CN1184482A - 糖聚合物的合成 - Google Patents
糖聚合物的合成 Download PDFInfo
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- CN1184482A CN1184482A CN96193865A CN96193865A CN1184482A CN 1184482 A CN1184482 A CN 1184482A CN 96193865 A CN96193865 A CN 96193865A CN 96193865 A CN96193865 A CN 96193865A CN 1184482 A CN1184482 A CN 1184482A
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- glycopolymers
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- transglycosylation
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Abstract
在含有机溶剂的介质中通过使用来自原玻璃蝇节杆菌的内-β-N-乙酰氨基葡糖苷酶(Endo-A)合成新糖缀合物及其合成所用的中间体。该方法能合成包括高甘露糖糖聚合物的新型糖缀合物。
Description
本发明涉及糖聚合物,合成糖聚合物的方法和组合物。具体而言,本发明涉及来自原玻璃蝇节杆菌(Arthrobacter protophormiae)的内-β-N-乙酰氨基葡糖苷酶的用途,用来生产含有高-甘露糖型链的新糖缀合物。
糖类具有重要的生物学功能,如细胞-细胞识别(Wassarman,1991;Patankar等,1993;及Lasky等,1992)、凝集素结合(Lee,1988;和Lee等,1991,Pure&Appl.Chem.)、病毒感染(Glick等,1991;和Toogood等,1991)。糖类功能的研究需要结构清楚定义的高纯化合物,这种化合物通常难以从自然资源中获得。因此,在过去的十年中(Lee,1994),新糖缀合物(用单糖或寡糖衍生的蛋白质、脂类和其它类型的化合物)的合成及构建很快赢得了人们的注意。新糖缀合物的化学合成已经取得了积极的进展,但化学合成通常包括复杂、繁琐的步骤。已证明高甘露糖型寡糖的合成特别困难,即使使用酶法合成。
来自原玻璃蝇节杆菌的内-β-N-乙酰氨基葡糖苷酶(Endo-A)是一种具有水解作用和转糖基作用两种功能的糖苷酶。此酶切割糖蛋白的高甘露糖型和杂合型N-联糖链的核心GlcNAcβ1、4GlcNAc残基中的糖苷键(Takegawa等,1989),并且还将寡糖转移至一些单糖和二糖(Takegawa等,1991a,1991b)。(高甘露糖型化合物是仅2-乙酰氨基葡残基与天冬酰胺紧邻、链的其余部分被分支且通常仅由甘露糖组成的化合物,但有时见到使用木糖和岩藻糖的进一步修饰。复合型化合物是一种由N-乙酰氨基葡、半乳糖、有时有岩藻糖和唾液酸组成的化合物。杂合型化合物是上述两种的杂合物。)
通过向反应溶液中加入有机溶剂如丙酮、二甲基亚砜(DMSO)和N,N-二甲基甲酰胺(DMF)能显著提高转糖基反应的效率。例如,当使用Man9-GlcNAc2Asn作为供体、GlcNAc作为受体测量Endo-A的转糖基活性时,在水溶液中转糖基作用与水解作用的比率为1∶2,但是当加入30%的丙酮时,进行的转糖化作用会接近完全。这一特点使得有可能合成具有高效高纯度的新糖苷和新糖缀合物。
使用这种方法,我们已经合成了几种用于新糖缀合物的功能中间体,其中一个已被转化成带有悬挂式Man9GlcNAc2链的糖聚合物。与单体寡糖相比,由此制备的糖聚合物对来自肝的甘露糖-结合蛋白的结合呈现出强得多的抑制作用。
本发明提供了新糖缀合物和新糖缀合物的新的功能中间体,它们是在含有机溶剂的反应混合物中用Endo-A合成的。
本发明还提供了具有高纯度的合成的高甘露糖型和杂合型糖聚合物。
本发明还提供了具有悬挂式Man9GlcNAc2链的糖聚合物,与单体寡糖相比,对来自肝的甘露糖结合蛋白(MBP)具有强得多的抑制作用。
本发明还提供了合成新糖缀合物及其功能中间体的方法。
图1.Endo-A转糖基作用条件的最优化。通过在含30%丙酮(A)或不同浓度丙酮(B)的10μl 10mM乙酸铵缓冲液(pH6.0)中11.6 nmolMan9GlcNAc2Asn(供体)、4μmol GlcNAc-NAP(受体)和不同量酶(A)或2.2mU酶(B)的混合物中进行反应,来确定用于转糖基作用的酶(A)和丙酮含量(B)的最佳水平。此反应混合物在37℃温育15分钟,产物用HPAEC-PAD分析。(◎):底物;(◆)转糖基产物;(▲):水解产物。
图2.通过Endo-A转糖基作用合成Man9GlcNAc2-NAP。在含35%丙酮的5ml 10mM乙酸铵缓冲液(pH6.0)中,5.75μmol Man9GlcNAc2Asn、2mmol GlcNAc-NAP和1.1U的酶于37℃反应15分钟。冷冻干燥后,相当于0.7nmol的Man9GlcNAc2寡糖的样品被注射入HPAEC-PAD系统进行分析。用100mM NaOH和20分钟内0至10%NaOAc线性梯度进行洗脱。A:转糖基产物Man9GlcNAc2-NAP;B:水解产物Man9GlcNAc;C:残留底物Man9GlcNAc2Asn。
图3.GlcNAc-NAP(A)和Man9GlcNAc2-NAP(B)的1H-NMR(300MHz)分析。在测量之前,将样品溶解于D2O中随后冻干,重复此循环三次,用氘置换出样品中不稳定的氢。在25℃使用丙酮(2.225ppm)作为内标在D2O中进行分析。
图4.在Sephadex G-50上进行的糖聚合物凝胶过滤。将样品(1ml)加到Sephadex G-50柱(2.5×90cm)上,用水洗脱。流速约30ml/hr,收集了4ml级分。通过苯酚-H2SO4法测定中性糖(虚线,480nm下的吸光度),用220nm下吸光度监测GlcNAc(实线)。a:表示结合为糖聚合物的级分。
图5.糖聚合物的1H-NMR(600MHz)分析。在60℃D2O中测量的化学位移基于4.441ppm处的HDO信号。★:表示来自聚合物骨架的信号。
图6.具有Man9GlcNAc2糖链的糖聚合物。
图7.通过HPGFC确定糖聚合物的分子量。使用一个大小排阻柱(7.5×600mm)和含0.3M NaCl的0.1M磷酸盐缓冲液(pH7.0)作为洗脱剂在1.0ml/min流速下进行HPGFC。通过220nm处吸光度监测流出物。○:糖聚合物;◇:参比化合物;1:蓝色葡聚糖(2,000,000);2:β-淀粉酶(200,000);3:醇脱氢酶(150,000);4:白蛋白牛血清(66,000)和5:碳酸酐酶(29,000)。
图8.糖聚合物对血清-MBP-CRD和肝-MBP-CRD结合的抑制作用。使用ALLFIT程序(DeLean等,1978)获得拟合曲线。根据Man9GlcNAc2表示SBA和糖聚合物浓度。■:SBA+血清MBP-CRD;□:SBA+肝MBP-CRD;◆:糖聚合物+血清MBP-CRD;◇:糖聚合物+肝MBP-CRD。
材料和方法
本说明书中使用下列缩写:
Bn:苄基;BSA:牛血清白蛋白;CRD:糖识别区域;DMF:N,N-二甲基甲酰胺;DMSO:二甲基亚砜;Endo-A:来自原玻璃蝇节杆菌的内-β-N-乙酰基-D-氨基葡糖苷酶;GlcNAc:N-乙酰-D-葡糖胺;1H-NMR:1H-核磁共振谱法;HPAEC-PAD:带有脉冲电流检测器的高效阴离子交换色谱;HPLC:高效液相色谱;HPGFC:高效凝胶过滤色谱;Man:甘露糖;MBP:甘露糖-结合蛋白;4mU:4-甲基繖形基(4-methylumbelliferyl);NAP:3-(N-丙烯酰氨基)-丙基;pNP:对-硝基苯基;SBA:大豆凝集素。
所用单糖均为D-构形。
实验步骤材料
根据Takegawa等人(1989)所述方法纯化Endo-A。从大豆凝集素中通过彻底的链霉蛋白酶消化、随后Sephadex G-50上凝胶过滤和进一步使用石墨化炭柱进行HPLC纯化(Fan等,1994)制备出Man9GlcNAc2Asn。糖酰胺酶A来自Seikagaku America,Inc.(Rockville,MD)。GlcNAc购自Pfanstiehl Laboratories,Inc.(Waukegan,IL)。3-(N-丙烯酰氨基)-丙基β-D-GlcNAc(GlcNAc-NAP)和GlcNAc-O-(CH2)3CH=CH2是日本Hokkaido University的Shin-Ichiro Nishimura博士所赠。使用Nishimura等人(1990)和Nishimura等人(1994a)所述的方法可以合成这些化合物。在该实验室根据Lee等人所述方法(1992)合成了苄基β-GlcNAc、4-甲基繖形基β-GlcNAc、对-硝基苯基β-GlcNAc、GlcNAc-S-(CH2)6NH2、GlcNAc-O-CH2CH=CH2、GlcNAc-O-(CH2)3NHCOCH=CH2、GlcNAc-S-CH2CN、GlcNAc-S-(CH2)3CH3、(GlcNAc-S-CH2CH2CH2)2和GlcNAc-S-CH2CONHCH2CH(OMe)2。根据Quesenberry和Drickamer所述方法(1992)使用Columbia University的Kurt Drickamer博士所赠的含表达质粒的细菌株表达和纯化来自血清和肝的重组大鼠MBP-CRD。方法酶反应
用于转糖基的典型的酶反应是在总体积20μl的含30%丙酮的25mM乙酸铵缓冲液(pH6.0)中的3nmol Man9GlcNAc2Asn、4μmol受体和0.9mU Endo-A混合物中进行。(也可使用其它有机溶剂如DMSO和DMF,通过常规实验调节浓度使反应最优化。)在37℃温育15分钟后,在沸腾水浴中3分钟使反应终止。使用真空泵用一Speedvac移去缓冲液。使用一个HPAEC-PAD系统分析反应混合物(参见下面所述)。高效阴离子交换色谱(HPAEC)
使用由装配有一个脉冲电流检测器(PAD-II)的Bio-LC(DionexCorp.,Sunnyvale,CA)组成的HPAEC系统进行反应产物分析。使用一个AI-450色谱软件(Dionex)分析色谱数据。在一Dionex CarboPac PA-I柱(4×250mm)上以1.0ml/min的流速用100mM氢氧化钠和梯度乙酸钠(30分钟内30mM至80mM展开)洗脱,分离Endo-A反应产物。在操作之间用100mM氢氧化钠/200mM乙酸钠溶液洗涤柱5分钟,并使柱平衡15分钟。PAD灵敏度设为1K。通过与Endo-A完全消化Man9GlcNAc2Asn和糖酰胺酶消化Man9GlcNAc2AsnPhe获得的标准物质相比较,进行Man9GlcNAc和Man9GlcNAc2的定量测定。通过从起始底物中减去残留底物和水解产物,评估以N-乙酰-葡糖胺之外物质为受体的转糖基作用产物的量。使用GlcNAc-NAP为受体的Endo-A转糖基作用
由5.8μmol Man9GlcNAc2Asn、2mmol GlcNAc-NAP和1.1U酶于5ml含35%丙酮的10mM NH4OAc缓冲液(pH6.0)中组成的混合物在37℃下温育15分钟。置于沸水浴中3分钟终止反应后,将样品加到Sephadex G-25柱(2×140cm),并用0.1M醋酸洗脱。通过229nm处紫外吸收监测流出物,用苯酚硫酸法(Mckelvy和Lee,1969)测定中性糖。将含高分子量物质的级分合并,并冷冻干燥,得10.5mg白色粉末。具有高甘露糖型寡糖悬链的糖聚合物的制备
凝胶过滤获得的白色粉末用作聚合反应的起始原料,无需进一步纯化。将少量此白色粉末(7.2mg,约3.25μmol Man9GlcNAc2-NAP)溶解于0.3ml H2O中,随后用水吸气器脱气30分钟。加入丙烯酰胺(8.4mg,118pmol)、过硫酸铵(APS,0.14μmol)和N,N,N’,N’-四甲基乙二胺(TEMED,6.6μmol),该混合物在室温下搅拌三天,此其间,每天向反应混合物中加入同样量的APS和TEMED,加2天,通过将混合物于55℃温育3小时,最终完成反应。反应混合物被加在Sephadex G-50柱(2.5×90cm)中,并用H2O洗脱。将含糖聚合物的级分合并并冷冻干燥得到5.3mg白色粉末。通过HPGFC评估糖聚合物分子量
用装配有一大小排阻柱(TSK-Gel G2000 SW,7.5×600mm,TosoHaas,ND)和紫外检测器(Model V4,ISCO)的Gilson HPLC系统进行HPGFC。洗脱剂为含0.3M NaCl的0.1M磷酸盐缓冲液(pH7.0),于220nm下监测流出物。用于分子量评估的标准化合物为i)蓝色葡聚糖(MW=2,000,000);ii)β-淀粉酶(MW=200,000);iii)醇脱氢酶(MW=150,000);iv)牛血清白蛋白(MW=66,000)和v)碳酸酐酶(MW=29,000)。糖聚合物的MBP结合
大致根据Quesenberry和Drickamer所述方法(1992),经少许改变进行固相结合研究。所有步骤在4℃时进行。简而言之,将CRD(50μl)涂膜于单独的聚苯乙烯小孔上(Immulon 4 Removawell Strips byDynatech,来自Fisher Scientific)。温育一昼夜后,加入1.25M NaCl/25mMCaCl2/25mM Tris(pH7.8)中的1%BSA的封闭溶液(blockingsolution),使反应进行2小时。配体和抑制剂存在于上述Tris缓冲液中的0.5%BSA中用来结合和抑制。使用的参比配体是根据氯胺T法(Lee等,1991,J.Biol Chem.(生物化学杂志))放射标记的125I-〔甘露糖30-BSA〕(约2000 cpm/μg)。大约500cpm/孔参比配体温育20小时,在各个浓度时含或不含抑制剂。然后,移出孔内容物,洗涤,在PackardMinaxiγ计数器上计数。计数值用本底(在未涂CRD的封闭孔中残余的数值)校正,使用程序ALLFIT(De Lean等,1978)分析数据,使用曲线拟合的对数方程确定I50值(50%抑制所需的测试配体浓度)。1H核磁共振谱
300MHz NMR谱被记录于Bruker AMX 300分光计(spectrometer)上,并在Brucker AM-600分光计上进行600MHz NMR的测量。化学位移基于丙酮(δ=2.225ppm)作为内标。通过溶解于D2O中、冷冻干燥循环三次,然后在测量前立即将残留物溶解于0.5ml高纯度D2O(99.96%D)中制备样品。在25℃记录300MHz数据、60℃记录600MHz数据。
结果Endo-A向水可混醇的转糖基作用
测试以Man9GlcNAc2Asn为供体、通过Endo-A向各种水可混醇的转糖基作用。在20μl25mM乙酸铵缓冲液(pH6.0)中、含3nmolMan9GlcAc2Asn(供体)和3mU酶的30%的醇(v/v)中于37℃进行表1反应10分钟。使用Man9GlcNAc和Man9GlcNAc2作参比化合物、通过HPAEC测定产物。
表1.通过Endo-A从Man9GlcNAc向醇的转糖基作用
醇(30%v/v) | 水解作用a)(%) | 转糖基作用a)(%) |
H2O | 94.1 | 0.0 |
MeOH | 33.2 | 64.0 |
EtOH | 45.5 | 46.9 |
PrOH | 4.5 | 8.0 |
iProH | 72.4 | 9.6 |
烯丙醇 | 0.0 | 0.0 |
甘油 | 27.8 | 56.5 |
a)基于初始供体底物。
正如表1所示,酶向甲醇(MeOH)和乙醇(EtOH)转移寡糖,产率分别为64%和47%,水解作用产率为33%和46%。通过1H-NMR发现与甲醇产物的异头构形为β(数据未示出)。丙醇(PrOH)(8%产率)和异丙醇(10%产率)也可用作转糖基作用的受体,但烯丙醇不能作为受体。酶在30%甲醇和乙醇中显示是稳定的,但在30%丙醇和烯丙醇中是不稳定的(甲醇和乙醇中酶总活性(水解作用和转糖基作用结合活性)显示出与H2O中活性相近,但在高级醇中要低得多)。发现甘油是与甲醇或乙醇一样好的受体,转糖基作用产率高达57%。Endo-A向各种GlcNAc糖苷的转糖基作用
Endo-A向一些官能化GlcNAc糖苷的转糖基作用是高效的,如表2所示。当受体浓度为0.2M时,Endo-A向GlcNAc-O-(CH2)6NH2(为被转化底物的93%)、GlcNAc-O-CH2CH=CH2(99%)、GlcNAc-O-(CH2)3CH=CH2(90%)和GlcNAc-O-(CH2)3NHCOCH=CH2(78%)转移Man9GlcNAc的产率分别为初始底物的81%、81%、84%和70%。这些反应在含3nmol Man9GlcNAc2Asn、0.88mU酶和所指定受体的30%丙酮的20μl 25mM乙酸铵缓冲液(pH6.0)中于37℃进行15分钟。使用100mM NaOH和在30分钟内浓度从30mM增至80mM的线性梯度的NaOAc通过HPAEC进行分析。
表2:通过Endo-A把Man9GlcNAc向GlcNAc糖苷的转糖基作用
受 体 | 浓度a)(M) | 产率b) | 转糖基作用率c)(%) | |
水解作用(%) | 转糖基作用(%) | |||
GlcNAc | 0.2 | 4.1 | 84.7 | 95.4 |
Bn-GlcNAc | 0.05 | 15.0 | 66.7 | 81.6 |
4mU-GlcNAc | sat.d) | 19.9 | 65.5 | 76.7 |
pNP-GlcNAc | sat. | 45.8 | 32.8 | 41.7 |
GlcNAc-O-(CH2)6NH2 | 0.2 | 6.5 | 81.1 | 92.6 |
GlcNAc-O-CH2CH=CH2 | 0.2 | 1.2 | 80.8 | 98.5 |
GlcNAc-O-(CH2)3CH=CH2 | 0.2 | 9.8 | 83.6 | 89.5 |
GlcNAc-O-(CH2)3NHCOCH=CH2 | 0.2 | 20.2 | 69.8 | 77.6 |
GlcNAc-S-CH2CN | 0.2 | 11.3 | 83.4 | 88.1 |
GlcNAc-S-(CH2)3CH3 | 0.2 | 12.4 | 77.5 | 86.2 |
(GlcNAc-S-CH2CH2CH2)2 | sat. | 42.0 | 42.6 | 50.4 |
GlcNAc-S-CH2CONHCH2CH(OMe)2 | 0.2 | 4.0 | 81.0 | 95.3 |
a)受体浓度
b)基于初始底物的产率
c)转糖基作用产物占总消化底物的百分率
d)sat.:饱和溶液
由于溶解度低,所用苄基β-GlcNAc浓度为0.05M,使用4mU β-GlcNAc和pNP β-GlcNAc在饱和条件下(低于0.05M)。即使在这些浓度时,酶也能向它们分别转移67%、66%和33%的起始寡糖链,且发现转糖基作用率(转糖基作用产物占消化底物的百分率)分别为82%、77%和42%。GlcNAc的硫-糖苷是Endo-A转糖基作用的良好受体。当0.2M的GlcNAc-S-CH2CN、GlcNAc-S(CH2)3CH3和GlcNAc-S-CH2CONHCH2CH(OMe)2用作受体时,转糖基作用率分别为88%、86%和95%,产率为83%、78%和81%。GlcNAc的二价硫糖苷即(GlcNAc-S-CH2CH2CH2)2在低浓度时(低于0.05M)也可用作Endo-A转糖基作用的受体,转糖基作用率为50%,产率43%。用于Endo-A更大规模转糖基作用的反应条件的最优化
为大规模进行转糖基作用,在较高浓度底物时检测用于转糖基作用的最佳水平的酶和丙酮用量。正如图1A所示,水解产物随酶量成正比增加。加入的酶上升至2.2mU时转糖基作用产物的产率增加,然后再加入更多的酶时,产率下降。当使用2.2mU的酶时,仅剩余5.6%的底物。另一方面,随丙酮含量增至35%,转糖基作用产物增加,水解产物减少(图1B)。在35%丙酮中,通过HPAEC分析发现有86%的转糖基作用和7%的水解作用。虽然在40%丙酮介质中没有发现水解产物,但与在其它介质中的反应相比,反应效率较低,因为剩余的底物量较多(为起始底物的64%)。通过Endo-A转糖基作用活性合成Man9GlcNAc2-NAP
为制备一定量的能用于聚合反应的Man9GlcNAc2-NAP,反应规模比上述最佳条件中所述的提高了500倍。通过HPAEC,转糖基作用产物Man9GlcNAc2-NAP多于90%。几乎没有检测到水解作用产物和起始供体底物。通过在Sephadex G-25柱上进行凝胶过滤回收未反应的受体,用1H-NMR分析测定Man9GlcNAc2-NAP,将它用于聚合反应无需进一步纯化。
1H-NMR用于鉴别转糖基作用产物。正如图3A所示,通过去偶技术受体信号被完全确定。在3.436ppm处发现GlcNAc的H-4信号,在4.495ppm附近发现异头质子(anomeric proton)信号。另一方面,转糖基作用产物的1H-NMR分析显示出10个新的异头质子信号,表明高甘露糖型糖链被转移至受体上。基于参比值(Vliegenthart等,1983)的1H-NMR信号的确定值列于表3。在D2O中25℃时使用丙酮作内标(δ=2.225ppm),GlcNAc-NAP和Man9GlcNAc2-NAP的1H-NMR数据记录于300MHz波谱仪上。在D2O中60℃时Man9GlcNAc2-聚合物的化学位移记录于600MHz波谱仪,以HDO(δ=4.441ppm)作对照。表3:GlcNAc-NAP,Man9GlcNAc2-NAP和具有Man9GlcNAc2悬链的糖聚合物的1H-NMR数据
残基号a) | Man9GlcNAc2-Asnb) | GlcNAc-NAP | Man9GlcNAc2-NAP | Man9GlcNAc2-聚合物 |
H-1 of 1 | 5.092 | 4.495 | 4.475 | 4.510 |
2 | 4.610 | - | 4.579 | 4.611 |
NAc of 1 | 2.015 | 2.032 | 2.021 | 2.041 |
2 | 2.067 | - | 2.060 | 2.070 |
H-1 of 3 | -4.77 | - | 4.744 | 4.763 |
4 | 5.334 | - | 5.324 | 5.322 |
4′ | 4.869 | - | 4.859 | 4.874 |
A | 5.404 | - | 5.395 | 5.379 |
B | 5.143 | - | 5.135 | 5.122 |
C | 5.308 | - | 5.300 | 5.290 |
D1 | 5.049 | - | 5.034 | 5.057 |
D2 | 5.061 | - | 5.034 | 5.073 |
D3 | 5.042 | - | 5.034 | 5.057 |
CH of a | - | 6.163 | 5.739 | 1.701 |
a′ | - | 5.748 | 6.161 | 1.701 |
b | - | 6.278 | 6.262 | -2.307 |
c | - | 3.301 | 3.291 | 3.136 |
d | - | 1.802 | 1.791 | -1.701 |
e | - | 3.944 | u.k.c) | u.k. |
e′ | - | 3.630 | u.k. | u.k. |
a)编号与图3所述相同
b)引自公开报道(17)
c)u.k.:未知
异头信号与自Man9GlcNAc2Asn上发现的相一致,除了两个GlcNAc异头质子外,这两个质子出现在比参照化合物中的质子更高的区域处。这是因为前者中GlcNAc和糖苷配基之间的连接键是N-酰胺键,后者中是O-糖苷键。GlcNAc-2异头质子的偶合常数是7.8Hz,表明由Endo-A转糖基作用形成的新的连接呈β-构型。没有再发现“还原末端”3.436ppm处GlcNAc的H-4信号,与用甲基α-GlcNAc获得的结果一致,表明连接发生在GlcNAc的4-OH处。质谱分析给出了转糖基作用产物的预测分子量。Man9GlcNAc2-NAP与丙烯酰胺的聚合作用
利用TEMED和ASP做催化剂从Man9GlcNAc2-NAP和丙烯酰胺获得糖聚合物。收集Sephadex G-50柱空隙容积时洗脱的含聚合物的级分(图4)并冷冻干燥。通过1H-NMR分析指示聚合反应的完成(图5),表明6.2ppm和5.7ppm处信号的消失可归因于糖苷配基和丙烯酰胺单体的不饱和键。NMR还显示了11个异头质子信号的存在,且化学位移与从单体中发现的相似(表3),确证了聚合物含Man9GlcNAc2-糖链。通过苯酚-H2SO4法使用甘露糖作内标评估出聚合物中糖含量为37%。因此,糖侧链与丙烯酰胺残基的比率估计为1∶44,如图6所示。
在末端位置具有双键的其它化合物(如GlcNAc-O-CH2CH=CH和表2中所示的其它代表性化合物)可以用基本上相同的方法聚合。除丙烯酰胺外,其它单体(例如包括苯乙烯衍生物、乙烯基、环氧化物和氮丙啶型化合物和其它具有不饱和键的化合物)也可被聚合。糖聚合物分子量的确定
使用蓝色葡聚糖、β-淀粉酶、醇脱氢酶、牛血清白蛋白和碳酸酐酶作参比化合物,通过HPGFC评估糖聚合物的分子量。该聚合物出现在空隙容积附近,保留体积比蓝色葡聚糖(分子量=2,000,000)稍大。根据校准曲线(图7),分子量在1,500,000和2,000,000之间。糖聚合物对甘露糖结合蛋白的抑制作用
使用含有相同Man9GlcNAc2的Man9GlcNAc2糖聚合物和大豆凝集素(SBA),在血清-MBP-CRD和肝-MBP-CRD上进行固相结合分析。分析结果示于图8。在测试的SBA浓度范围内,没有发现对血清-MBP-CRD的显著抑制作用。但是对于肝-MBP-CRD,基于Man9GlcNAc2或0.4mg/ml的SBA得到I50值为13.2μM。而糖聚合物显示出对血清-MBP-CRD的I50为3.5μM,对肝-MBP-CRD的I50为74.5μM。就整个糖聚合物而言,I50值将分别为:对血清-MBP-CRD大约2.0×10-2mg/ml,对肝-MBP-CRD约为3.8×10-4mg/ml。糖聚合物比前体对血清形式MBP-CRD抑制效力的增强值不能被确切计算,因为Man9GlcNAc2几乎不抑制这种MBP-CRD。但是对于肝形式,增强值基于Man9GlcNAc2时约为180倍,基于分子时约1000倍,虽然糖聚合物的糖含量比SBA仅高5.6倍。
讨论
Endo-A在30%丙酮中显示出高效的转糖基作用活性(>90%),比所报道的其它糖苷酶的10%-30%(Bardales等,1989;Sakai等,1992;Cartacuzene等,1991;Nilsson,1987和1989;Usui和Murataa,1988;和Usui等,1994)要高得多。这一发现可被用于合成新糖缀合物中间体,中间体能继续进行下步反应。
Endo-A还将Man9GlcNAc转移至醇,如甲醇、乙醇和丙醇。向甲醇(64%产率)和乙醇(47%产率)的转糖基作用比那些由各种来源的β-木糖苷酶、α-和β-葡糖苷酶和β-半乳糖苷酶(Shinoyama等,1988;Shinoyama和Yasai,1988)所进行的转糖基作用(20-60%)好。但是,向丙酮和异丙酮的转糖基作用效果不如甲醇和乙醇的情况。有趣的是,虽然在丙醇中总酶活力低于在异丙醇中的总酶活力,但向丙醇的转糖基作用高于向异丙醇的转糖基作用。甘油也是Endo-A转糖基作用的良好受体。已有报道,来自肺炎双球菌(Diplococcus pneumoniae)的内-β-N-乙酰氨基葡糖苷酶F(Trimble等,1986)和内-α-N-乙酰氨基半乳糖苷酶(Bardales和Bhavanandan,1989)将一寡糖转移至甘油的Cl(3)羟基上。
将能用于新糖缀合物合成的具有官能化糖苷配基的几个GlcNAc衍生物作为Endo-A转糖基作用的受体进行测试。使用0.2M受体时发现基于初始供体底物的产率高于80%,当在我们的系统中使用0.05M或更少时,产率约50%。若采用更高的受体浓度,则转糖基作用产率可进一步提高。
在反应物浓度较高时,Endo-A转糖基作用也是有效的,如表2所示。在向GlcNAc-NAP的较大规模转糖基作用中,转糖基作用产率(>90%)甚至比分析规模反应中的产率还要高。在相似规模(4μmol)的向GlcNAcα-OMe的转糖基作用中可获得相似的产率(89%)。
Endo-A转糖基作用产物Man9GlcNAc2-NAP进一步与丙烯酰胺聚合,形成糖聚合物。最近,已通过化学方法或化学-酶法(kochetkov,1984;Nishimura等,1991;Nishi mura等,1994a和1994b;Kobayashi等,1994;和Fukase等,1994)合成了具有二糖或三糖的糖聚合物,但据我们所知,这是第一次合成了具有高度复杂糖链的糖聚合物。Endo-A转糖基作用的高效率为合成这种新糖缀合物提供了一条便捷之路。
通过在一个简单的带侧链肽上连接上一串单糖能增强某些C-型凝集素的抑制效力(Lee和Lee,1987;和Lee等,1992)。通过单价配体上的多价配体获得的亲和力的增强被称为“糖苷群效果(glycoside clustereffect),这一效果高于预期的来自局部浓度增加的简单效果。糖聚合物的形成是提供糖苷群的便捷途径(Lee和Lee,1994)。在本发明中,在与含相同Man9GlcNAc2寡糖的天然糖蛋白(SBA)时,显示出对MBP-CRD抑制作用的戏剧性的提高。在肝-MBP-CRD情况中,由糖聚合物所获得的抑制效力比天然糖蛋白(SBA)增强了约180倍。与此相似,虽然没有观察到SBA对血清-MBP-CRD显著的抑制作用,但由其寡糖衍生的糖聚合物证明了惊人强的抑制效力(I50=3.5μM)。这是“大群(macro-clustering)”相对于“小群(micro-clustering)”的良好的例子(Lee,1993)。〔“小群”描述了一种情况,其中靶糖的空间排列使结合位点间的距离很小,如1.5至3.0nM;“大群”描述了一种情况,其中空间排列距离大得多(如此处为50-100nM)〕。
显然,这里所述的化合物具有许多潜在的作用。除了它们能应用于糖功能和代谢作用研究之外,各种化合物还可以用于诊断和治疗目的,例如,作为抗原,或用于测量或分离特异性糖结合蛋白。在血清样品中测量MBP
MBP是在应答侵染微生物或其它外来物质时由肝产生的急性期蛋白之一(Reid,1983,Sastry等,1991)。MBP与这些物质结合,将它们直接消灭或通过巨噬细胞的参与将它们消灭。本发明的Man9GlcNAc2糖聚合物比含甘露糖的天然产物具有高得多的亲和力,由此,能以C-反应蛋白相似方式(Oyamada等,1992;Ohtake,1993)用于诊断。
为测量血清样品中MBP的量,可使用下述步骤:
1)图6中Man9GlcNAc2糖聚合物与ELISA分析中常用的酶结合,如碱性磷酸酶或β-半乳糖苷酶。
2)一种不影响MBP结合甘露糖能力的抗MBP单克隆抗体被放于一孔中,以涂膜在孔表面。使用本领域技术人员已知的标准技术如Quesenberry和Drickamer所述的方法(1992)制备这种抗体。将要测试的血清样品放入已涂膜的孔中并在利于MBP与抗体结合的条件下温育。
3)适宜洗涤除去未结合的物质,将(1)中的糖聚合物-磷酸酶复合物放在此小孔中。在获得磷酸酶活性同时,与抗体结合的MBP现在与糖聚合物结合。加入适宜底物,磷酸酶活力水平成为血清样品中MBP的测量尺度。
或者,可以用未结合的糖聚合物涂膜小孔,加入血清样品,结合MBP与抗-MBP反应,所述抗-MBP结合至磷酸酶或另一种适宜的酶上。然后通过前述的结合酶活力确定MBP水平。
尽管已结合目前认为最有效和最优选的实例对本发明进行了描述,显然本发明不限于所公开的实施例,而是意在包括所附权利要求精神和范围内的各种改变。
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Vliegenthart,J.F.G.,Dorland,L,和van Halbeek,H.,(1983)Adv.Carbohydr.Chem.Biochem.(糖化学生物化学进展)(Tipson,R.S.,andHorton,d.,Eds.),Vol.41,pp.209-373.Wassarman,P.M.(1991)Development(进展)108,1-17.
Claims (11)
1.一种由下述方法制备的新糖缀合物,该方法包括在含来自原玻璃蝇节杆菌(Arthrobacter protophormiae)的内-β-N-乙酰氨基葡糖苷酶和一种有机溶剂的反应混合物中合成糖苷的步骤。
2.一种用于新糖缀合物合成的单体或中间体,它是通过来自原玻璃蝇节杆菌的内-β-N-乙酰氨基葡糖苷酶在含一种有机溶剂的反应混合物中合成的,所述单体或中间体选自:
R-GlcNAc-O-(CH2)3NHCOCH=CH2,
R-GlcNAc-O-CH2CH=CH2,
R-GlcNAc-O-(CH2)3CH=CH2,
R-GlcNAc-S-CH2CN,
R-GlcNAc-S-CH2CONHCH2CH(OMe)2和
R-GlcNAc-O-(CH2)6NH2,
其中R代表ManxGlcNAc,x为3至12的整数,包括3和12。
3.权利要求2的单体,其中有机溶剂为丙酮。
4.权利要求2的单体,其中x是6至9的整数,包括6和9。
5.权利要求3的单体,其中x=9。
6.一种含高甘露糖型糖链的糖聚合物,它的合成包括:
i)将供体和受体在含来自原玻璃蝇节杆菌的内-β-N-乙酰氨基葡糖苷酶和有机溶剂的反应混合物中结合,
ii)在适宜条件下温育此反应混合物,以生产转糖基作用产物,
iii)在Sephadex柱上纯化所述反应混合物的转糖基作用产物,和
iv)聚合所述转糖基作用产物。
7.权利要求6的糖聚合物,其中通过使用丙烯酰胺达到聚合。
8.一种带有悬挂式Man9GlcNAc2链的糖聚合物,它抑制来自肝的甘露糖结合蛋白。
9.图6所示的糖聚合物。
10.一种合成含甘露糖的糖聚合物的方法,包括步骤:
i)在含来自原玻璃蝇节杆菌的内-β-N-乙酰氨基葡糖苷酶和有机溶剂的反应混合物中结合供体和受体,
ii)在适宜条件下温育此反应混合物,以生产转糖基作用产物,和
iii)聚合所述转糖基作用产物。
11.根据权利要求10的方法,其中所述糖聚合物是一种带有悬挂式Man9GlcNAc2链的糖聚合物。
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US08/445,865 US5663254A (en) | 1995-05-22 | 1995-05-22 | Synthesis of high mannose glycopolymers |
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EP (1) | EP0827506A4 (zh) |
JP (1) | JPH11505263A (zh) |
KR (1) | KR19990008468A (zh) |
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DE69721635T2 (de) * | 1996-06-19 | 2004-05-13 | Canon K.K. | Polymerverbindung mit Glykopolymer und ein Verfahren zu ihrem Abbau |
US6770468B1 (en) | 1999-09-14 | 2004-08-03 | Genzyme Glycobiology Research Institute, Inc. | Phosphodiester-α-GlcNAcase of the lysosomal targeting pathway |
US6534300B1 (en) | 1999-09-14 | 2003-03-18 | Genzyme Glycobiology Research Institute, Inc. | Methods for producing highly phosphorylated lysosomal hydrolases |
US6905856B2 (en) | 2001-12-21 | 2005-06-14 | Genzyme Glycobiology Research Institute, Inc. | Soluble GlcNAc phosphotransferase |
US6800472B2 (en) | 2001-12-21 | 2004-10-05 | Genzyme Glycobiology Research Institute, Inc. | Expression of lysosomal hydrolase in cells expressing pro-N-acetylglucosamine-1-phosphodiester α-N-acetyl glucosimanidase |
WO2003099835A1 (en) * | 2002-05-21 | 2003-12-04 | Emory University | Multivalent polymers with chain-terminating binding groups |
US6740509B2 (en) * | 2002-05-22 | 2004-05-25 | Ikuko Ishii Karakasa | Method for the production of mucin-type glycopeptide |
EP1937308A4 (en) * | 2005-09-14 | 2010-09-15 | Univ Maryland Biotech Inst | SYNTHETIC ADDED CARBOHYDRATES AS INGREDIENTS OF MICROBICIDES |
EP2138586A1 (en) * | 2008-06-24 | 2009-12-30 | Cognis IP Management GmbH | Process for the regioselective preparation of disaccharides or oligosaccharides |
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JP3532593B2 (ja) * | 1993-08-24 | 2004-05-31 | 麒麟麦酒株式会社 | 糖質又は複合糖質の製造方法 |
DE69521074T2 (de) * | 1994-03-30 | 2001-09-13 | Takara Shuzo Co | Transglykosylierungsverfahren zur Herstellung eines Kohlenhydrats oder eines Glykokonjugates |
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US5807943A (en) | 1998-09-15 |
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EP0827506A4 (en) | 2002-05-22 |
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