NHS esters

Activated NHS esters of carboxylic acids are prepared by reacting the acid with NHS in the presence of DCC (Table 4, Figure 16). A-Hydroxysuccinimide esters are stable when kept under anhydrous and slightly acidic conditions, and they react rapidly with amino groups to form an amide in high yield. 

Figure 16 Conjugation of an amine and a carboxylic acid via the (V-hydroxysuccinimide (NHS)-activated ester method. NHS esters may be isolated and characterized and are stable to long term storage as the powder. Alternatively, the NHS esters may be used immediately upon formation without isolation. Details of the reaction are given in Table 4...

Fluorescent labels based on oxo-squaraines were described in numerous articles and patents [45, 52]. Mono-reactive hydrophobic (32a) and hydrophilic (32b) squaraine labels containing one NHS ester group were synthesized by Terpetschnig et al. [62]. The initially low quantum yields and short fluorescence lifetime of 32b in aqueous solutions significantly increase after covalent binding to proteins. 

Square-650-pH having a pKa in the physiological pH range (pKa = 7.1 for free dye and the pKa 6.1 when labeled to an antibody) was recently introduced by SETA BioMedicals [119]. This dye is commercially available as a free carboxylic acid and a mono-NHS ester. Square-650-pH has spectral properties similar to those of the CypHer dyes but is fluorescent in both the protonated and deproto-nated forms. This dye displays reasonable molar absorptivities (135,000 and 48,000 M-1cm-1) and quantum yields (16% and 9%) for the protonated and deprotonated forms, an extremely large Stokes shift of more than 100 nm for the deprotonated form, and enables excitation and emission ratiometric measurement... 

The heterobifunctional PEGs are very useful in linking two entities in cases where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimides, vinyl sulfones, pyridyl disulfides, amines, carboxylic acids, and /V-hydroxysuccinimide (NHS) esters. 

Figure 1.62 SATA can react with available amine groups in proteins and other molecules via its NHS ester end to form protected sulfhydryl derivatives. The illustrated protein is glutathione-S-transferase (E.C.2.5.1.18) (Ji et al., 1995).

Dissolve the protein to be thiolated at a concentration of l-5mg/ml in 50 mM sodium phosphate, pH 7.5, containing 1-10 mM EDTA. Other non-amine containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0, but environments closer to neutrality will limit the hydrolysis of the ester. 

Figure 1.65 SATP reacts with amine-containing proteins or other molecules via its NHS ester end to create protected sulfhydryl derivatives in a manner similar to that of SATA. Deprotection can be done with hydroxy-lamine to free the thiol.

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

Sulfo-LC-SMPT is not as stable as SMPT. The sulfo-NHS ester is more susceptible to hydrolysis in aqueous solutions and the pyridyl disulfide group is more easily reduced to the free sulfhydryl. Stock solutions of sulfo-LC-SMPT may be prepared in water, but should be used immediately to prevent loss of amine coupling ability. 

Carboxylic acids may be covalently modified with adipic acid dihydrazide or carbohydrazide to yield stable imide bonds with extending terminal hydrazide groups. Hydrazide functionalities don t spontaneously react with carboxylate groups the way they do with aldehyde groups (Section 4.5, this chapter). In this case, the carboxylic acid first must be activated with another compound that makes it reactive toward nucleophiles. In organic solutions, this may be accomplished by using a water-insoluble carbodiimide (Chapter 3, Section 1.4) or by creating an intermediate active ester, such as an NHS ester (Chapter 2, Section 1.4). 

Dissolve SANH, SHNH, or SHTH in DMF to prepare a stock solution at a concentration of 2.0-4.0mg in 100-200pi. Use highly pure and dry solvent (H20 content < 0.1 percent or treat with molecular sieves) to prevent hydrolysis of the NHS ester. 

Figure 1.113 The NHS ester-suberate derivative of lactose can be used to add lactose groups to amine-containing molecules. The reaction results in the formation of amide bonds containing terminal lactose groups.

Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a concentrated solution from which an aliquot may be taken and added to a final aqueous reaction medium. The compound is extremely soluble in DMF, and solutions of 100 mg/ ml may be prepared. The use of dry solvent is essential to prevent hydrolysis of the NHS ester. However, make only enough of this stock solution so that a small amount added to the protein reaction will provide the appropriate molar excess desired for the modification reaction. 

Prepare the protein to be modified in a non-amine-containing buffer at a slightly basic pH (i.e., avoid Tris or imidazole). The use of 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2 works well for NHS ester reactions. The concentration of the protein in the reaction buffer may vary from pg/ml to mg/ml, but highly dilute solutions will result in less efficient modification yields. A protein concentration from 1 to 10 mg/ml works well in this reaction. 

Figure 2.1 Three types of fluorophenyl esters have been used for coupling to amine-containing molecules. The PFP and TFP esters are relatively hydrophobic and typically have better stability toward hydrolysis in aqueous solution than NHS esters. The STP ester is water-soluble due to the negatively charged sulfonate group, and it provides better solubility to associated crosslinkers or bioconjugation reagents similar to that of a sulfo-NHS ester group.

Fluorophenyl esters react with amine-containing molecules at slightly alkaline pH values to give the same amide bond linkages as NHS esters (Reaction 15). However, in most cases, the fluorophenyl ester compound will display better stability toward hydrolysis in aqueous solution. It has been reported that a TFP ester has over twice the half-life in basic pH buffers (pH 8) than a corresponding NHS ester on the same compound (Molecular Probes). 

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