An Update: July 24th 2019

Hi everyone,

This website has been put back online as an educational resource for Leigh Revers' current chemistry students. The two most important resources are the practice problem sets which can be accessed using the header link above and the daily blog posts along the left-hand sidebar. The links in the individual blog posts are broken, so if you're looking for a particular problem set it's going to be in the problem set folder.

If anyone has any questions or suggestions, I can be contacted by email at: ramis.nazir@mail.utoronto.ca.

Good luck!


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A Quick Update!

Hi everyone,

I'm a bit late to posting this, but I think all my readers should be aware that as of right now I am not in any chemistry courses, and so I don't have any content to write about for the time being.

However, I am regularly updating my Instagram page @chemistryfamous, where I showcase the structure and functions of different compounds in the universe. Check out my work there.

To those of you who are currently using the blog for the CHM243 series, I hope that my content has been of value to you. If you can, please get in touch with me and give me your feedback! My email is ramisnazir@gmail.com. You can also find me on Facebook.

Good luck!

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.)

Preliminary Carbohydrate Protection

In my relatively limited studies of carbohydrates, I've gotten to know a fair bit about these cyclic buggers.

When dealing with carbohydrates, a fair bit of it involves selectively protecting the various substituents stemming from the cyclic carbon backbone.

Why bother? Well, it's because when you're making your own carbohydrates you're interested in coupling specific substituents with other carbohydrates. The only way you can achieve specificity in your coupling reactions is with protection.

So, I'm going to list the most important carbohydrate protective methods I've learned in my CHM347 class:

Acetylation
There are three ways you can go about acylating any available OH substituents on your carbohydrate.

1. Acetic Anhydride & Pyridine
2. Acetic Anhydride & Sodium Acetate @ 100C
3. Acetic Anhydride & ZnCl2
Deprotection: Zemplen Degredation -- NaOMe / HOMe

Why are there so many methods: I might want to acylate some free OH groups while preserving my protection at another site. A lot of protection chemistry involves versatility because, well, you need it. Depending on what the carbohydrate of interest has elsewhere, I might selectively pick one of the three techniques available to me.


Fischer Glycosidation
1. MeOH in acidic conditions (dry - anhydrous - hydrochloric acid)
This method specifically protects the anomeric (C1) hydroxy group of the carbohydrate.
Deprotection: Acid


Ether Protection
Ethers are good protecting groups because they use sterics. Additionally, there are different types of ethers, each with unique methods for deprotection.

1. NaH & BnBr
This converts all open OH groups into "OBn".
Deprotection: Catalytic Hydrogenation (H2 Pd/C) -- good if you've got other groups that are not sensitive to catalytic hydrogenation..

2. Ph3CCl (Trityl Chloride) 
This reagent selectively protects the free OH of the 6th most accessible carbon atom of the carbohydrate.
Deprotection: Mild acid (AcOH)

3. TMSCl (Trimethyl Silyl Chloride) 
Converts all available OH groups into "OTMS".
Deprotection: TBAF or acid

4. TBDMSCl (T-butyl Dimethyl Silyl Chloride)
Much like TMSCl, but with more steric hindrance provided. As such, this protecting method is also selective for the OH on the most accessible 6th carbon of the carbohydrate.
Deprotection: TBAF or acid


Ester Protection
There are two ways to protect carbohydrates through esterification.

1. BzCl (Ph-CO-Cl)
This method protects every free OH group on the carbohydrate, since it is a reasonably small protecting group.
Deprotection: Zemplen Degredation (see above) or acid.

2. TsCl (Tosyl Chloride) + Pyridine 
TsCl is a bulky protecting group, and as such, it is selective for the most accessible 6th carbon of the carbohydrate.
Deprotection: Acid

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The next post: A more complicated approach to selectively protecting more than one OH group in one step.

Carbohydrate Chemistry Series

Hello,

I haven't mentioned this in the past but I am actually enrolled in an Organic Chemistry of Biological Compounds course.

I want to make a directory of all the topics I've covered in my class on this blog. Personally, it will be more for my own use than yours, but I hope that people who Google the subject topics will also find utility in the content I'm about to begin posting.

I'm going to create a series on the sidebar dedicated to the subject matter that pertains to Carbohydrate Chemistry. Later in the month, I might create another series for Peptide Chemistry; corresponding to the units covered in the semester.

Lidocaine Synthesis

Synthesizing this compound was actually really tricky. It looks simply on paper but finding a way to have all three benzene substituents be right next to one another was a challenge.

I learned about Lidocaine in my Neurobiology course. It's an anesthetic that blocks the influx of sodium cations through the membrane of nerve cells. If less sodium cations enter the nerve cells, the interior can't depolarize as much, and there less action potentials are fired.


Check my complete synthesis in the image below:

Ritalin Synthesis

Ritalin is a medicine that contains methylphenidate, a chemical that interacts with the central nervous system; specifically nerves responsible for impulse control and hyperactivity. It's common for Ritalin to be administered to patients with ADD, ADHD, and narcolepsy.

Methylphenidate's structure is fairly simple, actually. It's got two cyclic structures and an ester group. To synthesize this molecule, we'll need to obtain a benzene ring, a heterocyclic group containing nitrogen, and a malonic ester. Let's get started:

Step 1: Treat a malonic ester with sodium ethoxide (so as not to disrupt the ester group) and mix in some bromopiperidine (shown as the reagent in Step 1). The reaction will proceed in an SN1-type fashion, and I reckon we might get a mixture of products because sodium ethoxide can attack bromopiperidine... I think it's unlikely though because C=N is softly electrophilic, and enolates are softer than ethoxides, so soft goes with soft and the ethoxide would rather act as a base.

Step 2: Next, use H30+ and heat to decarboxylate. We're now left with an amino acid.

Step 3: Use two equivalents of triethylamine and TMSCl to create an enol equivalent of the amino acid. I used to equivalents because the acidic oxygen in the amino acid might compete for the trimethylsilyl group.

Step 4: Prepare a benzene ring electrophile stabilized by N2 through nitration, reduction, and subsequent treatment of benzene with NaNO2 and HCl.

Step 5: Mix the enol equivalent of the amino acid with the benzene electrophile to facilitate an attack.

The reason I used an enol equivalent instead of an enolate equivalent is because I'm worried that an enolate equivalent might attack from the harder oxygen end since the benzene electrophile is most likely hard, too. Using an enol equivalent using TMSCl would protect the oxygen, making attack from the carbon more likely. I'm not 100% sure whether the benzene electrophile is considered hard or soft.

Step 6: Work-up to hydrolyze any TMS-protected acidic oxygens from Step 3.

Step 8: Add methanol to the product and keep removing water to drive the esterification forward.

Ta-Da. Done. We've successfully prepared the main component of Ritalin, methylphenidate.