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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]

Chemical structure of (+)-biotin N-hydroxysuccinimide ester (biotin-NHS).

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].

Productive aminolysis and non-productive hydrolysis as competing reaction pathways in protein labeling reactions with NHS esters in aqueous media.

Being amine reactive, NHS esters undergo an S_N2t reaction with primary or secondary amines to afford an amide bond as well as free NHS (Scheme 1, left). 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 1, right), 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.^[4] An important benefit of NHS esters is that, in their crystalline form, they are relatively resistant to hydrolysis and thus the concomitant inactivation, meaning that they can be stored for an extended period of time under dry conditions without special precautions while retaining their activity.^[3] However, this comes with a caveat: solutions of NHS esters are susceptible to hydrolysis especially if prepared in N,N-dimethylformamide or dimethyl sulfoxide. These hygroscopic solvents tend to draw moisture, promoting hydrolysis of the NHS ester; such solutions should thus be prepared immediately before use.^[6]
In our work, NHS esters containing a biotin moiety, biotin-NHS among them, are used to biotinylate inanimate protein components of bioink or reservoir, for instance PAS-Lysine. For the printed mesh to be stable, this covalently attached biotin must not be cleaved. In this respect, however, we now encounter a challenge: our printing system involves live cells. These cells require cell culture media, which commonly contain serum – and serum contains the potential downfall to mesh stability: the enzyme biotinidase.

How Stable is Biotin Coupled to Proteins?

Hydrolytic Cleavage

In the human body, biotin is a vital cofactor for several carboxylases, where it is attached to the ε-amino group of a lysine residue within the conserved sequence AMKM.^[7][8] To liberate this covalently attached biotin, biotinidase, or biotin-amide amidohydrolase (EC 3.5.1.12), naturally hydrolyzes biocytin (ε-N-biotinyllysine) to biotin (vitamin H) and lysine.^[7][9] The monomeric enzyme of size 76 kDa possesses a catalytic sulfhydryl group.^[7][10] It not only serves to recycle biotin for renewed usage as well as to make protein-bound biotin in food bioavailable,^[9] but also acts as a shuttle for biotin within the body^[11]. Biotinidase deficiency thus causes multiple carboxylase deficiency, which, for one, entails serious developmental retardation because the body lacks the free biotin necessary to replenish its holocarboxylase pool; effective treatment is easily accomplished by administering daily doses of free biotin.^[8] Biotinidase is ubiquitous throughout the human body. Its specific activity is highest in serum (5.80±0.89 nmol min^–1 mL^–1[12]), followed by other organs including the liver, where the enzyme is produced.^[8]

Stabilizing the Biotin Peptide Bond

Design

Experiments

Proof of concept

Demonstrate

Discussion

References

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 3.2} 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. ↑ ^{4.0 4.1} 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.
6. ↑ Savage, M. D. (1996). An Introduction to Avidin-Biotin Technology and Options for Biotinylation. In T. Meier & F. Fahrenholz (Eds.), A Laboratory Guide to Biotin Labeling in Biomolecule Analysis. Basel: Birkhäuser.
7. ↑ ^{7.0 7.1 7.2} Craft, D. V., Goss, N. H., Chandramouli, N., & Wood, H. G. (1985). Purification of biotinidase from human plasma and its activity on biotinyl peptides. Biochemistry, 24(10), 2471-2476.
8. ↑ ^{8.0 8.1 8.2} Wolf, B. (2010). Clinical issues and frequent questions about biotinidase deficiency. Molecular Genetics and Metabolism, 100(1), 6-13.
9. ↑ ^{9.0 9.1} Wolf, B., Hymes, J., & Heard, G. S. (1990). [11] Biotinidase. In W. Meir & A. B. Edward (Eds.), Methods in Enzymology (Vol. 184, pp. 103-111): Academic Press.
10. ↑ Knappe, J., Bruemer, W., & Biederbick, K. (1963). Reinigung und Eigenschaften der Biotinidase aus Schweinenieren und Lactobacillus casei. Biochemische Zeitschrift, 338, 599-613.
11. ↑ Chauhan, J., & Dakshinamurti, K. (1988). Role of human serum biotinidase as biotin-binding protein. Biochemical Journal, 256(1), 265-270.
12. ↑ Wolf, B., Grier, R. E., Secor McVoy, J. R., & Heard, G. S. (1985). Biotinidase deficiency: A novel vitamin recycling defect. Journal of Inherited Metabolic Disease, 8(1), 53-58.