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Carbanion generation and aldol condensation between glyoxylate and glycine or glycinamide
ARCHIVES
OF
BIOCHEMISTRY
Carbanion
AND
HIOPHYSICR
Generation Glyoxylate
193,
and and
WILLIAM
212-217 (1971)
Aldol
Glycine
Condensation or
Between
Glycinamide
A. WARREN
The Mary Imogene Bassett Hospital,1 Cooperslmn,
Xew York ii%926
Received October 6, 1970; Accepted December 17, 1970 Glyoxylate and glycine or glycinamide react under moderately alkaline conditions even in the absence of metal ions to generate stable intermediates which absorb in the wavelength range 250-280 nm. The intermediates appear to be M-glyoxylylideneglycine and N-glyoxylylideneglycinamide. Carbanions, formed from these intermediates, react with glyoxylate Do produce the aldol condensation products, fi-hydroxyaapartic acid and 8-hydroxyaapartamide. After react,ion of glyoxylate and glycine in *HzO, t.ritium was found in glyoxylate, glycine, and fi-hydroxyaapart.ic acid in a ratio of specific activity of 1:2:2. The formation of the @-hydroxyaapartic acids from glyoxylate and glycine was followed at pH 10.3, 25’. The reaction reached completion at 50 hr after 18% of the reactants had condensed. The Ihreo+hydroxyaspart,ic acid isomers composed 72% of the total. &Hydroxyaapart.amide formation from glyoxylate and glycinamide proceeded rapidly at pH 9.3 and 25”, and 587, of the reactants were converted into ,9-hydroxyaapartamides after 6 hr. Transaminat.ion between glyoxylate and glycine occurred very slowly at pH 10.2 and 25”. Complete exchange between glyoxylate and PC-glycine required 25 days.
It has been shown that glyoxylat,e transaminates noneneymat,ically in met,al-catalyzed reactions with several amino acids, including glycine, to produce glycinc and the corresponding wketo acids (l-6). These reactions are believed to proceed by t,he generation of an intermediate metal-stabilized carbinolamine or imine 1vit.h glyoxylate assuming a pyridoxal-like function (7-9). In addition to transaminat,ion products, the four isomers of /%hydroxyaspartic acid have been obtained from react,ion mixtures of glyoxylate, glycinc, and metal ions (3, 4, 10, ll), and in similar reactions, mixtures of other aldehydes or ketones, metal ions, and appropriate amino acids yield t,hc t.ransamination products and the aldol condensation adducts, including threonine (U-14)) P-methyl-&hydroxyaspartic acid (15)) serine (lG), cu-(hydroxymethyl) serine (17), CYmethylserine (17)) cr-ethylserine (17), &hy1 Affiliated
with Columbia
University.
droxyleucine (X3-20), &hydroxynorleucine (IS), and @-paru-nitrophenylserine (18). Formation of the hydroxy-a-amino acids appears to occur via an aldol condensation between the carbonyl compound and the amino acid, implying that, a carbanion is generated in t.he course of the reaction. Evidence for t.he occurrence of carbanions in the reaction of glyoxylate with glycine or gl@lamide is presented in the present work. MEturea of glyoxylate and glycine or of glyoxy1at.e and glycinamide, in the absence of divalent metal cations, were found to generate intermediates with unique ultraviolet absorption spectra. These intermediates appear to be the imines, N-glyoxylylideneglytine and N-glyoxylylideneglycinamide, which are partitioned to t,he transamination products and the aldol edducts. The rates of t.he aldol condensat,ion reactions and of the glyoxylate-glycine t.ransamination reaction were determined.
212
CARBANION ;\lATERIALS
GENERATION
AND METHODS
Isolation of j%hytEroxyaspartic a.cids. Glyoxylic acid (10 mmoles) and glyciue (10 mmoles) were dissolved in water, and the solution was adjusted to pII 11 with NaOTI altd made to a final volume of 10 ml. After 24 hr at 3i”, the pTI was carefully lowered to 6.7 with IICI, and a solution containing 2.5 mmoles of BaC12 was added. A heavy white precipitate formed immediately. After stirring for 30 min, t.he precipitate was collect,ed by ccntrifugat ion and wsshcd seveu t.imes with water. The barium precipitate was completely dissolved in aqucous HCI at a final volume of 10 ml, pII 2.0. Barium was precipitated ss the sulfate by addition of 2 mmoles of sodium sulfate. After centrifugat.ion the volume of the light yellow supernatant was rcduced to 3 ml by evaporation under reduced pressure at 45’. The solution was kept at 4” for 48 hr during which time three-fl-hydroxyaspartic acid crystallized. The mother liquor contained predominantly erythro-b-hydroxyaspartic acid which crystallized at 4” after addition of au equal volume of ethanol. The threo isomers were recrystdlized at 4” from 4 ml of a hot aqueous solut.ion. The crystals were dried under reduced pressure over silica gel. The yield was 0.55 mmoles. Anal. Calcd for C4HT06N: C, 32.21yo; H, 4.697,; N, 9.39%. Foutld: C, 31.19%; H, 4.70%; N, 8.96%. Glyoxylic acid (10 mmoles) and glycinamide (10 mmolesj were reacted in t.he same way. Upon lowering the pH of the reaction solution to 6.7, a white precipitate formed. The precipit,ate was collected by centrifugation, washed six times wit.h water, and dried over silica gel. The yield was 415 mg (2.8 mmoles of P-hydroxyaspartamide). Amino acid analysis revealed a compound which produced a single peak clearly different from t.he hydroxyaspartic acids, glycine, and glycinamide. Anal. Calcd for C,H~O,N’B: C, 32.44%; H, 5.44%; N, 18.89%. Found: C, 31.49%; H, 5.73y0; N, 18.29%. Kinetics of the formation of /3-hydroxyaspartic acids and &hydroxyaspartanaide. Equimolar amounts of glyoxylate and glycine or glycinamide (1 mmole each) were dissolved in aqueous solution at a final volume of 10 ml at 25”. The p1-I of the glycine solution was 10.3, and the pH of the glycinamide solution was 9.3. Aliquots (0.5 ml) were removed from the reaction mixtures at appropriate intervals, and the pH was lowered t,o 2.0 with HCl. Portions of each aliquot were quantitatively analyzed on an automatic amino acid analyzer for the threo and erythro isomers of @-hydroxysspartic acid and for B-hydroxyaspartamides. Standards were prepared from the known compounds. Tritium exchange. An aqueous solution (1 ml) containing 1 mCi of ‘H,O, 1 mmole of glyoxylate,
AND ALDOL
CGNI)ENS.QIGN
41.7
aud 1 mmole of glycine wss adjusted to pH 11 wit.h NaOH. The solution was kept in a stoppered tube at 37” for 24 hr. The pH of t.he solut,ion was lowered to 6.7 with HCl, aud the hydroxyaspartic acids were obtained by precipitation as the barium salts. The fhreo isomers were crystallized as the free acids. The supernat,ant from the barium precipit.at ion, containing glyoxylate and glycine, was charged onto a column (2.4 X 35.0 cm) of AG5OW-X2 (II+). Glyoxylate was eluted with water in t.he 60-lM-ml ellluent. Glycine was elutcd wi t.h 150 ml of 2 s HCl. Both producm were concentrated by evaporation under reduced pressure at 45”. Removal of exchangeable t.rit,ium was accomplished by repeated (6 times) solution in 40 ml of wat.er and evaporation. The final product.s were dissolved in a small volume of wat.er, aliquots were added to liquid srintillation fluid, and tritium w&as determined in a liquid scintillatiou spectrometer using the channels ratio met.hod. Glyoxylate-glycine transamination. Sodium glyoxy1at.e (1 mmole) and l-14C-glyciur (1 mmole, 10 pCi) were dissolved in water, aud the solution was adjusted to pII 10.2 with NaOH and made to a final volume of 10 ml. The solution was kept at 25”, and 0.5-ml aliqunts were taken at. intervals. The pH of each aliquot was lowered to 6.4 with HCl, and the entire amount was transferred to a column (0.8 X 4.5 cm) of AGSOW-12 (H+). Glyoxylate was eluted with 15 ml of water (KKI~~ recovery). Aliqunts of each glyoxylate solution wverc assayed for radioactive carbon and for glynxylate cnnceutration. Analytical methods. Glyoxylate was determined by reaction with 2,4-dinitrophenylhydrazine. Glytine, hydroxysspartic acids, and hydroxyaspartamide were determined by reaction wit.h ninhydrin employing the calorimeters, reagents, and recorder of an amino acid analyzer. Identificat.ion of the hydroxysspartic acids and of the hydroxysspartamides by ion exchange chromatography was performed on an automatic amino acid analyzer using a column (0.9 X 54.0 cm) of AG50-X8 (BioRad, Aminex A-G) at 55”. Ultraviolet spectra were recorded with a Hitachi-Coleman spectrophotometer, Model 124. The pH measuremeuts were made with a Beckman, Model G, pH meter. Reagents. Sodium glyoxylate and glycinamide, obtained from Sigma Chemical Co. were used in a11 tritium exchange and kinetic experiments. For t.he preparation of large amounts of the hydroxyaspart.ic acids, glyoxylic acid and glycinamide from Aldrich Chemical Co. were used. Sodium iminodiacetate was obtained from Aldrich. “Baker grade” glycine was used throughout.. The commercial hydroxyaspartic acids (99% based on N) were purchased from Calbiochem. Trit.iated water and
214
WARREK
I
I
I xx / X I
/*GNcINAMloE-
I”
l IX .’ dX .’ /
,i
fX-WC,,, XX 9’ 0’. .e -e*-
XjX
d X’ ,x-
6
7
I
8
9
I
10
11
PH
2. Absorbauce at 26-L nm of glyoxylat,eglycine and glyoxylate-glycinamide solutions as a function of pH. All solutions were in 1 ml containing either 200 pmoles of pot.aasium phosphate (pII fS8) or 200 pmoles of sodium carbonate-bicarbonate (pH 8.5-11.5). The net absorbance wae measured 3 min after mixing the react.ants. Glyoxy1at.e (20 rmoles) plus glycine (20 pmoles), X . Glyoxylate (20 pmoles) plus glycinamide (20 pmoles), 0. FIG.
WAVELENGTH FIG. 1. tine and 10.0. Glycine late (----), glyoxylate
h-d
Absorption spectra of glyoxylate-glyglyoxylate-glycinamide solutions, pH or glycinamide (a.. e), 0.02 N; glyoxy0.02 N; glycine (or glycinamide) plus (-), 0.02 M each.
l-l%-glycine were Nuc1ea.r Corp.
obtained
from
New
England
RESULTS
Formation
of glyoxylate-glyciue
adduct.
The ultraviolet spect!ra of glyoxylate, glytine, and a mixture of glyoxylate and glycine are shown in Fig. 1. Aqueous solutions of glyoxylate, glycine, or /3-hydroxyaspartic acids showed no appreciable absorbance in the wavelength range 250-250 nm. A mixture of glyoxylate and glycine, however, generated a molecular species which absorbed in this range. The magnitude of the absorbance at 264 nm waa proportional to the concent,ration of each reactant and increased with increasing pH from 7 to 11 (Fig. 2). The intermediate was assumed to be the imine formed between glyoxylate and glycine, and although it could not be isolated, iminodiacetate was identified by paper chromatography in a reaction mixture which included potassium borohydride. Glycinamide reacted with glyoxylate to
give the same spectrum as glycine-glyoxylate solutions (Fig. 1). The absorbance at 264 nm M a function of pH (Fig. 2). however, was shifted to a pH range from 5.5 t,o 9.0. Iclentijkation of &hydroxya.spartic acids and /34ydroxyaspartamide. The P-hydroxyaspartic acids were identified by comparison with known compounds. Since uL-three-phydroxyaspartic acid was the easier t.o purify, it was studied more extensively. The isolated product was ident,ical by paper chromatography to the known compound in four solvent systems, and it was eluted from the amino acid ion exchange column in the same position.2 The eqthro+-hpdroxyaspartic acid in the mother liquor of the first crystallization of the three isomers was identical to known erythro acids by amino acid ion exchange chromatography. Elemental analysis was that expect,ed for m-th.reo$hydroxyaspartic acid. 2 The order of elution from the amino acid ion exchange column is: DL-fhr8o-&hydroxyaapart,ic acids, 32-34 min (in two partially resolved peaks) ; DL-q&o-,9-hydroxysgpartic acids, 42-44 min (one peak); fl-hydroxyaspartamides, 5%54 min (two peaks).
215 1
1
I
I
HYDROXYASPARTAMIDES
0 E
x HYDROXYASPARTIC
ACIDS
.
I X .ey
HOURS FIG. 3. Rates of aldol condensation reactions from glyoxy1nt.e :tt~d glycinc or glycinamide. Conditions given in experimental section. Products: glyoxylrtle and glycine, fi-hydroxyaspartic acids (0) ; glyoxylate and glycinamide, fl-hydroxyaspartamides (X).
The compound crystallized from the glycinamide-glyoxylate rea&ion was eluted in a single peak after the erythro-&hydroxyaspartic acids.3 Elemental analysis was that calculated for C.J&O&,. After hydrolysis of 33 pmoles of the compound in 5.S N HCI, llO”, 24 hr, only three-p-hydroxyaspartic acid (84 rmolcs) and e~yt~~.~o-P-h-drox!aspartic acid (S pmoles) were found by amino acid analysis. Rate arid exterd oj P-l@rozyaspartic acid and 8-/Ly~~o~yaspartallz.~~e fonnatioj/ . Aldol condensation of glyoxylate and glycine into /3-hpdroxyaspnrt,ic acids reached a mwimum after 50 hr at pH 10.3 and 2;‘,” (Fig. 3). Eighteen per cent of the rea&ants were converted into hydroxyaspartic acids. The distribution into threo and erythro isomers at each time point w:~smeasured and remained approximately const,ant throughout, the rcact.ion. The fkeo isomers composed 71.9 f 2.5% of t,he total. The aldol condensation of glycinamidc8 Amino acid analysis of the complete reaction solution produced two peaks representing isomers of fl-hydroxyaapartamide. The compound which crystallizes from the reaction solution at plI 6.7 was the isomer that produced the first, peak.
glyoxylattc solutions proceeded rapidly (Fig. 3). The reaction at pH 9.3 and 25” reached completion in (i hr with 5X% of t#he react.ant,s appcnring as fi-hydroxyaspartamides:’and 4 % as /3-hgdroxyaspartic acids. .ITrom a reaction of 1 mmole of glycinamide and 1 mmolo of glyoxylatc, 0.575 mmoles of p-hydroxysspartamides, 0.037 mmolcs of p-ll?rdrox~:wp~~rt,ic acids, 0.344 mmoles of glycinamldc, and 0.034 mmoles of glycine were found in the reaction mixture aft,cr (i hr (0.9!10 mmoles total). l’ritiunr emhauye. IZvidcnce that, a carbanion was generated in thr reaction between glyoxylatc and glycino was t,he incorporation of t,ritium from the solvent, into all stable react ion products. (+wxylate and glycine were reacted in “HZ0 for 24 hr at 37”. Glyoxyla:tc, glycinc, and tlweo+?-hydroxyaspartic acids were isolated, and all exchangeable trit*ium was rcmovcd. Tritium was incorporAed into all products (Table I). The specific activit,y rat,io was very nearly 1: L?:2 for glyoxylate, glycine, and threo-/3hydroxyasparbic acid, respectively. When glyoxylate or glycine was individually reacted at the samepH, temperature, :md time, no residual tritium was found in t,he isolated compound.
Specilic activity (dpm;mmole)
Product
--
--..--
.~
(:lyoxylat.c (3ycirle rlt.rco-8-IIydroxyassp:rrt.ic, acid ~. -. ..-.. *I The reaction condii,icJns l.C!Xl .
..-. --.--.._ :WP give11 ill
-_ l.hc
l’~arrsamitlatiorr. The rate of t,r:uwnmination between glyoxylato and glycine was estimated by following the incorporation of radioactive carbon from lJ4C-glycine (st.arting at 10 &i/mmole) into glyoxylat,e. The reaction at pH 10.2 proceeded slowly for many days (Fig. 4). Glyoxylatc was maximally labeled (5 &i/mmole) in 25 days. 1)Iscuss ION The initial rapid reactions between glyoxylate and glycine or glycinamida to form the N-glyoxylylidene intermediat,cs may bc formulated as shown in Eqs. (1) and (2). OHC-COO-
+ H,N-
--
Hf
C H,-
OO--.CONH,
H+
-H,O
+kH, &
COO-
OHC-C
The intermediate imines partition int,o transamination products via slow t.aut,omcrization and aldol condensation adducts by carbanion formation at. the methylenc car-
CHOH-COO-
+ H,N-CH,--
FIG. 4. Rate of transamination between glyoxylate and glycine. Nonradioactive glyoxylatc and 1-1°Cglycine were rcactcd at p11 10.2, 25”. Aliquot.8 were taken at intervals, and the &oxylate wss separated and assayed for specific activity as described in the experimental section.
coo-
F”OH-COOH&-
CONH,
The absorbance at 264 nm is proportional t.o the concent,ration of both glyoxylat,c and the amino compound. That the free amino groups of glycine (p& = 9.6) and glycinamide (pK = 7.9, Ref. 21) participate in the formation of t,he intermediates is implied by the pH dependencies of the conccnkations of intermediates. The imine formed with glycine was trapped by reduction with borohydride to give iminodiacetate, thus providing structural evidence of the occurrence of N-glyoxylylideneglycinc.
t
+YH H&-COO-
HC-COO-H,O
+NH,
HF-COO-
5+&H
(2)
H,kCONH,
bons bonded to the nit,rogcn. The existence of the carbanion was dcmonstrat.ed by the incorporat.ion of tritium from the solvent into glyoxylate, glycine, and the hydroxyaspartic acids. The carbaniona formed from N-glyoxylylideneglycine [IQ. (l)] are identical so that the only aldol condensat,ion products with glyoxylate are t.he p-hydroxyaspartic acids. The bwo carbanions from N-glyoxylylideneglycinamide pq. (2)], however, are not identical, and two types of products, /Shydroxya+spartic acids and /I-
CARBANION
GENERATION
hydroxyaspart,amides, occur. Product analysis demonstrated t,hat the P-hydroxyaspartamides are the major type of product so that reaction wi;ith t,he carbanion adjacent, to the carboxamide group must predominate. In the synthesis of &h?;droxTaspartic acids from chloromalic acid, L>akm found 50 !Z ftj:reeoand SO% erythw diastereoisomers (22). Kornguth and Sallach reported 56% t//.rec>and 44%: erythro isomers from coppercat,ulyzed reactions of gl?cine and glyoxylate in very strong alkali (11). Under the mild conditions used in the present work, the three isomers composed 72% of the total ,8hydroxyaspart.ic acids. Metal ions are not required to stabilize the intermediate imines or t,o generate t,he carbanions in the reactions bet,ween glyoxylate and glycine or glycinamide. In ot.her work carbinolamines and imines between glyoxylate or pyruvate and several amino acids, all st.abilized by divalent cations, were studied (S, 9, 2X-23, while cobaltous ion was shown to facilitate carbanion generation in strongly basic solutions of either glycine or alanine alone (26-28). Cupric ions produce a marked increase in the rate of glyoxylate-glycine transamination at pH 7 (5). It, is important to establish the rates of taut.omerization and aldol condensat,ion since the imine carbanions generat.ed from glyoxylate and glycine or glycinamide will react wit.h other elect.rophilic centers at, rates much faster than occur wit,h glyoxylate (W. A. Warren, unpublished). In such cases the amounts of hydroxyaspartic acids and hydroxyaspartamides formed are greatly reduced. ACKNOWLEDGMENTS This investigation was supported by Grants AM-12440 and FR-05498 from The United States Public 1Iealt.h Service and by The Stephen C. Clark Research Fund of The Mary Imogene Bassett Hospital. The technical assistance of Caroline Brown and the secretarial assistance of Charlene Stevens are gratefully appreciated. REFERENCES 1. NAK.~~, H. I., IND WEINHOUBI.:, Chem. 384, 831 (1953). 2. METZLER, D. E., OLIVU~D, E. E.,
J.
Amer.
Chem.
Sot.
AXD
J.,
76,
.T. Bhl.
.~ND
SNELL,
G44 (1954).
CONDENSATION
3. MIX, H.,
217
Hoppe-Seyler’s
Z. Ph.ysiol.
(‘ham.
333,
173 (1961). 4. MIS, H., Hoppe-Seyler’s 106 (1961).
Z. Physiol.
Chctu.
326,
W.,
BiO-
5. li ’ 7.
PLEYING,
chim.
I,.
8.
CVIJ~I.I.O~ PL.~Z:\,
10.
11 . 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25.
27.
28.
AND
J.,
Ada
13.wrr,ro,
W.. dwh.
CIloSlHE,
G.
43,
139 (1969).
C.,
PRAJOVX,
Biochem.
Biophlls.
V.,
AND
100, 512
(1963). kkTZTA:H, LIXJASISG,
1). t:.,
lK:\V.\,
Chsrrt. D.?
.\SD
Sot.
hf.,
AND
79, 648
STANFIELD,
SNELL,
E. E.,
(1954). C. K.,
J. Amer.
(1964). LEUSSISG, D. L., ,\xo IIASXa, IC. M., J. Smer. Chem. Sot. 88, 69(j (19G6). METZLFX, D. E., LONGENECKER, J. B.. AND SSELL, E. I<., J. Amer. Chem. Sot. 76, 639 (1954). KORNGUTH, 11. L., END S.ILL.XH, TT. J.. drch. Biochem. Bi0ph.y.r. 91, 39 (1960). &TO, M., Os.\w.\, K., .\ND AKATIORI, S., BUZZ. CAem. Sot. Jap. 30, 937 (1957). IKUT.HI, Y., OKUDI, T., &TO, M., .\SD AK-IBORI, S., BUZZ. Chem. Sot. Jup. 33,203 (1959). M~R.~KAMI, M., AND TIKAHASHI, K., Bull. Chem. Sot. Jap. 32, 308 (1959). BENOITON, T,., WINITZ, M., COLX.\S, R. F., RTRXBU?M, S. M., AND GREENSTEIN, J. P., J. Amer. Chem. Sot. 81, 1726 (1959). AK~~BORI, 8.. OT.\SI, T. T., MARSHALL, R., WIXITZ, M., .\NJ> (hr:F.NSTEIN, J. P., Arch. Biochem. BiopAys. 83, 1 (1959). OTHI, T. T., .\ND WINITZ, M., Arch. Biochem. Biophys. 80, 251 (1960). OT~NI, T. T., AND WI~.ITZ, M., Arch. Biochem. Biophys. 102, 464 (1963). WIELUD, T., CORJ)S, II., .\sJ) KEC~, E., Chem. Ber. 87, 1312 (1954). IK~T~~NI, Y., OK~ID.\, T., ;~ND AK~BORI, S., Bull. Chem. Sot. Jap. 33,582 (1960). MEISTEIZ, A., “Biochemistry of the Amino Acids,” p. 22. Academic Press, New York (1957). D~KIN, H. D., .I. Biol. C.‘hem. 48, 273 (1921). LEUSSISG, D. L., ;ZND ST~~NFIELD, C. K., J. Amer. Chem. Sot. 88, 5726 (1966). Lauss~~o, D. L., :WD HANN.~, E. M., J. A,mer. ChmL. Sot. 93,693 (1966). LEUSSIXG, D. I,., :\z;D SHULTZ, D. C., J. Amer. Chem. Sot. 88, 4846 (19&l). WILLI.~, D. I-I., 2lND Buscrr, D. H., J. Amer. Chem. Sot. 87, 4644 (1965). BUCKINGHU, D. A., M:IRZILLI, L. G., AND SARGESON, ,4. Al., J. Amer. Chem. Sot. 89, 5133 (1967). BERNDT, D. C., J. Org. Chem. 36, 1129 (1970). Ch.ettr.
9.
W.,
Bioph.ys.
J. Amer.
26. S.,
ALDOL
SW.
88, 2805
OF
BIOCHEMISTRY
Carbanion
AND
HIOPHYSICR
Generation Glyoxylate
193,
and and
WILLIAM
212-217 (1971)
Aldol
Glycine
Condensation or
Between
Glycinamide
A. WARREN
The Mary Imogene Bassett Hospital,1 Cooperslmn,
Xew York ii%926
Received October 6, 1970; Accepted December 17, 1970 Glyoxylate and glycine or glycinamide react under moderately alkaline conditions even in the absence of metal ions to generate stable intermediates which absorb in the wavelength range 250-280 nm. The intermediates appear to be M-glyoxylylideneglycine and N-glyoxylylideneglycinamide. Carbanions, formed from these intermediates, react with glyoxylate Do produce the aldol condensation products, fi-hydroxyaapartic acid and 8-hydroxyaapartamide. After react,ion of glyoxylate and glycine in *HzO, t.ritium was found in glyoxylate, glycine, and fi-hydroxyaapart.ic acid in a ratio of specific activity of 1:2:2. The formation of the @-hydroxyaapartic acids from glyoxylate and glycine was followed at pH 10.3, 25’. The reaction reached completion at 50 hr after 18% of the reactants had condensed. The Ihreo+hydroxyaspart,ic acid isomers composed 72% of the total. &Hydroxyaapart.amide formation from glyoxylate and glycinamide proceeded rapidly at pH 9.3 and 25”, and 587, of the reactants were converted into ,9-hydroxyaapartamides after 6 hr. Transaminat.ion between glyoxylate and glycine occurred very slowly at pH 10.2 and 25”. Complete exchange between glyoxylate and PC-glycine required 25 days.
It has been shown that glyoxylat,e transaminates noneneymat,ically in met,al-catalyzed reactions with several amino acids, including glycine, to produce glycinc and the corresponding wketo acids (l-6). These reactions are believed to proceed by t,he generation of an intermediate metal-stabilized carbinolamine or imine 1vit.h glyoxylate assuming a pyridoxal-like function (7-9). In addition to transaminat,ion products, the four isomers of /%hydroxyaspartic acid have been obtained from react,ion mixtures of glyoxylate, glycinc, and metal ions (3, 4, 10, ll), and in similar reactions, mixtures of other aldehydes or ketones, metal ions, and appropriate amino acids yield t,hc t.ransamination products and the aldol condensation adducts, including threonine (U-14)) P-methyl-&hydroxyaspartic acid (15)) serine (lG), cu-(hydroxymethyl) serine (17), CYmethylserine (17)) cr-ethylserine (17), &hy1 Affiliated
with Columbia
University.
droxyleucine (X3-20), &hydroxynorleucine (IS), and @-paru-nitrophenylserine (18). Formation of the hydroxy-a-amino acids appears to occur via an aldol condensation between the carbonyl compound and the amino acid, implying that, a carbanion is generated in t.he course of the reaction. Evidence for t.he occurrence of carbanions in the reaction of glyoxylate with glycine or gl@lamide is presented in the present work. MEturea of glyoxylate and glycine or of glyoxy1at.e and glycinamide, in the absence of divalent metal cations, were found to generate intermediates with unique ultraviolet absorption spectra. These intermediates appear to be the imines, N-glyoxylylideneglytine and N-glyoxylylideneglycinamide, which are partitioned to t,he transamination products and the aldol edducts. The rates of t.he aldol condensat,ion reactions and of the glyoxylate-glycine t.ransamination reaction were determined.
212
CARBANION ;\lATERIALS
GENERATION
AND METHODS
Isolation of j%hytEroxyaspartic a.cids. Glyoxylic acid (10 mmoles) and glyciue (10 mmoles) were dissolved in water, and the solution was adjusted to pII 11 with NaOTI altd made to a final volume of 10 ml. After 24 hr at 3i”, the pTI was carefully lowered to 6.7 with IICI, and a solution containing 2.5 mmoles of BaC12 was added. A heavy white precipitate formed immediately. After stirring for 30 min, t.he precipitate was collect,ed by ccntrifugat ion and wsshcd seveu t.imes with water. The barium precipitate was completely dissolved in aqucous HCI at a final volume of 10 ml, pII 2.0. Barium was precipitated ss the sulfate by addition of 2 mmoles of sodium sulfate. After centrifugat.ion the volume of the light yellow supernatant was rcduced to 3 ml by evaporation under reduced pressure at 45’. The solution was kept at 4” for 48 hr during which time three-fl-hydroxyaspartic acid crystallized. The mother liquor contained predominantly erythro-b-hydroxyaspartic acid which crystallized at 4” after addition of au equal volume of ethanol. The threo isomers were recrystdlized at 4” from 4 ml of a hot aqueous solut.ion. The crystals were dried under reduced pressure over silica gel. The yield was 0.55 mmoles. Anal. Calcd for C4HT06N: C, 32.21yo; H, 4.697,; N, 9.39%. Foutld: C, 31.19%; H, 4.70%; N, 8.96%. Glyoxylic acid (10 mmoles) and glycinamide (10 mmolesj were reacted in t.he same way. Upon lowering the pH of the reaction solution to 6.7, a white precipitate formed. The precipit,ate was collected by centrifugation, washed six times wit.h water, and dried over silica gel. The yield was 415 mg (2.8 mmoles of P-hydroxyaspartamide). Amino acid analysis revealed a compound which produced a single peak clearly different from t.he hydroxyaspartic acids, glycine, and glycinamide. Anal. Calcd for C,H~O,N’B: C, 32.44%; H, 5.44%; N, 18.89%. Found: C, 31.49%; H, 5.73y0; N, 18.29%. Kinetics of the formation of /3-hydroxyaspartic acids and &hydroxyaspartanaide. Equimolar amounts of glyoxylate and glycine or glycinamide (1 mmole each) were dissolved in aqueous solution at a final volume of 10 ml at 25”. The p1-I of the glycine solution was 10.3, and the pH of the glycinamide solution was 9.3. Aliquots (0.5 ml) were removed from the reaction mixtures at appropriate intervals, and the pH was lowered t,o 2.0 with HCl. Portions of each aliquot were quantitatively analyzed on an automatic amino acid analyzer for the threo and erythro isomers of @-hydroxysspartic acid and for B-hydroxyaspartamides. Standards were prepared from the known compounds. Tritium exchange. An aqueous solution (1 ml) containing 1 mCi of ‘H,O, 1 mmole of glyoxylate,
AND ALDOL
CGNI)ENS.QIGN
41.7
aud 1 mmole of glycine wss adjusted to pH 11 wit.h NaOH. The solution was kept in a stoppered tube at 37” for 24 hr. The pH of t.he solut,ion was lowered to 6.7 with HCl, aud the hydroxyaspartic acids were obtained by precipitation as the barium salts. The fhreo isomers were crystallized as the free acids. The supernat,ant from the barium precipit.at ion, containing glyoxylate and glycine, was charged onto a column (2.4 X 35.0 cm) of AG5OW-X2 (II+). Glyoxylate was eluted with water in t.he 60-lM-ml ellluent. Glycine was elutcd wi t.h 150 ml of 2 s HCl. Both producm were concentrated by evaporation under reduced pressure at 45”. Removal of exchangeable t.rit,ium was accomplished by repeated (6 times) solution in 40 ml of wat.er and evaporation. The final product.s were dissolved in a small volume of wat.er, aliquots were added to liquid srintillation fluid, and tritium w&as determined in a liquid scintillatiou spectrometer using the channels ratio met.hod. Glyoxylate-glycine transamination. Sodium glyoxy1at.e (1 mmole) and l-14C-glyciur (1 mmole, 10 pCi) were dissolved in water, aud the solution was adjusted to pII 10.2 with NaOH and made to a final volume of 10 ml. The solution was kept at 25”, and 0.5-ml aliqunts were taken at. intervals. The pH of each aliquot was lowered to 6.4 with HCl, and the entire amount was transferred to a column (0.8 X 4.5 cm) of AGSOW-12 (H+). Glyoxylate was eluted with 15 ml of water (KKI~~ recovery). Aliqunts of each glyoxylate solution wverc assayed for radioactive carbon and for glynxylate cnnceutration. Analytical methods. Glyoxylate was determined by reaction with 2,4-dinitrophenylhydrazine. Glytine, hydroxysspartic acids, and hydroxyaspartamide were determined by reaction wit.h ninhydrin employing the calorimeters, reagents, and recorder of an amino acid analyzer. Identificat.ion of the hydroxysspartic acids and of the hydroxysspartamides by ion exchange chromatography was performed on an automatic amino acid analyzer using a column (0.9 X 54.0 cm) of AG50-X8 (BioRad, Aminex A-G) at 55”. Ultraviolet spectra were recorded with a Hitachi-Coleman spectrophotometer, Model 124. The pH measuremeuts were made with a Beckman, Model G, pH meter. Reagents. Sodium glyoxylate and glycinamide, obtained from Sigma Chemical Co. were used in a11 tritium exchange and kinetic experiments. For t.he preparation of large amounts of the hydroxyaspart.ic acids, glyoxylic acid and glycinamide from Aldrich Chemical Co. were used. Sodium iminodiacetate was obtained from Aldrich. “Baker grade” glycine was used throughout.. The commercial hydroxyaspartic acids (99% based on N) were purchased from Calbiochem. Trit.iated water and
214
WARREK
I
I
I xx / X I
/*GNcINAMloE-
I”
l IX .’ dX .’ /
,i
fX-WC,,, XX 9’ 0’. .e -e*-
XjX
d X’ ,x-
6
7
I
8
9
I
10
11
PH
2. Absorbauce at 26-L nm of glyoxylat,eglycine and glyoxylate-glycinamide solutions as a function of pH. All solutions were in 1 ml containing either 200 pmoles of pot.aasium phosphate (pII fS8) or 200 pmoles of sodium carbonate-bicarbonate (pH 8.5-11.5). The net absorbance wae measured 3 min after mixing the react.ants. Glyoxy1at.e (20 rmoles) plus glycine (20 pmoles), X . Glyoxylate (20 pmoles) plus glycinamide (20 pmoles), 0. FIG.
WAVELENGTH FIG. 1. tine and 10.0. Glycine late (----), glyoxylate
h-d
Absorption spectra of glyoxylate-glyglyoxylate-glycinamide solutions, pH or glycinamide (a.. e), 0.02 N; glyoxy0.02 N; glycine (or glycinamide) plus (-), 0.02 M each.
l-l%-glycine were Nuc1ea.r Corp.
obtained
from
New
England
RESULTS
Formation
of glyoxylate-glyciue
adduct.
The ultraviolet spect!ra of glyoxylate, glytine, and a mixture of glyoxylate and glycine are shown in Fig. 1. Aqueous solutions of glyoxylate, glycine, or /3-hydroxyaspartic acids showed no appreciable absorbance in the wavelength range 250-250 nm. A mixture of glyoxylate and glycine, however, generated a molecular species which absorbed in this range. The magnitude of the absorbance at 264 nm waa proportional to the concent,ration of each reactant and increased with increasing pH from 7 to 11 (Fig. 2). The intermediate was assumed to be the imine formed between glyoxylate and glycine, and although it could not be isolated, iminodiacetate was identified by paper chromatography in a reaction mixture which included potassium borohydride. Glycinamide reacted with glyoxylate to
give the same spectrum as glycine-glyoxylate solutions (Fig. 1). The absorbance at 264 nm M a function of pH (Fig. 2). however, was shifted to a pH range from 5.5 t,o 9.0. Iclentijkation of &hydroxya.spartic acids and /34ydroxyaspartamide. The P-hydroxyaspartic acids were identified by comparison with known compounds. Since uL-three-phydroxyaspartic acid was the easier t.o purify, it was studied more extensively. The isolated product was ident,ical by paper chromatography to the known compound in four solvent systems, and it was eluted from the amino acid ion exchange column in the same position.2 The eqthro+-hpdroxyaspartic acid in the mother liquor of the first crystallization of the three isomers was identical to known erythro acids by amino acid ion exchange chromatography. Elemental analysis was that expect,ed for m-th.reo$hydroxyaspartic acid. 2 The order of elution from the amino acid ion exchange column is: DL-fhr8o-&hydroxyaapart,ic acids, 32-34 min (in two partially resolved peaks) ; DL-q&o-,9-hydroxysgpartic acids, 42-44 min (one peak); fl-hydroxyaspartamides, 5%54 min (two peaks).
215 1
1
I
I
HYDROXYASPARTAMIDES
0 E
x HYDROXYASPARTIC
ACIDS
.
I X .ey
HOURS FIG. 3. Rates of aldol condensation reactions from glyoxy1nt.e :tt~d glycinc or glycinamide. Conditions given in experimental section. Products: glyoxylrtle and glycine, fi-hydroxyaspartic acids (0) ; glyoxylate and glycinamide, fl-hydroxyaspartamides (X).
The compound crystallized from the glycinamide-glyoxylate rea&ion was eluted in a single peak after the erythro-&hydroxyaspartic acids.3 Elemental analysis was that calculated for C.J&O&,. After hydrolysis of 33 pmoles of the compound in 5.S N HCI, llO”, 24 hr, only three-p-hydroxyaspartic acid (84 rmolcs) and e~yt~~.~o-P-h-drox!aspartic acid (S pmoles) were found by amino acid analysis. Rate arid exterd oj P-l@rozyaspartic acid and 8-/Ly~~o~yaspartallz.~~e fonnatioj/ . Aldol condensation of glyoxylate and glycine into /3-hpdroxyaspnrt,ic acids reached a mwimum after 50 hr at pH 10.3 and 2;‘,” (Fig. 3). Eighteen per cent of the rea&ants were converted into hydroxyaspartic acids. The distribution into threo and erythro isomers at each time point w:~smeasured and remained approximately const,ant throughout, the rcact.ion. The fkeo isomers composed 71.9 f 2.5% of t,he total. The aldol condensation of glycinamidc8 Amino acid analysis of the complete reaction solution produced two peaks representing isomers of fl-hydroxyaapartamide. The compound which crystallizes from the reaction solution at plI 6.7 was the isomer that produced the first, peak.
glyoxylattc solutions proceeded rapidly (Fig. 3). The reaction at pH 9.3 and 25” reached completion in (i hr with 5X% of t#he react.ant,s appcnring as fi-hydroxyaspartamides:’and 4 % as /3-hgdroxyaspartic acids. .ITrom a reaction of 1 mmole of glycinamide and 1 mmolo of glyoxylatc, 0.575 mmoles of p-hydroxysspartamides, 0.037 mmolcs of p-ll?rdrox~:wp~~rt,ic acids, 0.344 mmoles of glycinamldc, and 0.034 mmoles of glycine were found in the reaction mixture aft,cr (i hr (0.9!10 mmoles total). l’ritiunr emhauye. IZvidcnce that, a carbanion was generated in thr reaction between glyoxylatc and glycino was t,he incorporation of t,ritium from the solvent, into all stable react ion products. (+wxylate and glycine were reacted in “HZ0 for 24 hr at 37”. Glyoxyla:tc, glycinc, and tlweo+?-hydroxyaspartic acids were isolated, and all exchangeable trit*ium was rcmovcd. Tritium was incorporAed into all products (Table I). The specific activit,y rat,io was very nearly 1: L?:2 for glyoxylate, glycine, and threo-/3hydroxyasparbic acid, respectively. When glyoxylate or glycine was individually reacted at the samepH, temperature, :md time, no residual tritium was found in t,he isolated compound.
Specilic activity (dpm;mmole)
Product
--
--..--
.~
(:lyoxylat.c (3ycirle rlt.rco-8-IIydroxyassp:rrt.ic, acid ~. -. ..-.. *I The reaction condii,icJns l.C!Xl .
..-. --.--.._ :WP give11 ill
-_ l.hc
l’~arrsamitlatiorr. The rate of t,r:uwnmination between glyoxylato and glycine was estimated by following the incorporation of radioactive carbon from lJ4C-glycine (st.arting at 10 &i/mmole) into glyoxylat,e. The reaction at pH 10.2 proceeded slowly for many days (Fig. 4). Glyoxylatc was maximally labeled (5 &i/mmole) in 25 days. 1)Iscuss ION The initial rapid reactions between glyoxylate and glycine or glycinamida to form the N-glyoxylylidene intermediat,cs may bc formulated as shown in Eqs. (1) and (2). OHC-COO-
+ H,N-
--
Hf
C H,-
OO--.CONH,
H+
-H,O
+kH, &
COO-
OHC-C
The intermediate imines partition int,o transamination products via slow t.aut,omcrization and aldol condensation adducts by carbanion formation at. the methylenc car-
CHOH-COO-
+ H,N-CH,--
FIG. 4. Rate of transamination between glyoxylate and glycine. Nonradioactive glyoxylatc and 1-1°Cglycine were rcactcd at p11 10.2, 25”. Aliquot.8 were taken at intervals, and the &oxylate wss separated and assayed for specific activity as described in the experimental section.
coo-
F”OH-COOH&-
CONH,
The absorbance at 264 nm is proportional t.o the concent,ration of both glyoxylat,c and the amino compound. That the free amino groups of glycine (p& = 9.6) and glycinamide (pK = 7.9, Ref. 21) participate in the formation of t,he intermediates is implied by the pH dependencies of the conccnkations of intermediates. The imine formed with glycine was trapped by reduction with borohydride to give iminodiacetate, thus providing structural evidence of the occurrence of N-glyoxylylideneglycinc.
t
+YH H&-COO-
HC-COO-H,O
+NH,
HF-COO-
5+&H
(2)
H,kCONH,
bons bonded to the nit,rogcn. The existence of the carbanion was dcmonstrat.ed by the incorporat.ion of tritium from the solvent into glyoxylate, glycine, and the hydroxyaspartic acids. The carbaniona formed from N-glyoxylylideneglycine [IQ. (l)] are identical so that the only aldol condensat,ion products with glyoxylate are t.he p-hydroxyaspartic acids. The bwo carbanions from N-glyoxylylideneglycinamide pq. (2)], however, are not identical, and two types of products, /Shydroxya+spartic acids and /I-
CARBANION
GENERATION
hydroxyaspart,amides, occur. Product analysis demonstrated t,hat the P-hydroxyaspartamides are the major type of product so that reaction wi;ith t,he carbanion adjacent, to the carboxamide group must predominate. In the synthesis of &h?;droxTaspartic acids from chloromalic acid, L>akm found 50 !Z ftj:reeoand SO% erythw diastereoisomers (22). Kornguth and Sallach reported 56% t//.rec>and 44%: erythro isomers from coppercat,ulyzed reactions of gl?cine and glyoxylate in very strong alkali (11). Under the mild conditions used in the present work, the three isomers composed 72% of the total ,8hydroxyaspart.ic acids. Metal ions are not required to stabilize the intermediate imines or t,o generate t,he carbanions in the reactions bet,ween glyoxylate and glycine or glycinamide. In ot.her work carbinolamines and imines between glyoxylate or pyruvate and several amino acids, all st.abilized by divalent cations, were studied (S, 9, 2X-23, while cobaltous ion was shown to facilitate carbanion generation in strongly basic solutions of either glycine or alanine alone (26-28). Cupric ions produce a marked increase in the rate of glyoxylate-glycine transamination at pH 7 (5). It, is important to establish the rates of taut.omerization and aldol condensat,ion since the imine carbanions generat.ed from glyoxylate and glycine or glycinamide will react wit.h other elect.rophilic centers at, rates much faster than occur wit,h glyoxylate (W. A. Warren, unpublished). In such cases the amounts of hydroxyaspartic acids and hydroxyaspartamides formed are greatly reduced. ACKNOWLEDGMENTS This investigation was supported by Grants AM-12440 and FR-05498 from The United States Public 1Iealt.h Service and by The Stephen C. Clark Research Fund of The Mary Imogene Bassett Hospital. The technical assistance of Caroline Brown and the secretarial assistance of Charlene Stevens are gratefully appreciated. REFERENCES 1. NAK.~~, H. I., IND WEINHOUBI.:, Chem. 384, 831 (1953). 2. METZLER, D. E., OLIVU~D, E. E.,
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CONDENSATION
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Hoppe-Seyler’s
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CVIJ~I.I.O~ PL.~Z:\,
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AND
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AND
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hf.,
AND
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88, 2805