ARTICLE pubs.acs.org/JPCA
Microwave Spectra and Gas Phase Structural Parameters for N-Hydroxypyridine-2(1H)-thione Adam M. Daly,†,‡ Erik G. Mitchell,† Daniel A. Sanchez,† Eric Block,*,§ and Stephen G. Kukolich*,† †
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States Department of Chemistry, University at Albany, SUNY, Albany, New York 12222, United States
§
ABSTRACT: The microwave spectrum for N-hydroxypyridine-2(1H)-thione (pyrithione) was measured in the frequency range 618 GHz, providing accurate rotational constants and nitrogen quadrupole coupling strengths for three isotopologues, C5H432S14NOH, C5H432S14NOD, and C5H434S14NOH. Pyrithione was found to be in a higher concentration in the gas phase than the other tautomer, 2-mercaptopyridine-N-oxide (MPO). Microwave spectroscopy is best suited to determine which structure predominates in the gas phase. The measured rotational constants were used to accurately determine the coordinates of the substituted atoms and provided sufficient data to determine some of the important structural parameters for pyrithione, the only tautomer observed in the present work. The spectra were obtained using a pulsed-beam Fourier transform microwave spectrometer, with sufficient resolution to allow accurate measurements of the 14N nuclear quadrupole hyperfine interactions. Ab initio calculations provided structural parameters and quadrupole coupling strengths that are in very good agreement with measured values. The experimental rotational constants for the parent compound are A = 3212.10(1), B = 1609.328(7), and C = 1072.208(6) MHz, yielding the inertial defect Δ0 = 0.023 amu 3 Å2 for the C5H432S14NOH isotopologue. The observed near zero inertial defect clearly indicates a planar structure. The leastsquares fit structural analysis yielded the experimental bond lengths R(OH) = 0.93(2) Å, R(CS) = 1.66(2) Å, and angle — (NOH) = 105(4) for the ground state structure.
’ INTRODUCTION N-Hydroxypyridine-2(1H)-thione (1, Figure 1; pyrithione) exhibits an interesting tautomeric equilibrium with 2-mercaptopyridine N-oxide (2; MPO).1 The two tautomers are shown in Figure 1. Recent UV studies2 have shown this tautomeric equilibrium is solvent-dependent. Pyrithione is the main structure in polar and protic solvents whereas the MPO form dominates in nonpolar solvents.2 Calculations predict that in the gas phase the pyrithione tautomer has a higher thermodynamic stability than the MPO form by nearly 6 kcal/mol.1 This is in agreement with our experimental observations of only the pyrithione tautomer. One of us3 recently used DART mass spectrometry to demonstrate the formation of pyrithione upon cutting Allium stipitatum, a plant in the onion (Allium) family consumed as a food in Central Asia.4 Pyrithione had also been previously identified in Polyalthia nemoralis, a shrub found in Vietnam and southern China, whose extracts are used in herbal medicine for treatment of malaria.5 Pyrithione, first synthesized in 1950 as an antibiotic drug candidate,6 is highly active against fungi and Gram-negative bacteria69 and moderately cytotoxic toward human tumor cell lines.7 Pyrithione salts are also widely used in marine antifouling paints, as mildewcides in wallboards, as preservatives in paints and for odor control in textiles. The zinc salt of pyrithione, also found in Polyalthia nemoralis,5 is the active ingredient in several antidandruff shampoos.8,9 r 2011 American Chemical Society
Microwave spectroscopy can unambiguously differentiate between isomers 1 and 2 in the gas phase using differences in rotational constants and dipole moments. We report the first microwave study that unambiguously detects the pyrithione tautomer (1) in the gas phase. The first step in this collaboration was to measure and assign the microwave spectrum and to identify the species in a pure sample to give us a “fingerprint” unique to the molecule, which could later be used in the detection of pyrithione from a plant sample. However, repeated attempts to detect microwave transitions of 1 from extracts of the crushed bulbs of Allium stipitatum were not successful. We note that (E)propanethial S-oxide (CH3CH2CH=S+O) from the volatiles from crushed onion (Allium cepa) was successfully detected in earlier work using microwave spectroscopy.10,11
’ CALCULATIONS Electronic structure calculations were performed to obtain a starting structure for microwave frequency predictions. The predicted structure and derived rotational constants were accurate and reduced the effort in searching for transitions. The calculations were done using the Gaussian 0912 suite running on the Received: August 20, 2011 Revised: November 8, 2011 Published: November 09, 2011 14526
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Table 2. Calculated Bond Lengths (Å) and Interbond Angles (deg) Obtained Using MP2 with 6-31+G(d,p)a parameter
Figure 1. Two tautomers, pyrithione (1), and MPO (2).
Table 1. Summary of the Calculated Rotational Constants and Quadrupole Coupling Constants MP2/6-31+G(d,p) MPO
MP2/6-31+G(d,p) pyrithione
experiment 32 1 S/ H
A/MHz
3200.02
3197.71
3212.10(1)
B/MHz
1586.64
1596.93
1609.328(7)
C/MHz
1060.07
1065.05
1072.208(6)
χaa/MHz
0.048
0.97
1.15(1)
χbb/MHz
0.71
1.75
2.00(1)
χcc/MHz μa/D
0.75 5.0
2.72 3.8
3.09(1)
μb/D
0.3
3.0
N
61
σ/kHz
8
calculated
experimental
R(OH)
1.008
0.93(2)
R(C2S) R(NO)
1.682 1.376
1.66(2)
R(C2C3)
1.423
R(C3C4)
1.381
R(C4C5)
1.410
R(C5C6)
1.377
R(C6N)
1.355
R(NC2)
1.387
— (NOH) — (NC2S)
100 119
105(4) 118 173.0(5)
— (C5C2S) (180-ϕ)
174
— (NC2C3)
113
— (C6NC2)
127
a
All R(CH) very close to 1.08 Å. Internal angles not listed are very close to 120.
University of Arizona ICE high performance computing cluster. Structures for the two tautomers were optimized using MP2 with 6-31+G(d,p)13 basis set and the results are given in Table 1. The calculated bond lengths and interbond angles are given in Table 2. The predicted quadrupole coupling constants for nitrogen and predicted dipole moments are significantly different for the two tautomers and helped establish that the tautomer detected in our measurements is N-hydroxypyridine-2(1H)-thione. A calculated electrostatic map of the charge distribution for this molecule is shown in Figure 2.
’ EXPERIMENTAL SECTION The compound labeled “2-mercaptopyridine N-oxide” (CAS Reg. No. (supplied by author): 304675-7803) (mp 6972 C) was purchased from Sigma Aldrich and used without purification. This compound is believed to be in the pyrithione form in the gas phase. Because this compound is stable under normal conditions, no special precautions were used during the loading and unloading of the sample onto the spectrometer. The sample was placed into a cell that allows the flow of neon carrier gas over the sample as it is heated to produce a vapor pressure of a few torr. The pressure of the carrier gas ranged from 0.7 to 1 atm. The sample cell was attached to a Bosch automotive fuel injector and the mixture of carrier gas and sample were pulsed into the cavity at ∼2 Hz, transverse to the cavity axis. The cavity of the spectrometer was maintained at a pressure between 105 and 106 Torr while data were being acquired. The first few experiments were run at 70 C and resulted in serious degradation of the sample with color changes noticed within a couple of hours of testing. A
Figure 2. Electrostatic map of pyrithione showing the more electron rich area around the lone pairs of sulfur.
sample temperature of 45 C was determined to give sufficient vapor pressure without significant sample degradation. The molecular FID signals were transmitted via a microwave SPDT switch and passed on to a Miteq 618 GHz low-noise amplifier followed by a mixer and RF circuitry for further signal processing. Once the initial structure was determined, it was then confirmed through the use of different isotopologues with substitutions of 34 S and 2H. The 34S isotopologue was measured in natural abundance and the OD isotopologue was made by placing 0.250 mg of 2-mercaptopyridine N-oxide in 1 mL of methanol-d4 and allowing the solution to stir overnight. Only the hydroxyl group was deuterated, leaving the carbon-bonded hydrogen atoms unchanged. The solution was then transferred to a sample cell and the methanol-d4 was then pulled off using a vacuum. Strong signals were observed for the singly deuterated sample with signals being observable with only a single beam pulse (“single shot”). Details 14527
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Table 3. Measured Rotational Transitions (MHz) for Pyrithione and Fit Deviations (Observed Calculated in kHz) C5H432S14NOH
obs
C5H432S14NOD
obs
J0 KaKc
F
J00 KaKc
F
measured
calc
measured
calc
C5H432S14NOH
obs
C5H432S14NOD
obs
J0 KaKc
F
J00 KaKc
F
measured
calc
measured
calc
9463.119
1
9371.144
0.2
212
1
111
1
4824.697
1.2
414
5
313
4
212
3
111
2
4825.825
1.8
414
3
313
2
212
2
111
1
4826.247
7.6
414
4
313
3
9463.258
212
2
111
2
4826.819
1.4
414
4
313
4
202
1
101
1
5248.434
1.4
404
3
303
3
202
3
101
2
5249.107
0.6
404
5
303
4
202 202
2 2
101 101
1 2
5249.347 5249.688
2.5 2.7
404 404
4 3
303 303
3 2
9803.757
7.1
211
1
110
0
5899.131
0.4
404
4
303
4
9804.922
1.2
9705.338
1.4
211
2
110
2
5899.571
16.5
423
5
322
4
10636.49
13
10540.804
29.5
211
3
110
2
5900.198
23.9
423
4
322
3
10636.71
0.5
10541.031
22.6
211
2
110
1
5900.481
1.8
423
4
322
4
10636.71
0.5
10541.031
22.6
211
1
110
1
5901.435
8.2
432
3
331
2
10914.66
0.7
313
2
212
2
7172.46
6.1
313 313
4 2
212 212
3 1
7173.86
0.8
313
3
212
2
7174.056
313
3
212
3
7175.04
303
2
202
2
7618.24
303
4
202
3
7619.122
303
3
202
2
9371.221
3.1
6.7
9371.296
3.9
9464.446
3.1
9372.504
9.1
9802.572
2.1
9702.933
2.7
9803.757
2.5
9704.151
4.7
9704.435
8.4
432
5
331
4
10914.78
6.7
10821.461
7.3
7105.54 7105.486
7.5 11.8
432 431
4 3
331 330
3 2
10915.04 11004.83
0.2 4
10821.731
2.7
3
7105.544
17.4
431
5
330
4
11004.94
7.7
3.6
7106.562
2.4
431
4
330
3
11005.12
6.1
1.1
7543.638
10.2
413
3
312
2
11521.37
18.3
11418.868
19.6
7.1
7544.572
0.7
413
5
312
4
11521.48
5.2
11418.98
3.9
7619.452
20.9
7544.868
7.2
413
4
312
3
11521.66
5.8
11419.158
0.6
7545.484
6.2
413
3
313
3
11419.493
2.3
7973.236 7973.486
26.6 26.6
422 422
5 4
321 321
4 4
11557.13 11556.56
1.6 11.7
422
4
321
3
11466.21
11.1
7973.486
26.4
422
3
321
2
11466.231
3.8
11583.465
2.6
11583.588
3
303
3
202
3
322 322
2 3
221 221
1 3
8044.316 8044.52
5.7 1.6
322
4
221
3
8044.52
1.5
322
2
221
3
8044.52
1.4
322
3
221
2
8044.897
3.7
515
6
414
5
322
2
221
2
8044.897
3.5
515
5
414
4
11698.95
0.8
321
3
220
3
8469.475
5
8401.934
515
5
414
5
11700.29
5.6
11903.47
5.4
11781.472
18.4
11781.546 11781.718
34.6 17.8
11.3
321
4
220
3
8469.977
2.4
8402.451
23.1
505
6
404
5
321 321
2 3
220 220
1 2
8469.832 8470.07
2.3 5.6
8402.264
3.9
505 505
4 5
404 404
3 4
11903.69
13.7
11904.86
17
321
2
220
2
8470.739
0.2
505
5
404
5
11782.879
11.4
312
2
211
1
8769.579
2.8
616
5
515
4
13756.095
35.9
312
4
211
3
8769.762
2.1
8694.602
3.9
616
6
515
5
13756.209
9.4
312
3
211
2
8769.919
1.6
8694.785
16
606
7
505
6
13999.16
15.2
312
2
211
3
8769.919
7.3
8694.785
21.4
606
6
505
5
13999.32
27.2
13856.543
16.8
312
2
211
2
8770.549
3
8695.374
2.6
514
6
413
5
14090.21
29.3
414
3
313
3
9461.592
1.8
514
5
413
4
14090.41
1
of the homodyne mixing and detection system and the spectrometer have been given previously.14
’ MICROWAVE SPECTRUM The measured microwave spectrum obtained consisted of only a-type dipole transitions that corresponded to the predicted spectrum of N-hydroxypyridine-2(1H)-thione. The measured transitions are listed in Tables 3 and 4. For the normal isotopologue, 61 transitions were assigned to one species that exhibited a rich spectrum and could be fit using Herb Pickett’s SPFIT program. The “best fit” molecular parameters are listed in Table 5. The spectra displayed well-resolved nitrogen quadrupole coupling splittings. Well-determined values for the nitrogen
quadrupole coupling constants were obtained by fitting the measured transitions. The predicted nitrogen quadrupole coupling constant along the a-principal axis, χaa, is 20 times smaller in 2-mercaptopyridine N-oxide than N-hydroxypyridine-2(1H)thione and this is helpful in determining which tautomer is being observed. The corresponding ratios for χbb and χcc are 2.5 and 3.6. The lack of b-dipole transitions and the measured magnitude of the quadrupole coupling constants provide strong evidence that the molecule observed is the N-hydroxypyridine-2(1H)thione (pyrithione) tautomer. When signal averaging is employed, the acquisition time required for detection (at some fixed S/N ratio) signal is inversely proportional to the square of the relative concentration of a given molecular species. If the assumption is made that the concentration 14528
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Table 4. Measured Rotational Transitions (MHz) for C5H434S14NOH and Fit Deviations (Observed Calculated in kHz) J0 KaKc
F
J00 KaKc
F
measured
obs calc
313 313 313
2 4 3
212 212 212
2 3 3
7020.763 7022.167 7023.353
2.5 3.7 2.7
303 303 303 303 312 312 312 414 414 404 404 404 413 413
2 4 3 3 2 4 3 5 4 5 4 4 3 5
202 202 202 202 211 211 211 313 313 303 303 303 312 312
2 3 2 3 1 3 2 4 3 4 3 4 2 4
7463.234 7464.101 7464.396 7464.945 8552.205 8552.386 8552.538 9268.916 9269.041 9618.251 9618.528 9619.38 11249.05 11249.14
3 6.9 7.4 1.5 1 3.5 1.2 12.3 3.8 1.4 6.3 6.8 2.4 0.4
515 505
4 6
414 404
3 5
11464.9 11682.79
6.6 16.4
Table 5. Experimental Molecular Parameters for the Three Isotopologues C5H432S14NOH, C5H432S14NOD, and C5H434S14NOH 32
S/1H
32
S/2H
34
S/1H
A/MHz
3212.10(1)
3166.49(2)
3200.81(4)
B/MHz
1609.328(7)
1596.440(1)
1566.132(2)
C/MHz
1072.208(6)
1061.412(1)
1051.632(2)
DJ/kHz
0.021(8)
0.01(1)
0.23(8)
DJK/kHz
0.25(9)
0.4(2)
5(2)
χaa/MHz
1.15(1)
1.21(2)
1.09(3)
χbb/MHz χcc/MHz
2.00(1) 3.09(1)
1.91(2) 3.12(2)
1.93(3) 3.08(3)
N
61
47
20
σ/kHz
8
14
5
of the MPO tautomer is only 104 times that of pyrithione, it would have taken 108 times as much time to detect it. This is a reasonable explanation as to why the MPO tautomer was not observed.
’ STRUCTURE FIT The measured transitions for the three isotopologues were used in a least-squares fit to determine four of the more important structural parameters. The adjustable parameters were ROH, θ, RCS, and ϕ (Figure 3). A planar structure was assumed and this is consistent with the experimental inertial defect, Δ0 = 0.023 amu 3 Å2 for the C5H432S14NOH isotopologue. The “best fit” values obtained for the parameters are R(OH) = 0.93(2) Å, R(CS) = 1.66(2) Å, θ angle — (NOH) = 105(4), and ϕ angle — (C5C2S) = 7.0(5). This strategy allowed the hydrogen and the sulfur coordinates to be adjusted independently and results in an angle — (NCS) = 118. As predicted from the calculations using MP2/6-31+G(d,p), the sulfur atom is drawn slightly toward the hydroxyl group by approximately 7.
Figure 3. Four adjustable parameters, ROH, θ, RCS, and ϕ, used in the structure fit and the resulting structure obtained in the least-squares fit to the nine rotational constants.
’ DISCUSSION The primary predicted differences between the two tautomers are the dipole moments and quadrupole coupling constants. For pyrithione the predicted dipole moments are μa = 5.0 D and μb = 0.3 D whereas for MPO dipole moments are μa = 3.8 and μb = 3.0. Because no b dipole transitions were observed, this alone is a strong argument for the pyrithione tautomer being dominant in the gas phase. This project was initiated to identify and confirm the structure of a volatile component of a genus Allium plant, Allium stipitatum. One of us (E.B.) has had a long-standing interest in the chemistry of sulfur compounds in various Allium species and related plants. He has been studying the sulfur-containing molecules in garlic and other alliums for many years10 and recommended searching for 2-mercaptopyridine N-oxide or its tautomer N-hydroxypyridine-2(1H)-thione using microwave spectroscopy, because the presence of one or both of these tautomers was indicated by DART mass spectrometry.3 The first step in this collaboration was to measure and assign the microwave spectrum to identify the species in a pure sample that would give us a “fingerprint” that is unique to the molecule which could later be used in the detection of the molecule in plant sample. The second part of this project was to detect either form, Nhydroxypyridine-2(1H)-thione or 2-mercaptopyridine N-oxide, in extracts from crushed Allium stipitatum bulbs (crushing Allium plants is necessary to initiate enzymatic conversion of amino acid-based precursors to smaller molecules, such as pyrithione). Because N-hydroxypyridine-2(1H)-thione is more likely to be present, the frequencies of its known transitions were used. Several different isolation methods were employed including Soxhlet extraction using dichloromethane, freeze-drying, and simple extraction using anhydrous ether. Thus far we have been unable to detect known N-hydroxypyridine-2(1H)-thione transitions in extracts of the crushed bulbs. ’ CONCLUSION This paper provides experimental transition frequencies for three isotopologues of pyrithione, a commercially important, naturally occurring pyridine heterocycle. These data have been used to determine four important structural parameters for this 14529
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compound. Isotopic studies with 34S and 2H provide definitive evidence that the structure is N-hydroxypyridine-2(1H)-thione. The prediction of the sulfur atom “leaning” toward the hydroxyl group has been experimentally verified.
’ AUTHOR INFORMATION Present Addresses ‡
Grupo de Espectroscopía Molecular, Universidad de Valladolid, E-47005 Valladolid, Spain.
’ ACKNOWLEDGMENT This material is based upon work supported by the National Science Foundation under Grants No. CHE-0721505 and CHE0809053 at the University of Arizona (S.G.K.) and CHE 0744578 (E.B.) at the University at Albany, SUNY. ’ REFERENCES (1) Kaszynski, P. Phosphorus, Sulfur, Silicon Relat. Elements 2009, 184, 1296–1306. (2) Aveline, B.; Kochevar, I.; Redmond, R. J. Am. Chem. Soc. 1996, 118, 10113–10123. (3) Block, E.; Dane, A. J.; Cody., R. B. Phosphorus, Sulfur, Silicon Relat. Elements 2011, 186, 1085–1093. (4) Krzymínska, A.; Gawzowska, M.; Wolko, B.; Bocianowski, J. J. Appl. Genet. 2008, 49, 213–220. (5) Lewis, K.; Ausubel, F. M. Nat. Biotechnol. 2006, 24, 1504–1507. (6) Shaw, A.; Bernstein, J.; Losee, K.; Lott, W. A. J. Am. Chem. Soc. 1950, 72, 4362–4364. (7) (a) Han, G.-Y.; Shen, Q.; Chiang, W.; Li, Y.; Wang, G.; Zhang, C. Tianran Chanwu Yanjiu Yu Kaifa 1999, 11, 3336 [Chem. Abstr. 1999, 132, 47528]. (b) M€oller, M.; Adam, W.; Saha-M€oller, C. R.; Stopper, H. Toxicol. Lett. 2002, 136, 77–84. (8) Futterer, E. J. Soc. Cosmet. Chem. 1981, 32, 327–338. (9) Marks, R.; Pearse, A. D.; Walker, A. P. Br. J. Dermatol. 1985, 112, 415–422. (10) Block, E. Garlic and Other Alliums: The Lore and the Science; Royal Society of Chemistry: Cambridge, U.K., 2009. (11) Block, E.; Gillies, J. Z.; Gillies, C. W.; Bazzi, A. A.; Putman, D.; Revelle, L. K.; Wang, D.; Zhang, X. J. Am. Chem. Soc. 1996, 118, 7492–7501. (12) Frisch, M. J.; Trucks, G. W.; Schlegel, H.s B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, € Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; S.; Daniels, A. D.; Farkas, O.; Fox, D. J.; et al. Gaussian 09, revision A.2; Gaussian, Inc.: Wallingford, CT, 2009. (13) (a) Møller, C.; Plesset, M. S. Phys. Rev. 1934, 46, 618–622. (b) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem. Phys. 1980, 72, 650–654. (14) Tackett, B.; Karunatilaka, C.; Daly, A.; Kukolich, S. Organometallics 2007, 26, 2070–2076. 14530
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