Enzyme-linked click chemistry assay for GOAT

cat-ELCCA, enzyme-linked click chemistry assay, was developed by Janda Group for high througput assay of GOAT, Ghrelin O-Acetyl transferase. The methodology is depicted in the drawing. The following chemistries are elegantly combined for the assay: biotin-straptavidin binding, CuAAC click chemistry, HRP catalysis, O-acetyltransferase catalysis, and fluorogenic reaction.

The method should accelerate identification of inhibitors for GOAT, which activates ghrelin by esterification with a C-8 fatty acid unit. It intimately relates to the effort of seeking agents that lower ghrelin levels as obesity or diabetes treatments.

Much like the cat-ELISA assays in immunology, cat-ELCCA, should find applications in other acyltransferases.

Angew. Chem. Int. Ed. 2010, 9630.
                                                     

Click chemistry meets Cysteine

Ah Cysteine!

How many things happen with it: enzyme activity function, posttranslational modification, crosslinking, redox chemistry, Glutathione, GST the phase II detoxiciation, oxidative stress relief, etc. Everything happens around this unique one amino acid due to its R-SH reactivity.

Benjamin Cravatt and his group at Scripps Research Institute reported a click chemistry approach to obtain a proteome-wide quantitative analysis of native cysteine reactivity, in particular, the identification of hyper-reactive cysteine sites.

The appraoch is termed isoTOP-ABPP (isotopic tandem orthogonal proteolysis–activity-based protein profiling) and shown in the graph. The identified hyper-reactive cysteines correspond well to those of functional and posttranslational modification sites.

Benjamin Cravatt is well known for ABPP and I listened to his talk in Atlanta, 2006. He is one of the pioneers applying click chemistry to chemical biology research. See JACS 2003, 4686. "Activity-Based Protein Profiling in Vivo Using a Copper(I)-Catalyzed Azide-Alkyne [3 + 2] Cycloaddition"

Wonderful walking in the cold air on a Holiday morning - as well as surveying click chemistry progresses with a cup of hot coffee!
                                 

Labeling of live cells via Copper-catalyzed click chemistry

Copper-free click chemistry developed by Bertozzi Group allowed in vivo labeling and imaging of glycans without copper toxicity. This was reported widely by many media and scientific magazines. A summary on this topic will be given elsewhere.
 
Now two reports appeared using Copper-catalyzed click chemistry for the same purpose. The key is the identification of good ligands for Copper (1+) ion which allow for rapid labeling and minimal toxicity.
 
Vu Hong, M. G. Finn et al, "Labeling Live Cells by Copper-Catalyzed Alkyne-Azide Click Chemistry", Bioconj. Chem. received 6/17/2010.
Rapidly labels mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine (Bertozzi) followed by CuAAC reaction with dye-alkyne  

David Soriano del Amo, Peng Wu et al, "Biocompatible Copper(I) Catalysts for in Vivo Imaging of Glycans", JACS received 7/23/2010.
Rapid labeling and imaging of fucosylated glycans during zebrafish early embryogenesis without apparent toxicity. Alkyne-bearing  GDP-fucose microinjected and CuAAC reaction with dye-azide. 
                                            

First Thanksgiving of Click Chemistry

It is this time of the year and Click Chemistry has seen its first Thanksgiving. We celebrate it and express great respect and gratitude to the readers and workers in the field of click chemistry.

It is not only useful and convinent chemistry, it is fun and exciting chemistry. Only the future will tell how widespread and unique a certain type of reaction (such as click chemistry) actually penetrates into many other branches of sciences. We are very positive because it is only the starting point of the endless possibilities. 

Happy Thanksgiving everyone! 
                                                        

BASF press release on DNA labeling via click chemistry

BASF press release 10/4/2010

Baseclick GmbH, a spin-off from BASF SE and Ludwig-Maximilians-Universität Munich, has signed licensing agreements with Integrated DNA Technology (IDT), Ella Biotech and metabion to provide click chemistry labeling of DNA. European Patent Office has granted the this patent to baseclick GmbH effective on 9/1/2010.

Based on Sharpless click chemistry and developed by Prof. Thomas Carell of Ludwig-Maximilians-Universität Munich, Germany, the baseclick technology allows labeling multiple dyes on DNA strands in a sequence-specific manner. Baseclick GmbH has obtained a worldwide license for click chemistry from The Scripps Research Institute.

Applications include analyses for pathogens and detection of mutations in human, animal and plant genomes. A related report is available "Notable oligonucleotide modification using click chemistry".
                 
                          

Stable Phosphohistidine Analogues by Click Chemistry

Protein phosphorylation has been a hot spot in both academic and industrial research for possible drug targets. Instability and isomerism of Phosphohistidine however presents great difficulties in such studies.


Now a stable version of PhosphoHistidine has been developed using click chemistry by Muir and coworkers from The Rockefeller UniVersity and Active Motif, Inc.
JACS 2010, 14327. Published on Web 9/29/2010.


See "Active Motif offers click chemistry reagents" Tuesday, October 5, 2010


The preparation uses the two regio-selective click chemistries, Cu1+ and Ru2+ catalyses, for two stable isomers. They succesfully used these stable analogues in SPPS (solid phase peptide synthesis) to synthesize peptides. Also succesful was the development of the first antibody specifically for pHis.


Two small, good-looking molecules out of click chemistry!




                                                                

Active Motif offers click chemistry reagents

Active Motif offers research kits, assays, and the TimeLogic® biocomputing systems. Now it has extended to click chemistry reagents such as Chromeo™ Dyes for bioorthogonal labeling.

Active Motif was founded in 1999 and is headquartered in Carlsbad, California, and has international offices in Tokyo, Japan and Brussels, Belgium.

A collection of vendors with similar offerings was compiled previously:
Click Chemistry: Reagent Vendors Matter (February 8, 2010)

                                         

M.G. Finn to head ACS Combinatorial Science

September 1, 2010

Laboratory address http://www.scripps.edu/chem/finn/index.shtml

M.G. Finn, Ph.D., of Scripps Research Institute, was appointed as Editor-in-Chief of "ACS Combinatorial Science", the former "Journal of Combinatorial Chemistry". The revision calls for its expansion to include biological and materials combinatorial and high throughput studies.

Finn’s research interests include traditional and combinatorial synthesis of biologically active compounds, traditional and combinatorial development of transition metal catalysis, and development of reactions for organic synthesis and materials science. He was part of the team who developed the first click chemistries, including copper-cataluzed triazole click chemistry.

Sticky mussels and click chemistry

Messersmith group contributed significantly in revealing the mechanism how mussels stick firmly to a variety of surfaces (Science 2007, 318, 426). Early 2010, this nature-inspired, dopamine-based polymerization was further developed by the same group into a surgical glue now in trials. 

Now Muller group married dopamine chemistry and click chemistry for the functionalization of Fe3O4 nanoparticles. The dopamine linker with an alkyne group allows for supersticky coating AND superb subsequent modifications via click chemistry. 

Macromolecular Rapid Communications, first published online: 8 JUL 2010

DOI: 10.1002/marc.201000193

Indeed the strategies can pave the way for a simple and universal functionalization for any kind of surface.
                     

Allozyne licenses Click Chemistry from Scripps

One of the busy phone rings on the desk of Scripps Tech Transfer Office?

On July 15, 2010 Allozyne Inc. announced the signing of a worldwide licensing agreement with The Scripps Research Institute. It provides Allozyne with a license to apply click chemistry for exclusive development in key therapeutic fields in addition to a non-exclusive license for diagnostic applications. Allozyne’s biociphering platforms, CAESAR and VIGENÈRE, enable the site-specific incorporation of these “Click” components into virtually any protein and thereby provide a unique site at which any number of payloads can be securely attached - polymers, small molecules, antibodies, toxins, additional proteins and peptides - for building therapeutics not previously possible.

Notably, among the Scientific Advisory Board of Allozyne, are Barry Sharpless and David A. Tirrell, both of whom are listed in this blog's Click Chemistry Links.

Click chemistry in protein fatty acylation and prenylation

Nature Chemical Biology, Published online June 17, 2010 

The above and many other click chemistry reagents were chemically synthesized, metabolically or enzymatically introduced into target proteins to effect extremely sensitive detection, by western blotting or in-gel fluorescence. The applications of these probes are: identifying new lipid-modified proteins, analyzing the lipid content of cellular proteins, imaging lipid-modified proteins in cells, and tools for protein labeling in vitro. 

The success lies on the qualities of click chemistry: reaction efficiency, functional group and reaction condition tolerance,  small size of azido and alkenyl groups, etc.   

         

Click chemistry for conjugated molecular wire

J. Am. Chem. Soc., Article ASAP, DOI: 10.1021/ja103239b
Publication Date (Web): June 15, 2010

The article demonstrates a beautiful example of click chemistry for the synthesis of long (>4nm) pi-conjugated wires (all-aromatic backbone structure) bonded to metal electrodes.

Elegantly designed, nicely executed, fully characterized, and convincingly presented.

Aromatic and rigid triazole structure from click chemistry is explored to deliver the unique property for the applications in materials science and nanotechnology.  

The measurement confirms the length-dependent conductance and mechanism transition.
                                

Carolyn Bertozzi awarded Lemelson-MIT Prize for work including click chemistry

http://www.mit.edu/press/2010/500k-lemelson.html

By MIT press release of June 2nd, 2010, Carolyn Bertozzi was selected for the prestigious 2010 $500,000 Lemelson-MIT Prize.

Her contribution in the field of biotechnology includes: Bioorthogonal reactions, or biocompatible click chemistry, or the copper-free version; Glycobiology innovations and imaging glycans; Genetically-encoded aldehyde tags; Cell nanoinjector.

The bioorthogonal click chemistry yielded in vivo imaging of sugar molecules in live animals. The aldehyde tag technology is the basis for the company Redwood Bioscience. 

Her response? "This came completely out of the sky - it's totally stunning news."  
               

Click chemistry in medical imaging

http://pubs.acs.org/toc/chreay/110/5
May 12, 2010, Chemical Reviews, 110 (5), Pages 2575-3298  
2010 Medical Imaging and Diagnostics special issue

This issue highlights the current status of medical imaging with two dozen review papers. What a role has click chemistry played in this specific field? An examination shows that seven of the articles cited click chemistry in a wide range of applications:

Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications;
Activatable Photosensitizers for Imaging and Therapy;
Technetium and Gallium Derived Radiopharmaceuticals: Comparing and Contrasting the Chemistry of Two Important Radiometals for the Molecular Imaging Era;  
Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics; 
Peptides and Peptide Hormones for Molecular Imaging and Disease Diagnosis;
Multimodality Imaging Probes: Design and Challenges;
Phage Display in Molecular Imaging and Diagnosis of Cancer

Click chemistry in most cases is used as an improvement or addition to the developing medical imaging field. Some are included in patents or patent applications; some are under clinical studies. 
                                 

Click chemistry with Big Pharma

There is the click chemistry report by Luke Timmerman from Xconomy (5/19/2010).

It tells the pharma's take on click chemistry and the phone rings on the desk of technology transfer leader of Scripps: Integrated Diagnostics, Life Technologies, Roche, Waltham, ImmunoGen, Seattle Genetics, and Millennium: The Takeda Oncology Company.

Click chemistry as a philosophy existed before a really wonderful reaction was in place. After the miracle reaction was discovered, there is still more convincing work to do. "We (chemists) are trained as most experts to do the hardest things and do them well", stated Dr. Finn, but "we 're going to find or create the easiest reactions". Giving some thoughts, one might just agree that "it's a lot harder to invent a process that works all the time, than it is to make the process that's really difficult work a few times".  

The goal is to obtain function or properties through structures. But our ability (or inability) is poor in predicting what structure would deliver a desired function. The solution is libraries of compounds and screening aided by some "designing". This is THE reason why easier reactions are needed and applied, by big pharm. 

What makes the report more fun is really the other things - scientists, personality, truthful thinking, straight talk, and a lot of character.

By Sharpless, the "sincere-but-absent-minded professor": “I’m sort of the old mad dog who doesn’t believe in a lot of stuff. I have a hard time with biotechnology and green chemistry. It’s hard for me.” 

More: “The point of chemistry is in the middle where everything gets connected. Function is what matters. To me, it wouldn’t matter if you can make malignancies regress with three different types of inorganic salts as long as they weren’t toxic."
   
Smile, or laugh.
                       

Click chemistry into undergraduate labs

I have to say it is somewhat disappointing to see the currrent status of click chemistry in undergraduate labs. One has yet to identify a laboratory in which click chemistry is in use for undergraduate organic chemistry experiment. A few publications in Journal of Chemical Education should give a good starting point.

Back in 2008 researchers from Scripps Institute and other colleges published a wonderful click chemistry paper (J. Chem. Edu. 2005, 1833). It was tailored and tested to be robust undergruduate procedures in click chemistry. It should be an excellent opportunity for touching on some uptodate organic chemistry. Click chemistry between azide and terminal acetylenes can be introduced with Diels-Alder reaction.

Another publication covers alternative reaction media of Diels-Alder reaction as green chemistry solutions (J. Chem. Edu. 2009, 488). Both solventless-room temperature and water-as-solvent conditions are close to click chemistry standards.

A third paper deals with "Synthesis of Imidazolium Room-Temperature Ionic Liquids" (J. Chem. Edu. 2009, 856). It intends to use the synthesis to demonstrate the concepts of green chemistry and click chemistry.  

We look forward to the change. 
               

Craig Hawker named to Royal Society for macromolecules work including click chemistry

May 20th, 2010, Craig Hawker  was elected to Britain's prestigious Royal Society. Currently the society includes more than 60 Nobel Laureates.

The right-side bar takes you to Dr. Hawker's laboratory page - he is the director of the Materials Research Lanboratory and a professor of chemistry, biochemistry and materials in USCB.

By the Royal Society: "Craig Jon Hawker is one of the world's foremost pioneers in the design and synthesis of functionalized macromolecules." His publication of dendrimer synthesis using click chemistry demonstrated a beautiful example of click chemistry application in extremely demanding context - one of the most influential publications from Sharpless group.

Peng Wu, Alina K. Feldman, Anne K. Nugent, Craig J. Hawker,* Arnulf Scheel, Brigitte Voit, Jeffrey Pyun, Jean M. J. Frechet, K. Barry Sharpless,* and Valery V. Fokin*, Efficiency and Fidelity in a Click-Chemistry Route to Triazole Dendrimers by the Copper(i)-Catalyzed Ligation of Azides and Alkynes, Angew. Chem. Int. Ed. 2004, 3928.
                            

Click chemistry and green chemistry

Click chemistry, in a broader sense, is about using easier reactions to make compounds for certain functions, drugs or materials or anything else. Let us say it is almost purely a scientific term. Green chemistry has been in place for long as a scientific term without much significane until reacent times when everything wants or needs to be "green". To be scientific, not fancy here, I try to connect and compare these two, using the "Twelve Principles of Green Chemistry", by Anastas and Warner. http://www.epa.gov/greenchemistry/pubs/principles.html

1.Prevention - It is better to prevent waste than to treat or clean up waste after it has been created.

Click chemistry can do a good job in here since the reaction is addition and high-yielding.
2.Atom Economy - Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.
Click chemistry is particularly good at this.
3.Less Hazardous Chemical Syntheses - Wherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment.
Azide and Copper(1+) do not fit the bill but there are other good reactions in click chemistry - and the reactions are growing.
4.Designing Safer Chemicals- Chemical products should be designed to effect their desired function while minimizing their toxicity.
EPA recently launched ToxRefDB (Toxicity Reference Database) but obviously this type of testing takes time. 
5.Safer Solvents and Auxiliaries - The use of auxiliary substances (e.g., solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.
Click chemistry is good for this, especially the tolerance of water as the solvent.
6.Design for Energy Efficiency - Energy requirements of chemical processes should be recognized for their environmental and economic impacts and should be minimized. If possible, synthetic methods should be conducted at ambient temperature and pressure.
Click chemistry is good here - a lot of reactions can be done without much heating.
7.Use of Renewable Feedstocks - A raw material or feedstock should be renewable rather than depleting whenever technically and economically practicable.
Yet to see the relevance.
8.Reduce Derivatives - Unnecessary derivatization (use of blocking groups, protection/ deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste.
Click chemistry wins big for its superior selectivity and tolerance of most functional groups.
9.Catalysis - Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
Click chemistry does well, either by chemical or light catalysts.
10.Design for Degradation - Chemical products should be designed so that at the end of their function they break down into innocuous degradation products and do not persist in the environment.
Yet to establish the relevance.
11.Real-time analysis for Pollution Prevention - Analytical methodologies need to be further developed to allow for real-time, in-process monitoring and control prior to the formation of hazardous substances.
Yet to establish the relevance.
12.Inherently Safer Chemistry for Accident Prevention - Substances and the form of a substance used in a chemical process should be chosen to minimize the potential for chemical accidents, including releases, explosions, and fires.
Azides do not play well but there are other types of click reactions such as ene - thiol etc.

Well, green chemistry right now is as big as one can imagine, although many doubts exist among chemists. Let us wait and see.
             

Notable oligonucleotide modification using click chemistry

Glen Research, the oligo company, has made some notable claims, finally. In the recent "The Glen Report" (Vol 22, Nnumber 1, May 2010), the company announced its move towards oligonucleotide modification using click chemistry. 

It mentioned the earlier concerns and reluctnace and today's final launch in collaboration with the German company Baseclick whose IP is based on the technology developed by Thomas Carell et al in University of Munich (see the right-side bar?). 

This move is based on the maturation of the nucleic acid click chemistry and the availabilty of necessary reagents, such as alkyne phosphoramidites and catalyst solutions.  

Some noteworthy claims are made: " we now enthusiastically endorse click chemistry. We also expect that it will rapidly become the premiere mothod for oligonucleotide conjugation."

Let us see how soon it will knock out the common post-synthetic labeling methods like amine - NHS ester or thio - iodoacetamide / maleimide. 
            
                               

Click chemistry in journal special issues

The purpose is to provide a list of journal special issues on the topic of click chemistry, with links following the titles. They are invaluable resources where a lof of information about click chemistry is compiled in one place. Hope it helps both active practitioners and beginners.

Macromolecular Rapid Communications, 2008, Vol 29, Issue 12-13, special issue: click chemistry in polymer science, issue edited by WH Binder
http://www3.interscience.wiley.com/journal/120736131/non-graphissue
Australian Journal of Chemistry, 2007, Vol 60, Nomber 6, research front: click chemistry
http://www.publish.csiro.au/issue/3737.htm
Chemical Society Reviews, 2010, 39, 1223, highlights the latest applications of click chemistry, guest edited by Finn nand Fokin
http://www.rsc.org/Publishing/Journals/CS/Article.asp?Type=Issue&JournalCode=CS&Issue=4&Volume=39&SubYear=2010
QSAR & Combinatorial Science, 2007, Vol 26, Issue 11-12, special issue: click chemistry
http://www3.interscience.wiley.com/journal/117356113/issue
Molecules, 2010, special issue click chemistry, in development
http://www.mdpi.com/journal/molecules/special_issues/click-chemistry/
Polymers, 2010, special issue click chemistry in polymer science, in development
http://www.mdpi.com/journal/polymers/special_issues/polymers-click-chemistry/

Enjoy!
                  

Click chemistry in automated synthsis of 96-product library with solid-supported catalyst

First published on April 28, 2010

Girard et al, Molecules, doi:10.3390/molecules15053087

After a 2006 report (Org. Lett. 8, 1689) on a solid phase supported Copper (1+) catalyst for alkyne-azide click reaction ), this article showcases the use of the catalyst system in an automated synthesis of a 96 product-sized triazole library.

Several interesting things. First, they use the automated chemical synthesis station for high-throughput (ChemSpeed ASW-2000). Second, HNMR monitoring of the click reaction was an elegant kinetics measurement. Third, the stability and recycled use of the catalyst are impressive – 5 cycles over 5 days!

At least two questions can be asked. First, Cu(1+) leaching may still be a problem in certain applications / systems / solvents. Second, the resin swelling was not considered in the solvent effect for the click reaction.
An ideal solid-supported catalyst needs have the following: Cu(1+) should be chelated in a kinetically / thermodynamically stable complex for zero leaching yet highly reactive in catalysis; the immobilization should allow reaction components to access the catalyst easily; the resin should be insensitive to solvent choice, preferably including water.
A long way to go!
                          

Click chemistry reagents offerings from Novabiochem / EMD

Novabiochem Letters 2010, No. 1/10

The peptide giant, Novabiochem Brand from EMD, opened its click chemistry reagents offerings in peptide synthesis proucts lines. Newly availabe are a range of suitably protected amino acids bearing azide or terminal alkyne functionalities, in addition to some intermediates bearing these functional groups. They are designed for click chemistry ligations which possess the following features and benefits:
*High yielding ligation in aqueous media
*Orthogonal to standard methods of ligation
*Building blocks compatible with standard Fmoc SPPS methods

Notably, one caution was given that thiols lead to azide reduction.

Previously a list of click reagents vendors was given -
http://clickchemistry.blogspot.com/2010/02/click-chemistry-reagent-vendors-matter.html

This list is expected to grow - and that is only good news to our click chemistry users!
                                                                 

Click chemistry blog listed in Nature.com Blog

April 26th, 2010.

Click chemistry blog is listed now in Nature.com Blog.

Sincere thanks to the Nature.com Bloggers for the evaluation.  

And thanks to the readers - you are the purpose.

Hope the blog would be a good service to the community.

Believe me, click chemistry has a lot more to offer.
                      

Click chemistry between Nitrile N-Oxide and alkyne for polyrotaxanes

Macromolecules, DOI: 10.1021/ma100262g First published: April 2, 2010 Toshikazu Takata et al.

Click chemistry is more than alkyne-azide reaction - this article demonstrates a click chemistry using unstable and stable homoditopic nitrile N-oxides. Nitrile N-oxide was generated in situ through the reaction of the corresponding hydroxamoyl chloride with 4-Angstrom molecular sieves and was subjected to click reaction with alkynes. A kinetically stabilized homoditopic nitrile N-oxide was also developed for the same purpose. 

Polyrotaxane, a new class of polymer characterized by the mechanical linkage of its components, shows unique physical, chemical, mechanical, and rheological properties. Preparation of these sophisticated supramolecular systems requires powerful, highly reliable, and selective reactions. The above click chemistry is applied for the preparation of main-chain-type polyrotaxanes. The rotaxanation - polymerization by this click chemistry provides remarkable advantage of avoidance of catalyst contamination which exists in alkyne-azide click chemistry.

41683db89073a95b33ef8372d9df0f2c              

Click chemistry assembled HNO sensor in live cells

Joel Rosenthal, Stephen J Lippard
J. Am. Chem. Soc. 
DOI: 10.1021/ja909148v
First published: March 31, 2010

Unlike nitric oxide (NO) which earned prominent position in biological and medical world, other RNS (reactive nitrogen species) received less attention - least in the case of nitroxyl (HNO). However, HNO displays important biological roles with potential pharmacological applications related to K+ channels in mammalian vascular systems. A biologically compatible probe fo HNO would help the current status significantly. 

The authors came up with the Cu(2+)-complex shown in the figure. BODIPY dye - azide and tridentate amine - alkyne undergoes the click chemistry to form the triazole linkage which holds the sensing fluorophore and chelating metal complex in close proximity. One nitrogen atom from the triazole ring participates in the chelation of Cu(2+) as well. The molecules was characterizaed by ESI MS, titration, and spectroscopy.

HNO, when present in aqueous buffer or live HeLa cells, reduces Cu2+ to Cu1+ (EPR evidence and others) and fluorescence rises significantly at 526nm (4-fold) by removing Cu2+ quenching of BODIPY dye. Controls demonstrate that the other RNSs do not interfere: NO3(3-), NO2(1-), ONO2(1-), OCl(1-), H2O2, NO, SNOC. 

Interesting case where click chemistry and triazole moiety are ued in fluorogenic detection, fluorophore - quencher linker, metal-chelation, probing molecules of biological or pharmocological significance, etc.                           
                      

Post-assembly click chemistry in DNA origami structure

Kurt Vesterager Gothelf et al, Nature Nanotechnology, 2010, Vol 5, 200. First published online on February 28th.

Seeman and others have turned DNA nanostructures or DNA origami into a fascinating science with extraordinary structural features and with developing applications and potentials in many fields. This publication demonstrates that chemical reactions with single molecules can be performed and imaged at a local position on a DNA origami scaffold by atomic force microscopy via direct detection of Streptavidin anchored onto the nanostructure by biotins.


The biotins are incorporated into the DNA origami via acetylene-azide click chemistry and others. The high yields and chemoselectivities achieved in these reactions demonstrate the feasibility of post-assembly chemical modification of DNA nanostructures. This report opens the door for many applications in biotechnology and materials science since the design and formation of DNA origami is getting mature enough and now post-assembly reactions allow workers to reliably conjugate / insert the molecules of interest in a spatially controlled manner.

One of the many good uses of Huisgen–Sharpless–Meldal copper(I)-catalysed click chemistry. 
        

Click chemistry book review - 1,3-dipolar cycloaddition reactions and unplanned click chemistry implications

Padwa & Pearson
Synthetic Applications of 1,3-Dipolar Cycloaddition Chemistry Toward Heterocycles and Natural Products (Chemistry of Heterocyclic Compounds: a Series of Monographs)

Click chemistry is soaring and the most prominent ones are cycloaditions such as Diels-Alder and alkyne-azide reactions which often meet the stringent requirements for click reactions. In this background it is interesting to read this 1,3-dipolar cycloaddition book which was finished right before the birth of click chemistry concept.

In 940 pages, the many types of 1,3-dipolar cycloaddition reactions (1984-2000) were covered, with emphasis on the applications in real organic synthesis, either natural products or otherwise. Two other features are well covered as well: the transformation of cycloaddition products into other forms of products and stereochemistry of these cycloaddition reactions.

There might be an unplanned rewarding outcome - we are almost certain that some of reactions described here will be brought in for click chemistry candidates.
                                       

Click chemistry book review - Cumulenes in Click Reactions

By an industrial chemist, this book has collected, cataloged, classified and summarized many types of click reactions under the umbrella of cumulene click chemistry. The author, being a true expert monitoring the field since 60s, has extensively covered the reactions in a very balanced manner, by describing the main features of the reactions and giving carefully selected examples. They all point to original journal publications for further reading. The book clearly demonstrates how vast the field is and how many reactions can potentially be regarded and / or developed into true click chemistry. Although the author states no intention of exclusiveness, it is quite exclusive in my opinion. And it is updated as well - even if we admit that the click chemistry field grows extremely fast these days.

Click chemistry highlighted in Chemical Society Reviews Special Issue

Chem. Soc. Rev., 2010, DOI: 10.1039/c003740k

I would say it is a feast - a colletion of the most representative pieces of click chemistry. It  is certainly a must-read for both active practitioners and the ones who are looking to step in.  

Edited by Drs Finn and Fokin and writen by leading groups, this special issue highlights the most recent status of click chemistry and its applications in several major fields. By doing so it effectively reinforces the importance of click chemistry concept. 

The three fields are covered: biomolecules, materials, and different environments. In each of them click chemistry has been demonstrated to be a powerful and growing tool. As the editors pointed out (again): "the more tolerant the connection reaction between molecular building blocks, the more diverse the blocks that can be brought to bear on any problem, and the more likely it will be that solutions to the problem can be found and produced in quantity. If indeed it is useful function that is the goal, then keeping chemistry simple is a useful rule to remember."

"In this deliberate eschewing of more subtle synthetic techniques is an implicit challenge to organic chemists that make complex structures by complex methods (can complex structures be made by simple methods?)......." Quite a challenge to say the least.

Click chemistry: function follows form.   
                       

Click chemistry builds molecular library for antituberculosis agents

The authors identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. They demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics.

This is another good example that a small library, 80 in this case, built by click chemistry, yields an inhibitor with IC50 at 1.26 uM in a single selection round. Notably, the products were collected by simple centrifugation after 48 hr reaction, assessed by liquid chromatography–mass spectrometry to be at least 70–100% pure and used directly for screening without further purification.

Also, the effective strategy is used again here for the acquisition of active site-directed, potent, and selective PTP inhibitors  - tethering a nonhydrolyzable pTyr mimetic to an appropriately functionalized moiety in order to engage both the active site and a unique nearby subpocket. This strategy of utilising click chemistry has been reported a few times and proves to be productive.

PNAS, 2010, 4573.
                

BARAC - yet another strain-promoted copper-free click chemistry

Believe it or not, this one is called BARAC. 

Click chemistry is certainly not "done". This time, instead of electronic properties, Bertozzi group looked at the strain energy by introducing some level of double bond nature into the octyne ring structure. The choice was amide bond which displays well-known partial double bond nature (C=N). "Brushing against the line between stability and reactivity without crossing it", this is another beautiful example of "rational design" in physical organic chemistry. "Gratifying", The click reaction rate was superior, 12-fold faster than DIFO the difluoro-version. The group then evaluated BARAC derivatives in their "traditional" cell glycobiology imaging where cell surface carries glycan azide. It demonstrates superior performance than DIFO and DIBO. Modular and scalable synthesis of BARAC offers more opportunities and wider applications of this new copper-free click chemistry. 

Champagne finished, time to work on the next click chemistry.

Journal of the American Chemical Society, 2010, DOI 10.1021/ja100014q

Click chemistry patent - cyclopeptide imaging agents

US 7666392 B2

A good example of the application of click chemistry in biomedicine and diagnostics.

This patent by Hartmuth Kolb et al, was issued on February 23, 2010.
The title reads as "Click chemistry derived cyclopeptide derivatives as imaging agents for integrins". It described radiolabeled cyclic peptides and their use in imaging, such as Positron Emitting Tomography (PET) or Single Photon Emission Computed Tomagraphy (SPECT). The graph shows one example where two units of cyclic peptides, via a PEG linker, are conjugated to the radiolabel by triazole click chemistry. It is obviously advantageous to introduce the radiolabels by the "near-perfect" click chemistry.

Note Dr. Kolb is one of the authors of the influential article "Diverse Chemical Function from a Few Good Reactions".
Kolb, Finn, Sharpless, Angew. Chem. Int. Ed. 2001, 40(11), 2004
                      

Thiol-yne click chemistry

Sounds familiar? "A perusal of the literature from 1940s to 1960s" prompted this new version of click chemistry.

As indicated in the scheme, the double thiol-addition happens in two consecutive steps with the second 3-times faster than the first. The reaction can be driven either by light or radical initiators. Not only it complements established click chemistries such as alkyne-azide and thiol-ene, but also it provides a new feature which enables installation of more functional groups at once. This click chemistry was demonstrated with a few recent examples, either individually or in combination with other click chemitries. It is best suited for construction of dendrimers, networked structures, polymers, and other functional materials. I would expect it to find applications in biotechnology, nanotechnology, surface chemistry,  etc. 

Andrew B. Lowe et al, J. Mater. Chem., 2010, DOI: 10.1039/b917102a
      

Bioorthogonal click chemistry - Carolyn Bertozzi the champion

Chem. Soc. Rev., 2010 DOI: 10.1039/b901970g

Carolyn Bertozzi et al clearly defined the bioorthognal click chemistry. They used this figure to illustrate a general bioorthogonal reaction. Best described: "we must first look at an organic chemistry textbook and remove any reaction that is sensitive to water. Second, with an abundant supply of thiols and amines in the cell, we must also remove reagents that are prone to nucleophilic attack. Third, because of the reducing environment in the cytosol, we have to remove reactions that are sensitive to redox chemistry. If a reaction requires heat (above 37 °C), pressure, or high concentrations to work then it is also unacceptable. Lastly, some functionalities can be digested by cellular enzymes that have an ase in their name (e.g., esterase, phosphatase, sulfatase, etc.). Add reagent toxicity to this list and you will find a select few reactions that remain viable for performance in living systems."

Indeed one needs to be very picky in picking a good bioothogonal reaction from the already- small click chemistry tool box. The review covers the most viable reactions up-to-date, lists their pros and cons, and potentials. The chemistry aspects are detailed, with great insights and perspectives from a mechanistic point of view.

And did I mention the secret - go back to the old dusty organic chemistry toolbox and find treasures, such as click chemistry?
                      

Definition of click chemistry: a reminder

Yes, click chemistry is hot - so hot that it becomes a difficult task to track the huge body of "click chemistry" publications. But let us see how many publication have somewhat misused the term "click chemistry". Some of those uses are unnecessary, to say the least. BTW, How many times have I used "click chemistry" in this short paragraph?

Let us look at the definition of "click chemistry" by Sharpless et al in their paper (Angew Chem. Int. Ed. 2001, 40, 2004):  

"We have termed the foundation of this approach "click chemistry," and have defined a set of stringent criteria that a process must meet to be useful in this context. The reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation. Purification - if required - must be by nonchromatographic methods, such as crystallization or distillation, and the product must be stable under physiological conditions."

So this is the reminder for proper use of the term "click chemistry".
                    
           

Click Chemitry patent applications

    
I did a USPTO patent application search (2/12/2010) and here is the result with "click chemistry" in the title:
1 20090306310 Method of using click chemistry to functionalize dendrimers
2 20090247651 Bioadhesive Composition Formed Using Click Chemistry
3 20090214755 CLICK CHEMISTRY SURFACE FUNCTIONALIZATION FOR RESONANT MICRO-CAVITY SENSORS
4 20090123372 DEVELOPMENT OF MOLECULAR IMAGING PROBES FOR CARBONIC ANHYDRASE-IX USING CLICK CHEMISTRY
5 20090069561 CLICK CHEMISTRY ROUTE TO TRIAZOLE DENDRIMERS
6 20090054619 Functionalization of polyglycolides by "click" chemistry
7 20080311412 Polymeric Materials Via Click Chemistry
8 20080213175 Click chemistry-derived cyclic peptidomimetics as integrin markers
9 20080170992 Click chemistry-derived cyclopeptide derivatives as imaging agents for integrins
10 20060269942 In situ click chemistry method for screening high affinity molecular imaging probes
11 20060263293 Click chemistry method for synthesizing molecular imaging probes

It would be interesting to keep an eye on the following - which ones will be finally issued and what the implications will be in regard to the original Sharpless patent.

The list can clearly tell us the scope click chemistry has extended into: dendrimer, functional materials, surface, imaging, polymer, biomolecules, biomedicine, etc.

Sharpless patent claims and click chemistry

This patent by Sharpless et al was issued on May 20, 2008, titled "Copper-catalysed ligation of azides and acetylenes ". It claims:

1. A Cu(I) catalyzed process for preparing a 1,4-disubstituted 1,2,3-triazole comprising: contacting an organic azide and a terminal alkyne with a catalytic quantity of Cu(I) ion for a time sufficient to form by cycloaddition a 1,4-disubstituted 1,2,3-triazole.
2. The process in accordance with claim 1 wherein the cycloaddition is carried out at ambient temperature.
3. The process in accordance with claim 1 wherein equimolar amounts of the organic azide and the terminal alkyne are contacted with a catalytic quantity of the Cu(I) ion.
4. The process in accordance with claim 1 wherein said catalytic quantity of the Cu(I) ion is generated in situ by reduction of Cu(II) to Cu(I) with a reducing agent.
5. The process in accordance with claim 4 wherein the reducing agent is sodium ascorbate.
6. The process in accordance with claim 4 wherein Cu(II) sulfate pentahydrate is reduced by sodium ascorbate.

It appears the claim is the Cu(+) catalysed reaction ITSELF. Already having some impact on biotechnology and materials science, evidenced by licensing and product launches, it is expected to have a larger influence on more industries such as medicinal chemistry, nanotechnology, biomedical science, etc.

Click Chemistry: some working experience

  
Click chemistry is not equal to Cu(+) catalyzed triazole formation although literature centers on it. At least two types of Cu-free exist, which comforts many workers in toxicity concerns. Another click reaction, ene-thiol reaction, may find the best practical use in industry.

Running a click reaction is not as easy as a piece of cake. Actually one may need quite some tweaking in starting trials, particularly in catalyst and solvent selection. There are very good reviews detailing all these aspects. Once you get it working for you, click chemistry can be very rewarding and productive.

In early days, one had to make all the reagents themselves before coming to the final "click". No doubt it did not help those who were not familiar with organic synthesis but could potentially be interested and would not mind in "clicking". Last several years has seen increasing reagent offering from a wide range of vendors, both domestic and international. One would see more and more reagents available in "clickable" forms: biotin, fluorophore, quenchers, amino acids, carbohydrate, PEG, nucleosides for biotechnology, and many precursors, dendrons, intermediates for material science. There are even more for thiols and terminal alkenes.

738P8M3BA82F

Click Chemistry: Reagent Vendors Matter

To some extent, the availability of reagents will decide how far the click chemistry can reach into the non-chemist community. Initially many efforts of using click chemistry would have to prepare many intermediates or starting materials, sometimes in multiple steps, before that beautiful “click” – by chemists.

In biotechnology and biomedical world, all kinds of “kits” are right there, bringing in the ultimate convenience and reliability for the end users, much like “plug-and-play” computer parts. This is the way to go – and chemists should make it happen before the biotechnology community turns away.

I have compiled some click chemistry vendors – the list will likely be updated from time to time.

Conventional organic chemical vendors: look for azides and acetylenes, thiols and alkenes, and other pairs

With click chemistry emphasis or specialization:
Aldrich
Invitrogen
Jena Biosciences
Berry Associates
Glen research
Anaspec
Creative PEGWorks
Quanta Biodesign
Primetech
GFS
Enamine
Lumiprobe
Click Chemistry Tools
Base Click
IBA BioTAGnology

Shall we do some click chemistry?

Book Review - Click Chemistry for Biotechnology and Materials Science by Joerg Lahann

Click chemistry as a concept has been received well enough in terms of ever-increasing publications. Applications mostly point to material sciences and biotechnology where beautiful examples were demonstrated (the topics of the book). On industrial level click chemistry has yet to offer anything practically important. Neither has it afforded much in pharmaceutical world which was initially targeted. Some small libraries and screening work have been published in search of enzyme inhibitors using click chemistry. It is possible that there is large body of work not disclosed by industry for IP reasons.

These should not deny the concept and some very good reactions hard to come by in organic chemists' eyes. Like anything else, there has to be a period in which theories, examples, commercial reagents are getting ready in place - and workers would doubt, hesitate, then start to try it out, overcome some difficulties, and finally like and adopt it. This will take much longer for intended biotechnology scientists than for professional chemists. The book comes in a very timely fashion and may help to significantly shorten this period.

The 411-page book has the traditional "Wiley-quality". Frontier research groups describe their own work in 16 loosely connected chapters and give a broad picture of "current" status of the application of click chemistry in two fields: biotechnology and material science. Expect 16 reviews or accounts in very recent time - perfect for one to survey and enter the field. Readers may find it useful in two ways. They may adopt the applications directly in their closely related work. Or they may be excited by the elegance of the chemistry and adopt the chemistries in their own fields. After all, it is a very, very good tool. Some readers may turn out to be interested in certain chapters, not the others, largely due to the nature of the chemistry as a tool and therefore the large scope it can be used. The index appears very thin and may be hard to use. The literature does not cover the patent world.

One would realize how fast the field is developing if I say that many significant progresses have already taken place since April 2009, the Preface date by the Editor Joerg Lahann. It is necessary to follow the most current publications in major journals to keep up with the field.