Linker for antibody-drug conjugates (ADCs)

An ADC cytotoxin is connected to an antibody by use of an ADC linker to form an ADC.

Lomant’s Reagent, or DSP (Dithiobis(succinimidyl Propionate)), is a homobifunctional, thiol-cleavable, membrane-permeable crosslinker. Its structure features an 8‑atom spacer arm terminated with amine-reactive N‑hydroxysuccinimide (NHS) esters. At pH 7–9, these esters selectively target primary amines, forming stable amide bonds that covalently conjugate proteins or other amine-containing molecules. The central disulfide bridge within the spacer can be cleaved under reducing conditions (e.g., with DTT, TCEP, or 2‑mercaptoethanol), enabling reversible crosslinking for subsequent analytical steps.
Although insoluble in aqueous buffers, DSP is readily soluble in polar aprotic solvents such as DMF or DMSO. Its ability to permeate cell membranes allows it to crosslink intracellular proteins, thereby stabilizing transient or weak protein–protein interactions for further investigation. The reagent is also useful for fixing protein complexes in tissue samples prior to immunostaining, immobilizing proteins on amine-functionalized surfaces, and preparing bioconjugates for various biochemical assays.
A closely related analogue, 3,3’‑Dithiobis(sulfosuccinimidylpropionate) (DTSSP, often called Sulfo‑DSP), possesses the same 8‑atom spacer and homobifunctional NHS‑ester reactivity. The key difference lies in its sulfonate groups, which render DTSSP water‑soluble and membrane‑impermeable. This property restricts its labeling to extracellular or cell‑surface proteins, preventing intracellular entry.
Taken together, the structural design of Lomant’s Reagent—its spacer length, cleavable disulfide bond, and membrane permeability—makes it a versatile tool for probing protein interactions both inside and outside the cell, mapping protein complexes, and generating reversible bioconjugates.

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Key Properties
NHS Ester Moieties: These functional groups selectively react with primary amines to form stable amide linkages, facilitating covalent cross-linking between proteins or other amine-containing molecules.
Disulfide Spacer: Incorporation of a disulfide bond enables cleavage under reducing conditions, thereby allowing DSP to serve as a reversible cross-linker. This feature is crucial for recovering original molecules after analysis.

Primary Applications
Protein Cross-Linking: DSP is extensively utilized to covalently conjugate proteins or peptides, finding key uses in structural biology, protein interaction mapping, and immunoassays.
Bioconjugation: The reagent is fundamental in bioconjugation strategies, supporting the synthesis of antibody-drug conjugates (ADCs) and other targeted therapeutic agents.
Reversible Cross-Linking: Due to its cleavable disulfide bond, DSP facilitates temporary cross-linking that can be precisely reversed with reductants such as DTT or TCEP.

Key Advantages
Controlled Reversibility: The ability to perform and then reverse cross-linking is invaluable in experimental workflows where the subsequent removal of the linker is required.
Efficient and Robust Conjugation: The NHS ester chemistry ensures rapid, high-yield conjugation with primary amines under physiological pH conditions, generating stable conjugates suitable for diverse biological and analytical contexts.

Summary
DSP Crosslinker is a versatile and indispensable reagent in biotechnology and pharmaceutical research, particularly valued for applications that demand precise control over the cross-linking and subsequent release of proteins or other biomolecules.

[1]. Probing antibody surface density and analyte antigen incubation time as dominant parameters influencing the antibody-antigen recognition events of a non-faradaic and diffusion-restricted electrochemical immunosensor. Anal Bioanal Chem. 2020 Mar;412(7):1709-1717.

Electrochemical sensors based on antibody-antigen recognition events are commonly used for rapid, label-free, and sensitive detection of various analytes. However, various parameters at the bioelectronic interface, namely those before and after probe (e.g., antibody) assembly onto the electrode, significantly influence the detection performance of analytes (e.g., antigens). This study delves into the relationship between bioelectronic interface properties and some parameters that have not yet been thoroughly investigated: antibody density and antigen incubation time on the electrode surface. To this end, we employed sensitive non-Radidatic electrochemical impedance spectroscopy. We found that with increasing incubation time of antigen-containing droplets, the solution resistance, the diffusion resistance of the reflective boundary element, and the capacitance of the phase-constant element all decreased, at rates of decrease of 160 ± 30 kΩ/min, 800 ± 100 mΩ/min, and 520 ± 80 pF × s(α-1)/min, respectively. Using atomic force microscopy, we also found that the electrode coating at high antibody density was thicker than that at low antibody density, with root mean square roughness values of 2.2 ± 0.2 nm and 1.28 ± 0.04 nm, respectively. Furthermore, we found that as antigen accumulates on the electrode, the solution resistance increases at high antibody densities and decreases at low antibody densities. Finally, antigen detection performance tests showed that the detection limit was superior at low antibody densities (0.26 μM vs 2.2 μM). In summary, this article elucidates the importance of these two factors and how changing one of these parameters can significantly affect the expected results. (Figure and text summary)

C14H16N2O8S2

404.415441513062

404.034

57757-57-0

93313

White to off-white solid powder

1.6±0.1 g/cm3

560.1±60.0 °C at 760 mmHg

128-133 °C

292.6±32.9 °C

0.0±1.5 mmHg at 25°C

1.625

-1.66

0

10

11

26

552

0

S(CCC(=O)ON1C(CCC1=O)=O)SCCC(=O)ON1C(CCC1=O)=O

FXYPGCIGRDZWNR-UHFFFAOYSA-N

InChI=1S/C14H16N2O8S2/c17-9-1-2-10(18)15(9)23-13(21)5-7-25-26-8-6-14(22)24-16-11(19)3-4-12(16)20/h1-8H2

Dithiobis[succinimidyl propionate]

DTSSP CrosslinkerDSP Crosslinker

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~123.63 mM)
H2O : < 0.1 mg/mL

Prior to use in aqueous cross-linking reactions, DSP must first be dissolved in an appropriate organic solvent, such as DMSO or DMF. The crosslinker exhibits solubility in several common solvents, including DMF, DMSO, acetone, and chloroform (at concentrations up to 50 mg/mL). Its membrane-permeable nature allows it to be effectively employed for cross-linking intracellular proteins, making it a frequent choice for applications such as immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP) assays. For applications specifically targeting extracellular cross-linking, the water-soluble analog DTSSP is recommended as a suitable alternative.

When preparing DSP for aqueous reactions, precipitation can sometimes occur upon dilution of the DMSO stock solution into phosphate-buffered saline (PBS). To mitigate this, it is advisable to add the DMSO stock to PBS in a dropwise manner while gently vortexing the PBS solution. Gentle warming of the final reaction mixture can also help maintain solubility. If precipitation is observed, reducing the concentrations of both the protein and DSP in the reaction may resolve the issue. Alternatively, increasing the final DMSO concentration in the reaction solution up to 20% can improve solubility and prevent precipitate formation.

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.18 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.18 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)