Protecting group

Ethylene glycol protects a ketone (as an acetal) during an ester reduction, vs. unprotected reduction to a diol

A protecting group or protective group is introduced into a molecule by chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction. It plays an important role in multistep organic synthesis.^[1]

In many preparations of delicate organic compounds, specific parts of the molecules cannot survive required reagents or chemical environments. These parts (functional groups) must be protected. For example, lithium aluminium hydride is a highly reactive reagent that usefully reduces esters to alcohols. It always reacts with carbonyl groups, and cannot be discouraged by any means. When an ester must be reduced in the presence of a carbonyl, hydride attack on the carbonyl must be prevented. One way to do so converts the carbonyl into an acetal, which does not react with hydrides. The acetal is then called a protecting group for the carbonyl. After the hydride step is complete, aqueous acid removes the acetal, restoring the carbonyl. This step is called deprotection.

Protecting groups are more common in small-scale laboratory work and initial development than in industrial production because they add additional steps and material costs. However, compounds with repetitive functional groups – generally, biomolecules like peptides, oligosaccharides or nucleotides – may require protecting groups to order their assembly. Also, cheap chiral protecting groups may often shorten an enantioselective synthesis (e.g. shikimic acid for oseltamivir).

Orthogonal protection

Orthogonal protection is a strategy allowing the specific deprotection of one protective group in a multiply-protected structure. For example, the amino acid tyrosine could be protected as a benzyl ester on the carboxyl group, a fluorenylmethylenoxy carbamate on the amine group, and a tert-butyl ether on the phenol group. The benzyl ester can be removed by hydrogenolysis, the fluorenylmethylenoxy group (Fmoc) by bases (such as piperidine), and the phenolic tert-butyl ether cleaved with acids (e.g. with trifluoroacetic acid).

A common example for this application, the Fmoc peptide synthesis, in which peptides are grown in solution and on solid phase, is very important.^[2] The protecting groups in solid-phase synthesis with regard to the reaction conditions such as reaction time, temperature and reagents can be standardized so that they are carried out by a machine, while yields of well over 99% can be achieved. Otherwise, the separation of the resulting mixture of reaction products is virtually impossible.^[3]

Schematic diagram of a solid-state peptide synthesis with orthogonal protecting groups X and Y
Fmoc solid state peptide synthesis with orthogonal protecting groups

The technique was introduced in the field of peptide synthesis by Robert Bruce Merrifield in 1977.^[4]

A further important example of orthogonal protecting groups occurs in carbohydrate chemistry. As carbohydrates or hydroxyl groups exhibit very similar reactivities, a transformation that protects or deprotects a single hydroxy group must be possible for a successful synthesis.

Cleavage categorization

With respect to protecting groups, many reaction conditions have been established, under which, in accordance with the concept of orthogonality, some protecting groups cleave. One can roughly distinguish between the following cleavage conditions:^[5]

Acid-labile protecting groups
Base-labile protecting groups
Fluoride-labile protecting groups
Enzyme-labile protecting groups
Reduction-labile protecting groups
Oxidation-labile protecting groups
Protecting groups cleaved by heavy metal salts or their complexes.
Photo-labile protecting groups
Double-layered protecting groups

A wide variety of groups are cleaved in acid or base conditions, but the others are much more unusual.

Flouride ions form very strong bonds to silicon. Thus silicon protecting groups are almost invariably removed by fluoride ions. Each type of counterion, i.e. cleavage reagent can also selectively cleave different silicon protecting groups depending on steric hindrance. The advantage of fluoride-labile protecting groups is that no other protecting group is attacked by the cleavage conditions.

Esters can often be removed with enzymes like lipases. As enzymes work at a pH value between 5 and 9 and at moderate temperatures around 30–40 °C, and can be very selective on what the carboxylic acid connects to, this method is quite rarely used, but a very attractive method for protecting-group removal.

Structure of dichlorodicyanobenzoquinone

Benzyl groups can be removed reductively through catalytic hydrogenation. For instance, benzyl groups in ethers, esters, urethanes, carbonates, or acetals can protect alcohols, carboxylic acids, amines, or diols.

Only a few protecting groups can be detached oxidatively are practicable. In general, they are as a rule methoxybenzyl ethers. They can be removed with ceric ammonium nitrate (CAN) or dichlorodicyanobenzoquinone (DDQ) to a quinomethide.

The double bond of an allyl group can be isomerized to a vinyl group with platinum group elements (like palladium, iridium, or platinum). The residual enol ether from a protected alcohol or enamine of a protected amine can be hydrolyzed in light acid.

Photolabile protecting groups bear a chromophore, which is activated through radiation with an appropriate wavelength and so can be removed.^[6] For examples the o-nitrobenzylgroup ought be listed here.

The double-layer protecting group presents an special kind of protecting group. These exemplify a high stability, for the protecting group must first be transformed to a removable one through a chemical transformation. This kind of protecting group finds application rarely, for here an additional activating step is important, which lengthens the synthesis by another reaction.

Common protecting groups

Alcohol protecting groups

Protection of alcohols:

Protection of alcohol as tetrahydropyranyl ether followed by deprotection. Both steps require acid catalysts.

Acetyl (Ac) – Removed by acid or base (see Acetoxy group).
Benzoyl (Bz) – Removed by acid or base, more stable than Ac group.
Benzyl (Bn) – Removed by hydrogenolysis. Bn group is widely used in sugar and nucleoside chemistry.
Methoxyethoxymethyl ether (MEM) – Removed by acid.
Dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT) – Removed by weak acid. DMT group is widely used for protection of 5'-hydroxy group in nucleosides, particularly in oligonucleotide synthesis.
Methoxymethyl ether (MOM) – Removed by acid.
Methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT) – Removed by acid and hydrogenolysis.
p-Methoxybenzyl ether (PMB) – Removed by acid, hydrogenolysis, or oxidation – commonly with DDQ .
p-Methoxyphenyl ether (PMP) – Removed by oxidation.
Methylthiomethyl ether – Removed by acid.
Pivaloyl (Piv) – Removed by acid, base or reductant agents. It is substantially more stable than other acyl protecting groups.
Tert-butyl ethers (tBu) – Removed by acid.
Tetrahydropyranyl (THP) – Removed by acid.
Tetrahydrofuran (THF) – Removed by acid.
Trityl (triphenylmethyl, Tr) – Removed by acid and hydrogenolysis.
Silyl ether (most popular ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS or TBS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers) – Removed by acid or fluoride ion. (such as NaF, TBAF (tetra-n-butylammonium fluoride, HF-Py, or HF-NEt₃)). TBDMS and TOM groups are used for protection of 2'-hydroxy function in nucleosides, particularly in oligonucleotide synthesis.
Methyl ethers – Cleavage is by TMSI in dichloromethane or acetonitrile or chloroform. An alternative method to cleave methyl ethers is BBr₃ in DCM
Ethoxyethyl ethers (EE) – Cleavage more trivial than simple ethers e.g. 1N hydrochloric acid^[7]

Amine protecting groups

Protection of amines:

Carbobenzyloxy (Cbz) group – Removed by hydrogenolysis
p-Methoxybenzyloxycarbonyl (Moz or MeOZ) group – Removed by hydrogenolysis, more labile than Cbz
tert-Butyloxycarbonyl (BOC) group (common in solid phase peptide synthesis) – Removed by concentrated strong acid (such as HCl or CF₃COOH), or by heating to >80 °C.
9-Fluorenylmethyloxycarbonyl (Fmoc) group (Common in solid phase peptide synthesis) – Removed by base, such as piperidine
Acetyl (Ac) group is common in oligonucleotide synthesis for protection of N4 in cytosine and N6 in adenine nucleic bases and is removed by treatment with a base, most often, with aqueous or gaseous ammonia or methylamine. Ac is too stable to be readily removed from aliphatic amides.
Benzoyl (Bz) group is common in oligonucleotide synthesis for protection of N4 in cytosine and N6 in adenine nucleic bases and is removed by treatment with a base, most often with aqueous or gaseous ammonia or methylamine. Bz is too stable to be readily removed from aliphatic amides.
Benzyl (Bn) group – Removed by hydrogenolysis
Carbamate group – Removed by acid and mild heating.
p-Methoxybenzyl (PMB) – Removed by hydrogenolysis, more labile than benzyl
3,4-Dimethoxybenzyl (DMPM) – Removed by hydrogenolysis, more labile than p-methoxybenzyl
p-Methoxyphenyl (PMP) group – Removed by ammonium cerium(IV) nitrate (CAN)
Tosyl (Ts) group – Removed by concentrated acid (HBr, H₂SO₄) & strong reducing agents (sodium in liquid ammonia or sodium naphthalenide)
Troc (trichloroethyl chloroformate ) group – Removed by Zn insertion in the presence of acetic acid
Other Sulfonamides (Nosyl & Nps) groups – Removed by samarium iodide, thiophenol or other soft thiol nucleophiles, or tributyltin hydride^[8]

Carbonyl protecting groups

Protection of carbonyl groups:

Acetals and Ketals – Removed by acid. Normally, the cleavage of acyclic acetals is easier than of cyclic acetals.
Acylals – Removed by Lewis acids.
Dithianes – Removed by metal salts or oxidizing agents.

Carboxylic acid protecting groups

Protection of carboxylic acids:

Methyl esters – Removed by acid or base.
Benzyl esters – Removed by hydrogenolysis.
tert-Butyl esters – Removed by acid, base and some reductants.
Esters of 2,6-disubstituted phenols (e.g. 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol) – Removed at room temperature by DBU-catalyzed methanolysis under high-pressure conditions.^[9]
Silyl esters – Removed by acid, base and organometallic reagents.
Orthoesters – Removed by mild aqueous acid to form ester, which is removed according to ester properties.
Oxazoline – Removed by strong hot acid (pH < 1, T > 100 °C) or alkali (pH > 12, T > 100 °C), but not e.g. LiAlH₄, organolithium reagents or Grignard (organomagnesium) reagents

Phosphate protecting groups

2-cyanoethyl – removed by mild base. The group is widely used in oligonucleotide synthesis.
Methyl (Me) – removed by strong nucleophiles e.c. thiophenole/TEA.

Terminal alkyne protecting groups

Propargyl alcohols in the Favorskii reaction,
Silyl groups, especially in protection of the acetylene itself.^[10]

Other

Photolabile protecting groups As a proof of concept orthogonal deprotection is demonstrated in a photochemical transesterification by trimethylsilyldiazomethane utilizing the kinetic isotope effect:^[11]
Due to this effect the quantum yield for deprotection of the right-side ester group is reduced and it stays intact. Significantly by placing the deuterium atoms next to the left-side ester group or by changing the wavelength to 254 nm the other monoarene is obtained.

Criticism

The use of protective groups is pervasive but not without criticism.^[12] In practical terms their use adds two steps (protection-deprotection sequence) to a synthesis, either or both of which can dramatically lower chemical yield. Crucially, added complexity impedes the use of synthetic total synthesis in drug discovery. In contrast biomimetic synthesis does not employ protective groups. As an alternative, Baran presented a novel protective-group free synthesis of the compound hapalindole U. The previously published synthesis^[13]^[14]^[15] according to Baran, contained 20 steps with multiple protective group manipulations (two confirmed):

Protected and unprotected syntheses of the marine alkaloid, hapalindole U.
Hideaki Muratake's 1990 synthesis using Tosyl protecting groups (shown in blue).	Phil Baran's protecting-group free synthesis, reported in 2007.

Industrial applications

Although the use of protecting groups is not preferred in industrial syntheses, they are still used in industrial contexts, e.g.:

Oseltamivir (Tamiflu, an antiviral drug) Roche synthesis
Sucralose (sweetener)

References

^ Theodora W. Greene; Peter G. M. Wuts (1999). Protecting Groups in Organic Synthesis (3 ed.). J. Wiley. ISBN 978-0-471-16019-9.
^ Chan, Weng C.; White, Peter D. (2004). Fmoc Solid Phase Peptide Synthesis. Oxford University Press. ISBN 978-0-19-963724-9.
^ Weng C. Chan, Peter D. White: Fmoc Solid Phase Peptide Synthesis, S. 10–12.
^ Merrifield, R. B.; Barany, G.; Cosand, W. L.; Engelhard, M.; Mojsov, S. (1977). "Proceedings of the 5th American Peptide Symposium". Biochemical Education. 7 (4): 93–94. doi:10.1016/0307-4412(79)90078-5.
^ Michael Schelhaas, Herbert Waldmann: „Schutzgruppenstrategien in der organischen Synthese“, in: Angewandte Chemie, 1996, 103, pp. 2195–2200; doi:10.1002/ange.19961081805 (in German).
^ V.N. Rajasekharan Pillai: „Photoremovable Protecting Groups in Organic Synthesis“, in: Synthesis, 1980, pp. 1–26.
^ Kamaya, Yasushi; T Higuchi (2006). "Metabolism of 3,4-dimethoxycinnamyl alcohol and derivatives by Coriolus versicolor". FEMS Microbiology Letters. 24 (2–3): 225–229. doi:10.1111/j.1574-6968.1984.tb01309.x.
^ Moussa, Ziad; D. Romo (2006). "Mild deprotection of primary N-(p-toluenesufonyl) amides with SmI₂ following trifluoroacetylation". Synlett. 2006 (19): 3294–3298. doi:10.1055/s-2006-951530.
^ Romanski, J.; Nowak, P.; Kosinski, K.; Jurczak, J. (September 2012). "High-pressure transesterification of sterically hindered esters". Tetrahedron Lett. 53 (39): 5287–5289. doi:10.1016/j.tetlet.2012.07.094.
^ Clayden, Jonathan; Greeves, Nick; Warren, Stuart; Wothers, Peter (2000). Organic Chemistry. Oxford University Press. pp. 1291. ISBN 978-0-19-850346-0.
^ Blanc, Aurélien; Bochet, Christian G. (2007). "Isotope Effects in Photochemistry: Application to Chromatic Orthogonality" (PDF). Org. Lett. 9 (14): 2649–2651. doi:10.1021/ol070820h. PMID 17555322.
^ Baran, Phil S.; Maimone, Thomas J.; Richter, Jeremy M. (22 March 2007). "Total synthesis of marine natural products without using protecting groups". Nature. 446 (7134): 404–408. Bibcode:2007Natur.446..404B. doi:10.1038/nature05569. PMID 17377577. S2CID 4357378.
^ Synthetic studies of marine alkaloids hapalindoles. Part I Total synthesis of (±)-hapalindoles J and M Tetrahedron, Volume 46, Issue 18, 1990, Pages 6331–6342 Hideaki Muratake and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96005-3
^ Synthetic studies of marine alkaloids hapalindoles. Part 2. Lithium aluminum hydride reduction of the electron-rich carbon-carbon double bond conjugated with the indole nucleus Tetrahedron, Volume 46, Issue 18, 1990, Pages 6343–6350 Hideaki Muratake and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96006-5
^ Synthetic studies of marine alkaloids hapalindoles. Part 3 Total synthesis of (±)-hapalindoles H and U Tetrahedron, Volume 46, Issue 18, 1990, Pages 6351–6360 Hideaki Muratake, Harumi Kumagami and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96007-7

External links

[1] Theodora W. Greene; Peter G. M. Wuts (1999). Protecting Groups in Organic Synthesis (3 ed.). J. Wiley. ISBN 978-0-471-16019-9.

[FMOC-2] Chan, Weng C.; White, Peter D. (2004). Fmoc Solid Phase Peptide Synthesis. Oxford University Press. ISBN 978-0-19-963724-9.

[3] Weng C. Chan, Peter D. White: Fmoc Solid Phase Peptide Synthesis, S. 10–12.

[4] Merrifield, R. B.; Barany, G.; Cosand, W. L.; Engelhard, M.; Mojsov, S. (1977). "Proceedings of the 5th American Peptide Symposium". Biochemical Education. 7 (4): 93–94. doi:10.1016/0307-4412(79)90078-5.

[5] Michael Schelhaas, Herbert Waldmann: „Schutzgruppenstrategien in der organischen Synthese“, in: Angewandte Chemie, 1996, 103, pp. 2195–2200; doi:10.1002/ange.19961081805 (in German).

[6] V.N. Rajasekharan Pillai: „Photoremovable Protecting Groups in Organic Synthesis“, in: Synthesis, 1980, pp. 1–26.

[7] Kamaya, Yasushi; T Higuchi (2006). "Metabolism of 3,4-dimethoxycinnamyl alcohol and derivatives by Coriolus versicolor". FEMS Microbiology Letters. 24 (2–3): 225–229. doi:10.1111/j.1574-6968.1984.tb01309.x.

[8] Moussa, Ziad; D. Romo (2006). "Mild deprotection of primary N-(p-toluenesufonyl) amides with SmI₂ following trifluoroacetylation". Synlett. 2006 (19): 3294–3298. doi:10.1055/s-2006-951530.

[9] Romanski, J.; Nowak, P.; Kosinski, K.; Jurczak, J. (September 2012). "High-pressure transesterification of sterically hindered esters". Tetrahedron Lett. 53 (39): 5287–5289. doi:10.1016/j.tetlet.2012.07.094.

[10] Clayden, Jonathan; Greeves, Nick; Warren, Stuart; Wothers, Peter (2000). Organic Chemistry. Oxford University Press. pp. 1291. ISBN 978-0-19-850346-0.

[11] Blanc, Aurélien; Bochet, Christian G. (2007). "Isotope Effects in Photochemistry: Application to Chromatic Orthogonality" (PDF). Org. Lett. 9 (14): 2649–2651. doi:10.1021/ol070820h. PMID 17555322.

[12] Baran, Phil S.; Maimone, Thomas J.; Richter, Jeremy M. (22 March 2007). "Total synthesis of marine natural products without using protecting groups". Nature. 446 (7134): 404–408. Bibcode:2007Natur.446..404B. doi:10.1038/nature05569. PMID 17377577. S2CID 4357378.

[13] Synthetic studies of marine alkaloids hapalindoles. Part I Total synthesis of (±)-hapalindoles J and M Tetrahedron, Volume 46, Issue 18, 1990, Pages 6331–6342 Hideaki Muratake and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96005-3

[14] Synthetic studies of marine alkaloids hapalindoles. Part 2. Lithium aluminum hydride reduction of the electron-rich carbon-carbon double bond conjugated with the indole nucleus Tetrahedron, Volume 46, Issue 18, 1990, Pages 6343–6350 Hideaki Muratake and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96006-5

[15] Synthetic studies of marine alkaloids hapalindoles. Part 3 Total synthesis of (±)-hapalindoles H and U Tetrahedron, Volume 46, Issue 18, 1990, Pages 6351–6360 Hideaki Muratake, Harumi Kumagami and Mitsutaka Natsume doi:10.1016/S0040-4020(01)96007-7

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