Absolute Stereochemistry Determination of Bioactive Marine-Derived Cyclopeptides by Liquid Chromatography Methods: An Update Review (2018–2022)

Cyclopeptides are considered as one of the most important classes of compounds derived from marine sources, due to their structural diversity and a myriad of their biological and pharmacological activities. Since marine-derived cyclopeptides consist of different amino acids, many of which are non-proteinogenic, they possess various stereogenic centers. In this respect, the structure elucidation of new molecular scaffolds obtained from natural sources, including marine-derived cyclopeptides, can become a very challenging task. The determination of the absolute configurations of the amino acid residues is accomplished, in most cases, by performing acidic hydrolysis, followed by analyses by liquid chromatography (LC). In a continuation with the authors’ previous publication, and to analyze the current trends, the present review covers recently published works (from January 2018 to November 2022) regarding new cyclopeptides from marine organisms, with a special focus on their biological/pharmacological activities and the absolute stereochemical assignment of the amino acid residues. Ninety-one unreported marine-derived cyclopeptides were identified during this period, most of which displayed anticancer or antimicrobial activities. Marfey’s method, which involves LC, was found to be the most frequently used for this purpose.


Introduction
Since more than two-thirds of the global surface area are covered by oceans, marine organisms represent more than half of the total biodiversity [1]. Therefore, oceans constitute a rich source of many unique and novel compounds [2,3]. Long-term evolution of marine organisms promoted the fruitful development of a great number of natural products when exposed to extreme conditions, such as pressure, light, temperature, and salinity [4,5]. From 2015 to 2018, 17 clinically available drugs, based on marine natural products or their derivatives, were reported being 28 drugs in Phases I-III clinical trials [6]. Since 2018, great efforts have been made to find new therapeutically potent chemical and biological entities and, currently, 68 patents from marine organisms have been filed [7].
In a continuation of our previous publication [110] and to analyze current trends, herein, recent studies concerning new bioactive marine cyclopeptides are analyzed regarding the absolute stereochemistry determination of the amino acid residues by LC methodologies. The chemical structures and biological/pharmacological activities of the marine-derived peptides are also described to provide an update for this field of research. Structural issues relevant to the biological/pharmacological activities are also emphasized when considered pertinent.

Liquid Chromatography Methods for Determination of Absolute Configurations of Peptides
LC is an indispensable technique in several research laboratories all over the world, especially when combined with different detectors such as ultraviolet-visible (UV-Vis), mass spectrometry (MS), and fluorescence detection [115]. LC is a method of choice associated to both Marfey´s and chiral LC methods [110].
The first step, in both methods, to determine the absolute configurations of the amino acids of marine peptides is a total or partial hydrolysis of peptide bonds to obtain individual amino acid residues ( Figure 2). Typically, the hydrolysis reaction is carried out in acidic condition using 6 N of HCl solution at 110-115 °C for 24 h [110]. In Marfey´s method, after the acidic hydrolysis of peptides, the amino acid residues are derivatized with suitable chiral derivatizing reagents (CDRs). The reagents 1-fluoro- In Marfey s method, after the acidic hydrolysis of peptides, the amino acid residues are derivatized with suitable chiral derivatizing reagents (CDRs). The reagents 1-fluoro-2,4dinitrophenyl-5-D,L-alanine amide (FDAA) and 1-fluoro-2,4-dinitrophenyl-5-D,L-leucine amide (FDLA) ( Figure 3A) are the most widely used [110].
Molecules 2023, 28,615 2,4-dinitrophenyl-5-D,L-alanine amide (FDAA) and 1-fluoro-2,4-dinitropheny leucine amide (FDLA) ( Figure 3A) are the most widely used [110]. The CDRs react stoichiometrically, without racemization, under gentle heating within 1-2 h in alkaline condition, with the amino group of L-and D-amino acids r yielding diastereomers ( Figure 3B) [116]. It is important to mention that this meth found to be ineffective for some amino acid residues, especially those containing ri α-methyl-substituted phenylalanine and phenylalanine amides [117]. Nevertheless approach, called the "O-Marfey method", was described for the derivatization of α-h acids with L-FDAA by increasing the nucleophilicity of the hydroxyl group for the to take place [118]. This method requires the addition of NaH (60% dispersed in solution of α-hydroxy acids in tetrahydrofuran at room temperature. Then, the C FDAA) is added to this solution and stirred under argon or nitrogen atmosphere other cases, there is a need for optimization, such as by using a solution of FDAA in as well as a higher reagent concentration and longer reaction times [119]. Of not diastereomers can also be obtained by microwave-assisted synthesis [120].
The synthesis of FDAA as a CDR to determine the stereochemistry of amin was described for the first time by Peter Marfey in 1984 [121]. FDAA was obtain difluoro dinitro benzene by substitution of one of its two fluorine atoms by L amide. Then, a nucleophilic substitution of the remaining reactive aromatic fluor performed with the free amino group on L-and D-alanine. From this r diastereomeric pairs were obtained, which were separated by RP-LC. Amide was because it is quite stable and neutral and does not undergo racemization [121 The CDRs react stoichiometrically, without racemization, under gentle heating at 40 • C within 1-2 h in alkaline condition, with the amino group of L-and D-amino acids residues yielding diastereomers ( Figure 3B) [116]. It is important to mention that this method was found to be ineffective for some amino acid residues, especially those containing ring-and α-methyl-substituted phenylalanine and phenylalanine amides [117]. Nevertheless, a new approach, called the "O-Marfey method", was described for the derivatization of α-hydroxy acids with L-FDAA by increasing the nucleophilicity of the hydroxyl group for the reaction to take place [118]. This method requires the addition of NaH (60% dispersed in oil) to a solution of α-hydroxy acids in tetrahydrofuran at room temperature. Then, the CDR (L-FDAA) is added to this solution and stirred under argon or nitrogen atmosphere [118]. In other cases, there is a need for optimization, such as by using a solution of FDAA in acetone, as well as a higher reagent concentration and longer reaction times [119]. Of note is that diastereomers can also be obtained by microwave-assisted synthesis [120].
The synthesis of FDAA as a CDR to determine the stereochemistry of amino acids was described for the first time by Peter Marfey in 1984 [121]. FDAA was obtained from difluoro dinitro benzene by substitution of one of its two fluorine atoms by L-alanine amide. Then, a nucleophilic substitution of the remaining reactive aromatic fluorine was performed with the free amino group on L-and D-alanine. From this reaction, diastereomeric pairs were obtained, which were separated by RP-LC. Amide was chosen because it is quite stable and neutral and does not undergo racemization [121]. Now, FDAA is universally known as Marfey's reagent and continues to be widely used. Over the years, other structural variants of FDAA have been developed [122].
Generally, after the derivatization reaction, the diastereomers are analyzed by an RP-LC, using suitable standards for comparison to accurately determine the absolute configurations of the peptides [119]. Additionally, other methodologies have also emerged, including LC-mass spectrometry (LC-MS), thin-layer chromatography (TLC), and gas chromatography (GC) [123,124]. The key advantages of Marfey s method include improved detection sensitivity compared to underivatized amino acids, the commercial availability of both enantiomers of FDAA, and the use of readily available LC technology.
Harada et al. [125] proposed a method named "advanced Marfey's method". This method uses LC-MS to determine the absolute configurations of the amino acid residues. Since this method does not necessarily require amino acid standards to detect and identify target amino acids, it can be used to determine the absolute configurations of unusual amino acids for which standards are not available. In this method, the absolute configuration of an amino acid is deduced from its LC retention times of the L-FDLA derivative of the original amino acid, and its enantiomer formed by racemization. Therefore, the racemization is essential for this method [125]. Later, the same group introduced "DL-FDLA derivatization", a procedure that does not require the conventional chemical racemization process [126]. Due to this D,L-FDLA system, the advanced Marfey's method has been improved to be simple, more rapid and reliable, and its applicability has been significantly extended [103,104].
In order to develop a highly sensitive analytical Marfey's method capable of rapidly and unambiguously determining the absolute configurations and enantiomer regiochemistry for all commonly found amino acids, Vijayasarathy et al. [102] developed the C3 Marfey's method. This method is a more refined variation on the existing Marfey's method, using the same CDRs (L-FDAA and D-FDAA) but with a C3 LC column held at 50 • C, and a H 2 O/MeOH gradient elution modified by isocratic 1% HCO 2 H in CH 3 CN, and taking advantage of both UV (340 nm) and (±)-ESIMS detection. This method was shown to be very effective, allowing the stereochemical assignments of all amino acids, including isoleucine stereoisomers. Moreover, the residual Marfey's reagent did not obscure any target amino acid [102].
The applicability and limitations of Marfey's method and its optimized methods have been extensively examined in several reviews [116,119,122]. These methods were also found to be promising for applications in several other research fields and for other chiral molecules/drugs [122].
Regarding the chiral LC method, determination of the absolute configurations is developed by a direct analysis of the amino acid residues present in the acid hydrolysate without prior derivatization. First, enantioseparation of standards amino acids is performed on a chiral stationary phase (CSP). Then, comparison and/or co-injection of hydrolysate with the amino acids standards is performed, under the same chromatographic conditions, leading to the identification of the amino acid constituents [110].
The success of an efficient enantioseparation is mainly determined by the chiral discriminative power of the CSP. Over the years, various types of CSPs have been described [127][128][129]; nevertheless, the development of new CSPs for LC continued to be an evolutionary subject, encompassing the preparation of new chiral selectors [130], the introduction of new chromatographic supports [131], and the application of different synthetic approaches for the preparation of CSPs [132]. Macrocyclic antibiotic-based, crown etherbased, and ligand exchange-type ( Figure 4) are the most-used CSPs for the enantiomeric separation of amino acids and primary amine-containing compounds [133][134][135][136]. Evidently, the same trend was observed for the stereochemical determination of the amino acids subunits of marine peptides [110]. A direct analysis of amino acid hydrolysates without further derivatization is the main advantage of chiral LC, since it is a faster and simpler procedure when compared to indirect methods. Its principal drawback is the high cost of the commercial chiral columns, which could be a hurdle for these analyses [137].
Although these methods are highly effective, one limitation of both is that racemization can occur for some amino acid residues during the acid hydrolysis [138,139]. For example, the analysis of the acidic hydrolysate of a cyclopeptide isolated from a marine sponge, Homophymia sp., showed that the racemization of lysine took place. Although this peptide contains only one lysine residue, both D-and L-enantiomers were detected in almost equal amounts. By performing an analysis of the hydrolysates with shorter reaction times of 4 and 8 h, it was found that racemization occurred during acidic hydrolysis ( Figure 5). The longer the reaction time, the higher the content of L-lysine, indicating a formation of this enantiomer by epimerization [138].  A direct analysis of amino acid hydrolysates without further derivatization is the main advantage of chiral LC, since it is a faster and simpler procedure when compared to indirect methods. Its principal drawback is the high cost of the commercial chiral columns, which could be a hurdle for these analyses [137].
Although these methods are highly effective, one limitation of both is that racemization can occur for some amino acid residues during the acid hydrolysis [138,139]. For example, the analysis of the acidic hydrolysate of a cyclopeptide isolated from a marine sponge, Homophymia sp., showed that the racemization of lysine took place. Although this peptide contains only one lysine residue, both D-and L-enantiomers were detected in almost equal amounts. By performing an analysis of the hydrolysates with shorter reaction times of 4 and 8 h, it was found that racemization occurred during acidic hydrolysis ( Figure 5). The longer the reaction time, the higher the content of L-lysine, indicating a formation of this enantiomer by epimerization [138].  A direct analysis of amino acid hydrolysates without further derivatization is the main advantage of chiral LC, since it is a faster and simpler procedure when compared to indirect methods. Its principal drawback is the high cost of the commercial chiral columns, which could be a hurdle for these analyses [137].
Although these methods are highly effective, one limitation of both is that racemization can occur for some amino acid residues during the acid hydrolysis [138,139]. For example, the analysis of the acidic hydrolysate of a cyclopeptide isolated from a marine sponge, Homophymia sp., showed that the racemization of lysine took place. Although this peptide contains only one lysine residue, both D-and L-enantiomers were detected in almost equal amounts. By performing an analysis of the hydrolysates with shorter reaction times of 4 and 8 h, it was found that racemization occurred during acidic hydrolysis ( Figure 5). The longer the reaction time, the higher the content of L-lysine, indicating a formation of this enantiomer by epimerization [138].  [156] Tutuilamides A-C (19)(20)(21) Cyclic hexadepsipeptides Marine cyanobacteria Schizothrix sp. (19)(20) and Coleofasciculus sp.  In this section, the marine-derived cyclopeptides are organized according to the main biological activity.

Anticancer Activity
One of the most described biological activities associated to marine-derived cyclopeptides is anticancer activity [185]. Consistent with similar findings in previous works, several isolated marine peptides exhibited cytotoxicity. Among the 91 new marine-derived cyclopeptides, 43 showed promising results regarding anticancer activity and 11 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) were isolated from marine-derived bacteria ( Figure 6). Marine-derived bacteria have been shown to be a rich source of natural products, including cyclopeptides [71,186]. Bacterial natural products constitute 29% of Food and Drug Administration (FDA)-approved natural product-derived drugs, which mostly function as antitumor or antibacterial agents [187]. Bacterial natural products constitute 29% of Food and Drug Administration (FDA)approved natural product-derived drugs, which mostly function as antitumor or antibacterial agents [187].  A new cyclic lipopeptide, bacilohydrin A (1), was isolated from a crude culture extract of Bacillus sp. SY27F, obtained from the Indian Ocean hydrothermal vent [148]. Compound 1 exhibited significant cytotoxicity against three cancer cell lines-specifically, DU-145, MCF-7, and HepG2-with IC 50 values of 50.3-175.1 nM. The positive control, paclitaxel, showed IC 50 values of 10.9-94.3 nM. The absolute configurations of the amino acids in 1 were determined by a modified Marfey's method revealing the presence of seven mixed D/L-amino acids. It is important to highlight that it was the first report of a determination of the absolute configuration of a D-aspartic acid residue in the surfactin class [148].
The advanced Marfey's method, in combination with 3 J HH and rotating-frame Overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis, was applied to establish the absolute configuration of the amino acid units of epoxinnamide (2), a new cinnamoyl-containing nonribosomal bicyclic decadepsipeptide ( Figure 6), which was isolated from the culture extract of an intertidal mudflat-derived Streptomyces sp. OID44 [149]. Cinnamoyl-containing nonribosomal peptides represent a unique family of actinobacterial metabolites associated with diverse bioactivities [188]. Compound 2 induced quinone reductase activity in murine Hepa-1c1c7 cells, at 5 µM, by 1.6-fold. Additionally, 2 displayed considerable antiangiogenesis activity in human umbilical vein endothelial cells, with an IC 50 of 13.4 µM. The positive control, sunitinib, showed an IC 50 value of 1.7 µM [149].
A new family of polypeptides, bathiapeptides A1 (3), A2 (4), and B-E (5-8), isolated from a marine biofilm-derived Bacillus sp. B19-2, exhibited high cytotoxicity against MGC-803, Hep G2, MCF-7, and HeLa cell lines. Compound 3 exhibited the highest cytotoxicity in all cell lines, with IC 50 values ranging from 0.5 to 4.1 µM. The advanced Marfey's method was able to establish the absolute configuration of isoleucine and phenylalanine of 3-8 [150]. Due to the presence of a rare polythiazole moiety in 3-8 ( Figure 6), the thiazole-derived alanine moiety could not be directly determined by Marfey's method (or chiral LC) since the epimerization of thiazole-based amino acids can occur during acid hydrolysis [189]. To solve this issue, ozonolysis was carried out to break the thiazole ring before derivatization by Marfey's reagents [190]. Moreover, since Marfey's method does not allow distinguishing L-isoleucine from L-allo-isoleucine [104], an X-ray analysis was performed to prove the structure of L-isoleucine [150].
Dentigerumycin E (9), a new cyclic hexapeptide containing three piperazic acids and a pyran-bearing polyketide acyl moiety ( Figure 6), was isolated from the extract of a coculture of marine-derived Streptomyces and Bacillus strains, which were simultaneously isolated from an intertidal mudflat. The absolute configurations of the amino acid residues of 9 were established by a combination of advanced Marfey's method with ROESY correlations. Compound 9 exhibited moderate antiproliferative and antimetastatic activities against several human cancer cell lines, including A549, HCT-116, MDA-MB-231, SK-HEP-1, and SNU638, with IC 50 values ranging from 27 to 39 µM. The positive control, etoposide, showed IC 50 values ranging from 0.42 to 6.21 µM. Compound 9 was also assayed against MCF-10A to determine its cancer cell-specific cytotoxicity and showed an IC 50 value higher than 50 µM, indicating that 9 did not exhibit significant cytotoxicity against normal human epithelial cells. Additionally, 9 also showed antimetastatic potential against MDA-MB-231 in wound healing and cell invasion assays. In the wound healing assay, 9, at 20 and 40 µM, inhibited cell migration by 20 and 48%, respectively, while in the cell invasion assay, 9 exhibited inhibitory activity by 10 and 34% at 20 and 40 µM, respectively. A structureactivity relationship (SAR) study suggested that 2-N-OH, 16-N-OH, and 37-OH (carboxylic acid) in 9 are essential for its antiproliferative and antimetastatic activities [151].
Another promising compound with anticancer activity is a cyclic depsipeptide, nobilamide I (10), isolated from the saline cultivation of a marine-derived bacterium, Saccharomonospora sp. strain CNQ-490, obtained from a 45 m deep-sea sediment sample at 2 km west of the Scripps pier, La Jolla Canyon, in California. Nobilamide I (10) decreased cancer cell motility by inhibiting epithelial-mesenchymal transition (EMT) effectors. At a concentration of 5 µM, 10 inhibited migrations of AGS, A549, and Caco2 cells by ca. 70, 50, and 60%, respectively, and their invasions by ca. 60, 30, and 60%, respectively. It was suggested that 10 modulated the protein and mRNA expression levels of EMT N-cadherin and E-cadherin markers by downregulating the transcription factors, Snail, Slug, and Twist. In addition, 10 modulated the protein and mRNA expression levels of the matrix metalloproteinase family (MMP2 and MMP9) in the three cell lines. The absolute configurations of the amino acid residues were assigned by using the C3 Marfey's analysis [152].
A new cyclic hexapeptide, nocardiotide A (11) (Figure 6), was isolated from a broth culture of an actinomycete, Nocardiopsis sp. UR67, associated with the marine sponge, Callyspongia sp., which was collected from the Red Sea, Ras Mohamed, Sinai, Egypt. The planar structure of the compound was elucidated by HRESIMS and a 1D and 2D NMR spectral analysis. The absolute configurations of the amino acid residues were solved by Marfey's method. Compound 11 displayed significant cytotoxicity towards human MM.1S multiple myeloma, human HeLa cervix carcinoma, and murine CT26 colon carcinoma cell lines, with IC 50 values of 8, 11, and 12 µM, respectively [153].
Cyanobacteria are also an important source of new marine-derived cyclopeptides [191,192]. concentration of 5 μM, 10 inhibited migrations of AGS, A549, and Caco2 cells by ca. 70, 50, and 60%, respectively, and their invasions by ca. 60, 30, and 60%, respectively. It was suggested that 10 modulated the protein and mRNA expression levels of EMT N-cadherin and E-cadherin markers by downregulating the transcription factors, Snail, Slug, and Twist. In addition, 10 modulated the protein and mRNA expression levels of the matrix metalloproteinase family (MMP2 and MMP9) in the three cell lines. The absolute configurations of the amino acid residues were assigned by using the C3 Marfey's analysis [152].
A new cyclic hexapeptide, nocardiotide A (11) (Figure 6), was isolated from a broth culture of an actinomycete, Nocardiopsis sp. UR67, associated with the marine sponge, Callyspongia sp., which was collected from the Red Sea, Ras Mohamed, Sinai, Egypt. The planar structure of the compound was elucidated by HRESIMS and a 1D and 2D NMR spectral analysis. The absolute configurations of the amino acid residues were solved by Marfey's method. Compound 11 displayed significant cytotoxicity towards human MM.1S multiple myeloma, human HeLa cervix carcinoma, and murine CT26 colon carcinoma cell lines, with IC50 values of 8, 11, and 12 µ M, respectively [153].
Cyanobacteria are also an important source of new marine-derived cyclopeptides [191,192]. Compounds 12-23 ( Figure 7) with anticancer activity were discovered in 2018.   Phyo et al. [154] described the isolation of a new cyclic decapeptide, trikoramide A (12) (Figure 7), isolated from samples of the marine cyanobacterium, Symploca hydnoides, which was collected from the intertidal shores of Trikora beach, Bintan Island, Indonesia. Compound 12 is a C-prenylated cyclotryptophan-containing cyanobactin whose absolute configuration was determined by Marfey's method and a NOESY correlations analysis. Compound 12 displayed cytotoxicity against the MOLT-4 and AML2 cancer cell lines, with IC50 values of 4.8 and 8.2 μM, respectively [154].
Later, the same group isolated another three cyanobactins, trikoramides B-D (13-15) Phyo et al. [154] described the isolation of a new cyclic decapeptide, trikoramide A (12) (Figure 7), isolated from samples of the marine cyanobacterium, Symploca hydnoides, which was collected from the intertidal shores of Trikora beach, Bintan Island, Indonesia. Compound 12 is a C-prenylated cyclotryptophan-containing cyanobactin whose absolute configuration was determined by Marfey's method and a NOESY correlations analysis.
Later, the same group isolated another three cyanobactins, trikoramides B-D (13-15) (Figure 7), from the same samples. Compounds 13-15 are analogues of 12. The main difference in their structures is in the C-prenylated cyclotryptophan unit. Marfey's method, in combination with the ECD spectra and NOESY correlations, were applied to determine their absolute structures. Compounds 13 and 15 showed cytotoxicity against the MOLT-4 cell line, with IC 50 values of 5.2 µM and 4.7 µM, respectively [155].
Very recently, the same group further isolated three new cyclic depsipeptides, triproamide (16), and pemukainalides A (17) and B (18) (Figure 7), from the same cyanobacterium, which was collected from the same location. Compound 16 features a unique structure containing a rare 4-phenylvaline and a β-amino acid, dolamethylleucine. Stereochemical analyses were carried out by utilizing a combination of Marfey's method, J-based configuration, and chiral LC analyses. For the absolute configuration of the 2-hydroxyisovaleric acid unit in 16, a chiral column was used with a ligand exchange-type CSP to establish the R-configuration. By using mass spectrometry-guided fractionation, together with molecular networking, Keller et al. [157] described the isolation of tutuilamides A (19) and B (20) (Figure 7), from the marine cyanobacterium, Schizothrix sp., which was collected from the main island of Tutuila in American Samoa, and tutuilamides C (21) (Figure 7), from a cyanobacterium, Coleofasciculus sp., which was collected from Palmyra Atoll. The structures of 19-21 contain a novel vinyl chloride-containing residue and several unusual residues, including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid. Marfey's method was used to determine the absolute configurations of their amino acid residues. Compounds 19-21 exhibited a moderate potency of cytotoxicity against H-460 lung cancer cell line. Interestingly, 19-21 displayed potent elastase inhibitory activity. Additionally, the binding mode to elastase was analyzed by X-ray crystallography, which revealed a reversible binding mode. Moreover, the amino acid backbone of the flexible side chain of 19 can establish an additional hydrogen bond in the elastase binding pocket, allowing enhanced inhibitory activity [157].
Two new cyclic lipopeptides, laxaphycins B4 (22) and A2 (23) (Figure 7), were isolated, along with the known peptide laxaphycin A, from the marine cyanobacterium, Hormothamnion enteromorphoides, which was collected at Garden Key, Dry Tortugas National Park [158]. Compound 22 displayed growth inhibition against HCT-116 cell line with an IC 50 of 1.7 µM, and a synergistic effect to inhibit the growth of this cancer cell line when 22 was used in combination with laxaphycin A. The absolute stereochemistry of the amino acid residues of 22 and 23 was determined by both chiral LC using a macrocyclic antibiotic-based CSP, and by advanced Marfey's method. In addition to those methods, 1D and 2D ROESY correlations were used to determine the geometric configuration of α,β-dehydro-aminobutyric acid [158].
Cyclopeptides have also been isolated from a myriad of marine-derived fungi, mainly from genera Aspergillus, Acremonium, Fusarium, and Penicillium [193]. Marine-derived fungi represent a potential source for bioactive natural products, and an increasing number of new fungal metabolites, including peptides, have been discovered over the past years [194,195]. In addition, peptides isolated from the association of fungi with other macroorganisms produced a valuable and extra-large chemical database, exhibiting relevant biological activities [196]. From 2018, six marine-derived cyclopeptides (24)(25)(26)(27)(28)(29) with anticancer activity were isolated from marine-derived fungi (Figure 8).  [159].
Two new N-methylated cyclopeptides, asperflomide (28) and asperflosamide (29 (Figure 8), were obtained from the culture extract of Aspergillus flocculosus 16D-1, isolated from the inner tissue of the marine sponge, Phakellia fusca, which was collected from Yongxing Island, China. The planar structures of 28 and 29 were elucidated by th interpretation of HRESIMS and extensive analysis of 1D and 2D NMR spectra. Th absolute configurations of the amino acid constituents in 28 and 29 were established b Marfey's method, using UPLC-HRMS for analysis. Although 28 and 29 did not exhibi cytotoxicity against A2780, HCT-8, PC-9, SW480, MDA-MB-231, and U251 cancer cel lines, and nonmalignant cells (human cardiomyoblast cell lines HaCaT and CCD-18Co) they showed weak tankyrase1/2 inhibitory activity at the centration of 40 µ M (XAV93 was used as a positive control) [160]. Tankyrase is a member of the poly(ADP were determined by a single crystal X-ray diffraction analysis [159]. Two new N-methylated cyclopeptides, asperflomide (28) and asperflosamide (29) (Figure 8), were obtained from the culture extract of Aspergillus flocculosus 16D-1, isolated from the inner tissue of the marine sponge, Phakellia fusca, which was collected from Yongxing Island, China. The planar structures of 28 and 29 were elucidated by the interpretation of HRESIMS and extensive analysis of 1D and 2D NMR spectra. The absolute configurations of the amino acid constituents in 28 and 29 were established by Marfey's method, using UPLC-HRMS for analysis. Although 28 and 29 did not exhibit cytotoxicity against A2780, HCT-8, PC-9, SW480, MDA-MB-231, and U251 cancer cell lines, and nonmalignant cells (human cardiomyoblast cell lines HaCaT and CCD-18Co), they showed weak tankyrase1/2 inhibitory activity at the centration of 40 µM (XAV939 was used as a positive control) [160]. Tankyrase is a member of the poly(ADP-ribose)polymerase family which mediates Wnt signal transduction and has emerged as a new molecular target for the therapy of different kinds of cancer. Consequently, tankyrase inhibitors are considered as promising therapeutics for cancer treatment [197].
A number of peptides and depsipeptides with intriguing structures and interesting biological activities have been discovered from marine sponges [198,199], some of which were shown to be produced by symbiotic microorganisms [27]. From 2018, 14 marinederived cyclopeptides (30-43) (Figure 9) were isolated from marine sponges and from sponge-associated microorganisms.
Molecules 2023, 28, 615 19 of 42 new molecular target for the therapy of different kinds of cancer. Consequently, tankyrase inhibitors are considered as promising therapeutics for cancer treatment [197]. A number of peptides and depsipeptides with intriguing structures and interesting biological activities have been discovered from marine sponges [198,199], some of which were shown to be produced by symbiotic microorganisms [27]. From 2018, 14 marinederived cyclopeptides (30-43) (Figure 9) were isolated from marine sponges and from sponge-associated microorganisms.    From a deep-sea marine sponge, Pachastrella sp., two new peptides, microsclerodermins N and O (30 and 31) (Figure 9), were isolated [161]. Compounds 30 and 31 have unique structures since they possess a p-ethoxyphenyl moiety instead of a typical p-methoxyphenyl moiety at the terminus of the ω-phenyl-polyhydroxylated β-amino acid unit. The absolute configurations of all the stereogenic centers were determined by Marfey's method and were found to be identical with other microsclerodermins. For a stereochemical study, a naphthalene-bonded stationary phase was used. Relevant cytotoxic activity was observed for both compounds against HeLa cells, with an IC 50  Theonellamide J (32), 5-cis-Apoa-theopalauamide (33), and theonellamide K (34) (Figure 9) were isolated from a red sea sponge, Theonella swinhoei, which was collected in the southern part of the Gulf of Aqaba. Compounds 33 and 34 exhibited a growth inhibitory activity against HCT-116 cell line, with an IC 50 of 21.8 and 3.5 µM, respectively. The positive control, cytochalasin D, showed an IC 50 value of 0.8 µM. This finding suggested that the 3-amino-4-hydroxy-6-methyl-8-phenyl-5E,7E-octadienoic acid (Apoa) subunit is relevant for the activity. The absolute configurations of the amino acids were established by Marfey's and advanced Marfey's methods. The samples were first analyzed by Marfey's method, which allowed the configurations of four amino acids to be elucidated, followed by LC-MS (advanced Marfey's method) for the assignment of the stereochemistry of the remaining amino acids [162].
Fuscasins A-D (35-38) (Figure 8) are new cyclic heptapeptides isolated from the marine sponge, Phakellia fusca, which was collected from the South China Sea. Compound 35 exhibited a remarkable cytotoxicity against HepG2 cells, with an IC 50 value of 4.6 µM, while 36-38 were inactive even at a concentration up to 20 µM. Cisplatin was used as the positive control with an IC 50 value of 4.2 µM. Interestingly, 35 did not exhibit cytotoxicity against the nonmalignant rat cardiomyoblast H9C2 cell line even at concentrations up to 100 µM, suggesting that this compound may have a selective growth inhibitory effect against HepG2 cells. Moreover, none of the compounds displayed cytotoxicity against NCI-H460, MCF-7, HeLa, PC9, and SW480 cell lines. Compound 35 is a structurally unique cyclic heptapeptide with a pyrrolidine-2,5-dione-bearing backbone. The absolute configurations of amino acid residues were determined by the advanced Marfey's method by derivatizing its acid hydrolysis products with L-FDLA, followed by analysis with UPLCESI-QTOF MS. Figure 10 shows the chromatograms obtained after appropriate selective ion monitoring channels of the amino acid standards (green peaks) and Marfey's derivatives (red peaks) for comparison of the retention times [163]. Theonellamide J (32), 5-cis-Apoa-theopalauamide (33), and theonellamide K (34) (Figure 9) were isolated from a red sea sponge, Theonella swinhoei, which was collected in the southern part of the Gulf of Aqaba. Compounds 33 and 34 exhibited a growth inhibitory activity against HCT-116 cell line, with an IC50 of 21.8 and 3.5 µ M, respectively. The positive control, cytochalasin D, showed an IC50 value of 0.8 µ M. This finding suggested that the 3-amino-4-hydroxy-6-methyl-8-phenyl-5E,7E-octadienoic acid (Apoa) subunit is relevant for the activity. The absolute configurations of the amino acids were established by Marfey's and advanced Marfey's methods. The samples were first analyzed by Marfey's method, which allowed the configurations of four amino acids to be elucidated, followed by LC-MS (advanced Marfey's method) for the assignment of the stereochemistry of the remaining amino acids [162].
Fuscasins A-D (35-38) (Figure 8) are new cyclic heptapeptides isolated from the marine sponge, Phakellia fusca, which was collected from the South China Sea. Compound 35 exhibited a remarkable cytotoxicity against HepG2 cells, with an IC50 value of 4.6 μM, while 36-38 were inactive even at a concentration up to 20 μM. Cisplatin was used as the positive control with an IC50 value of 4.2 μM. Interestingly, 35 did not exhibit cytotoxicity against the nonmalignant rat cardiomyoblast H9C2 cell line even at concentrations up to 100 μM, suggesting that this compound may have a selective growth inhibitory effect against HepG2 cells. Moreover, none of the compounds displayed cytotoxicity against NCI-H460, MCF-7, HeLa, PC9, and SW480 cell lines. Compound 35 is a structurally unique cyclic heptapeptide with a pyrrolidine-2,5-dione-bearing backbone. The absolute configurations of amino acid residues were determined by the advanced Marfey's method by derivatizing its acid hydrolysis products with L-FDLA, followed by analysis with UPLCESI-QTOF MS. Figure 10 shows the chromatograms obtained after appropriate selective ion monitoring channels of the amino acid standards (green peaks) and Marfey's derivatives (red peaks) for comparison of the retention times [163].  Marfey's method was also used to determine the absolute configurations of the amino acid residues of a new cyclic heptapeptide, ectyoplasin (39), which was isolated from the marine sponge, Ectyoplasia ferox, collected on Tuxpan reef, Veracruz, Mexico. Compound 39 possesses significant cytotoxic activity against DU-145, Jurkat, MM144, HeLa, and CADO-ES1 cell lines, with IC 50 values ranging from 2.9 to 23.5 µM, being more remarkable against DU-145 cell line with an IC 50 value of 2.91 µM (the positive control, doxorubicin, showed IC 50 values of 10 −7 -10 −8 M). Moreover, it was shown that 39 induced apoptosis in DU-145 cells [164].
Three new kynurenine-bearing cyclic heptapeptides, phakefustatins A-C (40-42) (Figure 9), were isolated from the marine sponge, Phakellia fusca, which was collected off Yongxing Island in the South China Sea at a depth of 25 m, by using a neutral-loss scanning method based on LC-MS/MS. The structures of 40-42 were elucidated by HRESIMS and extensive analysis of their 1D and 2D NMR spectra, and the absolute configurations of their amino acid residues were determined by the advanced Marfey's method. Compound 40 was identified as a retinoic X receptor-α (RXRα) modulator, which is an important nuclear receptor that could control various biological processes through inhibition of the PI3K/Akt pathway [200,201]. These findings suggested that 40 might inhibit cancer cell growth and induce apoptosis through the RXRα-mediated PI3K/Akt signaling pathway, and its pharmacophores could be kynurenine and guanidine groups [165].
A new cyclic lipopeptide, aciculitin D (43), was isolated from the marine sponge, Poecillastra sp., which was collected by dredging at a depth of 245 m near the seamount Gonsone, Japan. The structure of 43 was elucidated by HRESIMS and 1D and 2D NMR spectral analysis as well as chemical degradation. The absolute configurations of the amino acid residues were elucidated by Marfey's method, while the absolute configurations of the sugar (lyxose) and the fatty acid (2,3-dihydroxytetradeca-4,6-dienoicacid) were established by chemical degradation and chiral LC/MS analysis. In the case of 2,3-dihydroxytetradeca-4,6-dienoicacid (Dhtda) unit, a polysaccharide-based CSP was used, allowing the conclusion that the absolute configurations of C-2 and C-3 were 2S,3R [166].
Bacicyclin (44) (Figure 11), a new cyclic hexapeptide, was isolated from a culture broth of Bacillus sp. strain BC028, which is associated with Mytilus edulis, and this was collected from the Kiel Fjord (Baltic Sea, Germany). The planar structure of 44 was elucidated by an extensive analysis of 1D and 2D NMR and HRESIMS spectra. Marfey's method was used to determine the absolute configurations of the amino acid constituents and revealed that, in all cases, the amino acids of the building block have the L-configuration, except for alanine and phenylalanine, which had a D-configuration. Compound 44 showed antibacterial activity against the clinically relevant strains Enterococcus faecalis JH212 and S. aureus NTCT8325, with minimal inhibitory concentration (MIC) values of 8 and 12 µM, respectively. Streptomycin was used as a positive control and exhibited an MIC value of 5.24 mM against E. faecalis and 2.09 mM against S. aureus, respectively [167]. Bacicyclin (44) (Figure 11), a new cyclic hexapeptide, was isolated from a culture broth of Bacillus sp. strain BC028, which is associated with Mytilus edulis, and this was collected from the Kiel Fjord (Baltic Sea, Germany). The planar structure of 44 was elucidated by an extensive analysis of 1D and 2D NMR and HRESIMS spectra. Marfey's method was used to determine the absolute configurations of the amino acid constituents and revealed that, in all cases, the amino acids of the building block have the Lconfiguration, except for alanine and phenylalanine, which had a D-configuration. Compound 44 showed antibacterial activity against the clinically relevant strains Enterococcus faecalis JH212 and S. aureus NTCT8325, with minimal inhibitory concentration (MIC) values of 8 and 12 µ M, respectively. Streptomycin was used as a positive control and exhibited an MIC value of 5.24 mM against E. faecalis and 2.09 mM against S. aureus, respectively [167].
A nonribosomal lipodecapeptide, taeanamide A (45), was isolated from the culture of Streptomyces sp. AMD43, which was obtained from an intertidal-mudflat sample collected in Anmyeondo, Republic of Korea. The planar structure of 45 was elucidated by an analysis of HRESIMS and 1D and 2D spectra. The absolute configurations of the amino acid residues were determined by Marfey's method, except for two serine residues whose stereochemistry was determined by a bioinformatic analysis of the biosynthetic gene cluster of the taeanamides. Compound 45 showed antitubercular activity against Mycobacterium tuberculosis mc 2 6230, with a concentration at which 50% of the strains were inhibited (MIC50) of 27 µM. Bedaquiline was used as the positive control with an MIC50 = 0.4 µM [168].
Ogipeptins A-D (46-49) (Figure 11), isolated from the culture broth of the marine bacterium, Pseudoalteromonas sp. SANK 71,903, which was collected from Ogi-machi, Sadoshi, Niigata Pref., Japan, exhibited antimicrobial activity against Gram-negative bacteria. Compounds 46-49 blocked lipopolysaccharide (LPS) binding to the cluster of differentiation 14 (CD14), with IC50 values of 4.8, 6.0, 4.1, and 5.6 nM, respectively [169,205]. LPS is a component of the cell membrane of Gram-negative bacteria and is known to induce a strong immune response [169]. LPS is well known as a bacterial endotoxin, and it is a key component for triggering sepsis or septic shock [205]. The A nonribosomal lipodecapeptide, taeanamide A (45), was isolated from the culture of Streptomyces sp. AMD43, which was obtained from an intertidal-mudflat sample collected in Anmyeondo, Republic of Korea. The planar structure of 45 was elucidated by an analysis of HRESIMS and 1D and 2D spectra. The absolute configurations of the amino acid residues were determined by Marfey's method, except for two serine residues whose stereochemistry was determined by a bioinformatic analysis of the biosynthetic gene cluster of the taeanamides. Compound 45 showed antitubercular activity against Mycobacterium tuberculosis mc 2 6230, with a concentration at which 50% of the strains were inhibited (MIC 50 ) of 27 µM. Bedaquiline was used as the positive control with an MIC 50 = 0.4 µM [168].
Ogipeptins A-D (46-49) (Figure 11), isolated from the culture broth of the marine bacterium, Pseudoalteromonas sp. SANK 71,903, which was collected from Ogi-machi, Sado-shi, Niigata Pref., Japan, exhibited antimicrobial activity against Gram-negative bacteria. Compounds 46-49 blocked lipopolysaccharide (LPS) binding to the cluster of differentiation 14 (CD14), with IC 50 values of 4.8, 6.0, 4.1, and 5.6 nM, respectively [169,205]. LPS is a component of the cell membrane of Gram-negative bacteria and is known to induce a strong immune response [169]. LPS is well known as a bacterial endotoxin, and it is a key component for triggering sepsis or septic shock [205]. The absolute configurations of the amino acid building blocks of 46-49 were elucidated by the advanced Marfey's method. This method also allowed the presence of (2S,3S) and (2S,3R) β-hydroxy-α,γ-diaminobutyric acid isomers in a ratio of 2:1 to be proved [101]. This finding was very important because chiral natural product molecules are generally assumed to be biosynthesized in an enantiomerically pure form [206].
Trikoramides B (13) and D (15) (Figure 7), isolated from the marine cyanobacterium, Symploca hydnoides, were also evaluated for their quorum-sensing inhibitory activity using the Pseudomonas aeruginosa PAO1 lasB-gfp and rhlA-gfp bioreporter strains. Compound 13 not only exhibited moderate quorum-sensing inhibitory activity, but also did not show a dose-dependent response. On the contrary, 15 exhibited moderate to significant dose-dependent quorum-sensing inhibitory effects against PAO1 lasB-gpf and rhlA-gfp bioreporter strains, with IC 50 values of 19.6 µM and 7.3 µM, respectively. The authors hypothesized that the higher potency of 15 could be due to the brominated indole ring at the hydroxylated prenyl-tryptophan residue [155].
Three new cyclic lipopeptides, maribasins C-E (50-52) (Figure 11), were obtained from Aspergillus sp. SCSIO 41501, which was isolated from the Sea gorgonian, Melitodes squamata Nutting (Melithaidae), collected from the South China Sea, Sanya, Hainan Province. The absolute configurations of the amino acid residues were determined by Marfey's method. Compounds 50-52 showed significant antifungal activity against five phytopathogenic fungal strains, Alternaria solani, Curvularia australiensis, Colletotrichum gloeosporioiles, Fusarium oxysporum, and Pyricularia oryzae, with MIC values of 3.12-50 µg/disc. Actidione (50 µg/disc) was used as a positive control. Comparison of the structures of the tested lipopeptides revealed that the difference in the β-amino fatty acid side chain may considerably influence the potency of the antifungal activity of this type of cyclic lipopeptides [170].
Simplicilliumtides N (53) and O (54) (Figure 11) were obtained from the deep-seaderived fungal strain Simplicillium obclavatum EIODSF 020, which was isolated from a marine sediment sample collected in the East Indian Ocean. The planar structures of both compounds were established based on an analysis of HRESIMS and 1D and 2D NMR spectra. The absolute configurations of the amino acid constituents were determined by Marfey's method. Compounds 53 and 54 showed significant antifungal activity against two phytopathogenic fungi, Alternaria solani (MIC values of 6.250 and 1. 562 µg/disc, respectively) and Colletotricum asianum (MIC values of 3.125 and 0.195 µg/disc, respectively). The positive control, ketoconazole, displayed MIC values of 6.250 and 12.5 µg/disc, against A. solani and C. asianum, respectively, while amphotericin B showed MIC values of 0.195 and 25 µg/disc against A. solani and C. asianum, respectively [171].
Motobamide (59), a new cyclopeptide containing a C-prenylated cyclotryptophan residue (Figure 11), was isolated from a marine cyanobacterium, Leptolyngbya sp., which was collected at Bise, Okinawa Island, Okinawa Prefecture, Japan. The planar structure of the compound was established based on an extensive analysis of 1D and 2D NMR and HRESIMS spectra. The absolute configurations of all normal amino acids were determined by a chiral LC analysis (using a ligand exchange-type CSP) of the hydrolysate after acid hydrolysis. For a prenyl-tryptophan residue, the combination of NOESY correlations and comparison of the calculated and experimental ECD spectra were applied to determine the absolute configurations of its stereogenic carbons. Motobamide (59) was shown to inhibit the growth of bloodstream forms of Trypanosoma brucei rhodesiense strains IL-1501 (IC 50 of 2.3 µM), a causative agent of human African sleeping sickness [173].

Other Activities
Among the 91 new marine-derived cyclopeptides isolated from various marine sources, 15 (60-74) ( Figure 12) were tested for other bioactivities, some of which showed promising results.
shown to inhibit the growth of bloodstream forms of Trypanosoma brucei rhodesiense strains IL-1501 (IC50 of 2.3 μM), a causative agent of human African sleeping sickness [173].
Three new cyclic hexapeptides, petrosamides A-C (62-64) (Figure 12), were obtained from Aspergillus sp. 151304, which was isolated from the inner tissue of a marine sponge, Petrosia sp., collected from Yongxing Island, China. The planar structures of the compounds were elucidated by HRESIMS, and 1D and 2D spectral analysis. The configurations of the amino acid constituents were determined by the advanced Marfey's method. Compounds 62-64 inhibited pancreatic lipase-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis in a dose-dependent manner, with IC 50 values of 7.6, 1.8, and 0.5 µM, respectively. The positive controls, isoginkgetin and orlistat, showed IC 50 values of 2.90 µM and 0.75 nM, respectively. Further inhibition kinetics analyses showed that 64 inhibited pancreatic lipases in a non-competitive manner, while molecular dynamics simulation revealed that it could bind to pancreatic lipase at the entrance of the catalytic pocket [175].
A new proline-rich cyclopeptide containing prenylated tryptophan, croissamide (65) (Figure 12), was isolated from a marine cyanobacterium, Symploca sp., which was collected at Minna Island, Okinawa, Japan. The structures and sequence of amino acid residues were elucidated by interpretation of HRESIMS and 1D and 2D NMR spectra. The configurations of the amino acid residues were established by a chiral LC analysis, using a ligand exchangebased CSP column, of the hydrolysate resulting from the acid hydrolysis of 65. Compound 65 showed weak inhibitory activity against NO production in LPS-stimulated RAW 264.3 cells, with an inhibition of 41.5% at 30 mM [176].
Cystargamides C (66) and D (67) (Figure 12), new lipodepsipeptides bearing six amino acids with an epoxy fatty acid side chain, were isolated from a marine actinomycete strain Streptomyces sp. JMS132, isolated from tidal flat sediment samples which were collected at Beolgyo, South Korea. The planar structures of the compounds were established by an extensive analysis of 1D and 2D NMR and HRESIMS spectra. The absolute structures of 66 and 67 were determined by a comparison of their circular dichroism curves with that of the previously reported cystargamide B, whose absolute configurations of its amino acid residues were determined by advanced Marfey's methods. The absolute configurations of the stereogenic carbons of the epoxide ring of the fatty acid side chains in 66 and 67 were determined by ROESY correlations and the value of coupling constant of the vicinal protons. Compounds 66 and 67 were evaluated for their antioxidant properties through their 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6sulfonic acid) (ABTS) radical scavenging effects. Vitamin C was used as a positive control. Compounds 66 and 67 showed significant scavenging activity on DPPH free radicals in a dose-dependent manner, with 66 able to decrease DPPH free radicals by ca. 53% at a concentration of 200 mg/mL. Compound 67, at a concentration of 200 mg/mL, decreased ABTS free radicals by 100%, which is comparable to vitamin C [177].
By combining LC-MS/MS-dependent molecular networking and 1 H NMR techniques, it was possible to identify seven new cyclohexadepsipeptides, chrysogeamides A-G (68-74) (Figure 12), from a coral-derived fungus, Penicillium chrysogenum CHNSCLM-0003. Compounds 68-74 exhibited angiogenesis towards zebrafish embryo at 1.0 µg/mL, with 68 and 69 promoting the cavity of blood vessels without toxicity in embryonic zebrafish at 100 µg/mL. Chrysogeamide D (71) comprises a rare 3-hydroxy-4-methylhexanoic acid moiety, which was first discovered to derive from marine-derived organisms. The absolute configurations of the amino acid residues were established by an HPLC-DAD and UPLC-MS analysis of the acid hydrolysate derivatized with Marfey's reagent. Interestingly, isotope-labeling feeding experiments indicated that D-Leu could be isomerized from L-Leu [178].
A marine-derived fungus, Sesquicillium microsporum RKAG 186, obtained from a marine sediment collected at low tide from the intertidal zone of Frobisher Bay, Nunavut, Canada, furnished four new cyclic decapeptides, auyuittuqamides A-D (75)(76)(77)(78). The planar structure and the sequence of amino acids were established by HRESIMS and 1D and 2D NMR analysis. Marfey's method was used to determine the absolute configurations of the amino acid units, except for the D-allo-threonine residue due to a lack of availability of a commercial standard of N-Me-D-allo-threonine. The auyuittuqamides represent a unique class of the decapeptide family due to the presence of multiple N-methylated amino acids. Compounds 75-78 showed neither cytotoxic activity against MCF-7, HTB-26, and a human epithelial keratinocyte cell line (IC 50 values higher than 10 µM), nor antimicrobial activity at concentrations up to 128 µM [179].
Haloirciniamide A (79) (Figure 13), a dibromopyrrole cyclopeptide containing a chlorohistidine ring, was isolated from a marine sponge, Ircinia sp., which was collected in the Thousand Islands of Indonesia. The planar structure of the compound and its amino acid sequence were elucidated by an extensive 1D and 2D NMR spectral analysis as well as by (+)-HRESI-TOFMS and QTOFMS. The absolute configurations of its amino acid residues were determined by Marfey's methods, except for N-methyl-chlorohistidine due to the absence of the standard amino acid. Compound 79 neither showed cytotoxicity against four human tumor cell lines, A-549, HT-29, MDA-MB-231, and PSN-1 (concentration that was able to cause 50% cell growth inhibition (GI 50 ) > 1.2 × 1p −5 M), nor inhibited the enzyme topoisomerase I (at a concentration of 1.0 × 10 −5 M). Moreover, 79 was unable to impair the interaction between the programmed cell death protein PD-1 and its natural ligand PDL1 [180].
Unguisin G (80) (Figure 13) was obtained from the culture extract of a marine-derived fungus, Aspergillus candidus NF2412, which was isolated from sponge collected from the East China Sea at Ningbo, Zhejiang, Province, China. The planar structure of 80, established by comprehensive analyses of HRESIMS and 1D and 2D NMR spectra, revealed the presence of non-proteinogenic amino acid residues, kynurenine and γ-aminobutyric acid. The advanced Marfey's method was used to determine the absolute configurations of the amino acids of the hydrolysate obtained by acid hydrolysis of 80, revealing not only the presence of D-alanine, D-valine, and L-phenylalanine, but also a pair of signals for L-and D-kynurenine. As shown in Figure 14, the LC-MS analysis of D/L-FDAA derivatives of acid hydrolysates of 80 with those of authentic samples showed a pair of ion signals for L-and D-kynurenine in a 1:1 ratio, indicating that this residue in 80 possesses two configurations. Even though 80 was isolated as a pair of diastereomers, attempts to separate them failed. marine-derived organisms. The absolute configurations of the amino acid residues were established by an HPLC-DAD and UPLC-MS analysis of the acid hydrolysate derivatized with Marfey's reagent. Interestingly, isotope-labeling feeding experiments indicated that D-Leu could be isomerized from L-Leu [178].

No Demonstrated Biological Activity
Among the 91 new marine-derived cyclopeptides reported herein, 17 (75-91) ( Figure  13) were devoid of any activities in the assays performed. Consequently, these compounds should be evaluated for their potential biological effects in other available assays.
A marine-derived fungus, Sesquicillium microsporum RKAG 186, obtained from a marine sediment collected at low tide from the intertidal zone of Frobisher Bay, Nunavut, Canada, furnished four new cyclic decapeptides, auyuittuqamides A-D (75)(76)(77)(78). The planar structure and the sequence of amino acids were established by HRESIMS and 1D and 2D NMR analysis. Marfey's method was used to determine the absolute configurations of the amino acid units, except for the D-allo-threonine residue due to a lack of availability of a commercial standard of N-Me-D-allo-threonine. The auyuittuqamides represent a unique class of the decapeptide family due to the presence of multiple Nmethylated amino acids. Compounds 75-78 showed neither cytotoxic activity against MCF-7, HTB-26, and a human epithelial keratinocyte cell line (IC50 values higher than 10 μM), nor antimicrobial activity at concentrations up to 128 μM [179].
Haloirciniamide A (79) (Figure 13), a dibromopyrrole cyclopeptide containing a chlorohistidine ring, was isolated from a marine sponge, Ircinia sp., which was collected in the Thousand Islands of Indonesia. The planar structure of the compound and its amino acid sequence were elucidated by an extensive 1D and 2D NMR spectral analysis as well as by (+)-HRESI-TOFMS and QTOFMS. The absolute configurations of its amino acid residues were determined by Marfey's methods, except for N-methyl-chlorohistidine due to the absence of the standard amino acid. Compound 79 neither showed cytotoxicity against four human tumor cell lines, A-549, HT-29, MDA-MB-231, and PSN-1 (concentration that was able to cause 50% cell growth inhibition (GI50) > 1.2 × 1p −5 M), nor inhibited the enzyme topoisomerase I (at a concentration of 1.0 × 10 −5 M). Moreover, 79 was unable to impair the interaction between the programmed cell death protein PD-1 and its natural ligand PDL1 [180].   aminobutyric acid. The advanced Marfey's method was used to determine the absolute configurations of the amino acids of the hydrolysate obtained by acid hydrolysis of 80, revealing not only the presence of D-alanine, D-valine, and L-phenylalanine, but also a pair of signals for L-and D-kynurenine. As shown in Figure 14, the LC-MS analysis of D/L-FDAA derivatives of acid hydrolysates of 80 with those of authentic samples showed a pair of ion signals for L-and D-kynurenine in a 1:1 ratio, indicating that this residue in 80 possesses two configurations. Even though 80 was isolated as a pair of diastereomers, attempts to separate them failed.  Compound 80 was evaluated for its antibacterial activity, by the agar diffusion method, against a series of pathogens, including Xanthomonas oryzae pv. oryzicola Swings, Acidovorax avenae subsp. citrulli, Pseudomonas syringae pv. Lachrymans, Bacillus subtilis CICC 10283, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CICC 10351, Micrococcus luteus CMCC(B) 28,001, Candida albicans and S. aureus CMCC(B) 26003; however, it showed no activity against all the tested strains even at a concentration as high as 125 µg/disk [181].
By using MS/MS-based molecular networking guidance, four cyclic heptapeptides, named asperheptatides A-D (81-84), were isolated from a culture extract of Aspergillus versicolor strain CHNSCLM-0063, which was isolated from the gorgonian coral, Rumphella aggregata, collected from the Nansha Islands in the South China Sea. The planar structures and amino acid sequences of 81 and 82, which were major compounds, were elucidated by an interpretation of 1D and 2D NMR and HRESIMS spectra. The absolute configurations of the amino acid residues of 81 and 82 were established by an LC analysis of their acid hydrolysates after derivatization with Marfey's reagent. On the other hand, the planar structures of 83 and 84, which were isolated as trace compounds, were characterized only by ESI-MS/MS fragmentation experiments. Compounds 81 and 82 were tested for their antitubercular activity against M. tuberculosis H37Ra; however, none were active (MIC values ≥ 100 µM). Rifampin and isoniazid were used as positive controls, showing MIC values of 6.25 and 12.5 nM, respectively [182].
By using MS/MS-based molecular networking guidance, pagoamide A (85) (Figure 13), a thiazole-containing cyclodepsipeptide comprising 11 amino acids, was isolated from laboratory cultures of a marine green alga, Derbesia sp., which were collected from a water depth of 12 to 18 m in Fagatele Bay, America Samoa. The planar structure of the compound and the amino acid sequence were established by 1D and 2D NMR as well as HRESIMS spectral analysis. The absolute configurations of the amino acid constituents of 85 were resolved by the advanced Marfey's method, chiral LC, and ROESY correlations. The configuration of the N,N-dimethyl valine residue was determined as L by chiral LC using a ligand exchange-type CSP [(D)-penicillamine stationary phase] by comparison of the hydrolyzed product with synthetic D/L-N,N-dimethyl valine standards. Interestingly, 85 has two serine and two phenylalanine residues, and each comprises one L-and one D-residues. Density functional theory (DFT) calculations in the Gaussian program to calculate the lowest energy conformers in combination with ROESY correlations allowed the positions of D-and L-serine, and D-and L-phenylalanine residues to be assigned. The cytotoxicity of 85 against H-460 cancer cell line was evaluated; however, it was inactive at the maximum concentration tested (30 µM) [183].
By a comparative global natural product social molecular networking analysis of ×63 co-isolated fungi, new scopularides were isolated and identified: scopularides C-G (86-90) from Beauveria sp. CMB-F585, and scopularide H (91) from Scopulariopsis sp. CMB-F115. The chemical structures and absolute configurations of the amino acid residues were assigned by spectroscopic and C3 Marfey's analysis, together with X-ray analyses of 86 and 91, and biosynthetic considerations. Compounds 86-91 did not display significant growth inhibitory activity against a selection of Gram-positive and Gram-negative bacteria (E. coli ATCC 11775, S. aureus ATCC 9144, and three clinical isolates of methicillin-susceptible S. aureus, methicillin-resistant S. aureus, and vancomycin-resistant E. faecalis), a fungus (C. albicans ATCC 10231), and a panel of three human carcinoma cell lines (adherent SW620, NCI-H460, and HepG2) [184].

Final Remarks
Regarding the stereochemical determination of cyclopeptides amino acid residues by LC methods, Marfey's analysis was demonstrated to be the most widely used method (Figure 15), allowing the determination of the absolute configurations of 89 cyclopeptides, seven of which were associated with chiral LC. Intriguingly, there were only two marine cyclopeptides whose absolute configurations of their amino acid residues were determined only by chiral LC. As expected, the most used CDRs were FDAA, accounting for 68% of the reported analyses, while FDLA accounted for only 31% of the reported analyses. Considering the chiral LC method, the ligand exchange-type CSPs were used in most cases. There are a few reasons that may justify a massive application of Marfey's method. First, it is a simple method which offers a better resolution when compared with a chiral LC analysis. Moreover, several derivatizing agents, such as FDAA and FDLA, are commercially available.  It is important to highlight that from January 2018 to November 2022, there were several studies on the structural revision of marine-derived cyclopeptides discovered before 2018, reporting the reinvestigation of the stereochemistry by LC and other methods, for the establishment of complete absolute configurations [138,207,208]. The focus of this review is on marine cyclopeptides, even though LC methods were chosen for It is important to highlight that from January 2018 to November 2022, there were several studies on the structural revision of marine-derived cyclopeptides discovered before 2018, reporting the reinvestigation of the stereochemistry by LC and other methods, for the establishment of complete absolute configurations [138,207,208]. The focus of this review is on marine cyclopeptides, even though LC methods were chosen for the determination of the absolute configurations of the amino acid residues of the recently isolated linear peptides [209][210][211]. Surprisingly, there are still a number of reports describing the identification, structure characterization, and biological activity evaluation of novel marine cyclopeptides without stereochemistry assignments [212], or referring to only the relative configurations [213,214].
As shown in Figure 15, interesting bioactivities have been described for the marinederived cyclopeptides, mainly anticancer (47%) and antimicrobial (18%). Nevertheless, other activities were found to account for 16% of the reported marine-derived cyclopeptides.
Great structural diversity was found, including cyclopeptides, cyclodepsipeptides, and cyclolipopeptides, with both L-and D-configured amino acid residues. Nevertheless, for some peptides, such as tutuilamides A-C (19)(20)(21), petrosamide A (62), and croissamide (65), all the amino acids were in the L-configuration. In addition, some interesting stereochemical features were observed. For example, pagoamide A (85) and unguisin G (80) contain amino acids that appear twice in the molecule, but with opposite configuration.

Conclusions
Ninety-one cyclopeptides have been isolated from marine sources over the last five years (January 2018-November 2022), and Marfey's analysis demonstrated to be the most widely used method for the stereochemical determination of their amino acid residues. This review also describes several promising marine-derived cyclopeptides, some of which possess unique structural features, and with relevant biological activities, resulting from diverse marine sources. Despite being a promising area, the application of marine bioactive cyclopeptides is still lacking. Therefore, further research in bioactive cyclopeptides application is necessary to better understand their potential and applicability.