EPR Study of Nitroxides Formed from the Reaction of Nitric Oxide with Photolyzed Amides

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MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2003; 41: 647–659 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1229 EPR study of nitroxides formed from the reaction of nitric oxide with photolyzed amides

Fan Wang, Jing Jin and Longmin Wu∗

State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China

Received 17 March 2003; Revised 12 May 2003; Accepted 13 May 2003

Free radicals generated from UV irradiation of simple aliphatic amides in anaerobic and nitric oxide (NO)-saturated liquid mixtures or solutions gave EPR spectra of nitroxides. The application of isotopic effects to EPR spectra and the generation of radicals by transient radical attack on substrate molecules or by photolysing amine or acetoin were used to help identify photochemically produced radicals from the amides. The aliphatic amides used were formamide, acetamide and their N-methyl- or deuterium- substituted derivatives. Transient radicals used to attack the amides via hydrogen-atom abstraction were generated from the initiator AIBN or AAPH. The observation of various nitroxides indicates the reactivity of NO for trapping acyl, carbamoyl and other carbon-centered radicals. Possibly mechanistic pathways diagnosed with this trap are proposed. Copyright  2003 John Wiley & Sons, Ltd.

KEYWORDS: EPR; nitric oxide; nitroxide; amides; acyl; carbamoyl

INTRODUCTION ‘Non-’ acyl radicals can be generated by different processes and trapped by nitroso compounds, yielding acylalkyl It has been well established that nitric oxide (NO) is a highly nitroxides.9–11 The EPR spectroscopic features of acylalkyl stable free radical under chemical conditions. NO shows no nitroxides are characterized by a significantly smaller nitro- tendency to dimerize or disproportionate. It does not abstract gen hyperfine splitting constant (HFSC), generally in the a hydrogen atom nor add itself to an inactivated double bond. range 0.7–0.8 mT, and by a larger g-value than those of nor- However, increasing experimental facts have indicated that mal dialkyl nitroxides.10 Therefore, they will be important NO can be used as a long-lived paramagnetic free radical criteria determining whether an acyl group bears directly scavenger in certain cases.1–5 The range of reactions of NO the nitroxide function or not. Generally, the EPR data with alkyl radicals is complicated. The (RNOR) dimer or 2 bank, especially for acylalkyl nitroxides, seems limited. It R NOR was found to be its major product.6,7 This suggested 2 is well known that the UV photolysis of aliphatic amides can that an oxyaminyl radical or a nitroxide appeared, although generate aliphatic acyl radicals.12–14 Other radicals derived the reaction yielding oxyaminyl-type radicals was ruled from amides, carbamoyl radicals in particular, could be out in some cases.8 There were also indications of aminyl formed by various methods, such as sonolysis,15 radiolysis,16 formation.5,9 In previous studies,4,5 we found that NO tended radicals or excited triplet organic molecule attack17 and to couple with carbon (C)-centered and less stereo-hindered photo-oxidation,18 etc. The radicals so formed were directly alkyl radicals to give nitroso compounds. The compounds , observed in the matrix at a low temperature,18 19 by the spin formed could trap other C-centered radicals, sulfinyl radicals trapping technique9,15,17e or by the flow technique.17a,d or thiyl radicals to yield nitroxides (i.e. aminoxyls) or Amides have long been of practical importance and of oxyaminyls, but they did not seem to trap alkyloxy or phenyl fundamental interest. The amide group is a ubiquitous moi- 4,5 radicals. The long-lived nitroxides thus formed were ety in biologically important macromolecules. Amides can observed by the EPR spectroscopic technique. Furthermore, serve as a linkage in proteins and act as a building block we were encouraged to investigate the availability of NO for many polymers. For instance, the simplest formamide for trapping other kinds of transient radicals, particularly is the smallest model molecule of the peptide prototype 9 the ‘non- ’ aliphatic acyl radicals, although Forrester et al. NH—C O linkage. It has been reported that some dipep- 9a suggested that nitrosoacyls, which stemmed from acyloxy tide amides showed selective inhibition effects on nitric oxide amidyl radicals generated by hydrogen-atom abstraction synthases (NOS) and exhibited therapeutic potential in the from N-acyloxyamides, might be useful traps, especially for treatment of some diseases resulting from NO overproduc- nucleophilic radicals. tion, such as septic shock and inflammation.20 In particular, organic radicals derived from DMF played a significant ŁCorrespondence to: Longmin Wu, State Key Laboratory of role in cell killing, with possible implications for cancer Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, treatment.21 This is another reason why we are interested in China. E-mail: [email protected] Contract/grant sponsor: Natural Science Foundation of China; spin trapping reactions of NO with acyl and other radicals Contract/grant number: 20072013. generated or derived from aliphatic amides.

648 F.Wang,J.JinandL.Wu

Table 1. EPR parameters of nitroxides R1R2N(Ož)

No. R1 R2 Solvent HFSC (mT)a g-Valueb

1 O O Benzene 1N: 0.962 2.0062 H C C H

2 O O Benzene 1N: 0.983 2.0060 D C C D Aqueous 1N: 1.028

3 (CH3)2N N(CH3)2 Aqueous 1N: 1.678 2.0053 2N: 1.447 O 4 C(CH3)2CN Benzene 1N: 1.080 2.0061 H C

O 5 C(CH3)2CN Benzene 1N: 1.116 2.0060 D C

6 O O Acetoin 1N: 0.614 2.0064 H3C C CCH3 OH O 7 Acetoin 1N: 0.614 2.0068 H C C CCH 3 3 1H: 1.064 H O 8 H C(CH3)2CN Benzene 1N: 1.010 2.0062 N C H 1H: 0.098 1H: 0.047 O 9 D C(CH3)2C(ND2 NH D2O 1N: 0.962 2.0060 N C D O 10 H C(CH3)2C(NH2 NH Aqueous 1N: 0.970 2.0061 N C H 1H: 0.105

O 11 C(CH3)2C(NH2 NH Aqueous 1N: 0.956 2.0060 HN C

CH3 O 12 C(CH3)2C(ND2 NH D2O 1N: 1.035 2.0060 DN C

CH3 O 13 C(CH3)2CN Benzene 1N: 1.001 2.0060 HN C

CH3 O H 14 C(CH3)2CN Benzene 1N: 1.438 2.0061 H C N C 1N: 0.219 H C H 3 1H: 1.713 1H: unresolved O H 15 C(CH3)2C(NH2 NH Aqueous 1N: 1.476 2.0059 H C N C 1N: 0.218 H3C H 1H: 1.821 1H: unresolved O D 16 C(CH3)2CN Benzene 1N: 1.550 2.0061 D C N C 1N: 0.250 D C D 3 1D: 0.132 O D 17 C(CH3)2C(NX2 NH Aqueous or D2O 1N: 1.479 2.0060 D C N C (X D HorD) 1N: 0.220 D C D 3 1D: 0.137 1D: 0.127 O H 18 C(CH3)2C(NH2 NH Aqueous 1N: 1.400 2.0059 H C N C 1N: 0.229 H H 1H: 1.324 1H: unresolved

EPR study of nitroxides from NO and photolyzed amides 649

Table 1. (Continued)

No. R1 R2 Solvent HFSC (mT)a g-Valueb O 19 C(CH3)2CN Benzene 1N: 1.475 2.0059 H C C N CH 3 2 1N: 0.271 CH 3 2H: 0.685 O H 20 C(CH3)2CN Benzene 1N: 1.502 2.0059 H C C N C 3 1N: 0.132 H C H 3 1H: 2.196 1H: unresolved O 21 C(CH3)2C(NH2 NH Aqueous 1N: 1.462 2.0060 H C C N CCH 3 2 1N: 0.184 CH 3 2H: 1.027 O H 22 C(CH3)2C(NH2 NH Aqueous 1N: 1.651 2.0060 H C C N C 3 1N: 0.159 H C H 3 1H: 2.044 1H: unresolved O 23 C(CH3)2C(NX2 NH Aqueous or D2O 1N: 1.410 2.0056 X N C CH 2 2 (X D HorD) 2H: 0.796 (X=H or D)

O 24 C(CH3)2C(NX2 NH Aqueous or D2O 1N: 1.502 2.0059 H C C N CH 3 2 (X D HorD) 1N: 0.219 X (X=H or D) 2H: 0.915 O H 25 C(CH3)2C(NX2 NH Aqueous or D2O 1N: 1.461 2.0059 H C C N C 3 (X D HorD) 1N: 0.229 X H (X=H or D) 1H: 1.416 1H: unresolved a Absolute accuracy š0.003 mT. b Absolute accuracy š0.0001.

EXPERIMENTAL described previously.4,5 All the EPR determinations were carried out at ambient temperature. EPR parameters such as N,N-Dimethylformamide (DMF) (Xian Chemicals, AP) was HFSCs and g-values are given in Table 1. dried over magnesium sulfate for 24 h and then treated with potassium hydroxide to remove water and formic acid. It was distilled and the middle fraction was col- RESULTS AND DISCUSSION lected for use.22 N,N-Dimethylacetamide (DMA) (Tianjin Nitroxides generated from reaction of NO with Chemicals, AP) was distilled at reduced pressure from bar- photo-produced acyl radicals 22 ium oxide. Formamide (Xian Chemicals, AP) was treated The EPR spectrum shown in Fig. 1(a) was recorded during 22 according to the literature. Acetamide (Xian Chemicals, the UV photolysis of NO-saturated DMF mixed with benzene 22 AP) was recrystallized from acetone. N-Methylformamide (DMF : benzene D 4 : 1, v/v). This spectrum consists of a (NMF) (J&K Chemicals, 99%), N-methylacetamide (NMA) 0.962 mT 1 : 1 : 1 triplet with a broad linewidth and a g-value 0 (J&K Chemicals, 99%), 2,2 -azobis(2-methylpropionamidine) of 2.0062. It might be assigned to a nitroxide (1). The smaller dihydrochloride (AAPH) (Aldrich), dimethylamine (Shang- nitrogen HFSC (0.962 mT) implies that one or more electron- hai Chemicals, 33%) and 3-hydroxy-2-butanone (acetoin) withdrawing substituents are attached to the nitrogen atom 0 (Aldrich) were used as received. 2,2 -Azobisisobutyronitrile of the nitroxide 1. An EPR spectrum [Fig. 1(b)] having almost (AIBN) (4th Shanghai Chemicals, AP) was recrystallized the same features as the nitroxide 1 was obtained when an from ethanol. NO was prepared and puriﬁed fully according NO-saturated DMF-d7 solution mixed with benzene (DMF- 4 to the procedure described previously. d7 :benzeneD 4 : 1, v/v) was UV photolyzed. The radical Benzene for sample preparation was treated by standard species might also be assigned to a nitroxide (2). The EPR procedures.22 Water for aqueous samples was distilled spectrum of the nitroxide 1 differs from that of the nitroxide and deionized. Deuterium oxide (D2O, 95.5%) (DeuChem, 2 in linewidth, that of the latter being much narrower than Leipzig, Germany) was used as received. that of the former. The sample preparation, UV irradiation performance, It is well known that DMF primarily undergoes pho- EPR measurements and calculations on EPR spectra were todissociation after excitation via a Ł transition upon UV

650 F.Wang,J.JinandL.Wu

(a) competition with dimethylamino radical for coupling with NO. The observation of the nitroxide 3 indicates that a dialkylamino nitrogen atom, attached directly to the nitroxide function, would characteristically contribute to a larger nitrogen HFSC of ¾1.4 mT. Otherwise, it has been observed 1 mT that the aN value of an acylalkyl nitroxide has a magnitude 10 (b) of 0.7–0.8 mT. Nevertheless, to our best knowledge, the aN value of a formylalkyl nitroxide is still unknown. Misik and 15b Riesz assigned a broad triplet with aN D 0.95 mT to the 3tBNB (2,4,6-tri-tert-butylnitrosobenzene) spin adduct of the dimethylcarbamoyl radical when DMF was UV photolyzed

inthepresenceofH2O2 and the spin trap 3tBNB. As described (c) above, the linewidths of the nitroxides 1 and 2 are sharply different, so it seems to us that dimethylcarbamoyl does not build the nitroxides 1 or 2, because dimethylcarbamoyl-

d6 does not inﬂuence the triplet linewidth of a nitroxide. Therefore, the greatest possibility in the present case is that two formyl radicals react with NO in sequence to produce nitroxides 1 and 2. Their structures are assumed to be as illustrated. Figure 1. EPR spectra of radicals from the UV photolysis of

NO-saturated (a) DMF, (b) DMF-d7 mixed with benzene and O O (c) AIBN-containing (0.03 M)mixtureofDMFand benzene (1 : 1). X CNCX (H3C)2NN N(CH3)2 O . O .

(a) 1: X = H 3

1 mT 2: X = D (b)

The production of the nitroxides 1, 2 and 3 is suggested inSchemes1and2.Twoissuescouldbeconsideredfromthe Figure 2. (a) EPR spectrum generated by the UV irradiation of above observation: (a) formyl directly bearing the nitroxide

an NO-saturated aqueous solution of dimethylamine at function causes an aN value of ¾1mT,somewhatlarger ambient temperature and (b) its simulation. than those of other acyl radicals;9b and (b) NO reacts more favorably with formyl radical than with dimethylamino absorption,12,14 giving a formyl radical and a dimethylamino radical under the present conditions. radical. In order to confirm whether dimethylamino rad- Clearly, unresolved HFSCs of two formyl protons icals build the nitroxide 1 or not, the UV photolysis of contribute a broadening linewidth to the nitroxide 1,whereas an NO-saturated aqueous solution of dimethylamine (33%) the deuteron HFSCs of two formyls in the nitroxide 2,which wasconducted.ItgaverisetotheEPRspectrumshownin is about 1/6.5 times smaller than that of the corresponding Fig. 2(a). The EPR lines were not intense, but clearly charac- protons in the nitroxide 1, make the EPR lines of 2 reasonably teristicofa1.447mT1:2:3:2:1quintetsuperimposedona much sharper than those of 1. 1.678 mT 1 : 1 : 1 triplet [Fig. 2(b)] and with a g-value of 2.0053, In order to gain more evidence to identify the structures of which clearly manifested a hyperfine coupling to two equiv- the nitroxides 1 and 2, the UV photolysis of an NO-saturated alent nitrogen nuclei and to another nitrogen nucleus. The and AIBN-containing (0.03 M) mixture of DMF and benzene corresponding radical is assigned to the nitroxide 3.Ithas was tested. At a lower volume fraction of DMF in benzene been well established that UV irradiation of dimethylamine (DMF : benzene D 1 : 1, v/v), a 1.080 mT 1 : 1 : 1 triplet EPR yields a dimethylamino radical and a hydrogen atom.23 spectrum was generated with a g-value of 2.0061 and with Therefore, the nitroxide 3 may be formed from the addition a broad linewidth [Fig. 1(c)]. The corresponding radical is of dimethylamino radical to N-nitroso-N,N-dimethylamine, assigned to the nitroxide 4. Its significantly larger aN value which is formed from the coupling reaction of a photochem- shows that it should be different from the nitroxide 1.Three ically produced dimethylamino radical with NO. However, primary radicals should exist in the experiment involving it should be pointed out that N—N ( O) bond forma- AIBN: formyl, dimethylamino and 2-cyano-2-propyl. As tion is energetically less favorable than C—N ( O) bond discussed above, the dimethylamino moiety should be 24 formation. As a result, the formyl radical reacts periodically excluded from the nitroxide 4.ComparedwithaN values of with NO. Therefore, no nitroxide-containing dimethylamino the nitroxides 1 and 2, another substituent is supposed to bear moiety was observed in Fig. 1. In the experiments involv- the nitroxide function. Most likely, it is 2-cyano-2-propyl. ing the amine, the situation is that no formyl radical is in Therefore, the nitroxide 4 may be 2-cyano-2-propylformyl

EPR study of nitroxides from NO and photolyzed amides 651

O O masked [Fig. 3(b)]. The two triplets [open and solid circles hν . + (CX ) N . (1) in Fig. 3(b), respectively] with overlapping central lines may XC N(CX3)2 X C 3 2 be assigned to two different nitroxides with the same g- X = H or D value and different aN values. The aN values (0.983 and O O 1.116 mT) enable us to assign them to the nitroxides 2 and 5, respectively. X C . + NO N CX (2) O

O O O O O CH3 X C . +NCN CX X C X (3) D CNCCN . O O . O CH3 1 or 2 5 In the presence of AIBN

CH3 CH3 Similar experiments involving DMA were carried out. H3C hν N C CN (4) A very complicated and unresolved EPR spectrum with a NC C 2 NC C . + N2 N weak line intensity was obtained during UV irradiation of CH3 CH CH3 3 NO-saturated DMA mixed with benzene (DMA : benzene D 9 : 1, v/v). It seemed to consist of several nitroxides. The CH CH 3 3 assignment of these radicals is very difﬁcult. When an NO- (5) NC C . + NO NCC NO saturated mixture of DMA and benzene (DMA : benzene D

CH3 CH3

O CH3 O CH3 (a) H C . + ON C CN H CNCCN (6) . CH3 O CH3 4 Scheme 1

hν (CH3)2NH (CH3)2N. + H . (7) 1 mT

(CH3)2N. +NO (CH3)2N NO (8) . (b) (CH3)2N + N(CH3)2 (H3C)2N N N(CH3)2 (9) NO O . 3 Scheme 2 nitroxide. This implies that the photodissociation of DMF is a major process in the present case, although a hydrogen-atom abstraction from DMF by 2-cyano-2-propyl may occur.17e

O CH3 H CNCCN . O CH3

4 (c)

The UV irradiation of an NO-saturated benzene mixture Figure 3. (a) EPR spectrum of radicals from the UV photolysis of DMF-d7 and AIBN (0.06 M) gave the EPR spectrum shown of an NO-saturated benzene mixture of DMF-d7 and AIBN, in Fig. 3(a). It seemed to be a mixture of three signals: two (b) the EPR spectrum recorded when light was masked and sets of strong triplets and a set of less intense multilines. (c) the simulation for the lines marked with sticks in The latter [sticks in Fig. 3(a)] disappeared when light was spectrum (a).

652 F.Wang,J.JinandL.Wu

9 : 1, v/v) containing AIBN (0.03 M) was UV photolyzed, a complicated EPR spectrum was obtained (see below). EPR spectrum assignments indicated a lack of acetyl nitroxides. In order to provide more spectroscopic evidence for acetyl nitroxides, a supplementary experiment was performed using acetoin. The UV photolysis of NO-saturated acetoin displayed a mixed EPR spectrum (Fig. 4). Lines marked with circles in Fig. 4 are a 0.614 mT 1 : 1 : 1 triplet with a g-value of 2.0064. Lines with sticks in Fig. 4 exhibit a hyperﬁne coupling

to a nitrogen atom (aN D 0.614 mT) and a hydrogen atom

(aH D 1.064 mT) with a g-value of 2.0068. These two radicals are assigned to the nitroxides 6 and 7, respectively. Primary 1 mT photoreaction of acetoin quantitatively undergoes Norrish type I homolytic ˛-cleavage from its n* excited triplet state, giving an acetyl radical and an ˛-hydroxyethyl radical.25 The acetyl radical may secondarily decompose into a 13 ž methyl radical and CO. Probably, the radical CH3C(O)C( ) Figure 4. EPR spectrum generated by the UV photolysis of (OH)CH3 may be secondarily generated by radical attack on acetoin. However, there are a series of experimental facts, (a) NO-saturated acetoin. the lack of nitrosomethane adducts,4 (b) the larger g-values of the radicals 6 and 7 and (c) the existence of a single ˇ- (a) proton in the nitroxide 7, that indicate that the secondary processes may be ignored and that the primary process is the major pathway for acetoin photochemistry in the present case. Consequently, both acetyl and ˛-hydroxyethyl radicals 1 mT react with NO to produce nitroxides. They are easily assigned (b) to the nitroxides 6 and 7, respectively. The reason why NO is able to trap the acetyl radical in this case, but not in the case of DMA, may most likely be attributed to a higher concentration of acetyl radicals in the system. The dissociation of acetoin is induced by UV light at 313 nm with a quantum yield of (c) 1.0,25b whereas that of the acetamides is achieved by UV light at 254 nm with a yield of 0.1.12a Otherwise, the quantum ﬂux of the lamp at 313 nm is at least about ﬁve times stronger than that at 254 nm. This causes the acetyl concentration (d) from acetoin to rise higher than that from the acetamides.

O O OH O

H3C CNCCH3 H3C CNCCH3 O . H O .

6 7 (e)

Nitroxides generated from reaction of NO with photo-produced carbamoyl radicals The UV photolysis of NO-saturated pure formamide or a mixture of formamide and benzene (7 : 3) did not give an EPR spectrum. However, in the presence of AIBN (0.04 M), Figure 5. (a) EPR spectrum of the radical from the UV the EPR spectrum shown in Fig. 5(a) was obtained. The g- photolysis of NO-saturated formamide mixed with benzene in value is 2.0062. The simulation [Fig. 5(b)] exhibits a hyperﬁne the presence of AIBN, (b) its simulation, (c) the EPR spectrum coupling to one nitrogen nucleus (aN D 1.010 mT) and two of the radical from the UV photolysis of NO-saturated non-equivalent protons (a D 0.098 and 0.047 mT). Certainly, H AAPH-containing formamide mixed with D2Oand(d)mixed this radical species (8) could be assigned to a nitroxide. The with water and (e) the simulation for spectrum (d). primary step for free radical production in the photolysis ž 19 of formamide yields H-atoms and CONH2 radicals. However, it appeared that they did not produce the nitroxide production of the nitroxide 8. It abstracted a hydrogen-atom 17e,19 ž 8, because no nitroxide was observed in the absence of AIBN. from H—C(O), giving the radical CONH2 and coupled Therefore, 2-cyano-2-propyl radicals played a key role in the periodically with NO to give 2-cyano-2-nitrosopropane.4,5,26

EPR study of nitroxides from NO and photolyzed amides 653

Because of its less steric block, 2-cyano-2-nitrosopropane OO ν * trapped favorably carbamoyl radicals, giving the nitroxide 8. h (10) X2NHC X2NHC This assumption is supported by two facts: (a) there exist two X = H or D much smaller hydrogen HFSCs (aH D 0.098 and 0.047 mT) in the nitroxide 8, which indicates that these two hydrogen atoms should stand in a position far away from the nitroxide O O * . * function and at a similar but distinct position; and (b) the X2NXC H + R . RH + 2N C (11) double-bond character of the C(O)—N bond in the moiety

C(O)NH2 could make two amino hydrogen atoms non- R = C(CH3)2(CN), or equivalent in the nitroxide 8.17 Therefore, its structure may be drawn as illustrated. C(CH3)2(C(NH2)) = NH

O CH (12) H 3 NO + R . RNO N CNCCN H O O O . CH3 X2N C . + RNO X2NNRC (13) 8 O. 8, 9 or 10 It is surprising that N-HFSC, which have a magnitude of Scheme 3 0.05 mT,9b is not observed. In order to obtain more evidence to assign the structure, two supplementary experiments were performed: UV photolysis of NO-saturated formamide Five experiments were conducted on NMF. (a) In the mixed with (a) D2O (1 : 1, v/v) and (b) water (1 : 1, v/v) UV photolysis of NO-saturated NMF mixed with water containing AAPH (0.03 M). Experiment (a) gave a 0.962 mT (1 : 2, v/v) containing AAPH (0.04 M), a 0.956mT 1:1:1 1 : 1 : 1 triplet [Fig. 5(c)], each line of which had no further triplet with unresolved hyperﬁne splitting and apparently splitting, even at a very low instrumental modulation broadening linewidth was obtained [Fig. 6(a)]. The g-value amplitude, e.g. 0.005 mT. It has been well established was measured as 2.0060 (assigned to the nitroxide 11). After that only two amino hydrogen atoms of formamide are a few minutes, a 1.462 mT 1 : 1 : 1 triplet appeared [Fig. 6(b)], 17d 4 easily exchanged by two deuterium atoms in D2O. As which was bis(2-amido-2-propyl) nitroxide. After 15 min, expected theoretically, the D-HFSC is 6.5-fold smaller than lines [circles in Fig. 6(c)] of a new radical became more the corresponding hydrogen HFSC. This led to an unresolved and more intense (see below). (b) A similar experiment to

ﬁne splitting of each triplet line in Fig. 5(c). This experimental (a) was conducted but in D2O. A 1.035 mT 1 : 1 : 1 triplet result provided the evidence for assigning one group of the with unresolved ﬁne structures was obtained. The line nitroxides 8 and 9 to be carbamoyl. Experiment (b) gave the separation was estimated to be ¾0.005 mT. (c) The UV EPR spectrum shown in Fig. 5(d), in which each triplet line photolysis of an NO-saturated benzene mixture of NMF was split in two relatively broad lines spaced by 0.105 mT (1 : 1, v/v) containing AIBN (0.04 M) was carried out, giving

[Fig. 5(e)]. The corresponding radical species can be referred also a 1 : 1 : 1 triplet, but with a little larger aN value of to as the nitroxide 10 owing to its g-value of 2.0061. The 1.001 mT and unresolved ﬁne structures (assigned to the apparently single H-HFSC may be explained in the following: nitroxide 13). (d) In the UV photolysis of NO-saturated pure (a) the double bond character of the C(O)—N bond causes NMF containing AIBN (0.07 M),a1.001mT1:1:1tripletwas the two amino hydrogen atoms to be non-equivalent; the obtained. Very weak lines belonging to the other radical were H-HFSC arising from one of them is commonly much larger seen. (e) In the UV photolysis of NO-saturated pure NMF, no 17b,d,19 than that of the other, and like the nitroxide 8,oneaH radical was observed. The g-values of these radicals enable is double the other in the pure formamide or in a mixture of us to assign all of them to nitroxides. The photochemical formamide and benzene; and (b) probably the unobserved reactions of NMF have been considered to be similar to those H-HFSC of the other hydrogen is due to a stronger EPR of formamide.19 Based on the above experimental facts, it ž line broadening effect caused by a signiﬁcant decrease in the is most likely that the radical C(O)NH(CH3 generated spin–spin relaxation time of the nitroxide 10 in the water by hydrogen-atom abstraction from NMF constructs these medium. The generation of the nitroxides 8, 9,and10 is nitroxides. They are assigned to the nitroxides 11,12 and13, suggested in Scheme 3. respectively.

O CH X 3 O CH3 O CH3 N CNCC NH XN CNCC NH HN CNCCN X . NX . . O CH3 2 CH3 O CH3 NX2 CH3 O CH3

9: X = D 11: x = H 13 10: X = H 12: x = D

654 F.Wang,J.JinandL.Wu

(a) N-HFSC to the nitroxide EPR spectrum, respectively. Thus, the nitroxide 14 is assumed most likely to be as illustrated.

O H CH3 (b) H C N CNCCN

H3C H O . CH3

1 mT 14

(c) The representation of —C(H)(H)— in the structural drawing of the nitroxide 14 indicates that the two ˇ-protons are non-equivalent. A possible mechanism is suggested in Scheme 4. The lines indicated by question marks in Fig. 7(a) are unresolved because of the smaller amount of spectroscopic information. They may belong to a nitroxide- containing a moiety formed by hydrogen-atom abstraction from the N-methyl of DMF. The photolysis of an NO saturated aqueous solution of DMF (15%, v/v) containing AAPH (0.025 M)gavethe (d) EPR spectrum shown in Fig. 8(a). It clearly consists of two radical species: (a) the more intense 1 : 1 : 1 triplet (arrows), which is assigned to bis(2-amido-2-propyl) nitroxide;4 and (b) the nitroxide 15. The calculation [Fig. 8(b)] for the whole

Figure 6. EPR spectra recorded (a) during the UV photolysis of NO-saturated NMF mixed with water containing AAPH, (b) a (a) few minutes after irradiation and (c) ca 15 min after irradiation and (d) the simulation for spectrum (c).

Nitroxides generated from reaction of NO with C-centered amide radicals At a higher volume fraction of DMF in benzene (DMF : ben- 1 mT zene D 9 : 1, v/v) and a higher AIBN content (0.07 M), the experiment gave a mixed EPR spectrum [Fig. 7(a)]. It seems to be a superposition of four radicals with the same g-value of 2.0061. The main radical species is marked with a stick, (b) the EPR linewidth of which is relatively sharp. Its simulation [Fig. 7(b)] was completed by overlapping of three radical species in concentration proportions of 35 : 4 : 1 in the order of (a) the nitroxide 14 [Fig. 7c] which consists of two non-

equivalent nitrogen atoms (aN D 1.438 and 0.219 mT) and one hydrogen atom (aH D 1.713 mT), (b) the nitroxide 4 [arrows in Fig. 7(a)] and (c) bis(2-cyano-2-propyl) nitroxide [circles in Fig. 7(a)].4 The EPR spectroscopic features of (c) the nitroxide 14 are similar to those of an isomer of the t ž 17e nitroxide Bu N(O )CH2N(CH3C(O)H. Accordingly, the resolved large proton HFSC of 1.713 mT in the nitroxide 14 should arise from one of the two ˇ-protons of the moiety

CH2N(CH3C(O)H and the unresolved HFSC arise from the other ˇ-proton with a dihedral angle ( ³ 90°)oftheC—H

bond with respect to the p-orbital of the unpaired electron on Figure 7. (a) EPR spectrum generated by the UV irradiation of , the nitroxide nitrogen.17e 27 Both formyl and dimethylamino NO-saturated DMF in benzene (9 : 1) containing AIBN (0.07 M), moieties could be excluded from the nitroxide 14 because (b) its simulation and (c) the simulation for the EPR spectrum they would contribute a ca 1 mT N-HFSC and a ca 1.4 mT assigned to the nitroxide 14.

EPR study of nitroxides from NO and photolyzed amides 655

CH3 O CH3 . O NC C . + (H3C)2N CH NC CHCH3 + H2C N CH (14)

CH3 CH3

CH O H CH

O . 3 3 H C N CH2 + NO + . C CN H C N CNCCN (15) H C . CH3 CH3 3 H O CH3 14 Scheme 4

EPR spectrum was carried out by superimposing a 1.475 mT Theoretically, aD is about 1/6.5 times smaller than the

1 : 1 : 1 triplet on a set of lines with a concentration proportion corresponding aH assuming that no other changes occur. of 2 : 1. The latter, i.e. the nitroxide 15, exhibits hyperﬁne Its simulation [Fig. 3(c)] exhibits a hyperﬁne coupling to coupling to two non-equivalent nitrogen nuclei (aN D 1.476 three non-equivalent nuclei, all with I D 1(aID1 D 1.550, and 0.218 mT) and one proton (aH D 1.821 mT). These values 0.250 and 0.132 mT). Its EPR spectroscopic features should are very close to those of the nitroxide 14.Itsstructurecould be very similar to those of the nitroxide 14. Thus, two values be similarly assumed to be as illustrated. The mechanism for aID1 D 1.550 and 0.250 mT are assigned to two non-equivalent its generation is closely similar to that illustrated in Scheme 4, nitrogen atoms, whereas the other aID1 D 0.132 mT has to be replacing 2-cyano-2-propyl by 2-amido-2-propyl. assigned to the ˇ-D. Accordingly, the ˇ-D HFSC in the nitroxide 16 should be expected to be ca. 0.26 mT. In fact, a D 0.132 mT. It is half the predicted value of a . Obviously, O H CH3 D D the deuteron exhibits a distinct and additional effect on H C N C N C C NH HFSC. The much smaller aD value in the nitroxide 16 may be H C H O . CH NH 3 3 2 referred to as a different equilibrium conformation from the nitroxide 14.Thea assignment will be supported below. 15 D

The lines marked with sticks in Fig. 3(a) overlap other O D CH3 two triplets. Although some details are not clear enough D C N CNCCN owing to superposition, a principal triplet feature of the D C D O. CH nitroxide 16 is clear. Compared with experiments concerning 3 3 DMF described above, the nitroxide 16 should be logically 16 similar to the nitroxide 14, except that the deuteron HFSC in the former replaces the proton HFSC in the latter. The photolysis of an NO-saturated aqueous solution of

DMF-d7 (25%, v/v) gave a 1.028 mT 1 : 1 : 1 triplet, which is the nitroxide 2. Furthermore, the photolysis of an NO-

saturated aqueous or D2OsolutionofDMF-d7 (25%, v/v) (a) containing AAPH (0.015 M) gave an EPR spectrum [Fig. 9(a)] of a pure single radical (17). Its perfect simulation [Fig. 9(b)] manifests a hyperﬁne coupling to four nuclei with I D 1. They are assigned to two non-equivalent nitrogen atoms

(aN D 1.479 and 0.220 mT) and two non-equivalent deuterons 1 mT (aD D 0.127 and 0.137 mT). The two very similar deuterium atoms suggest that one moiety of the nitroxide 17 should be

CD2N(CD3C(O)D generated by deuterium-atom abstraction 15 from the N-methyl of DMF-d7. Hence the radical 17 is easily (b) assigned to the nitroxide 17, as depicted. It should be noted that two non-equivalent deuterium atoms of the methylene group are resolved, in contrast to the case of the nitroxides 14, 15 and 16.

O D CH3 DNCC NCC NH

D3C D CH NX Figure 8. (a) EPR spectrum of radicals from the UV photolysis O. 3 2 of an NO-saturated aqueous solution of DMF containing AAPH X = H or D and (b) its simulation. 17

656 F.Wang,J.JinandL.Wu

(a) (a)

1 mT

(b) 1 mT

(b)

(c)

Figure 9. (a) EPR spectrum of the radical from the UV

photolysis of an NO-saturated aqueous or D2Osolutionof DMF-d7 containing AAPH and (b) its simulation. (d) The simulation for the lines marked with circles in Fig. 6(c) manifests a hyperﬁne coupling to two non-

equivalent nitrogen atoms (aN D 1.400 and 0.229 mT) and

aproton(aH D 1.324 mT). Its g-value of 2.0059 indicates that it is a nitroxide (18). Its EPR spectroscopic patterns are Figure 10. (a) EPR spectrum of radicals from the UV photolysis very similar to those of the nitroxide 15. Reactions involving of an NO-saturated mixture of DMA and benzene containing the formation of the nitroxide 18 are matchable to the reac- AIBN, (b) a spectrum generated by an overlapping of spectra tion mechanism in Scheme 4, replacing DMF by NMF and (c) and (d), (c) the simulation for one component of spectrum (a) 2-cyano-2-propyl by 2-amido-2-propyl. and (d) the simulation for the other component of spectrum (a).

two non-equivalent nitrogen atoms and two equivalent O H CH3 hydrogen atoms (aN D 1.475 and 0.271; aH D 0.685 mT). HNCC NCC NH This suggests that one moiety of the nitroxide 19 should be H H O. CH NH 15b,16a,17d,e 3 2 CH2N(CH3C(O)CH3. The calculation [Fig. 10(d)] for the nitroxide 20 [sticks in Fig. 10(a)] shows a hyperﬁne 18 coupling to two non-equivalent nitrogen atoms and one

hydrogen atom (aN D 1.502 and 0.132; aH D 2.196 mT). A complicated EPR spectrum [Fig. 10(a)] was obtained Like the nitroxide 14, the large H-HFSC (2.196 mT) of the when an NO-saturated mixture of DMA and benzene nitroxide 20 arises from one of the two ˇ-protons. The (DMA : benzene D 9 : 1, v/v) containing AIBN (0.03 M)was nitroxides 19 and 20 are conformational isomers. It is well UV photolyzed. When light was masked, only a very weak known that DMA undergoes a primary photodissociation to 1.496 mT 1 : 1 : 1 triplet with a g-value of 2.0058 existed, yield alternatively (a) an acetyl radical and a dimethylamino which was bis(2-cyano-2-propyl) nitroxide.4 It seemed radical or (b) a methyl radical and a dimethylcarbamoyl that the spectrum shown in Fig. 10(a) consisted of two radical, following excitation via a Ł transition upon UV nitroxides with the same g-value (2.0059) but different absorption.12,13 Sequentially, the acetyl radical decomposes linewidths. Its simulation [Fig. 10(b)] was completed by into a methyl radical and carbon monoxide (CO) and the an overlapping of two nitroxides [Fig. 10(c) and (d), the dimethylcarbamoyl radical into a dimethylamino radical and nitroxides 19 and 20, respectively] with a concentration CO.13 As the N-methyl hydrogen is more easily abstracted,17d ž 15b,16a,17d,e proportion of 1 : 2.5. The calculation [Fig. 10(c)] for the the radical CH2N(CH3C(O)CH3 can be generated nitroxide 19 [broader lines in Fig. 10(a), i.e. all lines except by hydrogen-atom abstraction from an N-methyl of DMA. those marked by sticks] reveals that it is composed of It is well established that NO reacts favorably with the

EPR study of nitroxides from NO and photolyzed amides 657

methyl radical to produce nitrosomethane, which proves O CH3 to be a reactive spin trap in solution.4 The EPR spectrum H3CNCHC 2 N C C NH of a methyl nitroxide formed from nitrosomethane by CH O . CH NH trapping the other radical is characteristic of a 1 : 3 : 3 : 1 3 3 2 1 quartet separated by ca 1.1 mT. The lack of a methyl 21 nitroxide implies that methyl radicals are not likely to be produced in this photolysis. Therefore, we may conclude O H CH3 that the signiﬁcantly major step for free-radical production H3C C N C N C C NH in the photolysis of DMA is the scission of the C—N bond H3C H O . CH NH under the present conditions, giving an acetyl radical and 3 2 an amino radical. Thus, except for the photochemically 22 formed acetyl radical and dimethylamino radical, other radicals possibly present in the system are 2-cyano-2-propyl ž and CH2N(CH3C(O)CH3. Owing to the characteristic EPR The UV photolysis of an NO- and acetamide-saturated 10 spectroscopic features of the nitroxide 3 and acyl nitroxides, benzene solution did not give EPR signals, whereas in the both acetyl and dimethylamino moieties are excluded presence of AIBN (0.04 M), the system gave an EPR spectrum. from the nitroxides 19 and 20. In the structural drawing It consists of two sets of triplets with the same g-value of illustrated, —CH2 — denotes two equivalent ˇ-protons and 2.0060: one is a stronger 1.483 mT 1 : 1 : 1 triplet and the —C(H)(H)— indicates two non-equivalent ˇ-protons. other is a weaker 1.01 mT 1 : 1 : 1 triplet. The former is bis(2-cyano-2-propyl) nitroxide.4 The latter is likely to be H1 the nitroxide 8, although the splitting arising from two H H protons of H2N—C(O) is not observed because of the H2 much lower intensity. Various primary free-radical processes O R O R involving acetamide have been reported: (a) acetamide was photolyzed to produce methyl and carbarmonyl radicals in R' rigid matrices;19 (b) the photolysis of an aqueous acetamide R' ž 28 solution, however, yielded acetyl and NH2 radicals; and ž R = C(CH ) CN (c) the radical CH2CONH2 could be formed by radical attack 3 2 17a,d,e on acetamide. However, both acetyl and CH2CONH2 R' = N(CH3)C(O)CH3 could be ruled out as moieties in the above nitroxide with the spectroscopic pattern of a 1.01 mT 1 : 1 : 1 triplet, because of

O CH3 O H CH3 the lack of an aN value of about 0.7 mT and an HFSC arising from two ˇ-protons. H3CNC CH2 N C CN H3C C N C N C CN . The UV photolysis of an NO-saturated aqueous or D2O CH3 O . CH3 H3C H O CH3 solution of acetamide (0.3 M) containing AAPH (0.05 M)gave 19 20 an EPR spectrum which exhibited hyperﬁne coupling to two

equivalent protons (aH D 0.796 mT) and one nitrogen nucleus The photolysis of an NO-saturated aqueous solution of (aN D 1.410 mT). The corresponding radical is assigned to DMA (15%, v/v) containing AAPH (0.03 M) gave the an the nitroxide 23. Its EPR behaviors are similar to those of t ž 17e EPR spectrum shown in Fig. 11(a). Its simulation [Fig. 11(b)] the nitroxide Bu N(O )CH2C(O)NH2. Therefore, it is most indicates that the whole spectrum consists of two radical likely that the nitroxide 23 may be assumed to have the components with a concentration proportion of 3.5 : 1; (1) one structure shown. [Fig. 11(c), the nitroxide 21] manifests a hyperfine coupling to two non-equivalent nitrogen atoms and two equivalent O CH hydrogen atoms (aN D 1.462 and 0.184; aH D 1.027 mT); 3 and (2) the other one [Fig. 11(d), the nitroxide 22] exhibits X2NCHC 2 N CCNH a hyperfine coupling to two non-equivalent nitrogen atoms O . CH3 NX2 and a hydrogen atom (aN D 1.651 and 0.159; aH D 2.044 mT). X = H or D These EPR parameters are very similar to those of the 23 nitroxides 19 and 20, respectively. The unique difference between the above systems lies in the initial radical: 2-cyano- 2-propyl radical in the former and 2-amido-2-propyl radical Figure 12(a) shows the EPR spectrum obtained during in the latter. Accordingly, the structures of the nitroxides 21 the photolysis of an NO-saturated aqueous solution of NMA and 22 areassumedtobeasdepicted. (0.5 M) containing AAPH (0.025 M). Its perfect simulation Mechanisms for the generation of the nitroxides 21 and [Fig. 12(b)] fits the experiment very well. It is composed 22 are similar to that shown in Scheme 4, replacing DMF by of two radical species with a concentration proportion of DMA and 2-cyano-2-propyl by 2-amido-2-propyl. The above 2 : 1; (a) a 1.465 mT 1 : 1 : 1 triplet, which is bis(2-amido-2- observation implies that NO does not react favorably with propyl) nitroxide;4 and (b) the nitroxide 24, which exhibits both acetyl and dimethylamino radicals in this case. a hyperfine coupling to two equivalent hydrogen atoms

658 F.Wang,J.JinandL.Wu

(a) (a)

1 mT

(b)

1 mT

(b) Figure 12. (a) EPR spectrum of radicals from the UV photolysis of an NO-saturated aqueous solution of NMA containing AAPH and (b) its simulation.

completed by an overlapping of two radical components with a concentration proportion of 1 : 1.4; one is the nitroxide 24 and the other is the nitroxide 25 [Fig. 13(e)], which exhibits

a hyperﬁne coupling to one hydrogen atom (aH D 1.416 mT)

and two non-equivalent nitrogen atoms (aN D 1.461 and 0.229 mT). When the above experiment was carried out in (c) D2O instead of in water, the same EPR spectra as shown in Fig. 13 were obtained although the N-hydrogen atom

of NMA was replaced by the deuterium atom in D2O. This means that the deuteron does not contribute HFSC to EPR spectra. This phenomenon indicates that the structural assignment of the nitroxides 24 and 25 is reasonable, where the N-X (X D H or D) is far away from the nitroxide function. Reactions that occur here couple with that in Scheme 4, replacing NMF by NMA and 2-cyano-2-propyl by 2-amido- 2-propyl only.

(d)

O CH3

H3C C NCHN2 CC NH

Figure 11. (a) EPR spectrum of radicals from the UV X O. CH3 NX2 photolysis of an NO-saturated aqueous solution of DMA containing AAPH, (b) the calculated spectrum generated by X = H or D overlapping of spectra (c) and (d), (c) the simulation for one 24 component of spectrum (a) and (d) the simulation for the other O H CH component of spectrum (a). 3 H3C C N C N CC NH . X H O CH3 NX2 (aH D 0.915 mT) and two non-equivalent nitrogen atoms X = H or D (aN D 1.502 and 0.219 mT). When the above solution contained a smaller amount of AAPH (0.01 M), the EPR 25 spectrum shown in Fig. 13(a) was ﬁrst recorded. The simulation [Fig. 13(b)] for lines in Fig. 13(a) except those marked with a stick indicates that the radical species is the nitroxide 24. The EPR spectrum shown in Fig. 13(c) was Acknowledgements obtained in ca 10 min after UV irradiation. It implies the The authors to express their gratitude to the Natural Science appearance of a new radical. Its simulation [Fig. 13(d)] was Foundation of China for its ﬁnancial support (grant No. 20072013).

EPR study of nitroxides from NO and photolyzed amides 659

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