synthesis and characterization of the fluorescent productsderived from malonaldehyde and amino acids

8/7/2019 Synthesis and Characterization of the Fluorescent ProductsDerived from Malonaldehyde and Amino Acids

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Synthesis and Cha racterization of the Fluorescent ProductsDerived from Malonaldehyde and Amino Acids"K . S. Chio and A. L. Tappel

ABSTRACT: Amino acids or their esters and n-hexylamine reactwith malonaldehyde to yield conjugated Schiff bases. TheSchiff bases possess characteristic absorption in the ultra-violet and visible regions. In solutions, Schiff bases derivedfrom amino acids show spectral shifts on standing at roomtemperature, and isosbestic points are found at 40 140 4 mp.cis-truns isomerization about the C=C bond or C=N bondmay account for the observed spectral shift. The electronic

Dialdehydes, such as glutaraldehyde, can react bifunc-tionally with proteins to give inter- and intramolecular cross-linking. Crystals of carboxypeptidase A, when reacted withglutaraldehyde, becom e completely insoluble in 1 M NaCl andshow a marked increase in mechanical strength (Quiocho andRichard s, 1964). It was assumed that the reaction of the alde-hyde and the amino groups of the protein resulted in Schiffbase formation. A review of bifunctional protein reagents wasrecently given by Wold (1967). Other aliphatic dialdehydes,like glyoxal and malonaldehyde, have been shown to reactwith DNA (Brooks and K lamerth, 1968); malonaldehyde alsoreacts with proteins (Kwon an d Brown, 1965) and with amin oacids (Crawford et al., 1966). T he mechanism of the reactionbetween malonaldehyde a nd glycine has been investigated byCrawford et a/. (1966). It involves a 1,4 addition of the nucleo-philic nitrogen atom of glycine to th e enolic carbon a tom ofthe cu,P-unsaturatedcarbonyl system of the enol of malonalde-hyde to form the enamine, N-prop-2-enolaminoacetic acid.By condensation with malonaldehyde in 10 N HCI at 25O, ar-ginine was converted into &N-(2-pyrimidinyl)ornithine (King,1966). Aromatic, but not aliphatic, primary amines reactwith malonaldehyde to yield N,N'-disubstituted l-amino-3 -iminopropenes, which are fluorescent conjugated Schiff bases(Sawicki et al., 1963).This investigation further examines the interaction of ali-phatic primary am ines, such as amino acids and n-hexylamine,with malonaldehyde to produce N,N'-disubstituted 1-amino-3-iminopropenes and includes study of their spectroscopicproperties.Methods and Materials

Materials. Malonaldehyde bis(dimethy1 acetal), that is,1 , I,3,3-tetramethoxypropane,was obtained from Aldrich

* From the Department of Food Science and Technology, Universityof California, Davis, California 95616. Receiaed December 27, 1968.This investigation was supported by U. S. Public Health Service Re-search Grant A M 09993 from the National Institutes of Arthritis andMetabolic Diseases.

P R O D U C T S O F

absorption a nd fluorescence properties have been a ttributedto the chrom ophoric system NC=C C=N which contains sixa electrons. Infrared spectra contain a band in the 1650-cm-lregion indicating the presence of a C=N bond. Mass spectralanalyses are ca rried out after the Schiff bases are reduced withsodium borohydride to confirm that 1 mole of malonaldehydereacts with 2 moles of amino acid ester or n-hexylamine toyield N,N'-disubstituted I-amino-3-iminopropenes.

Chemical Co. Glycine, L-valine, L-leucine, L-valine methylester hydrochloride, and L-leucine ethyl ester hydrochloridewere products of Nutritiona l Biochem icals Corp. Triglycineand pentaglycine were from Mann Research Laboratories.n-Hexylamine and Spectrograde KBr were from MathesonColeman and Bell. Sephadex G-10 and Sephadex LH-20 werepurchased from Pharmacia Fine Chemicals Inc. Bio-Gel P-2(200-400 mesh) is a product of Bio-Rad Laboratories. So-dium borohydride was obtained from Sigma Chemical Co.Synth esis of N,N '-Disubs tituted I-Amino-3-iminopropenesfrom Amino Acids. Two types of synthesis of amino acid withmalonaldehyde were carried out. Firstly, an amino acid wasreacted with malonaldehyde to obtain solid derivatives forspectroscopic studies. Secondly, the derivative of the m ethylor ethyl ester of the amino acid and the aldehyde was prepared,so that after reduction by sodium borohydride, the product,N,N'-disubstituted 1,3-propanediamine, could be used formass spectrometric studies and elemental analysis. Elementalanalyses were performed at the Microanalytical Laboratory,University of C alifornia, Berkeley, Calif.A. GLYCINE.An amino acid reacts with malonaldehyde toform an enam ine, with an am ino acid to aldehyde ratio of 1 :1.These reactants also form a fluorescent N,N'-disubstituted1-amino-3-iminopropene; a product with an amino acid toaldehyde ratio of 2 :1. The fluorescent product obtained fromglycine and malonaldehyde is prepared by the same methodas for preparation of th e 1 :1 product (Crawford et a/., 1966).A so lution of malonaldehyde acetal (24.6 g, 0.15 mole) in 13.5ml of 1 N HCI was allowed to stand a t 40' with occcasionalshaking until miscible. After cooling to room temperature,the solution was added to 7.5 g (0.1 mole) of glycine in 20 mlof water. After stirring for 1 hr and then standing at 4'for 1 hr, the solid product (amino acid:aldehyde, 1 :1 ) wasremo ved by filtration. Th e yellow filtrate containing the prod-uct (amino acid: aldehyde, 2 :l) ,which fluoresced under ultra-violet light (366 mp), was extracted four times with equa l vol-umes of ether to remove excess malonaldehyde and then ly-ophilized. After lyophilization, 3.4 g (3 6 x yield) of crudeflu-orescent product was obtained; 200 mg of the crude productwas dissolved in 3 ml of water and applied to a Bio-Gel P-2

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O.l:0.4 AWAVELENGTH, rnp

FIGURE 1: Absorption spectrum of the 1-amino-3-iminopropenederivative of glycine in water (10 pglml; I-c m cuvet) at (1)0, (2) 65,an d (3) 240 min.

column (100 X 2.5 cm). The colum n was eluted with distilledwater a t a f low ra te of 68 d / h r using a Technicon proportion-ing pump. Th e 6.8-ml fractions were analyzed by absorbanceat 400 mp or by fluorescence at 450 mp when excited at 370mp. Th e major fluorescent fractions peak at fraction 66. Th eeffluent, abou t 80 ml, was pooled an d lyophilized (80 mg, 40%yield), and the process of chromatography and lyophilizationwas repeated. The yellow, fluorescent compound was hygro-scopic, turned brown on heating to 136-140", and the yellowcolor faded on standing at room temperature or when exposedto light.Th e molecular weight of this glycine-malonaldehyde deriv-ative was determined by its elution position from a calibratedSephadex G-10 column by the method of Whitaker (1963).The colum n w as calibrated with glycine, triglycine, and p enta-glycine. From this linear relationship between VJV, and thelogarithm of the molecular weight, the 1-amino-3-iminopro-pene derivative of glycine gave a molecular weight of 182(theoretical mol wt 186).B. L-LEUCINE.The L-leucine and malonaldehyde deriv-atives were prepared by the same method as for glycine. A so-lution w hich contained 2.6 g (0.02 mole) of leucine in 100 mlof H 2 0 was added to a s olution of malonaldehyde acetal (4.9g, 0.03 mole) in 2.7 ml of 1 N HCl and allowed to stir for 1 hr .The suspension was cooled, filtered, and excess malonalde-hyde was extracted with ether. The aqueous solution waslyophilized, acetone was added , and the u nreacted leucine wasremoved by filtration. Th e filtrate containing the fluorescentproduct was evaporated to dryness (0.7 g, 23% yield) andpurified by chrom atography as before. Maximum absorbancewas found a t fraction 46. The leucine derivative has the samecharacteristics as those of the glycine derivative, and decom-poses at 170-173'.C . L-VALINE.The L-valine derivative was prepared in thesame way, with 4.5 g of v aline in 50 ml of H 2 0 and 10 ml ofmalonaldehyde acetal in 5.5 ml of 1 N HC1. However, after ly-ophilization the produc t w as dissolved in absolute ethanol, theunreacted valine was removed by filtration, and the ethanolfrom the filtrate was evaporated under vacuum (1.6 g, 30%

I"

2000 1750 1500 1400 1300 1200 1100 1000WAVENUMBER, CM- I

FIGURE 2: Infrared spectra of the 1-amino-3-iminopropene deriva-tive of L-leucine ethyl ester . (A ) Before reduction with sodium boro-hydride (KBr disk) and (B) after rzduction with sodium borohydride(liquid, neat).

yield) and purified as before, with highest absorbance beingfound a t fraction 50 . Th e compound decomposes at 155-160".Synthesis of N , N ' -Disubstituted I-Amino-3-iminopropenesand Their Sodium Borohydride Reduced Compounds fromAmino Acid Esters and n-Hexylamine. A. L-LEUCINEETHYLESTER. To 2 g (0.01 mole) of L-leucine ethyl ester hyd roch lorid edissolved in 2 ml of H 2 0 was added 2.46 g (0.015 mole) ofmalonaldehyde acetal in 0.5 ml of 1 N HC1. Th e solution wasstirred for 1 hr, and excess aldehyde was removed by extract-ing four times with equal volumes of ether. The aque ous layerwas neutralized with 6 N NaO H, and the neutral fluorescentester derivative was extracted with ether. After evaporatingthe ether, the yield of the orange ester derivative was 1 g(60% yield). The fluorescent ester derivative was purified bypassing it through a Sephadex LH-20 column (45 x 2.5 cm)with absolute ethanol as eluent at a flow rate of 60 ml/hr. Th epeak was at an elution volume of 117 ml. For reduction, 100mg of the semisolid ester derivative was dissolved in 5 ml ofabsolute ethanol and four batches, 40 mg each, of sodiumborohydride were added. The reduction was completed whenthe solution did not fluoresce under ultraviolet light (366 mp).After the ethanol was removed by vacuum evaporation, 10ml of H20 was added to the residue and then the derivativewas extracted with ether. The ether solution was evaporatedto dryn ess (70 mg, 70% yield) and the residue which was N,N'-(1,3-trimethylene)bis(~-leucineethyl ester) was purified twiceby passing it through the Sephadex LH-20 column. Th e pu-rified compou nd eluted as a single peak and was a pale yellowliquid.Anal. Calcd for G9H38N204:C, 63.69; H, 10.61; N, 7.82.Found: C, 63.57; H, 10.64; N, 7.98.

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TABLE I : Electronic Absorption Spectral Characteristicsa of 1-Amino-3-iminopropene Derivatives of Amino Acids and n-Hexyl-amine.-__ -Absorption Maxima and (Molar ExtinctionCoefficients)

Compound Solvent 0 rnin 240 min Isosbestic pointRN HC H= CH CH =N R, HZO 240 (5,400) 403 (2,810)

R = CHLCOOH 256 (9,85 0) 256 (4,65 0)280 (3,65 0)b 280 (3,620)b370 (1, 360)b 370 (3,96 0)435 (4,65 0) 435 (1,45 0)'JR = CH(C OO H)C H(C H& H2O 240 (4,510) 404 (2,600)256 (6,31 0) 256 (4,57 0)290 (5,070)* 290 (5,450)373 (2,96 0) 373 (4,46 0)435 (2,89 0) 435 (1,120)b

240 (4,84 0) 401 (2,770)256 (6,58 0) 256 (4,62 0)282 (4,1 40) '~ 282 (4,62 0)373 (2,70 0) 373 (3,99 0)435 (2,810) 435 (1,050)bR (CHJjC Ha Ethanol 303 (27,800) 303 (27,800)393 (2,37 0) 393 (2,370)

K = CH(COOH)CHsCH(CH& HzO

a Wavelengths are given in millimicrons; the m olar extinction coefficients are in parentheses. Shoulder.

B. L-VALINEMETHYL ESTER. The reduced derivative of theprod uct of L-valine methy l ester (0.01 mole) and ma lonaldehy de(0,015mole) was prepared a nd purified in the sam e way as forL-leucine ethyl ester. The product, N,N'-(1,3-trimethylene)-bis(L-valine methyl ester), obtained was 820 mg (55% yield)of a pale yellow liquid.Anal. Calcd for C I ~ H ~ O N ~ O ~ :C, 59.60; H, 9.93; N, 9.27.Found : C, 60.53; H, 10.13; N, 8.31.c.n-HEXYLAMINE. Similar ly, instead of am ino acid, n-hexyl-amine (0.01 mole) was reacted with malonaldehyde (0.015mole) and after sodium boro hydride reduction an d purificationby Sephadex LH-20 column chromatography, yellow-whitesolids (410 mg, 34% yield) appeared upon evaporation of theethanol. Further purification was carried out by dissolvingthe solids in 1 N HCI and filtering. Th e filtrate w as neutral-ized with 4 N NaOH and the white precipitate was extractedinto ether. After evaporation of ether, the precipitate was re-crystallized in ethanol and white crystals of N,N'-di-n-hexyl-1,3-p ropa nedia mine with a mp 53-54' were obta ined.

Anal . Cal cd for C15H34N2: C , 74.38; H, 14.05; N, 11.57.Fou nd: C, 74.10; H , 13.80; N, 11.50.Absorption Spectroscopy. The electronic absorption spectrain the visible and ultraviolet regions were recorded at roomtemperature with a Beckman DB-G spectrophotometer on aSargent recorder (Model SRL). The spectrophotometer waspreviously calibrated using a holmium oxide standard.Infrared Spectroscopy. Infrared spectra of the derivativeswere obtained with a Beckman IR-5 infrared spectrophotom-eter. For a solid compound, a pellet was prepared from amixture obtained by grinding 100 mg of oven-dried Spectro-

grade KBr with 0.5 mg of the compound. Liquid sampleswere run neat with NaCl disks.Fluorescence Measurements. Fluorescence spectra wereobtained using an Aminco-Bowman spectrophotofluorom-eter attached to a Moseley Autograf X-Y recorder. The fol-lowing settings were used: sensitivity 40, meter multiplier0.03, slit arrangement no. 3, and 1P28 photomultiplier tube.Mass Spectroscopy. Mass spectra were determined on aVarian M66 mass spectrometer operating at an ionizationpotential of 70 eV and the electron current was 30 pA. Sam-ples were introduced through the solid probe at 30".Results

Absorption and Fluorescence Spectra. The electronic absorp-tion spectra in a queo us solution of the conjugate d Schiff basesobtained fro m glycine, valine, and leucine showed two m ainabsorption peaks, at 256 and 435 mp, with shoulders at 280and 370 mp. In absolute ethanol, the Schiff base of n-hexyl-amine has A,,, at 303 and 393 mp. On standing at room tem-perature the m aximum at 435 mp decreased in absorba nce andthe shoulder at 370 mp increased in absorbance, and at thesame time the peak at 256 mp decreased and a new peak at240 mp was formed. Th e spectral shift with time of 1-amino-3-iminopropene derivative of glycine is shown in Figure 1,and an isosbestic point a t 403 mp is observed. Table I showsthe extinction coefficients of the derivatives before and afterspectral shift. After the spectral shift, if the aqueous solutionwas lyophilized and redissolved in w ater, partial recovery ofthe original spectrum was obtained. Whether in 0.01 M so-

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TABLE 11 : Relative Fluorescence Intensity of 1-Amino-3-iminopropene Derivatives of Amino Acids and n-Hexylamine.

CompoundRelative Molar Intensity_ _ _ ~ _ _ _ _ _ _ _ -~

Am,, (mp) Excitation at Emissions a tSolvent Excitation Emission Amax* XrnHX'JQuinineRNHCH==CHCH=NR,R = CHzCOOHR = CH(COOH)CH(CH&R CH(COOH)CHsCH(CH&R = (CHe)sCH,

~~ 0.1 N HZS04 350 450 1 1HzO 370 450 0.250 0.250HsOH20Ethanol

370 450 0.432 0,432370 450 0 ,285 0 , 29 0396 462 0 , 2 0 0 0 , 2 0 05 Instrume nt set at emission wavelength maximum . * Instrument set at excitation wavelength maximum.

dium phosphate buffer (pH 7) or in absolute alcohol, the 1-amino-34minopropene derivative of glycine exhibited thesame pattern of spectral shift as in aqueous solution.The infrared spectra of the Schiff bases contain a band ofthe C=N bond at 1650-1655 cm-l, and a band of the C=Cbond in the 1610-1620-~m-~region. The infrared spectrumof the Schiff base formed between leucine ethyl ester and mal-onaldehyde is shown in Figure 2A. After reduction of theSchiff base by sodium boroh ydride, the band s a t 1650 and1610 cm-1 are diminished as shown by the spectrum of thereduced compound in Figure 2B. In Figure 2B the band at1600 cm-1 can be attributed t o the weak asymmetric N H bend-ing.The Schiff bases derived from glycine, valine, and leucinehave intense blue fluorescence in aqueous solution with anexcitation maximum at 370 mp and an emission maximumat 450 m p (Figure 3); that of n-hexylamine in absolute e thanolhas an excitation maximum at 396 mp and an emission max-imum at 462 mp (Figure 4).The results of the relative fluorescence intensity are tab-ulated in Table 11. The fluorescence spectra of the am ino acidderivatives were taken at a concentration of 1 pgjml or less.

At a higher concentration similar to that used for the elec-tronic absorption spectrum, the excitation maximum shiftedwith time from 430 to 370 mp, and the emission maximumshifted from 505 to 460 mp, respectively.Mass Spectra. The mass spectrum of N,N'-(l,3-trimethyl-ene)bis(L-leucine ethyl ester) (Figure 5 ) shows a parent peaka t mje 358 with additional peaks at mje 343 (M - 15)+, mje315 (M - 43)+, and m /e 301 (M - 57)+. Accurate mass spec-tral analysis gave mje 358.2771; the calculated value forCI9H38N2O4was 358.2831. The base peak at mje 286 corre-sponds to a loss of C(=O)OC2H5 from the parent molecule togive the amine fragment plus one H . This amine fragmenta-tion is typical of the ethyl esters of unsubstituted acids (Bie-mann et al., 1961). The intensities (83%) of the peak at mje169 and (78%) of the peak at m/e 171 are derived from [(CHr),-

NHCH(HC==CH2)COOCH2CH3]+which is from anotheramine fragmentation together with loss of the two methylgroups from the gem-dimethyl group.Figure 6 shows the mass spectrum of N,Nf-(1,3-trimethyl-ene)bis(L-valine meth yl ester) with a molecular weight of 302,and peaks at mje 273 (M - 29)+, m /e 259 (M - 43)+, and mje243 (M - 59)+. Accurate mass spectral analysis gave mje

60ih> 401z

20

WAVELENGTH, mp

F I G U R E 3: Excitation (curve A) and fluorescence (curve B) spectra of1-amino-3-iminopropenederivatives of L-leucine in aqueous solution( 1 wdml).

0WAVELENGTH, m@

FIGURE 4: Excitation (curve A) and fluorescence (curve B) spectra of1-amino-3-iminopropene derivatives of n-hexylamine in ethanolsolution (1.25 pg/ml).



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. A , ,-210( I 1

F I G U R E 5 : Mass spectrum of N,N'-(1,3-trimethylene)bis(~-leucineethyl ester).

302.2242; the calculated value for C1 5H 30N 20 4was 302.2205.The base peak at rnje 130 for [NHCH(CH(CH&)C(=O)-OCH3]+is a result of cleavage at the C-N bond.The mass spectrum of N,N'-di-n-hexyl-l,3-propanediamineis shown in Figure 7, with a p arent peak at m je 242 and addi-tional peaks a t mje 227 (M - 15)+,nzje 213 (M - 29)+, and mje199 (M - 43)'. Accu rate mass gave 242.26 69; the calculatedvalue for C15Hr2N 2was 242.2722. T he base peak at m /e 112 isprobably the result of a an d /3 cleavage of the secondary amineand rearrangement to give [CH2NH(CH?)3NHCH2CH2]+less2 H. a Cleavage gave m /e 114, which is [CH3(CH &NH CH$,an d a cleavage together with the formation of olefin gave mje70, that is CH8(C H& CH =CH 2 (Budzikiewiczet af.,1964).Discussion

The N,N '-disubstituted 1-amino-3-iminopropene deriv-atives have been synthesized with a stoichiometry of 2 m olesof an amino acid or its ester or n-hexylamine and 1 mole ofmalonaldehyde. The reaction m ay proceed as shown inScheme I.

SCHEMEI- H a 00-CHCH-CHOH + RNHg+O-CHCH=CHNHRmalonaldehyde amino acid enamine(ester) orn-hexylamine-HsOO-CHCH=CHNHR + RNHz+RN=CHCH=CHNHRN,N'-disubstituted1-amino-3-iminopropene

COOH COOCH,I iCH3~ /CH3R=CH,COOH, CHCH or CHCH ,\ \CH3 CH3COO H CH3 C OOCzH, CH,I / I /

\CHC HLH or CHCHKH , (CH2);CHJCHI CH3\

The electronic absorption is due to the conjugated iminesystem. The N,N'-disubstituted 1-amino-3-iminopropenes ofthe amino acids with absorption maxima at 256 and 435 mpand shoulders at abou t 280 and 370 mp can be compared w ithN-salicylidene valine which has absorption maxima at 257,278, 318, and 411 mp (Heinert and Martell, 1963). Th e spec-

m/ e

FIGURE 6 : Mass spectrum of N,N'-(1,3-trimethylene)bis(~-valinemethyl ester).

trum has been correlated with the p resence of enolimine andthe ketoenamine species of the Schiff base. The spectral shiftobserved for the aminoiminopropene of glycine can be ex-plained in terms of a cis-trans equilibrium as described formalonaldehyde dianils (Feldm ann e t a f . , 1967). The cis-transequilibrium was solvent and tem perature dependent. In polarsolvent (cis) malonaldehyde dianil had an absorption max-imum at abou t 373 mp, and in nonpolar solvent (trans) it hadits maximum at 343.5 mp. As the temperature was increasedfrom -42 to +20", the absorption maximum of rnalon-aldehyde dianil of ben zylamine at 281.6 mp decreased and tha tat 317.4 mp increased. Therefore, the glycine derivative canbe represented asCHc/H 'CH \

N "/I

'C C'/ BoHO "\O H Ocis0 0//b H a N CH NH CB / \\ / \CH CH CHz/H O trans

The fact that lyophilization of dilute aqu eous solutions ofthe glycine, leucine, or valine derivatives, which have beenstanding at room temperature for a few hours, yields onlypartial recovery of the original spectrum, indicates that thesolution can undergo thermal or photoisomerization. Fur-ther investigation is necessary to confirm this p henomenon.The isosbestic points in the ab sorption spectra of the aminoacid derivative s have a bou t th e same extinc tion coefficientsalthough th e extinction coefficients at absorption maxima a redifferent as shown in Table I. This could be due to the dif-ference in the methods used to purify the amino acid deriv-atives. The presence of isosbestic points further supports theexistence of cis-trans isomerization either about the C==Cbond or with regard to the C=N bond. Isosbestic points werefound in the spectra of diacetylindigo subjected to irradiationand the spectral changes were attributed to cis-trans isomer-ization abo ut the C=C bond (Brode et at . , 1954). Fischer andFrei (1957) reported the effect of irradiation on spectral shift

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m /e

FI GU R E 7 : Mass spectrum of N,N'-di-n-hexyl-l,3-propanediamine.

on anils as caused by cis-trans isomerism about the C=Nbond.The chromophoric system NC=C C=N, which containssix ?r electrons, could explain the fluorescence properties ofthe Schiff base derivatives. Sawicki et al. (1963) used the reac-tion of aroma tic amines with malonaldehyde to estimate mal-onaldehyde by fluorometric methods, The T electrons of theconjugated Schiff base were postulated to be d elocalized withthe aromatic rings.Th e absorption a t about 1650 cm-1 in the infrared spectra ofthe amino acid malonaldehyde derivatives suggests that thesecompounds are imines. Malonaldehyde dianil has its C=Nband a t abou t 1650 cm-' (Tsybina el al., 1966). Reduction ofth e C=N bond with sodium borohydride removes the imineadsorption band at 1650 cm-1 a s shown in Figure 2B.The presence of conjugated imine structure is confirmedwhen th e 1-amino-3-iminopropenederivative of leucine ethylester is reduced with sodium borohydride. Purificationthrough Sephadex LH-20 gives pure reduced com pound w hosemass spectrum is shown in Figure 5. The mechanism of thereduction process can be postulated to consist of two steps:(1) the reduction of the C = N bond by sodium borohydride toyield an enamine; and (2) the rearrangement of the enaminethe corresponding imine, which is then reduced to amine bysodium borohydride. The reduction is represented in Scheme11. The reduction of the conjugated Schiff bases of valinemethyl ester and n-hexy lamine is assumed to take place by th esame m echanism.Malonaldehyde could be used in the modification of pro-teins to give fluorescent products. Enzymes with lysine res-idues, such as RNase A, can be inactivated and cross-linkedby malonaldehyde (Chi0 and Tappel, 1969). The importanceof malonaldehyde in biological systems must be emphasized.Malonaldehyde is produced during the y-irradiation of aminoacids such as arginine, glutamic acid, methionine, and homo-cystine (Ambe and Tappel, 1961). It has been shown to bethe major constituent in water extracts of oxidized methylarachidon ate and methyl linolenate (Kwo n and O lcott, 1966a,-b). It interacts with D N A in vitro and in vivo as demonstratedby thermal denaturation profiles, chromatographic behavior,and incomplete degradation of the reaction product by de-oxyribonuclease (Brooks and Klam erth, 1968). Recently,malonaldehyde has been reported to be formed in the irradi-ation of glycerol solution (Scherz, 1968) and in the 6oCoy-irradiation of glucose solution (Scherz and Stehlik, 1968).The aldehyde formed could react with primary amines, such

SCHEMEI1

as amino acids, and could produce adverse effects during theradiation preservation of food. Imines derived from aliphaticaldehydes, for example, acetaldehyde, show a greater ten-dency toward polymerization and subsequent reactions thanthose derived from aromatic aldehydes like benzaldehyde(Roya ls, 1959). The imines produced may play imp ortantroles in the browning of foodstuffs. In the nonenzym ic brown-ing systems of glucose-glycine and of sucro se-glyc ine, fluo-rescence and browning were observed to increase with con-comitant decrease in the content of free a-amino groups (Bur-to n et al., 1963). Reaction products of malonaldehyde andamines appear to be responsible for the yellow color and thefluorescent characteristics of lipofuscin age pigments. Hum anneuron al lipofuscin has an abso rption peak at 375 mp and flu-orescence peaks at 440-460 mp (HydCn and Lindstro m, 1950).Hendley et al . (1963) found fluorescence peaks at 450-470mp when the lipid extract of human heart homogenate wasexcited at 365 mp.

ReferencesAmbe, K. S., and Tappel, A . L. (1961), J . Food Sci. 26, 448.Biemann, K ., Seibl, J., and Gapp , F. (1961),J . A m . Chem.SOC.Brode, W. R., Pearson, E. G., and Wyman, G. M . (1954),Brooks, B. R., and Klamerth, 0. L. (1968), European J .Budzikiewicz, H. , D jerassi , C., and Williams, D. H. (1964),

83, 3795.J . Am. Chem.SOC.76,1034.Biochem. 5,178.



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Feldmann, K., Daltrozzo, E., and Scheibe, G. (1967), Z.Naturforsch. 22B, 722.Fischer, E., and Frei, Y .(1957),J . Chem. Phys. 27,808.Heinert, D., and M artell, A. E. (1963), J . A m . Chem. S U C .85,Hendley, D. D., Mildvan, A. S., Reporter, M. C., andHydCn, H., and Lindstrom, 3. (1950), Discussions Faraday

183.Strehler, B. L. (1963), J . Gerontol. 18, 144.Sac. 9 , 436.

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Inactivation of Ribonuclease and O ther Enzymes byPeroxidizing Lipids and by M alonaldehyde"K . S. Chi0 and A . L. Tappel

ABSTRACT: Quantitative enzyme inactivation by lipid per-oxidation has been studied. Sulfhydryl enzymes are most sus-ceptible to inactivation by lipid peroxidation intermediates.Oxidation products of polyunsaturated lipids also inactivatenonsulfhydryl enzymes, for example, ribonuclease A . Con-comitan t with the loss of ribonuclease A activity is the app ear-ance of fluorescence in the enzyme-lipid reaction m ixture.The inactivated RNase A shows fluorescent monom er, dimer,and higher molecular weight species in the Sephadex G-100fractionation patt ern. Th e fluorescence maximum is at 470mp; and the excitation maximum is at 395 mp. Ribonuclease

Lipid peroxidation h as been considered to be a dam agingreaction in biological systems (Barber a nd B ernheim, 1967).Oxidation of polyunsaturated fatty acids is postulated as amechanism of disruption of biological membranes and hasbeen reviewed by Packer et a/. (1967). Inactivation of sulf-hydryl enzymes in mitochondria has been associated with lipidperoxidation (McK night and H unter , 1966). Tappe l (1965)has summarized the properties of protein damage in peroxi-dation reaction systems and showed that there were consider-able similarities between protein damage by lipid peroxida-tion and that caused by radiation. It is suggested that age

* From the Department of Food Science and Technology, Universityof California, Davis, California. Rece iwd D ecem ber 27 , 1968. This in -vestigation was supported b y U. S . Public Health ServiceResearch GrantA M 09933 from the National Institute of Arthritis an d MetabolicDiseases,

A, inactivated by malonaldehyde, has fluorescence and a gelfiltration pattern similar to that of the e nzyme inactivated byperoxidizing polyunsatura ted lipids. Malonaldehyde is prob-ably the agent responsible for the intra- and intermolecularcross-linking of ribonuclease A. The fluorescence producedfrom the cross-linking is attributed to the conjugated iminestructure formed in protein between two e-amino groups andmalonaldehyde. There are marked similarities between theribonuclease A-polyunsaturated lipid product a nd age pig-ment; cardiac age pigment is a protein-lipid complex and bothhave similar fluorescence characteristics.

pigments were derived from oxidized lipid constitutents ofdamaged mem branes. In the study of the mechanism of dama geof cytochrome c, a lipid peroxide-protein complex interme -diate is reported (Desai and Tappel, 1963). Andrews et al .(1965) showed that in an autoxidized lipid-protein system,lipid intermediates reacted with th e free amino groups of p ro-teins. On the basis of trypsin hydrolysis and hydrogen fluoridesolubility tests, they concluded that reactive lipid interme-diates are produced which insolubilize proteins fiia a cross-linking reaction. By gel filtration and hydrogen fluoride sol-ubility studies, Roubal and Tappel (1966) have shown thatoxidized lipid-protein reaction products resulted in protein-protein cross-linked polymers which a re formed by a free-radical chain polymerization mechanism.Malonaldehyde, one of the many carbonyl compounds de-rived from oxidation of polyunsaturated lipids, was shown toreact with bovine serum albumin (Kwon and Brown, 1965;

I N A C T I V A T I O N O F R I B O N U C L E A S E 2827

synthesis and characterization of the fluorescent productsderived from malonaldehyde and amino acids

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