新型高效多肽缩合剂的研究
作者简介：李鹏，男，1972年09月出生，1997年09月师从于中国科学院上海有机化学研究所徐杰诚教授，于2000年06月获博士学位。
摘 要
发展新型、高效的多肽缩合剂一直是多肽领域的研究热点，也是高效地合成结构越来越具有挑战性的生物活性肽和多肽类似物的保障。本论文先后设计并合成了亚胺正离子型、吡啶正离子型，噻唑正离子型和苯并咪唑正离子型等新型多肽缩合剂， 并通过模型反应测试和一系列寡肽与生物活性肽的合成证实了这些缩合剂的优异性能。
1 亚胺正离子型缩合剂的设计、合成与应用研究
在目前各方面性能最佳的脲正离子型缩合剂分子结构基础之上，通过对母体结构的改造与优化，来适度地提高碳正离子的反应活性，从而设计了一类新型的缩合剂?亚胺正离子型缩合剂，例如BOMI，BDMP，BPMP，BMMP，DOMP，AOMP，FOMP，SOMP和SOMI。这类缩合剂可以由DMF，N-甲基吡咯烷酮等廉价的酰胺合成得到，产物为室温下稳定的非吸湿性固体，具有潜在的商业化价值。
通过一系列的模型反应测试和的实际应用，我们发现HOBt衍生的亚胺正离子型缩合剂无论在反应活性还是在产物光学纯度方面都大大优于目前被普遍认为性能最佳且应用较广的脲正离子型缩合剂。这些结果充分证明了这类缩合剂的优异性能，也说明了我们缩合剂分子设计的合理性。通过X-ray衍射，UV，IR，1H NMR和13C NMR分析，我们也对这类缩合剂异构现象和参与酰胺键或酯键形成反应的机理进行了研究。
这类试剂可以广泛地用于各种酰胺、酯和活化酯，特别是多肽的合成，具有反应速率快，产物收率高、消旋小等优点，且反应可采用“一锅法”进行操作简便，反应条件温和。另外，这类试剂不仅可以用于液相合成，也可用于固相法多肽合成。采用这类缩合剂，我们分别用固相法和液相法成功地合成了一系列小肽和生物活性肽，进一步验证了这类新型的亚胺正离子型缩合剂的优异的反应性能和应用性能。
2 用于有空间位阻多肽合成的缩合剂的设计合成及应用研究
具有高空间位阻的多肽的合成至今仍是一个很赋挑战性的难题，采用常规试剂往往不能得到令人满意的结果。本论文在a-卤代脲正离子型缩合剂CIP和CDTP分子框架的基础上，结合半经验法分子轨道计算，通过合理的修饰适度提高分子的反应活性，兼顾作为多肽缩合剂对化合物的溶解性、稳定性和非吸湿性等方面的要求，设计并合成了a-吡啶正离子型缩合剂BEP、FEP、BEPH和FEPH和a-卤代噻唑正离子型缩合剂BEMT。这些新型缩合剂可以由2－卤代吡啶或硫脲等廉价原料方便的制得，具有潜在的商业化价值。
本文通过采用HPLC对模型反应Z-Gly-Phe-OH + Val-OCH3×HCl  Z-Gly-(L+D)-Phe-Val-OCH3和Z-MeVal-OH + MeVal-OCH3×HCl? Z-MeVal-MeVal-OCH3进行跟踪测试，发现这两类缩合剂的反应活性明显高于目前应用较广的PyBroP、PyClU、BOP-Cl和BTFFH等试剂。对于普通氨基酸间的缩合，在-10°C，DIEA为缚酸剂，二氯甲烷为溶剂的条件下，反应几乎是瞬间完成。产物的消旋亦远远小于PyBroP、PyClU和BTFFH等a-卤代磷正离子和脲正离子型缩合剂，当加入HOAt作为添加剂时，可以进一步抑制产物的消旋。以上结果充分证明缩合剂BEP、FEP、BEPH、FEPH和BEMT是两类无论在反应活性方面还是在产物消旋方面都具有优异的性能的新型多肽缩合剂。
这两类新型缩合剂可以高效地促进N-甲基氨基酸间或Ca,Ca-二烷基化氨基酸间的缩合，产物的收率一般在90%以上，反应可以采用“一锅法”进行，无须首先对羧基组份预活化。另外，这类试剂也可用于制备各种Na-保护氨基酸的活化酯。为进一步检验这类吡啶正离子型缩合剂在有空间位阻的多肽合成中的性能，我们以BEP和FEP为缩合剂，成功地合成了具有抗肿瘤活性的亲脂性酯肽Dolastatin 15分子中高N-烷基化的肽链部分；分别以BEMT和BEP为缩合剂合成了生物活性肽Cyclosporin A中空间位阻最大的四肽片断。这类缩合剂还可用于合成由普通氨基酸组成的多肽，反应无需预活化，可采用“一锅法”方便地合成，未发现任何副反应。
本文进一步测试了这些新型缩合剂在固相法有空间位阻的多肽合成中的性能。我们仍选取生物活性肽CsA中空阻很大的8-11片段为目标分子，以BEP为缩合剂对其进行了固相合成。所得粗产物未经任何纯化，经HPLC检测纯度已达95%。这一结果证明这些缩合剂在固相法有空间位阻多肽的合成中亦具有较好的性能。
此外，我们以Boc-Val-OH + 缩合剂 + NEt3  ? 和Fmoc-Val-OH + 缩合剂 + NEt3 ?为模型反应，采用IR和1H NMR对这类缩合剂参与的酰胺键形成反应的机理进行了初步的探索。研究发现，反应的主要活泼中间体是Na-保护氨基酸或多肽的酰卤，少量的羧基组份的对称酸酐和5(4H)-口恶唑酮也参与酰胺键的形成反应。
3 其它类型缩合剂的设计与合成及其在多肽合成中的应用研究
除了以上我们设计合成的三大类型缩合剂之外，本文还设计、合成并研究了其它一些新型的缩合剂，例如：缩合剂CMBI、PyCloPP、CMMM、FMDP和CDMS。
通过模型反应测试，我们发现缩合剂CMBI与相应的a-氯代脲正离子型试剂PyClU相比具有更高的反应活性和更有效地抑制消旋。本文采用CMBI为缩合剂合成了一系列小肽，反应可以采用“一锅法”进行，许多情况下目标产物的收率几乎是定量的。我们还采用此缩合剂合成了抗癌药物Actinomycin D中空间位阻较大的酯肽链部分。这些寡肽和多肽片段成功合成充分证明了缩合剂CMBI在有空间位阻的多肽合成中的高效性。缩合剂PyCloPP是在a-氯代磷正离子型缩合剂PyCloP的分子框架基础之上设计得到的，实验证明此缩合剂亦可以快速地促进N-甲基氨基酸间的缩合。缩合剂CMMM是在a-卤代脲正离子型缩合剂PyClU的分子框架基础上设计的，此缩合剂的反应活性和抑制消旋能力与PyClU相当，但这一试剂更容易制备，且原料廉价低毒，特别适用于有空间位阻的多肽的大规模制备。为进一步探索缩合剂的分子结构与反应活性和抑制消旋能力的关系，本文还设计并合成了新型缩合剂FMDP、CDMS和CBDO，并对缩合剂的结构活性关系进行了初步的探讨。
4 生物活性肽Cyclosporin O的全合成
Cyclosporin O是一个从真菌的二级代谢物中分离得到的富含N-甲基氨基酸的亲脂性环十一肽。这一有高空间位阻的环肽具有很强的免疫抑制活性。具有潜在的商业化价值，但对于CsO的化学全合成目前尚无文献报道，而这正是实现工业化大规模生产所必经的途径。在CsO的化学合成的基础上，我们可以进一步通过结构活性关系(SAR)研究来筛选出活性更高、毒性更小的多肽类似物。因此实现CsO的化学全合成的意义是不言而喻的。另外，实现这一具有高空间位阻的生物活性肽的化学合成，也是对我们设计的各类缩合剂性能的一个有力挑战。
为保证合成得到的目标产物具有高的光学纯度，各种非天然氨基酸及其衍生物均选取产物消旋最小的方法制备。本文选择我们设计的噻唑正离子型缩合剂BEMT和吡啶正离子型缩合剂BEP来合成CsO中有空间位阻的肽链。对于氨基组份为普通氨基酸的缩合反应，我们选取亚胺正离子型试剂中性能最佳的BDMP作为缩合剂。肽链的环化，本文分别以HAPyU 和BDMP为缩合剂来进行，最终我们从氨基酸衍生物出发以18-23%的总收率实现了生物活性肽Cyclosporin O的首次化学全合成，并通过1H NMR、13C NMR、COSY、TOCSY、NOESY、HMQC、HMBC、ESI-MS和IR等方法对最终产物CsO的结构进行了鉴定。
ABSTRACT
The development of novel and highly efficient peptide coupling reagents is always one of the most active and attention-getting topics in the peptide field. It also promotes the efficient synthesis of more and more structural challenging peptides and peptidomimetics. In this thesis, a series of novel peptide reagents, such as immonium-, pyridinium-, thiazolium- and 1H-benzimidazolium-type reagents, were designed and synthesized. The high efficiency of these reagents were investigated and proved by the tests of model reactions and the successful syntheses of series of oligopeptides and bioactive peptides.
1 Design, synthesis and application of immonium-type peptide coupling reagents
A new type of peptide coupling reagents, immonium-type reagents, was designed based upon the structural skeleton of the uronium-type reagents, the most efficient reagents developed so far, by modification of carbocation moiety of uronium salts to enhance their activity moderately. These immonium-type reagents, such as BOMI, BDMP, BPMP, DOMP, AOMP, FOMP, SOMP and SOMI, can be synthesized from the inexpensive amides, such as DMF and N-methyl pyrrolidine. These compounds are shelf stable and nonhygroscopic solids, and show strong commercializing potential.
The HOBt-derived immonium-type coupling reagents were shown to be more superior to the commonly used uronium-type reagents in terms of reactivity and racemization-suppressing capability, which was proved by model reaction tests and practical applications. The high efficiency of the immonium-type reagents also reflects the rationality of our molecular design. The isomerization of these reagents and the mechanism for these immonium salts mediated coupling reactions were studied by X-ray analysis, UV, IR, 1H NMR and 13C NMR.
These reagents can be not only used to the synthesis of various kinds of amides and esters, but also used to the efficient synthesis of peptides with fast reaction speeds, high yields and low racemization. The coupling reactions can be carried out by ￠one-pot￠ reaction procedure under mild reaction conditions. In addition, these reagents can also be used in solid-phase peptide synthesis. The high efficiency of these immonium-type reagents was further demonstrated by the successful synthesis of series of oligopeptides and bioactive peptides both in solution and solid-phase.
2 Design, synthesis and application of novel coupling reagents for sterically hindered peptide synthesis
The synthesis of sterically highly hindered peptide is still a challenging problem so far; only poor results were obtained using conventional coupling reagents to synthesize this kind of peptide. Based upon this concern, we designed and synthesized a-pyridinium-type reagents, such as BEP, FEP, BEPH and FEPH, and a-thiazolium-type reagent BEMT. The molecular design was done by rational modification of the structural skeleton of a-uronium-type reagents, CIP and CDTP, and guided by molecular orbital calculations using semi-empirical PM3 method, to enhance the reactivity with the consideration of solubility, stability and non-hygroscopic property. These reagents can be easily synthesized from cheap materials, such as 2-halopyridine and thiourea, consequently exhibit strong potential of commercialization.
The reactivity of these pyridinium- and thiazolium-type reagents is shown to be much higher than that of the commonly used PyBroP, PyClU, BOP-Cl and BTFFH by the tests of model reactions: Z-Gly-Phe-OH + Val-OCH3×HClZ-Gly-(L+D)-Phe-Val-OCH3 and Z-MeVal-OH + MeVal-OCH3×HClZ-MeVal-MeVal-OCH3. As for the coupling between coded amino acids, the reaction can be accomplished instantly using these reagents under –10 °C in CH2Cl2, and DIEA as base. The racemization of products is also much less than that of a-halophosphonium- and a-halouronium-type reagents, such as PyBroP, PyClU and BTFFH. This racemization can be further suppressed by the addition of HOAt. The above results demonstrate that the pyridinium- and thiazolium-type reagents are extremely efficient in terms of reactivity and racemization-suppressing capability.
These two types of coupling reagents can effectively promote the condensations between N-methyl or Ca,Ca-dialkyl amino acids, and the yields are usually more than 90%. The reactions can be carried out using the so-called ￠one-pot￠ procedure without the preactivation of the carboxylic components. In addition, these reagents can also used to synthesize various kinds of active esters. To further testify the high efficiency of these new reagents in the synthesis of sterically hindered peptides, we successfully synthesized the extensively -N-methylated peptide moiety of the antineoplastic and lipophilic depsipeptide Dolastatin 15 using reagent BEP and FEP. We also synthesized the highly hindered tetrapeptide segment of Cyclosporin A using reagent BEMT and BEP, respectively. These reagents can also be used to synthesize peptides only containing coded amino acid residues using the convenient ￠one-pot￠ reaction procedure, and no side reaction was observed.
To investigate the efficiency of these reagents in solid-phase peptide synthesis, we selected the highly hindered 8-11 tetrapeptide fragment of CsA as the target, and synthesized it using reagent BEP in solid-phase. The purity of crude product was up to 95% evaluated by HPLC. This result demonstrates that these novel reagents also exhibit high efficiency in solid-phase synthesis of hindered peptide.
The mechanism for these new reagents mediated coupling reactions was studied via the monitoring of the model reactions, Boc-Val-OH + coupling reagent + NEt3 ? and Fmoc-Val-OH + coupling reagent + NEt3 ?, by IR and 1H NMR. It was found that the key reaction intermediate was the corresponding acyl halide of Na-protected amino acid or peptide. A small amount of symmetric anhydride of carboxylic component and 5(4H)-oxazolone intermediates was also involved in the above coupling reaction.
3 Design, synthesis and application of other novel peptide coupling reagents
Besides the three types of novel peptide coupling reagents shown above, we also designed and synthesized some other novel coupling reagents, such as CMBI, PyCloPP, CMMM, FMDP and CDMS.
Model reaction tests indicate that reagent CMBI exhibits higher reactivity and better racemization-suppressing capability than a-chlorouronium-type reagent PyClU. The yields are almost quantitative using this reagent to synthesize oligopeptides using one-pot reaction procedure. We also accomplished the synthesis of highly hindered depsipeptide moiety of the anti-tumor drug Actinomycin D using CMBI. These results indicate the high efficiency of reagent CMBI in the synthesis of hindered peptide. Reagent PyCloPP was designed based upon the structural skeleton of a-chlorophosphonium-type reagent PyCloP, and proved to be very efficient in the synthesis of peptide N-methyl amino acid residues. Reagent CMMM was designed based upon the molecular structure of reagent PyClU, and demonstrates similar reactivity and racemization-suppressing capability with reagent PyClU. In comparison with PyClU, reagent CMMM is easier to prepare, and the starting materials are cheaper and less toxic. Therefore, it is very suitable for the synthesis of hindered peptides on large scales. We also designed and synthesized novel reagents, FMDP, CDMS and CBDO, to further investigate the relationship between the molecular structure of coupling reagents and their reactivity and racemization-suppressing ability.
4 The total synthesis of bioactive peptide Cyclosporin O
Cyclosporin O produced as secondary fungal metabolites by Cylindrocarpon lucidum Booth and Tolypocladium inflatum Gams, is lipophilic and immunosuppressive cyclic undecapeptide containing seven N-methyl amino acid residues. The chemical synthesis of Cyclosporin O has not been reported so far, while this is the most feasible way to prepare this prodrug on a large scale. Based upon the synthetic procedure, we can rationally modify the molecular structure of CsO to screen out the more active and less nephrotoxic peptidomimetics. Therefore, It is very significant to accomplish the total synthesis of this immunosuppressive and extremely hindered cyclopeptide. In addition, the synthesis of this structural challenging target can further testify the high efficiency of these novel coupling reagents.
To ensure the high optical purity of final product, all the unnatural amino acids and their derivatives were synthesize using the method caused the least racemization. The hindered peptide segments were synthesized using thiazolium-type reagent BEMT and pyridinium-type reagent BEP. Immonium-type reagent BDMP was used to synthesize the peptide segment containing only coded amino acid residues. The macrocyclization of the linear undecapeptide was achieved using reagent HAPyU and BDMP, respectively. Thus, the extremely hindered bioactive cyclopeptide CsO was synthesized in 18-23% overall yield, and characterized by 1H NMR, 13C NMR, COSY, TOCSY, NOESY, HMQC, HMBC, ESI-MS and IR.
原文见：http://www.cdgdc.edu.cn/yxbslw/pxjg/2002/lipeng.htm
若您有任何意见或建议，请及时E-mail通知我们，谢谢！ |
