12238
J. Am. Chem. SOC.1993,115, 12238-12250
Conformational Analyses of Thiamin-Related Compounds. A Stereochemical Model for Thiamin Catalysis Whanchul Shin,’ DongGweon Oh, Chong-Hak Chae, and Tae-Sung Yoon Contribution from the Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul I51 - 742, Korea Received June 7, 1993”
Abstract: Conformational analyses of some thiamin-related compounds have been performed in order to find the relationship between their conformational and biochemical properties. Relaxed 2-D potential energy maps of free thiamin, its antagonists, and C(2) adducts were obtained using the molecular mechanics (MM) method. The antagonists include 4’-deaminothiamin, oxythiamin, pyrithiamin, thiamin thiazolone, thiamin thiothiazolone, 6’-methyl-4H-thiamin and 6’-methylthiamin, and the C(2) adducts include 2-(a-hydroxyethyl)thiamin(HET), 2-(a-lactyl)thiamin (LT), 6’-methyl-4H-lactylthiamin and 6’-methyllactylthiamin. All of the local minima conformers were also identified for the active intermediates LT and HET. In numerous crystal structures, free thiamin assumes mostly the F form and less frequently the S form. The C(2) adducts assume only the S form. However, neither thiamin nor its active intermediates are found in the V form in crystal, but the cofactor assumes the V form in the active site of the protein. The MM map of free thiamin shows that the F form is truly the global minimum, while the S form occupies another minimum slightly higher in energy and lower in existence probability than the F form. The V form is also a local minimum but with very low existence probability. In addition, the availability of only one 4’-amino H atom for the intermolecular hydrogen bond makes it very unlikely that the thiamin molecule assumes the V form in solution and thereby in crystals. For various antagonists, either the V form is the global minimum or its existence probability is higher than that of thiamin. The V form instead of the crystalline S form is the global minimum conformer for both LT and HET. However, the V form of the C(2) adducts also would not be observed in the crystalline state due to the conformational characteristics of free thiamin; their V forms accordingly may be unique conformers available only inside the protein but not in solution. Based on the results of MM calculations and the crystal structures of holoenzymes, it is proposed that the active conformers of both intermediates are V forms with an intramolecular N-H-mO(hydroxy1) hydrogen bond. A putative stereochemical model for thiamin catalysis is presented in which N-Ha-0 hydrogen bonds contribute to the acceleration of the enzymic reaction by lowering the energies of the various species occurring along the reaction path. The principles of least motion and maximum orbital overlap which were originally applied to the decarboxylation reaction for the intermediates in the S form still hold for the V form.
Scheme I
Introduction Thiamin (vitamin B1; I), in the form of the diphosphate ester (TDP), is a coenzyme in a number of enzyme systems that catalyze decarboxylation of a-keto acids and the transfer of aldehyde or acyl groups.’ Since Breslow* clarified the essential features of thiamin catalysis (Scheme I) 35 years ago, many details have been clarified from studies of both model and enzyme systems, but some important questions remain to be answered. One of the controversial points, that has been clarified very recently, was the active conformation of thiamin, which in turn is directly related to the role of the essential 4’-amino group in the enzymic catalysis. From extensive biochemical studies employing various analogues of thiamin, Schellenberger concluded that the 4’-amino group actively participates in the catalytic reaction acting as an intramolecular acid and base alternatively and thus thiamin and its C(2) adducts should be in the V form in which the 4’-amino group is close to the C(2) active center.3 Sable et al. also proposed the stable V form of free thiamin from N M R studies on the model c o m p o ~ n d s .However, ~ the V form has never been found
* To whom correspondence should be addressed. *Abstract published in Aduance ACS Abstracts, December 1, 1993. (1) For leading references, see: (a) Krampitz, L. 0.Annu. Reu. Biochem. 1%9,38,213-240. (b) Gallo, A.; Mieyal, J. J.; Sable, H. Z.Bioorg. Chem. 1978, 4, 147-177. (c) Kluger, R. Chem. Reu. 1987, 87, 863-876. (2) Breslow, R. J. J . Am. Chem. SOC.1958,80, 3719-3726. ( 3 ) (a) Schellenberger, A. Angew. Chem., Int. Ed. Engl. 1967,6, 10241035. (b) Schellenberger, A. Ann. N . Y. Acad. Sci. 1982, 378, 51-62. (c) Schellenberger, A. Chem. Ber. 1990,123, 1489-1494. (d) Golbic, R.; Neef, H.;HGbner, G.; Kdnig, S.; Seliger, B.; Meshalkina, L.; Kochetov, G. A.; Schellenberner. A. BioorP. Chem. 1991. 19. 10-17. (4) BiagGw; J. E.; MLyal, J. J.; Suchy,’J.; Sable, H. Z.J. Biol. Chem. 1969,244, 40544062.
CH3 H,?0 -C-HI
CHgCHO
t
in any of the crystal structures of free thiamin or of its C(2) adducts, making the activeconformation of thiamin a controversial subject. Very recently, the crystal structures of transketolase,s pyruvate oxidase,6 and pyruvate decarboxylase (PDC)’ that require TDP as a cofactor have been reported at 2.5,2.1, and 2.8 8, resolutions, respectively. They vividly show that the active conformation of thiamin is the V form. A protein structure (.5 ,) (a) . , Lindavist. Y.: Schneider. G.: Ermler. U.:Sundstrdm. M. EMEO J . 1992, 11, 2373-2379.’ (b) Lindqvist,’Y.;Schneider, G. FEES Lett. 1992, 313.229-231. (6) Muller, Y. A.; Schulz, G. E. Science 1993, 259, 965-967. (7) Dyda, F.; Furey, W.;Swaminathan, S.; Sax, M.; Farrenkopf, B.;Jordan, F. Biochemistry 1993, 32, 6165-6170.
OOO2-7863/93/ 1515-12238$04.00/0 0 1993 American Chemical Society
A Stereochemical Model of Thiamin Catalysis containing an active intermediate such as 2-(a-lactyl)thiamin [lactylthiamin, LT; 1111 or 2-(a-hydroxyethyl)thiamin (HET; VI) has not been determined yet. However, the protein structures strongly suggest that the 4’-amino group somehow plays a direct role through some kinds of intramolecular interactions during the catalysis as suggested by Schellenberger. In numerous crystal structures, free thiamin assumes mostly the F conformation and with minor exceptions the S conformation despite the apparent rotational degree of freedom about the bridging methylene joining the two aromatic rings.8 Furthermore, all C(2)-substituted thiamins assume only the S conformation although the conformationsof the substituents are variable. They include HET? 2-(a-hydroxybenzyl)thiamin10and its Hg complex,” 2-(a-hydro~ybenzyl)oxythiamin,~~ and phosphalactylthiamin (PLT).” In both the F and S forms, the 4’-amino group is far from the C(2) active center and thus cannot be directly involved in intramolecular acid-base catalysis if one of these is the active form. In the crystal structures of thiamin-related compounds, the V conformation has been observed only in three cases, namely, oxythiamin,14 thiamin thiazolone (TT),15 and 4’deaminothiamin analogue@ all of which are antagonists of thiamin. The crystal structure of TT which contains an intramolecular N-He-0 hydrogen bond in the V form has remained as the only example that remotely resembles the Schellenberger’s active V model. However, thiamin thiothiazolone (TTT),” a structural and functional congener of TT, assumes the S conformation, exemplifying a notion15 that the crystal conformation of a thiamin compound is a result of an intricate balance of intramolecular interactions. An important question remaining to be answered is why the active V form has virtually never been found for free thiamin or for the active intermediates in the crystalline state. Despite prolonged controversy over the conformational properties of thiamin-related compounds, extensive theoretical studies have not been reported yet. Conformational energy maps of thiamin and HET were obtained in terms of torsion angles of C#IT and C#Ipbut without geometry optimization.ls In particular, the calculation for HET was performed for a fixed conformer of the C(2)-substituent so that it does not represent the available conformational surface adequately.lSb N o conformational studies have been done on the antagonists of thiamin although their biochemical behavior provided the basis of some important mechanistic proposals. In this work we have calculated relaxed 2-D conformational potential energy maps for free thiamin and (8) The conformation of the thiamin molecule is best expressed in terms of the two torsion angles, & and Qp, about the bonds from the methylene bridge carbon to the thiazolium and pyrimidinerings, respectively. The torsion angles & = C(S‘)-C(3,5‘)-N(3)-C(2) and Q p = N(3)-C(S,S’)-C(S’)-C(4’). The F, S,and V conformations have been specified by (& = Oo, Q p = 190°), (ha&loo0,Qp 3 tl5Oo), and (& = f90°, Q p = r90°), respectively. For more details, see: footnote 13 in ref 10. Their locations in the (&, Qp) map are specified in Figure 3a. (9) Sax, M.; Pulsinelli, P.; Pletcher, J. J . Am. Chem. SOC.1974,96, 155165. (10) Pletcher, J.; Sax, M.; Blank, G.; Wood, M. J . Am. Chem. SOC.1977, 99, 1396-1403. (1 1) Louloudi, M.; Hadjiliadis, N.; Feng, J. A.; Sukumar, S.;Bau, R. J . Am. Chem. SOC.1990, 112, 7233-7238. (12) Shin, W.; Pletcher, J.; Sax, M. J . Am. Chem. SOC.1979,101,43654371. (13) Turano, A.; Furey, W.; Pletcher, J.; Sax, M.; Pike, D.; Kluger, R. J . Am. Chem. SOC.1982, 104, 3089-3095. (14) (a) Shin, W.; Pletcher, J.; Sax, M.; Blank, G. J . Am. Chem. SOC. 1979,101,2462-2469. (b) Shin, W.; Pletcher, J.; Sax, M. Acta Crystallogr., Sect. B 1981, 37, 1719-1724. (15) Shin, W.; Kim, Y. C. J . Am. Chem. SOC.1986,108, 7078-7082. (16) (a) Shin, W.; Chae, C. H. Acra Crystallogr., Secr. C 1993,49,68-70. (b) Power, L.; Pletcher, J.; Sax, M. Acra Crystallogr., Sect. B 1970, 26, 143-148. (17) Shin, W.; Lim, B. C. Acta Crystallogr., Sect. C, in press. (18) (a) Jordan, F. J . Am. Chem. SOC.1974,96,3623-3630. Energy map replotted in ref 14a. (b) Jordan, F. J . Am. Chem. SOC.1976, 98, 808-813. Energy map replotted in ref 12. (c) Friedemann, R.; Richter, D.; Griindler, W. In Thiamin Pyrophosphate Biochemistry; Schellenberger, A., Schowen, R. L., Eds.; CRC Press: Boca Raton, FL, 1988; Vol. I, pp 11-22.
J . Am. Chem. SOC.,Vol. 115, No. 26, 1993
12239
Chart I
R
1
10
2
o;.-.*;o
6 7
8 9
11 CH2CH2CHJ
R
Mi.
R1
R2
R1
H H
H CH3 H CH?
CHI
R
CHI CH] CH]
4 0 5 s
lactyl
H
its biologically important antagonists and C(2) adducts using molecular mechanics (MM). The antagonists include 4’deaminothiamin (l),oxythiamin (2), pyrithiamin (3), TT (4), TTT (3, 6’-methyl-4H-thiamin (6),and 6’-methylthiamin (7). The C(2) adducts include HET, LT, 6’-methyl-4H-lactylthiamin (8), and 6’-methyllactylthiamin ( 9 ) . All of the local minima conformers of HET and LT were also identified. Although the diphosphate esters of the above compounds are biologically active, we have disregarded thediphosphate ester moiety in the calculation since it does not affect the conformational properties of the thiamin moiety and the C(2)-substituent. In the present study we find the reason why the V form has not been observed in crystal structures of free thiamin and C(2) adducts. In addition we correlate the conformational and biochemical properties of the various analogues. We also propose a possible stereochemical course of thiamin catalysis. Computational Methods In order to obtain the fully minimized conformational energy maps, we used an in-house MM program which has several salient features.19 The various potential energy functions and the parameters were taken from Allinger’s MM2(85)20with a couple of modifications. We included the charge/charge, instead of the dipole/dipole, interaction energy and explicit hydrogen-bonding interaction energy terms. The functional form of the charge interaction energy is qiqj/erij. Partial atomic charges were derived by application of the Mulliken population analysis routine in AMl.*l The distance-dependent dielectric constant of e = 4rij (rij in A) was used in this study. The hydrogen-bonding interaction energies were calculated with the Buckingham function which was modulated with angle factor terms takenfromYETLZ2 eo (well depth) andro (equilibrium distance) parameters for hydrogen-bonding energy were set at -3.483 kcal/mol and 1.877 A for N-H-0 and -2.50 kcal/mol and 2.30 A for N-H-S, r e s p e c t i ~ e l y .A~ ~switching function was not used because of themathematicalcomplexity in thederivationof thederivatives. Instead, the H.-O (or S) interaction was treated as a normal van der Waals interaction, when the distance between the H and the acceptor atoms exceeded 1.5ro. X-ray bond distances and angles involving the nonhydrogen atoms were used as 10 of the bond stretching and 00 of the angle bending terms. Those involving the hydrogen atoms were the normalized values. In our program, energy minimization is done using the conjugate gradient minimizer in the internal coordinate system rather than the ~~~
~
(19) Shin, W.; Oh, D. G., manuscrip’t in preparation. (b)Allinger, (20) (a)Allinger,N.L.Adu.Phys.Org.Chem.1976,13,1-17. N. L.; Kok, R. A,; Imam, M. R. J . Comput. Chem. 1988, 9, 591-595. (21) (a) Dewar, M. J. S.;Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J . Am. Chem. SOC.1985, 107,3902-3909. (b) Dewar, M. J. S.;Yuan, Y. Inorg. Chem. 1990, 29, 3881-3890. (22) (a) Vedani, A.; Dunitz, J. D. J . Am. Chem. SOC.1985, 107,76537658. (b) Vedani, A.; Dobler, M.; Dunitz, J. D. J . Compur. Ckem. 1986, 7 , 701-7 10.
Shin et al.
12240 J. Am. Chem. SOC.,Vol. 115, No. 26, 1993 orthogonal system. It is possible to fix any structural variable during minimization in this scheme, and thus the problems that are associated with the dihedral driver scheme originally implemented in MM2 can be av0ided.2~ The MM calculations of the conformational potential energy maps of thiamin and its antagonists were performed on 5 O grids of (&, $p) with full geometry optimization (except for the fixed torsion angles of @T and bp). Contouring was done with an in-house program. The 2D-energy maps of C(2)-substituted thiamins and analogues were obtained by the newly developed iterative four-way scanning method incorporated in our MM program. Individual local minima conformers of HET and LT were found by application of a systematic grid search method in which a unique minimization scheme is employed in the search process for computational efficiency. First, each structure that is generated varying the rotatable bonds is minimized with respect only to the torsion angles associated with the rotatable bonds, and the local minima unique in terms of the torsion angles are saved. Then, the structures are subject to further minimization of the bond angles and the torsion angles associated with the rotatable bonds and screened again. Finally, the local minima are identified from a comparison of the fully minimized structures. A conformer is defined as unique when its torsion angles show an rms difference larger than a certain value ( l 5 O in the present study) from those of the other conformers. This procedure gives better results than the grid search method with fixed geometry while keeping the computation feasible even on the fast PC. We could even distinguish the conformers that differed only in the orientation of the H atom of the hydroxyl group. All calculations were performed on either a PC 486 or an IBM RS/6000 computer at the Seoul National University Computer Center.
Analyses of Crystal Structures of Thiamin Conformation. In order to investigate the structural and conformational properties of thiamin in detail, we first made detailed analyses of the crystallographic data. Previously, Shin et al. had tabulated the occurrence of 17 thiamin-related compounds in various conformations.25 Since then, the number of thecrystal structures has almost quadrupled (62 crystal structures in 57 independent determinations), and several more cases of free thiamin occurring in the S form have been reported. It seemed timely to make an updated list (Table I) which could be informative for discussing the conformational properties of thiamin-related compounds. The compounds have been classified in terms of the chemical variations on various sites of thiamin as well as their conformations. For free thiamin, the F form is overwhelmingly preferable to the S form (43 versus 8 cases). The average 4~ and q5p angles for the F form are -4 f 7O and -83 f 6O, respectively. The first time that free thiamin was found in the S conformation, it was suggested that the F form is an artifact of crystal packing forces.26 Since then, eight cases of the S form have been reported. In most of them the thiamin molecule is associated with a polyhalogenicmetal anion except for the picrate complex. Actually, however, only four of the cases are unique since two salts (CdC14 and CoCl4) and four metal (Zn,Co, Cd) complexes represent two isomorphous structures, as can be seen in the very similar torsion angles and the same space groups. The average 4~ and (23) In general, the parameters for N-He-0 are rather well-defined and our present MM calculation with the above parameters gave the results consistentwiththecrystal structureandthe AMI results. However,parameters for the N-H.4 hydrogen bond have not been studied in detail. The €0 and ro values in YETI are -1.455 kcal/mol and 2.667 A, respectively, but those are for the thiol or thioether S atom and may not be suitable for TTT with the thioketo S atom. Therefore, we tentatively used the values of eo = -2.50 kcal/mol and ro 2.30 A to provide the most favorable condition for the formation of an intramolecular hydrogen bond. These values are based on the shortest H-.S distance of 2.27A observed in the crystal structures of thio derivatives of the nucleic acid components which have the intermolecular N-H.4 hydrogen bond (Saenger, V. W.; Suck, D. Acta Crystallogr., Sect. B 1971,27,1178-I186) and the hydrogen-bondingenergy of -2.50kcal/mol predicted for the CHONHrH2CS system (Kollman, P.; McKelvey, J.; Johansson, A.; Rothenberg, S. J . Am. Chem. SOC.1975, 97,955-965). (24)Burkert, U.;Allinger, N. L. J. Comput. Chem. 1982,3, 40-46. Pletcher, J.; Blank, G.; Sax, M. J. Am. Chem. SOC.1977, (25)Shin, W.; 99,‘3491-3499. (26)Richardson, M. F.; Franklin, K.; Thompson, D. M. J . Am. Chem. SOC.1975,97. 3204-3209.
4 p angles for this conformer of thiamin are -1 12 f 1 and -1 34 f 4’, respectively. These angles are significantly different from those of the S form of C(2)-substituted thiamin in which the averagehand4panglesare-95 f 7’ and 175 f 7’,respectively. Since 4p differs by -50’ and since there are other structural differences, notably in the bond angles, it seems better to distinguish these two conformations. We tentatively designated the conformation of free thiamin as S’. Thiamin-HgC14-H2027 and HET9 which assume the S and S’forms, respectively, are exceptional cases. In the S’ form, H(6’) is directed toward C(2), while it is right on top of N(3) in the S form. The crystallographic data suggest that chemical perturbations at the C(2) or the C(4’) position affect the conformation of free thiamin but perturbations at N(1’) or O(57) do not. In fact, the torsion angles [S(l)C( 5)-C( 5a)-C( sa)] and dss[C(5)-C( 5a)-C( 5&0(57)] involving the 5-hydroxyethyl side chain vary widely among the various crystal structures. Therefore, we can assume that the major conformational properties of TDP are identical to those of thiamin as far as the torsion angles ( b and ~ 4 p are concerned. As is evident in Table I, thiamin can be crystallized with a variety of counter ions or complexing agents and in different spacegroups, indicating that the crystal packing modes around the thiamin molecule are quite different. Therefore, these data are statistically convincing evidence that the preferred conformations (major F and minor S) of free thiamin represent an intrinsicconformational property of the molecule rather than an artifact of crystal packing forces. MM studies presented below prove that this interpretation is correct. They also show that the most stable conformation of C(2)-substituted thiamins is the V form and that their crystalline S conformations are the combined result of the conformational property of thiamin in solution, crystallization processes, and crystal packing forces, as shall be discussed later. Molecular Dimensions. It has been observed that the molecular dimensions of the thiamin molecule show some systematic variations that are associated with the chemical and conformational changes. Cramer et al. have observed that bond lengths and endocyclic bond angles of the pyrimidine ring moiety are subject to the protonation state of the ring.28 They also observed that the two exocyclic angles around N(3) are sensitive to the molecular conformation, that is, C(2)-N(3)-C(35’) is 1-3’ smaller than C(4)-N(3)-C(35’) for free thiamin in the S conformation, while the opposite trend is true for the F form.28 In addition, they concluded that for the angles around C(5’) there are no obvious systematic variations depending on conformation. However, we have found that there are indeed systematic variations in the exocyclic angles around C(5’) depending only on the conformation, that is, the average value of C(4’)-C(5’)-C(35’) is smaller by 3.5” than that of C(6’)C(5’)-C(35’) for C(2)-substituted thiamin in the S form but larger by 2.7’ for free thiamin in both F and S‘ form. The average molecular dimensions of thiamin which show significant differences depending on the protonation state and conformational classes are shown in Figure 1.
Atomic Charges of Thiamin AM 1 calculations for all compounds studied were performed mainly for obtaining the atomic charges for use in the MM energy calculations. The net atomic charges for protonated and unprotonated thiamins are shown in Figure 2. It has been found that the atomic charges in the thiazolium ring show different values depending upon the methods of cal~ulation.l*a~~-~~ Although the thiazolium ring is commonly drawn with a formal positive charge on N(3), the AM1 results show that S(1) carries nearly unit positive charge, while C(2) and C(4) carry considerable (27)Hadjiliadis, N.; Yannopoulos, A.; Bau, R. Inorg. Chim. Acta 1983, 69, 109-115. (28)Cramer, R.E.;Maynard, R. B.; Ibers, J. A. J. Am. Chem. SOC.1981, 103, 16-8 1.
A Stereochemical Model of Thiamin Catalysis
J . Am. Chem. Soc., Vol. 115, No. 26, 1993 12241
Table I. Torsion Angles (deg) and Space Groups in the Crystal Structures of Thiamin and Its Analogues’ form
6T
6P -85.3 -76.8 -76.0 -76.1 -77.1 -76.6 -82.0 -81.0 -83.1 -77.5 -83.2 -81.5 -82.4
(l’-methylT)I2 Cu(T)C12 Cu(T)Br2 Pt(T)ClyH20
(phosphalactylHT)C1.3H20 [2-(a-hydroxyethyl)HT]C12 [2-(a-hydroxybenzyl)HT]Clr3H20
SPgP
form
F Form (1) Free Thiamin Salt Pilja P~I/c
C~/C P21/~
C~/C P21/a P21/~ P21/n P1 P2l/n P21/n P21/n
(2) Free Thiamin Substituted at or Coordinated to N(1’) . . -3.1 -83.7 P21 Zn(T)(SCN)3 10.1 [Mn(T)C12.H20].(HT)ClrH20 -83.7 Pi -82.1 Pi 12.0 -5.3 Pi [Rh(T-PO3H)(acetato)2].0.75H20 -70.0 (3) Free-Thiamin Substituted at O(57) -6.6 -85.4 P1 (HT-P206H)*4.5H20 -9.0 -78.8 Pi [(HT-P206H2)Cu(H20)(phen)] (N03)2*H20 -93.1 P z ~ / c (HT-indole-3-propionate)(C104)2CH3OH -2.7 -5.3 -85.8 P1 catena-((T)-Cd(SCN),) -20.4 -100.5
-99.2 -100.3 -92.7 -103.0 -1 10.4 -112.6 -113.4
S Form (1) C(2)-Substituted (Oxy)thiamin, S -173.6 P21/c Hg(2-(ar-hydroxybenzyl)T)Cl~-H20 -145.6 P1 [2-(a-hydroxybenzyl)oxyHT]C1~3H20 167.2 C2/c (T thiothiazolone)J/2CH3COCH3 (2) Free Thiamin Salt, S’ 179.0 P 2 1 / ~ ( H T ) ( C O C ~ ~ ) . H ~ O -137.3 P21/n (T)(picrate) (3) Free Thiamin Coordinated to N(l’), S’ -129.8 C ~ / C Co(T)Cly0.4H20 -130.4 C2/c Zn(T)Brs-O.ZH20
(o~yHT)ClyH20(A) (B)
105.5 101.5
V Form (1) Oxythiamin (Substituted at C(4’)) -62.9 P21/n (oxyT)Clz.HzO -64.2
(T thiazolone)
104.1
-74.2
0
6T
6P
-0.5 -8.9 5.8 -4.6 -7.3 -1.3 -5.9 -20.9 -2.0 -14.1 -9.0 0.2 5.4
-83.2 -87.2 -86.6 -87.7 -90.9 -76.2 -82.4 -79.0 -77.4 -82.6 -76.7 -85.8 -83.7
-10.2 -10.0 -3.6 -3.3
-82.8 -85.6 -79.6 -80.7
-8.5 1.0 4.4 0.2
-92.4 -95.0 -75.9 -80.1
-100.4 -92.8 -82.9
172.7 165.9 176.1
SPgP
P21/n P2lla
G/c
P1
G/c P1 P212121
P1 P21/C
P1
PI P21/n Pll/a P1 Pi Pi Pzl/n
P! P1
P21/n P21/~ P2l/c
-111.6 -99.3
-135.5 -122.9
Pll/n P1
-111.8 -113.5
-128.9 -130.5
C~/C C~/C
103.4
-64.6
Pi
(2) C(2)-SubstitutedThiamin P21/n
T represents unprotonated thiamin and HT represents thiamin protonated on N(1’).
negative and N ( 3 ) slightly negative (nearly neutral) charges. W e also performed an MND030 calculation which shows positive (0.404) and negative (-0.187) charges for S(1) and N(3), respectively. These results are consistent with an a b initio computation for the 2-(cu-hydroxyethyl)-3-methylthiazoliumion in which S(1) has positive (0.459) and N ( 3 ) negative (-0.187) charges.1sb T h e positive nature of the S atom in the thiazolium moiety has been implicit in numerous crystal structures in which S(1) is nearly always involved in electrostatic interaction(s) with a negative atom or ion(s).Q I t has been noted that resonance structures in which the positive charge resides on S ( l ) but N ( 3 ) is neutral can be drawn for both the thiazolium ring and the ylide (II).Ib,@a Rigorous studies seem to be required to elaborate the electronic structure of thiamin. The bond lengths obtained in AM1 calculations show maximum differences of 0.05 8, [ N ( 1’)C(2‘) in protonated thiamin] and 0.06 A [C(4’)-N(4’cu) in unprotonated thiamin] from the average dimensions observed in the crystal structures. (29) (a) Haake, P.; Nelson, R. M. Ann. N . Y. Acad. Sci. 1982,378, 107115. (b) Aldrich, H. S.; Alworth, W. L.; Clement, N. R. J . Am. Chem. SOC. 1978, 100, 2362-2366. (c) Scheffers-Sap, M. M. E.; Buck, H. M. J . Am. Chem. SOC.1979, 101,4807-4811. (30) Dewar, M. J. S.;Thiel, W. J . Am. Chem. SOC.1977, 99,4899-4917.
Conformational Potential Energy Surfaces Thiamin. The MM conformational potential energy m a p of thiamin is presented in Figure 3a. There are substantial differences between the present relaxed m a p and Jordan’s rigid map.lSa T h e F, S, and V regions are energetically similar, while the conformational space for each region is different. When the crystal conformations of free thiamin are superimposed on the map, there are two clusters roughly centered a t the F and S’ regions with the single exception of thiamiwHgClcH20 which is situated in the S region. Interconversions of the F and S forms can occur through a concerted rotation of the two torsion angles in two directions, but there is virtually no energy barrier in one of the directions. T h e small V region is isolated from the others by a n energy barrier higher than 2 kcal/mol. I t has been suggested that there is an attractive interaction between the acidic H ( 2 ) and the ?r electrons in the pyrimidine ring.3’ This kind of interaction can be considered to be a hydrogen bond in which the aromatic ring acts as the acceptor, and its energy is estimated to be ca. -1 kcal/mo1.32 Therefore, the energy of the F form may actually be lower than the MM value since in the present (31) Turano, A.; Pletcher, J.; Furey, W.; Sax, M. Ann. N . Y. Acad. Sci. 1982, 378, 91-106. (32) Levitt, M.; Perutz, M. F. J . Mol. Biol. 1988, 201, 751-754.
Shin et al.
12242 J . Am. Chem. Soc., Vol. 115, No. 26, 1993
(4
N4’a
I
I 1 3378
a1 352
1
7
1319
~ 1 4 1 j 4a ,C3\ ’
~
1499 I 490 1311 331 I493
c2
ly
y
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Average (a) bond lengths (A) and (b) bond angles (deg) in thiamin showing variations depending on the protonation state and the conformation. “top) unprotonated N( 1’) plus metal-coordinatedN( l’), (bottom) protonated N(1’); b(top) F form, (bottom) S‘ form; ‘(top) F form plus S‘ form, (bottom) S form. A single average value is given when there is no variation. Figure 1.
AM1 atomic charges (X103) in unprotonated (top) and protonated (bottom) thiamins.
Figure 2.
calculations this attractive interaction was not treated as such but rather as a normal van der Waals interaction. The presence of the H(2)-r attractive interaction is circuitously supported by the fact that the global minimum (& = f40° and 4~ = f65’) is different from the clustered crystal conformations (& = 0’ and 4 p = *goo) in which its energy can be maximized. 4’-Deaminothiamin,Oxythiamin,and Pyrithiamin. The energy maps of 4’-deaminothiamin, oxythiamin, and pyrithiamin, which are strong antagonists of thiamin, are presented in Figure 3b-d. Since thiamin analogues without a chiral C(2) substituent are centrosymmetric, only half of each map is unique. For 4’deaminothiamin, only a quarter of the map is unique since the 2’-methylpyrimidinyl moiety is symmetric with respect to the thiazolium moiety. The gross features of the energy maps at 6 kcal/mol level are similar to each other, but the details inside the low-energy regions are quite different. The 4’-deaminothiamin map is more like the oxythiamin than the thiamin map. The crystal structure of 4’-deaminothiamin has not been determined but those of its structural congeners benzylthiamin chloride (10)16a and 3-benzyl-4-methylthiazolium bromidel6b have been. The global minimum coincides with the crystal structures (which can be classified as either V or S due to symmetry), and there is
virtually no energy barrier between the various conformers. The oxythiamin map is quite different from the thiamin map in the overall shape, and the global minimum V form is exactly the same as the crystal V c0mformation.1~The pyrithiamin map is very similar to the thiamin map except that the energy barrier to the V form becomes higher by 1 kcal/mol. As in the case of thiamin, the global minimum, although in the F region, is different from the crystal F conformation of amprolium (ll),a structural congener of ~yrithiamin.3~ The crystal structure of the latter has not been reported yet. Thiamin Thiazolone and Thiamin Thiothiazolone. Both compounds were originally proposed to be the transition-state analogues34 of thiamin although strong evidence found later indicated that these are intermediate analogues.35 The energy maps of TT (Figure 3e) and TTT (Figure 3f) show a clear difference in the conformational properties of these congeneric compounds. The TT map (Figure 3e) shows a distinct global minimum ( 4 T = * 9 5 O , 4 p = ~ 7 5 which ~ ) agrees well with the crystal V conformation with an N-H-0 intramolecular hydrogen bond.15 In TTT, the global minimum also appears in the V region ( 4 =~ f130°, 4 p = ~ 4 5 ’ ) . However, it is not so distinct as in TT, and only 1 kcal/mol lower in energy than the other minima, even though we have used the most favorable parameters for the formation of the N-H-4 hydrogen bond in order not to underestimate its energetic c o n t r i b u t i ~ n .The ~ ~ crystal S conformation occurs in the center of the widest S region. Interconversion of the V and S forms should be facile due to the lowenergy barrier. In both maps, the F form is located at a saddle point indicating that it becomes an energetically unfavorable form for thiamin analogues with a C(2)-substituent larger than a H atom. 6’-Methyl-4H-thiaminand 6’-Methylthiamin, 6‘-Methyl-4Hthiamin shows partial activity (ca. 20%) but 6’-methylthiamin shows no activity in PDC. These data were the crucial ones that led Schellenberger to propose the V as the active form of thiamin.38 The crystal structures of both compounds have not been determined. The 6’-methyl-4H-thiamin map (Figure 3g) shows two distinct F and V + S’ minima whose centers locate at (C$T = Oo; 4 p = ~ 9 0 and ~ )(& = f18Oo; 4 p = r90°),respectively. This symmetric nature occurs due to the steric equivalence of C(2) and C(4) both of which are bonded to the H atoms. Both regions are nearly isoenergetic and occupy similar space, although the energy of the F form might be slightly lower as discussed in thiamin. The 6’-methylthiamin map (Figure 3h) shows a wide F and two narrow V (& = f135’; 4 p = 750’) and S’ ( 4 = ~ f130°; C$p = f130’) minima with the energy barriers of - 5 kcal/mol. The V form is 1 kcal/mol higher in energy than the F and S’ forms. In both compounds, S forms with +p = 180° are no longer stable. 2-(a-Hydroxyethyl)thiaminand Lactylthiamin. There are four major rotatable bonds [N(3)-C(35’), C(35’)-C(5’), C(2)-C(2a), C(2a)-C(2/3)] in LT and three in HET. It is generally difficult to obtain smoothly contoured 2-D energy maps for compounds with many rotatable pendant groups such as disaccharides due to the multiple minimum problem.36 The 2-D energy map usually shows discontinuous contours with a sudden drop in the energy when it is calculated with respect to a specific conformer of the pendant group(s). One way to circumvent this problem is to make a composite map from several maps each of which is calculated with the different local minima conformer(s) of the pendant group(s) as the starting conformer. Our unique iterative four-way scanning method developed for such a system makes it (33) Shin, W.;Oh. D. G.Acta Crystallogr., Sect. C 1993,49, 282-285. (34)(a) Gutowsky, J. A.; Lienhard, G. E. J. Biol. Chem. 1976,251,28632866. (bl Butler, J. R., Pettit, F. H., Davis, P. F.; Reed, L. J . Biochem. Biophys. Res. Commun. 1977,74, 1667-1614. (35)(a) Kluger, R.; Gish, G.; Kauffman, G. J. Biol. Chem. 1984,259, 8960-8965. (b) Kuo, D.J.; Jordan, F. J. Biol. Chem. 1983, 258, 1341513417. (c) Jordan, F.;Kudzin, Z. H.; Rios, C. B.J . Am. Chem. SOC.1987, 109, 4415-4416.
a) Thlamin
b) 4'-Deaminothiamin
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B'-Me-4-H-thlamin @m
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160
I) Thiamin Thiothlazolone
e) Thiamin Thiazolone
c)
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Figure 3. The relaxed M M conformational potential energy maps of thiamin and its analogues. The contour level is 1 kcal/mol and the lowest 20 kcal/mol levels are drawn for each map. The dashed thick lines denote the 6 kcal/mol level from the global minimum. Inside the low-energy region, the contour lines become thicker the lower the energy. The + and X signs denote the global minimum and the crystal
-180
-120
-eo
0
conformation, respectively. The approximate locations of the F, S, and V forms are depicted in (a). The circled X signs in (b) represent the same molecules as those without the circle, reflecting the symmetric nature of the molecule.
h,
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Shin et al.
12244 J . Am. Chem. Soc., Vol. 115, No. 26, 1993 a) 2- (a- Hydroxyethyl)thiamin
am
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60
120
180
60
120
180
b) Lactylthiamin
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Figure 4. The relaxed MM 2-D conformational potential energy maps of the stable intermediates. Contouring was done in the same way as stated in Figure 3. The crystal structures of HET and its analogues including 2-(a-hydroxybenzyl)oxythiamin are marked in (a) and the crystal structure of PLT is marked in (b).
possible to obtain a smooth contour map starting from one arbitrary side-chain conformation in an automated single run of calculation. In the resulting map, the side-chain conformations may be different for each grid point. This efficient method gave better results for carbohydrates than the other commonly used methods.19~3~ The relaxed 2-D energy maps of HET and LT in terms of 4~ and q5p are presented in Figure 4a,b, respectively. The absolute configuration of the chiral C(2)-substituent of HET was fixed as R according to results of Kluger et a1.37 The absolute configuration of LT was arbitrarily fixed as S, which assumes that the stereochemistry is retained rather than inverted when (36) (a) Hwft, R. W. W.; Kanters, J. A,; Kroon, J. J. Comput. Chem. 1991,12,943-947. (b) French, A. D.; Tran, V. H.; Perez, S.In Computer Modeling of Carbohydrate Molecules, French, A,, Brady, J. W., Eds.; ACS Symposium Series 430, 1990;pp 191-212. (37) (a) Kluger, R.;Karimian, K.; Gish, G.; Pangborn, W. A.; Detitta, G. J. Am. Chem. SOC.1987, 209, 618-620. (b) Kluger, R.;Tiachsel, M. R. Bioorg. Chem. 1990,18, 136-143.
the enamine (V) is converted into HET. The present energy maps of LT and HET are not centrosymmetric, and the crystal structures correspond to only one location in the present maps even though there are enantiomers in the crystal structures with centrosymmetric space groups. As pointed out by Turano et al., the introduction of a chiral substituent on C(2) generates a second element of asymmetry by virtue of restricted rotation about the bonds at C(359.13 They also noted that the conformation of thiamin in the synthetic reaction is a stereochemical determinant of the resultant product producing essentially two of four possible isomers, since a specific enantiomeric form at C(2a) exists only with a specific “enantiomeric” form at C(35’). Jordan’s HET map showed six distinct local minima separated by more than 18 kcal/mol energy barriers.lsb There are also six minima in the present relaxed map, but they are separated with muchlower energybarriers (at most < -9 kcal/mol). Theglobal minimum is in the V(+-) region, where the signs in the parentheses denote those of the torsion angles 41and 4p. There are two wide S regions representing the two enantiomeric S forms. The crystal structures of HET9 and its analoguesls12 are in the S(+ +) and S(+ -) regions. The relaxed conformational energy map of LT is the first one ever obtained for this intermediate. The gross features of the LT map are similar to those of HET with the global minimum in the V(+ -) region. However, a local minimum at 4~ = -60°, 4 p = -50” in HET disappeared in the LT map, and a narrow S(+ -) local minimum became isolated. The available conformational space in LT is narrower and the energy barriers between the conformers is higher than in HET since the size of the LT substituent is bigger. PLT is the only analogue of LT whose crystal structure has been determined.l3 It is located at the center of the S(+ -) region. 6’-Methyl-4H-lactylthiamin and 6‘-Methyllactylthiamin. Energy maps of these compounds were obtained to test the validity of Schellenberger’s interpretation of the catalytic properties of 6’-methyl-4H-thiamin and 6’-methylthiamin. The crystal structures of these compounds have not been determined. There are large differences in the S regions between the maps of these compounds and LT. The 6’-methyl-4H-lactylthiamin map (Figure sa) shows that the two V regions are wider than those in the L T map with more rotational freedom in 4~ reflecting the conformational property of its parent compound. The global minimum V(+ -) form is lower in energy by 1 and 4 kcal/mol than the V(- +) and S(+ -) forms, respectively. The 6’-methyllactylthiamin map (Figure 5b) shows several isolated minima separated by high-energy barriers (
