Bypassing the proline/thiazoline requirement of the macrocyclase PatG† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc06550g

Macrocyclisation of fully non-peptidic compounds and non-heterocycle containing macrocycles using the peptidic ligase PatGmac.

(0.5 μL) were applied to the MALDI target along with alpha-cyano-4hydroxycinnamic acid matrix (0.5 μL, 10 mg/mL in 50:50 acetonitrile:0.1% TFA) and allowed to dry. MSMS data were acquired using a TripleTOF 5600+. The sample was subjected to chromatography on an Acclaim PepMap 100 C18 trap and an Acclaim PepMap RSLC C18 column (ThermoFisher Scientific), using a nano-LC Ultra 2D plus loading pump and nano-LC as-2 autosampler (Eksigent). The sample was injected at neutral pH. The trap was washed with 2% acetonitrile, 0.05% trifluoroacetic acid, and the desired peptide was then eluted with a gradient of increasing acetonitrile, containing 0.1 % formic acid (15-40% acetonitrile in 5 min, 40-95% in a further 1 min, followed by 95% acetonitrile to clean the column, before re-equilibration to 15% acetonitrile). The eluent was sprayed into a TripleTOF 5600+ electrospray tandem mass spectrometer (Sciex) operating with standard nanospray conditions, and analysed in Product Ion Scan mode isolating the m/z of interest. The collision energy was adjusted to give optimal fragmentation. The MSMS fragmentation pattern was interrogated for diagnostic peaks.

General procedures PatGmac cloning, expression and purification
The PatGmac enzyme was cloned from genomic DNA (Prochlon sp.) into the pHISTEV vector, expressed in Escherichia coli BL21 (DE3) cells grown on autoinduction medium, and purified as previously described by Koehnke et al. 2 However, subsequent to the Nickel column eluting with 250 mM imidazole, the remaining purification steps were replaced with dialysis in a bicine buffered solution [20 mM Bicine, 150 mM NaCl, pH 8.1] to remove the imidazole and the reducing agent.

Solid-phase peptide synthesis of peptides 1-26
The different precursor peptides were synthesized by standard automated solid-phase (SPPS) on a Chem-Matrix Rink amide resin (~ 0.5mmol/g) using the Fmoc strategy and Fmoc-protected amino acids (aa). A double coupling strategy using a 5-fold excess with HBTU/DIEA (HBTU, 0.5M in DMF and DIEA, 2M in NMP) and DIC/oxyma pure (DIC, 0.5M in DMF, Oxyma, 1M in DMF) was used for all amino acids for 30 minutes at 75 °C. The Fmoc deprotection was done in 20% piperidine/DMF for 12 min at rt.
The introduction of the triazole moiety was accomplished in two ways (Scheme S1): 1-Standard amide bond coupling of a pre-synthesized triazole containing dipeptide 2-Copper-catalyzed azide alkyne cycloaddition (CuAAC) on resin (1,4-triazole only) Scheme S1: Two SPPS strategies for the synthesis of triazole-containing peptides.
For the manual CuAAC reaction, the resin containing a terminal azide is washed with 5x THF and the corresponding amino alkyne (5 eq.) is diluted in THF and added to the resin. DIEA (10 eq.) is then added followed by CuI (5 eq.). The mixture is bubbled with dry nitrogen for 1 min then sealed, protected from light, and left overnight with shaking. After the reaction is complete (mini work up of a few beads; repeat the procedure if reaction not complete), the resin is washed with 4x THF, 4x The disulfide bond of peptides 19-26 was formed overnight using 10% DMSO in TFA after full deprotection of the precursor peptides. The reaction was either done on the purified precursor, evaporated and freeze dried, or on the crude precursor, which was purified afterwards to afford the pure peptide.

Pat Gmac macrocylization reaction of peptides 1-26
The reactions were conducted in 20 mM bicine buffer, 500 mM NaCl, and 5% DMSO solution, pH 8.1 and incubated at 37 °C (without shaking) until full consumption of the starting peptide occured (MALDI monitoring). The reaction set-up was prepared in the following order; final concentrations:  The crude azide mixture (2.53mmol) and triphenylphosphine (0.73g, 2.78mmol) were solubilized in THF (24mL) and H 2 O (6mL) (4/1, v/v) and the reaction stirred at room temperature overnight. The THF was evaporated under reduced pressure. Attempts to extract the amine into organic phase failed and the aqueous phase containing the amine was subsequently used for the next step.
To the amine (2.53mmol) in water (10mL) and THF (10mL) was added Na 2 CO 3 (1g) and fluorenylmethyloxycarbonyl chloride (FmocCl, 1g, 3.85mmol) and the reaction was stirred at room temperature overnight. EtOAc (30mL) was added to the reaction and the two phases separated. The aqueous phase was extracted with EtOAc (2x30mL), and the combined organic phases washed with brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The crude mixture was purified over silica gel (100% CH 2 Cl 2 to 2% EtOAc/CH 2 Cl 2 ) to afford the desired Fmocprotected amine 27 (0.4g) as a white solid in 47% yield over three steps. The NMR spectroscopic data were in agreement with those described in the literature. 31  Hydrazoic acid HN 3 : To a paste mixture of sodium azide (6.5g, 0.1mmol) in water (6.5mL) at -10°C was added toluene (40 mL) under rigorous stirring. Concentrated sulphuric acid (2.66mL) was added drop-wise and the temperature monitored to keep under 10°C. The reaction temperature was cooled to 0°C and the solution decanted.
The toluene fraction was dried over MgSO 4 and filtered. 1mL of the solution was diluted in 30mL of water and titrated with NaOH 1M to determine its concentration.

2-Experimental procedure for the synthesis of azido-acids and amino alkynes
Scheme S4: General synthetic scheme of azido acid analogues (1.7/2/1, v/v), ImSO 2 N 3 .HCl 4 (7.29g, 34.8mmol) was added and the pH adjusted to pH=9 with saturated aqueous K 2 CO 3 solution. The mixture was stirred vigorously for 18h at room temperature. CH 2 Cl 2 (52mL) was added to the reaction and separated.
The organic phase was extracted with saturated aqueous NaHCO 3 solution (2x75mL).
The combined aqueous phases were washed with Et 2 O (2x75mL) and then the pH adjusted to pH = 2 with concentrated HCl (the aqueous solution changes colour from blue to colourless through shades of green and yellow). The acidic aqueous phase was then extracted with Et 2 O (3x100mL). The combined organic layer (of the last three extractions) was dried over MgSO 4 , filtered, and concentrated to afford azido acid 29 (1.8g, quant) as pale yellow oil. The NMR spectroscopic data were in agreement with those described in the literature. 5 The corresponding azido acids were then used without any further purification. 1  The NMR spectroscopic data were in agreement with those described in the literature. 6  Purification over silica gel (20% pentane/CH 2 Cl 2 ) afforded the Fmoc-protected amino-alkyne 39 (1.6g, 88%) as an off-white solid. To a solution of the azide (1 eq.) and the alkyne (1 eq.) in anhydrous toluene under inert atmosphere was added RuCp*(cod)Cl (0.1 eq.) and the reaction left stirring at room temperature overnight. The reaction mixture was concentrated.

S12
A mixture of Anti-and Syn-Fmoc-Gly-Tz-Ala-OBn was synthesized following procedure A. The two regioisomers 40 and 41 were inseperable by silica gel chromatography and RP-HPLC.
Anti-Fmoc-Gly-Tz-Ala-OBn was synthesized following procedure B. Only 30% conversion was achieved in these conditions. Triazole 40 was used for NMR reference only.
Syn-Fmoc-Gly-Tz-Ala-OBn was synthesized following procedure C. Triazole 41 was used for NMR reference only. The regioisomeric mixture was inseparable by RP-HPLC and was used as a mixture for the synthesis of peptides 2/4 and 3/5, which were separated by HPLC at the peptide stage.

4-Disulfide bond reduction of cyclic peptide 18c
Scheme S6: TCEP reduction of disulphide bond Cyclic peptide 19c was solubilized in methanol. 10 eq. of TCEP solubilized in the same amount of water (as methanol) were added to the peptide and left stirring at room temperature. The reaction was monitored by LCMS and found complete after 40 minutes ( Figure S2). Peptide 19d was purified by HPLC and the CD spectra in methanol of both cyclic peptides were recorded ( Figure S3).     Purity assessed by analytical HPLC at 220 nm UV absorption. LCMS traces can be found in section XIII [b] The yield of the enzymatic transformation(s) leading to the compound.

III. MS and HPLC data of starting and final hybrid peptides
[c] The purity indicated relates to the cyclic peptide 11c + 11d; 68/32 indicates the ratio 11d/11c [d] The purity indicated relates to the cyclic peptide 16c + 16d; 70/30 indicates the ratio 16d/16c

IV. NMR data of final cyclic peptides
Copies of the proton NMR spectra for each compound depicting the different species, when present, by color code as well as copies of the HSQC spectra and EXSY spectra can be found in sections VI and VII.

VII. Maldi-MS traces of the PatGmac reactions of peptides 19
and 20 Figure

VIII. LC-MS traces of final cyclic peptides
For each of the cyclic hybrid peptides 1-7, the UV trace at 220 nm obtained by HPLC is complemented by its corresponding LCMS trace at the desired molecular weight (Single Ion Monitoring SIM mode) The HPLC methods used are described in section II.