Marine Natural Peptides: Determination of Absolute Configuration Using Liquid Chromatography Methods and Evaluation of Bioactivities

Over the last decades, many naturally occurring peptides have attracted the attention of medicinal chemists due to their promising applicability as pharmaceuticals or as models for drugs used in therapeutics. Marine peptides are chiral molecules comprising different amino acid residues. Therefore, it is essential to establish the configuration of the stereogenic carbon of their amino acid constituents for a total characterization and further synthesis to obtain higher amount of the bioactive marine peptides or as a basis for structural modifications for more potent derivatives. Moreover, it is also a crucial issue taking into account the mechanisms of molecular recognition and the influence of molecular three-dimensionality in this process. In this review, a literature survey covering the report on the determination of absolute configuration of the amino acid residues of diverse marine peptides by chromatographic methodologies is presented. A brief summary of their biological activities was also included emphasizing to the most promising marine peptides. A case study describing an experience of our group was also included.


Introduction
In recent years, it has become well known that the oceans represent a rich source of structurally unique bioactive compounds from the perspective of potential therapeutic agents [1,2]. Bioactive compounds can be isolated from a myriad of marine invertebrates such as mollusks, sponges, tunicates and bryozoans, in addition to algae and marine microorganisms, especially cyanobacteria, bacteria and fungi [3][4][5].
Over the last decades, novel bioactive compounds from marine organisms with important bioactivities, such as antifungal, antibacterial, cytotoxic and anti-inflammatory properties, have been widely explored, and many of them are considered as lead compounds for drug discovery as well as biologically useful agents in pharmaceutical research [6][7][8][9][10]. In fact, owing to their pharmacological A number of reviews on marine peptides have appeared in recent years, focusing mainly on their biological activities, applications and biosynthesis as well as isolation procedures [16,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. In this review, several works related to the methods used for determination of the absolute configuration of marine peptides by chromatographic methods are presented in different sections according to the source of the marine peptides. Diverse types of peptides such as cyclic peptides, cyclic depsipeptides and lipopeptides are reported. A literature survey covering all the reports on liquid chromatographic methods (Marfey's method and chiral HPLC) is presented (from 1996 to 2017). Furthermore, a case study describing an experience of our group is included.

Peptides from Marine Cyanobacteria and Other Bacteria
Cyanobacteria (blue-green algae), the most ancient known microorganisms on Earth, are a rich source of novel secondary metabolites possessing a broad spectrum of biological activities including antitumor, antibacterial, anticoagulant, antifungal, antiviral, antimalarial, antiprotozoal, and antiinflammatory activities [58]. Currently, cyanobacteria are one of the most interesting sources of novel marine compounds [59]. Actually, the number of biologically active cyclic peptides, depsipeptides, lipopeptides, and other acyclic or small peptides, many of which containing unusual amino acid residues or modified amino acid units, is impressive. In addition to cyanobacteria, this type of compounds has also been isolated from other marine-derived bacteria. A number of reviews on marine peptides have appeared in recent years, focusing mainly on their biological activities, applications and biosynthesis as well as isolation procedures [16,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. In this review, several works related to the methods used for determination of the absolute configuration of marine peptides by chromatographic methods are presented in different sections according to the source of the marine peptides. Diverse types of peptides such as cyclic peptides, cyclic depsipeptides and lipopeptides are reported. A literature survey covering all the reports on liquid chromatographic methods (Marfey's method and chiral HPLC) is presented (from 1996 to 2017). Furthermore, a case study describing an experience of our group is included.

Peptides from Marine Cyanobacteria and Other Bacteria
Cyanobacteria (blue-green algae), the most ancient known microorganisms on Earth, are a rich source of novel secondary metabolites possessing a broad spectrum of biological activities including antitumor, antibacterial, anticoagulant, antifungal, antiviral, antimalarial, antiprotozoal, and anti-inflammatory activities [58]. Currently, cyanobacteria are one of the most interesting sources of novel marine compounds [59]. Actually, the number of biologically active cyclic peptides, depsipeptides, lipopeptides, and other acyclic or small peptides, many of which containing unusual amino acid residues or modified amino acid units, is impressive. In addition to cyanobacteria, this type of compounds has also been isolated from other marine-derived bacteria.

Cyclic Peptides
Scattered publications concerning the stereochemistry determination of the amino acid residues of several cyclic peptides, isolated from marine cyanobacteria and other bacteria, were reported (Table 1). Marfey's method, using FDAA as derivatization reagent, allowed the successful determination of the absolute configuration of the amino acid residues of cyclic peptides 1-4 ( Figure 2).
For the new cyclic tetrapeptide 1 isolated from the bacterium Nocardiopsis sp. [60], the absolute configuration of all the amino acid residues was found to be L. Similarly, the absolute configuration of the amino acid residues of three novel anabaenopeptins labeled NZ825 (2), NZ841 (3), and NZ857 (4) [61], were successfully determined by Marfey's method combined with HPLC.
However, as Marfey's method was not accurate enough to determine the absolute configuration of all the amino acid residues of some cyclic peptides 5-16 (Figure 2), it was necessary to associate this method with chiral HPLC.

Cyclic Peptides
Scattered publications concerning the stereochemistry determination of the amino acid residues of several cyclic peptides, isolated from marine cyanobacteria and other bacteria, were reported (Table 1). Marfey's method, using FDAA as derivatization reagent, allowed the successful determination of the absolute configuration of the amino acid residues of cyclic peptides 1-4 ( Figure 2).
For the new cyclic tetrapeptide 1 isolated from the bacterium Nocardiopsis sp. [60], the absolute configuration of all the amino acid residues was found to be L. Similarly, the absolute configuration of the amino acid residues of three novel anabaenopeptins labeled NZ825 (2), NZ841 (3), and NZ857 (4) [61], were successfully determined by Marfey's method combined with HPLC.
However, as Marfey's method was not accurate enough to determine the absolute configuration of all the amino acid residues of some cyclic peptides 5-16 (Figure 2), it was necessary to associate this method with chiral HPLC.     Tetrapeptide (

Cyclic Depsipeptides
As mentioned above, there are many publications describing the isolation and characterization, including the determination of the stereochemistry of their amino acids, of new cyclic depsipeptides from marine cyanobacteria and other bacteria (Table 2). However, contrary to cyclic peptides, several works reported the use of chiral HPLC as the only method for determination of the configuration of amino acids. Figure 4 shows the structure of cyclic depsipeptides 18-46, isolated from marine cyanobacteria and other bacteria, whose stereochemistry of the amino acids was determined only by this method.
Zhou et al. [79] described the determination of the absolute configuration of new anti-infective cycloheptadepsipeptides marformycins A-F (35)(36)(37)(38)(39)(40), produced by the deep sea-derived Streptomyces drozdowiczii SCSIO 1014, using a ligand exchange type CSP containing the same chiral selector as the previous ones (N,N-dioctyl-L(or D)-alanine) but purchased from Mitsubishi Chemical Corporation (MCI GEL CRS10W). Another type of CSP, specifically the macrocyclic antibiotic-based Chirobiotic TAG, confirmed the presence of L-Pro and L-Val in an unusual cyclic depsipeptide, pitiprolamide (41), isolated from Lyngbya majuscula [80]. Interestingly, in some works, more than one CSP were employed to elucidate the configuration of all the amino acids contained in the hydrolysates of cyclic depsipeptides. For example, two different types of ligand exchange type CSPs were used to elucidate the stereochemistry of the amino acid residues of palau'amide (42), depsipeptide with strong cytotoxicity against KB cell line (IC 50 value of 13 nM) [81].
In the case of pitipeptolides C-F (43)(44)(45)(46), which were isolated from the cyanobacterium Lyngbya majuscula, the configuration of most of the amino acid residues was determined using the macrocyclic antibiotic-based Chirobiotic TAG under reverse phase elution conditions [82]. Then, the N,N-dioctyl-L-alanine ligand exchange CSP Chiralpack MA (+), under the same elution mode, was used for the assignment of S configuration for Hiva residue [82].                  The concurrent applicability of chiral HPLC and Marfey's methods for determination of the absolute configuration of all the amino acid residues of cyclic depsipeptides 47-78 ( Figure 5) was also described in several reports, among which ten described the use of ligand exchange-type CSPs to perform the analysis in association with Marfey's method [71,72,[74][75][76]78,79,81]. Furthermore, the use of macrocyclic antibiotic-based CSPs was reported by Montaser et al. [82].

Lipopeptides
To the best of our knowledge, there are only two reports describing simultaneously the isolation and characterization of lipopeptides from marine cyanobacteria ( Figure 7) as well as the stereochemistry determination of the amino acids present in their hydrolysates (Table 3).   The configuration of N-Me-Hph of the lipopeptide antillatoxin B (95), isolated from the cyanobacterium Lyngbya majuscula, was assigned as L using FDAA as Marfey's derivatization reagent [101]. Compound 95 exhibited significant sodium channel activation (EC50 = 1.77 µM) and ichthyotoxicity (LC50 = 1 µM) [101]. The hydrolysates of lipopeptides lobocyclamides A-C (96-98), isolated from the cyanobacterium Lyngbya confervoides, were analyzed by either direct chiral HPLC, using the D-penicillamine ligand exchange type CSP or by prior derivatization by Marfey's method and reverse phase HPLC [102]. Both compounds displayed modest in vitro antifungal activity against a panel of Candida sp., including two fluconazole-resistant strains. Interestingly, synergistic antifungal activity was also observed [102]. The configuration of N-Me-Hph of the lipopeptide antillatoxin B (95), isolated from the cyanobacterium Lyngbya majuscula, was assigned as L using FDAA as Marfey's derivatization reagent [101]. Compound 95 exhibited significant sodium channel activation (EC 50 = 1.77 µM) and ichthyotoxicity (LC 50 = 1 µM) [101]. The hydrolysates of lipopeptides lobocyclamides A-C (96-98), isolated from the cyanobacterium Lyngbya confervoides, were analyzed by either direct chiral HPLC, using the D-penicillamine ligand exchange type CSP or by prior derivatization by Marfey's method and reverse phase HPLC [102]. Both compounds displayed modest in vitro antifungal activity against a panel of Candida sp., including two fluconazole-resistant strains. Interestingly, synergistic antifungal activity was also observed [102].

Peptides from Marine-Derived Fungi
Marine fungi have been isolated from various marine sources like algae, marine invertebrates, sediment or water, mangroves and sponges. Most of the fungal species isolated from marine sponges are related to the genera Aspergillus and Penicillium [103]. Marine fungi are a rich source of structurally unique and biologically active compounds with a wide range of biological activities, such as antimalarial, anticancer, antifungal, antibacterial, cytotoxicity and among others [104]. More than

Peptides from Marine-Derived Fungi
Marine fungi have been isolated from various marine sources like algae, marine invertebrates, sediment or water, mangroves and sponges. Most of the fungal species isolated from marine sponges are related to the genera Aspergillus and Penicillium [103]. Marine fungi are a rich source of structurally unique and biologically active compounds with a wide range of biological activities, such as antimalarial, anticancer, antifungal, antibacterial, cytotoxicity and among others [104]. More than

Cyclic Peptides
A large number of cyclic peptides have been isolated from marine-derived fungi ( Figure 8) and Table 4 shows the marine fungal cyclic peptides whose stereochemistry of their amino acid residues were determined. To the best of our knowledge, only three reports described the use of FDAA and FDLA as Marfey's derivatization reagents, specifically for analysis of the peptides 99-112.

Cyclic Peptides
A large number of cyclic peptides have been isolated from marine-derived fungi ( Figure 8) and Table 4 shows the marine fungal cyclic peptides whose stereochemistry of their amino acid residues were determined. To the best of our knowledge, only three reports described the use of FDAA and FDLA as Marfey's derivatization reagents, specifically for analysis of the peptides 99-112.
Marine mangrove-derived fungi Phomopsis sp. K38 and Alternaria sp.   Both Marfey's method and chiral HPLC analysis were also used for the analysis of the absolute configuration of the amino acids of asperterrestide A (104), a cyclic peptide isolated from the marine-derived fungus Aspergillus terreus SCSGAF0162 which revealed the presence of D-Ala in its structure [108]. Nevertheless, it was not possible to distinguish between D-Ile and D-allo-Ile. Compound 104 showed promising inhibitory effects to the influenza virus strains A/WSN/33, and A/Hong Kong/8/68 (IC 50 values of 15 and 8.1 µM, respectively) as well as cytotoxicity against U937 and MOLT4 cell lines (IC 50 values of 6.5 and 6.2 µM, respectively) [108].
There are some reports describing the application of different types of CSPs, including crown ethers and macrocyclic antibiotics, for a chiral HPLC as the only method for analysis of the absolute configuration of the amino acids of peptides. Thus, the determination of the stereochemistry of the amino acids in the cyclic peptides sclerotides A (105) and B (106), isolated from the marine-derived fungus Aspergillus sclerotiorum PT06-1 [109], and cordyheptapeptides C-E (107-109), isolated from the marine-derived fungus Acremonium persicinum SCSIO 115 [110], was achieved via chiral HPLC analysis of the hydrolysates using the crown ether-based CSP Crownpak CR (+). Sclerotides A (105) and B (106) were found to comprise L-Thr, L-Ala, D-Phe, and D-Ser [109]. Moreover, the presence of N-Me-D-Gly, and L-Val in cordyheptapeptides C (107) and D (108) and N-Me-L-Gly, N-Me-D-Tyr, and L-allo-Ile in cordyheptapeptide E (109) was confirmed, in addition to the present of other amino acids common to the three cyclic peptides [110]. Sclerotides A (105) and B (106) displayed antifungal activity against Candida albicans, with MIC values of 7.0 and 3.5 µM, respectively. Furthermore, sclerotide B (106) also exhibited cytotoxicity against HL-60 cell line as well as antibacterial activity against Pseudomonas aeruginosa [109] whereas cordyheptapeptides C (107) and E (109) exhibited cytotoxic activity against SF-268 (IC 50 values of 3.7 and 3.2 µM, respectively), MCF-7 (IC 50 values of 3.0 and 2.7 µM, respectively), and NCI-H460 (IC 50 values of 11.6 and 4.5 µM, respectively) tumor cell lines [110]. Recently, the macrocyclic antibiotic-based CSP Chirobiotic T was employed in our group to determine the stereochemistry of amino acid residues of a new cyclic hexapeptide, similanamide (110), isolated from a marine sponge-associated fungus Aspergillus similanensis KUFA 0013 [111] which confirmed the presence of L-Ala, D-Leu, L-Val and D-pipecolic acid as its amino acids constituent. By using a similar approach, the absolute configuration of all the amino acids of two new cyclotetrapeptides, sartoryglabramides A (111) and B (112), isolated from the marine sponge-associated fungus Neosartorya glabra KUFA 0702, were assigned to be L-configuration in both cyclic peptides [112]. Further details are described in the case-study presented below.

Cyclic Depsipeptides
Most of the works describing the stereochemistry determination of amino acid residues of cyclic depsipeptides, isolated from marine fungus (Figure 9), employed Marfey's method coupled with HPLC, using FDAA or FDLA as derivatization reagents ( Table 5).
In the last few years, ultra-high-pressure liquid chromatography (UHPLC) is becoming an essential technique for ultra-fast separations, since it offers many benefits, including high efficiency in short analysis time and low solvent consumption [120,121]. Thus, the absolute configuration of the amino acid residues of oryzamides A-E (123)(124)(125)(126)(127), isolated from the sponge-derived fungus Nigrospora oryzae PF18, was achieved by Marfey's analysis with FDLA, combined with UHPLC [122]. In the last few years, ultra-high-pressure liquid chromatography (UHPLC) is becoming an essential technique for ultra-fast separations, since it offers many benefits, including high efficiency in short analysis time and low solvent consumption [120,121]. Thus, the absolute configuration of the amino acid residues of oryzamides A-E (123)(124)(125)(126)(127), isolated from the sponge-derived fungus Nigrospora oryzae PF18, was achieved by Marfey's analysis with FDLA, combined with UHPLC [122].   Exumolides A (113) and B (114) Fungus of the genus Scytalidium sp. Spicellamides A (128) and B (129), which were isolated from the marine-derived fungus Spicellum roseum, exhibited cytotoxicity against rat neuroblastoma B104 cell line, with an IC 50 value of 6.2 µg/mL for spicellamide B (129) [123]. It is interesting to note that Marfey's method was not suitable for the determination of the configuration of all amino acid residues of these two peptides. Therefore, a chiral HPLC approach was also employed, using a ligand exchange type CSP [123]. Furthermore, the chiral HPLC, using the crown ether-based CSP Crownpak CR (+), was used as the only method for determination of the configuration of the amino acids residues to confirm the presence of L-Tyr, L-Val, D-Leu, and (S)-O-Leu in the cyclic depsipeptides 1962 A (130) and B (131), isolated from the endophytic fungus Kandelia candel [124]. The cyclic depsipeptide 1962 A (130) exhibited growth inhibitory activity against the human breast cancer cell line, MCF-7, with IC 50 of 100 µg/mL [124].

Peptides from Marine Sponges
Marine sponges are an important source of new metabolites from the marine environment [125]. They are considered one of the most prolific sources of novel bioactive compounds, such as terpenoids, alkaloids, macrolides, nucleoside derivatives, polyethers, fatty acids, sterols, peroxides and other numerous organic compounds [17,126]. In addition, cyclic peptides and depsipeptides have also been isolated from marine sponges. Most bioactive compounds from sponges displayed myriad of biological activities including anti-inflammatory, antibiotic, antitumor, antimalarial, antiviral, antifouling, and immuno-or neurosuppressive [127]. However, a significant number of marine natural products isolated from sponges were tested for the anticancer activity, and many of them were successfully undergoing to preclinical and clinical trials [126,128]. More recently, among bioactive compounds discovered from marine sponges, bioactive peptides have aroused attention of many researchers [8,17].
Phakellistatins 15-18 (161-164) were analysed only by chiral HPLC, using the ligand exchange type Chirex 3126 D-penicillamine CSP, being able to identify that all the amino acids presented L-configuration. Furthermore, phakellistatins 15 (161)  The simultaneous application of Marfey's method, using FDAA as derivatization reagent, and chiral HPLC, using a ligand exchange type CSP, afforded the total assignment of the configuration of all the amino acid residues of reniochalistatins A-E (156-160) [141]. Reniochalistatins A-E (156-160), the cyclic peptides isolated from the marine sponge Reniochalina stalagmitis, were found to have all the amino acid residues with L configuration, including L-Asn and L-Trp in reniochalistatins A (156) and E (160) respectively [141]. The octapeptide reniochalistatin E (160) exhibited cytotoxicity towards myeloma RPMI-8226, and gastric MGC-803 cell lines (IC50 values of 4.9 and 9.7 µM, respectively) [141].

Cyclic Depsipeptides
A number of cyclic depsipeptides (Figures 12 and 13), have been reported from marine sponges and Marfey's method using FDAA as the derivatization reagent was the most used for the determination of absolute configuration of the amino acid residues. Table 7 gives some examples of the cyclic depsipeptides, isolated from marine sponges, whose stereochemistry of their amino acid residues was determined by Marfey's method. By application of this method, callipeltins B (165) and C (166), isolated from the marine lithistida sponge Callipelta sp., were found to have in their structure L-Ala, N-Me-L-Ala, L-Leu, L-Thr and D-Arg [143]. For halipeptins A (167) and B (168), isolated from the marine sponge Haliclona sp., the referred method was only able to determine the configuration for L-Ala [144]. Marfey's method was successfully used to determine the absolute configuration of the amino acid constituents of several marine sponge cyclic peptides including phoriospongin A (169) and B (170), isolated from the marine sponges Phoriospongia sp. and Callyspongia bilamellata [145], mirabamides A-D (171-174), isolated from the marine sponge Siliquarias-pongia mirabilis [146], and neamphamides B-D (175-177), isolated from the marine sponge Naemphius huxleyi [147]. Furthermore, the stereochemistry determination of amino acid residues in pipecolidepsins A (178) and B (179), isolated from the marine sponge Homophymia lamellose, confirmed the presence of several L and D amino acid residues, besides the (3S,4R) diMe-L-Glu and (2S,3S)-EtO-Asp present in both peptides [148]. Stellatolide A (180), a cyclic depsipeptide isolated from Ecionemia acervus, was found to have N-Me-D-Ser and D-allo-Thr, among other L-configured amino acids [149]. The amino acid constituents of the cyclic depsipeptides cyclolithistide A (181) and nagahamide A (182), both isolated from the sponge Theonella swinhoei, were all found to have the S or L-configuration, and the 3-amino-5-hydroxybenzoic acid (AHBA) residue in nagahamide A (182) was established to have 3S configuration [150,151].
A simultaneous use of Marfey's method and chiral HPLC analysis for stereochemical analysis of the amino acids of this type of peptides have been reported (Table 7). For examples, the absolute configuration of the amino acids of theopapuamides B (183) and C (184) and celebesides A-C (185-187), isolated from an Indonesian sponge Siliquariaspongia mirabilis, was successful assigned by HPLC-MS analysis of FDAA derivatives as well as via chiral HPLC analysis using a ligand exchange type CSP [152]. In the case of theopapuamide (188), isolated from a papua new Guinea Lithistid Sponge Theonella swinhoei, Marfey's method was used to confirm the presence of D-allo-Thr, whereas chiral HPLC using a ligand exchange type CSP, revealed the presence of N-Me-L-Leu, D-Asp, L-Leu and N-Me-L-Glu in its structure [153]. The absolute configuration of the amino acid residues of a new sulfated cyclic depsipeptide, mutremdamide A (189) and six new highly N-methylated peptides, koshikamides C-H (190)(191)(192)(193)(194)(195), isolated from different deep-water specimens of Theonella swinhoei and Theonella cupola, was also established by using both approaches. However, two different columns (C 12 and C 18 ) were used in Marfey's method. By using chiral HPLC, it was possible to identify the amino acid residue N-Me-allo-L-Ile in koshikamide H (195) [154]. These cyclic peptides showed interesting biological activities. While theopapuamide (188) was cytotoxic against CEM-TART and HCT cell lines (IC 50 values of 0.5 and 0.9 µM, respectively) [153], koshikamides F (193) and H (195) were active against a CCR5-using viral envelope, with IC 50 values of 2.3 and 5.5 µM [154].    Callipeltins B (165) and  Theopapuamide (

Lipopeptides
The absolute configuration of the amino acids of new N-sulfoureidylated lipopeptides sulfolipodiscamides A-C (196)(197)(198), isolated from the n-butanol fraction of the marine sponge Discodermia kiiensis (Figure 14), was determined by Marfey's method to be L-Uda and L-Gly (Table 8).
Compound 196 was found to be cytotoxic against the murine leukemia cell line P388 with a IC 50

Lipopeptides
The absolute configuration of the amino acids of new N-sulfoureidylated lipopeptides sulfolipodiscamides A-C (196)(197)(198), isolated from the n-butanol fraction of the marine sponge Discodermia kiiensis (Figure 14), was determined by Marfey's method to be L-Uda and L-Gly (Table 8).
Compound 196 was found to be cytotoxic against the murine leukemia cell line P388 with a IC50 value of 15 µM [155].

Peptides from Other Marine Invertebrates and Algae
A number of diverse bioactive peptides such as cyclic peptides, cyclic depsipeptides and linear peptides have been isolated from other marine invertebrates including ascidians, commonly called tunicates, mollusks, among others [17]. Moreover, the potential applications of many bioactive compounds from marine algae, mainly red and brown as well as some green algae, were reported [156].

Cyclic Peptides
To the best of our knowledge, only five works described the analysis of the stereochemistry of the cyclic peptides from marine invertebrates and algae ( Figure 15). In all reported works, Marfey's method was employed (Table 9). Among these, the determination of the absolute configuration of the cyclic hexapeptides didmolamides A (199) and B (200) and mollamides B (201) and C (202), isolated from the marine ascidian Didemnum molle from Madagascar and Indonesia, respectively, was performed by Marfey's method using FDAA as derivatization reagent [157,158]. These compounds showed interesting biological activities, particularly, cytotoxicity against A549, HT29 MEL28 tumor cell lines, with IC50 values ranging from 10 to 20 µg/mL for didmolamides A (199) and B (200) [157] while 201 showed antimalarial activity against Plasmodium falciprum, clones D6 and W2, with IC50 values of 2.0 and 21 µg/mL, respectively [158].
Furthermore, the stereochemical determination of antatollamides A (203) and B (204), isolated from the marine ascidian Didemnum molle, sanguinamide A (205), isolated from the sea slug

Peptides from Other Marine Invertebrates and Algae
A number of diverse bioactive peptides such as cyclic peptides, cyclic depsipeptides and linear peptides have been isolated from other marine invertebrates including ascidians, commonly called tunicates, mollusks, among others [17]. Moreover, the potential applications of many bioactive compounds from marine algae, mainly red and brown as well as some green algae, were reported [156].

Cyclic Peptides
To the best of our knowledge, only five works described the analysis of the stereochemistry of the cyclic peptides from marine invertebrates and algae ( Figure 15). In all reported works, Marfey's method was employed (Table 9). Among these, the determination of the absolute configuration of the cyclic hexapeptides didmolamides A (199) and B (200) and mollamides B (201) and C (202), isolated from the marine ascidian Didemnum molle from Madagascar and Indonesia, respectively, was performed by Marfey's method using FDAA as derivatization reagent [157,158]. These compounds showed interesting biological activities, particularly, cytotoxicity against A549, HT29 MEL28 tumor cell lines, with IC 50 values ranging from 10 to 20 µg/mL for didmolamides A (199) and B (200) [157] while 201 showed antimalarial activity against Plasmodium falciprum, clones D6 and W2, with IC 50 values of 2.0 and 21 µg/mL, respectively [158].
Furthermore, the stereochemical determination of antatollamides A (203) and B (204), isolated from the marine ascidian Didemnum molle, sanguinamide A (205), isolated from the sea slug Hexabranchus sanguineus, and gamakamide E (206), isolated from the oysters Crassostrea giga, was carried out by Marfey's method using FDLA as a derivatization reagent. The analysis demonstrated that most of their amino acids have the L-configuration, with the exception of D-Ala and D-Lys in antatollamides A (203) and B (204), and gamakamide E (206), respectively [159][160][161].

Cyclic Depsipeptides
To the best of our knowledge, only four works reported the determination of the stereochemistry of amino acid constituents of the cyclic depsipeptides from marine invertebrates and algae ( Figure 16). Among these, three employed only Marfey's method, specifically for peptides 207-216. However, for peptide 217, Marfey's method was not efficient and, as a consequence, a ligand exchange type CSP was also used for complete determination of the configuration of its amino acids (Table 10).
The determination of the absolute configuration of the amino acids in kahalalides A-F (207-212), isolated from the marine mollusk Elysia rufescens, was performed by using FDLA as the derivatization reagent and the presence of diverse residues of L-and D-Val in these peptides was confirmed [162]. Using FDAA as the Marfey derivatization reagent, the absolute configuration of tamandarins A (213) and B (214), isolated from an unidentified Brazilian marine ascidian of the family Didemnidae [163], and kahalalides P (215) and Q (216), isolated from green algae Bryopsis species [164] were elucidated. In the case of kahalalide O (217), the absolute configuration of its amino acid constituents was determined by Marfey's method and chiral HPLC analysis, using a ligand exchange type CSP [165]. Tamandarin A (213) was found to display cytotoxicity against BX-PC3, DU-145, and UMSCC10b human cancer cell lines, with IC 50 values of 1.79, 1.36, and 0.99 µg/mL, respectively [163].

Cyclic Depsipeptides
To the best of our knowledge, only four works reported the determination of the stereochemistry of amino acid constituents of the cyclic depsipeptides from marine invertebrates and algae ( Figure 16). Among these, three employed only Marfey's method, specifically for peptides 207-216. However, for peptide 217, Marfey's method was not efficient and, as a consequence, a ligand exchange type CSP was also used for complete determination of the configuration of its amino acids (Table 10).
The determination of the absolute configuration of the amino acids in kahalalides A-F (207-212), isolated from the marine mollusk Elysia rufescens, was performed by using FDLA as the derivatization reagent and the presence of diverse residues of L-and D-Val in these peptides was confirmed [162]. Using FDAA as the Marfey derivatization reagent, the absolute configuration of tamandarins A (213) and B (214), isolated from an unidentified Brazilian marine ascidian of the family Didemnidae [163], and kahalalides P (215) and Q (216), isolated from green algae Bryopsis species [164] were elucidated. In the case of kahalalide O (217), the absolute configuration of its amino acid constituents was determined by Marfey's method and chiral HPLC analysis, using a ligand exchange type CSP [165]. Tamandarin A (213) was found to display cytotoxicity against BX-PC3, DU-145, and UMSCC10b human cancer cell lines, with IC50 values of 1.79, 1.36, and 0.99 µg/mL, respectively [163].

Lipopeptides
For lipopeptides isolated from other marine invertebrates and algae, there are only two works which reported the use of a chiral HPLC for the stereochemistry determination of the amino acid residues (Table 11) of the peptides 218-221 ( Figure 17).  Chiral HPLC analysis by using a ligand exchange type CSP (Phenomenex Chirex Phase 3126) was used to determine the configuration of the amino acid residues in eudistomides A (218) and B (219), isolated from an ascidian Eudistoma sp. It was possible to verify the presence of L-Pro, L-Ala and L-Leu in both compounds as well as the presence of L-Cyp in eudistomide A (218) [166]. Similarly, a chiral HPLC analysis using a ligand exchange type CSP (CHIRALPAK (MA+)) was able to confirm  Chiral HPLC analysis by using a ligand exchange type CSP (Phenomenex Chirex Phase 3126) was used to determine the configuration of the amino acid residues in eudistomides A (218) and B (219), isolated from an ascidian Eudistoma sp. It was possible to verify the presence of L-Pro, L-Ala and L-Leu in both compounds as well as the presence of L-Cyp in eudistomide A (218) [166]. Similarly, a chiral HPLC analysis using a ligand exchange type CSP (CHIRALPAK (MA+)) was able to confirm the presence of four L-amino acid residues and D-Ala, D-Phe, and D-Ser in mebamamides A (220) and B (221), isolated from the green alga Derbesia marina [167].

Case-Study: Chiral HPLC in the Analysis of the Stereochemistry of Cyclopeptides Isolated from Marine Sponge-Associated Fungi
Recently, the determination of the stereochemistry of the amino acid residues of three bioactive marine natural products, by chiral HPLC analysis of their acidic hydrolysates, using appropriate D-and L-amino acid standards was achieved in our group [111,112]. The marine sponge-associated fungus Aspergillus similanensis KUFA 0013 was the source of the cyclohexapeptide similanamide (110) (Figure 8), while cyclotetrapeptides sartoryglabramides A (111) and B (112) (Figure 8) were isolated from the marine sponge-associated fungus Neosartorya glabra KUFA 0702. The enantioseparations of the amino acids were successfully performed on Chirobiotic T column under reverse phase elution conditions. Actually, the teicoplanin selector of this column has several characteristic features that make it suitable for amino acid analysis [168,169]. Figure 18 shows selected chromatograms of the enantioseparation of standard amino acids. the presence of four L-amino acid residues and D-Ala, D-Phe, and D-Ser in mebamamides A (220) and B (221), isolated from the green alga Derbesia marina [167].

Case-Study: Chiral HPLC in the Analysis of the Stereochemistry of Cyclopeptides Isolated from Marine Sponge-Associated Fungi
Recently, the determination of the stereochemistry of the amino acid residues of three bioactive marine natural products, by chiral HPLC analysis of their acidic hydrolysates, using appropriate Dand L-amino acid standards was achieved in our group [111,112]. The marine sponge-associated fungus Aspergillus similanensis KUFA 0013 was the source of the cyclohexapeptide similanamide (110) (Figure 8), while cyclotetrapeptides sartoryglabramides A (111) and B (112) (Figure 8) were isolated from the marine sponge-associated fungus Neosartorya glabra KUFA 0702. The enantioseparations of the amino acids were successfully performed on Chirobiotic T column under reverse phase elution conditions. Actually, the teicoplanin selector of this column has several characteristic features that make it suitable for amino acid analysis [168,169]. Figure 18 shows selected chromatograms of the enantioseparation of standard amino acids. The elution order of all the standard enantiomers of amino acids was confirmed by injecting solutions of the racemic or enantiomeric mixtures of amino acids and then each enantiomer separately. As an example, Figure 19 shows the chromatograms obtained during the method development for the determination of the elution order of Ala. As expected, the D-enantiomer was always more strongly retained than the corresponding L-enantiomer on Chirobiotic T column [168]. Mixed HPLC analyses of the acidic hydrolysates with appropriate standard amino acids (co-injection) (Table 12), confirmed the stereochemistry of the amino acids of the three cyclopeptides [111,112]. Chiral HPLC technique demonstrated to be decisive leading to the unambiguous elucidation of the amino acid constituents of the three marine natural products.
Additionally, the in vitro growth inhibitory activity against MCF-7, breast adenocarcinoma, NCI-H460, non-small cell lung cancer and A373, melanoma, cell lines, as well as antibacterial activity against reference strains and the environmental multidrug-resistant isolates (MRS and VRE) were evaluated for cyclopeptide 110. Only weak activity against the three cancer cell lines was observed [111]. Moreover, cyclopeptides 111 and 112 were tested for their antifungal activity against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte (Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231), as well as for their antibacterial activity against Gram-positive (Escherichia coli ATCC 25922) and Gram-negative (Staphyllococus aureus ATCC 25923) bacteria. None of them exhibited antibacterial or antifungal activities [112]. The elution order of all the standard enantiomers of amino acids was confirmed by injecting solutions of the racemic or enantiomeric mixtures of amino acids and then each enantiomer separately. As an example, Figure 19 shows the chromatograms obtained during the method development for the determination of the elution order of Ala. As expected, the D-enantiomer was always more strongly retained than the corresponding L-enantiomer on Chirobiotic T column [168]. Mixed HPLC analyses of the acidic hydrolysates with appropriate standard amino acids (co-injection) (Table 12), confirmed the stereochemistry of the amino acids of the three cyclopeptides [111,112]. Chiral HPLC technique demonstrated to be decisive leading to the unambiguous elucidation of the amino acid constituents of the three marine natural products.
Additionally, the in vitro growth inhibitory activity against MCF-7, breast adenocarcinoma, NCI-H460, non-small cell lung cancer and A373, melanoma, cell lines, as well as antibacterial activity against reference strains and the environmental multidrug-resistant isolates (MRS and VRE) were evaluated for cyclopeptide 110. Only weak activity against the three cancer cell lines was observed [111]. Moreover, cyclopeptides 111 and 112 were tested for their antifungal activity against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte (Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231), as well as for their antibacterial activity against Gram-positive (Escherichia coli ATCC 25922) and Gram-negative (Staphyllococus aureus ATCC 25923) bacteria. None of them exhibited antibacterial or antifungal activities [112].

Conclusions
In summary, concerning all the reported studies surveyed in this review, which are related to the determination of the absolute configuration of the marine peptides, their distribution according to the methods used, is shown in Figure 20. It is possible to conclude that Marfey's method is the most employed accounting for 52% of the reported studies, while only 21% of the studies described the use of chiral HPLC analysis. Moreover, 27% of the studies included the application of both methods. In fact, in some cases, the complementarity of both methods demonstrated to be crucial for the stereochemical analysis of all the amino acid residues.  Figure 21 compares the reported studies before and after 2007. Interestingly, it is possible to observe that in the last ten years, Marfey's method is still the most used for determination of the absolute configuration of amino acid residues in marine peptides. However, it is important to point out a notable increase of the number of studies related to a chiral HPLC analysis, either as the only method or in a combination with Marfey's method. In our opinion, the current trend is to use chiral HPLC for stereochemical analysis due to many advantages of this method. For examples, there is no need for prior derivatization, it requires much less sample manipulation and the results are more rapid to obtain. In contrast, Marfey's method involves time-consuming and labor-intensive procedure.
We believe that the reasons that can justify the actual low number of studies using chiral HPLC is due to the price of the commercially available CSPs and the fact that there is no universal CSP, i.e., one CSP can only separate a limited number of chiral compounds and, in many cases, the choice of CSP may become a very difficult task.  Figure 21 compares the reported studies before and after 2007. Interestingly, it is possible to observe that in the last ten years, Marfey's method is still the most used for determination of the absolute configuration of amino acid residues in marine peptides. However, it is important to point out a notable increase of the number of studies related to a chiral HPLC analysis, either as the only method or in a combination with Marfey's method.  Figure 21 compares the reported studies before and after 2007. Interestingly, it is possible to observe that in the last ten years, Marfey's method is still the most used for determination of the absolute configuration of amino acid residues in marine peptides. However, it is important to point out a notable increase of the number of studies related to a chiral HPLC analysis, either as the only method or in a combination with Marfey's method. In our opinion, the current trend is to use chiral HPLC for stereochemical analysis due to many advantages of this method. For examples, there is no need for prior derivatization, it requires much less sample manipulation and the results are more rapid to obtain. In contrast, Marfey's method involves time-consuming and labor-intensive procedure.
We believe that the reasons that can justify the actual low number of studies using chiral HPLC is due to the price of the commercially available CSPs and the fact that there is no universal CSP, i.e., one CSP can only separate a limited number of chiral compounds and, in many cases, the choice of CSP may become a very difficult task. In our opinion, the current trend is to use chiral HPLC for stereochemical analysis due to many advantages of this method. For examples, there is no need for prior derivatization, it requires much less sample manipulation and the results are more rapid to obtain. In contrast, Marfey's method involves time-consuming and labor-intensive procedure.
We believe that the reasons that can justify the actual low number of studies using chiral HPLC is due to the price of the commercially available CSPs and the fact that there is no universal CSP, i.e., one CSP can only separate a limited number of chiral compounds and, in many cases, the choice of CSP may become a very difficult task.