Bioinspired total synthesis of (+)-pleuromutilin

Domain: chemistry
Published: 2026-04-08
Editorial outcome: minor_revision

Abstract
(+)-Pleuromutilin, a natural antibiotic, possesses an intriguing and complex 5/6/8 tricyclic skeleton with
densely packed functional groups. It attracts extensive attention in the field of total synthesis due to its promising
antibacterial activity and distinctive structural framework. Here we report the total synthesis of (+)-pleuromutilin
inspired by its biosynthetic pathway, which expands the available toolbox, particularly by providing a practical
and efficient entry to (+)-12-epi-Tmutilin. The key steps of the synthesis include a t-BuOK-catalyzed Michael
addition to establish the AC9 quaternary center, a LiDBB-promoted reductive coupling to construct the five-
membered ring, and a bioinspired Zn-mediated Barbier reaction to connect C11 and C12, ultimately enabling
the construction of the eight‑membered ring. Starting from (+)-pulegone, we accomplish the total synthesis of
(+)-pleuromutilin in 18 steps and (+)-12-epi-mutilin in 16 steps, respectively.

Introduction
(+)-Pleuromutilin (1), a diterpene antibiotic, was first isolated from the fungus Clitopilus passeckerianus in
1951 (Fig. 1).1 (+)-Pleuromutilin exerts its mechanism of action by selectively binding to the highly conserved
peptidyl transferase center of bacterial ribosomes.2 Thus, the resistance to 1 is slow to develop, and it displays
minimal cross-resistance with existing antibiotics. So far, four semisynthetic derivatives of 1 have been approved
by the FDA.3-5 (+)-Mutilin (2), an interconvertible deacylated derivative of 1, was found during the spectroscopic
and degradation studies.6 Interestingly, 12-epi-pleuromutilins, derivatives of 12-epi-mutilin, have recently been
demonstrated to exhibit potent activity against Gram-negative pathogens which remain a global health concern.7-
8 Therefore, a flexible synthetic strategy enabling access to both pleuromutilin (1) and 12-epi-pleuromutilin
would be valuable.

Structurally, pleuromutilin (1) and mutilin (2) possess an intriguing and complex 5/6/8 tricyclic skeleton, and
contain eight contiguous stereocenters, three of which are quaternary carbon stereocenters. The densely
substituted 8-membered carbocycle is rare among natural products,9 and represents a pivotal challenge in the
total synthesis of pleuromutilin.10-11 To date, seven total syntheses of 1 have been reported, all of which involve
strategic construction of eight-membered ring.12-21 In 1982, Gibbons and coworker accomplished the first total
synthesis of racemic pleuromutilin, in which the 8-membered ring was generated by a Grob fragmentation.12 In
2013 and 2018, Procter’s group13-14 and Reisman’s group15 independently utilized a SmI2-mediated cyclization
to establish the 8-membered ring via the formation of C5−C14 bond, respectively. In 2017, Herzon’s group
creatively employed a nickel-catalyzed reductive cyclization to assemble the 8-membered ring by the formation
of C10−C11 bond.16-18 Furthermore, the groups of Boeckman,19 Pronin,20 and Luo21 have each developed distinct
ring-expansion strategies to construct the 8-membered ring.

The biosynthetic pathway of 1 has been well elucidated (Fig. 2A).22-24 Cyclisation of geranylgeranyl
diphosphate (GGPP) with the class II terpene synthase produced mutildienyl-PP (3), which was further catalyzed
by the class I terpene synthase and underwent formation of the 8-membered ring through ionisation-dependent
dephosphorylation, hydride shifts from C14 to C10, and then trapping with water to generate the tricyclic
premutilin (4). Premutilin (4) was converted to 1 via further oxidation and modification.

In connection with our ongoing studies towards bioinspired total synthesis of natural products,25 and inspired
by the biogenetic pathway of 1, we envisioned that the formation of C11−C12 bond could be used to assemble
the 8-membered ring, and the hydride shifts from C14 to C10 could be utilized to set the C10 stereocenter (Fig.
2B, 7 to 6). Thus, (+)-pleuromutilin (1) could be derived from compound 5 through an intramolecular Barbier
reaction. Compound 5 could be generated from 6 through the functional group transformation (FGT).
Accordingly, the C10 stereocenter of 6 could be generated from 7 through bromonium ion-promoted 1,5-hydride
shift. The cyclopentanone moiety of compound 7 could be derived from ketone-ester 8 by the formation of
C3−C4 bond via an intramolecular reductive coupling.26 In turn, compound 8 can be synthesized from
commercially available and inexpensive (+)-pulegone (10) through 9 by the construction of two quaternary
carbon stereocenters.

Results
Preparation of the 1,5-hydride shift precursor
Our synthesis commenced with the preparation of the key 1,5-hydride shift precursor 7 (Fig. 3). The sequential
acylation of (+)-pulegone (10) with ethyl cyanoformate (Mander’s reagent) followed by alkylation with methyl
iodide (MeI) in a single synthetic operation proceeded smoothly to provide the desired product 11 and its C5-
epimer (C5-epi-11) in 85% combined yield as a mixture of two inseparable diastereoisomers (5:1 at C5) in favor
of 11,27 thus constructing the first quaternary carbon stereocenter. Selective reduction of the ester group in 11 by
sequential deprotonation of 11 with LDA followed by reduction with LiAlH4 afforded the corresponding primary
alcohol,28 which was then protected with TBSCl to afford ketone 9a in 57% yield and ketone 9b in 6% yield by
two steps.

Initially, Michael addition of compound 9 to methyl acrylate with DBU in DMSO at 120 °C successfully
provided the desired ketone-ester 8 in 27% yield with 30% conversion (Fig. S1),29 which gave us strong
confidence in optimizing this reaction. The low conversion is partially due to the polymerization of methyl
acrylate. After extensive screening, we found that the Michael addition promoted by a catalytic amount of t-
BuOK (0.10 equiv) and t-BuOH (0.10 equiv) in THF at -20 °C to provide the desired product 8 as a single
diastereomer in 90% yield,30 thus constructing the second quaternary carbon stereocenters. It should be
mentioned that the reaction temperature dramatically affected the diastereoselectivity, and lower temperature
favored the desired C9 configuration. Michael addition of ketone 9b to methyl acrylate could also provide 8 in
the same yield, and the mixture of ketone 9a and 9b could be directly used in large scale. The excellent facial
selectivity of the reaction could be explained as depicted in Fig 4. Two possible transition states, TS-1 and TS-
2, could be considered. Because of the significant steric hindrance of the OTBS group in TS-2, the less hindered
TS-1 should be favored, allowing the formation of the desired 8.31-32 Notably, when ketone 11 was used in the
same Michael reaction, the facial selectivity is reversed (see SI).

With compound 8 in hand, we proceeded to strategically construct the cis-fused 5/6 ring system via a direct
reductive coupling between C4 ketone and C3 ester, which provided a step-economic transformation (Fig. S2).
After many experimental trials, we found that LiDBB (Lithium 4,4’-Di-tert-butylbiphenylide) gave the best
result, affording not only the desired -hydroxyketone 14, but also 21% of the over-reduction diol 13.33 Diol 13
could be readily converted to 14 by oxidation with SO₃·Py and TEA.34 Dehydroxylation of -hydroxyketone 14
with SmI2 furnished the desired ketone 7.35

Having secured the stereocenters of compound 7, we investigated the key 1,5-hydride shift (Fig. S3).
Treatment of compound 7 with NBS in ethanol induced an intramolecular 1,5-hydride shift to afford C10-epi-6
as the sole product in 70% yield, whose structure was confirmed by X-ray crystallography analysis.
Unfortunately, the C10 stereocenter of 6 was not the desired configuration. Two possible transition states, TS-3
and TS-4, could be considered (Fig. 5).36-37 Because of the significant steric hindrance of the C10 methyl group
in TS-4, the less hindered TS-3 should be favored, allowing the formation of the undesired C10-epi-6.

Total synthesis of (+)-pleuromutilin (1), (+)-mutilin (2), and 12-epi-mutilin (23).
We turned our attention to convert the olefin 7 into the corresponding primary alcohol 15 (Fig. 6). After several
trials, we found that hydroboration-oxidation of 7 with Thx-BH2 afforded the desired alcohol 15 in 60% yield
with a 2:1 diastereomeric ratio in favor of the desired C10 stereocenter.38-39 Acetylation of alcohol 15 with Ac2O
delivered compound 16 in 95% yield. Deprotection of the TBS group in 16 with HF/pyridine followed by
oxidation of the resulting alcohol with IBX provided aldehyde 17 in 90% overall yield. Inspired by Herzon’s
work and in order to induce the desired C14 stereocenter, titanocene-mediated addition of 3-bromo-2-
methylpropene to aldehyde 17 was conducted.16-18 Gratefully, the desired diastereomer 18 was formed with 10:1
stereoselectivity.

Removal of the acetyl group of 18 with Me3SnOH provided alcohol 19,40 and the structures of compounds 18
and 19 have been unambiguously confirmed by X-ray crystallography analysis. Subsequently, compound 19
underwent intermolecular olefin metathesis with cis-1,4-diacetoxy-2-butene to furnish compound 20.41-42 The
C11 primary hydroxyl group played a pivotal role in olefin metathesis reaction, as evidenced by the sluggish
conversion and negligible product formation when compound 18 was subjected to identical condition.
Subsequently, selective oxidation of primary hydroxyl of 20 with IBX afforded the desired aldehyde 21 in 82%
yield. X-ray crystallography analysis of compound 21 revealed that the olefin metathesis products exclusively
adopted the trans-alkene configuration.

With 21 in hand, attempts to assemble the eight-membered ring were investigated (Fig. S4). Initially, a
palladium-catalyzed allylation was explored.43-44 There was no reaction at low temperature, and the starting
material decomposed at high temperature. A SmI₂-mediated reductive cyclization of 21 was further conducted.
However, the aldehyde reduction product, compound 20, was exclusively obtained as the sole product. These
results indicated that the poor leaving group ability of the acetate moiety may account for this outcome. After
systematic evaluation, we replaced the acetate group with a halogen to enable Barbier reaction. Removal of the
acetyl group of 21 with Me3SnOH furnished alcohol 22. Treatment of allylic alcohol 22 with TsCl successfully
provided the corresponding allyl chloride 5.45 However, the allyl chloride 5 proved unstable and underwent
degradation during the concentration process. Thus, a one-pot procedure was implemented wherein the allyl
chloride was generated in situ and subsequently subjected to zinc-mediated Barbier reaction (Fig. 7).46-48 The
condition of Zn in DMF gave the best result (entry 5), successfully affording both (+)-mutilin and 12-epi-mutilin.
Surprisingly, under the NHK reaction condition,49 (+)-12-epi-mutilin was obtained as the sole product (entry 6).
We speculate that this outcome is presumably due to the steric hindrance of the two chlorine atoms attached to
the trivalent chromium, which prevents the formation of the six-membered ring transition state TS-5, thus
allowing only the TS-6 transition state to operate, leading exclusively to (+)-12-epi-mutilin (see Fig. S5). Finally,
(+)-pleuromutilin (1) was obtained in 74% yield by stepwise acylation of the C11 and C14 alcohols followed by
methanolysis of the corresponding trifluoroacetyl esters.11

In summary, we have achieved the total synthesis of (+)-pleuromutilin (1) in 18 steps and (+)-12-epi-mutilin
(23) in 16 steps from commercially available (+)-pulegone (10). The catalytic Michael addition followed by
LiDBB-mediated reductive coupling constructs the cis-fused 5/6 ring system concisely, while the bioinspired
Barbier reaction forges the C11−C12 bond of the 8-membered ring collectively representing previously
unreported annulation strategies for pleuromutilin (1). Concurrently, we explored a new strategy to selectively
synthesize (+)-12-epi-mutilin (23) via NHK reaction. This work demonstrates the efficiency and versatility of
bioinspired synthesis, providing new avenues for the derivatization of pleuromutilin-type antibiotics and thus
supplying new chemical entities for breakthroughs in combating antibiotic resistance.

Methods
All reagents were obtained from commercial suppliers unless otherwise stated. Tetrahydrofuran (THF), diethyl
ether (Et2O), toluene (PhMe), and dichloromethane (CH2Cl2) were purified by solvent purification system.
Flasks were flame-dried under vacuum and cooled under a stream of argon. Flash chromatography was
performed using silica gel (200–300 mesh) with solvents distilled before use.

Nuclear magnetic resonance (1H and 13C NMR ) spectra were recorded on Bruker AVIII–400 [1H NMR (400
MHz), 13C NMR (101 MHz)]. The solvent signal was used for 1H NMR (CDCl3, 7.26 ppm), 13C NMR (CDCl3,
77.16 ppm). The following abbreviations are used for the multiplicities: s: singlet, d: doublet, t: triplet, q: quartet,
m: multiplet, brs: broad singlet for proton spectra and carbon spectra. Coupling constants (J) are reported in
Hertz (Hz).

Mass spectra were recorded using a PE SCLEX QSTAR spectrometer. Optical rotations were measured on a
Rudolph Autopol VI Polarimeter, 1 cm, 0.2 mL cell. Melting points were measured on a Buch Melting Point M–
560. X-ray single crystal was selected on an XtaLAB Synergy R, HyPix diffractometer.

Data availability
CCDC 2464290, 2464291, 2464292, 2464293, contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033. Details about materials and methods, experimental procedures,
characterization data, and NMR spectra are available in the Supplementary Information. All data are available
from the corresponding author upon request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "(+)-Pleuromutilin has long been a focal point in natural product total synthesis due to its notable antibacterial activity and\nunique 5/6/8 ring system, making this a highly competitive field. Jia and coworkers describe in this manuscript a bioinspired\ntotal synthesis of (+)-pleuromutilin and (+)-12-epi-mutilin. The synthetic strategy is novel and distinct from previously reported\nworks. Both the strategic bond disconnections and the specific bond-forming methods employed here are novel. The\nsynthesis employs a Michael addition to methyl acrylate and a LiDBB-mediated reductive coupling to construct the 5/6 fused\nring system with two all-carbon quaternary centers, and a bioinspired Zn-mediated Barbier reaction to assemble the bridged\neight-membered ring which represents the first time this strategic bond disconnection has been applied in the total synthesis\nof (+)-pleuromutilin. The manuscript was well written and is easy to follow, and the Supporting Information is of high quality.\nIn short, this is an excellent piece of work on some very challenging targets. I believe that the present work deserves to be\npublished in Nature Communications after the following issues are properly addressed:\nComments/remarks:\n1. Reisman's 2018 work also utilized a 1,5-hydride shift, yet it yielded the correct C10 configuration. In this work, however,\nthe opposite configuration was obtained. Apart to the mechanistic explanation provided in the manuscript, are there other\nfactors that could influence the stereoselectivity of the 1,5-hydride shift reaction?\n2. The Nozaki-Hiyama-Kishi (NHK) reaction in the article stereospecifically affords (+)-12-epi-mutilin. Could one potentially\nattempt to epimerize all of the (+)-mutilin to (+)-12-epi-mutilin by subjecting it to a C12 configuration inversion under similar\nconditions using, for instance, CrCl3?\n3. In the attempts to construct the 8-membered ring, only Zn and Cr were successful, while In, Sn, and Sm—all common\nmetals for Barbier reactions—failed to yield the desired cyclization. Could the authors provide a rational explanation for this\nobserved metal-dependent reactivity?\nSuggested Revisions:\n1. Add the definite article \"the\" before \"fungus\" and \"FDA\" in Page 1.\n2. The hydride shift from C14 to C10, utilized to set the C10 stereocenter, is currently misreferenced as (Fig. 1B, 7 to 6). This\nshould be corrected to (Fig. 2B, 7 to 6) in page 3.\n3. The phrase \"NBS-induced 1,5-hydride shift\" should be changed to \"bromonium ion-promoted 1,5-hydride shift\" to more\naccurately describe the reaction mechanism in Page 3.\n4. The numeral \"1\" in \"ethyl c1yanoformate (Mander’s reagent)\" in Page 4 should be removed. The authors are advised to\ncarefully proofread the entire manuscript for similar errors.\n5. On page S6 of the Supporting Information (SI), the page title for page S7 appears erroneously at the bottom of the page. This and similar typesetting errors should be corrected throughout the manuscript and SI to ensure professional\npresentation.\n6. In the NOE spectra presented in Page S40 and S45 in SI, dashed lines could be added to connect the correlated protons.\nThis would greatly facilitate the reader's identification and interpretation of the key spatial interactions.\nApart from the issues raised above, the work meets the standards for publication in Nature Communications.", "reviewer_2": "This manuscript presents an outstanding and meticulously designed bioinspired total synthesis of (+)-pleuromutilin and\n(+)-12-epi-mutilin, starting from the commercially available, cost-effective feedstock (+)-pulegone—an achievement that\naddresses the long-standing challenge of constructing the complex 5/6/8 tricyclic skeleton with eight contiguous\nstereocenters (three of which are quaternary carbons) characteristic of these valuable diterpene antibiotics. The synthetic\nstrategy stands out for its innovative annulation tactics: the t-BuOK-catalyzed Michael addition efficiently establishes the C9\nquaternary center with excellent diastereoselectivity (90% yield as a single diastereomer), while the bioinspired Zn-mediated\nBarbier reaction (implemented via a practical one-pot procedure to overcome allyl chloride instability) cleverly forges the\nC11−C12 bond of the rare eight-membered carbocycle—representing previously unreported and step-economical\napproaches to pleuromutilin’s core. Complemented by the LiDBB-promoted reductive coupling for cis-fused 5/6 ring\nformation, the 18-step synthesis of (+)-pleuromutilin and 16-step synthesis of (+)-12-epi-mutilin are concise and absolutely\nbeautiful.\nNotably, this work’s value extends beyond synthetic elegance: by enabling flexible access to both pleuromutilin and 12-epi-\nmutilin derivatives, it provides a powerful platform for antibiotic derivatization and combating rising antimicrobial resistance.\nThe manuscript is well-organized, with clear retrosynthetic analysis, detailed reaction optimization (e.g., Barbier/NHK\nreaction screening), and thorough discussion of stereoselectivity mechanisms (e.g., transition state models for Michael\naddition and 1,5-hydride shift), reflecting rigorous scientific inquiry. Overall, this study represents a significant advance in\nbioinspired natural product synthesis and antibiotic research, showcasing the efficiency of biomimetic strategies in tackling\ncomplex molecular architectures. It is highly meritorious of publication following minor revisions to address the below\nclarifications.\nA few questions and suggestions for clarification:\n1. In the intermolecular olefin metathesis of compound 19 with cis-1,4-diacetoxy-2-butene to form compound 20, the\nmanuscript notes that products exclusively adopted the trans-alkene configuration (confirmed by X-ray of 21). Was any Z-\nisomer detected or observed during the reaction, even if not isolated?\n2. Would the Barbier reaction give a better yield if the C-14 hydroxyl group got protected or esterified?", "reviewer_3": "In this manuscript, Jia and co-workers describe a concise and stereocontrolled total synthesis of the antibiotic diterpenoids\n(+)-pleuromutilin, (+)-mutilin, and 12-epi-mutilin, a class of natural products known to target the bacterial ribosome. These\nmolecules are a high-value and synthetically challenging targets. The authors present a strategically innovative approach\nthat addresses key challenges in the synthesis of this class of molecules.\nThe highlights of this total syntheses including: a) a stereospecific intermolecular Michael addition to install the sterically\nhindered C9 quaternary stereocenter, b) an effective LiDBB-promoted reductive coupling to establish the 5/6 fused ring\nsystem; c) a bioinspired Zn-mediated Barbier reaction to form the bridged eight-membered ring and C12 quaternary carbon\nstereocenter in one step. Additionally, the authors initially pursued a bioinspired, bromonium-ion mediated 1,5-hydride shift\nto install the C10 stereocenter. While this approach delivered the undesired diastereomer, they adeptly implemented a\nreliable hydroboration-oxidation sequence to overcome this challenge.\nTo date, seven total syntheses of (+)-pleuromutilin have been reported. This work significantly enriches the synthetic toolbox\nfor this important class of natural products, particularly by offering a practical and efficient route to (+)-12-epi-mutilin and its\nanalogues. A major strategic advantage of this synthesis lies in its ability to access previously unmodifiable positions within\nthe molecular framework, enabling late-stage functionalization and facilitating the development of novel derivatives.\nOverall, the manuscript presents a compelling and well-executed total synthesis with broad implications for medicinal\nchemistry and natural product synthesis. Therefore, this reviewer strongly recommends the manuscript for publication in\nNature Communications after mirror revisions.\n1. The chemical bonds highlighted in color in Fig. 1 are too thin. Please thicken them or change their color.\n2. On page 3, the sentence \"Premutilin (4) was converted to 2 and 1 via further oxidation and modification\" should be revised\nto \"Premutilin (4) was converted to 1 via further oxidation and modification\" to maintain consistency with the compounds\nshown in Fig. 2.\n3. On page 4, Line 7: \"ethyl c1yanoformate (Mander’s reagent)\" should be corrected to \"ethyl cyanoformate (Mander’s\nreagent)\"\n4. Is it possible that the stereoselectivity of the bromonium ion-mediated 1,5-hydride shift stems from a single, defined\napproach direction of the NBS?\n5. Why was a mixed solvent system employed for the initial attempts at the ring-closing reaction of compound 22? A mixed\nsolvent system is not typically the first choice for such reaction trials."}, "author_response": "Responses to Reviewer 1:\n1. Reisman's 2018 work also utilized a 1,5-hydride shift, yet it yielded the correct C10\nconfiguration. In this work, however, the opposite configuration was obtained. Apart to the\nmechanistic explanation provided in the manuscript, are there other factors that could influence\nthe stereoselectivity of the 1,5-hydride shift reaction?\nA: Thank you for your valuable question! In the work of Reisman, the 1,5‑hydrogen shift was\nattempted when the eight‑membered ring had already been constructed, and the conformation\nwas tightly constrained. In contrast, when we attempted the 1,5‑hydrogen shift, the\neight‑membered ring had not yet been formed, and the two side chains could rotate freely, which\nintroduced considerable challenges to the stereoselectivity of the reaction. Therefore, whether\nthe eight‑membered ring is pre‑constructed or not has a crucial influence on the stereoselectivity\nof the 1,5‑hydrogen migration.\n2. The Nozaki-Hiyama-Kishi (NHK) reaction in the article stereospecifically affords (+)-12-epi-\nmutilin. Could one potentially attempt to epimerize all of the (+)-mutilin to (+)-12-epi-mutilin\nby subjecting it to a C12 configuration inversion under similar conditions using, for instance,\nCrCl3?\nA: Various trivalent chromium reagents, along with different solvents and temperatures, were\nattempted to invert the configuration at the C12 position; However, all conditions failed to yield\nthe desired result.\n3. In the attempts to construct the 8-membered ring, only Zn and Cr were successful, while In, Sn,\nand Sm—all common metals for Barbier reactions—failed to yield the desired cyclization.\nCould the authors provide a rational explanation for this observed metal-dependent reactivity?\nA: We speculate that the reaction outcome may be influenced by the size of the metal atom. Zinc\nand chromium are in the same period and possess similar atomic sizes, both having relatively small\natomic radii. In contrast, indium, samarium, and tin have larger atomic sizes, which may be\nunfavorable for the formation of the six-membered ring transition state.\n4. Add the definite article \"the\" before \"fungus\" and \"FDA\" in Page 1.\nA: “the” was added before “FDA” and “fungus” in page 1.\n5. The hydride shift from C14 to C10, utilized to set the C10 stereocenter, is currently\nmisreferenced as (Fig. 1B, 7 to 6). This should be corrected to (Fig. 2B, 7 to 6) in page 3.\nA: “Fig. 1B” was corrected to “Fig 2B”.\n6. The phrase \"NBS-induced 1,5-hydride shift\" should be changed to \"bromonium ion-promoted\n1,5-hydride shift\" to more accurately describe the reaction mechanism in Page 3.\nA: \"NBS-induced 1,5-hydride shift\" was changed to \"bromonium ion-promoted 1,5-hydride\nshift\".\n7. The numeral \"1\" in \"ethyl c1yanoformate (Mander’s reagent)\" in Page 4 should be removed.\nThe authors are advised to carefully proofread the entire manuscript for similar errors.\nA: “1” was removed in the word “cyanoformate”.\n8. On page S6 of the Supporting Information (SI), the page title for page S7 appears erroneously\nat the bottom of the page. This and similar typesetting errors should be corrected throughout\nthe manuscript and SI to ensure professional presentation.\nA: Thank you for your valuable suggestion! We have corrected this accordingly.\n9. In the NOE spectra presented in Page S40 and S45 in SI, dashed lines could be added to connect\nthe correlated protons. This would greatly facilitate the reader's identification and interpretation\nof the key spatial interactions.\nA: The labeling of the NOE signals have been made more prominent.\nResponses to Reviewer 2:\n1. In the intermolecular olefin metathesis of compound 19 with cis-1,4-diacetoxy-2-butene to form\ncompound 20, the manuscript notes that products exclusively adopted the trans-alkene\nconfiguration (confirmed by X-ray of 21). Was any Z-isomer detected or observed during the\nreaction, even if not isolated?\nA: During the olefin cross-metathesis reaction, the formation of the Z-alkene was not observed,\nwhich is likely due to the steric hindrance imposed by the 5,6-fused ring system, resulting\nexclusively in the E-alkene product.\n2. Would the Barbier reaction give a better yield if the C-14 hydroxyl group got protected or\nesterified?\nA: In fact, contrary to our initial expectations, protecting the hydroxyl group at the C14 position\nproved to be extremely challenging. We attempted to introduce protecting groups such as TMS\nand OAc, but the starting material remained largely unreacted. This is likely due to the highly\ncongested chemical environment surrounding the C14 secondary alcohol. However, thanks to\nthe excellent tolerance of the Barbier reaction toward hydroxyl groups, the ring-closure reaction\nwas successfully performed without protecting the C14 hydroxyl group.\nResponses to Reviewer 3:\n1. The chemical bonds highlighted in color in Fig. 1 are too thin. Please thicken them or change\ntheir color.\nA: The color-coded bonds have been thickened in Fig. 1.\n2. On page 3, the sentence \"Premutilin (4) was converted to 2 and 1 via further oxidation and\nmodification\" should be revised to \"Premutilin (4) was converted to 1 via further oxidation and\nmodification\" to maintain consistency with the compounds shown in Fig. 2.\nA: Thank you for your valuable suggestion! We have corrected this accordingly.\n3. On page 4, Line 7: \"ethyl c1yanoformate (Mander’s reagent)\" should be corrected to \"ethyl\ncyanoformate (Mander’s reagent)\"\nA: \"ethyl c1yanoformate (Mander’s reagent)\" was changed to \"ethyl cyanoformate (Mander’s\nreagent)\".\n4. Is it possible that the stereoselectivity of the bromonium ion-mediated 1,5-hydride shift stems\nfrom a single, defined approach direction of the NBS?\nA: We tentatively propose that the attack direction of the electrophile is not singular. The first\nreason is that the eight-membered ring has not yet been formed, allowing the olefin side chain\nto adopt relatively free conformations. The second reason is that, in the hydroboration-oxidation\nreaction, we have consistently been unable to achieve exclusive stereoselectivity.\n5. Why was a mixed solvent system employed for the initial attempts at the ring-closing reaction\nof compound 22? A mixed solvent system is not typically the first choice for such reaction trials.\nA: During our initial attempts to achieve the Barbier reaction in a one-pot procedure, we found\nthat the conversion of allylic alcohol to allylic chloride proceeded with difficulty in THF.\nConsidering that THF is a common solvent in Barbier reactions, we therefore attempted a mixed\nsolvent system of DCM/THF. However, as described in the manuscript, our subsequent solvent\nscreening revealed that DMF could effectively serve as a single solvent for the one-pot\nprocedure."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The reviewer is satisfied with the author's revisions. Suggest publishing.", "reviewer_2": "This excellent manuscript has satisfactorily addressed all the questions raised by the reviewers, and the revised version is\nstrongly recommended for publication in Nature Communications.", "reviewer_3": "The authors have satisfactorily addressed all reviewer comments. The revised manuscript is now of high quality and meets\nthe standards of Nature Communications. I recommend acceptance."}, "author_response": ""}]

Decision letter