An improved method for preparing aqueous solutions of diaquocobinamide without hydrolysis products is described. The pKa values have been determined in 0.2 mol dm-3 NaClO4 at 25 °C as pK1 = 5.9 ± 0.1 and pK2 = 10.3 ± 0.2 and are shown to involve one proton each. Evidence is presented that diaquo-, aquohydroxo-, and dihydroxo-cobinamide exist in solution as isomers depending on hydrogen bonding between the axial ligands and different amide side-chains.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of the Chemical Society, Dalton Transactions|
|State||Published - 1983|
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The chemistry of vitamin B12. Part 20. Diaquocobinamide : pK values and evidence for conformational isomers. / Baldwin, David A.; Betterton, Eric A.; Pratt, John M.In: Journal of the Chemical Society, Dalton Transactions, No. 2, 1983, p. 217-223.
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TY - JOUR
T1 - The chemistry of vitamin B12. Part 20. Diaquocobinamide
T2 - pK values and evidence for conformational isomers
AU - Baldwin, David A.
AU - Betterton, Eric A.
AU - Pratt, John M.
N1 - Funding Information: David A. Baldwin Eric A. Betterton John M. Pratt An improved method for preparing aqueous solutions of diaquocobinamide without hydrolysis products is described. The p K a values have been determined in 0.2 mol dm –3 NaClO 4 at 25 °C as p K 1 = 5.9 ± 0.1 and p K 2 = 10.3 ± 0.2 and are shown to involve one proton each. Evidence is presented that diaquo-, aquohydroxo-, and dihydroxo-cobinamide exist in solution as isomers depending on hydrogen bonding between the axial ligands and different amide side-chains. J. CHEM. SOC. DALTON TRANS. 1983 217The Chemistry of Vitamin BI2. Part 20.1 Diaquocobinamide : pK Valuesand Evidence for Conformational IsomersDavid A. Baidwin, Eric A. Betterton, and John M. Pratt *Department of Chemistry, University of the Witwatersrand, Jan Smuts Avenue, Johannesburg 200 1 , South AfricaAn improved method for preparing aqueous solutions of diaquocobinarnide without hydrolysis products isdescribed. The PKa values have been determined in 0.2 rnol drn-3 NaCIO4 at 25 "C as pK, = 5.9 f 0.1and pK2 = 10.3 f 0.2 and are shown to involve one proton each. Evidence is presented that diaquo-,aquohydroxo-, and dihydroxo-cobinarnide exist in solution as isomers depending on hydrogen bondingbetween the axial ligands and different arnide side-chains.Diaquocobinamide (dac) can be regarded as the ' parent 'complex of the corrinoids and the determination of equilib-rium constants for the binding of ligands to dac is importantboth for analysing metal-ligand interactions within the corrin-oid family and for comparing corrinoids with other non-corrinoid CO" ' complexes. However, because the only com-mercially available corrinoids are cobalamins and the prepar-ation of dac is rather inconvenient, very little work has beenreported on dac, ahc, and dhc,? which are connected by equil-ibrium (1) (only axial ligands are shown). The pKl of dac hasbeen determined as 6.0 but only an approximate value hasbeen reported for pK2 and the number of protons has not beendetermined .2 *3H~O-CO-OH~ HO-CO-OH~ + H+ +HO-Co-OH + 2H+ (1)H~O-CO-CN + H2O + H~O-CO-OH~ + CN- (2)The main problem in the preparation of dac stems from thefact that removing the cobalamin side-chain from vitaminBIL, whether by hydrolysis in concentrated HCl or withcerium(ii1) hydroxide in the presence of cyanide yieldscyanoaquocobinamide (Factor B) in which the cyanide is veryfirmly held; for equilibrium (2), logl& < - 14.2 The cyanidecan be displaced by photolysis but, in order to prevent rapidrecombination and to displace equilibrium (2) to the right, thephotolysis must be carried out in acid in a stream of N2 gas toremove the HCN formed; we have found that acid condi-tions always cause some hydrolysis of the amide side-chains,as shown by t.1.c. The cyanide can also be displaced fromFactor B by reduction (e.g. with NaBH4) to the Co' complexwhich reacts rapidly with Me1 to give methylcobinamide; thiscan easily be purified and readily photolysed in the presenceof air to give d a ~ . ~ , ~ Unfortunately, photolysis involves freeradicals which, in the presence of air, produce stable yellowcorrinoids, as shown by increased optical density at ca. 455nm.70*8 Attempts to avoid acid conditions by photolysingFactor B in the presence of AgN03 or precipitated Ag20 werealso unsuccessful. We have, therefore, devised a simpleapparatus (see Experimental section) for simultaneouslyincreasing the rate of photolysis of Factor B with an ordinarytungsten lamp while reducing the rate of hydrolysis; thisconverts aqueous solutions of Factor B into dac in a singlestep without detectable levels of hydrolysis products.We have previously termed those equilibria of corrinoidst Abbreviations: dac, ahc, and dhc = diaquo-, aquohydroxo-,and dihydroxo-cobinamide, respectively. Cobinamides lack thenucleotide side-chain ending in 5,6-dimethylbenzimidazole (dbzm)which is present in the cobalamins. Factor B is cyanoaquo/cyano-hydroxocobinamide.which involve changes in the nature of the axial ligands (in-cluding their relative orientation) ' major ' equilibria, incontrast to the ' minor ' equilibria involving steric, hydrogen-bonding, and hydrophobic interactions of the corrin ring andthe axial ligands, and we have pointed out that the majorequilibria may be split into a set of minor equilibria involvingvariants or sub-species of the main complexes which differ in,for example, their hydrogen-bonding interactions.' Goodexamples of the effect of minor equilibria on the majorequilibria are provided by (a) the cyanoaquocorrinoidspossessing carboxylic acid side-chains which have beenstudied by Friedrich and co-~orkers,~~ where the relative sta-bility of the two isomers (with cyanide in the upper and lowerco-ordination positions) depends on the number of ionisedcarboxylate side-chains, and (6) the organocobalamins, wherethe neutral five-co-ordinate ' base-off ' form includes a variantin which the heterocyclic base is apparently held against thecorrin ring by hydrophobic and/or charge-transfer interac-tions.I0The major equilibria involving dac, ahc, and dhc are rep-resented by equilibrium (l), together with the possible inver-sion of the axial ligands in the case of ahc. It turns out thatthese complexes also provide good examples of minor equil-ibria which, we shall conclude, depend on hydrogen bondingbetween the axial ligands (H20 and/or OH-) and differentamide side-chains, i.e. they are similar to the minor equilibriainvestigated by Friedrich and co-w~rkers.~~ The main evidencefor the additional equilibria is provided by kinetic studies ofthe co-ordination of the ligands CN-, I-, and [Co(CN),J3-.We have been studying the kinetics of the substitution ofco-ordinated H20 by cyanide in corrinoids where the transligand is H20/OH-, dbzm (vitamin B12a), CN-, HC=C-,CH2=CH-, and CH,- over the range of pH 4-14 and havefound that all the observed kinetic plots and rate constants canbe interpreted in terms of (a) the known pK values of HCNand the corrinoid complexes, (b) labile ahc and kineticallyinert dhc, (c) simple pseudo-first-order kinetics under each setof experimental conditions, and (d) the scheme used byReenstra and Jencks l1 in their study of the reaction of B12awith cyanide (i.e. reaction of both CN- and HCN, followed byloss of a proton and isomerisation in the latter case), exceptthat the kinetic traces observed with ahc are bi- or even tri-phasic, i.e. show the occurrence of two or three parallel orconsecutive reactions. Full details of all these kinetic studiesand their interpretation will be published later; here we areconcerned only with the unusual kinetics observed with ahc.To check whether these anomalous kinetics are observed onlywith the combination of ahc + CN-, we have also studiedthe kinetics of co-ordination of I- and [Co(CN),J3- by dacand of the latter by ahc.The main aims of this paper are, therefore, to develop animproved method for the preparation of solutions of dac, t21 8 J. CHEM. SOC. DALTON TRANS. 1983determine accurate values for pK1 and pK2 for use in subse-quent work on equilibrium constants involving dac, ahc, anddhc, and to provide evidence for the existence and nature ofthe additional minor equilibria in these cobinamides.ExperimentalMaterials.-Samples of vitamins B12 and Bl2a were kindlygiven by Mr. Domleo of Glaxo-Allenbury (Pty) Ltd, SouthAfrica. Cyanoaquocobinamide (Factor B) was prepared aspreviously described l2 and converted into dac as describedbelow. AnalaR reagents were used wherever possible (HC104,NaC104, NaOH, NaCN, Nal, 0.880 ammonia); exceptionsincluded K,[Co(CN),] (B.D.H., reagent grade) and Bu'OH(Merck, chemically pure).U.u.- Visible Spectra-These were recorded with a JASCOUVIDEC-1 spectrophotometer using 1 -cm cells and, unlessotherwise stated, at 25 "C. The kinetics of ligand substitutionwere studied with a Durrum D-110 stopped-flow spectro-photometer .Thin-layer Chromatography.-T.1.c. was carried out at roomtemperature on cellulose (Merck, 0.1 mm, pre-coated), usingthe following three solvents (numbered as in refs. 5 and 13):I, BUSOH-water (9.5 : 4); 111, Bu"OH-0.88 ammonia-water(9.5 : 0.675 : 4); V, Bu"OH-0.88 ammonia-water-KCN (1 rnoldm-3) (250 : 0.4 : 100 : 0.3). Vitamin B12 was used as a markerin order to obtain values of RB12 (i.e. Rf values relative tothat of vitamin B12).5913pH Determinations.-These were made with a MetrohmEM 147 micro glass electrode.Preparation of dac.-The apparatus designed for photolysisconsisted of an annular glass container with an outer diameterof 8.5 cm, a width of 3 mm between the two walls, and a heightof 10 cm, open at the top. The hollow centre accommodated a60-W tungsten lamp. The annular space contained approxi-mately 40 cm3 of solution, which was stirred and flushed witha fine stream of nitrogen bubbles emanating from the ends offour plastic capillary tubes. The container and bulb were allplaced in a water-bath held at 0 "C. The higher surface areanormal to the light and the small depth to which the light hadto penetrate through the intensely coloured solution, togetherwith the vigorous flushing to keep [HCN] low, all served toincrease the rate of photolysis; while the large contact areawith the cooling water and vigorous stirring of the solutionserved to increase the rate of removal of the heat generated byabsorption of the light, and hence to reduce the rate ofhydrolysis.In a typical experiment Factor B (30 mg) was dissolved inwater (40 cm3), HC104 (1 rnol dm-3) added to give pH 2-3,and the resultant solution photolysed in the annular cell untilthe reaction was complete (ca. 5 h). The extent of reaction wasmonitored by withdrawing a small sample of solution, dilutingto the necessary concentration in NaOH (0.1 rnol dmd3), andexamining the spectrum in the region of the y-band. At this pHany unphotolysed Factor B (pK = 11 .O) 7c is present as thehydroxocyano-complex which has a sharp y-band at 362 nm(emolar = 2.3 x lo4 dm3 rno1-l cm-I), whose presence canreadily be detected in the presence of photolysed dhc, thespectrum of which (see Figure 3) includes a broad shoulderat ca. 356 nm (with &362 ca. 1.7 x lo4 dm3 mol-' cm-I). Thefinal analysis was carried out by t.1.c. using solvent 111; foralmost all preparations this revealed no products other thandac.After photolysis was complete, the solution of dac wasI 1 I I J5 9 130.61PHFigure 1. Spectrophotometric pH-titration of dac (3.3 xmol d m 3carefully neutralised with NaOH (0.1 rnol dm-3), degassed byevacuation with a water-pump for 30 min, and usually storedwithout further treatment as a frozen solution at -20 "C; nochanges (spectra, t.l.c., or kinetics) were observed on storagefor over two months. The cobinamide dac cannot be separatedfrom the low concentration of electrolyte by extractionthrough either phenol-chloroform or benzyl alcohol, since thiscauses partial reduction ; however, separation can be accomp-lished, if necessary, by using an Amicon 52 ultra-filtration unitwith a Diaflo UM 2 ultra-filter. Solid dac (or, more probably,ahc) can be prepared by freeze-drying, but partial reductionsometimes occurred (as seen from the spectrum after dissolu-tion); the cause of this reduction is not known.ResultsDetermination of pK Values.-Initial experiments over therange of pH 2-14 confirmed (cf. ref. 2) that aqueous solu-tions of dac show two reversible pH-dependent equilibria withpKl ca. 6 and pK2 ca. 10.5. Recording changes in the spectrumover the range 300-400 nm as the pH was increased showedthat the two equilibria overlapped, but good isosbestic pointswere observed at 341 and 357 nm for the first part of thechange corresponding to pKl. The latter part of the changecorresponding to pK2 also gave a good isosbestic point at 377nm and a reasonable one at 341 nm, but superposition of thespectra produced caustic curves around 320 and 358 nm; thespectral changes in the 300-360 nm region were less regularthan might be expected, but nevertheless appeared to bereversible.The pK values were determined quantitatively at 25 "C inNaClO, (0.2 rnol dm-3) by spectrophotometric titration ofdac (3.3 x rnol dm-3) from pH ca. 3 to ca. 13, followingthe optical density at 348 nm (the wavelength of greatestoverall change); see Figure 1. Analysis of the results gaveexcellent linear plots (see Figure 2) corresponding to oneproton; pK1 = 5.9 i 0.1 and pK2 = 10.3 f 0.2. The reversetitration from pH 13 to 3 (now at higher ionic strength) gavethe same values within experimental error.Spectra.-The spectra at pH 3.94 (dac), 8.24 (mainlyahc with a little dac), and 12.64 (dhc), all at 25 "C with I = 0.2rnol dm-3 (perchlorate), are shown in Figure 3. Solutions of allthree complexes obeyed Beer's law under these conditionsfrom 3 x lod to 6 x lo-' rnol dm-3. A solution of dhc iJ. CHEM. SOC. DALTON TRANS. 19830.02rI IP2191.0 I /I I I 15 9 13PHFigure 2. Evaluation of data of Figure I to give pK, and pK,; bothslopes correspond to 1.0 protonNaOH (1 rnol dm -3) showed no significant change over therange 300-600 nm on varying the temperature from 23 to43 "C; higher temperatures caused irreversible changes. Thewavelengths of the main bands and shoulders (nm) and theirabsorption coefficients ( 10-4~molar in parentheses, determinedby conversion to the dicyanide with A,,,,. = 367 nm and&367 = 3.04 x lo4 dm3 mol-I cm-') 7d for the three species wereas follows (Soret bands italicised): dac, 520 (0.98), 494 (1.00),349 (2.73), ca. 310 (ca. 0.9); ahc, 519 (1.04), 499 (1.05), 349(2.27), ca. 318 (sh) (ca. 1.3); dhc, 531 (l.lO), 508 (l.lO), 356(sh) (ca. 1.9), 344 (2.04), ca. 328 (sh) (ca. 1.6). The previouslyreported data for the Soret bands of dac (347.5 nm, 2.8 xlo4 dm3 mol-' cm-') and dhc (342,2. I x lo4) can be comparedwith the present data.l4Thin-layer Chromatography.-A comparison of the t.1.c. offive corrinoids using solvent I showed that the relative diffuse-ness D of their spots (very approximate values based on D =1 .O for methylcobalamin) increased in the following order:methylcobalamin (RBI* = 1.37, very sharp spot D = 1.0) <BI2 (1.00, D = 1.2) < Factor B (slow isomer 0.89, D = 2.3;fast isomer 1.03, D = 2.3) < B12a (noticeable streak 0.05-0.29, D ca. 3.3) < ahc (very marked streak 0.05-1.3, D ca.15). The streaking of the last two has been noted bef01-e.'~Hydroxocobalamin, ahc, and hydroxocyanocobinamide wereidentified from their spectra as the main species present in thepure solvent in a spectrophotometer cell. Similar streakingwas observed with three different samples of ahc. The streaksfrom B,2a and ahc showed no obvious signs of fronting ortailing or of concentrating into separate spots. When Blzaand ahc were each run alone in solvent 1 until a long streakhad formed and then run at right angles in solvent V, thestreaks ran as thin purple lines (of the dicyanide) parallel tothe solvent front, i.e. the various species responsible forforming the streak had been converted into a single dicyanidecomplex.Equilibrium Constants for Ligand Substitution.-As apreliminary to the kinetic studies, equilibrium constants forthe substitution of co-ordinated H20 in dac by I - , [Co-(CN),I3-, and acetate (since these studies were carried out inacetate buffer) were determined spectrophotometrically.There was no detectable change in the spectrum of dac inHC104 rnol dm-9 on adding up to 2 rnol dm-" NaC104,i.e. CI04- does not appear to co-ordinate. All the equilibria1:h/nmFigure 3. Spectra of 4.8 x lo-' rnol dmP3 solutions of dac (--),ahc (. - *), and dhc (- - -)investigated were established ' instantaneously '. For the twosmallest equilibrium constants, where the end-point wasunattainable (namely acetate and the second iodide), the datawere evaluated by the method of Newton and Arcand."The co-ordination of acetate hardly affects the wavelengthof the y-band (349 nm in both cases); the only noticeableeffect over the whole range 300-600 nm was an increase inoptical density around 320 nm. The equilibrium constant wasdetermined at pH 4.25 by titrating 5.5 x lo-' rnol dm-j dac in0.2 rnol dm-3 NaC104 (adjusted to pH 4.25 with HC104) withacetate buffer pH 4.25 ( I variable, up to 0.30 rnol dm-7and following the changes in A320. Correction for the pK, ofdac (5.9) and the pK, of MeC02H (4.75) gave K I = 13.1 f0.4 dm3 mol-' for the substitution of one H20 by MeCOz-.There was no evidence for co-ordination of a second acetateby dac at pH 4.25 or for even one acetate by ahc at pH 8.0;but no maximum values can be quoted for either constant,because the likely change in the spectrum is not known andwould probably be rather small.Qualitative experiments showed that dac catalysed the oxid-ation of I - by O2 to 13- and that the rate increased withacidity ; cf. the analogous catalytic activity of aquocobalaminin acid solution.7e The equilibrium constants were thereforedetermined in deoxygenated solutions at the highest pHpossible (ca. 4) by following changes in optical density in thectp region (to avoid interference from the intense band of 13-at 350 nrn).l6 Two well separated equilibria are exhibited bydac, both of which were shown to involve one iodide. Thevalue of K , = (1.76 41 0.03) x lo3 dm3 mol'l was obtainedwith 2.5 x rnol dm-3 dac at pH 4.0 and I = 0.2 rnoldm-' (NaC104/1-) following changes in A 5 2 2 and after correc-tion for bound acetate. The value of K2 = 2.2 i 0.2 dm3mo1-' was obtained with 3 x mol dm-3 dac at pH 4.0 byfollowing changes in A580 but, because of the low value of K,,the ionic strength could not be kept constant (up to 2 rnoldm-' NaI). The binding constant for acetate trans to iodidehas not been determined but, because of the trans effect of I - ,is expected to be significantly lower than 13 ; K2 has, therefore,not been corrected for any bound acetate. The very unusualspectra of these two iodide complexes will be discussed later.The low value of K , means that I - will not readily react withahc to displace OH- and that the co-ordination of I - can beconveniently studied only with dac itself in the acid region.HI is a very strong acid (pK ca. -- 10).17Equilibrium constants for the co-ordination of [Co(CN),I3-by dac were determined semi-quantitatively (the exact stoi220 J. CHEM. SOC. DALTON TRANS. 1983Table. Kinetics of reaction of dac and ahc with CN-, [CO(CN),]~-, and I-Reagent Second-order rate constants (dm3 mol-' S-~)concentration I 7Reagent PH mol dm-j) k* k m kS k m l k s(a) Varying pH and [reagent]; [Co] = ca. 8 x lo-' mol dm-34.505.006.007.028.099.0110.001 1.0312.011 .002.005.005.937.058.0811.0311.981 .o2.04 4 04-404-402-201.5-1 50.1-1 .o0.1-1.00.2-2.00 . 4 4 . 01-101-1010.1-1.011010100.5-50.5-56.51.5 x lo2 161.1 x 103 2.2 x lo21.4 x 104 2.4 x 1032.8 x 1043.9 x 1044.2 x 1041.3 x 1042.2 x 1032.1 x 1031.9 x 1037.8 x 1039.8 x 1034 x 104 1.0 x 1045.2 x 1032.4 x 1031.9 x 1032126.96.36.199.2 x lo2534.9 x 1 0 31.5 x 1041.4 x 1043.1 x 1034.2 x lo26.1 x lo27.4 x lo22.8 x 1033.5 x 1033.8 x 1031.1 x 1036.62.42.9 x 10'2.4 x lo2(b) Varying [Co]; [NaCN] = mol dm-3CN- 6.12 1.9 x lodd 2.4 x 103 2.3 x lo2 61CN- 6.12 1.9 x 10-5 d 2.8 x 103 2.2 x lo2 62CN- 6.12 1.9 x l W d 2.2 x 103 2.4 x lOt 604444633453333354388444Salts used: NaCN, NaI, K3[Co(CN),]. * Where studied, varying [reagent] had no significant effect on the rate constants. Two or threerate constants obtained by ' curve-stripping ' of the kinetic trace (see text). In every case the reaction represented by k, appeared to accountfor 50-70% of the total reaction. Value is [Co].cheiometry was not established) with 1.1 x mol dm-3dac in 0.2 rnol dm-3 acetate buffer at pH 4.0 in a 10-cm cell,following changes in AS1, to give Kl > 2 x lo6 dm3 rno1-l andK2 = (1.5 f 0.3) x lo5 dm3 rno1-l. The spectrum of the firstproduct had bands at 352,499, and 529 nm, that of the secondat 357,515, and 546 nm. The pK of the mono-adduct was notdetermined. The relatively high binding constants allow thekinetics of co-ordination of [Co(CN),p- to be followed wellinto the alkaline region. The hexacyanide can apparently onlybe protonated below pH 1; 18*19 we failed to observe anychange in the d-d spectra of the complex on acidification evenwith 3 mol dmF3 H2S04.Relevant equilibrium constants involving cyanide are asfollows: dac + CN-, i.e. reverse of equilibrium (2), log,, K 214; cyanoaquocobinamide + CN- to give the dicyanide,log,, K = 8 ;7f cyanoaquocobinamide, pK = 1 1 .0;7c HCN,pK, = 9.3.17Kinetics of Ligand Substitution.-The kinetics of co-ordin-ation of CN-, I-, and [Co(CN),p- by ca. 8 x lo-' moldm-3 solutions of dac, etc., have been studied by stopped-flowspectrophotometry at 25 "C and I = 0.2 mol dm-3. Therange of pH and ligand concentration used are given in theTable. The concentration of dac was varied in only one case,namely with cyanide at pH 6 (see Table). Several differentpreparations of dac (including one prepared by the photoly-sis of methylcobinamide) were directly compared at pH 6.0with cyanide; all gave the same absolute and relative values(within experimental error) of k, and k, (k, not evaluated inthis case; see below). Several different preparations were usedto complete the pH profile for cyanide.For every experiment the kinetic traces were followed for atleast four half-lives and the nature of the starting species andproducts checked on a conventional spectrophotometer usingidentical solutions. With cyanide the product was usuallythe dicyanide; other experiments established that cyanoaquo-cobinamide reacts with cyanide much faster than either dac orahc (due to the trans labilising effect of CN-), so that therate-determining step is the co-ordination of the first cyanide.The only product observed with I- was the mono-iodide com-plex. The reaction with the hexacyanide gave a mixture of themono- and/or bis-adducts, depending on conditions ; weassume that here too the rate-determining step is the co-ordin-ation of the first molecule of hexacyanide.In every case complete formation of the product was at-tained and the end-point was steady, yet all the kinetic traceswere distinctly bi- or even tri-phasic and could be resolved by' curve-stripping ' 2o into two or three pseudo-first-order rateconstants (see Figure 4). The second-order rate constants (fromfastest k,, through k,, to slowest k,) are listed in the Table; therates of reaction with CN- are too fast to ascertain whetherk, still persists above pH 7. These rate constants are indepen-dent of cobalt concentration, at least with cyanide at pH 6 (seeTable). The variation with pH of k, and k, for CN- and[co(cN)6]3- is shown in Figure 5.DiscussionOur results show that the photolysis of cyanoaquocobinamide(Factor B) in the annular container described in the Experi-mental section provides a convenient method for preparingsolutions of dac without any significant formation of hydroly-sis products.The pK values of dac have been determined in NaC104 (0.2moldm-3) at 25 "CaspKl = 5.9 f 0.1 andpK2 = 10.3 f 0.2and are shown to involve one proton each, in agreement witJ. CHEM. soc. DALTON TRANS. 1983 2214 ro-3t -4 1 h , I I 1 1 J0 5 10 15 20 25 30time (10~s)Figure 4. Example of ‘ curve-stripping ’ of a kinetic trace (reactionof ahc at pH 7.0 with 1 x lo-’ rnol dm-3 NaCN) to give kf (A),k m (01, and k s (0)previous values of pK1 = 6.0 and pK2 = ca. 10.5 at lower ionicstrength.2The highly unusual spectrum of dhc (see Figure 3) suggestedthe possibility that it might be a monomeric 0x0-complex(with or without HzO as the sixth ligand) or a dimeric poxo-complex, or even consist of two or more species related ac-cording to the pH-independent equilibria (3) and (4) (axialHO-Co-OH O‘Co-OH, (or O=Co + HzO) (3)2 HO-CO-OH + HO-CO-O-CO-OH + HzO (4)ligands only given). The oxide ligand (Oz-) would be expectedto have a much greater trans labilising effect than OH- ; dhcis, however, kinetically inert towards cyanide compared toahc (see Table and Figure 5). Solutions of dac, ahc, and dhcall obey Beer’s law and no temperature-sensitive equilibriumwas detected at pH 14. We conclude that the only majorequilibria involved are those represented by equilibrium (l),together with the possible change in relative orientation of thetwo ligands in ahc. The kinetic and t.1.c. results, however,provide evidence for the existence of additional minor equilib-ria.The kinetics of substitution of co-ordinated HzO in dacand ahc by I-, CN-, and [Co(CN),I3- (see Table and Figure5 ) show two features which require an explanation, namelythe occurrence of bi- and even tri-phasic kinetics and thevariation of these rate constants with pH. The latter can bequalitatively explained as follows. Starting from the pH-independent reactions of dac at pH 1-2, the increase in rateabove pH 5 corresponds to an increasing conversion of dacinto the more labile ahc (and of HCN into CN-) and the fallin rate at higher pH reflects conversion into the kineticallyinert dhc. The pH profile for cyanide can be quantitativelyinterpreted according to a scheme analogous to that used byReenstra and Jencks in their study of the reaction of aquo-cobalamin with cyanide; full details will be published later.The pH profile for the hexacyanide, on the other hand, cannotbe quantitatively explained in terms of just the two pK valuesof dac and no pK for the hexacyanide. We suspect that, be-cause of the high charge on the hexacyanide, ion-pairing mayplay some role and that the apparent effects of varying the pHmay include effects due to changes in ion-pairing; but this hasnot been proved.It is mathematically impossible 2o to differentiate betweenparallel rate constants, where the incoming ligand reacts withFigure 5. pH-Dependence of k , (0, @) and k, (0, .) with[Co(CN),13- (open circles/squares) and CN- (filled)different forms of the cobinamide which are not in ‘ instan-taneous ’ equilibrium with each other, and consecutiue rateconstants, where the initial adduct undergoes successive stepsbefore yielding the final product; the latter possibility can,however, be excluded because similar anomalous kinetics areobserved with all three ligands. The ratio of these rate con-stants is independent of cobalt concentration (see Table), i.e.they all represent reactions of corrinoids having the samedegree of aggregation, hence presumably monomers. Theonly reasonable conclusion is that dac and ahc exist in solutionas a pH-independent mixture of two or three ‘ isomers ’ andthat the rate of interconversion between the isomers is slowcompared to the rate of reaction with the incoming ligand andalso to the rate of loss or gain of a proton. The fact that thesame biphasic kinetics are observed at pH > 10 (i.e. wherethe complex is present as dhc) strongly suggests that, althoughdhc is kinetically inert, it must also be present as two ‘ isomers ’which can each be rapidly converted into the analogousisomer of ahc but only slowly into the other isomer of dhc.We have pointed out l3 that one of the most importantfactors which determines the Rr values of corrinoids in t.1.c.is the hydrogen-bonding capacity of the axial ligand(s), whichimplies a significant effect on the conformation of the side-chains; this is supported both by the X-ray diffraction data z1for cobyricacid and by the I3C n.m.r. spectra of its two isomersin solution (see later). We also noted the marked streaking ofaquocobalamin and dac; l3 we have now confirmed theseobservations, identified the main complexes present as hydr-oxocobalamin and ahc, and shown that streaking does notinvolve any irreversible decomposition. The existence of ahcas a mixture of slowly interconverting isomers provides anexplanation for the unusual streaking, as well as the unusualkinetics, of ahc. The observation of two waves in the reductionof dac by cyclic voltammetry may be a related phenomenon.We therefore have to find some structural feature which canprovide the basis for a new type of pH-independent ‘ isomer-ism ’ common to dac, ahc, probably dhc, and possibly evenvitamin Blza. The structure of cobyric acid (Factor Via)provides a clue.In Factor V1a the upper (orb) co-ordination site is occupiedby HzO (though the possibility that the ligand is OH- cannotbe excluded) and the lower (or a) site by CN-. The terminal Natom of the cyanide is hydrogen-bonded to one water molecule,while the co-ordinated H20 is hydrogen-bonded to the car-bony1 0 atom of the acetamide side-chain of ring B and alsoto a water molecule, i.e. the co-ordinated HzO provides th222 J. CHEM. SOC. DALTON TRANS. 1983protons for both hydrogen bonds, while the co-ordinatedCN- provides the lone pair of electrons for its single hydro-gen bond. The bonding between the co-ordinated H20 andthe amide is apparently strong enough both to cause a detect-able displacement of the co-ordinated 0 atom and to affectthe pucker of the corrin ring.21 This same acetamide side-chain also forms a hydrogen bond to the co-ordinated cyanidein dry vitamin B12,22 but in wet B1223 and in the co-enzyme 24 itturns outward to form intermolecular hydrogen bonds. TheX-ray evidence therefore shows that the axial ligands canform hydrogen bonds to the side-chains; that the ability toform such bonds is probably greater with H 2 0 than with CN-and will obviously be non-existent with alkyl ligands; and thatthe conformation of the side-chains can be affected by thehydrogen-bonding capacity of the ligands.It appears that all cyanoaquo-/cyanoh ydroxo-corri no ids(including Factor Via) exist in solution as a mixture of two iso-mers which differ in the relative orientation of the CN- andH,O/OH- ligands. The ratio of the two isomers varies from1.5 : 1 to 0.25 : 1 at pH 2 5 (i.e. where the ligands are CN - andH20); the dependence of this ratio on the number of ionisedcarboxylate side-chains provides fairly direct evidence forhydrogen-bonding between the axial ligands and the side-chains in A comparison of the n.m.r. spectra ofthe two isomers of Factor V1, (apparently in neutral solutionwhere the ligands would be CN- and HzO) revealed surprisingdifferences, which extended even to some of the carbon atomsin the side-chains; it was concluded than these differences mustreflect ' some conformational changes of the corrin ring.' 2sAll the isomeric pairs of cyanocorrinoids show similar largedifferences in RB12 values, 7b which can again be ascribed tosignificant effects of the axial ligands on the conformation ofthe side-chains. In the case of Factor B the main speciespresent in the t.1.c. solvent has been identified as the cyano-hydroxo-complex (see Results section); this suggests that co-ordinated OH- has a similar effect to co-ordinated H20.Furthermore, the fact that Factor B exists as an approxi-mately equal mixture of two isomers and that the two t.1.c.spots have a similar diffuseness (see Results section), in spiteof very different R,,, values, strongly suggests that there islittle difference between the side-chains above and below thecorrin ring in their ability to form hydrogen bonds to co-ordinated H 2 0 and OH-. It therefore appears that in cyano-aquo-/cyanohydroxo-corrinoids the co-ordinated H 2 0 or OH -is hydrogen-bonded to some side-chain in both stereoisomers;the slight diffuseness observed in the t.1.c. of Factor B mightindicate that the H 2 0 or OH- can be hydrogen-bonded tomore than one side-chain, but the evidence is clearly not verydefinite.We therefore propose ( a ) that the different isomers ob-served with dac, etc. represent hydrogen-bonding betweenthe axial ligand(s) and different side-chains or combinations ofside-chains; (b) that co-ordinated H 2 0 and HO- show fairlysimilar behaviour, such that the isomers of dac, ahc, and dhchave analogous structures; and (c) that the relatively slowrate of interconversion of the isomers is due to the slow con-formational change of one or more side-chains. Isomers ofthis type would be expected to show only slight differences inproperties such as spectra, pK values, and rate constants; theresolution of only two or three rate constants does not there-fore necessarily mean that this is the maximum number of suchisomers present in solution. There is no evidence to suggestwhether the two or more isomers observed for ahc have OH-in the same co-ordination position or not. The existence ofsuch isomers with slight differences in spectra and pK pro-bably explains why the changes in the spectrum correspondingto pK2 were not as regular as expected in the 300-600 nmregion.The streaking of BIza in t.1.c. could indicate the existenceof isomers, but neither we nor others 11~26,27 have obtained anyevidence for such isomers from the study of ligand substitutionreactions. However, Kenyhercz et ~ 1 . ~ ~ have reported thathigh-pressure liquid chromatography of B,2a gave two closelyspaced yet distinct peaks, while Bt2 apparently gave only asingle peak; this interesting observation clearly requiresconfirmation. They also found that, under the conditions oftheir spectroelectrochemical experiments, the redox behaviourof the CO'~'/CO'' couple of B12a showed pronounced hysteresiswith two different waves for reduction. They were forced toconclude that Blza (in contrast to B12) existed in aqueoussolution pH 7.0 as a 65 : 35 mixture of two species and sug-gested that these were the ' base-on ' and ' base-off' forms,but admitted that this explanation was contradicted by theirown results on the Co"/Co' redox reactions. These observ-ations might conceivably be related to the existence of isomersof the type discussed above.AcknowledgementsWe wish to thank Mr. A. P. Donileo of Glaxo-Allenbury (Pty)Ltd. for the gift of samples of vitamin BIZ and Blza, andAfrican Explosives and Chemical Industries Ltd. and theCouncil for Scientific and Industrial Research for support (toE. A. B.).ReferencesI Part 19, S. M. Chenialy and J . M. Pratt, J. Chem. SOC., DaltonTrans., 1980, 2274.2 G. C. Hayward, H. A. 0. Hill, J . M. Pratt, N. J . Vanston, andR. J. P. Williams, J . Cliem. SOC-., 1965, 6485.3 D. Lexa, J. Saveant, and J. Zickler, J . Am. Chem. SOC., 1980,102, 4851.4 J. B. Armitage, J. R. Cannon, A. W. Johnson, L. F. J . Parker,E. Lester-Smith, W. H. Stafford, and A. R. Todd, J . Chem. SOC.,1953, 3849.5 R. A. Firth, H. A. 0. Hill, J. M. Pratt, and R. G. Thorp, J .Chem. SOC. A , 1968, 453.6 J. M Pratt, J . Chem. SOC., 1964, 5154.7 J. M. Pratt, ' Inorganic Chemistry of Vitamin BIZ,' AcademlcPress, London, 1972, (a) pp. 286-292; (b) pp. 119-124;( c ) p. 140; ( d ) p. 46; (e) p. 209; (f) p. 144.8 A. Gossauer, B. Gruning, L. Ernst, W. Bccker, and W. S.Sheldrick, Angew. Chem., Int. Ed, Engl., 1977, 16, 481.9 J. M. Pratt, in ' Vitamin BIZ,' ed. D. Dolphin, John Wiley, NewYork, 1982, vol. 1, p. 325.10 S. M. Chemaly and J . M. Pratt, J . Chem. SOC., Dalton Trans.,1980, 2267.11 W. W. Reenstra and W. P. Jencks, J . Am. Chem. SOC., 1979,101,5780.12 R. A. Firth, H. A. 0. Hill, B. E. Mann, J . M. Pratt, R. G. Thorp,and R. J. P. Williams, J . Chem. SOC. A , 1968, 2419.13 R. A. Firth, H. A. 0. Hill, J . M. Pratt, and R. G. Thorp, Anal.Biochem., 1968, 23, 429.14 G. C. Hayward, H. A. 0. Hill, J. M. Pratt, and R. J. P. Williams,J . Chem. SOC. A , 1971, 196.15 T. W. Newton and G. M. Arcand, J . Am. Chem. SOC., 1953,75,2449.16 C. Herbo and J. Sigalla, Anal. Chim. Acfa, 1957, 17, 199.17 L. G. Sillen and A. E. Martell, ' Stability Constants of Metal IonComplexes,' Spec. Pub]. No. 17, Chemical Society, London,1964.18 J. Brigando, Bull. SOC. Chim. Fr., 1957, 503.19 J. Jordan and G. J. Ewing, Znorg. Chem., 1962, 1, 587.20 G. M. Fleck, ' Chemical Reaction Mechanisms,' Holt, Rinehart,21 D. C. Hodgkin, Proc. R . SOC. (London), Ser. A , 1965, 288, 294.22 D. C. Hodgkin, J . Lindsey, R. A. Sparks, K. N. Trueblood, andJ. G . White, Proc. R. SOC. (London), Ser. A , 1962, 266,494.23 C. Brink-Shoemaker, D. W. J. Cruikshank, D. C. Hodgkin,and Winston, New York, 1971J. CHEM. SOC. DALTON TRANS. 1983 223M. J. Kamper, and D. Pilling, Proc. R. SOC. (London), Ser. A,1964, 278, 1.27 D. Thusius, J. Am. Chem. Soc., 1971,93, 2629.28 T. M. Kenyhercz, T. P. DeAngelis, B. J. Norris, W. R. Heineman,Received 10th May 1982; Paper 2176224 P. G. Lenhert, Proc. R. SOC. (London), Ser. A, 1968, 303,45.25 D. Doddrell and A. Allerhand, Chem. Commun., 1971, 728.26 W. C. Randall and R. A. Alberty, Biochemistry, 1967, 6, 1520.and H. B. Mark, J. Am. Chem. Soc., 1976,98,2469
PY - 1983
Y1 - 1983
N2 - An improved method for preparing aqueous solutions of diaquocobinamide without hydrolysis products is described. The pKa values have been determined in 0.2 mol dm-3 NaClO4 at 25 °C as pK1 = 5.9 ± 0.1 and pK2 = 10.3 ± 0.2 and are shown to involve one proton each. Evidence is presented that diaquo-, aquohydroxo-, and dihydroxo-cobinamide exist in solution as isomers depending on hydrogen bonding between the axial ligands and different amide side-chains.
AB - An improved method for preparing aqueous solutions of diaquocobinamide without hydrolysis products is described. The pKa values have been determined in 0.2 mol dm-3 NaClO4 at 25 °C as pK1 = 5.9 ± 0.1 and pK2 = 10.3 ± 0.2 and are shown to involve one proton each. Evidence is presented that diaquo-, aquohydroxo-, and dihydroxo-cobinamide exist in solution as isomers depending on hydrogen bonding between the axial ligands and different amide side-chains.
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UR - http://www.scopus.com/inward/citedby.url?scp=37049106967&partnerID=8YFLogxK
U2 - 10.1039/DT9830000217
DO - 10.1039/DT9830000217
M3 - Article
AN - SCOPUS:37049106967
SP - 217
EP - 223
JO - Dalton Transactions
JF - Dalton Transactions
SN - 1472-7773
IS - 2