues were (43 also), several synthesized 43 analogues by a similar were strategy also synthesized of the total synthesisby a similar of vanchrobactin strategy of the ( total)[88 ]. Thesynthesis results of suggest vanchrobactin the aromatic (43) [88]. ring inThe catecholate results suggest siderophores the aromatic is essential ring in for catecholate the binding siderophores of the outer membraneis essential receptors. for the binding The lack of the of stereochemistryouter membrane of re theceptors. amino The acid lack sca offf oldstereochemistry will affect the of siderophore the amino activityacid scaffold in V. anguillarum will affect the. Moreover, siderophore microbes activity are in not V. anguillarum always selective. Moreover, in the usemicrobes of siderophores, are not always they canselective utilize in several the use di offf siderophores,erent siderophores they can to transportutilize several Fe(III) different into microbial siderophores cells, to even transport siderophores Fe(III) into microbial cells, even siderophores with very different structures. The relatively low specificity of siderophore utilization may facilitate the design of drugs against different pathogens.

Mar. Drugs 2019, 17, 562 21 of 28

withMar. Drugs very 2019 diff,erent 17 structures. The relatively low specificity of siderophore utilization may facilitate21 of 4 theMar. design Drugs 2019 of drugs, 17 against different pathogens. 21 of 4

OH OBn OBn OH OH 1) SOCl /MeOH OBn OBn 2 OBn Ba(OH)2,THF,H2O OBn OH 1) SOCl /MeOH OBn OBn 2) 2 Ba(OH)2,THF,H2O CO2H BnBr/K2CO3 CO2Me CO2H 2,3-DHBA 2) 1b CO2H BnBr/K2CO3 CO2Me 2b OBn CO2H OBn 2,3-DHBA 1b 2b 1) TBTU, Et N, DMF OBn L-Ser-OtBu 3 H + OBnN L-Ser-OtBu 2)1)Ba(OH) TBTU, Et, THF,N, DMF H O DBTU/DMF, TEA + 2 3 2 H NHCbz 1) CuSO4, H2O, NaOH 2) O N CO H Ba(OH)2, THF, H2O 2 NHCbz DBTU/DMF, TEA NH2 NH2 2)1) NaOH,CuSO ,dioxane, H O, NaOH ClCbz 4b NH2 4 2 NHCbz O CO2H HO C NH2 NH 2 3)2) EDTA,NaOH, dioxane,1M HCl ClCbz MeO2C 2 NH NHCbz 4b HO C 2 3b 2 D-Ornithine 4)3)SOCl EDTA,2/MeOH 1M HCl MeO2C 3b D-Ornithine 4) SOCl2/MeOH

OBn OBn OH OBn OBn OH OH OBn 1) H , Pd-C, MeOH OBn NBoc TFA/DCM NH H 2 H OBn OBnH OHN N 2) (NHBoc)1) H , Pd-C,NTf, MeOHEt N, CHCl N NBoc TFA/DCM N NHNH H NHCbz 2 2 3 3 N NHBoc H 2 H H N H O N 2) (NHBoc) NTf, Et N, CHCl O N O N NH O NH NHCbz 2 3 3 N NHBoc O NH 2 O NH H H O O OHO HOO NH HOO NH O NHCOOH CO2tBu CO2tBu HO5bHO 6b HO CO tBu Vanchrobactin(COOH43) 2 CO2tBu 5b 6b Vanchrobactin(43) Scheme 8. Chemical synthesis of vanchrobactin (43). Scheme 8. Chemical synthesis of vanchrobactin (43). Scheme 8. Chemical synthesis of vanchrobactin (43). 4.3.4.3. PetrobactinPetrobactin 4.3. Petrobactin PandeyPandey R.K.R.K. etet al.al [[89]89] performed efficient efficient total total synthesis synthesis of of petrobactin petrobactin (72)(72) viavia antimony antimony triethoxidetriethoxidePandey mediated R.K. et couplingal coupling [89] performed (Scheme (Scheme 9)efficient9. ).It not It only nottotal onlyproved synthesis proved the ofstructure thepetrobactin structure of 72 , (72)but of alsovia72 ,antimonyprovided but also providedsufficienttriethoxide suyield ffimediatedcient for subsequent yield coupling for subsequent use(Scheme in the 9)ther use. Itapeutic innot the only therapeuticstudy. proved First, the study.the structure hydroxyl First, ofthe 72group, hydroxylbut of also N-dibenzyl- provided group of sufficient yield for subsequent use in the therapeutic study. First, the hydroxyl group of N-dibenzyl- Naminoalcohol-dibenzyl-aminoalcohol 1c was mesyl1c wasprotected mesyl to protected provideto compound provide compound 2c. Nucleophilic2c. Nucleophilic displacement displacement of O-Ms withaminoalcohol amine 3c 1c yielded was mesyl 4c. Debenzylation protected to provide of 4c under compound H2-Pd/C 2c . conditionsNucleophilic to yielddisplacement 5c. On the of O-Msother of O-Ms with amine 3c yielded 4c. Debenzylation of 4c under H2-Pd/C conditions to yield 5c. On the with amine 3c yielded 4c. Debenzylation of 4c under H2-Pd/C conditions to yield 5c. On the other otherhand, hand, esterification esterification of 6cof under6c under reflux reflux conditions conditions was wasperformed, performed, followed followed by benzyl by benzyl protection protection of hand, esterification of 6c under reflux conditions was performed, followed by benzyl protection of ofcatechol catechol unit unit to to yield yield 8c8c. Ester–amide. Ester–amide exchange exchange was was performed performed with with amine amine 5c5c andand acid acid 8c8c toto produce produce catechol unit to yield 8c. Ester–amide exchange was performed with amine 5c and acid 8c to produce 9c9c.. The TheN N-Boc-Boc groupgroup waswas removedremoved from 9c to obtain obtain amine amine 10c10c.. Coupling Coupling of of 10c10c andand 12c12cwaswas carried carried 9c. The N-Boc group was removed from 9c to obtain amine 10c. Coupling of 10c and 12c was carried outout using using trimethylaminetrimethylamine toto yieldyield protectedprotected petrobactin 12c12c.. Finally, Finally, 12c12c waswas debenzylated debenzylated to to yield yield out using trimethylamine to yield protected petrobactin 12c. Finally, 12c was debenzylated to yield petrobactinpetrobactin ( 72(72).). Moreover,Moreover, sidechain-modifiedsidechain-modified petrobactin de derivativesrivatives were were also also synthesized synthesized for for the the petrobactin (72). Moreover, sidechain-modified petrobactin derivatives were also synthesized for the studystudy of of iron iron binding, binding, thethe resultsresults suggestsuggest bothboth acylationacylation and alkylation of of the the spermidine spermidine sidechain sidechain basicallystudy of irondo not binding, affect ironthe resultsbinding suggest properties both of ac theylation siderophore and alkylation [90]. of the spermidine sidechain basically do not affect iron binding properties of the siderophore [90]. basically do not affect iron binding properties of the siderophore [90].

Scheme 9. Chemical synthesis of petrobactin (72). Scheme 9. Chemical synthesis of petrobactin (72). Scheme 9. Chemical synthesis of petrobactin (72).

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5. Conclusions and Perspectives Siderophores from marine microbes are an important class of secondary metabolites that have intrigued microbiologists, chemists, and biochemists due to their structural diversity and potential applications in diseases and medicinal agents. However, with widely stated estimate that less than 1% marine microbes have been brought culture [91], the great challenge includes the development of new methods to bring more marine microbes into culture. Moreover, further detailed understanding of chemical synthesis and biosynthesis of marine siderophores will provide valuable insight for their study of biological activities, and elucidation of mechanism. In total, marine siderophore research will continue stimulating a multidisciplinary research eﬀort that will facilitate their potential applications in medicine and health care.

Table 2. Siderophores from marine microorganisms.

Compd. Name PubChem CID PubChem Database Ref. 1 Marinobactin A – – [22] 2 Marinobactin B – – [22] 3 Marinobactin C – – [22] 4 Marinobactin D – – [22] 5 Marinobactin E – – [22] 6 Marinobactin F – – [22] 7 Marinobactin HG – – [23] 8 Amphibactin – – [24] 9 Amphibactin – – [24] 10 Amphibactin – – [24] 11 Amphibactin – – [24] 12 Amphibactin – – [24] 13 Amphibactin – – [24] 14 Amphibactin – – [24] 15 Amphibactin – – [24] 16 Amphibactin – – [24] 17 Amphibactin – – [24] 18 Moanachelin – – [25] 19 Moanachelin – – [25] 20 Moanachelin – – [25] https://pubchem.ncbi.nlm.nih.gov/ 21 Moanachelin 122223347 [25] compound/122223347 22 Moanachelin – – [25] 23 Amphibactin U – – [26] 24 Amphibactin V – – [26] 25 Alcaligin – – [27] 26 Bisucaberin – – [28] 27 Avaroferrin – – [29] 28 Putrebactin – – [30] 29 Bisucaberin B – – [34] 30 Thalassosamide – – [35] https://pubchem.ncbi.nlm.nih.gov/ 31 Fradiamine A 129008905 [36] compound/129008905 https://pubchem.ncbi.nlm.nih.gov/ 32 Fradiamine B 60151746 [36] compound/60151746 33 Albisporachelin – – [37] 34 Desferrioxamine A1 ––[38] 35 Desferrioxamine A2 ––[38] 36 Desferrioxamine B – – [38] 37 Desferrioxamine D1 ––[38] 38 Desferrioxamine D2 ––[38] 39 Desferrioxamine E – – [38] 40 Desferrioxamine N – – [38] Mar. Drugs 2019, 17, 562 23 of 28

Table 2. Cont.

Compd. Name PubChem CID PubChem Database Ref. https://pubchem.ncbi.nlm.nih.gov/ 41 Vibrioferrin 11102119 [39] compound/11102119 42 Nigribactin – – [40] 43 Vanchrobactin – – [45] 44 Trivanchrobactin – – [45] 45 Divanchrobactin – – [45] 46 Turnerbactin [46] 47 Dibenarthin – – [47] 48 Streptobactin – – [47] 49 Tribenarthin – – [47] 50 Pistillarin – – [48] https://pubchem.ncbi.nlm.nih.gov/ 51 Loihichelin A 101476230 [51] compound/101476230 https://pubchem.ncbi.nlm.nih.gov/ 52 Loihichelin B 101476231 [51] compound/101476231 https://pubchem.ncbi.nlm.nih.gov/ 53 Loihichelin C 101476232 [51] compound/101476232 https://pubchem.ncbi.nlm.nih.gov/ 54 Loihichelin D 101476233 [51] compound/101476233 https://pubchem.ncbi.nlm.nih.gov/ 55 Loihichelin E 101476234 [51] compound/101476234 https://pubchem.ncbi.nlm.nih.gov/ 56 Loihichelin F 101476235 [51] compound/101476235 57 Aquachelin A – – [22] 58 Aquachelin B – – [22] 59 Aquachelin C – – [22] 60 Aquachelin D – – [22] 61 Aquachelin I – – [23] 62 Aquachelin J – – [52] 63 Aquachelin HG – – [29] 64 Imaqobactin – – [53] 65 Aerobactin – – [54] 66 Ochrobactin A – – [56] 67 Ochrobactin B – – [56] 68 Ochrobactin C – – [56] https://pubchem.ncbi.nlm.nih.gov/ 69 Synechobactin 122377042 [57] compound/122377042 https://pubchem.ncbi.nlm.nih.gov/ 70 Synechobactin 122377043 [57] compound/122377043 https://pubchem.ncbi.nlm.nih.gov/ 71 Synechobactin 122377044 [57] compound/122377044 72 Petrobactin – – [58] 73 Petrobactin sulfonate – – [59] 74 Alterobactin A – – [61] https://pubchem.ncbi.nlm.nih.gov/ 75 Alterobactin B 101775921 [62] compound/101775921 https://pubchem.ncbi.nlm.nih.gov/ 76 Pseudoalterobactin A 11434714 [63] compound/11434714 https://pubchem.ncbi.nlm.nih.gov/ 77 Pseudoalterobactin B 11788080 [63] compound/11788080 78 Lystabactin A – – [64] 79 Lystabactin B – – [64] 80 Lystabactin C – – [64] 81 Anguibactin – – [42,45] https://pubchem.ncbi.nlm.nih.gov/ 82 piscibactin 136754132 [65–67] compound/136754132 83 Tetroazolemycin A – – [68] 84 Tetroazolemycin B – – [68] Mar. Drugs 2019, 17, 562 24 of 28

Table 3. The EC numbers of enzymes.

Enzymes EC Numbers EnzA 4.1.1.7 VabC 3.1.22.4 VabD 2.7.8.7 VabF 3.2.1.55 2.3.2.27 Irp1 4.2.1.3 Irp2 2.3.2.27 Irp3 2.3.2.27 Irp4 2.3.2.27 AngB 3.3.2.1 AngE 1.14.14.148 VabD 2.7.8.7 IucA 6.3.2.38 IucC 6.3.2.39 IucD 1.14.13.59 AsbF 4.2.1.118

Author Contributions: J.C. and Y.G. conceived and wrote the review; Y.L. and B.W. edited the chemical structures; J.S., B.W. and H.Z. offered important advice to improve the review; H.W. revised the paper. Acknowledgments: This project was supported by National Natural Science Foundation of China (No. 81773628, and 41776139), National Key R & D Program of China (No. 2017YFE0103100 and 2018YFC0311003),and 111-National Overseas Expertise Introduction Center for Green Pharmaceutical Discipline Innovation (No. D17012). Conflicts of Interest: The authors declare no conflict of interest.

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