20151005 Ireland, Japan, China Scientists Share Nobel Prize for Medicine

394
84.8
220
84.5
中国医大一附院
294
82.7
天津医大总院
293
77.1
四川大学华西医院
208
88.0
内蒙古医大一附院
42
88.1
宁波龙赛医院
87
74.7
浦东人民医院
36
75.0
湖北秭归人民医院
6
66.7
3411/4055
82.6
耐药率（％）
120
MSSA
MRSA 100
88.9 83.9
利福平 庆大霉素 左氧氟沙星 克林霉素 环丙沙星 复方磺胺甲噁唑
红霉素 青霉素 苯唑西林
MSCNS(631株)和MRCNS(3411株)对抗菌药的耐药率（%）
120 100
80 60 40 20
0
MSCNS MRCNS
100 100 87
48.3 40.1
30.3
69.2
54.6
58
56.1 58.3
226
81.0
96
84.4
154
77.9
同济医院
471
87.7
广州一附院
17
82.4
北京医院
198
87.4
上海儿科医院
468
83.5
上海儿童医院
361
87.0
浙江邵逸夫医院
255
83.5
甘肃省人民医院
36
91.7
合计
医院 新疆医大一附院
凝固酶阴性葡萄球菌 MR株数/总株数 （%）
193
88.6
安徽医大一附院 昆明医大一附院
+ 数据分析 – 采用WHONET5.6软件统计分析数据。

SINO-OVERSEAS GRAPEVINE & WINE贺兰山东麓酿酒葡萄需水量变化特征及影响因素分析尚艳1,2，张磊1,3*，王静1,3，李娜1,3，徐蕊1,3，姜琳琳1,3，郭伟4（1. 中国气象局旱区特色农业气象灾害监测预警与风险管理重点实验室/宁夏气象防灾减灾重点实验室，宁夏银川 750002；2. 宁夏气象台，宁夏银川 750002；3. 宁夏气象科学研究所，宁夏银川 750002；4. 西鸽酒庄，宁夏吴忠 751100）摘 要：明确贺兰山东麓酿酒葡萄生育期需水量的变化特征及其影响因素，可为合理调剂供水量、提高水资源利用效率、减少水土流失等提供科学依据，为区域酿酒葡萄品质及产量提升提供理论支撑。

研究基于1981—2020年贺兰山东麓产区6个基本气象站点的资料，采用Penman-Monteith 及有效降雨量公式计算了酿酒葡萄生育期的需水量、缺水量及有效降雨量，并通过通径分析研究风速、平均气温、日照时数、有效降水、最高气温等气象要素对酿酒葡萄需水量的影响。

结果表明：1981—2020年间，贺兰山东麓产区酿酒葡萄生育期需水量为458.04～546.74 mm ，总体呈现“低-高-低”的抛物线走势，表现为4月需水量最少，7月需水量最大；有效降雨呈增加趋势，需水量、缺水量均呈减少趋势；逐年水分盈亏指数为﹣87%～﹣49%，石嘴山、永宁、贺兰、青铜峡、银川产区水分盈亏指数均呈上升趋势，仅同心产区呈下降趋势；影响酿酒葡萄生育期需水量的要素有风速、平均气温、最高温度、日照时数和有效降雨，其中风速和平均气温对需水量的影响较大。

关键词：贺兰山东麓；酿酒葡萄；需水量；气象要素中图分类号：S663.1 文献标志码：A DOI ：10.13414/ki.zwpp.2023.05.001收稿日期：2022-10-29基金项目：国家自然科学基金项目（41965009）；宁夏自然基金项目（2020AAC03466）作者简介：尚艳，女，助理工程师，研究方向为农业气象。

Total Synthesis of 1-O -Methyllateriflorone**K.C.Nicolaou,*Pradip K.Sasmal,Hao Xu,Kenji Namoto,and Andreas RitzØnWith its unique spiroxalactone framework carrying a prenyl-ated dihydrobenzoquinone moiety and a trioxatetra-cyclo[7.4.1.02,7.02,11]tetradecane system,lateriflorone (1)rep-resents an unusual synthetic challenge.Reported in 1999,thisnovel natural product [1]was isolated from the stem bark of Garcinia Lateriflora Bl (Guttiferae )collected from Indonesia,and it exhibits potent cytotoxicity against the P388cancer cell line (ED 50=5.4m g mL À1).Its seemingly fragile structure was secured by spectroscopic and X-ray crystallographic analysis.The secret of its stability,particularly at the spiroxalactone–dihydroquinone junction,is probably due to the syn arrange-ment between the C2’proton and the C3’ester grouping which locks the leaving group in place,avoiding the b -elimination pathway that may lead to its rupture.The intriguing structural features of lateriflorone,coupled with its biological activity,prompted us to seek a possible pathway for its construction in the laboratory.Herein we report our findings thus far in this project,including the first total synthesis of 1-O -methyllateriflorone (2).We planned our synthesis of 1-O -methyllateriflorone (2)based on the expectation that the ester bond would be theeasier bridge to forge between the two domains of the molecules.The retrosynthetic analysis began with the dis-connection of the spiroxalactone moiety by rupturing its ethereal C ÀO bond,thus unraveling prenylated quinone 3as a possible precursor (Scheme 1).The next rational disconnec-tions,namely retro-aromatization of 3and disassembly of its precursor 4at the ester bond led to fragments 5and 6as suitable starting materials.Given our previous studies on forbesione,[2,3]a naturally occurring substance related to the present target,the cage ring system 6[4]can be traced to the benzenoid compound 7.The coupling partner 5can be obtained from a simple benzene derivative.The required prenylated 2,2’-dimethybenzopyran frag-ment 5was synthesized as summarized in Scheme 2.Thus,2,3-dihydroxybenzaldehyde (8)was selectively benzylated at the 3-position according to a literature procedure [5]to afford compound 9,which was converted into bromophenol 10in61%overall yield by the following sequence:a)bromination para to the phenolic group;b)protection as a MOM ether,c)oxidation with m -CPBA;and d)cleavage of the resulting formate ester with NaHCO 3(for abbreviations of reagents and protecting groups,see legends in schemes).Protection of the phenolic group in 10as a TIPS ether to form 11proceeded smoothly under standard conditions (TIPSCl,imid,96%).Boronation of 11required premixing of the substrate withB(O i Pr)3prior to addition of t BuLi in Et 2O at À788C.Theresulting borate derivative was then oxidized with H 2O 2under basic conditions (NaOH),and the resulting phenolic product was methylated to afford 12in 76%overallyieldfrom 11.Scheme 1.Retrosynthetic analysis of lateriflorone (1).[*]Prof.Dr.K.C.Nicolaou,Dr.P.K.Sasmal,H.Xu,Dr.K.Namoto,Dr.A.RitzØn Department of Chemistry andThe Skaggs Institute for Chemical BiologyThe Scripps Research Institute10550North Torrey Pines Road,La Jolla,CA92037(USA )Fax:(+1)858-784-2469E-mail:***************and Department of Chemistry and Biochemistry University of California,San Diego9500Gilman Drive,La Jolla,CA92093(USA )[**]We thank Dr.D H.Huang and Dr.G.Siuzdak for NMRspectroscopic and mass spectrometric assistance,respectively,andDr.R.K.Chadha for X-ray crystallographic analysis.Financialsupport for this work was provided by The Skaggs Institute forChemical Biology and the National Institutes of Health (USA).4225Angew.Chem.Int.Ed.2003,42,4225–4229DOI:10.1002/anie.2003518052003Wiley-VCH Verlag GmbH &Co.KGaA,WeinheimHydrogenolysis of the benzyl group in this new product followed by reaction with CH CC(Me)2OCOCF 3[6]in the presence of DBU and CuCl 2resulted in the formation ofpropargylic ether 13in 76%overall yield.Heating of the latter compound in refluxing xylene induced a Claisen rearrangement,[7]furnishing,after desilylation with TBAF,benzopyran derivative 14in 93%overall yield.Finally,a second propargyl ether was introduced in 14;selective reduction (H 2,Lindlar catalyst)of the acetylenic group to the corresponding olefin and Claisen rearrangement under thermal conditions provided the targeted prenylated benzo-pyran segment 5in 53%overall yield.The sequence devised and executed for the synthesis of precursor 7is shown in Scheme 3.Thus,2,3,4-trihydroxybenz-aldehyde (15)was perbenzylated (K 2CO 3,BnBr,KI)and then selectively debenzylated at the position adjacent to the aldehyde group by the action of MgBr 2·Et 2O.Subsequent para bromination (Br 2,NaOAc)and reduction of the aldehyde with NaBH 4afforded dihydroxy compound 16in 57%overall yield.Acetonide formation (Me 2C(OMe)2,TsOH)followed by lithium–halogen exchange (n BuLi),borate formation (B(OMe)3),and oxidation (H 2O 2,NaOH)led to phenolic substrate 17in 86%overall yield.Methylation of 17followed by hydrogenolysis of its benzyl ethers led to dihydroxy derivative 18in 97%overall yield.The dipotas-sium salt of 18,generated by addition of KO t Bu,was suspended in acetonitrile and treated with bromoisobutyr-aldehyde in the presence of 18[crown]-6to afford a mixture of regioisomeric lactols 19a ,b (ca.1:1,70%yield),which reacted with methylene phosphorane (MeP +Ph 3Br À,NaHMDS)to provide olefins 20a ,b (ca.1:1,75%yield).Reiteration of the last two steps furnished,via 21a ,b ,the required dialkene 7in 60%overall yield.Upon heating in DMF at 1208C,the methoxy derivative 7entered into the expected Claisen rearrangement/Diels–Alder cascade [3,8]leading through the indicated intermediate products to 6and 6’in 47and 42%yields,respectively (Scheme 4).X-ray crystallographic analysis of 6’[9](see ORTEP drawing,Scheme 4)revealed its structure,and,by extension,that of 6;both structures 6and 6’were also supported by NOE studies (see Scheme 4).Selective removal of the acetonide group from 6(TsOH,MeOH),followed by a two-step oxidation protocol (1)DMP ,2)NaClO 2),led to hydroxy carboxylic acid 22in 91%overall yield.An analogous sequence to that shown in Schemes 3and 4was originally employed to construct the dihydroxy acid 22a .Attempted coupling of this fragment,22a ),with the phenolic domain 5(see Scheme 2),however,proved futile,presumably as a result of complications instigated by the free hydroxy group adjacent to the ketone moiety.Thus,this problem led to the targeting and adoption of the methoxy counterpart of this compound,substrate 22,for our subsequent studies.The coupling of 22with benzopyran system 5(see Scheme 2)was smoothly realized under the influence of EDC and 4-DMAP (64%yield)to furnish the required advanced intermediate 4as shown in Scheme 4.Having assembled the entire lateriflorone carbon skeleton as expressed in compound 4,its MOM group was selectively cleaved upon exposure to acidic conditions (HCl,MeOH/Et 2O)leading to a rapidly and spontaneously equilibrating mixture of phenolic esters 23a ,b (ca.1:1,96%yield)(Scheme 4).Oxidation of this mixture under a varietyofScheme 2.Synthesis of prenylated 2,2’-dimethylbenzopyran fragment 5:a)NaH (2.5equiv),BnBr (1.0equiv),THF,258C,48h,72%;b)Br 2(1.1equiv),NaOAc (1.2equiv),AcOH,258C,2h;c)Et 3N (5.0equiv),4-DMAP (0.1equiv),MOMCl (3.0equiv),258C,16h;d)m -CPBA(1.1equiv),0!258C,CH 2Cl 2,6h;e)aqueous saturated NaHCO 3,16h,61%for four steps;f)TIPSCl (1.5equiv),imid (2.0equiv),DMF,258C,2h,96%;g)premix 11and B(O i Pr)3(2.2equiv)in diethyl ether;then t BuLi (2.2equiv)at À788C,2h,À78!08C,1h;then MeOH,aque-ous NaOH (10%;4.8equiv),H 2O 2(5.0equiv),08C,30min,86%;h)K 2CO 3(10equiv),MeI (10equiv),acetone,258C,16h,88%;i)Pd(OH)2/C (10wt %;10%),H 2(1atm),EtOAc/EtOH (1:1),258C,30min,100%;j)propargyl alcohol (1.3equiv),DBU (1.5equiv),TFAA (1.2equiv),MeCN,08C,30min;DBU (1.35equiv),CuCl 2(0.01%equiv),10min;then TFApropargyl ester,4h,08C,76%(90%based on 84%conversion);k)xylene,1408C,30min;l)TBAF (1.5equiv),THF,08C,5min,93%for two steps;m)propargyl alcohol (1.3equiv),DBU (1.5equiv),TFAA (1.2equiv),MeCN,08C,30min;DBU(1.35equiv),CuCl 2(0.01%equiv),10min;then TFApropargyl ester,4h,08C,80%(90%based on 86%conversion);n)Lindlar catalyst (10wt %),H 2(1atm),EtOAc,quinoline (3equiv),258C,2h,95%;o)DMF,1208C,1h,70%.Bn =benzyl,4-DMAP =4-dimethylaminopyr-idine,MOM =methoxymethyl,m -CPBA =m -chloroperbenzoic acid,TIPS =triisopropylsilyl,imid =imidazole,DMF =N ,N -dimethylforma-mide,DBU =1,8-diazabicyclo[5.4.0]undec-7-ene,TFAA =trifluoracetic anhydride,TBAF =tetra-n -butylammonium fluoride.42262003Wiley-VCH Verlag GmbH &Co.KGaA,WeinheimAngew.Chem.Int.Ed.2003,42,4225–4229conditions [10]did not lead directly to the desired lateri-florone molecular framework,but rather to an array of other products,among which the most interesting were those shown in Scheme 5.Thus,exposure of 23a ,b to PhI(OCOCF 3)2in CH 2Cl 2led to benzoquinone 3(Table 1)in 43%yield.Attempts to cyclize this substance undera variety of conditions failed to produce the desired lateriflorone skeleton,leading instead to a number of other compounds whose description will have to await the full account of this work.Having failed to construct the desired 1-O -methyl-lateriflorone (2)from quinone 3,we then proceeded to investigate the possibility of casting this structural motif from 23a ,b via benzoquinone monoketal 24.To this end,23a ,b was exposed to PhI(OCOCF 3)2in MeOH/CH 2Cl 2(1:1),which led to the formation of 24(60%yield)through the participation of a molecule of methanol.Upon subsequent treatment with PPTS in refluxing benzene under azeotropic conditions,24led to25Scheme 3.Synthesis of intermediate 7:a)K 2CO 3(5.0equiv),BnBr(5.0equiv),KI (0.1equiv),DMF,258C,24h,85%;b)MgBr 2·Et 2O (1.1equiv),diethyl ether,258C,10h,83%;c)Br 2(1.1equiv),NaOAc (1.15equiv),AcOH,258C,2h,89%;d)NaBH 4(1.5equiv),EtOH,0!258C,30min,91%;e)2,2-dimethoxypropane (5.0equiv),TsOH (0.01equiv),CH 2Cl 2,1h,95%;f)n BuLi (1.1equiv),diethyl ether,À788C,2h;then B(OMe)3(3.0equiv),1h,À78!08C;then aqueous NaOH (10%;4.8equiv),H 2O 2(5.0equiv),08C,30min,90%;g)K 2CO 3(5.0equiv),MeI (10.0equiv),DMF,0!258C,16h,99%;h)Pd/C (10wt %;10%),H 2(1atm),EtOAc,258C,45min,98%;i)t BuOK (2.2equiv),THF,08C;then concentrated and suspended in MeCN;then 18[crown]-6(2.2equiv),15min,bromoisobutyraldehyde (5.0equiv),0!258C,1h,70%;j)CH 3P +Ph 3Br À(3.0equiv),NaHMDS (3.0equiv),THF,08C,1h,75%;k)t BuOK (1.1equiv),THF,08C;then concentrated and suspended in MeCN;then 18[crown]-6(1.1equiv),15min,bromoisobutyraldehyde (5.0equiv),0!258C,1h,75%;l)CH 3P +Ph 3Br À(2.0equiv),NaHMDS(2.0equiv),THF,08C,1h,80%.Ts =p -toluenesulfonyl,HMDS =hexamethyl-disilazane.Table 1:Selected data for compounds 2,3,and 25.2:R f =0.19(silica gel,EtOAc/hexanes 3:7);m.p.=173–1748C (Et 2O/hexane,uncorrected);1H NMR (600MHz,CDCl 3):d =7.50(s,1H),6.49(d,J =9.8Hz,1H),5.78(d,J =9.8Hz,1H),5.32(br t,J =7.0Hz,1H),4.66(br t,J =7.0Hz,1H),3.53(s,3H),3.20(dd,J =8.3,3.1Hz,1H)2.83(m,1H),2.79(m,1H),2.56(m,1H),2.39(dd,J =14.5,7.0Hz,1H),2.16(d,J =13.0Hz,1H),1.89(d,J =9.9Hz,1H),1.69(s,3H),1.68(s,3H),1.60(dd,J =13.0,9.9Hz,1H),1.56(s,3H),1.55(s,3H),1.48(s,3H),1.45(s,3H),1.16ppm (s,3H),1.14ppm (s,3H);13C NMR (150MHz,CDCl 3):d =200.7,189.0,186.7,158.0,150.9,139.0,136.5,133.6,134.0,125.2,123.0,121.6,116.0,115.4,101.3,85.6,84.7,84.0,82.8,81.3,57.4,54.1,48.6,31.9,30.0,29.1,28.3,28.2,27.8,25.9,25.8,21.6,18.1,17.9ppm;HRMS (MALDI):calcd for C 34H 40O 9Na +:615.2564[M +Na +],found 615.25493:R f =0.32(silica gel,EtOAc/hexanes 3:7);1H NMR (600MHz,CDCl 3):d =7.50(s,1H),6.45(d,J =10.1Hz,1H),5.63(d,J =10.1Hz,1H),5.43(s,1H,OH,D 2O exchangeable),4.99(br t,J =7.5Hz,1H),4.84(br t,J =7.2Hz,1H),3.57(s,3H),3.09–3.17(m,2H),2.68(dd,J =14.0,6.9Hz,1H),2.61(dd,J =14.0,7.9Hz,1H),2.37(d,J =13.0Hz,1H),2.35(d,J =10.1Hz,1H),1.69(s,3H),1.68(dd,J =13.0,10.1,Hz,1H),1.67(s,3H),1.61(s,3H),1.60(s,3H),1.59(s,3H),1.51(s,3H),1.50(s,3H),1.27ppm (s,3H);13C NMR (100MHz,CDCl 3):d =202.8,182.8,174.7,162.5,149.7,146.7,144.2,135.6,135.4,135.3,130.6,128.7,118.4,117.2,116.1,115.0,84.6,84.2,83.5,82.7,81.2,54.0,50.1,31.4,30.1,29.2,28.54,28.53,28.3,26.1,25.9,23.5,18.1,17.9ppm;HRMS(MALDI):calcd for C 34H 40O 9Na +:615.2564[M +Na +],found 615.259425:R f =0.24(silica gel,EtOAc/hexanes 3:7);m.p.=1548C (Et 2O/hexane,uncorrected);1H NMR (500MHz,CDCl 3):d =7.47(s,1H),6.25(d,J =10.0Hz,1H),5.84(d,J =10.0Hz,1H),5.24(br t,J =6.5Hz,1H),4.75(br t,J =8.0Hz,1H),3.60(s,3H),3.53(s,3H),3.24(dd,J =15.0,8.0Hz,1H),2.99(dd,J =15.0,6.5Hz,1H),2.84(dd,J =13.5,8.0Hz,1H),2.40(dd,J =13.5,6.0Hz,1H),2.13(d,J =13.0Hz,1H),2.08(d,J =9.5Hz,1H),1.75(dd,J =13.0,9.5Hz,1H),1.68(s,6H),1.56(s,3H),1.49(s,3H),1.48(s,3H),1.44(s,3H),1.34(s,3H),1.16ppm (s,3H);13C NMR (125MHz,CDCl 3):d =201.5,188.7,159.0,149.2,140.4,136.6,136.1,135.6,132.6,126.2,124.7,123.4,121.7,117.1,116.2,97.3,85.2,84.9,84.0,82.8,78.0,60.4,54.0,48.4,31.9,29.9,28.3,28.0,27.7,27.2,26.0,25.8,24.5,17.9,17.8ppm;HRMS (MALDI):calcd for C 35H 43O 9:607.2901[M +H +],found 607.28864227Angew.Chem.Int.Ed.2003,42,4225–4229 2003Wiley-VCH Verlag GmbH &Co.KGaA,Weinheim(Table 1)in 69%yield,together with quinone 3,which was obtained in 11%yield (Scheme 5).Pleasantly,com-pound 25crystallized as beautiful yellow crystals whose X-ray crystallo-graphic analysis [9]revealed its lateri-florone-like molecular architecture,including the correct stereochemistry at C3’(see ORTEP drawing,Scheme 5).However,an attempt to hydrolyze the enol methyl ether within 25under acidic conditions (HCl (aq.)/THF)led to red quinone ether carboxylic acid 3’(80%),which after methylation with diazomethane,afforded red quinone methyl ester 26(81%,Scheme 6).The structurally intriguing isomeric qui-nones 3(Scheme 5)and 3’(Scheme 6)may well be related to biogenetic precursors and/or in vivo degradation products of lateriflorone.As such,these novel compounds,3and 3’,may exhibit biological activity,and so could 1,1’-O ,O -dimethyllateriflorone (25).At this juncture,we felt that ben-zoquinone carboxylic acid 3’presented a new opportunity for a final attempt to reach the lateriflorone framework.Indeed,exposure of 3’to PPTS in refluxing benzene under anhydrous conditions led,in 83%yield,to a single yellow substance whose spectral data strongly suggested the lateriflor-one-like structure 2(Scheme 6).Pleas-ingly,X-ray crystallographic analysis [9]of the crystalline 2(Table 1)confirmed its identity as 1-O -methyllateriflorone,ending the quest for this highly unusual molecular architecture.The com-pletely stereoselective manner in which both 3’and 24cyclize to the corresponding lateriflorone frame-works is noteworthy.It is not impos-sible that both reactions are under thermodynamic control,leading to the natural stereochemistry,which may represent the most stable configura-tion of this system.The described chemistry opens a facile entry into the novel lateriflorone molecular architecture and under-scores the importance of careful strat-egy design for its construction.Future studies in the field will include further methodological explorations and chemical biology investigations with the synthesized compounds.Received:May 5,2003[Z51805]Scheme 4.Construction of advanced intermediate 23a ,b :a)DMF,1208C,1h,6’(42%)and 6(47%);b)TsOH (20mol %),MeOH,258C,16h,98%;c)DMP (2.0equiv),NaHCO 3(2.0equiv),CH 2Cl 2,258C,30min,93%;d)NaClO 2(6.0equiv),NaH 2PO 4(6.0equiv),2-methyl-2-butene (75.0equiv),THF/t BuOH/H 2O (2:4:1),30min,100%;e)EDC (1.5equiv),4-DMAP (1.5equiv),5(2.0equiv),CH 2Cl 2,0!258C,16h,64%;f)HCl (0.25m )in MeOH/Et 2O (1:1),0!258C,1h,96%.DMP =Dess–Martin periodinane,EDC =1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.42282003Wiley-VCH Verlag GmbH &Co.KGaA,WeinheimAngew.Chem.Int.Ed.2003,42,4225–4229.Keywords:cascade reactions ·Claisen rearrangement ·Diels–Alder reactions ·electrocyclic reactions ·natural products ·quinones[1]S.Kosela,S.-G.Cao,X.-H.Wu,J.J.Vittal,T.Sukri,Masdianto,S.-H.Goh,K.-Y.Sim,Tetrahedron Lett.1999,40,157–160.[2]For the isolation and structure elucidation,see:Y.-W.Leong,L.J.Harrison,G.J.Bennett,H.T.-W.Tan,J.Chem.Res.Synop.1996,392–393.[3]For the total synthesis of 1-O -methyl forbesione,see:K.C.Nicolaou,J.Li,Angew.Chem.2001,113,4394–4398;Angew.Chem.Int.Ed.2001,40,4264–4268.[4]For related synthetic studies on lateriflorone,see:a)E.J.Tisdale,C.Chowdhury,B.G.Vong,H.Li,E.A.Theodorakis,Org.Lett.2002,4,909–912;b)E.J.Tisdale,H.Li,B.G.Vong,S.H.Kim,E.A.Theodorakis,Org.Lett.2003,5,1491–1494;c)E.J.Tisdale,B.G.Vong,H.Li,S.H.Kim,C.Chowdhury,E.A.Theodorakis,Tetrahedron ,in press.[5]K.A.Parker,A.T.Georges,Org.Lett.2000,2,497–499.[6]J.D.Godfrey,Jr.,R.H.Mueller,T.C.Sedergran,N.Soundar-arajan,V .J.Colandrea,Tetrahedron Lett.1994,35,6405–6408.[7]A.J.Quillinan,F.Scheinmann,J.Chem.Soc.Perkin Trans.11972,1382–1387.[8]CCDC-209801(6’),CCDC-209802(4),and CCDC-209803(2)contain the supplementary crystallographic data for this paper.These data can be obtained free of charge via /conts/retrieving.html (or from the Cambridge Crystallo-graphic Data Centre,12,Union Road,Cambridge CB21EZ,UK;fax:(+44)1223-336-033;or ****************).[9]A.J.Quillinan,F.Scheinmann,mun.1971,966–967.[10]For related applications of hypervalent iodine reagents todearomatize benzenoid systems,see:a)A.Varvoglis,Tetrahe-dron 1997,53,1179–1255;b)Y.Kita,H.Tohma,K.Kikuchi,M.Inagaki,T.Yahura,.Chem.1991,56,435–438;c)G.Scheffler,H.Seike,E.J.Sorensen,Angew.Chem.2000,112,4785–4788;Angew.Chem.Int.Ed.2000,39,4593–4596;d)A.Pelter,R.S.Ward,Tetrahedron 2001,57,273–282;e)H.Tohma,H.Morioka,S.Takizawa,M.Arisawa,Y.Kita,Tetrahedron 2001,57,345–352;f)S.Canesi,P .Belmont,D.Bouchu,L.Rousset,M.A.Ciufolini,Tetrahedron Lett.2002,43,5193–5195.Scheme 6.Synthesis and ORTEP drawing of 1-O -methyllateriflorone (2):a)aqueous HCl (1n ),THF,0!258C,16h,80%;b)CH 2N 2,diethyl ether,08C,30min,81%;c)PPTS (1.0equiv),benzene,reflux,4h,83%.Scheme 5.Synthesis and ORTEP drawing of 1,1’-O ,O -dimethyllateriflor-one (25):a)PhI(OCOCF 3)2(1.2equiv),CH 2Cl 2,À78!258C,2h,43%;b)PhI(OCOCF 3)2(1.2equiv),CH 2Cl 2/MeOH (1:1),À78!258C,2h,60%;c)PPTS (1.0equiv),benzene,reflux,4h,3(11%)and 25(69%).PPTS =pyridinium p -toluenesulfonate.4229Angew.Chem.Int.Ed.2003,42,4225–4229 2003Wiley-VCH Verlag GmbH &Co.KGaA,Weinheim。

Protein-mimicking nanoparticle (Protmin)-based nanosensor for intracellular analysis of metal ionsDan Zhu 1,2•Dong-Xia Zhao 2•Jia-Xuan Huang 2•Jiang Li 1•Xiao-Lei Zuo 1•Li-Hua Wang 1•Chun-Hai Fan 1Received:7June 2017/Revised:17September 2017/Accepted:22September 2017/Published online:21December 2017ÓShanghai Institute of Applied Physics,Chinese Academy of Sciences,Chinese Nuclear Society,Science Press China and Springer Nature Singapore Pte Ltd.2017Abstract In this study,we designed and applied protein-mimicking nanoparticles (Protmin)as an intracellular nanosensor for in vivo detection of lead ions (Pb 2?).Monodispersed gold nanoparticles (AuNPs)of 13nm in diameter were modified using poly-adenine-tailed Pb 2?-specific 8–17DNAzyme to form a spherical and functional Protmin.Substrate strands modified with a fluorophore at the 50end and a quencher at the 30end were bound to DNAzyme.Pb 2?facilitated cleavage of DNAzyme to release the fluorophore-modified short strands to generate fluorescence.We observed rapid kinetics of the Protmin nanosensor,for which the typical assay time was 10min.Further,we demonstrated the Protmin nanosensor could readily enter living cells and respond to Pb 2?in the intracellular environment.The broad of range of Protmindesigns will be useful for advancing biological and medical applications.Keywords Protmin ÁNanosensor ÁPoly-adenine ÁLead ion ÁIntracellular detection1IntroductionMetal ions play an important role in cellular activities and participate in a variety of physiological processes.The maintenance of proper concentrations of metal ions in cells or tissues fundamentally affects various processes,such as supporting the basic structures and functions of nucleic acids,proteins,and hormones [1–3].Recent studies demonstrated that various diseases,such as neural distur-bance,organic damage,and cancer or gene mutation,are related to abnormal levels of metal ions or the presence of toxic metal ions [4–6].Over the past few decades,many sensors have been designed to detect metal ions in envi-ronmental samples or biological samples with a high sen-sitivity and selectivity [7–12].In recent years,tracing metal ions in cells have generated concerns because of their fundamental roles in biology and health.Developing sen-sitive,fast,and responsive cellular sensors is very impor-tant for understanding different functions at the molecular level in cells.Proteins are among the most important components in cells and tissues in the body and participate in various physiological processes.Proteins also have a significant clinical value in disease diagnosis and as therapeutics [13].Therefore,developing proteins with specific functions has been widely examined.However,natural proteins regulate their catalytic activity by binding to scaffolding proteins orThis work was supported by the National Natural Science Foundation of China (Nos.21390414and 21605087),the Chinese Academy of Sciences (No.QYZDJ-SSW-SLH031),the China Postdoctoral Science Foundation funded project (No.BX201700123),theScientific Research Foundation of Nanjing University of Posts and Telecommunications (No.NY215058),and the Natural Science Fund for Colleges and Universities in Jiangsu Province (16KJB150032).&Chun-Hai Fanfchh@1Division of Physical Biology and Bioimaging Center,Shanghai Synchrotron Radiation Facility,Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai 201800,China2Key Laboratory for Organic Electronics and Information Displays,Institute of Advanced Materials (IAM),Jiangsu National Synergetic Innovation Center for AdvancedMaterials (SICAM),Jiangsu Key Laboratory for Biosensor,Nanjing University of Posts and Telecommunications,Nanjing 210023,ChinaNUCL SCI TECH (2018)29:5https:///10.1007/s41365-017-0348-yassembling at the membrane and do not effectively cross the membrane of cells to exert their functions.Thus,the utilization of proteins as diagnostic probes and therapeutic agents is limited because of their instability and poor transduction across cell membranes.Nanomaterials may be useful for overcoming these limitations.By combining nanomaterials with enzyme-like molecules,protein-mim-icking nanoparticles(Protmins)can be developed with multiple functions and favorable stability in the biological environment.Gold nanoparticles(AuNPs)have been widely investigated nanomaterials because of their unique chemical and physical properties.The high surface-to-volume ratio,favorable chemical stability,and easy mod-ification make AuNPs applicable for biosensing[14–17], nanophotonics[18,19],catalysis[20–22],and drug deliv-ery[23–25].Through assembly with DNA probes,AuNP conjugates can enter cells without the use of additional transfection agents[26],enabling the construction of pro-tein-mimicking nanostructures with excellent biocompatibility.In this study,we constructed and applied an AuNP-based Protmin as an intracellular nanosensor for efficient in vivo lead(Pb2?)detection.DNAzyme was employed as a recognition and sensing element in the design of Protmin. As an enzyme-like oligonucleotide,DNAzyme can effi-ciently recognize its specific target with excellent speci-ficity and possesses catalytic proteinase-like activity [27,28].DNAzyme assembled onto AuNPs efficiently recognized intracellular target metal ions,such as uranyl [29],copper,and zinc ions[30].Salaita et al.evaluated the activity of DNAzyme on AuNPs and found that it mostly depended on the conformation and density of DNAzyme on the nanointerface,while the active sites of DNAzyme might be hidden by the adsorption between DNA and the gold surface[31].To improve the activity of Protmin,a poly-adenine(polyA)-tailed probe was employed,which has been shown to have high activity in vitro[32–36].The polyA tail serves as an efficient anchoring block based on its preferential binding with the AuNP surface and prevents nonspecific adsorption between the active sites of DNA-zyme and gold surface,thus improving the sensitivity for Pb2?detection.The sensitivity,selectivity,and kinetics of the Protmin-based nanosensor were investigated in vitro. The existence of intracellular Pb2?was evaluated byflu-orescence imaging,which was an effective tool for inves-tigating subcellular structures and intracellular fluorescence[19].By transferring this Protmin nanosensor into cells,intracellular toxic Pb2?can be detected.2Experimental section2.1Apparatus and materialsOligonucleotides(Table1)were synthesized and puri-fied by TaKaRa,Inc.(Shiga,Japan).HAuCl4Á4H2O (99.9%),NaCl,NaH2PO4Á2H2O,and NaH2PO4Á12H2O were purchased from China National Pharmaceutical Group Corporation(Shanghai,China).Pb(OAc)2,6-mer-capto-1-hexanol(MCH),diethylpyrocarbonate(DEPC), trisodium citrate,ethyl acetate,and other chemicals were all obtained from Sigma-Aldrich(St.Louis,MO,USA). All solutions were prepared with ultrapure water (18.2M X cm).Transmission electron microscopy(TEM) imaging was performed using H-7500model(Hitachi, Tokyo,Japan).UV–Vis spectra were measured with a U-3900(Hitachi).Fluorescence measurements were per-formed on an F-4500(Hitachi).Cell imaging andfluores-cence imaging in cells were conducted using a laser confocal microscope(Leica TCS SP5,Wetzlar,Germany).2.2Preparation of13-nm AuNPsThirteen-nanometer AuNPs were synthesized following classical citrate reduction procedures[37].Briefly,3.5mL of1%trisodium citrate was quickly added to a boiling, rapidly stirring0.01%aqueous HAuCl4solution,which changed the solution color from pale yellow to burgundy red.After further boiling for20min,the solution was left to cool to25°C while stirring.The prepared AuNPs were stored at4°C until use,and the size of the AuNPs was determined by TEM imaging.2.3Preparation of polyA-tailed ProtminThe prepared AuNPs werefirst incubated with polyA-tailed DNAzyme(polyA10-17E)in a1:200ratio for16h. Next,1M of phosphate-buffered saline(PBS,1M of NaCl,100mM of NaH2PO4/Na2HPO4,pH7.4)was added to the mixture5times repeatedly at30-min intervals to achieve a0.1M phosphate concentration.The salt solution was incubated for an additional40h at room temperature. Next,the solution was centrifuged(13,500rpm,20min) and resuspended in0.1M PBS(0.1M of NaCl,10mM of NaH2PO4/Na2HPO4,pH7.4)three times.The prepared Protmin was resuspended in the reaction buffer(20mM tris,100mM NaCl,pH7.4,prepared in DEPC-treated water)until use.5Page2of8 D.Zhu et al.2.4Calculation of17E loading on AuNPsThe loading of17E was determined by attaching FAM-labeled17E(polyA10-17E-F)onto the13-nm AuNPs.The concentration of17E-AuNPs was determined by UV–Vis spectroscopy measurements using Beer’s law(for13-nm AuNPs,e520nm=2.79108L mol-1cm-1).FAM-la-beled17E was chemically displaced from the surface of AuNPs by MCH treatment(final concentration10mM in 0.3M NaCl,10mM phosphate buffer solution,pH7.4) after18-h incubation at37°C with shaking.The released fluorescent DNA was collected via centrifugation and measured with the F-4500.Thefluorescence was converted to molar concentrations of probes by comparison with a standard curve,which was prepared using known concen-trations of17E-F under the same conditions.The loading of DNA was calculated by dividing the concentration offlu-orescent17E-F by the concentration of AuNPs.Experi-ments were repeated three times using fresh samples.2.5Determination of activity of Protmin nanosensorin vitroThe RNA contained substrate strand‘‘Active17S’’was modified with Cy3label at the50end and BHQ-2at the30 end.The Protmin nanosensor was fabricated as follows. First,active17S was added to a Protmin solution to afinal ratio of2:117S to17E strand.The solution was heated at 65°C for5min and then cooled at25°C for1h.The solution was stored at4°C overnight to complete the reaction.Excess17S was removed by centrifugation and the prepared Protmin nanosensor was washed with reaction buffer three times.Finally,the Protmin nanosensor was diluted to a concentration of2nM in the reaction buffer.A series concentration of Pb(OAc)2stock solution was added to the Protmin nanosensor.Fluorescence spectra were collected at540-nm excitation.2.6Pb21cellular uptake and imagingActive or inactive Protmin nanosensors were prepared by hybridizing‘‘Active17S’’or‘‘Inactive17S’’with Protmin.HeLa cells were dispersed at a density of 1.09105cells per well in24-well microtiter plates to a total volume of500l L/well.The plates were maintained at 37°C in a5%CO2/95%air incubator for1day.After removing the original incubation medium,HeLa cells were incubated with Pb2?at afinal concentration of200l M for 12h to allow sufficient metal ion uptake.The cells were washed thrice using0.1M PBS.Next,600l L of Dul-becco’s modified Eagle medium(DMEM)culture medium containing2nM active or inactive A10-17ES-AuNPs was added.After2h of incubation,the cells were washed using PBS thrice and fresh DMEM was added.Lysotracker Green was added to the cells for another30min at afinal concentration of50nM.Fluorescence images were acquired with a confocal laser scanning microscope.2.7MTT assayAn MTT assay was carried out to investigate the cyto-toxicity of the nanosensor.HeLa cells(1.09105cells per well)were dispersed in24-well microtiter plates to a total volume of500l L/well.The plates were maintained at 37°C in a5%CO2/95%air incubator for1day.After removing the original medium,HeLa cells were incubated with unmodified AuNPs(1nM)or nanosensor(1nM and 3nM)for3,6,12,and24h.Next,the medium was replaced with another medium containing MTT(0.5mg/ mL)and cells were incubated for another4-h incubation at 37°C.After removing the medium,150l L DMSO was added to the cells to dissolve the formed formazan. Absorbance at490nm in each well was recorded.Cell viability was expressed as the ratio of the absorbance of the cells incubated with AuNPs or the nanosensor to that of cells incubated with only culture medium.3Results and discussion3.1Fabrication and working principleof the Protmin nanoprobeBased on the polyA-tailed DNAzyme specific for Pb2? cleavage,we constructed a Protmin nanosensor for Pb2? detection in cells.As shown in Fig.1,Protmin wasfirst prepared by attaching the polyA-tailed DNAzyme onto the surface of AuNPs through polyA-Au binding,extending the DNAzyme block for further reactions.Protmin was then hybridized with thefluorescent17S substrate strandsTable1Base sequences and their labeling of DNA oligonucleotide samples used in the study DNA name sequence(50–30)polyA10-17E AAAAAAAAAATTTTTCATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT polyA10-17E-F AAAAAAAAAATTTTTCATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-FAM Active17S Cy3-ACTCACTATrAGGAAGAGATG-BHQ-2Inactive17S Cy3-ACTCACTATAGGAAGAGATG-BHQ-2Protein-mimicking nanoparticle(Protmin)-based nanosensor for intracellular analysis of…Page3of85to fabricate 17ES-AuNP.The substrate strands were labeled with a fluorophore at the 50end and quencher at the 30end.After attachment to the AuNPs,the fluorophore was quenched by gold and a quencher,resulting in very weak fluorescence.In the presence of Pb 2?,the substrate strands were cleaved,resulting in the release of short fluorophore-labeled DNA and an increase in fluorescence.3.2Characterization of Protmin probeBecause the 13-nm AuNP was reported to be an efficient quencher [38]and intracellular nanocarrier [26],our design was based on this intracellular nanosensor.TEM images of AuNPs (Fig.2a)showed that the 13-nm AuNPs were suc-cessfully synthesized with good dispersion.The UV–Vis absorption spectra of AuNPs and polyA10-tailed Protmin (AuNPs functionalized with polyA10-tailed DNAzyme)shown in Fig.2b demonstrated that the maximum optical absorption was shifted from 520to 524nm after polyA-tailed DNAzyme assembly on the surface of AuNPs.Because the position of the plasmon band of metal nanoparticles was closely related to the size and surrounding environment,the red shift in spectroscopy confirmed that the AuNPs were successfully functionalized with polyA10-tailed DNAzyme [39].By using a displacement-based flu-orescence method established by Mirkin [40],each AuNP was estimated to carry 42±2DNAzyme,which was in accordance with our previous result [33].3.3Pb 21detection of Protmin nanosensor in vitro We first evaluated the feasibility of using the Protmin nanosensor for Pb 2?detection in vitro (Fig.3a).The curve a in Fig.3a shows the fluorescence spectrum of freeCy3-Fig.1(Color online)Schematic illustration of Pb 2?imaging in living cells based on polyA-tailed protein-mimicking nanoparticles(Protmin)Fig.2(Color online)a TEM images of 13-nm AuNPs;b UV–Vis spectra forunmodified AuNPs (black line)and the Protmin (red line)5Page 4of 8D.Zhu et al.and BHQ-2-labeled active 17E solution upon excitation at 540nm.After hybridization with Protmin,fluorescence was decreased significantly because of efficient quenching by AuNPs (curve b in Fig.3a).After adding Pb 2?,the substrate strand was cleaved and the Cy3-labeled short strand was released,increasing the fluorescence of the system (curve c in Fig.3a).These results indicated that the Protmin nanosensor functioned well in response to Pb 2?in vitro.The results of kinetics in Fig.3b showed that the Protmin nanosensor responded rapidly to Pb 2?and the fluorescence at 565nm reached a plateau after 10-min incubation with Pb 2?,which was twofold faster than that of a thiolated DNAzyme-based nanosensor [29].These fast kinetic results may be attributed to the elimination of nonspecific adsorption between gold and DNAzyme by using polyA tails.These results demonstrate the potential for rapid detection by using Protmin-based sensors.3.4Sensitivity of selectivity of Protmin nanosensorin vitro To test the sensitivity of the nanosensor,different concentrations of Pb 2?(0–2l M)were added and emis-sion fluorescence was recorded.The fluorescence signal at565nm increased as the concentration of Pb 2?increased (Fig.4a).Good linearity was obtained from 5–30nM.The linear equation was fluorescence (F 565nm )=0.6732C Pb 2þ(nM)?21.66,with a correlation coefficient of0.9143.Fig.3(Color online)a Fluorescence spectra of Cy3-and BHQ-2-labeled ‘‘Active 17S’’in solution (curve a ),Protmin-based nanosensor in the absence (curveb )and presence (curvec )of 100nM Pb 2?;b kinetics of thenanoprobe in the absence (black curve)and presence of 100nM Pb 2?.The excitation wavelength was 540nmFig.4(Color online)a Fluorescence intensity at 565nm in the presence of various concentrations of Pb 2?(0,5,10,15,20,30,50,100,200,500,1000,and 2000nM).Inset:Calibration curve for the fluorescence intensity versus corresponding Pb 2?concentration.b Specificity of the nanoprobe for Pb 2?compared to other metal ions (Mg 2?,Co 2?,Fe 3?,Cu 2?,Zn 2?,K ?,Ca 2?).The concentration of other metal ions was 50l M and concentration of Pb 2?was 100nM.The excitation wavelength was 540nmFig.5MTT assay of HeLa cells.HeLa cells were incubated with unmodified AuNPs (1nM),nanosensor (1and 3nM)for 3,6,12,and 24h.Error bars represent variations between three parallel experimentsProtein-mimicking nanoparticle (Protmin)-based nanosensor for intracellular analysis of …Page 5of 85The detection limit of Pb 2?was calculated to be 5.33nM.This assay is more sensitive or comparable to other intracellular Pb 2?assays [41–43]because of the high activity of polyA-mediated DNAzyme on AuNPs [33].To evaluate the selectivity of the nanoprobe for sensing Pb 2?,other biologically relevant metal ions,including Mg 2?,Co 2?,Fe 3?,Cu 2?,Zn 2?,K ?,and Ca 2?,were tested.As shown in Fig.4b,the Protmin nanosensor exhibited two-to threefold higher fluorescence signals for Pb 2?than for other metal ions at higher concentrations.Thus,the nanosensor is suitable for evaluation in complex biologi-cal systems.3.5Intracellular sensing of Pb 21using Protminnanosensor The application of the Protmin-based nanosensor for intracellular Pb 2?was tested.The cellular uptake and flu-orescence response of the sensor were evaluated using HeLa cells as a model.To evaluate the cytotoxicity of the nanosensor,an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay in HeLa cells was performed.The results indicated that the AuNPs (1nM)and nanosensor (1and 3nM)showed low cytotoxicity or side effects in HeLa cells (Fig.5).Therefore,theFig.6(Color online)a ,b Confocal microscopy images of HeLa cells treated a with or b without Pb 2?with active Protmin nanosensor.c ,d Confocal microscopy images of HeLa cells treated a with or b without Pb 2?with inactive Protmin nanosensor.The red channel is Cy3fluorescence and green channel is Lysotracker Green fluorescence.The scale bar is 25l m5Page 6of 8 D.Zhu et al.nanosensor is a reliable probe for detection in biological samples.Wefirst treated HeLa cells with200l M Pb2?for12h to allow sufficient metal ion uptake.According to a pre-vious study[29],the AuNP-DNAzyme nanoprobe can enter cells through receptor-mediated endocytosis without the use of transfection agents.As shown in Fig.6a,a red fluorescence signal corresponding to Pb2?was observed. As a control,HeLa cells without treated with Pb2?were also incubated with the nanoprobes and imaged under the same conditions and nearly nofluorescence signal was observed(Fig.6b).To further confirm that thefluorescence observed in Fig.6a was because of the activity of DNA-zyme,an inactive Protmin nanosensor was prepared by using‘‘Inactive17S,’’in which the adenosine ribonu-cleotide at the cleavage site was replaced with a deoxyri-bonucleotide.As shown in Fig.6c,d,the inactive probe-treated HeLa cells showed lessfluorescence than those with the active probe.Staining of lysosomes of cells (green)showed good colocalization of Lysotracker and Protmin nanoprobes,indicating that the nanoprobes were mainly transported to the lysosomes.These results are consistent with those for other nanoprobes constructed based on DNA-AuNPs[29,30].The results demonstrated the successful implementation of intracellular detection of Pb2?by using this Protmin nanoprobe.4ConclusionIn summary,we developed a novel intracellular nanosensor based on high-efficient polyA-tailed Protmin with fast kinetics for Pb2?detection in HeLa cells.Protmin not only possesses enzyme-like catalysis activity,but also has advantages of nanomaterials,such as excellent chem-ical stability and unique chemistry and it is easy to trans-duce across cell membranes.Additionally,the polyA tail added to Protmin decreased nonspecific adsorption between DNA and gold,thus enhancing the activity of DNAzyme at the nanointerface.PolyA contains natural nucleotides that are easy to synthesize and modify,and thus reduces synthesis costs.Fast kinetics of the Protmin nanosensor were observed and the detection time in vitro was decreased to10min,indicating that this system can be used for a rapid detection.In combination with DNAzyme specific for other metal ions,the polyA-tailed Protmin nanoprobes are a simple and general platform for devel-oping high-efficient intracellular nanosensors.The Protmin design is promising for 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