Biosynthesis and Synthesis of Griseoviridin and Viridogrisein

Abstract

Griseoviridin and viridogrisein are synergistic type A/B streptogramin antibiotics that cooperatively inhibit protein translation in Gram–positive pathogens. This review provides an overview of their discovery and bioactivity and highlights key biosynthetic and synthetic advances that have enabled analog development to address antibiotic resistance. The review is organized into five sections:

1. Introduction

2. Structural assignment and biological studies

3. Biosynthesis

4. Synthesis

5. Conclusion

Graphical Abstract

1. Introduction

Griseoviridin (GV) and viridogrisein (VG, also known as etamycin) are a pair of structurally distinct but functionally synergistic antibiotics produced by Streptomyces griseoviridis that were first isolated in Texas, USA in the 1950s.^1,2 These compounds belong to the streptogramin family of antibiotics, which are characterized by the cooperative action of the type A and B members in inhibiting Gram-positive bacterial growth.³ Griseoviridin belongs to type A streptogramins, which consist of cyclic polyunsaturated macrolactones derived from a hybrid peptide/polyketide pathway.⁴ Structural variation in type A streptogramins can arise from desaturation of the Pro residue and from its substitution with Ala or Cys.⁵ In addition to griseoviridin, type A streptogramins also include pristinamycin II, madumycin II, and the semisynthetic derivative dalfopristin (Figure 1). Viridogrisein belongs to type B streptogramins, which consist of cyclic hepta- or hexadepsipeptides. Beyond viridogrisein, type B streptogramins also include pristinamycin I, virginiamycin S, and the semisynthetic derivative quinupristin (Figure 1). Due to colocalization of their biosynthetic genes, type A and type B streptogramins are usually produced together by the same bacterial species in the ratio of approximately 70:30, which is also the ratio observed for the natural occurrence of GV/VG.⁶

Figure 1.

Chemical structures of streptogramin antibiotics.

In the mid-20^th century, often considered the golden age of antibiotic discovery, considerable efforts were spent on verifying the chemical structures of griseoviridin and viridogrisein. Later on, follow-up studies on their mode of antibacterial action and clinical applications were reported, in an attempt to offer new solutions for antibiotic resistance. At the same time, total synthesis campaigns began to offer efficient routes to these antibiotics to promote medicinal and structure-activity relationship studies. Comparing the structures of the two natural products, viridogrisein poses significantly less synthetic challenge than griseoviridin since it consists only of amino acids which could be linked together through peptide coupling and esterification. In contrast, progress in the synthesis of griseoviridin was considerably slower due to the difficulty in constructing the strained thioether lactone motif. Over 30 years of effort have been dedicated to the synthesis of griseoviridin, with a particular focus on the construction of the thioether fragment. Moreover, derivatives of GV/VG and other streptogramins have been designed and synthesized using both chemical and biological means to address the imminent threat of antibiotic resistance.

This review will discuss the structural and biological characterization, biosynthetic pathway elucidation, and total synthesis of griseoviridin and viridogrisein. In addition, a brief discussion of the future outlook of these two antibiotics and the streptogramin family will be mentioned.

2. Structural assignment and biological studies

The structure of griseoviridin was determined by X-ray crystallography in 1976⁷, but this initial work erroneously reported the stereochemical relationship of its 1,3-diol as trans. The correct configuration of the 1,3-diol moiety was later rectified as syn as shown in Figure 1.⁸ The structure of viridogrisein was revealed two decades earlier in 1958, during which the collective work of several groups⁹ assigned it as a macrocyclic depsipeptide containing seven amino acids. Only two (L-threonine and D-leucine) out of the seven amino acids are regarded as canonical amino acids.

Griseoviridin and viridogrisein are known for their independent and synergistic^10–12 potent activity against Gram-positive bacteria, and even some Gram-negative pathogens¹³. For example, viridogrisein demonstrated potent activity against hospital- and community-associated methicillin-resistant Staphylococcus aureus (HA- and CA-MRSA) in microbroth dilution assays, with minimum inhibitory concentrations (MICs) as low as 1–2 mg/L.¹⁴ Both griseoviridin and viridogrisein were also reported to have potent anti-mycobacterial activity against M. avium and M. intracellulare with MICs between 0.024 and 1.56 μg/mL, with markedly enhanced potency when used in combination.¹⁵

Like other streptogramins, griseoviridin and viridogrisein act by binding to the 50S subunit of the ribosome and interfering with peptidyltransferase activity, which is responsible for peptide chain elongation during translation.¹⁶ Studies have shown that type A streptogramins block substrate attachment to both the acceptor site and the donor site of the peptidyl transferase catalytic centre (PTC) on the ribosome¹⁷, while type B streptogramins prevent the extension of polypeptide chains and induce the detachment of incomplete protein chains¹⁸. The synergistic antibiotic activity is due to the irreversible increase in affinity of type B streptogramins for the ribosome in the presence of type A streptogramins, facilitated by the conformational change that occurs upon type A streptogramin binding.^19–22

Antibiotic resistance against streptogramins can occur through several different mechanisms, including ribosomal target modification^23,24, active efflux²⁵, enzymatic inactivation^26,27, and impermeability.²⁸ However, since type A and type B streptogramins have different structures and binding modes, they are inactivated by different mechanisms, and resistance against one type is usually insufficient to render the mixture inactive.¹³ For example, ribosomal target modification usually involves monomethylation of an adenine nucleotide of the ribosomal 50S subunit²⁹, leading to a conformational change that impacts the binding mode of only type B streptogramins; in contrast, type A antibiotics are not affected and synergy with type B antibiotics is retained.²³ Drug efflux pumps are membrane-associated proteins that export antibiotics out of the cell, mostly belonging to the ATP-binding cassette (ABC) transporter family or the major facilitator superfamily (MFS).^30,31 ABC transporters, commonly encoded by vga and lsa, are activated by ATP hydrolysis to pump out type A streptogramins.^32,33 Conversely, type B streptogramins are effluxed by MFS transporters, which are commonly encoded by msr and mef.^34,35 Enzymatic inactivation of type A streptogramin is commonly mediated by acetyltransferases, which acetylate the hydroxyl group, while type B streptogramins are inactivated by hydrolases that linearize the macrocyclic structure.^26,36,37

In recent years, much effort has been focused on overcoming resistance to streptogramin antibiotics. New antibiotic analogs have been designed and synthesized to address the aforementioned inactivation modes, and chimeric antibiotics have been developed to evade the resistance mechanism toward a specific type of streptogramin.^37–39 Moreover, efflux pump inhibitors have been designed based on efflux protein targets to overcome resistance.⁴⁰ The most successful example, Synercid, consisting of a combination of quinupristin and dalfopristin (Figure 1), was approved in 1999 for the treatment of Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) infections. Though it has been clinically used for over 20 years, the prevalence of resistance to quinupristin-dalfopristin remains low (<1%) in staphylococci.⁴¹

3. Biosynthesis

The biosyntheses of griseoviridin and viridogrisein were not fully understood until 2012, when the gene cluster responsible for the production of both molecules was identified within the Streptomyces griseoviridis (S. griseoviridis) genome. Subsequent gene inactivation and complementation experiments enabled confirmation of its function in GV/VG biosynthesis.⁴² In the genome, the sgv gene cluster is localized to a 105kb DNA region that consists of 36 open reading frames (ORFs). Within this region, enzyme-encoding genes for VG and GV biosynthesis are separated into distinct “halves” of the cluster (Figure 2a).

Figure 2.

A. Biosynthetic gene cluster of griseoviridin and viridogrisein. B. Key steps in the griseoviridin biosynthesis. C. Key steps in viridogrisein biosynthesis.

The griseoviridin backbone is assembled by a set of hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) modules encoded by sgvE1-E6, along with the acyltransferase SgvQ. sgvP was hypothesized to encode for a cytochrome P450 enzyme, which catalyzes a critical late-stage C–S bond formation. (Figure 2b) Inactivation of sgvP resulted in products lacking the C–S bridge, while trans-complementation of sgvP into the ΔsgvP mutant strain partially restored GV production.⁴³ By feeding the wild-type strain with [1-¹³C] labeled cysteine, it was verified that the C–S bond formation in GV occurs through direct coupling from the cysteine-derived free thiol of pre-griseoviridin.⁴³ Later on, the crystal structure of SgvP was revealed, which shed light on the potential roles of several loops and residues in catalysis.⁴⁴ In 2024, the Renata group provided the first direct evidence for the role of SgvP by using it to catalyze the direct C–S bond from the free thiol precursor (pre-GV) in vitro to produce GV.⁴⁵

In contrast, the viridogrisein scaffold is assembled by NRPS modules SgvD1-D4, wherein the thioesterase domain on SgvD4 is postulated to catalyze the final macrolactonization. SgvF, G, L-N, and S are related to syntheses of viridogrisein building blocks since it contains several noncanonical amino acids in its backbone. (Figure 2c) Among these enzymes, SgvM is a SAM-dependent C-methyltransferase, which catalyzes the C-3 methylation of 4-methyl-2-oxovalerate.⁴² Additionally, SgvM can also methylate unnatural substrates such as pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate.⁴⁶ In 2024, the X-ray crystal structure of SgvM was reported, revealing the incorporation of a Zn²⁺ ion in the active site and coordination effects of residues around it (His-296 and Asp-245).⁴⁷ In SgvM, the aliphatic β-residue of the substrates is located in an uncharged binding pocket formed by a number of hydrophobic residues (Met-144, Phe-149, Phe-241, Ala-276, Phe-288, Val-292, and Phe-329). These hydrophobic residues play a crucial role in substrate recognition by creating a lipophilic environment for the non-polar β-residue of the substrates, which offers valuable information for protein engineering of other α-keto acid C-MTs to expand their substrate scope.⁴⁷

Beyond the core biosynthetic genes, there are several resistance and regulatory genes located in the GV/VG BGC. A series of four genes: sgvA, C, and K, are found downstream, likely coding for the construction of a γ-butyrolactone (GBL) molecule. SgvT1, SgvT2, and SgvT3 are identified as three transporters in GV/VG BGC.⁴⁸ (Figure 2a) It has been revealed that SgvT1 is an MFS transporter which plays an important role in averting undesired intracellular GV/VG accumulation, whereas SgvT2 appears to serve as the sole ABC transporter and SgvT3 is an alternative MFS-based transporter that appears to serve as a compensatory protein in GV/VG biosynthesis. These features explained why ~75% of GV/VG were found in the supernatant during fermentation.

4. Synthesis

In 2000, the Meyers group accomplished the first total synthesis of (–)-griseoviridin and its C-8 epimer based on ring-closing olefin metathesis. Before that, a number of groups had attempted the synthesis of griseoviridin for over 20 years, and none had been successful.^49–53 In Meyers’ approach, griseoviridin was traced back to oxazole-containing diene 3 and thioether-containing allyl ester 9. Diene 3 was obtained in 3 steps from aldehyde 1, which was prepared from Weinreb amide 1’ as previously described.⁵⁴ Ester 9 was obtained from aldehyde 4 in 7 steps with 22% overall yield featuring electrophilic thiolation of ketoester 5 and Mitsunobu macrolactonization. Amide coupling and ring-closing metathesis were then applied to construct the core skeleton of the natural product and deprotection with PPTS completed the total synthesis.⁸ (Scheme 1)

Scheme 1.

Meyers’s synthesis of griseoviridin.

Conventional wisdom in retrosynthetic analysis suggests that griseoviridin be constructed convergently by breaking the molecule into two similarly-sized fragments, one containing the oxazole motif and the other containing the thioether lactone motif. (Scheme 2) Following this logic, numerous approaches have been developed toward the thioether-containing fragment since its strained 9-membered lactone offers many opportunities for creative route development.

Scheme 2.

General disconnections of griseoviridin for convergent synthesis.

In 1980, the Meyers group reported the synthesis of thioether 13, which features an alternative method for the formation of the key C–S bond was formed (Scheme 3).⁵⁵ Cysteine derivative 14 was converted to the corresponding sulfenyl chloride in situ and treated with olefin 15 to give a mixture of 16 and 17 in a 1:4 ratio. Further conversion of the undesired regioisomer 16 to 17 was achieved by heating in acetonitrile in the presence of epoxybutane as a proton scavenger. Ester 17 was next subjected to E1cB elimination to furnish the corresponding enoate as an E:Z mixture (70% Z, 30% E). This mixture was transformed into a single Z isomer (18) by the reversible addition of a phenyl thiyl radical. The MOM and t-Bu protecting groups were next removed, followed by Mitsunobu reaction to close the macrolactone, affording ring closure product 13 with the correct 5R configuration (Scheme 3).

Scheme 3.

Meyers’ initial route to thioether lactone 13.

Following Meyers’ seminal work, various synthetic approaches toward the sulfur-containing 9-membered ring adopted the Mitsunobu macrolactonization step.^56–59 One example can be found in Helquist’s synthesis of thioether lactone 25 in 1985.⁵⁶ The route began with 1,3-oxathiolanone 19, which was converted to the corresponding enolate upon treatment with LDA and condensed with aldehyde 20. The corresponding alkenyl sulfide 22 was obtained by treating 21 with MsCl and DBU to effect alcohol elimination, followed by NaOMe-mediated methanolysis. Although the dehydration gave a 1:2.5 mixture of Z/E isomers, NaOMe cleavage led exclusively to the Z isomer of alkene 22. S-alkylation with alkyl iodide 23 and THP removal generated 24, whereafter Mitsunobu lactonization was applied to close the ring to form the desired thioether lactone 25 (Scheme 4).

Scheme 4.

Helquist’s route to thioether lactone 25.

In the same year as Meyers’ first total synthesis, Marcantoni and Bartoli used cysteine derivative 26 to synthesize thioether lactone 30.⁵⁷ This starting material was converted to 27 by S-deprotonation and alkylation with ethyl α-bromoacetate. In turn, ester 27 was enolized in the presence of tBuMgCl and treated with aldehyde 28 to conduct aldol coupling. Upon concomitant dehydration and MOM deprotection with a combination of CeCl₃ and NaI, alkene 29 was obtained in high yield. Finally, Mitsunobu lactonization gave the targeted thioether lactone 30 in 68% yield (Scheme 5).

Scheme 5.

Marcantoni and Bartoli’s route to thioether lactone 30.

In 2003, Moreau and Campagne reported a model study using chiral alcohol 31 as their starting point, which was converted to vinyl iodide 32 by Horner–Wadsworth–Emmons olefination.⁵⁹ An intermolecular palladium-catalyzed cross-coupling between 32 and cysteine derivative 33 formed vinyl sulfide 34 in 63% yield, which was treated with TFA to remove the MOM-ether and the tert-butyl ester. Intramolecular Mitsunobu cyclization of the resulting intermediate afforded thioether lactone 35 in 43% yield over two steps (Scheme 6).

Scheme 6.

Moreau and Campagne’s route to thioether lactone 35.

In all the aforementioned strategies, Mitsunobu lactonization was performed as the last step of the sequence to achieve ring closure. In contrast, the Ardisson group in 2002 reported a different synthetic sequence in which thioalkynylation was performed first, followed by macrolactonization and hydrostannylation.⁵⁸ Alkyne 36 was reacted with thiosulfonate 37 in the presence of nBuLi to furnish the corresponding thioacetylene intermediate, which was deprotected with Amberlyst 15 to give hydroxy ester 38. Unsaturated nine-membered lactone 39 was formed from 38 under Mitsunobu conditions. Later, a vinyltin lactone intermediate was generated from 39 via palladium-catalyzed hydrostannylation. This intermediate was next converted to the corresponding iodolactone by Sn/I exchange, followed by Heck carbonylation to afford thioether lactone 40 in 47% yield over three steps (Scheme 7).

Scheme 7.

Ardisson’s route to thioether lactone 40.

In addition to Mitsunobu lactonization, several other strategies have been applied in the construction of the nine-membered thioether lactone. For example, in 2004, the Ardisson group reported a reductive cyclization strategy to close this ring.⁶⁰ Ethyl ynoate 43 was synthesized via esterification of cystine derivative 41 with alcohol 42 in 89% yield, followed by Zn/HOAc induced cyclization, which proceeded in 11% yield. It was observed that partial racemization occurred at C-8, leading to the S-configured product 44 in only 7% yield (Scheme 8).

Scheme 8.

Ardisson route to thioether lactone 44

All the synthetic strategies described above treat the nine-membered thioether lactone as an independent module to be constructed at an early stage of the synthesis before connecting it with the other oxazole-containing building block. However, in the biosynthetic pathway of griseoviridin, the nine-membered ring is proposed to be formed at the end of the biosynthetic sequence through an oxidative C–S bond formation catalyzed by SgvP. As previously mentioned, several reports offer indirect evidence that SgvP is responsible for the final C–S bond formation, but there has been no success in isolating or synthesizing the putative substrate of SgvP (known as pre-griseoviridin), or providing direct biochemical evidence for the function of SgvP. In 2024, the Renata group reported a chemoenzymatic synthesis of griseoviridin in nine steps (longest linear synthesis), which first prepared pre-griseoviridin chemically and subjected it to a direct C–S bond formation in the presence of SgvP.⁴⁵ Taking inspiration from previous synthetic approaches, they divided griseoviridin into two fragments, the oxazole-containing fragment 48 and the thiol-containing fragment 52. Syn-1,3-diol 45 was converted to vinyl iodide 46 with E-selective Takai olefination, which was followed by coupling with L-Ser-OMe to provide 47. Following treatment with Deoxo-Fluor and oxidation with BrCCl₃, the oxazole ring was formed, while LiOH unmasked the carboxylic acid to give fragment 48. In parallel, fragment 52 was obtained from alcohol 49 in 5 steps. Cross metathesis was applied to transform 49 to α,β-unsaturated alkene 50, which underwent ammonolysis and hydrostannylation to afford stannane 51. Condensation of 51 with protected cysteine and Fmoc deprotection yielded fragment 52. Pre-griseoviridin was formed from 48 and 52 via peptide coupling and Stille coupling, followed by deprotection of the diol moiety and the thiol group. Purified SgvP was successfully reconstituted with spinach ferredoxin (Fdx)/ferredoxin reductase (Fdr) as its redox partner, and the system was employed together with Opt13 (for NADPH regeneration) in the presence of NADP⁺ and TCEP, to give nearly full conversion of pre-griseoviridin to griseoviridin in 78% isolated yield. Beyond providing an efficient synthesis of griseoviridin, this work also provides direct evidence that the P450 SgvP indeed installs the key C–S bond during griseoviridin biosynthesis (Scheme 9).

Scheme 9.

Chemoenzymatic synthesis of griseoviridin by Renata.

SgvP is not the first reported P450 enzyme that can catalyze C–S bond formation.⁶¹ In 2018, Wu, Hong, and co-workers provided genetic and biochemical evidence for the role of CxnD in forging the C–S bond in chuangxinmycin biosynthesis⁶² (Scheme 10a) and in 2016, ThnC was implicated in the C–S bond formation in thienodolin biosynthesis⁶³. More recently, TleB, which normally catalyzes a C–N bond formation in teleocidin biosynthesis, was reported to mediate intramolecular C–S bond formation to generate 57 and 58 from 56.⁶⁴ Comparing the substrate structures across these enzymes and SgvP, it is worth noting that TleB, CxnD, and ThnC all act on electron-rich indole derivatives, whereas SgvP catalyzes C–S bond formation between a thiol group and a conjugated alkene. It has been hypothesized^61,64–70 that the C–S bond formation catalyzed by SgvP could proceed by way of alkene epoxidation, diradical recombination or formation of an S–X electrophile (Scheme 10b). To date, there has been no attempt to verify the mechanism either through computational analysis or spectroscopic means.

Scheme 10.

A. Prior reports of P450 catalysis for C–S bond formation. B. Plausible mechanisms for C–S bond formation catalyzed by SgvP.

The first total synthesis of viridogrisein was reported in 1973 by Sheehan and Ledis.⁷¹ In this synthesis, viridogrisein was achieved by the cyclization of linear peptide 61, which was obtained by linking a threonine-containing peptide 59 and a phenylsarcosine-containing peptide 60 through esterification. In turn, 59 and 60 were obtained through straightforward peptide couplings and deprotections. Here, the authors chose to perform macrocyclization on one of the peptide bonds instead of the lactone moiety because prior precedents had suggested that macrolactamization is more efficient than macrolactonization. Furthermore, the chosen disconnection provided better protecting group compatibility (Scheme 11).

Scheme 11.

Sheehan and Ledis’s route toward viridogrisein

In parallel, Okumura in 1978 investigated several analogs of viridogrisein through mutasynthesis, which was performed by adding modified amino acids to S. griseoviridis cultures to generate “neoviridogriseins,” viridogrisein analogs with one or more building blocks replaced.⁷² Feeding L-α-amino-n-butyric acid (an analog of alanine) led to neoviridogrisein III, where the alanine in VG was replaced by 2-aminobutyric acid, whereas feeding D-proline in place of allo-hydroxy-D-proline resulted in a molecule lacking the hydroxyl group on the pyrrolidine ring (neoviridogrisein I/II). (Figure 3) Interestingly, these neoviridogriseins retained strong antibiotic activity toward various Gram-positive bacteria such as Staphylococcus aureus FDA 209P, S. aureus Smith, Streptococcus pyogenes NY-5, Sarcina lutea, and Bacillus subtilis ATCC 6633.⁷³ Several additional analogs of viridogrisein containing 4-chloro-L-proline motif have been successfully produced using the same mutasynthesis strategy.⁷⁴

Figure 3.

Chemical structures of neoviridogriseins

Beyond viridogrisein, other antibiotics in the streptogramin family have also been used as starting points for analog design and production. In 2020, the Seiple group offered a versatile analog design to virginiamycin M2 based on its binding to E. coli 50S ribosome.³⁸ As mentioned before, one mode of antibiotic resistance to type A streptogramin is mediated by acetyltransferases, which acetylate the hydroxyl group on the backbone of family members such as virginiamycin M2. This knowledge informed the design of new analogs of virginiamycin M2 that would avoid capture by virginiamycin acetyltransferases (Vats). The synthetic approach hinged upon the modular use of seven building blocks (62–68), which could be diversified further to efficiently generate over sixty new analogs (Scheme 12). Fragment 71 and 74 were built from 3 building blocks each, and were combined through peptide coupling, followed by Stille coupling to close the macrocycle, and final deprotection to yield the target molecule. Further building block diversification led to a wide range of analogs, among which compound 76 showed measurable activity (32 μg/mL) against ABC-F-expressing E. faecalis and Gram-negative E. coli (16 μg/mL), species that are highly resistant to streptogramins. Moreover, 76 showed moderate-to-excellent activity (0.5–16 μg/mL) against clinically isolated strains resistant to type A streptogramins, β-lactams, tetracyclines, and aminoglycosides.

Scheme 12.

Seiple’s synthesis of virginiamycin M2 and its analogs.

This work demonstrates the inherent potential of combining the knowledge of resistance mechanisms and the power of synthetic chemistry to overcome antibiotic resistance. To date, no analogs of griseoviridin have been reported. It remains to be seen whether the same strategy could be expanded to other type A streptogramins and enable their adaptation for better clinical longevity.

5. Conclusion

Griseoviridin and viridogrisein, a synergistic pair of streptogramin antibiotics co-isolated in the mid-20th century, have remained the subject of sustained interest due to their potent antibacterial activity. While early research work emphasized structural elucidation and biological function, focus in recent decades has shifted toward shedding light on their biosynthetic gene clusters and the development of innovative synthetic approaches.

Notably, the advancements in mechanism-driven analog design and modular synthesis have contributed to new antibacterials that overcome resistance. Most antibacterial agents currently in clinical development are typically derived from well-established antibiotic classes and are selected to address class-specific resistance mechanisms.^38,75–77 In this context, “old” compounds such as griseoviridin and viridogrisein are worth revisiting as templates for next-generation antibiotics, aided by the development of modular synthetic routes. Looking ahead, we anticipate that advances in modern synthetic chemistry and biosynthetic studies will continue to fuel the design of further streptogramin analogs to keep pace with the ever-evolving modes of resistance to the antibiotics. Especially exciting is the possibility of combining chemical and biosynthetic approaches to generate novel analogs that are inaccessible using each approach alone.