Tag: Protecting Groups

What is the TBS Protecting Group?

TBS or TBDMS is short for tert–butyldimethylsilyl, a protecting group for alcohols.
TBS was introduced by the legendary E. J. Corey in 1972 [1] as an evolution to simpler silyl ethers which had already been known. Note this was right around the time of Fmoc discovery, and relatively late in the history of organic synthesis.

The research already covered protection and deprotection conditions still used today. It is one of the most cited JACS publications ever. A decade later, Corey also introduced triflate reagents for silyl ether synthesis [2].

TBS Protection Mechanism

The most common TBS protection conditions are TBS-Cl (forms hygroscopic white crystals) in DMF with imidazole or DMAP.
Corey encountered challenges using TBS-Cl for protection of tertiary or hindered secondary alcohols. Use of TBS-OTf triflate (with 2,6-lutidine as base in solvents like dichloromethane) proved more potent for such cases.

The classic mechanism contains generation of an imidazolium intermediate which acts as a silyl transfer reagent for the actual silylation.
The steps might look counterintuitive. Silicon, in contrast to carbon, can have five bonds at once! Both silylations likely proceed through associative substitutions with pentavalent silicon intermediates. However, you’re likely also fine drawing a concerted step.

Silyl ether protecting group Stability

Many different variants of silyl ethers exist. Despite their overall similarity, they can behave quasi-orthogonal due to different lability. Below, you can see the relative stability towards acidic and basic aqueous hydrolysis.

Silyl ether protecting group	Stability to acid	Stability to base
TMS (trimethylsilyl)	1	1
TES (triethylsilyl)	~60	~10-100
TBS (tertbutyldimethylsilyl)	~20’000	~20’000 (likely higher as basic stability > acidic)
TIPS (triisopropylsilyl)	~700’000	~100’000

As you can see, bigger silyl ethers are more stable than smaller ones. This stability directly shows – TBDMS ethers are stable to chromatography and survive various reaction conditions which smaller ethers do not. With different labilities, chemists can deprotect TMS or TES ethers in presence of TBS protecting groups (more below).

TBS Deprotection Mechanisms

There are three types of deprotection mechanisms for TBS and silyl ethers in general.

1. Acidic hydrolysis works through protonation of the protected oxygen atom, followed by associate hydrolysis with a pentavalent silicon intermediate.

2. Fluoride-mediated deprotection is based on nucleophilic attack onto silicon, giving a pentacoordinate siliconate intermediate that can release our deprotected product. This is like the imidazole catalysis we’ve seen during TBS protection.

The thermodynamic driving force is the formation of the exceptionally strong Si-F bond (>30 kcal/mol stronger than Si-O) bond. For fluoride-mediated deprotection, TBAF (tetrabutylammonium fluoride) is the archetypical fluoride source. However, other sources like HF or amine-HF complexes are also commonly used.

3. Basic hydrolysis can theoretically also occur with TBS ethers, even though have high stability towards bases. However, at very forcing basic conditions, TBS ethers can hydrolyse (e.g., excess LiOH, dioxane, EtOH, H₂O, 90 °C).
The mechanism is also based on nucleophilic addition to silicon, followed by Si-O bond breaking.

Selective Deprotections of silyl ethers

So, we have mentioned that selective deprotection of silyl ethers might be possible. The first category of reactions follows the steric stability we laid out above. Let’s look at some examples [4].

Pyridinium p-toluene sulfonate (PPTS) in protic solvents like MeOH is the mildest system for deprotection of TBS ethers. In this example, you can see that the conditions are controlled enough so that the bulkier TIPS group does not fall off. The same would go for TBDPS (tert-butyldiphenylsilyl).

The next step in the synthesis is addition of K₂CO₃ to the diol. What is the product?

This next example is sneaky. Don’t be fooled by seeing “TMS” and thinking it will immediately be less stable than TBS! Upon close inspection, we realize that this is actually a alkyl silyl group, part of the so-called SEM protecting group. It can be removed through fluoride anions or more forcing acidic conditions.

Another question: Propose a mechanism the deprotection of SEM with TBAF!

Welcome to chemistry, it’s sometimes random

The second category of selective reactions does not follow any obvious rules. It’s really experimental randomness. A commonly cited example is from the synthesis of zaragozic acid C [5].

During this effort, it was possible to differentiate between two very similar TBS protecting groups. The use of dichloroacetic acid in MeOH was mild enough to selectively (90% yield) cleave only one of the TBS groups.

That same synthesis actually also nicely demonstrated the concept of selective protection based on steric hindrance. After full consumption and protection of the primary alcohol with TBS-Cl, the chemists threw in TMS-Cl to protect the tertiary hydroxyl group as well.

There’s a recent example (2024) of TBS randomness [6]. In the last synthetic steps towards (+)-heilonine, the authors performed a Clemmensen reduction with Zn in HCl to reduce the ketone. It would have been nice if this one-two punch would have directly delivered the natural product. However, for no apparent reason, only one of the two TBS groups fell off. So, a separate deprotection step with TfOH was required.

That’s it for TBS. Learn more about other protecting groups, or watch my educational videos for advanced science content!

TBS Protection experimental procedure [7]

“To a solution of the diol (57.48 g, 0.354 mol) in DMF (354 mL) were added imidazole (96.52 g, 1.418 mol) and TBSCl (160.27 g, 1.063 mol) at rt. After stirring at 50 °C for 17h. H2O was added and the mixture was extracted with Et2O. The organic layer was washed with brine, dried over MgSO4, and evaporated in vacuo. The residue was purified by flash column chromatography (SiO2; n-hexane:EtOAc = 10:1) to give the diTBS ether (138.48 g, 100%).”

TBS deprotection experimental procedure [7]

“To a solution of the alcohol (45.2 g) in THF (350 mL) was added TBAF (1.0 M in THF, 184 mL, 0.184 mol) at rt. After stirring at rt for 18 h, the mixture was concentrated in vacuo. The residue was purified by flash column chromatography (SiO2; n-hexane:EtOAc = 4:6→EtOAc) to give diol (29.1 g, 97% yield, 2 steps).”

TBDMS Protecting Group References

• [1] Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972, 94, 6190 | Protection of hydroxyl groups as tert-butyldimethylsilyl derivatives
• [2] Corey, E. J.; Cho, H.; Rücker, C.; Hua, D. H. Tetrahedron Lett. 1981, 22, 3455 | Studies with trialkylsilyltriflates: new syntheses and applications
• [3] T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis (Wiley)
• [4] P. Kocienski, Protecting Groups (Thieme Verlag)
• [5] Carreira, E. M., Du Bois, J.; J. Am. Chem. Soc. 1995, 117, 8106 | (+)-Zaragozic Acid C: Synthesis and Related Studies
• [6] J. Am. Chem. Soc. 2004 ASAP | Convergent and Efficient Total Synthesis of (+)-Heilonine Enabled by C–H Functionalizations
• [7] J. Am. Chem. Soc. 2004, 126, 14374 | Total Synthesis of Brevetoxin-B

What is the Fmoc Protecting Group?

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

Fmoc was introduced by Carpino in 1972 [1]. At that time, 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.

Fmoc: ✅ check. Learn more about other protecting groups, or watch my educational videos for advanced chemistry & science content!

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)

What is the Cbz Protecting Group?

Cbz is a benzyloxycarbonyl group (formerly carboxybenzyl) and protects amines as carbamates. More rarely, chemists might use Cbz to protect alcohols as their carbonates.

Leonidas Zervas (no not the one from the movie “300”) introduced the Cbz group, thus also abbreviated as Z [1]. With this discovery, Leonidas and his advisor Bergmann spearheaded the field of controlled peptide chemistry. In the 1930s, Cbz unlocked the synthesis of previously inaccessible oligopeptides. Zervas continued to be a driving force in peptide chemistry, including development of other protecting groups.

Cbz Protection Mechanism

Cbz protection is typically performed with Cbz-Cl either under Schotten-Baumann conditions (carbonate base) or with an organic base. The mechanism is attack of the nucleophilic amine to the highly reactive chloroformate. As chloride is the leaving group, the reaction liberates HCl and requires some base.

Like we’ve seen for Boc, Cbz₂O or other activated agents (e.g., Cbz-OSu) can offer more optionality, depending on the system. If you are into exotic reagents, you might like reagents A or B in the next figure – basically, anything with an activated “Cbz+” synthon works.

As is common the case, we can protect amines selectively given their higher nucleophilicity. However, there have been some reports of challenging selective protection of secondary amines over secondary alcohols. As always, our rules of thumb depend on the specific system at hand.

Cbz DeProtecTION Mechanism With Hydrogenolysis

Hydrogenolysis deprotects Cbz protecting groups, usually in an easy and rapid manner. The mechanism is a reduction with H₂, releasing toluene and the free carbamate. Consequently, decarboxylation to the deprotected amine is very much favoured (particularly at elevated temperatures).

Besides molecular H₂, it is possible to use other “H₂ donors” through transfer hydrogenation reactions (e.g., see procedures below).

Cbz protecting group Orthogonality

Cbz is orthogonal to Boc, Trt, Fmoc and other common protecting groups. As mentioned, it was an essential part of the early days of peptide synthesis.

But beware thinking it’s fully orthogonal! Although Cbz tolerates some acid, harsh conditions can cleave it as well (e.g., excess HCl, HBr)! This mechanism includes protonation of the carbamate and liberation through S_N2 and decarboxylation.

Beyond hydrogenation, Cbz can be susceptible to other transition metal catalysis as well, for instance Ni(0) or Pd(0). This interesting case report demonstrated selective removal of double Cbz-protected histidine [2] . Compared to heteroaromatic nitrogen atoms, originally basic amines did not engage in any reaction.

This is a great question for you: Suggest a mechanism for the reaction (note the other reagent!) and propose an explanation for the selectivity!

Cbz use cases and tricks

As always, it would be boring to just look at the simplest case of nitrogen protection and deprotection. Let’s briefly discuss three additional topics [3].

First, Cbz protects other nucleophilic functional groups like alcohols, phenols, thiols… as well. In these cases, an organic base (not carbonate) in dichloromethane or some ether-based solvent is typically used. To increase reactivity, NaH as base allows for protection of deactivated, tertiary alcohols:

Notably, Cbz-Cl as a reagent can activate pyridines to regioselective nucleophilic attack. The use of electrophiles for these purposes is a key concept in heterocyclic chemistry. There is a nice collection on 1-acylpyridiniums by the Baran Lab.

Third and coolest, the Cbz group can serve as a masked N-methylamine. Treatment with LiAlH₄ exhaustively reduces the carbamate to the alkane.

✅ That’s it for Cbz! Learn more about other protecting groups, or watch my educational videos for advanced chemistry & science content!!

Cbz Protection experimental procedure [4]

“To the SM (1.70 g, 2.64 mmol) in THF/H2O (2:1, 15 mL) was added NaHCO3 (443 mg, 5.27 mmol) and Cbz-Cl (0.56 mL, 3.96 mmol) at 0 °C and the solution was stirred for 20 h at the same temperature. The reaction mixture was diluted with H2O and extracted with AcOEt. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (40% AcOEt/n-hexane) gave 20 (1.85 g, 2.38 mmol) in 90% yield as a white powder.”

Cbz deprotection experimental procedure

[4] Normal hydrogenolysis
To a solution of SM (20.7 mg, 15.0 micromol) in 2 mL of MeOH were added 5% Pd-C (6.4 mg), and the mixture was stirred at 60 °C for 40 h under atmospheric pressure of H2. Then, the catalyst was filtered of on pad of celite. The filtrate was concentrated in vacuo to give a crude material containing 27, which was used without purification for the next step.

[5] Deprotection of 5 Cbz groups in the final step via transfer hydrogenation.
To a solution of protected caprazamycin A (32) (6.5 mg, 3.21 micromol) in EtOH/HCO2H (1.9 ml, v/v = 20:1) was added Pd black (66.5 mg, 625 micromol) at 25 °C, and the resultant mixture was stirred for 1.5 h at 25 °C. After filtration through celite, the filtrate was concentrated under reduced pressure. The resultant residue was purified by C18 reversed phase. Caprazamycin A eluted at 11-18 min as an isolated peak. The eluent was collected and concentrated under reduced pressure to give a pure caprazamycin A (3.6 mg, 98%) as a colorless powder:

Cbz Protecting Group References

• [1] M. Bergmann, L. Zervas, Berichte der deutschen chemischen Gesellschaft. 1932, 65, 1192 | Über ein allgemeines Verfahren der Peptid-Synthese
• [2] B. H. Lipshutz, S. S. Pfeiffer, A. B. Reed, Org. Lett. 2001, 3, 4145 | Selective Cleavage of Cbz-Protected Amines
• [3] J. Pearson, W. R. Roush, Handbook of Reagents for Organic Synthesis: Activating Agents and Protecting Groups (Wiley)
• [4] Org. Lett. 2014, 16, 3364 | Catalytic Asymmetric Total Synthesis of (+)-Caprazol
• [5] Angew. Chem. Int. Ed. 2015, 54, 3136 | Total Synthesis of (−)-Caprazamycin A
• P. Kocienski, Protecting Groups (Thieme Verlag)
• P. Wuts, T. Greene, Greene’s Protective Groups in Organic Synthesis (Wiley)

What is the Boc Protecting Group?

Boc stands for tert-butyloxycarbonyl and protects amines as carbamates. More rarely, it is also used to protect alcohols as their carbonates.
Boc is resistant to basic hydrolysis, many nucleophiles as well as catalytic hydrogenation. The fact that it can be removed with mild acid makes it orthogonal to other key protecting groups (see below).

Boc Protection Mechanism

The main method of Boc protection is use of Boc anhydride. Base is not strictly needed for the reaction with Boc₂O (tert-butanol formed as product). However, bases like triethylamine or NaOH (amino acids) are sometimes used, depending on the system.

The mechanism is straight-forward: attack of the nucleophilic amine to the electrophilic anhydride. The carbonate leaving group can release CO₂, providing a strong driving force for the reaction. This step is the same as for other carbamate protecting groups such as Cbz.

As we have seen for PMB, many variants of activated agents can exist. The same is true for Boc. For instance, we can also use Boc-Cl (t-butyl chloroformate) but because it’s unstable and needs to be prepared freshly, Boc₂O is much more convenient. Boc-ON is another variant; it’s an “oxyimino-nitrile” reagent.

As a common theme for protecting groups: Remember that amines are more nucleophilicity than alcohols, so you can selectively protect them in many cases.

Boc DeProtecTION Mechanism WITH Acid

Acids like TFA, HCl… can deprotect Boc groups. Protonation of the oxygen triggers fragmentation into a stabilized tertiary cation (inductive effect). It later deprotonates to form gaseous isobutene.
The fragmented carbamate can decarboxylate, releasing CO₂ (here you have a parallel of Boc anhydride introduction and its removal) and giving the free amine.

Given the high steric hindrance of the carbamate, the Boc group is not base-labile like methyl esters, for instance.

A potential issue are intermolecular side reactions of the intermediary t-butyl cation. An example from peptide chemistry is alkylation of nucleophilic amino acids methionine or tryptophan. That’s why you might see conditions which employ scavengers like thiophenol, anisole, cresol… to remove the reactive intermediate.

Boc protecting group Orthogonality

Boc protection is a key tool for heterocycle and peptide synthesis. In solid phase peptide synthesis (SPPS), it is used as a protecting group for alpha-amino groups and amino acids lysine, tryptophan and histidine.

Due to its acidic deprotection, it is orthogonal to other important amino acid protecting groups:

• Fmoc (9-fluorenylmethoxycarbonyl) – removed with base
• Cbz / Z (benzyloxycarbonyl) – removed with H₂ reduction
• Alloc (allyloxycarbonyl) – removed with transition metal catalysis (Pd)

However, Boc is not stable to Lewis acids or oxidative conditions. We have seen that the O-PMB protecting group is removable with DDQ. In one case [1], treatment of the PMB ether did not result in any desired deprotection.

Here’s some homework: Can you explain why this side reaction and overoxidation might have occurred?

Boc side reactions

In organic chemistry, surprises wait around every corner. Don’t assume that protecting groups stay on forever, and only cleave on demand!

Before, we have mentioned that alcohols are less nucleophilic than amines, allowing selective protections. In this example [2], the authors observed a N-O Boc transfer when preparing a chiral oxazolidinone auxiliary. Upon deprotection of the TBDMS group, the highly nucleophilic alkoxide grabs the Boc group from the nitrogen!

Overman et al used a more conscious Boc-participation in their synthesis towards the diazatricyclic core of sarain marine alkaloids. [3]
Base generates the alkoxide which again attacks Boc. This time however, the group is not transferred (the negative charge would not be stabilized on the nitrogen, unlike the previous example). Instead, we see an intramolecular cyclization.

Boc in Peptide Synthesis

As alluded to above – particularly in the early days – the Boc protecting group proved very useful for solid-phase peptide synthesis (SPPS). However, in the late 20th century, the Fmoc protecting group started to replace Boc methodology.

Fmoc deprotection is generally milder than the moderate/ strong acidolysis steps used for Boc. More specifically, Fmoc proved more compatible with synthesis of amino acids that are susceptible to acid-catalysed side reactions.

A good example is the synthesis of the peptide gramicidin A which contains four acid-sensitive tryptophan residues. Using Boc chemistry, yields were in the range of 5-24%. Switching to Fmoc dramatically improved the yields, in some cases to 87% [4].

Closing Remarks

The Boc group is pretty cool, and its deprotection mechanism is a must-know for every organic chemistry student. The orthogonality to base- or reduction-labile protecting groups make it a top pick for many total and peptide syntheses.

However, like all protecting groups, it has its downsides. The carbamate carbon remains somewhat nucleophilic which opens avenues for surprising reactions, particularly intramolecular. Also, the acidic conditions and reactive tert-butyl-cations used can pose challenges to some systems. As always, smart planning and workarounds might be needed.

Boc: ✅. Learn more about other protecting groups, or watch my educational videos for for advanced chemistry & science content!

BOC Protection experimental procedure [5]

One pot Cbz->Boc switch
“To a solution of Cbz-carbamate SM (3.5 g, 6.81 mmol) in MeOH (25 mL) were added Pd/C (10 % w/w, 200 mg, 0.19 mmol) and Boc2O (2.17 g, 9.9 mmol) at room temperature. The reaction mixture was stirred under a hydrogen atmosphere (balloon) at room temperature for 6 h. The reaction mixture was filtered through a pad of celite, and then concentrated in vacuo. Flash column chromatography (silica gel, hexanes:EtOAc 10:1 → 7:1) afforded Boc-carbamate 42 (2.94 g, 90 %) as an oil.”

BOC deprotection experimental procedure [6]

“Boc-L-allo-End(Cbz)2-OtBu (597 mg, 1 mmol) was dissolved in a mixture of TFA (10 mL) and water (1.0 mL). The mixture was stirred at room temperature for 3 h, then concentrated to give a brown oil. The resulting crude oil was azeotroped with toluene (3 x 10 mL) and concentrated in vacuo to remove any residual TFA.”

BOC Protecting Group References

• [1] S. M. Bauer, R. W. Armstrong, J. Am. Chem. Soc. 1999, 121, 6355 | Total Synthesis of Motuporin (Nodularin-V)
• [2] S.P Bew, S.D Bull, S.G Davies, Tetrahedron Lett. 2000, 41, 7577 | Polymer supported oxazolidin-2-ones derived from L-serine – A cautionary tale
• [3] R. Downham, F. W. Ng, L. E. Overman, J. Org. Chem 1998, 63, 9096 | Asymmetric Construction of the Diazatricyclic Core of the Marine Alkaloids Sarains A−C
• [4] M. Stawikowski, G. B. Fields, Curr. Protoc. Protein Sci. 2002, 18.1 | Introduction to Peptide Synthesis
• [5] Chem. Asian J. 2008, 3, 413 | Total Synthesis of Thiopeptide Antibiotics GE2270A, GE2270T, and GE2270C1
• [6] Org. Chem. Front., 2018, 5, 1431 | Total synthesis of teixobactin and its stereoisomers
• C. Agami, F. Couty, Tetrahedron 2002, 58, 2701 | The reactivity of the N-Boc protecting group: an underrated feature
• P. Kocienski, Protecting Groups (Thieme Verlag)
• P. Wuts, T. Greene, Greene’s Protective Groups in Organic Synthesis (Wiley)

What is the PMB Protecting Group?

PMB is a para-methoxybenzyl group, introduced by Yonemitsu in 1982 [1] to protect alcohols and other nucleophilic functional groups. Although PMB ethers are less stable to acid than normal benzyl ethers, they can uniquely be cleaved oxidatively (see below). This allows selective deprotection protocols which becomes critical in complex organic synthesis.

PMB Protection Mechanism

The main method of PMB protection is the Williamson ether synthesis. This reaction uses a moderately strong base to generate an alkoxide which undergoes S_N2 substitution with an activated agent like PMB-Cl. Typical conditions include sodium hydride NaH in THF/DMF or DMSO. However, stronger bases like nBuLi work as well.

Beyond PMB-Cl, the reagent you will see most, there are also other methods of PMB protection. Beyond halide variants like PMB-I or PMB-Br, PMB-trichloroacetimidate with catalytic acid can also protect hindered tertiary alcohols. This is due to its higher reactivity. The PMB-pyridyl thiocarbonate with silver(I) is another example.

Interestingly, tetrabutylammonium iodide can be used catalytically for sluggish reactions as well. Here’s a question for you: Do you know how that catalysis works?

PMB DeProtecTION Mechanism WITH DDQ

This protective group differs from others in that it undergoes easy single electron transfer (SET) with DDQ (2,3-dichloro-5,6-dicyano-l,4-benzoquinone).
The electron-donating methoxy group stabilizes intermediary radical and oxonium ion. Normal benzyl protecting groups oxidize as well, but much slower than PMB. Obviously, this is due to the O-PMB methoxy group.

After some proton exchanges, water captures the carbocation. As with every redox reaction, the electrons removed from O-PMB (oxidation) end up in the reduced hydroquinone product. Compared to the quinone in DDQ, this system is aromatic.

The hemiacetal formed after water addition can fragment to give the deprotected hydroxy – as well as anisaldehyde. One drawback of is unintended side reaction of the aldehyde or intermediary PMB cations with nucleophilic functional groups, as well as polymerization. Thus, it is common to add nucleophilic scavengers (e.g., thiols) that capture these reactive species. This is a common thread for some deprotections (e.g., Boc).

By the way, other oxidants like cerium(IV) ammonium nitrate (CAN) or NBS might work when DDQ fails.

PMB protecting group Orthogonality

The DDQ reduction (typically 1.1-1.5 equivalents DDQ in dichloromethane-water mixtures) leaves several functional groups and other protecting groups (MOM, THP, TBS, Bz…) alone. This makes PMB an interesting orthogonal protecting group.

However, electron-rich groups like dienes or trienes can be unintended victims of DDQ. This makes complete sense: electron-rich groups are nucleophiles and thus like to react with oxidants. In some cases, conjugation of dienes with electron-withdrawing groups sufficiently deactivates them, avoiding DDQ interference.

Also, always be on the lookout for unique systems and reactivities!
The synthesis of sterepolide, an antibiotic fungal metabolite, exemplifies this concept in oxidative deprotections [2]. When using an excess of DDQ (8 equivalents), the authors found the allylic O-PMB over-oxidized directly to the ketone. In this case, this was convenient as it gave the target natural product, saving one step. However, this can complicate cases where we need deprotection only (most of the time).

Advanced Question: Special PMB Deprotection

As a twist on the previous information, you can ponder on this research [3]. Using 0.5 equivalents of oxalyl chloride led to efficient PMB deprotection of various substrates.

What could be a mechanism for this unique deprotection reaction?

Closing Remarks

PMB is a quite unique protecting group given the ability to remove it oxidatively (in addition to normal acid-mediated cleavage, not explicitly discussed here). In addition, PMB can also protect carboxylic acids, thiols, amines, amides… – or even phosphates! Basically, many other nucleophilic groups. The protection and oxidative removal make it a standard question in organic chemistry courses.

Learn more about other protecting groups, or watch my educational videos for advanced chemistry & science content!

PMB protection experimental procedure [4]

“To an ice-water cooled solution of SM (3.91 g, 15.2 mmol, 1 equiv) in THF-DMF (100 mL-30mL) was added NaH (2.43 g, 60.8 mmol, 4 equiv, 60% suspended in mineral oil) portionwise. After addition, the reaction was stirred at the same temperature until cease of gas releasing, then p-methoxybenzyl bromide (6.11 g, 30.4 mmol, 2 equiv) in THF (25 mL) was slowly added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, then quenched by slowly adding 1M solution of NaOMe in MeOH (15 mL). The reaction mixture was diluted with EtOAc (300 mL) and washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel flash chromatography (hexanes : EtOAc = 25:1-10:1) to give 5.28 g of product as a colorless oil in 92% yield.”

PMB deprotection experimental procedure [5]

“To a solution of SM (1.97 g, 3.95 mmol) in CH2Cl2:0.1 M pH 7 sodium phosphate buffer (18:1, 47 mL) at 0 °C was added 2,3-dichloro-5,6-dicyano-p-benzoquinone (1.17 g, 5.14 mmol) slowly as a solid. The reaction was warmed to rt and stirred for 1 h. The crude mixture was directly loaded onto a silica gel column with a top layer of MgSO₄:sand (1:1, 0.5 inches). Elution with 5% to 30% EtOAc in hexanes yielded the product (1.45 g, 97%).”

PMB References

• [1] Oikawa, Y.; Yoshioka, T.; Yonemitsu, O. Tetrahedron Lett. 1982, 23, 885 | A facile chemoselective deprotection of the p-methoxybenzyl group
• [2] Barry M. Trost, John Y. L. Chung, J. Am. Chem. Soc. 1985, 107, 15, 4586 | Unusual substituent effect on a palladium-mediated cyclization: a total synthesis of (+/-)-sterepolide
• [3] Tetrahedron Lett. 2015, 56, 1080 | Facile and selective deprotection of PMB ethers and esters using oxalyl chloride
• [4] J. Am Chem. Soc. 2016, 138, 3926 | Total Synthesis of Mannopeptimycins α and β
• [5] Angew. Chem. 2018, 130, 16092 | Total Synthesis of Divergolides E and H
• P. Kocienski, Protecting Groups (Thieme)
• P. Wuts, T. Greene, Greene’s Protective Groups in Organic Synthesis (Wiley)