"Chitin Leash": a polysaccharide heterobifunctional cross-linking agent which can be cleaved by lysozyme.
6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.
Guan
K
K
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115.
Cecchini
D J
DJ
Giese
R W
RW
D002240:Carbohydrate Sequence; D002686:Chitin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D048271:Chitosan; D003432:Cross-Linking Reagents; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism*; D009608:Nitrous Acid / Q000737:chemistry; D010084:Oxidation-Reduction; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D002686:Chitin; D048271:Chitosan; D012259:Ribonuclease, Pancreatic; D008836:Micrococcal Nuclease; D009113:Muramidase; D009608:Nitrous Acid