2-Pyridyl disulfide reactions

Dissolve a protein or macromolecule containing primary amines at a concentration of 10 mg/ml in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other non-amine-containing buffers such as borate, HEPES, and bicarbonate also may be used in this reaction. Avoid sulfhydryl-containing components in the reaction mixture as these will react with the pyridyl disulfide end of SPDP. The effective pH for the NHS ester modification reaction is in the range of 7-9, but hydrolysis will increase at the higher end of this range. 

React with mixing for 2 hours at room temperature. At the completion of the reaction, cysteine may be added at 50 mM to block excess pyridyl disulfide-reactive sites. 

A cyclohexadienyl Lewis adduct or salt formed by the reaction of a Lewis base with an aromatic compound. Such an adduct is apparently formed from the reaction of OH with 4-(A/-2-aminoethyl-2 -pyridyl disulfide)- -nitrobenzo-2-oxa-l,3-diazole (2PROD). 2PROD is a two-protonic-state electrophile used as a probe for enzyme active site nucleophiles and as a fluorescent re-... 

The 4 mM soln of the crude peptide [H-(Gly-Pro-Hyp)5-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu-Gly-Cys(Acm)-Cys-Gly-Gly-OH] 28 in degassed argon-sat. DMF/AcOH (95 5) was added dropwise to a 100 mM soln of di[5-nitro(2-pyridyl)]disulfide (5 equiv) in DMF/AcOH (95 5) with exclusion of air oxygen. The reaction was monitored spectroscopically at 430 nm, and after completion (1 to 2 h), the solvent was removed under reduced pressure. The resulting residue was dissolved in H20 and the excess reagent was filtered off. The H20 was removed under reduced pressure and the residue was reprecipitated from TFE with methyl /ert-butyl ether and purified by preparative HPLC to give 29 yield 13% the product was characterized by MALDI-TOF-MS, HPLC, and amino acid analysis. 

Alternatively, both peptide chains could be protected at one cysteine residue as a 5-Acm derivative and at the second cysteine residue by an acid-labile [Trt, Mob, Xan, or Bzl(4-Me)], base-labile (Fm), or reduction-labile (5-tBu) group. Both peptide chains may then be separately converted into the free thiol/Acm-protected form for selective activation of one chain as S-SPy or. S -Npys derivatives by reaction with di(2-pyridyl)disulfide or di[5-nitro(2-pyridyl)]disulfide, or as a sulfenohydrazide derivative by reaction with azodicarbocylic acid derivatives for formation of the first interchain disulfide bridge. 

For the synthesis of bombyxin IV (53) 192 regioselective formation of three disulfide bonds was achieved by exploiting the differentiated acid-stability of the 5-Trt vs the 5-tBu protection in 49 that allowed the air-mediated intramolecular disulfide formation in the A-chain (Scheme 23). For subsequent activation of the third cysteine residue from the precursor S-tBu derivative the rather drastic conditions of TfOH/TFA (1%) were applied in presence of di(2-pyridyl) disulfide which despite the strong acidity allowed a concomitant deprotection/ activation of this residue to give 50. Subsequent reaction of 50 with the B-chain derivative 51 established the intrachain heterodisulfide cross-link in 52 which on oxidation gave bombyxin IV (53). 

To a solution of ethyl 2-pyridyl disulfide (55 mg, 0.32 mmol) and DMAP (33 mg, 0.27 mmol) in dichloromethane (2ml) was added 2-mercaptohexadecanoic acid (77 mg, 0.27 mmol). The progress of the reaction was monitored by TLC (silica, CHCl,/MeOH 9 1). Upon completion (1 h) the product mixture was passed through a column (0.5 X 5 cm) of AG MP-50 (0.5 g, 3.5 meq (ml dry resin)1, Bio-Rad Laboratories). Elution with 3 ml of dichloromethane, followed by concentration of the filtrate under reduced pressure, afforded 2-(ethyldithio)hexadecanoic acid (91 mg, 98%) as a colourless oil. 

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