Synthesis of Functionalized N-Acetyl Muramic Acids to Probe Bacterial Cell Wall Recycling and Biosynthesis

Article abstract of DOI:10.1021/jacs.8b03304

Uridine diphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purification strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust chemoenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2-N-functionalized UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal 3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP synthesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored the utility of these derivatives in the fluorescent labeling of both Gram (-) and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionalized NAMs and UDP NAMs, which now can be used in tandem with other complementary bio-orthogonal labeling strategies to address fundamental questions surrounding PG's role in immunology and microbiology.

Full text of DOI:10.1021/jacs.8b03304

Subscriber access provided by University of South Dakota
Article
Synthesis of functionalized N-acetyl muramic acids to
probe bacterial cell wall recycling and biosynthesis
Kristen E. DeMeester, Hai Liang, Matthew R. Jensen, Zachary S. Jones,
Elizabeth A. D'Ambrosio, Samuel L. Scinto, Junhui Zhou, and Catherine L. Grimes
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03304 • Publication Date (Web): 09 Jul 2018
Downloaded from http://pubs.acs.org on July 9, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 10
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Synthesis of functionalized N-acetyl muramic acids to probe bacteri-
al cell wall recycling and biosynthesis
Kristen E. DeMeester1, Hai Liang1, Matthew R. Jensen1, Zachary S. Jones1, Elizabeth A. D’Ambrosio1,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Samuel L. Scinto1, Junhui Zhou1, and Catherine L. Grimes1,2*.
1Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
2Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
*Correspondence to: The University of Delaware, Department of Chemistry and Biochemistry, Newark, DE 19716, 302ꢀ831ꢀ
2985, cgrimes@udel.edu.
ABSTRACT: Uridine diphosphate Nꢀacetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG)
biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM interꢀ
mediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purificaꢀ
tion strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust cheꢀ
moenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2-N functionalized UDPꢀNAMs. In
addition, a synthetic strategy to access biorthogonal 3ꢀlactic acid NAM derivatives was developed. The chemoenzymatic UDP synꢀ
thesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM αꢀ1 phosphate
uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored
the utility of these derivatives in the fluorescent labeling of both Gram (ꢀ) and Gram (+) PG in whole cells using a variety of
bioorthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionꢀ
alized NAMs and UDP NAMs, which now can be used in tandem with other complementary bioorthogonal labeling strategies to
address fundamental questions surrounding PG’s role in immunology and microbiology.
Introduction
Organophosphates are among the essential molecular
rial species.10 Synthetic and naturally occurring small moleꢀ
cule units derived from bacterial PG with the NAM glycan
core are known to activate human innate immune responses.12ꢀ
components for life. In addition to their role in the scaffold of
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), energy
storage and postꢀtranslational modifications of proteins, these
phosphate groups hold valuable roles in glycobiology.1ꢀ2 From
the polymerization of energy storing molecules such as glycoꢀ
gen to glycosylation of lipids on the surface of cells, the addiꢀ
tion of a unique sugar molecule onto heteroꢀatom scaffolds via
phosphate chemistry can tune the chemical and physical propꢀ
erties of the biomolecule.3ꢀ8 While many phosphate shuttling
biomolecules are key intermediates for signaling, they also
serve as carriers in many essential biosynthetic pathways.
13
Interestingly, extending or truncating the peptide portion of
these intermediates results in a tunable immune response.14ꢀ16
Scheme 1. Peptidoglycan Recycling and Biosynthetic Pathꢀ
ways
Uridine diphosphates (UDPs) are organophosphate
carriers that play a critical role in the biosynthesis of bacterial
peptidoglycan (PG), a coat that surrounds each bacterial cell to
protect it from environmental changes and stresses.9ꢀ10,11 The
strength of the bacterial cell wall is directly correlated to its
molecular composition; a complex network of peptides and
two carbohydrates, Nꢀacetyl glucosamine (NAG) and Nꢀacetyl
muramic acid (NAM), which are stitched together through a
conserved biosynthetic pathway that utilizes UDP sugar carriꢀ
ers of NAG and NAM carbohydrates (Scheme 1).10
The ability to monitor the natural production and
breakdown of this polymer at both the glycan and peptide levꢀ
els is critical for deciphering how human hosts are recognizing
and responding to bacteria at the molecular level.17ꢀ20 The deꢀ
velopment of a variety of small molecule NAM probes as well
NAM serves as a core structural element exclusively
for bacterial cell wall. While bacterial PG is dynamic, conꢀ
stantly changing as bacterial cells grow and divide, the NAM
building blocks of this material are conserved across all bacteꢀ
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 10
as methodology to selectively label the NAM core of PG in
tives were treated with purified AmgK26 and product forꢀ
1
2
3
4
5
6
7
8
whole cells could help tease out the molecular fingerprint of
these interactions to reveal how different bacteria present their
PG to host cells or how host cells process this material.
mation was monitored by high resolution mass spectrometry
(HRMS) (Supporting Information Table 1). The corresponding
monoꢀphosphate NAM products were observed in almost all
of the intermediates with the exception of cyclopropene derivꢀ
ative 11, benzophenone 12 and fluorescein derivative 13 (Taꢀ
ble 1, Supporting Information Table 1), suggesting that these
modifications were not accepted by the kinase. 6ꢀazidoꢀNAM
(Supporting Information, Scheme 3) and 2ꢀaminoꢀmuramic
acid showed no conversion into the monophosphate, indicating
that they are not substrates.
All bacteria naturally build their PG utilizing a series
of conserved enzymatic steps beginning with the production of
UDP NAM through biosynthetic enzymes MurA and MurB.21
Recently, our laboratory developed methodology to selectively
install azide or alkyne bioorthogonal tags onto the NAM
backbone of bacterial PG in whole cells for fluorescent visualꢀ
ization via copper catalyzed azide alkyne cycloaddition (Cuꢀ
AAC).22ꢀ25 This strategy was achieved by taking advantage of
the alternative formation of UDP NAM via the NAM lactol
building block and cell wall recycling enzymes Murꢀ
NAc/GlcNAc anomeric kinase (AmgK) and NAM αꢀ1 phosꢀ
phate uridylyl transferase (MurU)26 (Scheme 1). Here, we
further explored the chemical space of AmgK and MurU subꢀ
strates to expand both the NAM and UDP NAM tools availaꢀ
ble to probe bacterial PG biosynthesis and recycling. We deꢀ
veloped the necessary synthetic routes to show that the system
is amenable to a variety of permutations and demonstrate the
utility of the method in labeling bacterial PG.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bioorthogonal
Substrate
AmgK
MurU
Reaction or
Chemical Utility
3, 4, 14,
Copper Catalyzed
Azide-Alkyne
Cycloaddition
Y, Y, Y,
N
Y, Y, Y,
N
6ꢀazido
NAM
Ketone Condensa-
tion
5
Y
Y
Photoactivating
Crosslinker
6, 7, 12
Y, Y, N
Y, Y, ꢀꢀꢀ
Results
Inverse Electron-
Demand Diel’s-
Alder
The investigation began with the design and syntheꢀ
sis of an expanded library of NAM derivatives to probe the
substrate specificity of AmgK. The modular synthesis of the
previously reported Nꢀacetyl azido and alkyne NAM derivaꢀ
tives (3, 4) gave access to large quantities of the precursor, 2ꢀ
amino muramic acid (Scheme 2). This molecule was poised to
functionalize using mild amide bond coupling conditions with
a variety of activated ester derivatives in order to generate a
library of NAM compounds (Scheme 2, Supporting Inforꢀ
mation). This library includes multiple bioorthogonal NAMs27
(Scheme 2, Table 1) ranging from ketone condensation interꢀ
mediates28 (5), to inverse electron demand diels alder
(IEDDA) compounds29ꢀ32 (8, 11). Photoactivatable intermediꢀ
ates with diazirine33ꢀ37 and benzophenone functionality38ꢀ40 (6,
7, 12) were also synthesized in addition to diꢀfluoro methyl41ꢀ43
(2) fluorescein (13) and biotin44ꢀ46 tags (9, 10). To assess if
modifications around the carbohydrate were tolerated, the
starting material, 2ꢀaminoꢀmuramic acid and a NAM derivaꢀ
tive with an azide modification at the 6ꢀposition, 6ꢀazidoꢀ
NAM, were synthesized (Supporting Information).
8, 11
2
Y, N
Y
Y, ꢀꢀꢀ
Y
NMR Characteri-
zation
Streptavidin Af-
finity
9, 10
Y, Y
N, N
Purification
Fluorescence
Visualization
Other
13
N
ꢀꢀꢀ
1, amino
Y, N
Y, ꢀꢀꢀ
Table 1. AmgK and MurU substrate specificity. Each comꢀ
pound was grouped based on common bioorthogonal reactiviꢀ
ty or utility. Turnover of AmgK and MurU are reported with
either (Y) representing formation of corresponding product,
(N) representing no formation of product and (ꢀꢀꢀ) representing
untested substrate due to no conversion of AmgK. Product
formation was monitored by highꢀresolution mass spectromeꢀ
try (HRMS).
Scheme 2. Library of 2ꢀ N NAM and UDP NAM probes
In order to compare AmgK’s substrate preferences,
the kinetic profile of AmgK with the NAM derivatives was
measured according to the ATPase enzyme coupled assay used
by Mayer and coꢀworkers.47 For 1, apparent Km and kcat were
30.41 ± 5.75 ꢁM and 6.58 ± 0.21 sꢀ1, respectively (Table 2,
Supporting Information). These data agree with the values
reported by Mayer and coworkers26. For substrates 3 and 4,
AmgK showed ~10 fold less efficiency than it had turning
over 1. The catalytic efficiencies were ~100 fold less with
other derivatives as substrates (Table 2, Supporting Inforꢀ
mation). Apparent kcat/Km values were acquired for substrates
whose rates did not plateau by fitting the experimental kinetic
parameters to a double reciprocal plot (Table 2, Supporting
Information). 5, 6, 7, 8 and 9 appear to have high Km values
based on this analysis, suggesting that the enzyme has low
affinity for these larger modifications (Supporting Inforꢀ
mation).
With the library of NAM derivatives in hand, the
substrate specificity of the bacterial cell wall recycling enzyme
AmgK was probed (Table 1 and 2). Each of the NAM derivaꢀ
ACS Paragon Plus Environment

Page 3 of 10
Journal of the American Chemical Society
The monophosphate NAM intermediates were then
subjected to uridylyl transferase MurU48 to generate the UDPꢀ
sugar (Scheme 2). The production of the UDP NAM derivaꢀ
tives was monitored by HRMS (Table 1, Supporting Inforꢀ
mation Table 1). MurU converted the monophosphate interꢀ
mediates of 1ꢀ8 to their respective UDP products as observed
by HRMS. Furthermore, intermediates 1, 3ꢀ5 were converted
in milligram quantities (i.e. > 10 mgs) and isolated by preparaꢀ
tory mass directed HPLC for complete NMR characterization
(Supporting Information).
1
2
3
4
5
6
7
8
Scheme 3. Synthesis of lactic acid N3 functionalized NAM
a. Ac2O, DMAP, pyridine (85%), b. PhSH, SnCl4, DCM, reꢀ
flux (54%), c. NaOMe, MeOH, r.t. (quant), d. PhCH(OMe)2,
TsOH, DMF, 70°C (72%), e. NaH, 15, DMF (57%), f. K2CO3,
MeI, DMF, r.t. (80%), g. DDQ, DCM/H2O (quant), h. MsCl,
pyridine/DCM (80%), i. 1. NaN3, DMF, 70°C; 2. IRA H+,
H2O, 95°C; 3. TCCA (12% over 3 steps).
9
Table 2. Kinetic Characterization of AmgK with NAM deꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
rivatives as substrates.
To generate the final unprotected NAM lactic acid
derivative, a mild deprotection strategy was utilized. Notably,
the thiolꢀprotecting group was removed under oxidative condiꢀ
tions with trichloroisocyanuric acid (TCCA)51, an environmenꢀ
tally friendly chemical reagent. . Global deprotection revealed
compound 14. 14 was shown to be accepted by recycling enꢀ
zymes AmgK and MurU to generate the corresponding UDP
NAM intermediate 14b by HRLCMS (Table 1 and Suppleꢀ
mentary Table 1). Kinetic characterization with AmgK was
performed and the data show that the kinase accepted the lacꢀ
tic acid modification with the same efficiency as the natural
NAM (Table 2).
Substrate
1a
kcat(s-1)
Km(mM)
kcat/Km (M-1s-1)
6.58 ±
0.21
(3.04 ± 0.58)
X 10ꢀ2
(2.16 ± 0.42) X
105
3a
4a
14.07 ±
0.72
1.12 ± 0.16
0.55 ± 0.09
(1.26 ± 0.19) X
104
(1.8 ± 0.3) X 104
9.74 ±
0.45
14a
4.67 ±
0.14
(3.03 ± 0.54) (1.54 ± 0.28)
X
X10ꢀ2
105
With the formation of the UDPꢀNAM derivatives at
the 2 and 3 position of the carbohydrate confirmed, the promꢀ
iscuity of the bacterial PG biosynthetic enzymes MurCꢀF was
explored (Scheme 1, Supplemental Scheme 1). All of the UDP
NAM derivatives subjected to MurCꢀF were able to be conꢀ
verted into the desired pentapeptide Park’s nucleotide derivaꢀ
tives and were observed by HRMS (Supporting Information
Table 1). The relaxed substrate specificity of the PG recycling
and biosynthetic enzymes motivated the expansion of the PG
glycan labeling method of whole bacterial cells. In order to
improve this methodology, three key areas were explored:
modification at the 3ꢀpostion of the NAM, clickꢀchemistry
utility and UDPꢀNAMꢀcarrier tolerance
5b
6b
7b
8b
9b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3.45 X 103
1.55 X 103
1.02 X 103
2.25 X 103
2.95 X 103
(a, kinetic parameters were calculated by fitting the experiꢀ
mental data into the MichaelisꢀMenten equation using proꢀ
gram GraphPad Prismꢀ6. , Apparent kcat/Km values were calꢀ
culated by fitting the experimental data to the Lineweaverꢀ
Burk equation). Standard Error (SE) were calculated based on
3 technical replicates of each sample concentration
b
First the lactic acid modified NAM derivative 14 was
tested for incorporation in whole bacterial cells. E. coli QKU,
a MurQ52 knockout E. coli strain that contains the recycling
enzymes AmgK and MurU, cells were pulsed with 14 for two
doubling times (45 minutes) in the presence of fosfomycin and
subsequently treated with CuAAC click chemistry conditions
as previously reported22. Cells were visualized with superresoꢀ
lution Structured Illumination Microscopy (SIM)53 (Fig 1,
Supplementary Fig 1, Supplemental Material). Fluorescent
signal was observed at the septal division ring of the bacterial
cells as compared to the positive control cells treated with 3
and the negative control cells treated with 1 in which only
background fluorescence was observed (Supplementary Fig. 2
and 3A, respectively). This positive labeling result suggests
that NAM derivative 14 can be utilized by the bacterial PG
biosynthetic enzymes, which is consistent with the in vitro
enzymatic conversion study (Supporting information Table 1).
In addition to the 2ꢀN functionalized NAM derivaꢀ
tives, modification at the 3ꢀOH lactic acid portion of the small
molecule was desired (Scheme 3). It is known that some bacꢀ
teria including Mycobacterium tuberculosis (Mtb), naturally
modify their bacterial PG to a N- glycolyl moiety at the 2-N
position via NamH.49ꢀ50 In order to avoid the loss of a 2ꢀN
biorthogonal probe through this pathway, we moved to install
the functionality at another location of the NAM sugar. As
these derivatives have not been made before, a synthesis of
lactic acid functionalized NAMs was designed and impleꢀ
mented (Scheme 3, Supporting Information).
To efficiently derivatize the 3ꢀOH position, the synꢀ
thesis of a novel azide modified lactic acid precursor, which
would be added to the NAG core, was developed (Scheme 3,
Supporting Information). We found that the addition reaction
proceeded most efficiently with the brominated lactic acid
scaffold 15. The paraꢀmethoxybenzyl (PMB) protecting group
was selected over other groups due to ease of installation and
removal. 15 was readily coupled to the suitably protected
NAG 1ꢀthioglycoside to yield the derivatized NAM. Subseꢀ
quent methyl esterification, PMB deprotection with DDQ reꢀ
vealed the free alcohol, which was activated for azido installaꢀ
tion.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 10
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. E. coli QKU cells fluorescently modified with the
lactic acid NAM derivative 14 and WGA co-staining. DIC
and maximum intensity projection 2D images from superꢀ
resolution SIM Zꢀstacks of E. coli QKU cells treated with 14,
fosfomycin, IPTG for 45 min and clicked with Alk488 (green)
and then stained with tetramethylrhodamine WGA 561 (red)
of whole cell (white arrow, top) and dividing cell (white arꢀ
row, bottom) (scale bars, 10ꢂꢁm). Images are representative of
a minimum of three fields viewed per replicate with at least
two technical replicates and the experiment was conducted in
three biological replicates.
Figure 2. E. coli QKU cells modified with 8 for tetrazine-
TCO ligation. DIC and 2D images from superꢀresolution SIM
Maximum Intensity Projection Images Zꢀstacks of E. coli
QKU cells treated with 8 and IPTG for 20 min and clicked
with TCOꢀTAMRA (yellow) (scale bars, 2ꢂꢁm). Images are
representative of a minimum of three fields viewed per repliꢀ
cate with at least two technical replicates and the experiment
was conducted in at least three biological replicates.
Other bioorthogonal “click” chemistries were probed
for tolerance by the NAMꢀPG labeling method. Previously,
copper catalyzed azideꢀalkyne cycloaddition (CuAAC) in
Finally, UDPꢀNAM modifications were tested for
whole cell incorporation. To date, two methods have been
used to incorporate NAM derivatives into the PG of whole
cells. One involves the genetic manipulation of the bacterial
cell to include the recycling enzymes AmgK and MurU22 . The
second method involved a laborious 15 step synthesis of uriꢀ
dine diphosphate muramyl tetrapeptide fluorescein derivaꢀ
tive59. The latter was a tour de force that demonstrated the
unnatural UDP peptide fluorophore derivative could be taken
up and embedded into the PG of Gram + lactic acid bacteria.
However, the method was limited due to the demanding chemꢀ
ical synthesis of the UDP probe. Therefore, we took advantage
of the scalable chemoenzymatic production of the uridine diꢀ
phosphate sugar derivatives 3b and 4b containing the azide
and alkyne functionality, respectively (Scheme 2). This methꢀ
od was amendable to milligram scale synthesis; the phosphate
and uridylyl steps were performed subsequently without puriꢀ
fication of the phosphate intermediate (Supporting Inforꢀ
mation). Yields for the multiple enzymatic transformations
ranged from 19% to 89%. Whole cell bacterial labeling with
these derivatives was explored using compound 3b.
whole cells was performed.
Here the tetrazineꢀ
transcyclooctene (TCO) ligation, the fastest click reaction to
date was assessed.32, 54 E. coli QKU cells were treated with
either 1 or NAM derivative 8 and IEDDA was performed with
TCOꢀTAMRA (Supporting Information). Cells were visualꢀ
ized with superresolution SIM. The positive control cells were
labeled with 3 and treated with CuAAc to ensure E. coli QKU
labeling competence (Supplementary Fig 4). The optimal
probe pulse length of 8 was assessed (Supplementary Fig. 5A)
in the presence and absence of fosfomycin. It was determined
that 20 min pulse lengths were optimal without fosfomycin
(Fig 2). It appeared that fluorescent labeling was only obtained
in select cells per field of view when cells were treated with 8
(Fig 2, Supplementary Figure 5B), indicating that the uptake
of the tetrazine probe was not uniform throughout cells or that
a subꢀpopulation of the cells were capable of accepting the
tetrazine modification. Cells that were treated with 1, the unꢀ
labeled control, showed no fluorescence (Supplementary Fig
3B), suggesting that this limited labeling is not due to the
fluorophore nonꢀspecifically associating to the bacteria.
UDP sugars exist as charged species at physiological
pH (Scheme 1 and 2), and diffusion through the bacterial cell
membrane could be challenging. Therefore, the whole cell
studies began with the bacterium Lactobacillus acidophilus, a
commensal60 Gram + organism commonly found in yogurt and
previously used by Nishimura and coworkers to label bacterial
PG using UDP fluorescein lysine derivatives.59 After incubaꢀ
tion with L. acidophilus and the azido UDP NAM 3b, SIM
imaging revealed fluorescent labeling as compared to the conꢀ
trol cells which were incubated with the natural UDP NAM
compound 1b (Supplementary Fig. 7 A and B). L. acidophilus
cells that were treated with 3b and CuAAC were then stained
with WGAꢀ561 and PG coꢀlocalization was observed (Fig. 3).
These data imply that the other UDP derivatives isolated on
scale (4b-6b) could be incorporated. We note that
Traditionally, we and others have utilized mass specꢀ
trometry evidence for probe PG incorporation.22, 55ꢀ56 However,
due to the low fluorescent probe incorporation with 8 and 14,
we desired a method other than mass spectrometry to confirm
PG labeling. Wheat germ agglutinin (WGA) coꢀlabeling, a
technique commonly used to stain the NAG backbone of the
bacterial PG was used.57ꢀ58 If coꢀlocalization was observed
with the TCOꢀTAMRA probe, then modification of the PG
could be considered. Coꢀlocalization with tetramethylrhodaꢀ
mineꢀWGA stain, in E. coli QKU cells treated with 14 was
observed suggesting labeling of the PG (Fig. 1, Supplementary
Figure 1). No coꢀlocalization with 488ꢀWGA stain in E. coli
QKU cells treated with 8 was observed (Supplementary Fig 6).
Cells that incorporated 8 appeared to have degraded cell morꢀ
phologies, indicating that tetrazine incorporation is not well
tolerated and thus has low WGA signal.
ACS Paragon Plus Environment

Page 5 of 10
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
Figure 3. L. acidophilus labeling with azido UDP NAM 3b and WGA costaining. DIC and 2D Maximum Intensity Projection
images from superꢀresolution SIM Zꢀstacks L. acidophilus cells treated with 3b, clicked with Alk488 (green) and treated with
WGAꢀ561 (red) (scale bars, 10ꢂꢁm). Images are representative of a minimum of three fields viewed per replicate with at least two
technical replicates and the experiment was conducted in at three biological replicates.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
bacterial labeling was only achieved while bacteria were acꢀ
tively growing (Supplementary Fig 8A,B).
modifications were well tolerated. Bulky modification to the
2ꢀposition resulted in a ~100ꢀfold decrease in the catalytic
efficiency of the enzyme (5ꢀ9, Table 2). Yet, modification at
the 3ꢀposition was comparable to the natural substrate (Table
2), suggesting that the enzyme is more tolerant to modification
at this position. Further experiments are necessary to test the
steric limitations of this position on the carbohydrate.
As the growth conditions are challenging for L. aci-
dophilus, other bacteria species were assayed for UDP NAM
2b labeling. S. aureus, B. subtilis and two strains of E. coli:
E. coli DH5a and E. coli RMF795, an E. coli mutant strain
with a semiꢀpermeable outer membrane.61 S. aureus, B. sub-
tilis, and the E. coli strains were treated with the azido UDP
NAM 3b. SIM imaging revealed no fluorescent labeling after
click incorporation of fluorophore as compared to the control
cells which were incubated with the natural UDP NAM 1b
(Supplementary Figure 9AꢀD). We hypothesize that a subset
of species may be capable of UDP NAM uptake or tolerant to
modification on NAM residues. These probes could help deꢀ
fine which bacteria have this capability.
We further explored the chemoenzymatic synthesis
of UDP NAM derivatives, of which until this work were preꢀ
viously limited through synthetic methodology and no UDP
NAM derivatives with modifications on the carbohydrate have
been reported. We optimized the purification and compound
characterization of these UDP derivatives and further explored
their utility in the conversion to the pentapeptide intermediates
of PG biosynthesis known as Park’s nucleotide (Scheme 1).
Substrates that AmgK converted to the monoꢀphosphate were
next used to assess substrate specificity for the other PG recyꢀ
cling enzyme, MurU, and the four PG biosynthetic enzymes
(MurCꢀF) (Scheme 1). The data show that all modifications
tolerated by AmgK were accepted by the subsequent enzymes,
except the biotin modifications, 9 and 10, which was not
turned over by MurU (Table 1, Supporting Information Table
1). This is the first report of a large collection of Park’s nuꢀ
cleotide derivatives and will be valuable probes in studying
bacterial cell wall biosynthesis in a variety of organisms.
Discussion
Traditionally, the synthesis of UDP and its sugar
cargos has been extremely demanding, and therefore underexꢀ
plored. As UDP sugars are critical for nearly every step of the
biosynthesis of bacterial PG, access to these phosphate interꢀ
mediates is valuable to understanding the vitality of this polyꢀ
mer. The modular synthesis of 2ꢀN functionalized NAMs alꢀ
lowed for the development of a library of NAM derivatives to
include substrates beyond the azide and alkyne compounds (3,
4). We chose to construct the library at the 2ꢀN-postion
(Scheme 1) as modification at the 6 position was not tolerated
by AmgK (Table 1, entry 1). We have expanded the NAM
probes to include derivatives with capabilities of Staudinger
ligations, IEDDA, photo activating, affinity tagging, as well as
fluorine NMR spectroscopy analysis (Scheme 2). With these
derivatives in hand, we were able to assess the substrate speciꢀ
ficity and kinetic profile of the recycling enzyme AmgK, a key
enzyme in the production line of uridine diphosphate NAM
sugars (Table 1 and Table 2). AmgK appears to tolerate modiꢀ
fications at the 2 and 3 positions of the carbohydrate, convertꢀ
ing modifications such as levulinic acid, biotin, tetrazine, and
diazirine to the respective phosphorꢀsugars. Compounds that
lacked the acetyl (Table 1, entry 8) or displayed a quaternary
center alpha to the carbonyl of the amide (11ꢀ13) were not
accepted by the enzyme (Table 1). As there is no crystal
structure for this kinase, we propose that AmgK “reads” the
carbonyl state of the NAM. Although AmgK was able to turnꢀ
over substrates containing atomistically large modifications at
the 2ꢀposition, kinetic characterization revealed that not all
Other types of bioꢀorthogonal chemistry were asꢀ
sayed; the tetrazine NAM derivative 8 was subjected to the
whole cell labeling methodology in which genetically engiꢀ
neered E. coli cells containing the recycling enzymes AmgK
and MurU were subjected to IEDDA with TCO TAMRA.
While few cells were labeled using this methodology, the ones
that were fluorescent have different morphologies (Fig 2) with
what appears to be a thinner cell wall compared to the healthiꢀ
er cells in the field of view. While it appears that the smaller
bioorthonoal functionality is more desirable to study global
whole cell NAM labeling, the tetrazine modification upon
optimization could prove useful in studying liveꢀcell UDP
NAM production and shuttling.
In order to test if the PG labeling method was ameꢀ
nable to modification at the 3ꢀpostion of the NAM, a new synꢀ
thesis of a modified 3ꢀmuramic acid was developed. The synꢀ
thesis of the 3ꢀ azido NAM derivative 14 was designed and
optimized to correctly set the muramic acid stereochemistry
while incorporating the unnatural bioorthogonal azide handle
(Scheme 3). Biochemical assays indicated that both the recyꢀ
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 10
cling and early PG biosynthetic enzymes are permissive to the veloped: bioorthogonal tetrazine ligation and lactic acid modiꢀ
1
2
3
4
5
6
7
8
azide modification (Table 1, 2 and Supporting Table 1). When
14 was used in the whole cell labeling of E. coli QKU cells,
labeling was concentrated to the PG septal division ring (Figꢀ
ure 1, Supplementary Fig 1). A small population of cells apꢀ
pears to be labeled beyond the septal division ring (Figure 1,
top row white arrow). This preliminary observation suggests
that the modification at the lactic acid is incorporated into lipid
linked PG intermediates62 and/or new PG but may fail to inꢀ
corporate effectively into the growing PG polymer, suggesting
that different transglycosylases have different substrate preferꢀ
ences (Fig 1 and Supplemental Fig 1). Further detailed mass
spectrometry analysis is necessary to quantify the pools of PG
that are modified. 14 and derivatives thereof will be extremeꢀ
ly valuable in studying the PG of bacteria that are known to
modify the 2ꢀposition, such as Mtb, which is known to Nꢀ
fication. The robust chemoenzymatic synthesis of the 2ꢀazido
UDP NAM derivative allowed a Gramꢀpositive commensal to
be labeled on the sugar backbone at unprecedented resolution.
The NAMꢀPG labeling method and the expanded substrate
scope will be used in tandem with other complementary
bioorthogonal labeling strategies55ꢀ56, 59, 68ꢀ78 to illuminate funꢀ
damental aspects of PG biosynthesis and immune processing.
These tools will allow the study of NAM production and
breakdown of a variety of pathogen and commensal bacteria to
reveal novel antibiotic targets and immune recognition eleꢀ
ments.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
glycolylate this position to evade an immune response49, 63ꢀ66
.
The Supporting Information is available free of charge on the
ACS Publications website. Supplementary Information includes
supplementary figures and tables, biochemical methods, synthetic
procedures and compound characterization and is available as a
single PDF file.
NAM derivatives containing modification at the 3ꢀposition
could be used to probe this resistance mechanism.
This work expands and improves the methodology to
label bacterial PG at the NAM residue. Previously, this methꢀ
odology was limited to the genetic manipulation of organisms
to include the recycling enzymes amgK and murU as well as
lengthy synthesis of UDP peptide probes. In order to eliminate
this genetic editing step and make the synthesis scalable and
accessible, we took advantage of the chemoenzymatic producꢀ
tion of the azide UDP NAM derivatives and developed the
methodology to produce these analogues. PG coꢀlabeling was
only achieved in L. acidophilus as observed by superresolution
microscopy (Figure 3). The result complements the previous
work of Nishimura and coworkers where fluorescein was synꢀ
thetically attached to the lysine of UDPꢀNAM pentapeptide to
label the PG of L. acidophilus.59 With the application of superꢀ
resolution microscopy, new details about the localization of
the NAM residues in L. acidophilus along the PGꢀdivision ring
and sidewall have been observed (Fig. 3).
AUTHOR INFORMATION
Corresponding Author
* cgrimes@udel.edu
The authors declare no competing financial interest.
AUTHOR CONTRIBUTION
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
ACKNOWLEDGMENT
We thank Professor Joseph Fox and the Fox Laboratory, speꢀ
cifically Yi Li, the Boyd Laboratory at the University of Delꢀ
aware, specifically Nathan McDonald. We thank Professor
Burnaby Munsen at the University of Delaware. We thank Dr.
Papa Nii AsareꢀOkai, the director of the MS facility as well as
Dr. Yang in the Chemistry and Biochemistry Department at
the University of Delaware, with assistance in MS. We thank
Dr. Jeffrey Caplan, the Director of Bioimaging and the Delaꢀ
ware Biotechnology Institute for assistance with superresoluꢀ
tion microscopy. For financial support, this project was supꢀ
ported by the NIH U01 Common Fund program with grant
number U01CA221230ꢀ01; Delaware COBRE program, with
a grant from the National Institute of General Medical Sciencꢀ
esꢀNIGMS P20GM104316ꢀ01A1. C.L.G. is a Pew Biomedical
Scholar and Cottrell Scholar, and thanks the Pew Foundation
and the Research Corporation for Science Advancement.
K.E.D. and M.R.J thank the NIH for support through a CBI
training grant: 5T32GM008550. For instrumentation support,
the Delaware COBRE and INBRE programs supported this
project with a grant from the National Institute of General
Medical Sciences–NIGMS (5 P30 GM110758ꢀ02,
It is worth noting, that the growth conditions of the
bacterial cells resulted in different labeling efficiencies for L.
acidophilus (Supplemental Figure 8A,B). L. acidophilus is a
facultative anaerobe and is notoriously difficult to grow under
laboratory conditions. We qualitatively note that when cells
were actively growing, labeling efficiency of the PG appeared
to be higher than when cells were dormant (Supporting Inforꢀ
mation, Supplementary Figure 8A,B), indicating that the cells
may have been under different stresses, and those that were
more actively growing and dividing are more likely to uptake
the UDP NAM derivative. We note that these NAM probes
may be used to explore bacterial nucleotide sugar transportaꢀ
tion. To date, there are not any known UDPꢀNAM transporters
that could allow for this event to occur. However, transporters
for UDP galactose (UGT), UDP NAG, GDP fucose (GFT) and
CMP sialic acid (CST) exist.67 The identification of UDP
NAM sugar transporters could be potential sources for selecꢀ
tive antibiotic targets.
In conclusion, we have developed two critical synꢀ
thetic methods: chemoenzymatic synthesis of UDPꢀNAM
sugar derivatives and modification chemistry for the 3ꢀ
position of the NAM lactic acid. These syntheses were used to
explore the substrate specificity of the PG recycling and bioꢀ
synthetic enzymes and revealed relaxed specificity at the 2 and
3 positions of the NAM. These probes allowed for new biologꢀ
ical applications of the PGꢀglycanꢀlabeling method to be deꢀ
P20GM104316ꢀ01A1 and P20 GM103446) from the National
Institutes of Health.
ACS Paragon Plus Environment

Page 7 of 10
Journal of the American Chemical Society
17.
Thaiss, C. A.; Zmora, N.; Levy, M.; Elinav, E., The microꢀ
ABBREVIATIONS
biome and innate immunity. Nature 2016, 535 (7610), 65ꢀ74.
1
2
3
4
5
6
7
8
18.
Hasegawa, M.; Yang, K.; Hashimoto, M.; Park, J. H.; Kim,
AmgK, MurNAc/GlcNAc anomeric kinase; CMP, cyotidine
monophosphate; CMP sialic acid transporter, CST; CuAAC, copꢀ
per catalyzed azide alkyne cycloaddition; DNA, Deoxyribonucleic
acid; GDP, guanosine diphosphate; GFT, GDP fucose transporter;
HPLC, HighꢀPerformance Liquid Chromatography HRMS, High
resolution mass spectrometry; IEDDA, Inverse electron demand
diels alꢀder; Mtb, Mycobacterium tuberculosis; MurU, NAM αꢀ1
phosphate uridylyl; NAG, Nꢀacetylglucosamine; NAM, Nꢀ
acetylmuramic acid; PG, Peptidoglycan; PMB, paraꢀ
methoxybenzyl; RNA, Ribonucleic acid; SIM, Structured illuꢀ
mination microscopy; TCO, transꢀcyclooctene; TCCA, triꢀ
chloroisocyanuric acid; TLC, thin layer chromatography; UDP,
Uridine diphosphate; UDP galactose transporter, UGT; WGA,
Wheat Germ Agglutinin.
Y. G.; Fujimoto, Y.; Nunez, G.; Fukase, K.; Inohara, N., Differential
release and distribution of Nod1 and Nod2 immunostimulatory moleꢀ
cules among bacterial species and environments. J Biol Chem 2006,
281 (39), 29054ꢀ63.
19.
Humann, J.; Lenz, L. L., Bacterial peptidoglycan degrading
enzymes and their impact on host muropeptide detection. J Innate
Immun 2009, 1 (2), 88ꢀ97.
20.
innate immune system. Cell Mol Life Sci 2003, 60 (9), 1793ꢀ804.
21. van Heijenoort, J., Recent advances in the formation of the
bacterial peptidoglycan monomer unit. Nat Prod Rep 2001, 18 (5),
503ꢀ19.
22.
Dziarski, R., Recognition of bacterial peptidoglycan by the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Liang, H.; DeMeester, K. E.; Hou, C. W.; Parent, M. A.;
Caplan, J. L.; Grimes, C. L., Metabolic labelling of the carbohydrate
core in bacterial peptidoglycan and its applications. Nat Commun
2017, 8, 15015.
REFERENCES
23.
Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemisꢀ
1.
Westheimer, F. H., Why nature chose phosphates. Science
try: Diverse Chemical Function from a Few Good Reactions. Angew
Chem Int Ed Engl 2001, 40 (11), 2004ꢀ2021.
24.
K. B., A stepwise huisgen cycloaddition process: copper(I)ꢀcatalyzed
regioselective "ligation" of azides and terminal alkynes. Angew Chem
Int Ed Engl 2002, 41 (14), 2596ꢀ9.
1987, 235 (4793), 1173ꢀ8.
2.
Hunter, T., Why nature chose phosphate to modify proꢀ
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
teins. Philos Trans R Soc Lond B Biol Sci 2012, 367 (1602), 2513ꢀ6.
3.
Chou, W. K.; Hinderlich, S.; Reutter, W.; Tanner, M. E.,
Sialic acid biosynthesis: stereochemistry and mechanism of the reacꢀ
tion catalyzed by the mammalian UDPꢀNꢀacetylglucosamine 2ꢀ
epimerase. J Am Chem Soc 2003, 125 (9), 2455ꢀ61.
25.
Tornoe, C. W.; Christensen, C.; Meldal, M., Peptidotriaꢀ
zoles on solid phase: [1,2,3]ꢀtriazoles by regiospecific copper(i)ꢀ
catalyzed 1,3ꢀdipolar cycloadditions of terminal alkynes to azides. J
Org Chem 2002, 67 (9), 3057ꢀ64.
4.
in glycogen storage disease. J Biol Chem 1952, 199 (2), 661ꢀ7.
5. Lomako, J.; Lomako, W. M.; Kirkman, B. R.; Whelan, W.
Cori, G. T.; Cori, C. F., Glucoseꢀ6ꢀphosphatase of the liver
26.
Gisin, J.; Schneider, A.; Nagele, B.; Borisova, M.; Mayer,
J., The role of phosphate in muscle glycogen. Biofactors 1994, 4 (3ꢀ4),
167ꢀ71.
C., A cell wall recycling shortcut that bypasses peptidoglycan de novo
biosynthesis. Nat Chem Biol 2013, 9 (8), 491ꢀ3.
27.
the right (bioorthogonal) chemistry. ACS Chem Biol 2014, 9 (3), 592ꢀ
605.
28.
modified Staudinger reaction. Science 2000, 287 (5460), 2007ꢀ10.
29. Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.;
Prescher, J. A., Functionalized cyclopropenes as bioorthogonal chemiꢀ
cal reporters. J Am Chem Soc 2012, 134 (45), 18638ꢀ43.
30.
imaging of cyclopropene tags with fluorogenic tetrazine cycloaddiꢀ
tions. Angew Chem Int Ed Engl 2012, 51 (30), 7476ꢀ9.
31.
of methylenecyclopropanes from chiral cyclopropene derivatives.
Angew Chem Int Ed Engl 2006, 45 (24), 3960ꢀ2.
32.
tion: fast bioconjugation based on inverseꢀelectronꢀdemand Dielsꢀ
Alder reactivity. J Am Chem Soc 2008, 130 (41), 13518ꢀ9.
33.
momultimeric complexes of CD22 in B cells revealed by proteinꢀ
glycan crossꢀlinking. Nat Chem Biol 2005, 1 (2), 93ꢀ7.
34.
sugars for capturing glycoprotein interactions. J Am Chem Soc 2008,
130 (11), 3278ꢀ9.
35.
6.
Torres, C. R.; Hart, G. W., Topography and polypeptide
Patterson, D. M.; Nazarova, L. A.; Prescher, J. A., Finding
distribution of terminal Nꢀacetylglucosamine residues on the surfaces
of intact lymphocytes. Evidence for Oꢀlinked GlcNAc. J Biol Chem
1984, 259 (5), 3308ꢀ17.
Saxon, E.; Bertozzi, C. R., Cell surface engineering by a
7.
Warren, L.; Felsenfeld, H., NꢀAcetylmannosamineꢀ6ꢀ
phosphate and Nꢀacetylneuraminic acidꢀ9ꢀphosphate as intermediates
in sialic acid biosynthesis. Biochem Biophys Res Commun 1961, 5,
185ꢀ90.
8.
Wells, L.; Vosseller, K.; Hart, G. W., Glycosylation of nuꢀ
Yang, J.; Seckute, J.; Cole, C. M.; Devaraj, N. K., Liveꢀcell
cleocytoplasmic proteins: signal transduction and OꢀGlcNAc. Science
2001, 291 (5512), 2376ꢀ8.
9.
Holtje, J. V., Growth of the stressꢀbearing and shapeꢀ
Yang, Z.; Xie, X.; Fox, J. M., Diastereoselective synthesis
maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol
Rev 1998, 62 (1), 181ꢀ203.
10.
Strominger, J. L.; Park, J. T.; Thompson, R. E., Composiꢀ
Blackman, M. L.; Royzen, M.; Fox, J. M., Tetrazine ligaꢀ
tion of the cell wall of Staphylococcus aureus: its relation to the mechꢀ
anism of action of penicillin. J Biol Chem 1959, 234, 3263ꢀ8.
11.
Schleifer, K. H.; Kandler, O., Peptidoglycan types of bacteꢀ
Han, S.; Collins, B. E.; Bengtson, P.; Paulson, J. C., Hoꢀ
rial cell walls and their taxonomic implications. Bacteriol Rev 1972, 36
(4), 407ꢀ77.
12.
immunoadjuvants. Nat Immunol 2007, 8 (12), 1269ꢀ71.
13. Rangan, K. J.; Pedicord, V. A.; Wang, Y. C.; Kim, B.; Lu,
Strominger, J. L., Bacterial cell walls, innate immunity and
Tanaka, Y.; Kohler, J. J., Photoactivatable crosslinking
Y.; Shaham, S.; Mucida, D.; Hang, H. C., A secreted bacterial peptiꢀ
doglycan hydrolase enhances tolerance to enteric pathogens. Science
2016, 353 (6306), 1434ꢀ1437.
Feng, L.; Hong, S.; Rong, J.; You, Q.; Dai, P.; Huang, R.;
Tan, Y.; Hong, W.; Xie, C.; Zhao, J.; Chen, X., Bifunctional unnatural
sialic acids for dual metabolic labeling of cellꢀsurface sialylated glyꢀ
cans. J Am Chem Soc 2013, 135 (25), 9244ꢀ7.
36.
illuminate interactions in living cells. Mol Biosyst 2008, 4 (6), 473ꢀ80.
37. Li, Z.; Hao, P.; Li, L.; Tan, C. Y.; Cheng, X.; Chen, G. Y.;
Sze, S. K.; Shen, H. M.; Yao, S. Q., Design and synthesis of minimalꢀ
ist terminal alkyneꢀcontaining diazirine photoꢀcrosslinkers and their
incorporation into kinase inhibitors for cellꢀ and tissueꢀbased proteome
profiling. Angew Chem Int Ed Engl 2013, 52 (33), 8551ꢀ6.
14.
Girardin, S. E.; Boneca, I. G.; Viala, J.; Chamaillard, M.;
Labigne, A.; Thomas, G.; Philpott, D. J.; Sansonetti, P. J., Nod2 is a
general sensor of peptidoglycan through muramyl dipeptide (MDP)
detection. J Biol Chem 2003, 278 (11), 8869ꢀ72.
Tanaka, Y.; Bond, M. R.; Kohler, J. J., Photocrosslinkers
15.
Inohara, N.; Ogura, Y.; Fontalba, A.; Gutierrez, O.; Pons,
F.; Crespo, J.; Fukase, K.; Inamura, S.; Kusumoto, S.; Hashimoto, M.;
Foster, S. J.; Moran, A. P.; FernandezꢀLuna, J. L.; Nunez, G., Host
recognition of bacterial muramyl dipeptide mediated through NOD2.
Implications for Crohn's disease. J Biol Chem 2003, 278 (8), 5509ꢀ12.
38.
Kumasaka, R.; Kikuchi, A.; Yagi, M., Photoexcited states
16.
Wolf, A. J.; Underhill, D. M., Peptidoglycan recognition by
of UV absorbers, benzophenone derivatives. Photochem Photobiol
2014, 90 (4), 727ꢀ33.
the innate immune system. Nat Rev Immunol 2018, 18, 243ꢀ254.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 10
39.
Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin,
peptidoglycan dynamics of an intracellular pathogen. ACS Chem Biol
2013, 8 (3), 500ꢀ5.
57. Yoshida, M.; Stadler, J.; Bertholdt, G.; Gerisch, G., Wheat
J. W., Photoꢀcrossꢀlinking interacting proteins with a genetically enꢀ
coded benzophenone. Nat Methods 2005, 2 (5), 377ꢀ84.
1
2
3
4
5
6
7
8
9
40.
Morin, B.; Cadet, J., Type I benzophenoneꢀmediated nuꢀ
germ agglutinin binds to the contact site A glycoprotein of Dictyosteliꢀ
um discoideum and inhibits EDTAꢀstable cell adhesion. EMBO J
1984, 3 (11), 2663ꢀ70.
cleophilic reaction of 5'ꢀaminoꢀ2',5'ꢀdideoxyguanosine. A model sysꢀ
tem for the investigation of photosensitized formation of DNAꢀprotein
crossꢀlinks. Chem Res Toxicol 1995, 8 (5), 792ꢀ9.
58.
Ursell, T. S.; Nguyen, J.; Monds, R. D.; Colavin, A.;
41.
Okaru, A. O.; Brunner, T. S.; Ackermann, S. M.; Kuballa,
Billings, G.; Ouzounov, N.; Gitai, Z.; Shaevitz, J. W.; Huang, K. C.,
Rodꢀlike bacterial shape is maintained by feedback between cell curvaꢀ
ture and cytoskeletal localization. Proc Natl Acad Sci U S A 2014, 111
(11), E1025ꢀ34.
T.; Walch, S. G.; KohlꢀHimmelseher, M.; Lachenmeier, D. W., Appliꢀ
cation of (19)F NMR Spectroscopy for Content Determination of
Fluorinated Pharmaceuticals. J Anal Methods Chem 2017, 2017,
9206297.
59.
Sadamoto, R.; Niikura, K.; Sears, P. S.; Liu, H.; Wong, C.
42.
Martino, R.; Gilard, V.; Desmoulin, F.; MaletꢀMartino, M.,
H.; Suksomcheep, A.; Tomita, F.; Monde, K.; Nishimura, S., Cellꢀwall
engineering of living bacteria. J Am Chem Soc 2002, 124 (31), 9018ꢀ9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interest of fluorineꢀ19 nuclear magnetic resonance spectroscopy in the
detection, identification and quantification of metabolites of anticancer
and antifungal fluoropyrimidine drugs in human biofluids. Chemotherꢀ
apy 2006, 52 (5), 215ꢀ9.
60.
Jiang, Y.; Lu, X.; Man, C.; Han, L.; Shan, Y.; Qu, X.; Liu,
Y.; Yang, S.; Xue, Y.; Zhang, Y., Lactobacillus acidophilus induces
cytokine and chemokine production via NFꢀkappaB and p38 mitogenꢀ
activated protein kinase signaling pathways in intestinal epithelial
cells. Clin Vaccine Immunol 2012, 19 (4), 603ꢀ8.
43.
Dalvit, C.; Fagerness, P. E.; Hadden, D. T.; Sarver, R. W.;
Stockman, B. J., FluorineꢀNMR experiments for highꢀthroughput
screening: theoretical aspects, practical considerations, and range of
applicability. J Am Chem Soc 2003, 125 (25), 7696ꢀ703.
61.
Sampson, B. A.; Misra, R.; Benson, S. A., Identification
and characterization of a new gene of Escherichia coli Kꢀ12 involved
in outer membrane permeability. Genetics 1989, 122 (3), 491ꢀ501.
44.
Sano, T.; Vajda, S.; Cantor, C. R., Genetic engineering of
streptavidin, a versatile affinity tag. J Chromatogr B Biomed Sci Appl
1998, 715 (1), 85ꢀ91.
45.
V.; Penzotti, J. E.; To, R.; Hyre, D.; Le Trong, I.; Lybrand, T. P.;
Stenkamp, R. E., Streptavidinꢀbiotin binding energetics. Biomol Eng
1999, 16 (1ꢀ4), 39ꢀ44.
62.
Qiao, Y.; Srisuknimit, V.; Rubino, F.; Schaefer, K.; Ruiz,
N.; Walker, S.; Kahne, D., Lipid II overproduction allows direct assay
of transpeptidase inhibition by betaꢀlactams. Nat Chem Biol 2017, 13
(7), 793ꢀ798.
Stayton, P. S.; Freitag, S.; Klumb, L. A.; Chilkoti, A.; Chu,
63.
manipulate the immune response. Immunity 2007, 26 (2), 149ꢀ61.
64. Bera, A.; Herbert, S.; Jakob, A.; Vollmer, W.; Gotz, F.,
Sansonetti, P. J.; Di Santo, J. P., Debugging how bacteria
46.
Laitinen, O. H.; Hytonen, V. P.; Nordlund, H. R.; Kulomaa,
M. S., Genetically engineered avidins and streptavidins. Cell Mol Life
Sci 2006, 63 (24), 2992ꢀ3017.
47.
acetylmuramic acid/Nꢀacetylglucosamine kinase of Clostridium acetoꢀ
butylicum. J Bacteriol 2011, 193 (19), 5386ꢀ92.
48.
Mayer, C.; Stehle, T., Crystal Structure of the NꢀAcetylmuramic Acid
alphaꢀ1ꢀPhosphate (MurNAcꢀalpha1ꢀP) Uridylyltransferase MurU, a
Minimal Sugar Nucleotidyltransferase and Potential Drug Target
Enzyme in Gramꢀnegative Pathogens. J Biol Chem 2015, 290 (17),
10804ꢀ13.
Why are pathogenic staphylococci so lysozyme resistant? The peptiꢀ
doglycan Oꢀacetyltransferase OatA is the major determinant for lysoꢀ
zyme resistance of Staphylococcus aureus. Mol Microbiol 2005, 55
(3), 778ꢀ87.
Reith, J.; Berking, A.; Mayer, C., Characterization of an Nꢀ
65.
bacterial peptidoglycan. FEMS Microbiol Rev 2008, 32 (2), 287ꢀ306.
66. Coulombe, F.; Divangahi, M.; Veyrier, F.; de Leseleuc, L.;
Vollmer, W., Structural variation in the glycan strands of
RennerꢀSchneck, M.; Hinderberger, I.; Gisin, J.; Exner, T.;
Gleason, J. L.; Yang, Y.; Kelliher, M. A.; Pandey, A. K.; Sassetti, C.
M.; Reed, M. B.; Behr, M. A., Increased NOD2ꢀmediated recognition
of Nꢀglycolyl muramyl dipeptide. J Exp Med 2009, 206 (8), 1709ꢀ16.
67.
Hadley, B.; Maggioni, A.; Ashikov, A.; Day, C. J.; Haselꢀ
49.
Raymond, J. B.; Mahapatra, S.; Crick, D. C.; Pavelka, M.
horst, T.; Tiralongo, J., Structure and function of nucleotide sugar
transporters: Current progress. Comput Struct Biotechnol J 2014, 10
(16), 23ꢀ32.
S., Jr., Identification of the namH gene, encoding the hydroxylase
responsible for the Nꢀglycolylation of the mycobacterial peptidoglyꢀ
can. J Biol Chem 2005, 280 (1), 326ꢀ33.
68.
Daniel, R. A.; Errington, J., Control of cell morphogenesis
50.
Hansen, J. M.; Golchin, S. A.; Veyrier, F. J.; Domenech,
in bacteria: two distinct ways to make a rodꢀshaped cell. Cell 2003,
113 (6), 767ꢀ76.
P.; Boneca, I. G.; Azad, A. K.; Rajaram, M. V.; Schlesinger, L. S.;
Divangahi, M.; Reed, M. B.; Behr, M. A., Nꢀglycolylated peptidoglyꢀ
can contributes to the immunogenicity but not pathogenicity of Mycoꢀ
bacterium tuberculosis. J Infect Dis 2014, 209 (7), 1045ꢀ54.
69.
wall biosynthesis in bacteria. Curr Opin Microbiol 2015, 27, 69ꢀ77.
70. Garner, E. C.; Bernard, R.; Wang, W.; Zhuang, X.; Rudner,
Gale, R. T.; Brown, E. D., New chemical tools to probe cell
51.
Basu, N.; Maity, S. K.; Chaudhury, A.; Ghosh, R., Triꢀ
D. Z.; Mitchison, T., Coupled, circumferential motions of the cell wall
synthesis machinery and MreB filaments in B. subtilis. Science 2011,
333 (6039), 222ꢀ5.
chloroisocyanuric acid (TCCA): an efficient green reagent for activaꢀ
tion of thioglycosides toward hydrolysis. Carbohydr Res 2013, 369,
10ꢀ3.
71.
Kocaoglu, O.; Calvo, R. A.; Sham, L. T.; Cozy, L. M.;
52.
Uehara, T.; Suefuji, K.; Jaeger, T.; Mayer, C.; Park, J. T.,
Lanning, B. R.; Francis, S.; Winkler, M. E.; Kearns, D. B.; Carlson, E.
E., Selective penicillinꢀbinding protein imaging probes reveal substrucꢀ
ture in bacterial cell division. ACS Chem Biol 2012, 7 (10), 1746ꢀ53.
MurQ Etherase is required by Escherichia coli in order to metabolize
anhydroꢀNꢀacetylmuramic acid obtained either from the environment
or from its own cell wall. J Bacteriol 2006, 188 (4), 1660ꢀ2.
72.
Kuru, E.; Hughes, H. V.; Brown, P. J.; Hall, E.; Tekkam,
53.
Gustafsson, M. G.; Shao, L.; Carlton, P. M.; Wang, C. J.;
S.; Cava, F.; de Pedro, M. A.; Brun, Y. V.; VanNieuwenhze, M. S., In
Situ probing of newly synthesized peptidoglycan in live bacteria with
fluorescent Dꢀamino acids. Angew Chem Int Ed Engl 2012, 51 (50),
12519ꢀ23.
Golubovskaya, I. N.; Cande, W. Z.; Agard, D. A.; Sedat, J. W., Threeꢀ
dimensional resolution doubling in wideꢀfield fluorescence microscopy
by structured illumination. Biophys J 2008, 94 (12), 4957ꢀ70.
54.
Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A., Teꢀ
73.
Kuru, E.; Tekkam, S.; Hall, E.; Brun, Y. V.; Van Nieuꢀ
trazineꢀbased cycloadditions: application to pretargeted live cell imagꢀ
ing. Bioconjug Chem 2008, 19 (12), 2297ꢀ9.
55.
VanNieuwenhze, M.; Maurelli, A. T., A new metabolic cellꢀwall
labelling method reveals peptidoglycan in Chlamydia trachomatis.
Nature 2014, 506 (7489), 507ꢀ10.
wenhze, M. S., Synthesis of fluorescent Dꢀamino acids and their use
for probing peptidoglycan synthesis and bacterial growth in situ. Nat
Protoc 2015, 10 (1), 33ꢀ52.
Liechti, G. W.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.;
74.
Nelson, J. W.; Chamessian, A. G.; McEnaney, P. J.; Murelꢀ
li, R. P.; Kazmierczak, B. I.; Spiegel, D. A., A biosynthetic strategy for
reꢀengineering the Staphylococcus aureus cell wall with nonꢀnative
small molecules. ACS Chem Biol 2010, 5 (12), 1147ꢀ55.
56.
Siegrist, M. S.; Whiteside, S.; Jewett, J. C.; Aditham, A.;
Cava, F.; Bertozzi, C. R., (D)ꢀAmino acid chemical reporters reveal
ACS Paragon Plus Environment

Page 9 of 10
Journal of the American Chemical Society
75.
Sadamoto, R.; Matsubayashi, T.; Shimizu, M.; Ueda, T.;
Kahne, D., Reconstitution of peptidoglycan crossꢀlinking leads to
improved fluorescent probes of cell wall synthesis. J Am Chem Soc
2014, 136 (31), 10874ꢀ7.
Koshida, S.; Koda, T.; Nishimura, S., Bacterial surface engineering
utilizing glucosamine phosphate derivatives as cell wall precursor
surrogates. Chemistry 2008, 14 (33), 10192ꢀ5.
1
2
3
4
5
6
7
8
76.
Shieh, P.; Siegrist, M. S.; Cullen, A. J.; Bertozzi, C. R.,
Imaging bacterial peptidoglycan with nearꢀinfrared fluorogenic azide
probes. Proc Natl Acad Sci U S A 2014, 111 (15), 5456ꢀ61.
77.
D. Z.; Walker, S., Imaging peptidoglycan biosynthesis in Bacillus
subtilis with fluorescent antibiotics. Proc Natl Acad Sci U S A 2006,
103 (29), 11033ꢀ8.
Tiyanont, K.; Doan, T.; Lazarus, M. B.; Fang, X.; Rudner,
9
78.
Lebar, M. D.; May, J. M.; Meeske, A. J.; Leiman, S. A.;
Lupoli, T. J.; Tsukamoto, H.; Losick, R.; Rudner, D. Z.; Walker, S.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 10
Insert Table of Contents artwork here
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10
ACS Paragon Plus Environment