Covalent Intermediates in Flavin-sensitized...

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This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution 4.0 International License. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung 4.0 Lizenz. 1032 W.-R. KNAPPE AND P. HEMMERICH Covalent Intermediates in Flavin-sensitized Photodehydrogenation and Photodecarboxylation W.-R. KNAPPE and P. HEMMERICH Department of Biology, University of Konstanz, Germany (Z. Naturforsch. 27 b, 1032—1035 [1972] ; received May 10, 1972) Covalent Photoadducts of Flavins The photoreactions of the flavin triplet with unsaturated hydrocarbons, aldehydes, sulphur compounds, and carbonic acids are reported. These reactions proceed by attack at the allylic C —H-, the RCO —H-, or the a-C —H-bond, respectively; in the case of carbonic acids decarboxy- lation occurs. In all the reported reactions covalent adducts are formed, whose structure and mode of decay is characterized. The flavin molecule in its oxidized state ["flavo- quinone", Fl ox ] is able to attack many classes of organic compounds under light catalysis. This photochemistry starts from the excited flavoquinone triplet [ 3 Fl*x] as proved by iodide quenching 1 and leads to the formation of "covalent adducts" R Fl re jH, whose formation is characterized by eqn. (1) : R- CO 2 - j + 3 F1 * R _ F 1 - + jco 2 (1) In some cases of substrates containing two oxi- dizable centers, as e. g. a-carbon and /S-sulphur in compounds of type R S CH2 C0 2 H, we find more complicated fragmentation processes re- placing this simple mechanism (2) : R _ S - CH 2 - C0 2 " + m l R — FLTD + S = CH 2 + C0 2 (2) A slightly different type of fragmentation mechanism has been discussed by YANG, KU and PRATT 2 for the flavin-sensitized photodegradation of methionine. The classes of compounds investigated and the types of covalent adducts isolated are summarized in Table I. We find that decarboxylation always pre- vails over dehydrogenation, if a C0 2 ~-group (but not C0 2 H or C0 2 R) is present 3 . The "covalent adducts" R —Fl re( iH can be sub- divided into three classes, whose prevalence is governed by i) the type of substrate, ii) the solvent polarity, iii) the proton activity (pH), and iv) the temperature employed: Class 1: N (5) -Alkyl-l,5-dihydroflavins (5-R- Fl redH, 1), which are characterized by a pK around Requests for reprints should be sent to Prof. P. HEMME- RICH, Fachbereich Biologie der Universität, D-7750 Kon- stanz, Postfach 733. 7, owing to the acidic N (1) H-function, and by their sensitivity towards oxygen in the dark. Their autoxidative decay yields the corresponding 5-RF1- radical, the quaternary "fully oxidized" ion 5-RF1+ 4 , or its "pseudobase" 5-RFl ox -4a-OH 5 ' 6 , whose stability is governed by the leaving group properties of the R + -residue, wdiich is finally hydro- lyzed to yield R - OH 6 . Class 2: C (4a)-Alkyl-4a,5-dihydroflavins (4a-R- FlredH, 2), which have no pK in the range 3 < p H <10, but are protonated around pH 1 at N(l) or N(5) according to the more or less efficient shielding of N(5) by the 4a-substituent 7 . This class is in general stable towards oxygen in the dark and is also formed upon alkyl migration from N(5) to- wards C(4a) according to the migrational aptitude of R. This rearrangement seems, in principle, to be reversible 3 as depending on pH. The dealkylation of class 2 compounds is possible by Fl ox-sensitized photooxidation 8 or by action of nitrous acid, while the residue R is split at the aldehyde level 6 . Class 3: Up to now this case could be verified only with phenylacetate as substrate at alkaline pH 9 owing to the extreme lability of the residue R at position C(8) in the cyclohexadienone-imine type C(8)-alkyl-l,8-dihydroflavin (8-R-Fl red H, 3). 8-R-Fl redH, however, is the only type of covalent adduct, to which the widely observed "action pK" in flavin-sensitized photooxidations 10-12 can be at- tributed and which shows a long wave optical ab- sorption suitable to explain the color of flavoprotein-

Transcript of Covalent Intermediates in Flavin-sensitized...

Page 1: Covalent Intermediates in Flavin-sensitized ...zfn.mpdl.mpg.de/data/Reihe_B/27/ZNB-1972-27b-1032.pdf · also formed upon alkyl migration from N(5) to-wards C(4a) according to the

This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

1032 W.-R. KNAPPE AND P. HEMMERICH

Covalent Intermediates in Flavin-sensitized Photodehydrogenation and Photodecarboxylation W . - R . KNAPPE a n d P . HEMMERICH

Department of Biology, University of Konstanz, Germany

(Z. Naturforsch. 27 b , 1032—1035 [1972] ; received May 10, 1972)

Covalent Photoadducts of Flavins

The photoreactions of the flavin triplet with unsaturated hydrocarbons, aldehydes, sulphur compounds, and carbonic acids are reported. These reactions proceed by attack at the allylic C —H-, the RCO —H-, or the a-C —H-bond, respectively; in the case of carbonic acids decarboxy-lation occurs. In all the reported reactions covalent adducts are formed, whose structure and mode of decay is characterized.

The flavin molecule in its oxidized state ["f lavo-quinone", Fl o x ] is able to attack many classes of organic compounds under light catalysis. This photochemistry starts from the excited flavoquinone triplet [3Fl*x] as proved by iodide quenching 1 and leads to the formation of "covalent adducts" R — Fl r e jH, whose formation is characterized by eqn. (1) :

R - C O 2 - j + 3 F 1 * R _ F 1 - + j c o 2 ( 1 )

In some cases of substrates containing two oxi-dizable centers, as e. g. a-carbon and /S-sulphur in compounds of type R — S — CH2 — C0 2 H, we find more complicated fragmentation processes re-placing this simple mechanism (2) : R _ S - C H 2 - C 0 2 " + m l R — FLTD

+ S = C H 2 + C 0 2 ( 2 )

A slightly different type of fragmentation mechanism has b e e n d i s c u s s e d b y Y A N G , K U a n d P R A T T 2 f o r the flavin-sensitized photodegradation of methionine.

The classes of compounds investigated and the types of covalent adducts isolated are summarized in Table I. W e find that decarboxylation always pre-vails over dehydrogenation, if a — C02~-group (but not — C 0 2 H or — C 0 2 R ) is present3 .

The "covalent adducts" R —Flre(iH can be sub-divided into three classes, whose prevalence is governed by i) the type of substrate, ii) the solvent polarity, iii) the proton activity ( p H ) , and iv) the temperature employed:

Class 1: N (5) -Alkyl-l,5-dihydroflavins ( 5 - R -FlredH, 1 ) , which are characterized by a pK around

Requests for reprints should be sent to Prof. P. HEMME-RICH, Fachbereich Biologie der Universität, D-7750 Kon-stanz, Postfach 733.

7, owing to the acidic N (1) H-function, and by their sensitivity towards oxygen in the dark. Their autoxidative decay yields the corresponding 5-RF1-radical, the quaternary "fully oxidized" ion 5 -RF1+ 4 , or its "pseudobase" 5 -RFl o x -4a -OH 5 ' 6 , whose stability is governed by the leaving group properties of the R+-residue, wdiich is finally hydro-lyzed to yield R - OH 6.

Class 2: C (4a)-Alkyl-4a,5-dihydroflavins ( 4 a - R -FlredH, 2 ) , which have no pK in the range 3 < p H < 1 0 , but are protonated around pH 1 at N ( l ) or

N ( 5 ) according to the more or less efficient shielding of N ( 5 ) by the 4a-substituent7. This class is in general stable towards oxygen in the dark and is also formed upon alkyl migration from N ( 5 ) to-wards C(4a ) according to the migrational aptitude of R. This rearrangement seems, in principle, to be reversible3 as depending on pH. The dealkylation of class 2 compounds is possible by Flox-sensitized photooxidation 8 or by action of nitrous acid, while the residue R is split at the aldehyde level 6 .

Class 3: Up to now this case could be verified only with phenylacetate as substrate at alkaline pH 9 owing to the extreme lability of the residue R at position C ( 8 ) in the cyclohexadienone-imine type C(8)-alkyl-l ,8-dihydroflavin (8-R-Fl r e dH, 3 ) .

8-R-FlredH, however, is the only type of covalent adduct, to which the widely observed "action p K " in flavin-sensitized photooxidations 1 0 - 1 2 can be at-tributed and which shows a long wave optical ab-sorption suitable to explain the color of flavoprotein-

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lab. 1. Covalent adducts. AH new adducts have been isolated in the crystalline state and have been characterized by elemental analyses, IR-, UV- and ^-NMR-spectra.

Class of substrate and point of attack

Solvent (pH) Relative rate

Adduct R-FlredH

Reoxidation with 02 (dark) 02(light) acid + N0+

Ref.

Hydrocarbons, e.g. H 3 C \ / H

> = C < I H 3 C / > C = C <

H / \

CH3

CH2 H

h 3 ( \ y CH3

H 3 C / N C H 2 . H t

Aldehydes, e.g C 6 H 5 - C = O

H cf. C 6 H 5 C O - C O ;

t Sulphur compounds, e. g.

< s > Me3CSCH2 7 CO 2

t

S(CH 2^C0 2 - ) 2

S2(CH2^-C0;)2

Carboxylates, e.g. Me3C . CO^

f CeHaOCHa^COj

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

CH3CN

+ + +

+ +

+ + +

+ +

+ + +

+ +

+ + +

+ + +

+ + +

+

+ + +

R = 4 a-CH2

CH: > H c=c w x

CH3

CH3

4 a-CH2 CH3 >c=c< H3C CH3

- O

5 - C O - C 6 H 5

5 - C O - C 6 H 5

5 - < s 4a-CH2SC(CH3)3

5-CH 2 C0 2 H

4 a-SCH 2C0 2H

5-C(CH3)3

4 a-CH2OC6H5

+

+

+ (autocat.)

+ +

+

very fast

fast

slow

fast (autocat.)

+

+

+ + [20 °C]

+ + [20 °C]

+ [15 min 100 °C]

+

+ + + + + +

n o < > H

a o H O > Ö Ö Ö

CeHa-CHa^-COO" t

CH3

C 6 H 5 - C H r C O O ~ t

C H 2 = C H - C H 2 A c o o t

IndolyI-3-CH 2 xCOO~ l

CH3CO -.-COO" t

H 2 0 , 20 °C, pH 4—7 H 2 0 , 50°C, pH 4—7 H 2 0 , 20°C, pH 8 - 1 2 CH3CN

H 2 0 , p H 4 — 1 0 2 0 °C

4 a - u n d 5-CH2C6H5 « 1 : 1 , 4 a- CH2C6H5 4 a-, 5- und 8 - C H 2 C 6 H 5 « 1 : 1 : 1 4 a-CH2C6H5

4 a-CH(CH3)C6H5

4 a - C H 2 C H = C H 2

4 a-CH2-3-Indolyl

5-CO —CH3

+ (5) + (5)

+ (4a) + (4 a)

+ + +

+ + +

5

18

5

19 O 00 CO

• this work

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1034 COVALENT PHOTOADDUCTS OF FLAVINS

substrate complexes1 3 . The directly observable 8-benzyl-FlredH 9 is only stable in the anionic state and undergoes at pH < 7 rearrangement to yield 5-R-Fl r e dH. Before rearrangement it is characteristi-cally stable against oxidation in agreement with the localization of the substrate electron pair at the saturated carbon center in position 8. We assume, therefore, that 8-R-Flre(iH plays an important role in flavin (photo) catalysis though in most cases it cannot be observed directly because of fast dis-sociation and/or isomerization.

While it remains doubtful, whether flavin-photo-chemistry has anything in common with flavin-bio-chemistry, it remains a surprising fact that the nature of C — H-substrates undergoing flavin-depen-dent dehydrogenation is very similar in photo-chemistry and enzymology. Obviously there are common structural features (though clearly no magnetic ones) between flavoquinone as modified by excitation to the triplet state, on the one hand, and by binding to apoproteins, on the other hand. The most obvious property of the "flavinophilic" CH-group is their C •<— H rather than C H ground state polarisation which is now taken care of enzy-mologically by way of carbanion mechanisms 14 as proposed by us many years ago 15.

Nothing can be said presently about the potential radical character of the photoaddition reactions de-scribed here. Under all circumstances, synchronous addition of R and H (from substrate R —H) to Flo x has to be considered seriously. This would render the question meaningless as to "carbanion" or "radical" character of the reactions. In any case no radical RFI has ever been ascertained as primary intermediate on the catalytic pathway, neither in biochemistry, nor in photochemistry, though the species RFI (R being alkyl or H) can in many cases be observed — chemically and enzymatically — during oxidative decay of the covalent adducts formed primarily.

Though the photochemical formation of the co-valent adducts may be biologically irrelevant, their structure and mode of decay are revealing for the hitherto unexplained "molecular b io logy" of flavo-coenzymes. The failure to observe intermediates in many flavoprotein catalyses is readily ascribed to the fast protolysis of R-Fl r e dH to yield ROH and HFl redH, owing to the excellent leaving properties of the "natural" residues R. On the other hand, R-Fl redH may be readily mistaken for HFl r e dH in UV-spectra (cf . Fig. 1 ) , since the critical range around 350 nm is in most biological systems ob-scured by "background" absorptions.

Y ^ N Y N - ^ O + H3C CH3

0 3

N^N^O

H HjCv̂ CH^ H3C^ch3

vPh

0 <k

0" -.

5"

c .0 "co-

c .0 "co- 15"

X=20" \ \ # 346\ |

1 1 1 1 " 180"

10'02 1'02

300 400 500 X

Fig. 1. Solid lines: Photoreaction of 3-benzyl-lumiflavin (0.75 x 10~4 M) in acetonitrile with 2.3-dimethyl-butene-(2) (0.067 M) under strictly anaerobic conditions [250 W/24 V tungsten-halogen lamp with a filter and lens system transparent from 300 to 800 nm]. Broken lines: Reoxidation of the adduct formed with air and nitrous acid, respectively. Dotted line: 1,5-Dihydroflavin, generated by the photoreaction between

3-benzyl-lumiflavin and 1,4-cyclo-hexadiene in acetonitrile.

We want to thank Mrs. M. G l adys for technical as-sistance.

B . HOLMSTRÖM a n d G . OSTER, J . A m e r . c h e m . S o c . 8 3 , 1 8 6 7 [ 1 9 6 1 ] , S . F . Y A N G , H . S . K U , a n d H . K . P R A T T , B i o c h e m . b i o p h y -sic. Res. Commun. 24, 739 [1969] ; J. biol. Chemistry 242, 5 2 7 4 [ 1 9 6 7 ] . W . HAAS a n d P . HEMMERICH, Z . N a t u r f o r s c h . 2 7 b . 1 0 3 5 [ 1 9 7 2 ] . P . HEMMERICH, S . GHISLA, U . H A R T M A N N , a n d F . MÜLLER, in: H. KAMIN (Editor), Flavin and Flavoproteins, p. 83, University Park Press, Baltimore 1971. P . HEMMERICH, V . MASSEY, a n d G . WEBER, N a t u r e [ L o n -d o n ] 2 1 3 , 7 2 8 [ 1 9 6 7 ] .

6 W . H . W A L K E R , P . HEMMERICH, a n d V . MASSEY, H e l v . chim. Acta 50, 2269 [1967].

7 S . GHISLA, P . HEMMERICH, U . H A R T M A N N , a n d F . MÜLLER, submitted to Helv. chim. Acta.

8 G. BLANKENHORN, Dissertation, Universität Konstanz 1971. 9 M . BRÜSTLEIN, W . - R . KNAPPE, a n d P . HEMMERICH. A n g e w .

Chem. 83, 854 [1971] ; Internat. Edit. 10, 804 [1971]. 1 0 G . R . PENZER, G . K . R A D D A , J . A . T A Y L O R , a n d M . B .

T A Y L O R , i n : R . S .HARRIS, P . L . M U N S O N , a n d E . D i c z -FALUSY (Editors), Vitamins and Hormons, Vol. 28, p. 441, Academic Press, New York 1970.

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FLAVIN-SENSITIZED PHOTOOXIDATIONS 1035

11 W. HAAS, Dissertation, Universität Konstanz 1972. 1 2 M . B . K A T A N , L . J . GILING, a n d J . D . W . V A N V O O R S T ,

Biochim. biophysica Acta [Amsterdam] 234, 242 [1971]. 1 3 P . HEMMERICH, i n : R . S . HARRIS , P . L . M U N S O N , a n d E .

DICZFALUSY (Editors), Vitamins and Hormons, Vol. 28, p. 467, Academic Press, New York 1970.

1 4 T . W A L S H , A . S C H O N B R U N N , a n d R . H . ABELES, J . b i o l . Chemistry 246, 6855 [1971].

15 P. HEMMERICH, in: 14. Mosbacher Colloquium der Gesell-schaft für Physiologische Chemie, p. 183, Springer, Heidel-berg 1963.

1 6 A . D E K O K , C . VEEGER, a n d P . HEMMERICH, i n : H . K A M I N (Editor), Flavin and Flavoproteins, p. 63, University Park Press, Baltimore 1971.

1 7 W . - R . KNAPPE a n d P . HEMMERICH, F E B S - L e t t e r s 1 3 , 2 9 3 [1971],

18 M. BRÜSTLEIN, Dissertation, Universität Konstanz 1971. 1 9 M . BRÜSTLEIN a n d P . HEMMERICH, F E B S - L e t t e r s 1 , 3 3 5

[1968].

pH-Dependence, Isotope Effect and Products of Flavin-sensitized Photodecarboxylation and Photodehydrogenation

W . HAAS and P. HEMMERICH

Department of Biology, University of Konstanz, Germany

(Z. Naturforsch. 27 b, 1035—1037 [1972] ; received May 10, 1972)

Flavin-sensitized photodecarboxylation and photodehydrogenation

The pH-dependence of flavin-sensitized photodehydrogenation and oxidative photodecarboxyla-tion shows a change of reaction rate with an apparent pK of about 5.5. This "photo-pK" cannot be assigned to any known ground state or excited flavin or substrate species, nor is it related to the kind of bond being broken (C —H or C —COO) . Therefore, it is attributed to the pK of a covalent flavin-substrate intermediate. Carboxylates R —COO - react by decarboxylation, the nature of R determining only the rate of reaction. The acids R —COOH themselves are unreactive, unless functionally substituted at the a-carbon. Hence X—CH(R)—COOH behaves as does X—CH2 —R,

+ + for X = O H , OR, NH2 , NR2 . R3N—CH(R) — COO" is unreactive, while H S N - C H ( R ) - C O O " is slowly dehydrogenated through its neutral tautomer which is favored in the flavin-substrate exciplex.

Photooxidations sensitized by flavoquinone (1) show a change of reaction rate between pH 4 and 7 1 _ 5 . This change, observed with numerous sub-strates, is sigmoid with an apparent pK of about 5.5 (Fig. 1 ) . This "photo-pK" cannot be attributed to a substrate pK nor to the pK of flavin species in any known redox 5 or even photoexcited7 ' 8 state. The "photo-pK" cannot be attributed to flavin-sub-strate JT-complexes since it is independent of sub-strate structure and the kind of bond being broken (C — H or C — C O O " ) . Hence we assign the "photo-p K " to a short-lived, covalent flavin-substrate inter-mediate as could be trapped and characterized in the case of phenylacetate (R = H) under alkaline conditions9 (2, Scheme I ) . The reaction pathway via 2 is not obligatory, since some substrates of flavoquinone-sensitized photooxidations show no "photo-pK" (Fig. 2 ) . Furthermore, kinetic and iso-tope-exchange data1 0 demonstrate a second, direct

Requests for reprints should be sent to Prof. Dr. P. HEM-MERICH, Fachbereich Biologie der Universität, D-7750 Konstanz, Germany.

pathway for the formation of 3 (R = H ) . The sub-strate residue at position C ( 8 ) in the cyclohexa-2,5-dien-l-imine type intermediate 2 readily migrates in an acid-catalyzed rearrangement to position N ( 5 ) yielding the well known 5-alkyl-l,5-dihydro-flavin (3) and subsequently the thermodynamically more stable 4a-alkyl-4a,5-dihydro-flavin (4 ) by ther-mal rearrangement n .

In the case of mandelate (R = OH) only the final 1,5-dihydroflavin (5) can be trapped. W e propose from the pH-dependence of the reaction rate showing the "photo-pK" = 5.5, that in this case a species of type 2 is also formed but decays very rapidly with liberation of benzaldehyde. The latter has been characterized quantitatively.

From this it seems that flavoquinone has two "photoactive sites", i.e. 4a,5-C = N and 8,9-C = C bonds. The choice between them depends on the nature of the substrate and on reaction conditions (pH, temperature and solvent polarity) which in-fluence the structure of non-covalent "preequili-brium complexes". These are typical "exciplexes"