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Introduction

Purpose

Any cell mesh produced based on our bioink will suffer from exposition to serum-containing media or, in the case of in vivo application, the patient’s body fluids because of biotinidase. This ubiquitous enzyme is able to cleave biotin off of lysine – at a lower rate also off of intact protein. We may now shift this hydrolysis in two directions: either to prevent it altogether and thus stabilize our cell mesh, or to accelerate it to make our mesh quickly disintegrable. To this end, we must consider how biotin is linked to the proteins in our bioink.

NHS Esters as Amine Coupling Reagents

N-hydroxysuccinimide (NHS) esters belong to the group of amine-reactive active esters and are thus of great use in labeling proteins. Typically, NHS esters react with the primary amino group in the side chain of surficial lysine, forming a stable peptide bond.[1]
The utility of NHS esters was first suggested by Anderson et al.[2] in the context of peptide synthesis; nowadays, a plethora of NHS esters are widely used in bioconjugate techniques and thus commercially available, including those of many fluorescent dyes as well as that of biotin, often referred to as biotin-NHS (Figure 1).[3] NHS esters are typically synthesized from the corresponding carboxylic ester and NHS via the one-pot DCC coupling procedure[1] and purified by crystallization[2][3].
Being amine reactive, NHS esters undergo an SN2t reaction with primary or secondary amines to afford an amide bond as well as free NHS (Scheme 1a). While this aminolysis pathway is also used in chemical synthesis, e.g. of peptides, it is key for the above-mentioned protein-labeling capability of NHS esters. The latter, however, strictly requires aqueous reaction media, so that the undesirable hydrolysis, i.e. reaction with water to give the no longer reactive carboxylic acid (Scheme 1b), becomes a considerable side reaction even though reaction with amines, being more nucleophilic, is favored. At pH 7.0 and 0 °C, for instance, hydrolysis half-life is 4 to 5 h. For this reason, protein labeling must needs employ a great excess of NHS ester.[1][2][4][5]

For applications routinely requiring derivatization of primary amines, NHS esters are advantageous for a number of reasons. Anderson et al.[2] describe that they are highly reactive and thus give good yields in amine couplings; moreover, being crystalline, they are easy to handle. Good yields persist even when conducting the coupling reaction in the presence of water, which sets NHS esters apart from several other active esters.Cite error: Closing </ref> missing for <ref> tag
  1. ↑ 1.0 1.1 1.2 Hermanson, G. T. (2013) Bioconjugate Techniques (3 ed.). Boston: Academic Press.
  2. ↑ 2.0 2.1 2.2 2.3 Anderson, G. W., Zimmerman, J. E., & Callahan, F. M. (1963). N-Hydroxysuccinimide Esters in Peptide Synthesis. Journal of the American Chemical Society, 85(19), 3039-3039.
  3. ↑ 3.0 3.1 Klykov, O., & Weller, M. G. (2015). Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC). Analytical Methods, 7(15), 6443-6448.
  4. ↑ Anderson, G. W., Zimmerman, J. E., & Callahan, F. M. (1964). The Use of Esters of N-Hydroxysuccinimide in Peptide Synthesis. Journal of the American Chemical Society, 86(9), 1839-1842.
  5. ↑ Kalkhof, S., & Sinz, A. (2008). Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters. Analytical and Bioanalytical Chemistry, 392(1), 305-312.