Isothiocyanate groups

The DELFIA assay, the first effective lanthanide-based immunoassay, was developed and commercialized by the early 1980s.108-112 DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) is a heterogeneous assay which uses a lanthanide complex based on aminocarboxylate ligands such as EDTA, EGTA, or DTPA, linked to the antibody by reaction of appended isothiocyanate groups (e.g., complex (45)) with nucleophilic residues, particularly amines, on the protein surface (Figure 11). 

In a darkened lab, dissolve FITC (Thermo Fisher) in dry DMSO at a concentration of 1 mg/ml. Do not use old FITC, as breakdown of the isothiocyanate group over time may decrease coupling efficiency. Protect from light by wrapping in aluminum foil or using amber vials. 

The reaction may be quenched by the addition of ammonium chloride to a final concentration of 50 mM. Some protocols also include at this point the addition of 0.1 percent xylene cylanol and 5 percent glycerol as a photon absorber and protein stabilizer, respectively. React for a further 2 hours to stop the reaction by blocking remaining isothiocyanate groups. 

TRITC is relatively insoluble in water, but it can be dissolved in DMF or DMSO as a concentrated stock solution prior to its addition to an aqueous reaction mixture. The isothiocyanate group is reasonably stable in aqueous solution for short periods, but will degrade by hydrolysis. TRITC also is more stable to photobleaching than FITC (Section 1, this chapter), and its absorption and emission spectra are less sensitive to environmental conditions, such as plT. It is best, however, to use only fresh reagent for modification purposes. Storage should be done under desiccated conditions, protected from light, and at -20°C. 

Figure 9.52 The isothiocyanate group of a TMT europium chelate can be used to label amine-containing proteins and other molecules to result in an isothiourea bond.

The TMT chelator containing an amine-reactive isothiocyanate group can be used to label antibodies, proteins, or other molecules using the following protocol (Figure 9.52). 

Figure 10.2 The isothiocyanate group of DTTA can react with amine-containing molecules to form isothiourea bonds.

The reaction to the SH derivative also proceeds electrochemically [53, 54], It is also possible to introduce isothiocyanate groups [12, 54]. 

Many dyes complex with their primary analyte due to attractive forces such as ionic charges. These charges are susceptible to nonspecific complexation with interfering analytes with characteristics similar to those of the primary analyte. Both the primary and interfering analytes compete for complexation with the same site. The NIR dyes may be synthesized with specific functional groups that will bond more specifically to an analyte. For instance, an isothiocyanate group forms very stable thiourea chemical bonds with proteins or an amino-modified DNA oligomer. The introduction of more specific and reversible functionalities on the dye molecule should minimize the interference of extraneous molecules or ions. 

R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, and A.S. Wagooner, Cyanine dye labeling reagents containing isothiocyanate groups, Cytometry 10, 11-19(1989). 

Attaching an isothiocyanate group to the aromatic ring of U-50488 has been reported to produce the first site-directed alkylating agent to bind irreversibly to kappa receptors in guinea-pig membranes [51]. 

The carbon in the isothiocyanate grouping is highly susceptible to nucleophilic attack by the peptide s free amino group. Overall addition to the C=N creates a thiourea derivative. Making the conditions strongly acidic then promotes nucleophilic attack by the sulfur of the thiourea on to the carbonyl of the first peptide bond, producing a five-membered thiazoline heterocycle. Proton loss occurs from the nitrogen, and this creates an intermediate that is equivalent to the addition product in simple acid-catalysed amide... 

Hydroxylamine intermediates wilt react with an adjacent nitrile function [100, 101]. Interaction with the isothiocyanate group can be achieved in acid solution... 

In neutral or alkaline solution the isothiocyanate group is also reduced. 

It is worth mentioning that fluorothiocarbonyl isothiocyanate, produced by the reaction of CSFCl with metal thiocyanates (52), combines with chlorine quantitatively at low temperatures at the C=S double bond, without suffering an attack on the isothiocyanate group 21) ... 

Only at elevated temperatures is the isothiocyanate group also attacked by further addition of chlorine with simultaneous elimination of sulfur dichloride. It is thus converted into the isocyanide dichloride group, without isolation of an intermediate ... 

Isothiocyanate 23 (X = CO), when treated with AICI3 in nitromethane undergoes ring closure by an intramolecular electrophilic substitution between C3 of the pyrrole ring and the isothiocyanate group to afford pyrrolo[3,2-c][l]benzazepine-10(lH)-one-4(5H)-thione 24 (Scheme 2 (2005BMCL3220, 1998MI197)). 

N-Alkyl isoindolo[2,l-fc][2,4]benzodiazepines 190 (R = alkyl. Scheme 38, Section 3.1.1.2) are synthesized by an intramolecular N-acyliminium ion-amide reaction (1997TL2985, 1998T1497). Isothiocyanates 23 undergo under basic conditions in DMF ring closure by an intramolecular substitution between N1 of the pyrrole ring and isothiocyanate group to afford benzo[/]pyrrolo[l,2-c] [l,3]diazepine-5-thiones 25 (Scheme 2, Section 2.1.1.1 (2005BMCL3220)). 

Bis(benzimidazol-2-yl)disulfide (214) treated with phenyl isothiocyanate gives the 1,2,4-dithiazole derivative (215) as a result of electrophilic attack by the isothiocyanate group on the nitrogen atom... 

The presence of a thiocyanate group can be very easily deduced from the IR spectrum, which contains a sharp, but weaker in intensity, IR band around 2150 cm-1 and an NMR carbon resonance between 112 and 115 ppm. In the case of psamaplin B (172), C-NMR additivity effects applied to the methylene carbon linked to the thiocyanate were used to distinguish between a thio- and isothiocyanate group [142]. Chemical proof can be provided by LAH reduction of the thiocyanate, which gives a thiol rather than a methylamine group as the reaction product of the reduction of the isothiocyanate [195]. 

---