nitrenes and nitrenium ions (falvey/nitrenes and nitrenium ions) || nitrenium ions and related...

3NITRENIUM IONS AND RELATEDSPECIES IN PHOTOAFFINITYLABELING

R. MARSHALLWILSON AND VALENTYNAVOSKRESENSKA

3.1 Introduction

3.2 Reactions of Nitrenium Ions with Biomolecules

3.2.1 Properties of Aryl Azides that Serve as Nitrenium Ion Precursors

3.3 Applications of Aryl Nitrenium Ions in Photoaffinity Labeling

3.3.1 4-Azido-2-nitrophenyl Amines

3.4 General Considerations

3.4.1 Purine Azides

3.5 Conclusions

References

3.1 INTRODUCTION

Photoaffinity labeling (PAL) has found increasing application as a tool for unraveling

the complex interactions of biological molecules. In general, a small, photoaffinity

labeling molecule is attached to a large biological molecule of interest (Fig. 3.1). It is

usually assumed that this small PAL molecule is unobtrusive and does not affect the

binding properties of the much larger biological molecule to which it is attached. The

labeled molecule is then exposed to a variety of other biomolecules with which it

might associate via various ephemeral noncovalent bonds such as hydrogen bonds,

electrostatic/ionic bonds, hydrophobic aggregation, and so on. Irradiation of this

Nitrenes and Nitrenium Ions, First Edition. Edited by Daniel E. Falvey and Anna D. Gudmundsdottir.� 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

mixture releases a highly reactive species from the PAL tag, which then reacts with

any suitable functional groups in its immediate vicinity forming a permanent

covalent bond. Ideally, the two relatively weakly associating molecules become a

single large molecule with the two constituents permanently attached to each other

by the newly formed strong covalent bond. This new cross-linked molecule can be

identified as a new component of a complex biological mixture, isolated, and

analyzed in order to determine the exact identity of the biomolecule to which the

molecule of interest bound. More detailed analysis might then determine the exact

site on the target molecule to which the molecule of interest became bound.

From a biochemical perspective, an effective photoaffinity labeling agent is one

that can be easily attached to the surface of a biomolecule, that does not significantly

perturb the natural interactions of the unlabeled molecules, and that cross-links the

molecule to which it is attached with other interacting molecules. These criteria

having been met, the photoaffinity labeling agent is usually applied without concern

for the mechanism through which the coupling or cross-linking proceeds. An

excellent review on this topic has been provided by Fleming,1 although it was

written before much of the present-day mechanistic information pertaining to azide

photochemistry and nitrene/nitrenium ion chemistry was available.

3.2 REACTIONS OF NITRENIUM IONS WITH BIOMOLECULES

It turns out that some of the more effective photoaffinity cross-linking agents

generate aryl nitrenium ions as their pivotal intermediates. Consequently, knowledge

of the reactivity of nitrenium ions with biological molecules becomes critical in

designing effective PAL strategies. Interestingly enough one of the best sources for

Protein 1

Protein 2

PAL = Photoaffinity label

Protein 1

Protein 2

Associated proteinshν

Protein 2

Protein 1

Cross-linked proteins

CL = Cross-linking bridge

FIGURE 3.1. General cross-linking strategy used in applying PAL agents in the study of

association of complex biological molecules (dashed lines indicate weak, noncovalent bonds,

and the solid lines strong covalent bonds of the cross-linking bridge). The association of two

proteins is used as an example here, but the same strategy can be used to study the association

of many types of biolmolecules.

78 NITRENIUM IONS AND RELATED SPECIES IN PHOTOAFFINITY LABELING

this type of information is in the reactions of carcinogenic aryl amines with

biological molecules.2–9

Aryl amines occur in a wide variety of industrial and consumer products, and

many of these have been considered as possible environmental carcinogens. In

addition, they are generated via amino acid pyrolysis when meat is barbecued, and to

a lesser extent, when it is cooked by other means. As a result, aryl amines have been

studied extensively in efforts to understand how they initiate carcinogenesis and

mutagenesis.2–5While it is beyond the scope of this review to address this fascinating

topic, it seems to be generally agreed that nitrenium ions play a pivotal role in the

initiation of carcinogenesis. The process by which aryl amines are transformed into

nitrenium ions in biological systems is outlined in Scheme 3.1. Thus, the aryl amines

are initially converted to hydroxylamines via cytochrome P450 and P448 oxidation.

Acetylation of the amine nitrogen either before or after hydroxylation leads to

hydroxylamine acetate or hydroxamic acid acetate, respectively. Finally, the

hydroxamic acids might be acetylated or sulfonated on the hydroxy group to

form the most efficient nitrenium ion precursors.

Aryl nitrenium ions react with a variety of biological molecules more rapidly than

they do with water. For example, N-acetyl-2-fluorenyl nitrenium ion reacts 8000

times more rapidly with dGuanosine than it does with water.9 Many types of

biological molecules apparently compete favorably with water for nitrenium

ions. An approximate order of reactivity for the nitrenium ion derived from

1-naphthylamine is as follows: DNA> polyguanylic acid> denatured DNA and

ribosomal RNA> serum albumin> transfer RNA> polyadenylic acid.7 Nitrenium

ion adducts with DNA can be detected with very high sensitivity. Using the32P-postlabeling method of detection, adduct detectability is 1.8–14 adducts/1010

nucleotides.10 However, more recently, the detectability levels have been increased to

Aryl-NH2 Aryl-NHAc

Cytochrome P450

Aryl-N

H Aryl-N

Aryl-N

Ac Aryl-N

OSO3–

AcAryl-N

Aryl-N H Aryl-N AcNitreniumions

SCHEME 3.1.

REACTIONS OF NITRENIUM IONSWITH BIOMOLECULES 79

1–10 adducts/1012 nucleotides using accelerator mass spectrometry techniques.11–14

As indicated earlier, guanosine seems to be one of the most reactive targets for aryl

nitrenium ions, and studies with several aryl nitrenium ions indicate that two types

of adducts are most commonly formed. Thus, the nitrenium ion 1 derived from

N-2-(acetylamino)fluorine undergoes both C- and N-alkylation with dG to form

2 (85%) and 3 (15%) shown in Scheme 3.2.

This pattern is repeated with other nitrenium ions in their reactions with dG and

dA (Scheme 3.3). Thus, the nitrenium ion 4 derived from 2-naphthylamine also

undergoes both C- and N-alkylation with dG to form the N-adduct at C-8, 5, its

hydrolysis product 6, and the C-adduct at the 2-amino group, 7. Even though

adenosine (dA) is significantly less reactive than guanosine, it forms analogous

C-adduct at the 6-amino group, 8.15,16

There are many aromatic amines and heterocyclic aromatic amines that, on

oxidative activation via hydroxylation, form nitrenium ion precursors.17–19 In

SCHEME 3.2.

dRibdRib

SCHEME 3.3.

most of these cases, the nitrenium ion nitrogen becomes attached to the 8-position of

the guanosine, 2 in Scheme 3.2, and 5 and 6 in Scheme 3.3. This is unusual in that

most aryl nitrenium ions tend to undergo nucleophile attack at an aromatic ring

carbon rather than at the nitrenium ion nitrogen itself, 3 in Scheme 3.2, and 7 and 8 inScheme 3.3. There are many other less common modes of attack of nitrenium ions on

guanosine that are not shown in Schemes 3.2 and 3.3. These include attack of the

nitrenium ion nitrogen on the carbonyl oxygen to form hydroxylamine ether or on

the amino group in the 2-position to form a hydrazine.

The mechanism of addition of the nitrenium nitrogen to the 8-position of

guanosine has generated the most debate, since this mode of reaction generally

predominates and constitutes an unusual pathway of nitrenium ion reactivity.

Theoretical calculations have been applied in attempts to account for this strange

mode of nitrenium ion reactivity. Early studies indicated that initial attack of

the nitrenium ion occurs at the nitrogen in the 7-position of guanosine followed

by a 1,2-shift to the 8-position via one of several possible mechanisms.20–23 More

recently, direct observation of the intermediates involved in this reaction using

transient laser spectroscopy and flash photolysis show that the nitrenium ion attacks

the 8-position directly without passing through any intermediate adducts (Scheme

3.4),24,25 and later high-level calculations support this conclusion.26

The application of nitrenium ions as PAL intermediates has been shown to be

viable through the photolysis of substituted aryl azides that are effective nitrenium

ion precursors (see below). The capability to observe these short-lived reactive

intermediates directly is proving to be a mechanistic tool of great value. McClelland

has pioneered this area of aryl nitrene/nitrenium ion chemistry,27,28 and shown that

aryl nitrenium ions survive in water into the millisecond time domain. Surprisingly,

in aqueous solutions of guanosine and its derivatives, nitrenium ions react with

guanosine at nearly diffusion-controlled rates, p-phenylphenyl nitrenium ion reacts

with dG in water with a rate constant of 2.0� 109M�1 s�1.25,29 Most intriguing is

the observation that the initial adduct of 2-fluorenylnitrenium ion with dG, 9, is

surprisingly acidic, pKa¼ 3.9, and loses a proton from N1 to form the iminoquinone

10, (Scheme 3.4). Thus, 10 would be the predominant species at physiological pHs

NH2dRib

pKa =3.9

NH2dRib

hν, –N2, +H+

SCHEME 3.4.

not the initial adduct 9, which ultimately loses a proton to form the observed

product 11.

3.2.1 Properties of Aryl Azides that Serve as Nitrenium Ion Precursors

As indicated by McClelland’s studies, aryl azides would seem to provide a

particularly versatile photochemical source of nitrenium ions.24,25,27–29 In recent

years, the photochemical behavior of aryl azides has been studied in detail by Platz

et al.30 However, nitrenium ion formation was not observed in most of the systems

that they studied. The typical photochemical behavior of most simple phenyl azides

is to initially undergo loss of nitrogen from the singlet excited state of the azide. This

process is very rapid occurring within a few hundred femtoseconds of excitation

(Scheme 3.5).31,32 The singlet nitrene, then, rapidly cyclizes to azirines 12, which is

also very short-lived, and rapidly undergoes ring expansion to the ketenimine 13. In

the case of 1-azidonaphthalene, the singlet excited state of the azide loses nitrogen

with a time constant of t¼ 730 fs, and the resulting nitrene cyclizes to the azirine

with a time constant of t¼ 12 ps.32 The phenyl nitrene closes to the azirine 12 then

ring expands to the ketenimine 13 much more rapidly with a time constant of

t¼�0.1–1.0 ns.33,34 Azirines related to 12 can be trapped with ethanthiol35 and

dimethylamine to form 14.30,36 While both 12 and 13 react with nucleophiles to form14 and 15, respectively, and thus, might be considered viable PAL agents, they do not

seem to have been widely used for this purpose. Since most of the aforementioned

mechanistic studies have been conducted in nonaqueous media, the suitability of 12

and 13 as cross-linking species in aqueous media is questionable. Furthermore, even

if these species were applied as cross-linking species under biological conditions, the

cross-linked molecules would be susceptible to hydrolysis, and probably would not

survive the analysis processes. Therefore, this chemistry had to be evolved further to

be useful in PAL applications.

When the aforementioned studies were expanded to acidic and aqueous media, a

new branch of aryl azide photochemistry was observed. This branch was first

–N2Singletnitrene

Azirine Ketenimine12 13

HN NNuc NucH2N

SCHEME 3.5.

reported by Baetzold and Tong in 1971,37 but was largely unrecognized by the

photochemical community for the next several decades. These workers observed that

p-aminophenyl azides afforded the corresponding quinodiimines on irradiation in

buffered weakly acidic aqueous solutions, Scheme 3.6. While they did not

unequivocally establish the structure of the unstable nitrenium ions formed in these

reactions, or the products of their hydrolysis, they did select an optimal system for

switching the aryl nitrene chemistry from its usual ring-expanding, ketenimine mode

to its nitrenium ion mode.

Ten years later, Takeuchi and Koyama investigated the photochemistry of phenyl

azide in the presence of acetic acid and ethanol.38,39 They found a new line of

products, 16 and 17, in these reactions, Scheme 3.7. They argued that these products

arose from a branch of nitrene chemistry that was in competition with the ketenimine

branch described in Scheme 3.5. They speculated that these new ring-substituted

products arose from collapse of a nitrenium ion pair which in turn arose from

protonation of the nitrene by the acidic solvent.

Only in 1995 did it become possible to directly observe the nitrenium ions formed

in these reactions.40 McClelland systematically characterized aryl azide photo-

chemistry to the limits of the capabilities of his time when he observed aryl

nitrenium ions in aqueous acetonitrile and water alone, and found that nitrenium

ions were formed within the laser pump pulsewidth of 20 ns via the rapid protonation

Singletnitrene

Nitreniumion

Quinodiiminiumion

SCHEME 3.6.

Keteniminebranch

Nitreniumion branch

NHAcOR

R = H, Ac

SCHEME 3.7.

by solvent. The nitrenium ion, then, undergoes a relatively slow reaction with water

to form either benzoquinone or 4-substituted-4-hydroxy-2,5-cyclohexadienones40

depending on substitution in the 4-position of the phenyl ring as outlined in

Scheme 3.8. From these studies, it became clear that some nitrenes are rather strong

bases, pKa> 12.4,41 capable of being protonated by water and alcohols. The rate

constants for decay of various nitrenium ions in water are listed in Table 3.1. In

addition to the systems listed in Table 3.1, a number of other nitrenium ions have

Nhν–N2

R = OH

H2O–NH3

OArylazide

Nitrene Nitreniumion

Dienoneimine

Iminoquinone Quinone

SCHEME 3.8.

TABLE 3.1. Absorption Maxima and Rate Constants for Decay of Substituted

Nitrenium Ions in Water

Substituent lmax (nm) kdecay (s�1) Reference

NHR R =��H �2� 1010 44

��Cl �2� 108 45

��OPh �2� 107 46

��OC6H4-p-OCH3 1.9� 0.2� 107 46

��Ph 460 2.84� 0.04� 106 41

��OCH3 305 2.7� 0.2� 106 46

��OCH2CH3 300 1.8� 1�106 46

��OCH(CH3)2 295 8.0� 0.1� 105 46

��OC(CH3)3 NH 290 6.4� 0.2� 104 46

525–550��Br 6.5� 0.3� 106 47

��H 6.3� 0.3� 106 47

��CH3 1.5� 0.1� 106 47

��N(CH3)Ac 5.6� 0.3� 105 47

��OCH3 6.1� 0.1� 104 47

3.4� 0.2� 104 41

While most constants were determined in ionically buffered aqueous media, it should be noted that some

less stable ions are better generated and stabilities enhanced if they are generated in slightly acidic media.

been studied by means of laser transient spectroscopy. Some of these include

8-azido-8-methoxypsoralen27,42 and 2-azido-1-methylimidazole.28 During this

same period, Platz investigated a series of fluorinated phenyl azides including

perfluorophenyl azide and a series of 4-substituted tetrafluorophenyl azides.43

Nitrenium ions could be observed in these fluorinated systems, if irradiations

were conducted in dilute aqueous solutions of sulfuric acid to aid in the protonation

of the nitrenes. Unfortunately, no products could be isolated from these reactions as

the nitrenium ions apparently polymerized.

The next generation of ultrafast laser transient spectroscopy has made it possible

to visualize the extremely rapid events that were obscured by the long-probe laser

pulses in the previous studies. Platz, Burdzinski, and Bally have examined unadorned

polycyclic aryl nitrenes and observed them to undergo protonation to form nitrenium

ions, but in order for protonation to be competitive with intersystem crossing to the

triplet, the nitrenes must be generated in acidic solvents. Thus, the following singlet

nitrene lifetimes were observed when the nitrenes were generated in 88% formic

acid: 1-napthtyl nitrene, t¼ 8.4 ps48; p-biphenylyl nitrene, t¼ 11.5 ps48; phenyl

nitrene, t¼ 12.0 ps (100% formic acid)49; and 2-fluorenyl nitrene, t¼ 10 ps.50 Much

less rapid protonation occurs in alcoholic solvents, for example, in methanol, where

2-fluorenyl nitrene has a lifetime of t¼ 250 ps.50

The valuable information gleaned from these transient studies was that, if properly

substituted, aryl nitrenes are surprisingly strong bases that can abstract protons from

water without the aid of added acid. In order for the nitrenium ion-forming branch of

aryl nitrene chemistry to be favored over the ring-expansion branch in Scheme 3.7,

the nitrene must be conjugated to strong electron-donating groups or extended

p-systems. Detailed theoretical studies of phenyl azides substituted in the para

position with a wide variety of substituents reinforces the observation that para-

electron-donating groups greatly stabilize the singlet state of nitrenium ions.51 The

nitrenium ion pairs that result from protonation of these nitrenes are surprisingly

stable and do not collapse quickly, as might be expected, but survive into the

microsecond and even millisecond time domains. Furthermore, the mode of collapse

observed in these early studies was nucleophilic attack of water at the para position,

which results in hydrolysis to a quinone with loss of the original aryl azide

substituents (Scheme 3.8).

Many of these properties of aryl nitrenium ion are not particularly favorable for

development of photoaffinity-labeling systems. Thus, an ideal photoaffinity label

will form a strong covalent bond with neighboring molecules, and this bond must

survive isolation and analysis. As can be seen from the aforementioned examples, the

ketenimine branch affords cross-linking bonds that are easily broken by hydrolysis,

Scheme 3.7, and the nitrenium ion branch often does the same. In addition, while the

nitrenium ion pair is formed very rapidly, it collapses to form the pivotal cross-

linking bond very slowly in the micro- to millisecond time domain. So information

about specific binding sites derived from such slowly formed cross-links may be

spurious.

Some of these problems can be circumvented through the modification of the aryl

azide system as shown in Scheme 3.9. Thus, the PAL agent might be attached to the

biomolecule of interest via either the electron-donating group (EDG) or the electron-

withdrawing group (EWG). The nitrene will be stabilized by the EDG group and

directed along the nitrenium ion pathway and the EWG will tend to direct the

nucleophile attack to the 2- or 6-positions instead of the 1- or 4-positions, which can

lead to hydrolysis of the cross-linking bridge. This general strategy has often been

implemented in biological studies. One such system that has been widely used in

PAL studies is shown in Scheme 3.10. This system is easily attached to available

nucleophiles on the surfaces of biomolecules and on irradiation leads to stable cross-

links in the 2-position.

We have studied the ultrafast processes that occur with the 4-azido-2-nitrophenyl

amine 18 using a pump-probe system with time resolution of 200 fs.52 When 18 is

irradiated in alcohol solvents, ring expansion is not observed. Only ring substitution

and reduction products, 19 and 20, respectively, are observed as shown in

Scheme 3.9. Analysis of the transient behavior of this molecule clearly shows

that the nitrene 21 is formed virtually immediately on excitation within a time

interval of several hundred femtoseconds. The protonation of this very basic nitrene

is one of the fastest protonation reactions observed to date53–56 occurring within a

time window of 5–25 ps depending on the structure of the alcohol used as solvent.

Table 3.2 lists the rates observed for protonation by various alcohols to form the

nitrenium ion 22, Scheme 3.9. It is particularly informative to compare the rates of

protonation by methanol and ethanol of the nitrenes 21 derived from azide 18

(Table 3.2) with that derived from 2-azidofluorene, kH¼ 4� 109.50 While this

comparison is rather crude, it does indicate that a para-amino group accelerates

protonation by a factor of at least 25–50-fold. The rates of protonation in aqueous

hν XH

EDG = –NEt2 EWG = –NO2

18 21 22 19, X = –OR20, X = –H

SCHEME 3.9.

Protein1 NH2

Protein1 NH

Protein2 NH2

Protein1 NH

Protein2HNhν

SCHEME 3.10.

solutions have not been measured to date, but presumably would be even faster. Here,

as in the previous examples listed in Table 3.1, collapse of the nitrenium ion pairs is

relatively slow requiring 100s of nanoseconds to microseconds.

Several aspects of these nitrene reactions are worthy of note. The profile for their

protonation closely parallels that observed for the extensively studied insertion of

singlet carbenes into ��O��H bonds.55,56 The azide 18 absorbs at visible wave-

lengths, which is a very desirable property for PAL agents, since it allows them to be

selectively excited in a matrix of biomolecules that absorb UV light. However,

transient work52 clearly shows that excitation in the visible region does not lead to

nitrene formation. Nitrene formation occurs from the second or higher excited state

that is accessed via excitation at wavelengths of about 350 nm or shorter. Further-

more, the electronic structure of singlet nitrenes is currently a topic of debate.57–59

Singlet nitrenes might exist in either a closed-shell or open-shell electronic configu-

ration as shown in Figure 3.2. In many cases, theoretical evaluation indicates that the

open-shell configuration is at lower energy. However, it is much more difficult to

evaluate the configuration in which the nitrene is born. If that happens to be the

closed-shell configuration, relaxation to the open-shell configuration will be highly

forbidden due to difficulty in conserving angular momentum, lack of spin-orbit

coupling,56 and consequently, the closed-shell nitrene will be slow to relax to the

lower-energy open-shell configuration.

These same spin-orbit coupling considerations indicate that the closed-shell

singlet nitrene will also undergo intersystem crossing to the triplet nitrene much

TABLE 3.2. Rates of Protonation of Nitrene 21 in Alcohol Solvents

Alcohol kH (s�1) Alcohol kH (s�1)

CH3OH 2.08� 1011 CH3(CH2)2CH2OH 8.06� 1010

CH3OD 1.39–1.49� 1011 (CH3)2CHOH 5.26� 1010

CH3CH2OH 1.05� 1011 CH3(CH2)6CH2OH 4.27� 1010

Aryl N

Closed-shellsinglet nitrene

Open-shellsinglet nitrene

Triplet nitrene

FIGURE 3.2. Relationships between aryl nitrene electronic configurations and inter-

conversion between those configurations.

more rapidly than will the open-shell singlet nitrene. Finally, protonation of the

closed-shell singlet nitrene will afford the ground-state nitrenium ion, while pro-

tonation of the open-shell nitrene should afford the excited state of the nitrenium ion,

a very unfavorable process. Consequently, it would seem that protonation of

electron-rich aryl nitrenes (Scheme 3.9) is so rapid that the closed-shell singlet

nitrenes are intercepted before they can relax to the open-shell configuration.

The photochemical behavior of fluorinated aryl azides is an area of significant

importance, since the resulting fluorinated nitrenes possess properties that have made

them desirable PAL agents. Specifically, fluorinated nitrenes undergo ring expansion

to ketenimines much more slowly than their hydrocarbon analogs that ring expand

about 170–1700 times more rapidly.60 Perhaps due to this slow ring expansion,

nitrenes derived from fluorinated aryl azides are thought to have extended singlet

lifetimes (200–250 ns),61 and to undergo insertion into C��H bonds of cyclohexane,

and what appears to be insertion into N��H bonds of secondary amines as indicated

in Scheme 3.11.61–68 This type of indiscriminate reactivity is thought to be ideal for

fixing the ephemeral associations of biomolecules into covalently bonded units that

can be readily identified and studied in detail via careful disassembly of the cross-

linked systems. For these reasons, many PAL applications have been based on

modified fluorinated aryl azides. In all cases studied to date, the cross-linking

processes of fluorinated nitrenes are attributed to direct nitrene insertion or ring

expansion followed by nucleophile addition to the resulting ketenimine. However,

one might consider the possibility of nitrenium ion formation in these systems as

well, especially when reactions are conducted in aqueous media, Scheme 3.11. This

possibility seems to be indicated by two observations reported for fluorinated aryl

azides. The first of these is that the transient absorption spectrum of pentafluor-

ophenyl azide is significantly altered by irradiation in methanol.60,69–71 This has been

interpreted as methanol-catalyzed intersystem crossing from the singlet to the triplet

NHNEt2

Singlet nitrene

pX-C6F4-N

pXC6F4

pX-C6F4

R-HConcertedinsertion

Hydrogen abstraction

and recombination

Nitrenium ionformationand collapse

pX-C6F4

SCHEME 3.11.

nitrene, a most surprising effect.69 An alternative, more straightforward interpreta-

tion of this effect might be that methanol serves as a proton source for nitrenium ion

formation, which is plausible since the reference spectra generated in this work were

acquired in ethanol-containing EPA matrices, and therefore, might also contain the

spectra of nitrenium ions.72 These two alternatives would be very difficult to

differentiate without subnanosecond transient studies, which has subsequently

been done with other nitrenes in methanol without observing the proposed catalyzed

intersystem crossing.50 In a related observation, the hydrazone 23, Scheme 3.12, and

related bases were studied in the >17 ns time domain and do not afford cyclohexane

insertion products.61 In this time frame, 23 did not exhibit a long-lived singlet nitrene

spectrum, but displayed only a weak spectrum, which was interpreted as arising from

the triplet nitrene via an enhanced rate of intersystem crossing due to the resonance

delocalization shown in Scheme 3.12. Here again, this could equally well be

attributed to nitrenium ion formation, since nitrenes with para-electron-donating

groups are particularly prone to protonation and nitrenium ion formation (see

Schemes 3.6 and 3.9). These alternative interpretations, again, might be resolved

by subnanosecond transient spectroscopy.

In summary, on the basis of the information available to date, there are at least

three possible mechanistic interpretations that might account for the formation of

fluorinated nitrene insertion products. These are outlined in Scheme 3.11. In the

case of hydrocarbon (cyclohexane) insertion, both pathways A and B would seem

to be viable alternative.63 Of particular relevance to this review is pathway C,

which is the most probably alternative with a para-electron-donating group

(Schemes 3.6 and 3.9) or when the nitrene is generated in a protic media.

Pandurangi has assembled an extensive list of fluorinated aryl azides and com-

pared their relative performance in C��H and N��H insertion.62 Pandurangi and

Platz61 have observed that the lifetimes of singlet fluorinated nitrenes are strik-

ingly different if the para-substituent is an electron-donating group. Thus, in

acetonitrile, the singlet nitrene in Scheme 3.11 with pX¼H has t¼ 220 ns, but if

pX¼��CH��NNHCH3, the lifetime drops to t¼ 5 ns. In addition, pX¼ F has a

greatly reduced lifetime, t¼ 65 ns, in methanol. Thus, while nitrenium ions have

yet to be confirmed as reactive intermediates in the photochemistry of fluorinated

aryl azides, it would seem that this class of azides might be a promising area for

future research in the subnanosecond domain.

SCHEME 3.12.

3.3 APPLICATIONS OF ARYL NITRENIUM IONS

IN PHOTOAFFINITY LABELING

3.3.1 4-Azido-2-nitrophenyl Amines

Aryl azides have been widely applied in photoaffinity labeling studies with virtually

ever class of biological molecules. Several informative reviews describe the many

applications of PAL reagents to specific biochemical questions.73–75 While aryl

azides are among the most widely used PAL reagents, most of these studies were

conducted before the nitrenium ion branch of nitrene chemistry was fully appre-

ciated, or understood. Thus, these studies tend concentrate on the biochemical

aspects of the work and assume a mechanism for cross-linking that was generally

accepted at the time that the studies were conducted. Consequently many cross-

linking studies that probably involve nitrenium ion chemistry attribute the cross-

linking to ketenimine or nitrene insertion chemistry. From our remote position in

writing this review, it is not possible to evaluate this question of cross-linking

mechanism in many cases, if such has not been done by the authors of the work.

Therefore, we provide the following examples of PAL studies using aryl azides

against the background of the earlier discussion, and in most cases leave the question

of cross-linking mechanism to the reader and future researchers.

In few cases, the relative performance of several different azide PAL agents has

been examined with several classes of biomolecules. A particularly interesting study

of relevance to this review was conducted early in the development of azide PAL

agents.76 In this work, the relative labeling yields of 24a and b with bovine serum

albumin (BSA), mammalian histone (MHs), ribosomal RNA (rRNA), and calf

thymus DNA (ct-DNA) were determined as measured by acridine fluorescence

(Table 3.3). The relative rates of BSA labeling were estimated to be 24a/24b¼ 4.5.

This is a particularly interesting comparison, since the azides related to 24b are now

known to proceed through nitrenium ion intermediates,52 and 24a probably proceeds

through a nitrene or ring-expanded intermediate. Diazo PAL agents used in this study

TABLE 3.3. Percentage Yields for Photoaffinity Labeling of Various Classes of

Biological Substrates with Aryl Azide PAL Agents

NH NHR 24a R =O

24b R = N3

Yield (%)

Reagent BSA MH rRNA ctDNA

24a 22.3� 11.3 35 40 18� 5

24b 9.3� 3.2 17 8 10� 1

usually gave lower yields of adducts, but proceeded more rapidly than these azide

PAL agents. Finally, these data also indicate the difficulty in extrapolating the

effectiveness of a particular PAL agent from one type of biological substrate to

another.

Falvey has reviewed the chemistry of nitrenium ions and studied their chemistry

extensively via their photochemical generation from N-aminopyridinium salt 25 as

shown in Scheme 3.13.77 Of particular relevance to this review is his characterization

of nitrenium ion reactivity with the common amino acids and readily accessible

proteins using transient spectroscopy (>4 ns, pumped at 355 nm).78 The second-

order rate constants for nitrenium ion 26 in 10% aqueous acetonitrile are summarized

in Table 3.4. As might be expected, amino acids with carboxylic acid, amide, and

hydrocarbon side chains, including phenylalanine, were found to be unreactive. It is

rather surprising that the amino acids threonine and proline were also found to be

hνPh N

NH3C CH3

25 26–

SCHEME 3.13.

TABLE 3.4. Second-Order Rate Constants for the Reaction of Nitrenium Ion 26 with

Amino Acids and Standard Protein Molecules

Amino Acid/Protein

Second-Order Rate

Constants kq (M�1 s�1) Reactivity

Reactive

Amino Acids (%)

L-Tryptophan (2.91� 0.1)� 109 High

L-Methionine (1.29� 0.7)� 109 High

L-Tyrosine (2.95� 0.7)� 108 High

L-Cysteine (2.79� 0.2)� 108 High

L-Lysine (8.04� 1.6)� 107 Moderate

L-Histidine (5.39 �0.5)� 107 Moderate

L-Arginine (3.04� 0.5)� 107 Moderate

L-Serine (7.93� 0.7)� 106 Low

Glycine Nonlinear about 2� 108

at 0.02M glycine

Chymotrypsin (7.71� 0.8)� 109 High 9.9

Insulin (8.96� 0.8)� 108 High 19.6

BSA (bovine serum albumin) (8.22� 0.9)� 108 High 10.5

Lysozyme (5.79� 0.6)� 108 High 14.7

BPR (bovine pancreatic

ribonuclease)

(4.59� 0.4)� 108 High 14.5

APPLICATIONS OFARYL NITRENIUM IONS IN PHOTOAFFINITY LABELING 91

unreactive at least to the extent that they reacted, if at all, with rate constants of

<105M�1 s�1, the limit of detection for the equipment used. Most surprising was the

observation that glycine displayed a nonlinear pseudo-first-order-decay rate depen-

dence on concentration with significant reactivity at high concentration. This effect

was not explained, but does indicate a problem with relative rate studies of this type

when applied to large biological molecules. If a PAL agent is photoactivated while in

the binding site of an enzyme, the effective concentrations of all amino acid residues

in that binding site will be extremely high, and the reactivity of the encased nitrene or

nitrenium ion much higher that might be expected based on dilute-solution mea-

surements of this type.

Several other interesting aspects of the data in Table 3.4 are that the proteins do not

display reactivities that parallel their reactive amino acid content. This is perhaps not

surprising, since only those amino acid residues on the surface of the proteins will be

available for PAL cross-linking. What strikes this observer as very interesting is that

the order of amino acid reactivity seems to be more consistent with their relative

electron-donating abilities rather than their nucleophilicities. Falvey and Thomas

made note of this same correlation, which is especially obvious for methionine,

which affords nitrenium ion reduction products.78 Therefore, the second-order rate

constants listed in Table 3.4 are the combined rate constants for both reduction and

nucleophilic substitution, and since electron transfer/nitrenium-ion reduction is

usually not a productive branch of nitrenium ion chemistry in PAL studies, these

rate constants do not necessarily reflect the cross-linking susceptibilities of the

various amino acids listed. Finally, the order of reactivity of nitrenium ions generated

directly, as in this study, does not accurately reflect the order of reactivity of

nitrenium ions generated from azides via nitrenes, since this latter process will

depend on the acidities of the conjugate acids of the nucleophiles that cross-link with

the nitrenium ions, and will proceed through ion pairs incorporating the cross-linking

nucleophiles.

There are a large number of PAL studies that have been conducted with aryl

azides, many or most of which may proceed via the ring expansion ketenimine

branch (Scheme 3.7), while only one class of aryl azides is known with certainty to

proceed via the nitrenium ion branch, 4-azido-2-nitrophenyl amines related to 24b

(Table 3.3). We shall focus this initial discussion on studies that have used these

nitrophenyl azides.

A very interesting comparison of the relative performance of these two classes of

azides has been done in an effort to link together the filaments of the protein F-actin.

F-actin is a protein composed of globular subunits, G-actin containing 375 amino

acid residues, that in conjunction with myosin molecular motors is responsible for

the contraction and relaxation of muscles. The studies in question sought to restrict

this molecular motion between F-actin strands by cross-linking G-actin subunits

together. The two phenyl azide-modified proteins 27 and 28 have been applied in

these studies (Scheme 3.14).

In the case of 27, one would expect the ketenimine branch of cross-linking to be

active, and it was noted that the cross-linked peptide chains were susceptible to

hydrolysis during the HPLC separation procedures used in their isolation.79

Furthermore, in spite of these difficulties, the cross-link between strands was

determined to be between amino acid residues Gln41 and Lys113. In a very extensive

and latter study, the cross-linking agent 28 was applied to the same system.80 In this

case, which proceeds via a reactive nitrenium ion intermediate, cross-linking was

determined to be between the amino acid residues Gln41 and Cys374. No mention was

made of any instability of the cross-link and the motility of the F-actin was greatly

restricted by the cross-linking process, while the F-actin fibers appeared

unchanged.81,82 It is perhaps of significance that even though the PAL reagent

was attached to the same glutamine residue in both studies, the primary cross-link

occurred between different amino acid residues, a lysine residue in the case of 27, the

ketenimine precursor, and a cysteine residue in the case of 28, the nitrenium ion

precursor. This pattern of reactivity might well reflect the characteristics of the two

types of cross-linking agents. The precursor nitrene for nitrenium ions is very basic

and will abstract a proton from cysteine to form a powerful sulfide nucleophile and a

sulfide–nitrenium ion pair, which on collapse will form a strong cross-link. In

contrast, the ketenimine derived from 27 probably reacts directly with nucleophiles

without prior protonation.

As indicated earlier, myosins constitute a large family of proteins that drive many

biological processes including muscular contractions in eukaryotic cells with the aid

of adenosine triphosphate (ATP). Photoaffinity labels based on 4-amino-2-nitro-

phenyl azide analogs of ATP and ADP, 29 and 30, respectively, have been used to

study these processes.83 Even though 29 and 30 do not seem to closely mimic the

structures of ATP and ADP, they function effectively in myosin regulation. Conse-

quently, they have been applied in mapping the ATP/ADP active site of myosin in an

ingenious, entrapment double cross-linking strategy. Thus, a “jawlike” cleft in the

Gln-41

Lys-113

(CH2)4

(CH2)2

Gln-41

(CH2)4

(CH2)2

hν hν

Gln-41

Cys-374

SCHEME 3.14.

myosin subfragment 1, SF-1, is closed in the presence of MgATP and MgADP.

This closure brings two thiol groups into close proximity to each other so that

they can be linked to each other with either N,N0-p-phenylenedimaleimide or

Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]3 permanently closing the cleft

as shown in Scheme 3.15.84 This process can be used to entrap the PAL agents 29 and30. Irradiation of entrapped 30 afforded a >50% of covalently cross-linked 30.85,86

Extensive proteolytic digestion and Edman analysis of SF-1 indicated that the

nitrenium ion had become cross-linked to Trp130 of the heavy protein chain of

rabbit skeletal myosin, which has the partial structure Val125–Asn–Pro–Tyr–

Lys(Me3)þ–Trp130–Leu–Pro–Val–Tyr134.87,88

These results are quite interesting, since the tryptophan residue that becomes

cross-linked to PAL 30 is immediately adjacent to a positively charged quaternary

salt of lysine, which the authors suggested serves to bind the phosphate groups, but

see Figure 3.3. Furthermore, the selective reaction of tryptophan in this work

correlates very nicely with the relative rates of reaction of other nitrenium ions

Mg-29/30SH

SF-1 SF-1

Mg-29/30

Thiol linking agent

Mg-29/30 hν

Entrapment bridge

SCHEME 3.15.

with amino acids determined by Falvey and shown in Table 3.4.78 This same

entrapment technique using 30 has also been applied in the study of scallop

myosin.89,90 In this case, the entrapment cross-linking agents were Mn2þ and

vanadate, since the aforementioned Co2þ procedure cause precipitation of the

protein. Here again a high yield of photolabeling was achieved, about 35%, and

the major cross-link was found to be with Arg128 in the protein sequence Arg127–

Arg128–Leu–Pro–Ile–Tyr–Thr133, which is the analogous position to that of the

Trp130 in the rabbit myosin that was specifically labeled in the previous work. In both

of these studies, the structures of the cross-linked amino acids were not determined,

and in the latter study, the Arg128 photolabeled product was found to be unstable

under the sequencing conditions. The authors speculated that this might be due to the

cross-linking bond being a nitrogen��nitrogen bond between the nitrene nitrogen

and arginine guanidinium nitrogen.89 However, it might just as well have been an

adduct analogous to those shown in Scheme 3.9, where X is the guanidine group.

Adducts of this type, triaminobenzenes, are susceptible to spontaneous air oxidation,

which if followed by hydrolysis could easily regenerate the arginine residue. Clearly

it would be most desirable to firmly establish the structures of the PAL amino acid

adducts for this widely used PAL agent. For that matter, this same problem presents

an issue with many PAL agents for which the literature is rife with kinetic, transient,

and labeling studies of products of unknown or presumed structure. In an extension

of this scallop myosin work, which was conducted in the absence of Mg2þ or Ca2þ

ions, addition of these ions to the photolabeling experiment shifted the predominant

labeling site from Arg128 to Cys198.90 This study indicates that Ca2þ plays an

FIGURE 3.3. Binding of azide PAL agents to active site of chicken skeletal myosin: (a)

Docking of PAL agent 31 in myosin based upon molecular dynamics modeling.91 (b) Docking

of PAL agent 29 in myosin-based reorientation of the conformation of the PAL agent. Major

tryptic fragments colored orange, blue, and dark green, triphosphate unit colored pale green,

nitrenium ion colored red, Lys681 colored yellow, and Trp130 (rabbit myosin), or Trp131

(chicken myosin) colored purple.

important role in altering the myosin conformation through binding in a regulating

domain of the protein.

A final installment of this classic work of Cooke and Yount has been the

attachment of spin labels to the myosin skeleton.91,92 In this work, the nitroxide

radical 31 was attached to rabbit skeletal myosin using the entrapment technique

described earlier. Amino acid sequence analysis of the photo-cross-linked products

showed that coupling had taken place with Lys681 in the carboxy-terminal 20 kDa

tryptic fragment rather than the 23 kDa amino-terminal fragment containing the

Trp130 (rabbit myosin) or Trp131 (chicken myosin) residue. A rendition of the

explanation of this chain swapping observation taken from molecular modeling

of the known structure of chicken skeletal myosin is shown in Figure 3.3.91 Thus, the

bulky nitroxide spin label causes 31 (red in Fig. 3.3a) to assume a conformation in

which the nitrenium ion is directed toward Lys681 (yellow) on the opposite side of the

active site cleft. In the absence of the bulky spin-label unit, the PAL agents 29 or 30

assume conformations that direct the nitrenium ion (red in Fig. 3.3b) toward Trp130

(purple) (rabbit myosin) or Trp131 (purple) (chicken myosin). A very interesting

aspect of this type of PAL experiment is that following the attachment of the spin

label 31, treatment of the enzyme with its natural substrate actin and MgATP

completely regenerates its normal enzymatic activity.92 Thus, it would seem that

PAL strategies not only serve to identify specific enzyme–substrate interactions

within active sites, but also serve to deliver probes for the monitoring of the active

site in action.

In the earlier example, the azide PAL agent becomes attached to one of the

nucleophilic amino acid side chains found to be reactive with nitrenium ions as

indicated in Table 3.4. It has been known for more than 40 years that aryl nitrene

reactivity is drastically affected by the electronic environment of the nitrene

nitrogen.93 More recently, it has become apparent that this behavior is due to a

mechanistic shift on going from an electron-rich nitrene nitrogen to an electron-

deficient nitrene nitrogen. In order for aryl nitrenes to be efficient, precursors for

reactive nitrenium ions, the nitrene nitrogen must be in electronic communication

with a strong electron-donating group such as the para-amino group in 21, 24b, and

28–31. In this environment, the nitrene nitrogen becomes a strong base, abstracts a

proton from available sources in its vicinity, and is transformed into a nitrenium ion

that will be selective in its reactivity, reacting only with nucleophilic sites on the

biomolecule. On the other hand, if the nitrene nitrogen is in communication with a

powerful electron-withdrawing group, a nitrenium ion probably will not result.

Reactions of this type will probably proceed via more traditional nitrene insertion or

ring-expansion chemistry. Unfortunately, detailed and systematic femto/picosecond

studies of the effect of electron-donating and electron-withdrawing groups on nitrene

chemistry in the samemedia have only recently become possible, and so have not yet

been conducted.

Knowles was the first to apply the 4-amino-2-nitrophenyl azide unit in PAL

studies94 when he applied them to antibody–hapten complexes. At the time of this

work, the mechanism-altering significance of the electronic coupling between the

azido and amino groups was not appreciated. An example of how this type of

mechanistic shift might affect the interpretation of PAL studies is provided by the

PAL labeling of antibodies for the photo-precursor of aryl nitrenes.95–97 In these

studies, rabbit antibodies for 32 were generated and bound to their hapten 32

(Scheme 3.16). This antibody-hapten complex was then irradiated and covalent

bonding between the two was determined to have occurred adjacent to the antibody

hypervariable region of the heavy protein chain possibly at cysteine91 and alanine93

to the extent on about 13%.96 The exact sites of the cross-links were not rigorously

established, and might not be completely accurate, since cross-linking to alanine is

surprising. The azide 32 is a nitrenium ion precursor, if proton sources are

available,52 and alanine is not a nucleophilic amino acid that one would expect

to react with nitrenium ions. Therefore, one must assume that (i) proton sources are

not readily available in this bonding site, or (ii) the amino acid residue to which

cross-linking occurs is misassigned. Another problem arises in an extension of this

study in which the modified azide 33 was reacted with the same rabbit antibody used

with 32 on the assumption that the slightly different orientation of the nitrene (azide)

group might facilitate bonding to different amino acid residues in the same antibody

binding site.98 In apparent confirmation of this hypothesis, the ratio of heavy to light

antibody chain attachments was found to be quite different, 2.5–5.096,97 with 32 and1.71 with 33. Unfortunately, no detailed analysis of the amino acid residues to which

33 had become attached was reported. Since 33 is not a nitrenium ion precursor, the

assumption that the two nitrenes will have comparable reactivities is not justified.

From these results, it appears that 33 is reacting more indiscriminately than 32, and

that both the differences in reactivity, as well as, the positioning of the reactive

nitrogen species governs the cross-linking processes with the antibody active site.

Since nitrenium ion formation will only occur with appropriately subsituted aryl

azides if the precursor nitrenes have access to fairly acidic proton sources such as

water, alcohols, amine salts, and thiols, and so on, studies with nitrenium ion PAL

agents in lipids environments are particularly interesting. In several studies, neuronal

plasma membrane gangliosides have been modified with the appropriate nitrophenyl

azide PAL units on both the hydrophobic and hydrophilic ends, 3499–101 and 35,102

respectively (Scheme 3.17). The PAL unit 34 might be expected to become

embedded in the hydrophobic core of the membrane where proton sources are

scarce, and thus, react mostly via conventional nitrene chemistry rather than

nitrenium ion chemistry. Nitrenes in such a hydrophobic environment might become

cross-linked to membrane-embedded proteins or perhaps even unsaturated hydro-

carbon chains via hydrogen atom abstraction and radical coupling. Alternatively, the

NO2NH[CH2]4CH

CO2–

NH[CH2]4CHCO2

N332 33

SCHEME 3.16.

PAL unit 35 would position the nitrophenyl azide unit on the surface of the

membrane where proton sources should be abundant, and therefore, it might be

expected to form nitrenium ions readily and become cross-linked to proteins and

saccarides lining the membrane surface. In these studies, both 34 and 35were labeled

with tritium in the terminal sugar unit, and cross-linking was measured by incorpo-

ration of radioactivity into membrane components. Consistent with this model shown

in Figure 3.4, 35 was found to afford much more efficient cross-linking than did

34.102 Furthermore, in the case of 34, the amount of cross-linking was found to be

dependent on the incubation time prior to irradiation.99 Thus, incubation of 34 with

the cells for only 2 h afforded stable incorporation of 34 into the cell membrane, but

very few proteins became radioactive on irradiation. On the other hand, when 34was

incubated for 24 h before irradiation, it underwent metabolism to several derivatives

and migrated from the surface membrane of the cell into the interior of the cell where

irradiation afforded many radioactive proteins.

A similar strategy has been used to study the structure of critical membrane-bound

enzymes such as cytochrome c oxidase, blue unit in Figure 3.4, which is bound to a

mitochondrial inner membrane.103 To function effectively, the cytochrome c subunits

must be bound together with two high-affinity functional and two low-affinity

structural molecules of cardiolipin, a diphosphatidylglycerol. This lipid contains

four hydrophobic octadec-9,12-dienoic acid ester chains and a hydrophilic trigly-

cerol diphosphate head group. Both the polar head group and lipophilic fatty acid

chains have been modified with aryl azide PAL agents. Two of the four hydrophobic

fatty acid chains have been appended to 4-azido-2-nitrophenyl amine PAL units that

will afford nitrenium ion in protic environments, but the availability of proton

sources in a hydrophobic interior membrane environment is problematic. Alterna-

tively, the polar head group has been appended to a 3-azido-4-nitrophenyl carbox-

amide PAL agent, which is probably not a nitrenium ion precursor, but is probing the

OH–O2C

HOAcHN

OH–O2C

HOAcHN

N3O2N 34

SCHEME 3.17.

polar environment ideally suited for nitrenium ion formation. This complex ensem-

ble consists of the aforementioned cardiolipin units and at least eight protein

subunits. In spite of the apparent mismatch of PAL agents with environments,

both agents afforded significant cross-linking to different protein subunits with

varying efficiencies, but in general, the PAL agents on the nonpolar tail of cardiolipin

were more efficient cross-linking agents than the PAL agents on the polar head

group. While the information gleaned from this type of experiment provides useful

correlations about the proximity of the various subunits, the cross-linking processes

are derived from a variety of very different reactive intermediates, most of which

require microseconds or longer to establish the critical cross-linking bond, and thus,

may wander from optimal binding sites. If PAL strategies are to be advanced to the

next level of identifying specific amino acid residues and other biological constitu-

ents involved in the optimal bonding sites, then the PAL agents will have to be

selected with specific target environments and amino acid residues in mind.

Sex hormones, 36,104 and vitamin D105–109 receptors have also been explored

using 4-azido-2-nitrophenyl amine PAL agents (Scheme 3.18). Two strategies have

been used in efforts to identify the binding sites of vitamin D3. One of these has been

to isotopically label either the Vitamin D3 moeity106 or the PAL moiety107 with

tritium, 37a and 37b, respectively, and look for radioactive proteins produced by

irradiation of the protein–vitamin D3 mixture. In the second and more recent study,

the 14C-double labeling reagent, 38, was used in which the proteins first became

Transmembraneproteins

Src-familykinase

proteins

GPI-anchoredproteins

FIGURE 3.4. PAL strategies for cross-linking within the core of a membrane using agents

such as 34, and on the outer surface of the membrane using agents such as 35.

attached to the bromoacetate moiety, and then, following irradiation, attached to the

PAL moiety. This technique has been used to identify previously unrecognized

proteins that bind to vitamin D3.109 A potential pitfall in designing PAL experiments

of this type is that ether- or amine-linking chains, 36 and 37, are more suitable than

are ester- or amide-linking chains, 38. The latter type of linking chains are

susceptible to hydrolysis either during the cross-linking experiment or during the

analysis that follows, and if these cross-linking chain are broken, it can becomemuch

more difficult or impossible to identify the cross-linked proteins.

Intact proteins and polynucleotides can be labeled by alkylation with reactive

halides at nucleophilic sites to form useful PAL reagents. One example of this type of

strategy is the selective attachment of 4-azido-2-nitrophenyl amine PAL units to

radio-labeled (125I) synthetic analogs of bovine parathyroid hormone at lysine

residues along the peptide chain by treatment with FNPA as indicated in Scheme 3.19

(gray Lys units).110

The conditions selected for this labeling minimized attachment to the N-terminal

amino group. On irradiation in the absence of competing hormone, this modified

parathyroid hormone became cross-linked to four–six components of the renal

receptor membrane, but in the presence of competing hormone, one of these

membrane components failed to become cross-linked indicating that this was the

primary hormone binding site. This general strategy of using the natural substrates

modified by incorporation of PAL units, and determining the degree to which the

natural substrate competes with the PALmodified substrate is widely used to identify

binding sites of biomolecules.

In another variation of PAL-modified natural molecules, tRNA of some micro-

organisms contains extremely nucleophilic bases that can be easily attached to PAL

HO (CH2)1-3NH

(3H) OH

37a and b

ONH N3

SCHEME 3.18.

units. Thus, Escherichia coli has a tRNAArg that contains a 2-thiocytidine, S2C, at

position 32, two bases away from the anticodon on the 50 side, Figure 3.5. This basehas been modified through attachment of a 4-azido-2-nitrophenyl amine PAL unit,

NAMA-I. The NAMA-I group probably attaches to the 4-amino group of the

2-thiocytidine, as shown in Scheme 3.20, although it might conceivably become

attached to the sulfur atom. On exposure to ribosomes, the modified tRNAArg moves

to the central P-site in a ribosome where irradiation cross-links it to the 30S

ribosomal subunit to the extent of 4–6%.111

H2N Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His LeuSer

SerNleGluTrp ArgValGluLeuArgLysLysLeuGlnAspValHis

5 10 15

202530

FNPA = Nle = Norleucine

SCHEME 3.19.

FIGURE 3.5. Transfer RNA showing location of 2-thiocytidine (S2C), red, and anticodon

loop, blue.

3.4 GENERAL CONSIDERATIONS

The foregoing discussion has been limited to 4-amino-3-nitrophenyl azides, since

this configuration of substituents is definitely known to afford nitrenium ions

exclusively, and these nitrenium ion collapse to form covalently bound derivatives.52

The electron-donating capacity of the para-amino group can be attenuated by amide

formation, but nitrenium ion formation and collapse still occurs effectively as shown

in Scheme 3.21 (V. Voskresenska and R.M. Wilson, unpublished work).24 Other

para-electron-donating substituents might also afford very basic nitrenes that

effectively abstract protons to form nitrenium ions. Thus, para-thioethers112 and

ethers113 have both been used successfully as PAL agents that might proceed through

nitrenium ion species.46 However, the question of mechanistic bifurcation between

nitrene ring expansion and proton abstraction chemistry as a function of electron-

donating capacity of the para-substituents has not been adequately studied.

The presence of the nitro group is also an important component in the generation

of effective PAL species, since this powerful electron-withdrawing group makes the

resulting nitrenium ion much more susceptible to the Michael addition that ulti-

mately produces the cross-linking covalent bond, Scheme 3.9. McClelland’s work

has shown that without this type of Michael-facilitating group, nitrenium ions tend to

undergo hydrolysis to the quinone, which does not produce a cross-linking covalent

bond, see Scheme 3.8.24,46. Even so aryl azides lacking such a Michael facilitating

nitro group have been used with success in PAL studies.113,114 Thus, a nitro group

ICH2CO2CH2CH2

CH2CO2CH2CH2

NAMA-I

SCHEME 3.20.

HNCO2Et

OCH(CH3)2hν

HOCH(CH3)248%

SCHEME 3.21.

Michael facilitator does not appear to be an essential element in an effective

nitrenium PAL agent. In fact, if the nitro group is replaced by a ring nitrogen

atom to produce a pyridinium aryl azide, cross-linking become significantly more

effective as indicated in Scheme 3.22 (V. Voskresenska and R.M. Wilson,

unpublished work). PAL agents of this type have not been investigated in biological

environments. However, for many applications, the ring nitrogen might be much less

intrusive than is the much larger nitro group.

The popular fluorinated aryl azides have received a great deal of attention, since

they do not undergo ring expansion and produce persistent singlet nitrenes, which

might insert into even unreactive biomolecules. However, there are few cases in

which fluorinated aryl azides with activating para-amino groups have been studied,

and what little mechanistic work that has been done has been in an aprotic

environment.115 Therefore, it was not possible to evaluate whether this type of

aryl azide might serve as a precursor for fluorinated nitrenium ions. In one case, the

farnesyl analog 39was prepared and used to study the process by which a Ras protein

incorporates a farnesyl unit (Scheme 3.23). The attachment of the hydrophobic

farnesyl unit is necessary for the Ras protein to become bound to the inner plasma

membrane where it is activated by guanosine triphosphate and regulates certain

critical metabolic processes. The enzyme that attaches the farnesyl unit to the Ras

protein, farnesyltransferase, accepts 39 as a farnesyl analogue and is inactivated to

the extent of 70% by irradiation.116 Again no studies of the mechanism of enzyme

inactivation were done. Fluorinated nitrenium ions of the type that might be

generated from 39 are particularly interesting PAL species, since they would be

expected to undergo Michael addition followed by fluoride ion elimination to

regenerate an active cross-linking agent.117 Thus, fluorinated nitrenium ion might

be expected to form two or more cross-links from a single activation event.

One final aspect of concern in the implementation of nitrenium PAL strategies is

the acidity of the nitrenium ion. In most biological applications of the aryl azide PAL

agents, the para-amino group is a secondary amine. This gives rise to a nitrenium ion

98–100%

SCHEME 3.22.

SCHEME 3.23.

GENERAL CONSIDERATIONS 103

that could be in equilibrium with its unprotonated diiminoquinone form,

Scheme 3.24. The diiminoquinone would be expected to be a much less effective

Michael acceptor than the nitrenium ion. Apparently, nitrenium ions are sufficiently

basic to remain protonated under biological conditions, and aryl azides with

secondary para-amino groups are effective PAL agents (V. Voskresenska and

R.M. Wilson, unpublished work). Nevertheless, it would be most informative to

better characterize this type of nitrenium ion/diiminoquinone equilibrium, and the

effects that different Michael facilitators such as fluorine atoms, nitro groups, and

pyridine nitrogens have on these equilibria.

3.4.1 Purine Azides

One of the most extensively applied classes of PAL agents is the purine azides that

include 8-azidoadenosine (40) and 8-azidoguanosine. The photochemical behavior

of 40 has been studied in detail,118 and found to parallel that of the nitrenium ion

family of reactive species discussed in the previous section. Thus, the 6-amino group

in 40 serves to activate the nitrene for protonation to form the nitrenium ion 41 as

outlined in Scheme 3.25. The nitrenium ion 41 will be in equilibrium with the

diiminoquinone 42, and one of these two intermediates undergoes nucleophile attack

SCHEME 3.24.

RNitrenium ion Diiminoquinone

–H +HNitrene

5 678 9

SCHEME 3.25.

at the 2-position to produce 43, which is the cross-linked product in PAL applications

of 8-azidoadenosine.118 Although it has not been as thoroughly investigated, 8-

azidoguanonsine probably affords related nitrenium species. In 8-azidoguanosine,

attack of the nucleophile would be expected to occur at the 5-position.119–127 Since

5-adducts of this type are not stable, 8-azidoguanosine would not be as effective a

PAL agent as is 40. Here again, as with the aforementioned aryl azide system, the pH

dependence of the nitrenium ion/diiminoquinone equilibrium has not been investi-

gated. Consequently, it is not certain which species is the electrophile involved in the

cross-linking processes. This question should be easily resolved by ultrafast transient

spectroscopy, and offers fertile territory for future investigations. At this point, it can

be said that of all the possible monoprotonated forms of 42, 41 is the most stable as

estimated by theoretical calculations (DFT/B3LY/6-31G�).The first application of the 8-azidopurines was that of Haley and Hoffman in the

study of ATP binding and hydrolyzing sites in human red blood cell membranes

(Scheme 3.26).128 They found that phosphorylation of membrane proteins with

[b,g-32P]8N3ATP could be inhibited by up to 95% by irradiation of their ATPase

complexes prior to introduction of the phosphorylation substrate. Haley129,130 et al.

later extended this study to guanosine triphosphate (8N3GTP),131–134 30,50-cyclic

adenosine monophosphate (8N3cAMP),135 and cyclic guanosine monophosphate

(8N3cGMP). The placement of the azido group at the 8-position seems to have the

minimal effect on the binding of these coenzymes to proteins.

Haley also applied [g-32P]8N3ATP and [g-32P]2N3ATP in a very nice study of

rabbit creatine kinase from skeletal muscle, heart, and brain.136 Photoinsertion was

observed into two peptide regions, the Val279–Arg291 region with [g-32P]8N3ATP

and the Val236–Lys241 region with [g-32P]2N3ATP. These authors reasoned that since

the azide groups were located on opposite side of the adenine ring, the nitrene

nitrogen inserted into different regions of the enzyme active site. However, as

O PO–

OO–O P

OPO–

O PO–

OO–O P

OPO–

H2N HN

8N3ATP

8N3GTP

8N3cAMP

8N3cGMP

α β γ

SCHEME 3.26.

indicated earlier in Scheme 3.25, the cross-linking with 8N3ATP does not involve the

nitrene nitrogen, but occurs on the opposite side of the adenine ring at the 2-position.

The behavior of 2N3ATP is less clear, since there is no electron-donating group to

interact with the nitrene formed from this azide. Even though mechanistic studies for

2-azidoadenosine are lacking, in an earlier study 2-azidoadenine (44) was observed

to undergo the chemistry outlined in Scheme 3.27.137 While the yields of the alcohol

adducts 45a and b are not as high as those observed for 40, the 8-methoxy adduct

(45a) is a signature product for a nitrenium ion intermediate. The companion amine

46 observed in these reactions is typically a product derived from radical hydrogen

abstraction. Therefore, it is likely that 44 reluctantly forms a nitrenium ion, since the

precursor nitrene has lower basicity than that derived from 40, and slowly forms 45

via the corresponding nitrenium ion. A slow rate of nitrene protonation would

provide time for more nitrenes to reach the triplet state and to undergo hydrogen

abstraction to form 46. Returning to the creatine kinase study, here again, as with 40

and 44, cross-linking would not occur at the nitrene nitrogen, but at an electrophilic

site across the adenine ring. Even so, the two isomers of N3ATP might very well

become attached to different sites within a protein complex, but if a detailed analysis

of the binding site for ATP were to be done, 8N3ATP should become cross-linked

to protein sites close to the 2-position of the adenine ring and 2N3ATP to sites

close to the 8-position. Finally all of these correlations are complicated by the fact

that the syn/anticonformations of the adenine ring with respect to the ribose ring

might be altered by the presence of the azide group and the particular protein

environment.138,139

A further complication encountered with azides such as 44, in which the azide

group is attached to a six-membered nitrogen heterocyclic ring, is that an azide-

tetrazole tautomerization occurs as shown in Scheme 3.28. This process has been

studied with 2-azidoadenine140 and with 2-azidoadenosine monophosphate.141,142

Two such ring tautomers are possible in the adenine system, and tautomer a seems to

be favored over tautomer b. The azide form is favored under acidic conditions, and

tetrazole form under basic conditions. In neutral media, both forms are present in

about equal amounts at equilibrium.138While both forms might be photoreactive, the

azide seems to be considerably more reactive than the tetrazole form, although both

forms might afford the same reactive intermediate. Here is another area where a

detailed ultrafast mechanistic study would be very informative.

44 45a, R = Me, 37%45b, R = Et, 11%

46, 37% 75%

SCHEME 3.27.

In spite of these potential complications, derivatives of 2-azidoadenosine have

been used successfully in several biological environments. In one such application,

the ADP mimic 30 was used to study the structure of rabbit skeletal myosin, see

above. However, 30 resembles ADP only in that it contains a diphosphate extension.

Therefore, this work has been repeated using 2-azidoadenosine diphosphate to afford

18–14% cross-linking to the same Trp130 residue observed with 30,143 thus,

reinforcing the previous results.83–88

Some proteins are not synthesized by the usual DNA/mRNA/tRNA/ribosome

route, but by specific enzyme systems. One such enzyme is tyrocidine synthetase that

catalyzes the first step in the synthesis of the decapeptide antibiotic tyrocidine. This

process has an ATP requirement and has been studied using both 8N3ATP and

2N3ATP.144 Apparently, 8N3ATP is not accepted into the active site of this enzyme,

but 2N3ATP is accepted, and successfully photolabels the enzyme. Three tryptic

peptides were isolated and characterized from the N-terminal half of the enzyme.

Apparently, 8N3ATP assumes the unacceptable syn-conformation.138 and therefore,

is not bound to the synthetase active site, but 2N3ATP retains the acceptable anti-

conformation139 and is bound to the active site.

This same syn/anti-problem also apparently comes into play in many of the

labeling strategies involving 8-azidoadenosine. Both [50-32P]p8N3Ap and [50-32P]p2N3Ap have been used to study the binding of tRNAPhe to the ribosome. The

30-terminal nucleotides of tRNAPhe are –A73–C74–C75–A76 (see Fig. 3.5 for reference

to tRNA structure). Both of these azido diphosphates indicated earlier have been

used to replace the adenosines in the 73 and 76 positions. If the 8-azido diphosphate

was located in position 76, aminoacylation was completely inhibited, but if it was

located in the 73 position, aminoacylation occurred normally.145 Alternatively, the

2-azido diphosphate could be located in the 76 position, and aminoacylation

proceeded normally.146 When either p8N3Ap or p2N3Ap were inserted into tRNAPhe

at the 73 and 76 positions, respectively, and irradiated in the ribosome in the presence

of the appropriate mRNA (polyU), which positioned the tRNAPhe in the middle or

P-site of the ribosome, the 50S subunit of the ribosome was covalently cross-linked.

SCHEME 3.28.

When N-acetylphenylalanyl-tRNA containing 8-azidoadenosine in the 73 position

was bound to the P-site using the codon provided by polyU, and irradiated, the 23S

ribosomal RNA was the main ribosomal component cross-linked.145 This study

provides an example of the dynamics of enzyme action and how the enzyme

geometry changes as the programmed reaction progresses. The tRNA enters the

P-site with an attached amino acid and the ribosome folds to accommodate this

situation, which apparently brings 23S ribosomal RNA into close proximity to the

tRNAPhe 30 terminus. Following transfer of the growing peptide chain to the tRNA in

the entrance or A-site, the unacylated tRNAPhe occupies the P-site, and ribosome

refolds and brings a portion the 50S subunit close to the tRNAPhe 30 terminus. In this

way, photoaffinity labeling can provide insights into the dynamics of biological

processes that cannot be obtain even in an X-ray structure where the system is frozen

in a single geometry.

In many PAL applications, a molecule with a single photoactivatable unit is

employed, and this PAL unit is attached to another unit that has an affinity for a

particular site or molecule of interest. Molecules of this type are applied to “map” the

molecular environment of the target site. In the azidoadenosine strategies outlined

above, the affinity units and the PAL units have been integrated into the same

molecule. However, for mapping proteins made up from several subunits, each of

which has a binding site for ATP, it is useful to apply molecules with two separate

PAL units built into the same affinity agent. The diPAL reagent 47 (Scheme 3.29) has

been successfully used in the study of phosphofructokinase-1 that is composed of

eight subunits, four a-subunits and four b-subunits. Each of these subunits has

regulatory binding sites for MgATP. When phosphofructokinase-1 was treated with

OPO–

O PO–

OP O PO

OPO–

O–O P O P

CH2–CH2–NHO

OPO–

SCHEME 3.29.

47 and irradiated, two different a–b cross-linked proteins with different mobilities on

SDS-polyacrylamide gel electrophoresis were observed.147

Another diPAL reagent used to map subunit binding sites is 48. This reagent

combines the two most widely used PAL regents, 8-azidoadenosine and 4-amino-2-

nitrophenyl azide, and has been used to cross-link the binding sites of F1-ATPase, a

protein used for both the synthesis and hydrolysis of ATP. F1-ATPase usually has five

different subunits, a, b, g, d, and e. Irradiation of 48 in the presence of F1-ATPase

lead to the formation of a stable cross-link between the a and b units.148,149 This

same PAL agent, 48, has also been used to study the homodimeric protein SecA,

which is an ATP-dependent force generator in E. coli.150

While the diPAL reagents 47 and 48 have found utility in cross-linking relative

remote sites, the long, flexible chains connecting the two reactive azides could bind

to multiple sites over a large part of the protein surface. Some applications would

benefit from having a diPAL reagent that would attach to two more intimately

associated sites. The diPAL reagent 49 has been designed with this purpose in mind

and used to study F1-ATP synthase from the thermophilic bacteria PS3.151 Here, as in

many other cases in this field, PAL reagents are applied without ever studying their

basic photochemistry. In the case of 49, it was assumed that the two azide groups

would function independently of each other, and form separate cross-links with

protein components in close proximity. However, since each azide unit blocks the

cross-linking site of the nitrene generated from the other azide, it is not likely that

the two azides function independently of each other. A detailed mechanistic study of

the photochemistry of this fascinating diazide would be most interesting.

One of the major hurtles in the implementation of PAL regents based on

nucleotides has been the inability to incorporate nitrene photoprecursors units

into polynucleotides. The azido phosphoamidites are not available and 8N3ATP

does not serve as an efficient substrate for bacteriophage RNA polymerase. Recently,

the high fidelity incorporation of 8N3AMP into RNA has been reported using T7

RNA polymerase for the preparation of RNA molecules with as many as 100–300

bases, and these modified polymers can be prepared by transcription or reverse

transcription.152 The utility of this very intriguing approach to the synthesis of PAL

molecules remains to be seen, but at this point, it can be noted that multiple PAL sites

within a single RNA molecule can be generated by this method.

3.5 CONCLUSIONS

Aryl azides have become the most widely used class of photoaffinity labeling agents

due to their ability to cross-link a variety of biological molecules under a wide range

of conditions. Aryl azides containing activating amino groups are particularly

versatile in this regard. This is probably due to the occurrence of two mechanisms

in their nitrene chemistry. Under protic, polar conditions, they undergo singlet

nitrene/nitrenium ion chemistry, and under aprotic, nonpolar conditions, they

undergo triplet nitrene/radical chemistry. However, there has been a significant

lack of communication between mechanistic chemists who study these molecules

CONCLUSIONS 109

under nonbiological conditions, and biochemists who apply these reagents making

the assumption that their mechanisms of action in aqueous biological environments

are well understood. This situation has been succinctly summarized by Russian

workers as follows, “The opinion dominates in the literature that, in the case of

reagents carrying aromatic azide groups, including 5-azido-2-nitrobenzoyl group,

the reactive particles modifying the protein functional groups are aryl nitrenes

formed under irradiation. These conclusions are based on the speculations concern-

ing the results of kinetic studies and the identification of stable products isolated after

the azide photolysis in organic media, i.e., under the conditions far from those used in

the protein affinity modifications”.153 It is to be hoped that mechanistic chemists will

rectify this situation by extending their studies into aqueous media with biological

molecules and that biological chemists will provide more detailed information about

the structures of the cross-linked products that they isolate from their diverse studies.

With such a synergy of effort, it should be possible to design PAL agents that are

custom designed for a wide variety of molecular situations.

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