Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1260-1264, March 1980 Biochemistry Molecular structure and intermolecular interactions of N1'-methoxycarbonylbiotin methyl ester: A model for carboxybiotin (ureido electronic configuration/evolution of a coenzyme/enzymatic carboxyl transfer/x-ray diffraction) WILLIAM C. STALLINGS*, C. T. MONTI*, M. DANIEL LANEt, AND GEORGE T. DETITTAt *Institute for Cancer Research, 7701 Burholme Avenue, Fox Chase, Philadelphia, Pennsylvania 19111; tDepartment of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and tMedical Foundation of Buffalo, Inc., 73 High Street, Buffalo, New York 14230 Communicated by David Harker, October 24, 1979

ABSTRACT The crystal structure of N1'-methoxycarbon- In the first half-reaction, enzyme-bound biotin is carboxylated ylbiotin methyl ester, a model for M'-carboxybiotin, has been at Ni', generating N1'-carboxybiotin (II). In the second half- determined. The ureido carbonyl bond has more double bond reaction, C02 is transferred, as a carboxyl group, from car- (keto) character than does.the corresponding bond in free biotin, which has single bond (enolate) character. In addition, there is boxybiotin to another molecule. Biotin enzymes are, in general, an interesting intermolecular interaction between the ureido high molecular weight, multisubunit structures; the half-re- carbonyl oxygen and a methyl group. Comrparison of the mo- actions generally occur at separate active sites which are located lecular structure and crystal packing with those of free biotin on different subunits of the enzymes. suggests. that the coenzyme may have evolved with the incor- Here, we report the molecular structure of N1'-methoxy- poration of the ureido moiety because the electronic configu- carbonylbiotin methyl ester (III), which is a model for Ni'- ration of this region of the molecule is sensitive to MN' carbox- carboxybiotin (II). Structural results were obtained by using ylation. On decarboxylation, the ureido carbonyl bond becomes more polarized (C-Os° thereby-facilitating the deprotonation the technique of single-crystal x-ray diffraction. Previous of NI' and increasing its nucleophilicity. As a result, carboxy- crystallographic studies have established the molecular struc- lation can occur readily. On carboxylation, the carbonyl bond tures of free biotin (I) (2), dethiobiotin (3), azabiotin (4), oxy- is depolarized (C==O), allowing the carboxylated coenzyme to biotin, selenobiotin, biotin methyl ester, biotin sulfone, and interact with nonpolar groups and carboxy ate them. Thus, the biotin-d-sulfoxide, and carbobiotin (5). Differences between carboxylation and decarboxylation of biotin appear to act as a the molecular geometries and therefore the electronic config- mechanistic switch, turning off and'on the polarization of the ureido ca'rbonyl bond as well as modulating the nucleophilicity urations of the ureido moieties of I and III are of great interest of NI'. because the ureido ring is the catalytically active portion of the biotin molecule. The previous studies have stressed the notion (+)-Biotin (I) is a coenzyme involved in carbon dioxide me- that, in free biotin and its'derivatives, the polarized forms of tabolism and carboxyl transfer. Structurally, the molecule the biotin resomers (those with 'formal charge separation) consists of a bicyclic ring system whose mirror symmetry is 16110 02'11 02 02' all /OCH3 >HN' HN3 1.NH HN3 1.N-COO HN3 1N-C 0 I S S iCH2A (CH2)4- COON COOH 0 OCH3 II III coax a b C broken by a valeric acid side chain; the top ring of the bicyclic system is a ureido ring and the bottom ring, containing sulfur, contribute significantly to their observed electronic structures. is a tetrahydrothiophene ring. Biotin is covalently attached to As will be pointed out, this is less the case for the carboxylated an enzyme by means of an amide linkage between the side derivative (III). In addition, we shall also detail some aspects chain carboxyl group of biotin and the E amino group of a lysine of the intermolecular interactions which occur in the crystal residue of the enzyme. structure and which have possible biochemical relevance. Biotin enzymes (1) (carboxylases, transcarboxylases, and decarboxylases) function by means of a two-step reaction se- quence: EXPERIMENTAL Nl'-Methoxycarbonylbiotin methyl'ester, C13H20N205S (III), Enz-biotin + donor-COy - Enz-biotin-CO- + donor was synthesized according to the procedure described by Enz-biotin-CO- + acceptor - Enz-biotin + acceptor- Knappe et al. (6); the crystals are clear, colorless needles. The dimensions of the crystal used were 0.35 X 0.07 X 0.08 mm. The CO2. space group is P212121 with a = 24.737(4), b = 26.997(4), and c = A with Z = there are two molecules in the The publication costs of this article were defrayed in part by page 4.6469(4) 8; charge payment. This article must therefore be hereby marked "ad- asymmetric unit. The calculated density is 1.35 g-cm-3. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Three-dimensional x-ray intensity data were collected on an this fact. automated diffractometer with the 6-20 scan technique and 1260 Downloaded by guest on September 25, 2021 Biochemistry: Stallings et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1261 Table 1. Positional parameters* for N1'-methoxycarbonylbiotin methyl ester Molecule A Molecule B Atom x y z x y z S 0.3661 0.0458 0.3300 0.2656 0.6212 0.4716 02' 0.5014 0.1660 0.183 0.4349 0.5519 0.675 Ni' 0.4206 0.1570 0.438 0.3620 0.5344 0.371 N1'-Methoxycarbonylbiotifn N3' 0.4846 0.1008 0.478 0.3977 0.6098 0.377 methyl ester C2' 0.4722 0.1437 0.346 0.4023 0.5647 0.497 C 0.3929 0.2007 0.385 0.3564 0.4837 0.391

0 0.3511 0.2094 0.509 0.3237 0.4620 0.247 O lOb 4 O' 0.4154 0.2289 0.194 0.3882 0.4633 0.587 C' 0.3893 0.2751 0.118 0.3813 0.4117 0.632 C2 0.4213 0.0315 0.576 0.3123 0.6520 0.230 C3 0.4443 0.0824 0.674 0.3553 0.6142 0.158

C4 0.3991 0.1225 0.648 0.3293 0.5616 0.161 1404 o IO& C5 0.3465 0.0977 0.559 0.2710 0.5661 0.252 C6 0.4622 -0.0043 0.444 0.3337 0.7010 0.351 C7 0.4365 -0.0558 0.388 0.2895 0.7394 0.391 C8 0.4741 -0.0916 0.234 0.3108 0.7878 0.500 C9 0.4460 -0.1398 0.185 0.2689 0.8264 0.545 Cdo 0.4748 -0.1755 -0.015 0.2914 0.8740 0.681 OlOa 0.5171 -0.1688 -0.127 0.3337 0.8735 0.804 OlOb 0.4470 -0.2174 -0.048 0.2651 0.9101 0.660 ClH 0.4694 -0.2546 -0.232 0.2859 0.9536 0.825 HN3' 0.521 0.088 0.46 0.421 0.634 0.36 HiC' 0.398 0.299 -0.11 0.412 0.400 0.71 H2C' 0.347 0.267 0.06 0.350 0.400 0.73 H3C' 0.377 0.289 0.25 0.390 0.395 0.48 HC2t 0.403 0.014 0.76 0.290 0.660 0.04

HC3t 0.459 0.080 0.89 0.373 0.622 -0.05 O l0b HC4t 0.394 0.140 0.86 0.332 0.545 -0.05 1207 HlC5t 0.325 0.084 0.75 0.246 0.571 0.06 o0 H2C5t 0.321 0.123 0.44 0.259 0.534 0.37 HlC6t 0.495 -0.009 0.60 0.364 0.715 0.21 Biotin H2C6t 0.478 0.010 0.25 0.352 0.693 0.56 FIG. 1. (Upper) Bond lengths determined for N1'-methoxycar- HlC7t 0.401 -0.050 0.26 0.260 0.725 0.55 bonylbiotin methyl eter. Values for molecule A are above those for H2C7t 0.425 -0.072 0.59 0.269 0.746 0.19 molecule B. The average estimated standard deviation is 0.009 A. HlC8t 0.510 -0.097 0.36 0.341 0.801 0.35 (Lower) Bond lengths determined for free biotin in a previous study H2C8t 0.484 -0.076 0.02 0.332 0.781 0.71 (2). HlC9t 0.405 -0.133 0.10 0.237 0.812 0.68 H2C9t 0.442 -0.158 0.40 0.252 0.836 0.34 at calculated positions and their positional and thermal pa- HlCll 0.446 -0.289 -0.19 0.249 0.969 0.84 rameters were not refined. The quantity minimized in the H2Cll 0.469 -0.240 -0.42 0.306 0.932 1.05 least-squares calculations was wf IIFoI - IFcI J2. The weights, H3Cll 0.501 -0.259 -0.21 0.322 0.961 0.77 w, were o-2(F), with zero weight for those reflections below the threshold value. Published atomic scattering factors (7, 8) * The estimated standard deviations for carbon, nitrogen, and oxygen were used in all calculations. The starting material in the syn- atoms average 3 X 10-4, 2 X 10-4, and 2 X 10-3 for x, y, and z, re- spectively. Equivalent values for the sulfur atoms are 1 X 10-4, 1 thesis of Nl'-methoxycarbonylbiotin methyl ester was X 10-4, and 4 X 10-4; for refined hydrogen atoms, these values av- biotin, whose absolute configuration has been established (9); erage 4 X 10-3, 3 X 10-3, and 2 X 10-2. the structure with correct absolute stereochemistry was refined t Parameters for this atom were not refined. by using anomalous scattering factors, listed by Cromer and graphite-monochromated CuKa radiation. Data for 352 in- Liberman (10), for the sulfur atoms. The final crystallographic dependent and nonsystematically absent reflections were col- residuals are R = 0.069 for observed data and weighted R = lected in the range sin0/A = 0-0.61 A-' (20 = 138°); 2446 of 0.079; the R for all data is 0.106. Final positional parameters these reflections had intensity I greater than the threshold value are listed in Table 1. Bond lengths are collected in Fig. 1. of 1.5 a(I) where a(I) was derived from counting statistics. Computer programs used were UCLALS (§), the Crysnet Values of o(F) were determined as a(F) = (F/2)jfu2(I)/12 + package (1I), and other programs written in the Institute for 6211/2 where 6 (0.065) is an instrumental uncertainty determined Cancer Research laboratory (1) experimentally. There is apparently some disorder in the methyl esterified The structure was determined by locating the sulfur atoms side chain of molecule B as evidenced by thermal motions from the Harker sections of the Patterson map and by using which, in the side chain of the molecule, are greater than those Patterson superposition and Fourier techniques. The structure for molecule A and which, especially in the cases of atoms CIO, was refined anisotropically. All hydrogen atoms were located in a difference Fourier synthesis but, because of the relatively § Gantzel, P. K., Sparks, R. A., Long, R. E. & Trueblood, K. N. (1969) low data-to-parameter ratio (5.6:1), only the positional pa- UCLALS4; Program in Fortran IV. rameters of the methyl and amino hydrogen atoms were re- I Carrell, H. L. (1975) ICRFMLS; modification of UCLALS4 and other fined, isotropically; the remaining hydrogen atoms were placed unpublished programs. Downloaded by guest on September 25, 2021 1262 Biochemistry: Stallings et at. Proc. Natl. Acad. Sci. USA 77 (1980) Or----b

FIG. 2. A view of the packing down the c axis. Molecules A are labeled; molecules B are not labeled. Hydrogen bonds are distinguished by dotted lines; the intermolecular close approach between 02' and the Cli methyl group is distinguished by a dashed line. The hydrogen bonding relationships are as follows: Donor Acceptor D...A Coordinates of acceptor N3' (A) 02' (B) 2.882 A 1 -x,-1/2 + y, 3/2 - z N3' (B) 02' (A) 2.934 A 1 - X, 1/2 + Y, 1/2- Z The position of the acceptor is related to its position in Table 1 by the equation given. The position of the donor is given in Table 1. A break (if) in the dotted lines (hydrogen bonds) indicates that the bond is to a molecule related to the one illustrated by a c axis translation. Hence, there is no complementary hydrogen bonding in this structure but rather a continuous spiralling of hydrogen-bonded molecules A and B up and down the c axis. OlOa, 01Ob, and ClI of molecule B (Fig. 1) are highly aniso- chain is fully extended in the all-trans planar zigzag confor- tropic; the root-mean-square amplitudes of vibration along the mation. longest axes of vibration of the thermal ellipsoids for these atoms Analysis of the molecular geometry in some related com- are, respectively, 0.48, 0.61, 0.40, and 0.49 A. This results in pounds brings to light the fact that there is a precedent for this irregular molecular geometry for the methoxycarbonyl group type of electronic rearrangement upon substitution at a urea- of the side chain of molecule B. In Fig. 1 note especially the like nitrogen atom by a trigonal carbon atom. A similar pattern differences in the C10-OlOa bond lengths in molecules A and of changes in lengths and, hence, in the electronic structures B; the value for molecule B is probably poorly defined because of the ureido C=O and C-N bonds of urea (13) and biuret of this disorder. (14) has been observed. Interestingly, in the case of monomethyl The Molecular Structure. The ureido rings of both molecules urea (15), where the nitrogen substituent is the tetrahedral and in the asymmetric unit are planar. The C2'=:02' bonds at 1.207 All and the C2'-Ni' bonds at 1.405 A are, respectively, shorter 0 0 and longer than the corresponding values determined for free | 22 320 Ill.- ,O7 C SPPCARJ BACH3 biotin (2) (Fig. 1). This indicates decreased contributions of the x 23S N "<9 N kfi\N polarized forms of the biotin resomers to the overall electronic I I I ~0 I structure of this Ni'-carboxybiotin derivative compared with H H H H I4N-\tylrH the contributions for free biotin.. This comparison of the car- Urea Biuret N-Methylu rea boxybiotin analogue with free biotin suggests that the ureido character electron-releasing methyl group (as opposed to the trigonal and carbonyl bond in biotin has enolate** (C-O0) electron-withdrawing carbomethoxy or carbamide group), the whereas the equivalent bond in carboxybiotin is keto (C=O) and in character. The C2'-N3' bonds, lengths 1.345 A, retain depolarization of the C=O bond is less severe neither The bond C-N bond appears to be affected by the substitution. considerable double-bond character. following The Crystal Structure. There are two unique hydrogen lengths are indicative of full single- and double-bond character This in C-N and C-0 bonds: C-N, 1.47 A; C=N, 1.25 A; C=0, bonds in the crystal structure as illustrated in Fig. 2. scheme 1.21 A; C-O, 1.43 A (12). of hydrogen bonding (N3'H of molecule A to 02' of molecule The Nl'-methoxycarbonyl group lies in the plane of the B and N3'H of molecule B to 02' of molecule A) results in a ureido ring with 02' of the ureido ring proximal to the methoxy helix of hydrogen-bonded molecules A and B along the c oxygen atom (Fig. 1). The bond between N1' and the meth- axis. oxycarbonyl group, 1.392 A, is nearly equal to that of the Ni'- The ureido carbonyl oxygen atom, 02', of molecule A is in- 1.405 A. The esterified valeric acid side volved in an interesting intermolecular interaction with the -C2' bond, methyl methyl group, C11, of the methyl esterified valeryl side chain molecule A. This inter- 11 Values reported in the text are average values determined for the two in a (symmetry-related) neighboring crystallographically independent molecules. action is shown in Fig. 2 and in greater detail in Fig. 3. The ** We refer to the enolate forms of biotin shown as biotin resomers b interaction is at 3.08A, considerably less than the sum of the and c above. oxygen-methyl van der Waals radii (16), 3.4 A, and its geom- Downloaded by guest on September 25, 2021 Biochemistry: Stallings et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1263 therefore a van der Waals interaction in which the ureido oxygen atom is nestled in the cavity between the hydrogen atoms of the neighboring methyl group. DISCUSSION The addition of the N1'-methoxycarbonyl group to biotin alters the electronic structure of the ureido moiety as evidenced by changes in the C2'=02' and C2'-N1' bond lengths. Past studies (2, 18) have shown that the ureido portions of some bi- otin derivatives are influenced by polarized resonance structures similar to those postulated for urea, with single-bond character in the carbonyl bond and double-bond character in the C-N bonds. The addition of the methoxycarbonyl group to Ni' of biotin diminishes the contribution of these resomers, restores full double-bond character to the carbonyl bond, and displaces much of the C2'-N1' double-bond character to the bond be- tween Ni' and the methoxycarbonyl group. Evidently the lone pair of electrons on Ni' is involved in bonding not only with C2' but also with the methoxycarbonyl group. This effect may contribute to the stability of carboxybiotin, a factor which is essential because the carboxyl-carrying species of biotin must be stable enough to survive translocation (5) between the active sites of the two half-reactions [a distance which, for the enzyme transcarboxylase, has been determined to be maximally 7 A FIG. 3. A view of the intermolecular interaction between 02' of (19)]. The C2'-N3' bond appears to be unaltered by the ad- molecule A and the C11 methyl group ofa symmetry-related molecule dition of the Nl'-methoxycarbonyl group. A. The symmetry relationship between these two molecules is as Of the possible mechanisms considered for second half- follows: reaction of biotin enzymes, the one that has received the most Atom Coordinates rela- attention is a concerted mechanism. In this step, the Ni'-car- tive to Table 1 is 02' (A) x, y, z boxyl group transferred from carboxybiotin to another mol- C11 (A) 1 - X, 1/2 + -1/2 - z ecule. In the concerted mechanism (Fig. 4), the formation of Y, the methylene-carboxyl bond is concomitant with the ab- The separations between 02' and the neighboring C11 methyl hy- straction of a methyl hydrogen atom. Several lines of evidence drogen atoms are approximately equidistant and average 2.93 A. support this mechanism (20-22). It has been suggested (21) that etry is such that 02' is approximately colinear with the the hydrogen-abstracting group is the ureido oxygen atom, 02', OlOa-C11 bond of the neighboring group. This colinearity of carboxybiotin; however, an alternative mechanism would would seem to preclude the possibility of that rare phenomenon envision the hydrogen abstraction to be by a basic residue of (17), the C-H-O hydrogen bond; the separations between 02' the enzyme. and the neighboring C1 methyl hydrogens are approximately The crystal structure reported in this study provides a model equidistant and average 2.93 A. The CiI methyl ester group for the interaction between carboxybiotin and a methyl group. of the neighboring molecule is coplanar with the N1'-meth- In this model (Fig. 3), the methyl carbon is coplanar with the oxycarbonyl ureido ring. The close approach of the methyl ureido ring and is 3.08 A from the ureido carbonyl oxygen (02'); group to the ureido carbonyl oxygen is not along the vector 02' is nestled among the hydrogen atoms of the neighboring defined by the C2'=C02' bond, and therefore it is asymmetric methyl group. The interaction is an asymmetric one and occurs with respect to the ureido ring, occurring on the N1'-methoxy- from the Ni'-methoxycarbonyl side of the molecule. The model carbonyl side of the molecule (Fig. 3). The interaction is does not immediately suggest a geometry for the actual tran- 0 H\H /C COO Enz-B: C- ? t PyrLivate 10)? HO 11 : I C Oxaloacetate HN N 0 FIG. 4. The concerted reaction - c + c"I 0 mechanism ofthe second half-reaction H2C of the enzyme, transcarboxylase. In this mechanism, hydrogen abstraction C-.O from the methyl group of pyruvate is concomitant with the carboxyl trans- 0/ fer to that methyl group, thereby generating oxaloacetate. It is unclear whether the hydrogen-abstracting group is the ureido oxygen, 02', of carboxybiotin, or a basic residue of the Enz enzyme. Downloaded by guest on September 25, 2021 1264 Biochemistry: Stallings et al. Proc. Natl. Acad. Sci. USA 77 (1980)

sition state of the carboxyltransfer reaction but rather suggests 4. Glick, M. D., Wormser, H. C. & Abramson, H. N. (1977) Acta one for the incipient biotin-substrate interaction; thus, the Crystallogr. Sect. B 32, 1095-1 101. model may lie somewhere on the pathway along the reaction 5. DeTitta, G. T., Parthasarathy, R., Blessing, R. H. & Stallings, W. coordinate. This indicates that a possible role for the depolarized C. (1980) Proc. Natl. Acad. Sci. USA 77,333-337. electronic configuration, observed in this structure, may be to 6. Knappe, J., Ringelmann, E. & Lynen, F. (1961) Biochem. Z. 335, facilitate the asymmetric approach between carboxybiotin and 168. a methyl group. Biochemically, this is an important interaction 7. (1962) International Tables for X-Ray Crystallography (Kynoch, Birmingham, England), Vol. 3, pp. 202-207. because a methyl group is one species with which the N1'- 8. Stewart, R. F., Davidson, E. R. & Simpson, W. T. (1965) J. Chem. carboxyl group of carboxybiotin reacts. Phys. 42,3175-3187. The results of this study suggest one reason for the bio- 9. Trotter, J. & Hamilton, J. A. (1966) Biochemistry 5, 713-714. chemical evolution of this coenzyme. Perhaps the ureido moiety 10. Cromer, D. T. & Liberman, D. (1970) J. Chem. Phys. 53, was incorporated into the coenzyme because its electronic 1891-1898. structure is sensitive to carboxylation at the N1' position. Hence, 11. Bernstein, H. J., Andrews, L. C., Berman, H. M., Bernstein, F. on decarboxylation, the ureido carbonyl bond becomes more C., Campbell, G. H., Carrell, H. L., Chiang, H. B., Hamilton, W. polarized (C-0-), thereby facilitating the deprotonation of C., Jones, D. D., Klunk, D., Koetzle, T. F., Meyer, E. F., Mori- N1' and therefore increasing its nucleophilicity. As a result, the moto, C. N., Sevian, S. S., Stodola, R. K., Strongson, M. M. & coenzyme can interact with polar species such as bicarbonate Willoughby, T. V. (1974) in Second Annual AEC Scientific Computer Information Exchange Meetings, Proceedings of the or carboxyl groups, and carboxylation can readily occur. Technical Program, Report BNL 18803 (Brookhaven National Likewise, on carboxylation, the carbonyl bond is depolarized Laboratory, Upton, NY), pp. 148-158. (C 0), allowing the carboxylated coenzyme to interact with 12. Pauling, L. (1960) The Nature of the Chemical Bond (Cornell nonpolar species such as methyl or methylene groups and car- Univ. Press, Ithaca, NY), pp. 228-229. boxylate them. Thus, the carboxylation and decarboxylation 13. Caron, A. & Donohue, J. (1969) Acta Crystallogr. Sect. B 25, of biotin seem to function as a kind of mechanistic switch, 404. turning off and on the polarization of the ureido carbonyl bond 14. Craven, B. M. (1973) Acta Crystallogr. Sect. B 29, 1525-1528. as well as modulating the nucleophilicity of Ni'. 15. Huiszoon, C. & Tiemessen, G. W. H. (1976) Acta Crystallogr. Sect. B 32, 1604-1606. We thank Drs. Jenny Glusker, Albert Mildvan, Jerry Donohue, and 16. Pauling, L. (1960) The Nature of the Chemical Bond (Cornell Robert Blessing for discussions, comments, and criticisms. This research Univ. Press, Ithaca, NY), p. 260. was supported by U.S. Public Health Service Grants CA-05322, CA- 17. Donohue, J. (1968) in Structural Chemistry and Molecular 10925, CA-06927, CA-22780, AM-14575, AM-19856, and RR-05539, Biology, eds. Rich, A. & Davidson, N. (Freeman, San Francisco), by American Cancer Society Grant BC-242, by National Science pp. 443-463. Foundation Grant AG-370, and by an appropriation from the Com- 18. Stallings, W. C. (1977) Arch. Biochem. Biophys. 183, 189- monwealth of Pennsylvania. 199. 19. Fung, C. H., Gupta, R. K. & Mildvan, A. S. (1976) Biochemistry 1. Moss, J. & Lane, M. D. (1971) Adv. Enzymol. Relat. Areas Mol. 15, 85-92. Biol. 35, 321-442. 20. Retey, S. & Lynen, F. (1965) Biochem. Z. 342,256-271. 2. DeTitta, G. T., Edmonds, J. W., Stallings, W. & Donohue, J. 21. Rose, I. A., O'Connell, E. L. & Solomon, F. (1976) J. Biol. Chem. (1976) J. Am. Chem. Soc. 98, 1920-1926. 251, 902-904. 3. Chen, C. S., Parthasarathy, R. & DeTitta, G. T. (1976) J. Am. 22. Mildvan, A. S., Scrutton, M. C. & Utter, M. F. (1966) J. Biol. Chem. Soc. 98,4983-4990. Chem. 241, 3488-3498. Downloaded by guest on September 25, 2021