Sulfonyl isothiocyanates

Several resins have been used frequently in reactant sequestration. Ami-nomethylpolystyrene 1 and the more highly functionalized polyamine resins 2 and 3 have been reported to sequester excesses of solution-phase electrophiles, including isocyanates, isothiocyanates, sulfonyl chlorides, acid chlorides, anhydrides, aldehydes, and imines. Cross-site reactivity is not an issue with the more densely functionalized sequestering resins so their use in an automated laboratory environment offers a significant resin and volume economy compared to less densely functionalized resins. 

Of the many known fluorophores, only a few are routinely employed for immunofluorescence. Spectral maxima for such fluorophores are provided in Table 1. Conjugation of the fluorophores to antibodies is typically achieved through reactive isothiocyanate, sulfonyl chloride, or succinimidyl ester groups, which provide bonding to protein amines. Since most investigators purchase fluorophores already conjugated to antibodies, the chemistry of these and other reactive groups will not be further addressed (see Chapter 6 for details). Additional fluorophore information is summarized below. 

Sulfonylamino-1 -thioethers Sulfonyl isothiocyanates Sulfonyl thiocyanates C-Sulfonylthioformamides N-Sulfonylthione S-imides... 

Azoles containing a free NH group react comparatively readily with acyl halides. N-Acyl-pyrazoles, -imidazoles, etc. can be prepared by reaction sequences of either type (66) -> (67) or type (70)->(71) or (72). Such reactions have been carried out with benzoyl halides, sulfonyl halides, isocyanates, isothiocyanates and chloroformates. Reactions occur under Schotten-Baumann conditions or in inert solvents. When two isomeric products could result, only the thermodynamically stable one is usually obtained because the acylation reactions are reversible and the products interconvert readily. Thus benzotriazole forms 1-acyl derivatives (99) which preserve the Kekule resonance of the benzene ring and are therefore more stable than the isomeric 2-acyl derivatives. Acylation of pyrazoles also usually gives the more stable isomer as the sole product (66AHCi6)347). The imidazole-catalyzed hydrolysis of esters can be classified as an electrophilic attack on the multiply bonded imidazole nitrogen. 

Fluorescein isothiocyanate (FITC) or Lissamine rhodamine B sulfonyl chloride (RBSC). 

Both sulfonyl chloride and isothiocyanate will hydrolyze in aqueous conditions therefore, the solutions should be made freshly for each labeling reaction. Absolute ethanol or dimethyl formamide (best grade available, stored in the presence of molecular sieve to remove water) should be used to dissolve the reagent. The hydrolysis reaction is more pronounced in dilute protein solution and can be minimized by using a more concentrated protein solution. Caution DMSO should not be used with sulfonyl chlorides, because it reacts with them. 

The red-emitting rhodamine derivatives are constructed around the same basic xanthene framework as is fluorescein (2). Tetramethylrhodamine isothiocyanate (TRITC) has been widely employed for immunofluorescence. Additional derivatives of rhodamine available for conjugation to antibodies include lissamine rhodamine sulfonyl chloride (RB-200-SC), rhodamine B isothiocyanate (RBITC), rhodamine X isothiocyanate (XRITC), and Texas... 

An important prerequisite in these reactions is the nucleophilicity of the imine nitrogen. Indeed, electron-withdrawing substituents in the aryl ring of (159) retard the reaction with isothiocyanates, whereas such substituents in the R group of the isothiocyanates enhance the reaction. No reaction occurs under the above chosen conditions (reflux in chloroform), when the aryl ring of (159) is replaced by a sulfonyl group <88BSB83>. 

Thioxo-1,2,4-dithiazolidines can be obtained from thioxo-l,2,4-thiadiazolidines by Dimroth rearrangement (Scheme 47) or reversibly by addition and elimination of acetone (Scheme 22). 1,2,4-Dithiazole type products are also obtained from thiocarbonyl isothiocyanates (154) by reaction with CI2 <84CHEC-I(6)897>, PhMgBr, Sj, or CS2. Other routes include reactions of carbonyl isothiocyanates and thiocarbonyl isocyanates with P4S10 or Sg <84Chec-I(6)897> and sulfonyl isocyanates with dialkyl thioketenes. Phenylisothiocyanate heated with N,C-disubstituted oxaziridines affords 3,5-diimino-l,2,4-dithiazolidines (226, 227). Other miscellaneous syntheses of 1,2,4-dithiazoline derivatives include cyclization of A7-thioacyl hexafluoroacetonimine to 5,5-bis(trifluoromethyl)-... 

L abbe and co-workers have found that 4-alkyl-5-sulfonylimino-A -l,2,3,4-thiatriazolines (58) are readily prepared by reaction of the highly reactive sulfonyl isothiocyanates with alkyl azides (Equation (19)) <73JOC2916, 74JA3973). 

Cycloaddition reactions of sulfonyl isothiocyanate 76 and, -disubsti-tuted enamines were studied by Schaumann et The mechanism of cycloaddition is postulated to consist of two steps, originating with the formation of the zwitterionic adduct 77. 

When 176 is treated with hydrochloric acid it gives the intermediate acyclic structure 265, which decomposes to sulfonyl isothiocyanates, isothiocyanates, and chlorformamidines. Solvolysis of 155 with ethanol, followed by addition of hydrogen bromide, leads to 266. Base hydrolysis of 156 leads to the zwitterionic structure 267. ... 

The 4-amino group of 145 is readily monoacylated with acyl or sulfonyl chlorides and isocyanates or isothiocyanates ethyl acetoacetate and diethyl malonate yield, respectively, 159 and 160. Hydrazides 161 undergo a re-... 

The active hydrogens of pseudoureas react with various reagents, such as sodium hypochlorite [72], acyl halides [73], sulfonyl halides [19], isocyanates [26], isothiocyanates [26, 74] cyanamide, [75] and esters [76], to give linear or cyclic derivatives as shown in Scheme 2. 

Azoles containing a free NH group react comparatively readily with acyl halides. yV-Acyl-pyrazoles, -imidazoles, etc. can be prepared by reaction sequences of either type (75) — (76) or type (79)—>(80) or (81). Such reactions have been carried out with benzoyl halides, sulfonyl halides, isocyanates, isothiocyanates and chloroformates. Reactions occur under Schotten-Baumann conditions or in inert solvents. 

When the more reactive sulfonyl isothiocyanates are used as 1,3-dipolarophiles 4-alkyl-5-sulfonylimino-A2-l,2,3,4-thiatriazolines are readily prepared at room temperature by reaction with alkyl azides.64,65 The thiatriazolines are obtained in 50-75% yield. Their structures are deduced from NMR, IR, and mass spectral data and degradation experiments. Thus thermal decomposition at moderate temperature (45°-80°) in inert solvents (dry toluene, CC14, acetone) furnished the corresponding carbodiimides [Eq. (22)]. These were identified by their... 

This approach has been shown to work with a number of different fluorescent probes such as the short-wavelength fluorophores dansyl sul-fonyl chloride and coumarin chloride and the long-wavelength fluorophores tetramethylrhodamine-5-(and-6)-isothiocyanate [5(6)-TRITC], 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester [5(6)-TAMRA, succin-imidyl ester] and lissamine rhodamine B sulfonyl chloride (each in conjunction with different binding functionalities on the SAM surface. 

Disubstituted iminothiatriazolines may be formed by reaction of alkyl azides and aryl isothiocyanates but the reaction is relatively slow and the thiatriazoline formed undergoes further reaction with the isothiocyanate present (Section 4.28.2.3.l(iii)). 4-Alkyl-5-sulfonyliminothiatriazolines (73) are readily obtained when the more reactive sulfonyl isothiocyanates are treated with alkyl azides at room temperature (equation 47) (73JOC2916, 74JA3973). 

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