Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine.

S A Martin,J R Pappenheimer,M L Karnovsky,J M Krueger,K Biemann

doi:10.1016/s0021-9258(18)90795-7

Abstract

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FSu), the nature of whose components had been reported earlier. Less than 1 nmol of the underivatized substance sufficed for the FABMS experiments. The major somnogenic constituent of the purified preparation was a peptidoglycan of Mr = 921 with the structure N-acetylglucosaminyl-N -acetylanhydromuramylalanylglutamyldiaminopimelylalanine. The anhydro linkage is between C-1 and C-6 of the muramyl entity. Two additional substances accompanied the above compound. These were the hydrated form (i.e. in which the muramyl entity had a free reducing end, and a free hydroxyl on C-6), and an anhydro analogue lacking the terminal alanine. The Mr values were 939 and 850, respectively. Methyl esters were prepared, and these were also acetylated. The mass spectra of the methyl ester of Mr = 921 displayed an increase in Mr of 42 (i.e. 3 X 14), indicating the presence, originally, of three free carboxyls. Acetylation increased Mr by a further 168 units (i.e. 4 X 42), indicating 4 hydroxyl or amino groups. These data are consistent with the structure cited above for the main entity of FSu. Similar confirmatory results were obtained for the two minor constituents described above. These operations were worked out on natural muramyl peptides of known structure, obtained from other sources, and the data are given for comparison.

Highlights

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FS,), the nature of whose components had been reported earlier
This wasbased onthe observation that somnogenic activity correlated better with the presence of muramic acid than with thatof glucosamine (7)
Examination of FS, by Fast Atom Bombardment-Mass Spectrometry-The FAB mass spectrum of less than 1 nmol of sleep-promoting FS, included three signals which corresponded to protonated molecular ions (MH’) at m/z 940, 922, and 851, (Fig. 1)indicating the presence of three discrete compounds of M, = 939, 921, and 850, respectively

Summary

Introduction

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FS,), the nature of whose components had been reported earlier. Two additional substances accompanied the above compound These were the hydrated form (Le. in which the muramyl entity had a free reducing end, and a free hydroxylon C-6),and an anhydro analogue lacking the terminal alanine. We believed that the final productof the purificationprocedure (7) consisted of a t least two components and that the active substance was a monosaccharide peptide (i.e. containingmuramatebutnot glucosamine) (7). This wasbased onthe observation that somnogenic activity correlated better with the presence of muramic acid than with thatof glucosamine (7). More recently we reported that several muramyl peptides, synthetic and natural, are active as sleep promoters, as well as being pyrogens, immunoadjuvants, and promotersof nonspecific hostresistance,dependingonstructure (9-11). Thisholds for the pyrogenic and immunostimulatory peptides (fora review see Ref. 10)

Methods

Results

Conclusion