SMCC, formerly known as succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate, is a heterobifunctional protein crosslinker. SMCC is an amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane-stabilized spacer arm (8.3 angstroms). SMCC conjugation occurs via a maleimide group that is sulfhydryl (thiol; -SH) reactive and a NHS ester group that is amine reactive and forms stable, covalent protein crosslinks.
Physicochemical Properties
| Molecular Formula | C16H18N2O6 |
| Molecular Weight | 334.328 |
| Exact Mass | 334.116 |
| Elemental Analysis | C, 57.48; H, 5.43; N, 8.38; O, 28.71 |
| CAS # | 64987-85-5 |
| Related CAS # | 64987-85-5; |
| PubChem CID | 125175 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 501.7±42.0 °C at 760 mmHg |
| Melting Point | 180-182 °C(lit.) |
| Flash Point | 257.2±27.9 °C |
| Vapour Pressure | 0.0±1.3 mmHg at 25°C |
| Index of Refraction | 1.598 |
| LogP | -0.39 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 24 |
| Complexity | 599 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O(C(C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])N2C(C([H])=C([H])C2=O)=O)C([H])([H])C1([H])[H])=O)N1C(C([H])([H])C([H])([H])C1=O)=O |
| InChi Key | JJAHTWIKCUJRDK-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C16H18N2O6/c19-12-5-6-13(20)17(12)9-10-1-3-11(4-2-10)16(23)24-18-14(21)7-8-15(18)22/h5-6,10-11H,1-4,7-9H2 |
| Chemical Name | N-Succinimidyl 4-(N-Maleimidomethyl)cyclohexanecarboxylate |
| Synonyms | SMCC; heterobifunctional cross-linker; 71875-81-5; 2,5-Dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate; TRANS-4-(MALEIMIDOMETHYL)CYCLOHEXANECARBOXYLIC ACID-NHS; N-Succinimidyl 4-(Maleimidomethyl)cyclohexane-1-carboxylate; N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; Mal-AMCHC-OSu; SMCC crosslinker |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Protein crosslinker |
| ln Vitro | OVA and keyhole limpet hemocyanin (KLH) can be conjugated using SMCC or sulfo-SMCC. 300 μl KLH or OVA (3 mg) and 30 μl SMCC or Sulfo-SMCC (final concentration of 0.1 mM) are added in PBS with a final volume of 3 ml to the conjugation system. [1] |
| ln Vivo | The SMCC-conjugation technique allows antigen-coupled syngeneic splenic mononuclear cells to be injected into mice, eliciting strong immune responses to whole protein or peptide antigens[1]. |
| Cell Assay | A synthesized PEI-based gene delivery system, wherein PEI was crosslinked with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) conjugating trans-activating transcriptional activator (TAT), yielding PEI-SMCC-TAT (PST), a novel non-viral vector for apoptosis-related gene PUMA (p53 up regulated modulator of apoptosis), was designed and evaluated. Sulfo-SMCC is a commonly used heterobifunctional crosslinker and is soluble in water, making the crosslinking easier without organic reagent like DMSO or chloroform. The PST/pDNA nanoparticles were 171.9 nm at the optimal N/P ratio (50:1). DNA complexes of all the PST conjugation had much lower toxicity and exhibited enhancement in transfection efficiency in comparison with single PEI vector. The results also showed that the transfection efficiency of PST/pEGFP nanoparticles into malignant melanoma A375 cell increased, and PST carrying PUMA gene induced the apoptosis of A375 cells. It was suggested that PST could be a promising melanoma tumor-targeting nanovector, and have a good potential in clinical application [2]. |
| Animal Protocol | In the present study, we report our recently developed new approach to inducing antigen-specific immune response. We use two nucleophilic substitution "click" chemistry processes to successfully couple protein antigens or peptides to mouse spleen cells or T cells by a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) or sulfo-SMCC. SMCC and its water-soluble analog sulfo-SMCC contain N-hydroxysuccinimide (NHS) ester and maleimide groups, which allow stable covalent conjugation of amine- and sulfhydryl-containing molecules in trans. Protein coupling to cells relies on the free sulfhydryls (thiols) on cell surfaces and the free amines on protein antigens. Although the amount of protein coupled to cells is limited due to the limited number of cell surface thiols, the injection of spleen cells coupled with antigenic proteins, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA), induces a potent antigen-specific immune response in vivo, which is even stronger than that induced by the injection of a large dose of protein plus adjuvants. In addition, short peptides coupled to purified splenic T cells also potently elicit peptide-specific T cell proliferation in vivo after injection. Further studies show that antigen-coupled spleen cell treatment leads to augmented IFN-γ-producing T cells. Our study provides a unique antigen delivery method that efficiently distributes antigen to the entire immune system, subsequently eliciting a potent antigen-specific immune response with enhanced IFN-γ production. The findings in the present study suggest that this antigen-cell coupling strategy could be employed in immunotherapy for cancers, infectious diseases as well as immune-mediated disorders [1]. |
| References |
[1]. Potent antigen-specific immune response induced by infusion of spleen cells coupled with succinimidyl-4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SMCC) conjugated antigens. Int Immunopharmacol. 2016 Feb;31:158-68. [2]. Li F, Wang Z, Huang Y, Xu H, He L, Deng Yan, Zeng X, He N. Delivery of PUMA Apoptosis Gene Using Polyethyleneimine-SMCC-TAT/DNA Nanoparticles: Biophysical Characterization and In Vitro Transfection Into Malignant Melanoma Cells. J Biomed Nanotechnol. 2015 Oct;11(10):1776-82. PubMed PMID: 26502640. |
| Additional Infomation | Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate is an N-hydroxysuccinimide ester derived from 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. It is a member of maleimides and a N-hydroxysuccinimide ester. |
Solubility Data
| Solubility (In Vitro) |
DMSO : 25 mg/mL (~74.78 mM ) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9911 mL | 14.9553 mL | 29.9106 mL | |
| 5 mM | 0.5982 mL | 2.9911 mL | 5.9821 mL | |
| 10 mM | 0.2991 mL | 1.4955 mL | 2.9911 mL |