General Procedures (G.P)

8.3.1 General procedure to synthesize β

-D-homo-amino acids

8.3.1.1 G.P -1: Synthesis of diazoketone from corresponding α-amino acids

Based on the method developed by Guichard et.al, the N-Fmoc/Boc protected α-amino acid (1.0 eq) was dissolved in dry THF under argon atmosphere with continuous stirring and was cooled to -25⁰C followed by the addition of triethylamnine (1.10 eq) and isobutylchloroformate (1.10 eq). After stirring the reaction mixture at -25⁰C for 45 minutes, diazomethane (0.6.0.7 M, 2.0 eq) was carefully added to the reaction vessel under complete exclusion of light and the mixture was stirred at 0⁰C for 30 minutes followed by stirring at room temperature for 4.5 hrs. The reaction progress was monitored by TLC in the interim period. After the reaction was completed with almost no starting material left, glacial acetic acid (2.0 eq) was added to quench the excess diazomethane. The reaction mixture was further washed with aqueous 6% NaHCO3 solution (approx. 3 × 80 mL) and extracted with EtOAc (3 × 100 mL). The combined organic phases were washed with saturated aqueous NH4Cl (3 × 60 mL), saturated aqueous brine solution (aq. Nacl, 3

× 60 mL) and dried over Na2SO4. The excess solvent was removed under educed pressure and

the resulting product (yellow oil/solid) was used in the subsequent step without further purification.

8.3.1.2 G.P -2: Synthesis of β³-D-amino acid from the corresponding diazo-ketone intermediate

According to the methodology developed by Guichard et al., the diazo-ketone intermediate (1.00 eq.) was dissolved in a solvent mixture composed of THF/water (9:1, v/v) and silver benzoate (0.10 eq.) was added under complete exclusion of light. The reaction vessel is cooled in an ice-bath while adding the silver benzoate and afterwards placed in an ultrasonication water-ice-bath for the reaction to continue for 2 H under sonication. After completion of the reaction, the mixture was diluted with water (approx. 50 mL); pH was adjusted to 2-3 by adding 1.0 M HCL (aq.) and extracted with EtOAc (3 × 80 mL). The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure. The final rude product was purified either by flash column chromatography (Table 1) or by precipitation (crude product taken in DCM and was poured drop-wise into ice-cold pentane. The precipitate was filtered off, washed further with cold pentane for two times and dried overnight under reduced pressure).

β³-D-Homo-Amino Acids Yield in % Method of Purification

Fmoc- β³-D-Lys(Boc)-OH 83

Fmoc- β³-D-Ala-OH 90

Table 8.1. Methods to purify different β³-D-homo-amino acid building blocks.

8.3.2 General procedure to synthesize β-peptides

8.3.2.1 G.P - 3: General procedure for Fmoc-deprotection in microwave assisted manual SPPS

The resin was washed thoroughly with NMP or DMF (5 x 5 mL) followed by addition of a cocktail of 20% piperidine in NMP. First deprotection was carried out in microwave with 30 sec/25W/50⁰c condition. Subsequently the second deprotection was performed by adding a deprotection cocktail of DBU/piperidine/DMF (1:5:44) into the resin and carried out with 3 min/ 35W/60 °C condition in the microwave. The resin was washed with DMF (7 x 5 mL), DCM (4 x 5 mL) and NMP (4 x 5 mL).

8.3.2.2 G.P - 4: General procedure for Fmoc-deprotection in manual SPPS without microwave

The resin was washed thoroughly with DMF (5 x 5 mL) followed by addition of a cocktail of 20%

piperidine in DMF and shaken in a shaker at room temperature for 15-20 min. The second Fmoc-deprotection was carried out by the subsequent addition of a cocktail of 20% piperidine and 2%

DBU in DMF and shaken at room temperature for 5-10 min. The resin was then washed thoroughly with solution of {DCM/MeOH/DIPEA (17/32/1)} (5 x 5 mL), DMF (5 x 5 mL), DCM (5 x 5 mL).

8.3.2.3 G.P - 5: General procedure to load first amino acid (β³-D-homo-amino acid) to rink-amide MBHA or NovaPEG rink-rink-amide low loaded resins

Rink amide MBHA resin with a loading capacity of 0.57 mmol/g (used for β³-D-Val peptide of 14-helical secondary structure) or rink amide NovaPEG resin with a loading capacity of 0.18 mmol/g (used for β³-D-Leu peptide of 12-helical secondary structure) (0.1 mmol, 1 equiv) was placed in a 10 mL PE-frit equipped Becton-Dickinson(BD) discardit II syringe (Heidelberg, Germany)and was swollen with NMP or DMF (6 mL) for 60 min. To the rink-amide MBHA resin was then added Fmoc-deprotection mixture (5 mL) (20% piperidine in NMP or DBU/piperidine/DMF (1:5:44) + LiCl 0.3 M) and the deprotection was carried out in microwave assistance in a CEM Discover apparatus. The Fmoc-deprotection was carried out in 2 steps; at first for 30 sec/25W/50⁰c and then for 3 min/ 35W/60 °C in the microwave. No deportation was required for rink amide NovaPEG resin. After the last step, the resin was washed successively with DMF (7 x 5 mL), DCM (4 x 5 mL) and NMP (4 x 5 mL). Afterwards the coupling of the first amino acid was performed by dissolving the first Fmoc-β³-D-homo amino acid (5 eq.) and anhydrous HOBt (5 eq.) in 1.8 mL NMP and to the solution was added DIC (5 eq.) The mixture was vortexed for 5 sec and transferred to the resin and the coupling reaction took place with microwave assistance (15 min/35W/65⁰C). Re-coupling of the first amino acid was carried out in microwave assistance (20 min/25W/50⁰C) using the amino acid (3 eq.), anhydrous HOBt (3 eq.) and DIC (3 eq.) in 1.5 mL NMP. After each coupling step, the resin was washed successively with DMF (7 x 5 mL), MeOH (4 x 5 mL) and DCM (4 x 5 mL). The resin was dried under vacuum overnight. The estimation of the exact loading efficacy was carried out by UV-spectroscopic method as described in 6.2.4 section.

8.3.2.4 G.P - 6: General procedure to cap free amino groups in microwave-assisted SPPS

For capping of free amino groups, acetic anhydride (10 eq.), DIPEA (15 eq.) and DMAP (0.1 eq.) were dissolved in 1.5 mL DMF in a 2.0 mL- eppendorf vial and the mixture was purged with Ar-stream for 3 min followed by transferring to the resin. The resulting mixture was shaken mechanically for 1.5 h at RT. Then the resin was washed successively with a solution of {DCM/MeOH/DIPEA (17/32/1)} (5 x 5 mL), DMF (5 x 5 mL), DCM (5 x 5 mL) and subsequently was dried and stored under vacuum.

8.3.2.5 G.P - 7: General procedure to load first amino acid (β³-D-homo-amino acid or β² -D-homo-amino acid) to 2-Chlorotrityl chloride resin ^[8]

The Fmoc-β-D-amino acid (3 eq. to the resin) and DIPEA (6 eq. to the resin) were dissolved in DCM (10 mL/g resin). The dry resin was taken into a BD-syringe equipped with a PE filter from Becton-Dickinson and pre-swollen in DCM for 1 hr. After the resin was properly swollen, the solution containing the amino acid was added and the resin was shaken at room temperature for 1 hr. The resin was thoroughly with a solution of {DCM/MeOH/DIPEA (17/32/1)} (5 x 3 mL), DMF (5 x 3 mL), DCM (5 x 3 mL). Deprotection of the Fmoc-group of the first loaded amino acid was carried out by applying a solution of 20% piperidine in DMF (2 x 30 min). After the deprotection, the resin was again washed with a solution of {DCM/MeOH/DIPEA (17/32/1)} (5 x 3 mL), DMF (5 x 3 mL), DCM (5 x 3 mL) subsequently.

8.3.2.6 G.P - 8: General procedure to coupling in microwave assisted manual SPPS ^{[9, 10]}