MAL-dPEG®8-TFP ester, product number 10552, is a crosslinking reagent that joins a sulfhydryl to a free amine. The sulfhydryl groups react with a maleimide group via a Michael addition reaction. The amines form amide bonds with the crosslinker by nucleophilic substitution of the 2,3,5,6-tetrafluorophenyl (TFP) ester of the terminal propionic acid group. The maleimide and TFP functional groups on the crosslinking compound sit at either end of a medium-length, discrete polyethylene glycol (dPEG®) chain. The single molecular weight dPEG® spacer is 38 atoms (37.9 Å) long.
Crosslinking ReactionsOne of the most popular, most useful crosslinking reactions in bioconjugate chemistry, is the conjugation of free amines to free thiols. These reactions require heterobifunctional reagents that bridge the two groups. Typical crosslinkers are hydrophobic. Quanta BioDesign's dPEG® crosslinking products are water-soluble, amphiphilic, single molecular weight PEG compounds with discrete chain lengths.With conventional hydrophobic crosslinking reagents, aggregation and precipitation of the conjugates occur frequently. These problems do not happen with our water-soluble, non-immunogenic dPEG® crosslinkers. For more information about our dPEG® products, please see our "What is dPEG®?" page. Also, please click here for answers to our most frequently asked questions.TFP Esters are Superior to NHS estersTFP esters are more stable in aqueous buffers than N-hydroxysuccinimidyl (NHS) esters. Moreover, TFP esters have higher reactivity with free amines than NHS esters. NHS esters hydrolyze readily in water or aqueous buffer. As the pH increases, the hydrolysis rate of the NHS ester increases. In 2017, a study by J. Wang, et al., on the performance of fluorophenyl esters concluded, "With regards to PEGylation, the TFP ester performed better than NHS ester." In-house research at Quanta BioDesign confirms the superior performance of TFP esters over NHS esters.How to Use MAL-dPEG®8-TFP esterTFP esters react under the same conditions as NHS esters. However, the optimal pH range for TFP esters (7.5 – 8.0) is slightly higher than for NHS esters (7.0 – 7.5).5 Amide bond formation between the TFP-activated propionic acid group of MAL-dPEG®8-TFP ester and a free amine will be slightly slower at a suboptimal pH compared to the reaction rate within the optimum pH range. Nevertheless, the reaction rate remains relatively rapid.The reaction of the maleimide end of MAL-dPEG®8-TFP ester, product number 10552, with a sulfhydryl proceeds best at pH 6.5 – 7.5. Conduct the conjugation at the lowest reasonable pH within this range. Above pH 7.5, free amines compete with free thiols at the maleimide reaction site, which can cause confusing results. Moreover, at higher pH values, the maleimide ring may open to form unreactive maleamic acid. For details about maleimide-thiol reaction chemistry, please click here.Uses of MAL-dPEG®8-TFP esterMAL-dPEG®8-TFP ester, product number 10552, can be used the same way and in the same applications as the equivalent NHS esters. A 2019 study used PN10552 from Quanta BioDesign to synthesize pyrrolobenzodiazepine dimer antibody-drug conjugates with dual β-glucuronide and dipeptide triggers. Other possible uses for this product include the following:
- Coating nanoparticle surfaces;
- Tethering antibodies to atomic force microscopy (AFM) probes;
- Increasing the water solubility and hydrodynamic volume of hydrophobic biomolecules;
- Building supramolecular constructs; and,
- Conjugating the TFP ester end of the molecule a liposomal surface and then using the free maleimide end of the molecule to attach a small molecule drug, peptide, or antibody for targeted delivery of a diagnostic or therapeutic package.