Caged compounds

Since the invention of the microscope by Robert Hooke in 1695, light has been to study cells. Most laboratories are satisfied with such passive observation, however we use not only to observe but also to stimulate cellular chemistry. This area of biophotonics is called “caged compounds” as synthetic organic chemistry is used to make biologically signaling molecules functionally inert; irradiation liberates the caged molecules, thus “switching on” a chosen signaling pathway.

We have developed many caged compounds for stimulating both intracellular and extracellular receptors. These molecules include caged calcium, IP3, glutamate, GABA, D-apartate, serotonin, dopamine, anisomycin, siphigosine, capsacin, nitric oxide, carbamoylcholine, BAPTA, etc.

Caged calcium

Calcium is the most important signaling molecule inside cells. Fluctuations in its concentration control gene transcription, insulin secretion, muscle contraction, neurotransmission, wound healing, etc. The laboratory has developed four useful calcium cages: DM-nitrophen; NP-EGTA; DMNPE-4 and NDBF-EGTA. Two of our calcium cages (DM-nitrophen and NP-EGTA) have been commercially available for some time now. So, literally hundreds of biological studies have been published by us and many other groups using these probes. In particular, Erwin Neher and Bob Zucker and their co-workers have used our cages to study secretion in many cells (they have published more than 60 caged calcium papers).

Recently we have developed a new caging chromophore (NDBF) that is much more efficient than the traditional nitrobenzyl one that has so widely used by many groups for the past 20 years. Our first application of this new chromophore has been to caged Ca (NDBF-EGTA). The table summarizes the two basic of physico-chemical properties of all the commercially available nitrobenzyl-caged compounds: the extinction coefficient ( ε ) and the quantum yield ( φ ). The product ( ε.φ ) is the measure of the efficiency of uncaging. (Significantly, the 2-photon cross section of NDBF-EGTA is also very high, being about 0.6 GM.)

Caged neurotransmitters

Glutamate is the major transmitter in the CNS, being responsible for about 80% of transmission. In 2000 we synthesized MNI-glutamate; the Mill Hill group, lead by John Corrie, independently made the same compound. We, however, were the first to use this molecule for 2-photon photolysis in brain slices, in collaboration Haruo Kasai. Recently we have made a new caged glutamate (MDNI-glu) that is 10 times better than MNI-glu. The table summarizes the properties of all the caged glutamates that have been made by various groups over the past 10 years

Chromophore	Φ	2-photon cross section (GM)	ε ( 350 nm)	Φ.ε
MDNI	0.47	0.06	8,600	4042
MNI	0.085	0.06	4,300	366
NI	0.043	No datum	2,700	116
Bhc	0.019	0.95	17,300	329
CNB	0.15	<0.001	500	75
pHP	0.08	No datum	200	16

New caged compounds

Our current efforts focus on two fronts: synthesis of of photochromic calcium chelators (moleculs we call “calcium switches”); and the development of caged compounds for in vivo 2-photon photolysis. The lab uses 2-photon microscopy to study calcium signaling in vivo. Rapid drug application is impractical is such circumstances, thus we will synthesize pro-drugs that are caged and can be loaded into the brain of the living animal, and uncaged using the dual 2P microscope, during calcium imaging.