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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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December 13, 2013

Erythropoetin From Scratch

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Posted by Derek

EPO.pngThe Danishefsky group has published their totally synthetic preparation of erythropoetin. This is a work that's been in progress for ten years now (here's the commentary piece on it), and it takes organic synthesis into realms that no one's quite experienced yet:

The ability to reach a molecule of the complexity of 1 by entirely chemical means provides convincing testimony about the growing power of organic synthesis. As a result of synergistic contributions from many laboratories, the aspirations of synthesis may now include, with some degree of realism, structures hitherto referred to as “biologics”— a term used to suggest accessibility only by biological means (isolation from plants, fungi, soil samples, corals, or microorganisms, or by recombinant expression). Formidable as these methods are for the discovery, development, and manufacturing of biologics, one can foresee increasing needs and opportunities for chemical synthesis to provide the first samples of homogeneous biologics. As to production, the experiments described above must be seen as very early days. . .

I can preach that one both ways, as the old story has it. I take the point about how synthesis can provide these things in more homogeneous form than biological methods can, and it can surely provide variations on them that biological systems aren't equipped to produce. At the same time, I might put my money on improving the biological methods rather than stretching organic synthesis to this point, at least in its present form. I see the tools of molecular biology as hugely powerful, but in need of customization, whereas organic synthesis can be as custom as you like, but can (so far) only reach this sort of territory by all-out efforts like Danishefsky's. In other words, I think that molecular biology has to improve less than organic chemistry has to get the most use out of such molecules.

That said, I think that the most impressive part of this impressive paper is the area where we have the fewest molecular biology tools: the synthesis of the polysaccharide side chains. Assembling the peptide part was clearly no springtime stroll (and if you read the paper, you find that they experienced the heartbreak of having to go back and redesign things when the initial assembly sequence failed). But polyglycan chemistry has been a long-standing problem (and one that Danishefsky himself has been addressing for years). I think that chemical synthesis really has a much better shot at being the method of choice there. And that should tell you what state the field is in, because synthesis of those things can be beastly. If someone manages to tame the enzymatic machinery that produces them, that'll be great, but for now, we have to make these things the organic chemistry way when we dare to make them at all.

Comments (37) + TrackBacks (0) | Category: Chemical Biology | Chemical News


COMMENTS

1. Curious Wavefunction on December 13, 2013 11:53 AM writes...

I have always wondered if it's "Danishefsky" or "Danishevsky". It's a tour de force for sure, but I agree that the future belongs to molecular biology rather than organic synthesis except in rare cases.

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2. biotech scientist on December 13, 2013 12:07 PM writes...

I worked for a company not too long ago that was producing EPO in an avian cell line...the post-translational modifications were terrible. Almost no Sialic acids, and that is what is(or was) regulated by the EU, without those you are injecting water cleared from the first pass. What is strange about the glycosylation story on EPO is CHO actually does a better job than human cells at glycosylation. Very impressive though, I would have started with a smaller molecule like insulin, unless someone has already done that.

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3. marcello on December 13, 2013 12:22 PM writes...

Professor Kenner in Liverpool was trying to synthesize chemically Lysozyme. Assembling the peptide chain was obviously not a trivial part, but at the end they could not remove protecting groups cleanly from the full sequence. Soon after that "failure" Kenner commited suicide...

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4. Hap on December 13, 2013 12:39 PM writes...

This seems like a painful way to spend a postdoc or Ph.D., and it seems not very generalizable or useful for drugs (the size of the pieces in solubility- and solid phase synthesis-limited, you have to figure out where you can cut the peptide into pieces and the order in which you can assemble it, none of the steps are generally easy or high-yielding, although that could change). It is early in the history of making molecules like this, though, and it could be useful for modifications you can't easily get something to make for you (do any fragments work as well as the whole protein? do you need the sugars, and if you do, can you use simpler ones? does it matter where the sugars are?). If biologics are where pharma wants to go, being able to do drug discovery on them would seem to be useful.

As a proof of concept, this is OK, but it seems unimaginably painful and not very useful in and of itself. On the other hand, decent methodolgy is likely to come from it. I'd rather see more of this than the Odyssey of Maitotoxin.

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5. Hap on December 13, 2013 12:44 PM writes...

I think Danishefsky has also done parathyroid hormone, which is somewhat smaller (80 residues?) and has less glycosylation. I assume that he chose targets based on the presence of sugars and lack of easy synthesis - Kent has made insulin and variants using his ligation methods, for example.

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6. Moneyshot on December 13, 2013 12:46 PM writes...

Waste of time......and few job options for the poor souls who did this. Really what does this actually teach or train anyone to do?

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7. Sam Danishefsky on December 13, 2013 1:11 PM writes...

Dear KC,

I am pleased to inform you that my penis is officially bigger than yours,

Merry Christmas,

Sam

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8. Modality agnostic on December 13, 2013 2:18 PM writes...

See GlycoFi for precisely tailored glycan production via yeast.

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9. Anonymous on December 13, 2013 2:23 PM writes...

What a waste of limited tax dollars. How many fresh young bright chemists were denied funding so this should-be-retired professor could dick around making a compound that has been readily available to patients for 20 years? How many grams of this stuff did his group make?

This epitomizes all that is wrong with academic chemistry and specifically organic synthesis. This guy was well into his career when I started college and I am 65 and now retired from industry. What funding agency thought this is a must spend money on project in times of limited funds? Were there no better ideas from todays bright young people?

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10. HT on December 13, 2013 2:39 PM writes...

The comments here are kind of funny -- I suppose this is what happens when pure small molecule guys comment on chemical protein synthesis papers?
Peptides/proteins and oligosaccharides will always be used and useful in some way, not only in biological and medical research but in materials and other areas, so methodologies for examining structure-property relationships, and ultimately controlling those properties have merit. Certainly the synthesis of a protein drug or drug target with mostly solid-phase methods is at least as useful as synthesizing some bizarre natural product secondary metabolite, all in solution. For those not familiar, this is by no means the first example...see the collected works of Kent, Dawson, Tam, etc...but Danishevsky's group has done important work on the glycoside aspect, and also significantly improved the desulfurization methodology for multiple segment ligations.

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11. HT on December 13, 2013 2:49 PM writes...

...significantly improved the desulfurization methodology, which can be especially useful for proteins without Cys and for multiple segment ligations.
(Last sentence got mangled)

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12. Lu on December 13, 2013 4:11 PM writes...

9. Anonymous on December 13, 2013 2:23 PM writes...
What a waste of limited tax dollars. How many fresh young bright chemists were denied funding so this should-be-retired professor could dick around

C'mon, I am sure nobody would approve funding for this project. He did what most current PIs do these days: funded "preliminary studies" for this project from previous grant.

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13. SteveM on December 13, 2013 4:15 PM writes...

Re #9 Anonymous. "How many fresh young bright chemists were denied funding so this should-be-retired professor could dick around making a compound that has been readily available to patients for 20 years?"

I migrated from Chemistry to Operations Research (OR), a domain which is supposed to be predicated on tangible utility. Speaking of dicking around, academic OR is now considered a barren wasteland of "Mathematical Masturbation".

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14. Stop doing total synthesis now on December 13, 2013 8:54 PM writes...

Totally useless molecule. Sad for the students who made that piece of crap. No nice slides, no jobs, that's how we hire.

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15. Scorned methods guy on December 13, 2013 10:19 PM writes...

I'd say this is better science, than the useless random multicomponent reaction papers that seem to be endlessly popping up. Especially if they are examining further manipulation of the structure.

The only truly bad thing about this reporting was the god awful writing. One of the non native authors must have written the paper because it is very hard to read. Most of the sentences just don't make any sense.

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16. Secondaire on December 14, 2013 1:05 AM writes...

To me, although it's an interesting academic/training exercise, it seems to be an awful lot of time and money and manpower to waste on what amounts to little more than a Bigger Penis Contest (or intellectual masturbation, or whatever phallic metaphor works best). What are they going to synthesize from scratch next, a P540 enzyme, complete with a functioning heme? Unless a LOT of broadly applicable methodology is coming out of this, what, ultimately, is this scientific brinksmanship accomplishing than making a lot of post-docs acrimonious toward their PI? How did they get the grant to even do this?

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17. Rock on December 14, 2013 2:15 AM writes...

1) It's "Danishefsky"
2) This work is a culmination of several decades of research in the synthesis of simple sugars=>poly saccharides=> carbohydrate-based antigens for cancer therapeutics=>new methods in peptide chemistry. To me it shows someone with a strong vision of how all this methodology can be combined to create greater and greater complexity.
3) Given the state of the industry, this training is probably more likely to find you a job (there still is a demand for peptide chemists, protein-drug conjugates etc) than a traditionally-trained synthetic organic chemist.

Permalink to Comment

18. dearieme on December 14, 2013 6:40 AM writes...

O/T: there's a remark in this short article that might interest you, Derek; and your readers.
http://ftalphaville.ft.com/2013/12/12/1721252/have-you-noticed-equities-are-the-new-bonds/

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19. anchor on December 14, 2013 6:49 AM writes...

@17-There is job for peptide chemists, peptide-drug conjugates etc. but in only academic surroundings where at best you are getting a post-doctoral salary. The fact that only few bio-conjugates have reached market potential can be a deterrent in this area. As a practicing organic and medicinal chemist in a small molecule area from a reputed company, I switched job (after lay off) to do exactly what you have opined. My experience suggests to me that these are very difficult reactions (deceptively simple) to accomplish with abysmal yields in most cases! I mean you get the paper out and that’s it! That said, in these troubling times I am keeping my job and do what we all love to do and that is doing some challenging chemistry.

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20. anony-mous on December 14, 2013 8:44 AM writes...

A few examples of (surprising and unusual) ignorance on this particular blog:
1. As pointed out by #8 - see GlycoFi technology for directed (biological) assembly of oligosaccharides
2. To #14 - Epo - USELESS???? Tell that to a host of people with Anemia who have benefited immensely from its availability.

#2 gets it !

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21. Orthogon on December 14, 2013 3:40 PM writes...

The Rube Goldberg Synthesis of EPO

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22. sky on December 14, 2013 10:08 PM writes...

Though making EPO seems to be more of a mountain climbing exercise, the methodologies that have been developed along the way are whats really key. Specifically MFD (metal free desulfurization). That ACIE paper probably should've been nature or science by itself.

Although I didn't find anything new in this paper, compared to every other protein thats been made by this lab, I don't have a problem with it. If Princeton can takeover the chemistry world, via JACS, for not so useful science, then this work can certainly make it into a high caliber journal.

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23. Jose on December 15, 2013 3:31 AM writes...

"2. To #14 - Epo - USELESS???? Tell that to a host of people with Anemia who have benefited immensely from its availability."

Pretty sure 'Stop doing total synthesis now' was referring to the synthesis and not the protein. I hope you are not seriously suggesting that this chemical route will compete with the biologics route currently in use....

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24. drug_hunter on December 15, 2013 10:58 PM writes...

Uh, guys, hello ??

What if you wanted to create a novel reagent to, you know, learn something new about complex disease biology, or to point the way towards new analogs of EPO that have better potency or residency time or PK or some other desirable trait?

And to do that you wanted to insert a totally non-natural AA into the sequence? Something you couldn't get to through the new synthetic biology approaches? Or you wanted to be able to fine-tune the post-translational modifications to adjust physical properties, reactivity, specificity, localization, ... ?

To call this work worthless is tantamount to saying you have absolutely no clue about the complexity of disease biology or drug discovery.

And, no, I never worked for Sam.

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25. Anonymous on December 16, 2013 2:48 AM writes...

This is a bit like walking 500 miles instead of taking a train or flight - impressive work, but completely and utterly pointless. Unfortunately, it was all those poor students that were forced to walk (and possibly sacrifice their careers) rather than the PI. Slave labor for the sake of one man's ego!

It would have been better for the students (and society) to learn to use protein engineering in order to make existing enzymes to do new things.

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26. Anonzymous on December 16, 2013 7:53 AM writes...

Next paper from the Danishefsky lab: total synthesis of hemoglobin.

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27. Hap on December 16, 2013 10:25 AM writes...

I don't think this can compete with biological methods for protein synthesis (solid-phase synthesis doesn't except for short peptides and where there isn't another way), but if you want to get to unnatural glycosylation patterns, there isn't another way yet.

I don't think this is as pointless as lots of total syntheses - Dansiefsky's group developed various non-native peptide ligation strategies during their synthesis (also desulfurization methods), internally protected amides to mitigate aggregation, and actually making glycopeptides. It also might be possible using these methods to look at shorter or variant glycopeptides - bacteria could make them quicker, but if they misfold or aggregate, you might not know if the bacterial method doesn't work or if the sequences give unstable, aggregated, or misfolded products.

Is there the capacity to do anything close to this with protein engineering? It seems like suggesting using protein engineering to this is akin to seeing that aliens visiting in the 1800's could transport things across the galaxy, so instead of working on cars we should start designing transporters, despite having nothing resembling the technology.

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28. Chemjobber on December 16, 2013 12:18 PM writes...

18: It's interesting to see what people see Sandwich as: "its Viagra lab." Ummmm...

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29. Curt F. on December 16, 2013 4:30 PM writes...

Derek wrote I see the tools of molecular biology as hugely powerful, but in need of customization, whereas organic synthesis can be as custom as you like, but can (so far) only reach this sort of territory by all-out efforts like Danishefsky's. In other words, I think that molecular biology has to improve less than organic chemistry has to get the most use out of such molecules.

I agree with that, in general. In the specific case of protein glycosylation, one person who made tremendous improvements in the molecular biology toolset is Matt DeLisa at Cornell. See http://www.cell.com/trends/biotechnology//retrieve/pii/S0167779913000644?cc=y for a recent review. Bacteria can be engineered to make site-specific, glycoform-specific glycosylations to a variety of proteins. Remaining challenges are (1) increasing the yield of glycosylated protein relative to total recombinant protein, and (2) expanding the specific glycoforms that can be produced recombinantly.

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30. Curt F. on December 16, 2013 4:36 PM writes...

@27 Hap.

It's also worth noting that the review I mentioned gives examples of biological synthesis of some "non-natural" glycosylation patterns through engineering of the glycosylation pathways present in recombinant bacteria. Of course, for any given arbitrary non-natural glycosylation, it is unlikely that a recombinant pathway could be engineered in a reasonable amount of time. But for a certain few non-natural glycoforms, recombinant bacteria can already do the job.

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31. Hap on December 16, 2013 4:49 PM writes...

Sorry.

I think that biosynthetic methods probably can work better for making proteins, but the ability to manipulate them synthteically might still be useful for SAR or for fundamental knowledge of how they work. That, and the methodology developed, which might be useful for molecules for which a synthetic approach might be more tractable than a biosynthetic one, make this seem more useful to me than, say, the (eventual) total synthesis of maitotoxin.

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32. Anonymous on December 16, 2013 5:22 PM writes...

One also has to ask whether it is really worth the effort to incorporate non-natural amino acids and glycosylation patterns, whether this is done biologically or chemically. Certainly, nature seems to get by with the standard set of 20 amino acids, plus natural glycosylation patterns, just fine. Also, with the existing cellular apparatus one can easily make 20^n variants of any protein in a single pot, so that the number of combinations rapidly exceed the number of atoms in the universe. With the right selection technique you should be able to enrich and isolate any function you want. So in summary, why bother with any chemical synthesis? Unless it's just to show that something nobody really needs can be done...

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33. Curt F. on December 17, 2013 7:50 PM writes...

@32. Anonymous.

I agree with Hap. Chemical synthesis will certainly be valuable in a lot of different contexts, including SAR. I also think that you are far too optimistic if you really do think that "any function you want" can be obtained through naturally occurring amino acids and their glycoforms. What if the function you want is bio-orthogonality? What if you want to do boron neutron capture therapy, or you want solubility in a fluorous phase? Or if you want thermostability to 200 C or beyond? There are plenty of good reasons to investigate chemical synthesis of "bio"molecules.

That said, in this particular case, I'm not up to speed on the state of the art in bioengineered production of variant forms of Epo, so I don't know if chemical synthesis is an attractive option for Epo specifically. Could be quite useful, or maybe not.

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34. MIke B. on December 17, 2013 9:13 PM writes...

Waste of time. Glycosylation and PTMs of glycoproteins are more finely tuned than just differences in glycan structure. Neuraminic acid also has multiple forms of acetylation, which can significantly impact protein function and cellular signaling. Glycans on glycoproteins, such as EPO, can also have different patterns of sulfation, which also affects biology. The point here is that there are further PTMs on top of glycosylation that may be even required for properly functioning biologics, and things like acetylation of sialic acid and sulfation at different points in glycan structures are notoriously labile, and likely wouldn't survive a total synthetic procedure. You need the machinery of whole cells to do the work for you.

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35. MIke B. on December 17, 2013 9:14 PM writes...

Waste of time. Glycosylation and PTMs of glycoproteins are more finely tuned than just differences in glycan structure. Neuraminic acid also has multiple forms of acetylation, which can significantly impact protein function and cellular signaling. Glycans on glycoproteins, such as EPO, can also have different patterns of sulfation, which also affects biology. The point here is that there are further PTMs on top of glycosylation that may be even required for properly functioning biologics, and things like acetylation of sialic acid and sulfation at different points in glycan structures are notoriously labile, and likely wouldn't survive a total synthetic procedure. You need the machinery of whole cells to do the work for you.

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36. KI on December 18, 2013 2:03 PM writes...

I wouldn't call this a waste of time. I think Sam states that the goal here isn't to overpower molecular biology. The goal is to learn about why nature glycosylates, the way it does...in an uncontrollable fashion.

He's developed routes, in order to develop tools, to answer biological questions.

Now, if he said this is the new way Amgen should make EPO...I'd say manure. But that's not his stated goal.

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37. Alisia on October 17, 2014 1:23 PM writes...

Make sure that you don’t end up looking out of proportion. As an advanced bodybuilder, protein should probably
be the number one food in your diet as it will
help to build additional muscle while generally not add to your waistline the way an over abundance of carbohydrates do.
It's best that you purchase these from the reliable sources over the Internet.
Doing so would set your bodybuilding plan back by days, if not weeks.
But this is reminiscent of the old analogy of "running east and looking for a sunset"; it's not going
to happen.

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