Thanks a lot erik..

Try methyl alcohol - it esterifies exposed Coo- rapidly; 5 min for slices or other thin samples, more for tissue blocks

Thanks andy... Do you have a protocol or paper on this... ?

Glad to see you are in Dublin. I am a member of ASGBI. And I am not very young. That was largely used tens of years ago in classical chemistry. Look in AGE Pearse or M Gabe or Chayene book on histochemistry and histoenzymology. It might be older, from Gomori, the classic author of histochemistry 1951.

You don't need a protocol, just wash thin slices or cover with a drop of alcohol / if not enough try 5 min or more. You don't need to wash to remove extra alcohol, just leave it to dry free or shaking the slide in the air. OK ?

Dear Mayank,

dear Erik&Andy (I am interested in your protocol too...thank you!),

not being an expert with this, but interested in „blocking agents“ for histochemical reactions I would like to add the following:

> cf.: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224342/pdf/169.pdf

(Barrnett R. J., Seligman A.M. “Histochemical Demonstration of Protein-Bound Alpha-Acylamido Carboxyl Groups", 1957);

…modification of carboxyl groups by a carbodiimide and nucleophile at acid ph

EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (cf.: http://www.sciencedirect.com/science/article/pii/000527367390343X ;

….glycine ethyl ester at low pH using carbodiimide reagent….1976, cf. http://www.jbc.org/content/251/8/2353 ;

….blocked carboxylic acid compounds obtained by blocking carboxyl groups with alkyl vinyl ethers, cf.

http://www.sciencedirect.com/science/article/pii/S0300944099000867, 2000;

….”Reversible Blocking of Amino and Carboxyl Groups” …http://link.springer.com/chapter/10.1007/978-3-642-78056-1_3#page-1

and last but not least:

http://pubs.acs.org/doi/abs/10.1021/bi00853a038?journalCode=bichaw

(Modification of the Carboxyl Groups of Ribonuclease by Attachment of Glycine or Alanylglycine, Wilcek et al, Biochemistry, 1967, 6 (1), pp 247–252)

…..Among the early methods for the blocking of carboxylgroups of proteins esterification with methanol in hydrochloric acid (Fraenkel-Colrat and Olcott 1945) and with diazomethane (Herriot 1947) should be mentioned…..

… A limited esterification of RNAse has been accomplished recently with diazoacetoglycinamide (Riehm and Scheraga, 1965). Sheehan&Hlavka (1956) have first suggested the use of watersoluble carbodiimide for the selective modification of carboxyl groups of proteins. Riehm and Scheraga, 1966 have treated RNAse with 1-cyclohexyl-3-(2-morpholinethyl)carbodiimide-metho-p-toluenesulfonate……( and isolated from the reaction product five chromatographically distinguishable components….).

What I have found too (perhaps as an analog to the ) was:

"Reduction of Carboxylic Acids"(with perhaps interesting reaction equations): “carboxylic acids can only be reduced by LiAlH4 and NOT by the less reactive NaBH4” (cf.: http://www.mhhe.com/physsci/chemistry/carey/student/olc/graphics/carey04oc/ref/ch19reactioncarboxylicacids.html ).

@Erik: Since you are expert in such, for the latter I grately should acknowledge a comment whether you think an incubation in hydrous LiAlH4 would/could help with this matter (always depending on the task). It could be that I am thinking wrong here…

Unfortunately I was not able to find any source with a definite short recipe for the deactivation/blocking of COOH-groups.

So hopefully you get more precise answers in addition to those which were posted so far.

Good luck,

Wolfgang

High guys, I have something close to this and of particular interest to me for some time and that is SPECIFICALLY modifying the -COOH terminus of a protein...so NOT all the acid side chains of the protein, but just the terminus. Any ideas? You can easily do this with the N-terminus through pKa differences from side-chains (labeling at a mid-range pH like 7.5-8.0) but I have not seen a word on the c-terminus...

Hi,

You can black carboxy groups in different ways depending on your needs. Generally speaking you can trasform with relative ease the carboxy group in stable amides or esters. The type of those you can choose following your strategy, but before that you need to activate the carboxy group as an acive ester one of the mostly used coupling reagent (activating) being the WSC (water soluble carbodiimide) and that usually is performed without separations or in one pot fashion way.

Regards

Dear Mayank

You can convert carboxyl groups to amides through reaction with one of several amines promoted by a water soluble carbodiimides (WSCs).

This is a two step reaction and carbodiimides react only with caboxylic acids.

1-CYCLOHEXYL-3-(2-MORPHOLINYL-4-ETHYL) CARBODIIMIDE METHO-P-TOLUENESULFONATE (CMC) and 1-ETHYL-3-(3-DIMETHYLAMINOPROPYL) CARBODIIMIDE (EDC) are the most used WSCs in this reaction.

Hi Mayank: One thing to consider is that it is commonly preferred to avoid "blocking" the protein carboxyls by chemistry, such as forming an ester or amide, that results in an ester or amide formed between the carboxyl of the protein and some of the hydroxyls (i.e. from Ser or Thr) and aminos (i.e. Lys) on the protein. This may be obviated somewhat by using an excess alcohol reagent (for esterification) or an excess amine reagent. However, even then, local hydroxyl or amino groups may still participate in reaction. As for your question of selective blockage of C-terminal carboxyl, there are a few options for doing that. One aspect, as was mentioned, is to use the typical difference in pKa of C-terminal carboxyl versus R-group carboxyls. As one possibility for you to use, I would refer you to a procedure for oxazolone ring formation. You can look at a procedure in a patent reference (which will list other references) to help you. It is U.S. patent # 7,294,707 and can be found at the website: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=7,294,707.PN.&OS=PN/7,294,707&RS=PN/7,294,707.

Carboxylic groups in proteins can be blocked by reacting them with a carbodiimide, in the prescence of a succinimide catalyst. The carbodiimide reacts with carboxylic groups of acid treated polyester disks. The reaction between a carboxyl group and carbodiimide results in a urea derivative (O-acylisourea), a nucleophile which can attack a primary amine resulting in an amide bond.

For more reading see Hermanson GT (2008). Chapter 3 - Zero-Length Crosslinkers. Bioconjugate Techniques (Second Edition). New York, Academic Press: 213-33.

Cheers Erik, I shall look into it

Thanks everyone...

@madhumita which reagent of aryl group are you talking..? Do you have a paper or protocol for this reversible modification... Sounds interesting...

@ Erik: thank you so much for your thorough explanation. I understand now that treating with methanol (or alcohol) is called kind of a Fischer reaction [ when carboxylic acid is treated with an alcohol and an acid catalyst: an ester is formed (along with water)].

All other reactions posted so far unfortunately are far beyond my chemical knowledge, so I apologize for not being able to add something helpful in this context). But I shall follow this thread interestedly to see the outcome....@ all others:

Very best regards and wishes, Wolfgang

Hello Mayank

Your question is very good as the carboxylic acid group in proteins is hard to access and difficult to alter.I agree with Erik Hinze idea that a metal radical

such as sodium or even more reactively and faster a fluorine combination

or slower a chlorine combination linked witha tetrahydrocarbon would give a

reactant which could penetrate the protein and cut the carboxylic link at the connection zone.However the difficulty is ensuring that the specific acid tail or linkage within the protein is obtained.To this end you have to know exactly the structure of the protein and how many and where the carboxylic linkages are.

To review - slower chlorinetetrahydrocarbon faster fluorine tetrahydrocarbon.

Let me know.

Shane

University of cambridge and Sci-Tech(South0 and CPFT.NHS.UK.

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