Instalación de un dispositivo Rosemount 5900 con el asistente

The dried resin was transferred to a fresh solid phase reactor and a solution of acetic acid/ trifluoroethanol/DCM (20%/20%/60%) was added. The mixture was shaken for 2 h at RT. After this time the solution containing the cleaved peptide was collected and the resin was washed once with the cleavage solution and with DCM (3x 2 min); these solutions were also collected. The solvent was removed and the remaining oil was co-evaporated twice with cyclohexane to remove residual acetic acid.

Method J: full deprotection of cleaved peptides (removing Trt, Boc and t-Bu)

The crude peptide was dissolved in trifluoroacetic acid/DCM/triisopropylsilane (95%/2.5%2.5%) (~1 mL) and stirred for 2 h at RT. The reaction mixture was added to cold ether resulting in the precipitation of the crude peptide. The suspension was centrifuged and the pellet was washed once with cold ether. After centrifugation the remaining pellet was dried by evaporation of the ether at RT and normal pressure.

Method K: Hydrogenation to remove the benzyl ether protection on the serine sidechain The crude peptide was dissolved in EtOH (1 mL/100 mg crude peptide) and acetic acid (0.1 mL/100 mg crude peptide) and transferred to a two-neck round bottom flask equipped with a stop-cock and a rubber septum under an argon atmosphere containing Pd/C catalyst (10 %, 0.4 mg catalyst/mg crude product). The flask was equipped with a hydrogenation balloon and flushed with hydrogen by opening the stop-cock from time to time. The hydrogenation was carried out overnight at RT and the reaction mixture was filtered through a short pad of Celite® which was washed intensively with MeOH. The solvent was removed and the residue was dissolved in the minimal amount of ethyl acetate (and optionally a few drops of MeOH to enhance solubility). This solution was added to cold ether, resulting in the precipitation of the crude peptide. The suspension was centrifuged and the pellet was washed once with cold ether and dried after centrifugation.

6.4.2 Solid-phase synthesis of the symplocamide A model Ahp cyclodepsipeptide 19 Coupling of (S)-9-(Allyloxy)-8-(bis(tert-butoxycarbonyl)amino)-9-oxonon-4-enoic acid (16) to NovaPEG amino resin

NovaPEG amino resin (0.66 mmol/g, 1.00 g, 0.66 mmol) was transferred to a glass solid- phase reactor under an argon atmosphere and swollen in a mixture of 10% anhydrous DMF in anhydrous DCM for 30 min under gentle shaking.

(S)-9-(Allyloxy)-8-(bis(tert-butoxycarbonyl)amino)-9-oxonon-4-enoic acid (16, 706 mg, 1.60 mmol, 2.4 eq.), HOBt (446 mg, 3.30 mmol, 5 eq.) and DIC (514 μL, 3.30 mmol, 5 eq.) were dissolved in a 10% mixture of anhydrous DMF in anhydrous DCM (10 mL) and stirred for 20 min. This pre-activated solution was added to the pre-swollen resin and shaken for 24 h at RT. The reaction solution was removed and the resin was washed with DMF (2x 1 min), MeOH (2x 1 min) and DCM (2x 1 min). To eliminate remaining amino groups on the resin, a capping reaction was carried out. To this end, the resin was shaken with a mixture of acetic anhydride/DIEA/DCM (1:1:3) for 3 h and washed with DMF (2x 1 min), alternately with MeOH and DCM (3x 1 min) and finally with DMF (2 x 1 min). A Kaiser test performed according to method G then indicated the complete capping of all unreacted amines.

The resin loaded with the Ahp precursor will be called NovaPEG-Ahp5COAll (17) in the following descriptions.

Coupling of Fmoc-Cit-OH to NovaPEG-Ahp5-COAll (17)

The resin 17 (0.66 mmol) was deprotected according to method A. The coupling of Fmoc-Cit-OH to the resin was performed using method D. The amounts of reagents used were:

Fmoc-Cit-OH: 1.05 g, 2.64 mmol HOBt: 332 mg, 2.64 mmol HBTU: 877 mg, 2.31 mmol DIEA: 327 μL, 1.98 mmol.

After the regular washing, an additional washing step was carried out using alternately anhydrous MeOH and DCM (3x 1 min) and finally anhydrous ether (3x 1 min). The resulting resin 18 was dried under high vacuum overnight.

The Fmoc determination carried out with 6.4 mg of resin according to method F revealed a resin loading of 0.20 mmol/g (30%).

Sequential coupling of Fmoc-Thr-OH and Fmoc-Gln(Trt)-OH to NovaPEG-Ahp5-COAll-Cit4- Fmoc (18)

NovaPEG-Ahp5-COAll-Cit4-Fmoc (18, loading of 0.20 mmol/g, 1.12 g, 0.22 mmol) was deprotected according to method B. The coupling of Fmoc-Thr-OH*H2O to the resin was

performed using method D. The following amounts of reagents were used: Fmoc-Thr-OH*H2O: 322 mg, 0.90 mmol

HOBt: 121 mg, 0.90 mmol HBTU: 298 mg, 0.78 mmol DIEA: 111 μL, 0.67 mmol.

The coupling reaction was performed for 2 h. A Kaiser test performed according to method G indicated a complete coupling reaction.

NovaPEG-Ahp5-COAll-Cit4-Thr3-Fmoc (0.20 mmol) was deprotected according to method B. The coupling of Fmoc-Gln(Trt)-OH to the resin was performed using method D. The amounts of reagents were:

Fmoc-Gln(Trt)-OH: 547 mg, 0.90 mmol HOBt: 121 mg, 0.90 mmol

HBTU: 298 mg, 0.78 mmol DIEA: 111 μL, 0.67 mmol.

The coupling was performed for 2 h. A Kaiser test following method G indicated a complete coupling reaction.

After the regular washing, an additional washing step was carried out using alternately anhydrous MeOH and DCM (3x 1 min) and finally anhydrous ether (3x 1 min). The resulting resin 19 was dried under high vacuum overnight.

The Fmoc determination carried out with 5.1 mg of resin according to method F revealed a resin loading of 0.17 mmol/g (77%, 2 couplings).

Coupling of butyric acid to NovaPEG-Ahp5-COAll-Cit4-Thr3-Gln2(Trt)-Fmoc (19) and esterification of the threonine sidechain with Fmoc-Val-OH

NovaPEG-Ahp5-COAll-Cit4-Thr3-Gln2(Trt)-Fmoc (19, loading of 0.17 mmol/g, 1.14 g, 0.20 mmol) was deprotected according to method B. The coupling of butyric acid to the resin was performed using method D. The amounts of reagents were:

butyric acid: 73 μL, 0.80 mmol HOBt: 108 mg, 0.80 mmol HBTU: 265 mg, 0.70 mmol DIEA: 99 μL, 0.60 mmol.

The coupling reaction was performed for 2 h and a Kaiser test performed according to

method G indicated a complete coupling reaction after this time.

The esterification of the threonine sidechain was carried out following method E. The amounts of reagents used for this reaction were:

Fmoc-Val-OH: 679 mg, 2.00 mmol, 10 eq. DIC: 312 μL, 2.00 mmol, 10 eq.

4-(dimethylamino)pyridine: 24 mg, 0.2 mmol, 1 eq.

After the washing protocol, the resin was washed additionally with anhydrous solvents: alternately MeOH and DCM (3x 1 min) and ether (3x 1 min). The resulting resin 20 was dried overnight under high vacuum.

An Fmoc determination performed according to method F with 4.9 mg of resin gave a loading of 0.15 mmol/g (79%, 2 couplings).

Sequential coupling of Fmoc-NMePhe-OH and Fmoc-Ile-OH to NovaPEG-Ahp5-COAll-Cit4- Thr3(O-CO-Val8-Fmoc)-Gln2(Trt)-But1 (20)

NovaPEG-Ahp5-COAll-Cit4-Thr3(O-CO-Val8-Fmoc)-Gln2(Trt)-But1 (20, loading of 0.15 mmol/g, 1.07 g, 0.16 mmol) was deprotected according to method B. The coupling of Fmoc-

NMePhe-OH to the resin was performed using method D. The amounts of reagents were:

Fmoc-NMePhe-OH: 199 mg, 0.64 mmol HOBt: 86 mg, 0.64 mmol