. Author manuscript; available in PMC: 2021 Mar 25.

Published in final edited form as: Chem Rev. 2019 Nov 27;120(6):3051–3126. doi: 10.1021/acs.chemrev.9b00450

Table 1.

Applications of NCL and EPL to protein semisynthesis.^a

Protein	Location of synthetic modification	Modification
Abl tyrosine kinase	Central	Fluorophore labeling¹⁰⁶
	n/a	Segmental Isotopic Labeling¹⁰⁷
Adapter protein crk-II	n/a	Segmental isotopic labeling^{108, 109}
Akt1 serine-threonine kinase	C-terminal	Ser473Ph¹¹⁰, Ser477Ph¹¹⁰, Ser479Ph¹¹⁰
β-Amyloid	C-terminal	Unmodified¹¹¹
Androgenic Gland Hormone	C-terminal	N-linked glycosylation¹¹²
Anthrax Lethal Factor	N-terminal	N-terminal acetylation¹¹³
Apolipoprotein E (ApoE)	n/a	Segmental Isotopic Labeling¹¹⁴
Arylalkylamine N-acetyltransferase (AANAT)	N-terminal	Thr31Ph, ¹¹⁵ Thr31Pma¹¹⁶
	C-terminal	Ser205Ph,^{115, 117} Ser205Pfa^{115, 117}
Aspartate transporter Glt_Ph	C-terminal	Arg397Citrulline¹¹⁸
Autophagy-related protein 3 (Atg3)	N-terminal	Lys19Ac¹¹⁹
	Central	Lys48Ac¹¹⁹
Azurin	C-terminal	Cys112Sec¹²⁰, Met121Nle¹²¹, Met121Sec¹²², Met121Sem¹²¹, Met121Hcy¹²³
Casein kinase II (CK2)	C-terminal	Thr344Ph¹²⁴, Ser347GlcNAc¹²⁴
Chemotaxis protein CheA	n/a	Segmental Isotopic Labeling¹²⁵
Checkpoint kinase 2 (CHK2)	N-terminal	Thr68Ph¹²⁶
Chorismate mutase	C-terminal	Arg90Cit,¹²⁷ βArg9¹²⁸
Chaperone protein ClpB	n/a	Segmental isotopic labeling¹²⁹
	N-/C-terminal	Fluorophore labeling¹³⁰
Chaperone protein DnaK	n/a	Segmental Isotopic Labeling¹³¹
Chemokine (C-X-C motif) ligand 8 (CXCL8)	C-terminal	C-terminal truncation¹³², fluorescein¹³³, benzoylphenylalanine¹³⁴, β-peptide mimetics¹³⁵, segmental isotopic labeling¹³⁶
Chemokine (C-X-C motif) ligand 12 (CXCL12)	C-terminal	6-Nitroveratryl (Nvoc) photocages^{137, 138}, fluorescein¹³⁹
γD-Crystallin	n/a	Segmental isotopic labeling¹⁴⁰
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase	n/a	Unmodified¹⁴¹
Eph receptor tyrosine kinase	C-terminal	Unmodified^{142, 143}
Erythropoietin (EPO)	N-terminal	N-linked oligosaccharide,^{144, 145} alkyne tag¹⁴⁶
Eukaryotic initiation factor-4A (elF4A)	C-terminal	Ser209Ph¹⁴⁷
Glutamyl-prolyl-tRNA synthetase	n/a	Segmental Isotopic Labeling¹⁴⁸
Glutaredoxin	N-terminal	Cys11Sec¹⁴⁹
Glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1)	N-terminal	Thr/SerGalNAc¹⁵⁰
	C-terminal	Thr/SerGalNAc¹⁵⁰
Glycogen synthase	C-terminal	Thr668Ph¹⁵¹
α-Hemolysin	Central	Thr117Pra¹⁵², Thr117LysAcac¹⁵³
Heterogeneous nuclear ribonucleoprotein L (hnRNPL)	n/a	Segmental isotopic labeling¹⁵⁴
Histone H2A	N-terminal	Lys5Ac¹⁵⁵, Lys9Ac¹⁵⁵, Lys13Ac¹⁵⁵, Lys13Ub⁸¹, Lys15Ac¹⁵⁵, Lys15Ub⁸¹, Lys15Ub (H2A.X)¹⁵⁶
	C-terminal	Ser139ph (H2A.X)¹⁵⁶, Lys118Ac¹⁵⁵ Lys119Ub^{157,81, 158}
Histone H2B	N-terminal	Ser14ph,⁶⁷ Lys5Ac,⁶⁷ Lys11Ac,⁶⁷ Lys12Ac,⁶⁷ Lys15Ac⁶⁷, Lys16Ac¹⁵⁵, Lys20Ac¹⁵⁵, Lys34Ub⁸¹
	C-terminal	Lys108Ac¹⁵⁵, Ser112GlcNAc¹⁵⁵, Lys116Ac¹⁵⁵, Lys120Ac¹⁵⁵, Lys120Ub¹⁵⁹, Lys120Su¹⁶⁰, Lys125Ac¹⁵⁵
Histone H3	N-terminal	Arg2me2¹⁵⁵, Lys4Ac¹⁶¹, Lys4me3^162–164, Gln5Sero¹⁶⁵, Lys9me3^{161–164, 166–169}, Lys9Ac^{77, 161}, Lys14Ac^{77, 161}, Lys18Ac^{77, 161, 170}, Lys18Cr¹⁷¹, Lys23Ac^{161, 170}, Lys27me3^{162–164, 172}, Lys23Ac¹⁷⁰, Lys36me3^{155, 164}
	Central	Lys79me2/3¹⁶⁴, Tyr41Ph¹⁵⁵, Arg42me2¹⁷³
	C-terminal	Lys115Ac¹⁷⁴, Lys122Ac¹⁷⁴
Histone H4	N-terminal	Arg3me2¹⁵⁵, Lys5Ac,^{77, 161, 170} Lys5Cr¹⁷¹, Lys8Ac^{77, 161, 170}, Lys8Cr¹⁷¹, Lys12Ac^{77, 161, 175} Lys12Cr¹⁷¹, Lys12Su¹⁷⁶, Lys16Cr¹⁷¹, Lys16Ac,^{77, 175, 177} His18pTza¹⁷⁸, Lys20Ac,⁷⁷ Lys20Cr¹⁷¹, Lys20me3¹⁶⁸
	C-terminal	Lys77Ac¹⁵⁵, Lys79Ac¹⁵⁵
Heterochromatin protein 1α (HP1α)	C-terminal	Atto532 labeling¹⁷⁹,¹⁶⁶ fusion with shigoshin peptide¹⁷⁹
Heat shock protein 27 (Hsp27)	C-terminal	Arg188Apy¹⁸⁰
Heat shock protein 90 (Hsp90)	n/a	Unmodified¹⁸¹
Huntingtin	N-terminal	Thr3Ph^{182, 183}, Lys6Ac¹⁸³, Ser13Ph¹⁸⁴, Ser16Ph¹⁸⁴
Immunity protein Im7	N-terminal	N-linked glycosylation¹⁸⁵
Interferon Response Factor 3 (IRF3)	n/a	Segmental isotop[ic labeling¹⁸⁶
Interleukin 6	Central	N-linked glycosylation¹⁸⁷
Interleukin 13	Central	N-linked glycosylation¹⁸⁸
Jarid2	N-terminal	Lys116me3^{189, 190}, Ser120Ph¹⁹⁰, Ser124Ph¹⁹⁰, Ser126Ph¹⁹⁰
KcsA potassium channel	C-terminal	Unmodified^{102, 191}, Gly77d-Ala^192–194, Gly79ester¹⁹³
	Central	Trp68Bta¹⁹⁵, Val76ester¹⁹⁶, Val76(¹³C¹⁸O)¹⁹⁷, Gly77ester,¹⁹⁶ Gly77(¹³C¹⁸O)¹⁹⁷, Gly79ester,¹⁹⁶ Gly79(¹³C¹⁸O)¹⁹⁷, Gly77d-Ala^{193, 196}
KH-type splicing regulatory protein	n/a	Segmental Isotopic Labeling¹⁹⁸
KvAP potassium channel	Central	G198dA,¹⁹²
Lacticin 481 precursor (LctA)	C-terminal	Unmodified¹⁹⁹, sequence variants²⁰⁰, Thr33Ph^{201, 202}, Cys38Sec²⁰³, Asn39Hse²⁰⁴, Asn39Nva²⁰⁴, Asn39Cba²⁰⁴, Asn39dAsn²⁰⁴, Met40Pra²⁰⁴, Met40Nle²⁰⁴, Met40Nva²⁰⁴, Met40dMet²⁰⁴, Asn41Cba²⁰⁴, Trp43Nal²⁰⁴, Ser28dCys²⁰⁴, Gly29Sar²⁰⁴, Ile31Sar²⁰⁴, His32Sar²⁰⁴, Ile34Sar²⁰⁴, Glu37Sar²⁰⁴
Low molecular weight protein tyrosine phosphatase (LML-PTP)	C-terminal	Tyr131Pmp,²⁰⁵ Tyr132Pmp²⁰⁵
Microcin B17 precursor (McbA)	C-terminal	Sequence variants²⁰⁶, Cys41Hcy
Microtubule-associated protein light chain 3 (LC3)	C-terminal	Unmodified⁶³, lipidation^207–210
Mitogen-inducible gene 6 (Mig6)	C-terminal	Tyr394Ph²¹¹
Mxe GyrA intein	N-terminal	Isotopic labeling²¹²
	C-terminal	Isotopic labeling²¹³, Branched isopeptide intermediate,²¹⁴ Val182hVal²¹⁴, Thr⁺¹Dap^{213, 214}, His187ThA^{213, 214}, Asn198Nva²¹³
NaK ion channel	C-terminal	Asp66Csa,²¹⁵ Asp66Hse²¹⁵
NEDD4-like E3 ubiquitin-protein ligase WWP2	N-terminal	Tyr369Ph²¹⁶
Neurosecretory protein GM	C-terminal	Unmodified²¹⁷
Notch (V1711 – α-secretase cleavage product)	N-terminal	N_α-Ac²¹⁸
Nucleolar protein 3 (NPL3)	n/a	Segmental isotopic labeling¹⁵⁴
Npu DnaE split intein	n/a	Segmental isotopic labeling²¹⁹
cGMP phosphodiesterase	C-terminal	Crosslinker (BPA)²²⁰
p300 HAT domain (WT and circular permutation)	C-terminal	Unmodified,²²¹ Lys1546Ac,²²² Lys1549Ac,²²² Lys1551Ac,²²² Lys1554Ac,²²² Lys1558Ac,²²² Lys1560Ac,²²²
p38α MAP kinase	C-terminal	Thr323Ph²²³
Paxillin	N-terminal	Tyr31Ph,²²⁴ Tyr31Ph(caged)²²⁴
Peroxiredoxin-1	C-terminal	Lys197Ac²²⁵
Phosphatase and tensin homolog (PTEN)	C-terminal	Ser380Ph,^226–228 Thr382Ph, ^226–228 Thr383Ph,^226–228 Thr385Ph,^226–228
Postsynaptic density protein 95 (PSD-95)	N-terminal	Backbone ester mutants^{229, 230}, Ser73Phos (PDZ1)²³¹
	C-terminal	Tyr236Ph (PDZ2)²³¹, Tyr240Ph (PDZ2)²³¹, Y397ph (PDZ3)²³¹
Polypyrimidine tract binding protein	n/a	Segmental Isotopic Labeling^{148, 232}
Prion Protein	C-terminal	Unmodified,²³³ GPI anchor^{234, 235}, Palmitoyl anchor^{234, 236}, PEGylation,²³⁷
Prochlorosin precursor (ProcA)	C-terminal	[²H]Ser13²³⁸
Protein Kinase A	C-terminal	ATP-conjugate²³⁹
Rab7 GTPase	C-terminal	Fluorophore labeling²⁴⁰, Cys(Ger)-OMe^241–244, diprenylation²⁴²
Rad53 serine-threonine kinase	N-terminal	Thr5Ph,²⁴⁵ Thr8Ph,²⁴⁵ Thr12Ph,²⁴⁵ Thr15Ph²⁴⁵
H-Ras GTPase	Central	Gln61GluOMe²⁴⁶, palmitoylation²⁴⁷
K-Ras4B GTPase	C-terminal	Cys(Ger)-OMe^248–250, Ser181Ph²⁴⁸
Rheb GTPase	C-terminal	Cys(Ger)-OMe²⁴⁹
Ribonuclease A (RNase A)	C-terminal	Unmodified⁹⁸, Cys110Sec²⁵¹, β-turn mimics^252–256, N-linked glycosylation^{257, 258}
Ribonucleotide reductase (RNR)	C-terminal	Tyr365Ni²⁵⁹, fluorinated Tyr365²⁶⁰, Tyr365Dopa²⁶¹
S-Adenosylhomocysteine hydrolase	C-terminal	Lys401Ac²⁶², Lys408Ac²⁶²
Selenoprotein M (SelM)	n/a	Unmodified (Sec-containing)²⁶³
Akt1 serine-threonine kinase	C-terminal	Ser473Ph¹¹⁰, Ser477Ph¹¹⁰, Ser479Ph¹¹⁰

SHP-1 phosphatase	C-terminal	Tyr536Pmp/Pfp²⁶⁴, Tyr580Pmp/Pfp²⁶⁴
SHP-2 phosphatase	C-terminal	Tyr542Ph,²⁶⁵ Tyr542Pmp/Pfp,²⁶⁵ Tyr580Ph,²⁶⁵ Tyr580Pmp/Pfp,^{265, 266}
Smad2	C-terminal	Ser465Ph^{267, 268}, Ser467Ph^{267, 268}, diazirine photocrosslinker²⁶⁹, photocages^270–272
Sortase A	C-terminal	Cys184Sec²⁷³, Cys184Hcy²⁷³, Arg197Cit²⁷⁴
Src tyrosine kinase	C-terminal	ATP-conjugate²⁷⁵, Tyr527(2-MeTyr)²⁷⁶, Tyr527(b-MeTyr)²⁷⁶, Tyr527Hty²⁷⁶, Tyr527(2,6-F₂Tyr)²⁷⁶
	Central	Lys57Orn²⁷⁷, Lys57Dab²⁷⁷, Lys57Dap²⁷⁷
Signal Transducer And Activator Of Transcription 6 (STAT6)	C-terminal	Tyr641Ph (+photocage)²⁷⁸
α-Synuclein	N-terminal	Backbone thioamide^{279, 280}, Nα-Ac²⁸¹, Lys6Ub²⁸² Lys12Ub₄²⁸³
	C-terminal	Tyr125Ph²⁸⁴, Tyr125Ni²⁸⁵, Ser129Ph^{286, 287}
	Central	Tyr39Ph²⁸⁸, Thr65GlcNAc²⁸⁹, Thr72GlcNAc^{289, 290}, Thr75GlcNAc²⁹⁰, Ser81GlcNAc²⁹⁰, Ser87GlcNAc^289–291
	N-/C-terminal	Fluorophore labeling²⁹²
Tau protein	C-terminal	Unmodified²⁹³, Ser396Ph⁴⁹, Ser400GlcNAc²⁹⁴, Ser404Ph^{49, 295}, Ser422Ph²⁹⁶,
	Central	Lys280Ac⁴⁹, Lys294Cm²⁹⁷, Ser293Ph²⁹⁷, Ser305Ph²⁹⁷, Tyr310Ph⁴⁹,
Thioredoxin reductase	C-Terminal	Unmodified (Sec-containing)²⁹⁸, Cys-to-Hcy and -Sec mutants^299–305
Thymidine monophosphate kinase	N-terminal	Lys25NBD-Dap³⁰⁶
Transforming growth factor beta (TGFβ) receptor 1	C-terminal	Thr185Ph,^{307, 308} Ser187Ph,^{307, 308} Ser189Ph,^{307, 308} Ser191Ph^{307, 308}
Trypsin	N-terminal	Unmodified³⁰⁹
Tyrosine-protein kinase Csk	C-terminal	TyrPh⁹⁷
Ubiquitin^b	C-terminal	Diazirine crosslinker³¹⁰, K6Ub⁸³, K48Ub³¹¹, Ser65Ph³¹², non-hydrolyzable AMP-ester mimic^{313, 314}
Uracil DNA glycosylase	N-terminal	Thr6Ph³¹⁵, Tyr8Ph³¹⁵
Zif268 Zinc finger transcription factor	C-terminal	Arg70IDAO,³¹⁶ Arg70Pra,³¹⁶ His85TACN,³¹⁶ Lys89Fl³¹⁶
β-Xylanase	N-terminal	Fluorinated Glu78³¹⁷
Yeast α-factor mating receptor	n/a	Segmental Isotopic Labeling³¹⁸
Ypt1 GTPase	C-terminal	prenylation^{319, 320}
Zinc finger protein (QNK-QDK-RHR)	C-terminal	Arg78Cit³²¹

Ac, acetyl; Acac, acetatoacetyl; Apy, argpyrimidine; Bta, β-(3-benzothienyl)-alanine; Cba, cyano β-alanine, Cit, citrulline; Cm, carboxymethyl; Csa, cysteine sulfonic acid; Dab, diaminobutyrate; Dap, diaminopropionate; Fl, fluorescein; GalNAc, N-acetylgalactosaminyl; Ger, geranyl; GlcNAc, N-acetylglucosaminyl; Hcy, homocysteine; Hse, homoserine; Hty, homotyrosine; hVal, hydroxylvaleric acid; IDAO, iminodiacetic acid ornithine; Nal, napthyl alanine; NBD-Dap, N₃-nitrobenzofurazane-l-1,3-diaminopropionic acid; Ni, nitro; Nle, norleucine; Nva, norvaline; Orn, ornithine; Pfa, phosphonodifluoromethylenealanine; Pfp, phosphonomethylenephenylalanine; Pmp, phosphonomethylenephenylalanine; Pra, propargylglycine; Ph, phosphoryl; Pma, phosphonomethylenealanine; pTza, phosphoryltriazoylalanine; Sar, sarcosine; Sec, selenocysteine; Sem, selenomethionine; Sero, serotonyl; Su, sumoyl; TACN, triazacyclononane; ThA, 2-thienylalanine; Ub, ubiquityl.

Including the assembly of protein sequences from recombinant and synthetic peptide and protein fragments. Does not include the use of NCL/EPL as a general bioconjugation strategy, e.g. for the generation of antibody conjugates.

Semisynthesis of the protein sequence of ubiquitin itself. Examples of semisyntheses of ubiquitin-modified proteins are listed under the protein of interest.