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1
Supporting Information
Cyclic Peptide Production Using a Macrocyclase with Enhanced Substrate Promiscuity and Relaxed Recognition Determinants
Cristina N Alexandru-Crivac1,2, Christian Umeobika1, Niina Leikoski3, Jouni Jokela3,
Kirstie A. Rickaby1, André M. Grilo1,2, Peter Sjö4, Alleyn T Plowright5, Mohannad
Idress1,2, Eike Siebs1, Ada Nneoyi-Egbe1, Matti Wahlsten3, Kaarina Sivonen3, Marcel
Jaspars1, Laurent Trembleau1*, David P Fewer3* & Wael E Houssen1,2,6*
1Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston
Walk, Aberdeen AB24 3UE (UK).
2Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD (UK).
3Microbiology and Biotechnology Division, Department of Food and Environmental Sciences,
P.O.Box 56, Viikki Biocenter, Viikinkaari 9, 00014, University of Helsinki (Finland)
4Medicinal Chemistry, Respiratory, Inflammation & Autoimmunity, Innovative Medicines and
Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 43183
(Sweden).
5Medicinal Chemistry, Cardiovascular and Metabolic Diseases Innovative Medicines and
Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, Mölndal, 43183
(Sweden).
6Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516
(Egypt).
*Correspondence should be addressed to
Wael Houssen ([email protected]), David P. Fewer ([email protected]) or
Laurent Trembleau ([email protected])
Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2017
2
Experimental Data
Peptide Synthesis
Fmoc amino acid derivatives, 2-(1H-benzotriazol-1-yl)-1, 1, 3, 3 - tetramethyluronium hexafluorophosphate (HBTU), 2-chlorotrityl chloride resin, Fmoc-Phe-Ser(ψMe,MePro)-OH, and Fmoc-thioproline were purchased from Novabiochem, Merck Biosciences, UK. Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF) and piperidine were obtained from Alfa Aesar, UK, and used without further purification. Fmoc-Ser(ψPro)-OH was prepared from L-serine, formaldehyde, Fmoc chloride, and N,N-diisopropylethylamine (all from Sigma-Aldrich) using an adapted literature procedure for the synthesis of Boc-Ser(ψPro)-OH [Vishnumaya, Monika Raj; Singh, Vinod K. J. Org. Chem., 2009, 74, 4289 - 4297].
The linear peptide substrates (1-18) were synthesized manually in-house using the standard Fmoc-based solid phase peptide synthesis (SPPS) strategy. The attachment of the first amino acid to the 2-chlorotrityl resin was conducted using a molar ratio of amino acid: DIEA: resin of 1.2: 4.8: 1 in DMF. Subsequent amino acids were sequentially coupled following removal of the Fmoc protecting group at each cycle. Fmoc deprotection steps were carried out with 20% piperidine in DMF (v/v) for 6 min (3x: 2min each); coupling reactions were performed in DMF using a molar ratio of amino acid: HBTU: DIEA: resin of 5: 5: 10: 1. Reactions were monitored using the Kaiser test. Pseudoproline residues were introduced using the corresponding Fmoc-Phe-Ser(ψMe,MePro)-OH dipeptide acid, Fmoc-Ser(ψPro)-OH or Fmoc-thioproline.
The peptides were cleaved from the support and side-chain protecting groups removed by treatment with a mixture consisting of 95% TFA, 2.5% triisopropylsilane (TIPS), 2.5% H2O (20 mL of mixture per g of peptide resin, 5 hours at room temperature). The resin was then filtered and washed with TFA. The combined filtrates were concentrated under reduced pressure. The peptide was precipitated with cold diethyl ether and recovered by centrifugation. The peptide sequence was verified by MS-MS analysis.
As expected, peptide 17 containing the Ser(ψMe,MePro) residue did not survive the traditionally used cleavage cocktail (95% TFA, 2.5% TIPS, 2.5% H2O), but it was successfully prepared on 2-chlorotrityl resin using side-chain protecting groups sensitive to mildly acidic conditions for the tyrosine and aspartic acid residues [Fmoc-Tyr(2-ClTrt)-OH and Fmoc-Asp(O-2-PhiPr)-OH, Novabiochem, Merck Biosciences, UK]. The peptide was cleaved from the resin under mild acidic conditions (1% TFA / 5% TIS in DCM) and precipitated in cold diethyl ether. The ψMe,MePro residue was not affected under these mildly acidic conditions (see Figure S47).
Substrate 19 is prepared from a precursor linear peptide by proteolytic cleavage with Tobacco Etch Virus (TEV) protease and subsequent purification using Nickel affinity chromatography. The percusor peptide is produced recombinantly in Escherichia coli BL21(DE3) and contains the substrate sequence fused with an N-terminal TEV-cleavable Small Ubiquitin-like Modifier (SUMO) tag.
3
Protein Purification
Full length codon optimised OscGmac (Oscillatoria sp. PCC 6506) with an N-terminal TEV protease-cleavable His6-tag cloned in pJexpress411 vector was purchased from Atum (Figure S4). PatG mutants were generated using Q5® Site-Directed Mutagenesis Kit (New England Biolabs) and following the manufacturer protocol. Proteins were expressed in E. coli BL21 (DE3) grown on auto-induction medium (Formedium Terrific broth base containing trace elements) for 48 h at 20 °C, with shaking at 200 rpm. Cells were harvested by centrifugation at 4,000 g, 4 °C, for 15 min, and re-suspended in lysis buffer (500 mM NaCl, 20 mM Tris, pH 8.0, 20 mM imidazole pH 8.0 and 3 mM β-mercaptoethanol (BME) with the addition of complete EDTA-free protease inhibitor tablets (Roche) and DNase (Sigma) at 0.4 mg g−1 wet cells). Cells were lysed by passage through a cell disruptor at 30 kPsi and each lysate was cleared by centrifugation at 40,000 x g, 4 °C for 45 min. followed by filtration through a 0.45 µm membrane filter. Each cleared lysate was loaded onto a Ni-sepharose 6 FF column (GE Healthcare) prewashed with lysis buffer. The column was washed with lysis buffer and the protein was eluted with elution buffer (500 mM NaCl, 20 mM Tris-HCl pH 8.0, 250 mM imidazole pH 8.0, 3 mM BME). Each protein was then passed through a desalting column (Desalt 26/10, GE Healthcare), into buffer containing 350 mM NaCl and 20 mM Tris, pH 8. It was further purified via size-exclusion chromatography, using a HiLoad 16/600 Superdex 75 column (GE Healthcare) and the same buffer. The proteins were concentrated using Vivaspin 20 MWCO 30 kDa concentrators (GE Healthcare) and stored at -70°C for enzymatic assays. The purity of each protein was confirmed by SDS-PAGE analysis (Figures S5 and S56) and its identity was confirmed by mass spectrometry (MS).
Macrocyclization reactions
Macrocyclization reactions were prepared for 150 µM peptide substrate and 35 µM enzyme in buffer containing 500 mM NaCl, 10 mM bicine pH 7.5 and 5% DMSO. Reaction mixtures were incubated for 120 h at 30°C. Samples were analysed by LC-ESIMS. Control samples were prepared by incubation of the peptide substrates in the aforementioned buffer. No cyclic peptides were detected in control samples, suggesting there was no spontaneous cyclization occurring independent of the enzyme.
4
Figure S1. LC-ITMS analysis of methanolic extract from the Oscillatoria sp. PCC 6506. A: Total ion current chromatogram (TICC). B: Extracted ion m/z 1896.7 and m/z 1276.5 chromatogram (EIC) showing the elution of oscillacyclamide A at 19.0 min and oscillacyclamide B at 19.9 min. C: Mass spectrum at 19.0 min showing single and double charged oscillacyclamide A ions with and without a prenyl (Pr) unit. D: Product ion spectrum from the protonated oscillacyclamide m/z 1896.6. D: Enlargement of the product ion spectrum showing more clearly the loss of water and prenyl from the protonated oscillacyclamide A. F: The mass spectrum at 19.9 min showing double charged oscillacyclamide B ions. G: The product ion spectrum from the double protonated oscillacyclamide m/z 1276.5.
5
Figure S2. LC-ITMS analysis of native (containing 14N) and labelled (with 98+ atom % 15N urea (ISOTEC) as the only nitrogen source) oscillacyclamides A (A) and B (B). Comparison of the mass spectra shows that the 15N labeling increased the mass of protonated oscillacyclamide A by 20.9 mass units and oscillacyclamide B by 29 mass units which corresponds to 21 and 29 N atoms in oscillacyclamide A and B, respectively. The table analysis shows that fully (m/z 1917.5) and partially 15N labeled oscillacyclamide A[M+H]+ peak heights match excellently to the heights of the peaks calculated by assuming 97.7 % labeling degree.
6
Figure S3. LC-ITMS analysis of native (containing 32S) and labelled (with 90 atom % 34S MgSO4 (ICON) as the only sulphur source) oscillacyclamide A and B. Comparison of the mass spectra shows that the 34S labeling increased the mass of protonated oscillacyclamides by 5.9 – 6.0 mass units which corresponds to 3 S atoms in both oscillacyclamides. The column chart shows that fully (m/z 1902.6) and partially 15N labeled oscillacyclamide A [M+H]+ and their isotope peaks heights match the heights of the peaks calculated by assuming 90 % labeling degree.
7
Figure S4: Map of OscGmac construct in pJexpress 411 (Atum, USA). Codon Optimised DNA sequence of OscGmac ATGCACCATCACCACCATCATGAGAATCTGTATTTCCAGGGTTCCGGCGAGCAAGAGTCACGTGAGGCTGGCGAGCAAGGCTCTATTGAGAGCAAGGGTAGCACCAGCTTCACCAGCAGCAACTTAGTGGTCCCGAATCTGACGAATTTCATTACGCCGAGCGCGGCACCGATGAACAACGCAGACAAACTGCCTGGTCTGTATGATCTGTGGGCGCATACCAAAGGCGACCACGAAATCACGATTGTTATCCTGGATGGTAACGCCGACCTGGAGCGTAGCTGCTTTCAGGGTGCGAATATCAGCAAGATTTTTCCGTACTGGCATGAAACCCCGGAACCGATCGCGTTGGAGTACTATGAAGCATTTCTGGAAATCGAGAAATCCGGTGAGAAAGGTGAGGCGAAAGCGAAAAAGTTGCAAGCGGCCGTGCCGGAAGCGATTCTGAATCGCCTGAAGGGTAACTTTCACGCCACCCACATCATCAGCACCATTATTGGTCAGCATGGTTCTCCGGTTCCGGGCATCGCCCCACGTTGTCGTGCGATCAATATCCCGATTAACACTACTGGTGATAACGGC
OscGmac_pJepress4115121 bps
1000
20003000
4000
5000
AflIIPasI
PsiIDraI
XbaI
BglIIXhoI
HindIIIStyI
BsmBIBsu36I
SrfISmaIXmaI
AsiSI
AscI
AclI
BclI
HpaI
pUC_ori
T7 promotor
Tagged OscGmac
T7 terminator
Kan resistance
lacI
8
GAGTTCATTAGCCCTATCAATCTGACCCGTGCGTTCGAGCTGGCCATGAAACTGGGCGCGAACATCATTCACTGCGCTGCTTGTTGTGCGACCCAGACGGGCATTGCACACGATTTGCTGGCACGCGCGGTTAAGAACTGCCAAGATAACAATATCCTGATCGTGGCGCCGACCGGTAACGACAAAGGTGAGTGCTGGTGCATTCCGGCCATTCTGCCGGGTGTCCTGGGTGCCGGCATGATGAAAGACAACGGTAAACCGGCGAATTACAGCAATTGGGGTGGCAATTACCAGCACGACGGCATTCTGGCACCGGGCGAGAATATTCTGGGTGCGCAGCCAACGACCGAAGAAACCAAGCTGAGCCAAGGTACGTCGTGCGCAGCACCGATCGTCACGGGTGTTAGCGCATTGTTCCTGTCCCTGCAACTGCAGCGTGGCGAGAAGCCAAATGCTGAAGCTGTGCGCCAGGCAATCCTGAACAGCGCGATCCCGTGTGATCCGGAAGAAATTGAAGAACCGGAGCGTTGTTTGCGCGGCAAGCTGAACATTCCGGGTGCGTACCAACTGCTCACCGGTAAGTAA
His-Tagged OscGmac
MHHHHHHENLYFQGSGEQESREAGEQGSIESKGSTSFTSSNLVVPNLTNFITPSAAPMNNADKLPGLYDLWAHTKGDHEITIVILDGNADLERSCFQGANISKIFPYWHETPEPIALEYYEAFLEIEKSGEKGEAKAKKLQAAVPEAIL*NRLKGNFHATHIISTIIGQHGSPVPGIAPRCRAINIPINTTGDNGEFISPINLTRAFELAMKLGANIIHCAACCATQTGIAHDLLARAVKNCQDNNILIVAPTGNDKGECWCIPAILPGVLGAGMMKDNGKPANYSNWGGNYQHDGILAPGENILGAQPTTEETKLSQGTSCAAPIVTGVSALFLSLQLQRGEKPNAEAVRQAILNSAIPCDPEEIEEPERCLRGKLNIPGAYQLLTGK
*Underlined sequence in red is the double helix insertion
9
Figure S5: SDS PAGE gel showing the purified OscGmac protein against SeeBlue® Plus2 Pre-Stained protein standard (left lane)
188
98
62
49
38
28 17
14
6
kDa OscGmac
10
Figure S6: HR LCMS of the peptide substrate H-Asn-Glu-Phe-Met-Gln-Thr-Gly-Ser-Tyr-Ser-Gly-Pro-Ala-Tyr-Asp-Gly-OH (1).
MS53 - Substrate 29_Ctrl #1193-1232 RT: 14.73-15.16 AV: 20 NL: 1.90E6F: FTMS + p ESI Full ms [200.00-1800.00]
840 850 860 870 880 890 900 910m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
862.3497z=2
C 74 H 104 O 28 N 18 S = 862.348932.0 RDBE0.9681 ppm
905.8653z=2
873.3391z=2
C 74 H 126 O 28 N 18 S = 873.434921.0 RDBE
-109.7829 ppm
890.8600z=2 914.3783
z=2897.8670z=2
851.4371z=?
C 74 H 126 O 27 N 16 S = 851.434420.0 RDBE3.1091 ppm
NH2
O
H2N
OHN
O
OHO
NH
O
HN
O
S
NH
O
OH2N
HN
O
OH
NH
O
HN
O
OH
NH
O
OH
HN
O
OH
NH
O
N
O
NH
OHN
O
OH
NHO
O
HOHN
O
HO
11
Figure S7: HR LCMS of the cyclic peptide product cyclo-[Asn-Glu-Phe-Met-Gln-Thr-Gly-Ser-Tyr-Ser-Gly-Pro-] (1) generated with OscGmac.
MS53 - Substrate 29_OscG #1344-1374 RT: 16.49-16.85 AV: 16 NL: 5.56E4F: FTMS + p ESI Full ms [200.00-1800.00]
1296 1298 1300 1302 1304 1306 1308 1310 1312 1314m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1299.5291z=1
C 56 H 79 O 20 N 14 S = 1299.531024.5 RDBE
-1.4988 ppm
1311.5152z=1
C 56 H 91 O 20 N 14 S = 1311.624918.5 RDBE
-83.6785 ppm
1302.5382z=1
C 56 H 82 O 20 N 14 S = 1302.554523.0 RDBE
-12.5377 ppm
1304.1119z=?
[M+H]+
NH
O
NH2
O
HN
O
O
OH
NH
O
HN
O
S
HN
O O
NH2HN
O
HO
NH
O
HNO OH
NH
OHO NHOHO
HN
O N
O
12
Figure S8: HR LCMS of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψPro)-Ala-Tyr-Asp-Gly-OH (2).
UC124 #456-480 RT: 6.73-7.00 AV: 8 NL: 2.14E5F: FTMS + p ESI Full ms [150.00-2000.00]
562.5 563.0 563.5 564.0 564.5 565.0 565.5 566.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
563.2640z=2
C 51 H 74 O 17 N 12 = 563.264221.0 RDBE
-0.4105 ppm
563.7654z=2
C 51 H 75 O 17 N 12 = 563.768120.5 RDBE
-4.8081 ppm
564.2667z=2
C 51 H 76 O 17 N 12 = 564.272020.0 RDBE
-9.4661 ppm 565.2681z=2
C 51 H 78 O 17 N 12 = 565.279819.0 RDBE
-20.7386 ppm562.9929
z=?
[M+2H]2+
NH2HN N
H
HN N
HNH
HN N
O
O
O
O
O
O O
O
O NH HN
O
NH
CHN
OH
OHO2C
O
OHO
13
Figure S9: HR LCMS of the cyclic peptide product cyclo-[Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψPro)-] (2) generated with OscGmac.
BB 1 - UC124_OscG #578-609 RT: 8.57-8.97 AV: 10 NL: 3.35E4F: FTMS + p ESI Full ms [150.00-2000.00]
680 685 690 695 700 705 710 715 720 725 730 735 740m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
701.3616z=1
C 33 H 49 O 9 N 8 = 701.361713.5 RDBE
-0.1211 ppm
707.9011z=2
699.9787z=6 718.3876
z=?730.9927
z=6743.5282
z=?726.6649z=?
734.8599z=?
690.1107z=?
709.9078z=2
697.1444z=?
[M+H]+
HN NH
HN
NH
HNNH
HN
N
O
OO
O
O
OOO O
14
Figure S10: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (3).
Seq 1_Ctrl #1047-1352 RT: 5.60-6.06 AV: 28 NL: 3.45E8T: FTMS + p NSI Full ms [500.00-3500.00]
758.0 758.5 759.0 759.5 760.0 760.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8622z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE0.6003 ppm
758.8643z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-6.9256 ppm
759.3658z=2
C 71 H 104 O 22 N 15 = 759.373527.5 RDBE
-10.0799 ppm
[M+2H]2+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
15
Figure S11: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (3).
Seq 1_Ctrl #1245-5667 RT: 5.89-26.25 AV: 17 NL: 4.36E7T: Average spectrum MS2 757.86 (1245-5667)
200 400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
179.0858
1161.5999279.0978 993.5028522.2200354.1300 1292.6544878.4817771.4362
1102.58721522.8503 1696.3872
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile
16
Figure S12: HR LCMS of the cyclic peptide product Cyclo-[Dpr(Fmoc)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-] (3) generated with OscGmac.
Seq1-OscG #699-731 RT: 10.04-10.46 AV: 11 NL: 7.94E4F: FTMS + p ESI Full ms [150.00-2000.00]
1090 1091 1092 1093 1094 1095m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1090.5556z=1
C 53 H 76 O 14 N 11 = 1090.556821.5 RDBE
-1.0461 ppm
1091.5590z=1
C 53 H 77 O 14 N 11 = 1091.564621.0 RDBE
-5.0861 ppm
1092.5615z=1
C 53 H 78 O 14 N 11 = 1092.572420.5 RDBE
-10.0273 ppm 1095.4556z=?
C 53 H 81 O 14 N 11 = 1095.595919.0 RDBE
-128.0437 ppm1094.0727
z=?
HNHN
NH
O
O NH
O
HN
O
NH HN
O
HN O
O
O
OH
NO
ONH2
H2N
HO
O
O
17
Figure S13: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (4).
OC1 - 2A_Ctrl #577-613 RT: 8.18-8.64 AV: 13 NL: 2.73E7F: FTMS + p ESI Full ms [150.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8618z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE0.0542 ppm
758.8628z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-8.9538 ppm759.3646
z=2C 71 H 104 O 22 N 15 = 759.3735
27.5 RDBE-11.6174 ppm
760.3821z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE1.0630 ppm
[M+2H]2+ NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
18
Figure S14: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (4).
CC9 - 2A Ctrl #896-898 RT: 8.12-8.14 AV: 2 NL: 2.48E5T: Average spectrum MS2 757.86 (896-898)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
993.5027
522.2175
720.3444221.8171
662.8304878.4706 1161.5913
447.1864294.5205
1292.6465637.2433354.1295 1420.31951102.5854
1482.9946771.4312
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Asp
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile
Fmoc-Ala(Amino)-Ser-Lys-Leu
Fmoc-Ala(Amino)-Ser-Lys
19
Figure S15: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (5).
OC2 - 2B_Ctrl #561-603 RT: 8.08-8.57 AV: 14 NL: 2.39E7F: FTMS + p ESI Full ms [150.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5 761.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8620z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE0.2917 ppm
758.8625z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-9.2481 ppm759.3641
z=2C 71 H 104 O 22 N 15 = 759.3735
27.5 RDBE-12.2988 ppm
760.3762z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE
-6.7548 ppm757.7420
z=2
[M+2H]2+ NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
20
Figure S16: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (5).
seq 2B_Ctrl #557-566 RT: 8.18-8.30 AV: 4 NL: 1.71E7T: Average spectrum MS2 757.86 (557-566)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
993.5020
522.2184
878.4745
720.3444447.1868 637.2460
221.4997 1161.5931
1102.5880354.1295 1324.6594765.3889 1521.8564
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Asp
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile
Fmoc-Ala(Amino)-Ser-Lys
21
Figure S17: HR LCMS of the cyclic peptide product cyclo-[Dpr(Fmoc)-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-] (5) generated with OscGmac.
OC2 - 2B_OscG #681-760 RT: 9.70-10.79 AV: 27 NL: 9.41E5F: FTMS + p ESI Full ms [150.00-2000.00]
1090 1091 1092 1093 1094 1095m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1090.5561z=1
C 53 H 76 O 14 N 11 = 1090.556821.5 RDBE
-0.5908 ppm
1091.5594z=1
C 48 H 87 O 25 N 2 = 1091.55926.5 RDBE
0.1297 ppm
1092.5627z=1
C 42 H 82 O 22 N 11 = 1092.56307.5 RDBE
-0.3470 ppm 1094.5689z=1
C 45 H 86 O 24 N 6 = 1094.56886.0 RDBE
0.0769 ppm
[M+H]1+
HNHN
NH
O
O NH
O
HN
O
NH HN
O
HN O
O
O
OH
NO
ONH2
H2N
HO
O
O
22
Figure S18: MS MS of the cyclic peptide product cyclo-[Dpr(Fmoc)-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-] (5).
OC2 - 2B_OscG #701-704 RT: 9.98-10.02 AV: 2 NL: 6.41E4T: Average spectrum MS2 1090.56 (701-704)
300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
332.0910
975.5307
862.4379716.3768562.9697
621.3116 809.6340487.8616360.3999 702.51631087.5698894.4742 998.5551734.3773469.6604
Dpr(Fmoc)-Ser-D-Lys-Leu-Gln-Ile
Dpr(Fmoc)-Ser-D-Lys-Leu-Gln
Dpr(Fmoc)-Ser-D-Lys-Leu
Dpr(Fmoc)-Ser-D-Lys
23
Figure S19: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (6).
CC11 - 2C Ctrl #586 RT: 8.30 AV: 1 NL: 6.83E6F: FTMS + p ESI Full ms [150.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8615z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE
-0.3708 ppm
758.8636z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-7.8668 ppm
759.3647z=2
C 71 H 104 O 22 N 15 = 759.373527.5 RDBE
-11.5672 ppm
760.3685z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE
-16.8662 ppm757.7532
z=?
[M+2H]2+ NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
24
Figure S20: MS MS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (6).
Seq 2C_Ctrl #566-806 RT: 8.29-11.74 AV: 8 NL: 8.84E6T: Average spectrum MS2 757.86 (566-806)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
ndan
ce993.5027
522.2188
878.4753
720.3450637.2462
447.1871221.4996
1161.5941
294.4046 1292.64681102.58801324.6594765.3922
Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-Asp-Pro-Ala
Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-Asp
Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile
Fmoc-Ala(Amino)-D-Ser-Lys-Leu
25
Figure S21: HR LCMS of the cyclic peptide product cyclo-[Dpr(Fmoc)-D-Ser-Lys-Leu-Gln-Ile-Asp-Pro-] (6) generated with OscGmac.
OC3 - 2C_OscG #623-672 RT: 8.89-9.51 AV: 16 NL: 4.46E5F: FTMS + p ESI Full ms [150.00-2000.00]
1090 1091 1092 1093 1094 1095 1096m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1090.5561z=1
C 53 H 76 O 14 N 11 = 1090.556821.5 RDBE
-0.6620 ppm
1091.5593z=1
C 53 H 77 O 14 N 11 = 1091.564621.0 RDBE
-4.8636 ppm
1092.5623z=1
C 53 H 78 O 14 N 11 = 1092.572420.5 RDBE
-9.2955 ppm
1092.1223z=?
1094.5676z=1
C 53 H 80 O 14 N 11 = 1094.588119.5 RDBE
-18.7466 ppm1089.9906
z=1
[M+H]1+
HNHN
NH
O
O NH
O
HN
O
NH HN
O
HN O
O
O
OH
NO
ONH2
H2N
HO
O
O
26
Figure S22: MS MS of the cyclic peptide product cyclo-[Dpr(Fmoc)-D-Ser-Lys-Leu-Gln-Ile-Asp-Pro-] (6).
OC3 - 2C_OscG #638-641 RT: 9.07-9.11 AV: 2 NL: 4.66E4T: Average spectrum MS2 1090.56 (638-641)
300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
975.5305862.4471
894.4691
849.4132
734.3864332.0777
1055.5184
960.4486
603.2981831.4012706.3918487.8415
360.3909 782.2906456.4320
Dpr(Fmoc)-D-Ser-Lys-Leu-Gln-Ile
Dpr(Fmoc)-D-Ser-Lys-Leu-Gln
Dpr(Fmoc)-D-Ser-Lys-Leu
27
Figure S23: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (7).
CC12 - 2D Ctrl #558-616 RT: 7.85-8.58 AV: 20 NL: 2.93E7F: FTMS + p ESI Full ms [150.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5 761.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8615z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE
-0.3562 ppm
758.8625z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-9.2742 ppm759.3643
z=2C 71 H 104 O 22 N 15 = 759.3735
27.5 RDBE-12.1272 ppm
760.3685z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE
-16.8244 ppm
[M+2H]2+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
28
Figure S24: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (7).
Seq 2D_Ctrl #554-557 RT: 8.12-8.17 AV: 2 NL: 2.98E6T: Average spectrum MS2 757.86 (554-557)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
993.5037
522.2195
720.3457
662.8326 1161.5956447.1875 878.4726
221.5126 637.2471 1102.5895354.1297 1292.6498279.0980 765.3922 1324.6635
Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala
Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile-D-Asp
Fmoc-Ala(Amino)-Ser-D-Lys-Leu-Gln-Ile
29
Figure S25: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (8).
CC13 - 2E Ctrl #610 RT: 8.53 AV: 1 NL: 2.42E7F: FTMS + p ESI Full ms [150.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8615z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE
-0.3708 ppm
758.8628z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-8.9124 ppm759.3647
z=2C 71 H 104 O 22 N 15 = 759.3735
27.5 RDBE-11.4868 ppm
760.3821z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE1.1143 ppm
[M+2H]2+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
30
Figure S26: MS MS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (8).
CC13 - 2E Ctrl #587-593 RT: 8.24-8.31 AV: 3 NL: 2.76E6T: Average spectrum MS2 757.86 (587-593)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
ndan
ce522.2176
720.3443
221.6413
993.5024447.1863 662.8307
294.5174
637.2440354.1288878.4717 1162.5955
1293.6504765.3937 1103.5916 1419.9498
Fmoc-Ala(Amino)-D-Ser-Lys-Leu Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln
Fmoc-Ala(Amino)-D-Ser-Lys-Leu-Gln-Ile-D-Asp
31
Figure S27: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (9).
Seq 2F_Ctrl #589 RT: 8.61 AV: 1 NL: 2.23E5F: FTMS + p ESI Full ms [100.00-2000.00]
1514 1515 1516 1517 1518 1519m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1514.7163z=1
C 71 H 100 O 22 N 15 = 1514.716229.5 RDBE0.0811 ppm
1516.7219z=1
C 71 H 102 O 22 N 15 = 1516.731828.5 RDBE
-6.5352 ppm
[M+H]1+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
32
Figure S28: MS MS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-Ala-Tyr-Asp-Gly-OH (9).
Seq 2F_Ctrl #554-770 RT: 8.12-11.24 AV: 5 NL: 1.41E7T: Average spectrum MS2 757.86 (554-770)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
993.5018
522.2183
878.4745
662.8310447.1868
1161.5929221.5134294.4122 1102.5887 1324.6576765.3898 1521.8634
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-Asp
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-Ala
33
Figure S29: HR LCMS of the cyclic peptide product cyclo-[Dpr(Fmoc)-D-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-] (9) generated with OscGmac.
OC6 - 2F_OscG #653-685 RT: 9.29-9.70 AV: 11 NL: 9.94E5F: FTMS + p ESI Full ms [150.00-2000.00]
1090 1091 1092 1093 1094 1095m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1090.5561z=1
C 53 H 76 O 14 N 11 = 1090.556821.5 RDBE
-0.5982 ppm
1091.5594z=1
C 53 H 77 O 14 N 11 = 1091.564621.0 RDBE
-4.7348 ppm
1092.5626z=1
C 53 H 78 O 14 N 11 = 1092.572420.5 RDBE
-9.0292 ppm
1092.1229z=1
1094.5685z=1
C 53 H 80 O 14 N 11 = 1094.588119.5 RDBE
-17.8422 ppm1093.1335
z=11091.0568
z=?
[M+H]1+
HNHN
NH
O
O NH
O
HN
O
NH HN
O
HN O
O
O
OH
NO
ONH2
H2N
HO
O
O
34
Figure S30: MS MS of the cyclic peptide product cyclo-[Dpr(Fmoc)-D-Ser-D-Lys-Leu-Gln-Ile-Asp-Pro-] (9).
OC6 - 2F_OscG #662-665 RT: 9.38-9.42 AV: 2 NL: 7.46E4T: Average spectrum MS2 1090.56 (662-665)
300 400 500 600 700 800 900 1000 1100m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
975.5294
716.3751
621.3030862.4443
1055.5173
849.4114332.0976
894.4683734.3872 957.5203
562.8968489.2090360.3556 699.1213
Dpr(Fmoc)-D-Ser-D-Lys-Leu-Gln-Ile
Dpr(Fmoc)-D-Ser-D-Lys-Leu-Gln
Dpr(Fmoc)-D-Ser-D-Lys-Leu
Dpr(Fmoc)-D-Ser-D-Lys
35
Figure S31: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Ala-Pro-Ala-Tyr-Asp-Gly-OH (10).
CC15 - 2G Ctrl #572-604 RT: 8.11-8.49 AV: 11 NL: 1.82E7F: FTMS + p ESI Full ms [150.00-2000.00]
735.0 735.5 736.0 736.5 737.0 737.5 738.0 738.5 739.0 739.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
735.8666z=2
C 70 H 101 O 20 N 15 = 735.866828.0 RDBE
-0.2708 ppm
737.3696z=2
C 70 H 104 O 20 N 15 = 737.378626.5 RDBE
-12.1767 ppm737.8711
z=2C 70 H 105 O 20 N 15 = 737.8825
26.0 RDBE-15.4649 ppm
[M+2H]2+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
ON
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
36
Figure S32: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Ala-Pro-Ala-Tyr-Asp-Gly-OH (10).
CC15 - 2G Ctrl #578-581 RT: 8.17-8.20 AV: 2 NL: 3.62E5T: Average spectrum MS2 735.87 (578-581)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
950.5162
522.2180 879.4783
698.8513593.2555221.5731
447.1865294.5416 1118.6067
765.3924 1249.6573 1420.2074354.1292 1059.5992
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-Ala
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln
37
Figure S33: HR LCMS of the cyclic peptide product cyclo-[Dpr(Fmoc)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-] (10) generated with OscGmac.
OC7 - 2G_OscG #739-791 RT: 10.53-11.22 AV: 18 NL: 1.06E6F: FTMS + p ESI Full ms [150.00-2000.00]
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1046.5665z=1
C 52 H 76 O 12 N 11 = 1046.566920.5 RDBE
-0.3898 ppm523.7868z=2
367.5003z=3
1814.5260z=?
1420.4048z=?1159.6506
z=11610.9888
z=?
[M+H]1+
[M+2H]2+
NHNH
HN
O
OHN
O
NHO
HNNH
O
NHOO
NO
OH2N
NH2
OH
O
O
38
Figure S34: MS MS of the cyclic peptide product cyclo-[Dpr(Fmoc)-Ser-Lys-Leu-Gln-Ile-Asp-Pro-] (10).
OC7 - 2G_OscG #749-752 RT: 10.67-10.70 AV: 2 NL: 1.17E5T: Average spectrum MS2 523.79 (749-752)
200 300 400 500 600 700 800 900 1000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
221.6711
202.1832
294.4987868.4899
824.4958332.0770
621.2999562.8683412.7483 1001.1237933.4920806.4918
Dpr(Fmoc)-Ser-Lys-Leu-Gln
39
Figure S35: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Ala-Pro-Ala-Tyr-Asp-Gly-OH (11).
CC16 - 2H Ctrl #586-616 RT: 8.31-8.70 AV: 11 NL: 1.18E7F: FTMS + p ESI Full ms [150.00-2000.00]
735.5 736.0 736.5 737.0 737.5 738.0 738.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
735.8667z=2
C 70 H 101 O 20 N 15 = 735.866828.0 RDBE
-0.2101 ppm
736.8677z=2
C 70 H 103 O 20 N 15 = 736.874627.0 RDBE
-9.4626 ppm737.3693
z=2C 70 H 104 O 20 N 15 = 737.3786
26.5 RDBE-12.5104 ppm
736.7347z=2
737.2356z=2
738.3043z=2
C 70 H 106 O 20 N 15 = 738.386425.5 RDBE
-111.1791 ppm736.2658z=?
[M+2H]2+NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
ON
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
40
Figure S36: MS MS of the peptide substrate Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Ala-Pro-Ala-Tyr-Asp-Gly-OH (11).
CC16 - 2H Ctrl #602-605 RT: 8.52-8.56 AV: 2 NL: 2.33E5T: Average spectrum MS2 735.87 (602-605)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
949.5127
522.2184
221.5348
698.35081117.6006
294.5863 878.4775 1248.6588447.1872593.2562354.1299 1419.99741058.5968
1280.6702
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Ala-Pro-Ala
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile-D-Ala
Fmoc-Ala(Amino)-Ser-Lys-Leu-Gln-Ile
41
Figure S37: HR LCMS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (12).
seq 2 Fmoc_Ctrl #625 RT: 8.19 AV: 1 NL: 1.47E8F: FTMS + p ESI Full ms [100.00-2000.00]
757.5 758.0 758.5 759.0 759.5 760.0 760.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
757.8635z=2
C 71 H 101 O 22 N 15 = 757.861729.0 RDBE2.2869 ppm
758.8631z=2
C 71 H 103 O 22 N 15 = 758.869628.0 RDBE
-8.5102 ppm759.3641
z=2C 71 H 104 O 22 N 15 = 759.3735
27.5 RDBE-12.3709 ppm
760.3882z=2
C 71 H 106 O 22 N 15 = 760.381326.5 RDBE9.0609 ppm
[M+2H]2+ NH
H2N
HN
O
ONH
ONH
OHN
HNO
NH
O
O
O
HO
N
HN
O
O NH
HN
O
HO
O O
HN
O OH
O
NH2
HO
NH2
OHO
O
42
Figure S38: MS MS of the peptide substrate Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala-Tyr-Asp-Gly-OH (12).
seq 2 Fmoc_Ctrl #617-626 RT: 8.09-8.20 AV: 4 NL: 6.16E6T: Average spectrum MS2 757.86 (617-626)
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
522.2188
993.5036
720.3460662.8319
221.3861447.1877
1161.5945878.4735
294.3045 637.2459
1102.5908 1292.6499 1417.4858765.3930
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-D-Asp-Pro-Ala
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile-D-Asp
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu
Fmoc-Ala(Amino)-D-Ser-D-Lys-Leu-Gln-Ile
43
Figure S39: HR LCMS of the peptide substrate H-His-Gln-Trp-Gly-Trp-Leu-Val-Gly-Gly-Thr-Asp-Pro-Phe-Ala-Gly-Asp-OH (13).
MS56 - Substrate PirG 1_Ctrl #1408-1451 RT: 17.33-17.80 AV: 22 NL: 9.38E6F: FTMS + p ESI Full ms [200.00-1800.00]
871.5 872.0 872.5 873.0 873.5 874.0 874.5 875.0 875.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
871.9001z=2
C 81 H 109 O 23 N 21 = 871.899738.0 RDBE0.5025 ppm
872.9008z=2
C 81 H 111 O 23 N 21 = 872.907537.0 RDBE
-7.6848 ppm
873.4023z=2
C 81 H 112 O 23 N 21 = 873.411436.5 RDBE
-10.4839 ppm 874.9353z=2
C 81 H 115 O 23 N 21 = 874.923235.0 RDBE
13.8799 ppm
[M+2H]2+
NH2
ONH
NHN
O
OH2N
NH O
HN
HN
O
NH O
NH
HN
O
NH O
HN
O
NH O
HN
O
HO
NH
O
O
OH
NO
HN
O NH
O
NH
O
HN
O
O
OHOH
44
Figure S40: HR LCMS of the cyclic peptide product cyclo-[His-Gln-Trp-Gly-Trp-Leu-Val-Gly-Gly-Thr-Asp-Pro-] (13) generated with OscGmac.
MS56 - Substrate PirG 1_OscG #1550-1571 RT: 18.97-19.20 AV: 11 NL: 1.06E5F: FTMS + p ESI Full ms [200.00-1800.00]
667.5 668.0 668.5 669.0 669.5 670.0 670.5 671.0 671.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
667.8173z=2
C 63 H 85 O 16 N 17 = 667.817530.0 RDBE
-0.2367 ppm
668.3189z=2
C 63 H 86 O 16 N 17 = 668.321429.5 RDBE
-3.6394 ppm
668.8202z=2
C 63 H 87 O 16 N 17 = 668.825329.0 RDBE
-7.6635 ppm 670.3160z=2
C 63 H 90 O 16 N 17 = 670.337027.5 RDBE
-31.3133 ppm
[M+2H]2+
NH
O
HN
N HN
O O
NH2HN
O
HN
NH
O
HNOHN
NH
O NHO
HN
OHN
O
NH
O
HO
HN
O
O OH
NO
45
Figure S41: HR LCMS of the peptide substrate H-Asp-Trp-Gly-Thr-Phe-Cys-Val-Gln-Glu-Asp-Gly-Glu-Gly-Asn-Cys-Lys-Glu-Trp-Tyr-Glu-Leu-Pro-Phe-Ala-Gly-Asp-Asp-Ala-Glu-OH (14).
MS57 - Substrate PirG 2_Ctrl #1397-1433 RT: 17.25-17.69 AV: 19 NL: 4.15E3F: FTMS + p ESI Full ms [200.00-1800.00]
1654.0 1654.5 1655.0 1655.5 1656.0 1656.5 1657.0 1657.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1655.1573z=2
C 145 H 197 O 52 N 34 S 2 = 1655.162364.5 RDBE
-3.0288 ppm
1654.6546z=2
C 145 H 196 O 52 N 34 S 2 = 1654.658465.0 RDBE
-2.3241 ppm
1656.6575z=2
C 145 H 200 O 52 N 34 S 2 = 1656.674163.0 RDBE
-9.9684 ppm
1654.1708z=?
[M+2H]2+
NH2
O
O
HOHN
O
HN
NHO
NH
O
HO
NHO
NH
O
S
HNO
NHO
ONH2
NH
O
O
HO
NH O
OOHHN
ONH
O
OHO
HNO
HN
ONH2
OHN
OS
NH O
H2N
HN
O
OOH
NH O
NH
HN
OOH
NHO
O
HOHN
ON
O
HN
O
NH
O
HN
O
NH
O
O
OHHN
O
O
OHNH
O
HN
O
OHO
OH
46
Figure S42: HR LCMS of the cyclic peptide product cyclo-[Asp-Trp-Gly-Thr-Phe-Cys-Val-Gln-Glu-Asp-Gly-Glu-Gly-Asn-Cys-Lys-Glu-Trp-Tyr-Glu-Leu-Pro-] (disulfide bond) (14) generated with OscGmac.
NHO
O OHNH
OHN HNO
HN
O
OH
NHO
HN
O
S
NH
O
HN
O
OH2N
NH
O
OOH
HN
O
O
OHNH
O NHO
O
OH
HN
ONH
O
H2NO NH
O
S
HNO
H2N
NH
O
O
OH
HN
O
HN
NH
O
HO
HN
O
OHO
NH
O
N
O
47
Figure S43: HR LCMS of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Pro-Ala-Tyr-Asp-OH (15).
ES01_Ctrl #450-503 RT: 6.62-7.26 AV: 18 NL: 8.62E7F: FTMS + p ESI Full ms [150.00-2000.00]
1066 1067 1068 1069 1070m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1066.5200z=1
C 50 H 72 O 15 N 11 = 1066.520420.5 RDBE
-0.4065 ppm
1067.5220z=1
C 50 H 73 O 15 N 11 = 1067.528220.0 RDBE
-5.8200 ppm
1068.5248z=1
C 50 H 74 O 15 N 11 = 1068.536019.5 RDBE
-10.4718 ppm
1067.0225z=1 1068.0320
z=1
1070.5316z=1
C 50 H 76 O 15 N 11 = 1070.551718.5 RDBE
-18.7752 ppm1066.0070
z=21069.0511
z=1
[M+H]1+
NH2
O
HN
O
NH O
HN
O
NH O
NHO
HNO
N
O NH
O
HN
O
OH
NH O
O
OHOH
48
Figure S44: HR LCMS of the cyclic peptide product cyclo-[Val-Gly-Ala-Gly-Ile-Gly-Phe-Pro-] (15) generated with OscGmac.
ES01_OscG #600-622 RT: 8.65-8.95 AV: 8 NL: 2.29E6F: FTMS + p ESI Full ms [150.00-2000.00]
697 698 699 700 701 702 703 704m/z
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
Inte
nsity
699.3832z=1
C 34 H 51 O 8 N 8 = 699.382413.5 RDBE1.1160 ppm
700.3862z=1
C 34 H 52 O 8 N 8 = 700.390313.0 RDBE
-5.8174 ppm
701.3891z=1
C 34 H 53 O 8 N 8 = 701.398112.5 RDBE
-12.8685 ppm700.1889z=2
698.5780z=?
C 33 H 76 O 8 N 7 = 698.5750-0.5 RDBE4.2753 ppm
703.8726z=?
[M+H]1+
NHO
HNO
HN
O
NHO
HN
ONHO
HN
O
N
O
49
Figure S45: HR LCMS of the peptide substrate H-Val-Ala-Gly-Ile-Gly-Phe-Pro-Ala-Tyr-Asp-OH (16).
ES01_2nd_Ctrl #469-496 RT: 6.85-7.18 AV: 10 NL: 1.95E6F: FTMS + p ESI Full ms [150.00-2000.00]
1008.5 1009.0 1009.5 1010.0 1010.5 1011.0 1011.5 1012.0 1012.5 1013.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1009.4997z=1
C 48 H 69 O 14 N 10 = 1009.498919.5 RDBE0.8082 ppm
1010.5028z=1
C 48 H 70 O 14 N 10 = 1010.506719.0 RDBE
-3.8817 ppm
1011.5056z=1
C 48 H 71 O 14 N 10 = 1011.514618.5 RDBE
-8.9011 ppm
1009.1034z=?
1009.9727z=?
[M+H]1+NH2
O
HN
O
NH
O
HN
O
NH
O
NH
O
N
ONH
O
HN
O
OH
NH O
OOH
OH
50
Figure S46: HR LCMS of the cyclic peptide product cyclo-[Val-Ala-Gly-Ile-Gly-Phe-Pro-] (16) generated with OscGmac.
ES01_2nd_OscG #642-671 RT: 9.26-9.66 AV: 10 NL: 4.02E4F: FTMS + p ESI Full ms [150.00-2000.00]
640.5 641.0 641.5 642.0 642.5 643.0 643.5 644.0 644.5 645.0 645.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
642.3607z=1
C 32 H 48 O 7 N 7 = 642.361012.5 RDBE
-0.4555 ppm
643.3640z=1
C 32 H 49 O 7 N 7 = 643.368812.0 RDBE
-7.4537 ppm
641.7801z=?
640.8255z=?
642.1423z=?
644.1821z=?
645.4148z=?
C 32 H 51 O 7 N 7 = 645.384411.0 RDBE
46.9969 ppm
[M+H]1+
NHO
NH
O
HN
O
NH
O NHO
NH
O
N
O
51
Figure S47: HR LCMS of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψMe,MePro)-Ala-Tyr-Asp-Gly-OH (17).
FE09 #730-836 RT: 11.90-13.20 AV: 35 NL: 1.48E8F: FTMS + p ESI Full ms [100.00-2000.00]
1120 1140 1160 1180 1200 1220 1240m/z
0
20000000
40000000
60000000
80000000
100000000
120000000
140000000
Inte
nsity
1153.5526z=1
C53 H77 O17 N12 = 1153.552421.5 RDBE0.1903 ppm
1175.5334z=1 1210.5734
z=11224.5895
z=11252.6215
z=1
HN
NH
HN
NH
HN
NH
H2NO
O
O
O
O
ON
O
O
O NH
HN
NH
O
OH
OHOOC O
HN COOH
52
Figure S48: MS MS of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψMe,MePro)-Ala-Tyr-Asp-Gly-OH (17).
FE09 #748-1027 RT: 12.14-16.08 AV: 4 NL: 3.01E4T: Average spectrum MS2 1153.55 (748-1027)
400 500 600 700 800 900 1000 1100m/z
0
5000
10000
15000
20000
25000
30000
Inte
nsity
1095.5105
782.4186
905.4507642.3607495.2921 963.4921
800.4312 1020.4795614.3662438.2708887.4407323.7482
H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψMe,MePro)-Ala-Tyr
H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Ser(ψMe,MePro)-Ala
53
Figure S49: HR LCMS of the peptide substrate H-Val- Ala-Gly-Ile-Gly-Phe-Cys(ψPro)-Ala-Tyr-Asp-OH (18).
AB22 - UC127_Ctrl #1357-1385 RT: 16.79-17.12 AV: 15 NL: 4.10E6F: FTMS + p ESI Full ms [200.00-1800.00]
1027 1028 1029 1030 1031 1032m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1027.4562z=1
C 47 H 67 O 14 N 10 S = 1027.455319.5 RDBE0.7934 ppm
1028.4587z=1
C 47 H 68 O 14 N 10 S = 1028.463219.0 RDBE
-4.3620 ppm
1029.4593z=1
C 47 H 69 O 14 N 10 S = 1029.471018.5 RDBE
-11.3221 ppm
1028.8922z=1
1027.0239z=2
1031.7286z=2
C 47 H 101 O 13 N 9 S = 1031.72342.0 RDBE
5.0494 ppm1027.9342
z=?
[M+H]+
NH2
ONH O
HN
ONH O
HN
ONH
O
NS
ONH
O
HN
O
OH
NH O
OOHOH
54
Figure S50: HR LCMS of the cyclic peptide product cyclo-[Val- Ala-Gly-Ile-Gly-Phe-Cys(ψPro)-] (18) generated with OscGmac.
AB 3 - UC127_OscG (should be UC129_OscG) #1776-1822 RT: 21.70-22.26 AV: 24 NL: 3.50E5F: FTMS + p ESI Full ms [200.00-1800.00]
660.0 660.5 661.0 661.5 662.0 662.5 663.0 663.5m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
660.3174z=1
C 31 H 46 O 7 N 7 S = 660.317412.5 RDBE
-0.0591 ppm
661.3208z=1
C 31 H 47 O 7 N 7 S = 661.325212.0 RDBE
-6.7507 ppm662.3233
z=1C 31 H 48 O 7 N 7 S = 662.3330
11.5 RDBE-14.7394 ppm661.0554
z=2660.0716
z=?660.5838
z=2
[M+H]+
HN
ONHO
NH
O
HN
O
NH
O NH
O
N
S
O
55
Figure S51: HR LCMS of the peptide substrate H-Gly-Val-Cys-Gly-Glu-Thr-Cys-Val-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Gly-Cys-Thr-Cys-Ser-Trp-Pro-Val-Cys-Thr-Arg-Asn-Gly-Leu-Pro-Ala-Tyr-Asp-Gly-Glu-Leu-Glu-His-His-His-His-His-His-OH (two disulfide bonds) (19).
AG102 #411-447 RT: 6.03-6.46 AV: 12 NL: 1.96E6F: FTMS + p ESI Full ms [150.00-2000.00]
913 914 915 916 917m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
914.5758z=5
C 189 H 281 O 61 N 61 S 6 = 914.581280.0 RDBE
-5.8246 ppm
915.7766z=5
C 189 H 287 O 61 N 61 S 6 = 915.790677.0 RDBE
-15.2764 ppm
913.1729z=5
C 189 H 274 O 61 N 61 S 6 = 913.170283.5 RDBE2.9336 ppm
56
Figure S52: HR LCMS of the cyclic peptide product Cyclo-[Gly-Val-Cys-Gly-Glu-Thr-Cys-Val-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Gly-Cys-Thr-Cys-Ser-Trp-Pro-Val-Cys-Thr-Arg-Asn-Gly-Leu-Pro-] (two disulfide bonds) (19) generated with OscGmac.
AG104 #585-636 RT: 8.42-9.04 AV: 17 NL: 1.56E5F: FTMS + p ESI Full ms [150.00-2000.00]
1474 1475 1476 1477 1478m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
1475.5904z=2
C 119 H 186 O 40 N 36 S 6 = 1475.597045.0 RDBE
-4.4758 ppm
1476.5866z=2
C 119 H 188 O 40 N 36 S 6 = 1476.604844.0 RDBE
-12.3697 ppm
1474.5904z=2
C 119 H 184 O 40 N 36 S 6 = 1474.589246.0 RDBE0.8184 ppm
57
Figure S53: Chemical structure of the anti-HIV depsipeptide homophymine A.
HO NH
HN
O
NH
OH
OH
O
NH2O
OO
HN
NH2
NH
OO
NO
NH
OHO O
O
OH
OH
HN
O
ONH
NH2
O
O
O
N
HN
O
H2N
O
Homophymine A
58
Figure S54: Alignment of the cyanobactin macrocylases PatGmac, LynGmac, TenGmac, TruGmac, McaGmac, TriKmac, PirGmac and OscGmac. Sequence similarity is indicated by shading. Residues in PatGmac that are critical for macrocylization activity are in red and in green frames. Numbering on top of sequences is according to the PatGmac sequence. In PatGmac, the side chains of the bulky residues in red protrude and
Helical insertion
59
prevent the substrate from adopting the relaxed elongated conformation. In OscGmac, the corresponding residues are much smaller and this may create enough space to allow binding and processing of longer substrates.
60
Figure S55: A) Crystal structure of PatGmac (PDB: 4AKS) showing the helical insertion (magenta) and the three bulky residues Met169, Phe193 and Arg195 that protrude into the substrate binding groove and thus restrict the binding to substrates that adopt a bent-back conformation. B) Phyre 2 (Nat Protocols, 2015, 10, 845-858) Model of OscGmac showing the helical insertion (red) and the three residues Ile183, Ala207 and Cys209 that correspond to the three residues highlighted above in PatGmac.
Met169 Phe193
Arg195
Ile183 Ala207
Cys209
A B
61
Figure S56: SDS-PAGE gel showing thepurified PatGmac mutants; 1 = M169I, 2 = F193A, 3 = R195C, 4 = M169I F193A, 5 = M169I R195C, 6 = F193A R195C, 7 = M169I F193A R195C against SeeBlue® Plus2 Pre-Stained protein standard (left lane).
188
98
62
49
38
28 17
14
6
kDa 1 2 3 4 5 6 7
62
Figure S57: HR LCMS of the cyclic peptide product cyclo[-Asn-Glu-Phe-Met-Gln-Thr-Gly-Ser-Tyr-Ser-Gly-Pro] (1) generated with PatGmac mutant
M169I.
PatG_M169I_Substrate29 #478-579 RT: 6.86-8.29 AV: 34 NL: 3.92E3F: FTMS + p ESI Full ms [150.00-2000.00]
1298 1299 1300 1301 1302 1303 1304m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1299.5261z=1
C 56 H 79 O 20 N 14 S = 1299.531024.5 RDBE
-3.8032 ppm
1300.5296z=1
C 56 H 80 O 20 N 14 S = 1300.538924.0 RDBE
-7.1116 ppm
1301.5307z=1
C 56 H 81 O 20 N 14 S = 1301.546723.5 RDBE
-12.2846 ppm
NH
O
NH2
O
HN
O
O
OH
NH
O
HN
O
S
HN
O O
NH2HN
O
HO
NH
O
HNO OH
NH
OHO NHOHO
HN
O N
O
63
Figure S58: Processing of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Pro-Ala-Tyr-Asp-OH (16) with PatGmac. Upper panel showing the total ion chromatogram and the lower panel is the MS data for the cyclic peptide.
G:\COMPUTER AT WORK\...\ES1_PatG_WT 11/17/16 23:57:47
RT: 0.00 - 32.01
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
7.02
6.635.133.76 12.67 31.198.78 25.23 26.1911.77 13.89 23.382.500.41 20.4116.8310.25 30.8915.54 18.24 27.9722.13
NL:3.73E7Base Peak F: FTMS + p ESI Full ms [150.00-2000.00] MS ES1_PatG_WT
ES1_PatG_WT #600-629 RT: 8.65-9.05 AV: 10 NL: 2.61E5F: FTMS + p ESI Full ms [150.00-2000.00]
698.0 698.5 699.0 699.5 700.0 700.5 701.0 701.5 702.0 702.5 703.0 703.5 704.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
699.3830z=1
C 34 H 51 O 8 N 8 = 699.382413.5 RDBE0.8061 ppm
700.3863z=1
C 34 H 52 O 8 N 8 = 700.390313.0 RDBE
-5.6256 ppm
703.8730z=?
701.3890z=1
C 34 H 53 O 8 N 8 = 701.398112.5 RDBE
-12.9691 ppm700.1895z=?
702.9436z=?
703.6428z=?
Cyclic peptide
Unprocessed substrate
[M+H]1+
NHO
HNO
HN
O
NHO
HN
ONHO
HN
O
N
O
64
Figure S59: Processing of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Pro-Ala-Tyr-Asp-OH (16) with PatGmac mutant M169I F193A. Upper panel showing the total ion chromatogram and the lower panel is the MS data for the cyclic peptide.
ES1_PatG_M169I_F193A 03/24/17 22:10:33
RT: 0.00 - 32.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
8.76
3.706.95 8.45 16.1815.01 18.00 30.4413.22 25.4619.324.182.24 6.61 23.3412.101.49 26.28 29.1222.259.54 20.56
NL: 1.36E7Base Peak F: FTMS + p ESI Full ms [150.00-2000.00] MS ES1_PatG_M169I_F193A
ES1_PatG_M169I_F193A #594-624 RT: 8.59-8.98 AV: 10 NL: 6.59E6F: FTMS + p ESI Full ms [150.00-2000.00]
696 697 698 699 700 701 702 703 704 705m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
699.3786z=1
C 34 H 51 O 8 N 8 = 699.382413.5 RDBE
-5.4506 ppm
700.3812z=1
C 34 H 52 O 8 N 8 = 700.390313.0 RDBE
-12.9773 ppm701.3841
z=1C 34 H 53 O 8 N 8 = 701.3981
12.5 RDBE-19.9633 ppm700.1845
z=2
698.5738z=2
C 33 H 76 O 8 N 7 = 698.5750-0.5 RDBE
-1.7231 ppm697.2359
z=2
703.3911z=1
C 34 H 55 O 8 N 8 = 703.413711.5 RDBE
-32.1520 ppm
Cyclic peptide
Unprocessed substrate
[M+H]1+
NHO
HNO
HN
O
NHO
HN
ONHO
HN
O
N
O
65
Figure S60: Processing of the peptide substrate H-Val-Gly-Ala-Gly-Ile-Gly-Phe-Pro-Ala-Tyr-Asp-OH (16) with PatGmac mutant M169I F193A R195C. Upper panel showing the total ion chromatogram and the lower panel is the MS data for the cyclic peptide.
E:\COMPUTER AT WORK\...\T3-ES1 08/04/2017 00:59:36
RT: 0.00 - 32.02
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
8.78
31.20
6.91 17.793.68 12.62 16.918.476.342.621.88 27.2719.4410.54 27.97 30.5222.5515.335.86 13.84 25.4121.03 24.10
NL:1.94E7Base Peak F: FTMS + p ESI Full ms [150.00-2000.00] MS T3-ES1
T3-ES1 #593-616 RT: 8.69-8.99 AV: 8 NL: 1.14E7F: FTMS + p ESI Full ms [150.00-2000.00]
697 698 699 700 701 702 703 704 705 706m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
699.3825z=1
C 34 H 51 O 8 N 8 = 699.382413.5 RDBE0.1400 ppm
700.3853z=1
C 34 H 52 O 8 N 8 = 700.390313.0 RDBE
-7.1461 ppm
702.3907z=1
C 34 H 54 O 8 N 8 = 702.405912.0 RDBE
-21.6511 ppm700.1901
z=?
698.5759z=2
C 33 H 76 O 8 N 7 = 698.5750-0.5 RDBE1.3497 ppm
700.7289z=2
697.2377z=?
706.1330z=?
705.6760z=?
704.7975z=?
Cyclic peptide
Unprocessed substrate
[M+H]1+
NHO
HNO
HN
O
NHO
HN
ONHO
HN
O
N
O
66
Table S1. Predicted function of the proteins encoded in osc gene cluster (Fig. 2) from Oscillatoria sp. PCC 6506
Protein Accession number
Length Predicted function Organism Max identity
ORF CBN56371 548 Hypothetical protein Microcystis aeruginosa PCC 9809
60
OscC CBN56372 75 Hypothetical protein Cyanothece sp. PCC 7822
55
OscB CBN56373 68 Hypothetical protein Nodularia spumigena CCY 9414
79
OscA CBN56374 659 N-terminal protease Anabaena sp. 90 75 OscE1 CBN56375 83 Precursor peptide Nostoc spongiaeforme 43 ORF CBN56376 48 Transposase Microcystis aeruginosa
T1-4 75
OscF CBN56377 310 Prenyltransferase Cyanothece sp. PCC 7425
57
OscE2 Precursor peptide OscD CBN56378 789 Heterocyclase Rivularia sp. PCC 7116 66 OscG CBN56379 1167 Oxidase/ C-terminal
protease Rivularia sp. PCC 7116 56
67
Table S2. Product ions of the protonated oscillacyclamide A or cyclo[-Trp-Gly-Asp-Lys-Tyr-Ala-Thz-Ser-Asn-Thr-Trp(Prenyl)-Gly-Asp-Thz-Val-Val-Thz] and the double protonated oscillacyclamide B or cyclo [-Gly-Gln-Phe-Gly-Ser-Thz-Asn-Thz-Ser-Val-Ser-Ser-Gly-Asp-Tyr-Trp-Ser-Thr-Phe-Asn-Leu-Gly-Pro-Leu-Thz]. Product ions 1 - 4 from the protonated oscillacyclamide A prove the macrocyclic nature of the peptide while ions 13 - 18 and 20 prove the presence of thiazoles and ions 22 - 23, 25 - 27, 29 - 30 and 32 prove the presence of the immonium ion forming amino acids in the molecule. Product ions 5 - 8 from the double protonated oscillacyclamide B prove the macrocyclic nature of the peptide while ions 13 - 14, 16 and 18 - 20 prove the presence of thiazoles and ions 22 - 24, 26 and 28 - 32 prove the presence of the immonium ion forming amino acids in the molecule.
No Fragment sequence Oscillacyclamide A Oscillacyclamide B
[M+H]+ Δ [M+H]+ Δ Calculated Measured (ppm) Calculated Measured (ppm)
1 Val-Thz-Trp-Gly-Asp-Lys-Tyr 832.3447 832.3461 1.7 - - -
2 Val-Thz-Trp-Gly-Asp 541.1864 541.1879 2.7 - - - 3 Val-Thz-Trp-Gly 426.1594 426.1590 -1.1 - - -
4 Val-Thz-Trp 369.1380 369.1366 -3.8 - - -
5
Gly-Pro-Leu-Thz - Gly-Gln-Phe / Pro-Leu-Thz - Gly-Gln-Phe-Gly - - - 683.2970 683.2976 0.8
6 Pro-Leu-Thz - Gly-Gln-Phe - - - 626.2755 626.2763 1.1
7 Pro-Leu-Thz - Gly-Gln - - - 479.2071 479.2071 -0.1
8 Gly-Pro-Leu-Thz / Pro-Leu-Thz - Gly - - - 351.1485 351.1479 -2.0
9 [Thr-Trp(Prenyl)] − H2O 338.1863 338.1861 -0.6 - - -
10 [Asn-Thr-Trp(Prenyl)] − H2O 452.2292 452.2273 -4.3 - - -
11 Trp(Prenyl)-Gly-Asp-Thz 510.1806 510.1778 -5.5 - - -
12 Trp(Prenyl) 255.1492 255.1479 -5.3 - - - 13 Ala/Leu-Thz 155.0274 155.0275 0.6 197.0743 197.0758 7.3
14 [Ala/Leu –Thz] − NH3 138.0008 138.0009 0.3 180.0478 180.0477 -0.6 15 Asp-Thz 199.0172 199.0160 -6.2 - - -
16 [Asp/Asn-Thz] − NH3 181.9906 181.9906 -0.5 181.0066 181.0072 2.9 17 Val/Ser-Thz 183.0587 183.0599 6.5 171.0223 171.0225 1.1
18 [Val-Thz] − NH3 166.0321 166.0320 -1.0
19 [Ser-Thz] − H2O - - - 153.0117 153.0112 -3.7 20 Thiazole-CO 111.9852 111.9854 2.1 111.9852 111.9852 -0.1
Immonium ions:
21 Trp(prenyl) 227.1543 227.1532 -5.0 - - - 22 Trp 159.0917 159.0913 -2.6 159.0917 159.0918 0.5 23 Tyr 136.0757 136.0758 0.4 136.0757 136.0752 -4.0
68
24 Phe - - - 120.0808 120.0805 -2.7 25 Lys 101.1073 101.1071 -2.7 - - - 26 Asp 88.0393 88.0391 -2.8 88.0393 88.0388 -6.2 27 Asn 87.0553 87.0549 -5.1 - - - 28 Leu/Ile - - - 86.0964 86.0962 -3.3
29 Thr 74.0600 74.0596 -6.6 74.0600 74.0602 2.2 30 Val 72.0808 72.0814 7.9 72.0808 72.0819 14.8
31 Pro - - - 70.0651 70.0660 11.7 32 Ser 60.0444 nd - 60.0444 60.0452 12.7
69
Table S3. Some cyanobactin macrocyclases and their corresponding natural products:
Macrocyclase Compound
class
No of residues in the natural
products
Natural recognition signal
Producer organism Accession # Ref.
PatGmac Patellamides,
Lissoclinamides,
Ulithiacyclamides
7, 8 Ala-Tyr-Asp Prochloron sp. AAY21156.1 S1
McaGmac Microcyclamides,
Aerucyclamides
6, 7 Ala-Phe-Asp Microcystis aeruginosa
NIES-298 and PCC7806
CAO82089 S2
TruGmac Trunkamide,
Patellins
6, 7, 8 Ser-Tyr-Asp Prochloron sp. ACA04494 S3
TenGmac Tenuecyclamides 6 Ala-Tyr-Asp Nostoc spongiaeforme var.
tenue str. Carmeli
ACA04486 S3
LynGmac Aesturamides 7 Ser-Tyr-Asp Lyngbya aestuarii PCC
8106
WP_009787128 S3,
S4
TriKmac Trichamide 11 Ser-Tyr-Asp Trichodesmium erythraeum
IMS101
YP_722058 S5
PirGmac Piricyclamides 9, 12, 14 Phe-Ala-Gly-Asp Microcystis aeruginosa
PCC7005
CCI37144 S6
70
References:
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S2. N. Ziemert, K. Ishida, P. Quillardet, C. Bouchier, C. Hertweck, N. T. de Marsac, E. Dittmann, Appl. Environ. Microbiol. 2008, 74, 1791-1797.
S3. M. S. Donia, J. Ravel, E. W. Schmidt, Nat. Chem. Biol. 2008, 4, 341-343.
S4. K. Sivonen, N. Leikoski, D. P. Fewer, J. Jokela, Appl. Environ. Microbiol. 2010, 86, 1213-1225.
S5. S. Sudek, M. G. Haygood, D. T. A. Youssef, E. W. Schmidt, Appl. Environ. Microbiol. 2006, 72, 4382-4387.
S6. N. Leikoski, D.P. Fewer, J. Jokela, M. Wahlsten, P. Alakoski, K. Sivonen, PLoS One 2012, 7, e43002.