3.7.2 Mercapto-PEG-Acids

S-Acetyl-PEG-acids and active esters provide a method for converting amino groups to a thiol, while incorporating PEG units. Conjugation with thiol reactive agents e.g., maleimides, vinyl sulfones or α-halo keto functionalized reaction partners increases the conjugation possibilities of the former amino group by the whole set of thiol reactive reagents. Mercapto-PEG-Acids are highly hydrophilic, non-antigenic, non-immunogenic and non-toxic.

Protocol for in-situ Activation of PEG-Acids to the NHS ester:

Add a methylene chloride solution of the acid to the dry reagents under dry conditions (10-20% molar excess of EDC and NHS in dry methylene chloride, dried over 3A molecular sieves). Stir for several hours or overnight, then evaporate the solvent and use. The reaction mixture can also be treated with a small amount of silica gel to adsorb the excess EDC and the urea by-product. Filter, then evaporate the solvent and use. NHS should be added together with EDC to prevent formation of the anhydride. DCC and DIC can also be used. Typically use about 1 equivalent, and add a solution of the carbodiimide to the acid and NHS (1.1 to 1.2 eq.). PfOH (pentafluorophenol), MSNT (1-(Mesitylene-2-sulfonyl)-3- nitro-1,2,4-triazole), HOCt (Ethyl 1-hydroxy-1H-1,2,3-Triazole- 4-carboxylate), HOPO (2-Hydroxypyridine-N-oxide) and a set of other coupling reagents/leaving groups can be used in place of NHS, if this is of any preference.

Reference and Protocols:

• Bioconjugate Techniques; G. T. Hermanson; 2nd Edition; Elsevier 2008; 71: general description and use; 795: modification of antibodies; 984 modification of amines on nucleotides and DNA probes; 90,909 and 919: modification of enzymes, reactions with avidin and streptavidin; ISBN 978-0-12-370501-3

OPSS Protected Mercapto-PEG Crosslinkers

The general application of OPSS protected mercapto-PEG crosslinkers is the controlled and selective conjugation of an amine containing target, which reacts first with the NHS ester and then subsequently with a sulfhydryl containing complementary target molecule to form another disulfide. Many biological molecules contain both the amine function and the complementary compounds. Peptides, oligonucleotides or other biologicals, for example, can be terminated with thiols or have thiols designed into them, and vice versa. The contrast with the maleimide containing crosslinkers is that the OPSS derivatives form a disulfide, stable under non-reducing conditions. Normally, thiols can be cleaved with a reducing agent or exchanged with another thiol. The OPSS group presents the potential of using the pyridine- 2-thione, released in the reaction with another thiol, to measure the level of the PEG-OPSS incorporation in the labeling step or to monitor the subsequent reaction with another thiol by measuring its absorption at 343 nm. The cleavable OPSS PEGylating reagents produce a disulfide bond with thiols, which can later be cleaved with a variety of reducing agents like DTT or TCEP, or react with another thiol.

References:

• Synthesis and Characterization of Insulin−Transferrin Conjugates; N. J. Kavimandan, E. Losi, J. J. Wilson, J. S. Brodbelt and N. A. Peppas; Bioconjug Chem 2006; 17: 1376-1384. doi:10.1021/bc050344k
• Bioconjugate Techniques; G. T. Hermanson; 2nd Edition; Elsevier 2008; 276-335; ISBN 978-0-12-370501-3 Differential Conjugation of Tat Peptide to Superparamagnetic Nanoparticles and Its Effect on Cellular Uptake; M. Zhao, M. F. Kircher, L. Josephson and R. Weissleder; Bioconjug Chem 2002; 13: 840-844. doi:10.1021/bc0255236
• Antibody Linking to Atomic Force Microscope Tips via Disulfide Bond Formation; A. S. M. Kamruzzahan, A. Ebner, L. Wildling, F. Kienberger, C. K. Riener, C. D. Hahn, P. D. Pollheimer, P. Winklehner, M. Hölzl, B. Lackner, D. M. Schörkl, P. Hinterdorfer and H. J. Gruber; Bioconjug Chem 2006; 17: 1473-1481. doi:10.1021/bc060252a
• Quantitative Analysis of Derivatized Proteins Prepared with Pyridyl Disulfide-Containing Cross-Linkers by High-Performance Liquid Chromatography; D. H. Na, B. H. Woo and K. C. Lee; Bioconjug Chem 1999; 10: 306-310. doi:10.1021/bc980029g
• Synthesis and in Vitro Testing of J591 Antibody−Dendrimer Conjugates for Targeted Prostate Cancer Therapy; A. K. Patri, A. Myc, J. Beals, T. P. Thomas, N. H. Bander and J. R. Baker; Bioconjug Chem 2004; 15: 1174-1181. doi:10.1021/bc0499127
• Preparation of Thiol-Reactive Cy5 Derivatives from Commercial Cy5 Succinimidyl Ester†; H. J. Gruber, G. Kada, B. Pragl, C. Riener, C. D. Hahn, G. S. Harms, W. Ahrer, T. G. Dax, K. Hohenthanner and H.-G. Knaus; Bioconjug Chem 2000; 11: 161-166. doi:10.1021/bc990107f
• Michael-Type Addition as a Tool for Surface Functionalization; M. Heggli, N. Tirelli, A. Zisch and J. A. Hubbell; Bioconjug Chem 2003; 14: 967-973. doi:10.1021/bc0340621
• Covalent Protein−Oligonucleotide Conjugates for Efficient Delivery of Antisense Molecules; S. B. Rajur, C. M. Roth, J. R. Morgan and M. L. Yarmush; Bioconjug Chem 1997; 8: 935-940. doi:10.1021/bc970172u ff Basic Studies on Heterobifunctional Biotin−PEG Conjugates with a 3-(4-Pyridyldithio)propionyl Marker on the Second Terminus; K. Kaiser, M. Marek, T. Haselgrübler, H. Schindler and H. J. Gruber; Bioconjug Chem 1997; 8: 545-551. doi:10.1021/bc970086u