. Author manuscript; available in PMC: 2018 May 15.

Published in final edited form as: Nat Protoc. 2016 Jun 23;11(7):1280–1298. doi: 10.1038/nprot.2016.067

Table 1.

A survey of reports for in vitro Sugar-PP-Und synthesis.

Reports	Woodward, et al⁹	Holkenbrink, et al³³	Lebar, et al³¹	Huang, et al³²	Huang, et al²⁹	Gale, et al³⁰
Target Compounds	RU-PP-Und & analogues	Trisaccharide-PP-Und	m-DAP Lipid II precursor	Lipid II analogues	Lipid II-PP-Und analogues	GlcNAc-PP-Und
Biosynthetic Pathway Involved	O-PS, LPS	S-layer Glycoprotein	Cell wall Peptidoglycan	Cell wall Peptidoglycan	Cell wall Peptidoglycan	Teichoic acid
Source of Starting Lipid(s)	Commercial Und; Synthetic lipids	Commercial Und-P	Synthetic Heptaprenol	Synthetic lipids	Extracted and commercial lipids	Commercial Und
Chemical Synthesis	Monosaccharide-1-phosphate and Und-P formation; pyrophosphate bond formation; final deprotection	Trisaccharide-1-phosphate formation; pyrophosphate bond formation; final deprotection	Monosaccharide-1-phosphate and lipid phosphate formation; pyrophosphate bond formation; final deprotection	Disaccharide-1-phosphate and lipid phosphate formation; pyrophosphate bond formation; final deprotection	N/A	Monosaccharide-1-phosphate and Und-P formation; pyrophosphate bond formation; final deprotection
Enzymatic Synthesis	4 GTs; Step-wise synthesis	NA	1 GT	N/A	Multi-enzyme; One-pot synthesis	2 GTs; Step-wise synthesis
Structure Confirmation (final product)	LRMS(ESI) or MALDI-MS	HRMS(ESI)	LRMS(ESI)	HRMS(ESI) ¹H NMR	HRMS(ESI)	HRMS(ESI); MS/MS
Scale	mg scale	1 mg	<100 µg	mg scale	mg scale	mg scale; MS detectable
Advantage	Synthetic yields; notably pyrophosphate bond formation (59%)	Avoided use of enzymes which need expression and purification	Use of a catalyst in pyrophosphate bond formation greatly improves rate (1/2 day)	---------	Avoided a lengthy chemical synthesis route	---------
Disadvantage	Pyrophosphate bond formation requires 3 days	Low overall yield; Pyrophosphate bond formation requires 1 week	Pyrophosphate bond formation yields using a truncated lipid were below those reported herein	Low overall yield	Enzymatic pyrophosphate bond formation limits the scope of this approach	Low synthetic yields and pyrophosphate bond formation (reaction time = 1 week)