Succinimidyl carbonate

The present report describes preparation and use of new protein modifying reagents, namely methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG) and its bifunctional analog, polyethylene glycol)-bis-N-succinimidyl carbonate (BSC-PEG). 

Scheme I Synthesis of succinimidyl carbonate derivatives of polyethylene glycol.

Figure 4.11 DSC can react with hydroxyl groups to create a succinimidyl carbonate intermediate that is highly reactive toward nucleophiles. In the presence of an amine-containing molecule, the active species can form stable carbamate linkages.

Figure 13.6 APTS-modified surfaces can be activated with DSC to form amine-reactive succinimidyl carbonates for coupling proteins or other amine-containing molecules.

Miron, T., and Wilchek, M. (1993) A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to proteins. Bioconjugate Ghent. 4, 568-569. 

Zalipsky, S., Seltzer, R., and Nho, K. (1991) Succinimidyl carbonates of polyethylene glycol Useful reactive polymers for preparation of protein conjugates. In Polymeric Drugs and Drug Delivery Systems (R.L. Dunn, and R.M. Ottenbrite, eds.), pp. 91-100. American Chemical Society, Washington, D.C. 

A Paquet. Introduction of 9-fluorenylmethoxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amino protecting groups into O-unprotected hydroxyamino acids using succinimidyl carbonates. Can J Chem 60, 976, 1982. 

It seems that a maximum of about 10 ST-PHPMA chains can be attached to 1 CT molecule. The conjugation degree was lower than that of the chy-motrypsin conjugates with PEG-SC (succinimidyl carbonate of mPEG up to 14 chains per one CT molecule) [20,33]. This might be attributed to the solution stmcture difference between the two polymers. PHPMA has a random coil stmcture in aqueous solution, whereas PEG possesses a more extended one. The coiled PHPMA may cover more of the enzyme surface, and the steric effect may prevent more PHPMA macromolecules from attaching to the enzyme. 

The neutral alkali metal salts of benzohydroxamic acids have been found to undergo an unprecedented rearrangement to A(,A( -diarylureas. 2 side reaction, producing /9-alanine derivatives by way of a Lossen rearrangement, has been observed to accompany the hydrolysis of alkyl succinimidyl carbonates in basic aqueous buffers (see Scheme 97). The development of a modified Lossen rearrangement, whereby... 

As alternatives to 4-nitrophenyl chloroformate, carbonyl diimidazole [100-102] or di-A-succinimidyl carbonate [103,104] can be used to convert polymeric alcohols into alkoxycarbonylating reagents suitable for the preparation of support-bound carbamates. Polystyrene-bound alkoxycarbonyl imidazole is less reactive than the corresponding 4-nitrophenyl carbonate, and sometimes requires heating to undergo reaction with amines. Additional activation of these imidazolides can be achieved by N-methylation (Entry 9, Table 14.7). 

Carbamates can also be prepared by treating support-bound amines with alkoxycarbonylating reagents such as chloroformates or aryl carbonates. Chloroformates or dicarbonates (e.g. Boc20) should not be used in large excess for the alkoxycarbonyla-tion of primary amines because double derivatization can occur [119]. Less reactive reagents include 4-nitrophenyl carbonates and A-succinimidyl carbonates (Entries 2 and 3, Table 14.8). 

Support-bound benzimidazoles can be N-arylated by treatment with arylboronic acids in the presence of Cu(OAc)2 (Entry 3, Table 15.15). Benzimidazolones, which can be prepared from resin-bound 1,2-diaminobenzenes and di-iV-succinimidyl carbonate (Entry 8, Table 15.14), can also be N-alkylated on insoluble supports (Entry 4, Table 15.15). Benzimidazole-2-thiones, on the other hand, are generally cleanly S-alkylated (Entry 5, Table 15.15). Examples of amination by aromatic nucleophilic substitution at resin-bound halopurines are given in Section 10.1.2. 

H-Gly-Pro-Hyp-OH (3.0 g, 10.5 mmol) was dissolved in 10% Na2C03 (54 mL) and stored at 4°C. N-Fluoren-9-ylmethyl succinimidyl carbonate (4.05 g, 12.0 mmol) was dissolved in DME (45 mL) and stirred at 4°C. The aq Na2C03 soln was added slowly to the DME soln, and the reaction proceeded for 2.5 h at 4°C and 21 h at rt. The soln was filtered, and H20 (360 mL) was added to the filtrate. The aqueous layer was extracted with Et20 (300 mL), acidified to pH 2 with coned HQ, reduced to halfvolume at 80 °C under reduced pressure, and stored at 4 °C for 24 h to induce precipitation. The aqueous layer was decanted from the oily precipitate, reduced to ca. 30 mL at 81 °C under reduced pressure, and stored at 4°C for 24 h. Again, the aqueous layer was decanted from the oily precipitate. Both oily precipitates were dissolved in MeOH (20 mL), then EtOAc (250 mL) was added. A white residue was recovered by evaporation at 73 °C for 1 h under reduced pressure yield 3.23 g (60%). The identity of the... 

Unlike the succinimidyl succinate or succinimidyl glutarate activation methods, succinimidyl carbonate chemistry does not suffer from the presence of a labile ester bond. The intermediate carbonate may hydrolyze in aqueous solution to release NHS and C02, essentially regenerating the underivatized PEG hydroxyl. After coupling to amine-containing molecules, however, the resultant carbamate linkage stabilizes the chemistry to the point that a modified molecule will not lose PEG by hydrolytic cleavage. For these reasons, the succinimidyl carbonate method of PEG activation and... 

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