Fmoc-Lys（5-Fam）构建基块的合成和应用,Peptide Science

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Fmoc-Lys（5-Fam）构建基块的合成和应用
Peptide Science ( IF 1.5 ) Pub Date : 2013-07-19 , DOI: 10.1002/bip.22222
Michal Tokmina-Roszyk ₁ , Dorota Tokmina-Roszyk ₁ , Gregg B. Fields ₁

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荧光共振能量转移（FRET）肽底物通常用于蛋白酶活性测定。这项研究检查了使用5-羧基荧光素（5-Fam）作为荧光团和4,4-二甲基氨基-偶氮苯-4'-羧酸（Dabcyl）作为猝灭剂的FRET三螺旋肽（THP）底物的制备。所述N个^α - （9-芴基）-N ^ε - （5-羧基） -升赖氨酸[Fmoc-Lys（5-Fam）]结构单元是使用两种不同的合成途径合成的。第一个涉及Lys的铜络合，而第二个涉及Fmoc-Lys与微波辐射。两种方法都可以以合理的成本方便地生产非常纯净的最终产品。使用自动固相肽合成方案，将Fmoc-Lys（5-Fam）和Fmoc-Lys（Dabcyl）掺入THP底物序列中。组装第二个底物，其中（7-甲氧基香豆素-4-基）-乙酰基（Mca）是荧光团，而2,4-二硝基苯基（Dnp）是淬灭剂。圆二色光谱用于确定荧光团/猝灭剂对对三螺旋稳定性的影响。用三种基质金属蛋白酶（MMP），MMP-1，MMP-13和MT1-MMP检验了两种底物的活性。5-Fam作为荧光团和Dabcyl作为淬灭剂的组合产生了三螺旋底物，与Mca / Dnp的荧光团/猝灭剂对相比，其三螺旋结构略微不稳定，但被MMP-1和MMP-13并具有更高的敏感性。©2013 Wiley Periodicals，Inc.生物聚合物（Pept Sci）100：347-355，2013年。

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The synthesis and application of Fmoc‐Lys(5‐Fam) building blocks

Fluorescence resonance energy transfer (FRET) peptide substrates are often utilized for protease activity assays. This study has examined the preparation of FRET triple‐helical peptide (THP) substrates using 5‐carboxyfluorescein (5‐Fam) as the fluorophore and 4,4‐dimethylamino‐azobenzene‐4'‐carboxylic acid (Dabcyl) as the quencher. The N^α‐(9‐fluorenylmethoxycarbonyl)‐N^ε‐(5‐carboxyfluorescein)‐l‐lysine [Fmoc‐Lys(5‐Fam)] building block was synthesized utilizing two distinct synthetic routes. The first involved copper complexation of Lys while the second utilized Fmoc‐Lys with microwave irradiation. Both approaches allowed convenient production of a very pure final product at a reasonable cost. Fmoc‐Lys(5‐Fam) and Fmoc‐Lys(Dabcyl) were incorporated into the sequence of a THP substrate utilizing automated solid‐phase peptide synthesis protocols. A second substrate was assembled where (7‐methoxycoumarin‐4‐yl)‐acetyl (Mca) was the fluorophore and 2,4‐dinitrophenyl (Dnp) was the quencher. Circular dichroism spectroscopy was used to determine the influence of the fluorophore/quencher pair on the stability of the triple‐helix. The activity of the two substrates was examined with three matrix metalloproteinases (MMPs), MMP‐1, MMP‐13, and MT1‐MMP. The combination of 5‐Fam as fluorophore and Dabcyl as quencher resulted in a triple‐helical substrate that, compared with the fluorophore/quencher pair of Mca/Dnp, had a slightly destabilized triple‐helix but was hydrolyzed more rapidly by MMP‐1 and MMP‐13 and had greater sensitivity. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 347–355, 2013.

更新日期：2013-07-19

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