Preparation of some long-chain N -acyl derivatives of essential amino acids for nutritional studies

Preparation of some long-chain N-acyl derivatives of essential amino acids for nutritional studies1 ALENKA PAQUET Food Researcll Dtstitute, Agriculture Canada, Ottarva, Ont., Cattada KIA OC6 Received December 7, 1979 Paquet, A. (1980) Preparation of some long-chain N-acyl derivatives of essential amino acids for nutritional studies. Can. J. Biocliem. 58, 573-576 Long-chain N-acyl derivatives of methionine, tryptophan, threonine, and lysine (N6) have been obtained by the reaction of succinimidyl esters of fatty acids with the unprotected amino acids. Their physical properties have been characterized. Paquet, A. (1980) Preparation of some long-chain N-acyl derivatives of essential amino acids for nutritional studies. Can. J. Biochem. 58, 573-576 Par reaction des succinimidyl esters des acides gras avec les acides amin6 non protiges, nous avons prepare des derives N-acyles il longue chaine de la methionine, du tryptophane, de la threonine et de la lysine (N6). NOUS avons caracterisk les proprietes physiques de ces derives. [Traduit par le journal] Introduction Substitution o n the amino group of essential amino acids which are considered for food supplementation in human nutrition protects the amino acids f rom degradative processes in food mixtures (Maillard re- action, Strecker degradation) ( 1-4). N-Substitution thus enhances the effectiveness of the fortification proc- ess and prevents the formation of off-flavors which are unpalatable to humans. One possibility for food fortification purposes is fatty acyl amino acids. Only few reports o n their syntheses have appeared in the literature. Low yields of fatty N - acylmethionine derivatives have been obtained by the acid chloride method ( 4 ) . N(;-Acyllysine derivatives have been prepared in unreported yields f rom the cop- per complex of lysine by lengthy procedures using the acid chloride o r mixed anhydride methods (2) . M o r e efficient methods for the preparation of fatty acyl amino acids would be valuable. W e have recently described the preparation of suc- cinimidyl esters of fatty acids (5) which react smoothly with amino acids to give high yields of fatty N-acyl amino acids ( 6 ) . This paper concerns the extention of this method to the preparation of N-acylmethionine, N-acyltryptophan, N-acylthreonine, and N6-acyllysine derivatives. Materials and methods Amino acids were obtained from Sigma Chemical Co. (St. Louis, MO) . Succinimidyl esters were prepared from the thallium salt of N-hydroxysuccinimide and the acid chlorides, or by the N,N'-dicyclohexylcarbodiimide method as described previously ( 5 ) . Melting points were deter- ABBREVIATIONS : tlc, thin-layer chromatography; nmr, nuclear magnetic resonance. 'Contribution No. 414, Food Research Institute, Agri- culture Canada, Ottawa, Ont., Canada. mined by the capillary method (unless stated otherwise) and are uncorrected. Optical rotations were measured on a Perkin Elmer model 141 polarimeter. Infrared spectra were obtained with a Beckmann-IR-20 spectrometer. Nuclear magnetic resonance spectra were recorded on a Varian T-60 spectrometer. N-Acy 1-L-nzethiotzirze derivatives (lb-1 f) (Table I) To a stirred solution of L-methionine (0.75 g, 5 mmol) and triethylamine (2.1 mL, 15 mmol) in water ( 7 mL) and acetone ( 7 m L ) , was added succinimidyl ester (5 mmol) in portions. The mixture was stirred for 1-4 h (until no ester remained, tlc). The solvents were removed with a rotary evaporator, 10 mL of water was added, and the mixture was acidified with concentrated hydrochloric acid to pH 2. The product which precipitated was separated by filtration, washed with water, and crystallized from ethanol. Ether or hexane was used to remove traces of nonpolar impurities. N-Bzctyry l-L-n~ethionitze (1 a) This compound was prepared as described for lb-lf , except that sodium bicarbonate ( 2 equiv.) was used in- stead of triethylamine. After the acidification step, the product was extracted into methylene chloride and the solution was dried over sodium sulphate. The solvent was removed and the residue crystallized from ether. N-Stearoyl-L-methionine ethyl ester (lk) T o a stirred solution of L-methionine ethyl ester hydro- chloride (2.1 3 g, 10 mmol) and triethylamine (2.8 mL, 20 mmol) in methylene chloride (16 mL) , was added suc- cinimidyl stearate (3.81 g, 10 mmol) in small portions. The mixture was stirred overnight and then evaporated to dry- ness with a rotary evaporator. The residue was dissolved in methylene chloride which was washed 3 times with water and dried over sodium sulphate. The solvent was removed and the residue crystallized from ethyl acetate. N-Stearoy l-L-methionylglycine This compound was obtained from L-methionylglycine and succinimidyl stearate as indicated in the general pro- 0008-4018/80/070573-04$01 .OO/O @ 1980 National Research Council of Canada/Conseil national de recherches du Canada C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y SA V A N N A H R IV N A T L A B B F on 1 1/ 10 /1 4 Fo r pe rs on al u se o nl y. 574 CAN. J. BIOCHEM. VOL. 58, 1980 TABLE 1. L-Methionine derivatives (1) (R'CONHCHRCOOR") Analysis (5) Melting C H N point [ Q I ~ ~ ~ ~ Molecular R'CO- R" ("c) (degrees) formula Calcd. Found Calcd. Found Calcd. Found l a Butyryl l b Hexanoyl l c Lauroyl I d Myristoyl l e Palmitoyl If Stearoyl l g Lauroyl lh Myristoyl l i Palmitoyl lj Stearoyl l k Stearoyl 74-76 + 43 oil 69 +28.6 78-79 +27.6 84b +23.5 89-90' + 22 86d + 16eJ 68.5-71 .O +17g 75-77 + 230 8 1-84 +24.1Q 112 +24.50 79.0-80.5 +29.5 NOTE: R = C H 3 S C H 2 C H 2 - - . Infrared and nmr spectra of all compounds were in accord with their structures. l a was obtained in 7 2 % yield: l b - I f . 75- 85%; l g - l j , 88-90%; l k , 7 6 y 0 . =Determined in chloroform unless stated otherwise. OLiterature (4) rnp 75-77°C. CLiterature (4) rnp 80-82°C. dDetermined on Koffler Block. aDetermined in acetic acid. fliterature (4) - 18.8' (acetic acid). gDetermined in ethanol. TABLE 2. L-Tryptophan and L-threonine derivatives (2 and 3) (R'CONHCHRCOOH) Analysis (5) R'CO- Tryptophan (2) 2a Lauroyl 26 Myristoyl 2c Palmitoyl Threonine (3) 3a Lauroyl 36 Palmitoyl 3c Stearoyl Melting point ("C) 112 106-107 99-102 69-70 87-88 89 Molecular (degrees) formula C H Calcd. Found Calcd. Found Calcd. Found NOTE: 2 , R = &T'"-; 3, R = C H 3 C H O H . Infrared and nmr spectra of all compounds were in accord with their structures. All com- pounds were obtained in 80-88yo yields. aDetermined in chloroform (2a-2c), in methanol (3a-3c). cedure, except that the reaction time was 23 h. The crude product was washed with water and crystallized twice from ethanol: yield 63%, mp 119"C, and [ ( r ]~~~-13 .4" (C 0.35, acetic acid) : Anal. calcd. for C25H16N201S: C 63.52, H 10.23, N 5.92; found: C 63.19, H 9.88, N 6.28. Sodium salts of N-acyl-L-methionine derivatives (1 g-lj) These compounds were prepared by neutralizing the cor- responding N-acylmethionine derivative in methanol with an equivalent amount of methanolic sodium hydroxide; l g was crystallized from methanol-acetone and lh- l j from and triethylamine (1.4 mL, 10 mmol) in water (7 mL) and acetone (7 mL), was added succinimidyl ester (5 mmol) in portions. The mixture was stirred for 2-10 h under a stream of nitrogen (until no ester remained, tlc). The solvents were removed with a rotary evaporator, water was added, and the pH was adjusted to 2 with hydrochloric acid. The mixture was chilled and the product was separated by filtration, washed with water, and crystallized from chloroform-hexane. Hexane was used to remove the small amount of remaining nonpolar impurities. ethanol. N-Acyl-L-threonine derivatives (3a-3c) (Table 2) N-Acyl-L-tryptophan derivatives (2a-2c) (Table 2) A mixture of L-threonine (0.595 g, 5 mmol), sodium To a stirred solution of L-tryptophan (1.02 g, 5 mmol) bicarbonate (0.420 g, 5 mmol) and succinimidyl ester (5 C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y SA V A N N A H R IV N A T L A B B F on 1 1/ 10 /1 4 Fo r pe rs on al u se o nl y. PAQUET 575 TABLE 3. L-Lysine derivatives (4) (R1CONH(CH2)4CHNH2COOH) Analysis (( ; ) Decom- position C H N point [QID 20a Molecular R'CO- ("c) (degrees) formula Calcd. Found Calcd. Found Calcd. Found 4a Butyryl 46 Hexanoyl 4c Octanoyl 4d Decanoyl 4e Lauroyl 4f Myristoyl 4g Palmitoyl 411 Stearoyl NOTE: Infrared (Nujol) spectra of all compounds were in accord with their structures. Compounds prepared by the triethylamine niethod were obtained in yields 74482% except fo r 4e, 65%. 4a, sodium hydroxide method, 57% l ie ld . 4d, sodium hydroxide method, 69% lield. aDetermined i n 0.5 N methanolic potassium hydroxide. mmol) in water (2 mL), and dimethoxyethane (5 mL) was refluxed for 0.5-2 h (until no ester present, tlc). Acidification to pH 2 with hydrochloric acid and chilling gave the product which was washed with water, dried, and crystallized from chloroform. Products were finally washed with hexane or hexane-ether which removed small amounts of nonpolar contaminants. NCAcyl-L-lysine derivatives ( 4 a 4 h ) (Table 3) The series of Ncacyl-L-lysine derivatives from N6- hexanoyl-L-lysine to Ne-stearoyl-L-lysines were synthesized in the presence of triethylamine. Ne-octanoyl-L-lysine was obtained as described for the preparation of NG-hexanoyl-L- lysine. All other compounds were prepared as indicated for NH-myristoyl-L-lysine. All compounds prepared by this method were obtained in yields of 74-82%, except for Ne- lauroyl-L-lysine (65% ). Ne-Butyryl-L-lysine was prepared in the presence of sodium hydroxide (9 ) in 57% yield. This method (9 ) was also used for an alternative preparation of Ntdecanoyl-L- lysine (69% ) . Analytical samples of all Ne-lysine derivatives were ob- tained by crystallization from acetic acid and gave correct elemental analyses (C, H, N ) (Table 3 ) . All compounds decomposed a t temperatures above 230°C and exhibited low positive specific rotations (+1.5"-+4", 0.5 N KOH- CH,OH). The ir spectra (Nujol) of Ne-palmitoyl-L-lysine and of the authentic compound (6) were identical. The ir spectra (Nujol) of all other N6-lysine derivatives were simi- lar to it (3300, 1640, 1580, 1520-1540 cm-l). NB-Hexanoyl-L-lysine (triethylamine method) T o a stirred solution of L-lysine hydrochloride (0.365 g, 2 mmol) and triethylamine (0.84 mL, 6 mmol) in water (2.4 mL) and acetone (2 mL) , was added succinimidyl hexanoate (0.64 g, 3 mmol) in portions. The mixture was stirred for 2 h and evaporated to dryness on a rotary evaporator. Water-acetone was added and the mixture was re-evaporated. This was repeated 2 more times. A small amount of water and acetone was added and the mixture was chilled. Crystalline product was isolated by filtration and washed with icy water and acetone. A second crop was obtained by ev~porating the filtrate to a thick oil and add- (1.82 g, 10 mmol) and triethylamine (4.2 mL, 30 mmol) in water (20 mL) and acetone (20 m L ) , was added suc- cinimidyl myristate (3.25 g, 10 mmol) in portions during 0.5 h. During the addition, more water-acetone-triethyl- amine (10: 10:2) was added in order to prevent the product from precipitating as a thick voluminous mass. Stirring was continued for an additional hour. The mixture was acidified with hydrochloric acid to pH 4 and chilled. The light foamy product was separated by filtration and washed with water and hot dioxane. No-Blctyryl-L-lysine (sodium hydroxide metlzod) T o a stirred solution of L-lysine hydrochloride (365 mg, 2 mmol) in 3 mL of 2 N sodium hydroxide and 3 mL of dioxane, was added succinirnidyl butyrate (1.29 g, 7 mmol) in portions and the mixture was stirred for 2 h. Dioxane was removed with a rotary evaporator and the mixture was stirred for an additional hour in the presence of 3 0 m L of ion exchange resin (AG 50W-X2, H + form). The resin was collected by filtration, washed several times with water, and stirred in 3 N ammonium hydroxide for 1 h. The resin was filtered off, washed with a small amount of 3 N am- monium hydroxide, and the filtrate and washings were evaporated to dryness giving 345 mg of the residue which was purified by repeated crystallization from water-ethanol. Yield: 246 mg (57% ) . N6-Decanoyl-L-lysine (sodium hydroxide method) To a stirred solution of L-lysine hydrochloride (502 mg, 2.75 mmol) in 2 N sodium hydroxide (5 mL) , was added succinimidyl decanoate (5.75 g, 21.4 mmol) in dioxane (20 mL) during 0.5 h. During this time the pH was kept a t 11.0-11.5 by further addition of 2 N sodium hydroxide. The stirring was continued for another 0.5 h. The mixture was acidified with concentrated hydrochloric acid to pH 3 and evaporated to a small volume on a rotary evaporator. A small amount of water was added, the pH was adjusted to 6, and the mixture was chilled. The precipitate was filtered off and washed with icy water and ethanol, giving 530 mg of the product. Another 40 mg of the product was obtained from the aqueous filtrate by evaporation and addi- tion of a small amount of acetone. Overall yield was 570 mg (69% ) . ing of a small xnount of acetone. Results and discussion No-Myristoyl-L-lysine (triethylamine method) T o a vigorously stirred solution of L-lysine hydrochloride T h e previously described method f o r the acylation C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y SA V A N N A H R IV N A T L A B B F on 1 1/ 10 /1 4 Fo r pe rs on al u se o nl y. 576 CAN. J. BIOCHEM. VOL. 58, 1980 of amino acids by succinimidyl esters of fatty acids (6) was adapted to the acylation of L-methionine, L- tryptophan, L-threonine, and L-lysine. Succinimidyl esters of fatty acids coupled smoothly with L-methionine, L-methionine ethyl ester, and L- methionylglycine in water-acetone medium in the presence of triethylamine to give acylmethionine derivatives in high yields (Table 1) . These yields were higher than those reported by Damico (4) for similar derivatives prepared from methionine ethyl ester via the acid chloride method, followed by saponification of the ester. In addition there are some discrepancies with regard to melting points, and the specific rotation of stearoylmethionine is of opposite sign compared to that reported (4) . For reaction of succinimidyl esters with L-trypto- phan, longer reaction times (up to 20 h) and a nitrogen atmosphere were required to obtain high yields of the products (Table 2) . The acylation of hydroxy amino acids with active esters of N-protected amino acids in the presence of a tertiary base were recently studied (7, 8) . It was shown that the undesirable 0-acylation with threonine was slower than with the other hydroxy amino acids. The advantage of the use of succiilimidyl estars over other active esters of N-protected amino acids for the preferential N-acylation of hydroxy amino acids was also reported (7 ) . In this study we have found that high yields of long chain N-acyl derivatives of threonine could be obtained by the reaction of succinimidyl esters of fatty acids with the unprotected threonine in the presence of sodium bicarbonate. Small amounts of 0-acyl deriva- tives were formed in some cases when triethylamine was used instead of sodium bicarbonate. Thus re- fluxing of the reaction mixture for 0.5-2 h in the presence of sodium bicarbonate gave satisfactory re- sults and the desired N-acylthreonine derivatives were obtained in high yields (Table 3 ) . The correct ele- mental analyses and the absence of the absorption at 1730-1750 cm-I (ester group) in the ir spectra (Nujol) of the compounds served as a proof that the hydroxy group remained free under the above de- scribed reaction conditions. The previously described acylation of the side chain amino group of lysine by succinimidyl palmitate (6) has been refined and adapted to the general use of succinimidyl esters of fatty acids for this reaction. Thus acylations of lysine by succinimidyl esters from decanoate up to stearate was carried out in water- acetone medium in the presence of an excess of triethvlamine. The ~ roduc t s formed almost imme- diate& with the first'addition of an ester and tended to form thick, voluminous precipitates. Further dilu- tion of the reaction mixture with water-acetone- triethylamine was therefore necessary in all cases. The best results were obtained when, as a result of appro- priate dilution, the product appeared as a light foamy precipitate instead of a bulky mass. Small amounts of disubstituted derivatives were isolated in some cases in less diluted mixtures. Further dilution of the reac- tion mixture was not necessary during the preparation of NG-hexanoyl- and NG-octanoyl-L-lysine. We did not detect (tlc and ir analysis) any sig- nificant amounts of disubstituted derivatives during these acylations except for dilauryllysine (5-10%) which was isolated in some cases during the acylation of L-lysine by succinimidyl laurate. Due to the decomposition of succinimidyl acetate and succinimidyl butyrate under the above conditions, we were unable to acylate successfully lysine with these esters. However, NG-butyryl-L-lysine could be prepared in moderate yields in the presence of sodium hydroxide essentially under the conditions described by Leclerc and Benoiton (9) for the acylation of lysine by p- nitrophenyl acetate. When working with longer suc- cinimidyl esters using the sodium hydroxide method we found that the inorganic salts greatly increased the solubility of otherwise water-insoluble NLsubstituted lysine derivatives, thus preventing efficient recovery of the products, particularly NG-hexanoyl- and N6- octanoyl-L-lysine. NLDecanoyl-L-lysine was prepared without difficulty in 69% yield using an excess of succinimidyl decanoate. No disubstituted derivatives were formed during these acylations (tlc and ir analy- sis). The sodium hydroxide method could not be used for acylations with succinimidyl acetate due to its de- composition under these conditions. Triethylamine was required for acylation by lipophilic ' long-chain suc- cinimidyl esters. Acknowledgements The author is grateful to Dr. L. Benoiton for dis- cussion during the preparation of this manuscript. The author thanks Mr. M. Bergeron for technical assistance, Mr. G. Morris (Analytical Services, Chemistry and Biology Research Institute, Agriculture Canada) for the elemental analyses, and Dr. J. Holme for his in- terest in this work. 1. Boggs, R. W. (1978) in Nzitritional Improvement o f Food and Feed Proteins (Friedman, M., ed.), pp. 571-586, Plenum Press, New York 2. Finot, P. A., Mottu, F., Bujard. E. & Mauron, J. (1978) in Nzitritional lmprover?lent of Food and Feed Proteins (Friedman, M., ed.), pp. 549-570, Plenum Press, New York 3. Hodge, J. E. (1967) in The Chemistry and Physiology o f Flavors (Schultz, H . W., Day, E. A. & Libbey, L. M., eds.), pp. 465-491, The Avi Publishing Co., Inc., Westport, CT 4. Damico, R. (1975) J. Agric. Food Chem. 23, 30-33 Paquet, A. (1979) Can. J. Chem. 57, 2775-2778 Paquet, A. (1976) Can. J. Chem. 54, 733-737 Girin, S. K. & Shvachkin, Yu. P. (1979) Zh. Obshch. Khim. 49,451-457 Martinez, J . , Tolle, J. C. & Bodanszky, M. (1979) Int. J. Pept. Protein Res. 13, 22-27 Leclerc, J. & Benoiton, L. (1968) Can. J. Chem. 46, 1047-1951 C an . J . B io ch em . D ow nl oa de d fr om w w w .n rc re se ar ch pr es s. co m b y SA V A N N A H R IV N A T L A B B F on 1 1/ 10 /1 4 Fo r pe rs on al u se o nl y.