Hi. Pierce has a very helpful page.

http://www.piercenet.com/browse.cfm?fldID=02030312

Thank you all for your support. I'm currently working on polymer nanaoparticles. If i want to conjugate a drug with polymer nanoparticle by means of EDC-NHS mechanism, what type of drug would be suitable for this purpose?

You would use the EC-NHS to link -NH2 and -COOH. For more a more indepth explanation of techniques and conjugation rationale I highly recommend the book "Bioconjugation Techniques" by Greg T Hermanson.

EDC/NHS compounds use to active COOH groups in organic solvents. Thus, drug must have NH2 groups.

Dear fellows 

I am going to conjugate a short peptide to the end groups of PLGA-b-PEG diblock copolymer, in order to produce a PLGA-PEG-peptide triblock chain that would be able to be self-assembled to form peptide decorated nano-micelles or nanoparticles..

I've been considering to use EDC/NHS coupling for this purpose through activating the terminal COOH of the peptide and then conjugating it to PLGA-PEG-NH2 ..

But I cant' find a reliable protocol for that, most of them explain conjugation of ligands to NP surface, not to the Polymer or copolymer end groups

I would really appreciate it if any body could comment and give hints???

Thank U all..

Hi Farzad, as far as I can tell PLGA--b-PEG refers to Poly(ethylene glycol)-b-Poly(L-lactic acid). Is this right?

This polymer has a methoxy at one end and a -OH group at the other end. If this is so, then EDC will not work.

Hi Steingrimur, nice to meet you..

Yeas that's it, but there are also diverse comercially available PEG or PLGA-PEG  polymers with functional end groups in PEG moiety, making further derivatizations and conjugations possible.. e.g. adding targeting ligands or fluorescent dyes..

Check here for example: 

https://akinainc.com/polyscitech/products/polyvivo/catalogue.php

Hi Farzad, great site! I bookmarked it.

Anyway, this company has PLGA-PEG polymers with a multitude of reactive groups to choose from.

They have Bromoacetamide and N-hydroxysuccinimide reactive groups, so the only thing you have to do is just mix your peptide with the polymer at pH 7.

You can also just do EDC coupling with either the amine or the -COOH terminal polymer and your peptide. It is easier and more efficient than going through the NHS step.

Hi Steingrimur

Thank you for the helpful comment and hint. Probably we'd use the latter you mentioned. But unfortunately the polymers and reagents are expensive for us in Iran, and because of international sanctions ordering process is complex and takes too long.. My supervisor is considering the feasibility though..

Thanks

Regards

What about adding EDC/NHS to PLGa for incorporation with atelocollagen?

The mechanism for EDC coupling to carboxylic acid is electron donation of the electron-rich R''C(=O)OH to the electron-deficient carbodiimide carbon on RN=C=NR' to form O-acylisourea adduct RN=C(OR'')NR'.

R''C(=O)OH  +  RN=C=NR'  - - - - - >  RN=C(OC(=O)R'')NR'  (protons hopping around)

At this point, a common rearrangement (isomerization) can happen (in organic solvent or in alkaline solution) from O-acylisourea to N-acylurea adduct.

RN=C(OC(=O)R'')NR'  - - - - - >  RN=CN(OC(=O)R'')R'

The N-acylurea is resistant to aminolysis--watch out for it! However, if all goes well (i.e. quickly), the O-acylisourea, in the presence of some H+, will be substituted off as an EDC-urea RNC(=O)NR' (taking one of the carboxylic O with it) by an incoming nucleophile. IF your nucleophile was an amine, you now have a new amide bond: R''C(=O)N.

The mechanism for NHS coupling goes by substitution: the NHS kicks off water from the carboxylic acid. Like EDC, NHS gets substituted off itself by an incoming stronger nucleophile.

The presence of NHS (or Sulfo-NHS), along with EDC, is thought to suppress the formation of N-acylureas. Some have also noted that the formation of the final urea actually drives the coupling reaction itself (so some protons are good).

Sulfo-NHS is expensive and from what I've read, was created to ensure protein solubility once the NHS ester was formed.

Follow the link I posted here and paste the following SMILES strings in the empty space to compare structures visually (displays in browser).

O-acylisourea and N-acylurea

CC(=O)(N(C(=O)NCC)CCCN[H+](C)C).CC(=O)O(C(NCC)=NCCCN[H+](C)C)

EDC and EDC final urea

CCNC(=O)NCCCN(C)C.CCN=C=NCCCN(C)C

http://cactus.nci.nih.gov/gifcreator/

NHS and Sulfo-NHS are used to prepare amine-reactive esters of carboxylate groups for chemical labeling, crosslinking and

solid-phase immobilization applications. Carboxylates (-COOH) may be reacted to NHS or Sulfo-NHS in the presence of a carbodiimide such as EDC, resulting in a semi-stable NHS or Sulfo-NHS ester, which may then be reacted with primary amines (-NH2) to form amide crosslinks. Although NHS or Sulfo-NHS is not required for carbodiimide reactions, their use greatly enhances coupling efficiency. Furthermore, using NHS or Sulfo-NHS makes it possible to perform a two-step reaction.

Hey folks,

ive got a short question regarding the water solubility of EDC/NHS and maybe some of you have experiences with that already. I would like to attach a carboxylic acid (CA) carrying metalcomplex to a polymer. Id rather do that in water as all compounds are very good soluble. the resulting polymer-complex has a MW over 3000 so that should be easy to seperate from unreacted material. But, as soon as I bring my carboxylic acid conjugated complex together with the primary amines carrying polymer (without coupling reagent) it forms the salt that i cannot seperate by centrifugation anymore. No I have two questions: Within the activation of the CA in water should I rather use an excess of EDC NHS to prevent the hydrolysis back to the CA resulting into the salt together with the poylmer? And how stable is it anyway? As mentioned in one of the provided articles above it says:

"However, the coupling reaction has to be carried out fast, as the reactive ester that is formed can be rapidly hydrolyzed in aqueous solutions. To increase the sta-

bility of this active ester, N-hydroxysuccinimide (NHS) or N-hydroxysulfoxuccinimide (sulfo-NHS) can be used (Jang and Keng, 2008). Key parameters that should be controlled when using EDC are pH (as hydrolysis is largely dependent on pH), the

amount of EDC so that NPs do not aggregate due to loss of electrostatic repulsive forces between NPs, and the ratio EDC/NHS (Nakajima and Ikada, 1995; Sam et al., 2009; Shen et al., 2009). "

Normally i wait a couple of minutes after adding the EDC before adding the NHS but after adding the polymer i leave it stirring overnight. Is that conterproductive? Or is it even better to prepare the activated CA in another solvent?

Every comment and every person i can talk about pamam with is highly appreciated!

Hi Nadine,

Im not a chemist, but I managed to get the thermofisher method of doing this process working to surface bind proteins on gold. I think waiting between EDC and NHS additions is not a good idea. The EDC activates the carboxylic group, and this is not stable for long, hence usually they add the proteins with the EDC. NHS has to be added with the EDC instead to create a stable ester. The protein then binds to through the NHS termination. Ratio of EDC to NHS (sulfo NHS) is important and the buffer pH of that solution should be slightly acidic. The buffer needs to be without amine, hence do not use TRIS buffers etc. I use the recommended MES buffer at 5.5-6 pH. Now I do not have my notes with me but I think the EDC-NHS ratio should be around 15:1 or 20:1 . I have attached the thermofisher instructions for this process. I hope this helps. 

Hey Affar,

wahh, cool, ja, that should help - thank you so much! I was just wondering if it would also work with water only, but apparently a buffer system is always needed due to stability.. but im afraid that the buffer will somehow interact with the polymer - is that even possible?!

have a nice evening and thanks again!

Hi Nadine, Jao

Woah...too much chemistry for an electronics guy! This is why the chemists told me to keep away from PEG :) . Not sure how you can use the process without the buffer, its highly important. Do you have to use PEG? I do nanostructures for plasmonic sensing, and I prefer to have the molecule stick close to my evanescent fields for maximum interaction. If thats what you want, then why not use a shorter PEG, from what we have done in the lab, that is always easier to work with than those large PEGs that coil up. But I guess it depends what you want to do.

Can anyone explain me paclitaxel loaded MSN decorated with amine groups in outer layer need to be attached with another molecule consists of acid group.If i use EDC/NHS it will affect the drug(PTX) inside the MSN or not?

Can we bind sulphar containing carbohydrate to a polymer by using EDC/NHS method ? 

 Davoud Afshar, can you use Bradford Assay for determinate the protein concentration, after washing.

Hi Sara, the pH 6.1 is more utilized (Buffer MES 0,1M. 

 Hello guys,

I am having trouble to optimize a coupling reaction between a COOH-drug and HSA. I don't know which ratio of EDC/sulfoNHS use, compare to my drug. Can somebody help me ?

Thanks! 

Hi Gouel, you can skip the NHS coupling step altogether.

Just mix your drug with HSA in MES pH~5.5 and add EDC.

 In the absence of any free amines or free COOH in your preparations, you will get  great coupling. You will have to determine the best conditions, but I have had good results with 10X molar excess of EDC over the limiting component.

Sorry, I forgot to add that EDC couples any primary amines to any COOH groups. So if you have a high concentration of HSA, you will get crosslinked HSA polymers that may be insoluble..

Aurélie,

Steingrimur Stefansson is correct that you may omit Sulfo-NHS and use a large molar excess of EDC. Sulfo-NHS acts as a catalyst.  Both EDC and Sulfo-NHS are available from ProteoChem.  Protocols can be found following the links below.

http://www.proteochem.com/edchcl25gramsedachcl-p-34.html

http://www.proteochem.com/sulfonhs500mg-p-32.html

Thanks so much for your answers ! I am going to try that !

How to couple tertiary amines to carboxylic acid?

Dear all,

I want to do esterification of 1,1,2,2-tetrayl tetrabenzoic acid with NHS. I just want to replace only one COOH group and rest three COOH groups remain on its place..Please give me any idea what ratio i should keep? Thank you

Dear all,

I want to know the alternative chemical of EDC for the activates carboxyl groups on the RO membrane or TFC surface?

Hello all,

I was wondering if anyone can help me out in knowing what ratio of EDC:NHS used to conjugate lactobionic acid with DSPE-PEG-NH2?

Thank you

Hi Rand, I'm always surprised when people think that they have to modify their COOH groups to NHS esters to have them react with NH2 groups.

In many cases you don't have to because EDC will make the COOH group reactive towards NH2 groups. The NHS step is not needed.

Since lactobionic acid has one COOH group and your compound has one NH2 group, this will be easy. Mix them together in MES pH 5.0, add 10X molar concentration of EDC and they will couple.

Hello Mr. Stefansson,

Thank you for your reply.

How long does the coupling reaction need to take?and do you recommend a certain form of filtration?

Hi Rand, the coupling reaction is fast if both your reactants are soluble in 0.1M MES pH 5.0 (less than 4 hours at RT).

What filtration?

Steingrimur Stefansson do you need MES buffer or you can just adjust (with for exple NaOH) the pH of your solution to 5?

MES is not necessary per se, but it is very helpful to buffer the reaction and keep it at pH ~5.

Hi Merwan Benhabib . You need a buffer in that pH range that does not have -NH2 or -COOH groups.

Any one knows how to create ester linkage between 'carboxylic surface' polymer with the 'OH' end of Deoxycholic acid?

Meanwhile there is a publication that states: " Carbodiimide-activated carboxylic acid beads only react with oligonucleotides under conditions that promote nonspecific interactions (low salt, low pH, no detergent), comparably immobilizing primers on beads, but yielding no detectable enzymatic extension product."

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