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

Article abstract of DOI:10.1016/bs.mie.2018.06.005

Thiopeptide natural products have gained interest recently for their diverse pharmacological properties, including antibacterial, antifungal, anticancer, and antimalarial activities. Due to their inherent poor solubility and uptake, there is interest in d

Full text of DOI:10.1016/bs.mie.2018.06.005

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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249
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251
251
254
259
262
262
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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265
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266
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.
All rights reserved.
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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 10ꢀ3 μg/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 tRNAGlu, 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
Cα–Cβ fragmentation–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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Bo Wang et al.
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

Synthesis of Thiazolyl Peptide Mimics
247
Table 1 Composition of M9 Minimal Medium
1L of 20× M9 Saltsa
g
Na2HPO4 (Anhydrous)
KH2PO4
136
60
10
20
NaCl
NH4Cl
Volumes for 4L of M9 Minimal Medium
20ꢂ M9 Saltsb
200mL
40mL
40% w/v Glucosec
c
1M CaCl2
0.4mL
8mL
c
1M MgSO4
ddH2Ob
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 OD600 of 2.0.
5. At an OD600 of 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 OD600 of 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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Bo Wang et al.
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 N2 before 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.
A
B
C
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
kDa
kDa
96
66
96
66
45
30
45
30
20
14
20
14
300
400
500
600
700
Wavelength (nm)
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 FeCl3 solution 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.

254
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.

258
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 Na2SO4 and 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 Na2SO4 and 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 Na2SO4 and 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 Na2SO4 and 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 Na2SO4 and 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.

260
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 Na2SO4 and 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 Na2SO4 and 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 Na2SO4 and 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 ꢀCH2 group, 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 (t3NosMSR) 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
A
B
2.0´106
684
1.5
1.0
0.5
0.0
i
ii
iii
iv
680 682 684 686 688 690
3.5 4.0 4.5 5.0 5.5 6.0 6.5
m/z
Retention (min)
C
D
120´103
170
+
CH3
696
70´103
60
100
80
60
40
20
0
O
N
50
40
CH3
+
HO
CH3
30
20
10
0
N
H
+
N
O
144
H
188
692
696
m/z
700
704
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 t3NosMSR (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 t3NosMSR. (D) MS/MS of the t3NosMSR.
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 16,000Mꢀ1 cmꢀ1 and 15,400Mꢀ1 cmꢀ1,
respectively, while Trp displays an absorption feature at 278nm with an

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Bo Wang et al.
extinction coefficient of 5560Mꢀ1 cmꢀ1 (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 NosMSR in 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
50
50
50
9
87.9
136
92
50-dA
252
298
684
696
205
63
119
92
MTA
136
106
106
100
100
100
100
63
119
MIA-t3NosM
NosMSR
L-Tryptophan
158
112
188
170
188
118
63
29

Synthesis of Thiazolyl Peptide Mimics
265
A
B
30´10–3
50
1000
800
600
400
200
0
25
20
15
10
5
40
30
20
10
0
0
0
50 100 150 200 250
Time (min)
0
50
100 150 200 250
Time (min)
C
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
50
100 150 200 250
Time (min)
Fig. 10 (A) Detection of SAH (closed circles) and NosMSR (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 NosMSR. (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.

266
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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