Supporting Information for Coupling of Functional ydrogen Bonds in Pyridoxal-5 -phosphate- Enzyme Model Systems bserved by Solid State NMR Shasad Sharif, David Schagen, Michael D. Toney, and ans-einrich Limbach Institut für Chemie, Freie Universität Berlin, Takustraße 3, D-14195 Berlin, Germany Department of Chemistry, University of California-Davis, 95616 Davis, United States Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Figure S8. Table of Contents Superposed experimental and calculated 30.41 Mz 15 N{ 1 } CP NMR static spectra solid powder samples of the aldenamine Schiff base and its 1 : 1 acid base complexes of, 1b, 1g, 1i, 1j and deuterated 1g-d, 1i-d, 1jd. Also presented is the theory of Dipolar 15 N Solid State NMR. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of the aldimine Schiff base (2a), and its 1 : 1 acid-base complex 2a-4-nitrobenzoic acid (2c). 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of 15 N labeled pyridoxal- 5'-phosphate (PLP), and its derivative pyridoxal (PL) in natural abundance. Also presented are the crystallographic structures. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of 15 N labeled pyridoxine (PN), pyridoxine hydrochloride (PN Cl). Also presented are the crystallographic structures. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of 15 N labeled in imino position pyridoxamine (PM) and pyridoxamine dihydrochloride (PN 2Cl) in natural abundance. Also presented are the crystallographic structures. Plot of the estimated intramolecular r N distances against the intrinsic 15 N chemical shift of the imino nitrogen. Secondary isotopic effect 1 N1(D) vs. intrinsic 15 N chemical shift. pk a values vs. proton transfer coordinate q 1 correlation. In addition are also listed the estimated geometrical parameters S2 S5 S6 S7 S8 S9 S1 0 S1 1 S1
(1b) (1b) (1g-d) D (1g-d) (1g) (1g) (1i-d) D (1i-d) (1i) (1i) (1j-d) D (1j-d) (1j) (1j) 280 240 200 160 120 500 400 300 200 100 0-100 δ( 15 N) ppm / 15 N δ( 15 N) ppm / 15 4 Cl N 4 Cl Figure S1. Superposed experimental and calculated 30.41 Mz 15 N{ 1 } CP NMR static spectra solid powder samples of the aldenamine Schiff base and its 1 : 1 acid-base complexes of, 1b, 1g, 1i, 1j and deuterated 1g-d, 1i-d, 1j-d, at room temperature. Additionally the MAS spectra of the mentioned samples are shown. S2
Determination of N.../N...D - Distances by Dipolar 15 N Solid State NMR The NMR theory of heteronuclear dipolar coupling 1,2,3 between 15 N nuclei and a deuteron has been described previously. 4,5 The N...L - Distances, with L =, D, are calculated from the dipolar frequency of ν d NL = D NL (1-3cos 2 θ d NL ) is the dipolar coupling constant D NL given by ν d NL = D NL (1-3cos 2 θ d NL ), D NL γ N γ L hµ = 0. 16π 3 3 rc (NL) (1) γ N and γ L are the gyromagnetic ratios of the two nuclei, h Planck s constant, µ 0 the magnetic permeability. r c (NL) represents the average reversal cubic internuclear distance N...L in expression in Å D N = 12158 r 3 c (N) [z], and D ND = 1868 r 3 c (ND) [z]. (2) θ d NL is the angle between the magnetic field and the vector NL. This angle can more conveniently be described by the Euler angles in the coordinate system of the CSA tensor - φ B and θ B for the magnetic field and α d NL and β d NL for the vector NL, respectively. It can be shown that (1) Mehring M. In NMR BasicPrinciples and Progress 11, igh Resolution NMR in Solids, Springer: Berlin-eidelberg-New York, 1978. (2) Seth, N. K.; Alderman, D. W.; Grant, D. M. Mol. Phys. 1990, 71, 217-238. (3) Zilm, K. W.; Grant, D. M. J. Am. Chem. Soc. 1981, 103, 2913-2922. (4) Lorente, P.; Shenderovich, I. G.; Golubev, N. S.; Denisov, G. S.; Buntkowsky, G.; Limbach,.-. Magn. Reson. Chem. 2001, 39, 18-29. (5) oelger, C.-G.; Limbach,.-. J. Phys. Chem. 1994, 98, 11803-11810. S3
cos NL NL B θ d sin β d sinθ cos d + B NL NL B ( φ α ) cos β cosθ =. (3) d The 15 N NMR spectrum of a polycrystalline powder is obtained from eqs. (1) - (3) by calculating ν N for all possible orientations by variation of φ B and θ B using increments of 1-2. For statistical reasons the signal intensity has to be weighted by a factor of sinθ B. Experimentally, only relative chemical shifts δ ii = δ ref - δ ii, ii = XX, YY, ZZ which are measured with respect to some standard reference, can be obtained. For the determination of the 15 N...D distances first the elements of the 15 N CSA tensor are determined by simulating the experimental solid state 15 N NMR spectrum of the protonated system, i. e. L =, under proton decoupling. The values obtained are then corrected for /D isotope effects on the elements of the 15 N CSA tensor. The new values have been used for a simulation of the proton decoupled 15 N NMR spectrum of the species deuterated in the hydrogen bond, by adapting the remaining parameters α ND d, β ND d and D ND. The determination of the 15 N... distances more difficult. 4 ence, we could obtain the distances N... by extrapolation from the observed strong dependence of the N...D distances on the isotropic chemical shifts. S4
(2a) 3 N1 δ 1 N1 δ+ δ 2 N2 δ+ (2c) dominant -from 2 N2 doubly 15 N labeled dominant N-from 1 N2(D) 2.9 ppm 3 C 3 N2 2 N1 C 3 (2a) 2 N2 singly imino 15 N labeled C 3 1 N2(D) N2-3.4 ppm 2 δ 2 N2 δ+ (2c) 3 N1 C 3 1 (2a-d) D2 N2 singly imino 15 N labeled (deuterated) δ D2 N2 δ+ (2c-d) N 2 300 250 200 150 100 50 0 300 250 200 150 100 50 0 δ( 15 N) ppm / 15 N 4 Cl δ( 15 N) ppm / 15 N 4 Cl 1 : 1 Acid Base Complex (2c) Figure S2. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of the aldimine Schiff base (2a), and its 1 : 1 acid-base complex 2a-4-nitrobenzoic acid (2c). From the top of the figure are shown the 15 N labeled doubly, selectively imino, and the selectively imino of the deuterated compounds 2a-d and 2c-d. : spinning sideband. S5
δ(n1- + ) 170.2 ppm Pyridoxal-5 -phoasphate (PLP) (hydrate form) X-ray 15 N-PLP r N 2.671(6) Å P NMR 1.08 Å 1.60 Å N C 3 r N1 2.68 Å P δ(n1- + ) 164.8 ppm Pyridoxal (PL) cyclic hemi acetale X-ray NMR PL (neutral) r N 2.669(1) Å 1.06 Å 1.76 Å N C 3 r N1 2.73 Å 300 200 100 0-100 δ( 15 N) ppm / 15 N 4 Cl Figure S3. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of 15 N labeled pyridoxal-5'-phosphate (PLP), and its derivative pyridoxal (PL) in natural abundance. For the presented crystallographic structures and values see text. The spectra of 15 N labeled (PLP) is obtained at 60.8 Mz with a spinning speed of 5 kz. : spinning sidebands. S6
δ(n1) 249.5 ppm δ(n1- + ) 158.3 ppm 15 N-PN Pyridoxine (PN) X-ray r N 2.729(5) Å NMR 1.55 Å 1.06 Å N C 3 r N1 2.61 Å δ(n1- + ) 158.3 ppm 15 N-PN Cl 300 200 100 0-100 δ( 15 N) ppm / 15 N 4 Cl Pyridoxine (PN Cl) hydrochloride neutron diffraction r N 1.040(8) Å NMR N C 3 r N1 1.04 Å Cl Figure S4. 15 N { 1 } CP MAS (30.41 Mz, 6 kz) spectra of 15 N labeled pyridoxine (PN), pyridoxine hydrochloride (PN Cl). For the presented crystallographic structures and values see text. S7
δ(n2) 10.9 ppm δ(n1) 170.6 ppm PM 2Cl Pyridoxamie monohydrochloride X-ray (PM Cl) Pyridoxamie dihydrochloride (PM 2Cl) NMR r N1 1.08 Å Cl N N C 3 Cl r N 0.84 Å 15 N-PM ( 15 N imino) δ(n2) -4.3 ppm Pyridoxamie (PM) X-ray (PM 2 2 ) N N C 3 300 200 100 0-100 δ( 15 N) ppm / 15 N 4 Cl r N 2.776(4) Å Figure S5. 15 N{ 1 } CP MAS (30.41 Mz, 6 kz) spectra of in imino position 15 N labeled pyridoxamine (PM) and pyridoxamine dihydrochloride (PN 2Cl) in natural abundance. For the presented crystallographic structures and values see text. We note that crystal structure is the pyridoxamine hydrochloride (PN Cl). Comment. The mono-hydrochloride of pyridoxamine (PM Cl) was studied by x-ray crystallography, for which a value of r N = 0.84 Å was reported, but the accuracy of the crystallographic finding is questionable. Also pyridoxamine dihydrate (PM 2 2 ) was studied by x-ray diffraction, and a heavy atom distance of r N = 2.776(4) Å between the hydroxyl group of the adjacent water molecule and the pyridine ring was reported. For PM 2Cl we obtained by NMR a value of r N = 1.08 Å for the pyridine-cl hydrogen bond, indicating a zwitterionic structure. We observed for the amino nitrogen a 15 N chemical shift of 10.9 ppm, which we assign to the amino group in the protonated form, hydrogen bonded to chloride Cl -. For neutral dihydrate of PM 2 2, where the ammonium group is hydrogen bonded to water, a high field shift to -4.3 ppm was observed. S8
3.2 2.8 2.4-2 N2 R N r N r N / Å 2.0 1f 2a 1b 2 N2 1.6 2-N2 1.2 Schiff bases 2c 0.8 0-20 -40-60 -80-100 -120-140 δ(2n2) ppm Figure S6. Plot of the estimated intramolecular r N distances against the intrinsic 15 N chemical shift of the imino nitrogen in the Schiff bases 1b and 2a as well as their acid-base adducts 1f and 2c. For the calculation of the solid line see the main text. S9
1 N1(D)=δ(DN)-δ(N) / ppm 14 10 6 2-2 -6-10 -14-1 N1 1 N1 1-N1 N C 3 / D 1j-d R 1i-d 1g-d aldenamine Schiff base coll-da 0-20 -40-60 -80-100 -120-140 δ(1n1) ppm Figure S7. Secondary isotopic effect 1 N1(D) vs. intrinsic 15 N chemical shift of the intermolecular 1N1 hydrogen bond. The open circles represented the aldimine Schiff base-adducts 1g-d, 1i-d, and 1j-d, the solid curve correspond to this data. The filled diamonds represented the collidine-da complexes which were obtained from literature, the dash curve correspond to this data. For further details see the main text. S10
pk a 14 12 10 8 6 4 2 0-2 -1 N1 N C 3 1 N1 R 1d 1c 1e aldenamine Schiff base aldimine Schiff base 2d 2c 1g,1h 2b 1f 1j 1-N1-0.8-0.4 0.0 0.4 0.8 q 1 = ½ ( r r N ) / Å Figure S8. pk a values vs. proton transfer coordinate q 1 correlation of the acid components in the organic solids of the aldenamine Schiff base-adducts 1b to 1j (open circles) and aldimine Schiff baseadducts 2b to 2e (filled circles). The pk a values is a function of q 1 with the relation pk a = A B q 1 by using the parameters A = 3.5 and B = 8. Table S1. NMR crystallographic values. a,b,c,d a pk a δ(1n1) a b r N b r c q 1 d q 2 / ppm / Å / Å / Å / Å 1c 3.14-60.2 1.32 1.21-0.05 2.53 1d 3.66-71.6 1.25 1.27 0.01 2.52 1e 3.44-77.0 1.22 1.30 0.04 2.52 1f 2.17-95.1 1.14 1.43 0.14 2.57 1g 2.82-104.6 1.10 1.51 0.20 2.62 1h 2.97-104.9 1.10 1.52 0.21 2.62 1i -7-112.2 1.08 1.60 0.26 2.68 1j 0.66-116.2 1.06 1.65 0.29 2.72 2b 3.99-90.1 1.16 1.39 0.11 2.55 2c 3.44-100.2 1.12 1.47 0.17 2.59 2d 2.82-101.4 1.12 1.48 0.18 2.60 2e -6.65-118.7 1.06 1.69 0.32 2.75 Values of the pyridine nitrogen are with respect to solid 1a, resonating at 282.5 ppm. b NMR crystallographic values are extrapolated from the experimental isotropic 15 N chemical shifts. b q 1 = ½ (r N - r N ). c q 2 = r N + r. S11