A diversity-oriented synthesis strategy enabling the combinatorial-type variation of macrocyclic peptidomimetic scaffolds

Macrocyclic peptidomimetics are associated with a broad range of biological activities.


Compound labelling in mauscript and Supplementary Information
For the sake of clarity, the compound labeling systems used here in the Supplementary Information is different to that used in the main manuscript. The following table lists the compounds given in the main manuscript that are labeled, and their corresponding labels as used in the Supplementary Information. Boc-L-Ala-OH (12a) Ala

Figure 2
Boc-L-Phe-OH (12d) Phe   Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer with internal referencing as neat films. Selected absorption maxima (ν max ) are reported in wavenumbers (cm -1 ).
Nuclear magnetic resonance (NMR) spectra were recorded using an internal deuterium lock on Bruker DPX 400 (400MHz), Bruker Avance 400 QNP Ultrashield (400 MHz), Bruker Avance 500 BB ATM (500 MHz) and Bruker Avance 500 Cryo Ultrashield (500 MHz) spectrometers. Chemical shifts (δ) are referenced to the solvent signal and are quoted in ppm to the nearest 0.01 ppm for δ H and to the nearest 0.1 ppm for δ C . Coupling constants (J) are reported in Hertz to the nearest 0.1 Hz. Assignments are supported by DEPT-135, 1 H-1 H COSY, HMQC, HMBC and NOESY spectra where necessary. Data are reported as follows: chemical shift, integration, multiplicity (app., apparent; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet; or as a combination of these), coupling constant(s) and assignment (corresponding atom in itallics). Diastereotopic protons are assigned as H and H, where H indicates the proton at higher chemical shift. The numbering schemes used on selected spectra do not follow the IUPAC naming system and are used for the clear assignment of 1 H and 13 C spectra.
Low resolution mass spectra (ESI) were recorded using an LCMS system (Agilent 1200 series LC with an ESCi Multi-Mode Ionization Waters ZQ spectrometer using MassLynx 4.1 software).
High resolution mass spectrometry (HRMS) was carried out with a Micromass QTOF or a Waters LCT Premier Mass Spectrometer using electospray ionisation [ESI] or electron ionisation [EI]. The calculated mass value relative to found mass value is within the error limits of ±5 ppm mass units.

General Procedures GP1: Amide formation
The azido-amine (1.0 equiv) was dissolved in anhydrous CH 2 Cl 2 and triethylamine (2.2 equiv), EDC.HCl (1.1 equiv) and HOBt.H 2 O (1.1 equiv) were then added. Upon dissolution, the alkyneacid (1.0 equiv) in anhydrous CH 2 Cl 2 was added and the reaction was stirred at rt for 18 h. The solvent was removed under reduced pressure and the residue was re-suspended in EtOAc and washed with H 2 O. The organic layer was separated and washed with saturated NaHCO 3 solution (×2). A second addition of EtOAc was made and the organic fraction was washed with 5% citric acid (×2) and H 2 O (×2). The organic phase was dried (MgSO 4 ) and the solvent removed under reduced pressure. The crude material was purified by column chromatography to yield the linear peptide.

GP2: CuAAC Macrocyclization to form 1,4-triazoles
DIPEA (3.0 equiv) was added to a solution of the linear peptide (1.0 equiv, 1.2 mM) in anhydrous THF. The reaction was degassed by bubbling Ar directly into the solution for 30 min. CuI (2.0 equiv) was then added and the reaction was refluxed for 18 h under N 2 . The solvent was removed under reduced pressure and the crude material purified by column chromatography or preparative HPLC if necessary.

GP3: RuAAC Macrocyclization to form 1, 5-triazoles
The linear peptide (1.0 equiv., 1.25 mM) was dissolved in anhydrous toluene and the reaction mixture was heated to 80 °C and then degassed by bubbling Ar directly into the solution for 30 min. [Cp*RuCl] 4 (0.1 equiv.) was added and the reaction was heated to reflux for 18 h. The solvent was removed under reduced pressure and the crude material purified by column chromatography or preparative HPLC if necessary.

GP4: Removal of Boc protecting group with TMSCl
The Boc-protected macrocycle was dissolved in MeOH and the suspension was cooled to 0 °C. TMSCl (0.3 ml per 0.035 mmol linear peptide) was added dropwise to the solution. at 0 °C with stirring. The reaction was allowed to warm to rt and stirred until TLC analysis indicated complete consumption of starting material (typically 3 h). The solvent was removed under reduced pressure and the crude material resuspended in CH 2 Cl 2 and washed with. NaHCO 3 . The aqueous layer was extracted with CH 2 Cl 2 (×2) and the combined organic fractions were dried (MgSO 4 ) and the solvent removed under reduced pressure. The crude material was purified by column chromatography (or preparative HPLC) if required to yield the macrocyclic peptidomimetic.

GP5: Removal of Boc protecting group with HCl
The Boc-protected macrocycle was treated with 4.0 M HCl/dioxane (1 ml HCl/dioxane per 20 mg sample) and the reaction was stirred at rt for 18 h. The solvent was removed under reduced pressure and the crude material resuspended in CH 2 Cl 2 and washed with NaHCO 3 . The aqueous layer was extracted with CH 2 Cl 2 (×2) and the combined organic fractions were dried (MgSO 4 ) and the solvent removed under reduced pressure. The crude material was purified by column chromatography (or preparative HPLC) if required to yield the macrocyclic peptidomimetic.

GP6: Synthesis of diketopiperazine
The macrocycle (0.3 equiv, used directly after the deprotection and used as the salt, without purification) and morpholinomethyl-polystyrene (1.0 equiv) were placed in a microwave (MW) tube. 2-Butanol (40 ml per mmol) and acetic acid (1.25 equiv) were added and the reaction was heated to 150 °C in a microwave (typically 2-3 h). The resin was filtered off and several washings with MeOH and CH 2 Cl 2 were performed. The filtrate was evaporated to dryness and the crude material was purified by column chromatography to yield the final DKP-containing macrocycle.

Synthesis of the Common Precursors (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (CP1)
Boc-Asn-OH (8.00 g, 34.4 mmol) was suspended in EtOAc (40 ml), CH 3 CN (40 ml) and H 2 O (20 ml) and the mixture was cooled to 15 °C. PIDA (13.3 g, 41.3 mmol) was added in a single portion and following 45 min of stirring at 15 °C, the reaction was allowed to warm to rt. TLC analysis after 4 h indicated that most of the starting material was consumed. The reaction mixture was heated to 70 °C for 5 min (until completely dissolved) and then cooled to 0 °C. The mixture was filtered and the precipitate was washed on the filter with cold EtOAc (2 × 10 ml) to afford the title compound 41 as an amorphous white solid (4.72 g, 67%).
Spectroscopic data is consistent with literature values. [3] OH

Synthesis of Azido-Amine Building Blocks Building Block A
Building block A was prepared by literature procedures. [6] Building Block B

Building block 6
Building block 6 was prepared by literature procedures. [6] O OH

Building block 9
A procedure analogue to the one used for the synthesis of building block 10, except using hexynoyl-OSu instead of pentynoyl-OSu afforded building block 9 as a white solid (1.42 g, 95% yield). [α] D 25 = -3.2 (c 0.49, MeOH).

Building block 10
To a suspension of Boc-L-Dap-OH (1.00 g, 4.91 mmol), in dry DMF (20 mL), was added a fraction of DIPEA (940 µL, 5.4 mmol). A solution of pentynoyl-OSu (1.15 g, 5.90 mmol) in dry DMF (10 mL) was then added dropwise over 10 minutes. After 5 minutes a second fraction of DIPEA (940 µL, 5.4 mmol) was added and the mixture was stirred at rt for 5 hours. The organic solvent was removed under reduced pressure and the slurry was diluted with H 2 O (25 mL). The pH was adjusted to 8-9 by addition of saturated Na 2 CO 3 and washings with Et 2 O (3 x 15 mL) were performed. The aqueous layer was then acidified to pH 2-3 with conc. HCl and extracted with EtOAc (3 x 20 mL). The organic extracts were dried (MgSO4) and evaporated to dryness under reduced pressure. After precipitations in Et 2 O, building block 10 was obtained as a white solid in 89% yield.

Building block 12
A procedure analogues to the one used for the synthesis of building block 4, except using common precursor 1 as starting material afforded building block 12 as a white foam after flash column chromatography (5% MeOH: 1% AcOH: 94% EtOAc) (1.19 g, 79% yield).

Building block 14
Building block 14 was prepared by literature procedures. [6] O OH

Chemoinformatic analysis Principal component analysis
Principal component analysis (PCA) was carried out using the Molecular Operating Environment (MOE) software package [11] . A total of 15 physicochemical properties (Table S1) were obtained for 222 macrocylic DOS library members and established reference sets of 40 top-selling brandname drugs, 60 diverse natural products and 24 macrocyclic natural products [12] .
The summary of the PCA is shown in Table S2. The first three principal components account for 87.3% of the variance in the dataset and were used to generate Figures 3a-c in the manuscript.

Principal moment of inertia calculations
We compared the molecular shape diversity of our DOS library with the same reference sets of 124 compounds used in the PCA.
The LowModeMD conformational search algorithm in the MOE software package [11] was used to generate low-energy 3D conformers for each compound. The MMFF94x force field was used with the generalized Born solvation model for the minimizations. Sampling and minimization parameters were implemented as follows: Only the conformer with the lowest energy was retained for principal moment of inertia (PMI) calculations. Normalized PMI ratios (I 1 /I 3 and I 2 /I 3 ) of these conformers were obtained from MOE and then plotted on a triangular graph, with the coordinates (0,1), (0.5,0.5) and (1,1) representing a perfect rod, disc and sphere respectively ( Figure Xd).