Table 1.

Selected chemical reporters for peptidoglycan (PG)

Target	Metabolite	Compound	Reporter group (X)	Bioorthogonal reaction(s)a	Proposed route	Speciesb	Applicationsc	Synthesis	Notesb	Refd
PG stem peptide	Park’s nucleotide	1 (Fig. 6)	Ketone	Carbonyl–α-effect nucleophile	Intracellulare	Lactic acid species (GP)	In vitro imaging; modulation of adhesion	Chemo-enzymatic	Not tested in GN bacteria	75
	Park’s nucleotide	2 (Fig. 6)	Fluorescein	--	Intracellulare	E. coli (GN), lactic acid species (GP)	Cell surface detection	Chemo-enzymatic	EDTA required for labeling GN	75
	D-amino acid	AlkDA 10 (Fig. 7)	Alkyne	CuAAC	Intracellular, extracellular	Broad (GP, GN, M)	In vitro and in vivo imaging (infected macrophages); PG stapling	Chemical; comm. available	Route of incorporation may be mixed, species-dependent	76,77
	D-amino acid	AzDA 11 (Fig. 7)	Azide	CuAAC; SPAAC	Intracellular, extracellular	Broad (GP, GN, M)	In vitro imaging; selective killing of MRSA	Chemical; comm. available	Route of incorporation may be mixed, species-dependent	76,77
	D-amino acid	OctDA 12 (Fig. 7)	Cyclooctyne	SPAAC	Extracellular	Broad (GP, GN, M)	In vitro imaging	Chemical	Reacts with fluorogenic azide dye via SPAAC	78
	D-amino acid	NADA 16 (Fig. 7)	7-Nitrobenzo-furazan	--	Extracellular	Broad (GP, GN, M)	In vitro imaging	Chemical; comm. available	Reports on TPase-mediated PG remodeling	76
	D-amino acid	HADA 17 (Fig. 7)	7-Hydroxy-coumarin	--	Intracellular, extracellular	Broad (GP, GN, M)	In vitro and in vivo imaging (colonized host organism)	Chemical; comm. available	Route of incorporation may be mixed, species-dependent	76
	D-amino acid	Rf470DL 21 (Fig. 7)	Molecular rotor dye	--	Extracellular	S. aureus (GP), S. venezuelae (GP), B. subtilis (GP)	Imaging; inhibitor characterization	Chemical	Fluorogenic; labels GN poorly	79
	D-amino carboxamide	TetDAC 25 (Fig. 10)	Tetrazine	Tetrazine ligation	Extracellular	S. aureus (GP)	In vivo imaging (infected host organism)	Chemical	D-amino carboxamide enhances labeling in some GP bacteria	80,81
	D-amino carboxamide	FDL-NH2 27 (Fig. 10)	Fluorescein	--	Extracellular	B. subtilis (GP)	In vitro imaging	Chemical	D-amino carboxamide labels B. subtilis but not E. coli	82
	D-amino acid dipeptide	AlkDADA 28 (Fig. 11)	Alkyne	CuAAC	Intracellular	Broad (GP, GN, M)	In vitro and in vivo imaging (infected macrophages)	Chemical	Reports on MurF-initiated synthesis of new PG	83
	D-amino acid dipeptide	AzDADA 29 (Fig. 11)	Azide	CuAAC; SPAAC	Intracellular	Broad (GP, GN, M)	In vitro imaging	Chemical	Reports on MurF-initiated synthesis of new PG	83
	D-amino acid dipeptide	x-DADA-Alk 32 (Fig. 11)	Alkyne and phenylazide	CuAAC	Intracellular	B. subtilis (GP)	Detection of PG precursor–protein interactions	Chemical	Phenylazide allows protein photo-cross-linking	84
	Stem tripeptide	AeK-NBD 33 (Fig. 11)	7-Nitrobenzo-furazan	--	Intracellular	E. coli (GN)	In vitro imaging	Chemical	Reports on Mpl-mediated recycling route	85
	Stem pentapeptide	FSPPM-488 34 (Fig. 13)	Fluorescein	--	Extracellular	S. aureus (GP)	In vitro imaging	Chemical	Reports on TPase-mediated PG cross-linking	86
	Stem tetrapeptide	FSTPM-488 36 (Fig. 13)	Fluorescein	--	Extracellular	E. faecium (GN), M. smegmatis (M), M. tuberculosis (M)	In vitro imaging	Chemical	Reports on L,D-TPase-mediated PG cross-linking	87
PG glycan core	GlcNAc-1-P	38 (Fig. 14)	Ketone	Carbonyl–α-effect nucleophile	Intracellulare	L. plantarum (GP), W. confuse (GP)	--	Chemical	Peracetylation required for incorporation	88
	MurNAc	42 (Fig. 15)	Alkyne	CuAAC	Intracellular	E. coli ΔMurQ-KU (GN), P. putida (GN), H. pylori HJH1 (GN), B. subtilis 3A38-KU (GP)	In vitro and in vivo imaging (infected macrophages)	Chemical	Requires AmgK/MurU; some species require fosfomycin; peracetylation abolishes labeling; D-Lac methyl ester enhances labeling	89

^aNot applicable for fluorescent reporters operating via one-step incorporation (--).

^bGram classification: GN, Gram-negative; GP, Gram-positive; M, mycobacteria.

^cIn vitro refers to bacterial labeling experiments performed in broth culture; in vivo refers to bacterial labeling experiments performed in a host cell or organism.

^dInitial publication(s) referenced. See text for references for follow-up studies and applications.

^eLimited evidence available to support proposed route of incorporation and/or identity of labeled target (see text).