, Matériel et méthodes 2, p.7

. La-7-carboxyéthylguanine-a-Été-synthétisée-au-laboraroire-selon-la-méthode-de-roberts and . Warwick, , 1963.

. La-7-Éthylguanine, la 7-hydroxyéthylguanine proviennent de Chemsyn Sciences Laboratories (Lenexa, Kansas, USA). la 7-méthylguanine provient des laboratoires Ftuka (Fluka Chemie- Bucks, Swiuerland) Tous ces produis ont une pur?té chimique superieure à98 Voen CLHp

, Chapitre 4 : dérivation des T,alkylsuanines oar la ohénvlmalondialdéhvde 2.1.2) Synthèse de la phénylmalondialdéhyde

. La-phénylmalondialdéhyde-aété-synthétisée-selon-la-méthode-de-moschel and . Leonard, , 1976.

. Brièvement, mole de diéthylformamide sont ajoutés à 0,3 mole de chlorure d'hydroxy phosphore (POClg) Le mélange est maintenu à 30 oC pendanr 5 minutes. 0,1 mole d'acide phénylacétique est alors ajouté

, laissée à 40 oC 2 heures afin de terminer la production de diméthylamine. La solution est ensuite extraite au toluène, séchée à I'aide de MgSO4 Le toluène est par la suite évaporé et le résidu est distillé. L'acroléine ainsi obtenu est hydrolysée pour obtenir la phénylmalondialdéhyde comme indiqué ci-apÈs. L'acroléine dans l'éthanol est traitée par une solution de soude ù207o et chauffée sous reflux. L'éthanol est distillé, le mélange est refroidi, exmit avec du dichlorométhane. Enfin, la phase aqueuse est amenée à pH = 2 (HCl) puis concentrée. Le précipité formé est filtré, lavé à I'eau et recristallisé dans I'ether. 2.1.3) Synthèse des dérivés fluorescents 7-alkylguanine : méthode générale, La réaction est chauffée une heure à 110 oC et les produits sont alors recristallisés

, la 7-méthylguanine phénylmalondialdéhyde (PhMal-7-MeGua) recristallisent spontanément dans de I'acide acétique à 4"C. Pour la guanine phénylmalondialdéhyde (PhMal-Gua), la 7- hydroxyéthylguanine phénylmalondialdéhyde (PhMal-7-HetGua3-DHP)-Gua), I'acide acétique est évaporé et le produit est redissous dans un mélange de I ml d'éthanoVeau (70130) dans des tubes à recristallisation de 2 ml, La 7-éthylguanine phénylmalondialdéhyde, pp.3-7

. La-7-carboxyméthylguanine-phénylmalondialdéhyde, PhMal-7-CMGua) et la 7-carboxyéthyl guanine phénylmalondialdéhyde (PhMal-7-CEtGua)

, la dérivation se fait dans des tubes coniques de 20o pl (Alltech-Deerfield, IL, USA) silanisés par du diméthyldichlorosilane (DMCS 20 Vo dans de I'héxane) Les rubes sont ensuite rincés au méthanol et séchés à I'acétone. Laes 7-alkylguanines sont solubilisées dans une solution d'acide chlorhydrique (HCl 0,1 M) à la concentration de I mg/ml (cette solution concenrrée est concervée en fractions aliquot à -20.C)

, eau ultra-pure (Milliporc) et ammenées à pH = 9 environ par de I'ammoniaque concentrée Une gamme de 2 à 25 pmoles de jalkylguanines en solution aqueuse est déposée dans les tubes coniques. La solution est évaporée, puis le résidu est repris par 10 pg de phénylmalondialdéhyde (500pg,/ml d'acide acétique glacial) et chauffé durant 40 min à 1l0oc. Après évaporation de I'acide acétique, le résidu est repris par un mélange composé d'acétonirrile/ I(H2PO A, Q0/80) et analysé par CLHP. 2.1.5) caractéristiqges chimigues des Ph Ma l-7-alkyl guani, I-es dilutions successives sont effectuées dans de

. Grenoble, Les spectres de masse ont été faits par Mr J.H. LAMB (MRC Toxicology Unit - Laboratoire du Dr P. FARMER; Carshalton, UK) er les spectres fluorescents (spectrofluorimétrie dbxcitation synchrone)

, Laes PhMal-7-alkylguanines ont été séparées pour I'analyse CLHP sur colonne phase inverse Nucleosil (CI8, l0 pm, 250x8x4 mm, Macherey Nagel-Diiren-Allemagne) munie d'une précolonne remplie de phase ODS (Pellicular ODS-Whatmann) à température ambiante er à un débit de 1,3 mVmin. Le gradient délution est composé de tampon phosphate (KH2PO4, l0 mM, pH -7,l) additionné de 27o de tétrahydrofurane (THF) pour le tampon A et d'acétonitrile (solvant B). l? gradient d'élution compone une étape

, La déaection de la fluorcscence e$ réalisée à I'aide d'un déaecaeur Spectroflow 980 (Applied Biosysteme) à une longueur d'onde d'excitation de246 nm muni d'un filne

, Chaoitre 4 : dérivation des 7-alkylguanines par la phénylmalondialdéhyde PhMal-7-MeGua 8

, CHt, tr):4,4 -4.53 GCU2-, q), pp.47-79

, so3

, 42 -7 fi 343 -3.49 (-CH2-OH. m):3.88 -3.97 GCHOH. m), pp.3-9

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