A PROCESS FOR THE PRODUCTION OF
LYSINE BY CHEMICAL AND MICROBIOLOGICAL SYNTHESIS B . S . G O R T O N , J.
N . COKER, H. P. BROWDER,' AND C. W . DEFIEBRE
Research Diuision, Electrochemicals Dejartment, E. I. du Pont de Nemours & Go., Inc., WiImington, Del. 1-Lysine for food supplementation can b e produced by a process which combines the efficiency of synthetic methods with the stereospecificity of the enzymic. Diaminopimelic acid is synthesized from ethoxydihydropyran by way of hydrolysis of a hydantoin. An enzymic preparation from Bacillus sphaericus decarboxylates part of the acid to 1-lysine. This method eliminates the need for the expensive resolution of Dl-lysine produced by purely synthetic methods.
advances have been made in the technology of amino acid syntheses leading to commercial processes for the production of glutamic acid and lysine. The earlier processes for producing lysine were completely synthetic ; more recently fermentation processes have been announced. Synthetic methods in general afford the best economics for preparing the racemic amino acid, but suffer from problems of resolution of the D- and L- isomers. Fermentation methods yield only the biologically active L- isomer, but are often more costly than a chemical synthesis. This study explored the possibility of combining these two types of synthesis, to take advantage of the best features of each. A simple four-step synthesis of diaminopimelic acid (V) from 2-ethoxy-3,4-dihydropyran (I) has been discovered in our laboratories, yielding diaminopimelic acid as the expected mixture of meso- (Va) and DL- isomers (Vb) (7). A procedure for the rapid decarboxylation of the meso- isomer to L-lysine ( V I ) by bacterial enzymes was also developed ( 4 ) . Epimerization of the unreacted DL-diaminopimelic acid followed by another decarboxylation gave a semicontinuous six-step process believed to be competitive with both synthetic and fermentation processes (Figure 1). ECENT
Equipment and Procedures
Equipment. 2-Ethoxy-3,4-dihydropyran (I) was hydrolyzed to glutaraldehyde (11) in standard laboratory glassware. The trimethylene bishydantoin (IV) was formed in a Hastelloy C-lined shaker tube. Each fermentation was run initially in a 2.8-liter Fernbach flask fitted with a rubber stopper bored to accommodate a three-bladed stirrer, sampling tube, exhaust port, and aeration device. Later experiments were done in a New Brunswick 7.5-liter fermentor drive assembly. A 40-liter Stainless Steel Co. vat fermentor was available for larger scale work. Both
Present address, Mead Johnson and Co., Evansville, Ind. Chicago, Ill.
* Present address, LTilson Laboratory, 308
I & E C PRODUCT R E S E A R C H AND D E V E L O P M E N T
latter pieces of equipment had automatic antifoam control systems. Methods of Analysis. Diaminopimelic acid (V) was assayed by the colorimetric method of Gilvarg (2) or by a manometric procedure carried out with diaminopimelic acid decarboxylase. Lysine ( V I ) was assayed either manometrically with lysine decarboxylase or titrimetrically after elution from an ion exchange resin. Piperidinedicarboxylic acid ( X I ) was assayed titrimetrically. Synthesis of Diaminopimelic Acid (Steps A to D)
Diaminopimelic acid (Va, b) has been prepared in 80 to 90% over-all yield from 2-ethoxy-3,4-dihydropyran( I ) in four steps, without isolating intermediates. In Step A 2-ethoxy-3,4dihydropyran (I) was converted to glutaraldehyde (11) by hydrolysis with dilute aqueous sulfuric acid. Step B involved reaction of the glutaraldehyde with hydrogen cyanide at p H 4 to 6 and a temperature near 0 OC. to form the biscyanohydrin (111). The latter upon reaction with excess ammonia and carbon dioxide by heating under autogenous pressure (Step C) gave a mixture of products in which the trimethylene bishydantoin (IV) was an important component. Finally a hydrolysis of this mixture with hot aqueous sulfuric acid (Step D) yielded meso, DL-diaminopimelic acid (Va. b) which was characterized by paper chromatography (8). bioassay. and colorimetric assay (2). Step A. 2-Ethoxy-3,4-dihydropyran (I) was hydrolyzed to glutaraldehyde (11) by heating a suspension cf the compound briefly at approximately 70' C. Nearly quantitative yields of the dialdehyde were obtained, if the hydrolysis mixture was quenched rapidly to near 0 ' as soon as the immiscible layer of the pyran derivative had disappeared ; the resulting cold solution was used immediately in the next step of the synthesis; and the concentration of glutaraldehyde was limited to 35%. Step B. The reaction of glutaraldehyde with hydrogen cyanide to form the biscyanohydrin (111) was both temperature- and pH-sensitive, and reaction conditions had to be carefully controlled. The acidic glutaraldehyde solution obtained from Step A showed little or no tendency to react
i CHO
‘OH I50
/CH-cN I OH
4
Irnl
I25
NH-C=O
I
>NH
1
1
,CH-C=O
‘CH-
C=O >NH
I
”-C=O “2 CH-CO2H I
icn2i3
‘CH-COz
H
75
IS! O,b I * DEC4RBOXYLASE
i
“42 hCI0,
STEP
I I
OLI
DOWEX 5 0 RESIN l H t 1
CONDITIONS
“2
I
I cn-cozn
ICH213 C ‘ H 1
- C0 2 H
lPbl
“2
2 0 4 wl% AQUEOUS 013 GLUTARPLDEHYDE SOLUTION REACTED WITH 4 N HCN:NHI:COZ MOLAR R4TlO O F 2 1:70:5.0. CONTOUR L I N E S REPRESENT MOL4R X YIELD V4LUES O F DI4MINOPIMELIC ACID
CH-NH2
I COLH
(HI
iLI
(DLi
25 TIME l H R . 1
*Xo:rnero; X b Z D L
Figure 1. melic acid
Synthesis of t-lysine via meso, Dt-diaminopi-
with hydrogen cyanide. However, adjustment of the p H 4 to 6 by addition of a srnall amount of dilute aqueous ammonia or ammonium bicarbonate solution brought about a vigorous exothermic reaction. High yields of diaminopimelic acid (V) \vcre obtained only if this exotherm was controlled by external cooling; the best results came from runs where the temperature of the reaction mixture was maintained a t 0’ or lower. Step C. After the exotherm had subsided, cold concentrated aqueous ammonia was added in one portion near 0 O KO give the desired biscyanohydrin-ammonia ratio. T h e necessary amount of carbon dioxide was then charged and the mixture was heated under autogenous pressure. Highest yields of diaminopimelic acid were obtained where the molar ratio of glutaraldehyde-hydrogen cyanide-ammonia-carbon dioxide approached 1.0:2.1:7.0:5.0. As might be expected, a more pronounced deleterious effect was observed when the ammonia-carbon dioxide ratio was below rather than above the optimum value. Moreover, the use of only a slight excess of hydrogen cyanide was found desirable to repress the polymerization of this reactant. The form in which the carbon dioxide and ammonia were charged did not appear to have any special significance. comparable results being obtained with ammonia-ammonium bicarbonate. ammonia-ammonium carbonate, ammonia-gaseous carbon dioxide, or ammonia-ammonium carbamate. Urea was ineffectual. The preferred source of carbon dioxide was ammonium bicarbonate which, unlike ammonium carbonate, is available in relatively pure form ( > 9 8 7 , NHIHCOP), facilitating the study of optimum reactant ratios.
Figure 2. Diaminopimelic acid yield as related to Step reaction conditions
C
The effect of time and temperature on the hydantoin-forming reaction has been appraised using a statistical scatter of experiments. Conditions were use of a 20.4y0 aqueous solution of glutaraldehyde, a 1.0:2.1:7.0:5.0 molar ratio of glutaraldehyde-hydrogen cyanide-ammonia-carbon dioxide, a premixing temperature of 0’ to - S o , and operation under autogenous pressure. .4s shown in Figure 2, highest yields of diaminopimelic acid were realized using a 3- to 5-hour reaction cycle at looo to 12s0 C. Step D. T h e products formed in Step C to diaminopimelic acid (\.a, b) were hydrolyzed using hot sulfuric acid. This reaction took place in essentially quantitative yield, if it was run with a considerable excess of acid a t a relatively high temperature. Operating a t 150 with 65% sulfuric acid under autogenous pressure, a 6 to 1 molar ratio of sulfuric acid to trimethylene bishydantoin equivalent gave essentially cornplete hydro1)sis in 2 hours, whereas the hydrolysis was only 80% complete after 10 hours using a 2 to 1 acid-bishydantoin ratio (Figure 3). As expected, the rate of reaction dropped off sharply as the reaction temperature was decreased-hydrolysis amounted to only 87Y0 in a run carried out for 96 hours a t 123O in an open vessel. Probable Reaction Intermediates in Steps B to
D
The four-step reaction sequence described above yielded predominantly one by-product-2,6-piperidinedicarboxylic acid (XI). Its most likely precursor is 2,6-dicyanopiperidine (VII), which precipitated when a n ammoniacal solution of VOL. 2
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CONDITIONS: l50'/65% HzS04 REACTANTS S E A L E D I N GLASS TUBE UNDER VACUUM
*_--___--------
H SO 2* 4 :
M O L A R RATIO
30
*10
EISHYDANTOIN I
________--_ 3 -: -_
O
y I
I
4
Figure 3.
I
5 T I M E IHR.1
I
I
I
I
I
6
7
8
9
10
Per cent hydrolysis of trimethylene bishydantoin vs. molar ratio of
glutaraldehyde biscyanohydrin (111) was allowed to stand for several hours (5). Though this material is sufficiently stable to permit recrystallization from either water or concentrated aqueous ammonia, it readily underwent alkaline hydrolysis to yield a mixture of 2,6-piperidinedicarboxylic acid (XI) and 2,6-piperidinedicarboxamide (X). Its ability to function as a precursor of trimethylene bishydantoin (IV) or one of its openchain analogs-e.g., XII-has also been demonstrated. Treatment of the dinitrile Izith ammonium bicarbonate and aqueous ammonia produced material Lvhich upon hydrolysis with sulfuric acid gave diaminopimelic acid (V) in excellent over-all yield. The key nature of 2,6-dicyanopiperidine (VII) in the synthesis was indicated further by the fact that 2,6-piperidinedicarboxylic acid ( X I ) invariably was formed a t the expense of diaminopimelic acid (V). The possibility that glutaraldehyde bisaminonitrile (VIII) acts as a precursor of diaminopimelic acid is not excluded. However, the apparent high lability of this bisaminonitrile has prevented its isolation and characterization. Likely intermediates in the synthesis starting with glutaraldehyde biscyanohydrin (111) are shown in Figure 4.
HzS04
,IcHNH
CH - C 0 2 H
NH-C=O
"e
I im1
IIPI
"1
Various strains of B. sphaericus, including an asporogenous variant of ATCC 10208 selected by culturing on the medium described by Gladstone and Fildes ( 3 ) ,were used. Cultures were preserved by lyophilization or by drying on fish spine beads over anhydrous calcium sulfate (6). Inocula for growth were started in trypticase-soy broth (Baltimore Biological Laboratory) in Erlenmeyer flasks agitated on a New Brunswick rotary shaker operated a t room temperature (25' (2.). The production medium selected for growth of the organism contained 2.5% beet molasses (50.7% sugar), 2.0% cornsteep liquor (507, solids), 0.5% N-Z Amine Type A, 1.0% BYF (yeast extract), and 0.25y0 diammonium hydrogen phosphate 310
l&EC P R O D U C T RESEARCH A N D DEVELOPMENT
H
I
1x1
CH
- COZH
I ICH213 CH
- C02H
I "2
1x1
1 x 1 Figure 4.
Probable reaction intermediates in Steps B to D
28.0 ;
513-
p
J
26.0
Y U
4.0
2u z
-
24.0 Y
22.0
3.0
-
2.0
-
8.Cl
r-
7.0
t
1.0
I
1
I
I
2
4
6
Figure
1
I
IO 8 T I M E 1HR.I
1
I
1
12
14
16
20.0
i
1
60;
14.0
12.C
5. Fermentation of 6. sphaericus 7054
made u p in tap water and adjusted to p H 6.8 with potassium hydroxide. Fermentation. All equipment was sterilized for 1 hour a t 121 O C. before use. A 5y0 inoculum was used generally. Cultures were grown a t 32-33' C. for 12 to 18 hours with a n aeration rate amounting to 2 mmoles of 0 2 per liter of medium per minute, using vigorous agitation. A silicone preparation (Dow Corning Antifoam AP) was employed to control foaming. Cells were harvested by centrifugation. Maximum cell yields (10 to 12 grams per liter) were obtained generally in 6 to 8 hours, although maximum enzyme activity was reached in 10 to 14 hours.
A typical fermentation involving B. sphaericus 7054 is described in Figure 5. In this run the cell yield, p H of the medium, and enzyme activity were followed for 18 hours. Small aliquots were taken hourly for cell yield determination while larger samples of cells were taken every other hour for enzyme activity determination. These cells were harvested by centrifugation, washed, and made into an acetone poirder to facilitate enzyme assay. The growth pattern observed is typical of bacterial growth. Maximum cell yield occurred after approximately 8 hours, peak enzyme activity being noted at 12 hours. The significance of maximum cell yield and a simultaneous change in slope of the p H plot at approximately 8 hours is not known. The high p H of 9 noted a t the end of the fermentation was observed in all fermentations involving the B. sphaericus organism, the odor of ammonia usually being strong when the fermentor \vas opened to harvest the cells. Since maximum cell growth took place a t p H 7.5 to 7.9, a fermentation was carried out at p H 6.8 to 7.8. N o significant improvement in cell yield or enzyme activity was observed. Enzymatic Decarboxylation of Diaminopimelic Acid (Step E)
Bacillus sphaericus cells have been examined under a variety of conditions for decarboxylating diaminopimelic acid. In general, the cells in the form of an aqueous slurry were mixed
with an aqueous solution of the amino acid and the p H of the mixture was adjusted quickly to 6.8 with the aid of a diammonium hydrogen phosphate buffer. The decarboxylation was then accomplished by shaking at 30' to 40' for at least 8 hours. Some decarboxylations required the addition of pyridoxyl phosphate for a maximum reaction rate, but if fresh samples of cornsteep liquor u e r e used, this cofactor uiually could be dispensed with. In some cases where the batch of cornsteep liquor failed to contribute sufficient cofactor to the cells. addition of pyridoxine to the culture 2 hours before harvesting produced cells having enhanced decarboxylation activity. T h a t B . sphaerms decarboxylates only the meso-isomer of diaminopimelic acid was established by examining the undecarboxylated acid recovered from the decarboxylation reaction. This material exhibited n o optical activity. Moreover. it could be resolved into two fractions by paper chromatography under conditions known to separate meso,DLdiaminopimelic acid into the L- isomer and a mixture of the meso- and D- isomers (8). The presence of the L- isomer i n the mixture along Ivith an equal amount of the D- isomer is therefore indicated. The possibility that both the D- and L- isomers could have been decarboxylated at an equal rate, leaving an optically inactive racemate. qtill had to be considered. However, an authentic sample of the D- isomer (a gift of the late J. P. Greenstein) ( 9 ) \\as sho\.vn not to decarboxylate. Hence it folloivs that neither the D- or L- isomer is decarboxvlated. This conclusion is confirmed by the fact that no more than SO'$& of the charged diaminopimelic acid could be decarboxylated (see Figure 6), the yield expected from synthetically produced diaminopimelic acid which ivould consist of 50% meso- and 50% DL- isomers. A study of the diaminopimelic acid racemase would have been valuable, but was not made. Rupture of the semipermeable membrane coating of the B. sphaericus cells \+as found necessary to obtain consistent decarboxylation results. Once this has been accomplished, transport of the diaminopimelic acid substrate was no longer VOL. 2
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50
-
45
-
z 0 4
A X
P 4
8
s
40-
35
-
CONDITIONS. 5.09 D l A M l N O P l M E L l C ACID I N l O O m l H p M J X E D W I T H 00006g.P Y R I D O X Y L P H O S P H A T E A N D INCUBATED AT SHAKING.
33' FOR 20 HR. W I T H
35 2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10 0
11.0
12 0
DIAMINOPIMELIC ACIO/DRY C E L L RATIO
Figure 6. Decarboxylation of a 5% diaminopimelic acid solution b y 6. sphaericus at various diaminopimelic acid-dry cell ratios
limiting, and enzyme activity was increased. Agents that accomplished this rupture without apparent damage to the enzyme include acetone? iso-octyl alcohol, and toluene. The activity of acetone-dried cells, for example, was often several times greater than that of fresh cells. Decarboxylation activity was also enhanced when various surface-active agents were added. Removal of fat from the bacterial cells by all of these agents could also contribute to the faster decarboxylation rates observed. The influence of p H and of diaminopimelic acid-dry cell ratio has been considered in determining optimum conditions for decarboxylation. An optimum p H of 6.8 is reported for decarboxylations carried out with B. sphaericus ( 7 ) . However, under the conditions used by us the activity reached a maximum at p H 7 . 5 . With regard to optimum reactant concentration, a series of decarboxylations was carried out in which the weight ratio of diaminopimelic acid to dry cells was varied from 2.5:l.O to 12.5:l.O (Figure 6). A significant decrease in yield was noted at ratios greater than 6 to l . A ratio of approximately 3 to 1 was used in the decarboxylations reported. Lysine was routinely recovered from the decarboxylation mixture by adsorption on an ion exchange resin and subsequent elution. After removal of bacterial cells by filtration (or centrifugation), the lysine-containing liquor was passed through Dowex 50 resin (NHd+ form). After a water wash, lysine was recovered from the resin by elution with aqueous ammonia. The ammonia was removed by boiling and the solution titrated with HC1 to a p H of 5.15. The amount of lysine present as the monohydrochloride could then be calculated from the HCI 312
I&EC PRODUCT RESEARCH A N D D E V E L O P M E N T
titer. Relatively pure L-lysine monohydrochloride was recovered from this solution by boiling off the solvent in vacuo. Epimerization of DL-Diaminopimelic Acid (Step F)
Inasmuch as the enzymes from B. sphaericus decarboxylate only the meso- isomer (Va). the DL-diaminopimelic acid (Vb) Mhich goes through Step E unchanged must be converted into the meso- isomer by epimerization. This can be accomplished by treatment with aqueous sulfuric acid at 150'. but the rate of reaction is slow. A more efficient procedure uses Dowex 50 ion exchange resin in place of sulfuric acid. By heating this resin for about 6 hours at 180' after it had been saturated with the DL-amino acid, the desired 50-50 mixture of the meso- and DL- isomers could be isolated. The rate of hydrolysis of the sulfonic acid groups in the resin was negligible under these conditions. provided the operation was carried out in an inert atmosphere-e.g., nitrogen. Experimental
Conversion of 2-Ethoxy-3,4-dihydropyran(I) to Diaminopimelic Acid (V) (Steps A to D). To 100 ml. of 0.1% aqueous sulfuric acid was added 25.8 grams of 2-ethoxy-3,4-dihydropyran with vigorous agitation. The resulting suspension was warmed rapidly to 65" to 7 5 O , a t which temperature the immiscible phase quickly disappeared. The clear solution which was formed was quenched immediately to 0 to - 5 and 16 ml. (11.7 grams) of hydrogen cyanide was added in a single portion with stirring. The temperature was then reduced quickly to - 5 o to -7 o. Five milliliters of cold 57, aqueous ammonia was added dropwise with external cooling to maintain the temperature below 0'. The exothermic reaction was over by the end of this addition, and 27 ml. of
cold 287, aqueous ammonia was then added in one portion. it was After the resulting mixture had been chilled to -5', charged into a cold Hastelloy C-lined shaker tube containing 79 grams of ammonium bicarbonate. T h e tube was sealed quickly, purged lightly \vith nitrogen, and brought rapidly t o 100' with shaking. After the tube had been shaken a t 100' for 5 hours under autogenous pressure. it was stored for about 16 hours a t 0' to 5'. It was then opened and the slurry of yellowish \vhite crystals discharged with the aid of methanol and water ivashings. The washings were combined with the slurry and the mixture was taken to dryness by heating under vacuum in a Rinco evaporator. The resulting salt cake weighed 36.6 grams and could be used without further purification in the hydrolysis step. To 0.200 gram of the salt cake in a micro-Carius tube was added 0.8 ml. of 657,aqueous sulfuric acid, the tube was sealed under vacuum, and the sample was heated a t 150" for 4 hours. The resulting mixture of products contained 0.142 gram of diaminopimelic acid ; this represents a 907, yield. Workup of Products from Step D. The acidic solution obtained from the hydrolysis of trimethylene bishydantoin was diluted i\-ith \vater until it contained about 3% aqueous sulfuric acid. then passed through 20- to 50-mesh Dowex 50-8s resin (H' form; pretreated by washing with 3y0 aqueous sulfuric acid). ,\pproximately 100 rnl. of resin was used for each gram of combined weight of diaminopimelic acid and 2.6-piperidinedicarboxylic acid in the sample. Excess sulfuric acid \cas removed by washing the resin \vith water, leaving the diaminopimelic acid and 2,6-piperidinedicarboxylic acid completely absorbed on the resin. Eluiion Jvith 207, aqueous ammonia removed the latter t\vo materials. After the excess ammonia had been removed by boiling. the sample \vas ready for assay for diaminopimelic acid. DETERMINATION OF 2,6-~IPERIDIYEDICARBOXYIIC ,4CID. An aliquot of the sample solution \vas evaporated by heating in vacuo and the resulting salt cake heated for 2 hours at 120'~. then disolved in 100 ml. of \vater. A second aliquot containacid ing about 0.02 to 0.10 gram of 2,6-piperidinedicarboxylic \vas taken and to it \vas added 2.0 ml. of I S S a O H . The basic solution ivas then boiled until it \vas free of ammonia, cooled, and back-titrated to p H 7.0 \vith O.lOAt' HCI, using a p H meter. 2.5-Piperidinedicarboxylic acid \vas determined using the folloiving equation : \ V t . i y . i of 2.6-piperidinedicarbosylic acid =
__ 1'3 [(ml~?;aOH)(~vr;aOH) - (ml.HClj(dx-Hcl)l
1000
Preparation of L-Lysine Monohydrochloride (VI) from Diaminopimelic Acid (V) (Step E). T o 29.5 grams of diaminopimelic acid lverc. added 10 grams (dry \?..eight) of B. sphaericus 7051 cells. 0.004 gram of pyridoxyl phosphate. 4 ml. of iso-octyl alcohol. and tap 'ivater to give a total volume of 335 nil. After agitation a t 33 ' for 20 hours, the cells \vex removed by filtration and the supernatant liquid was passed through Doicex 50 resin ( S H I T form). Lysine was recovered from the resin by elution ivith 207, aqueous ammonia. After removal of ammonia by boiling, the solution was titrated to p H 5.15 Lvith hydrochloric acid and taken to dryness by heating jn vacuo. This yielded 14.9 grams of L-lysine monohydrochloride. 52% based on the diaminopimelic acid charged. The HCI titer could also be used to determine the yield of L-lysine by the following equation. (A comparable yield figure was obtained \\.hen L-lysine was assayed manometricallv using lysine decarboxylase ; the manometric method was less convenient, however.)
5.; 1)-sinemonohydrochloride
=
ml. of HC1 X . Y H ~ ,X 0.146 X 100 wt. of sample Colorimetric analysis of the effluent indicated the presence of 12.0 grams of unreacted diaminopimelic acid, a 417, recovery. The total diaminopimelic acid charged was thus accounted for to the extent of 93%. The recovered diaminopimelic acid was recycled by initially absorbing it on Dowex 50 resin ( H + form) and heating the wet saturated resin a t 180' for 5 hours under nitrogen. Elution of the resin Lvith 2093 aqueous ammonia yielded 10.4
grams of mesopL-diaminopimelic acid. Incubation of the latter with 3.1 grams (dry \ in the presence of air gave a 947, recovery of diaminopimelic acid which had been epimerized to the extent of 887,. The DoLvex 50 resin underwent 77, hydrolysis in this experiment. In a second run a t 180' for 6 hours in the presence of air the resin undenvent 14.47, hydrolysis. TREATMENT WITH 40% A Q U E O C S S U L F U R I C A C I D . .A 0.25gram sample of DL-diaminopimelic acid was dissolved in 3.0 ml. of 40% (by weight) sulfuric acid and sealed in a micro-Carius tube under vacuum. The tube after heating at 180' for 16 hours was cooled and the contents were discharged. The product mixture was diluted ivith 27 ml. of water, decolorized with Darco G-60, and filtered. T h e filtrate was then passed through 250 ml. of Do\vex 50-8X resin (NH4+ form) and the VOL 2
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DECEMBER 1963
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absorbed m-diaminopimelic acid eluted with one bed volume of 20y0 aqueous ammonia. The eluate was boiled to remove ammonia and taken to dryness by heating a t 120' for 2 hours. Manometric assay of the resultin product indicated that it contained 37.5% meso- isomer, 7 5 6 epimerization. Determination of Diaminopimelic Acid. COLORIMETRIC ASSAY( 2 ) . Using a stock solution containing 0.0010 gram of diaminopimelic acid per ml., a series of color standards was prepared as follows. Ten test tubes were set u p containing from 0.00 to 0.09 ml. of the stock solution in 0.01-ml. incremental amounts. After 2 drops of concentrated hydrochloric acid had been added to each tube, color was developed by the addition of 0.50 ml. of 5% ninhydrin in methyl Cellosolve. Each tube was then brought to a volume of 5.5 ml. and a color comparison made immediately with the test solutions under consideration. The solution to be tested for diaminopimelic acid content was diluted or concentrated so that it contained approximately 0.0002 to 0.0010 gram per ml. of the amino acid. Sample tubes were then prepared a t four concentration levels. After 2 drops of concentrated hydrochloric acid and 0.50 ml. of the ninhydrin solution had been added, the tubes were heated in boiling water for '/3 hour to develop the color. After the samples had been diluted with 5.0 ml. of an equivolume mixture of 1-propanol and water, the absorbance a t 420 m,u was determined in a spectrophotometer. The amount of diaminopimelic acid in the sample was then calculated from the standard curve, using the appropriate dilution factor. No appreciable interference was caused by lysine, 2,6piperidinedicarboxylic acid, ammonia, or sulfuric acid, if these materials were present a t levels comparable to that of the diaminopimelic acid. MANOMETRIC ASSAY. One milliliter of solution containing 0.001 to 0.003 gram of meso,DL-diaminopimelic acid was added to the main chamber of a manometer flask together with 2.0 ml. of 0.2M diammonium hydrogen phosphate buffer solution (pH 6.8) and 20 ,ug. of pyridoxyl phosphate. Using acetonedried cells of Bacillus sphaericus lacking L-diaminopimelic acid racemase? 0.030 gram of this material, well slurried in 0.5 ml. of the buffer solution, was added to one arm of the flask. T o the other arm was added 0.25 ml. of 2M aqueous citric acid solution. The flask was degassed by shaking with nitrogen at 37" for 10 minutes. The assay was accomplished by first introducing the enzyme, shaking the resulting mixture for 3 hours, then introducing the citric acid solution to expel all of the carbon dioxide. The amount of meso- isomer contained in the sample was calculated using the equation :
yc meso-diaminopimelic acid
=
Ah,,,, X k X 190 22.4 X 10 X mg. ofsample
where k = flask constant for C O S Ah,,,, = correction of manometer readings for blank
314
l & E C P R O D U C T RESEARCH A N D D E V E L O P M E N T
Preparation of 2,6-Dicyanopiperidine (VII). To 0.1 ml. of concentrated sulfuric acid in 50 ml. of water was added 25.6 grams of 2-ethoxy-3,4-dihydropyranand the resulting mixture was brought rapidly to 65 O to 75 with vigorous stirring. As soon as the immiscible layer had disappeared. the solution was cooled to 0 " to 5 '. After the p H was adjusted to 8 with aqueous ammonia, 15.6 ml. (1 1.O grams) of hydrogen cyanide was added in one portion, followed by dropwise addition of 13.5 ml. of 28% aqueous ammonia over a 10-minute period. After stirring for 45 minutes the temperature of the mixture had risen to 31'. The reaction vessel was then surrounded by an ice bath and stirred for 2 hours. The crystalline product which had formed was filtered off and air-dried. It weighed 13.3 grams (49%) and melted a t 113-14" [reported m.p. 114-15' (5)]. Analysis. Calculated for CiH9N3: C, 62.22; H , 6.67; N, 31.11. Found: C, 62.36; H , 6.93; S , 31.00. Conversion of 2,6-Dicyanopiperidine (VII) to Diaminopimelic Acid (Va, b). In a Hastelloy C-lined pressure tube was placed 17.2 grams of 2,6-dicyanopiperidine. After 85 grams of ammonium bicarbonate, 25 ml. of 28% aqueous ammonia, and 50 ml. of water had been added, the tube was sealed, purged with nitrogen, and heated a t 100' for 4 hours under autogenous pressure with shaking. .4fter cooling, the reactants were discharged and hydrolyzed with aqueous sulfuric acid. Colorimetric assay indicated the presence of diaminopimelic acid in 81 yo yield. Titrimetric assay showed that 2,6-piperidinedicarboxylicacid had been produced in 9% yield. Acknowledgment
The authors acknowledge the contributions of R. D. Emmick, N. L. Hause, T. E. Londergan, W. H. Todd, and D. E. Tuites. literature Cited
(1) Dewey, D. L., Hoare, D. S., Work, E., Biochem. J . 58, 523 (1954). ( 2 ) Gilvarg, C., J . B i d . Chem. 233, 1501 (1958). (3) Gladstone, G. P., Fildes, P., Brit. J . Exptl. Pathol. 21, 161 (19401.
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