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Stop Ignoring the Sugars!

This paper (from two groups at Yale’s chemistry department) addresses several important things that fall into the “important irritants” category in synthesis and molecular biology – or maybe that should be “irritatingly important”. We spend a lot of time thinking about proteins in terms of their primary sequence and features of their three-dimensional structure (binding pockets, recognition domains, etc.) And that’s understandable. But it’s always important not to lose sight of the fact that the surfaces of proteins are decorated with all kinds of ornaments that are vital for their function, even if they’re synthetically hard to control.

Chief among those are phosphates, you’d have to say. Kinases, of course, phosphorylate things and phosphatases take those back off (and as is often the case in biology, those two classes of enzymes are rather unbalanced: there are a lot more specific kinases out there, compared to a relatively small number of jack-of-all-trades phosphatases). Phosphorylation state can be a powerful on/off switch on specific protein residues, but it’s worth remembering that proteins can have a whole long list of phosphoryation sites once you get past the heavy-duty ones. The number of kinase inhibitors out there is proof enough that this method of regulating protein function can be taken advantage of, but it’s worth keeping in mind that making specifically phosphorylated proteins can be a painful process if you don’t have a living cell or at least a tame enzyme to do it for you. That’s particularly true if you’re interested in one of the less-common phosphorylation sites.

Then you have a very different sort of polarity modification, prenylation. Farnesyl (or geranyl-geranyl) groups are small lipophilic chains that get attached to proteins to modify their function and/or cellular localization. Proteins also get decorated with even longer lipid chains via palmitoylation and myristoylation. It’s believed that these are generally involved in localizing things to membrane sites (as those greasy side chains settle into the lipid membrane). What’s for sure is that these modifications can make proteins rather tricky to work with (as is generally the case when you have a big polar/nonpolar structural separation).

After that you get to the proteins-modified-with-other-proteins class, with ubiquitination at the head of that class. Ubiquitin, as the name implies, is all over the place, and proteins get ubiquitinated (at lysine residues) to mark them for degradation, to mark them not to be degraded, to change their localization and interaction networks, and for who knows what else. It’s a huge topic, since there are lots of varieties of ubiquitin attachment, and if you think we’ve got them all figured out you are sadly mistaken. Then there’s SUMOylation, another add-a-small-protein process that’s clearly important but which we understand even less. It’s less common than ubiquitination (we think!), but there are several different sorts of SUMO proteins and places that they can be attached.

I’m skipping over several other post-translational modifications to get to a big class of them that I haven’t mentioned yet: glycosylations. Sugar molecules (and chains of them) are stapled on to residues like tyrosine, starting with good old glucose and going on from there. These things can also be linked through amine and sometimes even SH residues, and that is just the beginning. I did my graduate work using carbohydrates as starting materials for organic synthesis, and anyone who’s worked with them will have some appreciation of the complications that are available. With all those open hydroxyls available, carbohydrates have a lot of linking possibilities (some more common than others, naturally), and you have the alpha/beta anomeric centers for variety as well. Two naturally occurring amino acids can give you four peptides, but two naturally occurring sugars can give you a whole lot more disaccharides, at least in theory.

These modifications are hugely important in proteins and many other biomolecules (including small-molecule natural products), but when I was in grad school, a lot of this was, chemically and biologically speaking, dark matter. A lot of it still is. Sometimes people would prefer just to ignor that part – even now, you’ll see total syntheses of some natural product as “whateverol aglycon”, that is, minus those pesky sugars. And when it comes to proteins, you almost have to rely on a living cell to glycosylate them properly, because chemically it can be a beast. A lot of chemical glycosylations are variations on the Koenigs-Knorr reaction, with glycosyl halides, pseudohalides, and other leaving groups, and the number of variations on this is surely beyond counting. Holy cow, are there ever a lot of attempts to make glycosylation a better and more predictable reaction. My old “Lowe’s Laws of the Lab List” had one that went “When there are twenty different ways of running a reaction in the literature, it means that there is no good way to run the reaction“, and the Koenigs-Knorr was what I had in mind. But it’s true: every time you vary the substrate being glycosylated or mess with the structure of the sugar part in any way, you can expect to see a new mixture of alpha/beta glycosides at the very least.

The paper I linked to so many paragraphs ago is a good shot, though, at taming this stuff for plain glucose-style glycosylation. The authors have a glycosyl fluoride protocol worked out (calcium hydroxide turns out to be a key addition) that seems to give pretty solid results on tyrosines (and phenols in general). What’s really impressive is that it works under aqueous conditions, using an unprotected sugar, and works on native (unprotected) proteins and peptides. It’s not perfect – you still get some glycosylation on other residues (serine, etc.), but it’s certainly the best I’ve ever seen. It would be interesting to see what happens when you try it on a protein with several possible tyrosines or with competing Cys residues, and I get the impression that the authors are heading there next. But even as it stands, this looks like a significantly better option than anything else out there for fast one-step glycosylation without messing around with protecting groups.

The sorts of tools that are available for synthesizing, manipulating, and analyzing proteins and nucleic acids are (in general) just not available for carbohydrates. They either don’t exist anywhere or haven’t been domesticated from their biochemical forms. Only in recent years has the dream of automated polysaccharide synthesis come close to reality. Even in this paper, it’s worth noting that the authors weren’t really able to tell what all those other minor glycosylated side products were; it would be a major effort to run all those down. But this stuff is important, and every new technique that makes it easier to work in the area is welcome.

14 comments on “Stop Ignoring the Sugars!”

  1. Andy II says:

    Thanks for bringing this sweet spot up. Carbohydate bio/chemistry has a long history when all the stereochemistry of D-glucose had been “correctly” assigned. In addition to the importance of monosaccharides in the metabolic pathway in the body, polysaccharides in diet is also capturing the lots of attention due to their potential role in gut microbiomes.
    My experiences in working in chemical and enzymatic syntheses of complex oligosaccharides, our synthetic platform is sufficient to make the target molecules in purity and quantity. However, due to the important role the sugar moiety plays in therapeutic antibody, we really need an efficient/robust technology platform to tailor the sugar moiety if it would help improve the clinical efficacy or reduce the toxicity associated with antibody possessing a mixture of oligosaccharides on it.

  2. Gutdecipher says:

    For in vitro studies of phosphorylation states, I’ve seen signaling pathway work done where they clone in S->A or S->D,E to mimic conformational changes and, consequently, activity observed in, respectively, the constitutively inactive and constitutively active mutant forms of the substrate protein (see eLife article# 23966, Fig 3).

    As for this paper, I wouldn’t have imagined you’d avoid getting a mess of sideproducts doing a bioconjugation on large peptides. I guess the reaction will also be useful for generating a set of Tyr glycones for use in direct synthesis in peptide libraries, or NCL for chemical synthesis of protein glycones that can’t be accessed via direct bioconjugation for one reason or another.

  3. Wavefunction says:

    I remember when force fields for modeling carbohydrates were both so woefully underdeveloped as well as enormously challenging compared to those for proteins or even small molecules. I presume the situation hasn’t changed dramatically.

  4. Bicoid says:

    It’s a continuing shame that Merck closed Glycofi down two years ago. Their yeasts were modified to glycosylate proteins like those on humans.

    1. A Nonny Mouse says:

      This sort of yeast fermentation work is being done in the UK by Hypha Discovery, with me occasionally helping out on the chemistry.

  5. Peter Kenny says:

    I think stoichiometry can also be an issue for glycosylation. I recall asking one of the plenaries at EuroQSAR 2016 a question about how the sugars had been modelled and I got the impression that some considered the question to be rather uncouth.

  6. Mark Thorson says:

    I’m interested in what anyone may have to say about glycosation of very insoluble small molecules to improve absorption across the intestinal mucosa and past the liver into the bloodstream. Is that a good way to go or are there better avenues? If so, which ones? Judging from the patent literature I’ve seen, it appears that attaching an amino acid can be used for that too. Also, you have a larger palette of colors in the amino acids, as compared to the sugars, though a sugar’s plurality of hydroxyls is very attractive for solubility. There’s also the question of what enzymes are available to cleave the hydrophilic attachment from the hydrophobic core.

    1. Dr CNS says:

      Re: aminoacid prodrugs- Look for valbenazine

  7. Peter S. Shenkin says:

    Sounds like advances in synthesis will allow us to no longer iKnorr glycosylation…. :-}

    1. Me says:


    2. Scott says:

      15 yards and loss of down for the terrible pun.

      (You know it’s a bad pun when the non-chemist is calling you on it!)

  8. JB says:

    Indeed, ignore sugars and glycobiology at your own peril. There are examples that exist of proteins that have more total molecular weight from glycans and glycosylation than due to the peptide backbone itself. Ion channels, for example, have nearly 30% or more of their entire mw from glycosylation, and just by changing one sugar you can radically alter the gating properties of a channel. Immuno oncology is now a hot area, but there are big gaps in our understanding of why many tumors and patients won’t respond to a treatment. Well, what better to study than glycans and carbohydrates in this area since glycans are literally at the interface of all of this biology, and plus the fact that nearly one of the first hallmarks ever discovered in cancer are altered cell surface patterns of glycosylation. In fact, the overabundance of Sialic acids on nearly all tumors can have significant effects on tampering down immune response through SIGLEC-sialic acid binding. In fact, SIGLECS tamper down immune response literally through the same mechanisms with which PD1-PDL1 and CTLA4 biology works. See Bertozzi’s intriguing work on resensitizing tumor models to immuno treatments that previously didn’t work by manipulating Sialic acid-siglec bindng.

    Finally, drug makers have already known for years that glycosylation is profoundly important for the production of biologics. Half-lives, immunogenicity and potency can be radically altered by changes to glycosylation. Removal of core fucose (GlycoFI) on mABs can increase their potency by 100 fold. Introduction of Sialic acids on the glycans buried in the Fc region of mabs can basically kill all Fc gamma receptor binding, which may be bad for cancer treatments or may be good for indications related to inflammation. Amgen’s EPO is really only a patented form that has enhanced glycosylation so its half-life is longer.

    I remember one lab who spent years studying how pH can affect tumor growth yet couldn’t recapitulate their in vitro results in vivo. They eventually found that the reason was because the tumor environment, which contains all sorts of messed up GAGs like hyaluronic acids etc., Are completely different in vivo than in in vitro models (acidic glucuronic acids in vivo radically change tumor pH). There’s literally almost no disease, target, even methods for generating biologics where glycobiology isn’t important. Ignore at your own peril.

  9. gippgig says:

    Off topic but may be of interest:
    May 11 9PM EDT on CW – Life Sentence
    Stella goes on a road trip with Dr. Grant and Sadie to help get Sadie into a clinical trial.

  10. Synthon says:

    Don’t forget formylation either.

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