*1.1. Resources*

Various natural sources of TBPs and other heterocyclic rings containing cyclopolypeptides comprise cyanobacteria [8–40], ascidians [41–62], marine sponges [63–70], and sea slugs [71–73]. Moreover, actinomycetes, sea hare, red alga, and higher plants [74–80] were found to be other potential resources of TBPs.

#### *1.2. Linear vs. Cyclic Peptides*

In linear peptides with amino acid units between 10 to 20, secondary structures like α-helices and β-strands begin to form, which impose constraints that reduce the free energy of linear peptides. Compared to linear peptides, cyclopeptides are typically considered to have even greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectivity, and improved pharmacodynamic properties. Although peptide cyclization generally induces structural constraints, the site of cyclization within the sequence can affect the binding affinity of cyclic peptides. Cyclization is a well-known technique to increase the potency and in vivo half-life of peptide molecules by locking their conformation. Hence, both the biological activity and the stability of peptides can be improved by cyclization. The reduction in conformational freedom brought about by cyclization often results in higher receptor-binding affinities. Overall, cyclization of peptides is a vital tool for structure–activity studies and drug development because ring formation limits the flexibility of the peptide chain and allows for the induction or stabilization of active conformations. Moreover, cyclic peptides are less sensitive to enzymatic degradation [81].

The cyclization process often increases the stability of peptides, can prolong their bioeffect, and can create peptides with the ability to penetrate tumors in order to enhance the potency of anticancer drugs [82,83]. Cyclization is envisioned to enhance the selective binding, uptake, potency, and stability of linear precursors. The prolonged activity may even be the result of additional resistance to enzymatic degradation by exoproteases. Cyclic peptides are of considerable interest as potential protein ligands and might be more cell permeable than their linear counterparts due to their reduced conformational flexibility.

Further, cyclic nature of peptides was found to be crucial to their bioactivity in the case of depsipeptides. For example, corticiamide A is a member of a family of structurally related cyclic depsipeptides with tryptophan moiety that include the discodermins, halicylindramides, polydiscamide A, and microspinosamide A. However, corticiamide A is the only member of the family to contain a *p*-Br-Phe at residue 11 and an *N*-MeAsn. Microspinosamide A and polydiscamide A contained the unusual β-Me-Ile at residue 6, whereas the same amino acid is found at residue 5 in corticiamide A. All these peptides were known to be cytotoxic in the low μM range and to inhibit the growth of bacteria and fungi in addition to inhibition of the cytopathic effect of HIV-1 in mosaic human T cell leukemia cells-Syncitial Sensitive (CEM-SS) by microspinosamide A. Interestingly, the cyclic nature of these peptides was important for their bioactivity, with linear versions exhibiting a loss of activity of at least 1 order of magnitude [84,85].

#### **2. Chemistry**

#### *2.1. Structural Features of Thiazole (Tzl)-Containing Cyclooligopeptides*

Aestuaramides, banyascyclamides, ulongamides (**1**–**3**), guineamides (**4**,**5**), microcyclamides MZ602 and MZ568, trichamide, tawicyclamides (**6**,**7**), obyanamide (**8**), cyclodidemnamide and cyclodidemnamide B, lyngbyabellins, oriamide (**9**), scleritodermin A (**10**), haligramide A (**11**), waiakeamide (**12**), haligramide B (**13**), mollamide C (**15**), jamaicensamide A (**16**), myotamides, didmolamides, dolastatin 3, homodolastatin 3, sanguinamides, cyclotheonellazoles, aeruginazole A, aeruginazole DA1497, aeruginazole DA1304, and aeruginazole DA1274 are examples of heterocyclic thiazole-based polypeptides having diverse unusual structural features from marine organisms.

Cyanobactin cyclopolypeptide aestuaramide A contained valylthiazole (Val-Tzl) and prolylthiazole (Pro-Tzl) residues in addition to proline, valine, and methionine units and a reverse *O*-Tyr isoprene moiety (Ptyr). Aestuaramide B was found to be an unprenylated analogue of aestuaramide A, whereas aestuaramide C was found to be a forward C-prenylated derivative. Aestuaramide D–F and aestuaramide J–L were found to be the sulfoxide derivatives of aestuaramides A–C and aestuaramides G–I, respectively. Similarily, aestuaramides G−L were reverse O-prenylated, unprenylated, or forward C-prenylated congeners, with or without Met oxidation, but contained alanylthiazole (Ala-Tzl) instead

of a Val-Tzl unit of aestuaramides A–F. Cyclic peptides such as aestuaramides may be exceptionally widespread metabolites in natural ecosystems [10].

Banyascyclamides B and C are modified cyclopolypeptides, closely related in structure, and composed of two thiazole-alanine units. The cyclohexapeptide banyascyclamide C exhibited close structural similarity with banyascyclamide A but differed in having l-phenylalanyl-l-threonine moiety instead of l-Phe-mOzl residue of banyascyclamide A. Similarily, banyascyclamide B differed from banyascyclamide C in having l-leucyl-l-threonine moiety instead of l-phenylalanyl-l-threonine residue [11].

The cyanobacterium-derived ulongamide A (**1**) and other ulongamides B–F are alanine-derived thiazole carboxylic acid (l-Ala-Tzl-ca) containing cyclodepsipeptides which possessed a novel β-amino acid residue, 3-amino-2-methylhexanoic acid (Amha). Further, there was the presence of 2-hydroxyisovaleric acid (Hiva) in ulongamide D (**2**) and 2-hydroxy-3-methylpentanoic acid (Hmpa) in ulongamide E and ulongamide F (**3**), which had replaced the l-lactic acid moiety present in ulongamides A–C. Ulongamides A–E displayed weak in vitro cytotoxicity against ubiquitous KERATIN-forming tumor cell subline (KB) and LoVo cells [13] (Figure 1).

**Figure 1.** Structures of ulongamide A (**1**), ulongamide D (**2**), and ulongamide F (**3**) with alanylthiazole (Ala-Tzl) and 3-amino-2-methylhexanoic acid (Amha) moieties.

The cyanobacterium-derived guineamide A (**4**) contained the common l-alanine-disubstitutedthiazole unit, unique β-amino acid 2-methyl-3-aminopentanoic acid (Mapa), lactic acid (l-Lac), *N*-methylated amino acids viz. *N*-methylphenylalanine (l-*N*-MePhe), and *N*-methylvaline (l-*N*-MeVal), but guineamide B (**5**) deviated from guineamide A (**4**) in having 2-hydroxyisovaleric acid (l-Hiv) and 2-methyl-3-aminobutanoic acid (Maba) units instead of l-Lac and Mapa units. The absolute stereochemistry of the 2-methyl-3-aminopentanoic acid (Mapa) unit in guineamide A (**4**) was found to be 2*S*,3*R*. From a biosynthetic perspective, the guineamides were found to be interesting molecules because of the presence of unusual α-amino and β-hydroxy acid residues. Further, guineamide B (**5**) exhibited moderate cytotoxic activity against a mouse neuroblastoma cell line [14] (Figure 2).

**Figure 2.** Structures of guineamide A (**4**) and guineamide B (**5**) with Ala-Tzl and l-*N*-Methylated amino acid units.

Microcyclamides MZ602 and MZ568 contained isoleucylthiazole moiety in common but differed in having phenylalanine and glycine amino acids in the former and valine and alanine in the latter. Trichamide possessed serylthiazole and leucylthiazole moieties in addition to histidine amino acid [18].

The cyanobacterium-derived lyngbyabellin A is a significantly cytotoxic dichlorinated peptolide with unusual structural features, including a dichlorinated α-hydroxy acid and two functionalized thiazole carboxylic acid units. This depsipeptide was found to be a potent disrupter of the cellular microfilament network [27]. Lyngbyabellin B is related cyclic depsipeptide in which one thiazole unit was replaced by a thiazoline ring, with the placement of the ring between the glycine residue and the α,β-dihydroxyisovaleric acid rather than adjacent to the valine-derived unit, and the isoleucine-derived unit in lyngbyabellin A was replaced by a valine-derived moiety in lyngbyabellin B. Lyngbyabellin B displayed potent toxicity toward brine shrimp and the fungus *Candida albicans* and was found to be slightly less cytotoxic in vitro than lyngbyabellin A against KB and LoVo cells, respectively [86]. The structures of lyngbyabellin E and H showed the presence of two 2,4-disubstituted thiazole rings and differed7 in having the α,β-dihydroxyisovaleric acid (dhiv) unit in lyngbyabellin E replaced by the 2-hydroxyisovaleric acid (hiva) unit in lyngbyabellin H. Intriguingly, lyngbyabellin E and H appeared to be more active against the H460 human lung tumor cell lines. From the bioactivity results, it appeared that lung tumor cell toxicity is enhanced in the cyclic representatives with an elaborated side chain [28].

In addition to two thiazole rings and a chlorinated 2-methyloctanoate residue, lyngbyabellin N contained an unusual dimethylated valine terminus and a leucine statine residue. The planar structure of lyngbyabellin N was closely related to that of lyngbyabellin H except for the replacement of the polyketide portion with an *N,N*-dimethylvaline (DiMeVal) residue [29]. The cytotoxic lyngbyabellin J contained the *gem*-dichloro moiety as part of a 7,7- dichloro-3-acyloxy-2-methyloctanoate residue in addition to the α,β-dihydroxy-β-methylpentanoic acid (Dhmpa, C19–24) unit and two disubstituted thiazole rings [30].

Tawicyclamides A and B (**6**,**7**) represent a novel category of cyclooligopeptides, bearing alternative sequences of two thiazoles and one thiazoline amino acid but lacking the oxazoline ring, which is characteristic of ascidian-derived heptapeptides lissoclinamides and the octapeptides patellamides/ulithiacyclamides. Moreover, the presence of a *cis*-valine-proline amide bond facilitates an unusual three-dimensional conformation to ascidian-derived tawicyclamides A and B (**6**,**7**). Tawicyclamide B (**7**) differs from tawicyclamide A (**6**) in having a leucine moiety in place of the phenylalanine residue of tawicyclamide A [41] (Figure 3).

**Figure 3.** Structures of tawicyclamide A (**6**) and tawicyclamide B (**7**) with valylthiazole (Val-Tzl) and l-isoleucyl-thiazole (Ile-Tzl) moieties.

In the structure of depsipeptide–obyanamide (**8**), the alanylthiazole (Ala-Tzl) unit and 3-aminopentanoic acid (Apa) were present [12,42] whereas the sponge-derived cytotoxic cyclic peptide, oriamide (**9**), was found to contain a new 4-propenoyl-2-tyrosylthiazole amino acid (PTT) moiety. Further, a novel conjugated thiazole moiety viz. 2-(1-amino-2-*p*-hydroxyphenylethane)-4(4-carboxy-2,4-di-methyl-2*Z*,4*E*-propadiene)-thiazole (ACT) was found to be part of the structure of tubulin inhibitory sponge-derived cyclopolypeptide scleritodermin A (**10**), along with *O*-methyl-*N*-sulfoserine and keto-*allo*-isoleucine units [64] (Figure 4).

**Figure 4.** Structures of obyanamide (**8**) with Ala-Tzl moiety, oriamide (**9**) with 4-propenoyl-2-tyrosylthiazole amino acid (PTT) moiety, and scleritodermin A (**10**) with 2-(1-amino-2-*p*-hydroxyphenylethane)-4- (4-carboxy-2,4-di-methyl-2*Z*,4*E*-propadiene)-thiazole (ACT) moiety.

In the structure of the bisthiazole-containing macrocyclic peptide, cyclodidemnamide B, two thiazole moieties viz. prolylthiazole (l-Pro-Tzl) and leucylthiazole (d-Leu-Tzl) were found to be present. The ascidian-derived cyclodidemnamide was found to be similar to reverse prenyl substituted cytotoxic cycloheptapeptide mollamide only in possessing the same dihyrothiazole-proline dipeptide unit (C20–C27), but it also contained leucylthiazole and phenylalanyl-methyl oxazoline moieties [43,62].

The sponge-derived cytotoxic hexapeptides haligramide A and B (**11**,**13**) were found to contain the phenylalanylthiazole (Phe-Tzl) moiety in addition to three proline units. Haligramide A (**11**) was the bismethionine analogue of waiakeamide (**12**), bearing Phe-Tzl moiety. Haligramide B (**13**) contained both methionine and methionine sulfoxide residues in comparison to haligramide A (**11**) which contained only methionine residues and waiakeamide (**12**), another sponge-derived cyclohexapeptide that contained methionine sulfoxide residues only [63,66] (Figure 5).

**Figure 5.** Structures of haligramide A (**11**), waiakeamide (**12**), and haligramide B (**13**) with phenylalanylthiazole (Phe-Tzl) moieties.

A unique amino acid, 2-bromo-5-hydroxytryptophan (BhTrp), and an unusual ureido linkage were found to be present in the composition of sponge-derived peptide konbamide with calmodulin antagonistic activity [87]. Further, the cytotoxic depsipeptide polydiscamide A contained a novel amino acid 3-methylisoleucine in addition to heterocyclic tryptophan moiety [65,88].

The notaspidean mollusk-derived cytotoxic cyclic hexapeptide keenamide A (**14**) contained a leuylthiazoline (Leu-Tzn) unit together with serylisoprene residue in its structure and differed from mollamide C (**15**), a tunicate-derived cyclohexapeptide, in having thiazoline moiety instead of thiazole [72]. Trunkamide A contained a thiazoline heterocycle and two residues of Ser and Thr with the hydroxy function modified as reverse prenyl (rPr). The structure of jamaicensamide A (**16**), a sponge-derived peptide having β-amino-α-keto and thiazole-homologated η-amino acid residues, was found to contain 2-aminobutanoic acid (Aba), 5-hydroxytryptophan (HTrp), and a terminal 2-hydroxy-3-methylpentanamide (Hmp) unit [44,89] (Figure 6).

**Figure 6.** Structures of keenamide A (**14**) with leuylthiazoline (Leu-Tzn) moiety, mollamide C (**15**) with Leu-Tzl moiety, and jamaicensamide A (**16**) with Ala-Tzl and 2-hydroxy-3-methylpentanamide (Hmp) residues.

Myotamides A and B are ascidian-derived cycloheptapeptides that contained three unusual amino acids containing heteroatoms including one thiazole (Tzl) and two thiazoline (Tzn) rings in addition to valine, proline, isoleucine, and methionine. Mayotamide A embodied the same Val-Pro-Tzn sequence as was found in ascidian-derived cyclic heptapeptide cyclodidenmamide and also contained an additional thiazoline (Tzn) ring. Myotamide A differed from myotamide B in having isoleucine moiety, which was replaced by valine moiety in the latter. Both cyclopolypeptides exhibited cytotoxicity against tumor cell lines [45].

Didmolamide B is a thiazole-containing ascidian-derived cyclopolypeptide that contained two l-alanylthiazole residues, and l-phenylalanine and l-threonine moieties. The threonine residue of didmolamide B was modified to a methyloxazoline (mOzn) heterocycle in the case of didmolamide A. Didmolamide B was found to exhibit mild cytotoxicity against several cultured tumor cell lines [48].

Dolastatin 3 is a cyanobacterium- as well as sea hare-derived cyclopolypeptide that contained two l-glutaminyl-thiazole (l-Gln-Tzl) and glycyl-thiazole (Gly-Tzl) units in addition to l-valine, l-leucine, and l-proline residues. The cyanobacterium-derived homodolastatin 3 differed from dolastatin 3 by the addition of a methylene group, i.e., an l-isoleucine residue in place of the l-valine residue of dolastatin 3. The cyclopentapeptide dolastatin 3 was found to exhibit HIV-1 integrase inhibitory activity as well as P388 lymphocytic leukemia (PS) cell growth inhibitory activity. Kororamide is another cyanobacterium-derived polypeptide having two l-tyrosinyl-thiazole (l-Tyr-Tzl) and leucyl-thiazoline (Leu-Tzn) units in addition to l-leucine, l-isoleucine, l-serine, l-proline, and l-asparagine residues [9,90].

The sponge-derived cyclotheonellazoles A–C are unusual cyclopolypeptides containing nonproteinogenic acids, the most unique being 4-propenoyl-2-tyrosylthiazole (PTT), 3-amino-4-methyl-2-oxohexanoic acid (Amoha), and diaminopropionic acid (Dpr), along with two or three proteinogenic amino acids like glycine and alanine. Cyclotheonellazoles B and C shared the same basic structure with cyclotheonellazole A, in which leucine (in cyclotheonellazole B) and homoalanine (in cyclotheonellazole C) replaced the 2-aminopentanoic acid residue of cyclotheonellazole A. Cyclotheonellazoles were found to be nanomolar inhibitors of chymotrypsin and sub-nanomolar inhibitors of elastase [68].

The nudibranch-derived sanguinamide A is a modified heptapeptide containing a 2-substituted thiazole-4-carboxamide moiety. Structural analysis of this peptide indicated the presence of two residues, l-proline and l-isoleucine, present in alternative continuous sequences in addition to amino acid moieties phenylalanine and alanine with an l-configuration. In this cycloheptapeptide, azole-modified amino acid was found to be l-isoleucyl-thiazole (l-Ile-Tzl). In comparison to sanguinamide A, the cyclic octapeptide sanguinamide B was found to contain additional heteroaromatic oxazole and thiazole rings [73].

The cyanobacterium-derived polythiazole peptide aeruginazole DA1497 contained leuylthiazole (Leu-Tzl), alanylthiazole (Ala-Tzl), phenylalanylthiazole (Phe-Tzl), and valylthiazole (Val-Tzl) residues and exhibited bioproperties against Gram-positive bacterium *Staphylococcus aureus*. However, in related cyclopolypeptides, aeruginazole DA1304 and aeruginazole DA1274 moieties like asparaginylthiazole (Asn-Tzl), Leu-Tzl and isoleucylthiazole (Ile-Tzl) were found to be present. l-Asn-Tzl moiety was also observed in the polythiazole containing cyanobacterium-derived polypeptide aeruginazole A in addition to d-Leu-Tzl and l-Val-Tzl residues. This cyclododecapeptide was found to potently inhibit the Gram-positive bacterium *Bacillus subtilis* [8,91].
