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Formation of N-(Deoxyguanosin-8-yl)aniline in the in vitro Reaction of N-Acetoxyaniline with Deoxyguanosine and DNA.

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[4] In the molecular complex of nylon-6 with 1 the cdrbonyl of perfluoroglutaric acid appears as a sharp singlet in the "C NMR spectrum.
IS] J. W. Visser, J. Appl. Crystallogr. 2 (1969) 89.
161 C. W. Bunn, E. V. Garner, Pror. R. SOC.London A189 (1947) 39.
[7] Bond lengths and bond angles for perfluoroglutaric acid were taken from
the native glutaric acid structure [8J. Fluorine atoms were added with C-F
bond lengths of 1.3 A.
[8] J. D. Morrison, J. M. Robertson, J. Chem. Soc. 1949, 1001.
[9] L. Leiserowitz, Acta Crystnllogr. Sect. B32 (1976) 775.
Q
Sect. 836 (1980) 3196.
[lo] M. Fujinagd, M. N. G. James, A C ~Crysfullogr.
[ l l ] 0 . Ermer,J. Lex,99(1987)455; Angew. Chem. Int. Ed. Engl.26(1987)447.
1121 Rotations of the planes of carbon atoms and syn-unfi interconversion of
a carboxyl may contribute to the deviations. These have appeared in
molecular dynamic simulations by Dr. J. J. Wendoloski. personal communica tion.
[13] This is in contrast to the formation of stable complexes between nylon-6
and perfluorosuccinic, -adipic, and -glutark acids.
[14] Other structural variations might occur if, in order to effect better packing
of chains along the 4.7-8, c axis, the nylon adopts a helical conformation,
as in y-nylon. Then, the alternate amide groups are interlinked by
N-H . . . 0 (amide) bonds along the r axis. Furthermore, the interleaving
carboxyl groups. adjacent to such amide groups, could still form
0 - H . . ' 0 (amide) bonds, although distorted as in the crystal structure of
urea.
[15] H. D. Flack. J. Polym. Sci. Part A-2 10 (1972) 1799.
Formation of N-(Deoxyguanosin-8-yl)aniline
in the in vitro Reaction of N-Acetoxyaniline
with Deoxyguanosine and DNA **
By Michael Famulok and Gernot Boche *
The acute toxicity of aniline 1 is due to the formation of
methemoglobin; the metabolite phenylhydroxylamine 2 appears to play the crucial role in this process.". 21 In the Ames
test,[3a11 and 2 exhibit mutagenicity in the S. typhimurium
TA 98 strain only in the presence of norharmane (H-pyrido[4,3-b]indole = P-carboline) and rat liver h ~ m o g e n a t e . ' ~ ~ *
In animal experiments, no tumor induction in B6C3F1 mice
was observed even after feeding them high doses of aniline
hydrochloride (6000 and 12 000 ppm) for long periods
(103 weeks). By contrast, half of that amount of aniline
hydrochloride (3000 and 6000 ppm, respectively, 103 weeks)
caused Fischer 344 rats to develop sarcomas in the spleen.[41
Of interest, therefore, is the observation that 14C-labeled
aniline binds to DNA in the kidneys, colon, and spleen of
rats, whereas practically no DNA binding could be detected
in mice.151Both recent[6a1and earlier investigations[6b1have
further shown that N-acetoxyarylamines are key metabolites
in the carcinogenesis of aromatic amines. They are formed
by 0-acetylation of arylhydroxylamines or by N,O-transacetylation of the corresponding hydroxamic acidsL6]
(Scheme 1).
oxidation"'
I
H20, THF,
NEt,, 37OC, DNA
24 h
1 ) nuclease P i
DNA adduct
/
2) alkaline phosphatase
Scheme 2
We recently showed that O-acetyl-N-(4-biphenylyl)hydroxylamine (N-aceto~y-4-arninobiphenyl)~~~~
and O-acetylN-(2-naphthyl)hydroxylamine("N-acetoxy-2-naphthylamine")[8b1react with deoxyguanosine 5 to give adducts that
have been isolated by other authors['] from the DNA of the
bladder epithelium of dogs after they were treated with 4-biphenylamine and 2-naphthylamine, respectively. Here we
report that a corresponding metabolite of 1, compound 4,
which can form in vivo from 2 and 3,[61
reacts with 5 to give
an adduct, N-(deoxyguanosin-8-yl)aniline 6. Further, we
were able to show that 6 is also formed in the in vitro reaction
of 4 with DNA (Scheme 2).
Adduct 6 is a new compound; previous attempts using
"activated aniline derivatives" to prepare an adduct of aniline 1 and 5 by in vitro electrophilic substitution failed."']
Only adducts of 5 with aromatic amines known to be strong
carcinogens have been known so far.I8. '1
To show that 4 also reacts with DNA, the DNA was
cleaved hydrolytically after reaction and the DNA hydrolysate was analyzed by HPLC. Figure l a shows the
HPLC profile of the adduct 6; Figure 1 b shows that of the
hydrolysate after reaction of 4 with DNA under the same
conditions. A concentration determination[' 31 revealed that
0.26 % of the deoxyguanosine molecules present in the DNA
had undergone amination by 4 to form the deoxyguanosine
C8 adduct of the DNA.
In conclusion, 4, a potential metabolite and ultimate carcinogen of aniline 1 covalently binds in vitro to deoxyguanosine 5 and DNA to form the deoxyguanosine C8
adduct 6.
ii
ON,,,
2
1
/
0
0-acetytation
//
4
5
0
-
10
t[rninl
15
Fig. 1. a) HPLC profile of 6. b) HPLC profile of the DNA hydrolysate
after reaction of 4 with DNA. HPLC conditions: Nucleosil RP 18, 7 pm,
230.x6-mm column, UV absorption at 260nm. 35% CH,OB, flow rate
0.7 m L min-', retention time of 6, 15.4 mi, [12].
3
Scheme I
Experimental Procedure
[*] Prof. Dr. G. Boche, DipLChem. M. Famulok
Fachbereich Chemie der Universitat
Hans-Meerwein-Strasse, D-3550 Marburg (FRG)
[**I This work was supported by the Deutsche Forschungsgemeinschaft, the
Fonds der Chemischen Industrie, and BASF AG.
468
0 VCH
Verlugsgesellschafi mbH, 0-6940 Weinheim, 1989
Preparation of 4: Compound 4 was first prepared in solution by A . M . Loho et
al. [14]; we have now isolated and completely characterized 4. AcCN (2.07 g,
30.0 mmol) in 20 mL of Et,O was added to a solution of 2 (3.27 g, 30.0 mmol)
and NEt, (3.54 g, 35.0 mmol) in 50 mL of Et,O ( - 40 " C ,20 min). The solution
was stirred for 3 h at - 15 " C , dried with MgSO, at 0 " C , and filtered. The oil
0570-0833j89j0404-0468S 02.5010
Angeu. Chem. Int. Ed. Engl. 28 (1989) No. 4
remaining after removal of the solvent in V ~ C U Owas dissolved in 10 mL of
CH,CI,, and 120 mL of petroleum ether (40-60°C) was than added. The
solution was concentrated to 70 mL and cooled for 3 h at 30 "C. The crystals
that formed were collected and dried at - 20 "C. Yield: 2.95 g (65%); m.p. 2729°C (not corrected); correct C,H.N elemental analysis (the sample was
weighed and maintained at - 78°C until combustion). NMR (400 MHz, CDCI,, 230 K): 6('H) = 2.24 (s, 3H), 7.07 (d, ZH), 7.11 (1. lH), 7.35 (t, 2H), 8.86
( s , IH, NH); 6(*'C) = 19.22, 116.36. 123.89, 129.00. 146.08, 170.78; IR (Nujol): ?[cm-l] = 3253, 1758, 1603, 1494, 1378, 1220, 723.
Reaction of4 with 5 to give 6 : Compounds4 (605 mg, 4.00 mmoi) and 5 (1.07 g,
4.00 mmol) and NEt, (455 mg, 4.50 mmol) were stirred in a 7:3:4 mixture
(70 mL) of EtOH, CHCI,, and H,O under N, for 24 h a t 37 "C. After removal
of the solvent, the residue was dissolved in 400 mL of H,O and the resulting
solution wasextracted with 150mLofEt,O(6x)and lOOrnLofnBuOH(5x).
After removal of the nBuOH, the residue was dissolved in 10 mL of 50%
CH,OH and 6 was purified by HPLC (30% CH,OH, 230 x 20 mm), LiChrosorb, RP I X . 7 pm, 1400 psi = 98 bar). Yield: 6%. NMR (400 MHz,
[D,]DMSO): 6('H) = 2.02 (m, lH), 3.76 (m, 2H), 3.93 (m, lH), 4.43 (m, lH),
~
2-U-Benzylglyceraldehyde: A Synthetic Building
Block Available in Both Enantiomeric Forms
and Configurationally Stable Owing to Rapid
Oligomerization **
By Volker Jager* and Volkmar Wehner
Optically active C, building blocks of type 1 are of great
importance in organic synthesis.['] Thus far, the key part is
taken by 2,3-O-isopropylideneglyceraldehyde2, whose two
enantiomers are readily accessible['. 21 and which finds highly diverse uses due to the free aldehyde and the protected diol
function. However, 2 is not stable as a monomer and undergoes racemization on st0rage.1~1
5.36(s,lH,OH),5.98(s,lH,OH),6.33(q,lH),6.45(s,2H,NH,),6.90(t,lH),
7.25 (t. 2H). 7.73 (d. 2H), 8.63 (s, l H , NH), 10.83 (s, IH, NH), a sugar H is
overlapped by DMSO signals;6("C)('J(C, H)[Hz]) = 38.39(t, 131.28),61.20
(t. 141.33). 71.14 (d. 150.52). 82.72 (d, 163.03), 87.08 (d, 146.00), 112.14 (s).
117.21 (d. 162.10). 120.44(d, 160.00), 128.39(d, 158.10), 140.77(s), 143.11 (s),
149.39 (s). 153.01(s), 156.03 (s); IR (KBr): ?[cm-'] = 3343, 3206. 1681, 1600,
1562,1498,1355.1099,751;
UV (CH,OH): A,, [nm] = 283.0.204.0; FD-MS:
mi; 358 ( M e ,
26.1 "Yo), 359 (M'
H, loo), 381 ( M e + Na, 33.5).
1
+
Reaction of 4 with DNA to give 6: A solution of calf thymus DNA (30 mg), 4
(30 mg. 0.2 mmol), and NEt, (17 mg, 0.17 mmol) in 20 mL of H,O/THF (l/l)
was kept for 20 h at 37 "C. After removal of the THF, extraction with 25 mL of
Et,O ( 3 x ), and addition of 10 mL of H,O, the H,O phase was treated with
2 mL of a 3 M NaOAc solution and 50 mL of EtOH. The DNA was precipitated
at -20 'C, centrifuged at 10000 rpm for 10 min, and washed twice with 70%
EtOH. Alter addition of 30 mL of H,O, 1 mL of the solution contained 0.95 mg
of DNA (UV determination of concentration). Following the literature procedure [lS]. 0.95 mg of DNA was hydrolyzed with nuclease PI and alkaline
phosphatase and the enzymes were then precipitated by addition of 800 pL
of 3 M NaOAc solution and 20 mL of EtOH at -20°C and removed by centrifugation. The solvent was removed from the supernatant and the residue was
dissolved in 1 mL of 35% CH,OH and analyzed by HPLC.
Received: November 28, 1988 [Z 3067 IE]
German version: Angew. Chem. 101 (1989) 470
[I] M . Kiese: Ferrihemoglobinemia: A Comprehensive Trearise. CRC Press,
Cleveland, OH, USA 1974, pp. 3ff.
[2] D. Henschler (Ed.): Gesundheitsschudliche Arbeiisstoffu, 1st-4th issue,
VCH Verlagsgesellschaft, Weinheim 1987, Aniline (Supplement 1983).
[3] a) D. M. Maron, B. N. Ames, Mutat. Rrs. ff3 (1983) 173; h) M. Nagao,
M. Yahagi, Y. Honda, T. Senio, T. Matsushima, T. Sugimura, Proc. Jpn.
Acod. 53 (1977) 34; c ) J. Suzuki, N. Takahashi, Y Kobayashi, R. Miyamae,
M. Ohsawa, S. Suzuki, Mutot. Res. 178 (1987) 187.
[4] 5iousso.v of Aniline Hvdrochloride,for Possible Carcinogenrcity, Publication
NCI CG TR 130. Washington, DC, National Cancer Institute, NIH,
US PHS. Department of Health and Human Services 1978, pp. 1-53.
[5] D. J. McCarthy. W. R. Waud, R. F. Struck, D. L. Hill, Cancer Res. 45
(19x5) 174.
[6] a) C.-C. Lai. E. C. Miller, J. A. Miller, A. Liem, Carcoinogenesis (London)
9 (1988) 1295; b) Review: D. W. Hein, Biochim. Biophys. Acta 948 (1988)
37
[7] a) J. W. Gorrod, L. A. Damani (Eds.): Biofogical Oxidation of Nitrogen in
Orgunic Molecules, Ellis Horwood, Chichester 1985; b) J. W. Gorrod. D.
Manson, Xenobioiica 16 (1986) 933.
[8] a) M. Famulok, F. Bosold, G. Boche, Angew. Chem. 101 (1989) 349;
Angew. Chc.m. Int. Ed. Engl. 28 (1989) 337. and references cited therein;
b) Gtrahedron Lerf.30 (1989) 321, in press, and references cited therein.
191 a) F.F. Kadlubar, F. A. Beland, D. T. Beranek, K. L. Dooley, R. H.
Heflich. F. E. Evans in T. Sugimura, S. Kondo, H. Takebe (Eds.): Environm m t d Mutagens und Carcinogens, A. R. Liss, New York 1982, 385; b)
F. A. Beland, D. T. Beranek, K. L. Dooley, R. H. Heflich, F. F. KadIubar, EHP Envrron. Health Perspecr. 49 (1983) 125.
[lo] M. D. Jacobson, R. Shapiro, G. R. Underwood, S. Broyde, L. Verna, B. E.
Hingerty. Chem. Res. Taxied. f (1988) 152.
1111 Review: H.-G. Neumann, J. Cancer Res. Clm. Oncol. 112 (1986) 100.
[12] Variationofthe HPLCconditions(30% CH,OH, flow rate0.7 mL min-'.
[retention time of 6 : 26.3 min]), as well as addition of authentic 6 to the
DNA hydrolysate, confirmed the presence of 6 in the DNA hydrolysate.
[I31 The concentration of 6 in the DNA hydrolysate was determined from the
area below the HPLC profiles.
[14] A. M. Lobo, M. M. Marques, S. Prabhakar. H. S. Rzepa, J . Org. Chem.
52 ( I 987) 2925.
[lS] C. W. Gehrke, R. A. McCune, M. A. Gama-Sosa, M. Ehrlich, K. C. Kuo,
J . C'hromutogr. 301 (1984) 199.
Angpw. C'hem. IN. Ed. Ennl. 28 (1989) No. 4
2
3
In principle, glyceraldehyde derivatives with one free and
one protected hydroxyl group could offer new applications,
since 02/03
are more strongly differentiated and regio- and
stereoselectivities may be altered by action of the free hydroxyl group in the first or subsequent steps. We report here
a new, simple synthesis of both enantiomers of such a compound, namely, 3, as well as some transformations that confirm this and which, in addition, prove 3 to be configurationally stable at room temperature.
(R)-2-O-Benzylglyceraldehyde,(R)-3,was prepared earlier from D-mannitol (four steps, ca. 5 % overall yield) or
D-glucose (six steps, ca. 50 YO)and used directly without further analysis.[41Both enantiomers, (R)-3 and (9-3, are now
readily obtained, as shown in Scheme 1, from the corresponding tartrates 4 via the known 2-0-benzylthreitols 5 [ 5 1
(three steps, ca. 70% overall yield). The freshly prepared
aldehyde 3,obtained as a colorless oil upon distillation, initially has a fruitlike odor; upon standing, it turns sirupy,
then becomes a waxy, odorless solid. This behavior is reminiscent of the oligomerization and polymerization known
€or simiIar hydroxyaldehydes.161Indeed, neat 3 evolves into
a mixture of at least ten oligo-/stereoisomers. The I3C NMR
spectrum of a sample recorded after 17 h showed a small
peak at 6 = 202.4 (CHO), but at least 32 signals in the region
6 = 60-100 where three are expected for the monomeric
aldehyde 3.
What about the configurational stability (C-2) of 3, the
crucial factor determining its preparative utility? The rapid
oligomerization [to (hemi)acetals] gave fair promise that the
racemization observed €or monomeric relatives such as 2
might be obviated. The optical rotation of freshly distilled
(R)-3 in ethanol was found to be + 21.7 (c = 1.23), which
decreased rapidly (+ 16.1 after 6 h), but later increased slowly and finally remained constant at + 30.3 (* 0.8) after 19 d!
[*I
[**I
Prof. Dr. V. Jdger, DipLChem. V. Wehner
Institut fur Organische Chemie der Universitat
Am Hubland, D-8700 Wiirzburg (FRG)
This work was supported by the Fonds der Chemischen Industrie, the
Deutsche Forschungsgemeinschaft, and Bayer AG, Wuppertal-Elberfeld.
We thank the state of Bavaria for a Graduiertenforderungsstipendium ( K W ) , Dr. C. Hubschwerlen (Hoffmann-La Roche, Basel), Dr.
A . Kleemonn. and Dr. K . Drauz (Degussa AG, Hanau) for donation of
chemicals, and H . Sartkr for experimental assistance.
," VCH Verlugsgerellschafi mbH, 0-6940 Weinheim. 19x9
0570-0833lR9J0404-0469$02.50/0
469
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