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Chemo-enzymatic total syntheses of bis-tetrahydroisoquinoline alkaloids and systematic exploration of the substrate scope of SfmC

Open AccessPublished:March 25, 2022DOI:https://doi.org/10.1016/j.tchem.2022.100010

      Abstract

      Chemo-enzymatic hybrid process merging enzymatic cascade reactions of designed substrates with chemo- and site-selective synthetic manipulations allowed the chemo-enzymatic total syntheses of jorunnamycin A and saframycin A, in just 4 and 5 pots processes, respectively. Artificial variants with installation of various amino acids in place of l-Ala were systematically generated, demonstrating the substantially broader substrate scope of SfmC. This chemo-enzymatic hybrid process can considerably minimize the number of steps, labor, and time, and thus offer a rapid and versatile means to generate therapeutically important natural products and their derivatives.

      Graphical abstract

      Keywords

      1. Introduction

      Chemical and enzymatic synthesis have been developed as two essentially independent approaches for total synthesis of structurally complex natural products [
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      ]. The application of enzymatic reactions in organic synthesis has been almost exclusively limited to single-step functional group transformations, such as optical resolution by lipase and site-selective oxidation by P450 [
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      ]. In stark contrast to the conventional applications of enzymatic transformations, we sought to develop a hybrid process for the rapid and efficient construction of densely functionalized sp3-rich alkaloidal skeletons by streamlining the integration of chemical and enzymatic approaches [
      • Tanifuji R.
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      Total synthesis of alkaloids using both chemical and biochemical methods.
      ].
      Antitumor antibiotics, bis-tetrahydroisoquinoline (THIQ) alkaloids represented by saframycin A (1), cyanosafracin B (2), renieramycin M (3), jorunnamycin A (4) and ecteinascidin 743 (5) share a highly functionalized complex core scaffold consisting of two THIQ units (Fig. 1) [
      • Scott J.D.
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      ]. A family of these bis-THIQ alkaloids exhibits potent antitumor activity triggered by the formation of a covalent bond with DNA following interaction with the bis-THIQ alkaloidal skeleton with a GC-rich region of the DNA minor groove. Activation of either the hemiaminal or aminonitrile moiety at the C21 position generates an electrophilic iminium cation, which then alkylates the guanine N2 amino group with the sequence preference, 5′-GGC-3′ and 5′-GGG-3’ [
      • Ishiguro K.
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      Binding of saframycin A, a heterocyclic quinone anti-tumor antibiotic to DNA as revealed by the use of the antibiotic labeled with [14C]tyrosine or [14C]cyanide.
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      DNA sequence selectivities in the covalent bonding of antibiotic saframycins Mx1, Mx3, A, and S deduced from MPE·Fe(II) footprinting and exonuclease III stop assays.
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      ]. Ecteinascidin 743 (5), composed of three THIQ substructures, possesses a 10-membered lactone on the common bis-THIQ scaffold and has been commercialized as an anti-cancer agent for soft-tissue sarcoma and ovarian cancer [
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      ]. A semi-synthetic analog, lurbinectedin (6), bearing a spiro-fused β-tetrahydrocarboline moiety in place of the THIQ unit, was approved as a prescription drug for small cell lung cancer in 2020 by the US FDA [
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      Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision repair and show activity toward platinum-resistant cells.
      ].
      Fig. 1
      Fig. 1Representative bis-THIQ alkaloids.
      These densely functionalized complex structure with a unique molecular architecture as well as their very potent and promising anti-cancer therapeutic efficacy, have led to intense efforts over the past several decades directed at the total syntheses of the THIQ alkaloids, saframycin A (1) [
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      Total synthesis of (±)-Saframycin A.
      ,
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      Enantioselective synthesis of saframycin A and evaluation of antitumor activity relative to ecteinascidin/saframycin hybrids.
      ,
      • Myers A.G.
      • Kung D.W.
      A concise, stereocontrolled synthesis of (−)-Saframycin A by the directed condensation of α-amino aldehyde precursors.
      ,
      • Myers A.G.
      • Plowright A.T.
      Synthesis and evaluation of bishydroquinone derivatives of (−)-Saframycin A: identification of a versatile molecular template imparting potent antiproliferative activity.
      ,
      • Dong W.
      • Liu W.
      • Liao X.
      • Guan B.
      • Chen S.
      • Liu Z.
      Asymmetric total synthesis of (−)-Saframycin A from L-tyrosine.
      ,
      • Kimura S.
      • Saito N.
      A stereocontrolled total synthesis of (±)-Saframycin A.
      ], renieramycin M (3) [
      • Wu Y.C.
      • Zhu J.
      Asymmetrie total syntheses of (−)-Renieramycin M and G and (−)-Jorumycin using aziridine as a lynchpin.
      ,
      • Zheng Y.
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      • Yang H.D.
      • Wei K.
      • Yang Y.R.
      Asymmetric total syntheses of (−)-Fennebricin A, (−)-Renieramycin J, (−)-Renieramycin G, (−)-Renieramycin M, and (−)- jorunnamycin A via C-H activation.
      ], jorunnamycin A (4) [
      • Wu Y.C.
      • Zhu J.
      Asymmetrie total syntheses of (−)-Renieramycin M and G and (−)-Jorumycin using aziridine as a lynchpin.
      ,
      • Zheng Y.
      • Li X.D.
      • Sheng P.Z.
      • Yang H.D.
      • Wei K.
      • Yang Y.R.
      Asymmetric total syntheses of (−)-Fennebricin A, (−)-Renieramycin J, (−)-Renieramycin G, (−)-Renieramycin M, and (−)- jorunnamycin A via C-H activation.
      ,
      • Chen R.
      • Liu H.
      • Chen X.
      Asymmetric total synthesis of (−)-Jorunnamycins A and C and (−)-Jorumycin from L-tyrosine.
      ,
      • Liu H.
      • Chen R.
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      A rapid and efficient access to renieramycin-type Alkaloids featuring a temperature-dependent stereoselective cyclization.
      ,
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      • Allan K.M.
      • Virgil S.C.
      • Slamon D.J.
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      Concise total syntheses of (−)-Jorunnamycin A and (−) -jorumycin enabled by asymmetric catalysis.
      ] and ecteinascidin 743 (5) [
      • Corey E.J.
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      Enantioselective total synthesis of ecteinascidin 743.
      ,
      • Endo A.
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      Total synthesis of ecteinascidin 743.
      ,
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      Total synthesis of ecteinascidin 743.
      ,
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      Total synthesis of ecteinascidin 743.
      ,
      • He W.
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      A scalable total synthesis of the antitumor agents et-743 and lurbinectedin.
      ]. Meanwhile, PharmaMar in Spain developed a pioneering semi-synthetic process starting from cyanosafracin B (2), obtained by cultivation of the bacteria Pseudomonas fluorescens, thereby enabling large-scale supply of the marketed drugs ecteinascidin 743 (5) and lurbinectedin (6) [
      • Cuevas C.
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      ,
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      A concise and practical semisynthesis of ecteinascidin 743 and (–)-Jorumycin.
      ].
      The large-scale production of 2 through in vivo fermentation implied that the biosynthetic machinery to assemble bis-THIQ alkaloids has evolved in bacteria as an efficient and robust enzymatic process. A group led by Oikawa revealed that a biosynthetic assembly line composed of three modules (SfmA, SfmB and SfmC) of a non-ribosomal peptide synthase (NRPS) is responsible for the biosynthesis of the bis-THIQ scaffold of saframycin A (1) [
      • Li L.
      • Deng W.
      • Song J.
      • Ding W.
      • Zhao Q.F.
      • Peng C.
      • Song W.W.
      • Tang G.L.
      • Liu W.
      Characterization of the saframycin a gene cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribosomal peptide synthetase system for assembling the unusual tetrapeptidyl skeleton in an iterative manner.
      ,
      • Koketsu K.
      • Watanabe K.
      • Suda H.
      • Oguri H.
      • Oikawa H.
      Reconstruction of the saframycin core scaffold defines dual pictet-spengler mechanisms.
      ,
      • Koketsu K.
      • Minami A.
      • Watanabe K.
      • Oguri H.
      • Oikawa H.
      The pictet-spengler mechanism involved in the biosynthesis of tetrahydroisoquinoline antitumor antibiotics: a novel function for a nonribosomal peptide synthetase.
      ]. Unlike the canonical NRPS that catalyzes amide bond formation of amino acids, the key and unique NRPS module SfmC is capable of assembling two molecules of tyrosine derivative 7 and peptidyl aldehyde 8 bearing a long fatty acyl chain to construct the bis-THIQ scaffold 9, together with installation of sp3-stereogenic centers in place of amide bond formation (Scheme 1) [
      • Koketsu K.
      • Watanabe K.
      • Suda H.
      • Oguri H.
      • Oikawa H.
      Reconstruction of the saframycin core scaffold defines dual pictet-spengler mechanisms.
      ].
      Scheme 1
      Scheme 1SfmC-catalyzed in vitro enzymatic assembly of 7 and 8 to form the bis-THIQ scaffold 9.
      Herein we report development of a chemo-enzymatic synthetic process to gain concise and flexible access to the bis-THIQ scaffold by employing simple synthetic substrates with exploration of wide substrate scope for SfmC as a follow up comprehensive account of our previous communication focused on the total synthesis of jorunnamycin A and saframycin A (Fig. 2) [
      • Tanifuji R.
      • Koketsu K.
      • Takakura M.
      • Asano R.
      • Minami A.
      • Oikawa H.
      • Oguri H.
      Chemo-enzymatic total syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3.
      ]. The long fatty acyl chain condensed with the l-Ala component in peptidyl aldehyde 8 is critical. We thus verified the effect of the chain length of the fatty acid moiety on SfmC-catalyzed assembly with the tyrosine derivative 7 by employing synthetic substrates with C12, C14, and C16 chain lengths. To remove the fatty acyl chain via chemo- and site-selective bond-breaking reactions after the in vitro enzymatic conversions, we designed synthetic variants 2022 of peptidyl aldehyde 8 by replacing one of the two amide bonds in 8 with either an ester or an allyl carbamate linkage. The results of analytical-scale experiments to elucidate the correlation between substrate structure and enzymatic conversion efficiency allowed us to design suitable synthetic substrates and explore scale-up of the hybrid process. Two-pot chemo-enzymatic approaches involving cyanation at C21 and N-methylation improved both the yield and reproducibility of the key process, allowing synthesis of the pentacyclic products in a single day. Our hybrid approach also allowed subsequent site-selective chemical transformations to accomplish the rapid and collective total syntheses of jorunnamycin A (4), saframycin A (1), and N-Fmoc saframycin Y3 (35) in just 4–5 pots starting from simple synthetic substrates [
      • Tanifuji R.
      • Koketsu K.
      • Takakura M.
      • Asano R.
      • Minami A.
      • Oikawa H.
      • Oguri H.
      Chemo-enzymatic total syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3.
      ]. By applying a series of substrate variants by replacing the l-Ala moiety in 8 with eight different amino acids, we succeeded in substantially expanding the applicability of this chemo-enzymatic synthetic approach and gained insight into the correlation between substrate structure and enzymatic conversion efficiency.
      Fig. 2
      Fig. 2Outline for the systematic investigation for the SfmC-catalyzed conversion of a series of peptidyl aldehydes with (1) various chain lengths of the fatty acyl chain, (2) the incorporation of different cleavable functional groups, and (3) various amino acid components in place of the l-Ala unit. This approach provided insights into the correlation between substrate structure and enzymatic conversion efficiency and allowed the chemo-enzymatic total synthesis of jorunnamycin and saframycins with diverse side chains at the C1 position.

      2. Results and discussion

      2.1 Biosynthetic machinery composed of NRPS SfmA–C for the bis-THIQ scaffold

      The pentacyclic bis-THIQ scaffold of saframycin A (1) produced by Streptomyces lavendulae comprises of L-Ala, Gly, and two molecules of L-Tyr derivative. Biosynthetic machinery composed of the three NRPS modules (SfmA, B and C) assembles the amino acid components (Fig. 3) [
      • Koketsu K.
      • Watanabe K.
      • Suda H.
      • Oguri H.
      • Oikawa H.
      Reconstruction of the saframycin core scaffold defines dual pictet-spengler mechanisms.
      ]. This NRPS assembly line has three notable features: (1) attachment and detachment of a fatty acid acyl side chain before and after formation of the bis-THIQ scaffold, (2) catalysis by the reduction domain (Red) in SfmC of the reduction of three distinct thioesters on the peptidyl carrier protein (PCP) domain to liberate the corresponding aldehyde intermediates, and (3) catalysis by the Pictet-Spengler (PS) domain in SfmC to form the THIQ scaffold in place of the canonical peptide condensation reaction. The key module SfmC thus allows the iterative assembly of two molecules of the tyrosine derivative with two distinct aldehydes released upon reduction of the corresponding thioesters.
      Fig. 3
      Fig. 3Proposed NRPS biosynthetic assembly line of saframycin A (1).
      Despite the absence of fatty acyl chains in 1, biosynthesis begins with the loading of a long-chain fatty acid such as myristic acid, catalyzed by SfmA equipped with an acyl ligase (AL) domain (Fig. 3). The two NRPS modules SfmA and SfmB are responsible for amide condensation with l-Ala and Gly, respectively, to yield N-acylated alanyl-glycidyl thioester (intermediate A). The key module, SfmC equipped with the Red domain, catalyzes the reduction of thioester A instead of amide bond formation to release aldehyde 8. Imine formation between the amino group of the l-tyrosine derivative 7 loaded on the PCP domain and the released aldehyde 8, followed by regio- and stereo-selective Pictet-Spengler cyclization, furnishes mono-THIQ intermediate B with formation of a sp3-stereogenic center at C1, completing the first round of SfmC-catalyzed sequential conversions. The second round of the iterative assembly commences with reduction of the thioester intermediate B by the Red domain of SfmC to release aldehyde 10. Reloading of the l-tyrosine derivative 7 on the PCP domain and subsequent Pictet-Spengler cyclization, together with installation of a stereocenter at C11 with the released aldehyde 10, generates the thioester intermediate C bearing two THIQ units with linked adjacent stereogenic centers. Finally, reduction of the thioester moiety in C liberates aldehyde 11, followed by spontaneous intramolecular cyclization via nucleophilic attack of the secondary amine, affording the pentacyclic bis-THIQ scaffold 9 with incorporation of a hemiaminal moiety at C21. N-methylation of the secondary amine at the N12 position, followed by oxidation of the two phenols in the A- and E-rings to the corresponding quinones, affords 12 [
      • Peng C.
      • Tang Y.M.
      • Li L.
      • Ding W.
      • Deng W.
      • Pu J.Y.
      • Liu W.
      • Tang G.L.
      In vivo investigation of the role of SfmO2 in saframycin A biosynthesis by structural characterization of the analogue saframycin O.
      ]. Membrane-bound peptidase SfmE then removes the fatty acyl moiety in the C1 sidechain to secrete the primary amine 13 via a transmembrane efflux protein, SfmG [
      • Song L.Q.
      • Zhang Y.Y.
      • Pu J.Y.
      • Tang M.C.
      • Peng C.
      • Tang G.L.
      Catalysis of extracellular deamination by a FAD-linked oxidoreductase after prodrug maturation in the biosynthesis of saframycin A.
      ]. Extracellular oxidative deamination catalyzed by FAD-binding oxidoreductase SfmCy2 to install a ketone moiety at the C25 position furnishes saframycin S (14), and subsequent cyanation by treatment with KCN provides saframycin A (1) [
      • Song L.Q.
      • Zhang Y.Y.
      • Pu J.Y.
      • Tang M.C.
      • Peng C.
      • Tang G.L.
      Catalysis of extracellular deamination by a FAD-linked oxidoreductase after prodrug maturation in the biosynthesis of saframycin A.
      ].
      The unique mechanism involving the attachment and detachment of long-chain fatty acids in the biosynthetic assembly of saframycins is shared with related NRPS machineries of quinocarcin/SF-1739 [
      • Hiratsuka T.
      • Koketsu K.
      • Minami A.
      • Kaneko S.
      • Yamazaki C.
      • Watanabe K.
      • Oguri H.
      • Oikawa H.
      Core assembly mechanism of quinocarcin/SF-1739: bimodular complex nonribosomal peptide synthetases for sequential mannich-type reactions.
      ], naphthyridinomycin [
      • Pu J.Y.
      • Peng C.
      • Tang M.C.
      • Zhang Y.
      • Guo J.P.
      • Song L.Q.
      • Hua Q.
      • Tang G.L.
      Naphthyridinomycin biosynthesis revealing the use of leader peptide to guide nonribosomal peptide assembly.
      ,
      • Zhang Y.
      • Wen W.H.
      • Pu J.Y.
      • Tang M.C.
      • Zhang L.
      • Peng C.
      • Xu Y.
      • Tang G.L.
      Extracellularly oxidative activation and inactivation of matured prodrug for cryptic self-resistance in naphthyridinomycin biosynthesis.
      ], safracin [
      • Zhang Y.Y.
      • Shao N.
      • Wen W.H.
      • Tang G.L.
      A cryptic palmitoyl chain involved in safracin biosynthesis facilitates post-NRPS modifications.
      ], and ecteinascidin [
      • Rath C.M.
      • Janto B.
      • Earl J.
      • Ahmed A.
      • Hu F.Z.
      • Hiller L.
      • Dahlgren M.
      • Kreft R.
      • Yu F.
      • Wolff J.J.
      • Kweon H.K.
      • Christiansen M.A.
      • Håkansson K.
      • Williams R.M.
      • Ehrlich G.D.
      • Sherman D.H.
      Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743.
      ,
      • Schofield M.M.
      • Jain S.
      • Porat D.
      • Dick G.J.
      • Sherman D.H.
      Identification and analysis of the bacterial endosymbiont specialized for production of the chemotherapeutic natural product ET-743.
      ]. The attachment of the long fatty acyl chain may protect the N-terminal nitrogen of the l-Ala unit during sequential enzymatic conversions mediated by the three NRPS modules SfmA–C. The substantially increased lipophilicity of the biosynthetic intermediates by appendage of the long acyl chain is assumed to both prevent diffusion of the peptidyl aldehyde intermediates 8 and 10 liberated by reduction of the thioester linkage on the PCP domains of SfmB and SfmC, respectively, and to facilitate subsequent Pictet-Spengler cyclization by anchoring the hydrophobic reaction sites likely present in the supramolecular complexes of SfmB and SfmC. In addition to the pivotal roles of the fatty acyl group in sequential enzymatic conversions involving the non-covalently linked intermediates 8 and 10, the resulting bis-THIQ scaffold possessing a long acyl chain is likely trafficked to the cell membrane. Indeed, a membrane-bound peptidase, SfmE, catalyzes the hydrolysis of an amide bond between the fatty acyl group and the l-Ala unit in a highly site-selective manner. Furthermore, appendage of the long fatty acyl chain at the C1 side chain substantially attenuates the DNA alkylating abilities of the corresponding bis-THIQ scaffolds [
      • Tanifuji R.
      • Tsukakoshi K.
      • Ikebukuro K.
      • Oikawa H.
      • Oguri H.
      Generation of C5-desoxy analogs of tetrahydroisoquinoline alkaloids exhibiting potent DNA alkylating ability.
      ]. Thus, the NRPS biosynthetic machinery of saframycin (1), safracin, and naphthyridinomycin are likely to support the intracellular formation of prodrugs with lower toxicity in producer strains, and subsequent release of the antibiotics to the extracellular space upon scission of the fatty acyl chain.

      2.2 Influence of the length of the fatty acyl chain linked to the peptidyl aldehyde substrate on the SfmC-catalyzed assembly of the bis-THIQ scaffold

      As an initial effort to ascertain the correlation between substrate structure and enzymatic conversion efficiency, we verified the effect of fatty acyl chain length using a series of peptidyl aldehydes bearing a long hydrophobic acyl chain of different lengths. We designed and synthesized the three peptidyl aldehydes 15, 8, and 16, with C12, C14, and C16 acyl chain lengths, respectively, as substrates for SfmC-catalyzed conversions with the tyrosine derivative 7 (Fig. 4A) [
      • Tanifuji R.
      • Oguri H.
      • Koketsu K.
      • Yoshinaga Y.
      • Minami A.
      • Oikawa H.
      Catalytic asymmetric synthesis of the common amino acid component in the biosynthesis of tetrahydroisoquinoline alkaloids.
      ]. The NRPS module SfmC was co-expressed with phosphopantetheinyl transferase Sfp to provide as an active holo-form bearing the phosphopantetheinyl arm. The conversion efficiency of each peptidyl aldehyde (15, 8, and 16) was compared based on the UV absorbance of the corresponding pentacyclic products 1719 with an identical bis-THIQ scaffold as a chromophore (Fig. 4A). The substrate 8 (C14) bearing a myristoyl chain exhibited the highest conversion of the tested substrates: the conversion efficiencies of substrates 15 (C12) and 16 (C16), with two fewer or two more methylene moieties than 8 (C14), were significantly lower at 15 (27%), 16 (17%), respectively, relative to that of 8.
      Fig. 4
      Fig. 4A: Conversion rates of synthetic aldehydes (15, 8, and 16) with 7 to the corresponding pentacyclic scaffolds (1719) by SfmC-catalyzed enzymatic reaction followed by cyanation and N-methylation, based on UV absorbance of the identical bis-THIQ scaffold chromophore. Error bars represent the SEM (the standard error of the mean) calculated from three replicates. B: Comparison of the degree of cloudiness and precipitation for reaction mixtures of each substrate (15, 8, and 16) in the presence of SfmC.
      During the course of our investigation of these in vitro enzymatic conversions, we found that the solubilities of the three aldehydes 15, 8, and 16 added as 50 ​mM DMSO solution to aqueous reaction mixture in the presence of SfmC and co-factors were considerably different (Fig. 4B). The aldehyde 15 with a C12 acyl chain was completely dispersed as small particles, whereas 16 with the longest (C16) acyl chain of the three substrates precipitated as a large mass. In contrast, 8 with an intermediate chain length (C14) was suspended in aqueous solution and formed medium-sized particles. Thus, the relative solubilities of the three aldehydes 15, 8, and 16 were consistent with their conversion efficiencies under the conditions tested for the SfmC-catalyzed sequential reaction in vitro.
      The effects of acyl chain length were previously investigated by employing more water-soluble substrates having a thioester linkage with a hydrophilic CoA moiety as mimetics of the thioester intermediate A on SfmB [
      • Koketsu K.
      • Watanabe K.
      • Suda H.
      • Oguri H.
      • Oikawa H.
      Reconstruction of the saframycin core scaffold defines dual pictet-spengler mechanisms.
      ]. Treatment of the synthetic CoA-thioester with a C2 acyl chain resulted in no conversion, whereas the corresponding CoA-thioester substrates bearing a C12, C14 or C16 acyl chain did show conversion, with the C16 showing the highest conversion. This previous study using CoA-thioesters in place of a peptidyl aldehyde also indicated that the appendage of a long fatty acyl chain on the substrate is indispensable for SfmC-catalyzed sequential conversions to assemble the bis-THIQ scaffold. Furthermore, the involvement of C16 palmitoylated intermediates in the biosynthesis of safracin B was recently reported based on gene deletion experiments using Pseudomonas fluorescens A2-2, despite differences in Streptomyces and Pseudomonas microbial producers of saframycins and safracins, respectively [
      • Koketsu K.
      • Watanabe K.
      • Suda H.
      • Oguri H.
      • Oikawa H.
      Reconstruction of the saframycin core scaffold defines dual pictet-spengler mechanisms.
      ,
      • Zhang Y.Y.
      • Shao N.
      • Wen W.H.
      • Tang G.L.
      A cryptic palmitoyl chain involved in safracin biosynthesis facilitates post-NRPS modifications.
      ]. Overall, the experimental findings in the present study and in the previous reports indicate that the attachment of fatty acyl groups such as myristoyl (C14) and palmitoyl (C16) are critically important for the biosynthetic assembly of bis-THIQ scaffolds. Considering the relatively hydrophobic nature of the peptidyl aldehydes, we selected substrate analogs bearing a C14 unit as the optimal chain length that showed the best conversion and utilization as an aldehyde substrate in the following in vitro chemo-enzymatic reactions.

      2.3 Enzymatic conversions of substrate analogs bearing either an ester or a carbamate linkage in place of an amide bond

      To expand the substrate scope and conduct further chemical manipulation of the substituent at the C1 position by removing the fatty acyl chain from the pentacyclic bis-THIQ scaffold, we designed and synthesized the three peptidyl aldehydes 2022 bearing a cleavable linkage, such as an ester or allyl carbamate moiety, in place of the amide bonds in the biogenetic substrate 8 (Fig. 5) [
      • Tanifuji R.
      • Koketsu K.
      • Takakura M.
      • Asano R.
      • Minami A.
      • Oikawa H.
      • Oguri H.
      Chemo-enzymatic total syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3.
      ]. The membrane-bound peptidase SfmE is reported to be responsible for the chemo- and site-selective hydrolysis of the amide bond linked with the fatty acyl chain [
      • Song L.Q.
      • Zhang Y.Y.
      • Pu J.Y.
      • Tang M.C.
      • Peng C.
      • Tang G.L.
      Catalysis of extracellular deamination by a FAD-linked oxidoreductase after prodrug maturation in the biosynthesis of saframycin A.
      ]. However, chemical manipulation of the site-selective cleavage of one of the two amide bonds of the densely functionalized pentacyclic skeleton 18 is believed to be very difficult in a practical sense. We thus installed either an ester or a carbamate linkage in place of the amide bonds, which could be cleaved by base or palladium catalysis, respectively. While the modification of both amide bonds led to a decrease in SfmC-catalyzed conversions, the substrate analog 20 bearing an ester linkage installed closer to the aldehyde group exhibited optimal conversion efficiency leading to 23, at around 55% compared to the conversion of 8 into 18. The other two substrate analogs, 21 and 22, having either an ester or an allyl carbamate linkage in place of the amide bond adjacent to the fatty acyl chain in 8, were converted to the corresponding bis-THIQ scaffold 24 and 25, respectively, with essentially identical conversion efficiencies (approximately 14% compared to that for 8 into 18). These experimental results indicate that the two amide bonds installed in 8 play substantial roles in SfmC-catalyzed sequential reactions. Notably, compared to the amide linkage closer to the aldehyde, the amide bond that connects the fatty acyl chain appears to be more crucial for SfmC-catalyzed conversions with 7.
      Fig. 5
      Fig. 5Relative conversion efficiencies of peptidyl aldehydes (8, 2022) for SfmC-catalyzed sequential reactions with 7 and subsequent cyanation and N-methylation leading to the corresponding bis-THIQ scaffolds (18, 2325). Formation of the bis-THIQ scaffolds (18, 2325) was quantified based on UV absorbance of the identical bis-THIQ scaffold chromophore. Error bars represent the SEM calculated from three replicates.

      2.4 Chemo-enzymatic total synthesis of bis-tetrahydroisoquinoline alkaloids

      Having realized SfmC-catalyzed conversions of synthetic substrates 2022 with incorporation of a cleavable linker, we then sought to achieve the rapid and concise chemo-enzymatic total syntheses of saframycin A (1) and jorunnamycin A (4) [
      • Tanifuji R.
      • Koketsu K.
      • Takakura M.
      • Asano R.
      • Minami A.
      • Oikawa H.
      • Oguri H.
      Chemo-enzymatic total syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3.
      ]. The optimal conversion efficiency of the peptidyl aldehyde 20 into 23 bearing an ester linkage close to the aldehyde moiety led us to pursue the SfmC-catalyzed in vitro enzymatic assembly of 20 and 7 to produce 23. This was followed by chemical manipulations for removal of the fatty acyl chain and oxidation of the phenols in the A and E rings to the corresponding quinones of the targeted jorunnamycin A (4) (Scheme 2). SfmC-catalyzed enzymatic conversion of the simple substrates 7 and 20 to the pentacyclic compound 26 having a hemiaminal at C21, followed by treatment with KCN, generated an aminonitrile 27 in one pot. Given the labile nature of the secondary amine 27 upon concentration and experimental manipulations, reductive amination leading to stable tertiary amine 23 was immediately performed after removing SfmC by precipitation and filtration (Scheme 2). Treatment of the resulting supernatant containing 27 with formaldehyde and 2-picoline borane in acetonitrile and water (≈3:1) [
      • Sato S.
      • Sakamoto T.
      • Miyazawa E.
      • Kikugawa Y.
      One-pot reductive amination of aldehydes and ketones with α-picoline-borane in methanol, in water, and in neat conditions.
      ] resulted in complete N-methylation in only 30 ​min to provide 23. This streamlined chemo-enzymatic process afforded the appropriately functionalized pentacyclic core scaffold 23 (6.5 ​mg) in 18% yield in one day and a two-pot conversion from the substrate analogs 20 and 7. The isolated yield of 23 synthesized on a preparative scale using a falcon tube and round bottom glass flask with gentle stirring was greater than that on an analytical scale with little or no stirring. Unlike conventional multi-step syntheses of natural products, this chemo-enzymatic protocol does not require the isolation of any intermediate and thereby substantially minimizes laborious manipulations, allowing rapid production of 6.5 ​mg of 23. Removal of the fatty acid moiety of 23 was achieved by simple basic hydrolysis of the ester linkage with lithium hydroxide to afford primary alcohol 28 in 83% yield. Oxidation of the phenols in the A and E-rings with the cobalt-complex salcomine to form bis-quinone moieties enabled the chemo-enzymatic total synthesis of jorunnamycin A (4) just in 4 pots from the simple synthetic substrates 20 and 7. Whilst elegant and efficient total synthesis of jorunnamycin A has been achieved by Zhu, Chen, Stoltz and Yang [
      • Wu Y.C.
      • Zhu J.
      Asymmetrie total syntheses of (−)-Renieramycin M and G and (−)-Jorumycin using aziridine as a lynchpin.
      ,
      • Zheng Y.
      • Li X.D.
      • Sheng P.Z.
      • Yang H.D.
      • Wei K.
      • Yang Y.R.
      Asymmetric total syntheses of (−)-Fennebricin A, (−)-Renieramycin J, (−)-Renieramycin G, (−)-Renieramycin M, and (−)- jorunnamycin A via C-H activation.
      ,
      • Chen R.
      • Liu H.
      • Chen X.
      Asymmetric total synthesis of (−)-Jorunnamycins A and C and (−)-Jorumycin from L-tyrosine.
      ,
      • Liu H.
      • Chen R.
      • Chen X.
      A rapid and efficient access to renieramycin-type Alkaloids featuring a temperature-dependent stereoselective cyclization.
      ,
      • Welin E.R.
      • Ngamnithiporn A.
      • Klatte M.
      • Lapointe G.
      • Pototschnig G.M.
      • Mcdermott M.S.J.
      • Conklin D.
      • Gilmore C.D.
      • Tadross P.M.
      • Haley C.K.
      • Negoro K.
      • Glibstrup E.
      • Grünanger C.U.
      • Allan K.M.
      • Virgil S.C.
      • Slamon D.J.
      • Stoltz B.M.
      Concise total syntheses of (−)-Jorunnamycin A and (−) -jorumycin enabled by asymmetric catalysis.
      ], the present chemo-enzymatic approach could provide an alternative means for producing natural product 4.
      Scheme 2
      Scheme 2Details of the chemo-enzymatic synthetic process and total synthesis of jorunnamycin A (4).
      The chemo-enzymatic approach was then adopted to saframycin A (1) to demonstrate its flexible applicability for the total synthesis of several bis-THIQ natural products. We installed the terminal methyl ketone moiety in 1 via site-selective cleavage of the substituent at the C1 position by employing the substrate analog 21 bearing an ester linkage with the fatty acyl chain (Scheme 3) [
      • Tanifuji R.
      • Koketsu K.
      • Takakura M.
      • Asano R.
      • Minami A.
      • Oikawa H.
      • Oguri H.
      Chemo-enzymatic total syntheses of jorunnamycin A, saframycin A, and N-Fmoc saframycin Y3.
      ]. SfmC-catalyzed sequential conversions of 21 and 7, followed by cyanation and N-methylation of the resultant secondary amine 29, furnished pentacyclic 24 in 13% isolated yield. Whilst the conversion efficiency for 21 (14% relative to 8) on an analytical scale was less than half that for 20 (Fig. 5), iterative synthesis on a preparative scale repeated three times provided more than 10 ​mg of the bis-THIQ scaffold 24 (12.2 ​mg). Removal of the fatty acyl chain by basic hydrolysis of 24 afforded the secondary alcohol 30 in 91% yield. Salcomine-catalyzed oxidation of the phenols, followed by Swern oxidation of the resulting secondary alcohol without isolation of 31, allowed the chemo-enzymatic total synthesis of saframycin A (1) in just 5 pots from 7 and 21. The chemo-enzymatic approach described herein is distinct from previously reported total syntheses of historically important natural product 1 [
      • Fukuyama T.
      • Yang L.
      • Ajeck K.L.
      • Sachleben R.A.
      Total synthesis of (±)-Saframycin A.
      ,
      • Martinez E.J.
      • Corey E.J.
      Enantioselective synthesis of saframycin A and evaluation of antitumor activity relative to ecteinascidin/saframycin hybrids.
      ,
      • Myers A.G.
      • Kung D.W.
      A concise, stereocontrolled synthesis of (−)-Saframycin A by the directed condensation of α-amino aldehyde precursors.
      ,
      • Myers A.G.
      • Plowright A.T.
      Synthesis and evaluation of bishydroquinone derivatives of (−)-Saframycin A: identification of a versatile molecular template imparting potent antiproliferative activity.
      ,
      • Dong W.
      • Liu W.
      • Liao X.
      • Guan B.
      • Chen S.
      • Liu Z.
      Asymmetric total synthesis of (−)-Saframycin A from L-tyrosine.
      ,
      • Kimura S.
      • Saito N.
      A stereocontrolled total synthesis of (±)-Saframycin A.
      ], possibly allowing expeditious access to the medicinally intriguing complex bis-THIQ scaffold.
      Scheme 3
      Scheme 3Chemo-enzymatic total synthesis of saframycin A (1).
      We then extended the chemo-enzymatic approach to a congener of 1, saframycin Y3 bearing a terminal amino group on the l-Ala component (Scheme 4). Site- and chemo-selective cleavage of the fatty acyl chain with formation of the terminal amino group was achieved by employing the substrate analog 22 bearing an allyl carbamate moiety instead of the amide linkage in 8 for the SfmC-catalyzed chemo-enzymatic process. The two-pot chemo-enzymatic synthesis employing 22 and 7 was repeated twice to secure approximately 13 ​mg of 25 via the secondary amine 32. Cleavage of the allyl carbamate linkage in 25 proceeded smoothly in dichloromethane in the presence of a catalytic amount of Pd(PPh3)4, and phenylsilane, as a reducing agent for the resulting π-allyl palladium species [
      • Dessolin M.
      • Guillerez M.G.
      • Thieriet N.
      • Guibé F.
      • Loffet A.
      New allyl group Acceptors for palladium catalyzed removal of allylic protections and transacylation of allyl carbamates.
      ], to afford primary amine 33. Cobalt-mediated oxidation of the phenols on the A and E-rings in 33 is believed to provide straightforward access to saframycin Y3. However, it was very difficult to isolate saframycin Y3, which possesses both quinone rings and a primary amine in the vicinity of the A-ring, presumably due to the labile nature of the products. We thus protected the primary amino group prior to oxidation to generate quinone moieties to isolate N-Fmoc saframycin Y3 (35). Palladium catalyzed removal of the acyl chain and subsequent Fmoc protection of the resulting primary amino group produced 34 in 80% yield (2 steps). Salcomine-catalyzed oxidation of the two phenols gave rise to the N-Fmoc saframycin Y3 (35) in 59% yield. The chemo-enzymatic process allows the rapid generation of C5-desoxy variants such as 33 and 34 with lower oxidation states compared to the natural products. Notably, the C5-desoxy variant 33 bearing phenolic hydroxyl groups was demonstrated to exhibit greater DNA alkylating ability with a wide range of sequence variations compared to the naturally occurring and commercially available cyanosafracin B (2), accessible by Pseudomonas fluorescens fermentation [
      • Tanifuji R.
      • Oguri H.
      • Koketsu K.
      • Yoshinaga Y.
      • Minami A.
      • Oikawa H.
      Catalytic asymmetric synthesis of the common amino acid component in the biosynthesis of tetrahydroisoquinoline alkaloids.
      ].
      Scheme 4
      Scheme 4Chemo-enzymatic synthesis of N-Fmoc saframycin Y3 (35).

      2.5 Chemo-enzymatic synthesis of bis-THIQ scaffolds with installation of various amino acid components

      To expand the substrate scope of SfmC-catalyzed chemo-enzymatic sequential conversions, a series of substrate analogs (36ah) bearing various l- or d-amino acids in place of the l-Ala of 8 were designed and synthesized (Fig. 6). Of the tested synthetic variants, analog 36a bearing an l-Leu residue with increased steric hindrance of the sidechain, corresponding to the substrate with an attached isopropyl group to the methyl group of l-Ala in 8, showed the highest conversion efficiency (91% compared to that of the biosynthetic intermediate 8). The conversion of the synthetic substrates 36b and 36c having l-Met and l-Phe residues, respectively, was in 33% and 25% relative to that of 8. These results indicated that SfmC was tolerant of the appendage of a methylthio methylene or phenyl group on the methyl substituent in 8. In contrast, installation of a branched substituent on the l-Ala sidechain in 8 hampered enzymatic conversion of the synthetic substrates 36d and 36e bearing l-Val and l-Ile, respectively, leading to decreased efficiency (23% and 6%). Although increased steric hindrance on the methyl substituent of the l-Ala component in 8 had an adverse effect, we unexpectedly found that replacement with l-Pro was tolerated upon incubation of 36f with SfmC, providing the corresponding bis-THIQ scaffold 37f with modest conversion (12%). In addition, reversal of the stereogenic center was accommodated by SfmC to some extent. Despite the substantially decreased conversions compared to that for 8, 36g (d-Ala) and 36h (d-Val) were converted to the corresponding pentacyclic compounds 37g and 37h with 2% and 13% conversion efficiencies, respectively. Taken together, our results suggested that replacement of the l-Ala with either l-Leu, l-Met or l-Phe, with extension of the substituent on the methylene group attached to the alpha carbon of the l-Ala component, was relatively well accepted by SfmC. In contrast, a change to either l-Val or l-Ile, resulting in the incorporation of a branched substituent on the methylene group in 8, led to substantial reduction in the conversion efficiency. Installation of the l-Pro, d-Ala, and d-Val was shown to be feasible, despite the low efficiencies. Notably, preparative scale conversions in two-pot enabled us to produce more than 2 ​mg (2.16 ​mg) of the pentacyclic product 37b with a point mutation of the amino acid residue from l-Ala to l-Met (Scheme 5). This hybrid process can rapidly and conveniently secure the minimum sample quantity (ca. 1–2 ​mg) required to perform both structural analysis and in vitro assays of natural product-based new chemical entities that are difficult to obtain by other means. Therefore, this chemoenzymatic synthetic approach could provide a fundamental means to accelerate drug lead discovery of natural products and their analogs.
      Fig. 6
      Fig. 6Conversion efficiencies of the biosynthetic intermediate (8) and its variants (36a36h) with 7 to the corresponding pentacyclic scaffolds (18, 37a37h) by SfmC-catalyzed enzymatic conversion followed by cyanation and N-methylation, based on UV absorbance of the identical bis-THIQ scaffold chromophore. Error bars represent the SEM calculated from three replicates.
      Scheme 5
      Scheme 5Preparative scale chemo-enzymatic synthesis of the pentacyclic scaffold 37b bearing an l-Met component.

      3. Conclusions

      At the dawn of the merger of in vitro engineered enzymatic transformations and precise organic synthesis, we focused on the unique NRPS biosynthetic assembly line to produce the bis-THIQ alkaloidal scaffold with the installation of two stereogenic sp3 carbon centers via iterative Pictet-Spengler reactions. The indispensable roles of the long chain fatty acyl group appended to the l-Ala component in the peptidyl aldehyde intermediate 8 in the SfmC-catalyzed sequential conversions with the tyrosine derivative 7 was verified by systematically investigating the relationship between the structure of the synthetic analogs and enzymatic conversion efficiency. Analytical scale experiments monitored by LC-MS system indicated that the substrate bearing a C14 fatty acyl chain resulted in the optimal conversion efficiency of the three tested substrates possessing a C12, C14 or C16 chain length acyl group. We then investigated the SfmC-catalyzed conversions of the three substrates 2022 having a C14 acyl chain but various cleavable functional groups involving either an ester or an allyl carbamate in place of one of the two amide bonds in the peptidyl aldehyde intermediate 8. Despite reduced conversion efficiencies compared to 8, all three substrates were converted to the corresponding pentacyclic skeleton, and synthetic substrate 20 bearing an ester installed by replacement of the amide closest to the aldehyde in 8 resulted in greater conversion efficiency than the other two analogs 21 and 22. These experiments indicated that the substructure in 8, comprising the C14 fatty acyl chain and the amide bond that links to the acyl group, could play crucial roles in SfmC-catalyzed sequential conversions.
      Based on these insights into in vitro conversions with SfmC, we then explored whether the chemo-enzymatic process could achieve both the total synthesis of naturally occurring bis-THIQ alkaloids and divergent synthesis to generate analogs with a distinct sidechain at the C1 position. The implementation of sequential chemical manipulations (cyanation and subsequent N-methylation), without the need to handle labile intermediates with either an hemiaminal moiety at C21 or a secondary amino group in the C/D ring, was demonstrated to be vital to substantially improve both the reaction scale and the reproducibility of the chemo-enzymatic process. The improved two-pot hybrid process, achieved through streamlined integration of a seven-step enzymatic conversion followed by a two-step chemical transformation, enabled rapid and flexible access to appropriately functionalized pentacyclic core scaffolds in one day, with installation of an ester or allyl carbamate group in the substituent at the C1 position. Further chemical transformations involving removal of the fatty acyl chains and oxidation of the phenols to form quinones allowed the chemo-enzymatic total synthesis of jorunnamycin A (4) and saframycins (1, 35), in just 4 and 5 pot sequences, respectively, starting from simple synthetic substrates.
      Contrary to the very limited substrate tolerance of NRPS assembly lines in general, which strictly discriminate the amino acid building block by the cognate A domains, we demonstrated substantially wide substrate tolerance of SfmC. Notably, enzymatic conversions with the synthetic substrates 36ae, in which the l-Ala unit was replaced with a different amino acid (l-Leu, l-Met, l-Phe, l-Val or l-Ile), were shown to produce the corresponding bis-THIQ scaffolds with acceptable efficiency. The substrate scope was further expanded by employing the synthetic substrates 36f36h installed with l-Pro, d-Ala or d-Val in place of the l-Ala, despite the reduced conversions. The establishment of a unique and environmentally benign hybrid enzymatic process with wide substrate scope will not only greatly simplify synthetic processes to gain rapid access to therapeutically important complex molecules, but will also provide a versatile platform for the structural diversification of bis-THIQ alkaloids.

      Declaration of competing interest

      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have patent applications on the development of the chemo-enzymatic synthetic process to generate bis-THIQ scaffolds.

      Acknowledgements

      We appreciate Prof. Hideaki Oikawa and Prof. Atsushi Minami of Hokkaido University for the valuable discussion and continuous support to explore the chemo-enzymatic total synthesis by utilizing SfmC. We are grateful for financial support from the Japan Science and Technology Agency (JST) ACT-X Grant Number JPMJAX211A , The Naito Foundation , Japan Foundation for Applied Enzymology and JSPS KAKENHI (Grant No. 20K15454 ) to R. Tanifuji, and JSPS KAKENHI (Grant No. 17H05433 and 19H02847 ), Nagase Science and Technology Foundation , and Japan Foundation for Applied Enzymology to H. Oguri. This work was also inspired by the international and interdisciplinary environments of the JSPS Asian CORE Program, “Asian Chemical Biology Initiative” as well as the JSPS A3 Foresight Program.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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