Protein Crosslinkers for Biological Applications

Crosslinking refers to the process of chemically joining two or more molecules together through covalent bonds. Crosslinkers contain reactive ends that can react under mild conditions with specific functional groups (such as primary amines and thiols) located on proteins or other biomolecules (DNA, RNA, lipids, and sugars). Thus, crosslinkers are often used to modify nucleic acids, drugs, and solid phase carrier surfaces in a process called bioconjugation, which includes crosslinking, immobilization, surface modification, and biomolecular labeling.

1.The main classification of protein crosslinkers (terminal active groups ):

Protein crosslinkers generally need to meet PH sensitivity, specific enzyme sensitivity, cleavage, non-cleavage and other conditions, commonly used ligation bonds such as hydrazone bonds, disulfide bonds, peptide bonds, glucose bonds, thioether bonds and maleimide bonds and so on. Therefore, protein crosslinkers are mainly concentrated into the following categories according to their structure and application:

（1）SH-SH crosslinker

（2）NH2-SH crosslinker

（3）COOH-NH2 crosslinker

（4）NH2-NH2 crosslinker

5）SH-Saccharides crosslinker

（6）Photoreactive crosslinker

（7）Chemically selective linkage crosslinkers

To maintain the enzymatic bioactivity of enzymes and the immunobinding activity of antibodies during enzyme-antibody coupling, in addition to improving crosslinking methods, the development of various new structural and functional crosslinking agents is of great importance. For instance, short-chain heterobifunctional crosslinkers like Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) can form degradable disulfide bonds by reacting with cysteine thiol groups through NHS-esters and pyridyl dithiol reactive moieties. The combination of amine and thiol groups enables controlled crosslinking, enhancing selectivity and product uniformity.

Therefore, protein crosslinking agents with two different reactive groups have garnered more attention due to their ability to facilitate controlled crosslinking reactions.

CAS# 68181-17-9 SPDP 2,5-dioxopyrrolidin-1-yl?3-(pyridin-2-yldisulfaneyl)propanoate	CAS#158913-22-5 LC-SPDP 2,5-dioxopyrrolidin-1-yl?6-(3-(pyridin-2-yldisulfaneyl)propanamido)hexanoate
CAS# 1334177-95-5 SPDP-PEG4-NHS ester

2.Key Applications of Protein Crosslinking Agents:

Protein-Protein Interactions

Most protein-protein interactions are transient, and crosslinking methods can be employed to stabilize or permanently link components within interaction complexes, aiding in the identification of these momentary contacts. The study of protein-protein interactions is crucial for a better understanding of biological phenomena and holds significant biological relevance. The rapidly advancing Chemical Cross-Linking Mass Spectrometry (CL-MS) technique, achieved through the optimization of various crosslinkers, is used to analyze protein structures and protein-protein interactions.

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Photo-crosslinking well coupled with QCLMS[2]

Ligation of carrier proteins to haptens

A peptide sequence (hapten) of a virus-coated protein can be linked to a carrier protein to make a vaccine to stimulate an immune response. In addition, monoclonal antibodies are prepared and used to return to find antigen-binding sites on macromolecular proteins. The crosslinker is adjusted to result in a lower molar ratio of hapten-carrier proteins with higher antibody affinity.

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PCSK9 Multicopy Displayed Nanovaccine[3]

Solidification of proteins or other molecules

The protein pass is covalently bound to various crosslinkers with different properties on solid matrices such as magnetic beads, 96-well plates, glass, etc., to adapt to different target analysis or substances that need to be adsorbed or for storage, etc., while reducing losses during elution processes.

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The preparation of SC-coupled magnetic beads[4]

Labeling of antibodies, marker transfer

Antibodies are linked to enzymes, fluorophores or biotin for quantitative measurements or positional observations. Biotin-containing crosslinker substances and labeled streptavidins are widely used in bioanalytical and bioassays such as flow cytometry, ELISA, immunohistochemical staining, western blotting, etc.。

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Diagrammatic drawing OF Flow cytometry from Bioon

	CAS# 58-85-5 Vitamin H	CAS#66640-86-6 Biotin Hydrazied	CAS# 109276-34-8 Biotin-LC-Hydrazide
CAS# 129179-83-5 N-[6-(Biotinamido)hexyl]-3'-(2-pyridyldithio)propionamide		CAS# 35013-72-0 N-(D-Biotinyloxy)succinimide	CAS# 120550-35-8 Biotin-PFP

Research on Targeted Drugs

Antibodies that recognize tumor surface antigens and drugs are covalently linked through cross-linking agents to achieve targeted therapy, thereby reducing the killing of healthy cells by traditional chemotherapy methods. Drug-linker-carrier conjugate, well improve and control the transport and metabolism of drugs in the body, realize sustained release administration and directed administration, improve bioavailability and therapeutic index, and meet the requirements of vector drugs that need to be quantified and released in vivo.

In recent years, through the selection of specific cross-linkers and the clever use of the interaction between cross-linkers and proteins, new therapeutic methods such as ADC and PROTAC, which are mainly used to study cancer treatment, have achieved rapid results.

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Mechanism of Proteolysis Targeting Chimeras (PROTACs)[5, 6]

3.Targeted Drug Selection for Protein Crosslinking Agents Requires Meeting Two Criteria:

(1) Maintaining drug stability in the bloodstream.

(2) Ensuring effective drug release within target cells once it reaches tumor tissue.

CAS# 165454-06-8 3-Oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-oic acid	CAS# 1393330-40-9 Propargyl-PEG5-NHS Ester>
CAS# 560088-89-3 Fmoc-AEEA-AEEA	CAS# 1395786-30-7 DBCO-Maleimide

References

4.Protein Crosslinking Agent Product Series

Seebio Biotech is pleased to introduce an expanded range of protein crosslinking agent products. Users can conveniently explore further details on our official website.

Target group-1	Target group2	Linkers
NH2-	COOH	Carboxyl Group-(Spacer)-Amino Group	56-40-6	AAU0115A，Glycine，100G 1KG 5KG
			107-95-9	AAU0116A, β-Alanine,100G 1KG 5KG
			56-12-2	AAU0117A, 4-Aminobutyric acid, 100G 1KG 5KG
			660-88-8	AAU0118A, 5-Aminopentanoic acid, 100G 1KG 5KG
			60-32-2	AAU0119A, 6-Aminocaproic acid, 100G 1KG 5KG
			929-17-9	AAU0120A, 7-Aminoheptanoic acid, 100G 1KG 5KG
			693-57-2	AAU0121A, 12-Aminododecanoic acid, 100G 1KG 5KG
		NHS Ester-Boc Amino	51513-80-5	AAU0009A, 6-(N-Boc)caproic acid NHS, 5G 25G 100G
	SH-	NHS Ester-Maleimide	55750-62-4	AAU0122A, N-Succinimidyl 3-maleimidopropionate(BMPS), 25G 100G
			80307-12-6	AAU0123A, 4-Maleimidobutyric acid N-hydroxysuccinimide ester(GMBS), 25G 100G
			55750-63-5	AAU0124A, 6-Maleimidocaproic acid-NHS (EMCS), 25G 100G
			87981-04-2	AAU0038A, Maleimide-C10-NHS ester, 25G 100G
			64987-85-5	AAU0125A, N-SucciniMidyl 4-(N-MaleiMidoMethyl)cyclohexane-1-carboxylate(SMCC), 25G 100G
			92921-24-9	AAU0126A, 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt(Sulfo-SMCC), 25G 100G
			58626-38-3	AAU0127A, 3-Maleimidobenzoic acid N-hydroxysuccinimide ester, 1G 5G
			79886-55-8	AAU0060A,4-(4-Maleimidophenyl)butyric acid N-hydroxysuccinimide ester(SMPB), 1G 5G
			125559-00-4	AAU0177A, N-SucciniMidyl 6-[[4-(N-MaleiMidoMethyl)cyclohexyl]carboxaMido]hexanoate(LC-SMCC), 500MG 1G
		Carboxyl -Maleimide	7423-55-4	AAU0130A, 3-Maleimidopropionic acid(MPA), 100G 1KG 5KG
			57078-98-5	AAU0019A, 4-Maleimidobutyric acid, 100G 1KG 5KG
			55750-53-3	AAU0131A, 6-Maleimidocaproic acid(EMCA), 100G 1KG 5KG
			25021-08-3	AAU0023A, 2-Maleimido acetic acid, 100G 1KG 5KG
			68181-17-9	AAU0113A, 3-(2-Pyridyldithio)propionic acid N-succinimidyl ester,5G 25G
		Carboxyl -Disulfide	1077-28-7	AAU0001A, DL-α-Lipoic acid, 100G 1KG 5KG
	-C≡C-/Cyclooctyne	Carboxyl -Alkyne/Cyclooctyne	53293-00-8	AAU0065A, 5-Hexynoic acid, 100G 1KG 5KG
			2777-65-3	AAU0044A, 10-Undecynoic acid, 100G 1KG 5KG
			6089-09-4	AAU0031A, 4-Pentynoic acid, 100G 1KG 5KG
	-C≡C-/Cyclooctyne	Carboxyl--Azide	79583-98-5	AAU0003A, 5-Azidopentanoic acid, 100G 1KG 5KG
	Other	NHS Ester--Acrylic	38862-24-7	AAU0034A, N-Acryloxysuccinimide, 100G 1KG 5KG
			38862-25-8	AAU0033A, N-Succinimidyl methacrylate, 25G 100G
		NHS Ester-Other	42014-51-7	AAU0138A, Bromoacetic acid N-hydroxysuccinimide ester, 1G 5G 25G
			39028-27-8	AAU0091A, Iodoacetic acid N-hydroxysuccinimide ester, 1G 5G
			60444-78-2	AAU0039A, 4-甲酰苯甲酸 N-琥珀酰亚胺酯,25G 100G
-COOH	SH-	Amino -Maleimide	134272-64-3	AAU0097A, N-(2-Aminoethyl)maleimid trifluoroacetate salt, 5G 25G
		Amino-Thiol	60-23-1	AAU0068A, 2-Aminoethanethiol, 100G 1KG 5KG
	N3-	Amino -Alkyne/Cyclooctyne	1255942-06-3	AAU0139A, Dibenzocyclooctyne-amine(DBCO-Amine), 5G 25G
			2450-71-7	AAU0056A, Propargylamine,100G 1KG 5KG
	-C≡C-/Cyclooctyne	Amino -(Spacer)-Azide	88192-19-2	AAU0071A, 3-Azido-1-propanamine, 5G 25G
CHO-	SH-	Hydrazide -Maleimide	151038-94-7	AAU0142A, 6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide 2,2,2-trifluoroacetate, 5G 25G
	N3-	Maleimide Group-Alkyne/Cyclooctyne	1395786-30-7	AAU0013A, Dibenzocyclooctyne-maleimide（DBCO-maleimide）, 1G 5G
			209395-32-4	AAU0028A, N-Propargylmaleimide, 5G 25G
N3	Other	Alkyne-Hydroxy	927-74-2	AAU0144A, 3-Butyn-1-ol, 100G 1KG 5KG
			928-90-5	AAU0145A, 5-Hexyn-1-ol, 100G 1KG 5KG
			63478-76-2	AAU0146A, 6-Heptyn-1-ol,100G 1KG 5KG
			5390-04-5	AAU0147A, 4-Pentyn-1-ol, 100G 1KG 5KG
			871-91-0	AAU0148A, 7-Octyn-1-ol, 100G 1KG 5KG
			17643-36-6	AAU0149A, 9-Decyn-1-ol, 100G 1KG 5KG
			2774-84-7	AAU0150A, 10-Undecyn-1-ol, 100G 1KG 5KG
		Cyclooctyne-Hydroxy	1263166-90-0	AAU0151A,(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol(BCN-OH),1G 5G

5.Featured Product Recommendations

Copper-Free Crosslinking

AAU0103A; 1G 5G CAS# 1263166-93-3 CN-POE3-NH2

AAU0105A; 1G 5G CAS# 1426827-79-3 BCN-CO-NHS

Light crosslinking

AAU0095A; 5G 25G CAS# 92367-11-8 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzyl Bromide	AAU0062A; 5G 25G CAS# 87736-88-7 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzyl Alcohol	AAU0041A; 5G 25G CAS# 1258874-29-1 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzylamine Hydrochloride	AAU0040A; 5G 25G CAS# 85559-46-2 4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzoic Acid
AAU0109A; 1G 5G; CAS# 1253202-38-8 NHS-SS-Diazirine (SDAD)	AAU0108A; 1G 5G CAS# 1239017-80-1 NHS-Diazirine (SDA)

Associated reagents

AAU0152A; 100g 1KG 5KG CAS# 156-57-0 Cysteamine Hydrochloride	AAU0153A; 100g 1KG 5KG CAS# 3483-12-3 Cleland's Reagent	AAU0166A, 100g 1KG 5KG CAS# 3945-69-5 DMTMM	AAU0155A; 100g 1KG 5KG CAS# 56-17-7 Cystamine Dihydrochloride
AAU0156A; 100g 1KG 5KG CAS# 51805-45-9 Tris(2-carboxyethyl)phosphine Hydrochloride	AAU0133A; 100g 1KG 5KG CAS# 76931-93-6 N-Succinimidyl S-Acetylthioglycolate	AAU0092A; 100g 1KG 5KG CAS# 84271-78-3 N-Succinimidyl 3-(Acetylthio)propionate	AAU0157A;  100g 1KG 5KG CAS# 1195-16-0 3-Acetamidotetrahydro-2-thiophenone
AAU0158A; 100g 1KG 5KG CAS# 69-78-3 5,5'-Dithiobis(2-nitrobenzoic Acid)	AAU0159A; 100g 1KG 5KG CAS# 64987-82-2 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic Acid	AAU0160A; 100g 1KG 5KG CAS# 69907-67-1 trans-4-(N-Maleimidomethyl)cyclohexane-1-carboxylic Acid	AAU0161A; 100g 1KG 5KG CAS# 108-30-5 Succinic Anhydride
AAU0173A; 100g 1KG 5KG CAS# 6066-82-6 N-Hydroxysuccinimide	AAU0174A; 100g 1KG 5KG CAS# 106627-54-7 N-Hydroxysulfosuccinimide Sodium Salt	AAU0175A; 100g 1KG 5KG CAS# 5672-89-9 N-Succinimidyl Trifluoroacetate	AAU0176A; 100g 1KG 5KG CAS# 25895-60-7 Sodium Cyanoborohydride
AAU0162A 100g 1KG 5KG CAS# 530-62-1 1,1'-Carbonyldiimidazole	AAU0163A; 100g 1KG 5KG CAS# 2491-17-0 CMC	AAU0164A 100g 1KG 5KG CAS#25952-53-8 EDCI·HCl	AAU0165A 100g 1KG 5KG CAS# 1892-57-5 EDC

 

 

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