MS And NMR Spectroscopic Analysis Of Bisbibenzyls Isolated From Bryophytes And Their Application In High Throughput Screening

Bryophytes are higher plants from aquatic to terrestrial transition.Although the plants are widely distributed,it is difficult to collect enough amount for the conventional separation and isolation of the chemical compositions,due to the low density,mixed species,and difficult distinction.Mass spectrometry(MS) is an effective method to determine the structural information of organic compounds.Multi-stage MS-MS data not only provide molecular weight information,but also give the structural information,combined with the data elements.Therefore,it has played an important role in the new high-throughput screening of new leads.To provide a fast-track approach for discovering the known and new bibenzyl compounds form liverworts,guide the phytochemical study of bryophytes,prevent blindness,and enhance the purpose of separation and to improve the efficiency of separation,we established a method based on gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry(LC-MS) in the rapid screening of bibenzyl compounds from bryophytes.To achieve a rapid screening of known and unknown bibenzyls from liverworts,a method using gas chromatography(GC) electron impact time-of-flight(EI-TOF) and liquid chromatography(LC) ESI triple quadrupole mass spectrometry(ESI-TQ-MS) was developed to detailedly analysize mass spectrometry of bisbibenzyl compounds. Firstly,one bibenzyl and 15 structurally similar compounds as bisbibenzyl standards were analyzed to obtain the mass spectrometry fragmentation patterns,and the possible secondary fragment ions were also carried out,to find the possible fragmentation rules and mechanism.Fifteen bisbibenzyls were classified into three types according the connecting styles between two bibenzyl units:one C-O-C connection(group A);two C-O-C connections(group B);one C-O-C connection;and one C-C connection(group C).The above three types of bisbenzyls possessed different EI-MS and ESI-MS/MS fragments, and the connecting styles and substituents would be verified by MS fragments.The isomers were also distinguished,however,the connecting sites and C-O-C sites were unable to be assigned.On the basis of above results,a simple and rapid liquid chromatography diode array tandem mass spectrometry(LC-DAD/MS) method was developed for screening of benzyl compounds form the Bryophytes crude extracts in the ppm level.Three China liverworts Marehantia polymorpha,Pagiochasma intermedium,and Asterella angusta colleted from three different regions were screened using the LC-DAD/MS method.By comparing their MS data,UV absorption spectra and retention characteristics,7/12,8/5 and 8/9(known/unknown) bisbibenzyl compounds were detected in the above three liverworts.The bisbibenzyl,which comprise four benzene rings by methylene or methylene-linked,show some commonalities in its nuclear magnetic resonance spectroscopy.Chemical shift value in ¹H NMR(δ_H) presents ten to sixteen aromatic proton signals at about 5.10-7.40 ppm,with six to eight methylene proton signals atδ_H 2.0-3.0 ppm.In the ¹³C NMR spectrum,chemical shift values ofδ_C 110-160 ppm indicate aromatic carbons,and two to four methylene carbon signals often appear at aboutδ_C 30-40 ppm.The introduction of substituents on the benzene ring can cause changes in its spectral data.The chemical shift value of carbon linked to methoxy,hydroxyl,and biphenyl ether located at aboutδ_C 140-160 ppm,with a 5-10 ppm upfield shift to the carbon adjacent to it.Different substituted benzene ring has a different split coupling. Disubstituted benzene rings on the type of main-substituted with para-substituted appear AA'BB' coupling in ring A.Trisubstituted patterns on benzene rings,1,2,4-trisubstituted styles is encountered frequently and occurred mainly on the rings B and D. The pattern present two double peaks(d) and a double-double peak(dd) in the ¹H NMR spectrum.When there are more than three substituents,the pattern is easily identifiable with two singlets atδ_H 6.60-6.90 ppm or two doublets atδ_H 6.60-7.10 ppm.With the different connection,the bisbibenzyl compounds show different characteristics of the NMR spectrum.Compared to the presence of biphenyl linkage,the chemical shift values of carbon linked to the biphenyl ether shift downfield by 20-30 ppm.Of all the bisbibenzyls with 1-2 'ether oxygen bond in connection type,show a highfield-shifted characteristic resonance for H-3' withδ_H at 4.84-5.60 ppm.The text display the result with four parts which are biphenyl ether linkage between A,C ring (A-O-C),biphenyl linkage between them(A-C),biphenyl ether linkage between B,D ring(B-O-D),as well as biphenyl linkage of B,D ring(B-D).Based on the analysis of the structure of bisbibenzyl compounds and the observation of the spectral data of these compounds(¹H and ¹³C NMR),we could find a analysis approach for structure elucidation quickly and efficiently.