Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide

CHAPTER NINE

Using Peptide Mimics to Study

the Biosynthesis of the Side-Ring

System of Nosiheptide

Bo Wang*, Joseph W. LaMattina*, Edward D. Badding*,

Lauren K. Gadsby^†, Tyler L. Grove*, Squire J. Booker*^,†,‡,1

*Department of Chemistry, The Pennsylvania State University, University Park, PA, United States

^†Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park,

PA, United States

^‡The Howard Hughes Medical Institute, The Pennsylvania State University, University Park, PA, United States

¹Corresponding author: e-mail address: squire@psu.edu

Contents

1. Introduction

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2. Overproduction and Purification of NosN

2.1 Gene Cloning and Expression Strategy

2.2 Protocol for Expression of the nosN Gene

2.3 Purification of NosN

2.4 Chemical Reconstitution of as-Isolated NosN

2.5 Determining the Stoichiometry and Forms of Iron–Sulfur Clusters

3. Preparation of Peptide Substrates

3.1 Design and Synthesis of Peptide Mimics to Study NosN

3.2 Protocol to Synthesize Thiazolyl Amino Acid Building Blocks

3.3 General Method for Protection/Deprotection and Amide Coupling

3.4 Incorporation of MIA and Final Deprotection

4. Quantitative Analysis of NosN Turnover

4.1 NosN Enzyme Assays Using Synthesized Peptide Mimics

4.2 Protocol for Quantification of Reaction Products by HPLC Coupled to

Electrospray-Ionization Mass Spectrometry

5. Conclusions

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Acknowledgments

References

Abstract

Thiopeptide natural products have gained interest recently for their diverse pharmaco-

logical properties, including antibacterial, antifungal, anticancer, and antimalarial activ-

ities. Due to their inherent poor solubility and uptake, there is interest in developing

new thiopeptides that mimic these unique structures, but which exhibit better pharma-

cokinetic properties. One strategy is to exploit the biosynthetic pathways using a

#

Methods in Enzymology, Volume 606

ISSN 0076-6879

https://doi.org/10.1016/bs.mie.2018.06.005

2018 Elsevier Inc.

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Bo Wang et al.

chemoenzymatic approach to make analogs. However, a complete understanding of

thiopeptide biosynthesis is not available, especially for those molecules that contain

a large number of modifications to the thiopeptide core. This gap in knowledge and

the lack of a facile method for generating a variety of thiopeptide intermediates makes

studying particular enzymatic steps difficult. We developed a method to produce

thiopeptide mimics based on established synthetic procedures to study the reaction

catalyzed by NosN, the class C radical S-adenosylmethionine methylase involved in

carbon transfer to C4 of 3-methylindolic acid and completion of the side-ring system

in nosiheptide. Herein, we detail strategies for overproducing and isolating NosN, as well

as procedures for synthesizing substrate mimics to study the formation of the side-ring

system of nosiheptide.

1. INTRODUCTION

Thiopeptide natural products are ribosomally synthesized and post-

translationally modified peptides (RiPPs) that exhibit a variety of biologically

activities (Ortega & van der Donk, 2016). Most notably, these activities

include inhibition of protein synthesis either by binding to the 50S ribosome

or by interacting with elongation factor thermal unstable (EF-tu), and thus

can produce antibiotic effects (Bagley, Dale, Merritt, & Xiong, 2005). These

natural products originate from a precursor peptide before undergoing a

multistep transformation to achieve, among other modifications, multiple

thiazole and/or oxazole rings, dehydrated alanine (Dha) and dehydrated

threonine (Dht) side chains, and a central nitrogen-containing six-membered

ring of different oxidation states (Ortega & van der Donk, 2016; Zheng,

Fang, & Liu, 2017). The core macrocycle comprises a 26- or 29-membered

ring, which is related to the specific biological target. The larger thiopeptides

include thiostrepton and nosiheptide (NOS), which have been cocrystallized

with the large subunit of the ribosome (Harms et al., 2008). Such thiopeptides

interact with H44 and L11 in the A-site, thus confirming the observation that

thiostrepton prevents elongation factor G (EF-G) crosslinking (Cameron,

Thompson, March, & Dahlberg, 2002).

NOS from Streptomyces actuosus 40,037 (NRRL 2954) was one of the

first thiopeptides to be identified and characterized and has been used as a feed

additive to promote the growth of poultry livestock (Benazet & Cartier,

1980). NOS exhibits potent in vitro activity against bacteria of urgent clinical

relevance, such as methicillin-resistant Staphylococcus aureus, penicillin-resistant

Streptococcus pneumoniae, and vancomycin-resistant-enterococci (Benazet

et al., 1980). Although it exhibits a minimum inhibitory concentration in the

Synthesis of Thiazolyl Peptide Mimics

243

range of 10^ꢀ3μg/mL against a list of Gram-positive bacteria, NOS is inactive

when used on experimentally infected mice (Benazet et al., 1980). The inabil-

ity to act as an antibiotic in vivo is due to a large extent to its poor solubility in

aqueous solution and gastrointestinal absorption ( Just-Baringo, Albericio, &

Alvarez, 2014). Remarkably, the total synthesis of NOS has been achieved,

but with poor yields (Wojtas et al., 2016). One strategy for optimizing the

yields and for generating analogs that are better suited as therapeutic agents

is to use a chemoenzymatic approach ( Just-Baringo et al., 2014; Lin,

He, & Liu, 2017).

Prior to determining the complete structure of NOS by X-ray crystal-

lography, NMR spectroscopy and chemical hydrolysis helped to identify

key fragments of the molecule (Depaire, Thomas, Brun, Hull, et al., 1977;

Depaire, Thomas, Brun, & Lukacs, 1977; Depaire, Thomas, Brun, Olesker,

& Lukacs, 1977; Pascard, Ducruix, Lunel, & Prange, 1977; Prange, Ducruix,

Pascard, & Lunel, 1977). Notably, NOS carries a 3,4-dimethyl-2-indolic

acid (DMIA) bridge that connects the 4-methyl carbon of DMIA to Glu6

of the peptide framework and the carboxylic acid group of DMIA to

Cys8 by ester and thioester linkages, respectively, to form a second mac-

rocycle, termed the side-ring (Fig. 1). The presence of the DMIA moiety

can be confirmed by hydrolysis, which generates (4-hydroxymethyl)-3-

methyl-2-indolic acid (Depaire, Thomas, Brun, & Lukacs, 1977). The

biosynthesis of the side-ring system in NOS has been of general interest

because of the complex and unique chemistry that it entails.

Fig. 1 Chemical structure of nosiheptide. The side-ring system formed by DMIA was

highlighted in red color.

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Bo Wang et al.

Pioneering studies by Wen Liu and coworkers led to the identification of

the nosiheptide biosynthetic operon (nosA–nosP), and encoded gene prod-

ucts were assigned preliminary functions based on amino acid sequence

similarities to proteins of known function and gene knockout studies in

S. actuosus (Yu et al., 2009). A working model was proposed wherein NosA,

B, and C are responsible for terminal posttranslational modifications to

the peptide core (Liu et al., 2015; Wang et al., 2015), and NosD, E, F, G,

H, M, O are responsible for generating the core thiopeptide (Yu et al.,

2009). NosG contains a YcaO-like domain, which is found in Ser/Thr

cyclodehydratases involved in natural product biosynthesis (Dunbar, Melby,

& Mitchell, 2012). Recent work by Wen Liu and coworkers showed that

NosGHF modify Cys39, 42, 44, 46, and 48 to form thiazole rings on the linear

precursor peptide while selectively leaving Cys47 for incorporation of

3-methylindolic acid (MIA) by NosK (Qiu et al., 2017). Dehydrated

amino acids are presumably formed by NosD and NosE, which together

are homologous to NisB, a dehydratase involved in nisin biosynthesis. This

dehydration requires activation of the Ser or Thr residue by glutamylation

using tRNA^Glu, and subsequent elimination to form Dha and Dht, respec-

tively (Garg, Salazar-Ocampo, & van der Donk, 2013). Despite our under-

standing of these enzymes, the timing in which these modifications are

installed is unclear. In thiomuracin biosynthesis, thiazole rings are generated

before dehydrated amino acids (Hudson, Zhang, Tietz, Mitchell, & van der

Donk, 2015), but the sequence of events may be pathway specific. NosO is

annotated as a Diels–Alderase that catalyzes a [4+2] aza-cycloaddition and

two subsequent eliminations to remove water and the leader peptide to afford

the central pyridine ring of NOS (Wever et al., 2015).

NosI, J, K, L, and N are involved in the preparation and installation

of the 3,4-dimethylindolic acid moiety to form the side-ring of NOS.

Biosynthesis of the side-ring begins with NosL, which catalyzes the

C_α–C_βfragmentation–recombination of L-Trp to afford MIA (Bhandari,

Fedoseyenko, & Begley, 2016; Sicoli et al., 2016; Zhang et al., 2011).

AMP is then attached to the carboxyl group of MIA by NosI to allow

for transfer of MIA to the 4⁰-phosphopantetheine prosthetic group on NosJ

(Badding et al., 2017; Ding, Ji, et al., 2017; Qiu et al., 2017). NosJ then

shuttles MIA to NosK, an α/β hydrolase that attaches MIA onto a seryl

residue within its active site. The structure of NosK revealed that the

active site contains a cleft that is large enough to accommodate the struc-

tural peptide (Badding et al., 2017). Further studies showed that NosK

Synthesis of Thiazolyl Peptide Mimics

245

transfers MIA to Cys45 of NosM at some stage during NOS maturation

after the installation of the thiazole rings (Qiu et al., 2017).

NosN is annotated as a class C radical SAM methylase, a subgroup of the

radical SAM superfamily that is largely understudied (Bauerle, Schwalm, &

Booker, 2015; Zhang, van der Donk, & Liu, 2012). Other reported class

C radical SAM methylases appear to be involved in the biosynthesis of

complex natural products or the degradation of heme in pathogenic bacteria

(Hein, Klimmek, Polly, Kern, & Simon, 2017; LaMattina, Nix, &

Lanzilotta, 2016). We proposed that upon attachment of MIA to the pre-

cursor peptide, NosN attaches a carbon unit at C4 of MIA to form an elec-

trophilic exocyclic methylene intermediate. A glutamyl (Glu6) residue on

NosM then adds to this electrophilic species, forming the ester linkage in

NOS and completing the side-ring (Fig. 2). Previous studies of NosN

showed that MIA containing an N-acetylcysteamine group (MIA-SNAC)

is recognized as a substrate to form DMIA. However, considering the lack

of additional gene products that could transform DMIA into a closed side-

ring system, we posited that DMIA is formed as a side product when NosN

cannot complete its catalytic cycle (Ding, Li, et al., 2017; Ding, Wu, et al.,

2017; LaMattina et al., 2017). Only when using substrate analogs that better

mimic the predicted physiological one could we probe the full reaction

catalyzed by NosN. With the increased interest in thiopeptides and other

RiPPs, we thought that providing a synthetic strategy to generate good

substrate mimics might be useful to the community.

Fig. 2 Proposed mechanism of NosN.

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2. OVERPRODUCTION AND PURIFICATION OF NosN

Our strategy to purify NosN focused on using similar overproduction

and purification methods previously shown to be successful for other radical

SAM enzymes (Lanz et al., 2012). Below is a detailed description of the

methods used to overproduce NosN and purify it to homogeneity.

2.1 Gene Cloning and Expression Strategy

The nosN gene from S. actuosus was synthesized by Geneart (Thermo Fisher)

with codon optimization for Escherichia coli to aid in optimal protein pro-

duction. The synthesized gene incorporated NheI and XhoI restriction sites

at its N- and C-termini, respectively, and was delivered in a pMK DNA

production plasmid. The plasmid is digested with NheI and XhoI, and the

fragment containing the nosN gene is ligated into a pET28a expression plas-

mid digested with the same restriction enzymes. The resulting construct

encodes NosN with an N-terminal hexahistidine (His₆) tag linked by a

thrombin cleavage site for removal of the tag (LaMattina et al., 2017). The

pET-based cloning system, coupled with the strong T7 promoter, allows for

facile cloning and robust overproduction of proteins (Panavas, Sanders, &

Butt, 2009). The pET28a vector also contains a kanamycin (Kan) resistance

marker for in vivo selection, which, in addition, allows for coexpression of

genes contained on plasmid pDB1282, because this plasmid confers ampi-

cillin (Amp) resistance. pDB1282 harbors the isc operon from Azotobacter

vinelandii, which encodes proteins involved in FedS cluster assembly

(Cicchillo et al., 2004; Zheng, Cash, Flint, & Dean, 1998). The BL21(DE3)

strain of E. coli is transformed both with pDB1282 and with pET28a-nosN,

giving rise to a strain that confers resistance to Kan and Amp.

2.2 Protocol for Expression of the nosN Gene

1. Transform E. coli BL21 (DE3) with pDB1282 and pET28a-NosN using

standard procedures and at least 1μg of each plasmid. Select trans-

formants on Luria–Bertani (LB) plates containing Kan and Amp at final

concentrations of 50 and 100μg/mL, respectively.

2. Use a single colony to inoculate 5mL of LB medium containing Kan

and Amp at 50 and 100μg/mL, respectively. Incubate at 37°C and

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Table 1 Composition of M9 Minimal Medium

1L of 20× M9 Salts^a

g

Na₂HPO₄(Anhydrous)

KH₂PO₄

136

60

10

20

NaCl

NH₄Cl

Volumes for 4L of M9 Minimal Medium

20ꢂ M9 Salts^b

200mL

40mL

40% w/v Glucose^c

c

1M CaCl₂

0.4mL

8mL

c

1M MgSO₄

ddH₂O^b

3700mL

^aAfter salts dissolve, adjust the pH of the medium to 7.4 with HCl and KOH; adjust volume

to 4L with water.

^bMedium should be autoclaved after addition to ddH₂O in a 6-L Erlenmeyer flask.

^cSolutions should be sterilized by filtration and added just before inoculation.

shake at 250rpm for ꢁ12h. In addition, place four 6-L flasks, each

containing 4L of M9 media (Table 1), in an incubator-shaker to allow

them to equilibrate at 37°C.

3. Add 50μg/mL Kan and 100μg/mLAmp to each flask just prior to

inoculation.

4. Inoculate each 6-L flask with ꢁ200μL of the 5mL starter culture grown

to an OD₆₀₀of 2.0.

5. At an OD₆₀₀of 0.3 (ꢁ15h), add L-arabinose to a final concentration of

0.1% (w/v) to induce the expression of the isc operon.

6. At an OD₆₀₀of 0.6, cool flasks to ꢁ20°C in an ice bath and add ferric

ammonium citrate to a final concentration of 25μM before adjusting the

temperature in the incubator to 18°C. Once the medium is equilibrated

(about 30min), return the flasks to the shaker and induce expression of

the nosN gene by adding isopropyl-β-D-thiogalactopyranoside (IPTG)

to a final concentration of 200μM.

7. Allow cultures to incubate for ꢁ16h at 18°C while shaking at 180rpm.

8. Harvest the bacteria by centrifugation and freeze the resulting cell paste

(ꢁ1.5g/L) in liquid nitrogen and store in a liquid nitrogen dewar.

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2.3 Purification of NosN

One of the greatest challenges of isolating radical SAM enzymes is their

instability upon exposure to oxygen. To address this issue, purification of

NosN and all subsequent manipulations of the protein are performed in a

Coy anaerobic chamber under an atmosphere of 95% nitrogen and 5%

hydrogen, as has been previously described (Lanz et al., 2012). Additionally,

the purification process is completed without interruption while keeping the

protein chilled during each step. The following steps detail the protocol that

is used to isolate homogeneous NosN with an N-terminal hexahistidine

(His₆)-tag.

1. In an anaerobic chamber, prepare 500mL each of lysis buffer (50mM

HEPES, pH 7.5, 400mM KCl, 10% glycerol, 10mM BME, 10mM

imidazole), wash buffer (50mM HEPES, pH 7.5, 400mM KCl, 10%

glycerol, 10mM BME, 20mM imidazole), elution buffer (50mM

HEPES, pH 7.5, 400mM KCl, 10% glycerol, 10mM BME, 250mM

imidazole), and storage buffer (50mM HEPES, pH 7.5, 400mM KCl,

10% glycerol, 2mM DTT)with waterthatisoxygen free. Except forcen-

trifugation, all subsequent steps are performed in an anaerobic chamber.

2. In the anaerobic chamber, resuspend 40g of frozen cell paste in 120mL

lysis buffer containing lysozyme (1mg/mL) and DNase I (0.1mg/mL)

by stirring in a 250mL metal sonication cup inserted into a crystallizing

dish packed with ice.

3. Once the cell paste is resuspended, sonicate the mixture for 45s at

60% output using a Fisher 1550 sonic dismembrator. Repeat for at

least six cycles with an 8min interval between each cycle to allow

for temperature reequilibration (ꢁ6°C).

4. Pour the lysate into centrifuge tubes. Cap the tubes tightly and seal the

edges with vinyl tape before removing the tubes from the anaerobic

chamber.

5. Centrifuge the lysate at 50,000ꢂg for 1h at 4°C. During centrifuga-

tion, equilibrate a column of Ni-NTA resin with the chilled lysis buffer.

6. After centrifugation, reintroduce the centrifuge tubes into the anaero-

bic chamber and carefully decant the supernatant into a clean 250mL

bottle. Remove a 100μL sample of the supernatant and a sample of the

pellet for SDS-PAGE analysis.

7. Load the supernatant onto the column. Proceed to wash with 150mL

of prechilled wash buffer. Take 100μL samples of the flow-through

both from the loaded lysate and from the wash for SDS-PAGE analysis.

Synthesis of Thiazolyl Peptide Mimics

249

8. Elute the protein from the column with prechilled elution buffer.

Once the colored band begins to elute, collect fractions until all of

the protein (brown/black band) elutes from the column. Concentrate

the protein to a final volume of ꢃ2.5mL by centrifugal ultrafiltration

at 3500ꢂg at 4°C using an Amicon centrifugal filter device (10kDa

molecular weight cutoff ). While the protein is concentrating, equili-

brate a PD-10 column with prechilled storage buffer.

9. Collect a 20μL sample of the concentrated protein for SDS-PAGE

analysis. Exchange the protein into prechilled cleavage buffer using

the preequilibrated PD-10 column. Collect the flow-through and con-

centrate the protein by centrifugal ultrafiltration if desired. Aliquot and

flash-freeze the protein in liquid N₂before storing in a liquid N₂dewar.

10. Typical expression and purification gels for NosN are shown in Fig. 3A

and B, respectively.

2.4 Chemical Reconstitution of as-Isolated NosN

Although the overproduction and purification methods described above

result in NosN that contains 4.6 and 3.2mol of iron and sulfide, respectively,

reconstitution can enhance FedS cluster incorporation.

1. Introduce the following into the anaerobic chamber: 27mg of

FeCl₃ꢄ6H₂O (270.3g/mol), 24mg of Na₂Sꢄ9H₂O (240.2g/mol),

17.5μL of stock BME (14.3M), and a 50-mL Erlenmeyer flask.

2. Add NosN and 40mL of Storage Buffer to the Erlenmeyer flask and

let incubate on ice for 10min.

Fig. 3 (A) NosN expression on SDS-page gel. (B) NosN purification on SDS-page gel.

(C) UV–visible spectra for as-isolated (dashed line) and reconstituted (solid line) NosN.

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Bo Wang et al.

3. Dissolve the FeCl₃ꢄ6H₂O and Na₂Sꢄ9H₂O in 1mL of water. Add a

4:1M equivalent of the FeCl₃solution to NosN and place mixture on

ice for 30min.

4. While incubating on ice, add 1M equivalent of the Na₂S solution to

the reconstitution mixture every 20min until reaching a 4:1M ratio

of sulfide to NosN. Let the mixture sit on ice overnight.

5. Concentrate the reconstitution mixture to ꢃ2.5mL by centrifugal ultra-

filtration as described above.

6. Exchange the concentrated protein into Gel-Filtration Buffer using

a PD-10 column.

7. Concentrate the flow-through to ꢃ2mL by centrifugal ultrafiltration as

described above.

8. Load the protein onto an S-200 column equilibrated in Gel-Filtration

˚

Buffer and connected to an AKTA fast-protein liquid chromatography

system (housed in an anaerobic chamber) for further purification by

size exclusion chromatography.

9. UV–visible spectra for as-isolated (dashed line) and reconstituted

(solid line) NosN are shown in Fig. 3C.

2.5 Determining the Stoichiometry and Forms of Iron–Sulfur

Clusters

Determining the correct number of FedS clusters per polypeptide is critical

for accurate characterization of radical SAM enzymes, which is typically

performed in concert with Mo€ssbauer and electron paramagnetic resonance

(EPR) spectroscopies (Pandelia, Lanz, Booker, & Krebs, 2015). We follow

protocols that have been previously described for determining the amount

of iron and sulfide per protein in other RS enzymes (Lanz et al., 2012).

A brief description of these methods follows.

1. Following purification, the concentration of enzyme is determined by

the Bradford assay using bovine serum albumin, fraction V (BSA) as a

standard (Bradford, 1976).

2. Amino acid analysis is conducted to determine a correction factor for

NosN when BSA is used as a standard for Bradford analysis.

3. Quantification of the amount of iron and acid-labile sulfide per protein

is done using established procedures (Beinert, 1978, 1983).

Although determining the amount of iron and sulfide per protein is useful,

further characterization is required to determine the types of FedS clusters

bound to an enzyme, which is done using EPR and ⁵⁷Fe-Mo€ssbauer spec-

troscopies. Preparation of samples for Mo€ssbauer spectroscopy is carried

out using methods that we have previously described (Lanz et al., 2012).

Synthesis of Thiazolyl Peptide Mimics

251

3. PREPARATION OF PEPTIDE SUBSTRATES

3.1 Design and Synthesis of Peptide Mimics

to Study NosN

In our proposed mechanism, NosN transfers a carbon unit to the C-4 posi-

tion of MIA attached to the precursor peptide through a thioester bond. The

resulting electrophilic methide intermediate is attacked by the γ-carboxylate

from a glutamyl residue on the thiazolyl peptide to form a lactone bond

(Fig. 2). Therefore, we designed the peptide mimic, t₃NosM, as shown

in Fig. 4A to validate the proposed mechanism of NosN. In our design,

we maintained the thiazolylcysteine residue (structure in red) to carry the

MIA (structure in black), as well as the thiazolyl glutamic acid residue (struc-

ture in blue) to act as the nucleophile for ring closure. The C-terminus of

the peptide was capped with an amide to mimic a peptide bond and to

avoid potential competition with the glutamyl residue. Capping a free

carboxylic acid with an amide increases the stability of the entire molecule

and provides a facile method for synthesis and purification. The N-terminus

was capped with a thiazole-4-carboxylic acid (structure in pink) to extend

the length of the peptide mimic.

Our strategy involves connecting individual amino acid building blocks

through amide coupling reactions to create the desired substrate. The

synthetic route is illustrated briefly in Fig. 4B. Due to the presence of a

labile thioester bond, the protecting group should be chosen carefully.

The thioester linkage is more sensitive than its oxygen counterpart (ester).

Therefore, harsh conditions, such as the use of strong acids/bases and intense

heat should be avoided to prevent hydrolysis of the MIA thioester bond.

Benzyl ester/ether and Cbz protection groups, which are usually removed

by catalytic hydrogenation, are also not compatible, because the molecule

is sulfur rich, which is poisonous to the palladium catalyst used for catalytic

hydrogenation. Hence, we use an allyl ester as the protecting group on the

carboxylic acid, because it can be removed by (PPh₃)₄Pd(0) under very mild

conditions following MIA incorporation.

3.2 Protocol to Synthesize Thiazolyl Amino Acid Building

Blocks

For the synthesis of the NOS mimic, two thiazolyl amino acid building

blocks are needed, a thiazolyl cysteine residue (S1) and a thiazolyl glutamic

acid residue (S2); however, the methods we describe can be applied to the

synthesis of any other thiazolyl amino acid analogs. The method starts from

Fig. 4 (A) Chemical structure of nosiheptide mimic (t₃NosM) used for mechanistic studies of NosN. (B) Retro-synthetic analysis of

nosiheptide mimic.

Synthesis of Thiazolyl Peptide Mimics

253

Fig. 5 General procedures for the synthesis of thiazolyl amino acid building blocks.

amino acids with proper protecting groups on their side chains and amino

groups, most of which are commercially available from Chem-Impex Inter-

national Inc. Our three-step method includes amidation of the carboxylic

acid (step a), thioamide synthesis (step b), and cyclization/dehydration

(step c), as shown in Fig. 5.

3.2.1 Amidation of the Carboxylic Acid

1. In a round-bottom flask, add the substrate, DMF (5mL/1mmol), and

a magnetic stir bar. Stir the solution and add CDI (1.5mmol/1mmol

substrate) in three portions. Let react for 1h.

2. Bubble ammonia gas into the reaction slowly and monitor the reaction

by silica TLC until disappearance of the starting material. For separation,

use chloroform:methanol (5:1v/v) as the mobile phase.

3. After completion, dilute the reaction mixture with ethyl acetate

(20mL/1mmol substrate) and transfer to a properly sized separatory funnel.

4. Wash the organic phase thoroughly with 0.5M HCl, aqueous saturated

NaHCO₃, and brine. Dry the organic layer with anhydrous sodium

sulfate and concentrate by rotary evaporation with the bath water set

no higher than 45°C. The resulting residue is used for the next step

without further purification.

Caution: The reaction conditions of step a tolerates most of the protecting

groups for commercially available starting material, including Fmoc, Boc,

Cbz, Alloc for the amino group, as well as tBu and benzyl for side chains

of amino acids. However, excessive ammonia gas can cleave Fmoc. There-

fore, when using such a protecting group, silica TLC monitoring should

be done frequently and, once the starting material is gone, the bubbling

of ammonia gas should be ceased immediately.

3.2.2 Thioamide Synthesis

1. Weigh the resulting material from Section 3.2.1 in a round-bottom

flask with a magnetic stir bar. Add 50mL of THF per 1mmol of the sub-

strate and stir before the addition of Lawesson’s reagent (MW¼404,

0.5mmol per 1mmol substrate). Stir the resulting reaction mixture at

room temperature overnight.

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Bo Wang et al.

2. After completion, concentrate the reaction by rotary evaporation in a

fume hood and resuspend the resulting residue in ethyl acetate (20mL/

1mmol of substrate). Wash the organic phase thoroughly with 0.5M

HCl, aqueous saturated NaHCO₃, and brine.

3. Dry the organic layer with anhydrous sodium sulfate and concentrate

by rotary evaporation. The resulting residue is used for the next step

without further purification.

Caution: Lawesson’s reagent gives a strong unpleasant odor. Use in a fume

hood that functions well. Deodorization of glassware used for this step can

be done by rinsing with 5% potassium permanganate in water.

3.2.3 Cyclization/Dehydration

1. Weigh the resulting material from Section 3.2.2 in a round-bottom flask

with a magnetic stir bar and add THF (30mL/1mmol substrate).

2. While stirring, add potassium bicarbonate (MW¼100, 10mmol per

1mmol substrate) as well as methyl bromopyruvate (MW¼181, 3mmol

per 1mmol substrate). Stir the reaction at room temperature for 3h or

until disappearance of the starting material as determined by silica TLC.

3. After completion, dilute the reaction with ethyl acetate (30mL/1mmol

substrate) and wash thoroughly with 0.5M HCl, aqueous saturated

NaHCO₃, and brine. Dry the organic layer over anhydrous sodium sul-

fate and concentrate by rotary evaporation.

4. Dissolvetheresultingresidueinethylacetate(30mL/1mmolsubstrate)and

pyridine (MW¼79, 10mmol per 1mmol substrate). After cooling thor-

oughly in an ice-water bath, add trifluoroacetic anhydride (MW¼210,

3mmol per 1mmol substrate) dropwise using a dropping funnel.

5. After completion, as indicated by TLC, quench the reaction by adding

methanol (MW¼32, 2mmol per 1mmol trifluoroacetic anhydride).

6. Wash the reaction mixture thoroughly with 0.5M HCl, aqueous satu-

rated NaHCO₃, and brine.

7. Dry the organic layer over anhydrous sodium sulfate and concentrate

by rotary evaporation.

8. Purify the resulting residue using a silica gel column with hexanes

and ethyl acetate as the eluent.

3.3 General Method for Protection/Deprotection and Amide

Coupling

The synthesis of S1 (Fig. 6, step d), S2 (Fig. 7, step e), and the final product

(Fig. 8) requires several protection/deprotection and amide coupling steps.

Synthesis of Thiazolyl Peptide Mimics

255

Fig. 6 Synthesis of S1.

Here, we describe the detailed procedure. To avoid repetition, each subsec-

tion corresponds to a particular reaction step during the synthesis and can be

referred to at different stages in the synthesis. Each of the figures has section

references for each of the reaction steps to make it easier to follow.

3.3.1 Protection of the Amino Group and Side Chain Thiol Group

With Boc and Acetonide

1. Suspend L-cysteine in polyhydratein, a mixture of acetone (20mL/1g

substrate) and 2,2-dimethoxypropane (4mL/1g substrate), and reflux

the resulting suspension for 48h.

2. After concentrating by rotary evaporation, dissolve the remaining resi-

due in pyridine (12mL/1g substrate) and Boc₂O (1.5mmol per 1mmol

substrate).

3. Stir the reaction mixture overnight at room temperature.

4. Aftercompletion,dilutethemixturewithEtOAc(50mL/1gsubstrate)and

follow with a thorough washing of the organic layer with hydrochloric

acid (0.5M), saturated aqueous sodium bicarbonate, and then brine.

5. Concentrate the organic layer by rotary evaporation and use the resulting

viscous residue for the next step without further purification.

3.3.2 Deprotection of Boc and Acetonide and Protection of the Thiol

Group With Trt

1. Weigh the round-bottom flask to obtain the amount of starting material.

Add TFA (10mL per 1g substrate) and water (0.35mL per 1g substrate).

Stir the solution at 50°C overnight and then concentrate by rotary

evaporation.

2. Resuspend the resulting residue in TFA (5mL per 1mmol substrate) and

trityl chloride (1mmol per 1mmol substrate). Stir the reaction mixture at

room temperature for 2h and then concentrate by rotary evaporation.

Fig. 7 Synthesis of S2.

Fig. 8 Assembly of S1 and S2, incorporation of MIA, and deprotection.

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Bo Wang et al.

3. Resuspend the resulting residue in EtOAc (75mL/1g substrate) and

wash the organic phase thoroughly with aqueous saturated NaHCO₃,

and then brine.

4. Dry the organic layer over anhydrous Na₂SO₄and concentrate by

rotary evaporation. Purify the resulting residue by silica-gel flash-

chromatography (hexanes:ethyl acetate¼1:1 as the eluent).

3.3.3 Deprotection of Cbz and Reprotection With Alloc of Amino Group

1. Dissolve the starting material in methanol (20mL per 1g of substrate) and

add palladium on carbon (10% palladium on carbon, 200mg per 1g

substrate). Stir the reaction under a hydrogen atmosphere overnight.

2. Filter the palladium on carbon through a pad of Celite before concen-

trating the filtrate by rotary evaporation. The resulting residue is used

for the next step without further purification.

3. Add THF (20mL per 1g of crude product from previous step). Place the

round-bottom flask in an ice-water bath and stir the solution to chill.

4. Once chilled, add aqueous saturated NaHCO₃(20mL/1g substrate).

5. Add allyl chloroformate (1.5mmol per 1mmol substrate) dropwise to the

reaction while stirring.

6. Continue stirring the reaction at 0°C for 2h before diluting with EtOAc

(20mL/1g substrate). Wash the organic phase thoroughly with water

and then brine.

7. Dry the organic layer over anhydrous Na₂SO₄and concentrate by

rotary evaporation. Purify the resulting residue by silica-gel flash-

chromatography (hexanes:ethyl acetate¼2:1 as the eluent).

8. Follow steps outlined in Sections 3.2.2 and 3.2.3 to prepare the substrate

for the next step

3.3.4 Removal of Alloc

1. In a round-bottom flask containing the starting material, add dioxane

(10mmol per 1mmol substrate) and water (1mL per 1mmol substrate).

2. Add (PPh₃)₄Pd (MW¼1155, 0.05mmol per 1mmol substrate) and 1,3-

dimethylbarbituric acid (MW¼156, 1mmol per 1mmol substrate) to

the reaction and stir at room temperature for 3h. Dilute the reaction

mixture with EtOAc (30mL per 1g substrate) and wash the organic

phase thoroughly with aqueous saturated NaHCO₃, and brine.

3. Dry the organic layer over anhydrous Na₂SO₄and concentrate by rotary

evaporation. The resulting residue is used in the next step without

further purification.

Synthesis of Thiazolyl Peptide Mimics

259

3.3.5 Amide Coupling Reaction

1. Dissolve acid (1mmol) and amine (1mmol) in DCM (20mL) and chill

the reaction in an ice-water bath while stirring.

2. Add DIPEA (MW¼129, 3mmol per 1mmol acid) and HATU

(MW¼380, 1.5mmol per 1mmol acid). Let the reaction gradually

warm up to room temperature and continue stirring overnight.

3. After completion, transfer the reaction mixture to a separatory funnel

and wash the reaction mixture thoroughly with 0.5M HCl, aqueous

saturated NaHCO₃, and brine.

4. Dry the organic layer over anhydrous Na₂SO₄and concentrate by

rotary evaporation. Purify the resulting residue by silica-gel flash-

chromatography using hexanes and acetone as the eluent.

3.3.6 Hydrolysis of the Methyl Ester

1. In a round-bottom flask containing the starting material, add a cosolvent

of THF, methanol, and water (5mL of each solvent per 1mmol substrate).

2. Add lithium hydroxide monohydrate (MW¼42, 2mmol per 1mmol

substrate) and stir at room temperature for 5h.

3. After completion, as indicated by silica TLC, dilute the reaction with

EtOAc (30mL per 1mmol substrate) and wash thoroughly with 0.5M

HCl and then brine.

4. Dry the organic layer over anhydrous Na₂SO₄and concentrate by

rotary evaporation. The resulting residue is used for the next step with

no further purification.

3.4 Incorporation of MIA and Final Deprotection

As shown in Figs. 6 and 7, precursors S1 and S2 are prepared from the pre-

vious synthetic protocols described above. The two precursors are first

coupled using the amide coupling procedure in Section 3.3.5. After replace-

ment of the t-butyl protecting group with an allyl group on the carboxylic

acid of the glutamate, the Trt can be removed selectively to allow for the

incorporation of MIA on the thiol group via an acyl chloride intermediate

of MIA. Below is the detailed protocol to make the final peptide mimic used

to study NosN turnover.

3.4.1 Amidation of Carboxylic Ester

1. Resuspend the starting material in 7N ammonia in methanol (200mL/1g

substrate) and stir at room temperature overnight.

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Bo Wang et al.

2. After completion, as indicated by silica TLC, concentrate the reaction

mixture by rotary evaporation.

3. Purify the resulting residue by silica-gel flash-chromatography using

hexanes and acetone as the eluent.

3.4.2 Deprotection of t-Butyl Ester and Reprotection With Allyl Ester

1. Add 20mL of DCM per 1mmol substrate in a round-bottom flask.

2. While stirring, add TFA (1/4 in volume of DCM used) and continue

stirring for 1h at room temperature.

3. Concentrate the reaction mixture by rotary evaporation and use the

resulting residue for the next step without further purification.

4. Add DMF (10mL per 1mmol substrate) to the residue while stirring.

5. Add K₂CO₃(MW¼138, 5mmol per 1mmol substrate) in one portion

and stir for 10min.

6. Add allyl bromide (MW¼121, 2mmol per 1mmol substrate) dropwise

to the stirred reaction.

7. Stir the reaction mixture at room temperature for 2h before diluting

with EtOAc (30mL per 1mmol substrate).

8. Wash the organic phase thoroughly with 0.5M HCl, aqueous saturated

NaHCO₃, and brine.

9. Dry the organic layer over Na₂SO₄and concentrate by rotary

evaporation.

10. Purify the resulting residue by silica-gel flash-chromatography using

chloroform and methanol as the eluent.

3.4.3 Deprotection of Trt

1. Add DCM (20mL per 1mmol substrate) in a round-bottom flask that

contains the substrate from the previous step.

2. Add triethylsilane (4mL per 1mmol substrate) while stirring with a mag-

netic stir bar. Continue stirring for 5min before adding TFA (4mL per

1mmol substrate).

3. Stir the resulting clear solution for 30min at room temperature.

4. Concentrate the reaction mixture by rotary evaporation and use the

resulting thiol intermediate for the next step without further purification.

3.4.4 Synthesis of MIA Acyl Chloride and Thioesterification

1. Prepare MIA acyl chloride (2mmol per 1mmol of crude product from

previous step) for the subsequent thioesterification as described below.

2. Add DCM (10mL per 1mmol of MIA) to a round-bottom flask with

MIA and a magnetic stir bar.

Synthesis of Thiazolyl Peptide Mimics

261

3. While stirring, add oxalyl chloride (MW¼127, 3mmol per 1mmol

MIA) and DMF (1 drop).

4. Stir the reaction for 1h at room temperature.

5. After completion, concentrate the reaction mixture by rotary evapora-

tion. The resulting acyl chloride is used in subsequent steps without fur-

ther purification.

6. In the round-bottom flask with the thiol intermediate from

Section 3.4.3, add THF (10mL per 1mmol of thiol intermediate).

7. While stirring, add DIPEA (MW¼129, 5mmol per 1mmol thiol

intermediate).

8. Dissolve the MIA acyl chloride intermediate in THF (5mL per 1mmol

MIA acyl chloride).

9. While stirring, add MIA acyl chloride in THF from the previous step

dropwise to the round-bottom flask containing the thiol intermediate

and DIPEA.

10. Stir the resulting reaction mixture for 30min at room temperature.

11. After completion, dilute the reaction with EtOAc (50mL/1mmol thiol

intermediate). Wash the resulting mixture thoroughly with 0.5M HCl,

0.1M aqueous NaOH, and brine.

12. Dry the organic layer over anhydrous Na₂SO₄and concentrate by

rotary evaporation.

13. Purify the resulting residue by silica-gel flash-chromatography using

chloroform and methanol as the eluent.

3.4.5 Deprotection of Allyl Ester

1. Add dioxane (10mL per 0.1mmol substrate) and water (0.5mL per

0.1mmol substrate) in the round-bottom flask with the allyl ester starting

material.

2. While stirring, add (PPh₃)₄Pd (MW¼1155, 0.005mmol per 0.1mmol

substrate) and 1,3-dimethylbarbituric acid (MW¼156, 0.1mmol per

0.1mmol substrate).

3. Stir the reaction for 2h at room temperature.

4. Once complete, dilute the reaction mixture with EtOAc (50mL per

0.1mmol substrate).

5. Wash the organic layer thoroughly with brine before drying the

organic layer over anhydrous Na₂SO₄and concentrating by rotary

evaporation.

6. Purify the resulting residue by silica-gel flash-chromatography using

chloroform and methanol as the eluent.

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Bo Wang et al.

4. QUANTITATIVE ANALYSIS OF NosN TURNOVER

NosN has been stated to produce 5⁰-deoxyadenosine (5⁰-dA) and

either S-adenosylhomocysteine (SAH) or thioadenosine (tA), depending on

whether S-adenosylmethionine (SAM) or methylthioadenosine (MTA) acts

as a methyl donor, respectively (Ding, Li, et al., 2017; LaMattina et al.,

2017). SAM typically degrades to MTA during long periods of storage, which

is heavily dependent on the pH of the storage solution and the temperature.

When using unlabeled MTA and (¹³C-methyl) SAM in NosN assays, we only

observe formation of labeled product, indicating that MTA is not a direct

methyl donor in our reactions. These products can be quantified by LC–

MS/MS using multiple reaction monitoring, as described below for a

NosN-catalyzed reaction. The substrate for NosN is more difficult to quantify

due to a lack of synthetic protocols to make a peptide mimic that contains

the intact side-ring product. One alternative is to use MIA-SNAC as a sub-

strate analog for NosN assays and quench with sodium hydroxide to form a

hydroxymethyl adduct of MIA (LaMattina et al., 2017). However, such

methods result in a mixture of products formed due to a lack of specificity

with the poor substrate, making the quantification less meaningful.

The NosN peptide reaction products can be analyzed structurally by

MS, as well as by MS/MS. For the addition of a ꢀCH₂group, the mass will

only change 12units (684–696 as shown in Fig. 9B and C), which can be a

result of either the predicted exocyclic methylene intermediate or the closed

side-ring. For this reason, MS/MS is used to detect the fragmented molecule

and determine which product is produced. For NosN turnover, the side-

ring closed product (t₃NosM_SR) will fragment to an m/z of 188 as shown

in Fig. 9D, which corresponds to the hydroxyl adduct from the glutamate

residue. This fragment is only observed when the side ring is formed and

can be verified by¹⁸O-labeling of the carboxylate oxygens of Glu6 of the

peptide mimic (LaMattina et al., 2017).

4.1 NosN Enzyme Assays Using Synthesized Peptide Mimics

1. Assays should contain final concentrations of the following in a total

volume of 200μL: 50mM HEPES, pH 7.5, 100mM KCl, 5% glycerol,

1mM SAM, 1mM substrate, 10–50μM NosN, 100μM Trp (internal

standard), and 2mM sodium dithionite as a reductant. Note: We have

not observed NosN activity with the E. coli flavodoxin/flavodoxin reductase

reducing system.

Synthesis of Thiazolyl Peptide Mimics

263

2.0 ´10⁶

680 682 684 686 688 690

3.5 4.0 4.5 5.0 5.5 6.0 6.5

12 0 ´10³

CH₃

7 0 ´10³

CH₃

CH₃

100 120 140 160 180 200 220

m/z

Fig. 9 LC–MS of the NosN activity assay. (A) Chromatogram displaying the total ion

chromatography (TIC) for t₃NosM and t₃NosM_SR(i, no SAM; ii, t¼1min with 50μM NosN;

iii, t¼250min with 50μM NosN; iv, t¼250min with 100μM NosN. (B) MS scan of t₃NosM.

(C) MS scan of t₃NosM_SR. (D) MS/MS of the t₃NosM_SR.

2. Initiate the reaction by adding SAM, then remove 20μL aliquots at

specified times and quench the reactions with an equal volume of

100% MeOH.

3. Centrifuge the samples for 15min at 14,000 ꢂ g before removing

supernatants and placing them into MS vials.

4.2 Protocol for Quantification of Reaction Products by HPLC

Coupled to Electrospray-Ionization Mass Spectrometry

1. Prepare standards of SAH, 5⁰-dA, and Trp using extinction coefficients

for each molecule. SAH and 5⁰-dA display absorption features at 260nm

with extinction coefficients of 16,000M^ꢀ1cm^ꢀ1and 15,400M^ꢀ1cm^ꢀ1,

respectively, while Trp displays an absorption feature at 278nm with an

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Bo Wang et al.

extinction coefficient of 5560M^ꢀ1cm^ꢀ1(Wood, 1987). Using these

standards, generate a standard curve containing 0.5–250μM of each

molecule and 100μM Trp (internal standard).

2. Analyze samples on an Agilent Technologies Zorbax Extend-C18

RRHD column (4.6mm ꢂ 50mm, 1.8μm particle size) equilibrated

in 98% solvent A (0.1% formic acid, pH 2.6) and 2% solvent

B (methanol). Apply a linear gradient of 2%–10% solvent B from 0 to

0.5min, and then a linear gradient of 10%–50% solvent B from 0.5 to

2.5min. Next, increase solvent B to 100% from 2.5 to 3.5min and hold

constant from 3.5 to 4.5min before returning to 2% solvent B from 4.5

to 5min. Allow the column to reequilibrate for 2.5min under initial

conditions before subsequent sample injections.

3. Detect products using MS/MS with electrospray ionization in positive

mode. All products are detected in their +1 charge states. Table 2 displays

the m/z values (parent and daughter ions), fragmentor voltages, collision

energies, and retention times of all molecules that are monitored in a

NosN reaction.

4. Quantify product formation. Shown in Fig. 10 is the time-dependent

formation of SAH, 5⁰-dA, MTA, and NosM_SRin the NosN-catalyzed

reaction.

Table 2 Parameters for LC–MS/MS Analysis of Reaction Products

Compound

Precursor Ion

Product Ion

Fragmentor

Collision Energy

SAH

385

136

92

17

61

29

13

18

60

50

9

87.9

136

92

5⁰-dA

252

298

684

696

205

63

119

92

MTA

136

106

100

63

119

MIA-t₃NosM

NosM_SR

L-Tryptophan

158

112

188

170

188

118

63

29

Synthesis of Thiazolyl Peptide Mimics

265

3 0 ´10^–3

Fig. 10 (A) Detection of SAH (closed circles) and NosM_SR(open squares) production when

50μM NosN (black) or 100μM NosN (red) is used in the assay. Solid curves are the fit for

SAH production, while dashed curves are for NosM_SR. (B and C) Progress curves for 5⁰-dA

and MTA production, respectively.

5. CONCLUSIONS

Class C radical SAM methylases catalyze the appendage of a carbon

unit to sp²-hybridized carbon centers, resulting in the transfer of either a

methyl group (ꢀCH₃), as in the example of TbtI for thiazole methylation,

or a methylene group (ꢀCH₂), which appears to be the case for NosN

and YtkT (Bauerle et al., 2015; LaMattina et al., 2017). With the role that

these enzymes play in the biosynthesis of medically relevant natural prod-

ucts, determining their mechanisms of catalysis can lead to producing RiPP

analogs to combat antibiotic resistant bacteria. However, the previous

understanding of these biosynthetic pathways and the lack of substrate

syntheses have hindered the study of the class C radical SAM methylaes.

This method provides a facile synthetic approach to generate similar

thiopeptides as a means to probe the reactions catalyzed by some of the

unique enzymes within the respective pathway.

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Bo Wang et al.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (GM-122595 and AI-133318).

S.J.B. is an investigator of the Howard Hughes Medical Institute.

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Article Doi

Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide

DOI: 10.1016/bs.mie.2018.06.005

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