2-Methoxy-5-nitrophenyl phosphates

For each type of assay, the label or tag involved is listed along with typical substrates, type of absorbance, and type of instrument required. RIA radioimmunoassay IRMA immunoradiometric assay CPM counts per minute ELISA enzyme linked immunosorbent assay EIA enzyme immunoassay TMB 5,5 tetramethylbenzidine HPA p hydroxyphenylacetic acid AMPPD 4 methoxy 4 (3 phosphatephenyl) spiro(l,2 dioxetane) 3,2 adamantane p NPP p nitrophenyl phosphate 4 MUP 4 methylumbelliferyl phosphate ONPG o nitrophenyl P galactopyranoside MUG 4 methylumbelliferyl P D galactopyrano side AMPGD 3 (4 methoxyspiro( 1,2 dioxetane 3,2 tricyclo(3.3.1.1(3,7))decan) 4 yl)phenylgalacto pyranoside ECL electrochemiluminescence. 

On the other hand, phosphorane intermediates are not expected to be involved in the hydrolysis of phosphate monoesters, so the effective observed catalysis by the carboxyl group of salicyl phosphate 3.21 [51] (Scheme 2.26) is presumed to be concerted vith nucleophilic attack. (The hydrolysis reaction involves the less abundant tautomer 3.22 of the dianion 3.21, and the acceleration is >10 -fold relative to the expected rate for the pH-independent hydrolysis of the phosphate monoester dianion of a phenol of pK 8.52.) However, this system differs from the methoxy-methyl acetals discussed above, in that there is a clear distinction between neutral nucleophiles, which react through an extended transition structure similar to 3.16 in Scheme 2.23, and anions, which do not react at a significant rate, presumably because of electrostatic repulsion. This distinction is well-established for the dianions of phosphate monoesters with good leaving groups (p-nitrophenyl [52] and... 

The enzyme SC was modified with methoxy PEG (molecular weight 5,000) activated with p-nitrophenyl carbonate (Sigma, M-3903). The enzyme (240 mg) was dissolved in 100 mM potassium phosphate buffer pH 7.8 and stirred with 480 mg ( 10 x molar equivalent) PEG for a period of 2 h. The reaction was monitored by release of the p-nitrophenyl group (absorbance at 405 nm). The modified enzyme was purified from the solution by washing with phosphate buffer in an ultrafiltration cell (Amicon, Model 8050) using a 30,000-Da molecular weight cut-off membrane, over several hours. The modified enzyme was then lyophiUzed as described below. 

An alternative base labile 5 -protecting group, 2-dansylethoxycarbonyl (111) has been proposed for RNA synthesis utilising tetrahydro-4-methoxy-2H-pyran-4-yl for 2 -protection, 2-(4-nitrophenyl)ethyl and 2-(4-nitrophenyl)ethoxycarbonyl for phosphate and amino group masking. 

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