Hembras sobrevivientes

This alternative synthetic strategy required the synthesis of building blocks such as the 4-(S)- amino-(E)-pentenoic acid moiety. This compound was readily synthesized from Boc-Ala-OH by adapting a protocol introduced by Pollini and coworkers.[147] In the first step, Boc-Ala-OH was converted to the Weinreb amide by reaction with DCC and N,O-dimethyl hydroxylamine. After a short work-up protocol, the resulting Weinreb amide was directly transformed to the aldehyde by reduction with LiAlH4. The aldehyde was then transformed directly to the α,β-

unsaturated methyl ester 37 by a Wittig reaction with methyl-2- (triphenylphosphoranylidene)acetate. The desired 4-(S)-amino-(E)-pentenoic acid building block 37 was obtained after chromatographic work-up in a yield of 59% starting from Boc- alanine. In the next step, this building block was deprotected and coupled with alanine methyl ester hydrochloride to obtain the dipeptide intermediate 38.

38 was then converted to the tripeptides required for the synthesis of the Sym4 derivatives

as well as for the Sym4mmp→Ala _{derivatives. To obtain the tripeptide for the Sym4 derivatives} (i. e. the tripeptide with the methyl-methoxypyrrolinone residue), the dipeptide 38 was deprotected C-terminally using lithium hydroxide. The resulting free acid was then coupled with Meldrum’s acid to obtain a highly reactive enol intermediate that rearranges to form the pyrrolidine-2,4-dione upon refluxing in acetonitrile. The methylation was carried out under Mitsunobu conditions using DEAD, methanol and triphenylphosphine providing the methyl-methoxypyrrolinone-modified dipeptide 39 in 90% yield from the protected dipeptide 38.[142] _{The tripeptide 40 was accessed by sequential Boc deprotection and}

coupling with Boc leucine using HOBt/HBTU activation and acetonitrile as the solvent.[148]

The tripeptide 40 was obtained in 85% yield.

In order to access the Sym4mmp→Ala derivatives, the dipeptide 38 was coupled with Boc leucine in the next step. So, first the Boc group was removed using trifluoroacetic acid in dichloromethane and the free amine intermediate was then coupled with Boc leucine. The tripeptide 41 was obtained in 99% yield using the same coupling conditions as for the synthesis of the tripeptide 40 (Fig. 71).

Figure 71: Synthesis of the C-terminal tripeptides 40 and 41. Reagents and conditions: (a) N,O-

dimethylhydroxylamine hydrochloride, Et3N, DCC, CH2Cl2, rt, 1 h; (b) LiAlH4, Et2O, 0 °C, 20 min; (c)

Ph3P=CHCOOMe, CH2Cl2, rt, o/n (59%, 3 steps); (d) LiOH, THF/MeOH/H2O (1.67:1:0.67), rt, 5 h; (e) H-Ala-OMe,

HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, o/n (46%, 2 steps); (f) LiOH, THF/MeOH/H2O (1.67:1:0.67), rt, 5 h; (g) 2,2-

dimethyl-1,3-dioxane-4,6-dione, DMAP, EDC*HCl, CH2Cl2, -10 °C to rt, o/n; (h) CH3CN, reflux, 1 h; (i) Ph3P,

MeOH, DEAD, THF, rt, o/n (90%, 4 steps); (j) TFA/CH2Cl2 (1:1), rt, 3 h; (k) Boc-Leu-OH, HOBt, HBTU, DIEA, CH3CN,

0 °C to rt, o/n (85%, 2 steps); (l) TFA/CH2Cl2 (1:1), rt, 2 h; (m) Boc-Leu-OH, HOBt, HBTU, DIEA, CH3CN, 0 °C to rt,

o/n (99%, 2 steps).

With the two C-terminal tripeptides in hands, the two different N-terminal depsipeptides were next synthesized. The synthesis for both depsipeptides started from (S)-2-hydroxy- isocaproic acid which was first converted to its methyl ester using thionylchloride and methanol. The resulting methyl ester was directly submitted to the esterification reaction without a further purification because of its high volatility. The esterification with N- methylated Boc isoleucine was carried out using DCC as the coupling reagent and DMAP as the additive to enhance the reaction rate and the depsipeptide 42 was obtained in a yield of 55% from (S)-2-hydroxy-isocaproic acid. The moderate yield can be explained with the high volatility of the (S)-2-hydroxy-isocaproic acid methyl ester: during the removal of residual thionyl chloride via distillation under reduced pressure, a partial evaporation of the product also occurred.

the free secondary amine intermediate with methyl iodide in dimethylformamide using DIEA as the base; the desired compound 43 was obtained in a yield of 38%. In the case of the methyl and alkyne-modified depsipeptide 44, the free secondary amide was reacted with propargyl bromide in acetonitrile using DIEA as the base. The reaction was warmed to 80 °C and required an overnight reaction, providing the desired methyl- and alkyne-modified depsipeptide 44 in 63% yield (Fig. 72).

Figure 72: Synthesis of the two depsipeptide fragments 43 and 44. Reagents and conditions:

(a) SOCl2 (2 M in CH2Cl2), MeOH, 0 °C to rt, o/n; (b) Boc-NMeIle-OH, DMAP, DCC, CH2Cl2, 0 °C to rt, o/n (55%, 2

steps); (c) TFA/CH2Cl2, rt, 2 h; (d) CH3I, DIEA, DMF, rt, 2 h (38%, 2 steps); (e) propargyl bromide (80% wt in

toluene), DIEA, CH3CN, 80 °C, o/n (63%, 2 steps).

With all four fragments in hands, the final assembly of the basic symplostatin 4 derivatives was initiated. To this end, the C-terminal depsipeptides were deprotected in order to obtain the free acid intermediates whereas the N-terminal Boc protecting group of the tripeptides needed to be cleaved. According to the original synthesis plan, the different fragments were then coupled using HOBt/HBTU activation in order to obtain the four different symplostatin 4 derivatives (Fig. 73). After the reaction work-up, all derivatives with the exception of

Sym4mmp→Ala were first purified by silica gel chromatography. The crude Sym4mmp→Ala and smaller fractions of the three other pre-purified compounds were also purified by HPLC in order to obtain highly pure material for the biological investigations.

Figure 73: Synthesis of the symplostatin 4 derivatives. Reagents and conditions: (a) LiOH, THF/MeOH/H2O

(1.67:1:0.67), rt, 5 h; (b) TFA/CH2Cl2 (1:1), rt, 2 h; (c) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, 5 h (91% (from 43)

after silica gel chromatography, 51% after additional HPLC, 3 steps); (d) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt,

o/n (39% (from 43) after HPLC, 3 steps); (e) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, o/n (82% (from 44) after silica

gel chromatography, 64% after additional HPLC, 3 steps); (f) HOBt, HBTU, DIEA, CH3CN, 0 °C to rt, 5 h (73%

(from 44) after silica gel chromatography, 53% after additional HPLC, 3 steps).

With the two alkyne-modified derivatives ≡Sym4 and ≡Sym4mmp→Ala _{available, the additional} derivatives modified with tags for the utilization in an activity-based approach were synthesized. The conditions for the click reaction were adapted from the conditions developed by Cravatt and coworkers for in vivo labeling.[139] However, the in vitro reactions carried out required an optimization of reaction conditions; in particular the amount of CuSO4 was increased greatly in order to achieve a good conversion of the starting material.

The corresponding reactions were monitored by LC-MS and stopped upon sufficient product formation. The purification of the different products turned out to be rather tedious and required customized conditions: The rhodamine-modified derivatives Rh-Sym4 and Rh-

Sym4mmp→Ala were purified by AluOx chromatography; in the case of Rh-Sym4 a second chromatography was necessary to obtain pure material. For the purification of the

stationary phase. In analogy to C18 HPLC purification an increasing gradient of acetonitrile in

water was used and allowed their purification in a satisfying manner (Fig. 74).

Figure 74: Synthesis of the different probes for the chemical biology investigations. Reagents and conditions:

(b) Tag-N3, CuSO4 (aq. solution), TBTA (100 mM in DMSO), TCEP (100 mM in H2O), H2O, rt, reaction monitoring

by LC-MS, chromatographic work-up.

3.3.3 Biological investigation of symplostatin 4 in the model plant Arabidopsis thaliana