Thermo Scientific Crosslinking Technical Handbook

1 Thermo Scientific Crosslinking Technical Handbook Protein 1 NH2. O NH. HN. S. O. O. || SO 3 - ||. S S N Protein 1 + HO-N. H ||. O. Protein 2. easy molecular bonding Crosslinking technology Reactivity chemistries, applications and structure references table of contents Crosslinkers Technical Handbook introduction 1 Applications with Crosslinkers 14 25. What is Crosslinking ? 1 Creating protein conjugates 14 16. Chemical Reactivity of Crosslinkers Protein immobilization and Modification Reagents 2 9 onto solid supports 17 18. Amine-reactive chemical groups 3 4 Creation of immunotoxins 19. Carboxylic acid reactive Label transfer 19 21. chemical groups 4 5. Structure determination Sulfhydryl-reactive chemical groups 5 6 with heavy/light crosslinker pairs 22 23.

2 Carbonyl-reactive chemical groups 6 8 Metabolic labeling 24. Chemoselective ligation 9 Cell surface Crosslinking 25. Molecular Properties of Crosslinkers Cell membrane structural studies 25. and Modification Reagents 10 13. Subunit Crosslinking and protein Homobifunctional and structural studies 25. heterobifunctional crosslinkers 11. Crosslinkers at a Glance 26 31. General reaction conditions 11 12. Protein Modification Reagents Spacer arm length 12 at a Glance 32 35. Spacer arm composition 12 Appendix 1 - Structures and References 36 51. Spacer arm cleavability 13. Appendix 2 - Online Interactive Spacer arm structure 13 Crosslinker Selection Guide 52. Water solubility and cell Appendix 3 - Glossary Of membrane permeability 13 Crosslinking Terms 53.

3 What is Crosslinking ? Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. The availability of several chemical groups in proteins and peptides make them targets for conjugation and for study using Crosslinking methods. Crosslinkers also are commonly used to modify nucleic acids, drugs and solid surfaces. The same chemistry is applied to amino acid and nucleic acid surface modification and labeling . This area of chemistry is known as bioconjugation and includes Crosslinking , immobilization, surface modification and labeling of biomolecules. Crosslinking and modification reagents can be described by their chemical reactivity (page 2), molecular properties (page 10) or by their applications (page 14).

4 First, select a crosslinker with the functional groups to bind your biomolecules of interest. NHS ester reaction Maleimide reaction Select the Right Chemistry Amine-containing NHS ester Amine bond NHS Sulfhydryl- Maleimide Thioether bond molecule compound containing compound Carboxylate molecule containing EDC o-Acylisourea A. molecule OxidationHydrazide reaction of a carbohydrate (cis-diol) to an aldehyde EDC coupling reaction reactive ester Aldehyde-containing Hydrazide Hydrazone linkage molecule compound Carboxylate containing EDC o-Acylisourea Amide bond Urea A. molecule o-Acylisourea B. reactive reactive ester ester Second, choose which linker characteristics are important for your application. Length Composition Cleavability Space Arm Composition o-Acylisourea Amide bond Urea B.

5 Reactive ester AMAS BMH with hydrocarbon spacer DSP with disulfide linker for cleavage SM(PEG)12. Spacer Arm . [ ]. SM(PEG)12 BM(PEG)2 with polyethylene glycol spacer DSS with non-cleaveable spacer arm Crosslinkers are soluble in organic solvents such as DMSO or DMF. However, certain crosslinkers contain functional groups which make them soluble in aqueous buffers as well. Solubility DSS soluble in organic solvents BS3 soluble in aqueous buffers BM(PEG)5 soluble in aqueous buffers Select a package size based on the scale of your reaction. Our crosslinkers are available from mg to kg quantities. Single-Use Milligram Gram Large/Custom Package Size Bulk-size Packages Available Single-use tubes with 2mg crosslinker/vial Milligram quantities of crosslinker Gram quantities of crosslinker Large volume/custom packages To order, call or Outside the , contact your local branch office or distributor.

6 1. chemical reactivity of crosslinkers and modification reagents bioconjugation the core tools The most important property of a crosslinker is its reactive chemical group. The reactive group establishes the method and mechanism for chemical modification. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids (Figure 1). Protein modification reagents like PEGylation or biotinylation reagents have a reactive group at one terminus and a chemical moiety at the other end (PEG chain or biotin group, respectively). The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids (Table 1).

7 Coupling can also be nonselective using photo-reactive groups. O O O. H2N H2N H2N. OH OH OH. R. HS. Generic Cysteine Amino Acid NH2. Lysine O O O O O O O. H H H H H H. H3N N N N N N N. O O O O O O O. R H H HO H. SH. O. HO O. R Gly Glu Gly Asp Gly Cys Figure 1. Common amino acid functional groups targeted for bioconjugation. 2 For more information, or to download product instructions, visit Table 1. Popular crosslinker reactive groups for protein conjugation. N-hydroxysuccinimide esters (NHS esters). Reactivity class Target functional group Reactive chemical group NHS esters are reactive groups formed by EDC. O. O. activation of carboxylate molecules. NHS ester-activated Amine-reactive -NH2 NHS ester N. Imidoester R. O crosslinkers and labeling compounds react with primary O.

8 Pentafluorophenyl ester amines in slightly alkaline conditions to yield stable amide Hydroxymethyl phosphine NHS Ester bonds. The reaction releases N-hydroxysuccinimide Carboxyl-to-amine -COOH Carbodiimide ( , EDC) (MW 115g/mol), which can be removed easily by dialysis or desalting. reactive NHS-reactive chemistry Sulfhydryl-reactive -SH Maleimide Haloacetyl (Bromo- or Iodo-) NHS ester Crosslinking reactions are most commonly performed in phosphate, Pyridyldisulfide carbonate-bicarbonate, HEPES or borate buffers at pH for 30 minutes Thiosulfonate to four hours at room temperature or 4 C. Primary amine buffers such as Tris Vinylsulfone (TBS) are not compatible, because they compete for reaction. However in some Aldehyde-reactive -CHO Hydrazide procedures, it is useful to add Tris or glycine buffer at the end of a conjugation , oxidized sugars Alkoxyamine (carbonyls) procedure to stop the reaction.

9 O O. Photo-reactive , random Diazirine O O. P. nonselective, random Aryl azide O. N NH2 pH 7-9 N. N. insertion R + P R H + HO. O O. Hydroxyl (nonaqueous)- -OH Isocyanate NHS Ester Primary Amine Stable Conjugate NHS. reactive Reagent on Protein (amide bond). Azide-reactive -N3 Phosphine Figure 3. NHS ester reaction scheme for chemical conjugation to a primary amine. Amine-reactive chemical groups (R) represents a labeling reagent or one end of a crosslinker having the NHS ester reactive group; (P) represents a protein or other molecule that contains the target functional group ( , Primary amines ( NH2) exists at the N-terminus of each polypeptide chain primary amine). (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon-amine).

10 Because of its positive charge at physiologic conditions, Hydrolysis of the NHS ester competes with the primary amine reaction. The primary amines are usually outward facing ( , on the outer surface) of rate of hydrolysis increases with buffer pH and contributes to less efficient proteins, making them more accessible for conjugation without denaturing Crosslinking in less concentrated protein solutions. The half-life of hydrolysis protein structure. A number of reactive chemical groups target primary amines for NHS ester compounds is 4 to 5 hours at pH and 0 C. This half-life (Figure 2), but the most commonly used groups are N-hydroxysuccinimide decreases to 10 minutes at pH and 4 C. The extent of NHS ester hydrolysis esters (NHS esters) and imidoesters.