A recent paper in ChemBioChem co-authored by several groups describes their use of fluorous moieties to produce reversible inhibitors of glycosidases.  In this instance, the fluorous modified iminoalditols, sugars where the ring oxygen has been replaced by a nitrogen, which are known to be glycosidase inhibitors.  Shown above are some representative structures.  The authors write that alkyl substitution leading to lipophilic iminoalditols has already been shown to provide potent inhibitors of various glycosidases.  They hypothesized that by using highly hydrophobic perfluoro groups that they could achieve even better results.  With that in mind they synthesized eight different flourous modified iminoalditols, three of which are shown.  When appropriate, they reported using fluorous solid-phase extraction (FSPE) as a purification method for intermediates and products.  They then screened the compounds against various glycosidases and found that many of the fluorous iminoalditols had smaller Ki’s and greater selectivity than the corresponding unsubstituted iminoalditol.

Rather than go into detail on the assay results, I’d like to spend the rest of this post describing something that I found interesting.  As seen in the scheme below, one of the fluorous modified compounds they made started from hexafluoroisopropanol (HFIP).  Mitsunobu reaction with the protected aminohexanol shown actually provided a fluorous acetal from two Mitsunobu reactions in 82% yield rather than just the single substitution that they desired (Somewhat surprising). They used FSPE to purify the product (Interesting).  While the authors did not describe the exact FSPE wash conditions they used, it seems that the fluorous acetal is retained on the fluorous silica gel.  The stability of the acetal is seen in the last step where they are able to selectively hydrolyze the acetonide under acid conditions.  Not real surprising since cation formation at that carbon is going to more difficult.

One of the concerns of fluorous chemistry is the persistance and bioaccumulative properties of perfluorocarbon chains.  This has certainly dampened the prospects of fluorous chemistry as a green chemistry strategy as was originally envisioned.  What’s needed are alternative fluorous chains which are shorter and more easily degradeable, yet retain the “fluorousness” necessary to partition into fluorous environments.  So here we have a fluorous moiety which is relatively easy to obtain and presumably could be degraded through more stringent acid treatment.  Unfortunately, the MSDS of HFIP does not provide any information on the persistence, degradability, or bioaccumulative potential of HFIP.  Of course, even if those are non-issues there are other things that have to be sorted out.  For instance, how fluorous is the acetal?  Is it equivalent to a C6F13 chain?  How stable is it to a variety of reaction conditions?  I could see that proton on the end being a problem.  Can other related acetals be made?  All interesting questions which might deserve some investigation.

Does it really though?

There have been a few papers comparing fluorous methods to others in library production and they have validated the value of fluorous methods . Another way perhaps to judge is to look at FTI’s participation in the NIH Pilot-Scale Libraries (PSL) intiative.  Fluorous Technologies, Inc. (FTI) is one of only two industrial labs to have been awarded by NIH a Pilot-Scale Libraries grant.  The purpose of the grant is to augment the NIH’s compound collection, the Molecular Libraries Small Molecule Repository (MLSMR).  The MLSMR is part of NIH’s Molecular Library Program and is the source of compounds for another element, the Molecular Libraries Screening Center Network (MLSCN) which is tasked with developing assays and identifying small molecule probes which can be used to study various gene and cell functions to better understand disease states.

Over the last two and half years FTI has submitted over 1600 compounds into the MLSMR of which 1433 have been entered into PubChem.  All of these compounds are de novo compounds made by methodologies developed at FTI.  A PubChem search found that this was more compounds on a per annum basis than any other PSL grant awardee.  Out of the 30 or so awardees only two have submitted more compounds than FTI and both have had PSL grants for almost six years.   We’ve have never had a compound not accepted into the MLSMR due to purity or other issues.  So in terms of absolute number and purity of compounds FTI, and fluorous methodology, is doing pretty well.

Of course that’s only part of the story.  Anyone could make large number of compounds if they have little or no diversity, but that wouldn’t be of much value in finding new chemical probes for various assays.  Nor would libraries of molecules with very little chance of exhibiting biological activity.  Using PubChem’s search and structure clustering functions , I compared FTI’s submitted compounds with the group that submitted the most compounds over the lifetime of the PSL initiative, Prof. Stuart Schreiber’s group at the Broad Institute.

As seen in the table above, the numbers are actually quite similar.  The Broad Institute’s % of active compounds is somewhat higher, but that could just reflect the extra time which translates into more assays being run on their compounds.  What’s interesting is that the diversity as measured by compounds/similarity score cluster is almost identical at the 0.9 level; 46 vs. 47.  FTI’s diversity amongst the active compounds was actually little better, although how meaningful that difference may be is not clear.  At the 0.8 similarity score level (data not shown), however, the Broad Institute’s diversity was clearly higher than FTI’s for all compounds, but about the same for active compounds.

Purely based on number of compounds submitted, Prof. Schreiber’s group is one of the top performers amongst all PSL grant awardees.  He is probably the biggest proponent of diversity-oriented synthesis (DOS), so you would expect a high degree of diversity.  In addition, his labs are one of the best equipped in the world, far beyond FTI’s resources.  Yet, FTI has managed to deposit more compounds per year, achieve comparable activity rates and diversity.  There is the caveat that I have no idea of the budget or man-hours committed by the Broad Institute to the PSL grant, so perhaps the comparison isn’t valid.  On the face of it at least though, FTI has been able to produce almost as much in half the time.  Only through the use of fluorous techniques could we achieve this level of productivity with our resources.

PV reactions were first described in 2002 by Ryu and Curran.  Since that initial report, a wide variety of creative variations on the PV theme have been reported.  These include variations in the reagents, the number of phases, the fluorous solvents used, reaction apparatus, etc.  For example, while halogenations have been the most prevalent type of reaction, Simmons-Smith cyclopropanations, halolactonizations, and photochemical reactions have all been conducted in PV mode.  A recent short “Concept” paper from Dragojlovic in Chemistry – A European Journal describes many of the variations of PV reactions that have been developed.  Some of the more interesting ones include tetraphasic reactions, multi-reaction reaction vessels, and polytetrafluoroethylene (PTFE) screen reactions.

Prof. Ryu meanwhile has continued working in this area and has just published a photoirradiative PV reaction using bromine for the anti-Markovnikov addition of HBr to terminal alkenes through a radical addition process.   They found that the PV reaction worked quite well for monosubstituted terminal olefins providing 1-bormo-alkanes in >90% yield.  Just add this to the many different types of PV reactions.

In a recent ASAP paper in J. Am Chem. Soc. Prof Nicola Pohl and co-workers have used synthetic oligosaccharides produced using fluorous means to work in modulating immune response to pathogens.  Oligosaccharides are very common molecules on the surface of bacteria and viruses and are important in recognition and response by immune systems.  The researchers’ goal was to investigate the role of these oligosaccharides in eliciting an immune response.  Their approach was to produce what they call an “artificial pathogen”; something of similar size to a real pathogen with the appropriate oligosaccharides on the surface.  The oligosaccharide chosen was the trimannose molecule labeled 4.  This trisaccharide is the terminal cap for glycolipids in both Mycobacterium tuberculosis and Leishmania parasites.  The artificial pathogen would then be incubated with stimulated macrophages and the levels of cytokine ineterleukin-12 (IL-12) produced measured.  IL-12 is critical for an appropriate immune response.

The trisaccharide and a control lactososide were both prepared using fluorous methods previously developed within Prof. Pohl’s labs. Fluorous solid phase extraction (FSPE) purification is a prime component of this methodology.  The fluorous tag was removed and the sugars attached to 1 micron diameter latex beads to provide the pathogen mimics.  Incubation with the stimulated macrophages resulted in a dampened immune response as measured by IL-12 produced for macrophages exposed to the trimannose coated beads vs. the control lactososide beads.  The researchers, therefore, demonstrated that the carbohydrate cap alone can influence immune response providing a path to new immunomodulators or vaccines.

In the Fang paper a polymerizable acrylamide tag which is attached to the 5′-OH group through a silane is used.  Upon attachment to the final oligonucleotide and cleavage from the support, this tag is polymerized to form a new solid containing only the desired full sequence.  The capped sequences are then washed away.  The desired sequence can then be cleaved from the polymer with HF and the polymer filtered off.  Excess HF and silyl by-products can be removed during lyophilization.  The use of a polymerizable phase tag is certainly not new and has been used for various catalysts and reagents with ROMP based methods being particularly popular.

The method does have some nice advantages, since the purification is a simple filtration.  The disadvantage is, of course, you have to run one more reaction, the polymerization, which may or may not cause problems.  In this case, the authors tested that by subjecting each of the four nucleosides to the radical polymerization conditions and found no change.  In practice, they synthesized a 20-mer and reported 72% recovery of the tagged oligo based on UV absorbance at 260 nm.

So how does this compare with fluorous methods, specifically the use of F-DMT phosphoramidite as reported by Berry and Assoc. in 2005?  Well, to read this latest report, the polymerization approach is superior.  It leads to higher recoveries and doesn’t use expensive materials such as fluorous affinity columns or avidin-coated beads.  So the authors specifically tout the advantages of polymer-method over fluorous techniques.  As one might guess, I’m not as sure.

Let’s compare the Berry and Assoc. 2005 J. Org. Chem. paper using F-DMT with the Fang paper.  To be fair the Berry and Assoc. publication is a full paper while this latest on is a communication, so whatever criticisms I point out may very well be addressed in a forthcoming full paper.  The JOC paper synthesized a 30-mer, a 50-mer, a 75-mer, and a 100-mer multiple times.  Recoveries, also using absorbance at 260 nm ranged from 70-100% with even the 100-mer at quantitative recovery.  The Fang paper reported a single synthesis of a 20-mer with 72% recovery.  So not only were the recoveries higher, but the oligonucleotides produced more challenging in the fluorous paper. The longer oligos are an important distinction since as Fang mentions the polymerization could be hindered by steric bulkiness of the oligonucleotide.  In addition, Berry and Associates notes that their reported recoveries are ranges based on multiple runs rather than a single run of 72% recovery for Fang.

As for cost of materials, there is a cost for the F-DMT phosphoramidite and the FluoroPak cartridge for purification, but to lump it in with avidin-coated beads is not really fair.  Our own cost analysis revealed between fluorous methods and biotin based separations resulted in fluorous methods being 25-50x less expensive.  There is no way, fluorous methods can be grouped with biotin based methods in terms of cost.  So is this polymerization method less expensive than biotin-avidin based methods?  Probably.  Less then fluorous?  Unclear, especially when you consider that F-DMT seems to provide greater recovery.

There is no doubt some appeal to the simple polymerization method, but until some further validation it’s not so easy to say one method is superior.  If I had to guess, I’d say the longer the oligo, the better fluorous comes out.  Of course, the shorter the sequence the less need for any type of tagging strategy.  As pointed out in the Berry and Associates paper, regardless of the tag, any 5′-OH tagging method is going to suffer from certain shortcomings related to the synthesis and apart from the tag itself.

The bottom line is that there is probably room for polymerizable tags, fluorous tags, and other methods.  The best method will probably depend on the exact length and composition of the oligonucleotide.

Thank you for your understanding.

A new publication from Prof. Philippe Durand in J. Comb. Chem. describes a fluorous tagged approach to the parallel synthesis of these useful compounds.  The paper highlights some of the virtues of fluorous chemistry.  Of primary importance in this instance is the solution phase nature of the chemistry and the inertness of the tag.  This allowed the researchers to test and apply a number of conditions that failed in solid phase approaches.  Application of various Petrassi coupling conditions to a solid-supported N-hydroxyphthalimide failed as did the use of a ROMP based strategy. By combining the solution phase chemistry of a fluorous tag with the ease of fluorous solid phase extraction (FSPE), the authors were able to develop a general synthetic methodology amenable to parallel synthesis.

The route is shown above and uses modified Petrassi conditions for the coupling of the fluorous tagged N-hydroxyphthalimide with arylboronic acids.  Their initial efforts using standard Petrassi conditions afforded only modest yields (40-60%).  Optimization of the reaction conditions was conducted using a multi-variable design of experiments (DoE) based on Taguchi methods.  This allowed them to identify which variables, or combinations of variables, were most important.  This led to conditions which used 4 equivalents of the boronic acid,  Cu(OAc)2 as catalyst, and benzotrifluoride (BTF) as solvent.  Of course, using that much excess arylboronic acid also led to increased amounts of by-products and that’s where the fluorous tag comes in.  The researchers developed their own unique FSPE protocol involving three different fluorophobic washes to remove the various by-products followed by a single fluorophilic wash with acetone to isolate the arylated hydroxyphthalimide 3 in generally excellent yield and purity.  Aminolysis of 3 to provide the desired aryloxyamines was conducted using resin-bound amine.  The authors noted that they could perform the aminolysis with methylamine then collect the products using FSPE.  However, the volatility of some of the products made isolation from the fluorophobic wash difficult.  No problem.  The solution phase nature of the fluorous tag made the use of the resin-supported amine in a volatile solvent easy, once again showing how versatile the fluorous tag can be.  The authors went on to produce 25 different aryloxyamines.

An interesting communication which highlights some of the advantages and adaptability of fluorous tagged methods to meet the needs of a specific chemical scheme.

With this award, Prof. Pohl joins an ever expanding group of scientists who have been recognized for their work in fluorous chemistry.  Fluorous Technologies Inc. founder and University of Pittsburgh professor Dennis Curran was the recipient of the 2008 ACS Award for Creative Work in Fluorine Chemistry.  Prof. István Horváth was a 2006 Humboldt Research Award winner for his pioneering work in fluorous chemistry.  Our own Yimin Lu was honored in 2006 by the Organic Division of the ACS with a Technical Achievement Award.

Beyond those who have been specifically recognized for their work in fluorous chemistry, however, are the numerous award-winning scientists who have used fluorous methods in their research.  Prof. Peter Seeberger, the 2009 recipient of the ACS Carbohydrate Division’s Charles S. Hudson Award among many other accolades, has published several papers using fluorous methods in both carbohydrate and peptide chemistry.  Staying within the ACS Carbohydrate Division, Prof. Xuefei Huang was last year’s recipient of the New Investigator Award.  Prof. John Porco who has used fluorous reagents in several library syntheses was named an Arthur C. Cope Scholar last year, while Prof. Mukund Sibi who has utlized fluorous tags in his free radical research was bestowed that same honor in 2008.  Prof. Veronique Gouverneur was awarded the Royal Society of Chemistry’s 2008 Bader Award for her work in fluorine chemistry, which includes the use of fluorous tags and reagents.  A different Bader Award, this one for bioorganic or bioinorganic chemistry, was given to Prof. Stuart Schreiber in 2000.  Prof. Schreiber’s group has published one of preeminent papers on fluorous microarrays.

These are just a few recent examples of distinguished researchers that have used fluorous methods as part of their work.  Research that has been recognized and valued by peers.  We at Fluorous Technologies are genuinely honored to have played a role, no matter how small or large, in helping these and other scientists around the world advance their research.

Please be aware that this may not be a complete list of presentations using fluorous chemistry since the online search only identified those presentations with “fluorous” in the title or abstract.  If you are presenting at the meeting and would like your presentation to be added, please contact us and we can add it.   Also note that poster 96 will be available for viewing at two sessions, hence the double listing.

To read the abstracts, please click on the titles.

1.  96 – Synthesis of a fluorous tagged disaccharide for the enzymatic preparation of heparin oligosaccharides.
Authors: Sayaka Masuko, Smritilekha Bera, Robert J. Linhardt
Date/Time: Monday, August 23, 2010 - 08:00 PM
Session info: Sci-Mix (08:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Hall C

2. 96 – Synthesis of a fluorous tagged disaccharide for the enzymatic preparation of heparin oligosaccharides.
Authors: Sayaka Masuko, Smritilekha Bera, Robert J. Linhardt
Date/Time: Wednesday, August 25, 2010 - 08:00 PM
Session info: General Posters (08:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Hall C

3.  604 – Recyclable fluorous organocatalysts for asymmetric synthesis
Authors: Zijuan Zhang, Liang Wang, Hong Zeng, Wei Zhang
Date/Time: Tuesday, August 24, 2010 - 08:00 PM
Session info: Asymmetric Reactions and Syntheses, Biologically-   Related Molecules & Processes, Material, Devices & Switches and Peptides, Proteins & Amino Acids (08:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Hall C

4.  162 – Fluorous benzaldehyde based synthesis of heterocyclic systems
Authors: Asha Kadam, Shang Ding, Ryan Fitzgerald, Minhgiao Le-Nguyen, Wei Zhang
Date/Time: Sunday, August 22, 2010 - 08:00 PM
Session info: Heterocycles and Aromatics, Metal-Mediated Reactions & Syntheses, Molecular Recognition & Self-Assembly and Physical Organic Chemistry: Calculations, Mechanisms, Photochemistry & High-Energy Species (08:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Hall C

5.  983 – Fluorous benzaldehyde based multi-component reactions for heterocycles
Authors: Bruno Piqani, Shan Ding, Tao Xi, Wei Zhang
Date/Time: Wednesday, August 25, 2010 - 07:00 PM
Session info: Chemistry for Preventing & Combating Disease, New Reactions & Methodology and Total Synthesis of Complex Molecules (07:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Ballroom

6.  532 – Fluorous linker facilitated synthesis of heterocyclic systems
Author: Wei Zhang
Date/Time: Tuesday, August 24, 2010 - 02:20 PM
Session info: Heterocycles and Aromatics (01:00 PM – 05:20 PM)
Location: Boston Convention & Exposition Center, Room 203

7.  1083 – Fluorous mixture synthesis and spectroscopic analysis of the phytophthora mating hormone a1 and the corresponding bis-MTPA esters
Authors: Reena Bajpai, Dennis P. Curran
Date/Time: Wednesday, August 25, 2010 - 07:00 PM
Session info: Chemistry for Preventing & Combating Disease, New Reactions & Methodology and Total Synthesis of Complex Molecules (07:00 PM – 10:00 PM)
Location: Boston Convention & Exposition Center, Ballroom

8.  485 – Surface modification of poly(dimethylsiloxane) with perfluorinated alkoxysilanes: Zeta potential and the adsorption of perfluoro-tagged proteins and peptides
Author: Hugh Horton
Date/Time: Thursday, August 26, 2010 – 4:25 PM
Session info: Symposium in Honor of Richard Lambert (1:30-4:50 PM)
Location: Westin Boston Waterfront, Harbor Ballroom II

9.  89 – Di-fluorous tagging strategy for the automated solution-phase synthesis of plant oligosaccharides
Author: Sahana Nagappayya, Heather Edwards, Nicola Pohl
Date/Time: Sunday, August 22, 2010 – 2:00 PM
Session info: New Reactions and Methodology (1:00 – 5:20 PM)
Location: Boston Convention & Exhibition Center, Room 206A

10. – Arrays and automated oligosaccharide synthesis
Date/Time: Sunday, August 22, 2010 – 4:10 PM
Author:  Nicola Pohl
Session info: Wolfrom-Isbell-New Investigator Award Symposium (1:00 – 4:45 PM)
Location: Boston Convention & Exhibition Center, Room 251

11.  408 – Oxidation of olefins with hydrogen peroxide and polyoxometalates in ionic liquids and fluorinated alcohols as “magic solvents”
Authors: Mauro Carraro, Serena Berardi, Martino Gardan, Marcella Bonchio, Gianfranco Scorrano, Andrea Sartorel
Date/Time: Tudsday, Aogust 24, 2010 – 9:20 PM
Session info: Organometallic Chemistry (9:00 – 11:25 AM)
Location: Boston Convention & Exhibition Center, Room 257B

12. 919 – Tuning optical and electronic properties of arenebased organic semiconductors via perfluoroalkylation
Author: Haoran Sun, Michael Billion
Date/Time: Wednesday, August 25, 2010 – 5:00 PM
Session info: Material, Devices and Switches (1:00 – 5:25 PM)
Location: Boston Convention & Exhibition Center, Room 207

It doesn’t end there, however.  The fluorous domains on the dendrimers are obviously capable of complexing with each other.  The complexes formed in the presence of the dye self-assemble with each other to form micron-bubbles.  The average diameter of the bubbles could also be tuned.  When the complexes are formed the amount of complexation could be estimated by 19F NMR.  The more complexation observed, the smaller average diameter of the micron-bubbles.  They also propose the these micron-bubbles consist of air, a dendrimer layer and a dye shell since they found that degassing led to bubble collapse, but that the bubbles could be reformed by gassing.

This is another example of the use of fluorous partitioning in order to form supramolecular complexes.  I’ll call it micro-phase formation.  There’s plenty of examples of nanoparticle formation based on “fluorous interactions”.  One application using these nanoparticles was published last year for DNA detection that we commented on.  In that example, gold nanoparticles contained a DNA sequence complementary to a target sequence.  A second complementary sequence with a fluorous tag was introduced.  The nanoparticles with the target sequence thus became fluorous and aggregated causing a color change which indicated the presence of the target DNA sequence.