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Preparative liquid chromatography of 1:1 adducts derived from the reaction of malondialdehyde with amino acids
ANALYTICAL
117, 245-249 (1981)
BIOCHEMISTRY
Preparative
Liquid
Chromatography
Reaction
Derived
with
Acids
of Malondialdehyde
DONALD Department
of 1: 1 Adducts
J. PIETRZYK
of Chemistry.
Amino
from
the
AND JOHN STODOLA’
University
of Iowa,
Iowa
City.
Iowa
52242
Received March 3, 1981 Purification of 1: 1 adducts (enaminals) formed from the reaction of methyl esters of amino acids with either malondialdehyde or methylmalondialdehyde was accomplished by preparative liquid chromatography on the stationary phase Amberlite XAD-4. The enaminal was easily and rapidly separated from the by-products and starting materials by a EtOH-HZ0 mobile phase and then easily and rapidly recovered from the EtOH-H,O at good yield and high purity. Depending on column diameter, up to 50 mg of enaminal could be purified in one chromatographic step. Adducts derived from L-Arg, L-His, L-Tyr, and L-Cys were purified.
Malondialdehyde (MDA)’ is a naturally occurring three-carbon dialdehyde produced in the oxidation of polyunsaturated lipids (I). Recently, it has been suggested that MDA is toxic (2), carcinogenic (3) mutagenic (4) may even be involved in age related disorders (5), and its production in foods may alter their nutritive value (6). MDA is a very reactive molecule and its undesirable properties may result from its reactions with primary amino groups or even other functional groups that are found in the
many different types of biological macromolecules. These reactions can lead to a crosslink through formation of 1-amino-3imino-propene links. Although MDA is known to react with proteins and probably produce crosslinks, structural details of these reactions and linkages are not clearly understood (7,8). In order to understand these reactions, Nair et al. (9) focused their studies on model reactions using several different amino acids and MDA and methylmalondialdehyde
0
0 II
+
HCC =CHOH( Na) + Cl-HsNCHC02CHSI I R H(CH,)
BcetatC
HCC=CHNHCHCO$ZH, I I H(CH,) R
buffer
+ NaCI(HC1)
Enaminal MDA or MMDA
Amino acid methyl ester hydrochloride .
[II
(MMDA), which is less reactive than MDA. The reaction proceeds through the enol form of MDA or MMDA to form a 1: 1 adduct, hereafter referred to as an enaminal (9), according to reaction [I].
’ Present address: The Upjohn Company, Control 7824-41-l. Kalamazoo, Michigan 49001. * Abbreviations use4l: MDA, malondialdehyde; MMDA, methylmalondialdehyde; tic, thin-layer chromatography. 245
0003-2697/81/160245-05$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrved.
246
PIETRZYK
AND
Procedures for characterization of the products of reaction [ 1] are outlined elsewhere (9). However, these procedures were severely handicapped because of the inability to isolate the products readily from reaction [I] in a reasonable time period at an appropriate purity using techniques such as thinlayer chromatography (tic), extraction, and/ or recrystallization. The following discussion focuses on a preparative liquid chromatographic method for the purification using the reverse-phase adsorbent Amberlite XAD-4 as the stationary phase. The method is simple, rapid, handles large loadings, and employs a mobile phase that leaves the isolated product in a solvent that is readily removed. XAD-4 is a polystyrenedivinylbenzene copolymer and is stable throughout the entire pH range unlike alkyl-modified silica. Its applications as a stationary phase for analytical liquid chromatography have been investigated extensively and include studies on the retention of amino acids, peptides, and derivatives ( lo- 12). Recently, the variables in a preparative liquid chromatographic experiment with XAD-4 as the stationary phase in columns up to 20.5 mm id. were evaluated (13). Milligram-to-multigram samples of benzenesulfonic acids, carboxylic acids, phenols, anilines, amino acids, dipeptides, dipeptide diastereomers, and polyaromatics were successfully separated rapidly and in high purity. MATERIALS
AND
METHODS
Reagents and instrumentation. The Lamino acid ester hydrochlorides were obtained from Sigma Chemical Company. MDA and MMDA were prepared as described elsewhere (9). Distilled water and 95% EtOH were used for the mobile phase; composition is expressed as percent by volume. Amberlite XAD-4 was obtained from Mallinckrodt Chemical Works as 20- to 50mesh particles. Procedures for crushing, sizing, cleaning, and then packing the 37- to 44-pm (3255400 mesh) and 75- to 105~Frn ( 140-200 mesh) XAD-4 particles into columns were described previously ( 13). Stain-
STODOLA
less steel tubing of either 8 mm i.d. (3/8 in. o.d.) or 20.5 mm i.d. (1 in. o.d.) equipped with 15-pm end fittings (Jones Chromatography, Inc.) was used for the columns. Column lengths were 25 and 32 cm, respectively. The essential parts of the liquid chromatograph consisted of one 4-liter glass solvent reservoir, an Altex Model 100 pump equipped with preparative pump heads (28ml/min maximum flow rate), a Rheodyne injector (Altex 905-19) modified to hold a 10.2-ml sample loop made from 25 ft of 0.05-mm-i.d. (l/16-in.-o.d.) stainless steel tubing, either a Varian Instruments Varichrom or an Altex Model 153 variable-wavelength detector containing an ~-PI and a lcm path-length cell and a Texas Instruments Servoriter (lo-mV) recorder. Samples were introduced into the injector via a lo-ml syringe. Experimental procedure. Reactions to produce the enaminals are described elsewhere (9). The only variation was that the synthesis was carried out so that the final reaction volume would be about 4 to 6 ml and about 0.1 M relative to the amino acid ester. (This is the limiting reactant in the reaction.) Initial liquid chromatographic experiments were done at an analytical level to establish the level of retention. Small volumes ( lo- 100 ~1) of dilute (mg/ml) solution of the reaction products were injected into the 8.0-mm-i.d. XAD-4 column using 1:I 95% EtOH:H,O as the mobile phase. Depending on the retention, the percent EtOH was either increased or decreased. After optimizing the mobile phase relative to resolution of the enaminal from other reaction components, fractions were collected. For the simpler separation the major components were identified by obtaining uv spectra of the fractions. Subsequently, the procedure was scaled up to a preparative level using either an 8.0- or a 20.5-mm-diameter column depending on the desired amount to be loaded, peaks were collected, solvent removed, and structures and purity for the iso-
PREPARATIVE
3.0 ml
8 min
LIQUID
CHROMATOGRAPHY
{H
FIG. I. Scaleup of an analytical separation to a preparative-level separation. The separations were completed on an 8.0-mm-i.d. X 250 mm, 37-44 pm XAD4 column using a 100% H20 mobile phase at a flow rate of 2.8 ml/min. Detection was at 280 nm. The sample was taken from an L-Arg-OMe/MMDA reaction mixture. Chromatogram A is an analytical scale separation where the loading is 0.060 mg of enaminal. In the scaleup, chromatograms B-D, the weight load is 0.60,2.4, and 7.2 mg, respectively. The arrows correspond to the starting points for fraction collecting.
lated solid was verified. Techniques used for the latter procedure were uv, NMR, and carbon, hydrogen, nitrogen elemental analysis (9).
OF
I:1 ADDUCTS
241
umes of dilute solution or smaller volumes of more concentrated solution. A mass overload can be achieved by both methods but only the former can also lead to a volume overload. For the 8.0- and 20.5-mm-i.d. XAD-4 columns, the mass overload limits for k’ of about 2 to 8 are about 8 to 16 mg and 80 to 120 mg, respectively (13). Chromatograms B-D in Fig. 1 illustrate a scaleup to a preparative level by increasing the volume of a dilute solution. The solution injected corresponds to about 2.4 mg L-Argenaminal/ml assuming 100% reaction. Thus, the enaminal peak in chromatogram D, which is for a single pass through the column, represents about 7.2 mg. If the injection volume is increased further to increase the enaminal loading, the peaks broaden significantly due to a volume overload and the baseline resolution is lost. The arrows in Fig. 1 indicate the fractions that were collected. The leading and trailing edge were purposely omitted to ensure a high level of purity of the sample after removal
o. L-Arg-enuminal
RESULTS AND DISCUSSION
Initial studies suggested that the enaminals in reaction [l] would have the highest retention. This was verified by chromatography of the enaminal, unreacted amino acids, MDA or MMDA, and inorganic salts and acids on an 8.0-mm-i.d. XAD-4 column. The analytical scale separation is so favorable that a baseline resolution of the enaminal is easily achieved in a matter of minutes. A typical analytical scale chromatogram is shown in Fig. 1A. Scaling up the analytical separation to a preparative level is illustrated in Figs. 1 and 2. In these experiments, reaction [l] was carried out using either MDA or MMDA and L-Arg methyl ester. Either of two choices can be employed in scaling up the separation procedure to a preparative level. The sample can be introduced in larger vol-
FIG. 2. Preparative-level separation of an L-Arg-enaminal at or close to a mass overload condition. Separations A and B were done on an 8.0-mm-i.d. X 250 mm, 37-44 pm XAD-4 column while C was done on a 20.5 mm X 320 mm, 75-105 Frn XAD-4 column. The mobile phase was 100% Hz0 at a flow rate of 4.2 ml/ min in A and B and 16.8 ml/min in C. Detection was at 280 nm. The sample was taken from an t.-Arg-OMe/ MDA reaction mixture and diluted or used neat. Loading in chromatograms A-C was 2.4, 8.0, and 48 mg, respectively. The arrows correspond to the starting points for fraction collecting.
248
PIETRZYK
86 s ::
AND
t 0 4 6 95% ElOH:H20
2
I\/“;;
\, I 45min
FIG. 3. Preparative-level separation of an L-HisOMe/MMDA reaction mixture. The column used was a 20.Smm-i.d. X 320 mm, 75-105 pm XAD-4 column. The mobile phase at a 16.8-ml/min flow rate was 3:7 95% EtOH:H,O initially and then stepped up to a 4:6 mixture. Detection was at 280 nm. The arrows correspond to the starting points for fraction collecting.
of the solvent. Thus, the percent yield of purified material in chromatogram D, 3 mg, represented about 40% of the theoretical yield assuming 100% conversion in reaction [I]. In Fig. 2 the sample was introduced as a more concentrated solution so that the effect of volume overload would be minimized. In curves A and B the 8.0-mm-i.d. column was used and the loading was 2.4 and 8.0 mg, respectively, with the latter being at the mass loading limit. As can be seen from Fig. 2B, even at a modest mass overload enough plates are still available for the separation and much of the product can be obtained at a high level of purity. Use of a wider diameter column permits greater loading and subsequently purification of a greater amount of the enaminal in a single pass. This is shown in Fig. 2C, where the sample introduced is about 48 mg enaminal assuming 100% conversion in reaction [ 11, This is below the mass loading limit of the 20.5-mm column. In this experiment the fraction collected (arrows) yielded 10 mg purified L-Arg-enaminal after removal of the solvent. No attempt was made to recycle the leading and trailing edges in order to increase the yield of purified enaminal. Reaction [ 1 ] was repeated using L-His, L-
STODOLA
Tyr, and L-Cys methyl ester derivatives and their corresponding enaminals were recovered. Unlike the L-Arg reaction mixture, these mixtures were more complicated since they contained several different side products. Also, since these enaminals and the byproducts had a higher retention on the XAD4 in comparison to the L-Arg-enaminal, stronger eluting agents (increased percent EtOH) were used in a stepwise elution to reduce elution times. Preparative chromatograms illustrating these separations are shown in Figs. 3-5, where 4-ml aliquots of reaction mixture (0.1 M in amino acid methyl ester) were introduced into a 20.5-mm-i.d. XAD-4 column. Several different unidentified compounds appear in the chromatograms in Figs. 3-5. No attempt was made to identify these compounds; however, it is known that MDA and MMDA are very reactive and can undergo aldol-type reactions to produce a mixture of polymers. The desired enaminals fluoresce strongly under uv light and this property was used to aid in fraction cutting; it should also be noted that some of the MDA and MMDA polymeric by-products also fluoresce. The arrows in Figs. 3-5 indicate the fractions that were collected. Since the solvent mixture was an EtOH/water mixture, it was easily and rapidly removed from the enaT
I
4ml
Ih
FIG. 4. Preparative-level separation of an L-TyrOMe/MDA reaction mixture. Conditions are the same as in Fig. 3 except that the mobile phase was I:4 95% EtOH:H,O initially and then stepped up to a higher percent EtOH. The arrows correspond to the starting points for fraction collecting.
PREPARATIVE
LIQUID
CHROMATOGRAPHY
--!iGGz 2
I
Ih
4ml
FIG. 5. Preparative-level separation of an L-CysOMe/MDA reaction mixture. Conditions are the same as in Fig. 3 except that the mobile phase was 4:6 95% EtOH:H,O initially and then stepped up to 95% EtOH. The arrows correspond to the starting points for fraction collecting.
minal by evaporation. Subsequently, the enaminal structure was confirmed via spectroscopic and tic measurements. The results obtained for several different preparative-level separations are summarized in Table 1. The number of by-products appears to be the largest for the L-Tyr-enaminal reaction (see Fig. 4). Fractions 6 and 7 in Fig. 4 were combined to yield the amount of L-Tyr-enaminal listed in Table 1. The reaction of L-Cys and MDA according to reaction [ 1] appeared to be more complex than the reactions with the other amino acids. The product isolated from the chromatogram shown in Fig. 5 was shown to be a 1:1 adduct. However, its exact structure TABLE QUANTITIES
I
OF 1: 1 AMINO
ACID-
MALONDIALDEHYDE ADDUCTS RECOVERED BY PREPARATIVE HPLC 0.1 hl
Col-
Reaction mixture
Mixture (ml) processed
umn i.d. (mm)
1:l Adduct (mg) recovered
L-Arg-OMe + MDA L-Arg-OMe + MMDA r,-His-OMe + MDA L-Tyr-OMe + MDA t-Cys-OMe + MDA
5 5 4 4 4
20.5 8.0 20.5 20.5 20.5
25 9 28 43” 52
a Fractions 6 and 7 in Fig. 4.
OF I:1 ADDUCTS
249
has not yet been unequivocally defined (Nair, V., Vietti, D. E., and Cooper, C. S., unpublished results). The final recoveries of purified enaminal in Table 1 correspond to about 12 to 45% of the theoretical amount of enaminal expected based on the amount of amino acid methyl ester taken and assuming that the conversion in reaction [ 1J was 100%. Recovered amounts depart from the theoretical yield because the fraction cuts were chosen purposely to omit leading and trailing edges of the bands since the major goal was to obtain a highly purified sample. Also, reaction [ 1 ] is not a 100% conversion. Although possible, no attempts were made to recycle edge fractions in order to increase isolated yield. ACKNOWLEDGMENTS This investigation was supported by Grant CA 18555 awarded by The National Cancer Institute, DHEW.
REFERENCES 1. Pryor, W. A., ed. (1976) Free Radicals in Biology, Vols. I and II, Academic Press, New York. L.q Shamberger, R. J., Andreone, T. L., and Willis, C. E. (1974) J. Nat. Cancer Inst. 53, 1771-1773. 3. Munkres, K. D. (1979) Mech. Ageing Develop. 10, 249-260. 4. Marnett, L. J., and Tuttle, M. A. (1980) Cancer Res. 40, 276-282. 5. Bland, J. (1978) J. Chem. Ed. 55, 151-155. 6. Arya, S. S., Yaduziri, Y., Parihar, D. B. (1974) J. Food Sri. Technol. 11, 226-230. 7. Chio, K. S., and Tappel, A. L. (1969) Biochemistry 8, 2827-2832. 8. Reiss, U., Tappel, A. L., and Chio, K. S. (1972) Biochem. Biophys. Res. Commun. 48,921-926. 9. Nair, V., Vietti, D. E., and Cooper, C. S. (1981) J. Amer. Chem. Sot. 103, 3030-3036. 10. Pietrzyk, D. J., and Chu, C. H. (1977) Anal. Chem. 49, 757-764. I I. Pietrzyk, D. J., and Chu, C. H. (1977) Anal. Chem. 49, 860-867. 12. Kroeff, E. P., and Pietrzyk, D. J. (1978) Anal. Chem. 50, 502-5 11. 13. Pietrzyk, D. J., and Stodola, J. D. ( 1981) Anal. Chem. 53, 1822-1828.
117, 245-249 (1981)
BIOCHEMISTRY
Preparative
Liquid
Chromatography
Reaction
Derived
with
Acids
of Malondialdehyde
DONALD Department
of 1: 1 Adducts
J. PIETRZYK
of Chemistry.
Amino
from
the
AND JOHN STODOLA’
University
of Iowa,
Iowa
City.
Iowa
52242
Received March 3, 1981 Purification of 1: 1 adducts (enaminals) formed from the reaction of methyl esters of amino acids with either malondialdehyde or methylmalondialdehyde was accomplished by preparative liquid chromatography on the stationary phase Amberlite XAD-4. The enaminal was easily and rapidly separated from the by-products and starting materials by a EtOH-HZ0 mobile phase and then easily and rapidly recovered from the EtOH-H,O at good yield and high purity. Depending on column diameter, up to 50 mg of enaminal could be purified in one chromatographic step. Adducts derived from L-Arg, L-His, L-Tyr, and L-Cys were purified.
Malondialdehyde (MDA)’ is a naturally occurring three-carbon dialdehyde produced in the oxidation of polyunsaturated lipids (I). Recently, it has been suggested that MDA is toxic (2), carcinogenic (3) mutagenic (4) may even be involved in age related disorders (5), and its production in foods may alter their nutritive value (6). MDA is a very reactive molecule and its undesirable properties may result from its reactions with primary amino groups or even other functional groups that are found in the
many different types of biological macromolecules. These reactions can lead to a crosslink through formation of 1-amino-3imino-propene links. Although MDA is known to react with proteins and probably produce crosslinks, structural details of these reactions and linkages are not clearly understood (7,8). In order to understand these reactions, Nair et al. (9) focused their studies on model reactions using several different amino acids and MDA and methylmalondialdehyde
0
0 II
+
HCC =CHOH( Na) + Cl-HsNCHC02CHSI I R H(CH,)
BcetatC
HCC=CHNHCHCO$ZH, I I H(CH,) R
buffer
+ NaCI(HC1)
Enaminal MDA or MMDA
Amino acid methyl ester hydrochloride .
[II
(MMDA), which is less reactive than MDA. The reaction proceeds through the enol form of MDA or MMDA to form a 1: 1 adduct, hereafter referred to as an enaminal (9), according to reaction [I].
’ Present address: The Upjohn Company, Control 7824-41-l. Kalamazoo, Michigan 49001. * Abbreviations use4l: MDA, malondialdehyde; MMDA, methylmalondialdehyde; tic, thin-layer chromatography. 245
0003-2697/81/160245-05$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrved.
246
PIETRZYK
AND
Procedures for characterization of the products of reaction [ 1] are outlined elsewhere (9). However, these procedures were severely handicapped because of the inability to isolate the products readily from reaction [I] in a reasonable time period at an appropriate purity using techniques such as thinlayer chromatography (tic), extraction, and/ or recrystallization. The following discussion focuses on a preparative liquid chromatographic method for the purification using the reverse-phase adsorbent Amberlite XAD-4 as the stationary phase. The method is simple, rapid, handles large loadings, and employs a mobile phase that leaves the isolated product in a solvent that is readily removed. XAD-4 is a polystyrenedivinylbenzene copolymer and is stable throughout the entire pH range unlike alkyl-modified silica. Its applications as a stationary phase for analytical liquid chromatography have been investigated extensively and include studies on the retention of amino acids, peptides, and derivatives ( lo- 12). Recently, the variables in a preparative liquid chromatographic experiment with XAD-4 as the stationary phase in columns up to 20.5 mm id. were evaluated (13). Milligram-to-multigram samples of benzenesulfonic acids, carboxylic acids, phenols, anilines, amino acids, dipeptides, dipeptide diastereomers, and polyaromatics were successfully separated rapidly and in high purity. MATERIALS
AND
METHODS
Reagents and instrumentation. The Lamino acid ester hydrochlorides were obtained from Sigma Chemical Company. MDA and MMDA were prepared as described elsewhere (9). Distilled water and 95% EtOH were used for the mobile phase; composition is expressed as percent by volume. Amberlite XAD-4 was obtained from Mallinckrodt Chemical Works as 20- to 50mesh particles. Procedures for crushing, sizing, cleaning, and then packing the 37- to 44-pm (3255400 mesh) and 75- to 105~Frn ( 140-200 mesh) XAD-4 particles into columns were described previously ( 13). Stain-
STODOLA
less steel tubing of either 8 mm i.d. (3/8 in. o.d.) or 20.5 mm i.d. (1 in. o.d.) equipped with 15-pm end fittings (Jones Chromatography, Inc.) was used for the columns. Column lengths were 25 and 32 cm, respectively. The essential parts of the liquid chromatograph consisted of one 4-liter glass solvent reservoir, an Altex Model 100 pump equipped with preparative pump heads (28ml/min maximum flow rate), a Rheodyne injector (Altex 905-19) modified to hold a 10.2-ml sample loop made from 25 ft of 0.05-mm-i.d. (l/16-in.-o.d.) stainless steel tubing, either a Varian Instruments Varichrom or an Altex Model 153 variable-wavelength detector containing an ~-PI and a lcm path-length cell and a Texas Instruments Servoriter (lo-mV) recorder. Samples were introduced into the injector via a lo-ml syringe. Experimental procedure. Reactions to produce the enaminals are described elsewhere (9). The only variation was that the synthesis was carried out so that the final reaction volume would be about 4 to 6 ml and about 0.1 M relative to the amino acid ester. (This is the limiting reactant in the reaction.) Initial liquid chromatographic experiments were done at an analytical level to establish the level of retention. Small volumes ( lo- 100 ~1) of dilute (mg/ml) solution of the reaction products were injected into the 8.0-mm-i.d. XAD-4 column using 1:I 95% EtOH:H,O as the mobile phase. Depending on the retention, the percent EtOH was either increased or decreased. After optimizing the mobile phase relative to resolution of the enaminal from other reaction components, fractions were collected. For the simpler separation the major components were identified by obtaining uv spectra of the fractions. Subsequently, the procedure was scaled up to a preparative level using either an 8.0- or a 20.5-mm-diameter column depending on the desired amount to be loaded, peaks were collected, solvent removed, and structures and purity for the iso-
PREPARATIVE
3.0 ml
8 min
LIQUID
CHROMATOGRAPHY
{H
FIG. I. Scaleup of an analytical separation to a preparative-level separation. The separations were completed on an 8.0-mm-i.d. X 250 mm, 37-44 pm XAD4 column using a 100% H20 mobile phase at a flow rate of 2.8 ml/min. Detection was at 280 nm. The sample was taken from an L-Arg-OMe/MMDA reaction mixture. Chromatogram A is an analytical scale separation where the loading is 0.060 mg of enaminal. In the scaleup, chromatograms B-D, the weight load is 0.60,2.4, and 7.2 mg, respectively. The arrows correspond to the starting points for fraction collecting.
lated solid was verified. Techniques used for the latter procedure were uv, NMR, and carbon, hydrogen, nitrogen elemental analysis (9).
OF
I:1 ADDUCTS
241
umes of dilute solution or smaller volumes of more concentrated solution. A mass overload can be achieved by both methods but only the former can also lead to a volume overload. For the 8.0- and 20.5-mm-i.d. XAD-4 columns, the mass overload limits for k’ of about 2 to 8 are about 8 to 16 mg and 80 to 120 mg, respectively (13). Chromatograms B-D in Fig. 1 illustrate a scaleup to a preparative level by increasing the volume of a dilute solution. The solution injected corresponds to about 2.4 mg L-Argenaminal/ml assuming 100% reaction. Thus, the enaminal peak in chromatogram D, which is for a single pass through the column, represents about 7.2 mg. If the injection volume is increased further to increase the enaminal loading, the peaks broaden significantly due to a volume overload and the baseline resolution is lost. The arrows in Fig. 1 indicate the fractions that were collected. The leading and trailing edge were purposely omitted to ensure a high level of purity of the sample after removal
o. L-Arg-enuminal
RESULTS AND DISCUSSION
Initial studies suggested that the enaminals in reaction [l] would have the highest retention. This was verified by chromatography of the enaminal, unreacted amino acids, MDA or MMDA, and inorganic salts and acids on an 8.0-mm-i.d. XAD-4 column. The analytical scale separation is so favorable that a baseline resolution of the enaminal is easily achieved in a matter of minutes. A typical analytical scale chromatogram is shown in Fig. 1A. Scaling up the analytical separation to a preparative level is illustrated in Figs. 1 and 2. In these experiments, reaction [l] was carried out using either MDA or MMDA and L-Arg methyl ester. Either of two choices can be employed in scaling up the separation procedure to a preparative level. The sample can be introduced in larger vol-
FIG. 2. Preparative-level separation of an L-Arg-enaminal at or close to a mass overload condition. Separations A and B were done on an 8.0-mm-i.d. X 250 mm, 37-44 pm XAD-4 column while C was done on a 20.5 mm X 320 mm, 75-105 Frn XAD-4 column. The mobile phase was 100% Hz0 at a flow rate of 4.2 ml/ min in A and B and 16.8 ml/min in C. Detection was at 280 nm. The sample was taken from an t.-Arg-OMe/ MDA reaction mixture and diluted or used neat. Loading in chromatograms A-C was 2.4, 8.0, and 48 mg, respectively. The arrows correspond to the starting points for fraction collecting.
248
PIETRZYK
86 s ::
AND
t 0 4 6 95% ElOH:H20
2
I\/“;;
\, I 45min
FIG. 3. Preparative-level separation of an L-HisOMe/MMDA reaction mixture. The column used was a 20.Smm-i.d. X 320 mm, 75-105 pm XAD-4 column. The mobile phase at a 16.8-ml/min flow rate was 3:7 95% EtOH:H,O initially and then stepped up to a 4:6 mixture. Detection was at 280 nm. The arrows correspond to the starting points for fraction collecting.
of the solvent. Thus, the percent yield of purified material in chromatogram D, 3 mg, represented about 40% of the theoretical yield assuming 100% conversion in reaction [I]. In Fig. 2 the sample was introduced as a more concentrated solution so that the effect of volume overload would be minimized. In curves A and B the 8.0-mm-i.d. column was used and the loading was 2.4 and 8.0 mg, respectively, with the latter being at the mass loading limit. As can be seen from Fig. 2B, even at a modest mass overload enough plates are still available for the separation and much of the product can be obtained at a high level of purity. Use of a wider diameter column permits greater loading and subsequently purification of a greater amount of the enaminal in a single pass. This is shown in Fig. 2C, where the sample introduced is about 48 mg enaminal assuming 100% conversion in reaction [ 11, This is below the mass loading limit of the 20.5-mm column. In this experiment the fraction collected (arrows) yielded 10 mg purified L-Arg-enaminal after removal of the solvent. No attempt was made to recycle the leading and trailing edges in order to increase the yield of purified enaminal. Reaction [ 1 ] was repeated using L-His, L-
STODOLA
Tyr, and L-Cys methyl ester derivatives and their corresponding enaminals were recovered. Unlike the L-Arg reaction mixture, these mixtures were more complicated since they contained several different side products. Also, since these enaminals and the byproducts had a higher retention on the XAD4 in comparison to the L-Arg-enaminal, stronger eluting agents (increased percent EtOH) were used in a stepwise elution to reduce elution times. Preparative chromatograms illustrating these separations are shown in Figs. 3-5, where 4-ml aliquots of reaction mixture (0.1 M in amino acid methyl ester) were introduced into a 20.5-mm-i.d. XAD-4 column. Several different unidentified compounds appear in the chromatograms in Figs. 3-5. No attempt was made to identify these compounds; however, it is known that MDA and MMDA are very reactive and can undergo aldol-type reactions to produce a mixture of polymers. The desired enaminals fluoresce strongly under uv light and this property was used to aid in fraction cutting; it should also be noted that some of the MDA and MMDA polymeric by-products also fluoresce. The arrows in Figs. 3-5 indicate the fractions that were collected. Since the solvent mixture was an EtOH/water mixture, it was easily and rapidly removed from the enaT
I
4ml
Ih
FIG. 4. Preparative-level separation of an L-TyrOMe/MDA reaction mixture. Conditions are the same as in Fig. 3 except that the mobile phase was I:4 95% EtOH:H,O initially and then stepped up to a higher percent EtOH. The arrows correspond to the starting points for fraction collecting.
PREPARATIVE
LIQUID
CHROMATOGRAPHY
--!iGGz 2
I
Ih
4ml
FIG. 5. Preparative-level separation of an L-CysOMe/MDA reaction mixture. Conditions are the same as in Fig. 3 except that the mobile phase was 4:6 95% EtOH:H,O initially and then stepped up to 95% EtOH. The arrows correspond to the starting points for fraction collecting.
minal by evaporation. Subsequently, the enaminal structure was confirmed via spectroscopic and tic measurements. The results obtained for several different preparative-level separations are summarized in Table 1. The number of by-products appears to be the largest for the L-Tyr-enaminal reaction (see Fig. 4). Fractions 6 and 7 in Fig. 4 were combined to yield the amount of L-Tyr-enaminal listed in Table 1. The reaction of L-Cys and MDA according to reaction [ 1] appeared to be more complex than the reactions with the other amino acids. The product isolated from the chromatogram shown in Fig. 5 was shown to be a 1:1 adduct. However, its exact structure TABLE QUANTITIES
I
OF 1: 1 AMINO
ACID-
MALONDIALDEHYDE ADDUCTS RECOVERED BY PREPARATIVE HPLC 0.1 hl
Col-
Reaction mixture
Mixture (ml) processed
umn i.d. (mm)
1:l Adduct (mg) recovered
L-Arg-OMe + MDA L-Arg-OMe + MMDA r,-His-OMe + MDA L-Tyr-OMe + MDA t-Cys-OMe + MDA
5 5 4 4 4
20.5 8.0 20.5 20.5 20.5
25 9 28 43” 52
a Fractions 6 and 7 in Fig. 4.
OF I:1 ADDUCTS
249
has not yet been unequivocally defined (Nair, V., Vietti, D. E., and Cooper, C. S., unpublished results). The final recoveries of purified enaminal in Table 1 correspond to about 12 to 45% of the theoretical amount of enaminal expected based on the amount of amino acid methyl ester taken and assuming that the conversion in reaction [ 1J was 100%. Recovered amounts depart from the theoretical yield because the fraction cuts were chosen purposely to omit leading and trailing edges of the bands since the major goal was to obtain a highly purified sample. Also, reaction [ 1 ] is not a 100% conversion. Although possible, no attempts were made to recycle edge fractions in order to increase isolated yield. ACKNOWLEDGMENTS This investigation was supported by Grant CA 18555 awarded by The National Cancer Institute, DHEW.
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