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Ph
2
P
O
MeO
H
Asp
Tyr
Lys
Asp
Asp
Asp
Asp
Lys
CO
2
-
TAG
O
Flag-modified TAG
O
O
O
N
O
N
S
N
H
H
3
O
HN
Biotin-modified TAG
NH
HO
O
O
O
Fluorescein-modified TAG
N
NH
O
O
5
O
N
N
N
N
O
-
O
3
S
SO
3
-
Et
2
N
O
NEt
2
-
O
3
S
SO
3
-
Cy5.5-modified TAG
Rhodamine-modified TAG
FIGURE 8.2
Various triarylphosphine-TAG conjugates for Staudinger Ligation.
ManNAz. The Staudinger ligation protocol was extended to C57BL/6 mice which
were administered intraperitoneally daily doses of Ac
4
ManNAz (300 mg.kg
−
1
)for
7 days [69]. Next, a phosphine probe comprising a Flag peptide was administered
(Phos-Flag, 16
mol), and 90 minutes later, the mice were euthanized, spleno-
cytes isolated, and cell-surface Flag epitopes probed by FITC-anti-Flag antibody. As
expected, only splenocytes isolated from mice treated with Ac
4
ManNAz and Phos-
Flag displayed a significant increase in fluorescence intensity, indicating a successful
Staudinger ligation
in vivo
. However, the labeling intensity was significantly reduced
when splenocytes were harvested after prolonged exposure to Phos-Flag, (12 hours)
and no significant labeling was detected after 24 hours introduction time. The loss of
detection was attributed to degradation of the Flag peptide, and this problem could
be addressed by employing fluorescently labeled phosphine probes (Fig. 8.2) [70].
In particular, the near-infrared emitter Cy5.5-phosphine conjugate was found to be
superior compared to fluorescein- and rhodamine-phosphine conjugates, which was
attributed to a greater solubility allowing a more efficient removal of excess phosphine
conjugate thereby increasing the signal-to-noise ratio.
The Staudinger ligation has been extended to a phosphine that becomes fluo-
rescent upon reaction with azides (Scheme 8.3) [71]. In this case, the lone pair of
electrons of the phosphine moiety quenches the excited state of the fluorophore,
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