Thermo Scientific Pierce Crosslinking Technical Handbook

Crosslinker Reactivities
Diazirines
The succinimidyl-ester diazirine (SDA) reagents are a new class of
crosslinkers that combine proven amine-reactive chemistry with
an efficient diazirine-based photochemistry for photo-crosslinking
to nearly any other functional group. Diazirine-based photocrosslinkers
have better photostability than phenyl azide-based
photocrosslinkers and are easily activated with long-wave UV
light (330-370 nm). The SDA crosslinkers include six compounds
differing in spacer arm lengths, ability to cleave the crosslinked
proteins, and presence or absence of a charged group for
differential membrane permeability. These SDA reagents extend
the efficiency and range of interactions that can be explored by
this approach.
Photoactivation of diazirine with long-wave UV light (330-370 nm)
creates reactive carbene intermediates. Such intermediates can
form covalent bonds through addition reactions with any amino
acid side-chain or peptide backbone at distances corresponding
to the spacer arm lengths.
The NHS-ester diazirine derivatives (SDA, LC-SDA and SDAD) lack
a charged group and are membrane-permeable. This property
makes them ideal for intracellular and intramembrane conjugations.
By contrast, Sulfo-SDA, Sulfo-LC-SDA and Sulfo-SDAD
contain negatively charged sulfonate groups that improve their
water solubility and reduce cell membrane permeability, enabling
their use for extracellular protein crosslinking. SDAD and
Sulfo-SDAD also have a disulfide bond within the spacer that
can be cleaved with reducing agents.
Thermo Scientific Pierce Diazirine-based Photo-crosslinkers.
Reagent Product # Reactive Groups
SDA 26167 NHS, Diazirine
LC-SDC 26168 NHS, Diazirine
SDAD 26169 NHS, Diazirine
Sulfo-SDA 26173 Sulfo-NHS, Diazirine
Sulfo-LC-SDA 26174 Sulfo-NHS, Diazirine
Sulfo-SDAD 26175 Sulfo-NHS, Diazirine
Protein 1
Protein 1
NH 2
O
N N
O
N
H
N
N 2
UV 350 nm
O
N
N
O
O
Protein 2
Protein 1
H
N
O
H
Protein 2
Figure 15. Mechanism of NHS-ester diazirine crosslinking.
N-hydroxysuccinimide (NHS) esters react efficiently with primary amine
groups (–NH 2 ) in pH 7-9 buffers to form stable amide bonds upon release
of NHS. Photoactivation of diazirines with long-wave UV light (330-370 nm)
creates reactive carbene intermediates. Such intermediates can form
covalent bonds through addition reactions with any amino acid side chain
or peptide backbone at distances corresponding to the spacer arm lengths.
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