Development of a solid-phase strategy with Ahp precursor

With the Ahp precursor II (16) in hands, a solid-phase synthesis strategy using this building block could finally be investigated. To this end, a synthesis of the natural product symplocamide A was envisaged. This natural product was isolated in 2007 and elucidated as a potent chymotrypsin inhibitor.[24] _{Furthermore, at the beginning of this dissertation no}

synthesis of symplocamide A had been reported.

Initially, first model studies on a symplocamide derivative in which the 3-bromo-4-methyl N- methyl tyrosine was replaced by an N-methyl phenylalanine moiety were performed. To this end, the Ahp precursor was coupled to amino-methylated polystyrene resin and the peptide sequence was assembled solely with standard conditions (HOBt/HBTU peptide coupling; DIC/DMAP esterification; PyBrOP coupling to mPhe) to test for general problems in the synthesis route. The conditions for the oxidative cleavage from the solid phase were adapted from the protocol provided by Meldal and coworkers, using OsO4 and NaIO4 in a one-pot

reaction and adding DABCO to suppress the formation of hydroxymethyl ketones (Fig.54).[108]

The peptide was cleaved from the resin and the crude peptide was purified by HPLC. A putatively pure peptide fraction was obtained by HPLC purification. The subsequent analysis of this fraction by LC-MS revealed however that the fraction still contained a large quantity of impurities.[115]

The extent of impurities found after resin cleavage gave rise to the suspicion that the polystyrene core of the resin used in the synthesis was not stable under the cleavage conditions. This hypothesis was reinforced by a simple test reaction in which the amino- methylated polystyrene was acylated with acetic anhydride and base. The resin was then subjected to the reaction conditions for the oxidative cleavage. A LC-MS analysis of the cleaved material showed clearly the same impurities that had been observed after the cleavage of the model peptide, proving that the polystyrene core is indeed instable under the cleavage conditions.

To overcome this obstacle, two alternative resins (NovaPEG amino resin and aminoPEGA resin, both from Novabiochem) that consist of a polyethylene-glycol core instead of a polystyrene core were investigated under the same conditions as the amino-methylated resin. For both resins, no impurities were observed after the cleavage. For practical reasons, NovaPEG amino resin was therefore chosen as the solid support in subsequent syntheses. In a first study the model peptide that had previously been synthesized on the amino- methylated polystyrene was re-synthesized on the NovaPEG amino resin. Consequently, reaction conditions adapted to the different handling of the polyethylene-glycol-based resin had to be developed. For example, this resin shows a swelling behavior that is significantly different from the behavior of customary polystyrene-based resins and is especially difficult to dry to a constant weight. Therefore, tedious washings with anhydrous solvents and prolonged drying under high vacuum were performed prior to reactions that required a dry resin (for example an Fmoc determination). Furthermore, the coupling time for a regular peptide coupling was extended to four hours or sometimes even overnight to achieve complete conversions.

The coupling of the Ahp precursor to the NovaPEG amino resin was achieved with DIC/HOBt activation, followed by a capping step to block the remaining free amines. The removal of the Boc groups from the Ahp precursor was achieved by using a mixture of 50% TFA in dichloromethane. This was followed by a neutralization step with 10% triethylamine in dichloromethane. For the regular peptide couplings HOBt/HBTU activation was applied after

the deprotection of the N-terminus. For the removal of the Fmoc protection a mixture of 20% piperidine in DMF was used.

After the coupling of the P1 amino acid citrulline, the resin was dried thoroughly to enable

the determination of the initial loading by an Fmoc determination. A full loading of the resin was not intended to prevent side-reactions during the on-resin macrolactamization reaction. Instead, a lower loading to ensure pseudo-dilution conditions similar to the conditions used for cyclization reactions in solution was envisaged. This was achieved by using 2.4 equivalents of the Ahp precursor 16 in the loading reaction which resulted in an initial loading of efficiency of 30% (0.20 mmol/g resin).

Next, a threonine building block was incorporated that did not feature a protecting group on the side chain hydroxyl function. The secondary alcohol of the threonine side chain is much less reactive than a primary amine in coupling reactions and therefore no laborious protection/deprotection sequence prior to the esterification on this moiety is therefore required. In the next steps, Fmoc-Gln(Trt)-OH was coupled, followed by butyric acid.

The subsequent esterification needed different activating reagents and a higher excess of reagents than a standard peptide coupling. To this end, the conditions for the esterification of the threonine side chain with valine were optimized. The best protocol consisted of four repetitive short-time (2 hours) couplings, using 10 equivalents of Fmoc-Val-OH and the coupling reagent (DIC) but only 1 equivalent of coupling additive (DMAP) in a mixture of dichloromethane and dimethylformamide. This procedure provided excellent coupling yields that were superior compared to the yields obtained for example by a single 24 hour coupling as determined by resin loading determination after the esterification (Fig. 55).

Figure 55: First steps in the synthesis of a symplocamide A model peptide on amino NovaPEG resin. Reagents

and conditions: (a) i. amino NovaPEG resin (0.66 mmol/g), 16 (2.4 eq.), HOBt, DIC, CH2Cl2/DMF (9:1), 24 h; ii.

CH2Cl2/DIEA/Ac2O (3:1:1), 3 h (b) i. TFA/CH2Cl2 (1:1), 1 h; ii. Et3N/CH2Cl2 (1:9), 2x 10 min; iii. Fmoc-Cit-OH, HOBt,

HBTU, DIEA, DMF, 5 h (30%, by Fmoc determination, 2 steps); (c) i. piperidine/DMF (1:4), 2x 15 min; ii. Fmoc- Thr-OH, HOBT, HBTU, DIEA, DMF, 2 h; iii. piperidine/DMF (1:4), 2x 15 min; iv. Fmoc-Gln(Trt)-OH, HOBT, HBTU, DIEA, DMF, 2 h (88% by Fmoc determination, 2 couplings); (d) i. piperidine/DMF (1:4), 2x 15 min; ii. butyric acid, HOBT, HBTU, DIEA, DMF, 2 h; iii. Fmoc-Val-OH, DIC, DMAP, CH2Cl2/DMF (9:1), 4 x 2 h (79% by Fmoc

determination, 2 couplings).[115]

Next, Fmoc-N-methyl phenylalanine was coupled, followed by cleavage of the Fmoc group. The coupling of Fmoc-Ile-OH to the N-methylated phenylalanine required harsher coupling conditions and was achieved by using PyBrOP as the coupling reagent and prolonging the reaction time to 24 hours. PyBrOP is the coupling reagent of choice for the difficult coupling to sterically hindered and less reactive amino acids such as N-methylated amino acids. After the attachment of the final amino acid isoleucine, the Fmoc protection on this amino acid as well as the allyl protection on the Ahp precursor needed to be removed to set the stage for the cyclization reaction. The Fmoc group was cleaved using the standard protocol, the regular washing protocol was supplemented by a tedious washing protocol and thorough drying in order to prepare the resin for the removal of the allyl protection. The removal of the allyl group was carried out using the protocol established by Vaz, using Pd(PPh3)4 as

solvent.[105] In order to remove residual catalyst, a tedious washing protocol was again required after the cleavage reaction.

For the on-resin macrolactamization, the conditions established during the first synthesis on the amino-methylated polystyrene resin were employed. Consequently, PyBOP as the coupling reagent along with the additive HOBt and the base DIEA were used and the reaction time was extended to 24 hours to ensure a complete conversion. As a further optimization, first HOBt and DIEA were added as a solution in DMF to the resin for the pre-activation of the free acid, followed by addition of PyBOP dissolved in DMF; the resulting suspension was then shaken for 24 hours. After the customary washing of the resin, a Kaiser test was carried out to check for full conversion, revealing a complete reaction after 24 hours. In addition, no intermolecular coupling products could be observed by LC-MS analysis after the cleavage from the resin, indicating the applicability of the found reaction conditions (Fig. 56).

In the first attempt to synthesize a symplocamide A model peptide, cleavage of the crude peptide from the resin was achieved by using the reaction conditions reported by the Meldal group.[108] _{In this procedure, a precipitation of sodium periodate and DABCO from the water-}

THF mixture was, however, observed. To prevent the precipitation as well as to account for the different swelling properties of the PEG-based resin, the cleavage conditions were adjusted. Sodium periodate and DABCO were dissolved individually in water, using ultrasonication to promote dissolution of the reagents. The reagent solutions were then added to the dry resin, which was subsequently allowed to swell in this mixture for 10 minutes. After this time, THF was added to obtain a 1:1 solvent mixture and the resin was shaken for 2 minutes. After this time, a solution of osmium tetroxide in t-BuOH was carefully added and the resin was shaken for 20 hours. For work-up, the highly toxic osmium tetroxide was quenched with sodium metabisulfite. The extraction of the resulting aqueous solution with organic solvents turned out to be rather tedious and best results were obtained with ethyl acetate.

In order to investigate the efficiency of the cleavage protocol, the cleavage of the crude peptide from the resin was carried out twice. The resulting crude mixtures were analyzed separately to investigate the impact of a double cleavage procedure on the yield of the solid-

side chain was purified by HPLC, yielding the desired Ahp cyclodepsipeptide in 1.2% overall yield (based on 18).[115]

Next, a cleavage of the trityl protecting of the glutamine side chain was attempted. To this end, a solution containing 95% trifluoroacetic acid and 2.5% water and TIS, respectively, was used. However, when the Ahp cyclodepsipeptide 24 was submitted to these conditions, a rapid and almost complete decomposition of the peptide was observed, owing most probably to the strong acidic conditions (Fig. 56).[115] This finding indicates that Ahp cyclodepsipeptides are not stable to strongly acidic conditions, calling for an adjustment of the synthetic route (via the use of alternative building blocks with differently cleavable protecting groups).

Figure 56: Synthesis of a symplocamide A model peptide on amino NovaPEG resin (continued). Reagents and

conditions: (a) i. piperidine/DMF (1:4), 2x 15 min; ii. Fmoc-NMePhe-OH, HOBT, HBTU, DIEA, DMF, 2 h; iii. piperidine/DMF (1:4), 2x 15 min; iv. Fmoc-Ile-OH, PyBrOP, DIEA, DMF, 24 h (60%, by Fmoc determination, 2 couplings); (b) i. piperidine/DMF (1:4), 2x 15 min; ii. Pd(PPh3)4, morpholine, CH2Cl2, 30 min; (c). PyBOP, HOBt,

DIEA, DMF, 24 h; (d) i. NaIO4, OsO4 (0.1 M in t-BuOH), DABCO, H2O/THF (1:1), 20 h; ii. HPLC purification (1.2 %

Thus, in later syntheses of Ahp cyclodepsipeptides, protecting groups requiring strong acid for their removal should be avoided or cleaved before the formation of the Ahp moiety. Fortunately, the overall strategy for the synthesis of the Ahp cyclodepsipeptides however proved to be feasible.