Enzymatic Macrocyclization of 1,2,3‐Triazole Peptide Mimetics

Abstract The macrocyclization of linear peptides is very often accompanied by significant improvements in their stability and biological activity. Many strategies are available for their chemical macrocyclization, however, enzyme‐mediated methods remain of great interest in terms of synthetic utility. To date, known macrocyclization enzymes have been shown to be active on both peptide and protein substrates. Here we show that the macrocyclization enzyme of the cyanobactin family, PatGmac, is capable of macrocyclizing substrates with one, two, or three 1,4‐substituted 1,2,3‐triazole moieties. The introduction of non‐peptidic scaffolds into macrocycles is highly desirable in tuning the activity and physical properties of peptidic macrocycles. We have isolated and fully characterized nine non‐natural triazole‐containing cyclic peptides, a further ten molecules are also synthesized. PatGmac has now been shown to be an effective and versatile tool for the ring closure by peptide bond formation.


I.
Structures of precursor peptides P and cyclic peptides CP 1

II. General information and materials
The HPLC grade acetonitrile (MeCN) was purchased from Fisher. Aqueous buffers and aqueous mobile-phases for HPLC were prepared using water purified with an Elga ® Purelab ® Milli-Q water purification system (purified to 18.2 MΩ.cm). DMSO, THF and dichloromethane (CH 2 Cl 2 ) were purchased from Fisher. Amino acids and coupling reagents were purchased commercially from different sources. Other chemicals and reagents were purchased from Sigma and used without any further purification.
Reactions performed in the enzymatic media were monitored using MALDI-MS acquired using a 4800 MALDI TOF/TOF Analyser (ABSciex, Foster City, CA) equipped with a Nd:YAG 355 nm laser and calibrated using a mixture of peptides.

III. General procedures
PatG mac cloning, expression and purification: The PatG mac 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
The different precursor peptides were synthesized on solid-phase (SPPS) on a Rink amide resin (RAR) using the Fmoc strategy and Fmoc-protected amino acids (aa).
Both automatic (A) and manual (M) procedures were employed. The introduction of the triazole moiety was accomplished by first reacting azido acids to the growing peptide (A or M) and then by manually introducing the triazole through a coppercatalyzed azide alkyne cycloaddition (CuAAC) between the azide-terminal peptide and the Fmoc-protected alkyne amino acid analogue (Scheme S1).

1-Resin preparation
The rink amide resin (RAR) is weighed into the fritted PP-syringe then swelled in DMF for half an hour.

2-Automated SPPS
A double coupling strategy was used for all amino acids using the following conditions for each step: 5 eq. HBTU in DMF; 8 eq. DIEA in NMP; 5 eq. aa in DMF Reaction time of 30 min with mixing/shaking for 15 s every two minutes at rt followed by 4x DMF washing cycles.
Fmoc deprotection is done in 40% piperidine/DMF in two steps:

S7
3 min reaction with mixing/shaking for 10 s every minute at rt then 12 min reaction with mixing/shaking for 10 s every two minutes at rt followed by 6x DMF washing cycles.
Scheme S1: General SPPS strategy for the synthesis of triazole-containing peptides.

3-Manual SPPS
A double coupling strategy was used for all amino acids using the following conditions for each step: 5 eq. HBTU in DMF; 10 eq. DIEA in NMP; 5 eq. aa in DMF Reaction time of 1h 30 min with shaking at rt followed by 4x DMF, 4x CH 2 Cl 2 , and 4x DMF washing cycles.

4-Manual CuAAC reaction; solid phase (SP)
After the coupling of an azido acid to the growing peptidic chain (A or M), the resin is washed with 5x THF. 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.

S8
beads; repeat the procedure if reaction not complete), the resin is washed with 4x THF, 4x H 2 O, 4x THF, and 4x DMSO. The resin is swelled in DMSO for 10 min before the Fmoc deprotection step (if needed) with 20% piperidine/DMF for 10 min followed by 4x DMF, 4x CH 2 Cl 2 , and 4x DMF washing cycles.

5-Final cleavage
The resin is washed with CH 2 Cl 2 and dried for a minute on the vacuum manifold. The beads are then transferred into a flacon tube and the cleavage cocktail is added: 95% TFA, 3.5% H 2 O, 1.5% TIS. The mixture is vortexed to mix everything and the flacon tube is left on the shaker for 2h. The resin is then filtered, washed with 3x CH 2 Cl 2 and the filtrate is evaporated under vacuum until a few milliliters of TFA are left (< 2mL).

6-Peptide precipitation and HPLC purification
To a falcon tube containing cold diethyl ether, the remaining TFA solution is added dropwise. The peptide precipitates upon contact with the cold ether. The falcon tube is placed for 5 min in the cold and centrifuged at 4 °C and 4000 rpm for 10 min. The supernatant is decanted and the remaining solid left a few minutes under nitrogen flow to dry. The crude is then solubilized in a minimum volume of 20% acetic acid and MeCN (ultrasonic bath helps) and immediately purified by HPLC.

Pat Gmac macrocylisation reaction
The reactions are conducted in 20 mM bicine buffer, 500 mM NaCl, and 5% DMSO The large scale reaction mixture is then extracted 3 times with n-butanol (BuOH): BuOH is added to the aqueous reaction, vigorously mixed, and then centrifuged for 10 min at high speed to help separate the two phases. The combined BuOH fractions are evaporated under reduced pressure to dryness. The crude is solubilized in a minimum volume of H 2 O/MeCN and immediately purified by HPLC using system Pa.
A suspension of NaN 3 (2g, 0.03 mol) in anhydrous MeCN (30 mL) is cooled to 0 °C. Sulfuryl chloride (2.5 mL, 0.03 mol) is then added drop-wise and the mixture stirred overnight at room temperature. Imidazole (3.98 g, 0.06 mmol) is added slowly to the reaction at 0 °C and the mixture is stirred for 3 h at room temperature. EtOAc (60 mL) is added and the reaction is washed with H 2 O (2x 40 mL) and saturated aqueous NaHCO 3 (2x 40 mL). The organic fraction is then dried over MgSO 4 and filtered. In a separate flask, a solution of anhydrous HCl in EtOH is prepared at 0 °C by the dropwise addition of acetyl chloride (3.28 mL, 0.046 mol) to dry ethanol (7.5 mL).
The EtOAc solution is cooled to 0 °C and while stirring, the HCl solution in EtOH is added dropwise to precipitate a white solid. The precipitate is then filtered, washed with EtOAc (3x 15 mL), and dried under pressure to give imidazole-1-sulfonyl azide hydrochloride as white solid (5g, 76%). The NMR spectroscopic data were in agreement with those described in the literature. [3] Caution! Imidazole-1-sulfonyl azide and its salts (including chloride salt) are known to be energetic materials that are sensitive to chock and friction. Care should be taken while handling this material 2-HCl ImSO 2 N 3 .HCl S10 and proper safety precautions should be used. Plastic container and materials are recommended for use. [4] 2-General procedure for the synthesis of azido acids 1a, 1b, and 1d Scheme S3: Synthesis of azido acids 1.
To a solution of the amino acid (1 eq.), K 2 CO 3 (2.7 eq.), and CuSO 4 .5H 2 O (0.01 eq.) in a MeOH/H2O mixture (5/1, v/v), ImSO 2 N 3 .HCl is added and the mixture stirred vigorously for 18h at room temperature. The reaction is then concentrated under reduced pressure, diluted with H 2 O, acidified with 1M HCl to pH = 1, and extracted with 3x CH 2 Cl 2 . The organic layer is dried over MgSO 4 , filtered, and concentrated.

V. NMR data of final cyclic peptides
Copies of the proton NMR spectra for each compound depicting the different species by color code as well as copies of the HSQC spectra can be found in section V. Copies of the exchange spectroscopy (EXSY) spectra can be found in section VI.