N-Hydroxysuccinimide (NHS) esters are a family of conjugation reagents first used almost sixty years ago. Not only are they relatively easy to prepare, but they are also reactive, selective, stable for storage, and functional in an aqueous environment. These properties make NHS ester conjugation one of the most common labeling and bioconjugation strategies.
NHS esters selectively react with primary aliphatic amine groups. While NHS esters can also react with other nucleophiles, such as hydroxyl and sulfhydryl groups, the resulting esters and thioesters are not very stable and can be hydrolyzed or displaced by amines. Typical NHS esters consist of an NHS ester group, a label, and often a linker as well (Figure 1). In the reaction, the carbonyl of the ester group is attacked by a primary aliphatic amine, resulting in a tetrahedral intermediate and the elimination of the NHS as a leaving group. The coupling reaction causes NHS (a weak acid) to be released, and an amide (a very stable covalent linkage) to be formed.
This labeling strategy has been used extensively, and it is highly effective for oligonucleotides labeled with amino-modifiers. NHS esters can be used with any one of the 25 amino modifiers in the Glen Research catalog. Most of these are for the 5’-terminus, but other versions can introduce amine groups to the 3’-terminus, as well as to several nucleobases. Generally, the NHS ester is dissolved in a small amount of dry DMF or DMSO, and the solution is added to the amine-labeled oligonucleotide in a non-nucleophilic buffer (pH 7–9). After a short reaction time, the amine gets quantitatively labeled and no other part of the oligonucleotide is affected. If there are multiple amino modifiers present, each and every one can be completely labeled as well. A general protocol can be found below.
For a 0.2 µmole synthesis of an amine-modified oligo:
Although these reactions are usually pretty straightforward, there are a few areas to be mindful of. First and foremost, the amino-modified oligonucleotide needs to be of good quality, as previously detailed (TB_Avoid_Amine_Alkylation.pdf). In addition, NHS esters can react with water and they should be stored appropriately. The moment the NHS ester is added to the oligo, the water begins to react with the NHS ester, directly competing with labeling. For this reason, several equivalents are required. As primary amines are much better nucleophiles than water, hydrolysis of NHS ester is generally not an issue, but if low conjugation efficiency is observed, perhaps due to a bulkier NHS ester that is slower to react, lowering the volume of buffer by two- or four-fold will make the reaction with the amine more favorable. Finally, there should not be any other competing nucleophiles present in the reaction mixture, as these will interfere with labeling. Oligonucleotides that are deprotected with amine-based deprotection solutions and dried down are usually isolated as ammonium salts. These oligos need to be converted to the sodium salt, a process that can be achieved with ethanol precipitation or an equivalent method prior to labeling. Tris buffer and other nucleophilic modifications are also not compatible.
In terms of work flow, incorporating modifications using phosphoramidites over post-synthesis/deprotection modification with NHS esters is generally advantageous. This is particularly true for high throughput operations, in which manual/additional steps are not desirable. However, there are many situations in which additional steps are preferred. If a desired modification/label is not stable during oligonucleotide synthesis or deprotection, then a post-synthesis conjugation would be the only option. Even if stability is not an issue, custom phosphoramidite synthesis is often complicated and expensive, and the NHS ester functionalization route might be more efficient in the early stages of a project, as it allows access to the reagent much sooner, while saving synthesis costs on structures that ultimately prove to not be useful. This strategy can also be used to aliquot a single amine-functionalized oligo into multiple aliquots for conjugation to a series of different NHS esters. When all the details of the application are finalized, an amidite that would give the exact same chemical structure obtained via the NHS ester labeling method can be synthesized.
Glen Research offers several NHS ester products (Figure 2). Some of these are available only as the NHS ester while others are also available as a phosphoramidite. The latter products include methylene blue and DBCO. Aside from those on this NHS ester list, we have a couple of additional NHS ester phosphoramidites for a different and previously discussed application (Glen Report 19-19)