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Supplementary Figure 1
HyCoSuL synthesis and quality control exemplified with the P2 sublibrary.
P2 HyCoSuL sublibrary contains amino acids mixtures at P3 and P4 positions. To test whether the coupling of isokinetic mixture provided the equal distribution of amino acids in P3 (and P4) position an Edman degradation can be utilized. After coupling and de-protection of P3 position (steps 38-44) several beads are subjected for the analysis in order to determine the molar distribution of amino acids at the N-terminal end of a peptide. The same procedure can be applied to test the equimolar coupling of amino acids mixture to the P4 position.
Nature Protocols: doi:10.1038/nprot.2017.091
Supplementary Figure 2
HR-MS and RP-HPLC analysis of ACC-labeled legumain substrate containing unnatural amino acids.
Substrate was synthesized according to standard solid phase Fmoc/Boc strategy, and purified using reverse phase high performance (pressure) liquid chromatography (RP-HPLC) Waters system with semi-preparative C18 column. The purity of the substrate was confirmed using the analytical HPLC with C18 analytical column (UV detector, 220nm). The molecular mass of the substrate was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.
Nature Protocols: doi:10.1038/nprot.2017.091
Supplementary Figure 3
HR-MS and RP-HPLC analysis of biotin-6-ahx-DTyr(tBu)-Tic-Ser(tBu)-COOH.
Peptide was synthesized according to the strategy presented in the main protocol (Block A, steps 100-124), and used without further purification. The purity of the peptide was confirmed using analytical HPLC (UV detector, C8 column, 220nm). The molecular mass of the peptide was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.
Nature Protocols: doi:10.1038/nprot.2017.091
Supplementary Figure 4
HR-MS and RP-HPLC analysis of Boc-Asp(Bzl)-AOMK.
AOMK warhead was synthesized according to the strategy presented in the main protocol (Block B, steps 125-137), and purified via extraction. The purity of the product warhead was confirmed using analytical HPLC (UV detector, C8 column, 220nm). The molecular mass of the compound was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module.
Nature Protocols: doi:10.1038/nprot.2017.091
Supplementary Figure 5
HR-MS and RP-HPLC analysis of biotin-labeled legumain activity containing unnatural amino acids.
Probe was synthesized according to the strategy presented in the main protocol (Block C, steps 138-151), and purified using reverse phase high performance (pressure) liquid chromatography (RP-HPLC) Waters system with semi-preparative C8 column. The purity of the probe was confirmed using analytical HPLC with C8 column (UV detector, 220nm). The molecular mass of the substrate was confirmed using High Resolution Mass Spectrometer WATERS LCT premier XE with Electrospray Ionization (ESI) and Time of Flight (TOF) module. Since the probe is more hydrophobic than the substrate, we used C8 (instead of C18) column to purify and analyze.
Nature Protocols: doi:10.1038/nprot.2017.091
No Name and code Structure before synthesis Structure after
de-protection
1 L-alanine
L-Ala
2 L-arginine
L-Arg
3 L-asparagine
L-Asn
4 L-aspartic acid
L-Asp
5 L-glutamine
L-Gln
6 L-glutamic acid
L-Glu
7 glycine
Gly
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8 L-histidine
L-His
9 L-isoleucine
L-Ile
10 L-leucine
L-Leu
11 L-lysine
L-Lys
12 L-norleucine
L-Nle
13 L-phenylalanine
L-Phe
14 L-proline
L-Pro
15 L-serine
L-Ser
16 L-threonine
L-Thr
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17 L-tryptophan
L-Trp
18 L-tyrosine
L-Tyr
19 L-valine
L-Val
20 L-methionine
L-Met
21 D-alanine
D-Ala
22 D-arginine
D-Arg
23 D-asparagine
D-Asn
24 D-aspartic acid
D-Asp
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25 D-glutamine
D-Gln
26 D-glutamic acid
D-Glu
27 D-histidine
D-His
28 D-leucine
D-Leu
29 D-lysine
D-Lys
30 D-phenylalanine
D-Phe
31 D-proline
D-Pro
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32 D-serine
D-Ser
33 D-phenylglycine
D-Phg
34 D-threonine
D-Thr
35 D-tryptophan
D-Trp
36 D-tyrosine
D-Tyr
37 D-valine
D-Val
38 D-homophenylalanine
D-hPhe
39 beta-alanine
-Ala
Nature Protocols: doi:10.1038/nprot.2017.091
40 L-azetidine
L-Aze
41 L-4-hydroxyproline
L-Hyp
42
O-benzyl-L-4-
hydroxyproline
L-Hyp(Bzl)
43 L-thiazolidine
L-Thz
44 L-octahydroindole
L-Oic
45 L-indoline
L-Idc
46 L-piperidine
L-Pip
47
L-1,2,3,4-
tetrahydroisoquinoline
L-Tic
48 dehydrohomoalanine
dhAbu
49 dehydroleucine
dhLeu
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50 amino-L-alanine
L-Dap
51 amino-L-homoalanine
L-Dab
52
N--
(benzyloxycarbonyl)amino-
L-homoalanine
L-Dab(Z) or L-Dab(Cbz)
53 L-citrulline
L-Cit
54 L-homocitrulline
L-hCit
55 L-ornithine
L-Orn
56 N,N-dimethyl-L-lysine
L-Lys(Me)2
Nature Protocols: doi:10.1038/nprot.2017.091
57 N-trifluoroacetyl-L-lysine
L-Lys(TFA)
58 N-acetyl-L-lysine
L-Lys(Ac)
59
N-2-chloro-
benzyloxycarbonyl-L-lysine
L-Lys(2-Cl-Z)
60 guanidino-L-alanine
L-Agp
61 guanidino-L-homoalanine
L-Agb
62 N-nitro-L-arginine
L-Arg(NO2)
Nature Protocols: doi:10.1038/nprot.2017.091
63
N,N-
di(benzyloxycarbonyl)-L-
arginine
L-Arg(Cbz)2
64 L-homoarginine
L-hArg
65 N(im)-benzyl-L-histidine
L-His(Bzl)
66
N(im)-benzyloxymethyl-L-
histidine
L-His(3-Bom)
67 4-amino-L-phenylalanine
L-Phe(4-NH2)
68
4-guanidino-L-
phenylalanine
L-Phe(4-guan)
Nature Protocols: doi:10.1038/nprot.2017.091
69 2-methyl-L-tryptophan
L-Trp(Me)
70 L-dihydrotryptophan
L-Dht
71
L-aspartic acid methyl
ester
L-Asp(Me)
72
L-aspartic acid cyclohexyl
ester
L-Asp(Chx)
73 L-aspartic acid benzyl ester
L-Asp(Bzl)
74
L-glutamic acid methyl
ester
L-Glu(Me)
75
L-glutamic acid cyclohexyl
ester
L-Glu(Chx)
Nature Protocols: doi:10.1038/nprot.2017.091
76
L-glutamic acid benzyl
ester
L-Glu(Bzl)
77 L-homoglutamic acid
L-Aad or L-hGlu
78 2-fluoro-L-phenylalanine
L-Phe(2-F)
79 3-fluoro-L-phenylalanine
L-Phe(3-F)
80 4-fluoro-L-phenylalanine
L-Phe(4-F)
81
3,4-difluoro-L-
phenylalanine
L-Phe(3,4-F2)
82
2,3,4,5,6-pentafluoro-L-
phenylalanine
L-Phe(F5)
Nature Protocols: doi:10.1038/nprot.2017.091
83 2-chloro-L-phenylalanine
L-Phe(2-Cl)
84 3-chloro-L-phenylalanine
L-Phe(3-Cl)
85 4-chloro-L-phenylalanine
L-Phe(4-Cl)
86
3,4-dichloro-L-
phenylalanine
L-Phe(3,4-Cl2)
87 4-bromo-L-phenylalanie
L-Phe(4-Br)
88 4-iodo-L-phenylalanine
L-Phe(4-I)
89 4-methyl-L-phenylalanine
L-Phe(4-Me)
Nature Protocols: doi:10.1038/nprot.2017.091
90 3-pyridyl-L-alanine
L-3-Pal
91 4-pyridyl-L-alanine
L-4-Pal
92 2-thienyl-L-alanine
L-Ala(2-thienyl)
93
3-(benzothiazol-2-yl)-L-
alanine
L-Ala(Bth)
94 3-benzothienyl-L-alanine
L-Bta
95 L-homoalanine
L-Abu
96
3-(benzothiazol-2-yl)-L-
homoalanine
L-Abu(Bth)
Nature Protocols: doi:10.1038/nprot.2017.091
97 O-acetyl-L-serine
L-Ser(Ac)
98 O-benzyl-L-serine
L-Ser(Bzl)
99 L-homoserine
L-hSer
100 O-benzyl-L-homoserine
L-hSer(Bzl)
101 O-benzyl-L-threonine
L-Thr(Bzl)
102 S-benzyl-L-cysteine
L-Cys(Bzl)
103
S-4-methyl-benzyl-L-
cysteine
L-Cys(4-MeBzl)
Nature Protocols: doi:10.1038/nprot.2017.091
104
S-4-methoxy-benzyl-L-
cysteine
L-Cys(4-MeOBzl)
105 L-methionine sulfoxide
L-Met(O)
106 L-methionine sulfone
L-Met(O)2
107 6-benzyloxy-L-norleucine
L-Nle(O-Bzl)
108 L-phenylglycine
L-Phg
109 L-homophenylalanine
L-hPhe
110 L-cyclohexylglycine
L-Chg
111 L-cyclohexylalanine
L-Cha
Nature Protocols: doi:10.1038/nprot.2017.091
112 L-homocyclohexylalanine
L-hCha
113 2-indanyl-L-glycine
L-Igl
114 1-naphthyl-L-alanine
L-1-Nal
115 2-naphthyl-L-alanine
L-2-Nal
116 L-biphenylalanine
L-Bip
117 4-benzoyl-L-phenylalanine
L-Bpa
118 L-2-aminooctanoic acid
L-2-Aoc
119 L-homoleucine
L-hLeu
Nature Protocols: doi:10.1038/nprot.2017.091
120 L-neopentyl-glycine
L-NptGly
121 L-norvaline
L-Nva
122 L-hydroxynorvaline
L-Hnv
123 L-tert-leucine
L-Tle
124 4-methyl-L-tyrosine
L-Tyr(Me)
125
4-(2,6-dichlorobenzyl)-L-
tyrosine
L-Tyr(2,6-Cl2-Bzl)
126 4-benzyl-L-tyrosine
L-Tyr(Bzl)
127
4-(2bromobenzyl)-L-
tyrosine
L-Tyr(2-Br-Bzl)
Nature Protocols: doi:10.1038/nprot.2017.091
128 L-homotyrosine
L-hTyr
129 4-methyl-L-homotyrosine
L-hTyr(Me)
Table 1 Structure of amino acids used in HyCoSuL synthesis. P1 Asp HyCoSuL contains 129 amino acids (19 natural and 110 unnatural). In the table we present the structures of Fmoc-protected amino acids used in the synthesis (left) and the structures of amino acids after Fmoc de-protection and TFA-assisted cleavage (right). Several protecting groups are TFA labile, thus the amino acids structures in the peptide library differ from the structures used for the synthesis. These groups are: Pbf (Arg), Trt (Asn, Gln, His), tBu (Asp, Glu, Ser, Thr, Trp, Tyr, Hyp, hGlu, hSer, Hnv, hTyr), and Boc (Lys, Dap, Dab, Orn, Agp, Agb, hArg, Phe-4-NH2, Phe-4-guan).
Nature Protocols: doi:10.1038/nprot.2017.091
