1． Introduction

What we refer to as “Middle molecular strategy” is an emerging platform for the development of new drug leads. Its central theme is the use of medium─ sized molecules to interact speci�cally with challenging biological targets. Peptides are one particular example, and have attracted growing interest as their medium─ sized scaffolds confer on them a large interac-tive surface able to bind to protein─ protein interaction sites. Versatile and ef�cient methodologies such as solid phase pep-tide synthesis are already available to construct the peptidic framework, and are applicable to diverse peptidic medium─ sized molecules. Indeed, these synthetic technologies have cul-minated in a diverse array of peptidic molecules, some of which are promising drug leads and now under clinical trial. 1

However, peptide molecules have inherent disadvantages in terms of absorption, metabolism and pharmaceutical ef�cacy. They can be hydrolyzed and decomposed by proteases or pep-tidases after administration, resulting in low ef�cacy in vivo. The membrane permeability of intact, linear peptides is also generally low. These disadvantageous features can sometimes be overcome by the use of peptide isosteres, incorporation of D─ amino acids, and macrocyclization. Especially, cyclic pep-tides are expected to exhibit not only higher membrane perme-ability, but also restricted conformations suitable for high af�nity binding to their target molecules. 2

Natural product cyclic peptides are common, and are clas-si�ed into cyclopeptides and cyclodepsipeptides, with respec-tively amide and ester bonds at the junction. The cyclization mode is further divided into head─ to─ tail, head─ to─ side─ chain and even the rare side─ chain─ to─ tail. 3 Of these various cyclic peptide frameworks, we mainly focus on the head─ to─ tail cyclization (N─ C cyclization) in this review. Although a num-ber of excellent condensation reagents have already been deve-loped, this macrocyclization has proved a problem for organic synthesis. Longer, linear peptide sequences keep the N─ termi-

nal away from the C─ terminal, resulting in the competitive intermolecular coupling in preference to intramolecular reac-tion. Therefore, high dilution conditions are usually required for the macrocyclization reaction. Activation of the C─ termi-nal residue often induces racemization at the Cα position. Some protective groups are also required to prevent concomi-tant reactions in undesired positions. A general solution that overcomes these obstacles is highly desirable.

In contrast, natural cyclic peptides are readily biosynthe-sized by cyclases effectively matched to their cognate linear peptides. Therefore enzymatic peptide cyclization has been considered to be potentially useful in a wider context, and to have many advantages, including greater stereo─ and regiose-lectivity, reduced need for protecting groups, and mild reaction conditions. Indeed, some peptide ligases have already been developed as biocatalysts, and some of these are commercially available. 4 However, they have still not found widespread use in the laboratory. This is due to some disadvantageous features of enzymes such as their instability, narrow substrate speci�city and low tolerance to organic solvents. In this review, we sum-marize recent advances made in the enzymatic cyclization pro-cess and the discovery of a brand─ new cyclase family of non─ ribosomal peptides.

First we should note that natural peptides are biosynthe-sized by one of two pathways, ribosomally or non─ ribosomally. The particular features of natural peptide cyclases derived from both pathways are brie�y described in the following sec-tions.

2． Enzymatic Peptide Cyclization

2.1　 Ribosomally Synthesized and Post─ translationally Modi�ed Peptides (RiPPs)

The ribosomal peptide biosynthetic pathway essentially corresponds to protein biosynthesis, but also generates a con-siderable number of head─ to─ tail cyclopeptides. Well known examples are the cyanobactins and cyclotides from cyanobac-

A New Cyclase Family Catalyzing Head─ to─ Tail Macrolactamization of Non─ ribosomal Peptides

Kenichi Matsuda 1, Takefumi Kuranaga 2, and Toshiyuki Wakimoto 1＊

1＊Faculty of Pharmaceutical Science, Hokkaido University Kita 12, Nishi 6, Kita─ ku, Sapporo 060─ 0812 Japan

2Graduate School of Pharmaceutical Sciences, Kyoto University Sakyo─ ku, Kyoto 606─ 8051 Japan

(Received July 1, 2019; E─ mail: wakimoto@pharm.hokudai.ac.jp)

Abstract: Macrolactamization is one of the most important peptide─ modifying reactions. However, chemical macrocyclization is often hampered by several technical problems such as epimerization at the C─ terminal resi-due, and a requirement for high dilution conditions, as well as for protective groups. In contrast, natural cyclo-peptides are biosynthesized by ef�cient cyclases under mild conditions, suggesting these enzymes have potential as biocatalysts. However, the versatility of natural cyclases has not been fully exploited, mainly due to their strict substrate speci�city. During the course of our synthetic and biosynthetic studies on surugamides, we have discovered a new family of cyclases showing broad substrate speci�city. The new cyclase SurE, which is homologous to penicillin binding protein, of�oads two assembly lines of non─ ribosomal peptide synthetase, and remarkably, catalyzes the head─ to─ tail macrolactamization of two distinct peptides, thereby offering a new platform for the development of biocatalysts for macrolactamization.

（ 54 ） J. Synth. Org. Chem., Jpn.1106

teria and plants, respectively. 5 One class of representative cya-nobactins is the patellamides, originally isolated from marine ascidian Lissoclinum patella. 6 Later, metagenomic analysis revealed that the biosynthetic gene cluster pat was encoded by a symbiotic cyanobacterium Prochloron. 6 Structurally, patella-mides are N─ C cyclic peptides containing multiple heterocy-clized serine, threonine, and cysteine residues, yielding oxazole, oxazoline, thiazole, or thiazoline rings. Besides the cyclase for these �ve─ membered heterocyclic rings, the enzyme responsible for the macrocyclization is also encoded in the biosynthetic gene cluster, that is PatG, which is composed of an oxidase domain and a subtilisin─ like serine protease domain. 7 The lat-ter domain, PatGmac, is responsible for the N─ C macrocycli-zation of the precursor peptides, which comprise a core peptide �anked by recognition sequences for the modi�cation enzymes. PatGmac cleaves the follower sequence at the C terminus of the core peptide, and concurrently forms an acyl─ enzyme intermediate. This undergoes head─ to─ tail macrolactamization to generate the patellamides. Thus, the C─ terminal amino acid recognition sequence is required for the PatGmac─ catalyzed macrocyclization. 8

Butelase 1 is an asparaginyl endopeptidase originally iso-lated from seeds of the plant Clitoria ternatea, which is well─ known to be rich in cyclopeptides such as cyclotides. 9 Its sub-strate must contain C─ terminal Asn/Asp─ His─ Val residues, and the Asn/Asp remains included in the cyclized product. However, its high ef�ciency and broad substrate speci�city renders butelase 1 a promising biocatalyst for peptide macro-lactamization. 10 Peptiligase is another cyclase with broad sub-strate speci�city, which has been developed from a mutant of the Bacillus amyloliquefaciens subtilisin BPN’. 10 Peptiligase recognizes those substrates having a unique ester motif at the C─ terminus. The acyl─ enzyme intermediate formed simultane-ously with cleavage of the ester moiety undergoes head─ to─ tail macrolactamization. Therefore, no residue required for sub-strate recognition is retained in the product, which is a particu-lar advantage of this family of enzyme. Thus, some ef�cient and site─ speci�c ligases for head─ to─ tail macrocyclization of peptides and proteins have been developed from the RiPPs pathway. 11

2.2　 Non─ ribosomally Synthesized Peptides (NRPs)Non─ ribosomal peptides are biosynthesized by modular

type megasynthetases, the non─ ribosomal peptide synthetases (NRPS). Each module is generally composed of an adeny-lation domain (A), peptidyl carrier protein (PCP), and conden-sation domain (C). 12 The number of modules is usually consis-tent with the number of amino acids comprising the �nal product, except for the case of dimerization or polymerization of the peptide produced by a single assembly line. In contrast to RiPPS, NRPSs are able not only to recruit non─ proteino-genic amino acids, but also to work in conjunction with polyketide synthase (PKS) to generate a diverse array of struc-turally unique natural products. The nascent peptide generated by the assembly line is terminated by a key additional domain, the thioesterase domain (TE), which is fused to the C─ terminal of the megasynthetase. 13 The TE domain of the α, β ─ hydrolase family has a Ser residue at its active center, and this accepts the nascent peptide tethered to the �nal PCP domain to form an acyl─ enzyme intermediate. Hydrolysis of this intermediate generates a linear peptide. On the other hand, the TE domain can also catalyze either head─ to─ tail or head─ to─ side─ chain

macrocyclization. Thus, the NRPS─ TE domain can potentially catalyze the �nal step in the biosynthesis of structurally diverse peptides, and as such is a highly attractive enzyme for the bio-catalysis of peptide macrocyclization.

Walsh and co─ workers have previously investigated the potential of the TE domain as a biocatalyst. 14 This pioneering work was conducted with the TE domain involved in the bio-synthesis of antibiotic, cyclic decapeptide tyrocidine A. The TE domain is fused with other NRPS modules. This is a gen-eral feature of NRPS─ TE domains and disadvantageous for the development of a versatile single─ protein cyclase, since only the TE domain is required for the truncated protein to be dissected out, but this is not always both soluble and func-tional. 15 Moreover, one would anticipate that the protein─ pro-tein interaction between the TE and PCP cannot be neglected, since the TE recognizes the peptide bound to PCP via a thioes-ter. Despite these obstacles, Walsh and co─ workers succeeded in the preparation of a highly functional tyrocidine TE domain, which exhibited ef�cient cyclization activity even with peptides conjugated with SNAC (N ─ acetylcysteamine). 14 It is remarkable that this TycC TE domain is capable of liberating resin─ bound peptides synthesized by solid─ phase peptide syn-thesis. However, the substrate speci�city of TycC was stricter than expected. Together with further research, these results suggest that NRPS─ TE is not generally tolerant to modi�ca-tion of its intrinsic substrate, and would be dif�cult to develop into a biocatalyst with broad substrate speci�city.

3． Surugamide Family Peptides

3.1　 The Discovery of the SurugamidesSurugamides A─ E were originally isolated from the marine

actinomycete Streptomyces sp. JAMM992 and have also been reported to be produced by several other Streptomyces strains

Figure 1.　 Structures of the surugamides. a. Structure of surugamide A─ E. b. Structure of surugamide F.

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such as Streptomyces albidoflavus NBRC 12854. 16─ 18 These cyclic octapeptides showed inhibitory activity against the ser-ine protease, cathepsin. 16 Even though the amino acid compo-sition is proteinogenic, these cyclic octapeptides are half com-posed of D─ amino acids, implying that they are biosynthesized by a non─ ribosomal peptide synthetase. Indeed, a draft genome sequence analysis of the producer strain revealed that the biosynthetic gene cluster consists of 18 NRPS modules. 19 It encodes two NRPSs (SurA and SurD) likely responsible for the biosynthesis of surugamides A─ E based on the putative substrate speci�cities deduced from the eight A domain sequences. 20 Remarkably, two additional NRPSs (SurB and SurC) are inserted between these two NRPSs, SurA and SurD. These two intercalated NRPSs are composed of ten modules, and the putative amino acid composition suggested the exis-

tence of an unknown decapeptide containing aliphatic amino acids in the extract of Streptomyces sp. JAMM992. Reinvesti-gation of the extract identi�ed a new linear decapeptide, suru-gamide F, containing one unique non─ proteinogenic amino acid, 3─ amino─ 2─ methylpropionic acid (AMPA). 19 The bio-logical activity of surugamide F has not been reported to date. The details of structure elucidation in conjunction with the total syntheses of surugamides are described in the next sec-tion.3.2　 Total Synthesis of Surugamide F

The structure elucidation of surugamide F was performed by NMR and MS/MS analyses, which revealed the amino acid sequence to be Trp─ Leu─ Val─ Thr─ AMPA─ Leu─ Val─ Ala─ Val─ Ala. The two Leu and two Ala residues have the D─ abso-lute con�guration, while the other amino acids are L─ form

Figure 2.　 Biosynthesis of the surugamides. a. Biosynthetic gene cluster of the surugamides. The NRPS genes surA/surD for the octapep-tide biosynthesis, surB/surC for the decapeptide biosynthesis, and trans ─ acting thioesterase gene, surE, are colored in yellow, gray and orange, respectively. b. Proposed biosynthetic scheme for octapeptidic (upper) and decapeptidic (lower) surugamides. Each colored ball represents a functional domain: blue, condensation; red, adenylation; gray, peptidyl carrier protein (PCP); green, epimerization, orange; trans ─ acting TE (SurE).

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except for the AMPA residue, which was determined by Marfey’s analysis. The positions of the D─ amino acids were consistent with the positions of the E domains in the biosyn-thetic gene cluster. To corroborate the absolute con�guration of the AMPA residue, an epimeric mixture of surugamide F differing only in con�guration of this residue was prepared by Fmoc─ based solid─ phase peptide synthesis. The epimers were then separated, and their absolute con�guration was con�rmed by Marfey’s method. 21 Comparative analysis by HPLC and NMR led to the conclusion that natural surugamide F has an (S)─ AMPA residue. However, later on we were noti�ed by the supplier of mislabeling of the commercially available AMPA used as optically pure standards for Marfey’s analysis. We therefore recon�rmed the absolute con�guration of suru-gamide F by diastereoselective total synthesis (Scheme 1). 22 To prepare the optically pure AMPA residue, Evans’ asymmetric alkylation 23 of propionyl oxazolidinone with BOMCl was car-ried out. The removal of the benzyl group of 8a afforded a primary alcohol 9a, which was further converted to an amine 10a. Removal of the chiral auxiliary then yielded the optically pure AMPA residue 13a. Its epimer 13b was prepared by same synthetic route from enantiomeric starting material. The abso-lute con�gurations were con�rmed by using phenylglycine methylester (PGME) 24 introduced into the Boc─ protected AMPA. With these building blocks in hand, the initially pro-posed and the revised structures of surugamide F were both synthesized based on the Fmoc─ based solid─ phase peptide synthesis strategy (Scheme 1). Brie�y, the synthesis of suru-gamide F commenced with the treatment of Fmoc─ D─ Ala

loaded wang resin (14) with piperidine to liberate the amine 15. Four rounds of DIC/Oxyma─ mediated amide coupling and piperidine─ promoted Nα ─ deprotection were applied to 15 to yield 16. Optically pure building blocks 13a and 13b were cou-pled to 16 to give 17a and 17b, respectively. Successive four rounds of amide coupling and deprotection yielded 19a and 19b. Treatment of 19a and 19b with TFA/i ─ Pr 3SiH/H 2O (= 95:2.5:2.5) afforded crude surugamide F 6a and 6b, respec-tively.

After ODS─ HPLC puri�cation, 6a and 6b were obtained in 20 steps in respectively 38% and 36% yield. The synthesized 6a and 6b were then chromatographically compared with natural 6, enabling the absolute con�guration of the natural suru-gamide F (6) having an (R)─ AMPA residue to be ascertained.3.3　 Total Synthesis of Surugamide B

Of 1─ 5, only 2 does not require the expensive D─ Ile as a building block for its preparation, and thus 2 was targeted in the initial trial total synthesis of the cyclic surugamides. The synthetic challenge in this study arises out of the peptide cycli-zation, a long─ standing problem for synthetic chemists, 25 and a particular obstacle at this stage is Cα epimerization. This is especially true for synthetic studies toward N─ C cyclic peptides such as surugamides A─ E (1─ 5) consisting totally of epimeri-zable α ─ amino acids, since hybrid peptide/polyketide com-pounds such as the nannocystins (Figure 3c) can be cyclized at non─ peptidic residues by using various C─ C bond forming reactions. To minimize this isomerization the following three operations are fundamental to linear peptide synthesis: (i) α ─ amino acids are mildly activated by the use of the proper cou-pling reagents; (ii) carbamate (e.g. Fmoc and Boc) protected α ─ amino acids are used instead of amide─ capped α ─ amino acids to avoid epimerization; (iii) excess acid (with pre─ activa-

Scheme 1.　 Diastereoselective synthesis and structure revision of surugamide F (6) a. Synthesis of chiral AMPA.

Figure 3.　General problems in the synthesis of N─ C cyclic peptides.

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tion) is generally used in each acylation step to accelerate the reaction. Fmoc─ based solid─ phase peptide synthesis, the most developed and widely used technology, also utilizes these tricks and tips for high yielding synthesis as exempli�ed by the total synthesis of surugamide F (Figure 3a).

However, in the macrocyclization of N─ C cyclic peptides (Figure 3b), an equal amount of the C─ terminus amidated acid and the amine has to be employed, which can lead to generation of the isomerized peptide. The C─ terminus acid has to be activated in the presence of the N─ terminus amine (no pre─ activation), which often generates N─ capped by─ products. Furthermore, the macrolactamization should be performed under high dilution conditions to avoid oligomerization of the peptide, even though the use of concentrated acylation agent could accelerate the amidation. Thus, the synthesis of these cyclic peptides has to be attempted with careful optimization of the chemical reaction conditions, (e.g. coupling reagent, solvent, cyclization site), at a suitable coupling position. Despite these challenges to their synthesis, nature generally delivers the N─ C cyclic peptides stereoselectively and regiose-lectively without any protective groups. To examine the ef�-ciency of the biosynthetic machinery, we tried to synthesize 2 on the basis of its biosynthetic pathway.

The proposed biosynthetic pathway for 2 is illustrated in Figure 2b. The mechanism of the cyclization forming 2 has remained elusive due to the lack of a TE domain, but we can deduce the linear precursor peptide for macrocyclization according to the domain organization of SurA and SurD. Namely, the linear precursor peptide biosynthesis is initiated at the substrate speci�c recognition of L─ Ile by the �rst A domain (A1), and terminated by the attachment of L─ Leu in conjunc-tion with its stereochemical inversion to D─ Leu by the terminal E domain. Then intramolecular amidation between L─ Ile and D─ Leu gives 2 without concomitant epimerization at the origi-nal C─ terminus. In our total synthesis, the cyclization site was selected based on the biosynthetic pathway. The cyclic octa-peptide 2 was retrosynthetically acyclized to 32. This linear peptide 32 was to be provided by solid─ phase peptide synthesis.

The synthesis of 2 commenced with the treatment of Fmoc─ D─ Leu loaded 2─ chlorotrityl resin (30) with piperidine to liberate the amine 31. Then seven rounds of DIC/Oxyma─ mediated amide coupling and piperidine─ promoted Nα ─ deprotection were applied to 31 and subsequent intermediates, leading to 32. The cleavage of 32 from the resin was performed through the use of (CF 3) 2CHOH/CH 2Cl 2 (=3:7) without deprotection of the side─ chain of Lys, releasing the acyclic peptide into solution, where it was then cyclized using PyBOP and HOAt as coupling reagents to give cyclic peptide 34. Finally, treatment of 34 with TFA/i ─ Pr 3SiH/H 2O (=95:2.5:2.5) afforded crude 2. Notably, only trace isomerization (dr=＞25:1) was observed in the product. After ODS─ HPLC puri�-cation, 2 was obtained in 34% yield in 18 steps. The average yield per step in the total synthesis is 94%, which suggested macrolactamization at the biomimetic position is highly ef�-cient. 18

To con�rm the superiority of the biomimetic macrocycliza-tion position, a non─ biomimetic synthesis of 2 and its dia-steromer was implemented. First, the non─ natural diastereo-mer with a C─ terminal L─ Leu in the place of the D─ Leu of 2 was synthesized through three manipulations under the same conditions as laid out in Scheme 2. Even though the corre-

sponding macrolactam was obtained, the macrolactamization step with PyBOP and HOAt resulted in extensive concomitant racemization at the original C─ terminal residue. The lower diastereomer ratio of the cyclic product (4.7:1) suggested that the coupling between the D─ and L─ residues is preferred to that between the L─ and L─ residues (Scheme 3a). Similar phenome-non have also been reported in macrolactamization of several other peptides. 26,27 Hill et al. demonstrated that condensation between substrates of opposite con�gurations are favored in competitive activated amide coupling using racemic amino acid derivatives. 28 They postulated that substrates form six─ membered transitional state with chair conformation, and that D/L─ con�gured substrates would enable more favored di─ equa-torial disposition of side chains, leading to signi�cant prefer-ence on heterochiral coupling (ref. 3). This model may also explain heterochiral preference observed in head─ to─ tail mac-rolactamization of linear peptide 33. To further verify the superiority of the biomimetic macrocyclization position, the non─ natural cyclization position between D─ Val and L─ Lys was selected for the synthesis of 2. Lys is a β ─ mono─ substi-tuted amino acid, which would seem to be a less hindered nucleophile than a β ─ di─ substituted amino acid such as Ile, and so might be expected to facilitate cyclization. However, 10% of the cyclic product was C α─ epimerized during the mac-rocyclization step at this non─ natural site (Scheme 3b), con-�rming the signi�cant advantage of the ring closing position naturally selected by NRPS.3.4　 The Structural Revision of Surugamide A

These synthetic efforts set the stage for the next biosyn-thetic studies of the surugamides. Two main questions arose from our bioinformatics analysis of the biosynthetic gene clus-ter. The �rst concerns the presence of D─ Ile at position 4 in surugamides A, and C─ E (Figure 1, 2 is a derivative with D─ Val at position 4). The second is the lack of a TE domain in its NRPS module. This section focuses on the �rst issue, while the latter is described in section 4. The biogenesis of D─ Ile requires the epimerization of the two stereogenic centers at the C α─, and C β─ positions of L─ Ile. Epimerization at the C α─ position is commonly observed with NRP. In the case of surugamide bio-synthesis, the C α─ position of the Ile residue at position 4 (Ile─ 4) should be epimerized by the E domain in module 4 of SurA. On the other hand, the biosynthetic mechanism for the epimeri-zation at its C β─ position remained unclear. Epimerization at the C β─ position is relatively rare in nature, and to the best of our knowledge, only one type of biosynthetic machinery has been identi�ed for biosynthesis of L─ allo ─ Ile from L─ Ile, that in the desotamides and marformycin pathways. 29 However, the

Scheme 2.　Biomimetic total synthesis of surugamide B (2).

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homologous enzymes required for C β─ epimerization are not encoded in the surugamide biosynthetic gene cluster. Also, biosynthetic route for D─ Ile was unprecedented. Therefore, to con�rm the structure of 1, we synthesized D─ Ile─ containing 1 by taking advantage of the established route for the total syn-thesis of 2.

We conducted the solid phase peptide synthesis of 1 in the same manner as the synthesis of 2. However, HPLC analysis revealed that this synthetic 1a was not identical to natural surugamide A (Figure 4a). This result prompted us to compare synthetic and natural 1 by the C 3 Marfey’s method, 30 using which a D─ allo ─ Ile residue in the natural 1 was clearly detected (Figure 4b). Considering the position of the E domain in the surugamide NRPS, the C α─ epimerization of Ile occurs only at position 4, suggesting that 1 possesses a D─ allo ─ Ile residue instead of D─ Ile in position 4. To con�rm this, 1 with the sub-stitution of D─ Ile by D─ allo ─ Ile (1b) was synthesized in the same manner as 1a, as shown in Figure 4c. As expected, the HPLC analysis revealed that 1b and natural 1 are identical (Figure 4a), which was further corroborated by the C 3 Marfey’s method (Figure 4b). Our synthetic efforts clearly demonstrated that 1 possesses D─ allo ─ Ile at position 4, rather than D─ Ile. 31 Due to the commonality of their biosynthetic pathways, the D─ Ile residue originally thought to be present in other derivatives, such as 3─ 5, should also be corrected to D─ allo ─ Ile.

4． Discovery of a New Of�oading Cyclase, SurE

4.1　 Non─ enzymatic Cyclization of the Precursor PeptideA TE domain, or an alternative such as a C T domain, 32 are

generally required for the macrocyclization of non─ ribosomal peptides, but none were found in the modules of surugamide NRPS. As discussed in section 3.3, we found macrocyclization

was ef�cient at the biomimetic position during the total syn-thesis of 2. This allowed us to envision that octapeptide─ S ─ PCP, the biosynthetic precursor of 2, would be non─ enzymati-cally cyclized to 2 even in the absence of a TE domain. To con�rm this, SNAC bound octapeptide 38 was synthesized as a mimic of the precursor peptide tethered to a PCP domain. Since this octapeptide has a Lys residue mid─ way along the precursor linear sequence, two cyclization modes, head─ to─ tail and head─ to─ side─ chain, can compete. Whereas the side chain of the Lys residue was protected by Boc group prior to macro-lactamization in the total synthesis of 2, the SNAC thioester 38 was prepared without any protective groups. A coupling reagent─ free cyclization of 38 was implemented in the presence of Et 3N (pH 12) for 2 days (Scheme 3c). Contrary to our hypothesis, under these non─ enzymatic conditions hydrolysis of the thioester was faster than the expected cyclization, and furthermore, the regioisomeric isopeptide 39 was preferentially

Figure 4.　 Structure revision of surugamide A (1). a. LC─ MS com-parison of the natural surugamide A (1), and the synthetic 1a and 1b. b. Marfey’s Analysis of the natural 1, the syn-thetic 1a, 1b, and standard amino acids. Extracted ion chromatographs (EICs) for m/z 384.15 corresponding to derivatized DL─ Leu, DL─ Ile, and DL─ allo ─ Ile are depicted. c. Synthetic scheme for surugamide A (1).

Scheme 3.　 Non─ enzymatic macrolactamization at different ring closing residues or with the SNAC─ bound precursors.

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formed. This suggests that the thioester 38 does not spontane-ously transform into 2, and more importantly, that an uniden-ti�ed thioesterase is involved in the later stages of the biosyn-thesis of cyclic surugamides.

4.2　 Identi�cation of a New Of�oading Cyclase, SurEIn order to identify the enzyme responsible for head─ to─

tail cyclization we inspected the sur cluster. In the upstream region of the NRPS genes is encoded a putative enzyme SurE. It exhibits sequential similarity with penicillin─ binding protein (PBP), a group of enzymes responsible for the transpeptida-tion step in the biosynthesis of bacterial cell wall peptide gly-can. 33 Transpeptidation catalyzed by PBP proceeds via two steps. The �rst is the formation of an acyl─ enzyme complex in which substrate peptide is covalently bound to PBP through its catalytic serine residue. Next, the adjacent peptide chain attacks the acylated serine residue to form a new peptide link-age. This acylation─ deacylation mechanism is analogous to that of the TE domain responsible for of�oading NRPS. SurE possesses the catalytic tetrad (Ser63, Lys66, Asn156, His305) required for transpeptidation reactions. These observations led us to hypothesize that SurE might be responsible for chain─ termination and macrolactamization in the biosynthesis of surugamides.

To test this, we �rst conducted a gene knockout experi-ment. The gene coding for surE in S. albidoflavus NBRC 12854 was disrupted by the conventional double crossover method. This abolished the production of 1─ 5 in the resulting mutant (ΔsurE). Notably, as well as the production of cyclic peptides, that of linear decapeptide surugamide F (6a) was also aboli-shed. When the surE gene was complimented to the ΔsurE strain, the production of both groups of peptides was restored. These results indicate that SurE is involved in the biosynthesis of both cyclic octapeptides 1─ 5 and linear decapeptide 6a. Next, the function of SurE was investigated in vitro. The gene cording for SurE was cloned from genetic DNA of suru-gamides─ producing strain S. albidoflavus NBRC 12854. The recombinant SurE was expressed in E. coli BL21 (DE3) as an

N─ terminal His─ tag fused protein. When the recombinant SurE was mixed with octapeptidic SNAC thioester 38, it ef�-ciently converted 38 to cyclic peptide 2. In this reaction regioi-somer 39 was not generated. These results showed that SurE is a peptide cyclase responsible for head─ to─ tail macrolactamiza-tion in the biosynthesis of cyclic octapeptides 1─ 5. 18

4.3　 SurE Generates Cyclosurugamide FThe remaining question is the of�oading mechanism of

surugamide F (6a) from SurC. The results of previous genetic experiments led us to hypothesize that SurE is responsible not only for the cyclization step in the biosynthesis of 1─ 5, but also for the hydrolysis step in the biosynthesis of 6a. To test this, we synthesized decapeptidic SNAC thioester (46) mimicking the precursor of hydrolysis tethered on PCP. LC─ MS analysis of the reaction mixture containing 46 and SurE detected ef�cient consumption of SNAC, whereas generation of the supposed product 6a was not observed. However, closer inspection revealed the accumulation of a new compound (7) that is broadly eluted from the column. Its molecular ion peak was 18 mass units less than that of 6a, suggesting that this is a cyclic form of 6a. Surugamide F (6a) could cyclize in two different modes; head─ to─ tail or head─ to─ side─ chain through its inter-nal Thr residue. To clarify the cyclization mode, we �rst pro-tected this Thr hydroxy group with a benzyl group to give O ─ benzyl─ surugamide F─ SNAC (47), and hence prevent the head─ to─ side chain cyclization. SurE then ef�ciently converted Figure 5.　 Regiospeci�c cylization of octapeptidic thioester 26

mediated by SurE.

Figure 6.　 SurE─ mediated cyclization of decapeptidic substrate and derivatives. a. Schematic diagram for structure determina-tion of cyclosurugamide F (7) b. LC─ MS analysis of SurE in vitro reaction mixture with 46 as substrate. Extracted ion chromatographs of m/z 1157.8 (dashed line) corresponding to 46 and m/z 1056.8 (solid line) corresponding to 7 are depicted. c. SurE’s speci�city on con�guration of substrate C─ terminal residue.

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47 to head─ to─ tail cyclic peptide (48). Cleavage of the benzyl group of 48 by hydrogenation yielded a compound identical to 7, showing that 7 is a new surugamide F derivative, cyclosuru-gamide F, that is cyclized in head─ to─ tail manner. This com-pound was not described in previous reports. However, we detected the identical compound in a culture broth of S. albidoflavus NBRC 12854 at the early stage of fermentation. Based on these observations, we hypothesize that cyclosuru-gamide F (7) is the genuine product of the SurB/SurC assem-bly─ line. We also assume that 6a is generated via hydrolysis of 7 that is presumably mediated by a peptidase yet to be identi-�ed. Taken together, our genetic and biochemical analyses revealed that SurE is a trans ─ acting of�oading cyclase that is responsible for the cyclization steps of both octa─, and deca-peptidic surugamides’ biosynthesis. 34 SurE accepts two distinct non─ ribosomal peptides that share no sequential similarity, highlighting its broad substrate speci�city. The common fea-ture of the two substrates is the presence of a D─ amino acid at their C─ terminus; D─ Leu and D─ Ala in the octa─, and deca-peptide, respectively. To evaluate the importance of C─ termi-nal D─ amino acids on recognition by SurE, we synthesized the octapeptidic SNAC derivative with C─ terminal D─ Leu substi-tuted by L─ Leu. It turned out however, that under the reaction conditions this peptide spontaneously cyclizes between the C─ terminal L─ Leu and the side chain of Lys. To avoid this unde-sired reaction, Lys was substituted by Ala to give two SNAC derivatives 49 and 50 with respectively D─ Leu and L─ Leu at their C─ terminus. When these were subjected to SurE─ medi-ated cyclization, 49 was cyclized whereas 50 was not, showing that SurE is speci�c for peptidyl substrates with a D─ amino acid at the C─ terminus. 18

Phylogenetic analysis shows that SurE and its homologs form a clade that is distinct from canonical of�oading domains such as type I TEs, type II TEs, or atypical TEs found in poly-ether PKS biosynthesis (Figure 7). Since SurE and homologs are rather similar to PBPs such as DD─ carboxypeptidase and

alkaline D─ peptidase, we propose a new family of of�oading cyclases “PBP─ type TE” for these enzymes. A database search identi�ed several PBP─ type TEs encoded in the biosynthetic gene clusters of non─ ribosomal macrolactams such as mannnopeptimycins, 35 desotamides, 36 ulleungmycins 37 and noursamycins. 38 Bioinformatic analysis revealed that the NRPSs encoded within these gene clusters lack canonical of�oading domains, indicating that these PBP─ type TEs act as of�oading cyclases in the biosynthesis of these macrolactams. Notably, the cognate assembly─ lines commonly possess an epimerization domain at their last module. This fact indicates that, similar to SurE, these homologs speci�cally act on pepti-dyl substrates with a D─ amino acid at their C─ termini.

5． Conclusion

The biosynthetic gene cluster of surugamides has unusual module and domain organization, which motivated us to pre-pare these octa─ and decapeptides. Total syntheses of suru-gamides A and F were accomplished by ef�cient solid─ phase peptide synthesis and resulted in structural revision. In partic-ular, the stereochemical assignment of Ile or allo ─ Ile is usually not easy due to their close retention in Marfey’s analysis. Surugamide A and C─ E have D─ allo ─ Ile in place of D─ Ile, as clearly con�rmed by the total synthesis of surugamide A, and which is in fact consistent with the absence of any candidate genes for C β─ epimerization in the biosynthetic gene cluster. Needless to say, the exact structure of a natural product is important to correctly validate its biosynthetic pathway. The lack of a TE domain in the surugamide NRPS modules is also peculiar, and inspired us to examine the macrocyclization mechanism of 2 by utilizing the biosynthetic precursor for in vitro enzyme assay. Taking advantage of our biomimetic syn-thesis of surugamides, the substrates required for the biosyn-thetic study were readily obtained by chemical synthesis. Study of SurE function revealed that this enzyme, homologous to PBP, is one of a new family of TE catalyzing head─ to─ tail macrolactamization of non─ ribosomal peptides, and is shared by two different NRPS pathways, responsible for the biosyn-thesis of surugamide A─ E and cyclosurugamide F. Unlike the conventional TE domain, SurE is a stand─ alone enzyme cata-lyzing of�oading and cyclization of two NRPS assembly lines. This is unprecedented and has attracted the interest of other research groups. Indeed, after our original report, two other research groups independently reported the identi�cation of SurE as a new of�oading cyclase. 40 PBP─ type TE are more widely distributed among actinomycetes and other bacterial strains than expected, even though only �ve gene clusters have been associated with the metabolites, those for the suru-gamides, mannopeptimycin, desotamide, ulleungmycin, and norsamycin. All NRPS associated with PBP─ type TE are ter-minated by an E domain, which strongly suggests a substrate preference of PBP─ type TE for a D─ amino acid at the C─ ter-minus. Considering that SurE catalyzes the macrocyclization of two unrelated peptides, aside from the C─ terminal residue SurE likely shows broad substrate speci�city, as had been sug-gested by functional analysis. Thus, SurE is an ef�cient and site─ speci�c ligase for head─ to─ tail macrocyclization of pep-tides, which would be highly suitable for peptide engineering and drug discovery.

Figure 7.　 Phylogenetic analysis of canonical TE and PBP─ type TE. The tree (Neighbor─ Joining) was generated by MEGA 7. 39 Type I TE, type II TE, polyether PKS TE and PBP─ type TE are colored in green, blue, yellow and orange, respec-tively. Representative SurE homologs in the database are classi�ed as PBP─ type TE. TE─ less assembly lines are encoded in the neighboring region of these homolog genes.

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AcknowledgementsThe authors would like to express their sincere apprecia-

tion to all of the past and present members of the laboratory of natural product chemistry at Hokkaido University for their vital contributions. In particular, special thanks to Ms. Ayae Sano, Mr. Masakazu Kobayashi and Mr. Atsuki Fukuba for their enormous contributions to this project. This work was supported by the Japan Agency for Medical Research and Development (AMED Grant Number 18061402) as well as Grants─ in─ Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAK-ENHI Grant Numbers JP16703511 and JP18056499). The contributions from the Takeda Science Foundation, the Asahi Glass Foundation, the SUNBOR GRANT, the NOASTEC Foundation, and the Akiyama Life Science Foundation are greatly appreciated.

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PROFILE

Kenichi Matsuda received his B.Sc. in 2012 from the University of Tokyo under the guid-ance of Professor Makoto Nishiyama. He continued his work in the same group and re-ceived his PhD in 2017. After two months of postdoctoral training at the Technology Re-search Association for Next Generation Nat-ural Products Chemistry, he was appointed as an Assistant Professor of Pharmaceutical Sciences in Hokkaido University and joined the group of Professor Toshiyuki Wakimoto. His current research interests are focused on the identi�cation of new natural products and enzymes through a genome─ mining ap-proach.

Takefumi Kuranaga has been a specially ap-pointed associate professor of the Research Unit for Physiological Chemistry C─ PIER, and an assistant professor of the Graduate School of Pharmaceutical Sciences, Kyoto University since 2018. He received his B.Sc. and Ph.D. degrees from the University of Tokyo under the supervision of Prof. Kazuo Tachibana. After six months of postdoctoral research with Prof. Masayuki Inoue at the same University, he was next appointed as-sistant professor (2011─ 2015). He then car-ried out six months of postdoctoral research with Prof. Jörn Piel at ETH Zürich, and joined the group of Prof. Toshiyuki Waki-moto at Hokkaido University as a lecturer (2015─ 2018). His research interests are in natural product chemistry and synthetic or-ganic chemistry.

Toshiyuki Wakimoto has been a Full Profes-sor of Pharmaceutical Science at Hokkaido University since 2015. He received his BSc in 1996 from the University of Tokyo. He re-mained at the same University for his PhD, which he received in 2001 working in the lab-oratory of Professor Nobuhiro Fusetani. He moved to the University of Pittsburgh to work with Professor Peter Wipf. In 2003, he was appointed as an Assistant Professor to the School of Pharmaceutical Science at the University of Shizuoka and worked with Professor Toshiyuki Kan. Seven years later he joined the group of Professor Ikuro Abe at the University of Tokyo (2010─ 2015). He was promoted to Associate Professor at the University of Tokyo in 2014. His research in-terests are in natural product chemistry, and encompass the isolation and structure eluci-dation of new natural products, and the mechanisms of their biosynthesis.

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