X-ray and neutron diffraction studies of the crystal and molecular structure of the predominant monocarboxylic acid obtained by mild acid hydrolysis of cyanocobalamin. Part II. X-ray diffraction studies of wet crystals
JOYCE M WATERS* and T N M WATERS
Formerly Chemistry Department, University of Auckland, Auckland, New Zealand Present address: Massey University, Palmerston North, New Zealand
Abstract. The crystal and molecular structure of wet crystals of the O-monocarboxylic acid of cyanocobalamin has been determined. The molecule crystallizes in a monoclinic cell of a = 14.845, b = 17.435, c = 16.243 A, fl = 103"54 ~ space group P21. Intensity data were collected by diffractometry and the structure solved by Patterson and Fourier methods.
Refinement, initially by a least-squares process and latterly by Fourier methods, led to an R of 0-140. Disordered sites were found for the terminal atoms of side chain e; the identification of the acid grouping was not made with certainty. Sites for sixteen water molecules were determined.
Keywords. Vitamin BI2; wet crystals of monoacid; dry crystals; crystal data; hydrogen bonding.
1. Introduction
Crystals o f the wet form o f the BI2 monoacid were grown from aqueous acetone solution and suitable specimens were mounted in quartz capillaries in contact with their mother liquor. Initially a data set was collected by photographic means and the structure solved by the heavy atom method followed by a succession o f electron density syntheses. Although the process of atom placement was carried out conventionally it was o f course helped enormously by the knowledge o f the structure o f the air-dried crystals. All but three atoms o f one of the side chains (side chain e--see figure 1) and some of the sixteen water molecules (assumed from density measurements to be present) were located. Refinement by electron density difference syntheses and then by block-diagonal least-squares methods made it clear that the side chain containing the missing atoms was disordered as were also a number of the solvent atom sites.
Difficulties in interpretation eventually led to the recollection o f a more accurate data set by diffractometer methods (Hilger-Watts Y290 diffractometer). Details o f this latter data set are included here.
2. Crystal data
C63H87CoN13OlsP, x H 2 0 (wh~re x is approximately 16). M = 1356.4 (excluding water molecules), monoclinic a = 14.845(6), b = 17.435(12), c = 16.243(7)A,
* To whom all correspondence should be addressed.
( C h e m . Sci.) -- 3
219
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Figure 1. Vitamin B12 m o n o a c i d - - W e t crystals. The numbering system o f the molecule.
Table I. Comparison of the cell parameters for different crystals of B12 monoacid.
a b c ~ Volume
Air-dried crystals 14.51A 1 7 . 0 9 A 16.35A 103 ~ 3951A 3 Semi-dry crystals 14-785 17.331 16"215 103.17 4046
Wet crystals 14"845 17.435 16.243 103.54 4087
Neutron diffraction study 14-91 17.49 16-41 104.11 4150
fl = 103.54(1) ~ (obtained by a least-squares fit to the setting angles o f twelve reflections), U = 4 0 8 7 A a, Dc = l ' 3 3 6 g c m -1 for z = 2, space g r o u p P21, Mo-K~
radiation (k = 0-71073A),/~(Mo-K~) = 3.22 c m - 1 .
It was noticed that the cell parameters were all less than those determined by n e u t r o n diffraction indicating a crystal with a slightly lower water content. Table 1 c o m p a r e s these cell parameters together with those o f a crystal sealed in a capillary, but n o t in contact with m o t h e r liquor (semi-dry form), a n d with the fully air-dried crystals.
Intensity data were collected by a symmetric o9-20 scan o f 1"60 ~ at a scan rate o f 0.02 ~ sec- 1 with b a c k g r o u n d c o u n t e d for 10 sec at each end o f the scan. O f 5597 reflections examined with a Bragg angle o f less than 23 ~ 2069 independent reflections had I FI 2 > 3 (IFI2) 9 T h e size o f the diffracted b e a m collimator was 3.55 m m (incident beam collimator 0.5 mm). D a t a were processed according to the p r o c e d u r e o f Corfield et al (1967) with p assigned a value o f 0-04. N o a b s o r p t i o n corrections were applied.
Full-matrix least-squares refinement on the 2069 reflections with [F[ 2 >
3o(IFI ~)
assuming isotropic thermal m o t i o n for all a t o m s initially, a n d anisotropic m o t i o n f o r
Co and P later, lowered R to 0.178. In these calculations the three terminal atoms o f side chain e (i.e. C50, N 5 1 , 0 5 2 ) were excluded and only five water molecules were included with full site occupancy. F r o m the subsequent electron density and associated difference electron density maps two disordered sites were located for the three terminal atoms o f side chain e and a further ten sites for water molecules. M a n y o f these were associated with lower peaks in the maps and hence seven were assigned site occupancy factors o f 0-5.
Calculations o f the intra-molecular distances showed a n u m b e r o f long bond lengths (C-42 1-66-2-00A) suggesting, at first thought, substantial and unexpected inaccuracies in the data. However, inspection of an electron density m a p showed well-resolved peaks for most atoms and a relatively low level o f background noise. Consequently, further refinement was undertaken but this time by locating atoms on the tops o f peaks in a series o f electron density maps. Three such maps were calculated before all fractional co-ordinate changes were ~< 0-001. The thermal parameters were held at the values obtained at the end o f the last least-squares refinement cycle. A comparison between the bond lengths calculated at the end of this refinement by Fourier synthesis and those obtained at the end o f the last least-squares cycle showed a significant improvement in the former values. Fourteen C - C distances which were calculating in the range 1-66-2.00 A at the end o f the least-squares process had since refined to m o r e acceptable values.
Once the final atomic co-ordinates had been obtained further refinement o f the thermal parameters was undertaken by the least squares process and after two cycles R was 0" 141. Initially the assignment of a t o m s as oxygen or nitrogen was that determined in the neutron analysis and the B-values obtained after two cycles of refinement are given in table 2. As a check the oxygen and nitrogen atoms o f each side chain were interchanged and the B values re-examined after a further two refinement cycles. The
Table 2. BI2 Monoacid--wet crystals. Values of thermal parameters (A 2) of the terminal atoms of the side chains at different stages in the refinement.
O and N
Neutron B o t h interchanged Refinement
Side study assigned from neutron Assignment after 74 H
chain assignment as oxygen study made included
a N28 5.4(7) 028 7.8(7) 028 7.9(7) N28 5.9(7) 5-7(9) 029 6"7(6) 029 6.8(6) N29 4.1(6) 029 6.8(6) 6'6(8) b O 33 6.4 (7) O 33 6.3 (7) O 33 6-4 (7) O 33 6-7 (6) 6.7 (9) O 34 6-4 (7) O 34 6"5 (7) O 34 6.6 (7) O 34 6.6 (6) 6-8 (9) c 039 8.8(8) 039 8-6(8) N39 5-7(8) N39 6-2(8) 6'6(10)
N40 3-6(6) 040 6.1(6) 040 6.0(6) 040 6.3(6) 6.3(8) d 044 16(1) 044 16(1) N44 13(2) 044 18(2) 17(2) N45 13(1) 045 16(1) 045 16(1) N45 14(2) 15(2) e N151 9 ( 2 ) O 1 5 1 11(2) O 1 5 1 11(2) N151 8(2) 9(3) O152 20(2) O152 20(2) N152 19(2) O152 30(2) 26(5) e' N251 12(2) O 2 5 1 14(2) O 2 5 1 13(2) N251 12(3) 12(4) 0252 21(2) 0252 20(2) N252 22(2) 0252 48(6) 37(6) g 062 7.2(7) 062 7.2(7) N62 4.5(7) 062 7.8(7) 7.5(9)
N63 6-1(9) 063 9"1(8) 063 9"1(8) N63 6'3(8) 6"0(10)
R 0.141 0-141 0-140 0-137 0-137
Rw ff193 0.193 0'192 0.188 0-178
two disordered sets of sites for side chain e, not unexpectedly, gave large values for B.
The high values also observed for the O and N atoms of side chain d suggest that some disorder may well occur in this region of the molecule but inspection of the electron density map gave no clear indication of alternative sites for these atoms. Because of the uncertainty in location no attempt was made to distinguish O and N for side chains d and e. However for a, c and g the refined B-values suggest an assignment of N28/O29, N39/O40 and O62/N63. Two of these are in agreement with the conclusion reached from the neutron study but for side chain c the opposite assignment, N39/O40, is suggested. Consideration of the hydrogen-bonding contacts for this side chain does not clearly distinguish the two atoms and in the final refinement cycle the neutron assignment (O39/N40) was assumed. The identification of b as the monoacid (033/034) as concluded in the neutron study is consistent with the evidence from the B-values.
Sites for 74 hydrogen atoms were calculated assuming a C-H bond length of 1.09 A.
Before inclusion in a structure factor calculation all proposed positions were checked against a difference map and all were found to lie on, or close to, regions of positive density. One hydrogen site, H81, was later found to have subsequently been included incorrectly and in a new difference map was seen to lie in a small trough; the correct position for this atom was clearly marked by a small peak. The inclusion of these 74 hydrogen atom sites in the structure factor calculation had little effect on the refined B's and had no effect on the oxygen and nitrogen assignment.
A total of sixteen water molecules was finally located from the electron density maps.
A number of these were sited on smaller peaks and were consequently included in the calculations with a site occupancy factor of 0.5. Finally all water molecules where their thermal parameters refined to high values (B > 20A 2) were reduced to half site occupancy in the final structure factor calculation (R = 0.14).
The numbering system used for the molecule is shown in figure 1, final atomic parameters for the wet crystals are listed in tables 3, 4 and 5. Figure 2 contains the bond distances and bond angles.
3. Brief description and discussion of the molecule
The expected non-planarity of the corrin ring is illustrated in figure 3; the largest deviation from the plane calculated through the four nitrogen donor atoms occurs at C7 ( + 0.80 A). The three acetamide substituents (starting at C26, C37 and C60)all lie on the same side of the plane whereas the three longer propionamide or acid groupings are disposed on the other (C30, C41, C48) as has been found for vitamin B12 itself and its coenzyme form. The puckering of the corrin ring is identical with that found in the structure of the dry crystals of the monoacid but does show small differences from the geometry observed in the parent B12 molecule and its coenzyme form. However in all these molecules the greatest deviations from the plane of the four coordinated nitrogens is seen for the atoms C5, C6 and C7. The substituents in this region of the molecule show considerable crowding and for the monoacid (wet crystals) the closest non-bonding contact distance occurs between C36 and C41 (2.85A).
Within the corrin ring the bonding distances observed agree reasonably well with those found in other similar molecules and with the pattern expected for a system of six resonating double bonds. Table 6 shows the theoretical values for this system together with the distances determined in the monoacid and other related molecules.
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Table 4. BI2 Monoacid--Wet crystals. Parameters for the water molecules.
Atom x/a y/b z/c B (A 2) S.O.F.(*)
W 1 0"1616 0"2455 0"0148 10"2 1'0 W 2 0"0329 0"3958 0"8653 8.1 1-0 W 3 0"5703 0.2395 0"7191 19-9 1"0 W4 0"1954 0"1390 0-4610 14.2 1'0 W5 0"4170 0.3476 0"6866 17.9 1.0 W6 0"7399 0"4609 0'6711 13.9 I'0 W7 0"1859 0"3446 0.1514 14.0 1-0 W8 0"1192 0"2760 ff2979 18.5 1"0 W9 0"1178 0"4966 0"1112 12-2 0"5 W 10 0.1600 0"3861 0"5247 20-1 0-5 W 11 0.0609 0-3272 0"4301 16.2 0"5 W 12 0.2744 0.2932 0"4925 18-8 0"5 W 13 0"8555 0-0166 0-7050 16-8 0-5 W 14 0"2685 0.4970 0"4086 18.6 0-5
W 15 0.4954 0.3421 0.4761 19.7 0.5
W 16 0"2943 0.3541 0-3732 19-9 0-5
* site occupancy factor.
4. H y d r o g e n bonding c o n t a c t s
Figure 4 shows the packing of the molecules in the crystal lattice. In the wet crystals the molecule is involved in a number of hydrogen bonding contacts with neighbouring molecules and also with water molecules. These contacts for a single molecule are shown in figure 5 (one site only is illustrated for the terminal atoms o f side chain e);
relevant distances are listed in table 7. The criteria used to define a 'normal' hydrogen bond (Hamilton and Ibers 1968) are O - H . . . O, 2.6-2.8 A; O - H . . . N, 2.7-2.9 A;
N - H . . . O, 2.8-3.0 A; N - H . . . N, 3.0-3.2 A.
O f the side chains a-e and # only a has contacts exclusively with neighbouring molecules. The rest all show contacts with at least two water molecules. F o r side chain a the two contacts o f N28 are consistent with the assignment o f this a t o m as nitrogen.
The single short contact o f O 2 9 ( 0 2 9 . . . O81, 2.55 A) is in accord with the assignment o f this a t o m as oxygen. Side chain b which has been assigned as the acid grouping in the neutron analysis shows a 'normal' hydrogen bonding contact o f 2.62 A between 0 3 4 and W l ; 0 3 3 is involved in weaker interactions with W9 and N28 at distances o f 2"92 and 3.09 A respectively.
Two normal hydrogen bonding contacts are made between N40 and O71 (2.99 A) and between N40 and W5 (2-83 A) whereas the oxygen o f this amide grouping, 039, is involved in one normal and one weak contact with the water molecules W l and W2 at 2.72 and 3.07 A respectively. Thus this evidence favours slightly the oxygen-nitrogen assignment of these atoms as deduced f r o m the neutron analysis. Close contacts are made by 0 4 4 to the disordered sites o f side chain e but precise details are uncertain due to the imprecision in locating these latter atoms. At least one contact is likely between each of the disordered sites o f O152'/0252'. A further weak hydrogen bonding contact o f 2.88 A is noted to the half-weighted water molecule, W14. F o r N45, two close
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F i g u r e 3. Vitamin B12 m o n o a c i d - - W e t crystals. Projection o f the corrin n u c l e u s o n a cylinder o f radius 2.8 A around the cobalt atom. The molecule is s h o w n as it would be seen w h e n v i e w e d from the metal atom outward. Vertical displacement o f each a t o m c o r r e s p o n d s to the distance from the plane o f best fit t h r o u g h the four n i t r o g e n a t o m s , 21, 22, 23 and 24.
approaches are indicated, one at 2"82 A to W3 and the other at 3.11 A to N65'. Both distances lie within the normal range proposed for N . . . O and N . . . N.
For side chain e hydrogen bonding interactions are likely with the two alternate disordered sites. For N151 the contact with W6 is replaced by one with a further water molecule, W3, when the alternative site, N251, is occupied. As already noted the contact with 044 is retained for both oxygen sites, O152 and 0252. In addition, both sites make close approaches to the water molecule, W14. The contact with W15 which occurs between the atom O 152 is lost when the 0252 site is preferred. Although N251 and 044 are separated by 3.08 A this contact is probably unlikely to indicate a hydrogen bond since this latter atom makes a much closer approach to 0252.
Both 062 and N63 of side chain g each approach closely to two water molecules but the atom assigned as oxygen makes the closest approach (2.47 A from W8) supporting the distinction already made between O and N atoms. This contact indicates a strong hydrogen bonding interaction; the remaining three contacts (between the atom pairs 062 and W4, N63 and W6, N63 and W2 at 2.76, 2-86, and 2.92 A respectively) all indicate normal hydrogen bonding interactions.
The secondary amide nitrogen atom, N59, approaches to within 3-23 A of W4, a distance which suggests a very weak hydrogen bonding interaction. That one occurs at all is supported by the fact that the calculated site for H59 (assuming trigonal N with C-H of 1-09 A) lies close to the line joining N59 and W4 and approaches to within 2.32 A of this latter atom. The oxygen (058) of the secondary amide grouping is also involved in contacts with water molecules at distances (2.83, 2.97, 3-02 A to W14, Wl0, W13 respectively) suggesting only weak interactions. All three of these water molecules have been determined with partially weighted site occupancy.
The P-O bonds are seen to form two pairs according to the bond distance data, the longer bonds, not unexpectedly, being associated with the chain-linking oxygens. The two terminal oxygens each have contacts with two other atoms at suitable hydrogen bonding distances. In each instance one contact is with an adjacent molecule and one with a water molecule. The close approach between 072 and N28 has already been noted but in addition there is also the contact between 072 and W2 of 2.79 A. For O71 the close approaches to 040 (2.99 A) and Wl0 (2.83 A) are noted.
The two substituent hydroxyl groups associated with the sugar moiety both appear to be involved in hydrogen bonding interactions. The strong bond between O81 and 029 has already been commented upon but in addition two further weaker interactions are likely with the water molecules W3 and W6 at 2.82 and 3-01 A respectively. Only one contact is found between 080 and the partially weighted water molecule, Wl 1.
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All but three o f the water molecules (W7, W12, W16) are involved in at least one contact with either an O or N of the various side chains. The three exceptions do, however, form hydrogen bonding contacts with other water molecules. Some o f the half-weighted water molecules approach very closely to one another ( W l 0 . . . W l 1, 2.13; W 1 2 . . . W16, 2"29; W 1 3 . . . W14, 2-31 A) suggesting that both molecules o f the pair cannot be present simultaneously in the same unit cell.
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Figure 5. Vitamin B12 monoacid. The molecule is shown projected on to the plane through the four nitrogen atoms 21, 22, 23 and 24. Hydrogen bonding contacts to the side chain oxygen and nitrogen atoms of a single molecule are shown as dashed lines. (a) Wet crystals (b) Dry crystals.
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5. Comparison of the structures of the wet and dry crystals
Some differences between the wet and dry crystals o f the vitamin B12 monoacid are noted but these are mainly associated with the side chains (see figure 6). Side chains a, b and g are almost identical in the two crystals. O f these only a makes close approaches to identical atoms in both the wet and dry forms. As noted earlier these contacts are all to oxygen or nitrogen atoms o f adjacent molecules and none are to water molecules. With side chains b and g although the same conformations are preserved for the a t o m s in both the wet and dry crystals, the slight expansion of the unit cell for the former allows some variation in the water molecule sites. Thus for b the 0 3 3 . . . N28 contact is preserved but the contacts with W l and W9 which occur for the wet crystals are replaced by a single water molecule (W16) in the dry form as well as a new contact with a t o m 39~ F o r side chain g the terminal a t o m s in the wet crystals show contacts with four water molecules. Similar contacts, also with four water molecules, are preserved in the dry crystals but the particular water molecules show considerable variation in their positions.
In the dry crystals atoms 38, 39, 40 are found to occupy two sets of disordered sites whereas this is not so for the wet form. The differences seem to arise f r o m rotation about the C74237 bond. Inspection o f figure 6 shows the relevant a t o m s in the wet crystals occupying sites which lie between the two disordered positions for the dry. In spite o f these differences the contact with 0 7 1 is preserved for the two disordered sites.
However a new close approach to a t o m 34 is observed for one o f the oxygen sites o f the dry crystals; a corresponding approach to side chain b is not observed for 0 3 9 o f the wet form.
Figure 6. ComparisonofthcvitaminB12monoacidasdeterminedinthewetanddrycrystal forms. The molecule as determined for the wet crystals is illustrated projected on to the plane through the four nitrogen atoms 21, 22, 23 and 24. Those atoms in the molecule of the dry crystals which show differences are depicted as filled circles.
Alternate disordered sites were not found for the atoms of side chain d in either the wet or the dry crystal structures. However, for the wet crystals some disorder is suspected from the high values of the thermal parameters for atoms 43, 44, 45.
Comparison of the two structures does show small positional differences for these atoms which seem to result from a slight rotation about the C41-42 bond. Consequent differences are seen in the hydrogen bonding contacts these atoms make with water molecules in the two crystals. Contacts between the terminal atoms of this pro- pionamide group with N65 as well as with the atoms of side chain e are preserved. With this latter side chain two sets of disordered sites were located for the wet crystals whereas only a single set were found for the dry. The differences here appear to be related to rotation about C49-C50 particularly but may also involve some rotation about C48-C49. Most of the contacts made by the oxygen and nitrogen atoms of this side chain are to water molecules which occupy different sites in the two crystals but the one contact to an adjacent molecule via 044 is preserved not only for the two disordered positions in the wet crystals but also for the single site in the dry crystals as well.
Comparison of the molecule in wet and dry crystals also reveals small atom displacements along the length of the secondary amide loop, these becoming most noticeable at atoms C66, C67, c68. Despite these displacements all hydrogen bonding interactions between the atoms of the loop and adjacent molecules are retained and only those contacts with solvent water differ. Indeed the retention of the inter- molecular hydrogen bonds in the different-sized unit cells no doubt provides the driving force for the atom movement as the cell enlarges to accommodate further solvent. Similar differences between the atom sites in wet and dry crystals of the parent vitamin B12 have also been reported (Hodgkin et al 1962a, b; Shoemaker et al 1964).
For this molecule it is side chains e and g which occupy almost identical positions with side chains a, b, c and d exhibiting the flexibility. A similar displacement of the secondary amide loop f was also apparent.
References
Corfield P W R, Doedens R J and Ibers J A 1967 lnorg. Chem. 6 197
Hamilton W C and Ibers J A 1968 Hydrogen bonding in solids (New York: Benjamin) p. 16 Hawkinson S W, Coulter C L and Greaves M L 1970 Proc. R. Soc. (London) A318 143 Hodgkin D C, Lindsey J, Mackay M and Trueblood K N 1962 Proc. R. Soc. (London) A266 475 Hodgkin D C, Lindsey J, Sparks R A, Trueblood K N and White J G 1962 Proe. R. Soc. (London) A266 494 Hodgkin D C, Kamper J, Lindsey J, Mackay M, Pickworth J, Robertson J H, Shoemaker C B, White J G,
Prosen R J and Trueblood K N 1957 Proc. R. Soc. (London) A242 228
Hodgkin D C, Pickworth J, Robertson J H, Prosen R J, Sparks R A and Trueblood K N 1959 Proc. R. Soc.
(London) A251 306
Kopf J, yon Deuten K, Bieganowski R and Friedrich W 1981 Z. Naturforschung. C36 506 Lenhert P G 1968 Proc. R. Soc. (London) A303 45
Moore F H, Willis B T M and HodgkJn D C 1967 Nature (London) 214 130
Nockolds C E, Ramaseshan S, Waters J M, Waters T N M and Hodgkin D C 1967 Nature (London) 214 129 Shoemaker C B, Cruickshank D W J, Hodgkin D C, Kamper M J and Pilling D 1964 Proc. R. Soc. (London)
A278 1
Stoeckli-Evans H, Edmond E and Hodgkin D C 1972 J. Chem. Soc. Perkin Trans. 2 605
Venkatesan K, Dale D, Hodgkin D C, Nockolds C K, Moore F H and O'Connor B H 1971 Proc. R. Soc.
(London) A323 455
