J. Anal. Appl.

Pyrolysis 72 (2004) 153163

On the role of peptides in the pyrolysis of amino acids

Ramesh K. Sharma a , W. Geoffrey Chan a, , Jia Wang a , Bruce E. Waymack a , Jan B. Wooten a , Jeffrey I. Seeman b , Mohammad R. Hajaligol a
a

Philip Morris USA Research Center, 4201 Commerce Road, Richmond, VA 23234, USA b SaddlePoint Frontiers, 12001 Bollingbrook Place, Richmond, VA 23236, USA Accepted 6 March 2004 Available online 2 July 2004

Abstract The pyrolysis of aspartic acid, asparagine, glutamic acid, glutamine, and pyroglutamic acid were studied. Each amino acid was rst pyrolyzed at 300 C to obtain a low temperature tar (LTT) and a low temperature char (LTC). The LTC was then pyrolyzed at 625 C to obtain a high temperature tar (HTT) and a high temperature char (HTC). The LTT and HTT were analyzed by gas chromatography/mass spectrometry. Maleimide, succinimide and related compounds were observed from asparagine and aspartic acid. Glutarimide was the major pyrolysis product from glutamine and glutamic acid. A number of new products were identied. In order to explain the formation of reduction products such as succinimide and glutarimide, a disproportionation mechanism involving polypeptides, formed in the pyrolysis melt, is suggested. The product distributions appear to depend on the relative volatility and stability of the amino acids and their pyrolysis products. In addition, the thermal properties of asparagine and its pyrolysis products were studied by thermogravimetry/differential scanning calorimetry (TGA/DSC). On the basis of the appearance temperatures of various products including ammonia and water and the temperatures at which the lower molecular weight products evaporate, it was concluded that asparagine (and glutamine and possibly other amino acids) thermally rst polymerize to polypeptides which, at higher temperatures, subsequently degrade to lower MW products. 2004 Elsevier B.V. All rights reserved.
Keywords: Thermal decomposition; Pyrolysis; Amino acid; Peptide; Asparagine; Aspartic acid; Glutamine; Glutamic acid; Pyroglutamic acid

1. Introduction The pyrolysis chemistry of amino acids is dominated by their common functionality [1,2]. Amino acids can undergo dehydration, decarboxylation and deammination when pyrolyzed, although the extent of these reactions are dependent on structure [15]. Experimental conditions, the most important of which are temperature and residence time, can also control the products observed. For example, at lower temperatures (200300 C), low molecular weight heterocyclic compounds are generally observed [1,2], although in certain circumstances, well-dened polymers may be formed [5]. At pyrolysis temperatures above 500 C, polynuclear aromatic compounds (PACs) including nitrogen containing PACs are observed [2,68].

Corresponding author. Tel.: +1-804-274-5865; fax: +1-804-274-2160. E-mail address: w.geoffrey.chan@pmusa.com (W.G. Chan). 0165-2370/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jaap.2004.03.009

In addition to thermally-induced loss of small, stable molecules (e.g., water, ammonia and carbon dioxide), amino acids have been reported to form, via dehydration, dipeptides 1 as reactive intermediates (Scheme 1) [1]. These dipeptides can form 2,5-diketopiperazines 2, the most important pyrolysis product from many amino acids [1,5]. For example, pyrolysis of proline (3) at 300 C leads to high yields of the diketopiperazine 4 as the major isolable product, and its formation via the thermally labile dipeptide intermediate 5 has been proposed [1]. In this example, 5 is likely formed in the heated melt, leading via dehydration and cyclization to the rather stable and volatile (under the thermal conditions) diketopiperazine 4. Diketopiperazines have also been observed in the Curie-point pyrolysis of a series of glycyl dipeptides [9]. In principle, dipeptides will be nondetectable intermediates in amino acid pyrolyses because of their high thermal reactivity and low volatility which keeps them in the thermal zone until they react further. Recently, we reported the pyrolysis of a number of amino acids, including proline, tryptophan (6) and asparagine

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Scheme 1. (A) Intermediacy of dipeptides in the pyrolysis of amino acids and subsequent formation of diketopiperazines. (B) Major pyrolysis products from the pyrolysis of proline (3).

(7) [2]. While the product distributions from proline and tryptophan can be understood on the basis of unimolecular reactions, asparagine formed products that were at that time either not identied (one instance) or whose structure could not be explained by any previously described mechanism (a second instance). In order to gain additional insight into these reactions, the pyrolysis products from asparagine were further investigated; in addition, the pyrolyses of aspartic acid (8), glutamine (9), glutamic acid (10), and 2-pyrrolidone-5-carboxylic acid (11, also called pyroglutamic acid) were also investigated. We herein propose the generalization that di-, tri-, and most likely polypeptides can be intermediates in the formation of multiple products in the pyrolysis of some amino acids.

2. Experimental 2.1. Pyrolysis experiments A quartz tube heated by a metal block furnace was used as reactor. Each amino acid was rst pyrolyzed at 300 C to obtain a low temperature tar (LTT) and a low temperature char (LTC). The LTC was then pyrolyzed at 625 C to ob-

tain a high temperature tar (HTT) and a high temperature char (HTC). All experiments were done at atmospheric pressure in a helium atmosphere with a continuous ow of 120 mL/min. The furnace was set to the desired temperature. The exit line, maintained at ca. 200 C to minimize any condensation of the products, led directly to a Cambridge pad that collected the tar. The pad was a glass (borosilicate) ber lter pad stabilized by an organic binder and manufactured by the Cambridge Filter Corporation, Syracuse, New York (retaining power 0.3 nm). A sample of 200 mg of amino acid was placed in a ceramic boat that initially rested in the unheated section of the reactor tube. A sliding K-type thermocouple was used to transport the boat, containing the sample, to and from the reaction zone and to measure the sample temperature. After the desired temperature was reached, the boat was pushed to a pre-determined location in the preheated reactor and left in the oven for 60 min. At the end of the pyrolysis, the boat was pulled back to the unheated section of the tube, and the residual material on the boat was allowed to cool. The Cambridge pad was removed and extracted with 10 mL of methanol to dissolve the collected tar. Any product that condensed prior to the pad but outside the heating zone was also collected and added to the tar. The combined tar is designated as low temperature tar (LTT). The residual in the boat is designated low temperature char (LTC). The LTC was then pyrolyzed similarly at 625 C to obtain a high temperature tar (HTT) and a residual in the boat, named high temperature char (HTC). See Table 1. The LTT and HTT were analyzed by an Agilent 6890 GCMS that was equipped with a nitrogen-phosphorus detector (NPD). A 30 m long 0.25 mm diameter 0.025 m thickness ultra low-bleed 5%-diphenyl95%-dimethylsiloxane column (J&W Scientic #122-4232) was used. The oven was held at 55 C for three minutes, heated at 5 C/min, and then held at 275 C for 25 min. The sample leaving the column was spit into two lines, one leading to the MS and the other to the NPD. The dimensions of these

R.K. Sharma et al. / J. Anal. Appl. Pyrolysis 72 (2004) 153163 Table 1 Overall mass balance in pyrolysis of amino acids (wt.% of amino acid)a Starting substrate Mass balance (% conversion) 300 Cb Char (LTC) Proline (3)c Asparagine (7)c Aspartic acid (8) Glutamine (9) Glutamic acid (10) Pyroglutamic acid (11) 0 64 66 30 27 44 LTT 80 <0.5 <1 9 13 6 Gaseous products 20 35 33 61 60 50 625 Cb Starting substrate n.a. LTC LTC LTC LTC LTC Char (HTC) n.a. 23 17 22 16 21 HTT n.a. 12 6 3 2 5 Gaseous products n.a. 29 43 5 9 18 n.a. 64 76 66 69 68

155

Total gaseous products

a The low and high temperature chars (LTC and HTC) and the low and high temperature tars (LTT and HTT) are dened in the text. The weight percents are based on initial mass of substrate. The mass of gaseous products was calculated by difference. In each case, the char yield was calculated from the amount of residue remaining after pyrolysis, based on the initial weight of the amino acid. The amounts of LTT and HTT in each run were calculated from the differences in the weights of the Cambridge pad before and after the pyrolysis. n.a.: not applicable, as there was no LTC formed in the pyrolysis of the initial substrate. b Pyrolysis temperature. c Data from [2].

lines were adjusted to obtain an optimal signal from each detector. The compound identication was achieved using a comparison of the experimental mass spectra with standard mass spectral data from a commercially available library (NIST98), as well as by comparing the chromatographic retention times and mass spectra with those from the reference standards when standards were available. The relative yield of an individual component was calculated from the calibration curve based on the initial amount of amino acid. In cases where a reference standard was not available, the relative yield was calculated from the total ion current and the overall yield of the product tar. Further details of the experimental procedures have been provided previously [2]. 2.2. Thermogravimetric analyses Thermogravimetric analyses (TGA) were performed using a Netzsch STA409 skimmer TGA/DSC/MS instrument. Samples of 510 mg were heated at a constant rate of 20 C/min from 25 to 1000 C in 150 mL/min ow of helium. Sample weight loss, endothermic/exothermic events, and evolved volatile products were monitored continuously with temperature. In the TGA/MS studies, typically the MS was scanned over the range of 1300 amu as a survey scan in a preliminary run. Selected ion monitoring (SIM) data are reported herein on the primary masses of interest be-

cause SIM provides a better signal-to-noise ratio and more time-dense data. 2.3. Isolation and structural identication of the m/z 196 product from asparagine and aspartic acid In the previous work, the LTT and HTT from the pyrolysis of asparagine was shown to contain three major products, with parent ions at m/z of 97 (maleimide, 12), 99 (succinimide, 13) and m/z 196 (14) (see Scheme 2) [2]. The component with parent m/z 196 was not identied at that time. In this work, the m/z 196 compound was isolated from the asparagine pyrolysis tar by column chromatography and analyzed by high resolution mass spectrometry (HRMS) and by 1 H and 13 C NMR spectroscopy, as follows. To collect a sufcient amount of LTT, a sample of 0.5 g of asparagine was pyrolyzed. The system was cleaned and a duplicate pyrolysis was conducted. The LTT from these two pyrolyses were individually extracted from the Cambridge lter pad with methanol, and the extracts were combined and evaporated to dryness under vacuum. The residue (79.6 mg) was dissolved in ca. 0.75 mL of chloroform to which ve drops of methanol had been added. The resultant solution was loaded onto a Sep-Pak Vac silica cartridge (6cc, 1 g) and eluted by chloroform with increasing amounts of methanol: 1, 2, 3, 4, 5 and 10% (v/v) of methanol/chloroform. Each fraction (ca. 5 mL) was analyzed

O HO NH2 O 7 asparagine NH2 O N H 12 maleimide O

O O

N H 13

+
NH O

succinimide

14

Scheme 2.

300 C

pyrolysis products of asparagine.

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by GCMS, using the exact same procedure and conditions used to analyze the pyrolysates, as described above. The 100:3 chloroform:methanol fraction was heavily enriched (>95%) with the unknown compound and was used for analysis. The solution was rotary evaporated under vacuum to obtain ca. 8 mg of sample. 1 H and 13 C NMR spectra were obtained on a Varian Unity 400 spectrometer with tetramethylsilane as internal standard. Chemical shifts are recorded in ppm. 1 H NMR: 400 MHz (acetone-d6 ) 2.45 (br s, 4H), 2.80 (dd, J = 5.7 J = 17.8, 1H); 3.03 (dd, J = 9.7 J = 17.8, 1H); 5.20 (dd J = 5.7 J = 9.7, 1H). 13 C NMR: 100 MHz (acetone-d6 ) 28.8 (br s, 2, CH2 ), 34.5 (1, CH2 ); 49.1 (1, CH); 174.7(1, C=O); 175.1 (1, C=O), 177.0 (br s, 2, C=O). Mass Spectra (JEOL HX-110): Calculated, High Resolution C8 H9 N2 O4 , 197.05623 amu. Found, 197.05774 amu.

an oxidation and a reduction. Based on new experimental data discussed below, we propose a new pathway for the formation of these single ring heterocycles, via polymeric peptides (Scheme 3). 3.1.2. Multiple ring pyrolysis products and reaction mechanisms The previously unidentied third product in the pyrolysis of asparagine is 14 (m/z 196), formed in non-reproducible yields (655 mg/g) and on occasion being the major product. As described below, 14 was found to be thermally unstable; hence, minor variations within the pyrolysis conditions were responsible for variations observed in the yield of 14. This compound was also formed from aspartic acid (see below). Interestingly, 14 was shown to be a dehydro-dimer, rst by GCMS and conrmed subsequently by high resolution mass spectrometry of a puried sample. The 1 H and 13 C NMR spectra are consistent with 14 being a bis(succinimide). A single ABX coupling pattern with sharp resonances was observed in the 1 H NMR spectrum for the asymmetrically substituted ring in 14. The geminal pair of methylene protons exhibited a characteristic AB quartet that is additionally split by coupling to the methine proton. A broad resonance was observed for the four AABB protons on the second symmetrically substituted succinimide ring of 14. We attribute this broadness to be due to the nearly perpendicular conformation of the two rings relative to each other. From the symmetrically substituted rings perspective, all four of its geminal protons are in different environments, hence, the line broadening. Careful examination of the 1 H NMR spectrum of the 100:3 chloroform/methanol chromatographic fraction revealed evidence for at least two minor products, tentatively attributed to the isomeric meso- and d,l-isomers of 15. A pair of weak overlapping methylene resonances having the same coupling pattern as the methylene protons of 14 was observed at ca. 5.27 and 5.28. These protons, the AB portion of the ABX system, were slightly downeld of the corresponding protons of 14, a reasonable position due to the presence of the directly bonded nitrogen to the adjacent carbon. Unfortunately, these minor isomers are not resolved from 14 under the GC conditions, and independent MS data are not available to conrm the molecular weights or fragmentation patterns.

3. Results and discussion 3.1. Pyrolysis of asparagine (7) 3.1.1. Single ring pyrolysis products and reaction mechanisms During pyrolysis, gases, tar and char can be formed. In our experimental design [2], the char consists of all materials remaining in the ceramic boat following the thermal treatment, including unpyrolyzed substrate and high molecular weight non-volatiles formed from pyrosynthesis. The tar is the total condensable product that has moved from the heating zone to a downstream collection chamber. Tar can consist of distilled starting material as well as volatile or semi-volatile pyrolysis and/or pyrosynthesis products. Recently we reported that the pyrolysis of asparagine (7) at 300 C led to gases not trapped on the Cambridge lter, almost no LTT, and a substantial amount of LTC (see Table 1) [2]. Three major products were observed in the HTT. Two of these products were readily identied as maleimide (12) and succinimide (13), in yields of 8 mg/g of starting material and 48 mg/g, respectively (see Scheme 2 and Table 1); the structure and thermal properties of the third product, 14, unidentied at the time, is discussed below. Maleimide (12) is at least formally formed from asparagine via a dehydrative cyclization and loss of ammonia. The observation of succinimide (13) as an asparagine pyrolysis product is notable because its formation requires a reduction step. Independent pyrolysis of maleimide under the same conditions does not lead to succinimide, a not unexpected result for two reasons: rst, pyrolysis conditions typically lead to more oxidized products, e.g., formation of polyunsaturated compounds such as PACs from saturated compounds, not the reverse [10,11]; and second, maleimide is relatively stable and can transfer from the thermal zone prior to degradation. We previously proposed that a dipeptide is formed in the thermal melt which then decomposes and rearranges to form succinimide [2]. This sequence is essentially a disproportionation reaction, involving both

The m/z 196 pyrolysis product 14 can, in principle, be formed by the direct reaction of a succinimide with succinimide via one of two pathways: either an ene reaction

R.K. Sharma et al. / J. Anal. Appl. Pyrolysis 72 (2004) 153163

157

H2N HO2C H2N NH HO2C 7 Asparagine CONH2 O 14 CONH2 O N O O

N H 12

+
O N H 13 O

-H2O

A+B C+D

A
O CONH2 NH O CONH2 NH O CONH2 NH CONH2 NH -NH3 H+ O N O N

A
O N O H N O H N O

O H N O H O

B
O

C
O

16

17(PSI)

18

Scheme 3. Thermal formation of poly(amino acid)s from asparagine and possible thermal decomposition pathways to lower molecular weight heterocycles.

between the enol of succinimide and the double bond of maleimide or a Michael attack of succinimides nitrogen atom on the conjugated double bond of maleimide. Similar reactions can, in principle, form 15. The signicant volatility of 12 and 13 under the reaction conditions, however, suggests that the concentration of these monomers in the gas phase would be too low for signicant yields of the bimolecular product. Two alternatives exist: one, that bimolecular condensation reactions may be take place in the char prior to volatilization; second, that more complex non-volatile intermediates form in the char and subsequently decompose to 14. For example, 14 could have been formed via the intermediacy of peptides formed in the pyrolysis char during heating. As illustrated in Scheme 3, the polypeptide 16 or poly(succinimide) 17 (PSI) formed directly from asparagine could undergo a series of fragmentations and cyclizations (see, for example, 18) to form 12, 13, and 14. In Scheme 3, the amino group of asparagine is shown to attack rst the carboxylic acid group of another asparagine molecule, followed by attack of the newly formed amide onto the pendant amide and cyclization to the substituted succinimide. Of course, the reverse order would also ultimately lead to 17. It is difcult to predict which pathway, if either, dominates at these temperatures. Typically, a carboxylic acid group would be more reactive than the amide moiety toward an amino group, assuming that the carboxylic acid was not in its zwitterionic form. The -amino groups electronic withdrawing

effect would further accentuate the electrophilic nature of the carboxylic acid carbon. Based on the mechanisms presented in Scheme 3, 19 might have been expected to have formed. However, careful examination of the mass spectra of a few of the asparagine chromatograms did not reveal any m/z 194 for 19. Co-pyrolysis of an equimolar mixture of succinimide and maleimide at 300 C under the asparagine pyrolysis conditions led to only a trace of 14, as judged by GC analysis of the trapped pyrolysis tars. This is likely due to rapid evaporation of these two imides prior to the temperature needed to initiate chemical reactions [12].

3.1.3. Likely intermediacy of polypeptides in the pyrolysis of asparagine The relative concentration of 14 or any pyrolysis product in the thermal zone is a function of the concentrations and temperature-dependent rates of formation and rates of reaction of the species involved, coupled with vapor pressures

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O HO NH2 O 7 NH2 O N H 12 O

O O

+O

N H 13

+
NH O 14

Formation of CO2 when asparagine is heated Appearance of 12 & 13 when asparagine is heated Formation of H2O and NH3 when asparagine is heated Appearance of 12 and 13 when14 is heated Evaporation of 12 Evaporation of 13 Evaporation range of a 1:1 mixture of 12 and 13

100

200

300

400

Temperature (deg C)
Fig. 1. Graphic representation of temperatures of evaporation and formation of the pyrolysis products from asparagines (7). Also shown are the temperatures of formation of 12 and 13 from 14. Data from TGA/DSC/MS experiments.

and residence times, matrix effects, and possible gassolid interactions. The non-reproducible yield of 14 suggests that it is thermally unstable, its yield being very sensitive to minor changes in, for example, sample size and other mass transfer effects. A thermal retro-ene or retro-Michael reaction are a very reasonable decomposition pathways of 14 and of 19. To examine the thermal properties of asparagine and its pyrolysis products further and to gain a better insight into the mechanisms of formation of the pyrolysis products, a series of thermogravimetric (TGA)/differential scanning calorimeter (DSC)/mass spectrometry (MS) analyses were performed. DSC of ca. 9 mg of maleimide demonstrated melting at ca. 90 C and evaporation at ca. 135 C. DSC of ca. 9 mg of succinimide demonstrated melting at ca. 125 C and evaporation at ca. 210 C. These results and other related DSC and TGA results are summarized in Fig. 1. A 1:1 mixture of succinimide and maleimide melts ca. 80 C with a broad evaporation at ca. 120180 C. All of these transitions are endothermic. TGA/MS of the mixture shows that maleimide and succinimide evaporate independently, as judged by separate ion appearances of m/z 97 and 99, with nearly complete loss of mass, i.e., little formation of char. In addition, MS analysis of the TGA gases are lacking signals for m/z 196 and 125, the major fragments of 14. Hence, 14 does not appear to be formed in signicant yields from the monomers in the melt of the 12 + 13 mixture; rather, maleimide and succinimide evaporate from the thermal zone. These conclusions, however, do not take into consideration that the pyrolysis and the TGA experiments were performed under dif-

ferent conditions, i.e., different heating rates and residence times, the effects of which have yet to be evaluated. Regarding the thermal (in)stability of 14, a small sample of this compound puried from the total asparagine pyrolysate tar was subjected to TGA/MS analysis. GCMS analysis of the pyrolysis tars as well as of the puried sample of 14 indicated that m/z 125 is the strongest mass observed for 14. TGA/MS single ion monitoring of puried 14 showed two signicant m/z 97 and 99 signals, observed at ca. 230260 C (Fig. 1); only a trace of m/z 125 was observed. Clearly, 14 is thermally unstable and degrades to maleimide (m/z 97) and succinimide (m/z 99) at ca. 230260 C under these TGA conditions. Critical information regarding the pathways of asparagine pyrolysis can be evaluated using TGA and DSC. As shown in Fig. 2, two distinct regions of weight loss are observed at ca. 245 C (endothermic) and 379 C (slightly exothermic). The rst region of weight loss corresponds almost exactly to one mole equivalent of ammonia and one mole equivalent of water, as determined by the TGA and single ion monitoring analyses (Fig. 3). The second region corresponds to one mole equivalent of carbon dioxide. Far lower in ion intensity are m/z 97 and 99 (Fig. 4), maleimide and succinimide, the two major semi-volatile pyrolysis products of asparagine. The key TGA results are summarized in Fig. 1, plotted to illustrate the temperatures at which these interrelated reactions occur. Critically, the appearance temperature of maleimide and succinimide during the TGA-heating of asparagine is ca. 370 C, far greater than the appearance temperatures when the pure compounds are heated (ca. 135 and

R.K. Sharma et al. / J. Anal. Appl. Pyrolysis 72 (2004) 153163

20 20 10 0 DSC relative heat flow, microvolts ( endothermic = down)

159

TG, Weight Loss, (%)

-20 TG DSC -40

-10

-20

-60

-30

-40 -80 -50 1000

200

400

600

800

Temperature, oC
Fig. 2. TG/DSC of asparagine. Two distinct regions of weight loss are observed, one at 240245 C (endothermic) and a second at 375380 C (slightly exothermic). A very slight weight loss (<0.5%) is observed at ca. 100 C, possibly due to the loss of water from a small amount of asparagines hydrate present. See text for further discussion on this point.
1e-8 m/z 16 - NH3 8e-9 m/z 18 - H2O m/z 44 - CO2 6e-9

Relative Intensity, amps

4e-9

2e-9

200

400

600
o

800

1000

Temperature, C
Fig. 3. TG/MS of asparagine. Evolution proles for ammonia, water, and carbon dioxide, as followed with single ion monitoring at m/z 16, 18, and 44 amu, respectively. Water contributes <5% of the signal of m/z 16 amu.

210 C, respectively) and also 100 C higher than the evolution from asparagine of one mole equivalent of water and one mole equivalent of ammonia (ca. 245 C). In the pyrolysis of asparagine, 17 appears to form at ca. 245 C, followed by formation, evaporation and detection of maleimide and succinimide at ca. 370 C. The origins of the nitrogen atoms in the maleimide molecules depends on its route of formation. For the direct unimolecular route, there is direct atom integrity. The amide nitrogen from one molecule of asparagine remains with the

original four carbon atoms of that molecule of asparagine. In contrast, in the PSI-route (Scheme 3), the nitrogen atom in the maleimide stems from a different originating molecule that provides the four carbon atoms in the maleimide. This data suggests that a major pathway for the bulk of the mass of asparagine involves initial polymerization to higher molecular weight peptides (e.g., 16 and 17) as shown in Scheme 3. In these mechanisms and some discussed below, specic di-, tri- and tetrapeptides have been drawn; however, similar chemistry can be envisioned from larger peptides,

160
6e-11

R.K. Sharma et al. / J. Anal. Appl. Pyrolysis 72 (2004) 153163

5e-11

m/z 97 -maleimide m/z 99 - succinimide

Relative Intensity, amps

4e-11

3e-11

2e-11

1e-11

200

400

600
o

800

1000

Temperature, C
Fig. 4. TG/MS of asparagine. Evolution proles of maleimide and succinimide, as followed with single ion monitoring at m/z 97 and 99 amu, respectively.

all of which could form thermally in the solid/liquid heterogeneous phase during the thermal treatment. The TGA results, carried out under slow heating, suggest a signicant matrix effect, the intermediacy of peptides, the formation of poly(succinimide), or some combination of these possibilities. It is known that poly(succinimide) (17, PSI) with a weightaverage molecular weight of 9000 amu, is formed when aspartic acid is heated at 160 C for 6 h under a nitrogen atmosphere (Scheme 4) [5]. In our initial study of asparagine pyrolysis, we reported that at 300 C, ca. 64% of the mass remained as char in the pyrolysis boat, ca. 35% was untrapped gases, and a small amount (<0.5%) of trappable tars consisting of succinimide and maleimide were quantied (see Table 1) [2]. These results are consistent with the TGA results (Figs. 14). The observation of a small but nite amount of 12 and 13 at 300 C may suggest the operation of reaction pathways not involving peptides as a minor but real contributor to the total formation of 12 and 13. We note that the TGA and DSC of asparagine previously reported in the literature contain a decomposition stage

representing 12% weight loss at ca. 77100 C [13] or 8% weight loss at ca. 90125 C reported by Sikora, et al. [14], not seen in our work as illustrated in Figs. 2 and 3. This decomposition was ascribed to the loss of ammonia by Rodante, et al. [13]. In fact, we have found that thermal analysis of asparagine monohydrate (7H) reveals a weight loss of 12.1% at ca. 100 C which is absent in the TG of asparagine (7). Single ion monitoring at m/z 18 of the decomposition products from both 7 and 7H reveals a signicant peak at 99104 C from 7H, absent in the mass spectral analysis from the TG of 7. We, therefore, conclude that the thermal analyses reported by Rodante et al. [13,15] and Sikora et al. [14] were performed on asparagine hydrate and not on asparagine. 3.2. Pyrolysis of aspartic acid (8) Pyrolysis of aspartic acid (8) at 300 C led to almost no LTT but to a 66% conversion to a LTC (Table 1). This LTC was pyrolyzed at 625 C, leading to maleimide (34 mg/g) and succinimide (21 mg/g) as major products with lessor amounts of 14 (4 mg/g). These products must form via a peptide route (Scheme 4). The TGA/DSC of aspartic acid found in this work is identical to that reported by Rodante et al. [13]. While aspartic acid has ve carbon atoms and one nitrogen atom, as do maleimide and succinimide, the atom connectivity of aspartic acids is not compatible with a unimolecular transformation to the two imides; that is, a 15 nitrogencarbonyl carbon connectivity is required to form 12 and 13. At least two molecules of aspartic acid are necessary to form either maleimide or succinimide, as the nitrogen atom comes from a different molecule that produces the four chain carbon moiety.

O OH OH O

O OH OH O 8 -2H2O

O N O n

H2N

H2N

17, PSI

Scheme 4. Formation of poly(succinimide) 17 (PSI) in the thermolysis of aspartic acid [5].

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161

Two facts suggest that PSI is the common intermediate in the pyrolyses of both asparagine and aspartic acid: rst, the similarity in the pyrolysis product prole for these two amino acids; and second, that lower temperature heating of aspartic acid is known to form PSI [5]. At this time, we cannot account for the different yields of 12 and 13 from the pyrolyses of asparagine and aspartic acid and the slightly different yields from the pyrolysis of LTC (i.e., the char resulting from the 300 C pyrolysis) at 625 C, if they both proceed through the same common intermediate, 17 (PSI). 3.3. Pyrolysis of glutamine (9) Pyrolysis of glutamine (9) at 300 C leads to at least six single-ring nitrogen-containing heterocycles, as shown in Scheme 5 and Table 1. The major product is glutarimide (20) (20 mg/g). The formation of the saturated ring in glutarimide is analogous to the formation of succinimide from asparagine, both reactions requiring a reduction step as discussed above. The related 2,6-dihydroxypyridine (21) (3 mg/g) is also observed, via dehydrative cyclization, loss of ammonia and aromatization. In fact, as found for asparagine and aspartic acid, formation and subsequent degradation of poly(glutarimide) can also explain the formation

of glutarimide and 2,6-dihydroxypyridine. Lesser amounts of four other single-ring nitrogen-heterocycles are formed: pyrrolidinone (22) (6 mg/g) via a (possibly only formal) decarboxylation and cyclization from glutamine; dehydroglutarimide (23) (<1 mg/g) via deammination and cyclization from glutamine; and succinimide (3 mg/g) and maleimide (4 mg/g) by routes not yet understood. 3.4. Pyrolysis of glutamic acid (10) and pyroglutamic acid (11) At 300 C, glutamic acid pyrolysis leads to three major products (Scheme 6 and Table 1), pyrrolidinone (22), 2-pyrrolidone-5-carboxylic acid (11) (pyroglutamic acid), and glutarimide (20). The latter compound is likely formed via a peptide, e.g., poly(glutarimide). While 10 can lead to the stable glutarimide (20), its unsaturated co-product could lead to 2,6-dihydroxypyridine (21), though 21 is seen from glutamine but not from glutamic acid. Independent pyrolysis of pyroglutamic acid (11) also leads to 20 and 22, along with lower yields of the diketopiperazine 24 (Scheme 7). It is noteworthy that 24 is observed in the pyrolysis of pyroglutamic acid (11) but not in the pyrolysis of glutamic acid, even though glutamic

O HO NH2 9 glutamine

O NH2 O N H 12 O

N H 13

N H 20

HO

OH

N H 22

N H 23

21

Scheme 5. 300 C pyrolysis products of glutamine.

O HO NH2 10 glutamic acid O OH O N H 20 O

HO2C

N H 11

N H 22

Scheme 6. 300 C pyrolysis products from glutamic acid and possible pathways.
O N N O 24 O

HO2C

N H 11

+
O N H 20 O N H 22

Scheme 7. Pyrolysis products from 2-pyrrolidone-5-carboxylic acid (11, pyroglutamic acid).

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acid does thermally produce pyroglutamic acid. The formation of 24 requires two molecules of pyroglutamic acid; not seeing 24 from glutamic acid must be a consequence of signicantly lower concentration of pyroglutamic acid in the heated melt from glutamic acid than in the melt of pyroglutamic acid itself. As can be seen by a comparison of Schemes 5 and 6, glutamine (9) and glutamic acid (10) do not yield the same pyrolysis product prole, even though the mass balance distribution is essentially the same (Table 1). Products such as glutarimide (20) and pyrrolidinone (22) can derive from the same two intermediates, which can be formed from both 9 and 10. The pyridine and piperidene derivatives (21 and 23) can stem from glutamine, as the ring nitrogen is derived from the amide nitrogen in 9. 3.5. Comparison of the pyrolysis mass balance proles for 711 Asparagine and aspartic acid have much higher yields of LTC and much lower yields of low temperature gaseous products than are observed for glutamine, glutamic acid, and pyroglutamic acid (see Table 1). Interestingly, the total gaseous products from all ve of these compounds is approximately the same, ranging from 6476%. In fact, the total tar yields (LTT plus HTT) are also essentially the same for 711, with values of 12, 6, 12, 15 and 12% respectively. Hence, the differences observed at 300 C may reect kinetics happening in the 300 C region. Without additional data, e.g., TG/DSC experiments, we are reluctant to place too much signicance on any differences reported in Table 1.

In the pyrolysis of amino acids, the thermal conditions provide sufcient energy to form intermediate peptides as well as poly(amino acids) in the solid/liquid phase. The formation of diketopiperazines 2 are one experimentally observable manifestation of peptide formation. Peptide-forming reactions occur readily because they are simple dehydrations. Peptides can serve as key intermediates in the formation of both low molecular weight volatile products and other higher molecular weight (and sometimes volatile) products. Char can also be formed in the pyrolysis zone. In the case of amino acid pyrolysis, this char will stem initially from polymerization reactions, i.e., formation of larger non-volatile peptides. Eventually, the char can aromatize to higher molecular weight aromatics, sometimes containing heteroatoms [2,17]. In the cases described herein, maleimide, succinimide and derivatives thereof are formed, all likely involving peptide intermediates. A novel disproportionation-type reaction is proposed to account for the formation of reduced products, such as succinimide from asparagine and glutarimide from glutamine. The key driving factor is formation of highly oxidized small cyclic compounds including the reduced precursor of succinimide or glutarimide. A fundamental conclusion of this study of amino acid pyrolysis is the intermediacy of peptides that serve various functions. Known previously is the (1) formation of diketopiperazines from dipeptides. We now can add (2) disproportionation reactions to form reduced products as well as (3) addition-cyclization reactions that can form chain elongation reactions. What remains unclear at this time are the factors which control the partitioning between two reaction pathways for the initially formed dipeptide: cyclization to diketopiperazine, as found for proline; and further polymerization to higher chain peptides, as found for asparagine.

4. Summary and conclusions High temperatures can simultaneously degrade (i.e., pyrolyze) compounds, pyrosynthesize compounds, and remove substances from the thermal zone via evaporation and aerosol formation. Because of the difculty in analysis of thermolysis chars [16], pyrolysis chemistry is typically understood by evaluating the volatile tars and making inferences about what happens in the thermal zone [17]. The product distribution in these pyrolysis reactions appears to be controlled by a few critical features: (1) the ability of the pyrolysis substrate and/or its degradation products to thermally eliminate carbon dioxide, water, ammonia, and/or other low molecular weight, highly stable and volatile compounds; (2) the ease of formation of low molecular weight, stable organic compounds; (3) the relative volatility and thermal stability of such products; and (4) the important role of peptides as important pyrolysis precursors to a variety of low molecular weight tar constituents. Key to our ability to understand these reactions has been the use of TGA/DSC experiments to determine the appearance temperatures of stable, low molecular weight pyrolysis products. Acknowledgements The work by one of us (J.I. Seeman) was funded by Philip Morris USA Inc. We thank Professor Neil Castagnoli, Virginia Tech, Blacksburg, Virginia, for providing the high resolution mass spectrum of 14. We also thank Dr. Mark Nimlos for helpful technical discussions, Professor Ernest L. Eliel for input regarding the NMR analysis of 14, a reviewer for valuable suggestions and Lynn Downing, Alan I. Goldsmith and Kathy Mitchell for technical assistance.

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