Oligo-Nucleotide Synthesis

The accepted definition of an oligonucleotide is a chain of 13-25 nucleotides.

Those of you in Biology programs have probably come across these in your studies. In CHM347, we are studying how to synthesize them. 

In particular, we're focusing on a solid-phase synthesis. This means that the first nucleotide in the sequence is anchored to a solid surface while the chain is being built. 

Let's first recap on the distinction between DNA and RNA. The D in DNA stands for 

de-oxy. Take a look at the sugars that constitute DNA and RNA on the right. 

DNA has two hydrogen atoms on C2, as compared to a hydrogen atom and an OH on C2 in RNA. DNA lacks an oxygen at C2, hence the term de-oxy!

Now that we're familiar with the difference, let's consider what constitutes a single nucleotide. There are three parts:

1. Nitrogenous Base
• Guanine
• Cytosine
• Adenine
• Thymine
2. 5-Carbon Sugar 
3. Phosphate Group

When synthesizing oligonucleotides, we need not concern ourselves with how to build nucleotides from scratch. Let's look at a synthesis for a three-membered nucleotide strand - 5" GTC 3"

Sequence of Events

Step One: You must first begin by customizing the 3" nucleotide, and then make your way towards the 5" end. The first move is to protect both of the alcohol groups on cytosine simultaneously. This can be done using trimethylsilyl chloride (TMSCl).

Step Two: Protect the nitrogenous nucleophile. Free amino groups in the nitrogenous bases in nucleotides have specific protection techniques (also see below): 

• Adenine
• Bz (Benzoyl) protection
• Cytosine
• Bz (Benzoyl) protection
• Thymine
• No protection required.
• Guanine
• Isobutyryl group protection

Cytosine requires benzoyl protection. This can be done using Ph-CO-Cl, which should cap the nitrogen.

Step Three: With the nitrogen protected, we can deprotect the oxygens and do some interesting things with them without nitrogen interfering. To deprotect both of the oxygens, we use NH4OH, I believe. This won't disrupt the amide we made in Step Two, since you can't climb up the stability ladder going from amide to ester.  

Step Four: Now, recall that this is a solid-phase synthesis, and so the first nucleotide must be latched to a silica gel surface. This involves the use of the 3' alcohol, not the 5'. So, we'll cap the 5' alcohol with some protection. This can be done using DMTCl (take a look at this group on Google Images) -- a very bulky group that is selective for the most available oxygen, which in this case is the primary 5' oxygen.

Step Five: The last remaining oxygen can be used to latch the nucleotide onto a solid surface. But before it is capable of doing so, it must be modified through an esterification with succinimide (a cyclic di-ester of a sort that can connect two nucleophilic centres together). Succinimide is a bridge, metaphorically speaking. In its linear form, it has an ester on both ends, which can be used to capture two nucleophilic groups. It modifies our 3' OH group in a way similar to what is shown in the photo below, except the phenyl ester is an amide instead.

The silica gel surface is denoted simply as 'NH2(CH2)3Si-Silica', from which the nitrogen will attack the amide on now modified 3' carbon centre. This reaction is facilitated using DCC (a peptide coupling reagent) and DMF. 

At this point we've secured the first nucleotide onto a solid surface. We can now begin adding pieces to our chain. 

Step Six: The next step is to remove the DMT protection, unveiling the nucleophilic OH on the 5' carbon centre. This deprotection is performed using dichloroacetic acid in dichloromethane (DCM).

Now, our nucleotide that's attached to our silica surface is equipped with a nucleophile from which it can latch onto another receptive nucleotide.

Step Seven: Now we move our attention to the second nucleotide in our chain, which in this case is Thymine. Notice that thymine does not need any protection on the nitrogenous base at all. We can get straight to work on modifying the 3' oxygen after protecting the 5' oxygen with DMT just as we did in Step Four.

Step Eight: The 3' oxygen needs to be linked to a phosphate group (which is part of the phosphate backbone we see in DNA). We throw in some ((iPr)2N)2-P-(OCH2CH2CN). The oxygen straight up attacks phosphate and displaces one of the secondary nitrogen groups '(iPr)2NH'. The product is a phosphoramidite, shown on the far left in the figure below.

Step Nine: The coupling reaction between the phosphorylated Thymine and the silica-bound Cytosine is catalyzed by Tetrazole. Notice that the Phosphite is not yet what a normal phosphate group looks like in DNA. It must be first be oxidized and then hydrolyzed in the following steps. 

Step Ten: The oxidation reaction of phosphate is performed with I2, H2O, and THF:

Step Eleven: We must deprotect the 5' oxygen of Thymine, so that it can be linked to our final nucleotide. The DMT removal is performed in the same way as in Step Six.

We've attached the two nucleotides together now. One of them (the first one) is still attached to the silica surface. It's time to add the last one; Guanine. 

Step Twelve: Guanine's oxygens must be protected first, because we need to cap the nitrogen on the base. We can use the anhydride I listed earlier for Guanine. 

Step Thirteen: Unblock the oxygens using NH4OH just like before. 

Step Fourteen: Protect the 5C OH using DMTCl.

Step Fifteen: We need to add phosphate to the 3C OH. Same reagents as before, check step Step Eight. 

Step Sixteen: Couple Guanine with Thymine using a tetrazole catalyst in the same way as before. 

Step Seventeen: Oxidize the phosphate group -- I2, THF, H2O.

I think its way more important to understand why we have to take each one of these steps than to memorize the reagents. You can look up reagents in the blink of an eye if you need them. You can't always Google up an understanding in 2 seconds, so its better to have that under your belt. 

Shown on the left is a perfect example of how during the global deprotection all the protecting groups are removed, the first nucleotide is removed from the silica surface, and the phosphate group loses its long substituent in exchange for an OH substituent; now looking more like your typical phosphate group in DNA! 

Step Eighteen: Global deprotection, my favourite. Just nuke the whole town with aqueous NH3 and leave it for a day. 

All protection groups removed, and cytosine leaves from the solid surface. Done. 

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The illustrations in this post have been abstracted directly from Organic Chemistry by John McMurry (9 ed.)