The first six cytoplasmic intermediates of murein biosynthesis (2-7) have been prepared in high yield (>60%) and at preparative scale (10-100 mg) using purified enzymes of the murein biosynthetic pathway (MurA-MurF). For at least three of these compounds, 5, 6, and 7, this is the first high-yield synthesis and purification by any means that has been reported. Detailed spectroscopic and analytical data accompany each of the six compounds and serve as reference standards for future work. The availability of these intermediates will facilitate the study of the murein pathway enzymes MurA-MurF, all of which are validated antibacterial targets.