Richard J.
Spears
*,
Clíona
McMahon
and
Vijay
Chudasama
*
Department of Chemistry, University College London, London, UK. E-mail: r.spears@ucl.ac.uk; v.chudasama@ucl.ac.uk
First published on 17th August 2021
Protecting group chemistry for the cysteine thiol group has enabled a vast array of peptide and protein chemistry over the last several decades. Increasingly sophisticated strategies for the protection, and subsequent deprotection, of cysteine have been developed, facilitating synthesis of complex disulfide-rich peptides, semisynthesis of proteins, and peptide/protein labelling in vitro and in vivo. In this review, we analyse and discuss the 60+ individual protecting groups reported for cysteine, highlighting their applications in peptide synthesis and protein science.
Fig. 1 Examples of Cys-containing biomolecules of therapeutic interest. (a) Oxytocin, (b) Human insulin, (c) Prialt™/Ziconotide, (d) 68Ga-DOTATOC, (e) Adcetris™/Brentuximab vedotin. |
The reactivity profile of Cys enables a plethora of applications in chemistry and biology related to peptides and proteins. However, this reactivity also leads to significant challenges when employing the residue for peptide chemistry in particular. The reactive nature of the thiol side chain makes it prone to side reactions such as alkylation and oxidation.21 Particularly for disulfide-rich peptides, ensuring the regioselective formation/correct connectivity of disulfides presents a significant challenge in peptide synthesis.5,6 Ensuring racemisation of Cys at the α-carbon stereocenter does not occur during peptide synthesis also presents a notable challenge; this is especially true for synthesising peptides containing a C-terminal Cys.22 Other side reactions related to Fmoc SPPS include formation of 3-(1-piperidinyl)alanine by-products at the C-terminal Cys position formed by an elimination–addition reaction involving piperidine.23,24 Unsurprisingly, this has led to significant research into strategies to protect (and subsequently deprotect) the thiol side chain to overcome these challenges, and to further expand upon the synthesis of biomolecules. Indeed, protection of Cys during standard peptide synthesis is therefore often critical to avoid undesired alkylation/oxidation, allowing for synthesis of the desired peptide. In the case of disulfide-containing peptide synthesis, the development of “orthogonal” Cys protecting groups (which can be selectively deprotected in the presence of one another) has proved paramount to ensuring regioselective disulfide formation.6
Herein we review the different thiol protecting groups reported for Cys throughout the last 70+ years. We analyse and discuss each of the individual 60+ protecting groups that have been reported for Cys protection (and deprotection). For each group (and where applicable), this review will focus on (a) the conditions used for its deprotection, (b) use in Boc and Fmoc peptide synthesis, (c) its comparison to other related Cys protecting groups, and (d) notable applications of the Cys protecting group in peptide and protein chemistry.
Other common systems used for ligation of large peptides are N,S-acyl shift systems, one example of which is the cysteinylprolyl ester (CPE) system.44 Here, peptides containing a CPE unit at the C-terminus can undergo spontaneous N- > S transformations into diketopiperazine thioesters. The resulting thioester can then undergo NCL in a one-pot process (Fig. 3d). A related system that uses cysteinylprolyl imide (CPI) peptides has more recently been described and used in the synthesis of proteins such as ZHER2 affibody.45 In addition, peptides containing C-terminal hydrazides can be used as more chemically stable peptide thioester precursors.46 In this strategy the hydrazide group can be chemically converted to a C-terminal azide or acyl pyrazole using nitrous acid or acetyl acetone, followed by conversion to a thioester through addition of 4-mercaptophenylacetic acid (MPAA, Fig. 3(e)).47 This methodology has since been used in the synthesis of multiple proteins, including Centruroides suffusus suffusus toxin II protein (CssII)46 and α-synuclein.48 Peptide hydrazides can also be prepared through Cys cyanylation using 2-nitro-5-thiocyanatobenzoic acid (NTCB) followed by addition of hydrazine, as shown in the recently described activated Cys-directed protein ligation (APCL).49 Another ligation strategy is the thioester method, whereby a silver (Ag+) activated C-terminal alkyl thioester is directly displaced by an N-terminal amine to yield the desired amide bond.50 A silver-free protocol has also been developed for the thioester method, which involves the use of aryl, as opposed to alkyl, thioesters (Fig. 3f).51 To ensure a successful peptide synthesis both thioester methods require peptide fragments with protected amino groups, such as Fmoc protection of N-terminal α-amino groups, or Boc/Z protection of Lys side chain ε-amino groups; alternatively, Lys side chains can be protected by using an azido-lysine analogue during peptide synthesis, followed by azide reduction to give the desired ε-amino group post-thioester ligation.52 Thioester methods have since been utilised for the synthesis of proteins such as chemokine CCL2751and various glycoproteins.53 For further reading on ligation methods, we direct the reader to extensive reviews on the subject.53–57
Protecting group, abbreviations and location within review | Lability | Deprotection conditions | Stable to/compatible with | Structure |
---|---|---|---|---|
Benzyl (Bzl, Bn); Section 5.1 | Acid, reducing agents | Na/NH3 (liq.)67 | Standard Boc SPPS reagents4 | |
HF (25 °C)68 | ||||
TMSBr/TFA/thioanisole69 | ||||
Trityl (Trt); Section 5.2.1 | Acid, oxidising agents | Ag(I)70 | Standard Fmoc SPPS reagents74 | |
Hg(II)70 | ||||
HBr/AcOH70 | ||||
TFA/TIS (90:10)71 | ||||
HBF4/scavengers72 | ||||
I24 | ||||
CuSO4-cysteamine, Gdn·HCl/HEPPS buffer73 | ||||
Diphenylmethyl (Dpm, Bzh, Bh); Section 5.2.2 | Acid | TFA/TIS/H2O/DCM (90:2.5:2.5:5)75 | Cocktails of <25% TFA75 | |
Standard Fmoc SPPS reagents75 | ||||
Tetrahydropyranyl (Thp); Section 5.2.3 | Acid | Silver nitrate (aq., 0 °C)76 | Na/NH3 (liq.)76 | |
TFA/TIS/DCM (95:2.5:7.5)77 | 1% TFA in DCM77 | |||
Standard Fmoc SPPS reagents77 | ||||
tert-Butyl (tBu); Section 5.2.4 | Acid | NpsCl (2 h, RT), then NaBH478 | I272 | |
HF (20 °C)79 | TFA86 | |||
DTNP/TFA80 | AgOTf/TFA87 | |||
Hg(OAc)2/TFA/anisole81 | Na/NH3 (liq.)78 | |||
Silyl chloride-sulfoxide/TFA82 | Hydrazine78 | |||
Tl(TFA)383 | Standard Fmoc SPPS reagents88 | |||
DMSO/TFA84 | ||||
PdCl2, 50 mM Tris or urea buffer (37 °C)85 | ||||
4-Methoxybenzyl (Mob, MBzl); Section 5.2.5 | Acid | TFA (100 °C)89 | HBr89 | |
HF79 | TFA (without scavengers)86 | |||
DTNP or DTP/TFA/thioanisole90 | Standard Fmoc SPPS reagents75 | |||
TFA/TIS (12 h, 37 °C)86 | Standard Boc SPPS reagents (for small peptides)4,91 | |||
Hg(TFA)281 | ||||
Tl(TFA)383 | ||||
AgOTf/TFA/thioanisole87 | ||||
3,4-Dimethylbenzyl (DMB); Section 5.2.6 | Acid | HF (10 min, 0 °C)92 | 50% TFA in DCM (23 h, 24 °C)92 | |
Standard Boc SPPS reagents92 | ||||
Methylbenzyl (Meb, 4-MeBn, 4-MeBzl); Section 5.2.7 | Acid | HF/anisole (1 h, 0 °C)91 | AgOTf87 | |
Tl(TFA)383 | Standard Fmoc SPPS reagents74 | |||
DMSO/TFA (45 °C)74 | Standard Boc SPPS reagents91 | |||
1-Adamantyl (Ad, 1-Ada); Section 5.2.8 | Acid | Hg(OAc)2/TFA81 | TFA (2.5 h, 0 °C)93 | |
1 M TFMSA/anisole/TFA93 | AgOTf/TFA87 | |||
Tl(TFA)393 | Standard Boc SPPS reagents87 | |||
Standard Fmoc SPPS reagents4 | ||||
Benzyloxymethyl (Bom); Section 5.2.9 | Acid | AgOTf/anisole/TFA (1 h, 0 °C)94 | TFA (4 h, 0 °C)94 | |
1 M TMSOTf/thioanisole/TFA (1 h, 0 °C)94 | Hydrazine94 | |||
Tl(TFA)394 | Piperidine/DMF94 | |||
NaBO394 | ||||
Standard Fmoc SPPS reagents94 | ||||
2,4,6-Trimethoxybenzyl (Tmob); Section 5.2.10 | Acid | ≥6% TFA with TES or TIS (0.5%) in DCM (5 min, 25 °C)95 | Standard Fmoc SPPS reagents96 | |
≥30% TFA in DCM with phenol/thioanisole/H2O (5% each)95 | ||||
I2/DMF (0 °C)95 | ||||
Tl(TFA)3/DMF/anisole (0 °C)95 | ||||
4,4′,4′′,-Trimethoxy-triphenylmethyl (TMTr); Section 5.2.11 | Acid | 1% TFA in DCM96 | Standard Fmoc SPPS reagents96 | |
Pseudoprolines (ΨPro); Section 5.2.12 | Acid | TFA (ΨMe,Mepro, 32–36 h, sequence dependant)97 | Standard Fmoc SPPS reagents97 | |
TFMSA (15 min, 0 °C)98 | TFA (ΨH,Hpro)97 | |||
TFA (ΨMe,Mepro, 1–6 h, sequence dependant)99 | TFA/MeOH/TIS/H2O (80:15:2.5:2.5, ΨMe,Mepro, 1 h, cyclic peptides)101 | |||
TFA (ΨH,Dmppro, minutes)97 | ||||
TFA (ΨH,Dmppro, C-terminal resin-bound, 1.5 h)100 | ||||
4-Methyltrityl (Mtt); Section 5.2.13 | Acid | 1% TFA/scavengers75 | Standard Fmoc SPPS reagents75 | |
4-Methoxytrityl (Mmt); Section 5.2.13 | Acid | 1–3% TFA in DCM/TES (95:5)71 | Bases, e.g. 30% piperidine in DMF (24 h, 22 °C)71 | |
I271 | Very weak acids, e.g. AcOH/TFE/DCM (1:2:7, 30 min)71 | |||
Standard Fmoc SPPS reagents71 | ||||
9H-Xanthen-9-yl (Xan); Section 5.2.15 | Acid | TFA:DCM:TES (0.1:99.4:0.5)102 | Piperidine/DMF (≥24 h, 25 °C)102 | |
TFA:DCM:BME (10:85:5)102 | HOBt/DMF (24 h, 25 °C); | |||
TFA:DCM:TES (1:98.5:0.5) solid phase (25 C, 2 h)102 | AcOH102 | |||
I2/MeOH102 | Standard Fmoc SPPS reagents102 | |||
Tl(TFA)3102 | ||||
2-Methoxy-9H-xanthen-9-yl (2-Moxan); Section 5.2.15 | Acid | TFA:DCM:TES (0.1:99.4:0.5)102 | Piperidine/DMF (≥24 h, 25 °C)102 | |
TFA:DCM:BME (10:85:5)102 | HOBt/DMF (24 h, 25 °C); | |||
TFA:DCM:TES (1:98.5:0.5) solid phase (25 C, 2 h)102 | AcOH102 | |||
I2/MeOH102 | Standard Fmoc SPPS reagents102 | |||
Tl(TFA)3102 | ||||
4,5,6-Trimethoxy-2,2-dimethyl-2,3-dihydrobenzofuran-7-methyl (Tmbm); Section 5.2.16 | Acid | TFA/TES/DCM (1:5:94)103 | Standard Fmoc SPPS reagents103 | |
2,2,5,7,8-Pentamethylchroman-6-methyl (Pmcm); Section 5.2.16 | Acid | TFA/TES/DCM (1:5:94)103 | Standard Fmoc SPPS reagents103 | |
2,2,4,6,7-Pentamethyl-2,3-dihydrobenzofuran-5-methyl (Pbfm); Section 5.2.16 | Acid | TFA/DCM/TIS (1:5:94)103 | Standard Fmoc SPPS reagents103 | |
I2103 | ||||
HFIP or TFE in DMF (15 min)103 | ||||
4-Methoxybenzyloxymethyl (Mbom); Section 5.2.17 | Acid | Reagent K/MeONH2·HCl104 | Standard Fmoc SPPS reagents104 | |
2,6-Dimethoxybenzyl (2,6-diMeOBn); Section 5.2.18 | Acid | TFA:DCM:TIS:H2O (50:45:2.5:2.5, 1 h, 25 °C)105 | Standard Fmoc SPPS reagents75 | |
4-Methoxy-2-methylbenzyl (4-MeO-2MeBn); Section 5.2.18 | Acid | TFA:DCM:TIS:H2O (50:45:2.5:2.5, 1 h, 25 °C)105 | Standard Fmoc SPPS reagents75 | |
4,4′-Dimethoxydiphenylmethyl (Ddm); Section 5.2.19 | Acid | TFA:DCM:TIS:H2O (10:85:2.5:2.5, 1 h, 25 °C)105 | Standard Fmoc SPPS reagents106 | |
Hmbon/off; Section 5.2.20 | Acid | TFA/TIS/H2O (95:2.5:2.5, 2 h, 25 °C) in Hmbon form107 | TFA/TIS/H2O (95:2.5:2.5, 2 h, 25 °C) in Hmboff form107 | |
Standard Fmoc SPPS reagents in Hmboff form107 | ||||
Standard NCL/desulfurisation/HPLC reagents in Hmbon form107 | ||||
Acetamidomethyl (Acm); Section 5.3.1 | Oxidising agents | NpsCl (2 h, RT), then NaBH478 | TFA (25 °C)110 | |
Hg(II)108 Ag(I)87 Pd(II)109 | HBr/AcOH (25 °C)110 | |||
6 M HCl (20 h, 110 °C)110 | HCl/EtOH (25 °C)110 | |||
15 eq. DTNP in 97.5% TFA/thioanisole78,90 | HF (0 °C)110 | |||
98% TFA with scavengers86 | Pd(0)113 | |||
I2111 | TCEP114 | |||
Tl(TFA)3111 | Standard Fmoc SPPS reagents74 | |||
Silyl chloride-sulfoxide/TFA111 | ||||
PdCl2, aqueous/buffered conditions112 | ||||
CuSO4, aqueous/buffered conditions with aminothiol source73 | ||||
5-Dibenzosuberyl (Dbs, Sub); Section 5.3.2 | Oxidising agents | Hg(OAc)2115 | TFA115 | |
I2/MeOH or AcOH115 | ||||
Tl(TFA)383 | ||||
Benzamidomethyl (Bam); Section 5.3.3 | Oxidising agents | Hg(OAc)2 (1 h, RT)116 | 1 M HCl (25 °C)116 | |
Ag(OTf)/TFA/anisole (1 h, 0 °C)87 | 1 M NaOH (25 °C)116 | |||
Silyl chloride-sulfoxide/TFA82 | TFA (25 °C)116 | |||
6 M HCl (24 h, 110 °C)116 | 90% Zn/AcOH (0 °C)116 | |||
Standard Boc SPPS reagents116 | ||||
(2-Oxo-1-pyrrolidinyl)methyl (Pym); Section 5.3.4 | Oxidising agents | HF117 | Solution phase Boc reagents117 | |
NaBO3117 | ||||
Dimethylphosphinothioyl (Mpt); Section 5.3.5 | Oxidising agents | AgNO3 (2–4 equiv., 20 min, 1 h, 0 °C) in H2O118 | 2 M HCl/EtOAc118 | |
Hg(OAc)2 (1–3 equiv., 1 h, 0 °C) in H2O118 | 1 M HCl/MeOH118 | |||
TBAF in THF (free thiol)119 | 2 M HCl/AcOH118 | |||
TBAF in DMF (disulfide)119 | 1 M HCl/H2O118 | |||
TBAF in DCM (–S–CH2–S– formation)119 | TFA118 | |||
Solution phase Boc reagents118 | ||||
Trimethyl-acetamidomethyl (Tacm); Section 5.3.6 | Oxidising agents | Hg(OAc)2120 | HF (1 h, 0 °C)117 | |
AgBF4/TFA/thioanisole121 | TFMSA/thioanisole/TFA (2 h, 0 °C)117 | |||
I2/EtOH in AcOH120 | 0.05 M NaOH in MeOH (aq., 1 h, 0 °C)117 | |||
Hydrazine/MeOH (24 h, RT)117 | ||||
Zn in 90% AcOH (1 h, 25 °C)117 | ||||
Standard Boc SPPS reagents93 | ||||
9-Fluorenylmethyl (Fm); Section 5.4.1 | Base | NH3/MeOH122 | TFA93 | |
50% Piperidine in DMF (2 h, RT)123 | TFMSA/TFA93 | |||
HCl (110 °C)93 | ||||
HF/anisole (95:5, 1 h, 0 °C)93 | ||||
0.1 M I2/DMF123 | ||||
H2, Pd/C122 | ||||
Standard Boc SPPS reagents123 | ||||
2-(2,4-Dinitrophenyl)ethyl (Dnpe); Section 5.4.2 | Base | 50% Piperidine in DMF (30 min)124 | 5% DIEA in DCM (2 h)124 | |
Dilute DBU68 | 40% TFA in DCM (24 h)124 | |||
90% HF/p-cresol or anisole (1 h, 0 °C)124 | ||||
Tl(TFA)3/TFA124 | ||||
I2 in 80% AcOH (aq.)124 | ||||
Standard Boc SPPS reagents124 | ||||
9-Fluorenylmethyl-oxycarbonyl (Fmoc); Section 5.4.3 | Base | Et3N, then NH3/MeOH or | 4 M HCl in dioxane125 | |
50% Piperidine in DMF (2 h, RT)125 | ||||
Phenyl-acetamidomethyl (Phacm); Section 5.5.1 | Enzyme, oxidising agents | Penicillin G acylase (pH 7.9 buffer)126 | 5% DIEA in DCM (24 h, 25 °C)126 | |
Hg(II)127 Ag(II)127 | 40% TFA in DCM (24 h, 25 °C)126 | |||
I2127 | 25% piperidine in DMF (24 h, 25 °C)126 | |||
Tl(TFA)3127 | 0.1 M TBAF in DMF (24 h, 25 °C)126 | |||
5% DBU in DMF (24 h, 25 °C)126 | ||||
90% HF/anisole or p-cresol (1 h, 0 °C)126 | ||||
90% TFA/scavengers (2 h, 25 °C)126 | ||||
Standard Boc SPPS reagents126 | ||||
Standard Fmoc SPPS reagents126 | ||||
Hydroxyglycine-Acm (Hgm); Section 5.6.1 | Hydrazine | 5% hydrazine in H2O (pH 8.5, 3 days, 37 °C)128 | Standard Fmoc SPPS reagents128 | |
Standard Boc SPPS reagents128 | ||||
Hydroxyquinoline-Acm (Hqm); Section 5.6.1 | Hydrazine | 5% hydrazine in H2O (pH 8.5, 8 h, 37 °C)128 | Standard Fmoc SPPS reagents128 | |
I2 (30 min)128 | Standard Boc SPPS reagents128 | |||
AgOAc (30 min)128 | ||||
Allyloxycarbonyl (Alloc); Section 5.7.1 | Pd, base | Pd(0) cat./Bu3SnH/AcOH126 | TFA/DCM (24 h, 50 °C)129 | |
Piperidine (3 h, 30 °C)129 | Standard Boc SPPS reagents129 | |||
Allyloxy-carbonylaminomethyl (Allocam); Section 5.7.2 | Pd | Pd(0) cat./Bu3SnH/AcOH (10 min, RT)130 | Piperidine130 | |
Pd(OAc)2/NMM/AcOH in DMSO (2 h, disulfide)131 | Acids (partially)130 | |||
Standard Fmoc SPPS reagents130 | ||||
[N-[2,3,5,6-Tetrafluoro-4-(N′-piperidino)-phenyl], N-allyloxycarbonyl]-aminomethyl (Fnam); Section 5.7.3 | Pd, oxidising agents | Pd(0) cat./allyl scavenger then AcOH/BME132 | Acid132 | |
Heavy metal salts132 | Base132 | |||
Tl(TFA)3132 | Standard Boc SPPS reagents132 | |||
Standard Fmoc SPPS reagents132 | ||||
S-[N-[2,3,5,6-Tetrafluoro-4-(phenylthio)-phenyl], N-allyloxycarbonyl]-aminomethyl (Fsam); Section 5.7.4 | Pd, oxidising agents | Pd(0) cat./allyl scavenger then AcOH/BME133 | Acid133 | |
I2 and other oxidants133 | Base133 | |||
Standard Boc SPPS reagents133 | ||||
Standard Fmoc SPPS reagents133 | ||||
Allyl (Sac); Section 5.7.5 | Pd | Pd(tppts)4134 | Unnatural amino acid mutagenesis134 | |
S-Propargyl-cysteine (SprC); Section 5.7.6 | Pd | Pd(tppts)4135 | Unnatural amino acid mutagenesis | |
CuAAC, Sonagashira coupling (alkyne functionality can be used for conjugation without cleavage)135 | ||||
Succinimide (Suc); Section 5.7.7 | Pd | PdCl2 and MgCl2, 6 M Gdn.HCl/0.2 M phosphate buffer pH 5.5, 37 °C then DTT136 | Aqueous solution phase conditions136 | |
Desulfurisation conditions136 | ||||
Thiazolidine (Thz); Section 5.8.1 | N-terminal | H2O2137 I2137 | PdCl2/H2O85 | |
Pd, oxidising agents, Cu | Iodoacetic acid/benzyl chloride (pH 10-11, RT)137 | Standard NCL reagents139 | ||
Air/ferric chloride (trace, pH 10)137 | ||||
Excess methoxyamine at pH 4 (8 h)138 | ||||
Pd(II) and MPAA/TCEP or GSH/6 M Gdn·HCl (pH ∼6.5, 37 °C, 45 min)85,138 | ||||
CuSO4/sodium ascorbate/5 M Gdn·HCl/HEPPS buffer then DTT (pH 7.0, 1 h, 37 °C)139 | ||||
20 mM DPDS, 50% MeCN (0.1% TFA)140 | ||||
Ninhydrin (Nin); Section 5.8.2 | N-terminal | Excess Cys (pH 7.7, 30 min, 23 °C)141 | Standard Boc SPPS reagents141 | |
Reducing agents | Cysteine O-methylester/DMF/DIEA (on resin)141 | |||
Excess MPS, pH 7141 | ||||
10% TFA/H2O/Zn dust, 1 h141 | ||||
2-Nitrobenzyl (oNB); Section 5.9.1 | Light | hν ≥ 350 nm142 | Standard Fmoc SPPS reagents143 | |
Standard Boc SPPS reagents143 | ||||
[7,8-Bis(carboxymethoxy)coumarin-4-yl]methoxycarbonyl (7,8-BCMCMOC); Section 5.9.2 | Light | hν ≥ 325 nm144 | TFA144 | |
Thiolysis144 | ||||
[7-Bis(carboxymethyl)-amino-coumarin-4-yl]methoxycarbonyl (BCMACMOC); Section 5.9.2 | Light | hν ≥ 402 nm144 | TFA144 | |
Thiolysis144 | ||||
α-Carboxy-4-methoxy- 2-nitrobenzyl (CDMNB); Section 5.9.2 | Light | hν ≥ 325 nm144 | TFA144 | |
Thiolysis144 | ||||
Piperidine144 | ||||
2-Nitroveratryl | Light | hν = 350 nm (30 min)142 | 10% TFMSA/TFA/excess dipyridine disulfide142 | |
6-Nitroveratryl | Standard Fmoc SPPS reagents142 | |||
4,5-Dimethoxy-2-nitrobenzyl (oNV, DMNB); Section 5.9.3 | ||||
6-Bromo-7-hydroxycoumarin (Bhc); Section 5.9.4 | Light | hν = 365 nm in photolysis buffer (1 mM DTT in 50 mM PB, pH 7.2)145 | Standard Fmoc SPPS reagents145 | |
Nitrodibenzofuran (NDBF); Section 5.9.5 | Light | hν = 365 nm145 | Standard Fmoc SPPS reagents145 | |
6-Bromo-7-hydroxy-3-methylcoumarin (mBhc); Section 5.9.6 | Light | hν = 365 nm146 | Standard Fmoc SPPS reagents146 | |
Methoxy-nitrodibenzofuran (OMe-NDBF); Section 5.9.7 | Light | hν ≥ 350 nm147 | Standard Fmoc SPPS reagents147 | |
para-Nitrobenzyl (pNB); Section 5.10.1 | Reducing agents | Zn/AcOH, then I2 (in solution)111 | HF/p-cresol (9:1, 1 h, 0 °C)111 | |
SnCl2/HCl, then I2 (on resin)111 | TFA111 | |||
Excess CAN/Hopkins reagent111 | I2/AcOH/2 M HCl111 | |||
H2, Pd/C, then oxidant111 | Standard Boc SPPS reagents111 | |||
Carbomethoxysulfenyl (Scm); Section 5.10.2 | Reducing agents | DTT148 | Strong acids – anhydrous HF, TFMSA149 | |
Standard Boc SPPS reagents149 | ||||
(N′-Methyl-N′-phenylcarbamoyl)sulfenyl (Snm); Section 5.10.3 | Reducing agents | DTT/NMM/CDCl3149 | Strong acids – anhydrous HF, TFMSA149 | |
Standard Boc SPPS reagents149 | ||||
4-Picolyl; Section 5.10.4 | Reducing agents | Zn/AcOH150 | TFA151 | |
Electrolytic reduction in 0.5 M H2SO4151 | 32% HBr/AcOH (1 week, RT)151 | |||
Standard Boc SPPS reagents151 | ||||
Sulfonic acid/sulfonyl (SO3H/SO2R); Section 5.10.5 | Reducing agents | Thiols152 | Standard Fmoc SPPS reagents153 | |
PBu3153 | Standard Boc SPPS reagents153 | |||
3-Nitro-2-pyridinesulfenyl (Npys); Section 5.10.6 | Reducing agents | Aliphatic thiols154 | TFA (24 h, RT)155 | |
Tertiary phosphine/H2O155,156 | HF (1 h, RT)155 | |||
4 M HCl/dioxane (24 h)155 | ||||
DCM157 DMF157 MeOH157 | ||||
N,N-Dimethylacetaminde157 | ||||
N-Methylpyrrolidone157 Trifluoroethanol157 | ||||
Pentafluorophenol157 | ||||
Standard Boc SPPS reagents155 | ||||
5-Nitro-2-pyridinesulfenyl (5-Nyps); Section 5.10.7 | Reducing agents | Thiols158 | Standard Boc SPPS reagents (see Nyps)158 | |
tert-Butylsulphenyl (StBu); Section 5.10.8 | Reducing agents | Thiols159 | Acid159 | |
Phosphines114 | Base159 | |||
TFA/DTNP/thioanisole80 | TFA/DNTP80 | |||
Standard Fmoc SPPS reagents74 | ||||
N-Methyl-phenacyloxy-carbamidomethyl (Pocam); Section 5.10.9 | Reducing agents, acid | Zn/AcOH (aq.)160 | TFA (4 h, 4 °C)160 | |
TFA (1 h, 50 °C)160 | Standard Fmoc SPPS reagents160 | |||
Phenacyl (Pac); Section 5.10.10 | Reducing agents | Zn powder, AcOH161 | Mild acid161 | |
Standard Fmoc SPPS reagents161 | ||||
Standard NCL reagents162 | ||||
S-Iso-Propyl (SiPr); Section 5.10.11 | Reducing agents | TCEP in PBS pH 7.4 | Standard Fmoc SPPS reagents114 | |
37 °C114 | ||||
Dimethoxyphenylthio (S-Dmp); Section 5.10.12 | Reducing agents | NMM (0.1 M) then either 20% BME/DMF or 5% DTT/DMF (5 min)163 | 20% piperidine in DMF (4 h)163 | |
DABDT, DIEA/H2O/MeCN (3:3:94)164 | 95% TFA (1 h, RT)163 | |||
Standard Fmoc SPPS reagents163 | ||||
2,4,6-Trimethoxyphenylthio (S-Tmp); Section 5.10.12 | Reducing agents | NMM (0.1 M) then either 20% BME/DMF or 5% DTT/DMF (5 min)163 | 20% piperidine in DMF (4 h)163 | |
95% TFA (1 h, RT)163 | ||||
Standard Fmoc SPPS reagents163 | ||||
Sec-isoamyl mercaptan 3-methyl-2-butanethiol (SIT); Section 5.10.13 | Reducing agents | BME in DMF (1:4), 0.1 M DIEA165 | Standard Fmoc SPPS reagents165 | |
20 equiv. DTT, MeCN/DIEA/H2O (90:5:5)165 | ||||
5 equiv. DTT × 3, DMF/DIEA/H2O (95:2.5:2.5)165 | ||||
2-Methyloxolane-3-thiol (MOT); Section 5.10.13 | Reducing agents | BME in DMF (1:4), 0.1 M DIEA165 | Standard Fmoc SPPS reagents165 | |
20 equiv. DTT, MeCN/DIEA/H2O (90:5:5)165 | ||||
5 equiv. DTT × 3, DMF/DIEA/H2O (95:2.5:2.5)165 | ||||
2-Pyridinesulfenyl (S-Pyr); Section 5.10.14 | Reducing agents | Thiols4 | 1 M TFMSA in TFA-anisole (10:1, 2 h, 0 °C)4 | |
Standard Boc SPPS reagents4 | ||||
4,4-Bis(dimethylsulfinyl)benzhydryl (Msbh); Section 5.11.1 | Safety-catch | NH4I/DMS/TFA166 | Acid (TFA, HF)166 | |
Oxidants166 | ||||
Reductants166 | ||||
Standard Boc SPPS reagents166 |
Fig. 5 (a) Cys thiol protection with the benzyl (Bn/Bzl) protecting group (b) the synthesis of oxytocin using Cys(Bzl). |
Should the desired product of Trt cleavage be a new disulfide bond, oxidising agents such as I2 may be used.4 The rate of Trt removal and subsequent disulfide formation by I2 has previously been to shown to depend on the choice of solvent.171 It is common to remove Trt using weak acids (e.g. HBF4 or TFA) in the presence of scavengers such as triisopropylsilane (TIS) or triethylsilane (TES); these scavengers prevent the released Trt cations from adding back onto the synthesised peptide during cleavage and isolation.172 This method of Trt removal renders Trt orthogonal to other common protecting groups such as Acm or tBu, as shown in the regioselective synthesis of human insulin, via sequential disulfide formation.72 Other applications of Cys(Trt) in SPPS include synthesis of human relaxin,173 and α-melanocyte stimulating hormone (α-MSH) analogues.174 Additionally, macrocyclic peptides displaying in vitro anti-malarial properties have also been synthesised using Cys(Trt); in this example, the Trt protecting group is retained rather than removed in the final bioactive product (Fig. 6b).175 Cys(Trt) has also very recently been reported to undergo complete deprotection in model peptides when treated with CuSO4 and cysteamine in aqueous buffered conditions.73
Cys(Trt) is routinely used in Fmoc SPPS. It is worth noting that Trt is highly hydrophobic and that the Trt cation-scavenger adducts formed during deprotection may not be removed completely during final TFA cleavage however. The presence of Trt can then mask the quality of the crude peptide due to its high UV absorbance.103 To avoid incomplete detritylation, typical cleavage cocktails of >90% TFA with TIS/TES (typically ca. 5%) can be used. These conditions can, however, cause the reduction of the indole ring of Trp.71,172 Due to its lability to TFA, Trt is removed during cleavage of acid-labile resins (as commonly seen in Fmoc SPPS). It is still possible to use Trt as a protecting group when forming disulfide bonds in solution post-cleavage from the resin, however, the deprotected residues must be re-tritylated post-synthetically. This was exemplified in the regioselective syntheses of μ-conotoxin SIIIA (Fig. 6c) and human hepcidin, using combinations of StBu, Trt, Meb/Mob and Acm.74
The above methods of deprotection are harsh,128 and often result in the formation of side products and low yields.166 Additionally, incubation in neat TFA causes ∼20% deprotection.86 In 2018, PdCl2 in a 50 mM Tris or urea buffer at 37 °C was shown to cleave tBu, providing a much milder way to remove the protecting group.85 This is significant for tBu, as the harsh conditions required for deprotection can hinder its practical use. tBu is not removed by [Pd(allyl)Cl]2, making it orthogonal to Thz and Acm under those conditions. This method of removal was successfully used to synthesise an activity-based probe of ubiquitinated histone H2A (Fig. 9b).85
Cys(Mob) can undergo oxidation to give the corresponding Mob-protected sulfone, Cys(Mob(O)) (Fig. 10b) with either NaBO3185 or H2O2.186 Cys(Mob(O)) can in turn be incorporated into peptides via Fmoc-SPPS. Deprotection with TfOH:TFA:H2O (50:45:5) leads to removal of the Mob protecting group, generating peptides bearing Cys sulfinic acid, a known PTM of Cys-containing proteins that can regulate protein function.186 Additionally, Cys(Mob(O)) can be used for disulfide formation via sulfoxide-directed disulfide reactions187 in an I2-free manner (I2 can otherwise oxidise amino acids such as Trp).188 Example syntheses include oxytocin,189 chicken calcitonin-gene-related peptide (cCGRP),190 and human insulin-like peptide-6 (INSL-6) (Fig. 10c).188
We note to the reader that the DMB protecting group is different to that of the similarly abbreviated 2,4-dimethoxybenzyl (Dmb) group, which is used as an amide backbone protecting group to prevent peptide aggregation and off-target reactions during peptide synthesis.4
More recently, it has been reported that Acm can be removed under significantly milder conditions using transition metal catalysts, such as Pd(II) complexes.109,112For example, the synthesis of the two-Cys containing ubiquitin-like protein 5 (UBL5) could be achieved in a one-pot manner, with deprotection of Cys(Acm) in the final step performed using 30 equiv. PdCl2 within 15 min (followed by DTT addition).112 Treatment of Cys(Acm)-containing peptides with PdCl2 in H2O or guanidinium chloride (Gdn·HCl) pH 7 (30 min, 37 °C) followed by DTT addition led to total deprotection to yield the free thiol-containing peptide. Alternatively, using 100 equiv. of diethyldithiocarbamate (DTC) after Pd-mediated deprotection of multi-Cys(Acm)-containing peptides allows for oxidation to form disulfide bonds. Total synthesis of E. Coli thioredoxin (Trx-1) in a one pot manner has been reported using this strategy. First NCL was performed between an N-terminal fragment (bearing two Cys(Acm) residues and a C-terminal thioester, Trx(1-56)) and a C-terminal fragment (bearing an N-terminal Cys (Trx58-109)). This was then followed by desulfurisation, and finally sequential Acm deprotection and disulfide formation using PdCl2 and DTC respectively to yield the target protein (Fig. 20c).109 Moreover, whilst Acm and Thz are both labile to Pd(II) complexes, Acm is stable to the lower concentrations of [Pd(allyl)Cl]2 compared to Thz, giving the two groups some orthogonality to one another. Following Thz deprotection, Acm can be deprotected within 5 h following the addition of an extra 10 equiv. of [Pd(allyl)Cl]2. Additionally, Acm is orthogonal to tBu under these conditions.85 Acm is also stable to Pd(0)-mediated removal of N-terminal Alloc-protected α-amino groups. This, in tandem with a β-thiolactone-mediated NCL procedure,213,214 was demonstrated in the full chemical synthesis of histone H3 bearing an Nε trimethylated Lys residue ([Lys(Me3)9]H3.1).113 The use of PdCl2 for Cys(Acm) deprotection, with DTC for Pd quenching and disulfiram (DSF) for disulfide formation, has very recently been described in combination with photolabile Cys protecting groups for rapid deprotection/disulfide formation in multiple disulfide-rich peptides, including Linaclotide and Ecballium elaterium trypsin inhibitor II (EETI-II, Fig. 20d).215
In addition to Pd(II) chemistry, deprotection of Cys(Acm) using CuSO4 has been reported.73 Initially, it was noted that CuSO4-mediated deprotection of Cys(Thz) would also lead to deprotection of Cys(Acm) in the same peptide scaffold if ascorbate was not added, but would leave Acm intact if ascorbate was added. In model peptides, CuSO4 would only deprotect Cys(Acm) if the peptide contained an N-terminal Cys that was either unprotected or protected with Thz (which could undergo CuSO4-mediated deprotection to reveal an N-terminal Cys). This instance of double deprotection has been hypothesised to result from Cu-mediated oxidation of the deprotected/unprotected N-terminal thiol to sulfenic acid, which then undergoes nucleophilic attack from the Cys(Acm) sulfur, leading to disulfide formation and loss of Acm. Excess Cu can then be quenched with DTT (leading to the free thiol-containing peptide) or ethylenediaminetetraacetic acid (EDTA, leading to the disulfide containing peptide). This protocol was demonstrated in the synthesis of the two-disulfide containing peptide apamin. Following formation of the first disulfide bond via Cys(Trt) deprotection and air oxidation, sequential deprotection of Cys(Thz) and Cys(Acm) with CuSO4 (100 mM, 6 M Gdn.HCl, 0.1 M HEPPS) followed by quenching with EDTA (100 mM), could be used to engineer the second disulfide bond in a regioselective manner (Fig. 20e). Oxidation by-products were formed, however, if the order of disulfide bond formation in this procedure was reversed (with Cys(Mob) used as opposed to Cys(Trt)). In the case of peptides which lack an unprotected N-terminal Cys, Cys(Acm) can be deprotected by addition of CuSO4 in the presence of an equimolar amount of aminothiol additive, such as cysteamine.73
Care must be taken when using I2 in AcOH, as partial oxidation of methionine (Met) residues to methionine sulfoxide (Met(O)) can occur. Modification of Trp residues has also been observed.121 Despite its higher resistance to acid than Bam, Tacm can still show instability in HF and TFA/heat. It is therefore not particularly suitable for Boc SPPS,111 although it has been used successfully in Boc solution phase synthesis of pBNP using this strategy.120 Furthermore, Cys(Tacm) has been used in Fmoc SPPS to synthesise oxytocin in combination with the silyl chloride-sulfoxide system.220
Dnpe is stable to a wide variety of reagents, both basic and acidic. These include: 5% DIEA in DCM (2 h), 40% TFA in DCM (24 h), 90% HF in the presence of p-cresol or anisole (1 h, 0 °C), and TFMSA-p-cresol in TFA (1:3:10, 2 h, 25 °C). It is also stable to oxidative conditions, e.g. Tl(TFA)3 in TFA, I2 in 80% AcOH (aq.) and is thus orthogonal to protecting groups including Meb, Acm and StBu.124
After its initial documentation, the Allocam group saw little use as a protecting group for Cys in the literature. More recently, however, it has seen a revival as an orthogonal Cys protecting group. Here, alternative deprotection protocols that lead directly to the disulfide formation on-resin have been developed: Pd(OAc)2 (1.5 equiv.), 3% NMM and 5% AcOH in DMSO for 2 h. Under these conditions, complete removal of Allocam could be achieved to yield the disulfide-containing resin-bound peptide.131 The use of Cys(Allocam) has since been expanded towards higher-yielding, on-resin synthesis of α4/7-Conotoxin LvIA (α-LvIA, Fig. 25c).205 Orthogonality of Cys(Allocam) to other protecting groups was also further established. For example, treatment of peptides containing Cys(Trt) and Cys(tBu) with Pd(OAc)2 (1.5 equiv.), 3% NMM and 5% AcOH in DMSO for 2 h, followed by resin cleavage, led to no removal of either protecting group, whereas treatment of Cys(Mmt) with the same conditions gave a mixture of products. Peptides containing two Cys(Allocam) residues could be fully deprotected to give the corresponding disulfide when using I2, whereas the Allocam protecting groups remained intact when using conditions typically used for StBu/Mmt removal (20% BME, DTNP, 1% TFA, 5% TIS). Depending on the combination of Cys protecting groups used, Cys(Allocam) could be orthogonally deprotected first or last in a given sequence to perform regioselective synthesis of disulfide-containing α-LvIA on-resin.205
Fsam is stable to acidic and basic conditions, and is compatible with both Boc and Fmoc SPPS. Fsam is labile to similar oxidative conditions to Acm and Phacm. A side reaction was observed during SPPS, which was suggested to be due to N-allyl peptides forming via nucleophilic attack of the neighbouring α-amino function at the allyl group. This reaction was proposed to occur under the basic conditions present during Fmoc removal or during the coupling step. This phenomenon has also been observed with the Alloc group.133
More recently, alternative strategies for the deprotection of Thz have been described; these have primarily been transition-metal based.235 For example, deprotection of Thz can be achieved using water-soluble Pd(II) complexes, such as [Pd(allyl)Cl]2 followed by treatment with DTT to both obtain the free thiol and quench remaining Pd species (Fig. 30c). This is performed under native chemical ligation (NCL) conditions and in the presence of MPAA and TCEP, with complete removal obtained within 15 min.138 MPAA and TCEP appear to be crucial for efficient removal (100% removal in 15 min vs. 40% removal after 4 h). It has been hypothesised that this is due to MPAA and TCEP chelating to Pd(II), or possibly reducing it to Pd(0). To demonstrate the usage of this deprotection method, Lys34-ubiquitinated H2B and several other sample peptides have been synthesised using Pd(II) to deprotect Thz.138 In further work, it was found that [Pd(allyl)Cl]2 and GSH (1:1) in 6 M Gdn·HCl (pH 6.5, 37 °C, 45 min) were sufficient for full removal, along with its orthogonality demonstrated to both Acm and tBu.85 Pd-mediated Thz deprotection has also been successfully applied towards in vivo systems for triggered release/chemical activation of peptides/proteins.236 Furthermore, Thz analogues have also been employed in within the field of protein bioconjugation; Pd-mediated237 (or Ag-mediated)238 “unmasking” of unnatural Thz side chains leads to generation of an α-oxo aldehyde side chain, which can be used for downstream site-selective protein modification.239 Aside from Pd, Cu complexes can also be used to deprotect Thz.139,240 Thz is removed by CuSO4 in the presence of sodium ascorbate (critical for avoiding oxidation by-products), in 5 M Gdn·HCl in HEPPS buffer (pH 7.0, 1 h, 37 °C). This was demonstrated in the synthesis of CXCL14 (Fig. 30d).139 As discussed previously, the reaction can then be quenched with DTT or EDTA.73 No epimerisation or side products are observed using this method of deprotection, and the reaction can be performed under standard NCL conditions.139 Outside of transition-metal based strategies, 2,2′-dipyridyl disulfide (DPDS) in 50% MeCN (0.1% TFA) as a reagent for Thz deprotection has also been described.140,241
Protection using the Nin group avoids the formylation side reactions normally found during TFA and HF cleavage when His(Bom) groups are present, without the need for additional scavengers. Additionally, in thioester-containing peptides, the products will cyclise under deprotection conditions, enabling a one-step process. As proof of concept, a Nin-E2-SR peptide was synthesised and cyclised in one-step (Fig. 30f), using an excess of MPS (where E2 refers to a 40-residue long sequence of the chemokine receptor CCR5's second extracellular loop).141
The protecting group has since featured in the synthesis of a thioester-containing HCDLP pentapeptide; this peptide can then undergo NCL to semisynthesise a variant of a nickel-dependant superoxide dismutase (NiSOD). Critically, Cys(oNB) remained intact after acidic deprotection of other amino acid residues (95% TFA) of the pentapeptide, and subsequent NCL to a recombinant Streptomyces coelicolor NiSOD bearing an N-terminal Cys. Photochemical deprotection of oNB could then be achieved by irradiation at 365 nm (100 mM NaOAc, 20 mM TCEP, 10 mM semicarbazide, pH 5.8).244 Cys(oNB) can also be incorporated into proteins via unnatural amino acid mutagenesis245,246 as demonstrated with a photocaged human superoxide dismutase (nbC-33-hSOD, Fig. 31b),246 along with being used to study protein activity,247,248 As discussed previously, Cys(oNB) is also orthogonal to Pd-mediated deprotection of Acm, as recently displayed in the synthesis of Linaclotide and plectasin (Fig. 31c).215 The oNB protecting group is, however, a poor chromophore and displays low two-photon sensitivity. Coumarin-based protecting groups have since been described in an attempt to address some of these issues.145
oNV is fully compatible with Fmoc SPPS, including stability to 10% TFMSA/TFA in the presence of excess dipyridine disulfide, the conditions necessary to convert the tBu protecting group to the activated S-Pyr group. The two groups may, therefore, be used together – photolysis of the oNV is followed by thiolysis in the presence of Cys(S-Pyr), selectively forming a disulfide bond. This strategy has been used for the solid-phase synthesis of several Cys-rich peptides, including human insulin and α-conotoxin ImI (Fig. 33b).142 oNv photocaged Cys has since been utilised in the synthesis of multi-Cys-containing peptide fragments.252 The protected amino acid has also been incorporated into proteins via unnatural amino acid mutagenesis to yield photocaged eGFP (EGFPTyr39DMNB-Cys),253 and photocaged glutathione peroxidase 3 (caged Gpx3 Cys32); the latter can then be oxidised with H2O2 to generate the sulfenic acid analogue (caged Gpx3 Cys32O, Fig. 33c).254
Although incorporation into a peptide using Fmoc SPPS is not an issue, the photocleavage efficiency of Bhc-protected thiols is context-dependent. This is because the major product of irradiation is typically an unwanted photoisomer (a 4-methylcoumarin-3-yl thioether, Fig. 34b) instead of the free thiol, which limits the applications of Bhc as a Cys protecting group.
The NDBF protecting group has been additionally used for live cell applications. A fluorescent, farnesylated peptide which can undergo enzymatic palmitoylation by palmitoyl acyltransferase was synthesised and protected as a NDBF thioether (Fig. 34d).145 Incubation of this peptide with human ovarian carcinoma SKOV3 cells led to localisation of the peptide to the cytosol and the Golgi apparatus. Upon irradiation, the peptide migrated to the plasma membrane, indicating that enzymatic palmitoylation had occurred (Fig. 34e).
Cys(Scm) has recently used in protein bioconjugation to facilitate the modification of MB23-Cys (an Alphabody – a trihelical peptide with potential as an anti-cancer therapeutic) via disulfide linkages. MB23-Cys was first reduced using DTT (to remove any protein dimer), then modified with a folic acid, Cys(Scm)-containing peptide in 10 mM Tris–HCl, pH 7.4, 37 °C (Fig. 37c).148
Sulfonyl (SO2R) protecting groups (Fig. 38d) have recently found some use in the synthesis of enantiomerically-enriched α-hydroxy and α-chloro acid building blocks. The sulfonyl group is first introduced to the Cys residue, and diazotisation is subsequently used to generate the α-hydroxy or α-chloro acid (depending on whether H2SO4 or HCl is used).260
Npys is not compatible with Fmoc SPPS, as it is unstable to piperidine (81% decomposition following treatment with 50% piperidine in DCM for 10 min). It is also unstable to TBAF−, an alternative to piperidine for Fmoc deprotection. Additionally, while treatment with HF in the presence of anisole or p-cresol leaves Npys unaltered, HF in the presence of p-thiocresol or DMS (HF/DMS/p-cresol, 25:65:10 or HF/DMS/p-cresol/p-thiocresol, 25:65:5:5), has been shown to cause significant loss of the protecting group.157 Npys has been used in many syntheses.154 An early example of this is the synthesis of [Lys]8vasopressin, which was chosen as a model peptide to display the use of Npys in Boc SPPS.156 More recently, the Npys protecting group has been the focus of a solid phase disulfide ligation (SPDSL) system.262 Here, Npys-Bn is loaded onto a solid support and converted to resin-bound Npys-Cl through chlorosulfenylation. Cys(tBu)-containing peptides can then be loaded onto the resin via Npys-mediated displacement of the tBu protecting group. Peptide release, along with disulfide bond formation, can then be achieved through addition of a Cys-containing peptide. Subsequent intramolecular amide formation then yields a disulfide containing peptide, as demonstrated in the synthesis of oxytocin.262 A similar platform that replaces the Npys-Cl group for a more stable Npys-OPh(para-fluoro) group has very recently been reported in the literature.263
Cys(StBu) has been used in the synthesis of a range of different peptides, including μ-SIIIA (Fig. 39c),74 α-LvIA (Fig. 39d),205 and Linaclotide (Fig. 39e).276 The removal of StBu with reducing agents has previously been shown to be sequence-dependent and challenging;166 additionally, the lengthy times required for deprotection are undesirable when using the group in routine SPPS.163 Alternative disulfide-based protecting groups such as dimethoxyphenylthio (S-Dmp) and 2,4,6-trimethoxyphenylthio (S-Tmp) have since been reported as replacements for the StBu protecting group.163
The Pac protecting group has been employed in the semi-synthesis of histone H3 bearing an Nε trimethylated Lys residue ([Lys(Me3)9]H3.1, Fig. 40d).278 First, the C-terminal fragment of the peptide containing an N-terminal Cys and two internal Cys residues (H3.1(26-135)) was recombinantly produced by gene expression and subsequent peptide production in E. coli. The N-terminal Cys residue was then orthogonally protected with Thz, whereas both the internal Cys were protected with Pac. Conditions for deprotection of N-terminal Cys(Thz) using methoxyamine also lead to oxime ether formation at the ketone position of Pac, converting Pac to “PacN”; this, however, does not impact on downstream deprotection as PacN also shows lability to Zn/AcOH similar to Pac. Deprotection of the N-terminal Cys(Thz), followed by CPE ligation with a suitable H3 N-terminal fragment and subsequent desulfurisation gave the dual Cys(PacN) protected peptide. Critically, the Cys(PacN) groups remained resistant to desulfurisation. Deprotection with Zn powder and 15% 3-mercaptopropionic acid (MPA) in H2O with 6 M Gdn yielded the product [Lys(Me3)9]H3.1 histone.278 Similarly, it has been shown that Cys(Pac) can be used in conjunction with recombinantly generated protein segments for the traceless semisynthesis of human small heat shock protein (Hsp27) and a lipidated variant of murine prion protein (Prp).162 Pac proved compatible both with installation into thioester-containing peptides, and in radical desulfurisation steps. Cys(Trt) was also investigated; however, in this case, desulfurisation of Cys(Trt) containing Hsp27 proved lead to a mixture of products.162
NH4I/TFA treatment is incompatible with Trp-containing peptides, as Trp undergoes a number of side reactions (due to the presence of I2) unless the indole nitrogen is protected with a formyl group. This is standard in Boc SPPS and thus should not present a significant issue in this case. The formyl group is, however, removed by piperidine treatment so if Fmoc SPPS is being used alternative sulfoxide reduction methods or Trp protecting groups are needed.166 Msbh has been used in the regioselective synthesis of human hepcidin (Fig. 44b), providing an orthogonal strategy for the synthesise of peptides containing four disulfide bonds.166 It has also been theorised to be key to the regiospecific construction of peptides containing five or more disulfide bonds, a feat that is yet to be accomplished.5
Finally, there is an increasing desire to make the process of SPPS more “green”, which currently uses toxic and environmentally unfriendly reagents such as piperidine, DMF, and DCM.286 This will require protecting group chemistry that is compatible with greener reagents, such as water/aqueous-based solvent systems. Enzymatically labile protecting groups such as Phacm, or photolabile groups such as NDBF, have already been successfully utilised in this context; future protecting groups with similar properties will likely prove important in the “greening” of SPPS. We additionally anticipate the field of peptide chemistry and growing field of protein bioconjugation will likely benefit each other in the coming years. In contrast to conventional peptide synthesis, protein bioconjugation/deconjugation is, by design, carried out in benign/environmentally friendly aqueous systems in a site-specific manner. Additionally, there is a continuing search for new methodology for use in bioconjugation; although these must yield stable conjugates, strategies whereby the bioconjugate can be released in a controlled manner offers huge potential in applications such as controlled drug release. This interplay has already been demonstrated with Cys protecting groups that have since been applied to bioconjugation, such as Acm, Scm, Thz, and SiPr. Similarly, Suc, which is routinely used in bioconjugation, has very recently been demonstrated as a Cys protecting group in peptide synthesis. It is likely other bioconjugation strategies will also be applied to peptide synthesis in the coming years.
We have reviewed, analysed, and discussed over 60 individual protecting groups for the thiol group of Cys. We hope that this review provides a useful resource for peptide and protein chemists research, and encourages further research into both old and new Cys protecting groups.
2-Moxan | 2-methoxy-9H-xanthen-9-yl |
2,6-diMeOBn | 2,6-dimethoxylbenzyl |
4MeO-2MeBn | 4-methoxy-2-methylbenzyl |
5-Npys | 5-nitro-2-pyridinesulfenyl |
7,8BCMCMOC | [7,8-bis(carboxymethoxy)coumarin-4-yl]methoxycarbonyl |
Acm | acetamidomethyl |
AcOH | acetic acid |
Ad/1-Ada | 1-adamantyl |
ADC | antibody drug conjugate |
Alloc | allyloxycarbonyl |
Allocam | allyloxycarbonylaminomethyl |
AgOAc | silver acetate |
AgOTf | silver trifluoromethanesulfonate |
Arg | arginine |
Bam | benzamidomethyl |
BCMACMOC | [7-bis(carboxymethyl)-amino-coumarin-4-yl]methoxycarbonyl |
Bhc | 6-bromo-7-hydroxycoumarin |
BME | 2-mercaptoethanol |
Boc | tert-butyloxycarbonyl |
Bom | benzyloxymethyl |
BSA | bovine serum albumin |
Bu3SnH | tributyltin hydride |
Bzl/Bn | benzyl |
C4MNB | α-carboxy-4-methoxy-2-nitrobenzyl |
CAN | ceric ammonium nitrate |
CDMNB | α-carboxy-4-methoxy-2-nitrobenzyl |
CuAAC | copper(I)-catalysed alkyne-azide cycloaddition |
CPE | cysteinylprolyl ester |
CPI | cysteinylprolyl imide |
Cys | cysteine |
Dbs/Sub | 5-dibenzosuberyl |
DBU | 1,8-diazabicyclo[5.4.0]undec-7-ene |
DCC | dicyclohexylcarbodiimide |
DCM | dichloromethane |
Ddm/4,4′-diMeODpm | 4,4′-dimethoxydiphenylmethyl |
DIEA | N,N-diisopropylethylamine |
DMB | 3,4-dimethylbenzyl |
Dmbm | (4,6-dimethoxy-2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)methanol |
DMF | dimethylformamide |
DMS | dimethyl sulfide |
DMSO | dimethylsulfoxide |
Dnpe | 2-(2,4-dinitrophenyl)ethyl |
DPDS | 2,2′-dipyridyl disulfide |
Dpm | diphenylmethyl |
DSF | disulfiram |
DTC | diethyldithiocarbamate |
DTNB | 5,5′-dithiobis-(2-nitrobenzoic acid) |
DTNP | 2,2′-dithiobis(5-nitropyridine) |
DTP | 2,2′-dithiodipyridine |
DTT | dithiothreitol |
EDT | ethane-1,2-dithiol |
EDTA | ethylenediaminetetraacetic acid |
EETI-II | Ecballium elaterium trypsin inhibitor II |
EGFP | enhanced green fluorescent protein |
EtOH | ethanol |
Fm | 9-fluorenylmethyl |
Fmoc | 9-fluorenylmethoxycarbonyl |
Fnam | [N-[2,3,5,6-tetrafluoro-4-(N′-piperidino)-phenyl], N-allyloxycarbonyl]-aminomethyl |
Fsam | S-[N-[2,3,5,6-tetrafluoro-4-(phenylthio)-phenyl], N-allyloxycarbonyl]-aminomethyl |
GBP | growth-blocking peptide |
Gdn·HCl | guanidinium chloride |
GFP | green fluorescent protein |
GSH | glutathione |
H2O | water |
HEPES | (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) |
HEPPS | 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid |
HFIP | hexafluoro-2-propanol |
Hgm | hydroxyglycine-Acm |
Hg(OAc)2 | mercury(II) acetate |
His | histidine |
Hmb | 2-hydroxy-4-methoxy benzyl |
hNP2 | defensin human neutrophil peptide-2 |
HOBt | 1-hydroxybenzotriazole |
Hqm | hydroxyquinoline-Acm |
Lys | lysine |
MAA | 3-mercaptoacetic acid |
mBhc | 6-bromo-7-hydroxy-3-methylcoumarin |
Mbom | 4-methoxybenzyloxymethyl |
Meb/4-MeBn/4-MeBzl | 4-methylbenzyl |
MeCN | acetonitrile |
MeOH | methanol |
MeONH2·HCl | O-methylhydroxylamine |
MESNA | sodium 2-mercaptoethanesulfonate |
Met | methionine |
Mmt | 4-methoxytrityl |
Mob/MBzl | 4-methoxybenzyl |
MOT | 2-methyloxolane-3-thiol |
MPAA | 4-mercaptophenylacetic acid |
MPS | mercaptopropiosulfonic acid |
Mpt | dimethylphosphinothioyl |
Msbh | 4,4-bis(dimethylsulfinyl)benzhydryl |
Mtt | 4-methyltrityl |
NCL | native chemical ligation |
NDBF | nitrodibenzofuran |
NDMBA | 1,3-dimethylbarbituric acid |
Nin | ninhydrin |
NMM | N-methylmorpholine |
NpsCl | 2-nitrophenylsulfenyl chloride |
Npys | 3-nitro-2-pyridinesulfenyl |
OMe-NDBF | methoxy-nitrodibenzofuran |
oNB | 2-nitrobenzyl |
oNV | 2-nitroveratryl |
pAB | para-aminobenzyl |
Pac | phenacyl |
PAL | peptide-amide-linker |
PB | phosphate buffer |
Pbfm/Pmbf | 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-methyl |
pBNP | porcine brain natriuretic peptide |
PBS | phosphate-buffered saline |
[Pd(allyl)Cl]2 | allylpalladium(II) chloride dimer |
PdCl2(PPh3)2 | bis(triphenylphosphine)palladium(II) dichloride |
Pd(OAc)2 | palladium(II) acetate |
Pd(PPh3)4 | tetrakis(triphenylphosphine)palladium(0) |
PFTase | protein farnesyltransferase |
PGA | penicillin G acylase |
Phacm | phenylacetamidomethyl |
PhSiH3 | phenylsilane |
Pmcm | 2,2,5,7,8-pentamethylchroman-6-methyl |
pNB | para-nitrobenzyl |
Pocam | N-methyl-phenacyloxycarbamidomethyl |
PTM | post-translational modification |
Pym | 2-oxo-1-pyrrolidinyl)methyl |
RP-HPLC | reversed phase high-performance liquid chromatography |
Sac | S-allyl cysteine |
Scm | carbomethoxysulfenyl |
S-Dmp | dimethoxyphenylthio |
SIT | sec-isoamyl mercaptan/3-methyl-2-butanethiol |
SiPr | S-iso-propyl |
Snm | (N′-methyl-N′-phenylcarbamoyl)sulfenyl |
SO2R | sulfonyl |
SO3H | sulfonic acid |
SPPS | solid phase peptide synthesis |
SprC | S-propargyl-cysteine |
S-Pyr | 2-pyridinesulfenyl |
StBu | tert-butylsulphenyl |
S-Tmp | 2,4,6-trimethoxyphenylthio |
Suc | succinimide |
Tacm | trimethylacetamidomethyl |
TBAF | tetrabutylammonium fluoride |
tBu | tert-butyl |
TCEP | tris(2-carboxyethyl)phosphine |
TES | triethylsilane |
TFA | trifluoroacetic acid |
TFE | tetrafluoroethylene |
TFMSA | trifluoromethanesulfonic acid |
THF | tetrahydrofuran |
Thp | tetrahydropyranyl |
Thz | thiazolidine |
TIS | triisopropylsilane |
Tmbm | 4,5,6-trimethoxy-2,2-dimethyl-2,3-dihydrobenzofuran-7-methyl |
Tmob | 2,4,6-trimethoxybenzyl |
TMSBr | bromotrimethylsilane |
TMSOTf | trimethylsilyl trifluoromethanesulfonate |
TMTr | 4,4′,4′′-trimethoxytriphenylmethyl |
Tppts | 3,3′,3′′-phosphanetriyltris(benzenesulfonic acid) trisodium salt |
Trp | tryptophan |
Trt | trityl |
Trx | thioredoxin |
Tyr | tyrosine |
VA-044 | 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride |
UBL5 | ubiquitin-like protein 5 |
Xan | 9H-xanthen-9-yl |
ΨPro | pseudoproline |
μ-SIIA | μ-conotoxin SIIIA |
This journal is © The Royal Society of Chemistry 2021 |