This section describes two types of photoactivatable probes: products that form short-lived, high-energy intermediates that can chemically couple to nearby residues, and "caged" probes that are designed to be biologically inactive until UV light–mediated photolysis releases a natural product. Photolysis of each of these photoactivatable probes can be accomplished with high spatial and temporal resolution, releasing active probe at the site of interest.

Nonfluorescent Photoreactive Crosslinking Reagents

In contrast to chemical crosslinking reagents (Chemical Crosslinking Reagents—Section 5.2), which are often used to prepare bioconjugates, photoreactive crosslinking reagents are important tools for determining the proximity of two sites. These probes can be used to define relationships between two reactive groups that are on a single protein, on a ligand and its receptor, or on separate biomolecules within an assembly. In the latter case, photoreactive crosslinking reagents can potentially reveal interactions among proteins, nucleic acids and membranes in live cells. The general scheme for defining spatial relationships usually involves photoreactive crosslinking reagents that contain a chemically reactive group as well as a photoreactive group. These crosslinkers are first chemically reacted with one molecule, for example a receptor ligand, and then this modified molecule is coupled to a second molecule, for example the ligand's receptor, using UV illumination. Depending on the reactive properties of the chemical and photoreactive groups, these crosslinkers can be used to couple like or unlike functional groups.

We offer three types of photoreactive reagents for covalent labeling:

  • Simple aryl azides that upon illumination (usually at <360 nm) generate reactive intermediates that form bonds with nucleophilic groups (Figure 5.3.1)
  • Fluorinated aryl azides that upon UV photolysis generate reactive nitrenes, thereby producing more C–H insertion products than the simple aryl azides (Figure 5.3.2)
  • Benzophenone derivatives that can be repeatedly excited at <360 nm until they generate covalent adducts, without loss of reactivity (Figure 5.3.3)

Photoreactive crosslinking reaction of a simple aryl azide
Figure 5.3.1
Photoreactive crosslinking reaction of a simple aryl azide.

Photoreactive crosslinking reaction of a fluorinated aryl azide
Figure 5.3.2 Photoreactive crosslinking reaction of a fluorinated aryl azide.

Photoreactive crosslinking reaction of a benzophenone derivative
Figure 5.3.3 Photoreactive crosslinking reaction of a benzophenone derivative.

Simple Aryl Azide Crosslinker

The "transferable" aryl azide N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (PEAS; AET; P6317) is a unique reagent for assessing protein–protein or protein–nucleic acid interactions. This aryl azide undergoes disulfide–thiol interchange of its pyridyldisulfide groups with the thiol groups of biomolecules to form mixed disulfides in the same way as SPDP ref (S1531, Chemical Crosslinking Reagents—Section 5.2). UV photolysis induces covalent crosslinking to residues or biomolecules adjacent to the crosslinker. The mixed disulfide can then be cleaved with DTT or TCEP (D1532, T2556; Chemical Crosslinking Reagents—Section 5.2). If the phenolic PEAS reagent is radioiodinated before the coupling and photolysis steps, then only the resulting target biomolecule will be radioactive at the conclusion of the reaction.

Fluorinated Aryl Azides: True Nitrene-Generating Reagents

Although the simple aryl azides may be initially photolyzed to electron-deficient aryl nitrenes, it has been shown that these rapidly ring-expand to form dehydroazepines—molecules that tend to react with nucleophiles rather than form C–H insertion products.ref In contrast, Keana and Cai have shown that the photolysis products of the fluorinated aryl azides are clearly aryl nitrenes ref and undergo characteristic nitrene reactions such as C–H bond insertion with high efficiency. Moreover, conjugates prepared from the amine-reactive succinimidyl ester of  4-azido-2,3,5,6-tetrafluorobenzoic acid (ATFB, SE; A2522) may have quantum yields for formation of photocrosslinked products that are superior to those of the nonfluorinated aryl azides. An important application of the succinimidyl ester of ATFB is the photofunctionalization of polymer surfaces ref (Figure 5.3.4).

Schematic showing attachment of an amine-modified oligonucleotide to a surface 
Figure 5.3.4 Schematic showing attachment of an amine-modified oligonucleotide to a surface using the photoreactive crosslinking reagent 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (ATFB, SE; A2522).

Benzophenone-Based Photoreactive Reagents

Benzophenones generally have higher crosslinking yields than the aryl azide photoreactive reagents.ref Benzophenone maleimide (B1508) has been used for efficient irreversible protein crosslinking of actin,ref calmodulin,ref myosin,ref tropomyosin,ref troponin,ref ATP synthase ref and other proteins. The succinimidyl ester of 4-benzoylbenzoic acid (B1577) and benzophenone isothiocyanate (B1526) have proven useful for synthesizing photoreactive peptides ref and oligonucleotides.ref A benzophenone-labeled ATP probe (BzBzATP, B22358) is described in Other Photoreactive Reagents below.

Other Photoreactive Reagents

Ethidium Monoazide for Photoreactive Fluorescent Labeling of Nucleic Acids

Ethidium monoazide (E1374) can be photolyzed in the presence of DNA or RNA to yield fluorescently labeled nucleic acids, both in solution and in cells.ref The efficiency of the irreversible photolytic coupling of ethidium monoazide, which intercalates into nucleic acids like ethidium bromide, is unusually high ref (>40%). The membrane-impermeant ethidium monoazide is reported to label only those cells with compromised membranes and can therefore serve as a fixable cell viability probe. This property, allied to the blocking of transcription caused by photoreaction of ethidium monoazide with DNA, provides a method for suppressing PCR amplification of dead-cell DNA.ref Similarly, multiphoton-targeted photochemistry of vertebrate cells labeled with ethidium monoazide was used to selectively inactivate gene expression.ref A mixed population of live and dead cells labeled with ethidium monoazide retains its staining pattern after aldehyde-based fixation, thereby reducing the investigator's exposure to potentially pathogenic cells during cell viability analysis.ref

Bimane Azide for Photoaffinity Labeling of Proteins

Bimane azide (B30600) is a small blue-fluorescent photoreactive alkyl azide (excitation/emission maxima ~375/458 nm) for photoaffinity labeling of proteins. This reactive fluorophore's small size may reduce the likelihood that the label will interfere with the function of the biomolecule, an important advantage for site-selective probes.

Photoreactive ATP Derivative for Labeling Nucleotide-Binding Proteins

Functional ion channels can be assembled from both homomeric and heteromeric combinations of the seven P2X purinergic receptor subunits so far identified (P2X1–7). Due to the lack of specific agonists or antagonists for P2X receptors, it is difficult to determine which receptor subtypes mediate particular cellular responses. We offer one of the most potent and widely used P2X receptor agonists,ref BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, B22358). BzBzATP also has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling.ref

Caged Probes and Their Photolysis

Flash photolysis of photoactivatable or "caged" probes provides a means of controlling the release—both spatially and temporally—of biologically active products or other reagents of interest.ref The chemical caging process may also confer membrane permeability on the caged ligand, as is the case for caged cAMP ref and caged luciferin.ref Our extensive selection of caged nucleotides, second messengers (photo), chelators and neurotransmitters has tremendous potential for use with both live cells and isolated proteins. These caged probes provide researchers with important tools for delivering physiological stimuli by naturally active biomolecules with spatial and temporal precision that far exceeds that of microinjection or perfusion. A recent review by Ellis-Davies describes the optical and chemical properties of many of our caged compounds, as well as of several common caging groups.ref

Caging Groups

The caging moiety (Properties of six different caging groups—Table 5.2) is designed to maximally interfere with the binding or activity of the molecule. It is detached in microseconds to milliseconds by flash photolysis at ≤360 nm, resulting in a pulse (concentration jump) of active product. Uncaging can easily be accomplished with UV illumination in a fluorescence microscope or with a UV laser or UV flashlamp. Low-cost light-emitting diodes ref (LED) and 405 nm violet diode lasers ref are providing increased access to experimentation using caged compounds. The effects of photolytic release are frequently monitored either with fluorescent probes that measure calcium, pH, other ions or membrane potential, or with electrophysiological techniques.

Most of the caged reagents described in the literature have been derivatives of o-nitrobenzylic compounds. The nitrobenzyl group is synthetically incorporated into the biologically active molecule by linkage to a heteroatom (usually O, S or N) as an ether, thioether, ester (including phosphate or thiophosphate esters), amine or similar functional group. Both the structure of the nitrobenzylic compound and the atom to which it is attached affect the efficiency and wavelength required for uncaging. We currently use six different photolabile protecting groups in our caged probes.ref Their properties are summarized in Properties of six different caging groups—Table 5.2.

  • Probes caged with our α-carboxy-2-nitrobenzyl (CNB) caging group generally have the most advantageous properties. These include good water solubility, very fast uncaging rates in the microsecond range, high photolysis quantum yields (from 0.2–0.4) and biologically inert photolytic by-products. Although the absorption maximum of the CNB caging group is near 260 nm, its absorption spectrum tails out to approximately 360 nm, allowing successful photolysis using light with wavelengths ≤360 nm.
  • The 1-(2-nitrophenyl)ethyl (NPE) caging group has properties similar to those of CNB and can also be photolyzed at ≤360 nm.
  • As compared with CNB and NPE, the 4,5-dimethoxy-2-nitrobenzyl (DMNB) and 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) caging groups have longer-wavelength absorption (absorption maximum ~355 nm) and therefore absorb 340–360 nm light more efficiently. However, photolysis rates and quantum yields of DMNB- and DMNPE-caged probes are generally lower than those obtained for CNB-caged probes.
  • The 5-carboxymethoxy-2-nitrobenzyl (CMNB) caging group provides an absorption maximum of intermediate wavelength (absorption maximum ~310 nm), while imparting significant water solubility to the caged probe. Its photolysis rate and quantum yield are intermediate between those of CNB- and DMNB-caged probes.
  • The nitrophenyl (NP) caging group is available on the caged calcium reagent NP-EGTA (N6802), a photolabile Ca2+ chelator that can be used to rapidly deliver a pulse of Ca2+ upon illumination with ultraviolet light, with a high photolysis quantum yield of 0.23.

Experiments utilizing probes caged with any of the above caging groups, except the CNB caging group, may require the addition of dithiothreitol (DTT, D1532; Introduction to Thiol Modification and Detection—Section 2.1). This reducing reagent prevents the potentially cytotoxic reaction between amines and the 2-nitrosobenzoyl photolytic by-products.ref

Caged Nucleotides

Photoactivatable nucleotides and phosphates have contributed significantly to our understanding of cytoskeleton dynamics, signal transduction pathways and other critical cellular processes.ref Some of our caged nucleotides are available with a choice of caging group:

  • Caged ATP (A1048, A1049), which has been shown to release ATP in skinned muscle fibers,ref sarcoplasmic reticulum vesicles,ref submitochondrial particles ref and membrane fragments containing Na+/K+-ATPase ref
  • Caged ADP (A7056), which has been used to investigate the molecular basis of contraction of skeletal muscle fibers,ref as well as transport by an ADP/ATP carrier ref
  • Caged cAMP (D1037), which is cell-permeant and rapidly photolyzed to cAMP ref
  • Caged inositol 1,4,5-triphosphate ref (I23580) and caged cADP-ribose ref (C7074), which are important probes for second messenger studies (Calcium Regulation—Section 17.2)

NPE-caged Ins 1,4,5-P3 can be used to generate rapid and precisely controlled release of Ins 1,4,5-P3 in intact cells (photo) and is widely employed in studies of Ins 1,4,5-P3–mediated second messenger pathways.ref Our NPE-caged Ins 1,4,5-P3 (I23580) is a mixture of the physiologically inert, singly esterified P4 and P5 esters and does not contain the somewhat physiologically active P1 ester. NPE-caged Ins 1,4,5-P3 exhibits essentially no biological activity prior to photolytic release of the biologically active Ins 1,4,5-P3 (I3716, Calcium Regulation—Section 17.2).

Cyclic ADP-ribose (cADP-ribose) is a potent intracellular Ca2+–mobilizing agent that functions as a second messenger in an Ins 1,4,5-P3–independent pathway.ref Our NPE-caged cADP-ribose (C7074) induces Ca2+ mobilization in sea urchin egg homogenates only after photolysis, and this Ca2+ release is inhibited by the specific cADP-ribose antagonist 8-amino-cADP-ribose ref (A7621, Calcium Regulation—Section 17.2). Furthermore, when microinjected into live sea urchin eggs, NPE-caged cADP-ribose was shown to mobilize Ca2+ and activate cortical exocytosis after illumination with a mercury-arc lamp.ref

Caged Ca2+ Reagents: NP-EGTA and DMNP-EDTA

Caged ions and caged chelators can be used to influence the ionic composition of both solutions and cells, particularly for ions such as Ca2+ that are present at low concentrations under normal physiological conditions. Developed by Ellis-Davies and Kaplan,ref nitrophenyl EGTA (NP-EGTA) is a photolabile Ca2+ chelator that exhibits a high selectivity for Ca2+ ions, a dramatic increase in its Kd for Ca2+ upon illumination (from 80 nM to 1 mM) and a high photolysis quantum yield (0.23). NP-EGTA's affinity for Ca2+decreases ~12,500-fold upon photolysis. Furthermore, its Kd for Mg2+ of 9 mM makes NP-EGTA essentially insensitive to physiological Mg2+ concentrations. We exclusively offer the tetrapotassium salt (N6802) and the acetoxymethyl (AM) ester (N6803) of NP-EGTA. The NP-EGTA salt can be complexed with Ca2+ to generate a caged Ca2+ reagent that will rapidly deliver Ca2+ upon photolysis ref (Figure 5.3.5). The cell-permeant AM ester of NP-EGTA does not bind Ca2+ unless its AM ester groups are removed. This AM ester can serve as a photolabile chelator in cells because, once converted to NP-EGTA by intracellular esterases, it will bind free Ca2+ until photolyzed with UV light.

The first caged Ca2+ reagent described by Kaplan and Ellis-Davies was 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (DMNP-EDTA, D6814), which they named DM-Nitrophen ref (now a trademark of Calbiochem-Novabiochem Corp.). Because its structure more resembles that of EDTA than EGTA, we named it as a caged EDTA derivative (Figure 5.3.6). Upon illumination, DMNP-EDTA's affinity for Ca2+decreases ~600,000-fold and its Kd for Ca2+ rises from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed with Ca2+ results in a pulse of free Ca2+. DMNP-EDTA has a stronger absorbance at longer wavelengths than does NP-EGTA (Figure 5.3.7), which facilitates uncaging. Furthermore, DMNP-EDTA has significantly higher affinity for Mg2+ (Kd = 2.5 µM) ref than does NP-EGTA (Kd = 9 mM),ref making it a potentially useful caged Mg2+ reagent. Two reviews by Ellis-Davies discuss the uses and limitations of DMNP-EDTA.ref

NP-EGTA complexed with Ca2+ 
Figure 5.3.5
NP-EGTA (N6802) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the photoproducts for Ca2+ is ~12,500-fold lower than that of NP-EGTA.
DMNP-EDTA complexed with Ca2+ 
Figure 5.3.6
DMNP-EDTA (D6814) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The affinity of the photoproducts for Ca2+ is ~600,000-fold lower than that of DMNP-EDTA.
Spectral comparison of equimolar concentrations  
Figure 5.3.7 Spectral comparison of equimolar concentrations of the caged Ca2+ reagents NP-EGTA (N6802, red line) and DMNP-EDTA (D6814, blue line), illustrating the optimal wavelengths for photolysis and subsequent release of Ca2+ from these chelators. Spectra were taken in 100 mM KCl and 30 mM MOPS buffer containing 39.8 µM free Ca2+ at pH 7.2.

Diazo-2: A Photoactivatable Ca2+ Knockdown Reagent

In contrast to NP-EGTA and DMNP-EDTA, diazo-2 (D3034) is a photoactivatable Ca2+ scavenger. Diazo-2, which was introduced by Adams, Kao and Tsien,ref is a relatively weak chelator (Kd for Ca2+ = 2.2 µM). Following flash photolysis at ~360 nm, however, cytosolic free Ca2+ rapidly binds to the diazo-2 photolysis product, which has a high affinity for Ca2+ (Kd = 73 nM). Intracellular loading of NP-EGTA, DMNP-EDTA and diazo-2 is best accomplished by patch pipette infusion with the carboxylate salt form of the caged compound added to the internal pipette solution at 1–10 mM. These reagents are increasingly being applied in vivo for controlled intervention in calcium-regulated fundamental processes in neurobiology ref and developmental biology.ref

Caged Amino Acid Neurotransmitters

Once activated, caged amino acid neurotransmitters rapidly initiate neurotransmitter action (Figure 5.3.8), providing tools for kinetic studies of receptor binding or channel opening.ref We offer caged carbamylcholine ref (N-(CNB-caged) carbachol, C13654) and caged γ-aminobutyric acid ref (O-(CNB-caged) GABA, A7110), as well as two caged versions of L-glutamic acid ref (C7122, G7055), all of which are biologically inactive before photolysis.ref

CNB-caged L-glutamic acid
Figure 5.3.8
CNB-caged L-glutamic acid (G7055). The CNB-caging group is rapidly photocleaved with UV light to release L-glutamic acid.

Caged Luciferin

Luciferase produces light by the ATP-dependent oxidation of luciferin. The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase activity or ATP concentrations. DMNPE-caged luciferin (L7085) readily crosses cell membranes, allowing more efficient delivery of luciferin into intact cells.ref Once the caged luciferin is inside the cell, active luciferin can be released either instantaneously by a flash of UV light, or continuously by the action of endogenous intracellular esterases found in many cell types.

Caged Fluorescent Dyes

Photoactivatable fluorescent dyes, which are generally colorless and nonfluorescent until photolyzed with UV light,ref are particularly useful for investigating cell lineage ref and for spatiotemporal interrogation of fluid flows.ref In addition to CMNB-caged fluorescein ref (F7103), we prepare the succinimidyl ester of CMNB-caged carboxyfluorescein (C20050), which can be used to attach the caged fluorophore to primary amine groups of a variety of biomolecules. CMNB-caged carboxyfluorescein succinimidyl ester is a key starting material in the preparation of probes for super-resolution photoactivation microscopy.ref Furthermore, caged fluorescein probes are immunochemically cryptic; i.e., the probe is immunoreactive with anti–fluorescein/Oregon Green dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) after but not before photoactivation (Figure 5.3.9).

Schematic representation of photoactivated fluorescence combined with sample masking 


Figure 5.3.9 Schematic representation of photoactivated fluorescence combined with sample masking. Initially, no fluorescence is observed from samples stained with a CMNB-caged fluorescein-labeled secondary detection reagent (A). The desired mask is then placed over the sample (B), after which the sample is exposed to UV light. The mask is then removed; fluorescein molecules present in the unmasked portion of the sample are uncaged by the UV light and fluoresce brightly when viewed with the appropriate filters (C). Uncaged fluorescein may now also serve as a hapten for further signal amplification using our anti–fluorescein/Oregon Green dye antibody (Anti-Dye and Anti-Hapten Antibodies—Section 7.4). For example, probing with the anti–fluorescein/Oregon Green dye antibody followed by staining with the Alexa Fluor 594 goat anti–mouse IgG antibody can be used to change the color of the uncaged probe to red fluorescent (D).

Kit for Caging Carboxylic Acids

Using organic synthesis methods, researchers can cage a diverse array of molecules. One of the preferred caging groups is the 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) ester. Because the diazoethane precursor to DMNPE esters is unstable, we offer a kit (D2516) for the generation of 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane and the subsequent preparation of DMNPE esters. This kit includes:

A wide range of compounds containing a weak oxy acid (with a pKa between 3 and 7), including carboxylic acids, phenols and phosphates, should react with the diazoethane to form the DMNPE-caged analogs ref (Figure 5.3.10).

Caging of a carboxylic acid using the hydrazone precursor of DMNPE
Figure 5.3.10 Caging of a carboxylic acid using the hydrazone precursor of DMNPE, a reagent that is provided in the 1-(4,5-Dimethoxy-2-nitrophenyl)diazoethane Generation Kit (D2516).

Data Table

Cat #MWStorageSolubleAbsECEmSolventNotes
A1048700.30FF,D,LLH2O25918,000noneMeOH1, 2, 3
A1049760.35FF,D,LLH2O3514400noneH2O1, 2
A7056614.44FF,D,LLH2O25915,000noneMeOH1, 2, 3
A7110396.28F,D,LLH2O2624500nonepH 72, 3
B1508277.28F,DDMF, MeCN26017,000noneMeOH3, 4
B1526239.29F,DDDMF, MeCN30026,000noneMeOH3
B1577323.30F,DDMF, MeCN25627,000noneMeOH3
B223581018.97FF,LH2O26027,000nonepH 73, 5, 6, 7
C7074690.45FF,D,LLH2O25916,000noneH2O2, 3
C7122326.26F,D,LLH2O2664800nonepH 72, 3
C13654439.34F,D,LLH2O2644200noneH2O2, 3
C20050962.79F,D,LLDMSO2899500noneMeOH2, 8
D1037524.38F,D,LLDMSO3386100noneMeOH1, 2
D3034710.86F,D,LLpH >636918,000nonepH 7.22, 9
D6814473.39D,LLDMSO3484200nonepH 7.22, 10
E1374420.31F,LLDMF, EtOH4625400625pH 711
F7103826.81FF,D,LLH2O, DMSO33315,000noneDMSO2, 8, 12
G7055440.29F,D,LLH2O, DMSO2625100nonepH 72, 3
I23580872.82FF,D,LLH2O2644200noneH2O2, 3, 13
L7085489.52FF,D,LLDMSO, DMF33422,000noneMeOH2, 14
N6802653.81FF,D,LLpH >62603500nonepH 7.22, 3, 15
N6803789.70FF,D,LLDMSO2504200noneMeCN16, 17
  1. Caged nucleotide esters are free of contaminating free nucleotides when initially prepared. However, some decomposition may occur during storage.
  2. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
  3. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.
  4. Spectral data of the 2-mercaptoethanol adduct.
  5. The molecular weight (MW) of this product is approximate because the degree of hydration and/or salt form has not been conclusively established.
  6. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  7. This product can be activated by long-wavelength ultraviolet light (>300 nm) for photoaffinity labeling of proteins.
  8. This product is colorless and nonfluorescent until it is activated by ultraviolet photolysis. Photoactivation generates a fluorescein derivative with spectral characteristics similar to C1359.
  9. The Ca2+ dissociation constant of diazo-2 is 2200 nM before photolysis and 73 nM after ultraviolet photolysis. The absorption spectrum of the photolysis product is similar to that of B1204.ref
  10. Kd(Ca2+) increases from 5 nM to 3 mM after ultraviolet photolysis. Kd values determined in 130 mM KCl, 10 mM HEPES, pH 7.1.ref
  11. E1374 spectral data are for the free dye. Fluorescence is weak, but intensity increases ~15-fold on binding to DNA. After photocrosslinking to DNA, Abs = 504 nm (EC ~4000 cm-1M-1), Em = 600 nm.ref
  12. Unstable in water. Use immediately.
  13. Ultraviolet photolysis of I23580 generates I3716.
  14. L7085 is converted to bioluminescent luciferin (L2911) upon ultraviolet photoactivation.
  15. Kd (Ca2+) increases from 80 nM to 1 mM after ultraviolet photolysis. Kd values determined in 100 mM KCl, 40 mM HEPES, pH 7.2.ref
  16. This product is intrinsically a liquid or an oil at room temperature.
  17. N6803 is converted to N6802 via hydrolysis of its acetoxymethyl ester (AM) groups.
  18. The absorption spectrum of P6317 includes an additional shoulder at 306 nm (EC = 10,000 cm-1M-1).