Proton nmr Study of Metal Coordination to Biotin Derivatives

Hiroki Kondo, Katsuhito Miura, Shingo uno, and Junza Smamoto Department of Indusm~al Chemistry, Faculty of Engineering, Nagasaki University, Japan

ABSTRACT

Proton nuclear magnetic resonance (nmr) spectra of d-biotin methyl ester (scheme 1) and its

N-methoxycarbonyl derivative (scheme 2) were determined in CD&N in the absence and presence

of magnesium perchlorate or silver perchlorate. In the metal-free spectrum of 2, the methylene

A HN,’ r*NH

10 HN N-&OCHa

n CH~OJKH,),, s

protons of the tetrahydromiophene ring appeared equivalently, suggesting a rapid flipping of the

ring on the nmr timescale. Addition of magnesium ion to 1 or 2 gave rise to a down-field shift of

protons of varying degtee. The hydrogens at the 3 and 4 position (H, and H,) of the tetrahydro-

thiophene ring exhibited the largest shift, followed by the hydrogens at the 2 (H,) and 5 (Hs)

position. Similar experiments with silver ion gave opposite results; H, and H s underwent the larger

shift. These data am interpreted as indicating that the magnesium ion interacts primarily with the

ureido carbonyl. The lack of interaction between magnesium and the sulfide was verified by

experiments with a biotin model compound carrying no sulfur atom. The significance of these

results is discussed in connection with the possible involvement of a metal in the enolixation of the

ureido carbonyl of biotin.

INTRODUCTION

The biotin-dependent carboxylation reactions play an important role in the synthesis of long-chain fatty acids in biological systems [ 11. It has now been established that the

Address reprint requests to Dr. Hiroki Kondo, Department of Industrial Chemistry, Faculty of Engineering. Nagasaki University, Nagasaki 852, Japan.

Journal of InorgMic Biochemism 21.93-102 (1984)

@ 1984 by Elsevier Science Publishing Co.. Inc. 52 Vanderbilt Ave.. New York, NY 10017

93

0162-0134/tJ4/53.00

94 H. Kondo et al.

carboxylation by, for example, acetyl-coenzyme A(CoA) carboxylase takes place in two discrete steps, as shown in eqs. (la) and (lb) (21.

M”+ Ebiotm + ATP f HCOs- + E-bioti&Os- + ABP + pi (la)

E-biotin-CO,- t Acetyl-CoA f: E-h&m + Malonyl-CoA (lb)

(ATP: adenosine triphosphate; ADP: adenosine diphosphate). Although the structum of carboxybiotin intermediate has been established to be the carboxylate group being placed at the 1 ‘-N position of coenzyme [3], the detailed mechanisms of how it is produced and how the catboxyl group is transferred in the subsequent steps remain largely obscure. A study of low molecular weight components of the reaction such as metal ions and ATP is a necessary step to an understanding of the whole reaction mechanisms of biotin-dependent enzymes, since they play an indispensable role in the enzymatic carboxylation, particularly, in the first half of the reaction. Sigel and co- workers, using model systems, studied the interaction of metal ions with biotin and concluded that metal ions such as Mn*+ and Cu*+ coordinate to the sulfide moiety of biotin stereospecifically [4-6]. Neverless, much remains to be elucidated regarding the structure and function of the metal-coenzyme complex in the biotm-dependent car- boxylation reactions. For one thing, most previous investigators employed free biotin; however, that group is blocked by an amide linkage in biotm-dependent carboxylases [ 11. Since the carboxylate is a much better ligand for most metal ions than the ureido or sulfide group, the mode of metal coordination to biotin might be altered considerably should a biotin derivative with the carboxyl group unavailable for metal chelation be used. The current source material used is d-biotin methyl ester and its N-methoxycar- bony1 derivative. The methodology adopted in this article utilizes the ‘H nuclear magnetic resonance (nmr) chemical shift change of individual hydrogens of a biotin derivative upon association with magnesium ion. Acetonitrile was used as solvent, since the ureido carbonyl and sulfuie groups are relatively poor ligands and little significant interaction should occur in hydroxylic solvents such as water.

EXPERIMENTAL

d-Biotin, anhydrous magnesium perchlorate. and silver perchlorate wefe obtained from Wako Pure Chemical Ind. Co., Osaka. Methyl d-biotinate (1) was prepared by reaction of d-biotin with diazomethane in 89% yield, melting point (mp) 165-1665°C. ANAL.

CALC. forC,,H,,N20,S:C,51.14;H,7.02;N, 10.84~0~~~.C,51.01;H.7.08;N. 10.99. Methyl I-methoxycarbonyl-&biotinate (2) was synthesized according to the literature [7]. The crude product obtained was purified by column chromatography on silica gel with acetonitrile as eluant. mp 130-13l’C (lit [7]. 131-132°C). ANAL. CALC. for C,,H2,-,N20,S: C, 49.35; H, 6.37; N, 8.85 FOUND. C, 48.95; H, 6.44; N, 8.82. 4- Dodecyl-5-methyl-2-imidazolidinone (3) and I-methoxycarhonyl4dodecyl-5-methyl- 2-imidazolidinone (4) were the same as those used previously [8].

NMR of Metal-Biotin Complexes 95

’ H nmr spectra were recorded on a JEOL FK-200 spectrometer, with the exception of those shown in Figure 1 A and B, which were determmed on a JEOL JNh+f-MH- 100 and FYWOQ spectrometer, respectively. The experimental parametets for the first spec- trometer, operating at 199.50 MHz in a Fourier transform mode were: spectral width 2000 Hz with acquisition of k 16K data points, 7 psec 90’ pulse, and a repetition time between pulses of 7 sec. Each specuum was obtained by 50 spectral accumulations at either 20 or 23°C. A sample was dissolved in CD&N at 2040 mM with and without a metal salt. The chemical shift 6 is expressed in ppm down field from the internal standard tetramethylsilane (TMS).

RESULTS

The I H nmr spectra of biotin and its derivatives were analyzed in some detail by Glasel [9]. Recently, Kohn and co-workers made a thorough spectral investigation of a series of biotin analogues [ 10, 111. The data we obtained are mostly consistent with these earlier results, and hence only that part of the spectra that is relevant to our discussion is presented. Figure 1A showns part (2.5-3.5 ppm region) of the MU spectrum of biotin in D20. This region is made up of three hydrogen atoms attached to the 2 and 5 position of the tetrahydrothiophene ring. The signal assignment, also depicted in Figure 1, is based on the following rationalization. The high-field doublet arises from the endo hydrogen

FIGURE 1. Parts of the ‘H MU spectra of (A) d-biotin in D,O and (B) d-biotin methyl ester (1) in CD&N.

(A) ’

Cppm

a HNI’ INH

(B) 5 %X -1

iI ‘42x. ,,;“, /,!,I ;

!.i.,,’ ,‘: . \.I I. I.

3.5 2.5

J,Wm

H. Kondoetal.

(H,, D for endo) that couples with the geminal exo hydrogen (J 13 f: 1 Hz). Hst, is placed over the shielding zone of the ureido carbonyl, thus appearing at a higher field than the exo hydrogen. In addition, this and the vicinal (H,) hydmgens ate virtually at right angles, and the vicinal coupling constant is minimally small. The double doublet (J 13 f 1 and 4 Hz) is assigned to the exo hydrogen (H,, X forexo), the smaller coupling constant originating tiom coupling with the vicinal hydrogen. The low-field multiplet is assigned to another exo hydrogen at the 2 position (H,). The corresponding region of the nmr spectrum of biotin methyl ester (1) in CD,CN was essentially the same,as that of biotin, the only difference being a more intricate splitting of H,, (Fig. 1B).

Figure 2 showns the nmr spectrum of Wmethoxycarbonyl biotin methyl ester (2) in CD&N. There is a notable difference between this spectrum and those of 1 and biotin. First of all, the hydrogens at the 5 position of 2 appear as a doublet with a coupling constant of 3.1 Hz. That this is a real doublet was confirmed by a decoupling experiment in which H4 at 4.76 ppm was irradiated to saturation. This led to an alteration of the doublet to a singlet, in line with the above notion that the endo and exo hydmgens (H,) of 2 interact with H4 equivalently in this solvent. This was not the case in CDC13. The two hydrogens exhibit nonequivalance, and this was not changed significantly by raising the temperature to 55°C. The assignment of other signals for the hydrogens attached to the tetrahydrothiophene ring follows (Fig. 2, insert). The double triplet at 4.76 ppm is assigned to H, that spin couples with HJ (J 7.8 Hz) and H5 (13.1 Hz). The octet at 4.12 ppm is for Hj, whose spin coupling involves those with H4 (J 7.8 Hz), HU, (J 4.2 Hz), and HJ’ (J 1.4 Hz). H2x appears at 3.20 ppm as a complex octet due to coupling with H3 and methylene protons attached to the 2 position. Analysis of the coupling pattern reveals that the signal is composed of two sets of double doublets. The one set constitutes the first, second, fourth, and sixth lines as numbered from higher field, and the other set makes up the rest of the lines. The coupling constants involved are 4.2 Hz with H, and

FlGURE 2.200 MHz 1 H MU spectrum of Wnethoxycarbonyld-biotin methyl ester (2) in CD&N.

\ ‘fslh”$,

mx

I n50.n5x

c Solvent

,:‘, J. n ‘I

I, ‘.

L . ( -.__- __._~ . - _. -. ~. __ ._ ..- . . .

I 1

7 6 5 I 3 2 1 0

NMR of Metal-Biotin Complexes 97

6.2 and 8.3 Hz. The unequal figures of the latter two coupling constants, originating from coupling with the vicinal methylene protons, suggest that rotation about the C&, bond is not completely free under these conditions.

Effects of Metal Ions

In order to ascertain that a chemical shift change of a given proton brought about by a metal ion can be used as a criterion to determine the site with which the metal coordinates, the effects of magnesium and silver ions on the chemical shift of biotin derivatives have been compared. The soft silver ion was supposed to interact predomi- nantly with the soft sulfur atom of biotin [ 12). This notion was verified by the data given in Table 1. With 1, the down-field shift caused by the silver ion was most pronounced (0.094 12 ppm) on the hydrogens placed closest to the sulfur, i.e., H,,, HSx, and HSD, whereas the effect of the metal was insignificant for scheme 3, which lacks the

“4%”

XCH CM-H,),, 3

tetrahydrothiophene ring. On the other hand, the magnesium ion brought about a shift of H3 and H, (0.15 ppm) almost twice as large as that of Hzx and HSx (0.07-0.09 ppm), in line with a view that the hard metal ion has a higher aftinity for the ureido carbonyl than for the sulfide moiety. The results with 3 also support this notion (Table 1). All these data demonstrate that a chemical shift change induced by a metal ion is a good indicator of the site of metal coordination on the biotin molecule.

Based on the preceding argument, a more detailed study of the interaction of metal

TABLE 1. Effects of Metal Salts on the Chemical Shift 6 @pm) of Individual Hydrogens of 1 (Upper Portion) and 3 (Lower Portion) in CD&No

Hydrogen None &‘-JO,

((0.83)b WWO,),

(0.77)b

HSD

4, H2X H3, H4

H,,. H7. H,

H9

HI,

H,‘, H,’

H4. HS

SCH, -(CH&&H,

2.62 2.74 (0.12) 2.73 (0.1 I)

2.86 2.95 (0.09) 2.93 (0.07) 3.14 3.25 (0.11) 3.23 (0.09) 4.31 4.36 (0.05) 4.46 (0.15)

1.14-1.89 1.15-1.92 1.15-1.91

2.31 2.31 (0.00) 2.32 (0.01, 3.61 3.61 (0.00) 3.61 (0.00)

5.08 5.20 (0.12) 5.61 (0.53)

3.66 (m) 3.68 (0.02) 3.82 (0.16) 1.03 (d) I.(# (0.01) 1. IO (0.07)

0.88 (1) 0.88 (0.00) 0.88 (0.00)

aNumbers in pnteathesa ntfer to the ougnitudc of down-field shifibtuughtaboutbythemetalsalt. bNumbers in pahe!iaamthcmolarratioofmaalsaitto1or 3.

98 H . Kondo et al.

ions with N-methoxycarbonyl derivatives of 1 and 3 has been carried out. Magnesium ion brought about a large down-field shift of H,, Hq, and the methyl hydrogens of methoxycarbonyl moiety at the 1’ position of 2 (Fig. 3). The magnitude of shift for H3 amounts to as much as 0.26 ppm at the equimolar concentration of metal and the ligand. The shift of Hzx, HSx. and HSD was less marked, and the shift of other hydrogens, including H,,, was virtually none existent, thus indicating that the metal interacts primarily with the methoxycarbonyl-urea region of 2. This was supported by an observation that scheme 4, which lacks the tetrahydrothiophene ring, behaves in a way

quite analogous to 2; Ha, HS, and the methyl hydrogens of the methoxycarbonyl group suffered the largest down-field shift (Fig. 4). In addition, the titration curves for such hydrogens tend to level off in a molar ratio of magnesium to ligand 0.5, suggesting that each metal binds two ligands.

The effect of silver ion was more pronounced for HXr H,,, and H,, of 2 than for H, and H4 (Fig. 5). That the metal interacts preferentially with the sulfide moiety was suggested by the fact that neither hydrogens of 4 underwent a significant chemical shift change over the’silver concentration range studied (Fig. 6).

FIGURE 3. Changes of the proton chemical shifts of 2 with magnesium perchlorate co”- centration in CD&N.

L 4.0 - z m 7 .u

FIGURE 4. Changes of the proton chemical $ 3.6 _ shifts of 4 with magnesium perchlorate con- 5 :: centration in CD&N. I.4 -

0.6 0 0.25 0.50 0.75 1.00

rMgc1rn WI

ii d

H3

r Lo - FIGURE 5. Changes of the proton chemical Ed shifts of 2 with silver perchlorate concentra- .g tion in CD&IN.

S fl = e l'-ocn, _

41 2 - Ho a-

3.5 -

100 H. Kondoet al.

FIGURE 6. Changes of the proton chemical shifts of 4 with silver perchlorate concentra- tion in CD&N.

0.6 ’ I 0 0.5 1.0 1.5 2.0

tAg(

t41

DISCUSSION

Prior to the discussion on the structure of magnesium complex of biotin derivatives, one novel finding regarding the stmcture of 2 in solution should be mentioned. The HSD and HSx of 2 appeared as a doublet in CDjCN (Fig. 2), demonstrating that the two hydrogens are equivalent on the mm. One possible explanation of this would be that the methoxy- carbonyl group at 1 ‘-N exerts a shielding effect on H,, , just like the ureido carbonyl does on HSD (vide supra). Alternatively, introduction of the methoxycarbonyl group to the

1 ‘-N position induces an internal motion of the tetrahydrothiophene ring in such a way that the two hydrogens at the 5 position become equivalent. The former possibility may be ruled out on the basis of the fact that this equivalence of hydrogens was preserved even in the presence of magnesium perchlorate. It has already been established that the mag- nesium complex of 2 or related compounds forms a virtually planar chelate ring [8]. Hence, the latter explanation appears to be the more plausible one, although we are not certain why the methoxycarbonyl group facilitates such a motion. In the crystal state of biotin derivatives the tetrahydrothiophene ring was found to assume a conformation with the sulfide moiety bent toward the ureido group [ 13, 141. In principle, another conforma- tion with the sulfide bent backward, tentatively named a chair form, is possible. A rapid interconversion of the two conformations could provide the basis for iwo hydrogens at the 5 position becoming equivalent on the nmr. In contrast to the solid state [ 151, such a conformational interconversion of the biotin skeleton may be facilitated in solution under limited conditions where, for example, there is a substituent on the ureido moiety. This argument leads us to propose its possible relevance to the catalytic mechanism of biotin. If we assume that carboxylation of the coenzyme brings about a conformation change and that the resulting chair form has a lower affinity for the carboxylase. the carboxylated biotin tends to leave the active site of carboxylase for the distant trans-

NMR of Metal-Biotin Complexes 101

carboxylation site [2]. Although this hypothesis remains to be verified experimentally, it may be an al&mat& explanation for the possible role played by the sulfide moiety during biotin catalysis, since involvement of the sulfide in catalysis through cheleation to

the metal ion is untenable (vide infra). It has been noted that I H mm chemical shift is an excellent means for studying the

interaction of biotin derivatives with metal ions, at least in model systems. The physio- logically pertinent magnesium ion was found to bind preferentially to the urea portion of biotin, but not to the suIfide moiety. This conculsion partially contmdicts previous work showing that metal ions bind primarily to the sulfide [4,5], although it was later claimed that a metal can also coordinate to the ureido moiety [6]: It is obvious that this discrepancy arose, at least in part, from the question of whether the biotin derivative used

contains a free carboxyl group or not. Several metal-biotin complexes that have a metal-sulfur bond have been reported, but the metal ion involved was either palladium, platinum, or silver [ 12, 161. It is not surprising to see a bond to sulfur with these soft metal ions, but these metals are biologically irrelevent. By contrast, physiologically relevant metal ions, such as magnesium and manganese, constitute hard category metal ions and should less affinity for the sulfide than for the ureido carbonyl. This was deduced on the basis of the evidence shown above and the following arguments. First of all, the spatial arrangement of the uteido carbonyl and the sulfur and their separation distance of 3.68 &7] would preclude simultaneous coordination of any single metal ion

to both sites. In fact, Aoki and Saenger recently isolated silver-biotin complexes that have both metal-sulfur and metal-carbonyl bonds, but separate silver ions were bound to each site [ 161. In addition, the fact that oxybiotin and selenobiotin were almost as reactive as ordinary biotin, at least for ace@-CoA carboxylase from E. coli [ 18, 191, may be taken as evidence against metal coordination to sulfur, since if the metal chelation to the sulfide were the requisite factor in biotin catalysis, the substituted biotins should have shown a decreased reactivity.

It is, therefore, tempting to conclude that magnesium ion plays a direct role in the carboxylation of biotin through chelation to the ureido carbonyl. It is often argued that the ureido moiety of biotin needs to be “activated” somehow during its catalysis [20]. This notion was based on an observation in model systems that the ureido nitrogen of biotin is not nucleophilic enough toward electrophilic centers such as carbonyls to allow ready carboxylation on that nitrogen [21]. One of the ways of activating biotin may be metal chelation to the ureido moiety. This would create an enolic character in that region of the molecule [8]. That the imino nitrogen of the resulting enol is likely to have a higher reactivity than the ureido nitrogen was confirmed by the fact that a stable enol-biotin model like scheme 5 showed a much higher nucleophilicity toward carboxylates and

carbonates than the corresponding keto compound [22,23]. Another role of metal ions may be to bring together necessary reactants by forming a ternary complex composed of metal, biotin, ATP, and/or bicarbonate [5. 241. This would obviously facilitate forma- tion of the carboxybiotin intermediate at the enzyme active site by a proximity effect. The N-methoxycarbonyl derivative of biotin (2) binds magnesium ion even mote

102 H. Kotubet al.

strongly than the parent compound 1 (Table 1 and Fig. 3). It should be noted that the stoichiometry obtained for the magnesium complex of 2 applies only to the present model systems, because two molecules of protein-bound biotin are unlikely to associate via a metal ion in enzymatic systems. Another point to note is the diion form which the metal binds to the ureido group. Unfortunately, the present technique does not enable us to establish diction, but since the sulfide group is not involved in the metal complex formation, it may be safe to presume that the metal binds either from above or below the ureido ring with equal probability. It is, however, plausible that the metal coordinates in

one direction stereospecifically to the enzyme active site due to the asymmetric topo- graphy there.

The authors thank Prqfessor Y. Kodem of Fuknoka University for derermining rhc nmr specrra or200 MHz. This work was sapported by Grant-in-Aid 56lO%W7 for Special Project Research from the Ministry of

Education, Science and Culiure, and by a grant for Basic Chemical Research from the Japan Society for rhe

Promorion of Science.

REFJDtENCES

I. H. G. Wood and R. E. Barden, Annu. Rev. Biochem. 46,385 (1977).

2. J. Knappe, Annu. Rev. Biuchem. 39.757 ( 1970).

3. R. B. Guchhait, S. E. Polakis, D. Hollis, C. Fenselau, and M. D. Lane, J. Bid. Chem. 249.6646 ( 1974). 4. H. Sigel, D. G. McCormick, R. Greisser. B. Prijs, and L. D. Wright, Bio&misny 8,2687 (1969).

5. R. Griessser, B. Prijs, H. Sigel, W. Fory, L. D. Wright, and D. B. McCotmick, Biochemisny 9.3285

(1970).

6. R. Griessex, H. Sigel, L. D. Wright, and D. B. McCormick, Biochemiswy 12.1917 (1973). 7. J. Knappe. E. Ringelmann, and F. Lynen. Biochem. 2.335, 168 (1961). 8. H. Kondo, D. Horiguchi, S. Jkeda. J. Sunamoto. and K. Tsujii, 1. Org. Chem. 44.4430 (1979). 9. J. A. Glasel,BiachemisnyS, 1851 (1%6).

10. H. Kohn, M. J. Cravey, J. H. Atwneaux, R. L. Cravey, and M. R. Willcott III, J. Org. Chem. 42, WI (1977).

I I. H. Flastex and H. Kohn, J. Hereroqdic Chem. 18, 1425 (1981). 12. N. Hadjiliadis and G. Pneumatikakis, J. Inorg. Biochem. 10.2 I5 ( 1979). 13. C. Bonnemere, J. A. Hamilton, L. K. Steimauf. and J. Knapp, Biochemistry 4,240 (1%5). 14. G. T. DeTitta, R. Parthwuathy, R. H. Blessing, and W. Stallings. Proc. Nad. Acad. Sci. U.S.A. 77,333

(1980). 15. W. C. Stallings, C. T. Monti, M. D. Lane, andG. T. DeTitta, Proc. Nod. Acod. Sci. U.S.A. 77. 1260

(1980). 16. K. Aoki and W. Saenger, J. Inorg. Biochem. 19,269 (1983).

17. G. T. DeTitta. J. W. Edmonds, W. Stallings, and J. Donohue, J. Amer. Chem. SOE. 98. 1920 ( 1976).

18. P. Dimroth, R. B. Guchhait, E. Stall. andM. D. Lane, Proc. Natl. Acud. Sci. U.S.A. 67, 1353 (1970).

19. A. Maquet, PureA& Chem. 49, 183 (1977).

20. R. Kluger and P. D. Adawadker, J. Amer. Chem. Sot. 98,3741(1976).

2 I. M. Caplow, 1. Amer. Chem. Sot. 87.5774 (1965).

22. A. F. Hegarty, T. C. Bruice, and S. J. Benkovic, Chum. Co-. 1%9. 1173.

23. H. Kondo. K. Miura. and J. Sunamoto. manuscript in won. 24. H. Kondo. F. Moriuchi, and J. SPnamoto. Bull. Chem. Sot. Jap. 55, 1579 (1982).

Received March IS, 1983; accepted September 21, 1983