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Glen Report 35-12: New Products — NHS Esters

Glen Research offers several N-Hydroxysuccinimide (NHS) Esters for post-synthetic oligonucleotide labeling. Most of our options are fluorophores, due to their increasing popularity. Of our non-dye NHS esters, only click reaction handles are available: alkyne (50-1905), azide (50-1904), and DBCO (50-1941). As we continue to support our customers and provide diverse options for their oligonucleotide synthesis and labeling needs, we are introducing two different NHS Esters: PC Biotin NHS Ester (50-4950) and Maleimide NHS Ester
(SMCC) (50-1938).

PC Biotin 

PC Biotin is a photocleavable biotin tag that is already available as a phosphoramidite (10-4950). The structures of PC Biotin Phosphoramidite and PC Biotin NHS ester differ quite a bit (Figure 1). The first difference is the presence of an ethylene glycol linker in the NHS ester. In addition, the nitrophenyl photocleavable moiety of each product differs in substituents and regiochemistry. Lastly, the phosphoramidite contains a DMT group on the biotin, which requires cleavage and is not present in the NHS Ester. Regardless of these structural differences, the functionality of the biotin’s affinity for streptavidin remains unchanged.

 Figure 1. PC Biotin Products

Figure 1

Relative to other biotin products, a major advantage of PC Biotin is the ability to release the oligonucleotide into solution after biotin capture with streptavidin beads by cleaving the linker between the biotin tag and the oligonucleotide using light.1 Once cleaved, the oligonucleotide can be used for activity or further analysis. Our PC Biotin Phosphoramidite can only be used as a 5’-modifier and yields a 5’-phosphate after cleavage (~350 nm). PC Biotin NHS Ester reacts anywhere an amino modifier is placed in the sequence to form a carbamate linkage. Upon photocleavage and subsequent decarboxylation, the primary amine target is released (Figure 2). 

 

 Figure 2. Mechanism of PC Biotin NHS Ester

Figure 2

PC Biotin NHS ester can be used to label a protein or oligonucleotide. Upon cleavage, the target is released unmodified. For proteins, that is ideal. In the case of an oligonucleotide, the starting amino-oligonucleotide post-conjugation could potentially be detected as a result of non-quantitative conjugation or photocleavage. This has the potential to complicate the reaction yield. With that said, we found that PC Biotin NHS Ester was stable to ambient lighting. 

PC Biotin NHS Ester is compatible with our general protocol, previously described.2 For a 0.2 µmole synthesis of an amine-modified oligonucleotide:

  1. Dissolve oligonucleotide in 500 µL of 0.1 M sodium bicarbonate (pH 9).
  2. Dissolve 5–10 eq of NHS ester in 25 µL DMF or DMSO.
  3. Add NHS ester solution to oligonucleotide solution.
  4. Agitate the mixture and incubate at room temperature for 1–2 hrs.
  5. Separate oligo-conjugate from salts and excess label by size exclusion on a Glen Gel-Pak™ desalting column or equivalent.

Maleimide NHS Ester (SMCC) 

Maleimide NHS Ester (SMCC) is similar to our other non-dye NHS esters, in that it is a dual reactive molecule used in a subsequent conjugation reaction. An amine-containing target reacts with the NHS ester group before the maleimide reaction occurs, which selectively targets free thiols at neutral pH.

We already have a protected maleimide modifier available as a phosphoramidite (10-1938).3 This modifier amidite requires a two-step deprotection. First the oligonucleotide deprotection takes place in ammonia and then a retro Diels-Alder reaction must occur before the maleimide can be conjugated to a thiol. This retro Diels-Alder deprotection takes place in toluene at high heat (90 0C) or microwave irradiation. For those who wish to avoid this second deprotection scheme, the Maleimide NHS Ester is an excellent alternative as the maleimide is not introduced until after oligonucleotide deprotection. The Maleimide NHS Ester contains a cyclohexane linker, which decreases the rate of maleimide hydrolysis compared to similar compounds lacking this ring (Figure 3). 

 Figure 3. Maleimide Products

Figure 3

Maleimides are very susceptible to hydrolysis at high pH. This conflicts with our previous protocol for NHS ester reaction at basic pH. Therefore, we have an updated protocol for the use of Maleimide NHS Ester at neutral pH to prevent hydrolysis of the maleimide during the NHS ester conjugation reaction.

For a 0.2 µmole synthesis of an amine-modified oligonucleotide:

  1. Dissolve oligonucleotide in 500 µL of 0.1 M potassium phosphate buffer (pH 6.9-7.2).
  2. Dissolve 10 eq of Maleimide NHS ester (SMCC) in 250 µL DMSO.
  3. Add NHS ester solution to oligonucleotide solution.
  4. Agitate the mixture and incubate at room temperature for 30-60 min.
  5. Separate oligo-conjugate from salts and excess label by size exclusion on a Glen Gel-Pak™ desalting column or ethanol precipitation.

Due to maleimide instability to hydrolysis, we recommend maleimide conjugation soon after the NHS ester reaction. For the conjugation of the maleimide with small molecule thiols, our general protocol can be used. For a 0.2 µmole synthesis of a maleimide-containing oligonucleotide:

  1. Dissolve oligonucleotide in 450 µL of aqueous buffer* (pH 6.5-7.8).
  2. Dissolve 5-10 eq. of thiol compound in 50 µL of compatible solvent.
  3. Add thiol solution to oligonucleotide solution.
  4. Agitate the mixture and incubate at room temperature for 30-60 min.
  5. Separate oligo-conjugate from salts and excess label by size exclusion on a Glen Gel-Pak™ desalting column or equivalent.

*We have successfully used sodium phosphate buffer (pH 6.5) and triethylammonium acetate buffer (pH 7.0) for maleimide functionalization. 

References

  1. The Glen Report, 2001, 14.1, 8-9.
  2. The Glen Report, 2020, 32.2, 9-10.
  3. The Glen Report, 2011, 23.2, 6.

 

Product Information

PC Biotin NHS Ester (50-4950)

Maleimide NHS Ester (SMCC) (50-1938)