Michael-type thiol-based additive. A systematic evaluation of your control factors. (2023)


Volume 76, Number 47,

20 november 2020

, 131637

Author links open the overlay panel, , , ,


This article discusses the factors that determine the kinetics and product stability of this popular productBioconjugationResponse. We show that a) the pKa of the thiol, i.e. the amountTiolatos, the only determining factor is theReaction kineticsFor himnucleofiel; B)deterioration of the productmainly throughHydrolysis(no thiol exchange) and is mainly characterized by the fastest reactionElectrophile. As for molecular design,Acrylamideand low pKa thiols seem to be the reactants that offer the best compromise for stability and reaction speed.


Bioconjugation has been a major research topic since the 1970s and is best known for linking antibodies to drugs [1] or prodrugs or therapeutic proteins to polyethylene glycol (PEG) [2] or other polymers [3].

Michael-type addition is one of the most popular bioconjugation reactions [4], mostly using thiols as nucleophiles and electron-deficient double bonds as electrophiles. To clarify, Michael addition differs from Michael addition, where the nucleophile is a (stabilized) carbanion, and from thiol-ene reactions, where thiols are added to olefins via a free radical mechanism. They are not electron deficient. The popularity of the thiol-based Michael addition for bioconjugation and beyond (e.g. in thiol recognition [5], surface functionalization [6], polymer synthesis [4] or biomaterials [7] etc.) is due to a) attributed to the mild reaction conditions, b) the absence of by-products, and c) their bioorthogonal character, i. H. the reaction hardly competes with other nucleophiles of biological origin. This selectivity has a kinetic origin: thiols have considerable acidity, so anionic thiolates, which are therefore highly nucleophilic, are already present at neutral pH; The more acidic the thiol, the faster the reaction [8], a property it also shares with disulfide formation [9]. However, there are still areas of insufficient mechanistic understanding of this reaction, which so far often allow accurate predictions, e.g. B. Reactivity of the thiol [10] or the stability of the products. For example, we do not yet know whether the thiol pKa is one or the main factor controlling the kinetics of the reaction and how this depends on the acceptor structure. Another point to be clarified is which of the two major degradation pathways and to what extent it may affect the stability of the conjugation (Fig. 1); It is known that sulfur at the γ position [11], more so when oxidized as the sulfoxide or sulfone [12,13], accelerates the hydrolysis of the ester, but no quantitative relationship or extension with others appears to hold to give hydrolyzable groups. ; It is also known that retro-Michael addition can occur, allowing exchange with more reactive/concentrated thiols, but this has only been shown for maleimides [14,15].

Here we perform a comprehensive study of the effects of Michael-type donor and acceptor structures and the reaction environment on the rate constant and stability of the final Michael-type adducts.

(Video) Emory Cardiology Grand Rounds 04-06-2015

section cutouts

results and discussion

We used a small library of α,β-unsaturated acceptors (Fig. 2), variable strength of the electron withdrawing group (ester, amide, maleimide) and double bond (CH) hindrance3versus H) and the polarity of the side chain potentially binding a charge (alcohol versus amide). Because amino- or NHS-ester-terminated heterobifunctional linkers are commonly used in conjugation reactions leading to amide bond formation, five of the seven Michael-type acceptors (AcAEA, AcAEMA, AcAEAm, AcAEMAm) are not suitable


In summary, this study yielded two important design criteria: 1) Irrespective of the chemical structure of the thiol, acidity is the main controller of the reaction kinetics, 2) unsaturated amides confer the highest stability on their structures. Kinetics vs Hydrolysis They suffer from somewhat slow reaction kinetics (taking tens of hours). If this is a problem, it can be solved by lowering the pKa of the thiol by modifying the structure accordingly (e.g. adding ...).

Determination of rate constants

All addition reactions of type Michael van3-AMPONACin different acceptors were prepared in a 96-well plate at 30 °C and different pH values ​​(7.9, 8.1, 8.6, 9.0) using an 80:20% v/v solvent mixture of 100 mM Tris/EtOH buffer. and three different mole ratios of the thiol/Michael acceptor (1:3, 1:6, 1:9 for the most reactive HEA and AcAEA and 1:30, 1:60, 1:90 for the least reactive HEMA, AcAEMA, AcAEAm). and AcAEMAm). Note that the presence of EtOH is necessary to ensure that all reactants are present

Declaration of Competing Interests

The authors declare that they have no competing financial interests or known personal relationships that may have influenced the work described in this article.

expression of gratitude

Dr Wedgwood is much indebtedResearch Council for Engineering and Physics(EPSRC) for a PhD. Northwest Nanoscience PhD Training Center (NoWNano) grant (EPSRC Grant No.EP/G03737X/1). Dr Lallana and Dr. Gennari received funding from EU FP7 ReLiver (Grant Agreement #304961) and Univax (Grant Agreement #601738). The authors thank Ms. Anna Daghetti (University of Milan, Milan, Italy) for HPLC-MS analysis and Dr.


  • FMveroneset al.

    drug discovery

    Day off.


  • MJVincentet al.

    Trends in biotechnology.


  • BDcaseet al.

    Program Polym Science


  • S.Vermaet al.

    English J.Med.


  • CXYinget al.

    Chemie Soc. Rd.


  • METRO.heggliet al.

    Bioconjugate chemistry.


  • MPLutolfet al.

    (Video) Discover the first issue: EES Catalysis



  • MPLutolfet al.

    Bioconjugate chemistry.


  • METRO.Butet al.

    application of hot water. Mate. interfaces


For more references, see the full text version of this article.

Quoted by (12)

View all articles that cite in Scopus

Featured Articles (6)

  • research article

    A reduced matrix modulus of PEG hydrogels induces a vascular phenotype in human umbilical cord blood stem cells

    Biomaterials, volume 62, 2015, blz. 24-34

    Cardiovascular diseases, both congenital and adult, are serious health problems that are often treated with surgery. Bypass transplantation is a main therapy, but grafts often fail due to hyperplasia, fibrosis and atherosclerosis. Biocompatible cellular materials that reduce these complications and promote healthy microvascularity could reduce graft failure, but a better understanding of the effects of biomaterials on human stem cells is needed to achieve clinical benefit. Our group is investigating stem cell-loaded biomaterials for placement along the adventitia of compromised vessels and grafts. In this case, the effects of the substrate module on CD34+ stem cells from human umbilical cord blood were investigated. Cells were isolated by immunomagnetic separation and encapsulated in 3, 4 and 6 wt% PEG hydrogels containing 0.032% gelatin and 0.0044% fibronectin. The gels reached moduli of 0.34, 4.5 and 9.1 kPa. Cell viability approached 100%. Cell morphologies appeared to be similar in the gels, but proliferation was significantly lower in the 6 wt% gels. Expression profiles using stem cell signaling arrays revealed greater self-renewal and differentiation in the vascular endothelium between cells in the lower weight percent gels. Therefore, the modulus was associated with cell proliferation and function. Gels with moduli in the low kilopascal range may be useful for stimulating cell engraftment and microvascularization of the graft adventitia.

  • research article

    One-step thiol isocyanate dispersion polymerization: production of uniform, cross-linked and functional particles

    Chemical Engineering Journal, Volume 304, 2016, pp. 461-468

    Uniform, highly cross-linked functional group particles are very useful, but are difficult to achieve by conventional dispersion polymerization. Here we develop a one-step dispersion polymerization based on the thiol-isocyanate reaction to produce such particles. This polymerization takes place energy-efficiently at room temperature without additional catalyst/initiator or UV treatment, achieves a high monomer conversion and has few side reactions. It is shown that by adjusting the stabilizer concentration and the monomer/ethanol ratio, particles with a size ranging from 1.8 to 8 µm can be produced. The thiourethane bonds created give the particles high glass transition temperatures (Tg) and uniform networks. Moreover, due to the stepwise growth mechanism of thiol isocyanate, the particles can be intrinsically functionalized and available for further modifications. As an attempt to perform the thiol-isocyanate reaction in dispersion polymerization, this method offers the possibility to produce uniform, functional and highly cross-linked particles in a one-step dispersion polymerization.

  • research article

    Michael addition reaction of symmetrically halogenated bisalcones with reduced glutathione evaluated by RP-HPLC and RP-HPLC-ESI-MS

    Microchemical Magazine, Band 169, 2021, Artikel 106603

    Chalcones and their derivatives are promising drugs that have antiviral, anticancer, anti-inflammatory and antioxidant effects, among others. The biological activity of chalcones and derivatives is related to their reactivity with cellular thiols such as reduced glutathione (GSH), the major non-protein thiol compound found in mammals. Here three symmetrically halogenated bishalcones (SHBC) were synthesized and their Michael addition reaction withGSHwas studied to investigate whether the compounds' cytotoxic effects correlated with theirsGSHreactivity. Formation of two diastereoisomeric adducts (SG) was observed as a result of the addition ofGSHto one of the unsaturated carbon-carbon bonds and other diastereoisomeric adducts (Diploma) were formed by adding another oneGSHMolecule to the second carbon-carbon double bond. However, the reactivity of the compounds withGSHit did not correlate directly with its toxicity to cancer cells in vitro.

    (Video) Power Your Health Q&A with Dr. Fuhrman
  • research article

    Curing kinetics and characterization of dual-cure thiol-acrylate-epoxy thermosets with latent reactivity

    Reactive and Functional Polymers, Volume 122, 2018, pp. 60-67

    A new dual cure schedule for thiol-acrylate-epoxy blends has been developed. A photoinitiated latent catalyst system was used to perform the thiol-acrylate Michael addition at 35°C (step 1) followed by a thiol-epoxide click reaction (step 2) at 80-110°C. Intermediates showed a shelf life of several days. The use of a radical scavenger suppressed the radical-mediated homopolymerization of acrylates, which would otherwise leave unreacted thiols. The kinetics of stage 2 were mathematically analyzed using the isoconversion differential method and the Kamal regression model. glass transition temperatures (Tgram) of samples with different epoxy resin contents and different types of acrylates. The epoxy-rich formulations gave the highest resultsTgram. The use of higher functionality acrylate monomers and stiffer acrylate monomers, although not as big an impact as the epoxy content, resulted in a higher intermediate and final capacity.Tgram. The proposed curing schedule and resulting materials may be useful for applications such as adhesives, industrial coatings with high chemical resistance, and optical and electronic materials.

  • research article

    Nucleophilic thiol-yne reaction in macromolecular engineering: from synthesis to applications

    European Polymer Journal, Volume 137, 2020, Article 109926

    Developments in synthetic polymer chemistry aim to meet the great need to develop efficient, high-throughput and operationally simple chemical methods that can be easily scaled up to the macromolecular scale. In this sense, thiol-based reactions have experienced a renaissance in the past two decades and are widely used in polymer chemistry. Among the thiol-yne reactions, because of the fast reaction speed to form complex macromolecular structures, such as. linear/non-linear polymers and network structures, and the reactions can take place in both organic and aqueous media. More importantly, thiol-yne reactions can proceed via the free radical and nucleophilic pathways, providing a remarkable opportunity to tailor polymer synthesis to the desired applications. Today, nucleophilic thiol-yne reactions are often used at the macromolecular level, as the reactions are simply activated by the use of a base or proceed in a slightly basic medium without the need for an external controller (e.g., heat and light). In addition, recent studies have shown that nucleophilic thiol-yne reactions not only enable even faster polymerization, but also lead to the rapid construction of hydrogels with unique properties and the preparation of reaction-based dynamic bond exchange reactions. of thiol-alquinone. This overview summarizes the historical developments in nucleophilic thiol-yne reactions in terms of polymer synthesis, modification and applications.

  • research article

    Water-soluble polymeric photoinitiator for the dual polymerization of acrylates and methacrylates

    Journal of Photochemistry and Photobiology A: Chemistry, Band 389, 2020, Artikel 112288

    A new water-soluble polymeric (photo)initiator has been developed for the efficient double-curing of acrylate and methacrylate mixtures. The thioxanthone (10 wt%) functionalized initiator (PAATX) was synthesized from the aza-Michael addition reaction of a poly(amidoamine) (prepared from the reactions of 1,4-diaminobutane with ).N,N'-methylenebisacrylamide) to 9-oxo-9H-thioxanthen-2-yl acrylate. It was used for the polymerization of a mixture of 2-hydroxyethyl methacrylate (HEMA) and poly(ethylene glycol) diacrylate (PEGDA, Mnorte=575D). The first stage of healing is the addition of Aza-Michael between PEGDA and the PAATX amines; and the second is the visible light-initiated radical polymerization (PAATX has an absorption maximum in the near UV-Vis range, ~400 nm) of HEMA and the rest of the acrylates. The kinetics of the curing reactions were investigated using FTIR and Photo-DSC. Steady-state photolysis results show that PAATX/bis-(4-ThirdThe -butylphenyl)-iodonium hexafluorophosphate (iodine) system shows the highest rate of photolysis, which means that PAATX generates free radicals more efficiently via a photo-oxidation mechanism. The excited-state reactivity was studied using steady-state photolysis, steady-state and time-resolved fluorescence, and laser flash photolysis experiments. Surprisingly, the newly reported structure shows singlet-state reactivity, contrary to the usual behavior of thioxanthone derivatives.


Aktuelle adres: Servier Laboratories Ltd, Sefton House, Sefton Park, Bells Hill, Stoke Poges, Buckinghamshire SL2 4JS

See full text

© 2020 Elsevier B.V. All rights reserved.

(Video) Gregg Braden - The SECRET To Achieving ANYTHING YOU WANT In Life! | Rob Dial


1. Caffeine Blues | Wake Up to the Hidden Dangers of America's #1 Drug | Stephen Cherniske | Summary
2. Lightning Talks // Full Recording
(Texas Additive)
3. 3 Things That DESTROY Your GUT HEALTH | Mark Hyman
(Mark Hyman, MD)
4. Using Play to Rewire & Improve Your Brain | Huberman Lab Podcast #58
(Andrew Huberman)
5. Jake Tran’s FRAUDULENT Anti-Vegan Milk Video EXPOSED!
(Lifting Vegan Logic)
6. 2022 Low Temperature Sub-kW Power and Energy Storage - Lightning Talks
(APL Webinars and Events)


Top Articles
Latest Posts
Article information

Author: Edmund Hettinger DC

Last Updated: 06/08/2023

Views: 5742

Rating: 4.8 / 5 (58 voted)

Reviews: 89% of readers found this page helpful

Author information

Name: Edmund Hettinger DC

Birthday: 1994-08-17

Address: 2033 Gerhold Pine, Port Jocelyn, VA 12101-5654

Phone: +8524399971620

Job: Central Manufacturing Supervisor

Hobby: Jogging, Metalworking, Tai chi, Shopping, Puzzles, Rock climbing, Crocheting

Introduction: My name is Edmund Hettinger DC, I am a adventurous, colorful, gifted, determined, precious, open, colorful person who loves writing and wants to share my knowledge and understanding with you.