Structure Prediction and Synthesis of Pyridine-Based Macrocyclic Peptide Natural Products

Graham A. Hudson, Annie R. Hooper, Adam J. DiCaprio, David Sarlah, and Douglas A. Mitchell*

Letter

Structure Prediction and Synthesis of Pyridine-Based Macrocyclic

Peptide Natural Products

Cite This: https://dx.doi.org/10.1021/acs.orglett.0c02699

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sı Supporting Information

ABSTRACT: The structural and functional characterization of

natural products is vastly outpaced by the bioinformatic

identiﬁcation of biosynthetic gene clusters (BGCs) that encode

such molecules. Uniting our knowledge of bioinformatics and

enzymology to predict and synthetically access natural products is

an eﬀective platform for investigating cryptic/silent BGCs. We

report the identiﬁcation, biosynthesis, and total synthesis of a

minimalistic class of ribosomally synthesized and post-translation-

ally modiﬁed peptides (RiPPs) with the responsible BGCs

encoding a subset of enzymes known from thiopeptide biosyn-

thesis. On the basis of the BGC content, these RiPPs were

predicted to undergo enzymatic dehydration of serine followed by

[

4+2]-cycloaddition to produce a trisubstituted, pyridine-based

macrocycle. These RiPPs, termed “pyritides”, thus contain the same six-membered, nitrogenous heterocycle that deﬁnes the

thiopeptide RiPP class but lack the ubiquitous thiazole/thiazoline heterocycles, suggesting that thiopeptides should be reclassiﬁed as

a more elaborate subclass of the pyritides. One pyritide product was obtained using an 11-step synthesis, and the structure veriﬁed by

an orthogonal chemoenzymatic route using the precursor peptide and cognate pyridine synthase. This work exempliﬁes

complementary bioinformatics, enzymology, and synthesis to characterize a minimalistic yet structurally intriguing scaﬀold that,

unlike most thiopeptides, lacks growth-suppressive activity toward Gram-positive bacteria.

dvances in genome sequencing and analysis have rapidly

expanded our knowledge of natural product biosynthetic

contain a signiﬁcant number of ancillary modiﬁcations, the

class-deﬁning PTM is the six-membered N-heterocycle, which

arises from an enzyme-catalyzed [4+2]-cycloaddition of two

dehydroalanine (Dha) residues and the amide backbone.

space. This is especially true for the ribosomally synthesized

and post-translationally modiﬁed peptides (RiPPs), where

characterization of new compounds is outpaced by an

increasingly inundating number of biosynthetic gene clusters

We sought to determine if orthologs of the thiomuracin

15,16

pyridine synthase, TbtD,

occurred in contexts beyond

−6

(

BGCs).

Natural product discovery eﬀorts often involve

canonical thiopeptide BGCs. A bioinformatic search yielded

several RiPP-like BGCs that lacked an azoline-forming

cyclodehydratase while still possessing a split LanB, putatively

time-consuming and frequently unsuccessful techniques, such

as traditional “grind-and-ﬁnd” or heterologous BGC expres-

sion. Given suﬃcient knowledge of the biosynthetic enzymes,

responsible for Ser glutamylation and elimination, to yield

the structure of the ﬁnal product can be predicted, permitting

−11

17,18

access to the compound by chemical synthesis.

RiPP

Dha,

as well as a pyridine synthase (Figure 1A, Figure S1,

19,20

structures are often more readily predicted from genomic

and Supplementary Data Set 1).

No other local genes were

information compared to other natural product classes given

conserved, suggesting pyridine formation as the sole PTM for

these RiPP products. The neighboring precursor peptides also

generally lacked Cys, which would be required for thiazole/

thiazoline formation, a PTM ubiquitous to all known

12,13

their direct ribosomal origin.

Structural predictions can

enable directed synthetic eﬀorts, which oﬀer several advan-

tages, including an unparalleled capacity for analogue

generation (often with scalability) and obviating the need for

tedious isolation from a producing organism.

Received: August 12, 2020

Recently, we reported a comprehensive genomic landscape

survey of the thiopeptides, an extensively modiﬁed RiPP

class. All thiopeptides feature azole/azoline heterocycles

from Cys, Ser, and Thr), dehydroamino acids (from Ser/

Thr), and (dehydro)piperidine/pyridine post-translational

(

2,14,15

modiﬁcations (PTMs).

While certain thiopeptides

XXXX American Chemical Society

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

Letter

Figure 1. Biosynthesis of pyritides. (A) BGC from M. rosaria,

encoding two precursor peptides, split LanB dehydratase, and

pyridine synthase. (B) Proposed biosynthetic route to pyritide A1

Figure 2. MALDI-TOF mass spectra detailing the chemoenzymatic

synthesis of 1. Compound 4 (m/z 5978) was didehydrothiolated to 5

(1). (C) Structure of pyritide A2 (2). LP, leader peptide.

(

m/z 5910) followed by reaction with MroD. The resulting masses

correspond to the [M + H] ions for 1 (m/z 933) and the eliminated

leader peptide carboxyamide (6, m/z 4959). Asterisks denote Na /K

thiopeptides. Given the lack of sulfur (and thus, thiazole/

thiazoline rings), we termed these RiPPs “pyritides”.

ions.

We next tested our genome-guided structural prediction

using the pyritide BGC from Micromonospora rosaria owing to

strain availability. The C-terminal core regions of MroA1 and

MroA2 (precursor peptides) contained two Ser residues

congruent with the predicted structure of 1 and the leader

peptide (LP) bearing a C-terminal carboxamide, 6.

This chemoenzymatic route was then carried out on a larger

scale to produce a quantity of 1 that was suﬃcient for

spectroscopic comparison to the total synthesis product.

Approximately 30 L of MBP-MroA1 and 8 L of MBP-MroD

culture were used to produce 2.5 mg (post-HPLC yield) of 1.

Several analytical techniques were used to characterize the

synthetic and chemoenzymatic samples of 1, including high-

resolution and tandem mass spectrometry (HR-MS/MS), C

(

[

Figure 1), both presumably converted to Dha. A subsequent

4+2]-cycloaddition would result in 14- and 17-atom macro-

cyclic products, hereafter pyritide A1 (1) and pyritide A2 (2),

respectively. To assess the validity of these predictions, we

attempted to isolate 1 and 2 from the native producer;

however, we were unsuccessful in detecting either pyritide

from M. rosaria. Instead, an orthogonal, 11-step total synthesis

Figure S2) and chemoenzymatic route were devised to obtain

(Scheme 1 and Supplementary Methods).

reverse-phase HPLC retention time, and H NMR (Figures

S4 −S9 and Tables S2 and S3 ). Data from all three methods

conﬁrmed that the structure of 1 was correctly predicted and

the synthetic and chemoenzymatically prepared samples were

indistinguishable.

With respect to the chemoenzymatic approach, we

recombinantly expressed and puriﬁed the MroA1 precursor

peptide and MroD pyridine synthase as fusions to maltose-

binding protein (MBP) (Table S1 and Figure S3). To sidestep

the tRNA dependency of MroB/C, we carried out a

dehydrothiolation reaction using dibromohexanediamide (3,

Using the chemoenzymatic route, we obtained an equivalent

quantity of pyritide A2 (2). The principal diﬀerence between 1

and 2 is the presence of an Arg in 2 adjacent to the pyridine.

This position is conserved in several other bioinformatically

predicted pyritides (Figure S1 ). Analogous to MroA1, the two

Ser residues within the core region of MroA2 were substituted

with Cys prior to chemical dehydrothiolation by 3. This

resulted in the production of a mass consistent with the

0−23

DBHDA)

of an MroA1 variant with Ser1 and Ser6

replaced with Cys (4). The resulting didehydrated inter-

mediate (5) was detected by matrix-assisted laser desorption/

ionization time-of-ﬂight mass spectrometry (MALDI-TOF-

MS) (Figure 2). As the predicted substrate for the pyridine

synthase, 5 was then reacted with MroD, yielding masses

predicted structure of 2, which was veriﬁed by H NMR and

Scheme 1. Orthogonal Routes to 1, Including (A) Chemical Synthesis Beginning from Pyridine-2,5-dicarboxylic Acid and

Protected Tripeptides (see Figure S2) and (B) Chemoenzymatic Synthesis Using MroA1 Double-Cysteine Variant to Allow

DBHDA-Mediated (3) Dehydrothiolation and Subsequent Enzymatic [4+2]-Cycloaddition

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

orcid.org/0000-0002-8736-8953

Letter

HR-MS/MS (Figures S10 −S13 and Tables S2 and S3 ). The

LP byproduct of MroA2 was detected as a C-terminal

carboxamide (Figure S14 ). Puriﬁed 1 and 2 were then

subjected to a brief panel of microbial growth-suppression

assays; however, unlike the well-known activity of thiopeptides

Output from the bioinformatics program RODEO

detailing the BGC architecture for all identiﬁed pyritide

BGCs (Supplementary Data Set 1) (ZIP)

AUTHOR INFORMATION

Corresponding Author

■

toward Gram-positive bacteria, we did not observe any

antimicrobial activity for 1 or 2 individually or in combination

Douglas A. Mitchell − Department of Chemistry and Carl R.

Woese Institute for Genomic Biology, University of Illinois at

Urbana-Champaign, Urbana, Illinois 61801, United States;

orcid.org/0000-0002-9564-0953; Email: douglasm@

illinois.edu

(Table S4 ). Further studies are warranted to investigate other

potential bioactivities of pyritides.

Our data show that MroD catalyzes the formation of

pyridine-based macrocycles that are 14 or 17 atoms in size,

mirroring the smallest size reported using enzymes and

engineered substrates from the lactazole biosynthetic path-

Authors

way. MroD is unique among characterized RiPP-related

pyridine synthases in that it does not require preinstallation of

Graham A. Hudson − Department of Chemistry, University of

Illinois at Urbana-Champaign, Urbana, Illinois 61801, United

States; orcid.org/0000-0002-3715-4279

Annie R. Hooper − Department of Chemistry, University of

Illinois at Urbana-Champaign, Urbana, Illinois 61801, United

States

Adam J. DiCaprio − Department of Chemistry and Carl R.

Woese Institute for Genomic Biology, University of Illinois at

Urbana-Champaign, Urbana, Illinois 61801, United States

David Sarlah − Department of Chemistry, University of Illinois

at Urbana-Champaign, Urbana, Illinois 61801, United States;

16,26

azole heterocycles prior to yielding a macrocyclic product.

It should be noted that while a chemoenzymatic route oﬀers

convenient access to pyritide analogues, a drawback is that

installing Dhb from Thr-containing ribosomal peptides cannot

employ a dehydrothiolation route. However, alternate

strategies using unnatural amino acids to incorporate Dhb

27,28

have been developed.

In addition, total synthesis provides

a complementary avenue for producing Dhb-containing

pyritide analogues.

Another pitfall of the chemoenzymatic approach is that it

assumes one knows the extent of dehydration to a precursor

peptide. In the cases of 1 and 2, the predicted structures were

unambiguous as only two Ser residues were present and both

were required for pyridine formation. For other pyritide core

sequences that contain additional Ser, Thr, or Cys residues,

several distinct structures could result, which requires further

exploration.

Advances in genomics have led to a renaissance in natural

product discovery. Bioinformatic analyses oﬀer a means of

prioritizing BGCs predicted to give rise to novel compounds,

mitigating the rediscovery of known natural products. This

paradigm shift is further enhanced by the uniﬁcation of

biosynthetic enzymology and total synthesis as it provides a

dual platform for accessing new molecules. Here, we applied

this methodology by identifying an unusual genomic context

for RiPP-related pyridine synthases, leading to the structural

prediction, chemical synthesis, and enzymatic veriﬁcation of

the pyritides. Given that the only PTM present in pyritide A1

and A2 is a trisubstituted pyridine (formed via an aza-[4+2]-

cycloaddition), and the same PTM is class-deﬁning for the

thiopeptides, we propose that thiopeptides be reclassiﬁed as

thiazole/thiazoline-containing pyritides.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.orglett.0c02699

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was supported in part by grants from the National

Institutes of Health (GM123998 to D.A.M.). The authors

thank Andraz Ostrek from the Department of Chemistry at the

University of Illinois at Urbana-Champaign for assistance in

■

the synthetic scale-up of 1.

REFERENCES

■

(

1) Cimermancic, P.; Medema, M. H.; Claesen, J.; Kurita, K.;

Wieland Brown, L. C.; Mavrommatis, K.; Pati, A.; Godfrey, P. A.;

Koehrsen, M.; Clardy, J.; Birren, B. W.; Takano, E.; Sali, A.;

Linington, R. G.; Fischbach, M. A. Insights into Secondary

Metabolism from a Global Analysis of Prokaryotic Biosynthetic

Gene Clusters. Cell 2014, 158 (2), 412−421.

(2) Tietz, J. I.; Schwalen, C. J.; Patel, P. S.; Maxson, T.; Blair, P. M.;

Tai, H.-C.; Zakai, U. I.; Mitchell, D. A. A New Genome-Mining Tool

Redefines the Lasso Peptide Biosynthetic Landscape. Nat. Chem. Biol.

017, 13, 470.

3) de los Santos; NeuRiPP, E. L. C. Neural Network Identification

of RiPP Precursor Peptides. Sci. Rep. 2019, 9 (1), 13406.

4) Blin, K.; Shaw, S.; Steinke, K.; Villebro, R.; Ziemert, N.; Lee, S.

We anticipate future investigations into pyritides will yield

greater insight into the requisite enzymology and biological

activity, which will undoubtedly be augmented by comple-

mentary chemical synthesis. Moreover, we predict that the

interplay between natural product enzymology and chemical

synthesis will become an increasingly useful means of accessing

natural products and analogues thereof.

Y.; Medema, M. H.; Weber, T. AntiSMASH 5.0: Updates to the

Secondary Metabolite Genome Mining Pipeline. Nucleic Acids Res.

2019, 47 (W1), W81−W87.

(

5) van Heel, A. J.; de Jong, A.; Song, C.; Viel, J. H.; Kok, J.; Kuipers,

O. P. BAGEL4: A User-Friendly Web Server to Thoroughly Mine

RiPPs and Bacteriocins. Nucleic Acids Res. 2018, 46 (W1), W278−

W281.

ASSOCIATED CONTENT

■

sı Supporting Information

(6) Skinnider, M. A.; Johnston, C. W.; Edgar, R. E.; Dejong, C. A.;

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.orglett.0c02699.

Merwin, N. J.; Rees, P. N.; Magarvey, N. A. Genomic Charting of

Ribosomally Synthesized Natural Product Chemical Space Facilitates

Targeted Mining. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (42),

No. E6343.

Experimental methods and supporting ﬁgures (PDF)

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

Letter

7) Bachmann, B. O.; Van Lanen, S. G.; Baltz, R. H. Microbial

Formal [4 + 2] Cycloaddition. J. Am. Chem. Soc. 2015, 137 (10),

3494−3497.

Genome Mining for Accelerated Natural Products Discovery: Is a

Renaissance in the Making? J. Ind. Microbiol. Biotechnol. 2014, 41 (2),

(21) Chalker, J. M.; Gunnoo, S. B.; Boutureira, O.; Gerstberger, S.

(

75−184.

C.; Fernandez-Gonzalez, M.; Bernardes, G. J. L.; Griffin, L.; Hailu, H.;

́ ́

8) Vila-Farres, X.; Chu, J.; Ternei, M. A.; Lemetre, C.; Park, S.;

Schofield, C. J.; Davis, B. G. Methods for Converting Cysteine to

Dehydroalanine on Peptides and Proteins. Chem. Sci. 2011, 2 (9),

1666−1676.

Perlin, D. S.; Brady, S. F. An Optimized Synthetic-Bioinformatic

Natural Product Antibiotic Sterilizes Multidrug-Resistant Acineto-

bacter Baumannii-Infected Wounds. mSphere 2018, 3 (1), e00528−

(22) Wright, T. H.; Bower, B. J.; Chalker, J. M.; Bernardes, G. J. L.;

(

9) Chu, J.; Vila-Farres, X.; Inoyama, D.; Ternei, M.; Cohen, L. J.;

Wiewiora, R.; Ng, W.-L.; Raj, R.; Faulkner, S.; Vallee

, M. R. J.;

Phanumartwiwath, A.; Coleman, O. D.; Thezenas, M.-L.; Khan, M.;

Gordon, E. A.; Reddy, B. V. B.; Charlop-Powers, Z.; Zebroski, H. A.;

Gallardo-Macias, R.; Jaskowski, M.; Satish, S.; Park, S.; Perlin, D. S.;

Freundlich, J. S.; Brady, S. F. Discovery of MRSA Active Antibiotics

Using Primary Sequence from the Human Microbiome. Nat. Chem.

Biol. 2016, 12 (12), 1004−1006.

Galan, S. R. G.; Lercher, L.; Schombs, M. W.; Gerstberger, S.; Palm-

Espling, M. E.; Baldwin, A. J.; Kessler, B. M.; Claridge, T. D. W.;

Mohammed, S.; Davis, B. G. Posttranslational Mutagenesis: A

Chemical Strategy for Exploring Protein Side-Chain Diversity. Science

2016, 354, aag1465.

(

10) Chu, J.; Vila-Farres, X.; Inoyama, D.; Gallardo-Macias, R.;

(23) Galan, S. R. G.; Wickens, J. R.; Dadova, J.; Ng, W.-L.; Zhang,

X.; Simion, R. A.; Quinlan, R.; Pires, E.; Paton, R. S.; Caddick, S.;

Chudasama, V.; Davis, B. G. Post-Translational Site-Selective Protein

Backbone α -Deuteration. Nat. Chem. Biol. 2018 , 14 (10), 955 −963.

Jaskowski, M.; Satish, S.; Freundlich, J. S.; Brady, S. F. Human

Microbiome Inspired Antibiotics with Improved β -Lactam Synergy

against MDR Staphylococcus Aureus. ACS Infect. Dis. 2018, 4 (1),

(24) Vinogradov, A. A.; Suga, H. Introduction to Thiopeptides:

Biological Activity, Biosynthesis, and Strategies for Functional

3−38.

Reprogramming. Cell Chem. Biol. 2020, 27, 1032.

11) Chu, J.; Vila-Farres, X.; Brady, S. F. Bioactive Synthetic-

Bioinformatic Natural Product Cyclic Peptides Inspired by Non-

ribosomal Peptide Synthetase Gene Clusters from the Human

Microbiome. J. Am. Chem. Soc. 2019, 141 (40), 15737−15741.

(25) Vinogradov, A. A.; Shimomura, M.; Goto, Y.; Ozaki, T.;

Asamizu, S.; Sugai, Y.; Suga, H.; Onaka, H. Minimal Lactazole

Scaffold for in Vitro Thiopeptide Bioengineering. Nat. Commun.

(

12) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni,

2020, 11 (1), 2272.

T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G. L.;

Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.;

Donadio, S.; Dorrestein, P. C.; Entian, K.-D.; Fischbach, M. A.;

Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.;

Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars,

M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.;

Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller, R.;

Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.;

Patchett, M. L.; Piel, J.; Reaney, M. J. T.; Rebuffat, S.; Ross, R. P.;

Sahl, H.-G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.;

Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang,

G.-L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.;

Wenzel, S. C.; Willey, J. M.; van der Donk, W. A. Ribosomally

Synthesized and Post-Translationally Modified Peptide Natural

Products: Overview and Recommendations for a Universal

Nomenclature. Nat. Prod. Rep. 2013, 30 (1), 108 −160.

(

26) Bogart, J. W.; Kramer, N. J.; Turlik, A.; Bleich, R. M.; Catlin, D.

S.; Schroeder, F. C.; Nair, S. K.; Williamson, R. T.; Houk, K. N.;

Bowers, A. A. Interception of the Bycroft −Gowland Intermediate in

the Enzymatic Macrocyclization of Thiopeptides. J. Am. Chem. Soc.

27) Goto, Y.; Iwasaki, K.; Torikai, K.; Murakami, H.; Suga, H.

020, 142 (30), 13170−13179.

Ribosomal Synthesis of Dehydrobutyrine- and Methyllanthionine-

Containing Peptides. Chem. Commun. 2009, 23, 3419−3421.

(

N. F.; Ittuveetil, A.; Saikam, V.; DeGrado, W. F.; Wang, Q.; Wang, P.

G.; Wang, L. Genetically Introducing Biochemically Reactive Amino

Acids Dehydroalanine and Dehydrobutyrine in Proteins. J. Am. Chem.

Soc. 2019, 141 (19), 7698−7703.

28) Yang, B.; Wang, N.; Schnier, P. D.; Zheng, F.; Zhu, H.; Polizzi,

14) Schwalen, C. J.; Hudson, G. A.; Kille, B.; Mitchell, D. A.

13) Funk, M. A.; van der Donk, W. A. Ribosomal Natural Products,

Tailored To Fit. Acc. Chem. Res. 2017, 50 (7), 1577−1586.

15) Hudson, G. A.; Zhang, Z.; Tietz, J. I.; Mitchell, D. A.; van der

Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics. J.

Am. Chem. Soc. 2018, 140 (30), 9494−9501.

(

Donk, W. A. Vitro Biosynthesis of the Core Scaffold of the

Thiopeptide Thiomuracin. J. Am. Chem. Soc. 2015, 137 (51),

(

6012−16015.

16) Zhang, Z.; Hudson, G. A.; Mahanta, N.; Tietz, J. I.; van der

Donk, W. A.; Mitchell, D. A. Biosynthetic Timing and Substrate

Specificity for the Thiopeptide Thiomuracin. J. Am. Chem. Soc. 2016,

(

38 (48), 15511−15514.

17) Bothwell, I. R.; Cogan, D. P.; Kim, T.; Reinhardt, C. J.; van der

Donk, W. A.; Nair, S. K. Characterization of Glutamyl-TRNA −

Dependent Dehydratases Using Nonreactive Substrate Mimics. Proc.

Natl. Acad. Sci. U. S. A. 2019, 116 (35), 17245.

(

18) Ortega, M. A.; Hao, Y.; Zhang, Q.; Walker, M. C.; van der

Donk, W. A.; Nair, S. K. Structure and Mechanism of the TRNA-

Dependent Lantibiotic Dehydratase NisB. Nature 2015, 517 (7535),

(

09−512.

19) Cogan, D. P.; Hudson, G. A.; Zhang, Z.; Pogorelov, T. V.; van

der Donk, W. A.; Mitchell, D. A.; Nair, S. K. Structural Insights into

Enzymatic [4 + 2] Aza-Cycloaddition in Thiopeptide Antibiotic

Biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 12928.

(

20) Wever, W. J.; Bogart, J. W.; Baccile, J. A.; Chan, A. N.;

Schroeder, F. C.; Bowers, A. A. Chemoenzymatic Synthesis of

Thiazolyl Peptide Natural Products Featuring an Enzyme-Catalyzed