Bifunctional reagent

Bifunctional reagents enable intra- and inter-molecular cross-linking of proteins. Examples are bifunctional imidoester, fluoronitrobenzene, isocyanate derivatives and maleic acid imides  

Diazotized arsanilic acid reacts with tyrosine and with histidine, lysine, tryptophan and arginine  

Tetranitromethane introduces a nitro group into the ortho position  

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Bis-Pyndoxal Tetraphosphate. A second class of bifunctional reagents, described in 1988, involves two pyridoxal groups linked by phosphates of different lengths (89). As shown in Table 4, the yield of intramolecularly cross-linked hemoglobin increases dramatically with increasing length of the phosphate backbone. It is beheved that the site of reaction of (bis-PL) is between the amino-terminal amino group of one P-chain and the... 

Diimida.te Esters. Ditmidate esters are bifunctional reagents that have been used in cross-linking a variety of proteins including hemoglobin. 

Seven procedures descnbe preparation of important synthesis intermediates A two-step procedure gives 2-(HYDROXYMETHYL)ALLYLTRIMETH-YLSILANE, a versatile bifunctional reagent As the acetate, it can be converted to a tnmethylenemethane-palladium complex (in situ) which undergoes [3 -(- 2] annulation reactions with electron-deficient alkenes A preparation of halide-free METHYLLITHIUM is included because the presence of lithium halide in the reagent sometimes complicates the analysis and use of methyllithium Commercial samples invariably contain a full molar equivalent of bromide or iodide AZLLENE IS a fundamental compound in organic chemistry, the preparation... 

A bifunctional reagent such as ethanolamine can favor ortho substitution of azines due to hydrogen bonding as in 62. With a bifunctional nucleophile such as ethylene glycol anion, facilitation of... 

Thus, the reaction of 4-dialkylaminobut-3-yn-2-ones with various bifunctional reagents makes it possible to carry out a targeted synthesis of five- and six-membered nitrogen-, oxygen-, and sulfur-containing heterocycles having two heteroatoms. 

Difunctional reagents, for example the very cheap dimethyldichlorosilane 48, which is produced on a large technical scale, and the much more reactive and expensive dimethylsilyl bis(O-triflate) 49 [65-67] (Scheme 2.8) convert alcohols or phenols 11 in the presence of bases, for example triethylamine or DBU, into the silylated compounds 50. Thus 48 and 49 and other bifunctional reagents such as di-tert-butyldichlorosilane [68] or di(tert-butylsilyl)-bis(0-triflate) [69] and the subsequently described 51 and 52 combine two alcohols to silicon-tethered molecules 50, which can undergo interesting intramolecular reactions [70-74]. 

It has been found that DTBP cross-linking substantially increased the salt stability of the complexes. The salt stabilization is reversed upon the addition of DTT, which cleaves the bifunctional reagent, indicating that it is not due to the conversion of the amines to amidines and is dependent upon the cross-linking. Similar results were achieved with other polycations, including poly(allylamine), and histone HI. 

Recently the use of another bifunctional reagent, glutaraldehyde, has been described for the stabilization of DNA complexes with cationic peptide CWK18 [104]. The authors of this paper, however, limited the study to the protective effects toward nuclease degradation. 

Harris, W.E. and Stahl, W.L. (1980). Oiganisation of thiol groups of electric eel electric organ Na/K ion stimulated adenosine triphosphatase, studied with bifunctional reagents. Biochem. J. 185, 787-790. 

Bechet, J.-J. Dupaix, A. Blagoeva, I. Inactivation of a-chymotrypsin by new bifunctional reagents halomethylated derivatives of dihydrocoumarins. Biochimie 1977, 59, 231-239. 

Gaffney, B.J., Willingham, G.L., and Schopp, R.S. (1983) Synthesis and membrane interactions of a spin-label bifunctional reagent. Biochemistry 22, 881. 

Hartman, F.C., and Wold, F. (1966) Bifunctional reagents. Cross-linking of pancreatic ribonuclease with a diimido ester. J. Am. Chem. Soc. 88, 3890-3891. 

Hillel, Z., and Wu, C.W. (1977) Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents. Biochemistry 16, 3334-3342. 

Peeters, J.M., Hazendonk, T.G., Beuvery, E.C., and Tesser, G.I. (1989) Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates./. Immunol. Meth. 120, 133-143. 

Sato, S., and Nakao, M. (1981) Cross-linking of intact erythrocyte membrane with a newly synthesized cleavable bifunctional reagent./. Biochem. (Tokyo) 90, 1177. 

Smith, R.J., Capaldi, R.A., Muchmone, D., and Dahlquist, F. (1978) Cross-linking of ubiquinone cytoch-nome c reductase (complex III) with periodate-cleavable bifunctional reagents. Biochemistry 17, 3719-3723. 

Wells, J.A., Knoeber, C., Sheldon, M.C., Werber, M.M., and Yount, R.G. (1980) Cross-linking of myosin subfragment 1. Nucleotide-enhanced modification by a variety of bifunctional reagents./. Biol. Chem. 255, 11135. 

Wold, F. (1961) Reaction of bovine serum albumin with the bifunctional reagent p,p -dif[uoro-m,m -dinitrodiphenylsulfone. J. Biol. Chem. 236, 106. 

Aminoimidazole (64) has been reported to react with various bifunctional reagents to give bicyclic systems (Scheme 6). In some cases the initial N-addition-elimination product has been isolated (e.g., 68 and 69). 

The problem of slow turnover has been noted. Initial reaction of a nucleophilic group, e.g. imidazole or oximate, in a functional micelle, with a carboxylic or phosphoric ester, for example, gives an acylated or phosphory-lated imidazole or oxime, and these derivatives hydrolyze slowly to regenerate the nucleophile. Kunitake and Shinkai (1980) discuss a number of reactions in micelles which contain both nucleophilic and basic groups which are potentially capable of acting as bifunctional reagents (Tonellato, 1979, Kunitake and Shinkai, 1980 Bunton, 1984)... 

We have observed that such proteins as CaM and bovine serum albumin (BSA) can be developed at the air-water interface to form monolayer protein films. In previous works, the developed BSA monolayer was stabilized by cross-linking with a bifunctional reagent immediately after the preparation of protein monolayer. The BSA thin film thus prepared can be employed as a passive material, e.g., an ultrathin protein film for a matrix of enzyme-linked immunosorvent assays. 

All procedures for ring closure to the title ring system by cyclization of the six-membered ring utilize the reactivity of 3,4-diamino[l,2,4]triazoles 209 these compounds can easily react with bifunctional reagents bearing these functional groups in adjacent positions. 

A special approach to [l,2,4]triazolo[3,2-c][l,2,4]triazine derivatives is the transformation of 3-diazo[l,2,4]triazoles, easily available by diazotation of aminotriazoles. These compounds already contain the five nitrogen atoms in the correct sequence in order to form the desired bicyclic ring system and, thus, their reaction with proper bifunctional reagents can give rise the cyclized products in one single step. Such transformations are collected in Scheme 50. 

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