申请人:Tsien Y. Roger
公开号:US20050118669A1
公开(公告)日:2005-06-02
Fluorescent substrates for beta-lactamases having the general formula shown above are provided. Z includes a fluorophore or chromophore and includes a group that may link to the lactam-containing group; R
1
and R
2
are independently selected from H, aliphatic, aromatic, alkyl, and acyl; R
4
may be H and lower alkyl; B may be H, physiologically acceptable salts or metal, ester groups, ammonium cations, —CHR
5
OCO(CH
2
)
n
CH
3
, —CHR
5
OCOC(CH
3
)
3
, acylthiomethyl, acyloxy-alpha-benz, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R
5
is selected from the group consisting of H and lower alkyl; n is an integer between 0 and 10, inclusive; and A may be S, O, SO, SO
2
or CH
2
. In embodiments, the beta-lactam ring may be cleaved by a beta-lactamase enzyme effective to free a fluorophore. Methods of assaying beta-lactamase activity and monitoring expression in systems using beta-lactamase as a reporter gene are also disclosed.
提供了具有上述一般式的β-内酰胺酶荧光底物。Z包括荧光团或色团,并包括可以连接到含内酰胺基团的基团;R1和R2独立地选择自H,脂肪族,芳香族,烷基和酰基;R4可以是H和低碳基;B可以是H,生理上可接受的盐或金属,酯基,铵阳离子,-CHR5OCO(CH2)nCH3,-CHR5OCOC(CH3)3,酰硫甲基,酰氧基-α-苯,三丁基内酯,甲氧羰氧甲基,苯基,甲硫氧基甲基,β-吗啡啉乙基,二烷基氨基乙基,和二烷基氨基羰氧甲基,其中R5选择自H和低碳基的群;n是0到10之间的整数,包括10;A可以是S,O,SO,SO2或CH2。在实施例中,β-内酰胺环可以被有效释放荧光团的β-内酰胺酶酶裂解。还公开了检测β-内酰胺酶活性和使用β-内酰胺酶作为报告基因的系统中监测表达的方法。