Studies of the Solution Structure of the Bleomycin A2-Iron(II)-Carbon Monoxide Complex by Means of Two-Dimensional NMR Spectroscopy and Distance Geometry Calculations

7462 J. Am. Chem. Soc. 1990, 112, 7462-7474

Studies of the Solution Structure of the Bleomycin

A2-Iron(II)-Carbon Monoxide Complex by Means of

Two-Dimensional NMR Spectroscopy and Distance Geometry Calculations

Marcel

A. J. Akkerman,7

Eric W.

J. F. Neijman,7 Sybren ^S. Wijmenga,7 Cornells

W. Hilbers,*

^{7 and}

Wolfgang

^BermeV

Contribution

from

the Department

of

BiophysicalChemistry, Faculty

of

^Science, University

of

Nijmegen, Toernooiveld, 6525EDNijmegen, The Netherlands, andBruker Analytische

Messtechnik GmbH, Silberstreifen, D-7512Rheinstetten, ^West Germany. ReceivedMay ^{1, 1989}

Abstract: Aspart of^our program to delineate the solution structure of bleomycin-metalcomplexes and theDNA_complexes thereof,^we studied thebleomycin-iron(II)-carbonmonoxide complex. Withaidofvarious2D NMR _techniqueswe were able to assignthe and l3C NMR _spectra ofthis_complexcompletely. Vicinal couplingconstant analysis, ^13Cchemical shift information,^andNOEdata revealed theactiveparticipation of^fiveironbinding^sitesin the bleomycin molecule, namely, the secondary aminefunction oftheß-aminoalanine fragment, ^thearomatic pyrimidine, ^{theamide and}imidazole_groupof the/3-hydroxyhistidineresidue, and thecarbamoylgroupofthemannose sugar. A _studyofthe ,3CNMR _spectrumofthe corresponding enriched iron-57 complex enabled^usto establish the carbonmonoxideasthesixth ironbinding^siteviaa 30-Hz coupling constant betweeniron-57 andthecarbonmonoxide carbon-13 atom. Subsequently, NOEdata and thefiveiron coordinationsitesin the bleomycin molecule^{were usedas}interpoint^distancesin distance geometry calculations. In_{this way,} severalacceptable structures^were generatedthat_representthefirstthree-dimensional structure ofthebleomycin-iron(11)-carbon monoxide complex in solution. Also,^adiscussion ispresented ofsome aspects ofthestructure-activityrelationship.

Introduction

Bleomycin A2(BLM,Figure1)belongstoafamilyofantitumor metalloglycopeptides produced by strains

of

Streptomyces^ver- ticillus.

It

was firstisolated in 1966as acopper(II)complex;1,2 however, the

iron(II)-oxygen

^{adduct has}been proposed ^asthe biologically activespecies3-7andisbelievedtobe responsiblefor the cellular degradation of DNA.8 The iron-oxygen ^center produces reactive oxygen radicals,whichmediateoxidative_damage to

DNA.

The C4' _hydrogen of the deoxyribose moiety of ^a pyrimidinenucleotide adjacent to guanosine^hasbeen proposed as the site ofaction of these radicals.6,7,9,10 The bithiazole fragment and perhaps the positively charged tail of BLM ^are required for

DNA

_bindingand sequence-specific recognition.11,12

As partofour program to delineate the solution structure of bleomycin-metalcompounds^and the

DNA

adducts thereof,^we studiedthebleomycin-iron(II)-carbon^monoxide

(BLM-Fe-CO)

complex. This complex^isgenerally^{consideredasa}modelfor the putative active iron-oxygencomplex. The iron coordination in the

BLM-Fe-CO

_complexhasbeen the subjectofearlierstudies.

Takitaetal.13proposed^astructure for this complex in which both amine functions, thepyrimidine^andimidazolegroups, thehistidine amide, ^and thecarbon monoxide molecule ^are chelated to the

iron(II)

^ion. This structure proposal ^was mainly^{based on the} crystal structure ofthecopper(II)complexofP-3A,14^abiosyn- thetic precursorofbleomycin. In contrast, the group of_Oppen- heimer15 suggested,^on thebasisofthe proton chemical shiftsof the

BLM-Fe-CO

complex compared to^thoseofthemetal-free bleomycin, that, instead ofthe histidine amide, the ^mannose carbamoyl group^iscoordinated to the iron ion. In the_present paper,^a complete assignment^isgiven

of

the and ¹³C

NMR

spectraofthe

BLM-Fe-CO

_complexatpH^7. Furthermore,^an assessmentofthemetalcoordinationsitesispresentedalong with

adiscussionofthesolution structure ofthis complex^ascompared tothestructures ofthemetal-freebleomycin16 andthe bleomy- cin-zinc

(BLM-Zn)

molecule.17,18

Materialsand Methods

Materials. BleomycinA2sulfatewas purchasedfrom Nipponkayaku Co. (Tokyo, Japan). Samples ^were prepared under anaerobic (N2) conditions. A3-mg sampleofbleomycin A2 sulfate^was lyophilized^three

fUniversityofNijmegen.

•BrukerAnalytische MesstechnikGmbH.

timesfrom99.8%D20^anddissolved in 300pL of^99.95%D20. Sub- sequently, ¹⁰jrL of^a20mMsodiumdithionitesolutioninD20^and10 pL of^{a 0.195} M solution of_FeS04-7H20 in D20 ^{were added.} The iron(II) ^{sulfate was liberated} from crystalwater byheating^andthen dissolvedin D20. Next,^thepH^was adjusted^{to 7}with 30pL of ^a0.1 M NaOD solution in D20, whereby^thecolorofthesolution_changed fromcolorless tolightpink. Finally,the sample^was exposed to carbon monoxide_gas(Hoek-Loos),causing thecolorofthesolutionto change to bright yellow. Thesamples ^were stored under acarbon monoxide atmosphere in sealed NMR tubes (Wilmad, 5-mm diameter). They couldbekept^{at 277} K forseveral weekswithout_anynoticeabledisin- tegration. The final bleomycin concentration amounted ^{to 5.4} mM.

Samples prepared in H20solutions contained5% D20.

(1) Umezawa, H.; Maeda, K.; Takeuchi, T.; Okami, I. J.Antibiot. 1966, 19, 200.

(2) Umezawa, H.; Suhara, Y.;Takita,T.; Maeda, K.^J.Antibiot. 1966, 19, 210.

(3) Sausville,^E.A.;Peisach, J.; Horwitz,^{S. B.}Biochemistry 1978, 17, 2740.

(4) Sausville,E.A.; Stein,R.W.;Peisach, J.;Horwitz,S.B.Biochemistry 1978, 17, 2746.

(5) Burger, R.M.;Berkowtiz,A.E.; Peisach, J.; Horwitz,S. B.J.Biol.

Chem._1980, 255, 11832.

(6)Giloni,L;Takeshita, M.;Johnson,F.;Iden, C.;Grollman, A.^P. J.

Biol.Chem._1981, 256, 8608.

(7) Wu,^J.C;Kozarich,^J.W.; Stubbe,J.J.Biol.Chem. 1983, 258, 4694.

(8) Umezawa, H. In Bleomycin: Current Status and New Developments;

Carter,^S.K., Crooke,^S.T., Umezawa,H.,Eds.;AcademicPress: New York, 1978; p ^15.

(9) Wu,^J.C.;Kozarich,J.W.;Stubbe,^J.Biochemistry1985, 24, 7562.

(10) Rabow,L; Stubbe,J.;Kozarich,J.W.;Gerlt,J.A.J.Am. Chem.

Soc. 1986, 108, 7130.

(11)Kross, J.;Henner, D.; Haseltine, W.A.; Rodriguez, L.; Levin,M.D.;

Hecht,S.M. Biochemistry 1982,21, 3711.

(12) Fisher, L.M.; Kuroda, R.; Sakai, T.Biochemistry1985, 24, 3199.

(13) Takita, T.; Muraoka, Y.; Nakatani, T.; Fuji, A.; Iitaka, Y.; Umezawa, H.J.Antibiot.1978, 31, 1073.

(14)Iitaka, Y.; Nakamura,H.; Nakatani, T.;Muraoka, Y.; Fujii, ^A.;

Takita,T.; Umezawa, H. J.Antibiot. 1978, 31, 1070.

(15) Oppenheimer, N. J.; Rodriguez, L. O.; Hecht,^S. M. Proc. Natl.

Acad. Sci. U.S.A.1979, 76, 5616.

(16) Haasnoot, ^C. A. G.; Pandit, U. K.; Kruk,C; Hilbers, C. W. J.

Biomol.Struct._Dyn. 1984, 2, 449.

(17) Akkerman, M.A. _{J.; Haasnoot,} C.A. G.; Hilbers,^C. W. Eur. J.

Biochem.1988, 173, 211.

(18) Akkerman, M. A.J.;Haasnoot, C. A. G.;Pandit, U.K.;Hilbers,^C.

W. Magn.^Reson.Chem.1988, 26, 793.

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Bleomycin

A¡-Fe(I¡)-CO

^2D

NMR

J. Am. Chem. Soc., Vol. 112, No. 21, 1990 7463 A. ß-Arainoalanine

5

Figure^1. Structureof_bleomycinA2. Fragment abbreviationscorrespond to those inTable I. The numbers in boldfaceare referredtointhe text.

The numberingofthemetal binding^sitesin Figure ¹⁴correspond withtheitalic numbering in thisfigure.

The BLM-57Fe~CO complex^was prepared,with_{57FeCl2 as}theiron source. The samplepreparation^was analogoustothemethod described above. 57FeCl2 was synthesized from95% enriched 57Fe (Matheson) according^to the literature.15 A 34 mM _sampleofthis complex^was prepared in ^1.3 mLof99.95% D20 solution. It was stored under a carbon monoxideatmosphere in^asealedNMR tube(Wilmad, 10-mm diameter)^{andcould alsobe}kept at 277Kforseveral weekswithout_any noticeable disintegration.

NMRSpectra. The NMR_spectra^were recordedonaBrukerAM 500NMR spectrometer interfaced^toan Aspect³⁰⁰⁰computer. ^De- coupling power^is referencedindB attenuation relativetoamaximum of20W. Thesolvent (HDO^or H20)signal^{was used as an} internal reference

(

⁼ 4.76 ppm at 300 K; ⁼ 4.97 ppm at 277K) ^inthe spectra. The^l3C IDNMR_spectrawere recordedon aBrukerWM200

NMR spectrometer, interfaced to an Aspect ²⁰⁰⁰ computer, using broad-band decoupling(11 dBof20W). The 'H-,3Ccorrelated 2D experiments^were performed^{on a}BrukerAM600NMRspectrometer (Bruker, Rheinstetten, West Germany). The methanolsignal^was used as anexternal reference

(

⁼ 49.3 ppm at 300K)^{in the13C}spectra. The following^2DNMRmethodswere employedforthe spectral assignments discussedin this_paper.

Spin-Echo CorrelatedSpectroscopy (SECSY).20,21 The experiment was performed at²⁷⁷ K in D20. Prior to^thestandardSECSY_pulse sequence,during^therelaxationdelay(rd)period(0.8 s), theHDOres- onance wasselectivelyirradiated(30dB), resulting^{in the}followingpulse scheme:

[rd^{- 90° -} '/2t, ^- 90° ^- _y2r, ^- _z2]

Quadrature detection^{was usedinboth}directions. Foreachvalueof

112FIDs (2Kdata points,acquisition time^0.2048s) were acquired. The valueofr, wasvaried between 0.2 and 102.6msin_stepsof200µ$. Prior to Fourier transformation, the FIDswere multiplied ^{with a sine-bell} windowfunctioninbothdirections. _Spectrawere calculated inabsolute value mode.

Double-Quantum-Filtered COSY(DQFC).22,23 The experiment^was performed ^{at 277} Kon aHaOsolution. Priortothe DQFCpulse^se- quence,during^therelaxation delay period,^theH20^solventsignal^was selectively irradiated (1 s, 13dB):

[rd-90°

-t, ^{-90° - dl - 90° -} r2]

(19) InorganicSynthesis’,Jolly, W.L,Ed.;McCraw-HillBook Co.: New York, 1939; p 102.

(20) Nagayama, K.; Wuethrich, K.; Ernst,^{R. R.}Biochem. Biophys.^Res.

Commun. 1979, 90, 305.

(21) Nagayama, K.; Kumar,A.; Wuethrich,K.; Ernst,^R.R.J. Magn.

Reson. 1980, 40, 321.

(22) Shaka, A.J.;Freeman,^R.J. Magn. Reson. 1983, 51, 169.

(23) Bax, A.; Edwards,M. W. J. Am. Chem.Soc. 1986, 108, 918.

Quadrature detection^intheZ,directionwasachieved by using theTPPI method.24 Foreach valueof_(,,80FIDswere accumulated(2K^data points, acquisition time 0.2048 s). The value of _r, was varied ^over 0.003-51.203 ms in_stepsof 100_ms. The multiple-quantum evolution period(dl)^{amountedto 0.003ms.} Prior to Fourier transformation,the FIDswere multiplied with^asine-bellwindowfunctionin bothdirections.

NuclearOverhauserEnhancement Spectroscopy(NOESY) inD20.25,26

The experiment^was performed at²⁷⁷K. Quadrature detection in^the r,directionwas achieved by usingtheTPPImethod.24 During^there- laxationdelay period theHDOsolvent signal^was selectively irradiated (0.8s, 30dB):

[rd^- 90° ^- _r, ^- 90° -tm- ^90°- t2]

The_spectrawere recordedwitha400-msmixing time(zm). Thevalue of_r,wasvariedover0.003-51.203msin_stepsof100_ms. Foreachvalue of fi,^80FIDswere acquired(2K^datapoints,acquisition time0.2048 s).

Prior to Fourier transformation,^theFIDswere multipliedwith Gauss- Lorentz-like window functions in both directions. Subsequently,^the spectrum^was submittedtoabase-planeflattening procedureusing the baselinecorrection algorithmofPearson.27

NuclearOverhauserEnhancement Spectroscopy(NOESY) inH20.25,26 The experiment^was performed at²⁷⁷K. Inorderto suppress the H2Q solventsignal,^atime-shared long(TSL)observation_pulsewas usedin combinationwithadatashift accumulation(DSA-4),28,25resulting^{in the} followingpulse sequence:

[rd^- 90°^- dl ^- z, ^- 90°^- _Zm^- TSL_pulse^- _z2]

Single side-band detection^{was used}in the , direction (carrierfrequency atlow-fieldsideof_spectrum). Arelaxation_delayof0.8swasused. Prior toZ], an extra delay(dl)^{of25mswas usedfor better}water suppression.

Thetotal mixing time (rm)of400ms includedahomospoilingpulseof 50-ms duration.30 TheTSLpulse consistedof109°_pulseswith inter- mittent_delaysof39.0ms. Thevalueofz,was variedbetween0.025 and 51.225 ms in_stepsof¹⁰⁰ms. Foreachvalueofz,,96 FIDswere accu-

mulated (4Kdata points, acquisition time0.2048 s). Priorto Fourier

(24) Marion,D.;Wuethrich, K. Biochem. Biophys.^Res.Commun. 1983, 113, 967.

(25) Jeener, J.;Meier,B.M.;Bachmann,P.;Ernst, R.^{R. J.}Chem. Phys.

1979, 71,4546.

(26) Macura,S.;Ernst, ^{R. R.}Mol._Phys. 1980,41, 95.

(27)Pearson,G.A._{J. Magn.} Reson. 1977, 27, 265.

(28) Haasnoot, C. A. G.; Hilbers, C. W. Biopolymers 1983, 22, 1259.

(29) Roth, K.;Kimber,B. J.; Feeney, J.J. Magn.Reson. 1980, 41, 302.

(30)Bax,A.; Mehlkopf,A.F.;Schmidt,J.;Freeman, R. J. Magn.^Reson.

1980, 41, 502.

7464 J. Am. Chem. Soc.. Voi. 112, No. 21, 1990 Akkermanet al.

transformation,theFIDswere multiplied with^aGauss-Lorentz window functioninboth directions. Spectra^were calculated in absorption^mode31 and subsequentlysubmittedtoabase-planeflattening procedureusing abaselinecorrection algorithmofPearson.27

-Detected One-Bond l3C-'HCorrelatedSpectroscopy.32·33 The experiment^was performed at 298KinD20using TPPI.24 The following pulse sequence ^was used:

' :

^{rd - 90° - -} 180° ^{- -} 90°^- - , ^{- 180°-} —i, ^{- -} (,

2 ^l 2 ^{1 2}

l3C: rd ^{- -} 180° ^{- -} 90° ^{- -} 90° ^-

During^therelaxation_delayperiod,^theHDOsolvent signal ^was selec- tively irradiated(Is,^30dBrelativeto 20W). The^valueof amounted to1.666 ms (1/(4VCH)). ^Foreachvalueof 56FIDswere accumulated (2K^datapoints, acquisition time^{0.1617 s).} Thevalueof_{r, was} varied over 0.003-16.998msin_stepsof^16.6ms. PriortoFourier transforma- tion,theFIDs^were multiplied with Gauss-Lorentz-like window^functions in bothdirections.

-DetectedMultiple-Bondl3C-'HCorrelatedSpectroscopy.34 The experiment^was performed^{at 298}K in D20. A relaxationdelay period of Iswas applied prior^tothe followingpulse sequence:

'H; rd^- 90° ^-

,

^{- -} _{», ^{- 180° -} i-r,^{- -} ,,

l3C: rd^{- -} 90°^- 2 ^{- 90° - - 90° -}

The value of

,

(1/(2VCH)) ^{amountedto 3.4 ms.} In principle, ^the optimum^choiceof 2^is1/(2VCH),^whereVch^isthe long-rangecoupling constantofinterest. However, in practice,^asomewhatshorter_{value (80} ms)^isfound tobeoptimal^becausedecayofthe magnetization^occurs dueto transverserelaxationandunresolvedhomonuclear_couplings. For eachvalueof_{rt, 96}FIDswereacquired(2Kdata points,acquisition time 0.1716 s). Thevalueof_{r, was} variedover 0.003-32.768^ms in_stepsof

32_ms. Prior to Fourier transformation,^theFIDswere multiplied^with

asine-bellwindow functionin the 2 direction. No_digitalfiltering^was applied in^the , direction.

Distance Geometry Calculations.35 Thecalculationswere performed withaNAS9060computer. The^atomsconstituting^thebleomycin-iron moleculewere represented^as120points; the carbonmonoxidemolecule was notincorporated^inthecalculationssincethepositionofthis ligand in the complex could ^{not be} established (vide infra). Also, ^the bi- thiazole-aminopropyl^endofthemoleculewas notincluded inthis_rep- resentation since therewas noevidencethatit participatedⁱⁿiron binding (videinfra);^onthecontrary, allavailable evidence indicatesthatthispart isattached asaflexibletail to thecore ofthemolecule. Incorporated in the 120points, however,^were thehydrogen ^atomsattached tothe carbon andnitrogenatoms. Thestructuralinformation available forthe iron-bleomycin complex^{was converted} into distance constraintswith defined _{upper and} lower bounds. These constraints were derived as follows:

(a) All bond lengths and bond angles^were allowed to vary^{over a} rangeof1%from their standardvalues.

(b) Torsionangles derivedfromthevicinal couplingconstant analysis (videinfra)^werefixed_byconstrainingthe1,4interpoint^distances(within

1%range). ThiswasdonefortheC„-C6partofthed-hydroxyhistidine fragment (H).

(c) Several partsofthe bleomycin molecule have^arigid conformation.

Theseare thearomatic pyrimidine^andimidazole_groups. Also_{the guióse} and mannose sugars ^were considered rigid^{as forthe} bleomycin-zinc molecule.17 Theseconformationswere kept fixedbyconstraining all^the interpoint distances concerned (within ^{a 1%} range). Thus, _{the sugar} residueswere only allowed^{to rotate} aroundtheglycosidiclinkages, and inaddition,the C5-C6 bonds^were freeto rotate.

(d)All amidebonds were considered tobeplanar/trans.

(e) The bleomycin molecule contains¹⁹asymmetriccarbon atoms,but foreachtheconfiguration ^isknown. The normal distance constraints obtainedforthosechiralcentersdo notdiscriminatebetweenRandS configurations. Hence,^avector contributiontotheerror functionwas usedsuchthat optimizationofthiserror functionboth ensuredproperly bounded distances and the correct chirality ^{about each} asymmetric carbon.35 Thisvectorcontributionwas alsousedtomaintaintheplanarity ofallsp2centers inthe molecule.

(31) Keeler,J.;Neuhaus, ^D.J. Magn. Reson. 1985, 63, 454.

(32)Muller, L. J. Am. Chem. Soc.1979, 101,4481.

(33) Bax,A.;Subramanian,S.J. Magn. Reson. 1986, 67, 565.

(34) Bax,A.;Summers,M.F.J.Am. Chem.Soc. 1986, 108, 2093.

(35) Crippen, G.M.Distance Geometry and Conformational Calculations;

Bawden,D„ Ed.;ResearchStudiesPress: Chichester, England,^1981.

Figure ^2. Contour plotofthe 500-MHzSECSY spectrumofBLM- Fe-COdissolvedin D20recorded at 277K during^solventirradiation.

Connectivity pathways of threonine (T), /3-hydroxyhistidine (H), ^bi- thiazole (B),^and(y-aminopropyl)dimethylsulfonium (S)^are outlined.

Thesubscripts, numbers andgreek letters correspondwiththose in Figure

1.

(f) ^Theiron binding^{sites were} translatedintodistanceconstraints.

They include^theaminefunctionofthe/3-aminoalanine fragment, ^the pyrimidine ringN5nitrogen,^the/3-hydroxyhistidinyl^amidenitrogen,^the imidazole ringN, nitrogen,^andthecarbamoyl nitrogenofthemannose sugar(Figure 1). Thedistances betweentheiron ionand thesebinding siteswere set to0.19-0.23nm.

(g)^Theexperimental^datafromtheNOESYexperiments(videinfra) were included inthe distanceconstraints. The interpointdistance range between protonsexhibitingNOEeffectswas uniformly^setfrom0.24to 0.5 nm. Incase NOE^effectswere observedwith methyl protons, the interpoint distancerange^{was set}from0.28 to 0.6nm. Thiswas neces- sary because themethylgroups^were represented^aspointgroupswith

a0.1-nm radius.

(h)If_nothing^was known aboutthe distance betweentwopoints, the lowerboundwas uniformly^setto0.24nm and the upperboundto2.5 nm. Thelattervalue seemed reasonableforamolecule having^{the size} of_bleomycin.

Subsequently,all_{upper and}lowerboundsofthedistancematrixthus derivedwere smoothedaccording^tothetriangularinequality. At this point, distances between all smoothed upper and lower bounds were selected and embedded in three-dimensional _space. In this way ^a three-dimensionalstructure wasgenerated. Ingeneral, such^astructure willnotsatisfy theinitialdistance constraints,^andfinalrefinementhas tobeachieved inaminimalization routine. In this routine,^aself-cor- recting conjugate gradient algorithm36^isemployed.

In_everycalculation20structureswere generated. In orderto reduce the calculation time, only ⁵⁰⁰ iterationswere carried out in thefinal optimizingstepofthedistance geometry calculations.

Results and Discussion

Assignments. Thespin systems^one expectstoobservein

a

NMR

_spectrum for

BLM

dissolvedina D20^solutionare listedinTableI togetherwiththe fragmentsofthemoleculeand their abbreviations. Residues or groups of residues without (apparent) J_couplings have not been incorporated in this list.

The

NMR

_spectraof

BLM-Fe-CO,

^{recordedat298 and}

277K,^were studied, but in_general,thediscussionwillbefocused on the low-temperature spectrumbecause,below, thestructure ofthelow-temperature complexwillbedetermined. The strategy followed to perform the ^resonance assignments ^is completely analogous to the^{one weused}intheinterpretationofthe

NMR

spectraofBLM16 and BLM-Zn.17 Therefore, only ^a briefdis- cussion ofthe present assignments ^isgiven below.

Theconnectivity patternsthat form thebasis forthe identi- fication of the nonexchangeable protons ^are collected in the spin-echo correlatedspectra presentedin_Figures2-4. Thecross

(36) Perry, A.Int._{J. Comp.}Math. 1978,B6,327.

Bleomycin

A}-Fef¡Ij-CO

^2D

NMR

^{J. Am.} Chem. Soc., Vol. _112, No. 21, 1990 7465 Table I. Networksof_CoupledSpinsin Bleomycin A2in D20

abbr fragment spinsystem

T threonine

ch3-ch-ch

a3mx

P pyrimidinylpropionamide

ch2-ch

^ABX

V methylvalerate CH3-CH-CH-CH-CHj AjMPTXj

H /3-hydroxyhistidine CH-CH AX

A iS-aminoalanine

ch2-ch

^ABX

B bithiazole

ch2-ch2

^AA'XX'

S (7-aminopropyl)dimethylsulfonium

ch2-ch2-ch2

^AA'MM'XX'

G a-L-gulose

ch-ch-ch-ch-ch-ch2

M a-D-mannose

ch-ch-ch-ch-ch-ch2

Figure^3. High-field regionofthespectrum ^shownin Figure^2. The connectivity patternsofmethylvalerate (V),/3-aminoalanine (A),^and pyrimidinylpropionamide (P)^are indicated. Alsoshownare theH,-H2 correlationsof_{the sugarfragments, -L-gulose(G)}and a-D-mannose (M).

peak patterns could ^be easily recognized ^on the basis ofthe knowledgealready availablefromthe spectra of

BLM

and BLM-Zn16,17 exceptforthoseofthe guióse and^mannose moieties.

The connectivitypatternsofthesetworesiduesare drawn in Figure 4. Someofthe resonances were readilyassigned,namely, the mannose H2,H3, andH4 aswellasthe guióse H2 resonance,while otherswere more difficultto_assignmainly^becausetheir_positions are soclose together. For instance the^resonances G4, G5,and G6. are nearly overlapping (cf. Figure 4). Nevertheless,assign- mentscouldbemade byinvolving 'H-13C correlatedspectroscopy (videinfra). Wewillreturn to this_aspectin the following^section.

Obviously, ^{no cross} peaks^are observed fortheresonances

of

the isolated_spinsofthebithiazole aromaticprotons and themethyl groupsofthepyrimidine ring^andthe dimethylsulfonium^residue.

The methylgroups^are assigned^on accountoftheir intensity^and position in the spectrum while botharomaticbithiazole protons can beassigned via themultiple-bond ‘H-13C correlatedspectrum, as

will

be discussedbelow.

The amide_protonsofthethreonine, methylvalerate, bithiazole, andaminopropyldimethylsulfoniumfragments^{can be}identified with theaidofa double-quantum-filtered COSYspectrum ^re- corded in

HjO

^(Figure 5). Cross peaks ^are observedbetween the amide proton ^resonances andthe resonances

of

thenonex- changeable neighboring protons, which ^were identified in the procedure described^above. Also, the position

of

_{the single}sec- ondary amine proton^resonance (5.38 ppm)^{can be}determined in this spectrum via^cross peakswiththe /3-proton ^resonances of thepyrimidinylpropionamide^andaminoalanine fragments. The primaryamine functionsoftheaminoalanineandthepyrimidine fragment^can onlybe assignedina NOESYspectrum recorded

I

Figure^4. Connectivity “walks”ofthemannose and guiósefragments(M, G)^{in the}500-MHz SECSY_spectrumofBLM-Fe-COinD20^recorded at277K. TheH,-H2^cross peaks^are omitted in this figure (cf. Fig.3).

inH20^viacross peakswiththeaminoalanine

-proton

^andthe pyrimidinylmethylresonances, respectively (cf. Figure 6). The resonances ofthe amine groupofthe ^-aminoalanine^are visible separately,whiletheamine groupofthepyrimidinylmoiety^shows onlyone resonance. As inthe

‘H NMR

_spectrumof BLM-Zn,17 thelowestfieldresonance (12.25 ppm; Figure6) belongstothe imidazole

NH

function.

Atthispoint^{we havenot} yetidentifiedtheresonances ofthe secondary amideprotonsofthe aminoalanine, thepyrimidinyl- propionamide,^andthemannose moieties. Candidates forthese protons ^are the six largest ^resonances between 5.5 and 9ppm, whichwere not yet characterized. Theirassignments^were made in analogy with those in the

BLM-Zn

spectrum. Two ^sets of

resonances can be distinguished^on the basisofthe connecting cross peaks and^beassignedtothefreeamidesoftheaminoalanine andthepyrimidinylpropionamide ^residues. We could however not establish at thistimewhich

of

the twocross peakpatterns belongs to either the A or the P moiety (cf. Figure 6). The remaining^{two resonances} (6.19 and 6.43 ppm)^are assigned to thecarbamoyl protonsofthemannose residue. This assignment issomewhat ambiguous^sincein the 5.5-6.5 ppm region in the spectrum^severalbroadresonances are observed. Thesecouldbe due to hydroxyl protons ^whose exchangewith water has been sufficiently^{sloweddown in}the complex(as^a result

of

Hbonds) tomakethemobservable. The carbamoyl^{resonances are some-} what lesserbroadened than in the

BLM-Zn

spectrum, but^ex- changewiththesolventH20protons^isstillsufficiently rapid that in the 2D-NOE_spectrum only the exchange^cross peaksto the H20^{resonance andnot}the diagonalpeaks^are observed (Figure 6). Finally

it

isnoted thattheamide resonance

of

the hydrox- yhistidinyl ^{residuecould not be}traced in any

of

the spectra^as for the

BLM-Zn

_complex17 but in contrast to the free

BLM

spectrum.16

7466 J. Am. Chem. Soc., Vol. ¡12. No. 21, 1990 Akkerman etal.

Figure^5. Partofthedouble-quantum-filtered COSY spectrum

( ,

⁼

2.2-4.9 ppm, 2 ⁼ 5.3-9.2 ppm)recorded at 277KforBLM-Fe-CO

dissolved in H20. The ^solventwas irradiated moderately in order^to reducesaturationofthe exchangeable proton^resonances inthe spectrum.

Thecross peaksbetween the amide proton^resonances andtheresonances

ofthe nonexchangeable neighbors^are indicated. The_meaningsofthe symbols^are given inTable Iandare the same asin Figures^{2 and 3.}

Also, thecross peaks between the _secondary amine resonance ofthe /3-aminoalanine moiety (-4NH, 5.38 ppm)^andthe neighboring ^ßreso- nances are indicated.

All lH

assignments^are summarized inTable

II

_alongwiththe

BLM

and

BLM-Zn

data.

I3CAssignments. The^l3Cspectra^were recorded at 298 K in D20. On inspection ofthe

ID

13C spectrum of

BLM-Fe-CO

(Figures^{7 and}8), ^{one observes53} separatedresonances,while

BLM-Fe-CO

contains56carbonatoms. Sincethe methyl^car- bonsofthedimethylsulfonium group^are magnetically equivalent,

one expectsthat three other carbonresonances overlap in^the13C spectrum, which ^{is indeed} the case at 68.1 ppm.

All

carbon resonances can be assignedwiththe aidof

-detected

one-bond andmultiple-bond 'H-13C correlatedspectra(Figures7-9). The one-bondcorrelated spectrum^was recordedwithoutcarbonde- coupling, which^resultedinantiphase doublets(in the/2 direction).

The spectrum^was recorded inthis manner becausewe were also interestedin_{the VCH}couplings, which^{can be}extractedfromthe antiphase doublets.

The assignments of all carbon ^resonances except for ^those arising from^thequaternary^{carbonscan be}establishedinarather straightforward^manner (Figures⁷and 8) because everyproton resonance inthis spectrum^isconnectedtothecarbonresonance

ofthedirectlyattached carbon atom via the doublet^cross _peaks.

In the proton assignment^{sectionweshowed}thatthe assignment ofthe mannose H2, H3, and H4 and the_{guióse H2}protons ^was rather straightforward. ^{Here,we can see} that ^{in the} 'H-13C correlated spectrum in Figure^8,the positionsofthe sugar H6 and H6- resonances (as well ^as the correspondingC6 carbons) ^are readily derived via the characteristic^cross peakpatternsusually obtainedformethylene fragments (one carbon^resonance connected with two protonresonances). The_assignmentoftheseresonances to either the_guióseor mannose sugar moiety follows from the characteristic ./-coupling pattern of the mannose methylene fragment^intheprotonspectra,^asinthecase ofthezinc complex.

Now, only^themannose Hs andtheguióse H3, H4, and H5 protons are not yet assigned. Onaccount ofthe positionsofthe antiphase cross peaks in Figure 8, it isconcluded that one sugarproton resonance hasthesame chemicalshift_position^asboth_H2reso- nances. In combination with the connectivitypatternsinarelayed coherencetransfer(RCT)spectrum (not shown), this^resonance

isassignedto the guióseH3proton. Now,the positionof_{the guióse} H4proton followsdirectly ^{from the}SECSYspectrum shown in Figure ^4. It ^{is seen} that this resonance has nearly the ^same chemical shift _position as the mannose H6and the _{guióse H6.}

««76 PPM

Figure^6. Contour plotofthe relevant partoftheNOESY_spectrumof BLM-Fe-COrecorded inH20^{at 277 K. Thesolvent}signal^was sup- pressedwith aidofasemiselectiveobservation_pulse (TSLONG)^com- bined with adigital shift accumulation (DSA-4). ^{Asa result}ofthe applicationofthis method, saturationofthe exchangeableproton^reso- nances isavoidedbutresonances near thesolventsignal^{are somewhat} suppressed. Theidentificationofseveral exchangeable protons^isshown here. The freeamide _groups oftheaminoalanine and propionamide residuesare indicatedby^asubscriptCNH2^{whereasthemannose car-} bamoylgroup^isdenoted by NH2.

resonances. Subsequently, the positionsofboth_{sugar H5}protons can beestablishedvia thecross peaks in Figure⁸ with_{the yet} unassignedcarbonresonances. Note thattheseproton^resonances reside in the region

of

the proton spectrum where three sugar proton ^{resonances were} alreadyassigned (M4, M6, G6-). The assignmentsofcarbonresonances M4, M5, andG5 were established viaacomparisonwiththe 13Cspectraoffree

BLM

and

BLM-

Zn.18

The multiple-bond'H-I3Ccorrelated spectrum(Figure9) ^shows more complicated^cross peakpatternsthanthe one-bond spectrum.

Therefore,^adetailed descriptionofthe quaternary carbon^reso- nance assignments ^ispresented. As an example, ^we shall

first

considerthequaternary ^carbons ofthe carbonyl groupsofthe primary^and secondary amides and then^thoseofthe aromatic residues.

All

_primary and secondary amide carbonyl carbon resonances display^cross peakswithresonances ofnonexchangeable neighboring protons. The secondary amide carbonyl carbon resonances ofthepyrimidinylpropionamide^andhydroxyhistidinyl fragments^{can be}distinguished^becausethelatterexhibitsavery small cross peak(not shown) withthehistidinyl /3-proton ^reso- nance.

Subsequently, thearomaticcarbonresonances ofthepyrimidine, bithiazole, ^and imidazole _fragments can be recognized via characteristic connectivity patterns with^resonances ofthe protons

Bleomycin

A¡-Fe(II)-CO

^2D

NMR

J. Am. Chem. Soc.. Vol. 112, No. 21. 1990 7467

Figure^7. Contour plotofthe600-MHzproton-detected one-bond'H-13Ccorrelated spectrumofBLM-Fe-COrecordedinD20^{at 298}K. The spectrum was recordedwithout carbon decoupling. ^Asaconsequence,the 'H-13Ccorrelations_appearasdoubletsinwhich the components^are separatedby the VCHcoupling. Forreference purposes,^a200-MHzl3Cspectrum^ispresentedalong the , ^axis. Theconnectivitiesenclosed inthe boxare shown in more detailin Figure ^8.

attached to thesearomaticresidues. Typically,^thepyrimidine

C2,C3,andC4 resonances showcross peakswiththe resonance

ofthe methyl group

of

this residue (Figure 9). They ^{can be} distinguished ^on the basis

of

their chemical shift differences.

Furthermore,^theC6 resonance ofthisresiduecan beassigned^on account ofcross peaks with the a- and _/3-protons

of

_{the pro-} pionamide fragment^andasmallcross peakwiththepyrimidine methyl group protons (not shown). The imidazoleC5 resonance shows cross peaks with _{all proton} resonances

of

the hydroxy- histidine fragment. Characteristically,^thebithiazole

Cr

^resonance

can berecognized via^cross peakswiththe^a- and/3-proton ^res- onances ofthisresiduealongwith^aconnectivity to the aromatic H5- resonance. Asaconsequence, thearomatic protons (H5^and H50^{can now be}distinguished in the proton spectrum. Having attained this information

it

ispossible to assign the remaining quaternary bithiazole^{carbonresonances onthebasis}ofcrosspeaks tothe

Hj

^and Hs-resonances.

Finally, byelimination, the yet unassigned low-field carbon resonance (218.9 ppm)must belong to thecarbon monoxide. For

this assignment, additional proof^{was obtained}from a

ID

13C

spectrumof^an enrichedBLM-57Fe-COcomplex where^a VFcC coupling (30 Hz) ^{isobserved between} iron-57 and the carbon monoxide carbon atom (Figure 11). This establishesthatthis ligand ^isdirectly^{bound to theiron atom.}

Thel3C assignments^aresummarizedinTable

III

and compared to

BLM

and

BLM-Zn

data.

TheThree-DimensionalStructure. With theelucidationofthe and ,3Cspectraof

BLM-Fe-CO,

^{the basisis}provided for the three-dimensional structure determinationofthe complex. As iscustomary, this

will

beattempted by making^use ofavailable J coupling and NOE information. Thus, for D20 ^and H20 samples,NOESY_spectra^were recorded at277K witha mixing timeof400ms (Figures ¹²and 13).

At

this temperaturepositive cross peaks^are observed, howeverwith low intensity. This^reflects thesituation that, forthe complex, btc ^{isclose to}unity (at ^11.7 T).

All

nontrivialNOEs (i.e., excluding NOEs^betweengeminal andvicinal protons)^are numbered_{in Figures} 12and 13;they^are listed inTable V.

7468 J. Am. Chem. Soc., Vol. _112, No. 21, 1990 Akkerman etal.

Figure^8. Partofthe contour plot^shownin Figure^7. The_assignment ofthecross peaks^isindicated. Foranexplanation,^seetext. Around61.5 ppm,doubledoubletsare seenthatare typicalofthe methylenegroups ofthemannose and guiósefragments(M6and G6).

InspectionofTableVshowsthatmostoftheNOEsare identical withthoseobserved forthe

BLM-Zn

_complex.17 For instance, theNOEsobservedfor the sugar^residues (NOEs 9-12^and22 in TableV) ^{were also} recorded in the NOESY _spectra ofthe

BLM-Zn

_complex.

It

_appearsthereforethattheconformation andorientationof_{the sugar}moieties,with_respecttoeach other andtothehistidine_residue,isthesame in both_complexes. This

istrue for other_partsaswell. For_instance,the_secondaryamine group^and theimidazolering^are directed towardeachother,^as isindicated_byNOEs23-26 (TableV). ^{Thisisalsothecase in} the

BLM-Zn

complex. Furthermore, ^no nontrivial NOEsare observed for therest ofthebithiazole _fragment and the (ami- nopropyl)dimethylsulfonium partofthemolecule, reflecting the larger

flexibility

^{ofthese moieties.} Evidently, this partofthe molecule is attached to the core as a flexible

“tail”,

^as in the

BLM-Zn(II)

complex. On the other hand, there^{are alsosome} interestingdifferences. Forthe

BLM-Fe-CO

_complex,no NOEs

are observedbetweentheaminoalanyl -amine_groupand protons ofother residues. Furthermore, the methylvalerate-threonine fragmentgenerates^some interesting interresidue NOEs (20^and 34 in Table V),^whichare absentin theNOESYspectra

of

the

BLM-Zn

_complex.

Another important^source of structural informationisformed by the vicinal proton coupling constants. In our study ofthe

BLM-Zn

_complex17 we have shown that the torsional _angles involving the Ca-Cg ^bond

of

the aminoalanine and hydroxy- histidine residues exist in a fixed conformation in the metal complex. When the vicinal coupling constants ofthis complex are comparedwiththe corresponding coupling^constants in the

BLM-Fe-CO

_complex(TableIV, Figure 10),

it

isobviousthat

a major differenceexistsfortheCa-Cg conformationoftheam- inoalanine fragment. Whileinthe hydroxyhistine^residueinthe

BLM-Fe-CO

complex the vicinalcoupling constant ^isidentical (3.2± 0.2 Hz) with that in^the

BLM-Zn

complex, reflecting^the same conformation,^thevicinal coupling constantsof^theamino- alanine fragment ^increasesignificantly,^ascompared^{to those}of the

BLM-Zn

complex, and approach thosefoundinthemetal-free BLM.16 Theseresults suggestthat_{this part}ofthe moleculehas theconformational freedom

of

that inthemetal-freeBLM. In- deed, using^aKarplus-like equation, which^takesinto accountthe electronegativity of^the substituents at the Ca-C0 ^bond

of

the aminoalanineresidue,37^we couldrule outthepossibilitythatone (37) Haasnoot,^C.A.G.; DeLeeuw,^F.A. A.M.;Altona,C.Tetrahedron 1980.36, 2783.

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