Photoremovable Protecting Groups - Givens Research Group · 2013-01-25 · Photoremovable...

69

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0-8493-1348-1/04/$0.00+$1.50© 2004 by CRC Press LLC

69

Photoremovable

Protecting Groups

69.1 Introduction .....................................................................

69

-169.2 Historical Review..............................................................

69

-2

o

-Nitrobenzyl • Benzoin • Phenacyl • Coumaryl and Arylmethyl

69.3 Carboxylic Acids.............................................................

69

-17

o

-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin • Other

69.4 Phosphates and Phosphites ...........................................

69

-23

o

-Nitrobenzyl • Coumaryl • Phenacyl • Benzoin

69.5 Sulfates and Other Acids................................................

69

-2669.6 Alcohols, Thiols, and

N

-Oxides ....................................

69

-27

o

-Nitrobenzyl • Thiopixyl and Coumaryl • Benzoin • Other

69.7 Phenols and Other Weak Acids.....................................

69

-36

o

-Nitrobenzyl • Benzoin

69.8 Amines ............................................................................

69

-37

o

-Nitrobenzyl • Benzoin Derivatives • Arylsulfonamides

69.9 Conclusion......................................................................

69

-40

69.1 Introduction

Photoremovable protecting groups are enjoying a resurgence of interest since their introduction byKaplan

1a

and Engels

1b

in the late 1970s.

A review of published work since 1993

2

is timely and will provideinformation about several new groups that have been recently developed.

The scope of this review is,therefore, limited to recent developments in the field and will cover only the applications with majorfunctional groups that have been “protected” by a photoremovable chromophore.

The review is notintended to be comprehensive but focuses instead on a series of well-chosen examples of chromophoresthat were deployed as protecting groups with a select group of representative functional groups.

Becausethe focus of this review is the application of photoremovable protecting groups, emphasis is placed onsynthesis of the protected functionality and on the procedures employed for deprotection, including theprotection and photodeprotection yields, the deprotection reaction rates, and the quantum efficiencies,when available.

An attempt has been made to list the advantages and disadvantages of each photoremov-able protecting group as well as a brief discussion of the mechanism for the photodeprotection.

Richard S. Givens

University of Kansas

Peter G. Conrad, II


Abraham L. Yousef


Jong-Ill Lee


1348_C69.fm Monday, October 13, 2003 3:22 PM

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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition

When the literature is insufficient for providing a comprehensive treatment of applications of aphotoprotecting group, then only a brief discussion is provided.

An exhaustive list of applications forany of the chromophores is not included; these may be found by consulting other reviews or the originalliterature on a topic.

Several good reviews on photoremovable protecting groups have appeared sincethis topic was reviewed in 1993 (e.g., Adams and Tsien

3

and Corrie and Trentham

4

).

Notable among themore recent reviews are those by Wirz,

5

Bochet,

6

and Givens.

7

A volume of

Methods in Enzymology

devotedentirely to the chemistry and applications of photoremovable protecting groups, also termed “caged”compounds, that are employed in biochemistry and other biological studies has also appeared.

In general, photolysis reactions present a noteworthy and often ideal alternative to all other methodsfor introducing reagents or substrates into reactions or biological media.

The ability to control the spatial,temporal, and concentration variables by using light to photochemically release a substrate provides theresearcher with the ability to design more precisely the experimental applications in synthesis, physiology,and molecular biology. Among the many possible examples is the recently reported inhibition–reactiva-tion of protein kinase A by photolysis of the dormant enzyme.

8–10

In this demonstration, it is necessarythat the deprotection process be initiated by photolysis of the dominant chromophore of the protectinggroup.

Covalent blocking of the functional groups at the active site of an enzyme essentially suspends itsmode of action and virtually shuts down the catalytic cycle.

It is this feature that has attracted biochemiststo the use of protecting groups for the investigation of biological mechanisms.

In synthesis, the protecting group serves as a mask that renders a functional group inert to subsequentsynthetic reaction conditions,

11

except, of course, conditions that are required for the removal of theprotecting group.

Construction of combinatorial platforms with photoremovable linkers is just oneexample of the applications in synthesis.

Photorelease is sometimes termed a

traceless reagent process

because no reagents other than light are needed.

The advantage of a process that requires no furtherseparation of spent reagents is attractive.

There are several limitations to the use of commonly employed protecting groups in synthesis and formechanistic studies of biological processes.

The reactions for incorporating and subsequently removingprotecting groups often involve acid or base that may be too harsh and interfere with the normal processesor otherwise be incompatible with the chemistry or biology under investigation.

In mechanistic bio-chemistry, it is often the case that the typical hydrolysis deprotection reaction is far too slow to serve asa means of investigating the initial rates of reaction for rapid biochemical processes.

An ideal remedy to these limitations is a protecting group that could be removed under neutral bufferedaqueous conditions, thus avoiding any alterations to the substrate or to the natural biological environ-ment.

12

The release should occur on a time scale fast enough for kinetic analysis of any subsequent rapidbiological processes.

Such a group may be a photoremovable protecting group.

69.2 Historical Review

In 1962, Barltrop et al.

13

were among the first to report a photochemical deprotection reaction of abiologically significant substrate; here, glycine was released from

N

-benzyloxycarbonyl glycine:

(69.1)

This seminal discovery prompted the development of several additional photoremovable protectinggroups.

The success of many researchers in biology, particularly Kaplan,

1a

led to the description of thephotoactivatable group as a “cage” to describe its deactivating influence on the biological substrate towhich it is covalently attached.

14–17

Ideally, the cage detaches only through the action of light.

O NH

OOH

O

CH3H2N

OH

OCO2++

hν


Photoremovable Protecting Groups

69

-3

It is important that the photoremovable protecting group also possess several other desirable proper-ties. The properties were originally compiled by several researchers in the field, including Sheehan andUmezawa

12

and Lester and Nerbonne,

18

who provide a series of benchmarks for evaluating the efficacyof a photoremovable group in a given circumstance or for evaluating the potential of a new cagechromophore. A more useful adaptation of the Lester rules and Sheehan criteria includes the following:

1. The substrate, caged substrate, and photoproducts have good aqueous solubility for biologicalstudies. For synthetic applications, this requirement is relaxed.

2. The photochemical release must be efficient (e.g.,

Φ

> 0.10).3. The departure of the substrate from the protecting group should be a primary photochemical

process (i.e., occurring directly from the excited state of the cage chromophore).4. All photoproducts should be stable to the photolysis environment.5. Excitation wavelengths should be longer than 300 nm and must not be absorbed by the media,

photoproducts, or substrate.6. The chromophore should have a reasonable absorptivity (

a

) to capture the incident light efficiently.7. The caged compounds, as well as the photoproduct from the cage portion, should be inert or at

least benign with respect to the media, other reagents, and products.8. A general, high-yielding synthetic procedure for attachment of the cage to the substrate must be

available.9. In the synthesis of a caged substrate, the separation of caged and uncaged derivatives must be

quantitative.

This is also necessary for the deprotection process for synthetic applications.

While these are the desirable guidelines for an ideal photoremovable protecting group, a potential cagethat lacks one or two of these properties may still be very useful;

however, the absence of several of thesefeatures may militate against the use of that group as a photoremovable protecting group for a specificapplication.

Some representative examples of photoremovable protecting groups that qualify as meeting the Lesterand Sheehan criteria include

α

-substituted acetophenones, benzoins, benzyl groups, cinnamate esters,coumaryl groups, and, the most popular of them all, the

o

-nitrobenzyl esters and their analogs.

o

-Nitrobenzyl

It was also Barltrop et al.

19

who first reported the use of an

o

-nitrobenzyl group to release benzoic acid(see Eq. (69.2)).

The poor yield stemmed from the subsequent conversion of 2-nitrosobenzaldehyde (

3

),the initial photoproduct, into azobenzene-2,2

′

-dicarboxylic acid (

4

),

20

which then competed for theincident light.

Yields were dramatically improved with the use of

α

-substituted nitrobenzyl esters (75 to95% conversion), as seen from

5

in Eq. (69.3).

The resulting photoproduct from

o

-nitrobenzyl ester

5

was a less reactive nitroso benzophenone derivative.

(69.2)

(69.3)

NO2

O

O

NO

H

O

OH

O

NN

CO2H

HO2C

+hν

1 2, 17% 3 4

NO2

O

O

NO

Ph

O

+

Phhν

5

2

6


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CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition

The report of the release of ATP by this method appeared in a 1978 rate study reported by Kaplanand co-workers.

1a

Inorganic phosphate (

P

i

) and ATP were released from their 1-(2-nitrophenyl)ethyl(NPE) and 2-nitrobenzyl (NB) esters, respectively:

(69.4)

The results of the release of

P

i

from NPE and NB showed very similar quantum efficiencies of 0.58 and0.50, respectively;

however, the release of ATP from the two cages gave very different rates of conversion.NPE released 80% of the caged ATP in less than 60 s compared with 25% for release from the NB cagedATP.

These results further indicated that the

α

-substituted nitrobenzyl esters were better suited asphototriggers.

Kaplan’s investigation also explored the potential use of photoprotecting groups in a physiologicalenvironment.

Na

+

,K

+

-ATPase, the enzyme responsible for sodium/potassium transport through cell walls,served as the model for exploring the effect of the caged ATP (NPE-ATP) on the Na

+

:K

+

transportassociated with enzymatic activity.

The enzyme acquires ATP as the energy source through hydrolysis ofthe terminal

γ

-phosphate. The hydrolytic activity of the enzyme can be monitored by the detection of

P

i

generated from the free ATP consumed by the enzyme.

In the absence of photolysis, NPE-ATP wasshown to be resistant to hydrolysis by the enzyme.

Upon photolysis, the liberated ATP triggered theresponse of the enzyme and

P

i

release was observed.The successful introduction of

o

-nitrobenzyl caged ATP into physiological media instigated interestin expanding the applications of caged release to a wide variety of biochemical systems.

The list includesthe mechanism of release of

P

i

in skeletal muscle,

21

the function of cAMP in the relaxation of distalmuscle,

22

the ATP-induced mechanism of actomyosin in muscle contraction,

23

and the activation ofantitumor antibiotics to highly reactive pyrrolic-type intermediates responsible for DNA crosslinkingreactions.

24

Benzoin

Sheehan and Wilson

25

were the first to explore the photochemical rearrangement of certain benzoinderivatives to yield 2-phenylbenzofuran (

9

).

These rearrangements occurred with concurrent loss ofgroups attached

α

to the carbonyl just as in the case of the

α

-chloroacetophenones.

They suggested thatbenzoins, especially the 3

′

,5

′-dimethoxybenzoin chromophore, could serve as a photoremovable protect-ing group for carboxylic acids. In 1984, Givens et al.26 showed that phosphates were quantitatively expelledfrom the ungarnished benzoin cage, as shown in Eq. (69.5), thus extending the range of applications andthe nature of the parent chromophore. The only major product accompanying the released phosphate

NO2

R

OPO

OR'

NO

R

O

OPO

OR'+

h�, 342 nm

7a R = H, R' = O-

7b R = H, R' = O-ADP7c R = CH3, R' = O-

7d R = CH3, R' = O-ADP

3 R = H, CH3

Pi R' = O-

ATP R' = O-ADP


Photoremovable Protecting Groups 69-5

(69.5)

was 2-phenylbenzofuran 9. These reactions were quenched with naphthalene, piperylene, or, for aqueousstudies, sodium 2-naphthalenesulfonate, all well-known triplet quenchers; this established the short-livedtriplet (3 to 14 ns) as the reactive excited state for benzoin. Further information was revealed fromStern–Volmer quenching analyses which provided the rate of release of phosphate from the benzoin-caged ester. Extremely fast rates (kr > 108 s–1) were measured along with good efficiencies for the reaction,ranging from 28 to 38% (Table 69.1).27,28 Phosphorescence spectra supported the multiplicity assignmentsand also established the triplet energy at 73 ± 1 kcal/mol.

The efficiencies for the disappearance of the caged phosphates (Φdis), as well as the appearance of 2-phenylbenzofuran (Φfuran) and phosphate (Φphosphate), were determined to be pH dependent with higherefficiencies reported under acidic conditions. The greater efficiency at lower pH suggests that the proto-nated phosphate is a more favorable leaving group than its conjugate base. This study was extended tonucleotide release from the benzoin cage through the synthesis and photolysis of benzoin cAMP. Theefficient release of cAMP as the exclusive product with quantum efficiencies on the same order ofmagnitude as the model phosphate esters first demonstrated the application of benzoin as a cage for

TABLE 69.1 Quantum Efficiencies for Benzoin Phosphate Esters 8a–c

Phosphate Ester Solvent pH Φdis Φfuran Φphosphate

8a C6H6 nd 0.28 0.26 nd8b H2O/CH3CN 2.0 0.37 0.20 0.128b H2O/CH3CN 7.0 nd 0.07 0.0138c H2O/CH3CN 2.0 0.38 0.14 0.158c H2O/CH3CN 7.0 nd 0.08 0.01

Note: All reactions were run in 60% aqueous acetonitrile, except 8a, asindicated; phosphate esters were irradiated at 350 nm and monitored via31P NMR; nd = not determined.

Source: Adapted from Givens et al.27

O

OPO(OR)(OR')

OPh

+

h�

OPO

OR'OR

8a R = R' = Et8b R = iPr, R' = +Na or +H8c R = R' = +Na or +H

R = R' = Et R = iPr, R' = +Na or +HR = R' = +Na or +H, (Pi)

9

ST 38O

CF3CH2OH

O

OCH2CF3

110

3

ST

310, 670 ns

from laser flash studies

O

OH

H

PO(OR)(OR')

3

<20 ns

<5 ns

+


69-6 CRC Handbook of Organic Photochemistry and Photobiology, 2nd Edition

nucleotides (Table 69.2). 31P spectra of released cAMP demonstrated that cAMP was the only phosphatepresent after release. As Table 69.2 indicates, the quantum efficiencies remained relatively constantthroughout the pH range examined.

It has been determined that carboxylate derivatives are released more readily when there are electron-donating substituents at the meta positions of the benzyl ring.29 The absorption spectra of the benzoinesters along with the observations that cyclization was enhanced by meta electron donating groups ledobservers to believe that reaction was taking place through an n,π* singlet state (see Scheme 1). It wassuggested that the excitation of the phenacyl group led to a short-lived 1,3-biradical, followed by demotionto the ground state. The zwitterionic intermediate led directly to the loss of the leaving group. Aroma-tization through loss of a proton gave the benzofuran 11 as the principal photoproduct. The inability toquench the reactions with high concentrations of piperylene suggested that the reaction originated fromthe singlet excited state or, alternatively, from a very short-lived, unquenchable triplet.

TABLE 69.2 Quantum Efficiencies for Photolysis of Benzoin Adenosine Cyclic 3′,5′-Monophosphatea

Aqueous Buffer pH Φdis Φfuran ΦcAMP

Tris (D2O) 7.3 0.39 0.19 0.34Tris (H2O) 7.3 0.37 0.17 0.34Phosphate (D2O) 8.4 nd 0.17 ndPhosphate (H2O) 8.4 nd 0.17 ndPerchloric (D2O) 1.6 0.40 0.16 0.36

a Irradiations were carried out in 1:1 buffer: 1,4-diox-ane at 350 nm. Quantum efficiencies (Φ) were mea-sured using 31P NMR, except where indicated (nd).


SCHEME 1 Benzoin photorelease mechanism.

O

O R

O

hνO

O R

O

OH

O

O R

O+

O

OH

R

O

H

O

OH

R

O

H

*

*

demotion

OMe

OMe

OMe

MeO

OMe

OMe

OMe

OMe

OMe

OMe

OMe

MeO

11



Based on these observations Corrie and Trentham4 re-examined the photochemistry of several sub-stituted benzoin phosphates. They found that the 3′,5′-dimethoxybenzoin cage was best suited amongthose investigated for the release of Pi. The formation of 3′,5′-dimethoxy-2-phenylbenzofuran (11)occurred at a rate that exceeded 105 s–1 and a quantum efficiency of 0.78. While the rate of productformation was lower than that reported by Givens and Matuszewski,26 the efficiency for the substitutedbenzofuran analog was much higher. For the benzoin series, the primary photoproduct 11 is also astrongly absorbing chromophore and thus competes for incident light and forms photodimers alongwith several other unidentified products upon further irradiation. Yet another limitation of this systemis the presence of a chiral center alpha to the carbonyl, which engenders isolation and purificationproblems in the synthesis of benzoin-protected chiral substrates.

More recently, Rajesh et al.30 examined the earliest events in the photolysis of benzoin diethyl phosphate(8a). With nanosecond resolution, the LFP excitation of 8a in trifluoroethanol gave an immediate,permanent 300-nm absorption identified as the benzofuran photoproduct (Eq. (69.5)). A second tran-sient absorption at 570 nm was also observed which decayed with a first-order rate constant of ~2 × 106

s–1 in degassed acetonitrile or trifluoroethanol that was assigned to the triplet α-ketocation 10. Theintermediate could be trapped by trifluoroethanol, yielding trifluoroethyl benzoin ether. Evidence forthe intermediacy of the α-ketocation triplet came from experiments with added halide ion or azide inwhich electron transfer quenching of the transient was observed. Oxygen and naphthalene quenchingexperiments demonstrated that 310 was formed adiabatically on the triplet manifold from 38a. Temper-ature-dependent studies indicated an activation energy for decay of 310 of 8.6 kcal/mol to the singlet,which then reacted with trifluoroethanol to form the trifluoroethyl ether. Stern–Volmer analysis of thenaphthalene quenching gave a triplet lifetime for 38a of τ3 = 18 ns.

The formation of 2-phenylbenzofuran during the nanosecond laser flash experiment was corroborated bya picosecond study of 8a. A rise time of 2 to 4 ps was determined for the 340 nm transient. A rich fluorescenceemission obtained in the nanosecond study was shown to arise from 19 generated during the nanosecondlaser excitation pulse. Naphthalene also quenched the formation of 9 at the same rate as the formation of310, establishing that the two primary photoproducts came from the same triplet (i.e., 38a). Thus, for theunsubstituted benzoin phosphates, reaction proceeds exclusively through the triplet manifold.

Phenacyl

In a similar study, Sheehan and Umezawa12 employed a stripped-down version of the benzoin chro-mophore, the p-methoxyphenacyl group, for the release of benzoic acid, several amino acid derivatives,and peptides, as shown in Eq. (69.6) and Table 69.3.

(69.6)

In this case, the photoproduct was the p-methoxyacetophenone (13), a reduction product. The proposedmechanism (Scheme 2) was a simple homolysis of the carbon-oxygen bond. Ethanol serves as a hydrogenatom donor during this process, and in the presence of 1 M benzophenone or naphthalene the reactionwas completely quenched, indicating a triplet reaction pathway. Benzophenone and naphthalene areknown quenchers of acetophenones and have triplet energies of 68 and 62 kcal/mol, respectively.

Epstein and Garrossian31 reported the release of ethyl and phenyl phosphate esters from the corre-sponding p-methoxyphenacyl phosphates in 1,4-dioxane. The released phosphates were recovered in highyields (Et, 86%; Ph, 74%) along with 13 (91–84%) as the only observed photoproducts. The absence ofany rearranged ester products contrasted with reports by Anderson and Reese34 for substituted α-chloroacetophenones (vide infra). The rationale for the discrepancy advanced by Epstein was an altered

MeO

RO

MeO

O

+ free acid

hν, pyrex

ethanol ordioxane

1312



mechanism due to a change in solvent. The solvent 1,4-dioxane may not be sufficiently polar to supportthe formation of the zwitterionic precursor to the Favorskii-like rearrangement that was proposed byAnderson and Reese. This was required for the rearrangement to the spirodienedione intermediate.Reinvestigation of the photolytic cleavage using polar solvents would have been an interesting test of thishypothesis.

(69.7)

Givens et al.28 examined the photorelease of phosphate esters using t-butyl alcohol and methanol assolvents, the former being a poor hydrogen atom donor and both exhibiting increased polarity comparedwith 1,4-dioxane. The results correlated well with those of Anderson and Reese in that the majorphotoproduct was the rearranged ester, not the photoreduction product (Eq. (69.7)). The amount of 13was decreased to 21% in methanol and 14% in t-butyl alcohol. Further investigation of the solvent dependencyfor the release of phosphates revealed a solvent isotope effect with deuterated vs. protiated methanol as thesolvent (Table 69.4). The formation of p-methoxyacetophenone was suppressed by a factor of five when

TABLE 69.3 Percent Yield for the Release of Various Acidsa from the Corresponding 4-Methoxyphenacyl Esters (12)

R Solvent Irradiation Time (hr) Yield of ROOH (%)

PhCOO Dioxane 17 81PhCOO Ethanol 6 96Boc-L-Ala Dioxane 17 82Boc-L-Ala Ethanol 6 93Boc-Gly Ethanol 6 94Boc-L-Phe Dioxane 17 89Z-D, L-Ala Dioxane 6 84Phthaloyl-Gly Dioxane 17 80Tri-Gly Dioxane 17 58Z-L-Trp Ethanol 4 33Z-Gly-Gly Ethanol 5.5 77Z-L-Asp(OBz)-L-Ser Dioxane 9 49

a All reactions were carried out below room temperature at (5 × 10–3–10–2) M usinga Pyrex filter. Irradiations were complete in 6 hr in ethanol and 11–17 hr indioxane. Yields were determined from product isolation following photolysis.

Source: Adapted from Sheehan and Umezawa.12

SCHEME 2 Sheehan et al. mechanism



photolysis was carried out in CD3OD compared with either CH3OD or CH3OH, suggesting that a rate-determining hydrogen abstraction occurs in the photoreduction process. Sheehan first suggested thismechanistic pathway (vide supra).

Indeed, Dhavale et al.32 have shown that the ratio of rearrangement to reduction for substituted α-chloroacetophenones is solvent dependent [Eq. (69.8) and Table 69.5]. Dhavale’s group reported thatirradiation of substituted α-choroacetophenones in methanol resulted in more photoreduction, whereasin aqueous acetonitrile rearrangement to the phenylacetate esters became the major pathway. For a givensolvent, the ratio of rearrangement to reduction increased with the electron-donating power of thesubstituent.

(69.8)

Scheme 3 outlines Dhavale’s proposed mechanism for the chloride loss and subsequent rearrangement,beginning with carbon–chlorine bond homolysis. An electron transfer from the α-ketoradical to thechlorine atom leads to the ion pair 21. The ion pair is more susceptible to Favorskii-like rearrangementin polar solvents; therefore, the rearranged phenylacetic acid is favored in polar, protic solvents. Hydrogenabstraction, resulting in the formation of 17, prevails in those solvents that are good hydrogen atomdonors.

Sonawane et al.33 investigated the photorearrangement of several para-substituted propiophenones asa convenient entry for substituted α-arylpropionic acids, as shown in Eq. (69.9):

TABLE 69.4 Quantum Efficiencies and Solvent Isotope Effects (kH/kD) for Photolysis of 4-Methoxyphenacyl Diethyl Phosphate (14a)a

Solvent Φ14a Φ15 kH/kD15 Φ13 kH/kD

13

C6H6, t-BuOH (3:1) 0.036 0.026 — 0.074 —CH3OH 0.42 0.20 — 0.07 —CD3OD — 0.14 1.4 0.013 5.4CH3OD — 0.11 1.8 0.053 1.3

a Irradiations were performed in the indicated solvent at 300 nm. kH/kD is a relative efficiency for H vs. D abstraction. Error limits are ±10%.


TABLE 69.5 Photolysis of α-Chloroacetophenones: Yields Obtained for Photoproducts 17 to 20 Under Varying Conditionsa

Methanol Acetone(aq) Acetonitrile(aq)

16 Ar 17 19 17 18 20 17 18 20

a Phenyl 60 00 37 24 29 14 47 18b 4-Methyl phenyl 62 06 24 47 23 14 70 11c 4-Methoxy phenyl 35 34 16 43 14 09 54 08d 4-Chloro phenyl 60 00 10 14 31 06 35 18

a Photolysis of 2% degassed solution of 16a–d in methanol, 95:5 acetone/water, or 95:5 acetonitrile/water using 300-nm lamps was carried out inthe presence of propylene oxide as a halogen scavenger. Irradiations were4 hr and yields are isolated products.

Source: Adapted from Dhavale et al.32

ArCl Ar CH3

ArAr

ArO O O

OO

OR hν

solvent+ +

17 18 R = H19 R = CH3

2016



(69.9)

In almost every case, para-substitution promoted rearrangement (Table 69.6). Phenyl- and chloro-sub-stitutions in the para position were the only cited examples where rearrangement did not dominate inmethanol. These cases also showed significant reduced and hydrolyzed products.

SCHEME 3 The mechanism suggested by Dhavale et al.32

TABLE 69.6 Product Distribution from Photolysis of 22a–i in Aqueous Acetone or Aqueous Methanola

Substrate Acetone(aq) Methanol(aq)

22 X 23 24 25 23 24 26

a H 58 25 — 39 30 —b CH3 84 5 — 76 8 —c C2H5 82 6 — 74 9 —d n-C3H7 84 5 — 77 9 —e i-C4H9 74 10 — 65 15 —f t-C4H9 78 7 — 69 8 —g Cl 45 25 20 30 51 30h Ph 40 25 35 18 26 35i OCH3 32 10 50 80 12 70

a Irradiations were carried out in 95:5 solvent/watersolutions employing a Hanovia 200-W, medium-pressure mercury vapor lamp with a Pyrex filter untilthe complete disappearance of starting material. Thephotoreaction was monitored and yields were deter-mined with GLC and 1H NMR.

Source: Adapted from Sonawane et al.33

ClO O

Cl

CH3

O

O

H Cl Cl O

OH

H-abstraction

a Favorskii-like

1,2-aryl migration

single electrontransfer (SET)

21 18

17

H O

hν

hν

16

+ HClX X X

X X X

X

ClO

X

O

X

OH

OX

ORO

+ +

hν, 300 nm

solventpropyleneoxide22a-i 23 24

25 R = H26 R = CH3



While not purported to be a photoremovable protecting group, the study by Anderson and Reese34

on substituted phenacyl chlorides did reveal an interesting photochemical rearrangement for certainmembers of the series, particularly the report that the Favorskii-like rearrangement of p-hydroxyphenacylchloride (27) in 1% aqueous ethanol gave two major photoproducts: p-hydroxyacetophenone (28) andethyl p-hydroxyphenyl acetate (29), as shown in Eq. (69.10). The authors proposed a spiro intermediate 30.

(69.10)

Further examination of the proposed aryl participation hypothesis led to the observation that electron-donating groups in the ortho or para positions were necessary for the rearrangement to occur (Table 69.7).

The results of Anderson and Reese coupled with the efficacy of the benzoin chromophore for cleavageof α substituents attracted our interest in developing the p-hydroxyphenacyl group as a photoremovableprotecting group. We further rationalized that the introduction of a phenolic hydroxy group wouldenhance the aqueous solubility. The absence of the attendant α phenyl substituents on benzoin alleviatedthe stereogenic center problem present with benzoin derivatives. Furthermore, Anderson and Reesereported a Favorskii-like rearrangement of the chromophore, e.g., p-hydroxyphenacyl to p-hydroxyphe-nylacetate for 27 → 29 and for 16b,c, suggesting a significant hypsochromic shift in the chromophore.Thus the promise of p-hydroxyphenacyl as a possible phototrigger was too enticing to pass up.

In 1995, we began a comprehensive exploration of a variety of p-substituted phenacyl phosphates fortheir efficacy toward releasing phosphate.2,28 Among the substituents examined, the p-acetamido, methylp-carbamoyl, and n-butyl p-carbamoyl groups proved untenable because they gave a plethora of products,most of which resulted from coupling or reduction of an intermediate phenacyl radical [Eq. (69.11)]35,36

TABLE 69.7 Product Formation from Photolysis of Substituted Phenacyl Chloridesa

Aryl Substitution, X % Ethyl Aryl Acetate % Acetophenone

p-OH (27) 32 26p-OMe (16c) 32 30o-OH — 3o-OMe 32 16p-Me (16b) 4 58H — 53p-CO2Me — 48p-Cl (16d) — 55o-Cl — 45m-OMe — 15

a Photolyses were carried out in 1% alcoholic solutions using a500-W Hanovia mercury arc lamp. Reactions were carried out for1 to 2 hr. Products were isolated via vapor phase chromatography.

Source: Adapted from Anderson and Reese.34

HO

OCl hν

1% aqueousEtOH HO

O

HO

OEt+

O

27 28

O

O

29

30



(69.11)

Table 69.8 gives the disappearance efficiencies for several p-substituted phenacyl phosphates from whichit is evident that release of phosphate does occur very efficiently for the acetamido and carbamoylderivatives; however, the large array of products of the phototrigger discouraged our further interest inthese three electron-donating groups.

The methoxy substituent (14c) showed a much cleaner behavior, yielding only two products from thechromophore, p-methoxyacetophenone and the rearrangement product p-methoxyphenylacetic acid. Thep-hydroxyphenacyl phosphate (33) gave the rearranged p-hydroxyphenylacetic acid when photolyzed inmixed aqueous organic solvents (Eq. (69.12)); in fact, of all of the groups examined, only p-hydroxy andp-methoxy produced any rearranged phenylacetic acids.

(69.12)

The initial discovery that diethyl p-hydroxyphenacyl phosphate exclusively followed a rearrangementpathway was followed by an extension of our study to p-hydroxyphenacyl ATP (35). Irradiation of 35 at350 nm released ATP and p-hydroxyphenylacetic acid with a quantum efficiency of 0.37 ± 0.01 and arate constant for ATP appearance of 5.5 ± 1.0 × 108 s–1 [Eq. (69.13)].

TABLE 69.8 Disappearance and Product Efficiencies for Ammonium Salts of p-Substituted Phenacyl Phosphate in pH 7.2 Tris Buffer at 300 nm

p-Substituent Φdis Φ34 Φ32,13 Φother

31a NH2 <0.05 0.0 <0.05 Not available31b CH3CONH 0.38 0.0 0.11 Dimers31c CH3OCONH 0.34 0.0 Not determined Two unknowns14c CH3Oa 0.42 0.20 0.07 Not available33 HOb 0.38 0.12 0.0 0.035 HOc 0.37 0.31 0.0 0.0

a Solvent was MeOH and diethyl phosphate was the leaving group.b The diammonium salt of the mono ester; 10% CH3CN was added to the

solvent.c The ATP derivative.

X

O

OPO

O hn, ROH

X

PHO

OO

+

31a-c, 14c 32a-c, 13

O

+ other products

a) X = NH2, b) X = NHCOCH3, c) X = NHCO2CH3, 14c) X = OCH3

OO

HO

O

OPO

OROR

hn, H2O

HOO

OH

PO

HOOR

OR+

33, R = Et 34



(69.13)

While the mechanism of this process is unknown, the p-hydroxyphenacyl photorelease likely involvesan initial triplet state deprotonation of the phenolic hydrogen. In one scenario, the initially generatedtriplet intermediate partitions between loss of a proton and C-O bond cleavage, as pictured in Scheme 4.The exact course of the reaction depends greatly on the leaving group, solvent, and substituents attachedto the chromophore. Here, the triplet phenol undergoes the equivalent of a homolytic cleavage of thebond to the substrate. In this scenario, it was envisaged that initial homolysis of the C-Y bond might befollowed by a rapid single-electron transfer process; that is, the triplet phenol is essentially converted toits conjugate base before other competing processes for the radical pair can intervene. In this sequence,a spirodienedione is eventually generated by electrocyclic closure of the intermediate zwitterion or possiblythe diradical.

The conjugate base formed by the proton loss undergoes bond reorganization to the putative spiro-dienedione 30 accompanied by release of ATP. Further hydration of the spirodienedione and bondreorganization lead to the phenylacetic acid that is suggested for both pathways.

A second mechanistic scenario involves proton loss concomitant with direct neighboring group assis-tance for the release of the substrate and formation of the spirodienedione. The subsequent proposednucleophilic hydrolysis of the spirodienedione follows as above. Current evidence from subsequentsolvent and substituent studies favor the latter mechanism (vide infra).

The onset of the triplet-state phosphorescence emission of several p-hydroxyphenacyl esters indicatedtriplet energies of 68.9 to 70.6 kcal/mol. The phosphorescence emissions were quenched by sodium 2-naphthalenesulfonate or potassium sorbate. Quenching studies confirmed the reactivity of the tripletstate and further provided a lifetime of 5.5 ns for the triplet with a release rate of 1.82 × 108 s–1 in laterstudies (vide infra).

SCHEME 4 Proposed triplet state mechanisms for photorelease of substrates from the p-hydroxyphenacyl protectinggroup.

O

HO

h� O

OHOH

N

N

N

N

HO POPOP O

O

-O

O

-O

O

-O

35

NH2

O

OHOH

N

N

N

N

OPOPOPO

O O

-O

O

-O

NH2

-O

�= 0.37kr = 5.5 x 108 s-1

- 34 ATP

HO

OY

ST

HO

O

HO

OY -H+

O

O

O

OY

H2O

3 3

+ Y 29

34

hν, 300 nm

pKa3

khom

kH

ket

khetY = (R'O)2PO2-

- Y

28



The original proposal that the triplet state was the reactive state was challenged by Zhang et al.37 Anexcited singlet state or possibly a tautomeric ground state (e.g., 37*) was proposed as the reactionintermediate. In their studies, quenching by triplet quentchers was not observed during photolysis of p-hydroxyphenacylacetate (36), suggesting that the release of acetate occurred through an excited singletstate possibly involving an intramolecular proton transfer. They postulated that the excited singlet stateintramolecular proton transfer (ESIPT) mechanism would form the quinone methide 37* that couldeither continue on to the spirodienedione (30) or decay to ground state and subsequently undergo releaseof acetate to form 34. Such a mechanism has precedence in the earlier work of Wan37,38a and Yates.38b

Laser flash photolysis (LFP) studies by Givens and Wirz39 with diethyl pHP phosphate (Eq. (69.12))confirmed the intermediacy of the phenacyl triplet state. Energy transfer quenching to naphthalene gavea rate of formation of the naphthalene triplet of 7.8 × 109 M–1 s–1. The presence of dioxygen increasedthe decay rate of the pHP phosphate triplet (kq ≈ 3 × 109 M–1 s–1). It was estimated from this study thatpHP intersystem crosses with a rate constant of 3.1 × 1011 s–1 in aqueous acetonitrile. Quenching studiesof the photochemical release of substrates from a series of pHP derivatives employing potassium sorbategave excellent linear Stern–Volmer quenching results with lifetimes of 10–8 to 10–9 s for their pHP tripletstates. These combined results firmly established the triplet as the reactive excited state.

LFP studies on the parent chromophore proved revealing. The p-hydroxyacetophenone triplet under-goes a facile adiabatic proton tautomerization converting the phenol 28 into its conjugate base 38.

SCHEME 5 ESIPT singlet state photorelease mechanism of p-hydroxyphenacyl acetate.37

FIGURE 69.1 Transient absorption spectra obtained by LFP of p-hydroxyacetophenone in water (10% CH3CN)with various buffers. (From Conrad II, P. G. et al., J. Am. Chem. Soc., 122, 9346–9347, 2000. With permission.)

HO

OOAc

HO

OOAc

HOO

OH

OO

O

OO

OCH3

H

hn ESIPT

(H2O)n

-HOAcH2O

36

3034

1

*

* = singlet or reactive ground state intermediate

37*

350 400 450 5000.0

0.4

0.8

/ nm

pH 7.05

pH 5.32

pH 5.05

pH 4.66

pH 4.38

pH 4.04

pH 2

O

HO

–O

O

3839



Evidence for this unusual adiabatic proton transfer to solvent from the triplet p-hydroxyacetophenonecame from analysis of the transient triplet absorption spectra of 28 as a function of the pH (Figure 69.1).

These results were further supported by DFT calculations that provided the pKE values for the equi-librium 28 39 in their ground and triplet excited states. A pKa

3 for 283 of 3.6 was derived fromthese data and the triplet state pKa

3 of 39 (4.6) and the known ground state pKa of 28 (7.9) (Scheme 6).

These studies revealed an increase in acidity of over four pKa units relative to the ground state. The proton

tautomer 39 is a non-productive intermediate because it is thermodynamically incapable of cleaving theester C–O bond.

Incorporating the rapid deprotonation that results from the large adiabatic decrease in pKa of 28 as afeature of the p-hydroxyphenacyl mechanism suggests that the conjugate base 38 is an attractive precursorto the rate-limiting release of the substrate. Because the triplet is formed with a ST rate constant of 3.1× 1011 s–1, it is unlikely that there is any singlet state contribution to the deprotonation step. Rather, thisappears to be exclusively a triplet process occurring on the excited triplet surface; that is, the twoprotonated species and the unprotonated ion undergo adiabatic proton tautomerizations (Scheme 6).The electron-rich aromatic ring increases the potential for intramolecular neighboring group attack atthe α-carbon, leading to the release of the substrate and rearrangement of the chromophore. Thus, itbecomes prudent to carefully explore the change in the pKa of the phenolic protons transitioning betweenthe ground and excited triplet states as a key element in understanding the role played by aryl participationin the release step.

Coumaryl and Arylmethyl

Zimmerman’s early studies40 on the photosolvolysis of benzyl acetates in 50% aqueous dioxane set the stagefor a variety of studies that employ m,m′-dimethoxybenzyl as a photoremovable protecting group (69.14).In general, the photofragmentation reactions of benzyl acetates are quite rapid, with rate constants of 108 s–1

or higher and are primarily singlet-state processes. According to Zimmerman, meta activation of the excitedsinglet state of benzyl acetates occurs through the approach of the excited- and ground-state energy surfaces,funneling the excited state toward heterolysis of the benzyl–ester bond. Substituents, including electrondonors, in the para position lead primarily to homolytic fission and radical derived products.

Direct heterolytic fission of the substrate-photoprotecting group bond is the required course forphotorelease of most biologically important substrates. This process avoids the generation of destructiveradicals that could result in reactions such as decarboxylation, radical dimerization, or redox processes.Thus, the effect of m-substitution on the photochemistry of benzyl, naphthyl, and other aromaticchromophores has become the object of many studies in search of alternatives to the o-nitrobenzyl classof protecting group.

SCHEME 6 Tautomerization of p-hydroxyacetophenone in the ground state and triplet excited state (in parentheses).(From Conrad II, P.G. et al., J. Am. Chem. Soc., 122, 9346-9347, 2000. With permission.)

HO

O

O

OH

O

O

pKE = 16.4 (-1.0)

pKa = -8.5 (4.6)pKa = 7.9 (3.6)

28

38

39



(69.14)

Recent studies by both Zimmerman41 and DeCosta and Pincock42,43 on the nature of the meta effecthave raised concerns about whether heterolytic or homolytic cleavage should be considered the primaryphotochemical process. According to Pincock, the mechanistic pathway for all substituted arylmethylsubstrates begins with homolysis of the C–O ester bond to the substrate followed by a competitionbetween electron transfer to an ion pair or typical ground-state radical reactions (Scheme 7).43 For thosearylmethyl derivatives substituted with a meta electron-donating group, such as methoxy, the electrontransfer occurs more rapidly than competing radical processes due to favorable redox properties of theradical pair.

Normally, the details of the steps leading to the ion pair would not play a significant role in the outcomeof the photorelease process except in those circumstances where the radical pair precursors have rapid,favorable divergent pathways available. Such could be the case for carboxylate esters, such as C-protectedamino acid, peptides, and protein derivatives, where decarboxylation of the initially generated carboxy radicalmay compete with electron transfer to the ion pair. This deleterious process can become significant, leadingto destruction of a portion of the released substrate. By either of these mechanisms, however, the product-determining process for meta- and especially the di-meta-substituted arylmethyl chromophores leads prin-cipally to an ion pair, an intermediate arylmethyl carbocation, and the conjugate base of the leaving group.

The coumaryl chromophore is essentially another arylmethyl analog, which has the attractive featureof high yields of fluorescence emission, sometimes a useful property for following the course of substrate-chromophore processes. One of the earliest studies using coumarin as a chromophore was the photore-lease of diethyl phosphate from coumarylmethyl diethyl phosphate.26 The resulting coumarylmethylcation covalently attaches to a wide variety of nucleophiles, as shown in Eq. (69.15):

(69.15)

Furuta et al.44 have reported the application of the coumaryl chromophore as a phototrigger for therelease of cAMP (Eq. (69.16)); as shown in Table 69.9, the methoxy and hydroxy methylcoumarins gavethe best conversions.

SCHEME 7 Mechanistic scheme for arylmethyl ester photolysis in methanol.43

h�

40

H3COCO X

��

��

� ��

��

��

��

��

��

-CO2�

��

��

h�

��

��

OCH3H3CO

h�, � = 0.10

OCH3H3CO

40 41, 79%

OCOCH3

50% aq. dioxane

OH

OH3CO O

OP(OEt)2O

hν, 360 nm

Nu

OH3CO O

Nu

42 43Nu = CH3OH, piperidine, cysteine, tyrosine, α -chymotrypsin, HMT



(69.16)

The historical and mechanistic background for the most common photoprotecting groups were pre-sented above. Examples that employ photoremovable protecting groups are given to illustrate the rangeand variety of the applications in chemistry and biology. As noted earlier, these are a very limited set ofexamples of the numerous published applications in biology and, to a lesser extent, in chemistry. Furtherinformation can be obtained from the reviews listed in References 1 through 7.

69.3 Carboxylic Acids

o-Nitrobenzyl

Because o-nitrobenzyl derivatives have been the most widely applied photoremovable protecting groups,modifications of this chromophore have received considerable attention. A recent study45 employing 2,2′-dinitrobenzhydryl (DNB) for N-methyl-D-aspartate (NMDA) probed the NMDA receptor, which is oneof the general classes of known glutamate receptors. The carboxyl group of NMDA was esterified withDNB, a stronger UV absorber (λmax 350 nm: ε = 1.69 × 104 M–1 cm–1) than typical o-nitrobenzyl analogs.The poor aqueous solubility of DNB-NMDA, however, required addition of 20% DMSO to attaincomplete dissolution. A single 308-nm laser pulse was sufficient to release NMDA within 4.2 µs, with aquantum efficiency of 0.18, as shown in Eq. (69.17).

(69.17)

The time constant for the release of NMDA is pH dependent, occurring within 3.8 µs at pH 3.8 and 13.8µs at pH 10.6, consistent with the rate of decay of the aci-nitro intermediate. The relatively rapid releaserate from DNB suggests that this protecting group could be useful for further studies of the NMDAreceptor.

The versatility of the o-nitrobenzyl group has also been demonstrated in solid-phase synthesis. Thephosphate group of the nucleotide tethered to a carboxylic acid through an alkyl chain provides aconvenient link to the o-nitrobenzyl group, which is attached to a solid support. Synthetic manipulationof the oligonucleotide can be carried out under standard conditions and then release of the synthesized

TABLE 69.9 Percent Conversion of Coumarylmethyl cAMP After a 10 s Irradiation at 334 to 365 nm

Caged cAMP R = Acetyl R = Propionyl R = Hydroxy R = Methoxy

Conversion (%) 23 9 64 60

ORO O

O

hν, 334 - 365 nm

44

N

NN

N

NH2

O

OHO

O

P

O

HO

N

NN

NNH2

O

OHO

OP

O

O2N

NO2

O

ONHH3C

HO

O

β-O-DNB-NMDA

hν, 308 nm

NO2

NO

O

+

OH

ONHH3C

HO

O

NMDA

pH 7



oligonucleotide from the support is conducted photochemically.46 The carboxylate and substituted o-nitrobenzyl alcohol were coupled in 89 to 97% yields followed by the controlled pore glass (CPG) loading.Standard oligonucleotide synthesis was then carried out at the 3′-terminus to obtain 45a–d and 45′′′′a–d(Eq. (69.18).

(69.18)

Upon completion of the synthesis, the oligonucleotides were severed from the CPG solid support pho-tochemically using a 400-nm light source. The efficiency of the release process was as high as 92%, asshown in Table 69.10. This protocol is useful for elaborating the 3′-terminus of oligopeptides using mild,traceless reagent conditions at room temperature and neutral pH.

Coumaryl

The photochemical and photophysical behavior of 4-(hydroxymethyl)-7-methoxycoumarin (MCM)caged acids was studied under physiological conditions by Bendig et al.47 (Eq. (69.19)).

(69.19)

Photocleavage of the excited singlet state of MCM caged compounds is thought to proceed via a photo-SN1 mechanism (solvent-assisted photoheterolysis). Evidence favoring this mechanism was found fromirradiation of MCM caged derivatives in 18O-labeled water, which exclusively incorporated the 18O-labelin the MCM–18OH product (see Scheme 8). The deprotection process of the MCM derivatives was

TABLE 69.10 Isolated Yields of Completely Deprotected Oligonucleotides

Protection (%) Deprotection (%) 45 Deprotection (%) 45′′′′

a 96 70 77b 89 91 80c 97 92 71d 90 69 70

NO2

H3COO

O

HN

O

O

DMTrO

Oligo.HO PO

OO (CH2)n+1CO2H

1. std. oligonuceotide synthesis2. h�� 400 nm

3. detritylation4. NH4OH

DMTr = 4,4'-dimethoxytrityl

n

n 1 2 3 4 a b c d

45a-d (n = 1-4) Oligo = T20 45'a-d (n = 1-4) Oligo = TAC GCA ATC CTA GAT CTA AT

O

OX

OMeO

X = CH3

O O

R

46b: R = OCH346c: R = H46d: R = CN

46a

hn, 333 nm

O

OH

OMeO

+ HOX



dependent on the leaving group and the solvent polarity. The quantum efficiencies for the reactions of46a–d were low, in the range of 0.0043 to 0.0064 as listed in Table 69.11. MCM–OH is a highly fluorescentproduct that can be conveniently monitored during the course of the reaction. Furthermore, MCM cagedcompounds are very stable to the hydrolysis.

Phenacyl

The rapid release (~108 s–1) of substrates from the p-hydroxyphenacyl (pHP) group enables fast biologicalprocesses to be studied. p-Hydroxyphenylacetic acid (34) is generated with a quantum efficiency (Φrea)of ~0.18. In contrast, the presence of added electron-donating substituents on the aromatic ring of thepHP group makes the rearrangement a minor pathway for 47′′′′a–b and completely suppress it for 47′′′′′′′′a–b(Scheme 9).48 The quantum efficiencies for the disappearance of the various pHP esters, the appearanceof (34), and corresponding pHP-protected substrates are given in Table 69.12 .

A complication that could arise in such systems is the potential for decarboxylation of the releasedcarboxylate ion. However, no decarboxylation products were observed within the detection limits of 1H-NMR and HPLC.

With the production of a biologically benign photoproduct, the pHP protecting group has proven tobe an efficient tool for investigations of fast biological processes. For example, Givens et al.49 applied thepHP phototrigger to the investigation of the bradykinin BK2 receptor. It is known that bradykinin actsas an active pain-transducer when released during tissue damage. A major difficulty in studying thedetailed physiological mechanism of the action of bradykinin is a concomitant rapid enzymatic degra-dation of the nonapeptide immediately after its release from its precursor protein. Therefore, the pho-torelease from pHP bradykinin (48), which protects bradykinin from degradation of the agonist prior

TABLE 69.11 Data for 4-(Hydroxymethyl)-7-Methoxycoumarin (MCM) Caged Acids

Acid Protection (% Yield) Deprotection (Φ)

46a 82.6 0.004346b 90.0 0.004546c 98.0 0.005246d 83.8 0.0064

SCHEME 8 Reaction pathways for photolytic cleavage of MCM esters.

OX1 *

h�

MC CH2

OXMC CH2

MC = 7-methoxycoumarin-4-yl moiety

OX1 *

MC CH2

(trace)

OH3CO O

CH3

OXMC CH2

k1

k0

kset-kset

kesc

MC CH2OX+

OHMC CH2

OH(solvent)

H(solvent)

HOX

R-H



to release, facilitates the investigation of its action during the transduction process by allowing precisetemporal and spatial release of bradykinin to activate the bradykinin BK2 receptor (Eq. (69.20)).

(69.20)

pHP Bradykinin 48 was obtained in an overall yield of 84% by derivatizing the partially protectedbradykinin, obtained by cleavage of the C-terminus from the resin after sold-phase Merrifield synthesis.49

TABLE 69.12 Quantum Yields of pHP Esters for Irradiation at 300 nma

Ester Solvent (Water/AcCN) Φdis Φapp Φrea

47a pHP GABA Water 0.21 0.21 0.1947b pHP glutamate Water 0.14 0.14 0.0847c pHP cyclopropylacetate 6:4 ~0.2b — —47d pHP phenylacetate 6:4 0.18 0.17 0.1447e pHP pivalate 6:4 ~0.18b — —47f pHP oleate 7:3 0.24 0.23 0.1748 pHP bradykinin Water 0.21 0.22 0.19

a An NMR tube was charged with ~10 mg (~40 µmol) of the appropriate photopro-tected acid and 10 mol% of 1,2,3-benzenetricarboxylic acid as an internal standardin 2 mL of solvent. The quantum efficiencies were determined at less than 20%conversion of the starting ester.

b By comparison with 47d.

Abbreviations: dis = disappearance, app = appearance of acid, rea = rearrangementto phenylacetic acid.

SCHEME 9 Photoreaction for the pHP esters.

O

OOCOR'

OO

HO

OOCOR'

47a-f

1. hn2. ISC3. deprotonation

3

+

HO

OH

OHO

O

HO

O+OH R'CO2

H2O

47*

R R

RRRR

47 R = H 47' R = 3-OMe 47" R = 3,5-OMe

rearrangementreduction

H2N NH

NH

NH2

N

O

O

N NH

O

O

HN

O

NH

O

N

HO

O NH

O

HN

NH

COO

OH

O

NH

H2N

D2O, PyrexNH2-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-CO2

48

+ 34h�� 337 or 300 nm



Reaction of the C-termnus with p-hydroxyphenacyl bromide followed by treatment with 1% TFA gavepHP bradykinin 48, free of the other protecting groups employed during the solid phase synthesis.49

A single 337-nm flash (<1 ns) released sufficient bradykinin to excite the BK2 receptors on single ratsensory neurons, which dramatically increased the intracellular calcium concentration measured withIndo-1, a Ca2+-chelating fluorescent indicator. The quantum efficiency of bradykinin appearance wasindependently determined to be 0.22, as shown in Table 69.12.

Benzoin

There are a number of examples of benzoin and substituted benzoin esters that have been employed asphotoremovable protecting groups for carboxylic acids. Among these, the application of benzoin as atraceless linker for solid-phase synthesis of oligopeptides and introduction of Fmoc-protected aminoacids by Balasubramanian50 is instructive. As an example, the release of Fmoc-Ala shown in Eq. 69.21occurs upon photolysis at 350. Balasubramanian found that the maximum yield was obtained after a 2-h photolysis as determined by HPLC.

(69.21)

In order to avoid premature photolysis of the benzoin linker by adventitious room light during the courseof the synthesis, the dithiane-protected 3-alkoxybenzoin (49a) has been suggested as a “UV-inactive”linker. Dithiane 49 released less than 3% of the product after irradiation at 350 nm. The photosensitivityis restored by hydrolysis of the dithiane prior to photocleavage, as illustrated in Eq. (69.22). Photolysisof the deprotected linker resulted in a 75% yield of the product.

(69.22)

Other

A series of 2,5-dimethylphenacyl (DMP) esters were photolyzed in benzene or methanol (Eq. (69.23)).

(69.23)

O Ph

O

O

O

NHFmoc

hν, 350 nm

HO

O

NHFmoc

OO

Ph

+

O O

O

NHFmoc

PhS

S

(i) O O

O

NHFmoc

PhO

(i) (a) bis[(trifluoroacetoxy)iodo]benzene, (b) mercury (II) perchlorate, or (c) periodic acid (4 eq.), THF:water (10:1) (for a and c), THF (for b), ambient temperature, 18 h. >95% conversion

49a 49b

OO

O

R OHO R

O

85-95%

benzeneor methanol

+

50

hν, >300 nm

51



The formation of the corresponding carboxylic acids occurred with almost quantitative isolated yields,as shown in Table 69.13.51 In contrast with the structurally related p-hydroxyphenacyl esters, the releasemechanism from 50 occurs through an efficient intramolecular γ-hydrogen abstraction via the (n,π*)excited ketone.

(69.24)

Recently, Wirz and Klan52 reported LFP studies on several of the DMP esters in benzene and methanol(Eq. (69.24)). Quantum efficiencies (Φ) in benzene are 0.18 to 0.25, while those in methanol fell to 0.09to 0.14. Thus, this photoprotecting group appears to be better suited to applications in nonpolar mediasuch as benzene rather than methanol (Table 69.14).

A novel design of “orthogonal” protecting groups, i.e., the removal of protecting groups selectivelyfrom a multi-protected substrate, has been reported by Bochet53 in which the irradiation wavelengths

TABLE 69.13 Data for 2,5-Dimethylphenacyl Esters

RProtection (%Yield)

Photolysis Condition (nm)

Deprotection (% Yield)a

CH3 84 Benzene, >280 85b

C6H5 95 Benzene, >280 86C6H5 95 Methanol, >254 92C6H5CH2 84 Benzene, >280 91n-Pentadecyl 69 Benzene, >280 95N-(t-butoxycarbonyl)-L-phenylalanine 51 Benzene, >280 90

a Isolated yield of the crude acids (>95% purity).b Determined by gas chromatography.

TABLE 69.14 Quantum Yieldsa of DMP Esters

XProtection (% Yield)

Photolysis Condition

Deprotection (Φ)

–OCOPh 95 BenzeneMethanol

0.230.09

–OCOCH2Ph 84 BenzeneMethanol

0.180.11

–OCOCH3 84 BenzeneMethanol

0.250.14

a Quantum yield for ester release; valerophenone was used asan actinometer; irradiated at λ >300 nm. Error margins areapproximately 10%.

OX +

OMeO

+hν, >300 nm HX51



serve as the “orthogonal reagents”. A mixed diester of pimelic acid was capped with a dimethoxybenzoingroup at one end and an o-nitrobenzyl group at the other terminus. Despite the possibility that intramo-lecular energy transfer or equilibration could occur between the two chromophores, selective photolysisled to the sequential removal of each with high chemical yield, as shown in Eq. (69.25). Upon photolysisof 52 at 254 nm for 5 minutes, 92% of 53b was released, whereas irradiation at 420 nm for 24 hoursreleased 70% of 53a, as determined by 1H NMR.

(69.25)

69.4 Phosphates and Phosphites

o-Nitrobenzyl

The most notable example of o-nitrobenzyl (ONB) caged phosphates remains that reported by Trenthamand co-workers54 on the synthesis and photochemistry of caged ATP in the late 1980s. Caged ATP, P3-1-(2-nitrophenyl)ethyladenosine 5′-triphosphate (54), was synthesized in nearly quantitative yield in threesteps starting with commercially available o-nitroacetophenone. Reaction with hydrazine to give thecorresponding hydrazone, followed by oxidation with MnO2 provided the aryldiazoethane precursor thatwas used to alkylate ATP. The photolysis of caged ATP furnished ATP with an efficiency of 0.63, asillustrated in Eq. (69.26):

(69.26)

The rate of release of ATP was found to be dependent on the pH and the relative concentration ofmagnesium ion in solution. Pelliccioli and Wirz5 have shown that the rate-determining step is the decayof the hemiacetal (or hemiketal) intermediate between pH 4 and 8. In this region, the slow hydrolysis ofthe hemiacetal limits the mechanistic value of the o-nitrobenzyl protecting group to studies of relativelyslow reactions (e.g., kr < 103 s-1).

The nitroso byproduct has also proved problematic for spectroscopic analyses of ONB reactions dueto its reactivity with some substrates and with proteins. This problem was circumvented by conductingthe photolysis in the presence of dithiothreitol, a hydrophilic thiol and an excellent nucleophile thatreadily sequesters the nitroso byproduct.

O O

OO

O Ph

NO2

MeO

MeOOMe

OMe

70% 1. hν, 420 nm2. TMSCHN2

92%1. hν, 254 nm2. TMSCHN2

O O

OMeO

O Ph

OMe

OMe

O O

OMeO

NO2

MeO

MeO

52

53a 53b

NO2

O PO

O-O

NO

O+ATPP

OO

O-PO

O-O

N

NN

N

NH2

O

OHOH

hν, 320 nm

app = 0.63

TES buffer, KCl, MgCl2, pH 7.1

54 Φ



Coumaryl

Coumarylmethyl esters have been used as photoprotecting groups by several groups (e.g., Furuta et al.55,56

and Hagen et al.57) for deprotection of cAMP and cGMP. To overcome the limited aqueous solubility ofthese derivatives, Hagen has modified the coumaryl chromophore with carboxyl and amino substituents.57

Three new variants of the coumaryl system were synthesized and their photochemistry explored — (7-diethylaminocoumarin-4-yl)methyl (DEACM), (7-carboxymethoxycoumarin-4-yl)methyl (CMCM),and (6,7-bis[carboxymethoxy]coumarin-4-yl)methyl (BCMCM) as esters of cAMP and cGMP. Photolysisresulted in liberation of the free cyclic phosphate along with the hydrolyzed chromophores, i.e., 56a-c(Eq. (69.27)).

(69.27)

The caged coumaryl compounds were synthesized in 11 to 34% yield, clearly a limitation with thisphotoremovable protecting group. The addition of the carboxymethoxy groups in 55b and 55c dramat-ically enhanced the water solubility of these analogs as compared with the ester 55a, and their photoreleaseoccurred with good quantum efficiencies as seen in Table 69.15. On the other hand, the less water-soluble55a had the best quantum efficiency, and its absorption maximum was the most red shifted in the series.

Phenacyl

In light of its inherent advantages over other chromophores, the p-hydroxyphenacyl group has receivedrecent attention as a promising photoprotecting group for phosphates. Recent reports on p-hydroxyphen-acyl esters of phosphate, diethyl phosphate, and ATP by Givens et al.36 and on GTP by Du et al.58 indicatethat these phosphate esters undergo efficient photorelease of the phosphoric acid moiety along with therearranged p-hydroxyphenylacetic acid as the sole photoproducts of the reaction (Eq. (69.28)).

TABLE 69.15 Results of Photolysis of cAMP and cGMP Coumaryl Esters

Coumaryl Derivative Solvent Φdisa λmax (nm)

55a 80:20 HEPES-KCl buffer/MeOH 0.21 (0.25) 40255b HEPES-KCl buffer, pH 7.2 0.12 (0.16) 32655c HEPES-KCl buffer, pH 7.4 0.10 (0.14) 346

a For the cAMP ester; quantum efficiency for the cGMP derivative is in parentheses.

O

O

O

R1

R2

OP O O

HO

O

A (or G)

hν, 333 nm

O

O

OH

R1

R2

cAMP or cGMP +

55a R1 = H, R2 = Et2N55b R1 = H, R2 = OCH2CO2H55c R1 = R2 = OCH2CO2H

56



(69.28)

Derivative 57a was synthesized by reacting the phosphate directly with p-hydroxyphenacyl bromide.Derivative 57b was synthesized from hydrogenolysis of the pHP dibenzyl phosphate ester. For 35 and57c, the pHP monophosphate (57b) was first protected as the corresponding ketal that was then coupledwith ADP or GDP, respectively. The caged ATP and GTP analogs were then obtained by hydrolysis ofthe ketal. Yields and quantum efficiencies for the disappearance of the pHP phosphate esters are givenin Table 69.16.

pHP caged ATP was recently used as a probe in the study of Na,+ K+-ATPase, an enzyme involved inthe intracellular transport of sodium and potassium ions.59 Membrane samples possessing the ion-channel proteins were bathed in caged ATP which was activated by UV laser flash photolysis. Na,+ K+

channel transport was observed as a result of the activation of the enzyme by the released ATP. Directspectroscopic evidence of the release of ATP was obtained by time-resolved Fourier transform infrared(FTIR) spectroscopy (Figure 69.2). The changes in the characteristic absorptions of the prominent func-tional groups of the reactant and product include the disappearance of the γ-PO2

– ester band of the cagedATP at 1270 cm–1 and the appearance of the free γ-PO3

–2 band of the released ATP at 1129 cm–1.

Benzoin

In 1994, Pirrung and Shuey60 reported the protection of phosphates using dimethoxybenzoin. Resolved(R)3′,5′-dimethoxybenzoin (optically active) was converted to the phosphoramidite by treatment withdiisopropylaminocyanoethoxychlorophosphine. Subsequent reaction with the appropriate primary orsecondary alcohol followed by oxidation led to the series of phosphate esters 58a-e in moderate to highyields:

TABLE 69.16 Data for pHP Caged Phosphates

Caged Phosphatea

Protection (% Yield)

Deprotection(% Yield) Φdis

57a 87 Not determined 0.7757b 96 Quant 0.3835 42 Quant 0.37 (0.30)b

57c 20 Not determined Not determined

a Results shown for 57c are from Du et al.;58 derivative 57c wasphotolyzed at 308 nm.

b The value in parentheses for 35 is the quantum efficiency for theappearance of ATP.

OOP

HO

O

OR2

OR1 hν, 300 nm -O-P

O

OR2

OR1 +

57a R1 = R2 = Et57b R1 = R2 = NH4

+

57c R1 = GDP, R2 = NH4+

35 R1 = ADP, R2 = NH4+

10% CH3CN/TRIS buffer, pH 7.334



(69.29)

Photolysis of 58a-e shown in Eq. (69.29) led to release of the phosphate derivative along with thebenzofuran byproduct 11. The overall sequence from the phosphoramidites to phosphate esters 58a–ewas accomplished in moderate yields, and the unprotected phosphates were obtained in good yields uponphotolysis, as seen from Table 69.17.

FIGURE 69.2 Time-resolved FTIR difference spectrum for the photolysis of pHP caged ATP. The absorbance wasmeasured 10 ms to 11 s after the photolysis flash and subtracted from the absorbance prior to photolysis. (We thankProfessors Klaus Fendler and Andreas Barth for the TR-FTIR results with pHP ATP.)

TABLE 69.17 Yields for the Photolysis of DMB Phosphoramidites

RProtection (% Yield)

Deprotection (% Yield)

58a 45 8558b 82 8658ca 58 8558d 60 8358e 55 87

a The (R) enantiomer.

-0.010

-0.005

0.000

0.005

0.010

0.015

∆Abs

.

90010001100120013001400150016001700180019002000Wavenumber / 1/cm

CO2-of product

ATP PO32-

Caged ATP PO2-

PhO

O

OMe

MeO

POO

OR

hν, 350 nm-O

PO

O

ORCN +

58

11

R = CO2Me

NHBoc

CO2Me

NHBoc O

O

MeMe

OTrO T O

O

Me Me

58a 58b 58c 58d 58e

CH2CH2CN



69.5 Sulfates and Other Acids

Sulfonic acids have largely remained unexplored in terms of a functional group that is released from aphotoremovable protecting group. One recent example has been reported by Bendig et al.,47 in whichmethanesulfonic acid was protected as the corresponding methoxycoumarin derivative. (7-Methoxycou-marin-4-yl)methylmethanesulfonate 59 was synthesized from reaction of methanesulfonic acid with 4-(diazomethyl)-7-methoxycoumarin in refluxing chloroform. Photolysis at 333 nm resulted in the releaseof methanesulfonic acid, as shown in Eq. (69.30).

(69.30)

While the protection yield for 59 was low (26%), the quantum efficiency of 0.081 is reasonable whencompared with other leaving groups that were reported in this study. The chemical yield of the depro-tection was not provided; however, the photoproduct 60 was recovered in ≥95% yield, suggesting a highyield of the released sulfonic acid.

69.6 Alcohols, Thiols, and N-Oxides

o-Nitrobenzyl

Among the most common photoprotecting groups used for alcohols are the o-nitrobenzyl derivatives.Recently, Iwamura61 reported the synthesis and photochemistry of caged carbohydrates protected withan o-nitrobenzyl group at one or more positions within the molecule. Equation (69.31) illustrates atypical example in which photolysis of the caged glucopyranoside 61 at 350 nm in methanol led to therelease of the free methylglycoside 62 in 60% yield, along with the nitroso byproduct 3.

(69.31)

Derivative 61 was synthesized in 71% yield by reductive bond cleavage of the corresponding 4,6-O-o-nitrobenzylidene acetal of methyl 3,4-acetyl-β-glucoside with triethylsilane and boron trifluoride ether-ate, followed by deacetylation of the 3,4-diacetates with sodium methoxide in methanol.

o-Nitrobenzyl chemistry was also extended to the release of thiols as demonstrated by Smith et al.,62

who demonstrated that a cysteine congener was protected as the corresponding thioether 63 in 89%yield. Photocleavage occurred at 366 nm to give the liberated Boc-protected thiol 64 in 44% yield in thepresence of semicarbazide hydrochloride, a carbonyl scavenger (Eq. (69.32)).

O

O

OH3CO

SO

O CH3 hν, 333 nm

7:3 HEPES buffer/MeOHpH 7.2

HEPES = N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)

59

O

OH

OH3CO

SO

OCH3HO+

60

O

HOHO

OMe

OHO

NO2

hν, 350 nm O

HOHO

OMe

OHHO

+

61 62

3MeOH



(69.32)

Finally, the addition of ascorbic acid as an antioxidant to the reaction mixture gave a quantitative yieldof 64. No thiol was recovered when the photolysis was carried out in the absence of ascorbic acid, despiteexperimental evidence of the disappearance of 63.

While the o-nitrobenzyl system is well accepted as a photoremovable protecting group, it neverthelesssuffers the limitation of toxicity to the biological entity and a highly reactive nitroso functional groupformed as the byproduct. As was seen in the case of the cysteine cogener 63, the photodeprotection mayrequire a scavenging or reducing agent if thiols are to be generated in such a strategy.

In the late 1990s, Pfleiderer and co-workers63 developed a new, β-substituted variant of the o-nitroben-zyl chromophore, 2-(o-nitrophenyl)ethoxycarbonate. The 5′-O-2-(o-nitrophenyl)ethoxycarbonyl thy-midines were obtained from 2-(o-nitrophenyl)ethanol carbonates by reaction with diphosgene underbasic conditions, followed by treatment with thymidine in anhydrous methylene chloride at reducedtemperature. The synthetic yields ranged from 41 to 81%.

Photolysis of a 0.1-mM solution of the photoprotected thymidine 65 in a 1:1 methanol/water mixtureat 365 nm resulted in the release of thymidine (66), carbon dioxide, and the photolabile o-nitrostyrenederivative 67 (Eq. (69.33)).The photorelease is believed to occur through a β-elimination mechanism64,65

from the aci-nitro intermediate 67′′′′ shown in Scheme 10.

(69.33)

SCHEME 10 β-Elimination mechanism proposed for 2-(o-nitrophenyl)ethoxycarbonyl thymidines.

H2N

ONHBoc

S NO2

hν, 366 nm

1:1 CH3CN/0.05 M PBSH2N

ONHBoc

SH

+

63

64

3

pH 6

NO2

RO OR'

O hν N

RO OR'

O

OH

O

67'

NO2

R

+ R'OH

67

-CO2

RO

NO2

O

OH

HN

N

O

OO

O

hν, 365 nm

1:1 CH3OH/H2O O

OH

HN

N

O

O

HO +

65 66

R1

R2

R3

R

NO2

R1

R2

R3

67

-CO2

65a R = CH3, R1, R2,R3 = H65b R = o-nitrophenyl, R1, R2,R3 = H65c R = H, R1 = I, R2,R3 = H65d R = H, R1 = Br, R2,R3 = H

65e R = H, R1 = Cl, R2,R3 = H65f R = H, R1, R3 = Cl, R2 = H65g R = H, R1 = F, R2,R3 = H65h R = R1 = H, R2,R3 = OCH3



Some advantages of this nitrobenzyl variant include the lack of the nitroso byproduct and release ratesthat are relatively fast compared with the parent o-nitrobenzyloxycarbonyl derivative. For example, therelease of thymidine from 65b is reported to be twice as fast as that for the α-substituted derivative;however, the nitrostyrenes (i.e., 67) are photolabile and thus can compete with the starting material forincident light.

The quantum efficiencies for the release of thymidine varied depending on the substituents on 65.Substitutions at the benzylic carbon appeared to give the highest release efficiencies, whereas the chloroderivatives gave slightly better conversions, as seen in Table 69.18.

The high quantum efficiency and respectable protection and deprotection yields obtained for themethyl-substituted 2-(o-nitrophenyl)ethoxycarbonyl photoprotecting group prompted Pirrung et al.66 touse this protecting group for the solid-phase synthesis of oligodeoxynucleotides.

Thiopixyl and Coumaryl

Coleman and Boyd67 introduced the 9-phenylthioxanthyl or S-pixyl photoprotecting group for the fourprincipal nucleosides, thymidine and three other benzoyl protected nucleoside bases. The chromophorewas synthesized in three steps, starting with thioxanthone, through Grignard addition of phenylmagne-sium bromide, followed by dehydration with trimethylsilyl chloride and dimethylsulfoxide to give the 9-chloro-9-phenylthioxanthene. Treatment with the corresponding alcohol in a dry solution of pyridineand dimethylaminopyridine (DMAP) afforded the photoremovable protected hydroxy derivatives 68 ingood yields (Table 69.19). Irradiation of 68 in aqueous acetonitrile resulted in the release of the nucleosideor alcohol along with 69 (Eq. (69.34)).

(69.34)

TABLE 69.18 Yields for 2-(o-Nitrophenyl)-ethoxycarbonyl Thymidine Derivatives

DerivativeProtection (% Yield) Φ

Deprotectiona (% Yield)

65a 71 0.35 7665b 70 0.20 nd65c 68 0.10 nd65d 73 0.076 nd65e 80 0.070 8065f 81 0.072 nd65g 70 0.037 nd65h 41 0.0013 nd

a Based on the recovery of thymidine; Buhler, S., Giegrich, H.,and Pfleiderer, W., Nucleosides & Nucleotides, 18, 1281-1283,1999.

nd = not determined

S

Ph OR

S

Ph OH

ROHhν, 300 nm

CH3CN(aq)

+

68 69

R =CH2

O

OH

CH2

X

X = Thymine, N-Bz-Adenine, N-Bz-Cytosine, N-iBu-Guanine

CH2



Mechanistically, this photorelease reaction occurs via photosolvolysis of the aryl ether carbon–oxygenbond. The resulting resonance stabilized S-pixyl carbocation reacts with water to form 69. Controlexperiments showed that the photoprotected alcohols are stable under thermal conditions; that is, reflux-ing in aqueous acetonitrile resulted in no detectable decomposition. Each of the caged products wasobtained as a solid, a convenience for laboratory purification and manipulation. The best deprotectionyields were obtained with solvent mixtures containing the maximum concentration of water permissible,limited only by the solubility of the protected alcohol. Concentrations of water ranged from 40 to 60%,depending on the type of alcohol used. In most cases, excellent deprotection yields were obtained.

A factor that limits the versatility of the S-pixyl group is the wavelength range required for photolysis.The range of excitation wavelengths from 200 to 300 nm overlaps with a number of functional groupsthat could compete with the incident light. For example, the pyrimidine base cytosine has substantialabsorptivity at 300 nm. As a result, extended irradiation times were required to effect deprotection ofthe N-Bz-cytidine analog and a lower yield was obtained, as seen from Table 69.19.

Coumaryl photoprotecting groups have also been used for the protection of alcohols. A recent studyby Lin and Lawrence68 described the synthesis and photorelease of caged diols using a coumaryl acetalderivative 70 (Eq. (69.35)).

(69.35)

Acetal 70 was prepared in a two-step sequence starting with oxidation of 6-bromo-7-hydroxy-4-hydroxymethylcoumarin with manganese dioxide, followed by addition of the corresponding diol in

TABLE 69.19 Synthesis and Deprotection Yields for Various Derivatives of 68

RProtectiona (% Yield)

Deprotectionb (% Yield)

CH2-benzyl 80 93CH2-(E)styryl 94 95Thymidine 79 97N-Bz-adenosine 86 96N-Bz-cytidine 82 75N-iBu-guanosine 92 89

a Based on isolated yields after column chromatog-raphy.

b Substrate concentrations were approximately 0.1 mM; yields were determined by HPLC.

O O

OOH

RH

hν, 348 nm

1:1 CH3OH/buffer

pH 7.470

O O

O H

+R

OHOH

71

R = OPh SPhO

Me

Me

Me

N PhMe

70a 70b 70c 70d

Br

HO

Br

HO

Φ = 0.0041 Φ = 0.0053 Φ = 0.0051 Φ = 0.0278



anhydrous toluene. Protection yields ranged from 94% for 70a to 15% for 70d. Photolysis of 70 at 348nm in a methanol/aqueous buffer solution afforded the free diol accompanied by the aldehyde photo-product 71. The specific deprotection yields were not provided but in all cases were reported to exceed75%.

The photorelease mechanism is not well understood but is speculated to proceed through an intramo-lecular ion pair69 generated from photoheterolysis of the carbon oxygen bond, as shown in Scheme 11.Attack of a water molecule at the electrophilic carbon generates a hemiacetal that eliminates the alkoxygroup to afford the diol and byproduct 71.

This particular coumaryl variant offers a unique advantage in essentially being able to protect bothhydroxyl groups of a diol using only one equivalent of the protecting group. In addition, the derivativesin this study were shown to be stable to hydrolysis in aqueous solvents at neutral or basic pH. For example,incubation of compounds 70a–d in a 1:1 methanol/buffer solution (pH 7.4) resulted in no detectabledegradation after a period of two weeks. Despite these advantages, the range of applications of the coumaringroup is limited thus far to 1,2- and 1,4-diols (1,3-diols were found to be inert to photolysis). Like the o-nitrobenzyl group, the coumaryl group also has the disadvantage of producing a highly absorbing photo-product. In addition, further studies need to be carried out in order to elucidate the mechanism of thephotocleavage.

Benzoin

In 1995, Pirrung and Bradley70 reported the use of dimethoxybenzoin (DMB) carbonate to protect variousalcohols, including the 5′-hydroxyl group of nucleosides. The DMB carbonate was synthesized in threesteps, starting with methylation of carbonyldiimidazole with methyl triflate followed by addition of 2-(3,5-dimethoxyphenyl)-2-hydroxy-1-phenylethanone to form a relatively stable activated acylating agent.Treatment with an alcohol under basic conditions in nitromethane furnished the protected alcohol 72in yields that ranged from 42 to 95%.

Irradiation of 72 at 350 nm resulted in release of the alcohol and formation of dimethoxybenzofuran11 (Eq. (69.36)). A wide variety of alcohols, including a thiol, were explored in this protection/depro-tection scheme. Excellent deprotection yields were obtained, as high as 98% for the protected thymidinederivative. The mechanism as discussed earlier is postulated to proceed through intramolecular cycliza-tion followed by demotion to form a zwitterionic intermediate. Expulsion of the alcohol occurs eitherconcomitantly with the release of carbon dioxide or by a stepwise decarboxylation of the initially releasedcarbonate 73.

SCHEME 11 Mechanism proposed for the photorelease of caged diols from 70.

hν

O O

OO

R1 *

O O

OO

R

O O

O

O R

H2O

O O

O

HO R

HO

70

71 +OH

HO R

Br

HO

Br

HO

Br

HO

Br

HO



(69.36)

Attractive features of the DMB photoprotecting group are that the benzofuran photoproduct 11 isinert and exhibits strong fluorescence at 396 nm, allowing the deprotection conversions to be monitoredspectroscopically. In the same study, the DMB group was successfully used to synthesize two trinucleotidesbearing adjacent thymidine residues, demonstrating its potential in solid-phase DNA synthesis. Com-pared with nitrobenzyl photoprotecting groups, the rate of release of substrate is much faster for theDMB group70 (krelease ~ 108–109 s–1). The main disadvantages of the DMB group are the competition forincident light by the photoproducts and poor solubility in aqueous media.

Other

The anthraquinon-2-ylmethoxycarbonyl (Aqmoc) photoprotecting group is a relatively recent additionto the chromophores used as photoremovable groups. In a recent application for alcohols, Furuta et al.71

reported that the caged derivative could be synthesized in two steps from anthraquinonylmethanol bytreatment with 4-nitrophenylchloroformate and DMAP followed by coupling to the desired alcohol withDMAP to provide 74 in good yields (Table 69.20). Photolysis of 74 in 50% aqueous tetrahydrofuran(THF) at 350 nm resulted in the release of the alcohol (Eq. (69.37)).

TABLE 69.20 Yields and Quantum Efficienciesa for Aqmoc Derivatives

Aqmoc DerivativeProtection (% Yield) Φ

Deprotection (% Yield)

74a 76 0.1 6874b 86 Not determined 91b

a For the disappearance of starting material.b Determined by HPLC.

Ph

O

OMeO

OMe

RO O

ROHhν, 350 nm

72

(36)

73

RO O

O

THF -CO2

OH

HO

C8H17

PhCH2OH PhCH2SH thymidineN-Bz-adenosine N-iBu-guanosine

88% 95%

96% 94%

98%97% 96%

-11



(69.37)

The photoproducts from the anthraquinone moiety were not fully characterized; however, in the case of74a, an anthraquinon-2-ylmethanol tetrahydrofuranyl ether was isolated, probably the result of hydrogenatom abstraction from the solvent. Also, a small amount of bis(1,2,3,4-di-O-isopropylidene-D-galacto-pyranosyl) carbonate was obtained after photolysis of 74a, likely the result of attack of the released alcoholon the starting material.

Little is known about the photorelease mechanism of the Aqmoc group. Stern–Volmer analysis showedthat the triplet excited state is the reactive excited state that leads to release of the alcohol. The rateconstant determined for 74a (4.6 × 106 s–1) is consistent with rapid release of the carbonate followed bya slower, rate-limiting loss of carbon dioxide to give the free alcohol. Additional studies are needed toconfirm this mechanism.

Recently, it was shown that nitro-substituted aryl carbamates could be used as photolabile protectinggroups for alcohols.72 N-Methyl-N-(o-nitrophenyl)carbamate 75 (Eq. (69.38)) was synthesized in twosteps, beginning with acylation of N-methyl-2-nitroaniline with phosgene followed by nucleophilicaddition of the alcohol, either as an alkoxide or in the presence of DMAP and triethylamine. An alternativesynthetic route involved the generation of the corresponding chloroformate from the alcohol and phos-gene followed by nucleophilic addition of the nitroaniline. The carbamate derivatives 75a-d were syn-thesized in yields ranging from 58 to 94%.

Photolysis of carbamate 75 at various wavelengths led to deprotection of the alcohol, which wasaccompanied by formation of nitrosoaniline 76 as a byproduct. Deprotection yields for all derivativeswere not provided; however, the reported yields for 75c and 75d were 100% and 91%, respectively. Suchhigh yields are a definite advantage in terms of both synthetic and biological applications. Anotherattractive feature is the solubility in ethanol and water, solvents suitable for biological studies. Despitethese advantages, two main limitations are worthy of note. First, the carbamate cages are susceptible tohydrolysis, particularly in basic media,73 thus limiting their use to aqueous solvents with relatively neutralpH. Second, longer irradiation wavelengths were found to result in decreased deprotection yields; forexample, when 75c was irradiated at 312 nm, a quantitative deprotection yield was obtained. The yielddropped to 76% when the photolysis was carried out at 365 nm.

(69.38)

O

O

O OR

Ohν, 350 nm

THF/H2O+ photoproducts

74

R =O

O

O

OO

CH2

N

NN

N

NH2

O

OHOH

CH2

74a 74b

ROH

N OR

O

NO2

hν, 254-365 nm

EtOH/H2O

NH

NO

+

R = CH3 CH2Ph N O N CO2Me

O

PhH

ROH

I

75 76

75a 75b75c 75d



Silyl photoprotecting groups have recently been developed for primary and secondary alcohols.74 Thesilyl cage, (2-hydroxy-3-naphthylvinyl)-diisopropylsilyl ether 77, was synthesized in nine steps startingwith the commercially available naphthalene-2,3-diol. Irradiation at 350 nm in methanol triggered therelease of the alcohol, accompanied by the formation of a cyclic silyl byproduct 78 (Eq. (69.39)). Thisbyproduct is likely formed via intramolecular attack of the naphthol oxygen at silicon following atrans,cis-isomerization of the starting material. Synthetic and photochemical yields are listed inTable 69.21.

(69.39)

Byproduct 78 exhibits its strongest absorption in the region below 310 nm and, therefore, does notsignificantly compete with 77 for incident light. The yields from Table 69.21 are sufficiently high to enablethe practical use of the silyl photoprotecting group in synthetic applications; however, the silyl cages lackthe water solubility necessary for application in aqueous media. Like triisopropylsilyl ethers, the cagesare also susceptible to cleavage in the presence of acidic media or solutions containing fluoride such as1-N HCl or TBAF.75

The importance of nitric oxide (NO) in bioregulatory processes and other physiological functionsprompted the development of photoprotecting groups specifically designed for its release. A most recentexample is the synthesis and photochemistry of a series of naphthylmethyl and naphthylallyl diazenium-diolates.76 These derivatives, represented by 79 in Eq. (69.40), were prepared from reaction of 1-(N,N-diethylamino)-diazen-1-ium-1,3-diolate (81) with the corresponding alkyl bromide. Photolysis produceda mixture of products that resulted from two different reaction pathways. Path a is a nonproductivepathway that leads to the formation of nitrosamine 80 and oxime byproducts. Path b leads to diazeni-umdiolate 81, which collapses to give free NO, along with diethylamine and other photoproducts. Theextent to which the reaction follows one pathway over another is dependent on the substituents presenton the naphthyl ring. As Table 69.22 shows, the best deprotection yields were obtained with a methoxygroup at the 5 and 8 positions of the ring.

TABLE 69.21 Caged Silyl Derivatives and Their Corresponding Deprotection Yields in Methanol

Silyl EtherProtection(% Yield)

Deprotection(% Yield)

77a 82 94a

77b 80 8977c 77 9077d 79 9277e 81 86a

77f 85 90

a Based on the yield of the byproduct 78.

SiOR

OH

hν, 350 nm

CH3OHO

Si+ ROH

77 78

R = t-BuO

OTBS

TO

DMT-O TH3C

CH3CH3

i-Pr Me

77a 77b 77c 77d 77e 77f



(69.40)

The likely mechanism proceeds through photosolvolysis of the carbon–oxygen bond, resulting in aresonance-stabilized carbocation. Subsequent release of NO occurs from the diazeniumdiolate 81. Acidicconditions that protonated the amine greatly enhanced the rate of NO release. Derivative 79e undergoesclean deprotection and exhibits an excellent quantum efficiency, making it the most attractive of the NOcages studied thus far. Some additional advantages are its absorption beyond 300 nm (λmax = 336 nm)and its stability in acidic and basic solutions at room temperature for up to 24 hr. Unfortunately, it suffersfrom a low protection yield of only 5%, and its solubility is limited to 20 µM in 95% aqueous acetonitrile.Despite these shortcomings, its development may pave the way for similar methoxy-substituted naphth-ylallyl derivatives with increased solubility in aqueous media and higher protection yields.

A unique photoprotecting group for alcohols and thiols was reported in the mid 1990s.77 Benzoylben-zoate ester 82 (Eq. (69.41)) was synthesized in one step from DCC coupling of the corresponding alcoholor thiol to 2-benzoylbenzoic acid. Photolysis at ~300 nm in the presence of cyclohexylamine, an electrondonor, afforded 3-phenylphthalide 84 along with the free alcohol or thiol.

(69.41)

TABLE 69.22 NO Cages and Their Corresponding Deprotection Yields and Quantum Efficiencies

NO cageProtection (% Yield) Φ

Deprotectiona,b (% Yield)

79a 90 0.007 179b 25 Not determined 179c 30 0.12 2579d 36 0.12 4079e 5 0.66 95

a Based on the disappearance of starting material (using HPLC)and the amount of NO measured; thermal decomposition of81 was found to produce 1.5 equivalents of NO.

b For 79a–c, a wavelength of 300 nm was used; for 79d,e, thewavelength was 350 nm.

ON

NO

NEt2

X

X Y

Et2N NO

Et2NH

n

hν

79

CH3CN(aq)

otherphotoproducts

Et2NN

NO

O

Ar O Nn+ oximes

+ Ar CH2

n

n = 0,1

+

+

80

81

a

b

2 NO

79a n = 0, X = Y = H79b n = 0, X = H, Y = Me79c n = 1, X = Y = H79d n = 0, X = OMe, Y = H79e n = 1, X = OMe, Y = H

Ph

O CO2Rhν

cyclohexylamine

1:1 PhH:CH3CN

Ph

OH CO2R O

Ph

O

+ ROH

82 83 84

or RSH

R = n-C12H25-, c-C12H23-, cholesteryl-, geranyl-, 2',3'-isopropylidene uridinyl-, n-C12H25 (thioester)



The mechanism outlined in Scheme 1278 involves electron transfer from the amine to the ketone inthe excited state followed by intermolecular proton transfer to generate radical pair 85–86; a secondelectron transfer and proton exchange lead to the reduced alcohol 83, which lactonizes to form 84concurrently with release of the alcohol. In general, the benzoylbenzoate photoprotecting group workedwell for the particular substrates studied. Synthesis yields for the benzoylbenzoate cages were respectable,and the deprotection occurred in most cases with good recoveries of the alcohol (Table 69.23). Problemswere encountered in the photolysis of the caged thiol that led to the formation of side products and thusa lower overall deprotection yield.

While the benzoylbenzoate cages exhibit good deprotection yields for alcohols, their application islimited to organic solvents. The presence of an electron donor (i.e., aliphatic amine) is also required, anecessity that may complicate the reaction mixture in the presence of other sensitive functional groups.Finally, the release process must be inherently a slow one due to the ground state lactonization processinvolved.

69.7 Phenols and Other Weak Acids

o-Nitrobenzyl

The photolabile o-nitrobenzyl derivative was utilized to protect the phenolic OH group of serotonin.79

The serotonin type-3 receptor is the only ligand-gated ion channel in the 5-HT receptor family.80,81 Theprotection of the phenolic hydroxy group of serotonin required four steps, as shown in Scheme 13. Thesubstrate was released upon excitation with 337-nm laser pulses. The signal decay from pulsed laser

SCHEME 12 Proposed photoreduction mechanism of benzoylbenzoate esters.78

TABLE 69.23 Data for Benzoylbenzoate Esters

RProtection(% Yield)

Deprotectiona

(% Yield)

n-C12H25- 76 95c-C12H23- 50 85Cholesteryl- 67 100Geranyl- 63 90b

2′,3′-isopropylidene uridinyl 79 90c

n-C12H25 (thioester) 76 60

a Yield of the recovered alcohol (thiol) was determined with NMR.b sec-Butylamine was used as the electron donor; the solvent was

1:1 benzene/isopropanol.c sec-Butylamine was the electron donor; photolysis was carried

out with a uranium filter.

Ph Ar

Ohν

Ph Ar

ONH2

Ph Ar

ONH2

*3

Ph Ar

OHNH2

Ph Ar

OHNH2

Ph Ar

OHNH

+

85

ISC

86

Ar = o-benzoate ester

83



studies gave a time constant of 16 µs, and the quantum yield was determined to be 0.03. The rate ofdecay of the intermediate was observed to be pH dependent. The caged serotonin showed good solubilityin buffered aqueous media (in excess of 2 mM); however, the authors suggested that the caged compoundwas subject to hydrolysis in the dark on standing.

This photoremovable protecting group has been employed as a photocleavable linker to reagents boundto Au surfaces.82 4-Hydroxy-stilbene was linked to 6-bromohexyl-3-nitro-4-bromomethylbenzoate in51% yield, then thiolated by trimethylsilylthioxy dehalogenation in THF, followed by desilylation in situ.The self-assembled monolayers of long-chain alkyl thiolate on bulk polycrystalline gold were constructed.Upon irradiation at 350 nm, the Z,E-photoisomerization attained a photostationary state within 25 min,while the dissociation took about 60 min; however, sensitization with 1,4-dibromonaphthalene (ET =58.1 kcal/mol) produced a cleaner photoisomerization. The unidirectional isomerization, from cis totrans, by both direct irradiation and sensitization was followed by the release of a bound chain from themetal surface.

SCHEME 13 Protection and photochemical deprotection of serotonin.

SCHEME 14

NO2 COOHO

NH

NH2hν, 337 nm

NO COOH

O + Serotonin-H+

O-CNB-5HT

HO

NH

NH2NO2 COOCH3

O

NH

NH

N-Boc-OMeCNB-5HT

BOC

Serotonin

1) di-t-BOC anhydride (97%)

2) K+ t-butylate

1) 3.5% K2CO3MeOH/H2O 1:1 (85%)

2) FCH2COOHCH2Cl2/Acetone/H2O10:10:1 (90%)

NO2COOCH3

Br

Methyl-2-bromo-2-(2-nitrophenyl) acetate

(15%)

O2N

O

O O(CH2)6SH

O2N

O

O O(CH2)6SH

hν, 350 nm

hν, 350 nm

hν, 350 nmHO

ON

O

O O(CH2)6SH

HO ON

O

O O(CH2)6SH

+

+

hν, 350 nm



Benzoin

The ubiquinol oxidizing enzyme is a redox active enzyme that requires a fast two-electron reduction ofubiquinone. 3′,5′-Dimethoxybenzoin (DMB) caged ubiquinol83 was synthesized to study the detailedenzymatic mechanism of the fast electron-transfer process in redox active enzymes. The monosilylatedubiquinol was coupled to the protecting group to form the o-nitrophenylcarbonate ester of 3′,5′-dimethoxyphenyl(phenyldithiane) in 55% yield. Upon photolysis, DMB caged ubiquinol generatedubiquinone with a rate greater than 106 s–1(Eq. (69.42)).

(69.42)

Pirrung and Bradley70 also applied DMB (3′,5′-dimethoxybenzoin) to protect the phenolic group, dem-onstrating that 4-methoxyphenol was released upon irradiation at 350 nm in 90% yield (Eq. (69.43)).

(69.43)

69.8 Amines

o-Nitrobenzyl

There are few variations for effective amine photoremovable protecting groups. The o-nitrobenzyl groupremains the most popular group and among the many examples the studies by Cameron and Frechetare noteworthy.84 In general, o-nitrobenzylcarbamates of aliphatic amines upon photolysis release theamine in good yield. For example, cyclohexylamine is released from its o-nitrobenzyl carbamate 87 asthe corresponding free carbamate upon irradiation in THF at 254 nm (Eq. (69.44)).

(69.44)

Subsequent loss of carbon dioxide frees the amine. Quantum efficiencies varied depending on thesubstituents present at the ortho and benzylic positions (Table 69.24). The best efficiencies were obtainedwith two o-nitro groups on the aryl ring, likely increasing the probability for the hydrogen atom abstrac-tion by one of the nitro groups.

O

O

O

O

OH

H3CO

H3CO

CH3

OCH3

OCH3

O

O

H3CO

H3CO

CH3

+

O

OCH3

H3CO

hn, 355 nm

-CO2

11

O

O

H3CO

OCH3

H3COOH

H3CO+ O

Ph

OCH3

H3COhn, 350 nm

THF

90%11

CO O

-CO2

R2

R1

NO2

O NH

O hν, 254 nm

THF

R2

R1

NO

OH2N

+ CO2 +

87



A problem that is not entirely avoidable is the formation of imine byproducts via reaction of thereleased amine with the aldehydic group in the photoproduct. This occurrence could be suppressed withalkyl or aryl substitution at the benzylic position, leading to the formation of a less reactive ketone incomparison with the nitroso aldehyde formed with no substitution at the benzylic position. Iminebyproduct formation is also less likely to occur in relatively nonpolar solvent systems, such as THF, whichultimately limits the application of the o-nitrobenzyl carbamate photoprotecting group to nonaqueoussystems in this regard.

Benzoin Derivatives

In the mid-1990s, Pirrung and Huang85 extended the use of the benzoin photoprotecting group to therelease of amines by synthesizing m,m′-dimethoxybenzoin (DMB) carbamates. The DMB derivativeswere synthesized by coupling the corresponding amine with benzoin carbonyl chloride that had beenelaborated by reaction of carbonyldiimidazole with the methyl triflate of 88 followed by nucleophilicaddition of DMB. Irradiation in benzene or THF at 350 nm produced the free amine, carbon dioxide,and the benzofuran byproduct (Eq. (69.45)).

(69.45)

Five different amines were protected and recovered in moderate to good yields as shown in Table 69.25.The DMB carbamates appeared to work well for a variety of amines in the presence of other functionalgroups, such as alcohols or esters; however, the reaction is limited to secondary amines, as primary amineswere found to undergo intramolecular cyclization leading to byproducts that are inert to photolysis.

Arylsulfonamides

Corrie and Papageorgiou86 have reported the synthesis and photochemistry of various methoxy-substituted arylsulfonamides. Similar derivatives had been previously found to undergo single electrontransfer reactions in the excited state, leading to cleavage of the sulfur–nitrogen bond.87,88 It wasreasoned that such a process could be used for the rapid release of neurologically active amines.

The arylsulfonamide derivatives were synthesized in several steps, starting from 1,5-dimethoxynaph-thalene. Photolysis of 89 in phosphate buffer (pH 7.0) in the presence of ascorbate resulted in release of

TABLE 69.24 Protection Yieldsa and Quantum Efficiencies for Various Substituted o-Nitrobenzyl Carbamates

R1 R2

Protection(% Yield) Φ

H H 71 0.13NO2 H 78 0.62H Me 71 0.11NO2 Me 71 0.35H o-Nitrophenyl 79 0.26NO2 2,6-Dinitrophenyl 52 0.28

a Deprotection yields were not provided.

O

O

N

O

R2 R1

hν, 350 nm

benzeneMeO

OMe

R1NHR2 + + CO2

88

11



the free amine in low to moderate yields, accompanied by the reduced arylsulfonyl byproduct 90 (Eq.(69.46) and Table 69.26).

(69.46)

TABLE 69.25 Protection and Deprotection Yields for DMB Carbamates

Amine Protection (% Yield) Deprotection (% Yield)

85 89a

76 79

90 56

90 73

88 97a

a The amine was recovered as the corresponding hydrochloride salt.

TABLE 69.26 Synthesis and Photochemistry Yields of Arylsulfonamides

R1 R2

Protection(% Yield)

Deprotectiona

(% Yield)

(CH2)3OPO3–2 CO2

– 71 35(CH2)3OPO3

–2 CH2CO2– 61 22b

Me CO2Me 59 53

a Yields were determined using quantitative amino acid analysis; irradiationswere only carried out to approximately 50% conversion of the startingmaterial in the presence of 10-mM ascorbate.

b Photolyzed in buffer solution only.

NH

NH

NH

NH

CO2t-Bu

NH

HO Ph

OMe

OR1

SO2NH R2

hν

1:2 buffer/MeOH

OMe

OR1

SO2-

89

H2N R2+

ascorbate

90



The mechanism is thought to involve an excited state intramolecular single eletron trnasfer from theelectron-rich naphthalene to the sulfonamide group (Scheme 15); release of the amine then occurs viaassistance from the neighboring oxygen, leading to a radical cation that is subsequently reduced to 90by ascorbate.

It was speculated that, in the case of the glycine and β-alanine substrates, a competitive electron transferwas occurring from the carboxylate group to the radical cation of 90. Such a process would yield acarboxyl radical that would undergo subsequent decarboxylation, leading to a mixture of side productsand thus a lower yield of the free amino acid. Spectroscopic evidence using LFP combined with FTIRspectroscopy supported this hypothesis. Low yields were still encountered even in the presence ofincreased amounts of ascorbate, suggesting that the electron transfer was taking place within a solventcage.

Despite this shortcoming, the arylsulfonamide group may still hold promise as a photoremovableprotecting group for amines lacking a carboxylate moiety. Further studies would need to be carried outto fully establish its capabilities in this regard.

69.9 Conclusion

A wide variety of photoremovable protecting groups have been added to the veteran o-nitrobenzyl series.Each new group has been developed to address the shortcomings of the o-nitrobenzyl group or to addfeatures such as faster rates for release, extension of the absorption range into the near UV-visible region,improved solubility, higher efficiencies, improved conversions and yields, and more benign photoprod-ucts from the protecting group. Extensions and applications of this chemistry to two photon excitationprocesses, to traceless reagents in combinatorial chemistry and photolithography, as orthogonal reagentsin synthesis, and to time-resolved spectroscopic techniques wil make even more demands on the design,synthesis, and development of new photoremovable protecting groups.

Even now, however, no single photoremovable protecting group fulfills all nine criteria Sheehan andLester had suggested (outlined at the beginning of this chapter). Nevertheless, important progress hasbeen achieved as evidenced by the growing number of applications reported for many of these photore-movable groups, especially in biological studies. Applications in synthesis, combinatorial chemistry, microarrays, and photolithography are also forthcoming. These fields have benefited and will continue to drawthe interest of the science community as improvements of existing systems and discovery of new photo-active protecting groups are developed. This field of photoremovable protecting groups is still in itsinfancy.

Acknowledgments

We acknowledge the support of the Department of Energy, University of Kansas and the National ScienceFoundation.

SCHEME 15 Proposed mechanism for amino acid release from arylsulfonamides.

OMe

OR1

S

89 90hν

SET

OMe

OR1

S

ONHCH2R2

O

O

O

-R2CH2NH2

H2Oback electron transfer



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