[Methods in Enzymology] Affinity labeling Volume 46 || [25] Arylazido nucleotide analogs in a...

[25] PHOTOAFFIN1TY ARYLAZlDO NUCLEOT1DE ANALOGS 259

[25] Arylazido Nucleotide Analogs 1 in a Photoaffinity Approach to Receptor Site Labeling

By RICHARD JOHN GUILLORY and STELLA JYHLIH JENG

The ATP molecule contains three distinct structural groupings: the adenine base, the ribose sugar, and the triphosphate component. On the basis of the interaction of actomyosin with ATP analogs modified in these positions, a model of the enzyme substrate complex has been postu- lated, la,~ This model is based upon evidence suggesting that the amino grouping at the 6 position of the adenine base and the T-hydroxyl of the ribose ring are hydrogen bonded to the protein. The nucleotide binding is thought to involve an NH group at the trinitrobenzene sulfonate inter- acting site 3 as well as conjugation of triphosphate with asparagine via metal interaction 4 and binding of the terminal pyrophosphate to a sulf- hydryl and guanidinium group at the active site2 An important aspect of these actomyosin-nucleotide analog studies is the appreciation that the ribose ring can be drastically modified without affecting markedly the rate of myosin-catalyzed hydrolysis of the analog.

In view of the above facts it was decided to develop methods by which an active photosensitive adjunct could be attached to the ribose portion of nucleoside triphosphates. An additional restraint that we impose upon ourselves was that the photogenerated species be formed at wavelengths of light remote from regions of protein damaging radiation.

This chapter describes the successful chemical coupling of arylazido groupings to adenine nucleotides and the extension of this methodology

1 Abbreviations most commonly used: arylazido ATP or arylazido-fl-alanine ATP, 3'- O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}ATP; arylazido-f3-alanine NAD ÷, 3'-O-{3-N-(4-azido-2-nitrophenyl) amino]propionyl}NAD ÷ ; arylazido-fl-alanine, N- (4-azido-2-nitrophenyl)~-alanine; NMN, nicotinamide mononucleotide; CoQ1, coenzyme Q with one isoprenoid unit in the side chain; F1, coupling factor 1 ATPase.

1, y . Tonomura, S. Kubo, and K. Imamura, in "Molecular Biology of Muscular Contraction" (S. Ebashi, F. 0osawa, T. Sekine, and Y. Tonomura, eds). Igaku Shoin, Tokyo, 1965.

~Y. Tonomura, "Muscle Proteins, Muscle Contraction and Cation Transport," Chapter 9. Univ. Park Press, Baltimore, Maryland, 1973. S. Kubo, S. Tokura, and Y. Tonomura, J. Biol. Chem. 235, 2835 (1960).

4 N. Asuma, M. Ikegara, E. Otsuka, and Y. Tonomura, Biochim. Biophys. Acta 60, 104 (1962). T. Yamashita, Y. Soma, S. Kobayashi, T. Sekine, K. Titani, and K. Narita, J. Biochem. (Tokyo) 55, 576 (1964).

260 ENZYMES, ANTIBODIES, AND OTHER PROTEINS [25]

to the pyridine nucleotides. The synthesis of arylazido analogs of CoA and tetrodotoxin are mentioned.

The use of the adenine analogs in photodependent inhibition studies of myosin ATPase (EC 3.6.1.3) fragments and F1 (mitochondrial) ATPase as well as the interaction of the pyridine nucleotide analogs with alcohol dehydrogenase (EC 1.1.1.1) and complex I (NADH-CoQ reductase) are illustrated. The synthesis of radiolabeled analogs is described together with experiments illustrating the use of these labeled compounds in evaluating the covalent binding of the analogs to myosin fragments and F1 ATPase.

The general utility of our procedure for the synthesis of nucleotide analogs by substitution at the ribose hydroxyl group is indicated by the synthesis of spin-labeled analogs of the adenine and pyridine nucleotides having 3-earboxyl-2,3,5,5-tetramethylpyrroline-l-oxyl coupled to this position. The use of photoaffinity analogs with a variation in the distance of the azido group from the 3'-hydroxyl position on the nucleotide ribose is discussed as a tool in the mapping of nucleotide binding regions of proteins.

Because of their potential application to other systems, a number of reactions describing the reactivity of the 3'-hydroxyl of the nucleotide ribose are initially outlined together with attempted synthesis of diazo derivatives of ATP as potential precursors of active carbene analogs.

Arylazido Adenine Nucleotide Analogs

Carbodiimidazole-Catalyzed Chemical Synthesis o] Arylazido Adenine Nucleotide Analogs. The utilization of carbodiimidazole to facilitate the formation of activated carboxylic acids was elegantly applied by Gottikh and his group 6,7 to the synthesis of a wide spectrum of aminoacyl nucleo- sides, nucleotides, nucleoside di- and triphosphates, and tRNA derivatives. The formation of an imidazolide intermediate has been used in our work in ~he synthetic schemes leading to the esterification of adenosine and diphosphopyridine nucleotides.

It is known that amino acylation can take place at the 2'- or 3"- hydroxy position or at the 6-amino group of the adenine ring structure. The solvent systems, varying in polarity and solv~tion capacity, play an important role in orienting precisely where the reaction occurs. The

e B. P. Gottikh, A. A. Kraevskii, P. P. Peuygin, T. L. Tsilevich, Z. S. Belova, and L. N. Rudzite, Izv. Akad. Nauk SSSR, Set. KMm. (Engl.) 2453 (1967).

T B. P. Gottikh, A. A. Krayevsky, N. B. Tarussova, P. P. Purygin, and T. L. Tsilevich, Tetrahedron 26, 4419 (1970).

[ 2 5 ] PHOTOAFFINITY .kRYLA.ZIDO NUCLEOTIDE AN&LOGS 261

dimethyl formide: water (1: 5) solvent system we have adopted has been shown by Gottikh et al. 7 to be most advantageous in restricting the reaction center to the ribose hydroxyl groups rather than at the 6-amino group.

Chemical Reactivity of the 3 P-Hydroxyl Group of Adenosine Triphosphate

Condensation with p-N itrophenyl Ester o ] 3-C hloropropionic Acid

Our initial experiments utilized 2'-deoxyadenosine in attempting to acquire knowledge of the reactivity of the 3'-hydroxyl group for condensation reactions with the p-nitrophenyl ester of 3-chloropropionic acid with free imidazole as catalyst, s The choice of the 3-chloropropionic acid rested upon the feasibility of converting the halide first into an azide, then an amine, and finally a diazo derivative.

When this reaction was carried out according to the procedure outlined below, three products were detected by paper chromatography using ultraviolet (UV) light as a detecting agent. The infrared (IR) and nuclear magnetic resonance (NMR) spectrum of the major component indicated that during the reaction imidazole displaced the chlorine atom from the 3-chloropropionyl moiety. Structure (I) was tentatively assigned to this product. It was thus concluded that esterification of the T-OH of the ribose using imidazole catalysis can take place with good yields. On the other hand, our expectation that chlorine would be present in the product for subsequent transformation into the diazo compound was frustrated.

NI~

o

o H I

O~---C I / ~ S CH 2 CH2"----N ~

(I)

83. A. Secrist, J. R. Barris, and N. J. Leonard, Chem. Ind. (London) p. 1960 (1967).


Condensation with N-t-Butoxycarbonyl-a-alanylimidazole

An alternative procedure attempted was the utilization of N-t-butoxycarbonyl amino acids for the esterification of the 3'- (or 2'-) hydroxyl group of ATP with carbodiimidazole catalysis. Products of a similar esterification of the hydroxyl groups at the 2 p- or T-positions of nucleo- sides and nucleotides by amino acids have been investigated as analogs for an intermediate step in protein synthesis.

Experimentally, N-t-butoxycarbonyl-~-alanylimidazole formed in anhydrous dimethylformamide was allowed to react with ATP in aqueous solvent at room temperature. Descending preparative paper chromatography using n-butanol:water:acetic acid (5:3:2) as the migrating solvent afforded three well defined, UV-absorbing bands with R~ values of 0.37, 0.45, and 0.69. The fractions isolated by eluting each band from the paper with water were stored in powder form desiccated at reduced pressure at --20 °. Fractions with R~ values of 0.45 and 0.69 were re- examined after storage and were found to have been partially converted to that fraction with an Rr value of 0.37. Acyl migration to the thermally more stable T-OH substituted structure 9 is felt to be responsible for this interchange reaction. It was also noticed that, during storage in an aqueous medium at room temperature, all three fractions produced ATP.

A potassium bromide pellet prepared from the major component indicated the formation of an ester linkage, the presence of a phosphate, and an N-t-butoxycarbonyl group. A UV spectrum with a maximum absorption at 257.5 nm identical to that of ATP eliminated the possibility that aminoacylation may have taken place at 6-amino group of nucleotide. Nuclear magnetic resonance spectroscopy demonstrated the presence of tertiary butyl protons. On the basis of the above evidence, structure (II) has been assigned to this product.

The cleavage of the N-t-butoxycarbonyl group from the N-t-butoxy- carbonylamino-~-alanyl ATP (II) was performed by treating with tri- fluoroacetic acid briefly at ice bath temperature (Eq. 1).

CHs O OH I I

t-BOC-NH--CH--C-~O

CFsCOOH ~- T P ~ A

(1)

CF s COONtt 3 - C H - C - - O

CII) (111)

g C. S. McLaughlin and V. M. Ingram, Biochemistry 4, 1442, 1448 (1965).

[25] PHOTOAFFINITY ARYLAZIDO NUCLEOTIDE ANALOGS 263

Diazotization of the aminoacyl derivative of ATP (III) using sodium nitrite in 5% sulfuric acid was not successful. Successful diazotization is known to depend upon being able to extract the product into a water- immiscible phase immediately upon formation. The water solubility of the nucleotide would thus appear to limit the formation of the diazotization product.

Condensation with N-t-Butoxycarbonyl a-Alanine p-Nitrophenyl Ester and N-t-Butoxycarbonyl fl-Alanine p-Nitrophenyl Ester

An additional method attempted for the synthesis of diazo derivatives of ATP was via the condensation of diazotized a-alanine p-nitrophenyl ester with ATP under imidazole catalysis. The N-protecting group from N-t-butoxycarbonyl a-alanine p-nitrophenyl ester was removed by acid and the product diazotized prior to attempted condensation with ATP.

The first step worked out with quantitative yields. However, yields for the formation of the diazo ester vary from 8% to near 0 depending to a great extent upon how rapidly and how thoroughly the methylene chloride extraction of the diazo ester can be managed. The diazo ester once isolated from the reaction mixture by preparative thin-layer chromatography can be stored indefinitely at 0 ° without decomposition.

The condensation of the diazo ester and the ATP triethylammonium salt l° was attempted by allowing the reaction to proceed at room tern- perature for 4 hr in dimethyl sulfoxide under imidazole catalysis. How- ever, no products other than ATP and unchanged diazo ester were recovered. The lower activity of the diazo p-nitrophenyl esters is ex- plained by the electron distribution resulting from the presence of the a- diazo group. This arrangement makes it difficult for the carbonyl carbon to assume the partial positive charge required for the nucleophilic attack.

The difficulty encountered in carrying out the final step in the synthesis of the precursors of active carbene analogs of ATP directed us toward investigations of the synthesis of the diazo derivative of fl-alanine p-nitrophenyl ester, a compound in which the diazo group is not conjugated to the active ester group. Diazotization of fl-alanine p-nitrophenyl ester hydrochloride salt has, however, not been accomplished. Since the diazo group is not conjugated with a carbonyl, it is very reactive and tends to decompose and undergo further reaction as soon as it is formed.

Preparation of Arylazido ATP Analogs and Their Utilization

In view of the relative stability of nitrene precursors and the diffi- culties encountered in the final stages of synthesis of the diazo deriva-

loj. Zemlicka and S. Chladek, Collect. Czech. Chem. Commun. 33, 3293 (1968).


rives of ATP, it was decided to investigate the synthesis of ATP analogs containing nitrene precursor groups. ATP was reacted with p-azidobenzoic acid in the presence of carbodiimidazole. The reaction mixture was ehromatographed and was shown to contain, in addition to ATP and p-azidobenzoic acid, a new UV-absorbing material. This band with an Ry of 0.35 in the solvent system n-butanol:water:acetic acid (5:3:2) was eluted with water. The UV spectrum of the material recovered after lyophilization showed a maximum at 266 nm and a shoulder at 285 nm. A similar reaction carried out with NADP replacing ATP showed that the azidopyridine nucleotide analog could be formed.

The successful synthesis of these compounds showed the feasibility of using the carbodiimidazole coupling reaction in the formation of arylazido ATP analogs. For the general synthesis of the arylazidocarboxylic acids, 4-fluoro-3-nitroaniline is first diazotized in a concentrated hydro- chloric acid medium in the presence of sodium nitrite [Eq. (2)]. The diazonium salt is subsequently treated with sodium azide, which results in the formation of a light-sensitive 4-fiuoro-3-nitrophenyl azide (IV) [Eq. (3) ]. The structure of (IV) has been confirmed by its melting point of 52°-52.5 ° (recrystallized from petroleum ether) ; its NMR spectrum, 7.47 ppm (2 protons), 7.78 ppm (1 proton) ; and its mass spectrum, m/e 182.

_• HCI _ ~ F ~ NaNO, ~ F N +

NO, NO,

cV (2)

F--~ N+ CI- NaN' --~ F N. (3)

NO~ NO, (IV)

The resulting azide is then condensed with an amino carboxylic acid in an ethanolic aqueous solution containing sodium carbonate. N-4- azido-2-nitrophenylaminocarboxylic acids with varying hydrocarbon chain lengths separating the amino and carboxylic group have been synthesized [Eq. (4)], and their structures were identified via infrared and mass spectrometry.

N , ~ F + NH,(CI~).COOH Na~CO3 N s NH(CHz) n COOH

NO 2 NO. . = I (v); 2 (vi); 3 (vn); 4 (vm); s (ix); 10 Cx); 11 (x~ (4)


The N-(4-azido-2-nitrophenyl)aminocarboxylic acids are then condensed with ATP by means of carbodiimidazole catalysis [Eq. (5)].

_ ~ CDI T P C ~ A

N s NH(CH2)nCOOH + ATP DMF/I~ ~

OH (5) N NH(CI~)n--C---O

NO~

n = 2 CXTT); 3 (xm); 5 (xlv)

Attempts to prepare an ATP analog by condensation of the azido derivative of glycine with ATP under our standard conditions were unsuccessful. Presumably the nucleophilic reaction center, i.e.,

o II --C--OH

is sufficiently deactivated by its proximity to the a-amino group. The condensation reaction with the azido derivative of fl-alanine, 4-aminobutyric acid, or 6-aminocaproic acid, in which the reaction center is not

to the amino-deactivating group, was found to proceed satisfactorily. In the case of the synthesis of arylazido-fl-alanine ATP (XII), following the condensation with ATP, the crude reaction product was first dried under reduced pressure and then subjected to preliminary purification by repeated washing with acetone. This removes the excess carbodiimidazole and the azido derivative of the fl-alanine (VI). The solid residue was taken up in water and applied to Whatman No. 3 MM paper. The material from the band migrating at an Rf of 0.38 was recovered by elution with water and concentrated by lyophilization. This material (XII) gave a UV spectrum with maxima at 260, 290 (sh), and 480 nm. The increase in 260 nm peak intensity as compared with the starting material is due to the presence of the adenine ring structure.

Methods

Esterification o/ A TP with N-t-Butoxycarbonyl-a-alanine

Carbodiimidazole (71 mg, 0.43 mmole) and N-t-BOC-a-alanine (70.8 mg, 0.40 mmole) dissolved in 0.2 ml of dimethylformamide (dried over molecular sieves) are stirred for 10 min at room temperature. 11 The solu-

1, R. Paul and G. W. Anderson, J. Am. Chem. Soc. 82, 4596 (1960).

266 ENZYMES, A.NTIBODIES, AND OTHER PROTEINS [25]

tion is added to ATP (24.2 mg, 0.04 mmole in 1 ml of water), and the reaction mixture is allowed to react at room temperature for 3.5 hr. Solvent is evaporated under reduced pressure, and the resulting oil is washed with ether to remove imidazole and excess N-t-BOC-a-alanine. The residue from ether extraction (87.8 rag) is dissolved in water and chromatographed on 20 X 20-cm Whatman No. 42 paper using n-butanol: water :acet ic acid (5:3:2) as eluent. A resulting, well defined, UV absorbing band at R~ 0.37, a diffused band at RI 0.45, and a narrow band at Rs 0.69 are eluted from the paper with water and lyophilized, resulting in the collection of 7.3 mg, 1.0 mg, and 2.9 mg, respectively. The fractions with Rs values of 0.45 and 0.69 are the 2 r isomer and 2',3'-disubstituted compound. The fraction with R~ 0.37 is the 3 p isomer (II) .

Esterification o] ATP at the 3" Position with p-Azidobenzoic Acid

The 3'-p-azidobenzoyl ATP is prepared with p-azidobenzoic acid and A TP using carbodiimidazole as a catalyst as indicated above for the esterification of A T P with N-t-BOC-a-alanine. The reaction mixture is subjected to chromatographic resolution with n-butanol :water :acet ic acid (5:3:2) as solvent. The reaction product has a UV absorption band with an Rs of 0.37. The material is eluted from the paper with water and found to have absorption peaks at 266 and 285 (sh) nm. Irradiat ion of the aqueous solution with UV light shifts the 266 nm absorption maximum to 257 nm. The starting p-azidobenzoic acid has a maximum absorption peak at 271 nm which is broadened by UV irradiation.

Preparation o] 4-Fluoro-3-nitrophenyl Azide (IV) 12,12a

4-Fluoro-3-nitroaniline (4.38 g, 0.028 mole) dissolved in 30 ml of concentrated HC1 and 5 ml of water at 45 ° is filtered and then chilled to - -20 ° in a Dry Ice-acetone bath. Sodium nitrite (2.4 g, 0.034 mole) in 5 ml of water is added slowly to the well stirred acid medium while the flask temperature is kept at --15 ° to --20 ° . The reaction mixture after a 10-min stirring is filtered into a flask at --20 °. To this filtrate, sodium azide (2.2 g, 0.032 mole) dissolved in 8 ml of water is added drop- wise while the reaction mixture is stirred and kept in the dark at --15 °

~2 Azido derivatives of this nature are known to be light sensitive. They are ex- plosive in large quantities when dry, and caution is urged in their preparation. We always utilized a ventilated hood, and reactions were carried out in the dark.

~--a G. W. J. Fleet, J. R. Knowles, and R. R. Porter, Biochem. J. 128, 499 (1972).

[ 2 5 ] PHOTOAFFINITY ARYLAZIDO NUCLEOT1DE ANALOGS 267

to --20 ° . The light brown solid obtained by filtration is washed with ice water and dried in a vacuum desiccator. Recrystallization from petroleum ether yields straw-colored needles. Yield: 3.2 g, 63%; m.p.: 52°-52.5°; MS: m/e 182; NMR (CDCI3): 7.47 ppm (2 H), 7.78 ppm (i H).

Preparation o] N-4-Azido-2-nitrophenyl-fl-alanine (VI) :2a

To 5.4 ml of an aqueous solution of fl-alanine (534 mg, 6 mmoles) and sodium carbonate (1.08 g, 10 mmole) is added 4-fluoro-3-nitrophenyl azide (900 mg, 4.9 mmoles). Ethanol (6.75 ml), water (5.4 ml), and another portion of ethanol (13.5 ml) are added subsequently to enhance the homogeneity of the reaction mixture. The reaction mixture suspension is stirred at 52 ° overnight with an attached cooling condenser. The resulting dark red mixture is first concentrated under reduced pressure to about one-third of its volume and then diluted with 18 ml of water. Two extractions with 45 ml of ether remove all the excess starting azide. The aqueous layer is acidified with 3 N HC1 to pH 2 and extracted with three 90-ml portions of ether. The combined ether extract is washed three times, each with 50 ml of saturated NaC1 solution, and is dried over sodium sulfate and then evaporated to dryness. The residue is recrystallized from hot ethanol. Yield: 736 mg (59%) ; m.p. : 142.5°-145° ; MS: m/e 251; UV: )tCmn~ °H 260 (molar extinction coefficient 27.2 X 103), 280 (sh) and 460 (molar extinction coefficient 5.9 X 103) nm; IR: XKoR ~ 2.96, 3.43, 4.74, 4.80, 5.83, 6.13, and 6.37 ~m; NMR (chemical shift is expressed in ppm, J in cps, solvent = CD3OD): 7.79 (aromatic H-3, d, J - - 2 ) , 7.28 (aromatic, quartet, H-5, Js~ = 10, J3s = 2), 7.12 (aromatic H-6, d, J = 10), 3.66 (CH: fl to the carboxylic proton, t, J = 6).

Preparation of N-4-Azido-2-nitrophenyl Amino Carboxylic Acid Derivatives

The N-4-azido-2-nitrophenyl derivatives of glycine (VI), a-alanine (Via), 4-aminobutyric acid (VII), 5-aminovaleric acid (VIII), 6-aminocaproic acid (IX), ll-aminoundecanoic acid (X), and 12-aminododecanoic acid (XI) were prepared following the procedure as outlined for the preparation of N-4-azido-2-nitrophenyl-fl-alanine (VI), and the physical characteristics of these derivatives are indicated below. The Rj values given are those obtained on silica-gel plates with n-butanol saturated with water as the solvent.


N-4-Azido-2-nitrophenylglycine (V). UV: )~c~orI 260, 280 (sh), and 460 nm; IR: hK~ 3, 3.5, 4.7, 5.70, and 6.12 ttm; R:: 0.31; MS: m/e 237.

N-4-Azido-2-nitrophenyl-a-alanine (Via). UV: hCm~OI~ 263, 284 (sh), and 460 nm; IR: xK~ 3.0, 3.5, 4.71, 4.78, 5.79, 6.12, and 6.38 gm; R:: 0.41; 5~S: m/e 251.

N-$-Azido-2-nitrophenyl-4-aminobutyric Acid (VII). UV: hCm~OH 260, 280 (sh), and 460 nm; IR: )K~ 2.96, 3.36, 4.71, 5.88, 6.12, and 6.38 ttm; R:: 9.63; MS: m/e 265.

N-4-Azido-2-nitrophenyl-5-aminovaleric Acid (VIII). UV: hc~oH 257, 282 (sh), and 460 nm; R:: 0.68.

N-~-Azido-2-nitrophenyl-6-aminocaproic Acid (IX). UV: ~cm~3°H 259, 280 (sh), and 460 nm; IR: ) g ~ 2.96, 3.42, 4.75, 5.88, 6.13, and 6.38 ~m; MS: m/e 293; R:: 0.68.

N-$-Azido-2-nitrophenyl-11-undecanoic Acid ( X) . UV: ~c~,oR 261,285 (sh), and 460 nm; IR: hKm~ 2.96, 3.43, 4.72, 5.90, 6.13, and 6.39 ~m; MS: m/e 363; R:: 0.77.

N-$-Azido-2-nitrophenyl-12-aminododecanoic Acid (XI). UV: hCr~O~ 259, 282 (sh), and 460 nm; IR: hK~ 2.96, 3.44, 4.77, 5.87, 6.14, and 6.40 ~m; R:: 0.77; MS: m/e 377.

Preparation o] 3'-0-{3- [ N- ( 4-Azido-2-nitrophenyl) amino ] propionyl}- adenosine 5'- Triphosphate ( XII) , "Arylazido-fl-alanine A TP"

Carbodiimidazole (270 mg, 1.67 mmoles) and N-4-azido-2-nitrophenyl- fl-alanine (125.5 mg, 0.5 mmole) dissolved in 0.5 ml of dimethylformamide (dried over molecular sieves) are stirred for 15 min prior to introducing 60.5 mg of ATP (0.1 mmole) dissolved in 2.5 ml of water. The reaction is allowed to proceed overnight in the dark at room temperature. After the evaporation of the solvent to dryness, the residue is repeatedly washed with acetone followed by centrifugation. The acetone-washed residue obtained after drying under reduced pressure is dissolved in about 0.2 ml of water. This sample is then applied to Whatman No. 3 MM paper and eluted with n-butanol: water:acetic acid (5:3:2). Two major orange-colored UV-absorbing bands of R: values 0.91 and 0.38, in addition to a colorless UV-absorbing band with an R: value 0.05, are obtained. The front moving ,band (R: = 0.91) and the slow moving band (R: = 0.05) have been identified as N-4-azido-2-nitrophenyl-fl-alanine and ATP, respectively. The material (19.3 mg) recovered by eluting the band of R: value 0.38 with water followed by lyophilization gives a spectrum with maxima at 480 (molar extinction coefficient 4.2 X 103), 290 (sh), and 260 nm (molar extinction coefficient 35.4 X 103) attributed to the addition of the ATP and azido-fl-alanine


molecules. NMR (chemical shift is expressed in ppm, J in cps, solvent = D..,O) : 8.29 (H-8), 7.63 (H-2), 6.54 (H-I', d, J = 7), 5.00 (H-2', buried under water peak), 6.08 (H-3', quartet J = 7), 4.80 (H-4'), 4.64 (H-5'), 4.00 (CH~ fl to the carboxylic group, 5, J = 6), 3.30 (CH2 a to the carboxylic group, J = 6).

Azido-4-aminobutyric ATP (XIII) and azido-6-aminocaproic ATP (XIV) were prepared according to the procedure described for the preparation of azido-fl-alanine ATP (XII). The R: values given refer to paper chromatography. Attempts to prepare azidoglycine ATP and azido-a-alanine ATP by identical procedures gave no indication of product formation.

Azido-4-aininobutyric A TP (X I I I ) . R: = 0.40 with n-butanol:water: acetic acid (5:3:2) as the solvent; R: = 0.64 with isobutyric acid:0.5 N NH3 (5:3) as the solvent; UV: k~2~ 260, 290 (sh), and 480 nm.

Azido-6-aminocaproic A TP (XIV) . R: = 0.49 (solvent, n-butanol: water :acetic acid, 5:3:2) ; R: = 0.65 (solvent, isobutyric :0.5 N NH3, 5:3); UV: ),~,~ 260, 290 (sh), 480 nm.

Preparation o] Azido- [ 1-~4C] fl-alanine-ATP

(i) The [1-14C]fl-alanine (0.05 mC, in 0.2 ml of 0.01 N HC1, 0.5 rag, 6.2 t~moles) is concentrated under reduced pressure dissolved in 0.2 ml of water. Sodium carbonate (3.9 mg, 37.2 t~moles) is added followed by 0.2 ml of an ethanol solution containing 3.4 mg of 4-fluoro-3-nitrophenyl azide (18.6 t~moles). The resulting clear solution is maintained at 50 ° in a sand bath for 5.5 hr.

The reaction mixture, after evaporation to dryness, is extracted with acetone and then methanol and the combined solvents are concentrated to yield a solid residue. The residue is dissolved in a minimal amount of methanol and chromatographed on a 20 X 20-cm silica gel plate with n-butanol saturated with water as the solvent. Three colored bands are observed (orange to yellow color), and radioactivity can be detected at the band with an R: value of 0.41 (azido-[1-14C]B-alanine). The labeled azido-fl-alanine is recovered from the plate by elution with methanol) ~

(ii) The azido-[1-14C]fl-alanine diluted with 61.5 mg (0.25 mmole) of cold N3-fl-alanine is allowed to react with ATP (30.25 rag, 0.05 mmole) in the presence of carbodiimidazole (60.5 mg. 0.37 mmole) as a catalyst. The azido-[l:4C]fl-alanine ATP obtained by paper chromatography separation had a specific activity of 20,000 cpm/t~mole with UV maxima at 260, 290 (sh), and 474 nm.

1,~ R. A. G. Snlith and J. R. Knowles, Biochem. J. 141, 51 (1974).


Preparation o] Tritium and ~2P-Labeled Analogs o] Arylazido-fl-alanine A TP

Tritium-labeled arylazido-fl-alanine ATP (1.6 X 107 cpm//Lmole) is prepared by condensing 3- IN- (4 azido-2-nitrophenyl) ] aminopropionic acid with [2,8-3H]adenosine 5-triphosphate tetrasodium salt (1 mCi) obtained from New England Nuclear. The 3-°P-labeled ATP analog is prepared in the identical manner utilizing 7-labeled [32p]ATP. The synthesis of a number of labeled arylazido-fl-alanine nucleotides is schematically outlined in Fig. 1.

The esterification of ATP by N-4-azido-2-nitrophenyl-fl-alanine could result in the formation of either the 2' or 3' isomer. In the case of amino acid esters of nucleotides, such isomers can be resolved chromatograph- ically. In the esterification of ATP with N-t-butoxycarbonyl-a-alanine, the reaction products could be shown to be resolved into three components

7.s2p-ATP + Ns~NHCH~CI~COOH

H,~c~H

0 0 0 T

o $- 6-

O OH

N NHCHCI-I 2 Q --"----'O

NO2 / ®

ATP + N~ NHCHCH2COOH

NO2

2,8-3H-ATP + Ns-~NI-ICH2CI'I2COOH

NO2

ATP + N3~NHCI'I~CI-I214COOH

NO~

Fro. 1. Synthesis of radioactive arylazido-fl-alanine ATP analogs.


by paper chromatography using n-butanol:water:acetic acid (5:3:2) as eluent. The two minor components with Rs values of 0.45 and 0.69 were converted to the major component (RI 0.37) upon standing at room temperature. We assmne that the component with RI 0.69, because of its lesser polarity, represents the 2',3'-disubstituted ATP analog. The component with Rr 0.45 represents the 2' isomer, and the component with Rs 0.37 the 3' isomer. The possibility of conversion of the 2' to the 3' isomer by acyl migration is well supported by a number of studies. 9,1~ Evidence in the literature indicates that the 2'-hydroxyl groups of both adenosine and uridine are more open to electrophilic attack than the 3'-hydroxyl group. 1~ Thus the 2'-hydroxyl group is kinetically more reactive for substitution. However, such substitution is relatively less stable in comparison to the thermally more favorable esterification at the T-hydroxyl group . 1~

Our NMR studies indicate that in the case of arylazido-fl-alanine ATP a proton shift from 4.98 ppm, where H-3' normally absorbs to a position substantially down field at 6.08 ppm, occurs owing to esterification at the 3' position.

The accumulation of data indicates the 3' isomers to be the specific components isolated under our synthetic conditions. Further support for this conclusion is seen in the recent observation that the esterification of adenosine with fiuorosulfonylbenzoyl chloride results in the preferential formation of 3'-p-fluorosulfonylbenzoyladenosine. TM

Hydrolysis of Arylazido-fl-alanine ATP (XII)

The stability of (3'-0 [3-N (4-azido-2-nitrophenyl) amino] propionyl)- adenosine 5'-triphosphate (arylazido-fl-alanine ATP) is tested at 20 ° in neutral and basic solutions. Small amounts of the reagent are dissolved in (a) 0.5 M NH4HCO~; (b) 1.0 M NH4HC03; (c) H..,O; and (d) 5 mM Tris chloride pH 6.7. Incubation is allowed to continue, and at 0, 1, and 24 hr a small aliquot of the reaction mixture is spotted on Whatman No. 3 MM paper. Chromatography is conducted with N-butanol:water: acetic acid (5:3:2) as solvent. The paper is dried and examined for UV absorbing material. All four samples immediately upon mixing show a single clear UV-absorbing spot with an Rj of 0.38 corresponding to the arylazido-fl-alanine ATP analog and indicating that no hydrolysis has taken place. One hour later, sample (a) gives one major spot at Rf 0.90

14 p. C. Zamecnik, Biochem. J. 85, 257 (1962). 15 B. E. Griffin, M. Jarman, C. B. Reese, J. E. Sulston, and D. R. Trenthan, Bio-

chemistry 5, 3638 (1966). ~ P. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).

2 7 2 ENZYMES, ANTIBODIES~ AND OTHER PROTEINS [25]

corresponding to arylazido-fl-alanine and two spots at Rs 0.38 and 0.05, which correlate with the arylazido-fl-alanine ATP and ATP. Sample (b) is completely hydrolyzed, only ATP and arylazido-fl-alanine being detected. Overnight storage of sample (a) at room temperature results in complete hydrolysis to ATP and arylazido-fl-alanine. Samples (c) and (d) retain resistance to degradation following overnight incubation at 20 ° or at 4°C.

Spectral Characteristics and Photochemical Reactivity o] A ry lazido-fl-alanine A T P

In all the experiments described, photolysis was carried out in an air-cooled unit kept below 15 °. The sample in either 13 X 100 mm culture tubes or a glass cuvette was located equidistant between two tungsten- halogen projector lamps, 650 watts, DVY 3400°K, spaced 20 cm apart. The volume of the irradiated mixture varied from 0.1 to 3 ml. The sample

0 . 5

0 . 4

/,,,

i ~ 03- i~ 0 , 2 -

I I /

1

0.1

' I I I I I ! l I 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 50Q 5 2 0 .540 5 6 0

(A) WAVELENGTH in nm

Fro. 2. The absorption spectra of arylazido-#-alanine ATP as a function of the t ime of photoirradiat ion. Arylazido-fl-alanine A T P was dissolved in water to 14.25 ~M and subjected to photo i r rad iadon for periods of 0 min ( . . . . ), 6 min ( . ), and 10 min ( - - ) . (A) Absorbancc from 200 to 360 n m ; (B) absorbance from 350 to 600 nm.

[ 2 5 ] P H O T O A F F I N I T Y A R Y L A Z l D O N U C L E O T I D E A N A L O G S 273

0.25 -

0.2

0.15

0.1

0.05

I I I I |

400 450 ,500 550 soo

( B ) WAVELENGTH in nm

FIG. 2(B).

was removed from the photolysis apparatus, chilled, and mixed well at 30-sec intervals during the irradiation in order to prevent overheating.

Figure 2 shows the absorption spectra of arylazido-fl-alanine A TP (XlI) as a function of the time of photoirradiation. The A TP analog has maxima a t 260 and 480 nm. The increase in the 260 nm peak intensity above that of ATP is due to the combined presence of the adenine ring structure and (XI) . The absorption band at 470-480 nm is a consequence of the 4-azido-2-nitrophenyl adjunct. As can be seen when (XlI) is subjected to photoirradiation in an aqueous solution, there occurs a light-dependent decrease in the intensity of both the 260 nm and 470-480 nm absorption band.

Biochemical Interact ions

Inhibition o] Myosin Sub]ragment 1 ATPase with Arylazido-fl-alanine ATP (XII)

When subfragment 1TM was irradiated in the presence of arylazido-fl- alanine ATP, a 67% loss in ATPase activity was observed within 60 sec,

~7 S. Lowey, H. S. Slater, A. G. Weeds, and H. Baker, J. Mol. Biol. 42, 1 (1969). is p. Liu-Osheroff and R. J. Guillory, Biochem. J. 127, 419 (1972).


while the control dark sample with arylazido ATP remained stable. The extent of photoinaetivation has been found to approach maximal at irradiation of 2-rain duration. A variable degree of inhibition has been obtained with different protein preparations; a 2-min photolysis of myosin subfragment 1 in the presence of azido-fl-alanine ATP resulted in a 45-75% decrease in ATPase activity. There are indications that the age of the protein preparation influences the degree of photoinactivation. One sample was shown to be initially inhibited 75%, whereas 16.days later, under otherwise identical conditions, only 46% inhibition was obtained.

The effectiveness of arylazido-fl-alanine ATP as a photoinactivator of ATPase activity is influenced greatly by the presence of the natural substrate ATP. When equivalent amounts of ATP and arylazido-fl-alanine ATP were added to the subfragment, irradiation induced no inhibition. The additional fact that arylazido-fl-alanine (VI) without the nucleotide adjunct is not an effective photoinactivator strongly indicates that the arylazido ATP photoinactivation is due to its active site-directing ability.

The Dephosphorylation o] Arylazido-fl-alanine ATP by Myosin Sub]ragme~t 1 ATPase

The possibility that the arylazido analog might act as a substrate for subfragment 1 ATPase was tested initially with the EDTA-stimulated ATPase activity. The control assay system contained in 0.5 ml, 71 mM EDTA, 0.41 M NH4OH, 86 mM Tris chloride pH 8, and 10 mM ATP. 19 In the experimental vessel 3 mM arylazido-fl-alanine ATP replaced the 10 mM ATP. Dephosphorylation was allowed to proceed for 20 min at 30 ° in the dark, and the degree of ATPase activity was determined by the amount of inorganic phosphate liberated. The arylazido analog of ATP was found to be dephosphorylated at a rate equivalent to 17% of the control ATPase activity. (Control ATPase activity was 230 t~moles of phosphate liberated per milligram of protein per hour.)

Subsequent kinetic studies on the Ca~+-dependent ATPase of subfragment 1 were accomplished by using the ATP analog containing 3~p in the phosphate position. The ATPase activity was determined by measuring the amount of radioactivity extracted into an isobutanol phase in the presence of acid molybdate2 ° AKm of 6.15 ~M was evaluated in this way for the nucleotide analog, a value remarkably similar to that for the natural adenine nucleotide substrate.

~9 R. Yount, J. S. Frye, and K. R. O'Keefe, Cold Spring Harbor Syrup. Quant. Biol. 37, 113 (1972).

20 R. Yount, D. Ojala, and D. Babcock, Biochemistry 10, 2490 (1971).


When ATP hydrolysis is measured in the presence of different concentrations of arylazido-fl-alanine ATP in the dark, the analog is observed to be an effective competitive inhibitor. This is in contrast to the irreversible inhibition of ATPase activity following photoirradiation in the presence of the analog.

Covalent Labeling o] Myosin Sub]ragment 1 with Arylazido-1- [14C ] fl-alanine A TP

Photoinactivation of myosin subfragment 1 with arylazido-l-[14C]fl - alanine ATP resulted in the labeling of the protein with approximately stoichiometric amounts of radioactivity. Subfragment 1 (2.6 rag, 2 5( 10 -2 ~mole) in 5 mM Tris HC1, pH 6.7, was incubated with arylazido-1- [14C]fl-alanine ATP (0.925 mg, 2.2 X 104 cpm, 1.1 ~moles) for 15 rain at 0 ° followed by 3 min of irradiation. The protein was dialyzed against 5 mM Tris HC1, pH 6.7, and then against 25 mM Tris chloride, pH 7.6, for 24 hr. The dialyzate was then treated with 2.6 mg of Norit. The Norit-treated preparation (1.5 ml) was passed through a Sephadex G-75 column (1 X 15 cm), and 0.35-ml fractions were collected. Protein eluted at the void volume contained radioactivity. These fractions were combined, and the total protein was determined at 6.8 mmoles. A molecular weight of 1.2 X 105 was used for subfragment 1. ~1 Radioactivity measurements indicated 7.1 mmoles of the arylazido compound to be covalently bound to the protein. Such measurements indicate a label:protein ratio of 1.04.

A Comparison o] the Arylazido-fl-alanine ATP Labeling o] Myosin Sub]ragment 1 and Heavy Meromyosin alter Photolysis

Different concentrations of tritium-labeled arylazido-fl-alanine ATP were incubated with either subfragment 1 or heavy meromyosin. 22 After photolysis the protein was subjected to chromatography on Sephadex G-50. When the specific radioactivity of the protein eluting at the void volume was evaluated together with the remaining enzymic activity, total photodependent inhibition of subfragment 1 was accomplished at a ratio of 1 mole of arylazido-fl-alanine bound per mole of protein. In the case of heavy meromyosin, the ratio was 2 moles of nucleotide analog bound per mole of enzyme. Covalent binding of the arylazido-fl-alanine

:ID. M. Young, S. Himmelfarb, and W. F. Harrington, J. Biol. Chem. 240, 2428 (1965).

:~ P. D. Wagner and R. Yount, Biochemistry 14, 1900 (1975).


ATP is thus associated in a very precise manner with Ca~+-dependent ATPase activity.

Reactivity o] Other Arylazido-ATP Analogs

Two additional arylazido ATP analogs, arylazido-4-aminobutyric ATP (VII) and arylazido-6-aminocaproic ATP (IX), in which there are three and five methylene groups, respectively, bridging the distance between the ribose portion of the adenine nucleotide and the potential aryl nitrene, have been tested on myosin subfragment 1 ATPase activity. While the specificity of only the arylazido-fl-alanine ATP analog has been investigated extensively, all three arylazido ATP analogs inhibit subfragment 1 enzymic activity in a photodependent reaction. In each case the extent of photoinactivation is dependent upon the concentration of the arylazido ATP analog and shows saturation kinetics.

Interaction o] Arylazido-fl-alanine A T P with Mitochondrial ATPase 23,24

Arylazido-fl-alanine ATP brings about a sizable photodependent inhibition of the soluble mitochondrial F1 (ATPase). That the photodependent inhibition by the ATP analog is not a characteristic unique to the soluble enzyme can be seen from Table I, where the ATP analog is shown to bring about a greater than 80% photodependent inhibition of ATPase activity associated with a number of mitochondrial particles, all of varying specific ATPase activity. 25,26 The membrane-bound ATPase activity appears to be much more sensitive to the arylazido-fl-alanine ATP-dependent photoinhibition than the soluble ATPase. In separate experiments the presence of p-aminobenzoic acid during photolysis and up to 15 ~M did not affect ATPase activity and had only a slight influence upon the degree of photoinactivation due to arylazido-fl-alanine ATP. The presence of the natural substrate (ATP) prevents the photodependent inhibition by arylazido-fl-alanine ATP.

When arylazido-fl-alanine ATP containing 82p in the ~ phosphate is incubated with F1 (ATPase) in the dark, there is a sizable turnover of the enzyme. Within 15 sec, in excess of 40% of the added 2 mmoles of the analog one is hydrolyzed by 6.37 ~g of F1 (ATPase). Assay of the enzyme under these conditions, i.e., in the absence of a nucleotide re-

2~ A. F. Knowles and H. S. Penefsky, J. Biol. Chem. 247, 6617 (1972). 24 M. F. Pullman, H. S. Penefsky, A. Datta, and E. Racker, J. Biol. Chem. 235,

3322 (1960). 25 j. M. Fessenden and E. Racker, J. Biol. Chem. 241, 2483 (1966). 2~ E. Racker and L. L. Horstman, J. Biol. Chem. 242, 2547 (1967).

[25] PHOTOAFFINI~Y ARYLAZlDO NUCLEOTIDE ANALOGS 277

TABLE I ARYLAZIDO-~-ALANINE ATP-DEPENDENT PHOTOINHIBITION

OF TI-IE ATPAsE ACTIVITY OF SUBMITOCHONDRIAL

PARTICLES a

ATPase, % control b

Arylazido-~-alanine ATP ¢ ETPd ETPA a Sephadex *

1.28 38 - - 22 2.53 - - 33 - - 3.85 31 - - 16 6.40 29 - - - - 7.60 - - 22 - -

12.70 - - 15 - - 25.60 - - - - 10 50.70 - - 12 - -

a Submitochondrial particles (ETP, 1,015 mg; ETPA, 0.514 rag, and Sephadex particles, 1.01 mg) were incubated with various amounts of arylazido-B-alanine ATP (6.5-118.2 t~M) in a total volume of 0.2 ml containing 0.25 M sucrose and 10 mM Tris HC1, pH 7.5. Following a 4-min irradiation period, the ATPase activity was assayed with the ATP regenerating system described by M. F. Pullman, H. S. Penefsky, A. Datta, and E. Racker, J. Biol. Chem. 235, 3322 (1960).

b The specific activity (micromotes of ATP hydrolyzed per milligram of protein per minute) was for ETP (0.82), ETPA (2.0), Sephadex particles (4.2) ; the data are recorded as percentage of these values.

c The concentration of the arylazido-~-alanine ATP is presented as nanomoles of analog per milligram of protein. J. M. Fessenden and E. Racker, J. Biol. Chem. 241, 2483 (1966). E. Racker and L. L. Horstman, J. Biol. Chem. 242, 2547 (1967).

genera t ing system, bu t as s a tu r a t i ng levels of [3-~P]ATP (5 mM), resul ted

in an es t imated ac t iv i ty of 61 t~moles per mi l l ig ram of prote in per minute .

The low specific ac t iv i ty observed (0.51 ~mole per mi l l ig ram prote in per

minu te a t 15 sec) is felt to represent a l imi t a t ion due to the low level of subst ra te .

Photodependent Labeling of FI(ATPase) by Arylazido-fl-alanine A T P

T h a t the a ry laz ido- f l - a l an ine A T P is bound cova len t ly to the F1 (ATPase) following photolysis was indica ted from exper iments u t i l iz ing a t r i t i um- l abe l ed nucleot ide analog. :7

The labeled a ry laz ido- f l - a l an ine A T P associated with the F1 (ATPase) prote in following photolysis is s table to Nor i t t r e a t m e n t and extensive

:7 j . Russell, S. J. Jeng, and R. J. Guillory, Biochem. Biophys. Res. Commun. 70, 1225 (1976).


dialysis at neutral pH. Dissociation of the tritium-labeled protein by incubation with sodium dodecyl sulfate in the presence of mercapto- ethanol followed by extensive dialysis did not result in the liberation of a significant portion of the label. When the labeling was carried out as outlined by Russell et al. ~7 assuming a molecular weight of 3.6 X 105 for F1 (ATPase),2s 0.8 t~mole of arylazido-fl-alanine ATP was bound per mieromole of F~ (ATPase) protein.

Preliminary experiments indicate that the fl subunit of the F~ (ATPase) is the major site for labeling by arylazido-fl-alanine when photolysis is carried out as described. 2s~

Influence o] Arylazido-fl-alanine A T P on Reactions Associated with the Mitochondrial Membrane

Energy-Linked Transhydrogenation. The energy-dependent transhydrogenation reaction associated with the mitochondrial membrane can be driven independently by ATP or by respiratory energy. 29 Presumably the succinate-driven pathway utilizes the energized state of the mitochondrial membrane, independent of ATP-dependent reactions and thus presumably independent of F1 (ATPase) involvement. From Table II it can be seen that, after light irradiation of ETP in the presence of varying concentrations of arylazido-fl-alanine ATP, the ATP-driven transhydrogenation appears to be preferentially inhibited over that of transhydrogenation supported by succinate oxidation. No inhibition of transhydrogenation was observed for dark controls. The relatively constant and low-level inhibition of the succinate-driven reaction by irradiation in the presence of arylazido-fl-alanine ATP may be due to the presence of a small amount of endogenous nucleotide in ETP. It would appear that arylazido-fl- alanine ATP is a specific reagent in its interaction with the mitochondrial membrane, restricting itself to reaction with the bound F1 (ATPase). Although ATPase activity of the ETP particle in inhibited some 50% at 30.6 t~M arylazido-fl-alanine ATP, there is no maior inhibition of either respiration or the phosphorylation reaction. We believe it to be highly significant that those particles whose ATPase is most sensitive to photoinhibition in the presence of arylazido-fl-alanine are as well

~ A. R. Senior, Biochim. Biophys. Acta 3{)1, 249 (1973). ~ Ching-san Lai, unpublished observations. ~L. Ernster and C.-P. Lee, Annu. Rev. Biochem. 33, 729 (1964); C.-P. Lee and

L. Ernster, in "Regulation of Metabolic Processes in Mitochondria" (J. M. Tager, S. Papa, E. Quagliariello, and E. C. Slater, eds.), pp. 218-234. Am. Elsevier, New York, 1966.

[ 2 5 ] PHOTOAFFIN1TY ARYLAZIDO NUCLEOTIDE ANALOGS 279

TABLE II INFLUENCE OF ARYLAZIDO-~-ALANINE ATP ON THE

ENERGY-DEPENDENT TRANSHYDROGENATION OF ETP ~

Transhydrogenation (% control) Arylazido-fl-alanine ATP present during ATP Succinate

photoirradiation b dependent dependent

1.6 51 70 4.9 34 85 8.2 21 71

a Electron transport particles (ETP), 1.0 mg in 0.25 ml of solution containing 0.25 M sucrose and 10 mM Tris chloride pH 7.5, were subjected to 4-min photoirradiation in the presence of varying amounts of arylazido-~-alanine ATP. After photoirradiation, an aliquot containing 400 ~g of protein was taken for the fluorometric analysis of transhydrogenation in a total volume of 3 ml consisting of 140 mM sucrose, 40 mM Tris HC1 pH 7.5, 50 mM sucrose, 40 mM Tris HC1 pH 7.5, 50 mM ethanol, 10 mM MgSO~, 0.020 mM NAD +, 0.20 mM NADP +, 1 ~,g of rotenone, and 0.25 mg of yeast alcohol dehydrogenase. Control transhydrogenation driven by either 5 mM succinate or 3 mM ATP was 19.5 and 8.8 nmoles of NADPH formed per milligram per minute, respectively. The results are presented as percentages of these control activities.

b Nanomoles of analog per milligram of protein.

particles that have the lowest phosphorylation potential and highest specific ATPase activity.

Arylazido-B-alanine Pyridine i u c l e o t i d e Analogs

Chemical Synthesis and Structural Elucidat ion

The successful synthesis of ATP analogs containing a photoactive arylazido grouping esterfied to the T-hydroxyl of ATP offers the possibility for the synthesis of a wide variety of different nucleotide photoactive probes. In our search for reagents to assist in a comparative study of nucleotide binding regions of a variety of enzyme systems, we decided to attempt the synthesis of a photoactive arylazido pyridine nucleotide. The NAD ÷ molecule contains four potentially reactive hydroxyl groups within the nucleotide ribose positions.

Preparation of Arylazido-fl-alanine NAD ÷

The N-(4-azido-2-nitrophenyl)-f l-alanine is coupled to NAD* under the same conditions as those described for the synthesis of arylazido-fl-


alanine ATP except for the substituting of NAD ÷ for the adenine nucleotide. s° Chromatography of the reaction product on Whatman No. 3 MM paper as described for the adenine analog indicates the formation of two maior orange-colored UV-absorbing bands of Rr values 0.62 and 0.47 in addition to a band of RI 0.91 corresponding to arylazido-fl-alanine and one of 0.12 corresponding to NAD ÷.

Characterization o] the Material with R~ Value o] 0.47

The product with the Rs value 0.47 is eluted from the Whatman paper with H.~O, the solvent is removed by lyophilization, and the residue is redissolved in H20. Rechromatography indicates that this reaction product if free of NAD ÷ and arylazido-fl-alanine.

This material has an absorption spectrum with maxima at 475, 290 (sh), and 260 nm in H~O. The 475 nm band is characteristic of arylazido- fl-alanine analogs. The concentration of the material is determined from the absorbance at 275 nm by utilizing the molar extinction coefficient, 5.9 X 108, as previously determined for arylazido-fl-alanine at 460 nm2 °

The compound was found to undergo reaction with yeast alcohol dehydrogenase. The spectrum obtained following the reaction showed the development of a 340-nm absorption band in addition to the initial two maxima at 475 and 260 nm. The increment of absorbance at 340 nm was linearly related to the concentration of this material as measured by its absorption at 475 nm. In view of these experimental facts, the material has been designated arylazido-fl-alanine NAD ÷. The above experiments indicate as well that arylazido-fl-alanine NAD ÷ can substitute for NAD ÷ as a substrate for yeast alcohol dehydrogenase.

In the case of arylazido-fl-alanine ATP an arylazido-fl-alanine adjunct is attached to the hydroxyl group at the 3'-OH of the ribose moiety of ATP2 ° For NAD ÷ there are two ribose units and thus four hydroxyl groups that are potential esterification sites. Our results indicate that but a single arylazido residue has been esterified to NAD ÷ under the conditions used for its preparation.

The S~ructure o] Arylazido-fl-alanine NAD*

The arylazido-fl-alanine derivatives of NAD ÷ and AMP sl are well separated from NAD + and AMP by paper chromatography on Whatman

80 S. J. Jeng and R. J. Guillory, J. Supramol. Struct. 3, 448 (1975). 3~Arylazido-fl-alanine AMP was synthesized using the same procedure as that

described for the synthesis of arylazido-tS-alanine ATP except for the substitution of AMP for the ATP. One product with an R, value of 0.63 was obtained with the solvent system n-butanol-water-acetic acid (5:3:2).


TABLE I I I CHROMATOGRAPHY OF NAD +, AMP, NMN, AND ARYLAZIDO-~-ALANINE DERIVATIVES IN

SOLVENT SYSTEM n-BUTANOL--WATER-- ACETIC ACID (5:3 :2)

Compound RI

NAD + 0.12 Arylazido-~-alanine NAD + 0.47 AMP 0.30 Arylazido-/~-alanine AMP 0.62 NMN 0.2O Arylazido-~-alanine NAD +~

Product A 0.18 Product B 0.60

Chromatography following incubation with nucleotide pyrophosphatase; see text for incubation conditions.

No. 1 paper with n-butanol-water-acet ic acid (5:3:2). Their RI values are shown in Table I I I .

To determine the structure of arylazido-B-alanine NAD*, the analog is t reated with nucleotide pyrophosphatase isolated from potatoes as described by Kornberg and Pricer2 ~ After incubation of the arylazido- fl-alanine N A D + with pyrophosphatase, the products are separated on W h a t m a n No. 1 filter paper with n-butanol -water -ace t ic acid (5:3:2). A single UV absorbing spot with RI value of 0.18 and two orange spots with Rf values of 0.60 and 0.91 were evident (Table I I I ) . The orange mater ial with the highest RI value (0.91) was characteristic of arylazido-fl-alanine.

The UV-absorbing mater ial (Ry 0.18) is considered to be N M N owing to the similari ty of their RI value in this solvent system. When the material is eluted from the paper with H20, it is found to have an absorption peak at 260 nm. An additional absorption band develops a t 325 nm upon the addition of 1 M KCN, indicating the presence of a nicotinic base.33, 34 Further characterization of this material has not yet been at tempted.

The orange mater ial with an Rf of 0.60, when eluted from the paper with H20, was found to have an absorption band at 475-480 nm as well

a'~ A. Kornberg and W. E. Pricer, d. Biol. Chem. 182, 763 (1950). ~ Y. Nishizuka and O. Hayaishi, J. Biol. Chem. 238, 3369 (1963). ~4 R. M. Burton and N. O. Kaplan, Arch. Biochem. Biophys. 101, 139 (1963).


as one at 260 nm. This material could not, however, be reduced by yeast alcohol dehydrogenase in the presence of ethanol. On the basis of a similar RI value to arylazido-fl-alanine AMP (0.62) in this solvent, it was considered to be arylazido-fl-alanine AMP. Since the ester bonding of arylazido-fl-alanine to the ribose hydroxyl of ATP is known to be sus- ceptible to rapid and complete cleavage when incubated at basic pH, s° the yellow material with RI 0.60 was eluted from the paper and the aqueous solution was incubated for 12 hr at room temperature in 1 M NH4HC03 (pH 9). The reaction products were examined by chromatography on Whatman No. 1 paper with a solvent system consisting of 1 M ammonium acetate-ethanol (3:7). One of the products is a UV-absorbing material with an R~ of 0.10. In this solvent this material cochroma- tographs with AMP while NMN has an RI value of 0.27. ss The other reaction product of the alkaline hydrolysis is orange and has an Rf of 0.68, identical to that of arylazido-fl-alanine.

Together with nuclear magnetic resonance spectroscopy studies indicating that the 3'-OH of ATP is esterified during the synthesis of arylazido-fl-alanine ATP, the above data indicate that the esterification of NAD ÷ by arylazido-fl-alanine occurs on the 3'-OH of the ribose associated with the adenine portion of the nicotinamide adenine dinuc- leotide (see Fig. 3).

The reaction product with Rf value of 0.62 has not as yet been completely characterized; it is not, however, reduced by yeast alcohol dehydrogenase.

Biochemical Interactions

Alcohol Dehydrogenase

In addition to acting as a substrate for yeast alcohol dehydrogenase, arylazido-fl-alanine NAD + can bring about a photodependent inhibition of this enzyme. In order to assure ourselves that the photoinhibition resulting from irradiation of alcohol dehydrogenase in the presence of arylazido-fl-alanine NAD ÷ was due to a true photoaffinity effect of the NAD ÷ analog, irradiation was carried out in the presence of arylazido- fl-alanine. Incubation of arylazido-fl-alanine at molar excess of 291-fold over that of alcohol dehydrogenase resulted in only a minor inhibition of the enzyme during photoirradiation, whereas the photolysis of the enzyme in the presence of a 59-fold molar excess of arylazido-fl-alanine NAD ÷ resulted in a 63% photodependent inhibition of the dehydrogenase activity (Table IV).

N~

o o I II H I o ,o _ _Oo _ -OCH o

H0 0 HO OH I C = O

Nit

Ns

"Arylazido • NAD"

Nil,

~ ~o_~_o

HO O I C : O

(~H,)s NH

Ns

"Arylazido • AMP"

NH~

~ ~o-~-o

HO OH AMP

nucleotide I pyrophosphatase

+

O

HO OH

~ CONH~

I OH- +

NMN

~co~

O2N

,~ " h N H O - - C - - ( C I ' I ~ ) z - - H N ~ s

"Arylazido-~ -alanine"

FIG. 3. An i l lus t ra t ion of the p rocedure used to d e t e r m i n e the s t ruc tu re

a ry laz ido-~-a lan ine N A D .


TABLE IV A COMPARISON OF THE PHOTODEPENDENT INHIBITION OF

YEAST ALCOHOL DEHYDROGENASE BY ARYLAZIDO-~- ALANINE N A D + AND ARYLAZIDO-~-ALANINE a

Additions Molar ratio b Percent act ivi ty

Arylazido-t~-alanine NAD + 59 36.6 Arylazido ~-a|anine 291 94.8

a For photolysis, a mixture containing 0.5 M phosphate buffer, pH 7.0, 0.2 mg of yeast alcohol dehydrogenase, and 0.93 m M arylazido-~-alanine N D A + or 1.94 m M arylazido-B-alanine in 0.21 ml was irradiated for 10 min at 15 °. After irradiation, 10 t~l of the above mixture was diluted to 1 ml with 0.5 M phosphate buffer, pH 7.0. The assay was initiated by the addition of 5 ~1 of the diluted irradiated sample to a mixture containing 0.15 M phosphate buffer, pH 7.0, 1.34 m M NAD +, 17 m M ethanol, and 0.59 m M glutathione in 1 ml at 30 °. The rate of NAD + reduction was assayed by the increase in absorbance at 340 nm. The act ivi ty of a nontreated control was taken as 100% and is equal to 422 t~moles of N A D H produced per minute per milligram of protein.

b The molar ratio of the arylazido-~-alanine NAD + and arylazido-/3-alanine to yeast alcohol dehydrogenase is based upon an enzyme molecular weight of 141,000 [Y. Hatefi, in" Comprehensive Biochemistry" (M. Florkin and E. Stolz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam, 1966].

Reaction with NADH-CoQ Reductase 3~

When alcohol and alcohol dehydrogenase are incubated together with arylazido-fl-alanine NAD ÷, they act as a regenerating system capable of maintaining the analog in its reduced state. Under these conditions the analog can bring about a reduction of CoQ~ catalyzed by complex I at a rate of some 75% that of the natural substrate. The reduction of CoQz by arylazido-fl-alanine NADH catalyzed by complex I is as well rotenone sensitive.

Photodependent Inhibition of NADH-CoQ Reductase by A rylazido-fl-alanine N A D H

The fact that the reduced form of arylazido-fl-alanine NAD ÷ in the presence of complex I catalyzes the reduction of CoQ1 in a rotenone- sensitive reaction indicates clearly that the reduced analog is acting as a substrate for the NADH dehydrogenase of complex I. The pyridine nucleotide analog is as well a potent photodependent inhibitor of the enzyme complex. In fact the reduced form of the arylazido-fl-alanine NAD ÷ is a more effective inhibitor with greater than 95~ inhibition of

35 y . Hatefi, D. G. Haavik, and D. E. Griffiths, J. Biol. Chem. 182, 1676 (1962).

[ 2 5 ] PHOTOAFFINITY ARYLAZIDO NUCLEOTIDE ANALOGS 285

NADH-CoQ reductase after a 4-min period of photoirradiation, and an equal concentration of arylazido-fl-alanine NAD results in 75% inhibition. The photodependent inhibition is a function of both the time of irradiation and the concentration of arylazido-fl-alanine NADH.

Competitive and Noncompetitive Inhibition o] NADH-CoQ Reductase by Arylazido-fl-alanine NAD ÷

Arylazido-fl-alanine NAD ~ can bring about a sizable inhibition of NADH-CoQ reductase activity when incubated with the enzyme complex in the dark without prior photoirradiation. In this case the addition of higher substrate concentrations (NADH) results in a reversal of this inhibition. Reduced NAD at 53 t~M is able to completely reverse the inhibitory influence of 11 ~M arylazido-fl-alanine NAD ÷. On the other hand, when complex I is subjected to photoirradiation in the presence of 11 ~M arylazido-fi-alanine NAD ~, the addition of higher concentrations of the natural substrate (NADH) is not able to reverse the inhibition. It would thus appear that the arylazido-fl-alanine NAD ÷ acts as a competitive inhibitor of NADH-CoQ reductase in the dark and is converted to a noncompetitive irreversible inhibitor upon light irradiation in the presence of the protein complex.

Other Applications of the Carbodiimidazole-Catalyzed Esterification

Preparation of Spin-Label ATP, ADP, AMP, and NADP

3-Carboxyl-2,2,5,5-tetramethylpyrroline-l-oxyl (184 rag, 1 mmole) and carbodiimidazole (173 rag, 1.1 mmoles) dissolved in 0.5 ml of anhydrous dimethylformamide are allowed to react at room temperature for 10 min to form the imidazolide. ATP (60.5 rag, 0.1 mmole) in 2.5 ml of water is then added to the DMF solution, and the mixture is stirred at room temperature for 10 hr. The liquid sample obtained following lyophilization is washed with ether and methanol to remove the starting material. The solid residue (37.5 rag) is chromatographed with isobutyric acid:0.5 N NH3 (5:3) as the solvent. Material (XV) recovered from a UV-absorbing band appearing at R~ 0.58 weighed 12.3 mg and had UV maxima at 259 and 208.5 nm, corresponding to adenine nucleoside derivatives and an aft-unsaturated carbonyl group, respectively. The electron spin resonance spectrum of this material gave three sharp N-oxide peaks. Spin label ADP, spin-label AMP, and spin-label NADP are prepared according to the same procedure. Products isolated utilizing the same chromatography system were found with Rf values of 0.74, 0.84, and 0.63,

286 ENZYMES, ANTIBODIES, AND oTHER PROTEINS [25]

respectively, all three compounds having strong ESR signals. The UV spectrum of the spin-label ADP showed two maxima, one at 259 and the other at 208 nm.

COOH CDI

+ ATP DM F/H2 0 ~

o

o OH l O----C~

0

(xv)

Recently Westheimer and his group 3~ have reported the synthesis of 2-diazo-3,3,3-trifluoropropionyl chloride from trifluorodiazoethane and phosgene. The reagent is a powerful acylating agent and should react with ATP at the 2'- or 3'-hydroxyl group (XVI). ;o:

o OH r

FsC--C--C--O II N,

(XVI)

The diazo derivative itself is known to be stable to acid pH and readily undergoes photolytic decomposition generating a carbene.

D i s c u s s i o n

The carbodiimidazole-eatalyzed formation of activated carboxylie acids containing a photosensitive arylazido has been utilized in the synthesis of a number of photoreactive adenine and pyridine nucleotide analogs. These analogs have properties consistent with the requirements of photoactive site-directed reagents. Their usefulness has been demon-

V. Chowdhry, It. Vaughan, and F. H. Westheimer, Proc. Natl. Acad. Sci. U~8.A. 73, 1406 (1976).


strated in the investigation of the nucleotide binding regions of a number of enzyme systems. The photoaetive species arylazido-fl-alanine does not inhibit myosin or mitochondrial F1 (ATPase) in comparison to the extensive photodependent inhibition observed in the presence of arylazido- fl-alanine ATP. The ATP moiety is thus required to direct the nitrene precursor to the active site region of the enzyme. This conclusion is supported by the competitive nature of the interaction of the analogs in the presence of the natural ligands. Similar conclusions can be made for the pyridine nucleotide photoactive analogs and their interaction with alcohol dehydrogenase and with NADH dehydrogenase of complex I of the mitochondrial electron transport chain.

The arylazido-fl-alanine analog has been found to be as well a potent inhibitor of yeast hexokinase and firefly luciferaseY

The variation in the distance separating the photoactive azido group from the 3'-hydroxyl position using azido-fl-alanine, azido-4-aminobutyric, and azido-6-aminocaproic ATP and other potential analogs is anticipated to become a powerful test in the chemical mapping of the topographical components of nueleotide binding regions of proteins. Such analogs can be used for the scanning of the region about the binding site, revealing the three-dimensional alignment of the region without the necessity of a complete knowledge of the primary structure of the protein.

Because of the chemical nature of the analogs discussed in this paper, one must assume that the covalent insertion reaction does not occur directly at the ligand binding site. This may be one of the reasons for the less than 100% inhibition of myosin ATPase activity with neverthe- less reasonable stoichiometry of analog labeling following photolysis. Labeling occurs within the vicinity of the active site with inhibition of enzymic activity being due to the irreversibly bound nucleotide blocking access of substrate to this region in a flexible manner. Under these conditions there is no reason to assume that the inhibition of enzymic activity should be directly proportional to active site binding. This would be especially true for those analogs with long spacers between the photoactive azido and the nucleotide (i.e., ligand) binding site. Thus while one might not be able to specify precisely that covalent insertion occurs directly at the active site, since the reactive reagent is at a distance from the binding site, one does have an exact knowledge of the distance separating the point of insertion from the point of ligand binding.

The preparatory procedures outlined have great potential use in that they may be used as the basis for the synthesis of a wide variety of

,7 Unpublished observations of R. J. Guillory.

288 ENZYMES, ANTIBODIES, AND OTHER PROTEINS [2S]

different nucleotides 3s as well as nonnucleotide probes. In addition to the photoaffinity labels, spin labels and fluorescent adjuncts may also be coupled to the ribose while still allowing the ligand to retain its enzymic function.

That the point of esterification need not be the hydroxyl group of a nucleotide can be seen from some recent experimental findings with tetrodotoxin2 9 This toxin is a specific inhibitor of the transient conductance of the muscle (and nerve) membrane. 4° The specificity of action (at nanomole concentrations) has raised the possibility of tetrodotoxin being used in the identification and characterization of membrane sodium channels. While the binding of tetrodotoxin to these channels is rapid, it is reversible, and attempts at the further characterization of the channel components are limited. Tetrodotoxin when subjected to a carbodiimidazole-catalyzed esterification by arylazido-fl-alanine results in the formation of a number of analogs of tetrodotoxin containing the azido ligand. Testing of these materials revealed that one in particular had the biological characteristics of the natural tetrodotoxin. While this material could be adequately washed from the muscle tissue to restore the membrane's normal transient Na ÷ conductance, this reversibility was completely prevented when the muscle bathed in the tetrodotoxin analog- containing medium was subjected to photoirradiation. The material has all the characteristics of a photoaffinity active site directed reagent for the labeling of the specific Na + channels of excitable tissue. Carried to its reasonable extension, the use of the arylazido-fl-alanine tetrodotoxin analog shall enable us to characterize the components within the vicinity of the Na ÷ channel of excitable tissue.

The availability of the described photoaffinity reagents represent chemical tools for the examination of the ligand binding regions of a wide variety of biologically active systems.

Acknowledgments

Portions of this work were supported by a grant from the Hawaii Heart Asso- ciation. The authors would like to express their appreciation for the collaboration of Mr. S. Chen and Mr. J. Russell in a number of the experiments detailed in this work. A portion of this work was accomplished during the tenure of R. J. G. as an Established Investigator of the American Heart Association and of S. J. J. as a recipient of NIH Research Fellowship 1FO 2 GM 57060.

~SThe carbodiimidazole-catalyzed esterification reaction with arylazido-~-alanine has recently been successfully applied to the synthesis of a photoaffinity analog of coenzyme A. Unpublished observations of S. J. Jeng.

29 Unpublished observations of R. J. Guillory in collaboration with Dr. M. Rayner and Dr. J. S. D'Arrigo.

40 T. Narahashi, Physiol. Rev. 54, 813 (1974).

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