Supporting Information Peptide macrocycles featuring a backbone secondary amine: a convenient strategy for the synthesis of lipidated cyclic and bicyclic peptides on solid support Alberto ddo*, Lena Münzker and Paul R. Hansen* University of Copenhagen Department of Drug Design and Pharmacology Universitetsparken 2 2100 Copenhagen Denmark *Correspondence to: Alberto ddo, alberto.oddo@sund.ku.dk; Paul R. Hansen, prh@sund.ku.dk. Content: S2: List of abbreviations S3-S5: Materials & Methods S6: Summary of the peptide characterization data S7-S14: Reverse-phase HPLC setup and analytical chromatograms S15: LC-MS (Q-ToF) setup and spectrum of 1 S15-S20: MALDI-ToF-MS setup and spectra of 2-11 S1
List of abbreviations Boc, tert-butyloxycarbonyl group; CFU, colony-forming units; Chloranil, tetrachloro-p-benzoquinone; CMU, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate; Dap, 2,3-diaminopropionic acid; DCM, dichloromethane; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl group; DIC, dicyclohexylcarbodiimide; DIEA, diisopropylethylamine; Dmab, 2-{1-[4-(methylhydroxyl)phenylamino]-3-methylbutylidene}-5,5-dimethyl-1,3-cyclohexanedione group; DMF, dimethylformamide; Et 2, diethyl ether; EtH, ethanol; Fmoc, 9-Fluorenylmethoxycarbonyl group; HATU, -(7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate; HAt, 1-hydroxy-7-aza-benzotriazole; LB, lysogeny broth; MeCN, acetonitrile; MHB, Müller-Hinton broth; MIC, minimum inhibitory concentration; Mtt, 4-methyltrityl group; Mmt, 4-methoxytrityl group xyma, ethyl (hydroxyimino)cyanoacetate; PTFE, polytetrafluoroethylene; PyBP, (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid; TIS, triisopropylsilane; S2
Materials Disposable 5-ml polypropylene reactors fitted with a PTFE filter were acquired from Thermo Scientific. Tentagel S RAM resin, TFA, piperidine and most Fmoc-protected amino acids (Fmoc- Dap(Mtt)-H, Fmoc-Lys(Boc)-H, Fmoc-Lys(Dde)-H, Fmoc-Lys(Mmt)-H and Fmoc-Phe-H) were purchased from Iris-Biotech GmbH. Fmoc-Asp-Dmab and PyBP were purchased from Bachem GmbH. DIEA, ω-halocarboxylic acids, palmitic acid, decanoic acid, KI and TIS were from Sigma-Aldrich. HAt and HATU were from GL Biochem Shanghai. All solvents were from VWR: DMF (synthesis grade), DCM (optical grade), MeCN (optical grade). All reagents and solvents were used without further purification. Methods General procedure for solid-phase peptide synthesis. Peptides were synthesized manually in 5- ml polypropylene reactors fitted with a PTFE filter. The typical synthesis scale was 0.02 mmol. The resin (Tentagel S RAM 0.2 mmol/g) was allowed to swell overnight in DMF then washed with DMF (5x). Chain elongation was achieved with single couplings using 5 equivalents of amino acid, xyma and CMU each, and 10 equivalents of DIEA (based on declared resin loading). Fmocprotected amino acids were dissolved in DMF at a concentration of 0.6 M; they were then activated by the sequential addition of xyma/cmu (0.6 M in DMF) and of DIEA, and the mixture was immediately transferred into the reactor. xyma and CMU were kept dry until immediately before coupling. The reactor was placed on a shaker and couplings were allowed to proceed for 1:15h. The Fmoc-group was removed by treatment with a 20% v/v piperidine solution in DMF (3 x 4 min). Deprotection times were increased (2 x 5 min, 1 x 7 min) for the assembly of the exocyclic chain. Before and after the piperidine deprotection procedure, the resin was washed with DMF (3x), DCM (3x) and DMF again (5x). Coupling of ω-halocarboxylic acids. All halogenated carboxylic acids (20 eq.) were dissolved in DMF (0.9 M) and preactivated with DIC (22 eq.), then quickly transferred to the reactors. Bromoacetic acid was preactivated for 3 minutes and coupled once for 25 min. For 3-chloropropionic acid the preactivation time was 5 min; 4-chlorobutyric and 5-chlorovaleric were preactivated for 10 min. Couplings were allowed to proceed for 25 min and repeated once. Removal of Mtt/Mmt groups. After coupling the ω-halocarboxylic acid, the resin was abundantly washed with DMF and DCM. Mtt-groups were removed as previously recommended by Li & Elbert (J. Peptide Res. 2002, 60, 300-303) with a 1.8% TFA/DCM solution (9 x 3 min). Mmtgroups were removed with a 1% TFA/DCM solution (4 x 1 min). The intense coloration of Mtt/Mmt cations provides a visual confirmation of reached completion. Ring closure via on-resin halide displacement. The resin was abundantly washed with DCM, then with a 5% (v/v) DIEA/DCM solution (3x) and finally with DMF (5x). A 0.25 M DIEA in DMF was S3
added to the resin (approx. 1.9 ml per 100 mg of resin, corresponding to 20 eq. of base) and the reactor was placed on a shaker overnight. In the case of Cl-containing precursors, the DIEA solution also contained KI (1 M). Cyclization completion was assessed via MALDI-ToF-MS after cleaving the peptide from a small portion of resin. Coupling to the backbone secondary amine. The coupling on the backbone secondary amine was carried out with 4 eq. of Fmoc-protected amino acid, 4 eq. of HAt and HATU, and 8 eq. of DIEA (based on the declared resin loading). HAt and Fmoc-AA were dissolved together in DMF at a concentration of 0.4 M; HATU was dissolved separately at the same concentration. Immediately before coupling, the two solutions were mixed and DIEA was added. The activated mixture was immediately transferred to the reactor and left on a shaker for 1.5h. Coupling completion was assessed via the chloranil test as follows: reagent A was prepared by dissolving 20 µl of acetaldehyde in 0.98 ml of DMF; reagent B was prepared by dissolving 20 mg of chloranil in 0.98 ml of DMF. A few resin beads were placed in an Eppendorf tube and 1 drop of reagents A and B each was added: the appearance of a blue coloration within 5 min was interpreted as incomplete coupling. Couplings were repeated in the same conditions until the chloranil test resulted negative. After Fmoc-removal, following couplings were carried out using the general procedure (see above). Dmab- and Dde-group removal and bicyclization. The Dmab- and Dde-groups were removed by treating the resin (4 x 3 min) with a 3.1% v/v hydrazine monohydrate (approx. 2% hydrazine content) solution in DMF (approx. 3 ml per 100 mg of resin). The resin was then washed with DMF (5x), DCM (5x), 0.1% TFA/DCM (3x), DCM (5x), EtH (5x) and dried in vacuo. After re-swelling in DMF for 2h, HAt (3 eq. in minimal DMF) was added, followed by PyBP (3 eq. in minimal DMF) and DIEA (6 eq.). The reactor was placed on a shaker for 6h, then the resin was drained and washed. Fresh coupling reagents and base were added and the cyclization reaction was allowed to proceed overnight. Cyclization completion was assessed via MALDI-ToF-MS after cleaving the peptide from a small portion of resin. Release from the solid support. The resin was washed with DMF (5x), DCM (3x), EtH (5x) and dried in vacuo. The release of lipidated macropeptoids from the solid support was performed with a 95% TFA/H 2 /TIS solution for 2h (approx. 3 ml per 100 mg of resin). The cleavage solution was collected and concentrated down to ~300 µl with a gentle stream of N 2, then the peptides were precipitated and washed with Et 2 (3x). After spontaneous evaporation of the residual Et 2, peptides were dissolved in H 2 and minimal MeCN and freeze-dried before analysis. MIC determination. Antimicrobial activity was measured using the tube dilution method as previously described by Wiegand et al. (Nat. Protocols 2008, 3, 163-175). Three to five isolated colonies of freshly plated bacteria were suspended into 4 ml of MHB medium. The tubes were incubated until D 625 > 0,1 was reached. Then the suspension turbidity was adjusted to D 625 = 0,1 and subsequently diluted 1:200 in MHB. Stock solutions were prepared at 10 times the highest concentration to be tested by dissolving the peptides in water containing 0.01% (v/v) acetic acid and 0.2% (w/v) BSA. From 20 µl of peptide solution 2-fold dilution series were prepared using the same solvent (test range: 32-0.25 µm) in a S4
polypropylene 96-well microtiter plate. The adjusted bacterial suspension (90 µl, corresponding to an inoculum of approx. 5 10 5 CFU) was added to each well containing the peptides or the positive control (ciprofloxacin); 100 µl were added to the negative control wells and the same volume of fresh MHB was transferred to the sterility control wells. A 10 µl aliquot from the negative control was diluted 1:100 and 1:1000 in MHB; 100 µl of each of the two dilutions were plated in triplicate on LB-agar plates. Microtiter and agar plates were incubated for 16-20 hours at 37 C. MIC values were interpreted as the lowest concentration without visible growth; colonies on agar plates were counted to validate the initial inoculum. S5
Summary of the peptide characterization data All compounds were isolated in the form of white solid. Macropeptoid 1: t R = 20.9 min (RP-HPLC); purity = 100%; m/z calculated for C 34 H 55 N 8 7 [MH + ] = 687.41, found = 687.2. Yield = 15% (2.75 mg as TFA-salt). Macropeptoid 2: t R = 16.5 min (RP-HPLC); purity = 96.0%; m/z calculated for C 43 H 73 N 10 8 [MH + ] = 857.55, found = 857.20. Yield = 15% (3.63 mg as TFA-salt). Macropeptoid 3: t R = 17.8 min (RP-HPLC); purity = 96.1%; m/z calculated for C 49 H 76 N 11 9 [MH + ] = 962.57, found = 962.30. Yield = 21% (5.5 mg as TFA-salt). Macropeptoid 4: t R = 17.5 min (RP-HPLC); purity = 95.0%; m/z calculated for C 52 H 82 N 11 9 [MH + ] = 1004.62, found = 1004.54. Yield = 37% (10.05 mg as TFA-salt). Macropeptoid 5: t R = 21.5 min (RP-HPLC); purity = 96.9%; m/z calculated for C 73 H 115 N 14 11 [MH + ] = 1363.88, found = 1363.71. Yield = 21% (7.15 mg as TFA-salt). Macropeptoid 6: t R = 20.7 min (RP-HPLC); purity = 96.1%; m/z calculated for C 88 H 136 N 17 13 [MH + ] = 1639.04, found = 1639.58. Yield = 24% (10.05 mg as TFA-salt). Macropeptoid 7: t R = 21.2 min (RP-HPLC); purity = 95.3%; m/z calculated for C 89 H 138 N 17 13 [MH + ] = 1653.06, found = 1653.21. Yield = 37% (17.2 mg as TFA-salt). Macropeptoid 8: t R = 21.6 min (RP-HPLC); purity = 81.9%; m/z calculated for C 90 H 139 KN 17 13 K [MK + ] = 1705.03 found = 1703.95. Yield = 27% (12.6 mg as TFA-salt). Macropeptoid 9: t R = 21.5 min (RP-HPLC); purity = 95.3%; m/z calculated for C 91 H 141 KN 17 13 [MK + ] = 1719.05 found = 1718.82 [MK + ]. Yield = 45% (21.2 mg as TFA-salt). Macropeptoid 10: t R = 16.3 min (RP-HPLC); purity = 96.9%; m/z calculated for C 97 H 145 N 20 15 [MH + ] = 1831.12, found = 1830.83. Yield = 25% (13.2 mg as TFA-salt). Macropeptoid 11: t R = 15.8 min (RP-HPLC); purity = 100%; m/z calculated for C 112 H 166 N 23 17 [MH + ] = 2106.28, found = 2106.07. Yield = 24% (14.5 mg as TFA-salt). Bicyclic Macropeptoid 12: t R = 20.3 min (RP-HPLC); purity = 95.7%; m/z calculated for C 85 H 136 N 17 13 [MH + ] = 1603.04, found = 1603.27. Yield = 20% (9.08 mg as TFA-salt). S6
Reverse-phase HPLC Setup For all experiments: Buffer A: 99.9% Water, 0.1% TFA Buffer B: 89.95% MeCN, 9.95% Water, 0.1% TFA Analytical HPLC Pump: Waters TM 600 Pump Detector: Waters TM 2996 Photodiode Array Detector Column: Waters TM Symmetry TM C18, 4.6 x 250 mm, 5 µm Gradient: Time (min) Flow (ml/min) Buffer A Buffer B 0.0 1.50 100% 0% 20.0 1.50 30% 70% 22.0 1.50 0% 100% 27.0 1.50 0% 100% 30.0 1.50 100% 0% Equilibration delay between injections (both methods): 9 min with 100% Buffer A, 1.50 ml/min. The ApexTrack TM algorithm has been used for peak integration. Peaks having an area <0.01% were not included in the purity calculations. Spectra were processed as follows: the raw data were exported using the Waters Empower 3 software and imported in Microsoft Excel. The baseline (blank) was subtracted and negative absorbance values were corrected to 0 (zero). S7
Preparative HPLC Pump: Waters TM 600 Pump Detector: Waters TM 996 Photodiode Array Detector Column: Waters TM XBridge TM BEH130 C18, 10 x 250 mm, 5 µm Gradient: Time (min) Flow (ml/min) Buffer A Buffer B 0.0 4.00 100% 0% 1.0 6.00 100% 0% 3.0 8.50 100% 0% 5.0 8.50 90% 10% 20.0 8.50 45% 55% 26.0 9.00 0% 100% 27.0 9.00 0% 100% 30.0 9.00 100% 0% 39.0 9.00 100% 0% Purified fractions were collected manually. S8
Chromatograms Macropeptoid 1 Macropeptoid 2 S9
Macropeptoid 3 Macropeptoid 4 S10
Macropeptoid 5 Macropeptoid 6 S11
Macropeptoid 7 Macropeptoid 8 S12
Macropeptoid 9 Macropeptoid 10 S13
Macropeptoid 11 Macropeptoid 12 S14
LC-MS (Q-ToF) Setup Instrument: Agilent 1100 series with Esquire 3000 Plus ion trap. Column: Agilent Poroshell C18 (2.1 x 75 mm). LC-MS Spectra Macropeptoid 1 MALDI-ToF-MS Setup Instrument: Bruker Microflex TM Matrix: α-cyano-4-hydroxycinammic acid in MeCN:H 2 :TFA (50:47.5:2.5) (10 mg/ml). MALDI Spectra Macropeptoid 2 857.203 S15
Macropeptoid 3 Macropeptoid 4 S16
Macropeptoid 5 Macropeptoid 6 S17
Macropeptoid 7 1653.211 Macropeptoid 8 S18
Macropeptoid 9 1718.818 Macropeptoid 10 S19
Macropeptoid 11 Bicyclic Macropeptoid 12 S20
Adducts formed during ring closure reaction in the presence of DBU: N N + NH HN NH NH 2 NH 2 H 2 N NHH 2 N H N N H H N N H NH 2 Calculated m/z = 1311.81 NH 2 NH N H HN HN HN NH H 2 N H 2 N NH H N HN NH 2 Calculated m/z = 1158.68 S21