Fluorous Supported Synthesis of Teichoic Acid Fragments

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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Orthogonal Fluorous Mixture Synthesis (FMS)

University of Pittsburgh Professor and Fluorous Technologies, Inc. founder, Dennis Curran, has over the last several years dedicated a good portion of his research to fluorous mixture synthesis (FMS).  In FMS, libraries or isomers of compounds can be made in a minimum number of reactions by labelling the molecules with a specific length fluorous tag in a single reaction vessel.  The molecules can then be separated in a predictable fashion by fluorous HPLC (F-HPLC) based on their fluorine content.  So those molecules tagged with a C4F9 tag can be readily separated from those with a C6F13 tag or a C8F17 tag.

One issue with FMS is the limited number of tag lengths that are practically available.  In theory any perfluorocarbon chain length should be usable from CF3 to CxF2x+1.  In practice, though, anything above C9F19 introduces solubility problems in organic solvents which defeats the purpose, since they won’t react in a similar fashion as the soluble components.  In addition, the odd numbered lengthed perfluorocarbons (C3,  C5, C7, and C9) are considerably more expensive than the even numbered chain lengths.  So now we’re down to just four lengths.  That then lowers the overall value of FMS since it limits the total number of reactions saved over parallel synthesis.

In response to that Prof. Curran has previously reported two methods to increase the number of reactions that can be conducted in a single pot and still be able to deconvolute the mixture.  One is to use fluorous tags in conjunction with oligoethylene glycol (OEG) tags of different length.  In this strategy, a two dimiensional separation strategy is used; a fluorous based separation and a RP-HPLC based separation.  Another is to use a double tagging strategy and separate the mixtures based on the cumulative fluorine count.  The synthesis of the cytostatins which we’ve covered in F-Blog before is an example.  This binary encoding works well, but is limited by additive redundancies.  In other words, the C6 and C4 tags in combination add up the same as the C2 and C8 tags in combination.  So even if you can easily separate the two by F-HPLC (not at all guarenteed), it’s impossible to predict which one is which.

A new paper from Dr. Curran’s labs in Nature Chemistry describes another double tagging FMS strategy which increases the number of molecules that can be made and overcomes the redundancy issue.  In this approach the researchers once again use two fluorous tags, but in this instance orthogonal tags rather than the same tags as in the cytostatin case.  The researchers produced a 16 member steroisomer library of macrosphelides (shown above) using a F-PMB and a F-TIPS tag as the orthogonal tags.  Each tag had four variants, C2, C4, C6, and C8, so 4 x 4 provides the 16 members.  By utilizing two tags that can be selectively removed, they were first able to partially deconvulute by FHPLC based on the cumulative fluorous content of both tags to provide 7 fractions.  These fractions were selectively deprotected of one tag and subjected to another FHPLC to completely deconvolute the mixture.  The figure below from the paper shows the redundancies and the fractions.

The macrosphelides are a series of natural product macrocycles containing five stereocenters.  The authors went on to prepare 16 of the 32 stereoisomers, leaving C3 stereochemistry fixed.  Each of the 16 stereoisomers had a distinct 1H and 13C NMR and they were able to match one of the isomers with previously characterized macrosphelide A and one with macrosphelide E (Note that the molecule they produced was enantiomeric macrosphelide E), thereby confirming the structure of each.

A problem was encountered, however, in that none of the spectra matched that of the reported structure of macrosphelide D.  After considering the possible structural isomers they hypothesized that macrosphelide D was actually a the ring contracted isomer shown below.  They then confirmed this through independent total synthesis.In conclusion the authors demonstrated that orthogonal binary fluorous tagging with double F-HPLC separation is a viable strategy for miaximizing FMS efficiency.  In the synthesis of the macrosphelides they required only six chemical reactions once the tagged intermediates were in place vs. 72 reactions if one were to do this in parallel synthesis mode.  In addition they once again demonstrated how important the synthesis of all possible isomers can be in natural products chemistry since it led to a definitive structural assignment of macrosphelide D.

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Fluorous Organocatalyst for Aldol Reactions

Researchers at Gifu Pharmaceutical University led by Prof. Tsuyoshi Miura have been working on fluorous sulfonamide organocatalysts for some time now and have reported their results in various reactions.  These include asymmetric aldol reactions and Michael additions which we have covered in the past.  In these instances the fluorous tag was attached to one of the nitrogens of the phenylalanine derived organocatalyst through a sulfonamide.  The researchers found that either enantiomer of the aldol product could be made starting from the diamine derived from L-phenyalanine by moving the sulfonamide from one nitrogen to the other.  Organocatalyst 1 then leads to enantiomer A, while catalyst 2 produces enantiomer B.  This is certainly more desired than starting from the D-phenylalanine which is considerably more expensive than Interestingly, however, while they reported using a fluorous sulfonamide in the case of 1, they only reported using the triflate for 2 and never mentioned a fluorous verion.  So the fluorous sulfonamide 2 must not have provided very good results in forming the opposite enantiomer.  In addition, as seen below, even with the triflate, in order to form enantiomer B, it required double the organocatalyst loading compared to the enantiomer A.

The same authors now report a fluorous version which can provide enantiomer B directly with lower catalyst and substrate loading.   Instead of rendering the catalyst fluorous via the sulfonamide the fluorous tag is attached to the aromatic portion of the diamine catalyst.  So in this case the diamine is derived from L-tyrosine rather than L-phenylalanine to provide catalyst 4.  They then used 4 to catalyze the aldol reaction between cyclohexanone and p-nitrobenzaldehyde.  Compared to 2 they were able to use half the amount of catalyst and half the amount of cyclyhexanone(see entry 5) to achieve comparable results.  They then used fluorous solid phase extraction (FSPE) to recover and reuse the organocatalyst.  The authors went on to test a number of difference ketone and benzaldehyde combinations to test the generality of the reaction.  They found that while electron deficient benzldehydes are good substrates but that electron rich ones are not.  Other cyclic ketones were tried but none of them gave results that were as good as cyclohexanone.

The results themselves are good, but somewhat limited due to the structural requirements of the reaction.  From a fluorous perspective, however, there are some good takeaways here. First is that it demonstrates the flexibility of fluorous tagging.  The authors were able to change where the fluorous tag was on organocatalyst 2 quite easily in order to achieve a better catalyst in 4.  This was done without effecting the absolute stereochemistry.  Second is that the same general purification procedure, FSPE, was used regardless of where the fluorous tag was through a ether bond or a sulfonamide.  This then provides an opening for further refinements in the organocatalyst which will hopefully result in a more general reaction.

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Diversity Oriented Synthesis Using Fluorous Tags

Diversity-oriented synthesis (DOS) has been discussed here often and the benefits of employing fluorous tags and purification methods to the construction of such libraries has been effectively established.  Among these are the solution phase nature of the chemistry, the general purification method across varied structures, and the high degree of chemical compatibility.  All of these are important when one is trying to target a diverse set of chemical compounds which has a little different set of demands than a library of analogs (although fluorous is good for that too).

Just published in ACS Combinatorial Science is a paper from Park et al at Seoul National University describing their work at producing a diverse set of polyheterocyclic benzopyrans.  Within this paper they use six different reaction pathways to prepare 284 compounds.  Each reaction pathway produced a different core structure.  In five of the six pathways (paths II-VI) the authors used a fluorous tag while in the other pathway (path I) a solid phase support was used.  The structures are shown below with the authors starting from four different benzopyrans 3.

This paper is actually part II and in part I, published in 2010, the authors strictly used solid phase methods to produce 434 compounds.  In reading the current paper the advantages of fluorous methods become readily apparent.  First, the authors point out that they tried to use the solid phase strategy which worked reasonably well in the previous report, but found that the reactions were sluggish.  This resulted in either incomplete reaction or in high amounts of side products for pathways II-VI.  That is when they decided to turn to a fluorous strategy and use fluorous solid phase extraction (FSPE) as a purification method.  In this instance they used a fluorous silane to tag their four starting benzopyrans.  The solution phase nature of the chemistry clearly was needed in order to have a viable route to the desired compounds.  The average purity of the compounds derived from the fluorous pathways was ~87% without final HPLC purification.  That doesn’t seem too much higher than the 85% avg. purity reported from the previous paper using solid phase, but remember that the chemistry here was more demanding and that they couldn’t even get to the compounds in reasonable purity using solid phase methods.  The scheme below shows two of the pathways.  Note the use of FSPE for the purification of intermediates.

There are also some other more subtle aspects which were interesting.  For example, for pathway II the substituted triazolinediones were not all commercially available, so were prepared in-house by the authors.  If you have to make something, you don’t want to use more than you have to in any reaction.  Solid-phase techniques usually require large excesses of reagents which is not the case for fluorous methods.  There was also the manner in which the amount of compound is on the solid phase was determined by measuring the mass increase of the resin and then confirming that  after cleavage from the resin.  Let’s compare that to a fluorous tag which can be directly weighed and analyzed to NMR, LC/MS, or any other method to not only give you an amount, but also structural and purity information.

So once again we see how fluorous methods are applied to DOS and the value they provide.


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Automated Synthesis of Oligosaccharides

The automated synthesis of oligosachharides is a topic that we’ve devoted quite a bit of time on here at F-Blog.  This is largely due to the efforts of Prof. Nicola Pohl’s group who have pioneered the use of fluorous tags in an automated setting.

Unlike DNA or peptide synthesis, the efficient automated synthesis of oligosaccharides has been difficult to achieve due to the increased complexity of oligosaccharides vs oligonucleotides and peptides.  There are more monomer units, stereochemistry in the coupling reaction, additional protecting groups, linear vs. branched structures, etc.  All of these factors increase the degree of difficulty, so even decades after the automated synthesis of peptides and DNA has been not only commercialized but commoditized, oligosaccharides remain unsolved.  The relative inaccessibility of oligosaccharides has undoubtedly held back the study of oligosaccharides in biological systems compared to DNA, RNA, and proteins.

A new review in Angewandte Chemie from Hsu et al describes the latest efforts in automated oligosaccharide synthesis.  The approaches include one-pot methods, chemoenzymatic syntheses, and phase supported methods.  Fluorous tags and separations, primarily in the form of fluorous solid phase extraction (FSPE), has been used in each of these synthetic strategies.  In addition to the actual synthesis, this new review also has sections on glycan microarrays and vaccine development using synthetic oligosaccharides.  Fluorous immobilization of course has been shown to be an effective method for the formation of glycan arrays.  This current paper then provides a good review of the field and places the contribution of fluorous methods in great context.

One of the little details that I found interesting is the authors’ characterization of the fluorous separation which they described as a “specific solvophobic interactions”.  This is in contrast to most reports which use terms such a “fluorous affinity” or “fluorous-fluorous interactions.”  Partitioning based on solvophobic effects is exactly the way that we like to describe fluorous separations, since there is no intermolecular forces between fluorous molecules that explain the partitioning observed.  As Prof. Craig Wilcox once rhetorically asked me, “Which compound has a lower boiling point, perfluorohexane or hexane?”  The answer is perfluorohexane which clearly indicates a lack of any intermolecular interactions or affinity which would explain the high partitioning observed.  So solvophobicity is a great term to describe fluorous partitioning, because it’s the incompatibility of fluorous domains with aqueous or organic phases that drives the the molecules into the fluorous phase rather than any attraction.  Push vs. pull, if you will.  The addition of the word specific is great.  It emphasizes that it’s not simply hydrophobic or lipophobic, but rather that the solvophobicity is beyond the usual phobicities that we’re accustomed to seeing.

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