Ni(II) and Ni(I) forms of pentaalkylamide derivatives of cofactor F430 of Methanobacterium thermoautotrophicum

312

Biochimica el BiophysicaActa, I(174(1901)312-319 © l'0t~lElsevierScience PublishersRV. 031)4-41b5/qt/$03,511 ADONIS 031144165~I01_11~42W

BI~.AGEN 23548

Ni(II) and Ni(I) forms of pentaalkylamide derivatives of cofactor F430 of Methanobacterium thermoautotrophicum Cristi L. H a m i l t o n ~'*, Li M a ~, M a r k W. R e n n e r

2 a n d R o b e r t A. S c o t t ~

Depar#neuts ~f Chomi~tryaed Biochemistry and the Centerfor Melalloenzyme5flrdies, Unirel:~'ityof Georgia. Athens. GA (~ S.AA and 2 Deoartme*.tof Applied Science, Brool~hm'en,VutionaiLaborato~,, Uptown,NY ([LS.A.)

(Received 4 March It)91)

Key words: CoI~lclorF4311; Mas~speClfometr~;EPR~(M, thermaetuto~rophicmn) A series of pentaalkylamide forms of F43e and of its la,13-diepimer have been generated and characterized. Carbedilmide-assisted N-hydroxysnlfosuccinimide activation of all five peripheral earboxylates of the 1~'4~a macrocytie allows nucleephilic attack by a number of primary amines (RNHa, R- = CH3-, CH3CH±-, CF3CHa- , CH3(CH 2)3") generating the pentaalkylamide derivatives. The identity of each derivative has been verified by fast-atom bombardment mass spectrometry (FAB-MS). The solubility of these derivatives in apratic organic solvents varies as the amine aiky! snbstituent (R-) is changed, Electrochemical measurements have shown that the N i ( l l / I ) reduction potentials in N,N-dimethylformamide (DMF) are ~ - 1 V (Ag/AgCI). Reduction by sodium amalgam in THF generates the Ni(I) form of the F4~ diepimer pentabutylamide. The. visible and EPR spectra of ~his Ni(l) species are very similar to the corresponding spectra of Ni(l) F4~0M (Jaun, B, and Pfaltz, A. (1986) J, Chem. Soc. Chem. Commun. 1327-1329.).

Introduction Mcthanogens are a eluss of strictly anaerobic arehaebactcria, many of which are capable of living autotrophically on hydrogen and carbon dioxide [ 1]. These bacteria derive energy from the stepwise reduction of carbon dioxide to methane, a process in whleh the C t fragment is shuttled at different levels of reduction between a series of unusual eofactors [2,3], S-methyl coenzyme M reductase (methylreductase) is the terminal enzyme in this pathway, catalyzing the reduetive cleavage of 2-(methylthio)-ethanesulfonic acid (CH3SCoM) [4]. The cofaetor N-7-mercaptoheptanoylO-phospho-L-threonine (HSHTP) appears to act as the reductant in this reaction with the result that methane

* Present address: Departmeat MC772)Bldg.28/3, Lilly Corporate Center. Eli Lilly and Company, Indianapolis. IN 46285, U.S.A. Abbreviations: HSI--ITP, N.7-rnereaptoheplanoyl.O.phospho.Llhreonlne: HSCoM, 2-mercaptoethanes01fonJeacid; EDU, I-ethyl-3(3-dirnc:lhylaminopropyl}carhod[imide; DMF, dimelhylformamide; THF. letrahydrofuran: suno-Nl-tS, N-hydrox3,sulfoguceinimide;CV, cyclic vollamm¢lry:DPV, differential pulse vollammetry, Corresl~ofldence: R,A. Scott+ Department~ of Chemistry and Bio-

chemislry. Universityof Georgia, Athens, GA 30602, U.S.A.

and a mixed disulfide of HSHTP and 2-mercaptoethanesulfonic acid (HSCoM) are produced [5-7]. The mcthylreductase of Methanobacterium thermoau. totrophicum (strain 3 H ) is a large complex enzyme of molecular weight ~ 300000 and an aagD, 2 subunit structure [8-10], The isolated enzyme contains two molecules of an extractable nickel(ll) tetra0yrrol¢ cofactor F4,~t~(so named because of its absorption maximum at ~ 430 nm) that are tightly associated with the a subunits [4,11.12]. The structure of the (native) F43o cofaetor has been deduced by extensive spectroscopic investigations of the pentamethyl ester derivative [13,14] and is shown in Fig. 1. Native F43t) is thermally unstable to epimerizafinn of the acid side chains at g-carbon positions 12 and 13 of pyrrolidine ring C to form the I3-monoepimer and ultimatei~¢ the 12,13-diepimer (Fig. 1) [ 14]. It is also oxidatively unstable, being slowly oxidized in air to 12,13-didehydro-Fa.~0, which is termed ~ o because it exhibits an absorption maximum at 560 nm (Fig, 1) [141. The role of F¢~0 in the reductive cleavage of CH3SCoM is unknown, but it is thought to be the site of substrate reduction. Possible mechanistic roles for F43n in this reaction include substrale (CH3SCoM a n d / o r HSHTP) binding, mediation of electron tram-

313 F.I~O lsome f.s COON

0

/. /

H.NOC~.~y

"'

'~-'~",

"

I ,' ,,I,,r~,~ ~,IHI~~ O 0 0 a

,"

Ni

. .~.~'..~

C(~H

'-h-, "

.ooe

U,

.~

~"

I l --,.v.--~o

COON

court

..~...\,' ~ / "'" " ~ c

¢oo. O0~.

Fig. l, Structures of F~.~c~ and its configurulional isomers. The i,.umcr~ izaliOns involve C ~_)and Ct.~, the ~8-carbons of pyra}lidine ring C~

fer from HSHTP to CH3SCoM, or methyl-group transfer. Results of whole-cell EPR spectra indicate that at least one Ni(l) form of enzyme-bound F.t~0 participates in the production of methane from hydrogen and carbon dioxide [151. Multiple Ni(l) forms of the enzyme are observed under different conditions, and it was speculated that they may differ with respect to the nature of the axial ligands [15]. Characterizing Ni(l) forms of F430 spectroscopically as well as investigating its r~aefi;'i~, are therefore crucial to the understanding of the unusual chemistry of methylreductase. Such studies are hampered by the solubility properties of F4m as it is not soluble in aprotie solvents, such as acetonitril¢ or tetrahydrofuran, that allow access to the negative reduction potentials required to generate Ni(I). The pentamethyl ester of F~.~ (F.tnt)M) has the necessary solubility properties and has been reversibly reduced to the Ni(l) form under aproti¢ conditions, both chemically and electrochemically [I6-18]. We decided to develop an alternate series of Fa_~)pentaanaide derivatives that also exhibit solubility in apolar solvents. These pentaamide derivatives are relatively simple to ~ynthesize and are often prepared in nearly quantitative yields. The use of different primary alkvlamines also allows tuning of solubility and other properties. This work reports the synthesis and characterization of several pentaalkylamide derivatives of both Fa_~oand its 12,13-diepimer. Sodium amalga~u reduction of the pentabutylamide of the F430 diepimer generates the Ni(l) form quantitatively, yielding the first example of the Ni(l) oxidation state in the diepimer. Maleri~ls and Methods

Materials. Protein.sequencing grade l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride was purchased from Sigma. N-hydro~sulfosuceinimide

(sulfo-NHS) was obtained from either Pierce or Polysciences and used without further purification. [:H]:Methyiamine hydrochloride (Y8+atom f~ Dk [~C]mcthylamine hydroehloride (99 atom % ~"C), 2,2.2-trifluoroethytamine (99.5%), and butylamine (99%) were purchased from Aldrich. Methylamine (40% aqueous solution) and dibutylamine (>99%) were purchased from F',uka. Ethylaminc (anhydrous) was purchased from Eastman Kodak, dimcthylformamide (DMF) was purchased from either Baker (Photrcx grade) and tetrahydrofuran (THF) was Aldrich 99.5% spectrophotometric grade (inhibitor free) and was packaged and stored under nitrogen or argon in a Sure/Seal bottle. The THF used for sodium amalgam redt'ction was distilled from sodium/benzophenone and stored over Na/K alloy in vacuo. Tetraethylammonium perehlorate (EtaNCtOa) (polarography grade) was purchased from Eastman Kodak and tetrabutyl0mmonium tetrafluorobotate (BujNBF,) was obtained from Fluka (> 99% purity). Triply distilled mercury was obtained from Bethlehem Apparatus. F~3n preparation. M. rhermoautotrophicura (strain AH) was grown at 65°C on Hz and CO, as ener~ and carbon sources, harvested, and stored under N 2 at - 20 or - 70°C according to published procedure~ (19]. F4~0 was purified as previously described [20]. A final purification step using reversed-phase chromatography on either a PepRPC HR 5/5 column with a Pharmaeia FP!C system (Pharma¢ia LKB P,ietechnology) or a DeltaPak C,~ column (0.39 × t5 cm) with a Waters 6()0 multisolvent delivery HPLC system wan always performed to ensure sample homogeneity and to completely desalt the sample. The purified F4,, ~;,az lyophilized and subsequently redissoNed in water. The pH was adjusted to 4.0 with dilute HCI or NaOH. The resulting F43o concentration was between 9-12.10 s M, assuming E =23000 M t cm-~ [21,22]. General penraamide synthesis. The appropriate amount of the amine was added (straight from the bottle, as a pH 4 aqueous solution, or as the hydrochloride) to an atiquot of the pH 4 F4~ solution, and the pH readjusted to 4 with dilute HC1 or NaOH if neeessat3,. The desired amounts of st~tfo-NHS and then EDC were added, each dissolved in a small ~lume of water. No pH adjustments were made during the course of the reaction, which was carried out at ambient temperature. Alter the reaction had proceeded for the desired time, it was quenched by immersion in N2(1), The reaction can also be quenched by the addition of an EDC-reactive buffer such as sodium formate with no change [n product formation or distribution. However, salts with an ammonium cation must never be used (vide infra). After quenching, the reaction mixture was lyophilized to dryness to remove votatile components such as the amine and HCI. The mixture was then redissolvod in either water or 0.1% aqueous TFA and

314 analyzed/purified using reversed-phase chromatography on either the PepRPC HR 5/5 or the DeltaPak (0.39 × 15 era) column. Larger scale reactions were purified on a 0.78 × 30 cm DeltaPak preparative column.

electrode and a Ag/AgCI reference electrode were used. The supporting electrolyte was 0.1 M KNO3; the solutions were adjusted to pH 7.0 with either HNO3 or KOH. All experiments were carried out at room temperature.

Chemical reduction of 12,I3-diepimer pentabutylctmide. The reduction of the pentabutylamide deriva-

Results and Discussion

tive of 12,13-diepi-F43o was carried out in a vacuum-tight optical/EPR cell of published design [23]. Purified lyophilized pentabutylamide was placed in the sample chamber of the cell and 0.1% sodium amalgam was added to a reduction chamber separated by a coarse glass flit. The cell was attached to a vacuum line and evacuated. Dry degassed THF was distilled into the cell, the cell was sealed, and an initial optical spectrum was recorded. The reduction was initiated by contacting the THF solution of the F4an derivative with the sodium amalgam. Optical spectra were recorded periodically to monitor the reduction and the EPR spectrum was recorded on the same solution after the optical changes were complete. Mass spectrometry. Fast-atom bombardment (FAB) mass spectrometry (MS) wz~" used to characterize the amides, with ~.lte spectra collected on a ZAB-SE mass spectrometer ~ Richard Milberg ned associates in the Mass Spectrc,netry Laboratory, School of Chemical Sciences, University of Illinois. Spectroscopy. EPR spectra were recorded on a Braker-IBM ER200D X-band spectrometer. Spectral parameters are given in the legend to Fig. 5. EPR spectral simulations were carried out using a published procedure [24]. ©ptic.~l spectra were recorded on a Hewlett-Packard g452A diode array sp~.ctrophotometer. Eleclroehemistry. An EG&G Princeton Applied Research Model 264A Polarographic Analyzer/Stripping Voltammeter interfaced to a MFE 815M Plotamatic or a Recorder Company Model 200 XY recorder was used to perform the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments in nonaqueous solvents. Gold or grassy carbon disk electrodes (Bioanalytieal Systems) with working electrode surfaces of 1.6 and 3.2 mm2, respectively, were used as working electrodes. A platinum wire served as counter electrode and a Ag/AgC] electrode (homemade or Bioanalytical Systems) was employed as a reference. The supporting electrolytes used were 0.1 M EhNCIO 4 in DMF and 0.1 M BuaNBF4 in THF. Electrochemical measurements in aqueous solution were obtained using either a cybernetic potentiostat proto~pe of the BAS-100A Electrochemical Analyzer (Bioanalytical Systems), or the commercial instrument itself. Cyclic voltammograms were measured using a hanging mercury drop electrode (Model 303 SMDE, EG&G Princeton Applied Research) which was interfaced to the potentiostat. A platinum wire counter

Synthesis of the F430 pentaamides utilized carbodiimide-assisted coupling chemistry [25-28]. The water-soluble carbodiimide l.ethyl-3.(3-dimetbylaminopropyl)earbodiim[de (EDC) was used to activate the F43o carboxylate groups by formation of an Oacylisourea, The electrophiiic O-acylisourea intermediate was then attacked by a nucleophilic amine resulting in an amide plus an urea leaving group, Unfortunately, there are two competing side reactions [25-2g]. The O-acyl[sourea is subject to hydrolysis, regenerating the original carboxylic acid plus urea. The O.acylisourea is also subject to rearrangement to form a stable Naeylurea. Applying this type of chemistry to F43o yielded a complex product mixture with little if any of the pentaamide being formed. The products presumably varied in the type of modification (amide or N-aeylurea) as well as the distribution of the modifications among the five earboxylate groups. Stares et al. have reported increased yields in EDC coupling reactions when the enhancer N-hydroxysulfosuceinimide (sulfo-NHS) is added to the reaction mixture [29,30], Sulfo-NHS reacts readily with the Oacylisourea to form a more stable ester. This ester hydrolyzes very slowly and is not subject to rearrangement. Thus the two major side reactions are essentially eliminated. The ester, like the O-acylisourea, is a good eleetrophile and the nndeophUic amine can readily attack it to yield the amide. Scheme 1 illustrates the application of this coupling chemistry to the peripheral carboxylates of F4ao. Upon addition of suifo-NHS to the reaction mixture, the yields of F4~a pentaamide increased dramatically. In fact, upon optimizing the reaction conditions, the pentamethylamide and pentaethylamide were made in nearly 100% yield. These syntheses involved reacting 1 • 10 -4 M F43o with 0.1 M EDC in the presence of 0.1 M amine and 5 mM snlfo.NHS at pH 4~ (A typical reaction started with ~ l ml of F4aa solution.) These concentrations correspond to an excess (over F430 carboxylate groups) of ~ 200 for EDC and amine and ~ 10 for sulfo-NHS. Both the diepimcr and native isomers undergo this amidation. Decreasing the amine or EDC concentrations or increasing the sulfo-NHS concentration decreased the yield. No reaction occurred in the absence of EDC. On the other hand, a complex mixture of products resulted when the amine was eliminated, with no unreacted F4~0 being recovered under these conditions. The addition of sulfo-blHS to

315 H

fl,~NmG=NR~ F~"-~

pH

H"

F~'--~

p-o

~.-- c~

~-" C'~O

N"Rz

0

0

H

SO~

HON - ~ 0

I

? o

so=

Rt~ Scheme I

the F~30/amine solution caused the pH to drop by ~ 0.2 units; addition of the EDC then induced a pH increase of ~ 2 units. No pH adjustment was necessary. For poo~:c J,ucl~ophiles, the concentration of the amine and ElM2 were increased to obtain maximum yields. For example, when butylamine was used as nudeophile, 0.3 M amine and 0.2 M EDC were optimal, while with trifluoroethylamine, 0.2 M amine and 0.2 M EDC gave maximum yields. Lower yields with some sid~ product formation were seen with amines that are poorer nueleophiles than metbylamine. Larger scale reactions also gave slightly lower yields. The optimized reaction conditions and approximate yields for the different pentaamides are summarized in Table 1. The coupling reactions were typically allowed to proceed for 15 rain to 2 h. The reaction time giving

maximum yield varied for the different amines. Timedependent studies showed that for the diepimerie pentamethylamide, a reaction lime of 16 to 32 rain was optimal. Quenching the reaction too early trapped an intermediate which eluted prior to the metbylamid¢ on the reversed-phase columns. Longer reaction times (typically 1 to 2 It) were required for amines that are poorer nucleophiles than methylamine. For the trifluoroethylamide, a reaction time of greater than 12 h was necessary to eliminate the intermediate. The reactions were very reproducible when quenched by immersion in N2(]) or by the addition of a large excess of an EDC-reactiv¢ buffer. It was necessa~ to avoid ammonium salts which had adverse effects on the reaction. In fact, it was essential that the F4~ samples be 100% desaited (ideally by reversed-phase chromatography) from the ammonium formate used in the purification

TABLE 1 Reaction conditionsand yidds for the synthcse~o[ F4~ pentaamides

F4~ was in HzO (pH 4.9) at a concentrationof ~ 1× 1074 M F~o isomer

Amidc

methyl ethyl butyl trifluoroethy[

[Amine] (M) 0.1 0,1 0.3 0.2

[$uffo-NHS] (mM) 5.0 5.0 5.0 5.0

[EDCI (M) 0.1 0.1 0.2 0.2

t2,13-Dicpimer

Native

methyl butyl

O.I 0+3

5.0 5,0

0.1 0.2

Reactionlime

16-32rain 16-32 rain 2h > 13 h

Yield (%) 98 98 > 90 60

16-32 rain 3-5 h

90 > 90

316 of F4:c5 if any ammonium formate was present in the reaction, a complex mixture of poorly separated products formed (see, for example, Fig. 2). The product distribntion was dependent on ammonium formate concentration with higher concentrations giving more complex distributions. These reaction conditions appear to be limited to primary amines. Attempts to use the secondary amine dibutylamine to form a pentaamide resulted in a complex mixture regardless of the concentration of amine or EDC or the reaction time. These particular reaction conditions are also limited to the use of water-soluble amines as nueteophiles, The amides were purified very quickly and simply through the use of reversed-phase chromatography, The elution profiles of the amides are shown in Fig. 3. isotopically labeled ('3C or 2H) methylamides exhibited chromatographic elution behavior identical to the methylamide, The elutions were monitored at 405 nm (FPLC) or 430 nm (HPLC) so that only F~.~0-containing peaks were observed. The elution times were unexpected in some instances. For example, although the methylamlde would be predicted to be more hydrophobic than the pentaacid and thus elute later, it eluted earlier on both reversed-phase columns used (Fig. 3a). The ethylamide essentially eoeluted with the unreaetcd F4.~ctdiepimer on the DeltaPak reversed-phase column (Fig. 3b). while these two molecules were completely separated on the PepRPC reversed-phase column (Fig, 3b, inset). The amides were characterized toy FAB mass spectrometry (FABMS), with the results shown in Table I1, The methylamide, the J~C-labeled methylamide, the 2H-labeled methylam;de, the ethylamide, the butylamide, the trifluoroethylamide, and the native F4.~o methylamide all have molecular ion peaks corresponding to the correct calculated mass. As shown in Table 111, the amides are soluble in acetonitrilc and dimethylformamide (DMF), both of which have large negative reductlon-potential windows. The solubility properties can be altered by changing the nature of the amine reactant. Using more hydrophobic amines enhances

D I

O.Oa

'~ o.o~

00~

40

"° 20 °~

0 ~0 SO 40 Elulion V0rume(raL) Fig. 2. Revcrscd-pha~e elutiofl profile of a sulfo-NHS-activatt:d

lO

F4S~/methylamine reaction mixture in the presence of ammonium formate. Gradient elation was performed on an FPLC HR 5/5 PepRPC column with 0.1% aqueous TFA as solvent A and 0.1% TFA in 80:20 acetnnitri~e/water as solvent B. The grad[eat program was 0-12.5% B in 15 ml and 12.5-20% B in 50 ml, al I ml/min. The peak labeled D shows the elution position of dicpimgri¢ F4?a, in an identical reaction mixture spiked with diepimer,

the solubility in nonpolar solvents. The ultraviolet/ visible and CD spectra are essentially unchanged upon amidation. Thus the ~ system of the macrocycle and the conformation do not appear to bc altered upon conversion of F4aa to the pentaamide derivatives. Cyclic and differential pulse voltammetry (CV and DPV, respectively) were used to characterize the redox properties of the amides. The reduction potentials for dicpimeric F,30 and its methyl, butyl, and trifluoroethylamides are summarized in Table IV. In all cases, the redox process was more reversible on a glassy carbon than on a gold working electrode. For the dicpimcr and its methylamide, two reduction waves were seen, the first of which was completely irreversible and of unknewn origin. The second reduction wave, assigned to a Ni(II/I) couple, was reversible (peak separation ~ 70 mV). The first irreversible reduction wave was not seen for the butylamide and trifluoroethylamid¢. The nickel(ll/l) reduction potential was insensitive to the nature of the amide group, having a value of - 1.0 V vs, Ag/AgCI ( ~ -0.80 V vs. NHE) (glassy carbon or gold electrode in DMF with 0.1 M Et4NCIO 4 as supporting electrolyte) for all the diepimeric amides studied. Our measured Ez/2 for 12,13-diepi-F43a of - t.0 V

TABLE It

FAB-MS rexutr,~for the synOlesizedF4.m pentaaondes Molecuk:

Formula

Observed mass

Calculated mass

F~, 12,13.dicpimer Diepimer pentamethylamide Dleplmer penta[ t3C]methylaraide Diepimcr pk:nta[2H]3mcthylamidc Diepimgr pealacthylamide Diepimer pcalabulylamide Diepimer pcnta-2,2,2-t rifluoroethylamide Nat lye F~.~, Native pent ~.methylamidc

NIC4.~H~tNt,OL~ NiC4~H~N I los Nica2L3c~H h~Nt~Os NiC47HsIZlIIsNI]O8 NiC~2It rsNuOs NiC~:tI~NI ,Os NiCszH~INNONFt. ~ NiC~H~tN~Ola NIC4;H~NLtOs

905,28 970.44 975.4b 985.54 1(140.52

905,29 970.44 9"/5.47 985.54 1040.52 1180.68 1310.38 905.29 970.44

1180.68 1310.38 905.2 970.44

317 0.8

- -

8

2 . C - c

80

D 0.6

I00

D

~80

I.E

I

o .< f

0.2

0

0

10 20 E:lUt~OnVolumB (mL}

~

30

20

+,,j

0.6

o jl

+-I

1,(

i,+ °

I+°

5O

m

40 20

10 20 Elution Volume (mL)

1.5

O

4O

+ ,¢

4O ~

0.~

[

-~-~'-

to

J:

2'o

20 0 10 Elulion Vc~me {mL)

Elution Volume (mL)

Fig. 3. Reversed-phase elution profiles or dicpimuric F4~~ reaction mLxlures forming the lx:ntamelh,/lamide (a), the penlaethyDmide (b), ~hu pcma(2.2.2-trifluorocthy[amid~) (c), ~nd the p~:ntabutylamid¢ (d). The r¢;~cUon times were 32 rain (a), 1:5 rain (b), 13 h (¢), and 15 rain (d), All flow rates were 1 ml/mln and. except [or the in~dt |o (b), all eledons '.+ere I,'¢rformed un a Waters 0.39× 1:5 cm DeltaPak C=s column with walcr as solvenl A, acetonitrile as solvent I~ and l% TFA as solvent C ( Ill% solvenl C was maintained throughout all gradients). In all cases, the peak labeled D indicates the elulion position of unmodified diepimerie F4~n. The sample ia (b) was spiked with auh'~ntic diepimer, no unmodified diepimer was obs¢~¢d in an unspikud sample, The gradienls were programmed as follows: (a} 0--0% B in 2 mk O~13% B in 10 ml. 13 20% B in 20 ml; (b} 0~0% B in 2 ml. 0-13% B in lfl ml, 13-25% B in 26 mh (¢) 0-0% B in 2 mL 0-75% B in 30 ml: (d) 0-0% B in 2 mL 0-,50% B in 10 mL 50-55% B in 12.5 ml. The inset to (b) shows an elulion profile of the dicpimorig F4~[~pentaethylamide 2-h reaction mLxtnro oa an FPLC HR 5/5 PcpRPC Cls column with 0.t% aqueous "TFA as solvent A and 0.[% TFA in 80:20 acetonilrile/watcr us solvent B. The gradient program was 11-12.5% B in 15 ml. 12.5-20% B in 50 mL

DPV at ~ - 0 . 6 4 V vs. Ag/AgC1 (Table IV). The N i ( [ l / l ) process was most cleanly observed by DPV, yielding a potential of - 0 . 9 6 V vs. Ag/AgCI. Thus, the nickel in the native pentaamidc was reduced slightly marc readily (i.¢., at a more positive reduction potential) than pentaamides in the diepimerie conformation. This result is expected as the nickel ion in native F4.~, is

vs. Ag/AgCI (DMF, 0.1 M Et+NCIO+) compares with previously reported values of - 0 . 7 8 V vs. Ag/AgCI (DMF, 0,l M Et4NCIO 4) [31] and - 1,03 V vs. SCE (butyronitril¢, 0.1 M Bu4NCIO 4) [32]. The redox behavior of the native F430 methylamide was quite complex. As with the diepimeric methylamide, an irreversible reduction wave was seen by TABLE Ill

Solvabilitypropertws of the diepimeric F+~) penmamides " Sample

H~O

MeOH

iPrOH

DMF

MeCN

Acelone

THF

CHCI3

C-H2CI 2

ether

CCI+

To1

Diepimer MeAm EtAm BuAm FEIAm

vs vs vs s s

vs vs vs w vs

ND ND ND vs vs

vs vs ,~s vs vs

i s s vs vs

si s s ND ~ 'is

sl s ~1 s sl s vs vs

i sl s sl s vs sl s

i i i s sl s

i i i i i

i i i i i

i i ND sl s h i

a "l'h¢ abbreviations used are: MeAm, diepimcr pcnlamelhylamide; EIAm, diepimer pcntacthylamiclu: BuAm, dicpimcr pcntabutylaml"de; FEtAm. dicpimer penta-2.2.2-triflumoeth~lamide: MeOH. methanol; iPrOH, isopropanol; DMF. N,N-dimeth~lformamidc; MeCN, acetanRrile; THF, tctrahydrofuran; Tel, toluene; vs, very soluble; s, soluble: sl s. slightly soluble; J. insoluble; lqD, not determined, o The small amount of BuA~ that dissolved in toluene was lan rather than yellow in color.

318 TABLE IV

Reduction potemia~ (t.~. Ag / AgCI) for F4~~ pentaumides Cyclic wltarnmet~ (CV) and differential pulse voltammetry (DPV) measurements in DMT with 0.1 M EIaNCIOa supportiDg electrolyte

F4:~0 isomer

Amide

Electrode

El~ 2 IDPV)

trifu01oulhyl

glassycarbon gold glassycarbon gold gla~y carbon gold

El, 2(CV) (V. Ag/AgCI} -0.72(E~) ~, - 1.0 - 0.66, - 0.99 -0,72(Ep), 0.9~ - 1.0 - 1.0 I,I

methyl

gold

~ - 0.96

- 0.64, -0.96

F,~3a 12,13-diepimcr

melhyl butyl

Native F430

(V, Ag/AgCI) - 0.69, - I.I - 0,5l, - 1,0 0.63, -I.0 - I. 1 -

1,0

(ND) b

a CV peak potential, not E=t~, b hal delcrmined.

known to be more electrophJlic than that of the diepimer because it cannot readily achieve the stable S4-ruffled conformation [14]. Consequently, it should more readily undergo Ni reduction to relieve the strain in the macrocycle, The electroanalytical behavior of the native methylamide and the diepimeric butylamide on a mercury hanging drop electrode in aqueous solution (0,1 KNO 3, pH 7.0) was also characterized. (The electrochemical window in water extends to more negative potentials on mercury than on gold and glassy carbon,) For the native methylamJde, the CV revealed only one reduction and reoxidation wave. This wave, assigned to Ni(ll/l) reduction and reoxidation, was only partially reversible, and the CV was a classic example of molecular adsorption on the electrode surface [33,341. It exhibited a broad, rounded reduction wave associated with the redaction of redox-active molecules diffusing to the electrode followed by a very sharp 'spike' due to reduction of the adsorbed species. The potential of the adsorbed methylamide is - 1.14 V vs. A g / A g C l ( - 0 . 9 4

V vs. NHE). The CV [or the butylamide shows a broad irreversible reduction wave with Ep ~ - 1 . 1 4 V vs. Ag/AgC1. No adsorption o c c u r s for the hutylamide, suggesting that the presence of bulkier groups on the F4z0 maerocycl¢ p r ~ e n t s adsorption. Optical monitoring of the chemical reduction of the pentabutylamide of 12,13-diepi-F430 by sodium amalgam in T H F reveals bleaching of the 433-nm peak of the Ni(ll) form and the simultaneous appearance of peaks at 382 and 756 nm (Fig. 4). The isosbestic points are indicative of reduction to a single product. The l'¢snlting spectrum is virtually identical to that published for Ni(1) F43aM [17]. The EPR spectrum of this product (Fig. 5) confirms its identification as the Ni(1) form of the 12,13-diepi.F4~ o pentabutylamid¢, EPR spectral simulation of this nearly axial signal (Fig. 5) yield gz -- 2.244 and g~, gv = 2,076, 2.060 which agree

O8 D

I

2 2=14

/ 2,076

2.~16

0.2 2800

3000

3209

3400

3600

~00

Gau~s ~se

35o

4so 55o ~so Wavelength {nm)

750

a~o

Fig. 4. Time-dupendentoptical ~poctra re[lowing the reduction of Ni(ll) 12,13.diepi-F43~ penlabutylamideby sodiumamalgamin THF. The arrows ind.icatt: the time-dependents of each major p~ak. The isos'oesficpoints near 410 and 4fi0nm indicate a clean redttclion to a singleproduct.

Fig. 5. (a) I~PR specffumat" Ni(I) 12,13-diapi-F4~ peatabutylamide in THF recorded at 9.54 GHz, 2 row, 5 G modulationamptitod¢, and t13 K. The small signal at g=2.00 is a radical impurity representing ~ 0.5% of the total signal (b) Simulatio~ of EPR spectrum ill (a), yielding g.~= 2,244, g,, g~ = 2.076, 2.060; line widths (z, x, y)= |3.S, 14.0, 14,0 G:. A~=9,5 (3+ The n,iuogca h~pcrfine e.ouplin~ are hal lose|rod,

319 well with the g-values m e a s u r e d for Ni(I) F~.~oM ( g : = 2.250; g~, gy = 2.074, 2.065) [17]. Such an F_.PR spect r u m is expected for a d 9 system in which the u n p a i r e d electron resides in a d,,_ y.- orbital.

Conclusions Amines can be effectively coupled to the earboxylate g r o u p s on the F~o macrocycle to form a p e n t a a m i d e by using E D C a n d sulfo-NHS. T h e synthesis o f F43o pent a a m i d e s is a simple a n d high-yield r e a c t i o n a n d the purification is triviat, involving only o n e reversed-phase H P L C column. T h e solubility p r o p e r t i e s o f the pent a a m i d e s c a n be adjusted by c h a n g i n g t h e a m i n e used to synthesize the amide. T h e electrochemical behavior of the p e n t a a m i d e a p p e a r s to be m o r e complex t h a n F43oM since a simple C V with o n e s h a r p reduction a n d rcogidation wave was not o b s e r v e d for a n y o f the a m i d e s u n d e r the conditions e m p l o y e d , However, Ni(I) forms of the pent a a m i d e s c a n be g e n e r a t e d , indicating that these amides will he useful in chemically a n d spectroscopicatly c h a r acterizing Ni(l) F43o.

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