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The structure of tetramycin, a new polyene macrolide antibiotic
THE STRUCTURE OF TETRAMYCIN, A NEW POLYENE MACROLIDE ANTIBIOTIC
Tetramycin(l)isanantihmgal,tetmaiemacroMeanti-
&ctron attdment @A) massspectmmetry (MS) could fufniah tk exact mfkular weight for I. From its bioticproduadbyS&#omycwnwneiHaz..enand Brown,l!M var. jam& IKW.var.JA 3789.Earlier,we mkroalysis and that of derivatives,I was tentatively kgln?d the mok!allarformulflCvHnNO*r(molecular pbYsico-c*=d Now we descriibe weight 6!@).’Howeva subsequent‘% NMR analyses llug8adthattctramycinhaa35catoms.uvspectra the completestructaoftctramycinash&rredfrom dklouedtheprcscnceofatetracnechro~feachemkaltraMf~tions~~similarto -tic3 (A= thWerqnMedinthestlIIcWstadiesofpimaricin~ 280,2!&304and 25,200, 53,MO, 85,700 and 75,ooO) lucensomycia,’tctriasA6and B’ and kacidin,’ aa well !izrl? as from detailedMS, ‘H and UC NMR spectroscopic remi&ent~f relatedpolyene antSoti~s.~ Treatment UAanged, indkatexaminationsof I, its dcivativca and transformation with lunt& leaves the chromophcm? iagthcabscIKcofanyfrccallylicoHgroup.oncatalyproaucb(PkL1). tic hya I absorbsBve molarcquivakntaof hydrogen; four of these arc accounted for by t&e Gaerul &am&&&n of tetmych Tetramycinis tctreaemoktywhilcthelifthmoleii’consumuibyan l&ddkMl&llbkbondtKe8entinthefonnofana;8clo!3elyrelated to pimericin(II). hlceasomYh 09 pnsatmrtedesta pxlp. I%e nature of the latex Ub tMill9A(fV)lUldB(V).ThelWptiWstroctmes~ aatwakn followedfrom~noft&IRapectraof dkP&& kF@ 2.. I with the comspondtqo SpWtrllm of decanotaWlntbcmml~0fgly~idic polyene antiiitics, neitba &ctron impact (El), Mu hydrot&amycin.The IR SpectNmof I di!3cloaedthe
N-ACEIVL - IE lRANVUN MEEIHVL ESTER
T
C4 Nz ACETONE+ ACE TALDEHVDE
N-ACEIVL-IETRAWVCIN
METHVL GLVCOSIM OF UVCOSAMNE
Cn Hr Fi&1.Dthativad~pmdocbobcliDedflomI. 1851
The structure of tetramycin
1853
Table I. Periodate uptakeof I and its derivatives moles periodates
0.5 h
TETRAI.IYCIN
1 h
2 h
4 h
8 h
3.3
3.8
3.8
DECAHYDROTETRAlnYCIN N-ACETYL DECAHYDROTETRAMYCIN
0.02
during the reduction to the saturated hydrocarbon. Finally, the existence of four OH groups in the aglycone of I is confirmed by the IH NMR spectrum of the decahydrotetramycin acetate. Hence the molecular formula of I is C28H 3203+ C6H 13N04(mycosamine) + CO2+ 4H20 = C35H53N0I3, with a molecular weight of 695. These results have been confirmed by EI-MS of perhydrotetramycin (Fig. 4). The parent peak at m!e 506 arises from the loss of 1 mycosamine and 2 water molecules, establishing the molecular weight of perhydrotetramycin as 705. Compared with the fragmentation pattern of I this means the uptakeof 5 moles of hydrogen per molecule which is in accord with the results of the catalytic hydrogenation. The inaccuracy of analytical data from which the C~53N014 formula was calculated is due probably to the solvation of the antibiotic crystals. The molecular weight of I has been later supported by means of field desorption mass spectroscopy." This technique has been shown to be the method of choice in determining molecular weights of several antibiotics of low volatility or thermal instability." The molecular ion in the FD mass spectrum of I appears at mle 696 (M+H). Retroaldol cleavage. The location of the tetraene chromophore within the macrolide skeleton was deduced from retroaldol cleavage. Treatment of I with 2 N NaOH (Fig. 5) and subsequent continuous extraction with ether at ambient temperature afforded a yellow substance assigned as 12 - ethyl - 13 - hydroxy - 2,4,6,8,10 tetradecapentaenal (Fig. 5). Its high resolution mass spectrum showed the molecular peak at mle 246.1636 (CI~2202)' Characteristic peaks appeared at mle
hydrocarbon is represented by the cluster of ions at mle 411 (M+ +OH). This is in conformity with the molecular formula C28H58 (M = 394). Therefore, the basic skeleton of I contains 28 C atoms. The size of the carbon skeleton of tetraene macrolide antibiotics could be established also on the basis of mass spectral data.
Mass spectral studies Determination of the molecular formula and structural assignment of the aglycone. Recently, we reported on the mass spectral behavior of some tetraene antibiotics by means of EI-MS, EA-MS and DADI mass spectroscopy." On the basis of these results, we have determined the carbonskeleton of the algycones and the number of their OH groups, using the parent compounds. The major mass spectral fragmentations of the parent compounds are relatively straightforward and the respective ions are formed by elimination of mycosamine, decarboxylation of the carboxyl group and loss of a varying number of water molecules according to the number of OH groups in the aglycone moiety (M-mycosamine-COz-n-H20). The apparentdriving force for this fragmentation pattern is the energetically favorable extension of the conjugated polyene system and the production of neutral molecules. The EI- and EA-mass spectra of I showa parent peak at mle 416 for which accurate mass measurement gave the composition of C28H 3203 (Found: 416.2353. Calc: mle 416.2351). The spectra exhibit other intensive peaks at mie 434 and 452, corresponding to the compositions C28H 3404 and C28H3605 (416 + 2H20). Together with the carboxyl group the aglycone skeleton of I contains 29 C atoms. The carboxyl C is apparently splitoff by decarboxylation
+
202.1376 (M+ - CH3CHO), 201.1293 (M+ - CH3-CH+ OH-CH 3-CH=OH) and 173.0974 (M+ - CH3CHDCH2CH3). The UV spectrum exhibited a broad maximum at 377 nm characteristic of a conjugated pentaene carbonyl chromophore" while the IR spectrum showed OH and aldehyde CH stretching bands at 3620, 2855 and 2735 cm", and bands at 1680 and 1590cm-1 attributable, respectively, to a highly conjugated CO function and a polyene grouping. Further structural evidence was provided by the IH and 13C NMR data (Table 2). The 'n
Fig. 3. Fragmentation ion of 1.
1 -co
462 (C28H4605)
-
H
20
- H 0
•
444 (C28H4404)
506.3222 (C29H4607)
L - H
20
488.3129 (C29H4406)
0.01
~ 426.3123 (C28H4203)
-
H20
~
470 (C29H4205)
-l -
Fig.4. Ions observed in EI mass spectrum of decahydrotetramycin.
CO 2
1854
K.Domesaosn et al.
1
OH-
MO
H”YC”J
I
CHJ-CH J
Fw. 5. Rctroaldolcleavageof I.
NMR spectra disclosed the presence of an aldehyde fuoctioo attached to tile end of the peotaene chaio having its last double bond in c[s co&uratioo and provided unambigousinformationsabout the sequence of protons and proton groups in the aliphatic portion of the molecule. The number of C atoms, their chemicalshift values and off-resonancemultiplicities,on the other hand, were found to be in complete agreement with the assumed tetradecapeotaenalstructure. Reduction of the peotaenal with sodium borohydride gave a peotaeoe4liol(Fii 1). Its UV spectrum (AZ? 304,314,32# and 346nm) attested the presence of an isolated peotaeoe chromophore. The peotaenal is formed under conditions known to induce retroaldol reaction. The reakoaldd cleavage was found to be triggered by the 0x0 function: if treatment with sodium hydroxide was performed after the reduction of tetramycin with sodium borohydride, no pentaenal could be detected among the products.
Table2. ‘H and ‘y NMRparameters of 1221hyl-13-hydroxy-24,68,16tetradccapeatacn
Chemical
Shlfte. Yultlpllcity
and Couplln& Constants
Proton
9.46
(d, 9 Hz)
C-13H
6.14
(d,d, 9: 8)
C-14H
7.17
(d,d, 8; 15)
C-15H
6.3 - 6.7
8H, m
5.64
(d,d, 9; 15)
C-23H
2.08
(d,d,t, 9; 6.5; 6.5)
C-24H
3.74
(d,q, 6.5; 6.5)
E25H
1.14
OH, d, 6.5)
~-26~~
1.6
(2H, d,q, 6.5; 6.5)
C-27%
0.88
OH, t, 6.5)
C-28H3
Chemical Shift
C-16H - C-22H
Carbon
Chemical Shift
Carbon
c-13
130.47
C-18
151.90
c-15
130.36
c-16
142.94
c-17
129.33
c-14
139.12
c-19
69.61
C-25
138.82
C-21
52.95
C-24
136.88
C-23
23.50
C-27
132.04
c-22
20.77
c-26
130.92
c-20
11.96
C-28
193.21
a Meeaeucedet ambient temperature in CDC13 at 100.1 and 25.16 MHz. Chemical shifta are in ppm relative to internal Tlds. b The numbering of atoms followe
that of 1.
Tbs stNctum
of tetramycln
1855
In an altemativeba.matdysed hydrolysis, tctramycin wastrcatcdwithsodiwnhydroxidcalldthcnthcproduct was &eamdixtilkd into a soiution containing 2,4dinitrophenylhydrazinc to give the 2,4dinitrophenylhydrazones of acetahkhyde and acetone, aa evidenced by h& resotin ma.98 measurements. !3ubacqucnt acidification of the residue and further steamdistilktion yielded additional amounts of acetaldchyde idcntifkd again through its 2&di&vphenylhydrazone. This second portion of acetaldehyde indicates the occurrence of dccarboxylation of fomlyl acetic acid (HoaCCHzCHO) as shown in Iv and v. These fIndiD@ are in accord with observations made on relateI t&acne antiiiotics aad indkate the occurrence of similar structural ekmcnts in I. The proposed &ucture of tctramycin is in ac4%rdwith biosynthetic considerations assumiog polyacetate origin.’ &3rding to these cons&rations, oxygenation is expected to occur at carbons C-5, C-7, C-9, C-11, C-13 and C-15. However, alternative struchucs, e.g. formation of the 0x0 group at C-7, or hemiketal ring between C-9 and c-5, cannot be 0 p&f exchlded. Following the bioa~thetic studies of 1Cmembcred macrolidc antiiie tics,‘formationofEtllroupatC-24mayberationalized by assuming butyWe incorpora&. In c4mn&ion with the present studies, we have also performed a &ve “C NMR analysis of Nacetyhetramycin methyl ester and N-acctylpimaricin methyl e!StcrfeatWing higher stab@ and better sohlbilitkx in pyridin& and DM!W-& than the underivatixcd antibiotics. In this way, a complete assignment of the “C reaonnnccs in I could be achieved which provided an independent support for the correctnc.ssof the proposed structure of tctramycin. These latter results, together with the stereochcmical conclusions derived from the high frequency (27OMIfz) ‘H NMR spectra, wiU be dcai in a forthcoming report.”
G&NOI~CH,OH~HZO: C, 56.13; H. 7.71; N. 1.76. Calc. for C.JItrNO~CH.OH~H~ C. S7.94: H. 7.75: N. 1.77. Found: C. _. __ ~I 511.67;H, i.rr; N, 1.6796). . . ’ HqmatyUcah~mm~ A so4 of 2SOmg of decabydrotetramycin, 2.5 ml of pyridlne, and 2.5 ml of A@ stood at roomtemp.for48hr.Tbemixtunwuthencoded,dillltsdwith icewater.amIcxtractedwlthCHCl,threetlmes.Thecombkd chloroform extracts woe waskd with water, dtied over Na@,, coaceatrated in wcuo aml chtomatognpbsd on alumina (Wivity II. acidic). Elution with CHClf alforded a foamy r&due, which was dissolved in MeOH. treated with activated charcuel. ftltered and concentrated. Addition of hexane a&rded 127mg of an amorphws solid, q.p. 105-lm”; IR VT cm-‘: 3436(NH), 1740 (aretam). 1675(amhk); ‘H NMR (CD(&) d: 1.88,1.38,2.62(6H), 2.04.2.10 (6H). Convwsion of dccahy&ot&amy& to the parent hydrocarfmn Asolaof4gofdbcPhyQotetnmyciniaa~F~~~~er re8uxfor48hrwlth4gofIiAlH,.The~hangedhydrtdewas lks&oyUIwithAcOEtmdtbemktttteevPpMItedtodryaesS. Tbe nskIoc was dissolved in dibrte H&IO, aod the polyol extracted with n-BuOH. The combkd extracts were was&d with water and the n-BnOH was removed irr vacao. The oily residue was dissolved in a amall amount of t-BuOH and freeze drkd to ykld3gofanoil.
M.pwmdetamiaedonaKoikr&tatrpeandpnuacorr.IR sxcctrawererecordedonPerMn-Elmer325andZclssUR2OIR s&ctmpbotometers, uv spacba on z&i spcctrophotometer Sword UV VIS. ‘H NMR wcctrawae obtahal on Varian HA-188 and XL188 spectrometers. “C NMR spectra were rccordd on Vatian XLlO/lJ Fourier transform kstnunent at 25.16MHz. EA-MS wete obtained on EA-mass soecboxranh (M. v. Ardenoe. Dresden). ELMS were rktermioal0; AEI MS ti S and Joel IMS D 180 mass spectrometers. Termmyci& Wails of fermentation and kolation have been &sail by Dornbergu et eL’ &clshy&ut&rmy& 1p of I was hydrogenated with 58 mg of ~hrlOmlofHOAcfot3hrattoomtemp.ThecsEalystt removed by l&ration, the solvent by freeze-d&g. The residue was wash&iwith ether, dried to give Seontg of decahydro tetramvcfn (88% of t&owl: _ -_ -44.8 (c 0.5. _,. m.o. 2.W dec.: 1ulK DMk I&ti -74.P (c 0.5, p&diW); & 0.35 (UCou &a gei sheets Fo(. Merch, n-BuOH/HOA&O; 4: 1: 9. IR vz cm? 3438.2948. ~.~ . 2868.. 1728. . 1580. . 1m. (Cak. for C&&lO,L __ ._ c. 57.!lS;II, 8.89; N. 1.97. Calc. for Cr&rNOrr: C, 58.71; II. 9.06; N, 201. Cak. for C,&,NO,,H& C, 58.09; II, 8.99; N, 1.33. Found: C. 57a H. 8.90: N, 201%). N-AcetykmmycLr A mixture of 186mg of I and 0.67ml of HC~ialJmlof~MeOHwasstirradatoOfor2hrwhilethe antiitic gmdually dlssolvul. T& resulting soln was concentratedhwcaoandt&reslduewasp4ecipltatedwlthetherto give 88mg of N-ac@k@m ycin; q.p. 15ZlW (from AcOEt); I&= t262.5” (c 0.48, MeOH); uv A= Ml: 280,288.304. 318; IR vzcm-‘: 3438 (OH, NH), 1726 (COOH). 1655 and 1558cm-’ (amide, bends I and 19; II, 0.58 (tk on silica 84 sheet.9 Fm, Merck, a-BuOH/HOAc&O 4: 1:S); (Calc. for
Basr-catdysed hyddysis of I 1. Ixolotgm of 12 - tthyl - 13 - hydtvxy - 2.4.6.8.10- tdmktpenteerud.Asolnof5UJmgofIln25Omlof2NNaOHwas extracted cootinuously with Eta for 38hr at room temp. The Et&) extract was washed with water, drkd over Na#O, and concentrated. The yellow crods pentaenal(86mgl was puritkd by prqmratlve UCon alumina HP= (Merck) usb CHCl,/AcOEt (3 : 2) as solvent under argon atmosphere. The major yellow band was eluted with MeOH and the solvent evaporated to give t& pure pentrenal: q.p. 10~105°; [ah - 15.1”(c 0.8, MeOH); UV AK” 370run; IR vz cm-‘: 3628 (OH), 2855. 2735 WHO). 1688 (conjugated CO), 1590 (C=C sttetcbing). 1tltHcm” (CC tmru): (Calc. for C&nol: ._ - _ mol wt 246.1620.Founds mol wt 246.1636). 2. Idation of acetone and acctakhyde. A suspension of 288mxofIinajolnofZOOmxNaOHlnSOmlofwaterwas steam~distilkd into a satsoln if Z&DPNH in 2N HCl. Ilre distillate (1.5l) was left overnight at 50. t&n l&red, dried and identikd as a mixture of the acetone and acetaldehyde 2,4dlnhrophenylhydraxoaes by MS (6Zmg, mp. 105-1150).(Cak. for t&H&N,: mol wt 238.0702;Found: mol wt 238.0709,Cak. for C&N&: mol wt 224.0545;Fouad: mol wt 224.8549).The aqowusbasicr&duewasacidklthenwithHClamlsteam dlstllkd again Into 2,4-DPNH-saturated 2N HCl until no mote hydrazoae was formed. As before, the precipitate (3mg) was identlfkd as acetaldchyde 2,4db&q&nylhydraxone. Frspamtioa of 12 - ethyl - 2,4,6,8.10- tetmdecaptaau - 1,13 -did20m0ofpentsenalwosdk~lv#lin~mlofMcOH~f andmixedIlpwitb60meofNaBHI.Tbemixtmewasstimdat 00 for 38 mln and at room temp. fur another 36 min. Then was diluted with 28 ml of water and concentrated ia wcuo to remove
24hr 1110 ntl of-HI were removed by distiBathm~168ml of water wma&kdandtbemIxtnrewasextmctedtwlcewith1s8mlof CHCl,. The extracts were waskd with 2% Na&I& q. then with water, and drkd over NaW,. Removal of solvent gave an oil (1.53g) which was dissolved in 55 ml of abs THP and re8uxedfor24hrwith2gofIiAlH,.Theunchangedhydridewas destroyed with AcOEt. After evapomtion of the solvent, the kganicmaterialwasdlssolvedin28%HsSO~andorganic mat&al extracted with hexane. The hexane solution, after bell washed with 18% Na#O, aq. and dried over NatC4, was concentratedandhydrogenatedusIngPtqascatalyst.Ihecatalyst was sqmmtcd arul the concentrated soln chtomatognpbed onacolumnofneutralalumlna(activItyl9.Theftrstfracthms &ted with hsxane were combined, yielded 19omg (996 of theory); IR v~cm-‘: 29#),2850,1465,1380,720; GC WY, 1.5m column, 2.5% lIuhyr.oneon pyrolfth) showed a single peak
t8mdiol u 8 ii#liydimrrwidps; 109m& 8l.p. 13&1#p;‘w A== m (r) 347 (lOlao), 329 @.730& 314 @io.oal),301 m.m.ok.farC&&molwt21Al776.Poad:molwt
%~,D.v~w.I~~,J.vo~oRo~~H.T~~~~, Tdmhdvn 32,3069 (1976). ‘K. w, H. Thrumaad 0. Jb@mdt, Tamkdnm Ldrm 4469 (1976). 4O.c.uk!r,Ac&c%mscpndI126(1964). ‘a. Gmdbo. P. Bravo.A. Aaiko. B. T. Qoka a~6 R. W.
‘RCC.huby,V.F.Qunmn,Y.NhMtrvruxlK.LRins
Jr..J.Am. aem. SM. 93.3736119711. %..L Kblmrk Jr..w. P;.T&r r6d R c. Pm&Y, _- IM 93. 3747(1971). ac.~~KLRinsbut.Jr.,J.~~,l146(1917). ‘w.0r08b&rad~~.~~~t8~b.~k.~.~dit~ etofr 21,16(l!kG). 7& Pm&y ad K. L Rim&& Jr.,1. AM& 29, 103s
.
w-a--
sprialsrvah& Be& (1971). “K. L Kiakt, Jr.,1.C. Cook,Jr.,K. H. Maurerad U. Rapp, I. An&b& 27,1(1974). %k~~rk~.~*~.~~~b~.Miyrmsr,F.Pirbn md0.Lalucl,JJ&&a1(,2860(1977). ‘L.Ibdk&K.DombegmaodH.~tobpablbM.
Tetramycin(l)isanantihmgal,tetmaiemacroMeanti-
&ctron attdment @A) massspectmmetry (MS) could fufniah tk exact mfkular weight for I. From its bioticproduadbyS&#omycwnwneiHaz..enand Brown,l!M var. jam& IKW.var.JA 3789.Earlier,we mkroalysis and that of derivatives,I was tentatively kgln?d the mok!allarformulflCvHnNO*r(molecular pbYsico-c*=d Now we descriibe weight 6!@).’Howeva subsequent‘% NMR analyses llug8adthattctramycinhaa35catoms.uvspectra the completestructaoftctramycinash&rredfrom dklouedtheprcscnceofatetracnechro~feachemkaltraMf~tions~~similarto -tic3 (A= thWerqnMedinthestlIIcWstadiesofpimaricin~ 280,2!&304and 25,200, 53,MO, 85,700 and 75,ooO) lucensomycia,’tctriasA6and B’ and kacidin,’ aa well !izrl? as from detailedMS, ‘H and UC NMR spectroscopic remi&ent~f relatedpolyene antSoti~s.~ Treatment UAanged, indkatexaminationsof I, its dcivativca and transformation with lunt& leaves the chromophcm? iagthcabscIKcofanyfrccallylicoHgroup.oncatalyproaucb(PkL1). tic hya I absorbsBve molarcquivakntaof hydrogen; four of these arc accounted for by t&e Gaerul &am&&&n of tetmych Tetramycinis tctreaemoktywhilcthelifthmoleii’consumuibyan l&ddkMl&llbkbondtKe8entinthefonnofana;8clo!3elyrelated to pimericin(II). hlceasomYh 09 pnsatmrtedesta pxlp. I%e nature of the latex Ub tMill9A(fV)lUldB(V).ThelWptiWstroctmes~ aatwakn followedfrom~noft&IRapectraof dkP&& kF@ 2.. I with the comspondtqo SpWtrllm of decanotaWlntbcmml~0fgly~idic polyene antiiitics, neitba &ctron impact (El), Mu hydrot&amycin.The IR SpectNmof I di!3cloaedthe
N-ACEIVL - IE lRANVUN MEEIHVL ESTER
T
C4 Nz ACETONE+ ACE TALDEHVDE
N-ACEIVL-IETRAWVCIN
METHVL GLVCOSIM OF UVCOSAMNE
Cn Hr Fi&1.Dthativad~pmdocbobcliDedflomI. 1851
The structure of tetramycin
1853
Table I. Periodate uptakeof I and its derivatives moles periodates
0.5 h
TETRAI.IYCIN
1 h
2 h
4 h
8 h
3.3
3.8
3.8
DECAHYDROTETRAlnYCIN N-ACETYL DECAHYDROTETRAMYCIN
0.02
during the reduction to the saturated hydrocarbon. Finally, the existence of four OH groups in the aglycone of I is confirmed by the IH NMR spectrum of the decahydrotetramycin acetate. Hence the molecular formula of I is C28H 3203+ C6H 13N04(mycosamine) + CO2+ 4H20 = C35H53N0I3, with a molecular weight of 695. These results have been confirmed by EI-MS of perhydrotetramycin (Fig. 4). The parent peak at m!e 506 arises from the loss of 1 mycosamine and 2 water molecules, establishing the molecular weight of perhydrotetramycin as 705. Compared with the fragmentation pattern of I this means the uptakeof 5 moles of hydrogen per molecule which is in accord with the results of the catalytic hydrogenation. The inaccuracy of analytical data from which the C~53N014 formula was calculated is due probably to the solvation of the antibiotic crystals. The molecular weight of I has been later supported by means of field desorption mass spectroscopy." This technique has been shown to be the method of choice in determining molecular weights of several antibiotics of low volatility or thermal instability." The molecular ion in the FD mass spectrum of I appears at mle 696 (M+H). Retroaldol cleavage. The location of the tetraene chromophore within the macrolide skeleton was deduced from retroaldol cleavage. Treatment of I with 2 N NaOH (Fig. 5) and subsequent continuous extraction with ether at ambient temperature afforded a yellow substance assigned as 12 - ethyl - 13 - hydroxy - 2,4,6,8,10 tetradecapentaenal (Fig. 5). Its high resolution mass spectrum showed the molecular peak at mle 246.1636 (CI~2202)' Characteristic peaks appeared at mle
hydrocarbon is represented by the cluster of ions at mle 411 (M+ +OH). This is in conformity with the molecular formula C28H58 (M = 394). Therefore, the basic skeleton of I contains 28 C atoms. The size of the carbon skeleton of tetraene macrolide antibiotics could be established also on the basis of mass spectral data.
Mass spectral studies Determination of the molecular formula and structural assignment of the aglycone. Recently, we reported on the mass spectral behavior of some tetraene antibiotics by means of EI-MS, EA-MS and DADI mass spectroscopy." On the basis of these results, we have determined the carbonskeleton of the algycones and the number of their OH groups, using the parent compounds. The major mass spectral fragmentations of the parent compounds are relatively straightforward and the respective ions are formed by elimination of mycosamine, decarboxylation of the carboxyl group and loss of a varying number of water molecules according to the number of OH groups in the aglycone moiety (M-mycosamine-COz-n-H20). The apparentdriving force for this fragmentation pattern is the energetically favorable extension of the conjugated polyene system and the production of neutral molecules. The EI- and EA-mass spectra of I showa parent peak at mle 416 for which accurate mass measurement gave the composition of C28H 3203 (Found: 416.2353. Calc: mle 416.2351). The spectra exhibit other intensive peaks at mie 434 and 452, corresponding to the compositions C28H 3404 and C28H3605 (416 + 2H20). Together with the carboxyl group the aglycone skeleton of I contains 29 C atoms. The carboxyl C is apparently splitoff by decarboxylation
+
202.1376 (M+ - CH3CHO), 201.1293 (M+ - CH3-CH+ OH-CH 3-CH=OH) and 173.0974 (M+ - CH3CHDCH2CH3). The UV spectrum exhibited a broad maximum at 377 nm characteristic of a conjugated pentaene carbonyl chromophore" while the IR spectrum showed OH and aldehyde CH stretching bands at 3620, 2855 and 2735 cm", and bands at 1680 and 1590cm-1 attributable, respectively, to a highly conjugated CO function and a polyene grouping. Further structural evidence was provided by the IH and 13C NMR data (Table 2). The 'n
Fig. 3. Fragmentation ion of 1.
1 -co
462 (C28H4605)
-
H
20
- H 0
•
444 (C28H4404)
506.3222 (C29H4607)
L - H
20
488.3129 (C29H4406)
0.01
~ 426.3123 (C28H4203)
-
H20
~
470 (C29H4205)
-l -
Fig.4. Ions observed in EI mass spectrum of decahydrotetramycin.
CO 2
1854
K.Domesaosn et al.
1
OH-
MO
H”YC”J
I
CHJ-CH J
Fw. 5. Rctroaldolcleavageof I.
NMR spectra disclosed the presence of an aldehyde fuoctioo attached to tile end of the peotaene chaio having its last double bond in c[s co&uratioo and provided unambigousinformationsabout the sequence of protons and proton groups in the aliphatic portion of the molecule. The number of C atoms, their chemicalshift values and off-resonancemultiplicities,on the other hand, were found to be in complete agreement with the assumed tetradecapeotaenalstructure. Reduction of the peotaenal with sodium borohydride gave a peotaeoe4liol(Fii 1). Its UV spectrum (AZ? 304,314,32# and 346nm) attested the presence of an isolated peotaeoe chromophore. The peotaenal is formed under conditions known to induce retroaldol reaction. The reakoaldd cleavage was found to be triggered by the 0x0 function: if treatment with sodium hydroxide was performed after the reduction of tetramycin with sodium borohydride, no pentaenal could be detected among the products.
Table2. ‘H and ‘y NMRparameters of 1221hyl-13-hydroxy-24,68,16tetradccapeatacn
Chemical
Shlfte. Yultlpllcity
and Couplln& Constants
Proton
9.46
(d, 9 Hz)
C-13H
6.14
(d,d, 9: 8)
C-14H
7.17
(d,d, 8; 15)
C-15H
6.3 - 6.7
8H, m
5.64
(d,d, 9; 15)
C-23H
2.08
(d,d,t, 9; 6.5; 6.5)
C-24H
3.74
(d,q, 6.5; 6.5)
E25H
1.14
OH, d, 6.5)
~-26~~
1.6
(2H, d,q, 6.5; 6.5)
C-27%
0.88
OH, t, 6.5)
C-28H3
Chemical Shift
C-16H - C-22H
Carbon
Chemical Shift
Carbon
c-13
130.47
C-18
151.90
c-15
130.36
c-16
142.94
c-17
129.33
c-14
139.12
c-19
69.61
C-25
138.82
C-21
52.95
C-24
136.88
C-23
23.50
C-27
132.04
c-22
20.77
c-26
130.92
c-20
11.96
C-28
193.21
a Meeaeucedet ambient temperature in CDC13 at 100.1 and 25.16 MHz. Chemical shifta are in ppm relative to internal Tlds. b The numbering of atoms followe
that of 1.
Tbs stNctum
of tetramycln
1855
In an altemativeba.matdysed hydrolysis, tctramycin wastrcatcdwithsodiwnhydroxidcalldthcnthcproduct was &eamdixtilkd into a soiution containing 2,4dinitrophenylhydrazinc to give the 2,4dinitrophenylhydrazones of acetahkhyde and acetone, aa evidenced by h& resotin ma.98 measurements. !3ubacqucnt acidification of the residue and further steamdistilktion yielded additional amounts of acetaldchyde idcntifkd again through its 2&di&vphenylhydrazone. This second portion of acetaldehyde indicates the occurrence of dccarboxylation of fomlyl acetic acid (HoaCCHzCHO) as shown in Iv and v. These fIndiD@ are in accord with observations made on relateI t&acne antiiiotics aad indkate the occurrence of similar structural ekmcnts in I. The proposed &ucture of tctramycin is in ac4%rdwith biosynthetic considerations assumiog polyacetate origin.’ &3rding to these cons&rations, oxygenation is expected to occur at carbons C-5, C-7, C-9, C-11, C-13 and C-15. However, alternative struchucs, e.g. formation of the 0x0 group at C-7, or hemiketal ring between C-9 and c-5, cannot be 0 p&f exchlded. Following the bioa~thetic studies of 1Cmembcred macrolidc antiiie tics,‘formationofEtllroupatC-24mayberationalized by assuming butyWe incorpora&. In c4mn&ion with the present studies, we have also performed a &ve “C NMR analysis of Nacetyhetramycin methyl ester and N-acctylpimaricin methyl e!StcrfeatWing higher stab@ and better sohlbilitkx in pyridin& and DM!W-& than the underivatixcd antibiotics. In this way, a complete assignment of the “C reaonnnccs in I could be achieved which provided an independent support for the correctnc.ssof the proposed structure of tctramycin. These latter results, together with the stereochcmical conclusions derived from the high frequency (27OMIfz) ‘H NMR spectra, wiU be dcai in a forthcoming report.”
G&NOI~CH,OH~HZO: C, 56.13; H. 7.71; N. 1.76. Calc. for C.JItrNO~CH.OH~H~ C. S7.94: H. 7.75: N. 1.77. Found: C. _. __ ~I 511.67;H, i.rr; N, 1.6796). . . ’ HqmatyUcah~mm~ A so4 of 2SOmg of decabydrotetramycin, 2.5 ml of pyridlne, and 2.5 ml of A@ stood at roomtemp.for48hr.Tbemixtunwuthencoded,dillltsdwith icewater.amIcxtractedwlthCHCl,threetlmes.Thecombkd chloroform extracts woe waskd with water, dtied over Na@,, coaceatrated in wcuo aml chtomatognpbsd on alumina (Wivity II. acidic). Elution with CHClf alforded a foamy r&due, which was dissolved in MeOH. treated with activated charcuel. ftltered and concentrated. Addition of hexane a&rded 127mg of an amorphws solid, q.p. 105-lm”; IR VT cm-‘: 3436(NH), 1740 (aretam). 1675(amhk); ‘H NMR (CD(&) d: 1.88,1.38,2.62(6H), 2.04.2.10 (6H). Convwsion of dccahy&ot&amy& to the parent hydrocarfmn Asolaof4gofdbcPhyQotetnmyciniaa~F~~~~er re8uxfor48hrwlth4gofIiAlH,.The~hangedhydrtdewas lks&oyUIwithAcOEtmdtbemktttteevPpMItedtodryaesS. Tbe nskIoc was dissolved in dibrte H&IO, aod the polyol extracted with n-BuOH. The combkd extracts were was&d with water and the n-BnOH was removed irr vacao. The oily residue was dissolved in a amall amount of t-BuOH and freeze drkd to ykld3gofanoil.
M.pwmdetamiaedonaKoikr&tatrpeandpnuacorr.IR sxcctrawererecordedonPerMn-Elmer325andZclssUR2OIR s&ctmpbotometers, uv spacba on z&i spcctrophotometer Sword UV VIS. ‘H NMR wcctrawae obtahal on Varian HA-188 and XL188 spectrometers. “C NMR spectra were rccordd on Vatian XLlO/lJ Fourier transform kstnunent at 25.16MHz. EA-MS wete obtained on EA-mass soecboxranh (M. v. Ardenoe. Dresden). ELMS were rktermioal0; AEI MS ti S and Joel IMS D 180 mass spectrometers. Termmyci& Wails of fermentation and kolation have been &sail by Dornbergu et eL’ &clshy&ut&rmy& 1p of I was hydrogenated with 58 mg of ~hrlOmlofHOAcfot3hrattoomtemp.ThecsEalystt removed by l&ration, the solvent by freeze-d&g. The residue was wash&iwith ether, dried to give Seontg of decahydro tetramvcfn (88% of t&owl: _ -_ -44.8 (c 0.5. _,. m.o. 2.W dec.: 1ulK DMk I&ti -74.P (c 0.5, p&diW); & 0.35 (UCou &a gei sheets Fo(. Merch, n-BuOH/HOA&O; 4: 1: 9. IR vz cm? 3438.2948. ~.~ . 2868.. 1728. . 1580. . 1m. (Cak. for C&&lO,L __ ._ c. 57.!lS;II, 8.89; N. 1.97. Calc. for Cr&rNOrr: C, 58.71; II. 9.06; N, 201. Cak. for C,&,NO,,H& C, 58.09; II, 8.99; N, 1.33. Found: C. 57a H. 8.90: N, 201%). N-AcetykmmycLr A mixture of 186mg of I and 0.67ml of HC~ialJmlof~MeOHwasstirradatoOfor2hrwhilethe antiitic gmdually dlssolvul. T& resulting soln was concentratedhwcaoandt&reslduewasp4ecipltatedwlthetherto give 88mg of N-ac@k@m ycin; q.p. 15ZlW (from AcOEt); I&= t262.5” (c 0.48, MeOH); uv A= Ml: 280,288.304. 318; IR vzcm-‘: 3438 (OH, NH), 1726 (COOH). 1655 and 1558cm-’ (amide, bends I and 19; II, 0.58 (tk on silica 84 sheet.9 Fm, Merck, a-BuOH/HOAc&O 4: 1:S); (Calc. for
Basr-catdysed hyddysis of I 1. Ixolotgm of 12 - tthyl - 13 - hydtvxy - 2.4.6.8.10- tdmktpenteerud.Asolnof5UJmgofIln25Omlof2NNaOHwas extracted cootinuously with Eta for 38hr at room temp. The Et&) extract was washed with water, drkd over Na#O, and concentrated. The yellow crods pentaenal(86mgl was puritkd by prqmratlve UCon alumina HP= (Merck) usb CHCl,/AcOEt (3 : 2) as solvent under argon atmosphere. The major yellow band was eluted with MeOH and the solvent evaporated to give t& pure pentrenal: q.p. 10~105°; [ah - 15.1”(c 0.8, MeOH); UV AK” 370run; IR vz cm-‘: 3628 (OH), 2855. 2735 WHO). 1688 (conjugated CO), 1590 (C=C sttetcbing). 1tltHcm” (CC tmru): (Calc. for C&nol: ._ - _ mol wt 246.1620.Founds mol wt 246.1636). 2. Idation of acetone and acctakhyde. A suspension of 288mxofIinajolnofZOOmxNaOHlnSOmlofwaterwas steam~distilkd into a satsoln if Z&DPNH in 2N HCl. Ilre distillate (1.5l) was left overnight at 50. t&n l&red, dried and identikd as a mixture of the acetone and acetaldehyde 2,4dlnhrophenylhydraxoaes by MS (6Zmg, mp. 105-1150).(Cak. for t&H&N,: mol wt 238.0702;Found: mol wt 238.0709,Cak. for C&N&: mol wt 224.0545;Fouad: mol wt 224.8549).The aqowusbasicr&duewasacidklthenwithHClamlsteam dlstllkd again Into 2,4-DPNH-saturated 2N HCl until no mote hydrazoae was formed. As before, the precipitate (3mg) was identlfkd as acetaldchyde 2,4db&q&nylhydraxone. Frspamtioa of 12 - ethyl - 2,4,6,8.10- tetmdecaptaau - 1,13 -did20m0ofpentsenalwosdk~lv#lin~mlofMcOH~f andmixedIlpwitb60meofNaBHI.Tbemixtmewasstimdat 00 for 38 mln and at room temp. fur another 36 min. Then was diluted with 28 ml of water and concentrated ia wcuo to remove
24hr 1110 ntl of-HI were removed by distiBathm~168ml of water wma&kdandtbemIxtnrewasextmctedtwlcewith1s8mlof CHCl,. The extracts were waskd with 2% Na&I& q. then with water, and drkd over NaW,. Removal of solvent gave an oil (1.53g) which was dissolved in 55 ml of abs THP and re8uxedfor24hrwith2gofIiAlH,.Theunchangedhydridewas destroyed with AcOEt. After evapomtion of the solvent, the kganicmaterialwasdlssolvedin28%HsSO~andorganic mat&al extracted with hexane. The hexane solution, after bell washed with 18% Na#O, aq. and dried over NatC4, was concentratedandhydrogenatedusIngPtqascatalyst.Ihecatalyst was sqmmtcd arul the concentrated soln chtomatognpbed onacolumnofneutralalumlna(activItyl9.Theftrstfracthms &ted with hsxane were combined, yielded 19omg (996 of theory); IR v~cm-‘: 29#),2850,1465,1380,720; GC WY, 1.5m column, 2.5% lIuhyr.oneon pyrolfth) showed a single peak
t8mdiol u 8 ii#liydimrrwidps; 109m& 8l.p. 13&1#p;‘w A== m (r) 347 (lOlao), 329 @.730& 314 @io.oal),301 m.m.ok.farC&&molwt21Al776.Poad:molwt
%~,D.v~w.I~~,J.vo~oRo~~H.T~~~~, Tdmhdvn 32,3069 (1976). ‘K. w, H. Thrumaad 0. Jb@mdt, Tamkdnm Ldrm 4469 (1976). 4O.c.uk!r,Ac&c%mscpndI126(1964). ‘a. Gmdbo. P. Bravo.A. Aaiko. B. T. Qoka a~6 R. W.
‘RCC.huby,V.F.Qunmn,Y.NhMtrvruxlK.LRins
Jr..J.Am. aem. SM. 93.3736119711. %..L Kblmrk Jr..w. P;.T&r r6d R c. Pm&Y, _- IM 93. 3747(1971). ac.~~KLRinsbut.Jr.,J.~~,l146(1917). ‘w.0r08b&rad~~.~~~t8~b.~k.~.~dit~ etofr 21,16(l!kG). 7& Pm&y ad K. L Rim&& Jr.,1. AM& 29, 103s
.
w-a--
sprialsrvah& Be& (1971). “K. L Kiakt, Jr.,1.C. Cook,Jr.,K. H. Maurerad U. Rapp, I. An&b& 27,1(1974). %k~~rk~.~*~.~~~b~.Miyrmsr,F.Pirbn md0.Lalucl,JJ&&a1(,2860(1977). ‘L.Ibdk&K.DombegmaodH.~tobpablbM.