Fmoc Protecting Group: Fmoc Protection & Deprotection Mechanism

You can zoom and play around with this 3D model of the Fmoc protecting group!
Can you find the acidic C-H bond?

What is the Fmoc Protecting Group?

Fmoc is a fluorenylmethoxycarbonyl group that forms carbamates with amines. However, as a common theme, alcohols and other nucleophiles can also be protected.

Fmoc was introduced by Carpino in 1972 [1]. You will realize that this is pretty late in the game! Few base-sensitive amine protective groups were known. Chemists obviously already used protecting groups, but they were not as straight-forward. For example, the tosylethyloxycarbonyl group (base-labile with KOH/NaOEt) gave the stable carbamate salt which required a second step for decarboxylation.

Fmoc Protection Mechanism

The classic Fmoc protection is with Fmoc-Cl under Schotten-Baumann conditions (e.g. NaHCO₃/dioxane/H₂O or NaHCO₃/DMF), or with anhydrous conditions (e.g. pyridine/CH₂Cl₂).

If you have seen more than one protecting group, this will not surprise you:
The mechanism is attack of the nucleophilic amine to the highly reactive 9-fluorenylmethyl chloroformate. As chloride is the leaving group, the reaction liberates HCl which is neutralized by the base.

Fmoc-Cl can be handled easily (it’s a solid) – however, as it’s an acid chloride, is sensitive to moisture and heat. Thus (as for all protective groups), other “Fmoc+”-equivalent reagents offer more optionality.

Fmoc-OSu is most commonly used nowadays due to the increased stability of this succinimide carbonate. It also has lower unproductive formation of oligopeptides that can occur during preparation of Fmoc amino acid derivatives.

Additional options exist such as Fmoc-OBt or Fmoc-N₃. However, you would rather deal with some harmless solids than explosive azides…

Fmoc DeProtecTION Mechanism With Base

Fmoc is typically deprotected with secondary amines in DMF. The mechanism has some parallels to Boc. Instead of a stabilized (tertiary) carbocation as an intermediate, Fmoc proceeds through a fluorenyl anion. But why is it stabilized? The position might not seem acidic at first sight.

Upon closer inspection, we see the deprotonated system fulfils Hückel’s rule for n=3 (14 electrons) and is aromatic! That’s why the pKa of fluorenyl is around ~23 (DMSO). This is basically a cyclopentadiene anion (whose aromaticity you will know) sandwiched between two benzene rings.

The intermediary carbanion can eliminate the carbamate in a E1cb mechanism, releasing dibenzofulvene. This side product lead to byproducts (e.g., reaction with nucleophilic amino acid groups) or polymers. This is why secondary amines like piperidine or morpholine are particularly handy!

They hit two birds with one stone. They cleave Fmoc, and also form a stable adduct with the dibenzofulvene. The “secondary” part is quite important as ammonia does not add to the fulvene system [1].

Fmoc DeProtecTION Speed

There is another reason why piperidine is the most commonly used base to deprotect Fmoc. This table compares half-lives for Fmoc-ValOH in presence of various amine bases in DMF.

Amine base used for Fmoc deprotection	Half life t1/2
20% piperidine	6 seconds
5% piperidine	20 seconds
50% morpholine	1 minute
50% dicyclohexylamine	35 minutes
50% diisopropylethylamine	10 hours

Piperidine (and morpholine) deprotection is virtually instantaneous on the second-scale. In contrast, secondary or tertiary amines deprotect Fmoc more slowly (hours) and require higher amounts of base. If you wonder, going from DMF to other solvents like DCM reduces the reaction rate.

Fmoc protecting group Orthogonality

Fmoc is very stable towards acid and electrophiles, tolerating reactive reagents like HBr, trifluoroacetic acid, sulfuric acid and thionyl chloride. Thus, its orthogonal to Boc.

However, it is only quasi-orthogonal to Cbz as it undergoes hydrogenolysis as well! It is less reactive than benzyl groups, so selectivity can be achieved. The reduction can occur under traditional (Pd/C, H₂) or different transfer catalytic conditions. The final step of the synthesis of Enkephalin was triple-deprotection of O-Bn, CO₂-Bn and N-Fmoc.

Question for you: Enkephalin is a human neuropeptide which binds to the body’s opioid receptors. What is the amino acid sequence of the enkephalin form here?

Fmoc Variants

Let us again look at some more advanced concepts. There are Fmoc-related variants that are more base-labile. By attaching electron-withdrawing substituents like sulfonic acid or halides.

What are the effects? Specifically, Sulfmoc increased proton abstraction by a factor 30 in DCM (vs. Fmoc) using 10% morpholine in DCM, or factor ~10 for 10% piperidine [3]. In specific cases, such labile groups might be pretty useful.
By the way, Sulfmoc was introduced by Merrifield who won the Nobel Prize for inventing solid-phase peptide synthesis.

Evidently, this comes from acidification of the fluorenyl position. The 2,7-dibromo Fmoc analog has a pKa value of 17.9 or almost 5 units lower than normal Fmoc!

However, there’s an even cooler analog, also published by Carpino called Bsmoc. It can be cleaved under specific conditions which leave normal Fmoc in tact, but typical conditions with piperidine work as well [4].

Another test for your skills: What is the mechanism of N-Bsmoc cleavage with secondary amines? Don’t scroll to the answer below before thinking about it!

Fmoc in Peptide Synthesis

Fmoc was rapidly adopted in modern peptide chemistry [5]. Compared to the established Boc, it was easy to automate: no corrosive TFA is required, and reaction monitoring is easy due to the fluorene by-product (see deprotection). Fmoc SPPS machines were less expensive and avoided use of unpleasant hydrogen fluoride (HF). The conditions themselves were more compatible with modified peptides (e.g., modification with carbohydrates, phosphorylation…).

As another advantage, the Fmoc protecting group enables orthogonal combination of temporary and permanent protecting groups. During Boc SPPS, iterative use of TFA during each cycle leads to deprotection of small amounts of side-chain protecting groups and cleavage of peptide from polymer support.

Bsmoc solution

Let’s conclude with Bsmoc.

The innovative thing is that it functions as a protecting group and scavenger in one!
It’s introduction is analogous to normal Fmoc, using the chloroformate or H-hydroxysuccinimide ester. Instead of a deprotonation with piperidine, we have a Michael addition to the conjugated sulfone.

The free carbamate proceeds to decarboxylate as always, but the piperidine stays on the Bsmoc group (thus, it’s a direct scavenger). You might not expect it but it’s very logical: The initial adduct rearranges after some time to the isomer where the double bond is conjugated to the benzene ring.

The ‘quasi-orthogonal’ conditions for Bsmoc-Fmoc are tris(2-aminoethyl)amine as a base. This primary amine cleaves Bsmoc rapidly while keeping Fmoc in tact. On the flip side, use of more hindered bases like diisopropylamine do not react with Bsmoc but cleave the Fmoc group! This is a consequence of steric hindrance slowing down the nucleophilic Michael addition.

I hope you learned something new today!

Fmoc Protection experimental procedure [6]

D-Threonine (5.00 g, 42.0 mmol) and Fmoc-succinamide (14.9 g, 44.1 mmol) were dissolved in a 2:1 v/v mixture of THF:saturated aqueous NaHCO3 (100 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction was then diluted with water (50 mL) and the pH of the mixture was adjusted to pH 9 via addition of saturated aqueous NaHCO3. The mixture was extracted with diethyl ether (3 x 50 mL) and the aqueous layer was acidified to pH 1 via addition of 1 M HCl. The acidic aqueous mixture was extracted with ethyl acetate (3 x 100 mL) and the combined organic extracts were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford crude Fmoc-D-Thr-OH (14.3 g) as a white foam which was deemed to be sufficiently pure and used without further purification.

Fmoc deprotection experimental procedure [7]

In a vial, SM (2043 mg, 2.5mmol) was added and dissolved in 60 mL of acetonitrile. Then, morpholine (647 uL, 7.5mmol) was added while stirring. The reaction was stirred at room temperature for 24 hours, formation of product and full conversion was confirmed by LC-MS. The reaction was quenched by addition of water and extracted with DCM. The organic 9 phases were combined and washed with aqueous LiCl 5%, dried with sodium sulphate and filtered. The solvent was evaporated and crude product the crude product was purified by silica gel flash chromatography (0-5% MeOH in DCM).

Fmoc Protecting Group References

• [1] L. A. Carpino, G. Y. Han, J. Org. Chem. 1972, 37, 3404 | The 9-Fluorenylmethoxycarbonyl Amino-Protecting Group
• [2] P. Wuts, T. Greene, Greene’s Protective Groups in Organic Synthesis (Wiley)
• [3] R. B. Merrifield, A. E. Bach, J. Org. Chem. 1978, 43, 4689 | 9-(2-Sulfo)fluorenylmethyloxycarbonyl chloride, a new reagent for the purification of synthetic peptides
• [4] J. Am. Chem. Soc. 1997, 119, 41, 9915 | New Family of Base- and Nucleophile-Sensitive Amino-Protecting Groups. A Michael-Acceptor-Based Deblocking Process. Practical Utilization of the 1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc) Group
• [5] R. Behrendt, P. White, J. Offer, J. Pept. Sci. 2016, 22, 4 | Advances in Fmoc solid‐phase peptide synthesis
• [6] Org. Lett. 2016, 18, 2788 | Total Synthesis of Teixobactin
• [7] Org. Lett. 2016, 18, 3810 | Total Synthesis of Gombamide A
• J. Pearson, W. R. Roush, Handbook of Reagents for Organic Synthesis: Activating Agents and Protecting Groups (Wiley)
• P. Kocienski, Protecting Groups (Thieme Verlag)