Synthesis And Anti-Liver Homogenate Degradation Study Of DOPA-Containing Dipeptides

Levodopa(L-DOPA)is widely used as a dopamine prodrug to treat the Parkinson’s disease.However,this application is significant hampered by L-DOPA’s low bioavailability(1-3%)and harmful side-effects arising from long-time administration.A promising strategy is to chemically modify L-DOPA with proper functional groups to produce a relatively stable precursor that after digestion or metabolism slowly releases L-DOPA at a controlled speed in the body.Since on the small intestine membrane there exist oligopeptide transporters,short peptides(usually less than 4 amino acids)can be directly absorbed into the blood circulation without the necessity of digestion.Thus,an ideal L-DOPA prodrug may be obtained by screening a series of synthesized L-DOPA-containing oligopeptides with proper biochemical experiments.Using acetonide protected L-DOPA intermediates such as FmocDOPA(Acetonide)-OH,Boc-DOPA(Acetonide)-OH,a series of about 19 L-DOPAcontaining liner dipeptides and 4 cyclodipeptides were synthesized,mainly through the solution-phase peptide synthesis protocol.The synthesis of L-DOPA-containing linear dipeptides is divided into two categories.Boc-DOPA(Acetonide)-AA-OtBu with LDopa in the left side chain was prepared by reacting Boc-DOPA(Acetonide)-OH prepared in the laboratory with 20 common amino acid tert-butyl ester hydrochlorides purchased.Or use the purchased Boc-AA-OSu to react with the Tfa-DOPA(Acetonide)-OMe prepared in the laboratory to obtain the protected linear dipeptide Boc-AA-DOPA(Acetonide)-OH on the right side of Dopa.The obtained protected form are very stable,ready to be purified and stored for future application.Finally,the target dipeptide can be obtained by removing the side chain protecting group with a high concentration of trifluoroacetic acid.As for the synthesis of L-DOPA-containing cyclodipeptides,we use Fmoc-protected amino acid reacted with methyl amino acid to form a protected linear dipeptide.Then Fmoc was removed from the obtained linear dipeptide in 20%piperidine solution at one step and the L-DOPA-containing cyclodipeptides with protected side chain was synthesized.Finally,the target cyclodipeptide containing free catechol group was obtained by using trifluoroacetic acid to remove the side chain protection group of the protected cyclodipeptides.The purity and identity of these dipeptides and cyclodipeptides were confirmed by HPLC,MS and NMR analysis.The stabilities of these dipeptides in liver homogenate of Sprague Dawley rat were measured with HPLC and primitive data showed that the amino sequences do influence the stability of the oligopeptides,the half-life of degradation varies from 10 minutes to 180 minutes.The half-life of H-DOPA-Asp-OH is as long as 174 minutes,while the shortest half-life of H-DOPA-Met-OH is only 16 minutes.That is to say,the H-DOPAAsp-OH has stronger anti-degradation ability.It should be more stable in human gastrointestinal tract,blood circulation and liver,and may have stronger antiParkinson’s disease activity.At present,the cytotoxicity and neuroprotective effects of L-DOPA oligopeptides and free radical scavenging ability experiments are under way.The stability of gastrointestinal tract and the screening of related animal models of Parkinson’s disease will be carried out in the follow-up.It is hoped that L-DOPA prodrugs with good anti-Parkinson’s disease potential will be screened eventually.