72040-63-2

Basic information

• Product Name: Succinimidyl 6-(biotinamido)hexanoate
• Synonyms: 6-[5-(2-OXO-HEXAHYDRO-THIENO[3,4-D]IMIDAZOL-4-YL)-PENTANOYLAMINO]-HEXANOIC ACID 3-HYDROPEROXYSULFONYL-2,5-DIOXO-PYRROLIDIN-1-YL ESTER SODIUM SALT;6-((BIOTINOYL)AMINO)HEXANOIC ACID, SUCCINIMIDYL ESTER;6-[(+)-BIOTINYL]AMINOCAPROIC ACID N-HYDROXYSUCCINIMDE ESTER;D-BIOTIN-AMIDOCAPROATE-N-HYDROXYSUCCINIMIDE ESTER;D-BIOTINOYL-EPSILON-AMINOCAPROIC ACID-N-HYDROXYSUCCINIMIDE ESTER;D-BIOTINYL-EPSILON-AMINOCAPROIC ACID-N-HYDROXY-SUCCINIMIDE ESTER;BIOTINYL-EPSILON-AMINOCAPROIC ACID-N-HYDROXYSUCCINIMIDE ESTER;BIOTIN-X-NHS
• CAS NO: 72040-63-2
• Molecular Formula: C₂₀H₃₀N₄O₆S
• Molecular Weight: 454.55
• Product Categories: API intermediates;Biotin Derivatives;Cross Linking Reagents;Labeling and Diagnostics Reagents;Sulfur & Selenium Compounds;BiotinylationReagents;Biotinylation Reagents;biotinyl
• Mol File: 72040-63-2.mol
• Article Data: 2

Chemical Properties

• Melting Point: 169-171 °C
• Appearance: /powder
• Density: 1.35 g/cm³
• Refractive Index: 1.591
• Storage Temp.: −20°C
• Solubility: H2O: ≤2 mg/mL with sonication
• PKA: 13.90±0.40(Predicted)
• Stability: Moisture Sensitive
• BRN: 3577403
• CAS DataBase Reference: Succinimidyl 6-(biotinamido)hexanoate(CAS DataBase Reference)
• NIST Chemistry Reference: Succinimidyl 6-(biotinamido)hexanoate(72040-63-2)
• EPA Substance Registry System: Succinimidyl 6-(biotinamido)hexanoate(72040-63-2)

Safety Data

• Safety Statements: 24/25
• WGK Germany: 3
• F: 10-21
• Hazardous Substances Data: 72040-63-2(Hazardous Substances Data)

Usage

Description

Succinimidyl 6-(biotinamido)hexanoate, also known as Biotin-LC-NHS Ester, is a biotinylation reagent with an extended aminocaproyl spacer arm. Succinimidyl 6-(biotinamido)hexanoate is a white solid that helps minimize steric hindrance and allows for efficient reaction with primary amino groups to form stable, irreversible amide bonds. Its unique structure enables it to be used for various applications in different industries.

Uses

Used in Biochemical Research:
Succinimidyl 6-(biotinamido)hexanoate is used as a biotinylation reagent for attaching biotin molecules to other compounds, such as proteins or DNA, to facilitate their detection, purification, or immobilization.
Used in the Protein Avidin-Biotin Capture System:
Succinimidyl 6-(biotinamido)hexanoate is used as a biotinylation reagent for both the antibody and the carrier. Its aminocaproyl spacer arm reduces steric hindrance in binding avidin, allowing for better interaction and reducing the direct interaction of a captured antibody with solid surfaces, such as plastics, which may reduce antigenicity.
Used in Diagnostics:
Succinimidyl 6-(biotinamido)hexanoate is used as a detection enhancer for DNA on nitrocellulose, improving the sensitivity and accuracy of DNA detection in various diagnostic applications.
Used in Cell Surface Labelling:
Succinimidyl 6-(biotinamido)hexanoate is used as a cell surface labelling reagent, taking advantage of its membrane permeability and ability to form stable bonds with primary amino groups on cell surfaces.
Used in Pharmaceutical Development:
Succinimidyl 6-(biotinamido)hexanoate can be used in the development of drugs that require targeted delivery or enhanced interaction with specific biological molecules, thanks to its biotinylation capabilities and spacer arm design.

Biological Activity

nhs-lc-biotin (succinimidyl-6-(biotinamindo)hexanoate), also known as nhs-x-biotin is a derivative of d-biotin, a amine-reactive biotinylation agent, that contains a spacer arm off the valeric acid side chain of d-biotin with an nhs ester group at its end. the nhs ester group at the end of nhs-lc-biotin covalently binds to amine groups in proteins and other molecules forming a stable amide linkage and releasing the nhs group. the 6-aminocaproic acid spacer of nhs-lc-biotin greatly increases the length between a covalently modified molecule and the bicyclic biotin rings leading to a better binding potential for avidin or streptavidin probes. nhs-lc-biotin is insoluble in aqueous environments requiring the dissolution of organic solvents prior to the addition to a buffered reaction.bioconjugate techniques , 2nd ed. by greg t.hermanson (pierce biotechnology, thermo fisher scientific, rockford, il). academic press (an imprint of elsevier): london, amsterdam, burlington, san diego . 2008. isbn 978-0-12-370501-3.

Purification Methods

Dissolve ~400mg of the ester in dry propan-2-ol (~25mL) with gentle heating. Reduce the volume to ~10mL by gentle boiling and allow the solution to cool. Decant the supernatant carefully from the white crystals, dry the crystals in a vacuum over P2O5 at 60o overnight. This material gives one spot on TLC. [Costello et al. Clin Chem 25 1572 1979, Kincaid et al. Methods Enzymol 159 619 1988.]

InChI:InChI=1/C20H30N4O6S/c25-15(7-4-3-6-14-19-13(12-31-14)22-20(29)23-19)21-11-5-1-2-8-18(28)30-24-16(26)9-10-17(24)27/h13-14,19H,1-12H2,(H,21,25)(H2,22,23,29)

Synthetic route

1-hydroxy-pyrrolidine-2,5-dione (6066-82-6) + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid (72040-64-3) → 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2)

Conditions	Yield
Stage #1: 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid With pyridine; dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20℃; for 0.0833333h; Stage #2: 1-hydroxy-pyrrolidine-2,5-dione In N,N-dimethyl-formamide for 18h;	56.8%

biotin (4443-68-9, 5652-41-5, 57378-70-8) → 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2)

Conditions	Yield
Multi-step reaction with 3 steps 1.1: pyridine; dicyclohexyl-carbodiimide / N,N-dimethyl-formamide / 0.08 h / 20 °C 1.2: 24 h / 20 °C 2.1: N,N-dimethyl-formamide / 4 h / 20 °C / pH 8 / sodium bicarbonate buffer 3.1: pyridine; dicyclohexyl-carbodiimide / N,N-dimethyl-formamide / 0.08 h / 20 °C 3.2: 18 h View Scheme

biotin N-hydroxysuccinimide ester (85718-04-3) → 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2)

Conditions	Yield
Multi-step reaction with 2 steps 1.1: N,N-dimethyl-formamide / 4 h / 20 °C / pH 8 / sodium bicarbonate buffer 2.1: pyridine; dicyclohexyl-carbodiimide / N,N-dimethyl-formamide / 0.08 h / 20 °C 2.2: 18 h View Scheme

6-aminohexanoic acid (60-32-2) → 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2)

Conditions	Yield
Multi-step reaction with 2 steps 1.1: N,N-dimethyl-formamide / 4 h / 20 °C / pH 8 / sodium bicarbonate buffer 2.1: pyridine; dicyclohexyl-carbodiimide / N,N-dimethyl-formamide / 0.08 h / 20 °C 2.2: 18 h View Scheme

tert-butyl N-(6-aminohexyl)carbamate (51857-17-1) + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) → (6-{6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoylamino}-hexyl)-carbamic acid tert-butyl ester

Conditions	Yield
With triethylamine In N,N-dimethyl-formamide at 20℃;	93.8%

9-(3-aminopropyl)-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-6-amine (1333155-97-7) + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) → C31H40IN9O5S2 (1418215-40-3)

Conditions	Yield
With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 1h;	72%

8-(6-iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-isopropylamino-propyl)adenine + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) → C34H46IN9O5S2 (1418215-42-5)

Conditions	Yield
With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 35℃; for 6h;	41%

N-(4-azido-2-nitrobenzoyl)-1,7-diaminoheptane + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) → 4-Azido-2-nitro-N-(7-{6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-hexanoylamino}-heptyl)-benzamide

Conditions	Yield
In N,N-dimethyl-formamide

6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) + 5-(3-aminopropen-1-yl)-5'-O-(β,γ-diphosphoryl-α-phosphonomethyl)-2'-deoxyuridine → 5-[3-(N-biotinyl-6-aminohexanoylamino)propen-1-yl]-5'-O-(β,γ-diphosphoryl-α-phosphonomethyl)-2'-deoxyuridine

Conditions	Yield
With 1-methyl-1H-imidazole; triethylamine In N,N-dimethyl-formamide

4-(2-aminoethyl)benzenesulfonyl fluoride (34284-75-8) + 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (72040-63-2) → 4-(2-{6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-hexanoylamino}-ethyl)-benzenesulfonyl fluoride

Conditions	Yield
In dimethyl sulfoxide at 20℃;

Upstream product

• 60-32-2 6-aminohexanoic acid 
• 85718-04-3 biotin N-hydroxysuccinimide ester 
• 4443-68-9 biotin 
• 6066-82-6 1-hydroxy-pyrrolidine-2,5-dione 
• 72040-64-3 6-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoylamino]-hexanoic acid 

Relevant articles and documents

METHODS FOR CONJUGATING NUCLEIC ACIDS WITH SMALL MOLECULES

A method for conjugating a nucleic acid with a molecule is provided. The method includes steps of (a) reacting the nucleic acid having a 5′-monophosphate with an activating agent in a first buffer to form a solution; (b) mixing an alcohol with the solution formed in the step (a) to obtain an intermediate; and (c) dissolving the intermediate in a second buffer containing an ethylenediaminetetraacetic acid (EDTA) and adding a nucleophile thereinto to react the intermediate with the nucleophile.