Nuclear Magnetic Resonance Spectroscopy of Chlorophylls ......Nuclear Magnetic Resonance Spectroscopy of Chlorophylls and Corrins Joseph J. Katz'and Charles E. Brown* *Chemistry Division

REVIEWS

11

Nuclear Magnetic Resonance Spectroscopyof Chlorophylls and Corrins

Joseph J. Katz'and Charles E. Brown*

*Chemistry DivisionArgonne National Laboratory

Argonne, III. 60439 USA

* Department of BiochemistryThe Medical College of Wisconsin

Milwaukee, Wis. 53226 USA

i ntroduction

The ChlorophylIsA. Structural FeaturesB. Experimental AspectsC. Chemical Shift AssignmentsD. Applicat ions of NMR

Corr insA. Structural FeaturesB. *H NMR Chemical Shift Assignments and ApplicationsC. 13C NMR Chemical Shift Assignments and ApplicationsD. NMR Studies with other NucleiE. Summary

References

Page3

kk5520

323233U2

i. INTRODUCTION

The role of NMR in the study of com-pounds of biological importance iswidely recognized and appreciated.There can be few categories of suchsubstances, however, to which NMR hasmade such substantive contributions asit has to the understanding of thechlorophylls and the corrins. Thechlorophylls are indispensible agentsin the conversion of the energy oflight to chemical oxidizing and reduc-ing capacity. The natural corrins arecoenzymes for a number of enzyme

systems involved in important isomeri-zation and methyl group transfer reac-tions, and are intimately involved inprotein and probably also in lipid andcarbohydrate metabolism. Vitamin B12plays a very important part in hemo-poiesis (stimulation of red blood cellformation), and together with folicacid participates in the formation ofdeoxyribonucleotides from ribonucleo-tides. Applied to the chlorophylls,NMR has provided information relevantto such aspects as the sites ofexchangeable hydrogen, keto-enol tau-tomerism, the biosynthetic pathways of

Vol. 5, No. 1/2

chlorophyll formation, hyperfine inter-actions in chlorophyll cations andother paramagnetic chlorophyll species,as well as more conventional NMR infor-mation useful in establishing the chem-ical identities of several chlorophyllsof previously unknown structure. Per-haps the most important contributionfrom NMR to an understanding of chloro-phyll behavior is the delineation ofthe coordination donor-acceptor proper-ties of the chlorophylls that largelydetermine the state of chlorophyll j_nvivo. For the corrins, clarificationof the path of biosynthesis and thevariables that affect their coenzymeactivities has up to now been the mostsignificant contribution from NMR stud-ies.

It. JHE CHLOROPHYLLS

The chlorophylls constitute a smallgroup of closely related compoundswhose function in nature is to collectlight quanta and to use the subsequentelectronic excitation energy to effectcharge separation. The oxidizing andreducing power so produced is then usedto drive redox reactions that wouldotherwise not proceed spontaneously.Useful introductions to the role ofchlorophyll in photosynthesis have beenprovided by Govindjee and Rabinow itch(1), by Clayton (2), and by Govindjee(3).

A. Structural Features

The chlorophylls are cyclic tetra-pyrroles, and thus belong to the por-phyrin family. There are both importantsimilarities and differences betweenthe chlorophylls and the more widelystudied porphyrins. The side chains ofthe tetrapyrrole macrocycle , i.e.methyl, ethyl, vinyl, and propionicacid, are much the same in both porphy-rins and chlorophylls. The side chainpositions likewise for the most partappear to be identical, arguing forsimilar biosynthetic pathways. Thechlorophylls, however, all have an ali-cyclic 5~membered ring V (Figure 1),which contains a keto carbonyl functionat position C-9. Most of the chloro-phylls contain a carbomethoxy group at

Figure 1. Structure and numbering ofthe chlorophylls. J_, Chlorophyll a (Chia); 2, pyrochlorophyl1 a (Pyrochl a);2, chlorophyll b (Chi b); k, bacter-iochlorophyl1 a (Bchl a). The methylchlorophy11 ides have the same macrocy-cle as the chlorophylls, but the phytylmoiety is replaced by -CH3. The removalof the central Mg and its replacementby 2H from chlorophylls and methylchlorophyl1 ides forms pheophytins(Pheo) and methyl pheophorbides,respectively. Chlorophylls £1 and £2have an acrylic acid side chain atposition 7 in structure 1; in Chi £2, avinyl group is also present at positionk. Both Chi £1 and £2 lack any esterif-ying alcohol at position JZ. Bacter-iochlorophylI b has only an ethylidenegroup, =CH-CH3 at position k in struc-ture J». Protochlorophyl I a is identi-cal with J except for the absence ofprotons 7 and 8. The protons at posi-tions 7 and 8 are also missing in Chici and C2.

Bulletin of Magnetic Resonance

the 10 position, but in severalimportant chlorophylls the carbomethoxygroup is replaced by an H atom. Ring Vis a structural feature unique to thechlorophylls. It is this feature thatis mainly responsible for the rich andcomplex chemistry characteristic of thechlorophylls. The proton at C-10 inchlorophylls containing a carbomethoxygroup is part of a 3~keto ester system,which can undergo enolization. Enoliza-tion is associated with epimerizationat the chiral center at C-10 , and isimplicated in a very complicated set ofoxidation (a 1lomerization) reactions bymolecular oxygen that occur at positionC-10, which ultimately results in therupture of ring V. Excellent reviewson the chemical properties and reac-tions of the chlorophylls by Seely(^)and more recently by Jackson (5) areavailable.

The central magnesium atom chelatedby the chlorophyll macrocycle is a reg-ular rather than a transition metal ionsuch as is present in porphyrin-con-taining respiratory pigments, oxidases,and the like. The Mg atom of the chlo-rophylls has significant electrophi1icproperties found to a distinctly lesserextent in the corresponding transitionelement complexes. The keto C=0 groupat C~9 in ring V endows the chlorophyllmolecule with nucleophilic propertiesthat have no parallel in the porphy-rins. Esterification of the propionicacid by phytol, a long-chain aliphaticalcohol, makes for solubility propel—ties different from those of heme,which contains the free acid. Thechlorophylls are for all practical pur-poses insoluble in water and must bestudied in organic solvents. There are,of course, many other important differ-ences between chlorophylls and porphy-rins, but those indicated here are per-haps the most significant in terms oftheir consequences for NMR spectros-copy. By far the best studied chloro-phyll is chlorophyll a (Chi a) theprincipal chlorophyll in green plantsand blue-green algae (cyanobacteria),and most of the discussion in thisreview will deal with this chlorophyll.

B. Exper imental Aspects

The susceptibility of chlorophyllsto oxidation by molecular oxygen neces-sitates special precautions in record-ing NMR spectra. Reaction with oxygenin polar organic solvents, particularlymethanol , rapidly produces sufficientallomerized chlorophylls to complicatespectral interpretation. The allomer-ized chlorphylls are similar in chemi-cal structure to chlorophyll itself,and the spectrum of a mixture ofclosely related but not identical com-pounds may show broad, poorly resolvedresonance peaks. Even 1% by weight of acompound of low molecular weight canproduce an equimolar concentration ofan impurity resonance. Samples for NMRare preferably dissolved in purifiedand inert solvents, and the sampletubes sealed off in a high vacuum afterthorough degassing. The manipulationof chlorophylls is best carried out innitrogen-atmosphere gloved boxes. Chlo-rophyll samples for NMR kept in air maybe altered so rapidly that they can nolonger be safely used after only a fewhours, but NMR samples prepared frompure components in sealed tubes show nochanges for months or even years. XHand 13C chemical shifts are given in 6,ppm, relative to TMS, unless otherwiseindicated.

C. Chemical Shift Assignments

Despite the structural complexity ofthe chlorophylls, chemical shiftassignments are straightforward, andindeed, more readily accomplished thanin the case of many simpler appearingcompounds. There are many protons onthe chlorophyll macrocycle sufficientlyisolated not to experience spin-spininteractions sufficient to complicatethe spectra; the methine protons, theproton at position C-10, the methylgroups (in Chi cO at positions la, 3a,kb, and 10b are wel1-separated and thusappear as singlets. Where spin-spininteractions occur, as in the vinylgroup at position 2, the protons ofring IV, and the ethyl group at posi-tion k, the resonances are still wellseparated, and their multiplicity con-tributes to the assignment. Where ahigh-field resonance originating in amacrocycle side chain is overlaid by

Vol. 5, No. 1/2

resonances from aliphatic protons inthe phytyl chain, the phytyt group canbe replaced by transesterification withmethanol. The methyl pheophorbides(Mg-free derivatives) and methyl chlo-rophyll ides (obtainable in si tu enzy-matically) have simple spectra in whichall of the macrocycle proton resonancesare clearly visible.

The highly characteristic featuresof the chlorophyll XH NMR spectra are,however, to a considerable extent theresult of interatomic induced fieldsoriginating in the highly aromatic mac-rocycle. Such ring current effects havelong been known to be important in the*H NMR spectra of aromatic compounds,and were early recognized by Becker andBradley (6) and Abraham (7.7a) to haveparticular significance for porphyrinand chlorin NMR. The ring current cal-culations of Janson et al. (8) for oli-gomeric silicon and germanium phthalo-cyanins have been successful incalculating ring current shifts inthese compounds, but this method hasyet to be applied to the chlorophylls.Abraham et al. (7a) have advanced adouble dipole model of the macrocyclicring current in the dehydroporphyrinring of chlorophyll derivatives. Thismodel accounts reasonably well for XHchemical shift differences between cor-responding protons in methyl pyropheo-phorbide a and its- porphyrin analog2-vinyl-phylloerythrin methyl ester, inwhich the additional hydrogen atomspresent in Ring IV of the methyl pyro-pheophorbide a have been removed byoxidation. In a qualitative way ringcurrent effects account for the unusu-al ly broad range of chemical shift val-ues typical of the chlorophylls. The 1HNMR chemical shifts of Chi a have arange of 10 ppm, and the Mg-free pheo-phytins a range of 12 ppm. The methineprotons in the plane of the macrocycleare deshielded and appear at unusuallylow field. The ring methyl, vinyl, andpropionic acid protons are likewisedeshielded to a significant extent. Inthe pheophytins (chlorophylls in whichthe Mg is replaced by 2H) the H atomsattached to the pyrrole N are stronglyshielded by the ring current and comeinto resonance several ppm above TMS.The ring current model of Abraham et

al. (7a) suggests that the alicyclicring V has no appreciable effect on themacrocyclic ring current, but that theketo C=0 group at position 9» and theaddition of 2h atoms in ring IV in thechlorin both reduce the ring current byabout 6 and 10% respectively. The sen-sitivity of the ring current effects togeometry is primarily responsible forthe unusual amount of structural infor-mation that can be deduced from NMRdata on chlorophyl1-nucleophile andchlorophyl1-chlorophylI interactions(cf. sections I I.D.3 and h).

1. *H NMR Chemical Shifts of MethylPheophorbides

All of the macrocycle ring protonsof Chi a (33 of the 72 protons in themolecule) have been assigned. The com-plete spectral assignment of Chls a andb depends to a considerable extent onthe assignment of resonances of thecorresponding methyl pheophorbides(chlorophylIs in which the central Mgatom is replaced by 2H and the phytylchain by a methyl group). Partialassignment of the *H NMR of the chlor-phyll derivatives chlorin e6 (9). andrhodochlorin dimethyl ester (9). and ofthe methyl pheophorbides of the chloro-phylls from green photosynthetic bacte-ria (10) had been made prior to thefull assignment of the methyl pheophor-bide a and b chemical shifts by Closset al. (11). A review of chlorophyllNMR work prior to 1966 describes therationale of the chemical shift assign-ments of the methyl pheophorbides (12),and more recent reviews (13~15) coversubsequent developments.

Table I summarizes 1H NMR chemicalshift data for the methyl pheophorbidesderived from a number of importantchlorophylls. The low field chemicalshifts originate from the methinebridge protons and the proton of theformyl group in methyl pheophorbide b.The methine assignments for methylpheophorbide a are based on the consid-erations that the proton lies between apyrrole and a pyrroline ring andshould, therefore, be the most shieldedmethine proton (6,9)» and that the 3methine proton, because of its proxim-ity to the ring V keto carbonyl group


o

Table 1

Proton

'H NKR Chemical Sh1ftsaof Methyl Pheophorbides In C!HC1:.(12)

MethylPheophorbide a_

(O.O6M)

MethylPyropheophorbide a_

(O.O6M)

MethylPheophorbide b_(O.O8M)

MethylBacter 1 opheophorb 1de

(0.04M)

Methyl 2-v1nylBacter 1 opheophorbIde cb

(0.05M)

cx352a2b1087

'5-CHa10b5a7d4a1a3a8a4b

N-H9

9. 159.328.507.85

G. 12/G.046.224 .404. 13-

3.883.623.573.483.323. 151 .821 .60

-1 .75

9.209.328.507.98

6.25/65. 134.424.23--

3.583.583.503.353. 131 .721 .55

-1 .85

15

9.768.898.477.75

6.16/6.086.224.454. 15

3.953.463.623.373.2810.581 .881 .480.83

-2. 15

8.968.478.40-

3. 15C

6.084.28d

4.02e

3.843.483.572.203.441 .721 .791 . 100.46-0.96

9.449.49

7.906.22/6.095.234.57n. r.3.86

1 .96'3.601 .673.48

1 .481 .67

a) In <5, ppm, downfield from TMS. b) A mixture, with position 4 occupied by ethyl, n-propyl, and 1-butyl, and posi-tion 5 occupied by methyl and ethyl. The a-hydroxyethyl group normally present 1n Bchl d has been converted to vinyl(32). c) The methyl group of the acetyl group at position 2. d) Includes the proton at position 3. e) Includes theproton at position 4. f) The methyl group 1n an ethyl group at position 5. g) The pyrrole nitrogen atoms. The 2 pro-tons are not equivalent and may appear as two resonances. A minus sign Indicates a shift at higher field than TMS.

is more strongly deshielded than the aproton. In methyl pheophorbide b, theassignment of the a and @ protons isreversed on the presumption that theformyl group should strongly deshieldthe a proton so that its resonance inthe b series appears at the lowestfield for the methine protons. Inmethyl bacter iopheophorbide a_, allthree of the methine protons are posi-tioned between a pyrrole and pyrrolinering, and all three resonances are at arelatively high field. The a protonmust be the least shielded because of.the acetyl C=0 group at position 2, andthe assignments of the £ and <5 reso-nances are based on the arguments usedfor the assignment in methyl pheophor-bide a. These methine assignments areconsistent with the results of disag-gregation titration experimentsdescribed in section I I .D.I*.

The resonances in the region 5~8 ppmare assigned to vinyl or to otherstrongly deshielded substituents. Thevinyl group at position 2 in methylpheophorbides a and b and methyl pyr-pheophorbide a_ are easily recognized asan AMX spin-splitting pattern, fromwhich by a standard analysis, the fol-lowing coupling constants for methylpheophorbides a and b respectively (inHz) can be extracted: N H (x) H (A) I18.7, 18.3; MHJ[X)H(B)I 1 0-9. H . 2 ;and ! JH (A) H (B) I U o > ]-*>-

The C-10 proton in methyl pheophor-bide a and b, is assigned to a sharpresonance at ~6.2 ppm, which coincidesin many solvents with the high fieldportion of the vinyl proton resonances.Assignment is made on the basis of ringcurrent considerations and the proxim-ity of deshielding functional groups;the exchange behavior of this protonconfirms the assignment. In methylpheophorbide a and b_, there are twoprotons at the C-10 position, and theseare not magnetically equivalent, havingchemical shifts that differ by about0.12 ppm. These two protons yield ahighly distorted AB pattern (Ao 6 Hz, J~ 20 Hz) in which the two central reso-nances of the expected quartet are themost prominent features. The magneticnon-equivalence of the two proton sitesat the tetrahedral C-10 provides impor-tant information about the epimers of

the natural chlorophylls (sectionI I.C.3) •

The low intensity multiplets near4.35 ppm in all the methyl pheophor-bides have been assigned to the protonsat position 7 and 8 (and positions 3and k in Bchl a and its derivatives) onthe basis of shielding considerationsand the complicated splitting patternsexpected for these protons. Theseassignments have been confirmed bydecoupling experiments that show the7~proton is coupled to the methyleneprotons of the propionic acid sidechain at ~2.50 ppm, and the 8-proton tothe high field methyl group doublet at~1.80 ppm. A maximum coupling constantof ~2.8 Hz has been estimated for thespin-spin interaction between the 7"and 8-protons, which is consistent onlywith a trans relationship between theproton and the alkyl groups in ring IV.The chemical shift difference betweenthe 7" and 8-protons is surprisinglylarge. Originally it was attributed tothe deshielding effect of the adjacentcarbomethoxy function at position 10.However, the difference in chemicalshifts occurs also in the pyro-deriva-tives of chlorophyll, in which the car-bomethoxy group has been replaced by H.The chemical shift difference betweenthe 7~ and 8-protons probably arisesfrom the anisotropy of the macrocyclicring. As the macrocyclic ring is notplanar, the f- and 8-protons may occupypositions that place them at differentdistances from the ring and so subjectthem to differing ring current desh-ielding. Another possibility is thatthe chemical shift differences betweenthe ring IV protons are the result ofsubstitution at the y methine position,as Abraham et al. (16) have shown thatthe effects of methine substitution inporphyrin NMR spectra are much largerthan can be accounted for by theresulting changes in bond anisotropies.

The macrocycle ring methyl groups atpositions la, 3a, 5a, and 10b haveresonances located between 3 and h ppmand have been assigned with considera-ble certainty. In the methyl pheophor-bides, the resonance of the CH3-groupon the propionic side chains can bedifferentiated from the methyl group ofthe carbomethoxy function at position

8 Bulletin of Magnetic Resonance

10 by the synthesis of [ClH3]-2H methyl

pheophorbide by transesterification ofthe phytyl group of fully deuteratedChi a with methanol of ordinary isto-topic composition; under the usualtransesterification conditions only thepropionic ester function undergoesreaction. The isotope hybrid methylpheophorbide in which the position ofthe C1H3-group is independently estab-lished makes possible an unequivocalassignment of the ester methyl groupsin the methyl pheophorbides, and illus-trates one of the ways in which fullydeuterated chlorophylls (17) find usein NMR spectroscopy. The assignment ofthe remaining macrocycle methyl groupsis largely from the disaggregationtitration studies described below.

The assignment of the high-fieldproton resonances of the methyl pheo-phorbides is completely straightforwardand follows directly from double reso-nance experiments. The elimination ofthe large group of resonances from thephytyl moiety greatly simplifies thespectra and does not significantlyaffect the position of the macrocycleproton resonances. The chemical shiftsof the pheophytins are to a good firstapproximation the sum of the methylpheophorbide and phytol chemicalshifts. As the capabilities of modernNMR spectrometers have improved it hasbecome possible to see many more highlyresolved phytyl resonances. This isparticularly the case when chlorophyllsor pheophytins of suitable adjustedisotopic composition are used. In2H-Chl a containing 1% 1H, all themethyl resonances of the phytyl moietycan be seen under 2H-decoupling, aswell as a number of the -CH2- reso-nances. These are at present unas-signed, but there is no reason to sup-pose that an assignment will not beforthcoming in the future. The phytylresonances are given in Table 2.Because of the near identity of thechemical shifts of the pheophytins andthe methyl pheophorbides, those of thepheophytins are not tabulated here.

A solvent sometimes employed in NMRstudies because it contains no protonsis tr i f luoroaceti c acid~di, CF3C0.22H.This is an excellent solvent for thepheophytins, and as it is free of

non-exchangeable protons, finds use in1H NMR work. It should be pointed outthat the pheophytins are di-protonatedin this strong acid, and form dica-tions, pheoH2**. Trifluoroacetic acidhas also been employed as a solvent forthe chlorophylls, on the unfoundedassumption that the central Mg atom isretained. In fact, the chlorophyllsdissolved in trifluoroacetic acid losetheir Mg atom and are protonated andconverted to the dication of the corre-sponding pheophytin. Not only are theoptical properties of pheohh** remarka-bly similar to those of the correspond-ing chlorophyll, but the XH chemicalshifts of the two are also very similar(18).

Unlike the case of the Mg-containingchlorophylls, the chemical shifts ofthe Mg-free pheophytins and pheophor-bides are strongly concentration depen-dent. As the concentration increases,7r-7r stacking occurs to an increasingextent (11,12), but as stacking occurswith only partial overlap, selectivering-current chemical shifts areobserved. The coordination interactionsbetween chlorophylls yield products ofrather different geometry, and theeffects of concentration on chlorophyllNMR spectra are considerably smallerthan for the Mg-free derivatives.Brockmann et al. (19) have examined theconcentration dependence of the lH NMRspectra of methyl pheophorbides poss-essing an a-hydroxyethyl group (deriva-tives of Bchl £). At high concentra-tions (>0.1 M) doubling of many of theresonance lines is observed, which isinterpreted by Brockmann et al. to be aconsequence of aggregate formation.This conclusion is somewhat suprising,as aggregates produced by either ir-ir orcoordination interactions involving Mgform and disaggregate on a much fastertime scale than that of the NMR meas-urements. Consequently, at ambienttemperature only one set of lines hasbeen observed in these systems. Pheo-phorbides containing a-hydroxyethylgroups, however, appear to show linedoubling, and this has been attributedto hindered rotation around the C-Cbond attaching the hydroxyethyl groupto ring I. Whether hindered rotation isresponsible, or whether some other

Vol. 5. No. 1/2

Table 2

1H NMR Chemical Shifts3'" of Monomer Chlorophylls a, b, £1, £2and Pyrochlorophy11 a (13)

Proton Chi ac Pyrochl ad Chi bc Chi c i e Chi c i f

Methine a

P6

3-CHO

2-Vinyl HxHAH B

4-Vinyl Hx

7-Acry l i c7a7b

10-H(2)

78la3a4b5a8aI Ob7a7bP-lP-2P-3P-4P-CHs's

P-CH3's

9-239-508.28

7.925.976.13

9.229.468.37

7-995-99

6.224.14*4.273.283.251.72d

3.601.78d

3-972.0-2.52.0-2 .54.414.891.421.741.181.16O.780.750.740.710.68

4.334.214.093.223.161.583.221.64-

-2 .09~2.4O

4.384.971.451-751.171 .120.770.740.700.670.64

9.879.558.1810.92• 85.9815

7.5.6.

6.104.154.453.22

n.r .3.52n.r.3-95-2.35~2.35

(9-95)(9-90)(9.80)

8.286.346.04

8.896.616.72

(3^5-4)9

1.67(3-5-4)9

n.r.n.r.

(10.10)(10.00)( 9-92)

8.336.356.068.336.326.04

8.996.676.84

(3-5-4)9

(3-5-4)9

(3*.5-Mj

n.r.n.r.

a) Chemical shifts in 0, ppm relative to internal TMS. b) Chemicalshifts enclosed in parentheses have been assigned from intercomparisonwith other chlorophylls. c) In C2H3C1/C2H3O

2H (11). d) In acetone-2H6. e) In tetrahydrofuran-2He (22,23). f) In pyridine-2H5 (24,47).g) In TFA (22) .

10 B u l l e t i n of Magnetic Resonance

cause must be sought for the line doub-ling still is not clear.

2. JH NMR Chemical Shifts of the Chlo-rophylIs

Proton chemical shifts of Chi a,Bchl a and the important derivativePyrochl a are listed in Table 2. Therationale of the chlorophyll assign-ments is very much the same as for themethyl pheophorbides. As indicatedbelow, the XH NMR spectra of the chlo-rophylls are strongly solvent depen-dent, and the relationship between thespectra in polar (nucleophi1ic) andnon-nucleophi1ic solvents providedvaluable information for the assignmentof the resonances observed in nucleo-phi 1 ic solvents.

It should be noted that phytol is byno means the universal esterifyingalcohol in the chlorophylls. While allsamples of Chi a and b so far examinedappear to be esterified primarily byphytol, Bchl a derived from Rhodospi-ri1lum rubrum is esterified principallyby geranyl geraniol (a C20 alcohol withk double bonds) (20). It has long beenknown that the chlorophylls from greenphotosynthetic bacteria (Bchl c_) con-tain farnesol (a C15 alcohol with 3double bonds). The presence of addi-tional vinylic protons or methyl groupsin farnesol or geranyl geraniol pro-duces additional olefinic resonances inthe region 5 k~S ppm and this possibil-ity must be kept in mind in the analy-sis of pheophytin and chlorophyll spec-tra.

Some more recently characterizedchlorophylls merit comment. Chloro-phylls £1 and C2 are auxilliary acces-sory pigments in marine diatoms andbrown algae. These chlorophylls areclosely related to each other and toChi a. Unlike Chi a, however, they areporphyrins, not chlorins, although anintact ring V is present in both. Chici and £2 are both free acids, and lackan esterifying alcohol at the position7 side chain. The side-chain substit-uent at position 7 is a transacrylicacid group (AX pattern). Chi £1 and £2differ from each other in that £1 has avinyl and ethyl group at positions 2

and 4, as does Chi a, but Chi £2 hastwo vinyl groups at positions 2 and 4(21,22). Deconvolution and integrationof the methine proton region can beused to estimate the relative amountsof Chi £1 and £2 in a mixture of thetwo (23). Chemical shifts of Chi £1 and£2 are listed in Table 2.

BacteriochlorophylI b is present inRhodopseudomonas vi r idi s and a fewother photosynthetic bacteria. Thischlorophyll is responsible for theextreme long wavelength light absorp-tion in these organisms. The moststriking feature of the Bchl b struc-ture is the ethylidene sideposition 4, which replacesgroup present in Bchl a. Theferences in the XH NMR of Bchl b com-pared to Bchl a are the resonances ofthe ring II protons (13,24). Both the3a and 4a protons give rise to doubledoublets (J1 = 2 Hz, J2 = 7 Hz)field (6 = 4.93 and 6.84 ppm).resonance experiments show themcoupled to each other (J = 2 Hz)a high field methyl group eachHz). The double bondgroup shifts the

chain atthe ethylmain dif-

at lowDoubleto be

and to(J '•- 7

in the ethylidene(3-proton resonance to

resonances areBchl a (Table

lower field. All otheridentical with those ot _3 ) .

Green photosynthetic bacteria con-tain very complicated mixtures of chlo-rophylls whose exact structures arestill under active investigation.Referred to in early publications as"chlorobium" chlorophylls because oftheir isolation from Chlorobium species(25) they are more often called bacter-iochlorophylIs £, d, and e. These chlo-rophylls are unique among all naturalchlorophylls in that each appears to bea mixture of various homologs. Thus,Bchl c, d, and e are each families ofchlorophylls containing homologousalkyl groups at positions 4 and 5. Bchl£ and e in addition contain a CH3-groupat the 6 methine position. Recently,another series of Chlorobium chloro-phylls isolated from Chlorobium phaeo-vi roides has been investigated anddescribed by Brockmann and co-workers(26,27). This family of chlorophylls(designated Bchl e) contains a formylgroup (established by the appropriateCHO resonances in both the *H and 1 3C

Vol. 5, No. 1/2 11

Table 3

'H NMR Chemical Shiftsa of Bacter1ochlorophyl1s a and b,and the Methyl Pheophorbides of Bacter 1ochl orophyl 1 s c, d_, and e_

L.

;ti n

o~h

Qnet i c Resonance

Proton

f-J0

103478

2a4a1a2b3a4b . ' .5a8a10b5-CHa7a7b7d5b

Bchl a b

(0.06M)

9 . 23 b

9.5O8.286.444. 103.864 . 104.21-

~2.5(3.33)(3.OO)1.58 (d)n. r .(3.44)1.41 (d)(3.66) (d)

-~2 . 5~2.5

-

a) Chemical shifts in 6,with other chlorophylls.and 5,methylchains

and an ethyl groupgroup at positionat posi tion 4 (3).

MethylBchl b b Bacteriopheophorbide(0.06M)

9.418 .938. 396.434.93 (dd)-

4 . 104.21-

6.84 (dd)(3.34)(2.99)1.66 (d)2.01 (d)(3.45)1.41 (d)

(3.66) •-

~2.5-2.5

-

ppm relative to internalb) In pyridine-'Hi, (24) .at posit1on 4 (30). d)

5 (31). e) T n C ' HC 1 :..d) Includes the proton

(0.08M)

9.909.41-

5. 17--

4. 144.556.47 (q)3.68 (q)3.482.12 (d)3.261.68 (t)3.611.41 (d)-

3.85--3.58

Methylc c Bacteriopheophorbide d

(0.04M)

9.579.288.455.04--

4. 134.366.31 (q)1 .68 (t)3.382.083.19-

3.511 .75--

-3.62

TMS. Chemical shifts in paranthesesc) In C!

In C * HC 1 :..This sampleat position

MethylBacteriopheophorbide ee

(0.05M)

10.589.42-

5.20--

n. r .4.586.561 .723.532.151 1 .071 .204 .011.51-3.86--

3.621 .92

are assigned by interconversionHCl.i. This sample contained methyl groups at positions 3This sample contains an ethyl group at position 4 and acontained a mixture of ethyl, n-propyl or Isobutyl side3.

NMR spectra) and, thus, has the samerelationship to Bchl £ and d as doesChi b to Chi a. All of the Chiorobiumchlorophylls have an (a-hydroxy-ethyl)-substituent at position 2 char-acterized by a low-field quadruplet at6.1-6.6 ppm and a high-field doublet.These chlorophylls all lack a 10 CO2CH3group and are, thus, pyrochlorophyl1derivatives. The AB double doubletexpected for the IO-CH2 protons isoften only poorly resolved (10). Thepredominant esterifying alcohol in Bchlc (the chlorobium-650 of Holt (28)) isfarnesol, but Strouse et al. (29) haveshown that small amounts of at leastfive other esterifying alcohols arepresent. Risch et al. (30) have like-wise found that the chlorophylls fromChloroflexus aurantiacus contains stea-ryl (C18H37), phytyl (C20H39) , and ger-anyl geranyl (C20H33), and not farnesolas the esterifying alcohols.

To simplify the application of NMRand mass spectroscopy, it has been cus-tomary to eliminate the long-chainaliphatic alcohol by transesterifyica-tion with methanol, a procedure duringwhich the Mg is lost. The methyl pheo-phorbides of Bchl c, d, and e are moreeasily separated by chromatography thanthe chlorophylls themselves (31). Con-sequently, all of the available NMRdata on Bchl c_, d, and e is for themethyl bacteriopheophorbides. Selecteddata for some of the numerous homo logsof the bacteriomethylpheophorbides aregiven in Table 3-

The methyl pheophorbides of Bchl £and d have been used by Trowitzch (32)to assign the methine chemical shiftsin methyl pheophorbide a_ and methylpyropheophorbide a. In the Bchl £ and dderivatives, the a and 3 protons havedistinctly different neighbors, unlikethe situation in methyl pheophorbide aor methyl pyropheophorbide a, andassignment of the a and (3 protons isfacilitated. Conversion of the hydroxy-ethyl group to vinyl converts methylbacteriopheophorbide d to methyl pyro-pheophorbide a, and the spectralchanges support the original assigment

of the methine chemical shifts ofmethyl pheophorbide a.

Sanders and co-workers (33.3*0 havereported spin-lattice relaxation times(Ti), nuclear Overhauser enhancements(NOE), and long-range coupling con-stants for the chlorophylls. The T1values for the methyl protons dependlargely on distance from the macrocycleand steric crowding, but the T1 valuesfor the methine protons are dependenton the substitution pattern (Table k).In the absence of any information onthe T1 error limits it is difficult tojudge the usefulness of relaxationtimes that fall in a narrow range formaking chemical shift assignments. San-ders et al. (33) attribute line-widthvariations in the chlorophyll spectrato unresolved long-range acycliccouplings. The assignments of Sanderset al. made on the basis of T1, NOE,and long-range coupling effects agree,however, in all particulars with previ-ous chemical shift assignments madefrom ring-current and disaggregationconsiderations.

3. Chlorophyll Related Structures

A number of structures related tothe chlorophylls have been character-ized by lH NMR. These include the epim-ers, enol, and the Krasnovskii photore-duction product of Chi a, and this hascontributed significantly to the clari-fication of some longstanding problemsin chlorophyll chemistry.

It has long been known that Chi aand b, in the course of column chroma-tography on sugar, are accompanied bysmall, faster- running satellite bands,designated a' and b1 (35)• The two sub-stances are easily interconvertible,and it was suggested by Strain (35)that a and a1 were diastereomers, epim-eric at carbon-10. Experimental evi-dence for this interpretation was pro-vided by *H NMR studies (36)., whichshowed that the diastereotopic C-10protons of a and a/ had chemical shiftsclosely resembling those of the twoC-10 protons of pyrochlorphyll a_.

Vol. 5, No. 1/2 13

Table k

Spin-Lattice Relaxation Times (s) for Chlorophylls

Protons Chi a Chi b Methyl Methyl Bchl aChlorophy11ide a ChlorophylVide b

aP0

1078la2b3akb5a8a10b7d

1.00.91.0).ha

0.70.60.7-

0.60.70.70.41.0-

0.80.60.81.2-

0.60.6--

0.60.70.50.8-

1.31.11-3l.7

0.9

1.8

a) For Chi a1, Ti =s. c) Estimatedmethyl group of the

2.0 s. b) For methyl chlorophyby null point because of signalacetyl function at position 2.

1.51.11.31.6--

0.9

-0.081.20.51-31.8

1 ide a1,over 1ap.

1.00.70.60.80.5c

o.5c

0.51.3d

0.30.70.50.40.8

Ti = 2.0d) The

In Pyrochl a, the two protons at C-TOare in magnetically non-equivalentpositions (37) and mixtures of Chi a_and a_' have peaks in their 1H NMR spec-tra that can be assigned to analogousdiastereomeric C-10 protons. Recently,an alternative interpretation for thestructure of Chi a' was revived (38) ,which claimed that Chi a1 is the enolform of Chi a. To resolve the situ-ation, Chi a and Chi a_' were separatedchromatographically at 0°C, and XH NMRspectra recorded on the eluted compo-nents at low temperatures, where inter-conversion between a and a_' is veryslow. Chi a and a1 are clearly seen tohave C-10 protons with different chemi-cal shifts (Table 5 ) . thus disprovingthe enol hypothesis and establishingChi a' as the epimer of Chi a (i+0) . Infurther work, Hynninen and Sievers(40a) deduced from additional 1H NMRdata that conformational changes (puck-ering) of the whole macrocycle occurswith epimerization at C-10. It isinteresting to note that the methine

protons (and other resonances as well)in a' are easily resolved from those ofa, and can often be clearly distin-guished in the 1H NMR spectrum of anequilibrium mixture of Chi a_ and a_'. Inan XH NMR study, Ellsworth and Storm(41) have shown that the Mg-free methylpheophorbide a_' is much less prone toisomerization than is Chi a'. In chlo-roform solution at room temperature,methyl 10-epipheophorbide a appears tobe stable indefinitely. The differencein the rates of epimerization isattributed to conformational differ-ences between the chlorophyll and pheo-phorbide. As in the case of Chi a anda1, the methyl pheophorbides a and a'have significantly different chemicalshifts for the methine, C-10, carbome-thoxy, and la, 3a, and 5a methyl pro-tons.

Ring V in the chlorophylls containsa (3-keto ester function and is there-fore prone to enolization. In solution,the keto/enol equilibrium in all of thechlorophylls is strongly displaced


toward the keto form, and only a small,stationary concentration of enolappears to be present. The enol hasbeen implicated in the Molisch phasetest, which establishes the integrityof ring V (42) as an intermediate inthe allomerization reactions of chloro-phyll (4,5). and in hydrogen exchangeat position C-10 (43). Interest in theenol remains keen, for many models havebeen advanced involving enol participa-tion in photosynthetic oxygen evolution(44) and in the primary events of pho-tosynthesis (45) .

Peripheral complexes are formed frompheophytins or methyl pheophorbides andMg 2 (46). Peripheral complex formationwith Mg-containing chlorophylls doesnot occur to a significant extent. 1HNMR shows that peripheral complexationoccurs with the enol form of the3-ketoester system of ring V. The C-10proton is no longer to be seen in theXH NMR spectrum of the peripheral com-plex. The ring-current induced shiftsin these complexes are smaller than inthe free pheophorbides. The IO-COOCH3becomes more or less coplanar with themacrocyclic systems, and the incremen-tal low-field shift is unusually low,which is a direct result of the move-ment of this group into a deshieldingregion of the ring current. A compari-son of the chemical shifts of methylpheophorbide a and its peripheral Mgcomplex is shown in Table 6.

The enols of Chi a, Pheo a, andmethyl pheophorbide a have been trappedas the tetramethylsilyl ethers (40).The silyated enol of Chi a is labile,and easily reverts to the original Chia, or is converted to the silylatedenol of Pheo a. The XH NMR chemicalshifts of methyl pheophorbide a and itsenol trimethysilylether are compared inTable 7- The largest changes areobserved in the chemical shifts of themethine protons, which again implies alarge decrease in the ring current inthe stabilized enol.

The Krasnovskii photoreduction ofChi a was the first and is possibly themost widely studied photoreaction ofthe chlorophylls. Chi a dissolved inpyridine can be reversibly reduced inlight by ascorbic acid to a pink photo-product, which in the dark reverts to

Chi a (48). The structure of the photo-reduction product remains elusive, how-ever, despite much study (49). XH NMRstudies have now made possible a struc-ture assignment to the photo-product(50). The photoreduction of Chi a iscarried out with 1H2S or 2HaS directlyin a sealed NMR tube. When 2H2S isused as the reductant, the already sim-ple NMR spectrum of the photo-productis even further simplified. From the XHNMR spectra it is immediately evidentthat the photereduction of Chi aresults in the loss of the ring cur-rent, i.e., the conjugation in the mac-rocycle is disrupted. Most of the lowerfield resonances of Chi a are shiftedto substantially higher field, whilethe signals originating in the phytylmoiety remain substantially unchanged.The upfield sh*ift is most pronounced inthe resonances of protons closest tothe macrocycle. The upfield shifts areof the order of 1.0-1.7 ppm for thevinyl and ring methyl protons, andabout 6 ppm for the (3 and 5 methineprotons. As the integrated area ofthese two resonances indicates thepresence of 2 protons, it is concludedthat the Krasnovskii reaction productis P,5-dihydro-chlorophyll a. lH NMRalso establishes that the reversal ofthe photoreaction in the dark restoresthe original Chi a. The increased sen-sitivity of modern NMR spectrometersmakes it possible to study the effectof light irradiation on chlorophyllsolutions sufficiently low in concen-tration to permit photochemical inves-tigations in the spectrometer probe,and such investigations very likelywill open a new chapter in chlorophyllphotochemistry.

4. 1 3C NMR

All 55 carbon atoms in Chi a havehad their 1 3C NMR chemical shiftsassigned. General studies of the 13CNMR spectra of chlor ins have beenreported by Lincoln et al. (51)» andSmith and Unsworth (52), and Chi aitself has been studied by a number ofresearch groups (53~59) • Assignment ofthe quarternary carbon atoms was car-ried out by Boxer et al. (56), whileGoodman et al. (60) have assigned all

Vol. 5, No. 1/2 15

Table 5

NMR Chemical 5hiftsa of Methine and C-10 Protonsin Chlorophyll a and a1 (39)

Proton

In Pyridine-2Hs

Chi a Chi a1

In Acetone-2He

Chi a Chi a1

a(36

. C-10

9.1»29-588.286.44

9-399.568.266.30

9.079.408.265-99

9.049.378.225-87

a) 8, ppm, relative to internal hexamethyl disiloxane.

Table 6

NMR Chemical Shifts3 of Peripheral Mg Complex of Methyl Pheophorbide a (47)

Proton

a(36Vinyl

107810b7b5a3ala8a4a4bN-HN-H

HxHAHB

Per ipheralMg Complex"

8.839.018.007-776.065-87-

lt.654.103.833.383-112-952.831.73,3.291.392.442.04

MethylPheophorbide ac

9.479-758.718.086.236.056.614.294.423.763.523-423-213.081.663.541-53

+0.74-1.48

16

a) Chemical shifts in 0, ppm relative to internal TMS. b) Recorded on asolution of pheophytin or pheophorbide a (7*10 3 M) in a saturated solutionof Mg(C104)2 in pyridine-2Hs. c) Spectrum of the free methyl pheophorbide aregenerated by addition of 10 /nl of 'HhO.


Table 7

*H NMR Chemical Shiftsa of Methyl Pheophorbide aand the Trimethylsilylether of the Enol of

Methyl Pheophorbide a_ (40)

Proton Methyl Pheophorbide a TrimethylsilylEther of the Enolc

a36Vinyl

107810b7d5a4ala3a7a7b8a4bN-H

9-259,33

18• 71•98• 78.2016

.95• 37.261813• 91• 90.29.06.5.1.42

0.79-1.38

8.7.5-5-6.4.3-3-3-3.3.2.2.

2.2,11

8.198.24.0319.64

7.7.5-5.46

.66

.22

.60077398.4637.817327.22.26

2.14

a) Chemical shifts in 6, ppm. b) In benzene-2He. Chemical shifts rela-tive to internal hexamethyldisiloxane. c) In ben2ene-2H6. Chemicashifts relative to the trimethylsi1yl group of the compound.

of the carbon atom resonances in thephytyl moiety. Argonne studies usedChi a enriched to 15-20% 13C and Matwi-yoff et al. (58,59) used Chi a of 90%13C enrichment, in both instances pre-pared by biosynthesis with 13C02 of theappropriate isotopic composition.

Chemical shift assignments for thecarbon atoms of methyl pheophorbide a,methyl pyropheophorbide a, and Chi aare listed in Table 8. The originalassignment by Boxer et al. (56) of C-6,C-l6, and C-17 have been revised bySmith et al. (6l) , Wray et al. (62),

and Lotjonen and Hynninen (62a). Therevisions have been incorporated intoTable 8. Insertion of Mg into methylpheophorbide a produces downfieldshifts of carbon resonances in rings Iand III, and upfield shifts for theresonances associated with the carbonatoms in rings II and IV. Coupling con-stants (JI3£_H) f°r carbon atomsbearing protons are listed in Table 9-13C NMR spectra recorded on Chi a and bcontaining 90% 13C have been made itpossible to extract a number of 1 3C- 1 3Ccoupling constants, which are listed in

Vol.. 5, No. 1/2 17

Table 8

13C Chemical Shifts (<5, ppm)a of Monomeric Chlorophyll a,Methyl Pheophorbide a , and Methyl Pyropheophorbide a

Carbon No.

la2a2b3a4a4b5a7a7b7c7d788a91010a10ba3y6l234561112131415161718P-lf

P-2P-3P-3aP-4P-5P-6P-7P-7aP-8

Chiorophyl1 ac

14.9 or 15.0133.*•121.213.522.220.2

14.9 or 15.033-632.6175.1

-53-351.826.1191.968.2173-154.3103.0110.1108.495.2

137.6 or 136.4141 .7

137.6 or 136.4146.6

137.6 or 136.4164.5 (13C.9)9

156.8150.7154.1148.7150.6158.6 (162.4)

-174.5 (156.3)9

170.263.8122.1144.718.542.427-739.435.422.340.1

MethylPheophorbide ad

11.8128.3121.810.719.017.111.831.029.8172.651.451.049.922.8189.064.5168.952.696.4103.6104.892.6131.1135.7135-3144.2128.3160.5 (128.3)141.3135.3155.0150.7137.2149.0172.6 (160.5)171.4

MethylPyropheophorbide a&

11.8128.5121.610.819.117.211.851.431.029.8172.851.449.722.9195-247.8--

96.4103.2105.492.4130.7135.1135-2144.0127.4129.7140.7135.3154.1(149-7)136-9148.2159.5170.4


Table 8 (Continued)

13C Chemical Shifts (5, ppm)a of Monomeric Chlorophyll a,Methyl Pheophorbide a, and Methyl Pyropheophorbide a

Carbon No.b

P-9P-10P-llP-llaP-12P-13P-14P-15P-15aP-16

Chlorophy11 ac

27-340.135-522.340.1

27-742.130.725.225-2

MethylPheophorbide 3d

MethylPyropheophorbide ae

a) Relative to hexamethyldisilane for chlorophyll a; relative to tet-ramethylsilane (TMS) for methyl pheophorbide £. b) See Figure 1 fornumbering, c) In benzene/tetrahydrofuran solution, d) In C2HCl3 solu-tion (14). The values in parentheses are from the reassignment of Smithet al. (61). e) References 52, 61. f) Phytyl carbon assignments arebased on those of Goodman et al. (60) and are relative to internal TMS.g) Lotjonen and Hynninen (62a) have revised the assignments for carbonatoms 6, 16, and 17. Note that their 13C chemical shifts are relativeto TMS and for acetone-d6 solutions.

Table 10.Smith et al. (6l), in connection

with efforts to resolve the complicatedquestions surrounding the structure ofthe Bchis j:, have collected extensive13C NMR data on the methyl pheophor-bides derived from the Bchls c, andhave presented more difinitive 13C NMRassignments for methyl pheophorbide aand methyl pyropheophorbide a. Assign-ment is greatly facilitated by the useof chlorophylls containing . 15-20% 13C.For Chi a and b, this is not difficultto accomplish, but for the chlorophy11sfrom photosynthetic bacteria, 13Cenrichment is a rather complicatedtask, which up to now has been fullysolved only for Bchl a. Prospects forobtaining the other bacterial chloro-phylls sufficiently enriched in 13C to

make a full assignment possiblehowever, appear to be good.

do,

5. 15N and 2H NMR

Full assignment of the 1SN chemicalshifts in Pheo a and Chi a have beenmade by Boxer et al. (56) (Table 11).The Chi a and Pheo a derived from itcontained 95% 15N, incorporated bybiosynthesis. The 15N spectrum of Pheoa was recorded directly, but for Chi a_the 15N relaxation times were so longas to preclude direct observation ofthe spectra, and the l5N spectralparameters were obtained indirectly byINDOR. Long-range coupling between 15Nand the methine protons was observed inthe lH NMR spectra of both compounds,as well as the expected 15N couplingwith the inner protons in Pheo a. Anal-ysis of the 15N NMR spectrum of Pheo aalso yielded all of the 15N-15Ncoupling constants (Table 12).

2H NMR spectra (at 15.4 MHz) havebeen reported by Dougherty et al• (63)

Vol. 5, No. 1/2

Table 9

Coupling Constants Ji3c_i^ for Chlorophyll aand Methyl Pheophorbide aa

Ji3C_xH (Hz)b

Carbon atom Chlorophyll a Methyl pheophorbide a

VinylC-2aC-2bC-Phy-2

Methi nea36

Aliphatic-CHC-7C-8C-10

-CH2C-4aC-7aC-7b

-CH3C-Phy-1C-laC-3aC-4bC-5aC-8aC-lOb

a) Table from Janson and

ithyl pheophorbide a-d35

150158152

150 .148152

-130132

---

153128125

128-148

Katz (53) •

and Chi

155160-

155155157

129-130136

125130126

-129126160129125148

b) ±2.5 Hz.

C. Applicat ions of NMR

a-d72. The line widths of 2 to 7 Hz formethyl pheophorbide a-d35 were broa-dened by quadrupole relaxation, butwere still sufficently narrow to permitassignment of many of the 2H reso-nances .

20

Many applications of NMR to struc-ture determination and conformationstudies have been made to the chloro-phylls. In addition, NMR has beenlargely responsible for major advancesin the understanding of chlorophyllbehavior, and has been particularly


Table 10

Some Coupling Constants Jur-ifor Chlorophylls a and b°>b

Carbon atom

1-la2a-2b3-3a4-4a4a-4b5~5a7b-7c8-8a10-10a

Chlorophyl1 a

446845423444554658

Chlorophyll b

446850423444554658

a) In Hz. b) Table from Matwiyoff and Burnham (59)

Table 11

15N Chemical Shifts for Chlorophyll a and Pheophytin aa'b

Nitrogen Chlorophyll ac Pheophytin ad

N-lN-2N-3N-4

163.6183-5166.4224.0

102.5218.5110.9272.8

a) Table from Boxer et al. (56). b) In ppm relative to external 15NH.iClin 2 N HC1. c) In acetone-2He. d) In C2HCls.

effective in defining the nature ofchlorophyll-chlorophyll and chloro-phyll -nucleoph Me interactions. Some ofthe more significant applications ofNMR in chlorophyll chemistry are nowcons idered.

1. Exchangeable Hydrogen in Chlorophyll

A possible role for chlorophyll inthe light conversion step in photo-synthesis as a cyclic hydrogen donor

was for long a subject of speculation.Such an hypothesis implies exchangeablehydrogen in either the ground state orexcited states of chlorophyll. XH NMRhas successfully addressed both ofthese problems (64,65). Both the C-10and the o-methine protons are readilyexchangeable in Chi a, Chi b, and Bchla. With the hydroxyl group of methanolin pyridine, hydrogen exchange at C-10is rapid, whereas exchange at all meth-ine carbon atoms is much slower.Exchange at the 6 position is strongly

Vol. 5, No. 1/2 21

influenced by the presence of Mg.Removal of the Mg reduces the exchangerate at the methine bridge positions tovery low values. That the "extra"hydrogen atoms in ring IV do not func-tion as reducing agents in a cyclicprocess in photosynthesis is demon-strated by the failure to observehydrogen exchange when green algaegrown in 99-5% 2H20 are transferred to1H20 and allowed to conduct photo-synthesis. Chlorophyll extracted fromthese organisms is found to contain no,XH by 1H NMR, arguing against a rolefor exchangeable hydrogen at the C~7,C-8, and o-methine positions in photo-synthes i s (65) •

2. NMRSpeci es

of Paramagnetic Chlorophyll

The hypothesis that the primaryelectron donor in photosynthesis is aspecial pair of chlorophyll moleculeswas originally based on EPR lineshapeanalysis of the cation species that

Table 12

15N Coupling Constantsin Pheophytin aa

Coupling Constant Value (Hz)

...

2Jl

2Jl

2Jl

5N-HI

5N-methine H

5 N i - 1 5 N ?

5 N 2 -1 5 N 3

•N,-»N4

98

3-0

2.0

5-7

l . i f

2-5

a) Table from Boxer et al. (56)

remains after electron transfer (66). Acomparison of the hyperfine coupling

constants of j_n vi tro Chi a and Bchla*' and in vivo P700*" and P865*' byENDOR spectroscopy has provided consid-erable support for the original conclu-sions (67). ENDOR spectroscopy, how-ever, is not without its problems, andthe chemical manipulations required forthe preparation of selectively deuter-ated chlorophyll derivatves requiredfor ENDOR assignments are not trivial(68). Sanders and Waterton have,therefore, undertaken the determinationof the hyperfine coupling constants ofthe chlorophyll cations by NMR linebroadening in the fast exchange limit(69,70). NMR in principle is a farsuperior method for determination ofthe hyperfine coupling constants asassignment follows immediately from thechemical shift assignment, and no chem-ical manipulations are required. Onlyrelative hyperfine coupling constantscan be deduced by NMR, and a reliablevalue from ENDOR of at least a few of.the coupling constants is required forconversion to absolute values. Water-ton and Sanders (70) find that NMRresults with Chi a* and Bchl a* agreewell with ENDOR, but for Chi b*',agreement is poor. The experiments ofWaterton and Sanders were carried outat room temperature or above to insurethe chlorophyll species were in fastexchange. The Chi a_* cation has ahalf-life at room temperature of about20 min. and at 310 K about 5 min. Thevariation in Chi a_* concentration dur-ing the experiment may, thus, compli-cate interpretation of the line broa-dening data.

Brereton and Sanders (70a) havestudied the radical anion of bacter-iochlorophy11 a by observing differen-tial electron transfer line broadeningin the 1H NMR spectrum of diamagneticBchl a in the presence of a smallamount of chemically produced anion,formed by reaction with sodium sulfide.From the observed line broadenings, itwas concluded that the protons at thea,3.T,7»^»5a»la,2b,3a. and 8a positionshave significant hyperfine interactionswith the unpaired electron in Bchl a" ,but no hyperfine coupling constantswere calculated. In these experiments,no chemical evidence is presented thatunequivocally establishes the chemistry


of the reaction of Bchl a with S"(see reference 50).

Closs and Sitzmann (71) have suc-cessfully determined the hyperfinecoupling constants of radical cationsof chlorophylls and derivatives bytime-resolved CiDNP (chemically induceddynamic nuclear polarization) studies.In these experiments, polarized *H NMRspectra are recorded on a chlorophyll-benzoquinone system in which electrontransfer is induced by a nanosecondlaser light pulse. The hyperfinecoupling constants determined by thisprocedure are relative, and require atleast one ENDOR-determined value fornormalization; the absolute hfc valuesobtained by CIDNP (after normalization)agree well with the ENDOR values. Thisprocedure should have wide applicabil-ity. It would be desirable, however,because of the importance of thesestudies, to explore in more detail thechemistry of the reaction used. Benzo-quinone can oxidize Chi a by a darkreaction, and nucleophilic attack onthe cation can form alteration prod-ucts. The presence of methanol inthese experiments raises the possibil-ity that 10-methoxy Chi a could beforming in the system during the courseof the experiment, which would conceiv-ably complicate the interpretation ofthe observations.

Selective line broadening by lightirradiation has been shown by Boxer andCloss (71a) to occur in light-excitedmolecules in an NMR probe, and theyhave developed a method that success-fully extracts information on spin dis-tribution in photo-excited tripletstates from high resolution *H NMRdata. Some preliminary 1H NMR data onthe relative hyperfine coupling con-stants in light-excited methyl chloro-phyll ide a have been reported (72).Spin distribution in the Chi a* cationfree radical appears to be considerablydifferent from that of 3Chl a. Rela-tively little spin density is to befound at the methine positions in doub-let state Chi a* , whereas the methinepositions have the highest spin densi-ties in the chlorophyll triplet.

Paramagnetic chlorophyll species areformed in the primary light conversionevent in photosynthesis, and other

paramagnetic species appear to beinvolved in the oxygen-generating sideof photosynthesis. Wydrzynski and co-workers (73, Jk, 75) have initiatedresearch in which the effects of param-agnetic species on the lH and 1 70relaxation times of water are beingexplored in an effort to clarify theoxygen-evolving apparatus in greenplants. Proton and 1 70 relaxationrates (1/Ti and I/T2) have been meas-ured in chloroplast preparations, withresults that suggest that manganese ina mixture of oxidation states is nor-mally present in dark adapted chloro-plasts (74). The relaxation rates forXH and 1 70 are for the most part deter-mined by loosely bound Mn present inthe chloroplast membranes; it is esti-mated that from one-third to one-fourthof the loosely bound Mn is present indark-adapted chloroplasts as Mn (II),the remainder being in higher oxidationstates. The Mn appears to be located inthe interior of the photosynthetic mem-branes (73)- Experiments have also beencarried out in which the XH spin-spin(transverse) relaxation rate of chloro-plast suspensions has been measuredafter each of a series of 2.1* juseclight flashes. The sequence of relaxa-tion rates oscillates and has a maximumvalue after every fourth flash. Thishas been interpreted to indicate thatmanganese participates in the chargeaccumulation process during oxygen evo-lution (75). However, the interpreta-tion of the experiments of Wydrzynskiet al. (75) has been questioned by Rob-inson et al. (75a). These investiga-tors found that the changes in the pro-ton relaxation rate can be abolished byremoval of Mn(ll) from the chloroplastswith appropriate chelating agents with-out affecting the evolution of O2. Themanganese that is involved in therelaxation phenomena thus appears notto be the manganese involved in theliterature evolution of O2 during pho-tosynthesis. In any event, the appli-cation of NMR techniques would appearto be of considerable promise for thestudy of the oxygen side of photo-synthesis.

3. Chlorophyl1-Nucleophile Interactions

Vol. 5, No. 1/2 23

Chlorophyll NMR spectra areremarkably solvent dependent, beingvery different in nucleophilic andnon-nucleophi1ic solvents. This solventdependence was early recognized to be aconsequence of nucleophilic interac-tions at the central Mg atom of thechlorophylls (11,76). All lines ofspectroscopic investigation support theview that Mg with coordination number kin chlorophyll is coordinative!y unsa-turated, and that there is in conse-quence a driving force to acquire•electron donor functions (i.e., lonepair electrons on oxygen, nitrogen, orsulfur) in one or both of the Mg axialpositions. Chlorophylls dissolved intypical nucleophilic, polar (Lewisbase) solvents such as acetone, diethylether, pyridine, tetrahydrofuran andthe like occur as monomeric chlorophyllwith one or two molecules of solvent,depending on basicity, in the axialpositions of the Mg. In weak donor sol-vents such as acetone or diethyl etherthe Mg occurs largely with coordinationnumber 5. and in more basic solventssuch as pyridine, the coordination num-ber of the Mg approaches 6.

The coordination interaction at Mgpositions the ligand in the center ofthe chlorophyll macrocycle , where itis subject to the full force of thering current. Chlorophyll is in effecta natural NMR shift reagent. The chemi-cal shifts of the protons of a ligandbound to Mg will therefore experiencean upfield ring current shift to anextent determined by the distance of aparticular proton from the center andplane of the chlorophyll macrocycle.

Katz et al. (77) have made a quanti-tative study of chlorophyl1-nucleophileinteractions by observing the ring cur-rent effect on proton chemical shiftsof the ligands bound to chlorophyll.The use of fully deuterated chlorophyllsimplifies interpretation of the spec-tra. Pentacoordinate Mg (II) appears todominate the equilibrium with aliphaticalcohols, the equilibrium constant forthe formation of Chi a^CHsOH in CCUsolution being K1 = 56 1 mol"1 (Figure2). Georghe et al. (78) have studiedthe chlorophyll-water interaction by *HNMR. Water as a nucleophile is observedto have about the same base strength as

4.505 10 15 20 25

Mole ratio CH,OH/d- chlorophyll a

Figure 2. Chlorophyll a-methanol inter-action in carbon tetrachloride solu-tion. Chemical shifts of CHa(O) andC-10 (A) protons as a function ofCH3/

2H-Chl a (0.064 M) ratio. Addi-tional C-10 points (o) are derived froma methanoi titration of ordinary Chi a_.The solid lines are calculated curves.

methanol as measured by coordination tothe Mg atom of Chi a.

Quantitative observations have beenmade on coordination interactionsbetween chlorophyll and various com-pounds present in thylakoids and likelyto be near neighbors of chlorophyll(77) • As expected, (3-carotene does notappear to experience any interactionwith chlorophyll that results in plac-ing any of its protons near the chloro-phyll macrocycle. The XH NMR spectrumof (3-carotene in C2HCl3 solution is thesame in the presence or absence of2H-Chl a. With lutein, a dihy-droxy-3-carotene, the situation is verydifferent, for there are major


differences in the *H NMR of luteinwhen 2H-Chl a is present. These differ-ences are consistent with the coordina-tion of the hydroxy] groups to the Mgof Chi a, which results in a markeddifference in the magnetic environmentof the ring methyl groups of thelutein. Similar changes are observedwith other carotenoids carrying nucleo-philic groups. Another important classof chloroplast components are thegalactolipids, sulfolipids, and phos-pholipids. These compounds might rea-sonably be expected to coordinate to Mgby way of ester C=0, -OH, or -SO3Hfunctions present in these molecules.However, in C2HCl3 solution, plant lip-ids, particularly the sulfolipid char-acteristically present in green plantphotosynthetic membranes, show only aweak tendency to compete for coordina-tion to Mg. This may be due to thepresence in chloroform solution of boththe chlorophyll and the sulfolipid asinverted micelles, in which the polarregions of both substances are buriedin the center of the micelle. With cur-rent interest in the photosyntheticmembrane, further studies of chloro-phyll-lipid interaction would appear tomerit attention.

Larry and VanWinkle (79) have madean XH NMR study of the interactions ofChi a and b with sym-trinitrobenzene.The interactions were studied in chlo-roform solution containing methanol sothat the chemical shift changes must beattributed to generalized t:—n forcesrather than to coordination interac-tions at Mg. At a molar ratio of 1:1,the largest paramagnetic chemical shiftdifferences are observed for the methy-lene protons bound to the oxygen of thephytyl moiety and the diamagneticshifts observed for the a- and(3-methine protons. The methyl protonsat positions 3a and 5a both show diam-agnetic shifts while little or nochange is observed for the protons atpositions la, 10b, 10, or 0. The trini-trobenzene protons experience a largeupfield shift. These observations areconsistent with the formation of a Chi<|-tr i ni trobenzene complex in which thetrinitrobenzene lies on the surface ofthe macrocycle with two of its nitrogroups extended over the a- and

3-methine protons. The shift in thephytyl may then be due to displacementof the phytyl chain from the diamag-netic zone of the macrocycle.

k. Chlorophyll-Chlorophyll Interactions

XH NMR spectra of Chi a in non-nu-cleophilic solvents are very differentfrom those in nucleophilic solvents

10.0 6.0 4.08,ppm

Figure 3. XH NMR spectra of chlorophylla_ in nucleophilic and non-nucleophi1icsolvents. (A) in tetrahydrofuran (0.13M); (B) in carbon tetrachloride (0.06M) ; (C) , in n-octane-2Hia (O.Oit M) . Themonomer spectrum assignments are shownin A. Spectrum B is the spectrum of(Chi a)2.

(Figure 3). In the polar solvent tet-rahydrofuran, Chi a occurs as the mono-solvate, Chi a»THF, but in CCU orn-octane, Chi a occurs as a dimer or anoligomer, respectively. Evidently, inthese solvents a mobile equilibriumnChl a * (Chi a ) n exists. The extent

Vol. 5, No. 1/2 25

of aggregation is then determined bysolvent, chlorophyll concentration, andtemperature. The equilibrium constantfor aggregation is very large, probablygreater than 10' mol"1 1 for the dimer,so that the concentration of monomerChi a in systems free of extraneousnucleophiles is very small. In polari-zable, non-nucleophi1ic solvents suchas carbon tetrachloride, chloroform, orbenzene, Chi a occurs predominantly asthe dimer, whereas in difficultlypolarizable , non-nucleophi1ic solventssuch as aliphatic hydorcarbons, oligom-ers, (Chi a ) n , with n > 20 occur in0.1 M Chi a solutions. It is, there-fore, not surprising that the XH NMRspectra reflect the differences in theChi a species present in the differentsolvents. lH NMR spectra of even thedimer are distorted, and for largeroligomers are obliterated. However, JHNMR studies on dimeri2ed solutionsyield important information that bearson the structure of the dimer. Adetailed review of chlorophyll-chloro-phyll interactions can be found in ref-erence ll».

Addition of a nucleophile to a solu-tion of (Chi a) 2 in CC14 changes the

XHNMR spectra to an extent determined bythe molar ratios of Chi a/nucleophile(11). When a molar excess of nucleo-phile has been added, the XH NMR spec-trum in a non-nucleophi1ic solventbecomes identical to that of Chi a in aneat base. Because the chemical shiftsare fully assigned in the monomer spec-trum, it is possible to ascertain thepositions of the corresponding protonresonances in the self-aggregated (Chia) 2 by a titration procedure in whichXH chemical shifts are recorded as afunction of Chi a/nucleophile ratio.Such an experiment makes possiblestructural conclusions about the natureof the chlorophyll aggregate.

A typical titration experiment, inthis case on aggregated Bchl a, isshown in Figure *• (80) . The addition ofincremental amounts of the strong basepyridine to a solution of (Bchj_ a) nresults in a larger change for thea-methine resonance than for the 3 ando protons. The C-10 proton, the protonsof the methyl groups at positions la,5a, and 10b, and the CHa-group of the

1.0 2.0 3.0 4.0 5.0 6.0 8.0 9.0 10.0MOLE RATIO C5D5N/BACTERI0CHL0R0PHYU.

Figure b. Titration of bacteriochloro-phyll a (0.03 M) in benzene solutionwith pyridine-2Hs. Chemical shifts in5, ppm relative to internal hexamethyl-disiloxane. See Figure 1 for protonnumber i ng.

acetyl function at position 2 likewiseexperience a large downfield shift asbase is.added. All of the changes inchemical shift on disaggregation are tolower field, indicating that in theaggregate the resonances of many of theprotons are at higher fields than inthe monomer. A reasonable hypothesisfor this effect is a diamagnetic ringcurrent effect on the protons experi-encing a high field shift in the aggre-gate. The protons fall roughly intotwo classes, one in which the protonresonances are essentially the same inboth monomer and aggregate, and theother in which the protons are shiftedto varying degrees upfield. It follows


that the chlorophyll macrocycles areonly partially eclipsed in the aggre-gates. The downfield shifts observed inthe titration experiment can, there-fore, be interpreted to indicated thatthe protons experiencing the largestdownfield shift are the ones moststrongly shielded. The titrationresults can be presented in the form ofan aggregation map (Figure 5), in which

0.49

0.33"

C02CH3 0

c6 2 (c 2 0 H 3 9 ) a 6 2

Figure 5- Aggregation map of bacter-iochlorophy11 a from chemical shiftdifferences between aggregated andmonomeric bacteriochlorophy11 a. Thenumbers in the figure show the maximumdifferences in chemical shift (in ppm)between monomer and aggregate for theindicated protons as deduced from thetitration data of Figure k. The arcsindicate regions of macrocycle overlapin the aggregate resulting from coordi-nation interactions by both the2-acetyl and 9~keto carbonyl functionswith Mg atoms in adjoining bacterioch-lorophyll molecules.

the maximum differences in chemicalshift between the aggregate and themonomer for given protons are superim-posed on a structural formula. For Bchla aggregates, two regions of overlapare evident, one in the vicinity of theketo C=0 function, the other near theacetyl C=0 group. Both of these groupsmust be acting as nucleophiles to theMg atom(s) of other chlorophyll mol-ecules. The presence of two donor func-tions in Bchl a considerably compli-cates matters, and whether there aretwo populations of dinners in this sys-tem is still uncertain.

Although there is convincing evi-dence from 1R spectroscopy that it isthe keto C=0 group in ring V that isthe principal donor in the coordinationinteraction between Chi a molecules(12,81), Fong and Koester (82,83) pro-posed a symmetrical (parallel) struc-ture for (Chi a)2 in which the carbome-thoxy C=0 functions are used as donorsto Mg. Additional NMR evidence on therelative donor strengths of the oxygenfunctions in Chi a is now availablefrom a detailed comparison of theaggregation behavior of Chi a andPyrochl a (a Chi a derivative lacking acarbomethoxy group at C-10, see Figure1), from a comparison of the 13C chemi-cal shifts in Chi £«Li and (Chi a)2,and from an examination of the aggrega-tion behavior of desoxomesochIorophy11a (a Chi a derivative in which the ketoC=0 function at position 9 is replacedby 2H) .

A titration experiment on a chloro-phyll dimer or oligomer relates thechemical shifts of the protons (or 13C)in the aggregated species to that inthe monomer, where the chemical shiftsare fully assigned. The aggregation mapso constructed, thus, defines the ringcurrent shifts resulting from aggregateformation. The results of titrationexperiments with (Chi a)2and (Pyrochl a)2 are shown in Figures

6 and ~J, respectively. The numberssuperimposed on the monomer structuregive the chemical shift differencebetween the corresponding proton (s) inthe dimer and the monomer. A positivesign indicates that the chemical shiftof the particular proton in the dimeris at higher field than in the monomer.

Vol. 5, No. 1/2 27

0.02

H CH2 2.05/7a

bCH2 (0aC02

7cC02Phytyl |0bC H 30.6l

7cC02phytyl

Figure 6. 1H NMR titration of chloro-phyll a (0.06 M) in carbon tetrachlo-ride with pyridine-2Hs. Chemical shiftdifferences (<5, ppm) between dimer andmonomer [A(5) = 6 m o n o m e r- <5dimer]are positive for upfield shifts in thedimer (14) .

Figure 7• *H NMR titration of pyrochlo-rophyll a_ (0.06 M) in carbon tetrachlo-ride with pyridine-2Hs. Chemical shift(o, ppm) differences [A(<5) =

^monomer" 5dimer^ a r e Positive forupfield shifts in the dimer

In both Figures 6 and 7» all cf theshifts in the dimers are to higherfield, suggesting that on the average,all of the protons in the dimer are inthe shielding region of the partnermacrocycle. A carefully constructedmodel of the Fong dimer structure puttogether with carbomethoxy C=0 interac-tions shows that for such a structurethe la, 3a» 4a, 10b, a-methine, andvinyl protons are all in the deshield-ing zone of the adjacent macrocycle,which is not what is observed. Theresults from the titration experiments,therefore, provide no support for adimer with a parallel structure cross-linked by carbomethoxy C=0 interac-tions. A perpendicular structure for

the dimer generated by the ketoC=O***Mg interactions as suggested byShipman et al. (84) appears, however,to be consistent with the experimentalfindings. A similar conclusion has beenreached by Georghe et al. (78) .

The largest differences in chemicalshifts between dimer and monomer are inthe vicinity of ring V, and the ringcurrent effects in (Chi a) 2 and(Pyrochl a_) 2 are qualitatively similar.As Pyrochl a has no carbomethoxy group,it is difficult to avoid the conclusionthat it is the keto C=0 group that isthe principal donor function in bothcases. The incremental shift in theC-10 protons of (Pyrochl a) 2 is sub-stantially larger than in (Chi a) 2,which indicates that ring V in (Pyrochl


a) 2 comes closer to the center of thebinding macrocycle than it does in (Chia)2. Indeed, from the incremental chem-ical shifts, (Chi a)2 appears to beless stable than (Pyrochl a)2, and thesteric hinderance from the10-carbomethoxy group is a destabiliz-ing influence rather than the drivingforce in dimerization.

A Chi a derivativebomethoxy group is

in whichpresent

thebut

car-y the

9-keto group is reduced to -CH2 hasbeen synthesized by Scheer (85). Inthis synthesis, the vinyl group and thedouble bond in the phytyl chain areboth hydrogenated, but this is notexpected to have any significant effecton the donor properties of the mol-ecule. As judged from the ring-currentinduced chemical shift of the C-10 pro-

6

i(ppm)

4

i i i I | i i i i • T

V .1 1 1 I ) T 1 1 i 1

I ' 7

X~

5 10Molar Rotio MeOH : Chlorophyll

Figure 8. XH NMR titration experimentthat compares aggregation in (A), 2HChlorophyll a (0.15 M) ; (0), desoxome-sochlorophyll a (0.59 M ) ; and (x) ,2H-pyrochlorophyl 1 a (0.0*»9 M) . Onlythe chemi.ca! shift (<5, ppm) of the C-10protons is shown. Both deuterio-chlo-rophylls had lH at position C-10. The9-desoxomesoch1orophy11 a, which lacksa 9-keto function, requires the small-est ratio of nucleophile/chlorophy11for complete disaggregation.

tons (Figure 8) the aggregation

Vol. 5, No. 1/2

strength is greatest in (Pyrochl a)2,which lacks a carbomethoxy group, andweakest in 9~desoxomesoch1orophy11 a,which has a carbomethoxy group but noketo C=0 function.

1 3C NMR has provided direct evidencefor the participation of the ring Vketo function in dimerization (54,55)•Figure 9 shows the results of a 1 3C NMRtitration experiment on (Chi a)2. Theincremental 1 3C chemical shifts areagain a function of ring currenteffects in the dimer, but superimposedis a deshielding effect expected forthe carbon atom of any carbonyl func-tion participating in a coordinationinteraction. By far the largest desh-ielding is observed for the carbon atomin the keto function. The carbonylcarbon in the carbomethoxy group expe-riences an upfield shift in the dimer.This upfield shift in the carbomethoxycarbonyl carbon effectively excludesthis function from consideration as adonor group. The downfield shift in thecarbonyl carbon atom of the propionicacid side chain is also observed in allof its immediate neighbors, and this isconsistent with a ring current ratherthan a coordination origin. Althoughdeshielded, the propionic ester C=0resonance is sharp, suggesting that itenjoys freedom of motion and, thus,does not participate to a significantextent in dimer formation.

All of the available evidence thusfocusses on the keto group as the prin-cipal donor function in Chi a. A simi-lar conclusion has been reached by Ras-quain et al. (86) from an lH NMR studyof the aggregation of protochlorophyl1a in non-nucleophi1ic solvents. Therelative donor strengths in Chi b , andBchls a, b, £, d, and e, all of whichhave donor functions in addition to the9-keto group still remain to be estab-lished. As in the case of Chi a, 1 3CNMR is expected to be the method ofchoice.

In a system at room temperature con-taining both monomer and dimer Chi a,only one set of resonance lines can beobserved, which implies an averagingprocess rapid on the XH NMR time scale.For Pyrochl a, the XH NMR spectrumshows comparatively sharp resonances atroom temperature and above. Decreasing

29

H +1.04

\2b ,H

+ 0.26

phy =

H CH2 +1.6./+0.96H»

+0.45ST2 i o ° c e*\ -0.98 \

n l c 7cc°2P>>y IObCH3-0.16 + 0 4 0

P-l t P-3 "0.491J>

-0.48

Figure 9. 1 3C NMR titration of chloro-phyll a dimer. The incremental chemicalshifts [A(o) = 5 m o n o m e r - 5 d i m e r](<5, ppm) are shown for various carbonatoms in the molecule. A negative signindicates the 1 3C chemical shift of theindicated carbon atom is at lower fieldi n the dimer (55)•

temperatures lead first to increasedline broadening, but below -35° C thelines gradually sharpen and split intoa multitude of resonances. Such behav-ior is typical for an exchange process,and the line broadening at room temper-ature and somewhat below shows that(Pyrochl a)2 is close to coalescence inthis temperature range. The room temp-erature 1H NMR spectrum of (Pyrochl a)2represents an average conformation towhich at least two but probably severalmore conformations contribute. Anattempt to define more precisely thestructures of the conformers of (Chia_) 2 by a lanthanide-i nduced chemicalshift study of Chi a in CCI4 has metwith indifferent success because theshift reagent, itself a good

electrophile, seriously perturbs the(Chi a_) 2 equilibria and the dimer pres-ent in the system experiences changesin conformation (87).

5. Photoreaction Center Models

There is considerable evidence thatthe primary electron donor in the pho-toreaction center of both green plantsand photosynthetic bacteria is a spe-cial pair of chlorophyll molecules(88). Possible structures for the chlo-rophyll special pair have been sug-gested (89-93) and, despite the manyunresolved questions remaining aboutthe structure of the _i_n vivo photoreac-tion center, chlorophyll special pairmodels have been synthesized fromPyrochl a (91), Chi a (94), and Bchl a(95)• These particular models consistof two macrocycles covalently linkedthrough their propionic acid sidechains. In the absence of extraneousnucleophiles, the covalently linkedpairs occur in an open configuration,but in the presence of nucleophilessuch as water or ethanol, which arecapable of coordination to Mg andsimultaneous hydrogen-bonding to ring Vketo function, folding of the linkedpair occurs to form species that mimicthe salient features of j_n vivo photo-reaction centers. 1H NMR has been usedto demonstrate that in the folded con-figuration the linked pair has thestructure suggested in references 91and 92. Wasielewski et al. (95) haveshown that in the folded covalentlylinked Bchl a special pair model, the5a-CH3 and the C-10 proton experiencesubstantial upfield shifts, while thekb methyl group is shifted downfield.This behavior closely parallels thatobserved by Boxer and Closs (91) in thePyrochl a linked pair. As the chemicalshift in the methyl group of the acetylfunction in Bchl a_ is not perturbed byfolding, it appears that only the ketogroup is involved in the folding opera-tion (Figure 10). All of the *H NMRevidence, thus, supports the structuresproposed by Boxer and Closs (91) andShipman et al. (92) .

Recently, Boxer and Bucks (96) havelinked through a covalent bond a


6. Biosynthesis Studies

1.54IIA7)

C H , 3.95

0.6211.20]

1.8-2.4(1.8-2.4)

3.40(3.02)

2 2

4.00(4.3 I)

Figure 10. 1H NMR titration experimentthat compares the chemical shifts (5,ppm indicated in parentheses) in theopen (in 10% pyr idine-2rls in benzene-2He solution) configurations of thephotoreaction center bacteriochloro-phyllide a_ special pair modelbis (bacteriochlorophyl1ide a) ethyleneglycol diester. The 5a and 10b methylgroups are strongly shielded in thefolded configuration (94).

pyropheophytin macrocycle to a pair ofcovaiently linked Pyrochl a molecules.The chemical shift changes observed onfolding are consistent with a symmetri-cal, rapidly-averaging, folded configu-ration in which the pyropheophytin ringU positioned over the 5a-CH3 and3-proton of the metal-containing foldedpair. 1H and 1 3C NMR can be expected toplay an increasingly important role indefining the structures and propertiesof these and other model systems nowunder development.

In spite of the current interest inchlorin biosynthesis, NMR methods havenot been widely used. Presumably, thisreflects the lower sensitivity of NMRmethods as compared to 14C tracer tech-niques. NMR, however, is capable ofproviding biosynthesis information dif-ficult to obtain by conventional tracerprocedures. Applications of 1H NMR tcChi a_ and Bchl a_ biosynthesis have beendescribed by Katz and Crespi (97)-

All higher green plants contain Chia and b (Figure 1). Although it isgenerally accepted that Chi a is theprecursor of Chi b j_n vivo , the evi-dence from tracer experiments on thisimportant point is not as firm as mightbe desired. When green algae are grownin an 1H20-2H20 mixture, both hydrogenisotopes are incorporated. The XH/2Hratio at different sites in the chloro-phylls extracted from such organismscan readily be obtained by integrationof the 1H NMR spectra of the respectivemethyl pheophorbides. Except for the1H/ 2H ratio in the formyl group of Chib, and CH3 group in Chi a in position 3all other isotope ratios are identicalin the two chlorophylls (Figure 11).The identical isotopic composition ofChi a and b_ at all corresponding sitesprovides strong evidence for the forma-tion of Chi b from Chi a, as it wouldbe surprising for two independent bios-ynthetic pathways to have identicalisotope effects at all correspondingsites in the two molecules. Thereappears to be no significant branchingin the biogenetic pathways prior to theoxidation of the methyl group at pos-tion 3 in Chi a to the -CHO group inChi b. The fractionation factors alsoindicate some possible complications inthe biosynthetic pathway. If the iso-topic composition of the vinyl group iscompared to that of the ethyl group atposition h, no mechanism readily sug-gests itself whereby an ethyl group ofthe observed isotopic composition couldbe generated from a vinyl group of theobserved composition even if pure iHwere used as the reducing agent. Eitherthe vinyl groups at positions 2 and hare produced by different reactionmechanisms, or protoporphyrin IX is not

Vol. 5, No. 1/2 31

1.9 ± 0 . 3 / V H (CH0 0.4±0.2)Hy " V IO±Q2 CH3 1.7±0.3

0.52.3 +

1.7±0.4 H3C~-H

4.0 ±

CH3 1.5 ± 0.2

TC02CH3u

hx + m 2.8±0.5

C02CH3

INT.

Figure 11. Isotope discrimination fac-tors (KIH/K2|-|) as determined byintegration of the 1H NMR spectrum ofmethyl pheophorbides prepared from Chia_ and b extracted from green algaegrown in a 1:1 mixture (v/v) ofand 2H20 (97)•

the only intermediate. The isotopiccomposition of the methyl groups atpositions 3 and 8a as compared to thela and 3a methyl groups differ to anextent greater than the experimentalerror, suggesting the possibility thatthe methyl groups at positions la and5a had a different chemical historyfrom that of the 3a and 8a methylgroups, or that all of the methyl goupswere not formed at the same time. Simi-lar NMR experiments with Bchl a inmedia of mixed isotopic compositionindicate similar problems requiringresolution in the biosynthetic pathwaysto Bchl a (98,99).

Ahrens et al. (100) have used 13CNMR to study the biosynthesis of phy-tol. 13C-enriched acetate was fed toEuglena graci 1 i s, the chlorophyll wasextracted, and the esterifying alcoholsobtained by hydrolysis and thin-layerchromatography. 13C incorporation fromthe acetate precursor was

preferentially localized in 8 sites inthe phytol backbone. These results dem-onstrate specific incorporation of[l-13C]acetate, and the results areconsistent with the normal terpenoidpathway to geranyIgeranyl pyrophosp-hate, the precursor of phytol.

I I CORRINS

A. Structural Features

A corrin is a partially reduced tet-rapyrrole macrocycle in which two ofthe pyrole rings are bonded directly toeach other via their a-carbon atoms.Most research involving the corrins hasbeen performed with vitamin B12 and itsbiologically active analogues, and hashad the goal of determining how thecorrin ring is biosynthesized and howthe chemical and physical attributes ofthe corrin ring yield the biologicalfunctions of B12. For this reason, weshall stress the NMR spectroscopy ofthis special class of corrins and shallmention the literature relating to thesynthetic corrin complexes only when itapplies to their biologically importantcounterparts.

Vitamin B12 (Figure 12) has threedistinct components. These are the cor-rin ring, which binds a Co (I I I) ion viaits four pyrrole nitrogens, a sidechain that is attached to carbon-17 ofthe corrin ring and whose5,6-dimethyIbenzimidazole moiety bindsan axial coordination position of theCo (III) ion, and a cyanide ion bound tothe remaining coordination position ofthe Co(UI) ion. The cyanide is presentin the vitamin for the technical reasonthat it binds to the Co (III) ion moretightly than most other ligands and ,thus, permits isolation of the moleculeas a single species with cyanide as theonly ligand in this coordination posi-tion. This substance is properly namedcyanocobalamin. The coenzyme form ofthis vitamin in the body is5'-desoxyadenosylcobalamin, with a5'-deoxyadenosy1 group substituted forthe cyanide. The biological activity ofcobalamin arises from its ability toproduce a cobalt-carbon bond with avariety of ligands at the coordinationposition filled by cyanide in Figure


HJNOCHJC,*

HjNOCHjC-t'8

O C - C H J C H ^ C H J , ' C H 3

NH . ' *CH2

HO-CH2

Figure 12. Structure of vitamin B12.The coordination position of the CNabove the corrin ring is referred to asthe fifth coordination position andthat to which the benzimidazole nitro-gen atom is coordinated below the cor-rin ring is the sixth coordinationposition, in agreement with the nomenc-lature of Brodie and Poe (I 12). Theligands in these coordination positionsalso are referred to in the text as theaxial ligands. The indicated carbonatoms of vitamin B12 are derived fromcarbon atom-5 of 6-aminolevulinic acid(0) and from the methyl group of methi-oni ne (*) .

12. When the 5»6~dimethyIbenzimidazole-nucleotide is removed so that bothaxial coordination position of theCo(lll) ion are available for bindingexogenous ligands, the complex iscalled a cobinamide.

B. H\ NMR Chemical Shift Assignmentsand Appli cations

The 75 to 100 protons of cyanocoba-lamin and its various analogues giverise to a relatively large number ofwell resolved resonances (101-113)' Thevery early experiments were severelylimited by the sensitivity and resolu-tion of early spectrometers, and therather low solubility of the cobalaminsand cobinamides in available solventssuch as trifluoroacetic acid and deu-terated dimethylsulfoxide. By comparingthe spectrum of cyanocobalamin withthose of its various isolated substit-uents and of synthetic corrin com-plexes, it was possible, however, toassign several chemical shift ranges tospecific functional groups. The earlyobservations yielded a number of inter-esting results: i) the chemical shiftsof the protons of the5,6-dimethylbenzimidazole moiety dependon whether this ring is protonatedand/or coordinated to the Co (I I I) ion;ii) the chemical shift of the proton onthe methine bridge C-10 depends on theligands attached to the cobalt; iii)the proton on C-10 exchanges rapidly intrifluoroacetic acid; and iv) the cor-rin system does not exhibit an aromaticring current.

The advent of superconducting NMRspectrometers made it possible torecord useful spectra on dilute solu-tions in 2H20, and this made possiblethe assignment of the entire *H NMRspectrum and the characterization ofthe parameters that regulate the bind-ing of ligands to the fifth and sixthcoordination positions of the Co(lll)ion (Figure 12) (108-113). The protonon C-10 of both cobalamins and cobinam-ides, which readily undergoes electro-phi lie substitution reactions ( H A ) ,was demonstrated to exchange with 2H20under acid conditions at a rate that isintermediate between those of porphy-rins and chlorins (107). ln addition,it was demonstrated that the chemicalshifts of the protons on the axialligand in the sixth coordination posi-tion of the cobalt and on C-10 of thecorrin ring depend on the identity ofthe ligand in the fifth coordinationposition. The length of the Co-N bondbetween the Co (II!) ion and the dimeth-yIbenzimidazole appears to be a

Vol. 5 , No. 1/2 33

function of the nature of the otheraxial ligand, as does the electron den-sity on the cobalt. The electron den-sity of the cobalt appears to be delo-calized partially to the C-10 positionof the corrin ring, and indeed there isa correlation between the chemicalshift of the C-10 hydrogen and theenergy of the first electronic absorp-tion band (107,112).

Since the protons on the 5'"carbonof the 5'"deoxyadenosy1 moiety of thecoenzyme appeared to be involved inhydrogen transfer reaction and reduc-tion of the Co (III) ion of cobalaminappeared to occur in both hydrogen andmethyl transfer reactions (115~H8),the XH NMR spectra of these complexeswere studied. The S^protons of the5'-deoxyadenosy1 ligand in the coenzymewere found not to exchange readily with2H20 and to be magnetically nonequiva-lent (108,109,113), but the results ofa more recent experiment with[5'"x3C]-5'-deoxyadenosy1coba1 am i nraise the possibility that the earlierassignments for the 5'protons wereincorrect (119)- In any case, nonequi-valence of the protons of the cobalt-bound methylene group has been observedwith alkyl ligands other than51-deoxyadenosine in the fifth coordi-nation position (112). The cobalaminswith cobalt in the Co (I) and Co (I II)oxidation states were found to be diam-agnetic, but the *H NMR spectra werefound to be paramagnetically broadenedwhen cobalt was in the Co (I I) oxidationstate (112). Spectral line broadeningappears to be a consequence of the rel-atively long relaxation time of theunpaired electron of the Co (II) ion(120-122). Furthermore, it was foundthat the 5,6-dimethylbenzimidazolegroup does not bind to the sixth coor-dination position of the Co (I) cobala-min although it does so in the Co (III)cobalamins. The 5.6-dimethylbenz-imidazole group of Co (III) coenzyme B12is in a dynamic equilibrium with thecoordination and uncoordinated states;the first-order rate constant for thebreaking of the Co (I I I)- benzimidazolecoordination was estimated as somewhatlarger than 550 s x (113). This equi-librium is dependent upon pH and temp-erature, and does not appear to be

associated with a paramagnetic interme-diate when the benzimidazole is notcoordinated (113)- Methyl cobinamideappears to bind a molecule of water toits sixth coordination position,whereas 5'-deoxyadenosy1 cobalamide maynot (112,113)• Aquocyanocorrins canexist in two isomeric forms in whichthe water and cyanide occupy the fifthand sixth coordination positions ineither order (123,124). The onlyeffect on the XH NMR spectrum of thesetwo isomers appears to be a smallsplitting of the resonance of the pro-ton on C-10.

The resonance in the XH NMR spectraof the cobalamins at ~0.5 ppm from TMSwas assigned to the methyl group on C-lof the corrin ring, and the shieldingwas shown to arise from the aromaticring current of the 5.6-dimethylbenz-imidazole moiety coordinated to theCo (I I I) ion (Figure 13) (107). Methylgroups of alkyl ligands bonded to thecobalt were demonstrated to resonate ateven higher field than the C-l methylgroup (112,125)• Comparison of a largenumber of cobalamins and cobinamides(112) permitted almost complete assign-ment of the proton resonances of themethyl groups on the corrin ring (Table13) •

C. j_?_C NMR Chemical Shifts and Applica-tions

The application of 13C NMR spectros-copy to the investigation of B12 andits analogues with natural-abundance1 3C did not become feasible until thelate 196O's. The detection and assign-ment of the natural-abundance X3C reso-nances required large diameter sampletubes and pulse-Fourier transform tech-niques to increase sensitivity andfacilitate measurements of spin-latticerelaxation times (Ti). From chemicalshift comparisons, Ti values, 1 3C- 3 1Pspin-spin coupling constants, and theresults of off-resonance single-fre-quency proton decoupling experimentsDoddrell and Allerhand were able toprovide surprisingly complete assign-ments for the 1 JC NMR spectra of cyano-coba1 am i n, 5'-deoxyadenosy1coba1 am i nand a number of their analogues(126,127). The.region of the spectrum


Hf

Figure 13- The orientations of themethyl group of vitamin B12 withrespect to the corrin and5,6-dimethylbenzimidazole rings. Thepro-R methyl group at carbon atom 12lies below the plane of the corrin ringand the pro-S methyl group lies above.The acetamide and propionamide sidechains have been omitted for the sakeof clar i ty.

from about 85 to 100 ppm upfield fromCS2 was assigned to the methine carbonsthat bridge pyrrole rings A, B, and C.C-10 was assigned to the resonance at100 ppm, whereas C-5 and C-15 wereassigned to the resonances at 86 to 89ppm because they are bonded to methylgroups. The unsaturated carbon atoms ofthe corrin ring directly bonded to thepyrrole nitrogen atoms were assigned toa downfield spectral range that over-laps that of the amide carbonyl region,and the methyl groups were assigned tothe spectral lines above 170 ppm rela-tive to CS2. The cyanide carbon atoms

of dicyanocobalam in were assigned tothe broad resonance centered at 55 ppm.Broadening was suggested to be due toscalar coupling to the 5'Co nucleus,which has a quadrupole moment. Unlikethe XH NMR spectra of the two isomersof aquocyanocorrins, the 1 3C chemicalshifts of aquocyanocobyric acid werefound to be very sensitive to the isom-eric orientations in which the waterand cyanide can bind to the cobalt.The observed differences in chemicalshifts between the two isomers wereattributed to both electronic differ-ences and conformational changes of thecorr in r i ng.

Historically, the use of specific1 3C labelling and 13C NMR for detectionof the label that followed the originalassignments of Doddrel1 and Allerhandfell into two categories. One of thesewas concerned with establishing thebiosynthetic pathway to Bt2, and theother consisted of studies on theeffects of axial ligands on the biolo-gical activity of B12.

I. 13C NMR of the Corrin Ring

The early steps in the biosyntheticpathway to the corrins and to the por-phyrins had been shown to be quite sim-ilar (128-130). It was known that the5-carbon atom in 6-aminolevulinic acid(Figure ]k) was the precursor of atleast seven carbon atoms in the corrinring of 612, and of eight carbon atomsin the porphyrins. The mechanism bywhich pyrrole ring D is incorporatedinto the corrins and prophyrins was thesubject of much speculation, and theorigin of the methyl group on C-l ofthe corrin was uncertain. It was knownthat one of the two methyl groups onC-12 of the corrin ring arises fromdecarboxylation of an acetic acid sidechain and that at least six of theseven remaining methyl groups on thecorrin ring arise from the methyl groupof methionine, but the general concen-sus was that the methyl group on C-lshould arise from the 5~carbon atom ofo-aminolevulinic acid, as does the5-bridge carbon atom of the porphyrins(Figure 14). For technical reasons,this latter point could not be provenwith 14C-labei1 ing experiments (130).

Vol. 5, No. 1/2 35

P0RPHYR1NS

100I

PPM(CS2)200. l

200 100 0PPM(TMS)

Figure 14. The labelling pattern thatis expected when the ring structures ofporphyrins and corrins are biosynthes-i2ed from 6-aminolevulinic acid that islabelled isotopicaily in carbon atom 5(0). The curving dashed arrow repre-sents the early hypothesis that thecorrin ring was biosynthesized directlyfrom a linear tetrapyrrole intermedi-ate. It has since been demonstratedthat uroporphyrinogen III is an inter-mediate in the biosynthetic pathway tothe biological corrins (48-50).

Figure 15- The proton-decoupled 1 3C NMRspectra of vitamin B12 in water. Thespectrum of (A) vitamin B12 synthesizedfrom o-amino[5-13C]levulinic acid; and(B) vitamin B12 synthesized fromL-[methyl-13C]methionine. CS2 is theexternal reference, but the chemicalshift is presented in ppm relative toboth CS2 (top scale) and tetramethylsi-lane (bottom scale) to permit easiercomparison of spectra in the references(see text) .

To overcome this complication, we addedo-amino[5~13C] levulinic acid to a cul-ture of Propionibacter ium shermani i,isolated the B12 produced as cyanocoba-lamin, and recorded its 1 3C NMR spec-trum (Figure 15A) (131) • This spectrumconfirmed the labelling pattern discov-ered by radiotracer techniques and con-firmed the assignments for these carbonatoms by Doddretl and Allerhand (126).From the 1 3C- 1 3C spin-spin splittingpatterns, we distinguished between

carbon atoms 5 andand demonstratedresonates at lower1 3C- 1 3C spin-spinobserved for C-15

15 of cyanocobalaminthat carbon atom 5field. The complexspli tting pattern

provided direct evi-dence that the pyrrole ring is turnedover during its incorporation into thecorrin ring, as also occurs in porphy-rin biosynthesis. This experimentyielded the unexpected result that themethyl group on C-l of the corrin ringdid not contain an appreciable amountof 13C-label. Similar results were


obtained by Scott et al. (132), whoalso assigned the 1 3C resonances of themethylene carbon atoms in the acetamideand propionamide side chains of thecorrin ring of cyanocobalamin and themethyl group on C-12 of the corrin ringthat arises from decarboxylation of anacetic acid side chain (132,133). It isinteresting to note that this methylgroup on C-12, rather than the methylgroup on C-l as assumed by Doddrell andAllerhand, resonates at abnormally low-field compared to the other methyl car-bon atoms in cyanocobalamin. This hasbeen ascribed to the lack of a y effectfor this one methyl group (see below)(133). Specific 1 3C chemical shiftassignments were reported recently forthe carbon atoms of several derivativesof cyanocobalami n (13M.

Realization that the methyl group onC-l of the corrin ring does not arisefrom the 5~carbon atom of6-aminolevulinic acid led to the imme-diate assumption that seven, ratherthan six, methyl groups arise from themethyl group of methionine. When Scottet al. (132,133) added L-[methyl-13C]methionine to a culture of Propion-ibacter ium Shermani i and isolated theB-. 2 produced as cyanocobalami n, themethyl region of the 13C NMR spectrumwas found to exhibit only six enhanced1 3C resonances. However, addition ofexcess cyanide to the sample to producedicyanocobalamin resulted in theappearance of a seventh peak in theupfield 1 3C NMR spectrum, and this wastaken as proof that the methyl group onC-l of the corrin ring arises from themethyl group of methionine. Brown etal. (135) reproduced this work, andalso demonstrated that protonation ofthe benzimidazole ring and substitutionof a water molecL'le at the sixth coor-dination position of the cobalt alsoproduces seven enhanced resonances inthe upfield region of the 1 3C NMR spec-trum (Figures 15B and 16) . In addition,single-frequency proton-decoupled 1 3CNMR spectra were recorded for all threeof these cobalamin complexes, whichpermitted assignment of all sevenenhanced resonances on the basis of theexisting assignment of the methylgroups in the XH NMR spectrum of B12(Figure 17 and Table 13).

Figure 16. The proton-decoupled l3C NMRspectra of vitamin B12 synthesized fromL-[methyl-13C]methionine. The spectrawere obtained in water (A), in 0.1 MKCN (B), and in aqueous HC1 (pH > 1)(C) . The chemical shift values aregiven in ppm downfield from tetrame-thylsi 1ane.

This latter experiment pointed outone of the apparent anomalies in the XHNMR spectrum of cobalamins. The labora-tories of both Scott and Battersby dem-onstrated that the methyl group ofmethionine gives rise to the pro-Rmethyl group on C-12 of the corrin ring(i.e., the methyl group cis to the pro-pionamide side -chain on C-13» markedby an asterisk in Figure 12). Scott etal. (133) produced dicyanocobinamideand dicyanoneocobinamide, in which theconfiguration of C-l3* in ring C isreversed (136), from cyanocobalaminv

that had been biosynthesized withL-[methyl-i3C]methionine. The l3C reso-nance of the enriched methyl group of

Vol. 5, No. 1/2 37

the dicyanocobinamide was found toresonate at higher field than that ofthe dicyandneocobinamide. This shield-ing was attributed to the steric inter-actions between the labelled methylgroup and the adjacent propionamideside chains, i.e., the y effect(137,138). Battersby et al. (139,UO)performed the same label 1 ing experimentbut isolated the imide of ring C byozonolysis and compared the XH and 1 3CNMR spectra of this isolated ring withthose of a chemically synthesized stan-dard. By combining the assignment ofScott and Battersby for the 1 3C reso-nances of the methyl groups on C-12with the single-frequency proton-decou-pling experiments, it is possible toassign the proton resonances of thesetwo methyl groups, as shown in Table13. The assignment of the peak at 120ppm for the protons of the pro-S C-12methyl group, which lies above the cor-rin ring in Figure 13, agrees with theobservation that this peak does notchange position when cyanide is substi-tuted for the benzimidazole ring in thesixth coordination position of thecobalt below the corrin ring. However,this assignment leads to the unexpectedconclusion that of the two methylgroups on C-12 of cyanocobalamin, theone lying above the aromatic benzimida-zole ring is the less shielded. In theXH NMR spectrum of dicyanocobalamin,the C-l methyl group is deshielded bydisplacement of the benzimidazole fromthe cobalt, and one other methyl group(which is the C-2, C~7, pro-R C-12, orC-17 methyl group) is more shielded, sothat its chemical shift is the same asthat of the pro-S C-12 methyl protons(Table 13). These apparent anomaliesmay be explained by anisotropic shield-ing properties of the bound cyanide,but this has yet to be demonstratedfully.

The subsequent 1 3C NMR experimentsinvolving the corrin ring have utilizedthe spectral assignments presentedabove for studying the incorporation ofvarious 13C-labelled intermediates intothe corrin ring. This work has led tothe discovery that the methyl groups ofthe corrin ring that arise from methio-nine are incorporated without protonexchange (l4l-li»3), and that both

PPMN N N —

Figure 17. The *H NMR spectra of vita-min B12. A spectrum of vitamin B12 withnatural abundance 1 3C; vitamin B12 syn-thesized from L-[methyl-13C]methionine.The chemical shift values are given in6, ppm downfield from tetramethyIsi-lane. The two arrows about the reso-nance 0.46 ppm indicate the satellitepeaks due to l3C-1H spin-spin split-ting.

uroporphyrinogen III and sirohydochlo-rin are intermediates in the biosyn-thetic pathway to corrins (144-147) .Since this work has been the subject ofrecent reviews (148-150), it is notfurther discussed here.

2. 1 3C NMR of the Axial Ligands

to

1 3C NMR spectroscopy has been usedinvestigate - 1 3CN, - 1 3CH 3 (125),g , (5

- 1 3CH 2-1 3COOH (151) and [5'-13C]-5'"de-

oxyadenosine (119,152) bonded to thefifth and sixth coordination positionsof coba cobinamide, and


o

•z.o

Table 13

'H NMR and 1]C NMR Chemical Shifts of Vitamin B i ? and 'HChemical Shifts at which Nuclear Overhauser Enhancement (NQE) 1s maximum for ' 3C Resonances.

Chemical Shifts in 5, ppm from TMS in H?0, O.1M KCN and HCL aq (pH<1)

Position of Methyl Group 1nVitamin Bi? (see Figs. 12,13)

C-1toC-12 (Methyl group trans'"

propionamide on C-13Methyl of aminopropanolC-2d , C-7d, C-12, (c1s«= topropionamide on C-13), andC-17d

Methyl groups of benzimidazole

C-5 and C-15

1

Assigned'H NMR

0.46b

1 .20

1.26b

1 .391 .411 .451 .872.26b

2.54b

2.58b

2

1>C NMR

20.059

17.689

16.719

20.0519.84

16.28h

15.95h

3

NOE

0.46

1 .391 .411 .481 .88

2.542.60

KCN

4

'H NMR '

1 .441 .26

1.311 .22®1 .371 .461 .742.362.382.262.30

5

'C NMR

22.53

18. 119

17.259

19.4019.73

15.6316.06

6

NOE

1 .44

1 .241.411 .461 .74

2.232.59

HC1

7

'H NMR

1.811.15

1 .201 .311 .541 .591.812.35

2.382.40

8

1'C NMR

24.589

19.4O9

18. 339

20. 1621 .56

16.0616 .60

9

NOE

1 .8

(1.3-1.6)'

1.8e

2 .402.44

a) The nuclear Overhauser enhancement data were obtained by single-frequency experiments by Brown et al. (135). b~5 Chemi-"cal shifts assigned by Hill et al. (106,107). c) ']C chemical shifts assigned by Scott et al . (133) and Battersby et al .(139). d) Individual assignments cannot be made among the 'H NMR peaks of these methyl groups. However, the ' 'C-resonancesare related to the 'H NMR assignments as determined by NOE. >H chemical shifts assigned by Brodie et al. (112). e) We can-not differentiate the assignments of these two proton resonances. f) Three chemical shifts were found 1n the region of1.3-1.6 ppm. However, no assignments of the proton resonances to the ''C resonances can be made. g) The 1]C resonances ofthese three methyl groups appear to shift to lower field when the dimethyl benzimidazole group 1s replaced by a less bulkyCN or H?0 1igand in the sixth coordination position. h) Hogenkamp et al. (152) have assigned 516.28 to the C-5 carbonatom.

epicobalamin (153)• The use of13C-labelled cyanide confirmed theassignment of Doddrell and Allerhand(126) for the cyanide carbon in thenatural abundance spectrum of cyanoco-balamin and provided evidence thataquocyanocobinamide binds the cyanideand water ligands in two isomeric ori-entations, as does aquocyanocobyricacid (127). 13C-cobalamin was observedto yield one relatively sharp enhanced1 3C NMR resonance in the upfield regionof the spectrum, with a chemical shiftdependent on the pH of the solution.This pH dependence was attributed todisplacement of the 5,6-dimethy1-benzimidazole moiety by water at lowpH. I3CH3-cobinamide gives rise to twoenhanced 1 3C resonances of equal inten-sity in the upfield region of the spec-trum. These have been assigned tomethyl groups bound to two differentisomers in the fifth and sixth coordi-nation positions of the cobalt, and thefact that the two resonances are ofequal intensity has been taken to meanthat there is no steric interferenceagainst the binding of a methyl groupto either coordination site.

In contrast to the observation ofDoddrell and Allerhand (126), the 13Cresonances of both the - 1 3CN and - 1 3CH3ligated to cobalamins or cobinamideswere found to be relatively narrow.This was attributed to a quadrupolarcontribution that dominates the spin-lattice relaxation rate of s'Co andthereby obliterates the cobalt-carbonspin-spin splittings. Chemical exchangeof the methyl group of methylcobalaminand methylcobinamides was found not tocontribute to the line width of themethyl 13C resonance, but chemicalexchange between the "base on" and"base off" forms of methylcobalam in atpH values near the pKa of the dimeth-ylbenzimidazole moiety was found tohave a marked effect on the line widthof the methyl 1 3C resonance.

From the long T1 value of the methylgroup of methylcobaI am in it was con-cluded that rotation about the carbon-cobalt bond is rapid and unrestricted.The same is not true, however, for thecarboxymethyl (-13CH2-

13C00H) and5'-deoxyadenosyl ligands. These havevery short T1 values, which indicate

severe restriction to rotation aboutthe carbon-cobalt bond for both thesecoordinated ligands. The pKa of thecarboxymethyl ligand is unusually high(pKa = 7.2), and both.the restrictedrotation and the high pKa value havebeen attributed to hydrogen bondingbetween the carboxyl group and the ace-tamide side chains on the periphery ofthe corrin ring. The restricted motionof the 5'-deoxyadenosyl moiety wasattributed to a combination of stericinteractions and intramolecular hydro-gen bonding. It is of interest to notethat the Ti values of theE13C]methylcorrinoids became shorterwhen the benzimidazole moiety isremoved from the coordination sphere ofthe cobalt.

The chemical shifts of the axialligands were found to be highly depen-dent on the identity of the other axialligand (the trans effect) in both thecobalamins and cobinamides. Replace-ment of the dimethylbenzimidazolemoiety of methylcobalamin by water atlow pH causes the methyl 13C resonanceto shift to higher field as notedabove. By investigating a number ofsubstituted methylcobinamides it waspossible to make the more generalobservation that substitution of a weakligand by a strong field ligand in thesixth coordination position leads to asubstantial downfield shift and reduc-tion of the 13C-1H coupling constant ofthe methyl group in the fifth coordina-tion position. The highest chemicalshift and JQ - H °f the methyl ligandwere observed with a molecule of H2O inthe sixth coordination position, andwere found to decrease in the order H2O> pyridine > benzimidazole > CN" > CH3upon substitution of these ligands inthe sixth coordination position. Boththe chemical shift of the 13C resonanceand the 1 3C- 1H coupling constant of themethyl group in the fifth coordinationposition are linearly correlated withthe energy of the (3-vibrational compo-nent of the first electronic absorptionband.

The chemical shift of the proton onC-10 of the corrin ring (the only pro-tonated methine bridge) correlated withthe energy of the first electronicabsorption band as discussed above


(107,112), and this was taken to sug-gest that the charge density of thecobalt might be delocalized to thismethine bridge. Delocalization ofcharge from the cobalt has been demon-strated by 1 3C NMR to occur with allthree methine bridges (cis effect).When a weak field ligand in the sixthcoordination position is replaced by astrong field ligand, all of the methine1 3C resonances shift to higher field.In the methylcobalamide series themethine 1 3C resonances shift to highfield in the order H2O < pyridine <cyanide, and similar correlations havebeen noted with cyanocorrinoids andalkylcobalamins. The chemical shifts ofthe 3-vibrational component of thefirst electronic absorption band, as inthe case of the 1 3C NMR resonance ofthe methyl ligand in the fifth coordi-nation position. However, introductionof ligands in the sixth coordinationposition that cause the 1 3C resonanceof the methine bridges to shift tohigher field cause the 1 3C resonance ofthe methyl ligand in the fifth coordi-nation position to shift to lowerfield.

In the epicobalamins the5,6-dimethylbenzimidazole moiety iscoordinated more strongly to thecobalt, the cobalt is more electronega-tive, and the carbon-cobalt bond in thefifth coordination position is strongerthan in the cobalamins. These differ-ences have been attributed to bothsteric and electronic changes that mayresult from reversing the configurationby which the propionamide side chain isbonded to C-l3 of the corrin ring, butthe exact mechanism appears to be ster-icaily blocked since the intensities ofthe two methy) resonances arising fromthe two isomers of aquo[ 1 3C]-cyanocobinamide are in a ratio of 95^5-

In contrast, the chemical shifts ofthe 1 3C nuclei on the remainder of theperiphery of the corrin ring appear tobe much less affectedstate of the cobalt, orvariablesroles.chemical shifts of the 1 3C atoms on theperiphery of the corrin ring, but themechanism by which the effects arisemay be different. For example,

by the chargeat least other

appear to play more prominentAxial ligands do affect the

substitution of a small ligand for thebulky dimethylbenzimidazole ring in thesixth coordination position of thecobalamins results in downfield shiftsof the 1 3C resonances of four of themethyl groups on the periphery of thecorrin ring. The most shifted of theseresonances arises from the C-l methylgroup (Table 13) (135) and the leastshifted has been assigned to the methylgroup on the C~5 methine bridge (152).The two remaining shifted resonances(Table 13) may arise from the other twomethyl groups that extend from the cor-rin ring on the same side as thedimethylbenzimidazole moiety (i.e., themethyl groups on C-7 and C-12 (pro-R)in Figure 13). These downfield shiftsresulting from substitution of a lessbulky ligand in the sixth coordinationposition have been attributed to areduction in steric compression (the yeffect) (152) .

On the basis of comparisons of thelH and 1 3C NMR spectra of the variouscobalamins and cobinamides that havebeen studied to date, it appears thatthere are at least three parametersthat determine the biological activityof coenzyme B12 (51-deoxyadenosyl-cobalamin) and methyl cobalamin. Theseare as follows: i) the charge densityon the cobalt and its delocalizationover the two axial ligands and themethine bridges of the corrin ring(15^-160); ii) steric interactionsbetween the dimethylbenzimidazolenucleotide and the propionamide sidechains at C-8 and possibly C-13, andthe methyl groups at C-l, C-5, C-7, andC-12 (pro-R) that project from theperiphery of the corrin ring on thesame side as this nucleotide(133,135,152,161); and, iii) hydrogenbonding between the axial ligand in thefifth coordination postion and the ace-tamide side chains on this side of thecorrin ring. When the dimethylbenzimi-dazole group of the cobalamins isreplaced by a weaker field ligand, theoverlap between the sp3 orbitals of thecarbon atom in the fifth coordinationposition and the ks and 3d z

2 orbitalsthat are localized in the axial bondsof the cobalt ion is expected todecrease (155). Decreased overlap isexpected to increase the polarizabi1ity

Vol. 5, No. 1/2 k)

of the cobalt-carbon bond, with elec-tron density shifted from the cobaltion to the carbon atom, and therebyweaken the cobalt-carbon bond. Mecha-nisms that have been suggested to causethe observed change in chemical shiftsof the axial ligand in the fifth coor-dination position to higher fieldinclude increased electron density onthe carbon atom, variations in spinpairing between p electrons, the extentof p orbital occupation, changes inradial distance of electrons fromscreened nuclei, and variations inexcitation energies for mixing groundwith excited state wave functions(125). The decrease in electron den-sity on the cobalt ion that is expectedto occur upon substitution of thedimethyibenzimidazole group by a weakerfield ligand increases the demand bythe cobalt ion for the electron densitythat is delocalized over the methinebridge carbon atoms of the corrin ring,which in turn decreases the charge den-sity of these methine carbon atoms andshifts their 1 3C NMR resonances tolower field. Steric interactionsbetween the dimethyibenzimidazolenucleotide and the side chains areexpected to reduce the strength of thecobalt-nitrogen bond with the benzimi-dazole ring, and, by the mechanismdescribed above, to weaken the cobalt-carbon bond to the ligand in the fifthcoordination position. Thus, a reasonfor the methylation of seven carbonatoms on the corrin ring may be to pro-vide sufficient destabi1ization of thecobalt-carbon bond to the trans ligandin the fifth coordination position tomake possible the biological roles ofB12. This possibility is supported bythe observation by XH and 1 3C NMR thatsuch steric factors have a rather pro-nounced effect on the rate of benzimi-dazole dissociation in methyl cobalamin(162). Hydrogen bonding between theligand in the fifth coordination posi-tion of the cobalt and the acetamideside chains that project on this sideof the corrin ring appear to be negli-gible in methylcobalamin, but can beexpected to play some role in determin-ing the stability of the cobalt-carbonbond in coenzyme B12.

D. NMR Studies with Other Nuclei

We are aware of only one investiga-tion of cobalamins by 15N NMR (I63) andthree with 3 1P NMR (164-166) . The 15NNMR spectrum of cyanocobalamin exhibitsseven resolved amide nitrogen reso-nances in the region 256.8-268.2 ppm,and a cyano group nitrogen resonance at8O.9 ppm upfield from external 0.1 M2H 1 SNO3 in 2H20. The four pyrrole ringnitrogen atoms were not observed, pos-sibly because of long relaxation times,small nuclear Overhauser enhancementvalues, and coupling to cobalt.

The first 3 1P NMR studies (164,165)are an attempt to use the 3 XP atom inthe cobalamins as a probe of eventsthat occur at the corrin site of rela-tively low molecular weight,Bi2-dependent enzymes such as the ribo-nucleotide reductases. The chemicalshift of the 3 1P atom of cobalamins insolution was found to depend on thecoordination of the dimethyibenzimida-zole moiety to the cobalt atom, but itis relatively insensitive to the iden-tity of the ligand in the fifth coordi-nation position. The line width of the31P resonance increases when the cobaltatom is reduced with d,1-penici1lamine.It was demonstrated that 31P NMR can beused to investigate the pH-dependentproperties of cobalamin and the coordi-nation state of cobalamins when boundto bovine serum albumin and the deter-gent SDS.

3 1P NMR spectroscopy also has beenused (166) to characterize a recentlydiscovered isomeric form of vitaminB12. The UV-visible absorption spectrumof this new isomer is the same as thatof native B12, but Mossbauer and 1H NMRresults (167) suggest that the newisomer has a small out-of-plane dis-placement of the cobalt and concomit-tant change in the conformation of thecorrin ring and benzimidazole group.Spin-lattice relaxation measurements ofthe 3 1P nucleus in both isomeric formsof the paramagnetic cob (I I)alamins anddiamagnetic cob (I I I)alamins appear tocorroborate these conformational dif-ferences (166). The spin-lattice relax-ation times of the 3 1P nucleus in thecob(!l)almin isomers are dominated bydipolar interaction with the


paramagnetic cobalt (I I) ion. Differ-ences in the measured Ti values indi-cate that the Co(ll)-31P distancebecomes longer when the new isomer isformed. In the diamagneticcob (I I I)alamins, the spin-latticerelaxation time of the 3 1P nucleus isdetermined mainly by dipolar interac-tion with protons on the neighboringribose and aminopropanol groups. Varia-tions in Ti values of the 3 1P nucleusin these diamagnetic complexes supportthe contention that the "puckering" ofthe corrin ring is different in the newisomer. These conformational differ-ences appear not to alter the elec-tronic environment of the 3 1P nucleussince its chemical shift in the newisomer is the same as that in nativeB12.

The s'Co NMR spectrum of a cobalaminhas been reported (165)• Furthermore,it has been suggested that 5'Co nuclearquadrupole resonance spectroscopy couldhave great value in probing the envi-ronment about the cobalt in vitaminB12, methyIcobalamin, and coenzyme B12(169) •

E. Summary

The investigations of the biosyn-thetic pathway to B12 and the chemicaland enzymatic activities of B12, whichstimulated the application of NMR spec-troscopy to the corrins, cannot be con-sidered complete. Now that the basicNMR techniques have been developed, theassignments of the 1 3C resonances ofthe corrin ring have been made, and thechemical synthesis of potential meta-bolic intermediates with specific 1 3Clabels is possible (148,167), rapidadvances in our understanding of hisbiosynthetic pathway can be expected.There continue to be reports andreviews in which chemical analogues ofcobalamins are characterized with NMRspectroscopy (171-17*0 • Complete inter-pretation of the 1 3C NMR spectrum ofdicyanobyrinic acid heptamethyl esterhas been reported (175)- A more flexi-ble form of cobalamins with a differentconformation of the corrin has beendiscovered (167), and analogues ofcobalamins have been characterized inwhich various metal ions have been

substituted for cobalt(104,148,176,177) . Furthermore, thered and yellow, metal-free corrins thatwere first isolated from photosyntheticbacteria by Toohey (178,179) have nowbeen subjected to NMR analysis(180-182). The predominant metal-freecorrins that are excreted into the cul-ture media by Rhodopseudomonas spher-oides are hydogenobyrinic acid c-amideand hydrogenbyrinic acid a,c-diamide(180)• These descobaltocorrinoids areformed when there is a deficiency ofcobalt in the culture medium and haveno known function. Broken cell prepa-rations of Propionibacterium shermani iand R. spheroides do not insert cobaltinto hydrogenobyrinic acid a,c-diamide(180). However, chemical insertion ofcobalt into hydrogenobyrinic acid c-am-ide yields, in addition to cobyrinicacid c-amide and 13~epi-cobyrinic acidc-amide, small amount of a bluecobalt-containing corrin. This bluecorrin has been identified to be18,19-didehydrocobyrinic acid c-amide(l8l) and may be an intermediate in thebiosynthesis of vitamin B12 (180).

Newer departures include characteri-zation of the chemical reactivities ofvitamin B12 and its derivatives. Theeffects of various ligands on the pho-to I ability of alkylcobinamides andcoenzyme B12 have been determined(183-185). It has been demonstratedthat mercuric ion and platinum com-plexes such as cis-diami nodiaquopiati-num(ll) displace the benzimidazole ringfrom the cobalt of a IkyIcobalaminsand/or coenzyme B12. Methylcobalaminhas been shown to be capable of methy-lating mercuric ion and PtCl6~2

(186-188). No chemical reactivities ofvarious corino id complexes were com-pared in a recent review (189)•

Another area of ongoing interest isactivity of B12 as an enzyme cofactor.The relative binding of coenzyme andsubstrate to ethanolamine ammonia-lyasehas been investigated (190). Nonenzy-matic modelling of the coenzymeB12-dependent isomerization of methyl-ma I onyl coenzyme A to succinyl coenzymeA has been demonstrated (191-193) andthe reversible cleavage of the cobalt-carbon bond of coenzyme B12 by methyl-ma I onyl CoA mutase has been observed

Vol. 5, No. 1/2

(191*) • Coenzyme B12 that is stereospe-cifically deuterated in the 5'-position(19*4,195) was found to lose deuteriumto the solvent and to undergo scram-bling of deuterium between the twodiastereotopic 5'-positions in thepresence of the mutase (195)• Thereaction appears to involve cleavage ofthe cobalt-carbon bond and conversionof the 5'-carbon atom into a torsiosym-metric group. The exchange reaction iscatalyzed by the methylmalonyl-CoAmutase but occurs without the partici-pation of the substrate. It has beensuggested that the mechanism of diol-dehydrase might also be investigatedwith the use of coenzyme B12 stereospe-cifically deuterated in the 5'-position(196). These enzyme studies depend onthe recent assignment of the two5'-protons of coenzyme B12 to XH reso-nances at 0.6 ppm and I.5 ppm09^i195)• These assignments were madepossible by the improved resolution ofthe new higher-field superconductingNMR spectrometers. One might expectthat these investigations of enzymemechanisms will be facilitated by othertechnical advances, such as, for exam-ple, the recent report that the XH NMRspectrum of as little as 2.5 mM vitaminB12 can be measured in 95% H2O with amultipulse sequence called the 2-1-^pulse (197)- It seems safe to say thatmany more applications of NMR spectros-copy to the investigation of the cor-rins can be expected.

ACKNOWLEDGMENTS

Work performed under the auspices ofthe Office of Basic Energy Sciences,Division of Chemical Sciences, U.S.Department of Energy.

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