Visual Monitoring of Fluorous Solid Phase Extraction (FSPE)

Posted on February 27, 2012 by Marv

The fluorous solid-phase extraction (FSPE) process is a very robust and  general process.  It’s utility and applicability have been demonstrated over a range of substrates and molecular classes.  One of the great things is the ease of operation.  One wash to remove non-fluorous components and then a wash to elute the fluorous portion.  A great demonstration of this is the fluorous dye experiment.  FTI has made a mixture of two dyes, a blue non-fluorous dye and an orange fluorous tagged dye, which are easily separated by FSPE as seen below.  It’s a great little demonstration.  However, when you’re making a library or fluorous tagging a substrate, your molecules of interest are not generally visible.  In that case you’re more or less trusting that the FSPE will work as advertised.  Thankfully, in the vast majority of cases that trust is well-founded and the FSPE will perform as advertised as evidenced by the number of papers that have been published by independent labs all over the world.  Sometimes, though, there are cases where the fluorous components are a little harder to get elute, primarily highly basic materials which can interact with some of the free silanols on fluorous silica gel.  Using an acidic or basic buffer usually solves that problem easily enough, but now you’ve gone off the reservation a little and it would be nice to be able to see what was going on.

Dr. Christopher Blackburn at Millennium Parmaceuticals has just published a Technology Note in ACS Combinatorial Chemistry to address this issue. Essentially he made a tag, pictured above, which contains three principal components; a) a fluorous domain, b) an azo dye, and c) a reactive functional group, in this instance an aldehyde.  The concept for the molecule is quite similar to what Lo reported using fluorous rhenium complexes although the application is different.  He was then able to tag amines through a reductive amination and purify the tagged compound by FSPE.  Due to presence of the dye, he was also able to follow the FSPE process visually.    He then demonstrated the utility of the tag in two applications; one a solution phase peptide synthesis and the other in sulfonamide synthesis.  The tag was then easily cleaved using TFA to provide the desired products after FSPE.

At FTI, we actually have received more than one request for a visual fluorous tag such as this one, but we’ve never developed one.  One of the reasons is that to develop a whole suite of visible tags which would encompass all the possible ones that people would want was always considered a daunting task.  Another reason is that the FSPE is really quite a robust process so how much value would it really add?  A tough question to answer.  Finally, the azo tag does put some additional constraints on the fluorous tag.  Dr. Blackburn notes that unlike most fluorous tag, the added dye makes the compound insoluble in MeOH, CH2Cl2, and CH3CN.  It took some heating in THF to get it into solution.  The dye portion also adds some functionality, and therefore reactivity, that limits some of the chemical compatibility.  These issues certainly takes away from some of the appeal of fluorous chemistry.

Even with the caveats above, a visible fluorous tag could certainly have it’s place in certain applications and it’ll be interesting to see what the response to the Note is.

 

 

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Isotope-coded Fluorous PALs

Posted on February 21, 2012 by Marv

Prof. Qisheng Zhang at UNC-Chapel Hill has published several papers about the synthesis and use of fluorous tagged photoaffinity labels (PALs) and has just published another report in Chemical Communications.  The general strategy is to attach a photosensitive reactive group on a probe small molecule, incubate it with the biological sample of interest, and then expose the mixture to light.  Whatever proteins or other molecules that the probe is bound to will then form an irreversible covalent bond through the photo-generated reactive group.  The classic photosensitive functionality if a diazirine which upon irradiation decomposes to a reactive carbene which will usually then react with whatever bond is nearby.  By using a fluorous tagged diazirine, Prof. Zhang has been able to then enrich samples for the fluorous tagged compounds through fluorous solid phase extraction (FSPE).  Tagging through PALs followed by enrichment is an often used approach with the enrichment being performed primarily through affinity tags such as biotin or His-tags.  Using fluorous enrichment, however, confers all of the usual advantages such as low non-selective binding, ease of elution, and excellent MS characteristics.

Besides PALs and other cross-linking strategies, another popular strategy in proteomics is isotope-coding in order to be able to directly compare different samples.  For example, if you wanted to compare protein levels in healthy cells vs. diseased cells, you could add the appropriate protein labeling agent to the healthy cells and the same protein labeling agent, except with some deuteriums of 13C’s in it, to the diseased cells.  Mix the two labeled samples together in equal amounts then compare the ratio of labeled proteins by MS.  Most of the time, you’d see a 1:1 ratio for the proteins, but every now and then you’d see a protein overexpressed or underexpressed in the disease cell state vs the healthy state.  This would then indicate that that protein may be important in the disease process.

In his group’s latest report, Prof. Zhang combines these two strategies by using a pair of isotope-encoded fluorous PALs.  As seen in the figure above, the difference in the two PALs is proton vs. deuterium substitution on the aromatic ring.  In practice, the authors used not the benzyl alcohol, but the NHS benzyl carbonate (not shown here) to first label a small peptide (RKRSRAE) through the side chain amine of the lysine. They took an equimolar ratio of the two in MeOH and irradiated the sample and found a near 1:1 ratio of the O-H bond insertion product of the carbene with MeOH.  This demonstrated that the isotope-encoded PAL’s behaved similarly and thus suitable for their intended use.

Next the authors looked at the effectiveness of the FSPE separation.  They took a 1:1 and a 2:1 mixture of the isotope-encoded insertion products from above and added them to a BSA tryptic digest.  As can be seen the amount of fluorous labeled peptides before FSPE is quite small in comparison to the BSA peptides.  The difference after FSPE, however, is quite dramatic and demonstrates the power of the FSPE in the sample enrichment.  They also found that the original isotope ratio was largely maintained, so no preferential enrichment of the deuterated over the protonated labels.

Next step:  a real mixture looking for some real answers.

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Using Fluorous Tags to Mass Differentiate Stereospecific Reactions

Posted on February 14, 2012 by Marv

Trent Northen and co-workers have been using fluorous immobilization in conjunction with nanostructure initiated mass spec (NIMS) in a number of different applications.  Dr. Northen was one of the inventors of NIMS while working in Gary Siuzdak’s labs at The Scripps Research Institute.  NIMS uses a fluorous siloxane as a initiator and is analogous to MALDI.  Nimzyme is what they dubbed the process by which a fluorous tagged substrate is immobilized on the fluorous siloxane, undergoes a reaction on the surface, and the products then analyzed by MS.  In all the previous cases that have been published the probe molecules have all been tagged with the same fluorous tag, so different substrates lead to different products that are recognized by their mass differences.

But what do you do if your different substrates all lead to the same product?  How then do you differentiate between them? You use a different fluorous tag for each substrate, of course.  Now the mass differentiation comes not from the substrate, but from the fluorous tag which encodes for each substrate and that’s exactly what Northen et al have done in their latest publication in Rapid Communications in Mass Spectrometry.  They encoded three different disaccharides, maltose (S1), lactose (S2), and cellobiose (S3) with a unique, and mass differentiated, fluorous tag as seen below.  Note that the mass (427.2) of the disaccharide portion is the same for each substrate and that they only differ in stereochemistry, but that after tagging each has a unique mass.  Without the fluorous tag there would not be a simple way to differentiate between these species by MS.

The authors took their mixture of substrates and incubated it with three different enzymes and a mixture of all three.  An aliquot of the reaction was then spotted on a NIMS surface with the fluorous tagged compounds being retained.  As expected, each enzyme reacted stereospecifically with only one of the substrates; a substrate that was easily identified by its unique mass courtesy of the fluorous tag.  When using the mixture of the enzymes, all three were hydrolyzed to a uniquely fluorous tagged glucose.  The authors went on to demonstrate the use of cell lysates rather than purified enzymes and found essentially the same results.  The fluorous NIMS surface is critical in this since the fluorous tagged substrates and products and retained on the surface once spotted and an on-surface enrichment conducted.

The use of different fluorous tags for the encoding of substrates is the key element in fluorous mixture synthesis (FMS), where mixtures of substrates  can be prepared at once and later demixed using fluorous HPLC (F-HPLC) based on fluorous tag length.  In this work, the application is not synthetic, but analytical, and the compounds can be unequivocally indentified through mass, even when working with mixtures of substrates and enzymes.  As the authors note given the vast array of combinations of fluorous tags and linkers that could be made, each with distinct masses, this could be a general method by which to encode enzyme substrate libraries.

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Perfluoropolyethers as Fluorous Tags

Posted on February 1, 2012 by Marv

For the most part fluorous domains used in fluorous biphasic systems have been linear perfluoroalkyls, such as perfluorooctyl moieties.  Branched perfluoralkyls such as perfluorinated t-butyl groups have also been used as tags to facilitate fluorous based separations.  In the 2004 “Handbook of Fluorous Chemistry”, the authors define a fluorous tag as the “portion of domain of a colecule that is rich in sp3 carbon-fluorine bonds and exerts primary control over the separability characgteristics of the molcule in fluorous separation techniques.”  So within that definition does lie moieties that are not necessarily perfluoroalkyls.  These would include perfluoropolyethers, generally in the form of perfluorinated oligoethyleneglycols.

At FTI we have in the past done some cursory examination into the fluorous separation behavior of perfluoropolyethers as fluorous tags and compared them to perfluoroalkyl tags.  What we found, if I recall correctly, was that the oxygens basically functioned much like a CF2, so that a CF2OCF2CF2OCF3 tag had a similar F-HPLC retention time as a C6F13.  This was somewhat surprising since we always used fluorine count as a first approximation of fluorophilicity.  What the F-HPLC experiments told us, however, is that perhaps we should consider the size of the fluorous domain instead, in this case a 6 atom chain that is “rich in sp3 carbon-fluorine bonds”.

A just available paper from Kvicala et al at the Institute of Chemical Technology in Prague attempts to explain the fluorous behavior of perfluoropolyethers, at least as tags on NHC ligands in silver complexes.  The authors first prepared a series of substituted NHC ligands, either with traditional perfluoroalkyl tags or with perfluoropolyether tags.  They then formed the silver salts of these ligands.  Fluorous partition coefficients were then measured for the ligands and the complexes by partitioning between toluene and perfluoromethylcyclohexane.  The results were fairly striking as seen in the table below.  

Compounds 9a-h were all perfluoroalkyl tagged compounds which essentially had fairly equivalent partition coefficients, something in the single digits.  All of these compounds had a total of 12-16 CF2 or CF3 groups.  Compounds 12b, c, and f, however, were polyfluoroether tagged and possessed dramatically higher partition coefficients.  12b contains only 8 perfluorinated carbons, 12c has 12, and 12f has 10, so the overall fluorine content for compounds 12 is less than 9 yet they are more fluorophilic.  So what’s going on?  Well, if you consider the FTI results mentioned earlier and regard the oxygens in the perfluoropolyether as CF2′s than you get a pseudo-fluorous count for 12b, c, and f of 12, 18, and 15.  That’s now in the range of compounds 9, but still doesn’t account for the greater fluorous partition coefficient.

The authors then turned to computational chemistry.  DFT calculations were conducted on both the NHC ligands and the imidazolium salts.  What these calculations showed was that the most stable conformation of the perfluoroalkyl chain had the chain directed  straight out from the imidizole ring and beasically planar to it.  The perfluoropolyether chains on the other hand were twisted at the carbon-oxygen bond resulting in the chains being out of plane with the heterocyclic ring.  The authors hypothesize that fluorophobic ionic center of the imidizolium salts and the silver complexes are more shielded with the perfluoropolyether chains resulting in a significantly more fluorophilic compounds.  It’s certainly a reasonable hypothesis, particularly when one considers that fluorophilicity is based on solvophobicity more than on any attractive forces.

It’s an interesting paper which shows how structural elements can have profound effects on fluorophilicity.  Something we should certainly keep in mind more often.

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Fluorous Supported Synthesis of Teichoic Acid Fragments

Posted on January 24, 2012 by Marv

Oligonucleotides, proteins, peptides, and carbohydrates are the best known biopolymers and one doesn’t have to explain how important each is to a number of biological functions.  The synthesis of these materials, or segments of them, are important in order to elucidate form and function.  Without access to various fragments of these molecules the study of them becomes difficult.  That’s one reason why genomics is ahead of proteomics which it ahead of glycomics, since oligonucleotides are easier to make that peptides which are easier to make than oligosaccharides.

In general, synthetic biopolymers are made either by solution phase chemistry of solid phase chemistry.  Solution phase chemistry has the advantage of better reactivity and the ability to monitor reactions and purify intermediates.  This means that you can use less amounts of monomer and reagents.  This also means, however, that syntheses can be quite labor intensive since purification is not always the easiest.  Solid phase methods, on the other hand, are highly labor efficient, but often times require large amounts of reagents and monomers due to the heterogeneous nature of the chemistry.  So the choice of solid phase or solution phase depends on a number of factors; accessibility to monomers, scale of synthesis, length of synthesis, etc.

Fluorous methods have been used in the synthesis of all three major classes of biopolymers.   Three major methods for the incorporation of fluorous techniques have emerged; fluorous capping, fluorous tagging, and fluorous supported synthesis.  The first two, capping and tagging, are used in conjunction with solid-phase methods.  The latter, fluorous supported synthesis, is a solution phase method where the solid phase is replaced by a fluorous tag.  The growing oligomer can then be purified each step along the way by either fluorous solid phase extraction (FSPE) or fluorous liquid-liquid extraction (FLLE).  Fluorous supported synthesis therefore tries to combine the main advantage of solution phase chemistry (lower stoichiometries) and the main advantage of solid phase chemistry (ease of purification).  This has been used to great effect in carbohydrate synthesis where just getting to the monomers can be difficult, so you don’t want to be using 5 equivalents of them.

A new ASAP paper in Organic Letters from researchers at Leiden University led by Codée and Van der Marel describe their use of fluorous supported synthesis in the preparation of fragments of teichoic acid, the best known of cell wall glycoproteins found in Gram-positive bacterial cell walls.  Not surprisingly these cell wall glycoproteins play an important, but not very well understood role in immunology and bacteriology.  Teichoic acid consists of repeating glycerol phosphate or ribitol phosphate units which are decorated with glycosyl and/or D-alanyl substituents.  Ready access to fragments of teichoic acid would allow further study of these interesting molecules to take place.  These same researchers have also reported traditional solution phase synthesis and solid phase synthesis of teichoic fragments and now extend that work to fluorous supported methods.

As the fluorous support they chose the F-Psc group as a phosphate protecting group.  The same authors developed the F-Psc group as a hydroxyl protecting group for carbohydrate synthesis a couple of years back and found it to be suitable as a phosphate protecting group.  They first then demonstrated it’s utility in techoic acid synthesis by producing glycerol phosphate oligomer shown below.  The synthesis was a 4 step cycle (phosphoramidite coupling, oxidation, DMT removal, and FSPE) that was repeated until the desired length was obtained after which the F-Psc group was removed by base and the secondary hydroxyls debenzylated by hydrogenation.  It proved to be quite successful and the FSPE was easily conducted for all of the compounds even as the oligomer grew longer.  Interestingly, the authors did note that as the oligomer grew longer that increasing amounts of phosphoramidite were required, so that once you got to an 18-mer you lost that advantage of solution phase chemistry.  Why that should be is unclear.

After demonstrating the value of the fluorous supported synthesis in the preparation of a unmodified teichoic acid, they then prepared a more complex version; a glycosylated hexamer 28 which also proved to be quite successful.  As a final demonstration the produced the fully glycosylated hexameric glycerol phosphate 39.  In each of these FSPE played a critical role as the last step of the 4 step coupling cycle.

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