Synthesis of radiometal-labeled and fluorescent cell-permeating Peptide -: PNA conjugates for targeting the bcl-2 proto-oncogene
Synthesis of radiometal-labeled and fluorescent cell-permeating Peptide -: PNA conjugates for targeting the bcl-2 proto-oncogene
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DOI：
10.1021/bc034084n
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发表时间：
2003-11-01
影响因子：
4.7
通讯作者：
Lewis, MR
中科院分区：
文献类型：
--
作者：
Gallazzi, F;Wang, Y;Lewis, MR
B细胞淋巴瘤/白血病-2（bcl-2）原癌基因与许多类型癌症中良性病变向恶性病变的转化、疾病进展、预后不良、生存率降低以及对放疗和化疗产生耐药性有关。这项工作的目的是合成一种与bel-2开放阅读框的前六个密码子互补的反义肽核酸（PNA），与透膜肽偶联用于细胞内递送，并用双功能螯合剂修饰，用于靶向成像和治疗过表达bcl-2的肿瘤的放射性金属。通过手动和自动化逐步延伸技术的组合合成了四种肽-PNA构建体，包括与任何已知的哺乳动物基因或DNA序列没有互补性的bcl-2反义缀合物和无义缀合物。PNA序列通过固相9-芴甲氧羰基（Fmoc）技术人工合成。然后，将用1，4，7，10-四氮杂环十二烷-N，N ′，N ″，N ″ ″-四乙酸（DOTA）修饰用于放射性金属螯合的完全保护的赖氨酸单体手动偶联至每个PNA序列。DOTA-PNA缀合物的合成之后是用已知介导不可渗透的效应分子的细胞内化的肽序列（PTD-4-甘氨酸，PTD-4-G）或其逆-反转类似物（ri-PTD-4-G）自动延伸。四种缀合物的制备需要创新的合成策略，使用温和的酸性条件来产生疏水的、部分脱保护的中间体。通过半制备型反相HPLC纯化这些中间体，并完全脱保护，以6%至9%的总产率产生纯肽-PNA缀合物。使用该合成策略的修改，使用四甲基罗丹明（TMR）的赖氨酸衍生物制备bcl-2反义PNA的ri-PTD-4-G缀合物用于荧光显微镜。血浆稳定性研究表明，In-111-DOTA标记的ri-PTD-4-G-抗bcl-2 PNA在37 ℃下稳定168小时，与含有亲本肽序列的缀合物不同。在Raji淋巴瘤细胞中TMR标记的ri-PTD-4-G-抗-bcl-2 PNA的扫描共聚焦荧光显微镜显示，反转肽在膜渗透中是活性的，并且介导反义PNA内化到细胞质中，其中预期存在高浓度的bcl-2 mRNA。
The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bel-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that In-111-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degreesC, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.