Linkage Isomerization in Nitrosothiols (RSNOs): The X-ray Crystal Structure of an S-nitrosocysteine and DFT Analysis of its Metastable MS1 and MS2 Isomers

Density functional theory calculations reveal that the previously unreported nitrosothiol MS1 and MS2 linkage isomers are metastable species with energies similar to those determined for other experimentally observed XNO linkage isomers. Nitrosation of protein cysteines to yield S-nitrosocysteine (cySNO) is an important component of nitric oxide biology. However, high-resolution structural data of cySNO in the absence of protein environment effects is lacking, as is information on cySNO linkage isomers. We report the ordered X-ray crystal structure of a cySNO compound. We employ density functional theory calculations to probe the ground-state and linkage isomers of cySNO, CH3SNO, and CF3SNO. The HOMO of CH3SNO contains an intramolecular CH···O interaction that helps rationalize the stability of the cis conformation; this interaction is absent in CF3SNO, which favors the trans conformation. We show that the isonitroso MS1 (RSON) and side-on MS2 (RS(η2-ON)) linkage isomers are metastable species, with MS1 and MS2 energies similar to those of other experimentally observed nitroso linkage isomers. GRAPHICAL ABSTRACT

The ordered molecular structure of 1.HCl is shown in Figure 2. The compound is present in the crystal as the cis isomer with a ffC-S-N-O torsion angle of 1.7(5)°and a ffN-C-C-S torsion angle χ 1 of 75.6(3)°. The packing diagram ( Figure S1) reveals no hydrogen-bonding interactions that affect the conformation of the CSNO functional group. The re-determined crystal

Linkage Isomerization in RSNOs
structure of the related cySNO-ethyl ester (Figures S2 and S3) reveals disorder with three CSNO conformations, two of them in the cis form at 53% and 29% occupancies, and a third in the trans form at 18% occupancy.
The geometrical parameters for the CSNO groups in the previously published X-ray crystal structures of small molecule alkyl/aryl RSNOs (Table S1) [13][14][15][16][17][18][19][20][21][22] and protein RSNOs (Table S2) [5,[8][9][10][11][12] are tabulated in the Supporting Information. Importantly, the crystal structure of 1.HCl represents the first reported structure of an ordered 1°RSNO of a non-protein nitrosocysteine ( Figure 2). While most other structurally characterized alkyl RSNOs display trans CSNO conformations (Table S1), it is important to note that a cis conformation is present as a minor component (33% occupancy) in the disordered structure of the 3°compound Ar 3 CSNO (Ar = 3,5-bis(2,6dimethylphenyl)). [19] Further, the two aryl RSNOs (R = aryl) that have been structurally characterized contain cis CSNO moieties. [20,21] These data, combined with our crystallographic determination of a trans conformation in the non-protein 1°cySNO ethyl ester ( Figure S2C), suggest a greater cis/trans conformational variation of the planar CSNO moiety in the solid-state structures of RSNO compounds than was previous appreciated. Although the majority of observed cySNO conformations are cis in the handful of protein RSNO crystal structures reported (Table S2), the trans CSNO conformation is observed as a minor component of the nitrosated Cys69 residue of human thioredoxin, [9] and in the A21V mutant of Cimex nitrophorin (PDB access code 4L20). In both latter cases, the CSNO (of cySNO) planes in these protein environments remain essentially planar. Indeed, the previously assigned nonplanar CSNO moiety (dihedral angle of~80°) in the X-ray crystal structure of "HbSNO" [37] has been subsequently modified to include the possibility of the product being HbSNO(H) [38] based in large part on the theoretical prediction of a planar CSNO group in RSNO compounds by Houk and coworkers. [17,39] Further, the non-planar CSNO group (of the nitrosated Cys302 residue of hypoxia-inducible factor prolyl hydroxylase domain 2) with a dihedral angel of~90°has been similarly attributed to a cySNO(H) species. [40] To probe the effects of substituents on the -CSNO conformations in 1°R SNO compounds and the relative energies of their isonitroso (-CSON) and side-on -CS(η 2 -ON) linkage isomers, we resorted to density functional theory (DFT) calculations on the nitrosocysteine methyl ester (and its parent nitrosocysteine), CH 3 SNO, and CF 3 SNO (Table 1).
Our choice of the B3P86/6-311+G(2df,p) method and basis set combination is based on our determination that the C-S-N(O) bond parameters obtained using this combination gave an overall better agreement with the X-ray crystal structure of ordered 1 (Table S3); due to the disorder and inherent uncertainty in bond parameters of the CSNO moiety for the ethyl ester derivative, we focus the discussion on the ordered nitrosocysteine methyl ester 1. The choice of B3P86/6-311+G(2df,p) method and basis set has also been evaluated and Table 1: Selected geometrical data (Å, deg) from the DFT computed RSNO compounds at the B3P86/6-311+G(2df,p) level 1.HCl cis 1.797 (4) 1.819 (4) 1.171 (6) 117. Computed as the methyl ester in the cis form. [b] X-ray structural data (this work). [c] A scale factor of 0.93 was used for the IR data based on the experimentally measured for MeSNO. [41,42] The numbers in brackets refer to the relative intensities of the IR bands. [d] Relative to each GS (cis), and in kcal/mol. considered appropriate for RSNO compounds. [43] We note that, although there are some reports on DFT calculations on RSNO compounds (including cySNO [43,44] ), [39,43,[45][46][47][48][49] calculations on RSNO nitroso linkage isomers have not been reported to date. The DFT results show the cis conformation of 1 to be lower in energy (by +0.49 kcal/mol) than the trans form, consistent with the reports by others for 1°R SNO compounds, with a calculated rotational barrier of 14.6 kcal/mol. It is important to note that the presence of the methyl ester moiety did not alter the relative energies of the cis vs trans conformations for cySNO (ΔE = 0.53 kcal/ mol; not shown), with the cis isomer being lower in energy. A substituent effect on the relative energies of the cis and trans forms in 1°alkyl RSNO compounds is noted (Table 1). [45] While the cis form of CH 3 SNO is calculated to be slightly lower in energy than the trans form (ΔE = 1.2 kcal/mol; with a rotational barrier of 15.5 kcal/mol), [50] it is the trans conformation for CF 3 SNO that is lower in energy [45] than its cis form (ΔE = 1.5 kcal/mol; with a rotational barrier of 9.4 kcal/mol). We note that the HOMO of cis CH 3 SNO reveals an intramolecular H 2 CH. . .O interaction between the CH 3 substituent and the nitroso group that is absent in the cis CF 3 SNO analog (Figure 3; left entries). [51,52] The substituent effect is also evident in the vibrational stretching frequencies. The calculated υ NO of cis CH 3 SNO is 29 cm À1 lower than that for the corresponding trans isomer, whereas the calculated υ NO of cis-CF 3 SNO is only slightly higher (by 3 cm À1 ) than that of trans CF 3 SNO. [52][53][54][55] The calculated cis-isonitroso linkage isomer of 1, namely cySON (first metastable state MS 1 ), is +31 kcal/mol higher in energy than the ground state. This energy difference of +31 kcal/mol is less than that calculated for the experimentally observed isonitrosyl (porphine)Fe(η 1 -ON) (ΔE = +36.7 kcal/ mol) [56] and isonitroso FON (ΔE = +46.7 kcal/mol) [28] metastable states. In the calculated isonitroso cySON linkage isomer, the S-ON bond is~0.5 Å longer than the precursor S-NO bond ( Table 1), indicative of an overall weaker interaction between the S atom and the nitroso moiety. Indeed, whereas the HOMO of the ground state cis-1 is more evenly distributed along the SNO fragment, the HOMO of the MS 1 cySON is primarily (>85%) localized on the S atom (Figure 3 (top) and Figure S4).
The υ NO of MS 1 cySON is 176 cm À1 higher than that of the ground state 1, consistent with the slight shortening of the N-O bond length in the MS 1 linkage isomer. The calculation of a higher υ NO in the isonitroso isomer is consistent with that observed for the experimentally generated isonitroso F-ON (Δυ NO +34.5 cm À1 ), [28] isonitrosyl cyanide NC-ON (Δυ NO +338.4 cm À1 ), [30] and Cl-ON (Δυ NO +36 cm À1 ) [57] isomers from their ground state nitroso precursors.
The calculated MS 1 linkage isomers CH 3 SON and CF 3 SON reveal that they are also minima (Table 1). These MS 1 isomers display bond parameter changes similar to those mentioned earlier for cySNO vs cySON. However, whereas the trans CH 3 SON MS 1 isomer is 6 kcal/mol higher in energy than cis CH 3 SON, the difference between the trans and cis forms for CF 3 SON is only 0.3 kcal/mol. A substituent effect is also evident, where the υ NO of trans CH 3 SON is higher in energy (by 7 cm À1 ) relative to the cis linkage isomer, in contrast to that for CF 3 SON, where the υ NO of the trans isomer is 11 cm À1 lower in energy than the cis form.
Interestingly, we also find from the calculations that the cyclic side-on MS 2 linkage isomers are minima for these 1°RSNO compounds, with the sum of angles around the S atoms in the MS 2 species being~270°. The side-on isomer of 1 is calculated to be +58 kcal/mol higher in energy than ground state 1 ( Table 1); similar energy differences are seen in the MS 2 linkage isomers of CH 3 S(ON) and CF 3 S(ON). It is important to note that the calculated energy difference of 27 kcal/mol between the first metastable state MS 1 and the sideon MS 2 isomer of cis-1 corresponds to a wavelength of 1058 nm that is surprisingly close to the wavelength of 1064 nm of the infrared radiation used by one of us (P.C.) for the experimental conversion of the MS 1 isomer of sodium nitroprusside, Na 2 [Fe(CN) 5 NO].2H 2 O, to its crystallographically determined MS 2 nitrosyl linkage isomer. [23] Figure S4 for %-orbital contributions for both the HOMOs and LUMOs calculated at the B3P86/6-311+G(2df, p) level.

Linkage Isomerization in RSNOs
It is not surprising that for the cyclic "S(ON)" moieties in the calculated MS 2 linkage isomers, the N-O bonds are significantly longer than in either the GS or the MS 1 isomers. The HOMOs of the MS 2 isomers of all three species (Figure 3 and Figure S4) are localized primarily (>90%) on the S(ON) moieties, with >85% contribution on the nitroso groups. The IR data for the side-on MS 2 isomers show more than one band associated with υ NO ; for example, the MS 2 cyS(NO) isomer displays its υ NO at 950 cm À1 (major) and 507 cm À1 (minor) ( Table 1). Such a feature is consistent with a strong vibrational coupling within the -S(ON) moiety, as also observed with the matrix-isolated Sc(ON). [31] In summary, we report the first ordered non-protein cySNO X-ray crystal structure (i.e., not constrained by a protein environment). DFT calculations reveal significant substituent effects in the geometry and properties of CH 3 SNO vs CF 3 SNO and help explain the relative energies of the conformations of the cis vs trans isomers of GS CH 3 SNO, whereas the order is reversed for CF 3 SNO. Importantly, the DFT calculations show that the isonitrosyl MS 1 and MS 2 linkage isomers are metastable states with energies within the range of other experimentally observed metal and non-metal isonitrosyl MS 1 and MS 2 systems. Our results thus raise the intriguing possibility that RSNO MS 1 and MS 2 linkage isomers may, in some cases, play a role in the kinetics of nitrosothiol biology. EXPERIMENTAL L-Cysteine methyl ester hydrochloride and L-cysteine ethyl ester hydrochloride were purchased from Sigma-Aldrich. The red S-nitroso derivatives were prepared by nitrosation of the precursors in methanol using ethyl nitrite as the nitrosating agent. [58,59] Single crystals of the S-nitrosocysteine products were obtained from their acetone solutions, after numerous trials over a one-year period using other solvent combinations and methods. Crystallographic details for 1.HCl: formula = C 4 H 9 ClN 2 O 3 S, M w = 200.64 g mol À1 , P2 1 2 1 2 1 , a = 5.084 (2) Density functional theory (DFT) calculations were performed using the Gaussian 09 package accessed using the WebMO interface (www.webmo.net).
Geometry optimizations were performed using both B3P86 and B3LYP methods utilizing the 6-31+G(d,p), 6-311+G(2df,p), and 6-311++G(3df,3pd) basis sets. All optimized geometries were checked by vibrational frequency analyses; all of the structures shown did not exhibit imaginary frequencies, indicating that they are minima. The B3P86/6-311+G(2df,p) combination of method and basis set gave the best match with the X-ray crystallographic data for the C-S-N(O) moiety in the ordered structure of 1; hence, we focused the discussion using this combination, although the trends for the other combinations were similar. Representative data for the other combinations are collected in the Supporting Information (Table S3).