579

Index

a acyl donors 63 Abl SH3 domain 241 γ-acyloxy Gln variant 139 AB variant of GM2 gangliosidosis 75 acytylacetone (acac) 94 AcA thioester 231 additive-free diselenide-selenoester acetamidomethyl (Acm) 121, 233 ligation 174 acetamidomethyl (Acm) Cys protecting African swine fever virus polymerase X group 196 (ASFV pol X) 113 acetamidomethyl group (Acm) 262 aldehyde-derivatized agarose 229 acetoacetyloxime (AcA) linker 272 aldehydes 328 acetoacetyl (AcA) purification 231 ALFALFA 39 acetonitrile (ACN) 185 27 acetylated histone H4 501 p-alkoxybenzyl-oxycarbonyl hydrazide acetylation 393, 499, 502 resin 91 N-acetylation 212 alkyl/aryl thioesters 61 acetylcholine 344 alkyl/aryl thiols 61 N-acetylglucosamine 137 N-alkylated 2-bromoethylamine p-acetyl-L- 333 derivative 111 acidic sodium acetate buffer 261 N-alkylated peptide hydrazide 240 acid-sensing ion channels (ASIC) 101 N-alkylcysteine 61 acid-sensitive CTC-resin 95 alkyl prolylthioesters 164 acid-stable C-terminal glycolamide-ester alkyl thioesters 71 193 alloc-protected Phacm linker 197 ACP(65-74) 39, 49 alloc-protected phenylactamidomethyl ACP(65-74) peptide 27 group 196 activity-based probes (ABPs) 403 allo- 367 acyl azide 88 allosteric effects 377 acyl azide-based coupling method 88 allyl alcohol 139 acyl azide-based solid phase peptide allyl iodoacetate 214 synthesis 88 N-allyloxycarbonyl (Aloc) group 263 acyl azide-based solution phase peptide allyloxy carbonyl (Alloc) protecting group synthesis 89 189 N-acyl-benzimidazolinone (Nbz) 169, Aloc-protected Aoa 266 295 amide-cyclized 99

Total Chemical Synthesis of , First Edition. Edited by Ashraf Brik, Philip Dawson, and Lei Liu. © 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH. 580 Index

amide-to-ester substitutions 359–361 aryl-selenoester functionalized fragment amide-to-thioester modification 359 147 amine 191 aryl thioesters 162 amine-containing sulfonylethylcarbonate arylthiol catalyst 4 building block 270 135 α-amine peptide bond 188 89, 122, 139, 147 aminoacetaldehyde dimethylacetal 237 aspartic acid ligation 298 Boc-hydrazine 88 aspartyl aldehyde (Asa) linkage 245 135 amino acid HA-tagged human histone Asx/Glx residues 197 2B (H2B) derivative 265 asymmetric disulfide 133 84-amino acid human parathyroid ATP synthase 442 187 automated fast flow 19 28 amino acid knottin EETI-II 265 first generation 21–24 D-amino acid peptidase 189 fourth generation 45–49, 50 61 amino acid polypeptide 266 second generation 24–32 d-amino acids 519 third generation 32–45 L-amino acids 519 automated fast flow peptide synthesizer amino acid sequence 1, 2, 4, 9 (AFPS) 19 aminoacyl-N-hydroxyl group 302 autophagosomal marker LC3-II γ-amino alcohol 298 195 γ-amino alcohol-based ligation 302 autoubiquitination 311 1, 2-aminoalcohols 329 auxiliary salicyaldehdye 302 amino-containing peptide segments 90 avidin-functionalized sepharose beads 8-amino-3,6-dioxaoctanoate (ADO) 194 268 aminomethyl ChemMatrix® resin 70 axinastatin 1 168 aminooxy-derivatized resin 227, 233 azide-protected N-terminal 295 aminooxy functionalization 335, 337 1-azido-5-[1,3-dimethyl-2,4,6(1H,3H,5H)- aminooxy-functionalized cellulose solid trioxopyrimidine-5-ylidene]pentyl

support 266 (N3-Dtpp) 272 aminooxy-functionalized SPCL solid 2-(2-azido-ethoxy)-ethanol 241 support 272 (2-[2-(2-azido-ethoxy)-ethyl-sulfonyl]-2-

aminooxy resin 231 ethoxycarbonyl linker (N3-Esoc) β-amino thioester 268 271 amyloid beta (Aβ) 193 amyloid ‘switch’ peptides 189 b animal toxins 101 Bacillus amyloliquefaciens 317 anti-diastereomer 133, 149 Bacillus amyloliquefaciens RNase 178 antifreeze glycoprotein (AFGP) 432, 433 backbone-amide-linker (BAL) strategy antigenic peptide 101 241 anti-HIV therapeutic Enfuvirtide 137 backbone engineering antimicrobial peptide RTD-1 176 approach 522 139 β-sheet assembly 520

Arg3 solubilizing tag 196 consequences of chirality 517–520

Arg4-tagged RBM group 298 control folding 520–522 aryl aldehyde esters 289 facilitate synthesis 516–517 Index 581

protein mimetics 522–525 bis(acetoxy)iodobenzene (BAIB) 151 base-catalyzed Henry reaction 131 1-[Bis(dimethylamino)methylene]-1H-1,2, base-labile 4-hydroxymethylbenzoic acid 3-triazolo[4,5-b]pyridinium 3-oxid (HMBA) linker 267 hexafluorophosphate 39 base-labile sulfonylethoxycarbonyl moiety bisphosphorylated p62 to K63 diubiquitin 221 105 benzoylglycine 88 bis-(2-sulfanylethyl)amido group (SEA) benzyl alcohol 149 270 benzyl cinnamate 127 bis-sulfoxide 266 benzyloxycarbonyl (Cbz) group 189 Boc-aminooxy acetic acid 336 bicyclic thiolactone-promoted internal Boc-Asp-OtBu 130 activation strategy 169 Boc-Asp(OtBu)-OAll amino acid 147 bicyclic turndipeptide (BTD) 521 Boc-Asp(OAll)-OH 135 bicyclononyne-functionalized resin 242 Boc-Asp(OAll)-OMe 137 bifunctional middle fragment 147 Boc-based SPPS 167 bifunctional peptide 71, 78 Boc/Bzl-solid phase peptide synthesis bifunctional SEAoff and SeEAoff (SPPS) 360 containing peptides 69 Boc-carbamate 151 bifunctional SEA peptide 72 Boc chemistry 2 binding assays 311 Boc-Glu(OtBu)-OAll 124 bioactive human chemokine hCCL21 99 Boc-Lys-OH 93 bioactive peptide LacZα 94 Boc-N-methyl-N-[2-(methylamino)ethyl] bioconjugation 297 carbamoyl (Boc-Nmec) 195 biologically active glycoproteins 105 Boc-N-methyl-N-[2-(methylamino)-ethyl]- biomimetic polymers 17 carbamoyl group 194 biopolymers 17 Boc-Sec2-OH dimer 147 biosourced oligosaccharide-based Boc-SPPS 438, 449, 450, 453, 454, 559, polymers 276 562, 563, 566 biotinylated analogue of the HGF/SF NK1 9-borabicyclononane (9-BBN) 133 domain 71 p-boronobenzyloxycarbonyl (Dobz) 502 biotinylated Cys peptide 74 p-boronobenzyloxycarbonyl biotinylated HGF 231 (Dobz)-protected Cys 96 biotinylated Lys residue 268 bovine pancreatic ribonuclease 90 bis(2-sulfanylethyl) amide (SEA) system bovine pancreatic trypsin inhibitor (BPTI) 63, 164 191, 369, 476 bis(2-selanylethyl)amido group (SeEA) Bowman Birk inhibitors (BBIs) 559 63 BPN 317, 318 bis(2-selanylethyl)amine 68 β-branched Thr and Leu 174 bis(2-sulfanylethyl)amine 68 β-branched 161 bis(2-selanylethyl)amine diselenide bromoacetic acid 268 precursor 68 N-bromosuccinimide (NBS) 125 bis(2-sulfanylethyl)amine trifluoroacetic building blocks 195 acid salt 68 bulky amino acid side chains 18 bis(2-sulfanylethyl)-amino (SEA) ligation bulky C-terminal residues 161 method 189 bulky valinylthioesters 173 582 Index

butelase-1 316, 317 covalent tethering 371–373 tert-butoxy carbonyl (Boc) 554 discovery, protein folds 373 tert-butoxycarbonyl hydrazide 89 foldamers 375–377 n-butyl nitrite 88 glycoproteins 411–413 tert-butyl nitrite 90 hydrazine functionalization 335–337 tert-butyloxycarbonyl (Boc)-protecting the hydrazone ligation 342–344, 346–348 imidazole ring (im) 20 oxidative folding 368–371 oxime ligation 337–342, 346–348 c Pictet-Spengler reaction 344–345 canonical α-amino acids 359 protein backbone amides 358–361 capillary isoelectric focusing (CIEF) protein backbone and side-chains 3–5, 9 362–366 capture and release purification 212 and semisynthesis 515 carbamate linkers 195 site-specific labelling 373–375 carbohydrates 18 TM peptides 456 carbonyl (aldehyde or ketone) 328 transmembrane (TM) peptides 449 Nα-carbonyl 169 ChemMatrix 19 carbonyl functionalization 328, 335 ChemMatrix® resin 68 carbonyl moieties 334 ChemMatrix PEG Rink-Amide resin 46 αN-carbonyl oxygen 161 ChemMatrix resin 27 carboxamidomethyl ester (CAM) 266 ChemMatrix Rink amide resin 40, 41 carboxyamidomethyl (CAM) protecting chemoenzymatic synthesis 413–414 group 233 chemokine CCL2 (MCP-1) 7 α-carboxylate of Boc-Asp(OtBu)-OH 122 chemokine CXCL14 75 carboxylic acid 127 chemokine interleukin 8 (IL8) 120 3-carboxypropane sulfonamide 215 chemokine receptor CXCR1 145 catch linker 212 chemokines 102 CCL1 424, 425 chemoselective KAHA peptide ligation CD spectroscopy 417 245 cell nucleus 489 chemoselective peptide ligation 285 cell-penetrating peptides (CPPs) 27 chemoselective reaction 338, 359 cell signaling 384 chimeric anti-protein EGFR nanobody chaotropic agents 229 94 chemical analogues 10 chiral inactivation 365 chemical domain of fractalkine (CDF) chitin-binding domain (CBD) 494 423, 424 2-chlorotrityl-chloride resin 293, 420, methods 2 555, 557 chemical ligation reactions 260 chlorotrityl hydrazide 27 chemical principles 20 chlorotrityl PS resin 68 chemical protein synthesis (CPS) 185 2-chlorotrityl resin 291 aminooxy functionalization 335–337 chromatin immunoprecipitation (ChIP) β-turn mimetics 361–362 497 carbonyl functionalization 328–335 Chymotrypsin inhibitor 2 (CI2) 359 cis-trans isomerization circular/concatenated polypeptide chains 366–368 10 Index 583 circular dichroism (CD) 105 C-terminal proline residues 166 CK2-catalyzed phosphorylations 310 C-terminal “safety-catch” linker 193 14C-labeled polyamines 339 C-terminal salicylaldehyde ester 287, classical alkyl thioester 64 289, 302 classical Fmoc SPPS 68 C-terminal SECmide 70 cleavable benzylidene acetal moiety 289 C-terminal SeEAoff peptides 68 2-Cl-(Trt)-Cl resin 91 C-terminal (Sec) 63 2-Cl-(Trt)-NHNH-Fmoc resin 92 C-terminal Ser thioester 289

2-Cl-(Trt)-NHNH2 resin 91 C-terminal synthetic XH3 (112-135) β-conotoxins 193 fragment 130 continuous flow chemistry 18 C-terminal thioesters 120, 262, 285 continuous flow solid phase peptide C-terminal thio-salicylaldehyde ester synthesis 18 289 controlled pore glass (CPG) solid support C-terminal valine-containing peptide 272 164 convergent covalent condensation 4 C-termini of peptides 168 copper-catalyzed alkyne/azide C-to-N ligation 294 cycloaddition (CuAAC) 260 C-to-N peptide ligations 221 copper(I) ligands 261 C-to-N solid-phase chemical ligations CoREST 315 235 covalent C-terminal-linked 327 cubic lipidic phases (CLPs) 443 covalent tethering 371, 373 Cu(I)-catalyzed azide-alkyne crambin 221, 421, 422 cycloaddition reaction (CuAAC) Crk-II adapter protein 268 241 crosslinked PEG resin 19 Curtius rearrangement 88 crude polypeptide product 267 cyclic conotoxins 561, 562 crude synthetic peptides 4 cyclic cystine knot (CCK) 565 cryo-electron microscopy (cryo-EM) 111 cyclic cystine ladder (CCL) 563 C-terminal alkyl thioester 144, 147 cyclic disulfide(diselenide) 66 C-terminal β-thiolactone 168 cyclic disulfide bis(2-sulfanylethyl)amido C-terminal carbonyl 331 (SEA) polystyrene resin 231

C-terminal His6 tag 221 cyclic peptides 59, 302, 553 C-terminal histidyl thioester 76 classes of 554 C-terminal hydrazide 99, 336 conotoxins 561–563 C-terminal isoleucine-containing peptide cyclotides 563, 565–567 170 orbitides 557–558 C-terminally-amidated peptides 266 paws-derived peptides (PDPs) 35-mer C-terminal peptide fragment 218 559–560 C-terminal peptide hydrazides 93, 94 θ-defensins 563, 564 C-terminal peptide hydroxylamine (HA) synthesis 99, 554–556 244 cyclic proline 161 C-terminal peptide segments for NCL cyclic selenopeptides 472 217 cyclic sulfate 127 C-terminal peptide thioester 87 cyclization linker 214 C-terminal polypeptide hydrazide 93 cyclotides 99, 563, 565–567 584 Index

Cys-based NCL 173 dengue 17 Cys-functionalized sepharose solid de novo design 366 support 267 de novo proteins 371 Cys-Gly 265 deoxy-D-ribose 5-phosphate aldolase Cys-masking strategy 262 (DERA) POI 93 Cys- or Met-containing peptides 187 L-deoxyribonucleotide triphosphates Cys-peptides 4, 64, 163 (L-dNTPs) 113 Cys-rich proteins 477 deprotected SEAoff peptide 68 cysteamine nitrogen 63 depsipeptides 187 cysteamine scaffold 63 Dess-Martin periodinane (DMP) 140 (Cys) 61, 463 desulfurization chemistries properties 464 arginine 139–140 cysteine-aminoethylation assisted asparagine 135–137 chemical ubiquitination (CAACU) aspartic acid 122–124, 147–148 strategy 111 124–125, 148–149 cysteine-cystine redox system 416 139 cysteine modifications 311 isoleucine 127–130 dehydroalanine (DHA) generation 133–135, 151 312–313 130–133 Lys mimics 313–314 metal-catalyzed hydrogenation protein semisynthesis 312 reactions 121 cysteine side chains 89 phenylalanine 125–127, 149–151 cysteine walk 223 proline 138–139, 151–153 cysteinyl MUC1peptide hydrazide 248 selenocysteine 146–147 cysteinyl-peptide 59, 61, 172, 221 140–142 19-mer cysteinyl peptide 268 144–146 N-cysteinyl peptide 167, 178 valine 142–144

cysteinyl peptide H-CSPGYS-NH2 176 deubiquitinase (DUB) 109, 111 cysteinyl prolyl ester (CPE) / imides (CPI) deubiquitinating module (DUBm) 498 61 3,4-diaminobenzoic acid (Dbz) Cys-to-Sec substitution 466, 475, 476 derivatized Rink amide resin 291 Cys-trityl solubilizing tag 197 3,4-diaminobenzoic acid (Dbz) linker 193 d diastereomers 27 DAGK 454–455 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) data analysis method 20 127 DawsonDbz linker 422 α,α′′-dibromo-adipyl(bis)-amide (DBAA) Dawson’s safety-catch strategy 291 471 Dbz-linked Asp/Glu residue 197 dichloroacetone (DCA) 334 θ-defensins 563, 564 2,3-dichloro-5,6-dicyano-1,4-benzo- dehydroalanine (DHA) 403, 405, 468, quinone (DDQ) 135 470 digitoxin 338 deiodinases (DIOs) 466 dihydroxylated compound 127 denaturants 4 diisobutylaluminium hydride (DIBAL-H) dendrimers 516 143 Index 585

N,N-diisopropylcarbodiimide (DIC) 504 n-Dodecyl β-D-maltoside 187 diisopropylethylamine (DIEA) 21, 498 dodecylphosphocholine 187 β-diketone 333 double-stranded DNA 490 dimedone 197 doxorubicin 343 2,4-dimethoxy-benzaldehyde (Dmb) 439 D-peptide inhibitors 101 N,N-dimethylethylenediamine (DMDA) drosocin 338 195 DUBs 390, 400–404 dimethylformamide (DMF) 20, 189 Duchenne muscular dystrophy 17 dimethylimidazolidinone (DMI) 20 dimethylimidazolidone (DMI) 43 e dimethyl(methylthio) sulfonium Ecballium elaterium trypsin inhibitor II tetrafluoroborate 124 (EETI-II) 365 dimethyl sulfoxide (DMSO) 187 E. coli strain 317 dimethylthiazolidine 441 EETI-II analogs 28 2,4-dinitrophenyl (Dnp) 403 EETI-II diastereomers 27 2,4-dinitrophenylsulfenyl chloride 145 Eglin C 153 dipeptidyl peptidase IV (DPPIV) 189 electrophilic sulfenylating reagent 122, diphenyldiselenide (DPDS) 153, 172 124 4-(diphenylhydroxymethyl)benzoic acid d-enantiomer 517, 518 197 L-enantiomer 517, 518 direct head-to-tail cyclisation 555 endogenous phospho-amino acids direct hydrazinolysis 93 phospho-arginine 544–545 direct infusion electrospray ionization phospho-aspartate 543–544 mass spectrometry (ESI-MS) 3–5 phospho-glutamate 543–544 diselenide 148, 153 phospho- 545–547 diselenide peptide dimer 175 phospho-serine 541–543 diselenide-selenoester ligation (DSL) phospho-threonine 541–543 146, 153, 181 phospho- 541–543 distal Ub-hydrazide 109 pyrophosphorylated serine 547 1,4-disubstituted 1,2,3-triazole products endoplasmic reticulum (ER) 427, 466 260 endoplasmic reticulum-associated disulfide bond 400 degradation (ERAD) 384 disulfide bond formation 554, 566, 567 5-endo-trig cyclization 302 disulfide-containing proteins 4, 5 6-endo-trig process 298 disulfide cross-linked protein molecule 5-endo-trig ring-chain tautomerization 4 287, 298 disulfide rich miniprotein EETI-II 275 6-endo-trig ring-chain tautomerization dithiol(diselenol) 66 302 dithiothreitol (DTT) 73, 146 enhanced green fluorescent protein divinylbenzene crosslinked polystyrene (eGFP) 367 resin 27 enzymatic assays 311 DNA-barcoded nucleosome library (DNL) enzymatic transglycosylation 431 platform 110 enzyme-catalyzed EPL 319 L-DNA promers 113 enzyme-catalyzed expressed protein DNA sequencing methods 1 ligation 318, 319 586 Index

enzyme-catalyzed peptide ligation flow-based solid phase peptide butelase-1 316–317 synthesizer 175 sortase 314–315 9-Fluorenylmethoxycarbonyl (Fmoc-) subtiligase 317–318 497 trypsiligase 318 fluorenylmethyloxycarbonyl enzyme DapA 259 (Fmoc)-protected amino acids 20 Eph tyrosine kinase receptor 193 fluorescence resonance energy transfer epimerization issue 289 (FRET) 403 γ-epimers 138 fluorophores 10, 373–375 EPO glycoforms 105 (4S)-fluoroproline 367 erythro-diastereomer 134 N-Fmoc-amido-PEG2-acid 197 erythro isomer 122 N-Fmoc amino (EPO) 105, 127–166, 193, acyl-N-sulfanylethylaniline unit 327, 328, 426, 430 70 erythro/threo diastereomers 122 Fmoc-Arg(Pbf)-OH 43 estimated purity 21 Fmoc-Asn(Trt)-OH 43 1-ethyl-3-(3-dimethylaminopropyl) Fmoc-Asp-OtBu 143 carbodiimide (EDC) 130 Fmoc-based SPPS 90 euchromatin 490 Fmoc chemistry 2 eukaryotic cell development 489 Fmoc-Cys(AcmNHAlloc)-OH 197 193 N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo- exogenous additive-promoted ligations hexylidene)-3-[2-(2-aminoethoxy) 162 ethoxy]-propan-1-ol exogenous additives 167 (Fmoc-Ddae-OH) 197 exopeptidases 189 Fmoc-Glu(OAll)-OH 215 expressed protein ligation (EPL) 107, Fmoc-γ-thiol Pro 138 193, 268, 308, 309, 494, 495 Fmoc-His(Trt)-OH 27, 30 development method 308–309 Fmoc-L-Cys(Aapam)-OH (Iris Biotech) N-hydroxysuccinimide-esters 311 protein post-translational modifications 197 309–311 Fmoc-NHNH2 92 expressed proteins 92 Fmoc-Pro-OH 47 expression protein ligation (EPL) 92 Fmoc protected amino acids 24 Fmoc-protected N-terminal serine 295 f Fmoc-protected proteogenic amino acids fatty acids 437 43 Fc-III mimetic (DCAF) 101 Fmoc-SPPS 75, 188, 418–420, 422, FDG 340 424–426, 439, 445, 453, 455, 504, 18F-fluorination reactions 338 535, 536, 537, 538, 541, 544, 558, first generation automated fast flow 568 peptide synthesis segment synthesis 267 characterization 23–24 foldamers 375–377, 522 design 21–23 folded protein molecule 4 18F-labeling techniques 338 folded synthetic protein, purification of flow-based peptide synthesizer 18 5 Index 587

(2′-formyl-[1,1′-biphenyl]-3-yl)methyl gluthatione/glutathione disulfide redox esters 289 system 74 formylglycine 333 (Gly) 363, 364 formylglycine generating enzyme (FGE) glycine-containing peptide 93 332 glycine-dependent amide-forming ligation Förster resonance energy transfer (FRET) 302 373, 374 glycopeptides 334 fourth generation automated fast flow glycopeptide synthesis 412 peptide synthesis glycophorin A (GpA) 443 key findings 45 glycoprotein assembly 338 solvent effect 45 glycoprotein mimetics 10 18F radiolabeling 340 glycoprotein modification 335 full length chromosomes 18 glycoprotein oximation 339 full-length C-terminal peptide segments glycoprotein quality control (GQC) 427 212 glycoproteins 10, 334, 335 antifreeze glycoprotein (AFGP) full-length GM2-AP protein 75 432–433 full-length hormone 127 CCL1 424, 425 full-length mambalgin-1 toxin 102 chemical domain of fractalkine (CDF) full-length peptide 212, 225 423–424 full length protein 179 chemical protein synthesis 411–413 full-length synthetic polypeptide chain 4 chemoenzymatic synthesis 413–414 full-length Tat(1-86) protein 135 crambin 421–422 fully protected amino acid 122 erythropoietin (EPO) 426–430 functional GM2-AP protein mimics 76 hirudin P6 (HP6) 415–416 functionalized cellulose 19 interleukin 2 (IL-2) 417, 418 functional protein molecule 4 interleukin 6 (IL-6) 424–426 furnished carbamate 130 interleukin 8 (IL-8) 425–427 interleukin 25 (IL-25) 417–419 g mucin 1 (MUC1) 419–421 ganglioside 75 saposin D (SapD) 416–417 Garner’s aldehyde 149 semisynthesis 413 genes 18 α-synuclein 414–415 genetically encoded orthogonal protection tau protein 422–423 and activated ligation (GOPAL) trastuzumab 430–432 389, 390 glycosylated β-thiol Asn building block genetic hypercholesterolemia 17 137 genome 1, 18 glycosylated cyclic peptides 99 Gibbs free energy 360 N-glycosylated IL-6 protein 103 GlcNAc-modified membrane protein glycosylation 87 Tim-3 195 glycosylation-modified full-length Human α-globin 398, 400, 401, 405 interleukin-6 103 globular protein molecule 1 glycosylphosphatidylinositol (GPI) 437 glutamic acid 89, 124, 148 Gly49-Ile50 junction 359 glutamine 139 (Gly)3-labeled barnase 178 588 Index

Gly-loaded aminomethyl PS resin 215 hexahistidine 221 glyoxylic acid 164 β-hexosaminidase A 75 glyoxylyl (Gly) 329 9H-fluoren-9-ylmethoxycarbonyl (Fmoc) glyoxylyl moiety 329 554 Gly thioesters 142 highly activated selenophenyl esters 61 GM2 activator protein 75 high-performance liquid chromatography GM2-AP protein 75 (HPLC) 211, 495 GM2 ganglioside activator protein high throughput screening (HTS) 401, (GM2AP) 137 402 G-protein coupled receptors (GPCRs) hirudin P6 (HP6) 415

437, 447, 456, 457 His6-labelled MUC1 thioester 248

granulocyte colony-stimulating factor His6–Ni(II) interaction 240

(G-CSF) 185–186 His6 tag guanidine 191 and hydrazide tags for sequential guanidine hydrochloride (GnHCl) 186 N-to-C capture and release guanidine thiocyanate 187 225–227 guanidinium-hydrochloride (Gdn-HCl) and hydrazide tags for sequential n-to-c 442 capture and release 223–225 immobilization for assembly of proteins h on microtiter plates 222–223 H-AEGSQAKFGX-salicylaldehyde ester immobilization for C-to-N assembly of 294 crambin 221–222

Haemophilus Influenzae DNA ligase 194 reversible HIS6-based capture tags 220 handshake motif 495 histidine degradation studies 30 “hard acid” reactivity 286 histidine monomer Fmoc-His(Trt)-OH “hard base” reactivity 286 20 H-Asp(OtBu)-OMe 123 histidine racemization studies 33 HATU 557 histone proteins 393 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram- histone synthesis ethyluronium hexafluoro- acetylation 499–502 phosphate (HBTU) 20, 30, erasers 491 559 methylation 502–505 HBV Cp149 protein 197 native chemical ligation (NCL) HCTU 557 492–493 head-to-tail cyclisation 555, 557, 558 octamer and nucleosome core particle hemorrhagic fevers 17 assembly 494–496 hepatocyte growth factor 1 (HGF1) 272 phosphorylation 497–499 hepatocyte growth factor (HGF) 71, 231, post-translational modifications 232, 272 490–492 hepatocyte growth factor/scatter factor proteins 489–490 (HGF/SF) 71 readers 491 heterochromatin 490 sumoylated histones 505–506, hexafluoroisopropanol (HFIP) 187, 197 505–507 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) writers 491 442 histone tails 490, 491 Index 589

HIV entry-associated chemokine receptor hydrazine 212 CXCR4 123 hydrazine functionalization 337 HIV-1 protease 193 hydrazone formation H4K16ac 98 immobilization for assembly of proteins H3K4me3 98 on microtiter plates 239–241 Hmb(Ac) auxiliary group 298 immobilization for N-to-C solid-phase Hmb(Ac)Gly96-containing peptide 298 peptide ligations using a protected Hmb linker 194 alkyne 241, 242–243 HMGA1a protein 298 immobilization for N-to-C solid-phase homogeneously modified CXCR4 (1-38) peptide ligations using a sea group peptide 123 242–243, 243–244 homogeneous phosphorylated p62 105 reversible hydrazone-based capture tags homogeneous synthetic protein molecule 238–239 7 hydrazone ligation 342, 344, 346, 348 homoserine isoacyl bond intermediate hydrogen bonding 359, 520 189 hydrogenolysis 90 HPLC-free preparation hydrophobic membrane 438 of C-terminal peptide segments for proteins 187 NCL 217 hydrophobic peptides 438, 441, 442, 445, of N-terminal peptide segments for 450, 453, 454 NCL 213 β-hydroxyamic acids 189 HPLC-free synthesis, of proteins using the 1,2-hydroxyamine 287 KAHA LIGATION 245–246 1,2-hydroxyamine bifunctionality 287 H-SPKALTFG-OH 294 4-hydroxybenzoate 91 human anaphylatoxin C5a 229, 231 hydroxybenzotriazine (HOOBt)-mediated human cytomegalovirus protein UL22A coupling 137 151 1-hydroxybenzotriazole (HOBt) 130, 195 human erythropoietin segment (hEPO) γ-hydroxy Gln variant 139 195 β human group V secretory phospholipase -hydroxy Leu 133 1-hydroxyl-1H-1,2,3-triazole-4-carboxylate A2 (GV-PLA2) 266 human (hGH) 469 (HOCt) 90 human interleukin-6 (IL-6) 103 5-R hydroxylysine 133 human (hPTH) 4-hydroxy Lys precursor 130 143, 179 β-hydroxy Lys variant 133 human thryroid-stimulating hormone 2-hydroxy-3-mercaptopropionic acid (hTSH) β-subunit 197 194 Hyalomin 3 (Hya3) 148 2-hydroxy-4-methoxybenzaldehyde 2-hydoxy-nitrobenzyl (Hnb) 446 (Hmb) 439 hydrazide-based native chemical ligation N-(2-hydroxy-4-methoxy benzyl) (Hmb) 101 194 hydrazide functionalization 335, 337 (2-hydroxy-4-methoxybenzyl) auxiliary hydrazide resin 91 group 298 hydrazide tags for sequential N-to-C 2-hydroxy-4-methoxy-5-methylsulfinyl capture and release 225–227 benzyl (Hmsb) 441 590 Index

4-hydroxymethylbenzoic acid (HMBA) influenza BM2 peptide 197

193 influenza BM2 proton channel 194 4-(hydroxymethyl)phenoxyacetic linker inorganic matrixes 276–277 (HMPA) 266 in situ-formed heterobicyclo[2.2.1]septane 4-(hydroxymethyl)phenoxypropionic structure 169 linker (HMPP) 266 -degrading enzyme (IDE) 369 hydroxymethyl salicylaldehyde 302 insulin-like growth factor 1 (IGF-1) 364 N-hydroxy moieties 302 intein 92–93, 103, 130, 308–310, 317, N-(2-hydroxy-5-nitrobenzyl)-cysteinamide 319, 375, 414, 422, 425, 457, 467, (N-Hnb) group 63 474, 494–495, 505 β-hydroxyphenylalanine variant 125 intein-mediated protein splicing 61 hydroxy-phosphorylated amino acids, into interferon-induced transmembrane peptides and proteins 534, 537 protein 3 (IFITM3) 453 trans-β-hydroxy Pro derivative 138 interleukin 2 (IL-2) 417, 418 trans-γ-hydroxyproline 151 interleukin 6 (IL-6) 103, 104, 424–426 N-hydroxysuccinimide (NHS) esters 311 interleukin 8 (IL-8) 120, 425–427, 475, hydroxytrityl ChemMatrix® resin 68 520, 523 hygroscopic amino acid solutions 26 interleukin 25 (IL-25) 294, 296, 417–419 intermediate SEAoff peptide 72 i intermediate SEAon species 270 imidazole 167 intermolecular O→S peptidyl transfer groups 191 170 phosphonate reagent 541 internal Alloc-protected Aoa-MUC1 imine ligation 327–329 segment 237 immobilization for assembly of proteins internally activated peptide Nbz on microtiter plates 222–223, derivatives 179 239–241 intra-and inter-molecular ligation immobilization for C-to-N solid-phase reactions 317 chemical ligations 233–237 intramolecular iodocyclization 124 immobilization for N-to-C solid-phase intramolecular S,N-acyl shift 87 chemical ligations 227–233 intrinsically disordered proteins (IDPs) immobilization for N-to-C solid-phase 7, 357, 377 peptide ligations using a protected iodoacetic allylester 215 alkyne 242–243 iodoacetyl-N-acetylglucosamine 78 immobilization for N-to-C solid-phase cis-γ-iodoproline 151 peptide ligations using a sea group ion channels 1, 101, 112, 365, 446, 450, 243–244 453, 456, 561 immobilized metal ion affinity ionizable picolyl ester group 212 chromatography (IMAC) 221 irreversible S-to-N acyl transfer 285 immobilized peptide segment 212, 214 isoacyl α-amine 189 immunoglobulin G (IgG) 101, 365, isoacyl 188, 198 430–431 isoacyl peptides 187, 189 immunoglobulin variable (IgV) domain of isocyanate byproducts 187 PD-L1 (D-PD-L1IgV) 101 isoleucine (Ile) 127, 129, 130, 161, 365, inactive cyclic diselenide 72–73 367, 495 Index 591 isonicotinyloxycarbonyl (iNoc) 195, 417 latent acyl donor 72, 285–287 isopeptide bond 383, 384, 387, 389, 390, latent thioesters 59, 61, 66–67, 75, 79, 393, 395, 401, 403, 404 114, 139, 231, 270, 417 isopeptide chemical ligation (ICL) 390, LC/MS purity of peptides 21 392, 395, 399–401 leucine 133–135, 151, 366, 495 isopeptide ligation 387, 389, 391 Leu thioesters 142 isopeptide-linked Ub isomer 109 levulinic acid 227, 266, 270, 334 isopropyl bromide 151 ligation(s) 60, 64, 71, 76, 94, 142, 146, isoxazolidine tag 245, 246 161–181, 189, 212–213, 217, 221, 227–238, 242–246, 261–266, j 268–270, 273, 294, 296, 314–318, Jones oxidation 334 346–348, 470, 474 JR-10mer 41, 43, 45, 49 ligation reactions 53, 59, 61, 63, 64, 67, 74, 96, 103, 105, 125, 145, 169, k 172–173, 187, 194, 245, 259, 260, kaliotoxin 164 276, 278, 317, 443, 453–457, 492, K+ channel protein Kir5.1 112 516–517 KcsA K+ channel 193 linear N-Cys-tetrapeptide hydrazide keratins 1 precursor 99 α-ketoacid-hydroxylamine (KAHA) α2,6-linked sialylglycopeptide (SGP) 431 ligation 453 lipoic acid ligase (LpIA) 332, 337 ketoacid-hydroxylamine (KAHA) ligation β- (β-LPH) analogue 169 strategy 187, 189 lispro insulin 342 ketoamide 329, 330 lithium enolate 122, 124, 147, 149 ketones 227, 234, 250, 266, 270, 328–331, lithium hexamethyldisilazide (LiHMDS) 333–335, 337, 342–343, 348, 413 122 ketoproline 333–334 LL-37 48, 51 K19, K48 bi-acetylated Atg3 protein 105, low molecular weight peptidic products 107 27 K27/K29/K33-linkage selective lumbricin-1 175, 179, 181 deubiquitinase OTUD2 109 lysine 108, 130–133, 247, 289, 329, 334, K27-linkage isopeptide bond 108 338, 443, 445, 449, 492, 500, K27-linked diUb 108, 109 504–505, 533 K27-linked tetra-Ub and lysine acetylation 492 K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs 109 m K27-linked triUb 109 maltose-binding protein (MBP) 474 K27-linked ubiquitin chains 108 mambalgin-1 101, 102 K27-modified proximal Ub 109 m-chloroperbenzoic acid (mCPBA) 142 Knauer pumps 24, 26 mechanical principle 20 Knorr pyrazole synthesis 94 12-membered cyclic tetrapeptide 99 13-membered thiolactone intermediate l 99 LabVIEW software package 38, 39 membrane protein 112, 438 ladder sequencing 4 dysfunction 112 592 Index

1,2-mercaptoamine bifunctionality 285 methylthiocarbonyl-aziridine (MTCA) δ-mercaptolysine 389, 390, 392, 393, 313 395, 398, 400 methyl thioglycolate (MTG) 162 γ-mercaptolysine 389, 390 MET tyrosine kinase receptor 71 4-mercaptophenylacetic acid (MPAA) Michael acceptor 132 72, 130, 162, 261, 399–400, 400 microfluidics-promoted NCL 175 trans-4-mercaptoproline derivative 169 β2-microglobulin (β2m) 362, 363, 367, 4-mercaptoprolyl thioester-based ligation 371 169 microtiter plates 222-223 3-mercaptopropionic acid (MPA) 243, mirror-image biological systems 112 387 mirror-image biomacromolecules 113 derived thioester 71, 164 mirror-image protein(s) 10, 362, 363 mercaptopropionic acid (MPA) 232 misfolded polypeptide chains 4 β-mercato amides 268 7–12 μm Porex UHMWPE membrane 46 35-mer C-terminal peptide fragment 218 model peptides 39 mercury(II) acetate 263 46-mer model protein 221 modified N terminal peptide hydrazide metagenome 1 107 metal-free desulfurization approach modified nucleosomes 169, 274 cysteine-aminoethylation assisted metal-free dethiolation (MFD) 492 chemical ubiquitination strategy methanol (MeOH) 185 111 121 DNA-barcoded nucleosome library methionine-containing peptides 215 (DNL) platform 110 methionine residues 215 ubiquitylated histones 111 7-methoxy-coumarin-4-acetic acid (MCA) modified RNA 17 403 molecular dynamics (MD) simulations 2-methoxy-4-methylsulfinylbenzyl 358, 361, 364 alcohol 193 molecular feature extraction (MFE) 21, 4-methoxy-5-nitrosalicyladehyde 194 27 methylated histone H3 504 mono-and diubiquitin (Ub) 111 methylation 393, 502, 505 monoclonal antibodies 339 N-methylation 373, 520 monoglycosylated prolyl thioester 78 α-methylcysteine 61 monoisotopic mass 5 methyl ester 127 monomers 327 ′ ′ (2 -formyl-[1,1 -biphenyl]-3-yl)methyl monoubiquitinated H2B 396 esters 289 monoubiquitinated proteins 393, 397 5-methyl isoxazole-carboxamide (MICA) mouse thioredoxin reductase 3 (Mtr3) 130 474 methyl methanethiosulfonate (MMTS) MPAA-derived thioester 178 133 MPA-thioester 72 methylsulfonylethyloxycarbonyl (MSC) 7-12 μm Porex UHMWPE membrane 46 262 Msz (p-(methylsulfinyl)benzyloxy- methyl/t-butyl methanethiosulfonate carbonyl) group 295 (tBMTS) 133 Msz-Ser(tBu)-OH 295 Index 593 mucin 1 (MUC1) 419, 421 NK1 protein 71 MUC1 protein 243 [Nle8,18]hPTH 179 9 MUC1 proteins 225 N-linked glycosylation 411 multi-dimensional NMR fingerprinting N-(2-mercaptoethoxy)amide scaffold 5 61 multiple acidic sialyloligosaccharides N-methylpyrrolidone (NMP) 20 105 N-monomethyltrityl (Mmt) 215 NMR spectroscopy 358, 361, 371, 423 n N,N-dimethyl barbituric acid (NDMBA) native amide-bond metathesis reactions 237, 264 63 N,N-dimethylethylenediamine (DMDA) native chemical ligation (NCL) 59, 120, 195 162, 185, 211, 259, 308, 319, 372, N, O-acetal intermediate 302 387, 412, 414–416, 424, 442, 445, N,O-benzylidene acetal 287 446, 449, 450, 469, 470, 473, 474 N,O-benzylidene acetal ligation product chemoselective approach 555 289 development of 516, 517 N,O-benzylidene acetal moiety 287 histone synthesis 492–494, 493 nonapeptide PPAHGVSTA 265 native NK1 74 non-canonical amino acid 366, 367, 374, native triphosphorylated tau peptide 247 375 natural N-terminal thioester 103 non-canonical phosphorylation 538 natural peptide bond 308 non-coded amino acids 10 natural RNA 17 non-enzymatic ubiquitination natural Sec-containing proteins 173 isopeptide ligation 387, 391 neopentyl (nP) ester 151 Ub building blocks 387 Ni(II)-loaded microtiter plate 222 non-ionic detergents 187 N-im Boc-protection 24 nonproteinogenic amino acid canaline N-im trityl-protected histidine 24 198 ⋅ Ni NTA agarose resin 225 p-NO2-phenol 170 15N-isotope-labeling (SGI) 423 N-or C-terminal solubilizing “tails,” nitro alcohol 131 192–194 p-nitrophenol (ONp) derivatives 227 N,S-acyl transfer systems 61 p-nitrophenol-derived ester 170 N,S-and N,Se-acyl transfer devices 4-nitrophenyl chloroformate 291 biotinylated analogue of the HGF/SF nitrophenylester-based ligation 170 NK1 domain 71–75 p-nitrophenyl ester protocol 170 chemical protein synthesis 61 p-nitrophenylsulfonyl (Nosyl) chloride cyclic peptides 59 134 cysteinyl peptide 59 6-nitroveratryloxycarbonyl (Nvoc) group general presentation 61 274 GM2 activator protein 75–79 nitroveratryloxycarbonyl (NVOC) latent thioesters 59 photo-cleavable amino protecting native chemical ligation 59 group 189 peptide thioester 59 NK1-B polypeptide 74 protein scaffolds 59 NK1-B protein 75 relative reactivity 63 594 Index

N,S-and N,Se-acyl transfer devices N-to-C ligation 296 (contd.) nucleation 362 SEA and SeEA peptides 68–70 α-nucleophiles 335 statistical overview 64–68 nucleosome core particle assembly 494, N,S-andN,Se-acyl transfer devicesSEA 496 and SeEA peptides 70 N-selenocysteinyl peptide 174 o N-substituted cysteamine 61 o-amino(methyl)aniline (MeDbz) linker N-substituted glycine residues 343 193 N-(2-sulfanylethyl)amide motif 61 octamer 494, 496 N-sulfanylethylaminooxybutyramide Octyl-β-glucoside 187 (SEAoxy) 63 O-glycoproteins 272 N-sulfanylethylanilide (SEAlide) 63 O-glycosylated serine residues 272 N-(2-sulfanylethyl)cysteine 63 O-glycosylated variants 193 N-terminal aldehydes or ketones 330 O-glycosylation 411, 415, 419, 427 N-terminal amino acid 285 oligonucleotide (ODN) 18, 341 N-terminal aminooxy group 237 oligosaccharide structure 105 N-terminal aminoxyacetic acid (Aoa) O-linked β-N-acetyl-glucosamine 237, 266 (O-GlcNAc) 310 N-terminal bifunctional amino acids 286 O-linked glycopeptide 334 N-terminal cysteine 99, 221, 262, 285 O-mesitylenesulfonylhydroxylamine N-terminal cysteine residue 120 312 N-terminal cysteine thiazolidine (Thz) O-methylhydroxylamine 218 76 one-pot litigation 96 N-terminal diselenide 147 one-pot sequential N-terminal g-thiol Glu residue 124 NCL/SEAlide-mediated ligation N-terminal homoserine 302 process 75 N-terminal ketone 330–331 one-pot Ser/Thr ligation 296 N-terminally cysteinyl peptide segments O-phosphoseryl-tRNA[Ser]Sec(pSer- 220 tRNA[Ser]Sec) 464 N-terminal MASSRVDGGRSDLIEGRC orbitides 557, 558 appendage 268 organic solvents 442, 449 N-terminal model peptide 294 ortho-nitrobenzyl linker 196 N-terminal oxazolidinone 227 O-to N-acyl shift 189 N-terminal peptide α-ketoacid (KA) 245 kinetics 189 N-terminal peptide segments 213, 237 O-to-N acyl transfer 287, 298, 302 N-terminal peptide thioester 96 OTUD2 mutagenesis assays 109 N-terminal peptide thioester fragments 1,2,5-oxadiazinane ring 302 222 oxazolidine 149, 151, 287 N-terminal serine 329 trans-oxazolidinone 125 N-terminal Ser/Thr 287 oxazolidinone intermediate 125 N-terminal Ser/Thr residue 287 oxazolidone-based strategy 217 N-terminal (Gly)3-tagged peptide 178 oxazolones 289 N-terminal triphosphorylated tau oxidative folding 368, 370, 371 segment 247 oxidizable amino acids 293 Index 595 oximation 337 acyl azide-based solution phase peptide oxime 212 synthesis 89–90 oxime bond 337 2-Cl-(Trt)-Cl resin 91–92 formation 402 convergent ligation 96 oxime formation conversion to peptide azide 88 C-to-N solid-phase chemical ligations cyclic peptide synthesis 99 237–238 DCAF 101 immobilization for C-to-N solid-phase D-peptide inhibitors screening 101 chemical ligations 233–237 erythropoietin (EPO) 105 immobilization for N-to-C solid-phase expressed proteins 92–93 chemical ligations 227, 230–233 glycosylation-modified full-length reversible oxime-based capture tags Human interleukin-6 103 227 homogeneous phosphorylated p62 oxime ligation 337, 338, 342, 346, 348 105 oxo-esters 170 isopeptide-linked Ub isomer 109 isopeptide-linked Ub 76-mer 109 p K19, K48 bi-acetylated Atg3 protein 105–107 1-palmitoyl-2-oleoyl-3-glycero-phosphati- K27-linked ubiquitin chains 108–109 dylcholine 187 Knorr pyrazole synthesis 94 partially protected ribonuclease 90, T1 ligation sites 95 paws-derived peptides (PDPs) 554, 559, membrane proteins 112 560 mirror-image biological systems Pd(0)-catalyzed deprotection 214 112–113 PD-L1-binding D-peptide molecules 101 modified nucleosomes 110–111 PEG 338 native chemical ligation 90 PEGA1900 resin 272 N-to-C sequential ligation 96 PEG-based synthetic polymers 276 one-pot litigation 96–99 PEG-polyacrylamide PEGA resins 276 peptide toxins 101–102 PEG-poly(oxetane)copolymer 276 sortase-mediated hydrazide generation pentafluorophenyl (Pfp) ester 137 93–94 36-mer peptide 178 transesterification of acyl azide 90 42-mer peptide 91 two-photon activated chemokine CCL5 62-mer peptide 181 103 peptide aggregation 185 peptide insolubility 185 peptide azide 88 peptide ligations peptide bond formation 327 exogenous additive-promoted ligations peptide-CONHNH2 4 162–167 peptide C-terminal 2-formylbenzyl ester internal activation strategy 169–170 289 microfluidics-promoted NCL 175 peptide C-terminal glycolaldehye esters oxo-esters 170 286 reactive thioesters 167–168 peptide cyclisation 553 representative applications 178–181 peptide hydrazides selenoesters 170–175 activation in TFA 94–95 peptide prolyl hydrazide 167 596 Index

peptide prolyl selenoester 172, 174 Pfp-active ester 137 peptides 18 L-Phe methyl ester 125 30-mer peptides 46 phenylacetamido (PAM)-substituted peptide salicylaldehyde esters 289, 291, phenylalanine linker 193 293, 294, 296 phenylalanine 125, 149 via Boc-SPPS 291 9-(9-phenylfluorenyl) (PhFl) peptide seleno-salicylaldehyde esters functionalized amino acid 143 289 phenylisopropyl ester 137 peptide sequence 559 2-phenyl-isopropyl (PhiPr)-ester 215 peptide solubility phenylisopropyl trichloroacetimidate isoacyl peptides 187–191 135 semipermanent solubilizing tags phenylselenoester 174 191–198 phospho-arginine (pArg) 537, 538, 544, solvent manipulation 185–187 545 peptide therapeutic teriparatide 124 phospho-aspartate 543, 544 peptide thiocarboxylate segments 268 phospho-cytesine (pCys) 539, 541 peptide thioesters 4, 59, 75, 91, 99, 189, phospho-glutamate 543, 544 213, 309 phospho-histidine (pHis) 538, 539, 545, peptide thio-salicylaldehyde esters 289 547 peptide toxins 101 phosphohistidine phosphatase 1 (PHPT1) peptidic Xaa-Ser/Thr linkage 287 470 peptidomimetic triazole ligation (PTL) phospholamban 447, 449, 450 260 phospho-lysine (pLys) 539 strategy 270 phosphonates 541–543 peptidyl alkyl thioesters 167, 179 phosphonodifluoromethyl (Pfa) peptidyl azide-based ligation protocol 542 178 phosphono-difluoromethylene alanine peptidyl azides 166 (PFA) 310 peptidyl C-termini 163 phosphonomethyl alanine (Pma) 542 peptidyl hydrazines 167 phosphoramidates 533, 537, 538, 543 peptidyl impurities 214 phosphorodiamidate morpholino peptidyl MPA-thioester 72 oligomers 17 peptidyl N-alkylcysteine 61 phosphorothioates 533, 539 peptidyl Pen-Nbz 179 phosphorylated histone H2A 499 peptidyl p-nitrophenyl ester 170 phosphorylated nucleophilic amino acids peptidyl prolylselenoester 172, 175 into peptides and proteins peptidyl prolyl thioesters 163, 169 phosphoarginine (pArg) 537–538 peptidyl selenoester 173 phosphocytesine (pCys) 539–541 peptidyl thiazolidine thioester 164 phosphohistidine (pHis) 538–539 peptidyl thioester intermediate 164 phospholysine (pLys) 539 peptidyl thioesters 161, 169, 172 pyrophosphorylation (PTM) 541 peptidyl valinyl αthiophenolester 167 serine and threonine 541 peptidyl valinyl thioesters 163, 169 phosphorylation 393, 395, 398, 497, 499 oligomer 297 phosphorylation assays 369 PF4 327 phospho-serine 541, 543 Index 597 phosphoseryl-tRNAkinase (PSTK) 464 prenyl groups 437 phospho-threonine 541, 543 preparative HPLC 8 phospho-tyrosine 541, 543 PreSciss linker 268 photocleavable biotin-based purification primary alcohol 149 tag 246–247 Proliferating Cell Nuclear Antigen photocleavable His6-based purification (PCNA) 311 tag 247–249 Proline 138, 151 picolyl esters (OPic) 195 cis-trans proline isomerization 366–368 Pictet-Spengler reaction 329, 344, 345 proline-rich peptide submaxillary gland piperidine 21, 195 androgen regulated protein 3B plant homeodomain (PHD) 503 (SMR3B) 153 p-methoxybenzyl (PMB) proline-rich polypeptides 175 diselenide 147 prolyl ester carbonyl 161 thiol 135 prolyl SEA peptide 166 p-nitrophenol (ONp) derivatives 229 prolyl thioester 78, 164, 166, 169 p-nitrophenol-derived ester 170 propargyloxycarbonyl (Proc) 196 p-nitrophenyl ester protocol 170 17-mer propeptide sequence 263 p-nitrophenylsulfonyl (Nosyl) chloride proteasome 400 134 protein 411 p-NO2-phenol 170 aggregation 357 POI-sortase thioester intermediate 93 conjugates 344 polar PEG-based polymers 276 design 376 polyethylene glycol (PEG) 191 diastereomers 10 functionalized polystyrene resin 19 function 357, 364, 377, 522 polypeptide chain 358 mimicry 522 backbone 10 misfolding 357 polystyrene 19 polypeptide chain 1 solid supports 18 scaffolds 59 polyubiquitination 383 semisynthesis 307, 311 positively charged amino acids 192 splicing 308 positively charged moieties 191 stability 359 positron emission tomography (PET) structure 358, 369, 373, 377 338 thioester 317, 319 post-ligations solid-supported ubiquitination 108 transformations protein backbone biochemical transformations 275 alteration 521 chemical transformations 274 amides 358, 361 posttranlational phosphorylations 534 side-chains 362, 366 post-translational modifications (PTMs) protein farnesyltransferase (PFTase) 213, 309, 383 332 potassium selenocyanate 149 protein folding 357–359, 465 potassium thioacetate yielded g-thiol Lys GSeSeG 478 variant 130 selenium 475–478 potassium toluene thiosulfonate 122 protein mimetics, backbone engineering premade peptidyl thioesters 164 522–525 598 Index

protein molecules recombinant C-terminal protein atomic structure 7 hydrazine 93 characterization of 8 recombinant expression 358 chemical synthesis 2 recombinant polypeptide covalent structure 9 α-N-acetyl-galactosaminyl defined folded structure 7 transferase 1 (pp-GalNAc-T1) function of 1 275 homogeneity 7 redox-controlled latent thio-and protein of interest (POI) 92 selenoesters 66 proteinogenic amino acids 121, 266 redox homeostasis 465 protein synthesis re-immobilization of the C-terminal “click” chemistry 242–244 fragment 266 His6 tag 221–222 removable-backbone modification (RBM) KAHA ligation 244–246 strategy 112, 194, 298, 446 oxime formation 225–238 Replication Protein A (RPA) 311 180-residue biotinylated NK1 domain photocleavable tags 246–249 (NK1-B) 231, 232 via hydrazone formation 238–241 86-residue HIV regulatory protein TAT proton donor 347 135 pseudoproline dipeptides 188 118-residue human group V secretory PTMs 490, 491, 494–497, 500, 506 phospholipase A (GV-PLA ) PylRS-tRNACUAPyl system 93 2 2 protein 233 pyrazole 546 136-residue (15 kDa) polypeptide 243 pyridinium dichromate (PDC) 127, 149 residue polypeptides 101 pyridoxal 5′-phosphate (PLP) 329 84-residue protein 143 pyridylmethoxycarbonyl (iNoc)-protected 66-residue protein CssII 96 isoacyl peptides 189 71-residue target protein 147 pyroGlu-ADan 189 160-residue triazole-derivative 241 pyroglutamate aminopeptidase 189 reverse-phase high performance liquid pyrophosphorylated serine 547 chromatography (RP-HPLC) 185, pyrophosphorylation (PTM) 541 416, 494, 496 pyruvic acid 334 reverse polarity protection 195 reversible backbone modification 194 q reversible HIS6-based capture tags 220 quasiracemic protein 518, 519 reversible hydrazinyl tag 238 reversible hydrazone-based capture tags r 238 racemic protein 517–519 reversible oxime-based capture tags 227 crystallography 7 reversible transthioesterification 285 radiometric assays 339 R-Garner’s aldehyde 127 radiosyntheses 338 5-R hydroxylysine 133 RANTES 327, 338 ribonuclease T1 89 reactive peptidyl αthiophenylesters 167 ribosomal protein S25 96 reactive peptidyl thioester SEAE 176 ribosomal RNA 494 reactive thioesters 167 ring-chain tautomerization 298 Index 599

Rink amide linker 27, 267 selenoamino acid-derived peptides 174 Rink amide-PEGA solid support 265 β-seleno Asp variant 147 Rink linker cleavage 265 selenocysteine (Sec) 63, 64, 146, 173, RiPPs 554 368–370, 463, 472 RP-HPLC 559, 563 properties 464 reactive handling 469–473 s selenocysteine insertion sequence (SECIS) safety catch acid-labile linker (SCAL) 463 266 selenoester 64, 66, 148, 170 salicylaldehyde derivative 194 selenoester/diselenide-based ligation salicylaldehyde dimethyl acetal 291 174 saposin D (SapD) 416 seleno-insulin 370 Schiff base 346 selenol-based ligation 173 SEA and SeEA peptides 68, 70 selenol ligation auxiliary 153 SEA ChemMatrixr resin 68 SELENON gene 466 SEAlide chemistry for protein total β-seleno Phe 149 synthesis 75 selenophenol 61, 172 SEAlide-derived thioester 67 selenophosphate synthetase 2 (SPS2) SEAlide-mediated one-pot process 75 466 SEAlide peptides 70 trans-γ-selenoproline 175 SEAlide system 66, 67 selenoprolyl peptides 173 SEAoff peptides 68 selenoprotein M (SELENOM) 470 SEAoff/SEAlide latent thioesters 67 selenoprotein P (SELENOP) 466 SEAoff/SeEAoff latent thio-or selenoester selenoproteins 463–466 precursors 71 expression 466–469 SEAon peptide 74, 176 synthesis and semi-synthesis of SEA polystyrene resin (SEAPS) 68 473–475 SEA/SeEA and SEAlide-based ligations selenoprotein S (SELENOS) 466 64 selenoproteinselenophosphate synthetase SEA/SeEA system 66 2(SPS2) 464 SEA trityl solid supports 68 selenoprotein W (SELENOW) 466 Sec-based ligation 173 seleno-salicylaldehyde esters 289 selenylated amino acid 149 Sec biosynthesis (SPS) 465 selenylating reagent 147 second generation automated fast flow self-purification effect 274 peptide synthesis self-purified peptide thioesters 239 amino acid stocks design 26 semicarbazone 293 characterization and use of 26 semipermanent “helping hand” linker key findings 24 197 pump design 24, 26 semipermanent solubilizing tags valve design 24 building blocks 195 Seebeck group 537 extendable side-chain-based segment D delivered SeEAoff peptide 74 solubilizing tags 195 selenazolidine (Sez) 470, 471, 474 N-or C-terminal solubilizing “tails” selenium, tool for protein folding 192 475–478 reversible backbone modification 194 600 Index

semisynthesis, glycoproteins 413 S-methyl asymmetric disulfide variant semi-synthesized diacetylated Atg3 107 124 semisynthetic H3 proteins 315 S-methyl methanethiosulfinate 130 sequential N-to-C ligation 96 SMR3B 175 Ser63-Gln106 298 snow flea anti-freeze protein (sfAFP) serine 153, 541 373, 374 serine/threonine ligation 287 sodium azide afforded azide 130 aryl aldehyde esters 289 sodium benzenesulfinate 147 bioconjugation 296–297 sodium 2-mercaptoethanesulfonate C-to-N ligation 294–296 (MESNa) 64 epimerization issue 289 sodium thioacetate 142 extension of 298–302 solid phase carbohydrate synthesis 18 N-to-C ligation 296 solid-phase peptide ligations (SPCL) 212 one-pot ligation 296 chemical ligation reactions 260–261 peptide salicylaldehyde ester 289–294 N-to-C direction 268 scope and limitations 294 post-ligations solid-supported solubility issues 298 transformations 274–275 promises of 259–260 Ser64-phosphorylated M2 proton channel requirements 261–262 112 solid support 275–278 Ser/Thr ligation (STL) 418 solid phase peptide synthesis (SPPS) 18, S-ethyl ethanethiosulfinate 127 59, 87, 119, 211, 333, 438, 439, 442, Shiga toxin subunit B 198 445–447, 449, 455, 456, 473, short linear peptides 302 535–537, 547 sialyloligosaccharides 105 solid-supported oxidative folding 275 side-chain aldehyde 139 solubility issues 298 sidechain deprotection 557, 558, 563, solubility tags 565 backbone tags 445–449 side-chain-protected peptide acid 291 side chain tags 445 silyl ester 133 terminal tags 443–445 silyl-protected alkyne 243 solvent manipulation 185 single defined molecular species 5 sortase 93, 314, 315 single-molecule fluorescence resonance sortase-A 314 energy transfer (smFRET) 109 sortase-mediated hydrazide generation site-specific labeling with isotopes 10 93 site-specific labelling 373, 375 sortase mediated protein hydrazide site-specific protein labeling 311 ligation technique 105 six-membered tetrahydro-1,3-oxazine spherilose resin 276 structure 302 sp3-hybridized nitrogen 287 small molecules 18 spinal muscular atrophy 17 small protein EETI-II 27 split inteins 309 small ubiquitin-like modifier (SUMO) Spt-Ada-Gcn5 acetyltransferase (SAGA) 111 complex 498 small ubiquitin-like protein (SUMO) Staphylococcus aureus sortase A (SrtA) 505 314 Index 601

Staudinger-phosphite reaction 539, 543 template assembled synthetic proteins stepwise solid phase synthesis (SPPS) 2 (TASPs) 450 stepwise synthesis of polypeptide chains TentaGel 19 10 terminally-protected monomer 18 L-stereochemistry 362 2-(tert-butyldisulfany)ethyloxycarbonyl sterically hindered amino acid sites 166 (Tbeoc) 96 sterically hindered C-terminal amino acid tertiary amide, 124 residues 170 tetrakis(triphenyl phosphine) palladium, S-(thiobenzoyl)thioglycolic acid 123 195 S-to-N acyl transfer 285 2,2,6,6-tetramethylpiperidine-1-oxyl stopped-flow techniques 358 radical (TEMPO) 151 strain-promoted azide-alkyne tetramethyl urea 30 cycloaddition (SPAAC) 243, 271 tetra-n-butylammonium fluoride (TBAF) subtiligase 317, 318 131 sulfenylation reagent 135, 137 tetrapeptide 89 sulfhydryl group 311 17 kDa modular tetratricopeptide repeat Sulfolobus solfataricus P2 DNA protein 221 polymerase IV 188 tetra-Ub sulfonamide 134 chains 390–392 sulfonamide resin 215 expressed proteins with 400–401 sulfonylethoxycarbonyl-based linker 227 tetra-Ub-α-globin-(HA) 399 sulfonylethoxycarbonyl moiety 238 tetra-ubiquitinated α-globulin 259 sulfonylethyl carbamate 272 tetraubiquitinated proteins 395, 400 sumoylated histones 505, 506, 507 TFA-labile Wang-type linker 265 superoxide dismutase (SOD) 195 TFA-stable allylic ester 195 superpermeable organic combinatorial thermodynamic stabilities 359 chemistry (SPOCC) resin 265 thermostable DNA polymerase IV (Dpo4) Swiss-Prot protein database 197 mutants 113 swollen Merrifield resin 91 thiazolidine (Thz) 135, 196, 221, 287, Syk tyrosine kinase 142 470, 471 symmetric and unsymmetrical peptide protected building block 134 diselenides 174 protected cysteine residues 121 syn-alcohol 127 L-thiazolidine-4-carboxylic acid (Thz) synthesized EPO glycoforms, 105 96 synthetic L-DNA templates 113 thiazolidine/oxazolidine 212 synthetic phosphotyrosine 308 trans-thiazoline 124 synthetic polypeptide chain 2 thioamide isosteres 522 synthetic protein molecule 5 thioamino acid-based procedure 173 synthetic proteins 2 thiobenzamidomalonic ester 123 α-synuclein (α-Syn) 376, 393, 414 syn-thiocyanate 127 thiodepsipeptide 317 t thioester 135, 153, 359 tau protein 422, 423 α-thioesterification 495 Tbeoc-Thz residue 96 thioester/thioamino acid ligations 174 TCEP-derived selenophosphine 68, 74 thioether 212 602 Index

thioether-containing isopeptide bonds temperature studies 41 111 threonine (Thr) 140, 161, 329, 365, 541 thioethylbutylamide (TEBA) 63 Thr38-Gly62-salicylaldehyde ester 298 β-thiolactone moiety 168 Thr69Xaa analogues 75 β-thiol Arg building block 139, 140 Thz-peptide 4 β-thiol Arg variant 140 Thz-protected segments 267 β-thiol Asp 135 tobacco etch virus (TEV) 474 β-thiol aspartate 123–124 protease 193 β-thiol aspartic acid 122 transcriptional regulator YAP65 139 thiolated amino acid 122, 130 transesterification of acyl azide 90 γ-thiolated Lys building block 131 transient receptor potential (TRP) 437 thiolated-proline derivative 138 transient thio-or selenoester 61 β-thiol GlcNAc Asn building block 137 transitional state 360–362 γ-thiol Gln variant 139 transmembrane (TM) domain 437 γ-thiol Glu 135 1 TM 449–450 γ -thiol glutamate 124 2 TM 450–453 γ-thiol Ile variant 127, 130 3 and more TM 454–456 δ-thiol Lys variant 131–133 transmembrane (TM) peptides 112 β-thiol Phe building block 127 backbone tags 445–449 β-thiol phenylalanine 121 chemical protein synthesis 449–456 γ-thiol Pro 138 purification and handling strategies γ-thiol Pro configuration 138 442–443 thiol-protected cysteinyl peptide thioester side chain tags 445 segment 235 solid phase peptide synthesis 438–442 γ-thiol Pro variant 138 terminal tags 443–445 thiol-selenoester interchange process 63 transmembrane potassium channel KcsA thiol-thioester 64 186 2-thiol Trp 144 trastuzumab 430–432 β-thiol Val 142 γ-thiol Val building block 143 triazole 212, 546 thio-mercurate 212 1,2,4-triazole 167 thioproline-dervied peptides 173 triazole-containing MUC1 polypeptide thioredoxin reductase 464 272 thiourea 212 tri-Boc-hydrazino acetic acid 336 third generation automated fast flow trifluoroacetamidomethyl 98 peptide synthesis trifluoroacetic acid (TFA) 185, 438 general lab use 41 trifluoroethanethio (TFET) 162 key findings 32–34 2,2,2-trifluoroethanol (TFE) 442 model peptides 39 trifluoroethanol (TFE) 187 new reactor design 36 trifluoroethanol thiol (TFET) 127 reagent reservoirs 38 trifluoroethyl thioester reagent stability study 43 Ac-LYRANX-SCH2CF3 176 separation of amino acid and activator trifolitoxin 91 35 triisopropylsilane (TIS) 197 software 38 tri-isopropylsilyl group (TIPS) 270 Index 603 trimethoxybenzyl alcohol (Tmob-OH) ubiquitination 383–387, 393, 395, 398, 122 401 2,4,6-trimethoxybenzyl isocyanide 139 ubiquitin-binding domains (UBDs) 384, trimethoxybenzyl (Tmob)-protected 390, 401 β-thiol Asp derivative 122 75-mer ubiquitin (Ala46Asa) protein trimethoxyphenyl thiazolidine 137 245–246 trimethylsilyldiazomethane (TMSCHN2) ubiquitin thioester 131 reagent 215 ubiquitinylated peptide 131 tris(3-hydroxypropyltriazolylmethyl)amine ubiquitylated histones 111 (THPTA) 261 Ub-thioester 388 tris-(2-carboxyethyl)phosphine (TCEP) uncleavable pseudoproline structure 274 287 tris-carboxyethylphosphine (TCEP) UniProt 2 146 unnatural amino acid (UAA) 92 tris-(2-carboxyethyl)phosphine (TCEP) unnatural protein 328 67, 72, 274, 503 N-α-unprotected amino acids 20, 43 trityl-based linker 197 unprotected glutamic acid 131 trityl-protected β-thiol aspartate 123 unprotected peptide salicylaldehyde ester trityl-protected β-thiol aspartate variant 291 124 unreacted C-terminal peptide segments Trp, and Boc-sarcosinyl-sacrcosinyl 212 (Boc-Sar-Sar) 195 urea 227 truncates, 21 urea-containing peptides 88 truncation products 27 user interface principles 20 trypsiligase 314, 318 USP51 deubiquitinase 111 tryptophan 121, 144 Tsuji-Trost reaction 263 v tubulin tyrosine ligase (TTL) 331 Valco ChemInert 10-position selector β-turn mimetics 361–362 valves 24 two-photon activated chemokine CCL5 valine 142 103 valinyl-Nbz peptides 169 Tyr, Boc-(N-methylamino)butanoyl valinyl thioesters 168 (Boc-Nmbu) 195 Φ-value analysis 360, 361 variable number tandem repeat (VNTR) u sequence 140 Ubiq[2-76] Ala46Asa 245–246 Varian Prostar 210 HPLC pumps 47 Ubiq[46-76] hydroxylamine 246 Varian 200 series HPLC pumps 24 Ubiq[2-45] ketoacid 246 vascular endothelial growth factor ubiquitin (Ub) 364, 383, 386 (VEGF) signaling 524 building blocks 387 vicinal diols 329 ubiquitinated histone analogs 111 vinyl glycine derivative 142 ubiquitinated proteins vMIP I chemokine 267 monoubiquitinated proteins 393, 397 voltage-gated sodium channels (VGSCs) tetraubiquitinated proteins 395, 400 475 604 Index

w X-ray crystallography 7, 109 Wang resin 336, 337 X-ray structural data 7 water-soluble radical initiator VA-044 121 y Weinreb amide resin 331 yes kinase-associated protein (YAP 65) 361 x Xenopus histone H3 (XH3) 130 XH3 (1-111) thioester 130