Coupling of functional hydrogen bonds in pyridoxal-5′-phosphate- enzyme model systems observed by solid-state NMR spectroscopy

Published on Web 03/20/2007

Coupling of Functional Hydrogen Bonds in

Pyridoxal-5′-phosphate-Enzyme Model Systems Observed by

Solid-State NMR Spectroscopy

‡

†

,‡

Shasad Sharif, David Schagen, Michael D. Toney, and Hans-Heinrich Limbach*

Contribution from the Institut f u¨ r Chemie und Biochemie, Takustrasse 3,

Freie UniVersit a¨ t Berlin, D-14195 Berlin, Germany, and the Department of Chemistry,

UniVersity of California-DaVis, DaVis, California 95616

Received August 28, 2006; E-mail: limbach@chemie.fu-berlin.de

Abstract: We present a novel series of hydrogen-bonded, polycrystalline 1:1 complexes of Schiff base

models of the cofactor pyridoxal-5′-phosphate (PLP) with carboxylic acids that mimic the cofactor in a

variety of enzyme active sites. These systems contain an intramolecular OHN hydrogen bond characterized

by a fast proton tautomerism as well as a strong intermolecular OHN hydrogen bond between the pyridine

ring of the cofactor and the carboxylic acid. In particular, the aldenamine and aldimine Schiff bases

N-(pyridoxylidene)tolylamine and N-(pyridoxylidene)methylamine, as well as their adducts, were synthesized

15

1

and studied using N CP and H NMR techniques under static and/or MAS conditions. The geometries of

1

15

the hydrogen bonds were obtained from X-ray structures, H and N chemical shift correlations, secondary

15

2

15

H/D isotope effects on the N chemical shifts, or directly by measuring the dipolar H- N couplings of

static samples of the deuterated compounds. An interesting coupling of the two “functional” OHN hydrogen

bonds was observed. When the Schiff base nitrogen atoms of the adducts carry an aliphatic substituent

such as in the internal and external aldimines of PLP in the enzymatic environment, protonation of the ring

nitrogen shifts the proton in the intramolecular OHN hydrogen bond from the oxygen to the Schiff base

nitrogen. This effect, which increases the positive charge on the nitrogen atom, has been discussed as a

prerequisite for cofactor activity. This coupled proton transfer does not occur if the Schiff base nitrogen

atom carries an aromatic substituent.

Introduction

Scheme 1. (a) Vitamin B6: Pyridoxine (PN), (b) Pyridoxal (PL),

(

c) Pyridoxal Hemiacetal, (d) Pyridoxal-5′-phosphate (PLP), (e)

Schiff Base, (f) Pyridoxamine-5′-phosphate (PM)

Vitamin B6 or pyridoxine (PN)¹plays an important role in

-3

living systems (Scheme 1). After conversion to pyridoxal-5′-

4

,5

phosphate (PLP), it is incorporated into a variety of enzymes

that catalyze reactions of amino acids such as transamination,

decarboxylation, and racemization.⁶During transamination,

-9

10

pyridoxamine-5′-phosphate (PMP) occurs as an obligatory

intermediate form of the cofactor.¹¹^-¹³According to the crystal

‡

Freie Universit a¨ t Berlin.

University of CaliforniasDavis.

†

(

1) Longo, J.; Franklin, K. J.; Richardson, M. F. Acta Crystallogr., Sect. B

1

982, 38, 2721-2724.

(

2) Bacon, G. E.; Plant, J. S. Acta Crystallogr., Sect. B 1980, 36, 1130-1136.

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3

62.

(

5) Fujiwara, T. Bull. Chem. Soc. Jpn. 1973, 46, 863-871.

(

6) Christen, P.; Metzler, D. E. Transaminases, 1st ed.; Wiley: New York,

_s_t_r_u_c_t_u_r_e_s_o_f_P_L_P_-_d_e_p_e_n_d_e_n_t_e_n_z_y_m_e_s_,14-18 PLP is covalently

bound in their active sites as a Schiff base. Binding can occur

either as an “internal aldimine” with the ꢀ-amino group of a

1

985; pp 37-101.

(

7) Spies, M. A.; Toney, M. D. Biochemistry 2003, 42, 5099-5107.

(

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3

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(

9) Zhou, X.; Toney, M. D. Biochemistry 1999, 38, 311-320.

(

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285-305.

Press: New York, 1970; Vol. 2.

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28, 8161-8167.

2

61-263.

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458.

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34.

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10438-10445.

2

1

(18) Jansonius, J. N. Curr. Opin. Struct. Biol. 1998, 8, 759-769.

4440

9

J. AM. CHEM. SOC. 2007, 129, 4440-4455

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

Scheme 2. Overview of the First Step of the Catalytic Cycle

Scheme 3. Schematic View of the Keto-Enol Tautomerism of the

Aldenamine Schiff Base (R ) Tolyl) and Aldimine Schiff Base

(

R ) CH3) and the Influence of Protonation of the Pyridine

Nitrogen. Only the Zwitterionic Resonance Structure is Shown for

Tautomer b

lysine residue or as or an “external aldimine” with the amino

acid substrate (Scheme 2). Both aldimines are stabilized by an

intramolecular OHN hydrogen bond. The ring nitrogen is

involved in an intermolecular OHN hydrogen bond with an

aspartic acid side chain in many PLP-dependent enzymes. The

phosphate group contributes to PLP binding by hydrogen

bonding but plays no direct role in the mechanism.

The protonation states of the two functional OHN hydrogen

4

,6

bonds have a large influence on enzymatic function. In the

first step of the catalytic cycle, it is assumed that the bridging

proton of the intramolecular OHN hydrogen bond has to be

transferred from the phenolic oxygen to the imino nitrogen, a

process which may be assisted by protonation of the pyridine

model the intermolecular OHN hydrogen bond, some of us have

studied various acid-pyridine or 2,4,6-trimethylpyridine (colli-

dine) complexes using liquid- and solid-state NMR tech-

34-44

niques.

Hydrogen bond geometries were obtained as a

6

,19

ring (Scheme 2).

function of the position of the proton in the bridge. Moreover,

solvent effects on the intermolecular OHN hydrogen bond

In order to elucidate the properties of the intramolecular OHN

hydrogen bond, model studies have been reported for several

Schiff bases of aromatic ortho-hydroxylaldehydes dissolved in

45

geometries were studied.

However, the interaction between the functional OHN

hydrogen bonds is still open to debate. Therefore, using solid

2

0-27

organic liquids or embedded in the organic solid state.

Using UV-vis,²⁸¹³C,

29,30 17

O,

31,32

and

15 20,33

N

NMR spectros-

1

15

state H and N MAS NMR techniques, we have examined

the coupling between these hydrogen bonds. This necessitated

copy, it has been shown that these compounds exhibit a keto-

enol tautomerism between the enolimine form (OH form) and

the ketoamine form (HN form) that interconverts very rapidly

via proton transfers, as illustrated in Scheme 3. Many factors,

such as the local polarity and specific solvation, influence this

15

the design of N-labeled model Schiff bases that mimic the

interactions of PLP in enzyme active sites. As the phosphate

group is not directly involved in the mechanisms of PLP-

15

catalyzed reactions, we prepared the singly and doubly N-

2

0

15

equilibrium. In particular, the analysis of the N chemical

shifts δ(OHN) provides information about the equilibrium

constants of the tautomerism. On the other hand, in order to

labeled Schiff bases 1b (R ) tolyl, aldenamine) and 2a (R )

11

methyl, aldimine) of pyridoxal (PL) to vary the basicity of

the imino nitrogen, as illustrated in Scheme 3. Several novel

1

:1 acid-base complexes of benzoic acid derivatives with these

(

19) Bach, R. D.; Canepa, C. J. Am. Chem. Soc. 1997, 119, 11725-11733.

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4101.

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M. M.; Schowen, R. L.; Limbach, H. H. Can. J. Chem. 1999, 77, 943-

949.

(

21) Benedict, C.; Langer, U.; Limbach, H. H.; Ogata, H.; Takeda, S. Ber.

Bunsen-Ges. Phys. Chem. 1998, 102, 335-339.

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Chelmieniecka, A. Solid State Nucl. Magn. Reson. 2000, 16, 285-289.

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Soc. 1988, 110, 7221-7223.

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11720.

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G.; Smirnov, S. N.; Golubev, N. S.; Denisov, G. S. Chem.sEur. J. 2004,

10, 5195-5204.

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003, 287, 113-124.

29) Filarowski, A.; Koll, A.; Rospenk, M.; Krol-Starzomska, I.; Hansen, P. E.

J. Phys. Chem. A 2005, 109, 4464-4473.

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Albrecht, J.; Golubev, N. S.; Findenegg, G. H.; Limbach, H. H. J. Phys.

Chem. B 2003, 107, 11924-11939.

30) Rozwadowski, Z.; Dziembowska, T. Magn. Reson. Chem. 1999, 37, 274-

2

78.

(

31) Zhuo, J. C. Magn. Reson. Chem. 1999, 37, 259-268.

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(

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4441

A R T I C L E S

Sharif et al.

Scheme 4. (a) Aldenamine Schiff Base and its Derivatives Studied in this Paper. (b) Aldimine Schiff Base and its Derivatives Studied in

this Paper

1

Schiff bases were designed (Scheme 4). In previous studies,

some of these complexes (i.e., 1b, 2a, and their adducts with

echo delay of 20 rotor periods and a recycle time of 10 s. All H spectra

were referenced to an external sodium salt of trimethylsilylpropionic

acid (TSP).

2

-nitrobenzoic acid 1f and 4-nitrobenzoic acid 2c) were

The static ¹⁵N CP powder spectra of 1g, 1i, 1j, and their deuterated

46

investigated using X-ray crystallography.

34,35

analogues were calculated as described previously for other systems,

using procedures described by Hoelger et al.⁴⁷From the H- N dipolar

couplings, mean cubic ND distances were derived. The distances were

not corrected for vibrational averaging. One needs to distinguish

between temperature effects arising from the population of excited

vibrational states and anharmonic vibrational effects, which are

operative also in the vibrational ground state. It is difficult to take these

effects quantitatively into account. In a case where the anharmonic

vibrational ground state wave function was known, an error of the ND

distances of a few percent was calculated.³⁴Therefore, vibrational

averaging effects were neglected in this study.

2

15

Experimental Section

A. NMR Measurements. Solid-state ¹⁵N NMR spectra of ¹⁵N-

labeled 1a-1j and 2a-2e were measured at 30.41 MHz using a Bruker

MSL 300 spectrometer (7 T) equipped with a 5 mm Chemagnetics

1

MAS probe. The H spectra were recorded on a Varian Infinity Plus

6

00 MHz (14 T) solid-state NMR spectrometer. Standard cross-

1

5

1

polarization N { H} CP NMR experiments were performed under

static or magic angle spinning (MAS) conditions. In the latter case,

the spinning rates were about 6 kHz. The 90° pulse width was about

8

µs, the cross-polarization contact times were 3 ms, and the recycle

B. Materials. ¹⁵N-labeled pyridoxine (PN), pyridoxamine (PM),

pyridoxal-5′-phosphate (PLP), pyridoxal (PL), N-(pyridoxylidene)-

tolylamine (1b) (1a is the protected 5′-triisopropylsilyl ether of 1b),

N-(pyridoxylidene)methylamine (2a), and p-toluidine were synthesized

1

5

times were 20 s. All N spectra are referenced to external solid

_a_m_m_o_n_i_u_m_c_h_l_o_r_i_d_e_,15_N_H

15

1

4

Cl (95% N-enriched). Standard H ECHO

MAS NMR experiments were performed at a spinning rate of 24 kHz.

The 90 and 180° pulse widths were 2 and 4 µs, respectively, using an

from 98% ¹⁵N-enriched NH

15

Cl (Deutero GmbH), 95% N-enriched

15

4

(

46) Sharif, S.; Powell, D. R.; Schagen, D.; Steiner, T.; Toney, M. D.; Fogle,

E.; Limbach, H. H. Acta Crystallogr., Sect. B 2006, 62, 480-487.

(47) Hoelger, C. G.; Limbach, H. H. J. Phys. Chem. 1994, 98, 11803-11810.

4442 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

Scheme 5. Overview of the Synthetic Route

methylamine hydrochloride (Chemotrade), and/or 95% ¹⁵N-enriched

hydroxylamine hydrochloride (Chemotrade). 3,5-Dichlorobenzoic acid,

which was hydrolyzed to pyridoxine hydrobromide.⁵⁶^,⁵⁷The pyridoxine

hydrobromide was converted to pyridoxine hydrochloride (V) using

5

6,57

3

-nitro-4-chlorobenzoic acid, 4-nitrobenzoic acid, 2-nitrobenzoic acid,

,5-dinitrobenzoic acid, 3,5-dinitro-4-methylbenzoic acid, hydrochloric

silver chloride.

After the neutralization of pyridoxine hydrochloride,

it was purified by column chromatography. The ¹⁵N-ring-labeled

aldenamine Schiff base (VIa) was prepared by oxidization of N-

labeled V with manganese dioxide to PL, followed by reaction with

p-toluidine according to the method of Iwanami. Using the N-labeled

p-toluidine (IX),⁵the N-doubly-labeled VIb was accessible. The

1

5

acid, trichloroacetic acid, 4-chlorobenzoic acid, 4-tolylsulfonic acid,

pyridoxamine dihydrochloride, PLP, and pyridoxal hydrochloride were

purchased from Aldrich. The following deuterated solvents were

58

15

9,60

15

purchased from Deutero GmbH: deuteriumoxide (D

2

O), dichloro-

15

methane-d

form-d

2

, dimethylsulfoxide-d

6

(DMSO), methanol-d /-d , chloro-

1

4

direct reaction of PL and N-labeled p-toluidine (IX) forms the N-

imino-labeled VIc. In addition, the 5′-hydroxyl group of the aldenamine

Schiff base was protected by the formation of the 5′-triisopropylsilyl

1

, and DCl solution in D

2

O.

C. Syntheses. The synthesis of ¹⁵N-labeled PN, Schiff bases ¹⁵N-

ether,⁶leading to the protected N-labeled VII. The aldimine Schiff

1,62

15

labeled at different positions, and PLP and its analogues is depicted in

1

5

15

Scheme 5. As a starting point, the N-labeled amino acid D,L-alanine

I) was prepared using a halogen exchange of 2-bromopropionic acid

base was prepared by condensation of methylamine and N-labeled

V, which was oxidized to PL and then transformed to ring N-labeled

VIIIa; the doubly N-labeled VIIIb was accessible by using the N-

labeled methylamine. The N-imino-labeled VIIIb was obtained by

condensing PL with N-labeled methylamine hydrochloride in aqueous

solution, following the unlabeled preparation.⁶

1

5

(

1

5

48-50

15

with ammonia, NH

preparing N-labeled amino acids, a method described by Block for

unlabeled amino acids was employed. The N-labeled I reacts with

formic acid in ethanolic solution in an autoclave to give ethyl-N-formyl-

D,L-alaninate (II); the preparation procedure followed the description

3

. Instead of the established methods

for

1

5

51

15

1

5

15

3-66

The 5′-triisopropyl-

silyl ether of the aldimine Schiff base was unstable.

5

2

¹⁵N-labeled PM (X) was prepared from ¹⁵N-enriched hydroxylamine

hydrochloride according to the procedures described in refs 67-70.

by Maeda et al. The dehydration of II to 5-ethoxy-4-methyloxazole

3-55

(

III) can be obtained using different methods;⁵

we employed the

1

5

15

last method using phosphorus pentoxide to give N-labeled III. The

reaction of III with 2,5-dihydrofuran catalyzed by trichloroacetic acid

in a autoclave gave N-labeled 2-methyl-3-hydroxy-4,5-epoxydi-

After acidification of the N-labeled aldimine Schiff base, N-labeled

PL (XI) was obtained. The ¹⁵N-labeled cofactor PLP (XII) was made

1

5

methylpyridine (IV). The cleavage of the epoxydimethyl group, using

8% hydrobromic acid, forms the dibromide hydrobromide of PN,

(56) Harris, S. A.; Folkers, K. J. Am. Chem. Soc. 1939, 61, 3307-3310.

(

57) Harris, S. A.; Folkers, K. J. Am. Chem. Soc. 1939, 61, 1245-1247.

58) Iwanami, M.; Numata, T.; Murakami, M. Bull. Chem. Soc. Jpn. 1968, 41,

4

(

161-165.

(

48) Baumgartner, F. J.; Barrio, J. R.; Henze, E.; Schelbert, H. R.; MacDonald,

N. S.; Phelps, M. E.; Kuhl, D. E. J. Med. Chem. 1981, 24, 764-766.

49) Ogo, S.; Uehara, K.; Abura, T.; Fukuzumi, S. J. Am. Chem. Soc. 2004,

(59) Lewis, E. S.; Holliday, R. E. J. Am. Chem. Soc. 1969, 91, 426-430.

(60) Lewis, E. S.; Insole, J. M. J. Am. Chem. Soc. 1964, 86, 32-34.

(61) Corey, E. J.; Venkateswarlu, A. J. Am. Chem. Soc. 1972, 94, 6190-6191.

(62) Cunico, R. F.; Bedell, L. J. Org. Chem. 1980, 45, 4797-4798.

(63) Van Genderen, M. H. P.; Van Lier, P. M.; Buck, H. M. Recl. TraV. Chim.

Pays-Bas 1989, 108, 418-420.

1

26, 3020-3021.

50) Dimitrijevich, S. D.; Scanlon, M. D.; Anbar, M. J. Labelled Compd.

Radiopharm. 1982, 19, 573-584.

(

51) Block, R. J. Chem. ReV. 1946, 38, 501-571.

(64) Heyl, D.; Luz, E.; Harris, S. A.; Folkers, K. J. Am. Chem. Soc. 1952, 74,

414-416.

(

52) Maeda, I.; Asai, S.; Miyashiki, H.; Yoshida, R. Bull. Chem. Soc. Jpn. 1972,

4

5, 1917-1918.

(65) Heyl, D.; Luz, E.; Harris, S. A.; Folkers, K. J. Am. Chem. Soc. 1948, 70,

3669-3671.

(

53) Maeda, I.; Togo, K.; Yoshida, R. Bull. Chem. Soc. Jpn. 1971, 44, 1407-

1

410.

(66) Metzler, D. E. J. Am. Chem. Soc. 1957, 79, 485-490.

(67) Tanenbaum, S. W. J. Biol. Chem. 1956, 218, 733-743.

(68) Harris, S. A.; Heyl, D.; Folkers, K. J. Am. Chem. Soc. 1944, 66, 2088-

2092.

54) Maeda, I.; Takehara, M.; Togo, K.; Asai, S.; Yoshida, R. Bull. Chem. Soc.

Jpn. 1969, 42, 1435-1437.

55) Harris, E. E.; Firestone, R. A.; Pfister, K., III; Boettcher, R. R.; Cross, F.

J.; Curie, R. B.; Monaco, M.; Peterson, E. R.; Reuter, W. J. Org. Chem.

(69) Ikawa, M.; Snell, E. E. J. Am. Chem. Soc. 1954, 76, 637-638.

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1

962, 27, 2705-2706.

J. AM. CHEM. SOC.

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VOL. 129, NO. 14, 2007 4443

A R T I C L E S

Sharif et al.

by phosphorylation of the 5′-hydroxyl group of ¹⁵N-labeled VIa⁷¹^-⁷³

with phosphorus pentoxide in phosphoric acid. It was purified with a

cation exchange column. The procedures for obtaining the unlabeled

Schiff bases were described previously.⁴⁶

5. ¹⁵N-Labeled Pyridoxine (PN) (V). A solution containing 1.9 g

(12 mmol) of ¹⁵N-labeled 2-methyl-3-hydroxy-4,5-epoxydimethyl-

pyridine (IV) in 12 mL of 48% hydrobromic acid was heated under

reflux for 1 h. Upon cooling in ice water, crystals separated and were

filtered and washed with diethylether. The hydrochloride was prepared

by boiling the hydrobromide for 30 min in 100 mL of water and

removing the bromide ions with freshly prepared silver chloride. The

filtrate was evaporated to dryness. The pyridoxine hydrochloride was

1

. ¹⁵N-Labeled d,l-Alanine (2-Aminopropionic acid) (I). Eight

grams (52 mmol) of 2-bromopropionic acid was dissolved in 250 mL

15

74

of water by adding an approximately 20% solution of NH

3

in water.

The product reacted positive to the ninhydrin test for the presence of

amino groups (1% in water, followed by heating). Yield: 3.0 g (64%).

2 2 3

purified by column chromatography (silica, CH Cl /CH OH, 10:1). The

1

15

H NMR (500 MHz, D

2

15

O, room temperature) (ppm/TMS): δ 1.50 (dd,

product was checked by mass spectroscopy, and the N isotopic

3

1

3

1

3

1

J( H, H) ) 7.0 Hz, J( N, H) ) 3.0 Hz, 3H, -CH

3

), 3.8 (q, J( H, H)

7 Hz, 1H, -CH-). C { H} NMR (125 MHz, D O, room

), 50.74 (d, -CH-,

enrichment was estimated as >90%. Yield: 2.1 g (84%). H NMR

1

3

1

2

15

1

)

2

(500 MHz, D

2

O, room temperature) (ppm/TMS): δ 2.65 (d, J( N, H)

), 4.81 (s, 2H, 5′-CH -), 5.01 (s, 2H, 4′-CH -),

8.17 (d, J( N, H) ) 3.2 Hz, 1H, 6-CH). C { H} NMR (125 MHz,

temperature) (ppm/TMS): δ 16.40 (s, -CH

3

) 3.2 Hz, 3H, 2′-CH

3

2

13

2

1

15

13

2

15

1

J( N, C) ) 5.6 Hz), 175.95 (s, -COOH).

1

5

D O, room temperature) (ppm/TMS): δ 14.56 (s, 2′-CH ), 57.00 (s,

2

.

N-Labeled Ethyl-N-formyl-d,l-alaninate (II). A mixture of

2

3

1

5

4′-CH -), 58.3 (s, 5′-CH -), 130.00 (d,

1

J(15N,13C) ) 12.7 Hz, C6),

4

g (45 mmol) of N-labeled d,L-alanine (I), 6.3 g (137 mmol) of abs

2

1

15

13

formic acid, and 140 mL of abs ethanol was allowed to react in an

autoclave at 220 °C overnight and was then chilled in an ice bath. The

ethanol and water formed in the reaction were distilled off under reduced

pressure. Next, 140 mL of ethanol was added to the residue and

removed under reduced pressure. This treatment was repeated three

times. The residue was distilled in a bulb tube at 80-100 °C and 0.3

136.96 (s, C5), 140.79 (s, C4), 142.95 (d, J( N, C) ) 14.4 Hz, C2),

152.93 (s, C3). These ¹C NMR data are similar to those reported

previously for unlabeled pyridoxine.⁷

3

5,76

6. ¹⁵N-Labeled (Ring and Doubly) N-(Pyridoxylidene)tolylamine

1

5

(VIa,b). For preparation of the ring or doubly N-labeled product,

0.4 g (1.9 mmol) of ¹⁵N-labeled pyridoxine hydrochloride (V) in

mbar. The product was stored under inert gas at -23 °C. Yield: 3.3 g

aqueous solution was added to MnO that was freshly prepared from

2

1

(

50%). H NMR (500 MHz, CDCl

3

, room temperature) (ppm/TMS):

0.3 g of potassium permanganate, 0.4 g of sodium bisulfite NaHSO₃,

and 1 mL of 50% sulfuric acid in 20 mL of water. The solution was

3

1

3

1

δ 1.27 (t, J( H, H) ) 7.2 Hz, 3H, -CH

)

2

-CH

), 4.20 (q, J( H, H) ) 7.2 Hz,

), 4.60 (q, J( H, H) ) 7.2 Hz, 1H, -CH-), 6.52 (ddd,

3

), 1.42 (dd, J( H, H)

3

15

1

3

1

15

7.2, J( N, H) ) 2.7 Hz, 3H, -CH

3

stirred for 4 h after adding 0.3 g (2.2 mmol) of p-toluidine and/or N-

3

1

2

H, -CH

2

-CH

3

labeled p-toluidine (IX). After diluting the solution to 400 mL with

1

15

1

3

1

3

1

J( N, H) ) 92.0, J(H, H) ) 7.6, J( H, H) ) 1.6 Hz, 1H, HN), 8.16

water, the pH was adjusted with 1 M NaHCO solution to 7.5. The

3

2

15

1

13

1

(

d, J( N, H) ) 16.1 Hz, 1H, -CHO). C { H} NMR (125 MHz,

CDCl , room temperature) (ppm/TMS): δ 14.24 (s, -CH -CH ), 18.62

), 47.06 (d, J( N, C) ) 11.7 Hz, -CH-), 61.87 (s, -CH -),

60.82 (d, J( N, C) ) 13.37 Hz, -CHO), 172.81 (s, -COOH).

Schiff base that precipitated from solution was filtered and washed

with water. The product was dried under vacuum. By using unlabeled

3

2

3

1

15 13

15

(s, -CH

3

2

p-toluidine, the singly ring N-labeled Schiff base was obtained.

1

15

13

15

1

Yield: 0.18 g (37%). VIa ( N-ring-labeled): H NMR (500 MHz,

DMSO-d , room temperature) (ppm/TMS): δ 2.36 (s, 3H, Ar-CH ),

2.43 (d, 3H, J( N, H) ) 2.9 Hz, 2′-CH ), 4.77 (d, 2H, J( H, H) )

-), 5.42 (t, 1H, J( H, H) ) 5.5 Hz, 5′′-OH), 7.30-

1

5

3

.

N-Labeled 5-Ethoxy-4-methyloxazole (III). Freshly prepared

6

3

1

5

2

15

1

3

1

(4 g; 27 mmol) N-labeled ethyl-N-formyl-d,l-alaninate (II) was

3

1

dissolved in 250 mL of dist dichloromethane, and 17 g of P was

O

2 5

5.5 Hz, 5′-CH

7.45 (m, 4H, -C

(s, 1H, H4′), 14.06 (s, 1H, H3′).

2

15

1

added slowly. The reaction mixture was heated for 24 h under reflux.

The solution was chilled to 0 °C, and 250 mL of 20% aqueous NaOH

was slowly added. The suspension was extracted four times with

6 4

H -), 7.99 (d, 1H, J( N, H) ) 11.0 Hz, H6), 9.18

_.15N-Labeled (Imino) N-(Pyridoxylidene)tolylamine (VIc). The

imino N-labeled product was prepared as follows. An aqueous solution

7

3

00 mL of dichloromethane and dried over NaHCO

3

; the solvent was

15

removed under reduced pressure. The residue was distilled in a bulb

tube at 39 °C and 25 mbar. The product was stored under inert gas at

of 0.37 g (2.2 mmol) of pyridoxal hydrochloride was added to 0.3 g

15

(

2.2 mmol) of N-labeled p-toluidine (IX). The resulting mixture was

1

-

23 °C. Yield: 2.7 g (78%). H NMR (500 MHz, CDCl

3

, room

3

brought to a pH 7.5 by adding 1 M NaHCO solution. The mixture

was kept on ice for 5 h, after which crystals were filtered off, washed

with cold water and diethylether, and dried under vacuum. Yield:

3

1

temperature) (ppm/TMS): δ 1.34 (t, J( H, H) ) 7.1 Hz, 3H, -CH -

2

3

15

1

3

1

CH

3

), 2.03 (d, J( N, H) ) 2.3 Hz, 3H, -CH

3

), 4.15 (q, J( H, H) )

), 7.37 (d, J( N, H) ) 12.6 Hz, 1H, -CH).

C { H} NMR (125 MHz, CDCl , room temperature) (ppm/TMS): δ

0.18 (d, J( N, C) ) 6.4 Hz, -CH ), 15.19 (s, -CH -CH ), 70.32

-), 112.5 (d, J( N, C) ) 1.6 Hz, C4), 142.36 (d, J( N, C)

2

15

1

7

.1 Hz, 2H, -CH

2

-CH

3

1

0

(

.32 mg (57%). H NMR (500 MHz, DMSO-d

6

, room temperature)

), 4.77 (d,

-), 5.42 (t, 1H, J( H, H) ) 5.5 Hz,

-), 7.98 (s, 1H, H6), 9.18 (d, 1H,

1

3

1

3

ppm/TMS): δ 2.36 (s, 3H, Ar-CH ), 2.43 (s, 3H, 2′-CH

H, J( H, H) ) 5.5 Hz, 5′-CH

′′-OH), 7.30-7.45 (m, 4H, -C

3

2

15

13

1

3

2

3

1

3

1

2

5

2

1

15 13

15

1

15 13

(s, -CH

2

6

H

4

2

13

)

1.2 Hz, C2), 154.56 (d, J( N, C) ) 0.9 Hz, C5).

N-Labeled 2-Methyl-3-hydroxy-4,5-epoxydimethylpyridine

IV). A solution of freshly prepared, 2.5 g (20 mmol) of N-labeled

2

15

1

J( N, H) ) 3.4 Hz, H4′), 14.06 (s, 1H, H3′).

1

5

4

.

_.15N-Labeled 5′-Triisopropylsilyl Ether of N-(Pyridoxylidene)-

8

15

(

15

tolylamine (VII). This synthesis was used to protect the selective N-

5

-ethoxy-4-methyloxazole (III) in a 20-fold molar excess of 2,5-

labeling in the imino or in the ring position, depending on the

dihydrofuran containing 1% trichloroacetic acid was heated in an

autoclave for 5 h at 190 °C. After cooling to room temperature

overnight, the precipitate was washed with diethylether. Excess 2,5-

dihydrofurane was removed under high vacuum, and the product was

15

N-labeling of the starting material. In 2 mL of abs DMF, 140 mg

15

(0.55 mmol) of N-labeled N-(pyridoxylidene)tolylamine was dissolved,

with stirring under an inert gas atmosphere. Then, 94 mg (1.38 mmol)

of imidazole was added. After a few minutes of stirring, 126 mg

purified by column chromatography (silica, CH

2

Cl

OD, room temperature)

ppm/TMS): δ 2.42 (d, J( N, H) ) 3.0 Hz, 3H, -CH ), 5.05 (m,

2

/MeOH, 10:1).

(0.66 mmol) of triisopropylsilyl chloride was added dropwise. The molar

1

Yield: 1.9 g (63%). H NMR (500 MHz, CD

3

ratio of reactant/chlorosilane/imidazole was 1:1.2:2.5. The solution was

stirred under an inert atmosphere for 3 days. The reaction was monitored

by TLC (silica, CH Cl /MeOH 10:0.3). The reaction was stopped by

2 2

3

15

1

(

3

2

15

1

4

H, -CH -), 7.77 (d, J( N, H) ) 9.2 Hz, 1H, CH).

2

dropping the mixture into 25 mL of 1 M sodium hydrogen carbonate

solution. The aqueous phase was extracted two times with 100 mL of

dist hexane. The combined organic phases were dried over NaHCO .

3

(

71) Chang, Y. C.; Graves, D. J. J. Biol. Chem. 1985, 260, 2709-2714.

72) Iwanami, M.; Numata, T.; Murakami, M. Bull. Chem. Soc. Jpn. 1968, 41,

(

1

61-165.

(

73) Muhlradt, P. F.; Morino, Y.; Snell, E. E. J. Med. Chem. 1967, 10, 341-

44.

74) Wehrle, B.; Zimmermann, H.; Limbach, H. H. J. Am. Chem. Soc. 1988,

10, 7014-7024.

3

(

(75) Harruff, R. C.; Jenkins, W. T. Org. Magn. Reson. 1976, 8, 548-557.

(76) Witherup, T. H.; Abbott, E. H. J. Org. Chem. 1975, 40, 2229-2234.

1

4444 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

The solvent was removed, leaving a yellow oil. The crude product was

purified by column chromatography (silica, CH Cl /CH OH, 100:1).

with platinic oxide in ethanolic hydrochloric acid to the ¹⁵N-labeled

product. The product reacted positively with ninhydrin (TLC, 1% in

2

3

It was necessary to use HPLC under the same conditions to purify the

acetone). Detailed procedures are described in refs 67-70. Yield:

200 mg (9%, based on used ¹⁵NH

OH‚HCl). H NMR (500.13 MHz,

1

final product. The products were stored under inert gas at 5 °C. All

2

1

5

products were analyzed by mass spectroscopy, and the N isotopic

enrichment was estimated in the case of the single ring to be >80%,

and for the imino-labeled case, it was >90%. Yield: 97 mg (42.5%).

D

4′-CH

2

3

O, 190 K) (ppm/TMS): δ 2.48 (s, 3H, 2′-CH ), 4.31 (s, 2H,

1

5

⁺), 4.71 (s, 2H, 5′-CH

-), 7.61 (s, 1H, H6).

2

- NH

3

2

13. ¹⁵N-Labeled Pyridoxal (PL) (XI). An amount of 180 mg

1

5

1

15

5

′-Triisopropylsilyl ether of VIb (ring N-labeled). H NMR

(0.99 mmol) of the N-labeled VIIIb was stirred for 1 h in 1 M

(

500 MHz, CDCl

3

, room temperature) (ppm/TMS): δ 1.05 (m, 21H,

hydrochloric acid. The crude product was purified two times by column

chromatography (silica, CH Cl /CH OH, 100:12). After the purification,

the pyridoxal hydrochloride was neutralized with sodium hydrogen

carbonate and was then lyophilized. The product was analyzed by

analytical HPLC (Luna C8(2) column; eluent acetonitrile/water/

trifluoroacetic acid, 95/5/0.1%). Yield: 70 mg (41%). (Purity according

2

15

1

Si(CH(CH

3

))

3

), 2.38 (s, 3H, Ar-CH

3

), 2.50 (d, 3H, J( N, H) )

-), 7.94

d, 1H, J( N, H) ) 10.5 Hz, H6), 9.19 (s, 1H, H4′), 14.07 (s, 1H,

2

3

(

.0 Hz, 2′-CH

3

1

), 4.95 (s, 2H, 5′-CH

2

-), 7.29 (s, 4H, -C

H

6 4

2

15

1

5

1

H3′). 5′-Triisopropylsilyl ether of VIc (imino N-labeled). H NMR

500 MHz, CD Cl , room temperature) (ppm/TMS):δ 1.05 (m, 21H,

), 2.38 (s, 3H, Ar-CH ), 2.50 (s, 3H, 2′-CH ), 4.95 (s,

-), 7.29 (s, 4H, -C -), 7.94 (s, 1H, H6), 9.21 (d, 1H,

(

2

1

Si(CH(CH

H, 5′-CH

3

))

3

to NMR <70%.) H NMR (500.13 MHz, D O, 190 K, PL in cyclic

2

63

2

6

H

4

hemiacetal form ) (ppm/TMS): δ 2.46 (s, 3H, 2′-CH ), 5.14 (s, 1H,

3

2

15

1

4

1

4

1

J( N, H) ) 2.9 Hz, H4′), 14.03 (s, 1H, H3′).

H5′a), 5.18 (d, J( H, H) ) 1.9 Hz, 1H, H5′b), 6.58 (d, J( H, H) )

1

5

2

15

1

9

.

N-Labeled (Ring and Doubly) N-(Pyridoxylidene)methyl-

1.9 Hz, 1H, H4′), 7.57 (d, J( N, H) ) 3.8 Hz, 1H, H6).

14. N-Labeled Pyridoxal-5′-phosphate (PLP) (XII). Under an

1

5

15

amine (VIIIa,b). To 90 mg (0.53 mmol) of N-labeled pyridoxine

V) dissolved in 10 mL of water was added a freshly prepared, 20 mL

(

inert gas stream, 6.50 g of conc phosphoric acid, H PO , and 5.00 g of

3

4

aqueous solution with manganese dioxide (prepared from 0.3 g of

potassium permanganate, 0.4 g of sodium bisulfite and 0.4 mL of 50%

sulfuric acid with stirring for 30 min). After standing overnight, the

2 5

phosphorus pentoxide, P O , were mixed. To the chilled mixture was

added 1.0 g (3.9 mmol) of VIa. The viscous mixture was mixed

thoroughly and heated for 9 h at 40 °C under a constant inert gas stream.

The reaction mixture was then chilled on ice, 5 mL of 0.1 M

hydrochloric acid was added slowly, and the solution was heated for

10 min at 60 °C, followed by standing overnight. The reaction mixture

was diluted with water and applied to a strong cationic exchanger

(Amberlite IR-120; 0.3-1.2 mm (15-50 mesh ASTM)) and eluted with

water. The product was isolated by using a flow rate of 1 drop/s. All

fractions were checked by TCL (silica gel, CH Cl /CH OH, 2:3). The

pH was adjusted with 1 M NaHCO

3

solution to 7.78. To the reaction

mixture was added dropwise a solution of 50 mg (0.75 mmol) of

15

methylamine hydrochloride or (95%) N-labeled methylamine hydro-

chloride. The pH was readjusted to 7.8. After stirring for several hours

at room temperature, the product was extracted with dichloromethane.

The combined organic phases were dried over NaHCO

was removed, and the product was dried under vacuum overnight. The

yellow crude product was dissolved in CH Cl and purified by column

chromatography (cellulose, CH Cl ) monitored by H NMR in the liquid

3

. The solvent

2

3

2

combined fractions were lyophilized, and a yellow powder was

obtained. The product was analyzed by mass spectrometry, and the

1

2

1

5

15

state. Thirty milligrams (0.22 mmol) of doubly N-labeled VIIIb was

N isotopic enrichment was estimated to be >80%. Yield: 0.2 g (21%).

1

obtained and analyzed by low-temperature, liquid-state H NMR.

H NMR (500 MHz, DMSO-d , room temperature) (ppm/TMS): δ 2.43

6

31

1

3

1

Yield: 0.22 mg (42%). H NMR (500.13 MHz, CD

2

Cl

2

, 230 K) (ppm/

(s, 3H, 2′-CH ), 5.19 (d, J( P, H) ) 6.8 Hz, 2H, 5′-CH -), 8.12 (d,

3

2

15

1

31

1

TMS): δ 2.39 (s, 3H, 2′-CH

3

), 3.14 (s, 1H, H5′′ (-OH)), 3.52 (s,

J( N, H) ) 10.8 Hz, 1H, H6), 10.3 (s, 1H, H4′). P { H} NMR

(202.46 MHz, D O, room temperature) (ppm/85% H PO ): δ 2.7 (s,

2

15

1

3

3 2

H, N-CH ), 4.69 (s, 2H, 5′-CH -), 7.63 (d, 1H, H6, J( N, H) )

2

3

4

1

15

1

2-

1

0.45 Hz), 8.82 (s, 1H, H4′), 14.60 (d, 1H, J( N, H) ) 5.61 Hz, H3′).

1P, -PO₃).

1

5

1

15

N { H} NMR (50.61 MHz, CD

2

Cl

), 166 ppm (s, N1 ring).

0. ¹⁵N-Labeled (Imino) N-(Pyridoxylidene)methylamine (VIIIb).

An aqueous solution of 300 mg (1.47 mmol) of pyridoxal hydrochloride

was neutralized with NaHCO

; 100 mg (1.47 mmol) of (95% ¹⁵N)

methylamine hydrochloride was added, and the reaction mixture was

adjusted with 1 M NaHCO to pH 7. After stirring for several hours at

room temperature, the product was extracted with dichloromethane.

2 4

, 190 K) (ppm/ NH Cl): δ

D. Preparation of NMR Samples. 1. Preparation of Polycrys-

1

5

1

71 ppm (s, N2, N-CH

3

15

talline Powders 1a-1j. Polycrystalline powders of the N-labeled

1

N-(pyridoxylidene)tolylamine (1b) and their 1:1 acid-base complexes

1

5

15

1b- N-3,5-dichlorobenzoic acid (1c), 1b- N-3-nitro-4-chloroben-

15

3

zoic acid (1d), 1b- N-4-nitrobenzoic acid (1e), 1b- N-2-nitroben-

zoic acid (1f), 1b- N-3,5-dinitrobenzoic acid (1g), 1b- N-3,5-

1

5

15

3

dinitro-4-methylbenzoic acid (1h), 1b- N-hydrochloric acid (1i), and

1

5

1b- N-trichloroacetic acid (1j) were prepared as follows. Solutions

of the purified compounds in dichloromethane were evaporated, washed

with diethylether, and dried under high vacuum overnight. The

compounds were deuterated by repeated distillation from methanol-d₁,

leading to 1b-d, 1g-d, and 1j-d, which were dried under high vacuum

15

The solvent was removed, and a yellow product, N-(pyridoxylidene)-

methylamine, was obtained in a yield of 193 mg (73%). The product

1

was analyzed by low-temperature liquid-state H NMR and mass

1

5

spectrometry; N isotopic enrichment was estimated to be >88%.

63,64

1

Yield: 193 mg (73.0%). Mp 145-146 °C (146-151 °C ). H NMR

500.13 MHz, CD Cl , 190 K) (ppm/TMS): δ 2.24 (s, 3H, 2′-CH ),

), 4.53 (s, 2H, 5′-CH -), 6.51 (s, 1H, H5′′ (-OH)),

.71 (s, 1H, H6), 8.78 (s, 1H, H4′), 14.85 (d, 1H, J( N, H) ) 10.3

overnight. Complex 1i-d was prepared from DCl solution in D O and

2

(

2

3

lyophilized. The extents of deuteration, reported in Table 2, were

1

3

.47 (s, 3H, N-CH

3

2

estimated by integration from H NMR liquid state spectra.

1

15

1

7

2

. Preparation of Polycrystalline Powders 2a-2e. Polycrystalline

1

5

1

Hz, H3′). N { H} NMR (50.61 MHz, CD

2

Cl

2

, 300 K) (ppm/

15

powders of the N-labeled N-(pyridoxylidene)methylamine (2a) and

their 1:1 acid-base complexes 2a- N-4-chlorobenzoic acid (2b),

2a- N-4-nitrobenzoic acid (2c), and 2a- N-3,5-dinitrobenzoic acid

(2d) were prepared as follows. Solutions of the purified compounds in

methanol were evaporated and dried. In the case of 2a- N-4-

tolylsulfonic acid (2e), dioxane was used instead, in order to avoid

decomposition, and was removed under reduced pressure at 40 °C.

Unlabeled samples of 2a to 2e for solid state ¹H and N NMR

measurements at natural abundance were prepared in a similar way.

The stability of 2a in the complexes was analyzed by H NMR in the

liquid state. Deuteration of the N-imino-labeled (2a) and the 2a-4-

15

NH

4

Cl): δ 252.36 (s, N-CH

1. ¹⁵N-Labeled p-Toluidine (IX). This was prepared from p-toluyl

3

).

15

1

5

15

chloride and NH

4

Cl followed by treatment of the N-labeled

1

5

p-toluamide obtained with sodium hypobromite solution. The synthesis

1

5

59,60

of N-labeled p-toluidine was described previously.

The product

was checked by mass spectrometry, and the ¹⁵N isotopic enrichment

was estimated to be 96%.

15

1

2. ¹⁵N-Labeled (Imino) Pyridoxamine (PM) (X). This was

1

prepared from 5 g (24.5 mmol) of pyridoxal hydrochloride and 1.0 g

15

(

14.2 mmol) of N-enriched hydroxylamine hydrochloride, which reacts

with sodium acetate to give the oxime. The oxime was hydrogenated

nitrobenzoic adduct (2c) was performed by repeated distillation of

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4445

A R T I C L E S

Sharif et al.

Table 1. NMR Parameters for the Aldenamine and Aldimine Schiff Bases and their 1:1 Acid-Base Complexes with Benzoic Acid Derivatives

a

(¹H)

O1H1N1

1.0

ppm

(¹⁵N)^b

O1H1N1

1.0

/ppm

(¹H)

(¹⁵N)^c

O2H2N2

1.0

¹∆N2(D) ^d

N2

acid−base complexes

pK

a

δ

−

OH1

O2H2N2

±

±2.0

/

/ppm

aldenamine Schiff base:

protected 1b

1a

1b

1c

1d

1e

1f

1g

1h

1i

282.5(0)

13.6

14.2

14.7

14.8

14.2

14.0

15.5

14.8

15.8

16.8

254.6(-21.4)

253.6(-22.4)

249.6(-26.4)

250.1(-25.9)

265.0(-11.0)

261.0(-15.0)

246.8(-29.3)

252.7(-23.3)

244.4(-31.6)

238.0(-38.0)

1

b (free base)

261.8(-20.7)

222.3(-60.2)

210.9(-71.6)

205.5(-77.0)

187.4(-95.1)

177.9(-104.6)

177.6(-104.9)

170.3(-112.2)

166.3(-116.2)

3.0

b + 3,5-dichloro b. a.

b + 3-nitro-4-chloro b. a.

b + 4-nitro b. a.

3.14

3.66

3.44

2.17

2.82

2.97

18.2

18.8

20.2

18.7

18.3

18.2

15.8

b + 2-nitro b. a.

b + 3,5-dinitro b. a.

b + 3,5-dinitro-4-methyl b. a.

b + hydrochloric acid

b + trichloroacetic acid

-7.0

0.66

1j

aldimine Schiff base:

f

13.6^e

14.3

14.1

14.4

14.0

2

a (free base)

a + 4-chloro b. a.

a + 4-nitro b. a.

a + 3,5-dinitro b. a.

a + 4-tolylsulfonic acid

2a

2b

2c

2d

2e

268.5(-14.0)

261.2(-18.8)

147.8(-132.2)

144.3(-135.7)

150.0(-130.0)

138.6(-141.4)

2.9

3.99

3.44

2.82

19.0

18.4

17.2

15.1

192.4(-90.1)^g

182.3(-100.2)

181.1(-101.4)

f

-3.4

g

-6.65

163.8(-118.7)^g

PLP and analogues:

pyridoxal-5′-phosphate

pyridoxine

pyridoxine hydrochloride

pyridoxal

pyridoxamine dihydrochloride

pyridoxamine

PLP

PN

PN‚HCl

PL

PM‚2HCl

PM

170.2(-112.3)

249.5(-33.0)

158.3(-124.2)

164.8(-117.7)

170.6(-111.9)

10.9^h

i

-4.3

a

b

pKa values of the acid proton H1 involved in the intermolecular O1H1N1 hydrogen bond, obtained from refs 83-86. Values of δ(O1H1N1) for the

c

20

pyridine nitrogen in brackets are with respect to solid 1a, resonating at 282.5 ppm. With respect to the methoxy-protected Schiff base, resonating at 276

and 280 ppm for the 1 and 2, respectively.

was assumed to be due to the small geometrical changes in the centrosymmetric dimer.

from spectra at natural abundance. Ammonium group -NH3 Cl . The amine group from N-imino-labeled PM. All H and N chemical shifts are with

respect to external solid TSP and NH4Cl, respectively.

9

4,95

d

1

40 e

The secondary isotope effect ∆N2(D) is defined as ∆N(D) ) δ(ODN) - δ(OHN).

The other signal

46

f

15

g

From the doubly N-labeled aldimine Schiff base. Obtained

h

+

-

i

15

1

15

1

5

Table 2. Chemical Shielding Parameters of Hydrogen-Bonded Acid-Base Complexes

b

calcd c

iso

exptl a

iso

¹∆N1(D)^d

2.0

e

f

solid state

:1 acid base

complexes

δ

±

xx

δ

±

yy

δ

±

zz

δ

D

DN

r

DN

1

−

1

±

3

±

1.0

±

75

±

0.08

/Å

% D

/ppm

/Hz

aldenamine Schiff base

1b

1g

1i

0

343

301

284

286

488

253

217

206

-37

-9

-11

0

265

182

163

164

261.8

177.9

170.3

166.3

1b + 3,5-dinitro b. a.

1b + hydrochloric acid

1b + trichloroacetic acid

1j

deuterated 1b

1b-d + 3,5-dinitro b. a.

1b-d + hydrochloric acid

1b-d + trichloroacetic acid

1g-d

1i-d

1j-d

60

100

305

284

286

233

217

206

-13

-11

0

175

163

164

172.9

168.5

165.5

-5.0

-1.8

-0.8

1286

1349

1481

1.13^g

1.11

1.08

h

a

δiso^e^x^p^t^l: N isotropic chemical shift from N CP MAS spectra and referenced to external solid ¹⁵NH4Cl. ^bδxx, δyy, δzz: the components of the CSA

15

c

calcd

d 1

1

e

tensor. δiso

) 1/3(δxx + δyy + δzz). ∆N1(D): secondary isotope effect is defined as ∆N1(D) ) δ(ODN) - δ(OHN). DDN: dipolar coupling constants.

g

f

rDN: cubic average distances, N‚‚‚D, obtained by line-shape analysis. Euler angles in the coordinate system of the CSA tensor were set to 0 and 90°. With

h

a predicted rOH distance of 1.45 Å. With a predicted rOH distance of 1.60 Å. For further details on the theory of the dipolar heteronuclear coupling, see

Supporting Information.

lations of hydrogen bonds of the general type A-H‚‚‚B.⁷

The bond valences pi of the A-H and of the H‚‚‚B bonds are

defined as described in the following, and in the valence bond

framework, it is assumed that the total bond order to hydrogen

8-81

methanol-d

1

from the sample at room temperature, leading to 2a-d and

2

c-d, which were dried under high vacuum overnight. The percent

deuteration in both compounds was >50%, as checked by integration

1

of H NMR liquid state spectra.

3

. Preparation of PLP and PLP Analogues. The syntheses of

82

is unity, for example, p1 + p2 ) 1.

1

5

the N-labeled PM, PLP, and PN in the neutral and hydrochloride

form are described in the preceding sections. Pyridoxal hydrochloride

o

2

p ) exp{-(r - r )/b } and p ) exp{-(r - r )/b } (1)

1

2

3

(PL‚HCl) from Aldrich was neutralized with 1 M NaHCO to PL and

then lyophilized. Pyridoxamine hydrochloride (PM‚HCl) from Aldrich

was used without further purification.

Here, r1 ) rAH represents the A‚‚‚H distance, r2 ) rHB is the

(

77) Pauling, L. J. Am. Chem. Soc. 1947, 69, 542-553.

78) Steiner, T. Angew. Chem., Int. Ed. 2002, 41, 48-76.

79) Steiner, T.; Majerz, I.; Wilson, C. C. Angew. Chem., Int. Ed. 2001, 40,

2651-2654.

Theoretical Section

Geometric and NMR Hydrogen Bond Correlations. In

(80) Steiner, T.; Wilson, C. C.; Majerz, I. Chem. Commun. 2000, 1231-1232.

81) Steiner, Th. J. Phys. Chem. A 1998, 102, 7041-7052.

previous work, it has been shown that the valence bond orders

(

7

defined by Pauling are useful for describing geometric corre-

(82) Brown, I. D. Acta Crystallogr., Sect. B 1992, 48, 553-572.

4446 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP

−

Enzyme Model Systems

A R T I C L E S

Scheme 6. (a) Parameters Characterizing Geometry of the

Hydrogen Bond: Distances rAH, rBH and the Angle R. (b) Definition

of q1 ) 1/2(rAH - rHB) and q2 ) rAH + rHB as the Divergence of the

Proton from the Center of the Bond and the Heavy Atom Distance,

Respectively

H‚‚‚B distance, and p1 and p2 are the corresponding valence

bond orders of the diatomic units. The parameters r1 and r2

are the equilibrium distances in the hypothetical free diatomic

units AH and HB, while b1 and b2 are the parameters describing

the decays of the bond order when the bond is stretched.

Therefore, it follows that r1 and r2 depend on each other. For

o

81

o

OHN hydrogen bonds, Steiner proposed the parameter set rOH

)

0.942, rHN^o) 0.992, bOH ) 0.371, and bHN ) 0.385 Å.

cal shifts of the hypothetical free molecular units into which

the OHN hydrogen bond can formally dissociate. The parameters

δ(OHN)* and δ(OHN)* are the “excess” terms that describe

the deviation of the quasisymmetric hydrogen bond from the

average of the hypothetical limiting molecular units. These

parameters were obtained by fitting the calculated data points

to the experimental ones, as discussed later in this paper.

Recently, it was assumed that these equations are only valid

for equilibrium geometries, as zero-point vibrations were not

3

9,40

taken into account.

In order to take the latter into account,

the following empirical bond order equations for the equilibrium

_b_o_n_d_o_r_d_e_r_s_w_e_r_e_p_r_o_p_o_s_e_d3

9,40

H

f

H

g

p₁) p - c (p p ) (p - p ) - d (p p ) )

1

2

1

2

1 2

H

o

exp{-(r₁- r )/b }

Results

1

H

f

H

g

The results of the solid state H and ¹⁵N NMR experiments

are depicted in Figures 1 and 2 and in Figures S1-S5 of the

Supporting Information. All parameters obtained are gathered

in Tables 1 and 2.

p₂) p + c (p p ) (p - p ) - d (p p ) )

2

1

2

1

2

1 2

H

o

2

^e^x^p^{^-⁽^r2 - r )/b } (2)

2

H

The parameters c ) 360, d ) 0.7, and f ) 5, g ) 2 were

used and have been adapted empirically to the case of OHN

hydrogen bonds to heterocyclic bases.⁴⁰Expressing the cor-

relation in terms of normal coordinates, defined as q1) 1/2(r1

A. The Aldenamine Schiff Bases and Their Acid-Base

Adducts. The H MAS NMR solid state spectra of 1a-1j were

observed using a fast-spinning speed of 24 kHz. Since fast

spinning removes homonuclear dipolar coupling, to a large

extent, we were able to observe the signals of the hydrogen-

bonded protons of the intra- and intermolecular OHN hydrogen

bonds depicted on the left side of Figure 1. When the acidity

1

-

r2) and q2 ) r1 + r2, is useful. For a linear hydrogen bond,

q1 represents the deviation of the proton distance from the

hydrogen bond center, while q2 represents the distance between

the heavy atoms A and B, as indicated in Scheme 6. Note that

the correlation is independent of the hydrogen bond angle.

Herein, A-H‚‚‚B corresponds to the intra- and intermolecular

OHN hydrogen bond in the Schiff base model complexes

83-86

1

of the carboxylic acid

intermolecular O1H1N1 hydrogen bond (labeled as H1 in Figure

) is shifted from 18.2 ppm to a maximum of 20.2 ppm and

is increased, the H signal of the

1

then back to 15.8 ppm. A similar behavior was observed

previously for 2,4,6-trimethylpyridine-acid complexes in the

organic solid state. At the same time, the proton of the

intramolecular O2H2N2 hydrogen bond (labeled as H2 in Figure

) is slightly shifted from 14 to 16 ppm. In the 1b-hydrochloric

(

Scheme 3); A is oxygen of the carboxylic acid or the phenolic

group, and B is the ring or imino nitrogen of the Schiff bases.

A typical correlation curve⁴⁰of q2 versus q1 will be presented

later (solid line in Figure 3a); it shows that, when the proton is

transferred from A to B, a hydrogen bond contraction occurs

that is maximal when the proton is located approximately in

the hydrogen bond center. In order to visualize the quantum

effects, a typical corrected⁴⁰q2 versus q1 curve is also presented

later (dashed line in Figure 3a). Throughout this paper, the

corrected bond valences are used in order to describe experi-

mental data because the equilibrium bond valences can be used

only as an approximation.

35

1

acid adduct (1i), only one signal at 15.8 ppm was detected for

both hydrogen-bonded protons.

The ₁₅_N_s_p_e_c_t_r_a_o_f_c_o_m_p_o_u_n_d_s₁_a_-₁_j_,_s_i_n_g_l_y15N-labeled in

the ring position, are presented in the center column of Figure

. The signals shifted monotonically to the high field with the

increasing acidity of the proton donor. Compound 1a resonates

at a lower field than 1b because of intermolecular O1H1N1

hydrogen bonds between the hydroxyl groups of 1b and the

pyridine ring. These measurements helped to assign the

signals of the intramolecular O2H2N2 hydrogen bond (imino

1

The bond orders are additionally useful for deriving correla-

tions between NMR parameters and hydrogen bond geom-

etries.³

46

15

N

4-40

The following relations were proposed for the

15

nitrogen N2) in the doubly N-labeled compounds, depicted

in the right column of Figure 1.

chemical shifts of OHN hydrogen bonds.

o

H

OH

o

H

In the corresponding deuterated compounds, 1g-d, 1i-d, and

j-d (see Figure S1 of the Supporting Information), high-field

δ(OHN) ) δ(N) p

+ δ(HN) p

+

HN

1

H

4

δ(OHN)* p_O_Hp_H_N(3)

shifts of the ring nitrogen signals were observed, indicating the

1

presence of secondary H/D isotope effects, ∆N(D) ) δ(ODN)

o

H

o

H

1

5

δ(OHN) ) δ(OH) p

+ δ(HN) p

+

- δ(OHN), on the N chemical shifts and hence changes in

the intermolecular O1H1N1 hydrogen bond geometries.⁴⁰

The chemical shifts of the imino nitrogen N2 are fairly

constant from 1a to 1d. Then, a small low-field shift occurs

OH

HN

H

4

δ(OHN)* p_O_Hp_H_N(4)

The pKa values associated with the acidic proton in the acid-

base complexes can be related to q1 .

H 35,41

(

83) Polansk, J.; Bak, A. J. Chem. Inf. Comput. Sci. 2003, 43, 2081-2092.

84) Kort u¨ m, G.; Vogel, N.; Andrussow, K. Dissociation Constants of Organic

Acids in Aqueous Soluions; Butterworth: London, 1961.

85) French, D. C.; Crumrine, D. S. J. Org. Chem. 1990, 55, 5494-5496.

86) Crumrine, D. S.; Shankweiler, J. M.; Hoffman, R. V. J. Org. Chem. 1986,

51, 5013-5015.

H

pK ) A - Bq₁

(5)

a

(

o

Here, δ(N) , δ(HN) , δ(OH) , and δ(HN) represent the chemi-

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4447

A R T I C L E S

Sharif et al.

Figure 1. Solid state spectra of the aldenamine Schiff bases 1b and their 1:1 acid-base complexes with benzoic acid derivatives 1a-1j. From the left are

1

15

1

15

1

shown H MAS ECHO NMR spectra, 600 MHz, 24 kHz; N { H} CP MAS spectra, 30.41 MHz, 6 kHz, N ring-labeled 1a-1j; N { H} CP MAS

1

5

spectra, 30.41 MHz, 6 kHz; N doubly labeled 1a-1j.

(

1e), followed again by small upfield shifts (1f-1j). For the

B. The Aldimine Schiff Bases and Their Acid-Base

Adducts. A collection of typical NMR spectra of the polycrys-

talline aldimine Schiff bases 2a and their 1:1 acid-base

complexes with benzoic acid derivatives 2a-2e are depicted

deuterated compound 1b-d, a 3 ppm shift to a lower field of

the imino nitrogen signal (not shown) was observed, indicating

a secondary H/D isotope effect, ∆N2(D), on the intramolecular

O2H2N2 hydrogen bond geometry.

1

in Figure 2. The H MAS spectra of 2a-2e are in the left

1

5

15

In order to estimate the hydrogen-nitrogen distances in the

column, the N CP MAS NMR spectra of the N selectively

1

intermolecular O1H1N1 hydrogen bond, we measured the H-

imino-labeled adducts are in the center column, and the

1

5

15

decoupled N CP NMR spectra of static deuterated powdered

compounds 1g-d, 1i-d, and 1j-d at 30.41 MHz (see Figure S1

of the Supporting Information). They contain information about

the chemical shift anisotropy of the pyridine nitrogen and the

dipolar H- N coupling. The spectra were analyzed as

described previously for 2,4,6-trimethylpyridine-acid com-

plexes.³⁵As in this reference, the spectra were simulated by

assuming that the N‚‚‚D axis coincides with the NC4 axis, that

is, that it is parallel to δyy. This assumption has been used and

justified using ab initio methods by Solum et al. in the case

of pyridine and pyridinium salts. Thus, only the values of the

dipolar coupling constants D were varied but not the Euler

angles relating the chemical shielding and the dipolar DN

coupling tensors.

corresponding spectra at natural N abundance are in the right

1

5

column, which helped to assign the N signals. We assign the

proton signal of aldimine Schiff base 2a at 13.6 ppm to the

intramolecular O2H2N2 hydrogen bond. Another low-field

proton signal of 2a at 15 ppm was observed, which was difficult

to assign. According to the X-ray crystallographic structure, the

aldimine Schiff base 2a forms cyclic centrosymmetric dimers

2

15

46

in the solid state, containing two intermolecular O1H1N1

hydrogen bonds between the side-chain hydroxyl groups and

the pyridine rings. As a consequence, the low-field proton signal

can be assigned to small geometrical changes in the intramo-

lecular O1H2N1 hydrogen bonds, which result in two different

proton signals. On the other hand, the possibility that this signal

derives from the intermolecular O1H1N1 hydrogen bond, where

the side-chain hydroxyl group is involved, is less possible since

87

DN

The parameters of the chemical shielding tensor and the cubic-

2

15

averaged H- N distances, rDN, obtained are listed in Table

2

NMR

20

the signal is located at too low field for such a group. In the

15

. Additional details on the theory of dipolar N solid state

adduct 2b with p-chlorobenzoic acid, the proton in the inter-

molecular O1H1N1 hydrogen bond was observed at 19.0 ppm

88-93

are given in the Supporting Information.

(

87) Solum, M. S.; Altmann, K. L.; Strohmeier, M.; Berges, D. A.; Zhang, Y.;

Facelli, J. C.; Pugmire, R. J.; Grant, D. M. J. Am. Chem. Soc. 1997, 119,

(90) Hoelger, C.-G.; Limbach, H. H. J. Phys. Chem. 1994, 98, 11803-11810.

(91) Bork, V.; Gullion, T.; Hing, A.; Schaefer, J. J. Magn. Reson. 1990, 88,

523-532.

9

804-9809.

(88) Mehring M. NMR Basic Principles and Progress 11, High Resolution NMR

in Solids; Springer: Berlin, Germany, 1978.

(92) Hughes, E.; Gullion, T.; Goldbourt, A.; Vega, S.; Vega, A. J. J. Magn.

Reson. 2002, 156, 230-241.

(89) Gullion, T.; Schaefer J. In AdVances in Magnetic Resonance; Warren, W.

S., Ed.; Academic Press: San Diego, CA, 1989; Vol. 13, p 57.

(93) Gullion, T. J. Magn. Reson. 2000, 146, 220-222.

4448 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

Figure 2. Solid state spectra of the aldimine Schiff bases 2a and their 1:1 acid-base complexes with benzoic acid derivatives 2b-2e. From the left are

1

15

1

15

1

shown H MAS ECHO NMR spectra, 600 MHz, 24 kHz; N { H} CP MAS spectra, 30.41 MHz, 6 kHz, N-imino-labeled 2a-2e; N { H} CP MAS

spectra, 30.41 MHz, 6 kHz, in natural abundance; 2a and 2c are ¹⁵N doubly labeled.

and was high field shifted to 14.1 ppm in 2e when the acidity

of the acid increased.

available for the compounds studied here by NMR. Finally, we

discuss potential biological implications arising from these

results.

In 2a, both the pyridine nitrogen signal N1 and the Schiff

base nitrogen signal N2 appear at a low field but abruptly shifted

to a high field, indicating protonation of both nitrogens when

the adducts were formed. Deuteration leads to a low-field shift

of 2.9 ppm of the N2 signal in 2a-d but leads to a high-field

shift of -3.4 ppm in the adduct 2c-d with 4-nitrobenzoic. The

A. Hydrogen Bond Distances in PLP and Its Derivatives.

Since it is difficult to obtain proton positions using X-ray

diffraction, in this section, we discuss ways to obtain this

15

1

information from the N and H NMR chemical shifts of PLP

and its derivatives.

1

5

N spectra of the deuterated compounds are given in Figure

1

5

1

. HN Distance- N Chemical Shift Correlation for the

S2 in the Supporting Information.

C. The Cofactors PLP and Their Analogues. Finally, we

Intermolecular O2H2N2 Hydrogen Bond. We start with a

discussion of the geometric correlation for OHN hydrogen bonds

obtained from low-temperature neutron crystallographic data

1

5

15

measured the N CP MAS NMR spectra of N-labeled PLP,

1

5

15

N-labeled PN, N-labeled PN‚HCl, N-labeled PM, as well

as PL and PN‚HCl at natural abundance. These spectra are given

in Figures S3-S5 in the Supporting Information. The

7

8-81

40

reported by Steiner

a. The solid line represents the correlation between the

hydrogen bond coordinates q2 ) rOH + rHN and q1 ) 1/2(rOH

rHN), which refer to the equilibrium geometries where zero-

point vibrations are not taken into account. According to eq 1,

and Limbach et al., depicted in Figure

3

1

5

N

isotropic chemical shifts are collected in Table 1.

-

Discussion

o

1

_W_e_h_a_v_e_c_a_r_r_i_e_d_o_u_t_s_o_l_i_d_s_t_a_t_e_H_a_n_d15N NMR experiments

the correlation line depends only on the values of r1 and r2 ,

on the equilibrium distances in the hypothetical free diatomic

units AH and HB, and on the values of b1 and b2, the valence

bond decay parameters. In the dashed line of Figure 3a, an

empirical correction for zero-point motions according to eq 2

on polycrystalline hydrogen-bonded, 1:1 complexes of Schiff

bases of the cofactor PLP with carboxylic acids, depicted in

Scheme 4. These complexes were designed to mimic the

cofactor in the active sites of a variety of enzymes. In particular,

we were interested in if and how the intermolecular O1H1N1

hydrogen bond and the intramolecular O2H2N2 hydrogen bond

interact which each other. In this section, we first correlate the

4

0

is included, which reproduces the experimental neutron data

well, as shown in Figure 3a.

The solid state, dipolar NMR studies on the polycrystalline

aldenamine Schiff base-acid complexes 1g-d, 1i-d, and 1j-d

allowed the inverse cubic-averaged N‚‚‚D distances to be

1

15

H and N solid state NMR parameters with the hydrogen bond

geometries, and then, we discuss the crystallographic structures

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4449

A R T I C L E S

Sharif et al.

and 2 from the corresponding intrinsic ¹⁵N chemical shifts even

in the cases where these distances could not be obtained by

dipolar NMR. Note that, in this correlation, we neglect the

difference between cubic and mean average distances. As

discussed in the Experimental Section, the error on rHN or rDN

increases when its value is increased. Assuming linear hydrogen

bonds and proceeding as described previously in this paper for

1

g-d and 1j-d, the corresponding values of rOH were estimated

from the correlation curve in Figure 3a. These are presented in

Table 3.

2

. HN Distance- ¹⁵N Chemical Shift Correlation of the

Intramolecular O2H2N2 Hydrogen Bond. The solid state

NMR spectra described above indicate that the proton in the

intramolecular OHN hydrogen bond is located near oxygen in

crystalline 1b, 1f, and 2a but near nitrogen in 2c. No signs of

a tautomerism between an OH and a HN form (Scheme 3) were

observed, as is usually found for these types of molecules in

2

0

the liquid state. Therefore, we did not need to take this

tautomerism into account in estimating the HN distances of the

intramolecular O2H2N2 hydrogen bonds of 1 and 2 by NMR.

We proceeded in a similar way as discussed in the preceding

sections for the intermolecular O1H1N1 hydrogen bond, that

is, estimated the hydrogen bond geometries by using a sim-

ilar correlation curve as that depicted in Figure 3b. The

difference was that we used the limiting chemical shift value

of -158 ppm for the protonated nitrogen (see eq 3), which was

estimated previously for the intramolecular O2H2N2 hydrogen

Figure 3. Geometric OHN hydrogen bond correlations. (a) q2 versus q1.

Equilibrium (solid line) and corrected (dashed line) correlation curves

calculated according to ref 40. The circles refer to neutron diffraction

8

1

geometries. The triangles represent the recently published neutron

7

8-80

diffraction data.

trimethylpyridine-acid complexes. The open circles correspond to crystal-

line aldenamine Schiff base-acid complexes. (b) DN distance versus ¹⁵

The diamonds correspond to crystalline 2,4,6-

35

20

bond of a model Schiff base. The distances obtained are

N

included in Table 3; a plot of the corresponding correlation curve

is presented in the Supporting Information. Table 3 also includes

the intramolecular heavy atom distances, rON, estimated using

a hydrogen bond angle R(O2H2N2) ≈ 150°. This value was

chemical shift correlation. The diamonds represents the rHN distances for

_,₄_,₆_-_t_r_i_m_e_t_h_y_l_p_y_r_i_d_i_n_e_-_D_A_c_o_m_p_l_e_x_e_s_f_r_o_m_d_i_p_o_l_a_r_,_s_o_l_i_d_s_t_a_t_e_N_M_R_,₃5

2

with respect to the frozen bulk 2,4,6-trimethylpyridine. The dashed line

o

35,40

corresponds to the latter complexes by using δ(HN) ) -130.

The open

circles correspond to crystalline aldenamine Schiff base-acid complexes,

with respect to solid 1a, resonating at 282.5 ppm. The solid line corresponds

to the latter complexes by using δ(HN) ) -140. For the calculation of

46

estimated from X-ray diffraction, which may exhibit a

o

systematic error.

the curves, see text.

B. Comparison between Distances Obtained by Diffraction

Methods and by Solid-State NMR. The known X-ray crystal-

estimated (cf. Table 2). The corresponding distances rOD of 1g-d

and of 1j-d were then varied such that the resulting hydrogen

_b_o_n_d_c_o_o_r_d_i_n_a_t_e_s_q₂D ₎_r_O_D₊_r_D_N_a_n_d_q₁D ) 1/2(rOD - rDN)

of 1g-d and of 1j-d fitted the dashed curve in Figure 3a. The

results indicate that the proton is slightly shifted beyond the

hydrogen bond center toward the nitrogen.

In Figure 3b, the cubic average rDN distances of pyridine

derivatives hydrogen bonded to acids are plotted as a function

_o_f_t_h_e_i_r_i_n_t_r_i_n_s_i_c15N chemical shifts. We chose 1a as a reference

1

,2,4,11,12,46

lographic structures

of PLP, PL, and PN and the

neutron structure of PN‚HCl are depicted in Figure 5. Figure 6

contains the X-ray crystallographic structures of the model Schiff

bases 1 and 2 and their acid-base adducts. Some hydrogen bond

distances obtained here by solid state NMR are included for

comparison.

1

. PLP and Its Analogues. PLP in the hydrated form (Fig-

ure 5a) forms an intermolecular O1H1N1 hydrogen bond

between the pyridine ring and the side-chain phosphate group

of an adjacent molecule, exhibiting a N1‚‚‚O1 distance of

for 1g-d, 1i-d, and 1j-d since it is not involved in an inter-

molecular O1H1N1 hydrogen bond and resonates at 282.5 ppm.

The graph also includes the corresponding distances rDN for the

polycrystalline 2,4,6-trimethylpyridine-acid complexes published

4

15

2

.671(6) Å. We predict by N NMR a value of rHN ) 1.08 Å

for the intermolecular O1H1N1 hydrogen bond and a value of

r_O_N= r_O_H+ r_H_N≈ 2.68 Å, which is close to the crystallographic

value. It is tempting to associate this coincidence to the presence

of a linear hydrogen bond. It follows that neutral PLP exhibits

a zwitterionic structure, where the proton comes from the

phosphate group which still carries a remaining proton.

_p_r_e_v_i_o_u_s_l_y_,₃5 where the intrinsic 15N chemical shifts refer to

the value of 268 ppm of neat frozen 2,4,6-trimethylpyridine.

The solid correlation curve in Figure 3b was calculated as

described in the Theoretical Section from eq 3, using a limiting

15

N chemical shift of the hypothetical “free” pyridinium nitrogen

at -140 ppm. This value was obtained by fitting the correlation

curve to the experimental data points in Figure 3b. The dashed

line was calculated using a value of -130 ppm proposed

The crystal structure¹¹of PL in the cyclic hemiacetal form is

presented in Figure 5b. The crystal packing shows an intermo-

lecular O1H1N1 hydrogen bond between the pyridine ring and

the phenolic group of a neighboring molecule, exhibiting an

N1‚‚‚O1 distance of 2.669(1) Å. By NMR, the distances rHN

) 1.06 and rOH + rHN ) 2.73 Å were obtained. The latter is

larger than the distance rON obtained from X-ray diffraction. A

35,40

previously for 2,4,6-trimethylpyridine-acid complexes.

We

1

associate the small difference in the limiting values to the

influence of the chemical structure. Thus, using the solid line

in Figure 3b, we were able to derive values of rHN or rDN for 1

4450 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

Table 3. Comparison of X-ray and NMR Crystallographic Data of the Inter- and Intramolecular OHN Hydrogen Bonds

Crystallographic Values

NMR Values

Intermolecular

r

HN/Å

rOH/Å

rON/Å

R(OHN)/

°

r

HN^h/Å

r

OH^h^,ⁱ/Å

r

ON^h^,^j/Å

O1H1N1 Hydrogen Bond

H1‚‚‚N1

O1‚‚‚H1

O1‚‚‚N1

O1‚‚‚H1‚‚‚N1

H1‚‚‚N1

O1‚‚‚H1

O1‚‚‚N1

PLP/analogue:

a

PLP

0.92

1.74

0.93

0.84

1.76

0.99

1.74

2.671(6)

2.729(5)

2.669(1)

168

177

170

1.08

1.55

1.06

1.08

1.04

1.60

1.06

1.67

2.68

2.61

2.73

b

PN

c

PL

d

PM‚2HCl

f

PN‚×b0HCl

1.040(8)

aldenamine Schiff base:

e

1

b

f

1.86

1.01

0.92

1.55

2.752(7)

2.549(3)

162

170

1.71

1.14

1.01

1.43

2.74

2.57

e

aldimine Schiff base:

e,g

2

a

c

1.95

0.95

0.92

1.71

2.865(2)

2.614(2)

171

160

1.88

1.12

0.99

1.47

2.86

2.59

e

Intramolecular

O2H2N2 Hydrogen Bond

r

HN/Å

r

OH/Å

r

ON/Å

R

(OHN)/

°

r

HN^h/Å

r

OH^h^,ⁱ/Å

r

ON^h^,^j/Å

H2‚‚‚N2

O2‚‚‚H2

O2‚‚‚N2

O2‚‚‚H2‚‚‚N2

H2‚‚‚N2

O2‚‚‚H2

O2‚‚‚N2

aldenamine Schiff base:

e

k

1

b

f

1.71

1.69

1.01

0.95

2.545(6)

2.553(3)

140

148

1.68

1.90

1.02

0.98

2.55

2.78

e

k

aldimine Schiff base:

e,g

k

2

a

c

1.73

0.93

0.92

1.81

2.570(2)

2.595(2)

152

141

1.81

1.05

1.00

1.72

2.73

2.62

e

k

a

X-ray values obtained from ref 4. ^bX-ray values obtained from ref 1. ^cX-ray values obtained from ref 11. X-ray values obtained from ref 12, the

d

crystal structure is the mono hydrochloride. X-ray values obtained from ref 46. From neutron diffraction analysis.²From the centrosymmetric dimer

involving the side-chain hydroxyl group.

rHN. rNO ) rHN + rOH. Estimated by using R(OHN) obtained from X-ray analysis and calculated by using the law of cosines.

e

f

g

4

6

h

NMR crystallographic values are extrapolated from the experimental isotropic N chemical shifts. ⁱrOH ) rON

15

j

k

-

Figure S4 in the Supporting Information). A value of rHN ≈

1

.55 Å was obtained by NMR, that is, rOH ≈ 1.06 Å. The sum

of the two distances, 2.61 Å, is smaller than the crystallographic

value. We assign this difference to different structures of the

amorphous and the crystalline forms. We note, however, that

the proton in amorphous PN is located close to the oxygen in

the intermolecular O1H1N1 hydrogen bond.

The neutron structure of PN‚HCl (Figure 5d) exhibits a rHN

2

15

distance of 1.040(8) Å. From the N chemical shift of

PN‚HCl, we obtained the same value of rHN ) 1.04 Å, which

indicates the reliability of the NMR method.

We performed some preliminary NMR experiments on PM

and its dihydrochloride (PM‚2HCl), see Scheme 1 and Tables

1

and 3. The interested reader can find the results and discussion

in the Supporting Information.

. Aldenamine and Aldimine Model Schiff Bases 1 and 2.

2

The aldenamine Schiff base 1b (Figure 6a) forms intermolecular

O1H1N1 hydrogen-bonded chains involving the hydroxyl side

groups and the pyridine rings of adjacent molecules, exhibiting

4

6

a N1‚‚‚O1 distance of 2.752(7) Å. For the intramolecular

O2H2N2 hydrogen bond, a heavy atom distance of 2.545(6) Å

4

6

was reported. We estimated, by NMR, a rHN distance of

.71 Å and a rON distance of 2.74 Å for the intermolecular

1

Figure 4. ¹H versus ¹⁵N chemical shift correlations for the aldenamine

Schiff base-adducts 1b-1j and the aldimine Schiff base-adducts 2a-2e.

O1H1N1 hydrogen bond, where the proton is located on the

oxygen of the neighboring hydroxyl group. In the intramolecular

O2H2N2 hydrogen bond, the proton is located near the oxygen

atom, as inferred by the estimated rHN distance of 1.71 Å from

NMR. This is expected for an enolimine form of the Schiff base.

The structure of the aldenamine 1b-2-nitrobenzoic acid

adduct (1f) is depicted in Figure 6b. The O1‚‚‚N1 distance of

the intermolecular O1H1N1 hydrogen bond between the car-

(

a) The intermolecular O1H1N1 hydrogen bond. (b) The intramolecular

O2H2N2 hydrogen bond. For the calculation of the curves, see text.

part of this difference arises probably from the margin of error

of NMR, but the presence of a slightly nonlinear H bond is not

excluded.

1

The crystallographic structure of PN is shown in Figure 5c.

PN forms an intermolecular O1H1N1 hydrogen bond between

the pyridine ring and the side-chain hydroxyl group of an

boxylic group and the pyridine ring in 1f is 2.549(3) Å.⁴⁶

similar rON distance for 1b of 2.553(3) Å was found for the

A

4

6

adjacent molecule, with a heavy atom distance of rON )

intramolecular O2H2N2 hydrogen bond. The estimated rHN

1

2

.729(5) Å. The sample studied by NMR was amorphous (see

distance of 1.90 Å by NMR shows that the Schiff base is still

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4451

A R T I C L E S

Sharif et al.

Figure 5. Crystallographic structures of PLP and its analogues. X-ray diffraction adapted (a) for PLP (hydrate form) from ref 4, (b) for PL (hemiacetal)

from ref 11, and (c) for PN from ref 1. (d) Neutron diffraction adapted for PN‚HCl from ref 2. The parameters shown are listed in Table 3.

Figure 6. Crystallographic structures of (a) the aldenamine Schiff base (1b), (b) 1b-2-nitrobenzoic acid (1c), (c) the aldimine Schiff base (2a), and (d)

2

a-4-nitrobenzoic acid (2c) obtained by X-ray diffraction, adapted from ref 46. The parameters are listed in Table 3.

in the enolimine form. Therefore, the bonding in the intramo-

lecular O2H2N2 hydrogen bond is not altered compared to that

in 1b. However, the pyridine ring is now involved in an inter-

molecular O1H1N1 hydrogen bond with the carboxylic group

of the acid added. NMR predicts a rHN distance of 1.14 Å and

a rON distance of 2.57 Å, which are in agreement with X-ray

diffraction. This represents a substantial compression of the

heavy atom rON distance compared to that of the weaker

intermolecular O1H1N1 hydrogen bonds of 1b. The proton is

located near the ring nitrogen N1, leading to a zwitterionic

structure.

dimers, involving intermolecular O1H1N1 hydrogen bonds

between the hydroxylic side group and the pyridine rings. The

O1‚‚‚N1 distance is 2.865(2) Å, which is substantially larger

4

6

than in the corresponding chains of 1b. Also, a O2‚‚‚N2

distance of 2.570(2) Å was reported for the intramolecular

4

6

O2H2N2 hydrogen bond. The basicity of the CH -N group

3

is expected to be larger than that of the corresponding tolyl-N

group in 1b. A N2‚‚‚H2 distance of 1.81 Å was estimated for

the proton in the intramolecular O2H2N2 hydrogen bond by

NMR. The proton remains on the oxygen atom, giving an

enolimine form. Also, we can predict, by NMR, an H1‚‚‚N1

distance of 1.88 Å and an O1‚‚‚H1 distance of 0.99 Å for the

intermolecular O1H1N1 hydrogen bond of 2a. This shows that

The X-ray structure of the aldimine Schiff base 2a is depicted

in Figure 6c. Compound 2a is packed as cyclic centrosymmetric

4452 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

the proton remains on the hydroxyl group in the packed cyclic

centrosymmetric dimers of 2a.

Table 4. Parameters for the NMR Hydrogen Bond Correlations of

the Schiff Bases

δ

(N)^o

δ

/ppm

(HN)^o^c

δ

(OHN)*

/ppm

δ

/ppm

(OH)^o

δ

/ppm

(HN)^o

δ

(OHN)*

The crystal structure of the aldimine 2a-4-nitrobenzoic acid

adduct (2c) is depicted in Figure 6d. As in 1f, an acid-base

complex exhibiting an intermolecular O1H1N1 hydrogen bond

between the carboxylic group and the pyridine ring is formed.

Again, the O1‚‚‚N1 distance is quite short at 2.6142(2) Å, and

/

ppm

/ppm

O1H1N1⁴

O2H2N2²

0,a

0,b

0

-140

-158

0

-5

-3

3

7

5

20

a

The ¹⁵N chemical shifts of the intermolecular O1H1N1 hydrogen bond

4

6

are referenced to solid 1a, resonating at 282.5 ppm with respect to solid

the intramolecular O2‚‚‚N2 distance is 2.595(2) Å. By

NMR, a O1‚‚‚H1 distance of 1.47 Å and a H1‚‚‚N1 distance of

1

5

NH4Cl. ^bThe N chemical shifts of intramolecular O2H2N2 hydrogen

15

20

bond are referenced to the methoxy-protected Schiff base, resonating at

1

.12 Å are estimated, which is in agreement with the crystal-

276 and 280 ppm for the 1 and 2, respectively, with respect to solid

15

94,95

c

o

NH4Cl.

experimental data points.

δ(HN) was obtained by fitting the correlation curve to the

lographic value; however, the presence of a zwitterionic structure

is demonstrated. As compared to that of 2a, the intramolecular

O2H2N2 hydrogen bond is substantially altered. The protonation

of the pyridine nitrogen leads to a shift of the proton in the

intramolecular O2H2N2 hydrogen bond, entirely stabilizing the

ketoamine form. This is evidenced by a shorter H2‚‚‚N2 distance

of 1.05 Å, predicted by NMR. Hence, using NMR, the coupling

of the intra- and intermolecular OHN hydrogen bonds is clearly

demonstrated.

partially from those reported previously for the model Schiff

base N-(3,5-dibromosalicylidene)methylamine, with adaptation

by fitting to the experimental data points in Figure 4b. We

associate the small difference with the influence of the chemical

structure of the Schiff bases since it is known that Schiff bases

1

5

containing different substituents exhibit different N chemical

9

4

shift values. For example, Naulet et al. observed a difference

1

15

C. H- N Chemical Shift Correlations and Geometric

Coupling of the Inter- and Intramolecular OHN Hydrogen

Bonds. We are now in a position to discuss the evolution of

the NMR parameters and hydrogen bond geometries of the two

functional OHN hydrogen bonds in the series of the Schiff bases

95

of 4 ppm, which was used for imines that carry a phenylic or

aliphatic group.

The solid correlation curves in Figure 4 give an acceptable

fit to the experimental data. The curves are similar to the q2

versus q1 curve of Figure 3a. The difference is that this curve

has no ends since q1 and q2 can be infinite. In contrast, the

1

and 2, which can be regarded as snapshots of a coupled

double-proton transfer. Instead of representing the data in terms

of a correlation diagram such as Figure 3a, we translate the

correlation curve q2 ) rOH + rHN versus q1 ) 1/2(rOH - rHN)

into the corresponding NMR parameter curve, that is, the

chemical shift of the H-bonded proton δ(OHN) as a function

of the ¹⁵N chemical shift δ(OHN). This correlation is expressed

implicitly by eqs 3 and 4. It arises from the observation that

the ¹⁵N chemical shifts are measures of the proton positions q1

1

15

H/ N chemical shift correlation curve has limiting values since

the chemical shifts of the separate moieties eventually become

independent of internuclear distance. Nevertheless, in the regions

where hydrogen bonds exist, both correlation curves display a

similar behavior, and one can discuss the data points in Figure

4

in terms of the geometric changes indicated in Figure 3a.

Different hydrogen bond geometries are obtained depending

on the acidity of the proton donor interacting with the pyridine

1

in the hydrogen bonds, while the H chemical shifts are measures

ring of the Schiff base. In contrast to collidine-benzoic acid

of the sum, q2, of the O‚‚‚H and H‚‚‚N distances.⁴⁰Such corre-

lations have been observed previously for pyridine- and 2,4,6-

40

complexes, which form molecular complexes of the type

O-H‚‚‚N, we could not obtain such a structure for solids 1

and 2, but rather, strongly hydrogen-bonded complexes of the

trimethylpyridine-carboxylic acid complexes in the solid and

liquid states.³

5-40

For a clearer comparison of different hydrogen

δ-

δ+

-

+

type O ‚‚‚H‚‚‚N or zwitterionic salts, O ‚‚‚H-N , were

obtained. The former exhibit low values of q2 (<2.6 Å) and

15

bonds, it is convenient to reference the N chemical shifts not

to a standard such as ammonium chloride but to the value of

the nitrogen atom in a non-hydrogen-bonded form, that is, to

1

40

low-field H chemical shifts in the range of 20 ppm. In the

zwitterionic states, the values of q2 increase according to Figure

1

a for the pyridine-type nitrogen atoms and to the methoxy-

1

3

a leading to a decrease of the H chemical shifts, as illustrated

20

substituted Schiff base N-(2-methoxybenzylidene)methylamine.

35,40

by the solid line in Figure 4a.

The results for the intermolecular and the intramolecular OHN

hydrogen bond of 1 and 2 are depicted in Figure 4. As described

in the theoretical section, the parameters used to calculate the

solid lines are the same as those used to calculate the hydrogen

bond correlation in Figure 3a and the NMR chemical shifts of

For the aldenamine Schiff base-acid adducts 1, comparison

of Figure 4a and 4b indicates that, when the proton is shifted

from the benzoic acid oxygen to the pyridine nitrogen, the intra-

molecular hydrogen bond is shortened in the series 1b-1j, but

its hydrogen bond proton remains closer to oxygen than to

o

the hypothetical limiting states listed in Table 4. Here, δ(N)

15

nitrogen. Because of the small low-field N chemical shift, 1e

to 1f are somewhat located outside of the correlation curve

represents the limiting chemical shift of the hypothetical free

o

nonprotonated nitrogen, and δ(OH) is that of the free OH group,

(Figure 1). These deviations are, however, so small that their

o

while δ(HN) and δ(HN) represent the chemical shifts of the

interpretation is difficult at present.

-

+

separate O and HN moieties. Additionally, δ(OHN)* and

δ(OHN)* are excess terms that describe the deviations of the

chemical shifts of the quasisymmetric strongest hydrogen bonds

from the average of the hypothetical limiting molecular moieties.

By contrast, the aldimine Schiff base adducts, 2, behave

differently; the formation of a hydrogen-bonded complex with

the pyridine nitrogen immediately shifts the proton in the

intramolecular hydrogen bond from oxygen to nitrogen, forming

δ-

The chemical shift parameters for the intermolecular hydrogen

bond were those reported previously⁴⁰for 2,4,6-trimethylpyri-

dine-acid complexes in the solid state. The chemical shift

parameters for the intramolecular hydrogen bond were taken

the zwitterionic state O2 ‚‚‚H2-N2 . Neither an intermediate

(

94) Neuvonen, K.; Fueloep, F.; Neuvonen, H.; Koch, A.; Kleinpeter, E.; Pihlaja,

K. J. Org. Chem. 2003, 68, 2151-2160.

(95) Naulet, N.; Martin, G. J. Tetrahedron Lett. 1979, 17, 1493-1496.

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4453

A R T I C L E S

Sharif et al.

state nor tautomerism between two protonation states was

2

0,28-33

observed, as was found with other Schiff bases in solution.

The origin of the H/D isotope effects on the ¹⁵N chemical

shifts of pyridine derivative-acid complexes,³

5-37,40

which

exhibit an intermolecular hydrogen bond, and of the model

2

0

Schiff base N-(3,5-dibromosalicylidene)methylamine, which

has an intramolecular hydrogen bond, has been discussed

recently. The latter is influenced by intrinsic effects arising from

isotope effects on the hydrogen bond geometry and by equi-

librium isotope effects on tautomerism.²⁰However, the values

that we have obtained here for the intramolecular H bond in

the solid state (cf. Table 1) are not precise enough for a detailed

discussion. Nevertheless, fair agreement is found with the values

obtained for the intermolecular H-bonds in complexes of 2,4,6-

Figure 7. The pKa values versus ¹⁵N chemical shift correlation for the

acid components in the organic solids of the aldenamine Schiff base-adducts

1

b-1j (open circles) and aldimine Schiff base-adducts 2b-2e (filled circles).

35,40

trimethylpyridine with various acids (Table 2).

A plot of

The diamonds represent the values for 2,4,6-trimethylpyridine-HA com-

plexes with respect to the frozen, bulk 2,4,6-trimethylpyridine. The pKa

values are a function of q1 , with the relation pKa ) A - Bq1 (see eq 5),

by using the parameters A ) 3.5 and B ) 8. For the calculation of the

curve, see text.

these isotope effects as a function of the ¹⁵N chemical shifts is

presented in Figure S7 in the Supporting Information.

D. The pKa Values and the Hydrogen Bond Geometries

in the Solid State. Here, acid-base interactions in nonaqueous

environments such as the solid state, aprotic polar solvents, or

the interior of an enzyme are addressed. Usually, the strength

of an acid in aqueous solution is expressed by its pKa value

defined by

35

H

average hydrogen bond geometry; an increase in the acidity or

proton donating power of the donor leads to a shift of the proton

position from the donor to the acceptor along a correlation curve,

such as depicted in Figure 3a. The hydrogen geometry obtained

depends on the molecular structure, environment, and the local

KAH

a

-

+

aq

9

6

AH_a_q{ } A + H

electric fields. In other words, both the pKa values and the

hydrogen bond geometry are measures of acid-base properties

of a proton donor-acceptor pair. It is, therefore, not astonishing

that, for pyridine- and 2,4,6-trimethylpyridine-acid adducts, a

near linear relation of pKa with the proton-transfer coordinate

aq

-

+

[

A ][H ]

AH

aq aq

AH

+

K_a

)

, -log K_a) pK_a) -log [H ] )

aq

[

AH ]

aq

-

aq

pH for [AH ] ) [A ] (6)

aq

H

35,41

q1 has been observed.

Since the ¹⁵N chemical shifts of intermolecular O1H1N1

and

hydrogen bonds in the absence of tautomeric equilibria are

KBH

H

a

convenient experimental measures of q1 , we have plotted, in

+

aq

+

BH { } B + H

aq

Figure 7, the pKa values of the acids forming the solid adducts

1

5

+

of 1 and 2 as a function of the pyridine N chemical shifts (cf.

[

B ][H ]

BH

aq

BH

+

K_a

)

, -log K_a) pK_a) -log [H ] )

Table 1). An alternative plot (cf. Figure S8) of the associated

aq

+

aq

[

BH ]

_p_K_a_v_a_l_u_e_s_a_s_a_f_u_n_c_t_i_o_n_o_f_t_h_e_p_r_o_t_o_n_-_t_r_a_n_s_f_e_r_c_o_o_r_d_i_n_a_t_e_q₁H

+

is given in the Supporting Information. The solid line was

calculated using eq 5, where the parameters were adapted in

such a way to fit the experimental data points best; see Figure

pH for [BH ] ) [B ] (7)

aq

Here, the acids and the bases all form hydrogen bonds to water

molecules, but there is no direct acid-base interaction. The acid-

base equilibrium is given by

7

. However, the scatter in the data obscures any direct relation

between the acidity in water and nonaqueous environments.

Moreover, comparison of the solid adducts, 1e and 2c, with

4-nitrobenzoic acid and of 1g and 2d with 3,5-dinitrobenzoic

acid does not clarify the relation. Whereas, in the first pair, the

aldimine seems to be more basic than the aldenamine, they

exhibit the same basicity in the second pair.

-

+

AH

[

A ][BH ] K_a

aq aq

K

9

-

aq

+

AH + B

8 A + BH , K )

)

aq

BH

[

AH ][B ]

aq

K_a

_p_K _aB H_-_p_K _aA H

(

)

∆pKa

10

) 10

(8)

As mentioned previously,³⁵eq 5 indicates that, in order to

By contrast, in nonaqueous environments, direct hydrogen bonds

between the acid and the base are usual, for example, in the

adducts of 1 and 2. In this case, eq 8 is no longer valid. If there

is an equilibrium between two forms such as

get a hydrogen bond with q1 ) 0, an acid with a pKa value of

97

∼3 is needed. Since the pKa value of pyridinium is 5.23, this

indicates that these substituted pyridines are ∼2 pKa units more

acidic than pyridinium, as has been shown previously for solid

1

:1 complexes of benzoic acids with pyridine derivatives by

K

35,98-101

-

IR spectroscopy.

acid with pyridine can be obtained only with collidine, which

is more basic than benzoate. In contrast to that in the solid

A solid molecular complex of benzoic

A`-H‚ ‚ ‚B {

\

} A`‚ ‚ ‚H-B

(9)

3

5

the equilibrium constant, K, characterizing the ratio of the two

tautomeric forms is not the same as the one in water. This

phenomenon is often incorrectly called a pKa shift. Generally,

direct acid-base hydrogen bonding may not lead to a conven-

tional equilibrium between two forms, but it may lead to a given

(96) Ramos, M.; Alkorta, I.; Elguero, J.; Golubev, N. S.; Denisov, G. S.;

Benedict, H.; Limbach, H. H. J. Phys. Chem. A 1997, 101, 9791-9800.

(97) Tamres, M.; Searles, S.; Leighly, E. M.; Mohrman, D. W. J. Am. Chem.

Soc. 1954, 76, 3983-3985.

4454 J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007

Hydrogen Bonds in PLP−Enzyme Model Systems

A R T I C L E S

state, in aqueous solution, the large dielectric constant of water

increases the acidity of a proton donor by favoring zwitterionic

forms exhibiting large dipole moments. In nonaqueous solution,

the acidity of a proton donor increases if it is involved in a

hydrogen-bonded network such as AH‚‚‚ (AH‚‚‚)nAH. Since

such association does not occur in water, the description of

acidity in terms of a single pKa value is not appropriate for

nonaqueous systems.

atom of PLP carries an aliphatic substituent, as in the internal

and external aldimines of PLP in the enzyme environment,

protonation of the ring nitrogen also shifts the proton in the

intramolecular OHN hydrogen bond from oxygen to the Schiff

base nitrogen, which increases its positive electric charge. This

charge on the Schiff base nitrogen may well be a prerequisite

3

6

1

9

for enzyme activity, as proposed by Bach et al.

Unfortunately, it was not possible to crystallize 2a with a

weak acid such as benzoic acid, which has a pKa value similar

to an aspartic acid side chain. If such a complex existed, from

Figure 7, we would expect a structure where the protons in both

the inter- and intramolecular OHN hydrogen bonds are close

to oxygen, that is, that a zwitterionic structure will not be

formed.

Conclusions

Using ¹⁵N CP MAS and H MAS NMR, we have studied a

series of specifically ¹⁵N-labeled hydrogen-bonded solids mod-

eling various forms (Schemes 1 and 2) of the cofactor pyridoxal-

1

5

′-phosphate (PLP) in enzymatic environments. In particular,

Schiff base formation with an ꢀ-amino group of a lysine side

chain or with amino acids, as well as hydrogen bonding of the

pyridine nitrogen to an aspartic acid residue, was modeled in

the series of novel 1:1 hydrogen-bonded solid complexes 1 and

In the solid state, the coupled double-proton shift could be

achieved by increasing the acidity of the benzoic acids via

electron-withdrawing substituents. On the other hand, in the

enzyme active site, the local polarity must be increased over

the solid states studied here. Specific interactions of the aspartic

acid residue with other proton/hydrogen bond donors is com-

monly observed in PLP enzyme structures, and increases in the

local dielectric constant due to the protein structure (for example,

R-helix macro dipoles) are probable. Combined, these effects

as well as specific interactions of active-site residues with the

Schiff base N and O make the pyridine ring and Schiff base

N-protonated forms readily accessible. Currently, studies on the

effects of the local polarity on the NMR parameters of the model

Schiff bases are underway, the results of which will be reported

elsewhere.

46

2, some of whose crystal structures were obtained previously.

The geometries of the intermolecular O1H1N1 hydrogen bonds

were estimated by establishing a correlation between nitrogen-

15

2

1

hydrogen distances obtained by dipolar N- H NMR and H

and ¹N chemical shifts and by well-established geometric OHN

hydrogen bond correlations. The latter also helped to locate the

proton in the intramolecular O2H2N2 hydrogen bond. However,

no thermal tautomerism was observed for the latter in the solid

state. When the acidity of the benzoic acids was increased, the

proton shifted continuously from the carboxylate oxygen to the

pyridine nitrogen, forming a zwitterionic state. During this

process, hydrogen bond compression was observed, which is

strongest in the quasisymmetric state where the proton is located

in the hydrogen bond center.

5

Acknowledgment. This work was supported by the Deutsche

Forschungsgemeinschaft, Bonn, and the Fonds der chemischen

Industrie, Frankfurt.

While similar features of pyridine and its derivatives have

been observed previously, the most important finding in this

paper is the coupling of the proton transfer in the intermolecular

O1H1N1 hydrogen bond to the proton transfer in the intramo-

lecular O2H2N2 hydrogen bond. When the Schiff base nitrogen

atom carries an aromatic substituent, protonation of the pyridine

ring only increases slightly the O2‚‚‚H2 bond length of the

intramolecular O2H2N2 hydrogen bond and compresses the

latter somewhat. However, whenever the Schiff base nitrogen

Supporting Information Available: The static powder ¹⁵N

spectra of the undeuterated and deuterated aldenamine Schiff

base and its acid-base adducts as well as the theory of the dipolar

1

5

15

N solid-state NMR are presented. N spectra and crystal-

lographic structures for PLP and its analogues are presented. A

1

15

plot of the isotopic effect, ∆N1(D), as a function of the

N

chemical shifts, δ(O1H1N1), is given. A plot of the intramo-

1

5

lecular rHN distances against the N chemical shift as well as

the alternative correlation of the associated pKa values with the

(

98) Majerz, I.; Malarski, Z.; Sobczyk, L. Chem. Phys. Lett. 1997, 274, 361-

3

64.

H

proton-transfer coordinate q1 is given. This material is available

(

99) Wolny, R.; Koll, A.; Sobczyk, L. J. Phys. Chem. 1985, 89, 2053-2058.

free of charge via the Internet at http://pubs.acs.org.

(

100) Hawranek, J. P.; Oszust, J.; Sobczyk, L. J. Phys. Chem. 1972, 76, 2112-

2

116.

(101) Johnson, S. L.; Rumon, K. A. J. Phys. Chem. 1965, 69, 74-86.

JA066240H

J. AM. CHEM. SOC.

9

VOL. 129, NO. 14, 2007 4455

Article Doi

Coupling of functional hydrogen bonds in pyridoxal-5′-phosphate- enzyme model systems observed by solid-state NMR spectroscopy

DOI: 10.1021/ja066240h

Source and publish data:

Authors:

Article abstract of DOI:10.1021/ja066240h

Full text of DOI:10.1021/ja066240h

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