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Topic: Synthesising dNTP with 3 prime phosphate  (Read 4520 times)

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Offline PeterGC

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Synthesising dNTP with 3 prime phosphate
« on: February 01, 2017, 08:04:53 PM »
So I have a long standing interest in diybio and especially the rather challenging tasks of sequencing and synthesising DNA. The basic process that can be used to do both these things is enzymatic polymerisation. Deoxynucleotide triphosphates (dNTPs) form the basic material for these reactions and are more or less readily available. Normally to sequence DNA a small number of modified dNTPs are used in which their hydroxyl group has been removed (ddNTPs). This irreversibly blocks DNA synthesis once a ddNTP has been incorporated into the strand. However this also makes ddNTPs very toxic to living cells, in fact it's used as a chemotherapy agent.

Now a lot of people in the diybio community would very much like do things like DNA sequencing but don't really want to handle supper toxic chemicals. It's also posable to replace the hydroxyl group with a phosphate group. These groups exist on the end of DNA strands in the body as a result of ordinary wear and tear and the cell has an enzyme to swap it back to a hydroxyl group as a repair mechanism. However as long as the phosphate group is there it blocks synthesis as well as removing the hydroxyl group all together. My supposition is a this phosphate group modified dNTP would be a lot less toxic than the ddNTP alternative. It would also allow the DNA polymerisation process to be stoped then restarted by removing the phosphate group. This way you could literally add a base at a time which facilitates synthesis of new specific DNA sequences from scratch.

So synthesising these phosphate group modified dNTPs from standard dNTPs is something I've been thinking about. So I've been reading up on organic synthesis and have come up with some ideas on how to do it, but honestly chemistry isn't my thing and I’m not quite there so I wanted to pick your brains.

My idea is to initially treat the dNTPs with Lucas' reagent to swap the hydroxyl group for a chlorine atom. After that I’m kind of stuck. The Michaelis-Arbuzov reaction with Phosphorous acid would replace the Cl with -PO3 but I need -O-PO3. I’m not sure what reaction would be suitable for that? Any sugestions?

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #1 on: February 03, 2017, 11:54:21 AM »
Maybe a reaction involving phosphorus pentoxide (P4O10)? This https://hal.inria.fr/hal-01186950/document seems to suggest that phosphorus pentoxide in combination with phosphoric acid can add phosphate groups to hydroxyl group sites. But I'm unsure what kind of solvent would be suitable since phosphorus pentoxide reacts violently with water. Any ideas?

Offline Yggdrasil

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #2 on: February 03, 2017, 04:32:16 PM »
I don't think ddNTPs are super toxic.  These are frequently used in molecular biology labs without much trouble.  Reversible chain terminators are frequently used in next generation sequencing methods (e.g. see http://www.sciencedirect.com/science/article/pii/S1672022913000077).

Artificial DNA synthesis is often performed via solid phase synthesis using the phosphoramidite method (see http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis#The-Phosphoramidite-method or https://en.wikipedia.org/wiki/Nucleoside_phosphoramidite), which is somewhat similar to what you would like to synthesize.

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #3 on: February 04, 2017, 09:07:14 AM »
Not really I mean Zalcitabine the very toxic aids drug is basicly converted in T cells to ddCTP which blocks HIV reverse transcription by terminating the reverse transcribing HIV DNA. https://en.m.wikipedia.org/wiki/Zalcitabine That's when the active ddCTP is only really found in one cell type. It's not something you want floating round your body.

If you go to buy some ddNTPs and look at the safety sheet you'll mostly see not tested or not known or some variation under most headings. I'm lead to believe it's pretty toxic. As a student they let me handle some fairly toxic stuff but they never let us touch that.

Be that as it may the dNTP-3'-OPO3 would have other uses. You can basicly forget any chance of getting hold of highly patented reversible chain terminating dNTPs for diybio use. It's hard enough to get ordinary dNTPs. Diy bio is building a culture of self reliance and ultimately this is going to mean doing some chemistry for our selves. Well a few of us who feel brave will and the community as a whole will benefit.

Which brings us back to putting phosphate (PO4) groups onto the 3' groups of dNTPs. The reagents used in 5' phosphorylation in the phosphoramidite method are Complex organic molecules that seem hard to acquire to me and probably very hard to synthesise. But the other issue is in the phosphoramidite method the DNA has protecting groups. I am concerned the standard reagents might interfere with the structure of unprotected nucleotides. Tbh I don't understand their chemistry enough to know if they would.

I'd like to know if they could be used but on balance I'm looking at processes which only involves access to well known simple compounds.

Offline Yggdrasil

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #4 on: February 04, 2017, 11:15:52 AM »
As long as you aren't eating the stuff, there's not too big a concern about working with ddNTPs.  Because they're highly charged, they're not going to be able to enter the body or cells very easily.  However, I could understand the worry about storing the materials at home.

Because they are patented, the details about the synthesis of reversible chain terminating nucleotides is publically available if you dig around for it.  You may also have better luck in the Organic Chemistry Forum with advice on the synthesis as most of the people with experience in synthesis don't drop by this board.

Offline rolnor

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #5 on: February 04, 2017, 01:31:07 PM »
There is a ot of phosphatases in the bodyfluids that will breakdown any triphosphate so toxicity is not a big problem. Triphosphates are very reactive so there is very little chemistry you can do without breaking them down am afraid.

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #6 on: February 04, 2017, 02:08:03 PM »
As long as there is one phosphate group left on the 5' end that's actually not a deal breaker. We can add 2 more on to remaining phosphate group enzymatically after we're through adding a phosphate group to the 3' end. Unfortunately there is no enzyme to add a group directly to the 3' end. Believe me if I could find an enzyme to do it that would suit me much better. I prefer to let them to the heavy lifting. So using a reagent to put the phosphate group onto the 3' end and then an enzyme to convert it from a mono to tri 5' phosphate would be fine.

Offline rolnor

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #7 on: February 05, 2017, 06:56:00 AM »
You can make a 3,5-diphosphate with phosphoroxychloride, I have experience with making 5-triphosphates enzymaticaly and it was messy, you get the triphosphate in a mixture with ATP. If you dont want to handle toxic or corrosive chemicals, then phosphoroxychloride is not sutable.

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #8 on: February 05, 2017, 12:41:15 PM »
I'm willing to accept a fair degree of toxicity so long as I don't have to store anything toxic long term. Now this is where I display my ignorence. When you say phosphoroxychloride do you mean POCl3? If so isn't there a risk of it bonding 2 or 3 nucleotides together through the same phosphor atom? Most reaction I've seen with POCl3 tend to bond 3 molecules together.

Offline rolnor

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #9 on: February 05, 2017, 03:33:25 PM »
POCl3 Containes thre chlorine atoms, if you react one of them with an alcohol the remaining two chlorines will be considerably less reactive then the first.
I have a fealing that this chemistry will be chalenging for you, you need to analyze and monitor the reaction, this will be best done by LC-MS. You will get byproducts and need to purify, this is done by DEAE-Sephadex-chromatography with a triethylaminehydrogencarbonate buffer as mobile-phase. It would probably be cheaper to buy the diphosphate, I think its commersialy available.

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #10 on: February 06, 2017, 09:02:07 AM »
Yeah realistically there's no chance of me getting hold of a mass spec. And for my self I think paying some one to make it for you makes sense if you can and the price isn't stupid. But a lot of DiyBio people find chemical / biotech companies won't sell to them or will only sell certain things. It's always good to have a garden shed friendly receipy in case you sudenly find you can no longer buy reagent X. Which of course means the the recipy has to work with the kind of gear you can buy off amazon and stuff you can get through non specialist suppliers.

It's a kind of 'think how a chemist in 1900 would have tried to do this' approach.

Offline rolnor

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #11 on: February 06, 2017, 09:37:36 AM »
I have looked for this diphosphate but I can not find a comersiall source, sorry.

Offline wildfyr

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #12 on: February 07, 2017, 05:08:52 PM »
Boom "One-pot synthesis of organophosphate monoesters from alcohols" http://www.sciencedirect.com/science/article/pii/S0040403913001068 This paper is full of references in case you don't like their approach.

For their procedure, which is in acetonitrile, I suggest you do the reaction and accept that conversion likely won't be 100% and youll have a mixture of phosphorylation that perhaps you can use some fancy biology tricks to separate out. or perhaps a large excess of reagents will get you full conversion of all alcohols to PO4. Do the reaction, gently evaporate the acetonitrile and trichloroacetonitrile after the reaction, then you can precipitate the DNA in something polar and protic to remove tetrabutylammonium hydrogen phosphate.

Unfortunately they do something some organic papers do and don't actually frickin give a specific example of their reaction with all the precise reagent ratios (I checked the supplementary info too)

Mod edit: Link modified due to copyright infringement. Dan
« Last Edit: March 26, 2017, 09:30:39 AM by Dan »

Offline PeterGC

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Re: Synthesising dNTP with 3 prime phosphate
« Reply #13 on: February 08, 2017, 12:00:06 PM »
you sir, rock, I doff my hat to you.

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