Table 3.

Synthetic methods used in chemical protein synthesis, indicating use in reported D‐protein synthesis and potential issues encountered with use.

Entry				Used in D‐protein synthesis
#	Synthetic method	Reagents	Y / N	Potential issues	Ref.
	Ligation method
1	Native chemical ligation	+ thiol catalyst	Y	Dependence on suitable cysteine or alanine residues.	[97a]
2	Serine/threonine ligation	+ acidolysis	N	Requires suitable Ser/Thr. Slower reaction kinetics than NCL.	[145]
3	KAHA ligation		N	Accessibility of enantiomeric reagent.	[146]
4	Selenocysteine NCL	+ thiol catalyst	N	Accessibility of enantiomeric reagent.	[123a]
	NCL reactive end
	Thioester surrogate
5	Hydrazides	Activation+NaNO2 + Thiol	Y	Oxidation incompatible with Thz. Low temperature activation needed (<−15 °C).	[100]
6	Dbz	Activation 4‐nitrophenyl chloroformate or NaNO2 or isoamyl nitrite +thiol	Y	Di‐acylation side product with excess Gly.	[16, 147]
7	MeDbz	Activation 4‐nitrophenyl chloroformate +thiol	N	Difficult to activate off‐resin.	[98]
8	SEA	Activation of SEA OFF TCEP+thiol	N	Latent SEAOFF thioester incompatible with TCEP during ligations.	[148]
	N‐cysteine protection
9	Thz	Deprotection MeONH2	Y	Incompatible with hydrazide oxidation.	[103]
10	Cys(Tfacm)	Deprotection pH 11.5	N	Accessibility of enantiomeric reagent.	[149]
11	TFA‐Thz	Deprotection Base then MeONH2	Y	Accessibility of enantiomeric reagent.	[41]
12	N3‐Cys	Deprotection TCEP	N	Accessibility of enantiomeric reagent.	[150]
13	Cys(Dobz)	Deprotection H2O2	N	Accessibility of enantiomeric reagent. Harsh deprotection conditions.	[151]
	Desulfurization
14	Metal‐based	Pd/Al2O3 Or Raney Nickel+ H2 (g)	Y	Removal of metal impurities can be problematic. Use of hydrogen gas. Potential side reactions with Trp and Met Quenched by thiol catalyst. Native Cys must be protected.	[108]
15	Metal‐free radical based	VA‐044 TCEP tert‐butylthiol	Y	Quenched by thiol catalyst. Native Cys must be protected.	[106]
16	Beta/gamma thiol amino acids	β‐thiol‐Phe β‐thiol‐Val β‐thiol‐Leu β‐thiol‐Asp β‐thiol‐Asn β‐thiol‐Arg γ‐thiol‐Val γ‐thiol‐Thr γ‐thiol‐Ile γ‐thiol‐Pro γ‐thiol‐Glu γ‐thiol‐Gln γ‐thiol‐Lys 2‐thiol‐Trp	N	Accessibility of enantiomeric reagent. Commercially available D‐Penicillamine (β‐thiol‐Val) could be used for D‐peptide ligation at Val, if directly following a glycine residue.	[152]
	Thiol catalysts for one‐pot ligation‐desulfurization
17	MPAA‐hydrazide	pKa=6.6 Removal Aldehyde‐resin capture	N	Preparation of MPAA‐hydrazide reagent coupled with use in large excess is uneconomical.	[153]
18	Trifluoroethanthiol	pKa=7.3 Removal Evaporation (bp=37 °C)	N	Malodorous and volatile, though could be used for D‐protein synthesis.	[154]
19	Methyl thioglycolate	pKa=7.9 Removal none	Y	Slower kinetics with C‐terminal beta‐branched residue.	[41]
	Solubility enhancers
20	Helping hand v1	Installation Amine labelling with lysine side chain Removal Hydrazine (aq)	N	Additional steps to incorporate and remove tag. Potential issues with stability.	[119a]
21	Helping hand v2	Installation Amine labelling with lysine side chain Removal Hydrazine (aq) Or Hydroxylamine (aq)	N	Additional steps to incorporate and remove tag.	[119b]
22	Removable backbone modification v1	Installation Standard Fmoc‐SPPS Removal pH 7 then TFA	Y	Limited to Gly only. Lengthy synthesis of building block. Additional steps to incorporate and remove tag.	[117, 155]
23	Removable backbone modification v2	Installation Reductive amination Acetylation Removal Deacetylation (Cys (aq)) then TFA	Y	Additional steps to incorporate and remove tag.	[77b, 118]
	Protein folding
		Disulfide #1 Removal TFA
		Disulfide #2 Removal Iodine or PdCl2
24	Cysteine orthogonal protection	Disulfide #3 Removal UV light (350 nm)	Y	Practically limited to two disulfide bonds. Accessibility of a third, orthogonally protected D‐Cys building block.	[52, 128]
25	“Ambidextrous” chaperone	GroEL/ES protein chaperone	Y	Mostly unnecessary for in‐vitro protein folding. Limited scope reported.	[121b]
	Post‐translational modifications
26	Lys ubiquitination	δ‐mercapto lysine for NCL followed by desulfurization	N	Accessibility of enantiomeric reagent.	[136a]
27		Solid‐phase isopeptide bond formation	N	Requires assembly of large fragments by SPPS – not cost‐effective with D‐amino acids	[156]
28		Installation Coupling to lysine side chain PTM NCL to Ub‐thioester Auxiliary removal TFA	N	Low efficiency of ligation. Glycyl auxiliary replaced with Cys in preparation of enantiomeric di‐ and tri‐ubiquitin proteins.	[28, 42]
29	Lys trimethylation	Fmoc‐Lys(Me3)‐OH	N	Accessibility of enantiomeric reagent.	[133a, 151]
30	Lys acetylation	Fmoc‐Lys(Ac)‐OH	N	Accessibility of enantiomeric reagent.	[133b]
31	Asn N‐Glycosylation	Fmoc‐Asn(Glycan)−OH or Boc‐Asn(Xan)−OH (and) further glycosylation on‐resin or in solution.	N	Accessibility of enantiomeric reagent (would also require L‐sugars).	[53, 157]
32		Oligosaccharide coupled directly to free Asn side chain during Boc‐SPPS.	N	Accessibility of enantiomeric reagent (would also require L‐sugars).	[158]
33	Thr O‐Glycosylation	Fmoc‐Thr(Glycan)−OH	N	Accessibility of enantiomeric reagent (would also require L‐sugars).	[131b]
34	Cys S‐palmitoylation	Fmoc‐Cys(Mmt)−OH Mmt removal on‐resin with 2 % TFA Reaction with palmitic anhydride	N	Incompatible with NCL. Potentially viable for D‐protein synthesis via STL or Sec NCL.	[137a]
35		Fmoc‐Cys(palmityl)‐OH	N	Incompatible with NCL. Fmoc‐D‐Cys(palmityl) must be synthesized.	[137b]
36	Tyr sulfation	Fmoc‐Tyr(OTBS)−OH Deprotection and sulfation on‐resin with:	N	Accessibility of enantiomeric reagent.	[131b]
		+DIEPA
37	Ser phosphorylation		Y	Accessibility of enantiomeric reagent.	[18, 130b, 159]
38	Tyrosine phosphorylation		N	Accessibility of enantiomeric reagent.	[131a]