Abstract: Tryprostatin A and B are prenylated, tryptophan-containing, diketopiperazine natural products, displaying cytotoxic activity through different mechanisms of action. The presence of the 6-methoxy substituent on the indole moiety of tryprostatin A was shown to be essential for the dual inhibition of topoisomerase II and tubulin polymerization However, the inability to perform late-stage modification of the indole ring has limited the structure-activity relationship studies of this class of natural products. Herein, we describe an efficient chemoenzymic approach for the late-stage modification of tryprostatin B using a cyclic dipeptide N-prenyltransferase (CdpNPT) from Aspergillus fumigatus, which generates novel analogs functionalized with allylic, benzylic, heterocyclic, and diene moieties. Notably, this biocatalytic functionalizational study revealed high selectivity for the indole C6 position. Seven of the 11 structurally characterized analogs were exclusively C6-alkylated, and the remaining four contained predominant C6-regioisomers. Of the 24 accepted substrates, 10 provided >50% conversion and eight provided 20-50% conversion, with the remaining six giving <20% conversion under standard conditions. This study demonstrates that prenyltransferase-based late-stage diversification enables direct access to previously inaccessible natural product analogs.
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
General procedure: All aza-peptidyl inhibitors and probes were synthesized by following the previously reported procedures 1, 2 with slight modifications. Fmoc protecting groups from Rink SS resin (0.75 mmol/g) were removed by treatment with 20percent piperidine in DMF for 15 min, followed by three washes with DMF. A 1.2 M solution of bromoacetic acid (10 eq) in NMP and DIC (10 eq) were added to the resin. The resin was shaken 1.5 hrs and washed three times. A solution of Mono-Fmoc protected hydrazide (3 eq) in NMP was added and shaken overnight. Resin loading was determined by Fmoc-quantification (0.2-0.3 mmol/g). A 0.5M solution of N-Fmoc-protected amino acid (3 eq.) and HOBt (3 eq.) in DMF and DIC (3 eq.) were added to the resin. The resin was shaken 1.5-2hrs. For each of the following steps, Fmoc-deprotection and coupling reactions were repeated as described above. Capping of N-terminal amine was achieved by shaking the resin with a 0.5 M solution of acetic anhydride (5 eq.) and DIEA (5 eq.) in DMF for 5 min.
General procedure: All aza-peptidyl inhibitors and probes were synthesized by following the previously reported procedures 1, 2 with slight modifications. Fmoc protecting groups from Rink SS resin (0.75 mmol/g) were removed by treatment with 20percent piperidine in DMF for 15 min, followed by three washes with DMF. A 1.2 M solution of bromoacetic acid (10 eq) in NMP and DIC (10 eq) were added to the resin. The resin was shaken 1.5 hrs and washed three times. A solution of Mono-Fmoc protected hydrazide (3 eq) in NMP was added and shaken overnight. Resin loading was determined by Fmoc-quantification (0.2-0.3 mmol/g). A 0.5M solution of N-Fmoc-protected amino acid (3 eq.) and HOBt (3 eq.) in DMF and DIC (3 eq.) were added to the resin. The resin was shaken 1.5-2hrs. For each of the following steps, Fmoc-deprotection and coupling reactions were repeated as described above. Capping of N-terminal amine was achieved by shaking the resin with a 0.5 M solution of acetic anhydride (5 eq.) and DIEA (5 eq.) in DMF for 5 min.
(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid[ No CAS ]
[ 58-85-5 ]
[ 198561-07-8 ]
C66H94N20O21S[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: 4.1.1. Peptide synthesis; 4.1.2; Solid-phase peptide synthesis (SPPS) was performed with standardFmoc chemistry on rink amide resin using an automated peptidesynthesizer (Syro I, Multisyntech). The resin was loaded into a5 mL reactor with a frit at the bottom. Swelling was performed bydispensing 1 mL DMF and incubating for 15 min (2) with 10 sshaking every minute. Fmoc deprotection was achieved by treatmentwith 40percent piperidine DMF for 3 min and 20percent piperidine inDMF for 12 min (10 s/min shaking). Peptide couplings were carriedout by double couplings with Fmoc-protected amino acids(5 equiv), HBTU (5 equiv), HOBt (5 equiv) and DIPEA (10 equiv) inDMF for 40 min (10 s/min shaking). At the respective position,Fmoc-F2Pmp-OH (3 equiv) was coupled in DMF (1 mL) by manualaddition using TBTU (3 equiv), HOBt (3 equiv) and DIPEA (6 equiv)for 3 h, after 3 min preactivation. In case of the sequences for which side-chain labeling with biotinor carboxyfluorescein was planned, an additional 4-methyltrityl-(Mtt-) protected lysine was coupled to the N-terminus. Toselectively remove the Mtt group the resin was washed for 1 minwith DCM (3), deprotection was then achieved by treatment with1.8percent TFA in DCM for 3 min (10). During the deprotection the DCMsolution turned yellow.For fluorescein-labeling of the amine side-chain 5(6)-carboxyfluorescein(3 equiv), HATU (3 equiv), HOAt (3 equiv) andDIPEA (6 equiv) were dissolved in DMF and pre-activated for3 min. The solution was aspirated and coupling was allowed toproceed for 1 h. This step was repeated 4 times.For biotin-labeling of the amine side-chain the resin waswashed for 1 min in NMP (3). D-(+)-Biotin (3 equiv), HATU(3 equiv), HOAt (3 equiv) and DIPEA (6 equiv) were dissolved inNMP and pre-activated for 3 min. The solution was aspirated andcoupling was allowed to proceed for 2 h. This step was repeated2 times. N-terminal acetylation (where applicable) was achieved by dispensing800 lL of a mixture of acetic anhydride/pyridine (1:9) andreaction twice for 5 min (10 s/min shaking). After each deprotection,coupling or acetylation step, 5 washings (1 min each) withDMF were performed (10 s/min shaking).After synthesis the resin was transferred in a 5 mL syringeequipped with a frit, washed with DCM for 1 min (3) and driedin high vacuum for at least 30 min. For cleavage 1 mL of a mixtureof TFA and TIS (20:1) was added. The syringe with the mixture waskept on a shaker for 3 h. Then the liquid phase was filtered into20 mL of ice-cold Et2O. Formed precipitate was centrifuged,washed with ice-cold Et2O (2 20 mL) and purified by HPLC. 4.1.2. Azide functionalization of the N-terminus; To the peptides with the longer carbon linker, 6-azidohexanoicacid was coupled (with standard coupling conditions) to the Nterminalamine.The N-terminal amine of the peptides with the shorter linkerwas converted to an azide functionality directly on solid support.Using the compound imidazole-1-sulfonyl-azide*HCl (synthesissee beneath) and modified conditions, which were reported forsolution phase chemistry from Goddard?Borger and Stick:8 Theresin was washed for 1 min each with DCM (2), DCM/MeOH(2) and MeOH (3). Then (for 40 mg resin, loading= 0.62 mmole/g) 1.4 equiv of imidazole-1-sulfonyl-azide*HClin 1 mL MeOH and 100 ll of a saturated and centrifuged solutionof CuSO4*5H2O was added. After 1 min, DIPEA (1.8 equiv) wasadded and the coupling was allowed to proceed for 1 h andrepeated once more with an intermediate washing with MeOH(3 1 min).
Chemistry
Pharmaceutical Intermediates
Inhibitors/Agonists
Material Science
For Research Only
120K+ Compounds
Competitive Price
1-2 Day Shipping
