Quenching of singlet oxygen by alkaloids and related nitrogen heterocycles

Phyrochemrstry, Vol 23, No IO, pp 2351-2354, 1984 Pnnted III Great Bntam

0

0031-9422/84 $3 00+0 00 1984 Pergamon Press Ltd

QUENCHING OF SINGLET OXYGEN BY ALKALOIDS AND RELATED NITROGEN HETEROCYCLES RICHARD A

Institute for Environmental

LARSON*

Studies, Environmental

and KAREN

A

MARLEY

Research Laboratory, Umverslty of Ilhnols at Urbana-Champatgn, Urbana, Illmols 61801, U S A

(Received2 February Key

1984)

Word Index-Photochemistry, singlet oxygen, alkaloids, mdoles, antloxldants, ultraviolet-B

Abstract-Fifteen plant alkaloids and related heterocychc compounds were tested for their ability to quench singlet oxygen Most of the compounds showed high activity, brucme and strychnine were especmlly effiaent quenchers Brucme, at a concentration of ca 2 6 x lo-’ M, IScapable of mactlvatmg half the singlet oxygen molecules it encounters This quenching may serve in nature to protect plants from the deleterious effects of singlet oxygen or other reactive oxidants

INTRODUCTION

Many physlologtcal functions for the plant alkaloids have been proposed, mcludmg roles as growth regulators, agents for lomc balance, nitrogen storage compounds, and feeding mhlbltors or toxicants for herbivores [l] However, despite the wrdespread use of mtrogencontaining antioxidants such as ethoxyqum (1) m mdustrial processes, the suggestion that alkaloids could play a role m protecting plant tissues from damage due to reactive oxygen species such as singlet oxygen (‘0,) or other agents has received httle attention A number of naturally occurrmg photosensrtlzers, such as chlorophyll degradation products, flavms and N-formylkynurenme (a metabolic product of tryptophan) may be involved m ‘02 generation in plant tissues [2] Unsaturated membrane hpids react rapidly with ‘02, affording peroxides and other oxldlzed denvatlves Recently, several alkaloid-related drugs have been shown to be potent mhlbltors of hpld peroxldatlon m in mtro systems [3-s] but the mechanisms of their mlubltlon are as yet undescrlbed Some ammo acids, such as hlstldme, tryptophan and methlonme, are also rapidly destroyed by ‘02, either m solution or as constituents of proteins [6] Accordingly, ‘02 quenchers may also be needed to protect enzymes from oxldatlve damage Unsaturated lipids and ammo acids may be oxidized by free radical processes as well as by ‘02 mechanisms, but there ISa high correlation between radical and ‘02 quenching activity [7], thus an agent showing strong activity toward one oxidant IS also likely to protect against other related forms High concentrations of alkaloids are very commonly found m plant epldermal cells, where light mtensltles are highest [l] Because many alkaloids absorb UV-B light strongly, it has been suggested that they, as well as other compounds with sun&r absorbance charactenstlcs located m the superficial cell layers, such as tlavonolds, may be acting as light filters, protecting the mesophyll from UV-B [lo] *To whom correspondence should be addressed

CK

1.

L

Nicotine (2) was demonstrated many years ago [8] to be a relatively efficient quencher of ‘O,, but only one alkaloid, dregamme (3), has since been tested as a ‘02 quencher [9] We report that several alkaloids of various structural types are effiaent quenchers of ‘02 at concentrations comparable to those m plant tissues

RESULTS AND DISCUSSION

Quenchmg rate constants and beta-values for the alkaloids and related compounds tested are listed m Table 1 Several of these compounds were much better quenchers than the tertq amme 1,4&aza[2 2 2]blcyclooctane (DABCO, 4), wluch has been widely used m ‘02 quenching studies and 1sknown to be highly actlve A number of other alkaloids were rather close to the actlvlty of DABCO

2351

R A LARSON and K A

2352

MARLEY

Table 1 Singlet oxygen quenchmg actlvlty of alkaloids and nitrogen heterocycles Compounds Brucme (So) Strychnine (Sb) 8-Hydroxyqumohne (129) DABCO (4) Atropme (9) 5, CDlmethoxymdole (&) Vincamme (7) Nicotine (2) Glaucme (11b) Reserpme (6) Boldme (1 la) Eucatropme (10) 5-Hydroxymdole (&I) 5-Methoxymdole (8b) 6-Methoxyqumohne (12b)

Rate constant*

t-J(M)t

(385fO62)xlOs (235fO28)xlOs (105 f 0 02) x 10s (584&085)x lo7 (432fO85)xlO’ (4 29 f 0 57) x 10’ (427&031)x 10’ (382+018)x10’ (308fO30)xlO’ (266fO31)xlO’ (239fO2l)xlO’ (190*043)x106 < 2 x lo5 .1 ” .1 w

26~10-~ 43~10-~ 9 5 x lo-’ 17 x lo+ 23~10-~ 23x10-“ 23 x lo+ 26~10-~ 32x10-* 38~10-~ 42~10-~ 53x1o-3 > 5 x lo-* ,, 3. ,, 3,

OCOHOH 6:I IO

lIncluding standard deviation tj ISthe ratio of the decay of ‘03 due to solvent quenchmg to its decay due to reaction with the quencher It represents the concentration of quencher necessary to inactivate half the molecules of ‘02 m a gwen solvent tndoles Bruclne and strychnine (5a and Sb) were particularly effective quenchers These complex mdole alkaloids have

two nitrogen atoms, but that of the dihydromdole portion of the structure 1s m an amide linkage and 1s only very weakly basic Amides m general are unreactive toward ‘O,, so it 1s a reasonable assumption that the quenchmg activity of brucme and strychnine 1s associated with the other (basic) nitrogen atom Two other mdole alkaloids, reserpme (6) and vmcamme (7), with tertiary basic mtrogen atoms and fully conJugated mdole nuclei, quenched ‘0, efficiently The reactlvlty data for a related mdole alkaloid, dregamme (3), also fall mto this range (2 7 x lo-7) [9]

OR Ilo

R=H

IIb R=CH3

1

Snnple monosubstltuted mdoles reacted much more slowly Hrlth ‘02, as would be expected for a weakly basic heterocycle (Indoles with 2,3_double bond substltutlon, however, are much more reactwe [ll] ) The difference between the simple heterocycles 5-methoxymdole (Bb) and S$-dlmethoxymdole (SC), however, Illustrates the importance of electron-donating groups to IO1 quenching activity Electron-nch phenols and phenol ethers quench ‘02 Hrlth high efficiency [12] It is also likely that the dnnethoxy substltuents of brucme help to make It a somewhat better quencher than strychnme, which lacks such substltutlon, by provldmg an alternative site for ‘02 attack

Other alkalords The large (ca x 20) difference m quenchmg activity between atropme (9) and eucatropme (10) may be explained by the extremely hindered stenc environment of the basic nitrogen atom of the latter compound Slmllar effects were noted m 2,6- and 2,2,6,6-methylplpendmes by Monroe [ 131 Two aporphme alkaloids, boldme (lla) and glaucme (llb), showed comparable actlvltles, m the range of the other tertiary ammes tested Our quenching value for rucotme m chloroform was far higher than that obtained 25 years ago for this compound m methanol (44 x 105) [8] More recently, however, Smith recalculated the quenching constant relative to that of DABCO (4) m pyndme and obtained an approximate value 4 3 x lo6 [14] We are uncertam whether solvent effects, expenmental design, or other factors are responsible for the wide varlablhty of the data relative to this compound The differences m quenching activity between 8-hydroxyqumohne (12a) and 6-methoxyqumohne (12b) are not easy to interpret It 1spossible that the sterlc proximity of the phenohc hydroxyl group with the lone pan on nitrogen helps to promote ‘02 deactlvatlon by faclhtatmg orbital overlap m the transltlon state

2353

IO2 quenchmg by alkaloids

l&,

R,=H

l&,

R,=CCl+,

f$=OH b=H

RI /

\‘i XP

RZ

#Q, R,=OH, R2=H f&, R,-0CH3, &,

R2=H

R,: R2 =OCH,

Quenchtng mechantsms

Ammes quench ‘02 by both physical and chenucal mechamsms [7] In the former, a colhslonal deactivation of IO2 back to the tnplet state has been suggested, perhaps via a charge-transfer complex ‘02 + NR3 + [‘02 - + NR,] + ‘02 + NR, However, many mstances of quenchmg of ‘02 with accompanying reactions are also known For example, some tertiary ammes may be demethylated upon attack by ‘02, with the production of formaldehyde and secondary ammes [9] We have not exammed the quenching reaction mtxtures for the possible presence of reaction products However, the geometry about the tertiary basic nitrogen atom of brucme and strychnme (cf Fig 1) 1s quite suggestive of a site where both physical quenchmg (due to the ngld, cagelike environment of the nitrogen atom) and chemical quenching by reactlon at the nearby electron-nch double bond could occur This sterlc environment may help to explain the unusually efficzent destruction of ‘02 by these alkaloids Ecophysrologtcal stgntficance

Many authors have noted that alkaloids are rapidly metabohzed, or at least turned over, m plant tissues For example, morphine concentrations are highest m the

Fig 1 Geometry around the tertiary basic mtrogen atom of brucme and strychnme

morning and fall durmg the day [15] A conceivable explanation may be that alkaloids are destroyed by oxldatlve reactions, either ablotlc or enzyme-promoted, and converted to other products For example, Catharanthus roseus tissue cultures, when exposed to light, accumulate the oxldlzed alkaloid serpentine (13) at the expense of the reduced alkaloid ajmahcme [16] If the prevention of photochemical oxldatlve damage 1s one possible rationale for the occurrence of alkaloids, they should be common m plant species inhabiting regions of high UV light mtensltles The short-wave ‘UV-B’ regon (290-320 nm) contams especmlly energetic and blologltally damaging wavelengths UV light does appear to play a role m the blosynthesls of some alkaloids, m potato plants, formation of the steroidal alkaloid solamne 1s stnnulated by UV [17] Alkaloids occur with greatest frequency m species of tropical ongm, where UV and UVB mtensltles are much higher than they are m temperate regons [ 181 UV and UV-B light mtensltles also vary with altitude, at 3000 m, UV-B mtensltles are ca 30% higher than they are at sea level [19] There are fewer data on alkaloid concentrations as a function of altitude In two out of three Himalayan Berberts spectes, leaf berberme concentrations were significantly higher m populations from higher elevations [20] In addition, Lycoperstcon species growing at high elevations m Peru contained much higher concentrations of tomatme (a steroidal alkaloid) than related species from lower elevations [G CooperDriver, personal commumcatlon] Although not many North American alpme plants have been mvestlgated for then alkaloid content, 30 (26 %) of the 113 genera which occur m the southern Rocky Mountam alpme tundra regon [21] Include alkaloid-bearing species, compared to ca 9 % for higher plant genera as a whole [22] Although alkaloids are often stored m cell vacuoles, they are actively translocated, and it 1s conceivable that small equlhbrmm concentrations exist m other parts of the cell, such as cell or organelle membranes, where they would be effective m mhlbltmg oxldatlve damage This posslblhty requires further research

EXPERIMENTAL Chemtcols Aikalolds and other heterocycles were obtamed commercially and were pun&d by recrystalhzatlon Theu structures Include examples of mdole, aporphme, qumohne, pyndme and tropane nuclei Detemnnatton of quenchtng rate constants The method of ref [13] was used In this techmque, the ablhty of an added substrate to mhlblt the self-sensmxcd photo-oxidation by vlstble hght of a colored polycychc hydrocarbon sensltlzer, rubrene, IS determined Rubrene ISadvantageous m that the wavelengths which are effective m generating ‘02 occur well mto the visible (> 500nm), so that photochenucal processes due to absorption of light by the alkaloids do not interfere In these experiments, typical alkaloid concns were 1 mM (The mean concn for alkaloids in the leaves of alkaloid-bearmg plants IS0 62 % dry wt [23], or ca 4 mM assummg on average MW of 300 and 80 % Hz0 content ) The solvent used m the quenching studies was CHQ, m which the deoly constant for ‘02 IS ca 1 x l@/sec [24]

Acknowledgements-We thank Carl Palmer, PankaJ Pate1 and Gear@ L Wmter for expenmental assistance, Gllhan CooperDrwer for permission to quote from unpublished results, May R

R A

2354

LARSONand

Berenbaum for helpful dlscusslons and the National 1nstltute.s of Health for partial financml support (Grant No ROl ES02426)

K

A MARLFZY

13 Monroe, B M (1977) J Phys Chem 81, 1861 14 Smith, W F Jr (1972) J Am Chem Sot 94, 186 15 Falrbalrn, J W and Wassel, G M (1964) Phytochemutry 3, 253

REFERENCES

I 8 9 10 11

McKey, D (1974) Am Nat 108, 351 Foote, C S (1976) m Free Radicals m Btology (Pryor, W A, ed) Vol 2, p 85 Academic Press, New York Malvy, C , Paolettl, C , Searle, A J F and Wdlson, R L (1980) Bzochem B~ophys Res Commun 95, 734 Shmashl, N, Anma, T, Aono, K , Inouye, B , Monmoto, Y and Utsunu, K (1980) Physlol Chem Phys 12,299 Koreh, K , Sehgman, M , Flamm, E S and Demopolous, H (1981) Blochem B~ophys Res Commun 102, 1317 Foote, C S (1981) m Oxygen and Oxyradrcals m Chemutry and Btology (Rodgers, M A J and Powers, E L, eds) p 425 Academic Press, New York Bellus, D A (1979) Ado Photochem 11, 105 Schenck, G 0 and Gollmck, K (1958)J Chum Phys 55,892 Herlem, D and Ouannes,C (1978)Bull Sot Chum Fr Suppl I, 451 Robbexecht, R , Caldwell, M M and Blllmgs, W D (1980) Ecology 61,612 Gorman, A A, Lovermg, G , and Rodgers, M A J (1979) .I Am Chem Sot 101, 3050

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16 Knobloch, K H, Bast, G and Berhn, J (1982) Phytochemzstry 21, 591 17 Conner, H W (1937) Plant Phystol 12, 79 18 Levm, D A (1976) Am Nat 110,261 19 Caldwell, M M (1971)m Photophyslology (Geese, A C , ed) Vol 6, p 131 Academic Press, New York 20 Chandra, P and Purohlt, A N (1980) Bwchem Syst Ecol 8, 379

21 Zwmger, A H and Willard, B E (1972) Land Abooe the Trees Harper & Row, New York 22 Raffauf, R F (1970) A Handbook of Alkaloads and Alkalozdcontatnmg Plants Wdey, New York 23 Levm,D A andYork,B M Jr (1978)Blochem Syst Ecol 6, 61 24 Wdkmson, F and Brummer, J G (1981)5 Phys Chem (Ref Data) 10, 809

NOTE ADDED IN PROOF A A Gorman et al petrahedron Letters 25, 581 (1984)] have independently shown that strychnme 1s a very fast physaxl quencher of ‘02 Then quenchmg constants were determmed m different solvents, but are m good agreement with ours